Effect of the mobile phase on antibody-based enantiomer separations of amino acids in high-performance liquid chromatography

Hofstetter, Oliver; Lindstrom, Heather; Hofstetter, Heike

doi:10.1016/j.chroma.2004.08.002

Cited by 13 publications

(8 citation statements)

References 32 publications

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“…Since low pH values may destroy the insulating layer on the surface of the electrode and cause the formation of pinhole structures, which impair capacitance measurements, all following experiments were carried out at pH 7.0. It is noteworthy, furthermore, that it was known from previous studies that the interaction of anti- d -AA with d -amino acids is strongest around neutral pH.…”

Section: Resultsmentioning

confidence: 91%

Development of a Highly Enantioselective Capacitive Immunosensor for the Detection of α-Amino Acids

et al. 2006

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This work describes a highly enantioselective and sensitive immunosensor for the detection of chiral amino acids based on capacitive measurement. The sensor was prepared by first binding mercaptoacetic acid to the surface of a gold electrode, followed by modification with tyramine utilizing carbodiimide activation. The hapten 4-amino-D-phenylalanine was then covalently immobilized onto the electrode by diazotization. Stereoselective binding of an anti-D-amino acid antibody to the hapten-modified sensor surface resulted in capacitance changes that were detected with high sensitivity by a potentiostatic step method. Using capacitance measurement, detection limits of 5 pg of antibody/mL were attained. The exquisite stereoselectivity of the antibody was also utilized in a competitive setup to quantitatively determine the concentration of the analyte d-phenylalanine in nonracemic samples containing both enantiomers of this amino acid. Trace impurities of d-phenylalanine as low as 0.001% could be detected.

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Section: Resultsmentioning

confidence: 91%

Development of a Highly Enantioselective Capacitive Immunosensor for the Detection of α-Amino Acids

et al. 2006

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“…Various anti-α-amino acid antibodies have been used as chiral agents to separate aromatic and aliphatic amino acids (see Figure 6) [189,191,192]. Antibodies against α-hydroxy acids have been employed to resolve the enantiomers of several α-hydroxy acids, including both aromatic and aliphatic compounds (e.g., lactic acid) [193].…”

Section: Chromatographic Studies Of Protein-based Cspsmentioning

confidence: 99%

Chromatographic Studies of Protein-Based Chiral Separations

Zheng

Azaria

et al. 2016

Separations

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The development of separation methods for the analysis and resolution of chiral drugs and solutes has been an area of ongoing interest in pharmaceutical research. The use of proteins as chiral binding agents in high-performance liquid chromatography (HPLC) has been an approach that has received particular attention in such work. This report provides an overview of proteins that have been used as binding agents to create chiral stationary phases (CSPs) and in the use of chromatographic methods to study these materials and protein-based chiral separations. The supports and methods that have been employed to prepare protein-based CSPs will also be discussed and compared. Specific types of CSPs that are considered include those that employ serum transport proteins (e.g., human serum albumin, bovine serum albumin, and alpha1-acid glycoprotein), enzymes (e.g., penicillin G acylase, cellobiohydrolases, and α-chymotrypsin) or other types of proteins (e.g., ovomucoid, antibodies, and avidin or streptavidin). The properties and applications for each type of protein and CSP will also be discussed in terms of their use in chromatography and chiral separations.

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“…However, harsh elution conditions are necessary due to the high affinity of mAbs against target molecules. Eluting a tightly bound enantiomer while maintaining the stability of mAbs is problematic . In addition, optimization of the immobilization method of mAbs onto columns is necessary to retain the binding affinity of mAbs .…”

Section: Figurementioning

confidence: 99%

“…The low cross reactivities of these mAbs for N and BP suggest that these mAbs recognize the relative position of the two naphthyl rings of BN . Unlike previous reports where the processes to immobilize antibodies or release bound enantiomer must be optimized, the chiral separation methodology presented herein is operationally simple. We expect that enantioselective complex formation of mAbs with artificial chiral molecules will be expanded to new applications such as chiral biocatalysts.…”

Section: Figurementioning

confidence: 99%

Direct Chiral Separation of Binaphthyl Derivatives Using Atroposelective Antibodies

et al. 2017

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A simple procedure for chiral separation based on the difference in the molecular weights of the antigen-antibody complex and analyte is developed. We raise monoclonal antibodies (mAbs) highly specific for the R-or S-isomer of a binaphthyl derivative (BN) by immunizing mice with the corresponding BN enantiomer. Aqueous solutions of racemic BN are mixed with mAbs. The free BN is separated by ultrafiltration. The enantiomers of BN are obtained with a high e.e. using atroposelective antibodies.Chirality is crucial in various scientific fields such as pharmaceuticals, [1] chemistry, [2][3][4] and materials science. [5] For example, in pharmaceutical applications employing a specific enantiomer is especially important. Often one enantiomer displays the desired effect, while the other elicits serious side effects. Therefore, chiral separations on both industrial and laboratory scales are important. High-performance liquid chromatography is a popular chiral separation methodology. [6] To date, many researchers have devoted much effort to develop chiral stationary phases (CSPs) such as synthetic polymers, [7] saccharides, [8,9] biomacromolecules, etc. [10][11][12] However, trial and error is still required for developing CSPs. One promising approach toward a straightforward chiral separation strategy is to use antibodies that precisely recognize the chirality of the target molecule. [13] With the advent of cell technology, [14] it is now possible to prepare individual antibodies, monoclonal antibodies (mAbs), in large amounts and in a homogeneous form semi-permanently.Many researchers have developed mAbs against optically active compounds for various purposes. [15][16][17][18][19][20] Among them, the use of mAbs as CSP of immuno affinity chiral columns has been extensively studied. [21] However, harsh elution conditions are necessary due to the high affinity of mAbs against target molecules. Eluting a tightly bound enantiomer while maintaining the stability of mAbs is problematic. [22][23][24] In addition, optimization of the immobilization method of mAbs onto columns is necessary to retain the binding affinity of mAbs. [25] To fully utilize the characteristic of mAbs, a more convenient methodology without columns for chiral separation is necessary.Our research focuses on the basic properties of mAbs, including high specificity for the target molecule and the molecular weight. Unlike chromatographic methods, the high molecular recognition ability of mAb can separate enantiomers with a one-step physical event. The large difference in the molecular weights between the analyte and mAbs realizes the separation of the free analyte by ultrafiltration. In addition, both enantiomers can be obtained by a simple one-step procedure where mAbs are prepared for each enantiomer.Herein, we report a convenient column-free methodology for chiral separation. This study provides insight on how to avoid difficulties when using mAbs. We chose a binaphthyl derivative with axial chirality, which is a well known asymmetric ligand, as a target ...

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Effect of the mobile phase on antibody-based enantiomer separations of amino acids in high-performance liquid chromatography

Cited by 13 publications

References 32 publications

Development of a Highly Enantioselective Capacitive Immunosensor for the Detection of α-Amino Acids

Development of a Highly Enantioselective Capacitive Immunosensor for the Detection of α-Amino Acids

Chromatographic Studies of Protein-Based Chiral Separations

Direct Chiral Separation of Binaphthyl Derivatives Using Atroposelective Antibodies

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