256
Bioconjugate Chem. 1993, 4, 256-261
Synthesis and Binding of New Polyfluorinated Aryl Azides to a-Chymotrypsin. New Reagents for Photoaffinity Labeling N. Soundararajan, Shwu Huey Liu, S. Soundararajan, and M. S. Platz* Department of Chemistry, The Ohio State University, 120 West 18th Avenue, Columbus, Ohio 43210. Received November 23, 1992
The preparation of several new, water-soluble, polyfluorinated aryl azide reagents for use in photoaffinity labeling studies is described. The ability of some of these reagents and their nonfluorinated controls tononcovalently bind to a-chrymotrypsin is reported. It is found that polyfluorination does not interfere with ligand binding.
I. INTRODUCTION Photoaffinity labeling (PAL) is a technique for marking the binding sites of proteins (1). It was invented by Westheimer and co-workers in 1962 (2). In the seminal experiment the Westheimer group successfully labeled a-chymotrypsin,a serine-basedprotease with a diazo ester labeling reagent. As noted by the original workers, several diazo ester reagents suffer from photochemical Wolff rearrangement and the carbene intermediate which is generated by the action of light reacts rapidly with water. These two factors resulted in a low labeling efficiency. In 1969Knowles and Fleet introduced aryl azide reagents for PAL (3). Azides are more easily synthesized than their diazo counterparts and can better tolerate physiologically relevant pH's. It has been widely assumed by practitioners of the PAL technique that heat- and light-initiated decomposition of aryl azides efficiently generates aryl nitrenes which react rapidly and indiscriminately with a large variety of organic functional groups to give robust adducts in good yields. Unfortunately, the yields of the desired intermolecular adducts formed from aryl azides are generally either poor or zero (4). Recent work from these and other laboratories has demonstrated that the first trappable intermediate formed on photolysis of phenyl azide 1 is dehydroazepine 3 (Scheme I) (4). The dehydroazepine 3 reacts with amines to give azepines (e.g. 4) in good yield (5). However, in the absence of amines, dehydroazepine 3 reacts with phenyl azide, or another ketenimine to eventually produce polymeric tar. The rate of ring expansion of singlet phenylnitrene at ambient temperature is so rapid as to preclude external trapping of the singlet nitrene ( 4 ) . In 1972Banks reported that pyrolysis of polyfluorinated aryl azides generates nitrenes which can be intercepted in good yield (6). It was recognized (1)that this might lead to an attractive new type of PAL reagent because the small size of the fluorine substituent might not interfere with ligand binding, and furthermore that fluorine provides an NMR probe of the labeling process. This encouraged ourselves (7) and Keana's laboratory (8) to study azides of this type. We note that Watt (9),Katzenellenbogen (IO), and Capaldi (11) and co-workers have recently employed fluorinated aryl azides in PAL experiments, albeit with mixed results. It is now amply demonstrated that polyfluorination retards ring expansion of singlet nitrenes by many orders of magnitude (3, which in turn allows efficient trapping of the nitrene intermediate. In this paper we are pleased to report the preparation of new water-soluble polyfluorinated aryl azides and to compare their binding t o
Scheme I
Scheme I1 F
O
P
-
OCH,
a) (1)(COl,CIa (1)CH,OH
F
b) Na", DMF, 60'
5
8
F
O
CI
l)CH,OH, H O 21
O
(co12c12- N F 7
6
9 XI WN-CH, (b)O
10 X
-
la1 N-CH] (b) 0
a-chymotrypsin with that of their nonfluorinated control compounds. We have found that polyfluorination of aryl azides does not interfere with binding of the substrate and thus these reagents should be valuable new tools for PAL experiments. 11. SYNTHESIS
Commercially available pentafluorobenzoic acid (5) is readily converted to pentafluorobenzoyl chloride and then to its methyl ester. The azide moiety can be introduced by heating the methyl ester with sodium azide in DMF to give 6. The ester 6 can be converted to 4-azido-2,3,5,6tetrafluorobenzoic acid by gentle hydrolysis and converted to the acid chloride 7 by treatment with oxalyl chloride (Scheme 11). Compounds 8,9a, and 9b are prepared by treatment of the acid chloride with imidazole, N,Ndimethylaminoethanol or N,N,N-trimethylethylenediamine, respectively. Compounds 9a and 9b noncovalently bind a-chymotrypsin (videinfra)whereas 8, first prepared by Keana (8a) et al., is expected to be useful for coualent labeling of a-chymotrypsin at serine-195 in the aromatic binding pocket (12). The nonfluorinated control compounds 10a,bwere prepared from 4-azidobenzoylchloride (13).
