Photoaffinity labeling via nitrenium ion chemistry: Protonation of the nitrene derived from 4-amino-3-nitrophenyl azide to afford reactive nitrenium ion pairs

Article abstract of DOI:10.1021/ja902224m

Phenyl azides with powerful electron-donating substituents are known to deviate from the usual photochemical behavior of other phenyl azides. They do not undergo ring expansion but form basic nitrenes that protonate to form nitrenium ions. The photochemistry of the widely used photoaffinity labeling system 4-amino-3-nitrophenyl azide, 5, has been studied by transient absorption spectroscopy from femtosecond to microsecond time domains and from a theoretical perspective. The nitrene generation from azide 5 occurs on the S₂ surface, in violation of Kasha's rule. The resulting nitrene is a powerful base and abstracts protons extremely rapidly from a variety of sources to form a nitrenium ion. In methanol, this protonation occurs in about 5 ps, which is the fastest intermolecular protonation observed to date. Suitable proton sources include alcohols, amine salts, and even acidic C-H bonds such as acetonitrile. The resulting nitrenium ion is stabilized by the electron-donating 4-amino group to afford a diiminoquinone-like species that collapses relatively slowly to form the ultimate cross-linked product. In some cases in which the anion is a good hydride donor, cross-linking is replaced by reduction of the nitrenium ion to the corresponding amine.

Full text of DOI:10.1021/ja902224m

Published on Web 07/22/2009
Photoaffinity Labeling via Nitrenium Ion Chemistry:
Protonation of the Nitrene Derived from
4-Amino-3-nitrophenyl Azide to Afford Reactive Nitrenium Ion
Pairs
Valentyna Voskresenska,† R. Marshall Wilson,*,† Maxim Panov,†
Alexander N. Tarnovsky,*,† Jeanette A. Krause,‡ Shubham Vyas,§ Arthur H. Winter,§
and Christopher M. Hadad*,§
Department of Chemistry and the Center for Photochemical Sciences, Bowling Green State
UniVersity, Bowling Green, Ohio 43403, Department of Chemistry, UniVersity of Cincinnati,
Cincinnati, Ohio 45221, and Department of Chemistry, The Ohio State UniVersity,
Columbus, Ohio 43210
Received March 23, 2009; E-mail: rmw@bgsu.edu; atarnov@bgsu.edu; hadad.1@osu.edu
Abstract: Phenyl azides with powerful electron-donating substituents are known to deviate from the usual
photochemical behavior of other phenyl azides. They do not undergo ring expansion but form basic nitrenes
that protonate to form nitrenium ions. The photochemistry of the widely used photoaffinity labeling system
4-amino-3-nitrophenyl azide, 5, has been studied by transient absorption spectroscopy from femtosecond
to microsecond time domains and from a theoretical perspective. The nitrene generation from azide 5
occurs on the S2 surface, in violation of Kasha’s rule. The resulting nitrene is a powerful base and abstracts
protons extremely rapidly from a variety of sources to form a nitrenium ion. In methanol, this protonation
occurs in about 5 ps, which is the fastest intermolecular protonation observed to date. Suitable proton
sources include alcohols, amine salts, and even acidic C-H bonds such as acetonitrile. The resulting
nitrenium ion is stabilized by the electron-donating 4-amino group to afford a diiminoquinone-like species
that collapses relatively slowly to form the ultimate cross-linked product. In some cases in which the anion
is a good hydride donor, cross-linking is replaced by reduction of the nitrenium ion to the corresponding
amine.
direct nitrene insertion into protein residues,4 as shown in
Scheme 2, or attack of a nucleophilic protein residue on one of
Introduction
The photochemistry of aryl azides such as 1 has been studied
extensively and been found to be highly diverse. The most
commonly encountered mode of reaction is ring expansion of
the photochemically generated nitrenes to ketenimines (2) via
azirines (3), both of which react rapidly with a wide variety of
nucleophiles (Scheme 1).1 In highly acidic media or when
substituted with powerful electron-donating groups, aryl nitrenes
tend to protonate, thereby forming nitrenium ions,2 and when
appropriately substituted with electron-withdrawing groups, aryl
nitrenes will undergo insertion reactions into activated σ bonds.3
Thus, aryl azides are being widely applied as photoaffinity
labeling (PAL) and photo-cross-linking (PCL) agents in the
study of many types of biochemical interactions. The mechanism
of action of these reagents is usually considered to be either
the nitrene-derived intermediates related to 2 or 3. More
specifically, the PAL/PCL activating agent 4-fluoro-3-nitrophe-
nyl azide (4) has been applied extensively in such studies.5 The
mode of action of this PAL/PCL agent has been assumed to be
related to the chemistry outlined in Schemes 1 and 2, but no
detailed mechanistic study has been conducted to establish its
chemistry. In general, the azide-derivatized biomolecules used
(2) (a) Baetzold, R. C.; Tong, L. K. J. J. Am. Chem. Soc. 1971, 93, 1347–
1353. (b) Sukhai, P.; McClelland, R. A. J. Chem. Soc., Perkin Trans.
2 1996, 1529–1530. (c) McClelland, R. A.; Davidse, P. A.; Hadzialic,
G. J. Am. Chem. Soc. 1995, 117, 4173–1474. (d) McClelland, R. A.;
Kahley, M. J.; Davidse, P. A.; Hadzialic, G. J. Am. Chem. Soc. 1996,
118, 4794–4803. (e) Ramlall, P.; McClelland, R. A. J. Chem. Soc.,
Perkin Trans. 2 1999, 225–232. (f) Ruane, P. H.; McClelland, R. A.
Can. J. Chem. 2001, 79, 1875–1880. (g) McClelland, R. A.; Postigo,
A. Biophys. Chem. 2006, 119, 213–218. Unsubstituted aryl azides can
be induced to form nitrenium ions, but only under strongly acidic
conditions such as in the presence of HCl: (h) Wang, J.; Burdzinski,
G.; Zhu, Z.; Platz, M. S.; Carra, C.; Bally, T. J. Am. Chem. Soc. 2007,
129, 8380–8388. Or in formic acid: (i) Wang, J.; Burdzinski, G.; Platz,
M. S. Org. Lett. 2007, 9, 5211–5214.
† Bowling Green State University.
‡ University of Cincinnati.
§ The Ohio State University.
(1) (a) Platz, M. S. In ReactiVe Intermediate Chemistry; Moss, R. A., Platz,
M. S., Jones, M., Jr., Eds.; John Wiley & Sons: Hoboken, NJ, 2004;
pp 501-560. (b) Schnapp, K. A.; Poe, R.; Leyva, E.; Soundararajan,
N.; Platz, M. S. Bioconjugate Chem. 1993, 4, 172–177. (c) Schnapp,
K. A.; Platz, M. S. Bioconjugate Chem. 1993, 4, 178–183. (d) Cline,
M. R.; Mandel, S. M.; Platz, M. S. Biochemistry 2007, 46, 1981–
1987. (e) Rizk, M. S.; Shi, X.; Platz, M. S. Biochemistry 2006, 45,
543–551.
(3) (a) Keana, J. F. W.; Cai, S. X. J. Org. Chem. 1990, 55, 3640–3647.
(b) Liang, T.-Y.; Schuster, G. B. J. Am. Chem. Soc. 1987, 109, 7803–
7810. (c) Soundararajan, N.; Platz, M. S. J. Org. Chem. 1990, 55,
2034–2044.
(4) Chehade, K. A. H.; Speilmann, H. P. J. Org. Chem. 2000, 65, 4949–
4953.
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A R T I C L E S
Voskresenska et al.
Scheme 1
Scheme 2
Scheme 3
in PAL/PCL studies represent a class of aryl azides in which
the azide (nitrene) is in electronic communication with a
powerful electron-donating group such as an amino nitrogen
(5 in Schemes 2 and 3) or an ether oxygen. We have observed
that aryl azides of this class deviate from the usual azirine/
ketenimine pathway (Scheme 1), in that they form the ring
substitution products such as those formed from azide 5 shown
in Scheme 3. Consequently, the photochemistry of this widely
applied 4-amino-3-nitrophenyl azide PAL/PCL system6 has been
studied in this work in the presence of nucleophiles using
ultrafast pump-probe and theoretical techniques in an effort
to better understand its mechanism of reaction. The results of
those studies are presented below.
Experimental Section
Ultrafast Transient Absorption Spectroscopy. The ultrafast
transient absorption spectrometer used in this work is based on an
amplified Ti:sapphire laser system (Hurricane, Spectra Physics) that
pumps two computer-controlled pump and probe TOPAS-C optical
parametric amplifiers (Light Conversion Ltd.).7,8 Pump-probe
ultrafast transient experiments with azide 5 have been conducted
(5) (a) Escher, E. H. F.; Robert, H.; Guillemette, G. HelV. Chim. Acta
1979, 62, 1217–1222. (b) Addo, J. K.; Swamy, N.; Ray, R. Bioorg.
