David J Aitken

Sandrine Marie-Christine Thomas

Sylvie

Catherine Florence

Isabelle Great

Jean Pierre Baltaze

Julien

Ahmad Mathieu

for your help and support in the lab, such as HPLC, chemicals and solvents, consumables. I am so happy to work together with all of you, and you are

Aitken research group (currently in CP3A at ICMMO) is interested in various cyclopeptide natural products (for example cyclotheonamide C), and has already developed various synthetic methodologies for the preparation of β-and γ-amino acids and especially of some polyhydroxy-β-amino acids present in microsclerodermins (APTO, AETD). Microsclerodermins this latter, a class of marine cyclic peptides with novel structures and biological activities appeared to us as interesting and challenging targets.

The objective of this project is the total synthesis of microsclerodermin D (Figure 1), which features a 23-membered ring macrocyclic peptide, containing six amino acids, of which three are common: glycine (Gly), sarcosine (Sar) and (R)-γ-amino-β-hydroxybutyric acid (GABOB). The other three amino acids possess some structural variations, but they can be classified as a modified tryptophan (TrpAA), a polyhydroxylated β-amino acid (APTO) containing four contiguous stereogenic centers and an original 3-amino-4-hydroxypyrrolidone-4-acetic acid (PyrAA). We plan to develop a modular synthetic route to microsclerodermin D that could be applicable for the preparation of other microsclerodermin family members and analogues thereof. We are also looking forward to make some investigations on their biological activities or potential as anticancer drug. In Chapter 1, an overview is presented on bioactive natural products, peptides and marine cyclic peptides, as well as a very detailed introduction on microsclerodermins, including their structure and origin, biological activities and their previous syntheses. Subsequently, the objective of this project, the total synthesis of microsclerodermin D, is presented.

In Chapter 2, we focus on the synthesis of a key dipeptide, masked-PyrAA-Sar , required for the preparation of microsclerodermin D, including its synthesis and evaluation of the unmasking strategy. Firstly, an introduction of previous synthesis pyrrolidinoacetic acid derivatives, as well as dipeptide PyrAA-Sar is presented. Subsequently, the synthesis of racemic masked-PyrAA-Sar dipeptide is completed following our proposed synthetic strategy and evaluation of the unmasking strategy, deprotection of the dithioketal and spontaneous cyclization, is described. We then examine access to optically pure β-ketoester. After much effort, the optically pure β-ketoester was obtained via condensation of a N-Fmoc protected UNCA with a magnesium hemimalonate, facilitating the synthesis of the key dipeptide: masked-(S)-PyrAA-Sar and masked-(R)-PyrAA-Sar.

In Chapter 3, we describe the synthesis of the dipeptide Trp-Gly fragment required for the preparation of microsclerodermin D. Following a short introduction on the dipeptide Trp-Gly in microsclerodermins and an overview on the synthesis of tryptophan derivatives, the successful synthesis of H-[(R)-Trp (6-Cl)]-Gly-Ot-Bu is presented, as well as the synthesis of racemic dipeptide Trp-Gly.

In Chapter 4, we consider the synthesis of dipeptide GABOB-APTO. After a short introduction on the dipeptide GABOB-APTO fragment required for the preparation of microsclerodermin D, our synthetic strategy is proposed. The successful synthesis of GABOB-APTO is presented, including the synthesis of GABOB and protected APTO.

In Chapter 5, a short introduction is given on the strategy of macrocyclic peptide assembly: peptide coupling and macrocyclization. The installation of tetrapeptide masked-PyrAA-Sar-Trp-Gly and hexapeptide masked-PyrAA-Sar-Trp-Gly-GABOB-APTO is

Bioactive natural products

Nature has been an inexhaustible source of novel chemotypes and pharmacophores, 1,2 representing highly promising bioactive agents. Bioactive natural products [START_REF] Milshteyn | Accessing Bioactive Natural Products from the Human Microbiome[END_REF][4][START_REF]Bioactive Natural Products: Detection, Isolation, and Structural Determination[END_REF][6][7][START_REF] Clark | Natural Products as a Resource for New Drugs[END_REF][START_REF] Avendaño López | Natural Products as Sources of New drugs. A General Overview[END_REF][START_REF] Bohlin | Natural Products in Modern Life Science[END_REF][11] possess various biological activities and play a significant role in the discovery of novel therapeutics and medicinal agents, including a myriad of organic compounds, roughly classified into such as nucleic acid, carbohydrates, lipids, proteins, peptides, amino acids, alkaloids, terpenoids, polyketides, flavonoids and so forth, [START_REF] Dias | A Historical Overview of Natural Products in Drug Discovery[END_REF]13 which originate as primary and secondary metabolites produced by a variety of organisms, such as animals, plants, microorganisms, sponges, cyanobacteria, vertebrates and invertebrates. [START_REF] Irchhaiya | Metabolites in Plants and its Classification[END_REF][START_REF] Bérdy | Bioactive Microbial Metabolites: A Personal View[END_REF][START_REF]Studies in Natural Products Chemistry[END_REF] For thousands of years, natural products have played a significant role in human health care and leads for the development of drugs and valuable bioactive agents to treat various human diseases, 1,2,[START_REF] Butler | The Role of Natural Product Chemistry in Drug Discovery[END_REF][START_REF]Natural Products as Leads to Anticancer Drugs" M. Gordaliza[END_REF][START_REF] Butler | Natural Products -The Future Scaffolds for Novel Antibiotics[END_REF] such as artemisinin in the treatment of malaria, or paclitaxel and its analogs in the treatment of various cancers. Historically, several ancient civilizations, such as the ancient Chinese, Egyptians, Indians, Greeks and North Africans have provided written evidence for the use of natural products in traditional medicines as remedies, potions and oils for the treatment of many human illnesses. [START_REF] Dias | A Historical Overview of Natural Products in Drug Discovery[END_REF][START_REF]Phytochemistry and Medicinal Plants[END_REF] For instance, the first record on the use of natural products in medicine was written on clay tablets in cuneiform from Mesopotamia dating from approximately 2600 B.C., when people used oils of Cedrus species (cedar) and Cupressus sempervirens (cypress), Glycyrrhiza glabra (licorice), Commiphora species (myrrh), and Papaver somniferum (poppy juice), all of which are still in use today for the treatment of ailments ranging from coughs and colds to parasitic infections and inflammation. 1 In addition, the Chinese Materia Medica (Chinese herb guides) on the use of herbaceous plants has been extensively documented over the millenia. The Wu Shi Er Bing Fang A.D. 1 Even now, the World Health Organization (WHO) estimates that approximately 80% yield of the world's population relies primarily on traditional medicines as sources for their primary health care, especially for millions of people in the vast rural areas of developing countries. [START_REF] Farnsworth | Medicinal Plants in Therapy[END_REF] The early days of organic chemistry were closely connected to natural products chemistry.

For a long period up to the 1960's, the structural studies of natural products served as the primary driving force for the discovery of new chemical reactivity. [START_REF]Natural Products Chemistry in the Third Millennium" V. Bankova[END_REF] As Woodward said, "Organic chemistry has literally placed a new nature beside the old… There is excitement, adventure, and challenge and there can be great art in organic synthesis." [START_REF] Woodward | Synthesis[END_REF] It is noteworthy that an explosive and diversified growth has taken place in the interest of natural products and natural products chemistry, 2 which have made, and continue to make an essential contribution to the discovery and innovation of novel drugs and bioactive agents, being based on their diversity of structures and biological activities. 1,[START_REF]Natural Products as Leads to Anticancer Drugs" M. Gordaliza[END_REF][START_REF] Kingston | Modern Natural Products Drug Discovery and Its Relevance to Biodiversity Conservation[END_REF][START_REF] Kingston | Natural Products as Drug Leads: An Old Process or The New Hope for Drug Discovery?[END_REF] A huge number of natural products have been isolated and reported to possess diverse biological activities, such as antibiotic, antiviral, antifungal, anticancer, immunosuppressive and anti-inflammatory etc... [START_REF] Pham | A Review of the Microbial Production of Bioactive Natural Products and Biologics[END_REF] In the last 40 years, almost 65% yield of new drugs approved were classified as naturally derived (unmodified natural product, modified natural product, or semi-synthetic compound with a natural product as pharmacophore). [START_REF] Newman | Natural Products as Sources of New Drugs from 1981 to 2014[END_REF] Thus, an overwiew on some interesting and selective bioactive natural products with diverse structure and biological activities will be presented. It will help us to have a more comprehensive and detailed understanding on bioactive natural products and their discovery and isolation, as well as the importance in the discovery of novel drugs and medicinal agents.

Artemisinin (Figure 2), an endoperoxide sesquiterpene lactone isolated by Tu in 1972 from the Chinese herb Artemisia annua L., is currently recommended as a first-line antimalarial agent by the WHO against multi-drug resistant strains of Plasmodium falciparum malaria, cerebral malaria, and infant's malaria, and also used to treat infections caused by Schistosoma spp., Pneumocystis carinii, Toxoplasma gondii, human cytomegaloviruses, Herpes simplex viruses (HSV) and hepatitis B and C. [START_REF] Ziffer | Artemisinin: An Endoperoxidic Antimalarial from Artemisia Annua L[END_REF][START_REF] Wu | Qinghaosu (Artemisinin) -Revelations from History and Reality[END_REF][START_REF] Tu | The Discovery of Artemisinin (Qinghaosu) and Gifts from Chinese Medicine[END_REF][START_REF]Key Natural Products in Malaria Chemotherapy: From Quinine to Artemisinin and Beyond[END_REF][START_REF] Abdin | Artemisinin, A Novel Antimalarial Drug: Biochemical and Molecular Approaches for Enhanced Production[END_REF] It has already been described in traditional Chinese medicine as a remedy for chills and fevers, [START_REF] Wu | Qinghaosu (Artemisinin) -Revelations from History and Reality[END_REF][START_REF]A Brief History of Qinghaosu[END_REF][START_REF]Qinghaosu (Artemisinin): An Antimalarial Drug from China[END_REF][START_REF]The History of Qing Hao in the Chinese Materia Medica" E. Hsu[END_REF] and was approved in 1987 by the US Food and Drug Administration (FDA) for use the treatment of malaria as a safe drug with few side effects. [START_REF] Newman | Natural Products as Sources of New Drugs from 1981 to 2014[END_REF] Artemisinin and its synthetic derivatives have also been shown to be promising prototypes for the development of new antiproliferative agents against certain tumor cells. [START_REF] Da Silva | Artemisinin: A Promise for the Development of Potent Anticancer Agents[END_REF][START_REF] Chaturvedi | Artemisinin and Its Derivatives: A Novel Class of Anti-Malarial and Anti-Cancer Agents[END_REF] In 2015, Tu shared the Nobel Prize in Physiology or Medicine to for her discovery and development of artemisinin as a novel therapy against Malaria. [START_REF]Recognition of the Role of Natural Products as Drugs to Treat Neglected Tropical Diseases by the 2015 Nobel Prize in Physiology or Medicine[END_REF][START_REF] Wang | Haem-Activated Promiscuous Targeting of Artemisinin in Plasmodium Falciparum[END_REF] Figure 2: Artemisinin (+)-Amphidinolide T1 (Figure 3) is a 19-membered ring macrolide, one of amphidinolides series that was first isolated by Kobayashi in 2000 from the Y-56 strain of the marine dinoflagellate Amphidinium sp., [START_REF] Tsuda | Amphidinolide T, Novel 19-Membered Macrolide from Marine Dinoflagellate Amphidinium sp[END_REF] in addition to four other amphidinolides T2, T3, T4 and T5. [START_REF] Kubota | Amphidinolide T5, A New 19-Membered Macrolide from a Dinoflagellate and X-Ray Structure of Amphidinolide T1[END_REF][START_REF]Amphidinolides T2, T3, and T4, New 19-Membered Macrolides from the Dinoflagellate Amphidinium sp. and the Biosynthesis of Amphidinolide T1[END_REF] The amphidinolide family comprises cytotoxic macrolides of different size from 12 to 27 membered ring with diverse structural features and pronounced biological activities which have been isolated

by Kobayashi and his research group since 1986. [START_REF]Amphidinolides: Unique Macrolides from Marine Dinoflagellates[END_REF][START_REF] Chakraborty | Chemistry of Potent Anti-Cancer Compounds, Amphidinolides[END_REF] They show potent cytotoxic activity against murine lymphoma L1210 and human epidermoid carcinoma KB cell lines, and potent antileukemic, antineoplastic activity. [START_REF] Kobayashi | Bioactive Macrolides and Polyketides from Marine Dinoflagellates[END_REF][START_REF] Kobayashi | Bioactive Macrolides and Polyketides from Marine Dinoflagellates of the Genus Amphidinium[END_REF][START_REF] Kitagawa | Antitumor Marine Natural Products[END_REF] Figure 3: (+)-Amphidinolide T1

Paclitaxel (Figure 4), known as Taxol ® , is perhaps the most exciting plant-derived anticancer drug discovered in the last fifty years. This tricyclic diterpenoid was first isolated by Wall and Wani in 1971 from bark of the Pacific yew, Taxus Brevifolia, [START_REF] Wani | Plant Antitumor Agents. VI. Isolation and Structure of Taxol, a Novel Antileukemic and Antitumor Agent from Taxus Brevifolia[END_REF] and was found to promote the assembly of tubulin into microtubules in 1979 by Schiff and Horwitz. [START_REF] Schiff | Promotion of Microtubule Assembly in vitro by Taxol[END_REF] It also shows excellent and reproducible activity against human solid tumor (such as antileukemic, P388, P1534, L1210 and HCT116 cell lines), and possesses an unusually broad spectrum of potent anticancer activity to treat various types of cancers, including ovarian cancer, breast cancer, lung cancer, Kaposi sarcoma, cervical cancer, pancreatic cancer and others, although some side effects may include hair loss, bone marrow suppression, numbness, allergic reactions, muscle pains, heart problems, increased risk of infection, and lung inflammation. [START_REF]Recent Advances in Medicinal Chemistry of Taxol[END_REF][START_REF]Taxol Science and Applications[END_REF][START_REF]Taxol: From Discovery to Therapeutic Use" M. Suffness[END_REF][START_REF] Kingston | The Chemistry of Taxol, A Clinically Useful Anticancer Agent[END_REF][START_REF]Recent Advances in the Chemistry of Taxol" D. G. I. Kingston[END_REF][START_REF] Cragg | Paclitaxel (Taxol ® ): A Success Story with Valuable Lessons for Natural Product Drug Discovery and Development[END_REF] However, extraction from the bark of Pacific yew gives a very low yield Chapter 1 10 (< 0.0001% dry weight), and three mature 100 years old trees provide only 1 g of paclitaxel, whereas a given course of treatment need 2 g of the drug, so that entire forests would need to be sacrificed. In 1992, Taxol ® was approved by FDA and launched on the US market in the following year by Bristol-Myers Squibb for treatment of refractory breast cancer and ovarian cancer. [START_REF]Recent Advances in Medicinal Chemistry of Taxol[END_REF][START_REF]Recent Advances in the Chemistry of Taxol" D. G. I. Kingston[END_REF][START_REF] Cragg | Paclitaxel (Taxol ® ): A Success Story with Valuable Lessons for Natural Product Drug Discovery and Development[END_REF][START_REF]The Story of Taxol: Ecstasy and Torment" K. Kacprzak[END_REF][START_REF]Tubulin-Interactive Natural Products as Anticancer Agents" D. G. I. Kingston[END_REF] In 1995, FDA has approved docetaxel (Taxotere ® ), a semisynthetic analogue developed by Potier in

France [START_REF] Sénilh | Chemical Studies of 10-Deacetyl Baccatin III. Hemisynthesis of Taxol Derivatives[END_REF][START_REF] Gueritte-Voegelein | Taxol and Derivatives: A Biogenetic Hypothesis[END_REF][START_REF] Guéritte-Voegelein | Chemistry and Structure-Activity Relationships of Taxoids with Modified Skeletons[END_REF][START_REF] Guénard | Taxol and Taxotere: Discovery, Chemistry, and Structure-Activity Relationships[END_REF][START_REF] Denis | Highly Efficient, Practical Approach to Natural Taxol[END_REF] which can be prepared from 10-deacetylbaccatin III initially extracted by Potier and his research group in 1981 from the needles of the European yew, Taxus baccata L.. [START_REF] Chauvière | Structural Analysis and Biochemical Study of Isolated Products of the Yew: Taxus Baccata L. (Taxaceae)[END_REF] The success of paclitaxel spurred an enormous amount of work on the synthesis of analogues. Although paclitaxel and docetaxel are the only taxanes currently approved for clinical use, there are many analogues developed by various pharmaceutical companies and institutes in clinical trials. [START_REF]Phytochemistry and Medicinal Plants[END_REF][START_REF] Kingston | The Chemistry of Taxol, A Clinically Useful Anticancer Agent[END_REF][START_REF]The Story of Taxol: Ecstasy and Torment" K. Kacprzak[END_REF][START_REF] Denis | Highly Efficient, Practical Approach to Natural Taxol[END_REF][START_REF] Rothenberg | Taxol, Taxotere and Other New Taxanes[END_REF][START_REF] Nicolaou | Novel Chemistry of Taxol. Retrosynthetic and Synthetic Studies[END_REF][START_REF] Li | Chemistry of Taxol and its Analogues[END_REF][START_REF]Studies on the chemistry of taxol" D. G. I. Kingston[END_REF][START_REF]Investigations of the Chemistry of Taxol[END_REF][START_REF] Kingston | The Chemistry of Taxol and Related Taxoids[END_REF][START_REF]Taxol: The Chemistry and Structure-Activity Relationships of a Novel Anticancer Agent" D. G. I. Kingston[END_REF] Figure 4: Paclitaxel and derivatives Caminoside A (Figure 5) is a novel antimicrobial tetrasaccharide glycolipid, first isolated by Andersen in 2002 from the marine Caribbean sponge Caminus sphaeroconia. It showed potent activity as the first bacterial type III secretion inhibitor. [START_REF] Linington | Caminoside A, an Antimicrobial Glycolipid Isolated from the Marine Sponge Caminus sphaeroconia[END_REF] Three other Caminosides B-D were isolated by the same group in 2006, [START_REF] Caminosides | Antimicrobial Glycolipids Isolated from the Marine Sponge Caminus sphaeroconia[END_REF] and the first total synthesis of Caminoside A has been completed by Yu in 2005. [START_REF] Sun | First Total Synthesis of Caminoside A, an Antimicrobial Glycolipid from Sponge[END_REF] Figure 5: Caminoside A (+)-Chaetocin (Figure 6) is an epidithiodiketopiperazine alkaloid which is a completely symmetric dimer, first isolated by Sigg in 1970 from the fungus Chaetomium minutum. [START_REF] Hauser | Isolation and Structure Elucidation of Chaetocin[END_REF] It possesses various biological activities, such as antibacterial and cytostatic, [START_REF] Hauser | Isolation and Structure Elucidation of Chaetocin[END_REF] thioredoxin reductase-1 inhibitor, [START_REF] Tibodeau | The Anticancer Agent Chaetocin Is a Competitive Substrate and Inhibitor of Thioredoxin Reductase[END_REF] anticancer in several kinds of tumors (gastric cancer, anti-myeloma anti-leukemia, anti-hepatoma). [START_REF] Reece | Epidithiodiketopiperazines (ETPs) Exhibit in vitro Antiangiogenic and in vivo Antitumor Activity by Disrupting the HIF-1α/p300 Complex in a Preclinical Model of Prostate Cancer[END_REF][START_REF] Isham | Chaetocin: a promising new antimyeloma agent with in vitro and in vivo activity mediated via imposition of oxidative stress[END_REF][START_REF] Lai | The SUV39H1 Inhibitor Chaetocin Induces Differentiation and Shows Synergistic Cytotoxicity with Other Epigenetic Drugs in Acute Myeloid Leukemia Cells[END_REF][START_REF] Bernhard | The SUV39H1 Methyltransferase Inhibitor Chaetocin Causes Induction of Integrated HIV-1 without Producing a T Cell Response[END_REF][START_REF]Chaetocin Antileukemia Activity Against Chronic Myelogenous Leukemia Cells is Potentiated by Bone Marrow Stromal Factors and Overcomes Innate Imatinib Resistance[END_REF][START_REF] Han | Chaetocin Induces Apoptosis in Human Melanoma Cells Through the Generation of Reactive Oxygen Species and the Intrinsic Mitochondrial Pathway, and Exerts its Anti-Tumor Activity in vivo[END_REF][START_REF] Cao | Identification of Chaetocin as a Potent non-ROS-mediated Anticancer Drug Candidate for Gastric Cancer[END_REF] It is proposed for treatment of diabetes [START_REF] Xu | CN103550222A[END_REF] and heart disease, [START_REF] Xu | Application of Chaetocin in Manufacturing Drugs for Prevention and Treatment of Heart Disease" CN103356637A[END_REF] and applications are suggested as an anti-HIV drug [START_REF] Bouchat | Histone Methyltransferase Inhibitors Induce HIV-1 Recovery in Resting CD4+ T Cells from HIV-1-Infected HAART-Treated Patients[END_REF] or as a potent histone methyltransferases inhibitor SUV39H1. [START_REF] Greiner | Identification of a Specific Inhibitor of the Histone Methyltransferase SU(VAR)3-9[END_REF] Figure 6: (+)-Chaetocin Hibarimicinone (Figure 7), containing a quasi-dimeric tetracycline backbone (other family members hibarimicins A, B, C, D, E, G, H and I include a glycoside), was first isolated by Uehara in 2002 from Microbispora rosea subsp. hibaria (actinomycetes and culture broth), which are known to possess v-Src tyrosine kinase inhibitory activity and to induce growth and differentiation of Human Myeloid Leukemia HL60 Cells. [START_REF] Cho | Effects of Hibarimicins and Hibarimicin-Related Compounds Produced by Microbispora on v-Src Kinase Activity and Growth and Differentiation of Human Myeloid Leukemia HL-60 Cells[END_REF][START_REF] Tatsuta | The First Total Synthesis of Hibarimicinone, a Potent v-Src Tyrosine Kinase Inhibitor[END_REF][START_REF] Milgram | Total Syntheses of HMP-Y1, Hibarimicinone, and HMP[END_REF][START_REF] Tatsuta | Total Synthesis of Hibarimicinone, a v-Src Tyrosine Kinase Inhibitor Possessing the Pseudo-Dimer Structure of Tetracycline[END_REF] Figure 7: Hibarimicinone Hyperforin (Figure 8) is a polycyclic polyprenylated acylphloroglucinol, first isolated by Gurevich in 1971 from a flowering plant Hypericum perforatum L. (St. John's wort, Hypericaceae family), [START_REF] Gurevich | Hyperforin, an Antibiotic from Hypericum perforatum[END_REF] and whose structure has been determined by Bystrov in 1975. [START_REF] Brondz | The Absolute Configuration of Hyperforin, an Antibiotic from Hypericum perforatum L., Based on the Crystal Structure Determination of its p-Bromobenzoate Ester[END_REF][START_REF] Brondz | The relative stereochemistry of hyperforin, an antibiotic from Hypericum perforatum L" I[END_REF][START_REF] Bystrov | Structure of Hyperforin[END_REF] It has various therapeutics and biological activities. For example, it has been widely used as phytomedicines for depression treatment by a novel mechanism of action; [START_REF] Muller | Hyperforin -Antidepressant Activity by a Novel Mechanism of Action[END_REF][START_REF] Chatterjee | Hyperforin as a Possible Antidepressant Component of Hypericum Extracts[END_REF][START_REF] Singer | Hyperforin, a Major Antidepressant Constituent of St. John's Wort, Inhibits Serotonin Uptake by Elevating Free Intracellular Na +[END_REF][START_REF] Wurglics | Hypericum perforatum: a 'Modern' Herbal Antidepressant: Pharmacokinetics of Active Ingredients[END_REF] it has demonstrated anticancer [START_REF] Schempp | Inhibition of Tumour Cell Growth by Hyperforin, a Novel Anticancer Drug from St. John's Wort that Acts by Induction of Apoptosis[END_REF] and antitumoral activity in vivo, and inhibits the growth of leukemia K562 and U937 cells, brain glioblastoma cells LN229 and normal human astrocytes. [START_REF] Schempp | Inhibition of Tumour Cell Growth by Hyperforin, a Novel Anticancer Drug from St. John's Wort that Acts by Induction of Apoptosis[END_REF][100][101] It presents also antibiotic and antibacterial activity (against Gram-positive bacteria etc...), [START_REF] Gurevich | Hyperforin, an Antibiotic from Hypericum perforatum[END_REF][100][101][102][103] photoprotective and anti-inflammatory effect in vivo; 104 AIDS immunodeficiency immunomodulation phagocytosis 105 and acts as an angiogenesis inhibitor in vitro and in vivo. 106 Hyperforin is an extremely attractive compound for biotechnological and pharmacological research because of its novel activities and structure.

Figure 8: Hyperforin (-)-Haliclonin A (Figure 9), [START_REF] Jang | Haliclonin A, a New Macrocyclic Diamide from the Sponge Haliclona sp[END_REF] a novel macrocyclic diamide alkaloid with a novel an unprecedented 3-azabicyclononane skeleton, was isolated by Shin in 2009 from the marine sponge Haliclona sp. collected from Korean waters, which exhibited moderate cytotoxicity toward brine shrimp larvae (LC 50 260 ppm) and against the K562 leukemia cell line with an IC 50 of 15.9 µg•mL -1 ; antibacterial activity against diverse microbial strains such as Bacillus subtilis with a MIC value of 6.25 µg•mL -1 , and cytotoxicity. [START_REF] Jang | Haliclonin A, a New Macrocyclic Diamide from the Sponge Haliclona sp[END_REF]108 (-)-Haliclonin A is structurally related to sarains A-C, and its first total synthesis has been reported by Huang group in 2016. 109 Figure 9: (-)-Haliclonin A Strychnine (Figure 10), was the first terpene indole alkaloid isolated in crystalline form by Pelletier and Caventou in 1818 from the seeds of Strychnos nux vomica L. plant. 110 It is a colorless, bitter and white powder, and also found as a notorious poison and typically used intentionally for suicidal or homicidal means. Strychnine poisoning has a very strong stimulating effect (LD 50 : 0.4-3 mg•kg -1 ) on certain functions of the central nervous system. [111][112][START_REF] Setnikar | Toxicity of Central Nervous System Stimulants in Rats of Different Ages[END_REF][114][115] It acts as a competitive antagonist of the postsynaptic glycine receptor predominantly in the spinal cord, resulting in a sequence of predictable events: hyperreflexia, severe muscle spasms, convulsions (seizures) and respiratory paralysis. 111,[116][117][118] Although strychnine is highly toxic to both man and animals, it has been used for hundreds of years as a medicinal remedy for a broad range of ailments. For example, the seeds have long been used as a bitter-tasting gastric medicine, Vomica, for treatment of stomach ailments in Asia, and also as a pesticide against small vertebrates such as rodents in Europe, from the 17 th century until the present. 116,119 Strychnine has been proposed for applications in pharmaceuticals and therapeutics [115][116][117]120,121 such as antitumor and anticancer, 122-126 analgesic (gout) and anti-inflammatory, 120,121,[START_REF] Chen | Analgesic and Anti-Inflammatory Activity and Pharmacokinetics of Alkaloids from Seeds of Strychnos Nux-vomica after Transdermal Administration: Effect of Changes in Alkaloid Composition[END_REF] and antibacterial. 120,121,[START_REF] Clementi | The Antibacterial and Antitoxic Activity of Bile. III. The Variation of the Toxicity of Alkaloid Solutions by Acidification in Presence of Bile[END_REF]129 The structure of strychnine was first determined by Robinson in 1946 [130][131][START_REF] Openshaw | Constitution of Strychnine and the Biogenetic Relationship of Strychnine and Quinine[END_REF] and its first total synthesis was reported by Woodward in 1954, 133 which is considered one of the classic syntheses in the history of organic chemistry. Chlorophyll a (Figure 11), the major green pigment of plants, was first isolated in 1817 from Carapichea ipecacuanha by Pelletier and Caventou. 134,135 The chlorophylls are a class of very reactive, sensitive, and complicated substances related to porphyrins (found in hemoglobin) and binding a Mg 2+ at the center. 136,137 They are certainly the most widespread organic natural products, and their chemical structure as magnesium complexes was described in 1913, and then confirmed in 1928 by Willstatter. 138 Numerous chlorophylls are found in plants, algae and cyanobacteria, of which chlorophyll a is universal; chlorophyll b is present mostly in plants; chlorophyll c 1 and c 2 occur in various algae, while chlorophylls d and f are found in cyanobacteria. 139 The first total immunodeficiency virus type 1 (HIV-1 RT, IC 50 = 60 μg•mL -1 ). 150 Molinski reported that it also inhibits melanogenesis of Cryptococcus neoformans in vitro by blocking the biosynthesis of laccase in the melanin biosynthetic pathway with an IC 50 of 7.3 μM. [START_REF] Dalisay | Ptilomycalin A Inhibits Laccase and Melanization in Cryptococcus neoformans[END_REF] Figure 12: (-)-Ptilomycalin A (-)-Phomoidride A (CP-225,917) and (-)-Phomoidride B (CP-263,114) (Figure 13), are novel Ras farnesylation inhibitors isolated in 1997 by Dabrah from an unidentified fungus with the characteristics of a sterile Phoma species. Phomoidrides A and B showed inhibition of Ras farnesyl transferase from rat brain with IC 50 of 6 μM and 20 μM respectively and also inhibition of squalene synthase IC 50 of 43 μM and IC 50 of 160 μM respectively. 152 In 2001, Sulikowski reported the isolation and identification of two C 7 epimers of phomoidride A and phomoidride B, and named them as phomoidrides C and D, respectively. It was also suggested that phomoidride B is the first-formed secondary metabolite and the source of the remaining three phomoidrides. [START_REF] Spencer | Investigations into the Production and Interconversion of Phomoidrides A-D[END_REF] Figure 13: (-)-Phomoidrides A and B

Vinblastine and vincristine (Figure 14), two of the longest lasting bis-indole alkaloids in clinical use, were isolated by two independent groups from the Madagascar periwinkle, over a decade. It was early found to protect mice in vivo against P388 lymphoma, B16 melanoma, M5076 ovarian sarcoma, Lewis lung carcinoma, and the LX-1 human lung and MX-1 human mammary carcinoma xenografts. 170,173 A huge amount of research and investigation on trabectedin has been done by many groups, it was found to exhibit excellent safety and efficiency in the treatment of tissue sarcoma cell lines, with preferential activity in ovarian, breast, NSCLC, melanoma, and renal human cell lines explanted from patients. [174][175][START_REF] Carter | Trabectedin: A Review of its Use in the Management of Soft Tissue Sarcoma and Ovarian Cancer[END_REF][177][178][179] Trabectedin was considered as a potent anticancer drug and showed an innovative mechanism of antitumor activity: it interacts with guanine rich sequences of DNA, binds to the minor groove of DNA and inhibits cell proliferation by disrupting the cell cycle. [START_REF] D'incalci | A Review of Trabectedin (ET-743): A Unique Mechanism of Action[END_REF][180][START_REF] Van Kesteren | Yondelis (Trabectedin, ET-743): The Development of an Anticancer Agent of Marin Origin[END_REF][182][START_REF] Beumer | Trabectedin (Yondelis, Formerly ET-743), a Mass Balance Study in Patients with Advanced Cancer[END_REF][START_REF] Soares | Replication and Homologous Recombination Repair Regulate DNA Double-Strand Break Formation by the Antitumor Alkylator Ecteinascidin 743[END_REF][START_REF] Angelo | The Impairment of the High Mobility Group A (HMGA) Protein Function Contributes to the Anticancer Activity of Trabectedin[END_REF][186] Presently, trabectedin is under Phase II trials for the treatment of pediatric sarcomas as well as breast and prostate cancers. 2,[START_REF] Butler | The Role of Natural Product Chemistry in Drug Discovery[END_REF][START_REF] Kingston | Modern Natural Products Drug Discovery and Its Relevance to Biodiversity Conservation[END_REF]187 Trabectidin was approved from the European Commission as Yondelis ® in sales in Europe, Russia and South Korea for the treatment of ovarian cancer and advanced soft tissue sarcoma (STS) by Zeltia and Johnson & Johnson in 2007. Yondelis ® has been authorized by the European Commission and FDA for the treatment of women's relapsed platinum-sensitive ovarian cancer, soft tissue sarcomas and orphan drug status. 173,[START_REF] Van Kesteren | Yondelis (Trabectedin, ET-743): The Development of an Anticancer Agent of Marin Origin[END_REF][188][189][190][191] , of which Calicheamicin γ 1 I was first isolated and studied on its chemistry and structure in 1986-1989, 192,193 followed by Calicheamicin β 1 Br , γ 1 Br was isolated and characterized. [START_REF] Lee | Calichemicins, a Novel Family of Antitumor Antibiotics. 3. Isolation, Purification and Characterization of Calichemicins β1Br, γ1Br, α2I, α3I, β1I, γ1I, and δ1I[END_REF] Notably, their structure elucidation was reported by the same group in 1992. [START_REF] Lee | Calicheamicins, a Novel Family of Antitumor Antibiotics. 4. Structure Elucidation of Calicheamicins β1Br, γ1Br, α2I, α3I, β1I, γ1I, and δ1I[END_REF] The chemistry and activity of calicheamicin γ 1 I was first studied by Maiese, and found to exhibit maximal antitumor activity against P388 at 5.0 µg•kg -1 (T/C 232) and B16 at 1.25 µg•kg -1 (T/C 186) when tested in male BDFi mice against P388 leukemia and B16 melanoma in vivo, and significant activity against Gram-positive and Gram-negative bacteria in vitro. [START_REF] Maiese | Calicheamicins, a Novel Family of Antitumor Antibiotics: Taxonomy, Fermentation and Biological Properties[END_REF][START_REF] Maiese | LL-D49194 Antibiotics, a Novel Family of Antitumor Agents: Taxonomy, Fermentation and Biological Properties[END_REF] It is possibly one of the most potent antitumor compounds approved for clinical use, which is over 1000 times more potent than adriamycin, a clinically useful antitumor antibiotic, when tested in murine tumor models. Calicheamicin γ 1 I and its analogues possess remarkable biological properties, such as cytotoxicity, anti-bacterial, antitumor. 198 For example, it can bind in the minor groove of DNA and caues DNA double-strand breakage with a high degree of specificity; it is extremely toxic to all cells (such as HL60 leukemia cells, acute myeloid leukemia etc…). [START_REF] Hamann | Gemtuzumab Ozogamicin, A Potent and Selective Anti-CD33 Antibody-Calicheamicin Conjugate for Treatment of Acute Myeloid Leukemia[END_REF][START_REF] Hamann | An Anti-CD33 Antibody-Calicheamicin Conjugate for Treatment of Acute Myeloid Leukemia. Choice of Linker[END_REF] Although it possesses very excellent sub-picomolar level activity, it was too toxic to pursue for many years. It has been linked to a specific directed monoclonal antibody against chronic myelogenous leukemia and use in antitumor (potent antitumor effects in breast and ovarian tumor xenografts). [START_REF] Hamann | A Calicheamicin Conjugate with a Fully Humanized Anti-MUC1 Antibody Shows Potent Antitumor Effects in Breast and Ovarian Tumor Xenografts[END_REF][START_REF] Zhao | Preparation of Specific Conjugation Bridge Linkers and Immunoconjugates and Methods of Using Them[END_REF][START_REF] Zhao | Preparation of Novel Bridge Linkers Containing an Acetylenedicarbonyl Group for Specific Conjugation of a Cytotoxic Agent to a Cell-Binding Molecule[END_REF] Calicheamicin γ 1 I is the first FDA-approved antitumor antibiotic of microbial second metabolites (the first commercially available chemotherapeutic immunoconjugate for clinical use) as Mylotarg ® , which is carried to the site where needed and released in situ, thereby reducing the systemic toxicity but not its activity. Some in-depth research and investigation on antibody-calicheamicin conjugates for treatment of acute myeloid leukemia has been done by Hamann's and Zhao's groups and further developed into new drugs. 1,[START_REF] Hamann | Gemtuzumab Ozogamicin, A Potent and Selective Anti-CD33 Antibody-Calicheamicin Conjugate for Treatment of Acute Myeloid Leukemia[END_REF][START_REF] Hamann | An Anti-CD33 Antibody-Calicheamicin Conjugate for Treatment of Acute Myeloid Leukemia. Choice of Linker[END_REF][204][205][START_REF] Zhao | Preparation of Bridge Linkers used for Specific Conjugation of Two or More Cytotoxic Agents to a Cell-Binding Molecule[END_REF][207][208] deep-water sponge Lissodendoryx sp. 211 Halichondrin B and several of its family members are known to exhibit exceptional antitumor activities towards a variety of cell lines, and showed strong cytotoxicity to bind to tubulin and to inhibit tubulin polymerization, thereby inducing cell cycle arrest and apoptosis. 212,213 Halichondrin B is the most potent candidate of this family for treatment of breast cancer and other neoplasms, 214,215 which is a noncompetitive inhibitor of Vinca alkaloids, colchicine and paclitaxel, [216][START_REF] Halichondrin | Marine Natural Products Binding in the Vinca Domain of Tubulin. Discovery of Tubulin-Based Mechanism of Action by Analysis of Differential Cytotoxicity Data[END_REF][218] and its discovery and development and clinical application has been reviewed. [START_REF] Kitagawa | Antitumor Marine Natural Products[END_REF][START_REF]Tubulin-Interactive Natural Products as Anticancer Agents" D. G. I. Kingston[END_REF]209,[START_REF] Kawano | A Landmark in Drug Discovery Based on Complex Natural Product Synthesis[END_REF][START_REF] Simmons | Marine Natural Products as Anticancer Drugs[END_REF][START_REF] Dans Pacifichem | International Chemical Congress of Pacific Basin Societies[END_REF][222][START_REF] Nakamura | Biologically Active Marine Natural Products[END_REF][224][225][226] A major obstacle was the issue of supply since only miniscule amounts are available from its marine source. 227 Fortunately, the first total synthesis of halichondrin B was developed by Kishi in 1992. 228 Eribulin (Halaven), an anticancer drug developed by Eisai Research Institute, is a synthetic analogue of halichondrin B, and its mesylate salt (also referred to as E7389)

was approved by FDA in 2010 for the treatment of metastatic breast cancer, treatment of prostate, sarcoma, breast, bladder, and ovarian cancers etc…, which are investigated in phase III trials. [229][230][231] Figure 17: Halichondrin B

Peptides and Marine cyclic peptides

Bioactive natural peptides are ubiquitous in all life kingdoms. They are often characterized by short amino acid sequences, usually with less than 20 units of amino acids, and include cyclic and linear peptides, as well as depsipeptides. They are found either free or encrypted in proteins, and thus released during enzymatic proteolysis of proteins or directly isolated from organisms. [232][233][START_REF] Gogineni | Marine Natural Product Peptides with Therapeutic Potential: Chemistry, Biosynthesis, and Pharmacology[END_REF][235] Nature remains the largest source of bioactive peptides since plants, animals, fungi, microbes and their metabolites contain a vast number of peptides. 236 In recent years, bioactive peptides have received increased attention from the pharmaceutical and biotech industries, and play a pivotal role as a source for discovery and development of drugs and candidates. 237,[START_REF] Dang | Bioactive Peptide Natural Products as Lead Structures for Medicinal Use[END_REF] Bioactive peptides have a positive impact on body functions or conditions, which play an important role in metabolic regulation and modulation, 239,240 and may ultimately influence human health by affecting the digestive, endocrine, cardiovascular, immune and nervous systems. 241 It is thought that they may fill the gap between small molecules and peptides drugs.

Insulin, a peptide hormone produced by our pancreas to control the blood glucose levels at any given moment, helps to regulate our body's metabolism of carbohydrates, fats, and proteins, which has been widely used for treatment of type I and II diabetes. 242 Some natural peptides and their derivatives have high commercial value and success in pharmaceutical markets, and some of them are already in preclinical and clinical pipelines for treatment of various diseases, [START_REF] Prasasty | Natural Peptides in Drug Discovery Targeting Acetylcholinesterase[END_REF] for example enfuvirtide (HIV fusion inhibitor) and liraglutide for treatment of type II diabetes.

Currently among more than 1500 different bioactive peptides reported in a database, more than 1250 peptides present different functional significance. [START_REF]Computer-Aided Characteristics of Proteins as Potential Precursors of Bioactive Peptides[END_REF] Numerous bioactive peptides have been isolated from a great variety of marine and terrestrial organisms and species, such as sponges, bacteria, cyanobacteria, animals, plants, fungus and microbes, and are also known to exhibit diverse biological activities such as antithrombotic, anti-cancer, antitumoral, anti-proliferative, antimicrobial, anticoagulating, antiviral, anti-diabetic, anti-coagulant, anti-hypertensive, antioxidant, antiobesity, hypocholesteremic, analgesic and calcium-binding activities. [START_REF] Simmons | Marine Natural Products as Anticancer Drugs[END_REF][232][233][START_REF] Gogineni | Marine Natural Product Peptides with Therapeutic Potential: Chemistry, Biosynthesis, and Pharmacology[END_REF][235][236][START_REF] Dang | Bioactive Peptide Natural Products as Lead Structures for Medicinal Use[END_REF]240,241, Thus, a short introduction on some selective peptides with diverse structural features and pronounced biological activities will be presented. Similary, it will promote us to have a better understanding on peptides and marine cyclic peptides and their discovery and isolation, as well as the significant role in the exploration of novel peptide drugs and therapeutic agents.

