Methods
1
Methods Synthesis: β-lactams (1-12) were prepared by cycloaddition of chlorosulphonyl isothiocyanate to the corresponding cycloalkenes and cycloalkadienes [2], and β-lactams (13-19) according to the methods from published literature [3]. CE : Agilent 3D CE + ChemStation software rev.A0903 (Agilent Technologies, USA) Capillary : Fused silica, 32.5 cm (24 cm effective) length x 50 m I.D. (Agilent Technologies, Germany) Background electrolyte: 20 mM sodium acetate pH 5.0 Samples : (see Fig. 1) stock solution of β-lactams: 1mg/ml in acetonitrile, diluted 5-10 times with 30% methanol; No 2,4,7,8,9,10,12 spiked (ratio 1:2 or 1:3) Injection : 50 mbar, 3 sec. Detection : UV-vis diode-array detector at 200 nm Separation : +10 kV, 25 o C In order to calculate the apparent complex stability constant (K i ) of β-lactam enantiomers to the selector CD, the mobilities of the analytes in the absence (μ 0,i ) and in the presence (μ x,i ) of CD in five concentrations in the range of 2,5-20 mmol/L were determined. By plotting (μ x,i - μ 0,i ) vs (μ x,i - μ 0,i ) / c x , the absolute value of slope of the regression line equals the stability constant [12]. Applied CD-s : Single CD system : CMαCD (2,5-20 mM); CMβCD (2,5-20 mM); CMγCD (2,5-20 mM); SBE (2,5-20 mM) D ual system : a charged and a neutral CD derivative is added together. Charged CD-s: CMβCD (20 mM); SBE (20 mM) Neutral CD-s: βCD (15 mM); HPB (40 mM); RAMEB (40 mM); TRIMEB (40 mM) Separation of β-Lactam Enantiomers by Capillary Electrophoresis Using Cyclodextrin Derivatives Németh K. 1 * , Visy J. 1 , Iványi R. 2 , Szemán J. 2 , Szente L. 2 , Forró E. 3 , Fülöp F. 3 , Péter A. 4 , Simonyi M. 1 1 Institute of Biomolecular Chemistry, Chemical Research Center, Hungarian Academy of Sciences, H-1525 Budapest P.O.B.:17, Hungary *Corresponding author: [email protected] 2 Cyclolab R&D Ltd, Budapest, H-1097 Illatos út 7, Hungary 3 Institute of Pharmaceutical Chemistry, University of Szeged, H-6720 Szeged, Eötvös u. 6, Hungary 4 Department of Inorganic and Analytical Chemistry, University of Szeged, H-6720 Szeged, Dóm tér 7, Hungary 8 th Balaton Symposium on High Performance Separation Methods, Siófok, 2-4. September 2009. Aim The aim of this study was to select the most effective cyclodextrin derivative(s) for chiral separation of β-lactam enantiomers by CE. Introduction β-lactams are in the center of interest of both medicine and organic chemistry. Due to lethal inhibitory effect on the synthesis of pathogenic bacterial cell wall, β-lactams are widely applied as antibiotics. The additional pharmaceutical importance of these chiral drugs and their derivatives is, that as intermediates they play key role in the stereoselective synthesis of various nitrogen containing heterocyclic compounds, β-amino acids, alkaloids and short chain peptides. Enantiomers used to have different physiological and anti-bacterial properties. Since β-lactams are synthesized as racemates, the development of effective chiral separation methods is needed. Previously, capillary electrophoresis (CE) separation of β-lactams by negatively charged cyclodextrin (CD) derivatives was reported [1]. Results and Discussion -In a single CD system SBE was more effective than carboxymethyl CD derivatives in the separation of β-lactam enantiomers (see Table 1). -The stability of β-lactam complexes formed with CD derivatives is CMγ<CMα<CMβ in progressive order (excepted of BL/16,17,19) (see Table 1). -The enantioselectivity of CD derivatives was not dependent on the