Med. Chem. Lett. 2002, 12, 279–281. (c) Mauri, L.; Prioni, S.; Loberto,
N.; Chigorno, V.; Prinetti, A.; Sonnino, S. Glycoconjugate J. 2003,
20, 11–23. (d) Sedla´k, E.; Panda, M.; Dale, M. P.; Weintraub, S. T.;
Robinson, N. C. Biochemistry 2006, 45, 746–754.
(6) Lormann, M. E. P.; Walker, C. H.; Es-Sayed, M.; Braese, S. Chem.
Commun. 2002, 12, 1296–1297.
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Photoaffinity Labeling via Nitrenium Ion Chemistry
A R T I C L E S
in various solvents at excitation wavelengths of 420, 350, and 305
nm. The white-light continuum generated by focusing a small
portion of the 800 nm amplified output into a 3 mm CaF2 window
was used as a probe light source. Alternatively, for the 305 pump,
a portion of the 800 nm amplified output was delivered to the
TOPAS-C probe amplifier to produce UV-probe pulses tunable from
280 to 390 nm. In a typical ultrafast measurement, about 120 ∆A
data points were collected at each position of the delay line, and
this procedure was repeated about 10 times for averaging. All
transient absorption (∆A) spectra were corrected for the group
velocity dispersion of the probe light with an accuracy of (30 fs
by using the nonresonant or two-photon absorption signals from
neat solvent.9 The excitation pulse energy was attenuated to ensure
that the ∆A signal due to two (excitation)-photon absorption by
the solvent was minor compared with the single-photon ∆A signal
of the azide at delay times equal to or longer than 100 fs.10 The
sharp Gaussian transient absorption signal at time zero due to
stimulated Raman scattering from solvent11 yielded the instrument
response function (150 fs, fwhm). Linearity of the transient
absorption signals from the azide was verified by attenuating the
excitation light with neutral density filters up to one-fourth of the
typically used pulse energy; the extrapolated line passed through
the origin. Dissolved oxygen had no noticeable effect on the
transient absorption spectra as verified by degassing the azide
solution with argon (solvent, i-PrOH). The ∆A spectra (solvent,
i-PrOH, 350 nm excitation) were found to be independent of azide
concentration (up to 16 mM). Steady-state absorption spectra of
the azide solutions measured before and after the pump-probe
experiment indicated that the degree of the sample decomposition
was always less than 10%. All measurements are performed at
magic angle polarization conditions and 22 °C.8
(B3LYP)15 methodology and using second-order couple cluster
method with resolution-of-identity approximation (RI-CC2)16 with
triple-ꢀ valence polarized (TZVP) basis sets as developed by
Ahlrichs and co-workers.17 Other than the azide 5, all of the other
species were optimized at the B3LYP/TZVP level. Vertical
excitations were obtained at these geometries using time-dependent
density functional theory (TD-DFT).18 The open-shell and closed-
shell nitrenes were optimized at the CASSCF(4,4)/6-31G(d) level
using the Gaussian 0319 program. However, further CASSCF
optimizations and CASPT2 single-point energies of open-shell and
closed-shell nitrenes using larger active space and more active
electrons, such as CASSCF(10,10), were performed using MOL-
CAS 6.2.20 These CASPT2//CASSCF computations were ac-
complished using the pVDZ basis set of Pierloot et al.21
Results and Discussion
The UV-vis absorption spectrum of azide 5 in i-PROH
consists of two broad structureless bands, a weaker one located
at 450 nm and stronger one at about 280 nm (Figure 1A). The
absorption spectra in polar protic and aprotic solvent are similar
with ε (445 nm) ) 1390 M-1 cm-1 in CH3CN. In nonpolar
solvents, such as cyclohexane, a blue shift (1000-2000 cm-1
)
of the spectrum is observed. Calculations (TD-B3LYP) suggest
the lowest energy transition, S0 f S1, is centered at λmax ) 477
nm (f ) 0.0042). The S0 f S2 transition is centered at λmax
)
353 nm, but it carries no oscillator strength (f ) 0.0000). At
the same time, the RI-CC2 calculations locate the 352 nm
transition (f ) 0.0062) but do not show the 477 nm band.
Finally, it should be noted that azide 5 did not exhibit any
noticeable fluorescence in polar or nonpolar solvents (excitation
280-360 nm).
The temporal evolution observed was globally fitted12 to a sum
of exponential functions with the time constants τi: ∆A(λ,t) )
∑iεiexp(-t/τi), where λ is the probe wavelength, τi are the resulting
time constants, and εi are the decay-associated spectra reconstructed
from the resulting τi values based on the assumption of a
consecutive reaction mechanism. A decay-associated spectrum
defines the absorption, which contributes to the recorded ∆A spectra
and which is characteristic of a specific decay component obtained
by a global fit. A global fit assumes that the absorption of product
species changes only in their strength, not band shape, which may
affect the resulting time constants (τi) up to several picoseconds.
For each excitation wavelength used, a global fit was performed
on 512 kinetic traces within the 274 nm bandwidth of the white-
light continuum probe. The region from -125 to 125 fs was not
used in the global fit because of the solvent contribution to the
measured ∆A spectra.
Product Studies. Azide 5 was prepared as indicated in Scheme
26 and, upon irradiation in i-PrOH, found to undergo photo-
chemical reduction to amine 6 and ring substitution to form 7
and 8 (Scheme 3). Surprisingly, the regiochemistry of this
substitution reaction favors the formation of the more hindered
isomer 8 rather than 7, which can be isolated in only trace
amounts. The regiochemistry of this addition has been confirmed
by an X-ray crystal structure determination of the acetate 9
(Figure 2). Initial studies with a variety of nucleophilic solvents
under both high and low intensity irradiation conditions gave
the reduction/substitution ratios shown in Table 1.
Ultrafast Transient Absorption Spectra. Figure 1B-D shows
the typical ∆A absorption spectra of azide 5 measured in i-PrOH
upon 350 nm excitation at time delays starting from 100 fs. The
measurements of the ∆A spectra for neat i-PrOH using the identical
excitation conditions indicate that significant solvent signals occur
at short times of -50 and 50 fs. At 100 fs, following irradiation of
5 in i-PrOH at 350 nm, transient absorption (positive ∆A signal)
is extensive and consists of a broad UV absorption (350-400 nm)
and a bell-like visible absorption band centered at 580 nm (Figure
Computational Details. All of the calculations are performed
using Turbomole 5.9113 at the Ohio Supercomputer Center, with
the exception of the transition state calculations for formation of
adducts 13 and 14, which were carried out using Spartan.14 The
azide 5 was optimized using Becke’s three-parameter hybrid
exchange functional with Lee-Yang-Parr correlation functional
(7) El-Khoury, P. Z.; Tarnovsky, A. N. Chem. Phys. Lett. 2008, 453, 160–
166.
(8) See Supporting Information for further details.
(9) Kovalenko, S. A.; Dobryakov, A. L.; Ruthmann, J.; Ernsting, N. P.
Phys. ReV. A 1999, 59, 2369–2382. Rasmusson, M.; Tarnovsky, A. N.;
Åkesson, E.; Sundstro¨m, V. Chem. Phys. Lett. 2001, 335, 201–208.
(10) For an example of the i-PrOH solvent response following 305 nm
excitation, see Figure SM1 in the Supporting Information.
(11) Kovalenko, S. A.; Ernsting, N. P.; Ruthmann, J. Chem. Phys. Lett.
1996, 258, 445–454.
(15) (a) Becke, A. D. J. Chem. Phys. 1993, 98, 5648–5652. (b) Lee, C.;
Yang, W.; Parr, R. G. Phys. ReV. B 1988, 37, 785–789.
(16) (a) Ha¨ttig, C.; Weigend, F. J. Chem. Phys. 2000, 113, 5154–5161.
(b) Ha¨ttig, C.; Ko¨hn, A.; Hald, K. J. Chem. Phys. 2002, 116, 5401–
5410. (c) Ha¨ttig, C. J. Chem. Phys. 2003, 118, 7751–7761.
(17) Weigend, F.; Ha¨ser, F.; Patzelt, H.; Ahlrichs, R. Chem. Phys. Lett.
1998, 294, 143–152.
(18) Olivucci, M. Computational Photochemistry; Elsevier: Amsterdam,
2005; pp 92-128.
(12) van Stokkum, I. H.; Larsen, D. S.; van Grondelle, R. Biochim. Biophys.
Acta 2004, 1657, 82–104.
(19) Frisch, M. J.; et al. Gaussian 03, revision C.02; Gaussian, Inc.:
Wallingford, CT, 2004.
(13) (a) Ahlrichs, R.; Ba¨r, M.; Ha¨ser, M.; Horn, H.; Ko¨lmel, C. Chem.
