Bioconjugate Chem. 1995, 6,630-634
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High Efficiency Photolabeling of Human Serum Albumin and Human y-Globulin with [14C]Methyl4-Azido-2,3,5,6-tetrafluorobenzoate Raghoottama S. Pandurangi,t Srinivasa R. Karra,t Robert R. Kuntz,*,t a n d Wynn A. Volkertt Departments of Chemistry and Radiology and Research Service, H. S. Truman Memorial VA Hospital, University of Missouri, Columbia, Missouri 65211. Received March 28, 1995@ The efficiency of photolabeling of HSA and IgG with [14C]methyl4-azido-2,3,5,6-tetrafluorobenzoate has been studied using size exclusion chromatography in conjunction with liquid scintillation counting. Labeling efficiencies of 78% for HSA and 82% for IgG have been determined. The extent of bond insertion into proteins exceeds the C-H insertion efficiency in cyclohexane with less wastage into anilinium and azo side products. These results suggest that the photoprobe accesses hydrophobic regions of both proteins prior to photolysis.
INTRODUCTION
A systematic study of the fundamental photochemistry of aryl azides by Keana et al. (1-3) and Platz et al. (47 )has identified the perfluoroaromatic azides as potential photolabeling precursors. The photolabeling technique is an attractive alternative to chemical labeling techniques for the attachment of complexes of radionuclides (e.g., Io9Pd,99mTc,ls6Re) to proteins and antibodies (8, 9). In the photolabeling approach, photoactivable moieties produce, upon excitation, highly reactive intermediates (e.g., nitrenes or carbenes) capable of efficient C-H or N-H bond insertion (20-14). In more rigid environments such as liquid crystals or matrix isolated systems a t low temperatures, the probability of C-H bond insertion can be increased dramatically (15, 16). For this reason, it is expected that trapping of the photoactivable group in hydrogenic crevices of proteins might give very good efficiencies for covalent attachment of the photoprobe to the protein. For applications in biochemistry and nuclear medicine where the attached probelprotein ratio must be kept near unity the efficiency of attachment is a very important consideration in photolabeling (17-20). At these low ratios, use of a radioactive probe greatly facilitates the identification, isolation, efficiency determination, and in vivo stability studies of the product. Modification of the perfluoroaryl ring can also alter the efficiency of insertion reactions. For example, Keana et al. (21) studied photolabeling efficiency on model solvents using a perfluoroaryl azide and iodinated analogues. In that study the C-H insertion yields for the iodinated derivatives were much lower than those observed with the parent perfluoroaryl azide. These yields were still higher than those obtained with the fully hydrogenated photoprobes. Although the insertion reactions of the perfluoroaryl azides in model systems (e.g., cyclohexane, diethylamine, etc.) are well characterized, a systematic investigation of their conjugation with proteins is rarely attempted (22-24). In the development of photoprobes for protein ~~
~
* Author
to whom correspondence should be addressed: Department of Chemistry, 123 Chemistry Building, University of Missouri, Columbia, MO 65211. Tel: 314/882-2815. Department of Chemistry. *Department of Radiology and Research Service, H. S. Truman V.A. Hospital. Abstract published in Advance ACS Abstracts, September 1, 1995. A
@
labeling, it is important to determine the relationship between the insertion characteristics of a photoprobe on model solventholute systems to the labeling of complex macromolecules containing a variety of functional groups and environments. For heterobifunctional chelating agents (25) carrying a photoactivable terminus for attachment to proteins, a direct determination of covalent attachment of the photoprobe to proteins and antibodies is essential. Recently, we demonstrated the utility of size exclusion chromatography (SEC-HPLC) to monitor the covalent modification of human serum albumin (HSA) using a long-wavelength photolabel (26). It was important to determine if the extent of labeling observed for HSA could be extended to antibodies in general. Also, it is of interest for predictive purposes to determine the relationship between the efficiency of covalent attachment to model solvents and to proteins when utilizing the same photoprobe. Here, we report the synthesis of a [I4C1methylanalogue of 4-azido-2,3,5,6-tetrafluorobenzoate (ATFMB), its conjugation with HSA and human y-globulin (IgG), and a comparison of insertion efficiency between the proteins and cyclohexane. IgG was chosen as a model for the labeling of monoclonal antibodies (MAbs) which have extensive applications in antibody targeting therapy (27). The unlabeled ATFMB is known to give efficient C-H insertion in cyclohexane (1) and should serve as a n efficient photoprobe. Substitution of a [I4C1methyl group on the ester permits monitoring of the photoconjugation by both spectroscopic and radiochemical means using SEC-HPLC. EXPERIMENTAL PROCEDURES
