OPTICAL SENSORS FOR DETECTION OF ENANTIOMERIC EXCESS APPLICATION
Sara Sheykhi
A Dissertation
Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
May 2019
Committee:
Pavel Anzenbacher, Jr., Advisor
Yuning Fu Graduate Faculty Representative
H. Peter Lu
Alexis Dee Ostrowski
© 2019
Sara Sheykhi
All Rights Reserved iii ABSTRACT
Pavel Anzenbacher, Jr., Advisor
Chiral compounds play an essential role in drug synthesis, biological chemistry, and asymmetric catalysis. More specifically, chiral carboxylates are extensively utilized in drug development processes, and many are commercialized as drugs. Synthesis of single enantiomer together with research in methods for chiral drug separation is now a central subject for pharmaceutical development. This is because opposite enantiomers frequently possess different pharmacological benefits and toxicity. Thus, the ability to determine enantiomeric excess in chiral compounds is important for the development of new chiral drugs. Supramolecular optical chemosensors are potentially important methods in many emerging medical technologies. Due to the rapidly developing fields of supramolecular chemistry and optical detection, new sensing approaches have been developed for the recognition of many essential molecules in aqueous solutions. Because of high sensitivity and low cost, fluorescence based sensors are gaining wide popularity for chemical trace detection.
In this dissertation, we present optical methods to determine the enantiomeric composition of chiral carboxylates and α-hydroxycarboxylic acids. We used enantioselective Indicator
Displacement Assays (eIDAs) for the determination of enantiomeric excess of chiral carboxylates and α-hydroxycarboxylic acids. This chiral receptor-fluorophore sensing ensemble is shown to be useful in the determination of enantiomeric excess of α-hydroxycarboxylic acids and chiral carboxylates including non-steroidal anti-inflammatory drugs (NSAIDs). iv
To My Dearest Family
To my parents (Eghbaleh Paknezhad Sahneh and Mokhtar Sheykhi)
To my sisters (Sahar and Gelareh Sheykhi)
To my brother (Saba Sheykhi)
To my nieces (Mehdana Ghamari and Golsa Marjani)
Thank you for your love
And your constant support v ACKNOWLEDGMENTS
First of all, I would like to express the deepest appreciation to my dissertation advisor,
Professor Pavel Anzenbacher, Jr., Ph.D., for his continuous support, genuine caring, patience, and faith in me during my graduate studies at Bowling Green State University. Without his guidance and persistent help this dissertation would not have been possible.
I would like to thank my committee members, Professor Yuning Fu Ph.D., a Graduate
College representative, Professor H. Peter Lu, Ph.D., and Professor Alexis D. Ostrowski, Ph.D., for their support, supervising my scientific progress over the last five years of study and giving me valuable suggestions.
In addition, a thank you to Professor Malcolm D. E. Forbes, director of Center for Pure and
Applied Photosciences, who has been supportive of my career goals.
I would like to extend my sincere thanks to my former and current colleagues, friends who supported me and shared their knowledge and expertise with me. I would like to acknowledge especially Dr. Lorenzo Mosca and Dr. Göghan Caglayan, former postdoctoral research fellows at the Anzenbacher research group, who would encouraged me and advised me countless times throughout my studies at BGSU. I would like to thank current and former colleagues who helped me during my research namely Professor Jeremy K. Klosterman, Dr. sc. nat. Dr. Elena
Shcherbakova, Dr. Valentina Brega, Dr. Liubov Lifshits, Dr. Petr Koutnik, Dr. Ali Akdeniz, Ms.
Mariia Pushina, and Mr. Travis Green.
I would like to acknowledge all professors at Bowling Green State University for their support and guidance, and for sharing their enormous wealth of knowledge and academic expertise.
Nobody has been more important to me in the pursuit of my Ph.D than my family. I would like to thank my parents for all the support and love without which I would never be who I am. vi
TABLE OF CONTENTS
Page
CHAPTER I. INTRODUCTION ...... 1
1.1 Chemical Sensors ...... 1
1.2 Optical Chemical Sensors ...... 2
1.3 Optically Active/Chiral Chemical Sensors ...... 3
1.4 Indicator Displacement Assay ...... 4
1.5 The Binding Constants and Complex Formation ...... 8
1.6 Signaling Recognition Mechanism in Optical Sensing ...... 11
1.7 Cross-Reactive Sensor Array ...... 13
1.8 High-Throughput Screening Assay and Pattern Recognition
Techniques for Multivariate Data Analysis ...... 14
1.9 References ...... 19
CHAPTER II. SENSING OF ENANTIOMERIC COMPOSITION USING
FLUORESCENCE PROBES ...... 28
2.1 Abstract...... 28
2.2 Introduction ...... 29
2.3 Sensing of Enantiomeric Composition of Chiral Carboxylates Using
Fluorescence Probes ...... 30
2.3.1 Introduction ...... 30
2.3.2 EIDA for Chiral Carboxylates ...... 34
2.3.3 Chiral Sensors: Design and Synthesis...... 35
2.3.4 Materials and Methods ...... 37 vii
2.3.5 Synthesis Protocols ...... 40
2.3.6 Chiral Sensors for Carboxylic Acids: NMR Spectra ...... 44
2.3.7 Chiral Sensors for Carboxylic Acids: Mass Spectra ...... 52
2.3.8 Complex Stoichiometry Determination: Job’s Plot ...... 56
2.3.9 Photophysical Properties ...... 57
2.3.10 Fluorescence Titrations: Indicator–Chiral Sensors ...... 57
2.3.11 Competitive Fluorescence Titrations ...... 60
2.3.12 Determination of Binding Affinities: Indicator Titrations ...... 76
2.3.13 Determination of Binding Affinities: Competitive Titrations ...... 77
2.3.14 Summary ...... 80
2.4 Sensing of Enantiomeric Composition of Chiral α-Hydroxycarboxylic
Acids Using Fluorescence Probes ...... 81
2.4.1 Introduction ...... 81
2.4.2 Chiral α-Hydroxycarboxylic Acid Sensing: Design and Synthesis ...... 85
2.4.3 Materials and Methods ...... 86
2.4.4 Synthesis Protocols ...... 90
2.4.5 Chiral Sensors for α-Hydroxycarboxylic acid: NMR Spectra ...... 95
2.4.6 Chiral Sensors for α-Hydroxycarboxylic acid: Mass Spectra ...... 109
2.4.7 Complex Stoichiometry Determination: Job’s Plot ...... 113
2.4.8 Photophysical Properties ...... 116
2.4.9 Fluorescence Titrations: Zn(II)–Based Sensors ...... 116
2.4.10 Fluorescence Titrations: Indicator and α-Hydroxycarboxylic
Acids by Cu(II)-Based Sensor ...... 135 viii
2.4.11 Determination of Binding Affinities: Zn(II)–Based Sensors
Titrations ...... 143
2.4.12 Determination of Binding Affinities: Indicator–Cu(II)-
Based Sensor Titration ...... 145
2.4.13 Determination of Binding Affinities: α-Hydroxycarboxylic
Acids–Cu(II)-Based Sensor Titrations ...... 146
2.4.14 High-Throughput Array for Chiral α-Hydroxycarboxylic Acids ...... 146
2.4.15 Summary ...... 151
2.5 References ...... 152
CHAPTER III. DETECTION OF AMMONIUM NITRATE-BASED
EXPLOSIVE USING OPTICAL SENSORS ...... 163
3.1 Abstract...... 163
3.2 Introduction ...... 164
3.3 Explosives ...... 166
3.4 Improvised Explosive Devices (IEDs) ...... 168
3.5 Detection of IEDs ...... 168
3.5.1 Bulk Detection Systems ...... 168
3.5.2 Trace Detection Systems ...... 169
3.5.3 Detection of Trace Explosives Using Optical Methods ...... 170
3.6 Ammonium Nitrate-Nitromethane (ANNM) Explosives ...... 174
3.6.1 Ammonium Nitrate (AN) ...... 174
3.6.2 Nitromethane (NM) ...... 175 ix
3.6.3 Detection of AN-Based Improvised Explosives and Their
Component ...... 175
3.7 Paper-Based Sensors ...... 178
3.7.1 Fabrication Techniques ...... 179
3.8 Detection Methods in μPADs ...... 180
3.8.1 Detection of Explosives Using μPADs ...... 180
3.8.2 Fluorescence-Based Detection of Explosives in μPADs ...... 182
3.9 Wearable Sensors for Detection of Explosives ...... 183
3.10 Sensors Design ...... 186
3.10.1 Materials and Methods ...... 188
3.10.2 Sensors Preparation ...... 189
3.10.3 Analytes Preparation ...... 190
3.11 Complex Formation: Mass Spectrometry ...... 191
3.12 Photophysical Studies and Fluorescence Titrations ...... 192
3.12.1 Solid State Fluorescent Spectra of S1-S4...... 192
3.12.2 Quantum Yields of S1-S4 ...... 194
3.12.3 Fluorescence Titrations of S3 in Solution ...... 195
3.13 Qualitative Analysis ...... 197
3.14 Paper Microzone Plate-Based Qualitative Assay ...... 198
3.15 Paper Microzone Array-Based Vapor Sensing ...... 201
3.16 Paper Microzone Array-Based Sensing in Solution ...... 204
3.17 Hierarchical Clustering Analysis (HCA) ...... 206
3.18 Non-Woven Nanofiber Mats of S3 ...... 207 x
3.19 Wearable Nanofiber Sensor ...... 208
3.20 Summary ...... 210
3.21 References ...... 211 xi
LIST OF FIGURES
Figure Page
1.1 The operation principle of a typical optical chemical sensor with host and
guest complexation ...... 2
1.2 The mathematical expression commonly used to quantify ee...... 3
1.3 Schematic of ee determination using an enantiopure chiral host with the formation
of two different diastereomeric complexes of varying stability...... 4
* 1.4 Equilibria of chiral recognition. H = chiral host/receptor; GR, GS = analyte/guest; [G]t
= total concentration of guest; KR, KS = affinity/binding constant...... 4
1.5 Schematic of the enantioselective indicator displacement assays...... 6
1.6 Schematic equilibria of an enantioselective indicator displacement assay. H* = chiral
host/receptor; I = indicator; GR, GS = analyte/guest; [G]t = total guest concentration, KI,
KR, KS = affinity/binding constant...... 7
1.7 Structures of chiral receptors and chrome azurol S as an indicator used by
Anslyn69,70 for enantiomeric discrimination of amino acids...... 8
1.8 Equilibrium of optical molecular sensor. H = host/receptor; G = analyte/guest;
[G]t = total concentration of the guest; Ka = binding/affinity constant...... 9
1.9 The equilibrium between a host (H) and guests (G1) and (G2) described by the
association constant (Ka), is equal to the amount of the host-guest complex
divided by the product of the concentration of the individual guest and host when
the system is in equilibrium. Host selectivity is determined by the ratio of the Ks
for guests (G1) and (G2) ...... 10 xii
1.10 Frontier molecular orbitals representation of the mechanism of oxidative and
reductive photoinduced electron transfer (PET) ...... 12
2.1 Structures of 1,8-bis(3,3’-(3,5-dimethylphenyl)-9,9’-diacridyl)naphthalene (1) and
1,8-bis(3,3’-(3,5-dimethylphenyl)-9,9’-diacridyl)naphthalene N,N’-dioxide (2)
fluorescent sensors reported by Mei and Wolf72,82 ...... 30
2.2 Structure of the fluorescent chiral sensor designed by Zhang et al.98 for
enantioselective recognition of chiral carboxylic acids ...... 31
2.3 Structures of cinchona alkaloids quinine (S1), cinchonidine (S2), cinchonine (S3),
quinidine (S4) based fluorescent sensors for detection of ee in chiral carboxylic
acids...... 32
2.4 Structures of fluorescent macrocyclic sensors for ee detection of chiral
carboxylic acids reported by Akdeniz et al.100 ...... 32
2.5 Structures of chiral TPE macrocycles reported by Feng et al.101 for
enantioselective recognition of chiral acidic compounds and α-amino acids ...... 33
2.6 Structures of chiral fluorescence sensors developed by Wang et al.102 ...... 33
2.7 General operational principle of an indicator displacement assay ...... 35
2.8 Sensors- [CuSSL]2+ and [CuRRL]2+-and indicator (C343) used in this study ...... 36
2.9 Structures of the chiral analytes used in this study ...... 37
2.10 1H NMR (500 MHz) of (1S,2S)-diphenyl-N1,N2-bis(quinolin-2-ylmethylene)
ethane-1,2-diimine in CDCl3 ...... 44
2.11 13C NMR (125 MHz) of (1S,2S)-diphenyl-N1,N2-bis(quinolin-2-ylmethylene)
ethane-1,2-diimine in CDCl3 ...... 45 xiii
2.12 1H NMR (500 MHz) of (1S,2S)-diphenyl-N1,N2-bis(quinolin-2-ylmethyl)
ethane-1,2-diamine in CDCl3 ...... 46
2.13 13C NMR (125 MHz) of (1S,2S)-diphenyl-N1,N2-bis(quinolin-2-ylmethyl)ethane-
1,2-diamine in CDCl3 ...... 47
2.14 1H NMR (500 MHz) of (1R,2R)-diphenyl-N1,N2-bis(quinolin-2-ylmethylene)
ethane-1,2-diamine in CDCl3 ...... 48
2.15 13C NMR (125 MHz) of (1R,2R)-diphenyl-N1,N2-bis(quinolin-2-ylmethylene)
ethane-1,2-diamine in CDCl3 ...... 49
2.16 1H NMR (500 MHz) of (1R,2R)-diphenyl-N1,N2-bis(quinolin-2-ylmethyl)ethane-
1,2-diamine in CDCl3 ...... 50
2.17 13C NMR (125 MHz) of (1R,2R)-diphenyl-N1,N2-bis(quinolin-2-ylmethyl)ethane-
1,2-diamine in CDCl3 ...... 51
2.18 (A) MALDI spectrum of (1R,2R)-diphenyl-N1,N2-bisquinolin-2-ylmethylene)
ethane-1,2-diimine. (B) Calculated isotope pattern for C34H26N4 ...... 53
2.19 (A) MALDI spectrum of (1S,2S)-diphenyl-N1,N2-bis(quinolin-2-ylmethylene)
ethane-1,2-diimine. (B) Calculated isotope pattern for C34H26N4 ...... 53
2.20 (A) MALDI spectrum of (1R,2R)-diphenyl-N1,N2-bis(quinolin-2-ylmethyl)ethane-
1,2-diamine. (B) Calculated isotope pattern for C34H30N4 ...... 54
2.21 (A) MALDI spectrum of (1S,2S)-diphenyl-N1,N2-bis(quinolin-2-ylmethyl)ethane-
1,2-diamine. (B) Calculated isotope pattern for C34H30N4 ...... 54
2.22 (A) MALDI spectrum of [CuII(1R,2R)-diphenyl-N1,N2-bis(quinolin-2-ylmethyl)
2+ RR 2+ ethane-1,2-diamine] , [Cu L] . (B) Calculated isotope pattern for C34H30CuN4 .. 55 xiv
2.23 (A) MALDI spectrum of [CuII(1S,2S)-diphenyl-N1,N2-bis(quinolin-2-ylmethyl)
2+ SS 2+ ethane-1,2-diamine] , [Cu L] . (B) Calculated isotope pattern for C34H30CuN4 .. 55
2.24 Job’s plot for the determination of the stoichiometry of (A) [CuRRL]2+ and C343;
SS 2+ (B) [Cu L] and C343 in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) ...... 56
2.25 (A) Fluorescence titration spectra (B) and fluorescence titration isotherm of C343
(0.01 µM) upon the addition of an incremental amounts of [CuRRL]2+ (0–800 µM)
(C) Fluorescence titration spectra (D) and fluorescence titration isotherm of C343
(0.01 µM) upon the addition of an incremental amounts of [CuSSL]2+ (0–800 µM)
in MeCN : H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) and λex= 444 nm. (E) The
binding isotherms for chiral receptors [CuRRL]2+ and [CuSSL]2+ show the two
chiral receptors have same binding affinity to the achiral C343 ...... 59
2.26 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of
SS + [Cu L.C343] (212 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM)
upon the addition of an incremental amounts of (R)-phenylpropionic acid. λex =
444 nm, [(R)-phenylpropionic acid] = 0-3 mM ...... 62
2.27 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of
SS + [Cu L.C343] (212 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM)
upon the addition of an incremental amounts of (S)-phenylpropionic acid. λex =
444 nm, [(S)-phenylpropionic acid] = 0-3 mM ...... 62
2.28 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of
SS + [Cu L.C343] (212 µM) in MeCN:H2O (7/3 % v/v) at pH=6 (MES=50 mM)
upon the addition of an incremental amounts of (R)-ibuprofen. λex = 444 nm, [(R)-
ibuprofen] = 0-4 mM...... 63 xv
2.29 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of
SS + [Cu L.C343] (212 µM) in MeCN:H2O (7/3 % v/v) at pH=6 (MES=50 mM)
upon the addition of an incremental amounts of (S)- ibuprofen. λex = 444 nm, [(S)-
ibuprofen] = 0-3 mM...... 63
2.30 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of
SS + [Cu L.C343] (212 µM) in MeCN:H2O (7/3 % v/v) at pH=6 (MES=50 mM)
upon the addition of an incremental amounts of (R)-naproxen. λex = 444 nm, [(R)-
naproxen] = 0-3 mM ...... 64
2.31 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of
SS + [Cu L.C343] (212 µM) in MeCN:H2O (7/3 % v/v) at pH=6 (MES=50 mM)
upon the addition of an incremental amounts of (S)-naproxen. λex = 444 nm, [(S)-
naproxen] = 0-3 mM ...... 64
2.32 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of
SS + [Cu L.C343] (212 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM)
upon the addition of an incremental amounts of (3R,5R)-atorvastatin calcium. λex
= 444 nm, [(3R,5R)-atorvastatin calcium] = 0-2.5 mM ...... 65
2.33 (A)Fluorescence titration spectra and (B) fluorescence titration isotherm of
SS + [Cu L.C343] (212 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM)
upon the addition of an incremental amounts of atorvastatin related compound E.
λex = 444 nm, [atorvastatin related compound E] = 0-2.5 mM...... 65
2.34 Overlaid binding isotherms for two corresponding enantiomers based on the
change in fluorescence intensity at the maximum wavelength of [CuSSL.C343]+
(212 µM) show changes in fluorescence intensity upon the addition of xvi
incremental amounts of (R)- or (S)-enantiomer (A) phenylpropionic acid (B)
ibuprofen(C) naproxen (D) atorvastatin in MeCN:H2O 7:3 at pH = 6 (MES = 50
mM). λex = 444 nm ...... 66
2.35 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of
RR + [Cu L.C343] (212 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM)
upon the addition of an incremental amounts of (R)-phenylpropionic acid. λex =
444 nm, [(R)-phenylpropionic acid] = 0-3 mM ...... 67
2.36 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of
RR + [Cu L.C343] (212 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM)
upon the addition of an incremental amounts of (S)-phenylpropionic acid. λex =
444 nm, [(S)-phenylpropionic acid] = 0-3 mM ...... 67
2.37 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of
RR + [Cu L.C343] (212 µM) in MeCN:H2O (7/3 % v/v) at pH=6 (MES=50 mM)
upon the addition of an incremental amounts of (R)-ibuprofen. λex = 444 nm, [(R)-
ibuprofen] = 0-4 mM...... 68
2.38 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of
RR + [Cu L.C343] (212 µM) in MeCN:H2O (7/3 % v/v) at pH=6 (MES=50 mM)
upon the addition of an incremental amounts of (S)-ibuprofen. λex = 444 nm, [(S)-
ibuprofen] = 0-4 mM...... 68
2.39 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of
RR + [Cu L.C343] (212 µM) in MeCN:H2O (7/3 % v/v) at pH=6 (MES=50 mM)
upon the addition of an incremental amounts of (R)-naproxen. λex = 444 nm, [(R)-
naproxen] = 0-3 mM ...... 69 xvii
2.40 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of
RR + [Cu L.C343] (212 µM) in MeCN:H2O (7/3 % v/v) at pH=6 (MES=50 mM)
upon the addition of an incremental amounts of (S)-naproxen. λex = 444 nm, [(S)-
naproxen] = 0-3 mM ...... 69
2.41 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of
RR + [Cu L.C343] (212 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM)
upon the addition of an incremental amounts of (3R,5R)-atorvastatin calcium. λex
= 444 nm, [(3R,5R)-atorvastatin calcium] = 0-2.5 mM ...... 70
2.42 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of
RR + [Cu L.C343] (212 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM)
upon the addition of an incremental amounts of atorvastatin related compound E.
λex = 444 nm, [atorvastatin related compound E] = 0-2.5 mM...... 70
2.43 Overlaid binding isotherms for two corresponding enantiomers based on the
change in fluorescence intensity at the maximum wavelength of [CuRRL.C343]+
(212 µM) show changes in fluorescence intensity upon the addition of
incremental amounts of (R)- or (S)-enantiomer (A) phenylpropionic acid (B)
ibuprofen(C) naproxen (D) atorvastatin in MeCN:H2O 7:3 at pH = 6 (MES = 50
mM). λex = 444 nm ...... 71
2.44 Overlaid binding isotherms based on the change in fluorescence intensity at the
maximum wavelength of [CuSSL.C343]+ and [CuRRL.C343]+ (212 µM) and show
changes in fluorescence intensity upon the addition of incremental amounts of
(R)- or (S)-enantiomer of (A) phenylpropionic acid (B) ibuprofen(C) naproxen
(D) atorvastatin in MeCN:H2O 7:3 at pH = 6 (MES = 50 mM). λex = 444 nm ...... 72 xviii
2.45 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of
[CuSSL•C343]+ (212 µM) upon the addition of an incremental amounts of (R)-
mandelic acid. (C) fluorescence titration spectra and (D) fluorescence titration
isotherm of [CuSSL•C343]+ (212 µM) upon the addition of an incremental
amounts of (S)-mandelic acid in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50
mM) λex = 444 nm, [(S)-, (R)-mandelic acid] = 0-2 mM. (E) overlaid isotherms
based on the change in fluorescence intensity at the maximum wavelength of
[CuSSL•C343]+ (212 µM) and show the same changes in fluorescence intensity
upon the addition of incremental amounts of (R)- and (S)-mandelic acid ...... 74
2.46 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of
[CuRRL•C343]+ (212 µM) upon the addition of an incremental amounts of (R)-
mandelic acid. (C) fluorescence titration spectra and (D) fluorescence titration
isotherm of [CuRRL•C343]+ (212 µM) upon the addition of an incremental
amounts of (S)-mandelic acid in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50
mM) λex = 444 nm, [(S)- and (R)-mandelic acid] = 0-2 mM. Overlaid isotherms
based on the change in fluorescence intensity at the maximum wavelength of (E)
[CuRRL.C343]+ (F) [CuSSL•C343]+ and [CuRRL•C343]+ (212 µM) and show the
same changes in fluorescence intensity upon the addition of incremental amounts
of (R)- and (S)-mandelic acid ...... 75
2.47 Structures of BINOL boronate complexes reported by Munusamy et al.138 ...... 82
2.48 Structures of triazine-based thiazole derivatives as chiral receptors reported by
Halay and Bozhurt60 ...... 82 xix
2.49 Pseudoenantiomeric sensor pair with the opposite chiral configurations designed
by Pu139 for the determination of enantiomeric composition of mandelic acid ...... 84
2.50 Structure of the sensors used by Zhu et al.108 for ee determination of α-hydroxy
acids...... 84
2.51 Structure of chiral sensors, [ZnII(S,S)-L]2+, [ZnII(R,R)-L]2+, [CuII(R,R)-L]2+ and the
indicator Coumarin 343 used in conjunction with [CuII(R,R)-L]2+ ...... 85
2.52 Structures of α-hydroxy acids used in this study...... 86
2.53 1H NMR (500 MHz) of (1S,2S)-N1,N2-bis(quinolin-2-ylmethylene)cyclohexane-
1,2-diimine in CDCl3 ...... 95
2.54 13C NMR (125 MHz) of (1S,2S)-N1,N2-bis(quinolin-2-ylmethylene)cyclohexane-
1,2-diimine in CDCl3 ...... 96
2.55 1H NMR (500 MHz) of (1S,2S)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2-
diamine in CDCl3 ...... 97
2.56 13C NMR (125 MHz) of (1S,2S)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2-
diamine in CDCl3 ...... 98 100
2.57 1H NMR (500 MHz) of (1R,2R)-N1,N2-bis(quinolin-2-ylmethylene)cyclohexane-
1,2-diimine in CDCl3 ...... 100
2.58 13C NMR (125 MHz) of (1R,2R)-N1,N2-bis(quinolin-2-ylmethylene)cyclohexane-
1,2-diimine in CDCl3 ...... 100
2.59 1H NMR (500 MHz) of (1R,2R)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2-
diamine in CDCl3 ...... 101 103
2.60 13C NMR (125 MHz) of (1R,2R)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2-
diamine in CDCl3 ...... 102 104 xx
2.61 1H NMR (500 MHz) of [ZnII(1S,2S)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-
2+ II 2+ 1,2-diamine] , [Zn (S,S)-L] in CDCl3 ...... 103
2.62 13C NMR (125 MHz) of [ZnII(1S,2S)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-
2+ II 2+ 1,2-diamine] , [Zn (S,S)-L] in CDCl3 ...... 104
2.63 1H NMR (500 MHz) of [ZnII(1R,2R)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-
2+ II 2+ 1,2-diamine] , [Zn (R,R)-L] in CDCl3 ...... 105
2.64 13C NMR (125 MHz) of [ZnII(1R,2R)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-
2+ II 2+ 1,2-diamine] , [Zn (R,R)-L] in CDCl3 ...... 106
2.65 Partial 1H NMR (500 MHz) spectra of ligand [(1R,2R)-N1,N2-bis(quinolin-2-
ylmethyl)cyclohexane-1,2-diamine] [(R,R)-L] and complex [ZnII(R,R)-L] 2+ ...... 107
2.66 Partial 13C NMR (125 MHz) spectra of ligand [(1R,2R)-N1,N2-bis(quinolin-2-
ylmethyl)cyclohexane-1,2-diamine], [(R,R)-L] and complex [ZnII((R,R)-L] 2+ in
CDCl3...... 108
2.67 (A) ESI spectrum of (1S,2S)-N1,N2-bis(quinolin-2-ylmethylene)cyclohexane-1,2-
diimine. (B) Calculated isotope pattern for C26H24N4Na ...... 109
2.68 (A) ESI spectrum of (1R,2R)-N1,N2-bis(quinolin-2-ylmethylene)cyclohexane-1,2-
diimine. (B) Calculated isotope pattern for C26H24N4Na ...... 110
2.69 (A) ESI spectrum of (1S,2S)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2-
diamine. (B) Calculated isotope pattern for C26H29N4 ...... 110
2.70 (A) ESI spectrum of (1R,2R)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2-
diamine. (B) Calculated isotope pattern for C26H29N4 ...... 111
2.71 (A) ESI spectrum of [ZnII(1S,2S)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2-
2+ II 2+ diamine] , [Zn (S,S)-L] (B) Calculated isotope pattern for C26H28N5O3Zn ...... 111 xxi
2.72 (A) ESI spectrum of [ZnII(1R,2R)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2-
2+ II 2+ diamine] , [Zn (R,R)-L] .(B) Calculated isotope pattern for C26H28N5O3Zn ...... 112
2.73 (A) ESI spectrum of [CuII(1R,2R)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-
1,2-diamine]2+, [CuII(R,R)-L] 2+. (B) Calculated isotope pattern for
C27H28CuF3N4O3S ...... 112
2.74 (A) ESI spectrum of [ZnII(1R,2R)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2-
diamine]2+, [ZnII(R,R)-L]2+ and atorvastatin related compound E. (B) Calculated
isotope pattern for C59H62FN6O5Zn ...... 113
2.75 Job’s plot for the determination of the stoichiometry of (A) Zn(II) and chiral
ligand (R,R)-L; (B) [CuII (R,R)-L]+2 and Coumarin 343 (C) [ZnII (R,R)-L]2+ and
II +2 (R)-mandelic acid ; (D) [Zn (R,R)-L)] and (S)-mandelic acid in MeCN:H2O
7:3 at pH = 6 (MES = 50 mM) ...... 114
2.76 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII
2+ (R,R)-L] (20 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon
the addition of an incremental amounts of (R)-mandelic acid. λex = 315 nm, [(R)-
mandelic acid] = 0-8 mM ...... 119
2.77 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII
2+ (R,R)-L] (20 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon
the addition of an incremental amounts of (S)-mandelic acid. λex = 315 nm, [(S)-
mandelic acid] = 0-8 mM ...... 119
2.78 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII
2+ (R,R)-L] (20 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon xxii
the addition of an incremental amounts of (R)-lactic acid. λex = 315 nm, [(R)-lactic
acid] = 0-6 mM ...... 120
2.79 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII
2+ (R,R)-L] (20 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon
the addition of an incremental amounts of (S)-lactic acid. λex = 315 nm, [(S)-lactic
acid] = 0-6 mM ...... 120
2.80 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII
2+ (R,R)-L] (20 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon
the addition of an incremental amounts of (R)-2-hydroxy-3-methylbutanoic acid.
λex = 315 nm, [(R)-2-hydroxy-3-methylbutanoic acid] = 0-6 mM ...... 121
2.81 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII
2+ (R,R)-L] (20 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon
the addition of an incremental amounts of (S)-2-hydroxy-3-methylbutanoic acid.
λex = 315 nm, [(S)-2-hydroxy-3-methylbutanoic acid] = 0-6 mM ...... 121
2.82 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII
2+ (R,R)-L] (20 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon
the addition of an incremental amounts of (R)-3-phenyllactic acid. λex = 315 nm,
[(R)-3-phenyllactic acid] = 0-6 mM ...... 122 124
2.83 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII
2+ (R,R)-L)] (20 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon
the addition of an incremental amounts of (S)-3-phenyllactic acid. λex = 315 nm,
[(S)-3-phenyllactic acid] = 0-6 mM ...... 122 124 xxiii
2.84 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII
2+ (R,R)-L] (20 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon
the addition of an incremental amounts of (3R,5R)-atorvastatin calcium. λex = 315
nm, [(3R,5R)-Atorvastatin calcium] = 0-450 µM ...... 123
2.85 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII
2+ (R,R)-L] (20 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon
the addition of an incremental amounts of atorvastatin related compound E. λex =
315 nm, [atorvastatin related compound E] = 0-450 µM ...... 123
2.86 Overlaid binding isotherms for two corresponding enantiomers based on the
change in fluorescence intensity at the maximum wavelength of [ZnII (R,R)-L]2+
(20 µM) show changes in fluorescence intensity upon the addition of incremental
amounts of (R)-enantiomer and (S)-enantiomer (A) mandelic acid (B) lactic acid
(C) 2-hydroxy-3-methylbutanoic acid (D) 3-phenyllactic acid (E) atorvastatin in
MeCN:H2O 7:3 at pH=6 (MES=50 mM). λex = 430 nm ...... 124
2.87 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII
2+ (S,S)-L] (20 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon
the addition of an incremental amounts of (R)-mandelic acid. λex = 315 nm, [(R)-
mandelic acid] = 0-8 mM ...... 125
2.88 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII
2+ (S,S)-L] (20 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon
the addition of an incremental amounts of (S)-mandelic acid. λex = 315 nm, [(S)-
mandelic acid] = 0-8 mM ...... 125 xxiv
2.89 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII
2+ (S,S)-L] (20 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon
the addition of an incremental amounts of (R)-lactic acid. λex = 315 nm, [(R)-lactic
acid] = 0-6 mM ...... 126
2.90 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII
2+ (S,S)-L] (20 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon
the addition of an incremental amounts of (S)-lactic acid. λex = 315 nm, [(S)-lactic
acid] = 0-6 mM ...... 126
2.91 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII
2+ (S,S)-L] (20 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon
the addition of an incremental amounts of (R)-2-hydroxy-3-methylbutanoic acid.
λex = 315 nm, [(R)-2-hydroxy-3-methylbutanoic acid] = 0-6 mM ...... 127
2.92 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII
2+ (S,S)-L] (20 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon
the addition of an incremental amounts of (S)-2-hydroxy-3-methylbutanoic acid.
λex = 315 nm, [(S)-2-hydroxy-3-methylbutanoic acid] = 0-6 mM ...... 127
2.93 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII
2+ (S,S)-L] (20 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon
the addition of an incremental amounts of (R)-3-phenyllactic acid. λex = 315 nm,
[(R)-3-phenyllactic acid] = 0-6 mM ...... 128 130
2.94 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII
2+ (S,S)-L] (20 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon xxv
the addition of an incremental amounts of (S)-3-phenyllactic acid. λex = 315 nm,
[(S)-3-phenyllactic acid] = 0-6 mM ...... 128 130
2.95 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII
2+ (S,S)-L] (20 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon
the addition of an incremental amounts of (3R,5R)-atorvastatin calcium. λex = 315
nm, [(3R,5R)-atorvastatin calcium] = 0-450 µM ...... 129
2.96 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII
2+ (S,S)-L] (20 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon
the addition of an incremental amounts of atorvastatin related compound E. λex =
315 nm, atorvastatin related compound E] = 0-450 µM ...... 129
2.97 Overlaid binding isotherms for two corresponding enantiomers based on the
change in fluorescence intensity at the maximum wavelength of [ZnII (S,S)-L]2+
(20 µM) show changes in fluorescence intensity upon the addition of incremental
amounts of (R)-enantiomer and (S)-enantiomer (A) mandelic acid (B) lactic acid
(C) 2-hydroxy-3-methylbutanoic acid (D) 3-phenyllactic acid (E) atorvastatin in
MeCN:H2O 7:3 at pH=6 (MES=50 mM). λex = 430 nm ...... 131
2.98 Overlaid binding isotherms based on the change in fluorescence intensity at the
maximum wavelength of [ZnII(S,S)-L]2+ and [ZnII(R,R)-L]2+ (20 µM) show
changes in fluorescence intensity upon the addition of incremental amounts of
(R)- or (S)-enantiomer of (A) mandelic acid (B) lactic acid (C) 2-hydroxy-3-
methylbutanoic acid (D) 3-phenyllactic acid (E) atorvastatin in MeCN:H2O 7:3 at
pH = 6 (MES = 50 mM). λex = 430 nm...... 131 xxvi
2.99 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII
2+ (R,R)-L] (20 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon
the addition of an incremental amounts of (R)-ibuprofen. λex = 315 nm, [(R)-
ibuprofen] = 0-12 mM ...... 132
2.100 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII
2+ (R,R)-L] (20 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon
the addition of an incremental amounts of (S)-ibuprofen. λex = 315 nm, [(S)-
ibuprofen] = 0-12 mM ...... 132
2.101 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII
2+ (S,S)-L] (20 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon
the addition of an incremental amounts of (R)-ibuprofen. λex = 315 nm, [(R)-
ibuprofen] = 0-12 mM ...... 133
2.102 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII
2+ (S,S)-L] (20 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon
the addition of an incremental amounts of (S)-ibuprofen. λex = 315 nm, [(S)-
ibuprofen] = 0-12 mM ...... 133
2.103 Overlaid binding isotherms based on the change in fluorescence intensity at the
maximum wavelength of [ZnII(R,R)-L]2+ and [ZnII(S,S)-L]2+ (20 µM) show the
same changes in fluorescence intensity upon the addition of incremental amounts
of (R)- or (S)-ibuprofen in MeCN:H2O 7:3 at pH = 6 (MES = 50 mM). λex = 430
nm...... 134 xxvii
2.104 (A) Fluorescence titration spectra (B) and fluorescence titration isotherm of C343
(0.01 µM) upon the addition of an incremental amounts of [CuII (R,R)-L]+2 (0–3
mM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM). λex = 430 nm ...... 137
2.105 Fluorescence titration spectra of [CuII (R,R)-L.C343]+ (780 µM) with (A) (S)- or
(R)-phenylalanine; (B) (S)- or (R)-phenylpropionic acid in MeCN:H2O (7/3 %
v/v) at pH = 6 (MES = 50 mM). λex = 430 nm ...... 137
2.106 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [CuII
+ (R,R)-L.C343] (780 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM)
upon the addition of an incremental amounts of (R)-mandelic acid. λex = 430 nm,
[(R)-mandelic acid] = 0-6 mM ...... 138
2.107 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [CuII
+ (R,R)-L.C343] (780 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM)
upon the addition of an incremental amounts of (S)-mandelic acid. λex = 430 nm,
[(S)-mandelic acid] = 0-6 mM ...... 138
2.108 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [CuII
+ (R,R)-L.C343] (780 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM)
upon the addition of an incremental amounts of (R)-2-hydroxy-3-methylbutanoic
acid. λex = 430 nm, [(R)-2-hydroxy-3-methylbutanoic acid] = 0-8 mM ...... 139
2.109 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [CuII
+ (R,R)-L.C343] (780 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM)
upon the addition of an incremental amounts of (S)-2-hydroxy-3-methylbutanoic
acid. λex = 430 nm, [(S)-2-hydroxy-3-methylbutanoic acid] = 0-8 mM ...... 139 xxviii
2.110 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [CuII
+ (R,R)-L.C343] (780 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM)
upon the addition of an incremental amounts of (R)-3-phenyllactic acid. λex = 430
nm, [(R)-3-phenyllactic acid] = 0-8 mM ...... 140 142
2.111 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [CuII
+ (R,R)-L.C343] (780 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM)
upon the addition of an incremental amounts of (S)-3-phenyllactic acid. λex = 430
nm, [(S)-3-phenyllactic acid] = 0-8 mM ...... 140 142
2.112 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [CuII
+ (R,R)-L.C343] (780 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM)
upon the addition of an incremental amounts of (R)-lactic acid. λex = 430 nm,
[(R)-lactic acid] = 0-6 mM ...... 141
2.113 (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [CuII
+ (R,R)-L.C343] (780 µM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM)
upon the addition of an incremental amounts of (S)-lactic acid. λex = 430 nm, [(S)-
lactic acid] = 0-6 mM ...... 141
2.114 Overlaid binding isotherms based on the change in fluorescence intensity at the
maximum wavelength of [CuII (R,R)-L•C343]+ (780 µM) show enhancement of
intensity upon the addition of incremental amounts of (R)-enantiomers and (S)-
enantiomers (A) mandelic acid (B) 2-hydroxy-3-methylbutanoic aid (C) 3-
phenyllactic acid (D) lactic acid in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50
mM). λex = 430 nm ...... 142 xxix
2.115 A semiquantitative assay of enantiomeric composition of (A) mandelic acid (B)
lactic acid (C) 2-hydroxy-3-methylbutanoic acid and (D) 3-phenyllactic acid by
employing the microarray in MeCN: H2O (7/3 % v/v) at pH = 6 (MES = 50 mM).
