Methods for synthesizing polypeptides

Abstract

The present invention relates to a method for synthesizing a polypeptide based on solid phase peptide synthesis, wherein the polypeptide comprises a scaffold, the scaffold comprising one amino acid moiety Δ selected from diaminoalkanoic acids containing 3 to 10 carbon atoms and one peptide side chain covalently attached to the amino acid moiety Δ, wherein the amino acid moiety Δ is coupled to an amino acid moiety W of the scaffold, the method comprising at least one reaction cycle comprising providing an amino acid building block, activating the amino acid building block in the absence of a solid phase at a defined temperature and time, and coupling the amino acid building block to the scaffold.

Description

[Technical Field]

[0001] The present invention relates to a method for synthesizing branched polypeptides based on solid phase peptide synthesis. [Background technology]

[0002] Solid-phase peptide synthesis (SPPS) of polypeptides, first developed by Bruce Merrifield (Merrifield, 1963), involves the synthesis of a polypeptide bound to an insoluble solid phase and subsequent cleavage of the polypeptide from the solid phase. SPPS involves repeated reaction cycles in which amino acids are coupled to the nascent peptide bound to the solid phase through the formation of an amide bond (commonly referred to as a peptide bond). The use of a solid phase allows for the use of excess reagents and the removal of these and other reaction ancillary substances by simple filtration. To obtain a high final (global) yield of the polypeptide, it is desirable for the (local) yield in each coupling to be as high as possible. In each reaction cycle, it is preferable to drive peptide bond formation to completion. As the number of amino acids in a polypeptide increases, it becomes increasingly important to obtain a high local yield in each coupling. The peptide synthesis strategy of the present invention involves the preparation of peptides that contain at least amino acids (branch-inducing amino acids) that have functionalized amino acid side chains that can serve as sites for peptide side chain formation, such as lysine and lysine analogs.

[0003] Embodiments of the present invention relate to the synthesis of branched polypeptides, particularly polypeptides containing peptide side chains, such as glucagon analogs. The peptide side chains can be formed by applying several strategies. The peptide side chains can be formed during resin binding of the polypeptide and by sequential coupling of appropriate side chain amino acid moieties. Alternatively, the entire peptide side chain or a portion of the peptide side chain can be coupled to an appropriate amino acid moiety of the resin-bound polypeptide. Additionally, the peptide side chains can be assembled after coupling of the branching amino acid but before coupling of the next backbone amino acid. Alternatively, the peptide side chains are synthesized after completion of the polypeptide backbone. In the context of the present invention, the next amino acid of the polypeptide backbone is coupled after completion of peptide side chain synthesis, either by sequential coupling of individual side chain amino acid moieties or by coupling of a portion or the entire peptide side chain. The amino acid serving as the locus for branching must contain at least three reactive groups, including a group that contributes to the covalent attachment of the peptide side chain. In the context of the present invention, the locus for branching is an amino acid building block defined as PROT1-Δ(PROT2)-OH, where Δ is selected from diaminoalkanoic acids containing 3 to 10 carbon atoms, PROT1 constitutes the α-amine protecting group, and PROT2 is an amino acid side chain protecting group. When the peptide side chain is synthesized after coupling of the branching amino acid but before coupling of the next polypeptide backbone amino acid, the amino acid side chain protecting group must be cleavable without cleaving the α-amine protecting group or any other amino acid side chain protecting groups (class (II) protecting groups) present in the resin-bound polypeptide. Thus, the flexibility in selecting PROT1 and PROT2 is determined by the reaction scheme for synthesizing the polypeptide side chain.

[0004] The present invention seeks to optimize / improve the local (as well as global) yields in the coupling of amino acids that act as loci for branching for the synthesis of polypeptides comprising a backbone and peptide side chains, e.g., glucagon analogs, using an SPPS scheme in which the peptide side chains are assembled prior to the formation of the complete target polypeptide backbone, i.e., the complete target polypeptide.

[0005] WO2018069295 discloses a method for the preparation of peptides containing lipophilically modified lysine side chains. More specifically, the method relates to a procedural scheme for the removal of the trityl-based side chain protecting groups of lysine after formation of the target polypeptide backbone but before cleavage from the resin. WO2019120639 relates to the synthesis of polypeptides containing an alloc N-terminal protected lysine or lysine derivative, which comprises acylation of the lysine side chain, removal of the alloc group, and coupling of an amino acid or peptide to the N-terminus of the lysine. Chinese Patent No. 106478806 provides a method for synthesizing semaglutide. Lys is provided in the form of Dde-Lys(Fmoc)-OH. Chinese Patent No. 106478806 does not mention the temperature during the coupling cycle.

[0006] Object of the invention One object of the present invention is to improve the overall yield of polypeptides synthesized using SPPS. A further object is to improve the overall yield of branched polypeptides having backbones of more than 10 amino acids, which are polypeptides synthesized using SPPS. Another object is to improve the yield in coupling with lysine or lysine analogs, particularly in coupling of lysine with backbone tryptophan. A further object is to improve the local (as well as global) yield in the coupling of amino acids that act as loci for branching for the synthesis of polypeptides comprising a backbone and peptide side chains, e.g., glucagon analogs, using an SPPS scheme in which the peptide side chains are assembled prior to the formation of the target polypeptide backbone. A further objective is to provide improved conditions for coupling of amino acids (which act as loci for branching) with tryptophan residues. Another objective is to establish optimal conditions for coupling the side chain to a branch-point amino acid, such as lysine. Summary of the Invention

[0007] The present invention relates to a method for synthesizing a polypeptide based on solid phase peptide synthesis, wherein the polypeptide comprises a backbone, the backbone comprising one amino acid moiety Δ selected from diaminoalkanoic acids containing from 3 to 10 carbon atoms and one peptide side chain covalently attached to the amino acid moiety Δ, wherein the amino acid moiety Δ is coupled to an amino acid moiety W, and the method comprises: (i) providing an amino acid building block PROT1-Δ(PROT2)-OH, where PROT1 is an α-amine protecting group and PROT2 is an amino acid side chain protecting group; (ii) activating the amino acid building block PROT1-Δ(PROT2)-OH in the absence of a solid phase at an elevated temperature of about 20° C. to about 50° C. for less than about 25 minutes; (iii) coupling the activated amino acid building block PROT1-Δ(PROT2)-OH to the unprotected α-amine of the amino acid moiety W of the peptide covalently bound to the solid phase to form an amide bond; The method comprises at least one reaction cycle comprising:

[0008] definition A solid-phase peptide synthesis (SPPS) reaction cycle involves steps including deprotection of a protecting group attached to the α-amine of a peptide or amino acid covalently bound to a solid phase, and coupling of the carboxylic acid functional group of the α-amine-protected amino acid moiety with the (free or deprotected) α-amine of a peptide or amino acid bound to a solid support to form an amide bond. Prior to coupling, the amino acid must be activated. Additionally, a reaction cycle may also include capping of the (unreacted, deprotected) α-amine of a solid-phase-bound peptide or amino acid that has not formed an amide bond, which is referred to herein as capping. The steps may be repeated in a reaction cycle; for example, a reaction cycle may include multiple coupling and capping steps. Operations for removing compounds from a reaction solution are collectively referred to as washing. The steps in a reaction cycle may also be referred to as steps or stages. A procedure, step, or stage may be characterized by any one of the following non-exhaustive procedures (steps, stages): use of one or more key compounds (examples of key compounds: activating agent, capping agent, deprotecting agent, activated amino acid building block), operation between two washes, reaction procedure (e.g., activation of amino acid building block, formation of amide bond, cleavage of covalent bond). Also, some steps may proceed in parallel, e.g., activation and coupling may occur simultaneously. Thus, in the context of the present invention, a reaction cycle may include multiple procedures (steps, stages), deprotection, activation, coupling, and optionally, the key procedure (step, stage) of capping.

