Organic Synthesis Using Biocatalysis 1St Edition Goswami Download PDF Chapter

Organic Synthesis Using Biocatalysis
1st Edition Goswami

Visit to download the full and correct content document:
https://ebookmass.com/product/organic-synthesis-using-biocatalysis-1st-edition-gosw
ami/
Organic Synthesis
Using Biocatalysis
Edited by
Animesh Goswami
Chemical Development, Bristol-Myers Squibb,
New Brunswick, NJ, USA
Jon D. Stewart
Department of Chemistry, University of Florida,
Gainesville, FL, USA
Amsterdam • Boston • Heidelberg • London • New York • Oxford

Paris • San Diego • San Francisco • Singapore • Sydney • Tokyo
Elsevier
Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands
The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK
225 Wyman Street, Waltham, MA 02451, USA
Copyright © 2016 Elsevier Inc. All rights reserved.
No part of this publication may be reproduced or transmitted in any form or by any means, electronic
or mechanical, including photocopying, recording, or any information storage and retrieval system,
without permission in writing from the publisher. Details on how to seek permission, further
information about the Publisher’s permissions policies and our arrangements with organizations
such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our
website: www.elsevier.com/permissions.
This book and the individual contributions contained in it are protected under copyright by the
Publisher (other than as may be noted herein).
Notices
Knowledge and best practice in this field are constantly changing. As new research and experience
broaden our understanding, changes in research methods, professional practices, or medical
treatment may become necessary.
Practitioners and researchers must always rely on their own experience and knowledge in evaluating
and using any information, methods, compounds, or experiments described herein. In using
such information or methods they should be mindful of their own safety and the safety of others,
including parties for whom they have a professional responsibility.
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume
any liability for any injury and/or damage to persons or property as a matter of products liability,
negligence or otherwise, or from any use or operation of any methods, products, instructions, or
ideas contained in the material herein.
British Library Cataloguing in Publication Data

A catalogue record for this book is available from the British Library
Library of Congress Cataloging-in-Publication Data

A catalog record for this book is available from the Library of Congress
ISBN: 978-0-12-411518-7
For Information on all Elsevier publications

visit our website at http://store.elsevier.com/
List of Contributors
Samantha K. Au
School of Chemical and Biomolecular Engineering, Georgia Institute of
Technology, Parker H. Petit Institute of Bioengineering and Bioscience, vii
Atlanta, GA, USA
Andreas S. Bommarius
School of Chemical and Biomolecular Engineering, Georgia Institute
of Technology, Parker H. Petit Institute of Bioengineering and Bioscience;
School of Chemistry and Biochemistry, Georgia Institute of Technology,
Atlanta, GA, USA
Chen Cao
Department of Bioengineering, Graduate School of Bioscience and
Biotechnology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku,
Yokohama, Japan
Pere Clapés
Department Química Biológica y Modelización Molecular, Instituto de Química
Avanzada de Cataluña, IQAC-CSIC, Barcelona, Spain
Rodrigo O.M.A. de Souza
Biocatalysts and Organic Synthesis Lab, Organic Chemistry Department,
Chemistry Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
Brent D. Feske
Chemistry and Physics Department, Armstrong State University, Savannah,
GA, USA
Michael J. Fink
Vienna University of Technology, Institute of Applied Synthetic Chemistry,
Vienna, Austria
Animesh Goswami
Chemical Development, Bristol-Myers Squibb, New Brunswick, NJ, USA
Gideon Grogan
Department of Chemistry, University of York, Heslington, York, UK
Harald Gröger
Faculty of Chemistry, Bielefeld University, Universitätsstr,
Bielefeld, Germany
viii List of Contributors
Jonathan Groover
Chemistry and Physics Department, Armstrong State University, Savannah,
GA, USA
Melissa L.E. Gutarra
Escola de Química, Federal University of Rio de Janeiro, Pólo Xerém, Estrada
de Xerém, Xerém, Duque de Caxias, Rio de Janeiro, Brazil
Romas Kazlauskas
Department of Biochemistry, Molecular Biology & Biophysics and The
Biotechnology Institute, University of Minnesota, Saint Paul, MN, USA
Tomoko Matsuda
Department of Bioengineering, Graduate School of Bioscience and
Biotechnology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku,
Yokohama, Japan
Marko D. Mihovilovic
Vienna, Austria
Leandro S.M. Miranda
Biocatalysts and Organic Synthesis Lab, Organic Chemistry Department,
Chemistry Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
Thomas S. Moody
Department of Biocatalysis and Isotope Chemistry, Almac, Craigavon,
Northern Ireland, UK
Ramesh N. Patel
SLRP Associates, Consultation in Biotechnology, Bridgewater, NJ, USA
Laila Roper
Department of Chemistry, University of York, Heslington, York, UK
J. David Rozzell
Provivi, Santa Monica, CA, USA
Florian Rudroff
Vienna, Austria
Jon D. Stewart
Department of Chemistry, University of Florida, Gainesville, FL, USA
CHAPTER 1
Introduction, Types of
Reactions, and Sources
of Biocatalysts
1
Animesh Goswami*, Jon D. Stewart†

*
Chemical Development, Bristol-Myers Squibb, New Brunswick, NJ, USA
†
Department of Chemistry, University of Florida, Gainesville, FL, USA
1 INTRODUCTION
1.1 Enzymes and Their Roles in Nature
Enzymes are nature’s catalysts, facilitating the creation, functioning, mainte-
nance, and ultimately the demise of all living cells. Enzymes are proteins com-
posed of 20 natural amino acids joined together by peptide bonds, in some
cases augmented with additional organic or inorganic species known as cofac-
tors.1 In addition to their primary molecular structures dictated by the amino
acid sequence, enzyme catalytic function also depends upon subsequent fold-
ing into specific three-dimensional shapes that contain a variety of secondary
and tertiary structural elements. These architectures determine not only enzyme
function but also how they interact with the external solvent medium in which
they are dissolved or suspended. This can have important ramifications when
enzymes are employed under partially or completely nonaqueous conditions.
Like all catalysts, enzymes increase reaction rates by lowering their activation
energies. The most important difference between enzymes and simple catalysts
such as a proton or hydroxide is that the former are much more restrictive in the
range of acceptable substrates. The three dimensional structure of the enzyme
allows binding of only those starting materials (usually referred to as substrates)
whose structures are congruent with the size, shape and polarity of the catalytic
portion of the enzyme (the “active site”). Formation of this noncovalent complex
prior to chemical conversion is the key to the high selectivity of enzyme-catalyzed reac-
tions since it places the substrate into a specific location where its functional groups are
oriented precisely with those on the enzyme.
1
Some RNA molecules also possess catalytic abilities; however, their substrate and range of chemical
conversions seems rather limited and for this reason, catalytic RNA molecules lie outside the scope
of this book.
Organic Synthesis Using Biocatalysis. http://dx.doi.org/10.1016/B978-0-12-411518-7.00001-9
Copyright © 2016 Elsevier Inc. All rights reserved.
2 Organic Synthesis Using Biocatalysis
Noncovalent complex formation allows chemical reactions between specific

amino acids on the enzyme and functional groups on the substrate to occur
in an environment that is kinetically equivalent to a unimolecular process.
Transforming what would otherwise be bimolecular reactions into effectively in-
tramolecular conversions is a major reason that enzymes can accelerate reactions
by up to 23 orders of magnitude over the background (uncatalyzed) reaction [1].
Selectivity is the second benefit from forming a noncovalent enzyme–substrate
complex prior to chemical conversion. By focusing the catalytic attention of the
enzyme onto a specific area of the substrate, reactions can be restricted to a
single portion of the molecule that may or may not be the most reactive por-
tion of the overall substrate structure. This allows enzyme-catalyzed reactions
to be selective in many respects: chemoselective (carrying out only one specific
transformation while others are possible), regioselective (transforming only one
among several possible sites), and stereoselective (producing and/or consuming
one stereoisomer in preference to others).
2 DEFINITION OF BIOCATALYSIS
In nature, enzymes catalyze transformations of metabolites that occur within
and/or outside of living cells. Although some enzymes accept only a limited
variety of substrates, a large fraction is more tolerant and allows conversions
of nonnatural starting materials. The field of biocatalysis rests upon this partial
promiscuity. If enzymes were truly selective for only a single substrate, it would
be impossible to utilize them for synthesizing new molecules from nonnatural
substrates. The goal is to identify or engineer enzymes that are sufficiently gen-
eral to accept a variety of related substrates, but selective enough to yield single
products or stereoisomers. We use the term “biocatalysis” to describe the use of
enzymes (either native or modified) for synthetic transformations of nonnatural
starting materials. Enzymes used for in vitro synthetic transformations are called
biocatalysts, and the processes are called biocatalytic transformations.
3 SCOPE OF THIS BOOK

Some enzymes catalyze the reactions that build up large molecules from simple
building blocks, for example, complex carbohydrates from carbon dioxide and wa-
ter or the synthesis of steroids and terpenoids from acetate. Others are involved in
the degradation of large assemblies to small molecules, for example, hydrolysis of
proteins to amino acids and the oxidative degradation of lignin. Although some of
these native conversions are industrially important and practiced on large scales, the
use of enzymes to produce their normal primary and secondary products of cells
lies outside the scope of this book. Here, our focus is on preparing nonnatural com-
pounds using enzymes since this addresses the need commonly encountered in or-
ganic synthesis. However, it should be noted that the native reactions of an enzyme
can often be used as starting points for their applications to nonnative reactions.
The field of metabolic engineering also lies outside the scope of this book. These
efforts use two or more enzymes to catalyze sequential steps in a pathway that
Introduction, Types of Reactions, and Sources of Biocatalysts Chapter 1 3
links a simpler starting material such as glucose with a final intracellular target
product such as butanol or lysine. In some cases, the complete pathway already
exists within a single organism; in others, enzymes from different sources are
assembled into an artificial metabolic pathway in a suitable host cell. The key
difference between biocatalysis and metabolic engineering is that the molecu-
lar skeletons are provided in vitro in the former case and in vivo in the latter.
Although it is economically attractive to produce a target molecule by meta-
bolic engineering, this benefit must be balanced against the (usually) lengthy
optimization phase required for efficient production and the restriction that
intermediates and the final product should be nontoxic to the host cells.
4 KEY BENEFITS OF EMPLOYING ENZYMES

IN SYNTHESIS
Enzymes offer several attractive features as catalysts for organic synthesis. They
often show high selectivities, they can operate under mild conditions and they
are completely biodegradable catalysts constructed solely from renewable re-
sources. They are thus ideal strategies as chemistry embraces sustainability.
4.1 Selectivity: Chemo-, Regio- and Stereo-

Biocatalytic reactions can show very high selectivities in all respects (Figure 1.1).
When similarly-reactive functional groups are present in a molecule, the enzymes
often catalyze only the reaction of one while leaving the others intact. For example,
nitrile hydratases catalyze the partial hydrolysis of a nitrile group to yield a primary
amide without cleaving an ester moiety present in the same molecule or further
hydrolyzing the amide product, a property referred to as “chemoselectivity.”
“Regioselectivity” is another useful property displayed by many enzymes. This
refers to the transformation of one functional group while leaving other iden-
tical (or nearly identical) moieties at different locations within the molecule
untouched. For example, among three esters in a triacylglyeride, many lipases
hydrolyze only one position, and do not catalyze further hydrolysis of the dies-
ter product.
All but one of the amino-acid building blocks have at least one chiral center,2
and for this reason, enzymes are intrinsically asymmetric catalysts. The asymmet-
ric nature of enzymes results in enantioselectivity when biocatalysts convert a
prochiral starting material into a single product enantiomer, for example, in ke-
tone or imine reductions. The same asymmetric nature also causes the biocata-
lysts to preferentially transform only one stereoisomer of a starting material that
contains a mixture of diastereomers or enantiomers. Such a process is referred to
as a kinetic resolution and the ratio of rate constants for the fast- and slow react-
ing enantiomers is termed the enantioselectivity (E) ratio. E values higher than
100 are commonly observed for biocatalysts, and values in this range allow both
the residual starting material (the slow-reacting enantiomer) and the product
2
Glycine is the only achiral amino acid normally found in proteins.
FIGURE 1.1
Types of selectivity exhibited by enzymes. Biocatalytic processes can provide chemo-,
regio-, and stereoselective conversions. Representative examples can be observed in reactions
catalyzed by nitrile hydratases, lipases and dehydrogenases.
(from the faster-reacting enantiomer) to be obtained with high optical purities

from a single reaction run to 50% conversion.
4.2 Biocatalyst Reaction Conditions

