J. CHEM. SOC. PERKIN TRANS. 1 1995 Strong inhibitory effect of sugarobiphenylylboronic acid complexes on the hydrolytic activity of a-chymotrypsin Hikaru Suenaga, Masafumi Mikami, Hiromasa Yamamoto, Takaaki Harada and Seiji Shinkai * CHEMIRECOGNICS Project, ERA TO, Research Development Corporation of Japan, 2432-3 Aikawa-cho, Kurume, Fukuoka 830, Japan Boronic acids act as transition-state analogues for certain peptidases. The inhibitory effect of 2-, 3-and 4-biphenylylboronic acids (2a, 2b and 2c) on the hydrolytic activity of a-chymotrypsin has been investigated. These inhibitors were employed to monitor the binding event formation of covalent bond with either serine residue (195) or histidine residue (57) occurring in the active site by a fluorescence method.It was shown that the decrease in the fluorescence intensity, which is induced by the formation of a covalent bond with the boronic acid moiety, is well correlated with the inhibitory effect estimated by kinetic measurements. The inhibitory effect appeared in the order 2a 2c 4 2b (Ki = 1.6 x mol dm-3). Interestingly, the inhibitory effect was further intensified by added saccharides. In particular, the combined system of 2b and D-glucose strongly inhibited the enzyme reaction, the inhibitory effect (Ki = 1.1 x lop7 mol dmP3) being stronger than that of a specijic inhibitor, chymostatin (Ki = 4.8 x mol dm-3). Hence, saccharides act as a lsquo;co-inhibitorrsquo; in the boronic acid inhibition system. This is a novel and efficient inhibition system for r-chymotrypsin (and probably more generally for other peptidases).Introduction Boronic acids adopt an sp2-hybridized boron atom which easily reacts as a Lewis acid with nucleophiles such as hydroxide, alkoxide or imidazole to give a tetrahedral boron adduct.rsquo; This adduct formation also occurs in the active site of certain hydrolytic enzymes such as subtilisin and a-chymo- trypsin and the active site serine (or histidine) is usually the fourth ligand of the tetrahedral structure.rsquo; In fact, phenyl- boronic acid 1acts as a novel lsquo;transition-state analoguersquo; for these enzymes and the amp;-values are estimated to be 0.23-0.80 mmol dm-3 for subtilisin and 0.19 mmol dmP3 for a-chymotrypsin. lsquo;v2 We previously found that the inhibitory effect of compound 1is efficiently intensified by added saccharides.3,4 The synergistic effect is attributed to the generation of a more acidic boron atom through self-complexation with saccharides; this efficiently reacts with the active site serine (or histidine) in a-chymo- Totryp~in.~.~ obtain further insight into the inhibition mechanism we investigated several fluorescent boronic acids which act as photo-induced electron-transfer probes in the active site of a-chymotrypsin. We unexpectedly found that biphenylylboronic acids 2 show a remarkably strong inhibitory effect in the presence of certain saccharides, which is comparable with that of chymostatin, a specific inhibitor for a-chymotrypsin. This system is useful as an expeditious method to inhibit totally or control conveniently the activity of 01-c hymot rypsin.Results and discussion pH Dependence of the inhibitory effect It was recently demonstrated by several groups that boronic acids serve as a useful means of detecting saccharides in ~ater.~-rsquo;~For example, saccharides in water can be spectro- photometrically detected by boronic acid-appended porphyrins or boronic acid-appended fluorophores. 