LC Enantioseparation of b-Lactamand b-Amino Acid Stereoisomers and aComparison of Macrocyclic Glycopeptide-and b-Cyclodextrin-Based Columns

R. Berkecz1,2, R. Torok1, I. Ilisz1, E. Forro2, F. Fulop2, D. W. Armstrong3, A. Peter1,&

1 Department of Inorganic and Analytical Chemistry, University of Szeged, Dom ter 7, 6720, Szeged, Hungary;E-Mail: apeter@chem.u-szeged.hu2 Institute of Pharmaceutical Chemistry, University of Szeged, Eotvos utca 6, 6720, Szeged, Hungary3 Department of Chemistry, Iowa State University, Gilmann Hall, Ames IA, 50011, USA

Received: 11 October 2005 / Revised: 3 November 2005 / Accepted: 21 November 2005Online publication: 28 January 2006

Abstract

Direct reversed-phase high-performance liquid chromatographic methods were developed forthe separation of the enantiomers of tricyclic b-lactams, cis-3,4-benzo-6-azabicy-clo[3.2.0]heptan-7-one, cis-4,5-benzo-7-azabicyclo[4.2.0]-octan-8-one, cis-5,6-benzo-8-az-abicyclo[5.2.0]nonan-9-one and new bicyclic b-amino acids, the six- and seven-memberedhomologues of cis-1-amino-4,5-benzocyclopentane-2-carboxylic acid (benzocispentacin), cis-1-amino-5,6-benzocyclohexane-2-carboxylic acid and cis-1-amino-6,7-benzocycloheptane-2-carboxylic acid. The direct separations of the analytes were performed on chiral stationaryphase (CSP) columns containing the macrocyclic glycopeptide antibiotic teicoplanin (ChirobioticT), teicoplanin aglycone (Chirobiotic TAG), vancomycin (Chirobiotic V), vancomycin aglycone(Chirobiotic VAG), ristocetin A (Chirobiotic R) or a new dimethylphenyl carbamate-derivatizedb-cyclodextrin-based Cyclobond DMP. The results achieved with the different methods werecompared in systematic chromatographic examinations. The effects of an organic modifier andof the mobile phase composition on the separation and the separation efficiency of differentcolumns were investigated. The difference in enantioselective free energy between the aglyconeCSP and the teicoplanin CSP for these b-lactams and b-amino acids ranged between 0.3 and)1.1 kJmol)1. Better enantioseparations were attained in most cases on the aglycone CSP.

Keywords

Column liquid chromatographyChirobiotic T and TAG columnsCyclobond DMP columnb-Amino acidsb-Lactams

Introduction

Alicyclic b-lactams and b-amino acids are

very attractive compounds because of

their potential biological activity (e.g.

monobactams and cispentacin) [1–3] and

their utility in synthetic chemistry [4–6].

They can serve as building blocks for the

synthesis of modified peptides with in-

creased activity and stability [7–11], which

exhibit well-defined three-dimensional

structures similar to those of natural pep-

tides (e.g. b-peptides with possible antibi-

otic activity) [12–18]. Alicyclic b-amino

acids also can be applied in heterocyclic

[19,20] and combinatorial chemistry

[21,22].

Studies on synthetic or natural b-lac-tams and b-amino acids can be facilitated

by versatile and robust methods for

determination of the enantiomeric purity

of the starting materials and products.

High-performance liquid chromatogra-

phy (HPLC) is one of the most useful

techniques for the recognition and/or

separation of stereoisomers, including

enantiomers. For the direct HPLC enan-

tioseparation of b-amino acids, Davankov

et al. [23], Lindner andHirshbock [24] and

Yamazaki et al. [25] reported preparative

ligand exchange chromatographic meth-

ods, while D’Acquarica et al. [26], Peter

et al. [27,28] and Hyun et al. [29,30] sepa-

rated different alicyclic, cyclic and aro-

matic b-amino acids on new types of CSPs,

containing a macrocyclic glycopeptide

antibiotic, a quinine-derived chiral anion-

exchanger, a crown ether or an (R)-phe-

nylglycinol derivative as chiral selectors.

