3-Trifluoromethyl-3-phenyldiazirine

THEJOURNALOP BIOLOGICAL CHEMISTRY Vol. 255,No 8, Iswe of April 25, pp. 3313-3318. 1980 Prmted Ln 1I.S.A.

3-Trifluoromethyl-3-phenyldiazirine A NEW CARBENE GENERATING GROUP FOR PHOTOLABELING REAGENTS* (Received for publication, September 20, 1979)

Josef Brunner,S Hans Serin,§ and FredericM. Richards From the Department

of

Molecular Biophysics and Biochemistry, Yale Uniuersity, New H a r m , Connecticut 06520

The synthesis of 3-trifluoromethyl-3-phenyldiazirine insertion of an aryl nitrene into an aliphatic C-H bond is (TPD) is reported in an overall yield of 60% based on likely to be one of the slowest processes. In addition, it has 2,2,2-trifluoroacetophenone as starting material. TPD recently been recognized that aryl azides are rapidly reduced is rapidIy photolyzed on irradiation near 350 nm to to aminesby thiols a t room temperature andphysiological pH yield 35% of the diazoisomer and 65%of the correspond- (3,4).This may represent a disadvantage since thiolsare often ing carbene. No internal rearrangement of the latter added to buffer solutions used in biochemical studies. compound by fluorinemigration was detected. PhotolAfter the pioneering work of Singh et al. (5) on photochernysis in methanol yielded the product of a formal OH ical labeling with diazoacetates, it appeared that many carinsertion in greater than 95%yield. Photolysis in cyclo- benes suffered from marked competition between bimolecular hexane gave at least a 50% yield of the CH insertion insertion andinternalrearrangement reactions. Therearproduct at a diazirine concentration of15 mM. The rangement products, such as ketenes, were frequently very products were identified by gas chromatographic mass spectra and by I9F NMR spectra. In the dark the diazir- reactive and resulted in unintended labeling by paths unreine is stable in 1 M acid or base and at temperatures as lated to the original photochemical product. More recently, high as 75°C for at least 30 min. The diazoisomer is new carbene-generating reagents have been prepared. Howmuch less photolabile and is stable in 0.1 M acetic acid ever, some problems still remain. Diazo-3,3,3-trifluoropropionconditions for for at least 12 h. The synthesis of a derivative of TPD ates (6) suffer fromthe disadvantage that harsh containing the 2-hydroxyethyl[O-tosylate]group on the photolysis are required. Potential advantages are offered bv para position of the phenyl ring is described. This com- p-toluenesulfonyl diazoacetates( 7 ) since t,he molar extinction pound permits the easy attachment of the TPD function coefficient at 370 nm is approximately 10 times that for the to other molecules. It is suggested that the ease of long wavelength absorption of other diazoesters and this persynthesis, dark stability, rapid photolysis, and reactiv- mits reasonably rapid photolysis in the near ultraviolet. ity of the carbene may make this group useful in the As precursors for carbenes, the photolabile diazirine group preparation ofvarious photochemical probes. holds much promisesince,unlike most of the diazoesters, diazirines are relatively stable in acid. In addition, theycan be photolyzedveryefficiently at wavelengths around 360 nm where most of the biological molecules do not absorb radiaThe main properties desired of a photoactivatable group tion. Bayley and Knowles (8)introduced 3H,3-phenyldiazirine are chemical stability prior to photoactivation, rapid photol- and spiro-(adamantane-2,2’-diazirine) as probes for the hydroysis at appropriate wavelength, and a very high reactivity of phobic core of membranes and many of the advantages o f the photogenerated species (1, 2). None of the photolabile carbenes over nitrenes were illustrated. One complication groups currently in use meet these requirements fully and it discovered so far derives from the fact thatphotolysis generis clearly desirable to have severaldifferent photoactivatable ates the linear diazoisomer as well as the carbene. Although groups available to facilitate a judicious choice depending on these can be photolyzed at suitable wavelength to generate the particular application being considered. the carbene, diazo compounds are in general highly sensitive Inmuch of the work involving aryl azides, it has been to protonation and subsequent nucleophilic attack. Phenyldiexplicitly or implicitlyassumed that, once generated,the azomethane, the isomer of 3H,3-phenyldiazirine has indeed nitrene reacts rapidly andnonspecifically with its immediate been used to alkylate carboxyi groups of carbonic anhydrase environment including aliphatic residues. However, aryl ni- (9). A transient accumulation of the diazoisomer during photrenes mayin fact not react rapidly on a molecular time scale, tolysis could therefore lead to photochemically irrelevant many havinghalf-lives in the millisecond timerangeand labeling. Since photoisomerization of diazirines appears to be a gen* This work was supported by the United States Public Health eral phenomenon(lo),diazirines which generate diazoisomers Service through Grant GM-21714 to the Membrane Research Center more stable than phenyldiazomethane therefore deserve ina t YaleUniversity. Thecosts of publication of thisarticle were vestigation. This study describes the synthesis and an invesdefrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 tigation of such a reagent, 3-trifluoromethyl-3-phenyldiazirine U.S.C. Section 1734 solely to indicate this fact. and several of its phenyl-substituted analogues. The isomeric $ Supported by fellowship from the Swiss National Science Founhas diazo compound, l-phenyl-2,2,2-trifluorodiazoethane, dation. Present address, Eidgenossische Technische Hochschule ZUbeen found to be reasonably stable in acid solution ( 11) such rich Laboratorium fur Biochemie, Universitatstrasse 16, CH-8092 that side reactions notinvolving carbene intermediatesshould Zurich, Switzerland. 5 Present address, Eidgenossische Technische Hochschule Zurich- be markedly reduced. The investigation of this diazirine was Honggerberg, Institut fur Molekularbiologie und Biophysik, CH-8093 further encouraged by reports in literature which indicated a Zurich, Switzerland. simple synthetic route.