All synthetic procedures were carried out in a dry nitrogen atmosphere and with prepurified solvents. Reactions involving the synthesis of azide derivatives were carried out in subdued light by wrapping the reaction vessels with aluminum foil. All reagents except for [14Clmethanol were purchased from Aldrich. [l4C1Methanol (48.2mCi/mmol) was purchased from Sigma. HSA and IgG (Cohn Fractions I1 and 111)were purchased from Sigma. Both proteins were used without further purification. NMR spectra were taken in CDC13 with TMS as an internal standard for lH and in CFC13 for 19Fspectra on a 300 MHz Bruker instrument. Thin layer chromtography (TLC) was performed on precoated glass-silica gel plates (E. M. Science, GF254) with 10% EtOAc-hexane as eluent. The Rf value of the azido ester was 0.6. UV spectra were recorded in cyclohexane on a Hewlett-
1 043-1802/95/2906-0630$09.00/00 1995 American Chemical Society
Photolabeling of Human Serum Albumin
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Bioconjugate Chem., Vol. 6,No. 5, 1995 631
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containing 0.05 M sodium sulfate (pH = 6.8) a t a flow rate of 1mumin. Absorption of the HSA-unlabeled probe solutions was monitored at 254 nm to characterize retention times and establish photolysis conditions. For the labeled probe, the detector was removed and 1 mL fractions were collected for liquid scintillation counting. Photolysis. All photolysis experiments were conducted with a 200 W super pressure Hg lamp with or without a 320 nm cutoff filter. Solutions were purged with prepurified nitrogen for several minutes before photolysis. Protein Conjugation. Protein labeling experiments were conducted by mixing 5 mL of 4 x M solutions of the protein with 200 pL of a low3M solution of either [WI-ATFMB or [14C]-ATFMBin ethanol to give a 1:l mol ratio. The photolysis time for probe destruction was predetermined with a n equivalent concentration of unlabeled ATFMB in cyclohexane as monitored by C-18 reversed-phase HPLC as described earlier (28). These times were used for the protein-probe photolysis. Twenty pL samples of the [l4C1-ATFMB-protein mixtures were injected into the SEC-HPLC column before and after photolysis and the eluent collected in 1 mL fractions. Each fraction was combined with 10 mL of a scintillation cocktail (Optifluor-aqueous) and counted by liquid scintillation (Tracer Analytic Delta 300) with the energy window set for 14C. Synthesis of [l4C1Methyl4-Azido-2,3,6,6-tetrafluorobenzoate. [l4C1Methanol(250 pCi) stored in a vacuum break seal vial was cooled in a n ice-salt bath and the break seal ruptured under 2 mL of dry dichloromethane which filled the evacuated vial. The solution was transferred by syringe to a 25 mL flask as were 5 x 0.2 mL washings of the initial container. A solution of 13.2 mg of 4-azidotetrafluorobenzoyl chloride was dissolved in 10 mL of methylene chloride. Two mL of this stock (10.4 pmol) was mixed with 2 mL of a stock solution of triethylamine prepared by dissolving 5.2 mg of triethylamine in 10 mL of dry dichloromethane and the mixture added slowly via syringe to the [I4C1methanolsolution. The mixture was stirred for 1 h during which time the reaction progress was monitored by TLC (detected by UV and AMBIS) for the ester a t Rf = 0.6. After 1 h, the solution was concentrated to 0.1 mL by passing a stream of nitrogen over the solution. This material was deposited on a silica gel column and eluted with 6-8 mL of 3% ethylacetatehexane and then with 10% ethyl acetatel hexane. The ester, eluting between 20 and 22 mL, was collected and evaporated by flushing Nz gas through the solution. Radiochromatographic scanning indicated a single product with a radiochemical purity '95% by comparison with the sample's total activity. The I9F NMR of this product showed the expected AAXX' pattern reported for the unlabeled material (1). Synthesis of Methyl 4-(Cyclohexylamino)2,3,6,6tetrafluorobenzoate. A solution of methyl pentafluorobenzoate (4.52 g, 20 mmol) and cyclohexylamine (2.10 g, 21.2 mmol) in absolute ethanol containing 2 mL of triethylamine was refluxed overnight. The mixture was cooled, extracted with ether (3 x 50 mL), and dried over anhydrous MgS04. After filtration, ether was removed under vacuum to give light yellow crystals of the product. (2.11 g, 65% yield, mp 85 "C): IH NMR (CDCl3) 6 (ppm) 1.22 (m, 3H), 1.31 (m, 2H), 1.58 (m, lH), 1.76 (m, 2H), 2.03 (m, 2H), 3.61 (m, lH), 3.91 (s,3H), 4.02 (m, 1H); I9F NMR (CDC13) 6 (ppm) -144.2 (m, 2F) and -161.2 (m, 2F); 13CNMR (CD3CN)6 (ppm) aromatic region 149 (m), 147 (m), 136 (m), 134 (m), 132 (SI, aliphatic region 53.2 (t) (4J(13C-19F)4.3 Hz), 34.4 (51, 25.3 (SI, and 24.6 (SI.