For the separation of lactam stereoisom-

ers, Pirkle et al. [31] andLee et al. [32] used

an (R)-N-(3,5-dinitrobenzoyl)phenyl gly-

cine-based CSP. Different cellulose or

amylose polysaccharide-based CSPs were

applied for the enantioseparation ofb- andc-lactam stereoisomers by Okamato et al.

[33], Ficarra et al. [34], Cirilli et al. [35]

and Peter et al. [36]. Huang et al. [37]

applied b-cyclodextrin-based, while Peter

et al. [36] made use of macrocyclic glyco-

peptide-based CSPs. In summary, b-lac-tam and b-amino acid stereoisomers have

Presented at: 6th Balaton Symposium onHigh-Performance Separation Methods,Siofok, Hungary, September 7–9, 2005.

2006, 63, S37–S43

DOI: 10.1365/s10337-005-0701-x� 2006 Friedr. Vieweg & Sohn/GWV Fachverlage GmbH

Original Chromatographia Supplement Vol. 63, 2006 S37

so farmainly been separated on p-complex

and polysaccharide-based CSPs.

The aim of the present work was to

evaluate direct HPLC methods for the

separation of the enantiomers of tricyclic b-lactams and bicyclic b-amino acids (for

structures, see the Tables). Direct separa-

tions were performed on CSPs containing

macrocyclic glycopeptide antibiotics such

as teicoplanin, vancomycin and their agly-

cone and ristocetin A and dimethylphenyl

carbamate-derivatized b-cyclodextrin as

chiral selectors. The effects on the selectiv-

ity of parameters, such as the nature of the

organic modifier, the mobile phase com-

position, the flow rate and the structures of

the analytes in the different chromato-

graphic methods are examined and dis-

cussed. The separation of the stereoisomers

was optimized by variation of the chro-

matographic parameters. The efficiency of

the each of the methods and the role of

the molecular structure of the analyte on

the enantioseparation were noted. The

sequence of elution of the enantiomers was

determined.

Experimental

Chemicals and Reagents

The racemic tricyclic b-lactams cis-3,

4-benzo-6-azabicyclo[3.2.0]heptan-7-one

(1), cis-4,5-benzo-7-azabicyclo[4.2.0] oc-

tan-8-one (2) and cis-5,6-benzo-8-azabi-

Table 1. Chromatographic data, retention factor (k), separation factor (a) and resolution (RS) of separation of analytes 1–6 on different columns inreversed-phase mode

Compound CSP Mobilephase (v/v)

k1 k2 a RS )DDG�298 K

(kJ mol)1)Elution sequence

1 NH

O

T a 1.00 1.15 1.15 0.70 0.3 1R,5R<1S,5ST b 0.98 1.17 1.19 1.71 0.4 1R,5R<1S,5STAG a 1.27 2.23 1.76 4.57 1.4 1R,5R<1S,5STAG c 1.81 2.64 1.46 2.81 0.9 1R,5R<1S,5SDMP e 3.52 3.88 1.10 1.76 0.2 1R,5R<1S,5S

2

NH

O

T a 0.87 0.87 1.00 0.00 0.0 –T b 0.89 0.89 1.00 0.00 0.0 –TAG a 1.09 1.19 1.09 1.09 0.2 1R,6R<1S,6STAG c 1.57 1.57 1.00 0.00 0.0 –DMP e 4.77 5.45 1.14 2.46 0.3 1R,6R<1S,6S

3 NH

OT a 0.88 0.96 1.09 0.77 0.2 1R,7R<1S,7ST b 0.87 0.93 1.07 0.70 0.2 1R,7R<1S,7STAG a 1.11 1.75 1.58 3.56 1.1 1R,7R<1S,7STAG c 1.64 2.67 1.63 4.00 1.2 1R,7R<1S,7SDMP e 6.86 7.17 1.05 0.88 0.1 1R,7R<1S,7S

4

C

NH2

OOH T a 6.24 6.62 1.06 0.68 0.1 1R,2R<1S,2ST b 3.31 3.42 1.03 0.50 0.1 1R,2R<1S,2STAG a 6.33 7.22 1.14 0.67 0.3 1R,2R<1S,2STAG c 3.21 3.83 1.17 1.14 0.4 1R,2R<1S,2SR a 2.41 2.80 1.16 0.86 0.4 1R,2R<1S,2SDMP* d 0.72 0.89 1.24 1.49 0.5 1S,2S<1R,2R

