Bioconjugate Chem. 1093, 4, 172-177
172
Exploratory Photochemistry of Fluorinated Aryl Azides. Implications for the Design of Photoaffinity Labeling Reagents Karlyn A. Schnapp, Russell Poe, Elisa Leyva, N. Soundararajan, and Matthew S. Platz' Department of Chemistry, The Ohio State University, 120 West 18th Avenue, Columbus, Ohio 43210. Received October 13, 1992
A series of fluorinated aryl azides and fluorinated azidobenzoates were studied by laser flash photolysis techniques. Using the pyridine ylide probe method it was possible to determine whether a singlet nitrene or ring-expanded ketenimine ylide is the trappable intermediate that is generated at ambient temperature. It was determined that two fluorine substituents, ortho and ortho' substituted relative to the azide group, are required to retard ring expansion and allow bimolecular capture of the singlet nitrene. LFP of ortho, ortho' difluorinated aryl azides in methanol produces the ground triplet state of the nitrene. The results are consistent with chemical analysis of reaction mixtures. The implications of this data for the design of photoaffinity labeling reagents are discussed.
Scheme I
I. INTRODUCTION
The photochemistry of phenyl azide (la) has been described as wonderfully complex (1). Over the last decade the complexity of this system has been reduced to that of Scheme I. Photolysis of phenyl azide la produces singlet phenylnitrene (lb) which ring expands to ketenimine l c within 10-100 ps (2). The rate of rearrangement is sufficiently rapid to prevent bimolecular trapping of lb. The barrier to ring expansion is about 3 kcal/mol, which means that the rate of this process slows dramatically at cryogenic temperatures (3). Between 10 and 77 K, singlet phenylnitrene decays by intersystem crossing to triplet lf, which is the ground state of the nitrene ( 4 , 5 ) . When the singlet nitrene is generated in the gas phase, it is formed in a vibrationally excited state which rearranges to cyanocyclopentadiene and ultimately forms the cyanocyclopentadienyl radical, which has been observed by absorption and emission spectroscopy (6). Aromatic azides are widely used in photoaffinity labeling (PAL) experiments (7). PAL is a biochemical technique invented by Westheimer that is used to identify amino acid residues present in the binding sites of biological receptors (8). In this experiment, a light-sensitive moiety is appended to a natural substrate of an enzyme. Photolysis of the enzymesubstratecomplex releases a reactive intermediate which in an ideal experiment will react with the first bond of the protein it encounters to form a robust new linkage between the reactive intermediate and the target biomolecule. The experiment will be defeated if the photogenerated intermediate is chemically discriminating because it can exit the binding site and react aspecifically with the protein. Unfortunately, the trappable intermediate produced from phenyl azide and most of its derivatives are ketenimines (IC) (1, 2). These species are not ideal labeling reagents because they react rather sluggishly (9) ( 7 >> 10 ps) and only with nucleophiles. This laboratory (IO), Keana's group (11),and Bayley (7) have proposed polyfluorinated azides as superior reagents for PAL studies. The research groups of Watt (13) and Katzenellenbogen and Pinney (24)have since used fluorinated aryl azides as PAL reagents with interesting results.
@"
l
gas phase
oX3ON hv
la
solution phase > 173 K
~
~
lb
IC
1 10-77K
I
03N If
Predictions of a fluorine effect were based on Banks' findings that pyrolysis of polyfluorinated aryl azides in hydrocarbons leads to products from formal CH insertion of the nitrene in modest yields (12). Recently we have demonstrated that singlet (pentafluoropheny1)nitrene (2b) ring expands at least 170 times more slowly than does singlet phenylnitrene (lb) at ambient temperature (15). This gives singlet nitrene 2b sufficient time to react with alkanes, alkenes, aromatics, amines, ethers, sulfides, and sulfoxides to form adducts in reasonable yields. Furthermore, we found that pentafluoro ketenimine 2c reacts with pyridine by nucleophilic
F' IC
'F 2c
addition almost lo4 times more rapidly than its nonfluorinated counterpart IC.
