Quantitative 2D 1H, 13C HSQC NMR Spectroscopy for the Determination of Chondroitin Sulfate and Dermatan Sulfate Content in Danaparoid Sodium.

Objective:  Danaparoid sodium is a biopolymeric complex drug composed of the most abundant heparan sulfate (HS) followed in descending order by dermatan sulfate (DS) and chondroitin sulfate (CS). This composite nature explains its peculiar antithrombotic and anticoagulant properties and make it particularly advantageous when the risk of heparin-induced thrombocytopenia occurs. A specific control of the danaparoid composition is required by the Ph.

Enantioselective Synthesis of Pharmaceutically Active γ-Aminobutyric Acids Using a Tailor-Made Artificial Michaelase in One-Pot Cascade Reactions.

Chiral γ-aminobutyric acid (GABA) analogues represent abundantly prescribed drugs, which are broadly applied as anticonvulsants, as antidepressants, and for the treatment of neuropathic pain. Here we report a one-pot two-step biocatalytic cascade route for synthesis of the pharmaceutically relevant enantiomers of γ-nitrobutyric acids, starting from simple precursors (acetaldehyde and nitroalkenes), using a tailor-made highly enantioselective artificial "Michaelase" (4-oxalocrotonate tautomerase mutant L8Y/M45Y/F50A), an aldehyde dehydrogenase with a broad non-natural substrate scope, and a cofactor recycling system. We also report a three-step chemoenzymatic cascade route for the efficient chemical reduction of enzymatically prepared γ-nitrobutyric acids into GABA analogues in one pot, achieving high enantiopurity (e.

From Farm to Pharma: An Overview of Industrial Heparin Manufacturing Methods.

The purification of heparin from offal is an old industrial process for which commercial recipes date back to 1922. Although chemical, chemoenzymatic, and biotechnological alternatives for this production method have been published in the academic literature, animal-tissue is still the sole source for commercial heparin production in industry. Heparin purification methods are closely guarded industrial secrets which are not available to the general (scientific) public.

Engineering a Promiscuous Tautomerase into a More Efficient Aldolase for Self-Condensations of Linear Aliphatic Aldehydes.

The enzyme 4-oxalocrotonate tautomerase (4-OT) from Pseudomonas putida mt-2 takes part in a catabolic pathway for aromatic hydrocarbons, where it catalyzes the conversion of 2hydroxyhexa-2,4-dienedioate into 2-oxohexa-3-enedioate. This tautomerase can also promiscuously catalyze carbon-carbon bond-forming reactions, including various types of aldol reactions, by using its amino-terminal proline as a key catalytic residue. Here, we used systematic mutagenesis to identify two hotspots in 4-OT (Met45 and Phe50) at which single mutations give marked improvements in aldolase activity for the self-condensation of propanal.

Inter- and intramolecular aldol reactions promiscuously catalyzed by a proline-based tautomerase.

The enzyme 4-oxalocrotonate tautomerase (4-OT), which in nature catalyzes a tautomerization step as part of a catabolic pathway for aromatic hydrocarbons, was found to promiscuously catalyze different types of aldol reactions. These include the self-condensation of propanal, the cross-coupling of propanal and benzaldehyde, the cross-coupling of propanal and pyruvate, and the intramolecular cyclizations of hexanedial and heptanedial. Mutation of the catalytic amino-terminal proline (P1A) greatly reduces 4-OT's aldolase activities, whereas mutation of another active site residue (F50A) strongly enhances 4-OT's aldolase activities, indicating that aldolization is an active site process.

The Generation and Exploitation of Protein Mutability Landscapes for Enzyme Engineering.

The increasing number of enzyme applications in chemical synthesis calls for new engineering methods to develop the biocatalysts of the future. An interesting concept in enzyme engineering is the generation of large-scale mutational data in order to chart protein mutability landscapes. These landscapes allow the important discrimination between beneficial mutations and those that are neutral or detrimental, thus providing detailed insight into sequence-function relationships.

Mutations Closer to the Active Site Improve the Promiscuous Aldolase Activity of 4-Oxalocrotonate Tautomerase More Effectively than Distant Mutations.

The enzyme 4-oxalocrotonate tautomerase (4-OT), which catalyzes enol-keto tautomerization as part of a degradative pathway for aromatic hydrocarbons, promiscuously catalyzes various carbon-carbon bond-forming reactions. These include the aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde. Here, we demonstrate that 4-OT can be engineered into a more efficient aldolase for this condensation reaction, with a >5000-fold improvement in catalytic efficiency (kcat /Km ) and a >10(7) -fold change in reaction specificity, by exploring small libraries in which only "hotspots" are varied.

Using mutability landscapes of a promiscuous tautomerase to guide the engineering of enantioselective Michaelases.

The Michael-type addition reaction is widely used in organic synthesis for carbon-carbon bond formation. However, biocatalytic methodologies for this type of reaction are scarce, which is related to the fact that enzymes naturally catalysing carbon-carbon bond-forming Michael-type additions are rare. A promising template to develop new biocatalysts for carbon-carbon bond formation is the enzyme 4-oxalocrotonate tautomerase, which exhibits promiscuous Michael-type addition activity.

Aqueous oxidative Heck reaction as a protein-labeling strategy.

An increasing number of chemical reactions are being employed for bio-orthogonal ligation of detection labels to protein-bound functional groups. Several of these strategies, however, are limited in their application to pure proteins and are ineffective in complex biological samples such as cell lysates. Here we present the palladium-catalyzed oxidative Heck reaction as a new and robust bio-orthogonal strategy for linking functionalized arylboronic acids to protein-bound alkenes in high yields and with excellent chemoselectivity even in the presence of complex protein mixtures from living cells.

The isoprenoid-precursor dependence of Plasmodium spp.

Due to the increase in resistance of Plasmodium spp. against available antimalarials, there is a need for new, effective and innovative drugs. The non-mevalonate pathway for the biosynthesis of the universal isoprenoid precursors, which is absent in humans, is suggested as an attractive source of targets for such drugs with a novel mode of action.