Journal of the American Chemical Society
Communication
Table 1. Binding Parameters of Ligands to Fl-Labeled FR on Live Cells Determined by CLSM Study: Binding-Induced
Fluorescence Intensity Change (Imax/I0), Association Constant (Ka), and Association/Dissociation Rate Constants (kon, koff)
FA
H2FA
H4FA
MTX
PT
TMP
Imax/I0
1.4 0.1
>109
3.0 × 104
<3 × 10−5
1.6 0.1
1.3 × 108
2.8 × 104
2.2 × 10−4
1.7 0.3
1.1 × 108
1.9 × 104
1.7 × 10−4
1.4 0.1
1.9 × 105
4.5 × 102
2.4 × 10−3
1.2 0.1
3.8 × 105
3.5 × 103
9.2 × 10−3
1.3 0.2
1.9 × 104
ND
Ka/M−1
kon/s−1 M−1
koff/s−1
a
−
a
Values were calculated from the equation Ka = kon/koff using experimental kon and Ka values. ND = not determined.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Figures S1−S9, synthetic procedures, compound character-
izations, and protein labeling methods using LDAI reagents.
This material is available free of charge via the Internet at
AUTHOR INFORMATION
■
Corresponding Author
Notes
(24) Tsukiji, S.; Miyagawa, M.; Takaoka, Y.; Tamura, T.; Hamachi, I.
Nat. Chem. Biol. 2009, 5, 341.
The authors declare no competing financial interest.
(25) Tsukiji, S.; Wang, H.; Miyagawa, M.; Tamura, T.; Takaoka, Y.;
Hamachi, I. J. Am. Chem. Soc. 2009, 131, 9046.
ACKNOWLEDGMENTS
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(26) Takaoka, Y.; Sun, Y.; Tsukiji, S.; Hamachi, I. Chem. Sci. 2011, 2,
511.
S.F. acknowledges JSPS Research Fellowships for Young
Scientists.
(27) (a) Matthews, D. A.; Alden, R. A.; Bolin, J. T.; Freer, S. T.;
Hamlin, R.; Xuong, N.; Kraut, J.; Poe, M.; Williams, M.; Hoogsteen, K.
Science 1977, 197, 452. (b) Miller, L. W.; Sable, J.; Goelet, P.; Sheetz,
M. P.; Cornish, V. W. Angew. Chem., Int. Ed. 2004, 43, 1672. (c) Miller,
L. W.; Cai, Y.; Sheetz, M. P.; Cornish, V. W. Nat. Methods 2005, 2,
255.
(28) LDAI reagent 1 was gradually hydrolyzed in aqueous solution
and inactivated. The half-life of LDAI reagent 1 was determined to be
20 h at pH 6.0 by HPLC analysis (Figure S2), which may suppress the
completion of DHFR labeling during the reaction time (48 h, Figure
S1).
(29) Based on an X-ray crystal structure of the DHFR-methotrexate
complex (Figure S3), Lys32 is proximal to the ligand-binding pocket of
DHFR. The distance from the γ-carboxylic acid of MTX to the ε-
amino group of Lys32 is 7.3 Å, roughly equal to the length between
the ligand-end amide and the reaction site of LDAI reagent. Thus, it is
conceivable that the proximity effect worked well for selective Lys32
labeling.
(30) Kukowsa-Latallo, J. F.; Candido, K. A.; Cao, Z.; Nigavekar, S. S.;
Majoros, I. J.; Thomas, T. P.; Balogh, L. P.; Khan, M. K.; Baker, J. R.
Jr. Cancer Res. 2005, 65, 5317.
(31) The fluorescence signal was clearly detected even after cells
were washed with FA (25 μM) (Figure S4), indicating that FR on KB
cells was covalently labeled by 2.
(32) Dissociation constants of FR for FA and the reduced FA
derivatives (H2FA and H4FA) were reported to be <1 nM and 1−10
nM, respectively ( Keleman, L. E. Int. J. Cancer 2006, 119, 243 ). Since
the binding affinities for PT, MTX, and TMP were not reported, we
roughly evaluated them by the competitive labeling experiment
(Figure S8).
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