5

COOH

NH2

T a 5.02 6.28 1.25 1.95 0.6 1R,2R<1S,2ST b 3.12 3.96 1.27 2.85 0.6 1R,2R<1S,2STAG a 6.03 11.44 1.90 4.33 1.6 1R,2R<1S,2STAG c 3.58 6.07 1.70 5.00 1.3 1R,2R<1S,2STAG d 2.82 3.74 1.33 3.43 0.7 1R,2R<1S,2SR a 2.80 4.99 1.78 4.25 1.4 1S,2S<1R,2R

6NH2

COOHT a 5.42 7.93 1.46 3.46 0.9 1R,2R<1S,2ST b 2.89 3.96 1.37 4.00 0.8 1R,2R<1S,2STAG a 8.55 11.04 1.29 1.18 0.6 1R,2R<1S,2STAG c 3.80 5.07 1.33 2.57 0.7 1R,2R<1S,2SR a 3.55 3.72 1.05 <0.40 0.1 –V a 1.74 2.55 1.47 3.15 1.0 1R,2R<1S,2SV b 0.81 1.36 1.68 3.47 1.3 1R,2R<1S,2SVAG a 0.79 0.98 1.24 2.08 0.5 1R,2R<1S,2S

Column (CSP): Chirobiotic T (T), Chirobiotic TAG (TAG), Chirobiotic R (R), Chirobiotic V (V), Chirobiotic VAG (VAG) and Cyclobond DMP(DMP); mobile phase, a, MeOH 100%, b, 0.1% TEAA (pH 6.5)/MeOH=10/90 (v/v), c, 0.1% TEAA (pH 6.5)/MeOH=30/70 (v/v), d, 0.1% TEAA (pH6.5)/MeOH=55/45 (v/v), e, 0.1% TEAA (pH 4.1)/MeOH=60/40 (v/v); detection, 210 nm; flow-rate, 0.5 mL min)1; temperature, 25 �C; * 10 �C; dead-time, Chirobiotic T, tM=3.82 min, Chirobiotic TAG, tM=3.91 min, Chirobiotc V, tM=6.40 min, Chirobiotic VAG, tM=6.40 min, Chirobiotic R,tM=6.08 min and Cyclobond DMP, tM=5.85 min.

S38 Chromatographia Supplement Vol. 63, 2006 Original

cyclo[5.2.0]nonan-9-one (3) were pre-

pared by the cycloaddition of chloro-

sulfonyl isocyanate to the appropriate

cycloalkenes and cycloalkadienes [1]. The

new bicyclic b-amino acids, cis-1-amino-

4,5-benzocyclopentane-2-carboxylic acid

(4), cis-1-amino-5,6-benzocyclohexane-2-

carboxylic acid (5) and cis-1-amino-6,7-

benzocycloheptane-2-carboxylic acid (6),

were prepared according to [38]. A very

efficient enzymatic method was used for

the synthesis of the above-mentioned b-lactam enantiomers and benzocispentacin

homologs: Lipolase (lipase B from Can-

dida antarctica produced by submerged

fermentation of a genetically modified

Aspergillus oryzae microorganism and

adsorbed on a macroporous resin) catal-

ysis of the enantioselective (E> 200) ring

cleavage of b-lactams with H2O was done

in an organic solvent as previously re-

ported [38].

Acetonitrile (MeCN), methanol

(MeOH) and 2-propanol (IPA) of HPLC

grade were purchased from Merck

(Darmstadt, Germany), as were trieth-

ylamine (TEA), glacial acetic acid

(AcOH), trifluoroacetic acid (TFA) and

other reagents of analytical reagent

grade. The Milli-Q water was further

purified by filtration on a 0.45-lm fil-

tration, type HV, Millipore (Molsheim,

France).

0.1% triethylammonium acetate

(TEAA) buffer was prepared by titration

of 0.1% (by volume) aqueous solutions of

TEA with AcOH to a suitable pH.Mobile

phases for reversed-phase, normal-phase

and polar-organic chromatography were

prepared by mixing the indicated volumes

of buffers and/or solvents. The eluents

were degassed in an ultrasonic bath, and

helium gas was purged through them

during the analyses.

