Chemistry: analytical and immunological testing – Peptide – protein or amino acid – Amino acid or sequencing procedure
Patent
1988-02-08
1992-06-30
Warden, Robert J.
Chemistry: analytical and immunological testing
Peptide, protein or amino acid
Amino acid or sequencing procedure
436164, 436166, 436172, 530333, 530337, C07K 108
Patent
active
051262738
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to a method of monitoring the synthesis in particular (but not exclusively) by a solid phase technique of a linear combination of amino acid residues, and in particular to an optical monitoring technique which facilitates recirculating flow synthesis methods and automation of the synthesis procedure, and to such methods and procedures including the optical monitoring technique.
The solid phase synthesis of a linear combination of amino acid residues, linked via peptide bonds, generally starts with an amino acid residue covalently linked to a support and protected by an N-alpha-amino protecting group, and involves the steps: group, group, via a peptide bond, to the N-alpha-amino group obtained in step (a) by use of a reactive protected amino acid derivative and, where necessary, a catalyst, and been obtained.
BACKGROUND ART
Traditionally, such solid phase peptide synthesis has been carried out using t-butoxycarbonyl (boc) amino-acids activated in situ with an equivalent amount of dicyclohexyl-carbodiimide (DCCD). A significant advance was the introduction of preformed Boc and fluorenyl-mehtoxycarbonyl (Fmoc) amino-acid anhydrides in both polystyrene- and polyamide-based solid phase synthesis, avoiding contact of the reactive resin-bound amino group with the activating reagent. Acylation reactions are rapid, especially in polar media such as dimethylformamide.
Activated esters have also been used from time to time in solid phase synthesis but reaction rates may be low even in the presence of catalysts. Again polar reaction media are preferred, particularly in polyamide-based synthesis since this resin support is totally compatible with a wide range of aprotic polar and non-polar organic solvents. A particular synthesis method is disclosed in International Patent Publication No. WO86/03494, this method involving the use of activated Fmoc-amino-acid derivatives in which the acyl group used to form the peptide bond is activated as a pentafluorophenyl ester.
We have investigated the use of alternative activating agents in peptide synthesis, one of which is the compound: ##STR1##
This compound is properly described as 3-hydroxy-1,2,3-benzotriazin-4(3h)-one; derivatives thereof are described by Koenig and Geiger in Chemische Berichte 1970, 103, 2034. Authors have used several ring numbering systems in the past and this has led to the use of several different names for this compound. For example, Koenig and Geiger (ibid.) describe the compound as 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine and we ourselves described this compound in our U.K. Patent Application No. 86025866 as 1-oxo-2-hydroxy-dihydrobenzotriazine (DHBT). For ease of reference, this compound will be referred to hereinafter as "DHBT".
There is a ned to develop a fully automated system for synthesizing macromolecules such as peptides. In current so-called automated or semi-automated techniques, equipment is used in which control of the necessary liquid transfer operations, and consequently control of reaction times, is effected by somewhat inflexible computer software. This problem is an important one in peptide synthesis because rates of reaction can vary significantly as the synthetic procedure progresses and the length of the peptide chain increases. Reduced reaction rates can arise because of steric factors or, more seriously, because of sequence-dependent aggregation effects within the resin matrix in solid phase synthesis. These latter effects are generally unpredictable in their onset and can lead to failure of the desired synthesis reaction. Hitherto it has not been possible to provide automated synthesis apparatus or automated synthesis methods in which optimum reaction times can be determined for nay given reaction step in the course of a synthesis operation. After one such determination, it may be possible for the apparatus to be programmed to operate in accordance with the reaction times thus obtained.
Accordingly, there is a need for a motoring technique which is amenable to use in automated p
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Wissman, H. et al., "Peptide Synthesis using Propylphosphonic Acid Anhydride as Coupling Agent" Chemical Abstracts 98, 726 (1983).
Teetz, V. et al., "Synthesis and Biological Properties of B1-3,5-diiodo-tyrosine" Chemical Abstracts 88, 612 (1978).
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"Continuous flow methods in organic synthesis", R. C. Sheppard, Chemistry in Britain, May 1983, pp. 402, 405, 407, 410, 411, 413 and 414.
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Meldahl Morten
Sheppard Robert
Novobiochem AG
Snay Jeffrey R.
Warden Robert J.
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