Bioconjugate Chem. 1994, 5, 454-458
454
Fluorogenic N-Nitrosoamides: Active-Site Labeling Reagents for Chymotrypsin-like Proteases Min Lit a n d Emil H. White* Department of Chemistry, The Johns Hopkins University, Baltimore, Maryland 21218. Received February 17, 1994@
Two fluorogenic N-nitrosoamides, N-nitroso-N-((7-methoxycoumarin-4-yl)methyl)-N'-isobutyrylalaninamide (6a) and N-nitroso-N-((6-methoxyquinolin-2-yl~methyl)-N'-isobutyrylalaninamide (6b),were synthesized. Both N-nitrosoamides inhibited a-chymotrypsin irreversibly; they show promise as labeling reagents for the active sites of chymotrypsin-like proteases.
INTRODUCTION N-Nitrosoamide derivatives of amino acids are activesite-directed enzyme-activated inhibitors ("suicide inhibitors") for hydrolytic enzymes (White et al., 1975; 1977a; 1977b; 1981; 1990; Donadio et al., 1985; White and Chen, 1993). Because of the extremely high reactivity of the carbocations (White et al., 1968; 1973; 1978) released during enzyme-catalyzed hydrolyses, they can serve a s relatively undiscriminating active-site labeling reagents (eq 1, Scheme 1). The alkylation by the carbocations can occur a t the amide linkages as well as the side chains (except alkyl groups) within the active site of a targeted enzyme (Donadio et al., 1985; White et al., 1990). No other affinity or "suicide" reagents, except photoaffinity labeling reagents (White et al., 1978), have comparable reactivity. We report here the synthesis of two fluorogenic labeling reagents for chymotrypsin-like proteases based on the nitrosoamide functional group. Upon enzymatic activation (hydrolysis), these reagents deliver fluorescent labels (methoxycoumarin o r quinoline moieties) to the amide linkages andor the side chains of amino acid residues in the active site (eq 1). The identification of the alkylated sites should be facilitated by the facile detection of fluorescent peptides produced in a subsequent hydrolysis of the inhibited (labeled) enzyme. RESULTS AND DISCUSSION The general synthetic route employed is outlined in Scheme 2. In the "b' series, the bromomethyl intermediate required (2b)was obtained through bromination of 6-methoxyquinaldine (lb) with N-bromosuccinimide (NBS) in CC14. In addition to the desired mono-a-bromination, ring- and presumably di-a-bromination also occurred. The bromination was terminated, therefore, before the latter side reactions became important. In the second step of the synthesis, only the mono-a-brominated compound (2b) reacted with hexamethylenetetramine (Blazevic et al., 1979) to form a complex salt (3b),which precipitated as a white powder in rather pure form. The salts 3a,b, formed upon reacting 2a,b with hexamethylenetetramine, were hydrolyzed (Nodiff et al., 1974; Blazevic et al., 1979) to give the amines 4a,b in high yields (-90%). The subsequent coupling reactions ~
Present address: Department of Medicinal Chemistry & Pharmacognosy (WC 7811, University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612. Abstract published in Advance ACS Abstracts, August 1, @
1994.
