Podophyllotoxin is probably the most prominent representative of lignan natural products. Deoxy‐, epi‐, and podophyllotoxin, which are all precursors to frequently used chemotherapeutic agents, were ...prepared by a stereodivergent biotransformation and a biocatalytic kinetic resolution of the corresponding dibenzylbutyrolactones with the same 2‐oxoglutarate‐dependent dioxygenase. The reaction can be conducted on 2 g scale, and the enzyme allows tailoring of the initial, “natural” structure and thus transforms various non‐natural derivatives. Depending on the substitution pattern, the enzyme performs an oxidative C−C bond formation by C−H activation or hydroxylation at the benzylic position prone to ring closure.
Taming iron: Podophyllotoxin and congeners were prepared by chemoenzymatic total synthesis employing the 2‐oxoglutarate‐dependent iron enzyme from Podophyllum hexandrum. The reaction outcome was changed from an enantiodivergent to a kinetic resolution process by substrate engineering. Modifications of the substrate allowed the kinetic resolution of dibenzylbutyrolactones.
Chemoenzymatic and enzymatic cascade reactions enable the synthesis of complex stereocomplementary 1,3,4‐trisubstituted tetrahydroisoquinolines (THIQs) with three chiral centers in a step‐efficient ...and selective manner without intermediate purification. The cascade employs inexpensive substrates (3‐hydroxybenzaldehyde and pyruvate), and involves a carboligation step, a subsequent transamination, and finally a Pictet–Spengler reaction with a carbonyl cosubstrate. Appropriate selection of the carboligase and transaminase enzymes enabled the biocatalytic formation of (1R,2S)‐metaraminol. Subsequent cyclization catalyzed either enzymatically by a norcoclaurine synthase or chemically by phosphate resulted in opposite stereoselectivities in the products at the C1 position, thus providing access to both orientations of the THIQ C1 substituent. This highlights the importance of selecting from both chemo‐ and biocatalysts for optimal results.
Diverging possibilities: Tetrahydroisoquinolines can be produced by enzymatic and chemoenzymatic three‐step cascade reactions with a combination of carboligases, transaminases, and norcoclaurine synthases (NCSs) or, alternatively, phosphate as the catalyst. With NCS and phosphate, products with opposite stereoselectivity are obtained.
(S)-Strictosidine represents the first key intermediate in the biosynthesis of several pharmaceutically relevant monoterpenoid indole alkaloids. Optically pure C3-methyl-substituted strictosidine ...derivatives were prepared by setting up the two stereogenic centers at the β-carboline core via two enzymatic steps catalyzed by the enzymes transaminase and strictosidine synthase in a one-pot cascade fashion. The two enzymatic steps were performed simultaneously as well as in a stepwise fashion. The amination of the prochiral ketones led to optically pure amines with up to >98% enantiomeric excess. Depending on the enzyme used, the (S)- and (R)-enantiomers were prepared in most cases. Selected amines were then condensed with secologanin in a Pictet–Spengler reaction catalyzed by strictosidine synthase leading to diastereomerically pure products (>98% diastereomeric excess).
The Pictet–Spengler reaction is a valuable route to 1,2,3,4-tetrahydro-β-carboline (THBC) and isoquinoline scaffolds found in many important pharmaceuticals. Strictosidine synthase (STR) catalyzes ...the Pictet–Spengler condensation of tryptamine and the aldehyde secologanin to give (S)-strictosidine as a key intermediate in indole alkaloid biosynthesis. STRs also accept short-chain aliphatic aldehydes to give enantioenriched alkaloid products with up to 99% ee STRs are thus valuable asymmetric organocatalysts for applications in organic synthesis. The STR catalysis of reactions of small aldehydes gives an unexpected switch in stereopreference, leading to formation of the (R)-products. Here we report a rationale for the formation of the (R)-configured products by the STR enzyme from Ophiorrhiza pumila (OpSTR) using a combination of X-ray crystallography, mutational, and molecular dynamics (MD) studies. We discovered that short-chain aldehydes bind in an inverted fashion compared to secologanin leading to the inverted stereopreference for the observed (R)-product in those cases. The study demonstrates that the same catalyst can have two different productive binding modes for one substrate but give different absolute configuration of the products by binding the aldehyde substrate differently. These results will guide future engineering of STRs and related enzymes for biocatalytic applications.
Racemization in combination with a kinetic resolution is the base for a dynamic kinetic resolution (DKR). Biocatalytic racemization was successfully performed for a broad scope of sec‐alcohols by ...employing a single alcohol dehydrogenase (ADH) variant from Thermoanaerobacter pseudoethanolicus (formerly T. ethanolicus; TeSADH W110A I86A C295A). The catalyst employed as a lyophilized whole cell preparation or cell free extract, which tolerated various non‐water miscible organic solvents under micro‐aqueous or two‐phase conditions, whereby cyclohexane and n‐hexane suited best. Various concepts for combining the enzymatic racemization with an enzymatic kinetic resolution to achieve overall a bis‐enzymatic DKR were evaluated. A proof of concept showed a successful DKR with racemization in aqueous phase combined with acylation in the organic phase.
