Organic chemistry and biocatalysis

Scientific publications - Organic chemistry and biocatalysis


Prasad, A.K., Kalra, N., Yadav, Y., Singh, S.K., Sharma, S.K., Patkar, S., Lange, L., Olsen, C.E., Wengel, J., Parmar, V.S.

"Selective biocatalytic deacylation studies on furanose triesters: A novel and efficient approach towards bicyclonucleosides"

Organic and Biomolecular Chemistry, 5 (21), pp. 3524-3530. (2007)

Lipozyme® TL IM catalyses the deacylation of 4-C-acyloxymethyl-3,5-di-O-acyl-1,2-O-(1-methylethylidene)-β-l-threo- pentofuranose to form 3,5-di-O-acyl-4-C-hydroxymethyl-1,2-O-(1-methylethylidene) -α-d-xylo-pentofuranose in a highly selective and efficient manner. The rate of lipase-catalyzed deacylation of tributanoyl furanose is 2.3 times faster than the rate of deacylation of the triacetyl furanose derivative. In order to confirm the structure of the lipase-catalyzed deacylated product, it was converted to a bicyclic sugar derivative, which can be used for the synthesis of bicyclic nucleosides of importance in the development of novel antisense and antigene oligonucleotides. Further, it has been established that the monohydroxy product of the lipase-catalyzed reaction is the result of selective deacylation of the 4-C-acyloxymethyl function in the substrate and not of any acyl migration process. © The Royal Society of Chemistry.

Pernille Tofteng, A., Hansen, T.H., Brask, J., Nielsen, J., Thulstrup, P.W., Jensen, K.J.

"Synthesis of functionalized de novo designed 8-16 kDa model proteins towards metal ion-binding and esterase activity"

Organic and Biomolecular Chemistry, 5 (14), pp. 2225-2233. (2007)

De novo design and total chemical synthesis of proteins provides a powerful approach for biological and biophysical studies with the ability to prepare artificial proteins with tailored properties, potentially of importance for biophysical studies, material science, nanobioscience, and as molecular probes. In this paper, the previously developed concept of carbohydrates as templates is employed in the de novo design of model proteins (artificial helix bundles) termed 'carboproteins'. The 4-α-helix bundle is a macromolecular structure, where four amphiphilic α-helical peptide strands form a hydrophobic core. Here this structure is modified towards achieving metal ion-binding and catalytic activity. We report: (i) test of directional effects from different tetravalent carbohydrate templates, (ii) synthesis and evaluation of carboproteins functionalized with phenol, pyridyl or imidazolyl moieties as potential ligands for metal ion-binding as well as for catalysis. Our results include: (i) support of our previous 'controversial' finding that for some carboproteins the degree of α-helicity depends on the template, i.e., that there is, to some extent, a controlling effect from the template, (ii) demonstration of binding of Cu(ii) to tetra-functional carboproteins by electrospray ionization-time of flight-mass spectrometry (ESI-TOF-MS), UV-VIS absorption spectroscopy and size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS); (iii) a kinetic investigation of the esterase activity. © The Royal Society of Chemistry.

Prasad, A.K., Kalra, N., Yadav, Y., Kumar, R., Sharma, S.K., Patkar, S., Lange, L., Wengel, J., Parmar, V.S.

"Deacylation studies on furanose triesters using an immobilized lipase: Synthesis of a key precursor for bicyclonucleosides"

Chemical Communications, (25), pp. 2616-2617. (2007)

Lipozyme® TL IM immobilized on silica catalyses the deacylation of 4-C-acyloxymethyl-3,5-di-O-acyl-1,2-O-(1-methylethylidene)-β-l-threo- pentofuranose to form 3,5-di-O-acyl-4-C-hydroxymethyl-1,2-O-(1-methylethylidene) -α-d-xylo-pentofuranose in a highly selective and efficient manner. © The Royal Society of Chemistry.

Larsen, T.O., Lange, L., Schnorr, K., Stender, S., Frisvad, J.C.