1043-10Q2/93/29Q4-Q256$Q4.Q~lQ0 1993 American Chemical Society
B i o c o n j ~ t eChem., Vol. 4, No. 4, 1993 257
Synthesis and Binding of New Polyfiuorlnated Aryl Azides
Table I. Dissociation Contrants of Various PAL Reagents and a-ChymotrypsinDetermined at pH 5 at Ambient Temmrature
Scheme I11
11
1 2 X-WNCHl
reagent 9a 9b 10a 10b 13a
(b)O
1
NaN3 lDMF
KI (mM) 0.15 0.32
0.40 0.014
reagent 13b 14a 14b 16
KI (mM) 0.11 0.12 0.10 0.33
0.14
Fp .P CH,CH,N(CH;),
lCH$N(CH,)a
N
F
1 4 X - (a)NCH, lb)O
1 3 X-(r)NCH; (b)O
Scheme IV H-
COIH C-NH, I I CIH,OH c
17
18
d
I N,
I N3
19
20
21
Cinnamic acid derivatives are known to bind strongly to a-chymotrypsin (14,15). Commercially available pentafluorocinnamic acid 11 was converted to watersoluble (HCVH20,pH 3) compounds 13a and 13b (Scheme 111) using the same strategy described previously. For the sake of comparison, the nonfluorinated cinnamoyl azides 14a,4b were also prepared. o *H
'XCH,CHp(CH,),
N3 H
14
X-WNCH,
(blO
Pentafluorobenzylbromide 15 was converted to a watersoluble ammonium azide 16. +
15
valent binding of aryl azides to a-chymotrypsin was investigated. Indole Inhibition Study. The competitive inhibition of indole (I) determined against the hyconstant (KI) drolysis of acetyltyrosine ethyl ester (ATEE) catalyzed by a-chymotrypsin has been reported to be 0.8 f 0.2 mM at pH 8 (16). For the sake of comparison, we repeated this work at pH 8 and found KI to be 0.88 mM, in good agreement with previously published work (16).Indole inhibition of acetyltyrosine ethyl ester (ATEE) (17) hydrolysis was also studied at pH 5, where a-chymotrypsin will bind aryl azides, but this is a pH where the enzyme activity is minimal and there will be little enzyme-catalyzed hydrolysis of benzoate esters and amides (12). The reaction rates of ATEE hydrolysis catalyzed by a-chymotrypsin at pH 5 in the presence of inhibitor were determined. A first-order relationship was found between reaction velocity and ATEE concentration. The first-order catalytic constant and Michaelis constant are obtained by applying the Michaelis-Menten equation to the experimental data. In this manner, the catalytic constant kat,app and Michaelis constant Km,app with varying indole concentration were obtained (18). From the linear plot of (K,,Jkcat)appversus [I] the KIof indole is obtained where K,,Jkcat = 4.12 X le3 mM s and (K,Jkat) X ( ~ / K I=) 2.37 X 10-3 s-l, thus KI= intercept/slope = 1.7 mM, of indole, at pH 5. Determinations of KI in the PAL/a-Chymotrypsin Complexes. All of the PAL reagents of this work behave as competitive inhibitors of ATEE hydrolysis when they bind to a-chymotrypsin. The dissociation constants (Kd or KI)of a PAL reagent with a-chymotrypsin are then calculated from the competitive inhibition equation (18):
16
A racemic tetrafluoroazide analog of phenylalanine 21 was prepared by the sequence shown in Scheme IV. 111. DETERMINATION OF DISSOCIATION
CONSTANTS WITH (u-CHYMOTRYPSIN In order for a PAL experiment to be successful, the labeling reagent must bind tightly to the target biomolecule. Thus the effect of polyfluorination on the nonco-
where Km = 5.45 mM, kcat = 1.39 X 103 s-l, [SI = 1 mM ATEE at pH 5, [I] = concentration of PAL in the sample solution, [ET] = concentration of a-chymotrypsin in the sample solution, and V,,, = k,t[E~l. The binding constants of the PAL reagents synthesized in this work are given in Table I. The data indicates that all of the reagents bind a-chymotrypsin with almost equal affinity, with the exception of nonfluorinated ester lob. The origin of the unusual affinity of 10b for a-chymotrypsin is unclear. However it is clear that the ability of fluorine substitution to enhance the desirable reactivity patterns of arylnitrenes, in particular the yield of bimolecular trapping products, does not interfere with noncovalent binding with a model enzyme, a-chymotrypsin. It is concluded that these new reagents will be useful tools for PAL experiments. EXPERIMENTAL PROCEDURES A. Materials. General. Melting points are uncorrected and were recorded on an Electrothermal apparatus, infrared spectra were obtained on a Beckman IR 4250 or a Perkin-Elmer 1710 infrared Fourier transform spectro-