[ZnII (S,S)-L]2+ = [ZnII (R,R)-L]2+ = 20 µM, [analytes] = 2 mM ...... 149
2.116 Multivariate linear regression for determination of ee in the samples of (A)
mandelic acid (B) lactic acid (C) 2-Hydroxy-3-methylbutanoic acid and (D) 3-
phenyllactic acid. Root mean square errors of prediction (RMSEP) of 4.5 %, 5.2
%, 6.5 %, 5.1 % for mandelic acid, lactic acid, 2-hydroxy-3-methylbutanoic acid,
and 3-phenyllactic acid, respectively, relates to the error with two unknown
independent samples. [ZnII (S,S)-L]2+ = [ZnII (R,R)-L]2+ = 20 µM, [analyte] = 2
mM...... 150
3.1 A fluorescent chemosensor developed by Andrew and Swager9 to detect saturated
nitramine and nitrate ester explosives ...... 165
3.2 Detection strategy used by Germain and Knapp11 for peroxide-based explosives .... 166
3.3 Chemical structure of organic peroxides TATP, HMTD and nitro-substituted high
explosives PETN, RDX, HMX, and TNT ...... 167
3.4 It has been shown that the pentiptycene-derived conjugated polymers are an
excellent fluorescent chemosensor for the detection of electron-deficient
unsaturated species such as TNT by Yang and Swager.44 The rigid pentiptycene
groups provide cavities for analyte binding studies of polymers 1-4 that probe the
electronic and structural effects on fluorescence quenching with a variety of
analytes...... 173 xxx
3.5 The electronic tongue formed by non-noble electrodes designed by Campos et al66
for detection of ammonium nitrate ...... 176
3.6 Illustration of the portable explosive detector prototype. The first step which is
not shown in the figure includes inserting the calibration point between the UV
LED and the photodiode and turning the calibration knob until the green LED
flashes. The second step displayed in the figure shows the detection of explosives
on the µPAD (red LED flashes).128 ...... 181
3.7 Illustrates the steps involved in trinitro aromatic explosive analysis from sample
collection and colorimetric detection to identification. 1) µPAD, 2) direct
collection after spraying the surface with a methanol/water solution, 3) inactive
spot is punched out using a hole-punch, 4−5) the chad is moistened with the
electrolyte and extracted by mixing with the pipet for 30 s, 6) the extracted
sample is added on the microchip, and 7) analysis using the lab on a chip 2100
Bioanalyzer.129 ...... 182
3.8 (A) The wearable Forensic Finger exhibiting the three-electrode surface screen-
printed onto a nitrile finger cot (bottom left inset), and a solid, conductive ionogel
immobilized on a similar substrate (top right inset); (B) VMP detection of
samples using a portable electrochemical analyzer (CH Instruments model
1230A) interfaced with a notebook computer; (C) Voltammetric response
obtained at Forensic Finger sensor/ionogel interface in the absence (black) and in
the presence (red) of (A) GSR and (B) DNT.140 ...... 184
3.9 Ring-based sensor platform for detecting explosives and nerve agent threats in
both vapor and liquid phases ...... 185 xxxi
3.10 (A) Aldehydes used in this study and (B) polyethylene imine used to prepare
sensor systems (C) sensors composition ...... 187
+ 3.11 MALDI-TOF mass spectra of (A) [S1+NM-H2O] = 192.1, Inset: Calculated
+ + isotope pattern for [S1+NM-H2O] = 192.1, (B) [S2+NM-H2O] = 199.1, Inset:
+ + Calculated isotope pattern for [S2+NM-H2O] = 192.1, (C) [S3+NM-H2O] =
+ 273.1, Inset: Calculated isotope pattern for [S3+NM-H2O] = 273.1, (D)
+ + [S4+NM-H2O] = 237.1, Inset: Calculated isotope pattern for [S4+NM-H2O] =
237.1...... 192
3.12 Solid state fluorescence spectra of S1-S4, S1-S4 in presence of NM, and S1-S4 in
presence of AN at (A) λexc,S1 = 380 nm, (B) λexc,S2 = 364 nm, (C) λexc,S3 = 410
nm, (D) λexc,S1 = 354 nm in room temperature ...... 194
3.13 Fluorescence titration spectra and fluorescence titration isotherm (Insets) of S3
(0.1 µM) upon the addition of an incremental amounts of (A) [NM] = 0-32 mM
(B) [AN] = 0-30 mM in EtOH (96%)-H2O. lEXC = 346 nm ...... 197
3.14 A photograph of the fluorescence under a handheld UV-light of 1: PyrC (0.1
mM), 2: S3 (0.1 mM), 3: S3 (0.1 mM) + NM (30 mM), 4: S3 (0.1 mM) + AN (30
mM), and 5: S3 (0.1 mM) + ANNM (26 mM)...... 198
3.15 Fluorescence image from the paper microzone array of 1: PyrC, 2: S3, 3: S3 +
NM, 4: S3 + AN, and 5: S3 + ANNM as a sum of the RGB channels recorded for
the measurements ...... 199
3.16 The sensor array fluorescence under black light (365 nm) and different responses
to the presence of the analytes (NM, AN, and ANNM)...... 200
3.17 The designed paper microzone plate for five repetition experiments ...... 201 xxxii
3.18 LDA corresponding to the paper microzone array. The cross-validation shows
100% correct classification of all 8 analytes and a control. All analytes except AN
were applied as vapor. AN, because of the low vapor pressure was applied to the
array as a solution ...... 203
3.19 The designed paper microzone plate for linear discriminant analysis in solution
for four sensors (S1-S4), six analytes and repetition experiments ...... 204
3.20 Graphical output of qualitative LDA for sensors S1-S4 in EtOH (96%)-H2O with
various analytes. The array was achieved using 13 excitation/emission channels
for each sensor, 6 repetitions and provided 100% correct classification ...... 205
3.21 Hierarchical clustering analysis dendrograms (Euclidean Distance, Ward's
Linkage) for sensors S1-S4 and 10 analytes displays 100% correct classification ... 207
3.22 Nanofiber mat deposited on a microscope slide (A) S3 mat fluorescence under
black light (256 nm) (B) S3 mat exposed to NM; resulting in fluorescence
quenching as seen under black light. (C) S3 mat exposed to AN (in water);
resulting in fluorescence enhancement as seen under black light. (D) S3 mat
exposed to the ANNM. The picture was taken under a handheld UV-Vis lamp
using a smartphone ...... 208
3.23 (A) and (B) Nanofiber mat deposited on the finger of a nitrile glove. (C) The
wearable sensor displays dramatic changes in color in the presence of NM as well
as increase in fluorescence in the presence of AN and ANNM ...... 209
xxxiii
LIST OF TABLES
Table Page
2.1 Photophysical properties of chiral ligands SSL, and RRL and chiral sensors
SS 2+ RR 2+ [Cu L] , [Cu L] . Absorption (λA,max) and absolute quantum yields F were
acquired in MeCN:H2O 7:3 at pH = 6 (MES = 50 mM) solutions...... 57
-1 RR 2+ SS 2+ 2.2 The association constants (Ka, M ) corresponding to [Cu L] and [Cu L]
chiral sensors with selected chiral carboxylic acids and a-hydroxy acids...... 76
2.3 Parameters used in the affinity constant model for [CuRRL]2+, [CuSSL]2+, C343,
and chiral carboxylic acids ...... 80
2.4 Photophysical properties of chiral sensors [ZnII (R,R)-L]2+, [ZnII (S,S)-L]2+, and
II 2+ [Cu (R,R)-L] . Absorption maxima (λA,max) and fluorescence lifetimes tFL were
acquired in MeCN:H2O 7:3 at pH = 6 (MES = 50 mM) solutions ...... 116
-1 II 2+ II 2.5 The association constants (Ka, M ) corresponding to [Cu (R,R)-L] , [Zn (R,R)-
L]2+, and [ZnII (S,S)-L]2+ chiral sensors with selected chiral a-hydroxy acids...... 143
2.6 Parameters used in the affinity constant model for [CuII (R,R)-L]2+, C343, and
chiral analytes ...... 146
3.1 Absorption (lA, max) and absolute quantum yields (F ) of aldehyde parents and
sensor systems S1-S4 ...... 195
3.2 The jackknifed classification matrix of the qualitative assay for S1-S4 in vapor
phase...... 203
3.3 The jackknifed classification matrix of the qualitative assay for S1-S4 in solution.. 206 xxxiv
LIST OF SCHEMES
Scheme Page
2.1 Synthesis of (1S,2S)- and (1R,2R)-diphenyl-N1,N2-bis(quinolin-2-ylmethylene)
ethane-1,2-diimine (1) ...... 40
2.2 Synthesis of (1S,2S)- and (1R,2R)-diphenyl-N1,N2-bis(quinolin-2-ylmethyl)
ethane-1,2-diamine (SSL or RRL)...... 41
2.3 Synthesis of [CuII(1S,2S)- and CuII(1R,2R)-diphenyl-N1,N2-bis(quinolin-2-
SS RR ylmethyl) ethane-1,2-diamine] ([Cu L](OTf)2 or [Cu L](OTf)2)...... 43
2.4 Synthesis of (1S,2S)- and (1R,2R)-N1,N2-bis(quinolin-2-ylmethylene)
cyclohexane-1,2-diimine (1) ...... 90
2.5 Synthesis of (1S,2S)- and (1R,2R)-N1,N2-bis(quinolin-2-ylmethyl)
cyclohexane-1,2-diamine, (S,S)-L and (R,R)-L) ...... 91
2.6 Synthesis of [ZnII(1S,2S)- and (1R,2R)-N1,N2-bis(quinolin-2-ylmethyl)
cyclohexane-1,2-diamine]2+ ,[ZnII(S,S)-L]2+ and [ZnII(R,R)-L]2+ ...... 92
2.7 Synthesis of [CuII(1R,2R)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2-
diamine]2+, [CuII(R,R)-L] 2+ ...... 94
3.1 Henry reaction also known as nitro aldol reaction is a base-catalyzed carbon-carbon
bond forming reaction between nitroalkanes and aldehydes or ketones ...... 186 1
CHAPTER I. INTRODUCTION
1.1. Chemical Sensors
In 1991, Hulanicki and coworkers defined a chemical sensor as “a device that transforms chemical information which may originate from a chemical reaction of the analyte or from a physical property of the system, ranging from the concentration of a specific sample component to total composition analysis, into an analytically useful signal”.1 Chemical sensors are used in a wide range of scientific fields and disciplines such as critical care, food safety, process controls, product quality controls, drug detection, environmental protection, forensics, clinical diagnostics, homeland security.2–11 In these applications, chemical sensors have resulted in both economic and social benefits. In this dissertation, we performed detailed studies and development of different types of chemical sensors for sensing of enantiomeric excess in various chiral carboxylic acids.
Chemical sensors consist of two functional units: a receptor and a transducer: The receptor acts as a binding unit providing specific moieties for interaction with an analyte and transforms the chemical information into a form of energy. The receptor which is responsible for the molecular recognition of the analyte utilizes a variety of capture mechanisms like covalent interactions such as molecular self-assemblies, or non-covalent binding interactions such as hydrogen bonding, Van der Waals forces, hydrophobic, electrostatic, or different type of π-π interactions, among others.12 The transducer is transforming the chemical information about the analyte into a useful analytical signal which has to be measured and interpreted as the quantity of molecular action.
According to the recognition mechanism and the operating principle of the transducer, chemical sensors classified into three types: electrical, mechanical, and optical sensors.13 2
1.2. Optical Chemical Sensors
Optical chemical sensors utilize the interaction between light in the visible, near-IR, and ultraviolet regions and matter. These interactions cause a change in the properties of the light such as intensity, phase, polarization, and wavelength.
Synthetic receptors have been used as sensors for detection of a variety of analytes. In optical sensors, the signaling is originating from a change in the electronic structure that occurs upon binding between a host/receptor and a guest/analyte (Figure 1.1). The change in signal allows for the detection and quantification of the analyte.14
Host Guest Host-Guest Complex Figure 1.1. The operation principle of a typical optical chemical sensor with host and guest complexation.
Sensitivity is one of the most desirable characteristics of any sensor. Fluorescence is a preferred signal transduction mechanism for potential sensing applications due to its high sensitivity, fast analysis, operational simplicity and cost-effectiveness. Furthermore, the ability to detect analytes down to the single molecule level, adaptability, the possibility of naked eye detection, and the ability to operate on the microscopic scale and track the changes in optical properties of analytes are main advantages of fluorescent sensing. Fluorescence signaling enables the system monitoring in numerous detection modes such as fluorescence intensity, polarization, and anisotropy, all of which can be performed in a steady-state or time-dependent manner.15–17 3
Modulation of the photophysical properties of a fluorophore, such as introduction of photoinduced electron/energy transfer (PET), charge transfer (CT), etc. offers a wide number of mechanisms of fluorescence signal transduction.14,18–28
1.3. Optically Active/Chiral Chemical Sensors
Since the number of chiral drugs on the market is constantly increasing,29–32 development of fast, cost-effective methods that are not, labor-intensive and time-consuming are important to drug development. R. Noyori, W. S. Knowles, and K. B. Sharpless received the 2001 Nobel Prize for their work on asymmetric catalysis33 highlighting the importance and significance of enantioselective syntheses.
Enantiomeric excess (ee), defined as mathematical expressions shown in equation 1, is used to quantify the enantiomer enrichment and the efficiency of an asymmetric transformation. Enantiomeric excess values cover a range from −100 % (pure enantiomer) to +100% (pure opposite enantiomer). In order to quantify ee, the enantiomers must be distinguished or separated in a chiral environment.
Diastereomers will form upon the use of a chiral environment. These diastereomers are different in energy which result in enantiomeric discrimination and quantification of chiral analytes.34
[�] − [�] % �� = × 100 % [�] + [�] (Equation 1)
Figure 1.2. The mathematical expression commonly used to quantify ee.
In order to obtain ee by diastereomeric assemblies, the synthetic chiral host must interact with chiral analytes to form diastereomers (Figure 1.3). The formed diastereomers have different stabilities which result in a different affinity/binding constant (KR and KS) between the chiral host 4 and enantiomeric guests (Figure 1.4).34 This makes the optical signature of such diastereomers to be different, allowing for the quantification of the guest ee.
(R)
more stable (R) diastereomer
(R)
(R)-Host less stable diastereomer
Chiral Guest
Figure 1.3. Schematic of ee determination using an enantiopure chiral host with the formation of two different diastereomeric complexes of varying stability.
(Equation 2)
(Equation 3)
∆ ������ = ƒ ([� ], ��) (Equation 4)
* Figure 1.4. Equilibria of chiral recognition. H = chiral host/receptor; GR, GS = analyte/guest; [G]t = total concentration of guest; KR, KS = affinity/binding constant.
1.4. Indicator Displacement Assay
Traditionally, the chemosensors (chemical sensors) utilize the indicator–spacer–receptor
(ISR) design where the indicator (chromophore or fluorophore) is covalently attached to the receptor via a spacer (Figure 1.3).35 Introduction of an analyte that binds to the receptor moiety 5 modulates measurable change in fluorescence or absorbance signal of the indicator. These measurements can be used to obtain affinity constant of binding.36 Although ISR is the most popular, this traditional design has major limitation. The main disadvantage of this traditional design is often the need of complicated synthesis required to covalently attach the indicator to the receptor and cost of the final sensor. The indicator–displacement assay (IDA)35,37–39 overcomes this obstacle and offers many advantages over traditional sensing methods. In the
IDA, an indicator is first allowed to bind to a receptor forming a reversible noncovalent complex.
Then, upon introduction of a competitive analyte, the indicator is displaced from the receptor thereby changing its optical properties (Figure 1.3).40 In this setup, the binding affinity of indicator–receptor must be comparable to the one of the analyte-receptor complex. Also, the complexation equilibrium of the indicator must give a rise to significant and measurable change in its optical properties.
In general, several mechanisms can be employed in the signal modulation in an IDA such as fluorescence resonance energy transfer (FRET),41 photoinduced electron transfer (PET),42,43 electronic energy transfer (EET),16,44 or a change of ionic strength or pH.45 Hydrogen bonding46–
51 and electrostatic interactions52–63 are common interactions between the indicator or analyte and the receptor. Complexation with transition metal centers has also been investigated.64,65 These interactions are dependent on the geometry of the indicator or analyte, their charge, and the solvent system.66 IDAs have been classified in three types: The colorimetric IDA (C-IDA) which employs colorimetric indicators, the fluorescent IDA (F-IDA) which uses fluorescent indicators, and the metal complexing IDA (M-IDA) that utilizes a metal center with either a colorimetric or fluorescent indicator.35 A metal complexing IDA is a subsection of both, a C-IDA and a F-IDA.
In chapter II of this dissertation, we are utilizing M-IDA. In a M-IDA, a metal is complexed with 6 a receptor. Then, an indicator (chromophore or fluorophore) is allowed to coordinate with the metal center-receptor complex. Addition of an analyte to the system causes the displacement of the indicator from the complex. This results in optical changes that can be measured to derive binding affinity. Zinc and copper have been most effectively used which we are using these metal ions in our M-IDA as well.
Importantly, IDA-based sensing systems possess several advantages over the conventional sensing assays: (i) this method does not require the indicator to be covalently attached to the receptor, (ii) IDA allows to employ various indicators due to no covalent bonds between the receptor and the indicator, hence providing access to a library of different sensing ensembles (iii) the assay can be used in both organic and aqueous media (iv) the assay is easily adapted to different receptors.
Enantioselective indicator displacement assays (eIDAs)67–73 were introduced the first time by
Anslyn and co-workers for the enantioselective discrimination applications. In eIDAs (Figure
* 1.5) a chiral receptor/host (H ) associates with chiral analytes/guest (GR, GS) to form diastereomeric host-guest complexes (Equation 6 and 7). The difference in the equilibrium constants (KR and KS) yields differential displacement of indicator, and hence a difference in optical properties for the two solution of diastereomers (Equation 8). Therefore, the use of chiral host allows for ee determination of the reaction.
✽ ✽ OFF ✽ ON ✽ ON ✽ Incubation Incubation ✚ ✚ ✚
Chiral Receptor-Indicator Receptor-Analyte Indicator Receptor/Host Indicator Complex Chiral Analyte Complex
Figure 1.5. Schematic of the enantioselective indicator displacement assays. 7
(Equation 5)
(Equation 6)
(Equation 7)
(Equation 8) ∆ ������ = ƒ ([� ], ��)
Figure 1.6. Schematic equilibria of an enantioselective indicator displacement assay. H* = chiral host/receptor; I = indicator; GR, GS, = analyte/guest; [G]t = total guest concentration, KI, KR, KS = affinity/binding constant.
Generally, ee values produced by eIDAs have lower accuracy than chromatographic techniques. But, eIDAs are much faster and allow for high-throughput screening (HTS) using the microwell plate reader and automated liquid handlers.
Metal complexes have also been used in eIDAs to determine ee.34 The interaction of chiral analytes with chiral metal complexes is exploited for molecular sensing due to the large variety of metal-ligand affinity and complexation geometries. Also, because the binding of the guest
(indicator) to the chiral receptor (metal complexes) is commonly reversible, allowing for displacement of bound indicator with analyte from the chiral receptor. The advantages of using metal complexation-eIDAs are they can be used in a highly polar solvent such as aqueous ethanol or pure water.
In 2008, Anslyn group69,70 reported two chiral Cu(II)-diamine complexes for the enantiomeric recognition of a-amino acids (Figure 1.7). Chrome azurol S was used as an indicator. Free indicator showed a different absorbance band compared to the indicator bounded to the copper complex. The free indicator produced a yellow solution with an absorbance band at
429 nm, while the coordinated indicator to the Cu(II)-metal center produced a blue solution with 8 an absorbance at 602 nm. This change in the indicator’s optical properties allows for the detection of chiral analyte upon indicator displacement. They performed the UV-Vis titration for chiral receptors and a-amino acids. According to the result (R,R)-isomer of the receptors showed the higher selectivity to the L-amino acids while the (S,S)-isomer of the receptor in the assay showed an equal but opposite enantioselectivity which confirms the enantiomeric cross- reactivity. Then, direct determination of ee was performed using calibration curves from standard
UV-Vis spectra by monitoring change in max absorbance as a function of ee.
O 3Na O O O O H O * H HO O * N N CuII CuII * N N Cl Cl H O H * SO O 3
II 2+ II 2+ Indicator [Cu (L1)] [Cu (L2)] Figure 1.7. Structures of chiral receptors and chrome azurol S as an indicator used by Anslyn69,70 for enantiomeric discrimination of amino acids.
A number of colorimetric eIDAs were developed.67–75 However, to the best of our knowledge there has not been any eIDAs using fluorescence probes for detection of enantiomeric composition to date. The metal complexing-eIDA concept and its application in fluorescence sensing of chiral analytes will be further discussed in Chapter II.
1.5. The Binding Constants and Complex Formation
The formation of a host-guest complex is a fundamental process in supramolecular chemistry. The thermodynamic stability of a host-guest complex is determined by measurement 9
of the binding/affinity of the guest to the host and described by an association constant (Ka). In a simple case, for a 1:1 host-guest complex, the binding constant has units of dm3 mol-1, or M-1.
Ka H + G H : G (Equation 9)
∆ ������ = ƒ ([� ]) (Equation 10)
Figure 1.8. Equilibrium of optical molecular sensor. H = host/receptor; G = analyte/guest; [G]t = total concentration of the guest; Ka = binding/affinity constant.
The value of the binding constant can be measured by many experimental techniques that can provide information about the formation of a host-guest complex, [H:G], as a function of the guest concentration, [G].76 All experimental techniques for the determination of affinity/binding constants are based on the analysis of a binding isotherm. Fluorescence and UV-Vis spectrophotometric titrations are the most suitable techniques for the determination of Ka for the fluorescent hosts.77
To determine a binding constant in fluorescence titration, a binding isotherm is constructed from the change in fluorescence properties of the fluorophore as a function of the concentration of the analyte at a constant temperature. In a titration, the quantity of the fluorescent host is constant while the concentration of the guest is varied.78 The magnitude of spectroscopic signal is monitored during the titration, and the change in the signal is plotted as a function of the added guest concentration. Mathematical isotherm use in the determination of affinity/binding constants are solved by nonlinear curve fitting method described and developed by Hargrove et al.79 using commercial data analysis software program Origin.
The most important characteristics of any sensor are selectivity and sensitivity. The unique combination of these two parameters can allow for the description of the recognition and 10 quantification of target species. Selectivity of a sensor is defined by the receptor structure and also depends on the host-guest/analyte-receptor binding affinity.80 The equilibrium between the host (H) and two different guests/analytes (G1) and (G2) are described by the affinity constant
(Ka) for each analyte (Figure 1.9). The selectivity of the host defined as a ratio between two
80 independent equilibria Ka1 and Ka2 (Equation 15).
(Equation 11)
(Equation 12)
(Equation 13)
(Equation 14)
(Equation 15)
Figure 1.9. The equilibrium between a host (H) and guests (G1) and (G2) described by the association constant (Ka), is equal to the amount of the host-guest complex divided by the product of the concentration of the individual guest and host when the system is in equilibrium. Host selectivity is determined by the ratio of the Ks for guests (G1) and (G2).
On the other hand, the sensitivity of the sensor is associated with the magnitude of intensity change in sensor’s response upon complexation with each analyte.81 In the calculated isotherm, the slope of the isotherm corresponds to the sensitivity of the sensor. The sensor’s response is described as a function of the analyte concentration.82 A sensor that is displaying high sensitivity towards particular analyte allows a low limit of detection (LOD) defined as the quantity of 11 analyte that can be distinguished from the blank within a stated confidence limit (generally
1%).83
1.6. Signaling Recognition Mechanisms in Optical Sensing
If the sensor is equipped with a suitable fluorophore, change in the emission of light can be used as a signal in the recognition method. As explained before, fluorimetry is a relatively simple method; it has short analysis time, higher sensitivity, and is suitable for analysis of complex mixtures.
The response of a fluorescent chemical sensor is usually measured as a change in fluorescence intensity, fluorescence lifetime, or a shift of fluorescence wavelength.84 The major mechanisms responsible for a change of optical properties of a fluorophore upon binding of an analytes are photoinduced electron transfer (PET),85,86 fluorescence (Förster) resonance energy transfer (FRET),87–90 electronic energy transfer (EET),91,92 and photoinduced charge transfer
(PCT).93,94
The PET has been the widely used fluorescence signal transduction mechanisms to create a variety of turn-on fluorescent sensors.95 The frontier molecular orbital theory can be used in donor-acceptor systems involving PET-based mechanism. In the absence of an analyte in the receptor, the LUMO of the electron-poor acceptor (A) is in lower energy level than the LUMO of the electron donor in its excited state (D*). So that the acceptor effectively de-populates excited state of the donor (D*) through a non-emissive process (oxidative PET). But if an electron-rich acceptor is present in the sensor, the HOMO of the acceptor is energetically higher so that the acceptor can donate an electron to HOMO of D* resulting in fluorescence quenching
(reductive PET). However, in the presence of the analyte in the receptor, the HOMO energy level of the A is lowered and also its LUMO level is unavailable. This reduces the ability to transfer an 12 electron to/from the acceptor, resulting in fluorescence recovery—turn-on response—(Figure
1.10). This principle has been utilized in so-called turn-on fluorescence sensors.96,97
Absence of the Analyte: No Emission
Oxidative PET: h! D D* + A D+! + A-!
LUMO h! LUMO LUMO
HOMO HOMO HOMO
D D* A D+! A-!
electron-poor acceptor Reductive PET:
h! D D* + A D-! + A+!
LUMO LUMO LUMO h! HOMO HOMO
HOMO
D D* A D-! A+!
electron-rich acceptor
Presence of the Analyte: Emission
h! D D* + A D A+ + h!
LUMO LUMO LUMO h!
HOMO HOMO HOMO
D D* A DA
Figure 1.10. Frontier molecular orbitals representation of the mechanism of oxidative and reductive photoinduced electron transfer (PET). 13
An internal charge transfer (ICT) is another signal transduction mechanism that may be used to measure output resulting from the binding of the analyte (usually a charged species) to the receptor.98 In ICT mechanism, in order to obtain a change in optical signal, the chemical sensor is required to contain an electron-donating group (EDG) acting as charge transfer donor or electron-withdrawing group (EWG) acting as charge transfer acceptor.99
1.7. Cross-Reactive Sensor Array
Selective sensors are effective for the detection of a specific analyte of interest, while they are not particularly useful for the analysis of complex mixtures of structurally similar analytes.
Thus, an alternative, combinatorial approach to multianalyte sensing has evolved, which utilizes a cross-reactive sensor architectures.100–102 These types of sensors are not selective toward one specific analyte but respond to various analytes with different degrees of selectivity and sensitivity towards each analyte.
Identification of analytes using the cross-reactive sensor array cannot be accomplished from the response of a single sensor. The cross-reactive sensor array should contain non-specific chemical sensors with a different complementary affinity towards various analyte.103,104 This provides a complex response pattern created upon interaction of each sensor in the array with analytes. Each sensor-analyte generates a distinct analyte-specific response pattern. Such systems are capable of generating distinguishable optical ‘fingerprints' for a variety of structurally similar compounds. The composite multivariate response obtained as fluorescence intensity read-out is then evaluated employing pattern recognition techniques allowing to interpret, identify and classify the analytes of interest.
The selection of distinct sensors to be used in an array is important. Each sensor has to possess a different degree of interaction with each analyte to achieve a high discriminatory 14 ability of the array. Such an array responds to the broad cross-section of analytes. Highly cross- reactive chemosensors may not generate enough information in response pattern (may have a low discriminatory power). The discriminatory power of the sensor array might be improved by employing partially selective sensors with different degree of sensitivity for each analyte.
Additionally, the discriminatory power of an array can be improved by increasing the number of detection modes, optical channels (combination of excitation/emission wavelengths), or the number of conditions such as different pH, buffers, and solvent polarity used in the array. This procedure can generate a large multi-dimensional-response data space and improved resolution of the array.105
Chapter II, second section will focus on the application of cross-reactive sensor array for quantitative analysis of a chiral a-hydroxycarboxylates. Chapter II, the second part, highlights a new approach in developing chiral fluorescent sensors in the array, where the chiral sensors are capable of discriminate enantiomeric excess in chiral guests. We show that this approach allows for monitoring enantiomeric excess of a-hydroxy acids. In chapter III, we will describe how the optical chemosensor arrays were tested for the ability of the array to detect nitroaliphatics such as nitromethane, nitroethane, and 1- and 2-nitropropane, ammonium nitrate, bases such as ammonia and triethylamine, acids such as hydrochloric and sulfuric acid.
1.8. High-Throughput Screening Assay and Pattern Recognition Techniques for
Multivariate Data Analysis
High-throughput screening (HTS) is a well-established process in drug discovery for pharmaceutical research and development (R&D).106–108 It utilizes screening of large chemical libraries using the automated robotic systems, miniaturized assays, and large-scale data analysis. 15
Identification of suitable candidates from combinatorial libraries with high potential and activity for further evaluation using HTS can increase efficiency and minimize the time required for the discovery process.109–111
The HTS must be designed in a way that the compounds with the desired biological/chemical effect display low variability (CV) and high signal-to-background (S/B) ratio so that false negatives and false positives are minimized.
Due to the high sensitivity of fluorescence-based methods, they are among the most promising detection technique used for HTS.112 The correct choice of excitation/emission wavelength is also one of the most important factors to consider for developing fluorescence assay in HTS. Usually, short excitation wavelengths (below 300 nm) should be avoided to minimize interference posed by most organic and biological compounds. In HTS assay the number of tested samples has increased dramatically, therefore, volumes miniaturization has been continuously optimized from 96-well plates to 384-microwell plates, and then 1536 and more to reduce the experimental cost. Chapter II, the second part, of this dissertation will be focused on the development of the HT fluorescence-based assay for detection of enantiomeric excess of a-hydroxy acids.
The response patterns associated with sensor array provide a multivariate dataset which is impossible to be interpreted by visual inspection or by using basic calibrations like a simple linear regression.113 Hence, chemometric pattern recognition protocols are routinely used to reduce the dimensionality of the obtained dataset into a lower dimensional space (dimensionality reduction) and present the resulting data in a graphical form for interpretation.113–117 There are many statistical analysis methods for pattern recognition. In this dissertation, only three common methods were used: an unsupervised method such as hierarchical clustering analysis (HCA) and 16 supervised methods including linear discriminant analysis (LDA) and support vector machine
(SVM) for linear regression analysis.118–120
Supervised and unsupervised algorithms are used to serve different purposes.121 The unsupervised methods, also known as clustering analyses, require just independent variable information such as sensor responses to be provided in order to classify and separate the dataset into groups of clusters based on similarities in the response data. On the other hand, supervised methods require dependent variable information such as analyte classes to be provided to generate classification model that can later be used to assign the unknown samples.122 Thus, while unsupervised pattern recognition methods are primarily used for “pattern discovery” in a dataset, the supervised algorithms are used for a classification of the data and prediction of unknown samples.
Linear discriminant analysis (LDA) is the most commonly used supervised pattern recognition method for reduction of dimensionality and classification of the multivariate data.118,123 In this method, the data obtained from the array experiment, in our case fluorescence intensities, and the analyte identities are used as input dataset. Discriminant functions (DF) are then calculated from these input data to maximize the separation between individual classes while minimizing the variation within the individual clusters.81,118 Performing LDA is possible if the number of analyzed experiments is significantly higher than the number of variables in a multidimensional dataset. The LDA algorithm first develops a model prediction with part of the dataset (training set) which is later on tested to see how well the model predicts the dataset
(model validation). A training set is a combination of responses of all the analytes, and this set is used to teach the algorithm. Since LDA is a supervised method, the classes (analyte identities) in the training set are known. The predictive power of an array can be determined by assigning an 17 unknown analyte to one of the classes based on the similarity of its response to the responses of the analytes in the training set.
The predictive power can be tested using cross-validation methods.124–126 The most common cross-validation method is leave-one-out (or jackknife) approach which removes only one sample at a time from the training set and considers it as a validation/unknown set.127 This algorithm is run repeatedly until all samples have been left out and classified. This way, the data of unknown identity can be assigned to one of the classes based on the similarity of its responses to the responses of the samples in the training set. Thus, the predictive power of an array can be determined. In my dissertation, chapter III, LDA is used for qualitative and semi-quantitative analyses to validate the capability of the sensor array to perform analyte recognition.
Hierarchical clustering analysis (HCA),128 is an unsupervised method of multivariate analysis, seeking classification of the analytes by clustering data points based on relative distances between all samples in the n-dimensional space and groups them in a hierarchical manner. Since HCA is a very sensitive method, it uses the entire dataset dimensionality to represent the clustering result in a two-dimensional pattern. The results of the clustering depend on the clustering metric used and the similarity measure applied to the input dataset. A Euclidean distance metric is the most commonly used distance metric in sensor array applications. It calculates the distance between two data points within n-dimensions. Also, there are several methods available to define the association between the calculated clusters.128,129 A number of studies use Ward’s (minimum variance) method,130 which takes into consideration the minimum amount of the variance between the samples and analytes to define a cluster. The graphical result of HCA is presented as a dendrogram showing the quantitative differences or similarities between the individual analytes in a hierarchical manner based on how different or similar the 18 response patterns are for each analyte and group. In Chapter III of this dissertation, HCA used to classify a set of analytes in a hierarchical manner.
Support vector machine (SVM), is a set of supervised methods that are based on a statistical learning theory that can be used for both, classification and regression analysis of the data.131–133
SVM can be used for quantitative analysis of the analytes of interest and also multianalyte samples. It is a reliable method, especially when dealing with datasets containing several distinct classes that cannot be easily separated by linear boundaries.132,134,135 The SVM seeks to obtain the optimal boundaries which separate the analyte classes by maximizing the distance between them.136,137 The transformation of the nonlinear data into an n-dimensional vector space where the classes can be separated linearly can be achieved by using kernel functions or similarity function. Based on this function, the SVM generates the regression (calibration) model from the training set. Based on the kernel function selected for the SVM regression algorithm, the root means square error of calibration (RMSEC) is used to attest to the quality of the created model.
Next, the model is validated using k-fold cross-validation described by the root mean square error of cross-validation (RMSECV).138 Finally, the predictive ability of the developed calibration model is validated using unknown data set which produces the root mean square error of prediction (RMSEP). The root means square deviation (RMSD) is a measure of the difference between values predicted by a regression model and the actual values that are being modeled.
In Chapter II of this dissertation, SVM was used for a linear regression analysis for quantitative estimation of enantiomeric excess of a-hydroxy acids.
19
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CHAPTER II. SENSING OF ENANTIOMERIC COMPOSITION USING
FLUORESCENCE PROBES
2.1. Abstract
Here, we present fluorescence probes capable of detection of enantiomeric excess (ee) of chiral carboxylic acids and -hydroxy acids, the structural units of many natural products and drug molecules. In this study, the chiral fluorescent sensors, [CuII(1R,2R)-N1,N2-bisquinolin-2-methyl- diphenyl-1,2-diamine]2+ and [CuII(1S,2S)- N1,N2-bisquinolin-2-methyl-diphenyl-1,2-diamine]2+ are synthesized and in conjunction with Coumarin 343 (C343) as an indicator shown to be useful in determination of enantiomeric compositions of carboxylic acids including non-steroidal anti- inflammatory drugs (NSAIDs). Also, chiral fluorescent sensors [ZnII(1R,2R)- N1,N2-bis(quinoline-
2-ylmethyl)-cyclohexane-1,2-diamine]2+, [ZnII(1S,2S)-N1,N2-bis(quinoline-2-ylmethyl)- cyclohexane-1,2-diamine]2+, and [CuII(1R,2R)-N1,N2-bis(quinoline-2-ylmethyl)-cyclohexane-1,2- diamine]2+ were synthesis and in conjunction with Coumarin 343 (C343) utilized for ee detection of -hydroxy acids. In the case of copper-based chiral sensors, we used enantioselective indicator displacement assays (eIDAs). The fluorescence of the Coumarin 343 (C343) quenched upon complexation with the chiral Cu(II)-based receptors, but its fluorescence is recovered in the presence of an analyte that binds to the receptor. The fluorimetric titration of the individual probes shows highly variable enantiomer-induced fluorescence signals. The Zn(II)-based fluorescence probes were utilized in a competitive sensor array using 384 well-plate and a fluorescence plate reader in high-throughput screening setting to investigate affinity of model towards -hydroxy acids. The approach allows for the accurate determination of the ee value with errors of just 2%.
This work opens a new avenue for the development of enantioselective supramolecular assays. 29
2.2. Introduction
The recognition of chirality and enantiomeric excess (ee) has received considerable attention in recent years due to the importance of chirality in biology,1–8 pharmaceutical chemistry,9–13 and asymmetric catalysis.14–25 In the field of chirality sensing, the discrimination of the absolute configuration and the determination of the enantiomeric purity are crucial to application of chiral compounds. The traditional methods of enantiomeric composition determination such as chiral- phase high performance liquid chromatography (HPLC)26–30, chiral gas chromatography (GC)31–
35, circular dichroism (CD)36–41 are labor intensive, require expensive instrumentation or may involve derivatization or use of chiral shift reagents (NMR)42–44. Optical spectroscopy such as circular dichroism, UV-Vis, and fluorescence−based ee determination is of particular interest because of the superior sensitivity, a short analysis time and the possibility to circumvent the expensive chromatographic separation.45 Also, optical methods allow for a potential high- throughput screening (HTS) method for determining ee.45–51
Fluorescence is an optical signaling technique used for the rapid determination of ee.45,52–61
Fluorescence techniques have many advantages, such as its sensitivity, less expensive instrumentation, and the ability to be used in rapid analysis.62,63 Fluorescence has various detection modes available, such as intensity, polarization, and anisotropy, all of which can be performed in a steady-state or time-dependent manner.64–66 Fluorescence enhancement and fluorescence quenching are the two most commonly used signaling modes in fluorescence intensity measurments.67,68 Several research groups have demonstrated the ability to perform enantioselective discrimination using fluorescence via the use of a range of binding interactions and scaffolds.50,51,69–78
30
2.3. Sensing of Enantiomeric Composition of Chiral Carboxylates Using Fluorescence
Probes
2.3.1. Introduction
Chiral carboxylic acids are the structural motifs present in many natural products and drug molecules.79–81 Thus, development of simple, high-throughput methods for the determination of enantiomeric excess in chiral carboxylates is important for the development of new chiral drugs and also natural product synthesis. Although, a significant research effort has been devoted to the detection of chiral carboxylates, only a limited number of enantioselective sensors for detection of chiral carboxylic acids have been reported.73,82–87 These include chiral macrocycles,69,83,88,89 dendrimers,90 oligomers,91,92 and others62,82,93–97 some of which require multiple synthetic steps to obtain the receptor.
