Provided is a method for solid phase synthesis of Etelcalcetide, comprising synthesizing Etelcalcetide backbone peptide resin, removing the side chain protecting group of Cys in the peptide chain, and then activating the sulfydryl group of the Cys side chain on the peptide resin with 2,2-dithiodipyridine and constructing a disulfide bond with L-Cys, such that a crude Etelcalcetide peptide is obtained by cleaving. The method does not require undergoing multi-step purification, the yield and purity of the obtained crude peptide are relatively high, and the total yield of the refined peptide after purification is greatly increased.

Provided herein are polymeric -hydroxy aldehyde or -hydroxy ketone reagents which can be conjugated to amine-containing compounds to form stable conjugates in a single-step reaction. In selected embodiments, the polymeric reagent itself incorporates an internal proton-abstracting (basic) functional group, to promote more efficient reaction. The substituent is appropriately situated, via a linker if necessary, to position the group for proton abstraction, preferably providing a 4- or 5-bond spacing between the abstracting atom and the hydrogen atom on the -carbon. Also provided are methods of using the reagents and stable, solubilized conjugates of the reagents with biologically active compounds. In preferred embodiments, the polymeric component of the reagent or conjugate is a polyethylene glycol.

A method of conversion of thioimidates into amidines includes reacting a starting material compound including a thioimidate with a nitrogen-containing reagent to form a product compound including an amidine in place of the thioimidate in the starting material compound. The nitrogen-containing reagent includes a primary amine, a secondary amine, an ammonium salt, or a combination thereof. The thioimidate includes SR.sup.1, wherein R.sup.1 is substituted or unsubstituted (C.sub.1-C.sub.20)hydrocarbyl. A method of forming a cyclized product includes intramolecularly reacting an amidine with a carbonyl carbon to form the cyclized product including a 4H-imidazolone and/or a derivative thereof.

The present invention provides a coryneform bacterium having an ability to produce a heterologous fusion protein by secretory production, which has been modified to express a genetic construct for secretory production of the heterologous fusion protein encoding at least a heterologous fusion protein comprising an extein and an intein having an activity of acyl rearrangement. The method for producing proteins modified at the C-terminus is also provided.

Method for preparing a peptide assembly of n fragments and n1 amino acids bearing a thiol function, represented by the formula:
A.sub.1-C.sub.1-A.sub.2-C.sub.2-A.sub.3- . . . -C.sub.i1-A.sub.i- . . . -C.sub.n1-A.sub.n(I)
in which A.sub.1, A.sub.2, A.sub.3, . . . A.sub.i . . . , A.sub.n are peptide fragments, C.sub.1, C.sub.2, C.sub.3 . . . C.sub.i1 . . . C.sub.n1 are amino acid residues bearing a thiol function, n is comprised between 3 and 50, and i is 2 to n, in which a peptide-thioester is prepared of formula: A.sub.1-SR (II) in which A.sub.1 is a peptide fragment and SR is an alkyl thioester residue, R being alkyl optionally substituted, starting from a bis(2-sulphanylethyl)amino peptide.

An object of the present invention is to provide a method for producing a peptide thioester compound, a peptide hydrazide compound, and a peptide amide compound. The present invention provides a method for producing a peptide hydrazide compound or a peptide amide compound by using a compound represented by Formula (2): ##STR00001##
wherein R.sup.1, R.sup.2, R.sup.3, and X are as defined in the specification, and using a hydrazine compound or an ammonia compound as a reaction reagent. The present invention also provides a method for producing a peptide thioester compound from the peptide hydrazide compound.

A method is provided for the preparation of an N-acyl peptide, N-acyl polypeptide or N-acyl protein comprising reacting a peptide, polypeptide or protein with an acyl halide in the presence of the biologically compatible tertiary amine nicotinamide as catalyst, thus obtaining the desired N-acyl peptide, N-acyl polypeptide or N-acyl protein.

Provided herein are polymeric -hydroxy aldehyde or -hydroxy ketone reagents which can be conjugated to amine-containing compounds to form stable conjugates in a single-step reaction. In selected embodiments, the polymeric reagent itself incorporates an internal proton-abstracting (basic) functional group, to promote more efficient reaction. The substituent is appropriately situated, via a linker if necessary, to position the group for proton abstraction, preferably providing a 4- or 5-bond spacing between the abstracting atom and the hydrogen atom on the -carbon. Also provided are methods of using the reagents and stable, solubilized conjugates of the reagents with biologically active compounds. In preferred embodiments, the polymeric component of the reagent or conjugate is a polyethylene glycol.

The invention provides methods for producing a protein in a cell free protein synthesis system such that the protein does not contain an asparagine (Asn or N) residue at serine (Ser or S) positions. Also provided are compositions and nucleic acid templates for use in the methods described herein.

A process for the preparation of plecanatide. Specifically, an improved process for preparation of plecanatide using a non-linear solid phase peptide synthesis. In particular, the process for preparation of plecanatide involves solid phase synthesis of peptide fragments of five amino acid units using 2-chlorotrityl chloride (2-ClTrt) resin and eleven amino acid unit using Wang resin.