Ernesto Nicolás

Formation of aspartimide peptides in Asp-Gly sequences

Abstract The behaviour of protected H-Val-Lys-Asp-Gly-Tyr-Ile-OH towards imide formation was evaluated. The use of a carboxylic acid temporary protecting group is not a safe strategy for avoiding aspartimide formation during HF treatment. Piperidine showed a marked tendency to form aspartimides during Fmoc deprotection.

A study of the use of NH4I for the reduction of methionine sulfoxide in peptides containing cysteine and cystine

Methionine sulfoxide reduction by NH4I has been studied in some disulfide containing peptides. In general, this reagent has proved to be effective in neat TFA at 0°C, with the obtention of the unprotected peptides in more than 99% yields and without reduction of the disulfide bridge bond. The use of Me2S as an additive resulted in faster reactions and disulfide scrambling was not observed. Whereas the Acm group proved to be stable to the reaction conditions, unprotected cysteine containing peptides afforded the corresponding parallel dimers.

ENPDA: an evolutionary structure-based de novo peptide design algorithm

SummaryOne of the goals of computational chemists is to automate the de novo design of bioactive molecules. Despite significant advances in computational approaches to ligand design and binding energy evaluation, novel procedures for ligand design are required. Evolutionary computation provides a new approach to this design endeavor. We propose an evolutionary tool for de novo peptide design, based on the evaluation of energies for peptide binding to a user-defined protein surface patch. Special emphasis has been placed on the evaluation of the proposed peptides, leading to two different evaluation heuristics. The software developed was successfully tested on the design of ligands for the proteins prolyl oligopeptidase, p53, and DNA gyrase.

Analysis of recombinant human erythropoietin and novel erythropoiesis stimulating protein digests by immunoaffinity capillary electrophoresis-mass spectrometry

In this work, we demonstrate that detection of a specific peptide marker by immunoaffinity capillary electrophoresis-mass spectrometry (IA-CE-MS) could be used to confirm the presence of recombinant human erythropoietin (rhEPO) in solution. Besides the carbohydrate content, the amino acid sequence of novel erythropoiesis stimulating protein (NESP) differs from human erythropoietin (hEPO) at five positions (Ala30Asn, His32Thr, Pro87Val, Trp88Asn, and Pro90Thr). After digesting both glycoproteins in solution by trypsin and PNGase F, two specific proteotypic peptides, EPO (77-97) and NESP (77-97) which differ in three amino acids, were selected as rhEPO and NESP markers, respectively. Both digests and their mixtures were analyzed by IA-CE-MS. The IA stationary phase was prepared from a custom made polyclonal anti-EPO (81-95) antibody immobilized on a solid support of CNBr-Sepharose 4B and was packed in a microcartridge near the inlet of the separation capillary. As the antibody was directed to a synthetic peptide EPO (81-95), only the proteotypic peptide EPO (77-97) was retained. The retained peptide was eluted, separated by electrophoresis and detected by MS. The method was specific to confirm the presence of rhEPO in solution. Although the limits of detection for the peptide marker were similar to those obtained with CE-MS (a few mg/L), these results show the potential of this novel approach to detect in the future rhEPO and its analogues selectively and unambiguously at the levels expected in biological fluids.

REDUCTION OF METHIONINE SULFOXIDE WITH NH4I/TFA: COMPATIBILITY WITH PEPTIDES CONTAINING CYSTEINE AND AROMATIC AMINO ACIDS

Abstract The reduction of methionine sulfoxide with ammonium iodide in trifluoroacetic acid has been studied in peptides containing cysteine, histidine, tyrosine or tryptophan residues. While histidine and tyrosine have proved to be stable under the experimental conditions, cysteine is oxidized to cystine and tryptophan dimerizes to form 2-indolylindolenine derivatives. The use of methyl sulfide to increase the reduction rate minimizes the problem and protection of indole ring with the formyl group avoids the side reaction for this amino acid.

Convergent solid phase peptide synthesis. v. synthesis of the 1-4, 32-34, and 53-59 protected segments of the toxin ii of androctonus australis hector.

Abstract Two protected peptide segments corresponding to the sequence 32-34 and 53-59 of toxin II of the north African scorpion Androctonus australis Hector have been synthesized on a photolabile Nbb-resin using the TFA-labile Boc α-amino protection and HF-labile side chain protecting groups. A third protected peptide corresponding to segment 1-4 has been synthesized on the same resin but with a t-butyl group for β protection of aspartic acid and a Z group on the lysine side chain. For this last segment a combination of Boc and Fmoc groups for α -amino protection has been used successfully on the Nbb-resin. After photolysis the crude peptides have been treated by solvent extraction and semi-preparative HPLC to yield highly purified segments. These syntheses show the flexibility of the convergent solid phase approach and how segments with different and independent protecting groups can be assembled by solid phase peptide procedure.

Catalytic C–H Activation of Phenylethylamines or Benzylamines and Their Annulation with Allenes

Tetrahydro-3-benzazepines and tetrahydroisoquinolines are synthesized in one step from allenes and phenylethylamines or benzylamines, respectively. Mechanistically, it is assumed that activation of a C-H bond of an aromatic ring with Pd(II) occurs, directed by the primary amine, leading to the formation of a palladacycle into which an allene then undergoes insertion. The resulting π-allyl intermediate cyclizes to the products by an intramolecular allylic alkylation. The process is particularly useful with 2,3-butadienoates and amines having a quaternary carbon at the α-position.

A new approach to Hmb-backbone protection of peptides: Synthesis and reactivity of Nα-Fmoc-Nα-(Hmb)amino acids

Abstract The use of N -Fmoc- N -(Hmb)amino acids for the introduction of the Hmb backbone protection into peptides during solid-phase synthesis is described. An efficient two step synthesis of these derivatives is reported and their coupling to the peptide chain is studied.

THE USE OF THE NBB-RESIN FOR THE SOLID-PHASE SYNTHESIS OF PEPTIDE ALKYLESTERS AND ALKYLAMIDES. SYNTHESIS OF LEUPROLIDE

The use of nucleophiles for the cleavage of an o-nitrobenzylester peptide-resin bond in order to afford peptide alkylesters and alkylamides has been studied. With this purpose, three short peptide sequences anchored to a Nbb-resin were used as models. Peptides were cleaved from the polymeric supports by reaction with primary and secondary amines and by a transesterification process with yields that depended on the nucleophile and the C-terminal amino acid of the sequence. This methodology was applied to the synthesis of leuprolide, an ethylamide peptide of relevant pharmacological interest, which was obtained in a 70% overall yield.

Solid-phase synthesis: a linker for side-chain anchoring of arginine

Abstract A new linker based on a chroman system is described for the side-chain anchoring of Arg and other guanidine-containing molecules. The system is compatible with the Fmoc/tBu solid-phase strategy, because the release of the final product is achieved by treatment with TFA in the presence of scavengers.