A novel method for preparation of multivalent oligonucleotide glycoconjugates on a solid support has been described. A pentaerythritol-based phosphoramidite (1) bearing two masked aminooxy groups has been used as the key building block. After conventional chain assembly, the aminooxy functions have been deblocked by a hydrazinium acetate treatment and subsequently oximated with fully acetylated 4-oxobutyl alpha-D-mannopyranoside. The conjugates obtained have been shown to withstand standard ammonolytic deprotection and cleavage from the support. Four different oligonucleotide glycoconjugates containing two, four, or six alpha-D-mannopyranosyl units (12-15) have been prepared to demonstrate the applicability of the procedure. The glycosyl residues only moderately retards hybridization of the oligonucleotide moiety.

SUMMARYWe have examined the expression of the IgG Fc receptors (FcRI, FcRII, FcRIII) and complement receptors (CR1, CR3) in neutrophils from 42 patients with febrile bacterial infection, 20 patients with febrile viral infection and 69 non-febrile healthy individuals. Using receptor-specific MoAbs and immunofluorescence flow cytometry the relative fluorescence intensity (as a measure of receptor number) and the proportion of receptor-positive cells were determined in peripheral blood neutrophils exposed to minimal processing, consisting only of erythrolysis. Both the percentage of FcRI-positive neutrophils and FcRI number per neutrophil were significantly (P < 0 . 001 and P < 0 . 0001) increased in bacterial infected patients compared with controls, whereas in viral infected patients only the FcRI percentage was markedly elevated (P < 0 . 05). In addition, both FcRII and CR1 levels were significantly higher in the bacterial infection group than in the viral infection and control groups (bacterial versus control P < 0 . 001, bacterial versus viral P < 0 . 0001). No changes in expression of FcRIII or CR3 were found in the patient groups. The kinetic analysis of receptor expression in bacterial infection patients revealed a shift in the percentage of FcRI-bearing neutrophils towards normal values already on day 2 after the first analysis. On the other hand, the levels of FcRI, FcRII and CR1 remained clearly elevated in these patients during 1 week's follow-up period. We conclude that febrile infection may cause systemic activation of the entire pool of circulating neutrophils, resulting in alterations in cell surface receptor expression, some of which are characteristic of the nature of the infectious agent.

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Diethyl O,O'-(methoxymethylene)bis(hydroxymethyl)malonate (3) was observed to undergo a stepwise aminolysis when treated with 3-aminopropanol. This allowed convenient preparation of bis(hydroxymethyl)-N,N'-bis(3-hydroxypropyl)malondiamide bearing orthogonal levulinyl (Lev) and tert-butyldiphenylsilyl (TBDPS) protections at the two N-hydroxypropyl groups (8). One of the hydroxylmethyl functions was then protected with a 4,4'-dimethoxytrityl (DMTr) group, and the other one was phosphitylated to obtain a methyl N,N-diisopropylphosphoramidite (1). This building block was used for the synthesis of oligonucleotide glycoconjugates (25 and 26) carrying three different sugar units. After conventional phosphoramidite chain assembly of the sequence containing 1, the 5'-terminal DMTr group was removed and an appropriate glycosyl 6-O-phosphoramidite was coupled. The remaining protections of the branching unit were removed in the order of Lev and TBDPS, and the exposed hydroxyl functions were reacted one after another with the desired glycosyl 6-O-phosphoramidites. Global deprotection and cleavage of the conjugate from the support were achieved by conventional ammonolysis.

Synthetic glycoclusters are extensively used as mimetics of naturally occurring, multivalent carbohydrate ligands in various glycobiological applications. Their preparation, however, is far from trivial, and it still is a limiting factor in the study of carbohydrate binding. We herein report the synthesis of an orthogonally protected building block, N-Alloc-N'-Boc-N' '-Fmoc-alpha,alpha-bis(aminomethyl)-beta-alanine (1), and its use in the preparation of triantennary peptide glycoclusters (21-24) on a solid support. The assembly of the clusters involves removal of the amino protections of the solid-supported branching unit 1 in the order Fmoc, Boc, and Alloc, and subsequent coupling of peracetylated O-(glycopyranosyl)-N-Fmoc-L-serine pentafluorophenyl esters (galactose, glucose, mannose, and ribose) to each amino group exposed.