Phys. Lett. 1989, 162, 165. (b) For the current version of TURBO-
MOLE, see http://www.turbomole.de. (c) Treutler, O.; Ahlrichs, R.
J. Chem. Phys. 1995, 102, 346.
(14) Spartan ′06, Wavefunction, Inc., Irvine, CA, Shao, Y.; et al. Chem.
Chem. Phys. 2006, 8, 3172.
(20) Karlstrom, G.; Lindh, R.; Malmqvist, P.-A.; Roos, B. O.; Ryde, U.;
Veryazov, V.; Widmark, P.-O.; Cossi, M.; Schimmelpfennig, B.;
Neogrady, P.; Seijo, L. Comput. Mater. Sci. 2003, 28, 222.
(21) Pierloot, K.; Dumez, B.; Widmark, P. O.; Ross, B. O. Theor. Chim.
Acta 1995, 90, 87–114.
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Figure 2. X-ray crystal structure of acetylated isopropyl alcohol adduct 9.
Table 1. Reduction to Addition Ratio for Photoreaction of 5 with
Various Nucleophiles
product ratiosa b
,
nucleophiles
reduction (6)
substitution (8)c
MeOH
i-PrOH
n-BuOH
t-BuOH
10 (2)
5 (4)
7 (5)
90 (98)
95 (96) [7:8 ) 4:92]
93 (95)
(86)
(14)
HOCH2CO2CH3
HOCH(CH3)CO2C2H5
1,4-HOC6H4OH
(CH3)2NH2+Cl-
C6H5OH
(100)
(0)
0 (0)
0 (0)
90 (95)
97 (98)
100 (100)
100 (100)
10 (5)
3 (2)
a Determined by mass spectrometry of photoproducts from laser
transient studies. b Determined from preparative runs using a Rayonet
photochemical reactor in parentheses. c All substitution products are
2-substituted (8) rather than 6-substituted (7) unless noted otherwise.
grows and sharpens to a peak at 470 nm that dominates the ∆A
spectrum. The absorption changes from 50 to 1000 ps (Figure 1D),
slowly decaying in the 350-400 and 525-700 nm ranges, with
formation of a narrowed, long-lived band centered at 486 nm with
a broad shoulder at around 390 nm.22
Figure 1. (A) UV-vis absorption spectra of azide 5 in several solvents
(ε(445) ) 1390 M-1 cm-1 in CH3CN) normalized to unity at the visible
absorption maximum. The UV part of the spectrum is shown in the inset.
The arrows indicate the excitation wavelengths used in the current study.
(B-D) Transient absorption (∆A) spectra of a 16 mM solution of azide 5
in i-PrOH shown for various delay times between the 350 nm pump and
probe pulses. The solution was flowed through a 0.5 mm flow cell and
excited with 4 µJ pulses. The delay times in picoseconds are shown in the
frame legends: B (short times, 0.1-1 ps), C (intermediate times, 1-20 ps),
and D (long times, 20-1100 ps). Data in D represent smoothed spectra by
adjacent-averaging (bandwidth, 3.5 nm). The solvent contribution to the
∆A spectra is minor at delay times equal to or longer than 100 fs, except
for the 388.9 nm feature that corresponds to stimulated Raman scattering
from i-PrOH and yields11 the instrument response function, 150 fs (fwhm).
The aforementioned transient behavior was compared with
that upon excitation at a longer wavelength, 420 nm (Figure
3A,B). The 100 and 200 fs ∆A spectra display bands at 550
and 360-380 nm, which resemble those observed upon 350
nm excitation in their shape, signal amplitude, and decay
constants (major components, ca. 0.4 and 3 ps), albeit the
100-200 fs ∆A spectra upon 350 nm excitation are much
broader, indicating a larger excess of internal energy content
of the contributing species and possibly the formation of new
species. With 420 nm excitation, no discernible ∆A signal is
observed beyond 20 ps. Vanishing of both bleach (negative ∆A)
and transient absorption signals beyond 20 ps indicates the
quantitative reformation of the relaxed ground state of azide 5.
Thus, a 420 nm photon delivers photoexcitation into the lowest
energy band of the absorption spectrum of azide 5, most likely
1B). From 100 to 200 fs, the transient absorption signal rises, while
undergoing a small blue shift to 560 nm. At 300 fs, transient
absorption starts decaying and shifts further to the blue. Between
300 fs and 1 ps, the blue shift continues accompanied by narrowing
(570 cm-1) and decay. As time progresses from 1 to 6 ps (Figure
1C), the visible band blue shifts even more with the formation of
a broad absorption spectrum centered at about 495 nm, and the
entire ∆A spectrum decays. From 6 to 20 ps, the visible absorption
(22) The transient absorption spectrum of 5 pumped at 350 nm in
acetonitrile is provided in Figure SM2 in the Supporting Informa-
tion.
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Photoaffinity Labeling via Nitrenium Ion Chemistry
A R T I C L E S
Figure 3. Transient absorption (∆A) spectra of azide 5 (1.2 mM) in i-PrOH for various delay times (in picoseconds, shown in the legends) between the
probe and pump pulses. (A,B) Solution was flowed through a 0.5 mm flow cell and excited with a 420 nm, 3.5 µJ pulse. (C-E) Solution was flowed through
a 0.2 mm flow cell and excited with a 305 nm, 3.8 µJ pulse. The solvent contribution to the ∆A spectra is minor at delay times g100 fs; see Figure SM1
in Supporting Information. For 305 nm excitation, the UV region (280-375 nm) of the ∆A spectra was measured using the probe light generated by TOPAS
([azide 5] ) 1.7 mM, 3.1 µJ pump pulse) and subsequently scaled to the visible (360-665 nm) ∆A spectra measured using the white-light continuum probe.
The inset compares the ∆A kinetic traces recorded at probe wavelengths of 350 and 465 nm.
the S0fS1 electronic transition, and neither the 470 nm nor the
long-lived 486 nm intermediates were observed.
The validity of the fitting procedure is evident by comparison
of the reconstructed decay-associated spectra maxima with the
maxima observed in the time-evolving ∆A spectra (cf. Table 2,
entry 7 vs the maxima at 565, 539, 528, 469, and 486 nm in
Figure 1). Lower quality fits are obtained with a smaller number
of time constants, while the increase of a number of time
constants leads to no further improvement of ꢁ2 values. It is
particularly important to note that the decay of the 350 nm band
correlates very well with growth of the ∼465 nm band in this
model (see inset in Figure 3, τ3 ) 19 ps). Only two time
constants and a very small permanent background spectrum are
required for a satisfactory global fit of the spectrum generated
by 420 nm excitation. The resulting time constants are sum-
marized in Table 2, together with the maximum absorption
wavelengths (λmax) of the resolved component spectra.
These observations have been extended to longer times using
nanosecond spectroscopy (Figure 4F),8 which for i-PrOH
initially displays the narrow red-shifted band of the 486 nm
transient that decays relatively slowly (τ5 ) 557 ( 8 ns) to yet
another intermediate absorbing at 445 nm. The decay of the
486 nm band matches the growth of the 445 nm band (τ5 )
556 ( 28 ns). The 445 nm transient eventually decays to
baseline in the microsecond domain. This same general pattern
is repeated for most other alcohols, amines, ammonium salts,
and some carbon compounds with acidic hydrogens such as
dimethyl malonate and even acetonitrile (Vide infra). In nona-
cidic solvents, such as toluene (Table 2, entry 18), the
subpicosecond absorption is very broad, extending beyond 700
nm. The ensuing broad transients occur at 514, 543, and 492
nm. The latter 492 nm transient persists beyond a time delay
Excitation into to a higher lying excited electronic state was
achieved using 305 nm pump pulses (Figure 3C-E). As in the
case of 350 nm excitation, a ∼100 fs delayed rise of the 550
nm band is observed. However, the short-time ∆A spectra are
dominated by the 350 nm product absorption band, the formation
of which occurs in <100 fs. The 350 nm band decays slightly
over 1 ps and significantly over 50 ps (time constant, 19 ps)
with a 900 cm-1 blue shift. Over the time that the 350 nm band
has decayed by 50%, the 550 nm band vanished completely,
indicating that these bands are not related to each other. In
general, the short-time 200 fs ∆A spectra observed upon 305
and 350 nm excitation resemble each other (Figures 1B and
3A), albeit the ∆A signal amplitude is much larger in the 350
nm spectrum. This indicates that 350 and 305 nm excitation
leads to the formation of several species, which have similar
absorption spectra, but that 305 nm excitation produces mainly
the more weakly absorbing components of the 350 nm spectrum.
These components ultimately decay to the same species exhibit-
ing the narrow 487 nm band with the 390 nm shoulder that are
dominant in the 350 nm ∆A spectra at the longest time delays
(1100 ps in Figures 1D and 3C).
