IONIC LIQUIDS IN BIOTRANSFORMATIONS AND ORGANOCATALYSIS pptx

IONIC LIQUIDS IN
BIOTRANSFORMATIONS
AND ORGANOCATALYSIS
IONIC LIQUIDS IN
BIOTRANSFORMATIONS
AND ORGANOCATALYSIS
Solvents and Beyond
Edited by
Pablo Domínguez de María
A JOHN WILEY & SONS, INC., PUBLICATION
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Library of Congress Cataloging-in-Publication Data:
Ionic liquids in biotransformations and organocatalysis : solvents and beyond / edited by
Pablo Dominguez de Maria.
p. cm.
Includes index.
ISBN 978-0-470-56904-7 (hardback)
1. Proteins–Biotechnology. 2. Ionic solutions. 3. Catalysis. I. Dominguez de Maria, Pablo, 1974-
TP248.65.P76I59 2012
572.6–dc23
2011038032
Printed in the United States of America.
10 9 8 7 6 5 4 3 2 1
CONTENTS
Foreword by Prof. Dr. Roger Sheldon xiii
Preface xv
Contributors xvii
PART I FUNDAMENTALS 1
1 IONIC LIQUIDS: DEFINITION, APPLICATIONS, AND CONTEXT FOR
BIOTRANSFORMATIONS AND ORGANOCATALYSIS 3
Pablo Domínguez de María
1.1 Ionic Liquids: Defi nition, Development, and Overview of Current
Main Applications 3
1.2 On the Greenness of ILs: Toward the Third Generation of ILs
and DES 6
1.3 Context of ILs in Biotransformations and Organocatalysis 12
References 13
2 IONIC LIQUIDS AND PROTEINS: ACADEMIC AND SOME

PRACTICAL INTERACTIONS 15
Zhen Yang
Abbreviations for Ionic Liquid Cations 15
Abbreviations for Ionic Liquid Anions 16
Abbreviations for Ammonium Ionic Liquids 16
Other Abbreviations 17
2.1 Introduction 17
2.2 Ionic Liquids, Water, and Proteins 18
2.2.1 Ionic Nature of Ionic Liquids 18
2.2.2 Protic and Aprotic Ionic Liquids 19
2.2.3 Water Present in the Ionic Liquids 21
2.2.4 Interactions of Water and Ionic Liquids with Proteins 21
2.2.4.1 Effect of Water and Water Activity 22
2.2.4.2 Effect of Ionic Liquids 26
v
vi CONTENTS
2.3 Hofmeister Effects on Biocatalysis 26
2.3.1 Hofmeister Effects of Inorganic Salts 27
2.3.1.1 Quantifi cation of Hofmeister Series 28
2.3.1.2 Effect of Ions on Protein Stability 29
2.3.1.3 Effect of Ions on Enzyme Activity 30
2.3.2 Hofmeister Effects of Ionic Liquids 34
2.3.2.1 Effect of Ionic Liquid Ions on Enzyme
Performance in Aqueous Solution 34
2.3.2.2 Kinetic Studies of Enzymes in Ionic
Liquid-Containing Aqueous Solution 38
2.3.2.3 Enzyme Performance in Ionic
Liquid-Dominating Reaction Systems 39
2.4 Impact of Ionic Liquids on Enzymes and Proteins 41
2.4.1 Effect of Ionic Liquids on Enzyme Activity

and Stability 41
2.4.1.1 Hydrophobicity and Log P 41
2.4.1.2 Nucleophilicity and H-bond Basicity 44
2.4.1.3 Viscosity 45
2.4.2 Effect of Ionic Liquids on Protein Structure
and Dynamics 46
2.4.3 Effect of Ionic Liquids on Protein Refolding
and Renaturation 50
2.4.4 Effect of Ionic Liquids on Protein Crystallization
and Fibrilization 52
2.5 Protein Extraction by Means of Ionic Liquids 52
2.5.1 Aqueous/Ionic Liquid–Liquid
Extraction Systems 52
2.5.2 Ionic Liquid-Based Aqueous
Biphasic Systems 53
2.5.3 Water-in-Ionic Liquid Microemulsion
Systems 56
2.6 Proper Selection of Ionic Liquids for Biocatalysis 57
2.6.1 Amino Acid Ionic Liquids 57
2.6.2 Ammonium and Phosphonium Ionic
Liquids 58
2.6.3 Design of Ionic Liquids for Biocatalysis 59
2.6.4 Proposed Guidelines for Selecting/Designing
Biocompatible Ionic Liquids 64
2.7 Concluding Remarks 65
References 66
CONTENTS vii
PART II IONIC LIQUIDS IN BIOTRANSFORMATIONS 73
3 IONIC LIQUIDS IN BIOTRANSFORMATIONS: MOTIVATION
AND DEVELOPMENT 75

