Synthesis of photoaffinity probes for heme-containing proteins

Article abstract of DOI:10.1021/jo981095e

Aryldiazenes with a second, photoactivatable, azide substituent on the aryl ring have been synthesized as active site probes for heme proteins of unknown active site structure. The probes include most of the possible isomers of the phenyl, naphthyl, and biphenyl ring systems. A selection of the probes has been shown to react with the model protein P450(cam) to give (arylazido)iron (Fe-ArN₃) complexes, a prerequisite for subsequent affinity labeling of the protein.

Full text of DOI:10.1021/jo981095e

J . Org. Chem. 1998, 63, 9711-9715
9711
Syn th esis of P h otoa ffin ity P r obes for Hem e-Con ta in in g P r otein s
Richard A. Tschirret-Guth and Paul R. Ortiz de Montellano*,†
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California,
San Francisco, California 94143-0446
Received J une 9, 1998
Aryldiazenes with a second, photoactivatable, azide substituent on the aryl ring have been
synthesized as active site probes for heme proteins of unknown active site structure. The probes
include most of the possible isomers of the phenyl, naphthyl, and biphenyl ring systems. A selection
of the probes has been shown to react with the model protein P450cam to give (arylazido)iron (Fe-
ArN3) complexes, a prerequisite for subsequent affinity labeling of the protein.
Sch em e 1
The study of membrane-bound cytochrome P450 en-
zymes has been hindered by a lack of the structural
information that can usually be obtained by X-ray
crystallography or nuclear magnetic resonance. In an
effort to bridge this gap, we have introduced the use of
aryldiazenes to probe the active site of heme-containing
proteins.1-3 Aryldiazenes react with hemoproteins to form
stable σ-bonded Fe-aryl complexes.4,5 In the case of the
cytochromes P450, which have a thiolate ligand, this
complex can be oxidized in the intact active site resulting
in a regioselective shift of the aryl group to one or more
of the pyrole nitrogens of the heme (Scheme 1).6,7 The
direction of the shift is primarily governed by the steric
encumbrance within the active site. Extraction, HPLC
separation, and quantitation of the N-arylprotoporphyrin
IX adducts can thus be used to obtain a low-resolution
topological map of the active site. This method is limited,
however, since it does not give information on the
residues that interact with the aryl probes. We have
undertaken, therefore, to prepare aryldiazenes bearing
a photoreactive group that can cross-link to the protein
which, after oxidative shift of the aryl group to the heme,
would result in covalent attachment of the heme to the
protein. We have recently validated the potential of this
approach by successfully cross-linking His64 to the heme
in myoglobin using (m-azidophenyl)diazene or (p-azi-
dophenyl)diazene,8 and we here report the synthesis of
a series of (azidoaryl)diazenecarboxylate esters as po-
tential general probes for active site mapping of heme-
containing proteins.
Sch em e 2
late ester group as the protective group for the hydrazine
(see below). Initial attempts to prepare azide 2 (R ) t-Bu)
by diazotation of amine 1 in diluted HCl at 4 °C and
subsequent nucleophilic displacement by NaN3 (Scheme
2) gave a complex mixture containing less than 3% of the
desired product. Application of the method developed by
Melhado and Leonard,10 in which diluted HCl was
replaced by 80% AcOH, still gave a mixture but 2 and 3
were obtained in 15% and 14% yield, respectively. By
adding NaN3 prior to NaNO2 and carrying out the
reaction at room temperature, we obtained azides 2 and
3 in 10 and 40% yield, respectively, along with unreacted
amine 1 as the sole products of the reaction. This latest
result clearly indicated that (a) addition of NaN3 prior
to diazotation diminishes the number of products formed
and (b) NaNO2 oxidizes the hydrazine 2 to the diazene
3, and thus excess reagent is required to carry the
reaction to completion. To the best of our knowledge,
oxidation of acylhydrazines by sodium nitrite has not
been previously reported. Following these observations,
we devised a simple and rapid method for obtaining
(azidoaryl)diazenes from the corresponding (nitroaryl)-
hydrazines (Scheme 3). In this scheme the reaction is
carried out in AcOH and the intermediate amine is not
isolated but is immediately converted to the final product
by addition of NaN3 and 2-3 equiv of NaNO2. A range
of compounds was synthesized using this methodology
(Figure 1). Table 1 gives the yields obtained for the
synthesis of a series of phenyl, biphenyl, and naphthyl
Aryldiazenes are moderately unstable and are best
prepared immediately prior to use by base hydrolysis of
aryldiazenecarboxylate esters.9 The synthetic scheme for
these compounds was therefore designed with carboxy-
† Tel: (415) 476-2903. Fax: (415) 502-4728. E-mail: ortiz@cgl.ucsf.edu.
(1) Ortiz de Montellano, P. R. Biochimie 1995, 30, 581.
(2) Mackman, R.; Guo, Z.; Guengerich, F. P.; Ortiz de Montellano,
P. R. Chem. Res. Toxicol. 1996, 9, 223.
(3) Gerber, N. C.; Rodriguez-Crespo, I.; Nishida, C. R.; Ortiz de
Montellano, P. R. J . Biol. Chem. 1997, 272, 6285.
(4) Ringe, D.; Petsko, G. A.; Kerr, D. E.; Ortiz de Montellano, P. R.
Biochemistry 1984, 23, 2.
