J. Am. Chem. SOC.1995,117, 3084-3095
3084
2-(Tributylstannyl)-4-[3-(trifluoromethyl)-3H-diazirin-3-yl] benzyl Alcohol: A Building Block for Photolabeling and Cross-Linking Reagents of Very High Specific Radioactivity Thomas Weber? and Josef Brunner* Contribution from the Department of Biochemistry, Swiss Federal Institute of Technology Zurich (ETHZ), ETH-Zentrum, CH-8092 Zurich, Switzerland Received October IO, 1994@
Abstract: A general approach for the synthesis of novel, radioiodinated photolabeling and cross-linking reagents at no-carrier-added (nca) specific radioactivity (> 2000 Ci/mmol) is described. In this approach, 2-(tributylstanny1)4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzylalcohol 12 serves as a module which, through acylation of its alcohol function, is connected to other structuraVfunctional elements to make the tin-containing precursor forms of the reagents. The final, radioactive compounds are then conveniently prepared in yields varying from 15-50% by electrophilic aromatic substitution of the tributylstannyl group by 1251+ generated in situ from lZ5I- by peracetic acid. Using this approach, we have synthesized two analogues of phosphatidylcholine (PC) (20 and 21), an analogue of ceramide (36), as well as two heterobifunctional label-transfer cross-linkers (29 and 33). PC 21, examined more closely, is a substrate of the PC-specific phospholipid exchange protein from beef liver and of phospholipase D from cabbage. The latter was utilized to enzymatically convert PC 21 into two additional phospholipids, phosphatidylserine (PS) 25 and phosphatidic acid (PA) 26. Reagents that contain this iodinated photophor also combine desirable photochemical properties. Thus, evidence is presented that they undergo efficient photolysis to generate a (singlet) carbene intermediate which inserts efficiently into hydrocarbon CH bonds. In addition, we demonstrate that the diazirine can be photolyzed in a selective manner, that is, without photodeiodination to occur to a significant extent.
Introduction Photo(affinity) labeling and cross-linking refer to a variety of important biochemical techniques to investigate structural and functional properties of biological systems.' These techniques make use of reagents which, after targeting to a biological system or component, can be activated with UV light to generate highly reactive intermediates capable of forming covalent bonds with adjacent molecules. Labeling reagents can be divided into three main classes: first, probes designed to report on general properties of a system (for example, small apolar molecules and lipids which partition into membranes and, upon activation, label selectively integral proteins); second, affinity labeling reagents intended to interact with and label components (receptors) in a specific, functionally relevant manner; and, third, heterobifunctional (photo)cross-linkers, containing a photoactivatable and a conventional (thermal) reactive function, preferentially connected via a cleavable linker. These latter reagents are primarily used to study the spatial relationship between components in complex systems, or to make macromolecular affinity probes. Most of the photoreagents developed to date are based on aryl azide or nitroaryl azide chemistry. However, unlike what
* Author to whom correspondence should be addressed at Laborato-
rium fur Biochemie, ETHZ, Universitatstrasse 16, CH-8092 Zurich, Switzerland. Tel.: +41-1-632 3003. Fax: f41-1-632 1269. E-mail: bcgraf@
aeo1us.vmsmail.ethz.ch. Present address: Cellular Biochemistry and Biophysics Program, Rockefeller Research Laboratories, Sloan-Kettering Institute, 1275 York Avenue, New York, NY 10021. @Abstractpublished in Advance ACS Abstracts, March 1, 1995. (1) For reviews see: (a) Knowles, J. R. Acc. Chem. Res. 1972, 5, 155160. (b) Bayley, H.; Knowles, J. R. Methods Enlymol. 1977,46, 69- 114. (c) Peters, K.; Richards, F. M. Annu. Rev. Biochem. 1977, 46, 523-551. (d) Chowhdry, V.; Westheimer, F. H. Annu. Rev. Biochem. 1979, 48, 2923325. (e) Ji, T. H. Biochim. Biophys. Acta 1977,559,39-69. (f)Bayley, H. Photogenerated Reagents in Biochemistry and Molecular Biology; Elsevier Science Publishers B.V.: Amsterdam, The Netherlands, 1983.(g) Gaffney, B. J. Biochim. Biophys. Acta 1985, 822, 289-317. (h) Brunner, I. Annu. Rev. Biochem. 1993, 62, 483-514.