"Solistatinol, a novel phenolic compactin analogue from Penicillium solitum"

Tetrahedron Letters, 48 (7), pp. 1261-1264. (2007)

Solistatinol, a novel phenolic compactin analogue, has been isolated from Penicillium solitum using a UV-guided strategy. The structure and relative stereochemistry were determined by NMR spectroscopy and mass spectrometry. The absolute stereochemistry was determined by chemical degradation and comparison of CD data with literature data. © 2006 Elsevier Ltd. All rights reserved.

Coward-Kelly, G., Chen, R.R.

"A window into biocatalysis and biotransformations"

Biotechnology Progress, 23 (1), pp. 52-54. (2007)

Eight papers were presented in this year's symposium "Advances in Biocatalysis" at the 232nd ACS National Meeting, accentuating the most recent development in biocatalysis. Researchers from both industry and academia are addressing several fundamental problems in biocatalysis, including the limited number of commercially available enzymes that can be provided in bulk quantities, the limited enzyme stability and activity in nonaqueous environments, and the permeability issue and cell localization problems in whole-cell systems. A trend that can be discerned from these eight talks is the infusion of new tools and technologies in addressing various challenges facing biocatalysis. Nanotechnology, bioinformatics, cellular membrane engineering and metabolic engineering (for engineering whole-cell catalysts), and protein engineering (to improve enzymes and create novel enzymes) are becoming more routinely used in research laboratories and are providing satisfactory solutions to the problems in biocatalysis. Significant progress in various aspects of biocatalysis from discovery to industrial applications was highlighted in this symposium. © 2007 American Chemical Society and American Institute of Chemical Engineers.

Brennan, J.L., Hatzakis, N.S., Tshikhudo, T.R., Dirvianskyte, N., Razumas, V., Patkar, S., Vind, J., Svendsen, A., Nolte, R.J.M., Rowan, A.E., Brust, M.

"Bionanoconjugation via click chemistry: The creation of functional hybrids of lipases and gold nanoparticles"

Bioconjugate Chemistry, 17 (6), pp. 1373-1375. (2006)

A simple and versatile method for the preparation of functional enzyme-gold nanoparticle conjugates using "click" chemistry has been developed. In a copper-catalyzed 1,2,3-triazole cycloaddition, an acetylene-functionalized Thermomyces lanuginosus lipase has been attached to azide-functionalized water-soluble gold nanoparticles under retention of enzymatic activity. The products have been characterized by gel electrophoresis and a fluorometric lipase activity assay. It is estimated that the equivalent of approximately seven fully active lipase molecules are attached to each nanoparticle. © 2006 American Chemical Society.

Prasad, A.K., Mukherjee, C., Sharma, D., Rastogi, S., Mangalam, A., Jha, A., Olsen, C.E., Patkar, S.A., Parmar, V.S.

"Synthesis and lipase-catalyzed resolution studies on novel (±)-2-(2-acetoxyethyl)-4-arylmethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-6-carboxylates"

Journal of Molecular Catalysis B: Enzymatic, 40 (3-4), pp. 101-110. (2006)

Five novel methyl (±)-2-(2-acetoxyethyl)-4-arylmethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-6-carboxylates have been synthesized and their lipase-catalyzed resolution via stereoselective deacetylation of acetoxyethyl moiety present in the molecule studied. It has been observed that Novozyme®-435 in THF efficiently catalyses the enantioselective deacetylation of these acetoxyethyl dihydrobenzoxazines leading to the formation of optically enriched methyl (+)-4-arylmethyl-2-(2-hydroxyethyl)-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-6-carboxylates. The biocatalytic reaction was found to be chemoselective alongwith being enantioselective, because the lipase exclusively catalyses the deesterification of the ester function derived from the alcoholic hydroxy moiety in the molecule over the one derived from the aromatic carboxylic acid group. © 2006 Elsevier B.V. All rights reserved.

Jensen, K.J., Brask, J.

