Accepted Manuscript

Title: Cationic permethylated6-monoamino-6-monodeoxy-�-cyclodextrin as chiral selectorof dansylated amino acids in capillary electrophoresis

Author: Krisztina Nemeth Celesztina Domonkos ViragSarnyai Julianna Szeman Laszlo Jicsinszky Lajos Szente JuliaVisy

PII: S0731-7085(14)00316-1DOI: http://dx.doi.org/doi:10.1016/j.jpba.2014.06.028Reference: PBA 9631

To appear in: Journal of Pharmaceutical and Biomedical Analysis

Received date: 18-4-2014Revised date: 17-6-2014Accepted date: 18-6-2014

Please cite this article as: K. Nemeth, C. Domonkos, V. Sarnyai, J. Szeman, L.Jicsinszky, L. Szente, J. Visy, Cationic permethylated 6-monoamino-6-monodeoxy-rmbeta-cyclodextrin as chiral selector of dansylated amino acids in capillaryelectrophoresis, Journal of Pharmaceutical and Biomedical Analysis (2014),http://dx.doi.org/10.1016/j.jpba.2014.06.028

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Cationic permethylated 6-monoamino-6-monodeoxy-β-cyclodextrin as chiral selector of 1

dansylated amino acids in capillary electrophoresis 2

3

Krisztina Németh1*, Celesztina Domonkos1, Virág Sarnyai1, Julianna Szemán2, László 4

Jicsinszky2, Lajos Szente2, Júlia Visy1 5

1Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy 6

of Sciences, H-1519 Budapest P.O.B.:286, Hungary 7

2CycloLab R&D Ltd., H-1097 Budapest, Illatos út 7, Hungary 8

9

*Corresponding author: K. Németh 10

Postal address: 1Institute of Organic Chemistry, Research Centre for Natural Sciences, 11

Hungarian Academy of Sciences, H-1519 Budapest P.O.B.:286, Hungary 12

Tel.: +36 1 382 6659; E-mail address: nemeth.krisztina@ttk.mta.hu 13

14

15

Highlights 16 17 • Permethylated 6-monoamino-6-monodeoxy-βCD resolves dansylated amino acids. 18 • Maximal chiral resolutions could be achieved at pH 6 or pH 4. 19 • The separations improved with increasing concentration of the selector. 20 • Low CD concentration was enough for the separation of the most apolar Dns-AAs. 21 • Resolution power of PMMABCD was proved by the separation of Dns-AAs’ mixture. 22 23

24

25

Abstract 26

27

The resolution power of permethylated 6-monoamino-6-monodeoxy-βCD 28

(PMMABCD) - a single isomer, cationic CD derivative - developed previously for chiral 29

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analyses in capillary electrophoresis was further studied here. Dansylated amino acids (Dns-30

AA) were chosen as amphoteric chiral model compounds. Changes in the resolutions of Dns-31

AAs by varying pH and selector concentrations were investigated and correlated with their 32

structures and chemical properties (isoelectric point and lipophilicity). Maximal resolutions 33

could be achieved at pH 6 or pH 4. The separations improved with increasing concentration of 34

the selector. Baseline or substantially better resolution for 8 pairs of these Dns-AAs could be 35

achieved. Low CD concentration was enough for the separation of the most apolar Dns-AAs. 36

Chiral discrimination ability of PMMABCD was demonstrated by the separation of an 37

artificial mixture of 8 Dns-AA pairs. 38

39

Keywords: cationic CD derivative, dansylated amino acid, enantioseparation, isoelectric 40

point, lipophilicity 41

42

Abbreviations: 43

Dns-AA: dansylated amino acid; Dns-Abu: dansylated α-amino butyric acid; Dns-Asp: 44

dansylated aspartic acid; Dns-Glu: dansylated glutamic acid; Dns-Leu: dansylated leucine; 45

Dns-Met: dansylated methionine; Dns-Nle: dansylated norleucine; Dns-Nva: dansylated 46

norvaline; Dns-Phe: dansylated phenylalanine; Dns-Thr: dansylated threonine; Dns-Trp: 47

dansylated tryptophan; Dns-Val: dansylated valine; PMMABCD: permethylated 6A-48

monoamino-6A-monodeoxy- β-cyclodextrin; 49

50

51

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1. Introduction 51

52

Analytical methods particularly capillary electrophoresis (CE) using chiral selectors 53

are appropriate for stereoselective separations [1-6]. Cyclodextrin (CD) derivatives are one of 54

the most favorable selectors to be applied in CE [7]. Development of new chiral selectors, 55

including charged cyclodextrins is important due to the increasing demand to analyze a great 56

number of pharmaceutical, environmental, and nutritional compounds. The advantage of the 57

charged selectors is that they provide relatively large separation window in the case of the 58

oppositely charged analytes [8]. Great numbers of anionic and even cationic CD derivatives 59

have been synthesized and released in the last decades [9-21]. Amino substituted CDs are 60

among the most widely investigated new selectors. Application of cationic –principally 61

amino- CD derivatives for chiral separation of several underivatized amino acids (AA) or 62

their fluorescent derivatives - for more sensitive detection –has been demonstrated [8,13,14-63

16,18-25]. Iványi et al. [26] reported that permethylated 6-monoamino-6-monodeoxy-βCD 64

(PMMABCD) developed previously as stereoselective agent for CE [27] was a good chiral 65

selector for dansyl-phenylalanine and dansyl-leucine as well as for many other – acidic - 66

chiral substances [26,28-32]. This cationic (pKa = 9.05 [28]), single-isomer βCD derivative is 67

completely O-methylated and substituted by an amino group in C6 position of one of the 68

glucopyranoside units (Degree of substitution = 20+1). The advantage of single isomer 69

selectors over randomly substituted ones is their outstanding batch-to-batch reproducibility. 70

Our study aimed to demonstrate the applicability of PMMABCD in chiral separation 71

of further dansylated amino acids (Dns-AAs) as amphoteric model compounds. Using Dns-72

AAs is explained by the increasing importance of the evaluation of the enantiomer ratio of 73

amino acids in biological samples and in food analysis [33-38] since the biological activity, 74

pharmacology and toxicology of D-amino acids are widely studied [39-42]. The pH and 75

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selector concentration dependencies of chiral resolutions were characterized and correlated 76

with the charged states of the analytes and selector and with the structure and lipophilicity of 77

the analytes. The chiral resolution power of PMMABCD was further examined with an 78

artificial mixture of Dns-AAs. 79

80

2. Materials and methods 81

82

2.1. Materials 83

84

Background electrolyte (BGE) components phosphoric acid, sodium hydroxide, 85

glacial acetic acid, boric acid and ethanol were purchased from Merck GmbH (Darmstadt, 86

Germany), eleven racemic (Table 1) and nine L-Dns-AA were obtained from Sigma-Aldrich 87

(St.Louis, USA). PMMABCD was the product of CycloLab Ltd. (Budapest, Hungary). Its 88

synthesis route has been described in details [27]. 89

90

2.2. Capillary electrophoresis 91

92

Capillary electrophoresis was performed with an Agilent Capillary Electrophoresis 93

3DCE system (Agilent Technologies, Waldbronn, Germany) applying bare fused silica 94

capillary having a 64.5 cm total and 56 cm effective length with 50 μm I.D. (Agilent 95

