Manfred T. Reetz

Microbial lipases form versatile tools for biotechnology

Lipases are secreted into the culture medium by many bacteria and fungi. They catalyse not only the hydrolysis but also the synthesis of long-chain acylglycerols. Important uses in biotechnology include their addition to detergents, the manufacture of food ingredients, pitch control in the pulp and paper industry, and biocatalysis of stereoselective transformations. This makes them the most widely used class of enzymes in organic chemistry. Immobilization in hydrophobic sol-gel matrices and in vitro evolution are promising novel approaches to increasing the stability or enantioselectivity, respectively, of lipases.

Phosphane-Free Palladium-Catalyzed Coupling Reactions: The Decisive Role of Pd Nanoparticles

Nanosized palladium colloids, generated in situ by reduction of Pd(II) to Pd(0) [Eq. (a)], are involved in the catalysis of phosphane-free Heck and Suzuki reactions with simple palladium salts such as PdCl(2) or Pd(OAc)(2), as demonstrated by transmission electron microscopic investigations.

Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes

Iterative saturation mutagenesis (ISM) is a new and efficient method for the directed evolution of functional enzymes. It reduces the necessary molecular biological work and the screening effort drastically. It is based on a Cartesian view of the protein structure, performing iterative cycles of saturation mutagenesis at rationally chosen sites in an enzyme, a given site being composed of one, two or three amino acid positions. The basis for choosing these sites depends on the nature of the catalytic property to be improved, e.g., enantioselectivity, substrate acceptance or thermostability. In the case of thermostability, sites showing highest B-factors (available from X-ray data) are chosen. The pronounced increase in thermostability of the lipase from Bacillus subtilis (Lip A) as a result of applying ISM is illustrated here.

Lipases as practical biocatalysts

Lipases are the most used enzymes in synthetic organic chemistry, catalyzing the hydrolysis of carboxylic acid esters in aqueous medium or the reverse reaction in organic solvents. Recent methodological advancements regarding practical factors affecting lipase activity and enantioselectivity are reviewed. Select practical examples concerning the use of lipases in the production of chiral intermediates are also highlighted.

Laboratory Evolution of Stereoselective Enzymes: A Prolific Source of Catalysts for Asymmetric Reactions

Asymmetric catalysis plays a key role in modern synthetic organic chemistry, with synthetic catalysts and enzymes being the two available options. During the latter part of the last century the use of enzymes in organic chemistry and biotechnology experienced a period of rapid growth. However, these biocatalysts have traditionally suffered from several limitations, including in many cases limited substrate scope, poor enantioselectivity, insufficient stability, and sometimes product inhibition. During the last 15 years, the genetic technique of directed evolution has been developed to such an extent that all of these long-standing problems can be addressed and solved. It is based on repeated cycles of gene mutagenesis, expression, and screening (or selection). This Review focuses on the directed evolution of enantioselective enzymes, which constitutes a fundamentally new approach to asymmetric catalysis. Emphasis is placed on the development of methods to make laboratory evolution faster and more efficient, thus providing chemists and biotechnologists with a rich and non-ending source of robust and selective catalysts for a variety of useful applications.

Biocatalysis in organic chemistry and biotechnology: past, present, and future.

Enzymes as catalysts in synthetic organic chemistry gained importance in the latter half of the 20th century, but nevertheless suffered from two major limitations. First, many enzymes were not accessible in large enough quantities for practical applications. The advent of recombinant DNA technology changed this dramatically in the late 1970s. Second, many enzymes showed a narrow substrate scope, often poor stereo- and/or regioselectivity and/or insufficient stability under operating conditions. With the development of directed evolution beginning in the 1990s and continuing to the present day, all of these problems can be addressed and generally solved. The present Perspective focuses on these and other developments which have popularized enzymes as part of the toolkit of synthetic organic chemists and biotechnologists. Included is a discussion of the scope and limitation of cascade reactions using enzyme mixtures in vitro and of metabolic engineering of pathways in cells as factories for the production of simple compounds such as biofuels and complex natural products. Future trends and problems are also highlighted, as is the discussion concerning biocatalysis versus nonbiological catalysis in synthetic organic chemistry. This Perspective does not constitute a comprehensive review, and therefore the author apologizes to those researchers whose work is not specifically treated here.