(5) Raag, R.; Swanson, B. A.; Poulos, T. L.; Ortiz de Montellano, P.
R. Biochemistry 1990, 29, 8119.
(6) Tuck, S. F.; Aoyamal, Y.; Yoshida, Y.; Ortiz de Montellano P. R.
J . Biol. Chem. 1992, 2672, 13175.
(7) Tuck, S. F.; Ortiz de Montellano, P. R. Biochemistry. 1992, 31,
6911.
(8) Tschirret-Guth, R. A.; Medzihradszky, K. F.; Ortiz de Montellano,
P. R. J . Am. Chem. Soc. 1998, 120, 7404.
(9) Huang, P. C.; Kosower, E. M. J . Am. Chem. Soc. 1968, 90, 2354.
(10) Melhado, L. L.; Leonard, N. J . J . Org. Chem. 1983, 48, 5130.
10.1021/jo981095e CCC: $15.00 © 1998 American Chemical Society
Published on Web 12/03/1998

9712 J . Org. Chem., Vol. 63, No. 26, 1998
Tschirret-Guth and Ortiz de Montellano
Sch em e 4
Sch em e 5
was detected when the reactions were out carried above
0 °C.
A possible limitation in the use of aryl azide for
photoaffinity labeling is the short lifetime of the photo-
generated singlet nitrene and ring expansion to a
didehydroazepine.11a-d Substitution with fluorine at posi-
tions 2 and 6 relative to the azido group raises the barrier
of ring expansion to 8 kcal/mol and increases the life-
time of the nitrene from 10-100 ps to 10-100 ns at room
temperature.12,13 The fluorinated arylhydrazines 15a ,b
were therefore also prepared by reacting hydrazine with
2,4,6-trifluoronitrobenzene (14a) and pentafluoronitroben-
zene (14b), respectively, followed by immediate reaction
of the arylhydrazine with ClCOOEt to prevent further
reaction (Scheme 5). Addition of hydrazine ortho to the
nitro group predominated in both cases. The yields of the
desired para adduct could not be improved by varying
temperature or solvent or by replacing NH2NH2 with
NH2NHLi or NH2NHNa.14 Conversion of hydrazines
15a ,b to the corresponding (azidoaryl)diazene did not
suffer from the fluorine substitution, and 7a ,b were
obtained in high yields (Table 1).
Figure 2 shows the UV spectra of the reactions of the
diazenes generated from base hydrolysis of 6a , 10a , and
13b with camphor-bound cytochrome P450cam. The initial
Soret absorbance at 395 nm is lost and is replaced by a
less intense band at 478 nm characteristic for thiol-
ligated Fe-aryl complexes.5 These results show that
addition of the azido group did not hamper the formation
of the Fe-aryl complexes, a prerequisite if they are to
be used to photolabel hemoprotein active sites. The
reaction with 4′-azido-4-biphenyldiazene is slightly slower
(Figure 2B), a not unexpected result given the larger size
of the probe and the fact that the resulting aryl-iron
F igu r e 1. Structures of the starting materials, intermediates,
and products.
Sch em e 3
Ta ble 1. P r ep a r a tion of (Nitr oa r yl)h yd r a zin es a n d
Con ver sion to (Azid oa r yl)d ia zen e
yield
(%)
yield
(%)
from NO2ArNHNHCO2Et
N3ArNdNCO2Et
4a
4b
14a
14b
8a
8b
8c
8d
11a
11b
11c
11d
5a
5b
15a
15b
9a
9b
9c
9d
12a
12b
12c
12d
90
6a
6b
7a
7b
10a
10b
10c
10d
13a
13b
13c
13d
68
72
63
61
77
92
91
86
52
41
55
72
98
5a
18a
78b
70b
17b
1b
57b
85b
53b
3b
a
b
Scheme 5. Scheme 4.
azido-diazene probes. The procedure appears to be of
wide scope, and yields in the series of compound that
were examined were high irrespective of the structure
of the starting compound. The (nitrobiphenyl)hydrazines
9a -d and (nitronaphthyl)hydrazines 12a -d were pre-
pared from the corresponding (nitroaryl)amines 8a -d
and 11a -d , respectively, by diazotation in 6 N HCl
followed by reduction with SnCl2 and derivatization with
ClCOOEt (Scheme 4, Table 1). In the case of the naphthyl
derivatives, temperature control was found to be critical
during diazotation and reduction, and no desired product
(11) (a) Doering, W. von E.; Odum, R. A. Tetrahedron 1966, 22, 81.
(b) Schrock, A. K.; Schuster, G. B. J . Am. Chem. Soc. 1984, 106, 5228.
(c) Leyva, E.; Platz, M. S.; Persy, G.; Wirz, J . J . Am. Chem. Soc. 1986,
108, 3738. (d) Li, Y.-Z.; Kirby, J . P.; George, M. W.; Poliakoff, M.;
Schuster, G. B. J . Am. Chem. Soc. 1988, 110, 8092.
(12) Platz, M. S. Acc. Chem. Res. 1995, 28, 487.
(13) Smith, A. S.; Cramer, C. J . J . Am. Chem. Soc. 1996, 118, 5490.
