Bjorn Luning

Synthesis of mono- and disaccharide amino-acid derivatives for use in solid phase peptide synthesis

N-Fluorenylmethyloxycarbonyl-protected serine and threonine derivatives, carryingO-glycosidically α- or β-linked peracetylated β-d-Galp-(1–3)-d-GalNAcp carbohydrate chains, were prepared. These derivatives are intended for use in solid phase glycopeptide synthesis. Suitably protected mono- and disaccharide thioglycosides were used as carbohydrate intermediates. These were activated by treatment with bromine to give the glycosyl bromides, which were then used in silver triflate-promoted glycosidations ofN-fluorenylmethyloxycarbonyl amino-acid phenacyl esters. Removal of the phenacyl esters with zinc gave the target free acids.

Biochemical properties of tissue polypeptide antigen

Tissue polypeptide antigen (TPA) is a complex protein which has been originally identified in extracts of pooled tumors using horse antisera raised against the insolubles of human tumor cells. The antigen is now routinely detected and measured by a previously described hemagglutination inhibition assay. It has been shown by this method that the concentration of the antigen is higher in tumor tissues and in sera of cancer patients as compared to normal tissues or normal sera, respectively. In aqueous solutions, pH 2-12, TPA has a tendency to form high molecular weight aggregates. However they can be dissociated in sodium dodecyl sulfate into subunits, each appearing as a single chain peptide: B1 (Mr 4.3 x 10(4)), B2 (Mr 3.0 x 10(4)), C (Mr 1.7 x 10(4)). The subunits saturate anti-TPA serum indistinguishably from TPA. Amino acid composition of TPA and subunits is dominated by glutamic acid, aspartic acid and leucine, cysteine being absent in subunit B1. The isoelectric point of the main subunit, B1, is 4.4-4.6. Sedimentation and diffusion analyses indicate that pure subunit B1 in aqueous solution exists in distinct oligomeric states.

[6] Solid-phase synthesis of O-glycopeptides

Publisher Summary To study the influence of carbohydrate variation on the properties of a peptide or a protein, it has become important to have access to several different glycoforms. This can be achieved by separating a mixture of glycoforms into components or by using different biological sources that produce different glycoforms. Furthermore, enzymatic glycosylation or deglycosylation can convert one glycoform to another. However, the most generally applicable approach for obtaining defined glycoprotein fragments is chemical synthesis. Technically, this can be carried out either in solution or, preferably for larger fragments, on a solid phase. The 9-fluorenylmethoxycarbonyl (Fmoc) modification of the original Merrifield scheme for solid-phase peptide synthesis is well suited for this purpose. The literature (1988 and onward) describes numerous successful syntheses of glycopeptides using this technique. This chapter reviews work published until mid-1993 on the synthesis of O-glycopeptides, with special emphasis on solid-phase syntheses.

Solid phase synthesis of the fibronectin glycopeptide V(Galβ3GalNAcα)THPGY, its β analogue, and the corresponding unglycosylated peptide

The fibronectin fragment VTHPGY and the corresponding glycopeptides V(Galβ3GalNAcα)THPGY and V(Galβ3GalNAcβ)THPGY were synthesized by the FMOC/solid phase approach. FMOC derivatives of threonine, carrying O-linked, peracetylated Galβ3GalNAc chains were used for introduction (HOBt-mediated coupling) of the disaccharide moieties.

Solid phase synthesis of mono- and di-saccharide-containing glycopeptides

Derivatives of Fmoc-threonine (Fmoc = fluoren-9-ylmethoxycarbonyl) with O-glycosidically peracetylated β-D-Galp-(1 → 3)-α-D-GalNAcp or α-D-GalNAcp chains have been used in a solid phase synthesis of the oncofetal fibronectin sequence VTHPGY (benzyl protection was used on histidine and tyrosine); a super-acid sensitive resin was used, which enabled the isolation of the protected glycopeptide after synthesis, a feature that substantially facilitated verification of the structure by n.m.r. and m.s.

Solid phase phosphorylation of a peptide by the H-phosphonate method

Abstract The partially protected peptide SerValSerGluAla was selectively phosphorylated at Ser3 by benzyl H-phosphonate activated by pivaloyl chloride in pyridine, followed by in situ oxidation with iodine.

Synthesis of Glycosylated Amino Acids for Use in Solid Phase Glycopeptide Synthesis, Part 2: N-(9-Fluorenylmethyloxycarbonyl)-3-0-[2,4,6-TRI-0-Acetyl-3-0-(2,3,4-TRI-0-Acetyl-α-D-Xylopyranosyl)-β-D-Glucopyranosyl]-L-Serine

Abstract FMOC-serine phenacylester was glycosylated with a derivative of the disaccharide α-D-Xylp-(1→3)-β-D-Glcp, and the product was treated with zinc to remove the phenacyl group. The title derivative is useful for synthesis of blood clotting factor IX glycopeptide fragments by the solid-phase approach.

What is TPA?(Tissue Polypeptide Antigen): on the Relation between TPA and Cytokeratins

Tissue Polypeptide Antigen (TPA) was originally defined by Bjorklund (1957) from human carcinomas and has been extensively used in the monitoring of cancer. It has been inferred from histochemical data that TPA could be related to cytokeratins (Nathrath et al . 1983). Alignment of the published sequences of TPA (Redelius et al .1980) with intermediate filament(IF) proteins (Geisler and Weber 1982) showed a significant homology (Luning and Nilsson 1983). Further alignment of new BrCN-fragments of TPA with IF proteins shows the highest homology with an epidermal Type II human keratin(Hanukoglu and Fuchs 1983)presented in Fig 1. Characterization of TPA and rabbit anti-TPA using immunological and immunohistological methods showed that TPA is related to and probably derived from cytokeratins 8, 18 and 19 typically found in simple non-squamous epithelia and their malignant counterparts (Weber et al . 1984). The Mw of TPA, its high α-helix content and rod-like morphology (Luning et al .1980, Wiklund et al .1981) corresponds to known stable and soluble proteolysis products of IF proteins, which may also explain the findings of TPA in serum and other body fluids during infections or malignancy. Thus although TPA comprises a set of complementary cytokeratins, anti-TPA has a high biological specificity since it recognizes only a subclass of epithelial cells and the corresponding carcinomas.

Further Immunochemical Studies of Tissue Polypeptide Antigen (TPA)

Abstract Cross reaction studies using radiolabeled TPA in a double antibody RIA with swine anti-rabbit IgG serum as second antibody and the following rabbit antibodies against human proteins (Dako) as first antibody showed no reaction at any of the following concentrations: 1/90, 1/300. …. 1/300000. First antibodies: antihaptoglobin, anti-retinol binding protein, anti β 2-microglobulin, anti-Gc-globulin, anti-AFP, anti-CEA, anti-erythrocyte membrane proteins, anti-human serum proteins. Rabbit anti-TPA subtraction B1 gave 70% B/T and anti-human placenta (Dako) gave 20% B/T.