Stereo-separations of Peptides by Capillary Electrophoresis and Chromatography

doi:10.1038/protex.2014.042

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Stereo-separations of Peptides by Capillary Electrophoresis and Chromatography

https://doi.org/10.1038/protex.2014.042

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Small peptides (di-, tri-, tetra- penta- hexa etc. and peptides) control many chemical

and biological processes. The biological importance of stereomers of peptides is of great

value. The stereo-separations of peptides are gaining importance in biological and medicinal

sciences and pharmaceutical industries. There is a great need of experimental protocols of

stereo-separations of peptides. The various chiral selector used were polysaccharides,

cyclodextrins, Pirkle’s types, macrocyclic antibiotics, crown ethers, ligand exchange, etc. The

attempts have been made to develop stereo-separations protocols for peptides using capillary

electrophoretic and chromatographic techniques. In addition to these, the optimization

strategies of stereo-separations were also discussed in the details. The efforts are also made to

discuss the future perspectives of peptides stereo-separations.

Chemistry

Peptides

Stereo-separations

Capillary electrophoresis

Chromatography

Future perspectives

Protocols

Its 21st century scientists are attempting to provide the best lives to the society.

Medication is one of the most important aspects in our lives. Chirality in the drugs is a

complex phenomenon creating confusion in medications. The demand of chiral drugs is

increasing constantly due to different pahramaceutical activities of drugs enantiomers. One

enantiomers may be active while the other inactive, toxic or ballast; leading to various side

effects and problems [1]. It is because of chiral nature of our biological systems. Mostly

biological reactions are stereo-selective because of different enantioselective distribution

rates, metabolisms, excretion and clearances of enantiomers. Due to these facts, scientists,

clinicians, industrialists, academicians and government authorities are asking data for

optically active drugs and other biological important molecules. US FDA, Health Canada,

European Committee for Proprietary Medicinal Products and Pharmaceutical and Medical

Devices Agencies of Japan, have banned the marketing of all racemic drugs [2-4].

Small peptides (monomers n < 6) are of great importance because of contribution in

various biological processes. The biological activities of small peptides include protein

synthesis, fertility, neurotransmission, inflammation process, pathogenic microorganisms

activities and other functions of human beings. These functions made peptide vital molecules

in drug development and health care [5-7]. Besides, these peptides are also being used as

biological markers in the biological systems [8]. The small peptides are also considered as

important molecules in food and nutrition industries. For examples, aspartame, carnosine, etc.

are being prepared at industrial scale [7]. It is important to mention here that biological

functions of peptides are stereoselective; especially related to enzymatic reactions.

In view of these facts, stereo-separation of small peptides is very important in drugs

development and health care. Stereo-separation of peptides may be achieved by capillary

electrophoresis and chromatography. Literature has many papers on stereo-separation of peptides [9-12]. Recently, Ali et al. [13] reviewed stereo-separations of small peptides by

capillary electrophoresis and chromatography techniques. It was observed that all these

papers contain sufficient information on stereo-separations of peptides but no one describes

the experimental procedures, methods development and optimization strategies in details,

which are urgently required at laboratory level globally. Therefore, the attempts have been

made to describe a protocol for stereo-separations of peptides by capillary electrophoresis and

chromatography. The present article describes the state-of-the-art of stereo-separations of

peptides using capillary electrophoresis, chromatography. The efforts have been made to

discuss optimization strategies and future perspectives of stereo-separations of peptides.

All solvents and reagents should be of HPLC and AR grades

Optically active pure and racemic peptides standards

Deionised water

Acetonitrile

Methanol

Reagents for the preparation of phosphate, acetate and borate buffers.

