New chemical tools for chemical protein synthesis
The production of proteins by total synthesis is a growing field of research. This approach is very complementary to recombinant techniques for applications to the deciphering of biological mechanisms at the molecular level (chemical biology) or the development of new drugs. This alternative route is particularly useful for accessing modified proteins (post-translational modifications, selective labeling, introduction of probes, non-natural amino acids, modification of the peptide backbone, etc.) or difficult to produce (cytotoxic proteins, disulfide-rich miniproteins etc.).
Current technologies focus on the modular assembly of unprotected peptide fragments, through very selective reactions called "chemical ligation". This approach revolutionized the field some thirty years ago and is gradually being democratized for the synthesis of small proteins (50-100 amino acids). However, access to more ambitious targets in terms of size or molecular complexity remains a real challenge still reserved for rare specialists, often at the cost of considerable effort and investment. Three major technological bottlenecks remain to be overcome : (1) the assembly of numerous fragments by successive ligations, that requires repeated purifications steps, ultimately leading to very low overall yields, (2) the handling of poorly soluble or aggregation-prone fragments, a very frequently encountered problem, and (3) the development of more efficient ligation methods, from both the points of view of the nature of the chemical reactions used and of the synthesis of appropriately functionalized peptide fragments.
One of the main focuses of our group relies on the conception and development of original solutions to each of the three above-mentioned bottlenecks. We have thus gradually established a coherent molecular toolkit, aiming to be robust and versatile.
Development of N-terminal linkers
Particular emphasis has been placed on the development of techniques allowing a solid support assembly in order to avoid intermediate purifications. To this end, we have introduced a range of new linkers allowing the specific immobilization of a first pure and unprotected peptide fragment on a suitable solid support. Similar linkers have also been applied to the temporary modification of hydrophobic or aggregation-prone fragments, in order to render them soluble by the addition of charged residues (e.g., hexalysine label). The main challenge is to control the conditions necessary for the cleavage of these arms under very mild conditions, in order to release the protein assembled by successive ligations from the solid support or to eliminate the solubilizing groups.
The most commonly used reaction for protein synthesis was discovered some 20 years ago and is called native chemical ligation (NCL). It consists in the reaction of peptide fragments equipped respectively with a thioester function and an N-terminal cysteine. If the synthesis of the cysteinyl moiety can be carried out routinely, that of the thioester moiety is still very problematic. Indeed, the reaction conditions most commonly used in solid phase peptide synthesis (Fmoc / tBu strategy) are intrinsically incompatible with the thioester function.
Inspired by the spontaneous formation of a thioester during protein splicing, a non-enzymatic maturation process mediated by protein domains called inteins, we designed a molecular device programmed to in situ form a thioester under NCL conditions : so-called peptide crypto-thioesters. This bio-inspired process has the advantage of not requiring an additional chemical step after solid phase peptide synthesis. It can be totally automated at low cost, which makes its implementation by non-specialists very affordable. The peptide crypto-thioesters thus obtained are stable and can be handled, analyzed and purified by conventional techniques, and can be directly used in an NCL reaction, with very fast kinetics of transformation into a reactive thioester.
Peptidomimetic triazole ligation
While seeking alternative reactions to NCL, we pioneered the use of the copper(I)-catalyzed alkyne/azide cycloaddition (CuAAC) in the context of chemical protein synthesis. This highly chemoselective reaction leads to the formation of a 1,4-disubstituted 1,2,3-triazole which is recognized as an excellent mimic of a trans amide bond : CuAAC can thus be considered as a peptidomimetic ligation technique.
Additionally, triazoles cannot be hydrolyzed by proteases, and this feature has been exploited for the development of peptidomimetics whith enhanced in vivo stability for drug discovery purposes.
Synthesis of disulfide rich miniproteins
Disulfide rich peptides (DRP) are ubiquitous natural products composed of 10 to 90 amino acids, characterized by evolutionary-conserved cysteines reticulated by a dense disulfide bond network. These remarkably compact and stable compounds are active in adverse environments, exhibiting incredibly diverse host-defense or predation-related functions. DRPs are potent and selective binders of various targets, making them attractive compounds as pharmacological tools or new drug leads.
Our group has a long-lasting expertise in the synthesis of these compounds, including their oxidative folding to achieve the natural disulfide connectivity. The ligation-based synthetic methodologies we developed now allow us to synthesize very long DRPs, particularly difficult to access through standard synthetic means.
New strategies for cancer immunotherapy.
Glycan antigens carried by mucins present on cancer cells are tumor markers which are in part responsible for the immune system anergy regarding tumors. Structure and density of mucin glycans have an important impact on immune cells inaction. The mucins most often used in order to study those mechanisms are a mixture of heterogeneous glycoproteins with multiple glycan structures. But, for a better understanding of the relationship between glycan structure and immune response, it is mandatory to have access to mucins of variable size with well-defined glycan structures. Therefore, in order to produce those synthetic glycoproteins, we developed solid support synthesis methodologies combining chemical and enzymatic glycosylation steps. This allowed the preparation of homogenous and structurally well-defined mucins (up to 160 aminoacids and 24 short O-glycans) useful to study with precision the role of glycans carried by those glycoproteins in endocytosis and antigen presentation by dendritic cells and macrophages.
Many tumor antigens could in theory be used as targets for cancer immunotherapy. However, they usually don’t provoke a strong response because they have already been present on normal cells during embryonic development. In order to circumvent that problem, we use monosaccharide analogs of N-acetylgalactosamine or N-acetylmannosamine which can be readily incorporated into tumor cell glycans, due to the abnormal and very active carbohydrate metabolism of cancer cells, and exposed on cell surface. This approach targets specifically tumor cells and the reactive groups on the glycans can be used as handles to covalently link through bioorthogonal ligation reactions, strong immunostimulants specifically to the tumor cell surface where they will activate the anergic immune cells inside the tumor.
- Jacobsen M. T., Petersen M. E., Ye X., Galibert M., Lorimer G. H., Aucagne V. and Kay M. S.
A Helping Hand to Overcome Solubility Challenges in Chemical Protein Synthesisarticle. Journal of the American Chemical Society (2016) 138 (36) 11775-11782
- Terrier V. P., Adihou H., Arnould M., Delmas A. F. and Aucagne V.
A straightforward method for automated Fmoc-based synthesis of bio-inspired peptide crypto-thioesters. Chemical Science (2016) 7 (1) 339-345
- Galibert M., Piller V., Piller F., Aucagne V. and Delmas A. F.
Combining solid phase chemical ligations and enzymatic glycosylations for the synthesis of glycoproteins. Chemical Science (2015) 6 (6) 3617-3623
- Chuh K. N., Zaro B. W., Piller F., Piller, V. and Pratt M. R.
Changes in Metabolic Chemical Reporter Structure Yield a Selective Probe of O-GlcNAc Modification. Journal of the American Chemical Society (2014) 136 (35) 12283-12295
- Aucagne V., Valverde I. E., Marceau P., Galibert M., Dendane N. and Delmas A. F.
Towards the Simplification of Protein Synthesis : Iterative Solid-Supported Ligations with Concomitant Purifications. Angewandte Chemie International Edition (2012) 51 (45) 11320-11324
- Valverde I., Lecaille F., Lalmanach G., Aucagne V. and Delmas A. F.
Synthesis of a biologically active triazole-containing analogue of cystatin A through successive peptidomimetic alkyne–azide ligations. Angewandte Chemie International Edition (2012) 51 (3) 718-722
Martinez, G., Hograindleur, J.-P., Voisin, S., Abi Nahed., R., Abd El Aziz, T. M., Escoffier, J., Bessonnat, J., Fovet, C.-M., De Waard, M., Hennebicq, S., Aucagne, V., Ray, P.,F., Schmitt, E., Bulet, P. and Arnoult, C. (2017)
Spermaurin, an La1-like peptide from the venom of the scorpion Scorpio maurus palmatus, improves sperm motility and fertilization in different mammalian speciesMolecular Human Reproduction : Basic science of reproductive medicine (2017) 23 (2) 116-131
Is it possible to identify original compounds that are able to enhance sperm motility from the venom of the scorpion Scorpio maurus palmatus ?
We identified a potent disulfide-rich peptide (DRP) of 73 amino acids that significantly improved the motility of fresh and frozen-thawed sperm in different mammalian species, including human, and improved fertilization outcome in mouse IVF experiments.
