Design and synthesis of laser-activatable tetrazoles for a fast and fluorogenic red-emitting 1,3-dipolar cycloaddition reaction

Article abstract of DOI:10.1021/ol402645q

The design and synthesis of a new class of laser light activatable tetrazoles with extended π-systems is reported. Upon 405 nm laser light irradiation, these bithiophene-substituted tetrazoles underwent extremely fast 1,3-dipolar cycloaddition reactions with dimethyl fumarate with second-order rate constants approaching 4000 M^-1 s^-1. The resulting pyrazoline cycloadducts exhibited solvent-dependent red fluorescence, making these tetrazoles potentially useful as fluorogenic probes for detecting alkenes in vivo.

Full text of DOI:10.1021/ol402645q

ORGANIC
LETTERS
2
013
Vol. 15, No. 21
496–5499
Design and Synthesis of Laser-Activatable
Tetrazoles for a Fast and Fluorogenic
Red-Emitting 1,3-Dipolar Cycloaddition
Reaction
5
Peng An, Zhipeng Yu, and Qing Lin*
Department of Chemistry, State University of New York at Buffalo, Buffalo, New York
1
4260-3000, United States
qinglin@buffalo.edu
Received September 13, 2013
ABSTRACT
The design and synthesis of a new class of laser light activatable tetrazoles with extended π-systems is reported. Upon 405 nm laser light irradia-
tion, these bithiophene-substituted tetrazoles underwent extremely fast 1,3-dipolar cycloaddition reactions with dimethyl fumarate with second-
À1 À1
order rate constants approaching 4000 M
these tetrazoles potentially useful as fluorogenic probes for detecting alkenes in vivo.
s . The resulting pyrazoline cycloadducts exhibited solvent-dependent red fluorescence, making
3
localization, probing protein electrostatics, and studying
protein conformations.
4
Biomolecular imaging with fluorophores allows real-
time monitoring of cellular processes with high spatial
and temporal resolution. Compared with fluorescent pro-
teins, chemical fluorescent probes are less likely to perturb
the biological process under study because of their small
5
Recently, we reported the in situ formation of pyrazoline
fluorophores for labeling proteins in living cells via a
photoinduced tetrazoleÀalkene cycloaddition reaction
1
6
sizes and tunable photophysical properties. To minimize
(‘photoclick’ chemistry). Compared with direct fluoro-
the interference caused by autofluorescence, environment-
sensitive fluorescent probes with emissions in the red or
near-infrared region are highly desirable for diverse appli-
phore attachment, the advantages of our photoclick chem-
istry approach include the following: (i) a fluorophore can
be formed at any desired location within a protein of
interest through the use of the amber codon suppres-
sion technique; (ii) the fluorogenic nature of the reaction
allows fluorescent imaging without the washing step; and
2
cations in living systems, including detecting protein
(
(
1) Lavis, L. D.; Raines, R. T. ACS Chem. Biol. 2008, 3, 142.
2) (a) Egawa, T.; Hirabayashi, K.; Koide, Y.; Kobayashi, C.;
(iii) the necessary photoinduction step offers spatiotemporal
Takahashi, N.; Mineno, T.; Terai, T.; Ueno, T.; Komatsu, T.; Ikegaya,
Y.; Matsuki, N.; Nagano, T.; Hanaoka, K. Angew. Chem., Int. Ed. 2013,
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(4) Cohen, B. E.; McAnaney, T. B.; Park, E. S.; Jan, Y. N.; Boxer,
S. G.; Jan, L. Y. Science 2002, 296, 1700.
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Jan, Y. N.; Kobilka, B. K.; Isacoff, E. Y.; Jan, L. Y. Proc. Natl. Acad.
Sci. U.S.A. 2005, 102, 965.
(6) (a) Song, W.; Wang, Y.; Qu, J.; Madden, M. M.; Lin, Q. Angew.
Chem., Int. Ed. 2008, 47, 2832. (b) Song, W.; Wang, Y.; Qu, J.; Lin, Q.
J. Am. Chem. Soc. 2008, 130, 9654. (c) Lim, R. K.; Lin, Q. Acc. Chem.
Res. 2011, 44, 828.
Wang, J.; Cui, S.; Sun, S.; Peng, X. Chem. Commun. 2013, 49, 3890. (c)
Yuan, L.; Lin, W.; Yang, Y.; Chen, H. J. Am. Chem. Soc. 2012, 134,
1
200. (d) Lukinavi ꢀc ius, G.; Umezawa, K.; Olivier, N.; Honigmann, A.;
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1
0.1021/ol402645q r 2013 American Chemical Society
Published on Web 10/10/2013

