Bioconjugate Chem. 1993, 4, 545-548
545
Chlorinated Phenyl Azides as Photolabeling Reagents. Synthesis of an ortho,ortho'-Dichlorinated Arylazido PCP Receptor Ligand Sui Xiong Cai,t*l Denis J. Glenn,t Kyle R. Gee,tJ Mingdi Yan,? Ronald E. Cotter,* N. Laxma Reddy,* Eckard Weber,%and John F. W. Keana'J Department of Chemistry, University of Oregon, Eugene, Oregon 97403, Cambridge Neuroscience, Inc., Cambridge, Massachusetts 02139, and Department of Pharmacology, University of California, Irvine, California 92717. Received July 14, 1993"
The enhanced photolabeling properties of chlorinated phenyl azides are demonstrated by the synthesis and photolysis of methyl 4-azido-2,3,5,6-tetrachlorobenmate (3)and methyl 4-azido-3,5-dichlorobenzoate (4). Photolysis of azide 3 in 1M diethylamine/cyclohexaneas the trapping medium gave 34% NHinsertion product. Similar photolysis of azide 4 gave 35% NH insertion product. These results demonstrate that chlorinated phenyl azides are significantly better at undergoing NH insertion than nonhalogenated analogs and suggest that improvement of existing aryl azide-based photolabels might be achieved by introduction of chlorine atoms on either side of the azide group. As an application, 3-azido-2,4-dichloro-10,5-(iminomethano)-lO,ll-dihydro-5H-dibenzo[a,dl cycloheptene (19),an analog of the potent PCP receptor ligand IDDC (141,was synthesized and its affinity for the PCP receptor was determined to be 6.3 f 0.7 pM (ICs0 against [3HlMK801).
INTRODUCTION Photoaffinity labeling (PAL) is used to examine the spatial relationship between components in biological systems (1-3). Typically, the photolabel is a ligand modified with a photoreactive group which can form a stable covalent bond between the label and biological macromolecule of interest. The success of experiments using PAL reagents depends in part on the CH or NH bond insertion efficiencyof the label upon photolysis. Aryl azides are often employed as the photoactive group in PAL reagents (4). These reagents form a highly reactive nitrene intermediate upon photolysis which can then undergo NH or CH insertion at the site of labeling with formation of a covalent bond. However, low levels of labeling are sometimes observed with aryl azides. These low labeling levels are attributed to ring expansion of the nitrene intermediate to the less reactive dehydroazepine intermediate. This intermediate is incapable of undergoing a bond insertion reaction (5,6) and instead reacts with solvent or nearby nucleophiles. We (7-10) and others (11-15) have been developing a series of functionalized perfluorophenyl azides (PFPAs) as a new class of PAL reagents. Fluorophenyl azide (FPA) based photolabeling reagents have shown significant improvement in both CH and NH insertion efficiency in organic solvents over nonfluorinated analogs. However, introduction of the FPA functionality into existing aryl azide-based photolabels or their amine precursors is complicated by the difficulty of fluorinating the aryl ring (16). We were therefore prompted to investigate the behavior of representative chlorophenyl azides (CPAs) for potential use as photoaffinity labels (17). CPAs are more readily available than the FPAs owing to the variety of methods available for aromatic chlori+ University of Oregon.