WHAT IS KNOWN ALREADY
Any disturbance of sperm motility has a strong impact on fertilization and can lead to subfertility or infertility. Significant efforts have, therefore, been made to identify pharmacological drugs that might improve sperm motility. Such compounds are particularly useful in azoospermia to improve testicular sperm extraction and in the domain of cryopreservation because the motility of frozen-thawed sperm is reduced.
STUDY DESIGN, SIZE, DURATION
This was a basic science/medical research study aimed at identifying original compounds from a library of venoms able to enhance mammalian sperm motility, including human. We first identified in the venom of a scorpion S. m. palmatus a fraction able to potently activate sperm motility. We next purified and characterized the compound by liquid chromatography, mass spectrometry and peptide synthesis. Finally, the potency and toxicity of both purified and synthetic versions of the identified compound on sperm motility were assessed using different in vitro tests in different mammalian species.
PARTICIPANTS/MATERIALS, SETTING, METHODS
For human sperm, biological samples were collected from normozoospermic donors and subfertile patients attending a reproduction department for diagnostic semen analysis. Testicular sperm was collected from cynomolgus monkeys (Macaca fascicularis) euthanized for the needs of specific authorized research projects. The peptide was also tested on bovine and mouse epidydimal sperm. We measured different sperm motility parameters with a computer-assisted sperm analysis system in the presence or absence of the peptide.
MAIN RESULTS AND THE ROLE OF CHANCE
Size exclusion chromatography enabled us to isolate a fraction of the venom of S. m. palmatus able to increase sperm motility. By liquid chromatography and mass spectrometry, a peptide comprising 73 amino acids with 4 disulfide bridges was identified as responsible for the biological activity and called ‘spermaurin’. The identity of spermaurin was confirmed by chemical synthesis. We showed that the peptide increased the motility of fresh and frozen-thawed human sperm. We observed that the potency of the peptide was higher on fresh ejaculated spermatozoa with a low motility, achieving a 100% increase of curvilinear velocity in poorly performing sperm. We also demonstrated that peptide is effective on bovine and mouse fresh epididymal, bovine frozen-thawed ejaculated and fresh non-human primate testicular sperm. Finally, in mouse IVF, the production of 2-cell embryos was increased by 24% when sperm were treated with the peptide.
LIMITATIONS, REASONS FOR CAUTION
This work is an in vitro evaluation of the ability of spermaurin to improve sperm motility parameters. Another limitation of this study is the small number of human sperm samples tested with the natural (n = 36) and synthetic (n = 12) peptides. Moreover, the effect of the peptide on IVF outcome was only tested in mouse and further tests with human and bovine gametes are required to confirm and extend this result in other mammalian species.
WIDER IMPLICATIONS OF THE FINDINGS
This work confirms our initial study showing that venoms represent an interesting source of molecules that are able to modify sperm physiology. Moreover, this work presents the first demonstrated biological action of a venom peptide from the scorpion S. m. palmatus with sequence similarities to La1 peptide from Liocheles australasiae (Wood scorpion), a widespread family of DRPs.
STUDY FUNDING/COMPETING INTEREST(S)
This work is part of the project ‘LAB COM-14 LAB7 0004 01-LIPAV’, funded by the program LabCom 2014 from t e French Research Agency (ANR). Dr Arnoult reports grants from IMV Technologies during the conduct of the study. In addition, Drs Arnoult, Martinez, Ray and Schmitt have a patent EP16305642.7 pending containing some of the information presented in this manuscript.
Efficient synthesis of cysteine-rich cyclic peptides through intramolecular native chemical ligation of N-Hnb-Cys peptide crypto-thioestersOrganic and Biomolecular Chemistry (2017) 15 (2) 316-319
We herein introduce a straightforward synthetic route to cysteine-containing cyclic peptides based on the intramolecular native chemical ligation of in situ generated thioesters. Key precursors are N-Hnb-Cys crypto-thioesters, easily synthesized by Fmoc-based SPPS. The strategy is applied to a representative range of naturally occurring cyclic disulfide-rich peptide sequences.
Jacobsen M. T., Petersen M. E., Ye X., Galibert M., Lorimer G. H., Aucagne V. and Kay M. S. (2016)
Although native chemical ligation (NCL) and related chemoselective ligation approaches provide an elegant method to stitch together unprotected peptides, the handling and purification of insoluble and aggregation-prone peptides and assembly intermediates create a bottleneck to routinely preparing large proteins by completely synthetic means. In this work, we introduce a new general tool, Fmoc-Ddae-OH, N-Fmoc-1-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-3-[2-(2-aminoethoxy)ethoxy]-propan-1-ol, a heterobifunctional traceless linker for temporarily attaching highly solubilizing peptide sequences (“helping hands”) onto insoluble peptides. This tool is implemented in three simple and nearly quantitative steps : (i) on-resin incorporation of the linker at a Lys residue ε-amine, (ii) Fmoc-SPPS elongation of a desired solubilizing sequence, and (iii) in-solution removal of the solubilizing sequence using mild aqueous hydrazine to cleave the Ddae linker after NCL-based assembly. Successful introduction and removal of a Lys6 helping hand is first demonstrated in two model systems (Ebola virus C20 peptide and the 70-residue ribosomal protein L31). It is then applied to the challenging chemical synthesis of the 97-residue co-chaperonin GroES, which contains a highly insoluble C-terminal segment that is rescued by a helping hand. Importantly, the Ddae linker can be cleaved in one pot following NCL or desulfurization. The purity, structure, and chaperone activity of synthetic l-GroES were validated with respect to a recombinant control. Additionally, the helping hand enabled synthesis of d-GroES, which was inactive in a heterochiral mixture with recombinant GroEL, providing additional insight into chaperone specificity. Ultimately, this simple, robust, and easy-to-use tool is expected to be broadly applicable for the synthesis of challenging peptides and proteins.
Activation of TRPV2 and BKCa channels by the LL-37 enantiomers stimulates calcium entry and migration of cancer cellsOncotarget (2016) 7 (17) 23785-23800 - doi : 10.18632/oncotarget.8122
Expression of the antimicrobial peptide hCAP18/LL-37 is associated to malignancy in various cancer forms, stimulating cell migration and metastasis. We report that LL-37 induces migration of three cancer cell lines by activating the TRPV2 calcium-permeable channel and recruiting it to pseudopodia through activation of the PI3K/AKT pathway. Ca2+ entry through TRPV2 cooperated with a K+ efflux through the BKCa channel. In a panel of human breast tumors, the expression of TRPV2 and LL-37 was found to be positively correlated. The D-enantiomer of LL-37 showed identical effects as the L-peptide, suggesting that no binding to a specific receptor was involved. LL-37 attached to caveolae and pseudopodia membranes and decreased membrane fluidity, suggesting that a modification of the physical properties of the lipid membrane bilayer was the underlying mechanism of its effects.
The neuropeptide kisspeptin and its receptor, KiSS1R, govern the reproductive timeline of mammals by triggering puberty onset and promoting ovulation by stimulating gonadotrophin-releasing hormone (GnRH) secretion. To overcome the drawback of kisspeptin short half-life we designed kisspeptin analogs combining original modifications, triazole peptidomimetic and albumin binding motif, to reduce proteolytic degradation and to slow down renal clearance, respectively. These analogs showed improved in vitro potency and dramatically enhanced pharmacodynamics. When injected intramuscularly into ewes (15 nmol/ewe) primed with a progestogen, the best analog (compound 6, C6) induced synchronized ovulations in both breeding and non-breeding seasons. Ovulations were fertile as demonstrated by the delivery of lambs at term. C6 was also fully active in both female and male mice but was completely inactive in KiSS1R KO mice. Electrophysiological recordings of GnRH neurons from brain slices of GnRH-GFP mice indicated that C6 exerted a direct excitatory action on GnRH neurons. Finally, in prepubertal female mice daily injections (0.3 nmol/mouse) for five days significantly advanced puberty. C6 ability to trigger ovulation and advance puberty demonstrates that kisspeptin analogs may find application in the management of livestock reproduction and opens new possibilities for the treatment of reproductive disorders in humans.
A straightforward method for automated Fmoc-based synthesis of bio-inspired peptide crypto-thioestersChemical Science (2016) 7 (1) 339-345 - doi : 10.1039/C5SC02630J
Despite recent advances, the direct Fmoc-based solid phase synthesis of peptide [small alpha]-thioesters for the convergent synthesis of proteins via native chemical ligation (NCL) remains a challenge in the field. We herein report a simple and general methodology, enabling access to peptide thioester surrogates. A novel C-terminal N-(2-hydroxybenzyl)cysteine thioesterification device based on an amide-to-thioester rearrangement was developed, and the resulting peptide crypto-thioesters can be directly used in NCL reactions with fast N [rightward arrow] S shift kinetics at neutral pH. These fast kinetics arise from our bio-inspired design, via intein-like intramolecular catalysis. Due to a well-positioned phenol moiety, an impressive >50 fold increase in the kinetic rate is observed compared to an O-methylated derivative. Importantly, the synthesis of this new device can be fully automated using inexpensive commercially available materials and does not require any post-synthetic steps prior to NCL. We successfully applied this new method to the synthesis of two long naturally-occurring cysteine-rich peptide sequences.