Romidepsin (Figure 18) (Istodax ® , NSC 630176, FK288, FR901228), a structurally unusual bicyclic depsipeptide, was first isolated in 1994 by a research team of Fujisawa Pharmaceutical

Company (now Astellas Pharma) in Tsukuba from the culture broth of the terrestrial bacterium Chromobacterium violaceum. It shows potent in vivo antitumor activity against both human tumor xenografts and murine tumors. [START_REF] Ueda | A Novel Antitumor Bicyclic Depsipeptide Produced by Chromobacterium violaceum No. 968. III. Antitumor Activities on Experimental Tumors in Mice[END_REF][START_REF]FR901228, A Novel Antitumor Bicyclic Depsipeptide Produced by Chromobacterium violaceum No. 968. II. Structure Determination[END_REF][START_REF] Nishimura | A Novel Antitumor Bicyclic Depsipeptide Produced by Chromobacterium violaceum No. 968. I. Taxonomy, Fermentation, Isolation, Physico-Chemical and Biological Properties, and Antitumor Activity[END_REF] Recently, romidepsin has proved to act as a strong HDAC inhibitor and thus has been approved for the treatment of cutaneous T-cell lymphoma and other peripheral T-cell lymphomas by the FDA. [START_REF] Van Der Molen | Romidepsin (Istodax, NSC 630176, FR901228, FK228, depsipeptide): A Natural Product Recently Approved for Cutaneous T-Cell Lymphoma[END_REF][START_REF] Piekarz | Inhibitor of Histone Deacetylation, Depsipeptide (FR901228), in the Treatment of Peripheral and Cutaneous T-Cell Lymphoma: A Case Report[END_REF][START_REF] Goldsmith | The Histone Deacetylase Inhibitor FK228 Given Prior to Adenovirus Infection Can Boost Infection in Melanoma Xenograft Model Systems[END_REF][START_REF] Nakajima | FR901228, A Potent Antitumor Antibiotic, is A Novel Histone Deacetylase Inhibitor[END_REF][277][278] Figure 18: Romidepsin Vancomycin (Figure 19) is a branched tricyclic glycopeptide antibiotic [START_REF] Mccormick | Vancomycin, A New Antibiotic. I. Chemical and Biologic Properties[END_REF]280 first isolated in 1953 by Kornfeld at Eli Lilly from the Actinobacteria species Amycolatopsis orientalis, 281 which was used for treatment of a number of bacterial infections by inhibiting proper construction of cell wall in Gram-positive bacteria. [START_REF] Anderson | Vancomycin, A New Antibiotic. IV. Pharmacologic and Toxicologic Studies[END_REF][START_REF] Ziegler | Vancomycin, A New Antibiotic. II. In vitro Antibacterial Studies[END_REF][284] It is recommended intravenously as a treatment for complicated skin infections, bloodstream infections, endocarditis, bone and joint infections, and meningitis. 285,286 The rapid approval by the FDA in 1958 was prompted by penicillin-resistant

Staphylococcus aureus and oxacillin-resistant (methicillin-resistant) staphylococci. 287,288 Figure 19: Vancomycin Alamethicins are a class of well-known linear 19-membered peptides antiobiotics, primarily isolated by Meyer and Reusser from Trichoderma viride in 1967. [START_REF] Meyer | A Polypeptide Antibacterial Agent Isolated from Trichoderma viride[END_REF] However, it is revealed that this material was composed of two major groups neutral ALM F30 (about 85%) and acidic ALM F50

(about 12%), as well as a minor group ALM F20 (2%) and trace of ALMs (F40, F60, F70) by Melling and McMullen in 1975. 290,291 All alamethicin are structurally blocked at the N-terminus by an acetyl moiety and at the C-terminus by the phenylalaninol. To date, 23 members of natural mixture of alamethicin have been sequenced, of which two exampls are shown (Figure 20):

Alamethicin I (belonging to F50) and Alamethicin (belonging to F30). 292,293 They are also shown to possess potent biological activities as antibacterial agent, in vitro and in vivo against strains

Trypanosoma brucei brucei strain and Trypanosoma brucei rhodesiense. 294,295 Besides, alamethicins are also known to exhibit voltage dependent ion channel activity in membrane environments and induce excitability phenomena in model membranes and has served as an excellent model for the study of voltage sensitive transmembrane channels. [START_REF] Nagao | Structure and Orientation of Antibiotic Peptide Alamethicin in Phospholipid Bilayers as Revealed by Chemical Shift Oscillation Analysis of Solid State Nuclear Magnetic Resonance and Molecular Dynamics Simulation[END_REF][297][298] Figure 20: Alamethicin

The oceans stand a uniquely vast source for new bioactive natural products, which is consider as the largest remaining treasure of undiscovered natural products with an unprecedented variety of chemical classes possessing a wide range of biological activities. [START_REF] Gogineni | Marine Natural Product Peptides with Therapeutic Potential: Chemistry, Biosynthesis, and Pharmacology[END_REF][START_REF] Abou-Elela | Marine Natural Products and their Potential Applications as Anti-Infective Agents[END_REF][START_REF] Donia | Marine Natural Products and their Potential Applications as Anti-Infective Agents[END_REF] containing a myriad of marine peptides 2,[START_REF] Nakamura | Biologically Active Marine Natural Products[END_REF]269, Recently much more attention has been focused on marine cyclic peptides due to their novel chemical structures and diverse biological properties. A considerable number of eminent natural product chemists are interested in marine natural products (peptides), such as Kingston (USA), Faulkner (USA), Fusetani (Japan), Blunt (New Zealand),

Hamann (USA). A huge number of marine peptides have been isolated and reported with various biological activities like antitumor, antibacterial, antifungal, anthelmintic, insecticidal, antineoplastic and anti-inflammatory activities and isolated from a variety of marine organisms, such as sponges, bacteria, actinomycetes, fungi and corals over several decades. [233][START_REF] Gogineni | Marine Natural Product Peptides with Therapeutic Potential: Chemistry, Biosynthesis, and Pharmacology[END_REF][235][247][START_REF] Mi | New Peptides Isolated from Marine Cyanobacteria, an Overview Over the Past Decade[END_REF][249][251][START_REF] Kang | Marine Peptides and their Anti-Infective Activities[END_REF][START_REF] Jo | Marine Bioactive Peptides: Types, Structures, and Physiological Functions[END_REF][254][255]259,[START_REF] Sable | Peptides, Peptidomimetics, and Polypeptides from Marine Sources: A Wealth of Natural Sources for Pharmaceutical Applications[END_REF][START_REF] Ireland | Natural Product Peptides from Marine Organisms[END_REF]264,265,267,269,[326][327][328][329] Marine cyclic peptides are one of the underexplored classes of bioactive peptides that are promising to be a new perspective for new drugs. Various marine cyclic peptides with structural and biological diversity have increased our understanding of properties of novel chemical structures related to the action mechanisms of biological activities. 233,259,328 The tetradecapeptide discodermins [START_REF] Gogineni | Marine Natural Product Peptides with Therapeutic Potential: Chemistry, Biosynthesis, and Pharmacology[END_REF]268 are a family of novel linear and cyclic peptides first isolated from the sponges of genus Discodermia, of which discodermin A-H have been reported and characterized, and show potent inhibition activity of cell growth, and cytotoxic to P388 murine leukemia cells and A549 human lung cell-lines. Especially, discodermin A (Figure 21) is the first isolated marine cyclic peptide with antimicrobial activity extracted from sponge Discodermia kiiensis by Fusetani in 1984, 257,330 highly inhibiting the growth of Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa, and the development of starfish embryos. 268,330,[START_REF] Matsunaga | Bioactive marine metabolites, IV. Isolation and the amino acid composition of discodermin A, an antimicrobial peptide, from the marine sponge Discodermia kiiensis[END_REF] Figure 21: Discodermin A Theonellamides A-G are a class of bicyclic glycopeptides isolated from Theonella sp., of which theonellamides A-E isolated by Fusetani in 1995 have been found to possess cytotoxic activity towards P388 leukemia cell-line, [START_REF] Matsunaga | Theonellamides A-E, cytotoxic bicyclic peptides, from a marine sponge Theonella sp[END_REF] while a potent antifungal activity was reported for theonellamide F also isolated by Fusetani in 1989. [START_REF] Wada | Theonellamide F, a bicyclic peptide marine toxin, induces formation of vacuoles in 3Y1 rat embryonic fibroblast[END_REF] Recently, theonellamide G (Figure 22) was isolated by Youssef from Red Sea Lithistida sponge Theonella swinhoei located in Hurghada (Egypt). Its structure has been elucidated and it was reported to possess potent antifungal activity against two strains of Candida albicans, the wild type and amphotericin B-resistant of Candida albicans with IC 50 of 4.49 and 2.0 μM, respectively. Additionally, it showed cytotoxic activity against the human colon adenocarcinoma cell line (HCT16) with IC 50 of 6.0 μM. [START_REF] Youssef | Theonellamide G, a potent antifungal and cytotoxic bicyclic glycopeptide from the Red Sea marine sponge Theonella swinhoei[END_REF] Figure 22: Theonellamide G Aciculitins (Figure 23) are the first bicyclic glycopeptidolipids obtained from a marine source, the lithistid sponge Aciculites orientalisa at Siquijor Island (Philippines). Isolation and structural elucidation of aciculitins A-C was reported by Faulkner in 1996. [START_REF] Gogineni | Marine Natural Product Peptides with Therapeutic Potential: Chemistry, Biosynthesis, and Pharmacology[END_REF]268,[START_REF] Bewley | Aciculitins A-C: cytotoxic and antifungal cyclic peptides from the lithistid sponge Aciculites orientalis[END_REF] They show cytotoxic activity against the human-colon tumor cell line HCT116 with an IC 50 of 0.5 µg•mL -1 and inhibit the growth of Candida albicans at a loading of 2.5 µg•disk -1 in the standard disk assay. [START_REF] Bewley | Aciculitins A-C: cytotoxic and antifungal cyclic peptides from the lithistid sponge Aciculites orientalis[END_REF] Figure 23: Aciculitin Thiocoraline (Figure 24) is a well-known 2-fold symmetric or pseudosymmetric bicyclic thiodepsipeptide isolated by Romero in 1997 from the mycelium cake (fermentation broth) of a marine actinomycete strain, Micromonospora sp. L-13-ACM2-092 collected from soft coral collected at the Indian Ocean near the coast of Mozambique, which showed antimicrobial activities against strains of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus and Micrococcus luteu, and also presented a strong cytotoxic effect against P388, A549 and MEL28 cell-lines with an IC 50 of 0.002 µg•mL -1 , along with a strong anti-microbial activity against Gram-positive microorganisms, and as well as inhibiting DNA polymerase at an IC 50 of 6 µg•mL -1 and binding to supercoiled DNA, and inhibiting DNA synthesis at an IC 50 of 0.4 µg•mL -1 and RNA-synthesis at an IC 50 of 0.008 µg•mL -1 . [START_REF] Romero | Thiocoraline, a new depsipeptide with antitumor activity produced by a marine Micromonospora. I. Taxonomy, fermentation, isolation, and biological activities[END_REF][START_REF] Perez Baz | Thiocoraline, a novel depsipeptide with antitumor activity produced by a marine Micromonospora. II. Physico-chemical properties and structure determination[END_REF][338] Additionally, it was also found to inhibit the elongation activity of DNA polymerase α in both LoVo and SW620 human colon cancer cells and induce profound perturbations of cell cycle. [338][START_REF] Negri | Deciphering the biosynthesis pathway of the antitumor thiocoraline from a marine actinomycete and its expression in two streptomyces species" F. Lombo[END_REF][340][341] It has been shown

to bind to DNA by high-affinity bisintercalation analogous to echinomycin, but with little or no perceptible sequence selectivity and exhibit exceptionally potent activity in the L1210 mouse leukemia cytotoxic assay (IC 50 of 200 pM). 342

Figure 24: Thiocoraline

Microsclerodermins

Structure and origin of microsclerodermins

Microsclerodermins (Figure 25) are a family of macrocyclic peptides extracted from the lithistid sponges Microscleroderma sp., Theonella sp. and Pachastrella sp. [START_REF] Microsclerodermins | Antifungal Cyclic Peptides from the Lithistid Sponge Microscleroderma sp[END_REF][START_REF] Schmidt | Microsclerodermins C -E, antifungal cyclic peptides from the lithistid marine sponges Theonella sp. and Microscleroderma sp[END_REF][START_REF] Qureshi | Microsclerodermins F-I, antitumor and antifungal cyclic peptides from the lithistid sponge Microscleroderma sp[END_REF][346][347][START_REF] Tian | Microsclerodermins N and O, cytotoxic cyclic peptides containing a p-ethoxyphenyl moiety from a deep-sea marine sponge Pachastrella sp[END_REF] and also produced from three different genera of myxobacteria Chondromyces sp., Jahnella sp. and Sorangium sp. 263 They are characterized by a 23-membered ring peptide macrocycle, which contains six amino acids, of which three are common: glycine, sarcosine and (R)-γ-amino-β-hydroxybutyric acid (GABOB).

The other three amino acids present some structural variations but can be classified as a modified tryptophan, a polyhydroxylated β-amino acid with four or five contiguous stereogenic centers and an original 3-amino-4-hydroxypyrrolidone-4-acetic acid (PyrAA), which in some cases may be dehydrated to give an unsaturated dehydroxypyrrolidone-4-acetic acid (PyrAA) on the macrocyclic backbone. Faulkner. [START_REF] Qureshi | Microsclerodermins F-I, antitumor and antifungal cyclic peptides from the lithistid sponge Microscleroderma sp[END_REF] More recently, several new members of the family has been isolated and reported.

Microsclerodermins J-K were isolated in 2012 from the deep-water sponge Microscleroderma herdmani in Mauritius by Li. 346,347 In 2013, Müller discovered and isolated microsclerodermins L-M from terrestrial myxobacteria from three different genera Chondromyces sp., Jahnella sp., and Sorangium sp., respectively. 263 More recently, Matsunga isolated microsclerodermins N and O from the deep sea sponge Pachastrella sp.. [START_REF] Tian | Microsclerodermins N and O, cytotoxic cyclic peptides containing a p-ethoxyphenyl moiety from a deep-sea marine sponge Pachastrella sp[END_REF] This family also comprises pedeins A and B which were isolated from the cell mass of the myxobacterium Chondromyces pediculatus by Kunze in 2008. [START_REF] Kunze | Pedein A and B: production, isolation, structure elucidation and biological properties of new antifungal cyclopeptides from Chondromyces pediculatus (Myxobacteria)[END_REF] 29

Figure 26: The microsclerodermin family

Biological activities

Faulkner and his co-workers have been the pioneers in exploring the biological activities of microsclerodermins. Early investigations disclosed that they possess antifungal, antitumor and cytotoxic activities. 263,[START_REF] Microsclerodermins | Antifungal Cyclic Peptides from the Lithistid Sponge Microscleroderma sp[END_REF][START_REF] Schmidt | Microsclerodermins C -E, antifungal cyclic peptides from the lithistid marine sponges Theonella sp. and Microscleroderma sp[END_REF][START_REF] Qureshi | Microsclerodermins F-I, antitumor and antifungal cyclic peptides from the lithistid sponge Microscleroderma sp[END_REF][346][347] For instance, they have potential in vitro antifungal activity against a range of pathogenic fungal species: such as Candida glabrata, Candida krusei, Cryptococcus neoformans, Botrytic cinerea, Fusarium oxyspotum, Helminthosporium sativum, Pyricularia oryaze and Aspergillus fumigatus. All microsclerodermins possess antifungal activity against Candida albicans (Table 1), such as microsclerodermin A-B (MIC 2.5 µg•disk -1 ). 263,[START_REF] Microsclerodermins | Antifungal Cyclic Peptides from the Lithistid Sponge Microscleroderma sp[END_REF][START_REF] Schmidt | Microsclerodermins C -E, antifungal cyclic peptides from the lithistid marine sponges Theonella sp. and Microscleroderma sp[END_REF][START_REF] Qureshi | Microsclerodermins F-I, antitumor and antifungal cyclic peptides from the lithistid sponge Microscleroderma sp[END_REF][346][347][START_REF] Kunze | Pedein A and B: production, isolation, structure elucidation and biological properties of new antifungal cyclopeptides from Chondromyces pediculatus (Myxobacteria)[END_REF] Faulkner supposed that antifungal activity was attributed to the polyhydroxylated β-amino acid moiety, although direct supporting evidence was lacking. Investigations on microsclerodermin F-I demonstrate that they exhibit good antitumor activities against the HCT116 human colon carcinoma cell line, indicating their potential as anti-cancer agents. [START_REF] Qureshi | Microsclerodermins F-I, antitumor and antifungal cyclic peptides from the lithistid sponge Microscleroderma sp[END_REF] Recently, Wright disclosed that microsclerodermin A is a novel inhibitor of the nuclear factor kappa B (NFκB) and induces apoptosis in pancreatic cancer cells. [START_REF] Guzmán | The marine natural product microsclerodermin A is a novel inhibitor of the nuclear factor kappa B and induces apoptosis in pancreatic cancer cells[END_REF] In addition, pedein A was found to strongly inhibit against the growth of yeasts and fungi, to induce hemolysis of erythrocytes, and cause changes in membrane permeability of Rhodotorula glutinis. [START_REF] Kunze | Pedein A and B: production, isolation, structure elucidation and biological properties of new antifungal cyclopeptides from Chondromyces pediculatus (Myxobacteria)[END_REF] According to Müller's findings, both of microsclerodermins and pedeins were discovered from several myxobacteria, which strongly suggested that some natural products isolated from marine sources such as sponges or invertebrates, probably ultimately originate from associated microbes. However, these reported studies on biological activities are limited by the low natural abundance of microsclerodermins, about only 0.001% yield of the dry weight of the sponge, which means that only few mg of materials could be extracted and isolated by consuming lots of sponges and manpower. Their interesting biological activities and potential as anti-cancer agent have motivated organic chemists to solve the challenge of their total synthesis. In the next part, we will develop in detail the efforts toward their total synthesis.

Previous Syntheses

Shioiri's pionnering studies towards microsclerodermins A and B

Shioiri was the first to show an interest in the synthesis of microsclerodermins. Although he never completed a total synthesis, he prepared all the fragment of microsclerodermins A and B, and has reported some important features about their stability.

Preparation of a protected derivative of AMMTD

In 1997, Shioiri reported a stereoselective synthesis of a core building block of AMMTD (Figure 27). 351 This was the first report on efforts towards the total synthesis of microsclerodermins A and B since their isolation by Faulkner in 1994.

Figure 27: AMMTD fragment present in microsclerodermin A and B

The chiral ester 1 was converted into the unsaturated methyl ester 2 in 39% yield over 7 steps. Sharpless asymmetric dihydroxylation 352,353 followed by protection of the diol as an acetonide gave the intermediate 3 with an excellent diastereomeric ratio. Functional modification of the methyl ester group provided the nitrone derivative 4 in 3 steps in good yield. Addition of furyllithium according to Dondoni's procedure 354 gave the furyl intermediate 5 in 59% yield.

Protecting group transformation provided 6 in 3 steps, followed by oxidative cleavage of the furyl ring using RuO 2 /NaIO 4 and sequential esterification gave amino ester 7, featuring the core skeleton of AMMTD with 4 consecutive stereogenic centers, in 5.4% yield over 18 steps (Scheme 1).

Scheme 1: Construction of the core building block of AMMTD

In 1999, Shioiri 355,[START_REF] Sasaki | Shioiri Dans Peptide Science -Present and Future[END_REF] completed the synthesis of AMMTD in its protected form (Scheme 2).

The acetonide alcohol 8 obtained as a synthetic intermediate in his previous work 351 was first converted into the alkyne 9 in good yield through a 5-step functional group modification sequence.

Hydrozirconation of this alkyne using Schwartz's reagent (Cp 2 Zr(H)Cl), followed by palladium mediated cross coupling with p-iodoanisole for an introduction of the styrene moiety according to Negishi's protocol [START_REF] Negishi | Nickelor palladium-catalyzed cross coupling. 31. Palladium-or nickel-catalyzed reactions of alkenylmetals with unsaturated organic halides as a selective route to arylated alkenes and conjugated dienes: scope, limitations, and mechanism[END_REF] afforded the p-anisyl-(E)-alkene 10 in good yield. After deprotection of the TBS group to give an alcohol with TBAF, and its Swern oxidation into an aldehyde, followed by

Paterson's anti-aldol reaction 358 with the ketone 11, the β-keto alcohol 12 that contained the requisite five contiguous stereogenic centers was obtained efficiently as a single isomer. Reduction of both the keto and benzoyl groups using LiBH 4 gave a triol in which the 1,2-diol moiety was cleaved with sodium periodate. Subsquent reduction of the resulting aldehyde with NaBH 4 produced the alcohol 13 in good yield. Protection of the primary alcohol as a pivaloyl ester, followed by activation of the secondary alcohol with triflic anhydride and azidation using tetrabutylammonium azide, proceeded with inversion of configuration and gave the protected azido alcohol 14 with the required stereochemistry. Reduction of the azide and protection of the resulting amine by a Boc group gave the amino alcohol 15. Finally, a 2-step oxidation sequence of the alcohol (Parikh-Doering oxidation into an aldehyde followed by Pinnick oxidation into the acid)

and protection as a methyl ester gave the protected AMMTD derivative 16 in 2.4% yield over 31 steps from the chiral ester 1.

Scheme 2: Construction of a protected derivative of AMMTD

Preparation of the modified tryptophan derivative

Microsclerodermins A and B include a (R)-2-carboxytryptophan residue. Shioiri report the preparation of this tryptophan derivative in a protected form using hydroxypinanone glycine imine alkylation methodology (Scheme 3). [START_REF]Construction of Three Building Blocks for the Total Synthesis of Microsclerodermins[END_REF]360 Thus, indole-2-carboxylic acid 17 was first protected as its methyl ester 18, followed by a Mannich reaction with formaldehyde and dimethylamine and quaternization with methyliodide to form the gramine deivative 19. Then α-alkylation of the glycine t-butyl ester imine derived from (-)-hydroxypinanone 20 with the trimethylammonium salt 19

proceeded with high diastereoselectiviy to create the α-amino acid derivative 21. 361,[START_REF] Irako | A new asymmetric synthesis of (S)-dolaphenine and its heteroaromatic congeners utilizing (+)-2-hydroxy-3-pinanone and (-)-3-hydroxy-2-caranone as chiral auxiliaries[END_REF] Hydrolysis of the chiral auxiliary using citric acid and Boc-protection of the free amine gave the protected tryptophan derivative 22 with good enantiomeric excess (ee = 87%; recrystallization from a mixture of hexane/ethyl acetate improved the ee to 91%).

Scheme 3: Construction of a protected 2-carboxytryptophan

Preparation of a protected GABOB derivative

All microsclerodermins incorporate a GABOB residue. Shioiri proposed a synthesis of GABOB from dimethyl (R)-malate (Scheme 4). Regioselective reduction of the methyl ester of dimethyl (R)-malate 23 with borane-dimethyl sulfide complex yielded the diol 24 in good yield. [START_REF] Saito | A revised mechanism for chemoselective reduction of esters with borane-dimethyl sulfide complex and catalytic sodium tetrahydroborate directed by adjacent hydroxyl group[END_REF]364 Activation of the primary alcohol [START_REF] Bellamy | A new, short and efficient synthesis of both enantiomers of carnitine[END_REF] followed by nucleophilic substitution with NaN 3 gave the azide 25. One-pot azido group hydrogenolysis and amine protection by a Boc group gave the hydroxy ester 26. Finally, saponification of the methyl ester followed by protection as a trichloroethyl ester by coupling with 2,2,2-trichloroethanol and protection of the secondary alcohol as a TBS ester gave the fully protected GABOB derivative 27 in 25% yield over 7 steps.

Scheme 4: Construction of a protected GABOB derivative

Preparation of the protected PyrAA unit

Shioiri also proposed a synthesis of the pyrrolidinoacetic acid residue (PyrAA) from the L-aspartic acid derivative (S)-28 (Scheme 5). [START_REF]Construction of Three Building Blocks for the Total Synthesis of Microsclerodermins[END_REF]360 Activation of the α-carboxylic acid function with CDI followed by treatment with the magnesium salt of ethyl hemimalonate according to Scheme 5: Construction of a protected PyrAA derivative Masamune's protocol 366 afforded the β-keto ester 29 in 78% yield. The pyrrolidinone moiety was then installed by hydrogenolysis of the benzyl ester followed by amidation of the resulting acid function through activation as a mixed anhydride and treatment with aqueous ammonia. The resulting γ-ketoamide cyclized spontaneously to give the protected PyrAA residue efficiently as a mixture of diasteroisomers 30-1 and 30-2 in a 3:1 ratio. Significantly, this hemiaminal was easily dehydrated to give the corresponding α,β-unsaturated ester under various acidic or basic conditions.

The easy dehydration of 30-1 and 30-2 meant that it could not be installed in the microsclerodermin macrocycle in this form. To solve this problem, Shioiri proposed the use of a γ-lactone which should be a suitable precursor of PyrAA residue allowing its construction at the end of the total synthesis (Scheme 6). 360 Thus, reduction of the β-keto ester 29 into an alcohol followed by saponification of both ester groups and subsequent lactonization of the resulting hydroxy acid 32

using EDCI gave the γ-lactone 33. Finally, coupling of the lactone 33 with a sarcosine derivative using BopCl yield the dipeptide building block 35 which was expected to be useful for a total synthesis of microsclerodermin.

Scheme 6: Construction of a dipeptide comprised of Sar and a masked-PyrAA residue

Conclusion

Although Shioiri described the synthesis of different building blocks of microsclerodermins A and B between 1997 and 2003, he never reported on the macrocylic peptide assembly.

Nonetheless, his work remains important since he was the first to report on the intrinsic propensity of the PyrAA residue to dehydrate under mild conditions. He logically proposed that a precursor of this residue should be employed in the macrocyclic peptide assembly and that the PyrAA residue should be unmasked at the end of the synthesis.

Other syntheses of microsclerodermin fragments

Chandrasekhar's work towards AMMTD

In 2006, Chandrasekhar's group reported the stereoselective synthesis of a protected AMMTD derivative starting from (S)-citronellol. 367 The synthetic route is based on the deoxygenative rearrangement of a hydroxyalkynoate to generate a polyunsaturated ester followed by a highly enantio-and diastereocontrolled iterative dihydroxylation procedure to install the four contiguous stereocenters of AMMTD.

(S)-Citronellol was protected as a tetrahydropyranyl ether, followed by cleavage of the alkene by ozonolysis to provide an aldehyde which was then submitted to a Wittig reaction to give the α,β-unsaturated ester 36 in good yield (Scheme 7). Sequential reduction of the conjugated C=C bond and the ester function 368 yielded the alcohol 37. Protection of the alcohol as its benzyl ether followed by selective deprotection of the THP-ether and then oxidation with IBX [START_REF] Frigerio | A mild oxidizing reagent for alcohols and 1,2-diols: o-iodoxybenzoic acid (IBX) in DMSO[END_REF] produced the aldehyde 38 efficiently. This aldehyde was immediately treated with a lithiated ethyl propiolate to furnish the hydroxy alkynoate 39 as an inseparable mixture of two diastereoisomers in 86% yield.

The hydroxy alkynoate was then converted into the 1,4-dienoate 40 by Ph 3 P-mediated deoxygenative rearrangement which proceeds via an allene intermediate. [START_REF] Guo | A novel deoxygenation-isomerization reaction of 4-hydroxy-2-ynoic esters and γ-hydroxy-α,β-ynones[END_REF][START_REF] Lu | Reactions of Electron-Deficient Alkynes and Allenes under Phosphine Catalysis[END_REF] Then, selective deprotection of the terminal acetonide under mild acidic conditions with PPTS 379 followed by protection of the primary alcohol as a TBS ether yielded alcohol 49. The remaining secondary alcohol was protected as its MOM ether and then the primary alcohol was deprotected with TBAF to give the primary alcohol 50 ready for oxidation into an acid.

In this way, Chandrasekhar developed a stereoselective route for the synthesis of a protected AMMTD derivative from (S)-citronellol in 3.0% yield over linear 26 steps. Alternatively, this alkene 53 was obtained directly by cross metathesis of the allyl ester 51 with methyl acrylate using Grubbs' II catalyst in a more modest yield. 381 Sharpless asymmetric aminohydroxylation 373,382 of (E)-alkene 53 smoothly produced β-amino alcohol 54 as a single regioisomer with an excellent 97% ee. Further protection of this β-amino alcohol as an oxazlidinone using 2-methoxypropene furnished the protected product 55 efficiently. Subsequent removal of the 4-methoxybenzoyl group with Cs 2 CO 3 in methanol gave a primary alcohol which was readily oxidized into aldehyde 56 via Dess-Martin oxidation. [START_REF]A useful 12-I-5 triacetoxyperiodinane (the Dess-Martin periodinane) for the selective oxidation of primary or secondary alcohols and a variety of related 12-I-5 species[END_REF] A modified Julia olefination of the oxazolidine-protected aldehyde 56 with 1-phenyl-1H-tetrazol-5-ylsulfone [START_REF]A stereoselective synthesis of trans-1,2-disubstituted alkenes based on the condensation of aldehydes with metalated 1-phenyl-1H-tetrazol-5-yl sulfones[END_REF] and KHMDS in 1,2-dimethoxyethane gave the alkene 57 as a mixture of isomers with a moderate stereoselectivity (E:Z =2.7:1). Fortunately, radical mediated isomerization using thiophenol in the presence of AIBN improved the E:Z ratio 6.5:1. 385 Sharpless asymmetric dihydroxylation 386 of alkene 57 using DHQ-IND ligand gave the corresponding diol 58 as an inseparable mixture of diastereoisomers in a 7.4:1 ratio. Finally, the diol was protected as its acetonide 59 and the PMP ether was removed using CAN to give a primary alcohol which was oxidized into aldehyde 60 using Dess-Martin reagent. and 17% yield respectively over 13 steps.

Aitken's work on the synthesis of protected APTO and AETD

The host group for this thesis reported the synthesis of protected APTO and AETD derivatives in 2008, 387 starting from 2-deoxy-D-ribose acetonide 63, 388 through an unified methodology based on a completely diastereoselective two-carbon homologation of appropriately selected intermediate chiral sulfinimines. [389][START_REF] Ellman | N-tert-Butanesulfinyl Imines: Versatile Intermediates for the Asymmetric Synthesis of Amines[END_REF][391] The synthetic route started with a Horner-Wadsworth-Emmons reaction of 2-deoxy-D-ribose acetonide 63 with a phosphonate-stabilized carbanion formed by deprotonation of benzyl diethylphosphonate to furnish the pure (E)-alkene 64 in moderate yield (Scheme 11).

Subsequently, a modified Swern oxidation 392 of the primary alcohol gave an aldehyde which was submitted to epimerization at the α-position according to an adapted literature method 393 by treatment with K 2 CO 3 in MeOH to give aldehyde 65 with the requisite stereochemistry.

Condensation of this aldehyde with (S)-(-)-tert-butanesulfinamide in the presence of Ti(OEt) 4 gave the chiral sulfinimine 66 in 69% yield. [START_REF] Davis | Asymmetric Synthesis of Polyhydroxy α-Amino Acids with the Sulfinimine-Mediated Asymmetric Strecker Reaction: 2-Amino 2-Deoxy L-Xylono-1,5-lactone (Polyoxamic Acid Lactone)[END_REF] According to Qin's report, the addition of a bulky O-Boc protected glycolate to simple chiral sulfinimines should proceed with a high diastereoselectivity. [START_REF]Highly Diastereoselective Enolate Addition of O-Protected α-Hydroxyacetate to (SR)-tert-Butanesulfinylimines: Synthesis of Taxol Side Chain[END_REF] Thus, the two-carbon homologation was performed by addition of the lithium enolate of O-Boc methyl glycolate to the sulfinimine 66 to generate the protected APTO derivative 67 as a single diastereomer in excellent yield. It is noteworthy that the stereochemical outcome of this two-carbon homologation is completely controlled by the sulfinyl group and thus proceeds independently of the other neighboring chiral centers in the system. This chiral predominance phenomenon was previously noted for sulfinimine one-carbon homologation with cyanide. [START_REF] Davis | Asymmetric Synthesis of Polyhydroxy α-Amino Acids with the Sulfinimine-Mediated Asymmetric Strecker Reaction: 2-Amino 2-Deoxy L-Xylono-1,5-lactone (Polyoxamic Acid Lactone)[END_REF] Scheme 11: Synthesis of a protected APTO derivative

The same strategy was applied for the preparation of a protected AETD derivative (Scheme

Williams' work towards AMPTD

An asymmetric synthesis of the core of AMPTD, as the polyhydroxy--amino acid in microsclerodermins F-G, was reported by Williams' group in 2009. 396 The AMPTD residue has five contiguous stereogenic centers and a conjugated-trans triene.

Dauban and Dodd's work on synthesis of protected APTO

In 2009, an alternative pathway to the fully protected form of APTO was reported by Dauban and Dodd 398 on the basis of their previous research on 2,3-aziridino-γ-lactones. [START_REF] Tarrade-Matha | Enantiospecific Synthesis of a Protected Equivalent of APTO, the β-Amino Acid Fragment of Microsclerodermins C and D, by Aziridino-γ-lactone Methodology[END_REF][399][400][401][402][START_REF] Tarrade | A new straightforward synthesis of 2,3-aziridino-γ-lactones" C. de Saint-Fuscien[END_REF][404][405][406][START_REF] Valle | Reactivity of 2,3-Aziridino-2,3-dideoxy-D-lyxono-γ-lactone Derivatives, Rigid Analogs of Aziridine-2-carboxylic Esters, toward Soft and Hard Nucleophiles: Control of Lactone vs Aziridine Ring Opening and C-2 vs C-3 Regioselectivity[END_REF] The key step is a regioselective opening of an aziridine derived from L-gulose by a soft nucleophile. 408

L-Gulose was protected as a diacetonide under standard conditions, 409 and the remaining free hydroxyl group was protected as its PMB ether to give the fully protected derivative 81 (Scheme In 2003, the first total synthesis of a microsclerodermin family member, microsclerodermin E, was reported by Ma et al. 424 Microsclerodermin E presents a dehydrated pyrrolidinoacetic acid residue (PyrAA) which is therefore not sensitive to the dehydration process reported by Shioiri, [START_REF]Construction of Three Building Blocks for the Total Synthesis of Microsclerodermins[END_REF]360 making its synthesis somewhat easier. The synthesis consisted of the construction of the tetrapeptide H-PyrAA-Sar-Trp(2-CO 2 H)-Gly-OMe and the dipeptide N 3 -GABOB-AETD-OH, coupling of these two peptides and macrocyclization between the GABOB and the Gly residue (Figure 28). 108. 425 Hydrolysis of the acetonides using 1 M HCl followed by regioselective reprotection of the free hydroxyl groups gave the new diacetonide 109. Activation of the free secondary alcohol as a corresponding mesylate followed by treatment with sodium azide furnished the azide 110.

with compound 111 afforded the diene 112 as a mixture of diastereoisomers (E/Z = 5:1). Selective deprotection of the terminal acetonide using PPTS gave 113, 379 followed by protection of the primary alcohol using TBSCl and of the secondary alcohol as a MOM ether produced the fully protected derivative 114. Finally, the TBS ether was removed by treatement with TBAF and reductive cleavage 427 of the trifluoroacetamide with NaBH 4 provided the AETD derivative 115, ready for peptide coupling with GABOB.

To summarize, Ma's synthesis of a protected AETD derivative ready for peptide coupling was achieved in 14% yield in 19 steps starting from δ-gluconolactone. The four stereogenic centers came from δ-gluconolactone, and the unsaturated tail was installed in a classic Julia olefination.

Preparation of the dipeptide GABOB-AETD

Similarly to Shioiri, [START_REF]Construction of Three Building Blocks for the Total Synthesis of Microsclerodermins[END_REF]360 Ma started the synthesis of a GABOB derivative from the azido derivative 25 obtained from dimethyl (R)-malate (Scheme 17). Thus protection of the secondary alcohol by TBSCl gave the protected azide 116. Saponification of the methyl ester followed by coupling of the resulting carboxylic acid with hydroxysuccinimide produced the activated ester 117. 428 Then, coupling with the free amine of AETD derivative 115 gave the amide 118 efficiently.

Oxidation of the primary alcohol into an aldehyde with Dess-Martin reagent and further oxidation of this aldehyde with NaClO 2 gave the carboxylic acid 119 which was then activated as the dipeptide succinimidyl ester 120.

Synthesis of the protected deydropyrrolidinone residue

In a similar way to Shioiri, the PyrAA was prepared from aspartic acid. However, the stereochemistry of this residue is different in microscleordermin E and required the use of the D-enantiomer. Thus protection of the carboxylic acid group of (R)-28 as an allyl ester followed by introduction of a second Boc group on the amino group gave the fully protected D-aspartic acid derivative 121 efficiently (Scheme 18). The allyl group was then removed using Wilkinson's catalyst and treatment of the bis-Boc-aspartic acid derivative thus obtained with thionyl chloride resulted in the formation of the Leuchs' anhydride 122 (urethane N-carboxyanhydride, UNCA) 429 which is both a protected and activated form of an amino acid. Addition of the lithium enolate of Scheme 17: Preparation of dipeptide GABOB-AETD trimethylsilylethyl acetate yielded the β-keto ester 123 in excellent yield with no noticeable racemization (ee > 98%). Other activation modes of the amino acid were studied, such as N,N'-carbonyldiimidazole, N-hydroxysuccinimide and pentafluorophenol (using Masamune's or Shioiri's procedure, Nu: Li-enolate or Mg-reagent), [START_REF]Construction of Three Building Blocks for the Total Synthesis of Microsclerodermins[END_REF] but resulted in partially or fully racemized β-keto ester. Then, the benzyl group was removed by hydrogenolysis to give the γ-keto acid 124

which was activated as a mixed anhydride using IBCF, and treated with aqueous ammonia to give an intermediate hydroxypyrrolidone which was directly subjected to elimination through mesylation to provide the pyrrolidinone derivative 125 in good yield. Deprotection of the trimethylsilylethyl by TBAF in the presence of p-TsOH to suppress of racemization 430 gave the free acid with partial racemization (R/S ratio 4:1 in the best case) and coupling with pentafluorophenyl alcohol using EDCI provided the PFP-activated ester 126 ready for peptide coupling. [START_REF] Kovacs | Optical purity of peptide active esters prepared by N,N'-dicyclohexylcarbodiimide and complexes of N,N'-dicyclohexylcarbodiimide-pentachlorophenol and N,N'-dicyclohexylcarbodiimide-pentafluorophenol[END_REF]432 Herein, the dehydropyrrolidinone derivative 126 was obtained in 33% yield over 11 steps.

Donohoe's work on microsclerodermins B and J

Starting from 2013, Donohoe's group worked on the total synthesis of microsclerodermins B and J. [438][439][START_REF] Melikhova | Dehydromicrosclerodermin B and Microsclerodermin J: Total synthesis and structural revision[END_REF] The most challenging part of these two targets was the hydroxypyrrolidinoacetic acid residue which is extremely sensitive and whose hemiaminal moiety was easily dehydrated under basic or acidic conditions, and up to this point had not been targeted in a total synthesis of any microsclerodermin family member. After preliminary studies and observations, they proposed to install the sensitive PyrAA residue at a late stage of the total synthesis (Figure 29). In a similar way to Ma's work on microsclerodermin E, 424 the macrocycle was constructed by peptide coupling of the dipeptide Fmoc-GABOB-AMMTD-OH with the tetrapeptide H-PyrAA-Sar-Trp(2-CO 2 TMSE)-Gly-OFm, macrocyclization between the GABOB and the Gly residue, then installation of the hydroxyl group on the pyrrolidinoacetic acid residue and finally assembly of the styrene moiety. In 2013, the synthesis of a protected derivative of the polyhydroxy-β-amino acid (AMMTD fragment) present in microsclerodermins A and B was reported by Donohoe's group. 438 The synthesis relied on sequential Sharpless dihydroxylation 352,353 and tethered aminohydroxylation 441 of a diene derived from (S)-Roche ester to install the requisite four stereogenic centers. Extension of the aliphatic side chain required a S N 2 reaction of primary iodide with Lipshutz-type lithiocuprate reagent. 442,443 Since the installation of the hydroxyl group on the pyrrolidinoacetic acid residue by hydroxybromination-debromination was not compatible with the styryl moiety, the plan was to introduce this moiety latter by cross-metathesis at the end of the total synthesis (See § 4.3.2.3.). The precursor of AMMTD has a terminal alkene chain, and is referred to as AMMTD * .

The synthesis commenced with benzylation of the primary alcohol of the (S)-Roche ester using benzyl 2,2,2-trichloroacetimidate [START_REF] Imidoester | Die Umlagerung von Trichloracetimidaten zu N-substituierten Säureamiden[END_REF][START_REF] Patil | A simple access to trichloroacetimidates[END_REF][START_REF] Eckenberg | A useful application of benzyl trichloroacetimidate for the benzylation of alcohols[END_REF] mixture of regioisomers (TBS-migration/preservation = 9:1). Finally, the remaining free alcohol was protected as its TBS ether and both the amine and primary alcohol were deprotected with CSA to produce the β-amino alcohol 147 and at this stage the regioisomers were isolated as a single compound, ready for coupling with an appropriate GABOB derivative.

Preparation of the dipeptide GABOB-AMMTD*

The GABOB derivative was synthetized from the azido derivative 116 by adaptation of the route developed by Shioiri [START_REF]Construction of Three Building Blocks for the Total Synthesis of Microsclerodermins[END_REF]360 and Ma. 424 Saponification of the methyl ester followed by hydrogenolysis of the azide group and subsequent protection of the resulting free amine by a Fmoc group gave the protected γ-amino acid 148 which was then activated as the acyl fluoride 149 using cyanuric fluoride (Scheme 23). 448,449 Therefore, peptide coupling of this acyl fluoride with the protected AMMTD derivative 147 gave the amide 150 efficiently. Finally oxidation of the primary alcohol into an aldehyde with Dess-Martin reagent and further oxidation of this aldehyde with NaClO 2 gave the dipeptide acid 151 which was ready for further peptide coupling.

Scheme 23: Construction of the dipeptide Fmoc-GABOB-AMMTD*-OH

Preparation of the pyrrolidinoacetic acid residue

Based on the fact that the hydroxypyrroldinoacetic acid residue is sensitive to dehydration, Donohoe proposed an interesting strategy to construct the sensitive hydroxypyrrolidinoacetic acid residue by electrophilic addition on the double bond of PyrAA at a late-stage in the total synthesis (Scheme 24). 439

Scheme 24: Late-stage hydration strategy for the preparation of PyrAA residue

The dipeptide PyrAA-Sar was chosen as a model to study this strategy. Thus, the dehydropyrrolidinoacetic acid residue (±)-152 was prepared in its racemic form by modification of the chemistry reported by Shioiri [START_REF]Construction of Three Building Blocks for the Total Synthesis of Microsclerodermins[END_REF]360 and Ma. 424 Coupling with sarcosine trimethylsilylethyl ester, treatment with TFA to remove the Boc group and acylation with acetic anhydride provided the racemic model dipeptide (±)-153. Additionally, the enantiopure material was obtained in 8 steps from L-aspartic acid derivative (S)-28 (Scheme 25). Activation of the acid with ethylchloroformate followed by treatment with ammonia produced an amide which was dehydrated into the nitrile 154.

