Pseudo-native chemical ligation

Abstract

The present invention concerns methods and compositions for extending the technique of native chemical ligation to permit the ligation of a wider range of peptides, polypeptides, other polymers and other molecules via an amide bond. The invention further provides methods and uses for such proteins and derivatized proteins. The invention is particularly suitable for use in the synthesis of optionally polymer-modified, synthetic bioactive proteins, and of pharmaceutical compositions that contain such proteins.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. patent application Ser. No. 10 / 332,386 (filed Jan. 8 2003) and PCT / US01 / 21935, and claims priority to U.S. Patent Applications Ser. Nos. 60 / 231,339 (filed Sep. 8, 2000) and 60 / 236,377 (filed Sep. 29, 2000), all of which applications are herein incorporated by reference in their entirety.FIELD OF THE INVENTION [0002] The present invention relates to methods and compositions for extending the technique of native chemical ligation to permit the ligation of a wider range of peptides, polypeptides, other polymers and other molecules via an amide bond. The invention further provides methods and uses for such proteins and derivatized proteins. BACKGROUND OF THE INVENTION [0003] Over the past 30 years, medical attention has increasingly turned to the possibility of using naturally produced proteins as therapeutic drugs for the treatment of disease. [0004] Recombinant DNA techniques have become the prim...

AI Technical Summary

Benefits of technology

[0039] Typically, the synthesis of bioactive peptides and proteins employs the “Merrifield”-chemistry stepwise solid phase peptide synthesis protocol developed in the early 1960's, using standard automated peptide synthesizers. Such synthesis may employ solid or solution phase ligation strategies. While such chemistry may be readily employed to produce many polypeptides, it is unsuitable for the production of proteins or large polypeptides due to associated yield losses, by-product production, and incomplete reactions. To address these limitations, techniques of chemical ligation have been developed that permit one to ligate together preformed peptide fragments in order to achieve the synthesis of larger polypeptides and proteins.

Problems solved by technology

Unfortunately, the use of recombinant DNA techniques has not been universally successful.

Recovery of protein from these bodies has presented numerous problems, such as how to separate the protein encased within the cell from the cellular material and proteins harboring it, and how to recover the inclusion body protein in biologically active form.

There are unfortunately multiple disadvantages to the stepwise solid phase synthesis method, including the formation of solid-phase bound by products that result from incomplete reaction at the coupling and deprotection steps in each cycle.

The longer the peptide chain, the more challenging it is to obtain high-purity well-defined products.

The synthesis of proteins and large polypeptides by this route is a time-consuming and laborious task.

Often, however, technical difficulties are encountered in many of the steps of solid phase segment condensation.

For example, the use of protecting groups on segments to block undesired ligating reactions can frequently render the protected segments sparingly soluble, interfering in efficient activation of the carboxyl group.

Limited solubility of protected segments also can interfere with purification of protected segments.

Protected segments are difficult to characterize with respect to purity, covalent structure, and are not amenable to high resolution analytical ESMS (electrospray mass spectrometry) (based on charge).

Racemization of the C-terminal residue of each activated peptide segment is also a problem, except if ligating is performed at Glycine residues.

Moreover, cleavage of the fully assembled, solid-phase bound polypeptide from the solid phase and removal of the protecting groups frequently can require harsh chemical procedures and long reaction times that result in degradation of the fully assembled polypeptide.

Moreover, the ligation in solution does not permit easy purification and wash steps afforded by solid phase ligations.

Furthermore, the limitations with respect to solubility of protected peptide segments and protected peptide intermediate reaction products are exacerbated.

The primary drawback of the original native chemical ligation approach is that it requires an N-terminal cysteine, i.e., it only permits the joining of peptides and polypeptide segments possessing an N-terminal cysteine.

One limitation of this method is that the use of a mercaptoethoxy auxiliary group can successfully lead to amide bond formation only at a glycine residue.

As such, this embodiment of the method is only suitable if one desires the ligation product of the reaction to contain a glycine residue at this position, and in any event can be problematic with respect to ligation yields, stability of precursors, and the ability to remove the O-linked auxiliary group.

Although other auxiliary groups may be used, for example the HSCH2CH2NH-[peptide], without limiting the reaction to ligation at a glycine residue, such auxiliary groups cannot be removed from the ligated product.

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