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Biocon/ugate Chem., Vol. 4, No. 2, 1993
Photochemistry of Fluorinated Aryl Azides
Chart I1
Chart I
DN3
F
FN- \
2a
la
F
3a
4a
b
BN3 F
6a
+
d
P
F
7a
8a
C02CH3
C02CH3
N3
F
la
C02CH3
F~
N3
N3
%"x(nm)
lb
:*
-
340
13a
12a
le
IC
F
lla
f
e
9a
QF Q 10a
C
- +
Sa
3*F F F
F
F
2b
2c
g%&
L a x (nm)
F , @F .
16a
Thus two extremely reactive, but trappable, intermediates are produced upon photolysis of pentafluorophenyl azide (2a). Since the potential of using 2a itself in PAL work is quite limited, it is important to determine the number of fluorine atoms as well as their positions relative to the azide moiety that is required to retard ring expansion and to achieve bimolecular trapping of the nitrene. In this regard we have prepared azides la-16a and have studied these compounds by laser flash photolysis (LFP) techniques. We have also studied fluorinated azidobenzoates (15)by LFP techniques since many PAL reagents can be elaborated through the ester functional group. The results indicate that at ambient temperature two fluorine atom substituents positioned ortho and ortho', or less effectively ortho and para to the azide moiety are sufficient to achieve the desired reactivity properties.
RESULTS LFP Studies in CH2Clz. Azides la-16a (Chart I) were studied by LFP (XeCI excimer laser, 308 nm, 150 mJ, 17 ns) in CH2C12. Our earlier studies with 2a have identified CHzClz as an inert solvent toward singlet (pentafluoropheny1)nitrene (15).The results with azide 4a (Chart 11)are typical. The transient intermediate produced in this solvent is ketenimine 4c, which is clearly identified by its transient absorption spectrum (Figure 1) and by comparison with that of IC produced by LFP of phenyl azide (1, 3, 13). LFP Studies in Neat Pyridine. As reported elsewhere, LFP of phenyl azide (la) and pentafluorophenyl azide (2a)in neat pyridine produce very different transient spectra (Chart 11). In the perfluorinated system nitrene 11.
F
F N3
15a
310,370,500
Q -
F
2a
F F)+F
If
490-500
\
C02CH3
14a
-
F N\- ~ "
a
179
F
F F
L a x (nm)
F
\
&5H5
340-360
F
, F@
N
F
\
F
F
F
2e
2d
2f
520
390
310,370,500
ylide 2d is formed (Amm = 390 nm) whereas with the parent azide la it is ketenimine ylide le that is formed with ,A, = 520 nm. It is possible to trap perfluorinated ketenimine 2c with pyridine to form ylide 2e, but dilute pyridine must
le
2d
2e
be used to allow singlet nitrene 2b sufficient time to ring expand (Scheme 11). Ylide 2d is sufficiently stable to be isolated and its structure has been determined by X-ray crystallography (15). Ylides le and 2e could not be isolated. Representative transient spectra are given in Figures 2 and 3 which are derived from LFP of two difluorinated aryl azides, 7a and 9a (Scheme 111, in neat pyridine. The 2,3 difluorinated azide produces the ketenimine 7c (or 7c') upon photolysis which is trapped in neat pyridine; the 2,6 isomer produces the nitrene 9b which is trapped in pyridine. The results for azides la-16a are summarized in Table I. LFP of 9a in the presence of dilute pyridine
174
Schnapp et al.
Bioconjugate Chem., Vol. 4, No. 2, 1993
7a
7c
7c'
'F
20 0 300
500
400
nm
700
600
7e' F'
7e
WAVELENGTH Figure 1. The transient spectrum of 4c produced by LFP of 4a in CH&lz at ambient temperature.
!'7 220 200 .nn
9b
9a
I
9d
40 20 400
300
500
nm
700
600
WAVELENGTH
Figure 2. The transient spectrum of ketenimine 7c (or 7c') derived ylide 7e (or 7e') produced by LFP of 7a in neat pyridine at ambient temperature.