778912 Detection of .saccharides with boronic acid-appended porphyrins is based on the idea that absorption and fluorescence spectra of porphyrins change sensitively in response to a shift in the aggregation- deaggregation equilibrium and that the complexation of saccharides with the boronic acid moieties changes this 1 2a 2-B(OH)22b 3-B(OH)?2c CB(O equilibrium to deaggregation because of the enhanced hydro- philicity of the complexed porphyrin.7 Here, it occurred to us that phenylboronic acid working as an inhibitor in the enzyme active site should be withdrawn upon complexation with saccharides because of the enhanced hydrophilicity of the saccharide-complexed inhibitor.As a result the enzyme activity should be regenerated. To test this intriguing hypothesis we investigated the influence of added saccharides on the inhibition ability of compound 1 in a-chymotrypsin-catalysed hydrolysis of N-benzoyl-L-tyrosine p-nitroanilide (Sub). However, to our surprise, added saccharides efficiently intensified the inhibitory effect and in certain saccharides a distinct enantiomeric (D/L) discrimination in the inhibitory effect was observed.The pH dependence for compound 1is shown in Fig. 1. In the absence of compound 1 the maximum enzyme activity is observed at pH 7.5. In the presence of compound 1,on the other hand, the enzyme activity is suppressed over a wide pH range and the maximum appeared at pH -8.0.In the presence of both compound 1 and D-fructose the enzyme activity is strongly suppressed at pH 47: above pH 7 the activity gradually increases and at maximum activity (pH 9.0) it becomes higher than that in the presence of only compound 1. In the presence of both compound 1and D-glucose the inhibition at pH 4-7 is not 1734 J. CHEM. SOC. PERKIN TRANS. 1 1995 E-Nu-B-(OmI Ph amp;Nu lessreaCtiamp; phB(oH)2 - OH - h PhB -(OH), E-Nu 2'7-E -NU-? -(OH), Ph L OH OH sa( sa OH OH bsol; bsol; E-Nu-B, -00,,Sacbh 0 E-NU fl morereactive Ph-B 0 Sac 0 - OH- - 0m-i~ 'sacI 'oO OH E-Nu .+-E-Nu-iiOSac ko Scheme 1 E-Nu and Sac :::denote x-chymotrypsin and saccharide, respectively 100 80 20 0 3 4 5 6 7 8 9101112 PH Fig.1 Plots of activity vs. pH for compound 1: 37 "C, x-chymo-trypsin = 6.08 x lo-' mol dm-3, Sub = 7.56 x rnol dm-3, I = 6.31 x rnol dm-3, saccharide = 7.69 x rnol dm-3: control (0)in the absence of compound 1 and in the presence of D-fructose or D-glucose (7.69 x rnol dm-3), in the presence of compound 1 (@) and in the presence of both compound 1 and either D-fructose (0)or D-glucose (a) so conspicuous as that in the presence of both compound 1 and D-fructose but the activity does not increase even at pH 7-9.In any case the pH-activity profile in the presence of both boronic acid 1 and the saccharide is more or less the same irrespective of the saccharide used. How can we rationalize these pH dependences? The reaction processes involved in the present system are expressed as in Scheme 1. As shown in Fig. 1, the inhibitory effect is scarcely. seen above pH 9.5. This implies that anionic species generated at high pH { ie., PhB-(OH), and Ph(HO)B-10,-Sugar) do not react with the active-site serine (or histidine) in a-chymotrypsin (E--Nu:Nu denotes either serine or histidine acting as a nucleophile in the active site).On the other hand, it can react with PhB(OH), and PhB0,-Sugar to give the corresponding boron adducts. It is already known that the pK, for PhB0,- Sugar is lower by -2.5 pK-units than that for PhB(OH),: that is, PhB0,-Sugar is more acidic as a Lewis acid than is PhB(OH),.8.'1*12 He nce, the nucleophilic reaction between E-Nu and PhB0,-Sugar occurs in preference to that between E-Nu and PhB(OH),. This difference causes the large inhibitory effect at pH 4-9. Above pH 9.5, on the other hand, PhB0,-Sugar is totally converted into Ph(HO)B-10,-Sugar and cannot react with the nucleophile in the enzyme active site. This kinetic situation gives the maximum activity at around pH 9. It is known that, in complexation with compound 1, D-fructose has an association constant larger than that for D-glucose: 5*7*8*' 9' hence, D-fructose forms the inhibitory species PhB0,-Sugar more efficiently than does D-glucose at low pH, but the major complex species