M Pierre Champy

Mme Soizic

Museum Prado Professeur

M Grégory

Genta Maître

M Michel

Rohmer Professe

M Erwan

Poupon Professeur

M Olivier

P Thomas Professeur

National M Laurent

Evanno Maître De Conf

Nnt : 2017sacls214

Mme Siguara

Bastos De Lemos

Prado Professeur

M Grégory Genta-Jouve

M Michel Rohmer

M Erwan Poupon

Professeur P Thomas

M Laurent Evanno

Siguara Bastos De Lemos

Silva Président

Prof. Dr Fernando Luiz

Da Silva

Daniel Rodrigues

Dr Marie-Aude Tribalat

Aurelie Barats

Eva Ternon

Jean-Pierre Goudour

Anna Maria Orani

Blandine Seon-Meniel

Nathalie Lemière

Agnés Rasneur

Laurent Ferrié

Natacha Bonneau

Asmaa Boufridi

Kevin Cotet

David Lackar

Adam Skiredj

Xinming Zhang

Linh Nguyen

Sara Vallerotto

Thi Hong

Long Nguyen

Hazrina Hazni

Pedro Vasquez-Ocmin

Elvis Otogo

Charlotte Alcover

Alexander Enrique

Fox Ramos

Alexandre Farag

Maria Isabel Acosta

Ramona Galantonu

Christina Werner

Erika Garcia

Lucrezia Martino

Gabriela Ramos Chagas

Mauro Safir Filho

Maysa Puccinelli

Mathiew Bannwarth

Maëlle Rui Costa

Sabine Bossard I Kelner

Francisco De Assis Da

Silva Filho

Maria Das

Graças Bastos De Lemos

Estela Gomes

Elaine Cristina Patriota

substances naturelles hautement École doctorale n°569 Innovation thérapeutique: du fondamental à l'appliqué Composition du Jury

Serai Ural, some of them for

accepting me as their PhD student and for design this motivating and interdisciplinary project. It was a big pleasure to discover that I would be in collaboration with professors that were long term friends. I specially thank professor Erwan for all his help with the administration service, for his patience during the hard parts of the project, for his enthusiasm with the promising results, and for always be concerned with me on the personal level. I really thank professor Olivier for directly guiding me in the practical work, for collecting samples, for warmly receiving me in his laboratory in Nice and for all the opportunities and efforts that he continuously did to improve the quality of this work and my professional skills; like the big chance to be intern in IAEA-Monaco. It was a great experience to work with these two high level professors that have such an integrated view about science and that are so motivated. I want to acknowledge Dr. Laurent Evanno for the guidance on the laboratory, for all the great Chemistry suggestions (on the practical and theoretical fields), and for the pleasant quotidian relationship. I am indebted with Dr. Laurent for the patience that he had to always speak in French with me, improving my language skills. It was a wonderful opportunity to work with such a clever chemist and a generous person. I acknowledge all the staff of IAEA for the support to the biosynthesis project, specially to my supervisors Jean-Louis Teyssié and François Oberhänsli. I thank them for the training with radiolabelled compounds, for the innumerable results discussions, for all the kindness and, mainly from the part of Jean-Louis, for the concernment with my professional future. These three months in IAEA were an intensive experience. I want to specifically acknowledge Mr. Juan Carlos Miquel

1 Introduction 1 Natural products chemistry, alkaloids and the importance of Porifera on discovery of original structures

Natural products, also referred as "secondary metabolites" or better "specialized metabolites", are the result of years of adaptation and evolution of terrestrial and marine organisms to diverse biotic and abiotic stressing conditions. As a result, these compounds usually exhibit interesting bioactivity properties for pharmaceutical applications. 1 Traditional medicine has been empirically employing extracts of plants and animals in the treatment of diseases, with hunting purposes or as pesticides. Apothecaries and the seminal pharmaceutical companies were for years producing partially crude therapeutic formulations. 1,2 In the early 19 th century the isolation of morphine by Friedrich Sertürner started a new era to the drug formulations, were the active principle became based on pure natural products. This represented a great advance in the drug discovery and development processes, that led to a continuous improvement on isolation and characterization techniques.

In the early medicine, alkaloids were the major class of natural products present in drug formulations. Well known examples include: morphine, quinine, atropine, and ephedrine.

Nowadays, they are underrepresented in medicine market, in part because the lack of an unique definition concerning what exactly constitutes an alkaloid. 2 Indeed, alkaloids have been described

as nitrogen containing natural products, typically as primary, secondary or tertiary amines, a feature that usually confers basicity on the alkaloid. The name itself comes from the word "alkali".

The basicity of the nitrogen atom varies a lot, depending on the other functional groups of the molecule and on the location of the nitrogen functionalities. If the nitrogen is an amide function, for example, the alkaloid will be non basic. Alkaloids containing positive charges (quaternary ammonium species) can also be found in nature. [START_REF] Dewick | Medicinal Natural Products[END_REF] Alkaloids can be subdivided according to the nature of the nitrogen-containing structure: pyrrolidine, piperidine, quinoline, isoquinoline, indole, etc. As the nitrogen is usually derived from an amino acid, and the carbon skeleton is often retained, they can be subdivided according to the amino acid precursor. [START_REF] Dewick | Medicinal Natural Products[END_REF] A popular division (but somehow old fashioned) based on the amino acid, creates three categories of alkaloids: 1) true alkaloids (derived from amino acids and containing a heterocyclic ring with nitrogen); 2) protoalkaloids (derived from amino acid but without a heterocycle containing nitrogen); 3) pseudoalkaloids (compounds where the carbon skeleton is not derived from amino acids). 2 Frequently and historically the alkaloid structures are associated mainly to plants, but they can also be found in microorganisms and animals, such as sponges. The advent of SCUBA (Self-Contained Underwater Breathing Apparatus) diving technique after the Second World War (1950 -1960) made the access to deeper marine habitats and their biodiversity possible. This led the discovery of a plethora of interesting molecular structures, usually different from those found in the terrestrial environment. During the last 50 years, Porifera (sponges) became the most popular studied phylum, although its popularity has been decreasing since 1990 (Figure 1). Although they are simple animals that lack tissues differentiation and organs, sponges diversity is still surprising scientists. In the last 20 years, for example, carnivorous species were discovered. 5 Currently the phylum Porifera is sub-divided in four classes: 1) Hexactinellida ("glass sponges", sponges with a skeleton made of six-pointed siliceous spicules, called hexactines); 2) Calcarea (sponges with a skeleton of calcium carbonate spicules), 3) Demospongiae (sponges with a siliceous and/or spongin skeleton), and 4) Homoscleromorpha (the skeleton, if present, is composed by small siliceous spicules with caltrops shapes). 6,[START_REF] Van Soest | World Porifera Database[END_REF][START_REF] Gazave | [END_REF] The Demospongiae comprises about 81% of all living sponges, 9 including the sponges that will be studied in this thesis.

The "toxicity" of sponges has been documented since the Antiquity. As sessile animals, they need to produce specific metabolites for chemical communication and to defense against: predation, overgrowth by fouling organisms or to competition for space. Between the metabolites isolated from sponges, recent outstanding examples with application as pharmaceuticals are halichondrin B (1) and eribulin (2) (Figure 2). Eribulin is a synthetic compound partially biomimicry of the halichondrin B, isolated almost 30 years ago from the marine sponge Halichondria okadai.

Halichondrin B exhibited potent anti-tumoral activity but was present in very low concentrations (a ton-scale isolation allowed the isolation of just 300 mg of the compound). Meanwhile, Prof.

Yoshito Kishi from Harvard University developed a total synthesis of halichondrin B in his laboratory and years later his methodology was applied to the development of the analogue eribulin mesylate. Eribulin takes 62 steps to be synthesized but is more potent than halichondrin B and is smaller and easier to synthesize than the natural compound. In 2010 the US Food and Drug Administration (FDA) approved the use of this compound for the treatment of metastatic breast cancer. [8] E. Gazave, P. Lapébie, A. Ereskovsky, J. Vacelet, E. Renard, P. Cárdenas, C. Borchiellini, Hydrobiologia 2012, 687, 3-10. 9 [9] C. Morrow

Phorbas tenacior sponge -an incursion into the family of anchinopeptolides

Phorbas tenacior, previously known as Anchinoe tenacior (Topsent, 1925), is a deeply blue marine sponge found in the Mediterranean Sea and in the Atlantic Macaronesia (Figure 3). 7 In the 90s Minale and collaborators performed the first natural product studies on P.

tenacior discovering a family of cyclic peptide alkaloids: the anchinopeptolides A-D (3a-3d) and the interesting cycloanchinoptolide C (4c), clearly a cycloaddition derivative formed by a head-to-head [2 + 2] cycloaddition of the hydroxystyrylamido groups of anchinopeptolide C (Scheme 1).

Moreover, two minor compounds distinct of anchinopeptolides were identified: 5 and 6. 11,12,13 Minale's group also reported that anchinopeptolides B-D displace specific ligands from the 11 [11] A. Casapullo, E. Finamore, L. Minale, F. Zollo, Tetrahedron Lett. 1993, 34, 6297-6300.

12 [12] A. Casapullo, L. Minale, F. Zollo, Tetrahedron Lett 1994, 35, 2421-2422.

13 [13] A. Casapullo, L. Minale, F. Zollo, J. Lavayre, J. Nat. Prod. 1994, 57, 1227-1233.

receptors of somatostatin, human B2 bradykinin and neuropeptide Y. Anchinopeptolide A exhibited weaker activities on these biological essays. A biogenic hypothesis to the formation of anchinopeptolides is based on dimerization between two halves of a tripeptide holding residues of L-arginine, L-alanine or glycine, and hydroxystyrylamide, that might come from tyrosine (Scheme 2). The core hydroxy-pyrrolidinone is formed by an aldolization and a subsequent addition of the amide of one half of the tripeptide to the carbonyl group of the second unit. (11) alanine (10) and/or proposed biogenic formation of anchinopeptolides 7a (anchinopeptolides B-D) (anchinopeptolides A-C)

Scheme 2

Based on these works, Snider's group proposed a biomimetic synthesis of anchinopeptolide D in which the key-step is the aldol dimerization of 7b, a modified structure of tripeptide 7a (Scheme 3). The preparation of the unnatural cycloanchinopeptolide D (4d) was achieved by a [2 + 2] photocycloaddition of anchinopeptolide D under mild conditions, giving thereby strong evidence concerning the biosynthesis of natural cycloanchinopeptolide C. 14 14 [14] B. B. Snider, F. Song, B. M. Foxman, J. Org. Chem. 2000, 65, 793-800.

3 Crambeidae sponges and the guanidine polycyclic alkaloids crambescins, crambescidins and derivatives

Crambeidae is a family of sponges from the Poecilosclerida order. Among the four genera of this family, sponges from the genera Crambe and Monanchora have been widely studied for their production of a range of polycyclic guanidine alkaloids (PGAs), for instance: crambescins, crambescidins, urupocidins, and monanchocidins. These compounds are chemotaxonomic markers of the Crambeidae family used naturally as animals defense. 15 Therefore, they possess a plethora of biological activities [START_REF] Aron | [END_REF]17,18 , including in vitro HIV inhibition 19 . Representative PGAs structures produced by Crambeidae and related compounds produced by other natural sources are depicted on Scheme 4. [START_REF] Nagarajaiah | [END_REF]21 Crambe crambe (Schmidt, 1862) is a marine red incrusting sponge mainly found all across the Mediterranean Sea and now in Macaronesia (Figure 4). This sponge is already known to produce specialized metabolites from two families of alkaloids: crambescins and crambescidins.

The crambescidins are pentacyclic PGAs named according to their molecular masses. They display a broad range of biological activities, such as antifungal 22 , antimalaria 23 , and cytotoxicity against diverse human tumor cell lines 24 . The crambescins are mono-and bicyclic guanidine alkaloids classified into three sub-families: A, B, and C, which differs from the presence of a pyrrolidine ring (A), a spiro-aminal (B), and a 3-hydroxypropyl chain (C). Crambescins presented a more specific activity on ion channels. 25 Crambe tailliezi (Vacelet and Boury-Esnault, 1982) is an incrusting creamy sponge found in the Mediterranean and Macaronesian areas (Figure 5), where its abundance seems to increase along the French Riviera (between 15 and 40 m) 26 . Even if the sister species C. crambe has been largely chemically studied leading to bioactive guanidine alkaloids, there is no report concerning the isolation and identification of metabolites from C. tailliezi.

2 Objectives of this Project

This project aimed at the description of complex guanidine alkaloids produced by Poecilosclerida Mediterranean sponges in order to discovery new structures and fully characterize the already described compounds. We also performed a big scale purification of C. crambe sponge in order to provide crambescidins and crambescins for biological studies in collaboration with The general isolation procedure used in this project is depicted on Figure 6. The freezedried sponges were initially fractionated by Vacuum Liquid Chromatography (VLC) for desalting and the separation of hydrophilic compounds from lipophilic ones. All the fractions obtained from VLC were submitted to 1 H-NMR and to analytical Ultra Pressure Liquid Chromatography (UPLC)-DAD-ELSD to identify the class of compounds present in each sample. As our goal was to isolate alkaloids, only fractions with these compounds were purified by HPLC.

3 Results and Discussion

3. 1 Identification of specialized metabolites produced by P. tenacior

We decided to first perform the complete chemical study of the sponge Phorbas tenacior.

In addition to the compounds previously described (anchinopeptolides A-D, 3a-d) and 5, we found a new compound named epi-anchinopeptolide C (Figure 7, 3e). Curiously, we did not find cycloanchinopeptolide C (4c) during the purification process. The 1 H-NMR spectrum of this compound is similar to the one of anchinopeptolide C, with the main difference being in the signal of H-4' (dd at 2.72 ppm in spectrum of 3c and a dd at 2.37 ppm in 3e, Figure 8). The NOESY spectrum of 3e in DMSO-d 6 did not show the cross peaks between 5-OH and 5'-OH protons, neither the correlations between the 3'-methylene protons and both the OH protons that were described to anchipeptolides 11,12 , suggesting that 3e has a different relative configuration. In the NOESY spectrum of 3e (Figure 9) cross-peaks between H-4' and H-3, H-4' and H-2, as well as H-4' and 5'-OH were observed, confirming that H-4' is in cis relative configuration with the side chain at C-5 and with the OH at C-5'. Trying to epimerize 3c in acidic medium failed to give 3e then suggesting that this compound is not formed during the purification process. As the absolute configuration of anchinopeptolides is not described, we performed the circular dichroism analysis of anchinopeptolides A and C-D. The data analyses as well as the computational calculations of these molecules have been performed by our collaborator Dr.

Grégory Genta-Jouve (Université Paris Descartes). The results are currently being processed. 10). This product was purified by preparative HPLC, furnishing the desired cycloanchinopeptolide C (4c) as a white solid, in 17% of yield (2.3 mg). The attributions of the compound 4c obtained by hemisynthesis are in complete agreement with the literature data 13 available for the compound isolated from the natural source (Figure 11). The signals corresponding to the carbons of the cyclobutane appear in the region of 50 ppm and in the 1 resulted in similar profiles for F2 and F3. Applying the method previously described (Figure 6), we observed peaks in the region where alkaloids usually come out (R t : 6.5-8 min, Figure 13). , injection volume: 5 mL, concentration: 10 mg.mL -1

).

Considering the outstanding alkaloid diversity isolated from Crambeidae sponges, such as crambescins and crambescidins produced by C. crambe, we decided to purify the fractions F2 and F3. Some of the assayed conditions are showed on Table 1. The initial attempts were performed with standard methods and according to the preliminary results, column and elution were modified. ). Preparative columns -isocratic 0-2 min (90 : 10), gradient 2-15 min (from 90 : 10 to 0 : 100), isocratic 15-18 min (0 : 100), gradient 18-20 min (from 0 : 100 to 90 : 10) and isocratic 20-22 min (90 : 10) (flow: 10 mL.min ). Equipment: Jasco LC-4000. The chromatogram (Figure 14) was separated in 14 peaks. Peaks F2P3-P8 presented almost the same NMR spectra (Figure 15), with absence of the characteristic methyl groups present in crambescidins, crambescins and ptilomycalins identified by signals between 0.8 and 1.2 ppm. After comparing such spectra with literature, we concluded that C. tailliezi produces a distinct family of alkaloids. The peak F2P3 was submitted to a set of NMR experiments (1D: 1 H and 13 C, 2D: COSY, NOESY, TOCSY, HMBC and HSQC), leading us to recognize guanidine fragments similar to the ones present in batzelladines and especially the methyl doublet at 1.27 ppm 27 (Figure 16). This would be the first report of batzelladine derivatives in a Mediterranean sponge, as most of them are found in Caribbean species of the genus Batzella or Monanchora. Although the structure of this major compound seemed highly promising we could not go further due to the impossibility to obtain a pure compound from this mixture.

