Book Reviews

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Molecular Biology and Biotechnology, 4th Edition

J. M. Walker and R. Rapley, Royal Society of Chemistry, Cambridge, xxiv + 564 pp., price £39, ISBN 0-85404-606-2 Search PubMedThe editors of this book have taken on quite a daunting task in attempting to produce an up-to-date book in the field of molecular biology in the post-genome era. The dangers are that any such book cannot adequately cover such a wide-ranging area and appears out of date by the time it is published. It is gratifying to find that the 4th Edition of Molecular Biology and Biotechnology does not suffer this fate.

Over 19 chapters the excellent contributions from experts in each field provide the reader with a knowledge of subjects ranging from basic concepts in molecular biology, through to overviews of new areas such as genetically modified foods and bioinformatics. This edition convinced me that molecular biology is an exciting field to work in because of its constantly expanding number of techniques and applications.

It is no surprise that nearly a third of the book is devoted to the basic concepts of recombinant DNA technology and protein expression. After an opening chapter on fermentation technology, Chapters two and three cover DNA purification and the subsequent design and application of the most widely used molecular biology technique, the Polymerase Chain Reaction (PCR). The emphasis is firmly on cloning genes as a means to be able to study their protein products and the following four chapters deal with the concepts and techniques that are available to clone genes in bacteria, yeast (Ch. 5), mammals (Ch. 6) and plants (Ch. 7). One slight drawback is that, although the authors give excellent examples of various plasmids to use, it is unfortunate that they cannot give guides to specific manufacturers of state-of-the-art cloning and expression systems available in the highly competitive “molecular biology kit” market.

The emphasis changes roughly half-way through the book from a description of the various molecular biology techniques to their applications. Chapter eight deals with the impact of genomic research on our understanding of the molecular biology of disease and on how it has changed the drug discovery process—the “gene to drug” philosophy. It also discusses how research is geared to linking gene identification through to gene function and introduces the hot topics of genomics (studies on the total genetic make-up of an organism) and proteomics (analyses of all the protein components of a cell). Molecular biology now plays its part in identifying drug targets, deciphering complex disease mechanisms, and supporting biochemical screening and structural determination. Gene mutations or deletions can now be used to study their effects on the physiological function of the encoded protein within an animal model. Various total genome sequencing projects are listed which include the human genome, Escherichia coli, yeast and various pathogenic bacteria. It is hoped this information will lead to breakthroughs in the discovery of novel drug targets. Moreover, it will aid in the design of new drugs to treat deadly pathogens that are making a comeback—bacteria that have evolved resistance to many of the currently available antibiotics.

Complete genome sequences of organisms and three dimensional structure of proteins are becoming available on a weekly basis and this is maybe one of the main reasons why any text book in this field could be seen as out-of-date immediately after publication. Indeed, the human genome was published after this appeared but the book encourages the reader to keep abreast of latest developments through interactive www sites.

Chapter nine deals with the sensitive area of genetically modified foods and provides background information, legal issues and covers products already licensed and on the market to protect crops or enhance products. The enzyme chymosin (originally from calf rennet) is now produced by recombinant sources and its role in the cheese-making process is discussed. Future work will impact on food additives, flavourings, vitamins and colourings once the debate between scientists, governments and the consumer reach an understanding.

The theme of the direct impact that molecular biology has had on the public begun in the previous chapter is further developed over the next four chapters which focus on the diagnosis of inherited disease, DNA in forensic science, vaccination and gene manipulation and transgenesis. The first two chapters cover the background and techniques involved in determining the basis of genetic disease citing Huntington’s disease and Fragile X syndrome as examples. This area is under massive public scrutiny since the release of the human genome. Data from forensic science laboratories are now being used in court cases around the world and better ways of analysing and presenting this data, possibly using DNA chip microarrays, are being developed. The impact of vaccination against viral and bacterial infectious disease is covered and a list is given of numerous vaccines and their origin; live attenuated vaccine versus killed. Some diseases which were thought to have been eradicated are making a comeback. It is hoped that a greater understanding of the immune system coupled to the production of antigens via recombinant methods will aid in the design of novel vaccines. The chapter on transgenesis describes how this area has developed through early success with mice through to its consequences for the field of animal model production through nuclear transfer, gene therapy and the commercial production of protein drugs.

