Proc. Nati. Acad. Sci. USA
Vol. 73, No. 5, pp. 1406-1408, May 1976 Chemistry
2-Diazo-3,3,3-trifluoropropionyl chloride: Reagent for photoaffinity labeling (Wolff rearrangement/thioesters)
VINAY CHOWDHRY, RONALD VAUGHAN*, AND F. H. WESTHEIMERt The James Bryant Conant Laboratories of the Chemistry Department, Harvard University, Cambridge, Massachusetts 02138
Contributed by F. H. Westheimer, February 20,1976
ABSTRACT 2-Diazo-3,3,3-trifluoropropionyl chloride has been synthesized from trifluorodiazoethane and phosgene. Its
CF3CHOCH3CO2C2H5 (75%) A + CF3CHOCH3CO2CH3 (8%) B + CF3CHOC2H5CO2CH3 (15o) C
CF3CN2CO2C2H5 CH3OH
derivatives are acid stable, can be used to label enzymes, and undergo photolysis with substantially less rearrangement than do derivatives of other known diazoacyl reagents designed for photoaffinity labeling. In particular, the diazotrifluoropropionyl thioester of methyl N-acetylcysteine undergoes photolysis in methanol with about 40% insertion into the -OH bond of the solvent; by contrast, photolysis of other diazoacyl thioesters gives substantially quantitative Wolff rearrangement. The trifluoro compounds hold promise for the photoaffinity labeling of thiols. Photoaffinity labeling was initiated with the photolysis of diazoacetyl chymotrypsin, prepared from the enzyme and pnitrophenyl diazoacetate (1, 2). Subsequently, this and other photolabile reagents have been applied (3-14) to a wide variety of problems in biochemistry and biology. (For reviews, see refs. 15-18.) Diazoacetates and diazomalonates (6, 7), however, suffer from the disadvantages that they are not acid stable, and that photolysis leads in large measure to the products of the photochemical analog of the Wolff rearrangement (2, 19). Furthermore, attempts to use the diazoesters of thiols, either with glyceraldehyde-3-phosphate dehydrogenaset or model compounds (20), have led only to products of the photochemical Wolff rearrangement or to other products that are not informative from the point of view of photoaffinity labeling. In this paper, we report the synthesis and photolysis of diazotrifluoropropionyl derivatives that are not subject to these limitations. 2-Diazo-3,3,3-trifluoropropionyl chloride is easily prepared, and is purified by vacuum distillation; p-nitrophenyl diazotrifluoropropionate is a crystalline solid that can be purified by sublimation. Various diazotrifluoropropionyl derivatives (including that of chymotrypsin) can be made from these acylating reagents; ethyl diazotrifluoropropionate is stable at room temperature in 1 M hydrochloric acid! All of the diazo derivatives, however, readily undergo photolytic decomposition. Furthermore, photolysis of ethyl diazotrifluoropropionate in methanol gives insertion into the -OH bond of the solvent to the extent of about 85%, and only about 15% rearrangement. [The product B, separated by vapor phase chromatography and identified by its nuclear magnetic resonance NMR) and mass spectra, results from transesterification and ten insertion into the solvent.] We have obtained similar results with other model
Scheme 1 contrast to results with thiol derivatives of other photoaffinity reagents) photolysis does not lead exclusively to rearrangement. When the thioester is illuminated at 350 nm, the mixture of products contains about 60% of the thioether, D, which results from rearrangement, but also includes about 40% of the thioester, E, which arises from insertion of the carbene, formed on photolysis, into the -OH bond of the solvent. This result suggests that diazotrifluoropropionates may prove useful in the photoaffinity labeling of sulfhydryl compounds.
