Romas J. Kazlauskas

Quantitative screening of hydrolase libraries using pH indicators: Identifying active and enantioselective hydrolases

Rapid and quantitative screening in 96-well microplates can identify active and enantioselective hydrolases. Hydrolysis of esters releases a proton, which can be detected with pH indicators by colorimetry (figure). Using pure enantiomers, we measured the initial rates of enzyme-catalyzed hydrolysis. The relative initial rate approximates the enantioselectivity. This screening greatly speeds up selection of the best hydrolase for a synthesis.

Elucidating structure-mechanism relationships in lipases: Prospects for predicting and engineering catalytic properties

Organic chemists use lipases as catalysts in the synthesis of enantiomerically pure intermediates, to modify triglycerides, and to deprotect synthetic intermediates under mild conditions. They discovered most of these uses empirically, but the recent determination of the X-ray crystal structures of transition-state analogs bound to lipases may change this approach. These structures identified distinct binding regions for the acyl and alcohol portions of esters and suggested molecular-level explanations for the known enantiopreferences of lipases. In future, these structures may enable biotechnologists to design new substrates and reactions using molecular modeling, as well as to modify the activity and selectivity of lipases using site-directed mutagenesis.

Molecular modeling and biocatalysis: explanations, predictions, limitations, and opportunities

Rapid advances in structural biology have revealed the three-dimensional structures of many biocatalysts. Molecular modeling is the tool that links these structures with experimental observations. As a qualitative tool, current modeling methods are extremely useful. They can explain, on a molecular level, unusual features of reactions. They can predict how to increase the selectivity either by substrate modification or by site-directed mutagenesis. Quantitative predictions, for example the degree of enantioselectivity, are still not reliable, however. Modeling is limited also by the availability of three-dimensional structures. Most current modeling involves hydrolases, especially proteases and lipases, but structures for other types of enzymes are starting to appear.

Magnetic separations in biotechnology

Abstract Magnetic separation techniques provide probably the most rapid and convenient method of separating certain particles from dilute suspensions, especially those that might block columns or filters. This and other applications of magnetism, including cell sorting and product recovery are discussed.

Enantioselectivity of Candida Rugosa Lipase Toward Carboxylic Acids: A Predictive Rule from Substrate Mapping and X-Ray Crystallography

We used substrate mapping to develop a rule that predicts which enantiomer of chiral carboxylic acid esters reacts faster in hydrolyses catalyzed by lipase from Candida rugosa (CRL, triacylglycerol hydrolase, E. C. 3.1.1.3). This rule, based on the size of the substituents at the stereocenter, is not reliable for crude CRL. It predicts the favoured enantiomer for only 23 out of 34 examples, 68% reliability. However, this rule is completely reliable for purified CRL; it predicts the favoured enantiomer for all 16 examples correctly. The examples include arylpropanoicacids, aryloxypropanoic acids, α-halophenylacetic acids, mandelic acid and O-methylmandelic acid. Further, purified CRL did not catalyse the hydrolysis of N-CBZ-phenylalanine methyl ester and N-CBZ-norleucine methyl ester. These two substrates were exceptions to the rule with crude CRL as the catalyst. Besides eliminating several exceptions, purification also raised the enantioselectivity of CRL toward carboxylic acid esters. To provide a struc...

A structure-based rationalization of the enantiopreference of subtilisin toward secondary alcohols and isosteric primary amines

Abstract Lipases favor one enantiomer of secondary alcohols (HOCHRR′) and isosteric primary amines (NH 2 CHRR′) while subtilisin favors the other enantiomer. In both cases, simple rules based on the size of the substituents at the stereocenter predict which enantiomer reacts faster. Thus, lipases and subtilisin are a pair of complementary enantioselective reagents for organic synthesis. The success of these rules suggests that these hydrolases distinguish between enantiomer primarily by the size of the substituents. Previously, we proposed a molecular mechanism for the enantiopreference of lipases based on the X-ray crystal structure of transition state analogs bound to a lipase. Here we suggest that a similar mechanism can also account for the opposite enantiopreference of subtilisin. The catalytic machinery (catalytic triad plus the oxyanion-stabilizing residues) in lipases is approximately the mirror image of that in subtilisin. In both hydrolases, the protein fold, as it assembles the catalytic machinery, also creates a restricted pocket for one substituent in the substrate (‘M’ or medium-sized). However, the catalytic His residue lies on opposite sides of this pocket in the two hydrolases. We propose that enantioselection arises from (1) the limited size of this pocket, (2) and a required hydrogen bond between the catalytic His and the oxygen or nitrogen of the alcohol or amine. This mechanism for enantioselection differs from that proposed by Derewenda and Wei who focussed on which carbonyl face in the ester or amide is attacked. Lipases and subtilisin indeed attack opposite faces, but we propose that this difference does not set the enantiopreference toward secondary alcohols.

