processes

Article The Effect of a Peptide Substrate Containing an Unnatural Branched Amino Acid on Activity

Yuuki Yamawaki, Tomoki Yufu and Tamaki Kato *

Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, 2-4 Hibikino, Wakamatsu-ku, Kitakyushu 808-0196, Fukuoka, Japan; [email protected] (Y.Y.); [email protected] (T.Y.) * Correspondence: [email protected]

Abstract: 7-Amino-4-methylcoumarin (AMC) is a low molecular weight fluorescent probe that can be attached to a peptide to enable the detection of specific , such as chymotrypsin, expressed in certain diseases. Because this detection depends on the specificity of the toward the peptidyl AMC, the development of specific substrates is required. To investigate the specificity of chymotrypsin, peptidyl AMC compounds incorporating four different amino acid residues were prepared by liquid-phase synthesis. Two unnatural amino acids, 2-amino-4-ethylhexanoic acid (AEH) and cyclohexylalanine (Cha), were used to investigate the substrate specificity as these amino acids have structures different from natural amino acids. AEH was synthesized using diethyl acetamidemalonate as a starting material. The substrate containing Cha had high hydrophobicity and showed a high reaction velocity with chymotrypsin. Although the AEH substrate with a branched side chain had high hydrophobicity, it showed a low reaction velocity. The substrate containing the aromatic amino acid phenylalanine was less hydrophobic than the Cha and AEH substrates, but  chymotrypsin showed the highest specificity for this compound. These results demonstrated that the  substrate specificity of chymotrypsin is not only affected by the hydrophobicity and aromaticity, but Citation: Yamawaki, Y.; Yufu, T.; also by the structural expanse of amino acid residues in the substrate. Kato, T. The Effect of a Peptide Substrate Containing an Unnatural Keywords: unnatural amino acid; peptide; fluorescent probe; 7-amino-4-methylcoumarin; specificity; Branched Amino Acid on chymotrypsin Chymotrypsin Activity. Processes 2021, 9, 242. https://doi.org/ 10.3390/pr9020242 1. Introduction Academic Editor: Kenji Usui Proteases are proteolytic that are found extensively in organisms. Proteases Received: 28 December 2020 play a role in most physiological systems, including digestion, immune response, and Accepted: 25 January 2021 Published: 28 January 2021 cell proliferation [1–4]. However, some proteases are closely related with disease states and genetic abnormalities, including cancer [5,6]. Over expression of certain proteases is

Publisher’s Note: MDPI stays neutral known to cause specific diseases [7,8]. In the progression of cancer, the over-expression of with regard to jurisdictional claims in in tumor cells promotes the degradation of the cells and cell re-organization [9] published maps and institutional affil- and expression of matrix metalloproteinases in stromal cells assists in tumor cell growth [10]. iations. Over-expression of hepsin in prostate tumors promotes the metastasis and growth of the tumor cells, followed by weakening of the adhesion of the stroma [11]. Quantitative analysis of the protease causing a disease is essential in understanding the progression of the disease. In one study, protease activity related to a disease was successfully detected using synthesized substrates for which the protease had specific activity [12]. The structure Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. of the of proteases differs according to the constituent amino acid residues. This article is an open access article Chymotrypsin is a general protease that is well known structurally and mechanically. distributed under the terms and Chymotrypsin is not only important for living organisms but is also involved in many conditions of the Creative Commons diseases [13]; therefore, investigation of the activity of chymotrypsin may lead to more Attribution (CC BY) license (https:// accurate and simpler diagnoses of diseases. creativecommons.org/licenses/by/ Analysis of the interaction of proteases with substrates can assist in the development 4.0/). and analysis of the characteristics of protease inhibitors [14]. Such analyses are frequently

Processes 2021, 9, 242. https://doi.org/10.3390/pr9020242 https://www.mdpi.com/journal/processes Processes 2021, 9, x FOR PEER REVIEW 2 of 9

