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 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
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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
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 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 University of Technology, Institute of Applied Synthetic Chemistry,
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 University of Technology, Institute of Applied Synthetic Chemistry,
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.
4 Organic Synthesis Using Biocatalysis

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.
Introduction, Types of Reactions, and Sources of Biocatalysts Chapter 1 5

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.
6 Organic Synthesis Using Biocatalysis

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
Introduction, Types of Reactions, and Sources of Biocatalysts Chapter 1 9

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.
10 Organic Synthesis Using Biocatalysis

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.
Introduction, Types of Reactions, and Sources of Biocatalysts Chapter 1 11

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.
12 Organic Synthesis Using Biocatalysis

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.
Introduction, Types of Reactions, and Sources of Biocatalysts Chapter 1 13

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)
14 Organic Synthesis Using Biocatalysis

Table 1.2   Summary of Common Biocatalytic Transformations (cont.)

Starting
Reaction Type Material(s) Product(s) Common Uses Enzyme Types
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)
Introduction, Types of Reactions, and Sources of Biocatalysts Chapter 1 15

Table 1.2   Summary of Common Biocatalytic Transformations (cont.)

Starting
Reaction Type Material(s) Product(s) Common Uses Enzyme Types
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
16 Organic Synthesis Using Biocatalysis

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.
Introduction, Types of Reactions, and Sources of Biocatalysts Chapter 1 17

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
18 Organic Synthesis Using Biocatalysis

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.
Introduction, Types of Reactions, and Sources of Biocatalysts Chapter 1 19

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).
20 Organic Synthesis Using Biocatalysis

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
Introduction, Types of Reactions, and Sources of Biocatalysts Chapter 1 21

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.
22 Organic Synthesis Using Biocatalysis

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.
Introduction, Types of Reactions, and Sources of Biocatalysts Chapter 1 23

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).
24 Organic Synthesis Using Biocatalysis

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,
Introduction, Types of Reactions, and Sources of Biocatalysts Chapter 1 25

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.
26 Organic Synthesis Using Biocatalysis

FIGURE 1.17
Addition of HCN to carbonyl compounds.

6.13 Hydrogen Cyanide Addition to Carbonyls

Enzymes of the hydroxynitrilase class catalyze the addition of HCN to
aldehydes, producing cyanohydrins. Recently, the reaction has been extended
to a few ketones with modified hydroxynitrilase enzymes. In many cases, these
are formed with good optical purities and such reactions are the simplest type
of enzyme catalyzed carbon–carbon bond formation. By pairing hydroxyni-
trile lyases with nitrilases or nitrile hydratases, one-pot, multistep conversions
become possible, and this also shifts the equilibrium to favor the addition
products. Such concerns are particularly important when applying these cata-
lysts to ketones where the equilibrium generally favors the starting carbonyl
compound (Figure 1.17).

6.14 Aldol Reactions

The vast majority of carbon–carbon bonds formed in nature occur by either
aldol reactions or Claisen condensations. Aldolases catalyze the first type of re-
action, and they have proven very useful in synthetic applications. In general,
aldolases are highly selective for the enol(ate) partner (the aldol donor), but
often tolerate a good deal of structural diversity in the acceptor aldehyde. This
tolerance has been exploited in using aldolases to synthesize a wide variety of
targets with high stereochemical control. In favorable cases, aldolases with com-
plementary stereoselectivities are commercially available, which enables one to
prepare all four possible stereoisomeric products from a single pair of starting
materials and no need for protecting groups or chiral auxilliaries.
Aldolases are typically classified by enol(ate) donor, and four basic types are
currently known. Pyruvate decarboxylase and deoxyribose phosphate aldolase
generate one stereocenter (Figure 1.18).
Dihydroxyacetone phosphate aldolases and threonine aldolases generate two
asymetric centers. The former type has been explored extensively and many syn-
thetic examples exist (Figure 1.19).

