molecules Article A Fluorogenic Assay: Analysis of Chemical Modification of Lysine and Arginine to Control Proteolytic Activity of Trypsin Kunal N. More † , Tae-Hwan Lim †, Julie Kang and Dong-Jo Chang * College of Pharmacy and Research Institute of Life and Pharmaceutical Sciences, Sunchon National University, 255 Jungang-ro, Suncheon 57922, Korea; [email protected] (K.N.M.); [email protected] (T.-H.L.); [email protected] (J.K.) * Correspondence: [email protected]; Tel.: +82-62-750-3765 † These authors contributed equally to this study. Abstract: The chemical modification of amino acids plays an important role in the modulation of pro- teins 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 modifi- cation 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, Citation: More, K.N.; Lim, T.-H.; MeRho-Lys-Gly-Leu(Ac) (lysine-specific substrate) and MeRho-Arg-Gly-Leu(Ac) (arginine-specific Kang, J.; Chang, D.-J. A Fluorogenic substrate), which were designed, synthesized, and evaluated for chemoselective modification of Assay: Analysis of Chemical specific amino acids (lysine and arginine) using the fluorogenic assay. The results are summarized in Modification of Lysine and Arginine terms of half-maximal inhibitory concentrations (IC50) for the extent of modification and ratios of IC50 to Control Proteolytic Activity of values (IC50arginine/IC50lysine and IC50lysine/IC50arginine) as a measure of the chemoselectivity of Trypsin. Molecules 2021, 26, 1975. chemical modification for amino acids lysine and arginine. This novel fluorogenic assay was found to https://doi.org/10.3390/ be rapid, precise, and reproducible for determinations of the extent and chemoselectivity of chemical molecules26071975 modification. Academic Editor: Kun Li Keywords: fluorogenic peptide substrate; trypsin; fluorogenic assay; chemoselective chemical modification of amino acids Received: 19 February 2021 Accepted: 29 March 2021 Published: 31 March 2021 1. Introduction Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Protein modification is important in the fields of chemical biology, shotgun proteomics, published maps and institutional affil- and peptide therapeutics [1–5]. Post-translational protein modification (PTM) is a protein iations. modification process that occurs during protein biosynthesis subsequent to translation [2,6] and involves chemical transformations that produce chemically and functionally diverse proteins via the covalent addition of chemical moieties to the amino acid side chains within proteins [7]. Similar modifications can be achieved experimentally by exploiting the vast range of chemical reactions to facilitate the conjugation of amino acids including lysine and Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. arginine with specific chemical moieties [3,8,9]. A large number of studies have been con- This article is an open access article ducted to date, regarding specific modification of lysine with citraconic anhydride [10–13] distributed under the terms and and arginine with phenyl glyoxal [14–16]. Shapiro et al. have described the methods of conditions of the Creative Commons chemical modification of lysine and arginine with specific chemical reagents and evaluated Attribution (CC BY) license (https:// the effect of modification on ribonucleolytic activity of angiogenin [17]. Under optimal creativecommons.org/licenses/by/ reaction conditions, chemical reagents such as citraconic anhydride and phenyl glyoxal 4.0/). covalently react with nucleophilic amino or guanidine group of lysine and arginine in Molecules 2021, 26, 1975. https://doi.org/10.3390/molecules26071975 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 2 of 12 anhydride and phenyl glyoxal covalently react with nucleophilic amino or guanidine Molecules 2021, 26, 1975 group of lysine and arginine in substrates, which interrupts the proteolysis of 2substrates of 12 by trypsin. Reported chemical reagents for lysine modification belong to the class of an- hydrides (citraconic anhydride, acetic anhydride, and diethylpyrocarbonate) while the ar- gininesubstrates, modification which interrupts exploits the the chemistry proteolysis of of 1,2-dicarbonyl substrates by trypsin. compounds Reported (phenyl chemical glyoxal andreagents p-hydroxy for lysine phenyl modification glyoxal). belongAlthough to the a numb class ofer