1f50molecular Imprmipinting

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Chiral Separation Via Molecular imprinting

Laboratory of Nano Green catalysis

Mohd Bismillah Ansari

Abhishek Burri

Jin Hua

A method for Resolution

Of

Enantiomers


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Contents

Necessity of Chiral separation

History of Molecular Imprinting

Application of Molecular Imprinting

Principle of Molecular Imprinting

Imprinting Methodologies

Template Monomers Species

Emphasis on different examples

Advantages and disadvantages ofdifferent Imprinting methods

References/Further Reading & Links

Laboratory of Nano green catalysis


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Necessity for Chiral Separation

The tragic case of the racemic drug of n-

phthalyl-glutamic acid imide that was

marketed in the 1960s as the sedative

Thalidomide. Its therapeutic activity

resided exclusively in the R-enantiomer. It

was discovered only after several hundred

births of malformed infants that the S-

enantiomer was teratogenic.

Chiral Supports

Non steroidal Anti-inflammatory Drugs of

2-Methyl aryl propionic Acids (Profens)


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Pioneers In molecular imprinting

Professor Gnter Wulff

The man behind the molecular imprinting

The major area of research of G. Wulff is

covalent molecular imprinting.

The beginning of molecular imprinting

started by wulff research at university ofDusseldorf

Amidine-based molecularly imprinted polymersnew sensitive elements for chiral chemosensors

Anal Bioanal. Chem. (2003) 377 : 608613

Racemic Resolution of Free Sugars with Macroporous Polymers Prepared by Molecular Imprinting.

Selectivity Dependence on the Arrangement of Functional Groups versus Spatial Requirements

J. Org. Chem., Vol. 56, No. 1, 1991

His major works involve utilization of

covalent templates in molecular imprinting


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Molecular Imprinting for Chiral Separation

Klaus Mosbach

Klaus Mosbach is Pioneer in the Molecular

imprinting. He introduced the new technique

Called Non covalent molecular imprinting, the

separation of enzyme is found to be a big

hassle . To solve this problem , hesuccessfully developed a new technique

today known as affinity chromatography

and bimolecular recognition . The same

technique for small molecules is now referred

as molecular imprinting.

References

Non covalent molecular imprinting with emphasis on its application in separation and drug development

Journal of Molecular recognition 2006; 19: 248-259

Molecular imprinting used for chiral separations

Journal of chromatography A, 694 (1995) 3-13


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MIP

Applicationssensor

Membrane

separationSPE

Chiral

separationPharma

analysis

Chromatographic

separation

Artificialantibodies

Enzyme

catalysis

MIP APPLICATIONS


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Characteristic of MIP:

Predetermined selectivity (tailor-made)

Specific recognition

Wide applications

MIP Technology
http://www.smi.tu-berlin.de/story/Protocol.gif


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Molecular Imprinting - Principle

The template is choosed based upon the molecule to be analyzed. The functional monomer are choosed based upon the template properties.

The functional monomers are copolymerized with cross linkers to yield a highly

cross linked and rigid polymer.

The imprint molecules are subsequently removed from the polymer, leaving

recognition sites complementary to the imprint species in shape and in the

positioning of the functional groups. The recognition of the polymer constitutes an induced molecular memory,

which makes the recognition sites capable of selectively recognizing the imprint

species.


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LOGOSchematic of molecular imprinting

Template

assembly

(binding)

Template

a

c

b1- add cross-linker

2- polymerise(in porogenic

solvent)

Template

re-binding

template

removal

recognition siteLaboratory of Nano green catalysis


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Features of imprinted polymers

Advantages

Target defines own recognition site

Stability of synthetic materials

Specificity of natural systems

Adaptability/flexibility in use

Facile, one-pot synthesis

Use in non-aqueous media/aggressive environments

Disadvantages

Diversity of binding sites

Poor processibility

Analytically opaque/Black box chemistry


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Enantiomer resolution

Imprinted enantiomer retained on column


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Separation from mixtures

Whitcombe et al. J. Agric. Food Chem., 2001, 49 (5), pp 21052114


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Imprinted polymers-antibody binding site mimics


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Imprinting methodologies

Covalent

Reversible covalent linkage

Non-covalent

Monomer-template complexes

Sacrificial spacer

Covalent link during synthesis Non-covalent rebinding


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Molecular Imprinting: Covalent

Wulff & SchauhoffJ. Org. Chem., 1991, 56, 395-400.


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Covalent template-monomer species

Template Binding moiety Binding at equilibrium

Saccharides

Polyols

Glycoproteins

Aldehydes

Ketones

Disulfides


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Molecular Imprinting: Non-covalent


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Non-covalent template-monomer species

Template Binding moiety Binding at equilibrium

Acids

Bases

Polyamides

Carboxylates


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Molecular Imprinting: Spacer Approach

CVPC


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Sacrificial spacer template-monomers

Pyridine analogue imprints


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Imprinting for Hydrophobic Recognition

Asanuma et al. Supramolecular Science1998, 5, 417-421

-CD =

=

1. assemble

complex

2. crosslink, DMSO

extract


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A simple and reliable method for the preparation of an Open tubular moleculeimprinted polymer (MIP) in a Silica capillary format is presented.

A non-covalent molecular imprinting polymerization protocol was used to synthesize

the MIP where (S)-ketoprofen template molecule was incorporated in the

copolymerization of methacrylic acid (MAA) and ethylene glycol dimethacrylate

(EDMA).