CF3CN2COSCH2CHCO2CH3 NHCOCH3 Ih, (350 nm)
[CF3-C
SCH2fCH-CO2CH3l
CHOH
]
NHCOCH3
j CF3-CH-SCH2-CH-CO2CH3 (60o) D
I
CO2CH3
I
NHCOCH3
CF3CH-COSCH2-CH-CO2CH3 (40%) E OCH3
NHCOCH3
Scheme 2
Photolysis at short wavelengths (254 nm) leads initially to the same products, but the thioester, E, is rapidly destroyed by the light, so only small amounts can be isolated, and then only with difficulty. Prolonged photolysis at 254 nm eventually destroys even the thioether, D. Both D and E, however, are stable for long periods of time to light of long wavelength (350 nm). EXPERIMENTAL Preparations. 2,2,2-Trifluorodiazoethane was prepared from 27 g of trifluoroethylamine hydrochloride by the procedure of Gilman and Jones (21). A solution of this diazo compound in 300
compounds. The results obtained with the diazotrifluoropropionyl ester of methyl N-acetylcysteine are even more significant, since (in
ml of dichloromethane was treated at room temperature with 35 g of finely powdered dipotassium hydrogen phosphate and 15 ml of phosgene in 100 ml of methylene chloride. The mixture was allowed to warm to room temperature, was stirred overnight, and was then decanted through glass wool; the methylene chloride was evaporated and the residue was distilled at 59-60°/137 mm (18 kPa). Infrared (liquid film) peaks were
Abbreviation: NMR, nuclear magnetic resonance. * Present address: Department of Chemistry, Harvey Mudd College, Claremont, Calif. t To whom requests for reprints should be sent. t L. J. Crane, unpublished results. 1406
Proc. Natl. Acad. Sci. USA 73 (1976)
Chemistry: Chowdhry et al. at: 2165, 1750 (broad), 1135 (broad) cm-. Exact mass of the s5CI molecular ion: Calculated, 171.9651; found, 171.9662. Crude undistilled acid chloride from 6.75. g of trifluoroethylamine hydrochloride was stirred in dichloromethane and tetrahydrofuran with 4.0 g of anhydrous sodium p-nitrophenoxide for 24 hr. After the mixture had been filtered and the solvent evaporated, the residue was chromatographed on neutral alumina (100 g, Woelm grade V) and eluted with carbon tetrachloride. The ester, which came through with the solvent front, crystallized on evaporation of the solvent; it was sublimed at 50-70°/10-3 mm (0.13 Pa) and recrystallized from cyclohexane-ligroin; mp 630. Infrared (KBr) peaks were at 2165, 1740 cm-1. Elemental analyses for C, H, and N agree with theory to 40.2%. The ethyl ester§ was prepared from 1.0 g of distilled diazotrifluoropropionyl chloride and 0.5 g of methanol with 0.75 g of quinoline. The mixture was allowed to stand overnight; the ester was extracted into ether and washed with acidified brine and then with bicarbonate solution. After the solvent had been evaporated, the ester was chromatographed on grade III alumina in petroleum ether and then distilled at 66.5°/75 mm (10 kPa). Infrared (liquid film) peaks were at 2165, 1730 cm-1. Elemental analyses for C, H, N, and F agree with theory to ±0.2%. 2-Diazo-3,3,3-trifluoro-N-acetylcysteine methyl ester was prepared (although in very poor yield) from 0.575 g of Nacetylcysteine methyl ester (22, 23), 0.560 g of 2-diazo-3,3,3trifluoropropionyl chloride, and 0.348 g of 2,6-lutidine in 30 ml of tetrahydrofuran overnight at room temperature. The pale yellow crystals obtained from methylene chloride-hexane were sublimed at 85°/0.1 mm (13 Pa); mp 96-97'. Analysis for C, H, and N were within 0.2% of theory. The compound shows a strong diazo band at 2092 cm-1 and UV and NMR spectra consistent with the expected structure. Diazotrifluoropropionyl chymotrypsin was prepared by allowing 2.5 X i0-5 M enzyme to react with 5.5 X l0-5 M pnitrophenyl diazotrifluoropropionate at pH 5.95 at 300 for 1 hr. It proved to be 93% inactivated but reactivated completely on standing at pH 7.0 at room temperature for 10-12 hr. The diazoacyl enzyme solution has also been purified by passage over Sephadex G-25. (This preparation has been repeated by Dr. Richard Hudson in our laboratories.) Photolysis of Ethyl Diazotrifluoropropionate. The photolysis of 15-30 mg of ethyl diazotrifluoropropionate was carried out for 200-300 min in a Srinivasan-Rayonet reactor with RPR 2537 A lamps in 0.1-0.3 ml of methanol. After photolysis, the products were separated on a 120 X 0.31-cm column of 5% SF-1265 on Chromosorb G/AW/DWCS at 60° using a Hewlett-Packard HP5750 gas chromatograph. Alternatively, the methanol was evaporated, and the products were separated on a 360 X 0.31 cm column on 15% Carbowax 20M on Chromosorb P at 1150. The products of photolysis consisted of 75% or more of A (insertion into the solvent), 8% or less of B (ester exchange and insertion into the solvent), and about 15% of C (rearrangement). The structural assignments of A, B, and C are derived from (a) their NMR spectra, (b) their mass spectra, and (c) a transesterification experiment. The NMR spectrum of A showsT 6 1.21 (t, J = 7.0 Hz, 3 H), 3.47 (s, 3 H, ether OCH3), 4.04 §
A different preparation of this ester was reported at the American Chemical Society Middle Atlantic Regional Meeting, Fall, 1974, by A. Zampini and R. A. Shepard (Medicinal Chemistry and Bio-
chemistry Section, Paper no. 156). NMR resonances, 6, given in parts per million downfield from tetramethylsilane. For peaks, s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet.
