Selection of appropriate chiral selectors for chiral analysis by regression modeling

of spectral data

Selorm Modzabi, Marianna A. Busch and Kenneth W. BuschBaylor University

One Bear Place #97348Waco, TX 76798

Need for chiral analysis*• Pharmaceutical industry

– Drug development– Process control

• Agro-chemical industry• Food and beverage industry• Fragrance industry• Basic research

*Chiral Analysis, K. W.Busch & M. A. Busch Eds., Elsevier, 2006

CHIRAL ANALYSIS BY REGRESSION MODELING OF SPECTRAL DATA

(CARMSD)

Modern chemical instrumentation allows us to combine—•Multivariate (multi-wavelength) data collectionwith•Multivariate modelingto give a powerful combination that can extract latent information from the multivariate data that would not be possible with univariate measurements

Basic Strategy of CARMSD1. Calibration Phase

1) Prepare a set of calibration samples

• Same total concentration of chiral analyte

• Different known enantiomeric compositions

• Fixed concentration of chiral auxiliary

Basic Strategy of CARMSD1. Calibration Phase

2) Collect spectral data on the calibration set

3) Perform PLS-1 regression modeling

Basic Strategy of CARMSD2. Validation Phase

1) Prepare a new set of validation samples

2) Collect spectral data3) Enter the spectral data into the

regression model and predict the enantiomeric compositions

4) Compare the predicted enantiomeric compositions with the known values

CARMSD•Clearly the chiral auxiliary is at the heart of the CARMSD method.•Regression modeling depends on changes in the spectral signature with enantiomeric composition of the sample. •The larger these spectral changes are, the easier it is to develop robust regression models.

CARMSD

• Chiral Selectors used to date– Cyclodextrins– Modified cyclodextrins– Surfactants & mixed cyclodextrins– Chiral Surfactants– Chiral Ionic Liquids

Enantiomer-CD transient inclusion complexes

C

CH3

ClH

BrC

CH3

H Cl

Br

Enantiomeric pair

Diastereomeric pair(hypothetical)

CDCD

Enantiomeric discrimination by transient noncovalent complex

formation with cyclodextrins— An Example

C

a

d bc

C

a

b dc

Chiral selectors used to date

Chiral selector Analyte concentration Prediction error (RMSEP) range

Cyclodextrins (CDs)

3.75 – 7.50 mM 0.02 – 0.07

Modified cyclodextrins (MCDs)

7.5 mM 0.05 – 0.6

Chiral surfactants (CSs) 1.5 – 6 % w/v

1.0 x 10-4 - 5.0 x 10-6

M0.02 – 0.05

Chiral ionic liquids (CILs) 30 and 150 mM

5 mM 0.05 – 0.09

Analyte to selector (CDs, MCDs) mole ratio: = 1 : 2RMSEP = xip – xi)2/n]1/2 : xi = known mole fraction, xpi = predicted mole fraction & n = total samplespredicted

Chiral analytes: amino acids, pharmaceuticals, other organics

Problems with CDs

•Limited Solubility•Extent of interaction depends on formation constant of inclusion complex•Possibility of more than one complex in solution (R—CD, CD—R—CD, etc.)•Inclusion complex formation depends on size of analyte in relation to cyclodextrin

What about other possible chiral auxiliaries?

Formation of quaternary ammonium salts of carboxylic acids

Use of Chiral AminesIon-pair formation as a means of enantiomeric discrimination

R CO

OH+ RNH2

R CO

O- +NH3R

Chiral selector: (S)-1-phenylethylamine (S-PEA)

CARMSD with a Homochiral Amine

Determination of enantiomeric composition of Tyrosine with (S)-phenylethylamine using UV spectroscopy

Diastereomeric ion pairs

Zwitterion

+NH3

O

O-

HO

H2N CH3

H3N+ CH3

NH2

O

O-

HO

+D

S

SD

+NH3

O

O-

HO

H2N CH3

H3N+ CH3

NH2

O

O-

HO

+L

S

SL

Use of (S)-1-phenylethylamineDetermination of enantiomeric composition of Tyrosine

(Tyr to S-PEA ratio = 1 : 1)

Mean centered UV spectra for different compositions of D- and L-Tyrosine + (S )-PEA