(14) Selivanova, G. A.; Chuikova, T. V.; Shtark, A. A.; Shteingarts,
V. D. Zh. Org. Khim. 1988, 24, 2513.

Aryl Azide Hemoprotein Probes
J . Org. Chem., Vol. 63, No. 26, 1998 9713
2-(4-Nit r op h en yl)h yd r a zin eca r b oxyla t e, et h yl est er
(5b): obtained as a pale brown solid (90%), mp 195-196 °C
1
(dec); H NMR δ (DMSO-d6) 1.193 (t, J ) 6.9 Hz, 3H), 4.072
(q, J ) 6.9 Hz, 2H), 6.725 (d, J ) 9.0 Hz, 2H), 8.064 (d, J )
9.0 Hz, 2H), 8.961 (s, 1H), 9.379 (s, 1H); 13C NMR δ (DMSO-
d6) 14.495, 60.713, 110.259, 125.892, 138.038, 154.997, 156.388;
IR ν (KBr) 3342, 3301, 1682, 1506, 1332; HRMS (EI) calcd for
C9H11N3O4 (M+) m/e 225.0749, found m/e 225.0753.
P r oced u r e for th e P r ep a r a tion of o-F lu or in a ted 2-(Ni-
tr op h en yl)h yd r a zin eca r boxyla te Ester s. (2,4,6-Trifluo-
ronitro)benzene or pentafluoronitrobenzene (5-30 mmol) was
dissolved in 50-100 mL of acetonitrile and pyridine (2.2 equiv).
Hydrazine (1.1 equiv) was added and the mixture stirred for
an additional 10 min. After addition of ethyl chloroformate (1.1
equiv), the mixture was evaporated and the residue purified
by column chromatography.
2-(3,5-Diflu or o-4-n it r op h en yl)h yd r a zin eca r b oxyla t e,
eth yl ester (15a ): obtained as a beige solid (5%), mp 118-
120 °C; 1H NMR δ (CDCl3) 1.292 (t, J ) 7.2 Hz, 3H), 4.210 (q,
J ) 7.2 Hz, 2H), 6.458 (d, J H-F ) 11.1 Hz, 2H), 6.845 (s, 1H),
6.950 (s, 1H); 13C NMR δ (acetone-d6) 14.746, 62.220, 95.664
(d, J C-F ) 100.8 Hz), 121.348 (t, J C-F ) 39.5 Hz), 155.420 (t,
J C-F ) 52.8 Hz), 157,145, 157.860 (d, J C-F ) 1027.5 Hz); IR ν
(KBr) 3362, 3289, 1685, 1531, 1344; HRMS (EI) calcd for C9H9
F2N3O4 (M+) m/e 261.0561, found m/e 261.0565.
F igu r e 2. Reaction of P450cam with (A) (m-azidophenyl)-
diazene, (B) (4-(4-azidophenyl)phenyl)diazene, and (C) (6-
azido-2-naphthyl)diazene. The original Soret band of camphor-
bound P450cam (395 nm) is replaced by a band at 480 nm,
typical of Fe-aryl complexes.1
2-(3,5-Diflu or o-2-n it r op h en yl)h yd r a zin eca r b oxyla t e,
eth yl ester (16a ): obtained as a yellow solid (70%), mp 90-
92 °C; 1H NMR δ (acetone-d6) 1.235 (t, J ) 6.9 Hz, 3H), 4.158
(q, J ) 6.9 Hz, 2H), 6.668 (ddd, J ) 2.4, 8.7, 11.4 Hz, 1H),
6.801 (ddd, J ) 2.1, 2.4, 11.7 Hz, 1H), 8.586 (s, 1H), 8.789 (s,
complex extends to a height of approximatively 10 Å
above the heme plane.
In conclusion, we report the synthesis of a family of
chemical probes for mapping the active sites of heme-
containing proteins. The probes, which form (azidoaryl)-
iron complexes with the heme iron atom, place a photo-
activatable moiety at defined distances and orientations
with respect to the heme plane. Photocross-linking to the
protein should provide, as found in a test case,8 important
information on the residues that define the active site
and their topological arrangement.
1H); 13C NMR δ (acetone-d6) 14.708, 62.360, 95.738 (t, J C-F
)
105.9 Hz), 97.028 (d, J C-F ) 117.0 Hz) 122.784 (d, J C-F ) 38.7
Hz), 148.814 (d, J C-F ) 55.2 Hz), 157.173, 159.106 (dd, J C-F
)
64.4, 1035.6 Hz), 166.178 (dd J C-F ) 64.4, 1035.6 Hz); IR ν
(KBr) 3319, 1698, 1585, 1341; HRMS (EI) calcd for C9H9F2N3O4
(M+) m/e 261.0561, found m/e 261.0564.
2-(2,3,5,6-Tetr a flu or o-4-n itr op h en yl)h yd r a zin eca r box-
yla te, eth yl ester (15b): obtained as a beige solid (18%), mp
135-136 °C; 1H NMR δ (CDCl3) 1.287 (t, J ) 7.2 Hz, 3H), 4.127
(q, J ) 7.2 Hz, 2H), 6.245 (s, 1H), 6.772 (s, 1H); 13C NMR δ
(acetone-d6) 14.729, 62.326, 121.692 (t, J C-F ) 39.6 Hz),
134.225, 137.144 (dd, J C-F ) 54.9, 980.4 Hz), 143.044 (dd, J C-F
) 53.7, 1035.0 Hz), 157.476; IR ν (KBr) 3333, 3308, 1684, 1560,
1345; HRMS (EI) calcd for C9H7F4N3O4 (M+) m/e 297.0373,
found m/e 297.0362.
