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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. Activity screening of a focused library in which these two hotspots were varied led to the discovery of a 4‐OT variant (M45Y/F50V) with strongly enhanced aldolase activity in the self‐condensation of linear aliphatic aldehydes, such as acetaldehyde, propanal, and butanal, to yield α,β‐unsaturated aldehydes. With both propanal and benzaldehyde, this double mutant, unlike the previously constructed single mutant F50A, mainly catalyzes the self‐condensation of propanal rather than the cross‐condensation of propanal and benzaldehyde, thus indicating that it indeed has altered substrate specificity. This variant could serve as a template to create new biocatalysts that lack dehydration activity and possess further enhanced aldolase activity, thus enabling the efficient enzymatic self‐coupling of aliphatic aldehydes.

The enzyme 4-oxalocrotonate tautomerase (4-OT) promiscuously catalyzes the Michael-type addition of acetaldehyde to β-nitrostyrene derivatives to yield chiral γ-nitroaldehydes, which are important precursors for pharmaceutically active γ-aminobutyric acids. In this study, we investigated the effect of different substituents at the aromatic ring of the Michael acceptor on the catalytic efficiency and stereoselectivity of the 4-OT-catalyzed acetaldehyde addition reactions. Highly enantioenriched (R)- and (S)-γ-nitroaldehydes and 4-substituted chroman-2-ol could be obtained in good to excellent yields by applying different substituents at appropriate positions of the aromatic substrate. Stereochemical control of these enzymatic Michael-type additions by “substrate engineering” allowed the enantioselective synthesis of valuable γ-aminobutyric acid precursors. In addition, the results suggest a novel enzymatic synthesis route towards precursors for chromans and derivatives, which are valuable scaffolds for preparing biologically active natural products.

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. The hotspots were identified by systematic mutagenesis (covering each residue), followed by a screen for single mutations that give a strong improvement in the desired aldolase activity. All beneficial mutations were near the active site of 4-OT, thus underpinning the notion that new catalytic activities of a promiscuous enzyme are more effectively enhanced by mutations close to the active site.

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. Here we present mutability landscapes for the expression, tautomerase and Michael-type addition activities, and enantioselectivity of 4-oxalocrotonate tautomerase. These maps of neutral, beneficial and detrimental amino acids for each residue position and enzyme property provide detailed insight into sequence–function relationships. This offers exciting opportunities for enzyme engineering, which is illustrated by the redesign of 4-oxalocrotonate tautomerase into two enantiocomplementary ‘Michaelases'. These ‘Michaelases' catalyse the asymmetric addition of acetaldehyde to various nitroolefins, providing access to both enantiomers of γ-nitroaldehydes, which are important precursors for pharmaceutically active γ-aminobutyric acid derivatives., The Michael-type addition reaction is used for carbon-carbon bond formation; however biocatalytic methods for this reaction are rare. Here, the authors generate and exploit mutability landscapes of 4-oxalocrotonate tautomerase to direct the redesign of this promiscuous enzyme into enantio-complementary Michaelases.

Numerous enzymes have been found to catalyze additional and completely different types of reactions relative to the natural activity they evolved for. This phenomenon, called catalytic promiscuity, has proven to be a fruitful guide for the development of novel biocatalysts for organic synthesis purposes. As such, enzymes have been identified with promiscuous catalytic activity for, one or more, eminent types of carbon-carbon bond-forming reactions like aldol couplings, Michael(-type) additions, Mannich reactions, Henry reactions, and Knoevenagel condensations. This review focuses on enzymes that promiscuously catalyze these reaction types and exhibit high enantioselectivities (in case chiral products are obtained).

The enzyme 4-oxalocrotonate tautomerase (4-OT), which has a catalytic N-terminal proline residue (Pro1), can promiscuously catalyze various carbon-carbon bond-forming reactions, including aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde, and Michael-type addition of acetaldehyde to a wide variety of nitroalkenes to yield valuable γ-nitroaldehydes. To gain insight into how 4-OT catalyzes these unnatural reactions, we carried out exchange studies in D2 O, and X-ray crystallography studies. The former established that H-D exchange within acetaldehyde is catalyzed by 4-OT and that the Pro1 residue is crucial for this activity. The latter showed that Pro1 of 4-OT had reacted with acetaldehyde to give an enamine species. These results provide evidence of the mechanism of the 4-OT-catalyzed aldol and Michael-type addition reactions in which acetaldehyde is activated for nucleophilic addition by Pro1-dependent formation of an enamine intermediate.

