Bioconjugate Chem. 2007, 18, 231−237
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Fluorescent Ligands for the Hemagglutinin of Influenza A: Synthesis and Ligand Binding Assays V. Ranjit N. Munasinghe,† John E. T. Corrie,*,† Geoff Kelly,‡ and Stephen R. Martin† MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K., and MRC Biomedical NMR Centre, The Ridgeway, Mill Hill, London NW7 1AA, U.K. Received June 22, 2006; Revised Manuscript Received September 21, 2006
Two fluorescent conjugates of sialic acid have been prepared, with a convenient synthetic route that involves preparation of an unsaturated benzyl ester by cross-metathesis, followed by combined hydrogenation/ hydrogenolysis to provide a sialoside bearing a δ-carboxybutyl group, suitable for coupling with the chosen fluorophores. The fluorescent conjugates bound to bromelain-cleaved hemagglutinin (BHA) with affinities in the low µM range. Binding was accompanied by ∼4.5-fold fluorescence enhancement for the dansyl conjugate 1 and ∼3-fold fluorescence quenching for the pyrene conjugate 3.
INTRODUCTION The influenza A virus is a cause of significant human disease and can on occasion result in devastating pandemics, as in the 1918 outbreak that caused at least 20 million deaths worldwide (1). The infection process is initiated by hemagglutinin on the viral surface binding to terminal R-sialosides of glycoproteins and glycolipids on the surface of the target cell (see ref 2 for a recent review). It is believed that the aquatic bird population is a reservoir for the virus (3). Avian viruses bind preferentially to sialosides with an R2,3-linkage (4), whereas those subtypes of the virus that have caused human pandemics recognize R2,6linked sialosides (5), and changes in binding specificity as a result of mutations are likely required for human infection. As part of studies of viral infectivity, we wished to develop a fluorescence-based assay system that might have potential to evaluate affinities of hemagglutinins from different viral strains for relevant sialosides. Three types of fluorescent sialoside ligands have been previously described, one with a dansyl group attached via a linker to position 4 of the sialoside and an additional naphthyl group linked to the anomeric position to enhance the affinity for hemagglutinin (6), a second with a range of aromatic species (not all fluorescent) attached in the R-orientation to the glycosidic position via linkers of different lengths (7), and a third with a dansyl group linked via a spacer at the 9-position (8). Among these derivatives, only that with the 4-O-linked dansyl group has a report of its fluorescence properties (6) upon binding to hemagglutinin. It showed only a small (99:1 in favor of the required R-product 5b. Under our conditions, 20-30% of the total product was the salicylate ester 13, but this compound was easily removed during workup by washing the organic extract with dilute sodium hydroxide. The byproduct was identified as the ester 13 rather than the isomeric aryl glycoside principally because of a sharp singlet at δ 10.1 that exchanged with D2O and was consistent with a phenolic hydroxyl rather than a carboxylic acid. Both types of byproduct (i.e., ester or aryl glycoside) have been observed in other glycosylations mediated by silver salicylate (28). The olefin 5b was smoothly transformed to the acid 4 by the sequence of olefin cross-metathesis and hydrogenation/hydrogenolysis described above, and the crude acid was coupled with either dansyl hydrazine or 1-pyrenemethylamine in carbodiimide-mediated reactions. Subsequent alkaline hydrolysis gave the required conjugates 1 and 3 that were each purified by reverse-phase HPLC, and each compound was used in fluorescence titrations with BHA. The emission spectrum of the dansyl conjugate 1 (Figure 5A) has a single broad band with an emission maximum at approximately 524 nm. Its binding to BHA results in ∼4.5-fold intensification of the fluorescence at saturation (see below) and a small blue shift to ∼521 nm. The emission spectrum of 3 (Figure 6A) shows two sharp bands at 376 and 395 nm, with a broad long wavelength shoulder at ∼416 nm. Its binding to BHA quenches the fluorescence ∼3-fold at saturation (see below),
Figure 5. (A) The emission spectrum of the dansyl conjugate 1 (1.9 µM) in the absence (solid line) and presence (dotted line) of 12.5 µM BHA trimer. (B) Fluorescence intensity at 521 nm as a function of the concentration of BHA trimer. Excitation was at 321 nm.