The 305 and 350 nm photoexcitation lead to a complicated
spectral evolution, which exhibits several kinetically and
spectrally different features, namely, subpicosecond and pico-
second components, ∼350 and 465 nm bands, and a ∼490 nm
product band. Figure 4 illustrates a good-quality global fit using
a sum of four exponential functions and an offset (permanent
background spectrum) for azide 5 in i-PrOH excited at 350 nm.
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Voskresenska et al.
an initial broad absorption band centered at 562 nm (τ1 ) 333
fs, Table 2, entry 7 and Figure 4A) is observed in contrast with
the previously observed singlet excited state of para-biphenylyl
azide, which has an absorption maximum at 480 nm (τ ∼ 100
fs).23 In general, the lifetimes of aryl azide excited singlet states
fall between 100 and 600 fs. Consequently, the 333 fs lifetime
of the 562 nm species observed in this system is consistent with
the singlet excited state of the azide 5. However, this absorption
is assigned to the lowest excited singlet state of azide 5 (S1),
which does not afford the nitrene (Vide infra and Table 2, entry
10).
Theoretical Considerations. Active Excited State and Nitrene
Formation. Detailed calculations have been conducted on the
azide 5 and its associated nitrene. The first question to be
addressed was that of the excited state behavior of the azide 5
in an effort to determine which of the several low lying excited
states would be most likely to produce nitrogen extrusion (Figure
6). The lowest excited state, S1, is (π,π*) with a shift of electron
density from the aromatic ring and the amino group ortho to
the nitro group at the TD-B3LYP level of theory, λmax
)
477 nm. On the other hand, the second excited state, S2,
(π,π*in-plane), is localized on the azide group with a significant
loss of electron density between the first and second nitrogen
atoms of the azide moiety (TD-B3LYP, λmax ) 353 nm, f )
0.0000, Figure 6) and suggests subsequent elongation of the
-N-N2 bond. Furthermore, TD-B3LYP optimization of the S0,
S1, and S2 states of the di-N-methyl analogue of 5 indicates
that the -N-N2 bond lengthens appropriately for the loss of
nitrogen and formation of the nitrene in the S2 excited state but
not in the S1 excited state (Figure 7). These correlations clearly
indicate that S1 is not an effective precursor state for loss of
nitrogen and formation of the nitrene. Consequently, S2 or higher
excited states, but not S1, seem to be ideal candidates for the
extrusion of nitrogen and formation of the nitrene.
Figure 4. Reconstructed decay-associated spectra (εi) extracted from the
∆A transient absorption spectra of azide 5 in i-PrOH irradiated with 350
nm laser pulses. The time constants (τi) obtained from a least-squares global
fit of the 512 ∆A kinetic traces to a sum of four exponential functions and
a permanent offset are shown in the insets. (A) Absorption of excited
electronic state of azide 5; (B) hot ground state of 5 following internal
conversion; (C) hot singlet nitrene and probably residual hot ground state
of azide 5 at 528 nm; (D) triplet nitrene and nitrenium ion 10; (E) nitrenium
ion 10; (F) ns/µs decay of the nitrenium ion 10 (486 nm) and formation of
the adduct 14 (445 nm).
These calculated energy relationships correlate very nicely
with the observed transient behavior of 5. Thus, irradiation of
5 at 420 nm might selectively populate S1 but not S2 (calculated
λ(S0fS1) ) 477 nm, calculated λ(S0fS2) ) 353 nm). The
transient cascades outlined in Table 2 indicate that irradiation
at 420 nm (entry 10) leads to a broad absorption ca. 559 nm
that forms within 400 fs and returns to baseline a few
picoseconds later. In contrast, irradiation with 350 nm light
(entry 7), which should populate S2 or higher excited states,
leads to the complete cascade of transients out into the
microsecond domain. The simplest interpretation of these
observations is that 420 nm excitation populates S1 and that
this intensely absorbing species returns to the ground state, S0,
without loss of molecular nitrogen or nitrene formation. In
contrast, 350 nm excitation populates both S1 and S2 since
similar initial transient spectra are observed, but the transient
cascade extends beyond the decay of S1 with a stream of
intermediates arising from the loss of nitrogen and nitrene
formation. In this interpretation, the source of the spectra in
Figure 4A is excited state absorption from S1 and S2 or higher.
Both of these excited states may de-energize to hot S0 within 1
ps followed by vibrational relaxation (Figure 4B), but S2 also
extrudes N2 within this same time frame leading to formation
of the nitrene, which is the spectrum in Figure 4C (possibly
with a small contribution from the 528 nm band of moderately
hot S0 of azide 5), and is a precursor for the spectra in Figure
4D,E. Comparison of the τ1 entries in Table 2 for 420 and 350
nm excitation shows that the short-time constants are not
of 1 ns (Figure 5), and the longer-lived 465, 486, and 445 nm
bands usually observed never develop (Table 2, entry 18, and
Figure 4D-F). The full range of transients is observed when
the azide 5 is irradiated at 350 nm in acetonitrile (Table 2, entry
13), but irradiation at 420 nm in acetonitrile leads only to the
initial transients similar to those observed at this wavelength in
i-PrOH (Table 2, entry 10).
A detailed analysis of the formation of nitrenes that undergo
aromatic ring expansion, including ortho- and para-biphenylyl
azide and 1-naphthyl azide, has recently been reported.23
Comparing the transient spectra observed in this work with these
literature data, we note that the initial absorption spectrum,
presumably that of the singlet excited state of 5, occurs at
significantly longer wavelength. Thus, in the case of 5 in i-PrOH,
(23) (a) Burdzinski, G.; Hackett, J. C.; Wang, J.; Gustafson, T. L.; Hadad,
C. M.; Platz, M. S. J. Am. Chem. Soc. 2006, 128, 13402–13411. (b)
McCulla, R. D.; Burdzinski, G.; Platz, M. S. Org. Lett. 2006, 8, 1637–
1640. (c) Gritsan, N. P.; Polshakov, D. A.; Tsao, M.-L.; Platz, M. S.
Photochem. Photobiol. Sci. 2005, 4, 23–32.
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Photoaffinity Labeling via Nitrenium Ion Chemistry
A R T I C L E S
Table 2. Transient Cascade Characteristics Observed upon Irradiation of Azide 5 in Various Solvents (Frames A-E Refer to the
Corresponding Time Frames in Figure 4, Their Associated λmax Values, and Time Constants for the Solvents Indicated)
pump λ
(nm)
frame A
λmax (nm)
frame B
λmax (nm)
frame C
λmax (nm)
frame D
λmax (nm)
frame E
λmax (nm)c
solvent
τ1 (fs)
τ2 (ps)
τ3 (ps)
τ4 (ps)
τ5 (ns)
1
2
3
4
5
6
7
8
9
CH3OH
CH3OD
CD3OD
CH3CH2OH
n-C4H9OH
350
350
350
350
350
350
350
305
350
420
350
350
350
350
350
420
350
350
568
556
562
567
555
566
562
580-620
562
559
554
562
559
547
275
547
543
547
539
544
539
541
540
541
538
539
535
523
548
0.904
503
502
510
513
522
501
528
531
528
-
4.8
6.7
7.2
9.5
12.4
23.4
19
20.5
16.4
-
12.8
12.8
9.6
488
484
486
470
460
462
465
460
465
-
446
430
481
394
275
435
507
833
289
-
501
497
490
488
485
484
486
484
486
-
485
490
489
487
-
316
329
310
286
329
333
564
304
378
266
525
247
231
1.07
1.21
1.4
325
389
1.28
2.05
1.76
5.46
1.83
2.81
1.28
3.28
0.71
0.94
n-C8H17OH
(CH3)2CHOHa
(CH3)2CHOHb
(CH3)2CHOD
557
-
-
10 (CH3)2CHOH
11 (CH3)3COH
12 HOCH2CO2Et
535
476
482
533
470
474
484
479
833
714
238
204
1260
-
-
-
-
-
-
-
13 CH3CN (dry)c d
14 CH3CN + 2 M PhOH
15 C6H12
,
11.8
550 ( 10 422 ( 88 522 ( 5 3.6 ( 0.3 488 ( 3 80 ( 29 452 ( 4 666
16 C6H12
17 C6H12 + 2 M PhOH
18 C6H5CH3
570
562
569
430
276
686
517
540
514
6.26
1.8
23.8
-
-
-
-
-
476
543
17.8
314.5
456
492
183
466
-
c
a Plotted in Figure 4. b S1 absorption is difficult to measure at this exciting wavelength. c Persists to the limit of the time window (1.2 ns) of ultrafast
transient absorption experiments. d Well-fitted by three exponentials, but these listed data are for four exponentials in order to compare with other data
that require four exponentials for a satisfactory fit.
Figure 5. Long-time transient absorption (∆A) spectra of azide 5 in toluene
upon 350 nm excitation. The solution (16 mM) was circulated through a
0.5 mm path length flow cell.