9e
In contrast to 7a and 9a, LFP of 2,4-difluorophenylazide (6a) in neat pyridine produces the transient spectra of both the ketenimine 6e and nitrene-derived ylide 6d (Figure 5).
Scheme I1 6a I
F'
F
I 2a
I t was possible to resolve the rate of formation of the ketenimine-derived ylides (1-16e) by nanosecond spectroscopy. The rates were exponential and could be fit by the Marquardtl' algorithm to yield an observed rate constant, kobs. Scheme I predicts eq 1, where 7 is the
F ' 2h
2g
kQ
kobs
2b
F 6e
6d
=
1/7
+ k'pyRCPYR]
(1)
lifetime of the ketenimine in the absence of pyridine and k'pyR is the second-order rate constant for the reaction of ketenimines with pyridine (18). Indeed, plots of ktobs versus [PYRI are linear (Figure 6) and the data are summarized in Table 11. It is clear that increasing fluorine substitution dramatically increases the rate of nucleophilic attack on the ketenimine. This is in accord with earlier studies of Li ( 9 b ) . LFP Studies in Methanol. We have previously reported that LFP of pentafluorophenyl azide (2a) in methanol produces the triplet nitrene 2d (15). Methanol
2c
G3
& JN f.-
F F F
F
F
F 2d
2e
produces ketenimine-derived ylide 9e. The ratio of nitrene ylide 9d/9e is a linear function of pyridine concentration as predicted by Scheme I1 (Figure 4). It was not possible to use transient UV-vis spectroscopy to determine whether ketenimine 7c or 7c', or both species, were trapped with pyridine to form either ylide 7e or 7e' or a mixture of both species.
apparently catalyzes the rate constant (kIsc) of singlet 2b to triplet 2f intersystem crossing. This has been attributed to differential hydrogen bonding, which lowers the energy of 2b relative to that of 2f thereby increasing the rate constant of the radiationless transition.
Bioconjugate Chem., Vol. 4, No. 2, 1993
Photochemistry of Fluorinated Aryl Azides
175
m -
(7
1oo.i
400
300
WAVELENGTH Figure 3. The transient spectrum of nitrene (9b) derived ylide 9d produced by LFP of 9a in neat pyridine at ambient temperature. Table I. Reactive Intermediate Trapped upon LFP of the Azide at 25 O C
intermediate trapped in neat pyridine ketenimine l c nitrene 2b ketenimine 3c ketenimine 4c ketenimine 5c nitrene 6b, ketenimine 6c ketenimine 7c ketenimine 8c nitrene 9b ketenimine 1Oc ketenimine 1 IC ketenimine 12c ketenimine 13c nitrene 14c nitrene 15b nitrene 16b
azide la 2a 3a 4a 5a 6a 7a 8a 9a 10a 1 la 12a 13a 14a 15a 16a
intermediate detected in CH30H ketenimine IC triplet nitrene 2f ketenimine 3c ketenimine 4c ketenimine 5c ketenimine 6c5 ketenimine 7c ketenimine 8c triplet nitrene 9f ketenimine LOc ketenimine 1 IC ketenimine 1 2 ~ 5 ketenimine 13c ketenimine 14c triplet nitrene 15f triplet nitrene 16f
a
2
Thus, azides 3a-16a were studied by LFP techniques in methanol. The results are summarized in Table I. It was found that the singlet nitrenes 2b, 6b, and 9b, which are sufficiently long lived to be trapped by neat pyridine at ambient temperature, show methanol-catalyzed ISC. Figures 7 and 8 are representative. LFP of 2,g-difluorophenyl azide (9a) in methanol produces the transient spectrum of the triplet nitrene (Figure 7), but LFP of the 2,3 isomer 7a in methanol again gives a ketenimine. The methanol catalysis of ISC is not universal but is restricted to singlet aryl nitrenes, which do not ring expand more rapidly than they react with pyridine.
nm
WAVELENGTH
4.00x1O6
i
1
3.00~106
1/
1. O O X 1 0 ~
3/1 90
600
Figure 5. The transient spectra of ketenimine-pyridine ylide 6e and nitrene-pyridine ylide 6d produced by LFP of 6a in neat pyridine at ambient temperature.