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. This catalytic promiscuity of 4-OT could be exploited as starting point to create tailor-made, artificial aldolases for challenging self- and cross-aldolizations.

Pantetheine, a low molecular weight thiol, has been found to ameliorate symptoms in various disease models but specifically in Pantothenate Kinase-Associated Neurodegeneration (PKAN). Pantetheine is usually administered in its disulfide form (i.e. pantethine) since pantethine is commercially available and is reduced to pantetheine in biological systems. The applicability and efficacy of pantethine (therefore also pantetheine) as a clinical therapeutic however is hampered since both forms can be degraded by pantetheinases present in the body. Here, we report the synthesis of a masked form of pantetheine, namely 4-thiobutyltriphenylphosphonium-pantetheine (TBTP-pantetheine), following our hypothesis that this pantetheine-derivative might be more stable in the presence of pantetheinases than pantetheine itself. Higher stability would enhance transport into the cytoplasm where TBTP-pantetheine is metabolized into pantetheine which can subsequently execute its medicinal action. We find that TBTP-pantetheine is stable in aqueous solution, however it was found to be less stable in 10% fetal calf serum (which contains pantetheinases) compared to pantethine, the commercially availabile disulfide of pantetheine. We show that TBTP-pantetheine has improved lipophilicity, but equal passive membrane permeability/diffusion, as compared with pantethine.

Catalytic promiscuity and evolution: Many enzymes exhibit catalytic promiscuity--the ability to catalyze reactions other than their biologically relevant one. These reactions can serve as starting points for both natural and laboratory evolution of new enzymatic functions. Recent advances in the study of enzyme promiscuity in the tautomerase superfamily are discussed.

Exploiting catalytic promiscuity: The proline-based enzyme 4-oxalocrotonate tautomerase (4-OT) promiscuously catalyzes asymmetric Michael-type additions of linear aldehydes--ranging from acetaldehyde to octanal--to trans-β-nitrostyrene in aqueous solvent. The presence of 1.4 mol% of 4-OT effected formation of the anticipated γ-nitroaldehydes in fair to good yields with dr values of up to 93:7 and ee values of up to 81 %.

The enzyme 4‐oxalocrotonate tautomerase (4‐OT) promiscuously catalyzes the Michael‐type addition of acetaldehyde to β‐nitrostyrene derivatives to yield chiral γ‐nitroaldehydes, which are important precursors for pharmaceutically active γ‐aminobutyric acids. In this study, we investigated the effect of different substituents at the aromatic ring of the Michael acceptor on the catalytic efficiency and stereoselectivity of the 4‐OT‐catalyzed acetaldehyde addition reactions. Highly enantioenriched (R)‐ and (S)‐γ‐nitroaldehydes and 4‐substituted chroman‐2‐ol could be obtained in good to excellent yields by applying different substituents at appropriate positions of the aromatic substrate. Stereochemical control of these enzymatic Michael‐type additions by “substrate engineering” allowed the enantioselective synthesis of valuable γ‐aminobutyric acid precursors. In addition, the results suggest a novel enzymatic synthesis route towards precursors for chromans and derivatives, which are valuable scaffolds for preparing biologically active natural products.

Serendipitous switch: While exploring cis-nitrostyrene as a potential electrophile in Michael-type addition reactions catalysed by the enzyme 4-oxalocrotonate tautomerase (4-OT), it was unexpectedly found that 4-OT catalyses the isomerisation of cis-nitrostyrene to trans-nitrostyrene (k(cat) /K(m) = 1.9×10(3) M(-1) s(-1) ).

Double play: The enzyme 4-oxalocrotonate tautomerase (4-OT) catalyzes not only the initial cross-coupling of acetaldehyde and benzaldehyde to yield 3-hydroxy-3-phenylpropanal, but also the subsequent dehydration of this aldol compound to yield cinnamaldehyde as the final product. Mechanism-inspired engineering provided an active site mutant (F50A) with strongly enhanced aldol condensation activity.