Figure 7. Excited state energies (kcal/mol) and bond lengths for the
optimized geometries (TD-B3LYP) of the ground state, and the first and
second excited states of the N,N-dimethyl analogue of azide 5.
Figure 6. Electron density redistribution in the (left) S1 and (right) S2 states
of 5 calculated at the TD-B3LYP/TZVP level of theory. The green contours
depict the accumulation of electron density in the excited state, and the red
time frame of the chemistry occurring in the aforementioned
contours illustrate the loss of electron density from the S0 ground state.
open-shell singlet, or the closed-shell singlet. Considering the
transient section (Figure 4A-C), the triplet configuration can
be excluded since singlet aryl nitrenes usually require about
100 ps to 10 ns to reach singlet-triplet equilibrium,1a,24 and in
the systems studied here, the singlet nitrene (Figure 4C) proceeds
to the next intermediate (Figure 4D) in <20 ps.
Therefore, the remaining questions are whether the config-
uration of the ground state singlet nitrene is closed-shell or open-
shell, and which of these is the reactive intermediate in the
The contour values are (0.005 au.
affected; therefore, it seems likely that the S2 lifetime is
significantly shorter than the S1 lifetime.
Nitrene Electronic Configuration. The electronic configura-
tion of the nitrene derived from azide 5 becomes quite important
in interpreting the chemistry of this species. This nitrene might
exist in any of three electronic configurations: the triplet, the
(24) (a) Gritsan, N. P.; Tigelaar, D.; Platz, M. S. J. Phys. Chem. A 1999,
103, 4465–4469. Carbenes require a similar time in order to establish
singlet-triplet equilibration: (b) Wang, J.; Kubicki, J.; Hilinski, E. F.;
Mecklenburg, S. L.; Gustafson, T. L.; Platz, M. S. J. Am. Chem. Soc.
2007, 129, 13683–13690.
(25) (a) Zuev, P. S.; Sheridan, R. S. J. Am. Chem. Soc. 1994, 116, 9381–
9382. (b) Zuev, P. S.; Sheridan, R. S. J. Org. Chem. 1994, 59, 2267–
2269. (c) Zuev, P.; Sheridan, R. S. J. Am. Chem. Soc. 1993, 115, 3788–
3789. (d) Tomioka, H.; Komatsu, K.; Nakayama, T.; Shimizu, J. Chem.
Lett. 1993, 1291–1294.
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J. AM. CHEM. SOC. VOL. 131, NO. 32, 2009 11541

A R T I C L E S
Voskresenska et al.
chemistry to follow. Extensive CASSCF calculations using large
electron/orbital sets (CASSCF(10,8)/6-311+G(3df,3pd)//CASS-
CF(4,4)/6-31G(d)) indicate that the open-shell (OS) singlet is
about 6.6 kcal/mol lower in energy than the closed-shell (CS)
singlet, and that ∆ECS-OS is definitely <10 kcal/mol. Therefore,
one can say with a fairly high degree of certainty that the open-
shell singlet is the ground singlet state of the nitrene.
However, both ultrafast femto/picosecond and conventional
nanosecond spectroscopy, as well as the products observed in
the photochemistry of azide 5, seem to be more consistent with
the chemistry of a closed-shell nitrene intermediate than an open-
shell nitrene intermediate. Intuitively, one expects open-shell
singlet nitrenes to engage in single-electron radical or diradical
chemistry; in contrast, one expects closed-shell singlet nitrenes
to engage in two-electron, acid/base or nucleophile/electrophile
chemistry. This type of behavior has been observed by
Sheridan25a-c and Tomioka25d for p-phenylenebis(chlorometh-
ylene), a bis-open-shell dicarbene, and p-phenylenebis(fluo-
romethylene), a bis-closed-shell dicarbene, which bears an
intriguing resemblance to the nitrene under study in this work.
It should be noted, however, that detailed studies on the
differences in reactivity between an open-shell and closed-shell
nitrene have not been performed, and so these predictions
currently lack rigorous experimental validation.
Figure 8. (A) Conserved angular momentum in intersystem crossing (ISC)
of carbonyl group. (B) Relationships between ISC and open- to closed-
shell singlet nitrene interconversion (OCSI).
In the photochemistry of 5, the amine 6 (Scheme 3) might
conceivably arise via a radical process, but the substitution
products 7 and 8 would be more expeditiously formed via two-
electron, closed-shell nitrene chemistry (Vide infra).26 Further-
more, the transient species observed in toluene, conditions under
which the triplet nitrene and associated radical intermediates
should be formed, are distinctly different from those formed
under the conditions of the reaction in question (compare Figures
3 and 4 with Figure 5). Finally, the calculated CdN bond length
for the S2 excited state of the azide 5 is 1.365 Å (Figure 7),
which is significantly closer to the calculated CdN bond length
for the closed-shell nitrene, 1.364 Å, than to that calculated for
the open-shell nitrene, 1.447 Å.8 Thus, theoretical calculations
and experimental evidence can be reconciled if the Franck-
Condon excited state of the azide leads directly to the closed-
shell nitrene. Since interconversion between the closed- and
open-shell singlet nitrene states is forbidden and, thus, a
relatively slow process, relative rates of reaction, not singlet
nitrene energies, might well determine the course of subsequent
reactions.
In support of this relative rate scenario, there is a straight-
forward correlation between singlet-triplet intersystem crossing
(ISC) and closed-to-open-shell nitrene interconversion (COSI).
An example of which is shown in Figure 8A for the carbonyl
(1n,π*)-(3π,π*) ISC. In this well-understood system, ISC
(effective rate <109 s-1), only occurs rapidly if the change in
electron spin angular momentum is balanced by a corresponding
and opposite change in orbital angular momentum. Thus, the
spin flip of the electron is coupled with the redistribution of
electron density between orthogonal molecular orbitals. If
orthogonal orbitals of similar energy are not available for the
conservation of angular momentum, then the higher energy
singlet excited state will not readily undergo ISC to the lower
energy triplet state, and the singlet state can persist for hundreds
of nanoseconds. This is the case with the singlet (π,π*) excited
states of aromatic hydrocarbons.27 In Figure 8B, the relationships
between open- and closed-shell singlet and triplet nitrene states
are shown. Thus, interconversion between open- and closed-
shell singlet states (OCSI) is expected to be highly forbidden
since the change in orbital angular momentum cannot be
balanced by a corresponding change in electron spin angular
momentum. Likewise, the ISC of the open-shell singlet to the
corresponding triplet also should be highly forbidden since the
change in electron spin angular momentum cannot be balanced
by a change in orbital angular momentum. However, the closed-
shell singlet nitrene should readily undergo ISC to the triplet
nitrene since this is a spin-orbit-allowed process.28 Therefore,
even though the closed-shell singlet nitrene may be at higher
energy than the open-shell singlet nitrene, it can play a
significant role in the chemistry of 5 if it is populated directly
upon loss of molecular nitrogen from the singlet excited state
of the azide 5 and if it reacts more rapidly than it undergoes
interconversion to the open-shell singlet or ISC to the triplet
state.
Nitrene Formation. On the basis of these calculations and
the experimental data, nitrene formation arises from the second,
S2, or higher excited singlet states of azide 5. The S1 singlet
state decays back to the ground state without giving rise to any
transients that survive longer than a few picoseconds (see Table
2 for excitation at 420 nm in i-PrOH, entry 10, and in
cyclohexane, entry 16). The source of the spectra in time frames
A and B (Table 2 and Figure 4) is thought to be largely due to
the lowest excited singlet S1 state of azide 5 and the vibrationally
hot states of the S0 state formed upon internal conversion of
S1. In contrast, irradiation at 350 and 305 nm leads to population
of S1 and higher energy excited states (S1 + S2 +...) and a
cascade of nitrene-derived intermediates extending into much
longer time domains. For example, excitation of 5 in i-PrOH at
(26) (a) Falvey, D. E. In ReactiVe Intermediate Chemistry; Moss, R. A.,
Platz, M. S., Jones, M., Jr., Eds.; John Wiley & Sons: Hoboken, NJ,
2004; pp 593-650. (b) Winter, A. H.; Gibson, H. H.; Falvey, D. E.
J. Org. Chem. 2007, 72, 8186–8195.
(27) Liu, Y. S.; de Mayo, P.; Ware, W. R. J. Phys. Chem. 1993, 97, 5987–
5994.
(28) Borden, W. T.; Gritsan, N. P.; Hadad, C. M.; Karney, W. L.; Kemnitz,
C. R.; Platz, M. S. Acc. Chem. Res. 2000, 33, 765–771.
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11542 J. AM. CHEM. SOC. VOL. 131, NO. 32, 2009

Photoaffinity Labeling via Nitrenium Ion Chemistry
A R T I C L E S
Figure 9. Possible reaction pathways connecting excited states of azide 5, nitrene states, and nitrenium ion (10) states. The vertical energy axis is not drawn
to scale.