Christina Kohlmann and Lasse Greiner
3.1 First Uses of Ionic Liquids in Biotransformations 75
3.2 Motivation to Use IL in Biotransformations 80
3.3 Challenges for the Use of IL in Biotransformations 91
References 98
4 IONIC LIQUIDS AND OTHER NONCONVENTIONAL SOLVENTS
IN BIOTRANSFORMATIONS: MEDIUM ENGINEERING AND
PROCESS DEVELOPMENT 103
Pedro Lozano and Eduardo García-Verdugo
4.1 Introduction: Toward Greener Catalytic Processes 103
4.2 The Importance of the Medium Engineering
in Biotransformations 106
4.2.1 Enzymes in Nonaqueous Environments 106
4.3 Biocatalysis in Monophasic ILs Systems 110
4.3.1 Medium Engineering in Monophasic
ILs System 110
4.3.2 Isolation and Recyclability Issues in Monophasic
ILs System 115
4.4 (Bio)catalytic Processes in SCFs 118
4.4.1 Properties of SFCs 118
4.4.2 Medium Engineering in Supercritical
Biocatalysis 119
4.4.3 Processes Design for SCF Biocatalysis 122
4.5 Multiphase Biotransformations 124
4.5.1 Biocatalytic Processes in Biphasic
Fluorous Solvents 125
4.5.2 Bioprocesses in Water/scCO
2
Systems 126
4.5.3 Bioprocesses in Biphasic ILs System 128

4.5.3.1 Biphasic ILs/Water System 128
4.5.3.2 Phase Behavior of IL/scCO
2
Biphasic Systems 132
4.5.3.3 Bioprocesses in IL/scCO
2
Biphasic Systems 134
4.6 Prospects 140
Acknowledgments 140
References 141
viii CONTENTS
5 IONIC LIQUIDS AS (CO-)SOLVENTS FOR
HYDROLYTIC ENZYMES 151
Hua Zhao
Nomenclature of ILs 151
Cations 151
Anions 152
5.1 Introduction 152
5.1.1 Type of Hydrolases 152
5.1.2 Properties and Applications of ILs 154
5.2 State-of-the-art: Lipases, Esterases, Proteases in ILs as
(co-)Solvents 155
5.2.1 Effect of Physical Properties of ILs on Hydrolase
Activity and Stability 156
5.2.1.1 IL Polarity 156
5.2.1.2 Hydrogen-bond (H-bond) Basicity and
Nucleophilicity of Anions 157
5.2.1.3 IL Network 160
5.2.1.4 Ion Kosmotropicity 161
5.2.1.5 Viscosity 165

5.2.1.6 Hydrophobicity 165
5.2.1.7 Enzyme Dissolution 170
5.2.2 Other Factors Infl uencing Hydrolase Activity and Stability 171
5.2.2.1 Halide Impurities in ILs 171
5.2.2.2 Water Activity 172
5.2.3 Methods to Improve Hydrolase Activity and Stability 174
5.2.3.1 Enzyme Immobilization 174
5.2.3.2 PEG-Modifi cation 176
5.2.3.3 EPRP 177
5.2.3.4 Water-in-IL Microemulsions. 178
5.2.3.5 Coating Enzymes with ILs 179
5.2.3.6 Designing Hydrolase-Compatible ILs 179
5.3 Use of ILs for (dynamic) Kinetic Resolutions ((D)KRs) 183
5.3.1 Kinetic Resolutions via Hydrolysis in Aqueous
Solutions of ILs 183
5.3.1.1 Enantioselective Hydrolysis of Amino
Acid Esters 183
5.3.1.2 Enantioselective Hydrolysis of Other Esters 184
5.3.2 Kinetic Resolution via Synthesis in Nonaqueous
Solutions of ILs 187
CONTENTS ix
5.3.2.1 Evaluating Hydrolase’s Enantioselectivity via
the Kinetic Resolution of 1-phenylethanol 187
5.3.2.2 Kinetic Resolutions of Other Alcohols 188
5.3.2.3 Kinetic Resolutions of Amines 199
5.3.2.4 Kinetic Resolutions Integrated with Supported
IL Membranes (SILMs) or Microfl uidic Separation 199
5.3.2.5 Kinetic Resolution Using IL/scCO
2
Biphasic Systems 202

5.4 Hydrolase-Catalyzed Esterifi cations of Saccharides and Cellulose
Derivatives in ILs 205
5.5 ILs for Glycosidases 210
5.5.1 Glycosidase-Catalyzed Synthesis in ILs 210
5.5.2 Cellulase-catalyzed Hydrolysis in ILs 211
5.6 Prospects 212
Acknowledgments 213
References 213
6 IONIC LIQUIDS AS (CO-)SOLVENTS FOR NONHYDROLYTIC
ENZYMES 229
Daniela Gamenara, Patricia Saenz Méndez, Gustavo Seoane,
and Pablo Domínguez de María
Nomenclature of ILs 229
6.1 Ionic Liquids and Nonhydrolytic Enzymes 231
6.2 Use of ILs in Oxidoreductase-Catalyzed Enzymatic Reactions 232
6.2.1 Dehydrogenases 232
6.2.2 Laccases, Peroxidases, Oxidases, and Oxygenases 247
6.3 ILs in Lyase-Catalyzed Reactions 253
6.3.1 Aldolases 253
6.3.2 Oxynitrilases 254
6.4 Prospects 256
References 256
7 IONIC LIQUIDS AND WHOLE-CELL–CATALYZED PROCESSES 261
Danielle Dennewald and Dirk Weuster-Botz
Abbreviations 261
Abbreviations of Ionic Liquid Cations 261
Abbreviations of Ionic Liquid Anions 262
Abbreviation of Ionic Liquid 262
List of Abbreviations 262
x CONTENTS