between the amines 4a,b and isobutyrylalanine were effected by using 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC) (Sheehan et al., 1965). In attempts to make optically pure 5b, however, a racemized product was obtained. This was first detected by the unusually small values of the optical rotation for the amides formed: D-5b, [a123.5~ -1.5" (c 4.08, CHC13); ~ - 5 b[a]23,5D , +0.6" (c 2.14, CHCl3). The racemization was confirmed via the 'H-NMR spectrum of D-5b in the presence of a chiral shift reagent, tris[3-((trifluoromethy1)hydroxymethylene)-D-camphoratoleuropium(III) derivative (Goering et al., 1974); two methoxy peaks of approximately equal intensity were observed. It has been reported that carbodiimide type coupling reagents can lead to a substantial amount of racemization of the peptides formed (Anderson and Callahan, 1958; Williams and Young, 1963). In the nitrosation step (Scheme 2) (White, 1955), approximately equal amounts of the mononitroso (6a,b) and dinitroso (7a,b)products were formed. It appears that the difference in steric hindrance between the two potential nitrosation sites is not large enough to substantially reduce the formation of the dinitroso compounds. The mono and dinitroso compounds, however, were readily separated on silica gel columns. In the EDC coupling step to make the quinoline amide (5b), it was found that when the reaction mixture was not protected from air and the reaction time was long (33 h) the initially formed amide 6b underwent an oxidation reaction to give compound 8 with an imide grouping (eq 2). lH-NMR spectra showed that the signal
for the two methylene protons in 5b (4.71-4.65 ppm) disappeared as a result of the oxidation, while the signal for one of the amide protons shifted from 7.52 ppm to 10.93 ppm. The mass spectrum was even more informative (Figure l); in addition to the parent ion a t mlz 343, it showed fragmentation peaks a t 230,229,202,186,158, and 114. Preliminary labeling tests with a-chymotrypsin showed that 40% of the enzyme was irreversibly inhibited (or labeled) by a 70-fold molar excess of 6b,where timedependent inhibition was clearly demonstrated (Figure 2). There appears to be no doubt that the enzyme was
1043-1802/94/2905-0454$04.50/00 1994 American Chemical Society
Bioconjugate Chem., Vol. 5, No. 5, 1994 455
Labeling of Chymotrypsin-like Proteases
Scheme 1
+
L H \
P H
N
A
II
Ri
"OH
Alkylated (labeled) enzyme molecules HX = side group
Scheme 2. Synthesis of Fluorogenic Inhibitors
NBS
R-CH3 lb
2b
a
Note: "a" series:
0
R=
"b"series: R=
CH3O
cH30m
HexamethyleneR-CH2-Br
tetramine
*
2a,b
R-CH2NH3*Cl'
4a,b
R-CH2-(C6H,2N,)+Bi 3a,b
HCVEtOH
Reflux
-
201 + 1 229
Figure 1. MS cracking peaks for imide 8.
N-Bobutyrylalanind "t,N
CH
O
R
Sa,b
0°C
H 6a.b
7a,b
0
40
80
120
.
160 &
Time
irreversibly inhibited; no regeneration of enzyme activity was observed during an incubation period of -10 h. With the same amount of 6a,10% of the enzyme was labeled (Figure 3). It is assumed that in both cases only the D-isomers would be responsible for the observed inhibition, according to a previous study by White et al. (1977a). That is, in reality, the "active" nitrosoamidelenzyme ratio used was 35 rather than 70. Since 6a and 6b contain aromatic groups a t the P1 position and since it is expected that they will not be highly selective (no group is present beyond P2),it is reasonable for us to speculate that these two compounds would also label other proteases that have a specificity for aromatic side chains similar to that of chymotrypsin.
Figure 2. Inhibition of chymotrypsin with 6b. The inhibitor (70-fold total molar excess) in acetonitrile was divided equally into two portions and added dropwise at 0 and 46 min, respectively. The arrows indicate the beginning of each addition. The activities shown are corrected for the activity loss due to autolysis (control run). EXPERIMENTAL PROCEDURES
Materials and Methods. 7-Methoxy-4-(bromomethyl)coumarin, 6-methoxyquinaldine, and 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimidehydrochloride (EDC) were obtained from Aldrich Chemical Co. a-Chymotrypsin (type I-S) was purchased from Sigma Chemical Co. All other reagents were of the highest purity avail-
Li and White
456 Bioconjugate Chem., Vol. 5, No. 5, 1994
i
0
40
80
120
160
Time
Figure 3. Inhibition of chymotrypsin with 6a. The inhibitor (70-fold molar excess) in acetonitrile was added dropwise over a 45-min period. The arrow indicates the beginning of the addition. The activities shown are corrected for the activity loss due to autolysis (control run).
able. Melting points were measured on a Thomas/Hoover Unimelt apparatus and were not corrected. 'H- and 13C-NMRspectra were acquired on a Varian Associates XL-400 NMR spectrometer. Tetramethylsilane (TMS) was used as an internal reference in organic solvents. Infrared spectra were recorded on a PerkinElmer 1600 Series FTIR spectrometer. A Beckman Model 25 UV-vis spectrophotometer was used for recording UV-vis spectra. Mass spectra of organic compounds were acquired on a VG 7 0 3 mass spectrometer.
