Jan von Langermann

Uneven twins: comparison of two enantiocomplementary hydroxynitrile lyases with α/β-hydrolase fold.

Hydroxynitrile lyases (HNLs) are applied in technical processes for the synthesis of chiral cyanohydrins. Here we describe the thorough characterization of the recently discovered R-hydroxynitrile lyase from Arabidopsis thaliana and its S-selective counterpart from Manihot esculenta (MeHNL) concerning their properties relevant for technical applications. The results are compared to available data of the structurally related S-HNL from Hevea brasiliensis (HbHNL), which is frequently applied in technical processes. Whereas substrate ranges are highly similar for all three enzymes, the stability of MeHNL with respect to higher temperature and low pH-values is superior to the other HNLs with alpha/beta-hydrolase fold. This enhanced stability is supposed to be due to the ability of MeHNL to form tetramers in solution, while HbHNL and AtHNL are dimers. The different inactivation pathways, deduced by means of circular dichroism, tryptophan fluorescence and static light scattering further support these results. Our data suggest different possibilities to stabilize MeHNL and AtHNL for technical applications: whereas the application of crude cell extracts is appropriate for MeHNL, AtHNL is stabilized by addition of polyols. In addition, the molecular reason for the inhibition of MeHNL and HbHNL by acetate could be elucidated, whereas no such inhibition was observed with AtHNL.

Hydroxynitrile lyase catalyzed cyanohydrin synthesis at high pH-values

The application of unusual high pH-values within enzymatic cyanohydrin synthesis has been investigated. Usually enzymatic cyanohydrin synthesis in two-phase systems requires low pH-values within the aqueous phase to suppress the non-enzymatic side reaction. In contrast, we investigated the usage of pH-values above pH 6 by using the highly enantioselective (S)-selective hydroxynitrile lyase from Manihot esculenta. With these unusual reaction conditions also the unfavorable substrate 3-phenoxy-benzaldehyde can be converted by the wild type enzyme with excellent conversion and enantiomeric excess yielding pure (S)-3-phenoxy-benzaldehyde cyanohydrin with an enantiomeric excess of 97%. Although the variant MeHNL–W128A shows a higher activity with respect to this reaction, the enantioselectivity was reduced (85% e.e.(S)). Additionally, a new continuous spectroscopic cyanohydrin assay monitoring the formation of 3-phenoxy-benzaldehyde cyanohydrin was developed.

Microwave‐assisted covalent immobilization of enzymes on inorganic surfaces

Enzymes on carriers can be easily recycled or used in fixed bed reactors. The immobilization often results in an improved stability. Depending on the support used and the method of coupling, this is a time‐consuming process. While the wide applicability of microwaves (MWs) within organic synthesis is known since the 1980s, proteins (including enzymes) are generally considered as too sensitive toward MW irradiation. In this article, MW methods were investigated to improve the processing speed of covalent enzyme immobilization on inorganic supports. Herein two laccases from Trametes versicolor and Myceliophthora thermophilia (Novozyme 51003®) and the glucose oxidase from Aspergillus niger were immobilized onto samples of ceramic honeycomb and porous glass (TRISOPERL® 1000 AMINO). The enzymes showed different sensitivity to MW irradiation, but all were suitable for MW‐assisted immobilization. Subsequent stability tests were conducted to compare conventional immobilization methods with those with MW irradiation. The glucose oxidase provided the best results. For all cases, a successful MW irradiation assisted covalent enzyme immobilization on solid support was obtained with a total 20‐fold reduction of the time necessary.

The elusive cyanoformate: an unusual cyanide shuttle.