Complete 1H, 15N and 13C assignment of trappin-2 and 1H assignment of its two domains, elafin and cementoinBiomolecular NMR Assignments (2016) 10 (1) 223-226 - doi : 10.1007/s12104-016-9671-1
Trappin-2 is a serine protease inhibitor with a very narrow inhibitory spectrum and has significant anti-microbial activities. It is a 10 kDa cationic protein composed of two distinct domains. The N-terminal domain (38 residues) named cementoin is known to be intrinsically disordered when it is not linked to the elafin. The C-terminal domain (57 residues), corresponding to elafin, is a cysteine-rich domain stabilized by four disulfide bridges and is characterized by a flat core and a flexible N-terminal part. To our knowledge, there is no structural data available on trappin-2. We report here the complete 1H, 15N and 13C resonance assignment of the recombinant trappin-2 and the 1H assignments of cementoin and elafin, under the same experimental conditions. This is the first step towards the 3D structure determination of the trappin-2.
Native Chemical Ligation Strategy to Overcome Side Reactions during Fmoc-Based Synthesis of C-Terminal Cysteine-Containing PeptidesOrganic letters (2016 18 (5) 920-923 - doi : 10.1021/acs.orglett.5b03612
The Fmoc-based solid phase synthesis of C-terminal cysteine-containing peptides is problematic, due to side reactions provoked by the pronounced acidity of the Calpha proton of cysteine esters. We herein describe a general strategy consisting of the postsynthetic introduction of the C-terminal Cys through a key chemoselective native chemical ligation reaction with N-Hnb-Cys peptide crypto-thioesters. This method was successfully applied to the demanding peptide sequences of two natural products of biological interest, giving remarkably high overall yields compared to that of a state of the art strategy.
Beltramo, M., Robert, V., Galibert, M., Madinier, J.-B., Marceau, P., Dardente, H., Decourt, C., De Roux, N., Lomet, D., Delmas, A. F., Caraty, A. and Aucagne, V. (2015)
Rational Design of Triazololipopeptides Analogs of Kisspeptin Inducing a Long-Lasting Increase of GonadotropinsJournal of Medicinal Chemistry (2015) 58 (8) 3459-3470 - doi : 10.1021/jm5019675
New potent and selective KISS1R agonists were designed using a combination of rational chemical modifications of the endogenous neuropeptide kisspeptin 10 (KP10). Improved resistance to degradation and presumably reduced renal clearance were obtained by introducing a 1,4-disubstituted 1,2,3-triazole as a proteolysis-resistant amide mimic and a serum albumin-binding motif, respectively. These triazololipopeptides are highly potent full agonists of KISS1R and are >100 selective over the closely related NPFF1R. When injected in ewes with a quiescent reproductive system, the best compound of our series induced a much prolonged increase of luteinizing hormone release compared to KP10 and increased follicle-stimulating hormone plasma concentration. Hence, this KISS1R agonist is a new valuable pharmacological tool to explore the potential of KP system in reproduction control. Furthermore, it represents the first step to develop drugs treating reproductive system disorders due to a reduced activity of the hypothalamo–pituitary–gonadal axis such as delayed puberty, hypothalamic amenorrhea, and hypogonadotropic hypogonadism.
Combining solid phase chemical ligations and enzymatic glycosylations for the synthesis of glycoproteinsChemical Science (2015) 6 (6) 3617-3623 - doi : 10.1039/C5SC00773A
The solid-phase chemical assembly of a protein through iterative chemoselective ligation of unprotected peptide segments can be followed with chemical and/or enzymatic transformations of the resulting immobilized protein, the latter steps thus benefitting from the advantages provided by the solid support. We demonstrate here the usefulness of this strategy for the chemo-enzymatic synthesis of glycoprotein analogues. A linker was specifically designed for application to the synthesis of O-glycoproteins : this new linker is readily cleaved under mild aqueous conditions compatible with very sensitive glycosidic bonds, but is remarkably stable under a wide range of chemical and biochemical conditions. It was utilized for solid-supported N-to-C peptidomimetic triazole ligation followed by enzymatic glycosylation, ultimately leading to a very large MUC1-derived glycoprotein containing 160 amino acid residues, 24 α-GalNAc moieties linked to Ser and Thr, and 3 triazoles as peptide bond mimetics.
The development of mechanically interlocked molecular systems programmed to operate autonomously in biological environments is an emerging field of research with potential medicinal applications. Within this framework, functional rotaxane- and pseudorotaxane-based architectures are starting to attract interest for the delivery of anticancer drugs, with the ultimate goal to improve the efficiency of cancer chemotherapy. Here, we report an enzyme-sensitive -rotaxane designed to release a potent anticancer drug within tumor cells. The molecular device includes a protective ring that prevents the premature liberation of the drug in plasma. However, once located inside cancer cells the -rotaxane leads to the release of the drug through the controlled disassembly of the mechanically interlocked components, in response to a determined sequence of two distinct enzymatic activations. Furthermore, in vitro biological evaluations reveal that this biocompatible functional system exhibits a noticeable level of selectivity for cancer cells overexpressing [small beta]-galactosidase.
Meindre, F., Lelièvre, D., Loth, K., Mith, O., Aucagne, V., Berthomieu, P., Marquès, L., Delmas, A. F., Landon, C. and Paquet, F. (2014)
The Nuclear Magnetic Resonance Solution Structure of the Synthetic AhPDF1.1b Plant Defensin Evidences the Structural Feature within the γ-MotifBiochemistry (2014) 53 (49) 7745-7754 - doi : 10.1021/bi501285k
Plant defensins (PDF) are cysteine-rich peptides that are major actors in the innate immunity in plants. Besides their antifungal activity, some PDF such as Arabidopsis halleri PDF1.1b confer zinc tolerance in plants. Here we present (i) an efficient protocol for the production of AhPDF1.1b by solid-phase peptide synthesis followed by controlled oxidative folding to obtain the highly pure native form of the defensin and (ii) the three-dimensional (3D) nuclear magnetic resonance structure of AhPDF1.1b, the first 3D structure of plant defensin obtained with a synthetic peptide. Its fold is organized around the typical cysteine-stabilized α-helix ?-sheet motif and contains the ?-core motif involved in the antifungal activity of all plant defensins. On the basis of our structural analysis of AhPDF1 defensins combined with previous biological data for antifungal and zinc tolerance activities, we established the essential role of cis-Pro41 within the ?-core. In fact, the four consecutive residues (Val39-Phe40-Pro41-Ala42) are strictly conserved for plant defensins able to tolerate zinc. We hypothesized that structural and/or dynamic features of this sequence are related to the ability of the defensin to chelate zinc.
Metabolic chemical reporters (MCRs) of glycosylation are analogues of monosaccharides that contain bioorthogonal functionalities and enable the direct visualization and identification of glycoproteins from living cells. Each MCR was initially thought to report on specific types of glycosylation. We and others have demonstrated that several MCRs are metabolically transformed and enter multiple glycosylation pathways. Therefore, the development of selective MCRs remains a key unmet goal. We demonstrate here that 6-azido-6-deoxy-N-acetyl-glucosamine (6AzGlcNAc) is a specific MCR for O-GlcNAcylated proteins. Biochemical analysis and comparative proteomics with 6AzGlcNAc, N-azidoacetyl-glucosamine (GlcNAz), and N-azidoacetyl-galactosamine (GalNAz) revealed that 6AzGlcNAc exclusively labels intracellular proteins, while GlcNAz and GalNAz are incorporated into a combination of intracellular and extracellular/lumenal glycoproteins. Notably, 6AzGlcNAc cannot be biosynthetically transformed into the corresponding UDP sugar-donor by the canonical salvage-pathway that requires phosphorylation at the 6-hydroxyl. In vitro experiments showed that 6AzGlcNAc can bypass this roadblock through direct phosphorylation of its 1-hydroxyl by the enzyme phosphoacetylglucosamine mutase (AGM1). Taken together, 6AzGlcNAc enables the specific analysis of O-GlcNAcylated proteins, and these results suggest that specific MCRs for other types of glycosylation can be developed. Additionally, our data demonstrate that cells are equipped with a somewhat unappreciated metabolic flexibility with important implications for the biosynthesis of natural and unnatural carbohydrates.