Then, Blaise reaction 450 of a t-butyl bromoacetate derived zinc enolate reagent onto this nitrile and subsequent lactamization gave the dehydropyrrolidinoacetate (S)-155 with excellent stereoselectivity (ee 95%). Deprotection of both the t-buyl ester and the Boc group followed by reprotection of the free amine with Boc 2 O gave the protected derivative (S)-152 which was then coupled with sarcosine trimethylsilylethyl ester, treated with TFA to remove the Boc group and finally acylated with acetic anhydride provided the model dipeptide (S)-155 in 54% overall yield for 8 steps.

While direct hydration of the model dipeptide (±)-153 with either Brønsted or Lewis acids were not effective, the hydroxy group was easily introduced by hydroxybromination using various reagent (NBS, TBCD, [451][452][453][454][455] Br 2 ) (Table 2). Thus, the bromohydrin (±)-156 was obtained in good yield as a single diastereoisomer presenting the appropriate Reductive debromination of the bromohydrin derivative was performed under radical conditions using tributyltin hydride and a catalytic amount of triethylborane as an initiator at low temperature. 457 In this way, hydroxypyrrolidone derivative (±)-157 was obtained in excellent yield (Equation 1). During this study, Donohoe noted that the use of AIBN as an initiator required a higher temperature and resulted in dehydration, and hydrogenolysis using Pd/C also led to dehydration.

Equation 1: Radical-mediated debromination of bromohydrin (±)-156

Before commencing the total synthesis of microsclerodermin, the compatibility of this strategy with the AMMTD residue, which possesses an electron rich p-methoxystyrene moiety susceptible to react with the bromine source, was evaluated. 457 Thus a series of long-chain dipeptides (similar to the AMMTD fragment) and bearing the styrene moiety, an alkene or an alkyne which could be used as precursors of the styrene moiety, or an alkane (for comparison purpose) were prepared and submitted to the hydroxybromination-debromination sequence (Table 3). Upon treatment with NBS (or TBCD) (entry 1), the undesired bromination of the aromatic ring was observed which therefore excluded to perform in this reaction in the presence of the styryl moiety. Both terminal alkene (entry 2) and terminal olefin (entry 3) led to a regioselective hydroxybromination in good yields (73% and 65% respectively) comparable to the 61% yield obtained on the saturated substrat (entry 4). Radical-mediated debromination gave the corresponding hydroxypyrrolidones efficiently, although reaction on the olefin (entry 2) required the careful control of the amount of Bu 3 SnH employed (≤ 1.1 equivalent). To explore this strategy further, the olefin substrate was submitted to cross metathesis [START_REF] Trost | Ruthenium-Catalyzed Alkene-Alkyne Coupling: Synthesis of the Proposed Structure of Amphidinolide A[END_REF][START_REF] Chatterjee | A General Model for Selectivity in Olefin Cross Metathesis[END_REF][START_REF] Quigley | Ruthenium-catalysed Z-selective cross metathesis of allylic-substituted olefins[END_REF] with an excess of p-methoxystyrene using Grubbs I catalyst. The expected styrene derivative was obtained efficiently as a single diastereoisomer with the appropriate geometry and with no signs of dehydration of the hydroxypyrrolidone moiety.

In summary, thanks to these model studies, Donohoe provided support for his interesting strategy to create the hydroxypyrrolidinoacetic acid residue directly on the macrocyclic peptide in one of the very last steps of the synthesis of microsclerodermin B. 346,347 which is structurally closely related to microsclerodermin B, and thus suspected that the dehydropyrrolidinoacetic acid residue was (45S) instead of (45R) (Figure 31). The only difference between dehydomicrosclerodermin B and microsclerodermin J is that the 2-carboxy-D-tryptophan residue is replaced by D-tryptophan. Thus, the synthesis was adapted to the D-tryptophan derivative 170, and the tetrapeptide 171 was obtained in 6 steps following the same procedure (See § 4.3.2.4 and § 4.3.2.5) used to prepare the tetrapetide 162 (Scheme 32).

Finally, peptide coupling followed by Fm and Fmoc group deprotection and subsequent macrolactamization and deprotection of the side chained as described above (See § 4.3.2.7) gave the macrocycle 172.

Scheme 32: Synthesis of (45R)-microsclerodermin J 172

No rotamers were observed in the 1 H NMR spectra of (45R)-microsclerodermin J 172.

Comparison of the 13 C NMR spectra with that obtained by Li 346,347 for the natural microsclerodermin J revealed a complete superimposition of the two spectra match (Δδ less than 0.1 ppm). Donohoe concluded that the configuration of the dehydropyrrolidinoacetic acid residue had been mis-assigned, as had been the case microsclerodermin B.

In conclusion, Donohoe prepared dehydromicrosclerodermin B and microsclerodermin J and reassigned the configuration of the PyrAA residue in both cases. He developed highly efficient approaches to the unusual amino acids such as the modified tryptophan fragment via Negishi coupling and the pyrrolidinone unit through a Blaise reaction. Although he proposed an interesting strategy for the late stage introduction of the sensitive hydroxyl group of the pyrrolidinoacetic acid residue, which was validated using model substrates, the approach failed when applied to the hexapeptide macrocycle. Thus, to date, none of the microsclerodermins with the sensitive hydroxypyrrolidinoacetic acid residue have been synthetized.

Synthetic work: conclusion

Over the decades, many groups worked on the total synthesis of microsclerodermins or of fragments thereof, such as Shioiri, Ma, Donohoe, Aitken, Chandrasekhar, Williams, McLeod, Dauban and Dodd, Davies. As described above, much of the work has focused on the preparation of the unusual amino acids present in microsclerodermins. To date, only two total synthesis have been reported: first Ma described the preparation of microsclerodermin E in 2003, 424 and recently Donohoe reported the synthesis of microsclerodermin J in 2016. [START_REF] Melikhova | Dehydromicrosclerodermin B and Microsclerodermin J: Total synthesis and structural revision[END_REF] The work of Shioiri [START_REF]Construction of Three Building Blocks for the Total Synthesis of Microsclerodermins[END_REF]360 and later Donohoe [START_REF] Melikhova | Dehydromicrosclerodermin B and Microsclerodermin J: Total synthesis and structural revision[END_REF] underline that microsclerodermins are extremely sensitive to dehydration of the hydroxypyrrolidinoacetic acid residue. This raises the question if microsclerodermins E, J and K are true natural products or perhaps the results of dehydration during the extraction process. This proposal viewpoint could be evidenced by 3 reasons below:

-only 3 members (microsclerodermins E, J and K) of the microsclerodermin family are reported to have the dehydrated pyrrolidinoacetic acid unit -as described by Donohoe in the 2016, [START_REF] Melikhova | Dehydromicrosclerodermin B and Microsclerodermin J: Total synthesis and structural revision[END_REF] Li provide a sample of dehydromicrosclerodermin B which was obtained by dehydration of microsclerodermin B in the HPLC purification process -combining all results previously reported from Shioiri, [START_REF]Construction of Three Building Blocks for the Total Synthesis of Microsclerodermins[END_REF]360 Ma 424 and Donohoe, [START_REF] Melikhova | Dehydromicrosclerodermin B and Microsclerodermin J: Total synthesis and structural revision[END_REF] it is clear that the hydroxypyrrolidinoacetic acid unit is extremely sensitive to dehydration and therefor can't be maintained during the synthesis

Objectives

The host research group is interested in various cyclopeptide natural products (cyclotheonamide C [464][465][START_REF] Faure | Passerini Reaction-Amine Deprotection-Acyl Migration Peptide Assembly: Efficient Formal Synthesis of Cyclotheonamide C[END_REF] ), and derivatives (rhodopeptin [START_REF] Roy | A solution to the component instability problem in the preparation of peptides containing C2-substituted cis-cyclobutane β-aminoacids: synthesis of a stable rhodopeptin analog[END_REF] ) and has already developed various synthetic methodologies for the preparation of β-and γ-amino acids and notably of some polyhydroxy-β-amino acids present in microsclerodermins (APTO, AETD). 387 The total synthesis of microsclerodermins thereof appeared to us to be a stimulating challenge.

The objective of the research described in this thesis is the total synthesis of microsclerodermin D (Figure 32), [START_REF] Schmidt | Microsclerodermins C -E, antifungal cyclic peptides from the lithistid marine sponges Theonella sp. and Microscleroderma sp[END_REF] which features the familiar 23-membered ring macrocyclic peptide of the microscelrodermin family, and contains six amino acids of which three are common: glycine (Gly), sarcosine (Sar) and (R)-γ-amino-β-hydroxybutyric acid (GABOB). The other three amino acids possess some structural particularities, but they can be classified as a chlorinated tryptophan (6-Cl-Trp), a polyhydroxylated β-amino acid (APTO) containing four contiguous stereogenic centers and the sensitive 3-amino-4-hydroxypyrrolidone-4-acetic acid (PyrAA). We planned to develop a modular synthetic route to microsclerodermin D which implicated a non-dehydrated pyrrolidinoacetic acid residue that could be applicable for the preparation of other microsclerodermin family members and analogues thereof. The choice of microsclerodermin D as a target was based not only on the presence of the intact PyrAA residue, but on the fact that an efficient synthesis of a protected derivative of APTO is already available, thanks to an in-house methodology (See § 4.2.3.). 387 The APTO fragment also exists in microsclerodermin C [START_REF] Schmidt | Microsclerodermins C -E, antifungal cyclic peptides from the lithistid marine sponges Theonella sp. and Microscleroderma sp[END_REF] which present an additional aminocarbonyl group on the indole nitrogen of the tryptophan residue and in microscleordermin L 263 which possess an additional substituent on the pyrrolidinoacetic acid residue. Thus, it appeared to us that a synthetic route to microsclerodermin D should also provide a basis for a synthesis of microsclerodermins C and L.

We herein propose a convergent synthetic approach to microsclerodermin D in which the peptide backbone will be constructed from three dipeptide subunits instead of a stepwise manner, and macrolactamization will be performed between the glycine and GABOB residues, where there is little steric hindrance and since glycine can be activated without epimerization (non-chiral center).

The major difficulty to be faced is that the hydroxypyrrolidinoacetic acid unit is highly sensitive and dehydrates easily in acidic or basic media. Our proposal is therefore to maintain this residue in a masked form all along the synthetic process and to liberate it in the very last step of synthesis, after formation of the macrocycle and deprotection of the different hydroxyl groups.

Thus, microsclerodermin D would be obtained by final-step deprotection of the masked pyrrolidinoacetic acid residue (mPyrAA) which is expected to undergo spontaneous cyclization (Scheme 33). The protected macrocyclic peptide would be prepared by peptide coupling between three appropriately protected dipeptides: mPyrAA-Sar, Trp(6-Cl)-Gly and GABOB-APTO.

Glycine and GABOB residues would be advantageously protected by non-orthogonal groups in order to perform a single step deprotection before macrolactamization with a t-butyl ester and a t-butyl carbamate being the groups of choice. (R)-6-Chlorotryptophan would be prepared by adaptation of literature chemistry and few modifications of Shioiri's procedures for preparation of 2-carboxytryptophan, [START_REF]Construction of Three Building Blocks for the Total Synthesis of Microsclerodermins[END_REF]360 and a simple peptide coupling with glycine would produce the dipeptide Trp(6-Cl)-Gly. The synthesis of protected APTO residue has been successfully reported by the host group, 387 and the synthesis of GABOB is already known in the literature and may requires some adaptation of the protecting groups. [468][469][START_REF] Herkommer | Design, Synthesis, and Biological Evaluation of Simplified Side Chain Hybrids of the Potent Actin Binding Polyketides Rhizopodin and Bistramide[END_REF] The most challenging task would appear to be the construction of the dipeptide mPyrAA-Sar containing a masked equivalent of the pyrrolidinoacetic acid residue that can be carried all through the synthesis and then generate the pyrrolidinoacetic acid unit in the final-step of the synthesis without dehydration.

Introduction

The success of our proposed total synthesis of microsclerodermin D depends on the ability to introduce the 3-amino-4-hydroxypyrrolidinoacetic acid residue (PyrAA) which is highly sensitive and dehydrates easily in acidic or basic media. To date, efforts toward the synthesis of any microsclerodermin presenting this residue have failed [START_REF]Construction of Three Building Blocks for the Total Synthesis of Microsclerodermins[END_REF]360 and even if Donohoe was able to establish a masking/unmasking strategy that proved to be valid on a tripeptide model, he was not able to reproduce this transformation on the macrocyclic peptide. 439,[START_REF] Melikhova | Dehydromicrosclerodermin B and Microsclerodermin J: Total synthesis and structural revision[END_REF] Thus, only microsclerodermins E and J presenting a dehydrated PyrAA residue have been prepared. 424,[START_REF] Melikhova | Dehydromicrosclerodermin B and Microsclerodermin J: Total synthesis and structural revision[END_REF] Thus, at the outset of this thesis, establishment of a synthetic access to this unit amenable on the macrocyclic peptide remains an unsolved challenge.

Considering the pioneering work of Shioiri [START_REF]Construction of Three Building Blocks for the Total Synthesis of Microsclerodermins[END_REF]360 and of Donohoe,439,[START_REF] Melikhova | Dehydromicrosclerodermin B and Microsclerodermin J: Total synthesis and structural revision[END_REF] we propose to maintain the PyrAA unit in a masked form all along the synthesis process and to liberate it in the very last step after formation of macrocyclic peptide (See Chapter 1, § 5, Scheme 33). This strategy requires the use of a linear precursor of the pyrrolidinoacetic acid residue presenting a primary amide function and a masked keto group (Scheme 34). It was expected that following unmasking of this latter group, spontaneous cyclization of the γ-ketoamide would occur (See Chapter 1, § 4.1.4, Scheme 5) and will be then exploited to install the pyrrolidinone ring by simple deprotection of the keto group. The macrocyclic precursor of microsclerodermin D bearing the masked pyrrolidinoacetic acid residue (mPyrAA) will be obtained by coupling of three dipeptides. Among them, the dipeptide mPyrAA-Sar will be prepared by coupling between appropriately protected mPyrAA and commercially available sarcosine. The mPyrAA residue will be derived from a Scheme 34: Synthetic strategy to the pyrrolidinoacetic acid residue -amino-β-ketoester which will be obtained from D-aspartic acid through two-carbon homologation of the carboxylic acid function. Although the use of D-asparagin is appealing, it was exclude as starting material since both the keto group and the amide function can't be present at the same time to avoid an unwanted spontaneous cyclization.

The choice of the keto protecting group is crucial to the success of our strategy.

Transformations of ketones into ketals and dithioketals are among the most common methods to protect them. Since the keto group must be kept in a masked form all along the synthesis process and liberated in the very last step without the use of Brønsted base or acid (to avoid dehydration of the PrAA residue), dithioketals appeared as a good choice since they are stable in the presence of Brønsted base or acid (and therefore orthogonal to many protecting groups), and are usually deprotected by Lewis acids (such as HgCl 2 /HgO), NBS or PIFA (Figure 33).

Synthetic strategy

Inspired by Shioiri's work, [START_REF]Construction of Three Building Blocks for the Total Synthesis of Microsclerodermins[END_REF]360 we set out to prepare a suitably founctionalized derivative of mPyrAA from D-aspartic acid (Scheme 35). Thus, the dipeptide mPyrAA-Sar should be obtained by peptide coupling of a N-terminal protected mPyrAA derivative with sarcosine. This mPyrAA derivative should be obtained by transformation of the ester side chain into a primary amide and deprotection of the C-terminal ester group of an aminodiester derivative obtained by protection of the keto group as a dithioketal. The parent γ-amino-β-ketoadipate should be obtained via a two-carbon homologation of an appropriately protected D-aspartic acid derivative following Masamune's procedure. [START_REF] Brooks | C-Acylation under Virtually Neutral Conditions[END_REF] Scheme 35: Retrosynthetic analysis of the dipeptide mPyrAA-Sar

The proposed synthetic strategy excludes the use of certain protecting groups. Since formation of the dithioketal requires the use of Lewis acid, Boc and t-butyl ester which are removed in acidic medium can't be used. Moreover, since amide formation will be performed using ammonia, the Fmoc group is also excluded. Furthermore, orthogonal protecting groups for the two carboxylic acid functions are required. Taking into accounts all these parameters, our initial plan was to use a Cbz group to protect the amine function, a methyl ester for the aspartic acid side chain and a benzyl ester for second carboxylic acid function.

Synthesis of the N-Cbz protected pyrrolidinoacetic acid derivative

It was decided that the development of our synthetic strategy would be done using L-aspartic acid and once the synthesis had been optimized, the required pyrrolidinoacetic acid residue would be prepared from D-aspartic acid. As outlined above, the β-ketoester would be obtained via two-carbon homologation of an aspartic acid derivative under Masamune's conditions. [START_REF] Brooks | C-Acylation under Virtually Neutral Conditions[END_REF] This reaction employs the magnesium salt of a hemimalonate. Thus, monobenzylmalonate 173 was obtained quantitatively by treatment of Meldrum's acid with benzyl alcohol at 120 °C (Scheme 36). 471 The hemimalonate was then treated with 2 equivalents of isopropylmagnesium chloride to produce the corresponding magnesium enolate salt 174 which was used directly in the coupling reaction. [START_REF] Balducci | A Stereoselective and Practical Synthesis of (E)-α,β-Unsaturated Ketones from Aldehydes[END_REF] Scheme 36: Preparation of the monobenzylmalonate magnesium salt

Regioselective esterification of the side chain of L-aspartic acid was performed by treatment of a suspension of this amino acid in methanol to give the methyl ester as the ammonium salt 175 in good yield (Scheme 37). Then the amino group was protected by treatment with benzyl chloroformate in the presence of a base to give the protected L-aspartic acid derivative 176 in reasonable yield. 473 Activation of acid 176 using CDI followed by treatment with freshly prepared magnesium enolate 174 produced the -amino β-keto benzyl ester 177 in 82% yield (ee value undetermined). The next step consisted of the protection of the keto group as a dithioketal. We initially used classical conditions, using a slight excess of 1,2-ethanedithiol (1.5 eq.) and a catalytic amount of BF 3 •Et 2 O (0.4 eq.) in dichloromethane. 474 After some adaptions inspired by Fieser's conditions, 475,476 we found that the reaction worked better in acetic acid as solvent, and the use of larger amounts of 1,2-ethanedithiol (3 eq.) and of BF 3 •Et 2 O (5 eq.) added in 5 portions (every 2 days). Nevertheless, the reaction was sluggish and after one week at room temperature, the desired dithioketal derivative 178 was obtained in a poor 34% yield. All the starting material was consumed and we were able to detect about 40% yield of benzyl alcohol which clearly showed that extensive degradation occurred during the process. Heating the reaction mixture increased the degradation even over shorter reaction times. It appears that the Cbz group can also be deprotected under these conditions, in a similar way to the results of Thurston 477 who reported the Cbz deprotection with ethanethiol/BF

. Synthesis of the N-Fmoc--amino-β-ketoester

Since the Fmoc group is stable in acidic media and usually deprotected by organic bases (such as piperidine, pyridine, etc...), it should be stable under the dithioketal formation conditions

(1,2-ethanedithiol/BF 3 •Et 2 O). Thus, the L-aspartic acid derivative 175 was protected efficiently by a Fmoc group using FmocCl. 479 The resulting protected amino acid 179 480 was submitted to the Masamune's procedure to form the -amino-β-ketoester 180 in 82% yield after optimization.

Unfortunately, evaluation of the enantiomeric excess of this -amino-β-ketoester 180 by HPLC revealed an extensive racemization of the chiral center (ee = 0-28%), and despite many attempts, we

were not able to suppress the racemization phenomenon.

Scheme 38: Preparation of N-Fmoc -amino-β-ketoester

Origin of the racemization in the Masamune's protocol

Initially, this racemization was quite surprising since the Masamune's protocol has been the most commonly employed method for the synthesis of β-ketoesters, and among the thousands of papers and reports generated by many groups, no racemization was reported. In most cases, results

were evaluated on the basis of NMR data, and sometimes optical rotation was measured, but no enantiomeric excess values were given. In the specific case of Shioiri's work dealing with the preparation of the pyrrolidinoacetic acid residue via Masamune's protocol there is no evidence of the obtention of optically pure benzyl β-ketoester. [START_REF]Construction of Three Building Blocks for the Total Synthesis of Microsclerodermins[END_REF]360 Moreover, in his work on the total synthesis of microsclerodermin E Ma mentioned that addition of an enolate to an aspartic acid derivative activated by N,N'-carbonyldiimidazole (CDI), N-hydroxysuccinimide (NHS) or pentafluorophenol (PFP) led to partial or full racemization. 424 Thus, we came to the conclusion that the use of Masamune's protocol is not a reliable method for the preparation of optically pure β-ketoesters.

In Ma's published work, 424 there are no details and no discussion on the reasons leading to partial or full racemization. However, Zhu's PhD manuscript 481 (Ma's PhD student working on the total synthesis of microsclerodermin E) was quite informative and revealed that they initially followed the work of Shioiri and thus used the Masamune's procedure. The acid was firstly activated by CDI, then treated with a magnesium hemimalonate or a lithium enolate reagent, and in all cases partially or fully racemic -amino-β-ketoester 29 was obtained after many tests (Equation 2).

Synthesis of the masked PyrAA-Sar dipeptide

Although we were not able to access an enantiopure β-ketoester using Masamune's protocol, we decided to continue our development of this synthetic route to get the racemic dipeptide mPyrAA-Sar using partially racemized β-ketoester 180. We reasoned that our strategy to install the hydroxypyrrolidinoacetic acid residue should be validated first, and if indeed this was the case. Of course, in the meantime we also set out to explore various possibilities to prepare the β-ketoester 180 in an enantioselective manner.

Dithioketal protection of the N-Fmoc protected benzyl β-ketoester 4.3.1.1. Dithioketal protection of ketones

The dithioketal is a common protecting group for ketones. Since the 1950s, dithioketal protection of simple ketones was performed using 1,2-ethanedithiol/BF 3 •Et 2 O in acetic acid under Fieser's conditions, 475,476 and it has been widely employed by many groups, such as Smith 483,484 and Covey. [485][486][487] It has also proved its efficiency for the protection of keto-peptides by Powers 488,489 and Meldal. 490 Dithioketal formation can be performed using various acid catalysts such as BF 3 •Et 2 O, p-TsOH, SnCl 2 , TiCl 4 , Cu(OTf) 2 , Sc(OTf) 3 and others.

In one example, Powers 488,489 reported a highly efficient conversion of α-ketoester dipeptides into dithioketals in excellent yield using 1,2-ethanedithiol in the presence of BF 3 •Et 2 O in dichloromethane (Equation 3). These α-ketoesters dipeptides substrates appear to be really reactive, making the reaction efficient.

Equation 3: Powers's dithioketal protection of α-ketoesters dipeptides

In another example, Meldal 490 reported a highly efficient conversion of α-ketoamide dipeptides into the corresponding dithioketals in excellent yield using 1,2-ethanedithiol in the presence of BF 3 •Et 2 O in dichloromethane (Equation 4). Equation 4: Meldal's dithioketal protection of α-ketoamides dipeptides

Protection of N-Fmoc protected benzyl β-ketoester

We studied the protection of the racemic β-ketoester 180 using 1,2-ethanedithiol. An initial experiment with a slight excess of 1,2-ethanedithiol (1.2 equivalent) and a catalytic amount of BF 3 •Et 2 O (0.5 eq.) in dichloromethane 474 showed that the reaction was sluggish as it had been the case for the Cbz protected β-ketoester 177 and that although the desired product was observed, some degradation occurred. We varied the amount of 1,2-ethanedithiol and BF 3 •Et 2 O and the reaction temperature, but we obtained similar results. When the solvent was changed to acetic acid, we were able to obtain 50% yield of the expected dithioketal product 181 while 25% yield of starting material remained. In most cases, we often observed the formation of acetyl 1,2-ethanedithiol as a by-product. Further improvements were obtained by using a large amount of BF 3 •Et 2 O added in several portions, and finally we managed to get 75% yield of the product 181 by the use of larger excesses of 1,2-ethanedithiol (3 eq.) and of BF 3 •Et 2 O (6 eq.) added in 3 portions (every 2 days) (Equation 5). In this case almost all the starting material was consumed and less than 3% was detected by 1 H NMR. However, the reaction seemed to be difficult to reproduce and in some cases the reaction just stopped, even when excess of BF 3 •Et 2 O was added or after prolonged reaction time. Reaching a full conversion was really important since separation of the dithioketal 181 from the ketone 180 by column chromatography was extremely difficult. Equation 5: Dithioketal protection of the N-Fmoc benzyl β-ketoester

Access to the dipeptide mPyrAA-Sar

With dithioketal protected compound 181 in hand, access to the target dipeptide required a few more steps. First of all, the methyl ester should be transformed into a primary amide, the benzyl ester should then be deprotected to allow the final coupling with a protected derivative of sarcosine.

Alternatively, the transformation of the methyl ester into the primary amide could be performed after peptide coupling.

Hydrolysis of methyl ester

The success of the hydrolysis of the methyl ester depends on our ability to maintain the Fmoc protecting group. In 1998, Pascal The failure of the saponification reaction could be the result of a heterogeneous reaction mixture that impedes the reaction. Our substrate 181 possesses a large organic moiety that could considerably limit its solubility in an aqueous medium. Moreover, the methyl ester appears to be quite sterically hindered.

Conversion of the methyl ester into a primary amide

Since the hydrolysis of methyl ester 181 was unsuccessful, we then studied the direct conversion of the methyl ester into a primary amide. Direct ammonolysis of a methyl ester was usually achieved by treatment with NH 3 , NH 4 OH, or HCONH 4 , but under these conditions the Fmoc group will also be deprotected.

To examine this eventuality, the methyl ester 181 was treated with 28% NH 4 OH in THF or in acetone, with a solution of NH 3 in methanol or with gaseous ammonia (Equation 7). In all cases the starting material was completely consumed and we observed the deprotection of the Fmoc group (presence of DBF). However complex mixtures of products were obtained and we were not able to detect or isolat the expected primary amide 183.

Since these results were not encouraging, we decided to modify our strategy and to install the primary amide at the dipeptide stage. This required the deprotection the benzyl ester before the peptide coupling.

Deprotection of the benzyl ester

Since sulfur compounds often poison palladium catalysts, it was not reasonable to envisage the hydrogenolysis of the benzyl ester of dithioketal compound 181. Thus, we examined the hydrogenolysis of the benzyl β-ketoester 180 over Pd/C. Unfortunately, we obtained the methyl ketone product 185 instead of the expected β-ketoacid 184 due to spontaneous decarboxylation (Scheme 43).

Scheme 43: Hydrogenolysis of the benzyl β-ketoester 180

A literature search revealed that a benzyl ester can be deprotected using HBr in acetic acid. 497 In the event, treatment of the N-Fmoc benzyl β-ketoester 181 using a 33% HBr solution in acetic acid produce 85% yield of N-Fmoc β-ketoacid 186 (Equation 8). The reaction had to be carefully controlled since both reaction time and subtrate concentration affected the chemical selectivity between the benzyl ester and the methyl ester and traces of diacid by-product were often observed. Moreover, it should be noted that traces of ketone 180 as impurity in sample of 181 also gave some by-products that led to a difficult purification of 186. -when the reaction was incomplete, the ketone 180 and the dithioketal 181 were very difficult to separate.

-traces of ketone 180 as impurity in sample of 181 gave some by-products during the benzyl deprotection

In order to address these problems, the dithioketal formation reaction was performed without solvent (Scheme 44). The use of a larger excess of 1,2-ethanedithiol (10 eq.) and BF 3 •Et 2 O (3 eq.) allowed us to obtain 85% yield of the N-Fmoc β-dithioketal acid 186 after only 3 days at room temperature. The reaction was followed by TLC and after 24 h we observed the formation of the intermediate dithioketal 181, keto group protection therefore occurred before cleavage of the benzyl ester.

Scheme 44: One-pot dithioketal formation and benzyl deprotection required mPyrAA-Sar dipeptide. The acid 186 can be engaged in the peptide coupling with sarcosine followed by installation of the primary amide or alternatively, the methyl ester could be converted into the primary amide before the coupling with sarcosine (Scheme 45).

Scheme 45: Alternative routes for the preparation of the mPyrAA-Sar dipeptide 189

Peptide coupling and conversion of the methyl ester into a primary amide

Peptide coupling between acid 186 and H-Sar-Ot-Bu was examined using different coupling reagent (Table 4). When the coupling was operated using EDCI in the presence of DMAP, 60% yield of the expected dipeptide 187 was obtained (entry 1). However, when DIC was used instead of EDCI, the coupling gave only 40% yield of 187 together with diisopropylurea as an unseparable mixture (entry 2). Activation of the acid with HATU in the presence of DIEA gave 61% yield of the peptide 187 (entry 3) whereas the use of PyBOP in the presence of DMAP produced 187 in 69% yield (entry 4). Finally, the use of PyBroP in the presence of DIEA allowed the formation of the dipeptide 187 in an excellent 95% yield (entry 5 The next step was the conversion of the methyl ester into a primary amide. Dipeptide 187 was treated by various sources of ammonia (Table 5). In a first experiment (entry 1), the dipeptide 187 was treated with 28% NH 4 OH in THF (1:1) and a very obvious phenomenon was observed at the beginning of the reaction (5-10 min): a white solid appeared at the surface of the solution, which we identified as dibenzofulvene (DBF). However, the reaction provided only unknown products along with 98% yield of DBF. Changing the solvent from THF to methanol (entries 2-3) allowed the formation of the amide in low yields after prolonged reaction times and increasing the proportion of methanol improved the yield. These reactions gave numerous unidentified by-products and the highly polar primary amide 188 was difficult to isolate in a pure form. Dipeptide 187 was then treated with an ammonia solution in methanol (entry 4) and after 5 days a complex mixture of products in which traces of amide 188 were observed by Since deprotection of the Fmoc group occurred during ammonolysis of the methyl ester, the dipeptide 188 was protected again using FmocCl in the presence of a base to give the protected dipeptide 189 in modest yield which was difficult to isolate in a pure form (Equation 9). Equation 9: Protection of the aminodipeptide 188

In summary, although coupling of 186 with sarcosine was successful, selective saponification of the methyl ester was not possible. Installation of the primary amide function by direct ammonolysis of 186 was accompanied by Fmoc deprotection, so a re-protection step was required. Thus, we were able to obtain the key dipeptide 189 in its racemic form in a rather low yield of 6.2% over 8 steps from aspartic acid. The last two steps are really limiting, characterized by poor yields, messy reaction and arduous purification.

Conversion of the methyl ester into a primary amide and peptide coupling

The difficulties encountered during the formation of the primary amide at the dipeptide stage led us to reexamine the transformation of a methyl ester into a primary amide. In the literature, we noticed a facile synthesis of β-amino primary amides in good yield reported by Kim 498 in which the hydrochloride salts of β-amino methyl esters were treated directly with NH 4 OH (Scheme 46). We supposed that the reaction was facilitated by a good solubility of the substrate in NH 4 OH.

Scheme 46: Kim's conversion of methyl esters into primary amides

Inspired by Kim's results, we considered the ammonolysis of the acid 186 (Scheme 47).

Treatment of this compound with 28% NH 4 OH gave the expected primary amide 190 in quantitative yield. The Fmoc group was then reinstalled under standard conditions and the resulting protected amino acid 191 was then coupled with sarcosine using PyBroP in the presence of DIEA to produce the key dipeptide 189 in excellent yield.

Scheme 47: Synthesis of key dipeptide 189 by ammonolysis followed by peptide coupling

In summary, ammonolysis could be performed efficiently on the acid 186 and thus, after Fmoc re-protection, we were able to obtain the key dipeptide 189 in its racemic form in 22.6% yield over 8 steps from aspartic acid.

Deprotection of the dithioketal and spontaneous cyclization

Methods for the deprotection of thioketals into the corresponding carbonyl compounds have been extensively reviewed. [499][500][501][502][503][504][505] The deprotection of a dithioketal can be achieved by three main pathways:

-acidic or transition metal ions hydrolysis (such as Hg +2 , Ti +4 , Ag Scheme 48: Overview of the deprotection followed by spontaneous cyclization strategy First, some common conditions were attempted for deprotection of the dithioketal 189 (Table 6): the use of CAN (entry 1), iodine in the presence of NaHCO 3 (entry 2) 506,507 and PIFA (entry 3) 508 lead to extensive degradation and complex mixtures of unidentified compounds. We then turn our attention to use the mixture of mercury salts. The first report of the deprotection of a dithioketal into a carbonyl group using a mercury salt was presented by Fisher in 1894 in his work on sugars, 509 and remains the most common method, partially due to its low cost and wide versatility. The reaction requires exactly 1.0 equivalent of Hg 2+ per sulfur atom (i.e. 2 equivalent of HgCl 2 for the dithioketal), any less amount would not allow a complete reaction, whereas an excess could lead to undesired solvent-mercuriation formation. 505 Typically, the reaction is run in the presence of an excess of a mild base such as HgO or CaCO 3 , in order to neutralize the HCl formed during hydrolysis. 502 The commonly used solvent medium is a mixture of acetonitrile or THF and water, but numerous early works reported the use of methanol and water. [START_REF] Tosin | VI. N,N-Disubstituted 4-aminobenzophenones from 1,3-dithian-and 1,3-dithiolan-2-ylium tetrafluoroborates, respectively" I. Stahl[END_REF]511 When dipeptide 189

was treated with HgO (3 eq.) and HgCl 2 (2 eq.) in a mixture of methanol and water (9:1) for 5 days, only the starting material 189 was recovered (entry 4). When larger amount of HgO (8 eq.) and

HgCl 2 (12 eq.) were used 5 days, some degradation was observed but 60% of the starting material 189 remained intact (entry 5). 499,[START_REF] Tosin | VI. N,N-Disubstituted 4-aminobenzophenones from 1,3-dithian-and 1,3-dithiolan-2-ylium tetrafluoroborates, respectively" I. Stahl[END_REF]511 Additionally, the reaction was also run in THF and water (8:2) for 24 h, and 93% of the starting material 189 was recovered (entry 6). We attributed the failure of the use of mercury salts to a strong affinity of primary amide with mercury ion, which has been explained and evidenced by Leopold 512 and Sease. 513 This strong affinity probably led to the formation of a Hg-amide complex that precipitate resulting in no reaction. However, interestingly, when changing the solvent for a mixture of acetonitrile and water, the reaction provided 10% yield of the dehydrated compound 194 and 60% of a by-product, the nitrile 195, after 24 h (entry 7) (Equation 10). We proposed that the formation of 195 resulted from the dehydration of the primary amide catalyzed by HgCl 2 similarly to the PdCl 2 catalyzed dehydration of primary amides in aqueous acetonitrile proposed by Maffioli in 2005. [START_REF] Maffioli | Mild and Reversible Dehydration of Primary Amides with PdCl2 in Aqueous Acetonitrile[END_REF] Subsequently, NBS was used for the deprotection of dithioketal. When dipeptide 189 was treated with NBS in the presence of AgNO 3 and Na 2 CO 3 in a mixture of acetonitrile and water (entry 8) 515 no starting material was observed but an unidentified product was obtained. We supposed that a Hofmann rearrangement 516 of the amide occurred because of the presence of water in a weakly basic medium since Ivanović has recently reported a NBS-mediated Hofmann rearrangement of carboxamides. [START_REF] Jevtic | Hofmann Rearrangement of Carboxamides Mediated by N-Bromoacetamide[END_REF] Then, treatment with NBS

Access to optically pure β-ketoester

Since we had successfully validated our mPyrAA strategy using a model substrate, we returned to solve the problem of the preparation of β-ketoester 180 as enantiomerically pure material. Racemization occurred during the Masamune's procedure between the activated aspartic acid derivative and the magnesium hemimalonate although this latter reagent was reported to be neutral.

Racemization of α-amino acids derivative is a common issue in peptide synthesis and can occurred in the presence of various activating reagents, such as SOCl 2 , CDI, NHS, PFP, IPCF, DIC, DCC and even HATU, HBTU. However, in many cases the racemization can be controlled.

β-Ketoesters are commonly obtained via cross-Claisen condensation on N-Boc or N-Cbz protected amino acids, or by addition of a lithium enolate (or a magnesium hemimalonate) on N-Fmoc-amino acid derivatives. 518,519 In 2015 Maillard developed a short route to heterocyclic γ-amino acids from N-Fmoc protected γ-amino-β-ketoesters which were obtained via cross-Claisen condensation of lithium enolates on N-Fmoc-amino acids (Table 7). [START_REF] Mathieu | Cross-Claisen condensation of N-Fmoc-amino acids -a short route to heterocyclic γ-amino acids[END_REF] Compared to neutral magnesium hemimalonates, lithium enolates are more basic, and thus more likely to induce racemization of α-amino acid derivatives. 49). 518,[START_REF] Mathieu | Cross-Claisen condensation of N-Fmoc-amino acids -a short route to heterocyclic γ-amino acids[END_REF]522,[START_REF] Honda | An efficient synthesis of γ-amino β-ketoester by cross-Claisen condensation with α-amino acid derivatives[END_REF] Scheme 49: Common approaches to β-ketoester via activation of α-amino acids

The N-Fmoc protected amino acids are of increasing importance in peptide synthesis, especially solid-phase peptide synthesis. [START_REF] Mathieu | Cross-Claisen condensation of N-Fmoc-amino acids -a short route to heterocyclic γ-amino acids[END_REF][524][START_REF] Di Gioia | Synthesis of chiral nitrones from N-Fmoc amino acids and N-Fmoc dipeptides[END_REF][START_REF] Garcia | Evaluation of Solution and Solid-Phase Approaches to the Synthesis of Libraries of α,α-Disubstituted-α-acylaminoketones[END_REF][527][528][529] In his work on the total synthesis of didemnins, Schmidt 530,531 claimed that treatment of Fmoc-alloisoleucine chloride with the lithium enolate of methyl trimethylsilylmalonate furnished the corresponding enantiopure N-Fmoc protected β-ketoester, which was based on Carpino's results (Scheme 50). 532 However, only optical rotation data was provided, and the enantiomeric excess was not reported.

Scheme 50: Schmidt's preparation of N-Fmoc protected β-ketoesters

Several methods for preparation of the N-Fmoc β-ketoester 180 in its optically pure form were systematically explored in order to secure an alternative to the Masamune's procedure, as we presented below.

Direct condensation of a Weinreb amide with a lithium enolate

Historically, Weinreb amides have been used for synthesis of ketones through treatment with a Grignard reagent (or an alkyllithium or a lithium enolate), and few examples of the preparation of β-ketoesters have been reported. The two common approaches to convert Weinreb amides into ketones are:

-reduction of Weinreb amides into aldehydes with LiAlH 4 , [START_REF]An efficient synthesis of optically active α-(t-butoxycarbonylamino)-aldehydes from α-amino acids[END_REF][START_REF]A Practical Approach to the Synthesis of 2,4-Disubstituted Oxazoles from Amino Acids[END_REF][START_REF] Shindo | Paecilopeptin, a new cathepsin S inhibitor produced by Paecilomyces carneus[END_REF][536][START_REF] Yang | Evaluation of α,β-unsaturated ketone-based probes for papain-family cysteine proteases[END_REF] followed by treatment with a lithium enolate or a Grignard reagent, and final oxidation 538,[START_REF] Koseki | A Formal Total Synthesis of (±)-Cephalotaxine Using Sequential N-Acyliminium Ion Reactions[END_REF] -direct displacement using an alkyllithium reagent [START_REF]A Practical Approach to the Synthesis of 2,4-Disubstituted Oxazoles from Amino Acids[END_REF] or a Grignard reagent [START_REF] Nahm | N-Methoxy-N-methylamides as effective acylating agents[END_REF][START_REF]Tosylhydrazide-Promoted Diastereoselective Intramolecular 1,3-Dipolar Cycloadditions: Synthesis of Tetrahydropyrrolo[END_REF] In our case, since we required to keep the methyl ester on the side chain, reduction of the corresponding Weinreb amide into an aldehyde with LiAlH 4 seemed likely to pose the problem of selectivity between Weinreb amide and methyl ester. Thus, direct displacement by treatment with a lithium reagent or a Grignard reagen appeared to be a more promising approach.

After perusal of literatures, we found that most Weinreb amides derived from N-Cbz, N-Boc or N-Ac α-amino acids [START_REF]A Practical Approach to the Synthesis of 2,4-Disubstituted Oxazoles from Amino Acids[END_REF][START_REF]Tosylhydrazide-Promoted Diastereoselective Intramolecular 1,3-Dipolar Cycloadditions: Synthesis of Tetrahydropyrrolo[END_REF][START_REF] Myers | A New Family of Small Molecules To Probe the Reactivation of Mutant p53[END_REF][543][START_REF] Ripa | Discovery of a Novel Oral Glucocorticoid Receptor Modulator (AZD9567) with Improved Side Effect Profile[END_REF] were used to provide ketones by treatment with Grignard or alkyllithium reagents. In 1989, Turner [START_REF] Turner | Acylation of ester enolates by N-methoxy-N-methylamides: an effective synthesis of β-keto esters[END_REF] reported an effective synthesis of β-ketoesters by treatment of Weinreb amides of simple carboxylic acids with lithium enolates (Equation 11). However there 103 are no cases of the preparation of β-ketoesters by treatment of Weinreb amides derived from N-protected α-amino acids with lithium enolates. A few cases of Weinreb amides derived from N-Fmoc α-amino acids were found to have applications in solid-phase reactions. [START_REF] Garcia | Evaluation of Solution and Solid-Phase Approaches to the Synthesis of Libraries of α,α-Disubstituted-α-acylaminoketones[END_REF][527][528] Equation 11: Conversion of Weinreb amides into β-ketoesters

The protected amino acid derivative 179 was converted into the corresponding Weinreb amide 196 by treatment with N,O-dimethylamine hydrochloride using PyBroP in the presence of DIEA (Scheme 51). [START_REF] Yang | Evaluation of α,β-unsaturated ketone-based probes for papain-family cysteine proteases[END_REF] Treatment of this Weinreb amide with the lithium enolate of benzyl acetate gave a complex mixture of unidentified products, but cleavage of the Fmoc group was observed.