3313

3314

New Carbene Precursor for Photolabeling EXPERIMENTAL PROCEDURES' RESULTSANDDISCUSSION

Synthe.si.~-3-Trifluoromethyl-3-phenyldiazirineandanalogues which contain in para position various reactive derivatives of a hydroxyethyl groupwere synthesized. The reaction sequence is summarized in Fig. 1. The diaziridines 4 and 10 were obtained by treatment of the corresponding O-p-tosyloximes 3 and 9 with ammonia according to Zeifman et al. (12). Oxidation of the diaziridines with silveroxide gave the diaziroxidation reaction ines 5 and 11. Both the amination and resulted in high yields of the products. The ease with which 0-tosylates of trifluoroacetophenone oximes canbe converted to diaziridines is remarkable and in contrast to the behavior 3, R=H of 0-tosyloximes of benzaldehydes which are inert to further 9, R = X reaction with methanolic or liquid ammonia (10). The prepaR I x = +~,-O-CH2-CH2ration of 3H,3-phenyldiazirines requires alternative strategies to form the intermediatediaziridine, and in general very poor yields are obtained (10). 4, R-H Substituted 3-trifluoromethyl-3-phenyldiazirinescontain3 IO, R = X ing additional functional groups such as amine, sulfhydryl, carboxyl, etc. are of special interest since these canbe utilized for the covalent attachment of the TPD' moiety to other molecules. The tosylate13 represents sucha key intermediate 5, R=H which is useful for further derivatization (13). The synthesis II, R z X of this compound involved the initial protection of the hyR droxyl group of p-bromophenylethanol as the dimethyl-t-butylsilylether(14) and subsequently the introduction of the trifluoroacetyl group via the Grignard intermediate. The procedure for thislatterstephas beendescribed for various substituted benzenes (15)and was successfully adapted in this HO" 12 I3 case. It is noteworthy, however, that the Grignard reagent of p-bromophenylethanol could not be obtained when the hySCHEME 1 droxylfunction was protectedasthetetrahydropyranoyl FIG. 1. 3-Trifluoromethyl-3-phenyldiazirine. Reaction ether. scheme for the synthesis of 3-trifluoromethyl-3-phenyldiazirine,5, Properties of the 3-Trifiuoromethyl-3-phenyldiazirine and of phenyl-substituted analogues, 11 to 23. group-TPD has reasonable thermal stability.It can be stored in the dark a t room temperature for weeks or heated to75°C I.( 1 I I I 1 I 1 for at least 30 min with little or no apparent change. Solutions 0. : in methanol containing 1 M HCl or 1 M NaOH are stable at mmo'or room temperature for at least 2 h. Furthermore, TPD is inert 0.f toward mild reducing agents; thus, a 5 mM solution in a 0.2 M glycine buffer containing 50% methanol,pH 9.8, is not 0.i changed by 100 mM dithiothreitol overaperiod of24 h. Similar conditions, however, have been reported to rapidly $0.6 reduce the photosensitivephenylazide (3). It appears that c T P D is stable undera variety of conditions that indude those 0 0.5 anticipated for its use in biochemical labeling reagents. 0 ul Photolysis-The UV spectrum of T P D is shown in Fig. 2 2 0.4 together with the isomeric l-phenyl-2,2,2-trifluorodiazoethane (16). The characteristic absorption of the diazirine centered 0.3 at 353 nm ( E = 266) permits rapid photolysis with near UV 0.2 radiation. With a sample approximately 4 cm from the jacketed 450-watt mercury lamp, half of the diazirine disappeared 0.I within 25 s while the half-life of the diazoisomer was approximately 22 min. Photolysis of 3H,S-phenyldiazirine has been investigated previously (10) and the general photolytic pathway applies also forthe trifluoromethyl-substituted diazirine (Fig.3). PhoAbsorption specFIG. 2. 3-Trifluoromethyl-3-phenyldiazirine. toisomerization to the linear diazo compound is competitive tra of 3-trifluoromethyl-3-phenyldiazirine (solid 1zne.s) and theisomer