Processes 2021, 9, 242 Analysis of the interaction of proteases with substrates can assist in the development2 of 8 and analysis of the characteristics of protease inhibitors [14]. Such analyses are frequently performed with fluorescent probes for analysis of the protease activity [15,16]. Fluorescent performedprobes enable with investigation fluorescent probesof the protease for analysis activity, of the simply protease and activity at low-cost [15,16 ].[17–19]. Fluores- 7- centAmino-4-methylcoumarine probes enable investigation (AMC) of is the usually protease used activity, as the simplyfluorescent and atprobe low-cost for proteases [17–19]. 7-Amino-4-methylcoumarine[20–23]. AMC produces different (AMC) fluorescence is usually states used depending as the fluorescent on whether probe or fornot pro-it is teasesbound[ 20to– 23a peptide.]. AMC producesTherefore, different the degradat fluorescenceion of statespeptide depending substrates on can whether be observed, or not it iswhich bound can to provide a peptide. information Therefore, on the the degradation degradation of activity peptide of substrates the protease. can In be the observed, present whichstudy, canthe providefluorescent information probe AMC on the was degradation used because activity it is relatively of the protease. easy to In connect the present to a study,peptide. the fluorescent probe AMC was used because it is relatively easy to connect to a peptide.The aromatic ring of peptide substrates is recognized by chymotrypsin and then the carboxyl-terminusThe aromatic ringof the of pepti peptidede is substratesselectively ishydrolyzed recognized because by chymotrypsin the vicinity andof the then ac- thetive carboxyl-terminus site of chymotrypsin of theis hydrophobic peptide is selectively [24,25]. Leucine hydrolyzed (Leu) because and valine the vicinityresidues of with- the activeout an sitearomatic of chymotrypsin ring are also is often hydrophobic recognized [24 and,25]. the Leucine substrate (Leu) hydrolyzed and valine by residues chymo- withouttrypsin [26,27]. an aromatic Substrates ring are with also natural often recognizedamino acids and are thealso substrate known to hydrolyzed be hydrolyzed by chy- by motrypsinchymotrypsin [26, 27[28].]. Substrates In the present with study, natural substrate amino acids peptides are also containing known tounnatural be hydrolyzed amino byacids chymotrypsin were synthesized, [28]. In and the presentamino acid study, residues substrate for peptideswhich chymotrypsin containing unnatural was highly amino spe- acidscific were were explored. synthesized, Unnatural and amino amino acid acids residues enable investigation for which chymotrypsin of the specificity was that highly re- specificsults from were the explored. structure Unnatural of the side amino chain acids of compounds enable investigation because various of the specificitystructures thatand resultsproperties from can the be structureimparted ofto unnatural the side chain amino of acids. compounds The unnatural because amino various acids structures 2-amino- and4-ethylhexanoic properties can acid be (AEH) imparted and cyclohexylalanine to unnatural amino (Cha) acids. were The selected unnatural for use amino in the acids pre- 2-amino-4-ethylhexanoicsent study because phenylalanine acid (AEH) (Phe) and tends cyclohexylalanine to be recognized (Cha) as a were substrate selected of chymo- for use intrypsin. the present The fact study that because the activity phenylalanine of chymotrypsin (Phe) tends has been to be observed recognized to as be a high substrate for Phe of chymotrypsin.and low for Leu The in previous fact that studies the activity has generally of chymotrypsin been attributed has been to the observed aromatic to ring be high and forhigh Phe hydrophobicity and low for Leu of inPhe. previous To further studies examine has generally this phenomenon, been attributed we compared to the aromatic the ef- ringfects and of unnatural high hydrophobicity amino acids, of using Phe. Cha To further without examine aromaticity, this phenomenon, and AEH with we a comparedside chain the effects of unnatural amino acids, using Cha without aromaticity, and AEH with a side including a degree of freedom greater than Cha. Therefore, this investigation should pro- chain including a degree of freedom greater than Cha. Therefore, this investigation should vide information for the development of substrates that chymotrypsin has specificity for, provide information for the development of substrates that chymotrypsin has specificity which exceeds that of substrates containing natural amino acids. for, which exceeds that of substrates containing natural amino acids.

2.2. Materials and MethodsMethods 2.1.2.1. Design of Substrates ToTo comparecompare the the effects effects of of the the side-chain side-chain structures structures of of the the amino amino acid acid residues, residues, a simple a sim- andple and easy easy sequence, sequence, H2N-Ala-Ala-XXX-AMC, H2N-Ala-Ala-XXX-AMC, was was adopted adopted for thefor the design design of the of the peptide pep- substrates.tide substrates. Where Where XXX XXX is either is either AEH AEH1, Cha 1, Cha2, Phe 2, 3Phe, or 3 Leu, or 4Leu(Figure 4 (Figure1). The 1). tripeptides The tripep- weretides designedwere designed with twowith alanine two alanine (Ala) residues(Ala) residues to improve to improve the binding the binding to chymotrypsin to chymo- withouttrypsin without interfering interfering with the with effects the of effects the residues of the residues under investigation under investigation (XXX). (XXX).

