Discovery of long-wavelength photoactivatable diaryltetrazoles for bioorthogonal 1,3-dipolar cycloaddition reactions

Article abstract of DOI:10.1021/ol801350r

(Chemical Equation Presented) Several long-wavelength (365 nm) photoactivatable diaryltetrazoles were discovered by screening a series of substituted diaryltetrazoles and subsequently showed excellent reactivity in the photoactivated 1,3-dipolar cycloaddition reactions toward electron-deficient and conjugated alkenes in organic solvents as well as an alkene-containing protein in the aqueous buffer.

Full text of DOI:10.1021/ol801350r

ORGANIC
LETTERS
2008
Vol. 10, No. 17
3725-3728
Discovery of Long-Wavelength
Photoactivatable Diaryltetrazoles for
Bioorthogonal 1,3-Dipolar Cycloaddition
Reactions
Yizhong Wang, Wen J. Hu, Wenjiao Song, Reyna K. V. Lim, and Qing Lin*
Department of Chemistry, State UniVersity of New York at Buffalo,
Buffalo, New York 14260
qinglin@buffalo.edu
Received June 15, 2008
ABSTRACT
Several long-wavelength (365 nm) photoactivatable diaryltetrazoles were discovered by screening a series of substituted diaryltetrazoles and
subsequently showed excellent reactivity in the photoactivated 1,3-dipolar cycloaddition reactions toward electron-deficient and conjugated
alkenes in organic solvents as well as an alkene-containing protein in the aqueous buffer.
Photoactivatable reagents have been widely used as a
photocrosslinking tool in mapping specific ligand binding
regions within large proteins, characterizing receptor dynam-
ics in the lipid membrane, and capturing transient protein-
protein and protein-DNA interactions.1 Because photoac-
tivatable reagents are inert under normal conditions and
become activated upon UV irradiation, they offer a unique
advantage of allowing temporal and spatial control over
reactivity. The most commonly used photoactivatable groups
include aryl ketones,2 aryl azides,3 and diazirines,4 which
generally require photoirradiation at wavelength <365 nm
in generating their respective biradical, nitrene, and carbene
species (Scheme 1). These reactive intermediates then undergo
(1) (a) Bayley, H. Photogenerated Reagents in Biochemistry and
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Tetrahedron 1995, 51, 12479–12520. (c) Dorma´n, G.; Prestwich, G. D.
Trends Biotechnol. 2000, 18, 64–77.
Scheme 1
.
Representative Photoactivatable Groups and Their
Respective Photoactivation Wavelength
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5673. (b) Ogawa, Y.; Hahn, W.; Garnier, P.; Higashi, N.; Massotte, D.;
Metz-Boutigue, M.-H.; Rousseau, B.; Kodaka, M.; Sunamoto, J.; Ourisson,
G.; Nakatani, Y. Chem. Eur. J. 2002, 8, 1843–1849. (c) Chin, J. W.; Martin,
A. B.; King, D. S.; Wang, L.; Schultz, P. G. Proc. Natl. Acad. Sci. U.S.A.
2002, 99, 11020–11024.
(3) (a) Zettl, R.; Feldwisch, J.; Boland, W.; Schell, J.; Palme, K. Proc.
Natl. Acad. Sci. U.S.A. 1992, 89, 480–484. (b) Blanton, M. P.; Cohen, J. B.
Biochemistry 1992, 31, 3738–3750.
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(b) Chiara, D. C.; Kloczewiak, M. A.; Addona, G. H.; Yu, J.-A.; Cohen,
J. B.; Miller, K. W. Biochemistry 2001, 40, 296–304. (c) Suchanek, M.;
Radzikowska, A.; Thiele, C. Nat. Methods 2005, 2, 261–268. (d) Durek,
T.; Zhang, J.; He, C.; Kent, S. B. Org. Lett. 2007, 9, 5497–5500. (e) Vila-
Perello´, M.; Pratt, M. R.; Tulin, F.; Muir, T. W. J. Am. Chem. Soc. 2007,
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rapid C-H (or X-H) insertion reactions with the surrounding
biomolecules to form the cross-linked photoadducts.1b
(5) Wang, Y.; Vera, C. I.; Lin, Q. Org. Lett. 2007, 9, 4155–4158.
(6) (a) Song, W.; Wang, Y.; Qu, J.; Madden, M. M.; Lin, Q. Angew.
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10.1021/ol801350r CCC: $40.75
Published on Web 08/01/2008
2008 American Chemical Society