anhydrides of reports (citraconichave been anhydride, published re- gardingacetic the anhydride, modification and diethylpyrocarbonate) of amino acids including while thelysine arginine and arginine modification based exploits on the the nature of thechemistry side chain of 1,2-dicarbonyl groups [8,18,19], compounds the extent (phenyl and glyoxal chemoselectivity and p-hydroxy of modification phenyl glyoxal). are not wellAlthough documented, a number owing of reports to a lack have of been practical published and regarding reliable assay the modification methods. In of aminoan attempt acids including lysine and arginine based on the nature of the side chain groups [8,18,19], to quantify exact extent and chemoselectivity of reported blocking agents for either lysine the extent and chemoselectivity of modification are not well documented, owing to a lack of or arginine,practical and we reliable have planned assay methods. to design In an and attempt develop to quantify a highly exact reliable, extent and fast, chemoselec- and practical assaytivity method. of reported Fluorogenic blocking agentsassays for are either highly lysine sensitive, or arginine, precise, we haveand plannedrapid, can to designbe used for theand determination develop a highly of enzyme-substrate reliable, fast, and practicalreactions assay and method.fluorogenic Fluorogenic peptide assayssubstrates are are routinelyhighly sensitive,used to determine precise, and the rapid, activity can be of used proteases for the such determination as endopeptidases, of enzyme-substrate carboxypep- tidases,reactions and and aminopeptidases fluorogenic peptide [20–22]. substrates The arefluo routinelyrogenic usedpeptide to determine substrates the that activity have of been reportedproteases to date such asconsist endopeptidases, of a fluorophore-conjugated carboxypeptidases, andpeptide aminopeptidases with a specific [20– 22sequence]. The for enzymefluorogenic recognition. peptide The substrates cleavage that of have a specif beenic reported peptide to bond date consist by proteolytic of a fluorophore- enzymes as trypsinconjugated releases peptide the fluorophore, with a specific and sequence the corre forsponding enzyme recognition. increase in Thefluorescence cleavage ofis adeter- specific peptide bond by proteolytic enzymes as trypsin releases the fluorophore, and the minedcorresponding as a measured increase of in protease fluorescence activity. is determined We speculated as a measured that ofchemical protease modification activity. (blocking)We speculated of an thatamino chemical acid modificationincluding lysine (blocking) and ofarginine an amino at acid the including proteolytic lysine site and would renderarginine the atpeptide the proteolytic substrate site resistant would render to prot theeolysis peptide by substrate protease resistant enzymes to proteolysis as trypsin and thatby the protease fluorescence enzymes would as trypsin decrease and that correspondi the fluorescenceng to wouldthe extent decrease of chemical corresponding modification to (Figurethe extent 1). of chemical modification (Figure1). FigureFigure 1. A 1.schematicA schematic representation representation of ofthe the use use of of fluorogenic fluorogenic peptidepeptide substrates substrates in in assays assays to determineto determine the extentthe extent of of chemicalchemical modification modification of endogenous of endogenous amino amino acids. acids. (A (A)) An An increase increase inin fluorescence fluorescence on on proteolysis proteolysis of a of fluorogenic a fluorogenic peptide peptide substratesubstrate by a byprotease; a protease; (B) (AB) reduction A reduction in in fluorescence fluorescence following following chemicalchemical modification modification of anof endogenousan endogenous amino amino acid atacid at the proteolyticthe proteolytic site of site the of thefluorogenic fluorogenic peptide peptide substrate. substrate. To test our hypothesis, we designed fluorogenic tripeptide substrates containing a To test our hypothesis, we designed fluorogenic tripeptide substrates containing a lysine or arginine, which serves as a site for cleavage by a specific proteolytic enzyme, lysinetrypsin. or arginine, Within the which structure serves of as the a fluorogenicsite for cleavage peptide by substrate, a specific which proteolytic comprises