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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.
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.
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.
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.
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.
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.
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 :
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.
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.
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.
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.
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.
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.
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).
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.
Poly(Leu-Lys-Lys-Leu) and poly(Leu-Lys) are sequential amphiphilic peptide isomers that adopt respectively an a-helical conformation and a ß-sheet structure in saline solutions and at rite air/water interface. The surface active properties of LKKL and LK sequential isopeptides containing 16, 20, and n residues have been compared in order to evaluate the contributions of the a-helical and ß-sheet conformations. Both have a natural tendency to spread at the surface of a saline solution and the values of the equilibrium spreading pressure pi(e) lie in the same range. When dissolved in a saline solution, a-helical peptides diffuse faster and adsorb faster at the interface than the ß-sheet isomers.
We describe an efficient solid-phase synthesis of C-terminal peptide aldehyde. Making use of the stability of the PAM linker towards both acid and base conditions, a pentapeptide was synthesized starting from a PAM resin according to Fmoc/tBu chemistry. The side-chains were deprotected by TFA. The peptide was cleaved by aminolysis with aminoacetaldehyde-dimethylacetal leading to a C-terminal masked aldehyde. The unprotected peptide aldehyde was then coupled to amino-oxy derivatives by chemoselective ligation in aqueous solution. (C) 1998 Elsevier Science Ltd. All rights reserved.
We have previously shown that when administered to mice without adjuvant, a chimeric peptide consisting of the fusion peptide F from measles virus protein linked at the C-terminus of a cytotoxic T-cell epitope from the M2 protein of respiratory syncytial virus efficiently primes for an major histocompatibility complex (MHC) class-I restricted cytotoxic T lymphocyte (CTL) response. In this report, we demonstrated by microspectrofluorometry that the fusion-peptide moiety bound to the plasma membrane of living cells.
Synthetic peptides constructed with doublets of hydrophobic residues tandemly repeated with doublets of positively charged residues, (Leu-Lys-Lys-Leu)(n), were used as models for the study of protein-membrane interactions. Their behaviour has been compared with that of their strictly alternating iso peptides, (Leu-Lys)(n). Both peptides present a random coil structure in pure water. In saline solutions, (Leu-Lys-Lys-Leu)(n) peptides adopt an a-helical structure whereas (Leu-Lys)(n) transit into a ß-sheet structure.
An RNA cleaving catalyst combined to an antisense DNA may represent a new approach for gene targeted therapy. As cleaving agents, we used basic polypeptides under the ß-sheet or a-helix conformations, Molecular modeling studies were used in an attempt to design a second generation of artificial ribonucleases, taking into account the three-dimensional arrangement of functional groups in the peptide/RNA complexes. Such computer aid in rational design processes appears as an original and promising approach.
The peptide (TSP)(4) is an efficient substrate for GalNAc transferase.(1) To localize the active site of GalNAc transferase, Biotin-(TSP)(4)-Lys(ASA)-G was prepared by on-line solid phase synthesis using Fmoc strategy. The new protecting group, 1-(4,4-dimethyl-2,6-dioxocyclohexy-1-ylidene) (Dde) was used as a temporary protecting group for N-epsilon-lysine. The bi-derivatized peptide was characterized by mass spectrometry and FTIR spectrometry. This strategy can be generalized to other bi-derivatized peptides synthesized for ligand-receptor studies.
Vibrational CD (VCD) and electronic CD (ECD) spectra of some sequential Lys and Leu based oligo- and polypeptides were studied as a function of added salt and (for ECD) as a function of concentration in aqueous solution. For these samples, the VCD spectra can only be measured at relatively high concentrations under which the well-known salt-induced transition to a ß-sheet form can be observed for the KL based species, but only the end-state a-helical conformation is obvious for the LKKL based samples. ECD concentration dependence demonstrates that, at high concentration with no added or with added salt, LKKL based oligomers and polymers give a-helical spectra. These data provide evidence of aggregation induced secondary structure formation in an exceptionally simple peptide system. Similarly, the KL based oligomers and polymers give ß-sheet like spectra at high concentration or at high salt.
Ingénieur de recherche , Protéines de synthèse et chimie bioorthogonale