7 SOURCES OF BIOCATALYSTS
All animals, plants and microorganisms produce enzymes to catalyze the myr-
iad reactions responsible for their survival. It has long been recognized that ex-
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gente? ¿Soy ladrón o facineroso?... No; yo vengo aquí con móviles de
honradez... ¿Podrán todos decir lo mismo?
—No, aquí no ha entrado nadie, nadie más que usted.
—Puesto que usted lo dice, Elenita, lo creo —dijo el hombre oscuro
tomando una silla—. Con la venia de usted me sentaré. Estoy muy
fatigado.
—¡Y se sienta!
—Sí, porque tenemos que hablar. Atención, Elenita: yo tengo la
desgracia de estar prendado de usted.
—Pues mire usted, yo tengo muchas desgracias, menos esa.
Romo contrajo su semblante, expresando sus afectos, como los
animales, de una manera muy opaca, digámoslo así, por ser incapaz
de hacerlo de otro modo. No podía decirse si era el ruin despecho o la
meritoria resignación lo que determinaba aquel signo ilegible, que en
él reemplazaba a la clara sonrisa, señal genérica de la raza humana.
—Pues mire usted —dijo afectando candidez—, a otros les ha
pasado lo mismo, y al fin, a fuerza de paciencia, de buenas acciones y
de finezas se han hecho adorar de las que les menospreciaban.
—No conseguirá usted tal cosa de la hija de mi madre.
—Pues qué... ¿tan feo soy? —preguntó Romo, indicando que no
tenía la peor idea respecto a sus gracias personales.
—No, no; es usted monísimo —dijo Elena con malicia—, pero yo
estoy por los feos... ¿Quiere usted hacer una cosa que me agradará
mucho?
—No tiene usted más que hablar, y obedeceré.
—Pues déjeme sola.
—Eso no... —repuso frunciendo el ceño—. No pasa un hombre los
días y las noches oyendo leer sentencias de muerte, y acompañando
negros a la horca; no pasa un hombre, no, su vida entre lágrimas
suspiros, sangre y cuerpos horribles que se zarandean en la soga
para venir un rato en busca de goces puros junto a la que ama, y
verse despedido como un perro.
—Pero yo, pobre de mí, ¿qué puedo remediar? —dijo Elena
cruzando las manos.
—Es terrible cosa —continuó el hombre-cárcel con hueco acento—
que ni siquiera gratitud haya para mí.
—¿Gratitud?..., eso sí..., estamos muy agradecidos.
 —Se compromete uno, se hace sospechoso a sus amigos
intercediendo siempre por un don Benigno que mató a muchos
guardias del rey en el Arco de Boteros; trabaja uno, se desvive, se
desacredita, echa los bofes..., y en pago..., vea usted... ¡Rayo!, hay
una niña que en nada estima los beneficios hechos a su familia..
¿Qué le importan a ella la buena opinión del favorecedor de su padre
su honradez, su limpia fama en el comercio?... Todo lo pospone a
morrioncillo, a las espuelas doradas y al bigotejo rubio de un
mozalbete que no tiene sobre qué caerse muerto, hijo y hermano de
conspiradores...
Encendida como la grana, Elena se sentía cobarde. Pero si su valo
igualara a su indignación, y sus tijeras pudieran cortar a un hombre
como cortaban un hilo, allí mismo dividiera en dos pedazos a Romo.
—Cállese usted, cállese usted —exclamó sofocada.
—Y, sin embargo —añadió el hombre opaco poniéndose más
amarillo de lo que comúnmente era—, soy bueno, tengo paciencia, me
conformo, callo y padezco... Es verdad que tengo en mi poder un
instrumento de venganza..., pero no lo emplearé por razón de amor
no; lo emplearé tan solo por el decoro de esta familia, a quien estimo
tanto.
Elena tuvo un arranque de esos que se han visto alguna vez, muy
pocas, pero se han visto, en las palomas, en los corderos, en las
liebres, en las mariposas, en los seres más pacíficos y bondadosos, y
pálida de ira, con los labios secos, y los puños cerrados, apostrofó a
amigo de su familia, gritando así:
—Usted es un malvado, y si yo supiera que algún día había de cae
en el pecado de quererle, ahora mismo me quitaría la vida para que no
pudiera llegar ese día. Usted es un tunante, hipócrita y falsario, y si m
padre dice que no, yo diré que sí, y si mi padre y mi madre me
mandan que le quiera, yo les desobedeceré. Hágame usted todo e
daño que guste, pues todo lo que venga de usted lo desprecio, sí
señor, como desprecio su persona toda, sí, señor; su alma y su
cuerpo, sí, señor... Ahora, ¿quiere usted quitárseme de delante, o
tendré que llamar a la vecindad para que me ayude a echarle por la
escalera abajo?
Al concluir su apóstrofe, la doncella se quedó sin fuerzas y cayó en
una silla; cayó blanda, fría, muerta como la ceniza del papel cuando ha
concluido la rápida llama. No tenía fuerzas para nada, ni aun para
mirar a su enemigo, a quien suponía levantado ya para matarla. Pero
el tenebroso Romo, más que colérico, parecía meditabundo, y miraba
al suelo, juzgando sin duda indigno de su perversidad grandiosa e
conmoverse por la flagelación de una mano blanca. Su resabio de
mascullar se había hecho más notable. Parecía estar rumiando un
orujo amargo, del cual había sacado ya el jugo de que nutría
perpetuamente su bilis. Veíase el movimiento de los músculos
maxilares sobre el carrillo verdoso, donde la fuerte barba afeitada