Enzymes have generally evolved to function best under the conditions that ex-
ist within their respective cellular environments. This usually means ambient
temperatures (20–40°C), near-neutral pH values, and with water as the solvent.
Such conditions are particularly appropriate for sensitive starting materials and/
or products, and this constitutes an important reason to employ enzymes in
organic synthesis. It should be noted, however, that some enzymes have evolved
to function under extreme conditions. For example, thermophilic bacteria that
thrive at temperatures more than 100°C have been valuable sources of enzymes
with much higher-than-usual thermal stabilities. In addition, enzymes normally
found outside cells (extracellular enzymes) are also generally more stable since
their operating environment is less predictable and largely uncontrolled. This
diversity in thermal stabilities often allows one to choose a reaction tempera-
ture that balances good reaction rates with enzyme stability and also maximizes
space–time yields.
In nature, most enzyme-catalyzed reactions occur in an aqueous environment

and many synthetic applications, therefore, also utilize water as solvent. This is
often advantageous with regard to maximizing the sustainability of a chemical
process. Moreover, enzymes are usually most stable in water. These benefits,
however, must be balanced against two disadvantages of using water as a sol-
vent for biocatalytic processes. Water is a reactant in hydrolytic processes and
its concentration must be minimized when such reactions are run in reverse in
order to shift the equilibrium, for example, when using enzymes to synthesize
esters or amides from carboxylic acids and alcohols or amines, respectively. In
such cases, water-organic biphasic systems or completely organic solvents can
be used. The second complication associated with aqueous conditions is that
many of the starting materials and products of synthetic interest have very lim-
ited solubilities in water. This either requires the use of dilute solutions, which
lowers space–time yields and also generates large volumes of wastewater that
must be treated, or the use of organic solvents as additives or replacements for
water.
Biocatalysts are proteins and composed of natural amino acids. In some cases,
additional natural ligands (cofactors) are present. This means that biocatalysts
are inherently nonhazardous materials, although it should be noted that, be-
cause they are proteins, some enzymes may cause allergenic reactions in suscep-
tible individuals. Although such reactions are rare, normal care should be taken
when handling solid enzyme powders.
5 MECHANISM AND KINETICS

OF ENZYME-CATALYZED REACTIONS
Although it is not necessary to determine kinetic parameters in order to use
enzymes for chemical synthesis, this knowledge can often be useful in deciding
which avenues offer the best opportunities for process improvements. Likewise,
it is not essential – but often highly useful – to understand the mechanism
of the enzyme-catalyzed reaction for the same reasons that one often benefits
from knowing the mechanisms of any reaction employed in a synthetic route.
Although every individual enzymatic reaction has a unique combination of
kinetic properties and reaction mechanism, several useful generalizations are
summarized in the subsequent section.
5.1 Features of Enzyme Catalyzed Reactions

As noted previously, noncovalent association between the enzyme and its
substrate(s) is the essential first step in biocatalytic reactions. Although early
theories of enzyme catalysis focused primarily on interactions between the en-
zyme and substrate, it was later appreciated that maximizing selective, nonco-
valent interactions between the enzyme and the high-energy transition state(s)
that linked enzyme-bound complexes of substrates and products was the key
to efficient rate enhancements. A somewhat oversimplified view is that ground-
state interactions determine substrate specificity and transition-state interac-
tions yield rate enhancements.
In addition to noncovalent associations (by van der Waals forces, hydrogen

bonds, electrostatic and hydrophobic interactions), some enzymes also form
covalent bonds between the enzyme and portions of the substrate. Lipases are
a well-known example of this phenomenon. These enzymes utilize a specific
protein hydroxyl group (most commonly a serine side chain) to form an ester
intermediate with the substrate that is subsequently cleaved by an exogenous
nucleophile to form the final reaction product and regenerate the free protein
hydroxyl group, making the active site suitable for the next catalytic cycle.
5.2 Coenzymes
Although some enzyme-catalyzed reactions utilize only functional groups found
in the protein, the limited number and variety of amino-acid side-chain moieties
severely limits the range of accessible reactions. For example, there are neither
common amino acids with electrophilic side chains nor amino-acid functional
groups suitable for redox catalysis.3 Nature has circumvented this problem by
evolving a suite of coenzymes (also known as cofactors) that specialize in par-
ticular types of chemical conversions. Table 1.1 lists some common cofactors
for biocatalytic processes. In some cases, for example, biotin and some flavins,
these cofactors are covalently coupled to the enzyme within the active site. Other
cofactors such as nicotinamides are bound reversibly by noncovalent forces dur-
ing the entire catalytic cycle. Finally, a few cofactors such as pyridoxal phosphate
form reversible covalent linkages with the resting form of the enzyme that are
cleaved during the catalytic cycle, and then re-formed at the end. When needed
by specific enzymes, provision for cofactor supply must also be made. Because
of their expense, cofactors are normally supplied in substoichiometric quanti-
ties (usually ≪ 0.1 mole %). This means that they must be regenerated prior to
the start of the next catalytic cycle, and strategies for cofactor regeneration have
been developed as an essential adjunct for biocatalytic reactions, particularly for
reductions and oxidations.
5.3 Kinetics and Reaction Mechanisms

Reaction mechanisms describe the sequence of bond breaking and bond mak-
ing steps, whereas kinetics are concerned with the nature and timing of the
noncovalent complexes that form and break down during the catalytic cycle.
Depending on the number of substrates and products, kinetics can be simple or
complex. Single-substrate/single-product reactions are the most straightforward
schemes, although there are relatively few examples of such reactions in bioca-
talysis. More commonly, two or more molecules are bound and/or released. This
introduces the question of timing with respect to formation and breakdown of
enzyme – ligand complexes. Three common reaction mechanisms for a two-
substrate/two-product reaction are illustrated schematically in Figure 1.2. In an
ordered mechanism, the enzyme cannot productively bind the second substrate
3
The only exception is disulfide bond formation between a pair of suitably positioned cysteine
side-chains.
Introduction, Types of Reactions, and Sources of Biocatalysts Chapter 1
Table 1.1 Cofactors Commonly Encountered in Biocatalytic Reactions Applied to Chemical Synthesis
Name Cofactor Structure Chemical Function Enzyme
Nicotin- Donates a hydride for polar Alcohol dehydrogenase/
amide adenine reductions of functional ketoreductase; alkene
dinucleotide, groups such as C ═ O and reductase/enoate reductase;
reduced form C ═ N; acts as an electron amino acid dehydrogenase;
(NADH)a source for monooxygenases monooxygenase;
dioxygenase
Nicotinamide Donates a hydride for polar Alcohol dehydrogenase/

adenine dinucleotide reductions of functional ketoreductase; alkene
phosphate, reduced groups such as C ═ O and reductase/enoate reductase;
form (NADPH)b C ═ N; acts as an electron amino acid dehydrogenase;
source for monooxygenases monooxygenase;
dioxygenase
Flavin Forms a hydroperoxy Alkene reductase/enoate

mononucleotide intermediate from O2 that is reductase; amino acid
(FMN) used by monooxygenases; oxidase; Baeyer–Villiger
following 2 electron monooxygenase
reduction, donates a hydride
for reductions of electron-
deficient C ═ C bonds
(Continued)
7
8
Organic Synthesis Using Biocatalysis
Table 1.1 Cofactors Commonly Encountered in Biocatalytic Reactions Applied to Chemical Synthesis (cont.)
Name Cofactor Structure Chemical Function Enzyme
Flavin adenine Forms a hydroperoxy Alkene reductase/enoate
dinucleotide (FAD) intermediate from O2 that is reductase; amino acid
used by monooxygenases; oxidase; Baeyer–Villiger
following 2 electron monooxygenase
reduction, donates a hydride
for reductions of electron-
deficient C ═ C bonds
Thiamine Allows umpolung anion Pyruvate decarboxylase;

pyrophosphate formation by aldehyde benzoylformate
carbonyl groups; facilitates decarboxylase;
a-keto acid decarboxylations phenylpyruvate
decarboxylase
Pyridoxal phosphate Following reversible Schiff’s Transaminase; threonine

base formation, the cofactor aldolase
stabilizes an anion on the
carbon adjacent to the amine
Known as DPNH in the very old scientific literature

a
Known as TPNH in the very old scientific literature

b
FIGURE 1.2
Three possible kinetic mechanisms for a two-substrate/two-product reaction depicted in
Cleland notation. Substrates are designated as “A” and “B”, products as “C” and “D” while “E”
represents the enzyme and “E*” is a covalently modified enzyme that forms transiently as part
of the normal catalytic pathway in the ping-pong mechanism.
(B) until it previously bound the first (A). Enzymes that follow this scheme of-
ten show substrate inhibition when the concentration of B is very high (relative
to that of A) since this favors formation of a dead-end E · B complex that must
dissociate prior to re-joining the productive pathway. A random mechanism re-
moves this restriction and either substrate can be bound first. Product release
steps can be similarly described.4 From a kinetic standpoint, cofactors that do
not remain permanently bound to the enzyme are equivalent to substrates and/
or products. Cofactors that remain bound to the enzyme during turnover are
kinetically treated as part of the enzyme itself. In either an ordered or random
4
It should be noted that “substrate” and “product” are meaningful only when the direction of the
reaction is specified. Like all catalysts, enzymes accelerate both the forward and reverse reactions.
mechanism, the key species is the ternary complex of enzyme with both sub-
strates (E · A · B) since this must be formed in order for the reaction to occur.
Enzymes that form a covalent bond with a part of the substrate often follow a
ping-pong mechanism (Figure 1.2). By definition, these must be ordered mech-
anisms. Ping-pong mechanisms always share two essential features. First, they
involve a covalently modified form of the enzyme (or cofactor) as an obligate
intermediate.5 In addition, the first product must be released from the active site
before the second substrate binds. Together, these two properties of enzymes that
follow ping-pong mechanisms open the possibility that the covalent intermedi-
ate might be redirected into alternative products by restricting access to the nor-
mal second substrate. Employing lipases for synthetic (rather than hydrolytic)
acyl transfer reactions is a very common example of this strategy and others can
be found in the appropriate sections of later chapters.
Although the methods for determining steady-state kinetic parameters can be
complex and the details lie outside the scope of this book, a general understand-
ing of their meaning is useful for applications to biocatalysis. It should be borne
in mind that most substrates and products in biocatalytic reactions have limited
aqueous solubilities. This complicates kinetic studies since the actual concentra-
tion “seen” by the enzyme may not be the same as the concentration added to
the reaction mixture unless all of the material is fully dissolved. Therefore, one
must be cautious when applying kinetic constants measured under one set of
experimental conditions (often dilute aqueous solutions) to reactions run un-
der process conditions that may involve partially dissolved substrates, organic
cosolvents, etc.
5.4 Kinetic Constants