Scheme 51: Attempt of the preparation of β-ketoester by addition of an enolate on a Weinreb amide

Coupling with Meldrum's acid followed by treatment with benzyl alcohol

Early in 1982, Yonemitsu 546 reported that acylation of Meldrum's acid derivatives could be used for the synthesis of β-ketoesters by condensation of acid chlorides. In 1987, Jouin [START_REF] Jouin | Stereospecific synthesis of N-protected statine and its analogs via chiral tetramic acid[END_REF] reported a facile approach to N-protected (Cbz or Boc) statines and their analogues via chiral tetramic acid derivatives (Scheme 52). The main step consisted in the condensation of α-amino acids with Meldrum's acid using IPCF in the presence of DMAP. The unstable intermediate thus obtained underwent decarboxylation and cyclization in refluxing acetonitrile or ethyl acetate, and even in methanol to give the cyclic tetramic acid derivatives instead of the expected γ-amino-β-ketoesters.

Thus, reduction of the keto group followed by methanolysis or hydrolysis produced the corresponding γ-amino-β-hydroxyesters or γ-amino-β-ketoesters. In his total synthesis of microsclerodermin E, Ma attributed the low enantiomeric excess of the -amino-β-ketoester prepared via the Masamune's procedure on the CDI activated Boc-amino acid to the presence of a free proton on the carbamate, which led to racemization in basic medium. 424 To preclude this process, we envisaged the protection of the amino group of aspartic acid as a phthalimide.

The aspartic acid derivative 175 was treated with N-carbethoxyphthalimide to produce the phthalimido derivative 199 in excellent yield (Scheme 54). [START_REF] Zhang | Stereoselective synthesis of chiral α-amino-β-lactams through palladium(II)-catalyzed sequential monoarylation/amidation of C(sp(3) )-H bonds[END_REF] Then Masamune's reaction of 199

with magnesium enolate 174 gave the β-keto-γ-phthalimidoester 200 in modest yield. [START_REF] Lin | Stereoselective formation of a functionalized dipeptide isostere by zinc carbenoid-mediated chain extension[END_REF] The racemic material was also prepared via the same synthetic route. [START_REF] Gazvoda | Completely Stereocontrolled Aldol Reaction of Chiral β-Amino Acids[END_REF] However, we were unable to achieve the separation of the enantiomers upon chiral HPLC analysis of the racemic material (±)-200 and therefore we were unable to evaluate a possible racemization in sample 200 prepared from non-racemic 175.

Scheme 54: Use of a N-Phth protected substrate for the preparation of benzyl β-ketoester

Through Mukaiyama aldol condensation followed by oxidation

Alternatively, the β-ketoester could be installed by oxidation of the corresponding β-hydroxyester. Thus, a possible pathway would be the reduction of the acid into an aldehyde followed by an aldol condensation then oxidation of the alcohol into a ketone. α-Amino aldehydes are prone to racemization and the method to prepare them is really important, among the various methods developed to avoid epimerization, two possibilities emerge: reduction of a Weinreb amide with LiAlH 4 or Fukuyama reduction of a thioester with triethylsilane in the presence of palladium.

The aspartic acid derivative 179 was coupled with propanethiol using HATU in the presence of DIEA to give the protected thioester 201 in good yield (Scheme 55). 555 Fukuyama reduction [556][557][START_REF] Tokuyama | Reduction of ethanethiol esters to aldehydes[END_REF][559][START_REF]New odorless protocols for the synthesis of aldehydes and ketones from thiol esters[END_REF][561][562] of the thioester 201 was performed with Et 3 SiH over Pd/C produced the aldehyde 202 in excellent yield (ee undetermined). This aldehyde was then submitted to a Mukaiyama aldol reaction [563][564][START_REF]Mukaiyama reactions of a silyl enol ether with heteroatom-substituted β-formyl esters[END_REF][566][567][568][569] to provide the γ-amino-β-hydroxyester 203 in 72% yield (ee undetermined) [START_REF] Park | A concise synthesis of tubuphenylalanine and epi-tubuphenylalanine via a diastereoselective Mukaiyama aldol reaction of silyl ketene acetal[END_REF] which was then submitted to Dess-Martin oxidation [START_REF]A useful 12-I-5 triacetoxyperiodinane (the Dess-Martin periodinane) for the selective oxidation of primary or secondary alcohols and a variety of related 12-I-5 species[END_REF][START_REF] Hickmann | Protecting-Group-Free and Catalysis-Based Total Synthesis of the Ecklonialactones[END_REF] to furnish the desired benzyl γ-amino-β-ketoester 180 near-quantitatively. 572 However, chiral HPLC analysis revealed that complete racemization had occurred.

Homologation of an aldehyde through the Roskamp reaction

The Roskamp reaction [START_REF] Holmquist | A selective method for the direct conversion of aldehydes into β-keto esters with ethyl diazoacetate catalyzed by tin(II) chloride[END_REF] consists of a double homologation of an aldehyde with a diazo compound in the presence of a Lewis acid that generates a ketone. It has already been applied for the synthesis of -amino-β-ketoesters. [574][START_REF] Bandyopadhyay | A facile transformation of amino acids to functionalized coumarins[END_REF][576][START_REF] Kumar | Synthesis of tetrasubstituted symmetrical pyrazines from β-keto γ-amino esters: A mild strategy for self-dimerization of peptides[END_REF][START_REF] Malik | Helices with Additional H-bonds: Crystallographic Conformations of α,γ-Hybrid Peptides Helices Composed of β-Hydroxy γ-Amino acids (Statines)[END_REF][579][580][581] Notably, Gopi reported a synthesis of N-protected -amino-β-ketoesters through the Roskamp reaction using Tin (II) chloride. 574 derivatives. [586][587][588][589] To date, thousands of reports and applications of Boc-and Cbz-UNCAs have been described over the decades. 429,482,587,[590][591][START_REF] Chevallet | Synthesis of chiral N-protected amino acid esters by the use of UNCAs[END_REF][593][594][595][596][597][598][599] There are only a few reports about N-Fmoc protected UNCAs (Fmoc-UNCAs). One example was the preparation of chiral urethane N-alkoxycarbonyl tetramic acids by condensation of UNCAs with Meldrum's acid in the presence of a tertiary amine, and subsequent cyclization supplied tetramic acid derivatives. [START_REF] Chevallet | Synthesis of chiral N-protected amino acid esters by the use of UNCAs[END_REF] Martinez and his colleagues have reported an interesting example of the use of UNCAs for the synthesis of Cbz and Boc protected -amino-β-ketoesters (Equation 12). 429 However Fmoc-UNCAs were not used probably because this protecting group was incompatible with the strongly basic lithium enolates. Equation 12: Preparation of -amino-β-ketoesters from Boc-and Cbz-UNCA

The most common approach to UNCAs is the direct phosgenation of free amino acids to produce NCAs (Fuchs-Farthing synthesis) 600-604 followed by the protection of the free nitrogen by alkoxycarbonylation (Scheme 58). 594 Alternatively, Boc or Cbz protected amino acids can be treated by thionyl chloride to give NCAs (Leuch's method), or by phosgene or triphosgene to give directly the UNCAs. However, there is no report about direct phosgenation of Fmoc-amino acids into

UNCAs. The main application of UNCAs is peptide coupling but they can react with a broad Scheme 58: Common methods for the preparation of UNCAs and NCAs range of nucleophiles. Racemization of UNCAs happens only by the direct enolization through abstraction of the α proton. However, NCAs and UNCAs are sensitive and difficult to purify because of their high reactivity [START_REF] Fuller | A procedure for the facile synthesis of amino-acid N-carboxyanhydrides[END_REF][START_REF] Ishida | Purification of N-carboxy-α-amino acid anhydrides" JP43020181B[END_REF][START_REF] Yamaguchi | Purification of α-amino acid N-carboxylic acid anhydrides[END_REF][START_REF] Togo | Purification of α-amino acid-N-carboxylic acid anhydrides" JP49024957A[END_REF] leading to various side reactions including polymerization. 596,598,599,608,609 In light of the interesting reactivity of UNCAs, we decided to study the reaction of magnesium hemimalonates and silyl enolether which are less basic than lithium enolates with the Fmoc-UNCA derived from aspartic acid as the activated amino acid derivative in order to access optically pure benzyl -amino-β-ketoester 180

We embarked on the preparation of the Fmoc-UNCA 204 by phosgenation of the Fmoc protected aspartic acid derivative 179 using triphosgene in the presence of an HCl trapping reagent such as α-pinene, TEA, DIEA or NMM. 610,611 In a first experiment the Fmoc amino acid 179 was treated with triphosgene (1.2 eq.) in the presence of α-pinene (2.5 eq.) in THF. After 6 h at 45 °C, the mixture was poured in n-hexane to crystallize the UNCA, but the sticky semi-solid obtained after filtration was only starting material (94% was recovered). Increasing the amount of α-pinene (5.0 eq.) and refluxing the reaction mixture for 18 h had no effect on the conversion and even when more α-pinene was added, this still had no use. We supposed that α-pinene was not strong enough to motivate the UNCA formation. When α-pinene was replaced by DIEA (2.0 eq.) and after 16 h of reaction at 60 C the expected Fmoc-UNCA 204 was observed, as well as the starting Fmoc amino acid 179. However, we also noticed the presence of dibenzofulvene which indicated that partial Fmoc deprotection had occurred. Moreover, UNCAs are not especially stable and heating the reaction might be deleterious to UNCA formation. The reaction was repeated at room temperature and after 3 h, some starting material was still present (determined by TLC). Additional triphosgene (0.15 eq.) and DIEA (0.5 eq.) were added and after an additional 3 h at room temperature, the mixture was poured into n-hexane to crystallize the UNCA. 1 H NMR analysis of the crude product showed an incomplete reaction and partial Fmoc deprotection, but about 40% yield of the desired UNCA 204 was observed. The crude sticky solid obtained was therefore treated directly in one case with magnesium hemimalonate 174 and in another case with the trimethylsilyl enol ether of benzyl acetate. Treatment with the magnesium hemimalonate 174 for 1 h produced 11% yield of the benzyl -amino-β-ketoester 180 over 2 steps with an enantiomeric ratio of 67:33, whereas treatment with the silyl enolether for 16 h gave only a complex intractable mixture. The preparation of Fmoc-UNCA 204 was considerably improved by addition of a solution of triphosgene (1.2 eq.) in THF over 30 min to a solution of the Fmoc amino acid 179 and DIEA (2.4 eq.) in THF. After 3 h at room temperature, almost 100% conversion was observed by 1 H NMR (Scheme 59). Since the crude Fmoc-UNCA appeared to be steadily decomposed during work-up and crystallization, 594 we decided to limit the treatment to the strict minimum. The diisopropylethylammonium salt precipitated was filtered off and the filtrate (containing traces of the ammonium salt) was used directly in the condensation step with the magnesium hemimalonate 174. After 6 h the expected benzyl -amino-β-ketoester 180 was obtained in 26% yield with an enantiomeric ratio of 84:16.

Scheme 59: Sequential Fmoc-UNCA formation and condensation with magnesium hemimalonate This very encouraging result in term of enantiomeric excess prompted us to continue our investigations on the use of Fmoc-UNCA. Although DIEA is a good promotor for the formation of the UNCA, unfortunately it is also a strong enough base that leads to Fmoc-deprotection and partial racemization. Moreover, the presence of traces of diisopropylethylammonium salt in the solution of Fmoc-UNCA seemed to affect the condensation of the magnesium hemimalonate and thus we decided to explore over bases (Table 8). Replacing DIEA by N-methylmorpholine (NMM) (entry 1)

for the formation of the UNCA in the conditions described above gave 56% yield of the -amino-β-ketoester 180 with an enantiomeric ratio of 94:6, which was a significant improvement.

However, when the UNCA formation was performed at 0 °C for 3 h (entry 2), after treatment with the magnesiusm hemimalonate only the starting material 179 was observed (90% were recovered).

When the amount of NMM was reduced and added over a shorter period of time (entry 3), we obtained 44% yield of the -amino-β-ketoester 180 with a slightly reduced enantiomeric ratio of 93:7. It was deduced that a slow addition of the base over 30 min at room temperature appeared the most appropriate conditions. Using these conditions, other bases were tested. When On the basis of these results above, sym-collidine was selected for further optimization (Table 9). When the solution of base was added at 0 °C over 30 min and the reaction mixture maintained at this temperature for 1 h (entry 1), the reaction seemed to slow down considerably, thus additional triphosgene was added and the mixture was the stirred for 4 h at room temperature.

The precipitate was filtered off and the filtrate, a solution of UNCA was split in two parts. On the first part, the addition of the magnesium hemimalonate was performed at -10 °C to provide 39% of the -amino-β-ketoester 180 with an enantiomeric ratio of 98:2. On the second part, the addition of the magnesium hemimalonate was performed at 0 °C to give 40% of the -amino-β-ketoester 180 with an enantiomeric ratio of 97:3. In order to improve the reaction rate, half of the solution of sym-collidine was added directly at 0 °C and the second half was then added over 30 min at room temperature (entry 2). Work-up after 1.5 h followed by condensation gave 53% of the -amino-β-ketoester 180 with an enantiomeric ratio of 95:5. When the solution of sym-collidine in THF was added all at once at room temperature and the stirring maintained for 1.5 h, we observed a slight decrease in yield and enantiomeric ratio (entry 3). Using a slight excess of sym-collidine (2.2 eq.) provoked a partial deprotection of the Fmoc group, revealed by the presence of DBF (entry 4).

Consequently, after condensation, only 26% of the product was obtained with a similar enantiomeric ratio. We then tried to add some FmocCl to compensate for Fmoc deprotection (entry 5), however we noticed that the reaction slowed down considerably and more triphosgene and sym-collidine were added. Finally after a total of 6 h for the formation of the UNCA, work-up and condensation with magnesium hemimalonate, we obtained 52% yield of the expected product with an enantiomeric ratio of 95:5. The speed of addition of sym-collidine seemed to influence the yield and thus a solution of sym-collidine in THF was added at 0 °C over 5 min (entry 6). Finally, after 1.5 h reaction time, work-up and condensation, 36% yield of the -amino-β-ketoester 180 was obtained with an enantiomeric ratio of 95:5. To test the impact of the temperature, the solution of sym-collidine in THF was added at 0 °C over 20 min and the mixture was then stirred at 0 °C for 40 min and then at r.t. for 2 h (entry 7). After work-up and condensation we obtained only 36% yield of the expected product with a slightly reduced enantiomeric ratio of 93:7. In conclusion, we successfully achieved the preparation of benzyl -amino-β-ketoester 180 in acceptable yield over 2 sequential steps and good enantiomeric excess.

The limitation of the yield was attributed to incomplete conversion of the starting material, whose consumption was difficult to be followed by TLC, and to polymerization of the NCA resulting from partial deprotection of the Fmoc group. Nonetheless, the optimization efforts were considered to be a success.

Entry

Conclusions

Final synthesis of the key dipeptide: (S)-mPyrAA-Sar

In summary, the (S)-mPyrAA-Sar dipeptide (S)-189 was obtained in 20.1% yield over 8 steps through the synthesis developed (Scheme 60). Notably, from (S)-180 to (S)-189 over 4 steps, an erosion of 10% ee occurred. Optimization of the condensation of the magnesium hemimalonate to the Fmoc-UNCA derivative of L-aspartic acid allow the preparation of the key benzyl -amino-β-ketoester (S)-180 with a good enantiomeric excess above 90%.

Scheme 60: Synthesis of the (S)-mPyrAA-Sar from L-aspartic acid

Final synthesis of the key dipeptide: (R)-mPyrAA-Sar

Since D-aspartic acid (R-configuration) was required for the total synthesis of microsclerodermin D, the same synthetic route was then applied to prepare the (R)-mPyrAA-Sar dipeptide (R)-189 from D-aspartic acid in 19.3% yield over 8 steps (Scheme 61). Notably, from

Synthesis of tryptophan derivatives

L-Tryptophan (Trp), the only proteingenic an amino acid with an indole ring, is one of the essential amino acids for animals. 612 It is not synthesized by the human body but ingested from diets such as meat, fish, eggs, fruit, vegetables, nuts, etc... and it plays a significant role in the synthesis of proteins and muscle tissues. It also serves as a precursor of key functional biomolecules such as tryptamine, quinolinic acid, nicotinamide, serotonin and others. 613 Besides, Tryptophan is the precursor of indole-3-acetic acid, the most abundant auxin in higher plants (Figure 35). 614 Earlier in 1944, Snyder reported the first synthetic approach to 3-indolemethylene derivatives using quaternary ammonium salts (methiodide of gramine) as alkylating agent. It was discovered that quaternary ammonium salts could generate a benzyl type radical. [START_REF] Snyder | C-Alkylation with quaternary ammonium salts. A new approach to the synthesis of compounds containing the β-indolemethylene group[END_REF] Afterwards, this method was used for the synthesis of DL-tryptophan and its analogues. [START_REF] Howe | Application of a new alkylation reaction to the synthesis of tryptophan[END_REF][START_REF] Snyder | A Convenient Synthesis of dl-Tryptophan[END_REF][START_REF] Snyder | A synthesis of tryptophan and tryptophan analogs[END_REF] Interestingly, Albertson et al. reported the synthesis of tryptophan from gramine at the same time. 619,620 Alkylation of the methiodide of gramine, obtained in a 3-component Mannich reaction between indole, formaldehyde and dimethylamine by Kuhn's procedure, 621 was performed with various reagents: sodium enolates of acetoacetates or cyanoacetates, potassium silver cyanide dialkyl aminomalonate (Scheme 62). [START_REF] Snyder | C-Alkylation with quaternary ammonium salts. A new approach to the synthesis of compounds containing the β-indolemethylene group[END_REF][START_REF] Howe | Application of a new alkylation reaction to the synthesis of tryptophan[END_REF][START_REF] Snyder | A Convenient Synthesis of dl-Tryptophan[END_REF] Scheme 62: Snyder's synthesis of tryptophan derivatives Current methods employed for the synthesis of tryptophan derivatives mainly rely on either chemical or enzymatic approaches, as well as biosynthesis:

-chemical synthesis approaches including stepwise functional group conversion, [START_REF] Howe | Application of a new alkylation reaction to the synthesis of tryptophan[END_REF][START_REF] Snyder | A Convenient Synthesis of dl-Tryptophan[END_REF][START_REF] Snyder | A synthesis of tryptophan and tryptophan analogs[END_REF][619] phase-transfer catalytic alkylation, [START_REF] Todd | One-pot enantioselective synthesis of tryptophan derivatives via phase-transfer catalytic alkylation of glycine using a cinchona-derived catalyst[END_REF]646 and metal-catalysis 460,625,[START_REF] Ma | Efficient Asymmetric Synthesis of Biologically Important Tryptophan Analogues via a Palladium-Mediated Heteroannulation Reaction[END_REF]648 -enzymatic synthesis [START_REF] Decottignies | Continuous enzymic synthesis of tryptophan" FR2379603A1[END_REF][START_REF] Miyahara | Enzymic process for preparing L-tryptophan and process for preparing a stable aqueous enzyme solution[END_REF][651][652][653] (tryptophan synthase such as TrpB2, N-acylase enzymes)

-biocatalytic platform 653-664 using bacteria, protozoa, neurospora, RNA or cells Historically, multistep syntheses of racemic tryptophan have been reported by many groups including those of Snyder, [START_REF] Snyder | C-Alkylation with quaternary ammonium salts. A new approach to the synthesis of compounds containing the β-indolemethylene group[END_REF][START_REF] Howe | Application of a new alkylation reaction to the synthesis of tryptophan[END_REF][START_REF] Snyder | A Convenient Synthesis of dl-Tryptophan[END_REF][START_REF] Snyder | A synthesis of tryptophan and tryptophan analogs[END_REF]619,620 Rinderknecht, 635 Hellmann, 632,665 Kornfeld, [START_REF] Jones | Synthesis of tryptophan and derivatives thereof[END_REF] McLaughlin 622 and recently Fuller. 666 To date, numerous asymmetric synthetic methods for chiral tryptophan derivatives have also been reported by many groups, which could be classified in two categories: use of chiral starting materials and use of chiral auxiliary or chiral ligands. The shortest synthesis of tryptophan consists of the direct coupling of the C 3 -position of indole with serine or its equivalents mediated by an enzyme such as tryptophan synthase (TrpB2, N-acylase enzymes) (Equation 13). [START_REF] Miyahara | Enzymic process for preparing L-tryptophan and process for preparing a stable aqueous enzyme solution[END_REF][651][652][653][671][672][START_REF] Besnainou | Recovery of tryptophan or its derivatives and enzymic synthesis of tryptophan[END_REF] Equation 13: Synthesis of L-tryptophan using tryptophan synthase

Various strategies have been developed to mimic synthetic pathways without enzymes. An example was the ring opening of aziridines derived from serine with 3-H-indoles to prepare various tryptophan derivatives (Equation 14). 674 However, the yields were unsatisfactory arranging from 3% to 57% after optimization, especially with lower yields for substituted indoles. Equation 14: Synthesis of tryptophan derivatives by ring-opening of aziridines

In 1989, Jackson reported the use of Negishi cross-coupling for the synthesis of enantiomerically pure protected β-aryl-alanines from aryl halides and readily available organozinc reagents of iodoalanine (derived from serine), 675 and for the preparation of enantiomerically pure protected γ-keto-α-amino acids from acyl chlorides and readily available organozinc reagent of iodoalanine. [START_REF] Jackson | A new direct method for the synthesis of enantiomerically pure protected α-amino acids[END_REF] Although numerous applications of the synthesis of various chiral aromatic amino acids have been reported. Only a few have reported the synthesis of tryptophan using this method.

For example, Rainier prepared the tryptophan unit of kapakahine E and F following this method to complete their total synthesis. 677,678 Kalesse reported a selective Pd-catalyzed vinylation of 4-bromoindole with dehydroalanine, followed by enantioselective hydrogenation of olefin in the presence of DuanPhos ligand, to synthesize the tryptophan derivative unit to complete the total synthesis of argyrin F. 679 Yokoyama explored various asymmetric synthesis of substituted tryptophan derivatives (Scheme 64):

-selective Pd-catalyzed vinylation of 4-bromoindole with dehydroalanine, followed by asymmetric hydrogenation [680][681][682][683][START_REF]A study for palladium-catalyzed chemoselective vinylation at C-3 position of 4-bromo-1-tosylindole[END_REF] -one-step synthesis of N-acetyltryptophan from indole and serine in the presence of acetic anhydride using kinetic resolution with acylase 685,686 -using Negishi cross-coupling reaction of iodoalanine with 3-haloindole without loss of enantiomeric purity after optimization. The best result was obtained when the reaction of 123 N-tosyl-3-bromoindole with N-Cbz-iodoalanine methyl ester was carried out in the presence of Pd 2 (dba) 3 and the sterically hindered ferrocenyl ligand Q-PHOS. 460 Scheme 64: Yokoyama's asymmetric synthesis of substituted tryptophan derivatives A short, practical and versatile synthesis of optically active substituted tryptophan derivatives from a variety of iodoanilines, via a palladium-mediated heteroannulation reaction with a chiral alkyne obtained by diastereoselective alkylation of the Schölkopf chiral auxiliary, 687 was developed by Cook (Scheme 65). 688 This method have been applied by many groups, such as Cook, 625, [START_REF] Ma | Efficient Asymmetric Synthesis of Biologically Important Tryptophan Analogues via a Palladium-Mediated Heteroannulation Reaction[END_REF] Castle, 689 Björn, 690 and Hecht. [START_REF] Talukder | Efficient Asymmetric Synthesis of Tryptophan Analogues Having Useful Photophysical Properties[END_REF] Scheme 65: Synthesis of substituted tryptophan derivatives through heteroannulation corresponding indoles, using a chiral auxiliary-facilitated Strecker amino acid synthesis 691 strategy (Scheme 66). [START_REF] Chen | A facile approach to tryptophan derivatives for the total synthesis of argyrin analogues[END_REF] Using this method, eight optically pure (S)-tryptophan analogues were synthesized, and subsequently used for the total synthesis of argyrin A and its analogues.

Scheme 66: Synthesis of optically active substituted tryptophan derivatives

Recently Katkevics reported a general method for the diastereoselective synthesis of substituted tryptophan derivatives by alkylation of William's morpholinone chiral auxiliary with quaternary gramines as alkylating agents with good overall yields (Scheme 67). 692

Scheme 67: Katkevics's diastereoselective synthesis of substituted tryptophan derivatives 125 Asymmetric phase-transfer catalytic alkylation of glycine imine derivative 209 using the cinchona-derived catalyst 210 was reported by Hossain for the preparation of substituted tryptophan derivatives with ee up to 95% (Scheme 68). [START_REF] Todd | One-pot enantioselective synthesis of tryptophan derivatives via phase-transfer catalytic alkylation of glycine using a cinchona-derived catalyst[END_REF] A similar method was also used by Maruoka for the enantioselective synthesis of α,α-dialkyl-α-amino acids by chiral phase-transfer catalysis from the Schiff base of 209, including the preparation of a N'-Boc protected D-tryptophan derivative in 78% yield and 91% ee. 646 Scheme 68: Enantioselective synthesis of tryptophan derivatives by asymmetric phase transfer catalysis A widely used method for the enantioselective preparation of tryptophan derivatives consists of a tandem Friedel-Crafts/conjugate addition of substituted indoles to dehydroalanines. For example, Reisman reported the reaction of 2-substituted indoles with methyl 2-acetamidoacrylate catalyzed by a BINOL•SnCl 4 complex to prepared synthetic tryptophan derivatives in good yiels and high level of enantioselectivity (Equation 15). [START_REF] Kieffer | Enantioselective Synthesis of Tryptophan Derivatives by a Tandem Friedel-Crafts Conjugate Addition/Asymmetric Protonation Reaction[END_REF] In a similar manner, Chen described the reaction of indoles with nitroacrylates catalyzed by the chiral complex (4R,5S)-DiPh-BOX-Cu(OTf) 2 . [START_REF] Sui | Catalytic and asymmetric Friedel-Crafts alkylation of indoles with nitroacrylates. Application to the synthesis of tryptophan analogues[END_REF] Besides, a convenient synthesis of constrained β-substituted tryptophans by asymmetric catalytic reaction of glycine derivatives with sulfonylindoles using a catalyst derived from AgCl and a commercially available chiral monodentate phosphoramidite ligand was developed by Hou. 693 Equation 15: Reisman's diastereoselective synthesis of substituted tryptophan derivatives

Synthesis of Trp-Gly

Alkylation of chiral glycine imine derivatives

Asymmetric synthesis of α-amino acids by alkylation of the glycine tert-butyl ester Schiff base derived from 2-hydroxypinanone is a well-known methodology, firstly reported by Yamada in 1976, 694 and widely applied by many groups. 361,[START_REF] Irako | A new asymmetric synthesis of (S)-dolaphenine and its heteroaromatic congeners utilizing (+)-2-hydroxy-3-pinanone and (-)-3-hydroxy-2-caranone as chiral auxiliaries[END_REF][START_REF] Yamada | Optically active α-alkyl-α-amino acid derivatives[END_REF][696][697][START_REF] Tabcheh | Alkylation and protonation of chiral Schiff bases: diastereoselectivity as a function of the nature of reactants[END_REF][699][700][701][START_REF] Swango | A novel enantioselective synthesis of (S)-(-)and (R)-(+)-nornicotine via alkylation of a chiral 2-hydroxy-3-pinanone ketimine template[END_REF][START_REF] Sugiyama | Efficient total synthesis of pentosidine, an advanced glycation endproduct[END_REF][704][705][706][707][708][START_REF] Cai | A general, efficient and stereospecific route to sphingosine, sphinganines, phytosphingosines and their analogs[END_REF][710][START_REF] Guilloneau | Preparation of novel beta-hydroxy amino acid derivatives via enantioselective aldolization using (1R,2R,5R)-(+)-2-hydroxy-3-pinanone as a chiral auxiliary and their use as synthesis intermediates for obtaining derivatives of sphingosines, ceramides and sphingomyelins with high yields[END_REF][712] However, only a few applications to the enantioselective synthesis of tryptophan derivatives have been described. [START_REF]Construction of Three Building Blocks for the Total Synthesis of Microsclerodermins[END_REF]360 The origin of stereoselectivity of this reaction was explored in details by different groups (Figure 36):

-a possible transition state was proposed by Soladié-Cavallo who postulated a dimeric aggregate based on literature X-ray crystallographic data early in 1993 [START_REF] Solladié-Cavallo | Diastereoselective monoalkylation of lithium and potassium enolates of a chiral imine of ethyl glycinate: the role of added salts[END_REF][714][715] -Seebach described the formation of dimers and even higher aggregates in 1988 when reviewing the structure and reactivity of lithium enolates 716 -computational studies were used by Matsumoto to proposed a meso-dimer type model on half of the dimeric enolate in 1996 699,700 -Haufe suggested the formation of aggregates of intermediary enolates in solution and diastereomeric interactions of the enantiomers in 2000 704

Figure 36: Proposed transition state type-model of alkylation of 2-hydroxypinanone glycine imine using lithium enolates

Strategy

Based on the work of Shiori, [START_REF]Construction of Three Building Blocks for the Total Synthesis of Microsclerodermins[END_REF]360 we proposed the preparation of the Trp(6-Cl) residue through alkylation of a 2-hydroxypinanone glycine imine derivative. This method presents the advantage of allowing access to either enantiomers depending on the stereochemistry of the chosen 2-hydroxypinanone. To fit with our overall plan for the preparation of microsclerodermin D, a Fmoc group and a t-butyl ester were chosen to protect the amine and the acid functions, respectively. The target dipeptide would be obtained by coupling of Fmoc-Trp(6-Cl)-OH with t-butyl glycinate (Scheme 69). The appropriately protected 6-chloroindole would be prepared by alkylation of the 2-hydroxypinanone glycine imine with an ammonium salt derived from 6-chlorogramine which would be obtained from 6-chloroindole through a Mannich reaction followed by quaternarization.

Synthesis of H-[(R)-Trp(6-Cl)]-Gly-Ot-Bu

The quaternary ammonium salt 211 was prepared efficiently in two steps from to provide the imine 213 in 52% yield and a diasteroisomeric ratio of 95:5 (after chromatography purification, the ratio improved above 97:3). It appearred that the hydrolysis of imine 213 required care to avoid racemization; thus, upon treatment with 15% citric acid, we were able to obtain 95% yield of the α-amino ester 214. Afterwards, protection of the amine with a Fmoc group followed by TFA-mediated hydrolysis of the t-butyl ester gave the N-Fmoc-Trp (6-Cl) derivative 215 in excellent yield over 2 steps. Interestingly, when hydrolysis of the t-butyl ester was performed in the absence of triethylsilane, the reaction was dirty with lower yield and the product was difficult to purify. In order to evaluate the enantiomeric excess of 216, the racemic material was also prepared according to a similar strategy, but the chiral Schiff base was replaced by the benzophenone imine derivative 218 (Scheme 72). Thus, condensation of glycine t-butyl ester with benzophenone imine gave the Schiff base 218 quantitatively. 718,[START_REF] Fanelli | Access to α,α-Disubstituted Disilylated Amino Acids and Their Use in Solid-Phase Peptide Synthesis[END_REF] This compound was deprotonated and treated with the quaternary ammonium salt 212 to provide the imine (±)-219. After hydrolysis of the imine, protecting group manipulation and peptide coupling, the racemic dipeptide (±)-216 was obtained in good yield and used to establish a chiral HPLC analysis protocol to evaluate the enantiomeric excess.

Scheme 72: Racemic synthesis of dipeptide Fmoc-Trp(6-Cl)-Gly-Ot-Bu

Conclusion

We successfully synthesized the Fmoc-Trp(6-Cl)-Gly-Ot-Bu dipeptide fragment in 32.2% yield over 7 steps with an enantiomeric excess of 94%, starting from 6-chloroindole. The chiral center was constructed by a classic strategy for chiral α-amino acids preparation based on the diastereoselective alkylation of the chiral Schiff base derived from 2-hydroxypinanone and glycine t-butyl ester with a gramine type of ammonium salt.

CHAPTER 4 SYNTHESIS OF DIPEPTIDE GABOB-APTO

Introduction

According to our proposed strategy, GABOB-APTO (Figure 37) is the third dipeptide fragment required for the total synthesis of microsclerodermin D. It is also found in microsclerodermins C and L. This fragment is composed of (R)-γ-aminobutyric acid (GABOB)

which is present in all the microsclerodermin family members, and of APTO, a polyhydroxylated β-amino acid residue containing four contiguous stereogenic centers. Across the microsclerodermin family, the polyhydroxylated β-amino acid unit presents some variations, and other residues like AETD, AMMTD, AMPTD, which contains four or five contiguous stereogenic centers and various styrene moiety can be encountered.

Figure 37: Dipeptide Boc-GABOB-APTO-OH GABOB is a relatively common γ-amino acid whose preparation is well documented [468][469][START_REF] Herkommer | Design, Synthesis, and Biological Evaluation of Simplified Side Chain Hybrids of the Potent Actin Binding Polyketides Rhizopodin and Bistramide[END_REF]720,[START_REF] Hayashi | A new synthesis of (+)-negamycin and its derivatives as a potential therapeutic agent for duchenne muscular dystrophy treatment[END_REF]

Synthesis of GABOB-APTO

Strategy

We proposed to prepare the APTO fragment through the in-house strategy 387 (See Chapter 1, § 4.2.3.) and to install appropriate protecting groups on the hydroxy groups, and to synthesize the protected GABOB from ethyl (R)-4-chloro-3-hydroxybutanoate according to Hayashi's procedures 469,[START_REF] Hayashi | A new synthesis of (+)-negamycin and its derivatives as a potential therapeutic agent for duchenne muscular dystrophy treatment[END_REF] (Scheme 73). To fit with our proposed total synthesis strategy, the amino group of GABOB would be protected by a Boc group, and all the hydroxyl groups would be protected by a TBS group, with the intention of performing a single deprotection operation at the appropriate moment. Functional group modification of the known fully-protected ATPO derivative 67 would require the cleavage of sulfinyl, acetonide and O-Boc groups in acidic medium, followed by Fmoc protection of amine and O-TBS protection of all the free hydroxyl groups, and final Fmoc deprotection. After coupling with protected GABOB, the methyl ester would be hydrolyzed in order to be ready for further coupling.

Scheme 73: Retrosynthetic analysis of the dipeptide GABOB-APTO derivative

Synthesis of the protected GABOB

The preparation of GABOB started with azidation of commercially available ethyl (R)-4-chloro-3-hydroxybutanoate according to Hayashi's procedures (Scheme 74). 469,[START_REF] Hayashi | A new synthesis of (+)-negamycin and its derivatives as a potential therapeutic agent for duchenne muscular dystrophy treatment[END_REF] In a first experiment the chloro ester was treated with sodium azide (1 eq.) at 90 °C in DMF for 48 h to give the azido ester derivative 220. Although complete conversion was observed, only 66% yield of the product was obtained, and we suspected that the prolonged reaction time at high temperature could be the cause. To reduce reaction time we turned to microwave activation. [START_REF] Ju | Revisiting nucleophilic substitution reactions: microwave-assisted synthesis of azides, thiocyanates, and sulfones in an aqueous medium[END_REF] Thus the chloroester was treated with sodium azide (1.3 eq.) in water under microwave irradiation at 120 °C for 30 min.

Unfortunately, the reaction was not complete and about 15% yield of starting material was observed.

Increasing the quantity of sodium azide (2.5 eq.) in the same conditions allowed a complete conversion and furnished 82% yield of the azido ester 220 which could be used without purification.

In a single procedure, the azide was reduced into an amine which was directly protected by a Boc

Synthesis of protected APTO

The protected ATPO derivative was then prepared from 2-dexoy-D-ribose according to the procedure developed by Aitken's group. 387 We began by preparing 2-deoxy-D-ribose acetonide 63 following Horton's conditions and procedures ( [START_REF] Bolte | Modular Total Syntheses of the Marine-Derived Resorcylic Acid Lactones Cochliomycins A and B Using a Late-Stage Nozaki-Hiyama-Kishi Macrocyclization Reaction[END_REF] Finally, the use of 2-methoxypropene in the presence of anhydrous PPTS in dried ethyl acetate according to Robertson's conditions 726 allowed the preparation of the expected acetonide 63 in 66% yield.

With 2-deoxy-D-ribose acetonide 63 in hand, we followed the synthetic route as previously described. 387 The acetonide was treated with a phosphonate-stabilized carbanion obtained by deprotonation of benzyl diethylphosphonate with BuLi, to furnish the pure E-alkene 64 in moderate yield (Scheme 75). In our hands Swern oxidation of alcohol 64 was inefficient and we thus decided to turn to Dess-Martin oxidation. The alcohol 64 was oxidized quantitatively into an aldehyde by treatment with Dess-Martin periodinane in the presence of sodium bicarbonate. 109,727 This aldehyde was then epimerized at the α-position by treatment with K 2 CO 3 in MeOH 393 to give aldehyde 65.

Condensation of this aldehyde with (S)-(-)-tert-butanesulfinamide in the presence of Ti(OEt) 4 gave the chiral sulfinimine 66 in 69% yield, [START_REF] Davis | Asymmetric Synthesis of Polyhydroxy α-Amino Acids with the Sulfinimine-Mediated Asymmetric Strecker Reaction: 2-Amino 2-Deoxy L-Xylono-1,5-lactone (Polyoxamic Acid Lactone)[END_REF] which was then treated with the lithium enolate of O-Boc methyl glycolate to produce the protected APTO derivative 67 in excellent yield and complete diastereochemical control (dr > 99:0:0:0).

Scheme 75: Synthesis of a protected APTO derivative

With the protected APTO derivative secured, we needed to adapt the protecting group pattern to fit with our proposed total synthesis strategy. Thus, all the hydroxyl groups should be protected as TBS ether and the amino group should be protected by a Fmoc group. Since the tert-butanesulfinyl, the acetonide and the O-Boc group are all acid labile, we decide to try to perform a complete deprotection through the action of TFA. HCl, all protecting groups can be removed in a single operation to provide the ammonium salt 225.

Moreover, it seemed difficult to install more than one TBS on the free hydroxyl groups, probably because of steric hindrance.

In the literature, the selectivity difference between TFA and HCl in the deprotection of some substrates with both acetonide and tert-butylsulfinyl, or with Boc and tert-butylsulfinyl has been reported by several groups. [START_REF] Jiang | Practical Asymmetric Synthesis of α-Branched 2-Piperazinylbenzylamines by 1,2-Additions of Organometallic Reagents to N-tert-Butanesulfinyl Imines[END_REF]729 We reasoned that it should be possible to selectively remove the tert-butylsulfinyl group of APTO derivative 67 using a 4 M solution of HCl in dioxane. In the course of our studies on the deprotection of compound 67 with HCl, we observed that the selectivity was highly dependent on the temperature (Scheme 77). Warm conditions resulted in the formation of the fully-deprotected lactone product 225, whereas ambient temperature allowed partial removal of the tert-butylsulfinyl group. Thus, when the reaction was performed at -10 °C, we were able to obtain the free amine APTO derivative 229 in a completely selective transformation and in quantitative yield. this step, the excess of HCl and most of the t-butanesulfinyl chloride produced during the deprotection were removed. 730 After coupling with GABOB, we were able to obtain up to 72% of the dipeptide 234 (Table 11, entries 1-2). Additionally, a one-pot reaction was also attempted, whereby the ammonium salt obtained by treatment with 4 M HCl was neutralized with DIEA, and Boc-GABOB(TBS)-OH and PyBroP were added to give 53% of the expected dipeptide 234 (entry 3). Finally, when amine 232 was isolated by simple extraction and used directly in the coupling with GABOB, the dipeptide 234 was obtained in 68% yield (entry 4).

Entry

Step

Saponification of Boc-GABOB-APTO-OMe

With the dipeptide derivative GABOB-APTO 234 in hand, we undertook the saponification of the methyl ester in order to make this dipeptide ready for pepetide coupling (Table 12). Initially, dipeptide 234 was treated with LiOH (4 eq.) at 0 C and slowly warmed to room temperature for 4

h, but no reaction was observed and the starting material was recovered quantitatively (entry 1).

When the reaction was performed with a large excess of LiOH (10 eq.) at room temperature for 16 h, followed by treatment with 10% citric acid at 0 °C until pH = 2-3, the desired dipeptide acid 235 was obtained in 71% yield, accompanied by traces of the alcohol 236 (entry 2). However, when the treatment with 10% citric acid until pH = 2-3 was performed at 5-10 °C, the alcohol 236 was obtained in 80% yield as a single product (entry 3). Since the O-Boc group is very sensitive to acidic conditions we tried to control the quenching conditions. Thus, the treatment was performed with a limited amount of citric acid (15 eq.)

Conclusion

In summary, starting from acetonide 63, we were able to prepare the third dipeptide derivative 234 required for the proposed total synthesis of microsclerodermin D in 20.2 % yield over 7 steps ad assumed to be enantiomerically pure (Scheme 80).

GENERAL CONCLUSION

The objective of this PhD thesis was to acomplish the first total synthesis of microsclerodermin D (Figure 32), which features a 23-membered ring macrocyclic peptide comprised of six amino acids, of which three are common: glycine (Gly), sarcosine (Sar) and

(R)-γ-amino-β-hydroxybutyric acid (GABOB). The other three amino acids possess some structural variations, but they can be classified as a modified tryptophan (Trp(6-Cl)), a polyhydroxylated β-amino acid (APTO) containing four contiguous stereogenic centers and a highly unusual 3-amino-4-hydroxypyrrolidone-4-acetic acid (PyrAA). We planned to develop a modular synthetic route to microsclerodermin D that could be applicable for the preparation of other microsclerodermin family members and their analogues thereof.

We proposed a convergent synthetic approach to microsclerodermin D in which the peptide backbone would be constructed from three dipeptide subunits, then macrolactamized between the glycine and GABOB residues, where there is little steric demand and the C-terminal residue is achiral and can be activated without racemization issue. The major difficulty to which we have to face is that the PyrAA unit which is highly sensitive and dehydrates easily in acidic or basic media.

Our proposal is therefore to maintain this unit in a masked form all along the synthetic process and to liberate it in the very last step after formation of macrocyclic peptide and deprotection of the various hydroxyl groups.

