Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-07-25
2001-11-13
Geist, Gary (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S046000, C428S423100, C428S426000, C428S447000, C428S532000
Reexamination Certificate
active
06316613
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates generally to the use of cyclodextrins to separate optical isomers by capillary electrophoresis.
BACKGROUND OF THE INVENTION
Many chemical compounds used in pharmaceutical formulations possess an asymmetric center responsible for optical activity that can strongly influence their pharmacological properties. Examples of synthetic drugs in which one of two enantiomers possess a different pharmacological properties are well known. For example, (−)-propranolol is 100-fold more potent than (+)-propranolol. In addition one of two enantiomers of the same drug can be more toxic, e.g., thalidomide, ketamine. Accordingly, analytical methods that separate enantiomers with high resolution power and high efficiency are becoming increasingly important. The need for stereochemically pure drugs and careful testing of existing racemates necessitates the development of efficient, fast, sensitive and accurate chiral analysis methods.
Chromatography techniques, particularly high-performance liquid chromatography (HPLC), are commonly used for the analysis of enantiomers. More recently, capillary electrophoresis (CE) has been adapted for use in conjunction with chiral selectors. The most common strategy employed is the use of cyclodextrins for differential host-guest complexation of enantiomeric pairs [Fanali, S., J.,
Chromatogr
., 735:77-121 (1996); Nishi, H., and Terabe, S., J.,
Chromatogr
., 694:245-276 (1995); Guttman, A., “Capillary Electrophoresis Separation of Enantiomers by Cyclodextrin Array Chiral Analysis”, pp. 75-100 in Handbook of Capillary Electrophoresis, 2nd Ed., J. Landers, Ed., 1997, CRC Press Inc., Boca Raton, Fla.]. In contrast to HPLC separations, in which one must select carefully from the wide variety of available columns containing the resolving agent bound to the solid support, chiral CE analyses are greatly simplified by having chiral selectors such as cyclodextrin dissolved in the separation buffer.
Cyclodextrins (CD) are cyclic oligosaccharides consisting of six, seven or eight glucose units corresponding to the names &agr;-, &bgr;- or &ggr;-cyclodextrin. CD's form a truncated cone with a rim of secondary hydroxy groups at the opening with the larger diameter. The internal cavity contains no hydroxy functions and exhibits a hydrophobic character. This hydrophobic nature permits CD's to form highly selective inclusion complexes with aromatic or alkyl groups. Differences in the complex formation constants of a CD and its guest molecules can effect the relative electrophoretic mobilities of structurally similar optical isomers, resulting in a clear separation of enantiomers.
In chiral separations of acidic and basic compounds, the CD complex migrates under the influence of the charge of the ionic analytes. For neutral analytes, however, uncharged cyclodextrins are not applicable because the analyte, the cyclodextrin and the complex have no electrophoretic mobilities. The use of charged cyclodextrins was therefore conceived to solve the problem for chiral separation of neutral compounds.
Mayer et al. [
J. Microcol
. Sep., 6:43-48 (1994)] and Tait et al. [
Anal. Chem
., 66:4013-41018 (1994)] first reported the use of a sulfobutyl ether, &bgr;-cyclodextrin (SBE-&bgr;CD) for CE chiral separations. Subsequent reports have described the analysis of amines and neutral compounds using SBECD as chiral selector [Dette et al.,
Electrophoresis
, 15:799-803 (1994); Chankvetadze et al.,
Electrophoresis
, 15:804-807 (1994); Lurie et al.,
Anal. Chem
., 66:4019-4026 (1994)]. Vincent et al. (“A family of novel, single-isomer chiral resolving agents for capillary electrophoresis. Part 2: Heptakis-6-sulfato-&bgr;-cyclodextrin, submitted to Analytical Biochemistry, Apr. 15, 1997) adapted the selective protection and deprotection methods of Moriya et al. [
J. Med. Chem
., 36:1674-1677 (1993)] to synthesize a sulfated &bgr;-CD (7S&bgr;CD) having sulfate esters solely on the 6-positions at the narrow end of the bucket-shaped cyclodextrin molecule. This synthetic scheme was designed to retain the geometry of the cavity and the complexation properties of native &bgr;-cyclodextrin. Stalcup and co-workers [Stalcup, A., et al.,
Anal. Chem
., 68:1360-1368 (1996); Wu, W., et al.,
Chromatogr
., 18:1289-1315 (1995)] recently reported the use of a mixture of cyclodextrin sulfate esters for the separation of racemates of neutral compounds and amines in untreated fused-silica capillary, which was effective for 40 compounds investigated.