Mei and Wolf72,82 reported a series of 1,8-diacridylnaphthalene fluorescent sensors for chiral hydrogen bond donors such as chiral carboxylic acids, amines and amino acids. The sensors are synthesized in six steps, followed by resolution on a chiral HPLC. The fluorescence quenching of sensors was observed upon binding to analytes. Here, the fluorescence quenching was used to develop calibration curve method used for determination of ee.
1 2
Figure 2.1. Structures of 1,8-bis(3,3’-(3,5-dimethylphenyl)-9,9’-diacridyl)naphthalene (1) and 1,8-bis(3,3’-(3,5-dimethylphenyl)-9,9’-diacridyl)naphthalene N,N’-dioxide (2) fluorescent sensors reported by Mei and Wolf.72,82 31
In 2015, Zhang et al.98 reported a chiral fluorescent sensor (Figure 2.2) used 1,10- phenanthroline moiety as a fluorescent signaling site and binding site, with optically active β- amino acids as a chiral barrier site. The chiral sensor has been used to conduct the enantioselective recognition of chiral mono and dicarboxylic acids derivatives such as tartaric acids and proline. L- enantiomers quench the fluorescence of the chiral fluorescent sensor more efficiently than D- enantiomers due to the absolute configuration of the β-amino acid.
Figure 2.2. Structure of the fluorescent chiral sensor designed by Zhang et al.98 for enantioselective recognition of chiral carboxylic acids.
In 2015, Akdeniz et al.99 have demonstrated simple phase transfer catalyst based on fluorescent
N(1)-alkylated cinchona salts can act as fluorescent sensors for determination of ee in chiral carboxylates (Figure 2.3). Some non-steroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, ketoprofen, and naproxen are pairs of enantiomers and have been tested to evaluate the potential application of the N(1)-alkylated cinchona salts as fluorescent sensors in sensing of ee in chiral drugs. These cinchona ammonium salts are shown to bind chiral carboxylic acids while displaying an increase in fluorescence intensity that can be utilized in determination of ee.
Also in 2016, Akdeniz et al.100 reported fluorescent macrocyclic sensors (Figure 2.4) for enantiomeric determination of carboxylates, such as enantiomers of ibuprofen, ketoprofen, 2- phenylpropanoate, mandelate, and phenylalanine. Depending on the structure of the sensor, the
32
R OH BF 9 4 R=OMe R=H C8 C9 N 8 1 N H S1 (Quinine) S2(Cinchonidine) S R S4 (Quinidine) S3(Cinchonine) R S Fluorophore
Figure 2.3. Structures of cinchona alkaloids quinine (S1), cinchonidine (S2), cinchonine (S3), quinidine (S4) based fluorescent sensors for detection of ee in chiral carboxylic acids.
presence of carboxylate yields fluorescence quenching or amplification. This information-rich signal was used to determine the identity of the analyte including the sense of chirality.
Chirabite-AR : R = H
S1 : R = - Me
R R OO OO S2 : R = - OMe HN NH
N N S3 : R = - NMe2
NH HN S4 : R = - NMe2 O O
NO2 Figure 2.4. Structures of fluorescent macrocyclic sensors for ee detection of chiral carboxylic acids reported by Akdeniz et al.100
Feng et al.101 reported chiral tetraphenylethylene (TPE) macrocycles bearing optical pure amine groups (Figure 2.5) for enantiomeric recognition of chiral acidic compounds and also α- amino acids by utilizing enantioselective aggregation and aggregation-induced emission (AIE) effect.
Chiral acidic compounds such as mandelic acid and ibuprofen, also α-amino acids such as glutamic acid, tyrosine, methionine, phenylalanine, proline, arginine, pyroglutamic acid, histidine, 33 and tryptophan have been tested to evaluate the potential of the chiral TPE macrocycle as a fluorescence chiral receptor in sensing ee in desired compounds.
Figure 2.5. Structures of chiral TPE macrocycles reported by Feng et al.101 for enantioselective recognition of chiral acidic compounds and α-amino acids.
In 2017, Wang et al.102 reported chiral fluorescence sensors (Figure 2.6) incorporating N‐Boc‐ protected alanine and acridine moieties. There was a slight change in fluorescence spectra of chiral sensors in the presence of carboxylate anions such as D‐ or L‐dibenzoyl tartrate, benzene lactate, phenylalanine, alanine, mandelate, malate, tartrate (as tetrabutylammonium salts) in acetonitrile, except for tartrate. The sensors displayed good enantioselective fluorescent sensing ability toward enantiomers of tartrate anions and formed 1: 1 complexes by multiple hydrogen bonding interactions.
Figure 2.6. Structures of chiral fluorescence sensors developed by Wang et al.102
34
Many of the receptors discussed above require multiple synthetic steps. In these receptors, the chromophoric unit is covalently attached to the receptors. In these types of receptors, the ability to signal the binding event is not known until after the synthesis of the receptor. This is unfavorable due to the required re-synthesis of the scaffold in cases where the signal or detection is not optimal.
The re-synthesis can be time-consuming, especially when multiple synthetic steps, purifications, or resolution, are necessary. Even after modification, the new system may not produce the desired signal, thereby needing further modification. To overcome these disadvantages, the use of enantioselective indicator displacement assays (eIDAs)103–105 has been implemented for determining ee. Because of the noncovalent nature of the indicator-receptor complex, various indicators with the same receptor system enabling a secondary tuning of selectivity.106 Also, the receptors used in eIDAs have generally simpler structures, which may be obtained in a few synthetic steps from commercially available starting material. Therefore, we used eIDAs concept for the determination of ee of chiral carboxylic acids in this project.
2.3.2. EIDA for Chiral Carboxylates
A number of colorimetric eIDAs were developed.72,103,107–113 However, to the best of our knowledge there has not been any eIDAs using fluorescence probes for detection of enantiomeric composition in chiral carboxylates. Figure 2.7 shows the principle of operation of the assay: An enantiomerically pure receptor is incubated with a fluorescent dye (indicator) that forms a non- covalent assembly with the receptor to yield a non-fluorescent receptor-dye complex. Upon addition of the analyte, in our case the chiral carboxylate drug, the indicator is displaced from the receptor and its fluorescence is regenerated.
35
ON OFF ON
Incubation Incubation
Chiral Dye Receptor-dye Analyte Receptor-analyte Dye Receptor (indicator) complex complex (indicator)
Figure 2.7. General operational principle of an indicator displacement assay.
The interaction between chiral receptor and chiral analytes results in formation of diastereomeric complexes that differ in stability. The stability of a complex is reflected in the different binding constant between the chiral receptor and enantiomeric analytes. This frequently results in a different fluorescence signal for each enantiomer, allowing for the determination of ee.
2.3.3. Chiral Sensors: Design and Synthesis
We synthesized two chiral receptors [CuII(1R,2R)-N1,N2-bisquinolin-2-methyl-diphenyl-1,2- diamine]2+−[CuRRL]2+−and [CuII(1S,2S)-N1,N2-bisquinolin-2-methyl-diphenyl-1,2- diamine]2+−[CuSSL]2+−featuring 2-quinoline carboxaldehyde and chiral 1,2- diphenylethylenediamines (Figure 2.8). We have chosen the tetradentate ligand 1,2-diphenyl-
N1,N2- bis(quinoline-2-ylmethyl)-ethane-1,2-diamine—RRL and SSL—for ease of synthesis. The ligand can be obtained as a pure enantiomer by reductive amination of enantiopure 1,2- diphenylethylene-diimine. Copper(II), which favors a square planar coordination geometry, allows for the introduction of an apical ligand—the indicator or the analyte—in a square-pyramidal geometry, and provides the necessary redox chemistry to quench the indicator’s fluorescence by 36 photoinduced electron transfer.114 We have selected coumarin 343 (C343) as the fluorescent indicator in this study since C343 showed affinity to these sensors.
[CuSSL]2+ [CuRRL]2+ Coumarin 343 (C343)
Figure 2.8. Sensors− [CuSSL]2+ and [CuRRL]2+−and indicator (C343) used in this study.
Commercially available 2-quinoline carboxaldehyde and chiral 1,2-diphenylethylenediamines were first used to make enantiopure 1,2-diphenylethylene-diimine (a Schiff base) in methanol as a solvent. Then, the enantiopure 1,2-diphenylethylene-diimine was reduced to enantiopure amine ligand 1,2-diphenyl-N1,N2- bis(quinoline-2-ylmethyl)-ethane-1,2-diamine. Finally, chiral sensors were synthesized by complexation between enantiopure ligand 1,2-diphenyl-N1,N2- bis(quinoline-
2-ylmethyl)-ethane-1,2-diamine and copper(II).
In this project, we used eIDAs concept for the determination of enantiomeric composition of chiral carboxylic acids including NSAIDs commonly used for pain relief and fever reduction. We have selected a series of carboxylate analytes that includes the enantiomers of 2-phenylpropanoate, lactate, mandelate, 3-phenyllactate, ibuprofen, naproxen and atorvastatin. For example, ibuprofen is commonly marketed as a racemic mixture, where both enantiomers display anti-inflammatory activity, whereas only (S)-naproxen is used as analgesic, because (R)-naproxen is hepatotoxic.79,115
It has been shown that the (R,R)-atorvastatin is the most active isomer at inhibiting cholesterol synthesis in the liver.106,116–118 Atorvastatin is a blockbuster drug used for the treatment of hypercholesterolemia119,120 and the prevention of cardiovascular diseases.121,122 Atorvastatin exists 37 in four stereoisomers, but commercialized is only the enantiopure (3R,5R)-7-[2-(4-fluorophenyl)-
3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid form.123,124
2-Phenylpropionic acidMandelic acid 3-Phenyllactic acid
Ibuprofen Naproxen Lactic acid Atorvastatin
Figure 2.9. Structures of the chiral analytes used in this study.
2.3.4. Materials and Methods
Starting materials and all reagents were obtained from commercial suppliers without further purification. Methanol was purchased from EMD and used by further treatment with activated 4Å molecular sieves. Acetonitrile (CAS:75-05-8) was purchased from VWR and used as received. 2-
Quinolinecarboxaldehyde (CAS:5470-96-2) was purchased from TCI America. Sodium cyanoborohydride (CAS:25895-60-7) was purchased from BeanTown Chemical. (1R,2R)- 1,2-
Diphenylethylenediamine (CAS:35132-20-8) and (1S,2S)- 1,2-diphenylethylenediamine
(CAS:29841-69-8) were purchased from Alfa Aesar. Copper(II) trifluoromethanesulfonate
(CAS:55804-65-4) was purchased from Aldrich. (R)-Naproxen (CAS:2397-41-1) was purchased from TRC Canada and (S)-naproxen (CAS:22204-53-1) was purchased from Cayman Chemical.
(R)-2-Phenylpropionic acid (CAS:7782-26-5) was purchased from Ark Pharma and (S)-2- 38 phenylpropionic acid (CAS:7782-24-3) was purchased from Chem-Impex international, Inc. (R)-
Mandelic acid (CAS:611-71-2) and (S)-mandelic acid (CAS:17199-29-0) were purchased from
Alfa Aesar. (R)-Ibuprofen (CAS:51146-57-7) was purchased from Enzo life science and (S)- ibuprofen (CAS:51146-56-6) was purchased from Aldrich. Atorvastatin calcium-(3R,5R)
(CAS:344423-98-9) and atorvastatin related compound E-(3S,5S) (CAS:1105067-88-6) were purchased from Sigma Aldrich. MES buffer aqueous solutions were prepared from 2-morpholin-
4-ylethanesulfonic acid (CAS: 4432-31-9) purchased from Amresco (Solon, OH). pH value of the buffer solution (pH = 6) was adjusted by addition of sodium hydroxide (6 M) and/or hydrochloric acid (6 M) using SevenMulti pH meter with glass electrode (both by Mettler Toledo, Toledo, OH).
Mass spectrometry measurement. Mass spectrometry was performed using a SHIMADZU
LCMS-2020 electrospray instrument (ESI), and SHIMADZU MALDI TOF-TOF Mass
Spectrometer AXIMA Performance.
UV-Vis measurements. Absorption spectra were acquired using Hitachi U-3010 double beam
UV-Vis spectrophotometer (Tokyo, Japan).
Nuclear magnetic resonance (NMR) measurements. 1H NMR and 13C NMR spectra were recorded on a Bruker Avance III 500 (500MHz) spectrometer in chloroform-d (CDCl3) at 25° C.
1H and 13C NMR chemical shifts were reported in ppm relative to either the residual solvent peak or TMS as an internal standard. Spectra were processed using the MestReNova software.
Fluorescence measurements. Steady-state fluorescence emission and excitation measurements were performed on Edinburgh single-photon counting spectrofluorimeter
FLSP920-stm (Edinburgh Instruments Ltd., Livingston, UK) at room temperature using quartz cuvette with a path length of 1 cm and with a right-angle detection. Optically dilute solutions used 39
for all photophysical experiments were prepared using MeCN:H2O (7/3 % v/v) at pH = 6 (MES =
50 mM).
Absolute quantum yield measurements. Absolute quantum yields were obtained upon excitation at absorption maxima using Hamamatsu Quantaurus-QY C11347-11 Absolute PLQY
Spectrometer equipped with 150 W Xe lamp and multichannel detector/CCD sensor (Hamamatsu,
Japan). Optically dilute solutions used for all photophysical experiments were prepared using
MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) solvents.
Spectroscopy. Proton NMR (1H-NMR) and carbon-13 NMR (13C-NMR) spectra were recorded on Bruker Avance III spectrometer at 500 MHz or 125 MHz, respectively at 25 °C. Proton and carbon NMR chemical shifts () are reported in parts per million (ppm) relative to residual solvent signals in CDCl3 ( = 7.26, 77.16). Coupling constants (J) are reported in hertz (Hz) and refer to apparent multiplicities. The following abbreviations are used for the multiplicities: s (singlet), d
(doublet), t (triplet), q (quartet), p (pentet), m (multiplet). Mass spectra (MS) were obtained on
Shimadzu AXIMA Performance MALDI-TOF mass spectrometer in reflectron mode using 1,8,9- trihydroxyanthracene (DIT) (CAS: 480-22-8) as a matrix. Solution of C343 and solution of chiral sensor-C343 complex were excited at 444 nm. The emission spectra were recorded between 450 nm and 600 nm. The emission was scanned in 1 nm step with a dwell time 0.20 sec under ambient condition. Scans were taken under laboratory temperature. Fluorescence guest and competitive titrations were performed in non-degassed aqueous MES buffer (50 = mM) : MeCN (3:7) at pH =
6 at laboratory temperature. Titration isotherms were constructed from changes in the fluorescence maximum at 493 nm.
40
2.3.5. Synthesis Protocols
Scheme 2.1. Synthesis of (1S,2S)- and (1R,2R)-diphenyl-N1,N2-bis(quinolin- 2ylmethylene)ethane-1,2-diimine (1).
The Schiff base 1 was synthesized according to a slightly modified literature procedure.125 2-
Quinolinecarboxaldehyde (222 mg, 1.41 mmol) was suspended in 10 mL of methanol and (1S,2S)- diphenylethylenediamine or (1R,2R)-diphenylethylenediamine (150 mg, 0.706 mmol) was added.
The mixture was stirred and refluxed for 12 hours. After this time the reaction mixture was cooled down to room temperature and the yellow precipitate was collected by filtration and washed with diethyl ether. The pale-yellow solid was dried in air. The resulting crude material was recrystallized from ethyl acetate to give 1 as a white solid. (232 mg, 0.473 mmol, yield: 67%).
(1S,2S)-diphenyl-N1,N2-bis(quinolin-2-ylmethylene)ethane-1,2-diimine (SSL): 1H NMR
(500MHz, CDCl3): = 8.41 (s, 2H), 8.04 (d, J = 8.5Hz, 2H), 7.99 (d, J = 8.5Hz, 2H), 7.75 (d, J =
7.9Hz, 2H), 7.68 (m, 6H), 7.64 (ddd, J = 8.3, 6.9, 1.3Hz, 2H), 7.48 (m, 2H), 7.34 (m, 6H), 5.48 (s,
13 2H) ppm. C NMR (125 MHz, CDCl3): δ 162.50, 160.55, 147.42, 136.21, 135.71, 130.70, 129.24,
129.04, 128.42, 128.40, 127.62, 127.47, 126.05, 121.81, 82.11 ppm. MALDI-TOF (m/z): 491.20
[M+H]+ 41
(1R,2R)-diphenyl-N1,N2-bis(quinolin-2-ylmethylene)ethane-1,2-diimine (RRL): 1H NMR
(500MHz, CDCl3): 8.40 (s, 2H), 8.04 (d, J = 8.5 Hz, 2H), 7.99 (d, J = 8.4 Hz, 2H), 7.75 (dd, J =
8.1, 1.1 Hz, 2H), 7.68 (m, 6H), 7.64 (ddd, J = 8.4, 6.9, 1.4 Hz, 2H), 7.48 (ddd, J = 6.9, 5.1, 1.1 Hz,
13 2H), 7.33 (m, 6H), 5.48 (s, 2H) ppm. C NMR (125 MHz, CDCl3): δ 162.49, 160.55, 147.42,
136.21, 135.71, 130.69, 129.24, 129.03, 128.41, 128.40, 127.61, 127.47, 126.04, 121.81, 82.11 ppm. MALDI-TOF (m/z): 491.30 [M+H]+
** ** N N NaBH3CN NH HN
N N N N MeOH, r.t.
1 2: 96 %
Scheme 2.2. Synthesis of (1S,2S)- and (1R,2R)-diphenyl-N1,N2-bis(quinolin-2-ylmethyl)ethane- 1,2-diamine (SSL or RRL).
We developed a procedure for reduction of Schiff base 1 as follow: The Schiff base 1 (200 mg,
0.408 mmol) was suspended in 40 mL of methanol then 0.4 mL acetic acid was added to the solution. Sodium cyanoborohydride (128 mg, 2.04 mmol) was added to the reaction mixture portion wise (32 mg x 4 times) and the solution was stirred under a nitrogen atmosphere for 12 hours at room temperature. As the reaction progressed, the solids start dissolving. After 12 hours the reaction mixture was stirred and refluxed for additional 1 hour then cooled down to room temperature. Hydrochloric acid (1 mL, 2 M) was added to remove unreacted reducing reagent. The pH of the solution was adjusted (pH = 8) by the addition of concentrated sodium hydroxide. The solvent was removed under reduced pressure and the residue was dissolved in 20 mL chloroform 42 and filtered. The solvent removed in vacuo to yield yellow-brownish oil. (193 mg, 0.391 mmol, yield: 96 %).
(1S,2S)-diphenyl-N1,N2-bis(quinolin-2-ylmethyl)ethane-1,2-diamine (SSL): 1H NMR (500MHz,
CDCl3) : 8.11 (d, J = 8.4 Hz, 2H), 8.06 (d, J = 8.5 Hz, 2H), 7.80 (d, J = 8.1 Hz, 2H), 7.70 (ddd,
J = 8.4, 5.5, 1.4 Hz, 2H), 7.51 (ddd, J = 8.1, 5.7, 1.1 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 7.41 (m,
4H), 7.34 (m, 4H), 7.27 (dt, J = 4.3, 1.7 Hz, 2H), 4.13 (s, 4H), 3.92 (s, 4H) ppm. 13C NMR (125
MHz, CDCl3): δ 162.36, 147.38, 140.51, 136.21, 129.22, 128.77, 128.57, 128.15, 127.51, 127.37,
126.68, 125.87, 120.72, 55.92, 54.05 ppm. MALDI-TOF (m/z): 495.30 [M+H]+
(1R,2R)-diphenyl-N1,N2-bis(quinolin-2-ylmethyl)ethane-1,2-diamine (RRL): 1H NMR
(500MHz, CDCl3) : 8.10 (d, J = 8.5 Hz, 2H), 8.02 (d, J = 8.5 Hz, 2H), 7.79 (m, 4H), 7.67 (ddd,
J = 8.4, 6.9, 1.5 Hz, 2H), 7.48 (m, 2H), 7.39 (dd, J = 7.9, 1.0 Hz, 4H), 7.28 (dd, J = 10.7, 4.4 Hz,
13 6H), 7.20 (m, 2H), 3.92 (s, 4H), 3.65 (s, 4H) ppm. C NMR (125 MHz, CDCl3): δ 160.15, 147.72,
140.13, 136.41, 129.46, 128.96, 128.42, 128.32, 127.57, 127.29, 127.02, 126.06, 120.60, 55.06,
53.67 ppm. MALDI-TOF (m/z): 495.30 [M+H]+
43
Scheme 2.3. Synthesis of [CuII(1S,2S)- and CuII(1R,2R)-diphenyl-N1,N2-bis(quinolin-2-ylmethyl) SS RR ethane-1,2-diamine] ([Cu L](OTf)2 or [Cu L](OTf)2).
The copper complexes were prepared according to a slightly modified literature procedure.125
In a 100 mL round-bottom flask, 2 (150 mg, 0.303 mmol) was dissolved in 30 mL of methanol under nitrogen. Copper (II) trifluoromethanesulfonate (110 mg, 0.303 mmol) was dissolved in 2 mL ultrapure water in a small vial. The two solutions were mixed and stirred under nitrogen for
12 hours. The solvent was removed in vacuo and the resulting green-bluish solid was dried under high vacuum for 2 hours. The product was washed with diethyl ether, filtered and dried under vacuum (166 mg, 0.297 mmol, yield: 98 %). MALDI-TOF (m/z): [M]+: 557.20
44 2.3.6. Chiral Sensors for Carboxylic Acids: NMR Spectra
TMS CDCl3 i h a h h g g i g b k c d j f e a b
c f
d e
j k
h i H2O a b g c f ed
1 1 2 Figure 2.10. H NMR (500 MHz) of (1S,2S)-diphenyl-N ,N -bis(quinolin-2-ylmethylene)ethane-1,2-diimine in CDCl3. 45
CDCl3
11,12
13 12 3 4 7 6 8 13 2 12 11 10 5 11 10 15 14 2 1 3 4 9 8 11,12 5 6 7
5,6 1 3 7,8 2 15 13 TMS 14 9 10 4
13 1 2 Figure 2.11. C NMR (125 MHz) of (1S,2S)-diphenyl-N ,N -bis(quinolin-2-ylmethylene)ethane-1,2-diimine in CDCl3. 46
j i h h CDCl3 h g d g g k b j a e K, I c f i a l b
c f d e
h TMS g i d CDCl3 b a e c f
1 1 2 Figure 2.12. H NMR (500 MHz) of (1S,2S)-diphenyl-N ,N -bis(quinolin-2-ylmethyl)ethane-1,2-diamine in CDCl3. 47
CDCl3 13 12 12 11 11 10 15 11 14 12 2 1 3 7 8 5 6 4 2 4 9 13 5 8
6 7
11 12 5 8 13 3 7 4 14 2 15 6 9 TMS 1 10
13 1 2 Figure 2.13. C NMR (125 MHz) of (1S,2S)-diphenyl-N ,N -bis(quinolin-2-ylmethyl)ethane-1,2-diamine in CDCl3. 48
TMS
CDCl3
j i h h g h a g k i j e a b g b c f d
c f d e
k j H2O ahe gi b c f d
1 1 2 Figure 2.14. H NMR (500 MHz) of (1R,2R)-diphenyl-N ,N -bis(quinolin-2-ylmethylene) ethane-1,2-diamine in CDCl3. 49
CDCl3
11,12 13 12 12 11 3 4 11 7 8 6 13 2 10 10 5 14 15 2 1 3 4 9 5 8 11,12 6 7
8 TMS 5,6 15 1 3 7 2 4 13 14 9 10
13 1 2 Figure 2.15. C NMR (125 MHz) of (1R,2R)-diphenyl-N ,N -bis(quinolin-2-ylmethylene) ethane-1,2-diamine in CDCl3. 50
i h CDCl3 h g TMS g k a h j f g a l b CDCl e d i b 3 c
c f d e
j K, I
a h f g b c e d i
1 1 2 Figure 2.16. H NMR (500 MHz) of (1R,2R)-diphenyl-N ,N -bis(quinolin-2-ylmethyl)ethane-1,2-diamine in CDCl3. 51
CDCl3
13 12 12 11 12 11 11 7 8 5 4 6 10 15 13 14 2 1 3 4 9 5 8
11 6 7 12 5 13 8 3 4 2 15 14 7 6 1 9 10 TMS
13 1 2 Figure 2.17. C NMR (125 MHz) of (1R,2R)-diphenyl-N ,N -bis(quinolin-2-ylmethyl)ethane-1,2-diamine in CDCl3. 52
2.3.7. Chiral Sensors for Carboxylic Acids: Mass Spectra
We confirmed the formation of chiral sensors using MALDI TOF. Figure 2.18 and Figure 2.19 show MALDI spectra of the (1R,2R)- and (1S,2S)-diphenyl-N1,N2-bisquinolin-2- ylmethylene)ethane-1,2-diimine (Schiff-base products), respectively. Figure 2.20 and Figure 2.21 show MALDI spectra of the (1R,2R)- and (1S,2S)-diphenyl-N1,N2-bis(quinolin-2- ylmethyl)ethane-1,2-diamine (chiral ligands), respectively. Finally, MALDI spectra of the
[CuRRL]2+ and [CuSSL]2+ are shown in Figure 2.22 and Figure 2.23, respectively. The MALDI spectra were recorded in positive detection mode.
DIT (1,8,9-trihydroxyanthracene) (in THF) has been used as the matrix in this experiment.
The samples for MALDI-TOF experiment were prepared as follow: Spotted 0.5 μL DIT matrix in the well of MALDI target plate and let dry for a few minutes. Then, 0.5 μL of acetonitrile solution containing the [CuRRL]2+ or [CuSSL]2+ = 10 mM was added on top of the dried matrix. Right after that 0.5 μL DIT matrix were added and let dry for 10 minutes.
53
Figure 2.18. (A) MALDI spectrum of (1R,2R)-diphenyl-N1,N2-bisquinolin-2- ylmethylene)ethane-1,2-diimine. (B) Calculated isotope pattern for C34H26N4.
Figure 2.19. (A) MALDI spectrum of (1S,2S)-diphenyl-N1,N2-bis(quinolin-2-ylmethylene) ethane-1,2-diimine. (B) Calculated isotope pattern for C34H26N4. 54
Figure 2.20. (A) MALDI spectrum of (1R,2R)-diphenyl-N1,N2-bis(quinolin-2-ylmethyl)ethane- 1,2-diamine. (B) Calculated isotope pattern for C34H30N4.
Figure 2.21. (A) MALDI spectrum of (1S,2S)-diphenyl-N1,N2-bis(quinolin-2-ylmethyl)ethane- 1,2-diamine. (B) Calculated isotope pattern for C34H30N4. 55
Figure 2.22. (A) MALDI spectrum of [CuII(1R,2R)-diphenyl-N1,N2-bis(quinolin-2- 2+ RR 2+ ylmethyl)ethane-1,2-diamine] , [Cu L] . (B) Calculated isotope pattern for C34H30CuN4.
Figure 2.23. (A) MALDI spectrum of [CuII(1S,2S)-diphenyl-N1,N2-bis(quinolin-2- 2+ SS 2+ ylmethyl)ethane-1,2-diamine] , [Cu L] . (B) Calculated isotope pattern for C34H30CuN4.
56
2.3.8. Complex Stoichiometry Determination: Job’s Plot
Binding stoichiometries of the complex formation in the solution were determined from fluorescence titration data using method of continuous variation (Job’s method).126 We investigated complex formation stoichiometries for chiral receptors [CuRRL]2+ and [CuSSL]2+ with
C343 (Figure 2.24). The Job’s plots obtained confirm the 1 : 1 binding stoichiometry for the association of C343 with the copper complexes.
A B
1.2x105
5 2+
2+ 1.0x10
L] L]
SS 4
RR 8.0x10
Cu
Cu
[
[
4 X
X 5.0x10
4.0x104 Intensity Intensity 0.0 0.0
0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 SS 2+ [CuRRL]2+ [Cu L]
Figure 2.24. Job’s plot for the determination of the stoichiometry of (A) [CuRRL]2+ and C343; (B) SS 2+ [Cu L] and C343 in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM).
The Job’s plot experiments127,128 were accomplished as follows: two stock solutions, one of
RR 2+ SS 2+ [C343] = 5 μM and the other of [Cu L] or [Cu L] = 10 mM, are prepared MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM). A set of working solutions is then obtained by mixing VR mL
RR 2+ SS 2+ (volume of the receptor) of the stock [Cu L] or [Cu L] solution with (VT - VR) mL of the stock C343 solution, where VT is a fixed total volume (4 μL) and VR is a variable, 0 ≤ VR ≤VT.
These working solutions were added to the 2 mL solvent of MeCN:H2O (7/3 % v/v) at pH = 6
(MES = 50 mM) in the cuvette. The fluorescence of these solutions is then measured at a fixed
RR 2+ SS 2+ wavelength (λex= 444 nm), and plotted as a function of mole fraction of [Cu L] or [Cu L] 57
(VR/VT). The position of maximum fluorescence on this plot, in relation to the mole-fraction axis, gives the stoichiometry of the complex.
2.3.9. Photophysical Properties
Photophysical properties of chiral sensors [CuSSL]2+ and [CuRRL]2+ (Table 2.1) were measured in MeCH3:H2O 7:3 (50 = mM, pH = 6) at room temperature and without previous degassing.
According to the result, as we expected the absolute quantum yields of chiral ligands decreased after complexation with Cu(II).
Table 2.1. Photophysical properties of chiral ligands SSL, and RRL and chiral sensors [CuSSL]2+, RR 2+ [Cu L] . Absorption (λA,max) and absolute quantum yields were acquired in MeCN:H2O 7:3 at pH = 6 (MES = 50 mM) solutions.
a A, max Chiral Sensors [nm] [%] SSL 315 0.7 RRL 315 0.7 [CuSSL)]2+ 315 0.5 [CuRRL)]2+ 315 0.5 aAbsolute quantum yields were determined upon excitation at wavelength indicated for solutions with optical density A = 0.1. All measurements were carried out in non-deoxygenated solutions.
2.3.10. Fluorescence Titrations: Indicator-Chiral Sensors
First, we investigated affinity of chiral sensors [CuRRL]2+ and [CuSSL]2+ for C343 using fluorescence titrations. The control experiment (Figure 2.25) confirms the identical photochemical properties of the resulting enantiomeric complexes by steady-state fluorescence. Titration of a solution of C343 in aqueous buffer (pH = 6, MES = 50mM) with a solution of [CuRRL]2+ or 58
[CuSSL]2+ shows an eight-fold decrease in the fluorescence of C343, centered around 493 nm, which is attributed to the quenching of C343’s excited state in the presence of the copper- complexes (Figure 2.25. A and C). The indicator (C343) fluorescence emission is quenched through an intramolecular photoinduced-electron-transfer (PET) process129,130 when the indicator is coordinated to the chiral copper-based sensors. The fitting of the titration isotherms affords comparable association constants for both complexes (Figure 2.25. B and D) and shows that the two chiral sensors have same binding affinity to achiral C343.
Steady-state fluorescence spectrometry revealed that both chiral sensors and
C343−[CuRRL•C343]+ and [CuSSL•C343]+−enantiomers showed an equal change in fluorescence response due to the structure of the achiral indicator C343. Besides, the value of the apparent binding constant is also almost equal (very similar) for both complexes. In other words, the configuration of the achiral indicator C343 influences the stability of the resulting complexes in a same way, and this stability is reflected in the fluorescence output.
The titrations were accomplished as follows: The C343 solutions (3 mL, 0.01 M) in MeCN :
H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) in cuvette were titrated with stock solution of chiral sensors (10 mg/mL in MeCN:H2O (7/3 % v/v) at pH = 6). The fluorescence spectrum was subsequently recorded and titration isotherms were plotted and binding constants calculated using
1:1 binding model by Newton’s iterative method.131 Figure 2.25 shows normalized emission
RR + SS + spectra of chiral sensors and C343—[Cu L•C343] and [Cu L•C343] —in MeCN : H2O (7/3 % v/v) at pH = 6 (MES = 50 mM).
59
A B 1.0 1.0
) 0.8
0.8 0 R2 = 0.998 - I f 493 nm I 0.6
0 0.6
/ (
/ I
I )
0 0.4 0.4
( I - I ( 0.2 0.2 4 –1 0.0 Ka = 7.4 10 M 0.0 475 500 525 550 575 600 0.0 2.0x10-4 4.0x10-4 6.0x10-4 8.0x10-4 RR 2+ / nm [Cu L ] / M
C D 1.0 1.0
) 0.8
0.8 0 2
- I R = 0.994
f 0 I 0.6
0.6 493 nm
/ I
/ (
I )
0 0.4 0.4
( I - ( 0.2 0.2 4 –1 0.0 Ka = 7.3 10 M 0.0 475 500 525 550 575 600 0.0 2.0x10-4 4.0x10-4 6.0x10-4 8.0x10-4 / nm [ CuSSL ]2+ / M
E 0.8
0.6
0
/ I
)
0 0.4
( I - 0.2
[CuRRL.C343]+ 0.0 [CuSSL.C343]+
0.0 2.0x10-4 4.0x10-4 6.0x10-4 8.0x10-4 [Cu(L)]2+ / M Figure 2.25. (A) Fluorescence titration spectra (B) and fluorescence titration isotherm of C343 (0.01 μM) upon the addition of an incremental amounts of [CuRRL]2+ (0–800 μM). (C) Fluorescence titration spectra (D) and fluorescence titration isotherm of C343 (0.01 μM) upon the SS 2+ addition of an incremental amounts of [Cu L] (0–800 μM) in MeCN : H2O (7/3 % v/v) at pH = RR 2+ 6 (MES = 50 mM) and λex= 444 nm. (E) The binding isotherms for chiral receptors [Cu L] and [CuSSL]2+ show the two chiral receptors have same binding affinity to the achiral C343. 60
2.3.11. Competitive Fluorescence Titrations
We performed the titration of the chemosensing ensemble—[CuRRL]2+ and [CuSSL]2+ = 212
μM, [C343] = 0.01 μM, MeCN : H2O 7:3, pH = 6, MES—with the corresponding carboxylic acid enantiomer and we observed recovery of C343’s fluorescence.
Diastereomeric complexes of [CuRRL]2+ or [CuSSL]2+ with carboxylate enantiomers show different fluorescence intensities, as well as different binding isotherms and apparent association constants (Ka). The addition of carboxylic acids resulted in a fluorescence amplification. This amplification is attributed to displacement of indicator C343 with enantiopure carboxylic acid analytes. Steady-state fluorescence spectrometry revealed that depending on the structure of the chiral guest, one diastereomer showed a stronger change in fluorescence response than the other.
Also, the value of the apparent binding constant is in most cases different for each analyte enantiomer. In other words, the stereochemistry of the chiral carboxylic acid influences the stability of the resulting complex, and this stability is reflected in the fluorescence output. Table
−1 2.2 shows the apparent association constants (Ka, M ) for selected analytes. Fluorescence titration results show that [CuRRL]2+ has a larger affinity toward the (S)-enantiomers of ibuprofen, naproxen and phenylpropanoic acid; however, it displays a larger affinity toward the (3R,5R)- atorvastatin.
[CuSSL]2+ shows a larger affinity toward the (R)-enantiomers of ibuprofen, naproxen and phenylpropanoic acid; but, it displays a larger affinity toward the atorvastatin related compound E
(enantiomer of the (3R,5R)- atorvastatin). Upon fitting of the titration isotherms using a 1:1 competition model, binding constants between the analytes and either [CuRRL]2+ or [CuSSL]2+ were determined.
We did more investigation by utilizing analytes with similar structures, namely α- hydroxycarboxylates. The behavior of enantiopure α-hydroxycarboxylates was unexpected. In 61 fact, we did not observe enantioselectivity upon titration of either [CuRRL]2+ or [CuSSL]2+ with lactate, mandelate and 3-phenyllactate. While the binding constants that were determined from the titration isotherms lie in the same affinity range as other analytes, the difference in binding constant between the enantiomers is negligible and is not statistically relevant. Because of the presence of the α-hydroxy group, we expected coordination of the carboxylate to the CuII center to be different.
The enthalpic penalty associated with the desolvation process of α-hydroxy acids might be enough to nullify any enthalpic gain from the formation of a stable diastereomer between the copper-complex and an enantiopure α-hydroxycarboxylate. Atorvastatin and its enantiomer also contain a hydroxyl group; however, the sensors still display enantioselectivity toward one enantiomer—[CuRRL]2+ for (3R,5R)-atorvastatin and [CuSSL]2+ for the atorvastatin related compound E (enantiomer of the (3R,5R)- atorvastatin). In contrast with α-hydroxycarboxylate, the position of hydroxyls group in atorvastatin—β- and δ- ⎯ precludes for the formation of 5- membered chelate rings, while 6- and 8-membered chelate rings are formed that presumably have a lower thermodynamic stability.
62
The following figures 2.26 – 2.34 show fluorescence titration spectra and isotherms of
[CuSSL•C343]+ in the presence of desired enantiopure carboxylic acids:
A B 6.0x105 4x105
4.0x105
3x105
a. u. / a.
I 5 2.0x105 2x10
Intensity Fluorescence 5 1x10 4 -1 0.0 Ka = 1.42 × 10 M 475 500525550575600 0.00 1.50x10-3 3.00x10-3 / nm [(R)-Phenylpropionic acid] / M
Figure 2.26. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of SS + [Cu L.C343] (212 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (R)-phenylpropionic acid. λex = 444 nm, [(R)-phenylpropionic acid] = 0-3 mM.
A B 6.0x105
3x105 4.0x105
5
a. u. / a.
2x10 I 2.0x105
Fluorescence Intensity Fluorescence 1x105 3 -1 0.0 Ka = 5.71 × 10 M 475 500 525 550 575 600 0.0 1.0x10-3 2.0x10-3 3.0x10-3 / nm [(S)-Phenylpropionic acid] / M
Figure 2.27. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of SS + [Cu L.C343] (212 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (S)-phenylpropionic acid. λex = 444 nm, [(S)-phenylpropionic acid] = 0-3 mM. 63
A B 6x105
5 5x105 4x10
4x105
5 5 3x10
a. u. a. 3x10
/
I 2x105 2x105 5
1x10 Intensity Fluorescence 4 -1 Ka = 1.04×10 M 0 475 500525550575600 0.0 2.0x10-3 4.0x10-3 / nm [(R)-Ibuprofen] / M
Figure 2.28. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of SS + [Cu L.C343] (212 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (R)-ibuprofen. λex = 444 nm, [(R)-ibuprofen] = 0-4 mM.
A B 6x105 6x105
5 5x10 5 5x10
4x105 4x105
3x105
a. u. / a.
I 3x105 2x105
5 5 2x10
1x10 Intensity Fluorescence 3 -1 Ka = 1.39 × 10 M 0 1x105 475 500 525 550 575 600 0.0 2.0x10-3 4.0x10-3 / nm [(S)-Ibuprofen] / M
Figure 2.29. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of SS + [Cu L.C343] (212 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (S)- ibuprofen. λex = 444 nm, [(S)- ibuprofen] = 0-3 mM.
