diverse target molecules. These sugar-nucleotides will be useful as
fluorescent probes for GDP-mannose-utilising enzymes, and
investigations into their biological activity are currently underway.
We thank the University of East Anglia for a studentship (to A.
C.), the EPSRC for financial support (First Grant EP/D059186/1),
the EPSRC National Mass Spectrometry Service Centre, Swansea,
for the recording of mass spectra, and Rob Field and Mark
Searcey for helpful discussions.
Notes and references
{ 8-Phenyl-GDP-mannose (4a). To a solution of GDP-mannose (10 mg,
0.016 mmol) in 0.5 M NaOAc buffer (0.5 mL, pH 4.0) was added,
dropwise, saturated bromine water until the yellow colour persisted. After
stirring at rt for 20 min, the solution was extracted with CHCl3 (3 6 5 mL)
and the aqueous layer dried in vacuo. A solution of Na2Cl4Pd (0.1 mg,
0.4 mmol), triphenylphosphine trisulfonate (TPPTS; 0.6 mg, 1 mmol),
PhB(OH)2 (2.3 mg, 0.019 mmol) and K2CO3 (6.6 mg, 0.048 mmol) in de-
gassed H2O (3 mL) was added, and the yellow solution stirred under N2 at
80 uC for 1 h. Upon completion of the reaction, the pH was adjusted to 7
with 1% aqueous HCl and all solvents were removed in vacuo. The crude
residue was purified, first by ion pair chromatography (0–15% MeCN
against 0.05 M TEAB; triethylammonium bicarbonate), and then on an
anion exchange cartridge (0–100% 1 M TEAB against H2O). The glassy
residue obtained from repeated evaporations with MeOH was dissolved in
H2O and treated with Chelex 100 resin to give 4a as the sodium salt in 44%
yield (6.2 mg). dH (400 MHz; D2O, acetone): 7.38 (5H, m, phenyl), 5.61
(1H, d, J = 6.1 Hz, H-19), 5.35 (1H, d, J = 7.8 Hz, H-10), 5.12 (1H, t, J =
5.9 Hz, H-29), 4.36 (1H, dd, J = 3.3 and 5.6 Hz, H-39), 4.15 (1H, m, H-59a),
4.06 (2H, m, H-49 and H-59b), 3.89 (1H, m, H-20), 3.76 (1H, dd, J = 3.4 and
9.7 Hz, H-30), 3.70 (2H, m, H-50 and H-60a) and 3.54 (2H, m, H-60b and
H-40); dC (100 MHz; D2O, acetone): 160.7, 159.4, 153.7, 152.8, 150.1, 130.4,
Fig. 2 Fluorescence emission spectra for GDP-mannose analogues 3, 4a,
4b, 4d and 4e in H2O.
Table 3 Fluorescence spectroscopic properties of GDP-mannose
analogues
Compound
lex/nm
lem/nm
Stokes shifta/cm21
5
262
278
282
280
290
240
—
—
4a
4b
4c
4d
4e
a
398
402
396
382
488
10846
10585
10462
8305
21175
Stokes shift = (1/lexcitation 2 1/lemission).
129.2, 128.8, 127.8, 116.0, 96.2 (d, Jc,p = 7.9 Hz), 88.7, 83.0 (d, Jc,p
=
11.5 Hz), 73.4, 70.0 (d, Jc,p = 11.9 Hz), 69.6, 66.2, 65.0 (d, Jc,p = 7.1 Hz),
60.5 and 58.7; dP (121 MHz; D2O, H3PO4) 27.75 (d, J = 20.4 Hz), 210.18
(d, J = 21.4 Hz). m/z (ESI) 680.1012 [M]2; C22H28N5O16P2 requires
680.1012.
bromine with chloroform and removal of all solvents, the crude
residue was used for the Suzuki cross-coupling. When we applied
our standard conditions (1.5 equivalents of base), no reaction was
initially seen. We attributed this lack of cross-coupling reactivity to
the neutral pH of the aqueous solution. Indeed, upon increasing
the number of equivalents of base to a total of three (pH 9), the
cross-coupling reaction went to completion in 20 min. No
decomposition products were observed throughout, and purifica-
tion by ion pair chromatography gave 3 in 75% isolated yield. This
one-pot procedure was then also applied successfully to GDP and
GDP-mannose (Table 2, products 6 and 4a).{ The lower yields
obtained for 6 and 4a reflect the increased fragility of the GDP and
GDP-mannose substrates, and these reactions needed further
purification to remove minor decomposition products.