Exp er im en ta l Section
CAUTION: All naphthalene amino products are suspected
carcinogens and should be handled accordingly.
Gen er a l Com m en ts. Melting points are uncorrected. 1H
and 13C NMR spectra were recorded at 300 MHz using TMS
as a standard. IR spectra (cm-1) were obtained in KBr on a
FT spectrophotometer. Mass spectra were obtained by at the
University of California Mass Spectrometry Facility. Starting
materials were prepared according to standard procedures. All
reagents were purchased from chemical suppliers and used
without further purification. Chromatography was carried out
with Merck 60 (230-600 mesh) silica gel. Cytochrome P450cam
was expressed and purified as previously described.5
2-(2,3,4,5-Tetr a flu or o-6-n itr op h en yl)h yd r a zin eca r box-
yla te, eth yl ester (16b): obtained as a yellow solid (37%),
mp 106-107 °C; 1H NMR δ (CDCl3, tms) 1.179 (t, J ) 7.2 Hz,
3H), 4.078 (q, J ) 7.2 Hz, 2H), 7.854 (s, 1H), 8.658 (s, 1H); 13
C
NMR δ (acetone-d6) 14.660, 62.433, 126.843 (d, J C-F ) 42.9
Hz), 131.135 (d, J C-F ) 42.9 Hz), 135.105 (td, J C-F ) 57.3, 982.2
Hz), 139.068 (dd, J C-F ) 46.8, 1030.8 Hz), 142.463 (dd, J C-F
)
49.8, 1014.3 Hz), 143.889 (td, J C-F ) 55.8, 1016.1 Hz), 157.943;
IR ν (KBr) 3304, 1692, 1557, 1363; HRMS (EI) calcd for
C9H7F4N3O4 (M+) m/e 297.0373, found m/e 297.0361.
Ir on -Ar yl Com p lex F or m a tion w ith P 450ca m . The dia-
zenecarboxylate esters (0.5 mg) were dissolved in 40 µL of
methanol and hydrolyzed by addition of 2 µL of 2 N NaOH.
The diazene solution thus formed (2 µL) was then added to
500 µL of a solution of P450cam (10 µM in 50 mM phosphate
buffer pH 7.3).
P r ep a r a t ion of (Nit r op h en yl)h yd r a zin eca r b oxyla t e
Ester s. The (nitrophenyl)hydrazines were dissolved in 100 mL
of acetonitrile at room temperature. Pyridine (1.1 equiv) and
ethyl chloroformate (1.1 equiv) were added successively, and
the mixture was stirred for 10 min. The products were purified
by column chromatography (50:50 hexanes/ethyl acetate).
2-(3-Nit r op h en yl)h yd r a zin eca r b oxyla t e, et h yl est er
(5a ): obtained as a yellow solid (98%), mp 106-108 °C; 1H
NMR δ (CDCl3) 1.286 (m, 3H), 4.213 (q, J ) 6.9 Hz, 2H), 6.468
(s, 1H), 6.863 (s, 1H), 7.059 (dd, J ) 8.1, 1.8 Hz, 1H), 7.298 (t,
J ) 8.1 Hz, 1H), 7.611 (d, J ) 1.8 Hz, 1H), 7.658 (d, J ) 8.1
Hz, 1H); 13C NMR δ (CDCl3) 14.371, 62.316, 107.560, 115.254,
118.585, 129.767, 149.176, 149.351, 157.071; IR ν (KBr) 3369,
3297, 1696, 1528, 1350; HRMS (EI) calcd for C9H11N3O4 (M+)
m/e 225.0749, found m/e 225.0755.
Gen er al P r ocedu r e for th e P r epar ation s of (Nitr oar yl)-
h yd r a zin eca r boxylic Acid , Eth yl Ester s fr om th e Cor -
r esp on d in g Am in e. To a suspension of 100-300 mg of the
starting amine in 15 mL of 6 N HCl at -4 °C was added
sodium nitrite (1.1 equiv in 0.2-0.5 mL of water). The mixture
was allowed to react for 15 min before addition of stannous
chloride (2.5 equiv in 0.4-1 mL of 12 N HCl). Stirring and
cooling were maintained for another 15 min. The mixture was
then neutralized with saturated sodium carbonate and ex-
tracted three times with ethyl acetate. The organic layers were
combined, washed with brine, dried over magnesium sulfate,
and evaporated to dryness. The residue was immediately
redissolved in 15 mL acetonitrile, and pyridine and ethyl
chloroformate were added in 1.1 equiv each. The mixture was
stirred for 10 min, and after removal of the solvent under
vacuum, the residue was purified by column chromatography.
2-(4-(4-Nitr oph en yl)ph en yl)h ydr azin ecar boxylate, eth -
yl ester (9a ): obtained as an orange solid (59%), mp 210-
1
212 °C; H NMR δ (CD3CN) 1.223 (t, J ) 6.9 Hz, 3H), 4.113
(q, J ) 6.9 Hz, 2H), 6.572 (s, 1H), 6.882 (d, J ) 8.7 Hz, 2H),

9714 J . Org. Chem., Vol. 63, No. 26, 1998
Tschirret-Guth and Ortiz de Montellano
7.393 (s, 1H), 7.599 (d, J ) 8.7 Hz, 2H), 7.780 (d, J ) 8.7 Hz,
2H), 8.228 (d, J ) 8.7 Hz, 2H); 13C NMR δ (DMSO-d6) 14.557,
60.348, 112.003, 124.043, 126.129, 127.127, 127.943, 145.337,
146.803, 150.464, 156.789; IR ν (KBr) 3318, 1684, 1586, 1341;
HRMS (EI) calcd for C15H15N3O4 (M+) m/e 301.1063, found m/e
301.1058.