1407
:t=o/
LLJ
z
m
t=24 r
\
02
220
260
300
340 nm
FIG. 1. UV spectra of 7 X 10-5 M 2-diazo-3,3,3-trifluoropropionyl N-acetyl cysteine methyl ester at zero time and after photolysis with 3500 A lamps for 24 hr.
(q, JF-H = 7.0 Hz, 1 H), 4.22 (q, J = 7.0 Hz, 2 H, ester methylene group). The spectrum of B shows 6 3.57 (s, 3 H, ether methoxyl), 3.86 (s, 3 H, ester methoxyl), 4.16 (q, JF-H = 6.5 Hz, 1 H), and that of C shows 6 1.26 (t, J = 7.0 Hz, 3 H), 3.70 (m, 2 H, diastereotopic ether methylene group), 3.84 (s, 3 H, ester methoxyl), 4.24 (q, JF-H = 6.5 Hz, 1 H). The mass spectra are dominated by the ions obtained by the McLafferty rearrangement (24, 25). In analogy with the mass spectrometry of the esters of methoxyacetic.acid (26)the ether groups of A, B, and-C are lost, presumably as the corresponding aldehydes. Thus A, a methyl ether, lost CH2O to yield an ion of mass 156.03904 (calculated for CH7F302, 156.03982), while B, also a methyl ether, lost CH2O, and C, an ethyl ether, lost CH3CHO to yield ions of measured mass 142.02707 and 142.02685, respectively; calculated for C4H5F302, 142.02417. The formation of the products of the McLafferty rearrangement unambiguously confirms the NMR assignment of A as an ethyl ester, and of B and C as methyl esters, since these groups survive in the ion beam. The transesterification experiment was carried out by heating B in completely deuterated methanol and 1% sulfuric acid. The NMR peak at 6 3.4 in CD30D as solvent disappears, while that at 6 3.14 is unchanged; these peaks correspond to those at 6 3.86 and 3.57 in CDCI3. This experiment, too, confirms the structural assignments made above. Photolysis of Methyl 2-Diazo-3,3,3-trifluoropropionylN-acetylcysteine. Photolysis of 3-4 mg of methyl 2-diazo3,3,3-trifluoropropionyl-N-acetylcysteine, dissolved in 100,ul of dry methanol, was conducted in quartz tubes in a Srinivasan-Rayonet reactor with either RPR 2537 A or RPR 3500 A lamps. Gas chromatographic analysis of 2 gl samples was conducted with a Hewlett-Packard 5750 gas chromatograph on a 180 X 0.31-cm column packed with 3% Dexil 400 GC/ Chromosorb W/HP 80/100. About 20 injections yielded enough material to obtain 1H Fourier transform NMR spectra (about 2000 transients). Photolysis with 3500 A Lamps for 18 hr led to the disappearance of the diazo band at 256 nm, whereas the intensity of the thioester band decreased (Fig. 1) by 60% but then held constant. Gas chromatographic analysis of the product led to two well-separated compounds, D and E, in the ratio 6:4 (retention times, 6.2 and 6.8 min; 40 ml/min helium carrier gas; temperature programmed 110°-170° at 20°/min). Mass:charge ratio for D was 317.05337; for E, 317.05483; calculated for
1408
Proc. Natl. Acad. Sci. USA 73 (1976)
Chemistry: Chowdhry et al.
This work was supported by the Institute of General Medical Sciences of the National Institutes of Health under Grant no. GM04712. The 100 MHz NMR spectrometer was purchased under National Science Foundation facilities Grant GP30965X to the Department of Chemistry. The authors are indebted to the National Institutes of Health Mass-Spectrometry Facility (RR 00317 Division of Research Resources) for the exact masses quoted.
D
1. Singh, A., Thornton, E. R. & Westheimer, F. H. (1962) J. Biol.
Chem. 237, PC 3007-3008. 2. Shafer, J., Baranowsky, P., Laursen, R., Finn, F. & Westheimer, F. H. (1966) J. Biol. Chem. 241, 421-427. 3. Hexter, C. S. & Westheimer, F. H. (1971) J. Biol. Chem. 246,
2.0
4.0
3928-3933.
4. Hexter, C. S. & Westheimer, F. H. (1971) J. Biol. Chem. 246,
3934-3938.