Although bistability of molecular switches in solution is well established, achieving highly robust bistable molecular switching in self-assembled monolayers remains a challenge. Such systems are highly attractive as components in organic electronics and molecular-based photo and electrochromic devices. Here we report a remarkably robust surface confined bisthiaxanthylidene redox switch that shows excellent bistability, manifested in reversible changes in spectroscopic and electrochemical properties and in.. physical properties such as water contact angle changes (ca. 30 degrees difference in water contact angle between the two redox states of a bisthiaxanthylidene self-assembled monolayer). The effect of surface immobilization of bisthiaxanthylidene on its photochromic, thermal and electrochemical properties is described. Surface immobilization is achieved by incorporating thiol- and alkylsiloxy-terminated "legs" on one of the tricyclic aromatic units. The molecular switch in its neutral and dicationic state, generated by bulk electrolysis, was characterized in solution, in the solid state and on surfaces, by UV/vis absorption, Fourier transform infrared, X-ray photoelectron, and Raman spectroscopies and by cyclic voltammetry. In solution, the redox switching to the dicationic state is achieved by oxidation at 1.2 V versus SCE. Reduction of the dication at

Although chemists have made small-molecule rotary motors, to date there have been no reports of small-molecule linear motors. Here we describe the synthesis and operation of a 21-atom two-legged molecular unit that is able to walk up and down a four-foothold molecular track. High processivity is conferred by designing the track-binding interactions of the two feet to be labile under different sets of conditions such that each foot can act as a temporarily fixed pivot for the other. The walker randomly and processively takes zero or one step along the track using a 'passing-leg' gait each time the environment is switched between acid and base. Replacing the basic step with a redox-mediated, disulfide-exchange reaction directionally transports the bipedal molecules away from the minimum-energy distribution by a Brownian ratchet mechanism. The ultimate goal of such studies is to produce artificial, linear molecular motors that move directionally along polymeric tracks to transport cargoes and perform tasks in a manner reminiscent of biological motor proteins.

We deal with the issue of quantifying and optimizing the rotation dynamics of synthetic molecular motors. For this purpose, the continuous four-stage rotation behavior of a typical light-activated molecular motor was measured in detail. All reaction constants were determined empirically. Next, we developed a Markov model that describes the full motor dynamics mathematically. We derived expressions for a set of characteristic quantities, i.e., the average rate of quarter rotations or “velocity,” V , the spread in the average number of quarter rotations, D , and the dimensionless Péclet number, Pe = V/D . Furthermore, we determined the rate of full, four-step rotations (Ω eff ), from which we derived another dimensionless quantity, the “rotational excess,” r.e . This quantity, defined as the relative difference between total forward (Ω + ) and backward (Ω − ) full rotations, is a good measure of the unidirectionality of the rotation process. Our model provides a pragmatic tool to optimize motor performance. We demonstrate this by calculating V , D , Pe , Ω eff , and r.e. for different rates of thermal versus photochemical energy input. We find that for a given light intensity, an optimal temperature range exists in which the motor exhibits excellent efficiency and unidirectional behavior, above or below which motor performance decreases.

The introduction of dibenzo-cyclohepten-5-ylidene as part of a unidirectional light-driven molecular motor allows a more complete picture of the pathway of thermal helix inversion to be developed. The most stable conformation is similar to that found in related motors in that it has, overall, an anti-folded structure with the substituent at the stereogenic centre adopting an axial orientation. Photochemical cis/trans isomerisation at -40 degrees C results in the formation of an isomer in a syn-folded conformation with the methyl group in an axial orientation. This contrasts with previous studies on related molecular rotary motors. The conformation of the higher energy intermediate typically observed for this class of compound is the anti-folded conformation, in which the methyl group is in an equatorial orientation. This conformation is available through an energetically uphill upper half ring inversion of the observed photochemical product. However, this pathway competes with a second process that leads to the more stable anti-folded conformation in which the methyl group is oriented axially. It has been shown that the conformations and pathways available for second-generation molecular motors can be described by using similar overall geometries. Differences in the metastable high-energy species are attributable to the relative energy and position on the reaction coordinate of the transition states. Kinetic studies on these new molecular motors thus provide important insights into the conformational dynamics of the rotation cycle.

