Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Synthesis of peptides
Reexamination Certificate
1999-06-22
2001-10-23
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Synthesis of peptides
C530S338000, C530S339000, C530S345000
Reexamination Certificate
active
06307018
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to methods for chemically ligating oligopeptides. More particularly, the invention relates to methods for chemically ligating oligopeptides end to end using a peptide bond.
BACKGROUND OF THE INVENTION
The synthesis of peptides by conventional stepwise solid phase methodologies is limited by low yields when synthesizing long peptides. (Merrifield et al.
J. Am. Chem. Soc.
1963, 85, 2149-2154; Kent et al.
Ann. Rev. Biochem.
1988, 57, 957-989.) To overcome this limitation, smaller synthetic peptides may be joined to one another by chemical ligation to produce longer peptide products.
A method for chemically ligating peptides was disclosed by Schnölzer et al.. (Schnölzer et al.,
Science
1992, 256, 221-225; Rose et al.
J. Am. Chem. Soc.
1994, 116, 30-34; Liu et al.
Proc. Natl. Acad. Sci. USA
1994, 91, 6584-6588). The methodology disclosed by Schnölzer involves a chemoselective reaction of unprotected peptide segments to give a product with an unnatural backbone structure at the site of ligation. This methodology makes possible the synthesis of peptides of greater size than attainable by standard methods of peptide synthesis. (Canne et al.
J. Am. Chem. Soc.
1995, 117, 2998-3007; Baca et al.
J. Am. Chem. Soc.
1995, 117, 1881-1887; Williams et al.
J. Am. Chem. Soc.
1994, 116, 10797-10798). This methedology also makes possible the synthesis of peptides of unusual structure and topology. (Dawson et al.
J. Am. Chem. Soc.
1993, 115, 7263-7266; Rose et al.
J. Am. Chem. Soc.
1994, 116, 30-34; Muir et al.
Biochemistry
1994, 33, 7701-7708; Canne et al.
J. Am. Chem. Soc.
1995, 117, 2998-3007). The combined use of conventional stepwise solid phase peptide synthesis together with chemical ligation enables chemists to routinely make unprotected peptides of up to 60 amino acid residues in good yield and purity. (Schnölzer et al.
Int. J. Pept. Protein Res.
1992, 40, 180-193). The combination of these two methodologies may also be employed to achieve a total chemical synthesis of proteins.
Another chemical ligation technique has been reported for the preparation of proteins having a native backbone structure (Dawson et al.
Science
1994, 266, 776-779). This mode of chemical ligation is termed “native ligation.” In this technique, an unprotected synthetic peptide bearing a C-terminal &agr;-thioester is reacted in a chemoselective manner with an unprotected peptide containing an N-terminal Cys residue. Thiol exchange reaction yields an intial thioester-linked intermediate which spontaneously rearranges to give a native amide bond at the ligation site joining the two peptide segments, in the process regenerating the Cys side chain thiol. This version of native ligation uses chemistry first described by Wieland for reacting amino acids. (Wieland et al.
Liebigs Ann. Chem.
1953, 583, 129-149.) As originally described, native ligation is restricted to joining peptide segments at an X-Cys bond. (Dawson et al.
Science
1994, 266, 776-779.)
What is needed is a general method for joining a C-terminal &agr;-thioester peptide segment to an N-terminal amino acid peptide segment, wherein the N-terminal amino acid peptide segment need not have an N-terminal cysteine.
SUMMARY OF THE INVENTION
The invention is directed to a method for chemically ligating unprotected oligopeptides to form a product having all peptide linkages. In the first step, two oligopeptides are ligated to form a ligation product having an aminothioester linkage. In the second step, the aminothioester linkage rearranges to form a product having an N-substituted amide linkage. In an optional third step, the substitution on the amide bond is removed by facile treatment with Zn in acidic medium, to give a native peptide bond at the ligation site.