On this premise, three dipeptides were required: Fmoc-mPyrAA-Sar-Ot-Bu (R)-189 3) and Boc-GABOB(TBS)-APTO-OH 235 (Chapter 4), which will be assembled first into a tetrapeptide, then a hexapeptide which would be cyclized before last-step unmasking of the PyrAA unit. This thesis includes 5 chapters.

(Chapter 2), Fmoc-[D-Trp(6-Cl)]-Gly-Ot-Bu 216 ( Chapter
In Chapter 1, an overview on bioactive natural products, peptides and marine cyclic peptides, as well as a detailed introduction on microsclerodermins was presented. The important thesis results

we have achieved are summarized below from chapter 2 to chapter 5.

In Chapter 2, we focused on the synthesis of key dipeptide mPyrAA-Sar, including its synthesis and evaluation of the unmasking strategy. According to our synthetic strategy and route, a Optimization of the condensation of the magnesium hemimalonate to the Fmoc-UNCA derivative of L-and D-aspartic acid allowed the preparation of the key benzyl -amino-β-ketoester 180 with a good enantiomeric excess up to 90% (Table 8 and9). Additionally, evaluation of the unmasking strategy was performed using racemic dipeptide 189. Surprisingly, treatment of dipeptide 189 with NBS provided 33% yield of the hydroxypyrrolidone derivative 193, as well as 5% yield of the dehydrated compound 194 (Table 6, entry 10). This encouraging result cofirmed the potential of our strategy to install the sensitive hydroxypyrrolidinoacetic acid residue by the deprotection of dithioketal and cyclization sequence and it was therefore retained for the pursuit of our work the total synthesis of microsclerodermin D. Thus, (S)-mPyrAA-Sar dipeptide (S)-189 was obtained in 20.1% over 8 steps through the synthesis developed above (Scheme 60). The same synthetic route was then applied to prepare the target (R)-mPyrAA-Sar dipeptide (R)-189 from D-aspartic acid in 19.3% yield over 8 steps (Scheme 61). Similarly to the (S)-derivative, the key benzyl -amino-β-ketoester (R)-180 was obtained with a good enantiomeric excess above 90%.

In Chapter 3, we concentrated on the synthesis of the dipeptide Trp(6-Cl)-Gly fragment.

Based on Shiori's work, we have completed the synthesis of the Fmoc-[D-Trp(6-Cl)]-Gly-Ot-Bu dipeptide 216 in 32.2% yield over 7 steps with an enantiomeric excess of 94%, starting from 6-chloroindole (Scheme 71). The chiral center was constructed using a classic strategy for α-amino acids preparation based on the diastereoselective alkylation of the chiral Schiff base derived from 2-hydroxypinanone and glycine t-butyl ester with a gramine type ammonium salt. In order to evaluate the enantiomeric excess, the racemic material was also prepared according to a similar strategy, but the chiral Schiff base was replaced by the benzophenone imine derivative 218 (Scheme 72). The racemic dipeptide (±)-216 was obtained in good yield and used to establish chiral HPLC conditions to evaluate the enantiomeric excess.

In Chapter 4, firstly, the preparation of GABOB was completed over 4 steps in 45.2% yield In conclusion, it is expected that the first total synthesis of microsclerodermin D will be completed soon.

BIBLIOGRAPHY

Fourier-transform infrared (IR) spectra were recorded for neat liquid or solid samples on a FT-IR Perkin Elmer Spectrum using an ATR diamond accessory; maximum absorbances (ν) are given in cm -1 .

High-resolution mass spectrometry (HRMS) data were recorded using a Bruker

MicroTOF-Q spectrometer equipped with an electrospray ionization mass spectrometry (ESI-MS) in positive mode or negative mode (ESI (+) or ESI (-)).

Optical rotation was measured using a 10 cm quartz cell; values for [α] D T were obtained with the D-line of sodium at the indicated temperature T, using solutions of concentration (c) in units of g•100 mL -1 .

High performance liquid chromatography (HPLC) analysis was performed on a commercial

Agilent 1260 Infinity apparatus equipped with diode array detector. All solvents were HPLC grade and were filtered through a 0.45 μm PTFE membrane. Injected solutions were filtered through a 0.22 μm PTFE membrane.

3-(Benzyloxy)-3-oxopropanoic acid 173 472

To a flask containing Meldrum's acid (20.0 g, 138.8 mmol) was added benzyl alcohol (15.0 g, 14.4 mL, 138.8 mmol). The mixture was refluxed for 6 h (oil bath, 120 °C), then cooling down. After concentrated to remove acetone, monobenzylmalonate 173 (26.2 g, 98%) was obtained as a light colorless oil (stored in fridge into a waxy solid) and used without further purification.

TLC: R f = 0.10 (EtOAc = 100%) 1701, 1497, 1456, 1432, 1401, 1381, 1333, 1307, 1200, 1166, 1082 cm -1

IR (ATR) ν 2989,
1 H NMR (360 MHz, CDCl 3 ) δ 3.45 (s, 2H), 5.17 (s, 2H), 7.33-7.35 (m, 5H).

13 C NMR (90 MHz, CDCl 3 ) δ 40.99, 67.58, 128.38, 128.56, 128.64, 135.0, 166.5, 172.0.

(S)-2-Amino-4-methoxy-4-oxobutanoic acid hydrochloride 175 Following Reddy's Procedure 474

To an ice cold solution of L-aspartic acid (5.0 g, 37.50 mmol, 1 eq.) in MeOH (25 mL) was added dropwise thionyl chloride (SOCl 2 , 3.87 mL, 53.30 mmol, 1.4 eq.) over 5 min. The ice bath was then removed and the resulting solution was warmed to room temperature stirred for 25 min at room temperature, and poured into an ice cold ether (25 mL). Upon cooling in ice bath and shaking, a white solid was precipitated. The white solid was filtered immediately, and the filtered solid was washed with ice cold diethyl ether (10 mL), collected and dried to give the desired L-Asp(β-OMe)-hydrochloride 175 (5.352 g, 78%) as a white solid. , 3125, 2821, 1731, 1567, 1498, 1446, 1418, 1386, 1286, 1228, 1200, 1181, 1101, 998, 851 cm -1 To an ice cold solution of L-Asp(β-OMe)-hydrochloride 175 (5.33 g, 36.26 mmol, 1 eq.) in water (60 mL) and dioxane (30 mL) were added Na 2 CO 3 (3.48 g, 36.26 mmol, 1 eq.) at 0 °C, then followed by a solution of benzyl chloroformate (CbzCl, 6.25 g, 36.62 mmol, 1.01 eq.) in dioxane (30 mL) dropwise over 30 min. The resulting mixture was slowly warmed to room temperature and stirred for overnight (16 h). The solution was extracted with ethyl acetate (25 mL × 3). The aqueous layer was then acidified to pH = 2-3 with 4 M HCl, as white solid precipitated was extracted with ethyl acetate (25 mL × 3). The combined organic layer was washed with brine (50 mL), dried over Na 2 SO 4 and the solvent was evaporated under reduced pressure to give a pale-yellow semid-solid.

Mp. 190-191 °C

Short flash chromatography on silica gel (EtOAc = 100%) gave the desired L-(S)-Cbz-Asp(β-OMe)-H 176 (5.512 g, 68%) as a white waxy solid.

TLC: R f = 0.11 (EtOAc = 100%) 13 C NMR (90 MHz, CDCl 3 ) δ 36.3, 50.2, 52.3, 67.4, 128.2, 128.3, 128.6, 136.0, 156.3, 171.6,

1-Benzyl 6-methyl (S) 4-(((benzyloxy)carbonyl)amino)-3-oxohexanedioate 177

To a solution of L-(S)-Cbz-Asp(β-OMe)-H 176 (2.8126 g, 10.0 mmol, 1 eq.) in anhydrous THF (20 mL) at room temperature under argon were added CDI (1.7836 g, 11.0 mmol, 1.1 eq.). Stirring for 3 h at room temperature provided solution A

Preparation of magnesium salt 174: In a separated flask, to an ice cold solution of monobenzyl malonate 173 (2.3292 g, 12.0 mmol, 1.2 eq.) in anhydrous THF (24 mL) under argon was dropwise added isopropylmagnesium chloride (2.0 M in THF, 6.0 mL, 12.0 mmol, 1.2 eq.). After stirred for 30 min at 0 °C, then heating at 50 °C for 30 min, giving solution B.

To an ice cold solution B was then slowly added solution A over 30 min. The resulting mixture was stirred for overnight (16-18 h) and slowly warmed to room temperature. The reaction was quenched with 1 M HCl ( 10 , 2951, 1743, 1720, 1688, 1538, 1443, 1367, 1339, 1252, 1211, 1066, 973, 746, 13 C NMR (90 MHz, CDCl 3 ) δ 35.2, 46.0, 52.2, 56.5, 67.3, 67.5, 128.2, 128.4 (4C), 128.5, 128.6 (4C), 135.3, 136.0, 156.0, 166.8, 171.8, 200.9.

HRMS

Methyl (S)-3-(2-(2-(benzyloxy)-2-oxoethyl)-1,3-dithiolan-2-yl)-3-(((benzyloxy)carbonyl)amino)propan oate 178

To a solution of N-Cbz-β-ketoester 177 (83.0 mg, 0.20 mmol, 1 eq.) in AcOH (2 mL) were added 1,2-ethanedithiol (56.5 mg, 2.26 mL, 0.60 mmol, 3 eq.) at room temperature under argon, followed by addition of BF 3 •Et 2 O (141.9 mg, 1.00 mmol, 36 µL× 5, 5 eq.) during 5 days in 5 portions, and the resulting mixture was stirred for 7 days at room temperature. The reaction was diluted with EtOAc (20 mL) and washed with water (10 mL × 3), sat. NaHCO 3 (10 mL × 3) and brine (10 mL ×

3), respectively. The organic layer was then dried over MgSO 13 C NMR (90 MHz, CDCl 3 ) δ 37.6, 39.8, 40.7, 46.9, 52.1, 55.7, 66.9, 67.1, 71.3, 128.2 (2C), 128.3 (2C), 128.5, 128.6 (4C), 128.7, 135.7, 136.5, 156.0, 169.9, 171.5. To an ice cold solution of L-Asp(β-OMe)-hydrochloride 175 (9.60 g, 52.29 mmol, 1 eq.) in water (250 mL) and dioxane (50 mL) were added Na 2 CO 3 (11.56 g, 83.68 mmol, 1.6 eq.) at 0 °C, then followed by a solution of FmocCl (14.88 g, 57.53 mmol, 1.01 eq.) in dioxane (200 mL) dropwise over 30 min. The resulting mixture was slowly warmed to room temperature and stirred for overnight (16 h). The solution was evaporated under reduced pressure to remove most of dioxane, then extracted with ether (25 mL × 3) to give the aqueous phase, which was then acidified with 4 M HCl until pH = 2-3. The acidified solution was then extracted with ether (80 mL × 3), and the combined organic layer was washed with brine (50 mL × 3), dried over MgSO 4 and concentrated under reduced pressure to give the pure desired L-N-Fmoc Asp(β-OMe)-H (S)-179 (17.56 g, 91%) as a white solid and used without further purification. 3352, 3063, 2974, 1762, 1738, 1673, 1531, 1442, 1376, 1357, 1323, 1282, 1253, 1236, 11207, 1189, 1167, 1087, 1041, 991, 836, 737, 541 7.38-7.42 (m, 2H), 7.60-7.62 (m, 2H), 7.75-7.77 (m, 2H), 10.11 (s, 1H).

HRMS (ESI+

IR (ATR) ν 3377,
13 C NMR (90 MHz, CDCl 3 ) δ 36.3, 47.1, 50.3, 52.3, 67.5, 120.0, 125.5, 127.1, 127.8, 141.3, 143.7, 143.8, 156.3, 171.7, 175.3.

HRMS (ESI+)

Calcd for C 20 H 19 NNaO 6 [M + Na] + : 392.1105, found 392.1107.

6-Benzyl 1-methyl 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-oxohexanedioate 180

To a solution of L-N-Fmoc-Asp(β-OMe)-H (S)-179 (3.10 g, 8.39 mmol, 1 eq.) in anhydrous THF (25 mL) at room temperature under argon was added CDI (1.50 g, 9.24 mmol, 1.1 eq.). The mixture was stirred for 3 h as solution A.

Preparation of magnesium salt 174: In a separated flask, to an ice cold solution of monobenzyl malonate of monobenzyl malonate (2.45 g, 12.59 mmol 1.5 eq.) in anhydrous THF (24 mL) under argon was dropwise added a solution of 2.0 M isopropylmagnesium chloride in THF (7.8 mL, 12.59 mmol, 1.5 eq). After stirred for 30 min at 0 °C, then heating at 50 °C for 30 min as solution B.

To the ice cold solution B was slowly added solution A over 30 min. The resulting mixture was stirred for overnight (16-18 h) and slowly warmed to room temperature. T The reaction was quenched with 1 M HCl ( 10 , 3035, 2952, 1716, 1501, 1450, 1405, 1368, 1317, 1216, 1177, 1104, 1047, 992, 757, 738, 697 cm - 13 C NMR (90 MHz, CDCl 3 ) δ 35.1, 45.9, 47.3, 52.2, 56.6, 67.1, 67.3, 120.1 (2C), 125.0, 125.1, 127.2 (2C), 127.9 (2C), 128.5 (2C), 128.7 (2C), 135.3, 141.4 (2C), 143.6, 143.7, 156.0, 166.8, 171.8, 200.8.

HRMS (ESI+

2-yl)propanoate 181

To a solution of N-Fmoc-β-ketoester 180 (4.50 g, 8.97 mmol, 1 eq.) in AcOH (50 mL) was added 1,2-ethanedithiol (2.54 g, 2.26 mL, 9.24 mmol, 3 eq.) at room temperature under argon, followed by addition of BF 3 Et 2 O (2. 3 mL × 3, 53.84 mmol, 6 eq.) was added every 2 days in 3 portions. The resulting mixture was stirred for 6-7 days at room temperature. Then diluted with EtOAc (100 mL), washed with water (50 mL × 3), sat. NaHCO 3 (50 mL × 3) and brine (50 mL × 3), respectively. The organic layer was then dried over MgSO 4 , filtered, and concentrated under reduced pressure. Flash , 3032, 2950, 1727, 1505, 1450, 1437, 1333, 1283, 1220, 1166, 1050, 756, 13 C NMR (90 MHz, CDCl 3 ) δ 37.4, 39.6, 40.6, 46.8, 47.2, 52.0, 55.5, 66.7, 66.8, 71.3, 119.9 (2C), 125.0 (2C), 127.1 (2C), 127.6 (2C), 128.2, 128.4 (2C), 128.5 (2C), 135.6, 141.2 (2C), 143.7, 143.9, 155.9, 169.8, 171.4.

IR (ATR) ν 3340

HRMS

Methyl 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-oxopentanoate 185

To a solution of N-Fmoc-β-ketoester 180 (200.6 mg, 0.40 mmol, 1 eq.) in EtOAc (10 mL) with a balloon of Hydrogen (1 atm.) was added Pd/C (10% wt., 40.0 mg, 0.08 mmol, 0.2 eq.). After the resulting mixture was stirred for 3 h at room temperature, completed. The reaction solution was filtered through a celite pad, washed with EtOAc (10 mL). The filtered liquid was then concentrated under reduced pressure. Flash chromatography on silica gel (petroleum ether/EtOAc = 70/30) gave a methyl ketone 185 (106.0 g, 73%) as a colorless oil.

TLC: R f = 0.50 (petroleum ether/EtOAc = 60/40)

2-(2-(1-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methoxy-3-oxopropyl)-1,3-dithiolan-2 -yl)acetic acid 186

To an ice cold solution of N-Fmoc-dithioketal benzyl ester 181 (580.0 mg, 1.00 mmol, 1 eq.) in

HOAc (6 mL) was slowly added a solution of 33% HBr in HOAc (3.5 mL, 20.00 mmol, 20 eq.).

The ice bath was removed and the mixture was stirred for 5 h. The reaction solution was poured into EtOAc (50 mL), then washed water (20 mL× 3) and brine (20 mL) To a solution of N-Fmoc-β-dithioketal acid 186 (80.0 mg, 0.16 mmol, 1 eq.) in CH 2 Cl 2 (10 mL) under argon was added H-Sar-Ot-BuHCl (33.0 mg, .0.18 mmol, 1.1 eq.), PyBroP (84.0 mg, 0.18 mmol, 1.1 eq) and dropwise DIEA (64.0 mg, 0.49 mmol, 4 eq.), respectively. The resulting mixture was stirred at room temperature for overnight (16 h), then diluted with CH 2 Cl 2 (10 mL) and subsequently washed with 1 M KHSO 4 (10 mL), sat. NaHCO 3 (10 mL) and brine (10 mL) to give the organic layer, which was then dried over MgSO 4 , filtered and concentrated under reduced , 2978, 2927, 1732, 1649, 1503, 1450, 1403, 1367, 1327, 1227, 1155, 757

tert-Butyl N-(2-(2-(1,3-diamino-3-oxopropyl)-1,3-dithiolan-2-yl)acetyl)-N-methylglycinate 188

To an ice cold solution of N-Fmoc-dipeptide 187 (77.0 mg, 125 mol, 1 eq.) in ethanol ( 2 13 C NMR (90 MHz, CDCl 3 ) δ 28.2 (s, 9H), 37.0, 37.3 and (35.4, rotamer), 39.6, 40.1, 45.3, 50.9 and 52.9 (rotamer), 55.6 (rotamer) and 55.8, 72.2, 82.5 and 83.3 (rotamer)

tert-Butyl N-(2-(2-(1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-amino-3-oxopropyl)-1,3-dithiolan-

2-yl)acetyl)-N-methylglycinate 189

To an ice cold solution of dipeptide of free amine 187 (18.0 mg, 48 mol, 1 eq.) in dioxane (1 mL) and water (2 mL) was added K 2 CO 3 . (10.5 mg, 76 mol, 1.6 eq.), and followed by addition of a solution of FmocCl (13.5 mg, 52 mol, 1.1 eq.) in dioxane (1 mL). The resulting mixture was stirred at room temperature for overnight (16 h). The reaction was diluted with CH 2 Cl 2 (20 mL) and washed with water (10 mL) and brine (10 mL). The separated organic phase was then dried over , 3332, 3214, 2978, 2927, 1714, 1649, 1510, 1450, 1402, 1368, 1231, 1155 13 C NMR (90 MHz, CDCl 3 ) δ 28.2 (3C), 35.4 (rotamer) and 37.2, 39.4 and 39.5 (rotamer), 39.9, 40.3 (2C), 45.4, 47.3, 50.7 and 52.9 (rotamer), 56.4, 67.0, 72.3 and 72.4 (rotamer) 7, 39.4, 39.5, 49.2, 55.7, 69.3, 174.8, 177.2. To an ice cold solution of β-amino primary amide 190 (150.1 mg, 0.60 mmol, 1 eq.) in dioxane ( 5mL) and water (10 mL) was added Na 2 CO 3 (101.8 mg, 0.96 mmol, 1.6 eq.), then followed by addition of a solution of FmocCl (232.8 mg, 0.90 mmol, 1.5 eq.) in dioxane (5 mL) over 10 min.

HRMS

After the resulting mixture was stirred for overnight (18-24 h) at room temperature, sat. NaHCO 3 (10 mL) was added and extracted with EtOAc (20 mL× 2) to give the aqueous phase. The aqueous phase was then acidified with 4 M HCl until = 2-3, and extracted with EtOAc (20 mL× 3). The combined organic phase was dried over MgSO 4 , filtered and concentrated under reduced pressure to give a pure N-Fmoc-mPyrAA 191 (197.5 8, 38.54, 40.0, 46.7, 46.8, 55.2, 65.5, 72.4, 120.1 (2C), 125.3 (2C) 127.1 (2C), 127.6 (2C), 140.6 (2C), 143.7, 143.8, 155.9, 171.3, 172.0.

HRMS (ESI

tert-Butyl N-(2-(2-(1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-amino-3-oxopropyl)-1,3-dithiolan-

2-yl)acetyl)-N-methylglycinate 189

To a solution of N-Fmoc-mPyrAA 191 (42.0 mg, 0.89 mmol, 1 eq.) in CH 2 Cl 2 (20 mL) under argon was added H-Sar-Ot-BuHCl (17.8 mg, 0.98 mmol, 1.1 eq.), PyBroP (45.6 mg, 0.98 mmol, 1.1 eq) and dropwise DIEA (46.0 mg, 0.356 mmol, 4 eq.), respectively. The resulting mixture was stirred at room temperature for overnight (16 h). Diluted with CH 2 Cl 2 (20 mL) and the solution was subsequently washed with 1 M HCl (20 mL), sat. NaHCO 3 (20 mL) and brine (20 mL), then dried over MgSO 4 , filtered and concentrated under reduced pressure. Flash chromatography on silica gel (EtOAc =100, EtOAc/MeOH = 90/10) gave the pure desired dipeptide 189 (48.0 mg, 90%) as a white solid.

tert-Butyl

N-(4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-cyano-3-oxopentanoyl)-N-methylglycina te 195

Table 6, entry 7:

To a solution of dipeptide 189 (90.0 mg, 0.15 mmol, 1 eq.) in acetonitrile/water (5/0.5 mL) was added HgO (260.0 mg, 1.20 mmol, 8 eq) and HgCl 2 (488.7 mg, 1.80 mmol, 12 eq.). The mixture was stirred for 24 at room temperature, then filtered through a Celite pad and washed with EtOAc , 2979, 2938, 2247, 1729, 1640, 1502, 1451, 1407, 1369, 1247, 1157, 1045, Mixed with enol-form not presented.

tert-Butyl

N-(4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-amino-3,6-dioxohexanoyl)-N-methylglyc inate 192

Table 6, entry 9:

To an ice cold solution of dipeptide 189 (120.0 mg, 0.20 mmol, 1 eq.) in acetone (10 mL) was added N-Bromosuccinimide (180.0 mg, 1.00 mmol, 5 eq.), and the mixture was stirred for 40 min at 0 ˚C (from orange red to pale-yellow). Quenched with water (1 mL), then diluted with EtOAc ( 50mL), washed with water (30 mL × 3) and brine (30 mL× 2). The organic layer was dried with Mg 2 SO 4 , filtered, and concentrated under reduced pressure. Flash chromatography on a very short silica gel (EtOAc/petroleum ether = 50/50, EtOAc/MeOH = 95/5, 90/10) gave the γ-keto primary amide 192 (46.4 mg, 44%) as a colorless oil, as well as the dehydrated 194 (5.0 mg, 5%) as a colorless oil.

TLC: R f = 0.66 (EtOAc/MeOH = 90/10) , 2978, 2930, 2248, 1713, 1671, 1635, 1479, 1450, 1402, 1368, 1232, 1154, 1046, 910, 760, 737 cm -1 13 C NMR (100 MHz, CDCl 3 ) δ 28.1 (rotamer) and 28.2, 35.1 (rotamer) and 37.3, 36.4 and 36.5 (rotamer), 45.7, 47.3, 50.4 and 53.0 (rotamer), 57.2, 67.2 and 67.3 (rotamer), 120.0 (rotamer) and 120.1 (2C), 125.2 and 125.4 (rotamer) (2C), 127.8 (rotamer) and 127.9 (2C), 141.4 (2C), 143.8 and 143.9 (rotamer) (2C), 156.4, 167.8 and 168.0 (rotamer), 168.1, 172.6, 202.4 (rotamer)

IR (ATR) ν 3330

tert-Butyl

N-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-hydroxy-5-oxopyrrolidin-2-ylidene)ac etyl)-N-methylglycinate 193

Table 6, entry 10:

Step1: To an ice cold solution of dipeptide 189 (120.0 mg, 0.20 mmol, 1 eq.) in acetone (10 mL) was added N-Bromosuccinimide (180.0 mg, 1.00 mmol, 5 eq.), and the mixture was stirred for 40 min at 0 ˚C (from orange red to pale-yellow). Quenched with water (1 mL), then diluted with EtOAc (50 mL), washed with water (30 mL × 3) , 2926, 1706, 1658, 1633, 1533, 1450, 1404, 1368, 1232, 1155, 1041, 739 13 C NMR (100 MHz, DMSO-d 6 ) δ 27.6 (rotamer) and 27.7, 34.4 (rotamer) and 36.4, 34.7, 39.6, 46.6 (rotamer) and 46.7, 49.6 and 51.9 (rotamer), 53.4 (rotamer) and 53.4, 65.6 and 65.7 (rotamer), 80.8 and 80.5 (rotamer), 85.9 and 88.8 (rotamer), 120.1 (2C), 120. Step1: To an ice cold solution of dipeptide 189 (60.0 mg, 0.10 mol, 1 eq.) in acetone (5 mL) was added N-Bromosuccinimide 90.0 mg, 0.20 mmol, 5 eq.), and the mixture was stirred for 40 min at 0 ˚C. Quenched with water (1 mL), then diluted with EtOAc (50 mL), and washed with water ( 30mL× 3) and brine (30 mL× 2). The organic layer was dried with Mg 2 SO 4 , filtered, and concentrated under reduced pressure to give a pale-yellow oil (55 mg).

Step2: The crude was dissolved in acetone (5 mL) and added 4 M HCl (5 mL). The resulting mixture was stirred for 24 h at room temperature, then diluted with EtOAc ( 50 , 2932, 1720, 1656, 1595, 1523, 1479, 1368, 1331, 1284, 1247, 1156, 1128, 1046, 758, 741 cm -1 13 C NMR (100 MHz, CDCl 3 ) δ 28.2, 36.4, 36.9 (35.2, rotamer), 47.3, 49.4, 50.3 (52.7, rotamer), 67.2, 82.1, 89.2, 120.2 (2C), 125.0, 125.1, 127.2 (2C), 128.0 (2C), 141.5 (2C), 143.7 (2C), 156.1, 168.1, 168.6, 174.1. To an ice cold solution of L-H-Asp(β-OMe)-H•HCl 175 (917.9 mg, 5.00 mmol, 1 eq.) in water ( 15mL) was added Na 2 CO 3 (529.9 mg, 5.00 mmol, 1 eq.). N-Carbethoxyphthalimide (1.096 g, 5.00 mmol, 1 eq.) was then added in 3 portions at 0 °C, and the ice bath was removed. After stirred for 3 h at room temperature, the reaction was diluted with sat. NaHCO 3 (20 mL), extracted with EtOAc (20 mL × 2) to give the aqueous layer. The aqueous was then acidified with 4 M HCl until pH = 2-3, extracted with EtOAc (30 mL × 3). The combined organic phase was washed with brine (30 mL), , 2937, 1783, 1755, 1744, 1690, 1405, 1256, 1213, 1169, 1013, 885, 723 cm -1

dried
IR (ATR) ν 3176
1 H NMR (360 MHz, CDCl 3 ) δ 3.14 (dd, 1H, J = 16.9, 9.0 Hz,ABX), 3.35 (dd, 1H, J = 16.9, 5.8

Hz,ABX), 3.65 (s, 3H),5.43 (dd, 1H, J =9.0, 5.8 Hz, ABX), 7.66-7.89 (m, 4H), 10.88 (1H).

13 C NMR (90 MHz, CDCl 3 ) δ 33.6, 48.1, 52.4, 123.9 (2C), 131.7 (2C), 134.5 (2C), 167.3, 170.5, 173.8. To an ice cold solution of phthalimido derivative 199 (277.5 mg, 1.00 mmol, 1 eq.) in anhydrous THF (5 mL) under argon were added CDI (178.6 mg, 1.10 mmol, 1.1 eq.). The ice bath was then removed and stirred for 3 h at room temperature as solution A

HRMS (ESI+

Preparation of magnesium salt 174 as solution B: In a separated flask, to an ice cold solution of monobenzyl malonate (233.0mg, 1.20 mmol, 1.2 eq.) in anhydrous THF (5 mL) under argon was dropwise added a solution of 2 M isopropylmagnesium chloride in THF (1.2 mL, 2.40 mmol, 2.4 eq.). After stirred for 30 min at 0 °C, then heating at 50 °C for 30 min.

To an ice cold solution B was then slowly added solution A over 30 min. The resulting mixture was stirred for overnight (16-18 h) and slowly warmed to room temperature. The reaction was quenched with 1 M HCl (10 mL) at 0 °C and extracted with Et 2 O (25 mL × 3). The combined organic layer was washed with 1 M HCl (25 mL), sat. NaHCO 3 (25 mL) and brine (25 mL), then dried over 13 C NMR (90 MHz, CDCl 3 ) δ 32.6, 45.9, 52.2, 54.7, 67.5, 124.0 (3C), 128.5 (3C), 128.6 (2C), 131.6, 134.7 (2C), 135.1, 166.0, 167.3, 170.6, 196.4.

Methyl (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-oxo-4-(propylthio)butanoate 201

To an ice cold solution of L-N-Fmoc-Asp(β-OMe)-H (S)-179 (1.847 g, 5.00 mmol, 1 eq.) in THF/DMF (9:3 mL) under argon was added HATU (2.844 g, 7.50 mmol, 1.5 eq.), propanethiol (4.523 g, 20.00 mol, 4 eq.), respectively. DIEA (5.170 g, 20.00 mmol, 4 eq.) was added dropwise.

The resulting mixture was stirred for overnight (17 h). Without work-up, the reaction solution was , 2958, 1725, 1683, 1504, 1446, 1438, 1226, 1049, 759, 741 13 C NMR (90 MHz, CDCl 3 ) δ 13.4, 22.7, 31.1, 36.1, 47.2, 52.2, 57.2, 67.4, 120.1 (2C), 125.1, 125.2, 127.1 (2C), 127.8 (2C), 141.3, 143.7, 143.8, 155.9, 171.5, 200.2. , 2952, 1728, 1518, 1450, 1250, 1056, 760, 741 (20 mL). The filtered liquid was washen with sat. NaHCO 3 (10 mL), and brine (10 mL). The organic layer was then dried over MgSO 4 , filtered and concentrated under reduced pressure. Flash 1 H NMR (360 MHz, CDCl 3 ) δ 2. 47-2.83 (m, 4H), 3.67 and 3.69 (s, 3H, two diastereoisomers), 3.50 and 3.61 (brs, 1H, two diastereoisomers), 3.94-4.09 (m, 1H, two diastereoisomers) 4.17-4.21 (m, 1H), 4.39-4.43 (m, 2H), 5.11-5.15 (m, 2H), 5.37 and 5.61 (d, 1H, two diastereoisomers), 7.28-7.41 (m, 9H), 7.55-7.59 (m, 2H), 7.69-7.76 (m, 2H) 6 IR (ATR) ν 3360, 2953, 1722, 1514, 1451, 1316, 1248, 756, 742 cm -1

IR (ATR) ν 3332

HRMS (ESI+

1 H NMR (360 MHz, CDCl 3 ) δ 2. 80 (dd, 1H, J = 17.3, 4.7 Hz, ABX), 2.99 (dd, 1H, J = 17.3, 5.0 Hz, ABX), 3.62 (s, 2H), 3.66 (s, 3H), 4.21 (t, 1H, J = 6.5 Hz), 4.46 (dd, 1H, J = 10.8, 6.5 Hz), 4.55 (dd, 1H, J = 10.8, 6.5 Hz), 4.64 (ddd, 1H, J = 9.0, 5.0, 4.7 Hz, ABX), 5.11-5.21 (m, 2H), 6.00 (d, 1H, J = 9.0 Hz), 7.29-7.43 (m, 9H), 7.59 (d, 2H, J = 7.2 Hz), 7.76 (dd, 2H, J = 7.2, 2.9 Hz).

13 C NMR (90 MHz, CDCl 3 ) δ 35.0, 45.8, 47.3, 52.2, 56.5, 67.1, 67.3, 120.1 (2C), 125.0 (2C), 127.1 (2C), 127.8 (2C), 128.5 (2C), 128.6 (2C), 135.3, 141.4 (2C), 143.6 (2C), 156.0, 166.7, 171.7, 200.7. 13 C NMR (100 MHz, 323 K, CDCl 3 ) δ 37.6, 39.9, 40.6, 46.8, 47.4, 52.1, 55.7, 67.2, 71.3, 120.0 (2C), 125.3 (2C), 127.2 (2C), 127.7 (2C), 141.4 (2C), 143.9, 144.1, 156.3, 171.6, 173.5. , 3160, 2922, 1667, 1563, 1382, 1278, 1146, 1006, 679cm 3.05 (d, 1H, J = 16.9 Hz), , 3.16 (d, 1H, J = 16.9 Hz), 3.27-3.43 (m, 4H), 4.07 (dd, 1H, J = 10.4, 2.9

HRMS

HRMS (ESI+

Hz, rotamer, ABX) and 4.12 (dd, 1H, J = 10.4, 2.9 Hz, ABX).

13 C NMR (90 MHz, D 2 O) δ 35.7 and 36.6 (rotamer), 39.4, 39.5, 49.2, 55.7 and 56.4 (rotamer), 68.1 (rotamer) and 69.5, 174.9, 177.2. To an ice cold solution of L-β-amino primary amide (S)-190 (425.9 mg, 1.70 mmol, 1 eq.) in water (30 mL) was added k 2 CO 3 (375.9 mg, 2.72 mmol, 1.6 eq.), then followed by addition of a solution of FmocCl (483.8 mg, 1.87 mmol, 1.1 eq.) over 10 min. The resulting mixture was stirred for overnight (18-24 h) at room temperature, then diluted with sat. NaHCO 3 (10 mL), extracted with , 2926, 1709, 1661, 1514, 1448, 1411, 1334, 1245, 756, 741 1H, J = 16.9 Hz), 3.22-3.30 (m, 4H), 4.26 (t, 1H,), 5.90 (d, 1H, J = 7.2 Hz), 4.29 (dd, 1H, J = 11.6, 7.2 Hz), 4.41 (dd, 1H, J = 11.6, 7.2 Hz), 4.84-4.91 (m, 1H, combined with water), 7.32-7.34 (t, 2H, J =8.4 Hz), 7.39 (t, 2H, J = 8.4 Hz), 7.64-7.67 (m, 2H), 7.79 (d, 2H, J = 8.4 Hz).

HRMS (ESI+

13 C NMR (90 MHz, MeOD-d 4 ) δ 39.3, 40.4, 41.4, 47.7, 48.4, 57.1, 67.8, 72.9, 120.9 (2C), 126.2 (2C), 128.2 (2C), 128.7 (2C), 142.5 (2C), 145.1, 145.3, 158.5, 173.5, 176.0. To a solution of L-N-Fmoc-mPyrAA (S)-191 (472.5 mg, 1.00 mmol, 1 eq.) in CH 2 Cl 2 (20 mL) under argon was added H-Sar-Ot-BuHCl (199.8 mg, 1.10 mmol, 1.1 eq,), PyBroP (512.8 mg, 1.10 mmol,

HRMS (ESI

1.1 eq.) and dropwise DIEA (516.9 mg, 4.00 mmol, 4 eq.), respectively. The resulting mixture was stirred at room temperature for overnight (16 h). Diluted with CH 2 Cl 2 (20 mL) and the solution was subsequently washed with 1 M HCl (2 × 20 mL), sat. NaHCO 3 (2 × 20 mL) and brine ( 20 , 3326, 3214, 2977, 2928, 1721, 1661, 1512, 1450, 1403, 1370, 1243, 1157, To a solution of D-aspartic acid (6.655 g, 50.00 mmol, 1 eq.) in MeOH (50.0 mL, 1.23 mol, ~24.6 eq.) at -10 °C (ice-salt bath) under argon was dropwise added thionyl chloride (SOCl 2 , 5.10 mL, 70.0 mmol, 1.4 eq.). The cooling bath was then removed, and the resulting mixture was slowly warmed to room temperature and stirred for 25-30 min, and then diluted with ice cold diethyl ether (150 mL), upon cooling and shaking, precipitated as a white solid, which was filtered immediately and washed with ice-cold diethyl ether (20 mL). The solid was collected and dried to give the desired pure D-Asp(β-OMe)-hydrochloride (R)-175 (7.89 g, 86%) a white power and used without further purification. To an ice cold solution of D-Asp(β-OMe)-hydrochloride (R)-175 (1.836 g, 10.0 mmol, 1 eq.) in

water (20 mL) and dioxane (10 mL) was added Na 2 CO 3 (2.211 g, 16.0 mmol, 1.6 eq.) at 0 °C, then followed by a solution of FmocCl (2.846 g, 11.0 mmol, 1.1 eq.) in dioxane ( 10 , 2952, 1716, 1517, 1439, 1209, 1178, 1052, 758, 738 13 C NMR (90 MHz, CDCl 3 ) δ 36.3, 47.1, 50.3, 52.3, 67.5, 120.1 (2C), 125.2 (2C), 127.2 (2C), 127.8 (2C), 141.4 (2C), 143.7, 143.8, 156.3, 171.7, 175.3. 37.0, 39.2 and 39.3 (rotamer), 39.5, 40.1 (2C), 45.2, 47.1, 50.5 and 52.7 (rotamer), 56.3, 66.8, 72.3 and 72.4 (rotamer)

HRMS

1-(6-Chloro-1H-indol-3-yl)-N,N-dimethylmethanamine 211

To an ice cold solution of 37% HCHO aq. (1.13 mL, 15.00 mmol, 1.5 eq.) in AcOH (10 mL) was added 40% Me 2 NH aq . (2.14 mL, 17.00 mmol, 1.7 eq.). After stirred for 10 min at 0 °C, a solution of 6-Cl-indole (1.516 g, 10.00 mmol, 1 eq.) in AcOH (5.0 mL) was added. The resulting mixture was stirred for 4 h at 0 °C until completed. The resulting mixture was poured into ice water (50 mL).

Lots of pale-yellow solid was precipitated, filtered and washed with water (3 × 20 mL) , 2860, 2817, 2775, 1542, 1452, 1341, 1327, 1229, 1167, 995, 9020, 853, 821, 13 C NMR (90 MHz, CDCl 3 ) δ 45.3, 54.3, 111.2, 112.8, 120.0, 120.2, 124.8, 126.5, 127.8, 136.7. To an ice cold solution of 6-Cl-gramine 211 (834.7 mg, 4.00 mmol, 1 eq.) in THF (12 mL) was added MeI (851.6 mg, 6.00 mmol, 1.5 eq). The mixture was stirred for 5 min at 0 °C then warmed to room temperature for 1 h, completed. Diluted with 3 times volume of petane (36 mL), precipitated and filtered. The filtered solid was washed with petane (10 mL) to give the quanternary salt 212 (2.10 g) in quantative yield as a pale-yellow solid after dried. This compound was not stable and decomposed on storage. It was usually prepared and used immediately in the next step.

HRMS

Mp = 154 °C, decomposition.

IR (ATR) ν 3215, 1616, 1535, 1470, 1452, 1408, 1292, 1107, 1060, 906, 807 8, 120.2 and 120.3 (rotamer), 120.6, 126.6, 126.7 and 126.8 (rotamer), 131.4 and 131.6 (rotamer), 136.5 and 136.6 (rotamer). Step (1) Basification of H-Gly-Ot-Bu hydrochloride salt: To an ice cold solution of H-Gly-Ot-Bu hydrochloride salt (1.2070 g, 7.20 mmol, 1.2 eq.) in toluene (10 mL) was added Et 3 N (728.6 mg, 7.20 mmol, 1.2 eq.). After stirred for 1 h at 0 C, then filtered and washed with toluene (5.0 mL).

HRMS

Step ( 2): To a solution of (1S,2S,5S)-(+)-2-hydroxy-3-pinanone (1.0094 g, 6.00 mmol, 1 eq) in toluene (20.0 mL) was added BF 13 C NMR (90 MHz, CDCl 3 ) δ 23.0, 27.4, 28.19 (2C), 28.3, 38.3, 38.6, 50.4, 53.5, 76.6, 81.3, 169.

1]heptan-3-ylidene)amino)propanoate 213

Step (1) preparation of fresh LDA solution: To a solution of DIPA (178.1 mg, 1.76 mmol, 2.2 eq.) in THF (5 mL) at -78 °C was added dropwise a solution of 1.6 M n-BuLi (1.1 mL, 1.76 mmol, 2.2 eq.). The mixture was stirred at -78 °C for 30 min.

Step ( 2) preparation of Li-enolate: To a solution of Gly-pinanone imine 20 (225.1 mg, 0.80 mmol, 1 eq.) in THF (2 mL) was slowly added to the well-prepared LDA solution of step ( 2) at -78 °C. The mixture was stirred at -78 °C for 30 min.

Step ( 3): To a solution of fresh gramine quaternary ammonium salt 212 (420.7, 1.50 mmol, 1.5 eq.)

in THF ( 10 , 2978, 2920, 1726, 1649, 1456, 1369, 1230, 1154 9, 27.2, 27.9, 28.1 (4C), 28.6, 28.5, 28.6, 33.0, 38.1, 38.2, 50.0, 63.2, 76.6, 81.6, 11.1, 112.3, 119.7, 120.1, 124.0, 126.1, 127.8, 13.6, 170.8. , 3161, 2977, 2932,1722, 1620, 1456, 1368, 1249, 1152, 1062, 906, 845, 803 13 C NMR (90 MHz, CDCl 3 ) δ 28.1 (3C), 30.7, 55.5, 81.4, 111.2, 111.4, 119.7, 120.4, 123.8, 126.2, 127.8 136.7, 174.6. 7.28-7.32 (m, 3H), 7.39-7.43 (m, 2H), 7.51-7.57 (m, 3H), 7.76-7.78 (m, 2H), 8.36 (brs, 1H).

HRMS (ESI+

HRMS

13 C NMR (90 MHz, CDCl 3 ) δ 28.0 (4C), 47.2, 55.1, 67.0, 82.4, 110.5, 111.2, 119.8, 120 7.27-7.31 (m, 2H), 7.33 (d, 1H, J = 1.4 Hz ), 7.38-7.42 (m, 2H), 7.51-7.55 (m, 3H),, 7.75-7.77 (m, 2H), 8.14 (brs, 1H).

13 C NMR (90 MHz, CDCl 3 ) δ 28.1 (3C), 28.4, 42.2, 47.2, 55.6, 67.2, 82.6, 110.6, 111.3, 119.7, 120.1 (2C), 120.6, 124.1, 125.2 (2C) 13 C NMR (90 MHz, CDCl 3 ) δ 28.2 (3C), 30.7, 41.8, 55.5, 82.3, 111.3, 112.0, 120.0, 120.5, 123.9, 126.2, 128.3, 136.9, 169.2, 175.0. 7.65-7.67 (m, 2H).