258 Bioconlugate Chem., Vol. 4, No. 4, 1993
photometer, UV spectra were recorded on a Perkin-Elmer Lambda 3B UV/VIS spectrophotometer, and high resolution mass spectra were obtained with a VG 70-250 S or Kratos MS-30 mass spectrometer. Elemental analyses were performed by M-H-W Laboratories. lH NMR (CDC13) and l9F NMR (CDCl3) were recorded using a Bruker AM-250 spectrometer. lH NMR are reported in ppm from internal standard tetramethylsilane (TMS) on the 6 scale with splitting patterns, coupling constants, and relative integrated areas. The letter designates the multiplicity of the signal: s, singlet; bs, broad singlet; d, doublet; d,d, doublet of doublet; t, triplet, q, quartet; m, multiplet. ‘9F NMR chemical shifts are obtained from the proton decoupled spectra and are reported in ppm relative to internal hexafluorobenzene (-162.9 relative to CFCl3 = 0). The solvents and reagents used were dried and purified prior to use. Dimethylformamidewas distilled from NaH. Benzene, toluene, and tetrahydrofuran were distilled from sodium metal and benzophenone, methanol, and ethanol were distilled from their corresponding magnesium alkoxides. Column chromatography was performed, depending on the nature of the compound to be purified over either Brockman Activity 1 Basic or Neutral (80-200 mesh) alumina or silica gel. In places where essentially identical procedures for the synthesis and workup are involved, a general procedure is described and spectral and analytical data are given under each compound. Indole, pentafluorobenzoic acid, pentafluorocinnamic acid, N,N-dimethylethanolamine were obtained from Aldrich Chemical Co. N-Acetyl-L-tyrosine ethyl ester (ATEE), was purchased from Sigma. N,N,ZV’-Trimethylethylenediaminewas purchased from Morton Thiokol, Inc. TLC aluminum plates were purchased from EM Science. All the other chemicals were reagent grade and were commercially available. a-Chymotrypsin (92-97 % protein, salt free, three times recrystallized) was purchased from Worthington BiochemicalCorp. All enzyme activity assays were performed either on a Perkin-Elmer Lambda 3B UV/VIS or a HewlettPackard 8452A diode-array spectrophotometer. Lyophilizations were carried out with a Virtis Freeze mobile-6 lyophilizer. All kinetic data were calculated by the “Enzfitter”program purchased from Elsevier-Biosoft (68 Hills Road, Cambridge CB2, lLA, United Kingdom). B. Methods. 1. Determination of a-Chymotrypsin Concentration. The concentration of a-chymotrypsinwas determined by the UV absorbance of the protein at X = 280 nm with a value of 6 determined from Beer’s law to be 5 X lo4 M-l cm-l. Generally, the purity of the a-chymotrypsin was close to the value given on the label. 2. Enzyme Activity Assay. Enzyme activity was measured by the hydrolysis of ATEE (17) at pH 5. The ATEE substrate solution (1 mM) was prepared by dissolving 26.9 mg of ATEE in 1mL of acetone and 99 mL of 0.1 M sodium acetate buffer, pH 5. Into both the sample and reference cuvettes was separately pipetted 3 mL of ATEE solution. Ten microliters of native or modified enzyme solution (32 mg/mL) was added into the sample cell. The decrease in absorbance at 237 nm with time followed a straight line and was recorded for 3 min. The slope of this line indicated the activity of the enzyme. 3. Inhibition by Indole. Solutions of different concentrations of indole/ATEE (0-1.2 mM, pH 5) were prepared. Three milliliters of these solutions were pipetted out into both the sample and the reference cells of a UV cuvette. One hundred microliters of a-chymotrypsin stock solution (8.9 X lo6 M) was then added to the sample cell. The activities of enzyme solutions containing inhibitor
Soundararajan et al.