We recently reported a new class of photoactivatable
groups responsive to 302 nm handheld UV light based on
the diaryltetrazole moiety (Figure 1).5 Unlike other photo-
activatable groups which generally exhibit poor chemose-
lectivity, diaryltetrazoles showed selective reactivity toward
alkenes in the photoinduced 1,3-dipolar cycloaddition reac-
tion both in vitro and in living cells.6 Because of their
potential as chemical reporters in living systems,7 it is highly
desirable that their photoactivation takes place at longer
wavelength such that it causes minimum photodamage to
the living cells and organisms.8 Herein, we report the
discovery of several 365-nm UV photoactivatable diaryltet-
razoles and the subsequent characterization of their reactivity
in the photoactivated 1,3-dipolar cycloaddition reactions
toward a series of small-molecule alkenes in organic solvents
as well as an alkene-containing protein in the biological
buffer.
Table 1. Absorption Maxima and Molar Absorption Coefficients
for Various Diaryltetrazoles
The rate of photolysis at any given wavelength in a
photochemical reaction is proportional to quantum yield and
molar absorption coefficient at the irradiation wavelength.9
Since the quantum yields for the photolysis of diaryltetrazoles
are very high in the range of 0.5-0.9,10 we reasoned that
long-wavelength photoactivatability can be achieved by
placing auxochromic and conjugative substituents on the
N-phenyl ring to increase molar absorption at the long-
wavelength region. Thus, a series of diaryltetrazoles (1a-h)
were synthesized,11 and their UV-vis spectroscopic proper-
ties were collected in Table 1. Compared to simple diaryltet-
razole 1i, the attachment of either an auxochrome such as
-NH2 (1a) and -NMe2 (1c) or conjugative groups such as
styryl (1h) at the para position of the N-phenyl ring led to
significant bathochromic shifts in λmax (34 nm for 1a, 60
nm for 1c, 28 nm for 1h), with concomitant increases in the
molar absorption coefficients at 365 nm. On the other hand,
attachment of auxochromes at the ortho position caused
hypsochromic shifts in λmax (1d-g). However, the molar
absorption coefficients increased substantially for 1e and 1g
compared to 1i due to peak broadening.
To directly assess the long wavelength photoreactivity of
these diaryltetrazoles toward alkene dipolarophiles, we
incubated them with methyl methacrylate in ethyl acetate
and irradiated the mixtures with a handheld UV lamp at either
302 nm (UVP, model UVM-57, 0.16 AMPS) or 365 nm
(UVP, model UVGL-25, 0.16 AMPS) for 2 h. Under both
conditions, only one regioisomer of the pyrazoline cycload-
ducts (3) was observed in crude products based on the NMR
signals of the pyrazoline ring protons. Generally, 302-nm
irradiation afforded higher yields than 365-nm irradiation for
nearly all diaryltetrazoles tested (Table 2). This can be
a UV-vis was measured by dissolving tetrazoles in MeOH/H2O (1:1)
mixed solvent to derive the final concentrations of 25 µM.
attributed to the filtering effect12 as the pyrazoline products
absorb light strongly at longer wavelength region (λmax
≈
370 nm). Under 365-nm photoirradiation, diaryltetrazoles
bearing amino and dimethylamino at the para position of
the N-phenyl ring gave rise to excellent conversions (entries
1 and 3 in Table 2), consistent with their spectroscopic
properties (Table 1). By contrast, m-amino- (1b) and o-
dimethylamino-substituted (1d) diaryltetrazoles gave sig-
nificantly lower conversions (compare entry 2 to 1 and entry
4 to 3). Attachment of additional auxochromes at the ortho
position of the N-phenyl ring led to a substantial drop in
long wavelength photoreactivity but not in short wavelength
photoreactivity (compare entry 5 to 3 and entry 6 to 1). In
addition, placement of a strong electron-withdrawing group
on the N-phenyl ring significantly reduced the long wave-
length photoreactivity (compare entry 7 to 6). Furthermore,
placement of a styryl group at the para position of the
N-phenyl ring afforded 50% conversion upon 365-nm
photoactivation (entry 8). Taken together, it appears the
photoreactivity of diaryltetrazoles is largely dependent on
(7) (a) Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol. 2005, 1, 13–21.
(b) van Swieten, P. F.; Leeuwenburgh, M. A.; Kessler, B. M.; Overkleeft,
H. S. Org. Biomol. Chem. 2005, 3, 20–27. (c) Barglow, K. T.; Cravatt,
B. F. Nat. Methods 2007, 4, 822–827.
(8) Wells, R. L.; Han, A. Int. J. Radiat. Biol. 1985, 47, 17–21.
(9) Turro, N. J. Modern Molecular Photochemistry; Benjamin Cum-
mings: San Francisco, 1978.
(10) Lohse, V.; Leihkauf, P.; Csongar, C.; Tomaschewski, G. J. Prakt.
Chem. 1988, 330, 406–414.
(11) (a) Ito, S.; Tanaka, Y.; Kakehi, A.; Kondo, K. Bull. Chem. Soc.
Jpn. 1976, 49, 1920–1923. (b) Ito, S.; Tanaka, Y.; Kakehi, A. Bull. Chem.
Soc. Jpn. 1976, 49, 762–766.
(12) Keating, A. E.; Garcia-Garibay, M. A. In Organic and Inorganic
Photochemistry; Ramamurthy, V., Schanze, K., Eds.; Marcel Dekker: New
York, 1998; Vol. 2; pp 195-248, and references therein.
3726
Org. Lett., Vol. 10, No. 17, 2008