3. 3 Purification of C. crambe sponge

The purification of 24 g of freeze-dried C. crambe sponge was performed by the procedure showed on Figure 6. Crambescins and crambescidins were sent to diverse biological assays 17,28 in collaboration with professor Botana's group (University of Santiago de Compostela). In one of this studies, it was showed that crambescidins 800 (23f), 816 (23g), and 830 (23h) (Scheme 4) strongly inhibited colorectal tumor cell proliferation using in vivo zebrafish model. 17 Aside the biological application, crambescins C1 and A2 isolated by this project were used as standards for HPLC and NMR analyses required during our biosynthesis and biomimetic synthesis studies (chapters 2 and 3). 28

4 Conclusions and perspectives

In this part, we described the identification of a new compound isolated from the Mediterranean sponge Phorbas tenacior. The compound (3e) is an epimer of the previously described anchinopeptolide C (3c). We also performed the cycloaddition of anchinopeptolide C (3c) to access cycloanchinopeptolide C (4a), that was absent during our purification. These compounds will have their absolute configuration determined by circular dichroism analyses.

The purification of C. tailliezi was a very challenging task and, unfortunately, we were not able to isolate and identify any new derivative. However, we identified crambescidin 816 (23g) and crambescidin 800 (23f), compounds previously isolated from C. crambe, as well as a mixture of new alkaloids containing similar guanidine fragments found in batzelladines. To the best of our knowledge, it is the first time that batzelladines analogs are observed in a Mediterranean sponge.

The purification of C. crambe sponge furnished a range of crambescins and crambescidins that valorized the potential biotechnological application of these compounds and furnished standards for the biosynthesis and biomimetic synthesis studies that will be discussed on next chapters. Crambescidins 800 (23f), 816 (23g), and 830 (23h) were discovered as potent carcinoma inhibitors on a in vivo animal model. 17

1 Introduction

1. 1 Marine sponges and biosynthetic studies

Physical-chemical peculiarities of seawater such as buffered pH (between 8.2 to 8.5), high saline concentration (up to 40%), and osmotic pressure of 15-25 atm create special conditions to an evolution of specialized marine metabolites. 29,30 As sponges are sessile animals, their specialized metabolites are supposed to also have the role of chemical cues, being responsible for ecological communications with the environment and ensuring the development and defense of the whole organism 31 .

Despite the evident interest on the isolation, structural elucidation and application of sponge natural products, knowledge regarding their biosynthetic pathways is scarce. Conceptually a biosynthetic pathway is the sequence of chemical transformations that converts a substrate into a more complex product inside a living metabolism. [START_REF] Dewick | Medicinal Natural Products[END_REF] Sponges are early metazoans with a long evolutionary history and produce an outstanding chemical diversity. Hence, the understanding of sponges metabolites biogenetic origin can help in the identification of the genes involved in their specialized metabolic pathways, furnishing some clues about their evolution. [START_REF] Genta-Jouve | Advances in Marine Biology[END_REF] On pathways where enzymatic control is not crucial, research on specialized metabolite biosynthesis may inspire the development of biomimetic or biomimicry synthetic approaches based on spontaneous reactions. [START_REF] Gravel | [END_REF] Moreover the identification and isolation of a specific gene will allow its expression within a heterologous organism 35,36 , rendering feasible a commercial scale production of bioactive metabolites.

The starting point to a biosynthetic study is to imagine and to draw a sequence of reactions involved in the production of a target metabolite. This hypothesis can be based on previously approach to achieve this goal is to feed, inject or grow the host organism with an isotopic labelled precursor. After the incorporation time, the metabolite of interest is isolated, purified and analyzed to detect the presence of the isotope. Identification of a product labelled in specific positions will provide experimental data to understand how the precursor is incorporated into the final product. To evaluate the origin of each fragment from a target molecule, consecutive incorporation experiments may be necessary [START_REF] Dewick | Medicinal Natural Products[END_REF] , as well as genetic and enzymatic studies. As a consequence the complete elucidation of a biosynthetic pathway may demand long investigation and the involvement of researchers from diverse fields. 37

described

1. 2 Difficulties associated to sponge feeding experiments

Most of the available hypotheses on the biosynthesis of sponge metabolites arise from pathways already described on terrestrial organisms. Unfortunately, the peculiarities of marine environment and the difficulties to adapt and cultivate sponges in controlled aquaria conditions represent hurdles to experimental validation of biosynthetic hypotheses. The main challenges of the sponge biosynthetic studies are presented below 29,30,38 :

Sponges often produce a complex chemical diversity in which the desired metabolites are usually present in small quantities.

Environmental conditions can change animals metabolic profile and the concentration of a desired metabolite. In situ protocols are prone to weather and tide interferences, as well as seasonal modifications of seawater composition. Experiments in laboratory, even if more controlled, will subject the animals to stressing and artificial conditions during their collection, transport and confinement in aquaria.

The metabolic turnover in sponges is apparently slow, although no clear kinetic study proved this conclusion. 30 Thus biosynthetic experiments would require long feeding periods for a detectable precursors incorporation. However not all sponge species survive in aquaria for many days. 37 [37] A. L. Incorporation rate of precursors is usually low and they need to be detected among a high concentration of non-labelled material. It is important to emphasize that some precursors like amino acids can be involved in both primary and specialized metabolism, reducing even more the incorporation.

Precursors concentration should be maintained close to natural occurrence of aminoacids and sugars in oceans (around 0 -25 mg.dm -3 ) to avoid any modifications of metabolic pathways caused by an overload of substrate or an intoxication by an exogenous substance. Considering this precursors dilution requirement, the incorporation will take place against a concentration gradient.

Precursors must be available in a form that allows them to be transported intact into the cells where the metabolites will be synthesized. Considering that the seawater is a polar environment and that cellular membranes are lipid bilayers, differences in hydrophilicity can pose difficulties to the precursors transportation to metabolization sites. An alternative could be the addition of surfactants to facilitate the incorporation.

Sponges create symbiotic associations with micro-organisms and in many cases the specific metabolites arise, at least partially, from these associations. There are few descriptions of sponge specialized metabolites isolated from the cultured microbial symbiont 39 but several specialized metabolites share structural similarities with microbial fermentation products 40 .

Sponges can also accumulate metabolites produced by algae, microalgae or even other invertebrates as a mechanism of auto-defense. 40 Whilst all of the influences of the microbial population to the metabolism of sponges are still unclear, microbial associations should be maintained as near as possible from natural conditions during the feeding experiments in order to ensure the host fitness and its natural metabolic profile. The influence of so many factors makes difficult the repeatability of a biosynthetic experiment. Nevertheless, feeding experiments are still a direct way to obtain initial experimental evidences toward the elucidation of a metabolic biosynthetic pathway.

1. 3 Detection Techniques

The first element to consider when planning a feeding experiment is to decide the precursors labelling positions. Another important question is related to the choice of stable or radioactive isotopes. On one hand, stable isotopes are easier to manipulate and can be detected by routine techniques, such as NMR and HRMS. On the other hand, the techniques associated with the detection of stable isotopes have low sensitivity, demanding the incorporation of high concentrations of precursors that can be toxic or modify the metabolic pathways, as previously mentioned. To give an idea, experiments using stable isotopes require a precursor concentration of mg.L -1 whereas radiolabelled compounds are required on tracer amounts of µg.L -1 . For this reason, radioactive isotopes are a better choice for feeding experiments using sponges. 29,30 Radionuclides often employed in biological studies, namely 14 C, 3 H, 32 P and 35 S, are weak βemitters. A method broadly used to detect this kind of radiation is liquid scintillation counting (LSC). This method consists in dissolving the sample with a scintillation cocktail that undergoes an excitation by collision with β-particles and emits photons when returns to the ground state. LSC is highly automated, has low detection limits (ca. 370 Bq) 30 and allows the quick measurement of hundreds of samples, being possible also to count multiple isotopes on the same sample. Despite the advantages, the method has a low sensitivity due to quenching effects (interferences on the energy transfer between β-particles and the liquid scintillator), what in some cases can generate artifacts and a false results interpretation. [START_REF] Knoll | Radiation Detection and Measurement[END_REF] Searching for a higher sensitivity, our group developed a sponge feeding experiment protocol 30,[START_REF] Genta-Jouve | [END_REF] that applies detection methods based on gas ionization. One of the techniques is the radio-TLC on which a silica TLC plate containing sample deposits is submitted to a chamber filled with methane : argon (1 : 9) where the β-radiation causes gas ionization. [START_REF] Knoll | Radiation Detection and Measurement[END_REF] This technique possess 41 [41] G. F. a low detection limit (ca. 7.4 Bq) 30 and does not present the undesired quenching effects observed with liquid scintillation.

Another technique recently developed is the β-imager autoradiography. In comparison with the classical autoradiography, the β-imager possess the advantage of lower background and a higher sensitivity (ca. 37 mBq). 30,43 It is based on gas scintillation effect, therefore when β-particles deposited on TLC silica plate interact with the detection medium their energy is partially converted into photons. The light emitted by photons is recorded and intensified on a special camera, generating images on which the luminosity is related to the sample activity.

The combination of these two sensible techniques, radio-TLC and β-imager, offers the opportunity to work with precursors close to natural concentrations and constitutes on powerful tools to biosynthesis experiments with sponges.

1. 4 Biosynthesis hypothesis for crambescins and derivatives

Crambe crambe, introduced in Chapter 1, is a red incrusting Mediterranean sponge that produces guanidine alkaloids from two groups: crambescins and crambescidins.

In the early 90's Snider and Shi reported a biomimetic synthesis of crambescins (Chapter 3) in which the guanidine cyclic core was achieved by a Michael addition of an O-methylisourea on an enone 44 (Scheme 5). In the light of recently discovered guanidine alkaloids produced by Crambeidae sponges 45,21 our groups elaborated a biosynthesis hypothesis 46 reconsidering the origin of the guanidine core through a Mannich-type reaction between a guanidinylated pyrrolidinium derived from agmatine and a long β-keto fatty acid (Scheme 6).

O O OMe R 1 O N H transesterification R 1 : TBDMS O O O R 1 O N H R 2 : CBZ Knoevenagel condensation H O m O O O R 1 O 1) addition of H 2 N OMe NH HN NH NH 2 R 1 O O O N H cyclic guanidine core
It is worth noting that in our proposition crambescins C are intermediates for conversion of crambescins A into B. Among crambescins C, we will focus our discussion on crambescin C1 47,48 (Figure 18), a compound that possess a mono-cyclic-6-membered ring guanidine core substituted by three side chains: 1) a linear 3-hydroxypropyl chain; 2) an aliphatic nonanyl chain; 3) a 6guanidino-heptyl acetate chain (Figure 18). As many others C. crambe metabolites, crambescin C1 also exhibit biological activity 49 .

2 Objectives of this project

Herein we present a biosynthetic study that aims to demonstrate and understand the production of the guanidine alkaloid crambescin C1 by the sponge C. crambe using in vivo 14 The protocol 30,[START_REF] Genta-Jouve | [END_REF] used was adapted from the one developed for the biosynthetic study of oroidin in Axinella damicornis and was planned to circumvent as best as possible the main problems associated with sponge feeding experiments. A summary is depicted on Figure 20.

Chapter 2 │ 14 C-Feeding Experiments with

3 Results and Discussion

3. 1 Feeding Experiments with

The sponge specimens were collected and acclimated in aquaria to avoid stressful conditions that would interfere on their filtration activity and downturn the incorporation of labelled precursors during the feeding experiments.

cultivation of marine sponges 50,51 animals were daily fed with phytoplankton ( (two tanks of 20 L) filled with natural seawater supply was adjusted to 18 weeks of acclimation, the sponge morphology were healthy and well adapted to the laboratory conditions. 50 [50] R. Osinga, J. Tramper, H. R. Wijffels, 51 [51] R. Osinga, J. Tramper, R. H. Wijffels 52 [52] X. Turon, J. Galera, M. J. Uriz, J. Exp. Zool. 53 [53] E. H. Belarbi, M. R. Dominguez, G.

Feeding Experiments with C. crambe: First Insights into the Biosynthesis of Crambescin C1

. Workflow to in vivo sponge feeding experiments

Results and Discussion

Feeding Experiments with C. crambe specimens were collected and acclimated in aquaria to avoid stressful conditions that would interfere on their filtration activity and downturn the incorporation of ed precursors during the feeding experiments. The following conditions described for 51 and specially for cultivation of C. crambe animals were daily fed with phytoplankton (Isocrysis galbana) and the temperature of natural seawater supply was adjusted to 18 °C. After , the sponge morphology and especially opened oscules were healthy and well adapted to the laboratory conditions. Insights into the Biosynthesis of 36 specimens were collected and acclimated in aquaria to avoid stressful conditions that would interfere on their filtration activity and downturn the incorporation of conditions described for were applied 52,53 Based on our biosynthetic hypothesis, L-[guanidino-14 C]arginine, [guanidino- 14 C]agmatine, L-[U-14 C]lysine, and L-[5-14 C]ornithine were chosen as possible precursors of the guanidine core and [U- 14 C]acetic acid and [1-14 C]lauric acid were used as possible precursors of the aliphatic chain formation (Scheme 7). Our proposition is that both guanidine moieties may be derived from Larginine and the cyclic guanidine core formation can arise from two possible pathways: A) a transamination of L-arginine followed by decarboxylation and an intramolecular condensation, without the involvement of agmatine; B) a decarboxylation leading to agmatine, that by an intramolecular condensation generates the iminium. It is well known that L-ornithine is the precursor of L-arginine (way C) and together with its analogous L-lysine, these three aminoacids are usually involved in the biosynthesis of alkaloids [START_REF] Dewick | Medicinal Natural Products[END_REF][START_REF] Dewick | Medicinal Natural Products[END_REF] , what justifies our choice. Lauric acid was selected as it possesses the same number of carbons necessary to build the upper aliphatic chain of crambescin C1, our target metabolite. The use of acetic acid was intended to analyze if the aliphatic moieties were formed by a de novo process.

Scheme 7

During the 14 C-feeding experiments, the sponges were placed inside opened plastic boxes of 700 mL filled with seawater for incubation with the radiolabeled precursors during 18 h (Figure 21). The experiments were performed in triplicates, which means that for each precursor evaluated, three sponges were fed in the same conditions. To keep a low concentration of precursors, we performed eight to ten incubations, each one containing an activity of 37 kBq ( 1Chapter 2 │ 14 C-Feeding Experiments with µCi). To access the decrease of radioact tissues, the β-radioactivity of the water surrounding the specimens was monitored by liquid scintillation at the beginning of significant decrease of activity even for Assuming this decrease was caused by microbial activity, we set an experiment with acid and agmatine to monitor controls during a long term. The aim was to compare the water column activity of controls containing sterile medium (d hypochlorite solution) against controls with pure seawater. Graphic 1 (Figure 22, controls with lauric acid) is in of microbial activity on the precursor counting related with the controls of distilled water and sea water containing disinfectant were stable (red and green lines, controls 2 and 3), having virtually the same amount of activity of t contrast, the sample containing only seawater (blue line, control 1) presen the activity since the first days. containing a sponge rock support (purple line, control 4). In this case, there was a small decrease in the activity of the water column Feeding Experiments with C. crambe: First Insights into the Biosynthesis of Crambescin C1

of radioactivity in the seawater due to incorporation radioactivity of the water surrounding the specimens was monitored by liquid scintillation at the beginning of the incubation period and at the end. Surprisingly, we noticed a even for the controls (empty plastic boxes filled with seawater).

was caused by microbial activity, we set an experiment with acid and agmatine to monitor controls during a long term. The aim was to compare the water column activity of controls containing sterile medium (distilled water, and sea water with hypochlorite solution) against controls with pure seawater.