had been assumed earlier, aryl azides or nitroaryl azides do not react via short-lived nitrene intermediates, but instead rapidly undergo ring expansion to dehydroazepines. The latter tend to be trapped by nucleophiles such as amines, or, in the absence of nucleophiles, undergo poorly defined polymerization reactiom2 More suitable in this respect are fluoro- and chlorophenyl azide^.^ When activated in hydrocarbon solvents, the intermediate nitrenes insert intermolecularly into CH bonds, the most desired type of reaction also for establishing a covalent bond with a target biopolymer. Another class of reagents are those which upon activation generate a carbene intermediate. Among these, 3-(trifluoromethyl)-3-aryldiazirinesappear to come closest to satisfying the chemical and biological criteria required for most photo reagent^.^ This functionality is easily synthesized in high yield and, in the absence of UV light, is remarkably stable under a range of different physical and chemical conditions, including heat (80 "C), strong bases, strong acids, oxidizing conditions and mild reducing agents. Upon irradiation with light at around 350 nm, the diazirine is rapidly photolyzed to generate a carbene capable of reacting with the full range of functional groups occurring in biomolecules, including paraffinic ( 2 ) (a) Chapman, 0. L.; LeRoux, 1.-P. J. Am. Chem. SOC.1978, 100, 282-285. (b) Schrock, A. K.; Schuster, G. B. J. Am. Chem. Soc. 1984, 106,5228-5234. (c) Liang, T.-Y.; Schuster, G. B. J. Am. Chem. SOC.1987, 109, 7803-7810. (d) Li, Y.-Z.; Kirby, J. P.; George, M. W.; Poliakoff, M.; Schuster, G. B. J. Am. Chem. Soc. 1988, 110, 8092-8098. (3) (a) Keana, J. F. W.; Cai, S. X. J. Org. Chem. 1990, 55, 3640-3647. (b) Cai, S. X.; Glenn, D. J.; Keana, J. F. W. J. Org. Chem. 1992,57, 12991304. (c) Schnapp, K. A.; Poe, R.; Leyva, E.; Soundararajan, N.; Platz, M. S. Bioconjugate Chem. 1993, 4, 172-177. (d) Soundararajan, N.; Liu, S. H.; Soundararajan, S.; Platz, M. S. Bioconjugate Chem. 1993,4, 256-261. (e) Cai, S. X.; Glenn, D. J.; Gee, K. R.; Yan, M. Y.; Cotter, R. E.; Reddy, N. L.; Weber, E.; Keana, J. F. W. Bioconjugate Chem. 1993, 4,545-548. (4) (a) Brunner, J.; Senn, H.; Richards, F. M. J. Biol. Chem. 1980, 255, 3313-3318. (b) Nassal, M. J. Am. Chem. Soc. 1984,106,7540-7545. (c) Shih, L. B.; Bayley, H. Anal. Biochem. 1985,144, 132-141. (d) Platz, M.; Admasu, A. S.; Kwiatkowski, S.; Crocker, P. J.; Imai, N.; Watt, D. S. Bioconjugate Chem. 1991, 2, 337-341. (e) Delfino, J. M.; Schreiber, S. L.; Richards, F. M. J. Am. Chem. Soc. 1993, 115, 3458-3474.
0002-7863/95/1517-3084$09.00/0 0 1995 American Chemical Society
J. Am. Chem. Soc., Vol. 117, No. 11, 1995 3085
Photolabeling and Cross-linking Reagents CH bonds. While most of the covalent adducts thereby formed are stable under a wide range of conditions, insertion into NH and OH bonds may lead, in some cases, to less stable products, a point to consider in the analysis of labeled target molecules.4bsd For most applications, photolabeling reagents must be available in an isotopically labeled form of (very) high specific radioactivity. A widely employed strategy to realize this task is to introduce one of the common radioisotopes of iodine, 1251 or 1311, a y- and a strong /?-emitter, respectively. Unfortunately, however, UV-irradition of most of these reagents is accompanied by photolytic liberation of iodine. This is a severe shortcoming, since iodine (radicals), in turn, can be trapped by biological targets resulting in their unspecific labeling.5 Toward the development of photophors with improved properties, iodinated azidoperfluorobenzoates and 4-azido-3,5,6-trifluorobenzoates have recently been examined.3b When photolyzed in cyclogave the hexane, methyl 4-azido-2-iodo-3,5,6-trifluorobenzoate expected CH insertion product in 12% yield, together with substantial amounts (8%) of the deiodinated counterpart and other products. Placement of the iodine atom into a ring different from the phenyl azide ring has not led to a significant improvement. Another recent class of potentially useful reagents are iodinated 3-(trifluoromethyl)-3-(alkoxy)phenyldiazirines.6 However, their photochemical properties have not yet been determined. A further question concerning both iodinated peffluorophenyl azides and (trifluoromethyl)-3-(alkoxy)phenyldiazirines is whether the iodination reactionstconditions can be adopted to prepare reagents of high specific radioactivity. Radioiodine can hardly be obtained and manipulated at those (iodine) concentrations (molar range) used to prepare the nonradioactive materials. In this article, we now report the synthesis of several members of a novel familiy of radioiodinated photolabeling and crosslinking reagents which can easily be prepared at the nca specific radioactivity of lZ5I (> 2000 CUmmol) and which also display favorable photochemical properties. Central in this work was the development of the building block 12. Tin-containing photoreagents synthesized from this module can be converted into the final reagents by electrophilic displacement of the tributylstannyl group by lZ5I+. In contrast to the photochemical performance of iodinated aryl azides, the diazirine can be photolyzed without significant loss of iodine.