[0009] Activation, in the context of the present invention, begins when the α-amine protected amino acid building block and at least one activating substance are in solution and continues until the amide bond is formed. Activation and Activated Amino Acid Building Blocks: Carboxylic acids and amines are unlikely to participate in condensation reactions to form amide bonds, but instead neutralize to form carboxylate and ammonium ions. In the broadest sense, activation (or activation procedure) allows carboxylic acids and amines to form amide bonds. Activation can refer to the activation of the carboxylic acid functional group of an amino acid moiety with the participation of at least one activating reagent (also called a coupling reagent). The carboxylic acid functional group is usually activated by reaction with an electron-withdrawing reagent. The activated carboxylic acid functional group of an amino acid building block is capable of forming an amide bond with an amine. An activated amino acid building block is typically an amino acid building block containing a carboxylic acid / carboxylate bond bound to an activating reagent. The stability of the activated amino acid building block depends in part on the activating reagent and the reaction conditions.

[0010] In the context of the present invention, amino acid building blocks are amino acids in which the α-amine and any reactive groups of the amino acid side chain are protected by suitable protecting groups. The term "amino acid moiety" is used to refer to an amino acid present within a peptide / polypeptide. Activators are molecules that activate amino acid building blocks. Capping is the chemical modification of the unreacted α-amine remaining after the coupling step to render it incapable of chemical reaction in downstream coupling cycles. Capping typically involves acylation or acetylation of the α-amine. The capping reagent can be acetic anhydride. Amino acid moieties and amino acid building blocks include naturally occurring amino acids and any chemical modifications of naturally occurring amino acids, as well as any synthetic molecules that contain functional groups, particularly amines and carboxylic acids, that allow the use of synthetic molecules in SPPS, resulting in amide bonds.

[0011] A polypeptide contains at least two amino acids and any number of amino acids for the molecule to be called a protein. Human proteins have about 200 amino acids or more. In the context of the present invention, the term polypeptide is used to refer to the branched polypeptides formed by the methods disclosed herein, and the backbone of the branched polypeptide is also referred to as the polypeptide. The term peptide is used in conjunction with a polypeptide side chain. A peptide side chain can have any number of amino acid moieties, from at least two to less than the number of amino acid moieties in the polypeptide backbone. Thus, a peptide side chain is a side chain of a branched polypeptide. A peptide side chain can also include a carbohydrate moiety. Typically, the carbohydrate moiety contains about 10 to about 25 carbon atoms, preferably about 14 to 20 carbon atoms. An amino acid side chain is the side chain of an amino acid moiety. An amino acid side chain is attached to the α-carbon. Lysine is an example of an amino acid that contains a side chain. Diaminopropionic acid contains 3 carbon atoms and diaminodecanoic acid contains 10 carbon atoms.

[0012] Acid-labile protecting groups are protecting groups that are cleaved under acidic conditions. Base-labile protecting groups are protecting groups that are cleaved under alkaline conditions. Class (I) protecting groups refer to protecting groups that are attached to the reactive groups of amino acid building blocks and are not involved in amide bond formation, excluding PROT2 protecting groups. Therefore, class (I) protecting groups are preferably protecting groups that are covalently attached to the reactive groups of the side chains of amino acid building blocks. Examples of amino acid moiety side chain reactive groups are amines, carboxylic acids, alcohols, thiols, and thioethers. Class (II) protecting groups refer to protecting groups that are attached to the amine of an amino acid building block and participate in amide formation (α-amine), excluding the PROT1 protecting group. The terminology Class (I) protecting group and Class (II) protecting group is proposed to distinguish such protecting groups from the PROT1 and PROT2 groups of amino acid building blocks of formula PROT1-Δ(PROT2)-OH. Δ is an amino acid moiety selected from diaminoalkanoic acids containing from 3 to 10 carbon atoms. PROT1 is an α-amine protecting group. PROT2 is an amino acid side chain protecting group. Coupling refers to the formation of an amide bond (peptide bond). WLESA stands for the peptide fragment Trp-Leu-Glu-Ser-Ala. The term "about" is used with respect to several parameters disclosed herein, such as temperature, time, and yield. Unless otherwise indicated, the term "about" should be interpreted as allowing for deviation of the appropriate parameter by up to 5%. An amino acid side chain (e.g., a Δ side chain) is the side chain of an amino acid moiety / amino acid building block. Lys is an example of an amino acid moiety / amino acid building block having an amino acid side chain. A peptide side chain, i.e., a side chain of a polypeptide (peptide side chain), is a side chain that is covalently attached to a backbone polypeptide. The side chain may comprise an amino acid moiety and / or a fatty acid moiety.

[0013] Figures 1-6 all illustrate HPLC profiles of workup solutions containing the truncated peptide (Dde-Lys(Fmoc)-Trp-Leu-Glu-Ser-Ala-NH) synthesized as described in Example 3. The variables were preactivation time (5, 10, 15 min) and temperatures of 30 °C and 35 °C. The temperature during preactivation and coupling was kept constant. [Brief explanation of the drawings]

[0014] [Figure 1] Graph showing 30° C., 5 min preactivation, 45 min coupling. [Figure 2] Graph showing 30° C., 10 min preactivation, 45 min coupling. [Figure 3] Graph showing 30° C., 15 min preactivation, 45 min coupling. [Figure 4] Graph showing 35° C., 5 min preactivation, 45 min coupling. [Figure 5] Graph showing 35° C., 10 min preactivation, 45 min coupling. [Figure 6] Graph showing 35° C., 15 min preactivation, 45 min coupling. DETAILED DESCRIPTION OF THE INVENTION

[0015] The method is based on SPPS, comprising at least one reaction cycle (reaction cycle A) in which an amino acid building block selected from the group consisting of PROT1-Δ(PROT2)-OH is coupled to the amino acid moiety W of a peptide covalently attached to a solid phase. Δ is selected from diaminoalkanoic acids containing from 3 to 10 carbon atoms. PROT1 is an α-amine protecting group. PROT2 is an amino acid side chain protecting group.

[0016] According to one aspect, the diaminoalkanoic acid is preferably an unbranched diaminoalkanoic acid, where unbranched, in the context of the diaminoalkanoic acid, refers to the side chain. According to a further aspect, the diaminoalkanoic acid is selected from (α,ω)-diaminoalkanoic acids, preferably unbranched diaminoalkanoic acids, where ω represents the distal end of the amino acid side chain, an amine at the ω carbon, and a second amine at the α carbon. According to one embodiment, the diaminoalkanoic acid contains 4 to 8, preferably 5 to 8, more preferably 5 to 7 carbon atoms. According to a further aspect, the diaminoalkanoic acid is selected from ornithine (2,5 diaminopentanoic acid) and lysine (2,6 diaminohexanoic acid), preferably lysine.

[0017] PROT1 and PROT2 are α-amine and amino acid side chain protecting groups, respectively, that are cleaved under alkaline conditions, with the proviso that only one of PROT1 and PROT2 is preferably selected from protecting groups that are cleaved under the same reaction cycle. PROT1 and PROT2 are Fmoc (fluorenylmethyloxycarbonyl), Nsc (2-(4-nitrophenylsulfonyl)ethoxycarbonyl), Bsmoc (1,1-dioxobenzo[b]thiophen-2-ylmethyloxycarbonyl), α-Nsmoc ((1,1-dioxonaphtho[1,2-b]thiophen-2-yl)methyloxycarbonyl), Dde (N-[1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl]), ivDde (1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl), and ivDde (1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl). and 2,7-di-tert-butyl-Fmoc, 2-fluoro-Fmoc, 2-monoisooctyl-Fmoc, 2,7-diisooctyl-Fmoc, TCP (tetrachlorophthaloyl), Pms (2-phenyl(methyl)sulfonio)ethyloxycarbonyl tetrafluoroborate), Esc (ethanesulfonylethoxycarbonyl), and Sps (2-(4-sulfophenylsulfonyl)ethoxycarbonyl). PROT1 is preferably selected from Dde and ivDde. PROT1 is suitably Dde. PROT2 is preferably Fmoc.

