Proteases as catalytic agents in peptide synthetic chemistry: Shifting the extent of peptide bond synthesis from a “quantité négligeable” to a “quantité considérable”

Under ordinary conditions the equilibrium point in protease-catalyzed reactions is near to complete hydrolysis. Therefore, proteases are commonly known for their proteolytic rather than for their proteosynthetic activities. Nevertheless, the proteases have proved to be excellent catalysts in preparative peptide synthetic chemistry. A brief review is given of the historical development of protease-catalyzed peptide synthesis. The theoretical aspects of peptide bond formation are described and particular emphasis is given to techniques for favoring the synthesis of the desired peptide linkages. The applicability of these techniques is exemplified with selected syntheses and semisyntheses. The advantages as well as the problems associated with the enzymatic method are evaluated. A critical assessment is given of the present state of the art and the perspectives of the enzymatic approach to peptide synthetic chemistry.     

Non-coded amino acids as acyl donor substrates for peptide bond formation catalyzed by thermoase in toluene

The peptide bond formation of N-protected non-coded amino acids having different structures as acyl donor substrates that is catalyzed by thermoase in organic media was investigated. In these reactions, N-protected l--non-coded amino acids, including l-Orn, l-Cit, -aminobutyric acid (l--Abu) and phenylalanine homologues, were used as the acyl donors and phenylalanine derivatives were used as the acyl acceptors. This kind of enzymatic reactions cannot be carried out in an aqueous buffer due to the rigid specificity of proteases to coded amino acids in water. The results demonstrated that the substrate specificity of proteases could be broadened in organic solvents. In addition, the factors that influenced these protease-catalyzed reactions, including structures of the substrates, water content and the bases used, were systematically studied. Our work provided important evidence for broadening the application of protease in organic synthesis.     

Protease-catalyzed fragment condensation via substrate mimetic strategy: a useful combination of solid-phase peptide synthesis with enzymatic methods.

The concept of substrate mimetic strategy represents a new powerful method in the field of enzymatic peptide synthesis. This strategy takes advantage of the shift in the site-specific amino acid moiety from the acyl residue to the ester-leaving group of the carboxyl component enabling acylation of the enzyme by nonspecific acyl residues. As a result, peptide bond formation occurs independently of the primary specificity of proteases. Moreover, because of the coupling of nonspecific acyl residues, the newly formed peptide bond is not subject to secondary hydrolysis achieving irreversible peptide synthesis. Here, we report the combination of solid-phase peptide synthesis with substrate mimetic-mediated enzymatic peptide fragment condensations. First, the utility of the oxime resin strategy for the synthesis of peptide fragments in the form of substrate mimetics esterified as 4-guanidinophenyl-, phenyl- and mercaptopropionic acid esters was investigated. The study was completed by using the resulting N(alpha)-protected peptide esters as acyl donors in trypsin-, alpha-chymotrypsin- and V8 protease-catalyzed fragment condensations.     

Synthesis of Modified Peptides with C-Terminal α-Amino Aldehydes

Various synthetic approaches to modified peptides with the C-terminal aldehyde group, capable of inhibiting a number of proteolytic enzymes belonging to the classes of thiol, serine, and aspartyl proteases, are considered. Both chemical methods, including solid phase peptide synthesis now widely used, and biocatalytic synthetic methods for obtaining these substances are discussed in detail.     

Modified Proteases for Peptide Synthesis in Organic Media

We showed that modified proteases could catalyze synthesis of a wide variety of peptides of various lengths and structures both in solution and on solid phase in organic solvents. The following modified proteases were studied as catalysts for enzymatic peptide synthesis in polar organic solvents (acetonitrile, dimethylformamide, and ethanol): pepsin sorbed on celite, a noncovalent complex of subtilisin with sodium dodecylsulfate, and subtilisin or thermolysin covalently immobilized on a cryogel of polyvinyl alcohol. The use of the noncovalent complex of subtilisin with sodium dodecylsulfate and immobilized subtilisin is especially promising for the segment condensation of peptide fragments containing residues of trifunctional amino acids with unprotected ionogenic groups in side chains, such as Lys, Arg, His, Glu, and Asp.     