Present address: Acea Pharmaceuticals, Inc., Irvine, CA. Molecular Probes, Inc., Eugene, OR. Cambridge Neuroscience, Inc. 8 University of California, Irvine. 0 Abstract published in Advance ACS Abstracts, October 1, 1
11 Present address:
1993. 1043-1802/93/2904-0545~04.00l0
nation (18). Photolysis of m-or p-chlorophenyl azides in dimethylamine has been shown to give ring-expansion products (19, 20). Herein we report the synthesis and photochemical properties of the tetrasubstituted functionalized CPA methyl 4-azido-2,3,5,6-tetrachlorobenzoate (3),and the ortho,ortho'-disubstituted CPA methyl 4-mido-3,5-dichlorobenzoate (4). As an application of this technique, 3-azido-2,4-dichloro-10,5-(iminomethano)-10,1l-dihydro-5H-dibenzo[a,dlcycloheptene (191,aligand for the PCP receptor site (21) of the NMDA receptor in the mammalian central nervous system, was synthesized. EXPERIMENTAL PROCEDURES lH NMR spectra were measured at 300 MHz in CDC13 unless otherwise noted. IR spectra were recorded in CDCh. Mass spectra were obtained in the electron ionization mode, and relative intensities are reported in parentheses after the mlz value. Solvents were reagent grade unless otherwise specified. MgSO, was used to dry organic solutions. All reactions involving azides were run under subdued light by wrapping flasks with aluminum foil. Analytical thin-layer chromatography was performed on Merck silica gel 60 F2u with a fluorescent indicator. Flash chromatography was performed using 200-425 mesh (Aldrich) silica gel. Preparative TLC was performed on Analtech GF precoated silica gel glass-backed plates (20 X 20 cm). Photolyses were carried out at ambient temperature in a Rayonet photochemical reactor with 350nm lamps and the progress of photolysis was followed by IRat 2123 cm-l (azide absorption). Solutions were purged with argon for 1 min before photolysis. Methyl 4-Azido-2,3,5,6-tetrachlorobenzoate (3). To a stirred solution of 312 mg (1.08 mmol) of aniline 1 (22) in CF3COzH (20 mL ) at 0 "C was added 173 mg (2.51 mmol) of NaNO2 at once. The solution was stirred at 0 "C for 1 h and then 187 mg (2.88 mmol) of NaN3 was added. The solution was allowed to come to room temperature and stirred for 1 h. It was then added dropwise to ice water, forming a yellow precipitate. The suspension was filtered and washed with ice water to leave 97 mg (29%) of 3 as a yellow powder, which was purified by crystallization from CHCldpentanes and then acetone/ 0 1993 American Chemical Society
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Cai et al.
H20 to give a fine yellow powder: mp 137-139 OC; IR 203-206 OC; lH NMR (major isomer) 2.05 (s, 3H), 2.92 2127, 1744 cm-l; lH NMR 4.00 (9); MS mlz 315 (6, M+), (dd, J = 17.2,2.3 Hz, lH), 3.50 (dd, J = 17.2,4.5 Hz, lH), 3.58 (br s, 2H), 3.74 (dd, J = 10.4, 3.8 Hz, lH), 3.86 (m, 301 ( 5 ) ,287 (58),282 (18), 272 (43), 256 (ll),244 (40), 228 (100),194 (271,156 (691,121 (66),86 (50),58 (85);HRMS lH), 4.03 (dd, J = 10.3, 1.3 Hz, 1H) 5.86 (dd, J = 4.3, 2.6 312.8971 (312.8979 calcd for C8H335C14N302). Hz, lH), 6.52 (qd, J = 8,2 Hz, 3H), 7.2 (m, 4H); 'H NMR (minor isomer) 2.29 (s, 3H), 3.06 (dd,J = 16.9,2.3 Hz, lH), Methyl 4-Azido-3,5-dichlorobenzoate (4). Ester 4 3.42 (dd, J = 16.9, 4.4 Hz, lH), 3.58 (br s, 2H), 3.61 (dd, was prepared from aniline 2 (23) in a similar manner to J = 12.4, 4.1 Hz, lH), 3.86 (m, lH), 4.18 (d, J = 13.0 Hz, ester 3 and was isolated as an off-white powder (68%). lH), 6.80 (t,J = 7.2 Hz, 3H), 7.2 (m, 4H); 13CNMR (major The sample was purified by sublimation (0.003 Torr, 40 isomer) 22.4, 38.6, 47.6, 52.6, 52.9, 115.11, 115.17, 124.5, "C) to give a white microcrystalline powder: mp 70-72 125.2,126.1,127.8,128.1,132.8,137.8,141.0,141.4,145.0, OC; IR 2130, 2112, 1728 cm-'; 'H NMR 3.92 (s, 3H), 7.95 170.2; l3C NMR (minor isomer) 22.0,39.9,46.8,51.2,57.5, ( 8 , 2H); MS mlz 245 (M+, 18), 217 (loo), 202 (550), 174 114.9,115.1,123.1,125.4,125.7,127.5,128.5,132.5,137.1, (67), 158 (73);HRMS 244.9769 (244.9759calcdfor C&&,35141.4, 141.7, 145.3, 169.8; MS mlz 278 (M+, 9), 207 (25), ClzN30z). 206 (1001, 130 (17); HRMS 278.1407 (278.1419 calcd for Photolysis of Azide 3 in Diethylamine/CyclohexC18Hl&o). Anal. Calcd for Cl8Hl8N20.0.5Hz0: C, ane. A solution of 51 mg of 3 in 1 M diethylamine/ 75.23; H, 6.66; N, 9.75. Found: C, 75.47; H, 6.35; N, 9.90. cyclohexane (51 mL) was photolyzed for 2 h. The crude 12-Acetyl-3-amino-2,4-dichlort~ lO,&(iminomethano)photolysis mixture was concentrated to dryness in vacuo, l0,11-dihydro-5H-dibenzo[a,dJcycloheptene(17). To leaving an oily residue which was separated by preparative a solution of 16 (64 mg, 0.23 mmol) in DMF (1.0 mL, dried TLC (2:l CHC&/hexanes)to give aniline 1 (23)as a white over molecular sieves) at room temperature was added powder (35%,Rf = 0.53) and hydrazine 7 as a yellow oil NCS (64 mg, 0.48 mmol) in one portion. The resulting (34%,Rf = 0.66). For hydrazine 7: 'H NMR 1.06 (t, J = pale brown solution was stirred overnight and diluted with 7.2 Hz,6H),2.81 ( q , J = 7.2 Hz,4H),3.96 (s, 3H),5.25 (s, water (5 mL). The resulting mixture was extracted with 1H); MS mlz 360 (M+, 1001, 345 (431, 329 (291, 309 (30), 288 (30); HRMS 357.9809 (357.9809 calcd for C12H14~~- dichloromethane (3 X 4 mL). The extract was washed with water (3 X 5 mL) and brine (1 X 7 mL) and dried C14Nz02). (KzCO3). Evaporation of the solvent left 76 mg of a brown Photolysis of Azide 4 in Diethylamine/Cyclohexsyrup, which was purified by preparative TLC (silica gel, ane. A solution of 50 mg of 4 in 1 M diethylamine/ 1000pm, CHCldMeOH/NH40H 50:l:O.Ol) to give the title cyclohexane (50 mL) was photolyzed for 2 h. The crude compound as a colorless oil as a 4:l mixture of N-Ac photolysis mixture after evaporation of the solvent was rotational isomers (44 mg, 55% yield): IR (CDCl3) 1635, separated by preparative TLC (1:4 ethyl acetate/hexanes) 1618,1412 cm-l; 'H NMR (major isomer) 2.06 (s,3H),2.94 to give aniline 2 (23)as a white powder (45 % , Rf = 0.60) (dd, J = 17.3,2.3 Hz, lH), 3.50 (dd, J = 17.3,4.3 Hz, lH), and hydrazine 8 as an oily yellow solid (35%,Rf = 0.77). 