[0018] The present invention relates to the synthesis of polypeptides comprising a backbone and one peptide side chain, which may be referred to as a branched polypeptide. The backbone comprises an amino acid moiety and one amino acid moiety Δ selected from diaminoalkanoic acids containing 3 to 10 carbon atoms, coupled to W. The peptide side chain is covalently attached to the amino acid moiety Δ of the backbone. The backbone is formed from amino acid building blocks selected from PROT1-Δ(PROT2)-OH and comprises only one amino acid moiety Δ, which serves as a locus for the construction / synthesis / formation of the peptide side chain. The backbone may comprise an additional amino acid, e.g., Lys, selected from diaminoalkanoic acids containing 3 to 10 carbon atoms. W and WLESA and ΔWLESA form part of the peptide backbone. In one embodiment, the polypeptide comprises only one peptide side chain, which is covalently attached to an amino acid moiety defined by the methods of the invention, i.e., amino acid moiety Δ.

[0019] According to one embodiment, the PROT2 protecting group and the class (I) protecting group are selected from protecting groups that are simultaneously cleaved under the same reaction cycle. The PROT2 protecting group and the class (I) protecting group may be selected from base-labile protecting groups, such as Fmoc. The class (II) protecting groups of reactive groups of amino acid building blocks that are not involved in amide bond formation (which may be referred to as amino acid side chain protecting groups, e.g., amines) and the protecting groups of class (I) protected amines (α-amines) of amino acid building blocks that are involved in amide formation (α-amine protecting groups) are preferably selected so that only one class of protecting group is cleaved during the same reaction cycle. The class (I) protecting groups may have a tendency to be cleaved under alkaline conditions (alkali / base-labile protecting groups), while the class (II) protecting groups may have a tendency to be cleaved under acidic conditions (acid-labile protecting groups). PROT1 and PROT2 are suitably selected such that only one of PROT1 or PROT2 is cleaved during the same reaction cycle. Moreover, PROT2 is preferably selected from protecting groups that are cleaved, but not other protecting groups of class (II), during the same reaction cycle.

[0020] In one embodiment, class (II) protecting groups are acid labile and class (I) protecting groups are base labile. This protection scheme is also referred to as an orthogonal protection scheme. When the method relates to a polypeptide formed from amino acid building blocks containing a reactive group not involved in amide bond formation and protected by a class (II) protecting group, and said class (II) protecting group is acid labile, PROT1 and PROT2 are preferably base labile, indicating that said protecting group is cleaved under alkaline conditions. According to certain embodiments, the method further comprises the steps of removing PROT2 prior to association of the peptide side chains, and removing PROT1 after association of the peptide side chains and attaching additional amino acids to the backbone. According to a further embodiment, the amino acid moiety Δ is coupled to the amino acid sequence WLESA. According to yet a further embodiment, the amino acid moiety A of the amino acid sequence WLESA is covalently attached to a solid phase.

[0021] When it is said that an amino acid sequence is covalently attached to a solid phase, it is understood that the amino acid sequence may be attached to the solid phase via a suitable linker. According to one embodiment, the activation in step (ii) is carried out at a temperature ranging from about 20°C to about 45°C, preferably from about 20°C to about 40°C, and preferably from about 30°C to about 35°C. In one embodiment, the activation in step (ii) is carried out for less than about 22 minutes, preferably less than about 18 minutes. According to certain embodiments, activation is maintained until at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75% of the amino acid building blocks PROT1-Δ(PROT2)-OH are activated. According to one embodiment, Δ is selected from unbranched (α,ω)-diaminoalkanoic acids, suitably ornithine and lysine, preferably lysine.

[0022] According to one embodiment, PROT1 and PROT2 are selected from protecting groups that are cleaved under alkaline conditions, with the proviso that PROT1 and PROT2 are not cleaved under the same reaction cycle. According to one embodiment, the method further comprises selective removal of PROT2. In one embodiment, PROT1 is selected from Dde and ivDde, preferably Dde, and PROT2 is Fmoc. According to one embodiment, the method further comprises at least one reaction cycle involving the formation of a peptide side chain attached to the amino acid moiety Δ, wherein the coupling is carried out at a temperature above about 30°C, preferably between about 30°C and about 45°C, preferably between about 30°C and about 35°C. According to a still further aspect, the polypeptide comprises: His-X2-X3-GTFTSDYSKYL-X15-X16-X17-X18-A-X20-DFI-ΔWLESA is selected from: X2 is selected from Aib, Ac3c, Ac4c, and Ac5c, preferably Aib and Ac4c; X3 is selected from Gln and His; X15 is selected from Asp and Glu, preferably Asp; X16 is selected from Glu and Ψ, preferably Glu; X17 is selected from Arg and Ψ; X18 is selected from Ala and Arg; X20 is selected from Lys and His, preferably Lys; Ψ is selected from Lys, Arg or Orn; The peptide side chains have the formula -Z 2 -Z 1 and Z 1 has a polar group at one end of the chain and Z 2 a hydrocarbon chain having a bond to, -X- located at the end of the chain distal from the polar group; the polar group comprises a carboxylic acid or carboxylic acid bioisostere, a phosphonic acid, or a sulfonic acid group; -X- is a bond, -CO-, -SO-, or -SO2; -Z 2 - is a spacer in the following formula:

[0023] [ka] wherein each Y is independently —NH, —NR, —S, or —0, and R is an alkyl, a protecting group, or a spacer Z 2and each X is independently a bond, CO—, SO—, or SO—, with the proviso that when Y is —S, X is a bond, each V is independently a divalent organic moiety that is bonded to Y and X, and n is 1 to 10.

[0024] The method comprises at least one reaction cycle comprising activation of an α-amine protected amino acid building block in the absence of a solid phase to form an activated α-amine protected amino acid building block, and coupling the activated α-amine protected amino acid building block with an unprotected α-amine of a peptide or amino acid covalently attached to a solid phase to form an amide bond, wherein the activation time is less than 25 minutes at an elevated temperature of about 20°C to about 50°C.

[0025] Activation can be achieved in the presence of a solid phase, i.e., in the reaction vessel where the polypeptide is formed and bound to the solid phase (polymer solid phase). Activation in the presence of a solid phase is often referred to as in situ activation. An alternative is to initiate activation in the absence of a solid phase. Activation in the absence of a solid phase, and by extension activation in the absence of the nascent polypeptide bound to the solid phase, is preferably performed in an activation vessel separate from the reaction vessel where the polypeptide is formed. Activation of the α-amine-protected amino acid building block is necessary for the establishment of an amide bond (peptide bond) between the α-amine-protected amino acid building block and the nascent peptide bound to the solid phase. Activation begins with the provision (addition) of at least one activating substance (activating substances are within the field of SPPS and are also referred to as coupling substances) and the α-amine-protected amino acid building block, and typically continues until the amide bond is formed. The stability of the activated α-amine-protected amino acid building block depends to some extent on the activating substance and solvent composition. Activated α-amine-protected amino acid building blocks can be stable for hours or even days under favorable conditions, indicating that activated α-amine-protected amino acid building blocks can be activated from the time of activation until amide bond formation. Thus, when α-amine-protected amino acid building blocks are activated in the absence of a peptide-solid phase, they exist in activated form after transfer to the peptide reaction vessel until amide bond formation. According to one embodiment, activation begins in the absence of a solid phase, preferably in a solution containing at least one activating substance, preferably in an activation vessel (other than the reaction vessel containing the solid phase). According to a further embodiment, activation is carried out by application of two activating substances.