Figure 1. Design of peptide substrates with four different amino acid residues. Figure 1. Design of peptide substrates with four different amino acid residues. 2.2. Synthesis of Boc-AEH-OH L-AEH was synthesized as previously described [29]. The progress of the reaction was monitored with thin-layer chromatography, and high-performance liquid chromatog- raphy (HPLC) using the following system: pump (L-7100, Hitachi High-Tech Science Corporation, Japan); UV-VIS detector (L-7420, Hitachi High-Tech Science Corporation, Japan); integrator (D-7500, Hitachi High-Tech Science Corporation, Japan); and column (chromolith performance RP-18e 100–4.6 mm, Merck KGaA, Darmstadt, Germany). Diethyl Processes 2021, 9, x FOR PEER REVIEW 3 of 9

2.2. Synthesis of Boc-AEH-OH L-AEH was synthesized as previously described [29]. The progress of the reaction was monitored with thin-layer chromatography, and high-performance liquid chroma- tography (HPLC) using the following system: pump (L-7100, Hitachi High-Tech Science Processes 2021, 9, 242 3 of 8 Corporation, Japan); UV-VIS detector (L-7420, Hitachi High-Tech Science Corporation, Ja- pan); integrator (D-7500, Hitachi High-Tech Science Corporation, Japan); and column (chromolith performance RP-18e 100–4.6 mm, Merck KGaA, Darmstadt, Germany). Di- acetamidomalonateethyl acetamidomalonate5 (50 mmol, 5 1(50 eq.) mmol, as a starting 1 eq.) as material a starting was material mixed with was 20% mixed sodium with 20% ethoxidesodium ethanol ethoxide solution ethanol (50 mmol, solution 1 eq.) (50 and mmol, then 1 theeq.) mixture and then was the refluxed mixture for was 30 refluxed min in for 30 mL30 of min dehydrated in 30 mL ethanol of dehydrated (Figure2 ).ethanol Then, 1-bromo-2-ethylbutane(Figure 2). Then, 1-brom (150o-2-ethylbutane mmol, 3 eq.) (150 was addedmmol, to3 eq.) the was mixture, added which to the was mixture, refluxed which for was 3 h. refluxed After cooling for 3 h. the After mixture cooling in the an mix- ice bath,ture selective in an ice hydrolysisbath, selective was hydrolysis performed was by additionperformed of 25by mLaddition of 1 M of NaOH 25 mL aqueousof 1 M NaOH solutionaqueous and stirring solution for and 1 h. stirring After the for reaction, 1 h. After the the mixture reaction, was the evaporated mixture was under evaporated reduced un- pressureder reduced and then pressure washed withand then diethyl washed ether, with extracted diethyl using ether, ethyl extracted acetate using at pH ethyl 3 (twice), acetate at and thenpH 3 washed (twice), withand then brine. washed The organic with brine. layer The was organic dried over layer MgSO was dried4 and over the solvent MgSO4 and was evaporatedthe solvent underwas evaporated reduced pressure. under reduced The product pressure. racemic The monocarboxylic product racemic acid monocarbox-6 was obtainedylic acid as a white6 was solidobtained (28.0 as mmol, a white 56%). solid Compound (28.0 mmol,6 (11.4 56%). mmol) Compound was decarboxylated 6 (11.4 mmol) was by refluxingdecarboxylated in 100 mL by ofrefluxing toluene in for 100 5 mL h and of tolu convertedene for to5 h the and racemic converted acyl to amino the racemic ester. acyl Afteramino the reaction, ester. After the solvent the reaction, was removed the solvent under was reduced removed pressure under andreduced then pressure the resulting and then oil wasthe dissolved resulting inoil ethyl was acetate.dissolved This in solutionethyl acetate. was washedThis solution with 4% was NaHCO washed3 aqueous with 4% Na- solutionHCO (twice)3 aqueous and brine.solution The (twice) organic and layer brine. was The dried organic over layer MgSO was4 and dried the solventover MgSO was4 and removedthe solvent under reducedwas removed pressure under and reduced then the pressure product and (11.7 then mmol) the product was obtained (11.7 mmol) as an was oil. Theobtained oil was as hydrolyzed an oil. The by oil stirring was hydrolyzed for 2 h in a by mixture stirring of 40for mL2 h ofin ethanol a mixture and of 20 40 mL mL of of 2 Methanol NaOH and aqueous 20 mL solution.of 2 M NaOH The mixtureaqueous was solution. evaporated The mixture under reducedwas evaporated pressure under and thenreduced extracted pressure using and ethyl then acetate extracted at pHusing 3 (three ethyl times)acetateand at pH washed 3 (three with times) brine. and The washed organicwith layer brine. was The dried organic over MgSOlayer was4 and dried the solvent over MgSO was removed4 and the undersolvent reduced was removed pressure. under The productreduced (8.6 pressure. mmol, 87%)The product was obtained (8.6 mmol, as the 87%) racemic was acyl obtained amino as acid the7 racemic. To obtain acyl the amino L-aminoacid acid,7. To theobtain racemic the L-amino mixture acid, was the separated racemic by mixture deacylation was separated with 0.11 by g deacylation of L-amino with acylase and 0.011 mg of CoCl 6H O in 20 mL of water. The reaction was stopped by 0.11 g of L-amino acylase and2 0.0112 mg of CoCl2 6H2O in 20 mL of water. The reaction was additionstopped of 1 Mby HCl addition aqueous of 1 solution. M HCl aqueous The mixture solution. waswashed The mixture using was ethyl washed acetate using (three ethyl times) and then L-AEH 8 (1.3 mmol, 44%) was obtained in the aqueous layer. To use acetate (three times) and then L-AEH 8 (1.3 mmol, 44%) was obtained in the aqueous layer. L-AEH in the peptide synthesis, 40 mL of the L-AEH aqueous solution (1.3 mmol, 1.0 eq.) To use L-AEH in the peptide synthesis, 40 mL of the L-AEH aqueous solution (1.3 mmol, was modified with the Boc group by reaction with a mixture of Boc2O (2.6 mmol, 2.0 eq.), 1.0 eq.) was modified with the Boc group by reaction with a mixture of Boc2O (2.6 mmol, triethylamine (2.6 mmol, 2.0 eq.), and dioxane at room temperature for 24 h. The mixture 2.0 eq.), triethylamine (2.6 mmol, 2.0 eq.), and dioxane at room temperature for 24 h. The was evaporated under reduced pressure and washed with diethyl ether, then extracted mixture was evaporated under reduced pressure and washed with diethyl ether, then ex- using ethyl acetate at pH 3 (three times). The organic layer was dried over MgSO4 and the tracted using ethyl acetate at pH 3 (three times). The organic layer was dried over MgSO4 solvent was evaporated under reduced pressure. To remove impurities, the compound was and the solvent was evaporated under reduced pressure. To remove impurities, the com- dissolved in diethyl ether and recrystallized. Finally, Boc-L-AEH-OH 9 (1.0 mmol, 82%) pound was dissolved in diethyl ether and recrystallized. Finally, Boc-L-AEH-OH 9 (1.0 was obtained as a white solid. mmol, 82%) was obtained as a white solid.

