2461
Anal. Chem. 1984, 56,2461-2464
New Fluorogenic Reagent Having Halogenobenzofurazan Structure for Thiols: 4-(Aminosulfonyl)-7-f luoro-2,l ,3=benzoxadiazole Toshimasa Toyo'oka and Kazuhiro Imai* Faculty of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, J a p a n
4-(Amlnosulfonyl)-7-fluoro-2,1,3-ben~oxadiazole(ABPF) has been syntheslzed as a new fluorogenlc reagent for thiols. Its reaction rate to homocystelne Is over 30 llmes fader than that with ammonlum 7-fluoro-2,1,3-benzoxadiazoie-4-suifonate (SBD-F). The fluorogenlc reaction to thiol was completed quantltatlvely In 5 mln at 50 OC and pH 8.0. Alanlne, prollne, and cystine dld not react under the same condltlons. The fluorescence intensity of the fluorophor was pH dependent wlth the hlghest at pH 2. The ABD-thiols obtalned by the prelabeling technlque were separated and detected by reversed-phase HPLC. The detection limits ( S I N = 3) for cysteine, glutathione, N-acetylcystelne, homocysteine, and cysteamine were 0.6, 0.4, 1.9, 0.5, and 0.5 pmol, respectlvely.
High-performance liquid chromatography (HPLC) combined with fluorescence detection has been one of the suitable techniques for the sensitive and specific determination of thiols because of its effective separation and selective detection. A few types of reagents, dansylaziridine, bimane, and maleimide, have been used for the purpose. Dansylaziridine is fluorescent itself and gives a large interfering peak (1). Bimane is not selective for thiol because it also reacts with alcohol, phenol, and/or amino groups (2). The various N-substituted maleimides form multiple fluorescent products owing to the hydrolysis of the initial fluorophors (3). In a previous paper, we described a new reagent, SBD-F (ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate) (41, and its application as a precolumn derivatization reagent for the HPLC determination of glutathione and cysteine in normal human blood ( 5 ) and captopril (an antihypertensive agent) in dog plasma (6). SBD-F has some excellent features as regards to sensitivity, fluorescence characteristics, fluorophor stability, and solubility in water. However, the condition for the completion of reaction is rather drastic: at 60 "C for 1h in an alkaline medium (pH 9.5) with 100 mol excess of the reagent (7). The shortening of reaction time required higher temperature and higher pH. T o overcome the possible degradation of thiols under these conditions, we searched for a more reactive reagent with a benzofurazan structure having a long fluorescence wavelength like the SBD adduct, so that interference by endogenous fluorescent materials in biological samples can be avoided. Considering the electron negativity of the sulfonic acid group, aminosulfonyl was selected as a more electron withdrawing group. This paper reports the synthesis of 4-(aminosulfonyl)-7fluoro-2,1,3-benzoxadiazole(ABD-F) (Figure 1)through the intermediate 4-(chlorosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (CBD-F) which is also a bifunctional reagent. The reactivity among the reagents is compared and the application of ABD-F to the determination of thiols by use of HPLC is described. EXPERIMENTAL SECTION Materials. 4-Fluoro-2,1,3-benzoxadiazole was prepared by the method of Nunno et al. (8). SBD-F and SBD adduct of homo0003-2700/84/0358-2461$01.50/0