HRMS

tert-Butyl 3-(6-chloro-1H-indol-3-yl)-2-((diphenylmethylene)amino)propanoate 219

Step (1) preparation of fresh LDA solution: To a solution of DIPA (202.4 mg, 2.00 mmol, 1 eq.) in THF (5.0 mL) at -78 °C was added dropwise a solution of 1.6 M n-BuLi in hexane (1. 1 mL, 1.76 mmol, 1 eq.). The mixture was stirred at -78 °C for 30 min.

Step (2): To a solution of imine 218 (591.2 g, 2.00 mmol, 1.0 eq.) in THF (2 mL) was slowly added to the LDA solution of step (1) at -78 °C. The mixture was stirred at -78 °C for 30 min.

Step (3): To a solution of fresh gramine quaternary ammonium salt 211 (420.7, 1.50 mmol, 1.5 eq.)

in THF (10 mL) at -78 °C was slowly added the solution of step (2). The resulting mixture was stirred for overnight (12 h) 13 C NMR (90 MHz, CDCl 3 ) δ 28.1 (3C), 29.2, 66.6, 81.3, 111.0, 112.2, 119.7, 119.8, 124.0, 126.4, 127.5, 127.6 (2C), 128.1 (2C), 128.2 (2C), 128.3, 128.8 (2C), 130.3, 136.1, 136.4, 139.6, 170.6, 171.4.

HRMS

tert-Butyl 2-amino-3-(6-chloro-1H-indol-3-yl)propanoate (±)-214

To a solution of racemic imine (±)-219 (651.7 mg, 1.42 mmol, 1 eq.) in THF (11 mL) was added a solution of 15% citric acid (1.637 g, 8.52 mmol, 6 eq.) in water (11.0 mL). The mixture was stirred for 6 at room temperature. Then evaporated under reduced pressure and diluted with water (10 mL).

The aqueous phase was extracted with EtOAc (2 × 20 mL), subsequently then basified with sat. , 3330, 2979, 2933,1722, 1617, 1457, 1367, 1225, 1151, 1063, 850, 797 13 C NMR (90 MHz, CDCl 3 ) δ 28.0 (3C), 30.7, 55.5, 81.4, 111.1, 111.2, 119.6, 119.8, 123.8, 126.1, 127.7 136.7, 174.6.

tert-Butyl

2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(6-chloro-1H-indol-3-yl)propanoate

To an ice cold solution of H-Trp(6-Cl)-Ot-Bu (±)-214 (294.5 mg, 1.00 mmol, 1 eq.) in THF (10 7.28-7.32 (m, 3H), 7.39-7.43 (m, 2H), 7.51-7.57 (m, 3H), 7.76-7.78 (m, 2H), 8.27 (brs, 1H). 120.4, 123.5, 125.2 (2C), 126.5, 127.2 (2C), 127.8 (2C), 128.1, 136.5, 141.4 (2C), 143.9 (2C), 155.9, 171.1. Ethyl (R)-4-chloro-3-hydroxybutanoate (900 mg, 5.41 mmol, 1 eq.) was added to a solution of sodium azide (894 mg, 13.8 mmol, 2.55 eq.) in water (10 mL). The mixture was stirred for 30 min at 120 °C under microwave irradiation. The resulting aqueous mixture was then extracted with EtOAc (3 × 30 mL). The combined organic phases were washed with brine (20 mL), dried over Na 2 SO 4 , filtered and evaporated under reduced pressure to give the azido ester (+)-N 3 -GABOB-OEt 220 (770 mg, 82%) as a yellow oil. 13 C NMR (90 MHz, CDCl 3 ) δ 14. 0, 38.6, 55.6, 61.0, 67.3, 171.9.

tert-Butyl

Ethyl (R)-4-((tert-butyloxycarbonyl)amino-3-hydroxybutanoate 221

To a solution of (+)-N 3 -GABOB-OEt 220 (710 mg, 4.10 mmol, 1 eq.) in EtOAc ( 50 13 C NMR (90 MHz, CDCl 3 ) δ 13.8, 28.1, 38.8, 45.4, 60.3, 67.3, 79.0, 156.4, 171.9.

Ethyl (R)-4-((tert-butyloxycarbonyl)amino-3-hydroxybutanoate 222

To a solution of (+)-Boc-GABOB-OEt 221 (571 mg, 2.31 mmol, 1 eq.) in DMF (5 mL) was added imidazole (786 mg, 11.6 mmol, 5 eq.) and TBSCl (696 mg, 4.62 mmol, 2 eq.). The resulting mixture was stirred for 16 h at room temperature and then washed successively with 1 M KHSO 4 (2 × 15 mL), saturated NaHCO 3 (2 × 15 mL), water (15 mL) and brine ( 15 4.17-4.27 (m, 1H), 4.80 (bs, 1H).

13 C NMR (90 MHz, CDCl 3 ) δ -4.9, -4.6, 14.3, 18.0, 25.8, 28.5, 40.4, 46.1, 60.6, 68.5, 79.3, 156.0, 171.3. To a solution of (+)-Boc-GABOB(TBS)-OEt 222 (740 mg, 2.05 mmol, 1 eq.) in ethanol (15 mL) was added a solution of KOH (345 mg, 6.14 mmol, 3 eq.) in water (7.5 mL). The resulting mixture was stirred for 16 h at room temperature and ethanol was removed under reduce pressure. The 3.18-3.30 and 2.99-3.13 (m, 2H, rot.), 4.14-4.27 (m, 1H), 4.85 and 5.93 (bs, 1H, rot.), 9.18 (bs, 1H).

HRMS

13 C NMR (90 MHz, CDCl 3 ) δ -4.9, -4.7, 18.0, 25.8, 28.4, 40.2, 46.0 and 47.3 (rot.), 68.3, 79.6 and 81.0 (rot.) To a stirred solution of 2-deoxy-ribose (200 mg, 1.49 mmol) in EtOAc (7 mL) at -10 ˚C was added PPTS (37 mg, 0.15 mmol, 0.1 eq.) and 2-methoxypropene (0.19 mL, 1.94 mmol, 1.3 eq.). The resulting mixture was then stirred for overnight (16 h) and warmed up to room temperature. The reaction solution was quenched with triethylamine (0.1 mL), concentrated under reduced pressure. , 2984, 2936, 1456, 1371, 1243, 1212, 1106, 1056, 1031, 1002 cm -1 Major (α-anomer):

1 H NMR (360 MHz, CDCl 3 ) δ 1.34 (s, 3H), 1.49 (s, 3H), 1.77 (ddd, 1H, J = 14.8, 7.2, 4.3 Hz), 2.22 (dt, 1H, J = 14.8, 4.3 Hz), 3.68 (dd , 1H, J = 12.6, 3.6 Hz), 3.94 (dd, 1H, J = 12.6, 3.2 Hz), 4.16 (dt, 1H, J = 6.5, 3.2 Hz), 4.47(dt, 1H, J = 6.5, 4.3 Hz), 5.22 (dd, 1H, J = 7.2, 4.3 Hz).

13 C NMR ( 75 MHz, CDCl 3 ) δ 25.3, 27.2, 32.1, 61.9, 70.5, 71.5, 90.7, 108.7.

Minor (β-anomer):

1 H NMR (360 MHz, CDCl 3 ) δ 1.36 (s, 3H), 1.55 (s, 3H), 1.95-2.02 (m, 1H), 2.09-2.15 (m, 1H), 3..72 (dd, 1H, J = 13.0, 4.7 Hz), 3.99 (dd, 1H, J = 12.6, 5.4 Hz), 4.14-4.18 (m, 1H), 4.37 (dd, 1H, J = 5.8 Hz), 5.00 (t, 1H, J = 4.3 Hz).

13 C NMR ( 90 MHz, CDCl 3 ) δ 25.7, 27.9, 33.4, 60.9, 70.6, 71.2, 91.6, 109.3. For the synthesis of compunds 64-67, following in-house procedures previously reported by IR (ATR) ν 2988, 2937, 1732, 1495, 1450, 1382, 1245, 1218, 1160, 1069, 968, 693 cm -1

1 H NMR (300 MHz, CDCl 3 ) δ 1.45 (s, 3H), 1.65 (s, 3H), 2.42-2.60 (m, 2H), 4.38 (dd, 1H, J = 8.3, 3.6 Hz), 4.47-4.53 (m, 1H), 6.24 (ddd pseudo dt, 1H, J = 16.2, 8.3, 8.3 Hz), 7.22-7.41 (m, 5H), 9.74 (d, J = 3.6 Hz).

13

C NMR (75 MHz, CDCl 3 ) δ 25.3, 27.5, 35.5,78.4, 81.9, 110.7, 124.8, 126.2 (2C), 127.5 (2C), 128.6 (2C), 133.3, 137.0, 202.1. (4R,5S)-5-Cinnamyl-2,2-dimethyl-1,3-dioxolane-4-carbaldehyde 65 388 To a solution of aldehyde 64 (106.0 mg, 0.43 mmol, 1 eq.) in MeOH (2 mL) at rt under argon at room temperature was added K 2 CO 3 (53.5 mg, 0.39 mmol, 0.9 eq.) and the resulting mixture was stirred for 4 h at room temperature. The reaction mixture was then poured into water ( 10 , 2932, 1733, 1495, 1450, 1381, 1240, 1216, 1086,1066, 968, 747, 694 cm -1

1 H NMR (360 MHz, CDCl 3 ): δ 1.44 (s, 3H), 1.52 (s, 3H), 2.56-2.72 (m, 2H), 4.08 (dd, 1H, J = 7.6, 2.2 Hz), 4.18-4,24 (m, 1H), 6.23 (ddd pseudo dt, J = 16.2, 7.2, 7.2 Hz), 6.52 (d, 1H, J = 16.2 Hz), 7.20-7.38 (m, 5H), 9.76 (d, 1H, J = 2.2 Hz).

13 C NMR (90 MHz, CDCl 3 ): δ 26.3, 27.2, 36.6, 76.6, 84.1, 111.3, 124.2, 126.3 (2C), 127.5, 128.7 (2C), 133.7, 137.1, 201.3. To a solution of the aldehyde 65 (1.478 g, 6.00 mmol, 1 eq.) in CH 2 Cl 2 (60 mL) under argon at room temperature was added (S)-t-butyl sulfinamide (800.0 mg, 6.60 mmol, 1.1 eq.) and Ti(OEt) 4 (6.3 mL, 30.00 mmol, 5 eq.). The resulting mixture was stirred for overnight (16 h) at rt. The mixture was cooled down to 0 °C and ice-cold water (20 mL) was added. The resulting solution was then filtered through a celite pad, washing the pad with EtOAc (50 mL × 3). The filtrate was washed with brine (100 mL), dried over MgSO 4 , filtered, concentrated under reduced pressure. , 1624, 1454, 1381, 1365, 1240, 1215, 1160, 1086, 967, 749, 694 cm -1

1 H NMR (360 MHz, CDCl 3 ): δ 1.16 (s, 9H), 1.42 (s, 3H), 1.49 (s, 3H), 2.53-2.69 (m, 2H), 4.12-4.17 (m, 1H), 4.50 (dd, 1H, J = 7.6, 4.7 Hz), 6.23 (ddd pseudo dt, J = 15.8, 7.2, 7.2 Hz), 6.47 (d, 1H, J = 15.8 Hz), 7.18-7.35 (m, 5H), 8.09 (d, 1H, J = 4.3 Hz).

13

C NMR (90 MHz, CDCl 3 ): δ 22.4 (3C), 26.5, 27.2, 36.1, 57.0, 78.6, 81.2, 110.8, 124.5, 126.3 (2C), 127.4, 128.5 (2C), 133.3, 137.0, 167.7. To a solution of Boc-glycate (524.6 mg, 6.00 mmol, 4 eq.) in THF (20 mL) at -78 °C under argon was added a solution of 1 M LiHMDS in THF (6.0 mL, 6.00 mmol, 4 eq.) and the resulting mixture was stirred for 1 h. A solution of sulfinimine 66 (524.6 mg, 1.50 mmol, 1 eq.) in THF (5. , 2983, 1750, 1457, 1370, 1302, 1254, 1214, 1163, 1136, 15.8, 7.2 , 7.2 Hz), 6.50 (d, 1H, J = 15.8 Hz), 7.16-7.21 (1H, m), 7.25-7.30 (m ,2H), 7.37-7.39 (m, 2H).

Methyl

13 C NMR (90 MHz, CDCl 3 ) δ 22.7 (3C), 27.8 (3C), 27.9, 28.0, 38.3, 52.5, 56.9, 61.0, 74.5, 79.3, 79.9, 83.6, 110.1, 126.0, 126.4 (2C), 127.2, 128.5 (2C), 132.8, 137.6, 152.5, 168 (20 mL), the washed with water (20 mL) and brine (20 mL).

The organic layer was dried over MgSO 4 , filtered, and concentrated under reduced pressure. Flash chromatography on silica gel (petroleum ether/EtOAc = 70 /30, 50/50, 40/60) to give 226 (13.0 mg, 27%, impure). To an ice cold solution of impure 226 (13.0 mg, 27 µmol) in CH 2 Cl 2 (1 mL) was treated with 2,6-lutidine (11.5 mg, 107 µmol, 4 eq.) and TBSOTf (28.3 mg, 107 µmol, 4 eq.) dropwise. The mixture was stirred for 3 h at room temperature. Quenched by sat. NH 4 Cl (1 mL), diluted with with CH 2 Cl 2 (20 mL), washed with 1 M HCl (15 mL) and brine (20 mL) , 2982, 2956, 2932, 2857, 1750, 1717, 1681, 1511, 1369, 1251, 1165, 1106, 838 cm -1

1 H NMR (360 MHz, CDCl 3 ) δ 0.11 (s, 3H), 0.15 (s, 3H), 0.93 (s, 9H), 1.42 (s, 3H), 1.44 (s, 9H, NBoc and acetonide), 2.31-2.55 (m, 4H, two ABX mixed), 3.03-3.17 (m, 2H, ABX), 3.78-3.92(m, 1H), 4.14-4.21 (m, 2H, two ABX mixed), 4.30 (pseuso s, 1H)), 4. 58-4.60 (m, 1H), 5.06 (pseudo s, 1H), 6.27 (dt, 1H, J = 16.0, 6.8 Hz), 6.44 (d, J = 16.0 Hz ), 7.16-7.36 (m, 5H).

13 C NMR (90 MHz, MeOD-d 4 ) δ -4.5 (2C), 18.9, 26.5 (2C), 27.6, 27.8, 28.9 (2C), 38.9, 42.5, 46.4, 55.6, 69.7, 71.9, 80.1, 80.4, 80.6, 110.2, 126.9, 127.1 (2C), 128.2, 129.5 (2C), 133.7, 138.8, 158.4, 172.8, 178.0. , 2979, 2931, 2860, 1750, 1698, 1657, 1511, 1369, 1271, 1253, 1166, 1093 cm -1

HRMS

1 H NMR (400 MHz, MeOD-d 4 ) δ 0.09 (s, 3H), 0.13 (s, 3H), 0.89 (s, 9H), 1.41 (s, 3H), 1.44 (s, 9H, NBoc and one methyl of acetonide), 1.47 (s,9H), 2.33-2.57 (m, 4H, two ABX mixed), 3.05-3.18 (m, 2H, ABX), 3.79 (pseudo t, 1H, J = 6.4 Hz), 4.11-4.18 (pseudo m, 2H, two ABX mixed), 4.75-4.84 (m, 1H), 5.06 (pseudo s, 1H), 6.26 (ddd pseudo dt, 1H, J = 16.0, 6.8, 6.8 Hz), 6.47 (d, J = 16.0 Hz ), 7.17-7.21 (m, 1H), 7.26-7.32 (m, 2H), 7.33-7.37 (m, 2H).

13 C NMR (100 MHz, MeOD-d 4 ) δ -4. 4, -4.3, 19.0, 26.5 (3C), 27.7, 27.9, 28.1 (3C), 28.9 (3C), 39.0, 42.5, 46.6, 54.3, 69.7, 76.5, 79.7, 80.2, 80.8, 83.3, 110.7, 126.6, 127.2 (2C), 128.2, 129.5 (2C), 133.9, 138.8, 154.4, 158.3, 173.4, 175.0. Step1: To a solution of dipeptide D-mPyrAA-Sar (R)-189 (180 mg, 0.30 mmol, 1 eq.) in CH 2 Cl 2 (1 mL) under argon was added TFA (0.5 mL, 6.00 mmol, 20 eq.). The resulting mixture was stirred at room temperature for 4 h, then concentrated under reduced pressure to give a proposed TFA complex 237 (20 mg,  = 100%).

HRMS (ESI+

Step2: The proposed TFA complex 237 was dissolved in EtOAc ( 20 , 2922, 1708, 1636, 1506, 1448, , 1401, 1322, 1230, 1045, 759, 738 cm -1

1 H NMR (400 MHz, MeOD-d 4 ) δ 2.51 (dd, 1H, J = 25.2,12.0 Hz ABX), 2.90 (s, rotamer) and 2.92 (s, 3H), 2. 96-3.31 (m, 7H), 3..78-4.34 (m, 5H), 4.82-4.86 (m, 1H), 7.28-7.37 (m, 4H), 7.59-7.65 (m, 2H), 7.75 (d, 2H, J = 7.2 Hz ).

13 C NMR (100 MHz, MeOD-d 4 ) δ 35.5 (rotamer) and 37.3, 39.4 and 39.5 (rotamer), 39.9 (rotamer) and 40.0, 41.1 (rotamer) and 41.2, 46.3 (rotamer) and 46.5, 48.3, 50.7 (2C) and 53.0 (one rotamer of the two carbon), 57.3, 67.8, 73.7 and 73.8 (rotamer), 120.9 (2C), 126.2, 126.4, 128.2 (2C), 128.8 (2C), 142.5, 145.0, 145.5, 158.4, 172.4, 172.5, 176.1. , 2922, 1708, 1636, 1506, 1448, , 1401, 1322, 1230, 1045, 759, 738 cm -1

HRMS

1 H NMR (400 MHz, MeOD-d 4 ) δ 2.51 (dd, 1H, J = 25.2,12.0 Hz ABX), 2.90 (s, rotamer) and 2.92 (s, 3H), 2. 96-3.31 (m, 7H), 3..78-4.34 (m, 5H), 4.82-4.86 (m, 1H), 7.28-7.37 (m, 4H), 7.59-7.65 (m, 2H), 7.75 (d, 2H, J = 7.2 Hz ).

13 C NMR (100 MHz, MeOD-d 4 ) δ 35.5 (rotamer) and 37.3, 39.4 and 39.5 (rotamer), 39.9 (rotamer) and 40.0, 41.1 (rotamer) and 41.2, 46.3 (rotamer) and 46.5, 48.3, 50.7 (2C) and 53.0 (one rotamer of the two carbon), 57.3, 67.8, 73.7 and 73.8 (rotamer), 120.9 (2C), 126.2, 126.4, 128.2 (2C), 128.8 (2C), 142.5, 145.0, 145.5, 158.4, 172.4, 172.5, 176.1. , 2951, 2927, 2855, 1748, 1664, 1529, 1456, 1368, 1276, 1252, 1162, 1093

HRMS (ESI+

FRENCH SUMMARY

  GENERAL INTRODUCTION ..................................................... CHAPTER 1: INTRODUCTION .................................................

Figure 1 :

 1 Figure 1: Microsclerodermin D

  report 52 prescriptions dating from ab 1000 B.C., the Herbal Classic Shennong Herbal describes 365 substances from plants around 2800 B.C. and the Tang Herbal report 850 drugs around 659

Figure 10

 10 Figure 10: Strychnine

Figure 15

 15 Figure 15: Trabectedin

Figure 16 :γ 1 I

 161 Figure 16: Calicheamicin γ 1 I

Figure 25 :

 25 Figure 25: Structure of microsclerodermins

Scheme 10 :

 10 Scheme 10: Acces to protected APTO and AETD derivatives (2 nd part)

Figure 28 : 4 . 3 . 1 . 1 .

 284311 Figure 28: Retrosynthetic analysis of microsclerodermin E

Figure 29 : 4 . 3 . 2 . 1 .

 294321 Figure 29: Retrosynthetic analysis of microsclerodermin B

Scheme 22 :

 22 Scheme 22: Preparation of a protected derivative of AMMTD *

  Isolated yields; b Undesired bromination on the aromatic ring was observedTable 3: Hydroxybromination and debromination of microsclerodermin dipeptide mimics

4. 3 . 2 . 4 . 4 . 3 . 2 . 5 . 4 . 3 . 2 . 6 . 4 . 3 . 2 . 8 .

 324432543264328 Scheme 27: Construction of the protected derivative of the dipeptide Sar-Trp(2-CO 2 H)

Figure 30 :Figure 31 :

 3031 Figure 30: Structural reassignment of microsclerodermin B

Figure 32 :

 32 Figure 32: Microsclerodermin D, the target of the total synthesis in this thesis

Scheme 33 :

 33 Scheme 33: Retrosynthetic approach to microsclerodermin D

Figure 33 :

 33 Figure 33: Choice of the keto protecting group

Scheme 37 :

 37 Scheme 37: Preparation of N-Cbz protected dithioketal derivative

Equation 2 :

 2 Scheme 39: Mechanism of the racemization during Masamune's procedure

Equation 8 :

 8 Deprotection of the benzyl ester using HBr in acetic acid 4.3.2.4. One-pot dithioketal formation and deprotection of the benzyl ester While 2 sequential steps, dithioketal formation and benzyl ester deprotection with HBr, allowed the formation of the free acid 186 ready for coupling with sarcosine, the process presented different drawbacks:-the dithioketal formation generally requires 6 or 7 days to reach an acceptable conversion -the dithioketal formation gave variable results and sometimes even the use of a larger excess of BF 3 •Et 2 O or prolonged reaction time didn't allow full conversion, and the reaction stopped for no apparent reason.

  alone in acetone followed by quenching with water and purification on a very short chromatography gave 44% of the γ-ketoamide 192 accompanied with 5% of the dehydrated dipeptide 194 (entry 9).506 This result was encouraging since the formation of compound 194 indicated that deprotection of the dithioketal and cyclization occurred indeed, but slowly. This NBS procedure was repeated and a solution of the crude product mixture was stirred in the presence of silica gel for 24 h to provide gratifingly the desired PyrAA dipeptide 193 in 33% yield along with 5% of the dehydrated dipeptide 194 (entry 10). ROESY NMR experiment revealed a cis relationship between the carbamate and hydroxyl substituents in position 4 and 5 of the pyrrolidinone ring as observed by Dohonoe in 2017.439 This NBS procedure was repeated again and the crude product mixture was product (entry 12

Equation 10 :

 10 Dithoketal deprotection with HgO/HgCl 2 in aqueous acetonitrile Herein, the deprotection of dithioketal then cyclization sequence was successfully validated by treatment of dipeptide 189 with NBS. This encouraging result confirmed the potential of our proposed strategy to install the hydroxypyrrolidinoneacetic acid residue for the total synthesis of microsclerodermin D by the deprotection of dithioketal then cyclization sequence.

Table 7 :

 7 Maillard's preparation of N-Fmoc protected -amino-β-ketoesters Since Claisen condensation 521 was discovered more than a century ago, β-ketoesters have become a very significant class of intermediates in organic synthesis. To date, cross-Claisen condensations of activated carboxylic acids (or esters) with a lithium enolate or Meldrum's acid is still a classic and common pathway to generate β-ketoesters and γ-amino-β-ketoesters (Scheme

Scheme 52 : 4 . 5 . 3 .

 52453 Scheme 52: Jouin's condensation of α-amino acids with Meldrum's acid

Scheme 55 :

 55 Scheme 55: Preparation of benzyl -amino-β-ketoester through a thioester

4 . 5 . 6 .

 456 Scheme 56: Preparation of N-Fmoc -amino-β-ketoesters using Roskamp's procedure

  According to our proposed strategy, the second target required for the total synthesis of microsclerodermin D, is the dipeptide D-Trp(6-Cl)-Gly (Figure34). The uncommon D-6-chlorotryptophan residue also exists in microsclerodermin L and pedein A, while differently modified tryptophan derivatives exist in the other members of the microsclerodermin family: microsclerodermins F, H, J, K, M and pedein B present the D-tryptophan, which is commercially available; microsclerodermins G and I present dehydrotryptophan; microsclerodermins A, B, E, N and O present D-2-carboxytryptophan; microsclerodermin C is the only family member bearing D-N-formamidotryptophan.

Figure 34 :

 34 Figure 34: Dipeptide D-Trp(6-Cl)-Gly fragment present in microsclerodermin D

Figure 35 :

 35 Figure 35: L-Tryptophan and Auxin

121

  Scheme 63: Tsuchihashi's first synthesis of 5-hydroxy-L-tryptophan

6 -

 6 Scheme 69: Retrosynthetic analysis of dipeptide modified 6-Cl-Trp-Gly

  Scheme 74: Synthesis of the protected (R)-GABOB derivative 223

Thus, compound 67

 67 Scheme 76: Deprotection of the APTO derivative 67 with TFA or HCl

Scheme 77 : 2 . 4 .Scheme 78 :

 772478 Scheme 77: Deprotection of the APTO derivative 67 with HCl in dioxane

5 MACROCYCLICScheme 82 : 3 .

 5823 Scheme 80: Synthesis of the dipeptide Boc-GABOB-APTO-OMe from 2-dexoy-D-ribose

  from commercially available ethyl (R)-4-chloro-3-hydroxybutanoate according to Hayashi's procedures (Scheme 74). The protected ATPO derivative was then prepared from 2-dexoy-D-ribose according to an in-house the procedure. Starting from acetonide 63, we were able to access the dipeptide GABOB-APTO 234 requires for the synthesis of microsclerodermin D in 20.2% yield over 7 steps and assumed to be enantiomerically pure. A key transformation was the completely diastereoselective addition of the lithium enolate of O-Boc methyl glycolate on a chiral sulfinimine derived from 2-deoxy-D-ribose which provided the fully protected APTO as a single diastereisomer in 94% yield. Selective deprotection of the tert-butanesulfinyl group was ensured by the use of 4 M HCl in dioxane at low temperature. Finally, the ammonium salt 229 was directly engaged in the coupling with GABOB to give the dipeptide 234 in 72% yield in 2 steps and avoiding an unwanted Boc migration process (Scheme 80). In Chapter 5, with the required three dipeptides: Fmoc-mPyrAA-Sar-Ot-Bu (R)-189, Fmoc-[D-Trp(6-Cl)]-Gly-Ot-Bu 216 and Boc-GABOB(TBS)-APTO-OH 235 (Chapter 4) in hand, the macrocyclic assembly was initiated with deprotection of Fmoc-mPyrAA-Sar-Ot-Bu (R)-189 and Fmoc-[D-Trp(6-Cl)]-Gly-Ot-Bu 216. Peptide coupling of 238 and 217 gave the corresponding tetrapeptide Fmoc-mPyrAA-Sar-[D-Trp(6-Cl)]-Gly-Ot-Bu 239 in 82% yield (Scheme 82). The N-terminal Fmoc removal of 239 and then direct coupling with the third dipeptide Boc-GABOB(TBS)-APTO-OH 235 provided the linear hexapeptide 240 in 33% yield over 2 steps (Scheme 83). The hexapeptide 240 is ready for deprotection and macrocyclization, and should give rise to the macrocyclic peptide 242. The dithioketal group will be removed in the last step to liberate the γ-ketoamide which is expected to cyclize spontaneously to install the pyrrolidinoacetic acid residue to provide the target microsclerodermin D (Scheme 84).

[α] D 23 =

 23 +4.8 (c 0.50, D 2 O) IR (ATR) ν 3435

  mL) at 0 °C and extracted with Et 2 O (25 mL × 3). The combined organic layer was washed with 1 M HCl (25 mL × 3), sat. NaHCO 3 (25 mL × 3) and brine (25 mL× 3), then dried over MgSO 4 , filtered, and concentrated under reduced pressure. Flash chromatography on silica gel (petroleum ether/EtOAc = 90/10, 75/25, 70/30) gave a L-(S)-N-Cbz-β-ketoester 177 (3.3864 g, 82%, ee undetermined) as a colorless oil, which was stored in the fridge into a white solid). TLC: R f = 0.45 (petroleum ether/EtOAc = 75/25) Mp. 52-54 °C IR (ATR) ν 3292

694 cm - 1 1H

 1 NMR (360 MHz, CDCl 3 ) δ 2.78 (dd, 1H, J = 17.3, 5.0 Hz, ABX), 2.99 (dd, 1H, J = 17.3, 4.7 Hz, ABX), 3.63 (s, 3H), 3.67 (s, 2H), 4.64 (ddd, 1H, J = 9.0, 5.0, 4.7 Hz, ABX), 5.11-5.16 (m, 4H), 5.97 (d, 1H, J = 9.0 Hz), 7.28-7.39 (m, 10H).

  4 , filtered, and concentrated under reduced pressure. Flash chromatography on silica gel (petroleum ether/EtOAc = 90/10, 80/20, 70/30) gave a N-CBz-dithioketal 178 (33.0 mg, 34%) as a white solid. TLC: R f = 0.45 (petroleum ether//EtOAc = 70/30) 1 H NMR (360 MHz, CDCl 3 ) δ 2.63 (dd, 1H, J = 21.2, 14.0 Hz, ABX), 33.05-3.15 (m, 2H),3.24-3.31 (m, 5H), 3.61 (s, 2H), 4.85-4.92 (m, 1H), 4.84-4.91 (m, 1H, ABX), 5.03-5.09 (m, 2H), 5.17 (s, 2H),5.35 (d, 1H, J = 14.0 Hz), 7.26-7.38 (m, 10H).

  ) Calcd for C 24 H 27 NNaO 6 S 2 [M + Na] + : 512.1172, found 512.1191.

TLC: R f = 0 . 28 (

 028 CH 2 Cl 2 /MeOH = 85/15) Mp. 124-125 °C (lit. 481 122-125 °C) [α] D 26 = +34.3 (c 1.05, CHCl 3 ), (lit. 481 [α] D 23 = +14.6 (c 1.05, CHCl 3 ))

  mL) at 0 °C and extracted with Et 2 O (25 mL × 3). The combined organic layer was washed with 1 M HCl (25 mL × 3), sat. NaHCO 3(25 mL × 3) and brine (25mL), then dried over MgSO 4 , filtered and concentrated under reduced pressure. Flash chromatography on silica gel (petroleum ether/EtOAc = 90/10, 75/25, 70/30) to give a partially racemic N-Fmoc-β-ketoester 180 (3.46 g, 82%, extensive racemization 0-28% ee) as a colorless oil, which was stored in the fridge into a waxy solid. TLC: R f = 0.43 (petroleum ether/EtOAc = 70/30) Mp. 52-55 °C IR (ATR) ν 3354

1 1H

 1 NMR (360 MHz, CDCl 3 ) δ 2.79 (dd, 1H, J = 17.3, 4.3 Hz, ABX), 3.00 (dd, 1H, J = 17.3, 5.0 Hz, ABX), 3.60 (s, 2H), 3.68 (s, 3H), 4.20 (t, 1H, J = 6.5 Hz), 4.44 (dd, 1H, J = 10.6, 6.5 Hz), 4.54 (dd, 1H, J = 10.6, 6.5 Hz), 4.63 (ddd, 1H, J = 9.0, 5.0, 4.7 Hz, ABX), 5.13-5.21 (m, 2H), 5.93 (d, 1H, J = 9.0 Hz,), 7.29-7.43 (m, 9H), 7.59 (d, 2H, J = 7.6 Hz), 7.76 (dd, 2H J = 7.6, 3.2 Hz).

  ) Calcd for C 29 H 27 NNaO 7 [M + Na] + : 524.1680, found 524.1678.

  chromatography on silica gel (petroleum ether/EtOAc = 90/10, 80/20, 70/30) gave a partially racemic N-Fmoc-dithioketal 181 (3.88 g, 75%, 0-28% ee) as a white solid. TLC: R f = 0.45 (petroleum ether/EtOAc = 70/30)

-1 1 H

 1 NMR (360 MHz, CDCl 3 ) δ 2.65 (dd, 1H, J = 14.8, 9.7 Hz, ABX), 3.05-3.12 (m, 2H),3.18-3.28 (m, 5H),3.63 (s, 3H), 4.20 (t, 1H, J = 6.8 Hz), 4.34-4.43 (m, 2H), 4.84-4.91 (ddd pseudo td, 1H,

  pressure. Flash chromatography on silica gel (petroleum ether/EtOAc = 70/30, 50/50) gave N-Fmoc-dipeptide 187 (95.6 mg, 95%) as a white solid. TLC: R f = 0.45 (petroleum ether/EtOAc = 50/50) Mp. 68-70 °C IR (ATR) ν 3331

  mL) was added a solution of 28% NH 4 OH (3 mL). The resulting mixture was stirred at room temperature for 4 days, completed. The reaction solution was then concentrated under reduced pressure, diluted with CH 2 Cl 2 (20 mL) and washed with wter (20 mL). The separated organic phase was then dried over MgSO 4 , filtered and concentrated under reduced pressure. Flash chromatography on silica gel (CH 2 Cl 2 /MeOH = 95/5, 90/10) gave a dipeptide of free amine 188 (13.5 mg, 52%) as a pale-yellow oil. TLC: R f = 0.19 (CH 2 Cl 2 /MeOH = 90/10)

MgSO 4 ,

 4 filtered and concentrated under reduced pressure. Flash chromatography on silica gel (petroleum ether/EtOAc = 70/30, then CH 2 Cl 2 /MeOH = 95/5, 90/10) to give N-Fmoc-dipeptide 189 (13.5 mg, 52%) as a white solid.

  containing N-Fmoc-β-dithioketal acid 186 (150 mg, 0.31 mmol, 1 eq.) was added a solution of 28% NH 4 OH(8.5 mL, 61.40 mmol, ~200 eq.). The mixture was stirred for overnight(18-20 h) at room temperature, then filtered through a filter Buchner funnel to remove dibenzofulvene (DBF) and the solid was washed with 28% NH 4 OH (5 mL× 3) to give the filtrate, which was extracted with Et 2 O (10 mL× 2) to remove the residue of DBF. The aqueous phase was concentrated under reduced pressure to give a pure primary amide 190 (74.0 mg, 100%) as a white solid and used without further purification. TLC: R f = 0.10 (MeOH/CH 2 Cl 2 = 10/90) Mp. 105-108 °C IR (ATR) ν 3325, 3162, 2921, 1667, 1557, 1378, 1276, 851, 679 cm -1 1 H NMR (360 MHz, D 2 O) δ 2.56 (dd, 1H, J = 15.8, 10.4 Hz, ABX), 2.96 (dd, 1H, J = 15.8, 2.9 Hz, ABX), 3.02 (d, 1H, J = 16.6 Hz), 3.13 (d, 1H, J = 16.6 Hz), 3.26-3.41 (m, 4H), 4.01 (dd, 1H, J 1 = 10.4, 2.9 Hz, ABX) 13 C NMR (90 MHz, D 2 O) δ 35.

  -) Calcd for C 23 H 24 N 2 NaO 5 S 2 [M + Na] + : 495.1019, found 495.1009.

(

  40mL).The filtrate was washed with water (20 mL) and brine (20mL). The organic layer was dried with Mg 2 SO 4 , filtered, and concentrated under reduced pressure. Flash chromatography on silica gel (EtOAc/petroleum ether = 30/70, 50/50, EtOAc = 100%, then EtOAc/MeOH = 100/1) gave the nitrile 195 (47.0 mg, 60%) as a colorless oil, as well as the dehydrated compound 194 (8.0 mg, 10%) as a colorless oil. TLC: R f = 0.64 (EtOAc/petroleum ether = 50/50) IR (ATR) ν 3316

  mL), then washed with water (30 mL× 3) and brine (30 mL× 2). The organic layer was dried with Mg 2 SO 4 , filtered, and concentrated under reduced pressure to give a pale-yellow oil (40 mg). Flash chromatography on silica gel with normal speed (EtOAc/petroleum ether = 40/60, 50/50) gave the dehydrated 194 (20.0 mg, 40%) as a colorless oil. TLC: R f = 0.25 (petroleum ether/EtOAc = 50/50) IR (ATR) ν 3300

  ) Calcd for C 13 H 11 NNaO 6 [M + Na] + : 300.0478, found 300

MgSO 4 ,

 4 filtered, and concentrated under reduced pressure. Flash chromatography on silica gel (petroleum ether/EtOAc = 90/10, 75/25, 70/30) to give a β-keto-γ-phthalimidoester 200 (163.9 mg, 40%, ee undetermined) as a colorless oil. TLC: R f = 0.62 (petroleum ether/EtOAc = 70/30) (360 MHz, CDCl 3 ) δ 2.78 (dd, 1H, J = 17.3, 5.0 Hz, ABX), 2.99 (dd, 1H, J = 17.3, 4.7 Hz, ABX), 3.63 (s, 3H), 3.67 (s, 2H), 4.64 (ddd, 1H, J = 9.0, 5.0, 4.7 Hz, ABX), 5.11-5.16 (m, 4H), 5.97 (d, 1H, J = 9.0 Hz), 7.28-7.39 (m, 10H).

  directly loaded for flash chromatography on silica gel (petroleum ether/EtOAc = 90/10, 80/20, 70/30) to give the desired L-(S)-N-Fmoc thioester 201(1.846 g, 86%) as a colorless oil.TLC: R f = 0.72 (petroleum ether/EtOAc = 70/30)

  ) Calcd for C 23 H 25 NNaO 5 S [M + Na] + : 450.1346, found 450To an ice cold solution of L-(S)-N-Fmoc thioester 201 (293.0 mg, 0.68 mmol, 1 eq.) in acetone(6 mL) under argon was added Pd/C (10% wt., 72.4 mg, 0.07 mmol, 0.1 eq.), followed by addition of Et 3 SiH (318.6 mg, 2.74 mmol, 4 eq.) dropwise. The ice bath was then removed, and the resulting mixture was stirred for 30 min at room temperature, completed. The reaction solution was filtered through a Celite pad and washed with acetone (5 ml × 3). The filtered liquid was concentrated under reduced pressure. Flash chromatography on silica gel (petroleum ether/EtOAc = 80/20, 70/30) to give the desired N-Fmoc aldehyde 202 (230.0 mg, 95%) as a white solid.TLC: R f = 0.66 (petroleum ether/EtOAc = 70/30)

  chromatography on silica gel (petroleum ether/EtOAc = 80/20, 70/30, 60/40) gave the desired γ-amino-β-hydroxyester 203 (205.5 mg, 72%) as a colorless oil, with an inseparable mixture of two diastereoisomers (1.4:1, by 1 H NMR). TLC: R f = 0.31 (petroleum ether/EtOAc = 70/30)

  To an ice cold solution of γ-amino-β-hydroxyester 203(50.3 mg, 0.10 mmol, 1 eq.) in acetone(6 mL) under argon was added Dess-Martin periodinane(63.6 mg, 0.15 mmol, 1.5 eq.). The ice bath was then removed, and the resulting mixture was stirred for 20 h at room temperature, completed. The reaction was concentrated under reduced pressure, diluted with EtOAc (20 mL), washed with sat. NaSO 3(20 mL), sat NaHCO 3(20 mL) and brine(20 mL), respectively. Flash chromatography on silica gel (petroleum ether/EtOAc = 80/20, 70/30) gave a fully racemic N-Fmoc-β-ketoester 180 (50.0 mg, 99%, as racemic, 0% ee) as a colorless oil when stored in the fridge in turned into a waxy solid). Data see compound 180.One-pot Roskamp reaction:To an ice cold solution of L-(S)-N-Fmoc thioester 201 (236.3 mg, 0.50 mmol, 1 eq.) in CH 2 Cl 2 (5 mL) under argon was added Pd/C (10% wt., 53.2 mg, 0.05 mmol, 0.1 eq.), followed by addition of Et 3 SiH (232.5 mg, 2.00 mmol, 4 eq.) dropwise. The ice bath was then removed, and the resulting mixture was stirred for 30 min at room temperature, completed. The reaction solution was filtered through a Celite pad and washed with CH 2 Cl 2 (5 ml × 3). The filtered liquid was concentrated under reduced pressure to give the aldehyde 202 crude (300 mg,  = 100%) as a semi-solid. To a solution of aldehyde 202 crude was directly subjected to Roskamp reaction. To a solution of crude 202 in CH 2 Cl 2 (10 mL) under argon at room temperature was added SnCl 2 (37.9 mg, 0.20 mmol, 0.4 eq.). A solution of benzyl diazoacetate in toluene (10% wt., 1.2 mL, 0.60 mmol, 1.2 eq.) was added over 10 min. The resulting mixture was stirred for 8 h at room temperature and quenched with 1 M HCl (1 mL). Then diluted with CH 2 Cl 2(10 mL), washed with 1 M HCl (10 mL), sat. NaHCO 3(10 mL), and brine(10 mL). The organic layer was then dried over MgSO 4 , filtered and concentrated under reduced pressure. Flash chromatography on silica gel (petroleum ether/EtOAc = 90/10, 80/20, 70/30) gave the desired benzyl γ-amino-β-ketoester 180 (160.0 mg, 64%, 58% ee) as a white solid. Data see compound 180.

  containing L--amino-β-ketoester (S)-180 (1.004 g, 2.00 mmol, 1 eq.) under argon at room temperature was added1,2-ethanedithiol (1.7 mL, 20.0 mmol, 10 eq.), followed by addition of BF 3 •Et 2 O (0.74 mL, 6.00 mmol, 3 eq.) dropwise. The resulting mixture was stirred for 3 days at room temperature. The reaction was quenched with water (20 mL), extracted with EtOAc (20 mL × 3). The combined organic phase was washed with brine (20mL × 3), then dried over MgSO 4 , filtered, and concentrated under reduced pressure. Flash chromatography on silica gel (petroleum ether/EtOAc = 90/10, 80/20, 70/30, 50/50) to give the desired L-N-Fmoc-β-dithioketal acid (S)-186 (828.7 mg, 85%) as a white solid. TLC: R f = 0.10 (petroleum ether/EtOAc = 75/25) Mp. 59-62 °C [α] D 25 = -15.5 (c 0.50, CHCl 3 )

  ) Calcd for C 24 H 25 NNaO 6 S 2 [M + Na] + :510.1015, found 510.0992.2-(2-(1,3-Diamino-3-oxopropyl)-1,3-dithiolan-2-yl)acetic acid (S)-190To a flask containing L-N-Fmoc-β-dithioketal acid (S)-186 (830 mg, 1.70 mmol, 1 eq.) was added a solution of 28% NH 4 OH (48 mL, 336 mmol, ~200 eq.). The mixture was stirred for overnight(24 h) at room temperature, then filtered through a filter Buchner funnel to remove dibenzofulvene (DBF) and the solid was washed with 28% NH 4 OH(3 × 5 mL) to give the filtered liquid. The filtered liquid was extracted with Et 2 O (2 ×20 mL) to remove the residue of dibenzofulvene, and the aqueous phase was concentrated under reduced pressure to give a pure primary amide (S)-190 (425.9 mg, 100%) as a pale-yellow solid and used without further purification directly in next step.