The protein engineering chapter deals with techniques to analyse protein structure and function such as site-directed mutageneis (SDM). Novel methods have been developed which will enable a protein with a specific function to be engineered either through a detailed knowledge of its structure or by randomly shuffling the encoding gene, then selecting for a particular biological phenotype. Protein design is still at an early stage but with new gene and protein sequences being constantly discovered through whole genome efforts allied to powerful computing technology, the future looks to be very exciting. Then follows an excellent and very timely chapter (fifteen) on bioinformatics (the application of information technology to biology), and attempts to guide the reader through the enormous amount of information in countless databases throughout the world. It gives details on various www sites and demystifies the jargon associated with this new area of biology.

The last few chapters deal with immobilization of biocatalysts and protein purification on an industrial scale and their impact on the biotechnology industries that use biotransformation reactions. Chapter eighteen describes novel techniques to produce monoclonal antibodies and their use in the diagnosis and treatment of disease such as cancer—a major breakthrough would be achieved through the production of recombinant fully human antibodies to overcome problems with regard to immunogenicity. A final chapter looks into the future and introduces the area of biosensors and molecular machines. These devices, which convert biological actions into electrical signals, have great potential in the diagnostic kit and environmental monitoring field.

Overall, this book will be very useful to everyone with an interest in molecular biology and biotechnology and who wants to know more than just the basics. Experts in specific fields will probably find that the material does not cover each subject in enough depth but they will probably have access to text books dealing specifically with each area such as PCR methodology, antibody production and/or transgenic animals. There are some gaps—there is little mention of the recent developments on the mass spectrometric analysis of proteins and small molecules—but I expect these to be covered in the next edition.

Where it will find the most use will be in the hands of final year undergraduates and post-graduate students in chemistry (or any other non-purely-biological discipline) who wants to get information on a range of molecular biology techniques. It should also find use in many bio-organic/protein biochemistry research laboratories where it will be an excellent reference text for PhD students, post-docs and lecturers. The references cited and more importantly, the www sites listed are very useful for gaining more information on a specific topic or technique.

Dominic Campopiano
University of Edinburgh, UK

Chemistry for the 21st Century

E. Keinan and I. Schechter, Wiley-VCH, Germany, 2001, xiv + 293 pp., price £24.95, ISBN 3-257-30235-2 Search PubMedThe “Whither chemistry?” question is frequently posed and usually answered disappointingly. The road to the future is paved with dud predictions ¹ and in any case Maurice Maeterlinck ² has remarked that the future Century is a world limited by ourselves; in it we discover only what concerns us. Organised chemistry is only about a century and a half old and so looking ahead on a century time scale is brave indeed.

In Chemistry for the 21st Century, 28 authors have contributed to a useful volume but it is difficult to tell what was the Editorial brief with regard to the future. Some authors bravely try to look ahead; others give us state-of-the-art without state-of-the-future. In 1990, for a BASF Jubilee Symposium ³ a somewhat similar volume appeared; in it, George Whitesides was wisely tentative over even a 20-year period and when restricting himself to an 11-year period was remarkably prescient. He listed the pull–push factors such as national security driving materials chemistry, health care driving biological chemistry and the environment and energy driving computational chemistry and nano-miniaturisation. Many of these topics appear in the present volume, which overall is a worthwhile collection of up-to-date and readable accounts of many contemporary and futuristic aspects of chemistry which may be unfamiliar to the hard-pressed specialist. The coverage is very wide and there are no obvious gaps. This means that some Perkin 1 readers for example may struggle a little with the following from the chapter on quantum theory:

Space does not permit detailed comment on each chapter and so picking out some of the intriguing as well as more familiar aspects may perhaps give a flavour of this book.