magnitude of complex stability constants (see Table 1) -The dual systems could be more effective in chiral separation than single CD systems (compare Table 1 vs Table 2) if the applied CD derivatives has opposite enantiorecognition preference. - In combination with negatively charged CD derivatives (CMβCD, SBE) the effectiveness of enantioseparation was raised in an order of RAMEB≤HPβCD<βCD<<TRIMEB (see Table 2). -TRIMEB and SBE dual system separated each enantiomer pairs investigated (Fig 2). Conclusion Using CD derivatives (SBE and TRIMEB dual system) CE is suitable for the chiral separation of each β-lactam enantiomer pairs. References [1] C. Jiang et al.: J. Liquid Chromatogr. & Rel. Technol. 30:1709 (2007) [2] A. Peter et al.: Chirality 17:193 (2005) [3] R. Berkecz rt al.:Chromatographia 63: S29 (2006) Abbreviations BL β-lactam CD cyclodextrin CMα carboxymethyl-αCD CMβ carboxymethyl-βCD CMγ carboxymethyl-γCD HPBCD hydroxypropyl-βCD SBE sulphobuthyl-βCD RAMEB randomly-methylated-βCD TRIMEB permethylated-βCD Acknowledgements Bicyclic b -lactams: (1) cis-6-azabicyclo[3.2.0]heptan-7-one (2) cis-7-azabicyclo[4.2.0]octan-8-one (3) cis-7-azabicyclo[4.2.0]oct-3-en-8-one (4) cis-7-azabicyclo[4.2.0]oct-4-en-8-one (5) cis-8-azabicyclo[5.2.0]nonan-9-one (6) cis-9-azabicyclo[6.2.0]decan-10-one (7) cis-9-azabicyclo[6.2.0]dec-4-en-10-one Aromatic tricyclic b -lactams: (8) cis-3,4-benzo-6-azabicyclo[3.2.0]heptan-7-one (9) cis-4,5-benzo-7-azabicyclo[4.2.0]octan-8-one (10) cis-5,6-benzo-8-azabicyclo[5.2.0]nonan-9-one Aliphatic tricyclic b -lactams: (11) exo-3-azatricyclo[4.2.1.0 1.5 ]nonan-4-one (12) exo-3-azatricyclo[4.2.1.0 1.5 ]non-7-en-4-one 4-aryl-substituted b -lactams: (13) 4-phenyl-2-azetidinone (14) 4-(p-tolyl)- 2-azetidinone (15) 4-(o-chlorophenyl)- 2-azetidinone (16) 4-(m-chlorophenyl)-2-azetidinone (17) 4-(p-chlorophenyl)- 2-azetidinone (18) 4-(p-fluorophenyl)- 2-azetidinone (19) 4-(p-bromophenyl)- 2-azetidinone NH O NH O NH O NH O 1 2 3 4 NH O NH O NH O 5 6 7 NH O NH O NH O 8 9 10 NH O NH O 11 12 NH O NH O CH 3 NH O Cl NH O Cl 13 14 15 16 NH O Cl NH O F NH O Br 17 18 19 8 10 12 14 16 18 20 B L/1 B L/2 B L/3 B L/4 B L/5 B L/6 B L/7 B L/8 B L/9 B L/1 0 B L/1 1 B L/1 2 B L/1 3 B L/1 4 B L/1 5 B L/1 6 B L/1 7 B L/1 8 B L/1 9 1S,5S 1R,5R 1S,8R 1 R ,8S 1R ,2R ,5S ,6S 1S ,2S ,5R ,6R 1S,7S 1R ,7 R 1S,6S 1R,6R 1S ,6 R 1R,6S 1S,6R 1R,6S Tim e (m in u tes) Absorbance at 200 nm Figure 2: Enantioseparation of β-lactams by CE using TRIMEB/SBE dual system Figure 1: Structure of β-lactams Table 2: Resolution (Rs) of β-lactam enantiomers by CE using dual CD systems Table 1: Resolution (Rs) of β-lactam enantiomers by CE using single CD systems and complex stability constants (K’) of β-lactams formed with CD derivatives The Cyclodextrin Company
description
The Cyclodextrin Company. Table 1: Resolution (Rs) of β -lactam enantiomers by CE using single CD systems and complex stability constants (K’) of β -lactams formed with CD derivatives. Table 2: Resolution (Rs) of β -lactam enantiomers by CE using dual CD systems. - PowerPoint PPT Presentation
Transcript of Methods
Methods
Synthesis: β-lactams (1-12) were prepared by cycloaddition of chlorosulphonyl isothiocyanate to the corresponding cycloalkenes and cycloalkadienes [2], and β-lactams (13-19) according to the methods from published literature [3]. CE: Agilent 3DCE + ChemStation software rev.A0903 (Agilent Technologies, USA)Capillary: Fused silica, 32.5 cm (24 cm effective) length x 50 m I.D. (Agilent Technologies, Germany) Background electrolyte: 20 mM sodium acetate pH 5.0Samples: (see Fig. 1) stock solution of β-lactams: 1mg/ml in acetonitrile, diluted 5-10 times with 30% methanol; No 2,4,7,8,9,10,12 spiked (ratio 1:2 or 1:3) Injection: 50 mbar, 3 sec. Detection: UV-vis diode-array detector at 200 nmSeparation : +10 kV, 25oC
