Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Synthesis of peptides
Reexamination Certificate
2000-03-23
2001-08-21
Russel, Jeffrey E. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Synthesis of peptides
C525S054100, C525S054110, C530S334000, C530S335000, C530S338000, C530S339000, C530S345000, C558S250000, C558S254000, C558S256000
Reexamination Certificate
active
06277957
ABSTRACT:
BACKGROUND
1. Field of the Invention
The invention provides a method to facilitate the chemical synthesis of a wide range of conjugated molecules, connected together by amide bonds, useful for research, therapeutic and diagnostic applications.
BACKGROUND
2. Description of Prior Art
It is relevant to outline here methods of bioconjugate preparation, the background of various ligation strategies which have used alternatively generated thio-intermediates, and the development of iso-thiouronium salts themselves, and their limited application as synthetic reagents.
A variety of coupling agents have been developed to conjugate biomolecules via amide bonds by processes involving reactions in aqueous media, notably the water soluble carbodi-imides. These methods, however, can not promote the exclusive formation of an amide bond between any desired carboxylate-amine pair; they also proceed with poor efficiency resulting from side-reactions, such as hydrolysis or rearrangement. Defined conjugates can normally only be prepared by application of synthetic strategies which utilize specific protecting groups to block undesired couplings. Despite some suggestions for the usefulness of acyl thio derivatives (based on the “soft” electron distribution of the acylthio group); they have, until quite recently, been very infrequently used for synthetic applications. The reasons were highlighted in a study which compared the relative reactivity of 26 active esters in organic solution (D. Hudson;
Peptide Research
, 1990, 3, 51-55, incorporated herein for reference). Under these conditions, thiophenyl esters were found to be the least reactive of the whole group. The advantage of being able to perform coupling in aqueous conditions was disregarded, as conventional methods used appropriately protected peptide acid precursors (which are totally insoluble in water), and far more active esters, and coupling reagents, evolved for coupling in organic solvents. A discussion of such coupling agents can be found in a work of one of the inventors (D. Hudson,
J. Org. Chem
. 1988, 53, 617-614; which is incorporated herein for reference).
The following discussion relates to the chemical assembly of large peptides/proteins by coupling of smaller fragments. With some exceptions peptides of shorter chain length (typically<30 residues) may be routinely assembled by a variety of traditional solid-phase and solution based chemical methods. Longer chain length targets become increasingly more difficult to prepare because of several factors: poor solubility, secondary structure issues which diminish the accessibility and reactivity of the groups being coupled, and the heterogeneity of the products which result from multiple step processes involving incomplete transformations. As is well known, also, many products are accessible by the application of recombinant technology in which modified cells, or viruses, produce proteins foreign to their natural genes. It is not possible to provide a complete discussion of this alternative, however, it is sufficient to point out that this method has several limitations, it is (with a few exceptions) restricted to the production of products containing only naturally coded amino acids, that the products are included within other longer protein sequences (from which they have to be dissected, and purified), that large scale production requires complex plant, and that regulatory approval for a chemically synthesized product is usually much simpler to obtain than for a recombinant product.
When chemical coupling and conventional fragment assembly methods are used then total protection of all extraneous reactive groups is required, and the processes are limited by steric and solubility effects imposed by this requirement (reviewed by F. Albericio, P. Lloyd-Williams and E. Giralt,
Methods in Enzymology
1997, 289, 313-336). The real merits of acylthio derivatives for peptide ligations became apparent when they were applied, by Blake, Yamashiro and Li (J. Blake and C. H. Li,
Proc. Natl. Acad. Sci. USA
, 1981, 78, 4055-4058; D). Yamashiro and C. H. Li, Int. J. Pept. Protein Res., 1988, 31, 322-334) to the production of small proteins, by the assembly of partially protected aqueous soluble easily purifiable sub-units (individually prepared by solid-phase synthesis). The methodology developed in these innovative ground breaking studies, subsequently referred to as “direct chemical ligation”, forms the basis of more recent variations; and is schematically represented in FIG.
1
. The original Merrifield solid-phase peptide synthesis method was used to assemble the fragments (see Bruce Merrifield, “The Concept and Development of Solid-Phase Synthesis”, in D. Hudson, Perspective: Matrix assisted Synthetic Transformations,
J. Combinatorial Chemistry
, 1999, 1, 333-360). This method employs tBoc- protected amino acids for chain extension; cycles consist of:- i), TFA mediated tBoc removal; ii), washes with tertiary amine base to neutralize the free amine TFA salts produced, and iii), coupling with the next tBoc-AA. The process is repeated with further addition cycles until the desired sequence is assembled. Finally, a very strong acid, e.g. liquid HF at 0° C., is used to simultaneously cleave the peptide from the support, and remove side-chain protecting groups. This process, as applied by Blake, Yamashiro and Li, produces one component as its alpha-thioacid (
5
). This derivative is intrinsically reactive to aminolysis reactions, unlike the corresponding carboxylic acid. Addition of N-hydroxysuccinimide and silver ions to the conjugation reaction with
6
; leads to the production of an intermediate activated ester (not shown). These additives result in a significant enhancement of the rate of coupling, whilst maintaining its specificity. Other activation procedures, involving disulfide formation, followed later. The major enabling contribution of these seminal works was the development of a thiobenzhydryl linker,
1
, which is compatible with the chain assembly chemistry. Improvements to the preparation of this linker have been published (L. E. Canne, S. M. Walker, & S. B. H. Kent,
Tet. Lett
., 1995, 36, 1217-1220.) To summarize, then, the linker introduces the thiofunctionality into the peptide at the initiation of the synthesis process, the attachment is stable to the reagents used in the chain extension; and the peptide acylthio derivative is liberated by the standard HF cleavage. The main limitation of this method, however implemented, is the need to protect extraneous amino groups, in either component (e.g. any Lys side-chain), normally achieved by citraconylation of the peptides after cleavage.
The above described methodology provides most of the fundamental principles underpinning a burgeoning industry for the chemical production of proteins. The importance of this capability is impossible to overestimate! There is a growing appreciation of the fact that a direct consequence of the human genome sequencing project (which will have been completed a long time before this patent is issued) is the need to synthesize the myriad of elucidated protein sequences, for their functional significance to be fully evaluated. An important modification of the original direct thioligation scheme was developed independently by the groups of Kent (reviewed by T. W. Muir, P. E. Dawson and S. B. H. Kent,
Methods in Enzymology
, 1997, 289, 266-298), and Tam (reviewed by J. P. Tam and L. Zhang, in “Innovations and Perspectives in Solid Phase Synthesis”; Proceedings of the Fifth International Symposium, London, R. Epton, Ed., Mayflower, Birmingham, 1999, p. 1-6.). This variation, known as “native” chemical ligation, is shown schematically in FIG.
2
. The pattern of fragment assembly is designed so that Cys residues are positioned at the N-termini of fragments involved in the ligation, as with
10
shown. The first step of the ligation is activation of
8
, by a thio exchange mechanism, to give the more active acylthio ester form,
9
. Although the ability to modulate the reactivity of
8
by exchange
Hudson Derek
Thompson Stewart
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