Technologies, Santa Clara, CA, USA). On-line absorption at 220 nm was monitored by DAD 96

UV-Vis detector. The capillary was thermostated at 25°C. Between measurements, the 97

capillary was rinsed subsequently with 0.1 M HCl, 1.0 M NaOH, 0.1 M NaOH and distilled 98

water for 3 min each and with BGE for 5 min. Various BGE buffers were prepared either 99

from 82 mM boric acid (pH 9); or from 16 mM phosphoric acid (pH 8); or from 22 mM 100

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phosphoric acid (pH 7); or from 35 mM phosphoric acid (pH 6); or from 65 mM acetic acid 101

(pH 5); or from 255 mM acetic acid (pH 4); or from 50 mM phosphoric acid (pH 3) with 102

sodium hydroxide to attain the desired pHs as indicated and the same ionic strength (I= 44 103

mM). The appropriate amounts of PMMABCD were dissolved in the running buffers. Dns-104

AA samples were dissolved in 50% ethanol and further diluted with distilled water, final 105

concentrations were 0.1 mg/ml. The racemic Dns-AAs were spiked by the pure L-enantiomers 106

(cL-Dns-AA/cD-Dns-AA= 7/3) – with the exception of Dns-Nle and Dns-Abu, their pure ‘L’ 107

enantiomers were lacking. The artificial mixture was prepared by mixing the stock solutions 108

of the Dns-AA samples and further diluted with distilled water. The ethanol content of the 109

mixture was higher, the concentrations of Dns-AAs were 0.03 mg/ml and enantiomer ratios 110

were similar with the individual samples. Samples were injected by 5×103 Pa pressure for 3 111

sec. Runs were performed in the positive-polarity mode with 30 kV. Enantiomer separations 112

were carried out with 2.5, 5, 10, 20 mM concentrations of PMMABCD. The resolution (Rs) of 113

the enantiomers is given by the following equation [43]: 114

( )( ) ( )25.015.0

12s ww

tt18.1R+

−×= Eq. 1 115

where t is the migration time of the enantiomers (1, 2 in lower index), w(0.5) is the peak width 116

at half height. 117

Selectivity values are given as αt,migr = t migr,2/tmigr,1 , where t migr,1 and t migr,2 are the migration 118

times of the Dns-AA enantiomers, and are also given as αµ,eff = µeff,2/µeff,1 , where µeff,1 and µeff,2 119

are the mobilities of the Dns-AA enantiomers, respectively. 120

121

2.3. Estimation of pI values of Dns-AAs 122

123

pIs of the Dns-AAs were determined from the effective mobilities (μeff) measured at 124

several pHs. The effective mobilities were plotted against the applied pHs and the intercept of 125

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the fitted curve on the abscissa gave the pI of the given analyte (Fig. S1, Table S1) [44,45]. 126

Predicted logP values were calculated by ChemAxon’s Marvin Calculator Plugin 127

software (version 5.9.2; URL: http://www.chemaxon.com/marvin) called as “Marvin” in the 128

followings. 129

130

3. Results and discussion 131

132

Chiral separations of 11 Dns-AA enantiomer pairs (Table 1) were studied by the 133

cationic PMMABCD. Separations were carried out in the pH 3-9 range and by applying the 134

selector in the 2.5 mM to 20 mM concentration range. Results are summarized in (Table 2, 135

Table S2). Using these conditions eight enantiomer pairs can be resolved at least at baseline 136

or better and additional two pairs can be separated partially. Separations can be carried out in 137

reasonable running times. Changes in resolution of Dns-AAs as a function of pH and 138

PMMABCD concentration is discussed in details as follows. 139

140

[Table 1] [Table 2] 141

142

3.1. pH dependence of resolution of Dns-AA enantiomers 143

144

Changes in the chiral separations of the enantiomers of Dns-AAs were investigated by 145

varying the pH between pH 3 and pH 9. In order to evaluate charge relationships between the 146

host and guest molecules in this pH range the pI values of the analytes were measured by CE. 147

The pI values of the Dns-AAs are between 3.46-3.79 (Table 1) being in good agreement with 148

literature data (Table S1) [46]. Consequently, the Dns-AAs are positively charged below and 149

negatively charged above these values while the PMMABCD is positively charged 150

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permanently below pH 9. The effects of the pH on chiral separations of Dns-AAs were 151

studied by investigating several combinations of different charge states of the selector and 152

selectands (at pH 3, 4, 6 and 9). 153

Migrations of the Dns-AAs were accelerated (μcompl>μfree) by PMMABCD at pHs 154

above their pI values (Table S2). The negatively charged Dns-AAs if complexed with 155

PMMABCD migrate faster than the free analytes alone due to the decrease in their negative 156

charges and increase in their mass (i.e. hydrodynamic radius). Using this cationic selector 157

below the pIs of the Dns-AAs the complexes gain positive charges not only from the analyte 158

but also from the selector, in addition their sizes are increased. Therefore their mobility 159

changes are not substantial and could not be predicted well due to those counter-effects. 160

Resolutions show maxima at pH 4 or at pH 6 (Table 2, Fig. 1) where PMMABCD is 161

cationic and the Dns-AAs are at least partially negatively charged. The highest selectivities 162

can be observed for Dns-Abu, Dns-Nva, Dns-Nle, Dns-Leu, Dns-Phe, Dns-Trp and Dns-Thr 163

at pH 4 and for Dns-Met, Dns-Val and Dns-Asp at pH 6. Resolutions are decreased at pH 9 164

where PMMABCD is partially in uncharged form and the Dns-AAs are negatively charged. 165

Resolutions are weaker too at pH 3 where the selector is fully positively charged and the 166

analytes are partially cationic. 167

In general, the amount of the D-amino acids is substantially lower than that of their L 168

pairs in nature. In most of our cases the migration order of the enantiomers (EMO) is D,L in 169

the entire pH range investigated. So that EMO is advantageous because the evaluation of 170

peaks is more accurate when the smaller component migrates before the major one. 171

172

[Fig. 1] 173

174

3.2. CD concentration dependence of resolution of Dns-AA enantiomers 175

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176

Selectivities increase with increasing selector concentrations in most cases of the Dns-177

AAs investigated. Our finding that the maximal selectivities are in the high concentration 178

range of the selector corresponds [2,47,48] to the weak interactions between PMMABCD and 179

Dns-AAs (Kassoc ≤ 10 M-1) estimated by the well known non-linear regression of the 180

electrophoretic mobility shifts [49-53]. 181

Whereas, increasing the selector concentration results in the improvement of 182

separations usage of PMMABCD in concentrations substantially higher than 20 mM is not 183

recommended because of longer analysis times and peak distortions. In addition, due to the 184

elevated current other counterproductive effects of Joule heating can also occur. Furthermore, 185

it is noteworthy that the pH and selector concentration dependencies for Dns-Nle and Dns-186

Phe deviate from the others (Fig. 1) because in these two cases the migration time of the 187

peaks of the enantiomers overlaps with the time of the electroosmotic flow at high (15-20 188

mM) concentrations of PMMABCD at pH 4. 189

Baseline or better separation can be achieved for some of the enantiomer pairs of these 190