Efficient immobilization of lipases by entrapment in hydrophobic sol‐gel materials

The commercial application of lipases as biocatalysts for organic synthesis requires simple but efficient methods to immobilize the enzyme, yielding highly stable and active biocatalysts which are easy to recover. In this study, we present a novel method to achieve lipase immobilization by entrapment in chemically inert hydrophobic silica gels which are prepared by hydrolysis of alkyl‐substituted silanes in the presence of the enzyme. A typical immobilization procedure uses: an aqueous solution of lipase; sodium fluoride as a catalyst; and additives like polyvinyl alcohol or proteins and alkoxysilane derivatives like RSi‐(OMe)3 with R = alkyl, aryl, or alkoxy as gel precursors. The effect of various immobilization parameters like stoichiometric ratio of water, silane, type and amount of additive, type and amount of catalyst, and type of silane has been carefully studied. The new method is applicable for a wide variety of lipases, yielding immobilized lipases with esterification activities enhanced by a factor of up to 88, compared to the commercial enzyme powders under identical conditions. Studies on the stability of sol‐gel immobilized lipases under reaction conditions or storage (dry, in aqueous or organic medium) revealed an excellent retention of enzymatic activity. The possible reasons for the increased enzyme activities are discussed.

Addressing the Numbers Problem in Directed Evolution

Our previous contribution to increasing the efficiency of directed evolution is iterative saturation mutagenesis (ISM) as a systematic means of generating focused libraries for the control of substrate acceptance, enantioselectivity, or thermostability of enzymes. We have now introduced a crucial element to knowledge‐guided targeted mutagenesis in general that helps to solve the numbers problem in directed evolution. We show that the choice of the amino acid (aa) alphabet, as specified by the utilized codon degeneracy, provides the experimenter with a powerful tool in designing “smarter” randomized libraries that require considerably less screening effort. A systematic comparison of two different codon degeneracies was made by examining the relative quality of the identically sized enzyme libraries in relation to the degree of oversampling required in the screening process. The specific example in our case study concerns the conventional NNK codon degeneracy (32 codons/20 aa) versus NDT (12 codons/12 aa). The model reaction is the hydrolytic kinetic resolution of a chiral trans‐disubstituted epoxide, catalyzed by the epoxide hydrolase from Aspergillus niger. The NDT library proves to be of much higher quality, as measured by the dramatically higher frequency of positive variants and by the magnitude of catalyst improvement (enhanced rate and enantioselectivity). We provide a statistical analysis that constitutes a useful guide for the optimal design and generation of “smarter” focused libraries. This type of approach accelerates the process of laboratory evolution considerably and can be expected to be broadly applicable when engineering functional proteins in general.

