Chapter I Introduction

Interest of fluorinated peptidomimetics in medicinal chemistry

Fluorine has become an essential tool in drug discovery. Including fluorine atoms in potential medicines can have a variety of dramatic effects on the molecules' properties, perhaps making them more selective, increasing their efficacy, or making them easier to administer. So it is not a great surprise that around a fifth of all drugs on the market today contain at least one fluorine substituent [1] , and that fluorine-containing pharmaceuticals are still very attractive for pharmaceutical companies in various therapeutic areas. [2]

III

Acknowledgments

I would like to pay my deepest gratitude and respect to my supervisor, Prof. Sandrine Ongeri. She offers me sustained enthusiasm, creative suggestions, and continuous encouragement during my PhD period. I thank her for the great effort and enormous patience she put into training me. Without her guidance, I think I can not finish my PhD thesis.

I would like to thank my committee members Prof. Philippe Karoyan, Dr. Grégory Chaume, and Dr. Valérie Alezra for sparing their time for reading my thesis and for their invaluable comments. Some of the results described in this thesis would not have been obtained without a close collaboration with few laboratories. I owe a great deal of appreciation and gratitude to Dr. Isabelle Correia and Prof. Olivier Lequin (UMR 7203, UPMC), Dr. Carine Van. Heijenoort (UPR 2301, ICSN, Gif-sur-Yvette), Prof. Tâp Ha-Duong (BioCIS UMR 8076) and Dr. Guillaume Bernadat (BioCIS UMR 8076).

I would like to thank Dr. Thierry Milcent and Dr. Benoît Crousse for their instructions in the organic synthesis. I would like to thank Dr. Julia Kaffy for her work in evaluations of all the compounds with ThT assay. I would also like to thank Karine Leblanc for the mass spectra, HPLC analysis and elementary analysis, and Camille Dejean, Jean-Christophe Jullian for the NMR spectra, and Denèfle, Thomas in Prof. Philippe Karoyan's lab for preparative HPLC. Many thanks to Soulier Jean-Louis for helping me with my laboratory work. Many thanks to Dr. Guillaume Bernadat again for teaching me molecular modeling knowledge and sharing his experience in managing a large amount of documents in research.

I would like to thank my lab mates and also friends: Dimitri Brinet, Leila Vahdati, Marius Mamone, Nicolò Tonali, Jing Hao, Linh Thi-Thuy, Lucia Feni, Faustine Bizet, Marianna Munafò, and Renjin Tang. Special thanks to Nicolò Tonali, for his great humorous and friendly companion and always funny and sometimes crazy ideas, without him it is would be really difficult to pass these three years. Special thanks also to Faustine Bizet, Lucia Feni and Marianna Munafò, with who we share a lot of fun during tea and coffee time.

I am thankful to all the members of BioCIS, Faculté de Pharmacie who helped me in one or other way to carry out my work successfully.

I gratefully acknowledge the financial support from China Scholarship Council (CSC).

Finally, I would like to thank my family and friends for all their support and encouragement. Special thanks to my parents, for their unconditional love, support and encouragement to me. Groningen machine for chemical simulations GS gamma (γ-) secretase GSI gamma (γ-) secretase inhibitor GSM gamma (γ-) secretase modulator HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo [4,5-b]

Physical and Chemical Properties of Fluorine

Most of the effects induced by the presence of fluorine atoms in a molecule come from both the structure and the fundamental atomic properties of the fluorine atom (Table 1.1). [3] Fluorine is the most electronegative of all elements (value of 3.98 on the Pauling electronegativity scale), thus making the carbon-fluorine bonds highly polar and very stable. Its strongly localized electrons render the C-F bond very low polarizability and a relatively small size. The excellent match between the fluorine 2s and 2p orbitals with the corresponding orbitals of carbon are unique. Fluorine has a slightly larger size compared to the hydrogen atom (1.20 Å), but with a van der Waals radius of 1.47 Å, it occupies a smaller volume than a methyl, amino or hydroxyl group (1.52 Å). The short carbon-fluorine bond length (1.39 Å) is similar to the carbon-oxygen (1.43 Å) one. These key points are the reason why the replacement of some functional groups, including C-H, C-OH, C=O by fluorine are so attractive. The introduction of fluorine into organic compounds has been widely used in pharmaceutical and medicinal chemistry in the last 30 years. Fluorine substitution allows the simultaneous modulation of electronic, lipophilic and steric parameters. Physico-chemical properties such as metabolic stability, enzyme substrate recognition, bioavailability, basicity, hydrolytic stability can be fine tuned.

The effects of fluorination on the molecular properties stem from the combination of the atomic properties of the fluorine atom: strong electronegativity, small size, excellent overlap of the 2s or 2p orbitals with the corresponding orbitals of carbon, and very strong bond with carbon. 1.1 Atomic parameters of fluorine atom (Adapted from reference [2])

Fluorine substitution effects on pK a Changes in pK a can have effects on a number of different parameters in lead optimization including physicochemical properties (solubility, log D or log P), binding affinities (potency, selectivity), and absorption, distribution, metabolism, excretion (ADME). Depending on the position of the fluorine substituent relative to the acidic or basic group in the molecule, a pKa shift of several log units can be observed. Due to its very high electronegativity, the introduction of fluorine significantly lowers the pK a or pK b of proximal functional groups as listed below (Table 1.2). [4] Compound 1. 2 Selected pK a values of various fluorinated compounds (Adapted from reference [4])

The effect of the pK a on bioavailability can be seen in a series of piperidinyl and piperazinyl indoles compounds (Table 1.3). The nonfluorinated parent compound is a very potent receptor ligand, but has a very low bioavailability. Monofluorination lowers the pK a and increases the oral bioavailability. The potency of the difluoro compound with a pK a of 5.9 is as good as the parent compound, but the bioavailability is increased substantially. [5] Compound pK a IC 50 5-HT 1D Bioavailability 9.7 0.3 poor 8.7 0.9 Good 6.7 78 -5.9 9.5 Good Table 1. 3 Basicity and bioavailability in a series of piperidinyl and piperazinyl indoles

Fluorine substitution effects on molecular conformation

Fluorine atoms influence the conformation of organic molecules through dipole-dipole interactions, charge-dipole interactions and hyperconjugation effects. All of these influences can be rationalized by considering that the C-F bond is short, strong and highly polarized. [6] Strong electronic modification may also influence conformation. This is best illustrated with 1,2difluoroethane, a compound known to adopt preferentially a gauche rather than anti conformation. [7] In the preferential gauche conformation of 1,2-difluoroethane, both the electronegative fluorine atoms are antiperiplanar to a C-H bond, a conformation benefiting from double stabilizing hyperconjugative interactions (Figure 1.1). The presence of a fluorine substituent on a stereogenic centre positioned vicinal to another electronegative group may therefore significantly influence conformation and has important consequences in leadoptimization programmes. The most active syn, syn fluorinated analogue has an inhibition constant (K i ) of 2.0 nM indicating that this compound is as effective as the parent compound (K i 1.9 nM). The gauche effect stabilizes the fully extended conformation of the carbon backbone for the syn, syn fluorhydrin allowing for optimal binding. [8] Figure 1.2 Indinavir and its analogues It was found that the syn, anti fluorinated analogue was 8-fold more potent than the corresponding non-fluorinated parent compound C 17 -epi-Indinavir, with K i of 20 nM and 160 nM respectively (Figure 1.2). Notably, each of the fluorinated inhibitors was found to be slightly more lipophilic than Indinavir, with the exception of the stereoisomer anti, anti. These data suggest that fluorination of carbons adjacent to heteroatom substituents results in increased lipophilicity, a result consistent with literature data.

Fluorine substitution effects on lipophilicity

Lipophilicity is an important parameter that influences the in vivo distribution of the drug, for instance, it can enhance the binding affinity to the target protein. No common rule to explain how fluorine substitution affects lipophilicity can be provided. The change in lipophilicity after fluorine substitution is very much affected by the atoms and functional groups in close vicinity to the substitution site. [9] For example, the presence of fluorine close to an oxygen atom can increase the overall polarity of the molecule and thus enhances its solvation in polar medium. Likewise, fluorine may polarize the neighboring oxygen atom leading to stronger hydrogen bonding between oxygen and water molecules.

The Lipinski ''rule-of-5'', a set of empirical rules used as a guide to predicting good drug candidates, states that a log P > 5 will probably give poor absorption. [10] Excess lipophilicity is a common cause of poor solubility, leading to erratic and incomplete absorption following oral administration. Some general rules on the effects of fluorination on lipophilicity are summarized [12] . These rule are listed below as: (1) Fluorination usually but not always increases lipophilicity;

(2) Aromatic fluorination always increases lipophilicity;

(3) Per/polyfluorination increases lipophilicity; (4) Fluorination adjacent to atoms with π-bonds increases lipophilicity, with exceptions for some fluorinated α-carbonyl compounds [11] ; (5) Monofluorination and trifluoromethylation of saturated alkyl groups decreases lipophilicity. Due to the relatively polar character of monofluoro-and trifluoromethylalkanes with their strong C-F and C-CF 3 bond dipoles, monofluorination or trifluorination of saturated alkyl groups decreases lipophilicity (Table 1.4). [12] Compound Log P (octanol-water) CH 3 1. 4 the effect of fluorine substitution on lipophilicity (log P) (Adapted from reference [12]) Fluorine substitution effects on metabolism Low metabolic stability due to oxidation processes mediated by P450 enzymes is a common problem in drug discovery, but can be circumvented by blocking metabolically labile sites with fluorine substitution. Fluorine substitution can also block, or at least slow down, oxidation in the aromatic ring. This is typically accomplished by introducing fluorine at the 4-position of the phenyl ring. A good example of how fluorine substitution can be utilized to modify drug metabolism, is exemplified in the optimization of the cholesterol uptake inhibitor Ezetimib (Figure 1.3). [13] The lead compound SCH48461 was metabolized extensively and some metabolites were more potent than the drug itself. Fluorine was introduced into the para-position of the phenyl ring to prevent oxidation to a phenol. Furthermore, the 4-methoxy group was replaced by fluorine to avoid metabolic demethylation. These fluorinations, along with the addition of some supplemental functional groups, contributed to the "optimized" drug Ezetimib, which was 400 times more potent than the lead compound.

F NMR and 18 F PET

The natural occurring stable isotope 19 F has a nuclear spin of ½, a sensitivity of 0.83 relative to 1 H nucleus and a high gyro magnetic ratio (γ) (Table 1.5). Due to the characteristics of the 19 F nucleus, 19 F NMR is now finding increasing applications in biological chemistry, especially to study aspects in chemical biology. [14] Due to the high sensitivity of the fluorine nucleus, the detection of µM concentrations is possible for analysis at physiological concentrations. Analyses are possible without the need of purification, because the resonances of fluorinated organic compounds do not overlap with those of 13 C and 1 H, and because of the lack of background signal from biological sources (unlike 13 C and 1 H). The relatively large chemical shifts resulting from only minor changes in the chemical environment, lead to little or no peak overlap. This is why fluorinated organic compounds and consequently 19 F NMR have very important roles in research at the chemical biological interface. They are widely used as probes to explore enzyme mechanisms, metabolic pathways, bimolecular interactions and structural analysis of macromolecules. [14] nucleus spin γ [10 7 rad T - Positron emission tomography (PET) is a non-invasive, quantitative medical imaging technique that takes advantage of positron decay to offer in vivo imaging of regional tissue, it has also emerged as a powerful tool in the drug development industry as it has the capability of mapping out the function of a pharmaceutical compound in vivo. [14] Commonly used PET radioisotopes include 18 F (t ½ = 109.7 min), 11 C (t ½ = 20.4 min), 13 N (t ½ = 9.96 min), and 15 O (t ½ = 2.1 min), [15] The application of 18 F is extensive within the field of medical imaging as it can be incorporated into biomolecules and subsequently imaged in a living target. [15] For example, two of the most widely used 18 F-labelled PET radiopharmaceuticals are [ 18 F] 2-fluoro-2-deoxy-D-glucose ([ 18 F] FDG) and [ 18 F] 6-fluoro-L-DOPA. [ 18 F] FDG has become the gold standard in cancer imaging, images glucose uptake, and thus areas of high metabolism, in the body (Figure 1.4). [ 18 F] 6-fluoro-L-DOPA is a leading tracer for imaging intracerebral dopamine in the central nervous system (CNS), as dopaminergic activity is decreased in Parkinson's disease (PD). [16]

Fluorinated amino acids and peptides

Fluorination may change the properties of biological molecules in physiological process in several ways, such as peptidomimetics with fluorinated amino acid are used as reversible inhibitors (analogues of substrates) to interact with metabolic enzymes, and through the incorporation of fluorinated amino acid into the peptide backbone or side chain to modulate the conformation, the transport, and the metabolism of peptides and proteins. [2] These effects result from the impact of fluorination on the properties of the peptide at the level of (a) the lipophilicity/hydrophobicity balance and pK a ; (b) the conformation, because of the demanding volume of the fluorinated moiety; and (c) the hydrolytic and metabolic stability. [2] Of course, the importance of these different effects depends on the location and nature of the fluorine substitution. Lastly, when a fluorinated amino acid id incorporated into a peptide or a protein, the fluorine atoms can be used as probes in 19 F NMR, and in medical imaging in the case of 18 F. This provides tools for kinetic and structural studies, as well as for clinical investigations.

Peptides are today reasonable alternatives to small molecule pharmaceuticals. They often offer greater efficacy, selectivity, specificity and a reduced risk of unforeseen side reactions compared to small organic molecules. [17] The use of peptides in drug development is limited due to its low metabolic stability in vivo and poor bioavailability. These pharmacodynamic weaknesses can be circumvented by innovative formulation. Various chemical modifications can also be performed on the peptide chain to obtain peptidomimetics with enhanced metabolic stability: non-natural amino acids, N-methylated amino acids, cyclizations to increase the rigidity of peptide, peptide bond surrogate units, and retropeptides. One possible approach to avoid proteolytic degradation of peptide bonds is to replace them with hydrolytically stable groups. Several fluorinated groups are able to substitute the peptide bond and remain stable in the presence of hydrolytic enzymes. For example, fluorovinyl and trifluoromethylvinyl groups have been demonstrated to be hydrolytically stable replacements for a peptide bond and much better electronic mimics than the unfluorinated vinyl function. [18] In bioorganic and medicinal chemistry, judicious introduction of fluorine atoms or appropriate fluorinated functions into a molecule has become a method of choice in order to modify and tune its biological properties. [18] Thus, for example, a fluorine atom has been used with great success as a replacement for a hydrogen atom or a hydroxy group, and a CF 2 has been used as a mimic for an oxygen atom. In particular, the xenobiotic trifluoromethyl (Tfm) group is recognized in medicinal chemistry as a substituent of distinctive qualities. It is, in fact, at the same time highly hydrophobic, electron-rich and sterically demanding; moreover, it can provide high in vivo stability and features a good mimic of methyl, isopropyl, phenyl, etc. [18] Another important application consists in the incorporation of a CF 3 CO [19] , or COCF 2 CO function [20] in a key backbone position in order to achieve inhibition of proteolytic enzymes, in particular serine proteases, thanks to the ability of the fluoroalkyl group to stabilize the gem-diolic form, that is able to mimic the hydrolytic transition state (Figure 1.5). Recently, fluorinated amino acids (trifluoroleucine, trifluorovaline, trifluoromethionine, etc.) have been incorporated into protein structures, resulting in a deep modification of properties such as enhanced thermal and chemical stability, affinity for lipid bilayer membranes, stronger self-association, and so on. [21] Figure 1.5 Structure of natural peptides, its hydrolytic intermediate and fluorinated peptides (Adapted from reference [22 (b)])

The Tfm group has been often described as sterically very similar to an isopropyl group. However, the volume of the Tfm group is known to be larger, and intermediate between that of isopropyl and isobutyl groups [23] . Pepstatin A (Iva-Val-Val-Sta-Ala-Sta) (Figure 1.6) is a subnanomolar inhibitor of many aspartyl proteases, with the notable exceptions of HIV-protease and renin [24] . The two statine units are known to play a key role. Many structural modifications of Pepstatin A, including the statine isobutyl side chain, have been investigated. Matteo Zanda et al. demonstrated that bis-Tfm analogue of Pepstatin (Figure 1.6) was a much stronger inhibitory activity was found toward Plasmepsin II, an aspartic protease of Plasmodium falciparum, the protozoal that causes the most serious forms of malaria. Bis-Tfm-Pepstatin analogue was found to be a low nanomolar inhibitor of Plasmepsin II, having nearly the same potency of Pepstatin A. However, bis-Tfm analogue was considerably more selective toward closely related enzymes, such as Plasmepsin IV and, in particular, Cathepsin D. [24] Figure 1.6 Pepstatin A and its bis-Tfm-analogue [25] By far, there has been no effective cure for AD, and the cost of care for a patient with AD is increasingly a great burden to the individual and also for the society.

General Aspects of AD

Alzheimer's disease is a progressive neurodegenerative disorder characterized by age-related impairment in cognition and memory. Many AD patients show clinical symptoms of severe memory loss and progressive cognitive difficulty in the age of 60 or above. These clinical symptoms include loss of ability in learning, memory, problem solving, speaking, calculation, judgment and planning.

The development of AD is progressive and can sometimes last for over decades. The development of AD can be divided into three stages according to clinical symptoms. The first stage is the mild stage with the symptom of losing short-term memory and becoming passive for social activities gradually, but still can maintain normal daily activities. The mild stage usually lasts for 2-3 years.

The second stage is the moderate stage. In this stage, patients cannot recognize family members.

They are not able to communicate well, the daily activities, such as feeding, cooking, dressing and bathing become more and more difficult. In the late stage of AD, patients completely lose the abilities to speak, solve problems and make decisions. Daily activities can be affected greatly.

AD pathological hallmarks

Pathologically, AD is characterized by a loss of synapses, an increase in the number of extracellular amyloid beta-peptide (Aβ)-rich senile plaques (SPs) formed from the amyloidogenic processing of amyloid precursor protein (APP), and an increase in intracellular neurofibrillary tangles (NFTs) composed of aggregated hyperphosphorylated Tau. [26] AD affects neurons in the neocortex, including the frontal lobe and the temporal, the entorhinal cortex and the hippocampus. Subcortical limbic areas such as the cholinergic neurons in the basal forebrain and the neurons in the amygdala, the anterior nucleus of the thalamus, the raphe, and the locus coeruleus, are also affected. It is suggested that the first area affected in the brain is the entorhinal cortex and then neurodegeneration progresses to the hippocampus and then to the cortex (Figure 1.7). [27] In AD brains, another pathological feature is intracellular NFTs. The affected neurons often show intracellular accumulations of single straight filaments and paired helical filaments and neuropil threads (Figure 1.8). The major component of these poorly soluble filaments is hyperphosphorylated tau, a 68 kDa microtubule-associated protein. In AD, the tau protein is accumulated within neurons in the form of NFTs leading to the neurotoxicity and reduced cognition [28] . Tau phosphorylation as well as the interaction of tau and glycosaminoglycans may play a role in abnormal filament formation in vivo. Phosphorylated tau has reduced ability to bind microtubules, which changes the stability of microtubules. In addition, phosphorylated tau may also affect intracellular transportation, cellular geometry and neuronal viability. [29] Figure 1.8 Normal neuron cells (left) and cells with amyloid plaques and neurofibrillary tangles (right)

[picture obtained from BrightFocus Foundation @ http://www.brightfocus.org/alzheimers/infographic/amyloid-plaques-and-neurofibrillary-tangles]

Synaptic and neuronal loss

In AD, a significant synaptic loss is observed, which might be one of the first events in AD development. Evidences also show that even before the appearance of synaptic loss, there are decline of synaptic plasticity and synaptic excitation in familial Alzheimer's disease (fAD) mutant transgenic mice model. [30] Synaptic loss and degeneration induce neuronal dysfunction and cell body loss. Neuronal loss in the cerebral cortex and the hippocampus is a hallmark feature of AD.

[31] Some of AD patients at late stage of the disease can have a severe decrease in brain volume and weight due to either neuronal loss or atrophy. [32] The loss of cholinergic neurons in AD is widely studied. Decrease of choline acetyltransferase activity and acetylcholine synthesis correlate well with the degree of cognitive impairment in AD patients. [33] Current drugs for AD such as Donepezil, Galantamine, Rivastigmine and Tacrine are acetylcholinesterase inhibitors that have been developed based on cholinergic hypothesis. [34] Because these drugs can treat some symptoms but not cure the disease, the acetylcholine deficiencies may not be directly causal, but are a result of widespread brain tissue damage.

Besides the main pathology, some other pathologies of AD include granulovacuolar degeneration, cerebral amyloid angiopathy, blood-brain barrier disorder, white matter lesions, neuropil thread and gliosis. [35] Because of a lack of diagnostic markers for live AD patients, the definite diagnosis of AD depends on cognitive tests and a quantitative assessment of numbers of SPs and NFTs in the postmortem brain tissues. [36] The diagnosis by neuropsychological assays is mainly completed by imaging technique. Modern magnetic resonance imaging (MRI) techniques not only allow studying the volume reduction in the gray matter but also the entorhinal cortex atrophy. In addition to the structural MRI techniques, PET studies measuring a deposition with 11 C-PIB (Pittsburgh compound B, a radioactive analog of thioflavin T) or other PET ligands and functional techniques such as glucose utilization and functional MRI (fMRI) are used today to follow the progress of the disease. However, these types of exams do not allow to put in evidence the specific injuries caused by the amyloid plaques and neurofibrillary tangles. [37] 1.

Amyloid Cascade Hypothesis

The amyloid cascade hypothesis was first asserted by John Hardy and Gerald Higgins in 1992 [38] . The hypothesis proposed that Aβ is the causative agent in AD and that neurofibrillary tangles, cell toxicity and neurodegeneration occur subsequent to Aβ pathology (Figure 1.9). According to the hypothesis, Aβ is the 'driver' of a cascade of down-stream events that ultimately leads to the death of neurons and the cognitive symptoms of AD. There are several experimental evidences supporting hypothesis.

Figure 1.9

The amyloid cascade hypothesis. The sequence of pathogenic events that are thought to lead to AD is shown.

Genetic support for Aβ as the initiator of the cascade is the cognitive deficit and AD-like pathology seen in Down's syndrome patients. Down's syndrome occurs when three copies of Chromosome 21 are inherited. The gene for APP lies in Chromosome 21 and so Down's syndrome patients have three copies of the APP gene and produce excess amounts of APP. These patients develop Aβ plaques by the age of 40 and subsequent dementia is common [39] . This syndrome implicates APP and Aβ as causes of neurodegeneration and dementia.

Genetic studies of fAD have served as the strongest foundation for the amyloid hypothesis, showing that mutations or polymorphisms in APP or in genes that are directly involved in proteolytic processing of Aβ occur in all subjects with early-onset (familial) AD. [40] At the functional effect of Aβ, when it was applied to neuronal cells or slice culture, is toxic to cells and detrimental to LTP. A 35 kDa fragment of APP was shown to bind to DR6 (death receptor 6) and trigger axonal degeneration and neuronal death [41] .

In summary, the amyloid cascade is initiated by the generation of Aβ 1-42. In familial early onset AD, Aβ 1-42 is overproduced owing to pathogenic mutations. In sporadic AD, various factors can contribute to an increased load of Aβ 1-42 oligomers and aggregates. Aβ oligomers might directly injure the synapses and neurites of brain neurons, in addition to activating microglia and astrocytes. Tau pathology, which contributes substantially to the disease process through hyperphosphorylated tau and tangles, is commonly believed to be triggered by Aβ 1-42. [42] APP Proteolysis APP is an integral membrane protein expressed in many tissues and concentrated in the synapses of neurons. Its primary function is not known, though it has been implicated as a regulator of synapse formation, [43] neural plasticity [44] and iron export. [45] The amyloid beta region of the protein, located in the membrane-spanning domain, is not well conserved across species and has no obvious connection with APP's native-state biological functions. [46] Multiple alternate pathways exist for APP proteolysis. The most traditional model of APP proteolysis involves APP processing in either the non-amyloidogenic pathway, where equential cleavage by α-secretase and the γ-secretase liberates sAPPα and p3, or in the amyloidogenic pathway, where sequential cleavage by β-secretase (BACE1) and γ-secretase liberates sAPPβ and Aβ (Figure 1.10). [47] About 90% of secreted peptides is Aβ1-40, while Aβ 1-42 accounts for < 10% of secreted Aβ. Aβ 1-40 is the most abundant but Aβ 1-42 is more neurotoxic as it aggregates faster. Aβ itself is a 4-kDa peptide that is primarily produced in neurons [48] . The Aβ domain in APP is located toward the C-terminal of the precursor protein, and is released extracellularly following cleavage by β and γ-secretase. [49] The two most prevalent isoforms of Aβ are 40 and 42 residues in length. The two forms differ at the C terminal, where length is determined by the cleavage site of γ-secretase [50] . While Aβ 1-40 is more common, the 42 residue isoform is the primary component of senile plaques as is it highly prone to aggregation [51] . Once released, Aβ will accumulate to form first oligomers, then fibrils and finally plaques of protein [52] .

Aβ1-40 has the same sequence as Aβ 1-42 but without the last two residues. Aβ 1-42 and Aβ 1-40 have the same hydrophilic N-terminal extended from residue 1 to residue 15. The residues 17-21

form the so called central hydrophobic core, while the turn region includes residues 22-28. The hydrophobic C-terminal has 13 residues (30-42) and 11 residues (30-40) for Aβ 1-42 and Aβ1-40, respectively. In water Aβ peptides are intrinsically disordered and they aggregate rapidly forming cross-beta fibril structures.

Aβ peptide conformations

In vivo, the Aβ peptide is supposed to be helical when it is part of the APP and to have a β conformation in the plaques. Increasing evidence suggests that a conformational transition from random coil/α-helix to β-sheet structures is the crucial step that determines the fate of Aβ (1-42) under physiological or pathological conditions. Many conformational studies have documented that, depending on the solvent features, Aβ peptide can undergo a conformational transition from random coil/α-helix to β-sheet structures or vice versa. In vitro the Aβ conformation is strongly influenced by the environment. [53] For example, the resulting structures of Aβ (1-40) and Aβ 1-42 in 100% HFIP solutions are mostly α-helical (~50-70%) and random coil (~30-50%), with a little β-sheet secondary structures (<1%); [54] however in a 10% (v/v) HFIP solution, FT-IR spectroscopy indicated high levels of β-stranded conformation [55] . During fibrillogenesis, Aβ1-42 undergoes a conformational change from an α-helix to parallel β-sheets, which are connected by a bent structure encompassing residues 23-29, and the close distance between the side chains of Asp23 and Lys28 forms an electrostatic interaction [56] . The structural characterization of a partially folded intermediate in the α-helix to β-sheet transition opens many perspectives for the design of molecules that are able to interfere with the aggregation process. This has great potential for possible therapeutic applications.

Aβ fibrils structures

Aβ fibrils have a cross-β structure in which the individual β-strands are oriented perpendicular to the fibril axis. In Aβ 1-42, the N terminus is thought to be unstructured, and there are two largely hydrophobic β-strand segments within the fibrils that have a parallel and in-register orientation [57] .

In Aβ 1-40 fibril models, residues 1-10 are found to be unstructured, and residues 11-40 form a staggered β-turn-β unit stabilized by a salt bridge between Asp 23 and Lys 28 (Figure 1.11b). Phe 19 is in contact with Leu 34 , Gln 15 is in contact with Val 36 and His 13 is in contact with Val40. [52] The cross-section of Aβ 1-40 fibrils has recently been shown to consist of either two or three β-turn-β units depending on the morphology of the fibril. Fibrils with striated ribbon morphology have two-fold symmetry with intermolecular contacts between Met 35 and Gly 33 and between Ile 31 and Gly 37 . In contrast, fibrils with a twisted morphology have three-fold symmetry with intermolecular contacts between Ile 31 and Val 39 . In both fibril structures, the β-turn-β units pack in a parallel and in-register fashion with roughly the same conformation (Figure 1.11b).

In Aβ 1-42 fibrils, residues 1-17 may be unstructured, with residues 18-42 forming a β-turn-β fold. Molecular contacts have been reported within the monomer unit of Aβ 1-42 fibrils between Phe 19 and Gly 38 and between Met 35 and Ala 42 . In both Aβ1-40 and Aβ1-42, the turn conformation is stabilized by hydrophobic interactions and by a salt bridge between Asp 23 and Lys 28 . [58a] A growing body of literature has implicated the prefibrillar oligomers, but not the fibrillar form, as the primary pathological species of AD. [59] Of particular pathological significance is the Aβ 1-42 peptide. Aβ 1-42 can form stable trimeric and tetrameric complexes as well as globulomer structures (dodecamers) in the presence of SDS (sodium dodecyl sulfate) and can form pentamers and hexamers in solution. [60] Combination of single-touch AFM (atomic force microscopy), native gels, size-exclusion chromatography (SEC) and SDS gel electrophoresis shown that Aβ 1-42 (at low temperature and under low-salt solution) form stable disc-shaped pentamers with widths of 10-15 nm and heights of ~ 2 nm. [58] Figure 1.12b shows a molecular model of the Aβ 1-42 pentamer that agrees with the overall dimensions observed by AFM. Each monomer within the pentamer has a diameter of ~ 5 nm and a height of 2 nm. The hydrophobic C terminus of each monomer is oriented toward the center of the oligomer, where it is protected from hydrogen-deuterium exchange. The compact fold of the peptide is facilitated by solvent-accessible turns at His 13 -Gln 15 , Gly 25 -Gly 29 and Gly 37 -Gly 38 , which place Phe 19 in contact with Leu 34 (Figure 1.12a). The disc-shaped pentamers may associate to form oligomers with an average height of ~3-4 nm. The formation of decamers (and possibly undecamers and dodecamers) is favored at higher peptide concentrations, higher temperature and physiological salt concentrations [58] .

Solid-state NMR confirm that the C-terminal hydrophobic sequence of Aβ 1-42 folds into a βturn-β conformation (Figure 1.12d) and that Aβ 1-42 fibrils are composed of multiple β-turn-β units that polymerize in a parallel and in-register orientation. Dipolar-assisted rotational resonance (DARR) NMR measurements specifically show that the side chain packing registry within the βturn-β structure involves molecular contacts between Phe 19 and Leu 34 and between Gln 15 and Gly 37 . Additionally, the Gln 15 -Gly 37 contact is intermolecular in nature and arises from a staggering of the β-strands within the β-turn-β unit (Figure 1.12e). The core β-strand-turn-βstrand unit is similar for fibrils formed from Aβ1-40 and Aβ 1-42 peptides. [58a] The cross-β-subunit structures determined by Colvin et al. and Wälti et al. agree well with the findings of a previous study [61b] . However, this earlier work did not show how subunits interact to produce two-fold symmetry and did not define the conformation of amino-acid residues 34-36 unambiguously. The excellent overall agreement between these three independent studies, which used different strategies for labeling their samples, different combinations of solid-state NMR measurements and different molecular-modeling approaches, makes the results highly reliable.

Therapeutic Strategies for AD

Clinical situation

During the 2002 to 2012 observation period in the Clinicaltrials.gov database, 413 AD trials were performed: 124 Phase 1 trials, 206 Phase 2 trials, and 83 Phase 3 trials. Seventy-eight percent were sponsored by pharmaceutical companies. The largest number of registered trials addressed symptomatic agents aimed at improving cognition (36.6%), followed by trials of diseasemodifying small molecules (35.1%) and trials of disease-modifying immunotherapies (18%). A very high attrition rate was found, with an overall success rate during the 2002 to 2012 period of 0.4% (99.6% failure). The success rate for advancing from one phase to another is low, and the number of compounds progressing to regulatory review is among the lowest found in any therapeutic area. [START_REF] Cummings | [END_REF] Effective therapy for AD is a major unmet medical need. For the typical AD patient, current symptomatic therapies (acetylcholinesterase inhibitors and the NMDA receptor antagonist memantine), demonstrate only minimal to modest symptomatic benefit that is not sustained.

Although key aspects of AD pathogenesis remain enigmatic, scientific advances over the last 25 years have provided sound rationale for the development of potentially disease-modifying AD therapies. A large percentage of current therapeutic development in AD is focused on therapies that target the Aβ peptide or Aβ aggregates.

A detailed understanding of the proteolytic processing that releases Aβ from the APP and its subsequent aggregation in the brain has provided a number of approaches to what may be generically referred to as anti-Aβ therapy. To date, four general categories of anti-Aβ therapy have been developed: (1) agents that decrease or modulate Aβ production in a manner that is designed to prevent or slow Aβ aggregation and accumulation, (2) therapies that degrade or enhance clearance of Aβ aggregates, (3) therapies designed to block Aβ aggregates, and (4) therapies designed to neutralize toxic Aβ aggregates. [63] 1.

Tau Strategy

Tau protein has a causative role in Alzheimer's disease and multiple other neurodegenerative disorders exhibiting tau histopathology collectively termed tauopathies. [64] The primary function of tau protein is to facilitate assembly and maintenance of microtubules in neuronal axons. In the disease process tau protein becomes phosphorylated, loses its affinity to microtubules and accumulates in the cell body where it forms aggregates. The large neurofibrillary tangles formed from tau protein assembled into filaments were thought to be the pathological structure of tau. Development of therapeutics targeting tau oligomers may be achieved by several strategies. [65] Targeting tau oligomers. There are a number of strategies to develop therapeutics directly or indirectly targeting tau oligomers in neurodegenerative diseases that may be generally categorized as (1) tau phosphorylation reduction, (2) inhibition of tau oligomer formation, (3) disruption of tau oligomers, and ( 4) inhibition of neurotoxic mechanisms of tau oligomers (Figure 1.15). [66] Figure 1.15 Drug discovery approaches. Therapeutics may be directed at inhibition of tau oligomer formation (A), disruption of tau oligomers (B) or inhibiting neurotoxic mechanisms associated with tau oligomers (C). (Adapted from reference [66])

Inhibition of tau hyperphosphorylation. Hyperphosphorylation of tau reduces the affinity of tau to microtubules and leads to its redistribution to the somatodendritic compartment of neurons where tau can aggregate. For example, Michael Hutton et al. demonstrated that an orally bioavailable and blood-brain barrier penetrating analog of the kinase inhibitor K252a (Figure 1.16) was able to prevent the typical motor deficits in the tau (P301L) transgenic mouse model (JNPL3) and markedly reduce soluble aggregated hyperphosphorylated tau. [67] Their findings strongly suggest that abnormal tau hyperphosphorylation plays a critical role in the development of tauopathy and provide clear in vivo evidence that inhibition of pathological tau hyperphosphorylation can delay or prevent tau-related functional deficits and support the use of such inhibitors as an approach to developing a disease modifying treatment for tau-related neurodegenerative disease. Disruption of tau oligomers and inhibition of their formation. Phenothiazines such as methylene blue (Figure 1.17) have been found to inhibit tau aggregation in vitro [68]. Interestingly, methylene blue also inhibits oligomerization of Aβ but does so by promoting fibril formation. [69] Presently, methylene blue is being evaluated in clinical phase 3 trials for treatment of AD and frontotemporal dementia. [70] It may be likely that the observed neuroprotective activity of methylene blue derives from both its inhibition of tau aggregation and its inhibition of oxygen radical formation similarly to curcumin, which inhibits Aβ aggregation and oxidative damage. [70] Beside improved learning and memory, methylene blue-treated Tau(ΔK) mice showed a strong decrease of insoluble Tau, a reduction of conformationally changed and phosphorylated Tau species as well as an upregulation of protein degradation systems. These data support the use of Tau aggregation inhibitors as potential drugs for the treatment of AD and other tauopathies and highlights the need for preventive treatment before onset of cognitive impairments. [71] Figure 1.17 Chemical structure of methylene blue

Aβ Strategy

The prominent role played by Aβ in AD makes it an obvious therapeutic target. Various therapeutic strategies have been employed to produce small molecules, peptides (or peptidomimetics), and anti-bodies to reduce or eliminate Aβ-induced toxicity. [72] Inhibitors of Aβ aggregation. The amyloid hypothesis suggests a direct correlation of AD with Aβ aggregates; hence inhibiting Aβ aggregation is considered an effective therapeutic strategy. Aβ peptide transits from a non-toxic α-helical state to a toxic β-sheet conformation. Molecules that can (i) block β-sheet formation, (ii) prevent the fibrillogenesis, (iii) dissolve Aβ aggregates to nontoxic species, (iv) destabilize Aβ oligomers and (v) accelerate the conversion of Aβ oligomers to Aβ aggregates are considered as modulators of Aβ aggregation. [73] This section has been organized into peptide-based modulators and small molecule-based modulators depending upon their chemical structure.