Dns-AAs even with low concentrations of PMMABCD. In the presence of only 2.5 mM-10 191

mM concentrations of the selector Dns-Abu, Dns-Nva, Dns-Nle, Dns-Leu, Dns-Phe, Dns-Trp 192

and Dns-Met exhibited good resolution with further improvements with elevated CD 193

concentrations. In the group of Dns-AAs with nonpolar aliphatic sidechains the higher is the 194

lipophilicity the better is the resolution. 15 mM - 20 mM concentrations of PMMABCD are 195

needed for the resolution of the more polar (cf. the logP values in Table 1) Dns-AAs (namely: 196

Dns-Val, Dns-Thr and Dns-Asp). Unfortunately, enantiomers of the polar Dns-Glu can not be 197

separated at all with PMMABCD. 198

Limit of detection (LOD at signal to noise ratio S/N = 3) and the limit of quantitation 199

(LOQ at S/N = 10) were determined for several dansylated D- and L-amino acids (Table 3). 200

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The LOD and LOQ values were between 0.2 - 3 µg/ml and 6 - 10 µg/ml, respectively. Most 201

biological samples contain D- and L-amino acid enantiomers approximately in a ratio of 202

1:100 [54]. Accordingly, the sensibility of our system for the detection of the minor (D-Dns-203

AA) component followed by the major ‘L’ enantiomer is demonstrated here with some 204

examples (Dns-Phe, Dns-Trp, Dns-Leu, Dns-Val, Dns-Nva and Dns-Met) where the 205

enantiomer ratios is also 1 to 100. In these setups (20 mM PMMABCD at pH 6.0) the 206

concentrations of D-Dns-AAs is around at their LOQ values (10 µg/ml) and those of their ‘L’ 207

pairs were 1 mg/ml (Fig. 2). 208

209

[Table 3] [Fig. 2] 210

211

3.4. Resolution of an artificial mixture of Dns-AAs 212

213

The resolution power of PMMABCD is further studied by mixture of Dns-AAs. From 214

our enantiomer pairs we chose those eight Dns-AAs which were separated at baseline at least. 215

Migration times of the enantiomers were taken into account too in selecting optimal running 216

parameters (pH and CD concentration) in order to obtain separated peaks for each constituent 217

of the mixture. Despite that in some cases pH 4 can provide more improved resolution for the 218

individual Dns-AAs than pH 6 the number of the enantiomer pairs resolved is greater at pH 6 219

than at pH 4. Consequently, we have chosen 20 mM PMMABCD at pH 6.0 and 7 enantiomer 220

pairs separated well and two further peaks partially of that mixture containing 8 pairs (Fig. 3). 221

[Fig. 3] 222

223

224

225

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4. Conclusions 226

227

The amino and methyl substituted βCD derivative PMMABCD is introduced as an 228

excellent cationic selector for Dns-AAs having nonpolar aliphatic and aromatic side chains 229

and a usable selector for some Dns-AAs with polar uncharged or charged groups. The 230

advantage of using PMMABCD is that it consumes a small amount of CD derivative, the 231

separations can be carried out in reasonable running times, and it provides the preferable ‘DL’ 232

migration order for the resolution of Dns-AAs at pHs above their pI values. Furthermore, the 233

chiral recognition ability of PMMABCD is proved by the separation of a mixture of Dns-234

AAs. 235

236

Acknowledgements 237

238

We gratefully acknowledge Dr. Róbert Iványi, Katalin Tuza (Cyclolab R&D Ltd.) as well as 239

Prof. Miklós Simonyi, Dr. Gábor Tárkányi, Mrs. Ilona Kawka (RCNS, HAS) and Dr. 240

Zsuzsanna Lakatos for their helpful contributions. The authors thank for the financial support 241

from the following grants: NKFP_A3-2008-0211 NATURSEP, TECH-09-AI-2009-0117 242

NanoSEN9, the Hungarian Scientific Research Fund (OTKA, grant numbers K-100134 and 243

NN-110214) and the “Lendület” Program of the Hungarian Academy of Sciences (LP2013-244

55/2013). 245

246

Appendix A. Supplementary data 247

Supplementary data associated with this article can be found, in the online version, at doi: 248

249

The authors have declared no conflict of interest. 250

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251

5. References 252

[1] G. Gübitz, M.G. Schmid, Chiral separation principles in capillary electrophoresis, J. 253

Chromatogr. A 792 (1997) 179-225. 254

[2] S. Fanali, Enantioselective determination by capillary electrophoresis with cyclodextrins 255

as chiral selectors, J. Chromatogr. A 875 (2000) 89-122. 256

[3] G. Blaschke, B. Chankvetadze, Enantiomer separation of drugs by capillary 257

electromigration techniques , J. Chromatogr. A 875 (2000) 3-25. 258

[4] B. Chankvetadze, Enantioseparations by using capillary electrophoretic techniques. The 259

story of 20 and a few more years, J. Chromatogr. A 1168 (2007) 45-70. 260

[5] P. Jac, G.K.E. Scriba, Recent advances in electrodriven enantioseparations, J. Sep Sci. 36 261

(2013) 52-74. 262

[6] L. Qi, Y. Chen, M. Xie, Z. Guo, X. Wang, Separation of dansylated amino acid 263

enantiomers by chiral ligand-exchange CE with a zinc(II) L-arginine complex as the selecting 264

system Electrophoresis 29 (2008) 4277-4283. 265

[7] D.A. Tsioupi R-I. Stefan-van-Staden, C.P. Kapnissi-Christodoulou, Chiral selectors in CE: 266

Recent developments and applications, Electrophoresis 34 (2013) 178-204. 267

[8] B. Chankvetadze, G. Endresz, G. Blaschke, Charged cyclodextrin derivatives as chiral 268

selectors in capillary electrophoresis, Chem. Soc. Review, 25 (1996) 141-148. 269

[9] V. Cucinotta, A. Contino, A. Giuffrida, G. Maccarrone, M. Messina, Application of 270

charged single isomer derivatives of cyclodextrins in capillary electrophoresis for chiral 271

analysis, J. Chromatogr. A 1217 (2010) 953-967. 272

[10] Z. Juvancz, R. Bodáné-Kendrovics, R. Iványi, L. Szente, The role of cyclodextrins in 273

chiral capillary electrophoresis, Electrophoresis 29 (2008) 1701-1712. 274

Page 12 of 25

Accep

ted

Man

uscr

ipt

12

[11] Y. Tanaka, S. Terabe, Enantiomer separation of acidic racemates by capillary 275

electrophoresis using cationic and amphoteric beta-cyclodextrins as chiral selectors, J. 276

Chromatogr. A 781 (1997) 151-160. 277

[12] H. Yamamura, Y. Yamada, R. Miyagi, K. Kano, S. Araki, M. Kawai, Preparation of β-278

Cyclodextrin Derivatives Possessing Two Trimethylammonio Groups on Their Primary 279

Hydroxy Sides as Chiral Guest Selectors, J. Incl. Phenom. Macrocycl. Chem. 45 (2003) 211–280

216. 281

[13] W. Tang, I. W. Muderawan, S.-C. Ng, H. S. O. Chan, Enantiomeric separation of 8 282

hydroxy, 10 carboxylic and 6 dansyl amino acids by mono(6-amino-6-deoxy)-beta-283

cyclodextrin in capillary electrophoresis, Anal. Chim. Acta 554 (2005) 156-162. 284