Picture of sponges during incubation period

, controls with lauric acid) is in accordance with the hypothesis of of microbial activity on the precursor decrease from the medium. During the four first days, the th the controls of distilled water and sea water containing disinfectant were stable (red and green lines, controls 2 and 3), having virtually the same amount of activity of t contrast, the sample containing only seawater (blue line, control 1) presented a visible the activity since the first days. To evaluate the role of the support, we prepared a control containing a sponge rock support (purple line, control 4). In this case, there was a small decrease activity of the water column after the first days, probably due to residual microorganisms Insights into the Biosynthesis of due to incorporation in the sponge radioactivity of the water surrounding the specimens was monitored by liquid incubation period and at the end. Surprisingly, we noticed a the controls (empty plastic boxes filled with seawater).

was caused by microbial activity, we set an experiment with labelled lauric acid and agmatine to monitor controls during a long term. The aim was to compare the water stilled water, and sea water with sodium with the hypothesis of a role from the medium. During the four first days, the th the controls of distilled water and sea water containing disinfectant were stable (red and green lines, controls 2 and 3), having virtually the same amount of activity of t 0 . In ted a visible decrease of evaluate the role of the support, we prepared a control containing a sponge rock support (purple line, control 4). In this case, there was a small decrease after the first days, probably due to residual microorganisms present on the rock. After the 5 th day, there was a significant decrease of the radioactivity of the controls 2-4, probably due to a microbial proliferation, and after the 20 th day the activity left was minimum. Considering the control experiment results, the boxes used to store the sponges during the feeding were cleaned with sea water, dried with paper and let at air until the next usage. This simple procedure decreased the microbial proliferation and the depletion in activity observed with the controls during the feeding experiments. As NaClO can interfere with the activity of agmatine, we avoided the use of any antimicrobial substance to clean the boxes.

After the feeding and metabolism cycles, the animals were freeze-dried and extracted with MeOH. Extracts were submitted to desalting by C18 Solid Phase Extraction (SPE). Four fractions were generated, namely: F1 (100% H 2 O), F2 (H 2 O : MeOH, 1 : 1), F3 (H 2 O : MeOH, 1 : 3) and F4 (100% MeOH). Methanolic fractions were evaluated on radio-TLC in order to provide a preliminary result concerning their radioactivity. We observed a significant radioactivity (1.0 Bq or more, Table 2 and Figure 23) associated to the fractions of samples fed with agmatine, lauric acid, ornithine and arginine. Among the sponges fed with lysine, the fractions of specimens 1 and 2 presented a high activity but the fractions of specimen 3 had low values, that could be explained by a filtering problem of this individual in relation with physiological state. Regarding the sponges fed with acetate, only fractions F4 showed some radioactivity. From our previous experience on the isolation of crambescins and crambescidins by the protocol applied on this work, the higher content of such specific metabolites should be in fractions F2 and F3. Therefore, the acetate data suggest that the precursor might be metabolized into fatty acids, explaining why the most apolar fractions presented higher activities, being mainly metabolized into the primary metabolism.

To each set of three sponges fed with the same precursor, we selected fractions F2 of two animals to be purified on a phenyl-hexyl semi-preparative HPLC column. The peaks with retention time 24.5 min were identified by 1 H-NMR as being crambescin C1 (Figure 24). In order to avoid radioactive contaminants that could lead to a false positive result, the crambescin C1 samples were further purified by a final C18 analytical HPLC column. Both crambescins C1 obtained in the first and second HPLC purifications were deposited on a TLC plate and had their specific activities determined by the highly sensitive technique β-imager autoradiography (Table 3). conclude the incorporation position sure that the guanidine moiety is arising from this precursor bacteria 54 and fungi 55 are able to biosynthesize 4 the biosynthesis of the 6-guanidino microorganisms. The fact that agmatine was not incorporated into crambescin C1 can be either an 54 [54] H. Hong, T. Fill, P. F. Leadlay, Angew. Chem. Int. Ed. 55 According to our biosynthetic hypothesis, arginine can be incorporated into the two crambescin C1 guanidine moieties. Although the autoradiography results are insufficient to position, as the L-arginine is labelled on the guanidine position, it is sure that the guanidine moiety is arising from this precursor. Recent reports prove that strains of are able to biosynthesize 4-guanidinobutyric acid from guanidino-hexyl unit might be related to a symbiosis with agmatine was not incorporated into crambescin C1 can be either an indication that the organism cannot uptake this precursor from the diet into the specific metabolism either that the formation of the iminium does not involve agmatine as an intermediate. In the second scenario, the guanidine cyclic core would be derivate from the way A of Scheme 7.

The high activity of the samples fed with lauric acid indicates that this precursor was incorporated into the aliphatic chain participating on few other metabolic pathways. As lauric acid has the same number of carbons necessary for the construction of the upper crambescin C1 aliphatic chain, this result strongly suggests a direct incorporation of the fatty acid from diet. This hypothesis is in accordance with the variety observed on the upper aliphatic chains of crambescins A1, A2, A3, and analogues (Scheme 4). In nature, the providers for fatty acids might be either microorganisms from micro flora of sponge either exogenous sources from marine environment, allowing a variety of specialized metabolites that is probably related with the availability of substrate on sponges' habitat. Although the high concentration of PGAs in C. crambe (about 1% of dry mass) might suggest the sponge as the only responsible for the biosynthesis of its secondary metabolites, our present incorporation results in addition to the report of a predominance of a betaproteobacterium in the microflora of C. crambe 56 can suggest the implication of bacteria in the biosynthesis of crambescins and crambescidins.

The lack of activity associated with acetic acid suggests that the upper aliphatic chain is not produced by a de novo pathway, although it is important to notice that during the long experiment period the precursors might be involved in many other metabolism/catabolism steps, including primary metabolism.

4 Conclusions

For the first time, biosynthetic experiments with C. Crambe allowed the identification of two building blocks involved in the synthesis of crambescin C1: L-arginine and lauric acid. The incorporation of lauric acid or close fatty acids from diet could explain the variety observed on the upper aliphatic side chain of crambescins. The microorganisms apparently play a crucial role on these secondary metabolic pathways, which can be expanded to the biosynthesis of other crambescins and derivatives. The incorporation of L-arginine labelled at the guanidine indicate that the origin of the guanidine is clearly L-arginine. However agmatine does not seem to be involved in the biosynthesis (way A, Figure 26).

Once more our sponge feeding experiments protocol 30,[START_REF] Genta-Jouve | [END_REF] proved to be useful to give initial insights into sponge metabolites biosynthetic pathways. Further studies focusing on the genome and enzymes of sponges would help in the understanding of this intriguing and exciting biochemistry.

1 Introduction

1. 1 "Nature knows best": biomimetic synthesis of natural products

Observing nature to answer human questions is the oldest and most fundamental activity of philosophy and its younger sister: science. Biomimicry, biomimetic or bioinspiration are some terms used to describe the concept of taking advantage of years of evolution in natural materials, substances, and processes to human benefit. Such concept can be applied to all domains: arts, science, fashion, philosophy, survival tactics, etc. When applied to chemistry, topics such as supramolecular chemistry, material chemistry and biomimetic synthesis exploit the concept of biomimicry. [START_REF]Biomimetic Organic Synthesis[END_REF][START_REF] Hanks | Bioinspiration and Biomimicry in Chemistry[END_REF] On the brilliant paper "Nature knows best: an amazing reaction cascade is uncovered by design and discovery" 59 , Heathcock poses fundamental questions about the advances of organic chemistry synthesis field, hints biomimetic synthesis as the alternative to the development of efficient synthetic pathways and culminates by the description of biomimetic synthesis itself:

"One of the strategies we have been used to look for efficient synthetic routes to complex natural products is to try and figure out how nature has solved the problem. The basic assumption of this approach is that nature is the quintessential process development chemist. We think that the molecular frameworks of most natural products arise by intrinsically favorable chemical pathways -favorable enough that the skeleton could have arisen by a non enzymic reaction in the primitive organism. (...)

A corollary of the foregoing hypothesis is that the coexistence of two structurally related molecules in an organism implies some reasonable chemical pathway from one to the other. (...) A 'biomimetic' synthesis is a laboratory synthesis that is based on such reasoning".

During the development of a biomimetic synthesis strategy, experimental biosynthesis evidence or at least a reasonable biogenic hypothesis will be demanded. Therefore, biomimetic synthesis and biosynthesis are tethered fields on the process of "reverse engineering" nature: on one hand the biosynthesis will inspire putative precursors and reactions to a synthetic strategy, on the other hand the achievement of the biomimetic synthesis will corroborate that putative 57 [57] in Biomimetic Organic Synthesis (Eds.: E. precursors and intermediate species are prone to react even in a non-enzymatic way. An example of the symbiosis between biomimetic synthesis and biosynthesis is the synthesis of endiandric acids A, D, and E (3e, 3a, and 3c) (polyketides isolated from the Australian tree Endiandra introrsa)

achieved by Nicolaou and co-workers and based on the biogenic hypothesis of Black's group (Scheme 9). During these works, endiandric acid D (3a) structure was suggested by the biosynthesis hypothesis and biomimicry synthesized even before its detection in the natural source. 34

Scheme 9

The "toolbox" of biomimetic synthesis is composed by "classical and old fashion" organic reactions (often revised on a "type" style to replace dangerous chemicals or to include a catalyst) like aldolization, Claisen condensation, Mannich reaction, and Diels-Alder reaction. [START_REF]Biomimetic Organic Synthesis[END_REF] Cascade reactions have also been extensively exploited in biomimetic approaches; they basically consist in having a substrate with a required functionality to be converted into the desired species in the same pot. [START_REF] Nay | Biomimetic Organic Synthesis[END_REF] The combination between the number of steps and the increase in molecular complexity from starting materials to the target molecule is relevant to describe the success of a organic synthesis strategy. A pioneer index to calculate molecular complexity was proposed by Bertz [START_REF] Bertz | [END_REF] in 1981 and relies on a combination of graphic and information theories to translate what a synthetic chemist could intuitively describe as a "complex structure". In this approach, a molecule is described as a "skeletal" without hydrogen atoms (Scheme 10). The calculation is then based on the sum (C T ) of two coefficients defined as: Cη -the number of connections, which means the amount of adjacent lines; and Cε -the elemental diversity coefficient, namely the number of heteroatoms. The Cη coefficient increases with the presence of multiple bonds, rings, branches and molecular asymmetry. The range in complexity from reactants to products in the course of a reaction is called ∆C T . 61

Scheme 10

Bertz's index is the most popular manner to measure molecular complexity and a database containing the C T of many chemical compounds is publically available on PubChem. 62 Hendrickson proposed a simplified formula of Bertz's index that is adapted to evaluate the atom environments and not linear-three atoms systems (simplifying a studied molecule to units of propane) as in Bertz approach. 63 Besides the limitations, such as not considering the stereochemistry 62 , herein we will employ the Bertz-Hendrickson index as an estimation to access the molecular complexity range in the course of the biomimetic synthesis of crambescin A2.

1. 2 The guanidine bicyclic alkaloid crambescin A2 448 and analogues

Crambescin A2 448 64 (5a, Figure 27) is a bicyclic guanidine alkaloid isolated from the Mediterranean encrusting sponge C. crambe. This alkaloid was initially called "crambine A" however Rinehart's group proposed to rename the whole class of alkaloids to "crambescins" 48 in order to avoid misunderstandings with the protein "crambin" (sometimes spelled as "crambine"), isolated 25 years earlier from the plant Crambe abyssinica. purification process, crambescin A2 has often been isolated as a mixture of homologues on the upper alkyl side chain, that justifies the need to precise the mass of the molecule after its name (Figure 28). 45 Analogues of crambescin A2 448 had also been isolated from non Crambeidae sponges, as compounds 5e-h 66 , produced by Pseudaxinella reticulata (Axinellida, Axinellidae). Some crambescins A2 presented biological activities such as effect in the voltage-gated calcium channels of neurons 25 and antifungal activity with a potency dependent upon the length of the aliphatic chain 66 .

1. 3 Biomimetic synthesis of crambescins

The first and only synthesis of crambescins is a "biomimetic" approach proposed by Snider and Shi on early 90s. 44,67 The authors designed a retro(bio)synthesis of the crambescins bicyclic guanidine core based on structural similarities between crambescins and two polycyclic guanidine alkaloids previously studied by the team: ptilomycalin A and ptilocaulin (8a and 13), both isolated from the Caribbean sponge Ptilocaulis spiculifer (Axinellidae, Axinelida). 68 On their approach, the central moiety arises from a Michael addition of a guanidine derivative (O-methylisourea) to an enone, hence considering a fully polyketide biochemical pathway (Scheme 11). According to their hypothesis, crambescins B (6) would be formed by an acid or base cyclization of crambescins C (7).

In the same way, crambescins A (5) would arise from a conversion of hydroxyl from crambescins C into a good leaving group followed by base-catalyzed cyclization. Summing up, crambescins C would be intermediates towards crambescins A and B.

66

[66] M. T.

2 Objectives of this project

This project aims at the synthesis of crambescin A2 448 based on the results of 14 C-feeding experiments with the Mediterranean sponge C. crambe (Chapter 2). The retro(bio)synthetic strategy (Scheme 13) has as starting materials: L-arginine (19), 4-amino-butan-1-ol ( 22) and lauric acid (23). The key-step would be the condensation between the guanidine pyrrolidinium 18 and the enone 17a, that already encloses the lower ester guanidine moiety chain. The achievement of this synthesis will: i) confirm the intrinsic reactivity of the putative intermediates proposed in the biosynthesis hypothesis, providing additional insights into the biogenic origins of crambescins and derivatives; ii) fully dissect all mechanisms leading to this class of natural substances.

Scheme 13

3 Results and Discussion

1 Synthesis of aliphatic fragment 21

The convergent synthesis of crambescin A2 started with the preparation of the "upper aliphatic fragment", achieved through a C-acylation of lauric acid 23 with a malonate magnesium salt 69

2. 1 Preparation of alcohols 20a and 20c

In order to mimic as best as possible the biosynthetic hypothesis, our first attempts were to convert L-arginine 19 into alcohol 20a by an oxidative decarboxylation followed by a reduction of resulting carboxylic acid 31 into the corresponding alcohol (Scheme 17). This fragment corresponds to a constitutive unit of the "lower ester side chain" of crambescine A2.

Scheme 17. Retrosynthesis of alcohol 20a from L-arginine

For this purpose, L-arginine was oxidatively decarboxylated by dimethyl dioxirane generated in situ 70 A proposal of mechanism is presented on Scheme 19 70 . The authors claimed that intermediates 19a, 19b, and 32 were produced from the oxidation of amines with dioxirane.

Decarboxylation of 19b by a cyclic transition state leads to oxime 32, that undergoes a hydrolysis to aldehyde 33 and is further converted into carboxylic acid 31 by an oxidation.

Scheme 19

Compound 31 was submitted to reduction by LiAlH 4 . Desired alcohol 20a was identified by HRMS but the product was lost during the purification on Amberlyst® 15.

Scheme 21

2. 2 Attempts to the formation of fragment 17 by transesterification

At this stage the alcohol 20 had to be connected to ester 25 in order to produce a fragment containing the "lower" and the "upper" side chains as well as the reactive β-keto-ester function necessary for the biomimetic assembly of the central core of crambescin A2. The initial approach to prepare fragment 17a was the transesterification of 25a by alcohol 33 (Scheme 22) promoted by acidic Amberlyst® 15 73 . After 2 days at reflux, the crude mixture was submitted to HRMS analysis where a high intensity peak possessing a m/z close to the desired product was identified For this purpose Taber's transesterification 75 of 25b with 20c catalyzed by 4dimethylaminopyridine (DMAP) (Scheme 25) was studied, the same approach as used by Snider's group during their synthesis of crambescins 44 . Despite Snider and Shi had reported a low conversion of starting material into their desired product (77% of recovered starting ester and 33% of yield of expected ester), we decided to try this approach due to the structural similarities shared between 25b and 20c and the substrates used by Snider's group. Unfortunately the HRMS of the crude product did not present the peak corresponding to desired product neither its 74 [

1 Preparation of 18 by sequential reductions of L-arginine

The second task was to convert L-arginine onto the pyrrolidinium 18 necessary for the biomimetic assembly of crambescin A2. The initial strategy to prepare 18 was an oxydative decarboxylation of L-arginine 19 into the aldehyde 33 (Scheme 28). An intramolecular nucleophilic substitution of the amino group from guanidine on the carbonyl of aldehyde would furnish the guanidinated pyrrolidinium 18. This pyrrolidinium could be trapped by a cyanide or, better, be strategy to the synthesis of iminium 18

Scheme 28

The decarboxylation of L-arginine promoted by trichloroisocyanuric acid (TCCA) 77 furnished 41 quantitatively (Scheme 29).

Scheme 29

For the oxidative decarboxylation of L-arginine into nitrile, we propose a mechanism similar to the oxidation of alcohols by TCCA published by van Summeren and co-authors 78 Next, a reduction of nitrile onto an aldehyde was required. Some approaches often used to reduce nitriles into aldehydes are presented below: 79 1) The classical Stephen aldehyde synthesis employing HCl gas and anhydrous SnCl 2 ;

2) The catalytic partial hydrogenation promoted by a metal catalyst such as Pd, Pt or Raney nickel and hydrogen gas. The nitrile is converted into an intermediate imine and subsequently hydrolyzed to aldehyde. The addition of a trapping agent such as phenylhydrazine can avoid the overreduction of aldehyde to amine.

