Small-angle x-ray scattering investigation of the integration of free fatty acids in polysorbate 20 micelles.

A critical quality attribute for liquid formulations is the absence of visible particles. Such particles may form upon polysorbate hydrolysis resulting in release of free fatty acids into solution followed by precipitation. Strategies to avoid this effect are of major interest for the pharmaceutical industry. In this context, we investigated the structural organization of polysorbate micelles alone and upon addition of the fatty acid myristic acid (MA) by small-angle x-ray scattering. Two complementary approaches using a model of polydisperse core-shell ellipsoidal micelles and an ensemble of quasiatomistic micelle structures gave consistent results well describing the experimental data. The small-angle x-ray scattering data reveal polydisperse mixtures of ellipsoidal micelles containing about 22-35 molecules per micelle. The addition of MA at concentrations up to 100 µg/mL reveals only marginal effects on the scattering data. At the same time, addition of high amounts of MA (>500 µg/mL) increases the average sizes of the micelles indicating that MA penetrates into the surfactant micelles. These results together with molecular modeling shed light on the polysorbate contribution to fatty acid solubilization preventing or delaying fatty acid particle formation.

Exploring and Exploiting Acceptor Preferences of the Human Polysialyltransferases as a Basis for an Inhibitor Screen.

α2,8-Linked polysialic acid (polySia) is an oncofoetal antigen with high abundance during embryonic development. It reappears in malignant tumours of neuroendocrine origin. Two polysialyltransferases (polySTs) ST8SiaII and IV are responsible for polySia biosynthesis. During development, both enzymes are essential to control polySia expression. However, in tumours ST8SiaII is the prevalent enzyme. Consequently, ST8SiaII is an attractive target for novel cancer therapeutics. A major challenge is the high structural and functional conservation of ST8SiaII and -IV. An assay system that enables differential testing of ST8SiaII and -IV would be of high value to search for specific inhibitors. Here we exploited the different modes of acceptor recognition and elongation for this purpose. With DMB-DP3 and DMB-DP12 (fluorescently labelled sialic acid oligomers with a degree of polymerisation of 3 and 12, respectively) we identified stark differences between the two enzymes. The new acceptors enabled the simple comparative testing of the polyST initial transfer rate for a series of CMP-activated and N-substituted sialic acid derivatives. Of these derivatives, the non-transferable CMP-Neu5Cyclo was found to be a new, competitive ST8SiaII inhibitor.

Engineering the product profile of a polysialyltransferase.

Oligo- and polysaccharides have myriad applications as therapeutic reagents from glycoconjugate vaccines to matrices for tissue engineering. Polysaccharide length may vary over several orders of magnitude and is a critical determinant of both their physical properties and biological activities. Therefore, the tailored synthesis of oligo- and polysaccharides of defined size is a major goal for glycoengineering. By mutagenesis and screening of a bacterial polysialyltransferase (polyST), we identified a single-residue switch that controls the size distribution of polymeric products. Specific substitutions at this site yielded distributive enzymes that synthesize polysaccharides with narrow size distribution ideal for glycoengineering applications. Mechanistic investigation revealed that the wild-type enzyme has an extended binding site that accommodates at least 20 residues of the growing polymer; changes in affinity along this binding site allow fine-tuning of the enzyme's product distribution.

Chemical synthesis and enzymatic testing of CMP-sialic acid derivatives.

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.

A universal fluorescent acceptor for high-performance liquid chromatography analysis of pro- and eukaryotic polysialyltransferases.

Polysialyltransferases (polySTs) play critical roles in diverse biological processes, including neural development, tumorigenesis, and bacterial pathogenesis. Although the bacterial enzymes are presumed to have evolved to provide molecular mimics of the host-specific polysialic acid, no analytical technique is currently available to facilitate a direct comparison of the bacterial and vertebrate enzymes. Here we describe a new fluorescent acceptor, a 1,2-diamino-4,5-methylenedioxybenzene (DMB)-labeled trimer of α2,8-linked sialic acid (DMB-DP3), which primes both pro- and eukaryotic polySTs. High-performance liquid chromatography separation and fluorescence detection (HPLC-FD) of reaction products enabled the sensitive and quantitative detection of polyST activity, even using cell lysates as enzyme source, and revealed product profiles characteristic of each enzyme. Single product resolution afforded by this assay system revealed mechanistic insights into a kinetic lag phase exhibited by the polyST from Neisseria meningitidis serogroup B during chain elongation. DMB-DP3 is the first fluorescent acceptor shown to prime the mammalian polySTs. Moreover, product profiles obtained for the two murine polySTs provided direct biochemical evidence for enzymatic properties that had, until now, only been inferred from the analysis of biological samples. With DMB-DP3, we introduce a universal acceptor that provides an easy, fast, and reliable system for the comprehensive mechanistic and comparative analysis of polySTs.