64
A B 6x105 5x105
5x105 4x105 4x105
3x105 3x105
a. u. / a.
I 2x105 2x105 1x105 Intensity Fluorescence K = 9.96 × 103 M-1 a 0 1x105 475 500525550575600 0.0 1.0x10-3 2.0x10-3 3.0x10-3 / nm [(R)-Naproxen] / M
Figure 2.30. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of SS + [Cu L.C343] (212 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (R)-naproxen. λex = 444 nm, [(R)-naproxen] = 0-3 mM.
A B 6x105 4x105 5x105
4x105 3x105
3x105
a. u. / a.
I 5 2x10 2x105
5 1x10 Intensity Fluorescence 3 -1 Ka = 5.47 × 10 M 0 1x105 475 500 525 550 575 600 0.0 1.0x10-3 2.0x10-3 3.0x10-3 / nm [(S)-Naproxen] / M
Figure 2.31. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of SS + [Cu L.C343] (212 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (S)-naproxen. λex = 444 nm, [(S)-naproxen] = 0-3 mM. 65
A B 5x105
5 4x105 4x10
5 3x10 5
3x10
a. u. / a. 5 I 2x10 2x105 5 1x10 Fluorescence Intensity Fluorescence 5 3 -1 1x10 Ka = 5.07 × 10 M 0 475 500525550575600 0.00 1.20x10-3 2.40x10-3 / nm [(3R,5R)-Atorvastatin Calcium] / M
Figure 2.32. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of SS + [Cu L.C343] (212 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (3R,5R)-atorvastatin calcium. λex = 444 nm, [(3R,5R)-atorvastatin calcium] = 0-2.5 mM.
A 5 B 5x10 3x105
4x105
5 3x10 2x105
a. u. / a. 5 I 2x10
5 5 1x10 Intensity Fluorescence 1x10 4 -1 Ka = 1.35 × 10 M 0 475 500 525 550 575 600 0.00 1.20x10-3 2.40x10-3 / nm [Atorvastatin related compound E] / M
Figure 2.33. (A)Fluorescence titration spectra and (B) fluorescence titration isotherm of SS + [Cu L.C343] (212 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of atorvastatin related compound E. λex = 444 nm, [atorvastatin related compound E] = 0-2.5 mM. 5 5
6.0x10 6.0x10 5 6x10 A 5 5 B 5 4x10 5 6x10 A 5 3x10 6x10 B 4x10 5 5x10
5 3x10 5 5 4x105 5x10 5 5x10
5
5 5 3x10 2x10
5 4.0x10 2x10 4.0x10 5 3x10 5 5
4x10 5 4x10 Fluorescence Intensity Fluorescence 5 2x10 5
1x10 Intensity Fluorescence 5 2x10 Fluorescence Intensity Fluorescence Fluorescence Intensity Fluorescence 1x10
-3 -3 -3 -3 -3
a. u. / a. a. u. a.
/ 0.00 5 0.0 5 5
1.50x10 3.00x10 1.0x10 2.0x10 3.0x10 1x10 I
I -3 -3 a. u. a. 3x10 -3 -3 3x10 [(R)-Phenylpropionic acid] / M 0.0 [(S)-Phenylpropionic acid] / M 0.0 2.0x10 4.0x10
/ 2.0x10 4.0x10 a. u. a.
5 5 /
[(S)-Ibuprofen] / M 2.0x10 [(R)-Ibuprofen] / M I I 2.0x10 4 -1 3 -1 Ka = 1.42 × 10 M 5 Ka = 5.71 × 10 M 5 2x10 4 -1 2x10 3 -1 Ka = 1.04×10 M Ka = 1.39 × 10 M
1x105 1x105 0.0 0.0 66 475 500525550575600 0 475 500525550575600 0 / nm 475 500525550575600 / nm 475 500525550575600 / nm / nm
A C B C 3 3
5x105 5 5 5
0 4x10
A B 0 6x10 6x10 5 5 5 3x10 4x105 / I 2 5x10 5 2 5x10 4x10 / I ) A B
5
5 5 )
3x10 0
5x10 5x10 0
3x105
5 5 3x10 5 2x105
4x10 4x10 5 5 5 2x10 4x10 5 4x10 2x10 ( I - I 5 1
1 ( I - I Fluorescence Intensity Fluorescence
2x10 Intensity Fluorescence
5 5 5 Fluorescence Intensity Fluorescence 5 5 5 5 Intensity Fluorescence 1x10 1x10 3x10 5 1x10 3x10 -3 -3 -3 -3 -3 -3 3x10 0.0 1.0x10 2.0x10 3.0x10 SS 3x10 + 1x10 0.0 1.0x10 2.0x10 3.0x10SS +
SS + SS + a. u. a.
/ [Cu L . R-Phenylpropionic acid] a. u. a. [Cu L•(R)-phenylpropionic / acid] -3 -3 [Cu L . R-Ibuprofen]
[(R)-Naproxen] / M 0.00 1.20x10[(S)-Naproxen] 2.40x10 / M [Cu L•(R)-ibuprofen] I
SSSS I ++ SS + 0.00 -3 -3 a. u. a.
SS / + 1.20x10 2.40x10 a. u. a. [Cu / L . S-Phenylpropionic acid] [Cu L•(S)-phenylpropionic acid]
5 0 5 5 [(3R,5R)-Atorvastatin0 Calcium] / M [ Cu[Cu L L• (S). S-Ibuprofen]-ibuprofen]5 I 2x10 3 -1 I 2x10 2x10 3 -1 2x10 [(3S,5S)-Atorvastatin Calcium] / M Ka = 9.96 × 10 M Ka = 5.47 × 10 M -3 -3 -3 -3 -3 0.00 1.50x10 3.00x10 0.003 -1 1.50x10 3.00x10 4.50x10 4 -1 5 5 Ka = 5.07 × 10 M Ka = 1.35 × 10 M 5 1x10 5 1x10 [Phenylpropionic1x10 acid] / M [Ibuprofen]1x10 / M
0 0 475 500 525 550 575 600 0 475 500525550575600 0 / nm 475 500525550575600 / nm 475 500525550575600 / nm / nm C C D3 C
2 0
0 2
/ I
/ I
)
)
0 0 1
1
( I - I ( I - I
SS + SS [CuSS L . (3R,5R)-Atorvastatin Calcium]+ [[CuCuSSL L• (R). R-Naproxen]-naproxen]+ [Cu L•(3R,5R)-atorvastatin calcium] SSSS + + SSSS + [Cu [Cu L•atorvastatin L . (3S,5S)-Atorvastatinrelated compound Calcium] E] 0 [[CuCu L L• (S). S-Naproxen]-naproxen] 0
0.0 1.0x10-3 2.0x10-3 3.0x10-3 0.00 1.20x10-3 2.40x10-3 [Naproxen] / M [Atorvastatin] / M
Figure 2.34. Overlaid binding isotherms for two corresponding enantiomers based on the change in fluorescence intensity at the maximum wavelength of [CuSSL.C343]+ (212 μM) show changes in fluorescence intensity upon the addition of incremental amounts of (R)- or (S)-enantiomer (A) phenylpropionic acid (B) ibuprofen(C) naproxen (D) atorvastatin in MeCN:H2O 7:3 at pH = 6 (MES = 50 mM). λex = 444 nm.
67
The following figures 2.35 – 2.44 show fluorescence titration spectra and isotherms of
[CuRRL•C343]+ in the presence of desired enantiopure carboxylic acids:
A B 6.0x105 4x105
5
4.0x10 3x105
a. u. / a.
I 5 5 2x10 2.0x10
Fluorescence Intensity Fluorescence 3 -1 1x105 Ka = 5.89 × 10 M 0.0 475 500 525 550 575 600 0.00 1.50x10-3 3.00x10-3 / nm [(R)-Phenylpropionic acid] / M
Figure 2.35. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of RR + [Cu L.C343] (212 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (R)-phenylpropionic acid. λex = 444 nm, [(R)-phenylpropionic acid] = 0-3 mM.
A B 5 6.0x105 5x10
4x105 4.0x105
3x105
a. u. / a.
I 5 2.0x10 2x105
Fluorescence Intensity Fluorescence 4 -1 5 K = 1.44 × 10 M 1x10 a 0.0 0.00 1.50x10-3 3.00x10-3 475 500 525 550 575 600 / nm [(S)-Phenylpropionic acid] / M
Figure 2.36. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of RR + [Cu L.C343] (212 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (S)-phenylpropionic acid. λex = 444 nm, [(S)-phenylpropionic acid] = 0-3 mM. 68
A B 6x105 5x105 5x105
5 4x105 4x10
5
3x10 5 a. u. a.
/ 3x10 I 2x105 2x105 1x105
Fluorescence Intensity Fluorescence 3 -1 Ka = 1.40 × 10 M 0 1x105 475 500 525 550 575 600 0.0 2.0x10-3 4.0x10-3 / nm [(R)-Ibuprofen] / M
Figure 2.37. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of RR + [Cu L.C343] (212 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (R)-ibuprofen. λex = 444 nm, [(R)-ibuprofen] = 0-4 mM.
A B 6x105
5 5x105 4x10
4x105 3x105
3x105
a. u. / a. I 2x105 2x105 5 1x10 Intensity Fluorescence 4 -1 Ka = 1.02 × 10 M 0 475 500 525 550 575 600 0.0 2.0x10-3 4.0x10-3 / nm [(S)-Ibuprofen] / M
Figure 2.38. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of RR + [Cu L.C343] (212 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (S)-ibuprofen. λex = 444 nm, [(S)-ibuprofen] = 0-4 mM. 69
A B 6x105 4x105 5x105
4x105 3x105
3x105
a. u. / a.
I 5 2x10 2x105
1x105 Intensity Fluorescence 3 -1 Ka = 5.72 × 10 M 0 1x105 475 500525550575600 0.0 -3 -3 -3 1.0x10 2.0x10 3.0x10 / nm [(R)-Naproxen] / M
Figure 2.39. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of RR + [Cu L.C343] (212 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (R)-naproxen. λex = 444 nm, [(R)-naproxen] = 0-3 mM.
A B 5 6x105 5x10
5x105 4x105 4x105
5
3x105 3x10
a. u. / a.
I 2x105 2x105 5
1x10 Intensity Fluorescence 3 -1 Ka = 9.89 × 10 M 0 1x105 0.0 -3 -3 -3 475 500 525 550 575 600 1.0x10 2.0x10 3.0x10 / nm [(S)-Naproxen] / M
Figure 2.40. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of RR + [Cu L.C343] (212 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (S)-naproxen. λex = 444 nm, [(S)-naproxen] = 0-3 mM. 70
A B 5x105 3x105 4x105
3x105
2x105
5
a. u. / a.
I 2x10
5
1x10 Intensity Fluorescence 1x105 4 -1 Ka = 1.43 × 10 M 0 475 500525550575600 0.00 1.20x10-3 2.40x10-3
/ nm [(3R,5R)-Atorvastatin Calcium] / M
Figure 2.41. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of RR + [Cu L.C343] (212 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (3R,5R)-atorvastatin calcium. λex = 444 nm, [(3R,5R)-atorvastatin calcium] = 0-2.5 mM.
A B 5x105
5 4x10 3x105
3x105
5
2x10
a. u. / a. 5 I 2x10
5 1x10 5
Fluorescence Intensity Fluorescence 1x10 3 -1 Ka = 5.05 × 10 M 0 0.00 1.20x10-3 2.40x10-3 475 500 525 550 575 600 / nm [Atorvastatin related compound E] / M
Figure 2.42. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of RR + [Cu L.C343] (212 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of atorvastatin related compound E. λex = 444 nm, [atorvastatin related compound E] = 0-2.5 mM.
5x105 5 4x10 5 5 5 6x10 A 5 5 6x10 5x10 5 6.0x10 6.0x10 5 A B 4x10 B 4x10 5 3x10 5 4x105 5 5x10 5 5x10
3x10 5
3x10
5 5 3x10 2x10 5 2x105 5 5 4x10 5 4x10 5 5 4.0x10 Intensity Fluorescence 4.0x10 2x10
2x10 Intensity Fluorescence
5 5 Intensity Fluorescence
1x10 Intensity Fluorescence 1x10 5 5 0.00 -3 -3 1x105 0.00 -3 -3 1.50x10 3.00x10 3x10 1.50x10-3 3.00x10-3 3x10
0.0 2.0x10 4.0x10 0.0 2.0x10-3 4.0x10-3 a. u. a.
[(S)-Phenylpropionic acid] / M / a. u. a.
[(R)-Phenylpropionic acid] / M /
a. u. / a.
[(R)-Ibuprofen] / M I a. u. a.
/ [(S)-Ibuprofen] / M
I
I 3 -1 5 I 4 -1 5 5 5 2.0x10 Ka = 5.89 × 10 M 2x10 2.0x10 Ka = 1.44 × 103 M-1 2x10 4 -1 Ka = 1.40 × 10 M Ka = 1.02 × 10 M
1x105 1x105 71 0.0 0 0.0 0 475 500525550575600 475 500525550575600475 500525550575600 475 500525550575600 / nm / nm / nm / nm
A C B 3 C
5x105 4x105 3 5 5 A B 6x10 6x10 5 5 4x10 5 5 5x10 5x10
0 5 3x10 A 3x10 0 2 B 5 5 5 3x105 3x10
5x10 2 5x10
/ I / I
) )
5 5 5 0 2x10 4x10 0 5 4x10
5 5 5 2x10 5
Fluorescence Intensity Fluorescence 2x10 2x10 4x10
4x10 Intensity Fluorescence 1 5 1x105 1x10 1 5 -3 -3 -3 5 ( I - I -3 -3 -3 ( I - I 0.0
5 0.0 1.0x10 2.0x10 3.0x103x10 5 1.0x10 2.0x10 3.0x10 3x10 5 Fluorescence Intensity Fluorescence 3x10 3x10 Intensity Fluorescence 1x10 [(R)_Naproxen] / M 5 [(S)-Naproxen] / M
1x10
a. u. / a.
a. u. / a.
RR I + RR + 0.00 -3 -3
a. u. / a. I -3 -3 RR + 1.20x10 2.40x10
5 [CuRR L5 . R-Phenylpropionic5 acid]+ 0.00 1.20x10 2.40x10 [ Cu[Cu L L•(R) . R-Ibuprofen]-ibuprofen] 5
I a. u. a. 3 / [Cu-1 L•(R)-phenylpropionic acid] 3 -1 2x10 2x10 K = 9.89 ×RRRR10 M + + [(3S,5S)-Atorvastatin Calcium] / M Ka = 5.72 × 10 I M2x10RR + [(3R,5R)-Atorvastatin Calcium] / aM 2x10 0 [ Cu[CuRRL L•(S) . S-Phenylpropionic-phenylpropionic acid acid]]+ 0 [ Cu[Cu L L•(S) . S-Ibuprofen]-ibuprofen] 4 -1 3 -1 5 -3 5 K-3a = 1.43 × 10 M Ka = 5.05 × 10 M 1x10 0.00 5 1x10 -3 5 -3 1x10 1.50x10 3.00x10 0.0 2.0x10 1x10 4.0x10 [Phenylpropionic acid] / M [Ibuprofen] / M 0 0 475 500525550575600 0 475 500525550575600 0 / nm 475 500525550575600 / nm 475 500525550575600 / nm / nm C C D 3 C
2 0
0 2
/ I / I
) )
0 0 1 1
( I - I ( I - I
RR + [CuRR L . R-Naproxen]+ RR + [Cu L•(R)-naproxen] [[CuCuRRL L• (3R,5R). (3R,5R)-Atorvastatin-atorvastatin calcium] Calcium]+ RRRR + + [Cu L•(S)-naproxen] RR + 0 [Cu L . S-Naproxen] 0 [[CuCuRRL•atorvastatin L . (3S,5S)-Atorvastatinrelated compound Calcium] E]+
-3 -3 -3 0.0 1.0x10 2.0x10 3.0x10 0.00 1.20x10-3 2.40x10-3 [Naproxen] / M [Atorvastatin] / M
Figure 2.43. Overlaid binding isotherms for two corresponding enantiomers based on the change in fluorescence intensity at the maximum wavelength of [CuRRL.C343]+ (212 μM) show changes in fluorescence intensity upon the addition of incremental amounts of (R)- or (S)-enantiomer (A) phenylpropionic acid (B) ibuprofen(C) naproxen (D) atorvastatin in MeCN:H2O 7:3 at pH = 6 (MES = 50 mM). λex = 444 nm.
72
A B A B A B 3 A 3 B 3 A 3 B 3
3 3 3
0
0
0 0
2 2 2
/ I
/ I 0
0 2
/ I
/ I
)
)
0
0
)
)
2
0
0 0
2 0 2 / I
/ I 2
/ I
/ I
)
)
)
)
0
0 0 1 RRRR + + 10 RR +
1 RR + [ Cu[Cu L •L(R) . R-Phenylpropionic-phenylpropionic acid acid]] RR + [Cu L . R-Phenylpropionic acid] ( I - I
( I - I 1 ( I - I [Cu L . R-Ibuprofen] [CuRR L R-Ibuprofen]+ ( I - I RRRR + + [Cu L•(R) . -ibuprofen] RR + RR + 1 [ Cu[CuRRL •L(S) . S-Phenylpropionic-phenylpropionic acid acid]] + [Cu L . S-Phenylpropionic acid] [Cu L R-Phenylpropionic acid] RRRR + + 1 RR +
1 [CuRR L . S-Ibuprofen]+ SS . + [Cu [Cu L •L(S) . S-Ibuprofen]-ibuprofen] ( I - I
[Cu L . R-Ibuprofen] ( I - I SS + 1 RR + [CuSS L . R-Phenylpropionic acid]+ ( I - I SS + [ Cu[CuRRL •L(R) . R-Phenylpropionic-phenylpropionic acid] acid]+ [CuSS L . R-Ibuprofen]+ ( I - I [Cu L . R-Phenylpropionic acid] [CuRR L R-Ibuprofen]+ [Cu L . S-Phenylpropionic acid] SS + RR + . SS + [Cu [Cu L •L(R) . R-Ibuprofen]-ibuprofen] [CuSS L S-Phenylpropionic acid]+ [Cu L . S-Ibuprofen] 0 SS + RR + 0 . ss + [ Cu[CuSSL •L(S) . S-Phenylpropionic-phenylpropionic acid acid]] + [Cuss L . S-Ibuprofen]+ [Cu L . S-Phenylpropionic acid] [CuSS L . S-Ibuprofen]+ [Cu L . R-Phenylpropionic acid] [CuSS L . S-Ibuprofen]+ SS + 0 0 [Cu LSS•(S)-ibuprofen] + [Cu L . R-Phenylpropionic acid] [Cu L . R-Ibuprofen] SS + [Cu L . R-Ibuprofen] SS + ss + 0 [Cu L . S-Phenylpropionic acid] ss + 0 [Cu L . S-Phenylpropionic acid] 0 [Cu -3 L . S-Ibuprofen]-3 -3 -3 -3 -3 -3 -3 -3 -3 -3 -3 0.00 1.50x10 3.00x10 4.50x10 0.0 1.0x10 2.0x10 3.0x10 0 0.00 1.50x10 [Cu L3.00x10 . S-Ibuprofen]4.50x10 0.0 1.0x10 2.0x10 3.0x10
-3 -3 -3 [Phenylpropionic-3 acid]-3 / M -3 -3 -3 -3 [Phenylpropionic-3 acid]-3 / M -3 0.00 1.50x10[Ibuprofen]3.00x10 / M 4.50x10 0.0 1.0x10 2.0x10 3.0x10 0.00 1.50x10[Ibuprofen]3.00x10 / M 4.50x10 0.0 1.0x10 2.0x10 3.0x10 [Ibuprofen] / M [Phenylpropionic acid] / M [Ibuprofen] / M [Phenylpropionic acid] / M 3 C D 3 C D 3 3 C C D D C D
2 2 0
2 0 0 2 0
2 2
/ I
/ I
/ I
/ I
)
) 0
2 0 0
2 0
)
)
0
0
0
0
/ I
/ I
/ I
/ I
)
) )
) 1
1 1 0
0 RR + 0 1 RR + 0
RR + [Cu L . R-Naproxen] ( I - I
RR + [Cu L . R-Naproxen] ( I - I ( I - I ( I - I [Cu L . (3R,5R)-Atorvastatin Calcium] RR + [Cu L . (3R,5R)-Atorvastatin Calcium] 1 RR + RR + 1 [Cu L . S-Naproxen] 1 [CuRR L . S-Naproxen]+ 1 RR + [CuRR L R-Naproxen]+ [Cu L . (3S,5S)-Atorvastatin Calcium] SS RR + +
[CuRR L . (3S,5S)-Atorvastatin Calcium]+ [Cu SSL•(R). -naproxen] + RR + [Cu [Cu L . LR-Naproxen] . R-Naproxen]
( I - I
( I - I ( I - I SS + ( I - I [Cu L . (3R,5R)-Atorvastatin Calcium] [CuRR L . R-Naproxen]+ [CuRR L . (3R,5R)-Atorvastatin Calcium]+ RR + SS + [CuRR L S-Naproxen]+ [Cu[Cu LL •. (3R,5R)(3R,5R)-Atorvastatin-atorvastatin calcium] Calcium] SS + [CuRR L . (3R,5R)-Atorvastatin Calcium]+ [Cu SSL•(S). -naproxen] + RR + [Cu [Cu L . LS-Naproxen] . S-Naproxen] SSRR + + [Cu SS L . (3S,5S)-Atorvastatin Calcium] + [CuSS L . S-Naproxen]+ 0 [Cu L . (3S,5S)-Atorvastatin Calcium] 0 SS + 0 SS + [Cu[Cu LL•atorvastatin . (3S,5S)-Atorvastatinrelated compound Calcium] E] 0 [CuSS L . (3S,5S)-Atorvastatin Calcium]+ [ Cu[Cu L •L(R) . R-Naproxen]-naproxen] SS + [Cu L . R-Naproxen] [Cu L . (3R,5R)-Atorvastatin Calcium] SS + [Cu[CuSSL L•(3R,5R) . (3R,5R)-Atorvastatin-atorvastatin calcium] Calcium]+ SS + SS + [Cu L S-Naproxen] SS + [ Cu[Cu L •L(S) . -S-Naproxen]naproxen] SS -3 + -3 -3 . -3 -3 0 -3 -3 0 -3 -3 -3 0 0.00 SS + 0 0.0 0.00 [Cu L . (3S,5S)-Atorvastatin1.20x10 Calcium]2.40x10 0.0 1.0x10 2.0x10 3.0x10 [Cu[Cu L•atorvastatin L 1.20x10. (3S,5S)-Atorvastatinrelated compound Calcium]2.40x10 E] 1.0x10 2.0x10 3.0x10 [Atorvastatin] / M [Atorvastatin]-3 / M -3 -3 -3 -3 -3 -3 [Naproxen]-3 / M -3 -3 0.00 1.20x10 2.40x10 0.0 1.0x10[Naproxen]2.0x10 / M 3.0x10 0.00 1.20x10 2.40x10 0.0 1.0x10 2.0x10 3.0x10 [Atorvastatin] / M [Naproxen] / M [Atorvastatin] / M [Naproxen] / M
Figure 2.44. Overlaid binding isotherms based on the change in fluorescence intensity at the maximum wavelength of [CuSSL.C343]+ and [CuRRL.C343]+ (212 μM) and show changes in fluorescence intensity upon the addition of incremental amounts of (R)- or (S)-enantiomer of (A) phenylpropionic acid (B) ibuprofen(C) naproxen (D) atorvastatin in MeCN:H2O 7:3 at pH = 6 (MES = 50 mM). λex = 444 nm.
73
Figure 2.44 shows the overlaid isotherms for both enantiomers of phenylpropionic acid, ibuprofen, naproxen, and atorvastatin with chiral receptors [CuSSL]2+ and [CuRRL]2+. The formation of [CuSSL•(R)-enantiomer]+ and [CuRRL•(S)-enantiomer]+ diastereomeric complexes exhibit very similar isotherms and consequently, almost equal binding constants. [CuSSL•(S)-enantiomer]+ and
[CuRRL•(R)-enantiomer]+ diastereomeric complexes show also very similar isotherms and binding constants but different from the [CuSSL•(R)-enantiomer]+ and [CuRRL•(S)-enantiomer]+ diastereomeric complexes, confirming the enantiomeric cross-reactivity.
Fluorescence titration spectra and isotherms of [CuSSL•C343]+ and [CuRRL•C343]+ in the presence of (R)- or (S)-enantiomers of mandelic acid (α-hydroxycarboxylic acid) are shown in
Figure 2.45 and Figure 2.46, respectively.
According the fluorescence titration spectra and binding isotherms in Figure 2.45 and Figure
2.46, there is not any enantioselectivity upon titration of either [CuSSL•C343]+ or [CuRRL•C343]+ with mandelate. While the binding constants that were determined from the titration isotherms lie in the same affinity range as other analytes, the difference in binding constant is negligible and is not statistically relevant. Because of the presence of the α-hydroxy group, we expect coordination of the carboxylate to the CuII center to be different.
The Ka values in Table 2.2 suggest that in the cases where two analytes elicit similar signals,
SS + RR + upon fluorescence titration of either [Cu L•C343] or [Cu L•C343] the Ka values are different, thus enabling a reliable recognition. 74
A B 5 6x105 5x10
5x105 4x105 4x105
5 5 3x10
3x10
a. u. / a.
I 5 2x10 5 2x10
1x105 Intensity Fluorescence 4 -1 1x105 Ka = 1.12 × 10 M 0 475 500 525 550 575 600 0.0 1.0x10-3 2.0x10-3 / nm [(R)-Mandelic acid] / M
C D 6x105 5x105 5x105 4x105 4x105
3x105 3x105
a. u. / a.
I
2x105 2x105
5
1x10 Intensity Fluorescence 4 -1 5 Ka = 0.95 × 10 M 1x10 0 475 500 525 550 575 600 0.0 1.0x10-3 2.0x10-3 / nm [(S)-Mandelic acid] / M
E 3
0 2
/ I
)
0
1 ( I -
II (R)-Mandelic[CuSSL•(R) -acid_Cumandelic((S,S)-L)acid]+ II 0 (S)-Mandelic[CuSSL•(S) -acid_Cumandelic((S,S)-L)acid]+
0.0 1.0x10-3 2.0x10-3 [Mandelic acid] / M
Figure 2.45. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [CuSSL•C343]+ (212 μM) upon the addition of an incremental amounts of (R)-mandelic acid. (C) fluorescence titration spectra and (D) fluorescence titration isotherm of [CuSSL•C343]+ (212 μM) upon the addition of an incremental amounts of (S)-mandelic acid in MeCN:H2O (7/3% v/v) at pH = 6 (MES = 50 mM) λex = 444 nm, [(S)-, (R)-mandelic acid] = 0-2 mM. (E) overlaid isotherms based on the change in fluorescence intensity at the maximum wavelength of [CuSSL•C343]+ (212 μM) and show the same changes in fluorescence intensity upon the addition of incremental amounts of (R)- and (S)-mandelic acid. 75
A B5 6x105 5x10
5x105 4x105 4x105
5
3x105 3x10
a. u. / a.
I 2x105 2x105 1x105 Fluorescence Intensity Fluorescence 4 -1 5 Ka = 1.11 × 10 M 0 1x10 475 500 525 550 575 600 0.0 1.0x10-3 2.0x10-3 / nm [(R)-Mandelic acid] / M
C 5 D 6x10 5x105
5x105 4x105 4x105
5
3x105 3x10
a. u. / a.
I 2x105 2x105
1x105 Intensity Fluorescence 4 -1 1x105 Ka = 1.14 × 10 M 0 475 500 525 550 575 600 0.0 1.0x10-3 2.0x10-3 / nm [(S)-Mandelic acid] / M
E 3 F 3 0
0 2 2 / I
/ I II
)
)
(R)-MandelicRR acid_Cu ((R,R)-L)+
0 [Cu L•(R)-mandelic acid] 0 II (S)-Mandelic[CuRRL•(S)-mandelic acid_Cu acid]((R,R)-L)+ II 1 1 (R)-Mandelic[CuSSL•(R)-mandelic acid_Cu acid]((S,S)-L)+ II
SS + ( I - ( I - (S)-Mandelic[Cu L•(S)-mandelic acid_Cu acid]((S,S)-L)
RR + [Cu (R)-MandelicL•(R)-mandelic acid_Cu acid]II((R,R)-L) RR II + 0 [Cu (S)-MandelicL•(S)-mandelic acid_Cu acid]((R,R)-L) 0
0.0 1.0x10-3 2.0x10-3 0.0 1.0x10-3 2.0x10-3 [Mandelic acid] / M [Mandelic acid] / M
Figure 2.46. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [CuRRL•C343]+ (212 μM) upon the addition of an incremental amounts of (R)-mandelic acid. (C) fluorescence titration spectra and (D) fluorescence titration isotherm of [CuRRL•C343]+ (212 μM) upon the addition of an incremental amounts of (S)-mandelic acid in MeCN:H2O (7/3% v/v) at pH = 6 (MES = 50 mM) λex = 444 nm, [(S)- and (R)-mandelic acid] = 0-2 mM. Overlaid isotherms based on the change in fluorescence intensity at the maximum wavelength of (E) [CuRRL.C343]+ (F) [CuSSL•C343]+ and [CuRRL•C343]+ (212 μM) and show the same changes in fluorescence intensity upon the addition of incremental amounts of (R)- and (S)-mandelic acid. 76
-1 RR 2+ SS 2+ Table 2.2. The association constants (Ka, M ) corresponding to [Cu L] and [Cu L] chiral sensors with selected chiral carboxylic acids and -hydroxy acids.
[CuRRL]2+ [CuSSL]2+ Chiral Guests -1 * -1 * Ka (M ) Ka (M )
(R)-Ibuprofen 1.40 × 103 1.04 × 104
4 3 (S)-Ibuprofen 1.02 × 10 1.39 × 10 (R)-Naproxen 5.72 × 103 9.96 × 103 (S)-Naproxen 9.89 × 103 5.47 × 103
(3R,5R)-Atorvastatin Calcium 5.05 × 103 1.35 × 104
4 3 Atorvastatin related compound E 1.43 × 10 5.07 × 10 (R)-Phenylpropionate 5.89 × 103 1.42 × 104 (S)-Phenylpropionic acid 1.44 ×104 5.71 × 103
(R)-Mandelic acid 1.11 × 104 1.12 × 104
4 4 (S)-Mandelic acid 1.14 × 10 0.95 × 10 (R)-Lactic acid 1.66 × 104 1.61 × 104 (S)-Lactic acid 1.68 ×104 1.65 × 104
(R)-3-Phenyllactic acid 8.20 × 104 8.37 × 104 4 4 (S)-3-Phenyllactic acid 8.13 × 10 8.03 × 10
* All titrations were performed in acetonitrile/water (7/3 v/v) at pH = 6 (MES = 50mM). The Kas were calculated based on the change in fluorescence intensity (λEM: 444 nm) upon the addition of each chiral guests. The association constants were calculated using the non-linear least-square fitting; errors of the fitting were < 20%.
2.3.12. Determination of Binding Affinities: Indicator Titrations
In order to obtain binding affinities of the chiral sensors to indicator C343 the fluorescence titration isotherms obtained from the direct probe titrations by plotting normalized fluorescence response versus chiral sensors molar concentration were fitted by nonlinear fitting function for 1 :
1 binding model (Eqns. 1 and 2) using Newton’s iterative method as described by Anslyn and
Sessler.132–134
77
[퐼] 퐾 − [퐻] 퐾 − 1 1 푡 퐼 푡 퐼 2 Equation 1 [퐼] = − √([퐻]푡퐾퐼 − [퐼]푡퐾퐼 + 1) + 4[퐼]푡퐾퐼 2 퐾퐼 2퐾퐼
퐾퐼[퐼][퐻]푡 퐹 − 퐹0 [퐻 ∙ 퐼] = = Equation 2 1 + 퐾퐼[퐼] 퐹푖 − 퐹0
[I]t is total concentrations of indicator and [H]t is total concentrations of chiral sensor. KI is a binding constant of the indicator. [I] is unknown concentration of the indicator and [H·I] is unknown concentration of the complex (chiral sensor and indicator). F is the sensor concentration- dependent fluorescence intensity, F0 is fluorescence intensity without chiral sensor, and Fi is fluorescence intensity at an infinite chiral sensor concentration.
Eqn. 1 defines value of unknown [I] based on KI and experimentally obtained values [H]t and [I]t.
2.3.13. Determination of Binding Affinities: Competitive Titrations
Dr. Mehmet Gökhan Caglayan, a postdoctoral research fellow in Dr. Anzenbacher Jr. research group in the spring of 2016 developed and implemented this model and related non-linear fluorescence isotherm data fitting for Origin. To obtain the binding affinities for the guest to the host, the change in the fluorescence intensity versus guest concentration were plotted and fitted by nonlinear function for modified dye-displacement assay protocol using Newton’s iterative method as reported by Anslyn and Sessler.132 First, we recorded the fluorescence titration data for the indicator C343 with chiral sensors to calculate equilibrium concentration of [H·I] at the saturation point (Eqn. 3). Sensors affinity constant KS was obtained as described above in section 2.3.14.
[퐻 ∙ 퐼] [퐻] + [퐼] ⇌ [퐻 . 퐼] 퐾 = 퐼 [퐻][퐼] Equation 3 78
The equilibrium concentration of [H·I] at the saturation point was calculated from the quadratic equation (Eqn. 4) which can be simplified using a substitution (Eqn. 5) using Equation 6 (only the root [H·I] < [I]t was considered).
2 퐾퐼[퐻 ∙ 퐼] − [퐻 ∙ 퐼] (퐾퐼[퐻]푡 + 퐾퐼[퐼]푡 + 1) + 퐾퐼[퐼]푡[퐻]푡 = 0 Equation 4
퐴푥2 + 퐵푥 + 퐶 = 0 Equation 5
퐴 = 퐾퐼
퐵 = −(퐾퐼[퐻]푡 + 퐾퐼[퐼]푡 + 1)
퐶 = 퐾퐼[퐼]푡[퐻]푡
−퐵 ± √퐵2 − 4퐴퐶 [퐻 ∙ 퐼] = Equation 6 2퐴
Parameters ƒHI and ƒI are fluorescence constants of H·I and I, respectively, and were calculated from the direct probe fluorescence titration data for indicator (I) with each chiral sensor (H)
(Equations 7 and 8).
F0 and Fsat are the fluorescence intensities at the initial and saturation point, respectively.
퐹0 ƒ퐼 = Equation 7 [퐼]푡
퐹푠푎푡 ƒ = Equation 8 퐻∙퐼 [퐻 ∙ 퐼]
Then, we performed fluorescence titration of chiral sensor-indicator complex with chiral guest at saturated concentration of chiral sensor-indicator complex. We utilized the fluorescence titration 79 data for the complex (chiral sensor-indicator complex) with chiral guests to plot the change in the fluorescence intensity versus guest concentration.
Finally, all parameters KI, [H]t, [I]t, fI, fHI were used as initial conditions for Newton’s iterative method to calculate guest (chiral analytes) affinity constants KG, which is the only unknown in the cubic polynominal equation from the software. KG calculated using Origin curve fitting function
(Eqn. 9).
[퐼]푡(ƒ퐼푏 + ƒ퐻퐼푏퐾퐼[퐻]) 퐹 = Equation 9 1 + 퐾퐼[퐻]
Origin Script:
Ki, Kg, Ei, Ehi, Ht, It, x, and y represent KI, KG, ƒS, ƒHI, [H]t, [I]t, [G]t, and fluorescence intensity
(F), respectively. [G]t is a total guest (chiral analytes) concentration. Parameters KI, ƒS, ƒHI, were determined by the direct probe titration using 1:1 curve fitting (see above) of the indicator (C343) and chiral sensors alone prior to this analysis. Each of these values, however, was treated as adjustable in accordance with Newton’s fitting method. [H]t and [I]t were treated as constants, and the variables [G]t (x-axis) and fluorescence intensity (y-axis) were read from the data sheet by the 80
program. Given an initial value for KG (educated guess), the program iteratively found the value of KG that best fits the experimental data for each complex.
Table 2.3. Parameters used in the affinity constant model for [CuRRL]2+, [CuSSL]2+, C343, and chiral carboxylic acids.
Parameter Values Obtained Unit Obtained from
-4 [H]t 2.12 × 10 푚표푙⁄퐿 exp. data -8 [I]t 1.00 × 10 푚표푙⁄퐿 exp. data 5 F0 4.79 × 10 푐푛푡푠 exp. data 5 Fsat 1.06 × 10 푐푛푡푠 exp. data 4 −1 A = KI 7.33 × 10 푀 Eqn.2, 3 B −9.58 none Eqn.2, 3 C 8.58 × 10-8 퐿 Eqn.2, 3 [H·I] 8.96 ×10-9 푚표푙⁄퐿 Eqn.4 13 ƒI 4.79 × 10 푐푛푡푠 퐿⁄푚표푙 Eqn. 7 13 ƒHI 1.19 × 10 푐푛푡푠 퐿⁄푚표푙 Eqn. 8 b 1 푐푚 exp. setting
2.3.14. Summary
We propose two copper(II)-containing chiral receptors—[CuRRL]2+ and [CuSSL]2+—in conjuction with the fluorescent indicator coumarin 343, to establish an enantioselective indicator displacement assay (eIDA) for the determination of enantiomeric composition of chiral carboxylates. Carboxylates were chosen because of their good coordinating abilities toward tetra- coordinated CuII-ions. The two enantiomeric complexes [CuRRL•C343]+ and [CuSSL•C343]+ showed a marked enantioselectivity toward one isomer of chiral carboxylates, however, this selectivity was not observed for α-hydroxy acids, because of the presence of additional coordination sites. In particular, the indicator-displacement sensors were able to discriminate 81 between the enantiomers of ibuprofen and naproxene, two commonly used anti-inflammatory drugs, and atorvastatin—a hypercholesterolemia medication.
2.4. Sensing of Enantiomeric Composition of Chiral α-Hydroxycarboxylic Acids Using
Fluorescence Probes
2.4.1. Introduction
Optically active α-hydroxycarboxylic acids are important as chiral building blocks, biologically active substances, or intermediates for the asymmetric synthesis of natural products.135,136 Therefore, the determination of the enantiomeric composition of chiral α- hydroxycarboxylic acids is attracted significant attention.45,137 For this purpose, fluorescent sensors that can carry out an enantioselective recognition of α-hydroxycarboxylic acids and allow a rapid determination of their enantiomeric composition are highly desirable. During the past several years, there has been a growing interest in the enantioselective recognition of α- hydroxycarboxylic acids,138–153 especially using fluorescence spectroscopy.138–149
In 2017, Munusamy et al.138 reported BINOL based boronate esters as a enantioselective fluorescent sensor (Figure 2.47) for chiral recognition of enantiomers of α-hydroxycarboxylic acids (mandelic acid, lactic acid, and leucic acid) and amino acids (N-(tert-butoxycarbonyl) phenylalanine and N-(tert-butoxycarbonyl)-alanine). Chiral fluorescent sensor 1 exhibited enantioselective recognition towards desired chiral analytes. The other sensor, boronate ester 2, which do not have hydroxyl group in the minor groove of BINOL did not show any fluorescent chiral discrimination. When sensor 1 was treated with desired chiral acids, one enantiomer of the acid greatly quenched the emission intensity of the sensor 1 while the quenching due to the other 82 enantiomer was low. This effect results in enantioselective recognition of the desired chiral analytes.