control over the fluorophore generation. While we have
endeavored to tune the photoactivation wavelength to the
through the Heck-type cross-coupling between acryloni-
trile and the various aryl halides 17bÀe with 60À72%
yields. The nitriles were then converted to 2H-tetrazoles
19a-e in 51À70% yields by treating the arylacrylonitriles
7
8
long-wavelength region, including 405 nm laser light, the
emissions of the pyrazoline fluorophores are still restricted
9
to cyan-to-green colors. Therefore, the pyrazolines with
thered toinfraredfluorescenceare highly desirable. Tothis
with NaN in the presence of triethylammonium chloride
3
in toluene. Tetrazoles 3À4 and 6À8 were obtained through
2
end, here we report the design and synthesis of N -bithio-
II
the Cu -catalyzed cross-coupling between tetrazoles 19aÀe
phene-substituted tetrazoles that can be activated by a
4
and phenyl(bithiophen-2-yl)iodonium salt 20 with modest
yields. The amine-substituted vinylaryltetrazole 5 was
obtained in 57% yield by treating tetrazole 4 with SnCl2
(Scheme 1).
05 nm laser light and produce the red-emitting pyrazoline
fluorophores upon fast cycloaddition reactions with di-
methyl fumarate. Futhermore, we found that the in situ
formed pyrazoline fluorophores showed solvent-dependent
fluorescence, which may make them useful to probe the
polarity change in biological systems.
The trans,trans-phenylbutadiene-substituted tetrazole 9
was constructed by the Wittig reaction according to
Scheme 2. Briefly, cinnamyl alcohol 21 was converted to
the corresponding phosphonium bromide 23 in 66% yield
through successive treatment with CBr and PPh . The
In designing tetrazoles that yield red fluorescent pyrazo-
line cycloadducts, we considered the following recent
findings: (i) the subtitution of the bithiophene moiety at
4
3
cognate aldehyde 27 was synthesized through a four-step
procedure: (i) ethyl carbonocyanidate was reacted with
NaN to give tetrazole 25 in 70% yield; (ii) tetrazole 25 was
3
2
the N -position of tetrazole gave rise to not only 405 nm
8
photoactivatability but also excellent cycloaddition reac-
1
0
tivity; and (ii) extended π-conjugation, particularly at the
C -position of tetrazole, modulates the fluorescence of
the resulting pyrazolines. We hypothesized that the tetra-
treated with phenyl(bithiophen-2-yl)iodonium salt 20 in
the presence of Cu(OAc) to afford tetrazole 26 in 40%
5
2
yield; (iii) reduction of 26 by LiAlH ; and (iv) oxidation
4
2
zoles with a N -bithiophene substituent and an extended
with PCC generated aldehyde 27 in 90% yield over two
steps. Finally, a Wittig reaction was carried out by treating
phosphonium bromide 23 with n-BuLi followed by the
addition of 27 to give (2-bithiophen-2-yl)-5-trans,trans-4-
phenylbuta-1,3-dienyltetrazole 9 in 50% yield.
5
π-system at the C -position would generate red fluorescent
pyrazoline cycloadducts upon cycloaddition with a suita-
ble dipolarophile while at the same time retain 405 nm
photoreactivity and fast cycloaddition reaction kinetics.
Accordingly, we designed tetrazoles 1À9 incorporating
these two features (Figure 1).
Scheme 1. Synthesis of Tetrazoles 3À8
Figure 1. Tetrazole structures with bisthiophene as a trigger for
05 nm photoreactivity.
4
Diaryltetrazoles 1 and 2 were synthesized using the
With tetrazoles 1À9 in hand, their UVÀvis properties
were measured (Figure S1) and the data are collected in
Table 1. The maximum absorption wavelength ranges from
8
cross-coupling method we reported recently (Scheme S1).
Separately, a convergent route was developed for the syn-
thesis of styrenylaryltetrazoles 3À8 (Scheme 1). In brief,
the substituted (E)-arylacrylonitriles 18bÀe were prepared
352 to 364 nm. Tetrazoles with the electron-donating groups
(
5 and 6) provide greater molar absorption coeffcients than
those with the electron-withdrawing groups (4 and 7).
Subsequently, we examined the photoreactivities of the
tetrazoles toward dimethyl fumarate, a symmetrical elec-
(
7) (a) Wang, Y.; Hu, W. J.; Song, W.; Lim, R. K.; Lin, Q. Org. Lett.
008, 10, 3725. (b) Yu, Z.; Ho, L. Y.; Wang, Z.; Lin, Q. Bioorg. Med.
Chem. Lett. 2011, 21, 5033.
2
8,12
tron-deficient dipolarophile we used previously, in PBS/
ACN (1:1, v/v; PBS = phosphate buffered saline, pH 7.4)
13
with photoirradiation by a diode laser (405 nm, 24 mW).
(
8) An, P.; Yu, Z.; Lin, Q. Chem. Commun. 2013, 49, 9920.
(9) (a) Song, W.; Wang, Y.; Yu, Z.; Vera, C. I.; Qu, J.; Lin, Q. ACS
Chem. Biol. 2010, 5, 875. (b) Yu, Z.; Pan, Y.; Wang, Z.; Wang, J.; Lin, Q.
Angew. Chem., Int. Ed. 2012, 51, 10600.
(10) Wang, Y.; Song, W.; Hu, W. J.; Lin, Q. Angew. Chem., Int. Ed.
2009, 48, 5530.
(11) Wang, Y.; Vera, C. I.; Lin, Q. Org. Lett. 2007, 9, 4155.
Org. Lett., Vol. 15, No. 21, 2013
5497