-0.04

-0.03

-0.02

-0.01

0

0.01

0.02

0.03

0.04

285 305 325 345 365 385 405

Wavelength/nm

Mea

n ce

nter

ed A

bsor

banc

e

I sobestic point(339 nm)

-Two diff erent absorbing species: D- & L- Tyr quaternary salts-Solutions contain identical sum of Tyr species-Relative amounts determine by ratio of D- and L-Tyr -Tyr species have identical absorptivity at 339nm

-0.1

0.4

0.9

1.4

1.9

2.4

2.9

3.4

3.9

4.4

250 260 270 280 290 300 310 320 330 340 350

Wavelength/nm

Abs

orba

nce

Original spectra

Isosbestic point(343 nm)

Effect of pH on spectrum

Effect of varying PEA/Tyrmol ratios at neutral pH

Effect of varying PEA/Tyr mol ratios in acid solution

UV spectra: 2.5 mM L-Tyrosine + 2.5 mM PEA

-0.1

0.4

0.9

1.4

1.9

2.4

2.9

3.4

3.9

236 256 276 296 316 336 356

Wavelength/nm

Abs

orba

nce

2.5mM PEA

2.5mM Tyr

PEA1 : 8Tyr

PEA2 : 7Tyr

PEA3 : 6Tyr

PEA4 : 5Tyr

PEA4.5 : 4.5 Tyr

PEA5 : 4Tyr

PEA6 : 3Tyr

PEA7 : 2Tyr

PEA8 : 1Tyr

2.5 mM Tyr

PEA : Tyr 1 : 1

2.5 mM PEA

236 - 252 nm: hyperchromic effect

289 - 313 nm : hyperchromic effect

UV spectra: 2.5 mM L-Tyrosine + 2.5 mM PEA (Solution acidified with HCL)

-0.05

0.45

0.95

1.45

1.95

2.45

2.95

3.45

3.95

236 256 276 296 316 336 356

Wavelength/nmA

bsor

banc

e PEA1 : 9Tyr

PEA2 : 8Tyr

PEA3 : 7Tyr

PEA4 : 6Tyr

PEA5 : 5Tyr

PEA6 : 4Tyr

PEA7 : 3Tyr

PEA8 : 2Tyr

PEA9 : 1Tyr

236 - 252 nm : no hyperchromic effect

289 - 313 nm : no hyperchromic effect

PEA : Tyr 1 : 1

Job’s Plot

Job’s plot in neutral solution indicating 1:1 ion pair formation

Job’s plot in acid solution indicating lack of ion pair formation

Job's Plot: 2.5 mM Tyrosine + 2.5 mM (S)-PEA

0

0.5

1

1.5

2

2.5

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85

Volume of (S)-PEA/ml

Abs

orba

nce

at 2

43 n

m

Job's Plot: 2.5 mM L-Tyrosine + 2.5 mM PEA (solutions acidified with HCl)

0.49

0.5

0.51

0.52

0.53

0.54

0.55

0.56

0.57

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

Volume of S-PEA/mlA

bsor

banc

e at

243

nm

Determination of enantiomeric composition of Tyrosine with (S)-

phenylethylamine using UV spectroscopy— PLS1 Calibration Model

PlotsRandomly Selected Calibration Samples: 0.0500, 0.150, 0.300, 0.400, 0.500, 0.650, 0.850, 0.950

-5

-4

-3

-2

-1

0

1

2

3

4

5

317 367 417 467 517

Wavelength/nm

Reg

ress

ion

coef

ficie

nt

D-TyrL-Tyr

Cross validation of calibration samples: 0.0500, 0.150, 0.300, 0.400, 0.500, 0.650, 0.850, 0.950

Actual D-Tyr mole fraction

Predicted D-Tyr mole fraction

Predicted L-Tyr mole fraction

0.100 0.101 0.899 (0.900)0.250 0.250 0.750 (0.750)0.300 0.303 0.697 (0.700)0.400 0.401 0.599 (0.600)0.450 0.462 0.538 (0.550)0.650 0.659 0.341 (0.350)0.750 0.758 0.242 (0.250)0.800 0.802 0.198 (0.200)0.900 0.901 0.099 (0.100) RMSEP: D-Tyr & L-Tyr = 0.006Values in bracket = Actual mole fraction of L-Tyr