Two steady-state kinetic constants are most useful in evaluating biocatalytic re-
actions. “kcat” is often known as the turnover number and higher values indicate
more catalytically efficient enzymes. This first-order rate constant describes the
speed at which an enzyme converts bound substrates to products and re-forms
the free enzyme to prepare for the next round of catalysis. It includes both the
“chemical” steps (bond making and bond breaking) as well as the product re-
lease step(s). Note that it is not uncommon for product release to be the slowest
step. As a practical matter, one normally seeks enzymes with kcat ≥ 1 s−1 under
the process conditions to ensure reasonable space–time yields along with ac-
ceptable catalyst loading levels.
“KM”, also known as the Michaelis constant, is a composite of several microscopic
rate constants that primarily describes substrate/product binding and has units
of concentration (typically M). Consistent with normal biochemical convention,
the binding interaction is considered in the dissociation direction and smaller
numerical values of KM therefore signal tighter substrate/product binding. When
5
The covalently modified form of the enzyme has been variously notated as “F,” “E,*” or “ E’ ” in
the literature.
the chemical step is slow relative to substrate release,6 KM is approximately equal

to the actual thermodynamic dissociation constant (denoted as KD). KM is also
equivalent to the substrate concentration required to give a reaction velocity (V)
that is one-half the maximal value (denoted as Vmax = kcat · [enzyme]). If only
a single substrate is involved, only a single KM value is required to describe the
reaction. On the other hand, for enzymes that require multiple substrates, each
has an associated KM value. Because a complete kinetic characterization is labori-
ous in these situations, KM values are often determined for only the substrate of
interest by varying its concentration while holding all others constant. Such stud-
ies yield apparent KM values (KM,app). These are very common for nicotinamide-
dependent reactions since the cofactors are typically held at a constant value
during biocatalytic processes.
Although not a kinetic constant per se, the ratio kcat/KM is often used to char-
acterize enzyme performance in a biocatalytic process. This ratio is often re-
ferred as the specificity constant and has units of a second-order rate constant
(M−1 · s−1). The kcat/KM ratio reflects the difference in relative energies between
the free enzyme and free substrate compared to the rate-limiting transition
state(s). The maximum value is approximately 1 × 108 M−1 · s−1, which is
the diffusion limit of small molecules in aqueous solution. In approximate
terms, kcat/KM describes the performance of an enzyme-catalyzed reaction
under nonsaturating conditions whereas kcat reflects its behavior under satu-
rating conditions. The most appropriate descriptor for a given biocatalytic
process depends upon the enzyme/substrate affinity and the concentrations
actually employed.
5.5 Kinetic Constants and Kinetic Resolutions

When presented with a mixture of substrate isomers, enzymes often show a pref-
erence for transforming only one. When the isomers are enantiomers, the pro-
cess is referred to as a kinetic resolution. Continued conversion of the favored
enantiomer increases the optical purity of the residual starting material in favor
of the slow-reacting enantiomer whereas the product is drawn primarily from
the fast-reacting enantiomer. The enantioselectivity (E) value is defined as the
numerical ratio of rate constants for the faster- versus the slower-reacting enan-
tiomer. Whether kcat or kcat/KM values are more appropriate in determining the
E ratio for a given reaction depends on the substrate concentration employed
and the enzyme/substrate affinity. A simple means to calculate the E value from
experimentally observable values are available [2]. As noted previously, one
usually targets enzymes with E values ≥ 100 in order to achieve good kinetic
resolutions.
It is important to note that racemic mixtures are the thermodynamic minima
for all reactions. Because enzymes catalyze reactions in both directions, even
enzymes with very high stereoselectivities will ultimately yield racemic mixtures
6
This is not an uncommon situation in practice, particularly when enzymes catalyze reactions of
nonnatural substrates.
if reactions are allowed to reach equilibrium. This can be a problem if extended

reaction times and/or very high catalyst loadings are employed. Obtaining race-
mic products from an enzyme-catalyzed reaction does not necessarily mean that
the enzyme has low stereoselectivity; the reaction should be explored further by
reducing the reaction time and/or enzyme concentration.7
5.6 Kinetic Constants and Temperature

Like all chemical reactions, the rates of enzyme-catalyzed conversions increase
with increasing temperature as predicted by the Arrhenius relationship. This re-
lationship only applies to a limited range, however, since enzymes are deactivat-
ed by higher temperatures. In addition, substrates and/or products may degrade
at elevated temperatures, particularly in water. This means that temperature ef-
fects on reaction rate and stability are diametrically opposed and the best choice
is necessarily a compromise. In practice, most biocatalytic processes are run at
temperatures between room temperature and approximately 50°C. Somewhat
higher optimum reaction temperatures may be achievable with thermophilic
enzymes possessing higher deactivation temperature.
6 TYPES OF ENZYME-CATALYZED REACTIONS

COMMONLY USED IN BIOCATALYSIS
Though nature uses many different types of enzymes to catalyze a wide vari-
ety of different transformations, only a limited number of reaction types have
been widely used in biocatalysis. An overview of the most common varieties is
provided below, approximately arranged by decreasing number of published ex-
amples. Detailed descriptions are available in subsequent chapters dedicated to
specific reaction types. Table 1.2 summarizes reaction types, starting materials,
products, and enzymes required for the most common types of biocatalytic reac-
tions. This provides a quick reference for retrosynthetic planning and designing
syntheses of specific molecules.
6.1 Hydrolysis and Synthesis of Carboxylic Acid Derivatives

such as Esters and Amides
This is the most common application of biocatalysis in organic synthesis and
represents the majority of published examples. Enzymes that catalyze acyl trans-
fer reactions of esters and amides are widely distributed in nature and belong
to the lipase/esterase and protease/amidase families, respectively. They play key
roles in the metabolism of lipids and proteins and the choice of names, lipase
versus esterase, is subject to debate. Normally, acyl transfer occurs almost ex-
clusively to water, resulting in hydrolysis. This is particularly valuable for am-
ide hydrolysis that normally requires forcing conditions and strong acid or
7
Finding products that are completely racemic nearly always indicates that the catalyst concentration
and or reaction time is too great. Even poorly stereoselective enzymes typically afford some optical
enrichment and only reaching true thermodynamic equilibrium completely erases this preference.
Table 1.2 Summary of Common Biocatalytic Transformations

Starting
Reaction Type Material(s) Product(s) Common Uses Enzyme Types
Hydrolysis Esters Alcohols and Synthesis of alcohols Lipase, esterase,
carboxylic acids and carboxylic acids; protease
resolution of esters,
alcohols, acids
Amides Amines and Synthesis of amines Lipase, esterase,
carboxylic acids and carboxylic acids; protease
resolution of amides,
acids and amines
Nitriles Amides Synthesis of primary Nitrile hydratase
amides; resolutions of
nitriles
Nitriles Carboxylic acids Synthesis of carboxylic Nitrilase
acids; resolutions of
acids
Epoxides 1,2-Diols Synthesis of diols; Epoxide hydrolase
resolution of epoxides
Esterification Carboxylic acid Ester Synthesis of esters; Lipase, esterase,
and alcohol resolutions of protease
carboxylic acids and
alcohols
Amidation Carboxylic acid Amide Synthesis of amides; Lipase, esterase,
and amine resolutions of protease
carboxylic acids and
amines
Transesterification Alcohol and Alcohol and Synthesis of esters; Lipase, esterase,
ester ester resolution of esters, protease
alcohols
Transamination Ketone and Ketone and Synthesis of amines Transaminase
amine amine
a-Keto acid and a-Keto acid and Synthesis of a-amino Amino acid
a-amino acid amino acid acids transaminase
Dehydro- Halohydrin Epoxide Synthesis of epoxides; Halohydrin
halogenation resolution of halohydrins dehalogenase
and epoxides
Carbonyl Ketone Alcohol Synthesis of alcohols Alcohol
reduction dehydrogenase/
ketoreductase
Aldehyde Alcohol Synthesis of alcohols Alcohol
dehydrogenase/
ketoreductase
(Continued)
Table 1.2 Summary of Common Biocatalytic Transformations (cont.)

Starting
Activated alkene a,b-Unsaturated Saturated Asymmetric reduction Alkene reductase
reduction aldehyde, ketone, aldehyde, ketone, of C ═ C bonds (also known as
ester ester enoate reductase
and ene-reductase)
Reductive Ketone, 2-keto Amine, 2-amino Synthesis of amines Amino acid
amination acid acid dehydrogenase
Alcohol oxidation Secondary Ketone Synthesis of ketones Alcohol
alcohol dehydrogenase,
alcohol oxidase
Primary alcohol Aldehyde Synthesis of aldehydes Alcohol
dehydrogenase,
alcohol oxidase
Amine oxidation Amine Aldehyde, ketone Synthesis of aldehydes Amine oxidase
and ketones; resolu-
tions of amines
a-Amino acid a-Keto acid Synthesis of a-keto Amino acid
acids; resolutions of oxidase,
amino acids amino acid
dehydrogenase
Hydroxylation Saturated Alcohols Synthesis of alcohols Monooxygenase,
carbon–hydrogen peroxidase
bond
Aromatic Phenols Synthesis of phenols Monooxygenase,
carbon-hydrogen dioxygenase,
bond peroxidase
Oxidative Phenyl ethers Phenol, aldehyde Synthesis of phenols; Monooxygenase,
dealkylation dealkylation of methyl peroxidase
or other alkyl ethers
Alkylated anilines Amine, aldehyde Synthesis of amines; Monooxygenase,
dealkylation of methyl Peroxidase
or other secondary
amines
Baeyer–Villiger Ketone Ester Synthesis of esters Baeyer–Villiger
oxidation and lactones monoxygenase
Cyanohydrin Aldehyde or 2-Hydroxy Synthesis of Oxynitrilase
formation ketone nitrile 2-hydroxy nitriles (Hydroxynitrile
lyase)
Table 1.2 Summary of Common Biocatalytic Transformations (cont.)

Starting
Aldol Aldehyde 2-Keto acid Synthesis of Pyruvate
condensation 2-hydroxy ketones decarboxylase,
benzoyl formate
decarboxylase,
phenylpyruvate
decarboxylase
Aldehyde 2-Hydroxy Synthesis of Deoxyribose
aldehyde 3,4-dihydroxy phosphate aldolase
aldehydes
Aldehyde, 1-Phosphorylated Synthesis of Dihydroxyacetone
dihydroxyacetone 2-keto-3,4-diol polyhydroxylated phosphate aldolase
phosphate 2-ketones
Aldehyde 2-amino acids Synthesis of 3-hydroxy Threonine aldolase
2-amino acids
The table is organized by reaction type, rather than by enzyme type to facilitate use in synthetic applications.
base. The reverse reaction (ester and amide synthesis) cannot be carried out un-
der aqueous conditions due to the large molar excess of water (whose concentra-
tion is 55 M in pure water). Enzymes can catalyze the reverse reaction – ester and
amide synthesis from acids and alcohols or amines – under nonaqueous con-
ditions, although the yields are limited by thermodynamic constraints unless
steps are taken to remove the water formed during the reaction. This limitation
can be circumvented by using enzymes to catalyze transesterification and trans-
amidation, using an ester or amide as the starting material and relying on Le
Chatêlier’s principle to shift the equilibrium to the desired product (Figure 1.3).
Acyl transferase enzymes have been widely used to synthesize chiral esters, am-
ides, alcohols, and amines. In many cases, these conversions involve kinetic
resolutions of alcohols, acids, esters, amines, and amides. Of course, since each
enantiomer makes up half of the racemic mixture, kinetic resolutions can pro-
vide a maximum 50% yield. This limitation can be overcome by racemizing
or inverting the configuration of the unreacted substrate during the enzymatic
reaction. Such a scheme is referred to as a dynamic kinetic resolution and theoreti-
cally allows complete substrate conversion to product along with 100% chemi-
cal yield of a single product enantiomer.
6.2 Carbonyl Reductions to Alcohols

Another common biocatalytic route to alcohols involves enzyme-mediated
reductions of the corresponding aldehydes or ketones. Because the starting
materials are prochiral, such processes are not kinetic resolutions and are there-
fore not subject to the 50% yield limitation. Enzymes that catalyze carbonyl
FIGURE 1.3
Hydrolysis and synthesis of carboxylic acid derivatives such as esters and amides.
reductions have been variously named as alcohol dehydrogenases or ketoreduc-

tases.8 Because carbonyl reductions involve the formal addition of H2 (usually
in the form of a hydride ion along with a proton), a second cosubstrate that
supplies reducing equivalents must be included along with the aldehyde or ke-
tone of synthetic interest. Nicotinamides are the most common hydride sources
for enzymatic reactions and they occur in the form of nicotinamide adenine
dinucleotide (NADH) and its phosphorylated analog (NADPH). Some enzymes
are highly selective for one cofactor type or the other whereas others accept
both NADH and NADPH (a property referred to as dual specificity). Carbonyl
reduction converts the nicotinamides into their oxidized forms and these must
be reduced back to their original oxidation states prior to reuse in subsequent
rounds of catalysis. Several methods for in situ cofactor regeneration have been
developed in response to the high cost of nicotinamides and cofactor turnover
numbers of more than 1000 are commonly achievable. This usually makes the
cofactor contribution to the overall process costs negligible (Figure 1.4).
Alcohol dehydrogenases/ketoreductases are widely distributed in nature where
they play many roles in metabolism. Bakers’ yeast is a particularly prolific
FIGURE 1.4
Carbonyl reductions to alcohols.
8
The “alcohol dehydrogenase” nomenclature is more common in the biochemical literature where-
as “ketoreductase” is typically used in biocatalysis since this is the synthetically more useful direc-
tion for the reaction.
producer of carbonyl-reducing enzymes that accept a diverse range of substrates.