7.1 Ionic Liquids Compatible with Whole-Cell
Biocatalysis: Fundamentals and Design 263
7.1.1 Biocompatibility 264
7.1.2 Availability and Purity 265
7.1.3 Stability 265
7.1.4 Process Design Criteria 265
7.1.4.1 Viscosity, Density, and Corrosiveness 266
7.1.4.2 Water Miscibility 266
7.1.5 Monophasic versus Biphasic Reaction Mode 266
7.1.6 Hazard Potential 268
7.1.6.1 Ecotoxicity 268
7.1.6.2 Biodegradability 269
7.1.7 Recyclability 271
7.1.8 Availability of Information 271
7.2 Biocompatibility, Tolerance, and Accumulation in the Cell 272
7.2.1 Methods 272
7.2.2 Tolerance 273
7.2.2.1 Composition of the Ionic Liquid and
Organism Type 273
7.2.2.2 Other Factors of Infl uence 276
7.2.2.3 Comparison with Organic Solvents 277
7.2.3 Interaction Mechanism 278
7.2.3.1 Effect on the Cell Membrane 279
7.2.3.2 Accumulation inside the Cell 280
7.3 State of the Art 281
7.3.1 Asymmetric Reductions by Whole Cells in
Ionic Liquids 282
7.3.2 Other Whole-Cell Biotransformations in
Ionic Liquids 304
7.4 Prospects 308

References 310
8 NONSOLVENT APPLICATIONS OF IONIC LIQUIDS
IN BIOTRANSFORMATIONS 315
Pablo Domínguez de María and Christina Kohlmann
8.1 Introduction 315
8.2 Ionic Liquids as Additives in Biotransformations 316
8.3 Ionic Liquids for Coating Enzymes:
The ILCE Concept 318
CONTENTS xi
8.4 Ionic Liquids Combined with Membranes
and Biotransformations 321
8.5 Ionic Liquids Anchoring Substrates 321
8.6 Ionic Liquids and Bioelectrochemistry 324
References 329
PART III IONIC LIQUIDS IN ORGANOCATALYSIS 331
9 IONIC LIQUIDS AS (CO-)SOLVENTS AND CO-CATALYSTS
FOR ORGANOCATALYTIC REACTIONS 333
Štefan Toma and Radovan Šebesta
9.1 Nontraditional Media in Organocatalysis 333
9.2 Early Organocatalytic Reactions in Ionic Liquids 334
9.3 Ionic Liquids as Solvents for Organocatalytic Reactions 335
9.3.1 Aldol Reactions 335
9.3.2 Mannich Reactions 341
9.3.3 α-Amination and Aminoxylation of Carbonyl Compounds 342
9.3.4 Michael Additions 343
9.3.5 Miscellaneous Reactions 351
9.4 Ionic Liquids as Co-catalysts for Organocatalytic Reactions:
Toward New Reactivities and Selectivities 353
9.5 Key Factors in Choosing Ionic Liquids for Organocatalysis
and Prospects 355

References 356
10 “NONSOLVENT” APPLICATIONS OF IONIC LIQUIDS IN
ORGANOCATALYSIS 361
Michelangelo Gruttadauria, Francesco Giacalone,
Paola Agrigento, and Renato Noto
10.1 Introduction 361
10.2 Immobilizing Ionic Liquids and Organocatalysts 363
10.2.1 Strategy 1a: Covalently Attached “Ionic Liquid” Moieties
as Supports 363
10.2.2 Strategy 1b: Covalently Attached “Ionic Liquid” Moieties as
Linkers 369
10.2.3 Strategy 1c: Covalently Attached “Ionic Liquid”
Moieties as Organocatalysts 372
10.3 Anchoring of Organocatalyst to Ionic Liquids 378
10.3.1 Aldol Reactions 379
10.3.2 Michael Reactions 393
xii CONTENTS
10.3.3 Morita–Baylis–Hillman Reaction and
Claisen–Schmidt Reaction 405
10.4 Ionic Liquids as Organocatalysts 409
10.5 Conclusions 414
References 414
Index 419
xiii
When Pablo Dom í nguez de Mar í a invited me to contribute a chapter to a book that he
proposed on biocatalysis and organocatalysis in ionic liquids, I had to decline the offer
owing to other pressing commitments, but I agreed to write a foreword to the book.
Now that I see the impressive result of his endeavors I am rather sorry that I am not a
contributor.
My introduction to the subject dates back to June 1996 when I attended an inspiring

lecture on ionic liquids presented by Ken ( “ Mr. Ionic Liquids ” ) Seddon of the Queen ’ s
University Belfast at the Clean Tech ’ 96 conference in London. I was immediately
hooked. I was fascinated by the possible benefi ts to be gained by using ionic liquids as
reaction media for catalytic processes. While listening to the lecture it occurred to me
that it would be very interesting to try ionic liquids as solvents for conducting biocata-
lytic processes. I was motivated by the notion that ionic liquids, by virtue of their
anticipated compatibility with enzymes, could possibly exert a rate enhancing and/or
stabilizing effect, resulting in an improved operational performance compared with that
observed in organic solvents. Afterward I asked Ken if he knew whether anybody had
tried to use an enzyme in an ionic liquid. His answer was: “ No, but why don ’ t we try
it? I can supply the ionic liquids. ” So we decided to try reactions with Candida ant-
arctica lipase B (CaLB), as this robust enzyme was known to be thermally very stable
and tolerant toward organic solvents under essentially anhydrous conditions. It took a
while to fi nd a Ph.D. student, Rute Madeira Lau, to perform the experiments, but the
results were gratifying. We observed that Novozyme 435 (an immobilized form of
CaLB) was able to catalyze various reactions — esterifi cation, amidation, and perhy-
drolysis — under anhydrous conditions in the second - generation ionic liquids, [bmim]
[BF
4
] and [bmim] [PF
6
], with rates at least as high as those in organic solvents.
Following the publication of our results, in Organic Letters in 2000, the use of ionic
liquids as reaction media for catalysis in general and biocatalysis in particular has
undergone exponential growth. It was soon recognized that the use of second - genera-
tion ionic liquids on a large scale was seriously hampered by their high price coupled
with ecotoxicity and poor biodegradability. Consequently, attention was devoted to the
development of a third generation of ionic liquids that are greener, more sustainable,
and less expensive than the second generation. In particular, ionic liquids derived from
natural raw materials, such as carbohydrates and amino acids, are emerging as green

