Discovery of new photoactivatable diaryltetrazoles for photoclick chemistry via scaffold hopping

Article abstract of DOI:10.1016/j.bmcl.2011.04.087

We report the discovery of two long-wavelength (365 nm) photoactivatable diaryltetrazoles through screening a small library of diaryltetrazoles that were designed using a 'scaffold hopping' strategy. A naphthalene-derived tetrazole showed excellent reacti

Full text of DOI:10.1016/j.bmcl.2011.04.087

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5033–5036
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of new photoactivatable diaryltetrazoles for photoclick chemistry
via ‘scaffold hopping’
⇑
Zhipeng Yu, Lok Yin Ho, Zhiyong Wang, Qing Lin
Department of Chemistry, State University of New York at Buffalo, Buffalo, New York 14260, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the discovery of two long-wavelength (365 nm) photoactivatable diaryltetrazoles through
screening a small library of diaryltetrazoles that were designed using a ‘scaffold hopping’ strategy. A
naphthalene-derived tetrazole showed excellent reactivity in the photoinduced cycloaddition reaction
with methyl methacrylate under 365 nm photoirradiation in acetonitrile PBS buffer mixture. Besides,
the brightly fluorescent pyrazoline cycloadducts that were formed further increase the potential utility
of these new diaryltetrazoles as ‘photoclick’ reagents and as reporters in biological studies.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 18 March 2011
Revised 13 April 2011
Accepted 19 April 2011
Available online 24 April 2011
Keywords:
Bioorthogonal chemistry
Photoclick chemistry
Dipolar cycloaddition
Tetrazoles
Fluorophores
Light-induced chemical reactions regulate many important cel-
lular and organismal functions in nature. For example, prokaryotes
such as Brucella abortus employ a photoinduced flavin addition to
cysteine of a histidine kinase to regulate bacterial chemotaxis, pho-
totaxis, and virulence.1 In mammals, a photoinduced isomerization
of 11-cis-retinyl Schiff base in rhodopsin forms the chemical basis
for vision formation.2 To harness the power of light for cell biology
studies, natural sensory photoreceptors such as channel rhodop-
sins have been successfully used as optogenetic modules in order
to achieve photo-regulation of the function of neurons and other
cells.3 However, because fusion of large photo-sensing proteins
adds considerable mass to a target protein, an alternative approach
has involved the introduction of small photoreactive chemical moi-
eties, for example, a photocaged4 or a photoisomerizable group,5 to
a target protein site-specifically followed by subsequent light-
dependent functional manipulation.
Recently, we have developed a protein photo-modification pro-
tocol based on a bioorthogonal, photoinduced tetrazole-alkene
cycloaddition reaction (‘photoclick chemistry’).6 In this approach,
a bioorthogonal alkene7 or tetrazole8 is introduced site-selectively
into proteins, which can be subsequently modified with the cog-
nate reagents in live cells in a light-dependent manner. Since al-
kene structures are considerably smaller in size compared to
tetrazoles, it is more straightforward to encode alkenes in proteins
and use tetrazoles as the reagents to drive the protein photo-mod-
ification. To this end, robust tetrazole reagents need to be identi-
fied that show both high reactivity9 and long-wavelength
photoactivatability.10 In addition, it is also desirable that the pyraz-
oline cycloadducts exhibit bright fluorescence in order to facilitate
the monitoring of protein photo-modification in vivo. Here we re-
port the synthesis of a small library of tetrazoles and subsequent
identification of two new diaryltetrazoles that show 365 nm
photoactivatability and cycloaddition products with good fluores-
cent properties.
Our previous study has identified several 365 nm photo-reac-
tive tetrazoles containing either amino or styryl group at the
para-position of the N-phenyl ring.10 The long-wavelength photo-
reactivity was attributed to greater absorption at the long-
wavelength region in the UV–vis spectra. However, the pyrazoline
cycloadducts derived from the amino-substituted diphenyltetraz-
ole showed rapid photo-bleaching, limiting its utility in biological
studies. To overcome this problem, we borrowed a concept from
medicinal chemistry dubbed as ‘scaffold hopping’, where key phar-
macophores are allowed to ‘jump’ from one scaffold to another in
order to derive novel ligands with improved bioactivities.11 We
hypothesized that by ‘hopping’ the photoreactive tetrazole moiety
from aniline to other known long-wavelength UV absorbing scaf-
folds, we may obtain new tetrazoles with increased photoreactivity
and photostability.
Based on the long-wavelength UV absorbance and synthetic
accessibility, five chromophores were selected as alternative scaf-
folds for our new diaryltetrazoles 1–5 (Fig. 1). Starting from 2-
naphthalenediazonium salt, the 2-naphthoate tetrazoles 7 and 11
were efficiently prepared using the Kakehi procedure,12 with the
yield of 26% and 62%, respectively (Scheme 1). Following
⇑
Corresponding author.
E-mail address: qinglin@buffalo.edu (Q. Lin).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.087