"Carbohydrates in peptide and protein design"

Biopolymers - Peptide Science Section, 80 (6), pp. 747-761. (2005)

Monosaccharides and amino acids are fundamental building blocks in the assembly of nature's polymers. They have different structural aspects and, to a significant extent, different functional groups. Oligomerization gives rise to oligosaccharides and peptides, respectively. While carbohydrates and peptides can be found conjoined in nature, e.g., in glycopeptides, the aim of this review is the radical redesign of peptide structures using carbohydrates, particularly monosaccharides and cyclic oligosaccharides, to produce novel peptides, peptidomimetics, and abiotic proteins. These hybrid molecules, chimeras, have properties arising largely from the combination of structural characteristics of carbohydrates with the functional group diversity of peptides. This field includes de novo designed synthetic glycopeptides, sugar (carbohydrate) amino acids, carbohydrate scaffolds for nonpeptidal peptidomimetics of cyclic peptides, cyclodextrin functionalized peptides, and carboproteins, i.e., carbohydrate-based proteinmimetics. These successful applications demonstrate the general utility of carbohydrates in peptide and protein architecture. © 2005 Wiley Periodicals, Inc.

Sharma, A.K., Kumar, R., Canteenwala, T.C., Parmar, V.S., Patkar, S., Kumar, J., Watterson, A.C.

"Biocatalytic synthesis and characterization of copolymers based on poly(ethylene glycol) and unsaturated methyl esters"

Journal of Macromolecular Science - Pure and Applied Chemistry, 42 A (11), pp. 1515-1521. (2005)

Biocatalytic organic synthesis has proved to be a significant breakthrough in the area of polymer synthesis. Environmentally benign methodology and the use of mild reaction conditions are a hallmark of this approach. We have studied the biocatalytic synthesis of unsaturated polyesters under solventless conditions by the condensation copolymerization of dimethyl fumarate and dimethyl maleate with polyethylene glycol (PEG) catalyzed by Novozyme435 (immobilized Candida antarctica lipase B). The structures of the resulting polymers, poly(ethylene glycol)codimethyl fumarate and poly(ethylene glycol)codimethyl maleate were studied from their 1 H and 13 CNMR spectra. The molecular weights of polymers were determined by size exclusion chromatography. Copyright © Taylor & Francis, Inc.

Damager, I., Jensen, M.T., Olsen, C.E., Blennow, A., Møller, B.L., Svensson, B., Motawia, M.S.

"Chemical synthesis of a dual branched malto-decaose: A potential substrate for α-amylases"

ChemBioChem, 6 (7), pp. 1224-1233. (2005)

A convergent block strategy for general use in efficient synthesis of complex α-(1→4)- and α-(1→6)-malto-oligosaccharides is demonstrated with the first chemical synthesis of a malto-oligosaccharide, the decasaccharide 6,6″″-bis(α-maltosyl)-maltohexaose, with two branch points. Using this chemically defined branched oligosaccharide as a substrate, the cleavage pattern of seven different α-amylases were investigated. α-Amylases from human saliva, porcine pancreas, barley α-amylase 2 and recombinant barley α-amylase 1 all hydrolysed the decasaccharide selectively. This resulted in a branched hexasaccharide and a branched tetrasaccharide. α-Amylases from Asperagillus oryzae, Bacillus licheniformis and Bacillus sp. cleaved the decasaccharide at two distinct sites, either producing two branched pentasaccharides, or a branched hexasaccharide and a branched tetrasaccharide. In addition, the enzymes were tested on the single-branched octasaccharide 6-α-maltosyl-maltohexaose, which was prepared from 6,6″″-bis(α-maltosyl)-maltolhexaose by treatment with malt limit dextrinase. A similar cleavage pattern to that found for the corresponding linear malto-oligosaccharide substrate was observed. © 2005 Wiley-VCH Verlag GmbH & Co. KGaA.

Bywater, R.P., Poulsen, T.A., Røgen, P., Hjorth, P.G.

"De novo generation of molecular structures using optimization to select graphs on a given lattice"

Journal of Chemical Information and Computer Sciences, 44 (3), pp. 856-861. (2004)

A recurrent problem in organic chemistry is the generation of new molecular structures that conform to some predetermined set of structural constraints that are imposed in an endeavor to build certain required properties into the newly generated structure. An example of this is the pharmacophore model, used in medicinal chemistry to guide de novo design or selection of suitable structures from compound databases. We propose here a method that efficiently links up a selected number of required atom positions while at the same time directing the emergent molecular skeleton to avoid forbidden positions. The linkage process takes place on a lattice whose unit step length and overall geometry is designed to match typical architectures of organic molecules. We use an optimization method to select from the many different graphs possible. The approach is demonstrated in an example where crystal structures of the same (in this case rigid) ligand complexed with different proteins are available.