3) The partial hydrogenation by a metal hydride, for instance LiAlH 4 or NaBH 4 ;

4) The modified Stephen reduction using Raney nickel in aqueous solution of an acid (e.g.: formic acid, acetic acid, H 2 SO 4 ).

In view of these procedures, our first assays to convert 41 into 33 were adapted from the classical reductions promoted by DIBAL-H and Pd/C (entries 1 and 2, Table 6), which resulted in starting material recovery. It was observed that 41 was insoluble in organic solvents, therefore a modified Stephen reduction in aqueous conditions was considered (conditions described on entries 3 to 7). Finally conditions of entry 7 led to the expected aldehyde 33 mixed with iminium 18.

79 [79] B.

2 Preparation of 18 by oxidative decarboxylation of L-arginine

Alternatively, it was also tried to obtain aldehyde 33 in a single step by oxidative decarboxylation of L-arginine promoted by silver(II) picolinate 82,83 (Scheme 31). We were glad to notice that the reaction proceeded exactly as described, with red colour discharging indicating the reduction of silver complex and the end of the reaction. Formation of aldehyde 33 and iminium 18 was confirmed by HRMS.

Scheme 31

Efforts were made to convert and to isolate the iminium species as a cyanide (Scheme 32).

For this purpose, after the acidification and filtration of reaction mixture with Ag(pic) 2 to get rid of silver, KCN (2 eq) was carefully added to the filtrate and submitted to magnetic stirring for 3 h at

rt. An extraction with AcOEt and water was tried to separate the picolinic acid (by-product of the oxidative decarboxylation) and inorganic salts from 40b, but the desired product was identified in the aqueous layer. Both phases were joined and evaporated. Unfortunately the product was obtained mixed with picolinic acid after purification by either flash chromatography on silice 60 or LH-20 Sephadex column.

Having into account the difficulties faced to purify the iminium trapped with cyanide, the crude product of the reaction presented on Scheme 31 was then employed for the next steps without further purification.

Scheme 32

The oxidative decarboxylation of aminoacids promoted by silver(II) picolinate was found to pass by a cyclic intermediate 19c, consisting in a eletrocyclic (concerted) mechanism with reduction of silver(II) to its elementary state (Scheme 33). 82,84 trapping experiments. 84 Thereafter hydrolysis of 43 furnishes aldehyde 33.

Scheme 33

4 Key-step: formation of the bicyclic guanidine core

4. 1 Formation of structures 45a and 45b

Having prepared the two required fragments 18 and 25, the study of the biomimetic assembly of the central core of crambescin A2 could be started. Following the biosynthesis hypothesis presented in Chapter 2, Scheme 6, condensations between 18 and 25a were logically evaluated under Mannich or Biginelli-type conditions to access platform 45 (Table 7). Despite the long reaction time, entry 5 provides the best conditions to obtain 45. These conditions correspond to the tethered Biginelli condensation developed by Overman and co-workers 85,86,87 that became a classical approach to access compounds containing 3,4dihydropyrimidin-2(1H)-ones-type cores (DHPMs) such as crambescidins, batzelladines, and monanchocidins (Scheme 34). 88 Despite often mentioned as a possible tool to access crambescins, to the best of our knowledge this is the first time that the tethered Biginelli is employed to build the guanidine core of such family of compounds. It is important to stress out this fact because in comparison with the DHPMs, crambescins have a double bond at positions 7 and 8 (45b) that by the mechanism of tetrahedred Biginelli condensation 85 will be formed between positions 7 and 13 (45a, Scheme 35), being necessary a further proton displacement in order to access the desired structure 45b.

Scheme 34. Tethered Biginelli condensation applied to the construction of tricyclic guanidine core of DHPMs-type alkaloids

Scheme 35

The crude mixture of the Biginelli-type condensation ( chromatography on silica 60 column, giving a fraction constituted mostly by picolinic acid and a mixture of 45 and 49 and their putative respective aromatized structures 48a and 48b (Figure 29).

By the analysis of 1D and 2D NMR, it was inferred the formation of both isomers 45a and 45b. In order to obtain only desired 45b, the product was submitted to acid reflux in DCM (Scheme 36). As a result the counter ion of the guanidinium compounds was exchanged by a triflate and we got rid of the picolinate counter anion signals on NMR spectra (Figure 30).

However, a mixture of 45a and 45b was still observed. Preliminary assays to achieve an asymmetric isomerization of 45a into 45b catalysed by Rh(I) complex or a full aromatization of this mixture promoted by MnO 2 (Scheme 37) were performed using a small amount of substrates. The full aromatization could lead to an analogue of dehydrocrambescin A2 418 (48c), a compound recently isolated from a French Polynesian Monanchora n. sp. sponge 89 . Surprisingly, the aromatization with MnO 2 gave the starting materials and according to LC-MS, the Rh(I) complex promoted the full aromatization of 45 and 49 instead of As the transesterification did not proceed, the strategy was changed to form the carboxylic acid 50 and to obtain 5a by an esterification. Unfortunately the desired product 50 was not observed by this approach (Table 9). Finding good conditions for the esterification or the transesterification of mixture 45 and 49 was more challenging than expected and was consuming both iminium 18, β-keto-ester 25a, alcohol 20a and time, whereas the condensation takes 5 days to be accomplished. For this reason, we optimized the preparation of substrate 17b, that contained already the ester moiety, and performed the tethered Biginelli condensation between 18 and this substrate (Scheme 38). was deprotected, giving 17a. This assumption is based on the fact that unconverted 17a was isolated during the HPLC purification of the crude product and any 17b nor protected bis-Boc 5a or 5k were observed. The desprotection of 17b is showed on Scheme 39.

Scheme 39

The comparison of the 1 H-NMR spectra of 5k with crambescin A2 1 H-NMR spectra available in literature 45 differs mainly on the multiplicity of peak at 4.5 ppm (a triplet for crambescin A2 and a double doublet for compound 5k) as well as in the signals between 2.4 and 3.0 ppm. It is possible to note the signals of crambescin A2 5a at 4.4 ppm (t, H7) and at 3.1 ppm (dd, H9) integrating to 0.3. Unfortunately the mass of the mixture containing crambescin A2 was too low (around 6 mg) and this compound was not isolated and fully characterized to confirm, by instance, its stereochemistry. The obtainment of iso-crambescin A2 448 (5k) supports the mechanistic hypothesis concerning both the synthesis and biosynthesis issues. The next efforts will be focused on the conversion of 5k into crambescin A2 (5a) and derivatives, such as dehydrocrambescin A2 (48c), and improvement of the Biginelli condensation yield. Presence of crambescin A2 might be an indication of a spontaneous conversion of iso-crambescin A2 5k into the natural product.

Importantly, the isomerization occured spontaneously on the sample kept in the MeOH-d 4 solution. After three weeks, the ration risen from 7 : 3 to 6 : 4 (5k : 5a, respectively). This result had been observed at the end of this work and still under study. The kinetic of this conversion needs to be improved, either by acid catalysis or photo-indution. This hypothesis will be tested by irradiation of mixture containing 5k with an UV-vis lamp system that mimics sunlight.

4 Conclusions and perspectives

Inspired by our results on the biosynthesis experiments with C. crambe sponge (Chapter 2),

we developed an alternative biomimetic synthesis to access crambescin A2 448 and its derivatives.

Up to now, only 4 steps with 3% of overall yield were required to obtain a mixture 7 : 3 of isocrambescin A2 5k and crambescin A2 448 (5a) (Scheme 40). It is interesting to notice the increase in molecular complexity achieved: the final product is twice more complex than the starting materials and is 1.26 times more complex than the intermediates involved on the Biginelli-type condensation (Figure 33). Further efforts will be performed to enrich the crambescin A2 content on this mixture and to achieve the aromatization of 5a and 5k analogues in order to obtain the natural alkaloid dehydrocrambescin A2 (48c, Scheme 37).

General conclusions and perspectives

This work studied three aspects related with complex marine alkaloids produced by Mediterranean sponges: isolation and structural elucidation, biosynthesis, and biomimetic synthesis.

In the first chapter three species were purified: Phorbas tenacior, Crambe crambe, and Crambe tailliezi. Purification of P. tenacior yielded the already described anchinopeptolides A, B, C, and D, as well as the new structure epi-anchinopeptolide C. The absolute configuration of already described anchinopeptolides as well as epi-anchinopeptolide C will be determined by circular dicroism. Cycloanchinopeptolide C was not found on our purification but it was successfully produced by the photo-cycloaddition of anchinopeptolide C.

The purification of C. tailliezi was a challenging task and, unfortunately, we were not able to isolate any new compound. However, we identified crambescidin 816 and crambescidin 800, compounds previously isolated from C. crambe, likewise a mixture of new alkaloids containing similar guanidine fragments found in batzelladines, a family never before observed in a Mediterranean sponge.

The purification of C. crambe sponge furnished a range of crambescins and crambescidins that valorized the potential biotechnological application of these compounds and furnished standard compounds to our biosynthesis and biomimetic synthesis studies.

The biosynthetic experiments with C. Crambe allowed the identification of two building blocks involved in the synthesis of crambescin C1: L-arginine and lauric acid. The incorporation of lauric acid or close fatty acids from diet could explain the variety observed on the upper aliphatic side chain of crambescins. The microorganisms apparently play a crucial role on these secondary metabolic pathways, which can be expanded to the biosynthesis of other crambescins and derivatives. The incorporation of L-arginine labelled at the guanidine indicates that the origin of the guanidine is clearly L-arginine. However agmatine does not seem to be involved in the biosynthesis.

Inspired by our results on the biosynthesis experiments with C. crambe sponge, we developed an alternative biomimetic synthesis to access crambescin A2 448 and its analogues.

Only 4 steps with 3% of overall yield were required to obtain a mixture 7 : 3 of iso-crambescin A2 and crambescin A2 448. Further efforts will be performed to enrich the crambescin A2 content on this mixture and even if the crambescin A2 synthesis is not yet fully accomplished, the obtainment

Résumé en français

of the molecule scaffold confirms that the intrinsic reactivity of the biosynthetic precursors is totally suitable to lead to a common platform to this family of natural products.

Finaly, we hope that the set of results presented herein will contribute to increase the knowledge concerning the chemistry of marine cyclic guanidine alkaloids.

Introduction

L'objet de ce travail de doctorat est l'étude chimique et biosynthétique d'alcaloïdes guanidiniques d'origine marine provenant d'éponges méditerranéennes. Ce manuscrit est divisé en trois parties complémentaires : i) l'isolement d'alcaloïdes d'éponges marines de l'ordre des Poeciloscerida ; ii) l'étude de la biosynthèse d'alcaloïdes de type crambescine par des études de radiomarquage ; iii) la synthèse biomimétique de la crambescine A.

Étude chimique d'éponges marines de l'ordre des Poeciloscerida

1 Étude chimique de Phorbas tenacior

La première étude chimique de l'éponge P. tenacior a été réalisée par l'équipe du professeur Minale. De cette éponge, une nouvelle famille d'alcaloïdes de nature peptidique a été isolée: les anchinopeptolides (3a-d), ainsi que le cyclo-anchinopeptolide C (4c) comportant un cycle cyclobutane. En plus de ces structures, deux autres composés minoritaires (5 et 6), correspondant à des précurseurs ont été isolés. 11,12,13 Au cours de notre étude de cette même éponge basée sur un profilage métabolomique, les anchinopeptolides précédemment décrits (3ad) ainsi quele composé 5 ont été ré-isolés. Toujours au cours de cette même étude, un nouveau membre de cette famille l'epi-anchinopeptolide C (3e, Figure 34), épimère du composé 3c a été isolé et caractérisé. De manière intéressante, le composé 4c n'a pas été détecté au cours de l'étude chimique.

Pour comprendre l'origine du composé 4c, nous en avons réalisé son hémisynthèse par photocycloaddition à partir de l'anchinopeptolide C (3c). Après irradiation du composé 3c pendant 23 h par une lampe riche en UV-B mimant la lumière du soleil, le cyclo-anchinopeptolide C (4c) a été isolé avec un rendement de 23%. Les données spectrales sont en accord complet avec la littérature (Figure 35). À partir de ce produit la détermination de la configuration absolue par dichroïsme circulaire est en cours. Une origine artéfactuelle de ce composé a été émise. biologiques. 17,22,23,24 De cette éponge, les crambescidines 800, 816 et 830 été isolées en quantités suffisantes pour des évaluations biologiques dans le cadre d'une collaboration. Nos collaborateurs ont identifié pour ces trois molécules une activité importante d'inhibitionde la prolifération de cellules colorectales tumorales dans un modèle in vivo chez le poisson-zèbre. 17 De plus, la crambescidine 816 s'est révélée neurotoxique sur des cultures de cellules neuronales. 28 Les crambescines A2 et C1 isolées pendant cette étude ont été utilisées comme standards pour les études de biosynthèse et de synthèse biomimétique réalisées au cours de ce travail de thèse.

Schéma 2. Structures d'alcaloïdes marins de type crambesine et molécules apparentées

Élucidation de la biosynthèse de la crambescine C1 par des études d'incorporation in vivo de précurseurs marqués au 14 C

La synthèse des crambescines a été décrite dans les années 1990 par le groupe du professeur Snider. 44 L'approche a été dite "biomimétique", bien qu'aucune étude de biosynthèse n'ait confirmé l'hypothèse biosynthétique utilisée (Schéma 3). Selon l'approche de Snider et Shi, le squelette guanidinique serait issu de la condensation d'une l'urée sur un fragment d'origine polyacétique. Selon nos hypothèses, le squelette guanidinique central serait issu de la condensation d'un acide gras sur d'un pyrrolidinium dérivant de la L-arginine.

Schéma 3. Hypothèses comparées de biosynthèse des crambescines

Pour démontrer nos hypothèses, nous avons effectué des expériences d'incorporation isotopique par l'éponge C. Crambe en utilisant des précurseurs marqués par du carbone-14 (Figure 37). La crambescine C1 a été choisie comme métabolite cible pour étudier les incorporations de radio-éléments (Schéma 3). En s'appuyant sur les travaux antérieurs de l'équipe, nous avons utilisé le protocole développé pour l'étude de biosynthèse des métabolites l'éponge protocole a montré son efficacité pour contourner de manière optimale les problèmes liés à la manipulationdes éponges en dehors de leur milieu naturel en vue de l'étude d'incorporation. 38 Un résumé est présenté par la Précurseurs utilisés dans les expériences de radiomarquage En s'appuyant sur les travaux antérieurs de l'équipe, nous avons utilisé le protocole développé pour l'étude de biosynthèse des métabolites l'éponge Axinella protocole a montré son efficacité pour contourner de manière optimale les problèmes liés à la manipulationdes éponges en dehors de leur milieu naturel en vue de l'étude d'incorporation.

Résumé en français

Un résumé est présenté par la Figure 38.

pour les expériences de biosynthése in vivo avec les éponges En s'appuyant sur les travaux antérieurs de l'équipe, nous avons utilisé le protocole Axinella damicornis. 30,[START_REF] Genta-Jouve | [END_REF] Ce protocole a montré son efficacité pour contourner de manière optimale les problèmes liés à la manipulationdes éponges en dehors de leur milieu naturel en vue de l'étude d'incorporation. 30, 29, éponges Après les expériences d'incorporation, les éponges ont été extraites par un protocole classique. La crambescine C1 a été choisie comme modèle d'étude car ce métabolite est le plus facile à obtenir à l'état pur. Après deux purifications par HPLC, les échantillons de crambescine C1 purs ont été déposés sur les plaques de silice et ont été analysés par β-imager. L'incorporation de radioéléments a été notée pour les échantillons issus d'éponges alimentées avec de la L-arginine et l'acide laurique marqués (Tableau 10 et Figure 39). Ce résultat a démontré que la guanidine est biosynthétiquement issue de la L-arginine. Le fait que l'acide laurique possède le même nombre de carbones que la chaîne aliphatique ainsi que son incorporation indiquent une incorporation des acides gras nécessaires à la biosynthèse directement par le régime alimentaire. L'absence de radioactivité dans la crambescine C1 provenant des éponges alimentées avec de l'acide acétique marquéne permet pas de conclure. Soit effectivement elle n'est pas incorporée, soit l'acide acétique marqué a été incorporé dans les processus du métabolisme primaire. The IR experiments were obtained using a Bruker Vector 22 spectrometer. The samples were solubilized in MeOH or CHCl 3 , placed on the cell of the equipment and let to evaporate, forming a film. The wavenumbers (ν) are described in cm -1 .

