Peptide bond formation by aminolysin-A catalysis: a simple approach to enzymatic synthesis of diverse short oligopeptides and biologically active puromycins

A new S9 family aminopeptidase derived from the actinobacterial thermophile Acidothermus cellulolyticus was cloned and engineered into a transaminopeptidase by site-directed mutagenesis of catalytic Ser(491) into Cys. The engineered biocatalyst, designated aminolysin-A, can catalyze the formation of peptide bonds to give linear homo-oligopeptides, hetero-dipeptides, and cyclic dipeptides using cost-effective substrates in a one-pot reaction. Aminolysin-A can recognize several C-terminal-modified amino acids, including the l- and d-forms, as acyl donors as well as free amines, including amino acids and puromycin aminonucleoside, as acyl acceptors. The absence of amino acid esters prevents the formation of peptides; therefore, the reaction mechanism involves aminolysis and not a reverse reaction of hydrolysis. The aminolysin system will be a beneficial tool for the preparation of structurally diverse peptide mimetics by a simple approach.     

Investigations on the enzyme specificity of clostripain: a new efficient biocatalyst for the synthesis of peptide isosteres

To explore the ability of the cysteine protease clostripain as a biocatalyst for the synthesis of peptide isosteres, the S'-subsite specificity of this enzyme toward unnatural substrates was investigated. First, the function of clostripain for acylating aliphatic noncyclic and cyclic amines varying in chain length and ring size was analyzed using a standard acyl donor. Additionally, this series was expanded by use of aromatic amines, amino alcohols, derivatives of non-alpha-amino carboxylic acids, and symmetric and asymmetric diamines, respectively. The results obtained give a detailed picture of the unique reactivity of clostripain toward synthetic substrates, allowing insights into the basic enzyme-substrate interactions. Furthermore, the data provide a guideline for the use of clostripain as a biocatalyst for synthesis of peptide isosteres. The study was completed by the utilization of a model substrate mimetic enabling clostripain to react with noncoded and non-amino acid-derived amines as well as nonspecific acyl moieties. The results of this study indicate that this approach may extend the application range of clostripain as a biocatalyst outside of peptide synthesis.     

Toward fully synthetic glycoproteins by ultimately convergent routes: a solution to a long-standing problem

A method is disclosed for the convergent synthesis of multiply glycosylated peptides. The approach centers on a convergent technique for generating masked, complex glycopeptide-containing C-terminal acyl donors. Activation of the latent donor in situ and use directly in segment coupling with a second peptide bearing a complex carbohydrate produces a completely unprotected, bifunctional glycopeptide. The system demonstrates a minimum level of hydrolysis and epimerization at the C-terminal amino acid residue of the acyl donor during fully convergent segment coupling and is therefore a powerful tool for the synthesis of glycoproteins.     

Fmoc synthesis of peptide thioesters without post-chain-assembly manipulation

An operationally simple method for the synthesis of peptide thioesters is developed using standard Fmoc solid-phase peptide synthesis procedures. The method relies on the use of a premade enamide-containing amino acid which, in the final TFA cleavage step, renders the desired thioester functionality through an irreversible intramolecular N-to-S acyl transfer.     

Visible light photoredox catalysed amidation of carboxylic acids with amines

A visible-light promoted photoredox catalysed, green one-pot approach for the amidation of carboxylic acids with amines has been developed for the synthesis of diverse aliphatic and aromatic amides. The proposed strategy is extendable also to biologically active amides and could represent a low-cost alternative to the common synthetic pathways. The developed strategy may hold great potential for a comprehensive display of biologically interesting peptide synthesis and amino acid modification.     

An improved approach for the synthesis of alpha,alpha-dialkyl glycine derivatives by the Ugi-Passerini reaction

A general and simple strategy for routine peptide synthesis with alpha,alpha-dialkyl glycines taking advantage of the four-component Ugi-Passerini reaction is presented. The isonitrile required for the reaction can be relatively simple and its selection based on cost, as the group it generates is easily removed under acidic conditions: in addition, this removal is not visibly affected by the bulkiness of the alpha-alkyl groups. Being a good leaving group from the N-terminal amino group of the amino acid, 4-methoxybenzyl was the choice for the amine component of the reaction. The method is illustrated with the synthesis of a series of acyl derivatives of several alpha,alpha-dialkyl glycines. The preparation of the latter compounds is also reported.     