The vast majority of characterized oxygenases use bound cofactors to activate molecular oxygen to carry out oxidation chemistry. Here, we show that an enzyme of unknown activity, RhCC from Rhodococcus jostii RHA1, functions as an oxygenase, using 4-hydroxyphenylenolpyruvate as a substrate. This unique and complex reaction yields 3-hydroxy-3-(4-hydroxyphenyl)-pyruvate, 4-hydroxybenzaldehyde, and oxalic acid as major products. Incubations with H2(18)O, (18)O2, and a substrate analogue suggest that this enzymatic oxygenation reaction likely involves a peroxide anion intermediate. Analysis of sequence similarity and the crystal structure of RhCC (solved at 1.78 Å resolution) reveal that this enzyme belongs to the tautomerase superfamily. Members of this superfamily typically catalyze tautomerization, dehalogenation, or decarboxylation reactions rather than oxygenation reactions. The structure shows the absence of cofactors, establishing RhCC as a rare example of a redox-metal- and coenzyme-free oxygenase. This sets the stage to study the mechanistic details of cofactor-independent oxygen activation in the unusual context of the tautomerase superfamily.

The concept of bisthioxanthylidene biscrown ethers as potential stereodivergent chiral ligands in asymmetric synthesis is introduced. Substituted bisthioxanthylidenes may be chiral and can exist as stable enantiomers due to their folded structure. As a result, both a right-handed helix (P) and left-handed helix (M) are present in this type of molecule. This offers the unique possibility to construct two crown ether moieties, attached to the same molecule, of which one exhibits (P)-helicity and the other (M)-helicity. When the crown ether moieties differ in size they can be complexed selectively with a base containing a cation of appropriate diameter. In this manner the (P)-helix and the (M)-helix can be activated selectively to serve as a chiral environment for base catalyzed asymmetric synthesis. Thus, we envisioned the new concept of a single chiral ligand to separately synthesize two enantiomers of a chiral product just by varying the added base. For this purpose, four new bisthioxanthylidene monocrown ethers and two new bisthioxanthylidene biscrown ethers were synthesized. Two biscrowns and two monocrowns were separated into their respective enantiomers (HPLC) and optical data (UV and CD) were collected to ensure stability of enantiomers at ambient temperatures. Ion complexation of one mono- and two biscrown ethers with potassium and sodium cations was investigated.

Nine new molecular motors, consisting of a 2,3-dihydro-2-methylnaphtho[2,1-b]thiopyran or 2,3-dihydro-3-methylphenanthrene upper part and a (thio)xanthene, 10,10-dimethylanthracene, or dibenzocycloheptene lower part, connected by a central double bond, were synthesized. A single stereogenic center, bearing a methyl substituent, is present in each of the motors. MOPAC93-AM1 calculations, NMR studies, and X-ray analysis revealed that these compounds have stable isomers with pseudoaxial orientation of the methyl substituent and less-stable isomers with pseudoequatorial orientation of the methyl substituent. The photochemical and thermal isomerization processes of the motors were studied by NMR and CD spectroscopy. The new molecular motors all show two cis-trans isomerizations upon irradiation, each followed by a thermal helix inversion, resulting in a 360 degrees rotation around the central double bond of the upper part with respect to the lower part. The direction of rotation is controlled by a single stereogenic center created by the methyl substituent at the upper part. The speed of rotation, governed by the two thermal steps, was adjusted to a great extent by structural modifications, with half-lives for the thermal isomerization steps ranging from t(1/2)(theta) 233-0.67 h. The photochemical conversions of two new motors proceeded with near-perfect photoequilibria of 1:99.

Call me Michaelase: The enzyme 4-oxalocrotonate tautomerase (4-OT) promiscuously catalyzes the Michael-type addition of acetaldehyde to a collection of aromatic and aliphatic nitro-olefins with high stereoselectivity producing precursors of g-aminobutyric acid (GABA) analogues (see scheme).

Optisch aktive sterisch uberfrachtete Alkene wurden unter Verwendung von Bi-β-naphthol als chiralem Templat in einer intramolekularen Kupplungsreaktion synthetisiert. Das Hauptisomer 2 hat eine einzigartige helicale Struktur mit verdrillten und gefalteten Strukturelementen. Die Abspaltung des chiralen Templats lieferte das sterisch uberfrachtete Thioxanthyliden 3 mit 96 % ee, was darauf hindeutet, das keine Racemisierung oder Isomerisierung der Enantiomere stattfand.