E
00H3
(ester)_
5. Stefanovsky, Y. & Westheimer, F. H. (1973) Proc. Natl. Acad. Sci. USA 70,1132-1136. 6. Vaughan, R. J. & Westheimer, F. H. (1969) Anal. Biochem. 29,
OCH3 1(ether)
H20
IA 42 20 FIG. 2. Comparison of the 100 MHz 'H NMR spectra of compound D (the thioether produced by rearrangement of the 2-diazo3,3,3-trifluoropropionyl thioester of N-acetyl cysteine methyl ester) and compound E (the thioester produced by insertion into the solvent). The double peaks at high resolution arise because the products are mixtures of diastereomers.
CIoH14FSNO5S: 317.05449. The critical region of the 100 MHz
1H NMR spectra is shown in Fig. 2. Compound D shows two tightly spaced methoxy signals at 6 3.78 and 3.81; the further, barely resolved doubling of the signals in both spectra presumably arises because each structural formula represents two diastereomers. The spacing between the methoxyl signals from E (6 3.62 and 3.71) and comparison of the spectra with those for the products of the photolysis of ethyl diazotrifluoropropionate identifies E as the methyl ether methyl ester thioester shown in Scheme 2. This structural assignment is confirmed by UV spectroscopy; E shows the strong absorption at 236 nm typical of thioesters (27-29), whereas A shows only the end absorption of thioesters (30, 31). Both D and E are stable to prolonged irradiation at 350 nm. Photolysis with 2537 A Lamps for 125 min completely eliminated the absorptions of both the thioester and diazo functions. When, however, the photolysis was carried only partially to completion, both UV and gas chromatographic analyses showed that 3-8% of E was present; on further irradiation with 2537 A lamps, the thioester E is rapidly destroyed. To isolate the insertion product, then, photolysis of the diazotrifluorothiopropionate must be carried out at long wavelength. Stability. A solution of ethyl diazotrifluoropropionate is stable for 24 hr in 1 M HCl. [Trifluorodiazoethane is also remarkably acid stable (32).]
305-310. 7. Vaughan, R. J. & Westheimer, F. H. (1969) J. Am. Chem. Soc. 91,217-218. 8. Fleet, G. W. H., Knowles, J. R. & Porter, R. R. (1969) Nature 224, 511-512. 9. Richards, F. F. & Converse, C. (1969) Biochemistry 8,4431-4436. 10. Sonneberg, N., Zamir, A. & Wilchek, M. (1974) Biochem. Biophys. Res. Commun. 59,693-696. 11. Galardy, R. E., Craig, L. C., Jamieson, J. D. & Printz, M. P. (1974) J. Biol. Chem. 249,3510-3518. 12. Escher, E. & Schwyzer, R. (1974) FEBS. Lett. 46,347-350. 13. Katzenellenbogen, J. A., Johnson, H. J., Jr. & Meyers, H. N. (1973)
Biochemistry 12,4085-4092.
14. Guthrow, C. E., Rasmussen, H., Brunswick, D. J. & Cooperman, B. S. (1973) Proc. Nati. Acad. Sci. USA 70,3344-3346. 15. Singer, S. J. (1967) Adv. Protein Chem. 22, 1-54. 16. Knowles, J. R. (1972) Acc. Chem. Res. 5, 155-160. 17. Creed, D. (1974) Photochem. Photobiol. 19,459-462. 18. Cooperman, B. S. (1976) Proc. Int. Conf. on Proteins and Other Adducts to DNA, ed. Smith, K. C. (Plenum Press, New York), in press. 19. Chaimovich, H., Vaughan, R. J. & Westheimer, F. H. (1968) J. Am. Chem. Soc. 90,4088-4093. 20. Hixson, S. S. & Hixson, S. H. (1972) J. Org. Chem. 37,1279-1280. 21. Gilman, H. & Jones, R. G. (1943) J. Am. Chem. Soc. 65,14581460. 22. Jones, J. B. & Wigfield, D. C. (1966) Can. J. Chem. 44,25172523. 23. Kupchan, S. M., Gicobbe, T. J., Krull, I. S., Thomas, A. M., Eakin, M. A. & Fessler, D. C. (1970) J. Org. Chem. 35,3539-343. 24. Gilpin, J. A. & McLafferty, F. W. (1957) Anal. Chem. 29,
990-994.
(1967) MassSpectrometry of Organic Compounds (Holden-Day, Inc., San Francisco, Calif.), p. 155. Goulden, J. D. S. & Manning, D. J. (1970) Org. Mass Spectrom. 3, 1467-1469. Sjoberg, B. (1942) Z. Phys. Chem. 52,209-221. Koch, H. P. (1949) J. Chem. Soc. 387-394. Harding, J. S. & Owen, L. N. (1954) J. Chem. Soc. 1536-1545. Cumper, C. W. N., Read, J. F. & Vogel, F. A. I. (1966) J. Chem. Soc. A 239-242. Fehnel, E. A. & Carmack, M. (1949) J. Am. Chem. Soc. 71, 84-93. H6hrig, J. R. & Keegstra, K. (1967) J. Am. Chem. Soc. 89, 5492-5493.