solvents potentially suitable for large - scale applications. Furthermore, it is possible to
FOREWORD
xiv FOREWORD
design task specifi c ionic liquids, for example, bio - based chiral ionic liquids, that meet
not only environmental requirements but are also eminently suited to particular tasks.
Pablo is to be complimented on bringing this group of knowledgeable authors
together to review the state of the art in biocatalysis and organocatalysis in ionic liquids.
The subjects covered are wide - ranging, from fundamental aspects of interactions
between proteins and ionic liquids to their use as reaction media with both hydrolytic
and nonhydrolytic enzymes, whole cell bioconversions, and, as a bonus, organocatalytic
reactions. Importantly, practical aspects are highlighted, including process development
issues such as downstream processing. Why use ionic liquids as reaction media in the
fi rst place? An important motivation for their use as reaction media was that, based on
their negligible vapor pressure, they would be environmentally acceptable alternatives
to volatile organic solvents. However, the question still remained of how to separate
the product from the ionic liquid. An elegant solution to this problem was found in
continuous product extraction with supercritical carbon dioxide.
In addition to the enhanced operational performance through increased stability and/
or selectivity another important motivation for using ionic liquids as reaction media
was their ability to dissolve large amounts of highly polar substrates, such as carbohy-
drates and nucleosides. In particular, their ability to readily dissolve biopolymers such
as cellulose and lignin has become an important asset in the current drive toward the
bio - based economy, in which there is a need for effective and sustainable methods for
the primary conversion of renewable lignocellulosic raw materials. The challenges of
using ionic liquids as reaction media for biotransformations are also addressed. In order
to be sustainable they must meet stringent requirements regarding the greenness and
economic viability of their synthesis and their environmental footprint, which is gov-
erned by properties such as bioaccumulation, biodegradability, and ecotoxicity. In
addition to the various chapters on the use of ionic liquids as reaction media, there is
an extra treat for the reader: two chapters on nonsolvent applications, in biotransforma-

tions and organocatalytic conversions, respectively. This includes interesting concepts
such as the use of ionic liquid – coated enzymes and the anchoring of organocatalysts
to ionic liquids.
In short, I believe that this book is an important addition to the literature on ionic
liquids as reaction media for biocatalytic and organocatalytic processes. In addition to
its obvious value to practicing organic chemists in both industry and academia, its
educational value should not be underestimated. It should prove to be of great value
for advanced undergraduate and graduate students. Finally, I would like to thank Pablo
for giving me the opportunity to air my views on the merits of this book. I wish him
all the success that he has surely earned.
Roger A. Sheldon
Emeritus Professor of Biocatalysis and Organic Chemistry
Delft University of Technology
January 2012
xv
When I was approached by Wiley to edit a book on ionic liquids in biotransformations
and organocatalysis, the spontaneous question that quickly came to my mind was, is
there a gap for such a book ? The fi eld of ionic liquids applied to biotransformations
and organocatalysis has developed enormously during the last two decades. Therefore,
the realization of a book that could gather, categorize, and provide an updated and
complete state - of - the - art in these areas was clearly a demand. There are obviously
several comprehensive reviews in the fi eld, but I think none of them can cover the
topic(s) in their widest extent. Thanks to the outstanding chapters of many world - class
experts in the area, this book is now a reality that I hope will be a useful contribution
for researchers in the fi eld, both in academia and industry.
Since the beginning of my work as editor, I have made it clear in my mind that I
do not want a book just covering uses of ionic liquids as solvents, albeit, of course, this
topic is broad and very important (Chapters 3 – 7 and 9 ), nor a book regarding ionic
liquids as “ green solvents ” — an unfortunate label that has surely brought more prob-
lems than advantages to ionic liquids (Chapters 1 and 7 ). In fact, many ionic liquids