4-((Hexamethylenetetra"yl)methyl)-7-methoxycoumarin Bromide (3a). Hexamethylenetetramine (HMTA) (0.527 g, 3.76 mmol) in 80 mL of CHC13 and 15 mL of acetone was added dropwise into a solution of compound 2a (1.011 g, 3.76 mmol in 500 mL of acetone) within a period of -20 min a t room temperature. About 10 min after the complete addition of the HMTA solution, the reaction mixture turned cloudy and a white precipitate began to form. The reaction solution was stirred for 4 h a t room temperature and then allowed to stand overnight. A white fine powder (1.37 g, 3.35 mmol, 89%) was obtained, mp 200 "C dec. 'H-NMR (DMSO-&): b 8.08 (d, 1 H, J = 8.8 Hz), 7.14 (d, 1 H, J =2.5 Hz), 7.07 (dd, 1H, J1 = 8.8 Hz, Jz = 2.5 Hz), 6.60 ( s , 1HI, 5.20 (s, 6 H), 4.54 (AB quartet, 6 H, J1 = J Z = 12.6 Hz), 4.22 (s, 2 H), 3.90 (s, 3 H). IR (KBr): 1718, 1613, 1300, 1148, and 1010 cm-l. 4-(Aminomethyl)-7-methoxycoumarin Hydrochloride (4a). Compound 3a (500 mg, 1.22 mmol) was suspended in 27 mL of concd HC1 in EtOH made up in a 1/15 ratio (v/v). The reaction mixture was refluxed for 2.5 h and then cooled to room temperature. Fine white crystals formed a t room temperature; they were collected by filtration to yield the product (280 mg, 95%), mp 234 "C dec. NMR ( D M s 0 - d ~ )6: 8.75 (s, 3 HI, 7.72 (d, 1H, J = 8.8 Hz), 7.07 (d, 1 H, J = 2.5 Hz), 7.1 (dd, 1 H, J1 = 8.8 Hz, Jz = 2.5 Hz), 4.36 (s, 2 H), 3.88 (s, 3 H). IR (KBr): 3030 (broad), 1731, 1682, 1618, 1405, and 1149 cm-'. Anal. Calcd for CllHlzN03C1*0.5Hz0: C, 52.71; H, 5.23; N, 5.59. Found: C, 52.98; H, 5.06; N, 5.96. N-Isobutyrylalanine. The title compound was synthesized according to the procedures of Doherty and Popenoe (1951). 'H-NMR (DMSO-ds): 6 8.00 (d, 1 H, J = 7.2 Hz), 4.17 (m, 1H), 2.41 (m, 1 H), 1.25 (d, 3 H, J = 7.3 Hz), 0.99 (dd, 6 H, J1 = 7.0 Hz, Jz = 6.8 Hz). DLRacemate: mp 124-126 "C (lit. (Doherty and Popenoe, 1951) mp 129-130 "C). D-ISOmer: mp 150-152 "C.
[ a I z 7 ~+32.5" : (c 0.080, EtOH). L-Isomer: mp 147-149 "C. [ a I z 3 ~-33.2" : (C 0.266, EtOH). DL-N-( (7-Methoxycoumarin-4-yl)methyl)-N'-isobutyrylalaninamide (5a). To 10 mL of CHzClz was added compound 4a (150 mg, 0.621 mmol), N-isobutyryl-DLalanine (101 mg, 0.635 mmol), EON (9OpL, 0.647 mmol), and EDC (133 mg, 0.645 mmol). After being stirred at room temperature for 24 h, the reaction mixture was mixed with another 20 mL of CHzClz and then washed with HzO (2x), 3% HC1, 5% NaHC03, and HzO, respectively. The washed solution was dried over anhydrous Na2SO4,and the solvent was removed to give 145 mg (0.419 mmol, 67.5%)of crude product as a yellowish solid, which was recrystallized (90%)from ethyl acetatehexane for use in the next step, mp 205 "C dec. NMR (CDC13): 6 7.7 (s, 1 H), 7.48 (d, 1H, J = 8.8 Hz), 6.85 (dd, 1H, J1 = 8.8 Hz, J2 = 2.5 Hz), 6.79 (d, 1 H, J = 2.5 Hz), 6.4 (d, 1H, J = 7 Hz), 6.16 (s, 1H), 4.64 (m, 2 H), 4.51 (m, 1H), 3.87 (s, 3 H), 2.41 (m, H), 1.41 (d, 3 H), 1.14 (t, 6 H, J = 7.0 Hz). IR (KBr): 3278, 1726, 1637, 1618, 1542, and 1292 cm-'. Anal. Calcd for C18HzzO~Nz.0.25HzO: C, 61.63; H, 6.42. Found: C, 61.74; H, 6.22. Nitrosation of ~~-N-((7-Methoxycoumarin-4-yl)methyl)-N'-isobutyrylalaninamide(5a). Coumarin amide 