[14] W. Tang, I. W. Muderawan, T.-T. Ong, S.-C. Ng, Enantio separation of dansyl amino 285

acids by a novel permanently positively charged single-isomer cyclodextrin: Mono-6-N-286

allylammonium-6-deoxy-beta-cyclodextrin chloride by capillary electrophoresis, Anal. Chim. 287

Acta 546 (2005) 119-125. 288

[15] Sz. Béni, T. Sohajda, G. Neumajer, R. Iványi, L. Szente, B. Noszál, Separation and 289

characterization of modified pregabalins in terms of cyclodextrin complexation, using 290

capillary electrophoresis and nuclear magnetic resonance, J. Pharm. Biomed.Anal. 51 291

(2010) 842-852. 292

[16] A. Giuffrida, A. Contino, G. Maccarrone, M. Messina, V. Cucinotta The 3-amino-293

derivative of gamma-cyclodextrin as chiral selector of Dns-amino acids in electrokinetic 294

chromatography, J. Chromatogr. A 1216 (2009) 3678-3686. 295

[17] P. Liu, W. He, X-Y.,Qin, X-L. Sun, H. Chen, S-Y. Zhang, Synthesis and application of a 296

novel single-isomer mono-6-deoxy-6-((2S,3S)-(+)-2,3-O-isopropylidene-1,4-297

tetramethylenediamine)-beta-cyclodextrin as chiral selector in capillary electrophoresis 298

Chirality 22 (2010) 914-921. 299

Page 13 of 25

Accep

ted

Man

uscr

ipt

13

[18] A. Giuffrida, R. Caruso, M. Messina, G. Maccarrone, A. Contino, A. Cifuentes, V. 300

Cucinotta, Chiral separation of amino acids derivatised with fluorescein isothiocyanate by 301

single isomer derivatives 3-monodeoxy-3-monoamino-beta- and gamma-cyclodextrins: the 302

effect of the cavity size J. Chromatogr. A 1269 (2012) 360-365. 303

[19] Y. Dai, S. Wang, J. Zhou, Y. Liu, D. Sun, J. Tang, W. Tang, Cationic cyclodextrin as 304

versatile chiral selector for enantiomeric separation in capillary electrophoresis, J. 305

Chromatogr. A, 1246 (2012) 98-102. 306

[20] S. Wang, Y. Dai, J. Wu, J. Zhou, J. Tang, W. Tang, Methoxyethylammonium 307

monosubstituted β-cyclodextrin as the chiral selector for enantioseparation in capillary 308

electrophoresis, J. Chromatogr. A 1277 (2013) 84-92. 309

[21] J. Zhou, F. Ai, B. Zhou, J. Tang, S.-C. Ng, W. Tang, Hydroxyethylammonium 310

monosubstituted cyclodextrin as chiral selector for capillary electrophoresis, Anal. Chim. 311

Acta 800 (2013) 95-102. 312

[22] H. Wan, L.G. Blomberg, Chiral separation of amino acids and peptides by capillary 313

electrophoresis, J. Chromatogr. A 875 (2000) 43-88. 314

[23] V. Poinsot, M.-A. Carpene, J. Bouajila, P. Gavard, B. Feurer, F. Couderc, Recent 315

advances in amino acid analysis by capillary electrophoresis, Electrophoresis 33 (2012) 14-316

35. 317

[24] V. Poinsot, C. Bayle, F. Couderc, Recent advances in amino acid analysis by capillary 318

electrophoresis, Electrophoresis 24 (2003) 4047-4067. 319

[25] M. Chiari, M. Cretich, G. Crini, L. Janus, M. Morcellet, Recent advances in amino acid 320

analysis by capillary electrophoresis, J. Chromatogr. A 894 (2000) 95-103. 321

[26] R. Iványi, L. Jicsinszky, Z. Juvancz, Permethyl monoamino beta-cyclodextrin a new 322

chiral selective agent for capillary electrophoresis, Chromatographia 53 (2001) 166-172. 323

Page 14 of 25

Accep

ted

Man

uscr

ipt

14

[27] L. Jicsinszky, R. Iványi, Catalytic transfer hydrogenation of sugar derivatives, 324

Carbohydr. Polym. 45 (2001) 139-145. 325

[28] R. Iványi, L. Jicsinszky, Z. Juvancz, Chiral separation of pyrethroic acids with single 326

isomer permethyl monoamino beta-cyclodextrin selector, Electrophoresis 22 (2001) 3232-327

3236. 328

[29] A.M. Abushoffa, M. Fillet, A.C. Servais, P. Hubert, J. Crommen, Enhancement of 329

selectivity and resolution in the enantioseparation of uncharged compounds using mixtures of 330

oppositely charged cyclodextrins in capillary electrophoresis, Electrophoresis 24 (2003) 343-331

350. 332

[30] B. Tőkés, L. Ferencz, P. Buchwald, G. Donázh-Nagy, Sz. Vancea, N. Sántha, E. L. Kis, 333

Structural studies on the chiral selector capacity of cyclodextrin derivatives, J. Biochem. 334

Biophys. Methods 70 (2008) 1276–1282. 335

[31] A.G. Cârje; Z. Juvancz, R. Iványi, V. Schurig, B. Tőkés, Comparative study on chiral 336

separation of pyrethroic acids with amino and neutral cyclodextrine derivatives, Acta 337

Medica Marisiensis 57 (2011) 52-54. 338

[32] G. Tárkányi, K. Németh, R. Mizsei, O. Tőke, J. Visy, M. Simonyi, L. Jicsinszky, J. 339

Szemán, L. Szente, Structure and stability of warfarin-sodium inclusion complexes formed 340

with permethylated monoamino-beta-cyclodextrin, J. Pharm. Biomed.Anal. 72 (2013) 292-341

298. 342

[33] R.M. Callejón, A.M. Troncoso, M.L. Morales, Determination of amino acids in grape-343

derived products: A review, Talanta 81 (2010) 1143-1152. 344

[34] M. Wcisło, D. Compagnone, M. Trojanowicz, Enantio selective screen-printed 345

amperometric biosensor for the determination of D-amino acids, Bioelectrochemistry 71 346

(2007) 91-98. 347

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[35] X. He, X. Cui, M. Li, L. Lin, X. Liu, X. Feng, Highly enantioselective fluorescent sensor 348

for chiral recognition of amino acid derivatives, Tetrahedron Lett. 50 (2009) 5853-5856. 349

[36] H. Zahradníčková, P. Hušek, P. Šimek, P. Hartvich, B. Maršálek, I. Holoubek, 350

Determination of D- and L-amino acids produced by cyanobacteria using gas chromatography 351

on Chirasil-Val after derivatization with pentafluoropropyl chloroformate, Anal. Bioanal. 352

Chem. 388 (2007) 1815-1822. 353

[37] Y. Miyoshi, R. Koga, T. Oyama, H. Han, K. Ueno, K. Masuyama, Y. Itoh, K. Hamase, 354

HPLC analysis of naturally occurring free D-amino acids in mammals, J. Pharm. Biomed. 355