Three-dimensional NMR Structure of Hen Egg Gallin (Chicken Ovodefensin) Reveals a New Variation of the beta-Defensin FoldJournal of Biological Chemistry (2014) 289 (10) 7211-7220 - doi : 10.1074/jbc.M113.507046
Gallin is a 41-residue protein, first identified as a minor component of hen egg white and found to be antimicrobial against Escherichia coli. Gallin may participate in the protection of the embryo during its development in the egg. Its sequence is related to antimicrobial beta-defensin peptides. In the present study, gallin was chemically synthesized 1) to further investigate its antimicrobial spectrum and 2) to solve its three-dimensional NMR structure and thus gain insight into structure-function relationships, a prerequisite to understanding its mode(s) of action. Antibacterial assays confirmed that gallin was active against Escherichia coli, but no additional antibacterial activity was observed against the other Gram-positive or Gram-negative bacteria tested. The three-dimensional structure of gallin, which is the first ovodefensin structure to have been solved to date, displays a new five-stranded arrangement. The gallin three-dimensional fold contains the three-stranded antiparallel beta-sheet and the disulfide bridge array typical of vertebrate beta-defensins. Gallin can therefore be unambiguously classified as a beta-defensin. However, an additional short two-stranded beta-sheet reveals that gallin and presumably the other ovodefensins form a new structural subfamily of beta-defensins. Moreover, gallin and the other ovodefensins calculated by homology modeling exhibit atypical hydrophobic surface properties, compared with the already known vertebrate beta-defensins. These specific structural features of gallin might be related to its restricted activity against E. coli and/or to other yet unknown functions. This work provides initial understanding of a critical sequence-structure-function relationship for the ovodefensin family.
PA1b Inhibitor Binding to Subunits c and e of the Vacuolar ATPase Reveals Its Insecticidal MechanismThe Journal of Biological Chemistry (2014) 289 (23) 16339-16408 - doi : 10.1074/jbc.M113.541250
The vacuolar ATPase (V-ATPase) is a 1MDa transmembrane proton pump that operates via a rotary mechanism fuelled by ATP. Essential for eukaryotic cell homeostasis, it plays central roles in bone remodeling and tumor invasiveness, making it a key therapeutic target. Its importance in arthropod physiology also makes it a promising pesticide target. The major challenge in designing lead compounds against the V-ATPase is its ubiquitous nature, such that any therapeutic must be capable of targeting particular isoforms. Here, we have characterized the binding site on the V-ATPase of pea albumin 1b (PA1b), a small cystine knot protein that shows exquisitely selective inhibition of insect V-ATPases. Electron microscopy shows that PA1b binding occurs across a range of equivalent sites on the c ring of the membrane domain. In the presence of Mg.ATP, PA1b localizes to a single site, distant from subunit a, which is predicted to be the interface for other inhibitors. Photoaffinity labeling studies show radiolabeling of subunits c and e. In addition, weevil resistance to PA1b is correlated with bafilomycin resistance, caused by mutation of subunit c. The data indicate a binding site to which both subunits c and e contribute and inhibition that involves locking the c ring rotor to a static subunit e and not subunit a. This has implications for understanding the V-ATPase mechanism and that of inhibitors with therapeutic or pesticidal potential. It also provides the first evidence for the position of subunit e within the complex.
Peptide-based complex biomacromolecules are now optimally assembled by sequential ligation of unprotected peptide segments. However, this approach is still limited by the laborious chromatographic purification and handling steps needed for multiple successive chemoselective couplings, which leads to loss of material. An efficient alternative is solid phase chemical ligation (SPCL) initially developed for native chemical ligation. We report here an extension of this approach to iterative oxime ligation reactions, and describe a streamlined approach for the modular preparation of oxime-containing polypeptides. In particular, we determined optimal conditions to remove the Aloc group in the presence of aminooxy and oxime ether groups, and we extended the applicability of iterative C-to-N SPCL through simplification of the access to a C-terminally-grafted, unprotected peptide segment, using solid supported chemical transformations only. The high purity of the crude oxime-containing polypeptides highlights the efficiency of our approach.
Raibaut, L., Adihou, H., Desmet, R., Delmas, A.F., Aucagne and V. Melnyk, O. (2013)
Highly efficient solid phase synthesis of large polypeptides by iterative ligations of bis(2-sulfanylethyl)amido (SEA) peptide segmentsChemical Science 4 (10) 4061-4066
Up to now, the advantages of solid phase protein synthesis have been largely under-utilized due to the difficulty of designing a simple and efficient elongation cycle enabling the concatenation of unprotected peptide segments. The combination of selective N-terminal anchoring (N3-Esoc linker) with the blocked thioester properties of the SEAoff group enabled the solid phase concatenation of unprotected peptide segments by N-to-C sequential formation of native peptide bonds. The strategy was applied to the synthesis of a 60 amino acid-long latent peptide thioester or to the assembly of five peptide segments to give a 15 kDa polypeptide.
Mitogaligin is a mitochondrion-targeting protein involved in cell death. The sequence of the protein is unrelated to that of any known pro- or antiapoptotic protein. Mitochondrial targeting is controlled by an internal sequence from residues 31 to 53, and although this sequence is essential and sufficient to provoke cell death, the precise mechanism of action at the mitochondrial membrane remains to be elucidated. Here, by focusing on the [31–53] fragment, we first assessed and confirmed its cell cytotoxicity by microinjection. Subsequently, with the aid of membrane models, we evaluated the impact of the membrane environment on the 3D structure of the peptide and on how the peptide is embedded and oriented within membranes. The fragment is well organized, even though it does not contain a canonical secondary structure, and adopts an interfacial location. Structural comparison with other membrane-interacting Trp-rich peptides demonstrated similarities with the antimicrobial peptide tritrpcidin.
Aucagne, V., Valverde, I.E., Marceau, P., Galibert, M., Dendane, N., Delmas, A.F. (2012)
Towards the Simplification of Protein Synthesis : Iterative Solid-Supported Ligations with Concomitant PurificationsAngewandte Chemie International Edition, 51 (45) 11320-11324
Please release me : A new linker for the temporary tagging of peptides at their N-terminus after solid-phase elongation, and its potential for capture/release purification is demonstrated. This concept is extended to a remarkably efficient self-purifying N-to-C iterative triazole ligation strategy, which is applied to the synthesis of a polypeptide having 160 residues, in a high purity without the need for chromatographic purification (see picture ; orange blocks : peptide segments).
Ligand binding study of human PEBP1/RKIP : interaction with nucleotides and Raf-1 peptides evidenced by NMR and Mass SpectrometryPLoS ONE 7 (4) e36187
In the present study, we investigated human PEBP1 by NMR to determine the binding site of four different ligands : GTP, FMN, and one Raf-1 peptide in tri-phosphorylated and non-phosphorylated forms. The study was carried out by NMR in near physiological conditions, allowing for the identification of the binding site and the determination of the affinity constants KD for different ligands. Native mass spectrometry was used as an alternative method for measuring KD values. Our study demonstrates and/or confirms the binding of hPEBP1 to the four studied ligands. All of them bind to the same region centered on the conserved ligand-binding pocket of hPEBP1. Although the affinities for GTP and FMN decrease as pH, salt concentration and temperature increase from pH 6.5/NaCl 0 mM/20°C to pH 7.5/NaCl 100 mM/30°C, both ligands clearly do bind under conditions similar to what is found in cells regarding pH, salt concentration and temperature. In addition, our work confirms that residues in the vicinity of the pocket rather than those within the pocket seem to be required for interaction with Raf-1.
A reducing agent–free version of CuAAC able to operate
under open air conditions is reported. A readily–synthesizable, hydrophilic and highly stable CuI–NHC allows the clean ligations of unprotected peptides comprising sensitive side chains, at millimolar concentrations.