were obtained by the decrease in absorbance at 237 nm over 3 min. The first-order catalytic constant (kat,app) and Michaelis constant (Km,app) were obtained using the Michaelis-Menten equation:
In addition, competitive inhibition behavior was found by plotting l / V versus [SI. The dissociation constant of inhibitor was then obtained by the Lineweaver-Burk equation (18):
(~~m/k,tl,pp)(l/[Sl) + l / k (2) where [Km/kJapp = Km/kat (1+ [II/KIJ,[I1 = [concentration of indole], [SI = [concentration of ATEE], and [ET] = concentration of a-chymotrypsin, thus, KI = intercept/slope. 4. Binding Constant (KI) Determination of Various PAL’S and a-Chymotrypsin. Solutions with different concentrations of ATEE substrate (0.5-2 mM in pH 5 acetate buffer) were prepared. The PAL a-chymotrypsin complex was formed by dissolving 4 X 10-6 mol (100 mg) of a-chymotrypsin in 3 mL of water (pH 3, HC1) followed by addition of 8.5 X 10-5 mol of PAL reagent in 125 pL of acetonitrile and incubation in the dark for 1h. Three milliliters of ATEE solution of various concentration was pipetted into both the sample and reference cuvettes, separately. Ten microliters of enzyme complex solution (32 mg/mL) was added to the sample cell. The enzyme activity was recorded using standard procedures as previously described. The binding constants for the PAL/ a-chymotrypsin complex were calculated by the same procedure as the indole inhibition constant. C. Synthesis. 4-Azidobenzoic acid (13, 19), 4-azidobenzoyl chloride (191,methylpentafluorobenzoate (7g,201, ethyl 4-azido-2,3,5,6-tetrafluorocinnamate(201, I m i d o 2,3,5,6-tetrafluorobenzoic acid @a),methyl 4-azido-2,3,5,6tetrafluorobenzoate (6) (7g, 8a), and ethyl pentafluorocinnamate (20) have been described in the literature. N- (4-Azido be nz oy 1)-N,N’ ,N’- t r im e t h y 1e t hy 1e ne di amine (loa). N,N,N’-Trimethylethylenediamine(1.4 g, 13.5 mmol) was dissolved in dry THF (50 mL). K&03 (3 g) was added at room temperature followed by a solution of 4-azidobenzoyl chloride (13) (15 mL, 12.25 mmol) dropwise with stirring at room temperature. After 1h of stirring at room temperature, the solvent was evaporated and the residue was taken up in water. The ethyl acetate extract of the aqueous solution was concentrated and passed through a column of neutral alumina in ethyl acetate. The product was obtained as a yellow oil: yield, 99%; lH NMR 6 1.9 (s, 6H, N(CH32 protons), 2.42 (t, J = 7 Hz, 2H methylene protons), 3.0 and 3.1 (s,3H, NCH3 protons), 3.3-3.5 (m, methylene protons), 7.0 (d, J = 6 Hz, 2H, aromatic protons), 7.4 (d, J = 6 Hz, 2H, aromatic protons); IR (neat) 2129, 2096, 1625, 1607 cm-l. 2-(N,N-Dimethylamino)ethyl4-Azidobenzoate (lob). Compound 10b was prepared following the procedure described for loa, but substituting N,N-dimethylethanolamine in the place of N,N,”-trimethylethylenediamine. The product was obtained as a yellow oil: yield, 70%; lH NMR 6 2.3 (s, 6H, N(CH3)2 protons), 2.7 (t,J = 7 Hz, 2H, methylene protons), 4.4 (t, J = 7 Hz, 2H, methylene protons), 7.0 (d,J = 6 Hz, 2H, aromatic protons), 8.0 (d, J = 6 Hz, 2H, aromatic protons); IR (neat) 2125, 1705, 1605 cm-l.