Jean-Marie Lehn starts it off with an economically brief and characteristically philosophical overview of supramolecular chemistry. He loves quotations and tells us that chemistry is not only to discover but to invent and above all, create. By contrast, Danishefsky gives us a twenty page review of his group's recent work on the epothilones. Here is medium-sized molecule synthesis at its craftiest and most relevant but we are still behind the curtain of the future. Leo Paquette takes us into the world of spirotetrahydrofurans and looks ahead to specific ion binding, the possibility of ion–spirotetrahydrofuranyl ladder polymers and spirocyclic restriction of nucleosides and nucleotides in future therapeutic applications.

Materials chemistry and catalysis are well represented here. The development of new surface analytical techniques will change what we can imagine in this century. Already we can see, in the chapter by Gerhard Ertl, events at the atomic level. In the oxidation of CO by oxygen on a platinum surface, STM images show the dissociation of individual oxygen molecules with the atoms separated by several times their molecular interatomic distance. On the economic front, the importance of chemical catalysis looks rosy—world-wide sales have doubled in the last ten years and automotive catalysis more than tripled (what price Kyoto???). Combinatorial chemistry is now everywhere and what is good for drug discovery is cutting down discovery times for new materials as well. The chapter on this topic reveals that this technology is more than thirty years old but growing fast. It can be applied to new phosphors, fuel cell catalysts, superconductors and piezoelectric materials to name but a few.

At the other end of the spectrum, Snyder gives us an essentially non-molecular account of drugs for the new millennium. He discusses drugs of addiction, schizophrenia, drug psychoses and cell death, regarding these as the big picture issues that chemists should be aware of even at the acronym level. He concentrates on Alzheimer's disease just as part of his look ahead and points the directions in which future work might go.

Somewhat surprisingly, there are two chapters on nitrogen in biology—its fixation and bioavailability. One deals with the enzymology of fixation with a nice swipe at Liebig who apparently did not think it was necessary to do any experiments to debunk the pioneering work of Boussingault in this area. There are many interesting insights, not least the ratio of biological fixation to chemical fixation at only a factor of two—what environmental price??—but no look ahead. The second by Schnitzer is a straight account of the author's work on nitrogen content of soils and again there is little forward look.

Man-made supramolecular assemblies have quite a brief mention by Jerry Attwood in the specific context of notably of his own work on cavitand calixarenes and the topographies of the Archimedean and Platonic solids which, he suggests, will usher in an era of spherical host–guest chemistry.

The topical and difficult matter of protein folding is interestingly (and briefly) dealt with and the chapter has a strong resonance with the three chapters on quantum chemistry which round off the volume. These are tough going—unsurprisingly—for the mathematically less able. Marvin Cohen's chapter is intriguingly called quantum alchemy and takes us from J. J. Thompson's plum pudding atom through the assembly of carbon nanotubes and into the future—given good enough computers of pentaheptite and metallic hydrogen.

The most profound and puzzling question of all—What is the origin of Life?—is addressed in a lovely chapter by Frank, Bonner and Zare which, unlike some of the others is made comprehensible in large part to the non-chemist. The problem is three-fold given that life and molecular chirality are inseparable. How did an enantiomeric excess originate, how was it amplified and how was it preserved? They speculate, for example, that an excess of one enantiomer of an amino acid over another could result from circularly polarised irradiation from stars and arrive on earth via meteorites. These then seeded terrestrial stocks of amino acids produced notably in hydrothermal vents and the polypeptides then formed from them amplified the chiral selectivity by selecting only one chirality in the chain extension. These were then sequestered, surviving in the cavities of microporous minerals and becoming the molecules of life. The look ahead here is into the far distance—in this new century shall we really be able to understand where we have come from?

The chemical polymath will enjoy this collection; the specialist will learn a lot.