5a (24.5 mg, 0.071 mmol) was dissolved in a mixture of acetic acid (306 pL, 5.39 mmol) and acetic anhydride (1.52 mL, 16.1 mmol), and the solution was cooled in an ice-water bath. Sodium nitrite (114 mg, 1.65 mmol) was added to the amide solution a t once with strong magnetic stirring. M e r the starting material had disappeared (-60 min; TLC), the reaction mixture was diluted with 20 mL of CHzClzand then washed with cold 5% NaHC03 solution (2x) and HzO. The washed solution was dried over anhydrous NazS04, and the solvent was removed in vacuo. The residue was applied in ethyl acetate to a short silica gel column; elution occurred with ethyl acetateihexane (1/2, v/v). The fast-moving yellow band (6 mg, 21%) ( R f =0.7) was found to be the dinitroso compound (7a);no NH signals were observed in the 'HNMR spectrum (CDC13): 6 7.44 (d, 1H, J = 8.8 Hz), 6.88 (dd, 1 H, J1 = 8.8 Hz, Jz = 2.5 Hz), 6.83 (d, J = 2.5 Hz), 6.03 (9, 1H, J = 7.0 Hz), 5.51 (s, 1H), 4.95 (AB quartet, J1 = J Z = 16 Hz), 3.88 (s, 3 H), 3.78 (m, 1H), 1.47 (d, 3 H, J = 7.0 Hz), 1.26 (d, 3 H, J = 6.9 Hz), 1.23 (d, 3 H, J = 6.8 Hz). After the first band had been collected, ethyl acetate was used to elute the desired mononitroso compound 6a (Rf=. 0.4). Extra pressure was employed during the separation to ensure that the whole process was complete within 10-15 min, as longer times led to decomposition of the nitroso compounds. After removal of the solvents, a yellow oil (6a)was obtained (6.8 mg, 26%). 'H-NMR (CDC13): 6 7.37 (d, 1H, J = 8.8 Hz), 6.81 (dd, 1 H, J1 8.8 Hz, J z = 2.5 Hz), 6.76 (d, 1H, J = 2.5 Hz), 6.18 (d, 1H, J = 7 Hz), 5.77 (m, 1HI, 5.51 (s, 1 HI, 4.95 (s, 2 HI, 3.81 (s, 3 H), 2.42 (m, 1 HI, 1.55 (d, 3 H, J = 7.2 Hz), 1.14 (d, 3 H, J = 6.9 Hz), 1.13 (d, 3 H, J = 7.0 Hz). Nitrosations were also conducted at -13 "C [same ratio of acetic anhydridelacetic acid (vlv) as used above: 5/11, as well as a t different ratios of acetic anhydride/acetic acid such as 2/1 and 1/1(0 "C), in an attempt to minimize the amount of dinitroso compound. Under these conditions, however, the product distribution was essentially the same; i.e., the mononitroso/dinitroso ratio was -1. 2-(Bromomethyl)-7-methoxyquinoline(2b) and 24(Hexamethylenetetraminiumyl)methyl)-6-methoxyquinoline Bromide (3b). To 434 mL of CC14 was added 6-methoxyquinaldine (lb)(7.24 g, 41.8 mmol), NBS (7.44 g, 41.8 mmol), and benzoyl peroxide (290 mg,
Labeling of Chymotrypsin-like Proteases
Bioconjugate Chem., Vol. 5, No. 5, 1994 457
mp 74 "C (began to decompose), 97 "C (crystals collapsed and turned opaque), 119 "C, full melting. 'H-NMR (DMSO-&): 6 8.19 (d, 1H, J =8.4 Hz), 7.85 (d, 1H, J = 9.2 Hz), 7.55 (d, 1 H, J = 8.4 Hz), 7.38-7.32 (m, 2 H), 3.92 (s, 2 H), 3.87 (s, 3 H). IR (KBr): 3351, 1622, 1601, 1501, and 1233 cm-l. N - ((6-Methoxyquinolin-2-yl)methyl)-N'-isobutyrylalaninamide (5b). The DL isomers were synthesized from 4b and DL-isobutyrylalanine following the same method outlined for Sa. In attempts to synthesize optically pure 5b, D- and L-isobutyrylalanines were utilized in lieu of the DL racemate. Both D-5band ~ - 5 b , giving identical lH NMR spectra to that of DL-Bb, had very low values of optical rotation. D-5b. [a]23,5~: -1.5" (C 4.08, CHC13). L-5b. +0.6" (C 2.14, CHCl3). 