In order to calculate the apparent complex stability constant (Ki)
of β-lactam enantiomers to the selector CD, the mobilities of the
analytes in the absence (μ0,i) and in the presence (μx,i) of CD in
five concentrations in the range of 2,5-20 mmol/L were
determined. By plotting (μx,i - μ0,i) vs (μx,i - μ0,i) / cx , the
absolute value of slope of the regression line equals the stability constant [12].Applied CD-s: Single CD system: CMαCD (2,5-20 mM); CMβCD (2,5-20 mM); CMγCD (2,5-20 mM); SBE (2,5-20 mM)Dual system: a charged and a neutral CD derivative is added together. Charged CD-s: CMβCD (20 mM); SBE (20 mM)Neutral CD-s: βCD (15 mM); HPB (40 mM); RAMEB (40 mM); TRIMEB (40 mM)
Separation of β-Lactam Enantiomers by Capillary Electrophoresis Using Cyclodextrin Derivatives
Németh K.1*, Visy J.1, Iványi R.2, Szemán J.2, Szente L.2, Forró E.3, Fülöp F.3, Péter A.4, Simonyi M.1
1 Institute of Biomolecular Chemistry, Chemical Research Center, Hungarian Academy of Sciences, H-1525 Budapest P.O.B.:17, Hungary *Corresponding author: [email protected]
2 Cyclolab R&D Ltd, Budapest, H-1097 Illatos út 7, Hungary3 Institute of Pharmaceutical Chemistry, University of Szeged, H-6720 Szeged, Eötvös u. 6, Hungary
4 Department of Inorganic and Analytical Chemistry, University of Szeged, H-6720 Szeged, Dóm tér 7, Hungary
8th Balaton Symposium on High Performance Separation Methods, Siófok, 2-4. September 2009.
Aim
The aim of this study was to select the most effective cyclodextrin derivative(s) for chiral separation of β-lactam enantiomers by CE.
Introduction
β-lactams are in the center of interest of both medicine and organic chemistry. Due to lethal inhibitory effect on the synthesis of pathogenic bacterial cell wall, β-lactams are widely applied as antibiotics. The additional pharmaceutical importance of these chiral drugs and their derivatives is, that as intermediates they play key role in the stereoselective synthesis of various nitrogen containing heterocyclic compounds, β-amino acids, alkaloids and short chain peptides. Enantiomers used to have different physiological and anti-bacterial properties. Since β-lactams are synthesized as racemates, the development of effective chiral separation methods is needed. Previously, capillary electrophoresis (CE) separation of β-lactams by negatively charged cyclodextrin (CD) derivatives was reported [1].
Results and Discussion
-In a single CD system SBE was more effective than carboxymethyl CD derivatives in the separation of β-lactam enantiomers (see Table 1). -The stability of β-lactam complexes formed with CD derivatives is CMγ<CMα<CMβ in progressive order (excepted of BL/16,17,19) (see Table 1). -The enantioselectivity of CD derivatives was not dependent on the magnitude of complex stability constants (see Table 1)-The dual systems could be more effective in chiral separation than single CD systems (compare Table 1 vs Table 2) if the applied CD derivatives has opposite enantiorecognition preference. - In combination with negatively charged CD derivatives (CMβCD, SBE) the effectiveness of enantioseparation was raised in an order of RAMEB≤HPβCD<βCD<<TRIMEB (see Table 2).-TRIMEB and SBE dual system separated each enantiomer pairs investigated (Fig 2).