Apparatus

The HPLC measurements were carried

out on a Waters HPLC system consisting

of an M-600 low-pressure gradient pump,

an M-996 photodiode-array detector and

a Millenium32 Chromatography Manager

data system. A second Water’s Breeze

system consisted of a 1525 binary pump,

a 487 dual-channel absorbance detector,

a 717 plus autosampler and Breeze data

manager software (both systems from

Waters Chromatography, Milford, MA,

USA). Both chromatographic systems

were equipped with Rheodyne Model

7125 injectors (Cotati, CA, USA) with

20-lL loops.

The columns used for direct separations

were teicoplanin-containing Chirobiotic

T�, teicoplanin aglycone-containing Chi-

robiotic TAG�, vancomycin-containing

Chirobiotic V�, vancomycin aglycone-

containing Chirobiotic VAG�, ristocetin

A-containing Chirobiotic R� and

dimethylphenylcarbamate-derivatized b-cyclodextrin-based Cyclobond DMP�columns 250·4.6 mm I.D., with a 5 lmparticle size, all fromAstec (Whippany,NJ,

USA). The dead-times (tM) of the columns

were determined in reversed-phase and

polar-organic modes by injecting 20-lL of

0.001 M potassium bromide and in nor-

mal-phase mode by injecting hexane/IPA

in different compositions.

The columns were thermostated in a

water bath, with a cooling-heating ther-

mostat (MK 70, Mechanik Prufgerate,

Medlingen, Germany). The precision of

temperature adjustment was ± 0.1 �C.Stock solutions of analytes (1 mg

mL)1) were prepared by dissolution in

water or in the starting mobile phase.

Results and Discussion

The CSPs used for the direct enantio-

separation of the tricyclic b-lactam and

bicyclic b-amino acid analytes in this

study were macrocyclic glycopeptide and

b-cyclodextrin-based columns. The re-

sults of the separations of the enantio-

mers in reversed-phase mode (Table 1)

were evaluated by using CSPs with a

minimum of three of the mobile phases:

100% MeOH and 0.1% aqueous TEAA

(pH 6.5)/MeOH in different composi-

tions. To simplify the presentation,

mainly results relating to partial or

baseline enantiomeric separation are lis-

ted in Table 1. However, in a few cases,

for purposes of comparison, examples

are included where no separation

occurred.

The retention and selectivity on the

teicoplanin-containing CSP, could be

controlled by altering the nature and

concentration of the organic modifier.

An increase in the MeOH content led to

an increase in the retention factor for

b-amino acids, while for b-lactams a

decrease in k was observed. The unusual

behavior i.e., an increase in k with

increasing organic modifier content in

the reversed-phase mode was usual for

the teicoplanin CSPs [39,40]. A possible

explanation may be the decreased solu-

bility of the amino acids in a MeOH-rich

mobile phase. Previously, when more

hydrophobic a-amino acids were sepa-

rated on a teicoplanin CSP, more typical

reversed-phase retention behavior was

observed [39,41]. With 100% MeOH as

eluent on the teicoplanin-containing

CSP, Chirobiotic T, the retention factors

of the first-eluted stereoisomers (k1) of

analytes 1–3 were in the range 0.87–1.00,

while the values of k1 for analytes 4–6

were in the range 5.02–6.24. Similar

tendencies were observed on the tei-

coplanin aglycone CSP, Chirobiotic

TAG, where for analytes 1–3 k1 lay in

the range 1.09–1.27 and for analytes 4–6

in the range 6.03–8.55. The lower

retention factors of the b-lactam ste-

reoisomers revealed a difference in sep-

aration mechanism between the amino

acids and the b-lactams (see the sub-

sequent discussion).

