Chemistry: analytical and immunological testing – Peptide – protein or amino acid
Patent
1991-06-26
1993-11-16
Housel, James C.
Chemistry: analytical and immunological testing
Peptide, protein or amino acid
436 89, 436164, 436111, 525 5411, 530334, 530337, 530338, C07K 100, G01N 2100
Patent
active
052623319
DESCRIPTION:
BRIEF SUMMARY
The present invention is related to the field of peptide synthesis, especially solid phase synthesis, and more particularly to a method for monitoring the time required for attachment of each amino-acid or peptide residue during the chemical synthesis of a linear combination of amino-acid residues, joined by peptide bonds.
Peptide synthesis in general is based on the reaction between an amino acid or peptide derivative, having the N-terminal alpha amino group unprotected but the C-terminal carboxyl group rendered unreactive by appropriate derivatisation, with a second amino-acid or peptide derivative of which the N-terminal alpha amino group is protected but the C-terminal carboxyl group is activated so as to enable formation of a peptide bond between the two compounds. This reaction can be carried out in solution or with one of the reacting species, usually the amino component, attached to an insoluble matrix. Immobilisation of the growing peptide chain in this way offers considerable savings in time and labour since it facilitates separation of the products of the reaction from the remaining excess reagents and soluble by-products. At the conclusion of the synthesis the assembled peptide is liberated from the support by chemical treatment. The solid phase approach is now the method of choice when chemically synthesised peptides are required.
Solid-phase peptide synthesis generally, and specifically where the present invention can be applied, comprises the following steps.
a) Removing an N-alpha protecting group from an appropriately derivatised amino-acid attached via its carboxyl group to an immobilising matrix.
b) Adding a second amino-acid derivative itself having an N-alpha protecting group and with its carboxyl group activated in such a manner as to facilitate peptide bond formation with the exposed amino group of the previous residue attached to the matrix.
c) Repeating steps a) and b) until the desired combination, termed a peptide, is obtained.
An intrinsic feature of the solid phase technique in its present forms is that intermediate peptide products cannot be purified and freed from peptides which, for any reason, do not contain the correct sequence. The method, therefore, relies for its success upon achieving reactions, both in the deprotection step a) and acylation step b), which are effectively quantitative. Removal of the alpha amino group is in practice rapid and, when the deprotection reagent flows past the substrate, is easily driven to competion. The chain elongation reaction can be by contrast problematic: for a combination of chemical, physical, and economic reasons, which stem respectively from the reactivity, molar solubility and cost of the activated amino-acid derivatives available, forcing conditions cannot be conveniently employed. An added and important complication is that the rate, and hence the time taken to achieve effective completeness of the acylation reaction is found to vary not only with the amino-acid derivative being coupled but also and in a sometimes unpredictable way with the sequence of the immobilisied substrate peptide. This condition is thought to be due to conformational properties of the peptide chain which render the amino terminus inaccessible to reagents, and its onset can prove catastrophic if the reaction conditions are not adjusted to compensate. In solid phase synthesis, therefore, information about the progress of the acylation reaction can be of critical importance. Inefficient couplings lead to an accumulation of `deletion` sequences such that the final product can be a complex mixture with the desired peptide only a minor component.
In the manual solid phase technique (or through operator intervention in automatic equipment) the method of choice for confirmation of total reaction involves withdrawal of a sample of the support matrix for detection of remaining amino groups (unreacted sites), usually by the use of ninhydrin (see for instance Kaiser E et al, Anal Biochem.1970 34(2) 595-8). This procedure does not readily lend itself to mechanisation
REFERENCES:
patent: 4746490 (1988-05-01), Saneii
patent: 4755558 (1988-07-01), Kalbag
patent: 4800166 (1989-01-01), Horn et al.
patent: 4855486 (1989-08-01), Kalbag
Artherton, E. et al., "Solid Phase Peptide Synthesis using N.alpha.-Fluorenyl-methoxycarbonylamino Acid Pentafluorophenyl Esters", Journal of the Chemical Society, Chemical Communications, pp. 165-166 (1985).
Stewart, J. M. et al., "Solid Phase Peptide Synthesis", 2d ed., Pierce Chemical Company, Ill., pp. 29-30 (1984).
Krchnak et al., Int. J. Peptide Protein Res. 32, 415-416 No. 5, (1988).
Cameron et al., J. Chem. Soc. Perkin Trans., 1, 2895-2901 (1988).
Nielsen et al., Journal of Biochemical and Biophysical Methods, 20, 69080 (1989).
Davies John
Owen David
Salisbury Stephen
Tremeer John
Housel James C.
Snay Jeffrey R.
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