¹ I made this up.

² Maurice Maeterlink, Joyzelle, Act 1.

³Angew. Chem., Int. Ed. Engl., 1990, 29, 1177.

Charles Stirling
University of Sheffield, UK

Organic Electrochemistry

4th edn., H. Lund and O. Hammerich, Marcel Dekker, New York, 2001, vii + 1393 pp., price $275, ISBN 0-8247-0430-4 Search PubMedThis is the fourth edition of a book well-known and much used by electrochemists. The first edition appeared in 1973 under the editorship of the late M. M. Baizer. Since this date three editions have been published, including each time more reactions, more data and more references. This thick edition of nearly 1400 pages and about 7000 references (up to year 2000) gives a thorough coverage of the field and includes everything needed by the specialist. But at the same time this book is a must for anybody who wants to start practising in the field; he will find both what is needed to run and interpret the analytical methods and the bibliography about the different types of reactions and classes of compounds.

Each of the 32 chapters can be read by itself, but is, at the same time, well included in the whole scope of the book. The book starts with five chapters which describe the general background of electrochemistry: i) The chapter which describes the Basic concepts of electrochemistry is focused on those particularly needed to understand the field of molecular electrochemistry. ii) The chapter Methods for studies of electrochemical reactions describes the physico-chemical techniques (cyclic voltammetry, polarography . . .) used to analyze the electrochemical mechanisms. iii) The chapter Relations between micro and macro phenomena deals in particular with the optimization of preparative scale electrolysis (it shows how to construct and use zone diagrams to rationalize the choice of parameters). iv) The Comparison between chemical and electrochemical reductions and oxidations is the subject of a chapter which may help to decide which method should be used and v) the chapter Practical problems in electrolysis will permit the choice of the cell, electrodes, solvents . . . which are often determining in the success of an experiment.

The remaining chapters deal with the reduction or oxidation of classes of compounds: Hydrocarbons (both oxidation and reduction) including a new chapter on C₆₀, Halogenated compounds (it is surprising that the first scheme of the chapter writes the reduction of alkyl halides as involving radical anions, although it has been demonstrated that the electron transfer and the cleavage are concerted; the correct mechanism is written on page 970), Nitro compounds, Carbonyl and Azomethine derivatives, Carboxylic acids, their reduction and oxidation leading to the thoroughly investigated Kolbe reaction. The Anodic oxidation of nitrogen, oxygen, and sulfur containing compounds is described including TTF and their cation radicals (but the oxidation of pyrrole, thiophene . . . leading to conducting polymers is not included in the scope of this book). The Electrolysis of heterocyclic compounds is the subject of a chapter. It includes the formation of heterocycles (by creation of C–O, C–N . . . bonds) as well as their electrochemical reactions. The chapter Electrolysis of bioactive materials describes the synthesis of natural compounds which involve at least one electrochemical step. One finds for example Stork's synthesis of α-onocerin through a Kolbe reaction and the oxidation of a carbanion leading to an anisomycin derivative. This chapter is particularly demonstrative of the use and possibilities of electrochemistry in the synthesis of complex compounds. The electrochemical synthesis, oxidation (leading to radical chain reactions) and reduction of Organoelemental compounds is the subject of a chapter. Reductive couplings are the basis of Baizer's industrial synthesis of adiponitrile, the largest industrial organic electrochemical process. Electrooxidative couplings involves couplings via radicals and radical cations. Cleavage and deprotection describes the cleavage of a variety of bonds which can be synthetically useful. Other chapters include: Anodic substitutions and additions, Electrochemical partial fluorination (by oxidation of F⁻ but most often of compounds such as Et₃N·3HF), Stereochemistry of organic electrode processes (as stated by the authors, molecular electrochemists have not paid much attention to stereochemical problems and it is only rather recently that they have tried to gain stereocontrol of organic electrochemical reactions; obviously much has to be done in this field if one wants to maintain the interest of electrochemistry in organic synthesis). A new chapter is devoted to Electroenzymatic synthesis; it describes electroenzymatic reductions or oxidations which try to take advantage of the specificity of enzymes to perform reactions and of an electrode to regenerate the cofactors. As Amalgams function as electron donors in a manner similar to electrodes, a chapter has been devoted to their reactions. It is possible to produce Electrogenerated reagents by electron transfer (radical anions and cations but also solvated electrons) which can be used in redox catalysis and in radical chain reactions. Reduction of certain species leads to anions with strong basic properties; the chapter Electrogenerated bases, describes their synthetic use. The last chapter: Industrial electroorganic chemistry lists 68 industrial processes from 300 000 to a few tonnes per year which are now operating or under development through the world.