5034
Z. Yu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5033–5036
H
H
N
H
N
N
N
N
Boc
Boc
N
N
OH
N
N
N
N
1
2
Me
O
O
NH2
MeO
N
MeO
N
N
N
N N
O
N
N
O
3
NH2
MeO2C
MeO2C
N
N
N
N
N
N
N
N
4
5
Figure 1. Structures of diaryltetrazoles with alternative scaffolds.
N
SO2Ph
O
N
CO2Me
LiOH, reflux
CO2Me
Cl
N
H
N
N
N
OH
N
N
pyridine, -15 oC
THF:H2O (1:1)
N
N
N
N
N
7
8
H
N
H
N
(PhO)2PON3
Et3N, BuOH
TFA
O
MeOH, 55 o
NH2
N
N
N
Boc
N
N
OH
t
DCM
C
N
N
N
N
N N
Toluene, reflux
9
10
1
1) LiOH, reflux,
THF/H2O (1:1)
CO2Me
MeO2C
BocHN
MeO2C
Cl
pyridine, -15 o
C
NHBoc
CO2Me
N
N
N
N
N
N N
N
N
2) (PhO)2PON3, Et3N,
N
t
BuOH, PhMe, reflux
N
SO2Ph
N
2
11
H
Scheme 1.
LiOH-mediated hydrolysis, the tetrazole carboxylic acids were con-
verted to the Boc-protected-6-aminonaphthyl-tetrazoles 9 and 2
via Curtius rearrangement in 67% and 46% yield, respectively.
Subsequent deprotection of 9 with TFA followed by alkylation of
naphthylamine with ethylene oxide produced a water-soluble
tetrazole 1. However, deprotection of 2 generated a chemically
unstable compound so we decided to use the Boc-protected 2
directly in our reactivity studies.
Since coumarin is a blue fluorophore and absorbs strongly in the
UV region around 365 nm, we prepared a tetrazole-modified
coumarin 3 from the commercially available 7-amino-4-methyl-
coumarin with a yield of 81% (Scheme 2). Separately, since
diarylacetylene and diaryl-1,3-butadiene have been used in fluoro-
phore design,13 we appended the photoreactive tetrazole to these
scaffolds and generated tetrazoles 4 and 5. Tetrazole 4 was readily
prepared from the Sonogashira coupling between 2-p-iodophenyl-
tetrazole 13 and p-ethynylaniline (Scheme 2). On the other hand,
the 1,4-dipenylbutadiene-conjugated tetrazole 5 was synthesized
in four steps: (i) Kakehi tetrazole synthesis produced N-tolyltetraz-
ole 14 in 86% yield; (ii) bromination of 14 with NBS and catalytic
amount of AIBN gave the intermediate 15 in 80% yield; (iii) heating
of benzyl bromide 15 in triethylphosphite gave benzyl phospho-
nate 16 in 60% yield; and (iv) the Horner–Wadsworth–Emmons
reaction between 16 and cinnamaldehyde afforded the final prod-
uct tetrazole 5 in 39% yield (Scheme 3).
Since tetrazole photoreactivity at any given wavelength is
dependent on molar absorption coefficient at that particular wave-
length,14 the UV–vis spectra of tetrazoles 1–5 were taken (Fig. S1 in
Supplementary data) and the data were collected in Table 1. Com-
pared to p-aminodiphenyltetrazole, all five tetrazoles showed sig-
nificant bathochromic shift in kmax, ranging from 14 nm for
tetrazole 2 to 50 nm for tetrazole 5. The strong absorption bands
in 300–400 nm can be attributed to the p–
p⁄ electronic transition
between HOMO and LUMO orbitals of the tetrazole-conjugated
fluorophores. The extent of bathochromic shift appears to correlate
with the calculated HOMO–LUMO gap as tetrazoles 4 and 5 with
smaller gap showed larger kmax shifts.
To probe whether long-wavelength UV absorption leads to en-
hanced photoreactivity in biological systems, we initially at-
tempted to test the reactivity of all five tetrazoles in a mixed
acetonitrile/PBS buffer (1:1), pH 7.5, and found that tetrazoles 1
and 2 and their cycloaddition photoproducts were reasonably sol-
uble but tetrazoles 3–5 were insoluble. Therefore, we performed a
fluorescence-based screen of the photoreactivity of tetrazoles 1

