[3-(Trifluoromethyl)-3H-diazirin-3-yl] coumarin as a carbene-generating photocross-linker with masked fluorogenic beacon

Article abstract of DOI:10.1039/c3cc45780j

A coumarin-fused diazirine photolabeling agent exhibited dramatic enhancement in fluorescence after cross-linking with the target protein. Fluorescence emission from the coumarin moiety was efficiently quenched by the diazirine group, and was then intensively recovered from photolysis treatment by irradiation with light at a wavelength of 365 nm.

Full text of DOI:10.1039/c3cc45780j

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
[
3-(Trifluoromethyl)-3H-diazirin-3-yl]coumarin as a
carbene-generating photocross-linker with masked
fluorogenic beacon†
Cite this: Chem. Commun., 2013,
4
9, 11551
Received 29th July 2013,
Accepted 15th October 2013
Takenori Tomohiro,* Akito Yamamoto, Yoko Tatsumi and Yasumaru Hatanaka*
DOI: 10.1039/c3cc45780j
www.rsc.org/chemcomm
A coumarin-fused diazirine photolabeling agent exhibited dramatic
The photoaffinity labeling method (PAL) is a cross-linking
enhancement in fluorescence after cross-linking with the target technique that joins molecules by photo-irradiation, and has
1
6
protein. Fluorescence emission from the coumarin moiety was been used in the life sciences field for the specific imaging
1
7
efficiently quenched by the diazirine group, and was then intensively and profiling of binding proteins, including those previously
recovered from photolysis treatment by irradiation with light at a unidentified. PAL enables specific labeling at the molecular
wavelength of 365 nm.
interaction site without any modification of the protein in advance,
resulting in the elucidation or mapping of the ligand-binding
Specific tagging of target molecules has enabled the visualization surface, and allowing for potent ligand screening. Since aryl azides
of cell functions based on molecular recognition. In particular, the are known to be nitrene precursors, some azide-incorporated
1
8
19
selective incorporation of small fluorophores has progressed to fluorophores (coumarin and nitrobenzoxadiazole ) have been
1
reducing structural perturbations of the labeled protein by using examined as potential photocross-linkers. 3-Phenyl-3-trifluoro-
enzymatic systems, affinity-based chemical methodologies, and methyl diazirine, another photophore, has been recognized as a
2
3
4
5
biological machineries such as metabolism and translation.
useful photocross-linker in biological systems because of its
Recently, bio-orthogonal ligation, one of the most widely stability under general synthetic conditions and its ability to
used chemoselective tagging methods, has been significantly generate an electron-deficient carbene capable of forming a stable
developed for the highly selective fluorescent labeling of bio- covalent bond with various substrates through rapid photolysis by
20
molecules that cannot be genetically encoded with fluorescent long-wave ultraviolet light (365 nm). Although these properties
6
protein or tagged using multi-step labeling procedures.
are attractive in practical use, radioisotope (RI)-coded cross-linkers
In addition to high target selectivity, a high signal-to-noise have often been used to identify targets because of difficulties in
ratio and easy handling are necessary for site-specific imaging. handling the labeled protein in infinitesimal quantities. Generally,
Reaction-dependent on/off switching strategies for emissions, PAL-based fluorophore labeling agents are made up of a photo-
which trace the time-space distribution of target molecules, phore, a ligand, and a fluorophore via a branching structure.
have been improved by coupling with a quenching system that However, a bulky and unstable fluorophore sometimes hinders
contains a selective chemical- or enzyme-reactive group that the specificity of the ligand as well as preparation of the probe.
can function in cleavage, capturing active species, and coordi- Here, we describe a new fluorogenic diazirine-based cross-linker as
7
nating with protons or metal ions. The fluorescence switching a small, non-RI, and highly sensitive tagging agent (Fig. 1A).
property was initially investigated by Kanaoka in studies of
The 3-trifluoromethyldiazirinyl (DA) coumarin derivative 1a is
8
protein function, using maleimide reagents for thiols. Given designed as a relatively small compound with a ring fused to the
9
10
that ethynyl and azide groups efficiently quench the fluores- common phenyldiazirine cross-linker unit that forms a coumarin
1
1
12
cence of compounds such as coumarins, naphthalimides,
fluorophore. Compound 1a was easily synthesized by a Knoevenagel
1
3
14
boron–dipyrromethene, and fluorescein, these derivatives condensation reaction from 2-hydroxy-6-methoxy-4-[(3-trifluoro-
were investigated as ligation-dependent fluorogenic agents for methyl)-3H-diazirin-3-yl]benzaldehyde (see the ESI†). A carboxylate
1
5
the Staudinger, Huisgen, and other reactions.
group has been introduced into the compound for attachment of
the ligand, and a methoxy group has been included at position
C-7 of coumarin to increase the compound’s fluorescent property.