with no significant change in band shape. These changes in fluorescence were used to estimate affinities and stoichiometries for the binding of the compounds to BHA. However, to be able to determine binding stoichiometry it would be necessary to perform titrations at concentrations that are typically at least 3-fold higher than Kd for the ligand, in order that essentially all the BHA added in the early part of the titration is saturated with ligand. This was impractical in the present experiments, where for reasons of availability we were unable to use high enough concentrations of BHA to make unambiguous determinations of stoichiometry. We therefore analyzed the titration data for two plausible models: a one-site model in which there is a single binding site for sialic acid in the BHA trimer and a three-site model in which each monomer within the trimer contains a site for binding. In the latter case we assumed that these sites were identical and noninteracting. For 1, the fit using the one-site model (Figure 5B) gave Kd ) 4.7 ( 0.7 µM and F(BHA‚1)/F(1) ) 4.58 ( 0.45; the corresponding values for the three-site model were Kd ) 17.9 ( 2.4 µM and F(BHA‚1)/F1) ) 4.83 ( 0.59. The computed best fit for the one-site model (shown as the solid line in Figure 5B) is marginally better than that for the three-site model (not shown) as judged by χ2 values of 1.09 and 1.43, respectively. For 3, analysis using the one-site model gave Kd ) 2.3 ( 0.4 µM and a fluorescence intensity ratio F(BHA‚3)/F(3) of 0.31 ( 0.02. The corresponding values for the analysis using the threesite model were Kd ) 8.7 ( 1.7 µM and F(BHA‚3)/F(3) ) 0.28 ( 0.03. As above, the best fit for the one-site model (shown as the solid line in Figure 6B) is marginally better than that for the three-site model (not shown) as judged by χ2 values of 1.23 and 1.69, respectively, but the differences are too small to distinguish between the models.
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LITERATURE CITED
Figure 6. (A) The emission spectrum of the pyrenyl conjugate 3 (1.25 µM) in the absence (solid line) and presence (dotted line) of 12.5 µM BHA trimer. (B) Fluorescence intensity at 394 nm as a function of the concentration of BHA trimer. Excitation was at 340 nm.
The binding of BHA to the pyrene conjugate is only 2-fold tighter than for the dansyl one, which is somewhat different from expectations based on previous data (7), in which a trend to higher affinity was associated with an increased number of aromatic rings in the fluorophore (or related pendent group). However, two factors may explain this divergence from the earlier results. First, those data were derived from hemagglutination inhibition experiments, and it has been noted that such assays may not give identical results to fluorescence assays (20d). Second, although the dansyl conjugate 1 described here was identical to that in the earlier study, the series of aromatic hydrocarbon conjugates described previously had six-carbon linkers to the sialic acid rather than the four-carbon one in the present pyrene conjugate 3. The previous data suggest that the linker length can influence the binding constant for these compounds, but there are insufficient data to draw definitive conclusions. Both compounds show much tighter binding (low µM range) than simple R-methyl sialoside [Kd 2.8 mM (7)], implying that the fluorophores and/or linker have interactions with hemagglutinin. Although it seems most probable that the sialic acid moiety does bind to the specific binding site of hemagglutinin, positive demonstration of this will be required and studies to obtain structural evidence for specific binding are planned. These and further application of the binding assays will be described elsewhere.
ACKNOWLEDGMENT We thank the Division of Virology (NIMR) for provision of BHA. We are grateful to the EPSRC Mass Spectrometry Centre, Swansea, for low-resolution negative ion spectra, and to the MRC Biomedical NMR Centre for access to facilities.
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