Analytical TLC were carried on Merck silica gel 60 F 254 aluminium plates. After elution, the plates were observed under UV light at 254 nm and stained with sulfuric vanillin solution or Dragendorff's reagent followed by gentle heating. For silica gel chromatography, the flash chromatography was used, with Merck silica gel 60 (230-400 mesh) p.a. grade.

Isolation and Structure Elucidation of Specialized Metabolites from Poecilosclerida Mediterranean Sponges

1 Isolation of metabolites from P. tenacior sponge

Isolation of metabolites

The specimen of P. tenacior was collected manually by SCUBA diving on the Coast of Villefranche-Sur-Mer (France) on July of 2014 at 50 m deep.

The sample was freeze-dried and grated, furnishing a yellow solid (46.1 g). The powder was exhaustively extracted by MeOH : DCM (1 : 1, 3 x 150 mL) and sonication bath (15 min to each extraction) followed by filtration. The filtrates were combined and evaporated under reduced pressure at 40 °C, giving a brown solid (7.00 g). This solid was adsorbed over silica gel RP-18 (7.00 g) and desalted by VLC using the following elution gradient: H 2 O (400 mL, fraction F1, 1.67 g), obtained 14 fractions assigned as: F2P1 (r t = 1.5 -2.5 min, 300 mg), F2P2 (r t = 2.7 min, 5.4 mg), F2P3 (r t = 3.3 min, 8.0 mg), F2P4 (r t = 7.5 min, 66.4 mg), F2P5 (r t = 9.0 min, 131.9 mg), F2P6 (r t = 10. Data are consistent with those reported in literature.

2. Radioactivity TLC and autoradiography beta-imager analyses

F2, F3 and F4 (1 mg of each one) were adsorbed on small spots of a silica TLC plate and had the beta-radioactivity measured by a radio-TLC equipment was accumulated for 1 hour. To access the specific activity, a standard curve of L-[U-14 C]proline was used.

Pure crambescins C1 (0.2 mg) were adsorbed on small spots of a silica TLC plate and measured during 46 hours on a autoradiography beta-imager equipment. It was determined an efficiency of 12.7% applying L-[U-14 C]proline as standard.

Experimental Section

Control Experiments

Into a 700 mL box containing sea water, distilled water, sea water with 2 mL 10% NaClO solution or sea water with 2 mL 10% NaClO and a sponge rock support, it was introduced 37 kBq (1 µCi) of a radiolabelled precursor (lauric acid or agmatine). To avoid external contamination, the samples were let inside a covered box during 20 days and during this period had the betaradioactivity of the water column evaluated by liquid scintillation (1 mL of sample was collected to each essay). After 17 days, the sample containing sea water with NaClO and agmatine was split into two and to the second box was added 2 mL of 10% NaClO solution. The radioactivity of these samples were also analysed by liquid scintillation.

Biomimetic Synthesis of Crambescin A2 and Derivatives

1. Preparation of benzyl 3-oxotetradecanoate (25a)

A mixture of dodecanoic acid (23) (1.01 g, 5.00 mmol) and CDI (0.901 g, 5.50 mmol) in anhydrous THF (30 mL) was stirred under inert atmosphere during 1 h at 0 °C. In another round bottom flask a solution of mono-benzyl malonate (1.08 g, 5.50 mmol) and isopropyl magnesium chloride (2 M in THF, 5.00 mL, 11.0 mmol) in anhydrous THF (20 mL) was stirred 1 h at 0 °C under inert atmosphere. Afterwards the magnesium salt solution was cooled to -20 °C and the reaction containing 23 was poured dropwise into this solution. After 45 min, the mixture was brought to rt. To a solution of 22 (45.0 mg, 0.500 mmol) in DMF (1 mL) was added S-methylisothiourea hemisulfate salt (76.0mg, 0.500 mmol). The white suspension was stirred during 7 days. After evaporation, the crude was suspended in EtOH (2 mL) centrifuged and the supernatant was discarded. This procedure was repeated twice and the remaining solid was dried under reduced pressureto yield 20a * as a white powder (38.6mg, 0.294 mmol, 59%).

* The compound is described but there is any experimental spectroscopic data available.

10. Molecular complexity calculation

The molecular complexity index was calculated by Excel using the formula developed by The main focus of the thesis will be the family of cyclic-guanidine alkaloids "crambescins", produced by the red incrusting sponge Crambe crambe. These alkaloids were discovered in the early 90s and despite the large interest on their biological activities, ecological roles, and synthesis, their biosynthesis is still unknown. The only available biomimetic synthesis of crambescins was based on a fully polyketide origin hypothesis. Recently our groups suggested an alternative biosynthetic hypothesis in which the guanidine-core would be originated from a condensation between a guanidinated pyrrolidinium derived from arginine and a β-keto fatty acid. Based on this hypothesis, we designed a biosynthesis experiment that inspired a biomimetic synthesis route to access the crambescins and derivatives. The insights from these studies are the first experimental conclusions about the biosynthesis of crambescins. Finally, this work leads to a better comprehension of the biochemistry involved in guanidine marine alkaloids of complex structures.

4

 4

Figure 1 .

 1 Figure 1. The phylum-preferences of the marine natural product research community across a 50-year period from 1963 4

Figure 2 .

 2 Figure 2. Halichondrin B and eribulin structures

Figure 3 .

 3 Figure 3. P. tenacior sponge (left) and itsgeographic distribution (right)

13 A

 13 Scheme 1

  Scheme 3

Figure 4 .

 4 Figure 4. Crambe crambe (left) and its natural distribution (right)

Figure 5 .

 5 Figure 5. Sponge C. tailliezi (left) and its geographic distribution (right)

Figure 6 .

 6 Figure 6. General methodology used for the isolation of alkaloids from Poecilosclerida sponges

Figure 7 .

 7 Figure 7. Structure of the new compound epi-anchinopeptolide C isolated from P. tenacior

Figure 8 .between the 1 HFigure 9 .

 819 Figure 8. Comparison between the 1 H-NMR spectra of anchinopeptolide C and epi-anchinopeptolide C

  To finalize the structure and the absolute configuration of cycloanchinopeptolide C, we attempted the cycloaddition of anchinopeptolide C (3c) in similar conditions that Snider's group used to prepare cycloanchinopeptolide D. After irradiation of 3c with a UV-B enriched lamp that mimics sunlight (lamps ReptiSun 10.0 ZOOMED® UV-B (10%) 26W) for 23 h the 1 H-NMR signals correspondent to H-9, H-9', H-10 and H-10' (signals at 6.2 and 7.2 ppm) decreased and new signals on the region of 4 ppm appeared, what indicated the formation of the cycloaddition product (Figure

Figure 10 .

 10 Figure 10. Comparison of 1 H-NMR spectra of anchinopeptolide C (3c) after 23 h and 4 h of artificial sunlight irradiation (top and middle, in D 2 O) and before the irradiation (bottom, in D 2 O)

Chapter 1 │Figure 11 . 1 . 3 . 2

 111132 Figure 11. Comparison between the attributions of cycloanchinopeptolide C obtained by hemisynthesis with the data from the literature

  Scheme 4). The fractions F4-F6 were composed mainly by nonpolar compounds (R t : 9-12 min), probably fatty acids and steroids.

Figure 12 . 1 H 4 Figure 13 .

 121413 Figure 12.1 H-NMR of fractions F2 (top) and F3 (bottom) of C. tailliezi in MeOD-d 4

- 1 ,

 1 injection volume: 20 mL, concentration: 100 mg.mL -1

  Although no condition has provided a satisfactory resolution between the peaks, the purification of F2 was performed as indicated in entry 4, using the following elution: isocratic 0-3 min (65: 35 : 0.1), gradient 3-15 min (from 65 : 35 : 0.1 to 60 : 40 : 0.1), gradient 15-17 min (from 60 : 40 : 0.1 to 40 : 60 : 0.1), isocratic 17-20 min (40 : 60 : 0.1), gradient 20-22 min (from 40 : 60 : 0.1 to 65 : 35 : 0.1) and isocratic 22-25 min (65 : 35 : 0.1) (flow: 10 mL.min -1 , injection volume: 200 mL, concentration: 100 mg.mL -1 ). Similar results were obtained with F3.

Figure 14 . 1 H

 141 Figure 14. Chromatogram of F2 in preparative HPLC

2 NHScheme 5 .

 25 Scheme 5. Snider and Shi "biomimetic" synthesis of cyclic guanidine core of crambescins

Figure 18 .

 18 Figure 18. Detailed structure of crambescin C1

  Cfeeding experiments. In accordance with the biosynthetic hypothesis presented on Scheme 6, C. crambe specimens were fed with the following putative and commercially available precursors: L-[guanidino-14 C]arginine, [guanidino-14 C]agmatine, L-[U-14 C]-lysine, L-[5-14 C]-ornithine, [U-14 C]acetic acid, and [1-14 C]lauric acid.

Figure 19 . Precursors applied on 14 C

 1914 Figure 19. Precursors applied on 14 C-feeding experiments with C. crambe sponge

Figure 20 .

 20 Figure 20.

  : the temperature of the aquaria °C. After one up to three and especially opened oscules indicated that they Biomol. Eng. 2003, 20, 333-337.

Figure 21

 21 Figure 21

2 Figure 22 .

 222 Figure 22. Long term controls using labeled lauric acid (Graphic 1, top) and agmatine (Graphic 2, bottom) (CPMB: beta counts per minute)

Figure 25 and 43 Figure 23 .CChapter 2 │

 2543232 Figure 25 and Table3evidence the incorporation of arginine and lauric acid. Activities associated with agmatine, lysine and acetic acid are presumably contamination, as they are clearly different between the first and second purifications and between the duplicates in case of lysine and acetic acid.

  Angew. Chem. Int. Ed. 2013, 52, 13096-13099. G. Romagnoli, M. D. Verhoeven, R. Mans, Y. Fleury Rey, R. Bel-Rhlid, M. van den Broek, R. Maleki Seifar, A. Ten Pierick, M. Thompson, V. Müller, S. A. Wahl, J. T. Pronk, J. M. Daran, Mol. Microbiol. 2014, 93, 369-389. Insights into the Biosynthesis of 44 hexyl column at 210 nm. The peak at 24.5 min HPLC purification, P2: 2 nd HPLC According to our biosynthetic hypothesis, arginine can be incorporated into the two the autoradiography results are insufficient to arginine is labelled on the guanidine position, it is . Recent reports prove that strains of guanidinobutyric acid from L-arginine. Therefore hexyl unit might be related to a symbiosis with agmatine was not incorporated into crambescin C1 can be either an den Broek, R. Maleki Seifar, A. Ten Pierick, M.

Figure 26 .

 26 Figure 26. Final conclusions and further questions concerning the biosynthesis of crambescin C1

Figure 28 .

 28 Figure 28. Structure of crambescins A2

11 Finally 44 Chapter 3 │ 5 DMAP

 114435 Scheme 11

  Scheme 30

Table 7 .

 7 Condensations between 18 and 25a applying Mannich and Biginelli-type conditions* Entry Conditions Result H 2 SO 4 (5 % v/v), THF, 25a (1.0 eq), rt, 3 h 30 Starting material recovery AcOH (2.5 eq), 25a (1.0 eq), Et 2 O, MeOH, -20 °C -rt, 17 h Starting material recovery morpholine (1.1 eq), AcOH (1.1 eq), 25a (3.0 eq), Na 2 SO 4 (3.6 eq), TFE, 60 °C, 2 days Recovery of starting material morpholine (1.1 eq), AcOH (1.1 eq), 25a (3.0 eq), Na 2 SO 4 (3.6 eq), TFE, 140 °C, sealed tube, 5days Complex mixture morpholine (1.2 eq), AcOH (1.2 eq), 25a (3.0 eq), Na 2 SO 4 (3.6 eq), TFE, reflux, 5 days LC-MS: 45 + coupling products transesterified with CF 3 O-and MeOmorpholine (1.1 eq), AcOH (1.1 eq), 25a (3.0 eq), Na 2 SO 4 (3.6 eq), TFE, 100 °C, microwave (850 W), 1 h LC-MS: low intensity peak corresponding to desired product 45 morpholine (1.1 eq), AcOH (1.1 eq), 25a (3.0 eq), Na 2 SO 4 (3.6 eq), TFE, 150 °C, microwave (850 W), 2 h LC-MS: low intensity peak corresponding to desired product 45 morpholine (1.1 eq), AcOH (1.1 eq), 25a (3.0 eq), Na 2 SO 4 (3.6 eq), TFE, 300 °C, microwave (850 W), 2 h LC-MS: very low intensity peak corresponding to desired product 45 + many peaks of small intensity *The equivalents were estimated considering 100% of conversion of 19 into iminium 18

Figure 29 .

 29 Figure 29. LC-MS of a fraction of the Biginelli-type condensation reaction between 18 and 25a

89 [ 89 ]

 89 A. El-Demerdash, C. Moriou, M.-T. Martin, A. d. S. Rodrigues-Stien, S. Petek, M. Demoy-Schneider, K. Hall, J. N. A. Hooper, C. Debitus, A. Al-Mourabit, J. Nat. Prod. 2016,79, 1929-1937.

3 │

 3 Scheme 38

Figure 31 . 1 HChapter 3 │

 3113 Figure 31. Crambescin A2 448 1 H-NMR spectra 45 (bottom, 500 MHz in MeOH-d 4 ) and 1 H-NMR spectra of isocrambescin A2 448 (5k) (down, 400 MHz in MeOH-d 4 )

Figure 32 .

 32 Figure 32. Attributions of iso-crambescin A2 (5k) in comparison with attributions of crambescin A2 448

Figure 33 .

 33 Figure 33. Comparison between the molecular complexity index of species involved on the synthesis of crambescin A2 (5a) and its isomer (5k)

  Au cours de ce travail, trois éponges marines ont été étudiées : Phorbas tenacior, Crambe crambe et Crambe tailliezi. Ces trois éponges ont pour point commun la présence d'alcaloïdes comportant un motif guanidine. De Phorbas tenacior, différents membres de la famille des anchinopeptolides dont un nouveau composé ont été isolés. De plus, par hémisynthèse le cycloanchinopeptolide C a été synthétisé, ce qui permettra ainsi de déterminer sa configuration absolue. Des deux éponges de la famille Crambeidae, des alcaloïdes du type crambescines et des batzelladines ont été identifiés.

Schéma 1 .Figure 34 .

 134 Figure 34. Structure de l'épi-anchinopeptolide C isolé de P. Tenacior

Figure 35 . 1 . 2

 3512 Figure 35. Comparaison des attributions du cyclo-anchinopeptolide C (synthétique versus naturel)

Figure 36 . 1 . 3

 3613 Figure 36. Fragment de la batzelladine découverte dans la C. taillezi

Figure 37 .

 37 Figure 37. Précurseurs utilisés dans les expériences de radiomarquage

Figure 38 .

 38 Figure 38. Protocole

  les expériences de radiomarquage

Tableau 10 .Figure 39 .stratégie de retrosynthèse de la crambescine A2 448 basé sur nos études de biosynthèse Schéma 4 . 3 . 1 .Schéma 5 . 6 .Schéma 7 .Schéma 8 .

 10394315678 Figure 39. Résultat qualitatif des analyses par β-imager associé aux crambescines C1 (LY: lysine, AG: agmatine, LA: acide laurique, OR: ornithine, AR: arginine, AC: acide acétique, P1: 1 re purification par HPLC, P2: 2 de purification par HPLC)

Schéma 9 . 2 3. 2 .Schéma 10 . 3 . 3 .Schéma 12 .