A small amount of triplet nitrene is observed.
m 0 m
500
1
Schnapp et al.
176 Bioconjugate Chem., Vol. 4, No. 2, 1993
2,6 substitution pattern leads to ring expansion to ketenimines rather than chemical trapping of the singlet nitrenes. The more fluorines that are present in the aryl azide the larger the rate constant of reaction of the corresponding ketenimine with the nucleophile and the greater the chance of the addition of a second nucleophile (Nuc) to the initial adduct, and cross-linking.
300
350
400
450
500
550
600
nm
WAVELENGTH
Figure 7. The transient spectrum of triplet nitrene 9f produced by LFP of 9a in methanol a t ambient temperature.
IV. EXPERIMENTAL PROCEDURES
350
400
450
500
550
nn
600
WAVE LENGTH
Figure 8. The transient spectrum of ketenimine 7c produced by LFP of 7a in methanol a t ambient temperature. Table 111. The Combined Yield of Azo Compound (and Aniline) Type Products Formed on Photolysis of Fluorinated Aryl Azides in Benzene and Methonol at Ambient TemDer8turessb solvent solvent
azide 2a 3a 4a 5a
benzene 5d 58
methanol
or 65
0 0
Oe
0"
azide 6a 7a Sa 9a
benzene 58 8 0
9d
methanol 72 8 0' 46e
Samples were photolyzed in a Rayonet photoreactor with a 350nm light source for 2 h. * Azide concentration: 0.01 M. Diphenylmethane (internal standard)concentration: 0.005 M. Percent yields are absolute. A benzene insertion product is the major product observed. e Large amounts of ketenimine-methanol adducts were detected by GC-MS. f A 56% yield of pentafluoroaniline was observed. g A 20% yield of 2,6-difluoroaniline was observed.
unactivated CH bonds. Thus they should be effective reagents for photoaffinity labeling of amino acids in hydrophobic environments. It is proposed that azides 15a C0ZCH3
C02CH3
15a
16a
and 16a will be useful synthons for the development of new PAL labels. Photolysis of 2,6-difluoro-substituted aryl azides in hydrophilic environments will generate triplet nitrene intermediates by hydrogen-bonding catalysis of intersystem crossing. The triplet nitrenes will undergo free-radical-like reactions with amino acids which are predicted to lead to cleavage rather than to labeling of biomolecules. Photolysis of polyfluorinated aryl azides which lack the
General Methods. Melting points were recorded on an electrochemical capillary melting point apparatus and are uncorrected. lH NMR spectra were recorded on a Bruker AM-250 (250-MHz)spectrometer. All 'H chemical shifts are reported relative to TMS as an internal standard. 19FNMR spectra were recorded on a Bruker AM-250 (250MHz) instrument and were proton decoupled. 19Fchemical shifts are reported relative to hexafluorobenzene(162.9 ppm as an internal standard). The 19FNMR spectra were recorded on a Varian EM-360 (56.60 MHz) and the IgF chemical shifts are reported relative to hexafluorobenzene (96 ppm) as an internal standard. Infrared spectra were recorded on a Perkin-Elmer Model 1710 infrared Fourier transform spectrometer. Mass spectra and exact masses were obtained on a V6 70-2SOS or a Kratos MS-30 mass spectrometer. GC-MS spectra were obtained with a Hewlett-Packard S890 gas chromatograph equipped with a Hewlett-Packard 5970B mass Spectrometer detector and a 30 m x 0.254 nm J&W Scientific fused silica capillary