[0026] Activation of the amino acid building block PROT1-Δ(PROT2)-OH in the absence of a solid phase takes less than about 25 minutes, less than 22 minutes, less than 21 minutes, less than about 20 minutes, less than about 18 minutes, or less than about 15 minutes. Activation can be carried out for about 1 minute, about 2 minutes, or about 5 minutes up to about 25 minutes, about 20 minutes, about 18 minutes, or about 15 minutes. Any of the lower and upper time limits indicated can be combined with one another. To some extent, activation time is determined by the temperature during activation. According to further embodiments, activation in the absence of a solid phase is preferably maintained until at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75% of the activated α-amine protected amino acid building block (PROT1-Δ(PROT2)-OH) has been formed.

[0027] Preferably, the α-amine protected amino acid building block, e.g., PROT1-Δ(PROT2)-OH, is charged at a molar ratio greater than about 1.5, preferably greater than about 2.0, based on the solid phase loading, which is the theoretical number of amines available for amide / peptide bond formation. The activating agent is preferably loaded in a molar amount equal to or similar to the molar amount of the α-amine-protected amino acid building block. Activating agents consumed during the activation process, such as DIC, may be loaded in an overall molar ratio that exceeds the ratio of the appropriate α-amine-protected amino acid building block.

[0028] Activation, in the context of the present invention, is the time from mixing an α-amine protected amino acid building block with at least one activating substance in the absence of a solid phase to the point at which an amide bond is formed in response to the addition of a solution containing the activated amino acid building block to a solution containing the solid phase. The α-amine protected amino acid building block, activated in the absence of a solid phase, is transferred from the activation vessel to a polypeptide reaction vessel. The activated α-amine protected amino acid building block is transferred to a peptide reaction vessel containing the peptide or amino acid on the solid phase, and coupling is initiated. Activation, which is the formation of an amino acid ester residue, is possible as long as a suitable activating agent is present, and therefore activation can occur even after transfer of a solution containing the activated amino acid residue from the activation vessel to the peptide reaction vessel. Coupling refers to the formation of an amide bond (peptide bond). The rate of amide bond formation decreases over time and is partially correlated to the amount (molar ratio) of activated α-amine protected amino acid building block with respect to the amine available for peptide coupling to the solid phase.

[0029] Activators include diisopropylcarbodiimide (DIC), ethyl cyano(hydroxyimino)acetate (Oxyma, sometimes referred to as OxymaPure), dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC×HCl), optionally in the presence of a coupling additive, such as 1-hydroxybenzotriazole (HOBt), 6-chloro-1-hydroxybenzotriazole (Cl-HOBt), 1-hydroxy-7-aza ... Benzotriazole (HOAt), 2-hydroxypyridine-N-oxide (HOPO), ethyl cyanohydroxyiminoacetate (Oxyma), Oxyma-B, N-hydroxysuccinimide (HOSu), N-hydroxy-5-norbornene-2,3-dicarboxylic acid imide (HONB), hexafluorophosphate benzotriazole tetramethyluronium (HBTU), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate [O-[N,N ,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate] (TBTU), O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium hexafluorophosphate (HOTU), O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate (TOTU), O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate Tetramethyluronium hexafluorophosphate (HCTU), O-(6-chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TCTU), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, azabenzotriazole tetramethyluronium hexafluorophosphate (HATU), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-Tetramethyluronium tetrafluoroborate (TATU), 1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), 1-[(dimethylamino)(morpholino)methylene]-1H-[1,2,3]triazolo[4,5-b]pyridin-1-ium 3-oxide hexafluorophosphate (HDMA), HDMB, 6-chloro-1-((dimethylamino)(morpholino)-methylene)-1H-benzotriazolium (HDMC), benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate C (BOP), (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), [ethylcyano(hydroxyimino)acetato-O, 2 ]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim), 6-chloro-benzotriazol-1-yloxy-tris-pyrrolidinophosphonium hexafluorophosphate (PyClock), N,N,N',N'-tetramethylchloroformamidinium hexafluorophosphate (TCFH), N-(chloro(morpholino)methylene)-N-methylmethanaminium hexafluorophosphate (DMCH), chlorotripyrrolidinophosphonium hexafluorophosphate (Pyclop), tetramethylfluoroformamidinium hexafluorophosphate (TFFH), tetramethyl The alkyl ammonium salt may be selected from the group consisting of ammonium trifluoromethanethiolate ((MeN)SCF), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), 2,4-dichloro-6-methoxy-1,3,5-triazine (DCMT), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMMCl), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate (DMTMMBF), and 2-(4,6-dimethoxy-1,3,5-triazinyl)trialkylammonium salts (DMT-Am).

[0030] In one embodiment, activation is achieved using DIC and Oxyma, for example, by combining DIC and Oxyma and an appropriate α-amine protected amino acid building block in a suitable solvent.

[0031] Solvents generally suitable for SPPS, applicable to all steps of the reaction cycle, include methylene chloride, dichloromethane (DCM), N-methylpyrrolidone (NMP), N-ethylpyrrolidone (NEP), N-propylpyrrolidone (NPP), N-butylpyrrolidone (NBP), N-pentylpyrrolidone (NPeP), N-hexylpyrrolidone (NHP), N-heptylpyrrolidone (NHeP), N-octylpyrrolidone (NOP), dimethylformamide (DMF), diethylformamide (DEF), dipropylformamide (DPF), N-formylpyrrolidine (NFP), N-formylmorpholine (NFM), N-methylcaprolactam (MCL), 1,3-dimethylpropanol (DMF), methyl ... The following compounds are commonly used: 1,3-dimethyl-3,4,5,6-tetrahydro-2-pyrimidinone (DMPU), dimethyl sulfoxide (DMSO), diethyl sulfoxide (DESO), sulfolane, (1R)-7,8-dioxabicyclo[3.2.1]octan-2-one (dihydrolevoglucosenone, Cyrene®), N,N-dimethylacetamide (DMA), N,N,N',N'-tetraethylsulfamide (TES), 1-butyl-3-methylimidazolium chloride (BMIMCl), 1-butyl-3-methylimidazolium bromide (BMIMBr), and 1-butyl-3-methylimidazolium iodide (BMIMI).

[0032] Activation is carried out at a temperature of about 20°C to about 50°C. As detailed above, activation is a procedure initiated by combining at least one activating substance with an α-amine-protected amino acid building block. The activation process results in an activated α-amine-protected amino acid building block with a specific lifetime. The activated α-amine-protected amino acid building block reacts with an amine to form an amide (peptide) bond. When activation is performed in the absence of a solid-phase-bound peptide / amino acid, amide bond formation begins upon addition of the activated α-amine-protected amino acid building block to a suspension containing the solid phase. When activation is initiated in the presence of a solid phase (in situ activation), amide bond formation can be initiated rapidly, and the α-amine-protected amino acid building block is still activated.

[0033] In the context of the present invention, and for reasons detailed herein, activation can also be achieved while the activated amino acid building block is participating in amide bond formation (coupling). The amino acid building block can be activated even after transfer of a solution containing the activated amino acid building block to a reaction vessel containing a solid phase. The temperature of activation is preferably still the coupling temperature or at least a temperature less than the coupling time, as long as a useful concentration of the activated α-amine-protected amino acid building block is present in the reaction solution. According to a further embodiment, activation is carried out at a temperature of about 20°C to about 45°C, preferably about 20°C to about 40°C, about 30°C to about 37°C, for example, about 30°C to about 35°C. The lower activation temperature limit may be 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., and the upper activation temperature limit may be 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., and 50° C. Each of the lower and upper temperature limits may be combined to represent a temperature range. In one embodiment, activation and coupling are carried out at the same temperature.

