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A statistical view of protein chemical synthesis using NCL and extended methodologies
Chemical synthesis of proteins: Trends in Biotechnology
When compared with recombinant hsPLA2 and synthetic hsPLA2 obtained from a two-segment ligation (), the current four-segment hsPLA2 ligation product and refolded enzyme had the correct mass and activity and was enzymatically indistinguishable from the recombinant enzyme (Table ). The striking shift of maximal intensity from the 16th and 17th charged state in the reduced polypeptide chain to the 7th charged state in the refolded hsPLA2–Bta-47 (Fig. C) is characteristic behavior of folded globular proteins compared with their denatured counterparts (, ).
A number of synthetic peptides are significant commercial or pharmaceutical products, ranging from the dipeptide sugar‐substitute aspartame to clinically used hormones, such as oxytocin, adrenocorticotropic hormone, and calcitonin. This unit provides an overview of the field of synthetic peptides and proteins, including their purification. It discusses selecting the solid support and common coupling reagents. Additional information is provided regarding common side reactions and synthesizing modified residues.
The 5th international conference on Chemical Protein Synthesis ..
Modularity in synthesis allows the combination of multiple structural elements to allow recognition of larger protein surfaces and the synthesis of multifunctional "proteins." The post-genomic era requires novel tools to elucidate the identity, classes and modes of protein-protein interactions in disparate cell types, developmental stages, and intracellular environments, in addition to changes due to age and disease states.
Our work involves the development of appropriate, readily accessible organic scaffolds, in solution and on solid phase, using modern methods of organic synthesis.
New Methods for Chemical Protein Synthesis - …
hsPLA2 1 (–), 2 (28–58), and 3 (59–87) (Fig A) only were synthesized on TAMPAL resin (0.25-mmol scale) to yield C-terminal MPAL-activated thioesters; peptide 4 (88–124) was synthesized on Cys-phenylacetamidomethyl resin to yield a C-terminal carboxylic acid group after deprotection and cleavage of the peptide resin (Fig. A). Peptides 2 and 3 were N-terminally protected with a 2-(methylsulfonyl)ethyl carbonate (Msc) group by a 2-hr incubation with a 10-fold excess of activated Msc–nitrophenyl ester in a minimal volume of DMF/5% DIEA. In a parallel synthesis, Bta-47 replaced His-47 in peptide 28–58. After deprotection and cleavage from the resin, the polypeptides were HPLC-purified, lyophilized, and stored at −20°C until use.
After trityl deprotection, 1.05 mmol of TAMPAL resin was divided into 20 glass tubes (0.05 mmol), and each was incubated for 1 hr with each of the 20 preactivated amino acids (1.1 mmol of X preactivated with 1 mmol of HBTU in the presence of 3 mmol of DIEA). After coupling, the resins were combined in four pools (pool 1, W, E, D, T, S; pool 2, R, Q, N, P, G; pool 3, F, M, I, V, A; and pool 4, Y, K, L, C, H), and LYRAX syntheses were continued in a parallel fashion (0.25 mmol of resin each, by using 1.1 mmol of amino acid preactivated with 1 mmol of HBTU in the presence of 3 mmol of DIEA). After synthesis, the LYRAX pools were Nα-acetylated, cleaved from the resin, and lyophilized. Nα-Amino CRANK peptide was synthesized on a 0.7 mmol scale on MBHA resin by using 2 mmol of preactivated amino acid per coupling (2.2 mmol of amino acid activated with 2 mmol of HBTU in the presence of 6 mmol of DIEA). After HF treatment of the peptide-resin, CRANK was lyophilized and stored at −20°C.
New Methods for Chemical Protein Synthesis 183.
Chemical approaches to the synthesis of peptides and proteins
Solid phase peptide synthesis
New Methods for Chemical Protein Synthesis …
Outlines the major steps in the process of protein synthesis, ..
Chemical Synthesis of Peptides and Small Proteins
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Disulfide formation on the resin after deprotection of the trityl-protected thiol could decrease the synthetic yield. This side reaction has not been a problem, although care should be taken to avoid pulling air through the resin after trityl deprotection. Synthetic yields of hsPLA2 (all fragments synthesized on TAMPAL resin) were between 73% and 85% of the theoretical final peptide-resin weight, whereas the synthesis yield of fragment 88–124 (on Cys-phenylacetamidomethyl resin) was 72%. Moreover, when the resin was treated with 2-mercaptoethanol (5%) in DMF for 5 min after trityl removal, no increase in synthetic yield was observed.
Department of Chemistry | UMass Amherst
To facilitate the synthesis of C-terminal thioester peptides, a general and straightforward procedure has been developed that generates a resin from which any desired thioester peptide can be readily synthesized. This thioester resin is a generalized and significantly simplified variant of that described by Hojo et al. (). After chain assembly and deprotection, the resulting C-terminal activated thioester peptides can be used in native-chemical ligation without further modification. The efficacy of all 20 naturally occurring amino acids in native chemical ligation reactions has been demonstrated by synthesis and analysis of defined mixtures of peptides. The utility of this approach was demonstrated by the total chemical syntheses of 124-aa human secretory phospholipase A2 (hsPLA2) and an active-site chemical mutant in which His-47 was replaced by the isosteric β-thienyl alanine (Bta).
Gierasch was awarded the Ralph F
Native chemical ligation has been developed to facilitate the synthesis of proteins of moderate size (≈150 aa) (–). This ligation chemistry involves a chemoselective reaction between a C-terminal thioester and an N-terminal cysteine residue to yield a native peptide bond at the site of ligation (). One limitation to this approach has been the efficient synthesis of C-terminal thioester peptides. Traditionally, these peptides have been obtained in two steps through selective alkylation of thioacid peptides () generated from optimized SPPS () by using an HF-labile thioacid linker (, ). However, the need for an individual-solution synthesis of a tert-butoxycarbonyl (Boc)–amino–thioacid linker for each C-terminal amino acid desired has limited the general application of this approach (). Furthermore, deprotection and cleavage of the polypeptide chain from the resin by anhydrous HF yields the corresponding C-terminal thioacid (COSH) that requires alkylation and purification to obtain the activated thioester (COSR). As a result, only a limited set of ligation pairs has been examined by using Ala (), Leu (), or Gly (, ) as the C-terminal thioester residue from individual syntheses of amino acid thioacid linkers (). Other approaches can be used to generate C-terminal thioester peptides directly (, , ), for example Asn, but also suffer from the requirement for synthesizing an amino acid–thioester linker of each individual amino acid.
The Current State of Peptide Drug Discovery: Back to …
To facilitate the synthesis of thioester peptides with a wide variety of C-terminal amino acids, a general thioester peptide-producing resin linker for Boc chemistry–SPPS was designed. Starting with the HF-labile MBHA resin, leucine was coupled followed by S-trityl mercaptopropionic acid by using standard SPPS conditions (). The resulting TAMPAL resin can be used as a starting resin for polypeptide-chain assembly after removal of the trityl protecting group. The desired thioester bond can be generated directly on the resin () by using any amino acid and standard coupling conditions. This peptide–linker had been previously shown to be compatible with Boc–SPPS with glycine at the C terminus and with norleucine as a spacer to provide greater stability during synthesis (). After HF cleavage and purification, the C-terminal MPAL–thioester peptide can be used directly in native chemical ligation after in situ thioester exchange (Fig. A).
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