Synthesis and analytical investigation of C-terminally modified peptide aldehydes and ketone : application to oxime ligationJournal of Peptide Science 18 (3) 147-154
C-terminally modified peptides aldehyde (glycinal and alpha-oxo aldehyde peptides) and ketone (pyruvic acid-containing
peptide) were synthesised to get new insights into the mechanism of acido-catalysed oxime ligation. Their tetrahedral
hydrated forms were investigated in solution and in the gas phase, using NMR and in-source collision-induced dissociation mass
spectrometry, respectively, and the kinetics of the oximation reactions followed using analytical HPLC. The results obtained confirmed
that the first step of the oximation reaction was the limiting step for the pyruvic acid-containing peptides because of the
steric effect and of the carbon angular strain of the ketone. The second step is the determining step for the aldehyde peptides
because the basicity of the oxygen of the hydroxyl function of the tetrahedral form is greater for glycinal than for alpha-oxo
aldehyde. These data strongly suggest that the hydrated form of the aldehyde partner has to be considered when oxime
reactions are performed in aqueous buffer.
Numerous β-defensins have been identified in birds and the potential use of these peptides as alternatives to antibiotics has been proposed, in particular to fight antibiotic-resistant and zoonotic bacterial species. Little is known about the mechanism of antibacterial activity of avian β-defensins (AvBDs), and the present work was carried out to obtain initial insights into the involvement of structural features or specific residues in the antimicrobial activity of chicken AvBD2. Chicken AvBD2 and its enantiomeric counterpart were chemically synthesized. Peptide elongation and oxidative folding were both optimized. The similar antimicrobial activity measured for both L- and D- proteins clearly indicates that there is no chiral partner. Therefore the bacterial membrane is in all likelihood the primary target. Moreover, this work evidences that the three-dimensional fold is required for an optimal antimicrobial activity, in particular for Gram-positive bacterial strains. The three-dimensional NMR structure of chicken AvBD2 defensin displays the structural 3-stranded antiparallel β-sheet characteristic of β-defensins. The surface of the molecule does not display any amphipathic character. In light of this new structure and of the king penguin AvBD103b defensin structure, the consensus sequence of avian β-defensin′s family was analyzed. Well conserved residues were highlighted and the potential strategic role of the lysine 31 residue of AvBD2 emphasized. The synthetic AvBD2-K31A variant displayed substantial N-terminal structural modifications and a dramatic decrease in activity. Taken together, these results demonstrate the structural as well as the functional role of the critical lysine 31 residue in antimicrobial activity.
A rotaxane, in which the peptidic axle is protected from degradation by the macrocyclic sheath and terminated with a novel glycosidase-cleavable stopper, is rendered water-soluble by derivatisation with tetra(ethylene glycol) (TetEG) or glucosylated tetra(ethylene glycol) (Glc-TetEG) chains using the CuAAC ’click’ reaction. The Glc-TetEG-derivatised rotaxane propeptide is >50 000 times more soluble in aqueous media than the parent rotaxane. Activation of the water-soluble rotaxane propeptide with a β-galactosidase efficiently releases the parent peptide.
Synthesis of a biologically active triazole-containing analogue of cystatin A through successive peptidomimetic alkyne–azide ligationsAngewandte Chemie International Edition 51 (3) 718-722
“Click” protein : CuI-catalyzed cycloaddition of azides and terminal alkynes has been applied to the successive ligations of three unprotected peptide fragments. Peptidomimetic triazole ligation (PTL, see scheme) as a new method for the chemical production of bioactive proteins is applied for the synthesis of a triazole-containing analogue of the 97 amino acid protein cystatin A.
Hardouin, J., Cremer, A. G. & Delmas, A. F. (2011)
Investigation of in-source decay of oxime-linked peptide by matrix-assisted laser desorption/ionization time-of-flight mass spectrometryRapid Communications in Mass Spectrometry 25 (14) 2106-2112
Aucagne, V. (2010)
[957753-00-3] C14H12S (MW 212.32)
InChI = 1S/C14H12S/c15-9-14-12-7-3-1-5-10(12)11-6-2-4-8-13(11)14/h1-8,14-15H,9H2
InChIKey = MXHVORUHWLKKMZ-UHFFFAOYSA-N
(reagent used to introduce a masked thiocarboxylic acid through thioesterification)
Alternate Name : Fm SH.
Physical Data : no reported physical data other than NMR and MS.
Solubility : soluble in Et2O, CH2Cl2, and other common organic solvents ; insoluble in water.
Form Supplied in : pale yellow oil.
Analysis of Reagent Purity : 1H and 13C NMR.
Preparative Method : not yet commercially available. FmSH can be prepared in three steps from commercially available (9H-fluoren-9-yl)methanol (FmOH) by treatment with tosyl chloride and pyridine in chloroform at rt for 6 h,1 followed by displacement of the tosylate by potassium thioacetate and 18-crown-6 in DMF at rt for 2 h, and then reduction with DIBAL-H in Et2O at –78 °C and then at 0 °C for 30 min.2 Purification requires column chromatography.
Purification : flash column chromatography on silica gel : 98:2 hexane/ethyl acetate.
Handling, Storage, and Precautions : stable under normal pressure and temperature in the absence of air. Unpleasant odor. Store in a cool dry place under an inert atmosphere. Wash thoroughly after use and remove any contaminated clothing. The toxicological properties have not been studied.
Da Silva, P., Strzepa, A., Jouvensal, L., Rahioui, I., Gressent, F. & Delmas, A.F. (2009)
Click a la carte : robust semi-orthogonal alkyne protecting groups for multiple successive azide/alkyne cycloadditions.Tetrahedron 65, 7597-7602. Fernandes, A., Viterisi, A., Coutrot, F., Potok, S., Leigh, D.A., Aucagne, V. & Papot, S. (2009)
Histidine-rich peptide : evidence for a single zinc-binding site on H5WYG peptide that promotes membrane fusion at neutral pH.J. Mass Spectrom. 44, 81-89.
Aucagne, V., Tatibouet, A. & Rollin, P. (2008)
Thermodynamics versus kinetics in hetero-Michael cyclizations : a highly stereoselective approach to access both epimers of a C-D-mannopyranoside.Tetrahedron Lett. 49, 4750-4753. Lelièvre, D ; Barta, P ; Aucagne, V ; Delmas, AF (2008)
The Methanosarcina thermophila MC1 protein is a small basic protein that is able to bend DNA sharply. When this protein is submitted to oxidative stress through gamma irradiation, it loses its original DNA interaction properties. The protein can still bind DNA but its ability to bend DNA is decreased dramatically. Here, we used different approaches to determine the oxidations that are responsible for this inactivation. Through a combination of proteolysis and mass spectrometry we have identified the three residues that are oxidized preferentially.
Decostaire, IP ; Lelièvre, D ; Aucagne, V ; Delmas, AF (2007)
Synergic effect of hydride and proton donors in the Pd(0)-mediated deprotection of N-a-Aloc proline derivativesTetrahedron Letters 48 6523-6526
In this study, the challenging Pd(O)-catalyzed N-Aloc removal from a proline residue using Me2NH-BH3 or PhSiH3 as allyl scavengers has been investigated. Standard conditions led to a large amount of an allylamine byproduct. A careful study of the reactions allowed us to design the optimal conditions for the quick and quantitative formation of the desired product, while taking advantage of a synergic effect between hydride and proton donors. (c) 2007 Elsevier Ltd. All rights reserved.
We compare herein the scope of three copper (I) catalysts on the synthesis of various 1,4-disubstitued-1,2,3-triazolo-carbanucleosides through a microwave (and thermic) assisted Huisgen 1,3-dipolar cycloaddition. The tetrakis(acelonitrile)copper hexafluorophosphate ([Cu(CH3CN4]PF6), the imidazoline(mesythyl)copper bromide (Imes)CuBr, and the copper/copper sulfate Cu(0)/CuSO4 (II) mixture have been chosen for this study. Their influence in a catalytic amount will be analyzed according to the substituent of the alkyne, the solvent, or the heating method.
Catalytic "Active-Metal" template synthesis of rotaxanes, rotaxanes, and molecular shuttles, and some observations on the mechanism of the Cu(I)-catalyzed azide-alkyne 1,3-cycloadditionJournal of The American Chemical Society 129 11950-11963
A synthetic approach to rotaxane architectures is described in which metal atoms catalyze covalent bond formation while simultaneously acting as the template for the assembly of the mechanically interlocked structure. This "active- metal" template strategy is exemplified using the Huisgen-Meldal-Fokin Cu(l)catalyzed 1,3-cycloaddition of azides with terminal alkynes (the CuAAC "click" reaction). Coordination of Cu(I) to an endotopic pyridine-containing macrocycle allows the alkyne and azide to bind to metal atoms in such a way that the metal-mediated bond-forming reaction takes place through the cavity of the macrocycle-or macrocycles-forming a rotaxane.