Facile synthesis of peptide nucleic acids and peptide nucleic acid‐peptide conjugates on an automated peptide synthesizer

Peptide nucleic acids (PNAs) are DNA mimics with a neutral peptide backbone instead of the negatively charged sugar phosphates. PNAs exhibit several attractive features such as high chemical and thermal stability, resistance to enzymatic degradation, and stable binding to their RNA or DNA targets in a sequence‐specific manner. Therefore, they are widely used in molecular diagnosis of antisense‐targeted therapeutic drugs or probes and in pharmaceutical applications. However, the main hindrance to the effective use of PNAs is their poor uptake by cells as well as the difficult and laborious chemical synthesis. In order to achieve an efficient delivery of PNAs into cells, there are already many published reports of peptides being used for transport across the cell membrane. In this protocol, we describe the automated as well as cost‐effective semi‐automated synthesis of PNAs and PNA‐peptide constructs on an automated peptide synthesizer. The facile synthesis of PNAs will be helpful in generating PNA libraries usable, e.g. for high‐throughput screening in biomolecular studies. Efficient synthetic schemes, the automated procedure, the reduced consumption of costly reagents, and the high purity of the products are attractive features of the reported procedure. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Enzymatic catalysis in heterogenous mixtures of substrates: The role of the liquid phase and the effects of "Adjuvants"

The physicochemical mechanism of protease-catalyzed peptide synthesis in heterogenous etuectic mixtures of substrates has been examined by a combination of microscopic techniques. Using a number of model reactions of dipeptide amide synthesis, it was determined that liquid phase catalysis was mostly, if not exclusively, responsible for the observed conversion of substrates. Furthermore, the formation of liquid or semiliquid eutectics was an important requirement for the occurrence of those reactions where both substrates were solids in the pure state. The addition of small quantities of hydrophilic solvents (adjuvants) often resulted in significat improvements in the rates and yields of the reactions. This was due to the ability of these adjuvants to promote the formation of eutectics, thereby increasing the proportion, as well as affecting the composition the properties, as well as affecting the composition and properties of the liquid phase. (c) 1994 John Wiley & Sons, Inc.     

S' subsite mapping of serine proteases based on fluorescence resonance energy transfer.

A microassay based on fluorescence resonance energy transfer has been developed to determine the S' specificity of serine proteases. The protease-catalyzed acyl transfer from a fluorescing acyl donor ester to a P'1/P'2 variable hexapeptide library of nucleophiles labeled with a fluorescence quencher leads to an internally quenched peptide product and a fluorescent hydrolysis product. The amount of fluorescence quenching allows one to draw conclusions about the interaction of the nucleophile at the S' sites of the protease. o-Aminobenzoic acid and 3-nitrotyrosine were used as an efficient donor-acceptor pair for the resonance energy transfer. The P'1/P'2 variable hexapeptide library with the general structure H-Xaa-Ala-Ala-Ala-Tyr(NO2)-Gly-OH and H-Ala-Xaa-Ala-Ala-Tyr(NO2)-Gly-OH, where Xaa represents Arg, Lys, Met, Phe, Ala, Gly, Ser, Gln and Glu, was prepared by solid-phase synthesis. Investigations of the S' specificity of trypsin, chymotrypsin and trypsin variants show that this assay is a fast and sensitive screening method for S' subsite mapping of serine proteases and is suitable for a high throughput screening. The assay might be useful for the development of restriction proteases and the estimation of yields in enzymatic peptide synthesis.     

Advances in Fmoc solid‐phase peptide synthesis

Today, Fmoc SPPS is the method of choice for peptide synthesis. Very‐high‐quality Fmoc building blocks are available at low cost because of the economies of scale arising from current multiton production of therapeutic peptides by Fmoc SPPS. Many modified derivatives are commercially available as Fmoc building blocks, making synthetic access to a broad range of peptide derivatives straightforward. The number of synthetic peptides entering clinical trials has grown continuously over the last decade, and recent advances in the Fmoc SPPS technology are a response to the growing demand from medicinal chemistry and pharmacology. Improvements are being continually reported for peptide quality, synthesis time and novel synthetic targets. Topical peptide research has contributed to a continuous improvement and expansion of Fmoc SPPS applications. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Synthetic hFSH peptide constructs in the evaluation of previous studies on the hFSH receptor interaction

The human follicle-stimulating hormone (hFSH) belongs to a family of glycoprotein hormones which contains two non-identical subunits. This paper describes the design and synthesis of a series of synthetic hFSH constructs as putative ligands for the receptor. The design of these constructs is based on the crystal structure of hCG and molecular modelling using the program package Insight II/Discover. The designed constructs contain peptides ranging from 7 to 48 amino acid residues, disulphide bridges and glycan residues. All the synthetic peptides were synthesized by the stepwise solid-phase method using Fmoc chemistry. Two of the synthetic peptides contain the glycosylated amino acid, Asn(GlcNAc-GlcNAc) and both were prepared using fully protected glycosylated building blocks in the solid-phase peptide synthesis. The disulphide bridges were formed from acetamidomethyl-protected glycopeptides and peptides by a direct deprotection/oxidation method using thallium(III) trifluoroacetate. Mass spectroscopy and amino acid analysis were used for characterization of the synthetic hFSH glycopeptides and peptides. The synthetic hFSH constructs were tested for binding activity on FSH receptor assays but none showed improved binding properties compared with the naturally occurring hormone. It was finally demonstrated that non-related peptides showed non-specific binding at the same level as reported for specific peptides. © 1997 European Peptide Society and John Wiley & Sons, Ltd.