Moreover, because living yeast cells continuously regenerate nicotinamide co-
factors by metabolizing simple sugars such as sucrose or glucose, whole yeast
cells purchased from a local grocery store can be used directly as biocatalytic
reducing agents in the lab. Many successful examples of this strategy have been
disclosed and they constituted one of the earliest widescale applications of bio-
catalysis to organic synthesis. The major disadvantage of whole yeast cells is
the large quantity of extraneous biomass that accompanies the relevant ketore-
ductase. This often makes scale-up difficult. Several vendors have responded to
this problem by making individual ketoreductases available in purified form. In
addition to minimizing the amount of added biomass, the use of purified yeast
ketoreductases eliminates competition between enzymes with overlapping sub-
strate specificities but divergent stereoselectivities.
Ketoreductases can also be used to catalyze alcohol oxidations to the correspond-
ing aldehydes or ketones when provided with oxidized nicotinamide cofactors.
The use of enzymes for this conversion is less commonly employed in synthesis
for two reasons. First, alcohol oxidations often involve kinetic resolutions that
are subject to the 50% yield limitation. When the same reactions are run in re-
verse (enzyme-catalyzed reduction of the carbonyl compound), one can obtain
a single enantiomer with a theoretical yield of 100%. The second problem with
dehydrogenase-catalyzed alcohol oxidations is that provision for regenerating
oxidized nicotinamide cofactors must also be made. Until recently, few good
methods for this conversion were available, although Bommarius’ development
of water-producing NADH oxidases has helped to overcome this problem [3].
Another efficient way is coupling with glutamate dehydrogenase enzyme and
conversion of glutamate to alpha-keto glutarate [4].
6.3 Transamination
Optically pure amines are very common synthetic targets, either as final prod-
ucts or key intermediates. In fact, many optically pure alcohols were prepared
specifically to generate leaving groups for chiral amine synthesis by subse-
quent SN2 reactions. Directly converting prochiral carbonyl starting materials
to optically pure amines – the synthetic equivalent of a chiral reductive ami-
nation – would clearly be much more efficient. Transaminases catalyze these
reactions by transferring an amino group from a donor amine to an acceptor
ketone or aldehyde. Until recently, virtually all known transaminases required
an a-amino-acid donor and showed limited diversity in acceptor ketone struc-
tures. Although this allowed synthesis of many amino acids and related ana-
logs, it also limited the range of accessible products. Moreover, because the
reaction is reversible, a large excess of the amino acid was usually required
to shift the equilibrium toward the desired product and make appreciable
quantities of amines from ketones. The last problem has largely been solved
by developing transaminases that accept isopropylamine as the amine donor.
Not only is the amine donor inexpensive but the acetone by-product can be
also removed during the reaction by evaporation that allows the reactions
FIGURE 1.5
Transamination.
to be driven to completion without large molar excesses of the amine donor.

The same types of protein engineering efforts have also broadened the range of
allowable amine acceptors for transaminases to encompass synthetically impor-
tant structures. The only drawback to transaminases is that relatively fewer are
available “off the shelf” with synthetically useful substrate ranges as compared
to alcohol dehydrogenases/ketoreductases, although this situation is improving
rapidly (Figure 1.5).
6.4 Oxygen-Dependent Oxidations

A variety of enzymes catalyze substrate oxidations that involve atmospheric O2
as a reactant. Monooxygenases, dioxygenases, peroxidases, and laccases are the
most common enzymes in this class and synthetic applications of each type
have been developed. Although some enzymes in this class use only organic
cofactors, for example, flavins, others contain one or more tightly bound metal
ions in the active site. Typical metals are iron, copper, and manganese with the
first being most common. Regardless of their identity, the role of the cofactor is
to interact directly with O2 and form the actual oxidant.
6.5 Hydroxylation by C─H Bond Insertion

A variety of enzyme systems catalyze the insertion of oxygen into substrate
C─H bonds. Insertions into alkyl C─H bonds yield alcohols as the initial
oxidation products while aryl C─H bonds yield phenols. Enzymatic hydrox-
ylation of an otherwise inactive center of an alkane or an aromatic hydrocar-
bon can provide alcohols or phenols that are often difficult (if not effectively
impossible) to obtain by any other means. Moreover, most biological hydrox-
ylations occur with very high regio- and stereocontrol so that only one en-
antiomer of a single product is typically obtained. In fact, one of the earliest
applications of biocatalysis for organic synthesis in pharmaceutical industry
was the use of a highly selective enzymatic hydroxylation that greatly facili-
tated production of corticosteroids [5]. Biocatalytic hydroxylations have been
critically important components of the synthetic toolkit for steroid chemistry
ever since.
FIGURE 1.6
Enzymatic hydroxylation by C─H bond insertion.
Cytochrome P-450s are the best-known class of hydroxylation enzyme. Their

active sites contain a heme iron that forms a highly activated oxygenating
species that reacts by a radical mechanism. In higher animals, they function
primarily in metabolite degradation as part of pathways that clear unnatural
substances such as toxins and drugs. Hydroxylation increases polarity that fa-
cilitates further derivatization by other detoxification enzymes or excretion
of the hydroxylated products. Other P-450 family members are involved in
secondary metabolite biosynthesis, particularly in plants and microbial cells
(Figure 1.6).
With few exceptions, most C─H hydroxylating enzymes are composed of mul-
tiple protein subunits and many are membrane bound and unstable in purified
form. All of these properties conspire to make such enzymes difficult to handle
as isolated proteins. In addition, oxygen activation requires a pair of electrons
that are typically supplied by NADH, which introduces cofactor regeneration
as an additional complication. For all of these reasons, most preparative bio-
catalytic hydroxylations have been carried out by mixing intact microbial cells
with the substrate of interest (analogous to the way that Bakers’ yeast cells have
been employed in carbonyl reductions). The main difficulty is that many organ-
isms produce more than one P-450 enzyme and this can lead to multiple prod-
ucts from a single reaction. Several groups have therefore created recombinant
strains that express single P-450’s in “clean” hosts that minimize side reactions.
In addition, by overproducing large quantities of the relevant enzymes, the oxi-
dations are often more efficient with regard to space–time yields.
6.6 O- or N-Dealkylations
Enzymatic hydroxylation of a carbon adjacent to an oxygen or nitrogen usually
results in dealkylation by spontaneous hydrolysis of the initial hemiacetal or
hemiaminal product. These conversions are commonly employed for two syn-
thetic purposes: cleavage of methyl ethers and oxidative deamination of amines.
The latter is particularly useful in amino-acid chemistry. These reactions can be
catalyzed by P-450 monooxygenases or by flavin-containing monooxygenases
(which are typically metal-free enzymes). As in the previous example, these hy-
droxylations require two electrons that must be supplied by NADH or NADPH,
and most synthetic applications have relied on whole microbial cells rather than
the isolated enzymes (Figure 1.7).
FIGURE 1.7
O- or N-dealkylations.
6.7 Oxidative Deamination of Amines to Carbonyl

Compounds and the Reverse Reaction
Oxidative deamination by amine oxidases generates ketones or aldehydes. There
are two types of amine oxidases. Those of the Type I contain both copper and
a covalently attached topaquinone cofactors. In these enzymes, amine oxida-
tion first generates an enzyme bound imine that is subsequently hydrolyzed
to a ketone that is finally released from the enzyme. Type II amine oxidases are
metal-free enzymes and contain only a flavin cofactor that remains bound to
the enzyme throughout the catalytic cycle. Catalysis by these enzymes produces
an imine intermediate that is released from the enzyme and subsequently hy-
drolyzed to the ketone in aqueous medium. To complete the catalytic cycle, the
reduced flavin cofactor is oxidized by molecular oxygen generating hydrogen
peroxide. In both cases, hydride (or a hydride equivalent) is transferred to the
cofactor. Many amine oxidases are highly stereoselective, and this makes them
very useful in kinetic resolutions of amines. For example, enantioselective oxida-
tion of one antipode of 26 removes this stereoisomer from the reaction mixture,
leaving behind the unreacted enantiomer. As in all kinetic resolutions, however,
the maximum yield of the enantiopure product is 50% (Figure 1.8).
Several ingenious solutions to overcome the 50% yield problem inherent
in amine kinetic resolutions have been devised. The most practical pairs are an
amine oxidase with another enzyme or in situ chemical reaction that changes
the process into a dynamic kinetic resolution with a 100% theoretical yield.
The Turner group developed a very successful strategy in which a biocompat-
ible chemical reducing agent such as amino borane was added along with the
racemic starting amine and an enantioselective Type II amine oxidase [6]. En-
zymatic oxidation depleted the reactive amine enantiomer; the imine product
of this step was reduced in a racemic fashion by the amino-borane reagent. The
net result was to racemize the starting amine that yielded 50% of the unreactive
(desired) amine enantiomer along with another 50% destined for re-oxidation
by the enzyme. After several rounds of oxidation/reduction, essentially all of
the “wrong” enantiomer had been converted to the desired product. This is a
very powerful strategy, particularly when combined with protein engineering to
FIGURE 1.8
Amine oxidase – kinetic resolution of amines.
endow amine oxidases with the desired substrate- and stereoselectivities. Since
the imine is not released from the Type I amine oxidase enzymes, chemical re-
duction will reduce the released ketone to the corresponding racemic alcohol.
Thus dynamic resolution by combining in situ chemical reduction is possible
with Type II amine oxidase only. However, both Type I and II amine oxidases
can be combined with transaminase (transferring ketone to the desired amine)
to effect dynamic resolution (Figure 1.9).
Amino acid oxidase enzymes are similar to the Type II monoamine oxidase and
oxidize amino acids to imino acid and then to keto acid. These enzymes can be
used for the kinetic resolution of racemic amino acids.
Rather than using O2 as the ultimate electron acceptor for amine oxidation,
amine dehydrogenases transfer hydride from the amine a-carbon to a nicotin-
amide cofactor. This yields the corresponding imine that undergoes subsequent
FIGURE 1.9
Chemo-enzymatic dynamic resolution of amines.
FIGURE 1.10
Conversion of amino acid to keto acid and vice-versa by amino acid dehydrogenase.
hydrolysis. The best-known enzymes in this class accept amino acids as their
normal substrates. Amine dehydrogenases can catalyze the reactions in either
direction; imine reduction is mechanistically similar to chiral reductive amina-
tion and is generally more synthetically useful. This reaction has formed the
basis of several commercial processes to produce both natural and unnatural
amino acids (Figure 1.10).
From a synthetic standpoint, the major drawback to this class of enzymes is their
relatively narrow range of acceptable substrates. With very few exceptions, they
require an a-keto acid substrate. Recent protein engineering efforts by Bommar-
ius have overcome this limitation, raising the prospect of tailor-made enzymes
for asymmetric reductive amination [7]. This solves a key unmet need in synthe-
sis and details are covered in a later chapter (Figure 1.11).
Like amine oxidases, one can also combine amino acid dehydrogenases with
in situ chemical reduction, a transaminase, or an amino acid dehydrogenase to
effect dynamic kinetic resolutions of amino acids. Details of these more com-
plex processes are described in the appropriate later chapters.
6.8 Nitrile Hydrolysis

Biocatalysis offers two advantages over chemical methodologies for nitrile
hydrolysis. First, enzymes operate under mild conditions in the absence of
FIGURE 1.11
Chemo-enzymatic dynamic resolution of amino acids.
FIGURE 1.12
Hydrolysis of nitrile to amide and acid.
heat, concentrated acid, or base. Their second advantage is that hydrolysis can
be halted at the amide intermediate if desired. Two classes of enzymes cata-
lyze nitrile hydrolysis. Nitrilases catalyze the exhaustive hydrolysis of nitriles
to yield the corresponding carboxylic acids with no release of intermediates.
Nitrile hydratases catalyze partial hydrolysis of nitriles to the corresponding
primary amides. In nature, nitrile hydratases are almost always coproduced
with amidases so that the organism can convert the amide intermediate to
the corresponding carboxylic acid that is used for catabolism. For synthetic
purposes, however, it is more common to eliminate the amidase (either by pu-
rifying the nitrile hydratase or employing mutant cells that do not express the
amidase) so that the reaction can be halted at the amide stage. Because amides
are generally more reactive with respect to hydrolysis than the corresponding
nitriles, it is extremely difficult to effect such a chemoselective partial hydro-
lysis by nonenzymatic means. With enzymes showing chiral discrimination,
these enzymes can be used for kinetic resolutions of nitriles, amides, and acids
(Figure 1.12).
6.9 Epoxide Hydrolysis