  ) Calcd for C 8 H 15 N 2 O 3 S 2 [M + H] + : 251.0519, found 251.0519.

Et 2 O

 2 (50 mL× 2) to give the aqueous phase. The aqueous phase was acidified with 4 M HCl until = 2-3, then with EtOAc (50 mL× 3) to give the organic phase. The combined organic phase was dried over MgSO 4 , filtered and concentrated under reduced pressure to give a pure L-N-Fmoc-mPyrAA (S)-191 (560.7 mg, 70%) as a white solid and used without further purification.TLC: R f = 0.20 (MeOH/CH 2 Cl 2= 20/80)

  ) Calcd for C 23 H 24 N 2 NaO 5 S 2 [M + Na] + : 495.1019, found 495.1001.

  mL), then dried over MgSO 4 , filtered and concentrated under reduced pressure. Flash chromatography on silica gel (petroleum ether/EtOAc = 50/50, EtOAc =100, EtOAc/MeOH = 90/10) to give the desired dipeptide L-mPyrAA-Sar (S)-189 (538.9 mg, 90%) as a white solid.

  mL) dropwise over 10 min. The resulting mixture was slowly warmed to room temperature and stirred for overnight(16 h). The solution was evaporated under reduced pressure to remove most of dioxane, then extracted with ether(2 × 30 mL) to give the aqueous phase, which was acidified with 4 M HCl . until pH = 2-3.The acidified solution was then extracted with ether with Et 2 O (3 × 30 mL), and the combined organic layer was washed with brine (50mL), dried over MgSO 4 and concentrated under reduced pressure gave the pure desired D-Fmoc-Asp(β-OMe)-H (R)-179 (3.68 g, 100%) as a white solid and used without further purification directly in the next step. TLC: R f = 0.11 (EtOAc = 100), R f = 0.28 (CH 2 Cl 2 /MeOH = 85/15)

  ESI+) Calcd for C 11 H 14 ClN 2 [M + H] + : 209.0840, found 209.0831.

  ESI+) : found [C 9 H 7 ClN + ] 164.0246, decomposition.

  mL) at -78 °C was slowly added the solution of Li-enolate from step (2). The resulting mixture was stirred for overnight (> 12 h), slowly warmed to room temperature, then quenched with sat. NH 4 Cl (20 mL) in ice bath and extracted with EtOAc (50 mL× 2), The combined organic phase was then washed with brine and dried over MgSO 4 , then filtered and evaporated under reduced pressure (crude 213, dr = 95:5 by 1 H NMR intergration). Flash chromatography on silica gel (petroleum ether/EtOAc = 90/10, 80/20, 70/30, 60/40) gave the desired (R)-Trp(6-Cl)-pinanone imine 213 (186.0 mg, 52%, dr > 97:3 by 1 H NMR integration) as a white solid. TLC: R f = 0.40 (petroleum ether/EtOAc = 50/50)

  ) Calcd for C 25 H 34 ClN 3 O 3 [M + H] + :445.2252, found 445.2237. of Trp(6-Cl)-pinanone imine 213 (111.3 mg, 0.25 mmol, 1 eq.) in THF (2 mL) was added a solution of 15% citric acid (300.0 mg, 1.60 mmol, 6 eq.) in water (2 mL). The mixture was stirred at room temperature for 24 h, then evaporated and diluted with water (5 mL). The aqueous phase was extracted with EtOAc (5 mL× 2), subsequently then basified with sat. NaHCO 3 until pH > 8, then extracted with EtOAc (5 mL× 2). The combined organic phase was dried over MgSO 4 , then filtered and evaporated under reduced pressure to give the pure desired H-Trp(6-Cl)-Ot-Bu 214 as a colorless oil (70.0 mg, 95%) TLC: R f = 0.54 (MeOH/CH 2 Cl 2 = 5/95) [α] D 22 = -14.8 (c 0.50, CHCl 3 ) IR (ATR) ν 3363

NaHCO 3

 3 until pH > 8, then extracted with EtOAc(3 × 20 mL). The combined organic phase was dried over MgSO 4 , then evaporated under reduced pressure to give the racemic H-Trp(6-Cl)-Ot-Bu (±)-214 as a white solid(350 mg, 84%). TLC: R f = 0.54 (MeOH/CH 2 Cl 2 = 5/95) Mp = 106-108 °C IR (ATR) ν 3360

  of racemic Fmoc-Trp(6-Cl)-Ot-Bu (103.4 mg, 0.20 mmol, 1 eq.) in CH 2 Cl 2(2 mL) was added Et 3 SiH (46.5 mg, 0.40 mmol, 2 eq.), then followed by addition of TFA (0.62 mL, 8.00 mmol, 40 eq) at room temperature. The resulting mixture was stirred at room temperature for overnight (18 h), completed. Evaporated under reduced pressure. Flash chromatography on silica gel (petroleum ether/EtOAc = 80/20, 50/50, then MeOH/CH 2 Cl 2 = 10/90) gave the desired racemic Fmoc-Trp(6-Cl)-OH (±)-215 (92.0 mg, 98%) as a white solid, then used for coupling.To a solution of racemic Fmoc-Trp(6-Cl)-OH (±)-215 (92.0 mg 0.20 mmol, 1 eq.) in CH 2 Cl 2 (5 mL) at room temperature under argon was added PyBroP (102.6 mg, 0.22 mmol, 1.1 eq.) and Gly•HCl (36.9 mg, 0.22 mmol, 1.1 eq.), subsequently. Followed by addition of DIEA (103.4 mg, 0.80 mmol, 4 eq.) dropwise. The resulting mixture was stirred at room temperature for overnight (>16 h). The reaction was then diluted with CH 2 Cl 2(20 mL), and then washed with 1 M HCl (20mL), sat. NaHCO 3(20 mL) and sat. NaCl(20 mL), respectively. The combined organic phase was dried over MgSO 4 , then filtered and concentrated under reduced pressure. Flash chromatography on silica gel (petroleum ether/EtOAc = 80/20, 70/30, 60/40, 50/50) gave the desired racemic peptide Fmoc-Trp(6-Cl)-Gly-Ot-Bu (±)-216(93.3 mg, 81%) as a white solid. TLC: R f = 0.35 (petroleum ether/EtOAc = 60/40) HPLC: Racemic (±)-216: 0% ee. sample prepration: 1 mg/L in MeOH: column: A_NP_Lux Cellulose-2 (250 mm × 4.6 mm), normal phase: hexane/EtOH = 70/30, flow: 1 mL/min, 30 °C; injection: 5 L; results: RT = 7.391 (R, Area: 50%), 13.706 (S, Area: 50%).

  (360 MHz, CDCl 3 ) δ 1.29 (t, 3H, 3 J = 7.1 Hz), 2.53 (dd, 1H, 2 J = 16.5 Hz, 3 J = 3.7 Hz, ABX), 2.56 (dd, 1H, 2 J = 16.5 Hz, 3 J = 8.8 Hz, ABX), 2.26 (bs, 1H), 3.34 (dd, 1H, 2 J = 12.5 Hz, 3 J = 6.1 Hz, A'B'X'), 3.38 (dd, 1H, 2 J = 12.5 Hz, 3 J = 4.1 Hz, A'B'X'), 4.19 (q, 1H, 3 J = 7.2 Hz),4.18-4.25 (m, 1H, ABX and A'B'X').

  mL) was added Boc 2O (1.34 g, 6.15 mmol, 1.5 eq.) and Pd-C (107 mg, 15% w/w). The resulting mixture was stirred for 24 h under hydrogen atmosphere then filtered through Celite ® and the solvent was removed under reduced pressure. Flash chromatography of the residue (petroleum ether/EtOAc = 90/10) gave the amino ester (+)-Boc-GABOB-OEt 221 (728 mg, 72%) as a pale-yellow oil.TLC: R f = 0.41 (petroleum ether/EtOAc = 70/30) (360 MHz, CDCl 3 ) δ 1.28 (t, 3H, 3 J = 7.1 Hz), 1.45 (s, 9H), 2.47 (dd, 1H, 2 J = 16.6 Hz, 3 J = 9.2 Hz, ABX), 2.50 (dd, 1H, 2 J = 16.6 Hz, 3 J = 3.4 Hz, ABX), 3.07-3.17 (m, 1H),3-27-3.40 (m, 1H), 3.56 (bs, 1H), 4.06-4.15 (m, 1H, ABX), 4.17 (q, 2H, 3 J = 7.1 Hz), 4.98 (bs, 1H).

  mL). The organic layer was dried over MgSO 4 , filtered and evaporated under reduced pressure. Flash chromatography of the residue (petroleum ether/EtOAc = 95/5) gave the amino ester (+)-Boc-GABOB(TBS)-OEt 222 (734 mg, 72%) as a pale-yellow oil. TLC: R f = 0.62 (petroleum ether/EtOAc = 90/10) [α] D 23 = +11 (c 1.04, CHCl 3 ) IR (ATR) ν 3382, 1735, 1719, 1504, 1250, 1167 cm -11 H NMR (360 MHz, CDCl 3 ) δ 0.06 (s, 3H), 0.09 (s, 3H), 0.87 (s, 9H), 1.26 (t, 3H, 3 J = 7.1 Hz), 1.44 (s, 9H), 2.46 (pseudo d, 2H, 3 J = 6.0 Hz), 3.13-3.29 (m, 2H), 4.13 (q, 2H, 3 J = 7.1 Hz),

  (ESI+) Calcd for C 17 H 35 NNaO 5 Si [M + Na] + : 384.2177, found 384.2157.

  aqueous residue was then washed with CH 2 Cl 2(3 × 20 mL), acidified with 1 M HCl until pH = 1 and then extracted with CH 2 Cl 2(3 × 20 mL). The combined organic layers were dried over MgSO 4 , filtered and evaporated under reduced pressure to give the amino acid (+)-Boc-GABOB(TBS)-OH 223 (597 mg, 87%) as a white solid.

Flash

  chromatograph on silica gel (petroleum ether/EtOAc = 70/30) gave the desired 2-deoxy-D-ribose acetonide 63 (170.0 mg, 66%, as an inseparable mixture of α and β anomers ratio of 3:1) as a colorless oil. TLC: R f = 0.30 (petrum ether/EtOAc = 60/40)

  mL) and extracted with CH 2 Cl 2 (20 mL× 2). The combined organic phase was washed with brine (20 mL), dried over MgSO 4 , filtered, concentrated under reduced pressure. Flash chromatography on silica gel (petroleum ether/EtOAc = 100/1, 95/5, 90/10) gave the desired epimerized aldehyde 65 (88.0 mg, 83%) as a colorless oil. TLC: R f = 0.41 (petroleum ether/EtOAc = 75/25)

Flash

  chromatography on silica gel (petroleum ether/EtOAc = 90/10, 80/20, 70/30) gave the desired sulfinimine 66 (1.443 g, 69%) as a pale-yellow oil. TLC: R f = 0.22 (petroleum ether/EtOAc = 90/10)

(

  0 mL) was then added dropwise and the resulting mixture was stirred at -78 °C for 1 h. The reaction was quenched by sat. NH 4 Cl (10 mL) at 0 °C and diluted with water (20 mL). The resulting mixture was extracted with EtOAc (50 mL × 2) and the combined organic phases were dried over MgSO 4 , filtered, concentrated under reduced pressure. Flash chromatography (petroleum ether/EtOAc = 70/30) gave a fully-protected APTO 67 (760.3 mg, 94%) as a white solid. TLC: R f = 0.33 (petroleum ether/EtOAc = 70/30) Mp = 161-163 °C [α] D 26 = +32.5 (c 0.90, CHCl 3 ), (lit. 388 [α] D 21 = +33.7 (c 0.90, CHCl 3 )) IR (ATR) ν 3335

  -4.22 (m, 2H), 4.26-4.29 (m, 1H), ),4.50 (d, 2H, J = 11.2 Hz), 4.73 (d, 1H, J = 9.7 Hz ), 5.02 (d, 1H, J = 7.6 Hz ),6.21 (ddd pseudo dt, 1H, J = 16.6, 7.6 Hz), 6.50 (d, 1H, J = 16.6 Hz), 7.20-7.36 (m, 7H), 7.41 (t, 2H, J = 7.2 Hz), 7.56 (d, 2H, J = 7.2 Hz), 7.77 (d, 2H, of GABOB 223 (40.0 mg, 0.12 mmol, 1.2 eq.) in CH 2 Cl 2 (1 mL) was added HATU (0.15 mmol, 57 mg, 1.5 eq.) and DIEA(0.15 mmol, 20 mg, 1.5 eq.), respectively. After stirred for 1 h at room temperature(30 °C) as solution ATo a flask containing fully-protected APTO 67 (54.0 mg, 0.10 mmol, 1 eq.) was slowly added a HRMS (ESI+) Calcd for C 23 H 33 NNaO 7 [M + Na] + : 458.2149, found 458.2137. containing fully-protected APTO 67 (108.0 mg, 0.20 mmol, 1 eq.) at -5 °C under argon was slowly added a solution of 4 M HCl in dioxane (1.00 mL, 4.00 mmol, 20 eq.) and the mixture was stirred for 35 min between -5 °C until completed. Then diluted with 10 mL ether, and evaporated to give a colorless sticky oil, which was dissolved in CH 2 Cl 2 (10.0 mL). To the resulting solution was then added PyBroP (140.0 mg, 0.30 mmol, 1.5 eq.) and GABOB (100.0 mg, 0.30 mmol, 1.5 eq.) and DIEA (129.4 mg, 1.0 mmol, 5.0 eq.) dropwise, respectively. The resulting mixture was stirred at room temperature for 48 h. The reaction was then diluted with CH 2 Cl 2(50 mL), and then washed with water, sat. NaHCO 3 and brine, respectively. The organic phase was dried over MgSO 4 , filtered, concentrated under reduced pressure. Flash chromatography (petroleum ether/EtOAc = 70/30) gave the desired peptide GABOB-APTO 234 (108.2 mg, 72%)

  (ESI+) Calcd for C 37 H 61 N 2 NaO 11 Si [M + Na] + : 659.3334, found 659.3315. of GABOB-APTO 234 (19.0 mg, 25 mol, 1 eq.) in MeOH/THF/H 2 O (v/v/v, 0.4/0.4/0.6 mL) was added LiOH•H 2 O (10.5 mg, 250 mol, 10 eq.). After stirred for 4 h at room temperature, completed, then diluted with water (10 mL), cooling down at -10 C and carefully acidified with a solution of citric acid (72 mg, 370 mol, 15 eq.) (pH = 3-4). The acidic aqueous phase was extracted with CH 2 Cl 2 (10 mL × 3). The combined organic phase was dried over MgSO 4 and concentrated under reduced pressure. Preparative thin layer chromatography on silica gel (MeOH/CH 2 Cl 2 = 5/95) gave the desired acid 235 (10.0 mg, 54%) as a colorless oil, as well as 2-hydroxyl acid 236 (6.0 mg, 38%) as a clolorless oil. Data below for the desired acid 235: TLC: R f = 0.36 (MeOH/CH 2 Cl 2 = 10/90) [α] D 22 = -7.8 (c 0.55, CHCl 3 ) IR (ATR) ν 3330

  ) Calcd for C 37 H 61 N 2 NaO 11 Si [M + Na] + : 759.3826, found 759.3858.

  mL), then basified with DIEA (775 mg, 6.00 mmol, 20 eq.) in ice bath. The basified solution was washed with water(10 mL × 3) to give the aqueous phase, which was then acidified with 4 M HCl until pH = 2-3 and extracted with a mixture solvents of CH 2 Cl 2 /MeOH = 95/5(20 mL × 3). The combined organic phase was dried over MgSO 4 and concentrated under reduced pressure to give the pure desired the pure desired free dipeptide acid 238 (133.5 mg, 82%) as a white solid.TLC: R f = 0.50 (EtOAc/MeOH = 90/10) [α] D 21 = +23.1 (c 0.50, MeOH) IR (ATR) ν 3329

  of D-mPyrAA-Sar-OH (R)-238(8.5 mg, 15.6 mol, 1.1 eq.) in CH 2 Cl 2 (1 mL) under argon at room temperature was added PyBroP (8.0 mg, .17.0 mol, 1.2 eq,) and DIEA(7.3 mg, 4.00 mmol, 4 eq.), respectively. A solution of H-Trp-Gly-Ot-Bu 217 (5.0 mg, 14.2 mol, 1 eq.) in CH 2 Cl 2 (1 mL) was added. The resulting mixture was stirred at room temperature for 48 h. Diluted with CH 2 Cl 2(20 mL) and the solution was subsequently washed with 1 M HCl (20 mL), sat. NaHCO 3(20 mL) and brine(20 mL), then dried over MgSO 4 , filtered and concentrated under reduced pressure. Flash chromatography on silica gel (EtOAc/MeOH = 95/5, 90/10) gave the pure desired tetrapeptide D-Fmoc-mPyrAA-Sar-Trp(6-Cl)-Gly-Ot-Bu 239 (10.2 mg, 82%) as a waxy solid.

  ) Calcd for C 43 H 49 ClN 6 NaO 8 S 2 [M + Na] + : 899.2634, found 899.2614. -Sar-Trp(6-Cl)-Gly-Ot-Bu 239(9.0 mg, 10 mol, 1.1 eq.) in MeOH (1 mL) at room temperature was added Et 2 NH(29.3 mg, .0.40 mmol, 40 eq,). The resulting mixture was stirred at room temperature for overnight ( 16 h), completed. Then concentrated under reduced pressure, and evaporated with CH 2 Cl 2(2 mL × 3) to remove the residue of Et 2 NH cleanly, to give the crude of desired free tetrapeptide amine (9.2 mg,  = 100%, combined with DBF, TLC: R f = 0.10 (EtOAc/MeOH = 95/5)) as a pale-yellow waxy solid.Step(2) Coupling with GABOB-APTO derivative 236: To a solution of GABOB-APTO derivative 236 (8.1 mg, 11 mol, 1.1 eq.) in CH 2 Cl 2 (0.5 mL) under argon at room temperature was added PyBroP (5.6 mg, 12 mol, 1.2 eq.) and DIEA(5.2 mg, 40 mol, 4 eq.), respectively. A solution of free tetrapeptide amine crude 239 (9.2 mg, 10 mol, 1 eq.) in CH 2 Cl 2 (0.5 mL) was then added at room temperature. The resulting mixture was stirred for 16 h at room temperature. The mixture was diluted with CH 2 Cl 2(20 mL) and washed with brine(20 mL) to give the organic layer, which was dried over MgSO 4 , filtered and concentrated under reduced pressure. -APTO-mPyrAA-Sar-Trp(6-Cl)-Gly-Ot-Bu 240 (4.5 mg, 33%) as a colorless oil.TLC: R f = 0.50 (EtOAc = 100%) IR (ATR) ν 3330

Table 1 :

 1 Examples of inhibition activities and cytotoxicity of microsclerodermins

	263

4.2.2. McLeod's work on the synthesis of protected APTO and AETD

	In 2007, McLeod et al. reported the stereoselective synthesis of two protected
	polyhydroxy-β-amino acids: APTO, present in microsclerodermin C and D, and AETD, present in
	microsclerodermin E. 380 Starting from simple substrates, four contiguous stereocenters were
	installed using Sharpless asymmetric aminohydroxylation and dihydroxylation, while Julia
	olefination allowed the introduction of the styrene moiety.
	Oxydative cleavage of allyl 4-methoxybenzoate 51 with OsO 4 gave aldehyde 52 which was
	used directly in a Horner-Wadsworth-Emmons reaction with methyl diethylphosphonoacetate to
	provide the pure (E)-alkene 53 in excellent yield after flash chromatography (Scheme 9).

dihydroxylactone 86. The diol was treated with triflic anhydride in the presence of pyridine to afford the monotriflate 87 which reacted in the presence of

	14). The terminal acetonide was selectively hydrolyzed with AcOH/H 2 O 410 to give a diol of which
	the primary alcohol was converted into a bromide using CBr 4 /PPh 3 411 to give the vicinal
	bromoalcohol 82. Upon treatment with CsF in acetone, this bromoalcohol furnished the epoxide 83
	in excellent yield. 412 Next, regioselective addition of vinyl cuprate to this epoxide 413 followed by
	protection of the free secondary alcohol as a TBS ether gave the orthogonally protected derivative
	84. After removal of the PMB group with DDQ 414,415 and oxidation with TPAP/NMO, 401,408,416 the
	lactone 85 was obtained in good yield. Hydrolysis of the remaining acetonide provided the
	2,3-a slight excess of
	3,4-dimethoxybenzylamine (DMB-NH 2 ) to give the DMB-protected aziridino-γ-lactone 88 in
	moderate yield. Thereafter, the DMB aziridine 88 was converted into the Ts-protected substrate 89
	by sequential DMB removal using DDQ and tosylation of the free aziridine. Regioslective opening
	of the Ts-aziridine at C 2 position 408 was achieved by treatment with PBu 3 /Ac 2 O 417 under microwave
	irradiation to provide the fully protected lactone derivative 90 containing the requisite
	stereochemistry present in APTO. A Heck coupling with iodobenzene allowed introduction of the Scheme 14: Synthesis of a protected derivative of APTO
	styrene moiety and was accompanied by simultaneous removal of the N-acetyl group to give lactone
	91. Finally opening of the lactone moiety with benzylamine gave a mixture of mono acetylated In summary, the synthesis of a fully protected derivative of ATPO was completed in 0.5%
	protected products at C 2 and C 4 and subsequent treatment of the mixture with acetic anhydride in yield over 18 steps from L-gulose. This was first example of the use of an aziridino-γ-lactone for the
	pyridine furnished the fully protected APTO derivative 92. stereoselective synthesis of a β-amino acid derivative.

4.2.6. Davies' work on the synthesis of protected APTO and AETD

	More recently Davies et al. reported an asymmetric synthetic pathway to protected APTO
	and AETD derivatives from a simple conjugated diene ester using the conjugated asymmetric
	addition of a lithium amide, Sharpless asymmetric dihydroxylation and olefination as key steps. 418
	(-)-CSO-mediated	conjugate	asymmetric	addition	of	lithium
	(R)-N-benzyl-N-(α-methylbenzyl)amide 419 to the conjugated diene ester 93 provided the
	anti-α-hydroxy-β-amino ester 94 as a single diastereoisomer (dr > 99:1) (Scheme 15). The requisite
	stereochemistry at C 2 was obtained in a two-steps epimerization protocol by Swern oxidation of the
	hydroxyl group followed by diastereoselective reduction using NaBH 4 and yield the
	syn-α-hydroxy-β-amino ester 95. Then asymmetric dihydroxylation of the syn-α-hydroxy-β-amino
	ester 95 was performed under Donohoe's conditions (See § 4.3.2) through OsO 4 /TMEDA complex
	intermediate 96, 420-423 which was sequentially treated with P(CH 2 OH) 3 /SiO 2 to produce the lactone
	97 presenting all the requisite stereocenters as a single diastereoisomer in moderate yield.
	Three-steps functional group transformation by deprotection of the TIPS ether using HF•pyridine
	followed by one-pot hydrogenolysis of the N-protecting groups and protection of the resulting free
	amine by Boc 2 O gave the lactonetriol 99 which was unstable and was use fresh for next step.
	Subsequently, selective Mitsunobu reaction of the primary hydroxyl group of compound 99 with
	1-phenyl-1H-tetrazole-5-thiol (PTSH) by treatment with DEAD/PPh 3 gave the corresponding
	sulfide 100 quantitatively, although the product is contaminated with Ph 3 PO. The mixture was
	directly oxidized by treatment with (NH 4 ) 6 Mo 7 O 24 •H 2 O and H 2 O 2 to give the corresponding sulfone
	101 which was directly used in the next step without purification. Thus, treatment of the compound
	101 with 2,2-dimethoxypropane in the presence of p-TsOH produced the acetonide 102 as a single
	diastereoisomer (dr > 99:1 dr). Finally, Julia olefination of acetonide 102 with benzaldehyde or
	(E)-3-(4-ethoxyphenyl)acrylaldehyde allowed access to both protected APTO derivative 103 and
	protected AETD derivative 104.
	To summarize, the protected APTO and AETD derivatives were synthesized in 3.4 and 1.0%
	yields respectively over 14 steps from commercially available starting materials, using
	(-)-CSO-mediated conjugate addition for asymmetric aminohydroxylation, Sharpless asymmetric
	dihydroxylation and Julia olefination to introduce the styrene moiety. Scheme 15: Synthesis of protected APTO and AETD derivatives

4.3. Total synthesis of microsclerodermins 4.3.1. Ma's work: total synthesis of microsclerodermin E

4.3.1.5. Construction of the tetrapeptide derivative PyrAA

	-Sar-Trp-Gly
	Regioselective saponification of the α-methyl ester of dipeptide 130 and coupling with
	glycine trimethylsilylethyl ester gave the tripeptide 131 (Scheme 20). Boc-deprotection of the
	sarcosine residue by treatment with TFA produced a TFA salt which was directly coupled with the
	PFP-activated ester 126 to give the tetrapeptide 132 as a mixture of two diastereoisomers (ratio 4:1,
	from the partially racemized PFP-activated ester 126).
	Scheme 20: Preparation of the tetrapeptide derivative PyrAA-Sar-Trp-Gly

4.3.1.6. Construction of the hexapeptide and macrocyclization The tetrapeptide 132 was treated with TFA before coupling with the activated dipeptide 120

	Scheme 21: Construction of the hexapeptide and macrocyclization
	In summary, Ma reported the first total synthesis of a microsclerodermin family member:
	microsclerodermin E, which was obtained in 1% overall yield in 26 linear steps. It should be noted
	that the pyrrolidinoacetic acid unit is dehydrated making the synthesis relatively easier.
	by simple treatment with aqueous NaHCO 3 to provide the linear hexapeptide 133 in excellent yield
	(Scheme 21). Both TMSE and TBS protecting groups were removed using TBAF, then Staudinger
	reduction 434 of the intermediate azide using Me 3 P 435 gave a free amine. Macrocyclization of this
	linear hexapeptide mediated by diphenylphosphoryl azide (DPPA) 436 afforded the macrolactam 134
	in 40% yield after purification by HPLC. Finally, saponification of the methyl ester of the indole
	ring and cleavage of both the MOM ether and the acetonide with a mildly acidic resin
	Amberlyst-15 437 produced the target microsclerodermin E, whose analytical data were identical
	with those reported and thus confirming the structure proposed by Faulkner. 344

  3 •Et 2 O and Sánchez 478 who observed the deprotection with Me 2 S/BF 3 •Et 2 O or 1,3-propanedithiol/BF 3 •Et 2 O. Additionally, the keto group of the protected β-keto benzyl ester 177 is quite sterically hindered making its protection somewhat challenging.In summary, the use of the Cbz group to protect the amine function of aspartic acid was

compromised due to its sensitivity to 1,2-ethanedithiol/BF 3

•Et 2 O. Substrate 177 underwent extensive degradation and the desired dithioketal product was obtained only in low yield. It was decided to use a different protecting group.

  Based on Pascal's conditions491 and Sinha's report, 492 the Fmoc protected substrate 181 was treated with LiOH in the presence of CaCl 2 but no saponification occurred. Most of the starting material was recovered and it failed to give the acid 182 (Equation6). Replacing LiOH by NaOH gave the same results, and we observed complete degradation after prolonged reaction time. Even

	Scheme 41: Deprotection of methyl esters in the presence of a Fmoc group 495,496 gave similar results. Seeking an alternative, we used a weak base, potassium carbonate in a THF/water mixture, but once again most of the starting material was In 2019, Taylor the use of LiOH without CaCl 2 recovered.
	Equation 6: Methyl ester saponification attempts
	This method was applied by Sinha 492 in 2016 to N-Fmoc protected tripeptide esters which
	were efficiently hydrolyzed with LiOH and concentrated CaCl 2 in an isopropanol/THF/water
	mixture (Scheme 41). As a comparison, treatment of the N-Boc analog with LiOH gave the acid in a
	similar yield, demonstrating the efficiency of the method developed by Pascal.

[START_REF] Pascal | Preservation of the Fmoc protective group under alkaline conditions by using CaCl2. Applications in peptide synthesis[END_REF]

reported that C-terminal methyl esters could be easily removed without removing the Fmoc protecting group under alkaline conditions in the presence of CaCl 2 in an isopropanol/water mixture. The use of CaC1 2 dramatically increased the lifetime of the Fmoc protecting group and had little effect on the saponification processes with LiOH or NaOH. 493 reported the sequential transformation of a methyl ester into a primary amide on a N-Fmoc protected substrate by saponification of the methyl ester with LiOH and concentrated CaC1 2 in an isopropanol/THF/water mixture followed by activation of the resulting acid using BOP/HOBt/DIEA, and treatment with NH 4 Cl (Scheme 42). However, when the α-hydroxy group was replaced by a methoxy group, saponification of the methyl ester was very slow even at high CaCl 2 concentration, and did not work either with LiOH, trimethyltin hydroxide or AlCl 3 /DMA. Efficient deprotection was achieved in good yield by lithium iodide mediated dealkylation via lithium ion coordination,

[START_REF] Fisher | Iodide dealkylation of benzyl, PMB, PNB, and t-butyl N-acyl amino acid esters via lithium ion coordination[END_REF]

in degassed ethyl acetate at 80 °C. Scheme 42: Taylor's hydrolysis of N-Fmoc protected methyl esters

Table 4 :

 4 ). Optimization of the peptide coupling between acid 186 with H-Sar-Ot-Bu

	Entry	Conditions	Yield (%) a
	1	EDCI (1.1 eq.), DMAP (3.0 eq), CH 2 Cl 2 , r.t., 48 h	60%
	2	DIC (1.1 eq.), NMM (2.0 eq.), CH 2 Cl 2 , r.t., 8 h	~40% b + Urea
	3	HATU (1.5 eq.), DIEA (3.0 eq.), CH 2 Cl 2 /DMF, r.t., 8 h	61%
	4	PyBOP (1.1 eq.), DMAP (3.0 eq.), CH 2 Cl 2 , r.t., 64 h	69%
	5	PyBroP (1.1 eq.), DIEA (3.0 eq.), CH 2 Cl 2 , r.t., 20 h	95%
	Notes:

a Isolated yield; b Determined by 1 H NMR.

Table 5 :

 5 Optimization of amide formation from dipeptide 187

	1 H NMR. Using 28% NH 4 OH in a

3

for NMR experiment recording. The amino primary amide 188 appears to be unstable, which partially explained the dirty reaction and low yields obtained for long reaction times. However, making a balance between conversion and degradation appears very difficult.

  ).

	Entry	Conditions	Results/Yield a
	1	CAN (4.0 eq.), CH 3 CN/H 2 O (4:1) r.t., 3 h	Degradation Fmoc deprotection
	2	I 2 (7.0 eq.), NaHCO 3 (8.5 eq.), acetone/H 2 O (5:1) r.t., 48 h	Complex mixture Traces of 189
	3	PIFA (3.0 eq.), CH 3 CN/H 2 O (10:1) r.t., 15 min	Degradation
	4	HgO (3 eq.), HgCl 2 (2 eq.), CH 3 OH/H 2 O (9:1) r.t., 5 d.	No reaction 189 recovered
	5	HgO (8 eq.), HgCl 2 (12 eq.), CH 3 OH/H 2 O (9:1) r.t., 5 d.	Some degradation 189 60%
	6	HgO (8 eq.), HgCl 2 (12 eq.), THF/H 2 O (8:2) r.t., 24 h.	No reaction 189 93%
	7	HgO (8 eq.), HgCl 2 (12 eq.), CH 3 CN/H 2 O (10:1) r.t., 24 h	194 10% 195 60%
	8	NBS, AgNO 3 /Na 2 CO 3 , CH 3 CN/H 2 O (8:2) 0 ˚C, 10 min	Unknown product
	9	1. NBS, acetone, 0 ˚C, 40 min, then H 2 O 2. Very short flash on silica	192 44% 194 5%
	10	1.NBS, acetone, 0 ˚C, 40 min, then H 2 O 2. Silica, MeOH, r.t., 24 h	193 33% 194 5%
	11	1. NBS, acetone, 0 ˚C, 40 min, then H 2 O 2. Neutral aluminum chromatography	193 29% 194 traces
	12	1. NBS, acetone, 0 ˚C, 40 min, then H 2 O; 2. 4 M HCl, acetone, r.t., 24 h	194 40%
	Notes:

a Isolated yield.

Table 6 :

 6 Deprotection of dithioketal and spontaneous cyclization

Table 8 :

 8 (entry 5), full conversion of the Fmoc-amino acid 179 was observed and 42% yield of the -amino-β-ketoester 180 was obtained with an enantiomeric ratio of 96:4. When DTBMP was used (entry 6), reaction was slower and some starting material was observed. Although the intermediate Fmoc-UNCA was formed, no condensation product was obtained. Base screening for the preparation of the Fmoc-UNCA 204

		UNCA formation		Condensation
	Entry	Conditions	Results	Yield (%)	ee (%)
	1	Solution of NMM (2.2 eq.) in 0.5 mL of THF added at r.t. over 30 min then r.t., 2.5 h	τ ≈ 100% UNCA observed	56	88
	2 a	Solution of NMM (2.2 eq.) in 0.5 mL of THF added at 0 °C over 30 min then 0 °C, 2.5 h	τ > 95% UNCA observed	no product, 90% yield of 179 recovered
	3	Solution of NMM (2 eq.) in 0.5 mL of THF added at 0 °C over 5 min then r.t., 1.5 h	τ ≈ 90% UNCA observed	44	84
	4	Solution of 2,6-lutidine (2.2 eq.) in 0.5 mL of THF added at r.t. over 30 min then r.t., 2.5 h	τ > 98% UNCA observed	41	92
	5	Solution of sym-collidine (2.2 eq.) in 0.5 mL of THF added at r.t. over 30 min then r.t., 2.5 h	τ ≈ 100% UNCA observed	42	92
	6	Solution of DTBMP (2.2 eq.) in 0.5 mL of THF added at r.t. over 30 min then r.t., 4 h	τ ≈ 90% UNCA observed	no product, some SM
	Notes: a Addition performed at -20 °C.

2,6-lutidine was used (entry 4), 41% yield of the product 180 was obtained witg an enatiomeric ratio of 96:4. With sym-collidine

Table 9 :

 9 Optimization of the preparation of the Fmoc-UNCA 204 and condensation Retrospectively, in our results on the use of Fmoc-UNCA in the Masamune's procedure, and

		UNCA formation		Condensation
		Conditions	Results	Yield (%)	ee (%)
	1 a	Solution of sym-collidine (2.2 eq.) in 0.5 mL of THF added at 0 °C over 30 min then 0° C for 1 h, then r.t. for 3 h additional triphosgene (0.15 eq.), r.t. 1.5 h	τ > 95% UNCA observed	39 b 40 c	96 94
		Solution of sym-collidine (2.0 eq.) in 0.5 mL
	2	of THF added in two parts: 0.25 mL in one portion at 0 °C and 0.25 mL at r.t. over 30	τ > 95% UNCA observed	53	90
		min, then r.t., 1.5 h
	3	Solution of sym-collidine (2.0 eq.) in 0.5 mL of THF added in one portion at r.t. then r.t., 1.5 h	τ > 95% UNCA observed	49	88
		Solution of sym-collidine (2.2 eq.) in 0.5 mL	τ ≈ 90%
	4	of THF added at r.t. over 30 min	DBF	26	90
		then r.t., 1.5 h	UNCA observed
		sym-collidine (2.0 eq.), FmocCl (0.1 eq.),
	5	r.t., 3 h (no reaction) then additional triphosgene (0.3 eq.),	τ > 95% UNCA observed	52	90
		collidine (0.2 eq.), 3 h
	6	Solution of sym-collidine (2.0 eq.) in 0.5 mL of THF added at 0 °C over 5 min then r.t., 1.5 h	τ ≈ 90% UNCA observed	36	90
	7	Solution of sym-collidine (2.0 eq.) in 0.5 mL of THF added at 0 °C during 20 min then 0 °C, 40 min, then r.t., 2 h	τ ≈ 90% UNCA observed	36	86
	Notes:

a Solution of UNCA in THF was split in two part to run two condensation with magnesium hemimalonate. b Addition performed at -10 °C. c Addition performed at 0 °C. compared with the racemization results (See Chapter 2, §4.1., Scheme 38), it seems clear that racemization occurs during the activation stage with CDI in contrast to what was proposed by Ma and Zhu (See Chapter 2, §4.2., Scheme 39). 481

  Bu 216 in 67% yield; the efficiency was then improved by replacing PyBOP with PyBroP which gave 81% yield of the dipeptide 216. Chiral HPLC analysis of 216 revealed an enantiomeric excess of 94%. Final deprotection of the Fmoc group was initially performed under the common conditions using piperidine in dichloromethane, but it gave a modest

	50% yield as impure product. Alternative conditions were sought. Treatment of 216 with TBAF in
	the presence of benzylmercaptan in THF and 28% NH 4 OH in THF (1:1), gave 80% and 81% yield
	of the product 217, respectively. The best result was obtained by treatment with Et 2 NH (40 eq.) in
	MeOH which gave an excellent 90% yield of 217. Under these conditions, no degradation, no
	polymer of DBF and no adduct of DBF with Et 2 NH were formed and only DBF by-product was
	observed.
	Scheme 71: Enantioselective synthesis of dipeptide H-Trp(6-Cl)-Gly-Ot-Bu

Peptide coupling of 215 with glycine t-butyl ester with PyBOP in the presence of DIEA, gave the dipeptide Fmoc-Trp(6-Cl)-Gly-Ot-

Table 10 ,

 10 entry 1).388 Unfortunately, the reaction was sluggish and hardly gave the desired acetonide 63 when conducted under various common

	Entry	Conditions a	Results b
	1	Sikkon CaSO 4 , 2-MP (2.0 eq.),p-TsOH•H 2 O (0.1 eq.), DMF, 0 C, > 3 h	No reaction
	2	2,2-DMP/p-TsOH•H 2 O (0.1 eq.), DMF, -10 C, overnight	No reaction
	3	2-MP (1.3 eq.), PPTS•H 2 O (0.1 eq.), EtOAc, -10 C, > 6 h	No reaction
	4	CaSO 4 , 2-MP (1.3 eq.), PPTS•H 2 O (0.1 eq.), EtOAc, -10 C, > 6 h	No reaction
	5 c	2,2-DMP (1.3 eq.), PPTS•H 2 O (0.1 eq.), EtOAc, -10 C, > 6 h	Complex
	6 d	2-MP (1.3 eq.), PPTS (0.1 eq.), EtOAc, -10 C, > 6 h	66%
	7 d	2-MP (1.5 eq.), PPTS (0.1 eq.), EtOAc, -10 C, > 6 h	62%
	Notes:

a Conditions: dried DMF and EtOAc under argon; b Isolated yield or checked by TLC and 1 H NMR; c EtOAc not distilled; d PPTS was recrystallized in acetone.

Table 10 :

 10 Ketal protection of 2-deoxy-D-ribose conditions such as 2,2-dimethoxypropane/p-TsOH•H 2 O, 723 2-methoxypropene/PPTS•H 2 O, 724 CaSO 4 /2-methoxypropene/p-TsOH•H 2 O were tested with no more success (entries 2-6).

Table 12 :

 12 at -10 °C but 54% of dipeptide acid 235 and 38% of alcohol 236 were obtained (entry 4). An attempt to re-install the Boc group on alcohol 236 using Boc 2 O in the presence of DMAP failed and gave only a mixture of undefined products. Saponification of GABOB-APTO methyl ester with LiOH

	Entry	conditions	235	236
	1	1. LiOH (4 equiv.), MeOH/THF/H 2 O (0.2/0.2/0.3 mL), 0 C ~ r.t., 4 h 2. 10% citric acid until pH = 2-3 at 0 C	No reaction
	2	1. LiOH (10 eq.), MeOH/THF/H 2 O (0.2/0.2/0.3 mL), r.t., 16 h 2. 10% citric acid until pH = 2-3 at 0 C	71%	trace
	3	1. LiOH (10 eq.), MeOH/THF/H 2 O (0.2/0.2/0.3 mL), r.t., 16 h 2. 10% citric acid until pH = 2-3 at 5~10 C	0%	80%
	4	1. LiOH (10 eq.), MeOH/THF/H 2 O (0.2/0.2/0.3 mL), r.t., 16 h 2. citric acid (15 eq.) until pH = 3-4 at -10 C	54%	38%

  N-Cbz protection synthetic route was first attempted, but N-Cbz protected β-ketoester 177 was sensitive to 1,2-ethanedithiol/BF 3 •Et 2 O, and underwent extensive degradation; the expected dithioketal product 178 was obtained only in low yield (Scheme 37). We decided to replace the Cbz group by a Fmoc group. However, this failed to give optically pure β-ketoester 180 using

	Masamune's procedure with N-Fmoc protected substrate 179 (Scheme 38). Many other methods
	were tried, such as direct condensation of a Weinreb amide with a lithium enolate (Scheme 51),
	coupling with Meldrum's acid followed by treatment with benzyl alcohol (Scheme 53), use of
	N-Phth protected substrate in the Masamune's procedure (Scheme 54), Mukaiyama aldol
	condensation followed by oxidation (Scheme 55) and homologation of an aldehyde through the
	Roskamp reaction (Scheme 57). The optically pure β-ketoester was finally obtained successfully via

condensation of N-Fmoc protected UNCA with magnesium hemimalonate successfully.