[0034] Whether activation is initiated in the presence or absence of a solid phase, it is preferred that the time from activation, i.e., from formation of the activated α-amine protected amino acid building block to amide bond formation, is preferably less than about 50 minutes, less than about 40 minutes, less than about 35 minutes, less than about 30 minutes, less than about 25 minutes, and preferably less than about 20 minutes. The activation time for the amino acid building block PROT1-Δ(PROT2)-OH in the absence of a solid phase is less than 25 minutes, less than 22 minutes, less than 21 minutes, less than 20 minutes, less than 18 minutes, or less than 15 minutes. Activation can be performed from about 1 minute, about 2 minutes, or about 5 minutes to about 25 minutes, about 20 minutes, about 18 minutes, or about 15 minutes.

[0035] According to one embodiment, activation and coupling of the amino acid building block PROT1-Δ(PROT2)-OH is carried out at a temperature of about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C to about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, and 50°C, and activation lasts for less than 25 minutes, less than 22 minutes, less than 21 minutes, less than 20 minutes, less than 18 minutes, less than 15 minutes, and from about 1 minute, about 2 minutes, about 5 minutes to about 25 minutes, about 20 minutes, about 18 minutes, about 15 minutes, in the absence of a solid phase, preferably in an activation vessel. If activation begins in an activation vessel (in the absence of a solid phase) containing a solution comprising at least one activating agent and an α-amine protected amino acid building block, this solution is then transferred to a solution containing a solid phase, i.e., a polypeptide reaction vessel containing a solid phase.

[0036] The reaction cycle may further include capping of unreacted, unprotected α-amines of the solid phase-bound peptides or amino acids. In one embodiment, capping involves the addition of a capping composition containing a capping substance and an additional activating substance, such as DIC. Preferably, capping is initiated by adding the capping composition to the coupling solution. Each reaction cycle involves cleavage of the α-amine protecting group of a solid phase-bound peptide or amino acid prior to coupling.

[0037] It is contemplated that the methods of the present invention are feasible for the synthesis of a variety of polypeptides selected from glucagon analogs or glucagon-like peptide 1. Any of the polypeptides disclosed herein that can be synthesized by the methods of the present disclosure are preferably synthesized using amino acid building blocks as defined herein, including naturally occurring amino acids and any chemical modifications of naturally occurring amino acids, as well as any synthetic molecules that contain functional groups, particularly amines and carboxylic acids, that allow for the use of synthetic molecules in SPPS and result in amide bonds. Thus, amino acid building blocks can be added to a polypeptide by the reaction cycles disclosed herein to form the target polypeptide backbone or any peptide side chains that are attached to the target polypeptide backbone. Polypeptides may be formed by strategies involving the formation of several peptide fragments that are subsequently coupled. Polypeptides containing at least one peptide side chain may be formed by synthesizing at least a portion of the peptide side chain by SPPS and coupling such fragment to the amino acid moiety of a solid phase-bound scaffold.

[0038] According to one embodiment, the method relates to a polypeptide selected from polypeptides comprising one peptide side chain, particularly peptides comprising only one peptide side chain. According to a further embodiment, the entire peptide side chain of a polypeptide is synthesized by successive reaction cycles carried out after coupling of an amino acid building block that serves as a locus for covalent attachment of the peptide side chain (forming part of the backbone and typically the amino acid moiety Δ) and before coupling of the next amino acid building block of the polypeptide backbone (main chain). When synthesizing the peptide side chain before completing the synthesis of the backbone, PROT1 and PROT2 of the amino acid building block PROT1-Δ(PROT2)-OH should preferably be selected such that PROT2 can be selectively deprotected with respect to any other protecting groups (class (II) group protecting groups) of the amino acid side chains of the amino acids present between PROT1 and the solid phase and the amino acid that serves as the branch point. Therefore, PROT2 is selected so that only PROT2 is deprotected during the deprotection procedure. According to a further aspect, the method implements an SPPS strategy, where class (II) protecting groups are acid labile and class (II) are cleaved under acidic conditions, and class (I) protecting groups are base labile and cleaved under alkaline conditions. When class (II) protecting groups are selected from the group of acid labile protecting groups, PROT1 and PROT2 are preferably selected from the group of alkali (base) labile protecting groups, with the proviso that PROT1 and PROT2 are preferably selected such that only PROT2 is cleaved in a particular reaction cycle (including the deprotection step). According to a further aspect, the class (I) protecting group is Fmoc.

[0039] The peptide side chains have the formula -Z 2 -Z 1 and -Z 1 has a polar group at one end of the chain and Z 2 a fatty acid chain having -X- at the end of the chain distal from the polar group, wherein the polar group comprises a carboxylic acid or carboxylic acid bioisostere, a phosphonic acid, or a sulfonic acid group, -X- being a bond, -CO-, -SO-, or -SO2; 2 - is a spacer in the following formula:

[0040] [ka] wherein each Y is independently —NH, —NR, —S, or —0, and R is an alkyl, a protecting group, or a spacer Z 2 and each X is independently a bond, CO—, SO—, or SO—, with the proviso that when Y is —S, X is a bond, each V is independently a divalent organic moiety that is bonded to Y and X, and n is 1 to 10. According to one embodiment, -Z 1 may also be an acyl group of the formula AB-Alk-(CO)- or a sulfonyl group of the formula AB-Alk-(SO)-, A is a carboxylic acid or a carboxylic acid bioisostere, preferably a carboxylic acid, and B is a bond, C6 arylene or C6 arylene-O-; Alk is a saturated or unsaturated, preferably unbranched, optionally fluoro, C 1-4 Alkyl, trifluoromethyl, hydroxymethyl, amino, hydroxy, C 1-4 a fatty acid chain substituted with one or more of alkoxy, oxo, and carboxyl; -Z 2 -S A -, -S A -S B -or-S B -S A - and -S A is a single amino acid residue selected from γ-Glu, α-Glu, α-Asp, β-Asp, Ala, β-Ala (3-aminopropanoic acid (Dap)), and Gaba (4-aminobutanoic acid); -S B - is a linker of the general formula:

[0041] [ka] (wherein n is 1 to 10, and each Pu is independently Pu i and PU iii is selected from Each Pu iare independently natural or unnatural amino acid moieties, and each PU iii are independently residues of the general formula:

[0042] [ka] (wherein m is 0 to 5, and p is 1, 3, 4, or 5) According to a further aspect, -Z 2 -Z 1 is selected from (i) [17-carboxy-heptadecanoyl]-isoGlu-PEG3-PEG3, (ii) [17-carboxy-heptadecanoyl]-isoGlu, (iii) [13-carboxy-tridecanoyl]-isoGlu-PEG3-PEG3, (iv) [carboxyphenoxynonanoyl]-isoGlu-PEG3-PEG3, (v) [13-carboxy-tridecanoyl]-isoGlu-PEG4-PEG4, (vi) [17-carboxy-heptadecanoyl]-PEG3-PEG3-isoGlu, (vii) [17-carboxy-heptadecanoyl]-isoGlu-Gly-Ser-Gly-Ser-Gly-Gly, and (viii) [17-carboxy-heptadecanoyl]-Ala-Ala-PEG3-PEG3. According to one embodiment, Z 2 -Z 1 is [17-carboxyheptadecanoyl]-isoGlu-PEG3-PEG3 or [17-carboxy-heptadecanoyl]-isoGlu-Gly-Ser-Gly-Ser-Gly-Gly. PEG3 and PEG4 are oligomers of ethylene glycol containing three and four ethylene glycol units, respectively.