Figure 2.47. Structures of BINOL boronate complexes reported by Munusamy et al.138
Halay and Bozhurt,60 in 2017, designed fluorescent triazine-based thiazoles as chiral receptors
(Figure 2.48) for enantioselective recognition of α-hydroxycarboxylic acids. Enantioselective capabilities of these chiral receptors against chiral carboxylic acids namely 2‐chloropropionic acid,
2‐chloromandelic acid, mandelic acid, α‐methoxyphenylacetic acid, and 2‐phenylpropionic acid were tested in 95 % H2O/DMSO by fluorescence spectroscopy. The fluorescent triazine-based thiazoles chiral receptors showed enantioselective recognition for the enantiomers of 2‐ chloropropionic acid, 2‐chloromandelic acid, and mandelic acid. On the other hand, these chiral receptors did not show enantioselectivity to α‐methoxyphenylacetic acid and 2‐phenylpropionic.
Figure 2.48. Structures of triazine‐based thiazole derivatives as chiral receptors reported by Halay and Bozhurt60 83
They proposed that hydrogen bonding, steric hindrance, structural rigidity or flexibility, and
π‐ π interactions paly special role in the enantioselectivity of carboxylic acids containing both α‐ hydroxy group and benzene ring. The result was determined that the (R)-isomers were more selective than the (S)-isomers.
In 2017, Pu reported139 a pseudoenantiomeric sensor pair (Figure 2.49) for enantiomeric determination of α-hydroxy carboxylic acid namely mandelic acid. In a pseudoenantiomeric sensor pair of the BINOL-based amino alcohols, sensor 1 ((S)-isomer) showed greater fluorescence enhancement with (R)-mandelic acid at λ1= 374 nm and sensor 2 ((R)-isomer) shows greater fluorescence enhancement with (S)-mandelic acid at λ2= 330 nm. Using a mixture of this sensor pair allowed the determination of enantiomeric composition with fluorescence measurement.
Sensor 1 is derived from BINOL, and 2 is derived from the partially hydrogenated BINOL. They proposed that the fluorescence enhancement generated by the chirality matched sensor-substrate interaction is attributed to two major factors: First, protonation of the amine nitrogen of the sensors by the substrates disrupts their intramolecular hydrogen bonding with the aryl hydroxyl groups and inhibits the fluorescence quenching generated by the excited state proton transfer. Second, the chirality matched sensor-substrate interaction can form a structurally more rigid intermolecular complex, leading to greater fluorescence enhancement.
In 2003, Zhu et al.108 utilized enantioselective indicator displacement assays (eIDA) for determination of ee in α-hydroxycarboxylic acids by using absorption measurements. They exploited the binding of boronic acids to α-hydroxycarboxylic acids and catechols in aqueous media in their study. They used three chiral receptors with alizarin complexone (AC) and pyrocatechol violet (PV) as the indicators (Figure 2.50). As chiral α-hydroxycarboxylic acids in this study, they utilized phenyllactic acid, mandelic acid, hydroxyisovaleric acid, hydroxybutyric 84
Figure 2.49. Pseudoenantiomeric sensor pair with the opposite chiral configurations designed by Pu139 for the determination of enantiomeric composition of mandelic acid.
acid, lactic acid, and hexahedromandelic acid. All the receptors showed comparable affinities to
α-hydroxycarboxylic acids and PV or AC. The association between (S,S)-2 and other α- hydroxycarboxylic acids showed that samples with (S)-analyte enantiomers were generally favored and while (R)-enantiomers were bond weakly.
OMe OMe OMe N N N N B B B B HO OH OMe HO OH HO OH OMe HO OH
1 (S,S)-2 (R,R)-2 (S)-3
O OH O OH
OH CO2H
N CO2H
O OH SO3H AC OH PV
Figure 2.50. Structure of the sensors used by Zhu et al.108 for ee determination of α-hydroxy acids.
85
2.4.2. Chiral α-Hydroxycarboxylic Acids Sensing: Design and Synthesis
We synthesized chiral fluorescent ligands, (1R,2R)- N1,N2-bis(quinoline-2-ylmethyl)- cyclohexane-1,2-diamine, (R,R)-L, and (1S,2S)-N1,N2-bis(quinoline-2-ylmethyl)- cyclohexane-
1,2-diamine, (S,S)-L. We selected Zn(II) and Cu(II) as a metal ions for complexation with the chiral ligands because these ligands favor the coordination of d9 or d10 metal ions that adopt distorted tetrahedral geometry (Figure 2.51).154 In the case of Zn(II)-based sensors the signaling mechanism operates on ground of enhanced internal charge transfer (ICT) interaction from the conjugated quinoline donor to the metal ion Zn2+ bound ligand.67,155,156 In the case of Cu(II)-based sensor the emission from the quinoline moieties is quenched due to a photoinduced energy-transfer process. For this reason, when employing Cu(II)-based receptor we used the enantioselective indicator displacement assay (eIDA).103,108,112 Here, we selected Coumarin 343 (C343) as the fluorescent indicator (Figure 2.51). Coumarin 343’s fluorescence is quenched via photoinduced electron transfer (PET)114—from Cu(II)-based receptor to indicator (C343)—when the indicator is coordinated to the complex but, in the presence of an analyte, Coumarin 343 is displaced and its fluorescence is recovered.
2+ 2+ 2+
(S) (S) (R)(R) (R)(R) NH HN NH HN NH HN
II II II N Zn N N Zn N N Cu N
II 2+ II 2+ II 2+ [Zn (S,S)-L] [Zn (R,R)-L] [Cu (R,R)-L] Coumarin 343 (C343)
Figure 2.51. Structure of chiral sensors, [ZnII(S,S)-L]2+, [ZnII(R,R)-L]2+, [CuII(R,R)-L]2+ and the indicator Coumarin 343 used in conjunction with [CuII(R,R)-L]2+
86
Commercially available 2-quinoline carboxaldehyde and chiral 1,2-diaminocyclohexane were first used to make enantiopure ligands. Then, a methanolic solution of chiral ligands, (R,R)-L) or
(S,S)-L were reacted at room temperature with an aqueous solution of Zn(NO3)2. 6H2O or copper triflate to yield the chiral complexes, [ZnII(R,R)-L]2+ and [ZnII(S,S)-L]2+, and [CuII(R,R)-L]2+.
Enantioselective capabilities of these chiral receptors against α-hydroxycarboxylic acids (Figure
2.52) tested in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM).
2-hydroxyl-3-methylbutanoic acid Mandelic acid
Lactic acid 3-Phenyllactic acid Atorvastatin
Figure 2.52. Structures of α-hydroxy acids used in this study.
2.4.3. Materials and Methods
Starting materials and all reagents were obtained from commercial suppliers without further purification. Methanol was purchased from EMD and used by further treatment with activated 4Å molecular sieves. Acetonitrile (CAS:75-05-8) was purchased from VWR and used as received. 2- quinolinecarboxaldehyde (CAS:5470-96-2) was purchased from TCI America. Sodium cyanoborohydride (CAS:25895-60-7) was purchased from BeanTown Chemical. Sodium borohydride (CAS:16940-66-2) was purchased from Sigma-Aldrich. (1R,2R)- 1,2- diaminocyclohexane (CAS:20439-47-8) and (1S,2S)- 1,2-diaminocyclohexane (CAS:021436-03-
3) were purchased from Fluka. Copper(II) trifluoromethanesulfonate (CAS:55804-65-4) and zinc 87 nitrate hexahydrate (CAS:10196-18-6) were purchased from Sigma-Aldrich. D-lactic acid
(CAS:10326-41-7) and L-lactic acid (CAS:79-33-4) were purchased from Enamine. D-3-
Phenyllactic acid (CAS:7326-19-4) was purchased from AK Scientific and L-3-phenyllactic acid
(CAS:20313-36-1) was purchased from Oakwood Chemicals. (R)-2-Hydroxy-3-methylbutanoic acid (CAS:17407-56-6) and (S)-2-hydroxy-3-methylbutanoic acid (CAS:17407-55-5) were purchased from Ark-Pharm. (R)-Mandelic acid (CAS:611-71-2) and (S)-mandelic acid
(CAS:17199-29-0) were purchased from Alfa Aesar. Atorvastatin calcium-(3R,5R) (CAS:344423-
98-9) and atorvastatin related compound E-(3S,5S) (CAS:1105067-88-6) were purchased from
Sigma Aldrich. MES buffer aqueous solutions were prepared from 2-morpholin-4- ylethanesulfonic acid (CAS: 4432-31-9) purchased from Amresco (Solon, OH). pH value of the buffer solution (pH = 6) was adjusted by addition of sodium hydroxide (6 M) and/or hydrochloric acid (6 M) using SevenMulti pH meter with glass electrode (both by Mettler Toledo, Toledo, OH).
Mass spectrometry measurement. Mass spectrometry was performed using a SHIMADZU
LCMS-2020 electrospray instrument (ESI), and SHIMADZU MALDI TOF-TOF Mass
Spectrometer AXIMA Performance.
UV-Vis measurements. Absorption spectra were acquired using Hitachi U-3010 double beam
UV-Vis spectrophotometer (Tokyo, Japan).
Nuclear magnetic resonance (NMR) measurements. 1H NMR and 13C NMR spectra were recorded on a Bruker Avance III 500 (500MHz) spectrometer in chloroform-d (CDCl3) at 25° C.
1H and 13C NMR chemical shifts were reported in ppm relative to either the residual solvent peak or TMS as an internal standard. Spectra were processed using the MestReNova software.
Fluorescence measurements. Steady-state fluorescence emission and excitation measurements were performed on Edinburgh single-photon counting spectrofluorimeter FLSP920-stm 88
(Edinburgh Instruments Ltd., Livingston, UK) at room temperature using quartz cuvette with a path length of 1 cm and with a right-angle detection. Optically dilute solutions used for all photophysical experiments were prepared using MeCN : H2O (7/3 % v/v) at pH = 6 (MES = 50 mM). Fluorescence lifetime measurements for [ZnII (R,R)-L]2+, [ZnII (S,S)-L]2+ were carried out on Edinburgh FLS920-stm combined steady state and lifetime spectrofluorimeter (Edinburgh
Instruments Ltd., Livingston, UK). Laser radiation (λ = 355 nm) was used as an excitation source for fluorescence lifetime experiments for [ZnII (R,R)-L] 2+, [ZnII (S,S)-L] 2+.
Absolute quantum yield measurements. Absolute quantum yields were obtained upon excitation at absorption maxima using Hamamatsu Quantaurus-QY C11347-11 Absolute PLQY
Spectrometer equipped with 150 W Xe lamp and multichannel detector/CCD sensor (Hamamatsu,
Japan). Optically dilute solutions used for all photophysical experiments were prepared using using
MeCN : H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) solvents.
Spectroscopy. Proton NMR (1H-NMR) and carbon-13 NMR (13C-NMR) spectra were recorded on Bruker Avance III spectrometer at 500 MHz or 125 MHz at 25 °C. Proton and carbon NMR chemical shifts () are reported in parts per million (ppm) relative to residual solvent signals in
CDCl3 ( = 7.26, 77.16). Coupling constants (J) are reported in hertz (Hz) and refer to apparent multiplicities. The following abbreviations are used for the multiplicities: s (singlet), d (doublet), t (triplet), q (quartet), p (pentet), m (multiplet). Mass spectra (MS) were obtained on electrospray ionization mass spectrometry (ESI-MS) experiments were carried out on Shimadzu 2010A LCMS instrument. Solution of C343 and solution of chiral sensor-C343 complex were excited at 430 nm and solution of chiral zinc-based sensors were excited at 315 nm. The emission spectra were recorded between 455 nm and 600 nm for copper-based chiral sensor and between 335 nm and
500 nm for zinc-based chiral sensors. The emission was scanned in 1 nm step with a dwell time 89
0.20 sec under ambient condition. Fluorescence titrations were performed in non-degassed aqueous MES buffer (50 mM) : MeCN (3:7) at pH = 6 at laboratory temperature. Titration isotherms were constructed from changes in the fluorescence maximum at 492 nm for copper- based chiral sensor and 376 nm for zinc-based chiral sensors. 90
2.4.4. Synthesis Protocols
Scheme 2.4. Synthesis of (1S,2S)- and (1R,2R)-N1,N2-bis(quinolin-2-ylmethylene)cyclohexane- 1,2-diimine (1).
The Schiff base 1 was synthesized according to a slightly modified literature procedure.157 In a 25 mL round-bottom flask 2-quinolinecarboxaldehyde (413 mg, 2.63 mmol) was dissolved in 8 mL of methanol. (1S,2S)- or (1R,2R)-diaminocyclohexane (150 mg, 1.31 mmol) was added to the round bottom flask. After a few minutes, a pale-yellow precipitate was formed. The reaction mixture was stirred at room temperature for 2 hours. The yellow precipitate was collected by filtration and washed with diethyl ether (3 × 25 mL). The resulting solid was dried under vacuum
(432 mg, 1.10 mmol, yield: 84 %).
(1S,2S)-N1,N2-bis(quinolin-2-ylmethylene)cyclohexane-1,2-diimine 1H NMR (500MHz,
CDCl3): δ 8.51 (s, 2H), 8.07 (s, 4H), 8.03 (d, J = 8.5 Hz, 2H), 7.75 (dd, J = 8.2, 0.6 Hz, 2H), 7.68
– 7.63 (m, 2H), 7.50 (dd, J = 11.0, 3.9 Hz, 2H), 3.65 (p, J = 8.8 Hz, 2H), 1.90 (d, J = 11.6 Hz,
6H), 1.56 (d, J = 9.4 Hz, 2H) ppm. 13C NMR (125 MHz, CDCl3): δ 161.86, 154.92, 147.65, 136.39,
129.57, 129.44, 128.70, 127.63, 127.15, 118.56, 77.27, 77.02, 76.76, 73.86, 32.71, 24.37 ppm. ESI
(m/z): 415.20 [M+Na]+
(1R,2R)-N1,N2-bis(quinolin-2-ylmethylene)cyclohexane-1,2-diimine 1H NMR (500MHz,
CDCl3): 8.51 (s, 2H), 8.07 (s, 4H), 8.03 (d, J = 8.5 Hz, 2H), 7.76 – 7.73 (m, 2H), 7.66 (ddd, J = 91
8.4, 6.9, 1.4 Hz, 2H), 7.49 (ddd, J = 8.1, 6.9, 1.1 Hz, 2H), 3.68 – 3.62 (m, 2H), 1.95 – 1.83 (m,
13 6H), 1.60 – 1.52 (m, 2H) ppm. C NMR (125 MHz, CDCl3): δ 161.87, 154.92, 147.65, 136.42,
129.59, 129.44, 128.71, 127.65, 127.17, 118.57, 77.29, 77.04, 76.78, 73.88, 32.72, 24.38, 0.02 ppm. ESI (m/z): 415.20 [M+Na]+
Scheme 2.5. Synthesis of (1S,2S)- and (1R,2R)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2- diamine, (S,S)-L and (R,R)-L).
The chiral ligands were synthesized according to a slightly modified literature procedure.157
The Schiff base (1) (250 mg, 0.637 mmol) was suspended in 60 mL of methanol. Sodium borohydride (128 mg, 3.38 mmol) was added to the reaction mixture portion wise (32 mg × 4 times) and the solution was stirred for 5 hours at room temperature. As the reaction progressed, the precipitate starts dissolving. The solvent was removed under reduced pressure and the residue was dissolved in (3 × 10 mL) chloroform. The combined fractions were washed with brine and water. The organic phase was dried over MgSO4, filtered and the solvent removed in vacuo, yielding a yellow oil (242 mg, 0.610 mmol, yield: 96 %).
1 2 1 (1S,2S)-N ,N -bis(quinolin-2-ylmethyl)cyclohexane-1,2-diamine H NMR (500MHz, CDCl3) :
8.07 (d, J = 8.4 Hz, 2H), 8.03 (d, J = 8.4 Hz, 2H), 7.78 (d, J = 8.1 Hz, 2H), 7.69 – 7.64 (m, 2H),
7.60 (d, J = 8.4 Hz, 2H), 7.50 (t, J = 7.5 Hz, 2H), 4.25 (d, J = 14.6 Hz, 2H), 4.06 (d, J = 14.6 Hz, 92
2H), 2.57 (s, 2H), 2.42 (dd, J = 5.4, 3.7 Hz, 2H), 2.22 (d, J = 13.2 Hz, 2H), 1.76 – 1.70 (m, 2H),
13 1.26 (dd, J = 13.2, 6.5 Hz, 2H), 1.12 (d, J = 8.1 Hz, 2H) ppm. C NMR (125 MHz, CDCl3): δ
161.26, 147.64, 136.29, 129.30, 128.95, 127.56, 127.30, 125.91, 120.81, 77.28, 77.03, 76.78,
61.66, 53.20, 31.76, 25.02 ppm. ESI (m/z): 397.20 [M+H]+
1 2 1 (1R,2R)-N ,N -bis(quinolin-2-ylmethyl)cyclohexane-1,2-diamine H NMR (500MHz, CDCl3) :
8.08 (d, J = 8.4 Hz, 2H), 8.03 (d, J = 8.5 Hz, 2H), 7.78 (d, J = 8.0 Hz, 2H), 7.67 (dd, J = 11.2,
4.1 Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.50 (t, J = 7.4 Hz, 2H), 4.26 (d, J = 14.6 Hz, 2H), 4.06 (d,
J = 14.6 Hz, 2H), 2.43 (dd, J = 5.2, 3.8 Hz, 2H), 2.22 (d, J = 13.1 Hz, 2H), 1.76 – 1.70 (m, 2H),
13 1.25 (t, J = 9.8 Hz, 2H), 1.12 (d, J = 8.2 Hz, 2H) ppm. C NMR (125 MHz, CDCl3): δ 161.09,
147.59, 136.33, 129.32, 128.91, 127.56, 127.29, 125.93, 120.80, 77.30, 77.04, 76.79, 61.61, 53.10,
31.67, 24.99 ppm. ESI (m/z): 397.20 [M+H]+
Scheme 2.6. Synthesis of [ZnII(1S,2S)- and (1R,2R)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane- 1,2-diamine]2+ ,[ZnII(S,S)-L]2+ and [ZnII(R,R)-L]2+
The zinc complexes were prepared according to a slightly modified literature procedure.125,157
In a 10 mL round-bottom flask, (R,R)-L or (S,S)-L (120 mg, 0.303 mmol) was dissolved in 7 mL of methanol. Zinc nitrate (90 mg, 0.303 mmol) was dissolved in 0.5 mL ultrapure water in a small vial. The two solutions were mixed and stirred overnight. The solvent was removed to 1/3 total 93 volume in vacuo. The residue was mixed with diethyl ether till precipitate formed. The precipitate was filtered and dried under the high vacuum (127 mg, 0.275 mmol, yield: 91 %).
[ZnII(1S,2S)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2-diamine]2+, [ZnII(S,S)-L]2+, 1H
NMR (500MHz, CDCl3) : δ 8.46 (d, J = 8.5 Hz, 2H), 7.93 (d, J = 7.5 Hz, 2H), 7.80 (d, J = 8.7
Hz, 2H), 7.55 (t, J = 7.5 Hz, 2H), 7.52 (d, J = 8.5 Hz, 2H), 7.36 (ddd, J = 8.5, 7.1, 1.3 Hz, 2H),
4.89 (dd, J = 16.9, 6.5 Hz, 2H), 4.58 – 4.51 (m, 2H), 4.28 (dd, J = 16.9, 5.3 Hz, 2H), 2.65 (s, 2H),
2.40 (d, J = 12.9 Hz, 2H), 1.85 (d, J = 8.3 Hz, 2H), 1.46 – 1.36 (m, 2H), 1.31 (t, J = 10.0 Hz, 2H)
13 ppm. C NMR (125 MHz, CDCl3): δ 159.05, 144.49, 141.47, 131.88, 128.74, 128.29, 128.00,
+ 126.87, 120.57, 77.28, 77.02, 76.77, 61.63, 50.21, 30.74, 24.46 ppm. ESI (m/z): 522.20 [M+NO3]
[ZnII(1R,2R)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2-diamine]2+, [ZnII(R,R)-L]2+, 1H
NMR (500MHz, CDCl3) : δ 8.46 (d, J = 8.5 Hz, 2H), 7.93 (dd, J = 8.1, 0.9 Hz, 2H), 7.80 (d, J =
8.7 Hz, 2H), 7.57 – 7.53 (m, 2H), 7.52 (d, J = 8.5 Hz, 2H), 7.38 – 7.34 (m, 2H), 4.89 (dd, J = 16.9,
6.5 Hz, 2H), 4.55 (s, 2H), 4.28 (dd, J = 16.9, 5.3 Hz, 2H), 2.69 – 2.60 (m, 2H), 2.40 (d, J = 13.1
Hz, 2H), 1.85 (d, J = 8.3 Hz, 2H), 1.46 – 1.37 (m, 2H), 1.30 (t, J = 10.0 Hz, 2H) ppm. 13C NMR
(125 MHz, CDCl3): δ 159.04, 144.47, 141.46, 131.87, 128.73, 128.28, 128.00, 126.87, 120.56,
+ 77.28, 77.02, 76.77, 61.61, 50.20, 30.73, 24.45 ppm. ESI (m/z): 522.20 [M+NO3]
94
Scheme 2.7. Synthesis of [CuII(1R,2R)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2- diamine]2+, [CuII(R,R)-L] 2+.
The copper complexe was prepared according to a slightly modified literature procedure.125,157
In a 10 mL round-bottom flask, (R,R)-L (134 mg, 0.338 mmol) was dissolved in 7 mL of methanol.
Copper (II) trifluoromethanesulfonate (122 mg, 0.338 mmol) was dissolved in 0.5 mL ultrapure water in a small vial. The two solutions were mixed and stirred under nitrogen for 12 hours. The solvent was removed to 1/3 of total volume under reduced pressure. The residue was mixed with diethyl ether till precipitate formed. The precipitate was filtered and dried under high vacuum (147 mg, 0.319 mmol, yield: 95 %). ESI (m/z): [M]+ : 608.11 95
2.4.5. Chiral Sensors for α-Hydroxycarboxylic acid: NMR Spectra
a b c g , j d e TMS f CDCl3
j g f i e h i h g , j
b , a H2O
a d
c
f h i H2O e b , a c a
1 1 2 Figure 2.53. H NMR (500 MHz) of (1S,2S)-N ,N -bis(quinolin-2-ylmethylene)cyclohexane-1,2-diimine in CDCl3 96
CDCl3
8 11 1 9 10 2 12 3 4 5 6 7 13 8 12 9 11 10
3 7 8 6 5 12 9 2 1 11 10 4 13 TMS
13 1 2 Figure 2.54. C NMR (125 MHz) of (1S,2S)-N ,N -bis(quinolin-2-ylmethylene)cyclohexane-1,2-diimine in CDCl3 97
a
c b d k e f e d j g j g f h i i h
c a e d b b a f CDCl3 g k TMS j h i
c b b a a k
1 1 2 Figure 2.55. H NMR (500 MHz) of (1S,2S)-N ,N -bis(quinolin-2-ylmethyl)cyclohexane-1,2-diamine in CDCl3 98
CDCl3
1 3 2 11 12 9 10 4 5 6 7 8 13 8 12 9 11 10
12 9 6 7 11 8 3 4 1 10 2
5 13
TMS
13 1 2 Figure 2.56. C NMR (125 MHz) of (1S,2S)-N ,N -bis(quinolin-2-ylmethyl)cyclohexane-1,2-diamine in CDCl3 99
a b c d TMS e f
j g i h f e h i CDCl g , j 3
H2O b , a d a
c i f h e H O b , a 2 a c
1 1 2 Figure 2.57. H NMR (500 MHz) of (1R,2R)-N ,N -bis(quinolin-2-ylmethylene)cyclohexane-1,2-diimine in CDCl3 100
CDCl3
1 2 3 8 11 4 5 6 12 9 10 7 13 8 12 9 11 10
7 12 3 11 9 5 6 2 1 8 10 13 4 TMS
13 1 2 Figure 2.58. C NMR (125 MHz) of (1R,2R)-N ,N -bis(quinolin-2-ylmethylene)cyclohexane-1,2-diimine in CDCl3
101
3
TMS
f
g
h
e
a
a
i
d
a
j
b
a a diamine in CDCl
-
c
k
b b 1,2
-
b
c
b
c d
ylmethyl)cyclohexane d - 2
-
d d bis(quinolin - 2 ,N 1 N - )
1R,2R
3
i
CDCl
e
i
e
h
h
f
g
j f H NMR (500 MHz) of ( of MHz) (500 H NMR
1
g j Figure 2.59. Figure 102
CDCl3
1 3 2 11 4 12 9 10 5 6 8 7 13 8 12 9 11 10
12 9 6 7 11 8 3 1 4 2 10 5 13
TMS
13 1 2 Figure 2.60. C NMR (125 MHz) of (1R,2R)-N ,N -bis(quinolin-2-ylmethyl)cyclohexane-1,2-diamine in CDCl3 103
a b j e c g f h k d i e f
j g i h TMS
CDCl3
d d c b b k a
j e g f h i d d k c b b a
Figure 2.61. 1H NMR (500 MHz) of [ZnII(1S,2S)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2-diamine] 2+, [ZnII(S,S)-L] 2+ in CDCl3 104
CDCl3
1 2 9 12 3 6 11 10 4 5 6 8 7 13 8 12 9 11 10
9 7 12 3 4 1 11 6 2 5 10 13 8 TMS
Figure 2.62. 13C NMR (125 MHz) of [ZnII(1S,2S)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2-diamine] 2+, [ZnII(S,S)-L] 2+ in CDCl3 105
CDCl3 TMS
a e j c b h g f i k d e f
j g i h
d d c b b a k
e j f i g h d d k c b b a
Figure 2.63. 1H NMR (500 MHz) of [ZnII(1R,2R)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2-diamine] 2+, [ZnII(R,R)-L] 2+ in CDCl3 106
CDCl3 1 2 9 3 12 10 4 11 5 8 6 7 13 8 12 9 11 10
9 12 1 7 6 2 11 10 3 4 8 5 13 TMS
Figure 2.64. 13C NMR (125 MHz) of [ZnII(1R,2R)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2-diamine] 2+, [ZnII(R,R)-L] 2+ in CDCl3 107
CDCl3
e
j e h f g f i
j g i h
[Zn (R,R)-L]2+
e j CDCl3
g f i h
e f
j g i h
(R,R)-L
Figure 2.65. Partial 1H NMR (500 MHz) spectra of ligand [(1R,2R)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2-diamine] II 2+ [(R,R)-L] and complex [Zn (R,R)-L] in CDCl3 108
11 6 7 9 12 10
5 6 7 13 8 13 8 5 12 9 11 10 (R,R)-L
11 12 7 10 6 9 8
5 13 5 6 7 13 8 12 9 11 10
[Zn (R,R)-L]2+
Figure 2.66. Partial 13C NMR (125 MHz) spectra of ligand [(1R,2R)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2-diamine], II 2+ [(R,R)-L] and complex [Zn ((R,R)-L] in CDCl3 109
2.4.6. Chiral Sensors for α-Hydroxycarboxylic acid: Mass Spectra
We confirmed the formation of chiral ligands and chiral sensors by using ESI-Mass. Also, we confirm the complex formation between the analytes and sensors by measuring ESI-Mass of
[ZnII(R,R)-L]2+ and atorvastatin related compound E complex (Figure 2.74).
The samples for ESI-MS experiment were prepared by mixing 10 μL of the solution of each sensor (100 μM) in acetonitrile. The ESI spectra were recorded in positive detection mode.
Figure 2.67. (A) ESI spectrum of (1S,2S)-N1,N2-bis(quinolin-2-ylmethylene)cyclohexane-1,2- diimine. (B) Calculated isotope pattern for C26H24N4Na
110
Figure 2.68. (A) ESI spectrum of (1R,2R)-N1,N2-bis(quinolin-2-ylmethylene)cyclohexane-1,2- diimine. (B) Calculated isotope pattern for C26H24N4Na
Figure 2.69. (A) ESI spectrum of (1S,2S)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2- diamine. (B) Calculated isotope pattern for C26H29N4
111
Figure 2.70. (A) ESI spectrum of (1R,2R)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2- diamine. (B) Calculated isotope pattern for C26H29N4
Figure 2.71. (A) ESI spectrum of [ZnII(1S,2S)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2- 2+ II 2+ diamine] , [Zn (S,S)-L] (B) Calculated isotope pattern for C26H28N5O3Zn 112
Figure 2.72. (A) ESI spectrum of [ZnII(1R,2R)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2- 2+ II 2+ diamine] , [Zn (R,R)-L] .(B) Calculated isotope pattern for C26H28N5O3Zn
Figure 2.73. (A) ESI spectrum of [CuII(1R,2R)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2- 2+ II 2+ diamine] , [Cu (R,R)-L] . (B) Calculated isotope pattern for C27H28CuF3N4O3S
113
Figure 2.74. (A) ESI spectrum of [ZnII(1R,2R)-N1,N2-bis(quinolin-2-ylmethyl)cyclohexane-1,2- diamine]2+, [ZnII(R,R)-L]2+ and atorvastatin related compound E. (B) Calculated isotope pattern for C59H62FN6O5Zn
2.4.7. Complex Stoichiometry Determination: Job’s plot
Binding stoichiometries of the complex formation in the solution were determined from fluorescence titration data using method of continuous variation (Job’s method).126 We investigated complex formation stoichiometries for Zn(II) and chiral ligands (Figure 2.75.A) and also complex formation stoichiometries of chiral sensor [CuII (R,R)-L]+2 and C343 (Figure 2.75.B).
The Job’s plots obtained confirm the 1 : 1 binding stoichiometry for the association of Zn(II) with chiral ligand, and also C343 with the copper complex.
Furthermore, we investigated complex formation stoichiometries for chiral sensor [ZnII (R,R)-L]+2 and enantiomers of mandelic acid. Figure 2.75.C and Figure 2.75.D show the obtained Job’s plots which confirm the 1 : 1 binding stoichiometry for the association of chiral sensors [ZnII (R,R)-L)]2+ with (R)-mandelic acid and (S)-mandelic acid, respectively. 114
A B 1.5x105 1.5x105 II II 2+
(R,R)-L : Zn = 1:1 [Cu (R,R)-L] : Coumarin 343 = 1:1 2+
)-L] -L
) 5 5
1.0x10 1.0x10
(R,R
R,R
II
(
Cu
X
[
4 4
5.0x10 X 5.0x10
Intensity
0.0 Intensity 0.0
0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 II 2+ (R,R )-L [Cu (R,R)-L] C D II 2+ II 2+ [Zn (R,R)-L] : (R)-mandelic acid = 1:1 [Zn (R,R)-L] : (S)-mandelic acid = 1:1 1.5x105 1.0x105
1.0x105
- mandelic acid
4 - mandelic acid
5.0x10
(R) (S)
4
5.0x10
X X
0.0 0.0
Intensity Intensity 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 (R)-mandelic acid (S)-mandelic acid
Figure 2.75. Job’s plot for the determination of the stoichiometry of (A) Zn(II) and chiral ligand (R,R)-L; (B) [CuII (R,R)-L]+2 and Coumarin 343 (C) [ZnII (R,R)-L]2+ and (R)-mandelic acid ; (D) II +2 [Zn (R,R)-L)] and (S)-mandelic acid in MeCN:H2O 7:3 at pH = 6 (MES = 50 mM).
The Job’s plot experiments127,128 were accomplished as follows: Figure 2.75 (A): two stock solutions, one of [(R,R)-L] = 10 mM and the other of [Zn(NO3)2] = 10 mM, are prepared in
MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM). A set of working solutions is then obtained by mixing VL mL (volume of the ligand) of the stock (R,R)-L solution with (VT – VL) mL of the stock Zn(NO3)2 solution, where VT is a fixed total volume (4 μL) and VL is a variable, 0 ≤ VL
≤VT. These working solutions were added to the 2 mL solvent of MeCN:H2O (7/3 % v/v) at pH =
6 (MES = 50 mM) in the cuvette. The fluorescence of these solutions is then measured at a fixed wavelength (λex = 274 nm), and plotted as a function of mole fraction of (R,R)-L (VL/VT). Analysis 115 of the Job’s plot in Fig. 2.1.A shows that at 274 nm excitation wavelength, the intercept between the best two straight lines occurs at a molar fraction (휒(R,R)-L) around 0.5. The corresponding stoichiometry is 1:1.
Figure 2.75 (B): two stock solutions, one of [CuII(R,R)-L]2+ = 10 mM and the other of [C343] =
10 mM, are prepared in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM). A set of working
II 2+ solutions is then obtained by mixing VR mL (volume of the receptor) of the stock [Cu (R,R)-L] solution with (VT – VR) mL of the stock C343 solution, where VT is a fixed total volume (4 μL) and VL is a variable, 0 ≤ VR ≤VT. These working solutions were added to the 2 mL solvent of
MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) in the cuvette. The fluorescence of these solutions is then measured at a fixed wavelength (λex = 444 nm), and plotted as a function of mole
II 2+ fraction of [Cu (R,R)-L] (VL/VT). The Job’s plot in Fig. 2.1.B shows that at 444 nm excitation wavelength, the position of maximum fluorescence on this plot is located at the molar fraction
II 2+ II 2+ (휒[Cu (R,R)-L] ) around 0.5 which indicate stoichiometry of 1:1 between C343 and [Cu (R,R)-L] .
Figure 2.75(C and D): two stock solutions, one of [ZnII(R,R)-L]2+= 10 mM and the other of [(R)- or (S)-mandelic acid] = 10 mM, are prepared in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM). A set of working solutions is then obtained by mixing VR mL (volume of the receptor) of
II 2+ the stock [Zn (R,R)-L] solution with (VT – VR) mL of the stock (R)- or (S)-mandelic acid solution, where VT is a fixed total volume (4 μL) and VL is a variable, 0 ≤ VR ≤VT. These working solutions were added to the 2 mL solvent of MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) in the cuvette. The fluorescence of these solutions is then measured at a fixed wavelength (λex = 430 nm), and plotted as a function of mole fraction of (R)- or (S)-mandelic acid (VT – VR /VT).
The position of maximum fluorescence on this plot, in relation to the mole-fraction axis, gives the stoichiometry of the complex. Analysis of the Job’s plot in Fig. 2.1.C and D shows that at 430 116 nm excitation wavelength, the position of maximum fluorescence on this plot is also located at the
II molar fraction (휒(R)- or (S)-mandelic acid) 0.5 which indicate stoichiometry of 1:1 between [Zn (R,R)-
L]2+ and (R)- and (S)-mandelic aicd.
2.4.8. Photophysical Properties
Photophysical properties of chiral sensors [ZnII (R,R)-L] 2+, [ZnII (S,S)-L] 2+, and [CuII (R,R)-
2+ L] (Table 2.4) were measured in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) at room temperature and without previous degassing. The photophysical properties are summarized in
Table 2.4. The chiral sensors [ZnII (R,R)-L] 2+ and [ZnII (S,S)-L] 2+ showed double-exponential fluorescence lifetime decay. As expected, the fluorescence of the chiral ligands decreased after complexation with Cu(II), but increased after complexation with Zn(II).
Table 2.4. Photophysical properties of chiral sensors [ZnII (R,R)-L]2+, [ZnII (S,S)-L]2+, and [CuII 2+ (R,R)-L] . Absorption maxima (λA,max) and fluorescence lifetimes FL were acquired in MeCN:H2O 7:3 at pH = 6 (MES = 50 mM) solutions.
a A, max FL Chiral Sensors [nm] [%] [ns] (S,S)-L 274 0.92 (R,R)-L 274 0.92 [CuII (R,R)-L] 2+ 315 0.47 8.50 (75.33 %) [ZnII (R,R)-L] 2+ 315 1.32 27.90 (24.67 %) 8.50 (83.63 %) [ZnII (S,S)-L] 2+ 315 1.42 31.90 (16.37 %) aAbsolute quantum yields were determined upon excitation at wavelength indicated for solutions with optical density A = 0.1. All measurements were carried out in non-deoxygenated solutions.
2.4.9. Fluorescence Titrations: Zn(II)-Based Sensors
We performed the titration of chiral sensors [ZnII (R,R)-L]2+ and [ZnII (S,S)-L]2+ (20 μM), in
MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) with the corresponding α-hydroxy acid 117 enantiomers. The sensor–α-hydroxy acids diastereomeric complexes show different fluorescence intensities, as well as different binding isotherms and apparent association constants (Ka). The response of α-hydroxy acids exhibited fluorescence quenching. This quenching is attributed to blocking the internal charge transfer between quinoline moieties and the Zn(II). Depending on the structure of the chiral guest, steady-state fluorescence spectrometry revealed that one diastereomer shows a more substantial change in fluorescence response than the other. Also, the value of the apparent binding constant is in most cases different for each analyte enantiomer. In other words, the enantiomers of the chiral α-hydroxy acids influences the stability of the resulting complex, and this stability is reflected in the fluorescence output. Table 2.5 shows the apparent association
−1 constants (Ka, M ) for selected analytes. For example, fluorescence titration results show that
[ZnII(R,R)-L]2+ has a larger affinity toward the (S)-enantiomers of mandelic acid, lactic acid, 2- hydroxy-3-methylbutanoic acid, and atorvastatin calcium. Upon fitting of the titration isotherms using a 1:1 model, binding constants between the chiral analytes and either [ZnII(R,R)-L]2+ or
[ZnII(S,S)-L]2+ were determined. We observed a marked difference in magnitude of the binding constant between the enantiomeric complexes and the enantiopure forms of mandelic acid, lactic acid, 2-hydroxy-3-methylbutanoic acid, and atorvastatin calcium.
Furthermore, we investigated the sensing of chiral carboxylic acids by [ZnII(R,R)-L]2+ or
[ZnII(S,S)-L]2+. The behavior of enantiopure carboxylates was unexpected. In fact, we did not observe enantioselectivity upon titration of either [ZnII(R,R)-L]2+ or [ZnII(S,S)-L]2+ with enantiomers of ibuprofen. The difference in binding constant is negligible and is not statistically relevant. We expect coordination of the carboxylate to the ZnII center to be different.