For the a-amino acids, the teicoplanin

aglycone afforded a higher separation

capability than that with the native tei-

coplanin [42]. Table 1 lists the separation

factors (a) and resolutions (RS) of the ste-

reoisomers of the b-lactams and b-amino

acids. The highest separation factors ob-

tained on Chirobiotic T for b-lactams

1 and 3 were a=1.19 and a=1.09,

respectively (for analyte 2, no separation

was observed), while forb-amino acids 4, 5

and 6 they were a=1.06, a=1.27 and

a=1.46, respectively. The highest separa-

tion factors obtained on Chirobiotic TAG

were a=1.76, a=1.09 and a=1.63 for

analytes 1, 2, 3, and a=1.17, a=1.90 and

a=1.33 for analytes 4, 5, 6, respectively. In

general, in the reversed-phase mode, the

Chirobiotic TAG CSP seemed to be more

effective in the separation of the enantio-

mers of b-lactams and b-amino acids than

the native Chirobiotic T CSP. The highest

a values observed (analyte 5) correspond

to a difference in enantioselective free en-

ergy of around the )1.6 kJ mol)1.

For the b-lactam and b-amino acid

stereoisomers, the selectivity factor and

the resolution were higher on the Chiro-

biotic TAG column. The only exception

was analyte 6, where higher a and RS

factors were obtained on the Chirobiotic

T column than on the Chirobiotic TAG

column.

The hydrophobic character of the

analytes exerted a slight effect on chro-

matographic behavior. The most hydro-

phobic b-lactam analyte 3 and b-amino

Original Chromatographia Supplement Vol. 63, 2006 S39

acid 6 exhibited the highest retention

factors, but the differences were not sig-

nificant. It seemed that the steric hin-

drance had a larger effect on the retention

behavior than did the hydrophobic

character of the molecule.

As concerns the other macrocyclic

glycopeptide columns, Chirobiotic R, V

and VAG were in some cases effective in

the separation of the b-amino acid ste-

reoisomers. The interactions between the

free amino functions of the ristocetin A,

vancomycin and vancomycin aglycone

selectors and the free carboxy groups of

the analytes were probably involved in the

chiral recognition. On the other hand, the

Cyclobond DMP column was effective in

the separation of the stereoisomers of

analytes 1–3, but not for 4–6 (the ste-

reoisomers of 4 were separated at

subambient temperature). The formation

of an inclusion complex was more likely

with the tricyclic analytes.

The sequence of elution in the

reversed-phase mode was determined in

all cases. For the a-amino acids on the

Chirobiotic phases, with a few excep-

tions, the sequence S<R was observed

[40–42], whereas, both the sequences

S<R and R<S were found for the b-amino acids [27,36]. For the b-lactams

and b-amino acids, when the configura-

tion of the annelation carbon atom at-

tached to the CO group (C-1) was taken

into account, the sequence was R<S

(exceptions were analytes 4 and 5 on the

Cyclobond DMP and Chirobiotic R col-

umns, respectively (Table 1)).

Use of the polar-organic mode with a

MeOH/AcOH/TEA mobile phase system

generally led to good enantioresolution for

the a-amino acids on either native teicopl-

anin or teicoplanin aglyconeCSPs [43]. For

the b-lactam and b-amino acid stereoi-

somers, use of the polar-organic mode did

not furnish a significant improvement in

enantioresolution (Table 2). The same was

true for the Chirobiotic R, V and VAG

columns: application of the polar-organic

phases did not improve the enantiosepa-

ration. The Cyclobond DMP column in

the polar-organic mode was practically

ineffective in the separation of either the b-lactam or the b-amino acid stereoisomers.

As regards the chromatographic behavior

of the Chirobiotic T and TAG columns at

the same eluent composition, MeOH/

AcOH/TEA (100/0.01/0.01 (v/v/v)), the

retention factors for the first-eluting enan-

tiomer, k1, for the b-lactams differed only

slightly, k1 ranging between 0.95 and 1.36.