extendía su zona negruzca. Después miró a Elena de un modo que s
indicaba algo, era una especie de paciencia feroz o el aplazamiento de
su ira. La córnea de sus ojos era amarilla, como suele verse en los
hombres de la raza etiópica, y su iris, negro con azulados cambiantes
Fijaba poco la vista, y rara vez miraba directamente como no fuera a
suelo. Creeríase que el suelo era un espejo, donde aquellos ojos se
recreaban viendo su polvorosa imagen.
Levantose pesadamente, y dando vueltas entre las manos a
sombrero, habló así:
—Y sin embargo, Elena, yo la adoro a usted... Usted me insulta, y
yo repito que la adoro a usted... Cada uno según su natural; el mío es
requemarme de amor... ¡Rayo!, si usted me quisiera, aunque no fuese
sino poquitín, me dejaría gobernar como un perro faldero... Sería usted
la más feliz de las mujeres y yo el más feliz de los hombres, porque la
quiero a usted más que a mi vida.
Sus palabras veladas y huecas parecían salir de una mazmorra. Sin
embargo, hubo en el tono del hombre oscuro una inflexión que casi
casi podría creerse sentimental; pero esto pasó, fue cosa de brevísimo
instante, como la rápida y apenas perceptible desafinación de un buen
instrumento músico en buenas manos. Elena se echó a llorar.
—Ya ve usted que no puede ser —balbució.
—Ya veo que no puede ser —añadió Romo mirando a su espejo, es
decir, a los ladrillos—. Puede que sea un bien para usted. Mi corazón
es demasiado grande y negro... Ama de una manera particular..., tiene
esquinas y picos..., de modo que no podrá querer sin hacer daño... A
mí me llaman el hombre de bronce... Adiós, Elenita..., quedamos en
que me resigno..., es decir, en que me muero... Usted me aborrece..
¡Rayo, con cuánta razón!... Es que soy malo, perverso, y amenacé a
usted con hacer ahorcar a ese pobre pajarito de Seudoquis... No lo
haré... Si lo ahorcara, al fin le olvidaría usted, olvidándose también de
mí... Eso sí que no me gusta. Es preciso que usted se acuerde de este
desgraciado alguna vez.
Elena, no comprendiendo nada de tan incoherentes razones
vacilaba entre la compasión y la repugnancia.
—Además, yo había amenazado a usted con otra cosa —dijo Romo
retrocediendo después de dar dos pasos hacia la puerta—. Yo tengo
una carta, sí, aquí está..., en mi cartera la llevo siempre. Es una
esquela que usted escribió a esa lagartija. En ella dice que yo soy un
animal... Bien: puede que sea verdad. Yo dije que iba a mostrar la
carta a su mamá de usted... No; ¿a qué viene eso? Me repugnan las
intriguillas de comedia. ¡Yo enseñando cartas ajenas, en que me
llaman animal!... Tome usted el papelejo y no hablemos más de eso.
Romo largó la mano con un papel arrugado, del cual se apoderó
Elena, guardándolo prontamente.
—Gracias —murmuró.
En aquel instante oyose la campanilla de la puerta, y la voz de don
Benigno que gritaba:
—¡Hija mía, soy yo, tu padre!
Elena corrió a abrir, y el amoroso don Benigno abrazó con frenesí a
su adorada hija, comiéndose a besos la linda cara, sonrosada de
llorar. También él lloraba como una mujer.
—¿Quién está aquí?... ¿Con quién hablabas? —preguntó con
viveza el padre, luego que pasaron las primeras expansiones de su
amor.
Al entrar en la sala, don Benigno vio a Romo que iba a su encuentro
abriendo también los brazos.
—¡Ah! ¿Estaba usted aquí..., era usted...? ¡Amigo mío!
—No esperábamos todavía al señor Cordero —dijo Romo—
Desconfiaba de que le soltaran a usted.
—¿Por qué llorabas, hija mía, antes de yo entrar? —dijo el patriota
fijando en esto toda su atención.
—El señor Romo —repuso Elena muy turbada, pero en situación de
poder disimularlo bien— acababa de entrar...
—Yo creí que estaría aquí doña Robustiana —añadió el realista.
 —Y me decía —prosiguió Elena—, me estaba diciendo que usted...
pues, que no había esperanzas de que le soltaran, padre.
—Eso me dijeron esta mañana en la Superintendencia; pero por lo
visto las órdenes que se dieron la semana pasada han hecho efecto.
—Venga acá el mejor de los amigos, venga acá —exclamó don
Benigno con entusiasmo, abriendo los brazos para estrechar en ellos a
su salvador—. Otro abrazo..., y otro... A usted debo mi libertad. No sé
cómo pagarle este beneficio... Es como deber la vida... Venga otro
abrazo... ¡Haber dado tantos pasos para que no me maltrataran en
Zaragoza, haberme servido tan lealmente, tan desinteresadamente
No, no se ve esto todos los días. Y es más admirable en tiempos en
que no hay amigo para amigo... Yo liberal, usted absolutista, y sin
embargo, me ha librado de la horca. Gracias, mil gracias, señor don
Francisco Romo —añadió con emoción que brotaba como un torrente
de su alma honrada—. ¡Bendita sea la memoria de su padre de usted
Por ella juro que mi gratitud será tan duradera como mi vida.
Era la hora de comer; y cerrada la tienda, llegaron la señora, los
niños y el mancebo. Quiso don Benigno que les acompañase Romo a
la frugal mesa; pero excusose el voluntario y partió, dejando a la
hidalga familia entregada a su felicidad. Elena no respiró fácilmente
hasta que no vio la casa libre de la desapacible lobreguez de aque
hombre.
 XI