Peptide-based inhibitors of Aβ aggregation. Peptide-based inhibitors (or peptidomimetics) are a class of molecules designed upon the understanding of β-sheet-driven self-assembly involved in the aggregation of Aβ peptides. The hydrophobic core (KLVFF, Figure 1.18) of Aβ plays a central role in the initiation of Aβ aggregation and it act as a recognition unit for their elongation to fibrillar aggregates. [74] For the past two decades KLVFF has been the basis of designing most of the peptide-based modulators of Aβ aggregation. Hydrophobic peptides like KLVFF and LPFFD (iAβ5p, Figure 1.18) have been screened, and reported to be effective in the inhibition of Aβ fibrillogenesis both in vitro and in vivo. [75] Andrew J. Doig, et al. reported that SEN304 (Figure 1.18) was able to inhibit Aβ aggregation and toxicity, shown primarily by thioflavin T fluorescence and MTT [76] . How SEN304 affects Aβ 1-42 aggregation and toxicity was extensively characterized using biophysical assays, toxicity assays in cell culture and long-term potentiation in a rat hippocampal brain slice. Several all-D amino-acid-containing peptides based on Aβ residues 17-21 were also found to be effective aggregation inhibitors, including PPI-1019 (also named Apan, Figure 1.18), which has been completed phase I and II human clinical trials. [77] It was found PPI-1019 can reach the brain in quantities that are sufficient to block the aggregation of beta-amyloid molecules and alter the course of the disease. Transgenic mice treated with Apan show significant increases in Aβ levels in the CSF (cerebrospinal fluid), indicating that Apan is able to mobilize Aβ in the brain and may be facilitating its clearance. Small molecule-based inhibitors of Aβ aggregation. Modulation of Aβ aggregation using small molecules has been reported to be a highly effcient approach. [78] Most of the modulators control the fibrillogenesis of Aβ through blocking hydrophobic interactions. Natural products like curcumin (IC 50 ~ 13.3 μM), resveratrol (IC 50 ~15.1 μM), and EGCG (IC 50 2.4±0.4 μM) (Figure 1. 19) have been shown to be effective in decreasing the load of Aβ plaques in the brain through their antioxidant and aggregation inhibition properties. [79] Curcumin and resveratrol inhibit Aβ aggregation by binding to the N-terminal of the low molecular weight Aβ oligomers and prevent the formation of the more toxic, high molecular weight oligomers. [80] Wanker et al. have shown that EGCG (in phase III clinical trials) is not just an antioxidant but also perturbs the aggregation propensity of Aβ by binding to its monomeric state and rendering it into non-toxic species. [81] Brazilin, a natural product obtained from aesalpinia sappan, inhibited Aβ 1-42 aggregation and also remodeled Aβ 1-42 aggregates to prevent from acting as secondary nucleation centers for further fibrillogenesis. [82] The four-residue sequence H 13 HQK 16 in Aβ peptide promote oligomerization and fibril propagation, and also constitute the hairpin binding site for endogenous glycosaminoglycans (GAGs), which assist and additionally stabilize Aβ aggregates and increases their resistance to proteolysis. [83] As an enhancer of Aβ nucleation, the HHQK region is a viable target for Aβ aggregation inhibition. [84] Tramiprosate (Alzhemed TM , 3-amino-1-propanesulfonic acid, Figure 1.20) has reached clinical trials, with a mechanism of action mimicking GAGs and targeting the HHQK region. Tramiprosate showed promising in vitro inhibition of Aβ aggregation and fibrillogenesis, a decrease in Aβ 1-42-induced cell death in neuronal cell cultures, and the ability to cross the murine blood brain barrier (BBB). However, it did not reach the market (failure at phase III) due to a lack of in vivo activity, having demonstrated only a ~30% decrease in brain amyloid plaque load in a TgCRND8 murine model along with considerable side effects. [85] Meek et al. performed an in silico screen of endogenous ligands and identified L-phosphoserine and 3hydroxyanthranilic acid (Figure 1.20) as binding favorably to the HHQK stretch [86] ; these compounds were further validated experimentally by in vitro assays. Analysis of the binding orientations of the ligand-HHQK complex optimized in the solution phase revealed that Lphosphoserine interacts preferably at His 13 -Lys 16 , His 13 -His 14 , and Glu 11 -His 14 motifs, whereas 3hydroxyanthranilic acid shows a preferred binding orientation at His 13 -His 14 and Glu 11 -His 14 residues. To target this region collectively, a molecule with negatively charged moieties would be best for interaction with the cationic residues of the HHQK region. The nonpeptidic RS-0406 molecule (Figure 1.21) was reported to target the central core KLVFF motif. [87] It was demonstrated to inhibit the fibrillogenesis of Aβ 1-42 and ameliorate Aβ 1-42induced cytotoxicity and also reverse Aβ 1-42-induced impairment of long-term potentiation (LTP) in hippocampal neurons. Recently, Arai et al. [88] reported two novel potential non-peptide small lead molecules, 5 and 6 (Figure 1.21), with significant inhibitory properties against Aβ 1-42 aggregation. These compounds were cleverly designed based on the structure-activity relationship (SAR) data from the cyclo-[KLVFF] peptide, in which the side chains of Leu 2 , Val 3 , Phe 4 , and Phe 5 were identified as unique pharmacophore motifs responsible for Aβ 1-42 aggregation inhibition without the involvement of backbone amide bonds. and Ala 42 are the most important residues to target. It is well known that the C-terminal region, which contains hydrophobic segments, interacts with the cell's lipid bilayer; Aβ accumulation onto the surface of ganglioside-rich lipid membranes thus initiates and propagates fibril formation. [89] A glycolipid-derived sugar, scyllo-inositol (AZD-103; Figure 1.22), demonstrated promising Aβ 1-42 aggregation inhibitory activity by significantly decreasing the plaque burden in APP transgenic mice. [90] In another report, tanshinone I and tanshinone II (Figure 1. 22), were found to be capable of preventing the formation of, and disaggregating preformed amyloid fibrils, interacting primarily with the hydrophobic residues Ile 31 , Met 35 , and Val 39 of the C terminus β-sheet groove. [91] Similarly, sulindac sulfide (Figure 1. 22), was found to interact with Aβ 1-42 C-terminal residues Ile 32 , Leu 34 , Met 35 , and Val 39 at high concentrations (3:1 molar ratio). [START_REF] Richter | Proc. Natl. Acad. Sci[END_REF] In another study, this molecule was not found to inhibit the low-molecular-weight Aβ oligomer at a 1:1 molar ratio; rather, the authors suggested its interaction with protofibrils, fibrils, and high-molecular-weight oligomers, the formation of which is critically governed by C-terminal residues. [START_REF] Fu | [END_REF] Figure 1.22 Small-molecule inhibitors targeting the C-terminal region.

Enzyme inhibitors-Inhibition of Aβ peptide secretation

Since the recognition of their central role in APP processing, secretases have been identified as a possible target for therapeutic intervention of AD. α-secretase has not been targeted, as it cleaves APP in the Aβ region and does not lead to the formation of Aβ deposits. However, both β-and γsecretases have been suggested as targets to inhibit Aβ formation and thereby stop the progression of AD. [94] There are a large number of pathways guided by enzymes that affect the production and clearance of Aβ. In this section we focus on enzyme inhibitors of β-secretase and γ-secretase which are involved in APP processing to produce Aβ.

β-secretase inhibitors. β-site APP-cleaving enzyme 1 (BACE1) is a 501 amino acid type-I transmembrane aspartic protease. It controls nerve axonal myelination and muscle spindle formation via proteolytic processing of neuregulin 1. In vivo (mice) testing of β-secretase inhibitors have shown the least effect on normal physiological functions implicating that the use of β-secretase inhibitors (BSIs) may have minimum side effects. In recent times, LY2886721, MK-8931, NB-360 and AZD3839 have entered in clinical trials (Figure 1.23). [95] Recently three companies have published on BACE1 inhibitors derived from fragment screening which have entered clinical development (Figure 1.23) with the most advanced, MK-8931 from Merck being reported to be in Phase III trials [96] . This promising approach inspires further efforts to develop effcient β-secretase inhibitors. Aβ1-40 concentration in the brain. [97] Wood et al. performed high throughput screening to obtain sulfonamide based molecules as novel γ-secretase modulators. Further structural optimization on the lead molecule gave a better modulator (37) (Figure 1.24) [98] . Compound 37 showed improved cell potency, enhanced PKDM (pharmacokinetics and drug metabolism) and reduced Aβ 1-42 (IC 50 = 0.26 ± 0.10 μM) production in an in vivo model. However, γ-secretase inhibitors (GSIs) tend to produce adverse effects by interacting with Notch, an important pathway for cell proliferation and differentiation during embryonic development. Thus, inhibition of γ-secretase is likely to cause unwanted side effects.

Immunotherapy to Aβ aggregates clearance

Aβ immunotherapy is a promising approach to reduce Aβ burden, as illustrated by the large number of ongoing clinical trials. [START_REF]Clinical Trials NIH database[END_REF] Active Aβ-immunotherapy uses synthetic full-length Aβ peptide, or a fragment, to stimulate B cells to generate specific antibodies for sequestering amyloid from the brain in the peripheral system. [101] The main drawback of active immunotherapy is the difficulty of intervening whenever adverse effects occur. Passive immunotherapy, in other words, treating the patients with ex vivo produced monoclonal antibodies (mAbs), overcomes this problem. Eight Aβ-directed mAbs have been, or are being, tested in clinical trials: bapineuzumap, ponezumab, solanezumab, gantenerumab, crenezumab, aducamab, BAN2401 and SAR228810. [START_REF]Clinical Trials NIH database[END_REF] Passive immunotherapy is more advanced than active immunotherapy but its drawbacks, such as the occurrence of side effects, complicate its development. Although these studies will take several years, it can be concluded that immunotherapy shows glimmers of promise [102] to find a diseasemodifying drug to treat this devastating disease.

Aβ and IAPP peptides α-helical stabilizers

Most of the modulators of amyloid peptides (Aβ 1-42 or IAPP) aggregation are designed to interact with the peptides in their β-sheet, elongated conformations and thus to prevent their polymerization. Indeed, apart from the peptide-based inhibitors, which are a minor class of molecules modulating the amyloid peptide aggregation, the large numbers of small molecules described to inhibit Aβ and IAPP fibrillation are built on a planar aromatic scaffold such as resveratrol, epigallocatechin-3-gallate and curcumin [103] , for which the β-sheet structure represent the macromolecular target. An alternative strategy would be to target the native form or the α-helix conformation of these amyloid peptides before they switch to β-sheet conformation in order to trap them into their non-toxic forms. To our knowledge, very few examples of ligands designed to interact with the amyloid peptides (Aβ 1-42 or IAPP) in their α-helical conformation are described in the literature.

Nerelius et al. have reported peptidomimetic derivative Pep1b (Figure 1.25) that was able to maintain the helical conformation of Aβ (13-26). It has shown a reduction of the toxicity of Aβ 1-42 peptide on PC12 cells as well as beneficial effects in a model of Drosophila expressing human Aβ 1-42 peptide. [START_REF] Nerelius | Proc. Natl Acad. Sci[END_REF] Pep1b was found to bind and stabilize the 13-26 region of Aβ in an α-helical conformation. The stabilization of Aβ 13-26 decreases Aβ polymerization as well as the formation of toxic intermediate structures. The effects of Pep1b on the stability of the Aβ central helix (residues 15-24) were investigated by using MD simulations. [105] Pep1b was found to bend unwound Aβ by the charge-charge interactions (both the basic and acidic functional groups in Pep1b can simultaneously interact with the acidic and basic residues of Aβ respectively) and by interactions between the aromatic rings, thus disturbed the extension of β-sheets. These results prove the idea that stabilization of the central Aβ-helix counteracts polymerization into toxic assemblies and provide a strategy for development of specific inhibitors of Aβ polymerization. ). [106] The new ligand AEDab-Dab was designed by replacing the arginine residue in Pep1b with AEDab triamino acid, which carries an extra positive charge in the side chain. Molecular dynamics (MD) simulation of the stability of the Aβ central helix in the presence of the AEDabDab ligand shows further stabilization of the helical conformation of Aβ compared to Pep1b. The replacement resulted in an improved capability of the ligand to prevent the Aβ1-42 induced reduction of gamma (γ) oscillations in hippocampal slice preparation.

Fülöp et al. have described foldamer-dendrimer conjugate which adopts an H14 helix conformation in solution and shows a tight binding with Aβ oligomers (Figure 1.26). [107] Moreover, this compound rescues the long term potentiation from the toxic Aβ oligomers in an ex vivo model. The tetravalent foldamer-dendrimer conjugates were constructed by coupling ordered recognition segments (helical β-peptide foldamers with designed secondary structures) to a tetravalent G0-PAMAM dendrimer. They were designed to wrap the synaptotoxic β-amyloid (Aβ) oligomers with high affinity through their helical foldamer tentacles. In vitro biophysical and biochemical tests indicated the tight binding between the foldamer conjugates and the Aβ oligomers. Ex vivo electrophysiological experiments revealed that the new material rescues the long-term potentiation (LTP) from the toxic Aβ oligomers in mouse hippocampal slices at submicromolar concentration.

7 (ACHC-β 3 hArg-ACHC-ACHC-β 3 hAsp-ACHC-Gly-Gly-Cys) 4 -TMP Figure 1.26 Foldamer-tetra-maleimidopropionyl-G0-PAMAM (TMP) conjugate studied. ACHC stands for trans-2-aminocyclohexanecarboxylic acid. In the structure of 7, the H-14 helical conformation adopted by the foldamer segments in water is indicated (red), whereas the flexible linker is in an arbitrary conformation (green). (Adapted from reference [107]) Brandenburg et al. reported a helical coiled-coil-based inhibitor peptide engaged a coiled-coilbased amyloid-forming model peptide in a stable coiled-coil arrangement (Figure 1.27), [108] thereby preventing rearrangement into a β-sheet conformation and the subsequent formation of amyloid-like fibrils. Moreover, they show that the helix-forming peptide is able to disassemble mature amyloid-like fibrils. Oligoquinolines maintains IAPP in his α-helical conformation, delaying his conversion to β-sheetrich species which is not the case of EGCG and acid fushin. Andrew D. Miranker et al. reported the design of oligoquinolines based inhibitors OQ5 and ADM 116 (Figure 1.28). [109] These inhibitors have the capacity to affect both solution-and phospholipid bilayer-catalyzed IAPP selfassembly. Importantly, this activity is associated with the oligoquinoline's capacity to irreversibly adopt a noncovalent fold. This suggests that compact foldamer scaffolds, such as oligoquinoline, are an important paradigm for conformational manipulation of disordered protein state. The capacity of OQ5 to inhibit lipid-catalyzed assembly reflects the contribution of α-helical intermediate states to IAPP gains of function. Far-UV CD and NMR suggest that OQ5 is interacting directly with the membrane-bound helical substates of IAPP.

Figure 1.28 chemical structures of helix mimetic compounds OQ5, ADM116 and IS5

The tetraquinoline amide foldamer (ADM-116, Figure 1.26) docks specifically with intracellular IAPP and rescues β-cells from toxicity. [110] The far-ultraviolet CD spectrum of membrane-bound IAPP indicated that ADM-116 binds to and stabilizes the α-helical subdomain of IAPP. INS-1 viability assay showed that ADM-116 rescues IAPP-induced toxicity. Co-addition of ADM-116 at a stoichiometric ratio of 1:1 (IAPP: ligand) wholly restores viability.

A small molecule a helix mimetic, IS5 (Figure 1.26), built on a pyridine scaffold, is shown to inhibit bilayer catalysis of fibrillogenesis and to rescue IAPP-induced toxicity in cell culture. [111] IS5 inhibition was also found synergistic with insulin. Insulin was a weak inhibitor of lipid bilayer-catalyzed IAPP fiber formation. [112] However, the capacity of IS5 to act as inhibitor of lipid-catalyzed fiber formation was found increased in the presence of insulin. Helical structure stabilized by lipid bilayers has been implicated in the mechanisms of other amyloid diseases including Aβ from Alzheimer's and α-synuclein from Parkinson's. As IS5 rescues α-helical intermediate-induced cytotoxicity, such targeting represents a rich and novel area for the successful production of therapeutics.

Objectives of the PhD thesis

On one hand fluorine has demonstrated its major interest in clinical and pre-clinical drugs. On the other hand, peptides and peptidomimetics have gained interest during the last years even in pharmaceutical companies, because they often offer greater efficacy, selectivity, specificity and a reduced risk of unforeseen side effects compared to small organic molecules. What about fluorinated peptides and peptidomimetics? Yet a lot of space remains for interesting and challenging research. We know that fluorinated amino acids can have drastic effects on peptides or protein stability and on peptide-peptide interactions due to the unique stereoelectronic properties of fluorine, however only scarce examples of fluorinated peptides with potential therapeutic effect have been yet reported.

The objective of my PhD research has been to participate to this wide field of understanding the trifluoromethyl effect on peptide conformations and biological effect. I have introduced particular trifluoromethyl amino acid derivatives in peptides and foldamers. In chapter 2, we have studied the effect of (2S, 3R) and (2S, 3S)-CF 3 -threonine compared to natural L-serine and L-threonine introduced in the central (third) position of synthesized pentapeptides. In chapter 3, we have developed the diastereoselective synthesis of a CF 3 -1,4-disubstituted-1,2,3-triazole-based amino acid, synthesized oligomers (trimer and tetramer) of these new fluorinated units and studied their conformations.

Both classes of trifluoromethyl peptidomimetics have been evaluated as potential inhibitors of Aβ 1-42 and IAPP aggregation. The use of fluorinated amino acid-containing peptides in amyloid aggregation inhibition has rarely been studied. The rational of the possible interest of our compounds was based on their expected conformations. Indeed, the designed pentapeptides were chosen to adopt preferentially an extended structure so potentially be able to interact with β-rich structures of Aβ 1-42 oligomers in order to modulate their structure and modulate their aggregation and toxicity.

Recent work on peptidic homo-oligomers based on heterocycles such as imidazole, thiazole, and triazole report a capacity of these structures to adopt helical structures. Furthermore, our preliminary computational studies rather tended to predict helical structures with trimer and tetramers of CF 3 -1,4-disubstituted-1,2,3-triazole-based amino acid. We were very curious to study the ability of our possible helical structures on the inhibition of Aβ 1-42 and IAPP aggregation as only very few but promising results have been obtained by targeting helical structures of Aβ 1-42 monomer or small oligomers.

Chapter II Fluorinated Peptidomimetics

Design Strategy

β-sheet breaker strategy

Several approaches have been used to inhibit the aggregation of soluble Aβ monomers, either by stabilizing native conformations [1] or by preventing β-strand intermolecular interactions using βsheet breaker [1] (Figure 2.1). β-sheet breaker normally consists in two parts, one part forms intermolecular hydrogen bonding with the target peptide, while the other part works as disrupter that inhibits the monomer peptide (or oligomer peptide) further aggregation. (Adapted from reference [1])

Several research papers and review articles reported small peptides that are variants of the key recognition element (KLVFF) in the central region of Aβ peptide (residues 16-20) can stabilize intra-and intermolecular interactions involved in Aβ aggregation and amyloid formation. [2] For example, Soto et al. introduced β-sheet breaker peptides formed by a recognition motif and a βbreaker element (proline) (Ac-LPFFD-NH 2 ). [3] Other β-sheet breaker peptides with β-breaker elements such as N-methyl amino acids [4] , α-amino-isobutyric acids [5] , α/β hybrid peptides [6] and terminal blocking groups [7] are also reported (Figure 2.2). All these small peptides mimicking this hydrophobic sequence have proved to be valuable molecules to modulate the kinetics of Aβ aggregation. Recently, inspired by the strategies of β-sheet breaker peptides and peptidomimetics, our team reported a novel class of glycopeptides derivatives (Figure 2.4, compounds 2.1-2.4) as inhibitors of Aβ aggregation (Figure 2.3). [8] These compounds incorporated two short hydrophobic dipeptide units, Ala-Val and Val-Leu that were designed as recognition moieties and chosen to interact with the hydrophobic regions of Aβ peptide. The flexible and hydrophilic sugar moiety was designed to act as a β-sheet breaker element, which is a unit that can disorganize locally β-sheet structures in order to prevent protein-protein interactions involved in the aggregation. β-sheet interactions involve hydrophobic interaction and regular hydrogen bonds. The sugar moiety was believed to disrupt the regular hydrogen bonds and might engage uncontrolled and undesirable interactions. The polarity of the sugar might also prevent locally hydrophobic interactions. These glycopeptides (Figure 2.4) were able to modulate Aβ 1-40 and Aβ 1-42 aggregation, which were demonstrated by fluorescence thioflavin T (ThT) assay and transmission electron microscopy (TEM). The N-terminal deprotected derivatives 2.5 (C 1 = β configuration) [8b] was found to significantly increase the activity towards the aggregation of Aβ 1-42, compared to the activity of the protected compound 2.1 (C 1 = β configuration), suggesting that ionic interactions are likely to be established between the terminal amine group and acidic residues of the Aβ 1-42 amyloid peptide. While acetylated compounds of the sugar moiety showed no aggregation inhibition activity, highlighting the importance of polar hydroxyl groups in hydrogen bond donor ability for delaying the Aβ 1-42 aggregation activity. In order to further increase the affinity for Aβ 1-42, the valine residue of compound 2.5 was replaced by a lysine residue to further provide the molecules with the possibility of engaging electrostatic interactions with Aβ 1-42. At the same time the Leu residue of compound 2.5 was replaced by the 5-amino-2-methoxybenzhydrazide unit which is a part of the β-strand mimic ("Hao" unit) reported by Nowick and co-workers [8d] to increase the proteolytic stability of the molecules (Figure 2.5). Compound 2.6 was found not only able to increase the stability toward proteolytic degradation but also to dramatically increase the capacity of these pentapeptide analogues to inhibit fibrillization of Aβ 1-42. [8c]

Fluorinated amino acids and peptides

The incorporation of fluorine atoms into bioactive molecules proved as a useful way to improve the physical and biological properties, such as the metabolic stability, the lipophilicity, and the reactivity and the acido-basic properties of neighboring functional groups. [9] Short peptides modified both in the backbone and side chains with trifluoromethyl group are becoming very attracting target molecules. [10] The incorporation of trifluoromethylated amino acids into peptides has been reported to modulate the proteolytic stability and hydrophobicity [11] and to control the conformations of the peptides. [12] Fluorinated amino acids are also used as powerful 19 F NMR probes for the study of protein-ligand interactions and enzymatic activities. [13] Few examples of fluorinated peptides have been used as amyloid protein inhibitors. Rocha et al. [14] have reported peptides based on the central sequence of Aβ LVFFD, in which the valine was substituted by either 4,4,4-trifluorovaline or 4-fluoroproline, or the phenylalanine at position 3 was replaced by 3,4,5-trifluorophenylalanine. They demonstrated for the first time that fluorination of the hydrophobic residue valine or phenylalanine could be effective in preventing the Aβ aggregation. Brigaud et al. [15] have demonstrated promising results with tripeptides containing (R)-α-trifluoromethylalanine which is a fluorinated mimic of α-aminoisobutyric acid (Aib) as β-sheet breaker elements. Marianna Török et al. described a new class of organofluorine molecules (Figure 2.6) as Aβ fibrillogenesis inhibitors. [16] These compounds have been found to be active in the disassembly of the preformed fibrils as well. It was found that the trifluoromethylhydroxyl functionality had crucial importance in possessing activity; the removal of either the CF 3 or OH groups resulted in completely inactive compounds. Furthermore, they formulated a relationship between the inhibition activity with the acidity of the organofluorine inhibitors: the more acidic the OH, stronger the potency (Figure 2.7). And they also found that the mode of interaction was not stereospecific, as the inhibitory activities of both of the chiral isomer pairs of the same compounds were not significantly different.

Figure 2.6

Indol-3-yl, trifluoromethyl, hydroxyl, and carboxylic acid esters reported by Marianna Török et al.

Figure 2.7

The acid strength order of the major types of inhibitors used in Marianna Török papers.

Previous work in our group and objectives of my project

Based on the encouraging results of the glycopeptides containing the polar sugar β-sheet breaker element, and peptidomimetics or small molecules containing the trifluoromethylhydroxyl functionality to modulate Aβ peptide aggregation, we designed and synthesized other peptidomimetics that mimic the core nucleation fragment (KLVFF) of Aβ peptide (Figure 2.8). The goal of this project was to design, synthesize and evaluate new pseudo-pentapeptide molecules capable of interacting with the nucleation site of the Aβ peptide in its monomeric or oligomeric form so as to disrupt the self-assembly into toxic oligomeric form.

The designed pentapeptides contain both a core recognition element and a β-sheet breaker element. As a β-sheet breaker, we decided to introduce in the central position an amino acid bearing the trifluoromethylhydroxyl functionality, which then has been connected via peptide bonds with two hydrophobic dipeptides Ala-Val and Val-Leu, on each side. Indeed, it has already been demonstrated in the glycopeptides series (2.5, Figure 2.4), in our laboratory, that these two dipeptide arms interact with the peptide Aβ. The effect of trifluoromethylhydroxyl has been observed in comparison with the corresponding natural amino acids introduced in the pentapeptide sequence similar to that of compound 2.5. For that purpose, in the PhD work of Nadjib Kihal (PhD thesis of Université Paris-Sud, 2013), (2S, 3R)-CF 3 -threonine 2.7, 7-hydroxyl-7-di (trifluoromethyl)-L-phenylalanine 2.8, and (2S, 4S)-4-hydroxyl-4-trifluoromethyl-L-proline 2.9 were chosen and introduced in the pentapeptidic sequences (2.10, 2.16, 2.12 respectively, Figure 2.10). For comparison, L-proline, 4-hydroxyl-L-proline (both 4S and 4R), L-serine and L-tyrosine were also introduced (Figure 2.10, compounds 2.15, 2.13, 2.14, 2.11, 2.17 respectively). Thus, the Nand Ctermini containing Ala-Val and Val-Leu respectively were designed to interact with the hydrophobic regions of Aβ peptide on one side, while inserting polar and acidic trifluoromethylated alcohols was designed to disrupt intra-and intermolecular interactions involved in Aβ aggregation and amyloid formation on the other side (Figure 2.8). The activity of the synthesized pentapeptides 2.10-2.17 in modulating the aggregation of the Aβ 1-42 peptide was evaluated by Thioflavin-T (ThT) fluorescence assay, which is the standard method to monitor the kinetics of aggregation of amyloid peptide forming fibers. ThT assay has shown that compound 2.11 was able to delay the aggregation of Aβ 1-42 significantly at the ratio 1: 1 (10 μM 2.11: Aβ 1-42), and altered the kinetics of aggregation and thereby prevents fibril formation (Figure 2.11). In contrast, the benzylated compound of 2.11 at 10 and 100 μM had no effect on the aggregation of Aβ 1-42. Under the same conditions, compound 2.10 induced a maximum fluorescence signal alone, indicating that this compound has the ability to strong self-aggregate (Figure 2.12). Compounds 2.12 and 2.13 did not show inhibition of aggregation properties. Similarly, there was no inhibition of Aβ 1-42 aggregation in the presence of compounds 2.15 regardless of the concentration used, from 1 to 100 μM. There was no observed effect of compound 2.17 to inhibit Aβ 1-42 aggregation at rations 1:1, but it accelerated the kinetics of aggregation at higher ratio 10:1. In the presence of compound 2.16, there was no effect on Aβ 1-42 aggregation at 1:1 or 10:1 ratios.

Synthesis of pentapeptides with CF 3 -threonine 2.2.1 Synthesis of (2S, 3S)-CF 3 -threonine

Practical synthesis of various α-amino acids using the glycine equivalent, including fluorine-, phosphorus-, and sulfur-containing analogues as well as isotopically labeled derivatives, is wellreported by Belokon's group, [17] and Vadim A. Soloshonok's group [18] .

The synthesis of (2S, 3S)-CF 3 -threonine has been described in several publications. [19] Among these approaches we followed a general procedure to access to (2S, 3S)-CF 3 -threonine through the aldol reaction of CF 3 CHO with Ni (II)-complex of the chiral Schiff base of glycine which was introduced by YN Belokon et al. [20] The chiral auxiliary (S)-N-(2-benzoylphenyl)-1-benzylpyrrolidine-2-carboxamide 2.23 was obtained in good yield starting from N-benzylation of (L)-proline 2.22 with KOH as a base, then activation of the carboxylic acid functionality using SOCl 2 at low temperature, followed by condensation with 2-aminobenzophenone. Furthermore, complexation of 2.23 with nickel nitrate and glycine under basic condition gave the nickel Schiff base complex 2.24 in 85% yield as red crystal (Scheme 2.1).

The nucleophilic glycine equivalent 2.24 went through the aldol reaction with CF 3 CHO to give the complex 2.25 in moderate yield (66%). Further hydrolysis of complex 2.25 led to the recovery of the chiral auxiliary 2.23 and release of free (2S, 3S)-CF 3 -threonine whose diastereoselectivity was determined to be about 96% de by 19 F NMR. In most of the reported cases, the free amino acid was released into the aqueous phase, and it was then purified by ion-exchange chromatography. However, we tried to purify the free (2S, 3S)-CF 3 -threonine by another way. We first managed to remove the Ni (II) by addition of 2.0 equivalent NaSCN and 4.0 equivalent pyridine to form the the complex Ni(Py) 4 (SCN) 2 , which precipitated from the aqueous phase. After filtration, we protected the free amino acid using Boc 2 O under basic condition. The Boc-(2S, 3S)-CF 3 -threonine 2.26 was then purified by silica column chromatography and was obtained as a colorless liquid in 43% yield after three steps from 2.25 (Scheme 2.1).

Scheme 2.1 Synthesis of (2S, 3S) CF 3 -threonine

Synthesis of the fluorinated pentapeptides 2.18 and 2.19

With the Boc-(2S, 3S)-CF 3 -threonine 2.26 in our hands, we then tried to incorporate it into a pentapeptide sequence in order to prepare compound 2.18. The synthesis of pentapeptide 2.18 was efficiently realized as shown in Scheme 2.2. The dipeptide Boc-Val-Leu-OMe 2.27 was accessed by coupling Boc-Val-OH with Leu-OMe using IBCF/NMM in excellent yield (83%). As shown in Scheme 2.2, all the following peptide coupling reactions were performed in solution, using HBTU/HOBt/DIPEA or DMTMM/NMM as efficient coupling reagents. Treatment of 2.27 with TFA and then coupling of the free amine with (2S, 3S) CF 3 -threonine 2.26 in the presence of HBTU/HOBt/DIPEA, afforded the tripeptide 2.28 in good yield (73%). The same reaction was performed using DMTMM (BF 4 -)/NMM, however proved to be less efficient (< 60%) and gave inseparable impurities. Further elongation of the peptide chain using HBTU/HOBt reagent afforded lower yields (<40%) than using DMTMM (Cl -)/NMM which improved the yield of the coupling reactions and provided easier and cleaner workup. Following the DMTMM (Cl -)/NMM procedure, we obtained the tetrapeptide 2.29 in good yield (72%), and then the targeted pentapeptide 2.18 in excellent yield (83%). To test the ability of the type of pentapeptides containing (2S, 3R)-CF 3 -threonine to inhibit Aβ 1-42 peptide aggregation, we further cleaved the Boc group in the presence of HCl (4 M, in Dioxane) to give compound 2.19 in quantitative yield. Scheme 2.2 Synthesis of pentapeptides 2.18 and 2.19

Synthesis of the non-fluorinated pentapeptides 2.20 and 2.21

The synthesis of pentapeptide 2.20 was efficiently achieved as shown in Scheme 2.3. First, treatment of 2.27 with TFA and then coupling of Boc-Thr (Bzl)-OH in the presence of HBTU/HOBt/DIPEA, afforded the tripeptide 2.30 in good yield (83%). The tripeptide 2.30 was deprotected using TFA, and the free amine was coupled to Boc-Val-OH using HBTU/HOBt/ DIPEA, led to the tetrapeptide 2.31 in satisfactory yield (61%). The pentapeptide 2.32 was obtained in relatively low yield (28%), by deprotecting 2.31 with TFA and then performing the coupling reaction with Boc-Ala-OH in the presence of HBTU/HOBt/DIPEA. Finally, catalytic hydrogenation, using 10% Pd/C under H 2 atmosphere, gave the pentapeptide 2.20 in quantitative yield. After removal of the Boc group in the presence of TFA (30 eq.), the pentapeptide salt 2.21 was obtained in quantitative yield (Scheme 2.3). Both 2.20 and 2.21 were subjected to conformational analysis and biological evaluations.

