Communications
139.0, 138.8, 138.2, 130.0, 129.7, 129.3, 129.2, 128.6, 128.0, 127.5, 104.8,
Marx, Angew. Chem. 2002, 114, 3778 – 3780; Angew. Chem. Int.
Ed. 2002, 41, 3620 – 3622; c) S. Brakmann, S. Lobermann,
Angew. Chem. 2002, 114, 3350 – 3352; Angew. Chem. Int. Ed.
2002, 41, 3215 – 3217.
96.9, 89.4, 89.2, 86.7, 76.1, 73.8, 73.0, 72.0, 71.5, 71.4, 71.3, 71.2, 70.6,
63.6, 41.6, 30.2 ppm; FTMS: calcd for C39H47N5NaO9 [M+Na]+:
752.3266; found: 752.3291.
9: A solution of 8 (13 mg, 0.018 mmol) and proton sponge
(5.6 mg, 0.027 mmol) in PO(OMe)3 (0.18 mL) was stirred for 10 min
at 08C under N2. POCl3 (3.3 mL, 0.036 mmol) was added, and the
mixture was stirred for 2 h at 08C. A mixture of Bu3N (25 mL,
0.11 mmol) and anhydrous bis(tri-n-butylammonium) pyrophosphate
(40 mg, 0.089 mmol) in DMF (0.18 mL) was added at once. After
1 min, triethylammonium bicarbonate buffer (1.0m, 4 mL) was
added, and the clear solution was stirred at room temperature for
30 min and lyophilized overnight. The crude material was separated
by reversed-phase HPLC with a DEAE column (0.1m TEAB/MeCN)
to give 9 (9.0 mg, 52%) as a white solid. 31P NMR (140 MHz, Tris
(50 mm), EDTA (2 mm), pH 7.5 in D2O): d = ꢀ5.6 (d, J = 15.8 Hz),
ꢀ10.5 (d, J = 15.8 Hz), ꢀ21.6 ppm (t, J = 15.8 Hz); MALDI-FTMS:
calcd for C39H50N5O18P3 [M+H]+: 970.2436; found: 970.2477;
TEAB = tetraethylammonium bromide.
[4] a) M. Matsushita, K. Yoshida, N. Yamamoto, P. Wirsching, R. A.
Lerner, K. D. Janda, Angew. Chem. 2003, 115, 6166 – 6169;
Angew. Chem. Int. Ed. 2003, 42, 5984 – 5987; b) Q. Wang, K. S.
Raja, K. D. Janda, T. W. Lin, M. G. Finn, Bioconjugate Chem.
2003, 14, 38 – 43.
[5] D. W. Chen, A. E. Beuscher, R. C. Stevens, P. Wirsching, R. A.
Lerner, K. D. Janda, J. Org. Chem. 2001, 66, 1725 – 1732.
[6] A. Simeonov, M. Matsushita, E. A. Juban, E. H. Z. Thompson,
T. Z. Hoffman, A. E. Beuscher, M. J. Taylor, P. Wirsching, W.
Rettig, J. K. McCusker, R. C. Stevens, D. P. Millar, P. G. Schultz,
R. A. Lerner, K. D. Janda, Science 2000, 290, 307 – 313.
[7] F. Seela, M. Zulauf, Helv. Chim. Acta 1999, 82, 1878 – 1898.
[8] T. Kovacs, L. Otvos, Tetrahedron Lett. 1988, 29, 4525 – 4528.
[9] a) D. K. Braithwaite, J. Ito, Nucleic Acids Res. 1993, 21, 787 –
802; b) J. Filee, P. Forterre, T. Sen-Lin, J. Laurent, J. Mol. Evol.
2002, 54, 763 – 773.
Additional experimental details for the synthesis of reported
compounds, and 1H and 13C NMR spectra of compounds 7 and 8 can
be found in the Supporting Information.
[10] J. A. Moss, A. R. Coyle, J. M. Ahn, M. M. Meijler, J. Offer, K. D.
Janda, J. Immunol. Methods 2003, 281, 143 – 148.
Fluorescence spectroscopy: Stilbene-containing DNA was gen-
erated by using PCR methodology with a dATP/9 ratio of 7:3 and Vent
exoꢀ DNA polymerase. The PCR product was purified, and the
product DNA concentration was spectrophotometrically determined
to be 0.55 mm. This DNA was used in spectrofluorometric assays to
estimate the actual concentration of the stilbene fluorophore. The
fluorescence (lexcitation = 327 nm, lemission = 425 nm) of 9 was measured
at various concentrations before and after complex formation with
mAb EP2–19G2 in PBS (pH 7.4). Compound 9 by itself displayed
only negligible fluorescence at the concentrations used (1 mm–50 mm),
whereas complex formation with mAb EP2-19G2 resulted in strong
blue fluorescence comparable to that measured for the original
stilbene hapten complexed with the same antibody.[6] Stilbene-
modified DNA (0.1 mm) was incubated with excess (10 mm)
mAb EP2-19G2 for 30 min and its fluorescence was measured with
an SLM-AMINCO 8100 spectrofluorometer equipped with a 450 W
xenon lamp.
[11] a) A. F. Gardner, C. M. Joyce, W. E. Jack, J. Biol. Chem. 2004,
279, 11834 – 11842; b) A. F. Gardner, W. E. Jack, Nucleic Acids
Res. 2002, 30, 605 – 613.
Fluorescence microscopy: Fluorescence images were taken with a
DeltaVision deconvolution microscope (API, Issaquah WA) equip-
ped with
a Photometrics CH350 L liquid-cooled CCD camera
attached to an Olympus IX70 inverted microscope. These data were
collected with either a 60 ꢁ (1.4 NA) or a 100 ꢁ (1.35 NA) oil
immersion objective lens, and a DAPI 360/40 nm (excitation), 457/
50 nm (emission) filter set. All images were deconvoluted with
constrained iterative algorithms (10 iterations) of DeltaVision soft-
ware (softWoRx, v2.5). The deconvoluted images were subsequently
converted into tiff format using softWoRx, v2.5.
Received: July 1, 2004
Revised: November 22, 2004
Published online: March 2, 2005
Keywords: antibodies · DNA recognition · fluorescent probes ·
.
nucleotides · polymerase chain reaction
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2148
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Angew. Chem. Int. Ed. 2005, 44, 2144 –2148