Scheme la 1: R'= Br;
R1= B r ;
R2 = TDS
R1= C-CF3;
R2 = TDS
4
R'= C-CF3; N-OH
R2 = TDS
5:
R1= C-CF3; N- OTOS
R2 = TDS
6
R1= ,C,-CF3; HN-NH
R2 = TDS
7:
R'= ,C,-CF3; N-N
R2 = TDS
8
R1= ,C,-CF3 ; N-N
R2 = H
"'2 b C3 R' d L
eL fc
O 'R2
R2=H
(a) TDS chloride/imidazole/DMF; (b) 1. Mg/THF/reflux, 2. (trifluoracetyl)piperidine/O "C, 3. saturated NH&l; (c) hydroxylamine/ pyridineheflux; (d) tosyl chloride/DMAP/EJt3N/CHzClz; (e) NH3 under pressurelEtZ0; (f)Iz/triethylamine/MeOH; (g) HCl/MeOH.
Scheme 2"
R'
E
9
R'=I;
R2=H
10
R1=l;
R2 = C(0)Me
11:
R' = Sn(n-Bu)~;R2 = C(0)Me
12
R' = Sn(n-Bu)~;R2 = H
(a) T1(CF3S03)3/trifluoroaceticacid80 "C; (b) aqueous NaI; (c)
Results and Discussion Design and Synthesis of 2-(Tributylstannyl)-4-[3-(trifluoromethyl)-3H-diazirinin-3-yl]benzyl Alcohol (12). Our original idea was to incorporate radioiodine through aromatic thallationl iodination of an appropriately functionalized 3-phenyl-34trifluoromethyl)diazirine, an approach analogous to that taken recently for the preparation of (nonradioactive) 3-(akoxyiodophenyl)-3-(trifluoromethyl)diazirines.6Because of undesirable quenching reactions expected for ortho-substituted arylcarbenes,' we planned to position the substituents meta ortand para to the 3-(trifluoromethyl)-3H-diazirinyl group. As the parent molecule we have chosen 4-[3-(trifluoromethyl)-3Hdiazirin-3-yllbenzyl alcohol 8. Acylation of the alcohol function was viewed to offer a convenient way to introduce additional structural or functional elements and, specifically, to provide a basis for the preparation of alkali-cleavable label-transfer (5) (a) Watt, D. S.;Kawata, K.; Leyva, E.; Platz, M. S. Tetrahedron Lett 1989, 30, 899-902. (b) Koch, T.; Suenson, E.; Korsholm, B.; Henriksen, U.; Buchardt, 0. Bioconjugate Chem. 1994, 5, 205-212.
(6)Hatanaka, Y.; Hashimoto, M.; Kurihara, H.; Nakayama, H; Kanaoka, Y. J. Org. Chem. 1994, 59, 383-387. (7) Kirmse, W.; Konrad, W.; Schnitzler, D. J. Org. Chem. 1994, 59,
3821-3829.
Ac20/DMAP/THF; (d) hexabutylditinln-Bu$JF/KH~PO~/50 "C; (e) NH3/MeOH/Et20; (f) Na12SI/peraceticacidAcOH. reagents (see below). Diazirine 8 was prepared by means of a combination of reported procedures (Scheme 1). For transient protection of the alcohol the (1,1,2-trimethylpropyl)dmethylsilyl (TDS), rather than the previously used dimethyl-tert-butylsilyl (DTBS) group, was employed. This provided a significant advantage during purification of diazirinylbenzyl alcohol 8, which, following the deprotection reaction (Scheme 1, step g), can be more easily separated from TDS-OH than from DTBSOH. Functionalization of benzyl alcohol 8 was achieved through reaction with thallium(III) -(trifluoromethane)sulfonate,an agent applied previously to thallate strongly deactivated perfluoroarenes.* Treatment of the reaction product mixture with an excess of iodide then afforded 2-iodo-4-[3-(trifluoromethyl)3H-diazirin-3-ylIbenzyl alcohol 9 in 63% yield (Scheme 2). The position of the iodine atom meta to the diazirine group was confirmed by NMR spectroscopy. The 19F NMR spectrum showed a triplet with a coupling constant of J = 0.7 Hz, (8) Deacon, G. B.; Tunaley, D. J. Fluorine Chem. 1977, 10, 177-180.