are not green, but their versatility and tunability makes us optimistic that it will be
possible to combine greenness with the acquired know - how on advantages that ionic
liquids may bring, for example, leading to the third generation of ionic liquids. Like-
wise, emerging deep - eutectic - solvents represent a promising option, and fi rst uses in
biotransformations are briefl y discussed herein (Chapters 1 , 5 , and 7 ). In addition, an
extensive updated state - of - the - art on toxicity and (bio)degradability of commonly used
ionic liquids, together with protocols and rules applied for assessing these parameters,
is provided in Chapter 7 .
Quite remarkably, ionic liquids are more than mere solvents. They represent a
fantastic academic tool for studying and understanding interactions with proteins,
enzymatic mechanisms, and so on (Chapters 2 and 5 ); there are also a number of “ non-
solvent ” approaches for practical applications, such as catalyst immobilization or acti-
vation, downstream processing, and catalyst grafting or coating (Chapters 8 and 10 ).
Ionic liquids can also be smartly combined with other nonconventional solvents, such
as supercritical fl uids, or with innovative process design concepts (Chapter 4 ). Finally,
some ionic liquids can be employed in whole - cell biotransformations, providing novel
and promising approaches, including proof - of - principle for deep - eutectic - solvents and
whole cells (Chapter 7 ).
PREFACE
xvi PREFACE
I want to acknowledge a number of people who have made this book a reality.
First of all, the greatest credits go obviously to the authors of this book, the actual and
unique protagonists of this work (together with the ionic liquids!). Without their out-
standing efforts, professionalism, and excellent and readily updated chapters, this
project would have never been possible. Furthermore, I wish to thank Dr. Daniela
Gamenara, Dr. Fabrizio Sibilla, and Dr. Andreas Buthe for many fruitful and stimulating
discussions. Likewise, thanks are given to Prof. Dr. Roger Sheldon for writing the
Foreword of this book. I am also indebted to Ms. Anita Lekhwani, Senior Acquisitions
Editor at Wiley, for the interest and patience she has had and the hard work she has
done throughout the editing process. And my thanks go as well to Dr. Edmund H.

Immergut, Consulting Editor for Wiley and Wiley - VCH, for inviting me to edit this
book and his trust and support during this time.
I must say that, overall, this project has been for me a fascinating and unforgettable
adventure. I really hope that readers will fi nd this book an attractive and useful tool for
working in the fi eld of ionic liquids, biotransformations, and organocatalysis. Sugges-
tions for further improvements, data treatment, new topics, and so on are of course
welcome for future editions of this work.
Pablo Dom í nguez de Mar í a
Aachen, Germany, January 2012
xvii
Paola Agrigento, Dipartimento Chimica Organica “ E. Patern ò ” , Universit à di Palermo,
Viale delle Scienze, Palermo, Italy
Danielle Dennewald, Lehrstuhl f ü r Bioverfahrenstechnik, Technische Universit ä t
M ü nchen, Garching, Germany
Pablo Dom í nguez de Mar í a, Institute of Technical and Macromolecular Chemistry
(ITMC), RWTH Aachen University, Aachen, Germany
Daniela Gamenara, Physical - Organic Chemistry and Bioprocesses Group, Organic
Chemistry Department, Facultad de Qu í mica, Universidad de la Rep ú blica
(UdelaR), Montevideo, Uruguay
Eduardo Garc í a - Verdugo, Instituto de Cat á lisis y Petroleoqu í mica, CSIC, Campus
de la UAM, Cantoblanco Madrid, Spain
Francesco Giacalone, Dipartimento Chimica Organica “ E. Patern ò ” , Universit à di
Palermo, Palermo, Italy
Lasse Greiner, Institute of Technical and Macromolecular Chemistry (ITMC), RWTH
Aachen University, Aachen, Germany and DECHEMA e.V. Karl - Winnacker -
Institut, Frankfurt am Main, Germany
Michelangelo Gruttadauria, Dipartimento Chimica Organica “ E. Patern ò ” , Univer-
sit à di Palermo, Palermo, Italy
Christina Kohlmann, Institute of Technical and Macromolecular Chemistry (ITMC),
RWTH Aachen University, Aachen, Germany and Cognis GmbH, D ü sseldorf,

Germany
Pedro Lozano, Departamento de Bioqu í mica y Biolog í a Molecular “ B ” e Inmunolog í a,
Facultad de Qu í mica, Universidad de Murcia, Murcia, Spain
Patricia Saenz M é ndez, Physical - Organic Chemistry and Bioprocesses Group,
Organic Chemistry Department, Facultad de Qu í mica, Universidad de la Rep ú blica
(UdelaR), Montevideo, Uruguay and Computational Chemistry and Biology
Group, DETEMA, Facultad de Qu í mica, Universidad de la Rep ú blica (UdelaR),
Montevideo, Uruguay
Renato Noto, Dipartimento Chimica Organica “ E. Patern ò ” , Universit à di Palermo,
Palermo, Italy
CONTRIBUTORS
xviii CONTRIBUTORS
Radovan Š ebesta, Faculty of Natural Sciences, Comenius, University Bratislava,
Bratislava, Slovakia
Gustavo Seoane, Physical - Organic Chemistry and Bioprocesses Group, Organic
Chemistry Department, Facultad de Qu í mica, Universidad de la Rep ú blica
(UdelaR), Montevideo, Uruguay
Š tefan Toma, Faculty of Natural Sciences, Comenius, University Bratislava, Bratislava,
Slovakia
Dirk Weuster - Botz, Lehrstuhl f ü r Bioverfahrenstechnik, Technische Universit ä t
M ü nchen, Garching, Germany
Zhen Yang, College of Life Sciences, Shenzhen University, Shenzhen, Guangdong,
China
Hua Zhao, Chemistry Program, Savannah State University, Savannah, GA, USA
PART I
FUNDAMENTALS
3
1.1 IONIC LIQUIDS: DEFINITION, DEVELOPMENT, AND OVERVIEW
OF CURRENT MAIN APPLICATIONS
Ionic liquid s ( IL s) (low - temperature molten salts) are simply mixtures of cations and