(a)

Diethyl acetamidomalonate 5 Racemic Monocarboxylic acid 6 Acyl amino acid 7 yield: 56% yield: 87%

(b)

Acyl amino acid 7 L-AEH 8 Boc-L-AEH 9 yield: 44% yield: 82%

FigureFigure 2. The 2. synthesis The synthesis of the of unnatural the unnatural amino amino acid, 2-amino-4-ethylhexanoicacid, 2-amino-4-ethylhexanoic acid (AEH).acid (AEH). (a) Synthetic (a) Synthetic scheme scheme for the for the backbonebackbone of racemic of racemic AEH. AEH. (b) Optical (b) Optical resolution resolution of the of acyl-AEH the acyl-AEH racemic racemic mixture mixture and synthesis and synthesis of Boc-L-AEH. of Boc-L-AEH.

2.3. Synthesis of Peptide-MCA Substrates Boc-Leu-OH, Boc-Phe-OH, and Boc-Cha-OH were purchased from Watanabe Chem- ical Industries, Ltd., Hiroshima, Japan. The four Boc-amino acids, Boc-Leu-OH, Boc- Phe-OH, Boc-Cha-OH, and Boc-AEH-OH, were each reacted with water soluble car- bodiimide hydrochloride (WSC HCl, 0.5 eq.) as a condensing agent in 8 mL of N,N- dimethylformamide (DMF) in an ice bath for 4 h (Figure3). Then, these compounds Processes 2021, 9, 242 4 of 8

