Agnès F. Delmas

Synthesis and Biological Evaluation of a Multiantigenic Tn/TF‐Containing Glycopeptide Mimic of the Tumor‐Related MUC1 Glycoprotein

Glycoprotein glycans have been shown to play essential roles in many biological processes such as protein folding, cell–cell interactions, and tumor development. The isolation of native or recombinant glycoproteins yields heterogeneous carbohydrate moieties leading to different glycoforms, which may exhibit different properties. The chemical synthesis of O-glycoproteins presents the advantage of producing well-defined constructs. However, such syntheses are truly challenging due to the multifunctionality of polypeptides and carbohydrates. One of the principal synthetic strategies relies on the synthesis of peracetylated O-glycosyl amino acid building blocks, which are subsequently used in solid-phase glycopeptide syntheses. For more complex or longer glycopeptides containing native O-glycosidic bonds, convergent strategies based on protected glycopeptides or native chemical ligation have been proposed. O-Glycosylation is found on many soluble and membrane proteins. It is present in the variable tandem repeat part of mucins, which, like the membrane-associated mucin MUC1, are overexpressed as underglycosylated forms on the surface of tumor cells. This altered expression of carbohydrate moieties that expose well-characterized tumor-associated epitopes such as Tn (a-GalNAc), TF (GalbACHTUNGTRENNUNG(1,3)a-GalNAc), and their sialylated forms, as well as the APDTR peptide epitope from the repeat unit of MUC1, have been exploited in immunotherapy. Elegant and demanding syntheses have afforded carbohydratebased cancer vaccines which are currently being tested in clinical trials. However, carbohydrate antigens are mostly limited to IgM production. The switch to a carbohydrate-specific IgG response is possible if tumor-associated carbohydrate moieties are linked to a T-helper epitope. 15] However, the antibody response is not restricted to the carbohydrate moiety but may be directed against the aglycone backbone. It is therefore preferable to find synthetic constructs composed of tumor-related MUC1 glycopeptides linked to a T-helper epitope. To evaluate these new immunogens at the biological and immunological levels, it is crucial to produce them in substantial amounts. Thus, a highly convergent strategy is needed. Moreover, to be useful for human immunotherapy the construct has to be immunogenic under conditions applied in the clinic, where it may be associated only with mild adjuvants. However, it must be capable of overcoming the immunological tolerance often observed with tumor-associated self antigens. Therefore, in this study, we focused on the synthesis of a glycopeptide construct containing the universal Pan DR epitope (PADRE) and three MUC1 repeat units, two of them carrying the Tn and the TF carbohydrate epitopes, respectively, on the Thr residue of the immunodominant sequence PDTRP. Its immunogenic properties were evaluated in mice, and the antisera produced were characterized with synthetic and natural tumor antigens. Our synthetic methodology is based on a modular multiligation scheme with the synthesis of a stem peptide bearing two aldehydes masked as 2-amino alcohol and acetal. Successive unmaskings of the aldehyde groups with periodate oxidation and trifluoroacetic acid (TFA) were followed by the oximation reaction with an aminooxyacetic acid (Aoa)-containing peptide. To adapt this convergent strategy to the synthesis of glycopeptides, two points need consideration: 1) the partner, aldehyde or aminooxy, on which the carbohydrate moieties will be introduced; 2) the deacetylation step of the hydroxy groups of the carbohydrate moieties by mild methanolysis. Herein, we demonstrate the potential of our approach by synthesizing a tri-branched glycoprotein construct using Aoa-containing glycopeptide building blocks carrying unprotected carbohydrate moieties. Aoa-containing glycopeptides 1 and 2 were obtained by solid-phase peptide synthesis (SPPS, Scheme 1). The carbohydrate moieties were introduced at Thr12 of the APDTR epitope as peracetylated N-Fmoc-Thr ACHTUNGTRENNUNG(Tn/TF)-OH building blocks. The elongation was carried out with an automatic synthesizer using an Fmoc/tBu strategy starting from a p-benzyloxybenzyl alcohol resin (Wang resin) using (1-H-benzotriazoleN,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU)/ 1-hydroxybenzotriazole (HOBt)/iPr2NEt in N-methylpyrrolidine (NMP) as coupling reagents up to the O-glycosylated Thr. Peracetylated Fmoc-Thr(Tn)-OH and Fmoc-Thr(TF)-OH were introduced manually using only 1.1 equiv O-glycosyl amino acid/ O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluroniumhexafluorophosphate (HATU)/iPr2NEt in NMP. The remaining free amino groups were capped with acetic anhydride/HOBt/ iPr2NEt. After the introduction of O-glycosylated Thr12, the Nterminal amino acids were automatically coupled through activation with HBTU/HOBt/iPr2NEt (10 equiv). The Wang linker and the acid-labile side-chain protecting groups were then cleaved simultaneously with TFA/iPr3SiH/H2O (95:2.5:2.5). Subsequent transesterification with NaOMe/methanol at pH 8.5 and 9 afforded the desired Aoa-containing glycopeptides 1 and 2, respectively. It is notable that the unprotected Aoa group is compatible with the mild methanolysis treatment needed to remove the protecting groups from the carbohy[a] Dr. G.-A. Cremer, N. Bureaud, Dr. V. Piller, Dr. F. Piller, Dr. A. F. Delmas Centre de Biophysique Mol culaire, UPR 4301 CNRS affiliated with the University of Orl ans and INSERM rue Charles Sadron, 45071 Orl ans Cedex 2 (France) Fax: (+33)238631517 E-mail : delmas@cnrs-orleans.fr [b] Prof. Dr. H. Kunz Institut f:r Organische Chemie Johannes Gutenberg-Universit>t Mainz Duesbergweg 10–14, 55128 Mainz (Germany) Supporting information for this article is available on the WWW under http://www.chemmedchem.org or from the author.