anions that do not pack well among them, and therefore remain liquid at low to moder-
ate temperatures. The low melting points are often achieved by incorporating bulky
asymmetric cations into the structure, together with weakly coordinating anions. Arbi-
trarily it has been established that ILs that melt below 100 ° C fall into the category of
“ ionic liquids. ” On the other hand, those that are liquid at room temperature are often
regarded as “ room - temperature ionic liquid s ” ( RTIL s). Although some IL compositions
have been known for a long time, it has been in the last decades when an impressive
development in the fi eld has emerged, providing innovative applications in many areas
of chemistry. This interest is driven by the fact that by changing the cation or the anion
of a certain IL, the physicochemical properties of that IL can be fi nely tuned. Thus,
novel solvents can be defi ned and used for a specifi c tailored application. Obviously
this wide tunability cannot be reached with conventional organic solvents. For instance,
IL polarities can be modulated to design ILs that are immiscible with either low - polarity
1
IONIC LIQUIDS: DEFINITION,
APPLICATIONS, AND CONTEXT
FOR BIOTRANSFORMATIONS
AND ORGANOCATALYSIS
Pablo Domínguez de Mar ía
Ionic Liquids in Biotransformations and Organocatalysis: Solvents and Beyond, First Edition. Edited by
Pablo Domínguez de María.
© 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.
4 IONIC LIQUIDS
organic solvents or high - polarity solvents. This facilitates conventional extraction
methods to be employed in product separation and furthermore provides promising
entries in many areas of chemistry. Likewise, combinations of ILs with other solvent
systems, for example, supercritical fl uids, have provided promising synergies for chem-
ical process setups since properties of both systems can be easily modulated.
Not without discussion, it is usually assumed that the fi rst “ true ” IL was described
in 1914 by Walden. The IL was ethylammonium nitrate ( EAN ), [EtNH

3
][NO
3
], with a
melting point (m.p.) of 12.5 ° C. However, the fi nding attracted rather little attention at
that time.
1
Two decades later, in 1934, a patent reported on some pyridinium - based
molten salts that were able to dissolve certain amounts of cellulose.
2
Again, in this case,
the importance, potential, or utility of this fi nding was underestimated. Remarkably,
nowadays cellulosic biomass pretreatment by means of a wide number of ILs is an
important topic of research since some ILs enable the dissolution of different lignocel-
lulosic materials. Once dissolved in these ILs, cellulose can be subsequently depoly-
merized by, for instance, different hydrolytic enzymes (see also Chapter 5 , Sections 5.4
and 5.5 ).
3
In a broad sense, ILs started to attract interest in the 1960s. During several decades
on (1960 – 1990), the fi rst generation of ILs appeared and was widely described and
chemically characterized. Typical cations for fi rst - generation ILs were dialkylimidazo-
lium and alkylpyridinium derivatives. As anions, chloroaluminate and other metal
halide structures were used. As an important drawback for practical applications, fi rst -
generation ILs were found to be sensitive to water and air. These features clearly
hampered further applications of fi rst - generation ILs in different fi elds of chemistry. In
the 1990s, the second generation of ILs emerged. Herein, anions were substituted for
weakly coordinating anions such as BF
4
or PF
6

. These new ILs were air - and water -
stable and therefore led to much research and efforts in the area, as the enhanced stabil-
ity of ILs provided a much wider frame for operating with them under many different
processing conditions. More recently, the third generation of ILs has emerged. This
third generation comprises biodegradable and readily available ions, such as natural
bases (e.g., choline), amino acids, and naturally occurring carboxylic acids.
4,5
Together
with this third generation of ILs, so - called deep eutectic solvent s ( DES ) represent a
promising alternative because they are simple to prepare, biodegradable, and more
economical, compared with other ILs. In general, DES are mixtures of a solid salt with
a hydrogen - bond donor in different proportions. An example of DES is represented by
the combination of choline chloride (solid salt at room temperature, m.p. 302 ° C) with
urea (solid at room temperature, m.p. 132 ° C), which leads to a DES with a melting
point of 12 ° C.
5 – 7
Yet it is not clear if DES can be regarded as “ IL ” since some of the
structures (e.g., urea) are not charged and therefore subsequently produced solvents are
not entirely ionic. Despite this, it is believed that many properties of ILs can also be
more or less extrapolated to DES. In Figure 1.1 some selected milestones in the IL
history are depicted.
Until now, second - generation ILs have been the subject of enormous fundamental
research, providing interesting and novel applications in many areas of chemistry. Yet
their use at commercial scale is still limited to a few cases, presumably due to economic
aspects related to ILs. However, along with the development of the third generation of
Figure 1.1. Some selected milestones in the history of ILs.
4,5
NH
3
N

1914 1934 1984
1990-onwards 2000-onwards
1960−1990
N
N
N
N
OH
N
N
N
R
R
R
COOH
R
2
R
2
H
2
N
R
1
R
1
[CI]
[NO
3
]

AICI
4
−
NH
3
(In Water)
[NO
3
]
AI
2
CI
7
−
BF
4
−
PF
6
−
Me
2
CO
2
−
First RTIL reported
First cellulose
dissolution in ILs
Enzyme catalysis
in the presence of ILs