column (DB-1, 0.25 pm). Materials. Benzene, cyclohexene, hexane, ethyl acetate, diethylamine, 2-fluoroaniline, 3-fluoroaniline, 4-fluoroaniline, 2,3-difluoroaniline, 2,4-difluoroaniline, 2,5difluoroaniline, and 3,4-difluoroaniline were purchased from Aldrich and were used without further purification. Preparation of Compounds. The substituted aryl azides of this work were described previously ( l l b , 15) and prepared from the appropriate corresponding anilines. All azo compounds used in this study were prepared by the method of Birchall (18) and Leyva ( I l b ) . The crude product was purified by passage through a neutral alumina column using a 9:l mixture of hexane/ethyl acetate as described. Product Studies. Solutions of azides (le2 M) in the corresponding solvent were prepared. Samples were prepared by putting 0.5 mL of the azide solution in 6-mm Pyrex tubes which were prewashed with ammonium hydroxide and oven dried. The solutions were degassed using three freeze-pump-thaw cycles and then sealed under vacuum. Samples were photolyzed with a 350-nm light using Southern New England RPR 3500-A lamps for 2 h. The product mixtures were analyzed by GC-mass spectrometry. The yields and identities of the products were determined by coinjection of authentic samples and by GC-mass spectrometry. Laser Flash Photolysis. The laser flash photolysis system in use at The Ohio State University has been described (20)and standard protocols (15)were employed. ACKNOWLEDGMENT
Support of this work by the NIH (GM34823)is gratefully acknowledged. One of us (K.A.S.) is grateful to The Ohio
Photochemlstry of Fluorinated Aryl Azides
State University for an Ohio State University College of Arts and Sciences postdoctoral fellowship. LITERATURE CITED (1) Shrock, A. K., and Schuster, G. B. (1984) Photochemistry of Phenyl Azide: Chemical Properties of the Transient Intermediates. J. Am. Chem. SOC.106, 5228. (2) Schuster, G. B., and Platz, M. S. (1992) Photochemistry of Phenyl Azide. Adu. Photochem. 17, 69. (3) Leyva, E., Platz, M. S., Persy, G., and Wirz, J. (1986) Photochemistry of Phenyl Azide: The Role of Singlet and Triplet Phenyl Nitrene as Transient Intermediates. J. Am. Chem. SOC.108,3783. (4) Wasserman, E. (1971) Electron Spin Resonance of Nitrenes. Prog. Phys. Org. Chem. 8, 319-335. (5) Hayes, J. C., and Sheridan, R. S. (1990) Infrared Spectrum of Triplet Phenyl Nitrene on the Origin of Didehydroazepine in Low Temperature Matrices. J.Am. Chem. SOC.112,58795881. (6) (a) Wentrup, C. (1984) Gas Phase and Matrix Studies. In Azides and Nitrenes (E. F. V. Scriven, Ed.) p 395-430, Academic, New York. (b) Cullin, D. W., Soundararajan, N., Platz, M. S., and Miller, T. A. (1990) Reinvestigation of the Electronic Spectrum of the Phenyl Nitrene Radical. J.Phys. Chem.94,8890. (c) Cullin,D. W., Yu, L., Williamson, J., Platz, M. S., and Miller, T. A. (1990) Laser-Induced Fluorescence Spectrum of the Cyanocyclopentadienyl Radical. A Band System Long Attributed to Triplet Phenyl Nitrene. J.Phys. Chem. 94, 3387. (7) Bayley,H. (1983)Photogenerated Reagents in Biochemistry and Molecular Biology, Elsevier, Amsterdam. (8) (a) Singh, A., Thornton, E. R., and Westheimer, F. H. (1962) The Photolysis of Diazo-acetylchymotrypsin. J. Biol. Chem. 237, PC 3006. (b) Fleet, G. W. J., Porter, R. R., and Knowles, J. R. (1969)Affinity Labelling of Antibodies with Aryl Nitrene as Reactive Group Nature 224, 511. (9) (a) DeGraff, B. A., Gillespie, D. W., and Sundberg, R. J. (1974) Phenyl Nitrene. Flash Photolytic Investigation of the Reactin with Secondary Amines. J.Am. Chem. SOC.96,7491. (b) Li, Y.