Scheme 2. Synthesis of Tetrazole 9
Table 1. UVÀvis Absorption Properties of Tetrazoles 1À9 and
Characterization of Their Reactivities in the 405 nm Laser Light
Induced Cycloaddition Reaction
a
We found that tetrazoles 2, 3, 7, and 8 gave clean conver-
sions to the pyrazoline cycloadducts (Table 1) while tetra-
zoles 6 and 9 showed g90% yields along with small amounts
of side products based on HPLC traces (Figure S2). Tetra-
zole 1 gave only 58% conversion with the remainder being
the starting materials. Interestingly, tetrazoles 4 and 5
showed no reactivity despite their strong absorption at
4
NO -containing tetrazoles, but not the ester-containing
05 nm (Figure S1). The lack of photoreactivity for the
2
7
tetrazoles, was also observed in our previous studies.
a
11
On the other hand, tetrazole 5 was found to be weakly
fluorescent, emitting the incident light as the fluorescence
rather than breaking up the tetrazole ring.
To quantify tetrazole reactivity, we performed kinetic
analyses by following the cycloaddition reactions of tetra-
zoles with dimethyl fumarate in quartz tubes under 405 nm
laser irradiation with HPLC. The second-order rate con-
stants, k , were determined (Figure S3), and the data are
2
collected in Table 1. To our delight, tetrazoles 6, 8, and 9
showed extremely fast kinetics with k values approaching
2
À1 À1
4
000 M s , more than 3-fold faster than pure oligothio-
8
phene-based tetrazoles. This is probably due to the
HOMO-lifting effect (increases in the nitrile imine HOMO
energies) endowed by attachment of the extended elec-
tron-rich π-system to the in situ generated nitrile imine.
Meanwhile, when the electron-withdrawing ester group
1
0
was present, greater than 10-fold reduction in kinetic con-
À1 À1
stant was observed (k = 220 and 350 M
1
showed faster reaction kinetics than diaryltetrazoles
s
and 7, respectively). In general, styrenylaryltetrazoles
for tetrazoles
2
(
intramolecular 1,3-dipolar cycloaddition reactions
compare 1 to 7 and 2 to 6). Remarkably, the potential
1
4
among styrenylaryltetrazoles 3À8 and phenylbutadie-
nylaryltetrazole 9 were not observed, presumably due
to the geometric constraint posed by the trans-alkenes
in these tetrazoles.
a
Next, the pyrazoline cycloadducts 10À16 were isolated
For UVÀvis measurement, tetrazoles were dissolved in PBS/ACN
(
1:1, v/v) to obtain concentrations of 25 μM. For cycloaddition reactions, a
and their photophysical properties are collected in Table 2.
solution of 20 μM tetrazole and 1 mM dimethyl fumarate in 1 mL of PBS/
ACN (1:1) in a quartz test tube was photoirradiated with 405 nm laser for
b
0 s. Yields were determined by comparing the desired pyrazoline peak area
3
in the HPLC trace to that of a pyrazoline control with known concentration.
(
12) (a) Wang, J.; Zhang, W.; Song, W.; Wang, Y.; Yu, Z.; Li, J.; Wu,
M.; Wang, L.; Zang, J.; Lin, Q. J. Am. Chem. Soc. 2010, 132, 14812. (b)
Lim, R. K.; Lin, Q. Chem. Commun. 2010, 46, 7993.
c
See Figure S2 in the Supporting Information for details. Second-order rate
constant of the cycloaddition; see Figure S3 in the Supporting Information
for details. The desired pyrazoline product was not detected; the tetrazole
was found stable under the photoirradiation conditions.
(13) The light intensity value was recorded by placing a FieldMaster
d
GS energy analyzer equipped with an LM-10 HTD power meter sensor
in the light path of the laser beam.
(14) Yu, Z.; Ho, L. Y.; Lin, Q. J. Am. Chem. Soc. 2011, 133, 11912.
5
498
Org. Lett., Vol. 15, No. 21, 2013