Racemization—Yes it can: Investigation of the substrate scope of a thermophilic alcohol dehydrogenase variant demonstrated its applicability for racemization of sec‐alcohols. The racemization under various conditions (buffer, micro aqueous, biphasic) was evaluated with the aim of getting closer to the efficient bis‐enzymatic dynamic kinetic resolution of sec‐alcohols.
Driving the machinery: A biocatalytic redox‐neutral cascade for the preparation of terminal primary amines from primary alcohols at the expense of ammonia has been established in a one‐pot one‐step ...method (see picture). Applying this artificial biocatalyst network, long‐chain 1,ω‐alkanediols were converted into diamines, which are building blocks for polymers, in up to 99 % conversion.
The nonribosomal enterotoxin tilivalline was the first naturally occurring pyrrolobenzodiazepine to be linked to disease in the human intestine. Since the producing organism Klebsiella oxytoca is ...part of the intestinal microbiota and the pyrrolobenzodiazepine causes the pathogenesis of colitis it is important to understand the biosynthesis and regulation of tilivalline activity. Here we report the biosynthesis of tilivalline and show that this nonribosomal peptide assembly pathway initially generates tilimycin, a simple pyrrolobenzodiazepine with cytotoxic properties. Tilivalline results from the non‐enzymatic spontaneous reaction of tilimycin with biogenetically generated indole. Through a chemical total synthesis of tilimycin we could corroborate the predictions made about the biosynthesis. Production of two cytotoxic pyrrolobenzodiazepines with distinct functionalities by human gut resident Klebsiella oxytoca has important implications for intestinal disease.
The Klebsiella oxytoca enterotoxin tilivalline was the first naturally occurring pyrrolobenzodiazepine to be linked to disease in the human intestine. In the biosynthesis of tilivalline the nonribosomal peptide assembly pathway initially generates the cytotoxic pyrrolobenzodiazepine tilimycin. Tilivalline results from the non‐enzymatic reaction of tilimycin with biogenetically generated indole.
Abstract As water miscible organic co-solvents are often required for enzyme reactions to improve e.g., the solubility of the substrate in the aqueous medium, an enzyme is required which displays ...high stability in the presence of this co-solvent. Consequently, it is of utmost importance to identify the most suitable enzyme or the appropriate reaction conditions. Until now, the melting temperature is used in general as a measure for stability of enzymes. The experiments here show, that the melting temperature does not correlate to the activity observed in the presence of the solvent. As an alternative parameter, the concentration of the co-solvent at the point of 50% protein unfolding at a specific temperature T in short $${c}_{{U}_{50}}^{T}$$ c U 50 T is introduced. Analyzing a set of ene reductases, $${c}_{{U}_{50}}^{T}$$ c U 50 T is shown to indicate the concentration of the co-solvent where also the activity of the enzyme drops fastest. Comparing possible rankings of enzymes according to melting temperature and $${c}_{{U}_{50}}^{T}$$ c U 50 T reveals a clearly diverging outcome also depending on the specific solvent used. Additionally, plots of $${c}_{{U}_{50}}$$ c U 50 versus temperature enable a fast identification of possible reaction windows to deduce tolerated solvent concentrations and temperature.
To improve the efficiency and applicability of biocatalytic redox-reactions for asymmetric ketone-reduction and enantioselective alcohol-oxidation catalyzed by nicotinamide-dependent ...dehydrogenases/reductases, several achievements for cofactor-recycling have been made during the last two years. First, the use of hydrogenases for NADPH recycling in a two enzyme system. Second, preparative transformations with alcohol dehydrogenases coupled with NADH oxidases for NAD+/NADP+ recycling. Third, an exceptional chemo-stable alcohol dehydrogenase can efficiently use i-propanol and acetone as cosubstrates for reduction and oxidation, respectively, in a single-enzyme system. Novel carbonyl reductases and dehydrogenases derived from plant cells are particularly suited for sterically demanding substrates.
A lipase from
Thermomyces lanuginosus and cutinases from
Thermobifida fusca and
Fusarium solani hydrolysed poly(ethylene terephthalate) (PET) fabrics and films and bis(benzoyloxyethyl) terephthalate ...(3PET) endo-wise as shown by MALDI-Tof-MS, LC–UVD/MS, cationic dyeing and XPS analysis. Due to interfacial activation of the lipase in the presence of Triton X-100, a seven-fold increase of hydrolysis products released from 3PET was measured. In the presence of the plasticizer
N,
N-diethyl-2-phenylacetamide (DEPA), increased hydrolysis rates of semi-crystalline PET films and fabrics were measured both for lipase and cutinase. The formation of novel polar groups resulted in enhanced dye ability with additional increase in colour depth by 130% and 300% for cutinase and lipase, respectively, in the presence of plasticizer.
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