[0043] Preferably, the carboxylic acid bioisostere has a proton with a pKa similar to that of the corresponding carboxylic acid. Examples of suitable bioisosteres may include tetrazoles, acylsulfonamides, acylhydroxylamines, and squaric acid derivatives. The fatty acid chain may be derived from a fatty acid, for example, a medium-chain fatty acid (MCFA) having an aliphatic tail of 6 to 12 carbon atoms, a long-chain fatty acid (LCFA) having an aliphatic tail of 13 to 21 carbon atoms, or a very long-chain fatty acid (LCFA) having an aliphatic tail of 22 or more carbon atoms. Examples of linear saturated fatty acids from which suitable fatty acid chains may be derived include tridecyl (tridecanoic) acid, myristic (tetradecanoic) acid, pentadecyl (pentadecanoic) acid, palmitic (hexadecanoic) acid, and margaric (heptadecanoic) acid. Examples of linear unsaturated fatty acids from which suitable fatty acid chains may be derived include myristoleic acid, palmitoleic acid, sapienic acid, and oleic acid.

[0044] The fatty acid chains are connected to Z by amide, sulfinamide, sulfonamide or ester bonds, or by ether, thioether or amine bonds. 2 Thus, the fatty acid chain can be linked to Z at the ω position, i.e., distal to the polar group. 2 or an acyl (-CO-), sulfinyl (-SO-) or sulfonyl (-SO2-) group. Preferably, the fatty acid chain has an acyl (-CO-) group distal to the polar group and is connected to Z by an amide or ester bond. 2 Combine with.

[0045] The method relates to SPPS, which comprises a reaction cycle in which an α-amine protected amino acid building block selected from the group consisting of PROT1-Δ(PROT2)-OH is coupled to a solid phase-bound peptide or amino acid. The amino acid building block PROT1-Δ(PROT2)-OHc is used to form the peptide side chain. Thus, in one aspect, the method relates to the synthesis of a polypeptide comprising a backbone and peptide side chains covalently attached to the backbone. The reaction cycle involved in the formation of peptide side chains of a polypeptide is referred to as the peptide side chain reaction cycle. In more particular aspects, all amino acid moieties of the peptide side chains, including amino acid building blocks of the formula PROT1-Δ(PROT2)-OH and amino acid moieties attached to the side chains of the amino acid building blocks further characterized herein, are formed by a peptide side chain reaction cycle.

[0046] The reaction cycle involved in the formation of a peptide side chain is referred to as the peptide side chain reaction cycle or side chain reaction cycle. The reaction cycle involved in the formation of the polypeptide backbone is referred to as the backbone reaction cycle. The reaction cycle associated with the formation of a polypeptide backbone is referred to as the backbone reaction cycle. Thus, a reaction cycle that includes the coupling of an activated amino acid building block selected from PROT1-Δ(PROT2)-OH is a backbone reaction cycle. All amino acid moieties of a polypeptide backbone, i.e., including amino acid building blocks of formula PROT1-Δ(PROT2)-OH and those further characterized herein, are formed by a reaction cycle referred to as the reaction cycle associated with the formation of a polypeptide backbone, or simply the backbone reaction cycle. The reaction cycle involved in the formation (synthesis) of a peptide side chain attached to the amino acid moiety Δ is referred to as the peptide side chain reaction cycle.

[0047] In one embodiment, any one of activation, coupling, deprotection, and optionally capping, preferably all of the steps of one or more reaction cycles (i.e., peptide side chain reaction cycles) associated with the formation of a (peptide) side chain of a polypeptide, preferably all reaction cycles, are carried out at a temperature above about 30°C, preferably between about 30°C and about 45°C, preferably between about 30°C and about 35°C. According to a further embodiment, the deprotection step of the reaction cycle involved in the formation of the (peptide) side chain of a polypeptide (peptide side chain reaction cycle) is carried out in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). According to yet a further aspect, all of the steps of one or more reaction cycles (backbone reaction cycles), preferably all reaction cycles, except for one of the activation, coupling, deprotection, and optionally capping steps, preferably the reaction cycle steps relating to the amino acid moiety Δ, associated with forming the polypeptide backbone, are carried out at a temperature above about 30°C, preferably above about 35°C, preferably above 40°C, preferably above about 45°C, for example, from about 35°C to about 55°C, from about 40°C to about 50°C, or from about 45°C to about 50°C.

[0048] In connection with any reaction cycle, regardless of the type of polypeptide disclosed herein, and for peptide side chain reaction cycle or backbone reaction cycle, the concentration of suitable one or more deprotecting agents, for example, a base such as piperidine or DBU, can be varied in the deprotection procedure.Preferably, the initial concentration of the deprotecting agent is less than the final concentration.Therefore, the deprotecting agent is typically added (added) multiple times, and the concentration of the deprotecting agent in the reaction solution after the initial addition of the deprotecting agent is less than the concentration after the final addition of the deprotecting agent. One embodiment relates to the application of a base, preferably piperidine, as a deprotecting agent for the cleavage of α-amine protecting groups (class (I)) involved in the backbone reaction cycle.

[0049] Polypeptides synthesized by the method of the present invention According to a further aspect, the method relates to the synthesis of a polypeptide comprising a backbone and one peptide side chain covalently attached to the backbone. The peptide side chains of a polypeptide typically comprise an amino acid and a hydrophobic moiety in the terminal region of the side chain, which may be a hydrocarbon chain with a polar group at the distal end. The polypeptide backbone can contain from about 10 to about 50 amino acids. According to a further aspect, the method relates to the synthesis of an incretin analog, e.g., an incretin analog selected from a glucagon analog, a GLP-1 analog, a GIP analog, an oxyntomodulin analog, an exendin-4 analog, and a PYY analog. In particular, the method relates to the synthesis of a glucagon analog, e.g., glucagon-like peptide 1 (GLP-1).

[0050] According to yet a further aspect, the method comprises: His-X2-X3-GTFTSDYSKYL-X15-X16-X17-X18-A-X20-DFI-ΔWLESA For the synthesis of a polypeptide selected from the formula: X2 is selected from Aib, Ac3c, Ac4c and Ac5c, preferably Aib and Ac4c; X3 is selected from Gln and His; X15 is selected from Asp and Glu, preferably Asp; X16 is selected from Glu and Ψ, preferably Glu; X17 is selected from Arg and Ψ; X18 is selected from Ala and Arg; X20 is selected from Lys and His, preferably Lys; Ψ is selected from Lys, Arg or Orn; The peptide side chains have the formula -Z 2 -Z 1 and Z 1 has a polar group at one end of the chain and Z 2 a hydrocarbon chain, preferably a fatty acid chain, having a bond to, -X- located at the end of the chain distal from the polar group; the polar group comprises a carboxylic acid or carboxylic acid bioisostere, a phosphonic acid, or a sulfonic acid group; -X- is a bond, -CO-, -SO-, or -SO2; -Z 2 - is a spacer in the following formula:

[0051] [ka] wherein each Y is independently —NH, —NR, —S, or —0, and R is an alkyl, a protecting group, or a spacer Z2 and each X is independently a bond, CO—, SO—, or SO—, with the proviso that when Y is —S, X is a bond, each V is independently a divalent organic moiety that is bonded to Y and X, and n is 1 to 10. According to one aspect, X2 is selected from Aib and Ac4c, X3 is selected from Gln and His, X15 is Asp, X16 is Glu, X17 is selected from Arg and Ψ, X18 is selected from Ala and Arg, and X20 is Lys. Peptide side chain-Z 2 -Z 1 is covalently linked to an amino acid moiety selected from diaminoalkanoic acids containing from 3 to 10 carbon atoms, preferably the diaminoalkanoic acid contains from 4 to 8, preferably from 5 to 8, more preferably from 5 to 7 carbon atoms. Preferably the diaminoalkanoic acid is an (α,ω)-diaminoalkanoic acid, preferably an unbranched diaminoalkanoic acid.