The hydrolysis of epoxides to diols is catalyzed by epoxide hydrolase enzymes.
These usually show very high stereoselectivities and have been used for kinetic
resolutions of racemic epoxides to yield both chiral epoxides (unreacted enan-
tiomer) and chiral diol products (from reactive enantiomer). For most enzymes,
nucleophilic attack by water occurs at the less-substituted end of the epoxide,
resulting in retention of configuration at the more-substituted center. Interest-
ingly, for some epoxide hydrolases, the regioselectivity of nucleophilic attack de-
pends on the absolute configuration of the substrate and is opposite for the two
antipodes. This makes it possible for some racemic epoxides to be converted to
a single diol enantiomer in a process referred to as an enantioconvergent process.
Some epoxide hydrolases also accept other nucleophiles in addition to water,
for example, azide. This significantly increases the range of products that can be
formed from epoxide intermediates (Figure 1.13).
FIGURE 1.13
Hydrolytic/Nucleophilic opening of the epoxide ring.
FIGURE 1.14
Synthesis of epoxides from halohydrins.
6.10 Epoxide Synthesis from Halohydrins

Epoxides can be produced by elimination of HCl from vicinal-halohydrins by
halohydrin dehalogenase enzymes. These enzymes can be used for the resolu-
tion of racemic halohydrins to chiral halohydrins that can be converted easily
to chiral epoxides, an important building block for synthesis of variety of com-
pounds (Figure 1.14).
6.11 Baeyer–Villiger Oxidations of Ketones

Baeyer–Villiger monooxygenases catalyze the conversion of ketones to the cor-
responding esters by oxygen insertion. As the name implies, these enzymes uti-
lize O2 as a reactant, with one atom incorporated directly into the product and
the second lost as water. This process requires a pair of electrons that must be
supplied by a nicotinamide (almost always NADPH rather than NADH). The
mechanism of the enzymatic Baeyer–Villiger oxidation appears to be identical
to that of the usual peracid-mediated reaction; the key difference is that the
enzyme forms a hydroxyperoxy-flavin intermediate in situ whose reactivity is
analogous to that of a peroxycarboxylic acid. The flavin hydroperoxide adds to
the substrate carbonyl, forming a tetrahedral Criegee-type intermediate whose
breakdown follows the same stereoelectronic rules as the peracid-mediated re-
action. This makes it possible to predict the stereochemical outcomes of these
biocatalytic oxidations with some confidence. In most cases, migration of
the more-substituted (more electron-rich) carbon is observed (as is also true
for peracid-mediated oxidations). This intrinsic electronic preference can be
overridden in the enzymatic reaction by specific substrate structural features,
thereby producing “abnormal” oxidation products. The ability to determine
which carbon migrates by correct enzyme choice – along with the mild reac-
tion conditions and nontoxic nature of the oxidant – makes enzymatic Baeyer–
Villiger oxidations particularly valuable in synthesis. It is also noteworthy that
most Baeyer–Villiger monooxgenases can also catalyze heteroatom oxidations,
FIGURE 1.15
Baeyer–Villiger Oxidation.
converting amines into amine oxides and thioethers into sulfoxides. Such oxi-
dations often occur with high stereoselectivities and constitute useful routes to
these chiral building blocks. In this case, the flavin hydroperoxide acts as an
electrophilic oxidant, further underscoring the synthetic versatility of these oxi-
dation catalysts (Figure 1.15).
6.12 Alkene Reductions

Biocatalytic alkene reductions have recently grown in popularity. The best sub-
strates for these enzymes involve alkenes conjugated with one or more electron-
withdrawing groups, particularly, aldehydes, ketones, esters and, nitro moieties.
Reducing equivalents are supplied exogenously by a nicotinamide cofactor. In
most cases, the nicotinamide first reduces an active site flavin that subsequently
transfers hydride to the electron-deficient alkene b-carbon (a proton is added
concomitantly to the a-carbon to complete the reduction process). In nearly
all cases, the reaction involves net trans-addition of H2 across the alkene, which
makes the biocatalytic strategy nicely complementary to organometallic and or-
ganocatalysts for alkene reductions that proceed via cis-addition. To date, the
major limitations of this methodology have been a lack of stereochemical di-
versity in the commercially available set of enzymes and the inability to reduce
isolated double bonds. The first limitation has been addressed by protein engi-
neering and several successful programs have yielded enantiocomplementary
alkene reductase variants (Figure 1.16).
FIGURE 1.16
Reduction of alkene by enoate reductase.
Another random document with
no related content on Scribd:
of the government.’ ‘Ah,’ replied Freneau, welcoming the fight, ‘I will tell
a short story that will put the matter in a proper light. A pack of rogues once
took possession of a church ... held in high veneration by the inhabitants of
the surrounding district. From the sanctuary they sallied out every night,
robbed ... all the neighbors, and when pursued took shelter within the
hallowed walls. If any one attempted to molest them there, they deterred
him from the enterprise by crying, “Sacrilege,” and swearing they would
denounce him to the inquisition as a heretic and an enemy of the Holy
Mother Church.’[616] And Freneau persevered in his perversity. Right
joyously he returned to the scandal of speculation. ‘It is worthy of notice
that no direct denial has ever appeared of the ... multiplied assertions that
members of the general government have carried on jobs and speculations
in their own measures even while those measures were depending.’[617]
III
An interesting picture was presented the day after the appearance of this
attack: Emerging from the doorway of the Morris house was a distinguished
party. Washington himself, sober and stately, with his matronly spouse;
Hamilton, alert and suave, with little Betty; and a tall, loose-jointed man of
pleasing aspect whom spectators instantly recognized as Jefferson. Entering
carriages they drove away to visit Mr. Pearce’s cotton manufactory. No one
knew better than Washington that a crisis had been reached in the relations
of his ministers. But a few days before he had sat pondering over a letter
from Jefferson. It dealt with the reason for the growing distrust in
government, the fiscal policy of Hamilton, the disposition to pile up debt,
the corruption in Congress—and it announced a determination to retire
from the Cabinet.[618] Washington, greatly distressed, had earnestly
importuned him to remain. He had agreed to stay on awhile, but the
quarreling was becoming intolerable.
At the factory the little party entered, pausing to examine the machinery
and comment upon it, Hamilton the irreproachable gentleman, courteous,
amusing, pleasant, Jefferson observing all the amenities of the occasion. It
was their last social meeting in small company. But if Washington, who had
invited them, hoped thus to persuade them to drop their quarrel, he was
foredoomed to disappointment. The cause of their disagreement was
elemental and eternal. They returned to the Morris house after a pleasant
diversion—and the fight went on.
IV
In early June, Fenno and Freneau were lashing each other with much
shouting. But the editor of the Hamilton paper played constantly into the
hands of his opponent. He lamented the appearance of a ‘faction,’ meaning
party, because factions mean convulsions under a republican government. It
would not be so serious if there were a king, because ‘a king at the head of
a nation to whom all men of property cling with the consciousness that all
property will be set afloat with the government, is able to crush the first
rising against the laws.’[619] There must have been high glee among the
cronies of Freneau in the office on High Street when they read it. ‘King,’
‘men of property’—Freneau could not have dictated the comment for his
purpose better. ‘Your paper is supported by a party,’ charged Fenno. Yes,
agreed Freneau, if ‘by a party he means a very respectable number of anti-
aristocratic, and anti-monarchical people of the United States.’[620] But, not
to be diverted, the poet-editor returned persistently to his indictment.
‘Pernicious doctrines have been maintained’—‘Members of Congress
deeply concerned in speculating and jobbing in their own measures ... have
combined with brokers and others to gull and trick their uninformed
constituents out of their certificates.’[621]
‘The names—give us the names,’ demanded Fenno. ‘That reminds us,’
said Freneau, ‘of the impudence of a noted prostitute of London, who,
having a difference with a young man, was by him reproached for her
profligacy, and called by the plain name of her profession.... “I’ll make you
prove it or pay for it,” said she. Accordingly, she sued the young man for
defamation of character, and although half the town knew her character, yet
nobody could prove her incontinency without owning himself an
accomplice, and the defendant was lost for want of evidence and obliged to
pay heavy damages. Thus it is when any man talks of speculators—“prove
the fact, sir”—as if, indeed, the men who hired out the pilot boats and the
brokers who negotiated the securities would come forward to expose their
employers and themselves.’[622]
Thus with charge on charge, with sarcasm and satire, especially the
latter, Freneau constantly increased the intensity of his assaults. These
slashing and insidious attacks did not reach the citizens of Philadelphia only
—they were copied far and wide. The paper itself went into every State.
Men were discussing and quoting it on the streets, in the coffee-houses of
New York, on the stage-coaches jolting between the scarcely broken forests
of remote places, about the fireplace in the cabin in the woods. No one had
followed it with greater rage than Alexander Hamilton. One day Fenno’s
‘Gazette’ contained a short letter bearing the signature ‘T. L.,’ which started
the tongues to wagging all the way from O’Eller’s grogshop to Mrs.
Bingham’s drawing-room.
Mr. Fenno: The editor of the National Gazette receives a salary from the
Government. Quere—Whether this salary is paid him for translations, or
for publications, the design of which is to vilify those to whom the voice of
the people has committed the administration of our public affairs—to
oppose the measures of government, and by false insinuations to disturb the
public peace?
In common life it is thought ungrateful to bite the hand that puts bread in
its mouth; but if a man is hired to do it, the case is different.
Freneau’s paper had become dangerous, Fenno was unable to meet its
onslaughts, and thus, anonymously, Hamilton took up his pen.[623]
It was at this time that Hamilton first shocked his friends with the
disclosure of his temperamental weakness that was to destroy his
leadership. Persuaded that Freneau’s journal was established for the primary
purpose of wrecking him, he saw red, lost his customary poise and self-
control, and, throwing discretion to the winds along with his dignity as a
minister of State, he entered the lists as an anonymous letter-writer. We
search in vain through the correspondence of his friends for evidence of
approval.
The attack was met by Freneau with a certain dignity. Reproducing the
‘T. L.’ letter he wrote:
The above is beneath reply. It might be queried, however, whether a man
who receives a small stipend for services rendered as French translator to
the Department of State, and as editor of a free newspaper admits into his
publication impartial strictures on the proceedings of government, is not
more likely to act an honest and disinterested part toward the public, than a
vile sycophant, who obtaining emoluments from government, far more
lucrative than the salary alluded to, finds his interest in attempting to poison
the minds of the people by propaganda and by disseminating principles and
sentiments utterly subversive of the true republican interests of the country,
and by flattering and recommending every and any measures of government
however pernicious and destructive its tendency might be to the great body
of the people. The world is left to decide the motive of each.[624]
This controversy of mere journalists did not interest Hamilton. He was
out gunning for bigger game. Thoroughly convinced that Jefferson was
responsible for much of the contents of Freneau’s paper, he hoped to draw
his colleague into an open newspaper fight and, if possible, drive him from
the Cabinet. The relations of the two Titans had been growing more and
more hostile. They disputed across the table in the council room, and at rare
times seemed at the point of blows. Hamilton knew Jefferson’s opinions of
his policies—and similar opinions were appearing in the paper edited by a
clerk in his rival’s office. Nor were they slovenly, superficial articles. They
were the work of close observers and clever controversialists. Not only was
he ignorant of the fact that many of these were the work of Madison, that
Brackenridge wrote some, George Tucker, editor of the American edition of
Blackstone, some,[625] but he ridiculously underestimated the capacity of
Freneau. These articles were strong, stinging, effective, and therefore
Jefferson wrote or dictated them. He would drag Jefferson into the arena
and have it out.
Thus, in his letter of August 4, he contemptuously dismissed the editor
as ‘the faithful and devoted servant of the head of a party,’ and launched his
personal bitter attack on Jefferson. If he wished to attack ‘the Government,’
why didn’t Jefferson resign?[626] ‘Can he reconcile it to his own personal
dignity, and the principles of probity, to hold an office under it, and employ
the means of official influence in that opposition?’ Besides, he was an
enemy of the Constitution. He had been opposed to it and had written his
objections ‘to some of his friends in Virginia.’[627] Four days later, Freneau
denied in an affidavit published in Fenno’s paper that Jefferson had any
connection with the ‘National Gazette’ or had written or dictated a line. The
same day, in his own paper, he raised the curtain on Hamilton’s nom de
plume, with a comment that ‘all is not right with certain lofty-minded
persons who fondly imagined their ambitious career was to proceed without
check or interruption to the summit of their wishes.’ To which he added that
‘the devil rageth when his time is short.’[628] In his letter of August 11th,
Hamilton dismissed the denial unimpressively. At this moment he thought
himself hot on the trail. Elias Boudinot, he recalled, had once told him of
the part Madison had played. If he could get an affidavit from Boudinot!
Acting on an impulse, he wrote him that ‘a friend’ was writing the attacks
on Jefferson. He had mentioned the Boudinot conversation to that ‘friend’
who was anxious to have an affidavit. ‘It is of real importance that it should
be done,’ he wrote. ‘It will confound and put down a man who is
continually machinating against the public happiness.’[629] But Boudinot
does not appear to have had any stomach for the mess, albeit he, like every
one else, must have known that the ‘friend’ was Hamilton himself. No
affidavit was forthcoming.
While he was waiting vainly for the affidavit, an anonymous writer in
Freneau’s paper, referring to Hamilton’s assaults, made a counter-charge.
What about ‘the immaculate Mr. Fenno’? Did he not have the printing of
the Senate, ‘the emoluments of which office are considerable?’ Did he not
‘enjoy exclusively the printing of the Treasury department where it seems
he has rendered himself a particular favorite?’ Was he not already ‘making
his approaches to another office on Chestnut Street [the Bank],’ and in a fair
way to secure ‘if not already in possession of the business appertaining
thereto?’[630]
On August 18th, Hamilton appeared again to sneer at Freneau’s
announcement that he would pay no attention to the charges until the author
came forward to make them in the open. ‘It was easily anticipated that he
might have good reasons for not discovering himself, at least at the call of
Mr. Freneau, and it was necessary for him to find shelter.’
Freneau’s affidavit! scoffed a writer in Hamilton’s organ. He had no faith
in it. The editor had certainly not sworn upon the Bible. Had he taken the
oath on Jefferson’s ‘Notes on Virginia’?[631]
But Hamilton was already discovered. No one there was in public life
from Washington down who did not know the author. The amazing
spectacle was the talk of the taverns and the dinner tables, and was
beginning to assume the proportions of a scandal. Washington was shocked
and aggrieved. He would stop it.
VI
On August 26th he tried his art of conciliation, appealing to both