First-generation ILs
(Water-and air-sensitive)
Second-generation ILs
(Water-and air-sensitive)
Third-ganeration ILs
biodegradable, DES
5
6 IONIC LIQUIDS
ILs, which are more sustainable, biodegradable, and cheaper derivatives, it is antici-
pated that novel IL - based applications will reach the commercial level in the coming
years. A signifi cant number of companies already commercialize some ILs and perform
R & D - related activities aimed at identifying new market niches and business
opportunities.
The enormous potential of ILs is driven by their intrinsic feature (previously men-
tioned), which is that ILs can be fi nely tuned by carefully selection of anions and
cations. Thus, ILs can be tailored for a specifi c application, leading to the concept of
task - specifi c ionic liquid ( TSIL ). By choosing anions and cations, relevant examples
of protic ILs, chiral ILs, multifunctional ILs, supported ILs, and so on have been
reported. Some general applications for ILs have been put forward (see overview in
Figure 1.2 ). More information on the general applications of ILs can be found in recent
reviews and books devoted to various IL areas.
8 – 16
1.2 ON THE GREENNESS OF ILs: TOWARD THE THIRD
GENERATION OF ILs AND DES
Apart from the ample tunability of ILs (previous section), probably another aspect that
has triggered signifi cant interest and research in the fi eld of ILs is the common claim
Figure 1.2. Overview of possible applications of ILs in different areas .
8–16
-
-

-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Biotechnology
Biocatalysis
Protein Extraction
Biomass (Pre)treatment
Chemistry
Catalysis
Catalyst Immobilization
Polymerization
Organic Synthesis
Ionic
Liquids
Others

Analytical Sciences
Nanoparticles
Liquid Crystals
Coatings
Surfactants
Metal Deposition
Lubricants
Chemical Engineering
Liquid Membranes
Extraction
Separation
Process Development
Energy
Batteries
Fuel Cells
Thermofluids
1.2 ON THE GREENNESS OF ILs: TOWARD THE THIRD GENERATION OF ILs AND DES 7
that ILs are “ green solvents. ” This general assumption is based on several important
properties commonly attributed to ILs, namely that ILs pose negligible vapor pressure
and that they are nonfl ammable. In this section aspects related to the greenness of ILs
will be briefl y discussed.
First of all, it is usually reported that ILs do not exert measurable vapor pressure
since they are entirely composed of ions. Hence, they cannot be distilled without
decomposition and thus are nonvolatile. An obvious conclusion that may be drawn from
these postulates is that environmental advantages would be achieved by using ILs
instead of volatile organic compound s ( VOC s). This nonvolatility statement has been
challenged by Seddon and coworkers, who demonstrated that some ILs can in fact be
distilled at low pressures.
17
However, at ambient pressures most of the ILs indeed show

a negligible vapor pressure, and therefore from that viewpoint they may still be con-
sidered environmentally advantageous compared with VOCs.
The second important property to categorize ILs as environmentally - benign sol-
vents is their nonfl ammability compared again with VOCs. However, this statement
has also been challenged by Wilkes, Rogers et al., who showed that a wide number of
commonly used ILs were actually combustible since products formed during thermal
decomposition of ILs were found to be combustible. Experiments thus showed that it
is not safe to operate with ILs close to fi re or heat sources.
18
Therefore, although the
low fl ammability of ILs may certainly provide advantages compared with VOCs, it is
obvious that ILs should not be regarded as “ green solvents ” by the mere fact that they
are ILs. Traditionally, in publications dealing with ILs, there are not many distinctions
among ILs, and usually all ILs are generically regarded as “ green solvents ” or as “ non-
fl ammable ” solvents. This trend, however, is starting to change.
Apart from the two above - mentioned IL properties (low vapor pressure and
nonfl ammability), there are other aspects — surely more important in assessing the
greenness of ILs — that defi nitely challenge the “ green label ” of many often used ILs.
These aspects are related to the environmental impact that IL syntheses may have (e.g.,
the E - factor of producing ILs), as well as to the eco - toxicity and biodegradability
of the ions composing the ILs, and of metabolites formed thereof, when ILs are (acci-
dentally) spoiled in the milieu. These topics have only recently started to receive the
attention that they actually deserve.
19 – 22
In this chapter a brief discussion of these
issues is given. Furthermore, a detailed and updated discussion of topics such as bio-
compatibility, toxicity, and biodegradability of ILs is also available in Chapter 7 (Sec-
tions 7.1 and 7.2 ).
As any other chemical or solvent, the production of ILs clearly involves a synthetic
process in which some chemical steps are conducted. Therefore, during IL syntheses

some reagents are used and some wastes or by - products are formed together with the
IL. These waste and by - product formation is crucial from an environmental viewpoint —
green chemistry and green engineering —
23,24
and are often not mentioned or even
considered when ILs are claimed as “ green solvents. ” In Table 1.1 , the principles
labeled “ PRODUCTIVELY ” (green chemistry) and “ IMPROVEMENTS ” (green engi-
neering) are summarized.
24
Despite the importance of these green chemistry principles, it has not been until
recently that studies focusing on the environmental concerns of IL syntheses were
8 IONIC LIQUIDS
reported.
20
Therein, the widely used alkylimidazolium - based ILs were taken as a model,
and critical studies regarding their syntheses ( E - factor and atom economy), purifi cation
steps, discoloration, and source of energy applied were carried out. Overall it was
concluded that the production of those ILs is far less green than what is usually claimed
in the literature dealing with ILs. At laboratory - scale processes, quaternization synthetic
approaches may still provide some green footprints if processes are conducted with
either microwave or conduction as the energy source.
24
However, in the other cases,
conclusions clearly challenged the environmental label that ILs usually have in the
literature. It is clearly expected that more environmental studies on the ILs syntheses
will be carried out in the coming years and therefore a better picture will emerge.
Moreover, when assessing the greenness of ILs other important aspects include IL
release, eco - toxicity, biodegradability, bioaccumulation, and spatiotemporal range in
the milieu.
22