[0052] In some embodiments, Z1 is an acyl group of the formula AB-Alk-(CO)- or a sulfonyl group of the formula AB-Alk-(SO2)-, A is -COOH or a carboxylic acid bioisostere, B is a bond, C6 arylene, or C6 arylene-O-, and Alk is 6 to 18 carbon atoms in length, saturated or unsaturated, optionally fluoro, C 1-4 Alkyl, trifluoromethyl, hydroxymethyl, amino, hydroxyl, C 1-4 a hydrocarbon chain substituted with one or more substituents selected from alkoxy, oxo, and carboxyl; -Z 2 -S A -, -S A -S B -or-S B -S A - and -S A is a single amino acid moiety selected from γ-Glu, α-Glu, α-Asp, β-Asp, Ala, β-Ala (3-aminopropanoic acid), and Gaba (4-aminobutanoic acid); -S B - is a linker of the general formula:

[0053] [ka] (wherein n is 1 to 10, and each Pu is independently Pu i and PU iii is selected from Each Pu i are independently natural or unnatural amino acid moieties, and each PU iii are independently residues of the general formula:

[0054] [ka] (wherein m is 0 to 5, and p is 1, 3, 4, or 5) In some embodiments, m is 1 and p is 1. That is, PU iii is the residue of 8-amino-3,6-dioxaoctanoic acid (also known as {2-[2-aminoethoxy]ethoxy}acetic acid and HN-PEG-COOH). This residue is referred to herein as -PEG-. In some embodiments, m is 2 and p is 1. That is, PU iii is the residue of 11-amino-3,6,9-trioxaundecanoic acid (also known as HN-PEG-COOH). This residue is referred to herein as -PEG-. In some embodiments, -Z 1 contains 6 to 18 carbon atoms and has a polar group and Z at one end of the chain. 2 wherein the polar group is selected from a carboxylic acid or a carboxylic acid bioisostere.

[0055] In a further aspect, -Z 2 -Z 1 teeth, (i) [17-carboxy-heptadecanoyl]-isoGlu-PEG3-PEG3, (ii) [17-carboxy-heptadecanoyl]-isoGlu, (iii) [13-carboxy-tridecanoyl]-isoGlu-PEG3-PEG3, (iv) [carboxyphenoxynonanoyl]-isoGlu-PEG3-PEG3, (v) [13-carboxy-tridecanoyl]-isoGlu-Peg4-Peg4, (vi) [17-carboxy-heptadecanoyl]-PEG3-PEG3-isoGlu, (vii) [17-carboxy-heptadecanoyl]-isoGlu-GSGSGG, and (viii) [17-carboxy-heptadecanoyl]-Ala-Ala-PEG3-PEG3 is selected from.

[0056] In a further aspect, -Z 2 -Z 1 is [17-carboxyheptadecanoyl]-isoGlu-PEG3-PEG3 or [17-carboxy-heptadecanoyl]-isoGlu-GSGSGG. In some embodiments, -Z 2 -Z 1 is [17-carboxyheptadecanoyl]-isoGlu-PEG3-PEG3 or [17-carboxy-heptadecanoyl]-isoGlu-GSGSGG. In a further aspect, -Z 2 -Z 1 is [17-carboxy-heptadecanoyl]-isoGlu-GSGSGG.

[0057] In a further aspect, the polypeptide comprises: His-AibQGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA, His-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA, His-Ac4c-QGTFTSDYSKYLDERRAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA The compound has a sequence selected from the group consisting of:

[0058] According to a further aspect, the polypeptide has the sequence: His-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA or H-His-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2. In a further aspect, the present invention relates to a polypeptide obtainable by the method defined above.

[0059] Example 1 Disclosed herein is SPPS of a polypeptide having the sequence: H-His-Ac4c-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Ala-Ala-Lys-Asp-Phe-Ile-Lys[N-(17-carboxy-heptadecanoyl-γ-Glu-Gly-Ser-Gly-Ser-Gly-Gly-)]-Trp-Leu-Glu-Ser-Ala-NH2. The peptide side chain building blocks are in brackets. The polypeptide backbone contains 29 building blocks, numbered consecutively from 1 to 29, with His being number 1 and Ala being number 29. The polypeptide has a molar mass of 4231.6 g / mol.

[0060] The synthesis was carried out in a 600 L SPPS reactor and a 332 L pre-activation reactor. The synthesis was carried out on a 12.61 mol scale, resulting in 53.4 kg of the target polypeptide in 100% yield. All amino acids were coupled individually, apart from the consecutive Gly-Gly side chains, which were coupled as dipeptides. The peptides were synthesized using an Fmoc-based strategy. This means that the α-amines of all generated amino acid building blocks, i.e., individual natural amino acids, synthetic Ac4c amino acids, and Gly-Gly dipeptides, were protected with the Fmoc group. The one exception was Lys at position 24, which was generated in the form Dde-Lys(Fmoc)-OH, where Dde is the α-amine protecting group and Fmoc is the amino acid side chain protecting group.

[0061] The dicarboxylic acid fatty acid hydrocarbon at the distal end of the peptide side chain (distal to the polypeptide backbone) is tBu (tert-butyl), tBuOOC-C 16 H 32 It was generated by protecting the distal carboxylic acid residue with -COOH. The peptide side chains were assembled prior to coupling of Ile. All reactive amino acid side chain amines were protected with acid-labile protecting groups selected from Boc (tert-butyloxycarbonyl), Trt (trityl), tBu, OtBu, and Pbf (2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl). Thus, an "orthogonal" scheme was applied, in which Fmoc and Dde were removed under alkaline conditions, and all amino acid side chain protecting groups were removed under acidic conditions simultaneously with cleavage of the polypeptide from the resin to form the crude polypeptide.

[0062] List of building blocks: Skeleton: Fmoc-His(Boc)-OH; 1-((Fmoc)amino)cyclobutane-1-carboxylic acid; Fmoc-Gln(Trt)-OH; Fmoc-Gly-OH; Fmoc-Thr(tBu)-OH; Fmoc-Phe-OH; Fmoc-Thr(tBu)-OH; Fmoc-Ser(tBu)-OH; Fmoc-Asp(OtBu)-OH; Fmoc-Tyr(tBu)-OH; Fmoc-Ser(tBu)-OH; Fmoc-Lys(Boc)-OH; Fmoc-Tyr(tBu)-OH; Fmoc-L eu-OH;Fmoc-Asp(OtBu)-OH;Fmoc-Glu(OtBu)-OH;Fmoc-Arg(Pbf)-OH;Fmoc-Ala-OH;Fmoc-Ala-OH;Fmoc-Lys(Boc)-OH;Fmoc-Asp(OtBu)- OH;Fmoc-Phe-OH;Fmoc-Ile-OH;Dde-Lys(Fmoc)-OH;Fmoc-Trp(Boc)-OH;Fmoc-Leu-OH;Fmoc-Glu-OtBu;Fmoc-Ser(tBu)-OH;Fmoc-Ala-OH

[0063] Peptide side chains: Fmoc-Gly-Gly-OH; Fmoc-Ser(tBu)-OH; Fmoc-Gly-OH; Fmoc-Ser(tBu)-OH; Fmoc-Gly-OH; Fmoc-Glu-OtBu; Octadecanedioic acid mono tert-butyl ester Generally, each reaction cycle involved the steps of deprotecting the α-amine of the resin-bound amino acid or peptide, coupling an α-amine-protected amino acid, and acetylating (capping) the unreacted α-amine. Prior to coupling of amino acid 29, Ala, the following scheme was carried out to couple the Ramage linker (Fmoc-RMG-OH) to an aminoethyl-modified polystyrene-based resin (AM resin). 1) 2.5 vol% piperidine in DMF 2) 12.5 vol% piperidine in DMF 3) Wash with DMF 4) Fmoc-RMG-OH in the presence of DIC and Oxyma in DMF 5) Capping substances in the presence of DIC 6) Wash with DMF