Dejamos a don Patricio como aquellas estatuas vivas de hielo, a

cuya mísera quietud y frialdad quedaban reducidas, según confesión
propia, las heroínas de las comedias tan duramente flageladas po
Moratín. El alma del insigne patriota había caído de improviso en
turbación muy honda, saliendo de aquel dulce estado de serenidad en
que ha tiempo vivía. Dudas, temores, desconsuelo y congoja le
sobresaltaron en invasión aterradora, sin que la presencia de Sola le
aliviara, porque la huérfana habló muy poco durante todo aquel día, y
no dijo nada de lo que a nuestro anciano había quitado hasta la última
sombra de sosiego.
Mas por la noche, cuando la joven se retiraba, volvió a decir la
terrible frase:
—Si yo me fuera a Inglaterra, ¿qué harías tú, viejecillo bobo?
Don Patricio no pudo hablar, porque su garganta era como de
bronce, y todo el cuerpo se le quedó frío. No pudo dormir nada en toda
la noche, revolviendo en su mente sin cesar la terrible pregunta.
—¡Consagrar yo mi vida a una criatura como esta!... —exclamaba
en su calenturiento insomnio—. ¡Amarla con todas las fuerzas de
alma, ser padre para ella, ser amigo, ser esclavo, y a lo mejor oí
hablar de un viaje a Inglaterra!... ¡Ingrata, mil veces ingrata! ¡Te
ofrezco mi gloria; transmito a ti, bendiciéndote, los laureles que han de
ornar mi frente, y me abandonas!... ¡Ah, Señor, Señor de todas las
cosas!... ¡La ocasión ha llegado! El momento de mi sacrificio sublime
está presente. No espero más. ¡Adiós, hija de mi corazón; adiós
esperanza mía, a quien diputé por compañera de mi fama!... Tú a
Inglaterra, yo a la inmortalidad... ¿Pero a qué vas tú a Inglaterra
grandísima loca? ¿A qué?... Sepámoslo. ¡Ay!, te llama el amor de un
hombre, no me lo niegues; de un hombre a quien amas más que a mí
más que a tu padre, más que al abuelo Sarmiento... ¡Por vida de la
ch...! Esto no lo puedo consentir, no mil veces... Yo tengo mucho
corazón... Sola, Sola de mi vida..., ¿por qué me abandonas? ¿Por qué
te vas, y dejas solo, pobre, miserable, a tu buen viejecito que te adora
como a los ángeles? ¿De qué me acusas? ¿Te he faltado en algo?
¿No soy siempre tu perrillo obediente y callado que no respiraría si su
respiración te molestara?
Diciendo esto, sus lágrimas regaban la almohada y las sábanas
revueltas.
Al día siguiente notó que Sola estaba también muy triste, y que
había llorado; pero no se atrevió a preguntarle nada.
Por la noche, luego que cenaron, Sola, después de larga pausa de
meditación, durante la cual su amigo la miraba como se mira a un
oráculo que va a romper a hablar, dijo simplemente:
—Abuelito Sarmiento, una cosa tengo que decirte.
Don Patricio sintió que su corazón bailaba como una peonza.
—Pues, abuelito Sarmiento —añadió la joven, mostrando que le era
muy difícil decir lo que decía—, yo, la verdad... ¡tengo una pena, una
pena tan grande!... Si pudiera llevarte conmigo, te llevaría, pero me es
imposible, es absolutamente imposible. Me han mandado ir sola
enteramente sola.
Don Patricio dejó caer su cabeza sobre el pecho, y le pareció que
todo él caía, como un viejo roble abatido por el huracán. Lanzó un
gemido como los que exhala la vida al arrancar del mundo su raíz y
huir.
—Es preciso tener resignación —dijo Sola poniéndole la mano en e
hombro—. Tú, en realidad, no eres hombre de mucha fe, porque con
esas doctrinas de la libertad los hombres de hoy pierden el temor de
Dios, y principiando por aborrecer a los curas, acaban por olvidarse de
Dios y de la Virgen.
—Yo creo en Dios —murmuró Sarmiento—. Ya ves que he ido a
misa desde que tú me lo has mandado.
—Sí, no dudo que creerás; pero no tan vivamente como se debe
creer, sobre todo cuando una desgracia nos cae encima —dijo la
huérfana con enérgica expresión—. Ahora que vamos a separarnos
conviene que mi viejecito tenga la entereza cristiana que es propia de
su edad y de su buen juicio..., porque su juicio es bueno, y felizmente
ya no se acuerda de aquellas glorias, laureles, sacrificios
inmortalidades, que le hacían tan divertido para los granujas de las
calles.
—Yo no he renunciado ni debo renunciar a mi destino —repuso e
anciano humildemente.
—Ni aun por mí...
—Por ti tal vez; pero si te vas...
—Si me voy, será para volver —replicó Sola con ternura—. Yo
confío en que el abuelito Sarmiento será razonable, será juicioso. Si e
abuelito, en vez de hacer lo que le mando, se entrega otra vez o la
vida vagabunda, y vuelve a ser el hazmerreír de los holgazanes
tendré grandísima pena. Pues qué, ¿no hay en el mundo y en Madrid
otras personas caritativas que puedan cuidar de ti como he cuidado
yo? Hay, sí, personas llenas de abnegación y de amor de Dios, las
cuales hacen esto mismo por oficio, abuelito, y consagran su vida a
cuidar de los pobres ancianos desvalidos, de los pobres enfermos y de
los niños huérfanos. A estas personas confiaré a mi pobre viejecillo
bobo, para que me le cuiden hasta que yo vuelva.
Don Patricio, que había empezado a hacer pucheros, rompió a llora
con amargura.
—Soledad, hija de mi alma... —exclamó—. Ya comprendo lo que
quieres decirme. Tu intención es ponerme en un asilo... ¡Lo dices y no
tiemblas!
Después, variando de tono súbitamente, porque variaba de idea
ahuecó la voz, alzó la mano y dijo:
—¡Y crees tú que a un hombre como este se le mete en un
hospicio! Sola, Sola, piénsalo bien. Tú has olvidado qué clase de
mortal es el que tienes en tu casa. ¡Y me crees capaz de aceptar esa
vida oscura, sin gloria y sin ti, sin ti y sin gloria!, ¡ay!, los dos polos de
mi existencia... Mira, niña de mi alma, para que comprendas cuánto te
quiero y cómo has conquistado mi gran corazón, te diré que yo no soy
el que era; que si mis ideas no han variado, han variado mis acciones
y mi conducta.
Y luego, con una seriedad que hizo sonreír a Sola en medio de su
pena, se expresó así:
—Es evidente..., porque esto es evidente como la luz del día..., que
yo estoy destinado a coronarme de gloria, a adornar mi frente de rayos
esplendorosos, sacrificándome por la libertad, ofreciéndome como
víctima expiatoria en el altar de la patria, como el insigne general, m
compañero de martirio, que me espera en la mansión de los justos
allá donde las virtudes y el heroísmo tienen eterno premio... Pues bien
es tanto lo que te quiero, que por tu cariño he ido dejando pasar días y
días y hasta meses sin cumplir esto que ya no es para mí una
predestinación tan solo, sino un deber sagrado. ¿Me entiendes?
Soledad le pasó la mano por la cabeza, incitándole a que no
siguiese tocando aquel tema.
—Por ti, solo por ti... —prosiguió el viejo—. ¡Me da tanta pena
dejarte!... Así es que me digo: «Tiempo habrá, Señor...». ¿Creerás que
