Chemistry of bifunctional photoprobes. 3. Correlation between the efficiency of CH insertion by photolabile chelating agents and lifetimes of singlet nitrenes by flash photolysis: First example of photochemical attachment of 99mTc-complex with human serum albumin

Article abstract of DOI:10.1021/jo981458a

Systematic functionalization of perfluoroaryl azides with chelating agents capable of complexing transition metals produces a new class of bifunctional photolabile chelating agents (BFPCAs). The strategy to shield the azide functionality from the electronic and steric influence of the electron-rich metal Pd through ester and amide bridges raised CH insertion efficiency to unprecedented levels (>92%) in a model solvent (cyclohexane). In contrast, perfluoroaryl azides attached to chelating agents via hydrazones show no significant CH insertion in cyclohexane upon photolysis. Measurements of the lifetimes of the singlet nitrenes derived from these agents by flash photolysis techniques correlate well with the efficiency of CH insertion by demonstrating longer lifetimes (10-50 times) for singlet nitrenes derived from azidotetrafluorinated esters and amides compared with the related hydrazones, which failed to yield significant CH insertion. A representative BFPCA 12 is chelated to diagnostic radionuclide ^99mTc and covalently attached to human serum albumin via photochemical activation extending the favorable bimolecular insertion characteristics of BFPCA to tracer level concentrations in buffer conditions. Flash photolysis experiments correlate singlet nitrene lifetimes with the efficiency of intermolecular insertion reactions. This work provides new photo-cross-linking technology, useful in radiodiagnostics and radiotherapy in nuclear medicine.

Full text of DOI:10.1021/jo981458a

J . Org. Chem. 1998, 63, 9019-9030
9019
Ch em istr y of Bifu n ction a l P h otop r obes.1 3. Cor r ela tion betw een
th e Efficien cy of CH In ser tion by P h otola bile Ch ela tin g Agen ts
a n d Lifetim es of Sin glet Nitr en es by F la sh P h otolysis: F ir st
Exa m p le of P h otoch em ica l Atta ch m en t of 99m Tc-Com p lex w ith
Hu m a n Ser u m Albu m in
Raghoottama S. Pandurangi,*,†,‡ Przemyslaw Lusiak,† Robert R. Kuntz,† Wynn A. Volkert,§
J acek Rogowski, and Matthew S. Platz
Department of Chemistry, University of Missouri, Columbia, Missouri 65211, Radiology and Research
Service, H. S. Truman Memorial VA Hospital, University of Missouri, Columbia, Missouri 65211,
Department of Internal Medicine, University of Missouri, Columbia, Missouri 65212, and Department of
Chemistry, Ohio State University, Columbus, Ohio 43210
Received J uly 24, 1998
Systematic functionalization of perfluoroaryl azides with chelating agents capable of complexing
transition metals produces a new class of bifunctional photolabile chelating agents (BFPCAs). The
strategy to shield the azide functionality from the electronic and steric influence of the electron-
rich metal Pd through ester and amide bridges raised CH insertion efficiency to unprecedented
levels (>92%) in a model solvent (cyclohexane). In contrast, perfluoroaryl azides attached to
chelating agents via hydrazones show no significant CH insertion in cyclohexane upon photolysis.
Measurements of the lifetimes of the singlet nitrenes derived from these agents by flash photolysis
techniques correlate well with the efficiency of CH insertion by demonstrating longer lifetimes
(10-50 times) for singlet nitrenes derived from azidotetrafluorinated esters and amides compared
with the related hydrazones, which failed to yield significant CH insertion. A representative BFPCA
12 is chelated to diagnostic radionuclide 99mTc and covalently attached to human serum albumin
via photochemical activation extending the favorable bimolecular insertion characteristics of BFPCA
to tracer level concentrations in buffer conditions. Flash photolysis experiments correlate singlet
nitrene lifetimes with the efficiency of intermolecular insertion reactions. This work provides new
photo-cross-linking technology, useful in radiodiagnostics and radiotherapy in nuclear medicine.
In tr od u ction
receptors,6 and organic substrates carrying toxins or
radionuclides,7 resulting in the development of new
diagnostic and therapeutic agents. Although several
reported heterobifunctional photolabeling reagents in-
corporate aryl azide and 3H or 125I radiolabels at
termini,8-10 applications in nuclear medicine and antibody-
target cancer therapy require incorporation of transition
metals and their analogous radioisotopes.11 In particular,
Development of methods for covalent modification of
biomolecules produces new tools in biochemistry and
medicine.2-4 The technique of photolabeling has been a
valuable method for arming monoclonal antibodies,5
* To whom correspondence should be addressed: Raghoottama S.
Pandurangi, Ph.D., 601S, College Ave., 17, Chemistry Department,
University of Missouri, Columbia, MO 65211. Phone: (573) 884 2520.
Fax: (573) 882 2754. E-mail: raghu@ missouri.edu.
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(b) Haley, B. E. Methods Enzymol. 1991, 200, 477. (c) Neilson, P. E.
In Photochemical Probes in Biochemistry; Neilson, P. E., Ed.; NATO
ASI Series C, 272; Kluver Academic Publishers: Boston, 1989. (d)
Lewis, C. T.; Haley, B. E.; Carlson, G. M. Biochemistry 1989, 28, 9248.
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Bioconjugate Chem. 1990, 1, 132. (c) Meares, C. F.; McCall, M. J .;
Reardan, D. T.; Goodwin, D. A.; Diamanti, C. I.; McTigue, M. Anal.
Biochem. 1984, 142, 68. (d) Fritzberg, A. R.; Abrams, P. G.; Baumier,
P. L.; Kasina, S.; Morgan, A. C., J r.; Rao, T. N.; Reno, J . M. Proc. Natl.
Acad. Sci. U.S.A. 85, 4025. (e) Baidoo, K. E.; Lever, S. Z. Tetrahedron
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† Department of Chemistry, University of Missouri.
‡ Department of Internal Medicine, University of Missouri.
§ H. S. Truman Memorial VA Hospital.
Ohio State University.
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10.1021/jo981458a CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/30/1998

9020 J . Org. Chem., Vol. 63, No. 24, 1998
Pandurangi et al.
covalent attachment of 99mTc-based bifunctional photo-
labile chelating agent (BFPCA) to biomolecules produces
new diagnostic probes.
Sch em e 1
Perfluoroaryl azides constitute an important class of
photolabeling agents12-15 that are superior to conven-
tional aryl azide or nitroaryl azide reagents. Photolysis
of nonfluorinated aryl azides generates singlet aryl
nitrenes that are consumed rapidly by intramolecular
reactions to yield secondary intermediates (ketenimines,
triplet nitrenes) and which, unlike their polyfluorinated
analogues, do not insert into unactivated CH bonds
(Scheme 1).16,17
The efficiency of CH insertion by bifunctional meta-
lated photolabels into antibodies is the key requirement
for successful application of this method in targeted
cancer therapy.18 Labeling in the hydrophobic regions
of the antibody, primarily by insertion reactions, facili-
tates the preservation of immunoreactivity, which is
essential for transport of radioactivity to the antigen sites
(tumor).19 Recently, we reported the high retention of
immunoreactivity of the B72.3 antibody (>97%) after
photolabeling using a 14C-labeled photoprobe.20 The next
milestone is the demonstration of efficient photoconju-
gation by BFPCAs carrying 99mTc and other useful
transition metals (e.g 109Pd, 105Rh, 186Re, etc.).
To our knowledge, the intermolecular insertion chem-
istry of bifunctional photolabels carrying transition met-
als in model solvents and proteins is relatively unex-
plored,21 although introduction of an electron-rich metal
such as Pd has been shown to dramatically22 reduce the
CH insertion yield of perfluoroaryl nitrenes. The design
of useful photoprobes requires an understanding of the
effect of substituents on the rate of rearrangement,
intersystem crossing, and the bimolecular reactivity of
singlet aryl nitrenes which has prompted this investiga-
tion. Modifications of the tether linking perfluoroaryl
azides by transition metals may, therefore, affect both
the quantum yield for singlet nitrene formation and the
efficiency of the insertion reactions. Herein we report
the effect of electronic and steric separation of the
photolabile group and the metal chelate on the CH
insertion yield in cyclohexane and its correlation with
singlet nitrene lifetimes, as measured by laser flash
photolysis techniques. Our data show a direct correlation
between nitrene C-H insertion efficiency and singlet
lifetime. We report the largest CH insertion yield of a
singlet aryl nitrene in cyclohexane ever observed (>92%).
We also extend the results in model organic solvents to
buffer conditions at tracer level concentrations by cova-
lent attachment of the 99mTc-complex of BFPCA to human
serum albumin (HSA). This new technology may be
useful for hepatic imaging.7g
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Resu lts
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(19) Buchsbaum D. J .; Lawrence T. S. Antibody, Immunoconjugates
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Syn th esis. The general synthetic targets are hetero-
bifunctional photolabel agents with a specific ligating
(21) For a recent review see: (a) Pandurangi, R. S.; Karra, S. R.;
Kuntz, R. R.; Volkert, W. A. Photochem. Photobiol. 1997, 66, 208. (b)
Pandurangi, R. S.; Kuntz, R. R.; Volkert, W. A.; Barnes, C. L.; Katti,
K. V. J . Chem. Soc., Dalton Trans. 1995, 565. (c) Pandurangi, R. S.;
Karra, S. R.; Kuntz, R. R.; Volkert, W.A. Bioconjugate Chem. 1995, 6,
630. (d) Pandurangi, R. S.; Katti, K. V.; Barnes, C. L.; Volkert, W. A.;
Kuntz, R. R. Chem. Commun. 1994, 1841. (e) Pandurangi, R. S., Kuntz,
R. R., and Volkert, W. A. App. Rad. Isotopes, 1995, 46, 233. (f)
Pandurangi, R. S.; Katti, K. V.; Barnes, C. L.; Volkert, W. A.; Kuntz,
R. R. Inorg. Chem. 1996, 35, 3716.
(20) Pandurangi, R. S.; Karra, S. R.; Kuntz, R. R.; Volkert, W. A.
Photochem. Photobiol. 1996, 65, 106.
(22) Pandurangi, R. S.; Karra, R. S.; Katti, K. V.; Kuntz, R. R.;
Volkert, W. A. J . Org. Chem. 1997, 62, 2798.

