Chemical reagents for formation of disulfide bonds in peptides

Details Chemical reagents for formation of disulfide bonds in peptides Chemical reagents for formation of disulfide bonds in peptides

2000-05-26

2004-02-03

Brumback, Brenda (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

Cyclic peptides

C530S300000, C530S333000, C530S345000, C514S002600

Reexamination Certificate

active

06686443

ABSTRACT:

CROSS-REFERENCE TO RELATED APPLICATIONS
[Not Applicable]
FIELD OF THE INVENTION
This invention pertains to the field of peptide chemistry. In particular, this invention provides reagents that effectively form intramolecular disulfide bonds in peptides bearing sulfhydryl groups.
BACKGROUND OF THE INVENTION
The chemical assembly of suitably derivatized amino acids to produce chemically synthesized peptides has become a well developed art. Indeed, a number of machines for the chemical synthesis of peptides are commercially available (see, e.g.,
PerSeptive Biosystems, Applied Biosystems
, Advanced ChemTech Venture, etc.). Such chemical syntheses proceed by the stepwise addition of amino acid residues to produce a linear peptide. Frequently, however, it is desirable to introduce or alter secondary structure by the formation of intramolecular linkages in a particular peptide. Such intramolecular linkages typically take the form of disulfide linkages, most typically formed between two cysteines that have sulfhydryl (SH) groups available for reaction.
Although formation of intrapeptide disulfide bonds can often be achieved by oxidation of the free thiol or sulfur-protected precursors under varying reaction conditions, a continuing challenge in peptide synthesis has been to develop more efficient and selective methods (Andreu et al. (1994) Pages 91-169 In
Peptide Synthesis Protocols
; Pennington, M. W., Bunn, B. M., Ed.; Humana Press: New Jersey; Moroder et al. (1996)
Biopolymers
40: 207-234; Annis and Barany (1997)
Meth. Enzymol
., 289: 198-221; Tam et al. (1991)
J. Am. Chem. Soc
. 111:, 6657-6662; Munson and Barany (1993)
J. Am. Chem. Soc
. 115: 10203-10216; Shik (1993)
J. Org. Chem
. 58: 3003-3008; Annis and Barany (1998)
J. Am. Chem. Soc
., 120: 7226-7238; Shi and Rabenstein (1999)
J. Org. Chem
. 64: 4590-4595). A variety of oxidants have been explored for formation of disulfide bonds, however, all have drawbacks and some limitations in practical use, including formation of dimers/oligomers and/or side reactions on the side chains of methionine, tyrosine and Tryptophan (Id.). For instance, thallium(III) trifluroacetate is a mild oxidant that can be employed as an alternative to iodine, and it gives a higher efficiency in some cases (Fujii et al. (1987)
Chem. Pharm. Bull
., 35: 2339-2347). The major drawback of this reagent is its high toxicity, and moreover, thallium can be difficult to remove from sulfur-containing peptides due to its high affinity to sulfur.
SUMMARY OF THE INVENTION
This invention pertains to the discovery of a class of reagents that effectively form intramolecular disulfide bonds in peptides. Intermolecular disulfide linkage formation is low or essentially non-existent. In addition, preferred reagents of this invention are relatively mild and do not oxidize “vulnerable” residues in the subject peptide(s). In addition the reagents and reaction products are safe and essentially non-toxic.
Thus, in one embodiment, this invention provides methods of forming an intramolecular disulfide bond in a peptide. The methods preferably involve contacting a peptide comprising at least two sulfhydryl (SH) groups with disulfide-linkage-forming reagent as described herein (see, e.g., Formula I and species thereof). In preferred embodiments, the reagent concentration is sufficient to produce one or more intramolecular disulfide linkages in the subject peptide(s). Particularly preferred reagents used in this context include, but are not limited to trans-[Pt(CN)
4
Cl
2
]
2−
, and trans-[Pt(en)
2
Cl
2
]
2+
(en=ethylenediamine), although trans-[Pt(CN)
4
Cl
2
]
2−
is most preferably used with peptides lacking a methionine. Preferred peptides for use in these methods range in length from about 2 to 200 amino acids, preferably from about 2 to about 100 amino acids, more preferably from about 2 to about 80 amino acids, and most preferably from about 2 to about 50 amino acids. The amino acids that form the disulfide linkage can be adjacent to each other or at essentially any distance away from each other on the peptide backbone so long as they can be juxtaposed sufficiently close (e.g. by peptide folding) to form a disulfide linkage. In certain preferred embodiments, the two amino acids that form a disulfide bond are separated by no more than about 58 amino acids. In certain preferred embodiments, the peptide contains a methionine. The contacting is preferably at a pH ranging from about pH 0 or pH 1 to about pH 9. The peptide can be a chemically synthesized peptide, a recombinantly expressed peptide, a cleavage product of a larger protein or peptide, a peptide isolated from a biological sample (e.g. a tissue, cell, organ, bioreactor, etc.). The peptide may bear a protecting group (e.g. an Fmoc or a tBoc).
In another embodiment, this invention comprises a solution comprising a peptide comprising at least two sulfur groups; and a disulfide linkage forming reagent as described herein (see, e.g., formula I and associated species). Such a solution is useful as a positive control when evaluating the efficacy of particular reagent species. In addition, such a solution is useful as a sample solution when optimizing a purification protocol to “clean up” or isolate the desired reaction product (e.g. when optimizing an HPLC protocol or evaluating a particular chromatography column). In preferred embodiments, the peptide in the solution is a peptide as described above. The peptide can be in solution or attached to a solid support (e.g. a peptide synthesis resin). The solution is preferably a buffered solution (e.g. at a pH ranging from pH 0 or pH 1 to about pH 9).
This invention also provides a peptide synthesizer for the synthesis of a peptide having an intramolecular disulfide linkage. A preferred synthesizer comprises a plurality of vials, said vials containing amino acids derivatized for chemical peptide synthesis wherein at least one of said vials comprises an amino acid that, when fully deprotected, bears a sulfhydryl (SH) group; and a vial comprising a disulfide linkage-forming reagent as described herein (see, e.g., Formula I and associated species described herein).
In still another embodiment this invention provides a method of chemically synthesizing a peptide comprising a disulfide linkage. The method involves chemically coupling a plurality of amino acids to form a peptide comprising at least two sulfhydryl (SH) groups; and contacting the peptide with a disulfide linkage-forming reagent (e.g. Formula I and associated species) as described herein.
This invention also provides a kit for forming an intramolecular disulfide bond in a peptide. The kit preferably comprises a container containing a disulfide linkage forming reagent (Formula I and associated species as described herein) instructional materials teaching the use of the reagent to form disulfide linkages in a peptide. In certain embodiments, the reagent is provided as a dry powder, while in other embodiments the reagent is in a buffer at a pH ranging from about pH 1 to about pH 9.
In certain preferred embodiments the methods, compositions, and generic formulas of this invention expressly exclude [Pt(CN)
4
Cl
2
]
2−
. In certain preferred embodiments the methods, compositions, and generic formulas of this invention expressly exclude [Pt(CN)
4
X
2
]
2−
where X is Cl—, Br—, I—, or RCOO— (where R is alkyl).
Definitions.
The term peptide refers to a polymer of amino acid residues. Preferred peptides range in length from about 2 to about 100 amino acids. The term also applies to linear or branched amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The term also includes variants on the traditional peptide linkage joining the amino acids making up the polypeptide.
The term “alkyl” refers to a paraffinic hydrocarbon group which may be derived from an alkane by dropping one hydrogen f

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