Conclusion
Using CD derivatives (SBE and TRIMEB dual system) CE is suitable for the chiral separation of each β-lactam enantiomer pairs.
References
[1] C. Jiang et al.: J. Liquid Chromatogr. & Rel. Technol. 30:1709 (2007) [2] A. Peter et al.: Chirality 17:193 (2005)[3] R. Berkecz rt al.:Chromatographia 63: S29 (2006)
Abbreviations
BL β-lactamCD cyclodextrinCMα carboxymethyl-αCDCMβ carboxymethyl-βCD CMγ carboxymethyl-γCDHPBCD hydroxypropyl-βCDSBE sulphobuthyl-βCDRAMEB randomly-methylated-βCDTRIMEB permethylated-βCD
Acknowledgements
Authors thanks for the financial support of the following grants: GVOP-3.2.1.-2004-04-0210/3.0, Jedlik Ányos Grant 00180/2007, NKFP_07_A3_NATURSEP and OTKA TO49721.
Bicyclic b-lactams: (1) cis-6-azabicyclo[3.2.0]heptan-7-one (2) cis-7-azabicyclo[4.2.0]octan-8-one (3) cis-7-azabicyclo[4.2.0]oct-3-en-8-one (4) cis-7-azabicyclo[4.2.0]oct-4-en-8-one (5) cis-8-azabicyclo[5.2.0]nonan-9-one (6) cis-9-azabicyclo[6.2.0]decan-10-one (7) cis-9-azabicyclo[6.2.0]dec-4-en-10-one Aromatic tricyclic b-lactams: (8) cis-3,4-benzo-6-azabicyclo[3.2.0]heptan-7-one (9) cis-4,5-benzo-7-azabicyclo[4.2.0]octan-8-one (10) cis-5,6-benzo-8-azabicyclo[5.2.0]nonan-9-one Aliphatic tricyclic b-lactams: (11) exo-3-azatricyclo[4.2.1.01.5]nonan-4-one (12) exo-3-azatricyclo[4.2.1.01.5]non-7-en-4-one 4-aryl-substituted b-lactams: (13) 4-phenyl-2-azetidinone(14) 4-(p-tolyl)- 2-azetidinone(15) 4-(o-chlorophenyl)- 2-azetidinone
(16) 4-(m-chlorophenyl)-2-azetidinone
(17) 4-(p-chlorophenyl)- 2-azetidinone
(18) 4-(p-fluorophenyl)- 2-azetidinone
(19) 4-(p-bromophenyl)- 2-azetidinone
NH
O
NH
O
NH
O
NH
O
1 2 3 4
NH
O
NH
O
NH
O
5 6 7
NH
O
NH
O
NH
O
8 9 10
NH
O
NH
O
11 12
NH
O
NH
O
CH3
NH
O
Cl
NH
O
Cl 13 14 15 16
NH
O
Cl
NH
O
F
NH
O
Br 17 18 19
8 10 12 14 16 18 20
BL/1
BL/2
BL/3
BL/4
BL/5
BL/6
BL/7
BL/8
BL/9
BL/10
BL/11
BL/12
BL/13
BL/14
BL/15
BL/16
BL/17
BL/18
BL/19
1S,5S 1R,5R
1S,8R 1R,8S
1R,2R,5S,6S 1S,2S,5R,6R
1S,7S 1R,7R
1S,6S 1R,6R
1S,6R 1R,6S
1S,6R 1R,6S
Time (minutes)
Absorbance at 200 nm
Figure 2: Enantioseparation of β-lactams by CE using TRIMEB/SBE dual system
Figure 1: Structure of β-lactams
Table 2: Resolution (Rs) of β-lactam enantiomers by CE using dual CD systems
Table 1: Resolution (Rs) of β-lactam enantiomers by CE using single CD systems and complex stability constants (K’) of β-lactams formed with CD derivatives
The Cyclodextrin Company