Chemistry of Bifunctional Photoprobes
J . Org. Chem., Vol. 63, No. 24, 1998 9021
Sch em e 2
Sch em e 3
on the azidotetrafluorobenzamide (Scheme 3). For ex-
ample, harsh reaction conditions (refluxing for hours)
were necessary for PSCl3 to react with azidotetrafluo-
robenzamide in the presence of excess Et3N for the
formation of monosubstituted product. Addition of 2
equiv of methyl hydrazine at 0 °C in situ in the presence
of excess of Et3N produces 5 in good yield. 31P NMR of 5
shows a single phosphorus signal at δ 67 and a clean
doublet (3J (P-H) ) ∼11-12 Hz) at δ 2.97. 1H NMR
reveals the coupling of methyl protons with phosphorus
indicating the incorporation of methyl hydrazine.23a
The ester- and amide-linked reagents were prepared
as follows. Treatment of pentafluorobenzoyl chloride
with an excess of ethylene glycol in tetrahydrofuran
(THF), followed by refluxing overnight, resulted in the
formation of monosubstituted alcohol 7 in high yield
(Scheme 4). The unreacted glycol was separated easily
by flash column chromatography. Azidolysis of the
monoalcohol gave parasubstituted product 9, character-
ized by the AA′XX′ pattern of 19F NMR spectrum.
Refluxing P(X)Cl3 (X ) O, S) with 9 in the presence of
excess of Et3N can be monitored via 31P NMR to the
disappearance of 31P signals for PSCl3 or POCl3, and 2
equiv of methyl hydrazine was added in situ to obtain
the required perfluoroaryl azido-functionalized phospho-
rus hydrazides 11 and 12, respectively.
framework connected via different tethers. Phosphorus
hydrazides were chosen as ligating moieties because they
had been used successfully for chelation to diagnostic and
therapeutic radiometals23,24 99mTc and 109Pd). A simple
(
and convenient method that links the photolabel and the
chelating framework is Schiff base coupling between
perfluoroaryl azidobenzaldehyde and the amine group of
methyl hydrazine and similarly, with mono- and bishy-
drazidophosphorus compounds. Dropwise addition of an
equimolar solution of 4-azidotetrafluorobenzaldehyde in
absolute ethanol to a solution of methyl hydrazine or
diphenoxyphosphorylmethyl hydrazine 3 in ethanol at
room temperature forms hydrazones 1 and 4, respec-
tively, in high yields (>90%, Scheme 2). Coupling
perfluoroaryl azidobenzaldehyde with bishydrazidophos-
phorus sulfide was conducted at -70 °C to increase the
selectivity of monosubstitution, which is reacted with
PdCl2‚(PhCN)2 to give Pd complex 2 as reported earlier.21b
An alternate linker design introduces an electron-
withdrawing group para to the azide moiety in the form
of an ester or amide group. However, in this case the
reactivity of the P-Cl bond in PSCl3 toward nucleophilic
substitution is controlled by the less basic amide group
Similarly, the reaction of pentafluorobenzoyl chloride
with 1,4-butanediol, followed by azidolysis and treatment
with PSCl3 or POCl3 and 2 equiv of methyl hydrazine,
produced methylene-bridged perfluoroaryl azidophospho-
rus hydrazides 13 and 14. All the ligands were charac-
(23) (a) Katti, K. V.; Reddy, V. S.; Singh, P. R. Chem. Soc. 1995, 97
and references therein. (b) Katti, K. V.; Singh, P. R.; Barnes, C. L.
Inorg. Chem. 1992, 31, 4588. (c) Katti, K. V.; Singh, P. R.; Volkert, W.
A.; Ketring, A. R.; Katti, K. K. Appl. Radiat. Isot. 1992, 43, 1151. (d)
Katti, K. V.; Ge, Y. W.; Singh, P. R.; Date, S. V.; Barnes, C. L.
Organometallics 1994, 13, 541. (e) Katti, K. V.; Singh, P. R.; Barnes,
C. L. J . Chem. Soc., Dalton Trans. 1993, 2153. (f) Singh, P. R.; J imenez,
H.; Katti, K. V.; Volkert, W. A.; Barnes, C. L. Inorg. Chem. 1994, 33,
736. (g) Wang, M. W.; Volkert, E. W.; Singh, P. R.; Katti, K. K.; Lusiak,
P.; Katti, K. V.; Barnes, C. L. Inorg. Chem. 1994, 33, 1184.
(24) (a) Majoral, J . P.; Kramer, R.; Navech; Mathis, J . Tetrahedron
1976, 32, 2633 (b) Majoral, J . P.; Caminade. In Chemistry of Inorganic
Ring Systems. Studies in Inorganic Chemistry; V-14, Ed. Stuedel,
Elsevier: Amsterdam, 1992; pp 201. (c) Nicot, B. D.; Mathien, R.;
Montauzon de D.; Lavigne, G.; Majoral, J . P. Inorg. Chem. 1994, 33,
434. (d) Caminamide, A. M.; Majoral, J . P. Chem. Rev. 94, 1183.
1
terized by H and 31P NMR, and elemental analysis and
1H NMR showed characteristic doublets around δ 2.8-
3.0 due to the phosphorus coupling (3J (P-H) ) 10-12
Hz) with the N-methyl protons. The coupling constants
values are in the expected range for a number of similar
compounds.23a,24
Coor din ation Ch em istr y. The reaction of the ligands
(5, 11-14) with PdCl2(PhCN)2 was performed in CH2Cl2