Conformational Studies of pentapeptides with CF 3 -threonine 2.3.1 Previous example of effect of CF 3 -threonine on peptide conformation

To our knowledge, only one example of conformational studies of (2S, 3S)-CF 3 -theronine containing peptide has been conducted. Kitamoto, T et al. reported an interesting contrast between the enkephalin-related peptide derivative 3.35 and its fluorinated analogue 3.36 (Figure 2.15). [19f, 19g] It was noticed that the NOESY spectrum of peptide 3.35 revealed long-range through-space interactions, indicating a folded/compact conformation possibly induced by a Tyr-OH•••Thr-OH hydrogen bonding. However, the NOESY spectrum of the trifluorinated analogue 3.36 showed no long-range NOEs, which meant that the crucial Tyr-Thr hydrogen bonding disappeared and that an extended backbone conformation predominated. The chemical shift alteration under different solvent polarity revealed that HB forming protons were the amide protons of D-Ser, Gly, Phe, and the hydroxy proton of D-Ser in both 3.35 and 3.36. Moreover, the more acidic hydroxy proton of CF 3 -Thr in 3.36 was also found to participate, probably due to the strongly electron-withdrawing property of CF 3 group, which should effectively decrease the electron density of CF 3 -Thr-OH, and resulted in increase of acidity of this OH group. Temperature coefficients of these amide and hydroxy protons were in the range of -4.0 ~ -6.2 ppb/K revealed both of Gly-NH and CF n -Thr-OH (n =0 and 3) were expected to form intramolecular HB and in addition, Phe-NH and D-Ser-OH in 3. 35 were also suggested to be in a similar situation but with weaker interaction. The results from polarity and temperature dependence convinced the intramolecular hydrogen bond formations the intramolecular hydrogen bond formations of Gly-NH, Phe-NH, and D-Ser-OH in 3.35, and Gly-NH and CF 3 -Thr-OH in 3.36. Our objective was to study the influence of CF 3 -threonine when introduced in the central position of pentapeptides. For that purpose, we studied, by NMR and molecular modeling, and compared the conformation preferences of the N-Boc protected pentapeptides 2.11, 2.20, 2.18, and 2.10 containing in the third position a L-serine, L-threonine, (2S, 3S)-L-CF 3 -threonine and (2S, 3R)-L-CF 3 -threonine respectively. The N-deprotected pentapeptides analogues 2.34, 2.21, 2.19 and 2.33 were also studied.

NMR Conformational Studies

The NMR conformational studies were done in collaboration with Dr. Isabelle Correia and Prof. Olivier Lequin (UMR 7203, UPMC). All the NMR studies were carried out in CD 3 OH.

The 1 H and 13 C chemical shifts of the 7 pentapeptides (2.10, 2.11, 2.18, 2.19, 2.20, 2.21 and2.34) were unambiguously assigned using 1D 1 H, 2D 1 H-1 H TOCSY, 2D 1 H-1 H ROESY, 2D 1 H- 13 C HSQC, and 2D 1 H- 13 C HMBC spectra. The observed 1 H and 13 C chemical shift values, at 298K, of pentapeptide 2.10, 2.11, 2.18, 2.19, 2.20, 2.21 and2.34 (16 mM) are given in Table 2.1, 2.4, 2.7, 2.10, 2.13, 2.16 and 2.19 respectively. Only one set of chemical shift was observed for the deprotected pentapeptides, indicating that a single conformation or an average of conformations in rapid exchange on the NMR scale time was observed for all studied deprotected pentapeptides. For all Boc-protected pentapeptides, two chemical shift sets were observed for the tBu protons of the Boc group and the amide proton of the residue Ala 1 at 298 K due to the existence of the synand anti-rotamers of the carbamate group. The more stable forms (about 85% population) were ascribed to anti-rotamers based on literature results. [21] Different parameters were studied, including 1 H α and 13 C α chemical shift deviations (CSD), vicinal 3 J HN-Hα coupling constant, H α -NH ROE Correlations and Temperature Coefficient (Δδ HN /ΔT) of the amide proton. The 1 H α and 13 C α chemical shift deviations (CSD), defined as the differences between experimental chemical shifts and corresponding random coil values, are good descriptors of backbone conformational space for each residue. [22] 1 Hα and 13 Cα chemical shift deviations (CSD) of residues Ala 1 , Val 2 , Val 4 Leu 5 in all of the 7 pentapeptides indicated extended conformations, as supported by upfield shifted Cα carbons (negative CSD values in the range of -2.9 ppm to -0.6 ppm) and downfield shifted Hα protons (positive CSD values between 0.07 ppm and 0.22 ppm) except for Ala 1 Hα protons, suggesting higher flexibility at the N-terminus of the pentapeptides (Table 2. 2, 2.5, 2.8, 2.11, 2.14, 2.17 and 2.20). (Note that the chemical shift analysis is less reliable for residues at N and C-termini due to the absence of neighboring residue).

The vicinal 3 J HN-Hα coupling constant is a function of the dihedral angle φ, and it can yield direct information on the main chain φ dihedral angle, through the Karplus relationship, so it is a valuable descriptor of peptide backbone conformations. The coupling constants of the five residues exhibit large values (6.8 -9.2 Hz range), that are systematically higher than average values found in the coil library (6.1, 7.0, and 7.5 Hz for Ala, Leu and Val, respectively). [23] This clearly reflects a preference of all backbone dihedral angles φ for values in the range of -160 o ~ -110 o , as expected for extended conformations. The possible φ torsional angles can be calculated from 3 J HN-Hα values using the Karplus-type equation ( 3 J HN-Hα = 9.4cos 2 (φ-60) -1.1cos(φ-60) + 0.4) [24] , assuming a major "static" conformation. However a dynamic ensemble of conformers exploring a distribution of φ values is very likely, so the calculated φ values should only be taken as conformational trends. [25] The observed 3 J HN-Hα coupling constants and φ angles for the peptides are given in Table 2. 3, 2.6, 2.9, 2.12, 2.15, 2.18 and 2.21. All the 3 J HN-Hα coupling constants for the residues in these pentapeptides were higher than 7.0 Hz and were characteristic of a predominantly extended backbone conformation (Table 2.3, 2.6, 2.9, 2.12, 2.15, 2.18 and 2.21). The three central residues presented higher 3 J HN-Hα coupling constants than Ala 1 and Leu 5 residues, thus demonstrating stronger extended conformational propensities. Furthermore, we can notice that pentapeptide 2.11 bearing the L-serine has slightly weaker 3 J HN-Hα coupling constants than pentapeptides 2.20, 2.18, and 2.10 bearing the L-threonine and its fluorinated analogues. A slightly more extended conformation can be deduced from the higher 3 J HN-Hα coupling constants of the 3 central residues of the fluorinated pentapeptides compared to those containing natural Lthreonine. No major difference could be observed between the Boc protected pentapeptides and their deprotected analogues.

In peptide NMR, the chemical shifts of amide proton generally display temperature dependence. [26] The temperature dependence of amide proton chemical shifts has been widely used to predict hydrogen bond involvement; it can provide information on the network of intramolecular hydrogen bonds and their relative stabilities. [27] Amide protons that are engaged in intramolecular hydrogen bonds typically exhibit small temperature dependence (Δδ HN /ΔT > -4.6 ppb K -1 ) in aqueous and alcoholic solvents, while those that are not intramolecularly hydrogen-bonded usually exhibit largely negative values of their temperature coefficients (Δδ HN /ΔT < -6 ppb K -1 ). Analysis of the temperature coefficient of the amide bond NH (Δδ HN /ΔT) shows a negative value in the range of -9.0 ppb/K to -5.0 ppb/K for most protons, which indicates that they are not engaged in stable intra/inter-molecular hydrogen bonds (Table 2 .3, 2.6, 2.9, 2.12, 2.15, 2.18 and 2.21, respectively). Interestingly, residue Val 4 displays small temperature coefficient (around -4.5 ppb K -1 ) in pentapeptides 2.18 and 2.19. Residue Val 2 shows intermediate values between -5.5 and -5.9 ppb K -1 . This i/i+2 periodicity could be explained by transient intermolecular β-strand contacts involving hydrogen bonding through Val 2 and Val 4 residues.

The analysis of Hα-NH ROE correlations shows strong sequential Hαi-NHi+1 ROEs, exhibiting higher intensities than intra-residual Hαi-NHi ROEs, which confirms extended backbone conformations of the pentapeptides. No long-range ROEs could be observed in the 7 pentapeptides, indicating that transient intermolecular association, if any, is too fast to be detected by ROE magnetization transfer.

In Summary, NMR Analysis has shown that the pentapeptides with the sequence (Boc) H 2 N-Ala-Val-X-Val-Leu-OMe (X = Ser, Thr, (2S, 3R)-CF 3 -Thr and (2S, 3S)-CF 3 -Thr) have a predominantly extended backbone conformation in CD 3 OH at 25 o C. Pentapeptides 2.11 and 2.34 bearing Lserine are less extended than their analogues L-threonine and CF 3 -analogues. A slightly higher propensity to adopt extended conformation of the (2S, 3S)-CF 3 -threonine has been also observed, with a propensity to self-associate by forming intermolecular β-strand contacts, as suggested by temperature coefficients (Figure 2.16). 0.15 -1.9 Leu 5 0.09 -2.9 (The random coil values of H α protons, C α carbons were calculated using random coil values reported in water [22e] .) ΔδNH 2 /ΔT=-5.5 ppb K -1

δ H α (ppm) δ H β (ppm) δ H γ & H δ (ppm) δ CO (ppm) δ C α (ppm) δ C β (ppm) δ C γ & C δ (ppm)
δ H α (ppm) δ H β (ppm) δ H γ & H δ (ppm) δ CO (ppm) δ C α (ppm) δ C β (ppm) δ C γ & C δ (ppm)
δ H α (ppm) δ H β (ppm) δ H γ & H δ (ppm) δ CO (ppm) δ C α (ppm) δ C β (ppm) δ C γ & C δ (ppm)
ΔδNH 5 /ΔT=-7.9 ppb K -1

42 43 A1 1 T3-CF 3 1 V2 1 V2 1 A1 1 T3-CF 3 1 V4 1 V4 1 ? 3 J N-α = 8.9 Hz
Sequential Noes observed (strong) intra-residus Noes observed (small) 3 J N-α = 8.9 Hz 3 J N-α = 9.0 Hz 3 J N-α = 7.6 Hz 16 Anti-parallel β-sheet conformation in equilibrium with extended monomer of (2S, 3S)-2.18 Table 2.10 1 H NMR and 13 C NMR chemical shifts of pentapeptide 2.19 (16 mM) 25.8, 23.4, 21.7 Table 2.17 Chemical shift deviations (CSD) of peptide residues of 2.21 (16 mM) 0.07 -2.9 (The random coil values of H α protons, C α carbons were calculated using random coil values reported in water [22e] .) (2.20 and 2.21). This discrepancy between the NMR and MD 3 J HN-Hα coupling values for the central residue of AV-(3S)-CF 3 -VL (2.18 and 2.19) peptide indicates that its simulation explored extended conformations less frequently than in experiments. However, the 3 J HN-Hα coupling constants alone cannot unambiguously discriminate between α-or β-structures for each residues and, above all, cannot determine the peptide global structure. In that context, MD simulations can provide useful complementary structural informations. In our case, MD trajectories revealed clear differences between the conformations of the four sequences. Indeed, when their end-to-end distances are analyzed (Figure 2.18), it can be noted that both the BOC-protected (2.18) and non-protected AV-(3S)-CF 3 -VL peptide (2.19) have significantly larger populations of extended conformations than the other three sequences whose the distributions are broader and shifted toward lower values. This global structural characteristic is reflected at a local level when the distribution of the backbone ψ dihedral angle values are examined (Figure 2.19). Contrary to the three sequences AVSVL (2.11 and 2.34), AVTVL (2.10 and 2.21) and AV-(3R)-CF 3 -VL (2.10 and 2.33), all the three central ψ dihedral angles of the AV-(3S)-CF 3 -VL peptide (2.18 and 2.19) clearly have a higher propensity to populate the β-(90°; 180°) than the α-region (-70°; +40°), endowing it with the aforementioned extended conformations. More specifically, the probability of the each residue to be in α-or β-conformation can be quantified by calculating the area under the peaks of the ψ distribution functions centered around -30° or +140°, respectively. The probability of the three central residues to be in β-conformation is reported in Table 2.22 for all studied peptides. It can be seen that, except the (3R)-CF 3 -Thr residue in the BOC-protected peptide, all central residues predominantly adopt local β-conformations, with probabilities ranging from 50 to 92%, in agreement with the NMR 3 J HN-Hα coupling values which were found larger than 7 Hz. The probability of each peptide to have all its three central residues in β-conformation (which is equal to the product of the three central residue probabilities) is a good indication of its propensity to adopt a global extended structure. According to this criterion, almost 50 % of the AV-(3S)-CF 3 -VL peptide (2.18 and 2.19) conformations are globally extended, whereas less than 30 % of the other sequence conformations are in that case (Table 2 2.22 Probability (%) of the three central residues of the eight studied peptides to be in βconformation. The column P indicates the probability for each peptide to have all its three central residues in β-conformation.

L5 1 L5 1 ΔδNH 4 /ΔT=-4.6 ppb K -1 ΔδNH 3 /ΔT=-8.3 ppb K -1 Figure 2.
in CD 3 OH (298 K) Residue δ NH (ppm) δ H α (ppm) δ H β (ppm) δ H γ & H δ (ppm) δ CO (ppm) δ C α (ppm) δ C β (ppm) δ C γ & C δ (ppm) Ala 1 - 3.
δ H α (ppm) δ H β (ppm) δ H γ & H δ (ppm) δ CO (ppm) δ C α (ppm) δ C β (ppm) δ C γ & C δ (ppm)
δ H α (ppm) δ H β (ppm) δ H γ & H δ (ppm) δ CO (ppm) δ C α (ppm) δ C β (ppm) δ C γ & C δ (ppm) Ala 1 - 3.
δ H α (ppm) δ H β (ppm) δ H γ & H δ (ppm) δ CO (ppm) δ C α (ppm) δ C β (ppm) δ C γ & C δ (ppm) Ala 1 - 3.
The most prevalent conformations of each peptide were determined by clustering their conformational ensembles, using the "gromos" method implemented in GROMACS with a RMSD threshold of 0. 3C-protected n-protected uctures, we the peptide 3S)-CF 3 -VL sequences (2.18 and 2.19), between, on one hand, the two residues Val 2 and Val 4 side chains, and, on the opposite side (with respect to the backbone), between the modified threonine trifluoromethyl group and the Leu 5 side chain. These hydrophobic interactions, which are seen in a lesser extent in the other sequences, are probably responsible for the observed global extended conformations of both the BOC-protected (2.18) and non-protected AV-(3S)-CF 3 -VL ( 2.19) peptides.

Figure 2.22

Distribution function of the distances between the Ala 1 side chain and the methyl or trifluoromethyl group of the central residue X 3 (black), between Val 2 and Val 4 side chains (red) and between the methyl or trifluoromethyl group of the residue X 3 and Leu 5 side chains (green) within the three peptides AVTVL, AV-(3R)-CF 3 -VL and AV-(3S)-CF 3 -VL.

A question remains regarding the intriguing difference between the predominant conformations of the two sequences AV-(3R)-CF 3 -VL (2.10 and 2.33) and AV-(3S)-CF 3 -VL (2.18 and 2.19). Indeed, it is observed that the (3R) stereoisomer is significantly less extended than the (3S) peptide (Figure 2.20 and 2.21), which is probably related to the lower probability of its central residue to be in βconformation (Table 2.22) In order to provide an explanation, we examined the distances between the hydroxyl group of the central residue side chain and the neighboring polar atoms in the peptide backbones (Figure 2.23). In both stereoisomers, the trifluoromethyl group is oriented toward the Leu 5 side chain (toward the right side of Figure 2.23) and the hydroxyl oxygen makes favorable electrostatic interactions with the amide hydrogen (red lines in Figure 2.23). However, in the (3R) stereoisomer, the central residue hydroxyl oxygen is quite close to its carbonyl one (green lines), making unfavorable electrostatic interactions. Consequently, the ψ dihedral angle of the central residue can easily change from the β-(140°) to the α-conformation (-30°) so that its amide hydrogen comes close to the hydroxyl oxygen (blue lines). This results in a more compact global structure of the AV-(3R)-CF 3 -VL peptide (2.10 and 2.33) than AV-(3S)-CF 3 -VL (2.18 and 2.19). In the AVTVL sequences (2.20 and 2.21), the hydrophobic interaction between the threonine methyl group and the Leu 5 side chain is weaker than the one involving the CF 3 group of AV-(3S)-CF 3 -VL (2.18 and 2.19). The threonine χ dihedral angle can therefore rotate more freely and it hydroxyl group can find favorable electrostatic contacts with the neighboring amide hydrogens (red and blue lines), while keeping a ψ dihedral angle in a predominant extended β-conformation (Figure 2.19). All together, the theoretical study shows that the replacement of the methyl group of the threonine side chain in the AVTVL pentapeptide by a trifluoromethyl induces an increase of the population of global extended conformations. This higher propensity of the AV-(3S)-CF 3 -VL peptide (2.18 and 2.19) to adopt extended structures can be explain by a stronger hydrophobic interaction of its trifluoromethyl group with the Leu 5 side chain with respect to the one involving the threonine methyl group.

Effect of pentapeptide 2.11 (BocHN-Ala-Val-Ser-Val-Leu-OMe) on amyloid fibril formation of Aβ 1-42 and IAPP

Figure 2.24 shows that pentapeptide 2.11 doesn't show any tendency to self-aggregate at high concentration (100 μM) alone. The presence of peptidomimetic 2.11 at pentapeptide/Aβ 1-42 ratio of 10/1 (100 μM/10 μM), did not delay the lag phase time, but seemed to very slightly modify the nucleation phase because the intensity of fluorescence is slightly enhanced at the beginning of the kinetics. However, the final fluorescence intensity did not change relative to Aβ 1-42 alone (10 μM). The final fluorescence intensity was slightly reduced (about 30%) only in the presence of a substoichiometric concentration of 2.11 (ratio of 0.1/1). It is important to note that this new batch of pentapeptide 2.11 (prepared by myself) did not demonstrated at all the same inhibitory activity than the previous batch (prepared by Nadjib Kihal and evaluated in 2012, see figure 2.11). Unfortunately, we don't have any explanation to this lack of reproducibility.

For the effect of pentapeptide 2.11 on IAPP peptide (5 μM), an acceleration of the aggregation at pentapeptide/IAPP ratio of 10/1, and 1/1 was observed, the lag phase being shortened by about 50%, but the final fluorescence intensity has not been changed (Figure 2.25). showing the fi apeptide 2.34 M), buffer (grey

Conclusions

In summary, we have prepared (2S, 3S)-CF 3 -threonine (natural threonine mimic) through the aldol reaction of CF 3 CHO with Ni (II)-complex of the chiral Schiff base of glycine, then we have introduced it into pentapeptides to study its effect on peptide conformation and also the ability of these fluorinated pentapeptides to modulate amyloid fibril formation of Aβ 1-42 and IAPP peptides.

We have designed and synthesized pentapeptides with the sequence as BocHN-Ala-Val-X-Val-Leu-OMe, where X are Ser, Thr, (2S, 3R)-CF 3 -Thr and (2S, 3S)-CF 3 -Thr. The conformational analysis of these pentapeptides was conducted by the combined use of Accelerating the aggregation pathways is less intuitive but this strategy has more recently aroused interest. [29] Indeed, Methylene Blue (MB) which is in phase III clinical trials promotes fibrillization [30] . A few other preliminary studies using compounds that accelerate Aβ 1-42 fibril formation demonstrated a reduced toxicity for mammalian cells concomitantly with the reduction of the concentration of transient soluble Aβ 1-42 oligomers. [29] There are very few reports of compounds that accelerate the aggregation of IAAP. [31] Bisphenol A (BPA) was reported to dose-dependently promote the aggregation of hIAPP and enhance the membrane disruption effects of hIAPP, which explains a possible link between BPA exposure and Type II Diabetes. [31a] A series of oligoamide-based helix mimetics were also reported that can bind to a complementary helical motif in IAPP. These helical compounds accelerated IAPP amyloid formation under lipid-free conditions, but inhibited it under lipid-catalyzed conditions. [31b] In consequence, it could be interesting to further study the effect of accelerating IAPP aggregation by complementary techniques. In particular, the effect of pentapeptides 2.11, 2.19 and 2.34 on the presence of small toxic oligomers could be studied by capillary electrophoresis or IMS-MS, when these techniques will be fully developed (one PhD student is actually developing them in our laboratory). The effect on reducing the toxicity of IAPP on cells could be also evaluated (in collaboration with our colleagues at Lancaster University).

In perspectives, the pentapeptides bearing (2S, 3S)-CF 3 -Thr 2.19 can be introduced in β-hairpin mimics as it modulates Aβ 1-42 aggregation (as seen in Figure 2.29) and as we have previously reported in our laboratory that isolated natural pentapeptides (such as KLVFF or GLMVG) are much less active than β-hairpin mimics based on a piperidine-pyrrolidine β-turn inducer and containing those pentapeptides. [32] Chapter III Fluorinated Triazole Oligomers

Design Strategy

Introduction to Oligomeric Foldamers

The term "foldamer" was first introduced by Gellman, S. H. to describe any oligomer that adopts well-defined secondary structure (such as helix, sheet, turn, strand etc.) in solution, the structure of which is stabilized by a collection of noncovalent interactions between non-adjacent monomer units. [1] These noncovalent interactions usually include electrostatic interactions, H-bonds, π-π interactions, coordination to metal ions, steric interactions, and solvophobic effects. [2] The stable and ordered solution structures with unique conformations are most often involved with interesting functions such as molecular recognition, electron transfer, and catalysis. The research of foldamer not only helps scientists gain deeper insight into the functions of nature's biomacromolecules, but also expands the scope of functionalities into an area that are outside of those seen in nature. Chemists have endeavored to intentionally generate unnatural oligomeric sequences, and to date progress has been encouraging, and the field is developing rapidly as demonstrated by the various architectures that have been generated, schematically represented in Figure 3.1. [2] Figure 3.1 Illustrations depicting the different types of foldamer secondary structures.

(Adapted from reference [2])

The key for the successful application of foldamers, i.e. non-natural folded peptidomimetic oligomers, is basically finding compounds which can reproduce some of the fundamental properties of natural peptides and proteins, such as structural flexibility and water solubility. Moreover, easy access to diverse amino acid units by robust modular synthesis also plays an important role in facilitating the construction of such foldamers.

The structural and constitutional composition of most foldamers can be derived back to natural peptide backbones, where α-amino acids and their homologues, β-, γ-, δ-etc. amino acids, are used. Since these homologues result in increased backbone flexibility, both aliphatic and aromatic cyclic insertions have been applied to facilitate folding into various, mainly helical, secondary structures. [3]

Triazole in Foldamers

General introduction to 1,2,3-triazoles

The stability of natural peptides is one of the main limitations of their use as drug candidates due due to the fact that natural peptide bonds are subjected to proteolysis by various proteases. The replacement of enzyme cleavable amide bonds with non-natural stable structures [4] , while at the same time maintaining biological activity, can be useful for improving the drugability of peptides.

Many strategies have been used to increase the stability of peptide drug candidates, such as introducing non-natural amino acids, terminal protection, cyclization, and backbone modification. The 1,2,3-triazole can be regarded as a bioisostere of the amide bond moiety of peptides. [5] The physicochemical properties of 1,2,3-triazole have been reviewed [6] . The similarity of the two moieties can be seen in their sizes (distances between substituents are 3.8-3.9 Å in amides and 5.0-5.1 Å in 1,2,3-triazoles, Figure 3.2), dipole moment (amide ~ 4 Debye, 1,2,3-triazole ~ 5 Debye), and H-bond acceptor capacities [7] . The 1,2,3-triazole rings, with sp 2 -hybridized nitrogen atoms can function as weak hydrogen-bond acceptors. [8] And, the strong dipole moment of the 1,2,3-triazole ring polarizes the C( 5) proton to such a degree that it can function as a hydrogenbond donor similar to the amide NH. [9] Furthermore, the 1,2,3-triazole ring has a large dipole that could align with that of the other amides in a given peptide secondary structure. Importantly, 1,2,3-triazoles are extremely stable to hydrolysis. So, 1,2,3-triazoles can be potential surrogates of amide bonds for peptide modification. 1,2,3-triazoles offer a great structural diversity in peptidomimetic research because their structural and electronic characteristics are similar to those of a peptide bond and because general methods are now available for their synthesis. [10] Classically, 1,2,3-triazoles are synthesized by a thermal Huisgen cycloaddition reaction, [11] which generally produces mixtures of 1,4-and 1,5-substituted triazoles. However, the emergence of the copper-catalyzed azide alkyne cycloaddition (CuAAC, also known as the copper-catalyzed Huisgen cycloaddition) now provides ready and selective access to 1,4-substituted 1,2,3-triazoles for use in peptidomimetics and other areas of bioorganic chemistry. [12] The CuAAC reaction was first reported by the group of Meldal [13] and later developed by Sharpless and co-workers as a key example of the click chemistry concept. [14] Sharpless and co-workers later reported a ruthenium-catalyzed azide alkyne cycloaddition (RuAAC) that allows the synthesis of 1,5-substituted triazoles, however the reaction conditions are not compatible with the Click Chemistry. [15] Figure

Proposed mechanism for the CuAAC reaction

To better understand the potential use of triazole peptidomimetics as foldamers, Nina Kann et al.

have explored the structural properties and stability of the two triazole-based δ-amino acid mimic: the 1,4-and 1,5-substituted 1,2,3-triazole peptides, 4Tzl and 5Tzl [16] (Figure 3.4).

Figure 3.4 Structures of the 4Tzl and 5Tzl

They described a systematic analysis of the conformational space of the two basic units using quantum chemical calculations and NMR spectroscopy. Theoretical results show 9 conformers for 5Tzl derivatives with relative energies lying close to each other, which may lead to a great structural diversity (Figure 3.6). NMR analysis also indicates that conformers preferring turn, helix and zig-zag secondary structures may coexist in solution. In contrast, 4Tzl has a much lower number of conformers, only 4, and these lack strong intra-residual interactions (Figure 3.5). The structural information provided on these building units could be employed in future design of triazole foldamers. Surprisingly, the vast majority of 5Tzl conformers have a relative energy lower than 3 kcal mol -1 , and they are structurally more diverse than those of 4Tzl. Together with previous NMR results on their corresponding homooligomers, [17] this observation suggests that several secondary structures built from these may coexist in solution, a basic prerequisite for larger scale conformational diversity in the case of natural proteins.

General introduction to triazole-based foldamers

Replacing peptide bond with a triazole unit could result in interesting structures with unique conformational characteristics. Triazole unit formed by the click reaction can act as a helical component, a β-turn unit and a cis/trans-prolyl ratio modifier. Ghadiri's group has shown that a non-natural triazolo-amino acid can replace a dipeptide in an α-helical secondary structure [18] . They incorporated triazoles into peptides that form 4-helix bundles and solved the crystal structure of the bundled peptides. These findings show the potential use of click reactions in the non-native chemical synthesis of peptides and proteins. Researchers have assessed the ability of 1,5disubstituted triazoles as cis-proline mimics [19] .

Based on the molecular modeling results that a 1,4-substituted 1,2,3-triazole ring might be commensurate with the required geometry for β-turns, Zhibin Guan et al. described a new β-turn mimicking unit through intermolecular alkyne-azide cycloaddition. [20] They discovered that the tendency of β-turn formation for the triazole system strongly depends on the linker length on both sides and three-carbon linker is optimal for stable β-turn formation (Figure 3.7). Cycloaddition of peptide alkynes to peptide azides affords quick access to triazole-based β-turn units. The literature contains many examples of peptidomimetics that have multiple peptide bonds replaced with a triazole. Arora and co-workers were the first to report the synthesis of fully backbone-modified peptidomimetics, termed triazolamers (Figure 3.8). [21] A conformational analysis of triazolamer trimers by NMR, suggests that they adopt an anti-conformation (Figure 3.9) that is similar to a β-strand. Ab initio and molecular mechanics studies support this structure, with the trans-conformation being more stable than the syn conformation by up to approximately 4 kcal/mol (Figure 3.9). Dipole-dipole interactions between neighboring triazole rings appear to play a critical role in stabilizing the observed conformations in polar solvents, such as DMSO and acetone. Because proteases are known to have substrates in a β-strand conformation, the protease resistance conferred by triazole moieties has also been tested. Screening of a small library of such triazolamers led to the discovery of HIV-1 protease inhibitors. [21] Recently peptidic homo-oligomers with heterocycles such as imidazole, thiazole, and triazole and so on as backbone have increasingly drawn attention in the foldamer research community.

Abdulselam Adam et al. find that imidazole-containing pseudopeptides [22] adopt helix-like secondary structures in a temperature and solvent dependence manner. The screw sense of the helix can be determined by attaching a single chiral imidazole unit to the N terminus of the oligomer (Figure 3.10). Investigation by circular dichroism (CD) spectroscopy showed that the folding process of the helix depends on the water content of the solvent in a parabolic way. In a pure organic medium, the helix is stabilized by hydrogen bonds between the hydrogen atoms of the amide groups and the sp 2 -hybridized nitrogen atoms of the azole ring. In aqueous solution, the formation of the helix is driven by dispersion interactions. The formation of the helix is more pronounced in aqueous solution than in organic solvents. ). [23] The conformations of the ATC oligomers were analyzed in solution by CD and NMR spectroscopy and in the solid state by X-ray crystallography. The ATC sequences adapted a well-defined 9-helix structure in the solid state and in aprotic and protic organic solvents as well as in aqueous solution. reported a new set of peptidomimetics derived from δ-peptides, consisting of n (n = 2, 3, 4) units of a 1,5-disubstituted 1,2,3-triazole amino acid (5Tzl) (Figure 3.12). [24] Conformational analysis by 2D NOESY NMR and quantum chemical calculations indicated that for the trimer there were several conformations co-existing with helical and turnlike conformers being the most probable conformers in DMSO solution. However, the 2D NOESY data from the NMR study of tetramer was unfortunately not assignable due to extensive signal overlap between the amine methylene protons, as well as the carbonyl methylene protons, the triazole protons, and the amide protons. These extensive overlaps render the assignment of any plausible secondary structures unjustified. Nevertheless, this feature of homo-5Tzl may prove essential for mimicking natural peptides composed of α-amino acids, where the various secondary structures could be achieved by possible side chain effects but not by the rigidity of the peptide backbone.

Fluorine in Foldamers

As seen examples in chapter 1.1.2, the introduction of fluorine can modify various parameters (electronic distribution, conformation, lipophilicity, and acidity/basicity) of peptides. Amino acids bearing a perfluoroalkyl (R f ) group have attracted particular attention recently, owing to their strong electron-withdrawing nature, R f groups generally increase the acidity and hydrogen-bond donating ability of adjacent protic functionalities, including amide NHs (Figure 3.13). [25] Therefore, such modification can have a significant influence on the stability of intramolecular hydrogen bonds between amide NHs and carbonyls in a peptide backbone, which would lead to a novel methodology to control the secondary structure of peptides. Yasuhiro Ishida et al. showed that a 14-helix structure of oligo-β-peptides was significantly stabilized by direct attachment of CF 3 groups to their backbones (Figure 3.14). [25] The studies also indicate that this stabilization originates from the CF 3 -promoted increase in the intramolecular hydrogen-bonding ability of their backbone amides. bond unit can also act as a poor hydrogen bond acceptor. [26] Intramolecular N-H•••N and N-H•••O hydrogen bonds are typically used to encourage folded conformations aromatic amide foldamers, [27] Zhan-Ting Li et al. found that intramolecular N-H•••F hydrogen bonds are also strong enough to encourage aromatic oligoamides to form folded conformations such as crescent and helical conformations (Figure 3.15). [28] Figure 3.15 Intramolecular F•••H-N hydrogen bonds illustrated in an aromatic amide foldamer.

Design of CF 3 -1,4-disubstituted 1,2,3-triazole Oligomers

Aim of the project

It is known that fluorinated amino acids can have drastic effects on peptides or protein stability and on peptide-peptide interactions due to the unique stereoelectronic properties of fluorine. [29] Although the effect of the incorporation of side chain fluorinated α-amino acids into amyloid forming peptides have been recently investigated, [30] the use of fluorinated amino acids containing peptides in amyloid aggregation inhibition have increasingly draw attentions among researchers. [31] There have been reports describing the synthesis and application of α-CF 3 -containing triazolyl amino acids as potential neurotransmitters (Figure 3.16), [32] the synthesis and applications of CF 3 -1,4-disubstituted-1,2,3-triazole-based amino acid mimic such as compound 3.0 listed in Figure 3.16 have rarely been reported.

(1) We aimed at the synthesis of trifluoromethylated 1,4-disubstituted 1,2,3-triazole scaffolds 3.0 and also at the synthesis of oligomers of this kind of amino acid mimics (Figure 3.17). We envisioned that these oligomeric peptidomimetics could adopt well defined secondary structures such as helix. (2) The second aim was to apply this kind of oligomers to the field of medicinal chemistry. We aimed at the synthesis of CF 3 -1,4-triazole amino acid based trimer and tetramer derivatives (Figure 3.17) and the utilization of these compounds as potential modulator of amyloid protein aggregation.

Diastereoselective synthesis of CF 3 -1,4-triazole-based scaffold

In our laboratory, Julien Bonnamour et al. reported trifluoromethyl azido compounds undergo a Cu (II) catalyzed Huisgen 1,3-dipolar cycloaddition with alkynes to yield 1,4-disubstitued 1,2,3triazoles. The corresponding products were obtained in high yields (74-87%), which could potentially be used to design new trifluoromethyl pseudopeptides. [33] Later, Michela Martinelli et al. described a method to obtain enantiopure trifluoromethyl 1,4-disubstituted 1,2,3-triazole scaffolds. [34] In the approach, the trifluoromethyl-propargylamine 3 bearing (R)-phenylglycinol chiral auxiliary was coupled with azidoacetic acid methyl ester via copper (I)-catalyzed 1,3dipolar cycloaddition affording the cycloadduct in good yield (79%) and as a single isomer without any racemization (Scheme 3.1).

Scheme 3.1 Synthetic approach to the trifluoromethyl triazoles

The (R)-phenylglycinol chiral auxiliary being not so easy to cleave, we have decided to develop a new asymmetric synthesis by using the chiral (R)-tert-butylsulfinyl protecting group. The chiral amine reagent tert-butanesulfinamide is inexpensive to prepare on a large scale in enantiomerically pure form. The direct condensation of tert-butanesulfinamide with a wide range of aldehydes proceeds in high yields under mild conditions to give stable N-tert-butanesulfinyl aldimines, which are significantly more electrophilic than typical N-alkyl or aryl imines. This enhanced electrophilicity enables clean and high yield additions of a very wide range of diverse nucleophiles, including organo-magnesium, lithium, zinc, silicon, indium, cerium, and boron reagents, stabilized carbanions exemplified by enolates, and noncarbon nucleophiles such as phosphorus, boron, tin, and silicon species as well as numerous hydride reagents. The tertbutanesulfinyl group is capable of metal coordination, resulting in high diastereoselectivities for the addition of many different nucleophiles to imines. The tert-butanesulfinyl group also serves as an extremely versatile protecting group. It is also stable to a wide range of reaction conditions, including strong bases, nucleophiles, and a variety of transition metal catalyzed reactions. Convenient and high yielding cleavage of the N-tert-butanesulfinyl group can be accomplished by simple treatment with methanolic HCl to provide the amine hydrochloride products after precipitation with ethereal solvents, often in nearly quantitative yields and in analytically pure form. [35] In our aim to obtain CF 3 -1,4-disubstituted-1,2,3-triazole-based amino acid mimic such as compound 3.0, we use the commercial available (R)-2-methyl-N-(2, 2, 2-trifluoroethylidene)

propane-2-sulfinamide (CAS NO: 1219607-85-8) as chiral starting material. The terminal alkyne compound 3.3 was accessed by zinc mediated Barbier type reaction (Scheme 3.2). The zinc mediated Barbier type reaction was found complete and efficient in DMF within 2 hours under argon atmosphere by 19 F NMR monitor. When the reaction was run in THF, it needed longer time (8h) to finish, and it gave a messy mixture (TLC and 19 FNMR showed) which was difficult to purify. When the stoichiometry was 1.1 equivalent of Zn and 1.1 equivalent of bromoprop-1-yne, the reaction was finished in 2h with a moderate yield (53%) after column purification. When the reaction stoichiometry was 1.5 equivalent of Zn and 1.5 equivalent of bromoprop-1-yne, the reaction proceeded with a much improved yield (92%) after column purification (Scheme 3.2).