3.77 (dd, J = 10.8,4.2 Hz, lH), 3.96 (dd, J = 10.9,1.2 Hz, For hydrazine 8: lH NMR 1.06 (t, J = 6.9 Hz, 6H), 2.79 lH), 4.42 (br s, 2H), 4.99 (m, lH), 5.84 (dd, J = 4.1, 2.8 ( q , J = 6.9 Hz,4H),3.87 (s,3H),5.26 (s,lH),7.87 (s,2H); Hz, lH), 6.87 (e, lH), 7.25 (m, 4H);'H NMR (minor isomer) MS mlz 291 (M+, loo), 275 (45), 261 (23), 239 (60), 218 (95);HRMS 290.0561 (290.0585 calcd for C~~H1635C1~N20~).2.28 (s, 3H), 3.08 (dd, J = 17.1, 2.5 Hz, lH), 3.42 (dd, J = 17.5, 4.2 Hz, lH), 3.66 (dd, J = 13.1,4.7 Hz, lH), 4.10 Photolysis of Azide 10 in Diethylamine/Cyclohex(d, J = 10.2 Hz, lH), 4.38 (br s, 2H), 5.07 (t,J = 3.4 Hz, ane. A solution of 45 mg of 10 in 1 M diethylamine/ lH), 5.95 (t, J = 3.6 Hz,lH), 6.85 (e, lH), 7.25 (m, 4H); cyclohexane (45 mL) was photolyzed for 8 h. The crude 13CNMR (major isomer) 22.2,39.3,40.8,50.0,51.8,118.2, mixture after evaporation of the solvent was separated by 119.0,125.3,125.6,126.1,128.2,128.3,130.4,136.6,137.9, preparative TLC with ethyl acetate/chloroform/hexanes 138.7, 139.8, 170.1; 13C NMR (minor isomer) 21.9, 30.0, (1:l:l) as the developing solvent to give 11 as a yellow oil 40.0,40.5,56.9,118.3,118.6,124.4,125.9,126.2,128.0,128.7, (21 % ,Rf = 0.75) with a NMR spectrum identical to that 130.0, 137.3, 137.5, 138.9, 140.2, 169.4; mlz 346 (M+, 7), reported for 11 (241, and 12 as a yellow oil (16%, Rf = 311 (91, 303 (111, 282 (111, 274 (681, 130 (57), 43 (100); 0.58). The prep TLC plates were pre-eluted with the HRMS 346.0647 (346.0640 calcd for ClsH16Nz035C1z). developing solvent containing 2 % v/v of diisopropylamine 3-Amino-2,4-dichloro-10,5-(iminomethan0)-10,l l-difollowed by the developing solvent. For 12: lH NMR hydro-5H-dibenzo[a,dJcycloheptene(18). A mixture (&-acetone) 1.00 (t, J = 7.2 Hz, 6H), 3.03 (9,J = 7.2 Hz, 4H), 3.70 (s, 3H), 5.72 (d, J = 3.6 Hz, 1 H), 7.09 (d, J = of 17 (34.5 mg, 0.099 mmol), ethylene glycol (1.25 mL, 3.6 Hz, 1H), 7.16 (d, J = 7.2 Hz, 1H), 7.21 (d, J = 7.2 Hz, distilled from CaHZ), and hydrazine hydrate (1.25 mL, 1H), 7.43 (br, 1H); MS mlz 223 (20), 222 (M+, 1001, 207 85% solution) was heated a t reflux with stirring for 1.5 h. (17),193(28), 161(61),105(24);HRMS 222.1379 (222.1368 The resulting pale yellow solution was cooled, and KOH calcd for C12H18N202). (150 mg, 2.3 mmol) was added. Reflux was resumed for 18 h. After cooling, the reaction mixture was treated with 12-Acetyl-3-amino-10,5-(iminomethano)-lO,l l-dihy5 % NaOH (5mL), and the resulting mixture was extracted dro-5H-dibenzo[a,d]cycloheptene (16). The method with dichloromethane (3 X 8 mL). The extract was washed of Bellamy and Ou (25)was adapted. A mixture of 15 (26) with water (2 X 10 mL) and brine and dried (K2C03). (150 mg, 0.486 mmol), ethanol (2 mL), ethyl acetate (3 Filtration and concentration afforded the title compound mL), and stannous chloride dihydrate (549mg, 2.43 mmol) as a pale yellow-brown oil (21.7 mg, 72%). An analytical was refluxed for 3 h, and poured into 20 mL of ice water. sample was obtained by flash chromatography over silica Saturated NaHC03 (10 mL) was carefully added, and the gel using CHCl3/MeOH (151)to give a colorless oil: lH resulting mixture was extracted with ethyl acetate (2 X 15 NMR61.90(brs,lH),3.10(dd, J = 17.3,4.1Hz,lH),3.34 mL). The extract was dried and concentrated. The (dd, J = 21.1,5.8 Hz, lH), 3.41 (dd, J = 17.4,3.3 Hz, lH), residue was dissolved in CHCL and passed through a short 3.55 (d, J = 12.2 Hz, lH), 4.24 (t, J = 3.7 Hz, lH), 4.36 (br plug of silica gel and the eluant concentrated in vacuo to s,2H),4.82 ( d , J = 5.5 Hz,lH),6.87 (s,lH),7.15-7.29 (m, give 136 mg (100%) of the title compound as a solid pale 4H); 13C NMR 40.0, 41.2, 49.7, 54.9, 125.0, 126.4, 127.5, yellow foam. The NMR spectrum consisted of a 4:l 127.7, 127.9, 128.1,131.3,132.2,136.1,139.8,141.0,141.7; mixture of rotational isomers about the N-Ac group: mp
Bioconjugate Chem., Vol. 4, No. 6, 1993
Chlorlnated Phenyl Azides as Photolabels
Table 1.. Photolysis of Chloro- and Fluoro-Substituted Phenyl Azides C0pCH3
xyJx ‘
Y
ax C02CH3
_a_
Y
+
x)$(x Y
547
Scheme 1. NR
NH
C02CH3 2 Y
&
13
isolated product (%) model
3 4 5*
X c1 H F
Y c1 c1
F
aniline
NH insertion
l(35)
7 (34) 8 (35) 9 (65)b
2 (45) 6 (24)b
NR
Photolysis waa carried out in 1M diethylaminelcyclohexanefor Photolysis results w i t h 5 are taken
17, R=Ac L18,R=H
2 h a t 350 nm for azides 3 and 4. from Keana and Cai (8).
a Key:
NH
19
(a)SnC12QHz0,EtOAc, EtOH; (b) NCS, DMF (c) KOH,
MS mlz 304 (M+, 65), 275 (loo), 240 (30), 204 (17), 130 (Cl+0H)2; (d) (i) HCI, NaN02, (ii) NaN3. (55);HRMS 304.0519 (304.0534 calcd for C I ~ H I ~ N ~ ~ ~ C ~ ~NH2NH2+120, ). 3-Azido-2,4-dichloro-l0,5-(iminomethano)10,ll-dianiline 1 (35%) (Table I). Similar results were obtained hydro-5H-dibenzo[a,~cycloheptene (19). To a stirred for dichlorophenyl azide 4. In contrast, photolysis of the mixture of 18 (25.3 mg, 0.0829 mmol) in 3 N HCl(4 mL) nonhalogenated analog methyl 4-azidobenzoate (10) in that was being cooled in a brine/ice bath was added sodium cyclohexane with diethylamine as the trapping agent gave nitrite (28 mg, 0.41 mmol). The resulting yellow solution 5-carbomethoxy-2-(diethylamino)-3H-azepine (1 1) (21 7%) was stirred with cooling for 75 min, and sodium azide (40 (24) and 5-carbomethoxy-2-(diethylamino)-lH-azepine mg, 0.62 mmol) was carefully added. The resulting mixture (12) (16 % ) as the major products (eq 2). Both 11 and 12 was allowed to warm to room temperature with the cooling bath, and then it was stirred for 16h. The colorless mixture COzCH3 I was cooled in an ice bath and basified to pH > 10 by Et,NH/cyciohexane addition of N a ~ C 0 (6 3 mL), and the resulting mixture was hu,350 nm, 8 h extracted with dichloromethane (3 X 5 mL). The extract I I was washed with brine, dried (NazSOd),and concentrated CH3OpC N3 to a pale yellow oil, which was purified by flash chroma11 12 10 tography (CHCla/benzene 1:l to 1:O) to give the title 21 % 16% compound as a colorless oil (20.7 mg, 75%1: IR (CDCl3) 2127 (N3) cm-l; lH NMR 6 1.87 (br s, lH), 3.16 (dd, J = resulted from ring expansion of the nitrene intermediate 17.9,4.1 Hz, lH), 3.36 (dd, J = 12.2,5.8 Hz, lH), 3.48 (dd, derived from 10 upon photolysis. Li et al. (5) reported J = 18.0, 3.0 Hz, lH), 3.54 (d, J = 12.4 Hz, lH), 4.26 (t, that photolysis of 4-azidobenzoic acid and 4-azidoaceJ = 3.7 Hz, lH), 4.89 (d, J = 5.6 Hz, lH), 6.99 (e, lH), 7.16-7.30 (m, 4H); 13CNMR 6 40.0,41.2,49.7,54.9,125.0, tophenone in diethylaminelcyclohexane