Hamilton and Jefferson, albeit, as he knew, the latter had not written a line.
Both replied in September, Hamilton admitting the authorship of the
articles, and declared his inability ‘to recede now.’ He had been forced to
write. He had been ‘the object of uniform opposition from Mr. Jefferson’;
‘the object of unkind whispers and insinuations from the same quarter’; and
he had evidence that the ‘National Gazette’ had been instituted by Jefferson
‘to render me and all the objects connected with my administration odious.’
He had been most patient. In truth, he had ‘prevented a very severe and
systematic attack upon Mr. Jefferson by an association of two or three
individuals, in consequence of the persecution he brought upon the Vice-
President by his indiscreet and light letter to the printer, transmitting Paine’s
pamphlet.’[632]
Jefferson replied that in private conversation he had ‘utterly
disapproved’ of Hamilton’s system, which ‘flowed from principles adverse
to liberty and calculated to undermine and demolish the Republic by
creating an influence of his department over members of the legislature.’
He had seen this influence ‘actually produced’ by ‘the establishment of the
great outlines of his project by the votes of the very persons who, having
swallowed his bait, were laying themselves out to profit by his plans.’ Then,
too, Hamilton had constantly interfered with his department, particularly in
relation to England and France.[633] As to Freneau, he hoped he ‘would
give free place to pieces written against the aristocratic and monarchical
principles.’ He and Fenno, he said, ‘are rivals for the public favor. The one
courts them by flattery, the other by censure, and I believe it will be
admitted that the one has been as servile as the other has been severe.’
Then, turning again to Hamilton: ‘But is not the dignity and even decency
of government committed when one of its principal Ministers enlists
himself as an anonymous writer or paragraphist for either the one or the
other of them?’ As for criticism of governmental measures, ‘no government
ought to be without censors; and where the press is free no one ever will. If
virtuous, it need not fear the free operation of attack and defense. Nature
has given to man no other means of sifting out the truth, either in religion,
law, or politics. I think it is as honorable to government neither to know nor
notice its sycophants, as it would be undignified and criminal to pamper the
former and persecute the latter.’[634]
Thus ended Washington’s attempt to intervene. Hamilton had refused to
discontinue his attacks, and, within two days after replying to Washington’s
appeal, he was again appearing in the ‘Gazette of the United States.’
VII
Even while Hamilton and Jefferson were writing their letters, the fight
was proceeding merrily, if bloodlessly, in the papers. ‘Aristides,’ none other
than Madison, had gone to the defense of his leader in an article in Fenno’s
paper on Jefferson’s attitude toward the Constitution. No one was so well
qualified to know, unless it was Washington himself. He had sat in the
Convention, a leading figure, and listened to Hamilton’s speeches and
proposals, and had been in correspondence with Jefferson. It was not this
defense that made Fenno restive. It was a pointed attack. ‘It is said, Mr.
Fenno, that a certain head of a department is the real author or instigator of
these unprovoked and unmanly attacks on Mr. Jefferson—and that the time
of that gentleman’s departure from the city on a visit to his home was
considered as best suited to answer the design it was intended to effect.’
‘Unmanly attack’ and an insinuation of cowardice! Fenno took the
precaution to add a note warning that no further letters would be printed
containing ‘personal strictures’ unless the name of the author was furnished
‘in case of emergency.’ Coffee and pistols—was it coming to that?[635]
Freneau had no such concern, for on the same day a writer in his paper
referred to the ‘base passions that torment’ Hamilton, and called upon the
author of the anonymous articles to ‘explain the public character who on an
occasion well known to him, could so far divest himself of gratitude and
revolt from the spirit of his station as to erect his little crest against the
magnanimous chief who is at the head of our civic establishment, and has
on many free occasions since spoken with levity and depreciation of some
of the greatest qualities of that renowned character; and now gives himself
out as if he were his most cordial friend and admirer, and most worthy of
public confidence on that account.’[636]
Two days after refusing Washington’s request for a cessation, Hamilton
returned to the attack in answer to the charge of the ‘National Gazette’ that
he had not liked the Constitution, and had pronounced the British monarchy
the most perfect government. All this he stoutly denied. The records and
debates of the Constitutional Convention were then under secrecy, and
members who had heard his speeches were under the ban of silence. He felt
safe. This is the most amazing letter of the series.
And so the dismal affair dragged on. Another letter appeared reiterating
a connection between Jefferson and Freneau; another charging that
Jefferson was opposed to the Constitution and against paying the public
debt; still another complaining of Jefferson’s interference with the Treasury
Department. Then another on Jefferson and the Constitution, and finally,
two months after Washington’s appeal, demanding that Jefferson, who
remained in the Cabinet on the earnest solicitation of Washington,
withdraw. ‘Let him not cling to the honor or emolument of an office,
whichever it may be that attracts him, and content himself with defending
the injured rights of the people by obscure or indirect means.’
Meanwhile, Jefferson had refused to be drawn into the controversy
personally. The situation had become painful—the Philadelphia drawing-
rooms lifting their brows at him. His official associations were unpleasant,
but he never touched pen to a paper intended for publication. Only in his
personal letters did he pour forth his bitterness against his colleague. ‘The
indecency of newspaper squabbling between two public Ministers,’ he
wrote Edmund Randolph, ‘has drawn something like an injunction from
another quarter. Every fact alleged ... as to myself is false.... But for the
present lying and scribbling must be free to those who are mean enough to
deal in them and in the dark.’[637] He had hoped for an early retirement, and
the attacks had indefinitely postponed the realization of his desire. ‘These
representations have for some weeks past shaken a determination which I
had thought the whole world could not have shaken,’ he wrote Martha.[638]
Meanwhile, the small-fry partisans were busy in all the papers. The effect,
on the whole, had been favorable to Jefferson, making him the idol of the
democrats everywhere. ‘It gives us great pleasure,’ said a Boston paper, ‘to
find that the patriotic Jefferson has become the object of censure, as it will
have a happy tendency to open the eyes of the people to the strides of
certain men who are willing to turn every staunch Republican out of office
who has discerning to ken the arbitrary measures, and is honestly sufficient
to reveal them.’[639] To the ‘Independent Chronicle’ the ‘slander and
detraction’ of men like Jefferson seemed ‘a convincing proof of the badness
of the cause behind it.’[640] The onslaught had in no wise weakened
Jefferson’s faith in the effectiveness of the ‘National Gazette.’ The smoke
had not lifted from the field when he was rejoicing because it was ‘getting
into Massachusetts under the patronage of Hancock and Sam Adams.’[641]
Even Freneau found the democrats rallying around him.
It is a Fact [wrote a correspondent] that immense wealth has been
accumulated into a few hands, and that public measures have favored that
accumulation.
It is a Fact that money appropriated to the sinking of the debt has been
laid out, not so as most to sink the debt, but so as to succor gamblers in the
funds.
It is a Fact that a Bank law has given a bounty of from four to five
million dollars to men in great part of the same description.
It is a Fact that a share of this bounty went immediately into the pockets
of the very men most active and forward in granting it.
These, Mr. Freneau, are facts—...severe, stubborn, notorious facts.[642]
VIII
Thus Hamilton’s remarkable attack had only whetted the appetite of the
Jeffersonians for battle—and a national campaign was in progress. The
unanimous reëlection of Washington was universally demanded, but why
should the ‘aristocratic’ and ‘monarchical’ author of ‘The Discourses of
Davilla’ be chosen again? At any rate, efforts could be made to change the
political complexion of Congress.
There were mistakes, blunders, tragedies, that could be used to affect
public opinion. What more shocking than the humiliating collapse of the
General St. Clair expedition against the Indians in the western country?
Gayly enough had the unfortunate commander set forth with twenty-three
hundred regular troops and a host of militiamen. There had been a scarcity
of provisions and inadequate preparations. Hundreds of soldiers, consumed
with fever, shaken with chills, had vainly called for medicine. Many died,
hundreds deserted in disgust, and finally but fourteen hundred worn and
weary, sick and hungry men remained to face the enemy. It was easy
enough to blame St. Clair, and, as he passed through the villages en route to
the capital, the people flocked about to hiss and jeer.
But why the lack of proper preparations? Why the insufficiency of the
commissary? Even the officials in Philadelphia were prone to find
extenuations for the failure of St. Clair. A correspondent of the Boston
‘Centinel,’ dining with some of the first official characters where the tragic
collapse of the expedition had been discussed, found ‘not one expression
dropped to his prejudice.’[643] The Jeffersonians were aiming higher than
St. Clair. There was Knox, Secretary of War—what had he to say in defense
of the honesty of the army contractor, to the negligence of the
quartermaster? The House investigating committee bore heavily on these
two in its report—but who was responsible for the cupidity of the one and
the inefficiency of the other? Soon the Jeffersonian press was attacking
Knox with distressing regularity, picturing him as the ‘Philadelphia
Nabob.’[644] Was he not squandering public money on ‘splendor’ and
‘extravagance’? Soon the more irresponsible of the gossip-mongers were
whispering that he had profited financially. ‘Infamous!’ screamed the
Federalist press. ‘The public monies have never been in the hands of Mr.
Knox.’[645] ‘But who made arrangements with the dishonest contractor?’
replied the Jeffersonians. ‘Who selected the quartermaster who let the
soldiers starve?’
All through the summer and autumn this was the talk in the taverns and
coffee-houses, but with the bursting of the bubble of speculation a far more
effective weapon of assault was at hand. To this inevitable outcome of the
gambling mania Jefferson had looked forward with the utmost confidence.
He had seen money ‘leaving the remoter parts of the Union and flowing to
[Philadelphia] to purchase paper’; had seen the value of property falling in
places left bare of money—as much as twenty-five per cent in a year in
Virginia. Extravagance, madness everywhere.[646] As a result in the remoter
sections the hatred of the speculator had reached the stage of hysteria.
‘Clouds, when you rain, bleach him to the skin,’ prayed a Georgia paper.
‘When you hail, precipitate your heaviest globes of ice on his ill-omened
pate. Thunders, when you break, break near him, shatter an oak or rend a
rock full in his view. Lightning, when you burst, shoot your electric streams
close to his eyelids. Conscience, haunt him like a ghost.... Ye winds, chill
him; ye Frost, pinch him, freeze him. Robbers meet him, strip him, scourge
him, rack him. He starved the fatherless and made naked the child without a
mother.’[647] Even the Worcester correspondent of the orthodox Boston
‘Centinel’ complained that ‘as soon as one bubble bursts another is blown
up’ and ‘we are in the way of becoming the greatest sharpers in the
universe’—all ‘assuredly anti-republican.’[648] When a town meeting was
advertised for Stockbridge, a village wit penciled on the poster the purpose
of the conference: ‘To see if the town will move to New York and enter into
the business of speculation.’[649] While publishing these letters and stories
the Federalist organ in Boston did it with the sneer: ‘They who are in—
Grin. They who are out—Pout. They who have paper—Caper. They who
have none—Groan.’[650]
Then in April, with the failure of Colonel Duer in New York the crash
came. Many went to ruin in the wreckage, and New York became a
madhouse, with business paralyzed, and Duer taking to flight. He had been
among the most favored of the beneficiaries of Hamilton’s policies, rising
from opulence overnight, and he was among the first to fall from their
abuse.[651] The brutality and cowardice of the speculators intensified the
general contempt for the tribe. ‘Instead of exerting themselves to preserve
some kind of moral character,’ wrote a New York correspondent of the
‘Maryland Journal,’ ‘they are endeavoring to lower themselves still more by
descending to the mean level of fish women and common street
boxers.’[652]
All this was viewed by Hamilton with indignation and concern. He had
sought in every way to discourage the frenzy of speculation, and had used
his office to protect the public wherever possible. But it began with the
funding system—and with thousands that was enough. Instantly the
Jeffersonian press was hot on the trail. ‘Business has not been benefited by
Hamilton’s Bank,’ declared the ‘Independent Chronicle,’ ‘for a merchant
can scarcely venture to offer his note for $100, while a speculator can
obtain thousands for no other purpose than to embarrass commerce.’ Look
around and see who have obtained wealth. ‘Speculators, in general, are the
men.’ Thus, ‘the industrious merchant is forced to advance to the
government thousands, while the gambling speculator is receiving his
quarterly payments.’[653] A Maryland correspondent of Louden’s New York
‘Register’ ‘could not help thinking Mr. Madison’s discriminating
propositions would have prevented in great measure the exorbitant rage of
speculation.’[654] Meanwhile, Fenno was denouncing the critics as
‘anarchists’ and enemies of the Government, which only intensified their
rage. ‘Our objection is not to paying off the debt,’ protested an indignant
critic, ‘but to ... the excise, failure to discriminate, the play to speculation’;
and if all who shared these views could be assembled it ‘would make the
greatest army that ever was on one occasion collected in the United
States.’[655] In the Boston ‘Centinel,’ John Russell was taking a lighter
tone. ‘The suffering yeomanry burdened with taxes? Why not simply
eliminate all State and National debts and forget them?’[656] The storm?
What of it? ‘The Six Per Cents, a first rate, belonging to the fleet
commanded by Admiral Hamilton, notwithstanding several hard Country
gales, and a strong lee current setting out of the Hudson and Delaware is
still working to windward and bids fair to gain her destined port.’[657]
IX
With such attacks and counter attacks in the papers, the campaign of
1792 was fought, with the bitter gubernatorial battle between John Jay and
George Clinton in New York setting the pace in the spring. The Federalists
had set their hearts on the crushing of Clinton, and but for the frown of
Hamilton, Burr might have joined them in the attempt.[658] The campaign
was spectacular, and class feeling and prejudice played a part. Jay was an
aristocrat by birth and temperament, and this gave the Clintonians their cue.
Up, Plebs, and at ’em! An aristocrat against a democrat, the rich against the
poor. Had not Jay said that ‘those who own the country ought to govern it’?
Had not Jay’s Constitution disfranchised thousands on the score of their
poverty? Were not the speculators, the stock-jobbers, the bankers, the
gamblers, swindlers, and the forces of privilege supporting Jay?[659] The
result was the election of Clinton, on a technicality,[660] and instantly there
was an uproar, broken bones and bloody noses, coffee-house quarrels and
blows, wild talk of a revolutionary convention and the seating of Jay with
bayonets, and serious bloodshed was prevented only through the efforts of
Hamilton, Jay, and King. Never had party feeling run so high, and several
duels were fought in the course of a week.[661] The defeated or cheated
candidate was accorded the acclamations due a conqueror on his journey
from his judicial circuit to New York where he was given a testimonial
dinner.[662] The democrats were none the less jubilant because of the
questionable nature of their triumph, and at a dinner in honor of Clinton, the
Tammany braves rose to the toast, ‘Thomas Jefferson,’ and gave their war-
whoop.[663]
The bitterness in New York spread to various parts of the country where
the Jeffersonians were fighting brilliantly, with clever strategy, to gain seats
in the Congress. Some of the Federalists, who were to prove themselves
generally inferior except in a smashing charge, and incapable of
maintaining their morale in a siege or in reverses, were even then growing
pessimistic. ‘Perhaps you are not informed,’ wrote George Cabot to
Theophilus Parsons, ‘that in Pennsylvania and New York the opponents are
well combined and are incessantly active, while the friends discover a want
of union and a want of energy.’[664] And Parsons, in melancholy mood, was
convinced that the Government had ‘seen its best days.’[665] Woe to the
politician who enters the reminiscent stage when confronted by a virile
opponent looking to the future. There was little in the New England of 1792
to depress the Federalists. Only a little evidence that among the working-
men in Boston ‘heresies’ were making their way; only reports that ‘itinerant
Jacobins’ were haranguing the curious in the bar-rooms of Rhode Island
and Vermont; only the strange spectacle of ‘drill masters’ meeting with
people of no property or importance to organize them to battle for
democratic principles.[666] Only this, and a strange doctrine creeping into
Vermont papers. In choosing members of Congress who should be selected?
asked a ‘Land Holder’ of that State. ‘What class of people should they
represent? Who are the great body of the people? Are they Lawyers,
Physicians, Merchants, Tradesmen? No—they are respectable Yeomanry.
The Yeomanry therefore ought to be represented.’[667] In Maryland a
ferocious fight was waged under the eyes of both Hamilton and Jefferson,
for both were interested in the fate of Mercer who had slashed right lustily
at the policies of Hamilton, making no secret of his belief that they were
bottomed on corruption. He had vitalized the democrats of Maryland,
extending his interest into districts other than his own, and arranging for
candidates to oppose the sitting Federalists in the House. McHenry, who
kept Hamilton informed of the progress of the fight, hoped to array the
German Catholics against the obnoxious Mercer through the intervention of
Bishop Carroll, whom he thought more influential than the better known
Charles Carroll of Carrollton.[668] A man was employed by the energetic
McHenry to circulate bills against Mercer, who fought back, and gave blow
for blow. He was charged with having said that Hamilton had tried to bribe
him in the Assumption fight;[669] that he was personally interested in the
contract for supplying the western army, and privately engaged in the
purchase of securities. This, Mercer was to disavow, and Hamilton’s friends
were to show that the conversation between the Marylander and the
Secretary had been in the presence of company and in jest.[670] Even so we
may assume that Mercer had painted the incident black. He let it be
understood that Washington wished his reelection, and the celerity with
which the President issued a denial was probably due to the importunity of
Hamilton who did not scruple to use him without stint to further the cause
of his party.[671]
In North Carolina the Jeffersonians, under the crafty leadership of
picturesque Willie Jones, contested every inch of the ground, determined to
retire all the Hamiltonians from Congress, and before the impetuosity of
their charge the Federalists were forced to fight defensively and under a
cloud.[672]
In the new State of Kentucky the Jeffersonians were thoroughly
organized under the leadership of John Brown, a Virginian, educated at
Princeton and at Jefferson’s alma mater, who had fought through the War of
Independence. ‘Brown can have what he wants,’ Madison wrote his leader
in midsummer,[673] and he took the toga. In Virginia the Democrats were
strongly in the ascendancy. The influence of Jefferson had been
strengthened by the acquisition of Madison, and Hamilton, in the course of
the campaign, wrote his famous letter to Colonel Edward Carrington
attacking both in an effort to satisfy the Virginia Federalists of the justice of
his own position, but it was blowing against a tornado.[674] An amazing
campaign document—this letter.
Thus, in 1792, if the Jeffersonians had not yet perfected their
organization, they had forced sporadic fighting, and the result of the
congressional elections was greatly to strengthen them in the House.
It was clear quite early that the Jeffersonians would not permit Adams’s
reëlection to go unchallenged. The press had teemed with controversial
articles on his books for more than a year. As early as March his friends
took up the cudgels in his defense. ‘Homo’ in the Boston ‘Centinel’ warned
that ‘a detestable cordon of desperadoes’ were trying to destroy public
confidence in Adams by vilification.[675] Within three months, Hamilton
convinced himself that the opposition, in dead earnest, had concentrated on
Clinton, and hastened to warn Adams, who was enjoying the placidity of
his farm at Quincy.[676] It is interesting to observe that this plan to displace
Adams was interpreted by Hamilton as ‘a serious design to subvert the
government.’ If the candidacy of Clinton was annoying to Hamilton, the
warning he received in September of the possible candidacy of Aaron Burr
was maddening, and he fell feverishly to the task of denouncing the
ambitions of this ‘embryo Cæsar’ in letters to his friends.[677] Clinton ‘has
been invariably the enemy of national principles,’ he wrote General C. C.
Pinckney in ordering a mobilization for defense in South Carolina, and as
for Burr, he was a man of ‘no principles other than to mount, at all events,
to the full honors of the state, and to as much more as circumstances will
permit.’ Was Jefferson behind the conspiracy against Adams—Jefferson,
that man of ‘sublimated and paradoxical imagination, entertaining and
propagating opinions inconsistent with dignified and orderly
government?’[678] To John Steele in North Carolina he wrote in the manner
of a commander, to inform him ‘that Mr. Adams is the man who will be
supported by the Northern and Middle States.’ Of course, he had ‘his faults
and foibles,’ and some of his opinions were quite wrong, but he was honest,
and loved order and stable government.[679] Meanwhile, painful
complications were threatened in Maryland where a number of
notables[680] joined in a public letter rallying Marylanders to the support of
Charles Carroll of Carrollton.[681] This gave James McHenry, an idolater of
Hamilton, and still tortured by a persistent, and, as yet, ungratified itch for
office, his opportunity. He assumed the responsibility for whipping the
rebels back into line. These signers of the Carroll letter had been imposed
upon. The fight against Adams was a fight against the Constitution—in
keeping with the plan of the enemies of government to drive able men from
office. Had not Hamilton ‘whose attachment to the Constitution is
unquestionable’ been assailed with virulence? Yes, from ‘the master
workman in his craft down to the meanest of his laborers,’ all were engaged
in the dirty work. Thus the submission of Carroll’s claims at so late an hour
wore ‘a very doubtful and invidious aspect.’ Was it done ‘to get ten votes
against Adams or to promote Carroll’s election?’ Was any one so foolish as
to think that the Democrats in New York, Pennsylvania, and Virginia would
desert Clinton?[682] This letter, signed by ‘A Consistent Federalist,’ was
copied by all the Federalist papers of the country.
Meanwhile, Adams, lingering lovingly on his home acres, showed no
inclination to return to Philadelphia, and it was reported that he might not
appear to preside over the Senate until late in the session. This was an
appalling lack of tact. Hamilton, assuming the rights of the leader, did not
hesitate. ‘I learn with pain that you may not be here until late in the
session,’ he wrote the loiterer behind the firing lines. ‘I fear this will give
some handle to your enemies to misrepresent.... Permit me then to say it
best suits the firmness and elevation of your character to meet all events,
whether auspicious or otherwise, on the ground where station and duty call
you.’[683]
By November the press was hotly engaged in the controversy, but poor
Fenno was to have trouble with his correspondents who were to convert his
dignified journal into a cock-pit. Adams was both pelted and salved on the
same page. His writings proved him a monarchist at heart, wrote
‘Mutius.’[684] His writings would be appreciated more a century hence, said
a defender in the same issue. Had he not already been vindicated on one
point in the appearance of the ‘gorgon head of party’? Freneau cleverly
replied by quoting a laudatory article from an English paper paying tribute
to the governmental notions of ‘the learned Mr. Adams.’[685] Yes, wrote
‘Cornucopia’ in the ‘Maryland Journal,’ ‘it will require the whole strength
of the federalists to keep poor John Adams from being thrust out of the
fold.’[686]
And ‘poor John Adams’ was not entirely happy in his defenders. Why
not reëlect him, demanded ‘Philanthropos’ in a glowing tribute, for was he
not ‘a man of innocent manners and excellent moral character?’[687] ‘Why
not?’ echoed a scribe in Albany. He was ‘a reputed aristocrat, at the same
time an honest man, the noblest work of God.’[688] From ‘Otsego’ came a
more robust blow at Adams’s enemies as ‘the jacktails of mobocracy’
seeking the defeat of ‘the virtuous Adams’ because he was against ‘anarchy
and disorder.’[689] Wrong, wrote ‘Portius’ the next day, advocating Clinton.
‘Untinctured by aristocracy, and a firm republican, the patriots of America
look to him.’[690] ‘Titles, titles,’ sneered ‘Condorcet.’ ‘This rattle which so
peculiarly delights certain characters.... He never appears but in the full
blaze of office, as if every place he went was a Senate, and every circle
which he invited needed a Vice President.’[691] Thus, throughout the fall
and early winter the lashing and slashing went on, but when the time came
Adams was reëlected, albeit the result was a bitter humiliation to the proud,
sensitive spirit of the victor. Where Washington had been unanimously
reëlected, Adams had a margin of but twenty-seven votes. New York,
Virginia, North Carolina, and Georgia had moved en masse into the Clinton
camp, and Kentucky had cast her vote for Jefferson. Five States had gone
over to the Jeffersonians, and the Federalists had been unable to get a
unanimous vote in Pennsylvania. But if Adams was hurt, Hamilton could
bear his pains, for the brilliant, dashing chief of the party preferred that the
uncongenial man from Braintree should not become too perky.
Thus ended the first year of actual party struggle—Hamilton a bit soiled
by his descent to anonymous letter-writing, Jefferson greatly strengthened
by his silence under assault; the Hamiltonians triumphant, but not exultant
over the reëlection of Adams, the Jeffersonians, having tasted blood, and
tested their weapons, more than ever eager for combat and rejoicing in their
congressional gains.
Hamilton had tried to drive Jefferson from the Cabinet, and failed. It was
now the latter’s turn.
CHAPTER IX
HAMILTON’S BLACK WINTER
T HE winter of 1792-93 was notable in many ways. Not within the