Substrate modification to increase the enantioselectivity of hydrolases. A route to optically-active cyclic allylic alcohols.

Abstract The esterase-catalyzed resolution of the cyclic allylic acetates-1-acetyloxy-2-cyclopentene, 1-acetyloxy-2-cyclohexene, and 1-acetyloxy-2-cycloheptene - was not enantioseleclive. We hypothesized that this inefficiency stems from the similarity in size of the substituents at the stereocenter (CH 2 CH 2 vs. CHCH). To increase the enantioselectivity, we resolved precursors to these cyclic allylic alcohols: esters of trans -2-bromocycloalkanols (C 5 , C 6 , C 7 ). These esters had a larger difference in the size of the substituents (CH 2 vs. CHBr) at the stereocenter and were efficiently resolved by both cholesterol esterase and lipase from Pseudomonas cepacia (Amano P, PCL). A synthetic-scale resolution with PCL yielded the (1S,2S)-1-butanoyloxy-2-bromocycloalkanes in >98% ee. Heating with DBU to eliminate HBr, followed by reduction with LiAlH 4 to cleave the ester, yielded the allylic alcohols: (S)-(−)-2-cyclopenten-1-ol (65% ee), (S)-(−)-2-cyclohexen-1-ol (>99% ee), and (S)-(−)-2-cyclohepten-1-ol (>98% ee).

`Watching' lipase-catalyzed acylations using 1H NMR: competing hydrolysis of vinyl acetate in dry organic solvents

Abstract Lipase-catalyzed acetylations of 1-phenylethanol with vinyl acetate were monitored in situ by 1 H NMR spectroscopy. Surprisingly, even under dry conditions (no added water) the major reaction was hydrolysis of the vinyl acetate, not acetylation of the substrate. Because this competing hydrolysis consumes water and releases acetic acid, the reaction conditions in lipase-catalyzed acylations are not constant, but vary with the reaction time. Addition of a chiral shift reagent reveals the enantiomeric purity of the starting alcohol and allows calculation of the enantiomeric ratio, E , for the reaction.

Sequential kinetic resolution of (±)-2,3-butanediol in organic solvent using lipase from Pseudomonas cepacia.

Abstract Lipase from Pseudomonas cepocia (PCL, Amano FS) catalyzed the enantioselective diacetylation of (±)-23-butanediol in vinyl acetate. Both acetylation steps favored the (R)-enantiomer (E 1 = 12, E 2 = 34), thus the reaction is a sequential kinetic resolution. The enantioselectivities of the two steps reinforced one another because both steps proceeded at comparable rates (S = 3) yielding an overall enantioselectivity of approximately 200. A synthetic-scale resolution starting from 2.7 g of (±)-2,3-butanediol yielded the diacetate ester of (R)-(−)-butanediol with 96% ee (1.6 g, 30% yield) and (S)-(+)-butanediol with 99% ee (0.63 g, 23% yield). This preparation is carried out entirely in organic solvent, thereby avoiding the difficult and low yield extraction of 2,3-butanediol from aqueous aolution.

Photogeneration of reactive organometallic species

Abstract : A large number of thermally inert organometallic complexes can be photoactivated for stoichiometric and catalytic reactions using visible or ultraviolet light. In favorable cases, low temperature irradiation leads to the generation of spectroscopically detectable intermediates, e.g. (eta 5-C5H5)W(CO)2C2H5 or Fe(CO)eta(alkene)5-eta, that likely participate in catalytic cycles at higher temperatures where the steady state concentration is too low to detect. The dissociative process of CO or H2 loss and metal-metal bond rupture can lead to the coordinatively unsaturated intermediates that pervade homogeneous and heterogeneous catalytic systems. In some cases, the same catalyst as generated thermally at high temperature can be prepared photochemically at low temperature using the same precursor, as in the case of H4Ru4(CO)12 where loss of CO thermally or photochemically produces an active species. In other situations different catalysis results, as reflected in product ratios, when photo-excitation is used compared to thermal activation as in hydrogenation vs. isomerization using H2Os3(CO10. New spectroscopic techniques such as Fourier transform infrared photoacoustic spectroscopy will become important in the in situ analysis of heterogenizea catalyst precursors, where gas/solid interfacial photoreactions can be monitored.