9022 J . Org. Chem., Vol. 63, No. 24, 1998
Pandurangi et al.
Ta ble 1. P er cen t Yield s of CH In ser tion Ad d u cts
Obta in ed by P h otolysis of F u n ction a lized P er flu or oa r yl
Azid es in Cycloh exa n e, a n d th e Reten tion Tim es (in
Min u tes of th e CH In ser tion Ad d u cts on HP LCa
Sch em e 4
CH insertion
yielda (%)
retention time
(Rt) (min)
compound
1, 2, 4
5
6
11
12
13
14
16
<5
73
2.8
3.3
3.6
3.8
4.7
4.5
3.2
70 ( 5
90
93
92
89
75 ( 5
a
The solvent was CH3CN and H2O in a ratio of 2:1 (flow rate,
1 mL/min). b Measured by the integration of fluorine signals which
agrees well with the values obtained from chromatographic
separation of photoproducts using hexane:ethyl acetate:methanol
in 9:9:1 ratio on silica gel column. For NMR, a 2.5 × 10-3
M
solution of azide in cyclohexane was photolyzed in NMR tube for
1-2 h at room temperature at >320 nm.
ligand per metal center. Irradiation of ligands or com-
plexes did not affect the chelating backbone, as shown
by the lack of any appreciable change in the phosphorus
chemical shift of these compounds after photolysis.
P h otoch em ica l In ser tion . Inspection of Table 1
reveals that photolysis of tetrafluorinated aryl azides
containing a parahydrazone substituent in cyclohexane
fails to produce C-H insertion adducts. In each case,
the major products formed upon photolysis of 1, 2, and 4
were the corresponding anilines. Aniline-type reduction
products are usually associated with reactions of triplet
aryl nitrenes. This was confirmed by the finding that
triplet-sensitized photolysis of 1, 2, and 4, or direct
photolysis in methanol (a solvent that catalyzes inter-
system crossing of singlet nitrene to their triplet ground
states13) also forms the same aniline products.
at 25 °C to produce a series of Pd complexes. The
interaction of Pd(II) with phosphorus hydrazides can
occur in two possible ways, depending on the substitution
on phosphorus, although four coordination modes are
possible for other transition metals (for example, Cu,
Co).23a In general, the alkoxy group on phosphorus
makes the sulfur or oxygen atom less nucleophilic and
hence leads to the formation of a six-membered ring
through N-N coordination.23a However, the carbon
substituents on phosphorus (e.g. RP(X)(NMeNH2), R )
Me, Et, etc; and X ) S, O) are known to involve sulfur or
oxygen coordination with metals leading to a five-
membered ring.21b 1H NMR of the complexes (15-18)
revealed only one doublet around δ 3.7-3.2 assigned to
the NMe protons, which indicates that the 2 methyl
protons flanking phosphorus have similar environments.
This could be rationalized in terms of N-N coordination
with Pd, which affects NMe protons equally and is
confirmed by several X-ray crystal structures reported
on similar systems.23a In all the compounds the intense
absorption band around 2100-2150 cm-1 in the IR
spectrum indicates the photoactivable group is intact
during the chemical manipulations and is available for
photoreaction and bioconjugation with proteins and
antibodies. The 31P NMR of the complexes exhibit
moderate downfield shifts compared with the ligands and
exhibit a sharp single resonance indicating the formation
of a single species. The IR stretching frequency of PdS
for complexes did not show any significant shift compared
with the free ligands, indicating that sulfur is not
involved in the coordination with Pd. The C, H, and N
analyses of all the complexes reveal that they have one
Soundarajan and Platz25 reported that photolysis of 19
in cyclohexane leads to the formation of an adduct in 57%
yield, in contrast to our work with tetrafluorohydrazone
azides 1, 2, and 4.
We find that this is a general result. Compounds 5
and 11-16 are tetrafluorinated but have carboxamide
or ester functionality para to the azide substituent,
separated by methylene groups. Photolysis of these
azides in cyclohexane produces CH insertion adducts in
excellent yield (Table 1, Scheme 5).
The placement of an ester or amide group with a bridge
of methylene groups between the photolabel and the
chelating moiety improves upon our previously reported
results with similar compounds.21 For example, photoly-
sis of 11-14 in neat cyclohexane gives CH insertion
products (11a -14a , Scheme 5) in almost quantitative
yield, whereas the Pd complex 16 shows an insertion
yield 75 ( 5% compared with the Pd complex (45%)
without the methylene bridge.21 These are the most
efficient aryl nitrene C-H insertion reactions known with
cyclohexane.
(25) Soundarajan, M.; Platz, M. S. J . Org. Chem., 1990, 55, 2034.

Chemistry of Bifunctional Photoprobes
J . Org. Chem., Vol. 63, No. 24, 1998 9023
F igu r e 1. The transient absorption spectrum produced upon
LFP of 1 in CH3CN. The transient absorption spectrum of
ketenimine 1c and triplet nitrene 1e was recorded 1.4 µs after
the laser pulse over a 200-ns window at ambient temperature.
F igu r e 2. The transient absorption spectrum produced upon
LFP of 4 in CH3CN. The transient absorption spectrum of
ketenimine 4c and triplet nitrene 4e was recorded 1.4 µs after
the laser pulse over a 200-ns window at ambient temperature.
Sch em e 5
nm (Figure 1) and weak broad bands at 480 and 650 nm.
Upon LFP of 1 in acetonitrile containing 1 M diethyl-
amine, a known quencher of didehydroazepines, very
weak transient absorption was observed at 380, 450, and
650 nm. Thus, by analogy with the photochemistry of
other polyfluorinated aryl azides, the strongly absorbing
transient of Figure 1 is attributed to ketenimine 1c,13,16-17
and the weak bands at 380, 450, and 650 nm are
associated with triplet nitrene 1e. A weak transient
spectrum of triplet nitrene 1e was also detected in CH2Cl2
at -32 °C and in methanol.
La ser F la sh P h otolysis Stu d ies. Tetrafluorinated
azide ester 19 and its amide analogues 20 and 21 have
been studied by laser flash photolysis (LFP) techniques.26a
The singlet nitrenes derived from these precursors were
remarkably long-lived. Nitrenes derived from 1, 2, and
4 were also studied by LFP methodology to understand
their failure to give cyclohexane adducts.
LFP of 4, in acetonitrile in the absence of pyridine,
produced a transient spectrum (Figure 2) that contains
maxima at 345, 370, and 620 nm. The bands at 345 and
370 nm must be associated with different species because
they have different lifetimes (41 and 32 µs, respectively).
The two species observed in acetonitrile are attributed
to ketenimine 4c (370 nm) and triplet nitrene 4e (345
and 620 nm). Only triplet nitrene 4e is observed in
acetonitrile containing diethylamine (where 4c is trapped),
in methanol solvent [which is known to accelerate
intersystem crossing (ISC13)], and in CH2Cl2 at low
temperature where ISC is favored relative to the rear-
rangement of singlet nitrenes13 (Supporting Information,
Figures 1-3, respectively). The positions of these bands
LFP (XeCl excimer, 308 nm, 17 ns) of azide 1 in
acetonitrile at room temperature produces a transient
spectrum with a broad absorption maximum around 380
(26) (a) Marcinek, A.; Platz, M. S.; Chan, S. Y.; Floresca, R.;
Rajagopalan, K.; Golinski, M.; Watt, D. J . Phys. Chem. 1994, 98, 412.
(b) Marcinek, A.; Platz, M. S. J . Phys. Chem. 1993, 97, 12674.

9024 J . Org. Chem., Vol. 63, No. 24, 1998
Pandurangi et al.
Ta ble 2. Lifetim es of F u n ction a lized P er flu or oa r yl
Nitr en es in Differ en t Solven ts a t Am bien t Tem p er a tu r e
X
t (ns)
solvent
CH ) NNHCH3
5
4
CH3CN
CH3CN
CH2Cl2
CH2Cl2
n-C5H12
CH2Cl2
CH3CN
CH3OH
CH ) NNCH3PSNCH3NH2‚PdCl2
COX (X ) OCH3, NHCH3, N(CH3)2)
F27
20826
38
27
C6F5
C6F5
C6F5
C6F5
259
254
220
65
27
27
27
studied by chemical trapping and flash photolysis tech-
niques, results in the development of more efficient
photolabeling agents for cross-linking biomolecules. For
example, Marcinek et al.26 have studied azides 19, 20,
and 21 by LFP techniques and found that the corre-
sponding singlet nitrenes react with pyridine with abso-
lute rate constant kPYR ) 3 × 107 M-1 s-1 at ambient
temperature.
The absolute values of kPYR of singlet pentafluorophenyl
nitrene and perfluorobiphenyl nitrene are 6 × 107 M-1
s-1 at ambient temperature.27 It was not possible to
measure the lifetime of singlet nitrenes 1d and 4d
directly. Thus, we measured the optical yields (Ay) of
ylides 1f and 4f produced per laser pulse as a function
of pyridine concentration (Figure 4). Plots of 1/Ay versus
1/[pyridine] are linear (Figure 5). The ratio of the
intercept/slope of such plots is kPYR τ, where kPYR is the
absolute rate constant of reaction of singlet nitrene with
pyridine, and τ is the lifetime of singlet nitrene in the
absence of pyridine.13,26,27 However, if we assume that
these nitrenes react with pyridine with kPYR ) 3 × 107
M-1 s-1, we deduce that their lifetimes are 5 and 4 ns,
respectively, in acetonitrile.
F igu r e 3. (a) The transient absorption spectrum produced
upon LFP of 1 in CH3CN containing pyridine. The spectrum
of ketenimine 1c and nitrene-pyridine ylide 1f was recorded
10 µs after the laser pulse over a 200-ns window, at ambient
temperature. (b) The transient absorption spectrum produced
upon LFP of 4 in CH3CN containing pyridine. The transient
absorption spectrum of ketenimine 4c and nitrene-pyridine
ylide 4f were recorded 1.4 µs after the laser pulse over a 200-
ns window at ambient temperature.
are not unreasonable because pentafluoroketenimine has
a broad absorption between 380 and 400 nm13d and
pentafluoro triplet phenyl nitrene absorbs sharply at 320
nm, more broadly at 370 nm, and has a weak band in
the visible region of the spectrum extending to 550 nm.13a
Similarly, LFP of 2 in acetonitrile produces transient
absorption at 360, 570, and 610 nm. The same bands
are obtained upon LFP of 2 in methanol, a solvent that
catalyzes ISC. These transient spectra are associated
with triplet nitrene 2e.
Singlet aryl nitrenes decay by rearrangement, inter-
system crossing (kISC), and reaction with solvent (kRH[RH],
see Scheme 1).16,26 The lifetime τ in the absence of
pyridine is simply related to the absolute rate constants
of Scheme 1, by eq 1.26,27
1/τ ) kR + kISC + kRH[RH]
(1)
LFP of 1, 2, or 4 in acetonitrile containing pyridine
produces transients that absorb strongly around 500 nm
(Figure 3). Based on analogy with our previous work
with polyfluorinated azides,13,26 these transients are
attributed to nitrene-pyridine ylides 1f, 2f, and 4f,
respectively.
Values of the lifetime of various polyfluorinated singlet
arylnitrenes are given in Table 2. It is clear that
hydrazine singlet nitrenes 1d and 4d have lifetimes that
are 40-50 times shorter than those of ester and amide
nitrenes 19d and 21d .
For a singlet nitrene to be useful as an highly efficient
photolabel, eq 2 must hold
Discu ssion
The correlation of lifetimes of singlet nitrenes derived
from azides with CH insertion yields in organic solvents,
(27) Gritsan, N. P.; Zhai, H. B.; Yuzawa, T.; Karwick, D.; Brooke,
J .; Platz, M. S. J . Phys. Chem. 1997, 101, 2833.