1 J. Kordulakova, M. Gilleron, K. Mikusova, G. Puzo, P. J. Brennan,
B. Gicquel and M. Jackson, J. Biol. Chem., 2002, 277, 31335.
2 M. L. Schaeffer, K. H. Khoo, G. S. Besra, D. Chatterjee, P. J. Belisle
and J. M. Inamine, J. Biol. Chem., 1999, 274, 31625.
3 C. A. Munro, S. Bates, E. T. Buurman, H. B. Hughes,
D. M. MacCallum, G. Bertram, A. Atrih, M. A. J. Ferguson,
J. M. Bain, A. Brand, S. Hamilton, C. Westwater, L. M. Thomson,
A. J. P. Brown, F. C. Odds and N. A. R. Gow, J. Biol. Chem., 2005,
280, 1051.
4 T. K. Smith, A. Crossman, J. S. Brimacombe and M. A. J. Ferguson,
EMBO J., 2004, 23, 4701.
5 Y. D. Lobsanov, P. A. Romero, B. Sleno, B. Yu, P. Yip, A. Herscovics
and P. L. Howell, J. Biol. Chem., 2004, 279, 17921.
8-Alkynylated and -arylated purine nucleosides have been
reported as useful fluorescent probes.14 Similarly, our novel
8-substituted GDP-mannose analogues possess interesting fluor-
escent properties, which can be modulated by the nature of the
substituent in position 8 (Fig. 2 and Table 3). As expected,
8-bromo GDP-mannose (5) is non-fluorescent. The phenyl-
substituted analogues 4a–c show only subtle differences in their
fluorescence characteristics, with lem around 400 nm and similar
Stokes shifts. A slight blue-shift was observed in the fluorescence
emission of 8-furanyl GDP-mannose 4d, while the attachment of
the bulky, fluorescent pyrene group (4e) resulted in a pronounced
red-shift into the visible region.
6 M. Rejzek, B. Mukhopadhyay, C. Q. Wenzel, J. S. Lam and R. A. Field,
Carbohydr. Res., 2007, 342, 460; J. J. Li and T. D. H. Bugg, Chem.
Commun., 2004, 182.
7 T. Szumilo, R. R. Drake, J. L. York and A. D. Elbein, J. Biol. Chem.,
1993, 268, 17943.
8 A. Suzuki, Chem. Commun., 2005, 4759.
9 N. Amann and H. A. Wagenknecht, Synlett, 2002, 687; E. C. Western,
J. R. Daft, E. M. Johnson, P. M. Gannett and K. H. Shaughnessy,
J. Org. Chem., 2003, 68, 6767; N. Kohyama, T. Katashima and
Y. Yamamoto, Synthesis, 2004, 2799; A. Collier and G. K. Wagner,
Synth. Commun., 2006, 36, 3713.
10 P. Capek, R. Pohl and M. Hocek, Org. Biomol. Chem., 2006, 4, 2278.
11 A. Collier and G. K. Wagner, Org. Biomol. Chem., 2006, 4, 4526.
12 J. Lee, H. Churchil, W. B. Choi, J. E. Lynch, F. E. Roberts,
R. P. Volante and P. J. Reider, Chem. Commun., 1999, 729.
13 S. H. Chu, C. Y. Shiue and M. Y. Chu, J. Med. Chem., 1975, 18, 559.
14 A. G. Firth, I. J. S. Fairlamb, K. Darley and C. G. Baumann,
Tetrahedron Lett., 2006, 47, 3529; N. J. Greco and Y. Tor, Tetrahedron,
2007, 63, 3515.
In summary, we have developed a fast and flexible synthetic
route to novel 8-substituted GDP-mannose analogues with
interesting fluorescent properties. From
a single synthetic
precursor, our protocol provides easy access to a set of structurally
180 | Chem. Commun., 2008, 178–180
This journal is ß The Royal Society of Chemistry 2008