1 H

 1 NMR (360 MHz, D 2 O) δ 2.87 (dd, 1H, J = 6.1, 1.8 Hz, ABX), 3.71 (s, 3H), 4.34 (ddd pseudo td, 1H, J = 6.1, 1.8 Hz, ABX).

	13 C NMR (90 MHz, D 2 O) δ 33.7, 49.2, 52.8, 170.8, 171.8.
	HRMS (ESI+) Calcd for C 5 H 10 NO 4 [M + H] + : 148.0605, found 148.0604.
	(S)-2-(((Benzyloxy)carbonyl)amino)-4-methoxy-4-oxobutanoic acid 176 2
	Following Reddy's procedure 474

  (ESI+) Calcd for C 22 H 23 NNaO 7 [M + Na]

+ : 436.1355, found 436.1366.

  (ESI+) Calcd for C 31 H 31 NNaO 6 S 2 [M + Na]

+ : 600.1485, found 600.1498. HPLC: 28% ee. sample prepration: 1 mg/mL in MeOH, Column 1: A_NP_ChiralpakIC (250 mm × 4.6 mm), normal phase: hexane/EtOH = 60/40, flow: 1 mL/min, 30 °C; injection: 5 L; results: RT = 22.51 (S, Area: 63.8%), 16.044 (R, Area: 36.2%).

  to give the organic layer, which was dried over MgSO 4 and concentrated under reduced pressure. Flash chromatography on silica gel (MeOH/CH 2 Cl 2 = 5/95, 10/90) gave β-dithioketal acid 186 (416.7 mg, 85%) as a white solid.

	TLC: R f = 0.38 (MeOH/CH 2 Cl 2 = 10/90)
	Mp. 81-83 °C
	IR (ATR) ν 3296, 2950, 1706, 1510, 1451, 1435, 1330, 1282, 1224, 1174, 1050, 739 cm -1
	1 H NMR (360 MHz, CDCl 3 ) δ 2.64 (dd, 1H, J = 14.8, 9.7 Hz), 3.01-3.10 (m, 2H), 3.18-3.23 m, 5H),
	3.61 (s, rotamer) and 3.63 (s, 3H), 420 (t, J = 6.8 Hz), 4.41 (d, J = 6.8 Hz), 4.86 (ddd pseudo td, 1 H,
	J = 10.1, 9.7, 3.6 Hz) and 4.97 (pseudo t, rotamer), 5.40 (d, J = 10.1 Hz) and 5.80 (d, rotamer),
	7.28-7.40 (m, 4H), 7.56-7.66 (m, 4H), 7.73-7.75 (m, 2H).

13

C NMR (90 MHz, CDCl 3 ) δ

37.5 and 380.0 (rotamer), 39.7 and 40.1 (rotamer), 40.6 (rotamer)

and

40.7, 46.8, 47.1 (rotamer) and 47.3, 52.2, 55.4 and 55.9 (rotamer), 67.1 and 68.6 (rotamer), 71.1, 120.0 (2C), 125.1, 125.2, 127.2 (2C), 127.8 (2C), 141.4 (2C), 143.8, 144.0, 156.3, 171.7, 174.0

.

HRMS (ESI+

) Calcd for C 24 H 25 NNaO 6 S 2 [M + Na] + : 510.1015, found 510.5992.

  ABX), 6.00 (d, 1H, J = 10.1 Hz) and 6.04 (d, 1H, J = 10.1 Hz), 7.29-7.33 (m, 2H) , 7.39 (t, 2H, J = 7.2 Hz), 7.61 (d, 2H, J = 7.2 Hz), 7.75 (d, 2H, J = 7.2 Hz). HRMS (ESI+) Calcd for C 31 H 38 N 2 NaO 7 S 2 [M + Na] + : 637.2013, found 637.2028.

	, 741
	cm -1
	1 H NMR (360 MHz, CDCl 3 ) δ 1.45 (s, 9H) and 1.47 (s, rotamer), 2.70-2.78 (dd pseudo m, 1H, with
	rotmer, ABX ), 2.98-3.08 (m, 4H), 3.17 (d, 1H, J = 16.2 Hz), 3.17 (brs, 4H), 3.29 (d, 1H, J = 16.2
	Hz), 3.65 (s, 3H), 3.90 (d, 1H, J = 5.4 Hz) and 3.93 (d, 1H, J = 5.4 Hz, rotamer), 4.14-4.34 (m, 4H),
	4.80-4.86 (ddd pseudo m, 1H,

  ABX),2.31-2.40 (dd pseudo d, 1H, J = 15.5 Hz, ABX), 2.93 (s, rotamer) and 3.08 (s, 3H),3.23-3.39 (m, 6H, mixed with brs), 3.92-4.05 (m, 3H), 6.1 (brs, 1H) and 6.16 (brs, rotamer), 7.30 (brs, 1H).

1 H NMR (360 MHz, CDCl 3 ) δ 1.45 (s, 9H) and

1.48 (s, rotamer), 2.31-2.40 (dd pseudo m, 1H, with rotamer,

  Hz),3.85 (d, 1H, J = 16.9 Hz), 4.22 (d, 1H, J = 16.9 Hz), 4.18 (t, 1H, J = 6.8 Hz), 4.31-4.41(m, 2H), 4.75-4.84 (ddd pseudo m, 1H) and 4.95-5.01 (m, rotamer), 5.63 (brs, rotamer) and 5.75 (brs, 1H), 5.81 (dd, rotamer) and 6.32 (dd, 1H, J = 10.1 Hz), 6.55 (brs, 1H) and 6.85 (brs, rotamer), 7.30 (t, 2H, J = 7.2 Hz) , 7.37 (t, 2H, J = 7.2 Hz), 7.60 (t, 2H, J = 7.2 Hz),7.74 (d, 2H, J = 7.2 Hz).

	, 729
	cm -1

1 H NMR (360 MHz, CDCl 3 ) δ 1.43 (s, 9H) and

1.46 (s, rotamer)

, 2.49 (dd, 1H, J = 14.4, 10.8 Hz, ABX ), 2.91-3.04 (m, 4H), 3.17 (d, 1H, J = 16.9 Hz), 3.17 (pseudo brs, 4H),

3.30 (d, 1H, J = 16.9

  (ESI+) Calcd for C 8 H 15 N 2 O 3 S 2 [M + H] + : 251.0519, found 251.0516. HRMS (ESI-) Calcd for C 8 H 13 N 2 O 3 S 2 [M -H] -: 249.0373, found 249.0372.

  and 202.6.

	HRMS (ESI+) : Calcd for C 28 H 33 N 3 NaO 7 [M + Na] + : 546.2211, found 546.2212.

  and brine(30 mL× 2). The organic layer was dried with Mg 2 SO 4 , filtered, and concentrated under reduced pressure. Flash chromatography on silica gel with a normal speed (EtOAc/petroleum ether = 50/50, EtOAc = 100%, EtOAc/MeOH = 98/2, 95/5,

	90/10) gave a mixture of γ-keto primary amide 192 and cyclization product 193 (35.0 mg, 33%) as
	a colorless oil, as well as the dehydrated 194 (5.0 mg, 5%) as a colorless oil.
	Step2: The mixture was stirred with silica (400 mg) in MeOH (5 ml) for 24 h at room temperature,
	then evaporated. Flash chromatography on silica gel (EtOAc/MeOH = 98/2, 95/5, 90/10) gave a
	cyclization product 193 (34.0 mg, 33%) as a colorless oil.
	TLC: R f = 0.67 (EtOAc/MeOH = 90/10)
	IR (ATR) ν 3300

  HRMS (ESI+) Calcd for C 28 H 33 N 3 NaO 7 [M + Na] + : 546.2211, found 546.2191.

	1 (2C), 125.2 and 125.3 (2C),
	127.0 (2C), 1227.6 (2C), 140.7 (2C), 143.7 and 143.8 (2C), 156.0, 168.2 and 168.4 (rotamer), 170.7
	and 170.9 (rotamer), 173.1.

Table 6 ,

 6 entry 12:

  over MgSO 4 , filtered, and concentrated under reduced pressure. Flash chromatography on

	silica gel (MeOH/CH 2 Cl 2 = 90/10) gave a phthalimido derivative 199 (1.246 g, 90%) as a white
	solid.
	TLC: R f = 0.49 ((MeOH/CH 2 Cl 2 = 80/20)
	Mp. 132-133 °C
	[α] D 23 = -62.7 (c 0.53, CHCl 3 )

  cm -1 1 H NMR (360 MHz, CDCl 3 ) δ 2.87 (dd, 1H, J = 17.3, 4.7 Hz, ABX),3.00 (dd, 1H, J = 17.3, 4.7 Hz,

	ABX), 3.69 (s, 3H), 4.22 (t, 1H, J = 6.5 Hz), 4.41-4.47 (m, 3H), 6.01 (d, 1H, J = 7.9 Hz), 7.30-7.34
	(m, 2H), 7.41 (t, 2H, J = 7.2 Hz ), 7.60 (d, 2H, J = 7.2 Hz), 7.77 (d, 2H, J = 7.2 Hz), 9.63 (s, 1H).
	13 C NMR (90 MHz, CDCl 3 ) δ 34.1, 47.1, 52.2, 56.4, 120.1 (2C), 125.0 (2C), 127.1 (2C), 127.8
	(2C), 141.3 (2C), 143.6 (2C), 156.2, 171.5, 198.6.
	HRMS (ESI+) Calcd for C 20 H 19 NNaO 5 [M + Na] + : 376.1155, found 376.1141.
	6-Benzyl	1-methyl
	(3S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-hydroxyhexanedioate 203
	Mukaiyama aldol: To a solution of N-Fmoc aldehyde 202 (200.0 mg, 0.57 mmol, 1 eq.) in CH 2 Cl 2
	(6 mL) under argon was dropwise added BF 3 •Et 2 O (0.21 mL, 1.71 mmol, 3 eq) at -78 °C over 30
	min, and followed by addition of ((1-(benzyloxy)vinyl)oxy)trimethylsilane (507.0 mg, 2.28 mmol,
	4 eq.). The resulting mixture was stirred for overnight and slowly warmed to room temperature. The
	reaction was quenched with water (2 mL), filtered through a Celite pad and washed with CH 2 Cl 2

  (ESI+) Calcd for C 29 H 27 NNaO 7 [M + Na] + : 524.1680, found 524.1663.

	HPLC: 93% ee. sample prepration: 1 mg/mL in MeOH,; column: A_NP_5-AmyCoat (250 mm ×
	4.6 mm), normal phase: hexane/EtOH = 60/40, flow: 1 mL/min, 30 °C; injection: 5 L; results: RT
	= 21.822 (R, Area: 3.4%), 26.681 (S, Area: 96.6%).

  3.19-3.27 (m, 5H), 3.63 (s, 3H), 4.22 (t, 1H, J = 6.8 Hz), 4.41 (d, 2H, J = 6.8 Hz), 4.88 (ddd pseudo brs, 1H, ABX),5.43 (brs, 1H) and 5.82 (brs, rotamer), 7.30 (t, 2H, J = 7.2 Hz), 7.37 (t, 2H, J = 7.2

	Hz), 7.59 (m pseudo brs, 2H), 7.73 (t, 2H, J = 7.2 Hz), 9.02 (brs, 1H).

  ABX), 2.78 (dd, J = 16.6, 2.9 Hz, rotamer, ABX) and 2.99 (dd, 1H, J = 16.6, 2.9 Hz, ABX),

	-1
	1 H NMR (360 MHz, D 2 O) δ 2.48 (dd, J = 16.6, 10.4 Hz, rotamer, ABX) and 2.65 (dd, 1H, J = 16.6,
	10.4 Hz,

  HRMS (ESI+) Calcd for C 30 H 37 N 3 NaO 6 S 2 [M + Na] + : 622.2016, found 622.1993.

	HPLC: 80% ee. sample prepration: 1 mg/mL in MeOH,; column: A_RP_5-CelluloseCoat (250 mm
	× 4.6 mm), normal phase: CH 3 CN = 100%, flow: 1 mL/min, 5 °C; injection: 5 L; results: RT =
	5.265 (R, Area: 10%), 6.247 (S, Area: 90%).
	(R)-2-Amino-4-methoxy-4-oxobutanoic acid hydrochloride (R)-175
	Following Reddy's procedure 474
	758,
	742 cm -1
	1 H NMR (360 MHz, CDCl 3 ) δ 1.41 (s, 9H) and 1.43 (s, rotamer), 2.39 (dd, rotamer, ABX) and 2.50
	(dd, 1H, J = 14.4, 10.1 Hz, ABX ), 2.89-3.00 (m, 4H), 3.11 (d, 1H, J = 16.6 Hz), 3.17 (pseudo brs,
	4H), 3.26 (d, 1H, J = 16.6 Hz), 3.82 (d, 1H, J = 16.9 Hz), 4.12 (d, 1H, J = 16.9 Hz), 4.18 (t, 1H, J =
	7.2 Hz), 4.24-4.36 (m, 2H), 4.74-4.85 (ddd pseudo m, 1H, ABX) and 5.01-5.07 (m, rotamer), 5.78
	(brs, rotamer) and 5.88 (brs, 1H), 6.32 (dd, 1H, J = 10.1 Hz), 6.62 (brs, 1H) and 6.64 (brs, rotamer),
	7.23-7.26 (m, 2H) , 7.34 (t, 2H, J = 7.2 Hz), 7.57 (t, 2H, J = 7.2 Hz), 7.69 (d, 2H, J = 7.2 Hz).
	13 C NMR (90 MHz, CDCl 3 ) δ 28.0 (3C), 35.2 (rotamer) and 36.9, 39.2 (2C), 40.1, 45.2, 47.1, 50.5

and 52.7 (rotamer), 56.1(rotamer) and 56.2, 66.8, 72.3 and 72.4 (rotamer), 81.9 and 82.7 (rotamer), 119.8 (2C), 125.2, 125.3, 127.0 (2C), 127.5 (2C), 141.1 (2C), 143.8, 144.0, 156.8, 167.8 (rotamer)

and

168.2, 170.4, 170.5, 173.1.

  ESI+) Calcd for C 20 H 19 NNaO 6 [M + Na] + : 392.1105, found 392.1100.

	13 C NMR (90 MHz, CDCl 3 ) δ 28.0 (3C), 35.2 (rotamer) and

  CDCl 3 ) δ 2.29 (s, 6H),3.60 (s, 2H), 6.95 (d, 1H, J = 1.8 Hz ), 7.06 (dd, 1H, J = 10.1, 1.8 Hz),7.20 (d, 1H, J = 1.4 Hz), 7.57 (d, 1H, J = 8.3 Hz), 9.32 (brs, 1H).

	795,
	422 cm -1
	1 H NMR (360 MHz,

  3 •Et 2 O (80 L 0.06 mmol, 0.01 eq.), and a filtered solution of step(1). The resulting mixture was refluxed (oil bath, 135 C) for overnight(16 h), then cooling down and evaporated under reduced pressure. Flash chromatography on silica gel (ether/petroleum ether  10/90, 20/80, 30/70, 50/50) gave the desired Gly-pinanone imine 20 (950.3 mg, 57%) as a pale-yellow oil.

	TLC: R f = 0.30 (ether/petroleum ether = 30/70)
	[α] D 25 = +11.1 (c 1.48, CHCl 3 )
	IR (ATR) ν 3434, 2979, 2920, 1737, 1655, 1475, 1368, 1153 cm -1
	1 H NMR (360 MHz, CDCl 3 ) δ 0.84 (s, 3H), 1.29 (s, 3H), 1.45 (s, 9H), 1.48 (s, 1H), 154 (d, 1H),
	1.97-2.06 (m, 2H), 2.37-2.34 (m ,1H), 2.38-2.51 (m, 2H), 2.76 (brs, 1H), 4.00-4.10 (m, 2H).

-3-(6-chloro-1H-indol-3-yl)-2-(((1S,2S,5S,E)-2-hydroxy-2,6,6-trimethylbicyclo[3.1.

	tert-Butyl(R)
	3,
	179.7
	HRMS (ESI+) Calcd for C 16 H 27 NNaO 3 [M + Na] + : 304.1883, found 304.1872.

fluoren-9-yl)methoxy)carbonyl)amino)-3-(6-chloro-1H-indol-3-yl)propanoate

  ESI+) Calcd for C 15 H 20 ClN 2 NaO 2 [M + Na] + : 317.1027, found 317.1019.To an ice cold solution of H-Trp(6-Cl)-Ot-Bu 213(80 mg, 0.271 mmol, 1 eq,) in THF(5 mL) in ice bath was added FmocCl(77.6 mg, 10.30 mmol, 1.1 eq.), and Na 2 CO 3(46.0 mg, 0.43 mmol, 1.6 eq.) subsequently. The resulting mixture was stirred at room temperature for 24 h, then evaporated and diluted with water, extracted with EtOAc (30 mL × 3). The combined organic phase was washed with brine and dried over MgSO 4 , filtered, concentrated under reduced pressure. Flash chromatography on silica gel (petroleum ether/EtOAc = 90/10, 80/20, 70/30) gave the desired dipeptide Fmoc-Trp(6-Cl)-Ot-Bu (140.2 mg, 100%,  94% ee) as a white solid.

	tert-Butyl
	(R)-2-((((9H-Mp = 74-78 °C
	[α] D 25 = -9.6 (c 0.50, CHCl 3 )
	IR (ATR) ν 3413, 3333, 2977, 1699, 1620, 1511, 1450, 1368, 1335, 1226, 1151, 1060, 738 cm -1
	1 H NMR (360 MHz, CDCl 3 ) δ 1.41 (s, 9H), 3.20-3.32 (m, 2H), 4.19 (t, 1H, J = 7.2 Hz), 4.35-4.44
	(m, 2H), 4.62-4.67 (m, 1H), 5.46 (d, H, J = 7.9 Hz), 6.84 (d, 1H, J = 1.4 Hz), 7.09 (dd, 1H, J = 8.6
	Hz, 1.8 Hz ),

TLC: R f = 0.33 (petroleum ether/EtOAc = 80

/20)

fluoren-9-yl)methyl)amino)-3-(6-chloro-1H-indol-3-yl)propanoyl)glycinate 216

  To a solution of Fmoc-Trp(6-Cl)-OH 215 (70.0 mg 0.15 mmol, 1 eq.) in CH 2 Cl 2 (3 mL) at room temperature under argon was added PyBroP (77.0 mg, 0.17 mmol, 1.1 eq.) and H-Gly-Ot-Bu hydrochloride (27.6 mg, 0.17 mmol, 1.1 eq.), subsequently. Followed by addition of DIEA (77.5 mg, 0.60 mmol, 4 eq.) dropwise. The resulting mixture was stirred at room temperature for overnight (>16 h). The reaction was then diluted with CH 2 Cl 2(20 mL), and then washed with 1 M HCl (20mL), sat. NaHCO 3(20 mL) and sat. NaCl(20 mL), respectively. The combined organic phase was dried over MgSO 4 , then filtered and concentrated under reduced pressure. Flash chromatography on silica gel (petroleum ether/EtOAc = 80/20, 70/30, 60/40, 50/50) gave peptide

	tert-Butyl
	(R)-(2-(((9H-TLC: R f = 0.35 (petroleum ether/EtOAc = 60/40)
	Mp = 163-166 °C
	[α] D 27 = +9.5 (c 0.50, CHCl 3 )
	IR (ATR) ν 3437, 3299, 1731, 1695, 1651, 1529, 1451, 1258, 1234, 1161, 808, 757, 739 cm -1
	1 H NMR (360 MHz, CDCl 3 ) δ 1.42 (s, 9H), 3.17 (dd, 1H, J = 13.3, 6.8 Hz, ABX), 3.17 (dd, 1H, J =
	.1 (2C), 13.3, 9.7 Hz, ABX), 3.73-3.87 (m, 2H,), 4.18 (1H, t, J = 6.8 Hz), 4.39-4.46 (m, 2H), 4.50 (pseudo
	120.4, 123.5, 125.2 (2C), 126.5, 127.2 (2C), 127.8 (2C), 128.0, 136.5, 141.3 (2C), 143.8 (2C), brs, 1H, ABX'), 5.47 (brs, 1H), 6.20 (brs, 1H), 7.06 (s, 1H), 7.08 (dd, 1H, J = 8.6 Hz , J = 1.4 Hz ),
	155.9, 171.1.
	HRMS (ESI+) Calcd for C 30 H 29 ClN 2 NaO 4 [M + Na] + : 539.1708, found 539.1691.
	HPLC: 94% ee. Fmoc-Trp(6-Cl)-Ot-Bu sample prepration: 1 mg/L in EtOH,; column:
	A_NP_Lux Cellulose-2 (250 mm ×4.6 mm), normal phase: hexane/EtOH = 80/20, flow: 1 mL/min,

30 °C; injection: 5 L; results: RT = 8.025 (R, Area:97%), 9.467 (S, Area: 3%).

Racemic one: sample prepration: 1 mg/L in MeOH: column: A_NP_Lux Cellulose-1 (250 mm ×4.6 mm), normal phase: hexane/EtOH = 80/20, flow: 1 mL/min, 30 °C; injection: 5 L; results: RT = Fmoc-Trp(6-Cl)-Gly-Ot-Bu 216 (69.6 mg, 81%, > 94% ee) as a white solid.

  Fmoc-Trp(6-Cl)-Gly-Ot-Bu 215 sample prepration: 1 mg/mL in MeOH,; column: A_NP_Lux Cellulose-2 (250 mm × 4.6 mm), normal phase: hexane/EtOH = 70/30, flow: 1 mL/min, 30 °C; injection: 2 L; results: RT = 7.309 (R, Area: 97.2%), 13.706 (S, Area: 2.8%).

	Racemic ()-215: 0% ee. sample prepration: 1 mg/L in MeOH: column: A_NP_Lux Cellulose-2
	(250 mm × 4.6 mm), normal phase: hexane/EtOH = 70/30, flow: 1 mL/min, 30 °C; injection: 5 L;
	results: RT = 7.391 (R, Area: 50%), 13.706 (S, Area: 50%).
	tert-Butyl (R)-(2-amino-3-(6-chloro-1H-indol-3-yl)propanoyl)glycinate 217
	(MeOH/CH 2 Cl 2 = 5/95, 10/90) gave the desired H-Trp(6-Cl)-Ot-Bu 215 (31.0 mg, 90%) as a white
	solid.
	TLC: R f = 0.36 (MeOH/CH 2 Cl 2 = 5/95)
	Mp = 128-131 °C
	[α] D 27 = 21.1 (c 0.50, CHCl 3 )
	IR (ATR) ν 3311, 2978, 2929, 2854, 1736, 1656, 1524, 1456, 1366, 1234, 1157 cm -1
	1 H NMR (360 MHz, CDCl 3 ) δ 1.47 (s, 9H), 1.77 (brs, 2H), 2.93 (dd, 1H, J = 14.4, 9.0 Hz, ABX),
	3.34 (dd, 1H, J = 14.4, 4.0 Hz, ABX), 3.72 (dd, 1H, J = 9.0, 4.0 Hz, ABX), 3.93 (dd, 2H, J = 14.4,
	4.0 Hz, ABX), 7.08 (d, 1H, J = 6.8 Hz ), 7.07 (d, 1H, J = 6.8 Hz ), 7.08 (d, 1H, J = 1.8 Hz ), 7.35 (d,

, 126.2, 127.2 (2C)

,

127.8 (2C)

,

128.2, 136.6, 141.4 (2C), 143.8 (2C), 156.2, 168.6, 171.4

.

HRMS (ESI+) Calcd for

C 32 H 32 ClN 3 NaO 5 [M + Na] + : 596.1923, found 596.1895. HPLC: > 94% ee.

To a solution of Fmoc-Trp-Gly(6-Cl)-Ot-Bu 216 (57.4 mg, 0.10 mmol, 1 eq.) in MeOH

(5 mL)

was added diethylamine (146.2 mg, 2.00 mmol, 20 eq.) dropwise at room temperature. After stirred for 24 h, the mixture was concentrated under reduced pressure. Flash chromatography on silica gel 1H, J = 1.8 Hz ),

7.55 (d, 1H, J = 8.6 Hz ), 7.73 (t, 1H, J= 5.0m 4.7 Hz), 8.34 (brs, 1H)

.

  ESI+) Calcd for C 17 H 22 ClN 3 NaO 3 [M + Na] + : 374.1242, found 374.1231. Cl 2(10 mL), washed with water (30 mL). The organic phase was dried over MgSO 4, filtered and evaporated under reduced pressure. Flash chromatography on silica gel (petroleum ether/EtOAc = 95/5) gave the desired imine 218 (1.7701 g, 100%) as a white solid.

	tert-Butyl 2-((diphenylmethylene)amino)acetate 218 717
	Following Fanelli's procedure 717
	To a solution of H-Gly-Ot-Bu hydrochloride (1.0058 g, 6.00 mmol, 1 eq.) in CH 2 Cl 2 (20 mL) was
	added diphenylmethanimine (1.0874 mg, 6.00 mmol, 1 eq.). After stirred for overnight (16 h), then
	diluted with CH 2

TLC: R f = 0.26 (petroleum ether/EtOAc = 98/2) 1 H NMR (360 MHz, CDCl 3 ) δ 1.46 (s, 9H), 4.12 (s, 2H),

7.17-7.19 (m, 2H), 7.30-7.39 (m ,6H)

,

  , slowly warmed to room temperature, then quenched with sat. NH 4 Cl(25 mL) in ice bath and extracted with EtOAc (20 mL× 3), The combined organic phase was then washed with brine and dried over MgSO 4 , then filtered and evaporated under reduced pressure.

	Flash chromatography on silica gel (petroleum ether/EtOAc = 90/10, 80/20, 70/30, 60/40) gave the
	desired imine (±)-219 (770.3 mg, 84%) as a white solid.
	TLC: R f = 0.40 (petroleum ether/EtOAc = 50/50)
	Mp = 61-63 °C
	IR (ATR) ν 3422, 3349, 2977, 1717, 1618, 1455 1446, 1367, 1148, 694 cm -1
	1 H NMR (360 MHz, CDCl 3 ) δ 1.39 (s 1H), 3.18 (dd, 1H, J = 14.4, 8.6 Hz, ABX), 3.33 (dd, 1H, J =
	14.4, 4.3 Hz, ABX), 4.24 (dd, 1H, J = 8.6, 4.3 Hz, ABX), 6.59 (d, 2H, J = 6.8 Hz ), 6.87 (s, 2H),
	7.11-7.16 (m, 3H), 7.20-7.37 (m, 5H), 7.55 (d, 2H, J =7.7 Hz), 8.32 (brs, 1H).

  ESI+) Calcd for C 28 H 28 ClN 2 O 2 [M + H]

+ : 459.1834, found 459.1817.

  cm -1 1 H NMR (360 MHz, CDCl 3 ) δ 1.44 (s, 9H),1.70 (brs, 2H), 2.93 (dd, 1H, J = 14.4 Hz, J = 7.9 Hz,

	ABX), 3.21 (dd, 1H, J = 14.4, 4.7 Hz, ABX), 3.71 (dd, 1H, J = 7.9, 4.7 Hz, ABX), 6.91 (d, 1H, J =
	1.8 Hz), 7.05 (dd, 1H, J = 8.6, 1.8 Hz ), 7.22 (d, 1H, J = 1.4 Hz ), 7.50 (d, 1H, J = 8.3 Hz ), 9.19
	(brs, 1H).

  , 156.2, 176.2. HRMS (ESI+) Calcd for C 15 H 31 NNaO 5 Si [M + Na] + : 356.1864, found 356.1862.

HRMS (ESI-) Calcd for C 15 H 30 NO 5 Si [M -H] -: 332.1899, found 332.1890.

  1081 cm -1 1 H NMR (360 MHz, CDCl 3 ) δ 1.14 (s, 9H), 1.43 (s, 6H), 1.50 (s, 9H), 2.54-2.62 (m ,1H, ABX), 2.66-2.73 (m, 1H, ABX), 3.56 (d, 1H, J =10.1 Hz), 3.71 (s, 3H), 3.85 (dd, J = 8.3, 5.4 Hz), 3.95 (1H, J = 10.1, 8.3, 1.8 Hz), 4.50 (ddd, 1H, J = 10.1, 5.4, 5.4 Hz, ABX), 5.31 (d, 1H, J = 1.8 Hz), 6.31 (ddd pseudo dt, 1H, J =

  .9 HRMS (ESI+) Calcd for C 27 H 42 NO 8 S [M + H] + : 540.2626, found 540.2625. overnight (20 h), diluted with CH 2 Cl 2

  . The organic layer was dried over MgSO 4 , filtered, and concentrated under reduced pressure. Flash chromatography on silica gel (petroleum ether/EtOAc = 90/10, 80/20, 70/30) gave compound 227 (6.2 mg, 10%) as a colorless oil. TLC: R f = 0.22 (petroleum ether/EtOAc = 80/20) 1 H NMR (360 MHz, CDCl 3 ) δ 0.88 (s, 3H), 0.15 (s, 3H), 0.89 (s, 9H), 2.49-2.64 (m, 2H, ABX), 3.75-3.79 (m, 1H, ABX), 4.17

  (ESI+) Calcd for C 26 H 29 N 3 NaO 6 S 2 [M + Na] + : 566.1390, found 566.1378. HRMS (ESI-) Calcd for C 26 H 29 N 3 NaO 6 S 2 [M -H] -: 542.1391, found 542.1403.

  , 839 cm -1 1 H NMR (400 MHz, CDCl 3 ) δ 0.10 (s, 3H), 0.13 (s, 3H), 0.90 (s, 9H),1.35 (s, 3H),1.43 (s, 21H),1.50 (s, 9H), 2.36-2.68 (m, 5H), 2.89 (s, 3H), 2.97-3.06 (m, 2H), 3.17-3.34 (m, 9H), 3.66-3.81 (m, 3H), 3.90-4.02 (2H), 4.12-4.31 (m, 2H). 4.64-4.67 (m, 1H), 4.75-4.81 (m, 1H), 4.83-4.87 (m, 1H), 5.06 (brs, 1H), 5.24 (s, 1H), 6.07-6.14 (m, 1H),6.37 (d, 1H, J = 16.0 Hz ), 7.04-7.13 (m, 3H), 7.19-7.23 (m, 3H), 7.26-7.36 (m, 5H), 7.57 (d, 1H, J = 8.4 Hz), 7.93 (brs, 1H), 8.61 (brs, 1H).HRMS (ESI+) Calcd for C 65 H 97 ClN 8 NaO 16 S 2 Si [M + Na] + : 1395.5814, found 1395.5774.

eluted through a neutral alumina column, this time providing the desired PyrAA dipeptide 193 in 29% yield accompanied by only traces of dehydrated dipeptide 194 (entry 11). In order to obtain sufficient quantities of reference material, a sample of crude NBS reaction product was treated with

H NMR (400 MHz, 323 K, CDCl 3 ) δ

2.64 (dd, 1H, J = 14.8, 10.0 Hz, ABX), 2H),

C NMR(90 MHz, CDCl 3 ) δ 28.0 (4C),47.2, 55.1, 67.1, 82.4, 110.6, 111.2, 119.9, 120.1 (2C),

ACKNOWLEDGEMENTS

First, I would like to thank Dr Catherine GUILLOU who accepted to be the president of my jury, Dr Sophie FAURE and Dr Grégory CHAUME for accepting to be the reporters for my PhD thesis. Thanks also to Prof Peter GOEKJIAN for accepting to be the examiner of my thesis.

scientific research, thesis writing and revision, even application on scholarship from Chinese Scholarship Council and life in France.

2-Carboxytryptophan was prepared according to Danishefsky's protocol. 433 Thus, cyanation of the protected D-tryptophan derivative 127 was performed by chlorination using t-BuOCl followed by treatment with TMSCN in the presence of BF 3 •Et 2 O to furnish the cyanotryptophan derivative 128 (Scheme 19). Hydrolysis of the cyano group followed by sequential esterification gave the D-tryptophan derivative 129 in 56% over 4 steps. After removal of both N-benzyl groups by hydrogenolysis, the resulting amino ester was engaged in a peptide coupling with N-Boc-sarcosine to provide the dipeptide Boc-Sar-Trp(2-CO 2 Me)-OMe in 67% yield over 2 steps. Scheme 19: Synthesis of the dipeptide Sar-Trp CHAPTER 3

SYNTHEIS OF DIPEPTIDE Trp(6-Cl)-Gly

General Conditions

All reagents and solvents were of commercial grade and were used without further purification, with the exception of CH 2 Cl 2 which was dried over activated alumina, THF which was distilled from sodium/benzophenone, DIEA and DIPA which were distilled on CaH 2 and PPTS which was recrystallized in acetone. LDA was prepared and freshly used.

All reactions requiring anhydrous conditions were performed under argon in oven drying glassware. Organic extraction was dried over MgSO 4 or NaSO 4 before evaporation under reduced pressure.

Flash chromatography was performed on columns of silica gel (40-63 m) or Aluminium oxide (neutral activated, 50-200 mesh). Analytical thin-layer chromatography (TLC) was carried out on commercial 0.25 mm silica gel plates which were visualized by UV fluorescence at 254 nm then revealed using a phosphomolybdic acid solution (10% in EtOH), ninhydrin solution (0.3% in n-BuOH), acidic p-anisaldehyde solution (5% in EtOH) or KMnO 4 solution (7.5% in water). Splitting patterns for 1 H signals are designated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), brs (broad single) and coupling constants (J) are reported in Hz. To a solution of L-N-Fmoc-Asp(β-OMe)-H (S)-179 (250.0 g, 0.67 mmol, 1 eq.) in CH 2 Cl 2 (8 mL) under argon at room temperature was added N,O-dimethylhydroxylamine hydrochloride (17.8 mg, 0.98 mmol, 1.1 eq.), PyBOP (356.0 mg, 0.68 mol, 1 eq) and dropwise DIEA (218.7 mg, 1.69 mmol, 2.5 eq.), respectively. The resulting mixture was stirred at room temperature for overnight (16 h).

Retention factors (R

Then diluted with EtOAc (30 mL) and subsequently washed with 1 M HCl (20 mL), sat. NaHCO 3 (20 mL) and brine (20 mL) to give the organic layer, which was then dried over MgSO 4 , filtered and concentrated under reduced pressure. Flash chromatography on silica gel (petroleum ether/EtOAc = 80/20, 90/10) to give the pure desired Weinreb amide 196 (143.0 mg, 52%) as a white solid.

TLC: R f = 0.45 (petroleum ether/EtOAc = 80/20) 1 H NMR (30 MHz, CDCl 3 ) δ 2. 68 (dd, 1H, J = 15.3, 6.6 Hz, ABX ), 2.83 (dd, 1H, J = 15.3, 5.4 Hz, ABX ), 3.21 (s, 3H), 3.67 (s, 3H), 3.75 (s, 3H), 4.20 (t, 1H, J = 6.9 Hz), 4.36 (d, 1H, J = 6.9 Hz), 5.10 (ddd pseudo m, ABX), 5.96 (d, 1H, J = 8.7 Hz), 7.28 (t, 2H, J = 7.5 Hz), 7.37 (t, 2H, J = 7.5 Hz), 7.5-7.60 (m, 2H), 7.73 (d, 2H, J = 7.5 Hz ). (1.108 g, 3.00 mmol, 1 eq.) in anhydrous THF (20 mL) under argon was added triphosgene (356.1 mg, 1.20 mmol, 1.2 eq.). Then sym-collidine (727.1 mg, 6.00 mmol, 2 eq.) was added in two portions (~0. 8 mL), of which the first portion (~0.4 mL) was added over 10 min at 0 °C (immediately white solid precipitated). The ice bath was then removed, the left portion of sym-collidine (~0. 4 mL) was added over 20 min at room temperature. The resulting mixture was totally stirred 2 h, then filtered rapidly through a filter Buchner funnel to remove the collidine hydrochloride salt and the solid was washend with anhydrous THF (10 mL × 3) to give the filtered liquid as solution A (~50 mL).

Preparation of magnesium enolate salt 174 as solution B: In a separated flask, to an ice cold solution of monobenzyl malonate of monobenzyl malonate (815.6 g, 4.20 mmol 1.4 eq.) in anhydrous THF (15 mL) under argon was dropwise added a solution of 2.0 M isopropylmagnesium chloride in THF (4. 2 mL, 8.40 mmol, 2.8 eq.). After stirred for 30 min at 0 °C, then heating at 50 °C for 30 min as

solution B.

Condensation: To the ice cold solution B was then slowly added solution A at -10 °C over 30 min.

The resulting mixture was stirred stirred for overnight (16-18 h) and slowly warmed to room temperature. The reaction was then quenched with 1 M HCl (20 mL) at 0 °C, evaporated under reduced pressure to remove most of THF, then extracted with EtOAc (50 mL × 3). The combined organic layer was washed with sat. NaHCO 3 (50 mL) and brine ( 50 (1.847 g, 5.00 mmol, 1 eq.) in anhydrous THF (20 mL) under argon was added triphosgene (593.5 mg,2.00 mmol, 1.2 eq.). Then sym-collidine (1.2118 g, 6.00 mmol, 2 eq.) was added in two portions (~1. 3 mL), of which the first portion (~0. 6 mL) was added over 10 min at 0 °C (immediately white solid precipitated). The ice bath was then removed, the left portion of sym-collidine (~0.7 mL) was added over 20 min at room temperature. The resulting mixture was totally stirred 2 h, then filtered rapidly through a filter Buchner funnel to remove the collidine hydrochloride salt and the solid was washed with anhydrous THF (10 mL × 3) to give the filtered liquid as solution A (~50 mL).

Preparation of magnesium enolate salt 174 as solution B: In a separated flask, to an ice cold solution of monobenzyl malonate of monobenzyl malonate (1.359 g, 7.00 mmol 1.4 eq.) in anhydrous THF (20 mL) under argon was dropwise added a solution of 2.0 M isopropylmagnesium chloride in THF (7.0 mL, 14.00 mmol, 2.8 eq.). After stirred for 30 min at 0 °C, then heating at 50 °C for 30 min as

solution B.

Condensation: To the ice cold solution B was then slowly added solution A at -10 °C over 30 min.

The resulting mixture was stirred stirred for overnight (16-18 h) and slowly warmed to room temperature. The reaction was then quenched with 1 M HCl (20 mL) at 0 °C and concentrated under reduced pressure to remove most of THF, then extracted with EtOAc (3 × 50 mL). The combined organic layer was washed with sat. NaHCO 3 (50 mL) and brine ( 50 13 C NMR (900 MHz, CDCl3): δ 27.5 (3C), 52.1, 62.5, 82.9, 152.8, 168.2.

To a solution of alcohol 64 (496.7 mg, 2.00 mmol, 1 eq.) in CH 2 Cl 2 (20 mL) at room temperature under argon was added DMP (1.272 g, 3.00 mmol, 1.5 eq) in 3 protions. After 5 min, NaHCO 3 (840.1 mg, 10.00 mmol, 5 eq.) was added in portions. The resulting mixture was stirred for 50 min at room temperature. The reaction solution was filtered a Celite pad, then washed with CH 2 Cl 2 ( 10mL × 3). The filtrate was then washed with sat. NaHCO 3 (20 mL × 3), water (20 mL) and sat (20 mL), respectively. The combined organic phases were dried over MgSO solution 4 M HCl in dioxane (3.2 mmol, 0.8 mL, 32 eq.) at 0 °C. The mixture was stirred for 50 min at room temperature ( 30 °C), then evaporated to give a waxy solid, to which was added ether ( 5mL), a pale-white solid precipitated, filtered and dried to give our proposed lactone of amine-HCl salt 225 as a white solid (30.1 mg,  = 100%). To a solution of lactone of amine-HCl salt 225 in CHCl 2 (2 mL) was added with DIEA (0.45 mmol, 58 mg, 4.5 eq.) at 0 °C. Then diluted with water (20 mL), extracted with EtOAc (20 mL × 3). The organic phase was dried over MgSO 4 concentrated under reduced pressure to give our proposed free amine crude 230 (as a pale-yellow oil, 16 mg).

The crude was in in CHCl 2 (1 mL) as solution B.

To solution A was added solution B, the resulting mixture was stirred for overnight (16 h). Diluted with EtOAc (20 mL), then washed with 1 M HCl (10 mL) , sat. NaHCO 3 (10 mL) and brine ( 10 2956, 2929, 2854, 1784, 1686, 1658, 1529, 1367, 1253, 1172, 1108, 837 cm -1 13 C NMR (90 MHz, CDCl 3 ) δ -4.7, 18.1, 25.9 (3C), 28.5 (3C), 36.4 , 41.6, 44.8, 55.4, 68.8, 68.9, 72.4, 80.4, 80.8, 125.2, 126.3 (2C), 127.5, 128.7 (2C), 133.6, 137.2, 157.5, 172.6, 173 NBoc and one methyl of acetonide), 1.49 (s, 9H), 2.31-2.53 (m, 4H, two ABX mixed), 3.12-3.29 (m, 2H, ABX), 3.69-3.71 ( m and s, 4H), 4.10-4.17 (m, 2H, two ABX mixed), 4.70 (ddd pseudo td, 1H, J = 18.9, 8.6, 1.4 Hz), 4.89 (t, 1H, J = 5.8 Hz ), 5.19 (d, 1H, J = 1.4 Hz), 6.22 (ddd pseudo dt, 1H, J = 15.8, 6.8, 6.8 Hz), 6.47 (d, J = 15.8 Hz ), 7.16 (d, 1H, J = 10.0 Hz), 7.18-7.22 (m, 1H), 7.26-7.31 (m, 2H), 7.34-7.36 (m, 2H).

13 C NMR (90 MHz, CDCl 3 ) δ -4.7, -4.6, 18.2, 26.1 (3C), 27.3, 27.7, 27.8 (3C), 28.6 (3C), 38.0, 41.2, 44.5, 52.5, 52.8, 68.1, 73.1, 78.5, 79.7 (2C), 83.4, 109.8, 125.5, 126.3 (2C), 127.4, 128.6 (2C), 132.8, 137.6, 152.5, 156.2, 169.4, 17.6. See Table 12, entry 3:

HRMS (ESI+

To a solution of GABOB-APTO 234 (45.0 mg, 60 mol, 1 eq.) Our goal is to develop a modular synthetic route to microsclerodermin D that could be applicable for the preparation of other microsclerodermin family members and analogues thereof. We are also looking forward to make some investigations on their biological activities or potential as anticancer drug.