Optically active overcrowded alkenes were synthesized by employing bis-beta-naphthol as a chiral template during an intramolecular coupling reaction. The major isomer 2 has a unique helical structure with twisted and folded structural moieties. Removal of the chiral template afforded overcrowded thioxanthylidene 3 with 96 % ee, which indicates that no racemization or isomerization of the enantiomers took place.

Call me Michaelase: The enzyme 4-oxalocrotonate tautomerase (4-OT) promiscuously catalyzes the Michael-type addition of acetaldehyde to a collection of aromatic and aliphatic nitro-olefins with high stereoselectivity producing precursors of g-aminobutyric acid (GABA) analogues (see scheme).

Catalytic promiscuity and evolution: Many enzymes exhibit catalytic promiscuity--the ability to catalyze reactions other than their biologically relevant one. These reactions can serve as starting points for both natural and laboratory evolution of new enzymatic functions. Recent advances in the study of enzyme promiscuity in the tautomerase superfamily are discussed.

Intramolecular nucleophilic additions to oxycarbenium ions derived from 5-alkoxy-2(3)-dihydrofuranones gave substituted cyclopentanes or cyclohexanes in good yields. Stereoselectivity in these reactions was usually very high, while regioselectivity could be improved using silicon containing nucleophiles.

The synthesis and system dynamics of a series of small-molecule walker-track conjugates, 3,4-C(n) (n = 2, 3, 4, 5, and 8), based on dynamic covalent linkages between the "feet" of the walkers and the "footholds" of the track, is described. Each walker has one acyl hydrazide and one sulfur-based foot separated by a spacer chain of "n" methylene groups, while the track consists of four footholds of alternating complementary functionalities (aldehydes and masked thiols). Upon repeatedly switching between acid and base, the walker moiety can be exchanged between the footholds on the track, primarily through a "passing-leg gait" mechanism, until a steady state, minimum energy, distribution is reached. The introduction of a kinetically controlled step in the reaction sequence (redox-mediated breaking and reforming of the disulfide linkages) can cause a directional bias in the distribution of the walker on the track. The different length walker molecules exhibit very different walking behaviors: Systems n = 2 and 3 cannot actually "walk" along the track because their stride lengths are too short to bridge the internal footholds. The walkers with longer spacers (n = 4, 5, and 8) do step up and down the track repeatedly, but a directional bias under the acid-redox conditions is only achieved for the C(4) and C(5) systems, interestingly in opposite directions (the C(8) walker has insufficient ring strain with the track). Although they are extremely rudimentary systems, the C(4) and C(5) walker-track conjugates exhibit four of the essential characteristics of linear molecular motor dynamics: processive, directional, repetitive, and progressive migration of a molecular unit up and down a molecular track.

The synthesis and characterisation, including solid state structure, of a macrocycle containing both a hydrazone and a disulfide linkage is described. Selective ring-opening of the macrocycle under thermodynamic control could be achieved at either the disulfide or the hydrazone linkage by applying mutually exclusive sets of reaction conditions.

Overcrowded alkenes are a fascinating class of inherent dissymmetric molecules that attract considerable interest for instance as chiroptical molecular switches and unidirectionally rotary motors. A practical synthesis route towards enantiomerically pure overcrowded alkenes is an important goal. We report here the development of an asymmetric synthesis of bi(thio)xanthylidenes. The upper and lower part of the desired alkenes were first coupled to a chiral template. Intramolecular coupling with partial stereocontrol, separation of diastereoisomers and subsequent removal of the templates gave stable enantiomers of bithioxanthylidene. The absolute configuration was determined by X-ray analysis. In the case of bixanthylidene, stereocontrol during the intramolecular coupling reaction was complete, and it was found that the presence of a binaphthol bridging unit prevents the fast racemization process of the bixanthylidene, which it normally exhibited at ambient temperature. (c) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006.

Surprisingly, a new motor with a tetrahydronaphthalene upper part rotates slower than the original molecular motor with a tetrahydrophenanthrene upper part despite decreased steric hindrance.