Anal. 69 (2012) 42-49. 356

[38] M. Herrero, C., Simo, V. Garcia-Canas, S. Fanali, A. Cifuentes, Electrophoresis 31 357

(2010) 2106-2114. 358

[39] K. Hamase, Sensitive two-dimensional determination of small amounts of D-amino acids 359

in mammals and the study on their functions, Chem. Pharm. Bull. 55 (2007) 503-510. 360

[40] M. Friedman, Origin, Microbiology, Nutrition, and Pharmacology of D-Amino Acids, 361

Chem. Biodiversity 7 (2010) 1491-1530. 362

[41] K. Hamase, Analysis and biological relevance of D-amino acids and relating compounds, 363

J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 879 (2011) 3077-3077. 364

[42] H. Ohide, Y. Miyoshi, R. Maruyama, K. Hamase, K. R. Konno, D-Amino acid 365

metabolism in mammals: Biosynthesis, degradation and analytical aspects of the metabolic 366

study, J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 879 (2011) 3162-3168. 367

[43] S. Fanali, Controlling enantioselectivity in chiral capillary electrophoresis with inclusion-368

complexation, J. Chromatogr. A 792 (1997) 227-267. 369

[44] D. Koval, V. Kasicka, J. Jiracek, M. Collinsová, Determination of pK(a) values of 370

diastereomers of phosphinic pseudopeptides by CZE, Electrophoresis 27 (2006) 4648-4657. 371

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[45] S.K. Poole, S. Patel, K. Dehring, H. Workman, C.F. Poole, Determination of acid 372

dissociation constants by capillary electrophoresis, J. Chromatogr. A 1037 (2004) 445-454. 373

[46] A. Bianchi-Bosisio, P.G. Righetti, N.B. Egen, M. Bier, Isoelectric focusing of 374

dansylated amino-acids in immobilized pH gradients, Electrophoresis 7 (1986) 128-133. 375

[47] A. Rizzi, Fundamental aspects of chiral separations by capillary electrophoresis, 376

Electrophoresis 22 (2001) 3079-3106. 377

[48] S. Wren, The separation of enantiomers by capillary electrophoresis, Chromatographia 378

54 (2001) S1-S95. 379

[49] S. G. Penn, D. M. Goodall, J. S. Loran, Differential Binding of tioconazole enantiomers 380

to hydroxypropyl-beta cyclodextrin studied by capillary, J. Chromatogr. 636 (1993) 149-152. 381

[50] K. L. Rundlett, D. W. Armstrong, Examination of the origin, variation, and proper use of 382

expressions for the estimation of association constants by capillary electrophoresis, J. 383

Chromatogr. A 721 (1996) 173-186. 384

[51] S. A.C. Wren, Mobility measurements on dansylated amino acids, J. Chromatogr. A 768 385

(1997) 153-159. 386

[52] A. Salvador, E. Varesio, M. Dreux, J-L. Veuthey, Binding constant dependency of 387

amphetamines with various commercial methylated beta-cyclodextrins, Electrophoresis 20 388

(1999) 2670-2679. 389

[53] Y. Tanaka, S. Terabe, Estimation of binding constants by capillary electrophoresis, J. 390

Chromatogr. A 768 (2002) 81-92. 391

[54] Zs. Wagner, T. Tábi, T. Jakó, G. Zachar, A. Csillag, É. Szökő, Chiral separation and 392

determination of excitatory amino acids in brain samples by CE-LIF using dual 393

cyclodextrin system, Anal. Bioanal. Chem. 404 (2012) 2363-2368. 394

395

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Figure captions 395

396

Fig. 1. Changes in selectivities (αt,migr) of several Dns-AAs as a function of pH and selector 397

concentration 398

Fig. 2. Separations of D- and L-Dns-AA enantiomers in a concentration ratio of 1:100 (10 399

µg/ml and 1 mg/ml, respectively) in the presence of 20 mM PMMABCD at pH 6 400

Fig. 3. Separation of Dns-AA mixture by 20 mM PMMABCD at pH 6 401

402

403

404

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Table 1: Structure, isoelectric point (pI) and lipophilicity (logP) of dansyl-amino acids 404 405

406 407

408

R group pI logP

Nonpolar, aliphatic R group, linear sidechain:

Dns-DL-α-aminobutyric acid (Dns-Abu) -CH2CH3 3.68 -2.54

Dns-DL-norvalin (Dns-Nva) -(CH2)2CH3 3.67 -2.11

Dns-DL-norleucine (Dns-Nle) -(CH2)3CH3 3.67 -1.52

Nonpolar, aliphatic R group, branched sidechain:

Dns-DL-valine (Dns-Val) -CH(CH3)2 3.70 -2.26

Dns-DL-leucine (Dns-Leu) -CH2CH(CH3)2 3.63 -1.52

Nonpolar, aromatic R group:

Dns-DL-phenylalanine (Dns-Phe) -CH2C6H5 3.67 -1.38

Dns-DL-tryptophan (Dns-Trp) -CH2C8NH6 3.79 -1.05 Nonpolar, uncharged R group:

Dns-DL-methionine (Dns-Met) -(CH2)2SCH3 3.55 -1.87

Polar, uncharged R group:

Dns-DL-threonine (Dns-Thr) -CHCH3OH 3.66 -2.94

Polar, negatively charged R group:

Dns-DL-aspartic acid (Dns-Asp) -CH2COOH 3.46 -3.89

Dns-DL-glutamic acid (Dns-Glu) -(CH2)2COOH 3.50 -3.69

N

CH3

CH3

S

O

O

NHR

COOH

*

N

CH3

CH3

S

O

O

NHR

COOH

*

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Table 2: Migration times (t1), selectivities (α) and resolutions (Rs) of Dns-AAs at pH 9-3 in 408 the presence of 2.5-20 mM PMMABCD as chiral selector 409 410 pH t1 (min) αe Rs

2.5d 5 10 15 20 2.5 5 10 15 20 2.5 5 10 15 20

9 n.a. n.a. 4.63 4.79 5.23 n.a. n.a. 1.000 1.004 1.010 n.a. n.a. nr 0.5 1.56 5.90 6.54 6.83 7.33 7.79 1.000 1.012 1.025 1.042 1.073 nr 1.8 3.9 6.5 11.14 12.19 14.19 17.21 19.23 20.30 1.000 1.009 1.039 1.085 1.131 nr 1.0 4.0 8.1 11.8

Dns-Abu

3 n.a. n.a. 14.67 15.49 19.16 n.a. n.a. 1.000 1.008 1.000 n.a. n.a. nr nr nr

9 4.22 4.30 4.47 4.64 5.07 1.000 1.000 1.000 1.000 1.012 nr nr nr nr 1.76 5.59 5.94 6.32 6.51 7.58 1.000 1.013 1.029 1.042 1.092 nr 2.1 4.6 8.0 12.94 12.21 14.02 16.14 17.22 17.59 1.000 1.016 1.068 1.133 1.179 nr 1.6 6.8 13.2 17.3

Dns-Nva

3 12.56 12.65 20.52 22.62 26.37 1.000 1.000 1.000 1.000 1.016 nr nr nr nr 0.8

9 n.a. n.a. 4.45 4.55 4.89 n.a. n.a. 1.005 1.018 1.032 n.a. n.a. 0.6 2.5 4.96 5.49 5.68 5.92 6.05 6.40 1.006 1.032 1.068 1.088 1.160 0.9 5.2 10.1 14.0 19.54 12.14 13.20 13.42 14.84 17.52 1.015 1.065 1.197 1.149 n.a. 1.9 5.5 20.4 20.3 n.a.