Chemistry of Bifunctional Photoprobes
J . Org. Chem., Vol. 63, No. 24, 1998 9025
F igu r e 5. (a) A double-reciprocal treatment of the data of
Figure 4a. (b) A double-reciprocal treatment of the data of
Figure 4b.
perature-dependent, and kISC was found to be 3.0-3.3 ×
106 s-1 for nitrenes 19d -21d .26 This allowed Marcinek
et al. to deduce that kR ) 1.5-1.8 × 106 s-1 at ambient
temperature with Arrhenius barriers of A ) 1.2 × 1013
s-1 and Ea ) 9 kcal/mol for tetrafluorinated singlet
nitrene ester and amides.
F igu r e 4. (a) The yield of ylide (Ay) formed upon LFP of 1 in
CH3CN as a function of pyridine concentration. (b) The yield
of ylide (Ay) formed upon LFP of 4 in CH3CN as a function of
pyridine concentration.
Karney and Borden28 calculated the rearrangement of
singlet arylnitrenes to ketenimines and found that it is
a two-step process.
kRH[RH] g kR + kISC
(2)
Marcinek et al.26 showed that for nitrenes 19d - 21d
that kR + kISC ) 4.8 ( 0.5 × 106 s-1 at ambient
temperature. Intersystem crossing rates are not tem-
(28) (a) Karney, W. L.; Borden, W. T. J . Am. Chem. Soc. 1997, 119,
1378. (b) Karney, W. L.; Borden, W. T. J . Am. Chem. Soc. 1997, 119,
3347.

9026 J . Org. Chem., Vol. 63, No. 24, 1998
Pandurangi et al.
to the perfluoroaryl ring participates in the extended
conjugation, thereby releasing electron density into the
empty orbital of the singlet nitrene compensating for the
electron-withdrawing capacity of the parasubstituted
nitrile group. Similarly, electron release from the Pd
metal through back-bonding to iminophosphorano nitro-
gen enhances the electron flow into the empty orbital of
singlet reducing the electrophilicity of nitrene. These
conclusions agree well with the results on the trans-(4-
azido-2,3,5,6-tetrafluorophenyl)-3,7-dimethyl-2,4,6,8-non-
atetranal)
The first step is the slow step. It has a calculated
barrier of ∼6 kcal/mol, in agreement with the measured
value of rearrangement of parent singlet phenylnitrene.29
Karney and Borden28 found that ortho methyl and
ortho fluoro substituents raise the barrier to rearrange-
ment by 2-3 kcal/mol by a steric effect in the transition
state.28 The steric effects in 1d and 2d appear to be no
different from those in singlet nitrenes 19d -21d . Thus,
we expect that kR will be comparable throughout this
series. Fast rearrangement of singlet nitrenes 1d and
2d probably is not responsible for the short lifetimes of
these species relative to 19d -21d .
Photolysis of 1 and 2 gives the corresponding anilines
almost exclusively in good yield (Scheme 5). This indi-
cates that kISC of 1d and 2d is larger than in 19d -21d .
Unlike singlet phenylcarbene,29 singlet phenylni-
trene30 has an open-shell electronic configuration.31 Thus,
singlet nitrenes undergo concerted bond insertion pro-
cesses more slowly than do carbenes.28,32 Substitution
patterns which make closed-shell configurations more
accessible will accelerate concerted reactions, which lead
to productive photolabeling.
Spin-orbit coupling (SOC)-ISC is allowed in carbenes
where a closed-shell “ionic” configuration is coupled to
an open-shell “covalent” configuration.33 SOC-ISC is
forbidden between open-shell singlet and triplet states
as in a nitrene.32,33 This explains why ISC is 103-4 times
faster in carbenes34 than in nitrenes.30
which failed to yield any significant CH insertion in
cyclohexane.35 The addition of the extended polyene side
chain, which is in conjugation with the azido group,
apparently modifies the reactivity of the singlet nitrene
making it less likely to insert into either the CH or the
NH bonds of model solvents. Failure of this compound
to insert even in the seemingly favorable protein matrix
conditions confirms our earlier suggestion that there is
a need for the development of intermolecular CH/NH
insertion chemistry in model solvents which has a close
relationship with the success of perfluoroaryl azides as
photolabels on specific biomolecules. Our results are also
consistent with the work of Miura and Kobayashi36 with
nitrene 22d . The absolute rate constant of ISC in 22d
is 8.3 ( 0.2 × 109 s-1, which is 3 orders of magnitude
larger than kISC in parent singlet phenyl nitrene.30
In the Schiff base, the parahydrazone group can
conjugate with a singlet nitrene as shown below.
P h otola belin g of Hu m a n Ser u m Albu m in . The
efficiency of covalent modification of biomolecules by
photolabeling techniques depends on the optimization of
productive versus nonproductive avenues for perfluoro-
aryl nitrenes which is controlled by (a) electronic tuning
of perfluoroaryl azides para to azido group, (b) net
electrophilicity of singlet nitrene through steric factors
especially with electron-rich metals, and (c) the vicinity
of the biomolecule around which nitrene is generated.
The three types of synthetic designs for obtaining BFP-
CAs provide an opportunity to study the electronic as well
as the steric effects of metalated systems on the photo-
chemistry of perfluoroaryl azides. Although, BFPCAs
showed high CH insertion in model organic solvents, the
intriguing question is whether the long lifetime of singlet
nitrenes derived from amides and esters can be extended
for insertion into biomolecules under buffer conditions
and at tracer level concentrations. One of our long-term
objective is to demonstrate the feasibility of formation
of instant 99mTc-labeled antibody fragment (Fab) conjuga-
tion kits using photolabeling agents to target Fab to
specific sites of tumor. Use of HSA for attaching BFPCAs
constitute a good model for antibody fragments because
This conjugation stabilizes a closed-shell singlet con-
figuration and lowers the singlet-triplet gap, both factors
which accelerate ISC. Hence, we believe that kISC in 1d
and 2d exceeds that of 19d -21d . This shortens the
lifetimes of 1d and 2d and reduces the efficiency of CH
insertion of these species (kISC . kRH[RH], Scheme 1) and
reduces their utility as photolabeling reagents. Similarly,
orthosubstitution of perfluoroaryl azides by electron-
donating N-methyl amine group21e or phosphinimine
group21f has been shown to reduce CH insertion yield
compared with their precursor perfluoroaryl benzo-
nitrile.21d The iminophosphorano moiety (NdP) attached
(29) (a) Matzinger, S.; Bally, T.; Patterson, E. V.; McMahon, R. J .
J . Am. Chem. Soc. 1996, 118, 1535. (b) Schreiner, P. R.; Karney, W.
L.; Schleyer, P. V. R.; Borden, W. T.; Hamilton T. P.; Schaefer, H. F.,
III. J . Org. Chem. 1996, 61, 7030 (c) Wong, M. W.; Wentrup, C. J . J .
Org. Chem. 1996, 61, 7022.
(30) Gritsan, N. P.; Yuzawa, T.; Platz, M. S. J . Am. Chem. Soc. 1997,
119, 5059.
(31) (a) Kim, S. J .; Hamilton, T. P.; Schaefer, H. F., III J . Am. Chem.
Soc. 1992, 114, 5349. (b) Hrovat, D. A.; Waali, E. E.; Borden, W. T. J .
Am. Chem. Soc. 1992, 114, 8698. (c) Travers M. J .; Coules, D. C.;
Clifford, E. P.; Ellison, G. B. J . Am. Chem. Soc. 1992, 114, 8699.
(32) Platz, M. S. Acc. Chem. Res. 1995, 28, 487.
(33) Salem, L.; Rowland, C. Angew. Chem., Int. Ed. Engl. 1972, 11,
92.
(34) (a) Sitzmann, E. V.; Langan, J .; Eisenthal, K. B. J . Am. Chem.
Soc. 1984, 106, 1868. (b) Grasse, P. B.; Brauer, B.-E.; Zupancic, J . J .;
Kaufmann, K. J .; Schuster, G. B. J . Am. Chem. Soc. 1983, 105, 6833.
(35) Beischel, C. J .; Knapp, D. R.; Govindji, R.; Ebrey, T. G.; Crouch,
R. K. Photochem. Photobiol. 1994, 60, 64.
(36) Miura, A.; Kobayashi, T. J . Photochem. Photobiol A. 1990, 53,
223.