Synthesls and Binding of New Polyfluorinated Aryl Azides
Bioconjugate Chem., Vol. 4, No. 4, 1993 258
N(CHd2 protons), 2.47 (t, J = 6.6 Hz, 2H, methylene General Procedure for the Nucleophilic Displacement protons), 3.04 and 3.14 (s, 3H, NCH3 protons), 3.47 and of Fluorine by Azide. A solution of the pentafluoro compound (0.01052 mol) in dry dimethylformamide (15 3.54 (t,J = 6.6 Hz, 2H, methylene protons), 7.23 (d, J = mL) and powdered sodium azide (0.01157 mol) was stirred 16Hz, lH, methine proton), 7.57 (d,J = 16Hz, l H , methine under nitrogen at 55-60 "C in an oil bath for 18 h. The proton); 19FNMR-141.45 (m, 2F),-154.09 (m, 1F),-163.29 solvent was removed by distillation below 60 "C under (m, 2F); exact mass calculated for C14H15F~N20m/e reduced pressure. The residue was dissolved in ethyl 341.109, found 341.113. Anal. Calcd for C ~ ~ H I S F B N ~ O : acetate (10 mL) and purified by passing through a column C, 49.25; H, 4.43; N, 8.21. Found: C, 49.37; H, 4.38; N, of neutral alumina in hexanelethyl acetate (1:l). 8.16. GeneralProcedurefor the Synthesis ofthe Fluorinated 2-(N,N-Dimethylamino)ethyl2,3,4,5,6-PentafluorocinAzides 8,9a, and 9b from 6. A solution of the azidoflunumate (12b). Compound 12bwasprepared from 2,3,4,5,6orobenzoic acid 6 (0.01 mol) in fresh oxalyl chloride (10 pentafluorocinnamic acid following the general procedure mL) was heated under reflux for 8 h. Excess oxalyl chloride described for 3-6: pale brownish oil at room temperature; was removed by distillation under reduced pressure and yield, 92 % ;lH NMR 6 2.33 (s,6H, N(CH3)2 protons), 2.66 10 mL of benzene was added to the cooled residue. (t,J = 6.6 Hz, 2H, methylene protons), 4.33 (t,J = 6.6 Hz, Benzene was distilled under reduced pressure, leaving the 2H, methylene protons), 6.78 (d, J = 16.5 Hz, lH, methine azidofluorobenzoyl chloride. The acid chloride was used proton), 7.66 (d, J = 16.5 Hz, l H , methine proton); lgF immediately without further purification and characterNMR -162.74 (m, 2F), -152.29 (m, lF), -140.53 (m, 2F); ization in the next step. exact mass calculated for C13H12F~N02mle 328.077, found Imidazole, amino alcohol, or diamine (0.01 mol) was 328.091. Anal. Calcd for C13H12F~N02:C, 47.55; H, 3.69; dissolved in freshly distilled THF (50 mL) in a 100-mL N, 4.27. Found: C, 47.46; H, 3.58; N, 4.38. round-bottomed flask. Distilled triethylamine (0.011 mol) N-(4-Azido-2,3,5,6-tetrafluorocinnamoyl)-NJV'JV'-triwas then added via syringe to the clear solution. The methylethylenediamine (13a):pale yellowish oil at room azidofluoro acid chloride dissolved in THF (5 mL) was temperature; yield, 84%;lH NMR 6 2.25 (s,6H, N(CH3)2 then added dropwise to the contents of the flask with protons), 2.47 (t,J = 6.6 Hz, 2H, methylene protons), 3.04 stirring at 0-5 "C. The reaction mixture was allowed to and 3.14 (8,3H, NCH3 protons), 3.47 and 3.54 (t, J = 6.6 stir for 2 h at room temperature. Triethylamine hydroHz, 2H, methylene protons), 7.23 (d, J = 16 Hz, l H , chloride precipitated and was filtered with suction and methine proton), 7.57 (d, J = 16 Hz, lH, methine proton); washed with fresh THF (10 