-Z., Kirby, J. P., George, M. W., Poliakof, M., and Schuster, G. B. (1988) 1,2-Didehydroazepinesfrom the Photolysis of Substituted Aryl Azides: Analysis of Their Chemical and Physical Properties by Time-Resolved Spectroscopic Methods. J. Am. Chem. SOC.110,8092. (10) (a) Leyva, E., Young, M. J. T., and Platz, M. S. (1986) High Yields of Formal CH Insertion Products in the Reactions of Polyfluorinated Aromatic Nitrenes. J. Am. Chem. SOC.108, 8307. (b) Leyva, E., Munoz, D., and Platz, M. S. (1989) Photochemistryof Fluorinated Aryl Azides in Toluene Solution and in Frozen Polycrystals. J.Org. Chem.54,5938. (c) Young, M. J. T., and Platz, M. S. (1989) Polyfluorinated Aryl Azides as Photoaffinity Labelling Reagents; The Room Temperature CH Insertion Reactions of Singlet Pentafluorophenyl Nitrene with Alkanes. Tetrahedron Lett. 30,2199. (d) Young, M. J. T., and Platz, M. S. (1991) Mechanistic Analysis of the Reactions of (Pentafluoropheny1)nitrene in Alkanes. J. Org. Chem. 56, 6403. (e) Poe, R., Grayzar, J., Young, M. J. T., Leyva, E., Schnapp, K. A., and Platz, M. S. (1991)Remarkable Catalysis of Intersystem Crossing of Single (Pentafluoropheny1)nitrene. J. Am. Chem. SOC.113, 3209. (11) (a) Keana, J. F. W., and Cai, S. X. (1990) New Reagents for Photoaffinity Labelling: Synthesis and Photolysis of Functionalized Perfluorophenyl Azides. J. Org. Chem. 55,3640. (b) Keana, J. F. W., and Cai, S. X. (1989) Functionalized Perfluorophenyl Azides: New Reagents for Photoaffinity Labelling. J.Fluorine Chem.43,151.(c) Cai, S. X.,andKeana, J. F. W. (1989)4-Azido-7-Iodo-3,5,6-Trifluorophenyl Carbonyl Derivatives. A New Class of Functionalized and Iodinated Perfluorophenyl Azide Photolabels. Tetrahedron Lett. 30, 5409. (d) Cai, S. X., Glenn, D. J., and Keana, J. F. W. (1992) Toward the Development of Radiolabelled Fluorophenyl Azide-Based Photolabelling Reagents: Synthesis and Photolysis of Iodinated 4-Azidoperfluorobenzoates and 6-Azido3,5,6-trifluorobenzoates.J. Org. Chem. 57, 1299-1304. (e) Cai, S. X., and Keana, J. F. W. (1991) Dizo and Azido-
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Functionalized Glutaraldehydes as Cross Linking Reagents and PotentailFixatives for Electron Microscopy. Bioconjugate Chem. 2, 38-43. (12) (a) Banks, R. E., and Sparkes, G. R. (1972) Studies in Azide Chemistry. Part V. Synthesis of 4-Azido-2,3,5,6-tetrafluoro,4Azido-3-chloro-2,5,6-trifluoro-, and 4-Azido-3,5-dichloro-2,6difluoro-pyridine, and Some Thermal Reactions of the Tetrafluorocompound. J.Chem.Soc.,Perkin Trans. 12964-2970. (b) Banks, R. E., and Prakash, A. (1974) Studies in Azide Chemistry. Part VI. Some Reactions of Perfluoroazidobenzene and Perfluoro-4-azidotoluene. J. Chem. SOC.,Perkin Trans. 