is considered to be better for living cells. However, in this case,
TFMD probe 11 gave the best result among these four probes (Fig.
2b). In this case, the labeling efficiency of TFMD functionality may
be superior to those of BP and IA because there would be little
difference among the affinity of three photoaffinity probes (11–13)
with MTJG. The use of the BArL probe and screening for the best
reactive functionality realized as much as ca. 70-fold enhancement
in labelling efficiency compared to 2.
Conclusions
In summary, we have found important advantages of a “click”-
made BArL probe, which has remarkably high performance in
protein labelling, attributed to its unique T-shaped conformation.
The efficiency surpassed that of the stepwise tagging approach for
the MTJG protein. “Click”-made BArL probes promise to make
ABPP-based exploration of trace membrane targets for bioactive
compounds a powerful method.
Notes and references
1 N. Jessani and B. F. Cravatt, Curr. Opin. Chem. Biol., 2004, 8, 54–59;
M. J. Evans and B. F. Cravatt, Chem. Rev., 2006, 106, 3279; U. Hillaert,
M. Verdoes, B. I. Florea, A. Saragliadis, K. L. L. Habets, J. Kuiper, S.
V. Calenbergh, F. Ossendrop, G. A. van derMarel, C. Driessen and H.
S. Overkleeft, Angew. Chem., Int. Ed., 2009, 48, 1629; S. Tsukiji, M.
Miyagawa, Y. Takaoka, T. Tamura and I. Hamachi, Nat. Chem. Biol.,
2009, 3, 341.
Scheme 1 CuAAC synthesis of BArL probes: a = benzophenone; b =
trifluoromethyldiazirine; c = iodoacetyl; d = tosyloxyacetyl.
2 C. Wasternack and E. Kombrink, ACS Chem. Biol., 2010, 5, 63; S.
Fonseca, A. Chini, M. Hamberg, B. Adie, A. Porzel, R. Kramell, O.
Miersch, C. Wasternack and R. Solano, Nat. Chem. Biol., 2009, 5, 344;
B. Thines, L. Katsir, M. Melotto, Y. Niu, A. Mandakar, G. Liu, K.
Nomura, S. Y. He, G. A. Howe and J. Browse, Nature, 2007, 661, 448;
A. Chini, S. Fonseca, G. Fernandez, B. Adie, J. M. Chico, O. Lorenzo,
G. G.-Casado, I. L.-Vidriero, F. M. Lozano, M. R. Ponce, J. M. Micol
and R. Solano, Nature, 2007, 448, 666.
3 M. Ueda, M. Okazaki, K. Ueda and S. Yamamura, Tetrahedron, 2000,
56, 8101; M. Ueda and S. Yamamura, Angew. Chem., Int. Ed., 2000,
39, 1400.
4 Y. Nakamura, R. Miyatake, A. Matsubara, H. Kiyota and M. Ueda,
Tetrahedron, 2006, 62, 8805 and references cited therein.
5 Y. Nakamura, R. Miyatake and M. Ueda, Angew. Chem., Int. Ed., 2008,
47, 7289.
Fig. 2 (a) Photoaffinity labelling and chemiluminescence detection of
MTJG by a stepwise approach using 4 with 5 or the BArL approach using
6. (b) Chemiluminescence detection of labeled MTJG using 6, 11, 12 and
13.
6 N. Moran, FEBS Lett., 2007, 581, 2337.
7 B. J. F. Feys, C. E. Benedetti, C. N. Penfold and J. G. Turner, Plant
Cell, 1994, 6, 751; J. Yan, C. Zhang, M. Gu, Z. Bai, W. Zhang, T. Qi,
Z. Cheng, W. Peng, H. Luo, F. Nan, Z. Wang and D. Xie, Plant Cell,
2009, 21, 2220.
8 M. Hashimoto and Y. Hatanaka, Eur. J. Org. Chem., 2008, 2513–2523.
9 H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem., Int. Ed.,
2001, 40, 2004; C. W. Tornøe, C. Christensen and M. Meldal, J. Org.
Chem., 2002, 67, 3057.
10 Y. Manabe, M. Mukai, S. Ito, N. Kato and M. Ueda, Chem. Commun.,
2010, 46, 469.
11 T. R. Chan, R. Hilgraf, K. B. Sharpless and V. V. Fokin, Org. Lett.,
2004, 6, 2853.
among them. Alkylation/acylation of common intermediate 3 and
subsequent CuAAC with alkyne units gave apanelcomprisingfour
probes (Scheme 1). All the CuAAC couplings proceeded in good
yields (ca. 70–90%).
We compared the performance of these four BArL probes in
labelling efficiency for MTJG (Fig. 2b). Labelling reactions by
four BArL probes (6, 11–13) using living cells were carried out in
as mild conditions as possible. Chemical labelling using carbon
◦
electrophile3,18 12 or 13 was carried out at 4 C for 30 min with
12 Pharmacophore of 1 was identified from previous SAR studies: Y.
Nakamura, R. Miyatake, S. Inomata and M. Ueda, Biosci., Biotechnol.,
Biochem., 2008, 72, 2867.
gentle incubation, and photoaffinity labelling using 6 or 11 was
executed at 4 ◦C by UV irradiation (365 nm) for 30 min. As shown
in the chemiluminescence detection of SDS-PAGE, photoaffinity
groups gave better results than carbon electrophiles (Fig. 2b).
The ratio in intensity of chemiluminescence was 6 : 11 : 12 : 13 =
1.0 : 3.4 : 0.2 : 0.7. Two photoaffinity probes, BP 6 and TFMD
11, labeled MTJG selectively, whereas IA 12 largely gave non-
specific binding with 13 and 27 kDa proteins (Figure S8). Ts 13
gave a moderate result without non-specific binding. Generally,
photoaffinity labelling is considered to be harmful for living cells,
whereas gentle substitution labelling using carbon electrophiles
13 Gaussian 03 (Revision D.02), M. J. Frisch, et al. Gaussian, Inc.,
Wallingford CT, 2004. See SI ref. 11.
14 A. J. Link and D. A. Tirrell, J. Am. Chem. Soc., 2003, 125, 11164; A. E.
Spencer, G. C. Adam and B. F. Cravatt, J. Am. Chem. Soc., 2003, 125,
4686.
15 L. Ballell, K. J. Alink, M. Slijper, C. Versluis, R. M. J. Liskamp and R.
J. Pieters, ChemBioChem, 2005, 6, 291.
16 L. M. Gaetke and C. K. Chow, Toxicology, 2003, 189, 147.
17 E. M. Sletten and C. R. Bertozzi, Angew. Chem., Int. Ed., 2009, 48,
6974.
18 E. Weerapana, G. M. Simon and B. Cravatt, Nat. Chem. Biol., 2008, 4,
405.
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