Chemistry of Bifunctional Photoprobes
J . Org. Chem., Vol. 63, No. 24, 1998 9027
Ta ble 3. P er cen ta ge of Cova len t Atta ch m en t of 99m Tc Com p lex w ith HSA
% of activity
% of activity
% of activity
conditions
at 8.0-cm spot
at 13.2-cm spot
at 14.5-cm spot
HSA + 99mTc complex (before photolysis)
HSA + 99mTc complex (10 min of photolysis)
HSA + 99mTc complex (15 min of photolysis)
99mTc complex in buffer
6.63 ( 2
73.13 ( 2
79.98 ( 2
13.5 ( 2
86.14 ( 3
12.74 ( 3
6.04 ( 3
13.26 ( 2
15.83 ( 2
85.8 ( 2
a
A 2 x 10-6 M solution of 99mTc complex was photolyzed (320 nm) for 10 and 15 min at room temperature. The yield of covalently
attached photoprobe to HSA was measured by the integration of the peaks of radiochemical scans (bioscan) subtracted by the residual
radioactivity before photolysis (control experiment).
tolysis (control experiment). Sequential photolysis of the
mixture shows a considerable shift of radioactivity to the
origin of spot (8.0 cm, Figure 6B and C after 10 and 15
min, respectively) indicating that the complex is associ-
ated with HSA which cannot move in TLC. Previously,
we showed such an association to be covalent in nature
for similar complexes.21e,22 A new spot at 14.5 cm also
appears after photolysis, which coincides with the reten-
tion time of the photolytic product in the absence of HSA
(buffer, Figure 6D). This spot can be assigned to a triplet
nitrene-derived aniline product based on previous obser-
vations.21,22 The low fraction of radioactivity seen at the
origin in Figure 6D may be caused by the partial
oxidation of the complex to 99mTcO2, which is not mobile
in TLC when acetone is used as eluent. However, in the
control experiment (Figure 6A), very little oxidation
product is seen (6%). Based on the percentage of the
radioactivity at the origin with respect to the total
activity, we can reasonably assume the efficiency of
covalent attachment of the probe to be approximately 72
( 5% (Table 3). These results demonstrate that the
correlation of long lifetimes of singlet nitrenes with
intermolecular insertion efficiency in organic solvents can
be extended successfully to the covalent attachment of
biomolecules at tracer level concentrations. The complete
characterization of the radiochemical complex and the
quantification of the efficiency of covalent attachment of
99mTc complex with HSA using HPLC (dual detectors, UV
at 254 nm and a radiochemical detector) is described in
the Supporting Information which will be reported
elsewhere.
Con clu sion s
In view of the increasing need for the development of
highly efficient CH insertion molecular probes for cova-
lent attachment to receptors and antibodies in biochem-
istry and in nuclear medicine, we synthesized several
BFPCAs. This series of compounds exhibit a favorable
F igu r e 6. (A) TLC of 99mTc complex 12 + HSA before
intermolecular insertion chemistry, for example, the
photolysis in acetone, (B) 10 min of photolysis, (C) 15 min of
largest CH insertion yield of a nitrene ever reported with
photolysis, and (D) photolysis without HSA in phosphate-
cyclohexane (compounds 11-14 and 16). LFP studies of
BFPCAs establish that the singlet nitrene lifetimes of
hydrazone derivatives are an order of magnitude shorter
than those observed for perfluoroaryl nitrenes with ester
or amide groups in the para position. The longer
lifetimes correlate well with the higher yields of CH
insertion observed in model solvents with the ester- and
amide-substituted compounds. Moderate to high ef-
ficiency of photochemical attachment of 99mTc-labeled
BFPCA to HSA demonstrates that the observations in
model solvents can be extended to tracer levels in buffer
conditions. The predictable photochemistry of this class
of highly electrophilic nitrenes establishes a firm basis
for the attachment of radiometalated probes to biomol-
buffered saline.
HSA has been used for hepatic imaging.7g In this
connection, we characterized 99mTc complex of 12 using
paper chromatography (PC), thin-layer chromatography
(TLC), electrophoresis, high-performance liquid chroma-
tography (HPLC) and solvent extraction procedures (see
Supporting Information, Figures 5-7). The 99mTc com-
plex was incubated with HSA for an hour before pho-
tolysis. The elution profile of the unphotolyzed mixture
in TLC showed a major radiochemical spot at 13.2 cm
(origin of spot at 8.0 cm) corresponding to the radio-
chemical complex (Figure 6A) indicating that the photo-
labile complex can be separated from HSA before pho-