Hitherto unknown 1,4-disubstituted-[1,2,3]-triazolo-4’,4’-dihydroxymethyl-3’-deoxycarbanucleosides were synthesized based on a "click approach." Various alkynes were introduced on a key azido intermediate by the "click" 1,3-dipolar Huisgen cycloaddition. Their antiviral activities and cellular toxicities were evaluated on vaccinia virus. None of the synthesized compounds exhibited a significant antiviral activity.
Cremer, GA ; Bureaud, N ; Lelievre, D ; Piller, V ; Kunz, H ; Piller, F ; Delmas, AF (2006)
Controlling the outcome of overacylation of N-protected aminooxyacetic acid during the synthesis of an aminooxy-peptide for chemical ligationTetrahedron Letters 47 (39) 7057-7060
An amino oxy-containing peptide, the nucleophile partner for oxime ligations, is usually grafted ona NH2-peptide resin by activating a protected aminooxyacetic acid as an active ester. Here, we have shown that its subsequent coupling to NH2-peptide resin competes with the overacylation of the -NH-O-nitrogen and that the overacylation level increases with the basicity of the reaction mixture. Moreover, we found that overacylation is prevented when the COOH of the Aoa-derivatives is engaged in an amide bond. (c) 2006 Elsevier Ltd. All rights reserved.
Synthesis and biological evaluation of a multiantigenic Tn/TF-containing glycopeptide mimic of the tumor-related MUC1 glycoprotein (Publication ayant donné lieu à la couverture du numéro)Chemmedchem 1 (9) 965-968
The glycopeptide construct shown was synthesized using a convergent strategy based on oxime chemical ligation. It is composed of a universal T-helper and three tumor-related epitopes from the human mucin MUC1, an non-glycosylated repeat unit, and two units glycosylated with the Tn and TF epitopes, respectively. In association with a mild adjuvant suitable for human therapy, this construct elicited a strong specific immune response in mice, directed against natural tumor-associated structures.
Bromine-modified oligodeoxynucleotides (ODNs) were fragmented in the electrospray source to study the influence of brominated bases on fragmentation. Several 13-mer ODNs containing a brominated pyrimidine base, BrdU (5-bromodeoxyuridine) or BrdC (5-bromodeoxycytidine), were analyzed. Low cone voltage fragmentation yields a loss of the brominated base with a preferential loss for the brominated base closer to the 5-end (2-position > 4-position > 12-position) as well as a preferential loss of BrdU over BrdC. Higher cone voltage produces backbone fragmentation with complementary a(n)-base and w(m) ions close to the brominated base. On the basis of these observations, we located the brominated base in the sequence for all of the ODNs studied. Copyright (c) 2005 John Wiley & Sons, Ltd.
Identification of isoenzymes using matrix-assisted laser desorption/ionization time-of-flight mass spectrometryRapid Communications in Mass Spectrometry 20 (5) 725-732
The identification of isoforms is one of the great challenges in proteomics due to the large number of identical amino acids preventing their separations by two-dimensional electrophoresis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has become a rapid and sensitive tool in proteomics, notably with the new instrumental improvements. In this study, we used several acquisition modes of MALDI-TOFMS to identify isoforms of porcine glutathiones S-transferase. The use of multiple proteases coupled to the different acquisition modes of MALDI-TOFMS (linear, reflectron, post-source decay (PSD) and in-source decay, positive and negative modes) allowed the identification of two sequences. Moreover, a third sequence is pointed out from a PSD study of a tryptic ion revealing the modification of the amino acid tyrosine 146 to phenylalanine. Copyright (c) 2006 John Wiley & Sons, Ltd.
Combining a polar resin and a pseudo-proline to optimize the solid-phase synthesis of a ’difficult sequence’Journal of Peptide Science 12 (6) 437-442
This paper describes the optimization of a synthesis of a difficult sequence related to a 12-mer sequence of a Pan DR epitope (PADRE). Elongation was followed by on-line monitoring of the N-a-Fmoc removal adapted for the batch methodology. Studying the intrinsic factors related to the peptide-resin, such as substitution level, resin nature and backbone protecting group, has led to an increase in the elongation yield and purity of the crude peptide. Optimal elongation was obtained by combining a polar resin such as PEGA and a pseudo-proline as the backbone protecting group. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.
A methodology for the successive regiospecific "clicking" together of three components in one pot via two triazole linkages is reported. The protocol utilizes copper(I)-mediated alkyne-azide cycloaddition reactions combined with a silver(I)-catalyzed TMS-alkyne deprotection under mild hydroalcoholic conditions. We exemplify the approach with peptide-based components to illustrate its compatibility with polyfunctionalized biomolecules. The method constitutes a promising tool for peptide ligation. We also provide a procedure for directly using TMS-alkynes as the cycloaddition partner in classical "click" chemistry.
Catalytic "click" rotaxanes : A substoichiometric metal-template pathway to mechanically interlocked architecturesJournal of The American Chemical Society 128 (7) 2186-2187
Marceau, P ; Bure, C ; Delmas, AF (2005)
Synthesis of a C-terminal modified peptide with an a-amido methylketone was efficiently carried out using a backbone-amide-type linker loading with a monofunctionalized diamine, provided that no base such as piperidine or diisopropylethylamine or a reducing agent such as triisopopylsilane was used for the synthetic pathway. The ketoxime-forming chemoselective ligation between a methylketone and an aminooxy was quantitative in 5 h at pH 2. (c) 2005 Elsevier Ltd. All rights reserved.
Preparation of protected peptidyl thioester inter-mediates for native chemical ligation by N-a-9-fluorenylmethoxycarbonyl (Fmoc) chemistry : considerations of side-chain and backbone anchoring strategies, and compatible protection for N-terminal cysteineJournal of Peptide Research 65 (3) 395-410
Native chemical ligation has proven to be a powerful method for the synthesis of small proteins and the semisynthesis of larger ones. The essential synthetic intermediates, which are C-terminal peptide thioesters, cannot survive the repetitive piperidine deprotection steps of N-a-9-fluorenylmethoxycarbonyl (Fmoc) chemistry. Therefore, peptide scientists who prefer to not use N-a-t-butyloxycarbonyl (Boc) chemistry need to adopt more esoteric strategies and tactics in order to integrate ligation approaches with Fmoc chemistry. In the present work, side-chain and backbone anchoring strategies have been used to prepare the required suitably (partially) protected and/or activated peptide intermediates spanning the length of bovine pancreatic trypsin inhibitor (BPTI). Three separate strategies for managing the critical N-terminal cysteine residue have been developed :
Matrix-assisted laser desorption/ionisation mass spectrometry for the direct analysis of enzymatically digested kappa- iota- and hybrid iota/nu-carrageenansRapid Communications in Mass Spectrometry 19 (16) 2217-2226
Enzymatically digested oligosaccharides of kappa-, iota- and hybrid iota/nu-carrageenans were analysed using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry in the negative-ion mode. nor-Harmane was used as matrix. Depending on the stock concentration and the laser intensity applied, the oligosaccharides exhibited losses of sulphate units (neutralised by the Na+ ion, and thus non-stable), leaving the primary backbone structure in most cases with only the deprotonated sulphate groups (carrying the negative charge, stable).
Collision-induced dissociation of peptide thioesters : the influence of peptide length on fragmentationEuropean Journal of Mass Spectrometry 11 (1) 31-34
Five peptide thioesters of increasing length were fragmented using two processes, in-source and in-collision cell fragmentation, using an electrospray source coupled to a triple quadrupole mass spectrometer. Comparison of their fragmentations was made in regard to their length. The two fragmentation conditions show that the peptide length has no influence on structural information and that the fragmentation efficiency is higher for the smallest peptides than for the longest. The particularity of these peptide thioesters consists of the neutral loss of ethanethiol. The absence of the a(3) fragment ion and the presence of the (a(3)-17) ion on the collision-induced dissociation mass spectra are noted.
Synthesis of 5-haloethynyl- and 5-(1,2-dihalo)vinyluracil-nucleosides : Antiviral activity and cellular toxicityBioorganic & Medicinal Chemistry 13 (21) 6015-6024
In this article, we report the synthesis of hitherto unknown 5-haloethynyl and 5-(1,2-dihalo)vinyluracil nucleosides in the 2’-deoxy, 3’-deoxy- and ribosyl series, and we discuss their in vitro anti-HIV and anti-HCV activities and cellular toxicitites. As a result, on the basis of their selectivity index (SI) obtained with the HCV replicon system, but also on their cytotoxicity on peripheral blood mononuclear, CEM and VERO cell lines, the best compounds were the 5-bromoethynyluridine (SI = 3.2) and the 5-(1-chloro-2-iodo)vinyluridine (SI > 2.8). (c) 2005 Elsevier Ltd. All rights reserved.