Fig. 1B shows the fluorescence spectra of compound 1a (1 mM in
methanol) after irradiation with light at a wavelength of 365 nm,
which can initiate the photolysis of the diazirine group, using a
high-pressure mercury lamp (Asahi Spectra REX-250, Tokyo, Japan)
Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama,
630 Sugitani, Toyama 930-0194, Japan. E-mail: ttomo@pha.u-toyama.ac.jp,
yasu@pha.u-toyama.ac.jp; Fax: +81-76-434-5063; Tel: +81-76-434-7516
Electronic supplementary information (ESI) available: Materials and methods,
2
†
and additional spectroscopic data. See DOI: 10.1039/c3cc45780j
This journal is c The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 11551--11553 11551

View Article Online
Communication
ChemComm
Fig. 1 (A) Structure of coumarin-fused diazirine cross-linker (1a) and (B) fluorescence
spectra (lex 349 nm) of 5-DA-7-OMe coumarin 1a (1 mM in methanol) after irradiation
with 365 nm light through a bandpass filter (full-width at half-maximum = 10 nm)
using a 250 W high-pressure Hg lamp at 0 1C.
through a bandpass filter [full-width at half-maximum (fwhm) =
À1
10 nm]. The spectra were measured at a scan rate of 500 nm min
with the excitation light at 349 nm. These data indicated that
fluorescence emission from the coumarin moiety was initially
quenched by the diazirine moiety, but was extensively recovered
in response to its decomposition by irradiation with 365 nm
light (fluorescence quantum yields: F
and F = 0.28 for the methanol adduct in hexane, respectively).
The photoreaction was also monitored through NMR spectro-
f
= 0.03 for compound 1a
f
1
3
scopy (20 mM in CD OD, Fig. 2). H NMR signals from the
aromatic protons of the photoproducts clearly distinguished
the diazirine compound as a starting material, with signals
1
observed at d 9.12 (s), 7.35 (d), and 7.13 (d), and the methanol Fig. 2 H NMR spectra (aromatic protons) of compound 1a (A) and ethyl
-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoate (B), respectively. The irradiation
was performed in a CD OD solution of (20 mM) with 365 nm light through a
4
adduct, which showed signals at d 9.06 (s), 7.15 (d), and 7.04 (d)
Fig. 2A). The results indicated that the photolysis of diazirine
compound 1a by irradiation with 365 nm light mainly produced
3
(
bandpass filter at 0 1C over various periods of time. Panels A and B show the
aromatic signals of diazirine compounds (red signals), the corresponding diazo
the corresponding methanol adduct, whose structure was compounds (green), and methanol adducts (blue).
determined by NMR and HRMS (see the ESI†). Interestingly,
the photolysis did not incorporate a pathway yielding a corre-
sponding diazo intermediate. Diazo derivatives are usually was increased by irradiation. The photolysis of the diazirine
2
0
produced in the photolysis of common phenyldiazirines, and group of compound 1b was as rapid as that of compound 1a.
in the subsequent denitrogenation, to yield the corresponding However, its fluorescence was weak and was not completely
carbene species; the reaction proceeds slowly under irradiation quenched by the diazirine group, which had an initial intensity
with 365 nm light, but proceeds rapidly under irradiation with of about 62% of the photoproduct in methanol (see Fig. S2 in
313 nm light. The spectra clearly show conversion among the the ESI†). On the other hand, the 5-DA-7-OH coumarin deriva-
three species: the peak at d 7.36 (d) from the diazirine derivative, tive (2, 1 mM in methanol), a hydroxyl derivative, exhibited a
which decreased with irradiation with 365 nm light; the peak at fluorogenic phenomenon similar to that of compound 1a in
d 7.23 (d) from the diazo derivative, which increased with response to photolysis. Hence, the rate constant of photolysis
irradiation at 365 nm and then disappeared with irradiation was calculated as less than one twenty of the value of com-
at 313 nm; and the peak at d 7.57 (d) from the methanol adduct, pound 1a (see Fig. S3 in the ESI†). On the basis of these data,
which increased constantly. Hence, in panel A (Fig. 2), the compound 1a was employed as a cross-linker unit for the
1
H NMR spectra of compound 1a do not show the signal from further fluorogenic labeling of proteins (Fig. 3).