Expanded structural and stereospecificity in peptide synthesis with chemically modified mutants of subtilisin

Employing the strategy of combined site directed mutagenesis and chemical modification, we previously generated chemically modified mutant enzymes (CMMs) of subtilisin Bacillus lentus (SBL). We now report the use of these SBL-CMMs for peptide coupling reactions. The SBL-CMMs exhibit dramatically altered substrate specificity, including the acceptance of d-amino acid acyl donors, generating dipeptides containing d-Phe, d-Ala and d-Glu in up to 66% yield, which was not possible using wild-type SBL (WT-SBL). In addition, SBL-CMMs accommodate α-branched amino acids such as l-Ala-NH₂ as acyl acceptors in their S₁′ pockets, which WT-SBL will not.     

Solid-phase synthesis of peptide and glycopeptide thioesters through side-chain-anchoring strategies

An efficient new strategy for the synthesis of peptide and glycopeptide thioesters is described. The method relies on the side-chain immobilization of a variety of Fmoc-amino acids, protected at their C-termini, on solid supports. Once anchored, peptides were constructed using solid-phase peptide synthesis according to the Fmoc protocol. After unmasking the C-terminal carboxylate, either thiols or amino acid thioesters were coupled to afford, after cleavage, peptide and glycopeptide thioesters in high yields. Using this method a significant proportion of the proteinogenic amino acids could be incorporated as C-terminal amino acid residues, therefore providing access to a large number of potential targets that can serve as acyl donors in subsequent ligation reactions. The utility of this methodology was exemplified in the synthesis of a 28 amino acid glycopeptide thioester, which was further elaborated to an N-terminal fragment of the glycoprotein erythropoietin (EPO) by native chemical ligation.     

N-Silylation of amines and amino acid esters under neutral conditions employing TMS-Cl in the presence of zinc dust

An expedient synthetic approach to N-silylamines has been developed. The protocol, using TMS-Cl/zinc dust instead of BSA, is useful for the conversion of amines or amino acid esters to the corresponding silyl derivatives, followed by acylation with an acyl chloride or Fmoc-amino acid chloride to give the corresponding amide or peptide. This procedure, affording products in good to excellent yields, is also efficient for the coupling of sterically hindered amino acids like α,α-dialkylamino acids and NMe-amino acids. Further, the use of an equimolar quantity of organic base, such as Et₃N/pyridine, is circumvented.     

Ready protease-catalyzed synthesis of carbohydrate-amino acid conjugates

The protease-catalyzed synthesis of amino acid est-carbohydrate conjugates as glycopeptide analogues has been achieved in a highly regioselective and carbohydrate-specific manner using amino acid vinyl ester acyl donors and minimally or completely unprotected carbohydrate acyl acceptors, which together probed active sites of proteases to reveal yield efficiencies that are modulated by the carbohydrate C-2 substitutent, and that may be exploited to allow selective one-pot syntheses.     

Substrate mimetics in protease catalysis: characteristics,kinetics, and synthetic utility

This article reviews the latest developments in protease-catalyzed peptide synthesis focusing on the use of substrate mimetics. The substrate mimetics approach takes advantage of the characteristic of this novel type of substrates to direct the enzyme to recognize an alternative site on the acyl donor, i.e. the site-specific ester leaving group, mediating the acceptance of originally poorly reactive acyl moieties. At first the kinetics and catalytic mechanism of substrate mimetics-mediated reactions are discussed on the basis of hydrolysis, peptide synthesis, protein-ligand docking, and molecular dynamics studies. By the example of the Glu-specific V8 protease and the aromatic amino acid-specific chymotrypsin both the empirical and computer-aided design of specific substrate mimetics is described. The influence of the leaving group specifically recognized by the enzyme is also considered. The benefits of these artificial substrates over common acyl donor components are illustrated by selected synthesis reactions of small peptides, peptide isosteres, non-peptidic carboxylic acid amides, and the coupling of peptide fragments at non-specific ligation sites resulting in biologically active peptide products. Finally, this review focuses on critical syntheses that uses specific-amino acid-containing peptides as the reactants of ligation. Based on these, the restrictions of the substrate mimetics approach is critically discussed and techniques to their overcoming are presented.     