were each reacted with AMC (0.5 eq.) for 20 h. In addition, each compound was re- acted with WSC HCl (0.25 eq.) overnight. After the reaction, each mixture was evap- orated under reduced pressure and then dissolved in ethyl acetate. These solutions were washed using 10% citric acid aqueous solution (twice), brine, 4% NaHCO3 aque- ous solution (twice), and brine. Each organic layer was dried over MgSO4 and the solvent was evaporated under reduced pressure. The products were obtained as Boc- XXX-AMC. Peptide synthesis was performed by standard Boc liquid-phase synthesis. The products prepared above were each stirred in 15 mL of 4 M HCl/dioxane at room temperature for 30 min to remove the Boc group. The solvent was evaporated under reduced pressure and then the products H2N-XXX-AMC (XXX = AEH: 0.51 mmol, Cha: 0.78 mmol, Phe: 0.30 mmol, and Leu: 0.14 mmol) were obtained. These compounds were reacted with Boc-Ala-OH (1.2 eq.) using 1-hydroxybenzotriazole (HOBt, 1.2 eq.), 1- [bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate (HBTU, 1.2 eq.), and N,N-diisopropylethylamine (DIEA, 3.0 eq.) for 2 h in 10 mL of DMF. After the reaction, each mixture was evaporated under reduced pressure and then dissolved in ethyl acetate. The solutions were washed using 10% citric acid aqueous solution (twice), brine, 4% NaHCO3 aqueous solution (twice), and brine. Each organic layer was dried over MgSO4 and the solvent was evaporated under reduced pressure. The crude products Boc-Ala-XXX-AMC were obtained. Subsequently, these products (XXX = AEH: 0.36 mmol, Cha: 0.72 mmol, Phe: 0.32 mmol, Leu: 0.21 mmol) were condensed with Boc-Ala-OH and then extracted as described above to give the products H2N-Ala-Ala-XXX-AMC 1–4. These products were purified using HPLC column with a gradient of acetonitrile/0.1% trifluoroacetic acid from 0 to 100% for 15 min at a flow rate of 2.0 mL min−1 and identified by mass spectrometry (Figure S1). The values of the mass analysis for each compound + were as follows. XXX = AEH 1 m/z calcd. for [(M+Na) ]C24H34N4O5: 481.24269; found: + 481.24294, Cha 2 m/z calcd. for [(M+Na) ]C25H34N4O5: 493.24269; found: 493.24139, Phe + 3 m/z calcd. for [(M+Na) ]C25H28N4O5: 487.19574; found: 487.19662, Leu 4 m/z calcd. for + [(M+Na) ]C22H30N4O5: 453.21139; found: 453.21194.

Figure 3. Liquid phase synthesis of the peptide substrates.

2.4. Chymotrypsin Activity Assay Detection of the degradation of each synthesized substrate was performed with a fluorescent plate reader (Wallac ARVO SX 1420 multilabel counter, PerkinElmer Japan Co., Ltd., Kanagawa, Japan). Substrate solutions were prepared at a concentration of 1 mM in dimethyl sulfoxide. A 0.1 M Tris-HCl buffer was prepared at pH 7.8 and 25 ◦C. A chymotrypsin (EC 3.4.21.1, bovine pancreas, Sigma-Aldrich Japan, Tokyo, Japan) solution was prepared at a final concentration of 1 µM in the buffer. Each substrate solution was prepared at various concentrations to final concentrations of 4, 8, 16, 32, 48, 64, 96, and 128 µM in the buffer, and 190 µL of the prepared substrate solutions were put into the wells of a 96-well plate. The measurements were immediately started by addition of 10 µL of the chymotrypsin solution. The measurements were performed with linear stirring for 1 s between a delay of 10 s at an excitation wavelength of 370 nm and a detection wavelength of 460 nm. The obtained data was used to calculate the maximum reaction velocity (Vmax), Michaelis constant (Km), turnover number (kcat), and specificity constant (kcat/Km) based on the Michaelis-Menten equation. Processes 2021, 9, x FOR PEER REVIEW 5 of 9

between a delay of 10 s at an excitation wavelength of 370 nm and a detection wavelength of 460 nm. The obtained data was used to calculate the maximum reaction velocity (Vmax), Processes 2021, 9, 242 Michaelis constant (Km), turnover number (kcat), and specificity constant (kcat/Km) based5 of on 8 the Michaelis-Menten equation.