'HNMR (CDC13): 6 8.03 (d, 1 H, J = 8.6 Hz), 7.94 (d, 1 H, J = 9.2 Hz), 7.52 (s, 1 H, br), 7.38 (dd, 1H, J1 = 9.2 Hz, J z = 2.8 Hz), 7.28 (d, 1 H, J =8.6 Hz), 7.08 (d, 1 H, J = 2.8 Hz), 6.24 (d, 1 H, J = 6.8 Hz), 4.71-4.65 (m, 3 HI, 3.94 (s, 3 H), 2.43 (m, 1 H), 1.47 (d, 3 H, J = 7.2 Hz), 1.190(d,3H,J=6.8Hz),1.186(d,3H,J=6.8Hz).IR (KBr): 3270,1639,1547,1501,1235,and 832 cm-l. The elementary analysis for D-5b was satisfactory. Anal. Calcd for C18HZ3N3O3:C, 65.63; H, 7.04; N, 12.76. Found: C, 65.63; H, 7.06; N, 12.38. DL-N((6-Methoxyquinolin-2-yl)carbonyl)-N-isobutyrylalaninamide (8). In an attempted synthesis of DL5b during which the reaction was carried out in air for a prolonged time (33h), it was found that a fluorescent side product was formed [Rf 0.78 on a silica gel TLC plate eluted with BuOWAcOWHzO (4/1/1, v/v/v); compound DL5b had a n Rf of 0.37 under the same conditions]. The side product was separated from DL-5b on a silica gel column (elution was carried out with ethyl acetate until the first compound was eluted and then with acetone). 'H-NMR and EIMS spectra indicated that this side product stemmed from the oxidation of the 2-methylene (d,lH,J=9.2Hz),7.62(d,lH,J=8.4H~),7.50-7.47 carbon of the quinoline moiety. lH-NMR (CDC13): 6 (m, 2 HI, 5.23 (s, 6 HI, 4.52 (AB quartet, 6 H, J1 = JZ= 10.93 (s, 1 H), 8.28 (d, 1 H, J = 8.4 Hz), 8.24 (d, 1 H, J 12.6 Hz), 4.26 (s, 2 HI, 3.92 (s, 3 HI. IR (KBr): 1620, = 8.4 Hz), 8.05 (d, 1 H, J = 9.2 Hz), 7.47 (dd, 1 H, J1 1500, 1266, 1241, 999, and 812 cm-l. Anal. Calcd for 9.2 Hz, J2 = 2.8 Hz), 7.14 (d, 1H, J = 2.8 Hz), 6.29 (d, 1 C1,HzzN~0Br0.25Hz0: C, 51.46; H, 5.72; N, 17.65. H, J = 6.8 Hz), 5.47 (m, 1H), 3.98 (s, 3 H), 2.45 (m, 1H), Found: C, 51.42; H, 5.59; N, 17.97. 1.54 (d, 3 H, J = 6.8 Hz), 1.21 (d, 3 H, J = 6.8 Hz), 1.20 (d, 3 H, J = 6.8 Hz). EIMS: m/z 343 (15, M+), 230 (331, The bromination of l b with NBS was also tried in 229 (321, 202 (201, 186 (401, 158 (loo), and 114 (13) CHC13, instead of CCl4. After being refluxed for 4 h, the (Figure 1). reaction solution was checked by TLC; it showed a strong Nitrosation of Quinoline Amide DL-5b. The prononfluorescent spot a t Rf 0.18 (a ring bromination cedure described for the nitrosation of coumarin amide product) and a very weak spot a t Rf0.51(2b),in addition 5a was followed. Dinitroso- (7b)and mononitrosoquinoto the strong fluorescent spot of the starting material l b line amides (6b) were formed in approximately equal at Rf 0.27. Therefore, the bromination of l b in CHC13 quantities and separated on a silica gel column as appeared to favor ring bromination over bromination a t described for 7a and 6a. Dinitroso 7b. NMR (CDC13): the a position; it was thus not useful for the desired 6 7.96 (d, 1H, J = 8.4 Hz), 7.84 (d, 1H, J = 9.0 Hz), 7.32 synthesis. (dd, 1 H, J1 = 9.0 Hz, J z = 2.6 Hz), 7.08 (d, 1H, J = 8.4 2-(Aminomethyl)-6-methoxyquinoline (4b). Using Hz), 7.01 (d, 1 H, J = 2.6 Hz), 6.08 (9, 1 H, J = 6.8 Hz), a method similar to that used for the synthesis of 4a, 5.15 (AB quartet, 2 H, J1= J z = 15.2 Hz), 3.91 (s, 3 H), compound 3b (3.47 g, 8.85 mmol) was dissolved