25. Budzikiewicz, H., Djerassi, C. & Williams D. H. 26.
27. 28. 29. 30. 31. 32.
Vol. 73, No. 5, pp. 1406-1408, May 1976 Chemistry
2-Diazo-3,3,3-trifluoropropionyl chloride: Reagent for photoaffinity labeling (Wolff rearrangement/thioesters)
VINAY CHOWDHRY, RONALD VAUGHAN*, AND F. H. WESTHEIMERt The James Bryant Conant Laboratories of the Chemistry Department, Harvard University, Cambridge, Massachusetts 02138
Contributed by F. H. Westheimer, February 20,1976
ABSTRACT 2-Diazo-3,3,3-trifluoropropionyl chloride has been synthesized from trifluorodiazoethane and phosgene. Its
CF3CHOCH3CO2C2H5 (75%) A + CF3CHOCH3CO2CH3 (8%) B + CF3CHOC2H5CO2CH3 (15o) C
CF3CN2CO2C2H5 CH3OH
derivatives are acid stable, can be used to label enzymes, and undergo photolysis with substantially less rearrangement than do derivatives of other known diazoacyl reagents designed for photoaffinity labeling. In particular, the diazotrifluoropropionyl thioester of methyl N-acetylcysteine undergoes photolysis in methanol with about 40% insertion into the -OH bond of the solvent; by contrast, photolysis of other diazoacyl thioesters gives substantially quantitative Wolff rearrangement. The trifluoro compounds hold promise for the photoaffinity labeling of thiols. Photoaffinity labeling was initiated with the photolysis of diazoacetyl chymotrypsin, prepared from the enzyme and pnitrophenyl diazoacetate (1, 2). Subsequently, this and other photolabile reagents have been applied (3-14) to a wide variety of problems in biochemistry and biology. (For reviews, see refs. 15-18.) Diazoacetates and diazomalonates (6, 7), however, suffer from the disadvantages that they are not acid stable, and that photolysis leads in large measure to the products of the photochemical analog of the Wolff rearrangement (2, 19). Furthermore, attempts to use the diazoesters of thiols, either with glyceraldehyde-3-phosphate dehydrogenaset or model compounds (20), have led only to products of the photochemical Wolff rearrangement or to other products that are not informative from the point of view of photoaffinity labeling. In this paper, we report the synthesis and photolysis of diazotrifluoropropionyl derivatives that are not subject to these limitations. 2-Diazo-3,3,3-trifluoropropionyl chloride is easily prepared, and is purified by vacuum distillation; p-nitrophenyl diazotrifluoropropionate is a crystalline solid that can be purified by sublimation. Various diazotrifluoropropionyl derivatives (including that of chymotrypsin) can be made from these acylating reagents; ethyl diazotrifluoropropionate is stable at room temperature in 1 M hydrochloric acid! All of the diazo derivatives, however, readily undergo photolytic decomposition. Furthermore, photolysis of ethyl diazotrifluoropropionate in methanol gives insertion into the -OH bond of the solvent to the extent of about 85%, and only about 15% rearrangement. [The product B, separated by vapor phase chromatography and identified by its nuclear magnetic resonance NMR) and mass spectra, results from transesterification and ten insertion into the solvent.] We have obtained similar results with other model
Scheme 1 contrast to results with thiol derivatives of other photoaffinity reagents) photolysis does not lead exclusively to rearrangement. When the thioester is illuminated at 350 nm, the mixture of products contains about 60% of the thioether, D, which results from rearrangement, but also includes about 40% of the thioester, E, which arises from insertion of the carbene, formed on photolysis, into the -OH bond of the solvent. This result suggests that diazotrifluoropropionates may prove useful in the photoaffinity labeling of sulfhydryl compounds.