3. Results and Discussion 3. Results and Discussion 3.1.3.1. HydrophobicityHydrophobicity ofof thethe PeptidePeptide SubstratesSubstrates TheThe hydrophobicityhydrophobicity of of the the substrate substrate affects affect thes the interaction interaction with with chymotrypsin chymotrypsin because be- ofcause the highof the hydrophobicity high hydrophobicity around around the active the siteactive of site chymotrypsin. of chymotrypsin. To compare To compare the inter- the actioninteraction of the of four the synthesizedfour synthesized peptide peptide substrates substrates with chymotrypsin,with chymotrypsin, the hydrophobicity the hydropho- ofbicity each of substrate each substrate was investigated was investigated (Figure 4(F).igure The relative 4). Thehydrophobicity relative hydrophobicity of the peptide of the substratespeptide substrates could be determinedcould be determined based on the base retentiond on the times retention in the times HPLC in used the forHPLC purifica- used tion.for purification. The retention The times retention increased times depending increased on depending the hydrophobicity on the hy ofdrophobicity peptide. Substrate of pep- 2tide.with Substrate the unnatural 2 with amino the unnatural acid Cha amino had a acid retention Cha had time a ofretention 5.58 min time and of possessed 5.58 min theand highestpossessed hydrophobicity. the highest hydrophobicity. The peptide substrate The peptide with thesubstrate synthesized with the AEH synthesized residue 1 AEHhad aresidue retention 1 had time a retention of 5.34 min time and of 5.34 possessed min and the possessed next highest the next hydrophobicity. highest hydrophobicity. The Phe substrateThe Phe substrate3 and Leu 3 and substrate Leu substrate4 had retention 4 had retention times of times 4.75 of and 4.75 4.62 and min, 4.62respectively, min, respec- indicatingtively, indicating lower hydrophobicity lower hydrophobicity than the Chathan2 theand Cha AEH 2 and1 substrates. AEH 1 substrates. The presence The of pres- the πenceelectrons of the inπ electrons the Phe residue in the Phe with residue an aromatic with an ring aromatic was thought ring was to thought cause the to lower causehy- the drophobicitylower hydrophobicity of substrate of 3substrate. In contrast, 3. In it contrast, was thought it was that thought the Cha that2 and the AEHCha 21 andsubstrates AEH 1 exhibitedsubstrates relatively exhibited higher relatively hydrophobicity higher hydrop becausehobicity of because their aliphatic of their ring aliphatic and branched ring and chainbranched structures, chain structures, respectively. respectively.

FigureFigure 4.4.HPLC HPLC datadata ofof thethe fourfour synthesizedsynthesized substrates.substrates. TheThe valuesvaluesindicate indicatethe theretention retentiontimes. times.

3.2.3.2. ChymotrypsinChymotrypsin ActivityActivity ofof thethe PeptidePeptide SubstratesSubstrates TheThe specificityspecificity of chymotrypsin chymotrypsin for for the the four four peptide peptide substrates substrates was was compared compared at var- at variousious substrate substrate concentrations. concentrations. The The activity activity of of chymotrypsin chymotrypsin toward toward the substratessubstrates waswas measured based on the fluorescence intensity of AMC resulting from the degradation of measured based on the fluorescence intensity of AMC resulting from the degradation of the substrate. The fluorescence intensity produced by the degradation increased over time the substrate. The fluorescence intensity produced by the degradation increased over time for all the substrates. These results indicated that all the synthesized substrates can be for all the substrates. These results indicated that all the synthesized substrates can be recognized and degraded by chymotrypsin. The kinetics of all the synthesized substrates recognized and degraded by chymotrypsin. The kinetics of all the synthesized substrates could be fitted to a Michaelis-Menten graph (Figure5). To compare the preference of could be fitted to a Michaelis-Menten graph (Figure 5). To compare the preference of chy- chymotrypsin for the synthesized substrates, the Vmax, Km, kcat, and kcat/Km values, which motrypsin for the synthesized substrates, the Vmax, Km, kcat, and kcat/Km values, which char- characterize the interaction between an and a substrate, were calculated based acterize the interaction between an enzyme and a substrate, were calculated based on the on the Michaelis-Menten equation (Table1). The Cha substrate 2 showed the largest Vmax Michaelis-Menten equation (Table 1). The Cha substrate 2 showed the largest Vmax and kcat and kcat values, followed by the Phe substrate 3. These values broadly agreed with those previously reported for chymotrypsin under similar conditions [30]. In addition, the Vmax and kcat values were dependent on the hydrophobicity of the substrates, except for the Phe substrate 3. Because of the differences in these peptide substrates, the hydrophobicity and presence or absence of aromaticity may have affected the reaction velocity with chymotrypsin. The AEH substrate 1 showed approximately 6-fold lower Vmax and kcat values than the Phe substrate 3, despite showing a high hydrophobicity in the four peptide Processes 2021, 9, x FOR PEER REVIEW 6 of 9