cysteine (SBD-homocysteine)were synthesized and purified according to previous reports (4, 7). SBD-Cl(9) was obtained from Pierce (Rockford, IL). Homocysteine, glutathione, N-acetylcysteine, and cysteamineHC1 were purchased from Sigma (St. Louis, MO). Cysteine hydrochloride, alanine, proline, and cystine were obtained from Ajinomoto Co. (Tokyo, Japan). Mercaptoethanol (Tokyo Kasei, Tokyo, Japan), a-mercaptopropionylglycine (Fluka AG, Switzerland), coenzyme A (Boehringer Mannheim, West Germany), and ethylenediaminetetraaceticacid disodium salt (EDTA.2Na) (Kanto Chemical, Tokyo, Japan) were also used. l-(~-3-Mercapto-2-methyl-l-oxopropyl)-~-proline (captopril) (10) was kindly donated by the Sankyo Co. (Tokyo, Japan). All other chemicals used for analysis were of analytical reagent grade and used without further purification. Deionized distilled water was used. Apparatus. lH NMR spectra were recorded on a JEOL Model FX-100 spectrometer at 100 MHz using tetramethylsilane (Me,Si) as an internal standard (abbreviation: s, singlet; d, doublet; m, multiplet). leFNMR spectra were recorded on a JEOL Model FX-9OQ equipped with a fluorine-19 accessory at 84.3 MHz using trifluoroacetic acid (TFA) as a reference (-76.5 ppm) (11, 12). Infrared (IR) spectra were recorded using potassium bromide (KBr) disk with a JASCO Model DS 701G spectrometer. Electron impact mass spectra (EI-MS) were obtained with an LKB-9000 (Shimadzu, Kyoto, Japan) mass spectrometer. Ultraviolet (UV) spectra were measured with a UVIDEC 505 (JASCO, Tokyo, Japan). A Hitachi 650-10s fluorescence spectrophotometer was used with a 1-cm quartz cell for manual methods or an 18-pL flow cell for HPLC detector. Fluorescence maxima measurements were operated without spectral correction. A Waters high-performance liquid chromatograph, equipped with a U6K universal injector and a Model 6000A pump was used. A pBondapak C18(300 X 3.9 mm id., 8-10 pm) connected to a guard column of Bondapak C18-Corasil (20 X 3.9 mm i.d., 37-50 pm) was used at ambient temperature. Reaction temperature was controlled by a water bath JB 1 (Grant Instrument, Cambridge, England). Synthesis of 4-(Chlorosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (CBD-F).The reaction was protected from moisture F
".Q=l>O aH S02CI CBD-F
with a calcium chloride tube. Eleven milliliters of chlorosulfonic acid was dropped into a solution of 4-fluoro-2,1,3-benzoxadiazole (3 g, 21.7 mmol) in CHC13 (10 mL) at 0-10 "C. After the addition, the mixture was stirred for 1h at room temperature and then refluxed for 2 h. The solution was cooled and slowly poured into ice water (200 8). The CHC13layer was separated and the aqueous layer was extracted with CHC13. The combined CHC13 extract was washed, dried on anhydrous MgS04,and evaporated in vacuo. The residue was dissolved in 5 mL of benzene and chromatographed on a silica gel column (Kanto Chemical, 100-200 mesh, 15 X 3 cm, eluent: n-hexane-benzene (1:l)). The fractions corresponding to CBD-F were collected and evaporated in vacuo (pale yellow needles, yield 78%): mp 64-66 "C (uncorrected);lH NMR (in Me2SO-ds)6 7.80 (1H, dd, Jab= 7.4 Hz,JoF= 4.9 Hz, a), 7.44 (1 H, dd, Jab = 7.4 Hz,J ~ =F 10.6 Hz, b); 19FNMR (in Me2SO-d6)-116.8 ppm (12); EI-MS m / e 236 (M'). Anal. Calcd for C6H2N2O3ClFS: C, 30.46; H, 0.85; N, 11.84. Found: C, 30.48; H, 0.61; N, 11.70. 0 1984 American Chemical Society
2462
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984
Table I. Comparison of the Reaction Rate Constants of Homocysteine with Various Reagents (ABD-F, SBD-F, or SBD-CI)' ABD-F PH 7.0
40 OC
50 O C
SBD-F 60 O C
60 O
C
SBD-C1 60 O
C
2.54 x
7.40 X 1.16 X 10-1 3.55 x 10-3 NDb NDb NDb 2.31 X NDb 9.5 NDb ND~ NDb 8.01 X 2.39 x 10-3 "Homocysteine (5 pM) and the reagent (500 pM) were reacted in 0.1 M buffer (phosphate or borate) containing 1 mM EDTA.2Na. Fluorescence measurement: Ex, 390 nm; Em, 515 nm. Buffers: phosphate (sodium dihydrogen phosphate and disodium hydrogen phosphate), borate (borax and HCl or NaOH). bND,not determined. 8.0