2-(4-(3-Nitr oph en yl)ph en yl)h ydr azin ecar boxylate, eth -
yl ester (9b): obtained as a yellow solid (76%), mp 155-156
°C; 1H NMR δ (CDCl3) 1.300 (t, J ) 7.2 Hz, 3H), 4.226 (q, J )
7.2 Hz, 2H), 5.971 (s, 1H), 6.638 (s, 1H), 6.935 (d, J ) 8.1 Hz,
2H), 7.509 (d, J ) 8.1 Hz, 2H), 7.546 (t, J ) 8.4 Hz, 1 H), 7.838
(d, J ) 8.4 Hz, 1H), 8.121 (d, J ) 8.4 Hz, 1H), 8.375 (s, 1H);
13C NMR δ (acetone-d6) 14.911, 61.544, 113.652, 121.101,
121.513, 128.499, 129.991, 130.828, 132.952, 143.619, 149.778,
151.092, 157.80; IR ν (KBr) 3395, 3245, 1708, 1515, 1345;
HRMS (EI) calcd for C15H15N3O4 (M+) m/e 301.1063, found m/e
301.1062.
Hz, 2H), 6.466 (s, 1H), 6.852 (s, 1H), 7.076 (d, J ) 8.7 Hz, 1H),
7.256 (t, J ) 7.8 Hz, 1H), 7.667 (d, J ) 8.7 Hz, 1H), 7.898 (d,
J ) 7.8 Hz, 1H), 7.911 (s, 1H), 8.213 (d, J ) 7.8 Hz, 1H); 13C
NMR δ (CDCl3) 14.450, 62.258, 101.762, 116.450, 120.929,
125.268, 130.122, 130.233, 134.793, 144.634, 149.120, 157.071;
IR ν (KBr) 3352, 3308, 1688, 1632, 1517; HRMS (EI) calcd for
C13H13N3O4 (M+) m/e 275.0906, found m/e 275.0901.
Gen er a l P r oced u r e for th e P r ep a r a tion of (Azid oa r yl)-
d ia zen es. All azides were stored foil-wrapped at -20 °C and
showed no decomposition by TLC for 1-2 years. The parent
nitro compound was hydrogenated in 20 mL of acetic acid with
10% Pd/C in a Parr apparatus. Following reduction, the
solution was filtered and water was added to adjust the solvent
to 80% acetic acid. The flask was wrapped in aluminum foil,
and NaN3 and NaNO2, in 0.1 mg/µL solutions, were added
successively in 1-2 and 2-3 eq, respectively. The reaction
mixture was stirred at room temperature for 4-16 h and
monitored by TLC. The mixture was then evaporated to
dryness, redissolved in CH2Cl2, and purified by column chro-
matography (CH2Cl2).
2-(3-(4-Nitr oph en yl)ph en yl)h ydr azin ecar boxylate, eth -
yl ester (9c): obtained as a yellow solid (17%), mp 127-128
1
°C; H NMR δ (CD3CN) 1.278 (t, J ) 6.9 Hz, 3H), 4.234 (q, J
) 6.9 Hz, 2H), 6.093 (s, 1H), 6.739 (s, 1H), 6.882 (d, J ) 7.8
Hz, 1H), 7.019 (s, 1H), 7.087 (d, J ) 7.8 Hz, 1H), 7.314 (t, J )
7.8 Hz, 1H), 7.634 (d, J ) 8.7 Hz, 2H), 8.231 (d, J ) 8.7 Hz,
2H); 13C NMR δ (CDCl3) 14.447, 62.029, 111.676, 113.359,
119.887, 123.860, 127.696, 129.896, 139.717, 146.968, 147.541,
148.783, 157.171; IR ν (KBr) 3362, 3296, 1693, 1511, 1349;
HRMS (EI) calcd for C15H15N3O4 (M+) m/e 301.1063, found m/e
301.1061.
(3-Azid op h en yl)d ia zen eca r boxyla te, eth yl ester (6a ):
1
obtained as an orange oil (68%); H NMR δ (CDCl3) 1.496 (t,
J ) 6.9 Hz, 3H), 4.523 (q, J ) 6.9 Hz, 2H), 7.210 (dd, J ) 8.1,
1.8 Hz, 1H), 7.508 (t, J ) 8.1 Hz, 1H), 7.556 (d, J ) 1.8 Hz,
1H), 7.728 (d, J ) 8.1 Hz, 1H); 13C NMR δ (CDCl3) 13.976,
64.414, 112.413, 121.249, 123.821, 130.385, 141.533, 152.493,
161.810; IR ν (KBr) 2112, 1759; HRMS (EI) calcd for C9H9N5O2
(M+) m/e 219.0756, found m/e 219.0749.
