Kurt Nilsson

Immobilization of enzymes and affinity ligands to various hydroxyl group carrying supports using highly reactive sulfonyl chlorides

Abstract A facile method for the activation of hydroxyl group carrying supports such as agarose, cellulose, diol-silica, glycophase-glass or hydroxyethyl methacrylate gels with 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride) is described. On reaction of the resulting tresylate containing supports with different enzymes of affinity ligands at neutral pH, overnight and at 4°C, coupling yields and retained specific activities of up to 80 and 50%, respectively, were obtained. The bound ligands showed excellent affinity properties. Thus, N6-(6-aminohexyl)-AMP coupled to diol-silica completely separated a mixture of albumin, lactate dehydrogenase and alcohol dehydrogenase in less than 15 min when the technique of high performance liquid affinity chromatography was employed.

Imprinted polymers as antibody mimetics and new affinity gels for selective separations in capillary electrophoresis

Abstract Methacrylate-based imprinted dispersion polymers could be prepared in situ in a fused-silica capillary as agglomerates ( ca . 10 μm) of micrometer-sized globular particles, exhibiting antibody mimetic, molecular recognition properties. Thus, in one example, imprinted polymer particles selective for pentamidine (PAM), a drug used for the treatment of AIDS-related pneumonia, could be prepared in situ in the capillary. The retention could be varied predictably by changing the electrolyte pH. Thus, whereas no observable caution of PAM was achieved at near neutral pH, the PAM-selective capillary gave a retention time of 18 min for PAM and 7.8 min for benzamidine at pH 3.5, whereas the retention times were 6.6 and 6.1 min, respectively, with a reference capillary. Importantly, the electrolyte could by pumped hydrodynamically through the capillaries, allowing rapid phase changes and micro-chromatographic possibilities with high plate numbers.

[3] Tresyl chloride-activated supports for enzyme immobilization

Publisher Summary As enzymes are costly and quite unstable molecules, it is desirable that the method used for immobilization be efficient and mild, giving a high yield of bound active enzyme even at neutral pH and at low temperature. For engineering purposes, it is advantageous if the enzyme is stabilized by its immobilization. The method used should be easy to reproduce and should give consistent couplings. Sulfonate esters are suitable for affinity ligand and enzyme immobilization to hydroxyl group carrying supports, such as agarose, cellulose, glycophase glass, and glycerylpropyl-silica. Tresylated supports allow efficient immobilization at near neutral pH and at 4°. Such activated supports are now commercially available. Apart from the activation of agarose and glycerylpropyl-silica, cellulose, hydroxyethyl methacrylate, and glycophase glass have also been activated with tresyl chloride.

High-performance liquid affinity chromatography on silica-bound alcohol dehydrogenase

Horse liver alcohol dehydrogenase was immobilized on glycerylpropyl-silica (10 micron, 1000-A pores) activated with 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride). The coupling and activity yield was almost 100%. The coenzyme-binding sites were equivalent and virtually unaffected by the immobilization process, as judged from Scatchard plots and active-site titrations. The silica-bound enzyme, packed in steel columns, was integrated with HPLC equipment and then successfully used for chromatography of adenine nucleosides, adenine nucleotides, and triazine dyes. Dissociation constants were calculated from chromatographic data and found to correspond well with literature values. The dissociation constants for a number of nucleotide derivatives with potential application in affinity chromatography were also determined. The spaces were found to affect the binding strength of the nucleotides in a qualitatively predictable way. Theoretical plate heights were calculated and found to be in the range 0.01 to 0.1 cm. Attempts to correlate peak widths with the rate constants for the binary complexes involved were only partially successful.

Molecular imprinting of acetylated carbohydrate derivatives into methacrylic polymers

The non-covalent imprinting procedure was used for the preparation of polymers selective for various carbohydrate derivatives, i.e. peracetylated phenyl α- and β-d-glycosides of galactose. The selectivities of the resulting polymers were tested in a HPLC procedure. The selectivity was influenced by the anomeric configuration of the glycoside (α- or β-configuration). Thus, a polymer prepared with p-aminophenyl β-galactoside as print molecule, gave a relatively high selectivity (α = 1.27) for the β-galactoside over the α-galactoside, whereas a polymer prepared with the corresponding α-galactoside showed no or low selectivity towards the α-glycoside (α = 1.02). Moreover, the structure of the aglycon was important. Thus, the use of aminophenyl glycosides as print molecules resulted in polymers with induced selectivity, whereas the use of the corresponding nitrophenyl- and acetaminophenyl-galactosides gave no induced selectivity.