aquí en tu compañía se me han pasado semanas enteras sin
acordarme de semejante cosa?... Hay más todavía: yo estaba
dispuesto a hacer un sacrificio mayor..., ¿te espantas?, que es el de
sacrificarte mi sacrificio, ¿no lo entiendes?... Sí, poner a tus pies m
propia gloria, mi corona de estrellas... Sí, chiquilla, yo estaba dispuesto
a no separarme jamás de ti, y a no pensar más en la política..., ni en
Riego, ni en la libertad... ¡Oh, hija mía! Tú no puedes comprender la
inmensidad de tal sacrificio. Por él juzgarás de la inmensidad del amo
que te tengo. ¡Y cuando yo renuncio por ti a lo que es mi propia vida, a
mi idea santa, gloriosa, augusta, tú me abandonas, me echas a un
lado como mueble inútil, me mandas a un hospicio y te vas!...
Soledad veía crecer y tomar proporciones aquel problema de la
separación que le causaba tanta pena. Su alma no era capaz de
arrepentirse del bien que había hecho al desvalido anciano; pero
deploraba que por los misteriosos designios de Dios, la caridad que
hiciera algunos meses antes le trajese ahora aquel conflicto que
empezaba a surgir en su cristiano corazón.
—El Señor nos iluminará —dijo, remitiendo su cuita al que ya la
había salvado de grandes peligros—. ¡Si tú le pidieras con fervor
como yo lo hago, luz, fuerzas, paciencia y fe, sobre todo fe...!
—Yo le pediré todo lo que tú quieras, hija de mi alma; yo tendré fe..
Dices que tengo poca; pues tendremos mucha. Me has contagiado de
tantas cosas, que no dudo he de adquirir la fe que tú, solo con
mirarme, me estás infundiendo.
—Para adquirir ese tesoro —dijo Sola con cierto entusiasmo— no
basta mirarme a mí ni que yo te mire a ti, abuelo, es preciso pedirlo a
Dios, y pedírselo con ardiente deseo de poseer su gracia, abriendo de
par en par las puertas del corazón para que entre; es preciso que
nuestra sensibilidad y nuestro pensamiento se junten para alimenta
ese fuego que pedimos y que al fin se nos ha de dar. Teniendo ese
tesoro, todo se consigue: fuerzas para soportar la desgracia, valo
para acometer los peligros, bondad para hacer bien a nuestros
enemigos, conformidad y esperanza, que son las muletas de la vida
para todos los que cojeamos en ella.
—Pues yo haré que mi sensibilidad y mi pensamiento se encaminen
a Dios, niña mía —replicó el vagabundo participando del entusiasmo
de su favorecedora—. Haré todo lo que mandas.
—Y tendrás fe.
—Tendremos fe..., sí; venga fe.
—Con ella resolveremos todas las cuestiones —dijo Sola
acariciando el flaco cuello de su amigo—. Ahora, abuelito, es preciso
que nos recojamos. Es tarde.
—Como tú quieras. Para los que no duermen, como yo, nunca es
tarde ni temprano.
—Es preciso dormir.
—¿Duermes tú?
—Toda la noche.
—Me parece que me engañas... En fin, buenas noches. ¿Sabes lo
que voy a hacer si me desvelo? Pues voy a rezar, a reza
fervorosamente como en mis tiempos juveniles, como rezábamos
Refugio y yo cuando teníamos contrariedades, alguna deudilla que no
podíamos pagar, alguna enfermedad de nuestro adorado Lucas... Ello
es que siempre salíamos bien de todo.
—A rezar, sí; pero con el corazón, sin dejar de hacerlo con los
labios.
—Adiós, ángel de mi guarda —dijo Sarmiento besándola en la
frente—. Hasta mañana, que seguiremos tratando estas cosas.
Retirose Soledad, y el anciano se fue a su cuarto y se acostó
durmiéndose prontamente; mas tuvo la poca suerte de despertar a
poco tiempo sobresaltado, nervioso, con el cerebro ardiendo.
—Ea, ya estamos desvelados —dijo dando vueltas en su cama, que
había sido para él durante diez meses un lecho de rosas—. Voy a
poner por obra lo que me mandó la niña: voy a rezar.
Disponiendo devotamente su espíritu para el piadoso ejercicio, rezó
todo lo rezable, desde las oraciones elementales del dogma católico
hasta las que en distintas épocas ha inventado la piedad para da
pasto al insaciable fervor de los siglos. Sarmiento rezó a Dios, a la
Virgen, a los santos que antaño habían sido sus abogados, sin olvida
a los que fueron procuradores de Refugio, mientras esta les
necesitara.
Mas a pesar de ello, el anciano no advirtió que entrara gran porción
de calma en su espíritu; antes bien sentíase más irritado, más inquieto
con propensiones a la furia y a protestar contra su malhadada suerte
Como llegara un instante en que no pudo permanecer en el abrasado
lecho, levantose en la oscuridad y se vistió a toda prisa sin esta
seguro de ponerse la ropa al derecho. Sentía impulsos de sali
gritando por toda la casa, y de llamar a Sola, y echarle en cara la
crueldad de su conducta y decirle: «Ven acá, loca, ¿quién es el infame
que te llama desde Inglaterra?... ¿Qué vas tú a hacer a Inglaterra?..
¡Ah! Es un noviazgo lo que te llama. Y si es noviazgo, ¡vive Dios!
¿quién es ese monstruo? Dime su nombre, y correré allá y le
arrancaré las entrañas».
En la sala distinguió débil claridad: supuso que había luz en e
cuarto de su amiga. Paso a paso, avanzando como los ladrones
dirigiose allá; empujada suavemente la puerta, pasó a un gabinete
deslizose como una sombra, extendiendo las manos para tocar los
objetos que pudieran estorbarle el paso. La puerta de la alcoba estaba
entreabierta; había luz dentro, pero no se oía el más leve rumor
Alargando el cuello, Sarmiento vio a Sola dormida junto a una mesa en
la cual había papeles y tintero.
«Estaba escribiendo —pensó,— y se ha dormido. Veremos a
quién».
Entró en la alcoba, andando quedamente y con mucho cuidado
para no hacer ruido. Su rostro anhelante, su cuerpo tembloroso, sus
ojos ávidos y saltones, dábanle aspecto de fantasma; y si la joven
despertase en aquel momento, se llenaría de terror al verle. Dormía
profundamente, la cabeza apoyada en el respaldo del sillón. Delante
tenía una carta a medio escribir, y otra muy larga y de letra extraña, a
la cual sin duda estaba contestando cuando se durmió.
—Yo conozco esa letra —pensó Sarmiento, devorando con los ojos
el escrito, apoyado en un libro puesto de canto a manera de atril.
Conteniendo su respiración, el vagabundo examinó el pliego, que
abierto por el centro no presentaba ni el principio ni el fin. Después fijó
los ojos en la carta medio escrita por Sola. Don Patricio miraba y
fruncía el ceño apretando las mandíbulas. Tenía tal aspecto de
ferocidad aviesa, que si él mismo pudiera verse tuviera miedo de s
mismo. No tardó mucho en satisfacer su curiosidad, y era esta tan
intensa, que después de leer una vez, leyó la segunda. A la tercera no
estaba tampoco satisfecho; mas temiendo que la joven despertara, se
retiró como había venido. Al llegar a su cuarto se dejó caer en la cama
y dando un gran suspiro exclamó para sí:
«¡Bien lo decía yo: los emigrados...!».
 XII