Table 2. UVÀvis Absorption and Fluorescence Properties of
a
Pyrazolines 10À16
b
λmax (nm)
λem (nm)
Stokes
shift
Φ
f
À1 c
d
pyrazoline EtOAc PBS/ACN EtOAc PBS/ACN (cm
)
(%)
1
0
425
402
416
418
437
424
435
425
402
416
418
437
424
432
596
553
582
574
610
595
608
630
575
612
600
644
615
620
6750
0.3
1.5
0.4
0.4
0.2
0.3
0.2
11
6790
6860
6500
6490
6780
6540
1
1
1
1
1
2
3
4
5
6
a
Pyrazolines were dissolved in either EtOAc or PBS/ACN (1:1, v/v)
b
c
to obtain concentrations of 10 μM. λex = 405 nm. Based on the values
measured in EtOAc. Fluorescence quantum yields were determined in
EtOAc using DAPI dye as a reference.
d
Figure 2. UVÀvis absorption (left) and fluorescence spectra
right) of pyrazoline 12 measured at 10 μM in the various
(
solvents. For fluorescence measurement, λex = 405 nm.
All pyrazoline cycloadducts showed bathochromic shifts
in their absorption and emission maxima compared to
In summary, we have designed and synthesized a series
of biaryl and styrenylaryl tetrazoles containing both a
405 nm photoactivatable bithiophene moiety and an extended
π-system. The majority of these new tetrazoles participated in
the laser-triggered photoclick reaction with dimethyl fuma-
rate, giving rise to the red fluorescent pyrazoline cycloadducts
7
diaryltetrazoles, accompanied by large Stokes shifts
À1
(
6490À6860 cm ; Table 2). All pyrazoline cycloadducts
except 11 showed red fluorescence in PBS/ACN (1:1, v/v),
and pyrazoline 14 even reached the near-infrared region
with λem of 644 nm. However, the quantum yields of
the pyrazoline fluorophores were rather low (0.2À1.5%),
which can be attributed to their flexible structures and
thus their strong tendencyfor nonradiative decay. Another
observation was that the emission maxima of these pyr-
azolines depend critically on solvent polarity with signifi-
cant hypsochromic shifts (12À34 nm) going from polar
solvents to nonpolar ones, while the absorption spectra
showed little change. As an illustration, pyrazoline 12
gave an emission maximum of 612 nm in polar PBS/
ACN (1:1, v/v) solvent, but 582 nm in nonpolar EtOAc
along with a concurrent increase in fluorescence intensity
by more than 6-fold (Figure 2). This fluorescence intensity
with the fastest kinetics reported so far for the photoclick
À1 À1
chemistry (k up to 3960 M s ). Because the pyrazoline
2
cycloadducts showed environment-dependent “turn-on”
fluorescence, these new tetrazoles should offer a useful tool
to study protein electrostatics and protein conformations
involving changes in solvent accessibility and/or polarity.
Acknowledgment. We gratefully acknowledge the
National Institutes of Health (GM 085092) for financial
support. P.A. is a visiting graduate student from Lanzhou
University sponsored by the China Scholarship Council.
Supporting Information Available. Supplemental fig-
ures, experimental procedure, and characterization of all
new compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
“
turn-on” increased to 30-fold when organic cosolvent
ACN in PBS/ACN decreased to 20% (Figure S4), suggest-
ing that these red-emitting pyrazoline fluorophores may
serve as environment-sensitive probes to detect polarity
3À5
change in protein structures.
The authors declare no competing financial interest.
Org. Lett., Vol. 15, No. 21, 2013
5499