Larger differences were observed for the b-amino acids, where k1 ranged between 4.66

Table 2. Chromatographic data, retention factor (k), separation factor (a) and resolution (RS) of separation of analytes 1–6 on different columns inpolar organic-mode

Compound CSP Mobilephase (v/v)

k1 k2 a RS )aDDG�298 K

(kJ mol)1)Elution sequence

1NH

O

T a 1.00 1.16 1.16 1.17 0.4 1R,5R<1S,5STAG a 1.36 2.36 1.74 5.54 1.4 1R,5R<1S,5S

2

NH

O T a 0.95 0.95 1.00 0.00 0.0 –TAG a 1.12 1.21 1.08 0.50 0.2 1R,6R<1S,6SR a 0.19 0.23 1.21 0.52 0.5 –

NH

O

T a 0.95 1.03 1.08 <0.40 0.2 1R,7R<1S,7S3 T b 0.95 1.03 1.08 0.80 0.2 1R,7R<1S,7S

TAG a 1.13 1.81 1.60 1.79 1.2 1R,7R<1S,7STAG b 1.14 1.81 1.59 2.93 1.1 1R,7R<1S,7S

NH2

COOH

T a 6.41 6.81 1.06 0.76 0.1 1R,2R<1S,2STAG a 9.44 10.42 1.10 0.98 0.2 1R,2R<1S,2S

4 TAG b 8.20 9.08 1.11 1.00 0.3 1R,2R<1S,2SR a 1.92 2.26 1.18 1.08 0.4 1R,2R<1S,2SV a 0.99 1.08 1.09 0.91 0.2 1S,2S<1R,2RVAG a 1.05 1.19 1.13 1.67 0.3 1S,2S<1R,2R

COOH

NH2

T a 5.19 6.53 1.34 2.10 0.7 1R,2R<1S,2S5 TAG a 7.84 13.38 1.71 3.85 1.3 1R,2R<1S,2S

TAG b 6.77 11.96 1.77 3.92 1.4 1R,2R<1S,2SR a 2.32 4.46 1.92 4.00 1.6 1S,2S<1R,2R

NH2

COOH

T a 4.66 6.52 1.40 2.00 0.8 1R,2R<1S,2STAG a 8.55 10.87 1.27 1.65 0.6 1R,2R<1S,2S

6 TAG b 8.64 10.93 1.27 1.70 0.6 1R,2R<1S,2SR a 2.57 2.70 1.05 <0.40 0.1 1S,2S<1R,2RV a 1.32 2.06 1.56 3.18 1.1 1R,2R<1S,2SVAG a 1.60 1.90 1.19 2.50 0.4 1R,2R<1S,2S

Column (CSP): Chirobiotic T (T), Chirobiotic TAG (TAG), Chirobiotic R (R), Chirobiotic V (V) and Chirobiotic VAG (VAG); mobile phase,a, MeOH/AcOH/TEA=100/0.01/0.01 (v/v/v), b, MeOH/AcOH/TEA=100/0.1/0.01 (v/v/v); detection, 210 nm; flow-rate, 0.5 mL min)1; temperature,25 �C; dead-time, Chirobiotic T, tM=3.82 min, Chirobiotic TAG, tM=3.91 min, Chirobiotc V, tM=6.40 min, Chirobiotic VAG, tM=6.40 min andChirobiotic R, tM=6.08 min.

S40 Chromatographia Supplement Vol. 63, 2006 Original

and 9.44. Higher retention was not always

accompanied by better resolution. The se-

quence of elution in the polar-organic

mode followed the sequence observed in

the reversed-phase mode, with some

exceptions for the b-amino acids on the

Chirobiotic R, V and VAG columns

(Table 2).

The normal-phase mode separation

was used only for the b-lactams because of

the lack of solubility of the bicyclic amino

acids in hexane-containing systems. Ana-

lyte 2 was better separated in the normal

phase mode on the macrocyclic glycopep-

tide-based columns than in other modes.

On the Cyclobond DMP column in the

normal-phase mode, baseline separation

was achieved for analyte 1 and partial

separation for 2. The elution sequence was

similar to that mentioned above, with the

exception of analyte 3 on the Cyclobond

DMP column.

The Role of Carbohydrate Unitsin Enantiorecognition

The carbohydrate units are themselves

chiral, which can help in the enantiorec-

ognition process. Comparison of the

results obtained on the Chirobiotic T and

TAG (or V and VAG) CSPs may con-

tribute to an understanding of the role of

the pendant sugar moieties in chiral rec-

ognition. To quantify the effects of the

sugar units, the differences in enantiose-

lective free energies between the two

CSPs, DTAG)TD(DG�), listed in Tables 1–

3, were used [)D(DG�)=RT lna]. Fromthe D(DG�) value obtained for a given

compound on the teicoplanin aglycone

CSP, the D(DG�) value found on the native

teicoplanin CSP was subtracted

[D(DG�)aglycone)D(DG�)native teicoplanin =

DTAG)TD(DG�)] and the difference was

plotted as shown in Fig. 1. A negative

number means that the stereoisomers are

better separated on the aglycone CSP,

while a positive number means that the

stereoisomers are better separated on the

native teicoplanin CSP, which contains

the carbohydrate units.