Table 2. Photoactivated 1,3-Dipolar Cycloaddition of 1 and 2a
at Two Different Wavelengths
Table 3. Photoactivated 1,3-Dipolar Cycloaddition of 1 with
Various Alkene Dipolarophiles at 365 nm
yield (%)a
at 302 nm
conversion (%)b
at 365 nm
entry
diaryltetrazole
1
2
3
4
5
6
7
8
R ) p-NH2 (1a)
R ) m-NH2 (1b)
R ) p-NMe2 (1c)
R ) o-NMe2 (1d)
R ) o, p-(NMe2)2 (1e)
R ) o-OMe-p-NH2 (1f)
R ) o-OMe-p-NO2 (1g)
R ) p-styryl (1h)
84
26
93
72
95
91
92
94
90
<5
95
40
70
45
15
50
a Reactions were conducted by irradiating 20 mg of 1 and 5 equiv of
2a in 6 mL of EtOAc with a 302-nm UV lamp in quartz test tubes for 4 h.
The yields were isolated yields. b Reactions were conducted by irradiating
1 mg of 1 and 5 equiv of 2a in 2 mL of EtOAc with a 365-nm UV lamp
in quartz test tubes for 2 h. Conversions were estimated based on crude
NMR spectra after removal of excess 2a.
their UV-vis properties. It is noteworthy that 365-nm
photoirradiation of the parent diaryltetrazole 1i did not yield
any observable pyrazoline cycloadduct.
To examine the reactivity of the nitrile imine dipoles
derived from the long-wavelength photoactivatable diaryltet-
razoles, we performed the photoactivated 1,3-dipolar cy-
cloaddition reactions by incubating 1a and 1c with a range
of alkene dipolarophiles (Table 3). As expected, electron-
deficient alkenes, such as methyl methacrylate, N-phenyl-
methacrylamide, and acrylonitrile, reacted efficiently with
1a upon 365-nm photoirradiation, affording the 5-substituted
pyrazoline adducts (entries 1-3). Unexpectedly, reaction
with methyl acrylate gave rise to the oxidized pyrazole
product with 90% isolated yield (entry 4). Furthermore,
styrene derivatives generally serve as good dipolarophiles
for 1a, with the reactivity trend paralleling the electron-
withdrawing ability of the para substituents (entries 5-8).
For the dimethylamino-substituted diaryltetrazole (1c), electron-
deficient alkenes such as methyl methacrylate, acrylonitrile,
and N-methyl-maleimide reacted well (entries 9-11) while
styrene gave a modest yield (entry 12).
a Reactions were conducted by irradiating 20 mg of 1a or 1c and 5
equiv of 2 in 6 mL of benzene/EtOAc (1:2) in quartz test tubes for 4 h.
b Isolated yields. c Oxidized pyrazole product was isolated.
and 1c) at the para position to maximally interact with the
tetrazole π system, resulting in bathochromic shifts. It is
plausible that attachment of substituents at the ortho position
alters this planar arrangement, resulting in hypsochromic
shift. However, it remains unclear whether substituents on
the N-phenyl ring also directly weaken the N2-N3 σ bond,
the first step during the rupture of the tetrazole ring.13
To understand how the attachment of auxochromes leads
to long-wavelength photoactivatability, we solved the X-ray
structure of 2-(4′-methoxyphenyl)-5-(2′′-isopropoxy-4′′-
methoxyphenyl)tetrazole (4, Figure 1). A quick glance of
the structure revealed that three aromatic rings are perfectly
coplanar with the dihedral angles for N(1)-C(1)-C(2)-C(3)
and C(17)-C(12)-N(2)-N(1) to be 8° and -6°, respec-
tively. This is not surprising given that the tetrazole ring does
not bear a single hydrogen and thus has no steric repulsions
with hydrogens from the neighboring phenyl rings. This
arrangement allows the lone pairs of auxochromes (e.g., 1a
Figure 1. ORTEP representation of tetrazole 4 structure.
To examine whether long-wavelength photoactivatable
diaryltetrazoles can be used to label an alkene-containing
protein in biological buffer, we incubated six long-
wavelength photoactivatable diaryltetrazoles (1a,c-f,h) with
(13) Bianchi, G.; Gandolfi, R. 1,3-Dipolar Cycloaddition Chemistry;
Padwa, A., Ed.; Wiley: New York, 1984; pp 470-477.
Org. Lett., Vol. 10, No. 17, 2008
3727