IF

e

(dashed lines).The con' The "Experimental Procedures" are presented in miniprint at the l-phenyl-2,2,2-trifluoromethyldiazomethane centration for each curve is indicated. The solvent was cyclohexane. end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, Maryland with carbene formation. Basedon spectroscopic measure20014. Request Document No. 79M-1921, cite author(s), and include ments of the disappearance of the diazirine and the initial a check or money order for $1.00 per set of photocopies. - The abbreviationused is: TPD, 3-trifluoromethyl-3-phenyldiazir- accumulation of the diazo compound, we have estimated that upon photolysis 35% of the diazirine is converted to the linear ine.

Precursor Carbene New

for Photolabeling

3315

95% of the starting material. This estimationis based on the relative peak intensities of "'F NMR spectra (Fig. 4) and on gas chromatography. The structure of this compound corresponds to theformal 0 - H insertion product as shown in Fig. 3. An authentic sample of this compound synthesized by a L J different route gave an identical chemical shift when compared by "F NMR. The structure was finally confirmed by mass spectroscopy which gave the fragment patternshown in Fig. 5A. in cyThe photolysis of 3-trifluoromethyl-3-phenyldiazirine clohexane was of particular interest. Possible reactions of the carbene with the solvent are insertion intoa C-H bond and hydrogen abstraction followed by the recombination of the resultingradicals. The ability of a carbene (or nitrene) to insert into aliphaticC-H bonds is indicative of a n extremely reactive species and is commonly regarded as a prerequisite for photolabeling reagents. When a solution of TPD (15 mM) in cyclohexane is photolyzed for 90 min (essentially complete 95% -50% photolysis of the diazoisomer), the reaction product contains many components asrevealed by "F-NMR spectroscopy (Fig. "F-NMR. 0 I pprn 14.5ppm 6 a ) . Most of the resonances except one at 6 ppm (uersus (v5 CFJ COOH) trifluoroacetic acid) are split by 7 to 10 Hz indicating vicinal SCHEME 2 fluorine-proton coupling. That this proton originated from the FIG. 3. Photolysis scheme for 3-trifluoromethyl-3-phenyldi- solvent cyclohexane was confirmed with a sample of T P D azirine showing formation of the isomerl-phenyl-2,2,2-trifluowhich had been photolyzed in deuterated cyclohexane. The rodiazoethane and direct and indirect formation of the car- fluorine NMR spectrum observed with this sample is shown bene. The principal reaction products of the carbene with methanol in Fig. 6b. All doublets previously observed were absent, and cyclohexane are shown. consistent with the smaller coupling (-1 to 1.5 Hz) of fluorine with a vicinal deuterium. Estimation of the relative signal diazoisomer. This fraction appears to be independent of the intensities revealed that approximately 80% of the carbene solvent, since very similar numbers were obtained whether reacted in some way with the solvent. The main resonance at photolysis was performed in methanol or in cyclohexane. 14 ppm accounts for approximately 50%of the initial diazirine. In order to establish the structure of the principal product, We have measured thekinetics of the disappearanceof the diazirine (at 353 nm) and of an authentic sampleof l-phenyl- a sample of T P D photolyzed in cyclohexane was evaporated 2,2,2-trifluorodiazoethane(at 265 nm) upon photolysis. Three solvents were usedand the initial concentrations of the solutes varied between 0.5 and 3 mM for the diazirine and was approximately 0.05 mM for the diazo compound. In both 1,4dioxane andmethanol,firstorderkinetics were obtained. Marked deviation from first order was found however for the diazirine in cyclohexane. The curvature of the plot was dependent on the concentration of the diazirine. With decreasing concentration a better first order approximationwas obtained. Photolysis of the pure diazo compound at 0.05 mM ( t = 0 ) resulted in a time course for the disappearance indistinguishable from a first order process. Methanol and 1,4-dioxane differ from cyclohexane not only in their polarity but also in their chemical reactivity toward (singlet) carbenes. Singlet carbenes coordinate very rapidly with the occupied p-orbitals of the oxygens to form ylicis. These can subsequently rearrange to products which correspond to those of a formal 0 - H or C - 0 insertionor C - 0 displacement reaction (17). The lifetime of the carbene in cyclohexane is much longer due to lack of reactivity of the solvent. This however increases the probability for reaction with another molecule of unphotolyzeddiazirine. It seems plausible, therefore, that the deviation from afirst orderdecay in cyclohexane may simply be correlated with the formation of reagent condensation products which interfere with the absorption measurements at353 nm. 15 10 5 0 -5 -10 PPm Product Analysis-The reaction mixtures of photolysis of FIG. 4. '"F Nuclear magnetic resonance spectrum (Bruker the diazirine in methanol and cyclohexane have been anafrom the photolysis lyzed. Trifluoromethylphenylcarbene does not rearrange to CXPBOO) of the reaction products obtained form trifluorostyrene by fluorine migration. In no case did the of 3-trifluoromethyl-3-phenyldiazirine(-50 m ~ in) methanol. The principal signal a t 0.1 ppm was also recorded on an expanded 19 F NMR spectrum of the product mixture show any detect- scale (doublet, J = 7.4 Hz) andcorresponds to the formal 0 - H able quantity of trifluorostyrene (18). In methanol, one preinsertion product. Chemical shifts were calculated relative to 100%. dominantproduct is formed accounting for approximately CF.,COOH with DrO lock.

