Alpha and beta L-Fucopyranosyl oxyamines: key intermediates for the preparation of fucose-containing glycoconjugates by oxime ligation

We report herein the synthesis of new alpha and beta aminooxylated L-fucopyranosyl derivatives for the preparation of glycoclusters through oxime ligation. The glycosylation reaction between activated triacetylated L-fucopyranosyl fluoride and N-hydroxyphthalimide was carried out in the presence of boron trifluoride-diethyl etherate and the stereochemical outcome of glycosylation was compared in dichloromethane, acetonitrile or tetrahydrofuran. Interestingly, an unexpected alpha and beta anomer ratio was obtained in spite of the presence of an acetate participating group at the carbon 2, particularly the 1,2-cis glycosylation was largely favoured in acetonitrile. The resulting alpha and beta N-oxyphthalimido fucopyranosyl derivatives were finally deprotected with methylhydrazine to obtain the corresponding free aminooxylated fucopyranosyls. The structure of single-crystal alpha anomer 12 was analysed by X-ray diffraction.     

A new reagent for the preparation of glycoconjugates

The thioglycoside derivative 2-carbazoylethyl 1-thio-beta-D-galactopyranoside hydrochloride was synthesized. Conversion of the carbazoyl group into the reactive azidocarbonyl function leads to a well suited reagent for the preparation of glycoconjugates via amidation of proteins or synthetic carriers in aqueous media.     

A method for the preparation of Tetrahymena thermophila phospholipase A1 suitable for large-scale production

A rapid and economical method for the purification of phospholipase A1 (PLA1) from the extracellular medium of the ciliate Tetrahymena thermophila is presented. Essentially, the procedure, here designated as purification by selective interaction (PSI), entails the incubation of media containing PLA1 with liposomes made of soy bean phospholipids. The PLA1-lipid complexes are precipitated by the addition of CaCl2 and collected by centrifugation. Elution of the PLA1 is effected by treating the complexes with 40% dimethylformamide, a reversible inhibitor of this enzyme, which is easily removed by dialysis. In combination with DEAE cellulose ion exchange chromatography, PSI yielded homogeneous PLA1 preparations with a 14% recovery and a 416-fold increase in specific activity. This procedure, which can be completed within 1 day, may prove useful for the isolation of phospholipases from other sources. This practical method for the purification of a microbial PLA1 opens the way to large-scale production of these types of enzyme, which are not as yet commercially available.     

Identification method for twelve oligomannose-type sugar chains thought to be processing intermediates of glycoproteins

A mixture of the pyridylamino (PA) derivatives of 12 oligomannose-type sugar chains was fractionated into five fractions (mannose5N-acetylglucosamine2-PA approximately mannose9N-acetylglucosamine2-PA) by size-fractionation HPLC with a MicroPak AX-5 column. Each fraction thus obtained was then analyzed by reversed-phase HPLC with a Cosmosil 5C18-P column. In this way, the 12 PA-oligomannose-type sugar chains were completely separated from each other. The method was used to identify the structures of oligomannose-type sugar chains of human C3, the third component of human complement.     

A single-step sequencing method for the identification of Mycobacterium tuberculosis complex species

Background

The Mycobacterium tuberculosis complex (MTC) comprises closely related species responsible for strictly human and zoonotic tuberculosis. Accurate species determination is useful for the identification of outbreaks and epidemiological links. Mycobacterium africanum and Mycobacterium canettii are typically restricted to Africa and M. bovis is a re-emerging pathogen. Identification of these species is difficult and expensive.

Methodology/Principal Findings

The Exact Tandem Repeat D (ETR-D; alias Mycobacterial Interspersed Repetitive Unit 4) was sequenced in MTC species type strains and 110 clinical isolates, in parallel to reference polyphasic identification based on phenotype profiling and sequencing of pncA, oxyR, hsp65, gyrB genes and the major polymorphism tandem repeat. Inclusion of M. tuberculosis isolates in the expanding, antibiotic-resistant Beijing clone was determined by Rv0927c gene sequencing. The ETR-D (780-bp) sequence unambiguously identified MTC species type strain except M. pinnipedii and M. microti thanks to six single nucleotide polymorphisms, variable numbers (1–7 copies) of the tandem repeat and two deletions/insertions. The ETR-D sequencing agreed with phenotypic identification in 107/110 clinical isolates and with reference polyphasic molecular identification in all isolates, comprising 98 M. tuberculosis, 5 M. bovis BCG type, 5 M. canettii, and 2 M. africanum. For M. tuberculosis isolates, the ETR-D sequence was not significantly associated with the Beijing clone.