values, followed by the Phe substrate 3. These values broadly agreed with those previ- ously reported for chymotrypsin under similar conditions [30]. In addition, the Vmax and kcat values were dependent on the hydrophobicity of the substrates, except for the Phe substrate 3. Because of the differences in these peptide substrates, the hydrophobicity and Processes 2021, 9, 242 presence or absence of aromaticity may have affected the reaction velocity with chymo-6 of 8 . The AEH substrate 1 showed approximately 6-fold lower Vmax and kcat values than the Phe substrate 3, despite showing a high hydrophobicity in the four peptide substrates. Thissubstrates. result may This be result caused may by bethe causedside chain by theof the side AEH chain residue of the that AEH has residue a high structural that has a degreehigh structural of freedom. degree Therefore, of freedom. the reaction Therefore, velocity the reaction of chymotrypsin velocity of for chymotrypsin a substrate forwas a affectedsubstrate by was the hydrophobicity, affected by the hydrophobicity, aromaticity, and aromaticity, the side chain and structure the side of chain the recognized structure of aminothe recognized acid residues. amino acid residues.

0.030 PhePhe substrate substrate 3 0.025 ChaCha substrate substrate 2 AEHAEH substrate substrate 1 0.020 LeuLeu substrate substrate 4 ) −1 0.015 (μM s 0 v 0.010

0.005

0.000 050100 Substrate concentration (μM) FigureFigure 5. 5. Michaelis-MentenMichaelis-Menten graph graph of of the the four four synt synthetichetic substrates substrates with with chymotrypsin. chymotrypsin.

Table 1. TheNext, hydrophobicity the Km values, and kinetics which valuesindicate of thethe four affinity peptide between substrates. the peptide substrate and chymotrypsin, were compared. Smaller Km values indicate higher affinity. The Phe sub- HPLC Retention Time V K k k /K Sample strate 3 showed the lowestmax Km value, indicatingm the highest affinitycat for chymotrypsin.cat m The (min) (nM s−1) (µM) (s−1) (M−1 s−1) Cha 2 and AEH 1 substrates with higher hydrophobicity showed approximately two-fold −3 Leu substrate 4 larger4.62 Km values than 0.60 that± of0.01 the Phe substrate 72.83 ± 1.22 3, indicating(0.60 ± lower0.01) × affinity.10 The8.21 low± affinity0.03 −3 AEH substrate 1 of 5.34the Cha 3 and AEH 4.36 1 substrates± 0.09 may 107.79 be related± 3.12 to the(4.36 large± 0.09) structural× 10 degree40.67 of freedom± 0.43 Cha substrate 2 5.58 54.54 ± 1.62 140.48 ± 5.65 ± × −2 388.32 ± 4.54 that the side chains of the Cha and AEH residues possess.(5.45 In0.16) contrast,10 the structure of the Phe substrate 3 4.75 27.96 ± 0.71 61.97 ± 2.40 (2.80 ± 0.07) × 10−2 452.35 ± 6.62 Suc-F-pNA a Leu and Phe residues allows these substrates720 to easily enter 0.011 the hydrophobic pocket 15 near A-A-F-AMC a the active site of chymotrypsin. In addition,500 chymotrypsin 0.83tends to recognize 2000aromatic amino acid residues and is well known to recognize the aromatic ring of Phe residues. a Data from 30. Thus, chymotrypsin may preferentially recognize amino acid residues that possess aro- maticity or have a smaller structural degree of freedom. Next, the Km values, which indicate the affinity between the peptide substrate and chy- Finally, the kcat/Km values of each substrate were compared. The kcat/Km value repre- motrypsin, were compared. Smaller Km values indicate higher affinity. The Phe substrate 3 sents the specificity of the enzyme for the substrate. The Phe substrate 3 showed the high- showed the lowest Km value, indicating the highest affinity for chymotrypsin. The Cha 2 estand kcat AEH/Km value,1 substrates indicating with higherhigh specificity hydrophobicity of chymotrypsin showed approximately for substrate two-fold 3. The k largercat/Km values of the Cha and AEH substrates with higher hydrophobicity than the Phe substrate Km values than that of the Phe substrate 3, indicating lower affinity. The low affinity of the 3Cha were3 andlower AEH than1 thosesubstrates of the may Phe besubstrate related 3 to. These the large results structural suggested degree that of the freedom specificity that ofthe chymotrypsin side chains of for the a Chaparticular and AEH substrate residues may possess. be because In contrast, of the presence the structure of an ofaromatic the Leu aminoand Phe acid residues residue allows rather thesethan a substrates hydrophobic to easily amino enter acid the residue. hydrophobic The kcat/ pocketKm value near of the the Chaactive substrate site of chymotrypsin.2 with greater Inhydrophobicity addition, chymotrypsin than the AEH tends substrate to recognize 1 was aromatic 9-fold larger amino thanacid that residues of the andAEH is substrate well known 1. The to least recognize hydropho thebic aromatic Leu substrate ring of 4 Phe showed residues. the lowest Thus, kchymotrypsincat/Km value. The may kcat preferentially/Km values for recognize each substrate amino increased acid residues with that increasing possess aromaticityhydrophobi- or city,have except a smaller for the structural Phe substrate degree of3. These freedom. results indicated that the specificity of chymo- trypsinFinally, for a thesubstratekcat/K increasedm values ofwith each increa substratesing hydrophobicity were compared. of the The substrate.kcat/Km value Alt- houghrepresents of the the four specificity peptide ofsubstrates, the enzyme the for Phe the substrate substrate. 3 has The relatively Phe substrate low hydrophobi-3 showed the city,highest the highkcat/ specificityKm value, of indicating chymotrypsin high specificityfor substrate of 3 chymotrypsin may be because for of substrate the aromaticity3. The kcat/Km values of the Cha and AEH substrates with higher hydrophobicity than the Phe substrate 3 were lower than those of the Phe substrate 3. These results suggested that the specificity of chymotrypsin for a particular substrate may be because of the presence of an aromatic amino acid residue rather than a hydrophobic amino acid residue. The kcat/Km value of the Cha substrate 2 with greater hydrophobicity than the AEH substrate 1 was 9-fold larger than that of the AEH substrate 1. The least hydrophobic Leu substrate 4 showed the lowest kcat/Km value. The kcat/Km values for each substrate increased with Processes 2021, 9, 242 7 of 8