Dns-Nle

3 n.a. n.a. 15.91 17.24 21.66 n.a. n.a. 1.022 1.067 1.060 n.a. n.a. 1.8 2.8 3.6

9 n.a. n.a. 4.56 4.73 5.19 n.a. n.a. 1.000 1.000 1.000 n.a. n.a. nr nr nr6 5.62 6.07 6.62 7.01 8.45 1.000 1.000 1.000 1.000 1.016 nr nr nr nr 2.34 11.53 13.43 16.16 18.85 20.59 1.000 n.a. 1.000 1.000 1.000 nr n.a. nr nr nr

Dns-Val

3 n.a. n.a. 15.64 16.60 n.a. n.a. n.a. 1.000 1.000 n.a. n.a. n.a. nr nr n.a.

9 4.17 4.24 4.41 4.58 5.08 1.003 1.005 1.007 1.006 1.004 0.5 0.9 1.2 1.5 0.66 5.85 6.66 6.91 7.27 7.91 1.004 1.000 1.000 1.013 1.035 0.6 nr nr 2.0 4.44 11.99 13.96 16.89 18.46 19.17 1.000 1.010 1.059 1.137 1.220 nr 1.2 6.0 12.8 19.5

Dns-Leu

3 12.91 13.19 24.46 27.14 34.52 1.000 1.000 1.012 1.023 1.036 nr nr 1.0 1.7 6.9

9 4.15 4.21 4.23 4.25 4.58 1.142 1.000 1.019 1.039 1.059 0.9 nr 3.4 5.9 6.76 5.63 5.95 5.66 5.77 6.07 1.016 1.071 1.115 1.148 1.168 2.5 10.5 16.3 21.2 21.24 11.43 12.26 12.70 16.23 17.24 1.021 1.085 1.216 n.a. n.a. nr 7.4 27.1 n.a. n.a.

Dns-Phe

3 13.05 13.16 22.41 24.90 31.77 1.000 1.000 1.015 1.017 1.000 nr nr 1.0 1.4 nr

9 4.16 4.26 4.48 4.64 5.11 1.000 1.000 1.000 1.000 1.000 nr nr nr nr nr6 5.47 5.89 6.37 6.64 7.33 1.000 1.000 1.014 1.019 1.045 nr nr 2.1 1.4 7.44 n.a. 12.21 14.33 n.a. 17.53 n.a. 1.006 1.026 n.a. 1.092 n.a. 0.8 2.8 n.a. 8.7

Dns-Trp

3 12.49 12.69 17.33 20.05 23.72 1.000 1.000 1.000 1.000 1.006 nr nr nr nr nr

9 n.a. n.a. 4.59 4.70 5.14 n.a. n.a. 1.000 1.011 1.021 n.a. n.a. nr 1.3 3.26 5.96 6.78 6.92 7.20 7.94 1.000 1.021 1.044 1.071 1.134 nr 3.3 6.8 10.9 17.04 n.a. n.a. n.a. n.a. n.a. n.a. 1.000 1.000 1.000 1.000 n.a. nr nr nr nr

Dns-Met

3 n.a. n.a. 18.25 20.60 24.35 n.a. n.a. 1.000 1.000 1.010 n.a. n.a. nr nr 0.5

Dns-Thr 4 12.74 14.96 19.15 24.44 29.21 1.000 1.000 1.000 1.000 1.015 nr nr nr nr 1.0

Dns-Asp 6 9.88 11.43 13.31 16.10 19.79 1.000 1.000 1.000 1.011 1.016 nr nr nr 1.0 1.4

411 a) n.a. not available 412 b) nr not resolved; Rs < 0.5 413 c) t1 migration time of the faster enantiomer 414 d) Selector concentrations applied is given in mM in the second row of the header 415 e) Selectivity is given as follows: α = t migr,2/tmigr,1 416 f) The enantiomer migration order is D,L in the majority of the cases. The selectivity values 417 are underlined where the enantiomer migration order is reversed (L,D). 418 419

420

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Table 3: Limit of detection (LOD) and quantitation (LOQ) of D- and L-Dns AAs 420 421

422 423

D-Dns-AA L-Dns-AA

LOD (µg/ml) LOQ (µg/ml)

LOD (µg/ml) LOQ (µg/ml)

Dns-Abu 0.4 5.8 0.4 6.4

Dns-Nva 0.2 5.7 0.2 6.3

Dns-Nle 0.1 5.2 0.1 6.5

Dns-Val 1.7 7.9 1.7 7.7

Dns-Leu 1.5 7.4 1.5 7.5

Dns-Phe 1.3 6.2 1.3 7.5

Dns-Trp 1. 8 6.1 1.8 6.1

Dns-Met 3.0 10.0 2.9 10.2

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6.5 7.0 7.5 8.0 8.5 9.0 9.5

Migration time (min)

Ab

so

rba

nce

(2

20

nm

)

Dns-A

bu

Dns-D

-Leu

Dns-L

-Nva

Dns-L

-Leu

Dns-D

-Val

Dns-A

bu

Dns-L

-Val

Dns-L

-Met

Dns-N

le

Dns-L

-Phe

Dns-D

-Phe

Dns-D

-Nva

Dns-N

le

Dns-D

-Met

EO

F

Dns-D

-Trp

Dns-L

-Trp

(OCH3)6 and (NH2)1

C(3) C(2)

(CH3O)7 (OCH3)7

C(6) *Graphical Abstract

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3 4 5 6 7 8 9

5

10

15

20

pH

CD

co

nce

ntr

atio

n (

mM

)

1.000

1.025

1.050

1.075

1.100

1.125

1.150

1.175

1.200

1.225

1.250

3 4 5 6 7 8 9

5

10

15

20

pH

CD

co

nce

ntr

atio

n (

mM

)

1.000

1.025

1.050

1.075

1.100

1.125

1.150

1.175

1.200

1.225

1.2503 4 5 6 7 8 9

5

10

15

20

pH

CD

co

nce

ntr

atio

n (

mM

)

1.000

1.025

1.050

1.075

1.100

1.125

1.150

1.175

1.200

1.225

1.2503 4 5 6 7 8 9

5

10

15

20

pH

CD

co

nce

ntr

atio

n (

mM

)

1.000

1.025

1.050

1.075

1.100

1.125

1.150

1.175

1.200

1.225

1.250

3 4 5 6 7 8 9

5

10

15

20

pH

CD

co

nce

ntr

atio

n (

mM

)

1.000

1.025

1.050

1.075

1.100

1.125

1.150

1.175

1.200

1.225

1.2503 4 5 6 7 8 9

5

10

15

20

pH

CD

co

nce

ntr

atio

n (

mM

)

1.000

1.025

1.050

1.075

1.100

1.125

1.150

1.175

1.200

1.225

1.2503 4 5 6 7 8 9

5

10

15

20

pH

CD

co

nce

ntr

atio

n (

mM

)