CF3CN2COSCH2CHCO2CH3 NHCOCH3 Ih, (350 nm)
[CF3-C
SCH2fCH-CO2CH3l
CHOH
]
NHCOCH3
j CF3-CH-SCH2-CH-CO2CH3 (60o) D
I
CO2CH3
I
NHCOCH3
CF3CH-COSCH2-CH-CO2CH3 (40%) E OCH3
NHCOCH3
Scheme 2
Photolysis at short wavelengths (254 nm) leads initially to the same products, but the thioester, E, is rapidly destroyed by the light, so only small amounts can be isolated, and then only with difficulty. Prolonged photolysis at 254 nm eventually destroys even the thioether, D. Both D and E, however, are stable for long periods of time to light of long wavelength (350 nm). EXPERIMENTAL Preparations. 2,2,2-Trifluorodiazoethane was prepared from 27 g of trifluoroethylamine hydrochloride by the procedure of Gilman and Jones (21). A solution of this diazo compound in 300
compounds. The results obtained with the diazotrifluoropropionyl ester of methyl N-acetylcysteine are even more significant, since (in
ml of dichloromethane was treated at room temperature with 35 g of finely powdered dipotassium hydrogen phosphate and 15 ml of phosgene in 100 ml of methylene chloride. The mixture was allowed to warm to room temperature, was stirred overnight, and was then decanted through glass wool; the methylene chloride was evaporated and the residue was distilled at 59-60°/137 mm (18 kPa). Infrared (liquid film) peaks were
Abbreviation: NMR, nuclear magnetic resonance. * Present address: Department of Chemistry, Harvey Mudd College, Claremont, Calif. t To whom requests for reprints should be sent. t L. J. Crane, unpublished results. 1406
Proc. Natl. Acad. Sci. USA 73 (1976)
Chemistry: Chowdhry et al. at: 2165, 1750 (broad), 1135 (broad) cm-. Exact mass of the s5CI molecular ion: Calculated, 171.9651; found, 171.9662. Crude undistilled acid chloride from 6.75. g of trifluoroethylamine hydrochloride was stirred in dichloromethane and tetrahydrofuran with 4.0 g of anhydrous sodium p-nitrophenoxide for 24 hr. After the mixture had been filtered and the solvent evaporated, the residue was chromatographed on neutral alumina (100 g, Woelm grade V) and eluted with carbon tetrachloride. The ester, which came through with the solvent front, crystallized on evaporation of the solvent; it was sublimed at 50-70°/10-3 mm (0.13 Pa) and recrystallized from cyclohexane-ligroin; mp 630. Infrared (KBr) peaks were at 2165, 1740 cm-1. Elemental analyses for C, H, and N agree with theory to 40.2%. The ethyl ester§ was prepared from 1.0 g of distilled diazotrifluoropropionyl chloride and 0.5 g of methanol with 0.75 g of quinoline. The mixture was allowed to stand overnight; the ester was extracted into ether and washed with acidified brine and then with bicarbonate solution. After the solvent had been evaporated, the ester was chromatographed on grade III alumina in petroleum ether and then distilled at 66.5°/75 mm (10 kPa). Infrared (liquid film) peaks were at 2165, 1730 cm-1. Elemental analyses for C, H, N, and F agree with theory to ±0.2%. 2-Diazo-3,3,3-trifluoro-N-acetylcysteine methyl ester was prepared (although in very poor yield) from 0.575 g of Nacetylcysteine methyl ester (22, 23), 0.560 g of 2-diazo-3,3,3trifluoropropionyl chloride, and 0.348 g of 2,6-lutidine in 30 ml of tetrahydrofuran overnight at room temperature. The pale yellow crystals obtained from methylene chloride-hexane were sublimed at 85°/0.1 mm (13 Pa); mp 96-97'. Analysis for C, H, and N were within 0.2% of theory. The compound shows a strong diazo band at 2092 cm-1 and UV and NMR spectra consistent with the expected structure. Diazotrifluoropropionyl chymotrypsin was prepared by allowing 2.5 X i0-5 M enzyme to react with 5.5 X l0-5 M pnitrophenyl diazotrifluoropropionate at pH 5.95 at 300 for 1 hr. It proved to be 93% inactivated but reactivated completely on standing at pH 7.0 at room temperature for 10-12 hr. The diazoacyl enzyme solution has also been purified by passage over Sephadex G-25. (This preparation has been repeated by Dr. Richard Hudson in our laboratories.) Photolysis of Ethyl Diazotrifluoropropionate. The photolysis of 15-30 mg of ethyl diazotrifluoropropionate was carried out for 200-300 min in a Srinivasan-Rayonet reactor with RPR 2537 A lamps in 0.1-0.3 ml of methanol. After photolysis, the products were separated on a 120 X 0.31-cm column of 5% SF-1265 on Chromosorb G/AW/DWCS at 60° using a Hewlett-Packard HP5750 gas chromatograph. Alternatively, the methanol was evaporated, and the products were separated on a 360 X 0.31 cm column on 15% Carbowax 20M on Chromosorb P at 1150. The products of photolysis consisted of 75% or more of A (insertion into the solvent), 8% or less of B (ester exchange and insertion into the solvent), and about 15% of C (rearrangement). The structural assignments of A, B, and C are derived from (a) their NMR spectra, (b) their mass spectra, and (c) a transesterification experiment. The NMR spectrum of A showsT 6 1.21 (t, J = 7.0 Hz, 3 H), 3.47 (s, 3 H, ether OCH3), 4.04 §
A different preparation of this ester was reported at the American Chemical Society Middle Atlantic Regional Meeting, Fall, 1974, by A. Zampini and R. A. Shepard (Medicinal Chemistry and Bio-
chemistry Section, Paper no. 156). NMR resonances, 6, given in parts per million downfield from tetramethylsilane. For peaks, s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet.