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Figure 1. Absorption spectrum of 1.25 x low5M [l4CIATFMB in cyclohexane (a) before photolysis, (b) after 15 s exposure, and (c) after 2 min exposure to a 200 W super-pressure Hg lamp. Spectra b and c are nearly identical.
0 3 6 9 12 15 18 21 24 Figure 2. Size exclusion chromatogram (SEC-HPLC)of unlabeled ATFMB with and without HSA. Plots show relative 254 nm absorbance vs elution time (min) (a) before photolysis, (b) after 15 s exposure, (c) after 2 min exposure, and (d) after 2 min photolysis in buffer without HSA. Eluting solvent was a 0.02 M monobasic sodium phosphate buffer (adjusted to pH 6.8) containing 0.05 M sodium sulfate a t a flow rate of 1 mumin.
Packard (8452) diode array spectrometer with 1-cm pathlength cells. Protein-probe solutions were separated by HPLC using a BioRad Bio-Si1 SEC 250 column. The eluent was a 0.02 M sodium phosphate (monobasic)buffer
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Figure 3. SEC-HPLC radiochromatogram of 14C-labeled ATFMB with HSA (a) before photolysis, (b) after 15 s exposure, and (c) after 2 min exposure. One mL fractions of the effluent (1 m u m i n flow rate) were collected and counted by liquid scintillation. Buffer specifications are indicated in Figure 2 .
HPLC analysis (CH&N/H20, 2/1) 1 mumin, on a C-18 reversed-phase column gave a retention time which coincided with that for the adduct produced by photolysis of the photoprobe in cyclohexane.
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RESULTS AND DISCUSSION
with and without IgG. Plots show relative 254 nm absorbance vs elution time (min): (a) unlabeled ATFMB, (b) mixture of ATFMB and IgG, (c) mixture after 15 s exposure, (d) mixture after 2 min exposure, (e) photolysis in the absence of IgG. Conditions identical to those indicated in Figure 2. Additional peaks in e can be assigned to unphotolyzed ATFMB and residual materials from prior IgG chromatograms.
