33 results about "Native chemical ligation" patented technology

Compositions and methods for chemical ligation are provided. Methods for nucleic acid sequencing, nucleic acid assembly and nucleic acid synthesis are also provided.

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.

The invention discloses a cysteine functionalized hyaluronic acid conjugate, a synthetic method and an application in preparation of injectable in-situ hydrogel thereof. According to the method, a hydroxy of hyaluronic acid is modified to obtain the cysteine functionalized hyaluronic acid conjugate with a stable ether bond, and tetrabutyl ammonium hydroxide is introduced in the reaction process. The new method improves the solubility of hyaluronic acid and increases the reaction efficiency. The obtained cysteine functionalized hyaluronic acid conjugate has a sulfhydryl active group which not only improves the adhesion of hyaluronic acid, but also can be further functionalized. According to the invention, injectable in-situ hydrogel is generated by a native chemical ligation reaction or a Michael addition reaction of the prepared cysteine functionalized hyaluronic acid conjugate with a polyethylene glycol conjugate; rheological research shows that the generated hydrogel has good rheological properties, the gel performance is controllable, and the hydrogel has wide application prospects in the biological medicine field.

A method of synthesizing a biocompatible hydrogel by covalently cross-linking an effective amount of a first macromonomer including a cyclic thioester group with an effective amount of a second macromonomer including a terminal cysteine group is disclosed. In addition, the synthesis and use of the following specific cyclic thioester macromonomer that can be used in the method, as well as specific hydrogels made using this macromonomer are disclosed. The disclosed method produces a biocompatible hydrogel, while producing substantially no toxic free thiol by-product. Accordingly, the method can be used in making biomedical products, such as sutures and tissue replacement biomaterials, and for encapsulating therapeutic cells and pharmaceuticals.

The present invention provides a method for preparing polyfunctionalized peptides and / or proteins at non-adjacent designated sites via native chemical ligation. In certain embodiments, the inventive method is a method for preparing a polyfunctionalized peptide comprising a peptidic backbone made up of four or more amino acids, wherein two or more non-adjacent amino acids are independently subsituted with a moiety having the structure: wherein A and L1 are as defined herein. In certain other embodiments, the inventive method allows the preparation of polyfunctionalized peptides having the general structure: wherein A, RP0, RP1, PX1, RX2, L1, to, s, t and q are as defined herein.

The present invention provides methods, apparatus and kits for synthesizing assembled peptides and proteins on a solid phase with sequential ligation of three or more unprotected peptide segments using chemoselective and mild ligation chemistries in aqueous solution. Also provided are methods of monitoring solid phase sequential ligation reactions using MALDI or electrospray ionization mass spectrometry of reaction products.

The invention provides a method of assembling oligopeptide intermediates in a native chemical ligation reaction that eliminates self-ligation of bi-functional intermediates. An important aspect of the invention is a bi-functional intermediate with an N-terminal heterocyclic protecting group which effectively prevents self-ligation in the chemical assembly process. The present invention is useful in methods for convergent synthesis of polypeptides and proteins and improves the efficiency of native chemical ligation reactions, particularly where three or more peptide fragments are used to assemble a polypeptide or protein product.

The invention relates to methods for selectively converting a cysteine residue in a peptide or protein to the dehydroalanine (Dha) residue. The method also works on selenocysteine and substituted cysteine and selenocysteine residues, resulting in the Dha residue which may be converted to any natural or unnatural amino acid residue desired without the alteration of the remainder of the peptide or protein. The invention also allows ligation of a desired peptide at any point rather than at a point where there should be a naturally occurring cysteine, thereby allowing native chemical ligation to be used in the synthesis of peptides that do not contain cysteine. The methodology allows for the synthesis of very large peptides.

Novel compounds having a fluorescence quencher as a leaving group are disclosed. Nucleic acids and other molecules containing a fluorophore and a fluorescence quencher are disclosed as an embodiment of this invention. The use of the oligonucleotides in enzyme-free oligonucleotide ligation reactions results in an increase in fluorescence when the oligonucleotide also contains a nearby fluorophore. The ligation reactions can be performed in solution, on surfaces, or in cells.

A chemoselective chemical ligation method is disclosed. The method joins two peptide segments efficiently to produce a larger peptide or protein, by generating a natural peptide bond (Xaa-Ser and Xaa-Thr) at the ligation site (Xaa represents any 5 amino acid). The method requires two steps (FIG. 1 (a)): a) reacting the starting peptide(s) to form an acetal intermediate with an acetal group at the ligation site; b) converting said acetal intermediate to a desired peptide or protein with said natural peptide bond.

A chemoselective chemical ligation method is disclosed. The method joins two peptide segments efficiently to produce a larger peptide or protein, by generating a natural peptide bond (Xaa-Ser and Xaa-Thr) at the ligation site (Xaa represents any 5 amino acid). The method requires two steps (FIG. 1 (a)): a) reacting the starting peptide(s) to form an acetal intermediate with an acetal group at the ligation site; b) converting said acetal intermediate to a desired peptide or protein with said natural peptide bond.