Browsing this book, one is impressed by the number of organic reactions which have been achieved by electrochemistry and this excellent new edition should foster further developments in the use of electrons as reagents.

Jean Pinson
Université Paris, France

Enzymes in Nonaqueous Solvents, Methods and Protocols

Evgeny N. Vulfson, Peter J. Halling and Herbert L. Holland, Humana Press, 2001, 649 pp., price $139.50, ISBN 0-89603-929-3 Search PubMedBiocatalysts are most often regio- and stereo-selective, active in mild conditions, and catalyze reactions that may be inaccessible or difficult to achieve by traditional synthesis. These qualities have motivated the use of enzymes in the preparation of various chemicals. The increasing need for enantiopure chemicals in fields such as drug discovery and food science is partially responsible for the growing interest in enzymatic asymmetric synthesis. However, the use of biocatalysts still poses serious challenges such as product inhibition, substrate solubility, enzyme stability issues, and cofactor requirements. Water is also difficult to eliminate and may undergo unwanted side reactions. Unquestionably, the early belief that enzymes could work solely in aqueous media has restrained the scope of their applications. However, it is now well established that numerous enzymes can function perfectly well under nearly anhydrous conditions. Furthermore, enzymes in organic solvents often display useful properties such as highly enhanced stability, molecular imprinting, and modified selectivity. Numerous techniques have been developed to optimize the use of enzymes in nonaqueous media. The goal of Enzymes in Nonaqueous Solvents, Methods and Protocols is to present some theory and detailed protocols to the reader interested in using enzymes in synthesis.

The first chapter of this volume addresses the issues that affect the activity, specificity, and stability of low water enzymes, namely, temperature, substrate concentration, acid/base effects, solvent, and residual water. Suitable preparation of the biocatalyst is critical to the behavior and activity that it exhibits. For example, the precise conditions of lyophilization, which are rarely specified clearly, have a significant effect on activity and can be improved by drying in the presence of salts or a suitable imprinting agent. Small variations in the residual water of anhydrous systems can have dramatic effects on the enzyme behavior. A variety of methods to precisely quantify and control the water content are described at great length in this book. In addition, the authors point out many specific considerations that apply to the determination of enzyme activity and selectivity in organic solvents.

Proteins with increased activity and stability in organic media can be obtained by immobilization of the biocatalyst. The authors outline three important techniques to immobilize enzymes: 1) entrapment within a polymer network (e.g. polyethylene glycol), 2) microencapsulation within permeable polymeric microcapsules, and 3) the use of hydrogel supports, which are cross-linked polymeric structures that can imbibe large quantities of water. Because enzymes in anhydrous media form heterogeneous mixtures, finer dispersions generally translate into higher synthetic yields. More homogeneous solutions are often achieved by covalent attachment of polyethylene glycol or hydrophobic modifiers, or non-covalent modification with polyethylene glycol or synthetic lipids. Interestingly, the addition of crown ethers has been reported to enhance the activity of many enzymes in anhydrous conditions. Also addressed is the study of enzymes by calorimetric, fluorescence, and CD measurements, followed by specific examples of enzymatic synthesis in the second chapter.

