We isolated a cDNA encoding a novel glucuronyltransferase from human placenta cDNA with the use of the degenerate reverse transcriptase-polymerase chain reaction method. Degenerate primers were designed based upon the amino acid sequence alignment of rat glucuronyltransferase (GlcAT-P) involved in the biosynthesis of the carbohydrate epitope HNK-1 with putative proteins in Caenorhabditis elegans and Schistosoma mansoni. The new cDNA sequence revealed an open reading frame coding for a protein of 335 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 43% identity to the rat GlcAT-P, and the highest sequence identity was found in the COOH-terminal catalytic domain. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active glucuronyltransferase with marked specificity for a glycoserine Gal␤1-3Gal␤1-4Xyl␤1-OSer. In contrast, asialoorosomucoid, which contains the Gal␤1-4GlcNAc sequence and is a good acceptor substrate for the GlcAT-P, did not serve as an acceptor. The reaction product was sensitive to ␤-glucuronidase digestion and co-chromatographed with authentic GlcA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-O-Ser in high-performance liquid chromatography, suggesting that the enzyme is a ␤1,3-glucuronyltransferase. These results indicate that this new member of the glucuronyltransferase gene family is the enzyme previously described as glucuronyltransferase I that forms the glycosaminoglycan-protein linkage region, GlcA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-O-Ser, of proteoglycans.

An efficient catalytic protocol for the three-component assembly of benzyl bromides, carbon monoxide, and alkyl zinc reagents to give benzyl alkyl ketones is described, and represents the first nickel-catalyzed carbonylative coupling of two sp -carbon fragments. The method, which relies on the application of nickel complexed with an NN -type pincer ligand and a controlled release of CO gas from a solid precursor, works well with a range of benzylic bromides. Mechanistic studies suggest the intermediacy of carbon-centered radicals.

The relationship between sulfation and polymerization in chondroitin sulfate (CS) biosynthesis has been poorly understood. In this study, we investigated the specificity of bovine serum UDP-GalNAc: CS beta-GalNAc transferase responsible for chain elongation using structurally defined acceptor substrates. They consisted of tetra- and hexasaccharide-serines that were chemically synthesized and various regular oligosaccharides with a GlcA residue at the nonreducing terminus, prepared from chondroitin and CS using testicular hyaluronidase. The enzyme preparation was obtained from fetal bovine serum by means of heparin-Sepharose affinity chromatography. The preparation did not contain the alpha-GalNAc transferase recently demonstrated in fetal bovine serum (Kitagawa et al., J. Biol. Chem., 270, 22190-22195, 1995), that utilizes common acceptor substrates. The beta-GalNAc transferase used as acceptors, two hexasaccharide-serines GlcA beta 1-3GalNAc beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser and GlcA beta 1-3GalNAc(4-sulfate) beta 1-4GlcA beta 1-3Gal (4-sulfate) beta 1-3Gal beta 1-4Xyl beta 1-O-Ser, but neither the monosulfated hexasaccharide-serine GlcA beta 1-3GalNAc(4-sulfate) beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser nor tetrasaccharide-serines with or without a sulfate group at C-4 of the third sugar residue Gal-3 from the reducing end. The results indicated that the sulfate group at the Gal-3 C-4 markedly affected the transfer of GalNAc to the terminal GlcA. In addition, a sulfate group at C-4 of the reducing terminal GalNAc of regular tetrasaccharides remarkably enhanced the GalNAc transfer, suggesting that the enzyme recognizes up to the fourth saccharide residue from the nonreducing end. The level of incorporation into a tetra- or hexasaccharide containing a terminal 2-O-sulfated GlcA residue was significant, whereas there was no apparent incorporation into tetra- or hexasaccharides containing a terminal 3-O-sulfated GlcA or penultimate 4,6-O-disulfated GalNAc residue. These results indicated that sulfation reactions play important roles in chain elongation and termination.

Carbon monoxide represents the most important C1-building block for the chemical industry, both for the production of bulk and fine chemicals, but also for synthetic fuels. Yet its toxicity and subsequently its cautious handling have limited its applications in medicinal chemistry research and in particular for the synthesis of pharmaceutically relevant molecules. Recent years have nevertheless witnessed a considerable headway on the development of carbon monoxide surrogates and reactor systems, which provide an ideal setting for performing carbonylation chemistry with stoichiometric and substoichiometric carbon monoxide. Such setups are particularly ideal for the introduction of isotope labels such as carbon-11, carbon-13, and carbon-14 into bioactive compounds. This review summarizes this growing field and examines the large number of carbonylation reactions that can be exploited for the introduction of a carbon isotope.

We studied a glucuronyltransferase involved in chondroitin sulfate (CS) biosynthesis in a preparation obtained from fetal bovine serum by heparin-Sepharose affinity chromatography. This enzyme transferred GlcA from UDP-GlcA to the nonreducing GalNAc residues of polymeric chondroitin. It required Mn2+ for maximal activity and showed a sharp pH optimum between pH 5.5 and 6.0. The apparent Km value of the glucuronyltransferase for UDP-GlcA was 51 microM. The specificity was investigated using structurally defined acceptor substrates, which consisted of chemically synthesized tri-, penta-, and heptasaccharide-serines and various odd-numbered oligosaccharides with a GalNAc residue at the nonreducing terminus, prepared from chondroitin and CS by chondroitinase ABC digestion followed by mercuric acetate treatment. The enzyme utilized a heptasaccharide-serine GalNAc beta 1-4GlcA beta 1-3GalNAc beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser and a pentasaccharide-serine GalNAc beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser as acceptors. In contrast, neither a trisaccharide-serine Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser nor an alpha-GalNAc-capped pentasaccharide-serine GalNAc alpha 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser that is a model compound of the reaction product formed by the action of the alpha-GalNAc transferase recently demonstrated in fetal bovine serum (Kitagawa et al., J. Biol. Chem., 270, 22190-22195, 1995) was utilized as an acceptor. Besides, all nonsulfated odd-numbered oligosaccharides except for the trisaccharide GalNAc beta 1-4GlcA beta 1-3GalNAc served as acceptors and the transfer rates roughly increased with increasing chain length. Moreover, 6-O-sulfation of nonreducing terminal GalNAc markedly enhanced GlcA transfer, whereas 4-O-sulfation had little effect on it. These results indicated that at least two glucuronyltransferases are involved in the biosynthesis of CS and that sulfation reactions may play important roles in chain elongation.