is converted into the non-inhibitory species Ph(HO)B-10,-Sugar at high pH.Therefore, the distribution of pH-dependent species can well explain the pH-activity profiles in Fig. 1. Fig. 2 shows the pH-activity profiles for three biphenylyl- boronic acid isomers. Added 2-isomer 2a suppresses the enzyme activity to some extent but added saccharides scarcely intensify the inhibitory effect. The steric crowding around the boronic acid moiety probably hampers the efficient binding of saccharides. The pH-activity profiles for 4-isomer 2c are more or less similar to those for the phenylboronic acid 1: in the presence of compound 2c the enzyme activity is suppressed at all pH ranges and the co-existence of D-fructose gives rise to both the strong inhibitory effect at pH 4-7 and the recovery of the activity at pH 8-10.The results suggest that complexes Zc*saccharide are bound to the enzyme active site in a manner similar to l-saccharide complexes. On the other hand, 3-isomer 2b shows unique pH-activity profiles. Addition of fructose moderately intensifies the inhibitory effect of 2b at pH 4-7 but lessens the inhibition at pH 7-9, and the L-isomer is more effective than the D-isomer. Addition of glucose strongly intensifies the inhibitory effect and at pH 4-7 a-chymotrypsin totally loses its hydrolytic activity. Both L-and D-isomer are effective but at pH 8-10 the D-isomer is more effective than the L-isomer.Fluorescence change in compound 2b bound to the active site To obtain further insight into the complexation event occurring at the active site of a-chymotrypsin we measured the fluorescence spectra of compound Zb, which showed the strongest inhibitory effect. The excitation wavelength is 246 nm, which is an isosbestic point in the pH-dependent absorption spectrum. Typical fluorescence spectra are shown in Fig. 3. In Fig. 4 the fluorescence intensity at the emission maximum (324 nm) is plotted against pH. In the absence of a-chymotrypsin the fluorescence intensity decreases with increasing medium pH (Fig.4A), which occurs in response to a change from fluorescence Ph-Ph-B(OH), to non-fluorescent Ph-Ph-B-(OH),.t Hence, the apparent pK, is estimated to be 8.6. When D-glucose was added, the pH-1324 profile shifted slightIy to lower pH: the apparent pK, was then 8.2. In contrast, when D-fructose was added, a large PH-1324 profile shift was induced at pH 6-9 and the pH-I,,, did not descend to zero even Previously, Mohler and Czarnik proposed 2-anthrylboronic acid as a fluorescent receptor for saccharide sensing: ref. 12(6). The fluorescence change induced by addition of saccharide (I in the presence of saccharidell, in the absence of saccharide) is -0.7. As shown in Fig. 4, compound 2b shows I/I, = 0-0.3, indicating that compound 2b is much superior as a fluorescent receptor to 2-anthrylboronic acid.J. CHEM. SOC. PERKIN TRANS. I 1995 1735 100 90 80 t8 70 .60h Y7-50u $40 30 20 10 0 Fig. 2 Plots of activity us. pH for compounds 2: 37T, a-chymotrypsin = 6.08 x mol dm-', Sub = 7.56 x 10 rnol dm-', in the absence of compounds 2 and in the presence of D-fructose 121 = 6.31 x rnol dm-', saccharide = 7.69 x mol dm-': control (0) or D-glucose (7.69 x rnol dm-')I, in the presence of compounds 2 (0)and in the presence of both compounds 2 and D-fructose (0)or D-glUCOSe (a) 250 300 350 400 450 250 300 350 400 450 500 Wavelength A /nm Wavelength A /nm Fig. 3 Fluorescence spectra of compound 2b (A) and compound 2b plus D-fructose (B): 37 "C,2b = 3.35 x rnol dm-3, ~-fructose = 3.30 x rnol dm-', excitation 246 nm.Numbers in figures denote medium pH 80 60 40 20 0 2 4 6 8 10 12 4 6 8 10 12 PH PH Fig. 4 Plots of the fluorescence intensity of compound 2b in the absence (A) and the presence (B) of a-chymotrypsin us. pH: 37"C, a-chymotrypsin = 2b = 3.35 x mol dm-', saccharide =3.30 x lo-' rnol dm-' (when it