Z. Yu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5033–5036
MeO2C
5035
Me
Me
O
N
SO2Ph
MeO2C
N
H
N
Cl
N
N
O
N N
pyridine, -15 oC
N
O
O
3
MeO2C
N
N
H
SO2Ph
I
MeO2C
I
Cl
N
N
pyridine, -15 oC
N
N N
13
N
PdCl2(Ph3P)2,
CuI, NH3 (aq.)
THF
NH2
NH2
MeO2C
N
N
N N
4
Scheme 2.
MeO2C
Me
N
SO2Ph
N
H
MeO2C
Me
NBS, CCl4
Cl
N
N
N
pyridine, -15 oC
N
AIBN, reflux
N N
14
P(OEt)3
MeO2C
MeO2C
O
P
N
N
Br
N
N
OEt
EtO
neat, 150 oC
N N
N N
16
15
O
H
MeO2C
N
N
KOtBu, DMF
N N
5
Scheme 3.
Table 1
Absorption maxima, molar absorption coefficients and molecular orbital energy calculation for diaryltetrazolesa
e302 (MÀ1 cmÀ1
)
e365 (MÀ1 cmÀ1
)
e395 (MÀ1 cmÀ1
)
HOMOd (eV)
LUMOd (eV)
DLUMO–HOMO (eV)
b
d
Tetrazole
kmax (nm)
p-aminoc
310
338
324
332
346
360
20500
7103
16415
25838
28892
9035
3500
11908
1813
907
35177
30819
N.D.
À5.66
À5.64
À5.57
À6.57
À5.41
À5.60
À1.51
À1.40
À1.40
À2.22
À1.74
À1.99
4.15
4.24
4.17
4.35
3.67
3.61
1
2
3
4
5
1935
1042
682
6167
8184
a
b
c
UV–vis was measured by dissolving tetrazoles in CHCl3 to derive the final concentration of 25
kmax were derived from the wavelength region of 300–450 nm.
Data were taken from Ref. 10
l
M.
d
DFT calculation were performed at the B3LYP/6-31G⁄ level in vacuum using the SPARTAN’08 program.
and 2 under 302, 365, or 395 nm UV irradiation in the aqueous buf-
fer by taking advantage of the fact that pyrazoline cycloadducts are
fluorescent. We found that tetrazole 2 showed rapid ‘turn-on’ fluo-
rescence upon 302 and 365 nm photoirradiation (Fig. 2) while tet-
razole showed no change in fluorescence after the
photoirradiation even though tetrazole 1 itself was weakly fluores-
1

5036
Z. Yu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5033–5036
BocHN
NHBoc
BocHN
N
Me
CO2Me
NHBoc
N
N
N
Me
CO2Me
N
N
302 nm, ACN/PBS
or 365 nm, ACN/PBS
2
17
Me
Me
O
MeO2C
Me
CO2Me
MeO2C
N
N
N
N
N
O
Me
CO2Me
302 nm, CHCl3
or 365 nm, CH2Cl2
O
O
7
N
3
18
Scheme 4.
may prove critical for their use as ‘photoclick’ reagents in visualiz-
ing and perturbing the alkene-tagged proteins in living cells.7 De-
tailed characterization of these two compounds and additional
structural modifications are currently underway and will be re-
ported in due course.
Sample
UV (nm)
1
-
2
3
4
5
-
6
8
302
365
395
302
365
395
Acknowledgment
We gratefully acknowledge the National Institutes of Health
R01 GM 085092 for financial support.
Supplementary data
tetrazole
2
tetrazole
3
Figure 2. Fluorescence images of tetrazoles 2 and 3 after the photo-irradiation with
methyl methacrylate in acetonitrile/PBS buffer (1:1) and chloroform, respectively;
kex = 365 nm. The concentrations of tetrazole and methyl methacrylate were
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.04.087.
100 lM and 10 mM, respectively, and the duration of photoirradiation was 10 min.
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302, 365 or 395 nm irradiation. Interestingly, the coumarin-fused
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isolated in 68% yield after tetrazole 3 was irradiated at 302 nm in
chloroform for 1.5 h, and 13% yield when 365 nm UV lamp was
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In summary, we have identified two long-wavelength photoac-
tivatable diaryltetrazoles containing either naphthalene or couma-
rin chromophore. The naphthalene-derived tetrazole showed
excellent long-wavelength photoreactivity in an acetonitrile/PBS
mixed solvent. Additionally, both tetrazoles produced bright pyr-
azoline fluorophores upon the cycloaddition reactions, which
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California, 1978.