9028 J . Org. Chem., Vol. 63, No. 24, 1998
Pandurangi et al.
Syn th esis of Tr ip let P r od u ct of 1 via P h otolysis.
A
ecules of interest. In this connection, studies on the
identification of specific receptor-based molecular target
systems are in progress.37
solution of 1 (650 mg, mmol) in a mixture of CH3OH and H2O
(9:1) was photolyzed for 5 min in a quartz tube. The photo-
chemical reaction was monitored to the level of 95% disap-
pearance of the 19F signals of the starting material. The
solution was filtered and evaporated and redissolved in CH3CN
and cooled to yield crystalline material 1b (87% yield, Scheme
4). 1H NMR (CDCl3) δ, 7.5 (s, 1H), 4.2 (s, 2H), 3.2 (b, 1H),
2.86 (s, 3H). 19F NMR (CDCl3) δ, -84.8 (m, 2F), -101.6 (m,
2F). Anal. Calcd for C8H7N3F4; C, 43.43; H, 3.19; N, 34.38.
Found: C, 43.78; H, 3.10; N, 34.51.
Exp er im en ta l Section
All synthetic procedures were conducted in a dry nitrogen
atmosphere using standard Schlenk tube techniques and
prepurified solvents. Reactions involving the synthesis of
azides were carried out under subdued light by wrapping the
flasks with aluminum foil. Diphenoxyphosphoryl methyl
hydrazine 3 was prepared as reported previously.23g NMR
spectra were recorded in CDCl3 on a 250-MHz spectrometer,
and chemical shifts are reported in ppm downfield from SiMe4
for 1H NMR. The 31P NMR chemical shifts are reported with
respect to 85% H3PO4 as an external standard, and positive
shifts lie downfield from the standard. 19F NMR chemical
shifts are reported with respect to trifluorotoluene as an
external standard. Infrared spectra were recorded as KBr
pellets on a Fourier transform-IR spectrophotometer. Elemen-
tal analysis for the new compounds were performed by Oneida
Research Services, Inc., New York. UV-visible spectra were
recorded either in cyclohexane or in acetonitrile on a Hewlett-
Packard diode array spectrophotometer.
Syn th esis of Tr ip let P r od u ct of 4 via P h otolysis.
A
solution of 4 (500 mg, 1.04 mmol) in a mixture of CH3OH and
H2O (9:1) was photolyzed for 5 min in a quartz tube. The
photochemical reaction was monitored to the level of 95%
disappearance of the 19F signals of the starting material. The
solution was filtered and evaporated and redissolved in CH3CN
and cooled to yield crystalline material 4b (92% yield, Scheme).
3
1H NMR (CDCl3) δ, 2.92 (d, J ) 11.4 Hz, 3H), 4.2 (b, s, 2H),
7.0-7.5 (m, 10H), 7.63(s, 1H), 31P NMR, δ, -5.8, 19F NMR
(CDCl3) δ, -83.9 (m, 2F), -101.7 (m, 2F). Anal. Calcd for
C
20H16N3O3PF4, C, 52.98; H, 3.55; N, 9.27. Found: C, 52.61;
H, 3.60; N, 9.22.
Syn th esis of 5 a n d 6. A solution of 4-azido-2,3,5,6-
tetrafluorobenzamide prepared as reported earlier13a (1 g, 4.27
mmol), taken in dry CH3CN, was slowly added dropwise to a
solution of PSCl3 (2.169 g, 12.81 mmol) at room temperature
in the presence of excess triethylamine. The solution was
refluxed for 8 h and monitored by 19F NMR until the fluorine
signals due to the starting material disappear. The excess
PSCl3 was removed under high vacuum, the resulting oil was
dissolved in CHCl3, and 2 mol of methyl hydrazine in CHCl3
(0.543 g, 11.78 mmol) was slowly added and stirred for an hour.
The solution was filtered and the filtrate was evaporated in a
vacuum to yield brown solid 5. The solid was dissolved in hot
CH3CN and cooled at -5° C to obtain an analytically pure
sample (67% yield). 1H NMR (CDCl3), δ, 4.8 (b, 1H), 2.97 (d,
J (P-H) ) 11.2 Hz, 6H), 3.3 (b, 4H). 31P NMR δ, 67.8, 19F NMR
(CDCl3) δ, -75.3 (m, 2F), -87.4 (m, 2F). Anal. Calcd for C9-
Photolyses were carried out with a 200-W high-pressure Hg
lamp directed through water as a filter. The total lamp output
was measured by ferrioxalate actinometry to be 2 mE/h in the
range of 320-360 nm. The photolysis solution absorbs only a
small fraction of the incident light at the red edge of the
photoprobe absorption. Approximately 50% of the focused
beam was intercepted with a 1-cm2 quartz cuvette for most
experiments. For specific in situ NMR experiments, the
solution in the NMR tube was irradiated directly, intercepting
10% of the incident light. Irradiation of solutions was con-
tinued to the destruction of azide, monitored by C-18 reverse-
phase HPLC. Retention time (Rt) values for cyclohexane
adducts are reported in minutes using eluent CH3CN:H2O (2:
1). Solutions (10-5 M) were purged with prepurified nitrogen
for 5 min before photolysis. Solutions of higher concentrations
(10-3-10-2 M) were photolyzed (2-3 h) for chromatographic
analysis of the photoproducts. The experimental apparatus
for conducting LFP studies has been described elsewhere.13h
For bioconjugation experiments, 1 mg/mL of the ligand 12
(2.319 µmol) is incubated with HSA in a glass tube for 10-15
min (69 mg in 10 mL phosphate-buffered saline, i.e. 2 µmol,
approximately, in 1:1 ratio).
Syn th esis of 1 a n d 4. Dropwise addition of methyl
hydrazine dissolved in absolute ethyl alcohol (0.460 g, 9.9
mmol) into a solution of perfluoroazidobenzaldehyde (2.19 g,
9.9 mmol) also taken in alcohol at 0 °C was followed by stirring
for 30 min. The solution was filtered and evaporated to yield
a brown precipitate that was redissolved in methanol and
evaporated slowly to obtain crystalline material 1 (89% yield).
1H NMR (CDCl3) δ, 2.91 (s, 3H), 3.2 (b, 1H), 7.6 (s, 1H), 19F
NMR (CDCl3) δ, -84.4 (m, 2F), -92.8 (m, 2F). Anal. Calcd
for C8H5N5F4; C, 38.88; H, 2.04; N, 28.34. Found: C, 38.85;
H, 2.10; N 28.51.
H
11N8F4OPS; C, 27.99; H, 2.87; N, 29.01. Found: C, 27.71;
H, 2.75; N, 29.09.
Syn th esis of 11-14. To 245 mg (3.95 mmol) of ethylene
glycol in dry THF was added 1 g (3.95 mmol) of pentafluo-
robenzoyl chloride, also in THF. TLC of the mixture showed
2 spots presumably caused by the bis- and monosubstitution
products. The mixture was separated chromatographically on
a silica gel column using hexane, ethyl acetate and methanol
in a 9:3:1 ratio to produce a colorless oil 7 (87% yield). 1H
NMR (CDCl3) δ, 4.53 (t, 2J ) 4.6 Hz, 2H), 3.97 (t, 2J ) 4.7 Hz,
2H), 1.42 (b, 1H). 19F NMR (CDCl3) δ, -75.5 (m, 2F), -88.4
(m, 2F), -97.2 (m, 2F). Anal. Calcd for C9H5F5O3; C, 42.21;
H, 1.97. Found: C, 42.31; H, 1.90.
A solution of pentafluorobenzoyl chloride (2 mL, 13.88 mmol)
in dry THF was added to a solution of 1,4-butanediol in THF
(1.25 mL, 14.11 mmol) dropwise and refluxed overnight. The
solution was cooled and treated with Na2CO3 to neutralize the
acid byproduct. The solution mixture was extracted with ether
(2 × 10 mL), and the organic phase mixture was separated
chromatographically on a silica gel column using hexane, ethyl
acetate, and methanol in a 9:3:1 ratio to produce a colorless
oil 8 (yield 78%). 1H NMR (CDCl3) δ, 1.41 (b, 1H), 1.72 (m,
A solution of diphenoxyphosphoryl methyl hydrazine 3
reported earlier23g in absolute ethyl alcohol (2.78 g, 9.9 mmol)
was added dropwise into a solution of perfluoro azido benzal-
dehyde (2.19 g, 9.9 mmol) also taken in alcohol at 0 °C. The
mixture was stirred for 30 min, and the reaction was moni-
tored by 31P NMR spectroscopy. The solution was filtered and
the precipitate was washed with ethanol and dissolved in
CH2Cl2 and evaporated slowly to obtain crystalline material
4 (89% yield). 1H NMR (CDCl3) δ, 3.21 (d, 3J ) 11.2 Hz. 3H),
7.0-7.5 (m, 10H), 7.6(s, 1H), 31P NMR δ, -5.3, 19F NMR
2
2
2H), 1.82 (m, 2H), 3.75 (t, J ) 6.2 Hz, 2H) 4.41 (t, J ) 6.4
Hz, 2H). 19F NMR (CDCl3) δ, -75.8 (m, 2F), -88.7 (m, 2F),
-97.6 (m, 2F). Anal. Calcd for C11H9O3F5; C, 46.51; H, 3.21.
Found: C, 46.63; H, 3.28.
Azid olysis of Alcoh ol. About 1.1 g of alcohol 7 (4.3 mmol)
was taken in acetone and mixed with 300 mg of NaN3 (4.61
mmol) in acetone/water and refluxed for 8 h. Water was added
to the mixture and extracted into ether and dried over MgSO4
to produce yellow liquid 9 (yield, 79%). 1H NMR (CDCl3) δ,
4.63 (t, 2J ) 4.9 Hz, 2H) 3.87 (t, 2J ) 4.63 Hz, 2H), 1.43 (b,
1H). 19F NMR (CDCl3) δ, -75.6 (m, 2F), -87.3 (m, 2F). Anal.
Calcd for C9H5N3O3F4; C, 38.71; H, 1.81; N, 15.11. Found: C,
38.48; H, 1.85; N, 15.03.
(CDCl3) δ, -75.1 (m, 2F), -85.3 (m, 2F), Anal. Calcd for C20
14N5O3PF4; C, 50.12; H, 2.94; N, 14.61. Found: C, 50.22; H,
3.01; N, 14.53.
-
H
Similarly, the palladium complex 2 (Scheme 1) with bis
hydrazine phosphorus sulfide (BHP) was prepared as reported
earlier22b and used as such in flash photolysis studies.
(37) Pandurangi, R. S.; Rao, S. N. (unpublished results).