1407
:t=o/
LLJ
z
m
t=24 r
\
02
220
260
300
340 nm
FIG. 1. UV spectra of 7 X 10-5 M 2-diazo-3,3,3-trifluoropropionyl N-acetyl cysteine methyl ester at zero time and after photolysis with 3500 A lamps for 24 hr.
(q, JF-H = 7.0 Hz, 1 H), 4.22 (q, J = 7.0 Hz, 2 H, ester methylene group). The spectrum of B shows 6 3.57 (s, 3 H, ether methoxyl), 3.86 (s, 3 H, ester methoxyl), 4.16 (q, JF-H = 6.5 Hz, 1 H), and that of C shows 6 1.26 (t, J = 7.0 Hz, 3 H), 3.70 (m, 2 H, diastereotopic ether methylene group), 3.84 (s, 3 H, ester methoxyl), 4.24 (q, JF-H = 6.5 Hz, 1 H). The mass spectra are dominated by the ions obtained by the McLafferty rearrangement (24, 25). In analogy with the mass spectrometry of the esters of methoxyacetic.acid (26)the ether groups of A, B, and-C are lost, presumably as the corresponding aldehydes. Thus A, a methyl ether, lost CH2O to yield an ion of mass 156.03904 (calculated for CH7F302, 156.03982), while B, also a methyl ether, lost CH2O, and C, an ethyl ether, lost CH3CHO to yield ions of measured mass 142.02707 and 142.02685, respectively; calculated for C4H5F302, 142.02417. The formation of the products of the McLafferty rearrangement unambiguously confirms the NMR assignment of A as an ethyl ester, and of B and C as methyl esters, since these groups survive in the ion beam. The transesterification experiment was carried out by heating B in completely deuterated methanol and 1% sulfuric acid. The NMR peak at 6 3.4 in CD30D as solvent disappears, while that at 6 3.14 is unchanged; these peaks correspond to those at 6 3.86 and 3.57 in CDCI3. This experiment, too, confirms the structural assignments made above. Photolysis of Methyl 2-Diazo-3,3,3-trifluoropropionylN-acetylcysteine. Photolysis of 3-4 mg of methyl 2-diazo3,3,3-trifluoropropionyl-N-acetylcysteine, dissolved in 100,ul of dry methanol, was conducted in quartz tubes in a Srinivasan-Rayonet reactor with either RPR 2537 A or RPR 3500 A lamps. Gas chromatographic analysis of 2 gl samples was conducted with a Hewlett-Packard 5750 gas chromatograph on a 180 X 0.31-cm column packed with 3% Dexil 400 GC/ Chromosorb W/HP 80/100. About 20 injections yielded enough material to obtain 1H Fourier transform NMR spectra (about 2000 transients). Photolysis with 3500 A Lamps for 18 hr led to the disappearance of the diazo band at 256 nm, whereas the intensity of the thioester band decreased (Fig. 1) by 60% but then held constant. Gas chromatographic analysis of the product led to two well-separated compounds, D and E, in the ratio 6:4 (retention times, 6.2 and 6.8 min; 40 ml/min helium carrier gas; temperature programmed 110°-170° at 20°/min). Mass:charge ratio for D was 317.05337; for E, 317.05483; calculated for
1408
Proc. Natl. Acad. Sci. USA 73 (1976)
Chemistry: Chowdhry et al.
This work was supported by the Institute of General Medical Sciences of the National Institutes of Health under Grant no. GM04712. The 100 MHz NMR spectrometer was purchased under National Science Foundation facilities Grant GP30965X to the Department of Chemistry. The authors are indebted to the National Institutes of Health Mass-Spectrometry Facility (RR 00317 Division of Research Resources) for the exact masses quoted.
D
1. Singh, A., Thornton, E. R. & Westheimer, F. H. (1962) J. Biol.
Chem. 237, PC 3007-3008. 2. Shafer, J., Baranowsky, P., Laursen, R., Finn, F. & Westheimer, F. H. (1966) J. Biol. Chem. 241, 421-427. 3. Hexter, C. S. & Westheimer, F. H. (1971) J. Biol. Chem. 246,
2.0
4.0
3928-3933.
