Martina Schnölzer

In situ neutralization in Boc-chemistry solid phase peptide synthesis

Simple, effective protocols have been developed for manual and machine-assisted Boc-chemistry solid phase peptide synthesis on polystyrene resins. These use in situ neutralization [i.e. neutralization simultaneous with coupling], high concentrations (>0.2xa0m) of Boc-amino acid-OBt esters plus base for rapid coupling, 100% TFA for rapid Boc group removal, and a single short (30xa0s) DMF flow wash between deprotection/coupling and between coupling/deprotection. Single 10xa0min coupling times were used throughout. Overall cycle times were 15xa0min for manual and 19xa0min for machine-assisted synthesis (75 residues per day). No racemization was detected in the .base-catalyzed coupling step. Several side reactions were studied, and eliminated. These included: pyrrolidonecarboxylic acid formation from Gln in hot TFA-DMF; chain-termination by reaction with excess HBTU; and, chain termination by acetylation (from HOAc in commercial Boc-amino acids). The in situ neutralization protocols gave a significant increase in the efficiency of chain assembly, especially for “difficult” sequences arising from sequence-dependent peptide chain aggregation in standard (neutralization prior to coupling) Boc-chemistry SPPS protocols or in Fmoc-chemistry SPPS. Reported syntheses include HIV-1 protease(1–50,Cys.amide), HIV-1 protease(53–99), and the full length HIV-l protease(1–99).

In Situ Neutralization in Boc-chemistry Solid Phase Peptide Synthesis

Simple, effective protocols have been developed for manual and machine-assisted Boc-chemistry solid phase peptide synthesis on polystyrene resins. These use in situ neutralization [i.e. neutralization simultaneous with coupling], high concentrations (>0.2xa0m) of Boc-amino acid-OBt esters plus base for rapid coupling, 100% TFA for rapid Boc group removal, and a single short (30xa0s) DMF flow wash between deprotection/coupling and between coupling/deprotection. Single 10xa0min coupling times were used throughout. Overall cycle times were 15xa0min for manual and 19xa0min for machine-assisted synthesis (75 residues per day). No racemization was detected in the .base-catalyzed coupling step. Several side reactions were studied, and eliminated. These included: pyrrolidonecarboxylic acid formation from Gln in hot TFA-DMF; chain-termination by reaction with excess HBTU; and, chain termination by acetylation (from HOAc in commercial Boc-amino acids). The in situ neutralization protocols gave a significant increase in the efficiency of chain assembly, especially for “difficult” sequences arising from sequence-dependent peptide chain aggregation in standard (neutralization prior to coupling) Boc-chemistry SPPS protocols or in Fmoc-chemistry SPPS. Reported syntheses include HIV-1 protease(1–50,Cys.amide), HIV-1 protease(53–99), and the full length HIV-l protease(1–99).

Ion-spray tandem mass spectrometry in peptide synthesis : structural characterization of minor by-products in the synthesis of ACP (65-74)

Ion-spray triple quadrupole mass spectrometry was used to investigate the products from the solid phase synthesis of the decapeptide (H)-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-(OH) [acyl carrier protein(65-74)]. The target sequence was assembled in stepwise fashion from the C-terminal using Boc chemistry on a Bly-OCH2-Pam-copoly(styrenedivinylbenzene) resin. The product was deprotected and cleaved from the resin by treatment with HF/p-cresol for 1 h at 0 degrees C. The crude product was analyzed by reverse-phase HPLC and contained a single major peptide component, one significant minor (late-eluting) component and several trace-level peptide by-products. The components were separated by HPLC and the fractions directly analyzed by mass spectrometry and tandem mass spectrometry. The major product was confirmed as the desired ACP(65-74). The significant minor component was apparently from incomplete deprotection of Asp70, an artifact of this particular experiment. The trace by-products were found to arise from succinimide formation at Asp70, succinimide formation at Asn73, acylation of the Tyr71 side chain phenolic hydroxyl leading to a branched heptadecapeptide, and tert-butylation of the decapeptide. The possible origins of these by-products are discussed in light of known peptide chemistry. Also notable was the absence, to very low detection levels, of by-products frequently reported to occur in peptide synthesis, illustrating the high degree of refinement and the accuracy of currently used synthetic methods.

Synthesis of Proteins by Chemical Ligation of Unprotected Peptide Segments: Mirror-Image Enzyme Molecules, D- & L-HIV Protease Analogs

Publisher Summary This chapter discusses the synthesis of proteins by chemical ligation of unprotected peptide segments. In a study described in the chapter, protected d - and l -amino acids were obtained. The 99-residue monomer of the HIV-1 protease molecule was prepared by directed ligation of functionalized unprotected segments. These segments were assembled in separate syntheses by a highly optimized machine-assisted SPPS protocol using Boc-chemistry performed on a modified ABI 430A synthesizer. The protocol comprised removal of the Nα-Boc group with undiluted TFA (2-min total) followed by a DMF flow wash to give the TFApeptide-resin salt, and a single 10 min coupling step using HBTU activated Boc-amino acids and in situ neutralization with DIEA in DMF. [αCOSH]HIV-l PR(1–51) peptide was assembled on 4-[α-(Boc-Gly-S)benzyl]phenoxyacetamidomethyl-resin (10), and [Na-BrCH2CO]HIV-l PR(53–99) was prepared by bromoacetylation of [Aba67,95]HIV-l PR(53–99)-OCH2 Pam peptide- resin. All peptides were cleaved and deprotected by high HF treatment.

Breaking the shackles of the genetic code: engineering retroviral proteases through total chemical synthesis.

Since early this century one of the fundamental goals of organic chemistry has been to understand the molecular basis of enzyme action (1). These protein molecules are the machines of the cell, catalyzing essentially all of the chemical reactions in the biological world. The action of enzymes is characterized by extraordinary specificity and by the ability to effect, in aqueous solvent and at normal temperatures, chemical transformations that are otherwise imperceptibly slow. Early approaches to the study of enzymes involved the laborious and painstaking isolation of pure molecules from biological sources, such as tissue homogenates. Only indirect methods, such as selective chemical modification of amino acid side chains, could be used to study the functional imperatives of the protein molecule itself. Consequently, much more was known about the (usually low MW) substrates than about enzymes themselves.