1
9
the corresponding diazo compound during photolysis. F NMR
Finally, compound 1a was installed at position C-17 of
group confirmed this result (see Fig. S1 geldanamycin (GA), a potent inhibitor of heat shock protein
in the ESI†). These data indicate efficient production of the 90 (Hsp90), to yield a photo-activatable GA probe 3a according
2
1
signals from the CF
3
2
2
carbene species from compound 1a, and implicate it in forming to the synthetic method in a previous report. Hsp90 is a
a covalent bond with the target molecule without going through molecular chaperone responsible for managing the conforma-
the rather slow photolysis of the diazo intermediate.
Another derivative, the 7-DA-5-OMe coumarin derivative (1b), and hydrolysis, GA inhibits ATPase activity through competi-
showed different phenomena. It emitted longer fluorescence at tion for ATP binding (dissociation constant (K
tional maturation of multiple client proteins upon ATP binding
2
3
2
4
d
) = 1.2 mM).
469 nm with a large Stokes shift (lex 329 nm), and its intensity A PAL of Hsp90 (5 mM) with probe 3a (10 mM) was performed by
1
1552 Chem. Commun., 2013, 49, 11551--11553
This journal is c The Royal Society of Chemistry 2013

View Article Online
ChemComm
Communication
unstable fluorophore in the probe. We recently reported a fluoro-
genic cross-linking method using a diazirinylcinnamate derivative
capable of creating a coumarin molecule at the interacting protein
by a two-step photochemical reaction, which was useful for both
25
26
protein identification and imaging. Therefore, this technique
of installing a fluorophore at the interacting interface should also
be useful in identifying a ligand-binding domain within the target
protein by simplifying the purification processes for analysis.
This research was supported by Grants-in-Aid for Scientific
Research (20390032, 21310138) from the Ministry of Education,
Culture, Sports, Science and Technology of Japan.
Fig. 3 (A) Structure of 7-DA-5-OMe coumarin (1b) and 5-DA-7-OH coumarin Notes and references
derivatives (2), and (B) emission recovery of coumarin derivatives (1 mM in
1
(a) J. Zhang, R. E. Campbell, A. Y. Ting and R. Y. Tsien, Nat. Rev. Mol.
Cell Biol., 2002, 3, 906; (b) J. A. Prescher and C. R. Bertozzi, Nat.
Chem. Biol., 2005, 1, 13.
I. Chen and A. Y. Ting, Curr. Opin. Biotechnol., 2005, 16, 35.
T. Hayashi and I. Hamachi, Acc. Chem. Res., 2012, 45, 1460.
(a) Y. Tang and D. A. Tirrell, Biochemistry, 2002, 41, 10635; (b) A. J. Link,
M. L. Mock and D. A. Tirrell, Curr. Opin. Biotechnol., 2003, 14, 603;
methanol for compound 1a (filled circles), 1b (filled triangles), and 2 (open circles),
respectively) with 365 nm light through a bandpass filter using a 250 W high-
pressure Hg lamp at 0 1C.
2
3
4
(
c) J. A. Prescher, D. H. Dube and C. R. Bertozzi, Nature, 2004, 430, 873.
5
(a) C. J. Noren, S. J. Anthony-Cahill, M. C. Griffith and P. G. Schultz,
Science, 1989, 244, 182; (b) M. Suchanek, A. Radzikowska and C. Thiele,
Nat. Methods, 2005, 2, 261.
6
7
For a general review of bioorthogonal chemistry, see: E. M. Sletten
and C. R. Bertozzi, Angew. Chem., Int. Ed., 2009, 48, 6974.
For general reviews of switchable fluorescence chemistry, see:
(
a) G. Lukinavi ˇc ius and K. Johnsson, Curr. Opin. Chem. Biol., 2011,
5, 768; (b) H. Kobayashi, M. Ogawa, R. Alford, P. L. Choyke and
1
Y. Urano, Chem. Rev., 2010, 110, 2620.
Y. Kanaoka, Angew. Chem., Int. Ed. Engl., 1977, 16, 137.
Z. Zhou and C. J. Fahrni, J. Am. Chem. Soc., 2004, 126, 8862.
8
9
1
0 (a) Y. Kanaoka, A. Kobayashi, E. Sato, H. Nakayama, T. Ueno, D. Muno
and T. Sekine, Chem. Pharm. Bull., 1984, 32, 3926; (b) K. Sivakumar, F. Xie,
B. M. Cash, S. Long, H. N. Barnhill and Q. Wang, Org. Lett., 2004, 6, 4603.
1 T. L. Hsu, S. R. Hanson, K. Kishikawa, S. K. Wang, M. Sawa and
C. H. Wong, Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 2614.
1
1
1
1
2 M. Sawa, T. L. Hsu, T. Itoh, M. Sugiyama, S. R. Hanson, P. K. Vogt
and C. H. Wong, Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 12371.