gave the corre126.4,127.5,127.7,127.9,128.1,131.3,132.2,136.1,139.8, sponding anilines and 3H-azepines as the only isolated products. Thus, our results show that although CPAs are 141.0,141.7; MS mlz 330 (M+,15),302 (M+- N2,61), 273 not as efficient at undergoing NH insertion as are the (471, 267 (431, 238 (331, 130 (100); HRMS 330.0443 FPAs (Table I), the NH insertion yields derived from the (330.0439 calcd for C I ~ H I ~ N ~ ~ ~ C W . CPAs are nevertheless significantly better relative to those seen for nonhalogenated phenyl azide analogs. RESULTS AND DISCUSSION Synthesis of 3-Azido-2,4-dichloro-IDDC(19). The Synthesis. Azide 3 was prepared by diazotization of photoaffinity labeling and characterization of the PCP amino ester 1 (22)followedby treatment with sodium azide site in mammalian brain has been an ongoing project in (eq 1). Attempts to prepare 3 by reaction of sodium azide our laboratory (26-29). A problem encountered which is common to receptor photoaffinity labeling approaches has C02CH3 C0ZCH3 been a low efficiency of nitrene insertion of the activated photoaffinity label into the receptor protein (27). Thus 1. NaNO&F3COzH far photolabeling approaches have utilized aromatic azides CI 2. NaN3 such as 3-azido-MK801 (13) (27, 28) that do not have NH2 N3 halogen atoms ortho to the azido group. It was therefore of interest to synthesize dichloroazide 19, a derivative of l,X=CI 3,X=Cl 2,X=H 4,X=H the potent PCP receptor ligand 10,5-(iminomethano)10,11-dihydro-5H-dibenzo[a,dlcycloheptene (26)(IDDC, with methyl 2,3,4,5,6-pentachlorobenzoate were not suc14) (Scheme I) as a potentially efficient photolabel. cessful. This latter reaction was patterned after that used The nitro group of 3-nitro-N-acetyl-IDDC (15) (26)was for the preparation of 5 from methyl 2,3,4,5,6-pentaflureduced quantitatively to amine 16 using the method of Bellamy and Ou (25).Controlled ortho,ortho’-dichloriorobenzoate. Methyl 4-azido-3,5-dichlorobenzoate (4) was nation to give 17 was achieved by reaction of 16 with 2.1 prepared by diazotization of amino ester 2 (23) followed by treatment with sodium azide. equiv of N-chlorosuccinimide (NCS) in N,N-dimethylformamide (DMF) at room temperature. Acidic hydrolysis Photolysis. A solution of tetrachlorophenyl azide 3 in (refluxing HC1 or H2SO4) of amide 17 failed to provide the 1M diethylaminelcyclohexanewas photolyzed at 350 nm to give the NH insertion product hydrazine 7 (34% ) and diamine 18 in adequate yield. However, under strongly P
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basic conditions (hydrazine, KOH, ethylene glycol, reflux), 18 was obtained in good yield. Diazotization followed by treatment with sodium azide afforded the azide 19. T h e affinity of 19 for the PCP receptor was assessed in terms of ita ability to compete with 13H]MK801,a ligand with high selectivity and affinity for the PCP receptor (29). For 19, the ICs0 value was determined t o be 6.3 f 0.7 pM (n = 3) whereas that of the corresponding nonchlorinated 3-azido-IDDC was 0.5 pM. T h u s we find that in the specific case of 19 the presence of the chlorine atoms decreased the receptor