The present invention provides isolated homogeneous polyfunctionalized proteins (e.g., erythropoietin), isolated glycopeptides, and a method for preparing polyfunctionalized peptides and / or proteins via cysteine-free native chemical ligation. In certain embodiments, the invention provides an isolated homogeneous polyfunctionalized protein having the structure (I). In certain other embodiments, the invention provides an isolated glycopeptide having Formula (II). In certain other embodiments, the inventive method is a method for preparing a polyfunctionalized peptide comprising a peptidic backbone made up of four or more amino acids, wherein two or more non-adjacent amino acids are independently substituted with a moiety having the structure (III) -LH. wherein A and L1 are as defined herein.

Novel methods of native chemical ligation are provided. The methods involve reacting a thioacid (e.g. a peptide thioacid) with an aziridinyl compound (e.g. an aziridinyl peptide) or glycosylamine under mild conditions without the use of protecting groups, and without requiring that a cysteine residue be present in the ligation product. Initial coupling of the thioacid and the aziridinyl compound yields a ligation product containing an aziridinyl ring. Optional subsequent opening of the aziridinyl ring (e.g. via a nucleophilic attack) produces a linearized and modified ligation product. Coupling of a peptide thioacid and glycosylamine yields a glycosylated peptide.

The invention discloses a cysteine functionalized hyaluronic acid conjugate, a synthetic method and an application in preparation of injectable in-situ hydrogel thereof. According to the method, a hydroxy of hyaluronic acid is modified to obtain the cysteine functionalized hyaluronic acid conjugate with a stable ether bond, and tetrabutyl ammonium hydroxide is introduced in the reaction process. The new method improves the solubility of hyaluronic acid and increases the reaction efficiency. The obtained cysteine functionalized hyaluronic acid conjugate has a sulfhydryl active group which not only improves the adhesion of hyaluronic acid, but also can be further functionalized. According to the invention, injectable in-situ hydrogel is generated by a native chemical ligation reaction or a Michael addition reaction of the prepared cysteine functionalized hyaluronic acid conjugate with a polyethylene glycol conjugate; rheological research shows that the generated hydrogel has good rheological properties, the gel performance is controllable, and the hydrogel has wide application prospects in the biological medicine field.

A method of synthesizing a biocompatible hydrogel by covalently cross-linking an effective amount of a first macromonomer including a cyclic thioester group with an effective amount of a second macromonomer including a terminal cysteine group is disclosed. In addition, the synthesis and use of the following specific cyclic thioester macromonomer that can be used in the method, as well as specific hydrogels made using this macromonomer are disclosed. The disclosed method produces a biocompatible hydrogel, while producing substantially no toxic free thiol by-product. Accordingly, the method can be used in making biomedical products, such as sutures and tissue replacement biomaterials, and for encapsulating therapeutic cells and pharmaceuticals.

Biocompatible macromonomers, hydrogels, methods of synthesis and methods of use thereof are provided. The biocompatible hydrogels of the present invention are prepared using native chemical ligation (NCL), in which a thioester readily reacts with a N-terminal thiol (cysteine) through transesterification and rearrangement to form an amide bond through a five-member ring intermediate.

The invention provides a method of assembling oligopeptide intermediates in a native chemical ligation reaction that eliminates self-ligation of bi-functional intermediates. An important aspect of the invention is a bi-functional intermediate with an N-terminal cyclic thiazolidine protecting group which effectively prevents self-ligation in the chemical assembly process. The present invention is useful in methods for convergent synthesis of polypeptides and proteins and improves the efficiency of native chemical ligation reactions, particularly where three or more peptide fragments are used to assemble a polypeptide or protein product.

The invention relates to methods for selectively converting a cysteine residue in a peptide or protein to the dehydroalanine (Dha) residue. The method also works on selenocysteine and substituted cysteine and selenocysteine residues, resulting in the Dha residue which may be converted to any natural or unnatural amino acid residue desired without the alteration of the remainder of the peptide or protein. The invention also allows ligation of a desired peptide at any point rather than at a point where there should be a naturally occurring cysteine, thereby allowing native chemical ligation to be used in the synthesis of peptides that do not contain cysteine. The methodology allows for the synthesis of very large peptides.

The invention relates to methods for selectively converting a cysteine residue in a peptide or protein to the dehydroalanine (Dha) residue. The method also works on selenocysteine and substituted cysteine and selenocysteine residues, resulting in the Dha residue which may be converted to any natural or unnatural amino acid residue desired without the alteration of the remainder of the peptide or protein. The invention also allows ligation of a desired peptide at any point rather than at a point where there should be a naturally occurring cysteine, thereby allowing native chemical ligation to be used in the synthesis of peptides that do not contain cysteine. The methodology allows for the synthesis of very large peptides.