Biotinyl-L-3-(2-naphthyl)-alanine hydrazide derivatives of N-glycans: versatile solid-phase probes for carbohydrate-recognition studies

Christine Leteux Robert A.Childs Wengang Chai 0 Mark S.Stoll Heide Kogelberg Ten Feizi 0 Mass Spectrometry Group, The Glycosciences Laboratory, Imperial College School of Medicine, Northwick Park Hospital , Watford Road, Harrow, Middlesex HA1 3UJ, United Kingdom - Received on May 13, 1997; revised on July 4, 1997; accepted on August 29, 1997 2To whom correspondence should be addressed Biotinyl-oligosaccharides are a relatively new generation of saccharide probes that enable immobilization of desired oligosaccharides on streptavidin matrices for studies of carbohydrate-protein interactions. Here we describe the facile preparation of biotinyl-L-3-(2-naphthyl)-alanine hydrazide (BNAH) derivatives of oligosaccharides, containing a strong UV absorbing and fluorescent group, in which the ring of the reducing-end monosaccharide is nonreduced. We evaluate reactivities of immobilized BNAH-N-glycans with plant lectins that recognize aspects of the oligosaccharide core or outer-arms. We make some comparisons with 2-amino-6-amidobiotinyl-pyridine (BAP) derivatives obtained by reductive amination, and 6-(biotinyl)-aminocaproyl-hydrazide (BACH) derivatives which have a longer spacer-arm. N-Glycan-BNAH and-BAP derivatives have, overall, comparable reactivities with lectins which recognize N-glycan outer-arms or the trimannosyl core, but only BNAH and BACH derivatives are bound by lectins which recognize the non-reduced core. Moreover, with Pisum sativum agglutinin (PSA) which additionally requires the fucosyl-N-glycan-asparaginyl core for high affinity binding, the immobilized BNAH derivative (which is an alanine hydrazide b -glycoside) can substitute for the natural b -glycosylasparaginyl core, whereas the BACH derivative (aminocaproyl-hydrazide-b -glycoside) is less effective. BNAH is a derivatization reagent of choice, therefore, for solid phase carbohydrate-binding experiments with immobilized N-glycans. Introduction Techniques for the study of proteincarbohydrate interactions have become a focus of interest in glycobiology. This is due largely to the awareness that interactions of proteins with specific oligosaccharide sequences of glycoproteins and glycolipids are crucial steps in the cascade of events that constitute the inflammatory response (Bevilacqua and Nelson, 1993; Drickamer and Taylor, 1993; Feizi, 1993; Crocker and Feizi, 1996; Rosen and Bertozzi, 1996). Thus, it has become desirable to have microtechniques that enable binding studies to be performed with structurally defined oligosaccharide sequences. It is also clear that there is a need for multiple techniques, as the binding signals may be markedly influenced by different modes of oligosaccharide presentation in in vitro experiments; these differences may have a bearing on the behavior of oligosaccharides as ligands in vivo (Feizi, 1993; Green et al., 1995, and references therein). Mono- and oligosaccharides linked to proteins or synthetic cluster glycosides mimicking branched oligosaccharide structures have been valuable reagents for probing the carbohydrate binding specificities of proteins, and for assessing the cooperative effects of multivalence in the strength of the binding signal (Lee, 1992). Oligosaccharides linked to aminophospholipids (neoglycolipids) behave as cluster ligands when immobilized on plastic matrices; they have the additional advantage that they can be resolved on chromatograms, and the technology is applicable for the pinpointing of ligand-bearing sequences among mixtures of oligosaccharides derived from biological sources (Feizi et al., 1994). Biotinylated oligosaccharides have emerged as an additional class of promising probes that allow exploitation of the strong affinity of avidin and streptavidin for biotin (Gitlin et al., 1987). As each streptavidin molecule has four biotin binding sites, the streptavidin-biotinyl-glycan complexes may behave as cluster ligands. Applications have included the immobilization of biotinylated glycopeptides on microtiter wells for conventional binding experiments (Shao, 1992; Shao and Chin, 1992; Shao et al., 1990), and on the sensor microchip of the BIAcore surface plasmon resonance instrument for kinetic measurements of lectin-carbohydrate interactions (Shinohara et al., 1995, 1996). Biotinylated oligosaccharides have been prepared in various ways: for example, by coupling biocytin hydrazide to oxidized sialic acid or to oxidized galactose residues of glycoproteins (Bayer et al., 1988) or by a multistep procedure involving formation of glycosylamines (Manger et al., 1992a,b). Biotinylation of glycosylasparagines has been achieved by coupling activated biotin (N-hydroxy-succinimido-biotin) to the amino group of the asparagine (Shao and Chin, 1992). Drawbacks of these procedures include the chemical modification of oligosaccharide structure, low yields in the multistep procedure, and difficulties in obtaining homogeneous preparations of glycoasparagines, respectively. Three papers have described improved methods for biotinylation of oligosaccharides. In one, oligosaccharides were treated with the UV-absorbent/fluorescent BAP, under reductive amination conditions, and biotinylated oligosaccharides were obtained in good yield (Rothenberg et al., 1993; Toomre and Varki, 1994). In another, oligosaccharides were coupled directly, with preservation of the reducing end monosaccharide, to the weakly UV-absorbent but nonfluorescent BACH (Shinohara et al., 1995). In a more recent paper, oligosaccharides were coupled to a hydrazide reagent containing a moderately UV-absorbing, nonfluorescent group: 4biotinamidophenylacetylhydrazide, BPH (Shinohara et al., 1996). Here we describe the preparation of novel biotinylated oligosaccharide derivatives using the reagent BNAH, which has both UV-absorbing and fluorescent properties, and allows Fig. 1. HP-TLC of Man5 oligosaccharide and of reaction mixtures of Man5-BNAH and of Man5-BAP. In (A) lane 1 contains Man5 oligosaccharide, and lane 2 contains Man5-BNAH reaction mixture stained with orcinol to reveal hexose in Man5 and Man5-BNAH. In (B/B), lane 1 contains Man5 oligosaccharide, and lane 2 contains Man5-BAP reaction mixture; (B) is a 300 nm UV fluorescence image revealing Man5-BAP and excess BAP; (B) is the same chromatogram stained with orcinol to reveal hexose in Man5 and Man5-BAP. Approximately 1 nmol of oligosaccharide was applied per lane. Chromatography was upward with 2-butanone/methanol/water, 6:2:2, v/v (A) and butan-1-ol/acetone/water, 6:5:4, v/v (B/B). coupling to oligosaccharide under nonreductive conditions. We evaluate the binding of the immobilized BNAH derivatives by plant lectins that recognize aspects of the core region and outer chains of N-glycans. Comparisons with the binding to oligosaccharide-BAP and -BACH derivatives show considerable advantages of the BNAH derivatives as immobilized ligands for carbohydrate-binding studies with the lectins investigated. Fig. 2. RP-HPLC of reaction mixtures (...truncated)