α-Chymotrypsin-catalyzed peptide synthesis in frozen aqueous solution using N-protected amino acid carbamoylmethyl esters as acyl donors

A kinetically controlled peptide synthesis catalyzed by α-chymotrypsin was performed in frozen aqueous solution (ice, −24 °C). The yield of the peptide was significantly improved by the use of the carbamoylmethyl (Cam) ester as the acyl donor instead of the conventional ethyl ester. The peptide yield increased up to ca. 90% when N-benzyloxycarbonyl (CBZ)-Phe-OCam and H-Phe-NH₂ were used as the acyl donor and nucleophile, respectively. Such an improvement of the peptide yield in ice was also observed in the coupling of other CBZ-amino acid Cam esters as acyl donors. Furthermore, this approach was applied to the synthesis of peptides containing d-amino acids. The peptides such as CBZ-d-Phe-Phe-NH₂, CBZ-Phe-d-Phe-NH₂ and CBZ-d-Phe-d-Phe-NH₂ were also obtained in excellent to moderate yields in ice. A high diastereoselectivity towards the l–l peptide was observed when the racemic amino acid Cam ester was used as the acyl donor in ice.     

Zirconium-mediated coupling reactions of amines and enol or allyl ethers: synthesis of allyl- and homoallylamines

An easy and efficient zirconium-mediated synthesis of allylamines from simple amines and enol ethers is described. This strategy also allows the synthesis of amino alcohol derivatives containing a Z double bond in their structure when 2,3-dihydrofuran is used. Simple conventional modification of these amino alcohols leads to 2-substituted piperidine derivatives. By applying this approach, a formal total synthesis of the alkaloid coniine is easily achieved from a protected butylamine. Finally, the zirconium-mediated reaction of amines and allyl phenyl ether furnishes homoallylamines or amino ethers depending on the structure of the starting amine.     

Enzymatic coupling of specific peptides at nonspecific ligation sites: effect of Asp189Glu mutation in trypsin on substrate mimetic-mediated reactions

Two main drawbacks seriously restrict the synthetic value of proteases as reagents in peptide fragment coupling: (i) native proteolytic activity and, thus, risk of undesired peptide cleavage; (ii) limited enzyme specificities restricting the amino acid residues between which a peptide bond can be formed. While the latter can be overcome by the use of substrate mimetics achieving peptide bond formation at nonspecific ligation sites, the risk of proteolytic cleavage still remains and hinders the wide acceptance of this powerful strategy for peptide coupling. This paper reports on the effect of the trypsin point mutant Asp189Glu on substrate mimetic-mediated reactions. The effect of this mutation on the steady-state hydrolysis of substrate mimetics of the 4-guanidinophenyl ester type and on trypsin-specific Lys- and Arg-containing peptides was investigated. The results were confirmed by enzymatic coupling reactions using substrate mimetics as the acyl donor and specific amino acid-containing peptides as the acyl acceptor. The competition assay verifies the predicted shift in substrate preference from Lys and Arg to the substrate mimetics and, thus, from cleavage to synthesis of peptide bonds. The combination of results obtained qualifies the trypsin mutant D189E as the first substrate mimetic-specific peptide ligase.     

Synthesis of Aminoboronic Acid Derivatives from Amines and Amphoteric Boryl Carbonyl Compounds

Herein, we demonstrate the use of α‐boryl aldehydes and acyl boronates in the synthesis of aminoboronic acid derivatives. This work highlights the untapped potential of boron‐substituted iminium ions and offers insights into the behavior of N‐methyliminodiacetyl (MIDA) boronates during condensation and tautomerization processes. The preparative value of this contribution lies in the demonstration that various amines, including linear and cyclic peptides, can be readily conjugated with boron‐containing fragments. A mild deprotection of amino MIDA‐boronates enables access to α‐ and β‐aminoboronic acids in high chemical yields. This simple process should be applicable to the synthesis of a wide range of bioactive molecules as well as precursors for cross‐coupling reactions.     