4. Hexter, C. S. & Westheimer, F. H. (1971) J. Biol. Chem. 246,
3934-3938.
E
00H3
(ester)_
5. Stefanovsky, Y. & Westheimer, F. H. (1973) Proc. Natl. Acad. Sci. USA 70,1132-1136. 6. Vaughan, R. J. & Westheimer, F. H. (1969) Anal. Biochem. 29,
OCH3 1(ether)
H20
IA 42 20 FIG. 2. Comparison of the 100 MHz 'H NMR spectra of compound D (the thioether produced by rearrangement of the 2-diazo3,3,3-trifluoropropionyl thioester of N-acetyl cysteine methyl ester) and compound E (the thioester produced by insertion into the solvent). The double peaks at high resolution arise because the products are mixtures of diastereomers.
CIoH14FSNO5S: 317.05449. The critical region of the 100 MHz
1H NMR spectra is shown in Fig. 2. Compound D shows two tightly spaced methoxy signals at 6 3.78 and 3.81; the further, barely resolved doubling of the signals in both spectra presumably arises because each structural formula represents two diastereomers. The spacing between the methoxyl signals from E (6 3.62 and 3.71) and comparison of the spectra with those for the products of the photolysis of ethyl diazotrifluoropropionate identifies E as the methyl ether methyl ester thioester shown in Scheme 2. This structural assignment is confirmed by UV spectroscopy; E shows the strong absorption at 236 nm typical of thioesters (27-29), whereas A shows only the end absorption of thioesters (30, 31). Both D and E are stable to prolonged irradiation at 350 nm. Photolysis with 2537 A Lamps for 125 min completely eliminated the absorptions of both the thioester and diazo functions. When, however, the photolysis was carried only partially to completion, both UV and gas chromatographic analyses showed that 3-8% of E was present; on further irradiation with 2537 A lamps, the thioester E is rapidly destroyed. To isolate the insertion product, then, photolysis of the diazotrifluorothiopropionate must be carried out at long wavelength. Stability. A solution of ethyl diazotrifluoropropionate is stable for 24 hr in 1 M HCl. [Trifluorodiazoethane is also remarkably acid stable (32).]
305-310. 7. Vaughan, R. J. & Westheimer, F. H. (1969) J. Am. Chem. Soc. 91,217-218. 8. Fleet, G. W. H., Knowles, J. R. & Porter, R. R. (1969) Nature 224, 511-512. 9. Richards, F. F. & Converse, C. (1969) Biochemistry 8,4431-4436. 10. Sonneberg, N., Zamir, A. & Wilchek, M. (1974) Biochem. Biophys. Res. Commun. 59,693-696. 11. Galardy, R. E., Craig, L. C., Jamieson, J. D. & Printz, M. P. (1974) J. Biol. Chem. 249,3510-3518. 12. Escher, E. & Schwyzer, R. (1974) FEBS. Lett. 46,347-350. 13. Katzenellenbogen, J. A., Johnson, H. J., Jr. & Meyers, H. N. (1973)
Biochemistry 12,4085-4092.
14. Guthrow, C. E., Rasmussen, H., Brunswick, D. J. & Cooperman, B. S. (1973) Proc. Nati. Acad. Sci. USA 70,3344-3346. 15. Singer, S. J. (1967) Adv. Protein Chem. 22, 1-54. 16. Knowles, J. R. (1972) Acc. Chem. Res. 5, 155-160. 17. Creed, D. (1974) Photochem. Photobiol. 19,459-462. 18. Cooperman, B. S. (1976) Proc. Int. Conf. on Proteins and Other Adducts to DNA, ed. Smith, K. C. (Plenum Press, New York), in press. 19. Chaimovich, H., Vaughan, R. J. & Westheimer, F. H. (1968) J. Am. Chem. Soc. 90,4088-4093. 20. Hixson, S. S. & Hixson, S. H. (1972) J. Org. Chem. 37,1279-1280. 21. Gilman, H. & Jones, R. G. (1943) J. Am. Chem. Soc. 65,14581460. 22. Jones, J. B. & Wigfield, D. C. (1966) Can. J. Chem. 44,25172523. 23. Kupchan, S. M., Gicobbe, T. J., Krull, I. S., Thomas, A. M., Eakin, M. A. & Fessler, D. C. (1970) J. Org. Chem. 35,3539-343. 24. Gilpin, J. A. & McLafferty, F. W. (1957) Anal. Chem. 29,
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25. Budzikiewicz, H., Djerassi, C. & Williams D. H. 26.
27. 28. 29. 30. 31. 32.