Photolysis in Cyclohexane. Photolysis of ATFMB in cyclohexane has been reported to give 57% of the C-H insertion product along with 21% of the aniline derivative and 11%of the azobenzene derivative (I). Photolysis of the 14Cester in cyclohexane resulted in a red shift of the absorption maximum from 262 to 276 nm (Figure 1).The photoproduct spectrum coincides well with the absorption spectrum of the cyclohexane adduct synthesized independently from unlabeled methyl pentafluorobenzoate. Integration of the 19FNMR of the photolyzed mixture (28) indicated 55% of the C-H inserted product which agrees with earlier reports (I). Photolabeling of HSk Equimolar solutions of HSA and unlabeled ATFMB were incubated for 1 h before injection into the SEC-HPLC column. In the unphotolyzed mixture, the photoprobe elutes a t 15.3 min as compared to HSA at 8.2 min (Figure 2a). This control indicates a clean separation of the probe and protein before photolysis. Exposure of the mixture to the beam of a n unfiltered 200 W Hg lamp results in 90% destruction of the probe after 2 min photolysis (Figure 2b,c). Use of the 320 nm cutoff filter to protect against protein photolysis increases the photolysis time to 15-20 min because light is only absorbed in the tail of the ATFMB absorption (Amm = 268 nm). In the chromatogram, disappearance of the absorbance assigned to the photoprobe during photolysis is accompanied by an increase in the HSA absorbance. A new peak a t 17.2 min appears
in the chromatogram and persists even after >90% photoprobe destruction. This product is neither unphotolyzed probe nor attached probe and is possibly a n anilinium-type product resulting from reactions of the triplet nitrene which is formed competitively with bond insertion of the singlet nitrene. Support for this assignment comes from photolysis of the photoprobe in a buffer solution without added protein (Figure 2d). The primary product from azide photolysis in hydroxylic solvents is the anilinium product ( I ) which appears here a t 17.3 min consistent with the retention time of the minor product formed in the labeling studies. Comparable experiments with the 14Canalogue were analyzed with a different pump-injection system, but employing the same HPLC column. The eluent was collected in 1mL fractions and counted by liquid scintillation techniques. Peaks are shifted to somewhat longer times because a different injection system with longer dead time was employed. Figure 3a shows the radiochromatogram of the unphotolyzed mixture and indicates that the 14Cprobe (fractions 14-17) is not associated with the protein fraction prior to photolysis. As photolysis progresses (Figure 3b,c), the activity shifts to the protein (fractions 9-14) and a new product (fractions 19-23). On the basis of relative activity, the protein fractions contains 78 f 5% of the labeled probe which is believed to be covalently bound. The major part of the activity not
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Sioconjugafe Chem., Vol. 6,No. 5, 1995 633
Photolabeling of Human Serum Albumin
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Figure 5. SEC-HPLC radiochromatogram of 14C-labeled ATFMB and IgG (a)before photolysis and (b)after 2 min photolysis. One mL fractions of the eMuent (1 m u m i n flow rate) were collected and counted by liquid scintillation. Buffer specifications are indicated in Figure 2.
associated with HSA is found in the region believed to be associated with the anilinium type products. We have shown previously that denaturing HSA with sodium dodecyl sulfate (SDS)does not result in a release of additional products, indicating the covalent nature of the binding (26). Photolabeling of IgG. Photolabeling of IgG followed the same protocol established for HSA. The prephotolysis mixture of IgG and ATFMB (Figure 4b) shows two impurities which must be assigned to IgG since they do not appear in the ATFMB sample (Figure 4a). The higher molecular weight impurity (eluting before IgG) may be a n aggregate, but the smaller species is not identified. Photolysis for 15 s (Figure 4c) and 2 min Scheme 1
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CONCLUSIONS
The I 4 C analogue of methyl 4-azido-2,3,5,6 tetrafluorobenzoate provides a convenient radiochemical probe for monitoring the extent of conjugation of the photoactivable probe with proteins using size exclusion chromatography. The short photolysis time required (2 min) for attachment
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(Figure 4d) shows a progressive depletion of the photoprobe and a lower molecular weight product similar to the one observed in HSA. Photolysis of the photoprobe in buffer without protein (Figure 4e) gives only the lower molecular weight material which we believe to be the anilinium product. Photolysis appears to enhance absorption by the aggregate impurity, but shows no effect on absorption of the unidentified impurity. The radiochromatogram (Figure 5) shows far less resolution because of the larger fractions collected for analysis. However, Figure 5a shows a clean separation of the probe from the protein region before photolysis. After photolysis, the radioactivity is found primarily in the protein elution region (6-12 min). Integration of the activity indicates that 82 f 5% of the activity is bound to the protein. Unfortunately, the protein region of the radiochromatogram shows considerable broadness. Apparently, the peak assigned to the IgG aggregate is also labeled in this procedure. Currently, we are investigating this problem more carefully. It is interesting to compare the efficiency of insertion efficiency in cyclohexane with the higher values observed for HSA and IgG (Scheme 1). Since singlet nitrene is known to deactivate in hydroxylic media, it appears that the hydrophobic probe becomes trapped in hydrophobic crevices of the protein prior to photolysis. This more rigid environment should,favor insertion of the singlet nitrene over intersystem crossing to the triplet and also will tend to inhibit bimolecular reactions leading to azo products. It is not known whether the -20% of unattached probe is due to intersystem crossing within the protein matrix or exposure of the singlet nitrene to the aqueous solvent during photolysis. However, the high degree of protein labeling (> 78%) confirms the assumption that the insertion characteristics of 4-azidotetrafluoroaryl photoprobes in cyclohexane or cyclohexaneldiethylamine are good indicators of ability to label proteins. In every case tried with this photoprobe, the protein labeling efficiency has surpassed the efficiency for insertion in model solvents.