Identification of by-products from an orthogonal peptide ligation by oxime bonds using mass spectrometry and tandem mass spectrometry

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 (aminooxy)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. In addition to the target tri-branched conjugate, two other conjugates were characterized allowing documentation of transoximation reactions in peptide chemistry. A fourth conjugate was identified as a side product appearing after the first ligation. Data obtained by low-energy collision-induced dissociation led to a rapid and reliable identification of impurities observed in the (aminooxy)acetyl peptide despite a previous high performance liquid chromatography (HPLC) purification. This highlights the great reactivity of the aminooxy group towards carbonyl-containing compounds.

Influence of polar support for the synthesis of large C-terminal peptide aldehyde: application to chemoselective ligation

Abstract 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.

Synthesis of peptide di-aldehyde precursor for stepwise chemoselective ligations via oxime bonds

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.

Three-dimensional NMR Structure of Hen Egg Gallin (Chicken Ovodefensin) Reveals a New Variation of the β-Defensin Fold

Background: Ovodefensins are small peptides from eggs, related to avian antimicrobial defensins. Results: The first three-dimensional structure of ovodefensins (gallin) is solved, and its antimicrobial properties are screened. Conclusion: Gallin adopts a β-defensin fold, with significant variations. Its antibacterial spectrum was restricted to E. coli. Significance: The first structural features may be related to E. coli specificity and/or other yet unknown functions. 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 β-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 β-sheet and the disulfide bridge array typical of vertebrate β-defensins. Gallin can therefore be unambiguously classified as a β-defensin. However, an additional short two-stranded β-sheet reveals that gallin and presumably the other ovodefensins form a new structural subfamily of β-defensins. Moreover, gallin and the other ovodefensins calculated by homology modeling exhibit atypical hydrophobic surface properties, compared with the already known vertebrate β-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.

A folded and functional synthetic PA1b: An interlocked entomotoxic miniprotein

PA1b (Pea Albumin 1, subunit b) is a hydrophobic, 37‐amino acid miniprotein isolated from pea seeds (Pivum sativum), crosslinked by three interlocked disulfide bridges, signature of the ICK (inhibitory cystine‐knot) family. It acts as an entomotoxic factor against major insect pests in stored crops and vegetables, making it a promising bioinsecticide. Here we report an efficient and simple protocol for the production of large quantities of highly pure, biologically active synthetic PA1b. The features of PA1b oxidative refolding revealed the off‐pathway products and competitive aggregation processes. The efficiency of the oxidative folding can be significantly improved by using hydrophobic alcoholic cosolvents and decreasing the temperature. The homogeneity of the synthetic oxidized PA1b was established by reversed‐phase HPLC. The correct pairing of the three disulfide bridges, as well as the three‐dimensional structure of synthetic PA1b was assessed by NMR. Synthetic PA1b binds to microsomal proteins from Sitophilus oryzae with a Kd of 8 nM, a figure quite similar to that determined for PA1b extracted from its natural source. Moreover, the synthetic miniprotein was as potent as the extracted one towards the sensitive strains of weevils. Our findings will open the way to the production of PA1b analogues by chemical means to an in‐depth understanding of the PA1b mechanism of action.

Synthesis and analytical investigation of C-terminally modified peptide aldehydes and ketone: application to oxime ligation

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. Copyright

Histidine-rich peptide: evidence for a single zinc-binding site on H5WYG peptide that promotes membrane fusion at neutral pH

The histidine-rich peptide H5WYG (GLFHAIAHFIHGGWHGLIHGWYG) was found to induce membrane fusion at physiologic pH in the presence of zinc chloride. In this study, we examined the ion selectivity of the interaction of Zn(2+) with H5WYG. This investigation was conducted by using adsorption at air/water interface and mass spectrometry. We found that a peptide-metal complex is formed with Zn(2+) ions. Electrospray ionisation-mass spectrometry (ESI-MS) reveals that the [H5WYG + Zn + 2H](4+), [H5WYG + Zn + H](3+) and [H5WYG + Zn](2+) ions, appearing by increasing the amount of Zn(2+) equivalent, correspond to a monomolecular H5WYG - Zn(2+) complex. Tandem mass spectrometry (MS/MS) provides evidence for the binding of the single Zn(2+) ion to the H(11) and H(19) and probably H(15) residues.

Complete 1H, 15N and 13C assignment of trappin-2 and 1H assignment of its two domains, elafin and cementoin

AbstractnTrappin-2 is a serine protease inhibitor with a very narrow inhibitory spectrum and has significant anti-microbial activities. It is a 10xa0kDa 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.

The Addressing Fragment of Mitogaligin: First Insights into Functional and Structural Properties

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.