3 K. E. Beatty, J. Szychowski, J. D. Fisk and D. A. Tirrell, ChemBioChem,
011, 12, 2137.
4 P. Shieh, M. J. Hangauer and C. R. Bertozzi, J. Am. Chem. Soc., 2012,
34, 17428.
Fig. 4 Structure of geldanamycin (GA) photoprobe 3a and the competitive
inhibition assays for the photolabeling of Hsp90 (2 mM) with probe 3a (10 mM) in
the absence (lane 1) or presence of GA (lanes 2–5) at various concentrations. The
bands were detected by a fluorescence method (lex = 320 nm, lem = 420 nm,
through bandpath filters (fwhm = 10 nm)).
2
1
1
1
5 C. Le Droumaguet, C. Wang and Q. Wang, Chem. Soc. Rev., 2010, 39, 1233.
6 T. Nagase, E. Nakata, S. Shinkai and I. Hamachi, Chem.–Eur. J., 2003,
9
, 3660.
irradiation with 365 nm light in a 20 mM Tris-HCl (pH 7.5) 17 (a) L. Dubinsky, B. P. Krom and M. M. Meijler, Bioorg. Med. Chem.,
012, 20, 554; (b) T. Tomohiro, M. Hashimoto and Y. Hatanaka,
2
2
solution containing 50 mM KCl and 5 mM MgCl . Fig. 4 shows the
Chem. Rec., 2005, 5, 385.
8 S. Kellner, S. Seidu-Larry, J. Burhenne, Y. Motorin and M. Helm,
Nucleic Acids Res., 2011, 39, 7348.
9 S. J. Lord, H.-l. D. Lee, R. Samuel, R. Weber, N. Liu, N. R. Conley,
M. A. Thompson, R. J. Twieg and W. E. Moerner, J. Phys. Chem. B,
2
structure of the GA photoprobe 3a and part of the 10% SDS-PAGE
of the photoproducts. Labeled Hsp90 was detected by measuring
fluorescence, which increased in an irradiation-time- or probe-
concentration-dependent manner (see Fig. S4 in the ESI†), at
1
1
010, 114, 14157.
4
20 nm (fwhm = 10 nm). Since the labeled Hsp90 was clearly 20 (a) M. Nassal, Liebigs Ann. Chem., 1983, 1510; (b) J. Brunner,
Annu. Rev. Biochem., 1993, 62, 483.
inhibited in the presence of GA as a competitor (lanes 2–4), the
probe can be considered to selectively bind to the target Hsp90.
The HPLC profile of photoproducts showed the same inhibition
manner as PAL (see Fig. S5 in the ESI†).
In conclusion, diazirinylcoumarin derivatives were synthe-
sized as a new class of small fluorogenic cross-linkers. A photo-
activatable GA probe was specifically labeled, and was able to
visualize Hsp90 as a binding protein by brief irradiation. The
photocross-linker provided cross-link-dependent fluorogenic
labeling at the binding interface of the target protein. This
should be an important feature for fluorescent imaging of a
target protein without the necessity of pre-installing a large,
2
1 (a) J. Brunner, H. Senn and F. M. Richards, J. Biol. Chem., 1980,
255, 3313; (b) K. Fang, M. Hashimoto, S. Jockusch, N. J. Turro and
K. Nakanishi, J. Am. Chem. Soc., 1998, 120, 8543.
2 Y. Shen, Q. Xie, M. Norberg, E. Sausville, G. Vande Woude and
D. Wenkert, Bioorg. Med. Chem., 2005, 13, 4960.
3 Z. Nahleh, A. Tfayli, A. Najm, A. El Sayed and Z. Nahle, Future Med.
Chem., 2012, 4, 927.
4 (a) C. E. Stebbins, A. A. Russo, C. Schneider, N. Rosen, F. U. Hartl
and N. P. Pavletich, Cell, 1997, 89, 239; (b) S. M. Roe, C. Prodromou,
R. O’Brien, J. E. Ladbury, P. W. Piper and L. H. Pearl, J. Med. Chem.,
1999, 42, 260.
2
2
2
2
2
5 S. Morimoto, T. Tomohiro, N. Maruyama and Y. Hatanaka, Chem.
Commun., 2013, 49, 1811.
6 T. Tomohiro, K. Kato, S. Masuda, H. Kishi and Y. Hatanaka,
Bioconjugate Chem., 2011, 22, 315.
This journal is c The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 11551--11553 11553