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Chem., Vol. 6,No. 5, 1995
to the protein and the absence of external activating reagents necessary for conventional chemical labeling make this photolabeling technique attractive for use in biochemical labeling studies. Photolysis of the photoprobe in the absence of protein gives only the anilinium product which is cleanly separated from the protein by SEC-HPLC. The small yield of this side product in the presence of HSA or IgG suggests that the probe accesses hydrophobic regions of both proteins. This result, combined with earlier denaturation studies (26),strongly supports a covalent attachment between the probe and the protein. The high efficiency for covalent bond formation with both HSA and IgG suggests a general utility of the photochemical technique for antibody labeling. Investigation of the retention of immunoreactivity of labeled antibodies and their in vivo stability is currently under investigation, and results will be published elsewhere (29). ACKNOWLEDGMENT
This work was supported by funds provided by DOE Grant DE FG0289E R60875. NMR data were determined on instruments purchased with funds from NSF grants 8908304 and 9221835. The authors thank Mr. Tim Hoffman of the VA Hospital for helping with the HPLC setup and for useful discussions of methodology. LITERATURE CITED
(1) Keana, J. F. W., and Cai, S. X. (1990) New Reagents for Photoaffinity Labeling: Synthesis and Photolysis of Functionalized Perfluorophenyl Azides. J . Org. Chem. 55,3640. (2) Keana, J. F. W., and Cai, S. X. (1989) Functionalized Perfluoroazides: New Reagents for Photoaffinity Labeling. J . Fluorine Chem. 43, 151. (3) Cai, S . X., and Keana, J. F. W. (1989) 4-Azido-2-Iodo-3,5,6 Trifluorophenyl Carbonyl Derivatives. A New Class of Functionalized and Iodinated Perfluorophenyl Azide Photolabels. Tetrahedron Lett. 30, 5409. (4) Poe, R., Grayzar, J., Young, M. J. T., Leyva, E., Schnap, K. A., and Platz, M. (1991) Remarkable catalysis of Intersystem Crossing of Singlet (Pentafluorophenyl)Nitrene J . Am. Chem. SOC.113, 3209 and references cited therein. (5) Poe, R., Grayzar, J.,Young, M., Leyva, E., Schnapp, K. A., and Platz, M. S. (1993) Exploratory Photochemistry of Fluorinated Arylazides. Implications for the Design of Photoaffinity Labeling Agents. Bioconjugate Chem. 4, 172. (6) Marcinek, A,, Levya, E., Whitt, D., and Platz, M. S. (1993) Evidence for Stepwise Nitrogen Extrusion and Ring Expansion upon Photolysis of Phenyl Azide. J . A m . Chem. SOC.115, 8609. (7) Poe, R., Schnapp, K., Young, M. J. T., Grayzar, J.,and Platz, M. S. (1992) Chemistry and Kinetics of Singlet (Pentafluoropheny1)nitrene. J . A m . Chem. SOC.114, 5054. (8) For reviews see: Brunner, J. (1993). New Photolabeling and Cross Linking Methods. Ann. Rev. Biochem. 62, 483. (9) Bayley, H., and Staros, J. W. (1984) Photoaffinity Labeling and Related Techniques. In Azides and Nitrenes, Reactivity and Techniques (E. F. V. Scriver, Ed.) p 433, Academic Press, New York. (10) Bayley, H. (1983) Reagents for Photoaffinity Labeling. Photogenerated Reagents in Biochemistry and Molecular Biology (T. S . Work and R. H. Burdon, Eds.) p 26, Elsevier, Amsterdam. (11) Schurter, G. B., and Platz, M. S. (1992) Photochemistry of Phenyl Azide. Advances in Photochemistry (D. Volman, G. Hammond, and D. Neckers, Eds.) Vol. 17, p 69, Wiley/ Interscience, New York.