CO2 makes up just 0.04% of the earth\9s atmosphere and is essential for all life on earth. Nevertheless, it is the major anthropogenic greenhouse gas. Consequently, significant scientific effort has been devoted to establishing CO2 as a cheap and readily available carbon feedstock. The electrophilic nature of CO2 is exploited in its catalytic reduction to formates by either enzymes, well-defined metal complexes, or metal oxides. Nanoporous materials such as amine-grafted metal– organic frameworks (MOFs) can reversibly bind CO2 and thus allow regeneration of the sorbent. It comes as a surprise that CO2 complexes with small nucleophilic anions, such as halides or pseudo-halides (e.g. OH , CN , OCN , SCN , and N3 ) have not been thoroughly studied to date. The only wellcharacterized halide/pseudo-halide CO2 species are hydrogen carbonate (old: bicarbonate) [HO-CO2] and fluorocarbonate [FCO2] . In this context, a recent report by Clybourne, Tuononen and co-workers on the preparation of the elusive cyanoformate anion warrants particular attention (Scheme 1). The synthesis of [NC-CO2] was realized by exposing a concentrated [PPh4][CN] solution in CH3CN to an atmosphere of CO2. [PPh4][NC-CO2] precipitates as a colorless crystalline solid and its formation was confirmed by combined crystallographic, spectroscopic, and computational methods. As expected for a Lewis acid–base adduct, the CCN–CCO2 distance is 1.480(9) , whereas the C–N distance with 1.06(1) is relatively short, in the typical range of a triple bond. This view is supported by computational studies revealing the C C bond to be predominantly a s-type donor–acceptor interaction and hence, cyanide acts as a two-electron donor to CO2. In contrast to CO2, ketones are known to interact readily with cyanides to form cyanohydrins; this was reported first in Strecker s pioneering work in 1850. However, the reaction of CO2 with CN is considerably less favorable for several reasons: 1) Distorting the linear CO2 molecule is energetically not favorable (310 kJmol ); 2) the p bonds in CO2 are considerably stronger than p bonds at sp-hybridized carbons; 3) the poor overall entropy is not compensated by the small enthalpic preference for cyanoformate formation. This situation is nicely reflected by the relatively large O-C-O angle of 1338, which indicates that CO2 wants to escape this unfavorable situation. The stability of [NC-CO2] increases with decreasing solvent polarity (toluene). Thus, fragmentation into CO2 and CN is favored in polar reaction media. However, kinetics protects the anion from immediate breakdown as C C bond scission has a relatively high activation barrier of ca. 40 kJ mol . The authors carried out decomposition studies utilizing in situ generated [Bu4N][NCCO2] in an ionic liquid. This mixture was added to anhydrous toluene, tetrahydrofuran, and acetonitrile, respectively, to monitor the decay of the nas(CO2) vibration in [NC-CO2] and determine the half-lives (toluene: 110 min; acetonitrile: 17 min). Traces of water led to immediate decomposition, as CN is an excellent nucleophile and hydrogen carbonate is formed. Besides hydrogen carbonate (A), fluorocarbonate is the only example of a stable halide/pseudo-halide adduct of CO2 (Figure 1). Seppelt et al. reported the facile synthesis of [FCO2] (B) by exposing anhydrous solutions of ammonium fluoride salts to CO2. [4] These salts are poorly soluble in common solvents and decompose when traces of water are present. The main hurdle for the preparation seems to be the low affinity of CO2 towards F , which amounts to just 133 kJmol . [FCO2] is isoelectronic with [NO3] ; consequently neutral [NC-NO2] (C) is the isoelectronic nitrogen congener of the cyanoformate anion. In a recent report Christe et al. described the challenging synthesis and comprehensive characterization of nitryl cyanide from NO2BF4 and tBuMe2SiCN in nitromethane at 30 8C. Fractional condensation yielded pure [NC-NO2] which melted at 85 8C (boiling point 7 8C) and complete decomposition was obScheme 1. Synthesis of the labile cyanoformate according to Clyburne et al. (top) and depiction of its breakdown into CO2 and CN depending on the polarity of the reaction medium (bottom).

Synthesis of Aliphatic and α-Halogenated Ketone Cyanohydrins with the Hydroxynitrile Lyase from Manihot esculenta

The potential of the hydroxynitrile lyase from Manihot esculenta towards ketone substrates was investigated. It was observed that the length of the aliphatic chain is a key parameter for the conversion of aliphatic, non‐branched ketones. Smaller substrates are readily converted with high enantioselectivites, but the elongation of the chain length causes a significant loss in enzyme activity. For a number of halogenated, herein especially fluorinated, acetophenone derivatives the corresponding cyanohydrins have been synthesized with good to moderate enantioselectivities.

Preparation and Characterization of Enzyme Compartments in UV-Cured Polyurethane-Based Materials and Their Application in Enzymatic Reactions

The preparation and characterization of UV-cured polyurethane-based materials for the mild inclusion immobilization of enzymes was investigated. Full curing of the polymer precursor/enzyme solution mixture was realized by a short irradiation with UV-light at ambient temperatures. The included aqueous enzyme solution remains highly dispersed in the polymer material with an even size distribution throughout the polymer material. The presented concept provides stable enzyme compartments which were applied for an alcohol dehydrogenase-catalyzed reduction reaction in organic solvents. Cofactor regeneration was achieved by a substrate-coupled approach via 2-propanol or an enzyme-coupled approach by a glucose dehydrogenase. This reaction concept can also be used for a simultaneous application of contrary biocatalytic reaction conditions within an enzymatic cascade reaction. Independent polymer-based reaction compartments were provided for two incompatible enzymatic reaction systems (alcohol dehydrogenase and hydroxynitrile lyase), while the relevant reactants diffuse between the applied compartments.

Recent developments in biocatalysis in multiphasic ionic liquid reaction systems

Ionic liquids are well known and frequently used ‘designer solvents’ for biocatalytic reactions. This review highlights recent achievements in the field of multiphasic ionic liquid-based reaction concepts. It covers classical biphasic systems including supported ionic liquid phases, thermo-regulated multi-component solvent systems (TMS) and polymerized ionic liquids. These powerful concepts combine unique reaction conditions with a high potential for future applications on a laboratory and industrial scale. The presence of a multiphasic system simplifies downstream processing due to the distribution of the catalyst and reactants in different phases.