Trials

Conditions Yield 1 Zn (1.5 equiv.), propargyl bromide (1.5 equiv.), THF, 8h messy mixture 2

Zn (1.1 equiv.), propargyl bromide (1.1 equiv.), DMF, 2h 53% 3

Zn (1.5 equiv.), propargyl bromide (1.5 equiv.), DMF, 2h 92% Scheme 3.2 Diastereoselective Zinc mediated Barbier type propargylation The Zn-mediated propargylation reaction on (R)-tert-butanesulfinyl aldimine was conducted in DMF, the reaction gave target product with excellent diastereoselectivity (91% dr, crude 19 F NMR). Later, the copper catalyzed click reaction was conducted in acetonitrile at room temperature with 10 % CuI. When the reaction was run without base, 19 F NMR showed no product formed even after 20h reaction time. But when Et 3 N was added, the reaction can complete within 2h with a satisfactory yield of 76% after purification by flash column chromatography (Scheme 3.3). The protecting group (R)-tert-butylsulfinyl can be readily cleaved at room temperature in the presence of HCl-Dioxane (4M, 30 eq.) or CF 3 COOH (30 eq.) in 3h in quantitative yield (Scheme 3.4).

Scheme 3.4 The removal of the (R)-tert-butylsulfinyl protecting group

The saponification reaction of the C-terminal ester group was found to proceed well using NaOH (2M, 2.5 eq.) in EtOH at 0 o C for 1h with a yield of 92% without purification [after acidify to pH = 3-4, then extraction with EtOAc three times, Scheme 3.5]. Due to the strong electron withdrawing nature of CF 3 group, the acidity of the neighboring proton is relatively strong, when the saponification reaction occurred at room temperature overnight, we observed that epimerization happened ( 19 F NMR showed two doublet peaks compared with 3.6). When the saponification reaction was conducted with LiOH (1M, 2.5 eq.) in THF at 0 o C for 1h, we got the desired acid in a moderate yield of 73% without epimerization. Due to the strong electron-withdrawing effect of CF 3 group, the nucleophilicity of the amine of compound 3.5 was decreased compared to the non-fluorinated analogue. In order to evaluate the capacity to introduce our new CF 3 -triazole amino acid unit in peptide sequences in foldamers, we designed model coupling reactions. In particular, we suspected a low nucleophilicity of the amine in the alpha position of CF 3 group due to its strong electron withdrawing properties. The Nterminal deprotected amine 3.5 was tried to couple with natural amino acid using different coupling conditions. When the amine was coupled with Boc-Ala-OH using EDCI/HOBt/DIPEA in DMF, compound 3.7 was obtained in a poor yield of 30%. When HBTU/HOBt/DIPEA in DMF was used, the yield was improved (61%). To our delight, when DMTMM/NMM was employed, the coupling reaction was found more efficient with a much better yield (87%). The C-terminal ester of 3.7 was hydrolyzed and tried to couple with H-Ala-OMe using HBTU/HOBt/DIPEA in DMF, the yield of coupling was a little bit low (40%). The above reactions showed the feasibility of CF 3 -1,4-disubstituted 1,2,3-triazole based scaffold to couple with natural amino acids both at N and C terminal parts. It is a promising mimic of natural amino acid that can be used to modify peptide and peptidomimetic conformations and biological properties. Since we had already managed to couple the CF 3 -triazole based amino acid mimic with natural amino acid on both sides, then we were encouraged to couple the CF 3 -triazole based amino acid mimic with itself to form a dimer.

Synthesis of

We first tried to couple the monomer with DMTMM (BF 4 -

)/NMM as it has previously been shown to work well in the coupling reaction of this CF 3 -triazole based amino acid mimic with natural amino acid. The first trial allowed us to obtain the dimer 3.9 in 11% yield after three purifications by flash column chromatography with lot of product remaining in the column because of the poor solubility of the dimer in EtOAc and cyclohexane. Later trial of the coupling reaction using DMTMM (BF 4 -)/NMM showed better yield (53%) using CH 2 Cl 2 -MeOH (100:2) as eluent during column chromatography. When the coupling reaction was tried using HBTU/HOBt/DIPEA, it was sluggish, and the crude mass showed no dimer formed by TLC. Furthermore, when the coupling was conducted using EDCI/HOBt/DIPEA, the conversion was so low that almost no dimer was observed by TLC. It was more efficient when the coupling reaction was conducted using HATU/HOAt/DIPEA, and the yield was improved to 63%. Table 3.1 Coupling conditions for the synthesis of dimer 3.9 with CF 3 -1,4-disubstituted-1,2,3triazole-based amino acid unit However, when the purity was evaluated, LC-MS showed that an oxidation of the dimer 3.9 occurred. As the Figure 3.19 shown, the peak at t 12.83 showed the mass of [dimer+H] +1 peak (MW: 590); while the peak at t 14.53 showed the mass of [dimer+16+H] +1 , which means the oxidation of the tert-butylsulfinyl group to tert-butylsulfonyl group (compound 3.10, Scheme 3.7). Furthermore we also observed the trans-esterification of the ethyl ester to methyl ester (the peak at t 11.97 and t 13.72 both showed the methyl ester product, 4%). Integration of the two peaks at t 13.72 and t 14.53 showed the oxidized impurity was about 5% (based on 1 H NMR calculation, the oxidized impurity was about 10%). For this moment we don't know how the oxidation happened, we are not sure if it occurs during the coupling reactions, or the hydrolysis reactions or during the LC-MS and HPLC purity analysis, but at least the oxidized impurity can be reduced to 5% in a degassed and oxygen free atmosphere. With the dimer in hand, even it was not fully pure, we continued to synthesize the trimer and tetramer. The most efficient coupling condition was: HATU/HOAt/DIPEA in DMF.

The dimer was successfully deprotected at both sides in the same conditions as the deprotection of the monomer 3.4 (Scheme 3.4 and Scheme 3.5). The saponification reaction of the C-terminal was found to precede well using NaOH (2M, 2.5 eq.) in EtOH at 0 o C for 1h to give the dimer acid 3.11 in 96% yield. The tert-butylsulfinyl group was cleaved at room temperature in the presence of HCl-Dioxane (4M, 30 eq.) in 3h in quantitative yield. The trimer 3.12 was obtained using HATU/HOAt/DIPEA as coupling reagent in satisfactory yield (65%). But the solubility of the trimer in CH 2 Cl 2 and CH 3 OH decreased compared with the dimer compound 3.9.

Scheme 3.8 Synthesis of trimer 3.12 of CF 3 -1,4-disubstituted-1,2,3-triazole-based amino acid

As observed in the case of the dimer 3.9, when the purity was evaluated, LC-MS showed that an oxidation occurred. Figure 3.20 showed, the HPLC-MS peak at t 13.77 showed the mass of [trimer+H] +1 peak (MW: 810); while the peak at t 15.19 showed the mass of [trimer+16+H] +1 , which indicates the oxidation of the tert-butylsulfinyl group to the tert-butyl sulfonyl group. Integration of the two peaks at t 13.77 and t 15.19 showed the proportion of non-oxidized product was 85%, and of the oxidized impurity was 15%.

Figure 3.20 HPLC and LC-MS analysis of the purity of trimer

Since solubility of the trimer 3.12 was not so good, we managed to deprotect both the tert-butyl sulfite group and the ester group to enhance the water solubility (Scheme 3.9). The ester group was first removed using NaOH (2M, 2.5 eq.) in EtOH at 0 o C for 1h with an isolated yield of 88%. Further deprotection of the tert-butylsulfinyl group in the presence of HCl in dioxane (4M, 30 eq.) gave the crude product, which was subjected to preparative reverse-phase HPLC to afford highly pure trimer of CF 3 -triazole-based amino acid unit 3.13 (Figure 3.21). The trimer 3.13 was obtained in low yield (34%) after purification. As usual, when the purity was evaluated, LC-MS showed that oxidation occurred. As the Figure 3.22 showed, the peak at t 14.03 showed the mass of [tetramer +H] +1 peak (MW: 1030); while the peak at t 15.25 showed the mass of [tetramer +16+H] +1 , which means the oxidation of the tertbutylsulfinyl group to the tert-butyl sulfonyl group. Integration of the two peaks at t 14.03 and t 15.25 showed that the proportion of the non-oxidized product was 82% (76% + 6% methyl ester), and of the oxidized impurity was 18%. It was noticed that the solubility of the tetramer 3.15 was really poor (except in DMF and DMSO), which was in agreement with the reported phenomenon [36] in a previous paper saying that the peptidomimetics with 1,4-disubstituted-1,2,3-triazole-based amino acid mimic as backbone decreased gradually with increasing number of triazole units. To resolve the solubility problem, there exist two possible ways, one method requires the introduction of a bulky hydrophobic group (such as dendritic group) [36] to confer better solubility in nonpolar organic solvents, the other way is to deprotect both ends of the peptidomimetic 3.12 and 3.15 to increase its solubility in protic (water) solvents.

With the purpose to increase the solubility of tetramer 3.15 in protic solvents, we managed to deprotect both the ethyl ester on the C-end and the tert-butylsulfinyl group on the N-end. The ethyl ester was hydrolyzed in NaOH (2 M, 2.5 eq.) in 84% yield, and then tert-butylsulfinyl group was cleaved in the presence of HCl-Dioxane (4M, 30 eq.) in 3h (Scheme 3.11). The crude product 3.16 was finally purified by preparative reverse-phase HPLC with high purity in 5% yield (due to its poor solubility in water and acetonitrile, some material was lost during the purification process, Figure 3

Synthesis of Peptidomimetics of CF 3 -1,4-disubstituted-1,2,3-triazole-based amino acid mimic with natural amino acids (mimic of KLVFFA)

Based on the structural features of amyloid fibrils and the mechanism of their assembly, several peptides and their analogs have been designed and synthesized towards various protein aggregation diseases. It is commonly admitted that the central part K 16 LVFFA 21 is involved in the fibrils and oligomers structures of Aβ 1-42. [37] Furthermore, pentapeptides, like KLVFF (Lys-Leu-Val-Phe-Phe) and LVFFA (Leu-Val-Phe-Phe-Ala) have been used as binding elements to design inhibitors of fibrillization as they mimic some residues in the hydrophobic core of Aβ 1-42 fibrils. [38] We designed compound 3.23 with the sequence of Lys-CF 3 -1,4-triazole-Phe-CF 3 -1,4-triazole (Figure 3.25), that can be considered as a KLVFFA mimic in term of hydrophobicity and hydrogen bonding network (Figure 3.25). As we have mentioned in chapter 1.1.2 (Fluorinated amino acids and peptides), the trifluoromethyl (Tfm) group is recognized in medicinal chemistry as a good mimic of methyl, isopropyl, phenyl, etc. in terms of hydrophobic and sterically demanding. [39] And also the possibility of 1,2,3-triazole can act as hydrogen bond donor and acceptor. [7,8,9] Comparing the KLVFFA peptide chemical structure with 3.23, we envisaged that the CF 3 groups could be good mimics of both isopropyl and phenyl groups in the KLVFFA peptide in terms of hydrophobicity and space occupations. Most importantly, the 1,4-disubstituted-1,2,3-triazoles could be regarded mimic of the amide bonds since the sp 2 hybridized N (2) atoms can work like the amide carbonyl as hydrogen bond acceptors and the C-H ( 5) proton can be a hydrogen bond donor like the amide protons. Further, the ethyl ester of compound 3.25 was hydrolyzed under basic condition to give the acid 3.26 in 96% yield (Scheme 3.17). The Boc group of compound 3.24 was removed in quantitative yield by acidic treatment (TFA (30 eq.) in CH 2 Cl 2 ) in room temperature, the resulting compound was then followed by coupling reaction with compound 3.26 using DMTMM (Cl -)/NMM in dry DMF to give the desired compound 3.23 in good yield (74%) and purity (96%). In the trials of deprotection, we tried to remove the Cbz group employing catalytic hydrogenation using Pd/C (10% wt.), H 2 (1 atm) in trifluoroethanol (TFE) first. The result seemed good, but LC-MS showed a impurity with a mass of [M+28+H] + which was thought to be the N-ethylated product probably caused by the traces of CH 3 CHO in TFE allowing a reductive amination in the presence of Pd/C. Then we also tried to remove the Boc and Cbz group in a one-pot reaction under the condition of H 2 , Pd/C (10% wt.), using a solvent mixture in DMF/H 2 O instead of TFE, and then followed by HCl-dioxane (4M, 30 eq.) treatment in situ. But the result was discouraging, the resulting product was found by LC-MS to be a mixture of starting material, hydrochloride salt 3.27, and the N-ethylated product of 3.27. Finally, we tried the deprotection in two steps, first to remove Cbz group using H 2 , Pd/C ((10% wt.) in dry EtOH, which seemed to be able to avoid Nethylated product of 3.27. Secondly we removed the Boc group in HCl-dioxane (4M, 30 eq.) at room temperature. The crude product was further purified by recrystallization in EtOH and ether (the product was soluble in EtOH, when ether was added, the hydrochloride salt 3.27 precipitated first). Later both compounds 3.23 and 3.27 will be evaluated by ThT fluorescence assay to test its ability to inhibit the aggregation of Aβ 1-42 and IAPP peptides. When this protocol was applied to the trimer derivative, some structures could be observed, including helix-like conformations (Figure 3

C

NMR Conformational Studies

An attempt to perform conformational studies by 2D NMR on protected dimer 3.9 and 3.19; trimer 3.12 and 3.20, tetramer 3.15 and 3.22 was done from the spectra recorded on a Bruker 400 MHz Spectrometer in D 6 -DMSO. However, no information could be drawn, either because of (1) protons overlap; (2) no long range NOEs could be detected because of the low field of the spectrometer; (3) no long range NOEs could be detected because of the presence of extended conformations or because of the lack of stable conformations due to DMSO solvent that can prevent intramolecular hydrogen bonds. 2D NMR spectra of trimer 3.13 and tetramer 3.16 were recorded in CD 3 OH on an 800 MHz Spectrometer equipped with a cryoprobe and analyzed in collaboration with Dr. Carine Van. Heijenoort (UPR 2301, ICSN, Institut de Chimie des Substances Naturelles, Gif-sur-Yvette).

Conformational studies of fully deprotected trimer 3.13

The 1 H NMR and 13 C NMR of the fully deprotected trimer 3.13 was assigned with the help of 2D 1 H-1 H COSY, 2D 1 H-1 H TROSY, 2D 1 H- 13 C HSQC and 2D 1 H- 13 C HMBC spectra at 283K (Table 3.2 andTable 3.3). Unfortunately, too many protons have very closed chemical shifts, precluding unambiguous ROEs assignment (Figure 3.28). The similarility of the chemical shifts in the two last CF 3 -triazole units can be in favor of an extended conformation. We examined the temperature dependence, between 283 and 303K, of amide proton chemical shifts in trimer 3.13, as it can provide information on the network of hydrogen bonds and their relative stabilities (Table 3.4). The Δδ HN /ΔT value of NH a2 , NH a3 in trimer 3.13 were -5.0 ppb/K, -6.0 ppb/K respectively, displaying relatively large temperature dependence (Δδ HN /ΔT < -4.

Residue

Conformational studies of fully deprotected tetramer 3.16

The 1 H NMR and 13 C NMR of the fully deprotected tetramer 3.16 was assigned with the help of 2D 1 H-1 H COSY, 2D 1 H-1 H TROSY, 2D 1 H- 13 C HSQC and 2D 1 H- 13 C HMBC spectra at 283K (Table 3 In this case the chemical shifts of the corresponding protons in the 3 last CF 3 -triazole units (triazole 2, 3 and 4) were more exploded than in the trimer 3.13 (in particular 1 H chemical shifts of triazoles 3 and 4 are rather different from those of triazole 2, while 1 H chemical shifts in triazoles 2 and 3 were very similar) This observation can be due to a less extended conformation and a rather more folded structure conducting to different environment of the corresponding protons. Indeed, numerous long-range ROEs could be observed (Figure 3.29). In particular Roes between protons of the second CF 3 -triazole unit and protons of the third and fourth CF 3 -triazole units could be observed. Specifically, we can observe the long-rang ROEs between H e1 and H b2 Figure 3.31 showed that trimer 3.13 shortened the lag phase of IAPP aggregation, demonstrating a slightly property to accelerate IAPP aggregation process. At 3.13/IAPP ratios of 1/1 (5 μM/5 μM) and 0.1/1 (0.5 μM/5 μM), trimer 3.13 shortened the lag phase of IAPP by about 20% and 50% respectively, and increased the final fluorescence intensity by about 60% and 50% respectively. However at a higher ratio of 10/1 (50 μM/5 μM), trimer 3.13 only shortened the lag phase of IAPP aggregation by about 20% but had no effect in increasing the final fluorescence intensity. All these features indicated that trimer 3.13 had an ability to accelerate IAPP aggregation at lower concentrations (0.1/1 and 1/1). It is worth to note that the effect of acceleration of the aggregation is inversely proportional to the concentration, which is rather difficult to understand in the present state of our studies. The effect of tetramer 3.16 on the aggregation of Aβ 1-42 peptide and IAPP peptide Figure 3.32 and Figure 3.33 both showed that tetramer 3.16 didn't have any tendency to selfaggregate at high concentration (100 μM/50 μM, red curves) alone. The presence of tetramer 3.16 at 3.16/Aβ 1-42 ratios of 10/1 (100 μM/10 μM) and 1/1 (10 μM/10 μM) delayed the lag phase of Aβ 1-42 by about 30% and 40% respectively. However, the final fluorescence intensity was not reduced. At the ratio of 10/1 (100 μM/10 μM), the final fluorescence intensity was increased about 150%, and at the ratio of 1/1 (10 μM/10 μM) the final fluorescence intensity remained the same compared to the control. In this case, it could be interpreted that the presence of tetramer 3.16 altered significantly the Aβ 1-42 aggregation by modifying the early oligomerization kinetics of Aβ 1-42 aggregation, and modifying the morphology of the final fibrils because the ThT fluorescence is increased. This result is rather interesting because it can be useful to greatly modulate the aggregation pathway in order to avoid the presence of toxic oligomeric species. Further experiments should be conducted such as capillary electrophoresis, transmission electron microscopy and cellular evaluations, as usually performed in our laboratory in order to confirm this hypothesis.

The effect of trimer 3.13 on the aggregation of Aβ 1-42 peptide and IAPP peptide

Conclusions

In summary, new CF 3 -1,4-triazole-based amino acid mimic was diastereoselectively synthesized through a Zinc mediated Barbier type propargylation and CuAAC reaction sequentially. The absolute configuration of this CF 3 -1,4-triazole-based amino acid 3.4 and was confirmed by X-Ray crystallography. This novel amino acid mimic compound was used to prepare foldamers. For that purpose, it was assembled into tert-butylsulfinyl and Boc protected oligomers (trimer 3.15, 3.22 and tetramer 3.12, 3.20) and coupled to natural amino acids to prepare peptidomimetic compounds 3.23 and 3.27. Fully deprotected trimer 3.13 and tetramer 3.16 were also obtained.

The conformational studies of the oligomeric compounds were conducted by 2D NMR analysis and molecular modeling. Preliminary results obtained by Monte Carlo Multiple Minimum method in implicit solvent, then refinement by DFT method, for protected trimer 3.20 and tetramer 3.22, predicted the possibility to obtain helical structures. NMR analysis of the fully deprotected trimer 3.13 and tetramer 3.16 were performed in CD 3 OH. The overlap of many protons hindered some attributions and the full characterization of the conformations. However, while trimer Furthermore, the conformation analysis of the N-deprotected peptidomimetic 3.27 must be performed and 3.27 must be evaluated as it has been designed as aggregation inhibitor of Aβ 1-42, by mimicking its central part KLVFF.

Chapter IV Experiment Part

General Information

Pure products were obtained after liquid chromatography using Merck silica gel 60 (40-63 μm). TLC analyses were performed on silica gel 60 F250 (0.26 mm thickness) plates. The plates were visualized with UV light (λ = 254 nm) or revealed with a 5% solution of phosphomolybdic acid in EtOH. Melting points were determined on a Kofler melting point apparatus. Element analyses (C, H, and N) were performed on a PerkinElmer CHN, Analyzer 2400 at the Microanalyses Service of the Faculty of Pharmacy at Chatenay-Malabry (BioCIS, U-PSud, France). NMR spectra were recorded on an ultrafield Bruker AVANCE 300 ( 1 H, 300 MHz, 13 C, 75 MHz) or on a Bruker AVANCE 400 ( 1 H, 400 MHz, 13 C, 100 MHz). Chemical shifts δ are in ppm, and the following abbreviations are used: singlet (s), doublet (d), doublet of doublet (dd), triplet (t). Mass spectra were obtained using a Bruker Esquire electrospray ionization apparatus. HRMS were obtained using a TOF LCT Premier apparatus (Waters), with an electrospray ionization source. The purity of compounds was determined by HPLC using the 1260 Infinity system (Agilent Technologies) and a Sunfire column (C 18 , 3.5 μm, 100 mm × 2.1 mm); mobile phase, MeCN/H 2 O + 0.1% formic acid from 5 to 100% in 20 min; detection at 254 nm; flow rate 0.25 mL/min. A solution of (L)-proline (5.0 g, 43.4 mmol) and KOH (7.3 g, 130.3 mmol, 3.0 eq.) in i PrOH (30 mL) was prepared with stirring at 40 o C. As soon as the solution became transparent, slow addition of BnBr (6.2 mL, 8.9 g, 52.1 mmol, 1.2 eq) was commenced under stirring at the same temperature. The dropwise addition took 3h to complete, and the stirring was continued overnight at the same temperature. The reaction mixture was neutralized with concentrated aqueous HCl (about 2 mL) until pH 5-6 (indicator paper); then CH 2 Cl 2 (15 mL) was added to the reaction mixture with stirring. The mixture was left overnight without stirring, the KCl salt precipitated first. After filtration of the precipitate and evaporation of the CH 2 Cl 2 from the mother liquid, acetone (30 mL) was then added to the residue. The product precipitated slowly. Compound 2.22 (6.31g, 30.8 mmol) (filtered and dried in P 2 O 5 under vacuum) was obtained in 71% yield. 7, 132.1, 131.8, 131.1, 130.4, 69.5, 59.6, 55.5, 29.9, 23.9; To a solution of compound 2.22 (4.11 g, 20 mmol) in CH 2 Cl 2 (10 mL) was slowly added SOCl 2 (2.85 g, 24 mmol, 1.8 mL), with stirring, at -20 o C over a period of 10 min. The stirring was continued at -10 o C until the reaction mixture became almost transparent. Then a solution of 2aminobenzophenone (3.94 g, 20 mmol) in CH 2 Cl 2 (10 mL) was added to the reaction mixture at -30 o C with stirring. 5 min later NEt 3 (6.6 mL) was added. The stirring was continued at room temperature for 20 hours and then a solution of Na 2 CO 3 (10%, 30 mL) was added to the reaction mixture with stirring at 0 o C. The organic layer was separated, the aqueous layer extracted twice with CH 2 Cl 2 (2 x 50 mL) and the organic solutions were combined and evaporated under reduced pressure. The residue was purified by column chromatography with EtOAc-Cyclohexane 5:100.

Experimental Part for Fluorinated Peptidomimetics

ESI
Compound 2.23 (5.72 g, 14.9 mmol) was obtained as a yellow solid in 77% yield. 1, 174.8, 139.3, 138.6, 138.2, 133.5, 132.7, 132.6, 130.2, 129.2, 128.4, 128.3, 127.2, 125.4, 122.3, 121.6, 68.4, 59.9, 54.0, 31.1, 24.3 (8.4 g, 150 mmol) in MeOH (20 ml) was poured into a stirred mixture of compound 2.23 (5.76 g, 15 mmol), Ni (NO 3 ) 2 .6H 2 O (8.72 g, 30 mmol), glycine (5.63 g, 75 mmol) in MeOH (50 ml) under inert gas at 60°C. The resulting mixture was stirred at 60°C for 2 hours and then neutralized with AcOH (16 mL) and MeOH was evaporated, then EtOAc (50 mL) and NaHCO 3 (10%, 50 mL) were added. The organic phase was separated and washed with brine, and then dried over Na 2 SO 4 .The residue was purified by column chromatography with EtOAc-MeOH = 100:10. The Ni (II) complex 2.24 (5.34 g, 10.7 mmol) was obtained as a red solid in 70% yield. 5, 177.5, 171.8, 142.6, 134.7, 133.4, 133.3, 132.3, 131.8, 129.9, 129.7, 129.5, 129.2, 129.0, 126.4, 125.8, 125.3, 124.4, 121.0, 70.0, 63.3, 61.4, 57.6, 30.8, 23.8 To the mixture of Ni (II) complex 2.24 (5.12 g, 10 mmol) and MeONa (6.7 mL, 36 mmol) in the solution of MeOH (15 mL) was added CF 3 CHO (4.0 mL, 15 mmol). The reaction mixture was heated and refluxed for 1 hour, and then was quenched with 4 mL AcOH in 10 mL H 2 O. Then MeOH was evaporated, the residue was extracted with EtOAc (50 mL X 2). The organic phase was washed with 10% NaHCO 3 solution (50 mL) and brine, and then was dried over Na 2 SO 4. The residue was purified by column chromatography with EtOAc: MeOH = 100:10. The Ni (II) complex 2.25 (3.92 g, 6.58 mmol) was obtained as a red solid in 66% yield. 5, 178.2, 173.9, 143.2, 133.8, 133.6, 133.4, 133.3, 131.6, 130.8, 129.6, 129.1, 129.1, 127.9, 126.7, 126.3, 126.2, 123.8, 122.9, 120.9, 70.9, 69.8, 69.1, 68.7, 68.3, 66.6, 63.7, 57.4, 30.8, 22.8; A solution of the Ni (II) complex 2.25 (3.6 g, 6 mmol) in methanol (30 mL) was added to a diluted aqueous HCl solution (1 mL of conc. HCl and 2 mL H 2 O for 1g of the complex). The mixture was refluxed for 15-30 min and then cooled to room temperature. Most of the MeOH was then evaporated. EtOAc (50 mL) and water (50 mL) were added, the organic phase was washed with NaHCO 3 (10%, 50 mL), and brine, then dried over Na 2 SO 4 . The crude residue was the chiral auxiliary 2.23, which could be reused. To the aqueous phase were added 2.0 equivalent of NaSCN and 4.0 equivalent of pyridine. The Ni(Py) 4 (SCN) 2 precipitate was filtered and then to the filtrate were added dioxane (50 mL), Na 2 CO 3 (2.0 eq., 1. 27 g, 12 mmol) and Boc 2 O (1.2 eq., 1.57 g, 7.2 mmol), the reaction mixture was stirred overnight at room temperature. Dioxane was evaporated under reduced pressure, then CH 2 Cl 2 (50 mL) and water (50 mL) were added, the organic phase was washed with 10% NaHCO 3 solution (50 mL) and brine, then was dried over Na 2 SO 4 . The crude residue was purified by column chromatography (MeOH: EtOAc: CH 3 COOH = 5: 100: 1), Boc-CF 3 -(2S, 3S) CF 3 -threonine 2.26 (368 mg, 1.35mmol) was obtained as a colorless oil in 43% yield. To a solution of Boc-Val-OH (4.0 g, 18.4 mmol) in anhydrous CH 2 Cl 2 (20 mL) at -10 °C, were added N-methylmorpholine (4.4 mL, 22.1 mmol). After 30 min. of stirring, isobutylchloroformate (2.63 mL, 20.3 mmol) was added dropwise and the mixture was stirred at -10 °C for 45 min before the addition of H-Leu-OMe (3.7 g, 20.3 mmol). The reaction mixture was stirred at room temperature for an additional 24 h, the CH 2 Cl 2 was evaporated under reduced pressure, and the residue was dissolved in EtOAc (40 mL). The organic layer was successively washed with a 10% aqueous solution of citric acid (50 mL), a 10 % aqueous solution of K 2 CO 3 (20 mL) and brine (20 mL). The organic layer was dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The crude residue was purified by column chromatography on silica gel using EtOAc: Cyclohexane = 3:7 as eluent. The dipeptide 2.27 (5.2 g, 15.2 mmol) was obtained as a white solid in 83% yield. 1, 171.4, 155.8, 60.0, 52.2, 50.7, 41.6, 30.8, 28.3, 24.8, 22.8, 21.8, 19.2, 17.8; MS (ESI, ion polarity positive, MeOH): m/z 367.21 [M+Na] + (6S, 9S, 12S)-methyl 12-isobutyl-9-isopropyl-2,2-dimethyl-4,7,10-trioxo-6-((S)-2,2,2-trifluoro-1hydroxyethyl)- 3-oxa-5,8,11-triazatridecan-13-oate (2.28) Chemical Formula: C 21 H 36 F 3 N 3 O 7 Exact Mass: 499.2505 Molecular Weight: 499.5216

The dipeptide Boc-Val-Leu-OMe 2.27 (1.03 g, 3 mmol) was dissolved in CH 2 Cl 2 (20 mL) at 0 o C, then to the solution was added CF 3 COOH (9.3 mL, 120 mmol, 40 eq.). The reaction mixture was stirred at 0 o C for 30 min, then at room temperature for 2 hours. After that time, all the solvents were evaporated under reduced pressure, and then a little amount of Et 2 O was added to precipitate the product. Et 2 O of the mother liquid was evaporated and precipitation by addition of Et 2 O was repeated several times until the mass of the crude did not change any more. The resulting residue was obtained as a white solid (1.09 g, 3 mmol) with quantitative yield without any further purification.

(2S, 3S)-CF 3 -Threonine 2.26 (164 mg, 0.6 mmol, 1.1 eq.), HBTU (270 mg, 0.71 mmol, 1.3 eq.) and HOBt (81 mg, 0.6 mmol, 1.1 eq.) were dissolved in dry DMF (5 mL) at 0 o C. The solution was stirred for 1 hour, then to the solution were added the previous dipeptide salt (197 mg, 0.55 mmol, 1.0 eq.) and DIPEA (424 mg, 3.28 mmol, 6.0 eq.). The reaction was stirred at room temperature overnight. Then DMF was removed under vacuum pump. To the residue was added EtOAc (10 mL) and 10% aqueous citric acid (10 mL), the organic phase was washed with distilled water (10 mL) , 10% NaHCO 3 solution (10 mL) and then brine, and was dried over Na 2 SO 4 . The crude residue was purified by column chromatography on silica gel using EtOAc: Cyclohexane = 1:2 as eluent. The tripeptide 2.28 (198 mg, 0.40 mmol) was obtained as a white solid in 73% yield. 3, 142.4, 136.5, 126.7, 122.9, 62.6, 51.0, 50.1, 49.8, 37.6, 28.3, 24.9, 14. 3297, 2961, 2930, 1748, 1643, 1536, 1369, 1159, 1135. (6S, 9S, 12S, 15S)-methyl 15-isobutyl-6,12-diisopropyl-2,2-dimethyl-4,7,10,13-tetraoxo-9-((S)-2,2,2-trifluoro-1-hydroxyethyl)- 3-oxa-5,8,11,14-tetraazahexadecan-16- The tripeptide 2.28 (110 mg, 0.22 mmol) was dissolved in CH 2 Cl 2 (5 mL) at 0 o C, then to the solution was added CF 3 COOH (1.1 mL, 13.2 mmol, 60 eq.). The reaction mixture was stirred at 0 o C for 30 min, then at room temperature for 2 hours. After that time, all the solvents were evaporated under reduced pressure, and then a little amount of Et 2 O was added to precipitate the product. Et 2 O of the mother liquid was evaporated and precipitation by addition of Et 2 O was repeated several times until the mass of the crude did not change any more. The resulting residue was obtained as liquid with quantitative yield without any further purification.

The resulting residue (113 mg, 0.22 mmol) was dissolved in dry DMF (2 mL) at room temperature, and then to the solution was added DMTMM (Cl -) (68 mg, 0.24 mmol, 1.2 eq.), NMM (122 mg, 1.32 mmol, 6.0 eq.) and Boc-Val-OH (44 mg, 0.20 mmol, 1.0 eq.). The reaction mixture was stirred at room temperature overnight. Then DMF was removed under vacuum pump. To the residue was added EtOAc (5 mL) and 10% aqueous citric acid (5 mL), the organic phase was washed with distilled water (5 mL) , 10% NaHCO 3 solution (5 mL) and then brine, and was dried over Na 2 SO 4 . The crude residue was purified by column chromatography on silica gel using CH 2 Cl 2 : MeOH = 100:2 as eluent. The tetrapeptide 2.29 (86 mg, 0.14 mmol) was obtained as a white solid in 72% yield. 7, 171.7, 170.4, 167.8, 155.5, 126.6, 78.1, 68.3, 67.9, 59.8, 57.2, 51.8, 51.7, 50.3, 31.1, 30.1, 28.1, 24.2, 22.7, 21.2, 19.2, 19.0, 18.0, 17 (cm -1 ): 3279, 2961, 2930, 1753, 1639, 1547, 1368, 1176, 1160. (6S, 9S, 12S, 15S, 18S)-methyl 18-isobutyl-9,15-diisopropyl-2,2,6-trimethyl-4,7,10,13,16pentaoxo-12-((S)-2,2,2-trifluoro-1-hydroxyethyl)- 3-oxa-5,8,11,14,17-pentaazanonadecan-19-oate (2.18) Chemical Formula: C 29 H 50 F 3 N 5 O 9 Exact Mass: 669.3561 Molecular Weight: 669.7306 Elemental Analysis: C, 52.01; H, 7.52; F, 8.51; N, 10.46; O, 21.50 The tetrapeptide 2.29 (72 mg, 0.12 mmol) was dissolved in CH 2 Cl 2 (2 mL) at 0 o C, then to the solution was added CF 3 COOH (0.60 mL, 7.2 mmol, 60 eq.). The reaction mixture was stirred at 0 o C for 30 min, then at room temperature for 2 hours. After that time, all the solvents were evaporated under reduced pressure, and then a little amount of Et 2 O was added to precipitate the product. Et 2 O of the mother liquid was evaporated and precipitation by addition of Et 2 O was repeated several times until the mass of the crude did not change any more. The resulting residue was obtained as liquid with quantitative yield without any further purification.