Nitrogen was purged to open the lumen of capillary and to obtain the thin film of

polymer.

MIP showed the high chiral selectivity towards R,S- ketoprofen in the -liquid

chromatography (-LC) as well as in Capillary electro chromatography (CEC).

Baseline separation of R,S- ketoprofen was achieved with the resolution and

selectivity of 4.7 and 1.6, respectively.

Shabi Abbas Zaidi,Won Jo Cheong

Journal of Separation Science, 31 (2008) 2962-2970

Robust open tubular layer ofS-ketoprofen imprinted

polymer for chiral LC


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Si

Si

Si

OH

OH

Si

OH

Si

Si

Si

OH

OH

Si

OH

OH

NaOH, Water,

Hcl,N2purg ing

O

O

O

O

Anch oring reagent

-MAPS in AcOH

Template

Monomer

Cross l inker

Init iator

Polymer matr ix

Vacuum/N2 purging

Thin polym er fi lm

Capi l lary wal l

Schematic representation of MIP layer formation


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SEM Images

Scanning electron micrographs of the open-tubularS-ketoprofen MIP layer: the

cross-section of view of the interface between the MIP layer and the capillary

inner wall.


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pH. 4.5

pH. 3

pH. 5

pH. 6

pH. 2

pH. 4

Mobile phase is acetonitrile/Sodium acetate (50mM, 30/70 v/v%),

Injection: 5 s at 5 mbar, capillary temp: 25;Inlet pressure: 5 mbar, UV wavelength: 214nm.

Influence of pH on resolution of R,S-Ketoprofen


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of R,S-ketoprofen

Mobile phase is acetonitrile/Sodium acetate (50mM, pH-4.5)

Injection: 5 s at 5 mbar, capillary temp: 25;Inlet pressure: 5 mbar, UV wavelength: 214nm.

50%

40%

30%

20%

10%



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0 5 10 15 20 25

0

1

2

3

4

5

6

Values

Pressure (mbar)

Resolution

Selectivity

Variation of Resolution and selectivity with Inlet pressure

on MIP column


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LC, 5 mbar

Mobile phase is acetonitrile/Sodium acetate (50mM,30/70 v/v%),

Injection: 5 s at 5 mbar, capillary temp: 25,pH: 3UV wavelength: 214nm.

CEC, 10kv

Comparison of chiral separation of Ketoprofen enantiomers

between CEC and LC.


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Unique open tubular (S)-ketoprofen MIP layer inside the capillary wall.

The mobile phase composed of 30% acetonitrile and 70% acetate buffer of pH 4.3

was found to be the optimum.

Inlet pressure of 5 mbar provided the best resolution and higher number oftheoretical plates/meter ( > 31,000 for R-isomer and >10,000 for S-isomer).

Small dimension of capillary and three step procedure of MIP formation enabled a

versatile nature.

Conclusion


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Covalent Imprinting

Ability to fix template in place during polymerisation -lower dispersity in binding sites

Can be carried out in any solvent flexibility Can be difficult to remove template from polymer -

low recovery of valuable templates and low numberof binding sites

Limited number of chemistries for fixing template topolymer reversibly - reduction in number of templatesthat can be imprinted

Poor kinetics of re-binding

Advantages and disadvantages


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Advantages and disadvantagesNon-covalent imprinting Easy to remove template from polymer- good recovery

of valuable templates and accessible binding sites

Very large number of templates amenable to non-covalent imprinting

Rapid kinetics of re-binding

Inability to fix template in place during polymerisation -

polydispersity in binding sites, poor definitionGenerally requires low-polarity aprotic solvents -

incompatible with aqueous polymerisations


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Advantages and disadvantagesSacrificial spacer methodAbility to fix template in place during polymerisation -

lower dispersity in binding sites

Can be carried out in any solvent flexibility

Rapid kinetics of re-binding

Can be difficult to remove template from polymer - lowrecovery of valuable templates and low number of

binding sites

Limited number of chemistries for fixing template topolymer reversibly - reduction in number of templatesthat can be imprinted


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References/Further reading

Bioseparations; Downstream Processing For Biotechnology. Belter, P.

A.; Cussler, E. L.; Hu, Wei-Shou (John Wiley & Sons 1988). Smart polymers and what they could do in biotechnology and

medicine. Galaev, I.Y.; Mattiasson, B. TIBTECH.17, 335-340 (1999).

Smart polymers and protein purification. Mattiasson, B.; Dainyak, M.B.;

Galaev, I.Y. Polymer-Plast ics Techn ology and Engineer ing37, 303-

308 (1998). Molecular imprinting in cross-linked materials with the aid of molecular

templates - a way towards artificial antibodies. Wulff,G. Angew. Chem.

Int. Ed. Engl . 34, 1812-1832 (1995).

Polymer- and template-related factors influencing the efficiency in

molecularly imprinted solid-phase extractions. Sellergren, B. TRAC. 18,164-174 (1999).

Assembling the Molecular Cast. Alexander, C.; Whitcombe, M.J.;

Vulfson, E.N. Chem. Br. 33, 23-27 (1997).


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Links

Bioseparations (general)

http://www.biotech.wisc.edu/

http://www.tamu.edu/separations/psepars.html

Polymers (general)

http://www.polymers.com/

http://irc.leeds.ac.uk/irc/

http://www.irc.dur.ac.uk/main.html

Molecular Imprinting

Society for Molecular Imprinting

http://www.ng.hik.se/~SMI/


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1f50molecular Imprmipinting

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