Expanding the utility of proteases in synthesis: broadening the substrate acceptance in non-coded amide bond formation using chemically modified mutants of subtilisin

The strategy of combined site directed mutagenesis and chemical modification creates chemically modified mutants (CMMs) with greatly broadened substrate specificities. We have previously reported that the CMMs of subtilisin Bacillus lentus (SBL) are efficient catalysts for the coupling of both l- and d-amino acids. We now report that these powerful catalysts also allow amide bond formation between a variety of non-coded carboxylic acids, including β-alanine and β-amino homologues of phenylalanine, with both l- and d-amino acid nucleophiles. As a guide to enzyme efficiency, a hydrolysis assay indicating pH change has been employed. CMMs selected by this screen furnished higher yields of coupling products compared to the wild-type enzyme (WT). Furthermore, both WT and CMM enzymes allow highly stereoselective aminolysis of a meso diester with an amino acid amine. These results highlight the utility of CMMs in the efficient formation of non-coded amides as potential peptide isosteres.     

Preparation of linear oligosaccharides by a simple monoprotective chemo-enzymatic approach

A monoprotective approach, involving acetyl ester as unique protective group in oligosaccharides synthesis, has been developed. Starting from peracetylated monosaccharides and glycals, by using an efficient and selective chemo-enzymatic ‘one-pot’ strategy (a regioselective hydrolysis catalyzed by immobilized lipases followed by a chemical acyl migration), different carbohydrate acceptors, only protected with acetyl ester, can be achieved. If combined with the use of an acetylated glycosyl donor, the glycosylation reaction with these glycosyl acceptors leads to peracetylated oligosaccharides. These compounds can be directly used as intermediates for the synthesis of glycopeptides used as antitumoral vaccines and, at the end of the process, can be easily fully deprotected in only one step. Thus, these key building blocks have been successfully used in glycosylation reactions for an efficient construction of peracetylated disaccharides, such as the biological relevant lactosamine, in multigram scale. Subsequently, glycosylation with the 3OH-tetraacetyl-α-d-galactose, used as carbohydrate acceptor, allowed the synthesis of a peracetylated N-trisaccharidic precursor of the lacto-N-neo-tetraose antigen. Extending this strategy to a 3OH-di-acetyl galactal, one peracetylated precursor of the T tumor-associated carbohydrate antigen has been synthesized.This efficient approach, characterized by the use of the acetyl ester as only protecting group during all the synthetical steps expected, represents an easy and efficient alternative to the classical synthetic methods in carbohydrate chemistry that involve several protecting group manipulation.     

Design of thiol-containing amino acids for native chemical ligation at non-Cys sites

<正>Protein chemical synthesis usually relies on the use of native chemical ligation that couples peptide thioester with a Cys-peptide.A limitation of this method is the difficulty of finding an appropriate Cys ligation site in many synthetic targets.To overcome this problem,the ligation-desulfurization approach has been developed.This approach involves the use of a thiol-containing amino acid as the ligation partner.After the sequence assembly is completed,the thiol group is removed through a desulfurization reaction to generate the standard amino acids.Currently this strategy has been applied to the ligations at a number of amino acids including Ala,Phe,Val,Lys,Thr,Leu,Pro and Gln.The present article reviews the design and synthesis of these thiol-containing amino acids for native chemical ligation at non-Cys sites.     

Enzymatic Synthesis of Dipeptide Derivatives Containing Non-coded Amino Acids in Organic Solvents

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Lipase-mediated enantioselective acylation of alcohols with functionalized vinyl esters: acyl donor tolerance and applications

Enzymatic acylation is commonly used for the kinetic resolution of alcohols and amines. The simple acyl group introduced during the enzymatic reaction is usually removed or replaced by another group. Retention of more complex acyl moieties as part of the target structures would be a more efficient strategy. We have studied the enantioselective acylation of a model alcohol substrate, 1-phenylethanol, with vinyl esters bearing various functionality on the acyl moieties in the presence of three lipases (Candida antarctica, Candida rugosa and Burkholderia cepacia) frequently used in organic synthesis. C. antarctica lipase is the most versatile lipase for this type of biotransformations. We applied this strategy to the synthesis of a protein kinase C ligand and a natural product, phoracantholide.