is added), excitation 246 nm, emission 324 nm: no saccharide (O),with D-fructose (0)and with D-glucose (A). For comparison we show in B the fluorescence intensity without a-chymotrypsin by dotted lines, and changes induced by a-chymotrypsin addition are indicated by arrows at high pH. The apparent pK, was then 6.8. The difference in the for D-glucose (1 10mol-' dm3 for phenylboronic acid).' On the profile shift is accounted for by the difference in the affinity with other hand, it is not so easy to offer a rationale for the difference boronic acids: the association constant for D-fructose (4370 in the quenching efficiency at high pH. Two possible explan- mol-' dm3 for phenylboronic acid) l3 is much greater than that ations come to mind. The first rationale is related to the steric 1736 J. CHEM. SOC. PERKIN TRANS. 1 1995 0 10 20 30 1062/mol dm-3 Fig. 5 Plots of 2 us. activity: 2a (A), 2b (e),2c (0),2b + D-fructose (A), 2b + D-glucose (0)and chymostatin (m). The measurement conditions are similar to those recorded in the caption to Fig. 2 (37 "C;standard pH 8.0 with 50 mmol dm-3 phosphate buffer).1004 1 1 I 80 60 IE 40 a 20 0 0 10 20 30 10~2/rnol Fig. 6 Plots of 2b vs. activity: 2b (a),2b + methyl a-DglUCO- pyranoside (O),2b + D-glucose (m) and 2b + 6-deoxy-~-glucose(0). The measurement conditions are similar to those recorded in the caption to Fig. 2. effect. We recently found that in a photo-induced electron- transfer system of compound 3 the quenching efficiency is sensitively affected by a dihedral angle between the naphthalene (Ar) plane and the lone-pair orbital in the nitrogen atom.', This suggests that in an Ar(fluorescent)-B-system the quenching efficiency is affected by the angle of the sp3-hybridized orbital in the B- atom. Conceivably, D-fructose and D-glucose give different dihedral angles, which leads to the different quenching efficiencies.The second rationale is related to the electronic effect. Since ArB0,-Sugar is more acidic than ArB(OH),,8~"i'2 the electron density of the boron atom in Ar(HO)B-10,-Sugar should be lower than that in ArB-(OH),. In compound 2b, therefore, the quench- ing efficiency in Ph-Ph(H0)B-10,-Sugar is inferior to that in Ph-Ph-B-(OH),. The pH-1324 profiles in the presence of a-chymotrypsin are more or less similar to those in the absence of a-chymotrypsin. This indicates that compound 2b is converted into a non- fluorescent or less fluorescent molecule through the nucleo- philic attack of a functional group (serine or histidine) in the active site of a-chymotrypsin.Careful examination of a plot for compound 2b in the presence of a-chymotrypsin reveals that there are two independent pKa-values, 5.0 and 8.6. These two 3 values are attributed to the enzyme-bound acid 2b and free acid 2b, respectively. Although both D-fructose and D-glucose induce a shift of the PH-1324 profile to lower pH, the magnitude induced by addition of D-glucose is much greater than that induced by addition of D-fructose, and with D-glucose two independent pKa-values (3.2 and 8.2) are observable. With D-fructose compound 2b is predominantly converted into a less fluorescent 2b~-fructose complex because of the high affinity with D-fructose. Hence, the formation of a complex with a-chymotrypsin scarcely changes the fluorescence intensity.With D-glucose, in contrast, compound 2b predomi- nantly exists as the fluorescent free species because of its low affinity with D-glucose, and the binding of the 2b~-glucose complex to the active site causes the remarkable shift of the pH-I,,, profile to lower pH. The apparent pKa-values are 3.2 and 8.2. Since the higher pKa (8.2) is nearly consistent with that of compound 2b in the absence of a-chymotrypsin (8.6), it is assigned to the pKa-value of unbound acid 2b. The lower pK, (3.2) is attributed to a new species which is