TEA-BAG METHODOLOGY

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TEA-BAG METHODOLOGY:

Naureen Shehzadi M.Phil. (Pharmaceutical chemistry) University college of pharmacy, University of the Punjab, Lahore, Pakistan TEA-BAG METHODOLOGY

BACKGROUND:

BACKGROUND This technique was introduced in 1985 by Richard Houghten for rapid multiple peptide synthesis.

PRINCIPLE:

PRINCIPLE Tea-bags are used to make multimiligram (up to 500µM) quantities of single peptide sequence in each packet that is sufficient for full characterization and screening. To save time and work when making many peptides simultaneously, bags could be combined into the same reactors for common chemical steps.

WHAT IS A TEA-BAG?:

WHAT IS A TEA-BAG? According to Houghten, a tea bag is polypropylene mesh bag, with dimensions 15x20mm, which is filled with resin beads, sealed and labeled for later identification. The “tea-bag” mesh size is too small to allow resin beads to escape, but solvents and soluble reagents can readily enter. Each bag contains between 50 and 100 mg polymeric resin beads as supports, wherein the mean bead diameter has to exceed the mean pore diameter of polymeric membrane of the bag.

STEPS IN SYNTHESIS:

STEPS IN SYNTHESIS

STEPS IN SYNTHESIS:

STEPS IN SYNTHESIS AMINO ACID PROTECTION Terminal amino group of amino acid is protected using various protecting groups e.g. FMOC, BOC PREPARATION OF TEA BAGS Tea bags are charged with resin beads that bear a protected amino acid. Each and every bag is labeled for easy identification.

STEPS IN SYNTHESIS:

STEPS IN SYNTHESIS DEPROTECTION Protective group is cleaved using piperidine (20-50%) in DMF to expose the α-amino group for reaction with an incoming activated amino acid. NEUTRALIZATION This step is required when protecting group exposes non-neutral amine. WASHING Solvents e.g. DMF, methanol are used for washing the bags.

STEPS IN SYNTHESIS:

STEPS IN SYNTHESIS AMINO ACID COUPLING Tea bags are sorted into groups for addition of amino acid. Deprotection, washing and neutralization steps are repeated until required length of peptide is achieved. WASHING CLEAVAGE After appropriate number of washing steps, all bags are treated with HF/anisole to cleave the peptide from the beads. ISOLATION

NOTE:

NOTE Washing, coupling and deprotection is carried out in common container whilst the coupling of amino acid and final cleavage is carried out using different containers.

EXAMPLES :

EXAMPLES Characterization of the influenza haemagglutinin protein (HA1) and discovering the amino acid position that is critical important to the binding interaction (Houghten et al. 1986 ) Rapid "tea-bag" peptide synthesis using 9-fluorenylmethoxycarbonyl (Fmoc) protected amino acids applied for antigenic mapping of viral proteins. The use of tea-bag synthesis with paper discs as the solid phase in epitope mapping studies. Production of a small combinatorial library of urea analogues (Burgess et al. 1997)

ADVANTAGES:

ADVANTAGES Speed Effectiveness Flexible Information about the spatial localization of each reaction step is ensured by labeling the bags High amount of resulting peptides Low equipment investment Complete biological and structural characterization can be done

LIMITATIONS:

LIMITATIONS Intensity of manual lab work is high because of repeated washing, sorting, labeling etc. Number of manageable peptide shorten the scope of application

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