Kung-Tsung Wang

New developments in enzymatic peptide synthesis

This review article describes new enzymatic methods developed for the efficient and irreversible synthesis of peptides based on native and modified proteases, and for the synthesis of polypeptides containing D- and/or unnatural amino acids. Potential opportunities for future developments in the field based on new enzymes, tailormade catalytic antibodies, and on the technique of in vitro mutagenesis are also described.

Peptide and protein hydrolysis by microwave irradiation.

The recent advances in instrumentation for amino acid analysis and highperformance liquid chromatography (HPLC) have shortened the net analysis time for amino acid analysis to less than an hour for each individual sample. Therefore the rate-determining and most essential step of a successful amino acid analysis now lies in the skillful preparation of protein hydrolysates. The commonly used conventional protocol of Hirs et al. [l] utilizes 6 M hydrochloric acid and llO-120°C for more than 24 h and has been the method of choice for the past thirty years. Although the recent development of HPLC amino acid analysis [ 2,3] coupled with gas phase hydrolysis of protein samples [4] has been widely used, the reproducible and complete amino acid analysis data for most proteins are not easily obtained, except in some cases of short peptides. We have introduced a rapid method of microwave heating for the facile preparation of protein and peptide hydrolysates [5,6]. This paper reports refinements of the method, and establishes microwave irradiation as a rapid and novel means of protein hydrolysis with special regard to its general application to amino acid analysis. It provides a radical method of protein and peptide hydrolysis via commercial microwave ovens and specially designed TeflonPyrex tubes with the potential of on-line automation of protein hydrolysis and analysis.

Simplified protein hydrolysis with methanesulphonic acid at elevated temperature for the complete amino acid analysis of proteins

The importance of an accurate and convenient method of amino acid analysis for proteins and peptides is becoming increasingly obvious with the recent developments in biotechnology and protein engineering. The amino acid analyzer based on ion-exchange chromatography developed by Moore and Stein’,’ has remained the method of choice. The most commonly used conventional protocol of Hirs et aL3 utilizes 6 M hydrochloric acid and 11&120’%/24 h for hydrolysis of most proteins. A time-course study of hydrolysis extended to 72 h is always needed to ensure a complete hydrolysis of peptide bonds next to some hydrophobic amino acids such as valine or isoleucine, in addition to correction for losses of some labile amino acids such as cystine, tyrosine, serine and threonine residues in the protein samples. The rate-determining step in a successful determination of amino acid compositions is thus dependent on the careful anaerobic preparation of protein hydrolysates and timeconsuming calibration and extrapolation of the data from different time courses of hydrolysis for these amino acids. The determination of tryptophan by amino acid analysis is non-trivial. More than a decade ago, Simpson et aL4 introduced the non-volatile solvent, 4 M methanesulphonic acid containing 3-(2-aminoethyl)indole in place of hydrochloric acid for complete amino acid analysis from a single protein hydrolysate. However, it has not become a standard method compared to the 6 M hydrochloric acid/l 10°C protocol. Recently we have introduced rapid heating for protein hydrolysis using microwave irradiation’. During the refinement and application of this new technique, we have determined the temperature inside the microwave oven to be higher than 150°C. This high temperature offers the advantage of shortening the hydrolysis time from 24 h to several minutes. With this finding in mind, we feel that it is important to study the effect of temperatures higher than the conventional 110°C for hydrolysis upon the accuracy of amino acid analyses for some standard proteins. Previous reports on the application of higher temperatures (> 1 SO’C) in peptide and protein hydrolysis have emphasized the use of mixed-acid solvents such as hydrochloric acid-trifluoroacetic acid or -propionic acid ‘3’ . In this report the use of a high temperature and shorter time has been applied to the amino acid analysis of proteins with a single non-volatile acid, 4 Mmethanesulphonic acid, which yields amino acid composition data including tryptophan and half-cystine. Some simplification and increase in speed relative to the conventional protocol of employing vacuum-sealed tubes in the 6 M hydrochloric

Polyamide layer chromatography of DNP-amino acids

Abstract The separation of seventeen DNP derivatives of amino acids, 2,4-dinitroaniline and 2,4-dinitrophenol by polyamide layer chromatography is investigated. Convenient procedure for the preparation of durable polyamide layers are described. A table of RF values in ten solvent systems and ultraviolet contract photographs of three one-dimensional chromatograms and three two-dimensional chromatograms are given. The present method is better than paper on thin-layer chromatography in speed and efficiency.

Kinetic resolution of N-protected amino acid esters in organic solvents catalyzed by a stable industrial alkaline protease

SummaryAn industrial alkaline protease “Alcalase” has been found to be very stable in organic solvents and usable as a catalyst for resolution of N-protected amino acids, in both aqueous solution and organic solvent with high yield and optical purity. Only the L-amino acid ester has been hydrolysed.

CONFORMATION OF VESPA BASALIS MASTOPARAN-B IN TRIFLUOROETHANOL-CONTAINING AQUEOUS SOLUTION

Mastoparan-B, a tetradecapeptide isolated from the venom of the hornet Vespa basalis, belongs to the mastoparan analogs of vespid venom with the lysine residues common for all mastoparan family toxins at positions 4, 11 and 12. Here we use 1H-NMR spectroscopy and hybrid distance geometry-simulated annealing calculation to investigate its three-dimensional structure in trifluoroethanol-containing aqueous solution. The calculated structure shows that residues 3-14 adopt an amphiphilic alpha-helical structure in which the residues with hydrophilic side chains (i.e. Lys-4, Ser-5, Ser-8, Lys-11, Lys-12) are located on one side and the residues with hydrophobic side chains (i.e. Leu-3, Ile-6, Trp-9, Ala-10, Val-13, Leu-14) located on the other side of the molecule. The overall structural features a very similar to the conformation of mastoparan-X reconstituted in vesicle [Wakamatsu et al. (1992) Biochemistry 31, 5654-5660] in spite of the substitutions made for eight residues with distinctly different hydrophobicity. These substitutions lead to a larger hydrophobic moment for the alpha-helical segment and further mobilized N-terminal. This study will help reveal the conformational significance of mastoparan toxins with respect to their potency and activity in G protein regulation.

Specific Peptide-Bond Cleavage by Microwave Irradiation in Weak Acid Solution

A rapid and selective peptide-bond cleavage in weak acid, induced by microwave irradiation, has been developed. The specific cleavage sites of peptide bonds located only at the carboxyl-and amino-terminal ends of aspartyl residues along the peptide chain. A systematic study including the time course for the cleavage of various aspartyl-containing peptides, the effect of the acidity of the reaction solution on the completeness of peptide-bond cleavage, and the relationship between the power of microwave irradiation and the reaction time of cleavage are studied.

Direct solid-phase synthesis of octreotide conjugates: precursors for use as tumor-targeted radiopharmaceuticals

Somatostatin analogues, such as octreotide, are useful for the visualization and treatment of tumors. Unfortunately, these compounds were produced synthetically using complex and inefficient procedures. Here, we describe a novel approach for the synthesis of octreotide and its analogues using p-carboxybenzaldehyde to anchor Fmoc-threoninol to solid phase resins. The reaction of the two hydroxyl groups of Fmoc-threoninol with p-carboxybenzaldehyde was catalyzed with p-toluenesulphonic acid in chloroform using a Dean-Stark apparatus to form Fmoc-threoninol p-carboxybenzacetal in 91% yield. The Fmoc-threoninol p-carboxybenzacetal acted as an Fmoc-amino acid derivative and the carboxyl group of Fmoc-threoninol p-carboxybenzacetal was coupled to an amine-resin via a DCC coupling reaction. The synthesis of protected octreotide and its conjugates were carried out in their entirety using a conventional Fmoc protocol and an autosynthesizer. The acetal was stable during the stepwise elongation of each Fmoc-amino acid as shown by the averaged coupling yield (> 95%). Octreotide (74 to 78% yield) and five conjugated derivatives were synthesized with high yields using this procedure, including three radiotherapy octreotides (62 to 75% yield) and two cellular markers (72 to 76% yield). This novel approach provides a strategy for the rapid and efficient large-scale synthesis of octreotide and its analogues for radiopharmaceutical and tagged conjugates.

Reversible enantioselectivity of enzymatic reactions by media

Abstract The esterification of racemic 2-(4-chlorophenoxy) propionic acid and n-butanol by the lipase from Candida cylindracea in different reaction media showed reverse enantioselectivity, pro-R enantiomer in n-hexane, isooctane, cyclohexane, toluene and aqueous phase; and pro-S in dichloromethane, acetone, n-butanol and tetrahydrofuran.

Preparative scale organic synthesis using a kitchen microwave oven

A new and practical continuous flow procedure for preparative scale organic synthesis using a kitchen microwave oven as the heat source has been developed and successfully applied to five different organic reactions.