2-(3-(3-Nitr oph en yl)ph en yl)h ydr azin ecar boxylate, eth -
yl ester (9d ): obtained as an orange solid (19%), mp 119-
(4-Azid op h en yl)d ia zen eca r boxyla te, eth yl ester (6b):
obtained as an orange oil (72%); H NMR δ (CDCl3) 1.474 (t,
1
1
120 °C. H NMR δ (CDCl3) 1.268 (t, J ) 6.9 Hz, 3H), 4.203 (q,
J ) 7.2 Hz, 3H), 1.491 (t, J ) 7.2 Hz, 3H), 4.522 (q, J ) 7.2
Hz, 2H), 4.555 (q, J ) 7.2 Hz, 2H), 7.163 (d, J ) 8.7 Hz, 2H),
7.972 (d, J ) 8.7 Hz, 2H), 8.057 (d, J ) 9.0 Hz, 2H), 8.405 (d,
J ) 9.0 Hz, 2H); 13C NMR δ (CDCl3) 14.128, 64.425, 119.683,
125.750, 145.798, 148.635, 161.946; IR ν (KBr) 2219, 1755;
HRMS (EI) calcd for C9H9N5O2 (M+) m/e 219.0756, found m/e
219.0765.
4-Azid o-3,5-d iflu or op h en yld ia zen eca r boxyla te, Eth yl
Ester (7a ): obtained as an orange oil (63%); 1H NMR δ (CDCl3)
1.470 (t, J ) 7.2 Hz, 3H), 4.523 (t, J ) 7.2 Hz, 2H), 7.562 (d,
J H-F ) 8.7 Hz, 0.4H), 7.660 (d, J ) 8.1 Hz, 1.6H); 13C NMR δ
(CDCl3) 14.057, 64.712, 107.868 (d, J C-F ) 96.6 Hz), 122.804
(t, J C-F ) 57.6 Hz), 147.331 (t, J C-F ) 29.7 Hz), 155.523 (dd,
J C-F ) 13.3, 1008.6 Hz), 161.439; IR ν (KBr) 2140, 1768; HRMS
(EI) calcd for C9H8F2N5O2 (M+ + 1) m/e 256.0646, found m/e
256.0650.
J ) 6.9 Hz, 2H), 6.153 (s, 1H), 6.831 (s, 1H), 6.845 (d, J ) 7.8
Hz, 1H), 6.994 (s, 1H), 7.050 (d, J ) 8.1 Hz, 1H), 7.278 (t, J )
7.8 Hz, 1H), 7.523 (t, J ) 8.1 Hz, 1H), 7.792 (d, J ) 7.8 Hz,
1H), 8.135 (d, J ) 8.1 Hz, 1H), 8.316 (s, 1H); 13C NMR δ
(CDCl3) 14.405, 61.976, 111.413, 112.913, 119.491, 121.744,
121.890, 129.433, 129.839, 139.496, 142.690, 148.445, 148.800,
157.243; IR ν (KBr) 3318, 1681, 1522, 1345; HRMS (EI) calcd
for C15H15N3O4 (M+) m/e 301.1063, found m/e 301.1060.
2-(5-Nitr o-2-n a p h th yl)h yd r a zin eca r boxyla te, eth yl es-
ter (12a ): obtained as a yellow solid (57%), mp 145-146 °C;
1H NMR δ (acetone-d6) 1.303 (t, J ) 7.2 Hz, 3H), 4.234 (q, J )
7.2 Hz, 2H), 6.307 (s, 1H), 6.750 (s, 1H), 7.157 (s, 1H), 7.186
(d, J ) 9.3 Hz, 1H), 7.393 (t, J ) 7.5 Hz, 1H), 7.840 (d, J ) 7.5
Hz, 1H), 7.989 (d, J ) 7.5 Hz, 1H), 8.369 (d, J ) 8.369 Hz,
1H); 13C NMR δ (CD2Cl2 + 5% CD3OD) 10.912, 58.633,
103.112, 115.193, 116.638, 117.573, 120.752, 121.232, 129.602,
132.737, 143.128, 143.638, 153.908; IR ν (KBr) 3335, 3303,
1697, 1514, 1334; HRMS (EI) calcd for C13H13N3O4 (M+) m/e
275.0906, found m/e 275.0908.
2-(6-Nitr o-2-n a p h th yl)h yd r a zin eca r boxyla te, eth yl es-
ter (12b): obtained as yellow solid (85%), mp 239-242 °C (dec);
1H NMR δ (DMSO-d6) 1.241 (t, J ) 6.9 Hz, 3H), 4.112 (q, J )
6.9 Hz, 2H), 7.022 (s, 1H), 7.196 (dd, J ) 0.9, 8.7 Hz, 1H),
7.834 (d, J ) 9.3 Hz, 1H), 8.051 (d, J ) 8.7 Hz, 1H), 8.071 (dd,
J ) 1.8, 8.7 Hz, 1H), 8.636 (s, 1H), 8.777 (d, J ) 0.9 Hz, 1H),
9.340 (s, 1H); 13C NMR δ (DMSO-d6) 14.498, 60.524, 103.280,
117.251, 119.534, 124.780, 125.309, 126.904, 131.419, 137.774,
141.598, 150.842, 156.605; IR ν (KBr) 3317, 1683, 1624, 1330;
HRMS (EI) calcd for C13H13N3O4 (M+) m/e 275.0906, found m/e
275.0912.