different both from Zb-~-glucose and from 2ba-chymotrypsin (pK, 5.0). Hence, this should be a ternary complex 2b-~-glucose-a-chymotrypsin. The pKa-value is lower by 5.4 pK-units than that of free boronic acid 2b and by 1.8 pK-units than that for enzyme- bound acid 2b.The remarkably large pKa shift implies that complex 2b~-glucose is strongly bound to the active site of a-chymotrypsin. Strong inhibitory effect of compound 2b plus glucose which is stronger than that of chymostatin The foregoing fluorescence studies suggest that compound 2b would show a very strong inhibitory effect in the presence of D-glucose. We therefore compared the inhibitory effect with that of chymostatin, a specific inhibitor for a-chymotrypsin. ' As shown in Fig. 5, the combined inhibitor of compound 2b and D-glucose shows a remarkably strong inhibitory effect at (0-20) x mol dm-3 and which is euen stronger than that of chymostatin. In chymostatin the activity is entirely suppressed at 2.2 x mol dm-3.In the combined inhibitor, on the other hand, the activity gradually decreases to zero and it is totally suppressed at 7.8 x mol drn-,. The combined inhibitor system can be expressed in the form of three equations (1H3)in addition to the conventional enzyme-catalysed reaction system. Although the 2b-~-glucose complex strongly binds to the enzyme active site and inhibits the enzyme activity, equations (1) and (2), which have not so large binding constants, retard the abrupt decrease in the activity.' On the other hand, the acid 2b itself shows a moderate inhibitory effect, which is much weaker than that of chymostatin (Fig. 5). It is also seen from Fig. 5 that compounds 2a and 2c show a further, weak inhibitory effect.2b + D-glucose e2b-~-glucose (1) Enzyme + 2b Enzyme-2b (2) Enzyme + 2b-~-glucose Enzyme-Zb.~-glucose (3) 1737J. CHEM. soc. PERKIN TRANS. 1 1995 40 1 30 20 10 -4-64-20 2 4 6 8 0 I1 0 1 2 3 -6 -4 -2 0 2 4 6 1062b/mol dm-3 1042b/mol dm-3 1072b/mol dm-3 Fig. 7 Typical Dixon plots: 37 "C, a-chymotrypsin = 6.08 x rnol dm-3, Sub = 7.56 x rnol dm-3 (open symbols) and 1.51 x lo4 rnol dm-3 (filled symbols), saccharide = 7.69 x mol dm-3: (a) (0,m) 2b only, (b) (0,@) 2b + D-fructose and (A, A)2b + D-glucose Table 1 K,-values for inhibitors (37 "C;pH 8.0) Inhibitor lo7 Ki/mol dm-3 2b 16 2b + D-Fructose 130 2b + L-Fructox 11 2b + D-Glucose 1.1 Chymostatin 4.8 To obtain insight into the complexation site of D-glucose with acid 2b, we compared the activity of methyl a-D-glucopyranoside 4 with that of 6-deoxy-~-glucose 5 as a 'co-inhibitor'.In methyl a-D-glucopyranoside 4 the sole complexation site is the 4,6-diol moiety, whereas in 6-deoxy-~-glucose 5 the sole complexation site is the 1,Zdiol moiety. As shown in Fig. 6, the inhibitory effect in the presence of compound 2b and methyl a-D-glUCO- pyranoside 4 is consistent with that in the presence of only compound 2b. On the other hand, the inhibitory effect in the presence of the acid 2b and 6-deoxy-~-glucose 5 is comparable with that in the presence of both the acid 2b and D-glucose. The results clearly establish that the complexation site in D-glucose is the 1,2-diol functionality.HO 110 HO OH *OH Ho* 011 OMe 4 5 We have estimated Ki-values for acid 2b, 2b + D-fructose, 2b + D-glucose and chymostatin by use of a Dixon plot.16 Although the combined inhibitor systems may not necessarily result in a straight line in the Dixon plot, because of the presence of additional equilibria, the plots at 2b = 0.95 x to 7.81 x mol dm-3 gave satisfactory straight lines (Fig. 7). We therefore determined the apparent Ki-value from the intercept with the I/ V = 0 line. Also, as commercially available chymostatin is a mixture of (S)-Ile and (S)-Val species, one must consider that this also gives rise to the apparent Ki. The results are summarized in Table 1. Several