Synthesis and antiviral activity of novel acyclic nucleosides in the 5-alkynyl- and 6-alkylfuro[2,3-d]pyrimidine seriesBioorganic & Medicinal Chemistry 13 (4) 1239-1248
The synthesis of novel acyclic nucleosides in the 5-alkynyl and 6-alkylfuro[2,3-d]pyrimidine series is described. These compounds were evaluated against HIV and HSV in order to determine their spectrum of antiviral activity. Their cytotoxicities against PBM, CEM and VERO cells were also determined. Compounds 21d and 24b displayed moderate EC50S of 2.7 and 4.9 muM, respectively, against HIV-1 and of 6.3 and 4.8 muM, respectively, against HSV. Nevertheless, these compounds also showed cellular toxicity, suggesting that the antiviral effects are secondary to the toxic effects. (C) 2004 Elsevier Ltd. All rights reserved.
Regioselective Michael-induced cyclisation of ?- and d-hydroxy vinyl sulfides and vinyl dithiocarbamatesTetrahedron Letters 46 (25) 4349-4352
We herein describe the unprecedented use of heteroaryl vinyl sulfides and vinyl dithiocarbamates as hetero-Michael addition acceptors. Combined chelating and electron-withdrawing effects Lire Postulated to stabilise the transient anionic species and allow smooth Michael-induced ring closure to produce diversely functionalised C-glycosides. (c) 2005 Elsevier Ltd. All rights reserved.
Cremer, GA ; Bureaud, N ; Lelievre, D ; Piller, V ; Piller, F ; Delmas, A (2004)
Synthesis of branched oxime-linked peptide mimetics of the MUC1 containing a universal T-helper epitopeChemistry-A European Journal 10 (24) 6353-6360
Our goal was to develop mimics of MUC1, highly immunogenic to induce an efficient immune response against the tumor-associated form of MUC1, and sufficiently different from the natural antigen to bypass the tolerance barrier in humans. With the aim of obtaining a well-defined peptide construct as a means of evoking the precise immune responses required in immunotherapy, we synthesized artificial mimics of the MUC1 protein composed of two MUC1 repeat units of inverse orientation and a universal T-helper epitope. To synthesize these heteromeric peptide constructs, we followed a convergent approach using chemoselective ligation based on oxime chemistry. A stem peptide was first synthesized bearing two orthogonally masked aldehydes. After successive depfotection, two oxime bonds can be specifically generated. The proposed strategy proved to be concise and robust, and allowed the synthesis of the tri-branched protein in a very satisfactory yield. The different constructs were tested for their ability to generate antibodies able to recognize the MUC1 protein.
The fragmentation of peptide acetals and peptide diols, corresponding to the hydrated form of the peptide aldehyde, is dominated by the successive losses of two molecules of MeOH and water, respectively. Using model peptides, the fragmentation mechanism, with respect to the loss of methanol and water, was elucidated. The first loss was certainly charge-directed whereas the second probably occurred via the nucleophilic attack of the nitrogen of an amine on the C-terminal carbon leading to a cyclic ion. Copyright (C) 2004 John Wiley Sons, Ltd.
Wittig approach to carbohydrate-derived vinyl sulfides, new substrates for regiocontrolled ring-closure reactionsTetrahedron 60 (8) 1817-1826
Reaction of methyl- and phenylthiomethylidene phosphoranes 1 and 2 with a variety of reducing sugars has been explored. Furano-type carbohydrates afforded with good yields the corresponding open-chain vinyl sulfides, whereas pyrano derivatives produced elimination compounds together with the expected vinyl sulfides, depending on the nature of the protective groups. (C) 2004 Elsevier Ltd. All rights reserved.
In this paper, the solid-phase synthesis of various substituted pyrimidine nucleosides is described starting from 2’-deoxyuridine (1), which has been attached through a base labile linker 2 to polystyrene resins. The utility of the Pd(0) cross-coupling to functionalized pyrimidine nucleosides is expanded herein to include reactions of resin-supported 5-iodo-2’-deoxyuridine (8) under Sonogashira, Stille, Heck, and Suzuki conditions. Upon cleavage with MeONa, a library of 5-substituted pyrimidine nucleosides (10a-e, 11a-c, 12a-e) was obtained in good (under Sonogashira and Stille conditions) to moderate (under Heck or Suzuki conditions) yields and high purity. Except the Suzuki-type reactions, the presented methods exhibit a significant improvement and facilitate the synthetic procedure with respect to purification and yields (determined after filtration over silica gel).
An efficient method for the synthesis of substituted furanopyrimidine nucleosides is described. Upon treatment with catalytic AgNO3, 5-alkynyl uracil derivatives were almost quantitatively converted into their corresponding bicyclic nucleoside analogues.
Frison, N ; Meunier, L ; Marceau, P ; Quetard, C ; Strecker, G ; Roche, AC ; Mayer, R ; Monsigny, M (2003)
Lewis a and Lewis x oligosaccharides Galbeta3(Fucalpha4)GlcNAcbeta3Galbeta4Glc and Galbeta4(Fucalpha3)GlcNAcbeta3Galbeta4Glc are easily isolated as a mixture from biological fluids, including human milk. However, because they behave almost identically in most chromatographic systems, it is difficult to have each of them as a pure compound. Incidentally, we found that they were easily separated by HPLC as glycosynthons [Galbeta3(Fucalpha4)GlcNAcbeta3Galbeta4Glc-Glp-betaAla-OBzl and Galbeta4(Fucalpha3)GlcNAcbeta3Galbeta4Glc-Glp-betaAla-OBzl] after substitution of the terminal reducing sugar by a short peptide (pyroglutamyl-betaalanyl-O-benzyl ester) in a one-pot two-step reaction (Carbohydr. Lett. 1 (1995) 269 ; Bioconjug. Chem. 9 (1998) 268).
We compared the properties of two peptides of identical size and amino acid composition, Ac-(LKKL)(5)-NHEt and Ac-(KL)(10)-NHEt. Both are amphipathic, but only Ac-(LKKL)(5)-NHEt is a potent promoter of negative curvature. CD studies performed in the presence of lipids confirmed that under these conditions Ac-(LKKL)(5)-NHEt forms an a-helix, and Ac-(KL)(10)-NHEt adopts a ß structure. We studied their binding affinity by centrifugation and isothermal titration calorimetry techniques. The Ac-(LKKL)5-NHEt bound to zwitterionic and anionic liposomes, while Ac-(KL)(10)-NHEt interacted mainly with anionic liposomes.
This review is a general survey of the main physico-chemical properties likely to distinguish a-helical from ß-sheet structured peptides. After giving a few basic notions of protein structures, their hierarchy and the determination methods, it focuses on the secondary structures. A large part of the review is devoted to the parameters affecting the preferential folding of a peptide chain into an a-helix or a ß-sheet. The environmental conditions of peptide polymorphism in solution but also at interfaces are described. The interactions of both kind of peptides with lipid membranes and the ensuing biological properties are described in the last part of the review. The topic is illustrated, throughout the text, by the behaviour of natural or designed antimicrobial peptides.
Straightforward synthesis of labeled and unlabeled pyrimidine d4Ns via 2 ’,3 ’-diyne seco analogues through olefin metathesis reactionsEuropean Journal of Organic Chemistry (4) 666-671
The synthesis of dideoxynucleosides (ddNs) or didehydro-dideoxynucleosides (d4Ns) from nucleosides has been extensively reviewed. While previously described methods are based on the modification of the 2’- and/or T-OH group of the intact ribose moiety, the use of a ring-closing metathesis (RCM) for the formation of the unsaturated cyclic system of nucleosides could be a straightforward approach to the d4Ns. Thus, as part of our drug labeling program, this paper reports a straightforward synthesis of 2’,3’-didehydro-2’,3’-dideoxyuridine (d4U) and [1’,2’,3’,4’,5’-C-13(5),6-C-13,1,3-N-15(2)]d4T using the RCM protocol. This paper discusses the preparation of nucleoside dienes and the activity of ruthenium-based metathesis catalysts. ((C) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003).