Although it can be expected that environmental release of ILs could be
easily controlled compared with VOCs — by virtue of the almost negligible vapor pres-
sure and volatility of ILs — it is clear that sooner or later some appreciable amounts of
ILs will reach the environment (e.g., in wastewater effl uents). Therefore, it is crucial
to assess how these ILs are going to interact with living organisms. To this end, a
number of standardized tests and protocols have been established. They include studies
on inhibition of acetylcholinesterase enzymes, luminescence inhibition of the marine
bacterium Vibrio fi sheri , growth rate inhibition of the freshwater green alga Pseu-
dokirchneriella subcapitata , cell viability of IPC - 81 cells, growth inhibition of duck-
weed, Lemna minor , and an acute test with zebrafi sh, Danio rerio .
21
In addition,
products formed during the environmental degradation of ILs must also be considered.
It has been reported that some of these degradation products may be even more toxic
than the original ILs.
21
In Chapter 7 of this book (Sections 7.1 and 7.2 ), detailed infor-
mation on the state - of - the - art of these aspects is provided.
TABLE 1.1. Principles of Green Chemistry ( “ PRODUCTIVELY ” ) and Green Engineering
( “ IMPROVEMENTS ” ), as Reported in the Literature
24
“ PRODUCTIVELY ” “ IMPROVEMENTS ”
Green chemistry Green engineering
Prevent wastes Inherently nonhazardous and safe
Renewable materials Minimize material diversity
Omit derivatization steps Prevention instead of treatment
Degradable chemical products Renewable material and energy inputs
Use safe synthetic methods Output - led design
Catalytic reagents Very simple
Temperature, pressure ambient Effi cient use of mass, energy, space, and time

In - process monitoring Meet the need
Very few auxiliary substances Easy to separate by design
E - factor, maximize feed in product
Networks for exchange of local mass and energy
Low toxicity of chemical products Test the life cycle of the design
Yes, it is safe Sustainability throughout the product life cycle
A general conclusion that can be set herein is that commonly used ILs are far less
green than what they are usually claimed in publications. However, once again it has
to be mentioned that the huge versatility of ILs (tunability, tailored properties) might
be used to provide greener ILs than the current ones. In general, these environmental
concerns are starting to shift research in the fi eld to the production of more sustainable
ILs. An envisaged future challenge will be to design ILs that maintain their promising
physicochemical properties and potential applications while providing greener foot-
prints, both in terms of E - factors and in terms of degradability, toxicity, and so on.
19
In
this respect, an alternative that has emerged is the production of ILs starting from
natural sources as substrates such as amino acids, carboxylic acids, and sugar - based
structures.
25
Herein, natural amino acids have been used extensively
26
because of their
interesting tunable chemical properties, greater affordability, and high compatibility for
living organisms. In addition, amino acids incorporate chiral centers into the IL, which
may add other interesting properties with promising applications. Thus, amino acid -
based ILs represent interesting examples of chiral bio - based ILs.
27
Another alternative
is to use amino acids as starting materials for the synthesis of ILs. Herein, albeit sub-

strates are obviously environmentally friendly, attention to subsequent synthetic proce-
dures to afford the fi nal derivatives should be taken into account in a case - by - case
scenario. In Figure 1.3 some examples of ILs derived from amino acids are depicted.
In the quest for ILs that could deserve the label “ green solvents, ” another important
approach is represented by designing ILs that can be not only entirely composed of
biomaterials, but also involve derivatization steps that may add limited environmental
concerns to IL production. An example of this strategy is the use of available and
inexpensive choline hydroxide as staring material. The production of choline - based ILs
are conducted simply by substituting choline hydroxide with the correspondent (natu-
rally occurring) carboxylic acid at ambient temperature, producing water as the only
by - product of the process (Figure 1.4 ).
28,29
Therefore, the design of ILs provides an enormous possibility for tailored ILs, even
when environmental concerns are considered, and a useful but green solvent is envis-
aged. In conclusion, although ILs cannot be generally regarded as safe (green) solvents,
it is possible to design ILs that can meet environmental requirements. Given the current
trends in environmental processes and green chemistry, it is clear that this will be the
most important and sustainable line of development for ILs in the coming years. Fur-
thermore, the design of ILs that can be used for certain applications while maintaining
acceptable environmental footprints will be crucial. Some examples of these combina-
tions have just appeared in the fi eld of biocatalysis, including the design of enzyme -
friendly choline - based ILs that are able to dissolve cellulose at the same time
(AMMOENG 110 ™ , Figure 1.5 ).
30
This combination provides a promising frame to
undertake biocatalytic reactions in reaction media that can be compatible with the
substrates/products employed. This approach is discussed more extensively in Chapter
2 (Section 2.6 ) and Chapter 5 (Sections 5.4 and 5.5 ).
As stated in the previous section, together with bio - based ILs, another important
fi eld in the third generation of ILs are represented by DES.

5 – 7,9
DES are a combination
of a room - temperature salt (e.g., choline chloride) together with a hydrogen - bond -
forming molecule (e.g., urea, glycerol, and carboxylic acids). When these structures are
1.2 ON THE GREENNESS OF ILs: TOWARD THE THIRD GENERATION OF ILs AND DES 9