mL) and the solvent distilled 19FNMR 163.29 (m, 2F), -154.09 (m, lF),-141.45 (m, 2F); off under reduced pressure. The residue obtained was exact mass calculated for C14H15F4N50 mle 364.116, found further purified by recrystallization from appropriate 364.12. Anal. Calcd for C ~ ~ H ~ S F ~C,N46.14; ~ O :H, 4.15; solvent(s). N, 19.23. Found: C, 46.11; H, 4.18; N, 19.32. N-(4-Azido-2,3,5,6-tetrafluorobenzoyl)imidazole (8) 2-(N,N-Dimethylamino)ethyl4-azido-2,3,5,6-tetraflu(8a):colorless crystalline solid; mp 61-63 "C (benzene/ orocinnamate (13b): pale brownish oil at room temperhexane) (lit. (8a)mp 63-64 "C); yield, 87%; 'H NMR 6 ature; yield, 78% ;lH NMR 6 2.30 (s,6H, methyl protons), 7.98 (8, lH, imidazole H), 7.46 (s, l H , imidazole H), 7.18 2.63 (t, J = 6.6 Hz, 2H, methylene protons), 4.32 (t,J = (m, l H , imidazo1e.H); 19F NMR -139.71 (m, 2F), -149.72 6.6 Hz, 2H, methylene protons), 6.76 (d, J = 16.5 Hz, l H , (m, 2F); IR (CHCl3) 2130, 1705, 1690, 1620, 1500, 1420, 1380, 1120 cm-l; exact mass calculated for C I O H ~ F ~ N ~ Omethine proton), 7.65 (d,J = 16.5 Hz, l H , methine proton); 19F NMR -159.19 (m, 2F), -141.09 (m, 2F); exact mass mle 285.027, found 285.024. Anal. Calcd for C10H3calculated for C13H12F4N402 mle 332.089, found 332.097. F4N50: C, 42.10; H, 1.06; N, 24.56. Found: C, 42.36; H, Anal. Calcd for C13H12F4N402: C, 46.98; H, 3.64; N, 16.87. 1.08; N, 24.87. N-(4-Azido-2,3,5,6-tetrafluorobenzoyl)-N,",N'-trim- Found: C, 46.92; H, 3.78; N, 16.94. N-(4-Azidocinnamoyl)-N,","-trimethylethylenediethylethylenediamine (9a):colorless crystals; mp 32-33 amine (14a). This compound was prepared from 4-azi"C (hexane); yield, 87%; 1H NMR 6 2.13 and 2.23 (s,6H, docinnamic acid following the general procedure described N(CH& protons), 2.40and 2.57 (t, J = 6 Hz, 2H, methylene for loa: pale yellowish oil at room temperature; yield, protons), 2.98 and 3.15 (s,3H, N-CH3 protons), 3.27 and 86%; lH NMR 6 2.28 (s, 6H, N(CH3)2 protons), 2.48 (t,J 3.65 (t,J = 6 Hz, 2H, methylene protons);19FNMR-151.52 = 7 Hz, 2H, methylene protons), 3.07 and 3.18 (8, 3H, (m, 2F), -142.54 (m, 2F); IR (CHCl3) 2940, 2860, 2830, NCH3 protons), 3.53-3.60 (m, methylene protons), 6.87 2780,2130,1710,1460,1400,1520,1310,1120,990 cm-l; (d, J = 15 Hz, l H , methine proton), 7.36 (d, J = 6 Hz, 2H, exact mass calculated for C12H13F4N50 mle 319.107, found aromatic protons), 7.48 (d, J = 6 Hz, 2H, aromatic protons), 319.109. Anal. Calcd for C12H13F4N50: C, 45.13; H, 4.11; 7.61 (d, J = 15 Hz, l H , methine proton); exact mass N, 21.94. Found: C, 45.22; H, 4.18; N, 21.87. mle 273.159,found 273.148. Anal. calculatedforC14H19NEO 2-(N,N-Dimethylamino)ethyl4-azido-2,3,5,6-tetrafluCalcd for C14H19N50: C, 61.50; H, 7.01; N, 25.63. Found: orobenzoate (9b): pale amber colored oil at room temC, 61.43; H, 6.89; N, 25.69. perature; yield, 92 % ;lH NMR 6 2.30 (s,6H, CH3 protons), 2-(N,N-dimethylamino)ethyl4-Azidocinnamate (14b). 