(4-Azid o-2,3,5,6-t et r a flu or op h en yl)d ia zen eca r b oxyl-
a t e, et h yl est er (7b): obtained as an orange oil (61%); 1H
NMR δ (CDCl3) 1.474 (t, J ) 7.2 Hz, 3H), 4.530 (q, J ) 7.2
Hz, 2H); 13C NMR δ (CDCl3) 14.095, 64.059, 109.057 (t, J C-F
) 91.8 Hz), 124.364 (t, J C-F ) 48.0 Hz), 140.632 (ddd, J C-F )
28.2, 72.6, 992.7 Hz), 141.430 (ddd, J C-F ) 14.7, 53.4, 1060.2
Hz), 160.911; IR ν (KBr) 2157, 1762; HRMS (EI) calcd for
C7F4N5O (M+ - C2H5O) m/e 246.0039, found m/e 246.0038.
(4-(4-Azid op h en yl)p h en yl)d ia zen eca r boxyla te, eth yl
ester (10a ): obtained as an orange oil (77%); 1H NMR δ
(CDCl3) 1.485 (t, J ) 6.9 Hz, 3H), 4.536 (q, J ) 6.9 Hz, 2H),
7.132 (d, J ) 8.7 Hz, 2H), 7.650 (d, J ) 8.7 Hz, 2H), 7.721 (d,
J ) 8.7 Hz, 2H), 8.015 (d, J ) 8.7 Hz, 2H); 13C NMR δ (CDCl3)
14.165, 64.423, 119.613, 124.453, 127.526, 128.892, 136.173,
140.492, 145.492, 150.737, 162.111; IR ν (KBr) 2102, 1755;
HRMS (EI) calcd for C15H13N5O2 (M+) m/e 295.1069, found m/e
295.1057.
2-(7-Nitr o-2-n a p h th yl)h yd r a zin eca r boxyla te, eth yl es-
ter (12c): obtained as an orange solid (53%), mp 154-155 °C;
1H NMR δ (CD3CN) 1.217 (br, 3H), 4.113 (q, J ) 7.2 Hz, 2H),
6.782 (s, 1H), 7.21 (m, 1H), 7.234 (s, 1H), 7.494 (s, 1H), 7.792
(d, J ) 9.6 Hz, 1H), 7.831 (d, J ) 9.0 Hz, 1H), 7.896 (dd, J )
2.1 Hz, J ) 8.7 Hz, 1H), 8.555 (d, J ) 2.1 Hz, 1H); 13C NMR
δ (CD3CN) 14.912, 62.332, 107.821, 116.770, 118.276, 120.260,
123.360, 126.760, 123.521, 130.031, 130.247, 131.884, 134.478,
147.196, 149.514, 157.952; IR ν (KBr) 3371, 3310, 1696, 1344;
HRMS (EI) calcd for C13H13N3O4 (M+) m/e 275.0906, found m/e
275.0902.
(4-(3-Azid op h en yl)p h en yl)d ia zen eca r boxyla te, eth yl
ester (10b): obtained as an orange oil (92%); 1H NMR δ
(CDCl3) 1.487 (t, J ) 7.2 Hz, 3H), 4.539 (q, J ) 7.2 Hz, 2H),
7.091 (d, J ) 7.5 Hz, 2H), 7.272 (d, J ) 1.2 Hz, 1H), 7.729 (m,
2H), 8.022 (d, J ) 8.7 Hz, 2H); 13C NMR δ (CDCl3) 14.128,
64.435, 117.789, 118.808, 123.812, 124.346, 127.915, 130.303,
140.816, 141.359, 145.372, 150.974, 162.070; IR ν (KBr) 2105,
1752; HRMS (EI) calcd for C15H13N5O2 (M+) m/e 295.1069,
found m/e 295.1059.
2-(8-Nitr o-2-n a p h th yl)h yd r a zin eca r boxyla te, eth yl es-
ter (12d ): obtained as an orange solid (55%), mp 141-142 °C;
1H NMR δ (CDCl3) 1.298 (t, J ) 6.9 Hz, 3H), 4.225 (q, J ) 6.9
(3-(4-Azid op h en yl)p h en yl)d ia zen eca r boxyla te, eth yl
ester (10c). Obtained as an orange oil (91%); 1H NMR δ
(CDCl3) 1.481 (t, J ) 6.9 Hz, 3H), 4.535 (q, J ) 6.9 Hz, 2H),

Aryl Azide Hemoprotein Probes
J . Org. Chem., Vol. 63, No. 26, 1998 9715
7.110 (d, J ) 8.4 Hz, 2H), 7.585 (t, J ) 7.8 Hz, 1H), 7.614 (d,
J ) 8.4 Hz, 2H), 7.773 (d, J ) 8.1 Hz, 1H), 7.902 (d, J ) 8.1
Hz, 1H), 8.122 (s, 1H); 13C NMR δ (CDCl3) 14.1112, 64.470,
119.530, 121.780, 122.552, 128.382, 129.745, 131.814, 136.210,
139.950, 141.355, 152.050, 162.043; IR ν (KBr) 2124, 1757;
HRMS (EI) calcd for C15H13N5O2 (M+) m/e 295.1069, found m/e
295.1066.