The titrations were performed as follows: The [ZnII(R,R)-L]2+ or [ZnII(S,S)-L]2+ solutions (3 mL, 20 M) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) in cuvette were titrated with 118
stock solution of enantiopure analyte (10 mg/mL in MeCN:H2O (7/3 % v/v) at pH = 6). The fluorescence spectrum was subsequently recorded and titration isotherms were plotted and binding constants calculated using 1:1 binding model by Newton’s iterative method.131
119
The following figures 2.76 – 2.86 show fluorescence titration spectra and binding isotherms of [ZnII (R,R)-L]2+ in the presence of desired enantiopure α-hydroxycarboxylic acids:
A B 1.0 1.0
0.8 0.8
)
0 - I f 0.6
0.6 I
0
/ (
/ I
) I 0 0.4 0.4
I - I ( 0.2 0.2 3 -1 Ka = 1.73 10 M 0.0 0.0 350 375 400 425 450 475 500 0.0 2.0x10-3 4.0x10-3 6.0x10-3 8.0x10-3 / nm [(R)-Mandelic acid] / M
Figure 2.76. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII 2+ (R,R)-L] (20 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (R)-mandelic acid. λex = 315 nm, [(R)-mandelic acid] = 0-8 mM.
A B 1.0 1.0 0.8
) 0.8
0
- I f
0.6 I 0.6
0
/ (
/ I
) I 0 0.4 0.4
I - I ( 0.2 0.2 K = 3.31 103 M-1 0.0 a 0.0 350 375 400 425 450 475 500 0.0 2.0x10-3 4.0x10-3 6.0x10-3 8.0x10-3 / nm [(S)-Mandelic acid] / M
Figure 2.77. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII 2+ (R,R)-L] (20 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (S)-mandelic acid. λex = 315 nm, [(S)-mandelic acid] = 0-8 mM. 120
A B 1.0 1.0
0.8 0.8
)
0
- I f
0 0.6 0.6
I
/ I
/ ( I
0.4 ) 0.4 0
0.2 I - I ( 0.2 K = 6.53 102 M-1 0.0 0.0 a
350 375 400 425 450 475 500 0.0 2.0x10-3 4.0x10-3 6.0x10-3 / nm [(R)-Lactic acid] / M
Figure 2.78. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII 2+ (R,R)-L] (20 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (R)-lactic acid. λex = 315 nm, [(R)-lactic acid] = 0-6 mM.
A B 1.0 1.0
0.8 ) 0.8
0
- I
f 0
0.6 I 0.6
/ I
/ (
I )
0.4 0 0.4
0.2 I - ( 0.2 3 -1 0.0 0.0 Ka = 1.74 10 M
350 375 400 425 450 475 500 0.0 2.0x10-3 4.0x10-3 6.0x10-3 / nm [(S)-Lactic acid] / M
Figure 1.79. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII 2+ (R,R)-L] (20 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (S)-lactic acid. λex = 315 nm, [(S)-lactic acid] = 0-6 mM.
121
A B 1.0 1.0
0.8 0.8
)
0
- I f
0 0.6
0.6 I
/ I
/ (
I )
0.4 0 0.4
0.2 I - I ( 0.2 3 -1 Ka = 3.31 10 M 0.0 0.0
350 375 400 425 450 475 500 0.0 2.0x10-3 4.0x10-3 6.0x10-3 / nm [(R)-2-hydroxy-3-methylbutanoic acid] / M
Figure 2.80. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII 2+ (R,R)-L] (20 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (R)-2-hydroxy-3-methylbutanoic acid. λex = 315 nm, [(R)-2-hydroxy-3- methylbutanoic acid] = 0-6 mM.
A B 1.0 1.0
0.8 0.8
)
0 - I
f
0 0.6 0.6
I
/ I
/ ( I
)
0.4 0 0.4
0.2 I - ( 0.2 4 -1 Ka = 7.26 10 M 0.0 0.0
350 375 400 425 450 475 500 0.0 2.0x10-3 4.0x10-3 6.0x10-3 / nm [(S)-2-hydroxy-3-methylbutanoic acid] / M
Figure 2.81. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII 2+ (R,R)-L] (20 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (S)-2-hydroxy-3-methylbutanoic acid. λex = 315 nm, [(S)-2-hydroxy-3- methylbutanoic acid] = 0-6 mM.
122
A B 1.0 1.0
0.8 0.8
)
0
- I f
0 0.6 0.6
I
/ I
/ ( I
0.4 ) 0.4 0
0.2 I - I ( 0.2 3 -1 Ka = 9.10 10 M 0.0 0.0
350 375 400 425 450 475 500 0.0 2.0x10-3 4.0x10-3 6.0x10-3 / nm [(R)-3-phenyllactic acid] / M
Figure 2.82. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII 2+ (R,R)-L] (20 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (R)-3-phenyllactic acid. λex = 315 nm, [(R)-3-phenyllactic acid] = 0-6 mM.
A B 1.0 1.0
0.8 0.8 )
0 - I f 0 0.6 0.6
I
/ I
/ ( I
0.4 ) 0.4 0
0.2 ( I - I ( 0.2 K = 1.30 104 M-1 0.0 0.0 a
350 375 400 425 450 475 500 0.0 2.0x10-3 4.0x10-3 6.0x10-3 / nm [(S)-3-phenyllactic acid] / M
Figure 2.83. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII 2+ (R,R)-L)] (20 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (S)-3-phenyllactic acid. λex = 315 nm, [(S)-3-phenyllactic acid] = 0-6 mM.
123
A B 1.0 1.0
0.8 ) 0.8
0
- I
f I
0 0.6 0.6
/ (
/ I
I ) 0.4 0 0.4
0.2 I - I ( 0.2
4 -1 0.0 0.0 Ka = 3.53 10 M
350 375 400 425 450 475 500 0.00 1.50x10-4 3.00x10-4 4.50x10-4 / nm [(3R,5R)-Atorvastatin calcium] / M
Figure 2.84. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII 2+ (R,R)-L] (20 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (3R,5R)-atorvastatin calcium. λex = 315 nm, [(3R,5R)-Atorvastatin calcium] = 0-450 μM.
A B 1.0 1.0
0.8 ) 0.8
0
- I
f 0
0.6 I 0.6
/ I
/ (
I )
0.4 0 0.4
0.2 I - ( 0.2
4 -1 0.0 0.0 Ka = 4.15 10 M
350 375 400 425 450 475 500 0.00 1.50x10-4 3.00x10-4 4.50x10-4 / nm [Atorvastatin related compound E] / M
Figure 2.85. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII 2+ (R,R)-L] (20 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of atorvastatin related compound E. λex = 315 nm, [atorvastatin related compound E] = 0-450 μM.
124
A B 0.8 0.6
0.6
0 0.4
0 / I
)
/ I 0
) 0.4
0 0.2 0.2 ( I - I ( I - I
II + II + [Zn (R,R)-L (R)-Mandelic acid] 0.0 [Zn (R,R)-L (R)-Lactic acid] 0.0 [ZnII(R,R)-L (S)-Mandelic acid]+ [ZnII(R,R)-L (S)-Lactic acid]+
0.0 3.0x10-3 6.0x10-3 9.0x10-3 0.0 2.0x10-3 4.0x10-3 6.0x10-3 [Mandelic acid] / M [Lactic acid] / M C D 0.9 0.8
0.6
0.6 0
0
/ I
/ I )
) 0.4
0 0 0.3
( I - I 0.2 ( I - I
[ZnII(R,R)-L (R)-3-Phenyllactic acid]+ II + 0.0 [Zn (R,R)-L (R)-2-Hydroxy-3-methylbutanoic acid] 0.0 [ZnII(R,R)-L (S)-3-Phenyllactic acid]+ [ZnII(R,R)-L (S)-2-Hydroxy-3-methylbutanoic acid]+ 0.0 2.0x10-3 4.0x10-3 6.0x10-3 0.0 2.0x10-3 4.0x10-3 6.0x10-3 [2-Hydroxy-3-methylbutanoic acid] / M [3-Phenyllactic acid] / M
E0.5
0.4
0
0.3 / I
)
0 0.2
( I - 0.1
[ZnII(R,R)-L (3R,5R)-Atorvastatin calcium]+ 0.0 II + [Zn (R,R)-L Atorvastatin related compound E]
0.00 1.50x10-4 3.00x10-4 4.50x10-4 [Atorvastatin] / M
Figure 2.86. Overlaid binding isotherms for two corresponding enantiomers based on the change in fluorescence intensity at the maximum wavelength of [ZnII (R,R)-L]2+ (20 μM) show changes in fluorescence intensity upon the addition of incremental amounts of (R)-enantiomer and (S)- enantiomer (A) mandelic acid (B) lactic acid (C) 2-hydroxy-3-methylbutanoic acid (D) 3- phenyllactic acid (E) atorvastatin in MeCN:H2O 7:3 at pH = 6 (MES = 50 mM). λex = 430 nm. 125
The following figures 2.87 – 2.98 show fluorescence titration spectra and binding isotherms of [ZnII (S,S)-L]2+ in the presence of desired enantiopure α-hydroxycarboxylic acids:
A B 1.0 1.0
0.8 0.8
)
0 - I
0.6 f 0.6
I
0
/ (
/ I
I )
0.4 0 0.4
0.2 I - I ( 0.2 3 -1 Ka = 3.43 10 M 0.0 0.0
350 375 400 425 450 475 500 0.0 2.0x10-3 4.0x10-3 6.0x10-3 8.0x10-3 / nm [(R)-Mandelic acid] / M
Figure 2.87. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII 2+ (S,S)-L] (20 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (R)-mandelic acid. λex = 315 nm, [(R)-mandelic acid] = 0-8 mM.
A B 1.0 1.0
0.8 ) 0.8
0
- I
f I
0 0.6 0.6
/ I
/ (
I )
0.4 0 0.4
0.2 I - I ( 0.2 3 -1 Ka = 1.73 10 M 0.0 0.0
350 375 400 425 450 475 500 0.0 2.0x10-3 4.0x10-3 6.0x10-3 8.0x10-3 / nm [(S)-Mandelic acid] / M
Figure 2.88. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII 2+ (S,S)-L] (20 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (S)-mandelic acid. λex = 315 nm, [(S)-mandelic acid] = 0-8 mM. 126
A B 1.0 1.0
0.8 ) 0.8
0 - I
f 0 0.6 I 0.6
/ I
/ (
I )
0.4 0 0.4
0.2 I - I ( 0.2 3 -1 0.0 0.0 Ka = 1.60 10 M
350 375 400 425 450 475 500 0.0 2.0x10-3 4.0x10-3 6.0x10-3 / nm [(R)-Lactic acid] / M
Figure 2.89. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII 2+ (S,S)-L] (20 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (R)-lactic acid. λex = 315 nm, [(R)-lactic acid] = 0-6 mM.
A B 1.0 1.0
0.8 0.8
)
0
- I f
0 0.6 0.6
I
/ I
/ (
I )
0.4 0 0.4
0.2 I - I ( 0.2 2 -1 0.0 0.0 Ka = 6.47 10 M
350 375 400 425 450 475 500 0.0 4.0x10-3 8.0x10-3 1.2x10-2 / nm [(S)-Lactic acid] / M
Figure 2.90. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII 2+ (S,S)-L] (20 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (S)-lactic acid. λex = 315 nm, [(S)-lactic acid] = 0-6 mM.
127
A B 1.0 1.0
0.8 ) 0.8
0
- I f
I 0.6
0 0.6
/ (
/ I
I )
0.4 0 0.4
0.2 I - I ( 0.2 4 -1 Ka = 7.64 10 M 0.0 0.0
350 375 400 425 450 475 500 0.0 2.0x10-3 4.0x10-3 6.0x10-3 / nm [(R)-2-Hydroxy-3-methylbutanoic acid] / M
Figure 2.91. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII 2+ (S,S)-L] (20 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (R)-2-hydroxy-3-methylbutanoic acid. λex = 315 nm, [(R)-2-hydroxy-3- methylbutanoic acid] = 0-6 mM.
A B 1.0 1.0
0.8
0.8 )
0 - I
f 0.6 I
0 0.6
/ I
/ (
I )
0.4 0 0.4
0.2 I - ( 0.2 3 -1 Ka = 3.25 10 M 0.0 0.0
-3 -3 -3 350 375 400 425 450 475 500 0.0 2.0x10 4.0x10 6.0x10 / nm [(S)-2-Hydroxy-3-methylbutanoic acid] / M
Figure 2.92. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII 2+ (S,S)-L] (20 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (S)-2-hydroxy-3-methylbutanoic acid. λex = 315 nm, [(S)-2-hydroxy-3- methylbutanoic acid] = 0-6 mM.
128
A B 1.0 1.0
0.8 0.8
)
0
- I f
0 0.6 0.6
I
/ I
/ ( I
0.4 ) 0.4 0
0.2 I - I ( 0.2 4 -1 Ka = 1.18 10 M 0.0 0.0
350 375 400 425 450 475 500 0.0 2.0x10-3 4.0x10-3 6.0x10-3 / nm [(R)-3-Phenyllactic acid] / M
Figure 2.93. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII 2+ (S,S)-L] (20 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (R)-3-phenyllactic acid. λex = 315 nm, [(R)-3-phenyllactic acid] = 0-6 mM.
A B 1.0 1.0
0.8 ) 0.8
0
- I
f I
0 0.6 0.6
/ I
/ (
I )
0.4 0 0.4
0.2 I - I ( 0.2
4 -1 0.0 0.0 Ka = 1.11 10 M
350 375 400 425 450 475 500 0.0 2.0x10-3 4.0x10-3 6.0x10-3 / nm [(S)-3-Phenyllactic acid] / M
Figure 2.94. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII 2+ (S,S)-L] (20 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (S)-3-phenyllactic acid. λex = 315 nm, [(S)-3-phenyllactic acid] = 0-6 mM.
129
A B 1.0 1.0
0.8 ) 0.8
0 - I f
0.6 I 0.6
0
/ (
/ I
) I 0.4 0 0.4
0.2 I - I ( 0.2
4 -1 0.0 0.0 Ka = 4.15 10 M 350 375 400 425 450 475 500 0.00 1.50x10-4 3.00x10-4 4.50x10-4 / nm [(3R,5R)-Atorvastatin calcium] / M
Figure 2.95. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII 2+ (S,S)-L] (20 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (3R,5R)-atorvastatin calcium. λex = 315 nm, [(3R,5R)-atorvastatin calcium] = 0-450 μM.
A B 1.0 1.0
0.8 0.8
)
0
- I f
0 0.6 0.6
I
/ I
/ ( I
0.4 ) 0 0.4
0.2 I - ( 0.2
4 -1 0.0 0.0 Ka = 3.18 10 M
350 375 400 425 450 475 500 0.00 1.50x10-4 3.00x10-4 4.50x10-4 / nm [Atorvastatin related compound E] / M
Figure 2.96. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII 2+ (S,S)-L] (20 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of atorvastatin related compound E. λex = 315 nm, atorvastatin related compound E] = 0-450 μM. 130
A B 0.8 0.6
0.6
0.4
0 0
/ I
/ I )
) 0.4
0 0 0.2
0.2 ( I - ( I -
II + II + [Zn (S,S)-L (R)-Mandelic acid] [Zn (S,S)-L (R)-Lactic acid] II + 0.0 0.0 [Zn (S,S)-L (S)-Mandelic acid] [ZnII(S,S)-L (S)-Lactic acid]+
-3 -3 -3 -3 -3 -3 0.0 3.0x10 6.0x10 9.0x10 0.0 2.0x10 4.0x10 6.0x10 [Mandelic acid] / M [Lactic acid] / M C D 0.9 0.8
0.6
0.6 0
0
/ I
/ I
) )
0.4
0 0 0.3
( I - 0.2 ( I -
II + [ZnII(S,S)-L (R)-2-Hydroxy-3-methylbutanoic acid]+ [Zn (S,S)-L (R)-3-Phenyllactic acid] 0.0 0.0 II + [ZnII(S,S)-L (S)-2-Hydroxy-3-methylbutanoic acid]+ [Zn (S,S)-L (S)-3-Phenyllactic acid]
-3 -3 -3 0.0 2.0x10 4.0x10 6.0x10 0.0 2.0x10-3 4.0x10-3 6.0x10-3 [2-Hydroxy-3-methylbutanoic acid] / M [3-Phenyllactic acid] / M E 0.5
0.4
0 0.3
/ I
)
0 0.2
( I - 0.1
[ZnII(S,S)-L (3R,5R)-Atorvastatin calcium]+ 0.0 II + [Zn (S,S)-L Atorvastatin related compound E]
0.00 1.50x10-4 3.00x10-4 4.50x10-4 [Atorvastatin] / M
Figure 2.97. Overlaid binding isotherms for two corresponding enantiomers based on the change in fluorescence intensity at the maximum wavelength of [ZnII (S,S)-L]2+ (20 μM) show changes in fluorescence intensity upon the addition of incremental amounts of (R)-enantiomer and (S)- enantiomer (A) mandelic acid (B) lactic acid (C) 2-hydroxy-3-methylbutanoic acid (D) 3- phenyllactic acid (E) atorvastatin in MeCN:H2O 7:3 at pH = 6 (MES = 50 mM). λex = 430 nm. 131
A B 0.8 0.6
0.6
0
0 0.4
/ I
)
/ I
)
0 0.4
0
II + 0.2 ( I - I 0.2 [Zn (R,R)-L (R)-Mandelic acid] II + ( I - I II + [Zn (R,R)-L (R)-Lactic acid] [Zn (R,R)-L (S)-Mandelic acid] II + [Zn (R,R)-L (S)-Lactic acid] II + [Zn (S,S)-L (R)-Mandelic acid] II + II + [Zn (S,S)-L (R)-Lactic acid] 0.0 [Zn (S,S)-L (S)-Mandelic acid] II + 0.0 [Zn (S,S)-L (S)-Lactic acid]
0.0 3.0x10-3 6.0x10-3 9.0x10-3 0.0 2.0x10-3 4.0x10-3 6.0x10-3 [Mandelic acid] / M [Lactic acid] / M C D 0.9 0.8
0.6 0.6
0 0
/ I
) 0.4
/ I 0
)
0 0.3
0.2 II + ( I - I II + [Zn (R,R)-L (R)-3-Phenyllactic acid] [Zn (R,R)-L (R)-2-Hydroxy-3-methylbutanoic acid] II +
( I - I II + [Zn (R,R)-L (S)-3-Phenyllactic acid] [Zn (R,R)-L (S)-2-Hydroxy-3-methylbutanoic acid] II + II + [Zn (S,S)-L (R)-2-Hydroxy-3-methylbutanoic acid] [Zn (S,S)-L (R)-3-Phenyllactic acid] II + 0.0 II + 0.0 [Zn (S,S)-L (S)-2-Hydroxy-3-methylbutanoic acid] [Zn (S,S)-L (S)-3-Phenyllactic acid]
0.0 -3 -3 -3 0.0 -3 -3 -3 2.0x10 4.0x10 6.0x10 2.0x10 4.0x10 6.0x10 [2-Hydroxy-3-methylbutanoic acid] / M [3-Phenyllactic acid] / M
E 0.5
0.4
0 0.3
/ I )
0 0.2
( I - I 0.1 [ZnII(R,R)-L (3R,5R)-Atorvastatin calcium]+ [ZnII(R,R)-L Atorvastatin related compound E]+ [ZnII(S,S)-L (3R,5R)-Atorvastatin calcium]+ 0.0 II + [Zn (S,S)-L Atorvastatin related compound E] 0.00 1.50x10-4 3.00x10-4 4.50x10-4 [Atorvastatin] / M Figure 2.98. Overlaid binding isotherms based on the change in fluorescence intensity at the maximum wavelength of [ZnII(S,S)-L]2+ and [ZnII(R,R)-L]2+ (20 μM) show changes in fluorescence intensity upon the addition of incremental amounts of (R)- or (S)-enantiomer of (A) mandelic acid (B) lactic acid (C) 2-hydroxy-3-methylbutanoic acid (D) 3-phenyllactic acid (E) atorvastatin in MeCN:H2O 7:3 at pH = 6 (MES = 50 mM). λex = 430 nm. 132
The following figures 2.99 and 2.100 show fluorescence titration spectra and isotherms of
[ZnII (R,R)-L]2+ in the presence of (R)- or (S)-ibuprofen:
A 1.0 B 1.0 0.8
) 0.8
0
- I
f 0
0.6 I 0.6
/ I
/ (
I
)
0 0.4 0.4
( I - I ( 0.2 0.2 3 -1 0.0 Ka = 3.46 10 M 0.0 350 375 400 425 450 475 500 0.0 4.0x10-3 8.0x10-3 1.2x10-2 / nm [(R)-Ibuprofen] / M
Figure 2.99. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII 2+ (R,R)-L] (20 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (R)-ibuprofen. λex = 315 nm, [(R)-ibuprofen] = 0-12 mM.
A B 1.0 1.0
0.8 ) 0.8
0 - I f
I 0.6 0.6
0
/ (
)
/ I
0 0.4 I 0.4 ( I - I ( 0.2 0.2 3 -1 0.0 Ka = 3.75 10 M 0.0 0.0 -3 -3 -2 350 375 400 425 450 475 500 4.0x10 8.0x10 1.2x10 / nm [(S)-Ibuprofen] / M
Figure 2.100. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII 2+ (R,R)-L] (20 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (S)-ibuprofen. λex = 315 nm, [(S)-ibuprofen] = 0-12 mM. 133
The following figures 2.101 and 2.103 show fluorescence titration spectra and isotherms of
[ZnII (S,S)-L]2+ in the presence of (R)- or (S)-ibuprofen:
A B 1.0 1.0
) 0.8
0.8 0
- I f
I 0.6
0 0.6
/ (
/ I
I )
0 0.4 0.4
I - I ( 0.2 0.2 3 -1 Ka = 3.68 10 M 0.0 0.0 350 375 400 425 450 475 500 0.0 4.0x10-3 8.0x10-3 1.2x10-2 / nm [(R)-Ibuprofen] / M
Figure 2.101. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII 2+ (S,S)-L] (20 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (R)-ibuprofen. λex = 315 nm, [(R)-ibuprofen] = 0-12 mM.
A B 1.0 1.0
) 0.8
0.8 0
- I
f I
0 0.6
0.6
/ (
/ I
) I 0 0.4 0.4 I - I ( 0.2 0.2 3 -1 Ka = 3.70 10 M 0.0 0.0 350 375 400 425 450 475 500 0.0 4.0x10-3 8.0x10-3 1.2x10-2 / nm [(S)-Ibuprofen] / M
Figure 2.102. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [ZnII 2+ (S,S)-L] (20 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (S)-ibuprofen. λex = 315 nm, [(S)-ibuprofen] = 0-12 mM. 134
A B 0.8 0.8
0.6 0.6
0
0
/ I
/ I
)
)
0.4 II + II + 0 [Zn (R,R)-L (R)-Ibuprofen] 0 0.4 [Zn (S,S)-L (R)-Ibuprofen]
[ZnII(R,R)-L (S)-Ibuprofen]+ [ZnII(S,S)-L (S)-Ibuprofen]+ ( I - I 0.2 ( I - I 0.2
0.0 0.0
0.0 4.0x10-3 8.0x10-3 1.2x10-2 0.0 4.0x10-3 8.0x10-3 1.2x10-2 [Ibuprofen] / M [Ibuprofen] / M
Figure 2.103. Overlaid binding isotherms based on the change in fluorescence intensity at the maximum wavelength of [ZnII(R,R)-L]2+ and [ZnII(S,S)-L]2+ (20 μM) show the same changes in fluorescence intensity upon the addition of incremental amounts of (R)- or (S)-ibuprofen in MeCN:H2O 7:3 at pH = 6 (MES = 50 mM). λex = 430 nm.
Fluorescence titration spectra and binding isotherms of [ZnII(R,R)-L]2+ and [ZnII(S,S)-L]2+ in the presence of (R)- or (S)-enantiomers of ibuprofen (carboxylic acid) are shown in Figure 2.99 and Figure 2.103, respectively.
According to the fluorescence titration spectra and overlaid binding isotherms in Figure 2.103 there is not enantioselectivity upon titration of either [ZnII(R,R)-L]2+ or [ZnII(S,S)-L]2+ with enantiomers of ibuprofen (carboxylic acids). While the binding constants that were determined from the titration isotherms lie in the same affinity range as other analytes, the difference in binding constant is negligible and is not statistically relevant. We believe that hydrogen bonding, steric hindrance paly special role in the enantioselectivity of carboxylic acids containing α‐hydroxy group.
135
2.4.10. Fluorescence Titrations: Indicator and α‐Hydroxycarboxylic Acids by Cu(II)-
Based Sensor
First, we investigated affinity of chiral sensor [CuII (R,R)-L]2+ for C343 using fluorescence titrations. Titration of a solution of C343 in aqueous buffer (pH = 6, MES = 50mM) with a solution of [CuII (R,R)-L]2+ shows an four-fold decrease in the fluorescence of C343 centered around 493 nm which is attributed to the quenching of C343’s excited state in the presence of the copper- complex (Figure 2.104.A). The indicator (C343) fluorescence emission is quenched through an intramolecular photoinduced-electron-transfer (PET) process129,130 when the indicator is coordinated to the chiral copper-based sensors.
The titration was accomplished as follows: The C343 solutions (3 mL, 0.01 M) in MeCN :
H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) in cuvette were titrated with stock solution of chiral copper-based sensor (10 mg/mL in MeCN:H2O (7/3 % v/v) at pH = 6). The fluorescence spectrum was subsequently recorded and titration isotherm was plotted and binding constants calculated using 1:1 binding model by Newton’s iterative method. Figure 2.104 shows normalized emission
II 2+ spectra of chiral sensor [Cu (R,R)-L] and C343 in MeCN:H2O (7/3% v/v) at pH = 6 (MES = 50 mM).
Then, we performed the titration of the chemosensing ensemble ([CuII (R,R)-L]2+ = 780 μM,
[C343] = 0.01 μM, MeCN:H2O 7/3 % v/v, pH = 6, MES = 50mM) with the corresponding α- hydroxy acids enantiomer and we observed recovery of C343’s fluorescence.
Diastereomeric complexes show different fluorescence intensities, as well as different binding isotherms and apparent association constants (Ka). The presence of α-hydroxy acids resulted in fluorescence amplification. This amplification is attributed to displacement of indicator C343 with enantiopure α-hydroxy acid analytes. Depending on the structure of the chiral guest, steady-state 136 fluorescence spectrometry revealed that one diastereomer showed a more substantial change in fluorescence response than the other. The value of the apparent binding constant is in most cases different for each analyte enantiomer. Thus, the enantiomerism of the chiral α-hydroxy acids influences the stability of the resulting complex, and this stability is reflected in the fluorescence
−1 output. Table 2.5 shows the apparent association constants (Ka, M ) for selected analytes. For example, fluorescence titration results show that [CuII(R,R)-L]2+ has a larger affinity toward the
(S)-enantiomers of α-hydroxy acids; however, it displays the opposite selectivity toward (R)- enantiomers of α-hydroxy acids suggesting there is a marked difference in the thermodynamic stability of the diastereomeric complexes formed by the coordination of an enantiopure α-hydroxy acid to the coordinatively unsaturated [CuII (R,R)-L]2+. The fitting of the titration isotherms using a 1:1 competition model, allowed to calculate the binding constants for the analytes and [CuII
(R,R)-L]2+. We observed a different magnitude of the binding constant between the enantiomeric complex and the enantiopure forms of mandelic acid, 3-phenyllactic acid, and 2-hydroxy-3- methylbutanoic acid.
We did more investigation by utilizing analytes with similar structures such as amino acids
(phenylalanine) and carboxylic acids (phenylpropionic acid). The behavior of enantiopure aminoacids and carboxylic acids were unexpected. In fact, we did not observe any response upon titration of [CuII (R,R)-L]2+ with phenylpropionic acid and phenylalanine (Figure 2.105).
137
The following figure 2.104 show fluorescence titration spectra and isotherms of [CuII (R,R)-
L]2+ in the presence of C343:
A B 1.0 1.0
0.8 ) 0.8
0
- I
f 0
0.6 I 0.6
/ I
/ (
I )
0.4 0 0.4
0.2 I - I ( 0.2 3 -1 Ka = (4.72 0.27) 10 M 0.0 0.0
475 500 525 550 575 600 0.0 1.0x10-3 2.0x10-3 3.0x10-3 II 2+ / nm [Cu (R,R)-L] / M
Figure 2.104. (A) Fluorescence titration spectra (B) and fluorescence titration isotherm of C343 (0.01 μM) upon the addition of an incremental amounts of [CuII (R,R)-L]+2 (0–3 mM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM). λex= 430 nm.
The following figure 2.105 show fluorescence titration spectra of [CuII (R,R)-L•C343]+ in the presence of amino acid or carboxylic acid:
Figure 2.105. Fluorescence titration spectra of [CuII (R,R)-L.C343]+ (780 μM) with (A) (S)- or (R)-phenylalanine; (B) (S)- or (R)-phenylpropionic acid in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM). λex = 430 nm. 138
The following figures 2.106 – 2.114 show fluorescence titration spectra and isotherms of [CuII
(R,R)-L]2+ in the presence of desired enantiopure α-hydroxycarboxylic acids:
A B 5 4x10 4.0x105
5 3x10 3.5x105
5
2x10 5
a. u. / a.
3.0x10 I
1x105 5 Fluorescence Intensity Fluorescence 2.5x10 3 -1 Ka = 4.47 10 M 0 475 500 525 550 575 600 0.0 2.0x10-3 4.0x10-3 6.0x10-3 / nm [(R)-Mandelic acid] / M
Figure 2.106. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [CuII + (R,R)-L.C343] (780 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (R)-mandelic acid. λex = 430 nm, [(R)-mandelic acid] = 0-6 mM.
A B 5 4x10 4.0x105
3x105 3.5x105
5
2x10
a. u. / a.
I 3.0x105 1x105 4 -1 Fluorescence Intensity Fluorescence 5 K = 1.69 10 M 2.5x10 a 0 475 500 525 550 575 600 0.0 2.0x10-3 4.0x10-3 6.0x10-3 / nm [(S)-Mandelic acid] / M
Figure 2.107. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [CuII + (R,R)-L.C343] (780 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (S)-mandelic acid. λex = 430 nm, [(S)-mandelic acid] = 0-6 mM. 139
A B 5 4x10 4.0x105
3x105 3.5x105
5
2x10
a. u. / a.
I 3.0x105 1x105
Fluorescence Intensity Fluorescence 5 3 -1 2.5x10 Ka = 4.38 10 M 0 475 500 525 550 575 600 0.0 2.0x10-3 4.0x10-3 6.0x10-3 8.0x10-3 / nm [(R)-2-Hydroxy-3-methylbutanoic acid] / M
Figure 2.108. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [CuII + (R,R)-L.C343] (780 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (R)-2-hydroxy-3-methylbutanoic acid. λex = 430 nm, [(R)-2- hydroxy-3-methylbutanoic acid] = 0-8 mM.
A B 5 4x10 4.0x105
3x105 3.5x105
5
2x10
a. u. / a.
I 3.0x105 1x105
Fluorescence Intensity Fluorescence 5 3 -1 2.5x10 Ka = 6.76 10 M 0
0.0 -3 -3 -3 -3 475 500 525 550 575 600 2.0x10 4.0x10 6.0x10 8.0x10 / nm [(S)-2-Hydroxy-3-methylbutanoic acid] / M
Figure 2.109. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [CuII + (R,R)-L.C343] (780 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (S)-2-hydroxy-3-methylbutanoic acid. λex = 430 nm, [(S)-2-hydroxy- 3-methylbutanoic acid] = 0-8 mM. 140
A B 4x105 4.0x105
5 3x10 3.5x105
5
2x10 5
a. u. / a.
3.0x10 I
1x105 5 Fluorescence Intensity Fluorescence 2.5x10 3 -1 Ka = 4.64 10 M 0 475 500 525 550 575 600 0.0 2.0x10-3 4.0x10-3 6.0x10-3 8.0x10-3 / nm [(R)-3-Phenyllactic acid] / M
Figure 2.110. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [CuII + (R,R)-L.C343] (780 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (R)-3-phenyllactic acid. λex = 430 nm, [(R)-3-phenyllactic acid] = 0- 8 mM.
A B 5 4x10 4.0x105
3x105 3.5x105
5
2x10
a. u. / a. 5 I 3.0x10 1x105
5 3 -1 Intensity Fluorescence 2.5x10 Ka = 6.52 10 M 0 475 500 525 550 575 600 0.0 2.0x10-3 4.0x10-3 6.0x10-3 8.0x10-3 / nm [(S)-3-Phenyllactic acid] / M
Figure 2.111. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [CuII + (R,R)-L.C343] (780 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (S)-3-phenyllactic acid. λex = 430 nm, [(S)-3-phenyllactic acid] = 0- 8 mM. 141
A B 4.0x105 4x105
5 3x105 3.5x10
5
a. u. a.
/ 2x10 5
I 3.0x10
1x105 5 Fluorescence Intensity Fluorescence 2.5x10 3 -1 Ka = 4.52 10 M 0 475 500 525 550 575 600 0.0 2.0x10-3 4.0x10-3 6.0x10-3 / nm [(R)-Lactic acid] / M
Figure 2.112. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [CuII + (R,R)-L.C343] (780 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (R)-lactic acid. λex = 430 nm, [(R)-lactic acid] = 0-6 mM.
A B 5 4x10 4.0x105
3x105 3.5x105
5
2x10
a. u. / a. 5 I 3.0x10 1x105
Intensity Fluorescence 3 -1 2.5x105 Ka = 4.54 10 M 0 475 500 525 550 575 600 0.0 3.0x10-3 6.0x10-3 9.0x10-3 / nm [(S)-Lactic acid] / M
Figure 2.113. (A) Fluorescence titration spectra and (B) fluorescence titration isotherm of [CuII + (R,R)-L.C343] (780 μM) in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM) upon the addition of an incremental amounts of (S)-lactic acid. λex = 430 nm, [(S)-lactic acid] = 0-6 mM.
142
A B 0.8 0.8
0.6 0.6
0
0
/ I
/ I )
) 0.4 0.4
0 0
0.2
0.2 ( I - I ( I - I [CuII(R,R)-L (R)-Mandelic acid]+ II + [Cu (R,R)-L (S)-Mandelic acid] [CuII(R,R)-L (R)-2-hydroxy-3-methylbutanoic acid]+ 0.0 II + 0.0 [Cu (R,R)-L (S)-2-hydroxy-3-methylbutanoic acid]
-3 -3 -3 -3 0.0 3.0x10 6.0x10 0.0 3.0x10 6.0x10 [Mandelic acid] / M [2-Hydroxy-3-methylbutanoic acid] / M C 0.8 D 0.6
0.6
0 0
/ I 0.4
/ I )
0.4 )
0 0
0.2
( I - I 0.2 ( I - I II + [CuII(R,R)-L (R)-3-Phenyllactic acid]+ [Cu (R,R)-L (R)-Lactic acid] II + II + [Cu (R,R)-L (S)-3-Phenyllactic acid] [Cu (R,R)-L (S)-Lactic acid] 0.0 0.0 0.0 2.0x10-3 4.0x10-3 6.0x10-3 8.0x10-3 0.0 3.0x10-3 6.0x10-3 9.0x10-3 [3-Phenyllactic acid] / M [Lactic acid] / M
Figure 2.114. Overlaid binding isotherms based on the change in fluorescence intensity at the maximum wavelength of [CuII (R,R)-L•C343]+ (780 μM) show enhancement of intensity upon the addition of incremental amounts of (R)-enantiomers and (S)-enantiomers (A) mandelic acid (B) 2- hydroxy-3-methylbutanoic aid (C) 3-phenyllactic acid (D) lactic acid in MeCN:H2O (7/3 % v/v) at pH = 6 (MES = 50 mM). λex = 430 nm.
143
-1 II 2+ II Table 2.5. The association constants (Ka, M ) corresponding to [Cu (R,R)-L] , [Zn (R,R)- L]2+, and [ZnII (S,S)-L]2+ chiral sensors with selected chiral -hydroxy acids.
[CuII (R,R)-L]2+ [ZnII (R,R)-L]2+ [ZnII (S,S)-L]2+ Chiral Guests -1 * -1 * -1 * Ka (M ) Ka (M ) Ka (M ) (R)-Mandelic acid 4.47 × 103 1.73 × 103 3.43 × 103 (S)-Mandelic acid 1.69 × 104 3.31 × 103 1.73 × 103 (R)-Lactic acid 4.52 × 103 6.53 × 102 1.60 × 103 (S)-Lactic acid 4.54 × 103 1.74 × 103 6.47 × 102 (R)-3-Phenyllactic acid 4.64 × 103 0.91 × 104 1.18 × 104 (S)-3-Phenyllactic acid 6.52 × 103 1.30 × 104 1.11 × 104 (R)-2-Hydroxy-3-methylbutanoic acid 4.38 × 103 3.31 × 103 7.64 × 104 (S)-2-Hydroxy-3-methylbutanoic acid 6.76 ×103 7.26 ×104 3.25 × 103 (3R,5R)-Atorvastatin Calcium - 3.53 × 104 4.15 × 104 Atorvastatin related compound E - 4.15 × 104 3.18 × 104 * All titrations were performed in acetonitrile:water (7/3 % v/v) at pH = 6 (MES = 50mM). The II Kas were calculated based on the change in fluorescence intensity—λEM: 492 nm for [Cu (R,R)- 2+ II 2+ II 2+ L] and λEM: 376 nm for [Zn (R,R)-L] and [Zn (S,S)-L] —upon the addition of each chiral guests. The association constants were calculated using the non-linear least-square fitting; errors of the fitting were < 20%.
2.4.11. Determination of Binding Affinities: Zn(II)-Based Sensors Titrations
The binding affinities of the chiral analytes to chiral zinc-based sensors were obtained from
the direct fluorescence titrations by plotting normalized fluorescence response versus chiral
analytes molar concentration. These data were fitted by nonlinear fitting function for 1 : 1 binding
model (Eqns.15 and 17) using Newton’s iterative method as described by Anslyn and Sessler.132–
134
퐻 + 퐺 ⇌ 퐻퐺 Equation 10
The desired binding constant (K) is expressed in eqn (11).
[퐻퐺] 퐾 = Equation 11 [퐻][퐺] 144
Assigning the total concentrations of H and G, as [H]t and [G]t, respectively, gives mass balance eqn (12) and (13).
[퐻]푡 = [퐻] + [퐻퐺] Equation 12
[퐺]푡 = [퐺] + [퐻퐺] Equation 13
The first step is to derive an equation based on unknown concentration of [G]. Thus, begin with the modification of eqn (13). This modification first requires the definition of [HG] in terms of [G]. Eqn (11) is rearranged to define [HG] and then used to substitute for [HG] in eqn (12).
Solving for [H]t yields eqn (14).