ND~
F
SR
S02NI-Q
S02NI-12
ABD-F
ABD-SR
Flgure 1. Structure of ABD-F and its reactlon with thiols,
Synthesis of 4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). To 1g of CBD-F was added dropwise F
hH@;:O aH
S02NH2c ABD-F
100 mL of 6% NH40H. Then, the alkaline solution was neutralized with 10% HCI and evaporated under reduced pressure. Two hundred milliliters of acetonitrile was added to the residue and the suspension was filtered. The filtrate was evaporated and chromatographed on a silica gel column (100-200 mesh, 30 X 2 cm, eluent, CHC13). The fractions corresponding to ABD-F were collected, evaporated, and recrystallized from n-hexane-benzene (white needles, yield 20%): mp 145-146 O C (uncorrected); lH NMR (in Me2SO-ds)6 8.06 (1H, dd, J a b = 7.6 Hz, Ja = 4.4 Hz, a), 7.58 (1H, dd, Jab= 7.6 Hz,JbF= 10.0 Hz, b), 7.94 (2 H, s, c); '?F NMR (in MezSO-ds)-111.5 ppm (12). IR (v-*~, cm-') 3330, 313 nm; 3230 (SO,",); ELMS m / e 217 (M+);UV (X,(CH&N) t313m 4200. Anal. Calcd for C6H4N303FS:C, 33.18; H, 1.86; N, 19.35. Found: C, 33.11; H, 1.82; N, 19.27. Synthesis of ABD adduct of Homocysteine (ABDHomocysteine). To 0.22 g (1 mmol) of ABD-F in 30 mL of
ABD-homocysteine
CH3CN was added an equal volume of homocysteine (0.14 g, 1 mmol) in 0.1 M borate (pH 9.5, Na+)containing 1mM EDTA92Na After 30 min of heating at 60 "C, the reaction mixture was cooled on ice water and extracted with 300 mL of benzene to remove the excess reagent. The aqueous layer was evaporated under reduced pressure. The residue was chromatographed on a Bio-Gel P-2 column (200-400 mesh, 60 X 2 cm, eluent: HzO). The fluorescent fractions corresponding to ABD-homocysteine were collected, evaporated, and recrystallized from HzO (yellowneedles, decomposed at above 250 "C, yield 20%). lH NMR (in Me2SO-d,) 6 7.92 (1 H, d, J a b = 7.8 Hz, a), 7.56 (1 H, d, Jab= 7.8 Hz, b), 7.60-8.00 (2 H, c), 3.20-3.49 (5 H, m, d + f + g), 2.10-2.49 (2 H, m, e). Anal. Calcd for CloH11N,0SS2Na: C, 33.90; H, 3.13; N, 15.81. Found: C, 33.81; H, 3.41; N, 15.87. UV: X,,(H20) 384 nm; cum 7100. Fluorescence in HzO: XEx 389 nm, XEm 513 nm. Determination of Reaction Rate Constant. One milliliter of 1 mM reagent (ABD-F, SBD-F, or SBD-C1) in 0.1 M buffer (phosphate or borate) and 1.0 mL of 10 pM homocysteine in 0.1 M buffer (phosphate or borate) containing 2 mM EDTA.2Na were mixed in a 5-mL glass tube. The tube was capped and heated to the required temperature (40-60 OC) in a water bath. At fixed reaction time intervals, the tube was taken out and cooled on ice water. The fluorescence intensities were measured at ambient
temperature with emission at 515 nm (excitation at 390 nm). The reagent blank without thiol was treated in the same manner. Pseudo-first-order rate constant was calculated on the difference of the fluorescence intensity (sample fluorescence minus blank fluorescence) from that of authentic derivatives (ABD-homocysteine or SBD-homocysteine), Fluorescence Characteristics of ABD-Homocysteine in Various pHs and Solvents. Relative fluorescence intensities (RFI) of 2.3 pM of authentic ABD-homocysteine dissolved in the buffers of various pHs (0.05 M Britton-Robinson buffer for pH 2-10,O.l M HC1 for pH 1)were measured at 515 nm with excitation at 390 nm. Authentic ABD-homocysteine(4.5 pM) was dissolved in various solvents (HzO, MeOH, EtOH, CH3CN, acetone, DMF, and Me2SO) and ita maximal wavelengths and