H. J. Deussen; M. Zundel; M. Valdois; S. V. Lehmann; V. Weil V; C. M. Hjort; P.R. Ostergaard; E. Marcussen; S. Ebdrup.

"Process development on the enantioselective enzymatic hydrolysis of S-ethyl 2-ethoxy-3-(4-hydroxyphenyl)propanoate."

Organic Process Research and Development, 7 (1), 82-88 (2003).

A novel biocatalytic approach for the large-scale production of S-2-ethoxy-3-(4-hydroxyphenyl)propanoic acid S-1 from its racemic ethylester rac-2 by enantioselective hydrolysis has been developed. S-1 is an important building block in the synthesis of PPARalpha and -gamma agonists such as Ragaglitazar [NNC 61-0029 ((-)DRF2725)]. The development history comprises enzyme screening, biocatalyst and process optimization, and scale-up to pilot plant. The project was thereby highly interdisciplinary by combining biotechnology and chemistry technologies. The final process was successfully run on a 44-kg pilot scale in 43-48% yields and with high enantiomeric purities (98.4-99.6% ee).

O. Kirk; M.W. Christensen.

"Lipases from Candida antarctica - unique biocatalysts from a unique origin."

Organic Process Research & Development, 6, 446-451 (2002)

The specificity of the A-lipase from Candida antarctica (CALA) has been characterized to further clarify the scope of the biocatalyst. The lipase was foun d to exhibit an almost uniform activity towards various straight-chained primary alcohols and carboxylic acids, only exhibiting a low activity towards very short-chained acids. More interestingly, the enzyme was found to exhibit a high activity towards a surprising diversity of sterically hindered alcohols, including both secondary and tertiary alcohols. These results indicate that CALA can have a unique applicability for the conversion of highly branched substrates where most other lipases fail to display any activity. A new, potentially highly cost-effective, immobilization technology using silica-based granulation has been applied in the immobilization of the B-lipase from the same yeast (CALB). Highly stable particles were obtained with an activity comparable to that of the commercially available immobilized preparations of this enzyme.

T.L. Husum; C.T. Jørgensen; M.W. Christensen; O. Kirk.

"Enzyme Catalysed Synthesis in Ambient Temperature Ionic Liquids"

Biocatalysis and Biotransformation, 19, 331-338 (2001)

The activity of three different lipases, a glycosidase and a protease in ionic liquids has been studied. Ambient temperature ionic liquids are a new class of solvents that are nonvolatile and nonflammable and thus an interesting alternative to classical organic solvents. Monitoring the synthesis of a simple ester, all lipases were found to exhibit both excellent activity and stability in the non- polar ionic liquid 1-butyl-3-methylimidazolium hexaflurophosphate ([bmin][PF6], 1). Furthermore, beta - galactosidase from E. coli and the Subtilisin protease Savinase(TM) were both found to exhibit a hydrolytic activity in a 50% aqueous solution of the water-miscible ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([bmin][BF4], 2) comparable to the activity observed in 50% aqueous solutions of ethanol and acetonitrile.

E.M. Anderson; K.M. Larsson; O. Kirk.

"One Biocatalyst - Many Applications: The Use of Candida antarctica B-lipase in Organic Synthesis".

Biocatalysis and Biotransformation, 16, 181-204 (1998)

The application of the B-component lipase from the yeast Candida antarctica in organic synthesis is reviewed. This enzyme has been found to be a particularly efficient and robust lipase catalyzing a surprising diversity of reactions including many different regio- and enantio-selective syntheses. Furthermore, the C. antarctica B-lipase is an example of an enzyme for which its specificity has been predicted based on the crystal structure and modeling of the active site region. This prediction is compared to experimental observations and a very close correlation is found.