[퐻]푡 [퐻] = Equation 14 1 + 퐾[퐺]
Combining rearranged eqn (11) and (14) then gives eqn (15).
퐾[퐺][퐻]푡 퐹 − 퐹0 [퐻퐺] = = Equation 15 1 + 퐾[퐺] 퐹푖 − 퐹0
Substituting eqn (15) into eqn (13) yields a quadratic equation, which is rearranged to give eqn
(16).
2 퐾[퐺] + (1 − 퐾[퐺]푡 + 퐾[퐻]푡)[퐺] − [퐺]푡 = 0 Equation 16
145
The real root of eqn (16) is expressed in eqn (17), which defines [G] based on K and experimentally determined values ([H]t and [G]t).
2 −(1 − 퐾[퐺]푡 + 퐾[퐻]푡 + √(1 − 퐾[퐺]푡 + 퐾[퐻]푡) + 4퐾[퐺]푡 [퐺] = Equation 17 2퐾
[H]t is total concentrations of chiral zinc-based sensors and [G]t is total concentrations of chiral analytes. KS is a binding constant of the sensor. [H] is unknown concentration of chiral sensors and [G·H] is unknown concentration of the complex (chiral zinc-based sensors and chiral analytes). F is the sensor concentration-dependent fluorescence intensity, F0 is fluorescence intensity without chiral analytes, and Fi is fluorescence intensity at an infinite chiral analytes concentration. Eqn. 9 defines value of unknown [S] based on KS and experimentally obtained values [G]t and [H]t.
2.4.12. Determination of Binding Affinities: Indicator–Cu(II)-Based Sensor Titration
We utilized same method that has been explained in section 2.1.17. In order to obtain binding affinities of the chiral sensors to indicator C343 the fluorescence titration isotherms obtained from the direct probe titrations by plotting normalized fluorescence response versus chiral sensor [CuII
(R,R)-L]2+ molar concentration were fitted by nonlinear fitting function for 1 : 1 binding model
(Eqns. 1 and 2) using Newton’s iterative method as described by Anslyn and Sessler.132–134
146
2.4.13. Determination of Binding Affinities: α-Hydroxycarboxylic Acids–Cu(II)-Based
Sensor Titration
To obtain the binding affinities for the guest to the host, we implemented same model that has been explained in section 2.1.18.
Table 2.6. Parameters used in the affinity constant model for [CuII (R,R)-L]2+, C343, and chiral analytes.
Parameter Values Obtained Unit Obtained from
-4 [H]t 7.80 × 10 푚표푙⁄퐿 exp. data -8 [I]t 1.00 × 10 푚표푙⁄퐿 exp. data 5 F0 5.16 × 10 푐푛푡푠 exp. data 5 Fsat 2.50 × 10 푐푛푡푠 exp. data 3 −1 A = KI 4.72 × 10 푀 Eqn.2, 3 B −4.68 none Eqn.2, 3 C 3.68 × 10-8 퐿 Eqn.2, 3 [H·I] 7.86 ×10-9 푚표푙⁄퐿 Eqn.4 13 ƒI 5.16 × 10 푐푛푡푠 퐿⁄푚표푙 Eqn. 7 13 ƒHI 3.17 × 10 푐푛푡푠 퐿⁄푚표푙 Eqn. 8 b 1 푐푚 exp. setting
2.4.14. High-Throughput Array for Chiral α-Hydroxycarboxylic Acids
To confirm that the sensing system is suitable for ee determination in high-throughput setting, the fluorescence data from complexation of chiral zinc-based sensors and chiral -hydroxy acids were evaluated in an assay using 384-well microplates and a fluorescence plate reader. Semi- quantitative experiments focused on the determination of the enantiomeric purity of analytes with varying ratios of R- and S-enantiomers. To show that these chiral sensors differentiate between 147 different enantiomeric compositions of α-hydroxy acids, we performed a semi-quantitative analysis of enantiomeric mixtures of each analyte.
First, a standard curve method was utilized for obtaining absolute configuration and ee of reaction mixtures from fluorescence intensity (FI). A standard curve is a graph relating a measured fluorescence to ee of the substance of interest in standard/known samples prepared in a way to span the range from -100 % ee to +100 % ee. In a standard curve, a graph of FI of a complex of sensor-analyte is plotted on the Y-axis, and various ee of analyte along the X-axis. The graph of FI vs. ee were obtained by regression of points which referring to FI readings from a number of mixtures of known ee (Figure 2.115). The absolute configuration and ee of controls (validation samples) and unknowns (for example asymmetric reaction samples) can be determined by interpolation of their fluorescence reading on the graph.
The trend and predictable behavior of the data in the semiquantitative analysis indicated that the system had the potential for the determination of ee of the unknown enantiomeric composition.
We performed linear regression analysis utilizing support vector machine (SVM)158,159 algorithm by using 13 data points for calibration and 2 data points from arrays for validation/prediction of samples with unknown enantiomeric composition (Figure 2.116). SVM is a supervised classification method that seeks to separate classes by mapping the input into an n-dimensional vector space using kernel functions. The SVM regression method constructs calibration models serving to predict the ee values of unknown samples. We used 13 data points corresponding to various ee values to model the behavior of the data and two different % ee values to validate the model. The developed model was used to quantify two unknown samples. The quantitative assay yielded a very accurate % ee regression analysis of the α-hydroxy acids, as shown by the root- mean-square errors (RMSEs). 148
Each point for the calibration curve (regression model) should be prepared separately and in the same manner as analyzed samples according to the following procedure.
II 2+ II 2+ We prepared 25 µM of chiral sensors [Zn (S,S)-L] and [Zn (R,R)-L] in MeCN:H2O (7/3
% v/v) at pH = 6 (MES = 50 mM). In order to prepare standards which will cover the desired range of enantiomeric composition, different amounts of concentrated chiral analyte stock solutions of both enantiomers have been combined. The total concentration of mixed analytes was 2 mM.
HTP measurements: The array experiments were performed in 384-IQ-EB Imager Quality,
Evaporation Barrier Aurora Biotechnologies Microplates. The sensors and analytes solutions were dispensed using a high-precision 16-channel pipetting system Nanodrop Express, and BNX
1536™ liquid handling system. Each experiment was performed in 24 repetitions. First sensor solution was dispensed using a high-precision 16-channel pipetting system Nanodrop Express, and then analytes solution was dispensed to the sensor dispensed solution in 384-IQ-EB Imager Quality
Evaporation Barrier Aurora Biotechnologies Microplates. The plates were centrifuged (2 min,
2500 rpm, T = 294 K) and immediately read with a BMG CLARIOstar microplate reader.
For all experiments, the optic settings used for simultaneous measurements of fluorescence intensity (FI) changes are as follow:
FI Excitation Emission channel wavelength wavelength Gain No. [nm] [nm]
1 300 380 1174 2 320 370 1967
Data analysis. The resulting emission data were subjected to the Student’s T-test to exclude 4 data points out of 24 repetitions. The coefficient of variability within the class of same repetitions 149 was lower than 4%. The obtained data were analyzed using LDA without any further data pretreatment for semi-quantitative analyses.
A B
C D
Figure 2.115. A semiquantitative assay of enantiomeric composition of (A) mandelic acid (B) lactic acid (C) 2-hydroxy-3-methylbutanoic acid and (D) 3-phenyllactic acid by employing the II 2+ II microarray in MeCN: H2O (7/3 % v/v) at pH = 6 (MES = 50mM). [Zn (S,S)-L] = [Zn (R,R)- L]2+ = 20 μM, [analytes] = 2 mM.
150
A X-block Preprocessing: Autoscale B X-block Preprocessing: Median Center
% RMSEC: 2.72 RMSEC: 2.12
100 % 100
ee RMSECV: 3.49 RMSECV: 3.37 /
RMSEP: 4.79 ee RMSEP: 5.25
/ ] 50 Calibration Data Set 50 Calibration Data Set Validation Data Set Validation Data Set
0 0
rac-mix rac-mix
Lactic Acid Lactic Mandelic Acid Mandelic ] -50 -50
-100 -100 Predicted [ Predicted [ -100 -50 0 50 100 -100 -50 0 50 100 Actual [Mandelic Acid] / ee % Actual [Lactic Acid] / ee % C D
X-block Preprocessing: Class Center X-block Preprocessing: Groupscale %
RMSEC: 0.89 ee RMSEC: 1.68
100 / RMSECV: 1.19 100 RMSECV: 2.11
% RMSEP: 6.47 RMSEP: 5.05
ee Calibration Data Set Calibration Data Set / 50 50 ] Validation Data Set Validation Data Set
0
0
HMBA rac-mix rac-mix Phenyllactic Acid Phenyllactic
-50 ] -50 3-
Predicted [ -100 -100
-100 -50 0 50 100 -100 -50 0 50 100 Predicted [ Actual [HMBA] / ee % Actual [3-Phenyllactic Acid] / ee %
Figure 2.116. Multivariate linear regression for determination of ee in the samples of (A) mandelic acid (B) lactic acid (C) 2-Hydroxy-3-methylbutanoic acid and (D) 3-phenyllactic acid. Root mean square errors of prediction (RMSEP) of 4.5 %, 5.2 %, 6.5 %, 5.1 % for mandelic acid, lactic acid, 2-hydroxy-3-methylbutanoic acid, and 3-phenyllactic acid, respectively, relates to the error with two unknown independent samples. [ZnII (S,S)-L]2+ = [ZnII (R,R)-L]2+ = 20 μM, [analyte] = 2 mM.
151
2.4.15. Summary
In this study, we synthesized three receptor [ZnII(S,S)-L]2+, [ZnII(R,R)-L]2+, and [CuII(R,R)-
L]2+ to determine the enantiomeric composition of α-hydroxy acids. Direct titration expriments using fluorescent ZnII complexes were used to calculate enantiomeic excess of mandelic acid, lactic acid, 2-hydroxy-3-methylbutanoic acid, and 3-phenyllactic acid. An enantioselective indicator displacement assay (eIDA) was used in the case of Cu(II)-based reseptor for the discrimination of enantiomeric composition based on displacement f an coumarin 343 as indicator by a chiral α-hydroxy acids. Both approches are simple and practical because they rely on a simple analytical technique—fluorescence spectroscopy—which does not require substrate derivatization, and the preparation of the chiral receptors does not require lengthy syntheses.
Lastly, semi-quantitative and quantitative analyses aimed at the determination of the enantiomeric compositions of various chiral α-hydroxy acids in aqueous media. Support vector machine (SVM)-based method was used for quantitative determination of the enantiomeric composition of mandelic acid, lactic acid, 3-phenyllactic acid, and 2-hydroxy-3-methylbutanoic acid. The array-based assay correctly differentiated mixtures with different enantiomeric excess and. The array-based assay also correctly predicts the ee for unknown samples with a high accuracy
(a root mean square error of prediction, RMSEP < 6.5 %). Experiments described herein are among the most accurate determination of ee using fluorescent probes. 152
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163
CHAPTER III. DETECTION OF AMMONIUM NITRATE-BASED EXPLOSIVE USING
OPTICAL SENSORS
3.1. Abstract
This chapter describes a sensing approach for ammonium nitrate-nitromethane (ANNM) explosive using fluorescent sensors. Here, we report of an easy-to-use optical sensor for both vapor-phase and solution-phase identification of explosive mixtures that uses a cross-reactive fluorimetric sensor array comprising chemically responsive fluorimetric indicators composed of aromatic aldehydes and polyethyleneimine. We selected four different carboxaldehydes, p- dimethylaminobenzaldehyde (DMABC), 2-naphthaldehyde (NapC), and 1-pyrenecarboxaldehyde
(PyrC) and 2-fluorenecarboxaldehyde (FluoC). When mixed with polyethylene imine (PEI), these aromatic aldehydes form four different sensor systems S1-S4.
To demonstrate the utility of this sensing technique, an array-based vapor sensing experiment was carried out to detect and analyze chemically diverse species including nitroalkanes, acids, and bases as analytes and possible interferents. ANNM was analyzed by paper microzone arrays and nanofiber sensor mats. Paper-based microfluidics allow for portable, on-site rapid detection which is crucial in many applications such as in the clinical, food and environmental sectors where simple and practical analytical devices are highly desired. In this project we have developed the microfluidic paper-based analytical devices (µPADs) for the analysis of ammonium nitrate-based improvised explosives. Both inorganic-ammonium nitrate (NH4NO3)-and organic explosives- nitromethane (CH3NO2)-are detected.
To illustrate the potential of this sensor for the fabrication of wearable sensors, non-woven nanofiber mats were fabricated with sensor doped into electrospun polyurethane nanofibers and 164 deposited on a variety of substrates, including gloves. The wearable sensor displayed a strong and fast response to all the analytes comprising nitromethane (NM), ammonium nitrate (AN), and
ANNM while displaying strong changes in fluorescence color and intensity.
3.2. Introduction
Explosives are widely used in mining, military, and civil construction. However, explosives have been also misused to take lives and destruct. Terrorist attacks with explosive weapons across the globe have drastically increased in the past decades. For example, explosives used in the bombing of the federal building in Oklahoma City in 1995 killed 168 and injured more than 600 people,1 the Oslo bombing in 2011 killed and injured more than 200 people.2 Therefore, the need for compact and portable detection tools for the rapid and reliable identification of explosives is crucial for homeland security applications.
State-of-the-art detection in public and military structures is performed by using different methods such as trained canines3 and on-site analytical instrumentation, such as ion mobility spectrometry,4 Fourier transform infrared spectroscopy,5 and Raman spectroscopy.6,7 However, while these methods are considered highly efficient, their cost is generally high and portability are usually limited, particularly for stand-off detection. Therefore, the development of a simpler, cheaper, and quicker on-site detection method for the rapid and reliable identification of explosives is needed.
Optical detection methods, such as a change in color or fluorescence intensity, hold much promise for a robust, sensitive, and selective portable devices for the safe stand-off detection of explosives.8 Fluorescence sensors are simple to use, rapid, effective, portable, and relatively inexpensive. Fluorescence based spectroscopy has inspired highly sensitive molecular sensors which incorporate a wide range of chemical explosives including, nitroaromatics, nitroalkanes, 165 nitramines, nitrate esters, and peroxides.8 Andrew and Swager9 reported a turn-on fluorescence chemosensor that displays a direct fluorescent response to either (1,3,5-trinitro-1,3,5-triazinane)
RDX or (pentaerythritol tetranitrate) PETN explosives based on a photochemical reduction reaction. This detection assay used 10-methyl-9,10-dihydroacridine (AcrH2) to photolytically
+ reduce RDX and other oxidants by a net hydride transfer, forming AcrH , a green emitter ( lmax =
480nm).
Figure 3.1. A fluorescent chemosensor developed by Andrew and Swager9 to detect saturated nitramine and nitrate ester explosives.
Sanchez and Trogler10 reported a unique fluorescence mechanism for PETN detection by a series of new blue-emitting silafluorene-dihydrofluorene copolymer. Germain and Knapp11 developed a turn-on fluorescence detection for peroxide-based explosives, like (triacetone triperoxide) TATP. The detection strategy relies on oxidative deboronation to unmask H2Salen, which subsequently binds Zn2+ to form fluorescent Zn(Salen).
166
Figure 3.2. Detection strategy used by Germain and Knapp11 for peroxide-based explosives.
Optical and fluorescence-based sensors have been researched for portable applications due to their sensitivity and portability.
3.3. Explosives
Based on structure and performance, explosives have been classified into many types. All explosives can be classified as either high explosives or low explosives.12
Low explosives or propellants are combustible materials which burn (deflagrate) very rapidly.
They are also defined as a solid mixture of chemicals containing within themselves all oxygen needed for their combustion. They function by producing gas which produces an explosion. Low explosives consist typically of strong inorganic ion oxidizers, such as nitrates, chlorates, and perchlorates. Gun powder and most pyrotechnic materials such as the black powder used in firework are examples of this class, which differs in the rate at which they deliver their energy.13
High explosives consist of materials that combine the reacting elements in the same molecule which allows them to react faster. High explosives detonate. Detonation involves supersonic shock that passes through the material causing fast explosion. They do not function by burning. They include unstable peroxides such as TATP, hexamethylene triperoxide diamine (HMTD) and nitrated organics like RDX, high melting explosive (HMX) also called octogen, 2,4,6- 167 trinitrotoluene (TNT), and PETN. Figure 3.3 shows the structure of these explosives. High explosives are normally employed in mining, demolition, and military applications. High explosives can be subdivided into three explosives classes differentiated by sensitivity: primary, secondary, and tertiary explosives.14
Primary high explosives such as TATP and HMTD are extremely sensitive to mechanical shock, friction, and heat. They easily explode by an application of fire, spark, impact and friction.
They are used in primers, detonators and percussion caps.15
TATP HMTD PETN Triacetone triperoxide Hexamethylene triperoxide diamine Pentaerythritol tetranitrate
RDX HMX TNT 1,3,5-Trinitro-1,3,5-triazine 1,3,5,7-Tetranitro-1,3,5,7-tetrazocane 2,4,6-Trinitrotoluene
Figure 3.3. Chemical structure of organic peroxides TATP, HMTD and nitro-substituted high explosives PETN, RDX, HMX, and TNT.
Secondary high explosives for example, PETN, RDX, and TNT are relatively insensitive to mechanical shock, friction, and heat. They are used in military and civil applications.16 Tertiary high explosives or blasting agents such as ammonium nitrate-fuel oil (ANFO) are insensitive to shock. They cannot be reliably detonated with practical quantities of primary explosive. Tertiary high explosives require an intermediate explosive booster of secondary explosive. Tertiary high explosives are used in large-scale mining and construction.17 168
3.4. Improvised Explosive Devices (IEDs)
New forms of terrorist attacks are more sophisticated and more dangerous. The most common explosives used in terrorist actions are improvised explosive devices (IED). This is because of their low cost, ease of manufacture and subsequent use and difficulty to be detected. Therefore, the development of detection systems to detect IEDs is important.
In recent years, the explosives used for making IEDs have shifted from commercial and military explosives to the use of homemade explosives (HMEs).
The manufacture of HMEs is quite simple and is a matter of growing concern. In addition,
HMEs are even more freely available than commercial explosives. HMEs typically comprise of inorganic energetic oxidants including chlorate, per-chlorate or nitrate salts such as urea nitrate
(UN), AN and organic compounds with nitamine-, nitrate-, or nitro- group such as the accelerant
NM.6
3.5. Detection of IEDs
IED detection techniques19 can be roughly divided into two groups: 1) bulk detection of explosives and 2) trace detection of explosives which differ in their point of focus.
3.5.1. Bulk Detection Systems
In bulk detection, the mass of explosive material is detected within a device, usually by viewing images made by scanners or similar equipment. There are mainly useful when the explosive device is well sealed or when the explosive does not readily release molecules from its bulk. The bulk detectors include different techniques such as X-ray techniques, both transmission and backscatter;20 neutron activation in several techniques;21 g-ray excitation, in either transmission or 169 backscatter modes;22 and nuclear resonance techniques, either nuclear magnetic resonance
(NMR)23 or nuclear quadrupole resonance (NQR).24
3.5.2. Trace Detection Systems
In trace detection, the explosives are detected by chemical identification of molecules released from the bulk of the explosive material. These residues can be applied in either or both of two forms: vapor and particulate. In particulate detection, trace quantities of particulate of solid explosives are detected where they are most often found in the environment, absorbed to solid surfaces such as dust, plants, and the like. In case of vapor detection (gas-phase molecules emitted by a solid or liquid explosive), the concentration of explosives in the air is related to the vapor pressure of the explosive material and to other factors, such as temperature, air circulation in the location, etc.25,26
The concentration of explosives can be divided into three groups: high, medium, and low vapor pressure. High vapor pressure explosives have equilibrium vapor concentrations in air about one part per million (1ppm). These explosives include ethylene glycol dinitrate (EGDN), nitroglycerin
(NG), and 2,4-dinitrotoluene (DNT). Medium vapor pressure explosives have equilibrium vapor concentrations in air near one part per billion (1ppb), and they include TNT and AN. Low vapor pressure explosives have equilibrium vapor concentrations in air one part per trillion (1 ppt). These group include HMX (octogen), RDX (hexogen or cyclonite), and PETN. The vapor pressures mentioned above are related to pure materials. But in the case of mixtures containing these explosives the vapor pressure may be even lower. However, the vapor pressure of a substance increases exponentially with temperature rise. High vapor pressure explosives are quite easy to detect using sensors such as ion mobility spectrometers or electron capture detectors.27 Medium vapor pressure explosives can be detected using surface swiping.28 Low vapor pressure explosives 170 do not produce enough volatiles to be detected. Mostly these compounds are detected using trace technology such as wipe collections of particulates of explosives.
Trace chemical detectors use technologies that can locate these traces in situ and offer a very different way to detect the explosives. Vapor and traces are currently detected by three main sensors: Electronic/chemical sensors, optical sensors and biosensors.
Electronic/chemical sensors such as electronic nose (ENose),29 ion-mobility spectrometry
(IMS),4 gas chromatography (GC),23 chemiluminescence (CL),30 thermo-redox sensors, electron capture (ECD),31 and surface acoustic wave/gas chromatography (SAW/GC),32 optical sensors such as transmission and reflection spectroscopy, and biosensors33 such as dogs, bees, and rats are some techniques used in trace chemical sensors.
Since in the last decade as terrorists have increasingly targeted civilians with IED, the detection of trace levels of IED has become more important for to the homeland security and social stability.
Therefore, much efforts have been made in the field of trace explosives detection. Many of these detection methods are difficult to adapt for field use owing to the size of laboratory-scale instrumentation and the need for other accessories that limit portability. Here, optical methods are among the most promising approaches for explosives detection.
3.5.3. Detection of Trace Explosives Using Optical Methods
All optical methods determining the chemical composition rely on the interaction between light and material. The optical methods may employ linear or nonlinear optical phenomena. The linear optical phenomena include absorption, fluorescence, phosphorescence, polarization, interference, etc. and non-linear phenomena involve second harmonic generation. The choice of a particular optical method depends on the nature of the application and desired sensitivities. The optical methods are attractive because they are relatively simple to use, inexpensive, and can be used for 171 a wide variety of scales. Furthermore, optical methods have some advantages as they are easily adapted for stand-off distances. Stand-off detection of trace levels of explosives would be of great benefit in identifying the location of hidden explosive devices. Optical techniques used for detection of IEDs rely on traces of explosives that inevitably remain on the surface when preparing a bomb or, alternatively, on the outgassing of the explosives or their decomposition products.34
Since many explosives are highly nitrated organic compounds such as nitroaromatics or nitrates are electron-deficient. This electron deficiency is important in explosives detection because good electron acceptors can efficiently quench fluorescence by photoinduced electron transfer.35 The fluorescence quenching method has the ability to detect a wider range of organic and inorganic nitrated compounds. Therefore, the fluorescence quenching approach has been explored for the analysis of nitrated explosives.36,37
More selective quenching arises from specific interactions between a non-fluorescent species, such as an explosive, and a carefully chosen fluorophore. Dynamic quenching involves the transient collisional interaction between an excited state fluorophore with a ground-state quencher.38 In contrast, static quenching involves the formation of a ground state fluorophore– quencher complex. Both dynamic and static quenching are more selective and more analytically useful than the trivial mechanisms such as radiative energy transfer that consist of the emission of light by the donor excited state which is followed by absorption of the emitted photon by the acceptor.39 By careful choice of fluorophore, the quenching interaction can be tuned to provide selectivity for a variety of analytes and to reduce potential interferences. Fluorescent sensors for vapor detection have been demonstrated with both intrinsically fluorescent conjugated polymers40 and extrinsically fluorescent polymers doped with chromophores.41 172
Semiconducting polymers are excellent candidate for being used as fluorescent material due to their electron rich behavior. Rose et al.42 utilized this mechanism to detect the nitroaromatic explosive compounds. Nitroaromatic explosives being electron deficient bind to these electron rich semiconducting polymers and quench their fluorescence by an electron transfer mechanism.
In 1998, Yang and Swager43,44 used a fluorescence quenching transduction mechanism together with the amplifying nature of conjugated polymers, materials highly sensitive to TNT vapor (Figure 3.4). In this method the fluorescence property of the sensor will change in the presence of an analyte. The fluorescence quenching method also uses pyrene as fluorophore and is applied for the detection of RDX, HMX, TNT, nitromethane and ammonium nitrate.36 The response is based on the interactions of nitrated explosives as quenchers with excited state pyrene molecules, which stabilize the excited state and shift the vibronic bands to slightly lower wavelengths.13,45
Moreover, a bifurcated optical fiber based chemical sensor has been developed by Wang et al.46 in 2006. The sensor is based on the reversible chemical reaction between a novel functional poly (vinyl chloride) (PVC), which contains fluorescent moiety-curcumin-as the sensing material and 2,6-dinitrophenol (DNP) as the analyte. PVC containing fluorescent moiety reacts with 2,6-
DNP to form a complex with low fluorescence efficiency through hydrogen bonding. Formation of the complex gives significant fluorescence quenching which is suitable for signaling the occurrence of the host–guest interaction.
Also, fluorescent film sensors based on pyrene-functionalized film have been studied for
47 detecting nitroaromatics in aqueous solutions by Zhang et al. Fluorescence quenching studies showed that the film is sensitive and selective to the presence of nitroaromatics such as TNT, DNT, 173 and 2,4,6-trinitrophenol (picric acid) in aqueous solution due to their strong electron-withdrawing ability and show different fluorescence emission of the film.
1 2 3 4
Figure 3.4. It has been shown that the pentiptycene-derived conjugated polymers are an excellent fluorescent chemosensor for the detection of electron-deficient unsaturated species such as TNT by Yang and Swager.44 The rigid pentiptycene groups provide cavities for analyte binding studies of polymers 1-4 that probe the electronic and structural effects on fluorescence quenching with a variety of analytes.
Fluorescence method has many different features to determine the sensor response, including lifetime, emission wavelength, intensity, and spectral shape. An important feature of fluorescence- based detection methods is the ability to detect explosives or landmines at a distance. In fluorescence-based detection method, either fluorescent-based sensor material is spread over the suspected area to get the image of an object or excitation light is directly flashed onto the object or area so that the suspected area gets illuminated and gives a response for the presence of explosive compound.48 174
3.6. Ammonium Nitrate-Nitromethane (ANNM) Explosives
ANNM explosive is one of the most powerful improvised types of AN-based explosives; usually contains a 60:40 (60% ammonium nitrate, 40% nitromethane by mass) mix of AN and
NM.49,50 ANNM have some advantages over conventional explosives: the components are not explosives before mixing; The components do not have to be transported as explosives; The components do not have to be stored as explosives and do not require expensive storage. Also, they can be mixed on site just before using. Compared to ANFO, ANNM explosive is more sensitive to shock and is therefore easier to detonate. When ANNM detonates, the primary products are H2O, CO2 and N2. Because of a negative oxygen balance, other toxic gases such as
51 NOx are inevitably formed. The balanced equation is as follows:
3NH4NO3 + 2CH3NO2 → 4N2 + 2CO2 + 9H2O
The products of the detonation can be decidedly unstoichiometric, depending on the detonation impetus. ANNM is commercially available as Kinepak.
3.6.1. Ammonium Nitrate (AN)
AN is an ionic solid commonly used as a fertilizer.52 It is the most readily available and cheapest salt of HNO3. AN is generally found in the form of odorless, transparent, hygroscopic deliquescent crystals or white granules compound used as a solid oxidizer in explosive mixtures.
AN has been used as energetic material in liquid and solid explosive mixtures.53 AN is used also in commercial blasting applications, because AN is the cheapest source of oxygen available in a condensed form for commercial explosives. Frequently, AN is mixed with certain fuels to produce explosive compounds such as dynamite, ANFO54,55 and other nitrate-based explosives. The nitrate- 175 based slurries normally serve as borehole explosives or blasting agents i.e. for tunnel construction works and need a booster to explode.
The explosive properties of AN, such as sensitivity to detonation, impact and heat, rate of detonation, etc. were reported first in the beginning of the last century.56,57 Since AN is highly hygroscopic, it becomes liquid in humid air. The hygroscopicity of AN pose a great problem in the storage of mining explosives containing AN. AN begins to decompose as soon as it melts, it dissociates into its precursors, ammonia and nitric acid, resulting in a vapor pressure similar to that of trinitrotoluene, 1.93´10-3 Pa.58,59
3.6.2. Nitromethane (NM)
NM is not usually considered as an explosive, but its oxygen balance suggests otherwise.
Therefore, it can be utilized in the fabrication of explosive devices.60 NM is also classified as a hazardous substance for human health.
Due to the high availability of this compound, NM can be classified as a very significant target for
61–63 explosive detectors. NM is highly volatile with a vapor pressure of 37000 ppmv at 20°C and explosion limits of 7.3–63% (v/v) in air.64
Mixtures of nitromethane with oxidizers, such as nitric acid or ammonium nitrate enables to achieve a high energy evolved at explosion and also a high detonation velocity and pressure.
3.6.3. Detection of AN-Based Improvised Explosives and Their Component
Detection techniques for AN-based improvised explosives are also of great interest. To the best of our knowledge, there has been no research on optical detection of AN-based explosives done before. Current sensing methods for AN-based improvised explosives include non-optical methods. For example, Bright et al.65 developed a cross-reactive chemical sensing array, made 176 from a composite of quantum dot and different organic polymers for chemical vapor detection of
AN. Campos et al.66 designed an electronic tongue to monitor the presence of ammonium nitrate in aqueous solution (Figure 3.5). It was based on pulse voltammetry and consists of an array of eight working electrodes encapsulated in a stainless-steel cylinder. In 2016, Guo et al.67 demonstrate the development of an artificial olfactory system based on an individual optoelectronic Schottky junction for the discriminative detection of explosive vapors such as ammonium nitrate explosives.
Figure 3.5. The electronic tongue formed by non-noble electrodes designed by Campos et al66 for detection of ammonium nitrate.
Nazarian and Presser68 studied nitromethane and ammonium nitrate using laser-driven thermal reactor. They used thermal analysis methodology for thermal and chemical characterization of homemade explosives. They showed that liquid-fuel saturation of the internal pores of a solid particle oxidizer appear to be a limiting parameter for the total specific heat release during exothermic processes. They also indicated that the thermal signatures of these materials are dependent on sample mass and heating rate. 177
Zapata et al.69 used Raman spectroscopy technique for detection of ammonium nitrate explosives through the identification of the energetic salts, the respective anion by ion chromatography (IC), and organic component by reversed- phase liquid chromatography (RP-
HPLC).
The nuclear quadrupole resonance (NQR) method has been applied to AN-based explosives.70,71 Rudakov72 used NQR method to find an optimized multi-pulse technique for effective detection of ammonium nitrate-based explosives. He showed that the cross-polarization technique can significantly enhance the NQR signal for AN-based explosives.
Most trace explosive detectors such as ion mobility spectrometers, detect AN after collecting particulates on a wipe and flash heating the material. Upon flash heating the AN dissociates into ammonia and nitric acid vapors58. An experimental system has been developed by Schendel et al.73 wherein a pulsed laser beam photofragments molecules of a gaseous sample introduced into a vacuum chamber. They studied emission of photoproducts of the excimer laser photolysis of nitromethane.
Also, Wynn et al.74 used photodissociation followed by laser induced fluorescence (PD-LIF) technique for noncontact detection of the homemade explosive such as urea nitrate, nitromethane and ammonium nitrate. Their technique utilizes a single ultraviolet laser pulse (~7 ns) in wavelengths 200-250 nm region to vaporize and photodissociate the condensed-phase materials, and then to detect the resulting vibrationally-excited NO fragments via laser-induced fluorescence.
Electrochemical detection of nitromethane vapors has been study by Delile et al.75 They used a gold screen-printed electrode in a flow-cell. Detection tests with the Nebulex™ - Nebulex™ couples the solubilization of an analyte from the atmosphere by a nebulization process and in-situ detection- were carried out using a custom-made calibrated nitromethane vapor generator. 178
Nitromethane, as a volatile liquid, can be detected by trained canines,76 and is suitable for analysis with electronic noses. Peveler et al.77 applied electronic noses technique, a sensor array based on metal oxide semiconductors (MOS), which utilizes basic metal oxides and zeolites for detection of hidden explosive devices. Wang et al.78 developed a method for the detection of nitroalkanes such as nitromethane in mainstream cigarette smoke by a combination of simple sample collection steps and GC-GC/MS analysis. Flanigan et al.79 used laser electrospray mass spectrometry for determination of inorganic improvised explosives from a metallic surface.
However, this method has a high accuracy but it is costly and time-consuming.
NM is also detectable with commercially available combustible gas sensors such as semiconductor or metal oxide sensor technology or volatile organic compound detectors based on photoionization technology. Although all these devices explained in this section are sensitive but, they only can detect a large group of chemical species and are not able to recognize one specific target substance. Thus, more investigations need in order to produce a more selective response to the presence of NM both in liquid or gaseous phases.
Fluorescence-based detection technologies hold promise for future realization of robust, sensitive, and selective portable devices for the safe detection of chemical explosives.
3.7. Paper-Based Sensors
Paper based sensors are a new alternative technology which has many unique advantages over conventional microfluidic devices fabricated using silicon, glass, and polymer as material substrates.80 Power-free fluid transport using capillary action and evaporation of fluids, a high surface area to volume ratio which improves detection limits for colorimetric methods, and compatibility with chemicals/biochemicals are the main advantages of using paper as a sensing platform.81,82 Furthermore, paper is inexpensive, it can be easily modified, safely disposed, and it 179 is scalable. Paper based analytical devices can be used for many applications such as in food industry for quality control,83,84 clinical monitoring and diagnosis of diseases,85–88 and environmental safety and security.89–91 The fabrication methods and the analysis techniques can be modified depending on the application of paper-based sensors. Current paper-based sensors are focused on microfluidic delivery of solution to the detection site. Whitesides and coworkers92 designed µPAD for chemical analysis and the first to introduce the concept of µPADs. The unique aspect of this work lies in the use of a hydrophilic channel confined within a hydrophobic barrier.
3.7.1. Fabrication Techniques
Device fabrication is the core to the novelty of µPADs. There are ten techniques reported in the literature for fabricating µPADs: 1) photolithography,93 2) plotting with an analogue plotter,94
3) ink jet etching,95 4) plasma treatment,96 5) paper cutting,97 6) wax printing,98–100 7) ink jet printing,101,102 8) flexography printing,103 9) screen printing,104 and 10) laser treatment.105 The fundamental principle of these fabrication techniques is to pattern hydrophilic-hydrophobic contrast on a sheet of paper in order to create capillary channels on paper.
Wax printing is the most promising techniques in µPADs fabrication due to using commercially available wax printers, the low cost of patterning agents and easy, rapid fabrication process, high resolution. It is fast and automated, and there is no need of mask. This technique can produce multiple devices or multizones on a piece of paper in a short time. Carrilho et al.99 introduced the wax printing technique as a rapid, inexpensive and efficient process for prototyping a device in under 5 minutes. After printing, the wax printed pages are simply heated by the use of a heat source such as an oven, hot plate or heat gun to melt the wax through the paper to form hydrophilic-hydrophobic contrast.106,107 The heating process allows the wax to penetrate both vertically and horizontally within the paper matrix. The most popular paper used in sensor 180 fabrication is chromatography paper from Whatman. This type of paper is an ideal substrate for fabricating µPADs due to faster transfer of solutions, better analytical performance, smooth surface, uniformity on both sides, medium flow rate, and 0.18-mm thickness that allows printing in commercial machines. Also, it consists of 98% α-cellulose, with no additives such as strengthening or whitening agents reducing the possibility of interference.108–110
3.8. Detection Methods in µPADs
Detection methods in µPADs to detect the presence of an analyte involve colorimetry,111–114 electrochemistry,85,115–117 chemiluminescence,118 electrochemiluminescence,102 photoelectrochemical,119,120 and fluorescence121–124 techniques. The detection technologies are compatible with devices of common use such as cell phones, smartphones, wearable technology, or other imaging devices, scanners, optical drives, and strip readers.125–127
3.8.1. Detection of Explosives Using µPADs
There have been a number of attempts to produce µPADs for the analysis of explosives. For example, in 2013, Taudte et al.128 reported a portable explosive detector based on fluorescence quenching of pyrene deposited on colored wax-printed µPADs. In this study ten different organic explosives, RDX, PETN, TNT, 1,3,5-trinitrobenzene (TNB), 2,4-DNT, 4-amino-2,6- dinitrotoluene (4-A-2,6-DNT), 4-nitrotoluene (4-NT), 1,3-dinitrobenzene (DNB), nitrobenzene
(NB), and 2,4,6-trinitrophenylmethylnitramine (tetryl), were detected under ultra-violet (UV) illumination following sample collection from surfaces using a swab (Figure 3.6).
In 2014, Pesenti et al.129 developed an µPAD in conjunction with confirmation by a lab on chip analysis for detection of three trinitro aromatic explosives. They deposited potassium hydroxide on the µPADs creating a color change reaction in the presence of explosives. Detection of 181 explosives extracted from the µPAD was performed using an indirect fluorescence assay in which the explosive molecules quench the fluorescent dye (DNA dye) present in the buffer, producing a negative peak (Figure 3.7).
Figure 3.6. Illustration of the portable explosive detector prototype. The first step which is not shown in the figure includes inserting the calibration point between the UV LED and the photodiode and turning the calibration knob until the green LED flashes. The second step displayed in the figure shows the detection of explosives on the µPAD (red LED flashes).128
Salles et al.130 introduced a novel approach for the detection and discrimination of five explosives-TATP, HMTD, NB, picric acid , and 4-amino-2-nitrophenol (4-A-2-NP)-by using a paper sensor, a smartphone and chemometric tools. The colorimetric paper-based sensor comprised a disposable wax paper-based array and three reagents (KI, creatinine, and aniline) that produced a unique color pattern for each explosive based on chemical interactions between the explosive species and the chemical reagents. The extracted information from the colorimetric paper-based device consisted of the RGB values of each spot, evaluated using a smartphone and 182 custom software. In 2015, Peters et al.131 utilized µPAD to visually detect improvised explosive.
They applied wax-based µPAD to detect inorganic and organic explosives. In this study colorimetric detection technique was applied by spotting the explosives solution (water or acetone/water) on top of the dried colorimetric reagents at the sample zone. The color changes indicated a positive result. They used two instrumental procedures; CAMAG TLC Scanner 3 and a digital camera-followed by processing with Image J software.
Figure 3.7. Illustrates the steps involved in trinitro aromatic explosive analysis from sample collection and colorimetric detection to identification. 1) µPAD, 2) direct collection after spraying the surface with a methanol/water solution, 3) inactive spot is punched out using a hole-punch, 4−5) the chad is moistened with the electrolyte and extracted by mixing with the pipet for 30 s, 6) the extracted sample is added on the microchip, and 7) analysis using the lab on a chip 2100 Bioanalyzer.129
3.8.2. Fluorescence-Based Detection of Explosives in µPADs
Detection by optical methods is the most inexpensive, simplest method and is universal.132 A variety of detectors can be applied for optical sensing; the technology is compatible with scanners 183 or camera phones to more specialized devices, including spectrophotometers and fluorimeters, to sophisticated devices, e.g., gel documentation systems.