fluorescence intensities were measured. Relative Fluorescence Intensities for Thiols Using ABD-F. To 1.0 mL of 1 mM ABD-F in 0.1 M borate (pH 8.0, Na+) was added an equal volume of 10 pM thiol in 0.1 M borate (pH 8.0, Na+) containing 2 mM EDTA.2Na. The solution was immediately mixed and allowed to react at 50 OC for 5 min. After cooling on ice water, the reaction mixture was adjusted to pH 2 with 0.6 mL of 0.1 M HCl and the fluorescence intensity was measured at 510 nm with excitation at 380 nm. The reagent blank without thiol was treated in the same manner. HPLC Separation and Detection Limits of ABD-Thiols. To a 5-mL glass tube were added 1.0 mL of ABD-F (1mM) in 0.1 M borate (pH 8.0, Na+) and 1.0 mL of mixed thiols (cysteine, 2.16 pM; glutathione, 2.08 p M N-acetylcysteine, 2.57 pM; homocysteine, 1.92 p M cysteamine, 2.46 pM) in 0.1 M borate (pH 8.0, Na+) containing 2 mM EDTA-2Na. The reaction tube was vortex mixed, capped, and heated at 50 OC for 5 min. After the reaction tube was cooled on ice-water,0.6 mL of 0.1 M HCI was aliquot of the acidic added to the tube (final pH 2) and a 1 0 - ~ L solution was injected into the column for HPLC. The eluting solvent was acetonitrile-0.05 M potassium biphthalate (pH 4.0) (892). The flow rate was 1.0 ml/min. The eluate was monitored at 510 nm with excitation at 380 nm.
RESULTS Reaction Rates of Homocysteine with ABD-F, SBD-F, o r SBD-CI. The pseudo-first-order rate constants were measured by using 0.5 mM reagent (ABD-F, SBD-F, or SBD-C1) and 5 pM homocysteine under various conditions. As shown in Table I, the reaction rate with ABD-F was over 30 times faster than that with SBD-F, and was 3 orders of magnitude faster than that with SBD-C1. In the case of SBD-C1 at pH 7.0 or 8.0, the reaction rate was not calculated because of the low yield of fluorescence. When the organic solvent such as acetonitrile was added to the reaction medium, the rate was not increased remarkably. As depicted in Figure 2, the reaction of homocysteine with ABD-F was completed quantitatively in 5 rnin a t 40 or 50 OC and pH 8.0 without decrement of fluorescence intensities over the 60 min tested. The gradual hydrolysis of ABD-F at higher pH and longer reaction time necessitates the use of as short a reaction time as possible. Effects of pH on Fluorescence of ABD-Homocysteine. To obtain an optimum pH for the determination of thiols, the correlation of the pH with the fluorescence intensity was
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984
2483
Table 111. Relative Fluorescence Intensities a n d Maximal Wavelengths for Various Thiols Using ABD-Fa thiol
Ex
nm Em
Flgure 2. Time courses of reactlon yield obtained from homocysteine
cysteamine mercaptoethanol a-mercaptoprophmylglycine coenzyme A glutathione homocysteine N-acetylcysteine cysteine
377 389 377 389 381 382 375 374
504 519 505 513 512 511 508 500
with ABD-F: Homocysteine (5 pM) and ABD-F (500 pM) were reacted in 0.1 M borate (pH 8.0, Na') Containing 1 mM EDTAn2Na. The yields were calculated based on the fluorescence intensities (Ex 380 nm; Em 510 nm) of authentic ABD-homocystelne. Temperatures were (0)40 50 O C . OC and (0)
captopril cystine alanine proline
381
511
,,A,
RFIb 127 122
119 118
108 100 65 62 65c 58 NDd NDd NDd
"Thiol (5 wM) and ABD-F (500 wM)were reacted in 0.1 M borate (pH 8.0, Na+) containing 1 mM EDTA-2Na at 50 O C for 5 min. After the reaction, 0.6 mL of 0.1 M HC1 was added and measured (final pH 2). *EX,380 nm; Em, 510 nm. cEx, 375 nm; Em, 500 nm. ND, not detected. Fluorescence intensity in homocysteine was arbitrarily taken as 100.