Muy gozoso y satisfecho estaba don Benigno Cordero con e

suceso de su vuelta a la patria y al hogar querido, y resuelto a que e
contento le durase, hacía propósito firmísimo de no tornar a mezclarse
en política, ni vestir uniforme, ni menos hacer heroicidades en Boteros
ni en otro arco alguno. Verdad es que guardaba en su pecho, cua
tesoro riquísimo, o como los restos queridos de una persona amada
que se depositan en secreta urna, las mismas aficiones políticas a que
debió su destierro. Eso sí: antes creyera que el sol salía de noche que
dejar de ver en la libertad, en el progreso y en la soberanía del pueblo
la felicidad de las naciones. Mas era preciso poner una losa sobre
estas cosas, y don Benigno la puso.
—Desde hoy —dijo— Benigno Cordero no es más que un
comerciante de encajes. No adulará al absolutismo, no dirá una sola
palabra en favor de este; pero no, ya no tocará más el pito
constitucional ni la flauta de la Milicia. A Segura llevan preso. Yo tengo
ideas, sí, ideas firmes, pero tengo hijos. Es posible, es casi seguro que
otros, que también tienen mis ideas, las hagan triunfar; pero mis hijos
por nadie serán cuidados si se quedan sin padre. Atrás las doctrinas
por ahora, y adelante los muchachos. Ahora silencio, paz, retraimiento
absoluto..., cabeza baja y pico cerrado... Pero, ¡ay!, alma mía, allá
recogida en ti misma y sin que te oigan los oídos de la propia carne en
que estás encerrada, no ceses de gritar: «¡Viva, viva, y mil veces viva
la señora Libertad!».
Los muchos amigos del exjefe de milicianos le felicitaban
cordialmente, y sus parroquianos, así como sus compañeros de
comercio, recibieron gran contento al verle. Como era tan generoso, y
tenía un natural por demás expansivo, antojósele, ocho días después
del de su vuelta, obsequiar a los amigos con un modesto banquete
dedicado a grabar en la memoria de todos el fausto evento de su
liberación; pero doña Robustiana, cuyo sentido práctico igualaba a
peso de su cuerpo, le quitó de la cabeza la idea de aquel dispendioso
alarde, arguyéndole así:
—Desgraciadamente no estamos para fiestas. Acuérdate del dinero
que has gastado en congraciarte con esos pillos; que tiempo hay de
dar banquetes. Mañana domingo, 28 de agosto, haremos para la cena
un extraordinario de poca monta, y convidaremos a Romo, al señor de
Pipaón, que también nos ha servido, y a Sola. Total: tres convidados
Basta, hombre, basta. Tiempo hay de echar la casa por la ventana, y
no faltará un motivo para ello ni tampoco elementos, ¿me
entiendes?..., porque si siguen los frailes reponiendo la ropa de altar
no faltará venta de encaje blanco en todo el año que corre.
Don Benigno, como siempre, armonizó su opinión con la de su cara
esposa, y a consecuencia de tan dulce concordia, al día siguiente la
cocina de los Cordero despedía inusitado aroma de ricas especias, e
cual anunciaba a toda la vecindad la presencia de un extraordinario. A
la hora de la cena resplandecía el comedor con la luz de dos quinqués
colocados en contrapuestos sitios, y alrededor de la mesa se sentaron
el señor de Pipaón, Sola y los de Cordero, sin excluir los niños, que
ocupaban un extremo junto a su hermana. El puesto más preeminente
entre los de convite estaba vacío, lo cual causaba gran disgusto a don
Benigno.
—¿Por qué no habrá venido Romo? —decía—. Es particular: no le
hemos visto desde el día de mi llegada. ¿Estará enojado con
nosotros?
Se esperó un rato; pero viendo que no parecía, dio principio e
banquete. El digno anfitrión estaba intranquilo por aquella ausencia de
su amigo, y a cada instante miraba a su esposa como para preguntarle
qué opinaba ella de tan extraño caso. Ya doña Robustiana había
dicho:
—Estará muy ocupado en la Comandancia de Voluntarios. Se le
han mandado tres avisos al anochecer. Ustedes no saben bien la
calma que gasta el señor de Romo. Otra noche le convidamos a cena
y se descolgó aquí a las diez de la noche.
La señora presidía majestuosamente la mesa y gobernaba con
mucha destreza aquella maniobra de los banquetes antiguos
consistente en estar pasando platos de aquí para allí, y de derecha a
izquierda, como si los convidados, en vez de reunirse para comer, lo
hicieran para jugar al juego de sopla y vivo te lo doy. Descollaba su
hermoso busto por encima de la blanca mesa, a manera de un tronco
forrado en tela oscura sobre el cual colocaran su cabeza como
provisionalmente y mientras parecía el cuello perdido. Con la
estrechez del ajuste, los abundantes dones que en ella acumuló sin
tasa Natura formaban un circuito de tanta extensión, que una mosca
(esto puede asegurarse y lo certificaron testigos oculares), una mosca
decimos, que salió de uno de los brazos para ir al otro pasando po
delante, tardó no se sabe cuánto tiempo en dar la vuelta y llegar a su
destino.
En el otro extremo de la mesa, Primitivo y Segundo, que por ser día
de fiesta vestían de padres provinciales de la Orden dominica, estaban
bajo la vigilancia de Soledad y Elena respectivamente, las cuales no
podían probar bocado, entretenidas en enseñar a los frailescos
ángeles el modo de comer; y mientras el uno se rociaba con sopa los
hábitos, llevábase el otro la cuchara a los ojos, sin cesar de pedir
chillar y hacer comentos varios sobre cuanto desde la fuente a sus
platos pasaba.
Pipaón, cuyo apetito parecía crecer a medida que había menos
motivos aparentes para ello, amenizaba con chistes la comida. Estaba
elegantísimo, como de costumbre, el ingenioso cortesano, ataviado
con su calzón blanco, su levita polonesa de mangas jamonadas, su
corbata metálica destinada a anticipar la idea de la muerte en garrote
por si acaso algún día era el individuo condenado a ella. Revueltos los
cabellos con artístico desorden, parecía su cabeza una escoba, en lo