Despite these encouraging results, there is no universal system tor carrying out chiral separations by CE. This is because the CE separations are very dependent upon the extent of complexation with a particular cyclodextrin. In addition to the size of the CD cavity, interactions between the solute and the functional groups on the rim of the CD may influence chiral selectivity. Accordingly, the selection of a particular charged or neutral cyclodextrin best suited for the separation of a particular chiral pair remains an empirical process of trial and error.
For the foregoing reasons there is a need for novel set of charged cyclodextrins that can be used to resolve a broad range of chiral compounds by capillary electrophoresis. Ideally, the novel cyclodextrins can be synthesized using a simple procedure and give reproducible separations of enantiomers.
SUMMARY OF THE INVENTION
The present invention is directed to a set of charged cyclodextrins that satisfy the need to separate a broad range of chiral compounds by capillary electrophoresis. The preferred chiral selectors are highly sulfated &agr;-, &bgr;-, and &ggr;-cyclodextrins. These charged cyclodextrins have a higher degree of sulfation and narrower heterogeneity than commercially available cyclodextrins. CE analyses using highly sulfated cyclodextrins can improve the resolution of a wide variety of chiral drugs compared to other chiral selectors. Moreover, highly sulfated &agr;-, &bgr;-, and &ggr;-cyclodextrins produce separations which complement each other, permitting the resolution of an even broader range of neutral and basic drugs.
The charged cyclodextrins of the present invention can be an &agr;-cyclodextrin, a &bgr;-cyclodextrin, or a &ggr;-cyclodextrin having at least one hydrogen of a secondary hydroxyl replaced with a charged substituent. Preferred charged cyclodextrins have the following general formula:
For a cyclodextrin in which n is 6 or 8, at least about one R
1
group is a modified hydroxyl, and less than about n R
1
groups are hydroxyl. For a cyclodextrin in which n is 7, at least about 5 R
1
groups are modified hydroxyls, and less than about 3 R
1
groups are hydroxyl. The hydrogen of a modified hydroxyl is preferably replaced by a sulfate, carboxylate, phosphate, or a quaternary amine substituent. The most preferred substituent is sulfate.
Preferred highly sulfated alpha cyclodextrins possess at least about 7 sulfate substituents; preferred highly sulfated beta cyclodextrins possess at least about 12 sulfate substituents; and preferred highly sulfated gamma cyclodextrins possess at least about 9 sulfate substituents.
The charged cyclodextrins of the present invention can be used in a method of separating enantiomers of a small molecule by capillary electrophoresis. The method includes the steps of: a) filling a capillary with an electrophoresis buffer, which includes a charged cyclodextrin and an electrolyte; b) injecting a sample containing enantiomers of a chiral compound into the capillary; c) conducting an electrophoretic separation of the enantiomers; and d) detecting the enantiomers of the chiral compound. Preferably, the electrophoretic separation is conducted under conditions suppressing electroendoosmotic flow. For example, a coated capillary may be used or the electrophoresis buffer may be acidic. The sample is generally a small chiral molecule (MW<5,000), which is preferably a neutral or basic compound. Optionally, an internal standard, s
Chen Fu-Tai A.
Evangelista Ramon A.
Oh Chan S.
Shen Gene G. Y.
Beckman Coulter Inc.
Geist Gary
Grant Arnold
May William H.
Owens, Jr. Howard V.
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