36 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.

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 improve,s; the efficiency of native chemical ligation reactions, particularly where three or more peptide fragments are used to assemble a polypeptide or protein product.

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 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, tand q are as defined herein.

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.

The invention provides a synthetic method of polypeptide hydrazide containing cysteine residues and an application of the synthetic method. According to the synthetic method, a tetracysteine label isinserted into the C end of a target polypeptide, and a double-arsenic fluorescent dye labeling reagent is used for specific recognition and binding of the tetracysteine label sequence. Cysteine sulfydryl side chains in the target polypeptide are protected by Pac groups, then the double-arsenic fluorescent dye labeling reagent is removed, and cyano hydrazinolysis on the tetracysteine label sequenceis conducted to obtain the polypeptide hydrazide containing cysteine. The synthetic method is helpful for perfecting native chemical ligation reactions and more effectively synthesizing proteins.

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 novel biocompatible macromonomers, hydrogels, methods of synthesis and methods of use thereof. 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 relates to a technology of polypeptide synthesis, and belongs to the technical field of pharmaceutical chemistry. A preparation method of semaglutide is provided. Based on a method (STL)of natural chemical connection of serine and threonine, the semaglutide can be high in purity, high in yield, less in reaction step and simple in operation, so that large-scale preparation of the semaglutide can be realized.

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 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 present invention concerns methods and compositions for extending the technique of native chemical ligation of a wider range of peptides, polypeptides, other polymers and other molecules via an amide bond (see FIG. 1). 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 invention discloses a method for preparing semeglutide by using a natural chemical ligation method, which comprises the following steps: (1) synthesizing semeglutide in two segments, namely an N-terminal segment with carboxyl-terminal hydrazide and a C-terminal segment with amino-terminal cysteine; (2) connecting the two fragments through a natural chemical connection method under a buffer salt condition; and (3) performing desulfurization treatment to convert Cys into target amino acid, and performing purification and freeze-drying after complete treatment. According to the method, the semeglutide is divided into the C-terminal polypeptide fragment and the N-terminal polypeptide fragment, the C-terminal polypeptide fragment and the N-terminal polypeptide fragment are connected through natural chemical connection reaction after being prepared respectively, then desulfurization treatment is conducted, the high-purity and high-yield semeglutide is finally obtained, the problem of difficult sequences is solved, the synthesis efficiency and purity of the semeglutide are remarkably improved, and the method is suitable for industrial production. The method is suitable for large-scale production.

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 present invention relates to an active substance and hydroxyalklyl starch, preferably hydroxyalkyl starch, wherein the conjugates are prepared by native chemical ligation via covalently linking the hydroxyalkyl stach and the active substance by reacting a thioester group with an alpha-X beta-amino group, wherein X is selected from the group consisting of -SH and NH-N. The present invention also relates to the method of producing these conjugates and the use of the conjugates.

An object of the present invention is to provide a novel method for producing a peptide utilizing a ligation reaction in which ligation efficiency is excellent and side reactions to other functional groups in the peptide are hard to occur, in comparison with the conventional native chemical ligation methods utilizing the thiol auxiliary group. The present invention provides a method for producing a peptide which comprises a step of causing a first peptide and a second peptide to react in the presence of a reducing agent to obtain a ligated product of the first peptide and the second peptide, wherein the first peptide contains, at the C-terminal end, an amino acid derivative having a thioester group, and the second peptide contains, at the N-terminal end, a serine or threonine derivative having a thiol auxiliary group.

The invention belongs to the field of polypeptide chemical synthesis, and discloses a one-pot three-section natural chemical ligation reaction method. According to the method, a one-pot three-section polypeptide connection reaction is carried out by utilizing the rate difference of two aryl thioesters, so that the polypeptide compound with a relatively long amino acid sequence is prepared. The method provided by the invention is simple to operate, small in required sample amount and convenient to test. The method does not need a mercaptan additive, provides a new strategy for natural chemical connection efficiency, and is controllable in cost and convenient to popularize and use.