Enzypep B.v.

Dsm IP Assets B.v.

Fresenius Kabi Ipsum S.r.l.

Timo Nuijens

Walter Cabri

Peter Jan Leonard Mario Quaedflieg

Ana Toplak

Jan Metske Van Der Laan

Bian Wu

Harold Monro Moody

Antonio Ricci

Dirk Barend Janssen

Claudia Cusan

Theodorus Johannes Godfried Maria Van Dooren

Catharina Hubertina Maria Schepers

Rowin De Visser

Kerstin Wallraven

Elvira Wijker

Van Theodorus Johannes Godfried Maria Dooren

Peter Jan Quaedflieg

Jurgen Mateman

A method for synthesizing a peptide having the sequence His-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Z-Gly-Arg-Gly is disclosed. The method includes enzymatically coupling:

Chemo-enzymatic synthesis of semaglutide, liraglutide and GLP-1

Chemo-enzymatic synthesis of Semaglutide, Liraglutide and GLP-1

The invention relates to a method for preparing a coupling product having the sequence P-W-His-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Y-Glu-Phe-Ile-Ala-Trp-Leu-Val-Z-Gly-Arg-Gly. The method includes enzymatically coupling

The invention further relates to a process for the enzymatic synthesis of an (oligo)peptide. The invention relates to a method for designing an enzymatic synthesis process of an (oligo)peptide, comprising identifying two or more (oligo)peptide fragments of an (oligo)peptide, which fragments are (oligo)peptides suitable for preparing the (oligo)peptide by enzymatic condensation of the two or more peptide fragments using a ligase. The invention relates to a method for designing an enzymatic synthesis process of a cyclic (oligo)peptide, comprising identifying a non-cyclic (oligo)peptide from which the cyclic (oligo)peptide can be prepared by cyclisation, catalysed by a cyclase. The invention further relates to a process for the enzymatic synthesis of an (oligo)peptide.

Designing an enzymatic peptide fragment condensation strategy

The invention relates to a subtilisin BPN' variant or homolog thereof, having the following mutations compared to subtilisin BPN&#x2032; represented by SEQUENCE ID NO: 2 or a homolog sequence thereof:&#x2014;a deletion of the amino acids corresponding to positions 75-83;&#x2014;a mutation at the amino acid position corresponding to S221, the mutation corresponding to S221C or S221 selenocysteine, preferably S221C;&#x2014;at least one further mutation selected from the group consisting of amino acid positions corresponding to F189W, F189Y, S33D, S33T, N218D, N218T, N218E, N62D, N62S, N62W, and N62Y; preferably a mutation at the amino acid position corresponding to P225; wherein the amino acid positions are defined according to the sequence of subtilisin BPN&#x2032; represented by SEQUENCE ID NO: 2. The invention further relates to enzymatically synthesizing a peptide.

Subtilisin variants having a mutation in the S2 or S2# pocket

The invention relates to a method for enzymatically synthesizing an (oligo)peptide, comprising coupling (a) an (oligo)peptide C-terminal ester or thioester and (b) an (oligo)peptide nucleophile having an N-terminally unprotected amine, Further, the invention relates to an enzyme suitable for use as a catalyst in a method of the invention.

Peptide fragment condensation and cyclisation using a subtilisin variant with improved synthesis over hydrolysis ratio

The invention relates to a method for enzymatically synthesizing an (oligo)peptide, comprising coupling (a) an (oligo)peptide C-terminal ester or thioester and (b) an (oligo)peptide nucleophile having an N-terminally unprotected amine, wherein the coupling is carried out in a fluid comprising water, and wherein the coupling is catalyzed by a subtilisin BPN' variant or a homo-log thereof, which comprises the following mutations compared to subtilisin BPN&#x2032; represented by SEQUENCE ID NO: 2 or a homolog sequence thereof: a deletion of the amino acids corresponding to positions 75-83; a mutation at the amino acid position corresponding to S221, the mutation being S221C or S221 selenocysteine; preferably a mutation at the amino acid position corresponding to P225 wherein the amino acid positions are defined according to the sequence of subtilisin BPN&#x2032; represented by SEQUENCE ID NO: 2. Further, the invention relates to an enzyme suitable for use as a catalyst in a method of the invention.

Method for enzymatically synthesizing an oligopeptide, comprising the coupling of an (optionally N-protected) protected oligopeptide ester with an (optionally C-protected) protected oligopeptide nucleophile in an organic solvent or an organic solvent mixture having a water content of 0.1 vol % or less, by a subtilisin in any possible form.

Side-chain protected oligopeptide fragment condensation using subtilisins in organic solvents

The invention relates to a method for synthesizing a peptide by enzymatically preparing an ester or thioester from (i) an N-terminal protected amino acid or an N-terminal protected peptide where either can have a protected C-terminal ester group and (ii) an alcohol represented by the formula HO&#x2014;CX&#x2014;Z or a thiol represented by the formula HS&#x2014;CX&#x2014;Z, each X independently representing a halogen atom or a hydrogen atom; and Z represents an electron withdrawing group comprising at least one sp-hybridized carbon comprising at least two substituents comprising a heteroatom directly attached to the at least one sp-hybridized carbon or at least one sp-hybridized carbon comprising one or two substituents comprising a heteroatom directly attached to the at least one sp-hybridized carbon, and enzymatically coupling the prepared ester or thioester with an optionally C-terminal protected amino acid or with an optionally C-terminal protected peptide in a medium comprising 2 wt. % water or less.

Peptide synthesis using enzymatic activation and coupling

The invention relates to a method for preparing an optionally N-protected amino acid C-terminal ester or an optionally N-protected peptide C-terminal ester, comprising transesterifying the C-terminal t-alkyl ester of the amino acid or the C-terminal t-alkyl ester of the peptide with an alcohol (other than the t-alcohol corresponding to the t-alkyl group of the ester) in the presence of a hydrolytic enzyme (E.C. 3). The invention further relates to a method for preparing a peptide comprising coupling an activated, N-protected, amino acid C-terminal ester or an optionally N-protected peptide C-terminal ester with an optionally C-terminal protected amino acid or an optionally C-terminal protected peptide via a peptide bond, in the presence of an enzyme catalysing peptidic bond formation.

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Geleen, Netherlands

Timo Nuijens