Table 3. Vertical Transitions and Absorption Properties of Possible
and the other is a proton-dependent process, probably nitrenium
ion formation (Table 3).
The aforementioned femtosecond-picosecond spectroscopic
Reactive Species Derived from the Irradiation of Azide 5 as
Calculated at the TD-B3LYP/TZVP Level of Theory
species
λmax (nm)
oscillator strength (f)
observations are correlated by the reaction scheme outlined in
Figure 9. Clearly, the lowest excited singlet state of the azide
5 does not lead to nitrene formation, but higher (π,π*) excited
states localized mainly on the azide unit, particularly the S2 state,
do afford the nitrene. Since similar processes in carbene
chemistry have been shown to produce both ground and excited
carbene states,24b,29 it is quite possible that the excited closed-
shell nitrene is formed initially upon loss of molecular nitrogen
in this system. As indicated in the previous discussion, the
closed- and open-shell singlet nitrenes should not be readily
interconverted without some type of second-order vibronic
coupling that is thought to be weak at best.28 Therefore, an
initially formed closed-shell nitrene might survive long enough
to initiate chemistry unique to its electronic configuration. In
fact, the lifetime of this closed-shell nitrene may be governed
by ISC to the triplet nitrene, which is a spin-orbit-allowed
process, rather than by decay to the open-shell singlet nitrene,
which is a spin-orbit-forbidden process.24b,28 Finally, one
plausible reaction pathway of the closed-shell nitrene might be
protonation, which correlates with the ground state of the
nitrenium ion 10. In contrast, protonation of the open-shell
nitrene correlates with the excited state of 10 that is estimated
to be approximately 87 kcal/mol above the nitrenium ion ground
state. In this scenario, the nitrenium ion 10 would be the pivotal
reactive intermediate leading to the substitution products 7 and
8 (Scheme 3). Many alternative scenarios have been considered
for the nitrene reaction cascade.8 For instance, insertion of the
closed-shell singlet nitrene directly into the O-H bond of the
solvent alcohol to form a hydroxylamine ether might be a
possible alternative. While this possibility cannot be rigorously
excluded, we tend to discount it due to the lack of hydroxy-
closed-shell singlet nitrene
open-shell singlet nitrene
triplet nitrene
nitrogen radical
nitrenium ion 10
adduct 13
340
483
426
447
441
360
409
0.0065
0.0323
0.0828
0.0978
0.0019
0.0443
0.0524
adduct 14
305 nm (Table 2, entry 8, and Figure 3) leads to a new
absorption at 580-620 nm and intense absorption at 350 nm
(time delay, 100 fs), both of which decay over about 20 ps to
a species absorbing at 460 nm that in turn decays over about
833 ps to a species absorbing at 484-486 nm. Nanosecond
transient absorption spectroscopy shows that this 484-486 nm
species slowly (τ5 ) 557 ns) decays to the final species observed
that absorbs at 445 nm. The nature of the 350 nm species (Figure
3C,D) that is formed in <100 fs is of central importance. This
absorption band is not due to hot S0. Indeed, 420 nm excitation
produces a 360 nm absorption band that is spectrally different
and also decays with two time constants, 380 fs and 2.8 ps, but
leads to no further absorption bands. In contrast, the decay of
this 350 nm band occurs with a time constant of 19 ps and
parallels the growth of the 465 nm band. During this evolution,
the 350 nm band undergoes a blue shift to 340 nm and spectral
narrowing, indicating that the species responsible for the 350
nm band are produced with excess vibrational energy. This 350
nm species is assigned to the hot closed-shell nitrene (Figure 9
and Table 3). Excitation of 5 at 350 nm in cyclohexane (Table
2, entry 15) produces the same singlet nitrene that decays to
the triplet nitrene (τ3 ) 80 ps, λmax ) 452 nm). When the proton
source phenol is added to cyclohexane (Table 2, entry 17, also
see Figure 11C), a very conspicuous new transient is rapidly
formed in the 452-480 nm region over ca. 18 ps and survives
for several hundred nanoseconds. These two experiments
indicate that two processes compete for the closed-shell nitrene,
and that both of these processes yield species absorbing in the
452-480 nm region. One of these is triplet nitrene formation,
(29) (a) Chang, K. T.; Shechter, H. J. Am. Chem. Soc. 1979, 101, 5082–
5084. (b) Langan, J. G.; Sitzmann, E. V.; Eisenthal, K. B. Chem. Phys.
Lett. 1984, 110, 521–527. (c) Wang, Y.; Sitzmann, E. V.; Novak, F.;
Dupuy, C.; Eisenthal, K. B. J. Am. Chem. Soc. 1982, 104, 3238–3239.
(d) Sitzmann, E. V.; Wang, Y.; Eisenthal, K. B. J. Phys. Chem. 1983,
87, 2283–2285.
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J. AM. CHEM. SOC. VOL. 131, NO. 32, 2009 11543

A R T I C L E S
Voskresenska et al.
Scheme 4
lamine ether products in any of the alcohol reaction mixtures.
Another mechanistic possibility leading to the nitrenium ion
intermediate that we cannot rigorously exclude is that the
observed nitrene is the lower energy open-shell singlet state
and that protonation occurs with a simultaneous change in
electronic state (e.g., open-shell nitrene f closed-shell nitrenium
ion). Since fundamental spectroscopic studies of the mechanism
of protonation of nitrenes have not been performed, we cannot
exclude this concerted mechanism as a possibility.
timeconstantsof60-100pshavebeenobservedforsinglet-triplet
ISC in p-amino-substituted aryl nitrenes.30 Apparently, the short
wavelength component of the 465 nm band in Figure 4D is
due to small amounts of the triplet nitrene. In support of this
assignment, the amine radical was obtained by irradiation of
azide 5 in toluene (Table 2, entry 18, and Figure 5). Under these
conditions, a broad band at 543 nm persists and slowly shifts
to an intense and broad band centered at 492 nm over ca. 315
ps. In addition, the benzylamine corresponding to benzylation
of the nitrene nitrogen was isolated as the main product of this
reaction in toluene.8 Therefore, the source of this 492 nm band
is most reasonably attributed to the nitrogen radical. These
considerations indicate that ISC to the triplet and hydrogen atom
abstraction to form the nitrogen radical are relatively slow
processes compared to the process shown in Figure 4C,D, and
that the nitrene triplet would be expected to absorb on the short
wavelength shoulder of the nitrenium ion absorption band (Table
3). Finally, additional confirmation of the presence of the nitrene
triplet in these reactions has been obtained via the isolation of
the typical azo triplet dimer 11,1a as well as the unusual oxidized
alternative dimer 1231 (Scheme 4), from reactions in nonalco-
holic solvents such as acetonitrile. Therefore, the observed rate
constant coupling frames C and D, τ3, is the sum of the rate
constants for the two branches of singlet nitrene reactions, kISC
+ kH, shown in Figure 9. However, so long as the singlet-triplet
ISC branch develops much more slowly,2i this composite rate
constant will largely reflect the much faster rate of the
protonation branch.
In the model outlined in Figure 9, the closed-shell nitrene
(Figures 3D and 4C) undergoes nitrene protonation in competi-
tion with ISC to the triplet nitrene. If the triplet nitrene is
generated in this cascade of intermediates, then one must take
into consideration hydrogen atom abstraction to form the
corresponding nitrogen radical, which is a reasonable precursor
for the amine 6. TD-B3LYP calculations indicate that these
possible intermediates all absorb in the 340-480 nm region as
indicated in Table 3. Clearly, a combination of triplet nitrene,
nitrogen radical, and/or nitrenium ion 10 might give rise to the
transient spectrum observed in Figure 3D,E (20 ps) or 4D, which
is a broad absorption band centered at 465 nm. This band
narrows and shifts to longer wavelength, 486 nm (Figure 3E or
4D,E). The band in frame E is also shifted to the red by polar
solvents (compare λmax ) 484-501 nm in polar solvents, Table
2, entries 1-9, 11, 12, and 14 with λmax ) 466 nm in
cyclohexane, entry 17). It is interesting to note that the calculated
oscillator strengths of the triplet and radical are about 50 times
larger that that calculated for the nitrenium ion 10 in Table 3.