 922103312 Décarbolylation oxydante de l'arginine N NH NH Étape-clé: assemblage biomimétique du coeur des cambescines L'assemblage biomimétique du coeur central des crambescines a été réalisé par une condensation du type Biginelli modifiée entre le β-céto-ester 25a et le pyrrolidinium 18. Le produit a été obtenu sous la forme d'un mélange de deux isomères, soit avec la double liaison entre les carbones C7 et C13 soit avec la double liaison entre les carbones C7 et C8. De plus un phénomène de transestérification partiel avec le trifluoroéthanol a été observé (Schéma 10). Assemblage biomimétique du coeur des crambescines Les tentatives d'introduction de la chaîne latérale ester pour achever la synthèse de la crambescine A2 448 ont été infructueuses. Les essais ont été réalisés soit par transestérification du mélange de produits 45 et 49 avec l'alcool guanidinique 20a, soit par une séquence de saponification suivie d'une estérification (Schéma 11). La stratégie a été alors modifiée en utilisant directement le fragment 17b, contenant déjà la bonne chaîne latérale pour réaliser l'assemblage biomimétique de la crambescine A2 448. Assemblage biomimétique de la crambescine A2 448 et de son isomère En réalisant l'assemblage biomimétique directement entre le fragment 17b et l'iminium 18, la crambescine A2 448 (5a) a été obtenue en mélange avec l'isocrambescine A2 448 (5k) avec un ratio de 7 pour 3 en défaveur de l'isomère « naturel » (Schéma 12 et Schéma 13). Ainsi en une étape la structure complète comportant le coeur bicyclique et les chaînes latérales a été obtenue avec un rendement de 16 % et la synthèse totale de la crambescine A2 448 a été réalisée avec un total de 4 étapes et un rendement total de 3% (Schéma 14). Synthèse de la crambescine A2 448 mCi.mmol -1 in 2 EtOH : 98 H 2 O), L-[5-14 C]ornithine (American Radiolabeled Chemicals, 55 mCi.mmol -1 in 0.01 N HCl), L-[guanido-14 C]arginine (American Radiolabeled Chemicals, 55 mCi.mmol -1 in 0.01 N HCl), [guanido-14 C]agmatine (American Radiolabeled Chemicals, 55 mCi.mmol -1 in 0.01 N HCl) and [1-14 C]lauric acid (American Radiolabeled Chemicals, 55 mCi.mmol -1 in EtOH). During the feeding experiments, the activity of the water column surrounding the sponges was measured by a liquid scintillation analyser Tri-Carb 2900TR, Perkin Elmer. Sponge extracts were desalted by solid phase extraction (SPE) using silica cartridges Chromabond ® C18, 2000 mg. The samples for beta-radioactivity thin layer (TLC) and autoradiography beta-imager analyses were applied on Merck silica gel 60 F 254 aluminium plates 20 x 20 cm. The betaradioactivity TLC (radio-TLC) analysis was performed by Rita Star equipment (Raytest) and the autoradiography was recorded by a beta-imager Biospace Lab.

After 16 h 4 . 4 .

 1644 of stirring, the reaction was quenched with saturated aq. NH 4 Cl solution(30 mL) and H 2 O (30 mL). The aqueous layer was extracted with Et 2 O (3 x 50 mL). The combined organic phases were dried over Na 2 SO 4 , filtered and the solvent was removed under reduced pressure at 40 °C. The crude product was purified by flash chromatography (cyclohexane : AcOEt, 4 : 1) to furnish the Preparation of 4-guanidinobutanoic acid (31) A mixture of L-arginine (179 mg, 1.03 mmol) and acetone (2 mL) in phosphate buffer (pH= 8, 15 mL) was cooled to 0 °C. Then, a solution of OXONE® (1.85 g, 3.00 mmol) in water (10 mL) was added dropwise. During the addition the pH was maintained between 7.5 and 8.5 by addition of a 0.1 M aq. NaOH solution. The reaction mixture was stirred at rt for 17 h. The excess of oxidant was quenched by addition of Na 2 SO 3 (≈ 50 mg) and the pH was adjusted to 2 with a 2 M aq. HCl solution. The mixture was concentrated under reduced pressure and 45 °C and the residue was washed with BuOH (4 x 2.5 mL). The organic extracts were evaporated under reduced pressure to yield 31 70* as a white solid (70.5 mg, 49 %). *Reference 70 reports preparation of compound 31 but does not describe experimental spectroscopic data. IR, spectra provided by BIORAD Laboratories are available on SciFinder (access: 06/19/2017).

1 H 4 . 5 .

 145 -NMR (300 MHz, MeOH-d 4 ) δ: 3.25 -3.20 (t, J = 7.2 Hz, 2H, H-4), 2.41 -2.37 (t, J = 7.2 Hz, 2H, H-2), 1.91 -1.81 (quint, J = 7.2 Hz, 2 H, H-3) 13 C-NMR (75 MHz, MeOH-d 4 ) δ: 177.1 (C-1), 158.7 (C-5), 41.7 (C-4), 31.8 (C-2), 25.3 (C-3) HRMS (ESI) m/z, calcd for [C 5 H 12 N 3 O 2 ] + : 146.0930, found: 146.0927 Data are consistent with those reported in literature Experimental Section 123 Preparation of 1-(4-hydroxybutyl)guanidine (20a)

4 . 6 .

 46 1 H-NMR (300 MHz, D 2 O) δ: 3.61 (t, J = 6.0 Hz, 2H, H-1), 3.20 (t, J = 6.6 Hz, 2H, H-4), 1.69 -1.53 (br s, 4H, H-2, H-3) 13 C-NMR (75 MHz, D 2 O) δ: 156.7 (C-5), 61.1 (C-1), 40.9 (C-4), 28.4 (C-3), 24.5 (C-2) IR (film) ν/cm -1 1204 (CH 2 ), 1455 (N-H), 1612 (C=N), 2468 (O-H), 3415 (NH 2 ) HRMS (ESI) m/z, calcd for [C 5 H 14 N 3 O] + : 132.1137, found: 132.1132 Preparation of 4-(N,N'-di-t-butoxycarbonylguanidino)-butan-1-ol (20c)To a solution of 22 (0.100 g, 1.10 mmol) in a mixture of formamide (3.00 mL) and 1,4dioxane (3 mL) was added DIPEA (0.450 mL, 2.60 mmol) and 9a (0.484 g, 1.50 mmol). After 17 h of stirring at rt, the reaction mixture was acidified with a 10% aq. HCl solution (5 mL), extracted with AcOEt (4 x 10 mL) and the combined organic phases were washed with brine (10 mL), dried over Na 2 SO 4 , filtrated and evaporated under reduced pressure at 40 °C. The crude was purified by flash chromatography (DCM : MeOH : formic acid, 16 : 1 : 0.1) yielding 20c95 as a white solid (0.246 g, 0.75 mmol, 68%).

1 H 4 . 7 .

 147 -NMR (300 MHz, CDCl 3 ) δ: 11.48 (s, 1H, NH), 8.38 (s, 1H, NHBoc), 3.69 (t, J = 5.9 Hz, 2H, H-1), 3.44 (m, 2H, H-4), 1.70 -1.59 (m, 4H, H-2, H-3), 1.55 -1.44 (m, 18 H, tBu) 13 C-NMR (75 MHz, CDCl 3 ) δ: 163.0 (C-5), 156.4 (C=O), 153.4 (C=O), 83.6 (OC(CH 3 ) 3 ), 80.0 (OC(CH 3 ) 3 ), 62.2 (C-1), 40.6 (C-4), 29.2 (C-2), 28.3 (3C, CH 3 ), 28.2 (3C, CH 3 ), 25.8 (C-3) IR (film) ν/cm -1 1291 (C-O), 1646 (CH 2 ), 1720 (C=O), 2980 (N-H), 3330 (O-H) HRMS (ESI) m/z, calcd for [C 15 H 30 N 3 O 5 ] + : 332.2180, found: 332.2133 Data are consistent with those reported in literature Synthesis of 4-(2,3-bis(tert-butoxycarbonyl)guanidino)butyl 3-oxotetradecanoate (17b)To a solution of 21 (0.214g, 0.880 mmol) in DCM was added 20c (0.292g, 0.880 mmol) and DMAP (11.0 mg, 10 mol %). The temperature was cooled to 0 °C and DCC (0.206 g, 0.970 mmol) was added. The mixture was stirred 5 min at 0 °C and 4 h at rt. By-product urea was removed by filtration and the filtrate was concentrated under reduced pressure. The crude product was purified by flash chromatography (AcOEt : MeOH, 9 : 1) yielding 17b (0.281 g, 0.506 mmol, 54 %) as a colorless oil.

1 H

 1 -NMR (300 MHz, CDCl 3 ) δ: 11.49 (s, 1H, NH), 8.31 (m, 1H, NH), 4.15 (t, J = 5.9 Hz, H-1), 3.42 -3.48 (m, 4H, H-4, H-2'), 2.52 (t, J = 7.4 Hz, 2H, H-4'), 1.71 -1.56 (m, 6H, H-2, H-3, H-5'), 1.49 (d, J = 95 [95] V. Rerat, G. Dive, A. A. Cordi, G. C. Tucker, R. Bareille, J. Amédée, L. Bordenave, J. Marchand-Brynaert, J. Med. Chem. 2009, 52, 7029-7043.

4. 9 . 10 18

 910 Synthesis of crambescin A2 448 (5a) and iso-crambescin A2 (5k)To a solution of 18 (72.3 mg, 0.080 mmol * ) and 17b (59.2 mg, 0.12 mmol) in TFE (15 mL) was added Na 2 SO 4 (41.8 mg, 0.29 mmol), AcOH (6.0µL, 0.10 mmol), and morpholine (8.0µL, 0.10 mmol).The mixture was brought to reflux for 5 days. Sodium sulfate was removed by filtration and the filtrate was concentrated under reduced pressure. The crude product was purified by preparative HPLC [column: C18 Sunfire Waters 30 x 150 mm 5 µm; mobile phase: (H 2 O + 0.2% TFA) : ACN; method: gradient of 30-80% of ACN in 20 min; flow: 42 mL.min -1 ; volume of injection: 300 µL, concentration: 287 mg.mL -1 in MeOH, detection at 280 nm]. The fraction with r t = 9.25 min corresponded to a mixture 7 : 3 of 5k and 5a (5.7 mg, 0.013 mmol, 16%, white solid).* Estimated number of mols considering 100% of L-arginine (19) conversion into 18.

Bertz-Hendrickson 61 1 -

 611 , 63 :C = C η + C E C η = 2η log 2 η -Σ i (S k ) i => C η :coeficient related to skeletal complexity as a function of bond conectivities (η) η: sum of all bonds connectivities η = 1 / 2 (4-h)(3-h) h: number of hydrogens bonded to the atom S k : symmetry term according to symmetry type (k) k = (3h) (2h) + R R: number of repetitions of bonds to identical attached atoms on studied atom A (either σor π-bonds). E.g.: R= 0 if all bonds from A are to different atoms; R= 1 if all bonds goes to the same or to two identical attached atoms; etc Values of S k are available on reference 63 to help hand calculations. C E = E log 2 E -Σ j E j log 2 E j => C E : coeficient related to the diversity of elements (kinds of atoms) E: sum of non hydrogen atoms E j : term related to symmetry. If all atoms attached to a given atom A are of the same type, so C E = 0. NMR Spectra Anchinopeptolide B (3b) ( 1 H-NMR, 500 MHz, MeOH-d 4 ) Isolation and Structure Elucidation of Metabolites from Poecilosclerida Mediterranean Sponges Anchinopeptolide D (3d) ( 1 H-NMR, 500 MHz, MeOH-d 4 ) NMR Spectra -Chapter 1 -Isolation and Structure Elucidation of Metabolites from Poecilosclerida Mediterranean Sponges Anchinopeptolide D (3d) ( 13 C-NMR, 100 MHz, MeOH-d 4 ) NMR Spectra -Chapter 1 -Isolation and Structure Elucidation of Metabolites from Poecilosclerida Mediterranean Sponges 4-(2,3-bis(tert-butoxycarbonyl)guanidino)butyl 3-oxotetradecanoate (17b) ( 1 H-NMR, 300 MHz, CDCl 3 )

Table 1 .

 1 Conditions tested on the purification of F2 and F3

	Entry	Conditions*	Mobile phase
	1	XSelect CSH Prep Phenyl-Hexyl 5 µm OBD	H 2 O : ACN : TFA (0.1%)
	2	XSelect CSH Prep Phenyl-Hexyl 5 µm OBD	H 2 O : ACN : AcOH (0.05%)
	3	XSelect CSH Prep Phenyl-Hexyl 5 µm OBD	H 2 O : MeOH : AcOH (0.05%)
	4	XSelect CSH Prep C18 5 µm	H 2 O : ACN : TFA (0.1%)
	5	Symmetry C18 Prep 7 µM	H 2 O: MeOH : AcOH (0.05%)
	6	Nucleodur C18 Htec 5 µM	H 2 O : ACN
	7	Nucleodur C18 Htec 5 µM	H 2 O : ACN : TFA (0.01%)
	8	Nucleodur C18 Htec 5 µM	H 2 O : ACN: TFA (0.1%)
	9	Nucleodur C18 Htec 5 µM	H 2 O : MeOH : TFA (0.1%)
	10	Nucleodur C18 Htec 5 µM	H 2 O : ACN : Formic Acid (0.1%)
	11	XSelect CSH Prep Fluoro-Phenyl 5 µM	H 2 O : ACN : TFA (0.1%)

*Elution: Semi-preparative columns -isocratic 0-4 min (90 : 10), gradient 4-28 min (from 90 : 10 to 0 : 100), isocratic 28-30 min (0 : 100), gradient 30-32 min (from 0 : 100 to 90 : 10) and isocratic 32-34 min (90 : 10) (flow: 3-4 mL.min -1 , injection volume: 10 mL, concentration: 100 mg.mL -1

core of batzelladine C methyl substituted N NH NH 2 O O 4.55 46.7 1.27 22.6 107 149 152 3.00 2.79 28.7 2.04 1.98 22.0 H N H 2 N NH 3.21 41.7 1.67 29.4 1.75 29.4 4.20 65.1 Figure 16. Fragment of batzelladine discovered in F2P3

		The NMR and HRMS (Figure 17) analyses of F2P10-P14 allowed the identification of the
	already described compounds crambescidin 816 (23g) and crambescidin 800 (23f) (Scheme 4). It is
	worth noticing that C. crambe and C. tailliezi share common secondary metabolites.
	2+ = 401.3142) (above, F2P12) and 816 (23g, + = 817.6161) (bellow, F2P14) Figure 17.HRMS spectra of crambescidins 800 (23f, calcd for [C 45 H 82 N 6 O 6 ] calcd for [C 45 H 81 N 6 O 7 ]
	Conditions: UPLC Ultimate 3000 Dionex equipped with an autosampler and a Dionex Ultimate 3000 diode array dual absorbance
	wavelength detector (210 and 280 nm), linked to QT of mass spectrometer fitted with an electrospray ionization interface (Bruker
	Impact II). Mass spectra were recorded in the positive mode. UPLC conditions: column Nucleodur PolarTec column (100 x 2 mm, 1.8
	µm, Macherey Nagel). Linear elution gradient of H2O : ACN : formic acid to which was added 10 mM of ammonium formate from 80 : 20 : 0.1 (isocratic from 0 to 2 min) to 40 : 60 : 0.1 (isocratic from 8 to 10 min), flow rate: 0.45 mL.min -1 for a total of 14 min.
	Injected volume: 10 µL. Detection: 280 nm.
	27 [27]	R. Laville, O. P. Thomas, F. Berrué, D. Marquez, J. Vacelet, P. Amade, Journal of Natural Products 2009, 72, 1589-1594.

Table 2 .

 2 Specific activity of SPE fractions from C. crambe sponges fed with

	14 C-labeled precursors

14 C]Lauric Acid 0.58 7.81 49.06 1.18 3.85 66.47 4.90 7.64 96.88

Table 3 .

 3 Activities of pure crambescin C1 isolated from sponges fed with

	14 C-precursors

28 29 30 29 23 25 26 27 24 Mechanism -C-acylation of lauric acid promoted by CDI

															Scheme 14 Scheme 16
	The first reaction step is the nucleophilic addition of the hydroxy group from carboxylic acid 23 to carbonyl of carbonyldiimidazole (CDI, 26), forming a sp 3 intermediate 27 (Scheme 15). The 3. 3. 2 Synthesis of fragment 17
	N O sp O C 11 H 23	N H	O	N	N				C 11 H 23	O	O N	O N	H N	N	N	HO	O N	H	O C 11 H 23 10	O	O	O	N	N	CO 2	C 11 H 23	O	N	N
																	Mg 2+
	Cl	Mg			H	O	O	H	O	OR	-MgCl 2 -2 iPr		O	O	O	OR		C 11 H 23	O	N	H N	CO 2	C 11 H 23	O	O	OR
				Cl	Mg																				HN	N
															Scheme 15

(Scheme 14)

. Two malonates were employed: benzyl malonate and ethyl malonate, furnishing the desired β-keto-fatty ester 25 in similar yields. 69

[69]

D. W. Brooks, L. D. L. Lu, S. Masamune, Angew. Chem. Int. Edit. 1979, 18, 72-74. 2 hybridization is re-established with the departure of imidazole followed by deprotonation of oxonium ion. Subsequently intermediate 28 suffers an intramolecular nucleophilic attack of the imidazole nitrogen on carbonyl group. During this second nucleophilic attack there is a CO 2 liberation, a driving force that shifts all previous equilibriums to formation of 29, just as in the fundamental steps of polyketide biosynthesis. Suspension containing 29 is then added into malonate magnesium salt mixture and the enol 30 attacks the carbonyl-imidazole 29. Desired 25 is obtained after a decarboxylation and departure of imidazole as leaving group. The β-keto-fatty esters were converted into carboxylic acid 21 by hydrogenation of 25a catalyzed by Pd/C or saponification of 25b (Scheme 16). Both ways were effective, furnishing the desired product in excellent yields.