2.67 (t, J = 6 Hz, 2H, methylene protons), 4.45 (t,J = 6 This compound was prepared from 4-azidocinnamic acid Hz, 2H, methylene protons); 19FNMR -139.77 (m, 2F), following the general procedure described for lob: pale -152.21 (m, 2F); IR (neat) 2960, 2120, 1740, 1645, 1490, brownish oil at room temperature; yield, 82 % ; lH NMR 160 cm-'; exact mass calculated for CllH10F4N402 mle 306.073, found 306.075. Anal. Calcd for CllH10F4N402: 6 2.31 (s, 6H, methyl protons), 2.63 (t, J = 7 Hz, 2H, C, 43.13; H, 3.29; N, 18.30. Found: C, 43.28; H, 3.17; N, methylene protons), 4.30 (t, J = 7 Hz, 2H, methylene 18.38. protons), 7.62 (d, J = 16 Hz, l H , methine proton), 7.37 (d, N-(2,3,4,5,6-Pentafluorocinnamoyl)-NJV,"-trimeth- J = 6 Hz, 2H, aromatic protons), 7.752 (d, J = 6 Hz, 2H, aromatic protons), 7.62 (d,J = 16Hz, lH, methine proton); ylethylenediamine (1Za). Compound 12a was prepared exact mass calculated for C13H16N402 mle 260.298, found from 2,3,4,5,6-pentafluorocinnamicacid following the 260.22. Anal. Calcd for C13H16N402: C, 61.74; H, 5.93; N, general procedure described for 8-10: pale yellowish oil 20.58. Found: C, 61.65; H, 5.78; N, 20.52. at room temperature; yield, 95%; 'H NMR 6 2.25 (s, 6H,
200 Bioconlugete Chem., Vol. 4, No. 4, 1993
(4-Azido-2,3,5,6-tetrafluoro benzy1)triethylammonium Bromide (16). 2,3,4,5,6-Pentafluorobenzylbromide (15) was heated under reflux with a 10% molar excess of triethylamine in THF. The precipitated salt was filtered, washed with THF, and dried under vacuu.: colorless crystals;mp 89-91 "C; yield, 100%; lH NMR 6 1.28 (t, J = 7 Hz, 9H, methyl protons), 3.25 (q, 6H, methylene protons), 4.51 (s,2H, benzylic protons); 19FNMR -137.00 (m, 2F), -149.61 (m, 2F), -161.53 (m, 1F); exact mass calculated for C13H17F5NBr mle 361.046, found 181.064 [M+- (C2H&NHBrl. Anal. Calcd for C13H17F5NBr: C, 43.21; H, 3.88; N, 3.88. Found: C, 43.71; H, 3.68; N, 3.72. (4-Azido-2,3,5,6-tetrafluorobenzyl) triethylammonium bromide (16) was synthesized using the general procedure described for the displacement of fluorine by azide. After the distillation of the residual DMF, the residue was triturated with benzene and the resulting solid recrystallized from ethanollwater, yielding colorless powdery white crystals which started decomposing at 156-158 "C: yield, 94%; lH NMR 6 1.29 (t, J = 7 Hz, 9H, methyl protons), 3.34 (q, 6H, methylene protons), 4.68 (8, 2H, benzylic protons); 19FNMR -133.83 (m, 2F),-158.53 (m, 2F). Anal. Calcd for C13H17F4N4Br: C, 40.62; H, 4.46; N, 14.58. Found: C, 40.74; H, 4.54; N, 14.64. d 1-2,3,4,5,6-Pentaf luoro-N-(trif1uoroacetyl)phenylala nine Ethyl Ester (1%. dl-2,3,4,5,6-Pentafluorophenylalanine (17)21(12.76g,0.05 mol) suspended in dry benzene (50 mL) was treated with trifluoroacetic anhydride (14g, 0.075 mol) in drops with stirring under nitrogen at 15-20 "C during 15 min. The reaction mixture, which was originally a slurry, became a clear solution. The clear solution was heated under reflux under nitrogen on a steam bath for 45 min. The solvent and excess trifluroacetic anhydride were distilled off at reduced pressure. The residue was treated with dry ethanol (75mL) and refluxed on a steam bath for 45 min. Ethanol was distilled off a t reduced pressure and the pasty residue obtained was recrystallized from ethyl acetate to give 16.15 g (yield, 87 % ) of a colorless cryatalline solid, 19: mp 90-91 "C; lH NMR 6 1.32 (t,J = 7 Hz, 3H, ester CHd, 3.16-3.47 (m, 2H, benzylic protons), 4.22-4.35 (m, 2H, ester CHz),4.86 (q,J = 7 Hz, l H , methine proton), 6.94 (bs, l H , NH proton); 19F NMR -162.49 (m, 2F), -154.82 (m, lF), -143.40 (m, 2F), -77.20 (8, 3F); IR (CHC13) 3380, 1720, 1650, 1270, 1150, 1110, 980, 960 cm-l; exact mass calculated for C13HgFaN03 mle 379.045, found 379.015. Anal. Calcd for C13HgFaN03: C, 41.16; H, 2.39; N, 3.69. Found: C, 41.22; H, 2.28; N, 3.71. 4-Azido-dl-2,3,5,6-tetrafluoro-N(trifluoroacety1)phenylalanine Ethyl Ester (20). Colorless crystals were obtained by treatment of 19 with NaN3 as described in the general procedure: mp 106-107 "C; yield, 77%; lH NMR 6 1.32 (t,J = 7 Hz, 3H, ester CH3), 3.15-3.46 (m, 2H, benzylic protons), 4.20-4.34 (m, 2H, ester CHZ),4.85 (q, J = 7 Hz, lH, CH proton), 6.97 (bs, lH, NH proton); 19FNMR -152.66 (m, 2F),-143.88 (m, 2F), -77.23 (s,3F); IR (CHCl3) 3390,2120,1725,1650,1465,1280,1150,965 cm-l; exact mass calculated for C13HgN403F7 mle 402.056, found 402.015. Anal. Calcd for C13HgN403F7: C, 38.80; H, 2.26; N, 13.93. Found: C, 38.91; H, 2.18; N, 13.72. 4-Azido-dl-2,3,5,6-tetrafluorophenylalanine (21). The azido amide ester 20 was hydrolyzed following the procedure described for the preparation of methyl 4-azido2,3,5,6-tetrafluorobenzoate(6). The aqueous solution obtained after hydrolysis and extraction with methylene chloride was cooled to 0 "C and neutralized to pH 7.00. The precipitated solids were filtered, redissolved in the minimum amount of 10% hydrochloric acid, and neu-
Soundararajan et al.
tralized to pH 7.00 using dilute ammonium hydroxide solution. The reprecipitated solid was filtered, washed with ice water and dried in a vacuum desicator over phosphorus pentoxide: colorless powder; mp 190-195 "C dec; yield, 76%; lH NMR (CDsOD) 6 3.34-3.57 (m, 2H, benzylic protons), 4.42-4.47 (m, l H , CH proton), 7.62 (bs, 2H, NH2 proton), 12.09 (bs, l H , acid proton); 19F NMR (CD30D) -144.04 (m, 2F), -151.69 (m, 2F). Anal. Calcd for C ~ H ~ F ~ N C, ~ O38.84; Z : H, 2.17; N, 20.17. Found: C, 38.71; H, 2.24; N, 20.14. ACKNOWLEDGMENT Support of this work by the National Institutes of Health (GM 34823) is gratefully acknowledged. LITERATURE CITED (1) Bayley, H. (1983)PhotogeneratedReagents inBiochemistry and Molecular Biology; Elsevier, Amsterdam. (2) Singh, A,, Thornton, E. R., and Westheimer, F. H. (1962) The Photolysis of Diazoacetylchymotrypsin. J. Biol. Chem. 237, 3006. (3)Fleet, G. W. J.,Porter, R. R., and Knowles,J. R. (1969)Affinity Labelling of Antibodies with Aryl Nitrene as Reactive Group. Nature, 224, 511. (4) Schuster, G. B., and Platz, M. S. (1992)Photochemistry of Phenyl Azide. Adv. Photochem. 17,69. (5) Doering,W. Von, and Odum, R. A. (1966)Ring Enlargement in the Photolysis of Phenyl Azide. Tetrahedron, 22,81. (6) (a) Banks, R. E., and Sparkes, G. R. (1972)Studies in Azide Chemistry. Part V. Synthesis of 4-Azido-2,3,5,6-tetrafluoro,4 Azido-3-chloro-2,5,6-trifluoro-, and 4-Azido-3,5-dichloro-2,6-
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