1 1365-1371. (c) Banks, R. E., and Prakash, A. (1973) New Reactions of Azidopentafluorobenzene; Intermolecular Insertions into the N-H Bonds. Tetrahedron Lett. 99-102. (d) Banks, R. E., and Medany, I. M. (1990) Studies in Azide Chemistry. Part 14. Thermolysisof Perfluoro-4-azidopyridine and Perfluoro-4-azidotoluenein the Presence of Penta- and Hexa-Methylbenzene. J. Fluorine Chem. 47, 527-532. (e) Banks, R. E., and Venayak, N. D. Studies in Azide Chemistry. Part 13. Intermolecular Insertion of Azido-derived Polyfluorinated Aryl- and Heteroaryl-Nitrenes into Ring C-H Bonds of 1,3,5-Trimethyl and 1,3,5-Trimethoxybenzene.J. Chem. SOC.,Chem. Commun. 900-901. (f)Banks, R. E., and Madany, I. M. (1985) Decisive Evidence for the Occurrence of Ringexpansion During Pyrolisis of Azidopentafluorobenzxene: X-Ray CrystallographicAnalysis of Pentafluorophenyl-nitrene Dimer. J. Fluorine Chem. 30, 211-226. (13) (a) Crocker, P. J., Imai, N., Rajogopalen, K., Boggess, M. A., Kwiatkowski,S., Dwyer, L. D., Vanaman, T. C., and Watt, D. (1990) Heterobifunctional Cross-LinkingAgents Incorporating Perfluorinated Aryl Azides. Bioconjugate Chem.3,419. (b) Drake, R. R., Slam, J., Wall, K., Abramove, M., D’Souza, C., Elbein, A. D., Crocker, P. J., and Watt, D. S. (1992) Application of an N-(4-Azido-2,3,5,6-tetrafluorobenzoyl)tyrosine Substituted Peptide as a Heterobifunctional Crosslinking Agent in a Study of Protein 0-Glycosylation in Yeast. Bioconjugate Chem. 3,69-73. (14) (a) Pinney, K. C., and Katzenellenbogen, J. A. (1991) Synthesisof aTetrafluoro-substituted Aryl Azide and ItsProtio Analogue as Photoaffinity LabellingReagents for the Estrogen Receptor. J. Org. Chem. 56, 3125-3133. (b) Pinney, K. C., Carlson, K. E., Katzenellenbogen,B. S., and Katzenellenbogen, J. A. (1991) Efficient and Selective Photoaffinity Labelling of the Estrogen Receptor Using Two Nonsteroidal Ligands that Embody Aryl Azide or Tetrafluoroaryl Azide Photoreactive Functions. Biochemistry 30, 2421. (15) Poe, R., Schnapp, K., Young, M. J. T., Grayzar, J., and Platz, M. S. (1992) Chemistry and Kinetics of Singlet (Pentafluoropheny1)Nitrene. J.Am. Chem. SOC.114, 5054. (16) Soundararajan, N., and Platz, M. S. (1990) Descriptive Photochemistryof PolyfluorinatedAzide Derivatives of Methyl Benzoate. J. Org. Chem. 55,2034. (17) The Marquardt algorithm is described Bevington, P. R. (1983) Data Reduction and Error Analysis for the Physical Sciences, McGraw-Hill, New York. We utilized a program based on the Marquardt algorithm written by Shamim Ahmed. (18) The pyridine probe method is basedupon models developed by Scaiano to probe free-radical reactions. For examples, see: (a) Small, R. D., Jr., and Scaiano, J. C. (1977) Reaction of Type I1 Biradicals with Paraquat Ions Measurement of a BiradicalLifetime. J. Phys. Chem. 81,828. (b) Small, R. D., Jr., and Scaiano,J. C. (1977)Direct Detection of the Biradicals Generated in the Norrish Type I1Reaction. Chem.Phys. Lett. 50, 431. (c) Ibid. (1978) 59, 246. (19) Birchall, J. M., Hazeldine, R. N., and Kemm, J. E. G. (1970) Polyfluoroarenes. Part X. Polyfluoroaromatic Azo-compounds. J. Chem. SOC.449. (20) Soundararajan, N., Platz, M. S., Jackson, J. E., Doyle, M. P., Oon, S.-M., Liu, M. T. H., and Anand, S. M. (1988)Addition of Arylchlorocarbenes to a,@-UnsaturatedEsters. Absolute Rates, Substituent Effects and Variable Reactivities. J. Am. Chem. SOC.110, 7143.