Pandurangi et al. (12) Scriven, E. F. Y. (1982) Current Aspects of the Solution Chemistry of Aryl Nitrenes Reactive Intermediates (R. A. Abramovitch, Ed.) Vol. 2, p. 1, Plenum Press, New York. (13) Iddon, B., Meth-Cohn, O., Schriven, E. F. Y., Suschitzky, H., and Gallagher, P. T. (1979) Developments in Arylnitrene Chemistry: Synthesis and Mechanisms. Angew. Chem., Int. Ed. Engl. 18, 900. (14) Reiser, A,, and Wagner, H. M. (1971) Photochemistry of the Azido Group. In The Chemistry of the Azido Group (S. Patai, Ed.) p 441, Wileyhterscience, New York. (15) Mahe, L., Izuoka, A., and Sugawara, T. (1992) How Crystalline Environment can Provide Outstanding Stability and Chemistry for Aryl Nitrenes. J . Am. Chem. SOC.114, 7904. (16) Leyva, E., Young, M. J. T., and Platz, M. S. (1986) High Yields of Formal C-H Insertion Products in the Reactions of Polyfluorinated Aromatic Nitrenes. J . A m . Chem. SOC.108, 8307. (17) For reviews see: Cavalla, D., and Neff, N. H. (1985) Chemical Mechanisms for Photoaffinity Labeling of Receptors. Biochemical Pharm. 34, 2821. (18) Choudhry, V., and Westheimer, F. H. (1979) Photoaffinity Labeling of Biological Systems. Annual Rev. Biochem. 48, 293. (19) Fedan, J. A,, Hogaboom, G. K., and ODonnell, J. P. (1984) Photoaffinity Labels as Pharmacological Tools. Biochem. Pharm. 33,1167. (20) Ji. J.. and Ji. I. (19821 Macromolecular Photoaffinitv Labeling With Radioactive Photoactivable Heterobifunctionil Reagents. Anal. Biochem. 121, 286. (21) Cai, S. X., Glenn, D. J., and Keana, J. F. W. (1992) Toward the Development of Radiolabeled Fluorophenyl Azide Based Photolabeling Reagents: Synthesis and Photolysis of Iodinated 4-Azido Perfluorobenzoates and 4-Azido-3,5,6-trifluorobenzoates. J . Org. Chem. 57, 1299. (22) Pinney, K. G., Carlson, K. E., and Katzenellenbogen, J. A. (1990) [3H] DU41165: A High Affhity Ligand and Novel Photoaffinity Labeling Reagent for the progesterone Receptor. J . Steroid. Biochem. 35, 179. (23) Pinney, K. G., Carlson, K. E., Katzenellenbogen, B. S., and Katzenellenbogen, J. A. (1991) Efficient and Selective Photoaffinity Labeling of Estrogen Receptor Using Two Nonsteroidal Ligands That Embody Aryl Azide or Tetrafluoroaryl Azide Photoactive Functions. Biochemistry 30, 2421. (24) Pinney, K. G., and Katzenellenbogen, J. A. (1991) Synthesis of Tetrafluoro-substituted Aryl Azide and It's F'rotio Analogue as Photoaffinity Labeling Reagents for the Estrogen Receptor. J . Org. Chem. 56, 3125. (25) Pandurangi, R. S., Kuntz, R. R., Volkert, W. A,, Barnes, C. L., and Katti, K. V. (1995) Phosphorus Hydrazides as Building Blocks for Potential Photoaffinity Labels. Synthesis and Coordination Chemistry of Perfluoroarylazide Conjugates of Phenylphosphonothioic Dihydrazide. J . Chem. SOC.,Dalton Trans. 565. (26) Pandurangi, R. S., Kuntz, R. R., and Volkert, W. A. (1995) Photolabeling of Human Serum Albumin by 4-Azido-(I4Cmethy1amino)trifluorobenzonitrile. A High Efficiency, Long Wavelength Photolabel. Znt. J . Appl. Radiat. Isotopes 46,233. (27) Koppel, G. A. (1990) Recent Advances with The Monoclonal Antibody Drug Targeting for the Treatment of Human Cancer. Bioconjugate Chem. 1, 13. (28) Pandurangi, R. S., Katti, K. V., Barnes, C. L., Volkert, W. A., and Kuntz, R. R. (1994) High Yields of Nitrene Insertion into Unactivated C-H Bonds. First Example of X-RayCrystallographic and 19FNMR Analysis of the Photochemically Produced C-H Inserted Adduct. J . Chem. SOC.,Chem. Commun. 1841. (29) Pandurangi, R. S., Karra, S. R., Kuntz, R. R., and Volkert, W. A. (1995) Unpublished results. BC9500570