1.000

1.025

1.050

1.075

1.100

1.125

1.150

1.175

1.200

1.225

1.250

Dns-Abu

Dns-Leu Dns-Trp Dns-Phe

Dns-Nle Dns-Nva

Dns-Met Fig. 1

Figure(s)

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3 4 5 6 7 8 9

5

10

15

20

pH

CD

co

nce

ntr

atio

n (

mM

)

1.000

1.025

1.050

1.075

1.100

1.125

1.150

1.175

1.200

1.225

1.250

3 4 5 6 7 8 9

5

10

15

20

pH

CD

co

nce

ntr

atio

n (

mM

)

1.000

1.025

1.050

1.075

1.100

1.125

1.150

1.175

1.200

1.225

1.2503 4 5 6 7 8 9

5

10

15

20

pH

CD

co

nce

ntr

atio

n (

mM

)

1.000

1.025

1.050

1.075

1.100

1.125

1.150

1.175

1.200

1.225

1.2503 4 5 6 7 8 9

5

10

15

20

pH

CD

co

nce

ntr

atio

n (

mM

)

1.000

1.025

1.050

1.075

1.100

1.125

1.150

1.175

1.200

1.225

1.250

3 4 5 6 7 8 9

5

10

15

20

pH

CD

co

nce

ntr

atio

n (

mM

)

1.000

1.025

1.050

1.075

1.100

1.125

1.150

1.175

1.200

1.225

1.2503 4 5 6 7 8 9

5

10

15

20

pH

CD

co

nce

ntr

atio

n (

mM

)

1.000

1.025

1.050

1.075

1.100

1.125

1.150

1.175

1.200

1.225

1.2503 4 5 6 7 8 9

5

10

15

20

pH

CD

co

nce

ntr

atio

n (

mM

)

1.000

1.025

1.050

1.075

1.100

1.125

1.150

1.175

1.200

1.225

1.250

Dns-Abu

Dns-Leu Dns-Trp Dns-Phe

Dns-Nle Dns-Nva

Dns-Met Fig. 1 in grey

Figure(s)

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7.0 7.5 8.0 8.5 9.0

Absorb

ance (

220 n

m)

Migration time (min)

7.5 8.0 8.5 9.0

Absorb

ance (

220 n

m)

Migration time (min)

8.0 8.5 9.0 9.5 10.0

Absorb

ance (

220 n

m)

Migration time (min)

6.0 6.5 7.0 7.5 8.0

Migration time (min)

Absorb

ance (

220 n

m)

7.5 8.0 8.5 9.0 9.5

Absorb

ance (

220 n

m)

Migration time (min)

8.5 9.0 9.5 10.0

Absorb

ance (

220 n

m)

Migration time (min)

Dns-D

-Phe

Dn

s-L

-Ph

e

Dns-D

-Nva

Dn

s-L

-Nva

Dns-D

-Val

Dn

s-L

-Val

Dns-D

-Trp

Dns-L

-Trp

Dns-D

-Le

u

Dns-L

-Le

u

Dns-D

-Met

Dns-L

-Met

Fig. 2 Figure(s)

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6.5 7.0 7.5 8.0 8.5 9.0 9.5

Migration time (min)

Ab

so

rba

nce

(2

20

nm

)

Dns-A

bu

Dns-D

-Leu

Dns-L

-Nva

Dns-L

-Leu

Dns-D

-Val

Dns-A

bu

Dns-L

-Val

Dns-L

-Met

Dns-N

le

Dns-L

-Phe

Dns-D

-Phe

Dns-D

-Nva

Dns-N

le

Dns-D

-Met

EO

F

Dns-D

-Trp

Dns-L

-Trp

Fig. 3 (OCH3)6 and (NH2)1

C(3) C(2)

(CH3O)7 (OCH3)7

C(6) Figure(s)

Supplementary data

Cationic permethylated 6-monoamino-6-monodeoxy-β-cyclodextrin as chiral selector of

dansylated amino acids in capillary electrophoresis

Krisztina Németh1*, Celesztina Domonkos

1, Virág Sarnyai

1, Julianna Szemán

2, László

Jicsinszky2, Lajos Szente

2, Júlia Visy

1

1Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy

of Sciences, H-1519 Budapest P.O.B.:286, Hungary

2CycloLab R&D Ltd., H-1097 Budapest, Illatos út 7, Hungary

*Corresponding author: K. Németh

Postal address: 1Institute of Organic Chemistry, Research Centre for Natural Sciences,

Hungarian Academy of Sciences, H-1519 Budapest P.O.B.:286, Hungary

Tel.: +36 1 382 6659; E-mail address: nemeth.krisztina@ttk.mta.hu

Determination of the pI values of the dansylated amino acids (Dns-AAs)

According to Eq. S1 the effective mobilities (µeff) of the analytes are zero when their charges

(q) are also zero (Fig S1A). Therefore the intercept of the fitted straight line at the abscissa

gives the isoelectric points of the amphoteric Dns-AAs (Fig. S1B; Table S1). Our

experimental pI data are in a good agreement with data in the literature (Table S1).

r6

qeff Eq. S1

Where r is the Stokes radius of the analyte and is the viscosity of the electrolyte.

-3.5

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

3 5 7 9pH

μeff

(1

0-4

cm

2/V

s)

Dns-Asp Dns-Trp

Fig. S1A: Effective mobility of dansylated aspartic acid and tryptophan as a function of pH

-1.1

-0.6

-0.1

0.4

0.9

1.4

3.0 3.2 3.4 3.6 3.8 4.0

pH

μeff

(1

0-4

cm

2/V

s) Dns-Abu Dns-Nva

Dns-Nle Dns-Val

Dns-Leu Dns-PheDns-Trp Dns-Met

Dns-Thr Dns-Asp

Dns-Glu

Fig. S1B: Intercepts at the abscissa of the straight lines of the effective mobilities of

dansylated amino acids vs. pH give the isoelectric points.

Table S1: Comparison of calculated isoelectric points (pI) and data in the literature

a: A. M. Rizzi,L. Kremser, Electrophoresis 20 (1999) 2715-2722.

b: A. Bianchi-Bosisio, P.G. Righetti, N.B. Egen, M. Bier, Electrophoresis 7 (1986) 128-133.

pI

Litrerature 1a Litrerature 2b calculated

Nonpolar, aliphatic R group, linear sidechain:

Dns-DL-α-aminobutyric acid (Dns-Abu) 3.68

Dns-DL-norvalin (Dns-Nva) 3.67

Dns-DL-norleucine (Dns-Nle) 3.67

Nonpolar, aliphatic R group, branched sidechain:

Dns-DL-valine (Dns-Val) 3.68 3.76 3.70

Dns-DL-leucine (Dns-Leu) 3.69 3.63

Nonpolar, aromatic R group:

Dns-DL-phenylalanine (Dns-Phe) 3.65 3.72 3.67

Dns-DL-tryptophan (Dns-Trp) 3.64 3.81 3.79

Nonpolar, uncharged R group:

Dns-DL-methionine (Dns-Met) 3.56 3.55

Polar, uncharged R group:

Dns-DL-threonine (Dns-Thr) 3.67 3.66

Polar, negatively charged R group:

Dns-DL-aspartic acid (Dns-Asp) 3.43 3.46

Dns-DL-glutamic acid (Dns-Glu) 3.55 3.50

pH and CD concentration dependence of mobility and selectivity

Effective mobilities of the Dns-AA enantiomers have been calculated in the presence of

PMMABCD at several pHs (pH 9-3). Selectivity data given as a ratio of the migration times

(t,migr) of the Dns AA enantiomers (Table 2) are completed with selectivity data calculated

from the effective mobilities (µ,eff,) (Table S2). t,migr and µ,eff change similarly as a function

of pH and CD concentration, but in some cases values of µ,eff are extremely high because one

of the enantiomers migrates close to the EOF and the other one remains slower, so their

mobilities differ in an order of magnitude from each other and therefore their ratio gives so

extraordinary (e.g. µeff = 22) values.