lysozyme acylated with methacrylic anhydride. The mixtures
were photoirradiated with a handheld 365 nm UV lamp
for 20 min. Because the pyrazoline cycloadducts are
fluorescent,6a we performed an in-gel fluorescence analysis
to check for the cycloadduct formation (Figure 2). Pairwise
Table 2) showed the strongest fluorescent intensity in this
analysis. Interestingly, a modest photoactivatable diaryltet-
razole (1f) also gave rise to strong fluorescence on the SDS-
PAGE, which can be partly explained by its relatively higher
quantum efficiency during the ring opening step.14
In summary, we have identified several long-wavelength
photoactivatable diaryltetrazoles that showed excellent re-
activity toward electron-deficient and conjugated alkenes in
organic solvents and an alkene-containing protein in the
aqueous buffer. Compared to the 302-nm photoactivatable
diaryltetrazoles we reported previously,5 these new diaryltet-
razole derivatives should serve as safer and more convenient
photoactivatable reagents for applications of bioorthogonal
1,3-dipolar cycloaddition reactions in living systems.
Figure 2. Photoactivated 1,3-dipolar cycloaddition of diaryltetra-
zoles with either alkene-modified lysozyme (m) or wild-type
lysozyme (w) at 365 nm: top panel, in-gel fluorescence; bottom
panel, Coomassie blue staining.
Acknowledgment. We gratefully acknowledge the Petro-
leum Research Fund (PRF no. 45503-G4), SUNY Buffalo,
and New York State Center of Excellence in Bioinformatics
and Life Sciences for financial support. We thank Dr. M.
Pitak (SUNY Buffalo) for solving the X-ray structure
(Cambridge Structural Database accession no. CCDC
691647).
comparisons of the reactions involving either the alkene-
modified lysozyme or the wild-type lysozyme revealed that
three diaryltetrazole (1a, 1c, 1f) showed specific cycloadduct
formation. To our satisfaction, diaryltetrazoles that exhibited
the highest photoreactivity in the earlier study (1a and 1c;
Supporting Information Available: Experimental details
and characterization of all new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
(14) The quantum yields for 1a, 1c, and 1f were determined to be 0.006,
0.04, and 0.03, respectively; see the Supporting Information for details.
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