Molecular Recognition of Sialic Acid End Groups
FULL PAPER
spectra were measured at 75.48 MHz and tBuOH was used as an internal
reference with the methyl peak of the standard set at d=31.2 ppm.
11B NMR were measured at 128.33 or at 96.3 MHz, with a 0.1m solution
of boric acid in D2O(d=0.00 ppm) as external standard. The conversion
to the BF3·Et2O scale is as follows: d(H3BO3 scale)=d(BF3·Et2O
scale)ꢀ18.7. About 240 scans were collected using a delay and an acquisi-
tion time of 1 s. 17O NMR spectra were recorded at 40.67 MHz. A spec-
tral window of 29996 Hz and an acquisition delay of 0 s were applied.
17O NMR spectra of mixtures of 17O-enriched glycolic acid and PBA
were acquired at ꢀ158C to lower the exchange rates on the 17O NMR
time scale. The peak positions and the integrations of the resonances in
11B, 17O and quantitative 13C spectra were determined by fitting the ob-
served signals with Lorentzian line-functions.
Conclusion
From the results of the present study it can be concluded
that phenylboronic acid forms esters with Neu5Ac between
pH 2 and 12. At low pH (2–8), the a-hydroxycarboxylate
moiety at C1/C2 is involved in the binding, whereas at pH
>8 the binding of PBA takes place at the glycerol side
chain to give a five-membered ester at C8 and C9. The for-
mation of another five-membered complex at C7 and C8 is
limited by the unfavorable erythro configuration of the gly-
cerol tail. Molecular modeling studies confirm these conclu-
sions, and show the lowest energies for the above-mentioned
ester. The insight into the binding of PBA by Neu5Ac ob-
tained in this study may be useful for the design of artificial
receptors for sialic acid moieties in glycoproteins. On the
cell surface sialic acids represent the terminal sugar residue
of a glycan chain. That is, they are linked through C2 to po-
sitions 3 or 6 of the penultimate sugar or to position 8 of an-
other sialic acid molecule.[20] Consequently, the carboxyl
group is not available for interaction with the PBA receptor
and, at physiological pH, it is the carrier of negative charge
on the sugar molecule (pKa =2.2). The experiment with the
2-a-O-methyl derivative of sialic acid demonstrates that in-
teraction with PBA occurs even without carboxylic group
participation.
Determination of stability constants: NMR-titration experiments were
used to determine both the pKa values of the studied compounds and the
apparent stability constant (Kf) of the complexes. The pH values of the
samples were measured at ambient temperature with a Corning 125 pH
meter and a calibrated micro-combination probe purchased from Aldrich
Chemical Company. The pH values of the solutions were adjusted with
1m solutions of NaOH and HCl. The reported values are uncorrected for
isotope effects and the presence of methanol. It has been reported that
corrections for the solvent systems selected are negligible.[23]
Computer calculations were performed with the Micromath Scientist pro-
gram, version 2.0 (Salt Lake City, Utah, U.S.A).
Molecular modeling: Calculations were performed with the HyperChem
7.5 Professional program. The MM+ force field was used to optimize
the conformations of the possible complexes of Neu5Ac and PBA and to
calculate the energies (given in kJmolꢀ1). The conformations obtained
were re-optimized by using AM1 semiempirical quantum mechanics. The
default options (Restricted Hartree–Fock (RHF) spin pairing) were used
with a total charge of ꢀ2 and a spin multiplicity of 1. Molecular dynamics
at 1000 K and searches with the conformational search tool of the Hyper-
Chem software were performed to obtain the various conformational
minima.
More-selective and -effective artificial receptors for sialic
acid residues in glycoproteins, therefore, should contain
both a PBA unit and a group that is able to recognize the
negatively charged COOꢀ group of sialic acid. Recently, we
have successfully applied these principles in the design of a
potential sialic acid targeting MRI contrast agents.[21]
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Experimental Section
Compounds: D2O was obtained from ARC Laboratories BV (Amster-
dam, The Netherlands) and 17O-enriched water (25% 17O) from A.
Matheson, USA Company (Miamisburg, Ohio). The water used for the
preparation of samples was purified with a Milli-Q filtration system. Gly-
colic acid was purchased from Acros (Geel, Belgium) and N-acetylneura-
minic acid (Neu5Ac) was purchased from Rose Scientific Ltd. (Edmon-
ton, Canada). For some experiments Neu5Ac was converted into the
methyl ester 2-a-O-methyl-5-acetylneuraminic acid according to a pub-
lished procedure.[22] The structure was confirmed from the 13C NMR
spectrum (75.48 MHz, D2O, 25 8C, tBuOH, APT): d=174.85 (CO),
170.85 (CO), 100.38 (C), 72.27 (CH), 71.40 (CH), 70.19 (CH), 67.71
(CH), 65.30 (CH2), 53.80 (CH), 53.17 (CH3), 51.66 (CH3), 41.58 (CH2),
22.75 ppm (CH3). During the 11B NMR titration the ester group was hy-
drolyzed at high pH to form 5 in situ, as confirmed by the shift of the
CH3 peak at d=53.17 ppm in the 13C NMR spectrum to d=49.14 ppm.
Threonic and erythronic acids were obtained from Aldrich as their calci-
um salts and were converted into the corresponding water-soluble
sodium salts with a Dowex 50Wꢂ8 cation-exchange resin.
17O-Enrichement of glycolic acid was achieved by stirring a 0.81m aque-
ous solution of glycolic acid containing 4% of 17O-labeled water at 808C
and pHꢃ4 for a period of 16 h, followed by freeze-drying of the solution.
17O NMR (40.67 MHz, D2O): d=253 ppm (ref. H2O, pH 5).
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NMR spectroscopy: All NMR measurements were performed on Varian
INOVA-300 and VXR-400S spectrometers at 258C unless stated other-
wise using 5-mm sample tubes. All NMR spectra were recorded using a
water (10% D2O)/methanol (2:1 v/v) mixture as the solvent. 13C NMR
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Chem. Eur. J. 2005, 11, 4010 – 4018
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