in 195 3.80 (m, 1H), 1.50 (d, 3 H, J = 6.8 Hz), 1.24 (t, 6 H, J = mL of a solution of concd HCl in EtOH (1/15, v/v), and 7.0 Hz). Mononitroso 6b. NMR (CDC13): 6 7.98 (d, 1H, the solution was refluxed for 1.5 h. The precipitate J = 8.5 Hz), 7.77 (d, 1 H, J = 9.2 Hz), 7.30 (dd, 1H, J1 formed was collected by filtration (2.36 g); lH-NMR = 9.2 Hz, J z = 2.8 Hz), 7.14 (d, 1H, J =8.5 Hz), 7.02 (d, spectrum showed that it contained 87% of the desired 1H, J = 2.8 Hz), 6.32 (d, 1H, J = 7.0 Hz), 5.99 (m, 1H), compound (corresponding to an 89% yield) and 13% of 5.21 (AB quartet, 2 H, J1= Jz = 15.5 Hz), 3.91 (s, 3 HI, NH4C1(w/w). 'H-NMR (DMSO-&): 6 8.93 (s, 3 H, br), 2.48 (m, 1H), 1.68 (d, 3 H, J = 7.0 Hz), 1.21 (d, 3 H, J = 8.61 (d, 1 H, J = 8.4 Hz), 8.10 (d, 1H, J = 9.2 Hz), 7.87 7.0 Hz), 1.22 (d, 3 H, J = 7.0 Hz). (d, 1 H, J = 8.4 Hz), 7.58-7.55 (m, 2 H), 7.43 (t, 4 H, J Inhibition of a-Chymotrypsin with Coumarin = 50 Hz, NH4C1), 4.46 (s, 2 H), 3.90 (s, 3 HI. This solid mmol) Inhibitor 6a. Chymotrypsin (12 mg, 4.8 x (2.18 g) was dissolved in 20 mL of HzO and 2 N NaOH was dissolved in 3.24 mL of pH 7.8, 50 mM phosphate was used to adjust the solution to pH 14. The basic buffer with gentle magnetic stirring a t 23 "C and the solution was extracted with CHzClz (4 x , 150 mL total) resulting solution was designated the "enzyme solution". and the extract was dried over KOH. Removal of CH2mmol) in 0.3 Coumarin inhibitor 6a (10 mg, 2.7 x Clz yielded 1.18 g of amine (6.28 mmol, 71% from 3b),
1.20 mmol), and the mixture was refluxed for 2 h. After the reaction mixture was cooled to room temperature, the succinimide formed and unreacted NBS were removed by filtration. Removal of C C 4yielded a brown oil, which was dissolved in 45 mL of CHCl3. TLC [silica gel; ethyl acetatehexane, 1 4 , (v/v)] showed that four compounds were present in the solution with R p of 0.18, 0.27, 0.51, and 0.60. The compound with Rf 0.27 fluoresced very strongly; it was shown to be the starting material (lb). The compound with Rf0.51 appeared initially as a strong dark spot which quickly turned strongly fluorescent, suggesting that it was the desired product (2b). The spot with Rf 0.18 was very weak and never became fluorescent, indicating a possible ring bromination. The spot with Rf 0.60, almost equally weak, quickly turned fluorescent suggesting a possible dibromination on the methyl group. It has been known that a n introduction of heavy atoms such a s bromine into a fluorescent chromophore results in loss of fluorescence (heavy atom effect) (Wehry, 1973). Apparently, the bromination on the ring or a t the a position had the same effect in terms of quenching the fluorescence. The mixture was also analyzed by HPLC b-Bondapak C-18 column under isocratic conditions: 50% A solution (0.1%TFA in acetonitrile) and 50% B solution (50% MeOH in HzO)] with U V detection a t 333 nm; two peaks were observed a t Rf 3.7 and 4.6 min, respectively, with a relative ratio of 3/1. The relative area of the peak a t Rf4.6 min represented a 25% yield of compound 2b. Thus, to the above mixture was added 1.46 g of hexamethylenetetramine (25% x 41.8 mmol) in 30 mL of CHCl3. The resulting mixture was heated at 52 "C for 5 min; a white precipitate formed, which was collected by filtration (1.94 g). Condensation and cooling of the mother liquor yielded another 1.53 g of the white solid. Therefore, a total of 3.47 g of 3b was obtained (21% yield from compound lb), mp 172 "C dec. lH-NMR (DMSO-&): 6 8.41 (d, 1 H, J = 8.4), 8.00