Fluorescence detection method are highly sensitive. This detection technique is especially promising for applications that require a very low limit of detection. A fluorometry device can be used in order to quantify the luminescence.133 Although fluorescence detection in µPADs requires external instrumentation, it was demonstrated to provide good analytical performance in the detection of explosives.134
3.9. Wearable Sensors for Detection of Explosives
Due to the heightened level of national security currently prevalent because of increasing terrorist incidents, highly portable and miniaturized detection tools capable of detecting trace levels of explosives in a faster, simpler, and reliable manner are of the utmost importance.
Therefore, considerable attention has been given to the development of wearable sensor technologies. Electrochemical sensors have been developed widely in recent years.135,136 Textiles are a class of substrates for fabricating wearable electrochemical sensors. Chuang et al.137 examined the influence of textile substrates upon the behavior of wearable screen printed electrodes and sensing properties of these sensors towards the detection of nitroaromatic explosives. Wearable electrochemical sensors for in situ analysis in marine environments was developed by Malzahn et al.138 in 2011. Underwater wearable sensors could monitor the electrochemical response of explosive agents such as TNT in a continuous fashion and could alert the wearer of a potential threat. The sensors were printed on neoprene substrates-commonly used in underwater garments-and used for voltammetric detection of trace levels of Cu and TNT.
184
Chen et al.139 have demonstrated TNT chemical sensing based on ZnO nanowires and also aligned semiconductor single-walled carbon nanotubes (SWNTs) on fabric which can be used for wearable electronics.
In 2013, Bandodkar at al.140 reported a wearable fingertip sensor for voltammetric screening of gunshot residue (GSR) and 2,4-DNT explosive surface residues. They fabricated the Forensic
Fingers through screen-print a three-electrode setup onto a nitrile finger cot, and coat another finger cot with an ionogel electrolyte layer (Figure 3.8). Their detection methodology relies on voltammetry of microparticles (VMP). Voltammeric measurements of the sample are based on bringing the working electrode in direct contact with a second finger cot coated with an ionogel electrolyte.
Figure 3.8. (A) The wearable Forensic Finger exhibiting the three-electrode surface screen-printed onto a nitrile finger cot (bottom left inset), and a solid, conductive ionogel immobilized on a similar substrate (top right inset); (B) VMP detection of samples using a portable electrochemical analyzer (CH Instruments model 1230A) interfaced with a notebook computer; (C) Voltammetric response obtained at Forensic Finger sensor/ionogel interface in the absence (black) and in the presence (red) of (A) GSR and (B) DNT.140 185
In 2017, Sempionatto et al.141 developed a wireless electrochemical wearable ring-based multiplexed sensor platform for monitoring of explosive in vapor and liquid phases (Figure 3.9).
The ring-based sensor system consists of two parts: a set of printed electrochemical sensors and a miniaturized electronic interface which is based on a battery-powered stamp-size potentiostat, for signal processing and wireless transmission of data.
Figure 3.9. Ring-based sensor platform for detecting explosives and nerve agent threats in both vapor and liquid phases.
Eventhough the wearable electrochemical devices provide real-time sensing capabilities, major obstacles still remain that prevent the realization of practical, robust, low-cost wearable sensors suitable for routine use. These limitations include the lack of appropriate methods to effortlessly integrate electronics and wireless transmitters into the sensor package to lead to truly versatile and utilitarian monitoring devices. Therefore, further improvements in the field of wearable sensors are necessary. A substantial amount of effort is still required to further advance the capabilities of such devices in order to realize the promise of truly innocuous sensing in a broad range of applications such as security and detection of explosives. 186
3.10. Sensors Design
The a-CH bond in the lighter nitroalkanes (C1–C3) are considerably acidic (pKa ~17 in
DMSO) 142,143 which makes these nitroalkanes excellent nucleophiles for addition reactions to carbonyl compounds (such as Henry reaction). The nitro-aldol reaction, or Henry reaction,144 is a frequently used synthetic transformation of nitroalkanes. The nitro-aldol reaction was discovered by Belgian chemist Louis Henry in 1895.
OH NO2 O Base (cat.) + O2N R” R R’ H R” R R’
Scheme 3.1. Henry reaction also known as nitro aldol reaction is a base-catalyzed carbon-carbon bond forming reaction between nitroalkanes and aldehydes or ketones.
The reaction is combination of nitroalkane and carbonyl compounds -aldehydes or ketones- to yield b-nitro alcohol. The b-nitro alcohols may undergo water elimination when aryl aldehydes are used to give nitroalkenes and the yield of this reaction will be high in the presence of a catalytic amount of base.145 Base-catalyzed reactions of aromatic aldehydes with nitromethane give nitroalkenes directly. Even though, the Henry reaction between nitroalkane and aldehydes or ketones proceed very slowly; the reaction between nitromethane and aldehydes is particularly simple; just mixing aldehydes, base, and nitromethane. The reaction is generally conducted at room temperature in the presence of about 10 mol% of a base. An efficient base for the Henry reaction is a polyamine base such as polyethylenimine (PEI). The most popular solvents employed in the Henry reaction are water or ethanol.
Thus, we decided to utilize the Henry reaction for detection of nitromethane in ANNM explosives. We used arylaldehyde in the reaction as we believed that the reaction 187 products-nitroalkene-may cause a color change and fluorescence change in the parent aldehydes.
These changes in color and fluorescence can be used to detect nitroalkanes.
To this end, we selected four commercially available aromatic aldehydes, p- dimethylaminobenzaldehyde (DMABC), 2-naphthaldehyde (NapC), 1-pyrenecarboxaldehyde
(PyrC), and 2-fluorenecarboxaldehyde (FluoC). To make the sensors, we mixed these aromatic aldehydes with PEI to form sensor systems S1, S2, S3, and S4, respectively.
Here, the PEI has two roles. First, it anchors the aldehyde, preventing leaching, and makes it water soluble or dispersible through the formation of labile C=N imine bonds. Then, it also acts as the base required for the deprotonation of the nitroalkane and formation of the nitroaldol-Henry product-and drives the dehydration to the unsaturated nitroalkene.145
A
(DMABC) (NapC) (PyrC) (FluoC) p-dimethylaminobenzaldehyde 2-naphthaldehyde 1-pyrenecarboxaldehyde 2-fluorenecarboxaldehyde
B C
= S1 DMABC + PEI S2 = NapC + PEI S3 = PyrC + PEI
S4 = FluoC + PEI (PEI) polyethylene imine
Figure 3.10. (A) Aldehydes used in this study and (B) polyethylene imine used to prepare sensor systems (C) sensors composition.
Figure 3.10 shows the structure of the aromatic aldehydes and branched polyethyleneimine used in this study. S1-S4 comprise different aromatic structures mixed with branched 188
polyethyleneimine in EtOH (96%)-H2O as a solvent. A fluorescence change in the sensor systems upon interaction with desired analytes was expected, enabling the detection of the nitroalkanes.
3.10.1. Materials and Methods
All chemicals were analytical grade and were obtained from commercial suppliers. They were used without purification.
p-Dimethylaminobenzaldehyde (CAS: 100-10-7), 2-naphthaldehyde (CAS: 66-99-9), 1- pyrenecarboxaldehyde (CAS: 3029-19-4), and 2-fluorenecarboxaldehyde (CAS: 30084-90-3) were purchased from Aldrich. Poly(ethyleneimine) (CAS:9002-98-6) was purchased from TCI
(Tokyo, Japan). Nitromethane (CAS: 75-52-5), nitroethane (CAS: 79-24-3), 2-nitropropane (CAS:
79-46-9) were purchased from Aldrich. 1-nitropropane (CAS: 108-03-2) was purchased from Alfa
Aesar. Ammonium nitrate (CAS: 80395-03) was purchased from Science kit Inc (SK). Sulfuric acid (CAS: 7664-93-9), hydrochloric acid (CAS: 7647-01-0), and acetic acid (CAS: 64-19-7) were purchased from EMD. Triethylamine (CAS: 121-44-8) was purchased from Alfa Aesar.
Mass spectrometry measurement. Mass spectrometry was performed using a SHIMADZU
LCMS-2020 electrospray instrument (ESI), and SHIMADZU MALDI TOF-TOF Mass
Spectrometer AXIMA Performance.
UV-Vis measurements. Absorption spectra were acquired using Hitachi U-3010 double beam
UV-Vis spectrophotometer (Tokyo, Japan).
Fluorescence measurements. Steady-state fluorescence emission measurements were performed on Edinburgh single-photon counting spectrofluorimeter FLSP920-stm (Edinburgh
Instruments Ltd., Livingston, UK) at room temperature using quartz cuvette with a path length of
1 cm and with a right-angle detection. The concentration of the samples was adjusted so that the absorbance used for fluorescence measurements was equal to or below 0.1. 189
Absolute quantum yield measurements. Absolute quantum yields were obtained upon excitation at absorption maxima using Hamamatsu Quantaurus-QY C11347-11 Absolute PLQY
Spectrometer equipped with 150 W Xe lamp and multichannel detector/CCD sensor (Hamamatsu,
Japan). Optically dilute solutions used for all photophysical experiments were prepared using
EtOH (96%)-H2O solvents.
3.10.2. Sensors Preparation
Preparation of poly(ethyleneimine) solution 7.5%: Commercially available polyethyleneimine solution 30% in H2O (5 g) was diluted with water (5 mL) and absolute ethanol (10 mL) to prepare polyethyleneimine solution 7.5%.
Preparation of S1: Commercially available p-dimethylaminobenzaldehyde (DMABC, 268 mg,
1.79 mmol) was dissolved in absolute ethanol (17.4 mL). Then polyethyleneimine solution 7.5%
(2.6 mL) was added to get [S1] = 90 mM. The final concentration of polyethyleneimine was 1%.
Preparation of S2: Commercially available 2-naphthaldehyde (NapC, 305 mg, 1.95 mmol) was dissolved in absolute ethanol (17.4 mL) and 2.6 mL of polyethyleneimine solution 7.5% was added to get [S2] = 98 mM. The final concentration of polyethyleneimine was 1%. A milky suspension was obtained over the course of 24 hours.
Preparation of S3: Commercially available 1-pyrenecarboxaldehyde (PyrC, 73 mg, 0.317 mmol) was dissolved in absolute ethanol (17.4 mL). The solution was heated to complete dissolution of the solid and then cooled to room temperature. Polyethyleneimine 7.5% (2.6 mL) was added to get [S3] = 16 mM. The final concentration of polyethyleneimine was 1%. 190
Preparation of S4: Commercially available 2-fluorenecarboxaldehyde (FluoC, 61 mg, 0.314 mmol) was dissolved in absolute ethanol (17.4 mL). Polyethyleneimine 7.5% (2.6 mL) was added to get [S4] = 16 mM. The final concentration of polyethyleneimine was 1%.
3.10.3. Analytes Preparation
We used nitromethane (NM), nitroethane (NE), 1-nitropropane (1-NP), 2-nitropropane (2-NP), ammonium nitrate (AN), mineral acid (H2SO4), triethylamine (TEA), and acetic acid (AcOH) as analytes in this study. Solution of the analytes NM, NE, 1-NP, and 2-NP were prepared in 10.0 mL of a water / ethanol mixture (ethanol <5%), to guarantee solubility of the analytes. AN and
H2SO4 solutions were prepared in water (10.0 mL).
To prepare 10 mL of 485 mM stock solution of NM:
Weigh out 279 mg (4.57 mmol) of NM (MW=61.04) in a suitable vial and add 10.0 mL of a water
/ ethanol mixture solvent (ethanol <5%).
To prepare 10 mL of 513 mM stock solution of NE:
Weigh out 385 mg (5.13 mmol) of NM (MW=75.07) in a suitable vial and add 10.0 mL of a water
/ ethanol mixture solvent (ethanol <5%).
To prepare 10 mL of 503 mM stock solution of 1-NP:
Weigh out 448 mg (5.03 mmol) of NM (MW=89.09) in a suitable vial and add 10.0 mL of a water
/ ethanol mixture solvent (ethanol <5%).
To prepare 10 mL of 436 mM stock solution of 2-NP: 191
Weigh out 388 mg (4.35 mmol) of NM (MW=89.09) in a suitable vial and add 10.0 mL of a water
/ ethanol mixture solvent (ethanol <5%).
To prepare 10 mL of 273 mM stock solution of AN:
Weigh out 218 mg (2.72 mmol) of NM (MW=80.04) in a suitable vial and add 10.0 mL of a water.
To prepare 10 mL of 15.7 mM of H2SO4:
Diluted 62.8 µL of the H2SO4 (2.5 M) stuck solution in 10.0 mL of a water.
3.11. Complex Formation: Mass Spectrometry
We confirmed the formation of the nitroalkene products in sensor systems S1–S4 using
MALDI-TOF Mass Spectrometry. Figure 3.11 shows MALDI-TOF spectra of the NM with S1-S4 complexes. The spectrum suggests the binding stoichiometry of the complexes formed to be 1 : 1.
DHB (2,5-dihydroxybenzoic acid) has been used as the matrix in this experiment. The samples for MALDI-TOF experiment were prepared as follow. Spotted 0.5 µL of matrix in the well of
MALDI target plate and let dry for a few minutes. Then, 0.5 µL of [S1] = 90 mM, [S2] = 98 mM,
[S3] = 16 mM, or [S4] = 16 mM was added to the dried matrix. Right after that 4×0.5 µL of the nitromethane solution (200 mM), then 0.5 µL DHB were added to each well and let dry for 10 minutes.
192
+ Figure 3.11. MALDI-TOF mass spectra of (A) [S1+NM-H2O] = 192.1, Inset: Calculated isotope + + pattern for [S1+NM-H2O] = 192.1, (B) [S2+NM-H2O] = 199.1, Inset: Calculated isotope pattern + + for [S2+NM-H2O] = 192.1, (C) [S3+NM-H2O] = 273.1, Inset: Calculated isotope pattern for + + [S3+NM-H2O] = 273.1, (D) [S4+NM-H2O] = 237.1, Inset: Calculated isotope pattern for + [S4+NM-H2O] = 237.1.
3.12. Photophysical Studies and Fluorescence Titrations
Photophysical properties of sensors S1-S4 (Table 3.1) were measured in EtOH (96%)-H2O at room temperature and without previous degassing.
3.12.1. Solid State Fluorescent Spectra of S1-S4
Solid state fluorescence measurements were performed using a sample holder oriented at a 45o angle on the x-y plane between the excitation source and the detector and facing upward at a 45o angle from the x-y plane to minimize scattering and direct reflection. The sample was prepared by spotting 0.6 µL of S1 (90 mM), S2 (98 mM), S3 (16 mM), and S4 (16 mM) solutions onto chromatographic paper. AN (485 mM) and NM (273 mM) were added by spotting 0.5 µL of their solutions. 193
According to the solid-state fluorescence result (Figure 3.12), S1 did not respond in an appreciable manner to AN. S2 and S4 showed a detectable fluorescence increase, albeit lower in magnitude compared to S3. In the case of S3 the fluorescence spectra show an interesting non- monotonic behavior.
S1-S4 were excited at 380 nm, 364 nm, 410 nm, and 354 nm, respectively. In the case of S1 and S4, the band pass of the excitation and emission monochromator were set to 1 and the emission was scanned in 2 nm steps with a dwell time of 0.2 sec. For S3 the band pass of the excitation was set to 1 nm and the band pass of the emission monochromator were set to 0.5 nm and the emission was scanned in 2 nm steps with a dwell time of 0.2 sec. For S2 the band pass of the excitation was set to 0.5 nm and the band pass of the emission monochromator was set to 2 nm and the emission was scanned in 2 nm steps with a dwell time of 0.2 sec. All fluorescence titrations were performed under ambient conditions.
194
5 3x10 S1 S2 S1+NM 3x105 S2+AN S1+AN S2+NM
2x105 2x105
I / a. u. a. / I I / a. u. a. / I 5 1x10 5 1x10
0 0 400 450 500 550 600 650 400 450 500 550 600 l l / nm / nm 4x105 S3 S4 5 S3+AN S4+AN 6x10 S3+NM S4+NM 3x105 5x105
5
2x10 5 3x10 u. a. / I I / a. u. a. / I
5 5 1x10 2x10
0 0 450 500 550 600 650 700 400 450 500 550 600 l / nm l / nm
Figure 3.12. Solid state fluorescence spectra of S1-S4, S1-S4 in presence of NM, and S1-S4 in presence of AN at (A) λexc,S1 = 380 nm, (B) λexc,S2 = 364 nm, (C) λexc,S3 = 410 nm, (D) λexc,S1 = 354 nm in room temperature.
3.12.2. Quantum Yields of S1-S4
Absolute quantum yield measurements are summarized in table 3.1. The formation of sensor system S3 (PyrC + PEI) was accompanied with a dramatic decrease in quantum yield compared to other sensor systems. Sensor system S3 best fulfills the requirements as it displays a considerably higher quantum efficiency. Therefore, we have concentrated more on sensor system S3 for the detection of nitroalkanes.
195
Table 3.1. Absorption (lA, max) and absolute quantum yields (F ) of aldehyde parents and sensor systems S1-S4.
lA, max F lA, max F Aldehyde Sensor (nm) [%] (nm) [%] DMABC 338 0.43 S1 334 0.34 p-dimethylaminobenzaldehyde NapC 250 4.88 S2 250 4.56 2-naphthaldehyde PyrC 287 5.5 S3 346 1 1-pyrenecarboxaldehyde FluoC 269 0.96 S4 317 0.46 2-fluorenecarboxaldehyde Absolute quantum yields were determined upon excitation at wavelength indicated for solutions with optical density A = 0.1 (all errors < 4%). All measurements were carried out in non-deoxygenated solutions.
3.12.3. Fluorescence Titrations of S3 in Solution
Since we observed strong fluorescence amplification with AN for the sensor systems S3 and
S4 in the solid-state fluorescence experiments, we decided to do fluorescence study of sensor systems S3 and S4 in solution as well. The sensor system S4 did not show sufficient change in fluorescence emission in solution upon addition of desired analyte, but sensor system S3 showed significant change in fluorescence emission of the sensor in presence of AN and NM. The titrations were accomplished as follows: The sensor S3 (l = 346 nm) solution (0.1 µM) in EtOH (96%)-H2O in cuvette were titrated with stock solution of NM (0-32 mM) or AN (0-30 mM). The fluorescence spectrum was subsequently recorded and titration isotherms were plotted and binding constants calculated using 1:1 binding model by Newton’s iterative method.146,147
The shape of the spectrum of the pyrene in S3 displayed both the monomer emission (p - p*,
148–151 lmax = 410 nm) with a significant contribution of the excimer emission (lmax = 520 nm).
Fluorescence emission of sensor system S3 upon the addition of an incremental amounts of a NM solution (in ethanol) was quenched (Figure 3.13.A). The change in fluorescence intensity is due to 196 the formation of the imine and to the presence of free amino-groups which are known fluorescence quenchers via photoinduced electron transfer. The monomer and excimer emissions of S3 decrease upon formation of the non-fluorescent nitroalkene (Figure 3.13.A) and the fitting of the titration isotherms allowed us to determine an apparent affinity constant of 630 M-1. In contrast with the addition of NM, the addition of an incremental amounts of AN solution (96% ethanol–water) exhibited a dramatic increase in fluorescent intensity. This is likely due to the protonation of the amine moieties of PEI and hydrolysis of the imine bond between the fluorescent PyrC and PEI.
First, the excimer emission at 520 nm increases in intensity, then, a new spectral feature with a lmax = 455 nm emerges (Figure 3.13.B), while the excimer emission decreases. These dramatic changes arise from two different processes. Upon addition of the first aliquots of NH4NO3, protonation of the free amine-groups takes place, creating an ionic, more polar environment that
+ stabilizes the formation of excimer of the pyrene fluorophore. Then, with excess of NH4 the imine bond is hydrolyzed, and the fluorescence of the monomeric PyrC is restored. This is confirmed by comparing it to the fluorescence of free PyrC, with an emission centered at 455 nm.148–151 The corresponding apparent affinity constant is 13600 M-1.
Solution of S3 were excited at 346 nm. Fluorescence emission spectra were recorded between
360 nm and 650 nm. The band pass of the excitation monochromator was set to 0.4 nm for AN and 2 nm for NM. The band pass of the emission monochromator was set to 0.5 nm for both AN and NM. The emission was scanned in 2 nm steps with a dwell time of 0.2 sec under ambient conditions. Titration isotherms were constructed from the change in the fluorescence maxima at
398 nm.
197
A B
Figure 3.13. Fluorescence titration spectra and fluorescence titration isotherm (Insets) of S3 (0.1 µM) upon the addition of an incremental amounts of (A) [NM] = 0-32 mM (B) [AN] = 0-30 mM in EtOH (96%)-H2O. lEXC = 346 nm.
3.13. Qualitative Analysis
The first qualitative analysis has been done by taking a photograph of fluorescence under the black light (lEXC = 365 nm). Figure 3.13 shows the fluorescence of solutions of PyrC (1), sensor system S3 (2), S3 + NM (3), S3 + AN (4), and S3 + ANNM (5). As expected, an identical behavior to the one observed by fluorescence titration spectra was observed by naked eye. This experiment suggests that the fluorescence will be attenuated in the presence of NM and, amplified in the presence of AN.
198
1 2 3 4 5
lex=365nm
Figure 3.14.1: A PyrC photograph2: PyrC+ of thePEI fluorescence(S3) 3: S3 under+NM a 4:handheld S3+AN UV-light 5: S3 +ANNMof 1: PyrC (0.1 mM), 2: S3 (0.1 mM), 3: S3 (0.1 mM) + NM (30 mM), 4: S3 (0.1 mM) + AN (30 mM), and 5: S3 (0.1 mM) + ANNM (26 mM).
3.14. Paper Microzone Plate-Based Qualitative Assay
To increase the potential field applicability of this method, we performed a similar test in paper microzone plates by printing hydrophobic barriers on paper using a wax printer. The plates were printed on chromatography paper (Whatman) with a Xerox ColorQube model 8570 wax printer.
The diameter of a zone on the paper microzone plate was 2.95 ± 0.04 mm and the total size of the plate was 10.2 ± 0.36 × 22.1 ± 0.17 mm (Figure 3.15). After printing, it was baked in an oven for
4.5 minutes at 110 °C to allow for the penetration of wax into the paper. Finally, the back side of the paper was covered with transparent scotch tape (3 inch). The paper microzone plates were explained in more details in the introduction part of this chapter.
The array experiment was performed as follows. Solutions of sensor S3 (15.8 mM) were applied (500 nL per microzone) into paper microzone plate using 2 µL microsyringe (Hamilton,
Reno, NV) and the solvents let dry for 5 min. After that, the solutions of the analytes in EtOH
(96%)-H2O solution (500 nL, 200 mM) were pipetted into each single well of paper microzone plate and let dry at room temperature for 15 min. The sensor responses could be recorded as images 199 using an UV-scanner capable to read the fluorescence images. The fluorescence images were recorded using Kodak 440CF image station as a sum of the RGB channels recorded for the measurements.
1 2 3 4 5
Figure 3.15. Fluorescence image from the paper microzone array of 1: PyrC, 2: S3, 3: S3 + NM, 4: S3 + AN, and 5: S3 + ANNM as a sum of the RGB channels recorded for the measurements.
Figure 3.15 shows the paper microzone plate. In this experiment, the paper microzone plate consists of five microzone spots, where the spot 1 corresponds to PyrC, spot 2 corresponds to S3
(PyrC + PEI), spot 3 corresponds to S3 + NM, spot 4 to S3 + AN, and spot 5 to S3 + ANNM.
As expected from the solution experiments the fluorescence was quenched in the presence of
NM and strongly amplified in the presence of AN. Finally, a net amplification of fluorescence was observed for ANNM. Interestingly, a change in the emission color from excimer green (PyrC) to a monomer-like blue emission was observed (S3 + AN and S3 + ANNM).
The change in fluorescence color from green (lmax = 520 nm, excimer emission) is due to the dislodging of the monomers from the excimer in the presence of nitromethane (formation of the nitroalkene), or ammonium ions (protonation of amines/hydrolysis of the imine), thus restoring the monomer blue emission (lmax = 460 nm), as confirmed by the solution experiments.
Furthermore, optical sensing array was studied using fluorescence image under the black light.
In this experiment also, the plates were printed on chromatography paper (Whatman) with a Xerox
ColorQube model 8570 wax printer. The diameter of a zone on paper microzone plates was 2.95
± 0.04 mm and the total size of the plate was 23.1 ± 0.36 × 21.3 ± 0.17 mm. After printing, it was baked in an oven for 4.5 minutes at 110 °C to allow for the penetration of wax into the paper. The 200 back side of the microzone plate was covered with transparent scotch tape (3 inch). Next, 600 nL of the sensors ([S3], [S4] = 16 µM), Rhodamine B (3 µM, H2O), and Fluorescein (3 µM, EtOH) were added on the microzones, then 200 nL of NM, AN, ANNM (60:40) added on the microzones
(Figure 3.13). The picture was taken under a handheld UV-Vis lamp using a smartphone.
Figure 3.16. The sensor array fluorescence under black light (365 nm) and different responses to the presence of the analytes (NM, AN, and ANNM).
In addition to S3 and S4, two fluorescent indicators and pH dependent luminescent probes, rhodamine B and fluorescein, have been used to further probe the potential fluorescence response to pH of the tested analytes. Rhodamine B and fluorescein did not show significant change in their fluorescence in the presence of each analyte. Therefore, it seems that the fluorescence responses do not depend on pH. As it is obvious from the fluorescence responses in Figure 3.16 only sensor system S3 showed obvious different fluorescence responses to the presence of AN, NM, and
ANNM compare to the S4, rhodamine B, and fluorescein.
201
3.15. Paper Microzone Array-Based Vapor Sensing
An array-based vapor sensing experiment was designed to demonstrate the utility of this sensing technique for analytes in the vapor-phase. This array designed to detect and analyse chemically diverse species. In this study, we decided to employ a paper microzone plate approach.
The plate was printed on chromatography paper (Whatman) with a Xerox ColorQube model 8570 wax printer as explained before. The diameter of a zone on paper microzone plates was 3.95 ±
0.04 mm and the total size of the plate was 31.1 ± 0.36 × 8.3 ± 0.17 mm (Figure 3.17). After printing, it was baked in an oven for 4.5 minutes at 110 °C to allow for the penetration of wax into the paper. The back side of the microzone plate was covered with transparent scotch tape (3 inch).
1 2 3 4 5
Figure 3.17. The designed paper microzone plate for five repetition experiments.
We chose nitroalkanes, acids, and bases as analytes and possible interferents. The analytes used were ammonia, acetic acid, triethylamine, 1-nitropropane, 2-nitropropane, nitroethane, and nitromethane. In this qualitative analysis each array experiment was performed as follow. 600 nL of sensor (S1-S4) was applied on microzones of a paper array. A few drops of analyte were place in a jar on a piece of cotton and allowed to equilibrate with the air in the jar. The microzone plates were introduced in the jars and exposed to the vapors of the analyte for 5 minutes at 25°C.
The fluorescence images of the microzone plates exposed to analyte vapors were acquired. The sensor responses were then recorded as images using an UV-scanner (blue and green color 202 channels) capable to read the fluorescence images and the responses were evaluated using standard pattern recognition protocol. The linear discriminant analysis (LDA) experiment provided an excellent separation of the data clusters suggesting that the array is able to distinguish between eight analytes and a control (total of nine analytes). Excellent recognition capability of the sensors is reflected by the 100% correct classification of the acidic compounds, basic compounds, C1–C3 nitroalkanes and ammonium nitrate. Figure 3.18 shows the results of the linear discriminant analysis.
As expected, NM and NE cluster together while 1NP and 2NP form well separated clusters.
This behavior is presumed to be due to the inability to form the nitroalkene product from the corresponding nitroaldol. Furthermore, bases such as ammonia (NH3, as ammonium hydroxide) and TEA also cluster separately. Finally, AN forms a cluster separate from all other cluster groups.
The coefficient of variability among the data within the class of 5 repetitions was lower than 3%.
Validation of the analysis was confirmed with cross validation and 100% classification was achieved (Table 3.2).
In summary, the paper microzne array-based vapor sensing using S1-S4 provided 100% correct classification and showed capability to discriminate relevant analytes used in this experiment.
203
100% classification
AN NH 3 CTRL AcOH TEA 0 NM F3 (1.3%) 2NP NE 1NP AN -30 2NP NE 1NP -60 NM 200 AcOH
-700 150 NH -600 3 -500 100 -400 TEA F1 (92.1%)-300 50 -200 CTRL F2 (6%) -100 0
0 100 -50
Figure 3.18. LDA corresponding to the paper microzone array. The cross-validation shows 100% correct classification of all 8 analytes and a control. All analytes except AN were applied as vapor. AN, because of the low vapor pressure was applied to the array as a solution.
Table 3.2. The jackknifed classification matrix of the qualitative assay for S1-S4 in vapor phase. Jackknifed Classification Matrix 1NP 2NP AN AcOH Blank NE NH3 NM TEA %correct
1NP 5 0 0 0 0 0 0 0 0 100 2NP 0 5 0 0 0 0 0 0 0 100 AN 0 0 5 0 0 0 0 0 0 100 AcOH 0 0 0 5 0 0 0 0 0 100 Blank 0 0 0 0 5 0 0 0 0 100 NE 0 0 0 0 0 5 0 0 0 100 NH3 0 0 0 0 0 0 5 0 0 100 NM 0 0 0 0 0 0 0 5 0 100 TEA 0 0 0 0 0 0 0 0 5 100 Total 5 5 5 5 5 5 5 5 5 100
204
3.16. Paper Microzone Array-Based Sensing in Solution
To demonstrate the utility of the sensing technique in solution phase, we designed a qualitative assay. Toward this end, we fabricated a paper microzone plate to be used in solution phase analysis.
As analytes we selected amoniumnitrate, nitromethane, nitroethane, 1-nitropropen, and acid-sulfuric acid-as analytes.
The plates were printed on chromatography paper (Whatman) with a Xerox ColorQube model
8570 wax printer. The diameter of a zone on the paper microzone plate was 3.95 ± 0.04 mm and the total size of the plate was 62.1 ± 0.36 × 56.1 ± 0.17 mm. After printing, it was baked in an oven for 4.5 minutes at 110 °C to allow for the penetration of wax into the paper. Then, the back side of the paper was covered with transparent scotch tape (3 inch). These plates were utilized for qualitative classification of analytes using water as the solvent.
1 2 3 4 5 6 a b c d e f
Figure 3.19. The designed paper microzone plate for linear discriminant analysis in solution for four sensors (S1-S4), six analytes and repetition experiments.
The array experiments were performed as follows: 600 nL of S1, S2, S3, and S4 solutions were applied to the zones. For this qualitative analysis, [NM] = 458 mM, [NE] = 513 mM, [1NP] = 503 205 mM, [2NP] = 436 mM were prepared as described before. [AN] = 273 mM and [H+] = 15.7 mM
(H2SO4) were dissolved in H2O. Then 500 nL of each analyte were added on the zones and fluorescence intensities were collected using Kodak 4000MM Pro image station. The coefficient of variability among the data within the class of 6 repetitions was lower than 3%. Fluorescence intensities were classified by using LDA without any further pretreatment. Validation of the analysis was confirmed with cross validation and 100% correct classification was achieved (Table
3.3).
Figure 3.20. Graphical output of qualitative LDA for sensors S1-S4 in EtOH (96%)-H2O with various analytes. The array was achieved using 13 excitation/emission channels for each sensor, 6 repetitions and provided 100% correct classification.
206
Table 3.3. The jackknifed classification matrix of the qualitative assay for S1-S4 in solution. Jackknifed Classification Matrix 1NP AN Blank H+ NM NE %correct
1NP 6 0 0 0 0 0 100 AN 0 6 6 0 0 0 100 Blank 0 0 0 6 0 0 100 H+ 0 0 0 0 6 0 100 NM 0 0 0 0 0 6 100 NE 0 0 0 0 0 0 100 Total 6 6 6 6 6 6 100
The paper microzone array for sensing in solution phase using S1-S4 provided 100% correct classification and showed capability to discriminate relevant analytes used in this experiment.
3.17. Hierarchical Clustering Analysis (HCA)
HCA was performed to quantify differences among the analytes. HCA for the 10 different analytes can be seen in Figure 3.21.
Data were reduced to 5 repetitions in each class and subjected to HCA using Minitab.152 Unlike
LDA, HCA does not reduce dimensions of the data set. HCA seeks classification of the samples by clustering data points based on relative distances in the n-dimensional space to one another and groups them in hierarchical manner. In this research, we used the most common clustering criterion
(Ward’s minimum variance) method, which takes into consideration the minimum amount of variance between analytes to define a cluster. There is clear discrimination among all ten analytes.
The resultant dendrogram shows connectivity and distance between each analyte. (Figure 3.21)
As expected, NM and NE cluster together, 1NP and 2NP cluster together, bases ammonia and
TEA cluster together, and hydrochloric acid and sulfuric acids also cluster separately.
207
Control HCl NM NE 1NP 2NP TEA NH OH AcOH 4
Figure 3.21. Hierarchical clustering analysis dendrograms (Euclidean Distance, Ward's Linkage) for sensors S1-S4 and 10 analytes displays 100% correct classification.
3.18. Non-Woven Nanofiber Mats of S3
Electrospinning is an electrostatic fiber fabrication technique. They have a diverse application in tissue engineering, biosensors, filtration, drug delivery, etc.153–155 The nanoscale fibers are generated by the application of strong electric field on polymer solution or melt.
Electrospinning has been used to synthesize nanofibers for various applications as it allows a good control over the composition, structure, and mechanical properties of the resulting material.156
Electrospinning needs a high-voltage power supply, a syringe pump, a polymer solution to be spun, and a grounded collection surface. Polymer solutions are exposed to a high-voltage power supply and delivered with a syringe through a blunt-tip needle using a syringe pump. Nanofibers collected onto a specially designed grounded placed at a specific distance from the needle tip.156–158
For the preparation of non-woven nanofiber mats, S3 fibers were electrospun from a solution containing S3 (50 mM), tridodecylmethylammonium chloride (TDMACl) (50 µM) to increase the 208 conductivity and the mass flow,159 and 12% w/w of Tecoflex™ (EG-80A, from Lubrizol®) in
THF/EtOH 2:1. S3 solution was electrospun from an 8.5 cm height target-to-collector. A typical voltage applied was 5.5 kV and the injection rate was set to 0.198 mL/h. A shadow mask was utilized to electrospin S3 on top of four microscope slides (Figure 3.22).
Then, a few drops of nitromethane were applied to S3 fibers on the microscope slide. After 5 minutes, fluorescence of the S3 fibers under black light (256 nm) was quenched upon exposing to nitromethane (Figure 3.22.B). The S3 fibers exposed to the ammonium nitrate solution (in water)
(Figure 3.22.C) for 5 minutes, then the fluorescence of the S3 fibers under black light (256 nm) showed enhancement upon exposing to ammonium nitrate. Finally, a net amplification of fluorescence was observed upon exposing the S3 fibers on the microscope slide to ANNM. (Figure
3.22.D).
A B C D
l exc = 256 nm
Figure 3.22. Nanofiber mat deposited on a microscope slide (A) S3 mat fluorescence under black light (256 nm) (B) S3 mat exposed to NM; resulting in fluorescence quenching as seen under black light. (C) S3 mat exposed to AN (in water); resulting in fluorescence enhancement as seen under black light. (D) S3 mat exposed to the ANNM. The picture was taken under a handheld UV-Vis lamp using a smartphone.
3.19. Wearable Nanofiber Sensor
To test the utility of S3 in wearable sensing devices the S3 sensor material was doped into polyurethane and the resulting weakly fluorescent material was electro-spun into non-woven nanofiber mats which can be deposited to form a conformal coating on any kind of surface. 209
Depositing and electrospining conditions were the same as the conditions used in depositing the non-woven nanofiber mat on the microscope slides. S3 fibers were electrospun from a solution containing S3 (50 mM), TDMACl (50 µM), and 12% w/w of Tecoflex™ (EG-80A, from
Lubrizol®) in THF/EtOH 2:1. The height between target and collector was 8.5 cm. A 5.5 kV voltage was applied and the injection rate was set to 0.198 mL/h. The nanofiber mat of S3-doped polyurethane deposited onto a nitrile glove finger (Figure 3.23.A and B). Then, the wearable sensor was exposed to AN, NM, or ANNM explosive by simply touching a contaminated surface. The clearly observable changes in fluorescence color and intensity were also identical to those observed in paper analytical microdevices (Figure 3.23.C). The S3 fiber’s fluorescence was quenched upon exposing the wearable sensor to nitromethane contaminated surface. As expected, the fiber fluorescence was enhanced upon touching the ammonium nitrate contaminated surface. Also, the wearable sensor showed a different fluorescence change when it was exposed to ANNM. The pictures were taken under a handheld UV-Vis lamp using a smartphone.
A B
C
S3 S3+NM S3+AN S3+AN-NM (PyrC+PEI)
Figure 3.23. (A) and (B) Nanofiber mat deposited on the finger of a nitrile glove. (C) The wearable sensor displays dramatic changes in color in the presence of NM as well as increase in fluorescence in the presence of AN and ANNM. 210
3.20. Summary
In summary, we have developed an easy-to-use optical sensor for both vapor-phase and solution-phase identification of explosive mixtures that uses a cross-reactive fluorimetric sensor array comprising chemically responsive fluorescent indicators composed of aromatic aldehydes and polyethyleneimine. We have established a simple fluorescent sensor for nitromethane and ammonium nitrate. Sensor system S3 (PyrC + PEI) was found to be particularly sensitive. Its fluorescence is quenched by nitromethane, a component of explosive ANNM, via the formation of a non-emissive nitroalkene. In contradistinction, an increase of emission intensity is observed in the presence ammonium nitrate, a component of ANFO. The latter has been attributed to the protonation of the free amines and to the hydrolysis of the imine bond, as confirmed by solution studies. Paper microzone arrays comprising the four sensor systems showed a marked response to
AN, NM, and ANNM, and inspired us to prepare nanofiber-based sensors. Thus, non-woven nanofiber mats were fabricated with S3 doped into electrospun polyurethane nanofibers. This nanofiber sensor was deposited on a variety of substrates, including gloves, to illustrate the potential of this sensor for the fabrication of wearable sensors. The wearable sensor displayed a strong and fast response to all the analytes in the assay particularly AN, NM, and ANNM with strong changes in fluorescence color and intensity. Furthermore, a paper microzone array comprising sensor systems S1–S4, was tested for its capacity to discriminate between a set of 9 different analytes achieving 100 % correct classification.
211
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