Table S2: Effective mobilities (µeff,1), selectivities () and resolutions (Rs) of Dns-AAs at pH

9-3 in the presence of 2.5-20 mM PMMABCD as chiral selector

pH µeff,1 (10

-4cm

2/Vs)

c e Rs

0d 2.5 5 10 15 20 2.5 5 10 15 20 2.5 5 10 15 20

Dns-

Abu

9 -1.84 n.a. n.a. -1.65 -1.55 -1.42 n.a. n.a. 1.000 1.010 1.027 n.a. n.a. nr 0.5 1.5

6 -1.80 -1.72 -1.53 -1.38 -1.20 -0.93 1.000 1.023 1.054 1.095 1.196 nr 1.8 3.9 6.5 11.1

4 -0.61 -0.59 -0.56 -0.45 -0.32 -0.19 1.000 1.022 1.098 1.258 1.620 nr 1.0 4.0 8.1 11.8

3 0.83 n.a. n.a. 1.11 1.11 0.96 n.a. n.a. 1.000 1.009 1.000 n.a. n.a. nr nr nr

Dns-

Nva

9 -1.76 -1.73 -1.66 -1.54 -1.47 -1.31 1.000 1.000 1.000 1.000 1.035 nr nr nr nr 1.7

6 -1.80 -1.67 -1.50 -1.32 -0.96 -0.81 1.000 1.030 1.071 1.132 1.288 nr 2.1 4.6 8.0 12.9

4 -0.63 -0.59 -0.53 -0.38 -0.20 -0.04 1.000 1.043 1.213 1.719 5.883 nr 1.6 6.8 13.2 17.3

3 0.76 n.a. n.a. 0.72 0.71 0.66 1.000 1.000 1.000 1.000 1.019 nr nr nr nr 0.8

Dns-

Nle

9 -1.72 n.a. n.a. -1.49 -1.33 -1.16 n.a. n.a. 1.016 1.058 1.111 n.a. n.a. 0.6 2.5 4.9

6 -1.78 -1.60 -1.34 -1.07 -0.77 -0.41 1.014 1.084 1.209 1.362 2.129 0.9 5.2 10.1 14.0 19.5

4 -0.65 -0.60 -0.44 -0.12 -0.01 0.00 1.043 1.212 3.118 21.181 n.a. 1.9 5.5 20.4 20.3 n.a.

3 0.75 n.a. n.a. 0.98 0.91 0.79 n.a. n.a. 1.027 1.081 1.068 n.a. n.a. 1.8 2.8 3.6

Dns-

Val

9 -1.77 n.a. n.a. -1.62 -1.52 -1.42 n.a. n.a. 1.000 1.000 1.000 n.a. n.a. nr nr nr

6 -1.79 -1.68 -1.57 -1.44 -1.21 -1.09 1.000 1.000 1.000 1.000 1.037 nr nr nr nr 2.3

4 -0.53 -0.50 n.a. -0.40 -0.30 -0.22 1.000 n.a. 1.000 1.000 1.000 nr n.a. nr nr nr

3 0.75 n.a. n.a. 1.02 1.03 n.a. n.a. n.a. 1.000 1.000 1.000 n.a. n.a. nr nr n.a.

Dns-

Leu

9 -1.72 -1.67 -1.60 -1.48 -1.41 -1.31 1.008 1.014 1.020 1.018 1.011 0.5 0.9 1.2 1.5 0.6

6 -1.74 -1.62 -1.46 -1.35 -1.20 -0.99 1.008 1.000 1.000 1.032 1.091 0.6 nr nr 2.0 4.4

4 -0.63 -0.60 -0.55 -0.43 -0.28 -0.13 1.000 1.026 1.157 1.487 2.548 nr 1.2 6.0 12.8 19.5

3 0.69 n.a. n.a. 0.56 0.54 0.47 1.000 1.000 1.018 1.030 1.044 nr nr 1.0 1.7 6.9

Dns-

Phe

9 -1.76 -1.66 -1.55 -1.30 -1.09 -0.89 1.350 1.000 1.067 1.163 1.279 0.9 nr 3.4 5.9 6.7

6 -1.75 -1.52 -1.10 -0.75 -0.45 -0.10 1.036 1.207 1.499 2.026 6.203 2.5 10.5 16.3 21.2 21.2

4 -0.57 -0.50 -0.34 -0.01 0.00 0.00 1.072 1.381 22.109 n.a. n.a. nr 7.4 27.1 n.a. n.a.

3 0.67 n.a. n.a. 0.63 0.61 0.54 1.000 1.000 1.021 1.022 1.000 nr nr 1.0 1.4 nr

Dns-

Trp

9 -1.67 -1.80 -1.61 -1.55 -1.46 -1.36 1.000 1.000 1.000 1.000 1.000 nr nr nr nr nr

6 -1.69 -1.61 -1.48 -1.33 -1.10 -0.98 1.000 1.000 1.032 1.053 1.128 nr nr 2.1 1.4 7.4

4 -0.40 n.a. -0.33 -0.24 n.a. -0.06 n.a. 1.032 1.149 n.a. 2.716 n.a. 0.8 2.8 n.a. 8.7

3 0.84 n.a. n.a. 0.90 0.82 0.75 1.000 1.000 1.000 1.000 1.007 nr nr nr nr nr

Dns-

Met

9 -1.76 n.a. n.a. -1.64 -1.51 -1.38 n.a. n.a. 1.000 1.032 1.059 n.a. n.a. nr 1.3 3.2

6 -1.77 -1.69 -1.50 -1.34 -1.17 -0.86 1.000 1.042 1.094 1.165 1.365 nr 3.3 6.8 10.9 17.0

4 -0.78 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.

3 0.48 n.a. n.a. 0.84 0.80 0.73 n.a. n.a. 1.000 1.000 1.011 n.a. n.a. nr nr 0.5

Dns-

Thr 4 -0.65 -0.62 -0.61 -0.60 -0.56 -0.52

1.000 1.000 1.000 1.000 1.020

nr nr nr nr 1.0

Dns-

Asp 6 -3.17 -3.12 -3.03 -2.95 -2.85 -2.75

1.000 1.000 1.000 1.005 1.006

nr nr nr 1.0 1.4

a) n.a. not available

b) nr not resolved; Rs < 0.5

c) effective mobility of the faster enantiomer

d) Selector concentrations applied is given in mM in the second row of the header

e) Selectivity is given as follows: = µeff,2/µeff,1

f) The enantiomer migration order is D,L in the majority of the cases. The selectivity values

are underlined where the enantiomer migration order is reversed (L,D).