increasing hydrophobicity, except for the Phe substrate 3. These results indicated that the specificity of chymotrypsin for a substrate increased with increasing hydrophobicity of the substrate. Although of the four peptide substrates, the Phe substrate 3 has relatively low hydrophobicity, the high specificity of chymotrypsin for substrate 3 may be because of the aromaticity and the planar structure of the Phe residue, which has less steric hindrance than the AEH 1 and Cha 3 substrates. Therefore, high substrate specificity for chymotrypsin requires aromaticity, high hydrophobicity, and low steric hindrance at the recognition site for the substrate. These properties are important for controlling the specificity of chymotrypsin for a substrate, and unnatural amino acid residues are useful to investigate this substrate specificity.

4. Conclusions AEH was successfully synthesized using diethyl acetamidomalonate as a starting material and enzymatic optical resolution. The synthesized AEH was incorporated into a tripeptide as a novel peptide substrate. Novel peptide substrates with the unnatural amino acid residues, AEH and Cha, showed high hydrophobicity that contributed to increased chymotrypsin specificity. In particular, the Cha substrate 2 was the most hydrophobic and showed a high reaction velocity with chymotrypsin. On the other hand, chymotrypsin was less specific for the AEH substrate 1 because of the structural degree of freedom caused by the branched side-chain structure of the AEH residue. This degree of freedom increases the space in which this amino acid side chain can exist dynamically, which may have affected the enzyme-substrate interaction. From a comparison of the peptide substrates containing natural amino acids, the specificity of chymotrypsin was determined to be greatly affected by the substrate’s hydrophobicity, aromaticity, and the conformation of the amino acid residue at the recognition site. The unnatural amino acids in this study were used to investigate the specificity of chymotrypsin for substrates with side-chain structures with a branched chain or an aliphatic ring. Unnatural amino acids with structures different from natural amino acids can be used in further investigations into the specificity of protease enzymes, including chymotrypsin.

Supplementary Materials: The following are available online at https://www.mdpi.com/2227-9 717/9/2/242/s1, Figure S1: MS data of the four synthesized substrates H2N-Ala-Ala-XXX-AMC, where XXX is either AEH 1, Cha 2, Phe 3, or Leu 4. Author Contributions: Conceptualization, Y.Y. and T.K.; substrate synthesis, T.Y.; data analysis, Y.Y.; writing-original draft preparation, Y.Y.; writing-review and editing, T.K.; supervision, T.K. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available in article and supple- mentary material. Conflicts of Interest: The authors declare no conflict of interest.

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