Determination of enantiomeric composition of Tyrosine with (S)-

phenylethylamine using UV spectroscopy— Results of CARMSD

Cross validation plot

Determination of enantiomeric composition of Phenylalanine with (S)-

phenylethylamine using UV spectroscopy- Results of CARMSD

Cross validation of calibration samples: 0.0500, 0.100, 0.200, 0.392, 0.500, 0.527, 0.700, 0.950

Actual D-Phe mole fraction

Predicted D-Phe mole fraction

Predicted L-Phe mole fraction

0.150 0.185 0.823 (0.850)0.267 0.264 0.738 (0.733)0.352 0.348 0.649 (0.648)0.468 0.464 0.529 (0.532)0.486 0.483 0.515 (0.514)0.527 0.524 0.465 (0.473)0.600 0.597 0.397 (0.400)0.650 0.640 0.346 (0.350)0.819 0.814 0.161 (0.181)RMSEP: D-Phe = 0.013 & L-Phe = 0.011Values in bracket = Actual mole fraction of L-Phe

Determination of enantiomeric composition of Alanine with(S)-

phenylethylamine using UV spectroscopy- Results of CARMSD

Cross validation of calibration samples: 0.0500, 0.100, 0.250, 0.350, 0.500, 0.650, 0.750, 0.850

Actual L-Ala mole fraction

Predicted L-Ala mole fraction

Predicted D-Ala mole fraction

0.200 0.211 0.789 (0.800)

0.300 0.279 0.721 (0.700)

0.400 0.404 0.596 (0.600)

0.600 0.631 0.369 (0.400)

0.700 0.692 0.308 (0.300)

RMSEP: D-Ala & L-Ala = 0.018

Values in bracket = Actual mole fraction of D-Ala

Fischer Esterification

Use of Chiral Alcohols

Esterification results in the formation of true covalent diastereomers

+ ROH + HOHH+

heatC

O

R OHC

O

R OR

CARMSD with Homochiral (S)-(+)-1,2-propanediol

Determination of enantiomeric composition of Phenylalanine with (S)-1,2-propanediol using UV spectroscopy

NH2

O

OH

OH

OH

+NH3-Cl

O

O+NH3

-Cl

O

O+NH3

-Cl

O

O

70oC , HCL

OH

D or L-Phe

D- or L hydroxy monoester compound D- or L diester compound

excess

Possible products

Determination of enantiomeric composition of Phenylalanine with

1,2-propanediol using UV spectroscopy

Original UV spectra (15 samples)

Mean centered UV spectra (15 samples)

-0.01

-0.008

-0.006

-0.004

-0.002

0

0.002

0.004

0.006

0.008

0.01

222 242 262 282 302 322 342 362 382

Wavelength/nm

Mea

n C

ente

red

Abs

orba

nce

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

225 245 265 285 305 325 345

Wavelength/nm

Abs

orba

nce

Randomly selected calibration samples: 0.050, 0.150, 0.250, 0.300, 0.500, 0.750, and 0.950

Determination of enantiomeric composition of Phenylalanine with 1,2-propanediol using UV spectroscopy- PLS1 Calibration Model Plots

Randomly selected calibration samples: 0.050, 0.150, 0.250, 0.300, 0.500, 0.750, and 0.950

Determination of enantiomeric composition of Phenylalanine with

1,2-propanediol using UV spectroscopy- Results of CARMSD

Actual D-Phe mole fraction

Predicted D-Phe mole fraction

Predicted L-Phe mole fraction

0.103 0.0848 0.915 (0.897)0.400 0.407 0.593 (0.600)0.451 0.425 0.575 (0.549)0.597 0.596 0.404 (0.403)missing 0.773 0.227

(missing)0.801 0.801 0.208 (0.199)0.851 0.859 0.141 (0.149)missing 0.877 0.123

(missing) RMSEP: D-Phe & L-Phe = 0.014Values in bracket = Actual mole fraction of L-Phe

CARMSD with noncovalent diastereomers vs. CARMSD with covalent diastereomers

RMSEP figure of merit analysis of chiral discrimination strategies

LEL = lower RMSEP limit and UEL = upper RMSEP limit

0

0.1

0.2

0.3

0.4

0.5

0.6

Cyclodextrins ModifiedCyclodextrins

Chiral Surfactants Chiral Ionic Liquids Covalent/ionic Diatereomers

LEL

UEL

UEL - LEL