Weber and Brunner
3086 J. Am. Chem. SOC.,Vol. 117, No. 11, 1995 consistent with two protons ortho to the diazirinyl group. In addition, the HCC-correlation-2D-NMR spectrum of acetate 10 showed coupling between the benzylic protons and three aromatic carbons, of which only one bears a H atom. In turn, only one aromatic proton shows long-range coupling with the benzylic carbon atom. Although the above pathway could, in principle, be used to introduce 1251or 1311, at the low concentrations (xO.1 mM iodine) at which these isotopes are accessible, the reaction turned out to be too sluggish to be of practical utility. Moreover, this approach would not permit the radioisotope to be introduced in the very last step in the synthesis of actual cross-linking reagents (see below). The above results prompted us to examine an alternative approach, iododestannylation. The use of organotin chemistry for the preparation of radiohalogenated (and tritiated) organic compounds is well established, and for a variety of situations, in which high specific radioactivity, rapidity of incorporation and site-specificity of labeling are required, electrophilic destannylation is the method of c h ~ i c e .The ~ requisite [(trialkylstanny1)arylldiazirine (12) was synthesized in 36% yield by reaction in THF of aryl iodide 10 with hexabutylditin in the presence of n-Bu4NF as a catalyst.'O To prevent (extensive) cleavage of benzyl ester 10 during the stannylation reaction, solid KH2P04 was added to the reaction mixture." Treatment of 11 with methanolic ammonia then afforded the free alcohol 12 in 97% yield.
2-Iodo-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]be~yl Acetate: Radioisotopic Labeling. To evaluate the suitability of 12 as a building block for photoreagents, acetate 11 was used
300
350
400
4 io
Figure 1. UV-vis spectra of the reaction products from photolysis acetate (10). of 2-iodo-4-[3-(trifluoromethyl)-3H-diaz~n-3-yl]benzyl A solution (2mM) in cyclohexane was inradiated with filtered LJV light (1> 320 nm) for increasing periods of time (in seconds) as indicated with numbers. The spectra show the decay of the diazirine characteristic absorption with a maximum at 35 1 nm. The inset shows the decay as a function of time of photolysis in a semilogarithmic plot.
as a model compound. In the presence of peracetic acid as an oxidant, (radio)iododestannylation of 11 proved both efficient and experimentally very simple. In fact, 2 min after addition of peracetic acid to a dilute solution of 11 (1 mM) and [lZ5I]Scheme 3" NaI (0.05 mM) in AcOH, the reaction was essentially complete and gave 10 in 50-60% yield (Scheme 2, step f). This R R suggested that a broad diversity of photoreagents can be synthesized as stable, tin-containing precursor forms, which, prior to use, are then easily and rapidly converted into the final radioiodinated reagents. Photochemical Properties. Consistent with the spectral properties of other 3-(trifl~oromethyl)-3-phenyldiazirines$~~~~ 14: R = l 10 R = l the iodinated diazirines described here show a characteristic 15: R = H 13: R = H absorption at around 350 nm. This is depicted in Figure 1 for Cyclohexanelhv (1> 320 nm). model compound 10 (Scheme 3), which was chosen for some photolysis studies. cyclohexane (14) (Scheme 3). The isolated yield of 14 was When a 2 mM solution of 10 in cyclohexane was irradiated approximately 25%. This product also corresponds to the largest with the filtered light from a high-pressure mercury lamp, the peak (estimated 48%) in the gas chromatogram (Figure 2A). diazirine absorption at 351 nm decreased with a half-life period To examine whether photolysis of diazirine 10 is accompanied of ca. 14.6 s following apparent first-order kinetics (Figure 1). by loss of iodine, the reaction products were compared with Upon photolysis, the solution turned slightly orange (absorbance noniodinated reference compounds prepared by photolysis in maximum at around 460 nm), a result of the well-documented cyclohexane of a 2 mM solution of the (noniodinated) diazirine partial rearrangement of the diazirine to the diazo isomer!a,b,e benzyl acetate 13. As shown (Figure 2B), photolysis of 13 also After photolysis for 60 s, the solution was concentrated and gave rise to several components, among which the CH (cyclosubjected to TLC and GC/MS analyses. The most prominent, hexane) insertion product 15, being the most prominent, fastest migrating compound on TLC was identified as the accounted for approximately 58%.'* As revealed by the gas product from insertion of the carbene into a CH bond of chromatograms (Figures 2A and B), no common products were (9) (a) Seevers, R. H.; Counsell, R. E. Chem. Rev. 1982, 82, 575-590. (b) Moerlein, S. M.; Beyer, W.; Stocklin, G. J. Chem. Soc., Perkin Trans. 1 1988,779-786. (c) Hanson, R. N. New Trends in Radiopharmaceutical Synthesis, Quality Assurance, and Regulatory Control; Emran, A. M., Ed.; Plenum Press: New York, 1991; 303-315. (10) Bumagin, N. A,; Gulevich, Yu. V.; Beletskaya, I. P. Dokl. Akad. Nauk SSSR (Engl. Transl.) 1985, 280, 633-636; 1985, 280, 17-20. (1 1) Initial attempts to prepare 12 through stannylation of free benzyl alchol 9 resulted in the formation of 8 as the main product. Reduction of qlhalogenides during stannylation with hexaalkylditin is a side reaction reported in the literature; see ref 10.