memory of the oldest inhabitant of Philadelphia had one so mild been
known. As late as February there had been no interruption in the
navigation of the Delaware, and the papers, making much of the catching of
shad, were predicting that ‘a considerable school may soon be expected.’ In
this, however, the sons of Ike Walton were to be disappointed, for a
snowstorm and a northwester soon put an end to fishing.[692] Even so, the
weather continued, for the most part, mild beyond the usual. Never had
society adorned itself with more frills and furbelows, danced more
feverishly, or pursued its pleasures with greater zest. The elegant new
Chestnut Street Theater threw open its doors for the entertainment the
mimic world can give, and the aristocracy, along with the plebeians, flocked
to the play, despite the pouting of the uppish Mrs. Bingham who had been
refused a box on her own terms. Even the venomous bitterness of the
politicians failed to dim the lights of the great houses, albeit the followers
of Jefferson were more and more given to understand that they were not
wanted among the elect. The events, moving rapidly in France, were
making a distinct cleavage here among the aristocrats and democrats. The
members of the old French nobility, who had left their country for their
country’s good, were giving the tone to the most fashionable dinner tables.
Out in the streets the ‘people of no particular importance’ were vulgarly
vociferous over the trials and tribulations of the King and Burke’s beautiful
Queen—and the Jeffersonians were taking their tone from the howlings of
this ‘mob.’
It was evident from the moment Congress convened that a tremendous
party struggle was impending. The incidents of the preceding summer had
left their scars. The Jeffersonians were embittered against Hamilton because
of his anonymous attacks, and nothing could have done more to unsheathe
their swords. The truce was over. Washington had permitted Hamilton to
continue his attacks by disregarding his request; they would not now permit
even Washington to interpose to save Hamilton from their assaults. The
elections had given them a confidence they had not had before. The next
Congress would not be so subservient to ‘the first lord of the Treasury.’[693]
The supercilious assumption of superiority on the part of the Federalist
leaders would henceforth be resented. The war would begin in earnest.
The line the attack would take was shown early when Fitzsimons, one of
Hamilton’s henchmen in the House, offered a resolution calling for the
redemption of so much of the public debt as the Nation had a right to
redeem, and asking Hamilton ‘to report a plan for the purpose.’ This was in
accordance with the custom which had grown up. From the moment he had
taken office, Hamilton had considered the members of the House,
constitutionally charged with the duty of framing money bills, as his
automatons. He would determine upon the plans himself, prepare the bills,
and call upon the House to pass them without too much discussion. He
would manage the finances himself and he would not be plagued by foolish
questions. For many months the committees to which his measures had
been referred had been of his own choosing. They were his followers, and,
not a few of them, beneficiaries of his policies.
The Fitzsimons Resolution was instantly challenged by the Jeffersonians
as a rather high-handed proposal under a republican form of government,
and Madison rose to suggest that the House should know the exact state of
the finances before measures were taken for the reduction of the debt. After
all, it was with the House, not with the Secretary of the Treasury, that
money bills should originate. At any rate, the House could not act
intelligently without having the facts in its possession. All too long had it
been patient without definite reports.[694]
The feeling of the masses over the by-products of the funding system
had by this time become deep-seated. Men who had voted to create the
Bank had been made members of the board of directors. The ne’er-do-wells
of yesterday were riding in coaches and building pretentious houses.
Hamilton was urging bounties or protective duties for manufacturers one
day and running over to the Falls of Passaic on the next to assist the
directors of a corporation, that was to profit by his recommendations, in
selecting the sites for the factories. Not a few honestly believed that he was
personally profiting through governmental measures. Almost from the
beginning, Senator Maclay had been suspicious of his integrity. This utterly
false impression grew out of the positive knowledge that some of
Hamilton’s closest political associates were speculating in the securities.
‘Hamilton at the head of the speculators, with all the courtiers, are on one
side,’ Maclay wrote in his diary.[695] Only a month before at Mount
Vernon, where Washington had begged Jefferson to reconsider his
determination to resign, the latter had charged the head of the Treasury with
creating ‘a regular system for forming a corps of interested persons who
should be steadily at the orders of the Treasury.’[696] In 1790, William Duer
retired from Hamilton’s office to become the king of the money-chasers,
and, going down to ruin in the financial crash of the preceding summer, was
sending out dire threats of startling revelations from the debtors’ prison.
Many honest men were quite ready to believe that these threats were aimed
at Hamilton.[697] It was under these conditions that a miserable creature by
the name of James Reynolds, in prison for a crime against the Treasury,
sought to blackmail his way out. He had papers in his possession to prove
some financial transactions with Alexander Hamilton. An obscure person of
a low order of mentality, he hinted at his use as a dummy in business in
which a member of the Cabinet did not care to appear. These facts reached
some members of Congress.
II
On December 15th, two sober-faced members of the House and one