RFI
'n
Figure 3. Effect of pH on the fluorescence intensities of authentic ABD-homocysteine: concentration of ABD-homocysteine, 2.3 pM each; fluorescence measurement, Ex 390 nm, Em 515 nm; buffers, 0.05 M Brltton-Robinson (pH 2-10), 0.1 M HCI (pH 1).
Table 11. Comparison of Maximal Wavelengths a n d Relative Fluorescence Intensities of ABD-Homocysteine in Various Solvents" A,.
nm
solvent
Ex
Em
EtOH acetone MeOH CH&N H2O DMF Me2S0
386 389 385 389 389 393 397
492 491 495 493 513 493 502
RFI 330 200
190 140 100 92 25
a ABD-homocysteine (4.5 gM) was dissolved in various solvents. Fluorescence intensity in H20 was arbitrarily taken as 100.
studied. As can be seen in Figure 3, relatively high fluorescence intensities were observed from pH 2 to pH 7. The highest fluorescence intensities were obtained at about pH 2. On the other hand, the fluorescence intensity was increased in organic solvent, especially in EtOH, except for Me2S0. The spectral shift expected from the previous report (7),however, was not observed among the different polarity of solvents (Table 11). Relative Fluorescence Intensities and Maximal Wavelengths for Various Thiols Using ABD-F by the Manual Method. The fluorescence intensities of the reaction medium of the biologically important thiols or amino acids with 100 mol excess of ABD-F were measured. As shown in Table 111, the fluorescence intensity of cysteamine was the largest while that of captopril (an antihypertensive agent) was the smallest. The difference in the fluorescence intensities for all the thiols was less than a factor of 3. In contrast, amino acids (alanine or proline) and disulfide (cystine) did not react with ABD-F. The excitation and emission maxima of the thiols were in the range of 374-389 nm and 500-519 nm, respectively (Table 111).
I
60
40
Tirne(min)
20
0
Flgure 4. Chromatogram of biological thlols derivatized with ABD-F: (a) ABD-cysteine 8.3 pmol, (b) ABDglutathione 8.0 pmol, (c) ABD-Nacetylcystelne 9.9 pmol, (d) ABD-homocysteine 7.4 pmol, (e) ABDCysteamine 9.5 pmol, (f) ABD-F about 5 nmol, (UK) unknown; column, pBondapak C,! (300 X 3.9 mm l.d., 8-10 pm); eluent, CH,CN-O.O5 M potassium biphthalate (pH 4.0) (8:92); flow rate, 1.0 mL/mln; detection, Ex 380 nm, Em 510 nm.
