Molecular Imprinting

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Molecular Imprinting

• Cameron Alexander
• School of Pharmacy and Biomedical Sciences,
• University of Portsmouth, St Michael's Building,
• White Swan Road, Portsmouth, PO1 2DT, UK.
• www.sci.port.ac.uk/pharmacy

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Schematic of molecular imprinting
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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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Applications of imprinted polymers

• Chiral HPLC stationary phases

• Antibody mimics for polymer-based immunoassays

• Enzyme-type catalysis (printzymes)

• Chiral microreactors

• Sample enrichment

• Racemate resolution

• Robust sensors

• Crystallization mediators

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Enantiomer resolution

• Imprinted enantiomer retained on column

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Separation from mixtures

• Whitcombe et al J Food Agric. Chem.in press

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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 Schauhoff J. 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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Sacrificial spacer template-monomers
Pyridine analogue imprints
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Sacrificial spacer template-monomers
Pyridine and quinoline analogue imprints
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Imprinting for Hydrophobic Recognition
ß-CD
1. assemble complex 2. crosslink, DMSO
extract

Asanuma et al. Supramolecular Science 1998, 5,
417-421
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Imprinting methodologies - advantages and
disadvantages

• 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
  number of binding sites
• Limited number of chemistries for fixing template
  to polymer reversibly - reduction in number of
  templates that can be imprinted
• Poor kinetics of re-binding 

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Imprinting methodologies - advantages and
disadvantages

• Non-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 definition
• Generally requires low-polarity aprotic solvents
  - incompatible with aqueous polymerisations 

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Imprinting methodologies - advantages and
disadvantages

• Sacrificial spacer method
• Ability 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
  - low recovery of valuable templates and low
  number of binding sites
• Limited number of chemistries for fixing template
  to polymer reversibly - reduction in number of
  templates that can be imprinted

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Synthesis and catalysis at imprinted polymer
binding sites

• Synthesis
• Imprinted polymers as protecting groups
• Modifications of sterol hydroxyl groups
• Regioselective acylation at binding sites

• Catalysis
• Imprinted polymers with constrained binding sites
• Imprinting of transition state analogues (TSAs)
• Catalytic turnover in imprinted sites

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Regioselectivity of Modification

• What can the degree and position of reaction
  tell us about binding at the imprinted sites?
• Dependent on the fit of a ligand, chemical
  modification can be directed to different parts
  of the molecule
• Imprinted site geometry and chemistry can be
  inferred from product ratios

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Androstene templates
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Synthesis of androstene-based templates
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Template-monomer binding via boronate ester
chemistry
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Synthesis of androst-5-ene-3b-ol binding sites
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Synthesis of androst-5-ene-17b-ol binding sites
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Synthesis of androst-5-ene-3b,17b-ol binding sites
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Uptake measurements
Uptake by Androst-5-ene-17-ol Imprinted Polymers
250C CHCl3
Uptake of Androst-5-ene-3-ol 250C CHCl3
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Uptake measurements
Uptake by Androst-5-ene-3,17-diol Imprinted
Polymers 250C CHCl3
Uptake of Androst-5-ene-3,17-diol 250C CHCl3
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IR Spectra of Androst-5-ene-3b,17b-diol
Androst-5-ene-3b,17b-diol polymer
School of Pharmacy and Biomedical Sciences
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Kinetics of binding
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Synthesis at sterol-imprinted polymer binding
sites

• Androstene sterols used to imprint 3 different
  polymers
• Androst-5-ene-3b-ol-imprinted and
  androst-5-ene-17b-ol-imprinted polymers both bind
  similar amounts of their templates
• Androst-5-ene-3b,17b-ol-imprinted polymer binds
  its own template most strongly- co-operative
  interaction
• Can the three different binding sites be used for
  synthesis?

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Chemical Modification as Probe of Binding

• Reaction of functional groups within binding site
• If all functional groups on molecule are involved
  in binding interactions, no modification occurs
• Imprinted polymer acts as protecting group for
  its own template
• Partial interaction of template with polymer
  allows unbound groups to be modified
• Imprinted polymer directs chemistry in site

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Reactions in imprinted cavities
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Regioselective modification
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Synthesis in imprinted polymer binding sites
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Synthesis in imprinted polymer binding sites
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Synthesis in imprinted polymer binding sites
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Examples of imprinted polymer catalysts
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Examples of imprinted polymer catalysts
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Catalysis
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Catalysis
School of Pharmacy and Biomedical Sciences
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Catalysis
School of Pharmacy and Biomedical Sciences
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Catalysis
School of Pharmacy and Biomedical Sciences
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Catalysis
School of Pharmacy and Biomedical Sciences
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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-Plastics Technology and Engineering 37,
  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/