458 Bioconjugate Chem., Vol. 5, No. 5, 1994
mL of acetonitrile was then added to 2.70 mL of the “enzyme solution” (containing 10 mg of chymotrypsin, 4.0 x mmol) over a 45-min period. At the same time a control solution was prepared by mixing 0.27 mL of “enzyme solution” with 0.03 mL of acetonitrile. Aliquots (5 pL) were taken from both the inhibition and control solutions for enzymatic activity assay (Hummel, 1959) (Figure 3). Inhibition of a-Chymotrypsin with Quinoline mmol) was Inhibitor 6b. Chymotrypsin (5 mg, 2 x dissolved in 1 mL of pH 7.8, 50 mM phosphate buffer with gentle magnetic stirring a t 25 “C. After the enzyme had dissolved, three 5-pL aliquots were taken and added to three 1-mL volumes of pH 3 HC1 for the enzymatic activity assay (Hummel, 1959). Quinoline nitrosamide 6b (5 mg, 1.4 x mmol) was dissolved in 0.111 mL of acetonitrile, and half of the solution was added to the enzyme solution over a period of 4 min. The remaining half of the inhibitor solution was added 46 min later (inhibitodenzyme = 70). The decreasing enzymatic activity was followed by assaying aliquots (5 pL) from the inhibition solution (Figure 2). A 10-fold molar excess of DFP (10pL of 0.2 M solution in acetonitrile) was added 70 min after the second batch of 6b had been added. Immediately prior to the addition of DFP, two aliquots were taken out of the enzyme solution, and they were allowed to stand at 25 “C for another 8 h and then assayed. A control run in the absence of 6b was performed a t the same time. The DFP-treated 6binhibited enzyme was gravity filtered through Whatman #1 filter paper to yield a clear, strongly fluorescent solution. The filter paper was washed with -0.5 mL of water, and the washing solution was combined with the enzyme filtrate. The filtrate was transferred to dialysis tubing with a molecular weight cutoff of 3500 and dialyzed against pH 3 HC1 solution for 40 h a t 4 “C with four bath changes (500 mL every 10 h). The dialyzed enzyme solution was still strongly fluorescent. ACKNOWLEDGMENT
We would like to thank the Institute of General Medical Sciences of the U.S.Public Service for financial support (Grant 21450) and the D. Mead Johnson Foundation for a fellowship to M.L. LITERATURE CITED Anderson, G. W., and Callahan, F. M. (1958) Racemization by the dicyclohexylcarbodiimide method of peptide synthesis. J . Am. Chem. SOC.80,2902-2903. Blazevic, N., Kolbah, D., Belin, B., Sunjic, V., and Kajtez, F. (1979) Hexamethylenetetramine, a versatile reagent in organic synthesis. Synthesis 3, 161-176. Doherty, D. G., and Popenoe, E. A., Jr. (1951) The resolution of amino acids by asymmetric enzymatic synthesis. J . Biol. Chem. 189,447-460. Donadio, S., Perks, H. M., Tsuchiya, K., and White, E. H. (1985) Alkylation of amide linkages and cleavage of the C chain in
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