The ability to form esters and amides without competing hydrolysis, the manipulation of regio- and stereo-selectivity, and the higher solubility of substrates and products have all contributed to the spread of enzyme applications in nearly anhydrous conditions. Organic solvents can be advantageous for oxidation reactions which are severely limited by oxygen solubility in water. The series of empirical rules provided in this volume for selection of a hydrolase with optimum activity and selectivity is a highly valuable resource. Although most of the work in this field has involved hydrolytic enzymes, many other reactions are amenable to biocatalysis including reductions, oxidations, preparation of chiral alcohols by transesterification, as well as several transformations of hydrophobic organosilicon substrates.

Low water systems are equally useful with whole-cell biocatalysts. Organic solvents, water–organic solvent biphasic systems, and reverse micelles have been used successfully to optimize specific transformations by whole cells. A number of interface bioreactors adapted to nearly anhydrous mixtures are described in great detail. Yeast, one of the most popular biocatalysts, can catalyze a wide range of reactions. It is commonly used as an actively growing culture and, therefore, all of its biochemical pathways are active. This methodology is well established and often affords high selectivity, but the same problems encountered with enzymes in aqueous media apply. In addition, product isolation is impeded by the complex nature of the fermentation media. These can be overcome using a recently reported methodology which uses dried Baker's yeast in an organic solvent. Since the yeast is not fermenting, very few of the natural metabolites are produced and no food source is required.

Organic solvents are not the only alternative to aqueous media. Numerous other low water systems have been successfully used in enzymatic catalysis, and the third chapter focuses on them. Supercritical fluids, solvent-free, gas phase, solid-to-solid, and solid-to-gas systems are some of the extreme conditions reported. Also useful are frozen aqueous solutions, supersaturated substrate solutions, reverse micelles, and microemulsions. In general, the appropriate type of conditions to be used with the bioreactor is determined by the properties of the substrate.

Above the critical point of the phase diagram, a substance is said to be supercritical (SC) and is described as a dense gas or an expanded liquid. Close to the critical point, small changes in temperature or pressure can result in large changes in density and solvation ability of the fluid. These media also show lower viscosity and higher diffusivity as compared to organic solvents. This is a major advantage since diffusion is often rate-limiting when enzyme and substrates are not in the same phase. In addition, supercritical fluids represent a less toxic and less flammable alternative to organic solvents and can be easily separated from the product. As a result, SC-CO₂ and SC-SO₂ are increasingly popular media for enzymes that have been freeze-dried, immobilized, or crystallized.

Solid-to-solid peptide synthesis consists of up to 20% (w/w) of enzyme in water mixed with solid substrates. In all cases, some substrate remains undissolved. Similarly, enzymatic reactions can be performed in the liquid phase using low-melting-point eutectic mixtures of supersaturated substrates. Such systems can be prepared by mixing molten substrates together at elevated temperature followed by slow cooling to room temperature. This procedure has been employed in the preparation of numerous peptide derivatives, glycosides, and disaccharides. The advantages of using this technique include: a better volumetric productivity, higher yields of products, and change in stereoselectivity of glycosylations from primary to secondary hydroxy groups.

Micellar enzymology was initially developed as a model for biomembranes, yet reverse micelles are attractive media for many other reasons including: driving the equilibrium towards the formation of products, allowing for accurate control of the water content, and permitting the dissolution of both hydrophilic and hydrophobic substrates while providing an isotropic solution which can be monitored by spectroscopy.

Enzymes in Nonaqueous Solvents, Methods and Protocols includes detailed descriptions of the latest technologies used for biocatalysis in nearly anhydrous systems. Numerous specific enzymatic reactions, bioreactors, and useful techniques are carefully explained in a format intelligible to most readers in spite of the great diversity of the fields covered. This work of reference will be extremely valuable to those interested in enzymatic and asymmetric synthesis. One drawback however is its high cost.

Karine Auclair
McGill University, Montreal, Canada

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