(7-Azid o-2-n a p h th yl)d ia zen eca r boxyla te, eth yl ester
(13c): obtained as an orange solid (55%), mp 63-64 °C; 1H
NMR δ (CD2Cl2) 1.452 (t, J ) 7.2 Hz, 3H), 4.500 (q, J ) 7.2
Hz, 2H), 7.274 (dd, J ) 9.0 Hz, J ) 2.1 Hz, 1H), 7.604 (d, J )
2.1 Hz, 1H), 7.786 (dd, J ) 9.0 Hz, J ) 2.1 Hz, 1H), 7.858 (d,
J ) 8.7 Hz, 1H), 7.877 (d, J ) 8.7 Hz, 1H), 8.424 (s, 1H); 13C
NMR δ (CD2Cl2) 14.328, 64.821, 115.212, 117.799, 121.936,
129.795, 130.314, 130.517, 133.724, 134.191, 139.518, 150.396,
162.624; IR ν (KBr) 2115, 1751; HRMS (EI) calcd for C13H11N5O2
(M+) m/e 269.0913, found m/e 269.0907.
(8-Azid o-2-n a p h th yl)d ia zen eca r boxyla te, eth yl ester
(13d ): obtained as an orange solid (72%), mp 83-85 °C; 1H
NMR δ (CDCl3) 1.496 (t, J ) 7.2 Hz, 3H), 4.508 (q, J ) 7.2
Hz, 2H), 7.332 (d, J ) 7.2 Hz, 1H), 7.601 (t, J ) 7.2 Hz, 1H),
7.659 (d, J ) 7.2 Hz, 1H), 7.845 (d, J ) 9.0 Hz, 1H), 7.930 (d,
J ) 9 Hz, 1H), 8.869 (s, 1H); 13C NMR δ (CDCl3) 14.189,
64.457, 115.072, 116.464, 124.481, 126.140, 126.933, 128.942,
129.273, 136.860, 138.849, 149.300, 162.205; IR ν (KBr) 2116,
1748; HRMS (EI) calcd for C13H11N5O2 (M+) m/e 269.0913,
found m/e 269.0905.
(3-(3-Azid op h en yl)p h en yl)d ia zen eca r boxyla te, eth yl
ester (10d ): obtained as an orange oil (86%); 1H NMR δ
(CDCl3) 1.484 (t, J ) 7.2 Hz, 3H), 4.540 (q, J ) 7.2 Hz, 2H),
7.056 (d, J ) 6.9 Hz, 1H), 7.252 (s, 1H), 7.389 (d, J ) 7.5 Hz,
1H), 7.443 (t, J ) 7.5 Hz, 1H), 7.604 (t, J ) 7.8 Hz, 1H), 7.786
(d, J ) 7.5 Hz, 1H), 7.933 (d, J ) 7.8 Hz, 1H), 8.134 (s, 1H);
13C NMR δ (CDCl3) 14.134, 64.538, 117.666, 118.449, 122.214,
122.963, 123.678, 129.812, 130.293, 132.166, 140.759, 141.389,
151.990, 162.022; IR ν (KBr) 2109, 1756; HRMS (EI) calcd for
C
15H13N5O2 (M+) m/e 295.1069, found m/e 295.1078.
(5-Azid o-2-n a p h th yl)d ia zen eca r boxyla te, eth yl ester
(13a ): obtained as a yellow solid (52%), mp 68-69 °C; 1H NMR
δ (CDCl3) 1.496 (t, J ) 7.2 Hz, 3H), 4.552 (q, J ) 7.2 Hz, 2H),
7.387 (d, J ) 7.5 Hz, 1H), 7.578 (7, J ) 7.5 Hz, 1H), 7.793 (d,
J ) 7.5 Hz, 1H), 7.906 (d, J ) 9.0 Hz, 1H), 8.169 (d, J ) 9.0
Hz, 1H), 8.547 (s, 1H); 13C NMR δ (CDCl3) 14.122, 64.423,
116.637, 115.918, 124.287, 126.382, 127.108, 128.616, 130.781,
134.170, 137.070, 149.907, 162.139; IR ν (KBr) 3113, 1750;
HRMS (EI) calcd for C13H11N5O2 (M+) m/e 269.0913, found m/e
269.0907.
Ack n ow led gm en t. This research was supported by
the National Institute of Health Grant GM25515. Mass
spectrometric data were obtained at the Biomedical
Bioorganic Mass Spectrometry Facility (A. Burlingame,
director) of the University of California at San Francisco
supported by National Institute of Health Grants RR
01614 and Liver Core Center 5 P30 DK26743.
(6-Azid o-2-n a p h th yl)d ia zen eca r boxyla te, eth yl ester
(13b): obtained as an orange solid (41%), mp 66-68 °C; 1H
NMR δ (CDCl3) 1.491 (t, J ) 7.2 Hz, 3H), 4.546 (q, J ) 7.2
Hz, 2H), 7.231 (dd, J ) 1.8, 8.7 Hz, 1H), 7.466 (s, 1H), 7.777
(d, J ) 8.7 Hz, 1H), 7.928 (dd, J ) 1.8, 8.7 Hz, 1H), 7.988 (d,
J ) 8.7 Hz, 1H), 8.528 (s, 1H); 13C NMR δ (CDCl3) 14.164,
64.385, 115.965, 116.952, 120.001, 128.372, 130.470, 131.607,
131.932, 136.856, 140.913, 148.966, 162.153; IR ν (KBr) 2108,
1747; HRMS (EI) calcd for C13H11N5O2 (M+) m/e 269.0913,
found m/e 269.0904.
Su p p or tin g In for m a tion Ava ila ble: 13C NMR spectra for
the compounds described this paper (26 pages). This material
is contained in libraries on microfiche, immediately follows this
article in the microfilm version of the journal, and can be
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J O981095E