Organotitanium Reagents in Organic Synthesis

1. Introduction.- 1.1 Adjustment of Carbanion-Selectivity via Titanation.- 1.2 Other Uses of Titanium in Organic Chemistry.- References.- 2. Synthesis and Properties of Some Simple Organotitanium Compounds.- 2.1 Synthesis and Stability.- 2.1.1 Historical Aspects.- 2.1.2 Mono-Aryl- and Alkyltitanium Compounds.- 2.1.3 Polyalkyl- and Aryltitanium Compounds.- 2.1.4 h5-Cyclopentadienyltitanium(IV) Compounds.- 2.2 Bond Energies.- 2.3 Bond Angles and Lengths.- 2.4 Aggregation State.- 2.5 Spectroscopic and Theoretical Aspects.- 2.5.1 Introductory Remarks.- 2.5.2 Methyltitanium Compounds.- 2.5.3 h5-Cyclopentadienyltitanium Compounds.- 2.6 Conclusions.- References.- 3. Chemoseleetivity in Reactions of Organotitanium Reagents with Carbonyl Compounds.- 3.1 Aldehyde/Ketone Differentiation.- 3.1.1 Reagents of the Type RTi(OCHMe2)3.- 3.1.2 Organotitanium Reagents Bearing Other Ligands.- 3.1.3 Titanium Ate Complexe.- 3.2 Aldehyde/Aldehyde and Ketone/Ketone Differentiation.- 3.3 Chemo- and Regioselective Additions to ?,?-Unsaturated Carbonyl Compounds.- 3.4 Aldehyde/Ester and Ketone/Ester Differentiation.- 3.5 Reactions in the Presence of Additional Functionality.- 3.6 Addition to Enolizable Ketones.- 3.7 Limitations of Organotitanium Reagents.- 3.8 Hints on How to Use Organotitanium Compounds.- 3.9 Why Does Titanation of Carbanions Increase Chemoselectivity?.- 3.10 Comparison with Other Organometallic Reagents.- 3.11 Reversal of Chemoselectivity: Chemoselective in situ Protection of Carbonyl Compounds.- 3.12 Organotitanium Reagents from Non-Organometallic Precursors 107 References.- References.- 4. Rates of Reactions.- 4.1 Kinetics of the Addition of CH3Ti(OCHMe2)3 to Carbonyl Compounds.- 4.2 Other Kinetic Studies.- References.- 5. Stereoselectivity in the Addition of Organotitanium Reagents to Carbonyl Compounds.- 5.1 Titanation of Carbanions as a Means to Control Stereoselectivity.- 5.2 Diastereofacial Selectivity.- 5.2.1 The Cram/anti-Cram Problem.- 5.2.2 Chelation-Control in Addition Reactions of Chiral Alkoxy Carbonyl Compounds.- 5.2.2.1 1,2 Asymmetric Induction.- 5.2.2.2 1,3 and 1,4 Asymmetric Induction.- 5.2.3 Non-Chelation-Controlled Additions to ?-Chiral Alkoxy Carbonyl Compounds.- 5.3 Simple Diastereoselectivity.- 5.3.1 Titanium-Mediated Aldol Additions.- 5.3.2 Aldol-Type Additions of Titanated Heterocycles.- 5.3.3 Addition of Prochiral Allylic Titanium Reagents.- 5.3.4 Addition of Prochiral Propargyl-Titanium Reagents.- 5.4 The Problem of Equatorial vs. Axial Addition to Cyclic Ketones.- 5.5 Enantioselective Additions.- 5.5.1 Reagents with Chirally Modified Ligands at Titanium.- 5.5.2 Reagents with the Center of Chirality at Titanium.- 5.5.3 Titanation of Carbanions which Contain a Chiral Auxiliary.- References.- 6. Michael Additions.- References.- 7. Substitution Reactions.- 7.1 Titanium Enolates as Nucleophiles.- 7.2 Alkyltitanium Compounds as Nucleophiles.- 7.2.1 SN1-Active Alkyl Halides and Related Alkylating Agents.- 7.2.2 Direct Geminal Dialkylation of Ketones and Aldehydes and Exhaustive Methylation of Acid Chlorides.- 7.2.3 Acetals as Alkylating Agents.- 7.2.4 Metallated N,0-Hemiacetals as Alkylating Agents.- 7.3 Other Substitution Reactions: Present and Future.- References.- 8. Wittig-type Methylenation of Carbonyl Compounds.- References.

Suzuki and Heck reactions catalyzed by preformed palladium clusters and palladiumnickel bimetallic clusters

Abstract Soluble palladium clusters and palladium nickel bimetallic clusters stabilized by tetraalkylammonium salts or poly(vinylpyrrolidone) are effective catalysts in Suzuki and Heck reactions involving iodo-, bromo- or activated chloroaromatics, whereas chlorobenzene is not a suitable reaction partner.