HPLC Separation and Detection Limits for ABDThiols. The separation of ABD-thiols was studied by using a reversed-phase HPLC column with a binary eluent system. The retention times of ABD derivatives were, as expected, decreased with increased acetonitrile concentration and at higher pH. Figure 4 shows the complete separation of ABDcysteine, ABD-glutathione, ABD-N-acetylcysteine, ABDhomocysteine, and ABD-cysteamine in this elution order within 35 min by a simple binary mixture (CH3CN-0.05 M potassium biphthalate (pH 4.0) (8:92)). Peak f was confirmed as the excess ABD-F by injection of an ABD-F solution without thiols (Figure 4). The small unknown peak eluted a t about 24 min, slightly increased with the longer reaction time, was the same as that found in the blank solution without thiol. This peak is therefore attributed to the hydrolysis product of ABD-F. The ABD-thiols seem to be stable for more than 1week under refrigeration judging from the peak heights obtained by the repeated analysis of ABD-homocysteine by
HPLC. Under the selected condition, the detection limits (signal to noise ratio of 3) for ABD-cysteine, ABD-glutathione, ABD-N-acetylcysteine, ABD-homocysteine, and ABD-cysteamine were 0.6, 0.4, 1.9, 0.5, and 0.5 pmol, respectively. DISCUSSION Thiols are easily oxidized by dissolved oxygen in alkaline medium (13); mild conditions (neutral pH, low temperature, short reaction time) are therefore desirable for the derivatization. Although NBD-X (X = C1 or F, 4-halogeno-7-nito-
2464
ANALYTICAL CHEMISTRY, VOL. 56,
NO. 13, NOVEMBER 1984
ro-2,1,3-benzoxadiazole),which was developed as the fluoro-
-
genic reagent for amine (14-27), also reacts with thiol a t low pH (pH 4-7), the S N migration of the NBD group (18) renders it unsuitable.-As.doned above, the quantitative reaction of thiol with SBD-F required a rather drastic condition (at 60 OC and pH 9.5 for 1h) (7). A new reagent having the halogenobenzofurazan (2,1,3-benzoxadiazole) structure similar to SBD-F which affords a fluorescent adduct of long wavelength (SBD-homocysteine: XE= 385 nm; X E 515 ~ nm) is needed. To increase the reactivity, the presence of other functional groups at the 7 position of 4-fluorobenzofurazan seems to be important. According to the Hammett equation, the substituent constants (u) of para-substituted NO2and SOY groups were 0.78 and 0.38, respectively (19,20). Acetyl (0.31), cyano (0.66), carboxymethyl (0.501, and aminosulfonyl (0.62) groups were intermediates between the NOz and SO; groups (19). The aminosulfonyl group was selected since it is easily obtained from 4-fluorobenzofurazan (a precursor for SBD-F) through an intermediate, 4-(chlorosulfonyl)-7-fluorobenzofurazan which might be a useful bifunctional reagent for the conformational studies of enzymes. The ABD-F thus obtained reacted faster with thiols than SBD-F and the reaction was quantitatively completed in 5 min (more than 90%) at 50 “C and pH 8.0. The selectivity of ABD-F to thiol is superior to other fluorogenic reagents such as I-AEDANS (24, dansylaziridine (22),and bimane (2),since it does not react with amino and hydroxy groups under the above conditions. The highest fluorescence intensity of ABD-thiol (-homocysteine) is at about pH 2. The acidic solution is also suitable for the determination of thiols by a manual method. When the ABD-thiols are separated by reversed-phase HPLC, the use of acidic eluent at low pH is also favorable because of the higher peak heights and shorter retention times obtained. Under the proposed HPLC condition, picomole levels of thiols are detectable and the detection limits are comparable to those reported for SBD-F, N-[7-(dimethylamino)-4-methyl-3coumarinyl]maleimide (DACM), and bimane (7,23,24).The application of the reagent to biological samples is under investigation and the details will appear in elsewhere. In conclusion, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) is a useful reagent for the sensitive and specific detection of thiols in both the manual and HPLC methods because of its excellent reactivity, selectivity to thiol, low fluorescence background, and good stability of fluorophor. Its solubility in water may be also advantageous for the investigation of the reactive site of SH enzymes. ACKNOWLEDGMENT The authors thank T. Nakajima, University of Tokyo, for his valuable suggestions and discussion. Thanks are also due
to Y. Kawahara of Sankyo Co. for the donation of captopril, Y. Watanabe of Chugai Pharmaceutical for mass spectra measurements, N. Fujii of JEOL for NMR measurements, and C. K. Lim of Clinical Research Center for his kind elaboration of the manuscript. Registry No. CBD-F, 91366-64-2;ABD-F, 91366-65-3;SBD-C1, 81377-14-2;SBD-F, 84806-27-9;ABD-homocysteine,91366-66-4; ABD-mercaptoethanol,91366-67-5; ABD-a-mercaptopropionylglycine, 91366-68-6; ABD-coenzyme A, 91366-69-7; ABD-glutathione, 91366-70-0; ABD-N-acetylcysteine, 91366-71-1; ABDcysteine, 91366-72-2;ABD-captopril, 91366-73-3; 4-fluoro-2,1,3benzoxadiazole, 29270-55-1; chlorosulfonic acid, 7790-94-5; cysteine, 52-90-4;glutathione, 70-18-8; N-acetylcysteine, 616-91-1; homocysteine, 6027-13-0; cysteamine, 60-23-1;ABD-cysteamine, 91409-19-7.