Precursor Carbene New

3316

Photolabeling for

and theyellow residue subjected to a bulb-to-bulb distillation in uucuo. The volatile material was used for further analysis. Thin layer chromatography on silica gel (1igroin:methylene loo] 80 A chloride = 9:l) gave a t least five bands which were detectable by fluorescence quenching or in iodine vapor. The most intense band under the UV lamp (RF-0.9) turned purple in 60 iodineindicating an inert compound of low polarity. This material was extracted with ether and after evaporation of the a colorless liquid which upon rechromatograsolvent yielded 40phy appeared as a single spot. The 'H NMR spectrum was consistent with that expected for the product of insertion of the carbene into a C-H bond of cyclohexane. The benzylic proton a t 3.14 ppm was split with an intensity ratioof 1:4:6:4: 1, corresponding to overlapping quartets, due to the three fluorines of the CF3 group and the1-H of the cyclohexyl ring. and ,?JH.H were 8 to 10 The measuredcoupling constants :'JH.F Hz. Aromatic protons were found a t 7.15 to 7.41 ppm and protons of the cyclohexyl moiety between 0.67 and 2.07 ppm. a 'O01 The intensity ratioswere consistent with this structure. Final results toc o n f i i t h estructure of the principal product obtained from the carbenein cyclohexane were provided by electron impact mass spectroscopy of the purified sample. This fragmentation pattern is shown in Fig. 5B. The most abundant species of mass 83.1 corresponds to the cyclohexyl ion. The 'F NMRspectrum of this compound gave one doublet at 14 ppm (J = 8 Hz) corresponding exactly to the chemical shift measured for the major component in the crude mixture obtainedfrom the photolysis of T P D in cyclohexane. While this product analysisdoes not suggesta detailed mechanism for the formation of the formal insertion products, a mechanism involving a triplet carbene seemsunlikely. Since photolyses were performed under an air atmosphere oxygen m/e presumably would have quenched a triplet carbene species. FIG. 5. Mass spectra of trifluoromethylphenylcarbene.Elec- This is consistent with the observation that in methanol the tron impact mass spectra of the principal reaction products of trifluoromethylphenylcarbene with methanol (A) and with cyclohexane ( B ) . product of a formal 0-H insertion reaction was formed in The major components were analyzed on a Hewlett-Packard model almost quantitative yield (-95%) and that little or no C-H insertion product was obtained (17). 5985 GC/MS system.