The method employs two starting oligopeptides, viz., a first starting oligopeptide and a second starting oligopeptide. The first oligopeptide has a C-terminal auxiliary group with a thioester moiety, i.e., [peptide
1
]
&agr;
COSR, where R is selected from the group consisting of 3-carboxy 4-nitrophenyl and benzyl. The second oligopeptide has an N-terminal auxiliary functional group with an unoxidized sulfhydryl moiety, i.e., HSCH
2
CH
2
(O)-N
&agr;
[peptide
2
]. When the first and second starting oligopeptides are admixed under conditions promoting thioester exchange, they condense with one another to form an intermediate oligopeptide product wherein the first and second oligopeptides are linked via an amino-thioester bond. The amino-thioester bond then spontaneously rearranges intramolecularly to form a ligation product linked by an N-substituted amide bond. During the intramolecular rearrangement, the amino group of the N-terminal auxiliary functional group atacks the thioester to form an amide bond with the attached N-linked auxiliary functional group containing a displaced sulfhydryl moiety. The N-linked auxiliary functional group containing the displaced sulfhydryl moiety may then be optionally removed by chemical means to form a product having all native peptide linkages.
One aspect of the invention is directed to a method for ligating a first oligopeptide with a second oligopeptide end to end for producing an oligopeptide product. More particularly, the method comprises two steps with an optional third step.
The first step involves condensing the C-terminal thioester of a first peptide with the unoxidized sulfhydryl moiety of a second oligopeptide for producing an intermediate oligopeptide linking the first and second oligopeptides with an &dgr; or &ggr;-amino-thioester bond. The first oligopeptide includes the C-terminal thioester on a C-terminal residue and the second oligopeptide includes the N-terminal auxiliary functional group on an N-terminal residue having an unoxidized sulfhydryl moiety. If the C-terminal residue on the first oligopeptide is non glycine then the N-terminal residue on the second oligopeptide is glycine and if the N-terminal residue on the second oligopeptide is non glycine then the C-terminal residue on the first oligopeptide is glycine with the proviso that the non glycine residue is a non &bgr;-branched amino acid.
The second step rearranges the &dgr; or &ggr;-amino-thioester bond of the intermediate oligopeptide via intramolecular attack of the &dgr; or &ggr;-amino group onto the thioester moiety and displaces a sulfhydryl moiety as a byproduct from the thioester moiety thereby producing an oligopeptide product linking the first and second oligopeptides with an amide bond. The nitrogen of the amide bond contains the auxiliary functional group with the displaced sulfhydryl moiety.
An optional third step involves the removal of the auxiliary functional group on the amide nitrogen, from the oligopeptide product with a reducing agent for producing a native peptide bond. The amide nitrogen auxiliary functional group is
N
-&agr;-O—(CH
2
)
n
—SH wherein 1≦n≦2 and the reducing agent is Zinc (underlined nitrogen represents the coupled amide nitrogen).
Another aspect of the invention includes the oligopeptide intermediate comprising a first oligopeptide segment including a C-terminal thioester, a second oligopeptide segment including an N-terminal auxiliary functional group having an unoxidized sulfhydryl moiety, and an aminothioester linkage unit linking the C-terminal thioester and the sulfhydryl group of the auxiliary functional group.
REFERENCES:
patent: 5589356 (1996-12-01), Tam
patent: 5625030 (1997-04-01), Williams et al.
patent: 96/34878 (1996-11-01), None
patent: 98/28434 (1998-07-01), None
Gennari, Tetrahedron 46, 7289, 1990.*
Muir, Methods Enzymol. 289, 266-298, 1997.*
Dawson, J. Am. Chem. Soc. 119, 4325, 1997.*
Canne, J. Am. Chem. Soc. 121, 8720, 1999.*
Wilken, Curr. Opin. Biotechnol 9, 412-426, 1998.*
Liu, Proc. Natl. Acad. Sci. 91, 6584-88, 1994.*
Dawson, Science 266, 776-779, 1994.*
Wieland, et al., “Über Peptidsynthesen. 8. Mitteilung Bildung von S-haltigen Peptiden durch intramolekulare Wanderung von Aminoacylresten”,Liebigs. Ann. Chem. 583: 129-
Bannen Lynne Canne
Bark Steven J.
Dawson Philip E.
Kent Stephen B. H.
Muir Tom W.
Lewis Donald G.
Low Christopher S. F.
Lukton David
The Scripps Research Institute
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