The resulting residue compound (57 mg, 0.11 mmol) was dissolved in dry DMF (2 mL) at room temperature, and then to the solution was added DMTMM (Cl -) (60 mg, 0.22 mmol, 2.0 eq.), NMM (65 mg, 0.66 mmol, 6.0 eq.) and Boc-Ala-OH (22 mg, 0.12 mmol, 1.1 eq.). The reaction mixture was stirred at room temperature overnight. Then DMF was removed under vacuum pump, to the residue was added EtOAc (5 mL) and 10% aqueous citric acid (5 mL), the organic phase was washed with distilled water (5 mL) , 10% NaHCO 3 solution (5 mL) and then brine, and then was dried over Na 2 SO 4 . The crude residue was purified by column chromatography on silica gel using CH 2 Cl 2 : MeOH = 100:2 as eluent. The pentapeptide 2.18 (61 mg, 0.09 mmol) was obtained as a white solid in 83% yield. 3, 173.5, 173.1, 170.4, 80.7, 70.0, 69.6, 60.4, 60.2, 54.2, 52.6, 52.1, 51.6, 41.3, 32.4, 31.8, 28.7, 25.8, 23.3, 21.7, 19.8, 19.6, 18.8, 18.4, 18 IR ν max (cm -1 ): 3278, 2962, 2928, 1749, 1639, 1547, 1458, 1367, 1162. (2S, 5S, 8S, 11S, 14S)-methyl 14-amino-2-isobutyl-5,11-diisopropyl-4,7,10,13-tetraoxo-8- C, 47.56; H, 7.15; Cl, 5.85; F, 9.40; N, 11.56; O, 18.48 The pentapeptide 2.18 (25 mg, 0.037 mmol) was dissolved in CH 2 Cl 2 (1 mL) at 0 o C, then to the solution was added HCl-Dioxane (0.60 mL, 2.24 mmol, 60 eq.). The reaction mixture was stirred at 0 o C for 30 min, then at room temperature for 2 hours. After that time, all the solvents were evaporated under reduced pressure, and then a little amount of Et 2 O was added to precipitate the product. Et 2 O of the mother liquid was evaporated and precipitation by addition of Et 2 O was repeated several times until the mass of the crude did not change any more. The resulting residue (23 mg, 0.037 mmol) was obtained as a white solid with quantitative yield. 4, 173.3, 173.3, 171.3, 170.3, 70.2, 60.6, 60.0, 54.1, 52.7, 52.3, 50.3, 41.4, 32.8, 31.8, 26.0, 23.4, 21.9, 20.0, 19.7, 18.8, 18.9, 17.9 3285, 2962, 1748, 1636, 1554, 1455, 1186, 1138. (6S, 9S, 12S)-methyl -((R)-1-(benzyloxy)ethyl) -12-isobutyl-9-isopropyl-2,2-dimethyl-4,7,10trioxo-3-oxa-5,8,11-triazatridecan-13- (20 mL) at 0 o C, then to the solution was added CF 3 COOH (9.3 mL, 120 mmol, 40 eq.). The reaction mixture was stirred at 0 o C for 30 min, then at room temperature for 2 hours. After that time, all the solvents were evaporated under reduced pressure, and then a little amount of Et 2 O was added to precipitate the product. Et 2 O of the mother liquid was evaporated and precipitation by addition of Et 2 O was repeated several times until the mass of the crude did not change any more. The resulting residue was obtained as a white solid (1.09 g, 3 mmol) with quantitative yield without any further purification. To a solution of the previous dipeptide salt (1.0 g, 2.79 mmol) in anhydrous DMF (10 mL) at 0 °C, were added a solution of DIPEA (2.9 mL, 16.1 mmol), Boc-Thr (Bzl)-OH (950 mg, 3.07 mmol), HOBT (450 mg, 3.35 mmol) and HBTU (1.27 g, 3.35 mmol). The mixture was stirred at 0°C for 30 min and 2 days at room temperature. 50 mL of water were poured into the reaction mixture, and an extraction was performed with ethyl acetate (3x30 mL). The organic layer was washed with a 10% aqueous solution of citric acid (30 mL), water (20 mL), a 10% aqueous solution of K 2 CO 3 (30 mL) and brine (20 mL). The organic layer was dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The crude residue was purified by column chromatography on silica gel using EtOAc: Cyclohexane = 3:7 as eluent. The tripeptide 2.30 (1.23 g, 2.30 mmol) was obtained as a white solid in 83% yield. R f = 0.80 (EtOAc: Cyclohexane = 7:3); 1 H NMR (300 MHz, CDCl 3 ) δ 7. 35 -7.29 (m, 5H), 6.97 (d, J = 9.0 Hz, 1H, NH), 6.36 (d, J = 8.2 Hz, 1H, NH), 5.48 (d, J = 6.0 Hz, 1H, NH), 4.68 -4.53 (m, 3H), 4.30 -4.20 (m, 3H), 3.73 (s, 3H), 2.19 (s, 1H), 1.66 -1.50 (m, 3H), 1.48 (s, 9H), 1.22 (d, J = 6.3 Hz, 3H), 0.94 -0.82 (m, 12H); 13 C NMR (75 MHz, CDCl 3 ) δ 173.0, 170. 5, 170.1, 137.8, 128.4, 127.9, 127.8, 80.3, 74.7, 71.7, 58.7, 58.0, 52.2, 50.7, 41.2, 30.3, 28.2, 24.7, 22.7, 21.8, 19.2, 17.6, 15.3; (6S, 9S, 12S, 15S)-methyl 9-((R)-1-(benzyloxy)ethyl)- 15-isobutyl-6,12-diisopropyl-2,2-dimethyl-4,7,10,13-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-oate (2.31) Chemical Formula: C 33 H 54 N 4 O 8 Exact Mass: 634.39 Molecular Weight: 634.82

The tripeptide 2.30 (1.23 g, 2.30 mmol) was dissolved in CH 2 Cl 2 (30 mL) at 0 o C, then to the solution was added CF 3 COOH (10 mL, 130 mmol, 60 eq.). The reaction mixture was stirred at 0 o C for 30 min, then at room temperature for 2 hours. After that time, all the solvents were evaporated under reduced pressure, and then a little amount of Et 2 O was added to precipitate the product. Et 2 O of the mother liquid was evaporated and precipitation by addition of Et 2 O was repeated several times until the mass of the crude did not change any more. The resulting residue was obtained as a white solid with quantitative yield without any further purification.

To a solution of the previous tripeptide salt (600 mg, 1.09 mmol) in anhydrous DMF (10 mL) at 0 °C were added a solution of DIPEA (1.15 mL, 6.55 mmol), Boc-Val-OH (260 mg, 1.20 mmol), HOBt (162 mg, 1.20 mmol) and HBTU (497 mg, 1.31 mmol). The mixture was stirred at 0°C for 30 min and 1 day at room temperature. 50 mL of water were poured into the reaction mixture, and an extraction was performed with ethyl acetate (2x30 mL). The organic layer was washed with a 10% aqueous solution of citric acid (40 mL), a 10% aqueous solution of K 2 CO 3 (40 mL) and brine (40 mL). The organic layer was dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The crude residue was purified by column chromatography on silica gel using MeOH: CH 2 Cl 2 = 3:97 as eluent. The tetrapeptide 2.31 (420 mg, 0.66 mmol) was obtained as a white solid in 61% yield. R f = 0.60 (MeOH: CH 2 Cl 2 = 5:95); 1 H NMR (300 MHz, CDCl 3 ) δ 7. 35 -7.28 (m, 5H), 7.11-7.05 (m, 2H, 2 NH), 6.71 (d, J = 7.8 Hz, 1H, NH), 5.00 (d, J = 5.7 Hz, 1H, NH), 4.69 -4.54 (m, 3H), 4.40 (m, 1H), 4.23 (m, 1H) 1, 172.1, 170.8, 169.6, 156.1, 137.7, 128.4, 127.8, 127.8, 80.4, 74.1, 71.7, 60.3, 58.8, 57.0, 52.1, 50.7, 40.9, 30.7, 30.1, 28.2, 24.7, 22.8, 21.7, 19.4, 19.1, 17.6, 17.5, 15.6; (6S, 9S, 12S, 15S, 18S)-methyl 12-((R)-1-(benzyloxy)ethyl)- 18-isobutyl-9,15-diisopropyl-2,2,6trimethyl-4,7,10,13,16-pentaoxo-3-oxa-5,8,11,14,17-pentaazanonadecan-19-oate (2.32) Chemical Formula: C 36 H 59 N 5 O 9 Exact Mass: 705.43 Molecular Weight: 705.89

The tetrapeptide 2.31(0.420 g, 0.66 mmol) was dissolved in CH 2 Cl 2 (20 mL) at 0 o C, then to the solution was added CF 3 COOH (3 mL, 39.2 mmol, 60 eq.). The reaction mixture was stirred at 0 o C for 30 min, then at room temperature for 2 hours. After that time, all the solvents were evaporated under reduced pressure, and then a little amount of Et 2 O was added to precipitate the product. Et 2 O of the mother liquid was evaporated and precipitation by addition of Et 2 O was repeated several times until the mass of the crude did not change any more. The resulting residue was obtained as a white solid with quantitative yield without any further purification.

To a solution of the previous tetrapeptide salt (380 mg, 0.59 mmol) in anhydrous DMF (15 mL) at 0 °C, were added a solution of DIPEA (0.6 mL, 3.50 mmol), Boc-Ala-OH (122 mg, 0.64 mmol), HOBt (87 mg, 0.64 mmol) and HBTU (244 mg, 0.64 mmol). The mixture was stirred at 0°C for 30 min and 1 day at room temperature. 50 mL of water were poured into the reaction mixture, and an extraction was performed with ethyl acetate (2x30 mL). The organic layer was washed with a 10% aqueous solution of citric acid (40 mL), a 10% aqueous solution of K 2 CO 3 (40 mL) and brine (40 mL). The organic layer was dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure to give a crude product (115 mg, 0.163 mmol) as a yellowish powder (28%). Being very poorly soluble, the pentapeptide 2.32 was extracted with difficulty and could not be purified. R f = 0.85 (EtOAc: Cyclohexane = 7:3); 1 H NMR (300 MHz, DMSO) δ 8.28 (d, J = 7.5 Hz, 1H, NH): 8.01 (d, J = 8.7 Hz, 1H, NH), 7.65 (d, J = 9.3 Hz, 2H, 2 NH), 7.31 -7.27 (m, 5H), 7.00 (d, J = 7.3 Hz, 1H, NH), 4.56-4.23 (m, 5H),, 3.95 (m, 2H), 3.57 (s, 3H) 5, 172.7, 171.2, 170.8, 169.2, 155.0, 138.5, 128.0, 127.6, 127.2, 78.0, 75.0, 70.5, 57.2, 56.4, 51.7, 50.1, 49.7, 30.8, 28.1, 24.1, 22.6, 21.1, 19.1, 18.9, 17.9, 16.9, 16. (6S, 9S, 12S, 15S, 18S)-methyl 12-((R)-1-hydroxyethyl)- 18-isobutyl-9,15-diisopropyl-2,2,6trimethyl-4,7,10,13,16-pentaoxo-3-oxa-5,8,11,14,17-pentaazanonadecan-19- To a stirred solution of the pentapeptide (100 mg, 0.142 mmol) in a mixture of CH 3 OH/DMF (5 mL/0.5 mL) was added Pd/C (20 mg, 20% mass). The reaction flask was purged three times with hydrogen, and stirring was maintained under hydrogen atmosphere at room temperature for 20 hours. Upon completion of the reaction monitored by TLC, the solution was filtered through a pad of Celite which was washed several times with CH 3 OH. Then, the filtrate was concentrated under reduced pressure to provide the de-benzylated pentapeptide as a white-yellowish solid with a quantitative yield. 3, 173.6, 173.5, 172.1, 80.6, 68.4, 60.2, 59.9, 52.6, 52.1, 51.6, 41.2, 32.1, 28.7, 25.8, 23.3, 21.7, 19.8, 19.7, 18. (2S, 5S, 8S, 11S, 14S)-methyl 14-amino-8-((R)-1-hydroxyethyl)-2-isobutyl -5,11-diisopropyl-4,7,10,13-tetraoxo-3,6,9,12- 50, 173.70, 173.45, 172.14, 171.37, 129.51, 68.63, 60.83, 59.93, 52.72, 52.34, 50.25, 41.39, 32.34, 31.91, 26.02, 23.42, 21.91, 19.92, 19.74, 18.86, 18.64, 17. , 2874, 2665, 1627, 1562, 1431, 1367, 1271, 1222, 1172, 1123, 1062, 2, 142.4, 124.4, 125.0 (d, J = 285 Hz, CF 3 ), 62.5, 57.1, 57.4 (q, J = 30 Hz, CHCF 3 ), 50.9, 25.9, 22.3, 14 3274, 3147, 2978, 2943, 1760, 1466, 1397, 1380, 1262, 1204, 1176, 1116, 1067, 1059, 1043, 1027, 875, 833. Then to the solution was added NaOH aqueous solution (2M, 1.25 mL, 2.5 mmol, 2.5 eq.). The reaction was stirred at 0 o C for 1 hour. When the TLC showed the reaction completed, EtOH was removed under reduced pressure. Distilled water (5 mL) and EtOAc (5 mL) were added, the pH of the solution was then adjusted to 3-4 (pH paper) using HCl (1M) at 0 o C. After that the solution was extracted with EtOAc (10 mL ×3), the organic phase was dried over Na 2 SO 4 and concentrated to afford a white solid in 92% yield (315 mg), which was pure enough for the next reaction. 3235, 3140, 2968, 1741, 1562, 1464, 1433, 1376, 1343, 1297, 1264, 1208, 1172, 1118, 1039, 834, 809, 685; Ethyl 2-( 4 Compound 3.6 (342 mg, 1.0 mmol) in dry DMF (15 mL) at 0 o C were added HATU (494 mg, 1.3 mmol, 1.3 eq.), HOAt (218 mg, 1.6 mmol, 1.6 eq.), the resulting solution was stirred at 0 o C for 60 min, then to the solution were added compound 3.5 (302 mg, 1.0 mmol, 1.0 eq.) and DIPEA (388 mg, 0.3 mL, 3.0 mmol, 3.0 eq.) at 0 o C. The reaction mixture was stirred at room temperature overnight. When TLC showed the reaction finished, DMF was removed under vacuum pump; to the residue was added 10% aqueous citric acid (10 mL) and EtOAc (10 mL). The organic phase was washed with 10% NaHCO 3 solution (10 mL) and brine, and then was dried over MgSO 4 . The crude product was purified by column chromatography using CH 2 Cl 2 : MeOH = 98:2 as eluent.

The dimer 3.9 (360 mg, 0.63 mmol) was obtained as a white solid in 63% yield. 5.38 (s, 2H), 5.00-5.19 (dd, J = 27.9 Hz, 16.2 Hz, 2H), 4.74 (m, 1H), 4.11-4.21 (m, 2H), 3.34 (s, 2H), 2.95-3.34 (m,4H), 1.22 (t, 3H), 0.87 (s, 9H); 13 C NMR (75 MHz, DMSO-d 6 ) δ 167. 3, 165.9, 141.7, 141.2, 125.0, 124.7, 61.5, 58.3 (CHCF 3 ), 56.2, 51.1, 50.3, 49.9 (CHCF 3 ), 25.0, 23.9, 22.1, 13.9 3220, 2988, 1753, 1707, 1562, 1378, 1219, 1179, 1117, 1070, 1058, 1040, 874, 664. Then NaOH aqueous solution (2 M, 0.13 mL, 2.5 eq.) was added and the reaction mixture was stirred at 0 o C for 2 hours. Then TLC showed the reaction finished completely. MeOH was removed under reduced pressure, then to the residue was added H 2 O (5 mL) and EtOAc (10 mL) and acidified using HCl (1M, 0.5 mL) at 0 o C to make the pH was 3-4 (pH paper). Then quickly extracted with EtOAc (3×10 mL) and the organic phase was dried with anhydrous MgSO 4 and the solvent evaporated affording a white solid, which was pure enough for the next reaction. The dimer acid 3.14 (54 mg, 0.96 mmol) was obtained as a white solid in 96% yield. 4, 167.9, 143.6, 143.0, 126.9, 126.8 (d, J = 278 Hz, CF 3 ), 126.3 (d, J = 285 Hz, CF 3 ), 126.2, 60.2 (q, J = 30 Hz, CHCF 3 ), 58. 5, 52.8, 52.4, 52.1 (q, J = 30 Hz, CHCF 3 ), 26. 1, 25.2, 22.8 , 2928, 1704, 1560, 1466, 1422, 1364, 1267, 1227, 1208, 1174, 1124, 1056, 876, 682. The dimer acid 3.11 (50 mg, 0.089 mmol) in dry DMF (1.0 mL) at 0 o C was added HATU 49 mg (1.5 eq.), HOAt (22 mg, 1.8 eq.), stirred at 0 o C for 60min, then added compound 3.5 (36 mg, 0.12 mmol, 1.3 eq.) and DIPEA (39 mg, 3.4 eq.) at 0 o C. The reaction stirred at room temperature overnight. When TLC (CH 2 Cl 2 : MeOH:CH 3 COOH = 70:30:1) showed there remained no 3.11, then DMF was removed under vacuum pump, the residue was washed with EtOAc (5 mL ) and water (5 mL ), then the trimer 3.12 precipitated and was filtered (21 mg after dried under vacuum), and the mother liquid washed brine and concentrated to be purified by column chromatography using CH 2 Cl 2 : MeOH = 98:2 as eluent, and obtained the trimer 3.12 in 60% yield (47 mg, 0.058). 3, 166.3, 166.1, 141.7, 141.2, 141.0, 126.5, 125.0, 124.9, 124.8, 123.7, 118.5, 61.5, 57.8 (CHCF 3 ), 56.3, 51.2, 51.1, 50.4, 50.1 (CHCF 3 ), 50.0 (CHCF 3 ), 25.1, 24.0, 23.5, 22.1, 13.9; 3220, 2959, 1754, 1706, 1558, 1466, 1440, 1355, 1228, 1176, 1123, 1058, 846. 2,2,2-trifluoroacetic Compound 3.12 (36 mg, 0.045 mmol) was added to a round bottom flask with EtOH (5 mL) at 0 o C. Then 60μL of NaOH (2M, 2.5 eq.) aqueous solution was added and the reaction was stirred at 0 o C for 2h and at room temperature for 1h. Then TLC showed the reaction finished completely. MeOH was removed under reduced pressure, then to the residue was added H 2 O (5 mL) and EtOAc (10 mL) and acidified using HCl (1M, 0.3 mL) at 0 o C to make the pH was 3 (pH Paper). Then quickly extracted with EtOAc (3×10 mL) and the organic phase was dried with anhydrous MgSO 4 and the solvent evaporated affording a white solid, which was pure enough for the next reaction.

The resulting acid was (34 mg, 0.043 mmol) was dissolved in 2.0 mL dioxane at rt, then HCldioxane (4.0 M HCl in dioxane, 330 μL , 1.30 mmol, 30 eq.) was added. The reaction stirred at rt for 3h. After 3h reaction time, evaporated all the solvent as much as possible under reduced pressure, then the crude residue was subjected to reverse phase preparative HPLC purification and after lyophilization, the salt TFA 3.13 was obtained as white powder in 34% yield (12 mg, 0.015 mmol).

Preparative HPLC: 44 mg compound 3.13 was dissolved in 4 mL H2O + 1 mL MeCN A: H 2 O + 0.1% TFA, B: MeCN + 0.1% TFA, Column: C18, 150 × 20 mm id, 5 μm, 200 Å, Method Set: 20 min/ 20 mL, Injection Volume: 5.0 mL, Run Time: 30 min, Purification gradients: 20% -50% MeCN + 0.1% TFA;

Analytical HPLC: 5% -95% MeCN + 0.1% TFA in 10 min, UV-Vis wavelength: 220 nm, t R = 5.21 min, purity: 100 % 1 H NMR (800 MHz, CD 3 OH) δ 9.03 (t, J = 9.7 Hz, 2H, NH), 7.86 (s, 1H), 7.85 (s, 1H), 7.77 (s, 1H), 5.28 -5.19 (m, 6H), 4.44 (br, 3H), 3.39 (dd, J = 16.6, 3.7 Hz, 1H), 3.30 -3.25 (m, 2H), 3.14 (dd, J = 16.4, 10.4 Hz, 1H), 3.03 (td, J = 16.3, 11.9 Hz, 2H); 4, 167.0, 141.3, 141.0, 139.6, 125.9, 125.7, 125.2, 52.2, 51.1, 50.9, 50.8 (2C), 50.7, 22.8, 23.6, 23.9; (36 mg, 0.064 mmol) in dry DMF (1.0 mL) at 0 o C was added HATU (32 mg, 1.3 eq.), HOAt (14 mg, 1.6 eq.), stirred at 0 o C for 60 min, then added compound 3.14 (40 mg, 0.077 mmol, 1.2 eq.) and DIPEA (25 mg, 3.0 eq.) at 0 o C. The reaction stirred at room temperature overnight. When TLC (CH 2 Cl 2 : MeOH:CH 3 COOH = 70:30:1) showed there was no compound 3.15, then removed DMF under vacuum pump, the residue was washed with distilled water 5 mL, and EtOAc 5 mL. The product then precipitated and filtered, the precipitates was washed with H 2 O 2 mL three times and EtOAc 2 mL three times and then dried under vacuum pump with P 2 O 5 at 50 o C. The tetramer 3.15 was obtained as a white powder in 65% yield (43 mg, 0.042 mmol).

(R)-2-((R)-1,1-dimethylethylsulfinamido)-3,3,3- trifluoropropyl)-1H-1,2,3-triazol-1-yl)acetamido)-3,3,3-trifluoropropyl)-1H-1,2,3-triazol-1- yl)acetamido)-3,3,3-trifluoropropyl)-1H-1,2,3-triazol-1-yl)acetamido)-3,3,3-trifluoropropyl)-1H- 1,2,3-triazol-1-
HPLC: Sunfire C 18 -2.1 × 100 mm -3.5 μm; H 2 O + 0.1% formic acid / CH 3 CN -gradient 5%-100% in 20 min, t R = 13.87 min, purity: 74% + 4% (methyl ester) + 20% (Oxidized). 5.37 (s, 2H), 5.22 -4.98 (m, 6H), 4.75 (m, 3H), 4.20 -4.11 (m, 3H), 3.22 -3.13 (m, 3H), 3.04-2.92 (m, 5H), 1.21 (t, J = 6.0 Hz, 3H), 0.87 (s, 9H); 2, 166.2, 165.9, 141.6, 141.2, 140.9, 140.8, 126.9 (CF 3 ), 125.0, 124.8, 124.7, 123.1 (CF 3 ), 61.4, 58.0 (CHCF 3 ), 57.7 (CHCF 3 ), 56.2, 51.1, 50.3, 50.2 (CHCF 3 ), 49.9 (CHCF 3 ), 25.1, 24.0, 22.1, 13.9 , 2918, 1748, 1697, 1558, 1467, 1439, 1356, 1293, 1252, 1236, 1210, 1181, 1126, 1066, 1039, 1027, 878, 664. 2,2,2-trifluoroacetic acid The tetramer 3.15 (50 mg, 0.085 mmol) was added to a round bottom flask with EtOH (2 mL) at 0 o C. Then 105 μL of NaOH (2 M, 2.5 eq.) aqueous solution was added and the reaction was stirred at 0 o C for 2 hours. When TLC showed the reaction finished completely, MeOH was removed under reduced pressure, to the residue was added H 2 O (5 mL) and EtOAc (10 mL) and acidified using HCl (1 M, 0.5 mL) at 0 o C to make the pH was 3 (pH paper). Then quickly extracted with EtOAc (3×10 mL) and the organic phase was dried with anhydrous MgSO 4 and the solvent evaporated affording a white solid, which was pure enough for the next reaction. The resulting acid was obtained as a white solid 48 mg in quantitative yield. Then the corresponding acid (48 mg) was dissolved in 2.0 mL DMSO-d 6 at rt, then HCl-dioxane (4.0 M HCl in dioxane, 640 μL, 2.55 mmol, 30 eq.) was added. The reaction stirred at rt for 3 hours. After 3 hours reaction time, evaporated all the solvent as much as possible under reduced pressure, then added diethyl ether to the DMSO solution and compound 3.16 precipitated, the solvent was removed and 3.16 was subjected to reverse phase preparative HPLC purification, after lyophilization the salt 3.16 was obtained as white powder in 5% yield (4 mg, 0.004 mmol).

compound with 2-(4-((R)-2-(2-(4-((R)-2-(2-(4-((R)-2-(2-(4-((R)-2- amino-3,3,3-trifluoropropyl)-1H-1,2,3-triazol-1-yl)acetamido)-3,3,3-trifluoropropyl)-1H-1,2,3- triazol-1-yl)acetamido)-3,3,3-trifluoropropyl)-1H-1,2,3-triazol-1-yl)acetamido)-3,3,3- trifluoropropyl)-1H-1,2,3-triazol-1-yl)acetic
Preparative HPLC: 60 mg compound 3.16 was dissolved in 4 mL H2O + 1 mL MeCN A: H 2 O + 0.1% TFA, B: MeCN + 0.1% TFA, Column: C18, 150 × 20 mm id, 5 μm, 200 Å, Method Set: 20 min/ 20 mL, Injection Volume: 5.0 mL, Run Time: 30 min, Purification gradients: 20% -50% MeCN + 0.1% TFA;

After preparative HPLC: 5% -95% MeCN + 0.1% TFA in 10 min, UV-Vis wavelength: 220 nm, t R = 5.87 min, purity: 100 % 1 H NMR (800 MHz, CD 3 OH) δ 9.13 (d, J = 8.5 Hz, 1H, NH), 9.08 (d, J = 9.5 Hz, 1H, NH), 8.74 (d, J = 8.8 Hz, 1H, NH), 7.95 (s, 1H), 7.82 (s, 1H), 7.78 (s, 1H), 7.66 (s, 1H), 5.24-5.18 (m, 4H), 5.11-5.06 (m, 4H), 4.03 (m, 4H), 3.30 -3.19 (m, 4H), 3.07-2.99 (m, 4H); 13 C NMR (200 MHz, CD 3 OD) δ 170. 5, 140.8, 141.3, 141.2, 141.1, 125.7, 126.0, 126.4, 125.1, 52.5, 51.0 (2C), 50.9 (2C), 50.8 (2C), 24.1, 23.9, 23.8, 23.7;

F NMR (188 MHz, CD 3 OD) δ -75.65 (d, J = 7.5 Hz), -76.84 (d, J = 7.5 Hz), -76.88 (d, J = 7.5 Hz), -77.05 (d, J = 7.5 3232, 2926, 1690, 1561, 1439, 1391, 1251, 1203, 1180, 1124, 800, 721. (R)-ethyl 2- (4-(2-((tert-butoxycarbonyl) 4.0 M) in dioxane (30 mL, 120 mmol) was added. The reaction was stirred at 0 o C for 30 min, then at r.t for 3 hours. After the reaction finished, all the solvent was evaporated under reduced pressure, then added a little amount of diethyl ether to precipitate the product and evaporated the diethyl ether, repeated this process several times until the mass of the crude did not change any more. Compound 3.5 was obtained as white solid in quantitative yield.

The resulting crude compound 3.5 was dissolved in THF-H 2 O (5 mL-5 mL) at r.t, then to the solution was added Boc 2 O (1.75 g, 8 mmol, 2.0 eq.), Na 2 CO 3 (1.27 g, 12 mmol, 3.0 eq.). 30 min later, the reaction was stirred at r.t overnight. When TLC showed the reaction finished, THF was removed under reduced pressure; the residue was washed with EtOAc (10 mL) and distilled water (10 mL), then brine, and dried over MgSO 4 . The crude residue was purified by column chromatography on silica gel using EtOAc: Cyclohexane = 30:70 as eluent. Compound 3.17 was obtained as white solid in 80% yield (1.17 g, 3.2 mmol, 80% yield). 2,155.2,142.7,124.9 (d,J = 278 Hz,CF 3 ),123.5,80.8,62.6,51.69(q,J = 30 Hz,CHCF 3 ),51.0,28.3,25.3,14. 3349, 2986, 2360, 1760, 1693, 1521, 1375, 1349, 1312, 1251, 1207, 1151, 1120, 1052, 1022, 888, 663. (R)-2- (4-(2-((tert-butoxycarbonyl)amino)-3,3,3-trifluoropropyl)-1H-1,2,3-triazol-1-yl)acetic acid (3.18) Chemical Formula: C 12 H 17 F 3 N 4 O 4 Exact Mass: 338.1202 Molecular Weight: 338.2830 Compound 3.17 (366 mg, 1.0 mmol) was added to a round bottom flask with 2 mL EtOH at 0 o C. Then to the solution was added 1.25 mL of NaOH 2M aqueous solution (2.5 mmol, 2.5 eq.) and the reaction was stirred at 0 o C for 1h. When the TLC showed the reaction completed, solvents were removed and 5 mL distilled water and 5 mL EtOAc were added, then HCl (1M) was added at 0 o C until pH was 3-4. Then solution was extracted with EtOAc (3×10 mL) and the solution was dried with Na 2 SO 4 and the solvent was evaporated to afford a white solid in quantitative yield (360 mg, 0.98 mmol), which was pure enough for the next reaction. 7, 157.7, 143.7, 126.8 (d, J = 278 Hz, CF 3 ), 126.0, 81.2, 54.1 (q, J = 30 Hz, CHCF 3 ), 51.8, 28.7, 25.7; 2982, 2488, 1698, 1422, 1369, 1274, 1219, 1175, 1128, 1051, 813. To the compound 3.18 (212 mg, 0.63 mmol) in dry DMF at 0 o C was added HATU (342 mg, 1.3 eq.), HOAt (163 mg, 1.6 eq.) and the resulting reaction solution was stirred at 0 o C for 60 min, then to the reaction solution were added compound 3.5 (211 mg, 0.70 mmol) and DIPEA (271 mg, 3.0 eq.) at 0 o C, after the addition the reaction was stirred at room temperature overnight. When TLC showed the reaction finished, DMF was removed under vacuum pump, the residue was washed with 10% citric acid 10 mL, and EtOAc 10 mL. The residue was then extracted with EtOAc (10 mL × 3) and the organic phase washed with 10% NaHCO 3 10 mL and then brine, dried over MgSO 4 . The crude product was purified by column chromatography using CH 2 Cl 2 : EtOH = 98:2 as eluent and the dimer 3.19 (285 mg, 0.49 mmol) was obtained as a white solid in 77% yield. 2, 167.0, 156.2, 143.1, 142.7, 126.5 (CF 3 ), 126.1 (CF 3 ), 125.4, 125.2, 80.6, 62.9, 52.8 (CHCF 3 ), 52.6, 51.7, 51.3 (CHCF 3 ), 28.3, 25.4, 25.1, 14.4 3286, 2983, 1748, 1697, 1538, 1370, 1249, 1214, 1173, 1124, 1051, 1022, 847, 660. 2-(4- Compound 3.17 (117 mg, 0.2 mmol) was dissolved in MeOH (2.5 mL) at 0 o C. Then NaOH aqueous solution (2 M, 0.25 mL, 0.5 mmol, 2.5eq.) was added and the reaction was stirred at 0 o C for 2h and at room temperature for 1h. Then TLC showed the reaction finished completely. MeOH was removed under reduced pressure, then to the residue was added H 2 O (5 mL) and EtOAc (10 mL) and acidified using HCl (1M, 0.5 mL) at 0 o C to make the pH was 3 (pH Paper). The resulting aqueous phase was quickly extracted with EtOAc (3×10 mL) and the organic phase was dried with anhydrous MgSO 4 and the solvent evaporated affording a white solid, which was pure enough for the next reaction. The acid 3.21 was obtained as a white solid in quantitative yield (117 mg, 0.2 mmol). 1, 168.1, 157.7, 143.8, 143.3, 126.8 (d, J = 285 Hz, CF 3 ), 126.4 (d, J = 278 Hz, CF 3 ), 126.3 (2 HC=C), 81.2, 53.4 (CHCF 3 ), 52.7, 51.9 (CHCF 3 ), 51.8, 28.7, 25.7, 25.5; 3334, 3043, 1741, 1689, 1568, 1530, 1422 1363, 1306, 1238, 1155, 1122, 1055, 816. To the compound 3.18 (67 mg, 0.2 mmol) in dry DMF (1.0 mL) at 0 o C was added HATU (114 mg, 0.3 mmol, 1.5 eq.), HOAt (49 mg, 0.36 mmol, 1.8 eq.), stirred at 0 o C for 60 min, then compound 3.14 (105 mg, 0.2 mmol, 1.0 eq.) and DIPEA (155 mg, 1.2 mmol, 6.0 eq.) were added at 0 o C. The reaction stirred at room temperature overnight. When TLC (CH 2 Cl 2 : MeOH: CH 3 COOH = 70:30:1) showed there was no compound 3.18, DMF was removed under vacuum pump, the residue was washed with EtOAc (5 mL) and water (5 mL), then the organic phase was washed with 10% citric acid solution (5 mL) and 10% NaHCO 3 solution (5 mL) and was dried over Na 2 SO 4 . The concentrated crude residue (103 mg) was purified by column chromatography on silica gel using EtOAc: CH 2 Cl 2 =2:1 as eluent. The trimer 3.20 was obtained as a white solid in 34% yield (54 mg, 0.067 mmol). 3, 166.2, 166.1, 155.2, 141.5, 141.2, 141.0, 126.9 (CF 3 ), 124.9, 124.8, 124.4, 123.6 (CF 3 ), 123.2 (CF 3 ), 78.9, 61.5, 51.9, 51.5, 51.2, 51.1, 50.4, 49.9, 27.9, 24.0, 14. 3356, 3146, 2982, 2936, 1756, 1699, 1521, 1369, 1251, 1178, 1153, 1123, 1051, 1026; HPLC: X-Select C 18 -2.1 × 150 mm -3.5 μm; H 2 O + 0.1% formic acid / CH 3 CN -gradient 5%-100% in 20 min, t R = 16.04, purity: 97%. The dimer acid 3.21 (84 mg, 0.15 mmol) in dry DMF (1.0 mL) at 0 o C was added HATU (76 mg, 1.5 eq., 0.2 mmol), HOAt (28 mg, 1.6 eq.), stirred at 0 o C for 60 min, then added compound 3.11 (78 mg, 0.15 mmol, 1.0 eq.) and DIPEA (155 mg, 6.0 eq.) at 0 o C. The reaction stirred at room temperature overnight. When TLC showed there was no compound 3.21, and then DMF was removed under vacuum pump, the residue was washed with distilled water 5 mL, and EtOAc 5 mL. The product then precipitated and filtered, the precipitates was washed with H 2 O 2 mL three times and EtOAc 2 mL three times and then dried under vacuum pump with P 2 O 5 at 50 o C. The tetramer 3.22 was obtained as a white solid in 14% yield (21 mg, 0.02 mmol). 7, 167.3, 166.2, 166.1, 155.2, 141.5, 141.2, 141.0, 126.5, 124.9, 124.7, 124.1, 123.7, 78.9, 61.5, 51.6, 51.1, 50.4, 50.3, 50.0, 27.9, 24.0, 13.9; 3292, 2980, 1696, 1555, 1440, 1368, 1248, 1173, 1125, 1050, 1025; HPLC: X-Select C 18 -2.1 × 150 mm -3.5 μm; H 2 O + 0.1% formic acid / CH 3 CN -gradient 5%-100% in 20 min, t R = 16.40, purity: 96%. The Boc protected monomer 3.17 (92 mg, 0.25mmol) was dissolved in 2mL CH 2 Cl 2 at 0 o C, then to the solution at 0 o C was added CF 3 COOH (1.2mL, 15mmol, 60 eq.). The reaction was stirred at 0 o C for 30 min, and then was stirred at r.t for 2h. After that time, all the solvents were evaporated under reduced pressure, a little amount of diethyl ether was added to precipitate the product and the diethyl ether was evaporated. This process was repeated several times until the mass of the crude did not change any more. The resulting crude compound was obtained as liquid (95mg) with quantitative yield without any further purification. The resulting crude compound (95mg) was dissolved in dry DMF (2mL) at r.t, and then to the solution was added DMTMM (Cl -) 83mg (0.3mmol, 1.2 eq.), NMM 152mg (1.50mmol, 6.0 eq.) and Boc-Phe-OH 73mg (0.28mmol, 1.1 eq.). The reaction was stirred at room temperature overnight. Then DMF was removed under vacuum, the residue was washed with EtOAc (5mL) and 10% citric acid (5mL), distilled water (5mL) , 10% K 2 CO 3 (5mL) and then brine, and dried over Na 2 SO 4 . The crude residue was purified by column chromatography on silica gel using CH 2 Cl 2 : MeOH = 100:2 as eluent. The compound 3.24 was obtained as white solid in 87% yield (112mg). R f = 0.4 (CH 2 Cl 2 : MeOH = 50:1); 1 H NMR (300 MHz, CDCl 3 ) δ 7.51(s, 1H), 7.28-7.24 (m, 3H), 7.21 (d, J = 4 Hz, 1H, NH), 7.16-7.14 (m, 2H), 7.07 (d, J = 8 Hz, 1H, NH), 4.94 (br, 1H), 4.36(s, 1H), 4.25 (q, J = 7.1 Hz, 2H), 3.17-3.05(m, 4H), 1.38(s, 9H), 1.29 (t, J = 7.1 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 ) δ 172.0, 166. 3, 155.7, 142.4, 136.5, 129.4, 128.7, 127.0, 124.7 (d, J = 278 Hz, CF 3 ), 123. 6, 80.7, 62.5, 55.6, 50.9, 50.0 (q, J = 30 Hz, CHCF 3 ), 37.5, 28.3, 14. 3322, 2925, 1743, 1686, 1666, 1526, 1392, 1271, 1227, 1170, 1123, 1050, 1023, 699. The Boc protected monomer 3.17 (114 mg, 0.32 mmol) was dissolved in 5mL CH 2 Cl 2 at 0 o C, then to the solution at 0 o C was added CF 3 COOH (1.4 mL, 18.6 mmol, 60 eq.) and stirred at 0 o C for 30 min, then the reaction solution was stirred at r.t for 2h. After that time, all the solvents were evaporated under reduced pressure, then a little amount of diethyl ether was added to precipitate the product and evaporated the diethyl ether, repeated this process several times until the mass of the crude did not change any more. The resulting crude compound was obtained as liquid (118 mg) without any further purification.