  Chavan and co-authors74 described a procedure for the transesterification of β-ketoesters promoted by zinc and iodine that was applied to access 17a (Scheme 23). The HRMS of the crude mixture presented a peak that could correspond to 17a ([C 19 H 37 N 3 O 3 +H] + calculated= 356.2908, experimental= 356.2903). One interesting finding was a peak at m/z 338.2784, 18 units less than

	the m/z of 17a, that could be generated by a water elimination on 34 (Scheme 24). This
	elimination is possible if a formation of a terminal hydroxyl takes place, such as in 34, so this
	finding may indicate that the NH from guanidino function of 20a, instead of the OH, had attacked
	the carbonyl group. To assure the desired chemoselectivity it was decided to try the
	transesterification with protected substrate 20c.
	Scheme 23
	Scheme 24

Scheme 25. Attempt of Taber's transesterification of 25b and TLC result 3. 3. 2. 3 Formation of fragment 17

	Chapter 3 │ Biomimetic Synthesis of Crambescin A2 and Derivatives Biomimetic Synthesis of Crambescin A2 and Derivatives
	protected version. Instead, it was it was found a peak of starting ester 25b ([C 16 H
	293.2087, experimental: 293.2125) 293.2087, experimental: 293.2125) mixed with the aldol product 37 ([C 32 H 60
	563.4282, experimental: 293.4308 563.4282, experimental: 293.4308). Formation of aldol product can explain the appearance of a
	spot less polar than starting ester polar than starting ester 25b on TLC analysis.
		H
	RHN	N	OH
	NR	20a: R= H
		20c: R= Boc
	At this point, we consider
	carboxylic acid 21 instead of a transesterification reaction
	It was noticed that 20a
	experiments of entries 4-6. Also,
	eluents and at the end the stationary phase was always cleaned with AcOEt or MeOH,
	that did not avoid losing the product.
	Following the reaction evolution was another problem taking into account
	invisible under UV and did not furnish spots in any of tested TLC stains: I

74] S. P. Chavan, K. Shivasankar, R. Sivappa, R. Kale, Tetrahedron Lett. 2002, 43, 8583-8586. 75 [75] D. F. Taber, J. C. Amedio, Y. K. Patel, J. Org. Chem. 1985, 50, 3618-3619. sodium nitroprussate / potassium hexacyanoferrate(III), an product formation, we decided to keep trying coupling reactions but using protected substrate 20c. The Boc groups would also furnish a chromophore to . Attempt of Taber's

transesterification of 25b and TLC result Formation of fragment 17 promoted by coupling reagents

	Biomimetic Synthesis of Crambescin A2 and Derivatives
							H 30 O 3 + Na] + calculated:
							60 O 6 + Na] + calculated:
	ormation of aldol product can explain the appearance of a
	O	O				H	O
	HO	21	10	RHN	NR	N	O 17a: R= H
							17b: R= Boc
	we considered, as an alternative, a coupling between alcohol
	transesterification reaction (Table 5).
	20a was insoluble in most of organic solvents,
	, the chromatographic purifications were performed with different
	the end the stationary phase was always cleaned with AcOEt or MeOH,
	that did not avoid losing the product. Even Sephadex purifications did not allow
	Following the reaction evolution was another problem taking into account
	UV and did not furnish spots in any of tested TLC stains: I 2 , vanilline, Dragendorf,
							63

sodium nitroprussate / potassium hexacyanoferrate(III), and ninhydrin. As HRMS analysis indicated product formation, we decided to keep trying coupling reactions but using protected substrate furnish a chromophore to the desired β-ketoester and decrease its . Attempt

of Taber's transesterification of 25b and TLC result

  Biomimetic Synthesis of Crambescin A2 and Derivatives The mechanism of this reaction is presented on Scheme 27. The reactivity of the carboxyl function from 21 is enhanced to nucleophilic attack by N,N'-dicyclohexylcarbodiimide (DCC), forming O-acylisourea intermediate 38. Catalyst DMAP, a stronger nucleophile than alcohols, reacts with protonated intermediate 38 giving amide 39. The acyl group is then transferred to 20c by a nucleophilic substitution leading to 17b.

	Scheme 27
	O	O
	3. 3.	10
	H
	Boc
	coupling between alcohol 20a and
	most of organic solvents, explaining the
	the chromatographic purifications were performed with different
	the end the stationary phase was always cleaned with AcOEt or MeOH, a procedure
	did not allow a good separation.
	Following the reaction evolution was another problem taking into account that the product is
	, vanilline, Dragendorf,

d ninhydrin. As HRMS analysis indicated product formation, we decided to keep trying coupling reactions but using protected substrate ketoester and decrease its Chapter 3 │

3 Synthesis of guanidinated pyrrolidinium 18

Table 6 .

 6 Reduction of nitrile 41 into aldehyde 33

	Entry		Conditions	Result
	1	DIBAL-H (10 eq), DMSO : THF (1 : 5), -78 o C, 3 h	Starting material recovery
	2	Pd/C (10% w/w), EtOH, rt, H 2 , 42 h	Starting material recovery
	3 80	Et 3 NH w/w), THF : EtOH (2 : 1), 0 °C, 2 h 30 + H 2 PO 2-. 1.5 H 2 O, Ni-Raney (4 : 1,	Starting material recovery	( * )
	4	Et 3 NH w/w), THF : EtOH (1 : 2.5), 0 °C, 7 h + H 2 PO 2-. 1.5 H 2 O, Ni-Raney (25 : 1,	Starting material recovery
	5	Et 3 NH	+ H 2 PO w/w), EtOH, rt, H 2 , 42 h 2-. 1.5 H 2 O, Ni-Raney (25 : 1,	Starting material recovery
	6	Ni-Raney (5 : 1, w/w), HCOOH 75 % (v/v), reflux, 1 h	Checked by HRMS.
				[C 5 H 14 N 4 +H] + calculated: 131.1291,
				experimental: 131.1297
	81 7	Ni-Raney (1 : 1, w/w), HCOOH 75 % (v/v), reflux, 1 h	33 and 18 crude checked by 1 H-RMN and HRMS

* 1 H-NMR presented low intensity peaks corresponding to aldehyde 33

Table 7

 7

	, entry 5) was purified by flash

85

[85]

Z. D. Aron, L. E. Overman, Chem. Comm. 2004, 253-265. 86 [86] B. L. Nilsson, L. E. Overman, J. Org. Chem. 2006, 71, 7706-7714. 87 [87] S. K. Collins, A. I. McDonald, L. E. Overman, Y. H. Rhee, Org. Lett. 2004, 6, 1253-1255. 88 [88] C. O. Kappe, Acc. Chem. Res. 2000, 33, 879-888.

Table 8 .

 8 Transesterification of the mixture containing 45

	Entry	Conditions	Result
	1	Amberlyst 15 (10% w/w), 20a (1 eq), toluene, reflux, 18 h	Recovery of starting materials
	2	K 2 CO 3 (0.5 eq), 20a (3 eq), DMF, 100 °C, 1 day	Recovery of starting materials
	3	Amberlyst 15 (10% w/w), 20a (3.9 eq), toluene, 150 °C, sealed tube, 18 h	Complex mixture
	4	Amberlyst 15 (10% w/w), 20a (3.9 eq), DMF, reflux, 2 days	Complex mixture

Table 9 .

 9 Saponification of mixture containing 45b

	Entry	Conditions	Result
			LC-MS: 25a and
	1	KOH (4 M), MeOH, rt, 1 day
	2	LiOH (2.0 eq), MeOH : H 2 O (1 : 1), reflux, 2 h	25a

  35% of ACN in 30 min (column: C18 Sunfire Waters 19 x 150 mm, 5 µm; flow: 17 mL.min -1 ; concentration: 63.5 mg.mL -1 in MeOH, injection volume: 200 µL; detection at 254 nm).

	Experimental Section
	13 C-NMR (125 MHz, MeOH-d 4 ) δ: 177.0, 174.7, 172.1, 170.2, 159.1, 159.0, 158.0, 157.9, 129.3, HRMS (ESI) m/z, calcd for [C 33 H 45 N 10 O 8 ] + : 709.3416, found: 709.3365
	3 min, 129.2, 128.2 (2C), 128.1 (2C), 121.7, 121.5, 116.9 (2C), 116.8 (2C), 116.3, 115.3, 92.5, 77.2, 53.8,
	39.3 mg), F2P7 (r t = 11.0 -11.5 min, 29.4 mg), F2P7a (r t = 12.3 min, 7.8 mg), F2P7b (r t = 13.0 min, 5.6 52.1, 46.9, 42.8, 41.0, 34.8, 25.1, 24.7, 17.9, 15.0 Data are consistent with those reported in literature
	mg), F2P8 (r t = 15.5 min, 63.5 mg), F2P8a (r t = 17.0 min, 3.2 mg, anchinopeptolide A, 3a), F2P8b (r t =
	19.0 min, 18.2 mg), F2P9 (r t = 21.5 min, 17.7 mg), and F2P10 (r t = 23.3 min, 2.2 mg). Samples F2P4, [α] D 20 = -40 (c= 0.05, MeOH) (literature 11 : [α] D 20 = -103.6, c= 4, MeOH) anchinopeptolide D 13 (3d)
	F2P5, and F2P8 were purified on HPLC. Fraction F2P4 was purified using as eluent (H 2 O + 0.2% 1 H-NMR (500 MHz, MeOH-d 4 ) δ: 7.23 (d, J = 14.5, 1H), 7.21 (d, J = 14.5, 1H), 7.14 (d, J = 8.5, 4H),
	TFA) : ACN and applying a gradient of 15 -35% of ACN in 20 min (column: C18 Sunfire Waters 19 x HRMS (ESI) m/z, calcd for [C 34 H 47 N 10 O 8 ] + : 723.3573, found: 723.3627 6.18 (d, J = 14.5, 1H), 6.17 (d, J = 14.5, 1H), 4.22 (d, J = 16.5, 1H), 4.12 (d, J = 16.5, 1H), 3.91 (d, J =
	150 mm, 5 µm; flow: 17 mL.min -1 ; concentration: 66.4 mg.mL -1 in MeOH, injection volume: 200 µL; 16.5, 1H), 3.89 (d, J = 16.5, 1H), 3.49-3.43 (m, 1H), 3.27-3.22 (m, 1H), 3.21-3.15 (m, 2H), 2.68 (dd,
	detection at 254 nm). Pure anchinopeptolide A (9.0 mg, 3a) was eluted at r t = 8.2 min. Sample Data are consistent with those reported in literature J = 4.5 and 10.3, 1H), 2.10-2.01 (m, 1H), 1.89-1.83 (m, 2H), 1.82-1.74 (m, 2H), 1.58-1.50 (m, 1H)
	F2P5 was purified using as eluent (H 2 O + 0.2% TFA) : MeOH and employing a gradient of 30 -45%
	of MeOH in 20 min (column: C18 Sunfire Waters 30 x 150 mm, 5 µm; flow: 42 mL.min -1 , anchinopeptolide B 13 (3b) 13 C-NMR (100 MHz, MeOH-d 4 ) δ: 177.8, 173.6, 168.3, 168.0, 158.7, 157.8, 128.7, 128.7, 127.9 (2C),
	concentration: 131.9 mg.mL -1 in MeOH, injection volume: 200 µL, detection at 254 nm). Pure 127.8 (2C), 121.0, 120.8, 116.6 (4C), 116.1, 115.6, 91.1, 76.6, 46.7, 45.0, 43.4, 42.5, 40.8, 34.6,
	anchinopeptolide D (3d) (r t = 9.0 min, 36.1 mg) and anchinopeptolide C (3c) (10.2 min, 67.6 mg) 24.6, 24.0
	were isolated. Fraction F2P8 was purified using as eluent (H 2 O + 0.2% TFA) : ACN and applying a
	6.5, 3H) [α] D 20 = +86 (c= 0.07, MeOH) (literature 13 : [α] D 20 = +11.4, c= 1, MeOH) gradient of 15 -Pure epi-anchinopeptolide C (3e) (r t = 15.0 min, 10.6 mg; r t = 15.5 min, 8.6 mg) was obtained. HRMS (ESI) m/z, calcd for [C 32 H 43 N 10 O 8 ] + : 695.3260, found: 695.3225
	HRMS (ESI) m/z, calcd for [C 33 H 45 N 10 O 8 ] + : 709.3416, found: 709.3497
	(S,E)-4-amino-5-((2-((4-hydroxystyryl)amino)-2-oxoethyl)amino)-5-oxopentanoic acid 12 (5) Data are consistent with those reported in literature
	Data are consistent with those reported in literature 1 H-NMR (500 MHz, MeOH-d 4 ) δ: 7.25 (d, J = 14.5 Hz, 1H), 7.15 (d, J = 8.5 Hz, 2H), 6.74 (d, J = 8.5 Hz, epi-anchinopeptolide C (3e) 2H), 6.18 (d, J = 14.5 Hz, 1H), 4.25 (m, 1H), 3.96 (s, 2H), 2.53-2.40 (m, 2H), 2.36-2.29 (m, 1H), 2.19-2.13 (m, 1H) anchinopeptolide C 13 (3c) IR (film) ν/cm -1 3400 (O-H), 1673 (C=C and N-H), 1466 (C-N), 1242 and 1204 (C-O)
	Data are consistent with those reported in literature 1 H-NMR (500 MHz, MeOH-d 4 ) δ: 7.22 (d, J = 14.5, 1H), 7.21 (d, J = 14.5, 1H), 7.15 (d, J = 8.5, 1H), 7.12 (d, J = 8.5, 2H), 6.69 (d, J = 8.5, 2H), 6.68 (d, J = 8.5, 2H), 6.19 (d, J = 14.5, 1H), 6.16 (d, J = 14.5, HRMS (ESI) m/z, calcd for [C 33 H 45 N 10 O 8 ] + : 709.3416, found: 709.3445
	1H), 4.37 (q, J = 7.3, 1H), 4.17 (d, J = 17, 1H), 3.28-3.24 (m, 2H), 3.22-3.19 (m, 2H), 2.72 (m, 1H),
	anchinopeptolide A 11 (3a) 2.09-1.94 (m, 3H), 1.60-1.54 (m, 1H), 1.46 (d, J = 7, 3H) [α] D 20 = -54 (c= 0.5, MeOH)
	1 H-NMR (500 MHz, MeOH-d 4 ) δ: 7.15 (d, J = 15 Hz, 2H), 7.02 (d, J = 8.5 Hz, 4H), 6.63 (d, J = 8.5 Hz,
	2H), 6.62 (d, J = 8.5 Hz, 2H), 6.19 (d, J =14.5 Hz, 1H), 6.04 (d, J = 14.5 Hz, 1H), 4.42 (q, J = 7.0 Hz, 13 C-NMR (100 MHz, MeOH-d 4 ) δ: 177.9, 172.6, 172.0, 168.0, 158.7 (2C), 157.8, 157.7, 128.7, 128.9,
	1H), 4.12 (q, J =7.0 Hz, 1H), 3.28-3.16 (m, 2H), 3.14 (t, J = 5.0 Hz, 2H), 2.70 (t, J = 7.5 Hz, 1H), 1.99 -127.9 (2C), 127.8 (2C), 121.1, 120.8, 116.5 (4C), 116.1, 115.8, 90.9, 76.6, 51.3, 46.5, 44.9, 42.5,
	1.92 (m, 2H), 1.85-1.72 (m, 2H), 1.56 (d, J = 7 Hz, 3H), 1.49 (d, J =7.0 Hz, 3H), 1.46-1.40 (m, 1H) 40.6, 34.9, 24.6, 24.4, 17.7
	[α] D 20 = -35 (c= 0.09, MeOH) (literature 13 : [α] D 20 = -6.3, c= 1, MeOH)
	109

1 H-NMR (500 MHz, MeOH-d 4 ) δ: 7.26 (d, J = 15, 1H), 7.21 (d, J = 14.5, 1H), 7.16 (d, J = 8.5, 2H), 7.11 (d, J = 8.5, 2H), 6.71 (d, J = 8.5, 2H), 6.68 (d, J = 9, 2H), 6.20 (d, J = 14.5, 1H), 6.05 (d, J = 14.5, 1H), 4.28 (d, J = 16, 1H), 4.01 (q, J = 7, 1H), 3.90 (d, J = 16, 1H), 3.63-3.59 (m, 1H), 3.45-3.41 (m, 1H), 3.14-3.10 (m, 2H), 2.64 (dd, J = 5.5 and 9.0, 1H), 2.09-1.94 (m, 4H), 1.77-1.70 (m, 2H), 1.55 (d, J =

Table 12 .