LITERATURE CITED Lankmayr, E. P.; Budna, K. W.; Muller, K.; Nachtmann, F. Z. Anal. Chem. 1079, 295, 371-374. Kosower, N. S.; Newton, G. L.; Kosower, E. M.; Ranney, H. M. Blochlm. Biophys. Acta 1980, 622, 201-209. Kanaoka, Y. Yakugaku Zasshi 1980. 100, 973-993. Imai, K.; Toyo’oka, T.; Watanabe, Y. Anal. Blochem. 1983, 128, 471-473. Toyo’oka, T.; Imai, K. J. Chromatogr. 1983, 282, 495-500. Toyo‘oka, T.; Imai, K.; Kawahara, Y. J. Pharm. Blomed. Anal., In press. Toyo’oka, T.; Irnai, K. Analyst (London), In press. Nunno, L. D.; Florio, S.: Todesco, P. E. J. Chem. SOC. C 1970, 1433-1434. Andrews, J. L.; Ghosh, P.; Ternai, 6.; Whitehouse, M. W. Arch. 610chem. Biophys. 1982, 214, 386-396. Cushman, D. W.; Cheung, H. S.; Sabo, E. F.; Ondetti, M. A. Biochemistfy 1977, 76, 5484-5491. Emsley, J. W.; Feeney, J.; Sutcllffe, L. H. “Progress In Nuclear Magnetic Resonance Spectroscopy”; Pergamon Press: Oxford, 1971; Vol 7. Dungan, C.H.; Van Wazer, J. R. “Compilation of Reported F19 NMR Chemical Shifts”; Wiley-Interscience: New York, 1970. Jocelyn, P. C. “Biochemistry of the SH Group”; Academic Press: New York, 1972. Ghosh, P. B.; Whltehouse, M. W. Biochem. J. 1968, 108, 155-156. Fager, R. S.; Kutlna. C. B.; Abrahamson, E. M. Anal. Blochem. 1973, 53, 290-294. Imai, K.; WBtanabe, Y. Anal. Chim. Acta 1981, 130, 377-383. Watanabe, Y.; Irnai, K. J. Chromatogr. 1982, 239, 723-732. Blrkett. D. J.; Price, N. C.; Radda, G. K.; Salmon, A. G. FEBS Lett. 1970, 6,348-348. Hammett, L. P. “Physical Organic Chemistry”, 2nd ed.; McGraw-Hill: New York, 1970. RRchie, C. D.; Sager, W. F. “Progress in Physical Organic Chemistry”; Wlley: New York, 1964; Vol. 2. Hudson, E. N.; Weber, G. Biochemistry 1973, 12, 4154-4161. Scouten, W. H.; Lubcher, R.; Baughman, W. Blochim. 6iophys. Acfa 1974, 336, 421-426. Kagedal, B.; KBllberg. M. J. Chromatogr. 1982, 229, 409-415. Fahey, R. C.; Newton, 0. L.; Dorian, R.; Kosower, E. M. Anal. Blochem. 1981, 1 1 1 , 357-365.
RECEIVED for review April 10,1984. Accepted May 24,1984. Presented in part at the 104th Annual Meeting of the Pharmaceutical Society of Japan, Sendai, March 28-30,1984,