Amino acid ester synthesis by immobilized α-chymotrypsin

SummaryA screening of immobilized α-chymotrypsin preparations suitable for the synthesis of N-acetyl-L-tyrosine ethyl ester from N-acetyl-L-tyrosine and up to 99% ethanol was carried out. α-Chymotrypsin adsorbed to Sepharose LH-20 or covalently bound to Sepharose 4B (tresyl chloride activation) was found to be an efficient catalyst. A column packed with immobilized enzyme retained 60% of its initial activity after 6 days of operation in a cyclohexane-ethanol medium.

Synthesis of glycosylated serine and threonine derivatives employing glycosidases

The galactosyl derivatives Galβ-Ser(N-Boc) and Galβ-Thr(N-Boc) of N-Boc-protected serine and threonine were prepared with galactose or lactose as the glycosyl donor employing β-galactosidase as the catalyst. Similarly, the mannosyl derivatives Manα-Ser(N-Boc) and Manα-Thr(N-Boc) were prepared with mannose as the glycosyl donor (equilibrium reaction) employing α-mannosidase as the catalyst.

Production of glucosamine containing disaccharides of the Lewis-A and -X types employing glycosidases

Disaccharide derivatives of interest for inhibition studies and for synthesis of the blood group determinants Lewis-a and Lewis-x were obtained with glycosidases as catalysts. Thus, Fucα(1–4)(6-OBn)GlcNH2βSEt and Galβ1–3(6-OBn)GlcNH2β-SEt were produced employing (6-OBn)GlcNH2βSEt as acceptor and α-L-fucosidase and β-D-galactosidase, respectively, as catalysts. The phthalimido derivative of lactosamine, Galβ1-4GlcNPhthβSEt, was prepared from lactose employing GlcNPhthβSEt as the acceptor and a yeast β-galactosidase as catalyst. The reactions were both regio- and stereospecific, which allowed straightforward production of pure products on a g scale and higher.

Synthesis of α-mannosylated ergot alkaloids employing α-mannosidase

The ergot alkaloids elymoclavine, ergometrine and chanoclavine were α-mannosylated with α-mannosidase as catalyst. The kinetic reaction with p-nitrophenyl α-mannoside as glycosyl donor gave ca 28 % yield of chanoclavine α-mannoside, whereas the equilibrium reaction with mannose as the glycosyl donor gave ca 11 % yield. However, in the case of elymoclavine and ergometrine, higher yields of α-mannosides were obtained with the equilibrium approach (18 and 13 %).

Syntheses of Modified Carbohydrates with Glycosidases: Stereo- and Regiospecific Syntheses of Lactosamine Derivatives and Related Compounds 1

Abstract Different lactosamine derivatives, modified in the 2-N- and anomeric positions and suitable as intermediates for synthesis of Lewis-x and related compounds, were prepared with high specificity on a multigram scale directly from lactose, employing different D-glucosamine derivatives as acceptors and the abundant β-D-galactosidase from Bullera singularis as catalyst. Thus, methyl O-β-D-galactopyranosyl-(1→4)-2-azido-2-deoxy-β-D-glucopyranoside, ethyl O-β-D-galactopyranosyl-(1→4)-2-deoxy-2-phthalimido-1-thio-β-D-glucopyranoside and ethyl O-β-D-galactopyranosyl-(1→4)-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-1-thio-β-D-glucopyranoside were formed in 20-40 % yield as calculated based on added acceptor. The 2-phthalimido derivative was isolated in crystalline form without chromatography (extraction/crystallization procedure). The trisaccharide derivative ethyl O-β-D-galactopyranosyl-(1→4)-O-β-D-galactopyranosyl-(1→4)-2-deoxy-2-phthalimido-1-thio-β-D-glucopyranoside was also isolated. The correspon...