((R)-2-(2-(4-((R)-2-((tert-butoxycarbonyl)amino)-3,3,3-trifluoropropyl)-1H-1,2,3-triazol-1- yl)acetamido)-3,3,3-trifluoropropyl)-1H-1,2,3-triazol-1-
2-(4-((R)-2-(2-(4-((R)-2-(2-(4-((R)-2-(2-(4-((R)-2-((tert-butoxycarbonyl)amino)-3,3,3-triflu- oro propyl)-1H-1,2,3-triazol-1-yl)acetamido)-3,3,3-trifluoropropyl)-1H-1,2,3-triazol-1-yl)acetam- ido)-3,3,3-trifluoropropyl)-1H-1,2,3-triazol-1-yl)acetamido)-3,3,3-trifluoropropyl)-1H-1,2,3- triazol-1-
The resulting crude compound (118 mg) was dissolved in dry DMF (2 mL) at r.t, and then to the solution was added DMTMM (Cl -) (104 mg, 0.38mmol, 1.2 eq.), NMM (189 mg, 1.87 mmol, 6.0 eq.) and Boc-(Cbz)-Lys-OH (130 mg, 0.34 mmol, 1.1 eq.). The reaction stirred at room temperature overnight. Then DMF was removed under vacuum pump, the residue was washed with EtOAc (5 mL) and 10% citric acid (5 mL), distilled water (5 mL) , 10% K 2 CO 3 (5 mL) and then brine, and dried over Na 2 SO 4 . The crude residue was purified by column chromatography on silica gel using CH 2 Cl 2 : MeOH = 100:2 as eluent. The compound 3.25 was obtained as white solid in 76% yield (148 mg). R f = 0.3 (CH 2 Cl 2 : MeOH = 50:1); 1 H NMR (300 MHz, CDCl 3 ) δ 7. 56 (s, 1H) , 7.34-7.33 (m, 5H), 7.02 (d, J = 9 Hz, 1H, NH), 5.12-5.09 (m, 6H), 4.91(m, 1H), 4.25 (q, J = 7.1 Hz, 2H), 4.04(m, 1H), 3.27-3.14 (m, 4H), 1.72 (m, 1H), 1.52-1.45 (m, 3H), 1.41 (s, 9H), 1.28-1.24 (m, 5H); 13 C NMR (100 MHz, CDCl 3 ) δ 172. 6, 166.3, 156.9, 156.0, 142.4, 136.8, 128.7, 128.4, 128.2, 124.8 (d, J = 285 Hz, CF 3 ), 123.9, 80.3, 66.8, 62.6, 54.4, 51.0, 50.3 (q, J = 30 Hz, CHCF 3 ), 40.4, 31.2, 29.5, 28.4, 24.9, 22.4, 14. 3306, 2938, 1753, 1692, 1526, 1456, 1367, 1220, 1172, 1127, 1048, 1022, 875, 698 The ethyl ester 3.25 (101 mg, 0.16 mmol) was added to a round bottom flask with 2 mL EtOH at 0 o C. Then to the solution was added 0.20 mL of NaOH 2M aqueous solution (0.4 mmol, 2.5 eq.) and the reaction was stirred at 0 o C for 1h. When the TLC showed the reaction was completed, solvents were removed and 5 mL distilled water and 5 mL EtOAc were added, then HCl (1 M) was added at 0 o C until pH was 3-4. Then solution was extracted with EtOAc (3×10 mL) and the solution was dried with Na 2 SO 4 and the solvent was evaporated to afford a white solid in 96% yield (92 mg), which was pure enough for the next reaction.

1

H NMR (300 MHz, CD 3 OD) δ 7.83 (s, 1H), 7. 35-7.33 (m, 5H), 5.23 (s, 2H), 5.07 (m, 2H), 4.89 (br, 1H), 3.96 (br, 1H), 3.27-3.21 (m, 1H), 3.11-3.04 (m, 3H), 1.41-1.20 (m, 15H); 13 C NMR (75 MHz, CD 3 OD) δ 175. 6, 169.7, 158.9, 157.8, 143.4, 138.4, 129.5, 128.9, 128.8, 126.0, (d, J = 278 Hz, CF 3 ), 80.6, 67.4, 55.8, 51.6, 41.3, 32.8, 30.3, 28.7, 25.2, 23. 3337, 2933, 1677, 1524, 1457, 1367, 1230, 1167, 1124, 1063, 1019, 695. A mixture of 3.23 (20 mg, 0.02 mmol) and Pd/C (10% wt, 2 mg) in 0.5 mL DMF and 2 mL EtOH was stirred under 1 atm. of H 2 atmosphere at r.t. for 2h, when TLC showed the starting material disappeared, the reaction mixture was filtered through a celite pad, and the residue was washed with EtOH (4 × 1 mL), and the solvents were removed in vacuo to afford a crude residue. The crude residue was dissolved in dioxane (2 mL), and to the solution was added 0.3 mL HCldioxane (1.2 mmol) and the reaction was stirred at r.t. for another 3h. After that the solvents were removed in vacuo to afford a crude residue, which was washed with EtOH and diethyl ether for several times. The compound 3.27 was obtained as a white solid in 84% yield (14 mg, 0.017 mmol). 2,169.9,168.4,166.8,142.3 (2C),135.7,128.9,128.6,127.1,125.5 (2CH),63.2,53.3,51.7,50.7,50.1,38.8,37.1,30.0,26.2,23.5,20.7,13.1;19 F NMR (188 MHz, D 2 O) δ -76.08 (d, J = 7.5 Hz), -76.25 (d, J = 7.5 Hz); ESI-HRMS: m/z calcd for C 31 H 42 F 6 N 11 O 5 [M+H] + : 762.3275, found: 762.3275; IR (cm -1 ): 3388 (br, strong), 1749, 1650, 1549, 1440, 1388, 1349, 1265, 1217, 1181, 1123, 1062, 698.

ThT Assays

Fluorescence-detected Thioflavin-T binding assay Thioflavin T was obtained from Sigma. Aβ 1-42 was purchased from American Peptide. The peptide was dissolved in an aqueous 1% ammonia solution to a concentration of 1 mM and then, just prior to use, was diluted to 0.2 mM with 10 mM Tris-HCl, 100 mM NaCl buffer (pH 7.4). Stock solutions of glycopeptides were dissolved in DMSO with the final concentration kept constant at 0.5% (v/v).

Thioflavin T fluorescence was measured to evaluate the development of Aβ 1-42 fibrils over time using a fluorescence plate reader (Fluostar Optima, BMG labtech) with standard 96-well black microtiter plates. Experiments were started by adding the peptide (final Aβ 1-42 concentration equivalent to 10 µM) into a mixture containing 40 µM Thioflavin T in 10 mM Tris-HCl, 100 mM NaCl buffer (pH 7.4) with and without the tested compounds at different concentrations (100, 10µM) at room temperature. The Th-T fluorescence intensity of each sample (performed in duplicate or triplicate) was recorded with 440/485 nm excitation/emission filters set for 42 hours performing a double orbital shaking of 10 s. before the first cycle. The fluorescence assays were performed between 2 and 4 times on different days, with the same batch of peptide. The ability of compounds to inhibit/accelerate Aβ 1-42 aggregation was assessed considering both the time of the halflife of aggregation (t 1/2 ) and the intensity of the experimental fluorescence plateau (F). The relative extension (or reduction) of t 1/2 is defined as the experimental t 1/2 in the presence of the tested compound relative to the one obtained without the compound and is evaluated as the following percentage: t 1/2 (Aβ + compound) / t 1/2 (Aβ) x 100. The decrease (or increase) of the experimental plateau is defined as the intensity of experimental fluorescence plateau observed with the tested compound relative to the value obtained without the compound and is evaluated as the absolute value of following percentage : |F Aβ -F Aβ+compound | / F Aβ x 100 (a decrease is indicated with a (-) and an increase with a (+).

Thioflavin T assay on IAPP

IAPP was purchased from Bachem (batch 7001244). IAPP was dissolved in hexafluoroisopropanol (HFIP) at a concentration of 1 mM and incubated for 1 hour to dissolve any preformed aggregates. Next, HFIP was evaporated with dry nitrogen gas followed by vacuum desiccation for 3 hours. The resulting peptide film was then dissolved in DMSO to obtain stock solutions of IAPP (0.2 mM). Stock solutions of compound were dissolved in DMSO (10, 1 and 0.1 mM in DMSO). The concentration of DMSO was kept constant at 3% (v/v) in the final volume of 200 µL.

Thioflavin-T binding assays were used to measure the development of fibrils over time. A plate reader (Fluostar Optima, BmgLabtech) and standard 96-wells flat-bottom black microtiter plates in combination with a 440 nm excitation filter and a 485 nm emission filter were used. The ThT assay was started by adding 5 µL of a 0.2 mM IAPP stock solution and 1 µL of stock solutions of test compound to a mixture of 10 µM ThT and 10 mM Tris/HCl, 100 mM NaCl at pH 7.4. The concentration of IAPP was held constant at 5 µM and test compound was added to yield compound /IAPP ratios of 10/1 and 1/1. The ThT assay was performed in triplicate.

Molecular Dynamics Studies Methods

All-atom MD simulations were performed using the GROMACS 4.5 package [1,2] , with the OPLS-AA force field [3] in combination with the SPC/E water model [4] . The extended conformation of each of the eight studied pentapeptides, in which all residues were in β-conformations (Figure 1), was initially placed in a 4.3 x 4.3 x 4.3 Å 3 box filled with about 2600 water molecules and one chloride ion to neutralize the systems in the case of the protonated free amine forms. The nonbonded interactions were treated using the smooth PME method [5] for the electrostatic terms and a cut-off distance of 1.2 nm for the van der Waals potentials. The covalent bonds length was kept constant using the LINCS [6] and SETTLE [7] procedures, allowing the use of a 2 fs time step. The simulated systems were first equilibrated using two short MD simulations of a 2 ns duration, the first one with the V-scale and Berendsen coupling algorithms [8] to rapidly reach the target temperature T = 300 K and pressure P = 1 bar respectively, and a second one with the Nose-Hoover and Parrinello-Rahman coupling methods [9][10][11] to generate correct temperature and pressure fluctuations around the previous equilibrium values. Finally, the systems were simulated without any constraints during 500 ns in the NPT ensemble. Trajectories coordinates were saved every 10 ps for subsequent analysis. 11) u( 22) u( 33) u( 23) u( 13) u( 12 Title: Fluorinated Peptidomimetics: Synthesis, Conformational Studies and Evaluation as Amyloid Proteins Aggregation Modulators Keywords: fluorine, peptidomimetic, trifluoromethylated threonine, molecular modelling, NMR, conformational study, Aβ 1-42 peptide, IAPP, trifluoromethylated 1,2,3-triazole, foldamer.

Abstract: It has been widely recognized that Alzheimer's disease (AD) represents an unsettling, worldwide challenge for society. By far, there has been no effective cure for AD. Pathologically, AD is characterized by a loss of synapses, an increase in the number of extracellular Aβ plaques and an increase in intracellular aggregated hyperphosphorylated Tau (neurofibrillary tangles). It is commonly believed that AD is primarily linked to oligomerization and fibrillization of amyloid β peptides, and the soluble Aβ oligomers and fibrils are neurotoxic species. One strategy to reduce the Aβ fibrils is the use of peptides or peptidomimetics to inhibit the Aβ aggregation either by interaction with the Aβ oligomers to prevent its further aggregation into fibrils, or by stabilizing the transient Aβ α-helical conformations to prevent its transition to β-sheet structures.

In the first direction, based on the encouraging results of the glycopeptides containing the polar sugar β-sheet breaker element to modulate Aβ peptide aggregation and the unique properties of trifluoromethylhydroxyl group, we designed and synthesized pentapeptide mimics with the sequence (Boc) Ala-Val-X-Val-Leu-OMe (X = Ser, Thr, (2S, 3R)-CF 3 -Thr and (2S, 3S)-CF 3 -Thr) to interact with the nucleation site of Aβ peptide in its monomeric or oligomeric form so as to disrupt the self-assembly into toxic oligomeric form. Both 2D NMR and molecular modelling studies indicated that these peptides in polar solvent (water and methanol) adopt mainly extended backbone conformations. It is found both experimentally and theoretically that, the (2S, 3S)-CF 3threonine-containing pentapeptides are more extended than the L-serine and L-threoninecontaining pentapeptides. It is also observed that the (2S, 3S)-CF 3 -Thr pentapeptides have a propensity to self-associate of by forming intermolecular β-strand contacts. The ability of these pentapeptides to inhibit amyloid fibril formation was evaluated on Aβ 1-42 and IAPP peptides by ThT fluorescence assay. It was found that none of these pentapeptides have any inhibition effect in Aβ 1-42 peptide and IAPP aggregation. On the contrary, some compounds showed an acceleration effect in IAPP aggregation. Accelerating the aggregation pathways is less intuitive but this strategy has more recently aroused interest. It could be interesting to further study the effect of accelerating IAPP aggregation by complementary techniques.

In the other direction, we have assembled novel a CF 3 -1,4-triazole-based amino acid mimic into homo-oligomers (trimer and tetramer) and peptidomimetics. The fluorinated oligomers were investigated both by NMR conformational studies and molecular modelling simulations. Our preliminary modelling studies predict helical structures with trimer and tetramers of CF 3 -1,4disubstituted-1,2,3-triazole-based amino acid. NMR analysis of tetramer displayed very interesting NOE correlations, indicating a folded structure. The ability of these fluorinated oligomers to modulate the amyloid fibril formation of Aβ 1-42 and IAPP peptides were evaluated by ThT fluorescence assay. It was found that trimer was a weak inhibitor of Aβ 1-42 aggregation but also a promoter of IAPP aggregation. The tetramer was found able to modulate the aggregation of Aβ 1-42 and IAPP but not in a classical inhibition or promotion manner. In this aspect, we demonstrated that a possible helix-forming peptidomimetic could be used to modulate amyloid proteins aggregation.

Titre: Peptidomimétiques Fluorés: synthèse, études conformationnelles et évaluation comme modulateurs de l'agrégation de protéines amyloïdes Mots clés: fluor, peptidomimétique, thréonine trifluorométhylée, modélisation moléculaire, RMN, études conformationnelles, peptide Aβ 1-42, IAPP, 1,2,3-triazole trifluorométhylé, foldamère Résumé: La maladie d'Alzheimer représente un défi mondial pour la société. Il n'y a pas à ce jour de traitement efficace pour traiter ou ralentir les symptômes de la maladie d'Alzheimer. Cette maladie est caractérisée par une perte de synapses, une augmentation du nombre de plaques extracellulaires d'Aβ et une augmentation de Tau hyperphosphorylée agrégée intracellulaire (neurodégénérescence fibrillaire). Il est communément admis que la maladie d'Alzheimer est principalement liée à l'oligomérisation et à la fibrillation de peptides amyloïdes β, et que les oligomères Aβ solubles et les fibres sont des espèces neurotoxiques. Une stratégie pour réduire la présence de ces espèces toxiques de Aβ est l'utilisation de peptides ou de peptidomimétiques pour inhiber l'agrégation de Aβ, soit par interaction avec les oligomères d'Aβ pour empêcher davantage son agrégation en fibres ou en stabilisant les conformations hélicoïdales transitoires de Aβ pour empêcher sa transition vers des structures en feuillets β.

Dans un premier temps, sur la base des résultats encourageants de glycopeptides contenant un élément polaire casseur de feuillets β pour moduler l'agrégation du peptide Aβ, et des propriétés uniques du groupe trifluoromethylhydroxyle, nous avons conçu et synthétisé des mimes de pentapeptides contenant la séquence (Boc) Ala-Val-X-Val-Leu-OMe (X = Ser, Thr, (2S, 3R)-CF 3 Thr et (2S, 3S)-CF 3 Thr), pour interagir avec le site de nucléation du peptide Aβ dans sa forme monomérique ou oligomérique de manière à perturber l'auto-assemblage en oligomères toxiques. Des études conformationnelles par RMN 2D et par modélisation moléculaire ont indiqué que ces peptides adoptent des conformations étendues dans des solvants polaires (eau et méthanol). Nous avons constaté expérimentalement et théoriquement que les pentapeptides contenant l'acide aminé non naturel (2S, 3S)-CF 3 -thréonine sont plus étendus que les pentapeptides contenant les acides aminés naturels L-sérine et L-thréonine. Nous avons également observé que les pentapeptides (2S, 3S)-CF 3 -Thr ont une propension à s'auto-associer en formant des brins β intermoléculaires. La capacité de ces pentapeptides à inhiber la formation de fibres amyloïdes a été évaluée sur les peptides Aβ 1-42 et IAPP (impliqué dans le diabète de type II) par des essais de fluorescence à la thioflavine T (ThT). Il a été constaté qu'aucun de ces pentapeptides ont un effet d'inhibition de l'agrégation des peptides Aβ 1-42 et IAPP. Au contraire, certains composés ont montré un effet d'accélération de l'agrégation de IAPP. Accélérer l'agrégation est moins intuitif mais cette stratégie a plus récemment suscité un intérêt. Il pourrait être intéressant d'étudier plus en détail l'intérêt de cette accélération de l'agrégation de IAPP par des techniques complémentaires.

Dans une deuxième partie de cette thèse, nous avons assemblé une unité amino acide basée sur un motif CF 3 -1,4-disubstituted-1,2,3-triazole en homo-oligomères (trimères et tétramères) et en peptidomimétiques. Des études conformationnelles de ces oligomères fluorés ont été menées par RMN 2D et par des simulations de modélisation moléculaire. Nos études préliminaires de modélisation moléculaire prévoient des structures hélicoïdales avec des trimères et des tétramères d'acides aminés à base de ces CF 3 -triazoles. L'analyse par RMN 2D du tétramère affiche des corrélations NOE très intéressantes, ce qui indique une structure repliée. La capacité de ces oligomères fluorés à moduler la formation de fibres amyloïdes des peptides Aβ 1-42 et IAPP a été évaluée par test de fluorescence à la ThT. Nous avons constaté que le trimère est un faible inhibiteur de l'agrégation de Aβ 1-42, mais aussi un promoteur d'agrégation de IAPP. Le tétramère a été trouvé capable de moduler l'agrégation de Aβ 1-42 et de IAPP, mais non d'une manière classique d'inhibition ou de promotion. Nous avons ainsi démontré qu'une structure potentiellement hélicoïdale d'un peptidomimétique peut être utilisée pour moduler l'agrégation de protéines amyloïdes.

Figure 1 . 1

 11 Figure 1.1 Gauche effect of 1,2-difluoroethane

Figure 1 . 3

 13 Figure 1.3 Optimization of metabolic properties of Ezetimibe by the incorporation of fluorine.

Figure 1 . 4

 14 Figure 1.4 Structures of [ 18 F] 2-fluoro-2-deoxy-D-glucose ([ 18 F] FDG) and [ 18 F]6-fluoro-L-DOPA.

Figure 1 . 7

 17 Figure 1.7 Structural comparison of brain with AD and healthy brain [picture obtained from BrightFocus Foundation @ http://www.brightfocus.org/alzheimers/infographic/progressionalzheimers-disease]

Figure 1 . 10

 110 Figure 1.10 Production of amyloid-β protein from amyloid precursor protein (Adapted from reference [47])

Figure 1 . 11

 111 Figure 1.11 Sequence and structure of the monomer unit in Aβ 1-40 and Aβ 1-42 fibrils. (a) Sequence of Aβ 1-42 derived from human APP. (b) Structural constraints in Aβ 1-40 and Aβ 1-42 fibrils. (Adapted from reference [58a])

Figure 1 . 12

 112 Figure 1.12 Molecular models of Aβ 1-42 oligomers and fibrils. (a) Schematic of the monomer within the oligomer complex of Aβ 1-42. (b) Schematic of the Aβ 1-42 pentamer. (d) Schematic of the monomer within Aβ 1-42 fibrils. (e) Schematic showing the parallel and in-register packing and staggering of the individual β-strands within Aβ 1-42 fibrils. (Adapted from reference [58a])

Figure 1 .

 1 Figure 1.13 The 3D structure of a 35 Mox Aβ (1-42) fibril. Ribbon diagram of the protofilament core structure comprising residues 17-41 (Adapted from reference [58b])

Figure 1 . 14

 114 Figure 1.14 Molecular structures of amyloid fibrils. (a) Individual monomers form S-shapes that comprise short β-strand segments (arrows) linked by bends, and that stack in two parallel subunits to form (PDB id: 5KK3), (b) A comparison of one Aβ 1-42 monomer with a monomer from a

Figure 1 . 16

 116 Figure 1.16 Chemical structure of SRN-003-556. SRN-003-556 is an orally bioavailable CNSpenetrating synthetic analog of K252a.

Figure 1 .

 1 Figure 1.18 Peptide-based modulators of Aβ aggregation.

Figure 1 . 19

 119 Figure 1.19 Small molecule-based modulators of Aβ aggregation.

Figure 1 . 20

 120 Figure 1.20 Small molecules targeting the HHQK sub-region of the N terminus.

Figure 1 .

 1 Figure 1.21 Small-molecule inhibitors targeting the KLVFF hydrophobic core

Figure 1 .

 1 Figure 1.23 β-secretase inhibitors that in clinical trials

Figure 1 .

 1 Figure 1.24 γ-secretase inhibitors

Figure 1 . 25

 125 Figure 1.25 Chemical structures of Pep1b and AEDabDab. Arrows indicate the amino acids in Aβ with which the different groups are designed to interact. (Adapted from reference [106])

Figure 1 .

 1 Figure 1.27 (a) Helical wheel diagram and (b) sequences of peptides VW18, VW01, and VW01ran. The circle joins the b, c, and f positions occupied by valines that make peptide VW18 prone to β-sheet formation. The positively charged residues are marked in blue, the negatively charged residues in red. (Adapted from reference [108])

Figure 2 . 1

 21 Figure 2.1 Inhibition of amyloidosis by synthetic peptide derivatives. (i) Proline introduced as β-sheet breaker. (ii) Terminal blocking group [e.g. cholyl or poly(lys)]. (iii) N-methylated amides to block one edge of β strand.

Figure 2 . 2

 22 Figure 2.2 Peptide-based modulators of Aβ aggregation containing β-sheet breaker element.

Figure 2 . 3

 23 Figure 2.3 Hypothesis of the mechanism of Aβ1-42 aggregation inhibition by the designed glycopeptides.

Figure 2 . 4

 24 Figure 2.4 Structure of peptidomimetics containing D-glucopyranosyl sugar amino acid derivatives.

Figure 2 . 5

 25 Figure 2.5 Proposed model of interaction of compound 2.6 with Aβ 1-42.

Figure 2 . 8

 28 Figure 2.8 Interaction model of peptidomimetics containing trifluoromethylhydroxyl group with Aβ peptide.

Figure 2 . 9

 29 Figure 2.9 non-natural amino acids containing trifluoromethylhydroxyl group

Figure 2 . 10

 210 Figure 2.10 Structure of pentapeptide derivatives synthesized by Dr. KIHAL Nadjib.

  Figur 42 pept

Figure 2

 2 Figure 2.11 Th

FFigure 2 .

 2 Figure 2.13 natural and CF 3 -threonine-sawhorse projections

Figure 2 . 14

 214 Figure 2.14 Structure of target molecules

Scheme 2 . 3

 23 Scheme 2.3 Synthesis of pentapeptides 2.20 and 2.21

Figure 2 .

 2 Figure 2.15 Assigned NOESY comparison between the enkephalin-related peptide derivative 3.35 and its fluorinated analogue 3.36.

Figure 2 .

 2 Figure 2.18 Distribution of the peptide conformations as a function the end-to-end distance defined as the distance between the nitrogen of residue Ala 1 and the carbon of the C-terminal carbonyl.

Figure 2 . 19

 219 Figure 2.19 Distribution of the peptide dihedral angles ψ for the three central residues Val 2 (black), X 3 (red) and Val 4 (green).

Figure 2 . 23

 223 Figure 2.23 Distribution function of the distances between the hydroxyl group of the central residue X 3 and the neighboring polar atoms of the AVTVL, AV-(3R)-CF 3 -VL and AV-(3S)-CF 3 -VL backbone.

Figure 2 . 24

 224 Figure 2.24 ThT fluorescence assays: representative curves showing fibril formation of Aβ 1-42 (10 μM) in the absence (purple) or in the presence of the pentapeptide 2.11 at an 2.11:Aβ ratio of 0.1:1 (yellow), 1:1 (blue) and 10:1 (green); pentapeptide 2.11 alone (red, 100 μM), buffer (grey).

  Figur μM) in (blue)

  Figur(10 μM 0.1:1

  Figur (10 μM 0.1:1

Figure 3 . 2

 32 Figure 3.2 Comparison of 1,4-substituted triazoles with trans-peptide bonds and 1,5-substituted triazoles with cis-peptide bonds.

Figure 3 . 5 Figure 3 . 6

 3536 Figure 3.5 Conformers of 4Tzl obtained by molecular modeling (Adapted from reference [16])

Figure 3 . 7

 37 Figure 3.7 Triazole based β-turn mimetic. (Adapted from reference [17])

Figure 3 . 8 Figure 3 . 9

 3839 Figure 3.8 Development of nonpeptidic foldamers by replacement of the amide bond with triazole rings

Figure 3 .

 3 Figure 3.10 Imidazole-containing pseudopeptides form helix-like secondary structures

Figure 3 . 11

 311 Figure 3.11 Oligomers of 1,3-thiazole based γ-amino acid

Figure 3 . 12

 312 Figure 3.12 Structure of trimer and tetramer of 1,5-disubstituted 1,2,3-triazole amino acid (5Tzl) and its possible conformation.

Figure 3 . 13

 313 Figure 3.13 Effect of a perfluoroalkyl (R f ) group directly attached to a peptide backbone on the acidity of the adjacent amide NH and the hydrogen bonding ability.

Figure 3 . 14

 314 Figure 3.14 Hexametric β-peptides pep-FF and its X-ray crystal structure (CCDC 978602) viewed (a) along the helix axis and (b) from the side. (Adapted from reference [25])

Figure 3 .

 3 Figure 3.16 Published CF 3 -containing triazolyl α-amino acids and the target analogues 3.0

Figure 3 . 17

 317 Figure 3.17CF 3 -1, 4-triazole amino acid based trimer and tetramer

  Figure 3.17CF 3 -1, 4-triazole amino acid based trimer and tetramer

Scheme 3 . 3 19 F

 3319 Scheme 3.3 Copper (I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC) reactions

Figure 3 .

 3 Figure 3.18 X-Ray diffraction structure of single crystal of 3.4

6

 6 Model reactions of CF 3 -1,4-disubstituted 1,2,3-triazole based amino acid to couple with natural amino acid.

Scheme 3 . 7

 37 Scheme 3.7 Synthesis of dimer 3.9 (and 3.10) of CF 3 -1,4-disubstituted-1,2,3-triazole-based amino acid unit

Figure 3 .

 3 Figure 3.19 HPLC and LC-MS analysis of the purity of dimer 3.9

Scheme 3 . 9 S 12 Figure 3 .Scheme 3 . 10

 39123310 Scheme 3.9 S Deprotected reactions of the trimer of CF 3 -1,4-disubstituted-1,2,3-triazole-based amino acid scaffold 3.12

Figure 3 .

 3 Figure 3.22 HPLC and LC-MS analysis of the purity of tetramer 3.15

Figure 3 .

 3 Scheme 3.11 Deprotection of the tetramer of CF 3 -1,4-disubstituted-1,2,3-triazole-based amino acid scaffold

Figure 3 . 25

 325 Figure 3.25 Similarity of hydrogen bonding possibilities between KLVFFA and 3.23 (two possible orientations of the triazole rings are represented).

Scheme 3 . 17

 317 Scheme 3.17 Synthesis of peptidomimetics of CF 3 -1,4-triazole-based amino acid scaffolds and natural amino acids (mimic of KLVFFA)

Figure 3 .

 3 Figure 3.27 top (a) and side (b) views of trimer 3.20. Hydrogen bonds found are materialized by yellow lines.

Figure 3 .

 3 Figure 3.28 NOEs of trimer 3.13 (5 mM) recorded on an 800 MHz Spectrometer in CD 3 OH at 283K.

Figure 3 .

 3 Figure 3.30 shows that trimer 3.13 very slightly delayed the lag phase of Aβ 1-42 aggregation. At 3.13/Aβ 1-42 ratios of 10/1 (100 μM/10 μM) and 1/1 (10 μM/10 μM), trimer 3.13 reduced the final fluorescence intensity by about 40% and 20% respectively. This ThT fluorescence assay could indicate that trimer 3.13 probably is a weak inhibitor of Aβ 1-42 aggregation. No selfaggregation was observed even at high concentration (100 μM).

Figure 3 . 30

 330 Figure 3.30 ThT fluorescence assays: representative curves showing fibril formation of Aβ 1-42 (10 μM) in the absence (purple) or in the presence of trimer 3.13 at a 3.13/ Aβ ratio of 1/1 (blue) and 10/1 (green), trimer 3.13 alone (grey, 100 μM).

Figure 3 .

 3 Figure 3.31 ThT fluorescence assays: representative curves showing fibril formation of IAPP (5μM) in the absence (purple) or in the presence of trimer 3.13 at a 3.13/IAPP ratios of 0.1/1 (yellow), 1/1 (blue) and 10/1 (green); trimer 3.13 alone (red, 50 μM).

  Figur (10 μM and 10

16 .

 16 3.13 seems to display a rather extended conformation, tetramer 3.16 displayed very interesting long-range NOE correlations, indicating a rather folded conformation. Molecular dynamics should help us to identify the possible conformation of tetramer 3.The ability of these fluorinated oligomers to modulate the amyloid fibril formation of Aβ 1-42 and IAPP peptides were evaluated by ThT fluorescence assay. It was found that trimer 3.13 was a very weak inhibitor of Aβ 1-42 aggregation and an IAPP aggregation promoter. However, it was shown that tetramer 3.16 can modulate the aggregation of Aβ 1-42 and IAPP but not in classical inhibition or a promotion manner. It delayed the lag phase of Aβ 1-42 aggregation but increased the final fluorescence intensity. In the case of IAPP, tetramer 3.16 had no effect in delaying or decreasing the lag phase, but it increased the final fluorescence intensity, indicating the formation of aggregates of different morphology from IAPP control. Complementary experiments should be done to identify the possible interest of tetramer as potential inhibitor of Aβ 1-42 and/or IAPP toxicity.

1 H

 1 NMR (300 MHz, DMSO-d 6 ) δ 7.47-7.36 (m, 5H), 4.12 (dd, 2H), 3.69 (dd, J = 9.0, 6.2 Hz, 2H), 3.29-3.13 (m,1H), 2.86 (dd, J = 17.0, 9.2 Hz, 1H), 2.29-2.19 (m, 1H), 1.94-1.75 (m, 3H); 13 C NMR (75 MHz, CD 3 OD) δ 172.

1 H 13 C

 113 NMR (300 MHz, CDCl 3 ) δ 11.54 (s, 1H), 8.58 (d, J = 8.2Hz, 1H), 7.80-7.06 (m, 14H), 3.76 (dd, 2H), 3.35-3.22 (m, 2H), 2.41 (dd, J =16.4, 9.0 Hz, 1H), 2.30-2.24 (m, 1H), 2.00-1.78 (m, 2H); NMR (75 MHz, CDCl 3 ) δ 198.

1 H 13 C

 113 NMR (300 MHz, CDCl 3 ) δ 8.27(d, J = 8.6 Hz, 1H), 8.07 (d, J = 7.0 Hz, 2H), 7.50 (m, 3H), 7.42 (t, J = 7.5 Hz, 2H), 7.31 (m, 1H), 7.20 (ddd, J = 8.7, 6.9, 1.7 Hz, 1H), 7.09 (d, J = 7.9 Hz, 1H), 6.97 (m, 1H), 6.79 (dd, J = 8.2, 1.7 Hz, 1H),6.73-6.65 (m, 1H), 4.47 (d, J = 12.6 Hz, 1H), 3.82-3.62 (m, 4H), 3.47 (dd, J = 10.7, 5.5 Hz, 1H), 3.34 (dd, 1H), 2.62-2.35 (m, 2H), 2.14 (m, 2H); NMR (75 MHz, CDCl 3 ) δ 181.