Senator filed into Hamilton’s office in the Pemberton mansion. The
Secretary knew them all and knew two of them as enemies. Frederick
Muhlenberg had served as a Speaker in the first House and was to resume
that post in the third. A strong character, the recognized leader of the
Germans, the foremost American Lutheran minister of his time, he had
played a conspicuous part in the Revolution and in the constructive work
that followed. Abraham Venable was a Representative from Virginia. The
Senator was James Monroe whose fanatical devotion to Jeffersonian ideals
and ideas had long since made him the object of Hamilton’s contempt.
As they took seats facing the masterful little man at the desk, they had
the manner of judges confronting a victim. None of them were finished in
the art of tactful speech. Bluntly they blurted forth their mission—they had
evidence of a mysterious connection between the Secretary of the Treasury

Organic Synthesis Using Biocatalysis 1St Edition Goswami Download PDF Chapter

Uploaded by

Copyright:

Available Formats

Organic Synthesis Using Biocatalysis 1St Edition Goswami Download PDF Chapter

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Organic Synthesis Using Biocatalysis 1St Edition Goswami Download PDF Chapter

Uploaded by

Copyright:

Available Formats

Organic Synthesis Using Biocatalysis

1st Edition Goswami

Amsterdam • Boston • Heidelberg • London • New York • Oxford

Copyright © 2016 Elsevier Inc. All rights reserved.

British Library Cataloguing in Publication Data

Library of Congress Cataloging-in-Publication Data

For Information on all Elsevier publications

Animesh Goswami*, Jon D. Stewart†

Noncovalent complex formation allows chemical reactions between specific

3 SCOPE OF THIS BOOK

4 KEY BENEFITS OF EMPLOYING ENZYMES

4.1 Selectivity: Chemo-, Regio- and Stereo-

(from the faster-reacting enantiomer) to be obtained with high optical purities

4.2 Biocatalyst Reaction Conditions

In nature, most enzyme-catalyzed reactions occur in an aqueous environment

5 MECHANISM AND KINETICS

5.1 Features of Enzyme Catalyzed Reactions

In addition to noncovalent associations (by van der Waals forces, hydrogen

5.3 Kinetics and Reaction Mechanisms

Nicotinamide Donates a hydride for polar Alcohol dehydrogenase/

Flavin Forms a hydroperoxy Alkene reductase/enoate

Thiamine Allows umpolung anion Pyruvate decarboxylase;

Pyridoxal phosphate Following reversible Schiff’s Transaminase; threonine

Known as DPNH in the very old scientific literature

Known as TPNH in the very old scientific literature

5.4 Kinetic Constants

the chemical step is slow relative to substrate release,6 KM is approximately equal

5.5 Kinetic Constants and Kinetic Resolutions

if reactions are allowed to reach equilibrium. This can be a problem if extended

5.6 Kinetic Constants and Temperature

6 TYPES OF ENZYME-CATALYZED REACTIONS

6.1 Hydrolysis and Synthesis of Carboxylic Acid Derivatives

Table 1.2 Summary of Common Biocatalytic Transformations

Table 1.2 Summary of Common Biocatalytic Transformations (cont.)

Table 1.2 Summary of Common Biocatalytic Transformations (cont.)

6.2 Carbonyl Reductions to Alcohols

reductions have been variously named as alcohol dehydrogenases or ketoreduc-

producer of carbonyl-reducing enzymes that accept a diverse range of substrates.

to be driven to completion without large molar excesses of the amine donor.

6.4 Oxygen-Dependent Oxidations

6.5 Hydroxylation by C─H Bond Insertion

Cytochrome P-450s are the best-known class of hydroxylation enzyme. Their

6.7 Oxidative Deamination of Amines to Carbonyl

6.8 Nitrile Hydrolysis

6.9 Epoxide Hydrolysis

6.10 Epoxide Synthesis from Halohydrins

6.11 Baeyer–Villiger Oxidations of Ketones

6.12 Alkene Reductions

On August 26th he tried his art of conciliation, appealing to both

HAMILTON’S BLACK WINTER

T HE winter of 1792-93 was notable in many ways. Not within the

On December 15th, two sober-faced members of the House and one

You might also like