cual cumplía a maravilla con los conceptos de la moda corriente. ¡Oh!
era aquel un señor muy bondadoso y sencillo, que lo mismo se
sentaba a la mesa del rico que a la del pobre, con tal que en ellas
hubiera buenos manjares que comer; y sin dar privadamente excesiva
importancia a las ideas políticas, lo mismo fraternizaba con el negro
que con el blanco, siempre que ni el uno ni el otro le estorbasen en su
prodigioso medro. Menos alegre que su comensal a causa de la
ausencia de Romo, don Benigno conversaba con chispa y donaire
volviendo con graciosa movilidad el rostro hacia Pipaón, hacia su
esposa y hacia la silla vacía donde se echaba de menos la torva figura
del voluntario realista; y, ¡cosa singular!, aquella silla donde no se
sentaba el hombre oscuro, tenía cierto aspecto lúgubre. Romo no
estaba allí, y, sin embargo, parecía que estaba.
Esquivando entrar en el tema político a que la verbosidad importuna
y mareante de Pipaón quería llevarle, don Benigno dijo:
—Ya he manifestado cuál es mi propósito. Y qué, señor don Juan
¿cree usted que me será difícil cumplirlo? De ningún modo. Los que
necesitan de la política para vivir, porque si no hay bullanga no comen
difícilmente aceptarán esta oscura vida privada que es mi delicia. Quite
usted a los intrigantes la política, y será como si les cortaran las manos
a los rateros o los pies a las bailarinas. ¿Digo mal? Hoy con este
partido, mañana con el otro, ello es que siempre se les ve a flote...
A don Benigno se le cayó del tenedor un pedazo de calabacín que
en él tenía, aguardando a que la boca callase para entrar. La causa de
tan inesperado siniestro fue que doña Robustiana le estaba tocando e
codo, primero suavemente y después con fuerza, para que su marido
cayese en la cuenta de que estaba haciendo la sátira de Pipaón.
—Verdad es que no todos los que se ocupan de política son así —
dijo el honrado comerciante pinchando de nuevo la hortaliza—, ya se
comprende; pero ni a unos ni a otros quiero parecerme. La vida
privada es hoy mi sueño de oro... No quiere decir que en lo íntimo de
mi alma no exista siempre... Pero dejemos esto. Puede uno llevar en
su fuero interno el fardo que más le acomode, sin necesidad de
ponerse una etiqueta en la frente..., esto es claro como el agua. No
hay necesidad de meter ruido. En la vida privada puede tener el buen
ciudadano mil ocasiones de realizar fines patrióticos y de servir a la
patria. ¿Cómo? Cumpliendo lealmente esa multitud de pequeños
esfuerzos que en conjunto reclaman tanta energía como cualquier acto
de heroísmo: así lo ha dicho Juan Jacobo Rous..., tente, lengüita
Dejemos a ese caballero en su casa, pues hay palabras que ahorcan..
Yo me concreto a lo siguiente: vea usted mi plan, señor de Pipaón.
Antes que el plan de don Benigno, merecía la atención de Bragas
una lonja de ternera, cuyo especioso condimento bastaba a acredita
la ciencia culinaria de la señora de Cordero.
—Muy bien, señor don Benigno —gruñó Pipaón, engullendo—. Su
plan de usted me parece muy bien asado... No, no; quiero decir que la
ternera está muy bien asada, y que su plan de usted es excelente
sabrosísimo, es decir atinadísimo.
 —Mi plan es el siguiente: yo trabajo todo el día, con excepción de
los domingos; yo cumplo con los preceptos de nuestra Santa Madre la
Iglesia oyendo misa, confesando y comulgando como se me manda
yo cumplo asimismo mis obligaciones comerciales; yo no debo un
cuarto a nadie; yo educo a mis hijos; yo pago mis contribuciones
puntualmente; yo obedezco todas las leyes, decretos, bandos y
órdenes de la autoridad; yo hago a los pobres la limosna que m
fortuna me permite; yo no hablo mal de nadie, ni siquiera del gobierno
yo sirvo a los amigos en lo que puedo; yo no conspiro; yo celebro
mucho que todos vivan bien y estén contentos; en suma, yo quiero se
la más ordenada, puntual y exacta clavija de esta gran máquina que se
llama la patria, para que no dé por mi causa el más ligero tropezón..
¿Qué tal? ¿Me explico bien?
Conversación tan interesante hubo de interrumpirse, porque uno de
los chicos tuvo la ocurrencia de derramar sobre su hábito toda la salsa
que había en el plato, mientras el otro berrequeaba como un ternero
porque no le permitían comer con las manos. Calmada la agitación a
otro extremo de la mesa, don Benigno continuó:
—Siempre ha sido mi norma de conducta..., Segundito, cuidado...
ocupar el puesto que me señalaban las circunstancias. He sido y soy
esclavo de mi deber... Primitivo, que te estoy mirando; ¿cómo se coge
el tenedor?... Un día las circunstancias me dijeron: «es preciso que
seas valiente», y fui valiente. Heridas tengo que darán razón de ello
Hoy me dicen las circunstancias: «es preciso que seas pacífico», y
pacífico soy... Niños, ¿me enfado?... Mi conciencia está tranquila con
tan juicioso plan de conducta; a mi conciencia obedezco, y nada más.
En esto sonaron fuertes campanillazos en la puerta de la casa.
—A buena hora viene ese señor..., cuando ya estamos en los
postres —dijo don Benigno—. De seguro es Romo.
—No, no llama él de ese modo —observó la señora, poniendo
atención para oír en el momento que la criada abría.
—Puede que sea Romo —indicó Pipaón dirigiendo sus dedos en
persecución de una pera que rodaba por el mantel.
—Son dos señores, dos hombres —dijo la criada entrando en e
comedor—. Preguntan por el amo.
—Allá voy —dijo Cordero levantándose.
—Que esperen —manifestó doña Robustiana con mal humor—
¡Que siempre te has de levantar de la mesa...!
 Don Benigno salió con la servilleta sujeta al cuello. En la sala
encontró a dos hombres desconocidos.
—Una luz, Reyes —gritó a la criada.
La claridad de la vela que trajo la moza permitió al honrado patriota
