A new and efficient method for the synthesis of nucleoside di- and triphosphates as well as dinucleoside polyphosphates (Np(n)N') is described. 5-Acceptor-substituted (5-nitro and 5-chloro) cycloSal-nucleotides are used as starting material that were reacted with a variety of phosphate nucleophiles as pyrophosphate or nucleotides to the corresponding products in short times and very good yields. After consumption of the starting cycloSal-phosphate triester, first the protecting groups were cleaved and finally the products were isolated after RP-column chromatography. Examples are shown for all five pyrimidine and purine bases found in natural nucleosides as well as one non-natural pyrimidine base to prove that the method can be applied generally.

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The cycloSal approach has been used in the past for the synthesis of a range of phosphorylated bioconjugates. In those reports, cycloSal nucleotides were allowed to react with different phosphate nucleophiles. With glycopyranosyl phosphates as nucleophiles, diphosphate-linked sugar nucleotides were formed. Here, cycloSal-nucleotides were used to prepare monophosphate-linked sugar nucleotides successfully in high anomeric purity and high chemical yield. The method was successfully used for the synthesis of three nucleotide glycopyranoses as model compounds. The method was then applied to the syntheses of CMP-N-acetyl-neuraminic acids (CMP-Neu5NAc) and of four derivatives with different modifications at their amino functions (N-propanoyl, N-butanoyl, N-pentanoyl and N-cyclopropylcarbonyl). The compounds were used for initial enzymatic studies with a bacterial polysialyltransferase (polyST). Surprisingly, the enzyme showed marked differences in terms of utilisation of the four derivatives. The N-propanoyl, N-butanoyl, and N-pentanoyl derivatives were efficiently used in a first transfer with a fluorescently labelled trisialo-acceptor. However, elongation of the resulting tetrasialo-acceptors worsened progressively with the size of the N-acyl chain. The N-pentanoyl derivative allowed a single transfer, leading to a capped tetramer. The N-cyclopropylcarbonyl derivative was not transferred.

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Nucleoside diphosphate pyranoses 1 (NDP sugars; see Scheme 1) play a key role as glycosyl donors in the synthesis of oligo-and polysaccharides. [1,2] Moreover, they serve as precursors of deoxysugars, aminodeoxysugars, chainbranched sugars, uronic acids, as well as glycoconjugates. In biosynthetic pathways the energy-rich linkage between the C1 atom and the b-phosphate is cleaved, and thus the glycosyl part is enzymatically transferred to an oligosaccharide chain, releasing the nucleoside diphosphate (NDP) moiety. For biosynthesis studies of oligosaccharides (for example, of lipopolysaccharides) [3] an efficient access to this important class of compounds is needed. The classical method is the coupling of glycosyl 1-phosphates to nucleotide morpholidates (Moffat-Khorana method). [4,5] However, this reaction normally takes days, and the chemical yields are often low (5-25 %). Attempts to improve the reaction yields by using 1H-tetrazole as activator [6][7][8][9] are often not successful and have failed also in our hands. Instead of the morpholidates, also imidazolides have been used in the past but without improving the yields markedly. [10][11][12] Alternatively, Hindsgaul and Jakeman published a procedure starting from nucleoside diphosphates and glycopyranosyl bromides. [13,14] However, yields were also found to be low, and often the stereochemistry at the anomeric center could neither be controlled nor stereospecifically formed. Thiem and co-workers reported an enzymatic procedure starting from unprotected sugars that were first phosphorylated and then treated with a nucleoside triphosphate. [15,16] However, this reaction sequence is based on a three-enzyme pathway and depends on the availability of the needed kinases and NDP sugar pyrophosphorylases and on expensive nucleoside triphosphates. The yields obtained have seldom exceeded 30 %. Herein, we report on a conceptionally new chemical synthesis of NDP sugars that uses cyclo-saligenyl (cycloSal) nucleosyl phosphate triesters II as an active ester (Scheme 1).Originally, the cycloSal technique was developed to deliver biologically active nucleotides into cells.[17] The cleavage relies on a nucleophilic attack of the neutral phosphate triester by water or hydroxide and a subsequent selective hydrolysis pathway to yield the nucleotide (pathway a-c; Scheme 1). The technique has been applied successfully to a variety of nucleoside analogues, providing superior antiviral activity. [18][19][20] However, the same type of compounds may also be used as an active ester for synthetic applications. Here, cycloSal nucleotides like II were treated with glycopyranosyl 1-phosphate salts III-VI as nucleophiles with formation of the pyrophosphate bond in NDP sugars (pathway d,e; Scheme 1).As starting materials, 5-nitro-cycloSal-3'-O-acetylthymidine 2 and peracetylated glycopyranosyl phosphates 3-6 were prepared (see Scheme 3). Thus, thymidine was protected by silylation with tert-butyldimethylsilylchloride (TBDMS-Cl) at the 5-position. The product was treated with acetic anhyd...