Conclusions/Significance

ETR-D sequencing allowed accurate, single-step identification of the MTC at the species level. It circumvented the current expensive, time-consuming polyphasic approach. It could be used to depict epidemiology of zoonotic and human tuberculosis, especially in African countries where several MTC species are emerging.     

A chemoselective method for site-specific immobilization of peptides via aminooxy group

Site-specific modification of peptides and proteins is an important area of basic research for preparation of well-defined biosensors and probes. The unique properties of aminooxy group present an opportunity for chemoselective site-specific immobilization of peptides to prepare well-defined biosensors. We have prepared FLAG peptide derivatives containing L-epsilon-aminooxylysine (L-epsilon-AOLys, 1a) and L-lysine units in their sequence at the C- and N-terminals via solid-phase synthesis. Site-specific modification of peptides through aminooxy group was demonstrated in the preparation of biosensors and selective conjugation in the preparation of biotinylated probes. Effect of the incorporation of L-epsilon-AOLys (1a) into the peptide sequence and its subsequent labeling on the FLAG epitopic character was measured using a surface plasmon resonance detector. It was found that incorporation of L-epsilon-AOLys (1a) into the FLAG peptide and site-specific immobilization through aminooxy group preserved the integrity of FLAG epitope.     

An improved method for the single-step purification of streptavidin

A new method for the preparation of a more efficient, stable iminobiotin-containing resin for the isolation of streptavidin was developed. CL-Sepharose was activated with p-nitrophenyl chloroformate, and the resultant carbonate derivative was reacted with diaminohexane. Subsequent reaction of the amino-containing resin with iminobiotin-N-hydroxysuccinimide ester (in an organic solvent) yielded the stable affinity resin. The capacity of this resin for either avidin or streptavidin was 12 mg per ml resin, and streptavidin could be purified in one step directly from the culture broth of Streptomyces avidinii. The biotin-binding protein isolated in this manner exhibited a major band at about 75 kDa and a minor band at about 150 kDa. Under denaturing conditions, a spectrum of subunit molecular weights ranging between 15 and 19 kDa was detected, the distribution of which depended upon the specific preparation.     

A single-step chromatographic method for the purification of proteins devoid of exposed histidine residues

The principle of the immobilized metal affinity chromatography (IMAC) is based on the differences in the affinity of proteins for metal ions bound in a 1:1 complex of iminodiacetic acid (IDA) immobilized on a chromatographic support. A single step purification was carried out for luteinizing hormone (LH) on Cu2+, Zn2+, Ni2+, and Co2+ IDA Sepharose affinity columns. Highly purified LH was obtained with a Cu2+ IDA Sepharose column. SDS-PAGE and Western blot analysis were done to confirm the purity of the hormone. Biological activity has been evaluated by radio-immunoassay. This method was found economically viable and suitable for the recovery of biologically active hormone.     

A single-step gel-filtration method for the isolation of procollagens from cell culture conditioned medium

Procollagens are the major proteins secreted into the conditioned medium of cultured arterial smooth muscle cells. Methods for the isolation and quantification of these macromolecules have traditionally required preliminary salt precipitation of procollagens from the conditioned medium followed by cellulose ion-exchange chromatography. The method described here exploits the elongated conformation of soluble procollagens and allows the direct recovery of procollagens from culture medium by a single gel-filtration chromatographic step under nondissociating conditions. Procollagens are isolated in high yield and show minimal processing by procollagen N- or C-terminal peptidase activity. This method results in rapid recovery of highly purified procollagens, free of most proteoglycans of other products of smooth muscle cell metabolism.     

A new method for chemoselective conjugation of unprotected peptides to dauno- and doxorubicin

A new approach for chemoselective ligation of peptides to dauno- and doxorubicin through an oxime bond is presented. The method does not require protecting groups on the peptide moiety.     

Targeted liposomes: A method for preparation and analysis

A simple method for the preparation of antibody targeted liposomes is described. It consists of the covalent coupling of the F(ab′)₂ fragment of antibody to phosphatidyl-ethanolamine and subsequent association of this complex with dimyristoylphosphatidyl-choline liposomes. The side effects during the coupling are minimized with prior citraconylation. The association of the protein with the liposomes is demonstrated by gel filtration. Binding assays with antigen-coated Staphyloccus aureus are used to evalute the immunologically specific recognition and the membrane stability of the targeted liposomes.