As may be seen in Fig. 1 A and B, in

the reversed-phase and polar-organic

modes the effect of the lack of the sugar

units was more pronounced in the cases

of analytes 1–5. The free energy differ-

ences DTAG)TD(DG�) for analytes 1–6 in

the reversed-phase mode were )1.1,)0.2, )0.9, )0.2, )1.0, and +0.3 kJ

mol)1, respectively, while in the polar-

organic mobile phase the corresponding

values were )1.0, )0.2, )1.0, )0.1, )0.6and +0.2, respectively. From the aspect

of chiral separation, the sugar moieties

of the native teicoplanin may intervene

in the chiral recognition process in at

least three ways [40–42]: (a) steric

Fig. 1. Enantioselectivity differences, DTAG)TD(DG�), between the aglycone and native teicoplanin CSPs in different chromatographic modes. Forcompounds, see Table 1. Columns, Chirobiotic T and TAG; mobile phase, A, 100% MeOH, B, MeOH/AcOH/TEA=100/0.01/0.01 (v/v/v), C, hexane/IPA=10/90 (v/v); flow-rate, 0.5 mL min)1; detection, 210 nm

Original Chromatographia Supplement Vol. 63, 2006 S41

hindrance, the sugar units occupying

space inside the ‘‘basket’’, which limits

the access of other molecules to the

binding sites; (b) the blocking of possible

interaction sites on the aglycone, where

two sugars are linked through phenolic

hydroxy groups and the third sugar is

linked through an alcohol moiety; and

(c) the supply of competing interaction

sites, where the three sugars are them-

selves chiral and have hydroxy, ether

and amido functional groups.

In the reversed-phase and polar-or-

ganic modes for b-amino acids 4 and 5,

the free energy difference between the

two related CSPs may be due to the

effect of steric hindrance, but other

possibilities too should be considered.

The sugar moieties are thought to

‘‘dock’’ and bind inside the cleft of the

aglycone, near its amine (or ureido, if

attached to a linkage chain) functional

group. Besides steric hindrance, the two

phenols and the hydroxy group on the

aglycone seem to further enhance the

interaction with the amino acids. For

analyte 6, a positive DTAG)TD(DG�) and

DVAG)V D(DG�) values were calculated

on the teicoplanin and vancomycin-

based CSPs, (Tables 1 and 2). This

means that one of the stereoisomers of

analyte 6 associated more selectively

with the teicoplanin or vancomycin

selector containing sugar moieties than

on the aglycone one, the difference in

interaction energy of the two stereoi-

somers therefore being larger on the

CSPs containing sugar moieties.

The negative DTAG)TD(DG�) values forb-lactams 1–3, indicate that the interac-

tion on the teicoplanin aglycone CSP was

stronger. The relatively large free energy

difference was observed in spite of the lack

of the carboxy group on the b-lactams.

The carboxy groups generally participate

in the primary interaction with the amine

functional group of the selector, and for

the amino acids this resulted in a strong

interaction between the analyte and the

selector. It seemed that the tricyclic system

provided a better fit in the aglycone bas-

ket, while the sugar moieties limited the

access of the analyte molecules to the

binding sites.

In the normal-phase mode for b-lactams 2 and 3, similar negative DTAG)

TD(DG�) values were calculated as in the

reversed-phase and polar-organic modes

(Fig. 1, C). For analyte 1, the large

positive DTAG)TD(DG�) value indicates

that the separation of the stereoisomers

of 1 in the normal-phase mode occurred

via a different mechanism. Selected

chromatograms for the enantiosepara-

tion of analytes 1–6, evaluated by

different methods, are depicted in

Fig. 2.