Consequently, any mixture of (triplet + radical)/nitrenium ion
10 of ca. 2/98 would give overlapping signals of approximately
equal intensities as observed in Figure 4D. While it may be a
coincidence, this is about the same ratio that is observed for
product formation, amine 6/nitrenium ion adducts 7 and 8 )
5-2/95-98 (Table 1). Apparently, in neat protic solvents, the
rate constant for nitrene singlet-triplet ISC (τISC-1) is signifi-
cantly slower than that for protonation (τH-1 ) kH). This is also
indicated in cyclohexane (Table 2, entry 15), where protonation
is not possible, τ3 ) τISC ) ca. 80 ps, and entry 17 in
cyclohexane/phenol, where τ3 ) τISC+H ) ca. 18 ps. Similar
A factor that might influence the observed rates of protonation
is the initial formation of a vibrationally hot singlet nitrene
(30) Irradiation of p-(dimethylamino)phenyl azide in toluene leads to triplet
ground state formation in 120 ps: Kobayashi, T.; Ohtani, H.; Suzuki,
K.; Yamaoka, T. J. Phys. Chem. 1985, 89, 776–779, but time constant
may have involved competitive ISC to the triplet and proton abstrac-
tion. Lloyd-Jones, G. C.; Alder, R. W.; Owen-Smith, G. J. Chemistry
2006, 12, 5361–5375.
(31) (a) Nay, B.; Scriven, E. F. V.; Suschitzky, H.; Thomas, D. R. J. Chem.
Soc., Perkin Trans. 1 1980, 611–613. (b) Kvaskoff, D.; Mitschke, U.;
Addicott, C.; Finnerty, J.; Bednarek, P.; Wentrup, C. Aust. J. Chem.
2009, 62, 275–286.
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11544 J. AM. CHEM. SOC. VOL. 131, NO. 32, 2009

Photoaffinity Labeling via Nitrenium Ion Chemistry
A R T I C L E S
Table 4. Transition State Properties for Proton Abstraction of
Closed-Shell Singlet Nitrene 10 from Methanol (Geometries of
These Transition States are Shown in Figure 10)
transition state
for protonation ∆Hq (kcal/mol)
O-H bond N-H bond
distance (Å) distance (Å)
νi
A
A
B
4.81
4.43
i1119.48 56.81
i1107.93 41.78
1.22
1.22
1.21
1.21
nitrene. Since the nitrene is formed in a vibrationally hot state,
it is assumed that the expelled molecular nitrogen rapidly leaves
the immediate vicinity of the nitrene nitrogen and thus has little,
if any, influence on the reactivity of the nitrene. Furthermore,
since nitrenes are only attached to a single substituent, they
extend further into solvent space than do carbenes, which are
attached to two shielding substituents. As a result, nitrenes are
sterically more available to solvent molecules and more flexible
to geometric modifications during the course of reactions.
Theoretical analyses of the reactions of this nitrene with alcohol
have located two transition states for nitrene protonation, and
these are described in Table 4.8 These protonation trajectories
have small activation energies (ca. ∆Eq ) 4.81-4.43 kcal/mol)
and are quite exothermic reactions with ∆H ) ca. -40 kcal/
mol. In addition, they both have transition states that occur fairly
early along the reaction coordinate with the RO-H bond being
only ca. 28% broken. Therefore, we assign the 486 nm transient
in Figure 4E,F to the nitrenium ion 10 that results from simple
nitrene protonation. The small or lack of a KIE in this
protonation process would seem to be due to a combination of
factors including the availability of the nitrene nitrogen in
solvent space, the early and low activation energy barriers for
singlet nitrene protonation, and, at least in the case of methanol,
the very rapid protonation of a vibrationally hot nitrene shortly
after its formation.
Figure 10. Two regions of space surrounding the closed-shell singlet nitrene
nitrogen and alcohol approach geometries leading to protonation transition
states.
(Figure 3C,D), which subsequently undergoes a slight blue shift
of its spectrum as it cools.32 For example, the nitrene derived
from para-biphenylyl azide in acetonitrile has a cooling time
constant of 11 ps,23a which is coincident with the time constants
for the intermediates involved in Figure 3C,D. Consequently,
it seems highly likely that the protonations observed in this work
involve vibrationally hot singlet nitrenes to a significant extent,
which may help to account for the rapidity of these protonation
reactions.
The identity of the intense band at 486 nm in both E and F
of Figure 4 is of central importance in determining the pivotal
intermediates in this chemistry. The source of this intermediate
is thought to be the nitrenium ion 10 arising from protonation
of the nitrene and calculated to have λmax ) 441 nm (Table 3).
In an effort to evaluate this possibility, the effect of deuterium
on the rates of the nitrene protonation step with MeOD and
i-PrOD was investigated. These nitrene reactions display little,
if any, deuterium kinetic isotope effect (KIE) within experi-
mental error. Thus, kH/kD ) 1.4 for methanol (Table 2, entries
1 and 2), and kH/kD ) 0.9 for i-PrOH (Table 2, entries 7 and 9).
Similar small KIE values ranging from 1.0 to 1.7 are observed
for alcohol protonation of arylcarbenes.29d,33a,b Kirmse and co-
workers have discussed the lack of deuterium isotope effects
for the reactions of a variety of carbenes with alcohols and note
that it can be attributed to an “early” transition state with little
proton transfer from ROH to the carbene, and, correspondingly,
little charge development.33c This notion is consistent with the
involvement of vibrationally hot species for which an earlier
transition state might be expected.
In alcohol solvents, nitrene protonation occurs over a range
of τ3 ) 5-23.4 ps, with the most rapid being protonation by
methanol and the slowest being protonation by n-octanol. The
same general dependence on alcohol structure has been observed
in previous work with carbenes in which the most rapid
protonation occurs in methanol with other primary, secondary,
and tertiary alcohols protonating at slower rates that are similar
to each other.33a,b The protonation of the nitrene by methanol
observed in this work ranks with the fastest intermolecular
proton transfer processes known, which includes the protonation
of singlet diphenylcarbene by methanol, τ ) 9 ps,33a,b and the
protonation of water by several excited photoacids.34a,b
Variations on these suggestions have been explored from a
theoretical perspective for the case of the closed-shell singlet
nitrene derived from 5 at the B3LYP/6-31G* level as outlined
in Figure 10. While the possibility exists that hydrogen bonding
between alcoholic solvents and the azide nitrogen occurs prior
to photochemical expulsion of molecular nitrogen, no such stable
complex could be observed either experimentally (IR) or
theoretically. The question also arises as to the influence of the
departing nitrogen molecule on the reactivity of the incipient
Finally, immediately following protonation, a contact ion pair
(CIP) would result. This CIP will rapidly equilibrate to the
solvent-separated ion pair (SSIP) via a proton relay mechanism
(Grotthuss mechanism35) that will redistribute the alkoxide
anions in an equilibrated array surrounding the nitrenium ions.
Again, a parallel exists in the protonation of carbenes. Thus,
protonation of singlet di(p-methoxyphenyl)carbene yields a CIP
(λmax ) 470 nm) which red shifts to a SSIP (ca. 500 nm) over
ca. 700 ps.33b The time constant for this type of proton exchange
(32) (a) Laermer, F.; Elsaesser, T.; Kaiser, W. Chem. Phys. Lett. 1989,
156, 381–386. (b) Miyasaka, H.; Hagihara, M.; Okada, T.; Mataga,
N. Chem. Phys. Lett. 1992, 188, 259–264. (c) Schwarzer, D.; Troe,
J.; Votsmeier, M.; Zerezke, M. J. Chem. Phys. 1996, 105, 3121–3131.
(d) Elsaesser, T.; Kaiser, W. Annu. ReV. Phys. Chem. 1991, 42, 83–
107.
(33) (a) Peon, J.; Polshakov, D.; Kohler, B. J. Am. Chem. Soc. 2002, 124,
6428–6438. (b) Dix, E. J.; Goodman, J. L. J. Phys. Chem. 1994, 98,
12609–12612. (c) Kirmse, W.; Guth, M.; Steenken, S. J. Am. Chem.
Soc. 1996, 118, 10838–10849. (d) Wang, J.; Burdzinski, G.; Gustafson,
T. L.; Platz, M. S. J. Org. Chem. 2006, 71, 6221–6228. (e) Wang, J.;
Burdzinski, G.; Gustafson, T. L.; Platz, M. S. J. Am. Chem. Soc. 2007,
129, 2597–2606. (f) Wang, J.; Kubicki, J.; Hilinski, E. F.; Mecklen-
burg, S. L.; Gustafson, T. L.; Platz, M. S. J. Am. Chem. Soc. 2007,
129, 13683–13690.
(34) (a) Pines, E.; Pines, D.; Barak, T.; Magnes, B. Z.; Tolbert, L. M.;
Haubrich, J. E. Ber. Bunsen-Ges. Phys. Chem. 1998, 102, 511–517.
(b) Lima, J. C.; Abreu, I.; Brouillard, R.; Macanita, A. L. Chem. Phys.
Lett. 1998, 298, 189–195.
(35) (a) Cukierman, S. Biochim. Biophys. Acta 2006, 1757, 876–885. (b)
Voth, G. A. Acc. Chem. Res. 2006, 39, 143–150. (c) Markovitch, O.;
Chen, H.; Izvekov, S.; Paesani, F.; Voth, G. A.; Agmon, N. J. Phys.
Chem. B 2008, 112, 9456–9466. (d) http://www.lsbu.ac.uk/water/
grotthuss.html.