Frison, N ; Marceau, P ; Roche, AC ; Monsigny, M ; Mayer, R (2002)
In search of specific and highly selective sugar clusters for cell receptors, such as membrane lectins, various disaccharides were coupled to small peptide cores through an amide bond. In a first step, the reducing disaccharides, i.e. lactose and three different dimannoses, were converted into glycosyl-pyroglutamyl-ß-alanine derivatives. The free carboxylic group of these conjugates was then coupled to the a and epsilon amino groups of the core peptide (Lys(n)-Ala-Cys-NH2) with n = 1 to 5, with complete substitution leading to homogeneous glycoclusters. The thiol group of the cysteine residue was used to tag the glycosylated oligolysines upon reaction with fluorescein iodoacetamide. The affinity of these glycoclusters towards two plant lectins was assessed by surface plasmon resonance. The selectivity of their cell uptake was investigated by flow cytometry using two types of cells : a human hepatoma cell line (HepG2 cells) expressing the plasma membrane galactose-specific lectin, and monocyte-derived dendritic cells expressing the plasma membrane mannose-specific lectin. The glycoclusters containing four or five disaccharides were shown to bind plant lectins and cell surface membrane lectins with a narrow selectivity and with a high affinity.
Influence of polar support for the synthesis of large C-terminal peptide aldehyde : application to chemoselective ligationTetrahedron 58 (27) 5525-5533
Efficient conditions have been developed for the synthesis of large peptide aldehydes from solid support through nucleophilic displacement. Aminolysis of the ester bond between a deprotected peptide and the phenylacetamidomethyl linker with aminoacetaldehyde-dimethylacetal leads to a peptide aldehyde masked as an acetal. Besides the optimization of parameters such as solvents, workup procedure and temperature, the influence of the nature of the polymeric support was crucial. Among the solid supports tested, the poly(ethylene glycol)poly(acrylamide) resin proved to afford the best cleavage yield. This work underlines that the solid support has to be considered as a co-solvent rather than an inert carrier. Our methodology was further applied to the synthesis of a 33-mer with T-helper activity from the fusion protein of measles virus. The 33-mer peptide aldehyde was then chemoselectively ligated via an oxime bond to an (aminooxy) acetyl peptide with T-cytotoxic activity. (C) 2002 Elsevier Science Ltd. All rights reserved.
Leucine, a-methyl leucine and two peptides were exposed to space conditions on board the MIR station during the Perseus-Exobiology mission. This long duration space mission was aimed at testing the delivery of prebiotic building blocks. During this mission, two amino acids (leucine and a-methyl leucine) and two peptides (leucine-diketopiperazine and trileucine thioethylester) were exposed in Earth orbit for three months. Basalt, clay and meteorite powder were also mixed with the samples in order to simulate the effects of potential meteorite protection.
Sulfenic acids in the carbohydrate field. Synthesis of transient glycosulfenic acids and their addition to unsaturated acceptorsJournal of Organic Chemistry 67 (20) 6925-6930
A new method is described for building up anomeric glycosyl sulfoxides, via the formation of transient glycosulfenic acids and their addition to unsaturated acceptors. Thermolysis of alpha- and beta-3- [(2,3,4,6-tetra-O-acetyl-D-glucopyranosyl)sulfinyl]propanenitriles affords 1-glucosulfenic acids, which are reacted in situ with common substituted alkynes. The obtained (R-s,E)-2-[(2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl)sulfinyl] -2-butendioates are involved as enantiopure sulfinyl dienophiles in Diels-Alder reactions with 2,3-dimethyl-1,3-butadiene to evaluate the role that the sugar moiety plays in the steric control of the cycloaddition. This chemistry provides a direct synthetic strategy for the stereocontrolled connection between thioglycon and aglycon moieties, thus offering the basis for an easy elaboration of new molecules incorporating thiosugar residues.
Vallee, BS ; Tauc, P ; Brochon, JC ; Maget-Dana, R ; Lelievre, D ; Metz-Boutigue, MH ; Bureaud, N ; Schoentgen, F (2001)
Behaviour of bovine phosphatidylethanolamine-binding protein with model membranes - Evidence of affinity for negatively charged membranesEuropean Journal of Biochemistry 268 (22) 5831-5841
The ability of phosphatidylethanolamine-binding protein (PEBP) to bind membranes was tested by using small and large unilamellar vesicles and monolayers composed Of L-a -1,2-dimyristoylphosphatidylcholine, L-a -1,2-dimyristoylphosphatidylglycerol and L-a -1,2-dimyristoylphosphatidylethanolamine. PEBP only bound to model membranes containing L-a -1,2-dimyristoylphosphatidylglycerol ; the interaction was primarily due to electrostatic forces between the basic protein and the acidic phospholipids. Further experiments indicated that the interaction was not dependent on the length and unsaturation of the phospholipid acyl chains and was not modified by the presence of cholesterol in the membrane.
Knowing that human blood monocyte-derived dendritic cells express cell-surface mannose-specific lectins, we prepared various mannoses containing glycoconjugates with the aim of developing highly specific synthetic carriers of oligonucleotides and genes. Conjugates were prepared from oligosaccharides obtained by hydrazinolysis of Saccharomyces cerevisiae invertase glycopeptides.
To synthezise a triple-function branched peptide in a modular way, we present a new strategy based on orthogonal generation of two aldehyde functions from an acetal and a 2-amino alcohol. Successive unmaskings of aldehyde functions of the stem peptide affords stepwise chemoselective ligations of two (aminooxy)acetyl peptides via oxime bonds. (C) 2000 Elsevier Science Ltd. All rights reserved.
In-source fragmentation of peptide aldehydes and acetals : influence of peptide length and charge stateJournal of Mass Spectrometry 36 (10) 1149-1155
The use of in-source collision-induced dissociation (CID) was evaluated to generate structural information on peptide aldehydes, which represent an important class of compounds as inhibitors for serine and cysteine proteases and as key intermediates for protein engineering. By studying five peptide aldehydes of different lengths, and their peptide acetal counterparts, mass to charge (m/z) dependency of in-source fragmentation was established for peptides that differ only by their C-terminal functionalization. In-source fragmentation of peptide aldehydes and acetals leads to the same final ion, probably via a similar mechanism. Moreover, the gas-phase information obtained here reflects the equilibrium occurring in solution between the peptide aldehyde and its hydrated form, which was retained during the ionization process. The equilibrium constant was determined to be close to unity. Disturbance of this equilibrium should enable the stability of covalent hydration of a given series of aldehydes to be compared. Copyright (C) 2001 John Wiley & Sons, Ltd.
Comparative interaction of a-helical and ß-sheet amphiphilic isopeptides with phospholipid monolayersBiopolymers 59 (1) 1-10
The two sequential amphiphilic peptide isomers, (Leu-Lys-Lys-Leu)(4) and (Leu-Lys)(8), were chosen as models for a -helical and ß -sheet peptides, respectively. In order to evaluate the contribution of the secondary structure of a peptide to its penetration into cellular membranes, interactions of these isopeptides with L-a -dimyristoyl phosphatidylcholine (DMPC) monolayers were studied. Both isopeptides penetrate into DMPC monolayers up to an exclusion pressure of similar to 27 mN/m, but a discontinuity is observed in the penetration profile of the a -helical (LKKL)(4).
Bure, C ; Lelièvre, D ; Delmas, A (2000)
Identification of by-products from an orthogonal peptide ligation by oxime bonds using mass spectrometry and tandem mass spectrometryRapid Communications in Mass Spectrometry 14 (23) 2158-2164
Synthetic proteins with unusual architecture are obtained through chemoselective ligation, a method based on the condensation of unprotected peptides under mild aqueous conditions,. The last step of a new procedure leading to a tri-branched conjugate consists of the chemoselective ligation reaction between an (amioooxy)acetyl peptide and a peptide aldehyde resulting from a first ligation via an oxime bond. In order to optimize the reaction conditions, electrospray ionization mass spectrometry combined with Liquid chromatography and tandem mass spectrometry has been used.
Vasostatin-1, the natural N-terminal 1-76 chromogranin A (CGA)-derived fragment in bovine sequence, has been purified from chromaffin secretory granules and identified by sequencing and matrix-assisted laser desorption time-of-flight mass spectrometry. This peptide, which displays antibacterial activity against Gram-positive bacteria at micromolar concentrations, is also able to kill a large variety of filamentous fungi and yeast cells in the 1-10 mu M range. We have found that the C-terminal moiety of vasostatin-1 is essential for the antifungal activity, and shorter active peptides have been synthesized.
In these pages, short and efficient synthetic approaches to C-analogs of glucosinolates are described. Starting from D-glucose, C-glucotropaeolin (6) and C-glucocapparin (11) were synthesized in three steps. Preliminary enzymatic assays involving sulfatase and myrosinase have been performed. (C) 2000 Published by Elsevier Science Ltd. All rights reserved.