Chemistry of Bifunctional Photoprobes
J . Org. Chem., Vol. 63, No. 24, 1998 9029
The oil 8 (2.84 g, 9.9 mmol) was dissolved in acetone and
then NaN3 (0.650 g, 9.9 mmol) taken in water was added. The
solution was refluxed overnight, cooled and mixed with water
and extracted into ether, and dried over MgSO4; the solvent
was removed in a vacuum to produce yellow oil. The crude
oil was purified by chromatography on a silica gel column using
hexane and ethyl acetate in a 10: 3 ratio to obtain a yellow
oil 10 (yield 75%). 1H NMR (CDCl3) δ, 1.42 (b, OH, 1H), 1.73
(m, 2H), 1.85 (m, 2H), 3.74 (t, J ) 6.2 Hz, 2H) 4.42 (t, J )
6.4 Hz, 2H). 19F NMR (CDCl3) δ, -75.8 (m, 2F), -87.7 (m,
2F). Anal. Calcd for C11H9N3O3F4; C, 42.99; H, 2.99; N, 13.71.
Found: C, 42.64; H, 2.81; N, 13.64.
2F). Anal. Calcd for C11H14N7F4O3PSPdCl2; C, 21.71; H, 2.32;
N, 16.11. Found: C, 22.01; H, 2.39; N, 16.21. An a lyt ica l
3
d a ta for 16: (82% yield). 1H NMR (CD2Cl2) δ 2.76 (d, J (P-
2
H) ) 10.1 Hz, 6H), 3.62 (b, 4H), 4.25 (t, J ) 4.4 Hz, 2H) 4.57
2
(t, J ) 4.3 Hz, 2H). 31P NMR δ, 17.15 19F NMR (CD2Cl2) δ,
-75.5 (m, 2F), -88.4 (m, 2F). Anal. Calcd for C11H14N7F4O4-
PPdCl2; C, 22.31; H, 2.38; N, 16.55. Found: C, 22.47; H, 2.45;
N, 16.69. An a lytica l d a ta for 17: (68% yield). 1H NMR
(CD2Cl2) δ, 1.72 (m, 2H), 1.84 (m, 2H), 2.67 (d, 3J (P-H) )
12.8 Hz, 6H), 3.62 (b, 4H), 3.71 (t, 2J ) 6.3 Hz, 2H), 4.36 (t, 2J
) 6.2 Hz, 2H). 19F NMR (CD2Cl2) δ, -76.1 (m, 2F), -87.4 (m,
2F). 31P NMR, δ, 87.2 Anal. Calcd for C13H18N7F4O3PSPdCl2;
C, 24.52; H, 2.85; N, 15.41. Found: C, 24.31; H, 2.95; N, 15.67.
An a lytica l d a ta for 18: (84% yield). 1H NMR (CD2Cl2) δ, 1.68
2
2
An equimolar solution of azido alcohol 9 (980 mg, 3.43 mmol)
was slowly added to PSCl3 (580 mg, 3.43 mmol) in CHCl3 at
0° C with excess of Et3N and refluxed for 8 h and monitored
by 31P NMR to the disappearance of PSCl3 signals. The
reaction mixture was cooled at 0° C and 2 molar equivalents
of methyl hydrazine (360 µL, 6.86 mmol) were added slowly
at 0 °C with continuous stirring. The solution was filtered,
and the filtrate was evaporated and the mixture separated
chromatographically on a silica gel column using hexane, ethyl
acetate, and methanol in a 9:3:1 ratio to produce slightly yellow
oil 11, which solidifies on cooling (yield 75%). 1H NMR (CDCl3)
3
(m, 2H), 1.84 (m, 2H), 2.65 (d, J (P-H) ) 12.8 Hz, 6H), 3.12
2
(b, 4H), 3.76 (t, 2J ) 6.4 Hz, 2H), 4.36 (t, J ) 6.1 Hz, 2H). 19
F
NMR (CD2Cl2) δ, -75.4 (m, 2F), -88.7 (m, 2F). 31P NMR δ,
17.2. Anal. Calcd for C13H18N7F4O4PPdCl2; C, 25.16; H, 2.92;
N, 15.81. Found: C, 25.34; H, 2.88; N, 15.61.
P h otoch em ica l Syn th esis of Cycloh exyla m in e Ad -
d u cts 5a , 11a -16a . Separate solutions of perfluoroaryl azido
phosphorus hydrazide derivatives (5, 11-16) in a mixture of
CH2Cl2 and cyclohexane (1:1) were exposed to a beam of light
for 5 min in a quartz tube after bubbling with nitrogen for
2-3 min. The photochemical reaction was monitored by 19F
NMR spectroscopy to the destruction of the parent azide peaks.
The solution was concentrated by evaporating solvent partially
and dissolved in a minimum amount of methanol and sepa-
rated on silica gel column using hexane, ethyl acetate, and
methanol in a ratio of 9:2:1. In Pd complexes (6a , 15a , and
16a ), the solvent was partially evaporated and reprecipitated
using hexane. The precipitate was washed with hexane (3 ×
10 mL) and dissolved in hot CH3CN and evaporated slowly at
room temperature to yield an orange yellow powder. An a lyti-
ca l d a ta for 5a : (yield 73%). 1H NMR (CDCl3) δ, 4.7 (b, 1H),
3
2
δ, 2.84 (d, J (P-H) ) 11.1 Hz, 6H), 3.57 (b, 4H), 4.37 (t, J )
4.2 Hz, 2H) 4.59 (t, 2J ) 4.3 Hz, 2H). 19F NMR (CDCl3) δ,
-75.3 (m, 2F), -87.9 (m, 2F) 31P NMR δ, 86.1. Anal. Calcd
for C11H14N7F4O3PS; C, 30.63; H, 3.27; N, 22.73. Found: C,
30.52; H, 3.19; N, 22.86.
Compound 12 was prepared in a similar manner using
POCl3.
1
Analytical data for 12 (yield 65%) H NMR (CDCl3) δ, 2.84
3
2
(d, J (P-H) ) 12.3 Hz, 6H) 3.81, (b, 4H) 4.22 (t, J ) 4.3 Hz,
2H), 4.62 (t, J ) 4.5 Hz, 2H), 19F NMR (CDCl3) δ, -75.8 (m,
2
2F), -88.5 (m, 2F). 31P NMR δ, 18.5. Anal. Calcd for C11
-
H
14N7O4F4P; C, 31.84; H, 3.41; N, 23.61. Found: C, 31.71; H,
3
3.36; N, 23.52.
2.96 (d, J (P-H) ) 10.2 Hz, 6H), 3.3 (b, 4H), 1.05-1.48 (m,
A solution of the azido-modified alcohol 10 (1 g, 13.7 mmol)
was refluxed with PSCl3 (1.39 mL, 137 mmol) taken in CHCl3
in the presence of excess of Et3N and refluxed for 6 h,
monitored by 31P NMR spectroscopy. Two molar equivalents
of methyl hydrazine was added to the above mixture cooled to
0 °C and stirred for an hour. The solution was filtered, and
the filtrate was evaporated in a vacuum to yield pale yellow
solid 13 (72% yield). 1H NMR (CDCl3) δ, 1.74 (m, 2H), 1.85
5H), 1.55-1.73 (m, 3H), 1.93-2.01 (m, 2H), 3.53 (m, 1H), 4.02
(s, 1H), 13C (Dept), methine carbon δ, 51.8. 31P NMR δ, 85.3,
19F NMR (CDCl3) δ, -77.2 (m, 2F), -99.8 (m, 2F). Anal. Calcd
for C15H23N6F4O2PC; 40.72; H, 5.24; N, 18.99. Found: C,
40.31; H, 5.57; N, 19.45. An a lytica l d a ta for 6a : (yield 70%
( 5). 1H NMR (CD2Cl2) δ, 4.6 (b, 1H), 3.12 (d, 3J (P-H) )
11.5 Hz, 6H), 3.6 (b, s, NH2, 4H), 1.06-1.41 (m, 5H), 1.59-
1.76 (m, 3H), 1.95-2.02 (m, 2H), 3.56 (m, 1H), 4.06 (s, 1H),
13C (Dept), methine carbon δ, 53.4. 31P NMR δ, 87.8 19F NMR
3
(m, 2H), 2.85 (d, J (P-H) ) 12.3 Hz, 6H), 3.41 (b, 4H), 3.72
(t, 2J ) 6.5 Hz, 2H), 4.38 (t, 2J ) 6.3 Hz, 2H). 31P NMR δ,
86.2, 19F NMR (CDCl3) δ, -75.8 (m, 2F), -88.7 (m, 2F). Anal.
(CDCl3) δ, -77.25(m, 2F), -99.2 (m, 2F). Anal. Calcd for C15
-
H
23N6F4OPSPdCl2; C, 29.07; H, 3.74; N, 13.56. Found: C,
Calcd for
C
13H18N7F4O3PS; C, 33.99; H, 3.95; N, 21.34.
29.28; H, 3.57; N, 13.21. An a lytica l d a ta for 11a : (yield
3
Found: C, 33.88; H, 3.83; N, 21.25.
90%). 1H NMR (CDCl3), δ 2.84 (d, J (P-H) ) 11.1 Hz, 6H),
Compound 14 was prepared using a procedure similar to
the one described with POCl3. Analytical data for 14 (yield
3.52 (b, 4H), 4.37 (t, 2J ) 4.2 Hz, 2H) 4.59 (t, 2J ) 4.3 Hz, 2H),
1.05-1.45 (m, 5H), 1.56-1.77 (m, 3H), 1.91-2.04 (m, 2H), 3.56
(m, 1H), 4.05 (s, 1H), 13C (Dept), methine carbon δ, 52.4, 19F
NMR (CDCl3) δ, -76.5 (m, 2F), -99.8 (m, 2F) 31P NMR δ, 86.5.
Anal. Calcd for C17 H26 N5 O3 F4PS; C, 41.89; H, 5.38; N, 14.37.
Found: C, 41.85; H, 5.28; N, 14.33. An a lytica l d a ta for 12a :
1
82%) H NMR (CDCl3) δ, 1.78 (m, 2H), 1.81 (m, 2H), 2.81 (d,
2
3J (P-H) ) 12.4 Hz, 6H), 3.51 (b, NH2, 4H), 3.75 (t, J ) 6.1
Hz, 2H), 4.31 (t, 2J ) 6.3 Hz) 2H). 31P NMR δ, 18.8, 19F NMR
(CDCl3) δ, -75.6 (m, 2F), -88.2 (m, 2F). Anal. Calcd for C13
-
3
H
18N7F4O4P; C, 35.22; H, 4.09; N, 22.12. Found: C, 35.14; H,
(yield 93%). 1H NMR (CDCl3) δ, 2.83 (d, J (P-H) ) 12.3 Hz)
4.18; N, 22.25.
6H) 3.80, (b, 4H) 4.22 (t, 2J ) 4.3 Hz, 2H), 4.61 (t, 2J ) 4.5 Hz,
2H), 1.05-1.45 (m, 5H), 1.54-1.77 (m, 3H), 1.92-2.04 (m, 2H),
3.56 (m, 1H), 4.03 (s, 1H), 13C (Dept), methine carbon δ, 53.1.
19F NMR (CDCl3) -77.7 (m, 2F), -99.8 (m, 2F). 31P NMR δ,
18.9. Anal. Calcd for C17H26N5O4F4P; C, 43.31; H, 5.56; N,
14.86. Found: C, 43.35; H, 5.51; N, 14.88. An a lytica l d a ta
for 13a : (yield 92%). 1H NMR (CDCl3) δ, 1.73 (m, 2H), 1.83
Gen er a l Syn th esis of P d Com p lexes of 6, 15-18.
Equimolar concentrations of PdCl2 (PhCN)2 and each of the
amide-coupled perfluoroaryl phosphorus hydrazide 5 or per-
fluoroaryl azido functionalized methylene-bridged phosphorus
hydrazides in CH2Cl2 (11-14) were mixed by adding PdCl2
(PhCN)2 solution dropwise with slow stirring. The solution
was evaporated partially and treated with hexane to precipi-
tate the complex and filtered. The filtrate was washed with
hexane (5 × 50 mL), dried, and dissolved in hot dry acetonitrile
and cooled at -5 °C to yield orange red crystalline material.
An a lytica l d a ta for 6: (72% yield). 1H NMR (CD2Cl2) δ, 3.15
(d, 3J (P-H) ) 12.1 Hz, 6H), 31P NMR δ, 68.9 19F NMR
(CD2Cl2) δ, -74.8 (m, 2F), -87.5 (m, 2F). Anal. Calcd for C9-
3
(m, 2H), 2.83 (d, J (P-H) ) 12.3 Hz, 6H), 3.40 (b, 4H), 3.72
2
2
(t, J ) 6.5 Hz, 2H), 4.36 (t, J ) 6.3 Hz, 2H) 1.09-1.42 (m,
5H), 1.61-1.77 (m, 3H), 1.96-2.05 (m, 2H), 3.55 (m, 1H), 4.04
(s, 1H), 13C (Dept), methine carbon δ, 52.7. 31P NMR δ, 86.8,
19F NMR (CDCl3) δ, -77.1(m, 2F), -99.6 (m, 2F). Anal. Calcd
for C19H30N5F4O3PS; C, 44.27; H, 5.87; N, 13.59. Found: C,
44.31; H, 5.83; N, 13.61. An a lytica l d a ta for 14a : (yield
89%). 1H NMR (CDCl3) δ, 1.76 (m, 2H), 1.82 (m, 2H), 2.81 (d,
3J (P-H) ) 12.4 Hz, 6H), 3.56 (b, 4H), 3.75 (t, 2J ) 6.1 Hz,
H
11N8F4OPS. PdCl2; C, 19.18; H, 1.97; N, 19.88. Found: C,
19.14; H, 1.90; N, 19.74. An a lytica l d a ta for 15: (73% yield).
1H NMR (CD2Cl2) δ, 3.12 (d, 3J (P-H) ) 11.3 Hz, 6H), 3.68 (b,
4H), 4.36 (t, 2J ) 4.2 Hz, 2H), 4.52 (t, 2J ) 4.5 Hz, 2H) 31P
NMR δ, 87.8 19F NMR (CD2Cl2) δ, -75.8 (m, 2F), -87.9 (m,
2
2H), 4.31 (t, J ) 6.3 Hz) 2H), 1.08-1.45 (m, 5H), 1.56-1.75
(m, 3H), 1.95-2.04 (m, 2H), 3.59 (m, 1H), 4.05 (s, 1H), 13C
(Dept), methine carbon δ, 51.9. 31P NMR δ, 18.2, 19F NMR