interesting points can be made about the data in Table 1, First, system 2b + L-fructose has a Ki-value smaller than that for free acid 2b whereas system 2b + D-fructose has a K,-value larger than that for acid 2b.The difference is due to the medium pH (8.0) employed for the kinetic measurements: although both D-fructose and L-fructose similarly intensify the inhibitory effect at low pH and rather weaken it at high pH (Fig. 2), the inhibitory effect appears more strikingly in L-fructose. At pH 8.0 two isomers operate in opposite direction on the enzyme activity. Anyhow, it is interesting that the inhibitory effect of acid 2b reflects the absolute configuration of covalently bound saccharides. Secondly, D-glucose is more effective as a 'co- inhibitor' by two orders of magnitude than is D-fructose.As described previously, the affinity of D-fructose with boronic acids is much greater than that of ~-glucose.~~~~~,"*~~ This trend means that D-fructose-boronic acid complexes are relatively stable and therefore less reactive with a-chymotrypsin. In contrast, D-glucose*boronic acid complexes are relatively unstable and therefore active for nucleophilic attack of a-chymotrypsin. Of course, one has to take the shape selectivity of a-chymotrypsin for substrates into account but we believe that the stability of saccharide=boronic acid complexes is also operative for the determination of the inhibitory effect. Thirdly, and most importantly, the Ki-oalue for system 2b + D-glucose is smaller by more than 4-fold than that for chymostatin.It is very interesting that in spite of its simple structure the inhibitory effect of the 2b-~-glucose complex is stronger than that of the specific a-chymotrypsin inhibitor. At present the binding mode is not yet clear except for the fact that the boronic acid moiety forms a covalent linkage with either serine or histidine." We consider, however, that because of the superinhibitory effect the binding mechanism merits further investigation. We are now trying to understand the binding mode in the active site by computational methods and "B NMR spectroscopy. Conclusions The original aim of this study was to synthesize biphenylyl- boronic acids and to monitor the inhibition event occurring at the active site of a-chymotrypsin by fluorescence spectroscopy.Through this study we unexpectedly found that the inhibitory effect of the system 2b + D-glucose is comparable to that of a specific inhibitor, chymostatin. Since acid 2b itself only moderately inhibits the enzyme's activity, it follows that the effect is remarkably enhanced by complexation with D-glucose. Therefore, one may call D-glucose a 'co-inhibitor'. Although the detailed binding mode of the 2b-~-glucose complex is not yet clarified, the present findings suggest that the development of new boronic acid derivatives leads to exploitation of superinhibitors for nucleophilic hydrolytic enzymes. We are currently studying different boronic acids bearing different acidity functions, different saccharides possessing different stereostructures and different enzymes having similar mech- anisms, expecting that the large inhibitory effect will be reproduced by a simple, combined system of boronic acids and sugars.1738 Experimenta1 General procedures All experiments were carried out under a nitrogen atmosphere. Tetrahydrofuran (THF) was distilled from sodium-benzo- phenone immediately prior to use. 