 12

				1 H-and	13 C-NMR data of compound 3e in DMSO-d
	attribution	δ (ppm)	1 H	multiplicity	J (Hz)	integration	δ (ppm)	13 C
	1	-			-	-	-	157.4
	2	3.14-3.10		m	-		40.8
	3a	1.66-1.60		m	-		23.3
	3b	1.41-1.45		m	-		23.3
	4a	1.69-1.68		m	-		31.2
	4b	1.56-1.50		m	-		31.2
	5	-			-	-	-	76.1
	6	-			-	-	-	174.3
	7a	3.97			d	16		41.7
	7b	3.63			d	15.5		41.7
	8	-			-	-	-	165.9
	9	7.13			s	-		120.3
	10	6.11			d	14.5		112.6
	11	-			-	-	-	127.8
	12, 16	7.16			s	-		126.4
	13, 15	6.69			d	8.5		115.5
	14	-			-	-	-	157.0
	N A -H	7.56			br t	5.5		-
	N C -H	10.14			d	10		-
	5-OH	5.52			s	-		-
	14-OH	9.40			s	-		-
	1'	-			-	-	-	156.7
	2'	3.24-3.20		m	-		39.2
	3'a	1.56-1.50		m	-		23.4
	3'b	1.66-1.60		m	-		23.4
	4'	2.30			dd	5.5 and 10.5		48.4
	5'	-			-	-	-	90.7
	6'	-			-	-	-	171.0
	7'	4.33			t	6.5		49.2
	8'	-			-	-	-	169.5
	9'	7.17			s	-		120.6
	10'	6.14			s	15		112.9
	11'	-			-	-	-	127.8
	12', 16'	7.16			s	1		126.4
	13', 15	6.69			d	8.5		116.0

Table 14 .

 14 NMR shifts of synthetic and natural cycloanchinopeptolide (4c) in MeOH-d 4 .7, 67.1, 56.1, 54.7, 54.1, 43.8, 38.5, 38.0, 37.6, 37.4, 34.1, 32.8, 32.7, 32.4, 30.9, 30.8, 27.0, 26.6, 26.5, 24.5, 21.7, 19.0, 11.0 HRMS (ESI) m/z, calcd for [C 45 H 81 N 6 O 7 ] + : 817.6161, found: 817.6134

		Hemisynthetic	Natural product 13		Hemisynthetic	Natural product 13
	attribution	δ (ppm)	1 H	∆ ppm	δ (ppm)

1 H-NMR (500 MHz, MeOH-d 4 ) δ: 5.71 (br t, J = 9, 2H), 5.50 (br d, J = 11, 1H), 4.45-4.38 (m, 1H), 4.37-4.30 (m, 1H), 4.17

-4.11 (m, 2H), 3.97-3.90 (m, 1H), 3.69-3.59 (m, 1H), 3.50-3.59 (m, 1H), 3.44-3.36 (m, 1H), 3.16-3.06 (m, 2H), 2.97-2.94 (m, 1H), 2.91-2.84 (m, 1H), 2.64 (dd, J = 5 and 13, 1H), 2.51-2.37 (m, 4H), 2.19-2.13 (m, 1H), 2.05-1.80 (m, 9H), 1.73-1.56 (m, 10H), 1.43 (t, J = 12.5, 13 C-NMR (125 MHz, MeOH-d 4 ) δ: 177.5, 168.8, 149.7, 134.2, 131.2, 90.6, 85.2, 84.6, 72.4, 70.0, 91 [91] N. C. Gassner, C. M. Tamble, J. E. Bock, N. Cotton, K. N. White, K. Tenney, O. R. P. St, M. J. Proctor, G. Giaever, R. W. Davis, P. Crews, T. R. Holman, R. S. Lokey, J. Nat. Prod. 2007, 70, 383-390.

68

Isolation of metabolites from C. crambe sponge

  Specimens of Crambe crambe were manually collected by SCUBA diving on the coast of Villefranche-Sur-Mer (France) on November 2014 at deeps ranging from 10 to 25 m and kept frozen until used. The sponges were freeze-dried and grated, furnishing a red solid (24.2 g). This powder was purified according to the procedure described on literature45 . Compounds sent to biological assays were identified by NMR and HRMS (95% of purity, determined by HPLC).

3.

14 C-Feeding Experiments with C. crambe Sponge: Initial Insights into the Biosynthesis of Crambescin C1 3. 1. Sponges Collection and in vivo feeding experiments with 14 C-labelled precursors

  For the feeding experiments, the sponges were placed in 700 mL round plastic boxes in presence of the radiolabelled precursor (37 kBq= 1 µCi per spike, 10 µL of precursor each one) during 18 h. The activity of a 1 mL sample of the water column surrounding the sponge was analysed by liquid scintillation just after injecting the precursor and just before opening the water system to the metabolization period in order to follow the percentage of activity disappearance. After the incubation with the precursor, the sponges were removed from the plastic boxes, fed with phytoplankton and let in open water system aquaria during at least 6 hours. After 10 cycles of incubations followed by metabolization period (8 cycles for acetate), the sponges were freeze, lyophilized and extract 3 times with 30 mL of MeOH under ultrasound during 15 minutes. The samples were centrifuged and the supernatants were concentrated under reduced pressure at 40°C and fractionated by SPE by a step gradient of H 2 O (F1), H 2 O : MeOH 1:1 v/v (F2), H 2 O : MeOH 1:3 v/v (F3), and MeOH (F4). Fraction F2 was purified by semi-preparative HPLC (RP column

	Phenomenex Luna phenyl-hexyl, 250 x 10 mm, 5 µm) eluted with H 2 O/ACN/TFA as following:
	isocratic elution 0-3 min (90 : 10 : 0.1), gradient elution 3-5 min (from 90 : 10 : 0.1 to 65 : 35 : 0.1),
	isocratic elution 5-10 min (65 : 35 : 0.1), gradient elution 10-30 min (from 65 : 35 : 0.1 to 55 : 45 :
	0.1), isocratic elution 30-34 min (55 : 45 : 0.1), gradient elution 34-36 min (from 55 : 45 : 0.1 to 90 :
	10 : 0.1), and isocratic 36-45 min (90 : 10 : 0.1), applying a flow rate of 3 mL.min -1 , to give 7
	fractions, named as: F2P1 (r t = 9.0-11.0 min), F2P2 (r t = 26-27 min), F2P3 (r t = 28.0-29.5 min), F2P4
	(r t = 30-32 min), F2P5 (r t = 32-34 min), F2P6 (r t = 34-36 min) and F2P7 (r t = 36.5-39 min). The F2P2
	was identified by NMR as crambescin C1 45 and was submitted to further HPLC purification (RP
	column C18 HTec, 250 x 4.6 mm, 5 µm, analytical column) applying an isocratic elution of H 2 O :
	ACN : TFA (57 : 43 : 0.1) during 20 min, which afforded the pure compound (r t = 11 min).
	crambescin C1 45
	5,
	2H), 3.17 (t, J = 7, 2H), 2.88-2.76 (m, 2H), 1.81 (quint, J = 6.5, 2H), 1.73-1.70 (m, 2H), 1.62-1.58 (m,
	5H), 1.45-1.38 (m, 8H), 1.36-1.26 (m, 14H), 0.90 (t, J = 6.5, 3H).

C. Crambe sponges were collected at 50 m deep of the Coast of Villefranche-Sur-Mer (France) by SCUBA diving, on June 2015. The specimens were acclimatized in open system aquaria supplied with sea water during 3 weeks (average values for water parameters: pH= 8.11, T= 18°C, dissolved oxygen= 105.3%), being daily fed with phytoplankton Isochrysis Galbana.

1 H-NMR (500 MHz, MeOH-d 4 ) δ: 4.41 (dd, J = 4.5 and 7.0, 1H), 4.24-4.13 (m, 2H), 3.62 (t, J = 6.

Table 15 .

 15 IR (film) ν/cm-1 1135 (C-N), 1204 (C-O), 1681 (C=O and C=N), 2855 (N-H), 2926 (N-H) HRMS (ESI) m/z, calcd for [C 24 H 45 N 6 O 2 ] + : 449.3599, found: 449.3628

				1 H-and	13 C-NMR of compound 5k in MeOH-d 4
	attribution	δ (ppm)	1 H	multiplicity	J (Hz)	integration	δ (ppm)	13 C
	1	-		-		-	-	158.7
	2	3.22		t		7.0	2	42.0
	3a	1.63 -1.69	m	-	2	27.0
	3b	1.70 -1.78	m	-	4	27.0
	4	1.98 -2.10	m	-	2	23.0
	5	4.21		t		6.4	2	65.1
	6	-		-		-	-	166.2
	7	-		-		-	-	104.2

  4-(2,3-bis(tert-butoxycarbonyl)guanidino)butyl 3-oxotetradecanoate (17b) ( 13 C-NMR, 75 MHz, CDCl 3 ) Chimie et biosynthèse de substances naturelles hautement complexes de la biodiversité méditerranéenne Mots clés: substances naturelles, éponges marines, alkaloïdes, biosynthèse, synthèse biomimétique Résumé: Le but de ce travail de doctorat est l'étude chimique et biosynthétique de familles d'alcaloïdes guanidiniques d'origine marine provenant d'éponges de Méditerranée. Le travail est divisé en trois parties successives : 1) l'isolement d'alcaloïdes produits par des éponges marines de l'ordre des Poeciloscerida; 2) l'élucidation de la biosynthèse de la crambescine C1 par des études in vivo d'incorporation de précurseurs marqués au 14 C; 3) la synthèse biomimétique de la crambescine A2 448 et de dérivés proches. La famille des alcaloïdes guanidiniques cycliques des crambescines est au coeur de la thèse, ces substances naturelles sont produites par l'éponge incrustante Crambe crambe. Ces alcaloïdes ont été découverts dans les années 1990 et ont suscité beaucoup d'intérêt pour leurs propriétés biologiques et écologiques et leurs synthèses totales. Par contre, leur bio-synthèse était encore inconnue à ce jour. La seule synthèse biomimétique disponible était basée sur une hypothèse d'origine polyacétique. Les hypothèses récentes de nos groupes ont permis de mettre en avant une origine mixte: la partie cyclique guanidinique proviendrait d'un pyrrolidinium issu de l'arginine et d'un précurseur "céto-acide" proche des acides gras. Sur la base de cette hypothèse, nous avons mis au point une expérience d'incorporation qui a ensuite inspirée une voie de synthèse biomimétique pour l'accès aux crambescines et dérivés. Les premières conclusions quant à l'origine biosynthétique de ces molécules sont les faits les plus marquants de ce travail. Nous apportons une meilleure compréhension de la biochimie des alcaloïdes guanidiniques marins de structures complexes. This thesis aims at the study of the chemical and biogenetic origin of specialized guanidine-alkaloid metabolites produced by sponges from the Mediterranean Sea.

	Titre: The work is divided into three main parts:	BocHN	H N NBoc	17b	O	O	O
	1) isolation of alkaloids produced by sponges
	of the Poeciloscerida order;
	2) biosynthesis of crambescin C1 by in vivo
	14 C-feeding experiments with Crambe crambe
	sponge;
	3) biomimetic synthesis of crambescin A2 448
	and derivatives.

Title: Chemistry and biosynthesis of highly complex marine alkaloids from Mediterranean biodiversity

Keywords: natural products, marine sponges, alkaloids, biosynthesis, biomimetic synthesis Abstract:

 [4] J. W. Blunt, B. R. Copp, R. A. Keyzers, M. H. G. Munro, M. R. Prinsep, Nat. Prod. Rep. 2015, 32, 116-211.SCUBA diving Snorkeling

Scheme 4

[26] J. Vacelet, N. Boury-Esnault, Trav. Sci. Parc Natl. Port-Cros 1982, 8, 107-113.

Scheme 6

Résumé en Français

Schéma 11. Tentatives de transestérification de 45 et 49

Schéma 13. Analyse RMN de la crambescine A2 448 et de son isomère

Schéma 14. Bilan de la synthèse biomimétique de la crambescine A2 448

H-NMR (300 MHz, CDCl 3 ) δ: 4.19 (q, J = 7.2 Hz,

2H, OCH 2 ), 3.42 (s, 2H, H-2), 2.52 (t, J = 7.2 Hz, 2H,

Cycloanchinopeptolide C (4c) (HSQC-2D-1 H and13 C-NMR correlations, 400 MHz to 1 H nucleus, MeOH-d 4 )

1-(4-hydroxybutyl)guanidine (20a) (JMOD-13 C-NMR, 75 MHz, D 2 O)

4-(N,N'-di-t-butoxycarbonylguanidino)-butan-1-ol (20c) ( 13 C-NMR, 75 MHz, CDCl 3 )

Acknowledgements i Acknowledgements I acknowledge to Prof. Dr. Michel Rohmer, member of the Académie des Sciences, for the honour of accepting to participate of my PhD jury. I acknowledge to Prof. Dr. Soizic Prado and Dr. Grégory Genta-Jouve for accepting the invitation to evaluate this work as rapporteurs. I acknowledge to Prof. Dr. Pierre Champy for accepting the invitation to judge this thesis and for being the president of the PhD jury. I kindly thank you all members of the jury for their valuable contribution to improve this work through the discussion. I would like to acknowledge Prof. Dr. Erwan Poupon and Prof. Dr. Olivier Thomas for Anchinopeptolide A (3a) ( 1 H-NMR, 500 MHz, MeOH-d 4 ) NMR Spectra -Chapter 1 -Isolation and Structure Elucidation of Metabolites from Poecilosclerida Mediterranean Sponges Anchinopeptolide C (3c) ( 1 H-NMR, 500 MHz, MeOH-d 4 ) NMR Spectra -Chapter 1 -Isolation and Structure Elucidation of Metabolites from Poecilosclerida Mediterranean Sponges Anchinopeptolide C (3c) ( 13 C-NMR, 100 MHz, MeOH-d 4 ) NMR Spectra -Chapter 1 -Isolation and Structure Elucidation of Metabolites from Poecilosclerida Mediterranean Sponges crambescin A2 448 (5a) and iso-crambescin A2 448 (5k) (HMQC-1 H and 13 C-NMR, 400 MHz and 100 MHz, MeOH-d 4 ) References References

Abbreviations

Abbreviations

ACN : acetonitrile

Scheme 20

To reduce the number of steps, we decided to obtain 20a by guanylation of commercial amino alcohol 22 (Table 4). Two kinds of guanylating agents were tried: a thiourea 71 and a sulfinic acid 72 . Thiourea was the one that furnished desired 20a in higher yield. Predicting that protected guanidine could be necessary to obtain chemoselectivity or to simplify purification in the following steps, we also synthesized the N,N'-bis protected guanidine 20c by reaction of commercially available N, N' bis-Boc-1-guanylpyrazole 9a with amino alcohol 22 (Scheme 21).

71 [71] R. G. polarity facilitating the purification process. Even if the crambescin A2 synthesis is not yet fully accomplished, the obtainment of the molecule scaffold confirms that the intrinsic reactivity of the biosynthetic precursors is totally suitable to lead to a common platform to this family of natural products. Summing up, we hope that the set of results presented in Chapters 2 and 3 will contribute to increase the knowledge concerning the chemistry of marine cyclic guanidine alkaloids.

Conclusions and Perspectives

Conclusions and Perspectives

Experimental Section

Experimental Section MeOH (500 mL, fraction F2, 2.01 g), MeOH : DCM (1 : 1, 400 mL, fraction F3, 1.74 g) and DCM (400 mL, fraction F4, 0.472 g). Fractions F2 to F4 were analyzed by UPLC and 1 H-NMR and the fraction F2 was purified twice by HPLC.

On the first time, 300 mg of F2 was purified by HPLC on semi-preparative mode (column:

Hemisynthesis of cycloanchinopeptolide C (4c)

A sample of anchinopeptolide C (3c, 13.8 mg) in a quartz tube was dissolved into 0.70 mL of D 2 O and exposed to an irradiation chamber (lamps ReptiSun 10.0 ZOOMED® UV-B (10%) 26W).

The evolution of the reaction was followed by 1 The complex silver(II) picolinate was prepared according to the protocol available in the literature. 83 A suspension of 19 (179 mg, 1.02 mmol) and Ag(pic) 2 (709 mg, 2.00 mmol) in strictly degassed water (15 mL, ultrasound, 15 min, flushed with Ar) was brought to 70 °C in a flask protected from the light and under inert atmosphere. After 20 min the reaction color faded from red to pale yellow. After cooling to rt, the reaction mixture was acidified with aq. 2M HCl (5 mL), filtered and evaporated under reduced pressure. The resulting pink solid was employed without further purification.