1

0.00

0.00

0.01

0.01

0.02

0.02

13 18 23 28 33 38 43 48 53 58 63

Time (min)

A2

-0.01

0.05

0.10

0.15

0.20

0.25

8 10 12 14 16 18 20 22 24 26Time (min)

A

3

-0.01

0.02

0.04

0.06

0.08

0.10

0.12

8 13 18 23 28 33 38Time (min) Time (min)

A

4

0.00

0.00

0.01

0.01

0.02

0.02

0.03

0.03

0.04

5 6 7 8 9 10 11 12 13 14 15

A

5

-0.01

0.04

0.09

0.14

0.19

0.24

0.29

0.34

0.39

10 15 20 25 30 35Time (min)

A

-0.01

0.04

0.09

0.14

0.19

0.24

0.29

0.34

0.39A

6

10 12 14 16 18 20 22 24Time (min)

Fig. 2. Selected chromatograms of analytes 1–6. Column, Chirobiotic T for analytes 1, 2, 3 and 6,Chirobiotic TAG for 5 and Cyclobond DMP for 4; mobile phase, hexane/IPA=10/90 (v/v) foranalytes 1, 2 and 3, 0.1% TEAA (pH 4.1)/MeOH=55/45 (v/v) for analyte 4, 30/70 (v/v) for 5 and0.1% TEAA (pH 6.5)/MeOH=10/90 (v/v) for 6; flow-rate, 0.5 mL min)1; detection, 210 nm;temperature, 25 �C

Table 3. Chromatographic data, retention factor (k), separation factor (a) and resolution (RS) ofseparation of analytes 1-3 on different columns in normal-phase mode

Compound CSP Mobilephase (v/v)

k1 k2 a RS )DDG�298 K

(kJ mol)1)Elution sequence

NH

O

T a 6.11 10.72 1.75 3.42 1.4 1R,5R<1S,5S1 TAG a 10.46 10.46 1.00 0.00 0.0 –

DMP b 5.83 7.25 1.24 2.30 0.5 1R,5R<1S,5S

NH

O

T a 2.68 3.35 1.25 1.57 0.6 1R,6R<1S,6S2 TAG a 6.30 9.55 1.52 2.00 1.0 1R,6R<1S,6S

DMP b 5.82 6.49 1.12 1.33 0.3 1R,6R<1S,6S

NH

OT a 3.04 5.33 1.75 2.75 1.4 1R,7R<1S,7S

3 TAG a 4.62 10.97 2.37 2.86 2.1 1R,6R<1S,6SDMP B 4.34 4.50 1.04 0.67 0.1 1S,7S<1R,7R

Column (CSP): Chirobiotic T (T), Chirobiotic TAG (TAG) and Chirobiotic DMP (DMP); mobilephase, a, hexane/IPA=10/90 (v/v), b, hexane/IPA=90/10 (v/v); detection, 210 nm; flow-rate,0.5 mL min)1; temperature, 25 �C; dead-time, Chirobiotic T, tM=3.82 min, Chirobiotic TAG,tM=3.91 min, Cyclobond DMP, tM=5.85 min.

S42 Chromatographia Supplement Vol. 63, 2006 Original

Conclusions

HPLC methods were developed for the

separation of the enantiomers of tricyclic

b-lactam and bicyclic b-amino acids. The

direct separations were performed on

CSP columns containing macrocyclic

glycopeptide antibiotics (teicoplanin,

vancomycin and ristocetin A) and a

b-cyclodextrin. Of the macrocyclic gly-

copeptide-based CSPs, the teicoplanin-

based CSPs proved more applicable. Of

the teicoplanin-based CSPs, the teicopla-

nin aglycone proved more suitable for the

separation of the stereoisomers of the

tricyclic b-lactam and bicyclic b-amino

acids. By variation of the chromato-

graphic modes and parameters, the sep-

aration of the stereoisomers could be

optimized. In conclusion, baseline reso-

lution was achieved for the tricyclic b-lactams and bicyclic b-amino acids in at

least one of chromatographic systems

tested. The elution sequence was deter-

mined in all cases and a general rule was

established for the sequence of elution of

the stereoisomers.

Acknowledgements

This work was supported by OTKA

grants T 042451, T 049407 and by the

National Institutes of Health grant NIH

RO1 GM53825–08.

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