distinguir bien las fisonomías. Creía reconocer aquellas caras. Ninguna
de las dos despertaba grandes simpatías, y en cuanto a los cuerpos
eran de lo más sospechoso que puede imaginarse.
—¿Es usted don Benigno Cordero? —le preguntó uno de ellos
secamente.
—Para lo que gusten mandar. ¿Qué quieren ustedes?
—Que venga usted con nosotros.
—¿A dónde?
—¡Toma!..., a la cárcel —exclamó el individuo esgrimiendo su
bastoncillo, y admirado de que no se hubiera comprendido el objeto de
tan grata visita.
Don Benigno se quedó aturdido... Creía soñar... Estaba lelo.
—¡A la cárcel! —murmuró.
—Y pronto. Tenemos que hacer...
—A la cárcel... —dijo otra vez Cordero, como el delirante que repite
un tema—. Yo..., ¿por qué?..., yo..., ¿han dicho que a la cárcel...?
—Sí, señor, a la cárcel... Nosotros no tenemos que explicar... No
somos jueces —graznó el polizonte con desenfado y altanería
consecuente con el tono general de los pillastres que se dedican a
perseguir a la gente honrada.
—Aguarden un momento —dijo Cordero sin saber lo que decía—
Voy... Les diré a ustedes...
Dio varias vueltas, tropezó en una puerta. Parecía un hombre que
ha perdido la cabeza y la está buscando. Sin propósito deliberado, fue
al comedor, entró. Su esposa y su hija perdieron el color al ver su cara
que era la cara de un muerto.
—Son dos caballeros —murmuró Cordero con voz trémula—. Dos
amigos... No hay que asustarse... Tengo que salir con ellos... Pipaón
amigo, salga usted a ver qué es eso... Mi sombrero, ¿en dónde está m
sombrero?
Dio una vuelta alrededor de la mesa y salió otra vez. Sin duda había
perdido el juicio.
—Conque dicen ustedes que... ¡a la cárcel!... ¿Y se podrá saber...?
 —Si usted no viene pronto —dijo el polizonte con ira—, llamaremos
a los voluntarios que están abajo.
El otro bribón había encendido un cigarro y fumaba mirando los
cuadros de la sala.
—Pues vamos. Esto es una equivocación —dijo el comerciante
recobrando un poco su entereza.
—¿Pero su hija de usted no se presenta? —preguntó el prime
esbirro.
—¡Mi hija!
—¡Sí, señor, su hija! —exclamó el mismo abriendo las manos y
mostrando en dos abanicos de carne sus diez dedos sucios, negros
nudosos y con las yemas amarillas por el uso del cigarro de papel.
—¿Y para qué tiene que presentarse mi hija?
—¿Pues qué?... ¿No le dije que su hija tiene que venir también a la
cárcel?
—Usted no me ha dicho nada, y si me lo hubiera dicho, no lo habría
creído —afirmó Cordero sintiendo que su corazón se oprimía.
—Vea usted este papel —dijo el funcionario mostrando un volante
—. Benigno Cordero y su hija Elena Cordero.
—¡Mi hija! —exclamó don Benigno, lanzando un gemido de dolor—
¿Pues qué ha hecho mi hija?
—¡Eh! Que suban los voluntarios. Así despacharemos pronto.
Don Benigno se había vuelto idiota. No se movía. Pipaón, que
había oído algo desde la puerta, se acercó diciendo:
—Esto ha de ser alguna equivocación de la Superintendencia.
Al verle, los de policía le hicieron una reverencia, como suele
usarlas la infame adulación cuando quiere parecerse a la cortesía.
—¿No es usted el que le llaman Mala Mosca? ¿No me debe usted
su destino? —preguntó Pipaón.
—Sí, señor —repuso el infame, mostrando tras los replegados
labios una dentadura que parecía un muladar—. Soy el mismo para
servir al señor de Pipaón.
—A ver la orden.
Pipaón leyó a punto que entraban en la sala, sobrecogidas de
terror, las tres mujeres, los dos frailecitos y la criada.
—Nada, nada: esto debe de ser un quid pro quo —dijo Bragas con
disgusto evidente—; pero es preciso obedecer la orden. Desde este
momento empiezo a dar los pasos convenientes...
 Los de Cordero se miraron unos a otros. Se oía la respiración. En
aquel instante de congoja y pavura, Elena fue la que tuvo más valor, y
haciendo frente a la situación, exclamó:
—¿Yo también he de ir presa? Pues vamos. No tengo miedo.
—¡Hija de mi alma! —gritó doña Robustiana abrazándola con furo
—. No te separarás de mí. Si a los dos os llevan presos, yo voy
también a la cárcel y me llevo a los niños.
—Con usted no va nada, señora —dijo el polizonte—. El seño
mayor y la niña son los que han de ir... Conque, andando.
Arrojose como una hiena la señora sobre aquel hombre, y de
seguro lo habría pasado mal el funcionario de la Superintendencia, s
doña Robustiana, en el momento de clavar las manos en la verrugosa
cara de su presa, no hubiera quedado sin sentido, presa de un breve
síncope. Acudieron todos a ella, y el de policía gritó, poniéndose rojo y
horrible:
—¡Al demonio con la vieja!... Vamos al momento, o que suban los
voluntarios. No podemos perder el tiempo con estos remilgos.
Don Benigno, cuyo espíritu estaba templado para hacer frente a las
situaciones más terribles, elevose sobre aquella tribulación, como e
sol sobre la bruma, e iluminando la lúgubre escena con un rayo de
heroísmo que a todos les dejó absortos, gritó:
—Vamos, vamos a la cárcel. Ni mi hija ni yo temblamos. La
inocencia no tiene miedo, cobardes sayones... Vamos a la cárcel, a
patíbulo, a donde queráis, canallas, mil veces canallas... Yo había
vuelto la espalda a la libertad, y la libertad me llama... ¡Allá voy, idea
divino; aquí estoy; adelante!... Vamos, miserables, abandono a m
esposa, a mis hijos. Todo se queda aquí... Tan miserables sois
vosotros como Calomarde que os manda. Vamos a la cárcel, y ¡viva la
Constitución!
Salió bizarra y noblemente, lleno de entusiasmo y valor, rodeando
con su brazo el cuello de Elena, que al heroico arrojo de su padre
respondió diciendo también: «¡Viva la Constitución!».
Al salir encargó a Soledad que cuidase de su madre y de sus
hermanos. Algo más pensaba decir; pero los sayones no le dejaron. E
compañero de Mala Mosca se quedó para registrar la vivienda.
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