Bioconjugate Chem. 2005, 16, 722−728
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A Single-Step Method for the Production of Sugar Hydrazides: Intermediates for the Chemoselective Preparation of Glycoconjugates Nicholas S. Flinn,* Martin Quibell, Tracy P. Monk, Manoj K. Ramjee, and Christopher J. Urch† Amura Therapeutics Limited, Horizon Park, Barton Road, Comberton, CB3 7AJ, United Kingdom. Received February 15, 2005; Revised Manuscript Received March 10, 2005
The ability to selectively conjugate carbohydrate molecules to a protein is a key step in the preparation of conjugate vaccines, while facile methods for linking carbohydrates to polymers or solid surfaces to produce diagnostic probes and functional microarrays are also sought. Here, we describe a simple, single-step method of producing glycosylhydrazides from unprotected sugars, which were then linked in a controlled manner to a desired carrier, through an appropriate linker. The method was chemoselective and did not require coupling reagents, and the native pyranose form of the reducing end residue was retained. Initially, mono- and disaccharide hydrazides were produced from the corresponding reducing sugars and linked to BSA through a bifunctional linker. Final exemplification of the procedure was demonstrated by the preparation of a LewisY tetrasaccharide protein conjugate, which was recognized by a LewisY monoclonal antibody indicating the preservation of the natural conformation of the tetrasaccharide in the final construct. It is envisaged that this method will have general applicability to a variety of functionally diverse reducing sugars and provide a route to highly defined glycoconjugates, without the need for elaborate synthetic strategies.
INTRODUCTION
Bacterial derived polysaccharides presented as vaccines have already been effective in controlling certain types of pneumonia and meningitis (1, 2), while “tumorassociated” carbohydrates unique to certain cancers, have also proved attractive as candidates for incorporation into therapeutic cancer vaccines (3-7). The promise of such subunit vaccines has been limited by their low immunogenicity, and to make polysaccharide vaccines broadly more effective, the polysaccharides require conjugation to “carrier proteins” (8), which are often prepared from bacterial sources themselves. The resultant conjugate vaccines tend to be highly immunogenic and confer longlasting protection. Conjugation of polysaccharides to a carrier protein, however, is by no means a simple task, given the polyfunctional nature and complexity of the molecules. Nonspecific approaches include the use of cyanogen bromide (CNBr) (9, 10) and 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) (11, 12), which involve activation of hydroxyl groups on the polysaccharide and result in attachment through multiple sites. More controlled methods of conjugation utilize reductive amination, whereby the reducing end of the polysaccharide is reacted with an amine present on a bifunctional spacer molecule or on a protein to form a Schiff adduct, which is then reduced with sodium cyanoborohydride to form a physiologically stable bond (13, 14). However, the ring opening of the reducing end residue, which occurs during reductive amination, may have a detrimental effect on the biological activity or immunogenicity of the * To whom correspondence should be addressed. E-mail:
[email protected]. Telephone: +44(0)1223-264211. Fax: +44(0)01223-265662. † Current address: Glycoform Limited, Unit 44C, Milton Park, Abingdon, Oxon, OX14 4RU, U.K.
carbohydrate, because the loss of structure ultimately equates to the loss of function (15). A much improved approach involves the introduction of a primary amine at the reducing end terminus of an unprotected carbohydrate (16). The pyranose form of the reducing end terminus monosaccharide is preserved, and the new amine provides a useful handle for further elaboration that may lead to selectively defined glycoconjugates (15, 17, 18). Obviously, defined sugar conjugates may be obtained from a wholly synthetic approach; however, this chemistry is complex and is limited to short-chain-length oligosaccharides (