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J. AM. CHEM. SOC. VOL. 131, NO. 32, 2009 11545

A R T I C L E S
Voskresenska et al.
Figure 11. Spectroscopic properties in the nanosecond-microsecond time domain of azide 5 in (A) dimethyl malonate, (B) dimethylamine hydrochloride,
(C) phenol, and (D) hydroquinone with the last three spectra acquired in acetonitrile.
is estimated to be 55 ps for methanol/methoxide36 and is
expected to be about an order of magnitude slower in i-PrOH,33b
which is very close to the time constant observed for the
formation of the 486 nm, τ4 ) 507 ps, in that solvent (Figure
4D,E). Therefore, it seems likely that the red shift observed
upon going from frame D to E in Figure 4 is not entirely due
to decay of the short wavelength triplet component but may
have a significant contribution from the equilibration of the CIP
to the SSIP.
The nitrenium ion 10 is very long-lived to the extent that it
can be easily observed by nanosecond transient absorption
spectroscopy (Figure 4F). It slowly undergoes transformation
over several hundred nanoseconds to the final intermediate(s)
absorbing at 440-445 nm. In the i-PrOH reaction, the source
of this absorption seems to be the nucleophile adducts 13 and
14 (Scheme 4) arising from collapse of the SSIP. Upon the basis
of the structures of the observed products, 14 will be the major
contributor to this absorption, and its absorption is estimated
by TD-B3LYP to have λmax ) 409 nm, while that of the minor
contributor 13 is estimated to have λmax ) 360 nm (Table 3). It
is perhaps surprising that this ion pair collapse does not occur
immediately following the protonation step, but this is clearly
not the case, and the SSIP survives into the nanosecond-
microsecond time domains.
The aforementioned relationships are summarized in Scheme
4, where the absorption characteristics and lifetimes of the
intermediates are provided and related to Figure 4A-F, where
their spectra are shown. Several aspects of this general scheme
require further comment. The general spectroscopic pattern
shown in Figure 4F is repeated for many different types of
proton sources. Some of these are shown in Figure 11. Carbon
acids such as malonate esters, ammonium salts, and phenols
all clearly display the characteristic nitrenium ion absorption
in the 480-490 nm region. In addition, all of these proton
sources except hydroquinone (Figure 11D) clearly display the
characteristic absorption for the nucleophile adduct at about 440
nm. In the cases of phenol and dimethylamine hydrochloride,
the adducts 15 and 16, respectively, can be isolated. As indicated
above, hydroquinone is a proton source that forms the nitrenium
ion 10 (Figure 11D). However, the lack of a 440 nm band
indicates that no adduct is formed, and this observation is
supported by the fact that hydroquinone yields only the reduction
product amine 6 (Table 1). Exactly the same reduction pattern
is observed for the alcohol ethyl glycolate, which affords only
reduction to the amine 6 (Table 1). Consequently, in addition
to radical pathways for reduction of the nitrene to the amine 6,
there appear to be ionic reduction pathways, as well. Thus, 10
might undergo reduction to amine 6 via an internal oxidation-
reduction of the nitrenium ion pair via hydride transfer from
the hydroquinone anion, or ethyl glycolate alkoxide anion as
(36) Grunwald, E. J. Chem. Phys. 1982, 86, 1302–1305.
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11546 J. AM. CHEM. SOC. VOL. 131, NO. 32, 2009

Photoaffinity Labeling via Nitrenium Ion Chemistry
A R T I C L E S
shown in structure 17. In protic solvents, this ionic pathway
seems to be the main route for reduction of the nitrene to the
amine. However, one can not exclude small contributions to
reduction via radical pathways such as that outlined in
Scheme 4.
ring expansion to ketenimines. Instead, these electron-rich
nitrenes are strong bases that form nitrenium ions via very rapid
proton transfer from a wide variety of proton sources. The
nitrenium ions that result in these processes have iminoquinone
structures that in past studies have been observed to undergo
hydrolysis to quinones in aqueous media. However, when an
auxiliary electron-withdrawing group, such as a nitro group, is
attached to the aromatic ring, the nitrenium ion becomes a
powerful electrophile and usually undergoes nucleophilic aro-
matic substitution with the conjugate base of the acid that led
to its formation.
This photochemical cross-linking (PCL) reaction has been
used in recent biochemical studies to establish the interactions
between many classes of biomolecules. In studies of this type,
the two primary assumptions are that the introduction of the
photo-cross-linking agent, an amino azide related to 5, will not
perturb the natural interactions of the biomolecules, and that
when the photo-cross-linking agent is activated with light, it
will react rapidly with critical sites that participate in binding
the biomolecules together. Under this set of assumptions, the
activated PCL agent will react rapidly with sites in its immediate
vicinity before it has time to migrate to alternative sites. In the
present work, it has been established that, while the initial nitrene
protonation step is very rapid in the picosecond time domain,
the collapse of the nitrenium ion pair is surprisingly slow, often
requiring microseconds for the formation of the critical cross-
linking bond. While these critical site/nitrenium ion pairs might
be maintained over microseconds by salt bridges before they
collapse to form adducts, due caution should be exercised in
the interpretation of any PCL studies of biomolecules using this
system. This will be particularly true in cases where the cross-
linked sites are analyzed in detail in an effort to ascertain the
structural nature of associative interactions.
Another surprising aspect of these reactions is that the major
addition products were always the more hindered regioisomers
related to 8, 15, and 16 rather than the less hindered isomers
related to 7. This is the case even for the most hindered t-BuOH
adduct. Subsequent theoretical calculations, B3LYP/TZVP and
B3LYP/6-31G*, indicate that the more hindered i-PrOH adduct
14 (Scheme 4) is 6-8 kcal/mol more stable and has a 2 kcal/
mol lower transition state energy for formation than the less
hindered adduct 13. In addition, the possibilities that these
adducts can equilibrate with each other via 1,5-alkoxide shifts
or reversion to the starting nitrenium ion pair seem to be
excluded on theoretical grounds. Thus, determination of the
barriers for equilibration of 13 and 14 via a 1,5-alkoxide shift
was determined to be in the 34-42 kcal/mol range, and the
barriers for return to nitrenium ion were in the 41-47 kcal/
mol range (B3LYP/6-31G*).8 Therefore, it would appear that
the distributions of regioisomers in these reactions are deter-
mined kinetically.
Finally, the picosecond transient absorption behavior of the
nitrene derived from 5 in dry acetonitrile (Table 2, entry 13)
parallels that observed in methanol quite closely and that in
other alcohols reasonably closely. This may mean that the
nitrene derived from 5 is sufficiently basic to abstract a proton
from acetonitrile (pKa ) ca. 25).37 However, the nitrene
generation in dry acetonitrile does not give rise to the usual
adducts related to 7 and 8. Instead, good yields of the dimer 11
and the oxidized dimer 12 are obtained, 11/12 ) 65:35, both
of which are derived from triplet nitrenes.31 Analysis of the
transient data by any of several kinetic models (see Table 2,
entry 13) indicates that the intermediate is formed very rapidly,
<10 ps, and in the nanosecond time domain gives rise to a
complex of absorptions in the 440 nm region. Absorption in
this region would normally signal the formation of adducts
related to 14. However, in this system, the 440 nm absorption
apparently signals formation of the dimer 18 (Scheme 4) or some
related intermediate that carries the same chromophore as the
adduct 14.
Finally, the results of this study are being used to assist in
the development of more effective photo-cross-linking agents,
and while not emphasized in this work, photo-oxidizing agents
for targeting specific biochemical sites.
Acknowledgment. We would like to thank Dr. Eric T. Mack
(Harvard University) for suggesting that we investigate this system,
Dr. Torbjo¨rn Pascher (Lund University, Sweden) for his help with
spectral global analysis, Dr. Stephen Macha and Dr. Larry Sallens
of the Mass Spectrometry Facility at the University of Cincinnati
for providing us with detailed mass spectrometric analysis, and Dr.
Eugene Danilov of the Ohio Laboratory for Kinetic Spectroscopy
for assistance in the acquisition nanosecond transient absorption
data. A.N.T. and R.M.W. gratefully acknowledge partial financial
support from BGSU (RCE2008). C.M.H. acknowledges financial
support from the National Science Foundation and the National
Institutes of Health. Also, generous computational resources from
Ohio Supercomputer Center are gratefully acknowledged.
Conclusions
This work has confirmed the earlier observations4 that aryl
nitrenes conjugated to powerful electron-donating groups such
as amines do not undergo the usual collapse to azirines and
Supporting Information Available: Synthesis of starting
materials, characterization of photoproducts, and further spec-
troscopic and theoretical studies are included. This material is
available free of charge via the Internet at http://pubs.acs.org.
(37) Pearson, R. G.; Dillon, R. L. J. Am. Chem. Soc. 1953, 75, 2439–
2443.
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