1 H 13 C

 113 NMR (300 MHz, CDCl 3 ) δ 8.23(d, J = 8.7 Hz, 1H), 8.06 (d, J = 7.3 Hz, 2H), 7.55 (m, 3H), 7.36 (m, 3H), 7.19 (m, 2H), 6.92 (d, J = 7.4 Hz, 1H), 6.72-6.56 (m, 2H), 5.58 (d, J = 10.0 Hz, 1H), 4.37 -4.22 (m, 2H), 3.66 (d, J = 8.9 Hz, 1H), 3.58(d, J = 12.7 Hz, 1H), 3.51-3.34 (m, 3H), 2.88- 2.83 (m, 1H), 2.63-2.44 (m, 1H), 2.08-2.03 (m, 2H); NMR (75 MHz, CDCl 3 ) δ 180.

19 F

 19 NMR (188 MHz, CDCl 3 ) δ -72.84 (J = 7.6 Hz); ESI-HRMS: m/z calcd for C 29 H 27 F 3 N 3 NiO 4 [M+H] + : 596.1307, found: 596.1309; m/z calcd for C 29 H 26 F 3 N 3 NiO 4 Na [M+Na] + : 618.1127, found: 618.1145; Melting Point: 148-150 o C (2S, 3S)-2-((tert-butoxycarbonyl)amino)-4,4,4-trifluoro-3-hydroxybutanoic acid (2

1 H 13 C 19 F

 11319 NMR (300 MHz, CDCl 3 ) δ 7.77 (s, 1H), 6.32 (d, J = 9.5 Hz, 1H), 5.72 (d, J = 9.0 Hz, 1H), 4.67(d, J = 9.1 Hz, 1H), 1.43 (s, 9H); NMR (75 MHz, CD 3 OD) δ 172.7, 157.9, 125.9 (m, CF 3 ), 81.2, 70.4 (q, CHCF 3 ),54.7, 28.8; NMR (188 MHz, CDCl 3 ) δ -76.14 ( J = 7.5 Hz); ESI-HRMS: m/z calcd for C 9 H 14 F 3 NO 5 Na [M+Na] + : 296.0722, found: 296.0723;(S)-methyl 2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-4

1 H 13 C

 113 NMR (300 MHz, CDCl 3 ) δ 6.25 (m, 1H, NH), 5.05 (m, 1H, NH), 4.65 (m 1H), 3.90 (m, 1H), 3.74 (s, 3H), 2.11 (m, 1H), 1.66 (m, 3H), 1.46 (s, 9H), 1.00 -0.93 (m, 12H); NMR (75 MHz, CDCl 3 ) δ 173.

1 H 13 C

 113 R f = 0.35 (CH 2 Cl 2 : MeOH =20:1 ); NMR (300 MHz, CDCl 3 ) δ 7.41 (d, J = 8.9 Hz, 1H, NH), 7.01 (d, J = 7.6 Hz, 1H, NH), 5.61 (d, J = 8.4 Hz, 1H, NH), 4.96 (s, 1H), 4.68-4.50 (m, 3H), 4.25 (t, J = 7.6 Hz, 1H), 3.71 (s, 3H), 2.14 (m, 1H), 1.67-1.55 (m, 3H), 1.45 (s, 9H), 0.94-0.90 (m, 12H); NMR (75 MHz, CDCl 3 ) δ 172.0, 166.

2 ; 19 F

 219 NMR (188 MHz, CDCl 3 ) δ -77.19 (J = 7.5 Hz); ESI-HRMS: m/z calcd for C 21 H 37 F 3 N 3 O 7 [M+H] + : 500.2584, found: 500.2584; m/z calcd for C 21 H 40 F 3 N 4 O 7 [M+NH 4 ] + : 517.2865, found: 517.2842; IR ν max (cm -1 ):

R f 1 H 13 C

 f113 =0.20 (CH 2 Cl 2 : MeOH =20:1); NMR (300 MHz, DMSO-d 6 ) δ 8.31 (d, J = 7.2 Hz, 1H), 8.16 (d, J = 9.0 Hz, 1H), 7.48 (d, J = 8.8 Hz, 1H), 6.84 (d, J = 8.7 Hz, 2H), 4.86 (s, 1H), 4.73 (d, J = 8.7 Hz, 1H), 4.52 -4.40 (m, 1H), 4.29-4.21 (m, 2H), 4.10-4.05 (m, 1H), 3.91 (t, J = 8.1 Hz, 1H), 3.59 (s, 3H), 1.98-1.95 (m, 2H), 1.64 -1.44 (m, 3H), 1.37 (s, 9H), 0.91 -0.77 (m, 18H); NMR (75 MHz, DMSO-d 6 ) δ 172.

R f 1 H

 f1 =0.20 (CH 2 Cl 2 : MeOH =20:1 ); NMR (300 MHz, CD 3 OD) δ 4.83 (d, J = 2.3 Hz, 1H), 4.54 (qd, J = 7.1, 2.5 Hz, 1H), 4.45 (dd, J = 9.3, 5.6 Hz, 1H), 4.28 (m, 2H), 4.15 (q, J = 7.2 Hz, 1H), 3.70 (s, 3H), 2.24 -2.07 (m, 2H), 1.72 -1.58 (m, 3H), 1.46 (s, 9H), 1.33 (d, J = 7.1 Hz, 2H), 1.03 -0.89 (m, 18H); 13 C NMR (75 MHz, CD 3 OD) δ 174.

1 H 13 C

 113 NMR (300 MHz, CD 3 OD) δ 4.84(m, 1H), 4.52(d, J = 6.5 Hz, 1H), 4.46 -4.39 (m, 1H), 4.34 (d, J = 7.7 Hz, 1H), 4.29 (d, J = 6.4 Hz, 1H), 4.01 (q, J = 6.6 Hz, 1H),3.69 (s, 3H), 2.25 -1.97 (m, 2H), 1.67-1.60 (m, 3H), 1.51-1.44 (m, 3H), 1.01-0.88 (m, 18H); NMR (100 MHz, CD 3 OD) δ 174.

13 C

 13 , 4.03 (m, 1H), 3.72 (s, 3H), 2.23 (s, 2H), 1.61 (m, 4H), 1.41 (s, 9H), 1.19 (d, J = 6.3 Hz, 3H), 1.02-0.86 (m, 18H); NMR (75 MHz, CDCl 3 ) δ 173.

13 C

 13 , 1.90 (m, 2H), 1.47 (m, 2H), 1.35 (s, 9H), 1.21 (m, 2H), 1.15 (d, J = 7.2 Hz, 3H), 1.05 (d, J = 5.7 Hz, 3H), 0.88-0.74 (m, 18H); NMR (75 MHz, DMSO) δ 173.

1 H 13 C

 113 NMR (300 MHz, MeOD) δ 4.46-4.09 (m, 5H), 3.70 (s, 3H), 2.10 (m, 3H), 1.65 m (3H), 1.44 (s, 9H), 1.30 (d, J = 7.2 Hz, 3H), 1.16 (d, J = 6.4 Hz, 3H), 0.99-0.88 (m, 18H); NMR (75 MHz, MeOD) δ 176.0, 174.

  mg, 1.0 mmol) was added to a round bottom flask with EtOH (2 mL) at 0 o C.

13 C 19 F

 1319 (300 MHz, DMSO-d 6 ) δ 7.91 (s, 1H), 6.06 (d, J = 9.9 Hz, 1H, NH), 5.09 (s, 2H), 4.15 (m, 1H), 3.03-3.06 (d, J = 7.2 Hz, 2H), 0.89 (s, 9H); NMR (75 MHz, DMSO-d 6 ) δ 168.5, 141.5, 124.9, 125.6 (d, J = 285 Hz, CF 3 ), 58.0 (d, J = 30 Hz, CHCF 3 ), 56.2, 50.2, 25.1, 22.2; NMR (188 MHz, DMSO-d 6 ) δ -72.25 (J = 7.5 Hz); ESI-HRMS: m/z calcd for C 11 H 18 F 3 N 4 O 3 S [M+H] + : 343.1052, found: 343.1059; m/z calcd for C 11 H 17 F 3 N 4 O 3 SNa [M+Na] + : 365.0871, found: 365.0873; IR (cm -1 ):

  -((R)-2-(2-(4-((R)-2-((R)-1, 1-dimethylethylsulfinamido)-3, 3, 3-trifluoropropyl)-1H-1, 2, 3-triazol-1-yl) acetamido)-3, 3, 3-trifluoropropyl)-1H-1, 2, 3-triazol-1-yl) acetate (3.9) Chemical Formula: C 20 H 28 F 6 N 8 O 4 S Exact Mass: 590.1858 Molecular Weight: 590.5429

1 H

 1 NMR (300 MHz, DMSO-d 6 ) δ 9.15 (d, J = 9.0 Hz, 1H, NH), 7.97 (s, 1H), 7.86 (s, 1H), 6.08 (d, J = 9.9 Hz, 1H, NH),

  mg, 0.1 mmol) was added to a round bottom flask with EtOH (2.5 mL) at 0 o C.

R f 1 H 13 C

 f113 = 0.30 (CH 2 Cl 2 : MeOH: CH 3 COOH= 90:10:5); NMR (300 MHz, CD 3 OD) δ 7.85 (m, 2H), 5.24 (s, 2H), 5.10(dd, J = 15 Hz, J = 9 Hz, 2H), 4.90(m, 1H), 4.21(m, 1H), 3.25-3.06 (m, 4H), 1.01 (s, 9H); NMR (75 MHz, CD 3 OD) δ 178.

  triazol-1-yl)acetamido)-3,3,3-trifluoropropyl)-1H-1,2,3-triazol-1yl)acetamido)-3,3,3-trifl-uoropropyl)-1H-1,2,3-triazol-1-yl)acetate (3.12) Chemical Formula: C 27 H 35 F 9 N 12 O 5 S Exact Mass: 810.2430 Molecular Weight: 810.6948

1 H 13 C

 113 NMR (400 MHz, DMSO-d 6 ) δ 9.33 (d, J = 9.0 Hz, 1H, NH), 9.17(d, J = 9.0 Hz, 1H, NH), 7.96 (s, 1H), 7.83 (s, 2H), 6.05 (d, J = 9.0 Hz, 1H, NH), 5.37 (s, 2H), 5.26-5.01 (m, 4H), 4.74 (s, 2H),4.17 (m, 3H), 3.71 (s, 2H), 3.44 (m, 2H), 3.16-2.99 (m, 4H), 1.21 (s, 3H), 0.88 (s, 9H); NMR (100 MHz, DMSO-d 6 ) δ 167.

19 F

 19 NMR (188 MHz, DMSO-d 6 ) δ -76.65(d, J = 7.5 Hz), -76.83 (d, J = 7.5 Hz), -76.90 (d, J = 7

  acid compound with 2-(4-((R)-2-(2-(4-((R)-2-(2-(4-((R)-2-amino-3,3,3trifluoropropyl)-1H-1,2,3-triazol-1-yl)acetamido)-3,3,3-trifluoropropyl)-1H-1,2,3-triazol-1yl)acetamido)-3,3,3-trifluoropropyl)-1H-1,2,3-triazol-1-yl)aceticacid (1:1) (3.13) Chemical Formula: C 21 H 23 F 9 N 12 O 4 Exact Mass: 678.1822 Molecular Weight: 678.4709

13 C

 13 NMR (200 MHz, CD 3 OH) δ 169.0, 167.

19 F

 19 NMR (188 MHz, CD 3 OD) δ -77.41 (d, J = 7.5 Hz), -77.61 (d, J = 7.5 Hz), -77.71 (d, J = 7

1 H

 1 NMR (300 MHz, DMSO-d 6 ) δ 9.15 (d, J = 9.0 Hz, 1H, NH), 9.11 (d, J = 9.0 Hz, 1H, NH), 8.98 (d, J = 6.0 Hz, 1H, NH), 7.93 (s, 1H), 7.82 -7.70 (m, 3H), 6.03 (d, J = 6.0 Hz, 1H, NH),

13 C

 13 NMR (75 MHz, DMSO-d 6 ) δ 167.

1 H

 1 NMR (300 MHz, CDCl 3 ) δ 7.59 (s, 1H), 5.25 (d, J = 9 Hz, 1H, NH), 5.14 (s, 2H), 4.59 (s, 1H), 4.56 (m, 1H), 4.27 (q, J = 7.1 Hz, 2H), 3.25-3.10 (m, 2H), 1.42 (s, 9H), 1.31 (t, J = 7.1 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 ) δ 166.

1 H 13 C

 113 NMR (300 MHz, CD 3 OD) δ 7.83 (s, 1H), 5.24 (s, 2H), 4.53-4.48 (m, 1H), 3.21-3.15 (m, 1H), 3.02-2.93 (m, 1H), 1.37(s, 9H); NMR (75 MHz, CD 3 OD) δ 169.

19 F

 19 NMR (188 MHz, CD 3 OD) δ -76.14 (J=7.5 Hz); ESI-HRMS: m/z calcd for C 12 H 18 F 3 N 4 O 4 [M+H] + : 339.1280, found: 339.1281; Melting Point: 146-148 o C; IR (cm -1 ):

1 H 13 C

 113 NMR (300 MHz, CD 3 CN) δ 7.69 (s, 1H), 7.59 (s, 1H), 7.23 (d, J = 9 Hz, 1H, NH), 5.87 (d, J = 9 Hz, 1H, NH), 5.19 (s, 2H), 4.99(m, 2H), 4.89 (m, 1H), 4.54 (m, 1H), 4.23 (q, J = 9 Hz, 6 Hz, 2H), 3.23-2.90 (m, 4H), 1.34 (s, 9H), 1.26 (t, J = 6 Hz, 3H); NMR (75 MHz, CD 3 CN) δ 168.

R f 1 H 13 C

 f113 = 0.30 (CH 2 Cl 2 : MeOH: CH 3 COOH= 90:10:5); NMR (300 MHz, CD 3 OD) δ 7.85 (s, 1H), 7.75 (s, 1H), 5.27 (s, 2H), 5.24-5.08 (m, 2H), 4.89 (m, 1H), 4.48 (m, 1H), 3.28-2.91 (m, 4H), 1.36 (s, 9H); NMR (75 MHz, CD 3 OD) δ 170.

19 F

 19 NMR (188 MHz, CD 3 OD) δ -79.14(d, J = 7.5 Hz), -79.86 (d, J = 7.5 Hz); ESI-HRMS: m/z calcd for C 19 H 25 F 6 N 8 O 5 [M+H] + : 559.1852, found: 559.1854; Melting Point: 172-176 o C; IR (cm -1 ):

1 H 13 C

 113 NMR (300 MHz, DMSO-d 6 ) δ 9.21 (d, J = 9 Hz, 1H, NH), 9.10 (d, J = 9 Hz, 1H, NH), 7.95 (s, 1H), 7.83 (s, 1H), 7.73 (s, 1H), 7.63 (d, J = 9 Hz, 1H, NH), 5.37 (s, 2H), 5.24-5.00 (m, 4H), 4.75 (m, 2H), 4.40 (m, 1H), 4.17 (q, J = 9 Hz, 6 Hz, 2H),3.18-2.96 (m, 6H), 1.31 (s, 9H), 1.21 (t, J = 6 Hz, 3H); NMR (75 MHz, DMSO-d 6 ) δ 167.

0; 19 F

 19 NMR (188 MHz, DMSO-d 6 ) δ -72.54 (d, J = 7.5 Hz), -72.60 (d, J = 7.5 Hz), -73.08 (d, J = 7.5 Hz); ESI-HRMS: m/z calcd for: C 28 H 36

  ethyl

1 H 13 C

 113 NMR (300 MHz, DMSO-d 6 ) δ 9.16 (d, J = 9 Hz, 1H, NH), 9.12 (d, J = 9 Hz, 1H, NH), 9.05 (d, J = 9 Hz, 1H, NH), 7.94 (s, 1H), 7.82 (s, 1H), 7.80 (s, 1H), 7.72 (s, 1H), 7.60 (d, J = 9 Hz, 1H, NH), 5.37 (s, 2H), 5.24-4.99 (m, 6H), 4.74 (br, m, 3H), 4.39 (br, m, 1H), 4.17 (d, J = 9 Hz, 6 Hz, 2H), 3.23-2.92 (m, 8H), 1.32 (s, 9H), 1.21 (t, J = 6 Hz, 3H); NMR (100 MHz, DMSO-d 6 ) δ 169.

19 F

 19 NMR (188 MHz, CD 3 CN) δ -76.04 (d, J = 7.5 Hz), -76.59 (d, J = 7.5 Hz), -76.70 (d, J = 7.5 Hz), -77.39 (d, J = 7.5 Hz); ESI-HRMS: m/z calcd for C 35 H 43

ethyl 2 -

 2 (4-((R)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-phenyl-propanamido)-3,3,3-trifluoropropyl)-1H-1,2,3-triazol-1-yl)acetate (3

1 ; 19 F

 119 NMR (188 MHz, CDCl 3 ) δ -75.44 (d, J = 7.5 Hz); ESI-HRMS: m/z calcd for C 23 H 31 F 3 N 5 O 5 [M+H] + : 514.2277, found: 514.2273; Melting Point: 176-178 o C; IR (cm -1 ):

ethyl 2 -

 2 (4-((R)-2-((S)-6-(((benzyloxy)carbonyl)amino)-2-((tert-butoxycarbonyl)amino)hexane mido)-3,3,3-trifluoropropyl)-1H-1,2,3-triazol-1-yl)acetate (3

1 ; 19 F

 119 NMR (188 MHz, CDCl 3 ) δ -75.78 (d, J = 7.5 Hz); ESI-HRMS: m/z calcd for C 28 H 40 F 3 N 6 O 7 [M+H] + : 629.2911, found: 629.2902; Melting Point: 140-144 o C; IR (cm -1 ):

  .

2 -

 2 (4-((R)-2-((S)-6-(((benzyloxy)carbonyl)amino)-2-((tert-butoxycarbonyl)amino)hexanamido)-3,3,3-trifluoropropyl)-1H-1,2,3-triazol-1-yl)acetic acid (3

7 ; 19 F

 719 NMR (188 MHz, CD 3 OD) δ -77.56 (d, J = 7.5 Hz); ESI-HRMS: m/z calcd for C 26 H 36 F 3 N 6 O 7 [M+H] + : 601.2598, found: 601.2591; Melting Point: 166-170 o C; IR (cm -1 ):

1 H

 1 NMR (300 MHz, D 2 O) δ 7.85 (s, 1H), 7.76 (s, 1H), 7.30 (m, 3H), 7.09 (m, 2H), 5.28 (m, 4H), 5.14-5.12 (m, 2H), 4.59 (m, 2H) 4.17

  -4.15 (m, 2H), 3.92 (s, 2H), 2.71 (s, 1H), 3.34 (m, 2H), 3.07 (m, 2H), 2.88-2.85 (m, 2H), 1.61-1.52 (m, 2H), 1.20 (m, 3H), 1.01 (m, 2H); 13 C NMR (75 MHz, D 2 O) δ 173.

Figure 1 :

 1 Figure 1: Initial conformations of the eight studied pentapeptides: Boc-AVXVL-OMe and NH 3 -AVXVL-OMe, where X = Ser, Thr, (2S, 3R)-CF 3 -Thr and (2S, 3S)-CF 3 -Thr).

  CH 2 CO 2 H 4.87 (CH 3 ) 2 CHOH 17.1 CH 3 CH 2 NH 2 10.6 CH 2 FCO 2 H 2.59 CF 3 CH 2 CO 2 H 3.06 (CF 3 ) 2 CHOH 9.3 CF 3 CH 2 NH 2

		pK a	Compound	pK a	Compound	pK a	Compound	pK b
	CH 3 CO 2 H	4.76 CH 3 5.7
	CH 2 ClCO 2 H 2.87	C 6 H 5 CO 2 H	4.21	(CH 3 ) 3 COH 19.0	C 6 H 5 NH 2	4.6
	CH 2 BrCO 2 H 2.90	C 6 F 5 CO 2 H	1.70	(CF 3 ) 3 COH	5.4	C 6 F 5 NH 2	-0.36
	CHF 2 CO 2 H 1.34	CH 3 CH 2 OH	15.93	C 6 H 5 OH	9.99
	CF 3 CO 2 H	0.52	CF 3 CH 2 OH	12.39	C 6 F 5 OH	5.5
	Table

Table 1 . 5

 15 Overview of spin characteristics of the most important NMR-active nuclei

			1 S -1 ]	γ	S abs	Natural abundance [%]
	1 H	1/2	26.75	1	1	99.99
	19 F	1/2	25.18	1	0.83	100
	13 C	1/2	6.73	0.25	0.000176	1.07
	15 N	1/2	-2.71	0.1	0.00000385	0.37
	31 P	1/2	10.84	0.41	0.0663	100

  It has been widely recognized that alzheimer's disease (AD) represents an unsettling, worldwide challenge for society. AD is the most common form of dementia, accounting for over half of all diagnosed cases. AD is estimated to afflict more than 35 million people worldwide. An estimated 5.4 million Americans of all ages have alzheimer's disease in 2016. This number includes an estimated 5.2 million people age 65 and older and approximately 200,000 individuals under age 65 who have younger-onset alzheimer's. By 2025, the number of people age 65 and older with Alzheimer's disease is estimated to reach 7.1 million, and by 2050, the number may nearly triple, from 5.2 million to a projected 13.8 million.

1.2 Alzheimer's Disease (AD)

Table 2 .

 2 11 H NMR and13 C NMR chemical shifts of pentapeptide 2.10 (16 mM) in CD 3OH (298 K)

	Residue δ NH
	(ppm)

Table 2 . 2

 22 Chemical shift deviations (CSD) of peptide residues of 2.10 (16 mM) in CD 3 OH (298 K) The random coil values of H α protons, C α carbon were calculated using random coil values reported in water[22e] .)

	Ala 1	6.89	4.10	1.28		175.8	51.7	18.0
	Val 2	7.78	4.21	2.04	0.91 , 0.90	173.0	59.7	32.5	19.7, 18.4
	Thr 3 /CF 3	8.59	4.83	4.23	6.98	170.6	54.0	70.6
	Val 4	8.19	4.29	2.15	0.95, 0.92	173.5	59.8	31.8	19.7, 18.2
	Leu 5	8.20	4.41	1.59, 1.59	1.99	174.4	52.4	41.3	25.8,
					0.94, 0.89				23.3, 21.8
		Residue			H α (ppm)			C α (ppm)
		Ala 1			-0.22			-0.8
		Val 2			0.09			-2.5
		Thr 3 /CF 3			-			-
		Val 4			0.17			-2.4
		Leu 5			0.07			-2.7
	(

Table 2 . 3

 23 Temperature coefficients and coupling constants for NH protons of 2.10 in CD 3 OH

	Residue	Δδ/ΔT (ppb K -1 )	3 J HN-Hα (Hz) (271K)	φ torsional angles (271K)	3 J HN-Hα (Hz) (298K)	φ torsional angles (298K)
	Ala 1	-6.5	7.1	-158	7.0	-159
	Val 2	-5.3	8.9	-147	8.9	-147
	Thr 3 /CF 3	-7.4	9.1	-145	8.6	-149
	Val 4	-7.4	9.2	-144	overlap	-
	Leu 5	-5.0	7.6	-155	overlap	-

Table 2 .

 2

	4 1 H NMR and 13 C NMR chemical shifts of pentapeptide 2.11 (16 mM) in CD 3 OH (298 K)
	Residue δ NH
	(ppm)

Table 2 . 5

 25 Chemical shift deviations (CSD) of peptide residues of 2.11(16 mM) in CD 3 OH (298 K)

	Ala 1	6.94	4.09	1.30		176.2	51.9	18.0
	Val 2	7.82	4.21	2.10	0.97, 0.95	173.6	60.2	32.2	19.8, 18.5
	Ser 3	8.12	4.48	3.78,		172.4	56.7	63.2
				3.74
	Val 4	7.87	4.27	2.15	0.97, 0.95	173.7	60.1	31.9	19.7, 18.3
	Leu 5	8.31	4.43	1.60,	1.68,	174.4	52.3	41.3	25.8,
				1.60	0.94, 0.89				23.3, 21.8
		Residue			H α (ppm)			C α (ppm)
		Ala 1			-0.23			-0.6
		Val 2			0.09			-2.0
		Ser 3			0.01			-1.6
		Val 4			0.15			-2.1
		Leu 5			0.09			-2.8
	(The random coil values of H α protons, C α carbon were calculated using random coil values
	reported in water [22e] .)

Table 2 . 6

 26 Temperature coefficients and coupling constants for NH protons of 2.11 in CD 3 OH

	Residue	Δδ/ΔT (ppb K -1 )	3 J HN-Hα (Hz) (298K)	φ torsional angles (298 K)	3 J HN-Hα (Hz) (271 K)	φ torsional angles (271 K)
	Ala 1	-7.8	broad	-	6.8	-160
	Val 2	-6.4	7.7	-154	8.2	-151
	Ser 3	-7.7	broad	-	7.5	-156
	Val 4	-8.0	7.1	-158	8.9	-147
	Leu 5	-7.4	7.4	-156	7.8	-154

Table 2 .

 2

	Ala 1	6.86	4.13	1.30		176.4	51.7	18.1
	Val 2	7.95	4.25	2.15	0.95, 0.98	173.6	60.6	31.8	18.3, 19.8
	Thr 3 /CF 3 Val 4	-7.69	4.80 4.27	4.52 2.11	0.95, 0.97	170.5 173.2	54.3 60.3	69.9 32.4	18.8, 19.6
	Leu 5	8.31	4.43	1.61	1.67, 0.94, 0.88 174.3	52.2	41.3 25.8, 23.3, 21.7
									3 OH (298K)
		Residue			H α (ppm)			C α (ppm)
		Ala 1			-0.19			-0.8
		Val 2			0.13			-1.6
		Thr 3 /CF 3 Val 4			-			-

8 Chemical shift deviations (CSD) of peptide residues of 2.18

(16 mM)

in CD

Table 2 . 9

 29 Temperature coefficients and coupling constants for NH protons of 2.18 in CD 3 OH

	Residue	Δδ/ΔT (ppb K -1 )	3 J HN-Hα (Hz) (271 K)	φ torsional angles (271 K)	3 J HN-Hα (Hz) (298 K)	φ torsional angles (298 K)
	Ala 1	-7.4	6.8	-160	5.8	-165
	Val 2	-5.9	7.7	-154	broad	-
	Thr 3 /CF 3 Val 4	-8.9 -4.5	8.6 9.0	-149 -146	n.d. broad	--
	Leu 5	-9.0	7.7	-154	7.8	-154

Table 2 .

 2 The random coil values of H α protons, C α carbons were calculated using random coil values reported in water[22e] .)

			97	1.48		171.7	50.4	18.1
	Val 2	8.42	4.34	2.15	0.96, 0.99	173.3	60.2	31.7	18.5, 19.9
	Thr 3 /CF 3 8.24 Val 4 7.67	4.84 4.29	4.52 2.07	0.95, 0.98	170.3 173.2	54.1 59.8	70.0 32.8	18.6, 19.5
	Leu 5	8.51	4.42	1.60	1.66, 0.93, 0.87 174.3	52.3	41.3 25.9, 23.3, 21.8
								3 OH (298 K)
		Residue			H α (ppm)			C α (ppm)
		Ala 1			-0.35			-2.1
		Val 2			0.22			-2.0
		Thr 3 /CF 3 Val 4			-0.17			--2.4
		Leu 5			0.08			-2.8
	(

11 Chemical shift deviations (CSD) of peptide residues of 2.19

(16 mM) in CD

Table 2 .

 2 12 Temperature coefficients and coupling constants for NH protons of 2.19 in CD 3 OH

	Residue	Δδ/ΔT (ppb K -1 )	3 J HN-Hα (Hz) (271 K)	φ torsional angles (271 K)	3 J HN-Hα (Hz) (298 K)	φ torsional angles (298 K)
	Ala 1	-	-	-	-	-
	Val 2	-5.5	8.9	-147	broad	-
	Thr 3 /CF 3	-8.3	9.0	-146	8.9	-147
	Val 4	-4.6	8.9	-147	8.8	-147
	Leu 5	-7.9	7.6	-155	7.6	-155

Table 2 .

 2 131 H NMR and13 C NMR chemical shifts of pentapeptide 2.20(16 mM) in CD 3 OH (298 K)

	Residue δ NH
	(ppm)

Table 2 .

 2

	Ala 1	7.09	4.10	1.29		176.1 51.6	18.1
	Val 2	8.04	4.25	2.07	0.96, 0.95	173.7 60.2	32.1	19.8, 18.6
	Thr 3	8.17	4.39	4.07	1.13	172.2 60.0	68.5	19.9
	Val 4	8.08	4.23	2.08	0.98, 0.96	173.7 60.0	32.1	19.7, 18.6
	Leu 5	8.59	4.40 1.63,1.57 1.67, 0.94, 0.88 174.3 52.2	41.2	25.8, 23.4, 21.7
								3 OH (298 K)
		Residue			H α (ppm)		C α (ppm)
		Ala 1			-0.2			-0.9
		Val 2			0.13			-2.0
		Thr 3			0.04			-1.8
		Val 4			0.13			-2.2
		Leu 5			0.11			-2.9
	(The random coil values of H α protons, C α carbons were calculated using random coil values
	reported in water [22e] .)

14 Chemical Shift deviations (CSD) of peptide residues of 2.20

(16 mM) in CD

Table 2 .

 2 15 Temperature coefficients and coupling constants for NH protons of 2.20 in CD 3 OH

	Residue	Δδ/ΔT (ppb K -1 )	3 J HN-Hα (Hz) (298K)	φ torsional angles (298K)
	Ala 1	-7.4	6.9	-159
	Val 2	-5.8	8.3	-151
	Thr 3	-7.5	8.4	-150
	Val 4	-7.6	8.8	-147
	Leu 5	-7.9	7.8	-154

Table 2 .

 2

	16 1 H NMR and 13 C NMR chemical shifts of pentapeptide 2.21 (16 mM) in CD 3 OH (288 K)
	Residue δ NH
	(ppm)

Table 2 .

 2 18 Temperature coefficients and coupling constants for NH protons of 2.21 in CD 3 OH

	Residue	Δδ/ΔT (ppb K -1 )	3 J HN-Hα (Hz) (271 K)	φ torsional angles (271 K)	3 J HN-Hα (Hz) (298 K)	φ torsional angles (298 K)
	Ala 1	-	-	-	-	-
	Val 2	-5.5	broad peak	-	-	-
	Thr 3	-8.1	8.4	-150	8.3	-150
	Val 4	-8.0	8.7	-148	8.7	-148
	Leu 5	-7.7	7.7	-154	7.8	-154

Table 2 .

 2

	19 1 H NMR and 13 C NMR chemical shifts of pentapeptide 2.34 (16 mM) in CD 3 OH (298K)
	Residue δ NH
	(ppm)

Table 2 .

 2 20 Chemical shift deviations (CSD) of peptide residues of 2.34(16 mM) in CD 3OH (298K)

			97	1.51	-	171.7 50.4	18.1
	Val 2	8.33 b	4.26	2.12	0.99, 0.97 173.3 60.5	32.0	19.8, 18.6
	Ser 3	8.15	4.51	3.78, 3.74	-	172.3 56.5	63.3
	Val 4	7.91	4.27	2.13	0.98, 0.94 173.7 60.0	32.0	19.6, 18.3
	Leu 5	8.39	4.42	1.61	1.67,	174.4 52.3	41.3	25.9,
					0.94, 0.89			23.3, 21.8
	b broad signal
		Residue			H α (ppm)		C α (ppm)
		Ala 1			-0.35		-2.1
		Val 2			0.14		-1.7
		Ser 3			0.04		-1.8
		Val 4			0.15		-2.2
		Leu 5			0.08		-2.8
	(The random coil values of Hα protons, Cα carbon were calculated using random coil values
	reported in water [22e] .)

Table 2 .

 2 21 Temperature coefficients and coupling constants for NH protons of 2.34 in CD 3 OH Overall, the theoretical 3 J HN-Hα coupling values are in fair agreement with the experimental ones, indicating that the peptide conformational ensembles were sampled quite faithfully by the MD trajectories. Excepting the first residue Ala 1 , all the theoretical coupling constants have high values above 7 Hz, confirming that the pentapeptides have locally extended backbone conformations. It could be noted that the 3 J HN-Hα experimental value of the central residue is

	Residue	Δδ/ΔT (ppb K -1 )	3 J HN-Hα (Hz) (298 K)	φ torsional angles (298 K)
	Ala 1	-	-	-
	Val 2	-6.1	8.3 b	-151 b
	Ser 3	-7.8	7.6	-155
	Val 4	-7.8	8.6	-149
	Leu 5	-7.0	7.6	-155
	b at 271 K

Table

  .22).

			BOC-Protected			Non-Protected
	peptide
		Val 2	X 3	Val 4	P	Val 2	X 3	Val 4	P
	AVSVL	59.9	51.4	55.8	17.2	76.5	50.3	64.9	25.0
	AVTVL	53.5	91.1	61.0	29.7	64.8	73.5	59.1	28.2
	AV-(3R)-CF 3 -VL	59.7	31.7	69.9	13.2	63.3	59.4	74.7	28.1
	AV-(3S)-CF 3 -VL	67.1	82.7	75.6	42.0	77.9	85.6	74.8	49.9

  2 nm. Visual inspections of the representative structure of the most populated clusters(Figures 2.20 and 2.21) confirm that the AV-(3S)-CF 3 -VL (2.18 and 2.19) sequence visits extended β-strand-like structures more frequently than the other three which have higher propensities to form compact α-helix-like conformations.

	Figur re 2.20 Repre	sentative struc ctures of the	five most pop pulated cluste	rs of the BOC
	peptid des.
	Figur re 2.21 Repre esentative stru uctures of the	five most po opulated clust ers of the no
	peptid des.
	In ord der to investiga ate in more de etail on the ph hysical factors s that favor the e extended stru
	compu uted the distri ibution functio ons of several l distances be etween nonpol lar groups of
	side c chains (Figur re 2.22). The ese data high hlight importa ant contacts i in the AV-(
			67

11, 2.18, 2.19, 2.20, 2.21 and 2.34) in

  2D NMR spectroscopy and molecular dynamics simulations. NMR conformational studies indicate that the 7 pentapeptides (2.10, 2.CD 3 OH adopt mainly extended backbone conformations. The MD simulated conformations were in good agreement with the NMR results.

It is found both experimentally and theoretically that, pentapeptides 2.11 and 2.34 bearing L-serine are extended to a lesser extent than their analogues containing L-threonine (2.20 and 2.21) and CF 3 -analogues (2.10, 2.33, 2.18 and 2.19); and the BOC-protected

(2.18)

and non-protected AV-(2S, 3S)-CF 3 -VL

(2.19)

are more extended than L-threonine-containing pentapeptides (2.20 and 2.21) and (2S, 3R)-CF 3 -Thr pentapeptides (2.10, 2.33). It is also observed that the (2S, 3S)-CF 3 -Thr pentapeptide (2.18 and 2.19) have a propensity to self-associate of by forming intermolecular β-strand contacts, as suggested by temperature coefficients. This higher propensity of 2.18 and 2.19 to adopt extended structures can be explain by a stronger hydrophobic interaction of its trifluoromethyl group with the Leu 5 side chain with respect to the one involving the threonine methyl group, as observed in MD. Thus, both conformational studies demonstrated the trifluoromethyl effect on peptide conformations that promotes an extended conformation in order to mimic a β-strand structure. Interestingly in the MD results, we find that the deprotected pentapeptides

(2.19, 2

.21, 2.33 and 2.34) all

show increased propensities to adopt extended conformations compared to the Boc-protected counterparts

(2.18, 2

.20, 2.10 and 2.11, respectively

).