Required chiral selectors

Acids and bases for pH adjustment

Capillary Electrophoresis Instrument

. Personal computer (PC) for data acquisition

. Fused silica capillaries (~ 50 cm effective length with 50 or 75 μm inner diameter)

. Special capillary cutting blade

. pH meter

. UV–Vis. Spectrometer

. Degasification unit

. Filtration unit

. Micro balance

Prepare stock solutions of peptides (optically active pure and racemic) in water (0.1 mg/mL).
Prepare required BGE and dissolve suitable and appropriate amount of chiral selector in it.
Protocols given in Figure 2 should be used before selecting and preparing BGE.
Filter through 0.45 μm membrane and degas by sonication.
Rinse the capillary for 5 min. with 0.5 M NaOH followed by 10 min with deionize water.
Fill BGE in CE reservoirs and dip the ends of capillary into them.
Rinse capillary for 5 min with BGE.
Inject racemic peptides sample solutions.
Apply appropriate potential and run CE instrument. 10.. Among the measurements, rinse capillary for 2 min with BGE from time to time.
Optimize the stereo-separations.
Identify the resolved enantiomers by running standard pure optically active stereomers.
Wash capillary by deionised water before stopping HPLC instrument.
Calculate capillary electrophoretic parameters using standard equations [14].
Determine the qualitative and quantitative stereo-separations.

Rinse the capillary for 5 min. with 0.5 M NaOH followed by 10 min with deionize water.

Rinse capillary for 5 min with BGE

Among the measurements, rinse capillary for 2 min with BGE from time to time.

CE is gaining importance in stereo-separations of peptides.
CE instrument has two injection modes i.e. electrokinetic and pressure injection modes.
pH meter should be calibrated using pH 4.0 and pH 10.0 standards.
First filter about 10 mL of deionized water in order to remove any impurities from filtration unit.
pH of electrolyte is a crucial parameter and should be adjusted as per the requirements.
The addition of organic modifiers improves the stereo-separations.
Generally, organic modifiers are toxic to health.
Care should be taken to avoid skin contact, inhalation and swallowing.
Organic modifiers should be handled with cautions using gloves, glasses, etc.
These organic modifiers should be stored in cool, dry and well ventilated places.
Both ends of capillary should be sealed by heating; if instrument is kept for long time.

Stereo-separation of small peptides is a growing research area since early 1990s. The

metabolism of D-amino acid containing peptides stimulated stereo-separations of peptides

[55,56]. The stereo-separations methods are gaining advancement with respect to time. For

example, switching from indirect methods to direct stereo-separations and a gradual

replacement of protocols requiring derivatization of samples to the analysis of underivatized

peptides are the advancements. The cyclodextrines and macrocyclic antibiotics are the most

commonly used chiral selectors. However, in recent time polysaccharides and other chiral

selectors have been used. During last few years, hyphenation of CE and HPLC with MS

detectors, 2D-LC [57] and the development of miniaturized analytical devices are other

developments [58]. Reducing analysis time and complication of analyzed samples have

economic impact [59]. Fast speed UPLC instrument has not been used in chiral

chromatograph1 of peptides. But UPLC is used for stereo-separations of derivatized amino

acids [60]. Therefore, it is expected that UPLC may acquire a good position in stereoseparations

of peptide. The stereo-separation of peptides diastereomers is important in

physiological researches and industries. This is due to the fact that multi-components, multistereoisomer

mixtures are found in the food and pharmaceutical industries. Such situation

will expand industries with respect to enantiopure peptides.

Keeping these facts into consideration, the stereo-separations of peptides at

preparative scale is the requirement of today. Literature survey indicates only one report at

preparative scale [61]. Therefore, there is a great need for stereo-separation of peptides at

preparative scale. Really, optically active pure di- and tri-peptides can be obtained by chiral

synthetic methods but not larger peptides. This is a niche for preparative chromatographic

methods. It is assumed that more attention will be drawn to the area in pharmaceutical

industries for peptide stereo-separations

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The author would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University, Riyadh, Saudi Arabia for its funding this research group No. RGP-150

The author declare no competing financial Interest.

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Stereo-separations of Peptides by Capillary Electrophoresis and Chromatography

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