9030 J . Org. Chem., Vol. 63, No. 24, 1998
Pandurangi et al.
(CDCl3) δ, -77.4 (m, 2F), -99.3 (m, 2F). Anal. Calcd for C19
-
P h otoch em ica l Rea ction of 1, 2, a n d 4 w ith P yr id in e.
All compounds were dissolved in a mixture of CH2Cl2 and
pyridine (8:2) and degassed by bubbling with nitrogen for 5
min. The solution was photolyzed for 2-5 min. The solution
turned from light yellow to a persistent amber color shifting
the absorption maximum to the visible range 390-415 nm for
all the compounds. This is a typical absorption of a pyridine
ylide.15
H
30N5F4O4P; C, 45.69; H, 6.05; N, 14.02. Found: C, 45.63; H,
6.08; N, 14.08. An a lytica l d a ta for 15a : (yield 74 ( 5%). 1H
NMR (CD2Cl2) δ, 3.12 (d, 3J (P-H) ) 12.1 Hz, 6H), 3.67 (b,
2
2
4H), 4.25 (t, J ) 4.2 Hz, 2H) 4.57 (t, J - 4.3 Hz, 2H), 1.03-
1.45 (m, 5H), 1.61-1.79 (m, 3H), 1.94-2.05 (m, 2H), 3.56 (m,
1H), 4.07 (s, 1H), 13C (Dept), methine carbon δ, 53.1. 31P NMR
δ, 87.3 19F NMR (CD2Cl2) δ, -77.1 (m, 2F), -99.2 (m, 2F). Anal.
Calcd for C17H26N5F4O3PSPdCl2; C, 30.72; H, 3.94; N, 10.53.
Found: C, 30.85; H, 3.99; N, 10.62. An a lytica l d a ta for 16a :
(yield 75 ( 5%). 1H NMR (CD2Cl2) δ, 2.75 (d, 3J (P-H) ) 10.1
Hz, 6H), 3.60 (b, 4H), 4.25 (t, 2J ) 4.2 Hz, 2H) 4.56 (t, 2J ) 4.3
Hz, 2H), 1.07-1.45 (m, 5H), 1.57-1.78 (m, 3H), 1.91-2.06 (m,
2H), 3.55 (m, 1H), 4.06 (s, 1H), 13C (Dept), methine carbon δ,
53.1. 31P NMR δ, 17.3 19F NMR (CD2Cl2) δ, -77.9 (m, 2F),
-99.7 (m, 2F). Anal. Calcd for C17H26N5F4O4PPdCl2; C, 31.48;
H, 4.04; N, 10.79. Found: C, 31.42; H, 4.08; N, 10.81.
Ack n ow led gm en t. The work was supported by
funds provided by DOE Grant DEFG0289ER 60875.
NMR data were collected on instruments purchased
with funds from National Science Foundation Grants
8908304 and 9221835. We appreciate the help of Dr.
Guo and Mr. Gao of the NMR facility with the decou-
pling experiments and Dr. Karra for reviewing the
manuscript. P.L. acknowledges Prof. Karl T. Weber of
Internal Medicine for partial support. The authors
acknowledge Prof. K. V. Katti for the useful discussions
in the initial stages of this project, especially on the
inorganic part. Support of the National Science Founda-
tion for the work performed in Columbus (CHE-
8814950) is gratefully acknowledged.
P h otoch em ica l Rea ction of Hyd r a zon es 1, 2, a n d 4 in
Cycloh exa n e. All the hydrazones 1, 2, and 4 were dissolved
in either neat cyclohexane or in a mixture of cyclohexane and
CH2Cl2 (1:1) and photolyzed in NMR tube for 30-60 min. The
photochemical reactions were monitored by 19F NMR spec-
troscopy. All the Schiff base compounds yielded triplet-derived
amines in high yields after photolysis. An a lytica l d a ta for
1b: (yield 87%). 1H NMR (CDCl3) δ, 2.93 (d, 3H), 3.3 (b, 1H),
4.2 (b, 2H), 7.5 (s, 1H), 19F NMR (CDCl3) δ, -84.6 (m, 2F),
-101.8 (m, 2F). An a lytica l d a ta for 2b: (yield 82%). 1H
3
3
NMR (CDCl3) δ, 2.95 (d, J ) 11.8 Hz, 3H), 3.25 (d, J ) 8.4
Hz, 3H), 3.3 (b, 2H), 4.3 (b, 2H), 7.1-7.8 (m, 5H) 7.7 (s, 1H),
19F NMR (CDCl3) δ, -82.3 (m, 2F), -101.6 (m, 2F), 31P NMR
(CDCl3) δ, 82.5. An a lytica l d a ta for 4b: (yield 82%). 1H
Su p p or tin g In for m a tion Ava ila ble: Radiochemical char-
acterization and purity of 99mTc complex, additional flash
photolysis data are available as supplementary information
(8 pages). This material is contained in libraries on mi-
crofische, immediately follows the article in the microfilm
version of the journal, and can be ordered from the ACS. See
any current masthead page for ordering information.
3
NMR (CDCl3) δ, 2.94 (d, J ) 11.6 Hz., 3H), 4.2 (b, 2H), 7.0-
7.5 (m, 10 H) 7.6 (s, 1H), 19F NMR (CDCl3) δ, -82.1 (m, 2F),
-101.7 (m, 2F), 31P NMR δ, -5.3. The analytical data for the
compound above coincides well with the triplet-derived product
from the photolysis of 4 in methanol.
J O981458A