'H NMR spectra were recorded on a Bruker ARX-300 spectrometer. Mass spectro- metry was performed on a Hitachi M-2500 instrument. IR spectra were obtained as KBr disks using a Shimadzu FT-IR 8100 spectrometer. UV spectra were measured on a Shimadzu UV-2200 spectrometer. Melting points were determined on a Yanaco (MP-5OOD) micro melting point apparatus and are uncorrected.Materials Compounds 1 and 2a were purchased from Aldrich. Compounds 2b and 2c were synthesized from the corresponding biphenylyl bromides (vide post). Chymostatin was purchased from Sigma. Biphenyl4ylboronic acid 2c. To a tetrahydrofuran (THF) solution (30 cm3) of 4-bromobiphenyl (2.33 g, 10.0 mmol) at -78 "C was added a hexane solution (10 cm3) of butyllithium (16 mmol) and the reaction mixture was stirred for 1 h under nitrogen. The obtained lithium reagent was added to another flask which contained a THF (50 cm3) solution of trimethyl borate (6.41 g, 61.6 mmol) via a cannula by nitrogen pressure at -78 "C. The reaction mixture was stirred for 1 h, and then was gradually warmed to room temp.After addition of 2 mol dm-3 HCl (10 cm3), the mixture was stirred at room temp. for 15 h. After removal of the solvent under reduced pressure, diethyl ether (50 cm3) was added and the organic phase was washed twice with water. The organic layer was separated, and dried over anhydrous magnesium sulfate. Concentration of the solution resulted in an oily product, which was triturated with hexane. Finally, the solid was reprecipitated from methanol- water to give title compound 2c (40), mp 263-266deg;C; vmax(KBr disk)/cm-' 3393 (OH); dH(3O0 MHz; CD,OD), 7.24 (1 H, d, ArH), 7.33 (2 H, t, ArH), 7.46-7.53 (4 H, m, ArH), 7.59 (1 H, d, ArH) and 7.73 (1 H, d, ArH) (Found: C, 72.5; H, 5.6. CI2H,lBOzrequiresC, 72.78; H, 5.60);m/z(SIMS+, glycerol) 254(M + glycerol -2H,O+).Biphenyl-3ylboronic acid 2b. This compound was synthesized in a similar manner to its 4-isomer 2c: yield 43, mp 203- 205 "C; v,,,(KBr disk)/cm-' 3274 (OH); dH(300 MHz; CD,OD), 7.24 (1 H, d, ArH), 7.33 (3 H, t, ArH), 7.50-7.55 (4 H, m, ArH) and 7.73 (1 H, s, ArH) (Found: C, 72.9; H, 5.6); m/z (SIMSf, glycerol) 254 (M' + glycerol -2H,0+). Estimation of the a-chymotrypsin activity a-Chymotrypsin was purchased from Sigma (Type 11: MW 25100). The hydrolytic reaction was carried out according to Kouzuma's method l9 37 "C; standard pH 8.0 with 50 mmol dm-3 phosphate buffer, 0.3 vol methanol plus 0.8 vol dimethyl sulfoxide (DMSO) and the progress of the reaction was followed by monitoring the appearance of the absorption band at 410 nm (p-nitroaniline: P) (see Fig.7). The activity was estimated from the liner A,,, us. time plots for the initial 10 min. Fluorescence measurements A DMSO solution (3.3 cm3) containing compound 2b (1.01 x rnol dm-3), an aqueous solution (3.3 cm3) containing saccharide (1 .Ornol dm-3) and an aqueous solution (4.22 cm3) containing a-chymotrypsin (7.97 x rnol dm-3) were added to a buffered aqueous solution (100 cm3) adjusted J. CHEM. soc. PERKIN TRANS. 1 1995 with 1.0 rnol dm-3 HCI and 1.0 mol dm-3 NaOH. The final concentrations were 3.35 x mol dm-3 for compound 2b, 3.30 x rnol dmP3 for saccharide and 3.35 x rnol dm-3 for a-chymotrypsin. After the solution was stored for 5 min at 25 "C, the fluorescence spectra were measured in a 1 cm cell with a Perkin-Elmer Model LS5OB fluorescence spectro- photometer.References 1 (a) J. C. Powers and H. J. Wade, Proteinase Inhibitors, Elsevier Science Publishers, Amsterdam, The Netherlands, 1986; V. K. Antonov, T. V. Ivanina, I. V. Berezin and K. Martinek, FEBS Lett., 1970,7,23; (b)M. Philipp and M. L. Bender, Proc. Natl. Acad. Sci. USA, 1971, 68, 478; (c) C. A. Kettner and A. B. Shenvi, J. Biol. Chem., 1984,259,15106;W. W. Bachovchin, W. Y. L. Wong, S. F. Jones, A. B. Shenvi and C. A. Kettner, Biochemistry, 1988,27, 7689. 2 The mechanistic controversies on trigonal borons us. tetrahedral borons and serine adducts us. histidine adducts are discussed in E. Tsilikounas, C. A. Kettner and W.W. Bachovchin, Biochemistry, 1993,32,1265 1. 3 H. Suenaga, K. Nakashima and S. Shinkai, J. Chem. SOC.,Chem. Commun., 1995, 29. 4 H. Suenaga, K. Nakashima, M. Mikami and S. Shinkai, Chem. 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