4

1

60140i

b

a I7HI

,

FIG. 6. Nuclear magneticresonancespectra of photolysis products. The "F NMR spectra (Bruker

I

CXPSOO) of the products of photolysis (90 min) of 3-trifluoromethyl-3-phenyldiazirine in cyclohexane ( a )and deuterated cyclohexane ( b ) .The principal signals were also recorded on an expanded scale and are shown above the corresponding resonances in the full trace. Chemical shifts were calculated relative to 100% CF,L!OOH with dr2-cyclohexane lock.

cc

m 1 6 14 12

10 8

642

0 -2 -4

ppn

16l4 1210 8

6

4

2 0 -2-4

Precursor Carbene New CONCLUSIONS

TPD appears to meet most of the criteria required for a photochemical labeling reagent for biological systems: 1) It is easily synthesized in high yield. 2) In the absence of light, it is stable under expected conditions of use, and under those likely to be required for synthetic incorporation into other types of reagents. 3) It is rapidly photolyzed in the near-UV where most cellular components do not absorb and yields the carbene as the principal initial species. 4) The diazoisomer which is the major side product of photolysis is reasonably stable in acid (11)and is expected not togive significant dark reaction products (for example, no reaction in cyclohexane containing 0.1 M acetic acid was detected in 24 h). 5) The photolytically produced carbene is very reactive isand capable of providingahigh yield of CH insertionproduct in the absence of competing reaction paths. Selectivity ratios have not yet been established, but it is alreadyclear that, as with all otheravailable photochemical reagents, totallack of specificity has not yet been attained. Acknoudedgments-Our sincere thanks to Dr. Ian Armitage for to David help in recording and interpreting the "F-NMR spectra and Pearson for recording the mass spectra. We also acknowledge the help of J. Mouning andM. Lane in the preparationof the manuscript.

REFERENCES 1. Bayley, H., and Knowles, J. R. (1977) Methods Enzymol. 46, 69-

114

for Photolabeling

3317

2. Chowdhrv. V..Westheimer. and F. H. (1979) Annu. Biochem. Rev. 48,293'1325 3. Staros, J. V., Bayley, H., Standring, D. N., and Knowles, J. R. (1978) Biochem. Biophys. Res. Commun. 80, 568-572 4. Czamecki, J., Geahlen, R., and Haiey, B. (1979) Methods Enzymol. 56,642-653 5. Singh, A,, Thornton, E. R., and Westheimer, F. H. (1962) J. Biol. Chem. 237, PC3006-3008 6. Chowdhry, V., Vaughan, R., and Westheimer, F. H. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 1406-1408 7. Chowdhry, V., and Westheimer, F. H. (1978) Bioorg. Chern. 7, 189-205 8. Bayley, H., and Knowles, J. R . (1978) Biochemistry 17,2420-2423 9. Foelsch, G. (1974) Chem. Scr. 6, 32-34 10. Smith, R. A. G., and Knowles, J. R. (1975) J . Chem. SOC. Perkin I I 686-694 11. Diderich, G. (1972) Hetv. Chim. Acta 55,2103-2112 12. Zeifman, Yu. V., Abduganiev, E. G., Rokhlin, E. M., and KnunSer. Khim. 12,2737yants, I. L. (1972) Izv. Akad. Nauk. SSSR 2741 13. Brunner, J., and Richards, F.M. (1980).J . Bioi. Chem.255,33193329 14. Corey, E. J., and Venkateswarlu, A. (1972) J . Am. Chem. Soc. 94, 6190-6191 15. Bergmann, E. D., Pelchowicz, Z., and Shani, A. (1963) Isr. J . Chem. 1, 129-135 16. Shepard, R. A., and Wentworth, S. E. (1967) J . Org. Chem. 32, 3197-3199 17. Kirmse, W. (1964) Carbene Chemistry, AcademicPress, New York 18. Dewar, M. J. S., and Kelemen, J. (1968) J. Chem. Phys. 49, 499508

3318

New Carbene Precursor

for Photolabeling