formed from 10, and 13 (the minor component at 15.32 min (Figure 2B)) has not been identified, but must be distinct from the (iodinated) CH-insertion product 14 having nearly the same retention time (15.40 min). It should be noted that these results do not rule out the possibility that loss of iodine from 10 leads to the destruction along pathways resulting in products different (12) At an initial concentration of 13 of 30 mM, the CH insertion product 15 was produced in 28&5% yield, as determined by quantitative GC analysis.
Photolabeling and Cross-linking Reagents
J. Am. Chem. SOC., Vol. 117,No. 11, 1995 3087
A
14
- 15.40
B 5
That this type of iodinated phenyldiazirines make promising photolabeling reagents is inferred also from a number of studies ([ 1251]with 3-(trifluoromethyl)-3-(3-[125I]iodophenyl)diazirine TID), a reagent widely used in the past for labeling of the apolar phase of membranes.14 The superb selectivity of this reagent in labeling integral (versus peripheral) proteins may be a direct reflection of the C-I bond stability during diazirine photolysis. The lower CH insertion efficiency measured by TLC as compared to GC can be attributed, in part, to losses during product isolation by TLC and is likely to be due to thermal decomposition of the diazo isomer present in the photolysis reaction mixture upon injection of the sample into the GC system, resulting in a larger proportion of the CH insertion product.15 Although further studies are needed to elucidate the details of the photochemistry of iodinated diazirines, we, nonetheless, note that the efficiency of 13 and 10 to insert into cyclohexane CH bonds, as determined by GC analysis, is in a range comparable to that reported for the nonsubstituted 3-(trifluoromethyl)-3-phenyldiazirine( 250%)4aor for N-BOC4-[3-(trifluoromethyl)-3H-diazi1in-3-yl]phenylalanine methyl ester (73%).4b The high efficiency of CH insertion reactions is suggestive of a mechanism involving a singlet carbene, implicating that the heavy atom iodine has, if any at all, only a moderate effect in promoting triplet photochemical reactions.
Design, Synthesis, and Biochemical Properties of Phospholipid Photolabels. Over the last 20 years, numerous
I.
I
. 11.92l 15.32l
1
LL
10
17.31 15.92
20
Retention Time (min) Figure 2. Gas chromatograms of reaction products derived from photolysis of 2 mM solutions of (A) 2-iodo-4-[3-(trifluoromethyl)-3Hdiazirin-3-yllbenzyl acetate (10)and (B) 4-[3-(trifluoromethyl)-3Hdiazirin-3-yllbenzyl acetate (14) in cyclohexane. After irradiation with fdtered W light (1> 320 nm) for 60 s, the solutions were concentrated to approximately one third of the original volume and 1 p L each was analyzed by GC (SPB-S Supelco 2-4034 column (30 m, 0.25 mm), injection block at 250 "C, 1:lO split mode) using FI detection. The main product peaks (retention times 15.40 min (A) and 11.92 min (B)) correspond to the CH insertion products 14 and 15, respectively. The arrow marks the retention time (11.92 min) for compound 15. For more details see main text and Experimental Section. from those in Figure 2B.13 To further examine this point, we also investigated whether iodine is liberated upon photolysis of 10. Following photolysis of a solution (5 mM) of tracelabeled (lZ5I) 10 in cyclohexane, any free iodine was reduced and extracted into aqueous NazS205. Less than 0.5% (nonphotolyzed control samples:
