Abstract
The chemical modification of amino acids plays an important role in the modulation of proteins or peptides and has useful applications in the activation and stabilization of enzymes, chemical biology, shotgun proteomics, and the production of peptide-based drugs. Although chemoselective modification of amino acids such as lysine and arginine via the insertion of respective chemical moieties as citraconic anhydride and phenyl glyoxal is important for achieving desired application objectives and has been extensively reported, the extent and chemoselectivity of the chemical modification of specific amino acids using specific chemical agents (blocking or modifying agents) has yet to be sufficiently clarified owing to a lack of suitable assay methodologies. In this study, we examined the utility of a fluorogenic assay method, based on a fluorogenic tripeptide substrate (FP-AA1-AA2-AA3) and the proteolytic enzyme trypsin, in determinations of the extent and chemoselectivity of the chemical modification of lysine or arginine. As substrates, we used two fluorogenic tripeptide probes, MeRho-Lys-Gly-Leu(Ac) (lysine-specific substrate) and MeRho-Arg-Gly-Leu(Ac) (arginine-specific substrate), which were designed, synthesized, and evaluated for chemoselective modification of specific amino acids (lysine and arginine) using the fluorogenic assay. The results are summarized in terms of half-maximal inhibitory concentrations (IC50) for the extent of modification and ratios of IC50 values (IC50arginine/IC50lysine and IC50lysine/IC50arginine) as a measure of the chemoselectivity of chemical modification for amino acids lysine and arginine. This novel fluorogenic assay was found to be rapid, precise, and reproducible for determinations of the extent and chemoselectivity of chemical modification.
Highlights
Protein modification is important in the fields of chemical biology, shotgun proteomics, and peptide therapeutics [1,2,3,4,5]
We found that both anhydrides and cyclic anhydrides showed chemoselective blocking activity for lysine compared with arginine, whereas sulfonic anhydrides proved to be ineffective in blocking the activity of either of the two amino acids (Table 1)
We investigated the reactivity of arginine-specific blockers toward the two fluorogenic peptide substrates (Table 2), and found that α-dicarbonyl compounds such as phenyl glyoxal derivatives were highly arginine-selective in our fluorogenic assay, which is consistent with the findings of previous studies [15]
Summary
Protein modification is important in the fields of chemical biology, shotgun proteomics, and peptide therapeutics [1,2,3,4,5]. Similar modifications can be achieved experimentally by exploiting the vast range of chemical reactions to facilitate the conjugation of amino acids including lysine and arginine with specific chemical moieties [3,8,9]. A large number of studies have been conducted to date, regarding specific modification of lysine with citraconic anhydride [10,11,12,13] and arginine with phenyl glyoxal [14,15,16]. Shapiro et al have described the methods of chemical modification of lysine and arginine with specific chemical reagents and evaluated the effect of modification on ribonucleolytic activity of angiogenin [17]. Reported chemical reagents for lysine modification belong to the class of anhydrides (citraconic anhydride, acetic anhydride, and diethylpyrocarbonate) while the arginsiunbesmtraotdesi,fiwcahtiicohninetxeprrlouipttsstthhee cphroetmeoislytrsyis ooff 1su,2b-sdtricaatersbboynytrlycposminp. Tflhueocrleesacveangceeoifsadeterminscpoeredrceiafispscopanedpmitniedgaeisnbucorrneeddasbeoyifnpprflorutoeotoreleaysstcieecneacnceztiiyvsmidteyest.earWsmteriynpsepdsienacsrueallaemtaeesdeassutthhreeadtfloucfhopreormpothiecoaarslee,maancotdidvtiihftiyec.ation (bloWceksinpegc)uolafteadnthaamt icnhoemaicciadl minocdliufidcaintigonly(bslioncekianngd) oaf ragninaminienoaat ctihdeinpcrluodteinoglylytiscinseitaendwould renadregrintihnee aptetphteidperosteuoblysttricatseiterewsiosutaldnrtetnodperrothteeopleypstiisdbeysupbrsotrtaetaesreeseinstzaynmt teosparostteroylypssiisn and thabtythperoftleuaosreeesnczeynmceeswaosutrlydpdsiencarenadstehcaot rthresflpuoonredsicnegncteowthoeueldxtdeenctreoafscehceomrreiscpaol nmdoindgiftiocation (Figthuerex1te).nt of chemical modification (Figure 1)
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