Combinatorial chemistry and autosynthesis:

COMBINATORIAL CHEMISTRY - definition - Combinatorial chemistry can be defined as the synthesis of different chemical compounds as ensembles ('libraries') and the immediate (maybe 'in-situ') screening of them for desirable properties. Combinational  chemistry is potentially an efficient route to new drugs, catalysts, and other compounds and new materials.

Protein Synthesis

Two theoretical combinations of two different alpha amino acids, differing only in the R and R' groups to form a dipeptide.

H₂N-CHR-COOH + H₂N-CHR'-COOH

{H₂N-CHR-CO-NH-CHR'-COOH or H₂N-CHR'-CONH-CHR-COOH} + H₂O

• Note:

  1. This is a condensation reaction, because a small molecule (water) is eliminated as the two molecules join to form a bigger molecule, a 'natural condensation polymer' and hence the name 'polypeptide' for a protein structure.

  2.  -NH-CO- is the secondary amide 'peptide linkage' and the reaction is illustrated below with aminoethanoic acid, (glycine) and 2-aminopropanoic acid (alanine) showing the two possible di-peptide products (GlyAla or AlaGly).

  3. In this case, both dipeptides have one chiral carbon, >CH-CH₃ and >CH-NH₂ respectively.

  4. Since each of the two possibilities is stereochemically different, not surprisingly, you would expect a different enzyme (with cofactor?) for each alternative.

These days it is possible to 'automatically' synthesise polypeptides in a 'peptide synthesiser' into which, the required reactants are fed in a pre-programmed way, so all sorts of permutations can be prepared.

The polypeptides can be prepared or 'grown' on polystyrene beads - an example of solid phase chemistry.

This has positive 'efficiency' implications for the pharmaceutical industry e.g. some polypeptides are used as drugs and their structure is effectively a 'mini-protein'.

It is possible to synthesise a huge variety of permutations using this combinational chemistry, analyse them to validate their structure and then test them for their biological/pharmacological activity.

This idea can be used for a variety of organic synthesis, not just for amino acids ===> polypeptide, and this, since the 1990's is speeding up the development of drugs and other biologically active molecules.

Protein hydrolysis - broken down into their constituent amino acids - analysis

Proteins-polypeptides can be broken down by hydrolysis.

In principle, the complete hydrolysis reaction for a polypeptide of n residues is ...

-(HN-CHR-CO-)_n- + nH₂O ==> n H₂NCHRCOOH (but remember, R varies from amino acid to amino acid residue)

The complete hydrolysis into the constituent alpha-amino acids can be achieved by heating the protein with 5-6M hydrochloric acid in a sealed tube at 100-120^oC, for 10-24 hours.

A partial and selective/specific hydrolysis into short peptide sequences can be done under much milder conditions using enzymes such as the protease trypsin, found in the small intestine.

There are other much more elaborate methods involving chemical modification of the terminal amino acid residue, to obtain specific amino acid residue sequencing, but these are well beyond the scope of this web page (i.e. UK GCE courses). The resulting hydrolysis mixture can be analysed by e.g.

(i) paper/thin layer chromatography, often in two dimensions using two different solvents (paper rotated 90^o between solvents). The colourless amino acids can be made visible by spraying the chromatography paper/plates with ninhydrin reagent. Purple spots show up from the coloured compound formed from the combination of the amino acid with ninhydrin.

For more details see 6.13 Amino acids - molecular structure, preparation and reactions including chromatography

(ii) electrophoresis, in which the ionic forms of the amino acids are separated by movement in a buffered aqueous gel medium under the influence of an applied electric field (from d.c. voltage electrodes). The different amino acid mobilities depend on the average total +ve or -ve charge in a particular buffer.

The amino acids form bands which can be detected-analysed by using staining techniques or uv light fluorescence. The technique can also be applied to the analysis of protein or nucleic acid mixtures, and the latter can be detected using a radioactive phosphorus tracer ³²P (in the laboratory you should only deal with stable ³¹P).

In all cases the techniques can be calibrated using pure samples of known amino acids, proteins or nucleic acids etc.

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