binding affinity. Since in general it is not possible to predict t h e effect of adding o-chlorine atoms to other aryl azide-based receptor ligands, in other instances the chlorinated ligands may well show satisfactory receptor affinity. For example, Hawkinson et al. (17) report that 1-(4-azido-3,5-dichlorophenyl)-4tert-butyl-2,6,7-trioxabicyclo[2.2.2locte is a 16-fold more potent inhibitor of PHITBOB binding than the corresponding nonhalogenated analog for the PHI TBOB binding site of the GABAA receptor complex. I n conclusion,we have developed t h e chlorinated phenyl azides 3 and 4 as models for a new class of photolabeling reagents. In photolysis experiments these models showed significant improvement in NH insertion in 1 M diethylamine/cyclohexane relative to the nonhalogenated analog 10. In addition, the relative ease by which amine precursors of existing aryl azide-based photolabels may be chlorinated should make application convenient. By way of illustration, the dichloroazido analog 19 of the PCP receptor ligand IDDC (14) was synthesized and its affinity for the PCP receptor was determined. ACKNOWLEDGMENT This work was supported by NIH Grant GM-27137, by the National Institute on Drug Abuse (DA 06726), a n d by Cambridge Neuroscience, Inc. LITERATURE CITED (1) Bayley,H. (1983) Photogenerated Reagents in Biochemistry and Molecular Biology Elsevier, New York. (2) Knorre,D. G., andvlassov, V. V. (1989)Affinity Modification of Biopolymers CRC Press, Boca Raton, FL. (3) Schuster, D. I., Probst, W. C., Ehrlich, G. K, and Singh, G. (1989) Photoaffinity Labeling. Photochem. Photobiol. 49,785, (4) Bayley, H., and Staros, J. (1984) Azides and Nitrenes (E. F. V. Scriven, Ed.) Chapter 9, Academic Press, San Diego, CA. (5) Li, Y.-Z., Kirby, J. P., George, M. W., Poliakoff, M., and Schuster, G. B. (1988) 1,2-Didehydroazepines from the Photolysis of Substituted Aryl Azides: Analysis of Their Chemical and Physical Properties by Time-Resolved Spectroscopic Methods. J.Am. Chem. SOC. 110, 8092. (6) Schuster, G. B., and Platz, M. S. (1992) Photochemistry of Phenyl Azide. Adv. Photochem. 17,69. (7) Keana, J. F. W., and Cai, S. X. (1989) Functionalized Perfluorophenyl Azides: New Reagents for Photoaffinity Labeling. J.Fluorine Chem. 43, 151. (8) Keana, J. F. W., and Cai, S. X. (1990) New Reagents for Photoaffinity Labeling: Synthesis and Photolysis of Functionalized Perfluorophenyl Azides. J. Org. Chem. 55, 3640. (9) Cai, S. X., Glenn, D. J., and Keana, J. F. W. (1992) Toward the Development of Radiolabeled Fluorophenyl Azide-Based PhotolabelingReagents: Synthesis and Photolysis of Iodinated 4-Azidoperfluorobenzoates and 4-Azido-3,5,6-trifluorobenzoates. J. Org. Chem. 57, 1299. (10) Aggelar, R., Chicas-Cruz, K., Cai, S. X., Keana, J. F. W., and Capaldi, R. A. (1992) Introduction of Reactive Cysteine Residues in the t Subunit of Escherichia coli FI ATPase, Modification of These Sites with Tetrafluorophenyl AzideMaleimides, and Examination of Changes in the Binding of the t Subunit When Different Nucleotides Are in Catalytic Sites. Biochemistry. 31, 2956.
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