The ability of compounds 2

.11, 2.18, 2.19, 2.34, 2.20 and 2.21 to

  inhibit amyloid fibril formation was evaluated on Aβ 1-42 and IAPP peptides by ThT fluorescence assay. It was found that none of these pentapeptides have any inhibition effect in Aβ 1-42 peptide and IAPP aggregation. Compounds 2.11, 2.19 and 2.34 showed an acceleration effect in IAPP aggregation at the pentapeptide/IAPP ratio of 10/1 and 1/1 (except 2.19 at 10/1). The Boc protected pentapeptides containing (2S, 3R)-CF 3 -Thr and (2S, 3S)-CF 3 -Thr (2.10 and 2.18) were found to self-aggregate at high concentration (100 μM), probably forming β-sheet rich structures because the aggregates are able to bind ThT (Which is consistent with NMR observations at least for compound 2.18.).

Table 3 .

 3

		a (ppm)	b (ppm)	c (ppm)	e (ppm)	f (ppm)
	triazole 1	-	4.54	3.16, 3.42		7.87	5.10, 5.26
	triazole 2	9.13	4.91	3.04, 3.28		7.81	5.08, 5.25
	triazole 3	9.15	4.96	3.06, 3.33		7.90	5.29
		2 1 H chemical shifts for trimer 3.13 (5 mM) in CD 3 OH at 283K
	Residue	b (ppm)	c (ppm)	d (ppm)	e (ppm)	f (ppm)	g (ppm)
	triazole 1	52.2	22.8	139.6	125.9		51.1	167.4
	triazole 2	50.9	23.6	141.3	125.7		50.9	167.0
	triazole 3	50.8	23.9	141.5	125.2		50.7	169.3

Table 3 .3 13

 3

C NMR chemical shifts for trimer 3.13 (5 mM) in CD 3 OH at 283K

Table 3 .

 3 6 ppb K -1 ), which probably can preclude the existence of stable intra/inter-molecular hydrogen bonds.

	temperature	NH a1	NH a2	NH a3
	283 K	-	9.11	9.13
	293 K	-	9.05	9.07
	298 K	-	9.02	9.04
	303 K	-	9.01	8.99
	Δδ/ΔT (ppb K -1 )	-	-5.0	-6.0

4

Temperature coefficients of NH protons of trimer 3.13

(2 mM)

in CD 3 OH at 800M Hz.

Table 3 .

 3 .5 and 3.6).

	Residue	a (ppm)	b (ppm)	c (ppm)	e (ppm)	f (ppm)
	triazole 1	8.97, 9.06	4.15	3.02, 3.27	7.78	4.88, 5.09
	triazole 2	8.77	4.80	3.06, 3.28	7.62	5.08, 5.20
	triazole 3	9.26	4.79	3.00, 3.23	8.03	5.13, 5.25
	triazole 4	9.18	4.93	3.02, 3.31	7.83	5.08

5 1 H chemical shifts for trimer 3.16 (2 mM) in CD 3 OH at 283K

Table 3 .

 3 613 C NMR chemical shifts for trimer 3.16 (2 mM) in CD 3 OH at 283K

Table 3 . 7

 37 , H e4 and NH a4 , H e3 and NH a3 , and H e2 with other protons. The most important and distinct long-rang ROEs are the H e2 and NH a2 , H e2 and H b3 , H e2 and H e3 , H e2 and H b4 , H c2 and H b4 . These distinct long-rang ROEs indicate that probably tetramer 3.16 adopts a rather folded conformation.The temperature coefficients of the amide protons in tetramer 3.16 were also conducted between 278 and 313K (Table3.7), and the values were more dispersed than those of the trimer 3.13. The Δδ HN /ΔT value of NH a2 (-2.0 ppb K -1 ) was very different from the other NH Δδ HN /ΔT values (-9.0 and -6.0 ppb K -1 ). The relatively small Δδ HN /ΔT value of NH a2 (-2.0 ppb K -1 ) indicated that it could be engaged in hydrogen bonding. The high values for NH a3 and NH a4 indicate that they are not engaged in stable intra/inter-molecular hydrogen bonds. Temperature coefficients of NH protons of tetramer 3.16(2 mM) in CD 3 OH at 800M Hz.Molecular dynamics simulations using water model are in course (by Prof. Tap Ha-Duong, BioCIS), with or without NMR constrained in order to identify the possible folded conformations of tetramer 3.16.

	Figure 3.29 Long-range ROEs of tetramer 3.16 (2 mM) recorded on an 800 MHz Spectrometer in
	CD 3 OH at 283K.

  -HRMS: m/z calcd for C 12 H 16 NO 2 [M+H] + : 206.1181, found: 206.1183; m/z calcd for C 12 H 15 NO 2 Na [M+H] + : 228.1000, found: 228.0999;

	(S)-N-(2-benzoylphenyl)-1-benzylpyrrolidine-2-carboxamide (2.23)
	Chemical Formula: C 25 H 24 N 2 O 2
	Exact Mass: 384.1838
	Molecular Weight: 384.4703
	Melting Point: 178-180 o C

; ESI-HRMS: m

  /z calcd for C 25 H 25 N 2 O 2 [M+H] + : 385.1916, found: 385.1917; Melting Point: 96-98 o C.

	Chemical Formula: C 27 H 25 N 3 NiO 3
	Exact Mass: 497.1249
	Molecular Weight: 498.1991
	A solution of KOH

; ESI-HRMS: m

  /z calcd for C 27 H 26 N 3 NiO 3 [M+H] + : 498.1328, found: 498.1348; m/z calcd for C 27 H 25 N 3 NiO 3 Na [M+Na] + : 520.1147, found: 520.1164;

	Chemical Formula: C 29 H 26 F 3 N 3 NiO 4
	Exact Mass: 595.1229
	Molecular Weight: 596.2230
	Melting Point: 218-220 o C

oate (2.29)

	Chemical Formula: C 26 H 45 F 3 N 4 O 8
	Exact Mass: 598.3189
	Molecular Weight: 598.6527

  ;

	19 F NMR (188 MHz, CD 3 OD) δ -78.65 (d, J = 7.5 Hz);
	ESI-HRMS: m/z calcd for C 24 H 43 F 3 N 5 O 7 [M+H] + : 570.3115, found: 570.3105;
	m/z calcd for C 24 H 42 F 3 N 5 O 7 Na [M+H] + : 592.2934, found: 592.3028;
	Elementary Analysis: For C 24 H 43 ClF 3 N 5 O 7 .2H 2 O,
	Calculation: C, 44.89%; H, 7.39%; O, 10.91%;
	Experiment: C, 44.69%; H, 7.13%; O, 10.85%;
	IR ν max (cm -1 ):

oate (2.30)

	Chemical Formula: C 28 H 45 N 3 O 7
	Exact Mass: 535.33
	Molecular Weight: 535.68
	The dipeptide 2.27 (1.03 g, 3 mmol) was dissolved in CH 2 Cl 2

  Cl 2(5 mL), then to the solution was added CF 3COOH (300 µL, 3.92 mmol, 53 eq.). The reaction mixture was stirred at room temperature overnight. After that time, all the solvents were evaporated under reduced pressure, and then a little amount of Et 2 O was added to precipitate the product. Et 2 O of the mother liquid was evaporated and precipitation by addition of Et 2 O was repeated several times until the mass of the crude did not change any more. The resulting residue was obtained as a white solid with quantitative yield.

	tetraazapentadecan-1-oate 2,2,2-trifluoroacetate (2.21)
	Chemical Formula: C 24 H 45 N 5 O 7
	Exact Mass: 515.33
	Molecular Weight: 515.65
	Elemental Analysis: C, 55.90; H, 8.80; N, 13.58; O, 21.72
	The pentapeptide (45 mg, 0.073 mmol) was dissolved in CH 2 1 H NMR (400 MHz, D 2 O) δ 4.44-4.41 (m, 2H), 4.27-4.24 (m, 2H), 4.09 (t, J = 8 Hz, 1H), 4.01 (q,
	J = 8 Hz, 1Hz, 1H), 3.68 (s, 3H), 2.14-2.07 (m, 2H), 1.67-1.64 (m, 1H), 1.62-1.58 (m, 2H), 1.49 (d,
	J = 4 Hz, 3H), 1.14 (d, J = 4 Hz, 3H), 0.98-0.87 (m, 18H);
	13 C NMR (100 MHz, D 2 O) δ 174.

  To the solution of CF 3 -imine 2.02 g(10 mmol) and propargyl bromide 2.24 g (15 mmol) in dry DMF (10 mL) was added Zn (960 mg, 15 mmol) under N 2 at room temperature, the reaction stirred for 3 hours under N 2 . Saturated NH 4 Cl solution (10 mL) was added to quench the reaction, and then diethyl ether (20 mL) was added, the organic phase was dried over MgSO 4 and then was concentrated under reduced pressure. The crude residue was purified by column chromatography using cyclohexane: EtOAc = 80:20 as eluent. The product 3.3 was obtained as colorless oil in 92% yield.

	4.3 Experimental Part for Fluorinated Triazole Oligomers
	(R)-2-methyl-N-((R)-1, 1, 1-trifluoropent-4-yn-2-yl) propane-2-sulfinamide (3.3)
	Chemical Formula: C 9 H 14 F 3 NOS
	Exact Mass: 241.0748
	Molecular Weight: 241.2738
	R f = 0.25 (Cyclohexane: EtOAc = 80:20);
	Yield: 92%
	1 H NMR (300 MHz, CD 3 Cl) δ 3.85-3.81 (m, 2H), 2.70 (dd, J = 4.6, 2.7 Hz, 1H), 2.63 (dd, J = 6.7,
	2.6 Hz, 1H), 2.04 (m, 1H), 1.19 (s, 9H);
	91; C NMR (75 MHz, CD 3 Cl) δ 124.5 (q, J = 281.3 Hz, CF 3 ), 77.7, 72.2, 57.5, 56.1 (q, J = 30.0 Hz, 13
	ESI-HRMS: m/z calcd for C 24 H 45 N 5 O 7 Na [M+Na] + : 538.3217, found: 538.3220; CHCF 3 ), 22.5, 20.7;
	m/z calcd for C 24 H 46 N 5 O 7 [M+H] + : 516.3397, found: 516.3384; F NMR (188 MHz, CD 3 Cl): δ -75.3 (d, J = 7.5 Hz); 19
	ESI-HRMS: m/z calcd for C 9 H 15 F 3 NOS [M+H] + : 242.0826, found: 242.0831;
	m/z calcd for C 9 H 14 F 3 NOSNa [M+Na] + : 264.0646, found: 264.0658;
	IR (cm -1 ): 3242

  The reaction was stirred at room temperature until completion (TCL). The solution was diluted with brine (20 mL) and the aqueous layer was extracted with AcOEt (20 mL x3). The combined organic layers were dried over MgSO 4 and then concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using cyclohexane: EtOAc = 1:2 as eluent. Compound 3.4 (2.81 g, 7.6 mmol) was obtained as a white solid in 76% yield.

	Ethyl 2-(4-((R)-2-((R)-1, 1-dimethylethylsulfinamido)-3, 3, 3-trifluoropropyl)-1H-1, 2, 3-triazol-1-
	yl) acetate (3.4)
	Chemical Formula: C 13 H 21 F 3 N 4 O 3 S
	Exact Mass: 370.1286
	Molecular Weight: 370.3910
	To a solution of azide (1.56 g, 12 mmol) and trifluoromethyl propargylamine (2.41 g, 10 mmol) in
	acetonitrile (10 ml) was added a catalytic amount of CuI (190 mg, 10 mol%) and NEt 3 (1.01 g, 10
	mmol). R f = 0.20 (Cyclohexane: EtOAc = 1:2);
	[α] D = + 65 (32 o C, 0.01, MeOH);
	1 H NMR (300 MHz, CDCl 3 ) δ 7.68 (s, 1H), 5.15 (s, 2H), 4.24-4.35 (m, 2H), 4.21 (m, 1H), 3.21-
	3.31 (m, 2H), 1.29 (t, J = 7.2 Hz, 3H), 1.13 (s, 9H);
	13 C NMR (75 MHz, CDCl 3 ) δ 166.
	687;

  .1;

	19 F NMR (188 MHz, CDCl 3 ) δ -74.8 ( J = 7.6 Hz);
	ESI-HRMS: m/z calcd for C 13 H 22 F 3 N 4 O 3 S [M+H] + : 371.1365, found: 371.1366;
	m/z calcd for C 13 H 21 F 3 N 4 O 3 SNa [M+Na] + : 393.1184, found: 393.1194;
	Melting Point: 132-134 o C;
	IR (cm -1 ):

  ;

	19 F NMR (188 MHz, DMSO-d 6 ) δ -72.24 (d, J =7.5 Hz), -72.52 (d, J =7.5 Hz);
	ESI-HRMS: m/z calcd for C 20 H 29 F 6 N 8 O 4 S [M+H] + : 591.1937, found: 591.1949;
	m/z calcd for C 20 H 28 F 6 N 8 O 4 SNa [M+Na] + : 613.1756, found: 613.1760;
	Melting Point: 234-238 o C;
	IR (cm -1 ):

  ;

	19 F NMR (188 MHz, CD 3 OD) δ -76.6(d, J=7.6 Hz), -76.9(d, J=7.6 Hz);
	ESI-HRMS: m/z calcd for C 18 H 25 F 6 N 8 O 4 S [M+H] + : 563.1624, found: 563.1624;
	m/z calcd for C 18 H 24 F 6 N 8 O 4 SNa [M+Na] + : 585.1443, found: 585.1443;
	IR (cm -1 ): 3230

  C 27 H 36 F 9 N 12 O 5 S [M+H] + : 811.2509, found: 811.2517; m/z calcd for C 27 H 35 F 9 N 12 O 5 SNa [M+Na] + : 833.2328, found: 833.2342; m/z calcd for C 27 H 35 F 9 N 12 O 5 SK [M+K] + : 849.2277, found: 849.2286;

	Melting Point: 240 -244 o C;
	IR (cm -1 ):

  yl)acetate(3.15)

	Chemical Formula: C 34 H 42 F 12 N 16 O 6 S
	Exact Mass: 1030.3002
	Molecular Weight: 1030.8467
	Compound 3.11

  ;

	19 F NMR (188 MHz, DMSO-d 6 ) δ -72.29, -72.59 (1:3, 4 CF 3 );
	ESI-HRMS:
	m/z calcd for C 34 H 43 F 12 N 16 O 6 [M+H] + : 1031.3081, found: 1031.3059;
	m/z calcd for C 34 H 42 F 12 N 16 O 6 Na [M+Na] + : 1053.2900, found: 1053.2864;
	Melting Point: 268-272 o C;
	IR (cm -1 ): 3229

  acid (1:1)(3.16)

	Chemical Formula: C 28 H 30 F 12 N 16 O 5
	Exact Mass: 898.2393
	Molecular Weight: 898.6228

  Hz); ESI-HRMS: m/z calcd for C 28 H 31 F 12 N 16 O 5 [M+H] + : 899.2472, found: 899.2465; m/z calcd for C 28 H 30 F 12 N 16 O 5 Na [M+Na] + : 921.2291, found: 921.2292; IR (cm -1 ):

  (1.48 g, 4.0 mmol) was dissolved in CH 2 Cl 2 (30 mL) at r.t, and then a solution of HCl (

	amino)-3,3,3-trifluoropropyl)-1H-1,2,3-
	triazol-1-yl)acetate (3.17)
	Chemical Formula: C 14 H 21 F 3 N 4 O 4
	Exact Mass: 366.1515
	Molecular Weight: 366.3361
	Compound 3.4

; 19 F

 19 NMR (188 MHz, CD 3 CN) δ -76.64(d, J = 7.5 Hz), -76.34 (d, J = 7.5 Hz); ESI-HRMS: m/z calcd for C 21 H 29 F 6 N 8 O 5 [M+H] + : 587.2165, found: 587.2166; m/z calcd for C 21 H 28 F 6 N 8 O 5 Na [M+Na] + : 609.1985, found: 609.1989;

	Melting Point: 208-210 o C;
	IR (cm -1 ):

  yl)acetic acid (3.21)

	Chemical Formula: C 19 H 24 F 6 N 8 O 5
	Exact Mass: 558.1774
	Molecular Weight: 558.4349

  F 9 N 12 O 6 [M+H] + : 807.2737, found: 807.2736; m/z calcd for: C 28 H 35 F 9 N 12 O 6 Na [M+Na] + : 829.2557, found: 829.2557;

	Melting Point: 248-250 o C;
	IR (cm -1 ):

  F 12 N 16 O 7 [M+H] + : 1027.3309, found: 1027.3334; m/z calcd for C 35 H 42 F 12 N 16 O 7 Na [M+Na] + : 1049.3128, found: 1049.3142;

	Melting Point: 240-242 o C;
	IR (cm -1 ):

Table II : Fractional atomic coordinates for C 13 H 21 F 3 N 4 O 3 S

 II

	Atom	x/a	y/b	z/c	U(eqv)
	S(1)	0.43427(7) 0.82603(7) 0.50369(5)	0.0263
	C(1)	0.6124(3) 0.79164(14) 0.6723(2)	0.0314
	C(2)	0.8767(4) 0.79016(19) 0.6570(2)	0.0465
	C(3)	0.5644(5) 0.85331(16) 0.7810(3)	0.0450
	C(4)	0.5224(4) 0.71211(15) 0.7103(2)	0.0396
	C(5)	0.3155(4) 0.74273(13) 0.2666(2)	0.0311
	C(6)	0.4049(5) 0.77886(16) 0.1370(2)	0.0440
	C(7)	0.2607(4) 0.65623(14) 0.2384(2)	0.0370
	C(8)	0.1355(4) 0.61959(13) 0.3500(2)	0.0308
	C(9)	0.2075(4) 0.55998(13) 0.4422(2)	0.0324
	C(10)	-0.0080(4) 0.48859(14) 0.6193(3)	0.0405
	C(11)	0.0197(4) 0.52209(15) 0.7686(3)	0.0390
	C(12)	-0.0451(5) 0.48787(19) 1.0045(3)	0.0579
	C(13)	-0.2703(7) 0.5268(2) 1.0234(4)	0.0692
	O(1)	0.5505(3) 0.90036(11) 0.46955(18)	0.0412
	O(2)	0.0865(4) 0.58641(13) 0.8041(2)	0.0626
	O(3)	-0.0442(3) 0.46702(12) 0.85597(19)	0.0498
	N(1)	0.4837(3) 0.75380(11) 0.39659(18)	0.0266
	N(2)	-0.0894(4) 0.64061(13) 0.3681(2)	0.0442
	N(3)	-0.1602(3) 0.59560(13) 0.4670(3)	0.0454
	N(4)	0.0202(3) 0.54666(12) 0.5118(2)	0.0343
	F(1)	0.4639(4) 0.85306(11) 0.15674(18)	0.0610
	F(2)	0.5964(3) 0.74178(12) 0.10366(17)	0.0618
	F(3)	0.2354(3) 0.77561(13) 0.02128(15)	0.0711

Table S1 : Anisotropic thermal parameters for C 13 H 21 F 3 N 4 O 3 S

 S1

	Atom	u(

Table S2 : Hydrogen atoms fractional atomic coordinates for C 13 H 21 F 3 N 4 O 3 S

 S2

	Atom	x/a	y/b	z/c	U(iso)		)
	S(1) H(1)	0.02667(19) 0.0193(2) 0.0336(2) 0.6350 0.7414 0.3989 0.0550(18) 0.00016(18) 0.00678(14) 0.00015(17)
	C(1) H(21)	0.0312(9) 0.0358(10) 0.0274(9) 0.0027(8) 0.9681 0.7717 0.7441 0.0550(18)	0.0047(7)	-0.0006(8)
	C(2) H(22)	0.0327(11) 0.0643(15) 0.0416(11) 0.0027(11) 0.9269 0.8406 0.6363 0.0550(18)	0.0028(9)	-0.0034(10)
	C(3) H(23)	0.0546(13) 0.0450(14) 0.0370(11) -0.0073(9) 0.9000 0.7552 0.5812 0.0550(18)	0.0123(10) -0.0100(10)
	C(4) H(31)	0.0476(12) 0.0339(12) 0.0383(11) 0.0064(9) 0.6501 0.8397 0.8728 0.0550(18)	0.0099(9)	0.0023(10)
	C(5) H(32)	0.0363(9) 0.0281(11) 0.0287(9) 0.0025(8) 0.6156 0.9022 0.7523 0.0550(18)	0.0047(7)	0.0046(8)
	C(6) H(33)	0.0581(14) 0.0407(16) 0.0344(11) 0.0093(9) 0.3979 0.8543 0.7857 0.0550(18)	0.0103(10) 0.0075(11)
	C(7) H(41)	0.0443(11) 0.0339(11) 0.0328(10) -0.0064(8) 0.6142 0.6936 0.7972 0.0550(18)	0.0065(8) -0.0011(9)
	C(8) H(42)	0.0330(9) 0.0243(10) 0.0344(10) -0.0074(8) 0.3588 0.7152 0.7220 0.0550(18)	0.0036(7) -0.0024(8)
	C(9) H(43)	0.0282(9) 0.0241(10) 0.0461(12) -0.0029(8) 0.5378 0.6755 0.6355 0.0550(18)	0.0093(8)	0.0023(8)
	C(10) H(51)	0.0445(12) 0.0248(11) 0.0553(13) 0.0011(9) 0.1703 0.7674 0.2784 0.0550(18)	0.0176(10) -0.0009(9)
	C(11) H(71)	0.0314(10) 0.0323(12) 0.0536(13) 0.0027(10) 0.0075(9) -0.0050(9) 0.1619 0.6505 0.1478 0.0550(18)
	C(12) H(72)	0.0703(18) 0.0537(17) 0.0484(15) 0.0037(11) 0.0047(12) -0.0038(13) 0.4073 0.6287 0.2366 0.0550(18)
	C(13) H(91)	0.090(2) 0.3570	0.061(2) 0.5328	0.0591(18) -0.0044(14) 0.0182(16) 0.0063(16) 0.4547 0.0550(18)
	O(1) H(101)	0.0516(9) 0.0223(8) 0.0499(9) 0.0036(6) -0.1631 0.4655 0.5970 0.0550(18)	0.0090(7) -0.0082(7)
	O(2) H(102)	0.0817(14) 0.0397(11) 0.0661(12) -0.0071(9) 0.1096 0.4484 0.6171 0.0550(18)	0.0105(11) -0.0228(10)
	O(3) H(121)	0.0608(10) 0.0382(9) 0.0517(10) 0.0016(7) 0.0849 0.5215 1.0348 0.0550(18)	0.0132(8) -0.0082(8)
	N(1) H(122)	0.0281(8) 0.0221(8) -0.0271 0.4411	0.0302(8) 0.0003(6) 1.0613 0.0550(18)	0.0068(6)	0.0033(6)
	N(2) H(131)	0.0392(10) 0.0377(11) 0.0558(12) 0.0058(9) -0.2660 0.5391 1.1217 0.0550(18)	0.0082(9)	0.0100(8)
	N(3) H(132)	0.0360(9) 0.0358(12) 0.0674(14) 0.0054(9) -0.2890 0.5723 0.9672 0.0550(18)	0.0177(9)	0.0086(9)
	N(4) H(133)	0.0336(8) 0.0223(9) -0.4011 0.4919	0.0490(10) -0.0012(7) 0.9937 0.0550(18)	0.0126(7)	0.0023(7)
	F(1)	0.0936(12) 0.0397(9)	0.0537(9) 0.0161(6)	0.0233(8) -0.0021(8)
	F(2)	0.0797(10) 0.0621(10) 0.0521(9) 0.0150(7)	0.0372(8)	0.0184(8)
	F(3)	0.0915(13) 0.0849(14) 0.0327(7) 0.0175(7)	-0.0038(7) -0.0008(10)

Molecular Modeling Studies

The molecular dynamics (MD) simulations were done in collaboration with Prof. Tâp Ha-Duong (BioCIS UMR 8076). All-atom MD simulations were performed using the GROMACS 4.5 package, with the OPLS-AA force field in combination with the SPC/E water model. (For a complete description of the method, see experimental part Chapter 4.5 Molecular Dynamics Studies.) The conformational ensembles generated for each of the eight peptides were first characterized by the average coupling constants 3 J HN-Hα of their five residues and then compared to available NMR measurements (Figure 2.17).

Figure 2.17

Comparison between the theoretical (black) and the NMR measured (red) 3 J HN-Hα coupling constants as a function of the peptide residues.

Evaluations

The ability of compounds 2.11, 2.18, 2.19, 2.10, 2.21 and 2.34 to inhibit amyloid fibril formation was evaluated on Aβ 1-42 peptide (10 μM) by fluorescence-detected thioflavin-T (ThT) binding assay, which is the standard method used to monitor the time course of fibril formation. The final ThT fluorescence intensity is dependent on the amount of fibrillar material formed, the binding constant for ThT, and the quantum yield of the bound dye. The fluorescence assay of Aβ 1-42 alone at a concentration of 10 μM shows a typical fibrillization process with a lag phase of 4 ~ 5 h followed by an elongation phase and a final plateau reached after 10-12h (Figure 2.23).

We also evaluated the ability of compounds 2.11, 2.18, 2.19, 2.34 to inhibit amyloid fibril formation of another amyloid peptide, IAPP peptide, which is involved in type II diabetes mellitus. [28] This peptide shares similar features in length (37 residues), sequence (~20% identity) and morphology of amyloid fibrils. Fluorescence data show a faster fibrillization process for IAPP (5 μM) with a final plateau reached after 7-8h (Figure 2.24).

However, the ThT assays showed that none of the pentapeptides (Ala-Val-X-Val-Leu-OMe, where X are Ser, Thr, (2S, 3S)-CF 3 -Thr and (2S, 3R)-CF 3 -Thr) had an inhibition effect on Aβ 1-42 and IAPP aggregation. The lag time and the final fluorescence intensity were not significantly modified in the presence of the pentapeptides at different peptide/Aβ 1-42 or peptide/IAPP ratios. In addition, our results showed that pentapeptides (2.11, 2.19, and 2.34) slightly promoted IAPP aggregation as evidenced by a decrease in the lag time and an increase in the equilibrium plateau relative to the control.

The effect of each compound (2.11, 2.18, 2.19, 2.10, 2.21 and 2.34) on the aggregation of Aβ 1-42 peptide (10 μM) and IAPP peptide (5 μM), evaluated by ThT fluorescence spectroscopy, is described in the following section.

Fluorinated triazole-based oligomers -Boc group

To avoid the side product from oxidation of the tert-butylsulfinyl group, we also tried to obtain the oligomers (trimer and tetramer) with Boc as N-terminal protection group.

After removal of the tert-butylsulfinyl group using HCl-Dioxane (4M, 30 eq.) in CH 2 Cl 2 at room temperature for 3h in quantitative yield, the crude residue was protected directly in the presence of Boc 2 O under basic condition. The Boc protected scaffold 3.17 was obtained in good yield (80%) (Scheme 3.12). Scheme 3.12 Synthesis of Boc protected CF 3 -1,4-disubstituted-1,2,3-triazole-based amino acid mimics

The Boc protected scaffold 3.17 also went saponification efficiently in NaOH (2 M, 2.5 eq.)/EtOH to afford the acid 3.18 as a white solid in quantitative yield (Scheme 3.12). The acid 3.18 was then coupled with the N-terminal deprotected monomer 3.5 using HATU/HOAt/DIPEA to give the Boc protected dimer 3.19 in good yield (77%) (Scheme 3.13), while using DMTMM/NMM to give the dimer in 73% yield. The Boc-monomer acid 3.18 was coupled with dimer amine 3.14 using HATU/HOAt/DIPEA as coupling reagent to afford the Boc protected trimer 3.20 in moderate yield (34%) as a white solid (Scheme 3.14), and in 94% purity (Figure 3.24). Further improvement using DMTMM/NMM gives the trimer in 53% yield (97% purity). The Boc protected dimer 3.17 was hydrolyzed in NaOH (2M, 2.5 eq.)/EtOH to give the acid 3.21 in quantitative yield. Afterwards, the acid 3.21 was coupled with the dimer 3.14 using HATU/HOAt/DIPEA to give the tetramer 3.22 as a white solid in low yield (14%, crude yield), and with a purity of 80% as indicated by HPLC analysis, while using DMTMM/NMM gives the tetramer in moderate yield (40%) in 96% purity. It was noticed again that, as the same situation with the tert-butylsulfinyl protected trimer 3.12 and tetramer 3.15, the solubility of these Boc protected trimer 3.20 and tetramer 3.22 was so poor that they are only soluble in DMF and DMSO, which prevented its conformational studies by NMR in 105 protic solvents such as D 2 O and CD 3 OD.

Compared with the tert-butylsulfinyl protected trimer 3.12 and tetramer 3.15, the Boc protected trimer 3.20 and tetramer 3.22 were obtained in lower yields, but with much easier treatment than in the case of the tert-butylsulfinyl (the oxidized compound could not be separated). The Bocprotected trimer 3.20 and tetramer 3.22 will later be cleaved at the N-termini to give the free amine and then they will be evaluated by ThT fluorescence assay to test their ability to inhibit the aggregation of Aβ 1-42 and IAPP peptides.

Evaluations

The ability of trimer 3.13 and tetramer 3.16 to inhibit amyloid fibril formation was evaluated on Aβ 1-42 peptide (10 μM) and IAPP peptide (5 μM) by ThT binding assay.

ThT binding assay showed that trimer 3.13 was a weak inhibitor of Aβ 1-42 aggregation as evidenced by very a slightly delayed lag phase of Aβ 1-42 aggregation and minor reduced final fluorescence intensity. It also showed that trimer 3.13 had a promotion effect in IAPP aggregation as evidenced by a decrease in the lag time and an increase in the equilibrium plateau relative to the control.

ThT binding assay showed that tetramer The compound 3.25 (62 mg, 0.12 mmol) was dissolved in 2 mL CH 2 Cl 2 at 0 o C, then to the solution at 0 o C was added CF 3 COOH (0.60 mL, 7.2 mmol, 60 eq.) and stirred at 0 o C for 30 min, then at r.t for 2h. After that time, all the solvents were evaporated under reduced pressure, then a little amount of diethyl ether was added to precipitate the product and evaporated the diethyl ether, repeated this process several times until the mass of the crude did not change any more. The resulting crude compound (62 mg) was obtained as liquid without any further purification.

The resulting crude compound (62 mg, 0.12 mmol, 1.2 eq.) was dissolved in dry DMF (2 mL) at r.t, then to the solution was added DMTMM (Cl -) (42 mg, 0.15 mmol, 1.5 eq.), NMM (61 mg, 0.60 mmol, 6.0 eq.) and the compound 3.26 (60 mg, 0.10 mmol, 1.0 eq.). The reaction stirred at room temperature overnight. Then DMF was removed under vacuum pump, the resulting residue was washed with EtOAc (5 mL) and 10% citric acid (5 mL), distilled water (5 mL) , 10% K 2 CO 3 (5 mL) and then brine, and dried over Na 2 SO 4 . The crude residue was purified by column chromatography on silica gel using CH 2 Cl 2 : MeOH = 100:2 as eluent. The compound 3.23 was obtained as white solid in 74% yield (74 mg). 5, 171.4, 166.9, 165.1, 156.0, 155.8, 141.3, 141.0, 137.4, 137.1, 129.1, 128.3, 128.0, 127.7, 126.3, 124.3, 124.1, 78.1, 65.1, 61.4, 54.2, 53.8, 51.4, 50.2, 49.8, 40.1, 37.9, 31.1, 28.9, 28.1, 24.1, 22.6, 13.9;

Molecular Modeling Studies

Experimental Procedures 100,000 initial random conformations were generated and minimized using MCMM (Monte Carlo Multiple Minima) method [1] with OPLS_2005 force field [2] and GBSA implicit solvation model (with water parameters) [3] as implemented in MacroModel v11.0 software package [4] . 3967 Unique conformations were obtained, and clustered. A 1.73 Å (atomic RMSD) value for similarity threshold provided 67 clusters. The closest conformation to the centroid of each cluster was retained as representative.

The representative helical conformation of tetramer 3.22 was subjected to energy minimization without constraint using DFT method [5] with the hybrid Becke3LYP functional [6] and the 6-31G* base [7] as implemented in Gaussian 09 software package [8] . PCM model was used to take into account water salvation [9] . Vibrational analysis within the harmonic approximation was performed at the same level of theory upon geometrical optimization convergence and the local minimum was characterized by the absence of imaginary frequency.

Chapter V Conclusions

The objectives of this thesis were to participate to the field of fluorinated peptides to understand the trifluoromethyl effect on peptide conformations and its biological effect, especially in its ability to modulate amyloid peptides (Aβ 1-42 and IAPP) aggregation.

In the first chapter, we introduced the unique physicochemical properties of fluorine and its potential use in the modifications of amino acids and peptides conformations. We also presented the background of AD, the amyloid cascade hypotheses and therapeutic strategies for AD especially the Aβ strategy to reduce the Aβ toxic oligomers and Aβ fibrils.

In the second chapter, we have prepared (2S, 3S)-CF 3 -threonine (natural threonine mimic) through the aldol reaction of CF 3 CHO with Ni (II)-complex of the chiral Schiff base of glycine, then we designed and synthesized pentapeptides with the sequence as Boc-Ala-Val-X-Val-Leu-OMe, where X are Ser, Thr, (2S, 3R)-CF 3 -Thr and (2S, 3S)-CF 3 -Thr. The conformational analysis of these pentapeptides was conducted by 2D NMR spectroscopy and molecular modeling studies.

NMR conformational studies showed that the 7 pentapeptides (2.10, 2.11, 2.18, 2.19, 2.20, 2.21 and 2.34) in the polar solvent CD 3 OH adopt mainly extended backbone conformations. The MD simulated conformations were in good agreement with the NMR results. It is found both experimentally and theoretically that, the (2S, 3S)-CF 3 -threonine-containing pentapeptides are more extended than the L-serineand Lthreonine-containing pentapeptides. It is also observed that both the Boc-protected and deprotected (2S, 3S)-CF 3 -Thr pentapeptides (2.18 and 2.19) have a propensity to self-associate of by forming intermolecular β-strand contacts, as suggested by temperature coefficients. This higher propensity of 2.18 and 2.19 to adopt extended structures can be explain by a stronger hydrophobic interaction of its trifluoromethyl group with the Leu 5 side chain with respect to the one involving the threonine methyl group, as observed in MD. Thus, both conformational studies demonstrated the trifluoromethyl effect on peptide conformations that promote an extended conformation in order to mimic a β-strand structure. Interestingly in the MD results, we find that the deprotected pentapeptides (2.