[0064] The backbone amino acids 1-22 and 24-29 (excluding amino acid Ile (23)) were coupled to the resin-bound amino acids or peptides by cycles according to the following scheme: 1) 2.5 vol% piperidine in DMF (N,N-dimethylformamide) 2) 12.5 vol% piperidine in DMF 3) Wash with DMF 4) Fmoc-AA-OH (coupling) in the presence of DIC and Oxyma and DMF 5) Acetic acid capping in the presence of DMF (capping) 6) Wash with DMF

[0065] The peptide side chain building blocks were coupled by cycles according to the following scheme. 1) 0.1 vol% DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) in DMF 2) 0.1 vol% DBU in DMF 3) 0.7 vol% DBU in DMF 4) Wash with DMF 5) Fmoc-AA-OH and tBuOOC-C in the presence of DIC, Oxyma, and DMF 16 H 32 -COOH coupling 6) Wash with DMF 7) Capping material acetic acid in DMF 8) Wash with DMF

[0066] The coupling of Ile (23) was carried out according to the following scheme: 1) 5 vol% piperidine in DMF 2) Wash with DMF 3) NH2OH × HCl in the presence of DIPEA (N,N-diisopropylethylamine) and DMF 4) Wash with DMF 5) Repeat steps 1 to 4 6) 10 vol% piperidine in DMF 7) Repeat step 6 twice. 8) Coupling of Fmoc-Ile-OH in the presence of DIC, Oxyma, and DMF 9) Repeat step 8 10) Capping substances in the presence of DIC 11) Wash with DMF

[0067] After coupling of His, the polypeptide-resin was subjected to the following scheme. 1) 2.5 vol% piperidine in DMF 2) 12.5 vol% piperidine in DMF 3) Wash with DMF 4) Wash with IPA (isopropanol) 5) Drying All Fmoc-protected amino acids were preactivated in separate preactivation reactors, typically in the presence of DIC and Oxyma in the presence of DMF for less than 10 minutes before being added to the SPPS reactor.

[0068] All deprotection, preactivation, coupling, and capping procedures related to the backbone polypeptide were carried out at temperatures ranging from 45 to 50 °C, with the exception of the deprotection of the Fmoc α-amine protecting group of Trp prior to coupling of Dde-Lys(Fmoc)-OH, and the deprotection, activation, and coupling of Dde-Lys(Fmoc)-OH, which were carried out at temperatures ranging from 30 to 35 °C. All deprotection, preactivation, coupling, and capping procedures involving peptide side chains were carried out at temperatures between 30 and 35°C. Coupling times were typically 30-50 minutes, and capping was performed for 15-30 minutes. The polypeptide was cleaved from the resin using TFA (trifluoroacetic acid) / scavenger and precipitated with MTBE (ethyl tert-butyl ether). The crude polypeptide was dissolved in aqueous buffer and IPA. The polypeptide was then purified in three separate reversed-phase chromatography (RPC) steps, followed by concentration by ultrafiltration and removal of isopropanol by diafiltration. The final product was isolated by lyophilization.

[0069] The SPPS yield was 93.9 kg of peptide resin (before cleavage). Based on the resin, 208 g of target polypeptide was produced per kg of resin. The quantitative yield was obtained by multiplying 93.9 kg of peptide resin by 0.208 to obtain 19.5 kg of target polypeptide on the resin, i.e., 4.6 mol. The quantitative yield (target polypeptide on the resin) before cleavage was 4.6 / 12.61, approximately 36.5%. After cleavage, 21.2 kg of target polypeptide was obtained, i.e., approximately 5.0 mol, for a yield of 109%. The RPC yield (yield over all three RPC steps) was 65% (13.6 kg / 21.2 kg). After concentration and lyophilization, 12.1 kg of net target polypeptide was obtained (2.86 mol). The total overall yield after concentration and lyophilization (based on a theoretical amount of 53.4 kg of target polypeptide for a synthesis scale of 12.61 mol) was 22.7% (12.1 kg (net target polypeptide corresponding to 2.86 mol) divided by 53.4 kg).

[0070] Example 2 In this example, the activation of the model amino acid Dde-Lys(Fmoc)-OH in the presence of Oxyma and DIC as activators and benzylamine was investigated. If the amino acids are not activated, the carboxylic acids and amines of the two amino acids neutralize rather than condensing to form an amide / peptide bond. Activation is the process of converting an amino acid into a species that readily reacts with amines to form an amide bond, and the reactivity of the activated amino acid must be balanced so that it reacts preferentially with the desired amine. There are many activators. Here, DIC and Oxyma were used to convert amino acids into activated esters. The activated ester of Dde-Lys(Fmoc)-OH reacts readily with benzylamine to liberate Oxyma according to the following scheme.

[0071] [ka] The activated ester of Dde-Lys(Fmoc)-OH is unstable in aqueous mixtures and decomposes during HPLC analysis, but it readily reacts with benzylamine. Therefore, the benzylamide of Dde-Lys(Fmoc)-OH acts as a surrogate for the activated ester of Dde-Lys(Fmoc)-OH.

[0072] Activation of Dde-Lys(Fmoc)-OH in the presence of DIC and Oxyma was carried out at 30°C, 35°C, 40°C, 45°C, 50°C, and 55°C for 0.5, 5, 10, 15, 20, 25, and 30 minutes, respectively, followed by reaction with benzylamine.

[0073] In Table 1, unreacted starting material (SM) refers to Dde-Lys(Fmoc)-OH and activated ester (AE) refers to the benzylamide of Dde-Lys(Fmoc)-OH. [Table 1]

[0074] From Table 1, the following can be derived: - As the temperature increases, the amount of AE increases up to an optimum and then decreases at a given time. - As the temperature increases, the amount of impurities increases from a given time (shown by the grey shading). The percentage of impurities at 30°C and 30 minutes is 1-(0.7416+0.2406)=1.78%. The percentage of impurities at 50°C and 30 minutes is 1-(0.4392+0.1223)=43.85%.

[0075] Example 3 In this example, the peptide Dde-Lys(Fmoc)-Trp-Leu-Glu-Ser-Ala-NH2 was synthesized using the protocol set forth in Example 1. Lys was generated as Dde-Lys(Fmoc)-OH and activated in separate preactivation reactors at 30°C or 35°C for 5, 10, and 15 minutes, respectively. After activation, the solution containing the activated ester of Dde-Lys(Fmoc)-OH was transferred to an SPPS reactor. The activated ester of Dde-Lys(Fmoc)-OH was reacted with the α-amine of tryptophan (Trp) under amide bond formation at the same temperature as during activation, i.e., 30°C or 35°C, for 45 minutes. After 45 minutes, the unreacted α-amine of Trp was capped (acetylated) by adding 2 molar equivalents of capping agent in the presence of 2 molar equivalents of DIC.

[0076] After capping, the peptide was cleaved from the resin with TFA, neutralized (NH4OAc:IPA:HO), and diluted with DMF. After workup, the solution containing the cleaved peptide was analyzed by HPLC. Figures 1-6 show HPLC chromatograms of the workup solution containing the cleaved peptide (Dde-Lys(Fmoc)-Trp-Leu-Glu-Ser-Ala-NH). The eluent was a mixture of purified water (Milli Q) in acetonitrile (ACN) in the presence of 0.1% TFA. The ratio of ACN to Milli Q was 90%-10% initially and 10%-90% at the endpoint, and the flow rate was 1.0 ml / min.

[0077] [Table 2]

[0078] Example 4 In Example 4, the coupling conditions for Dde-Lys(Fmoc)-OH were further investigated. The peptide Trp-Leu-Glu-Ser-Ala was synthesized using the protocols described in Examples 1 and 3. For this purpose, preactivation of Dde-Lys(Fmoc)-OH was performed in separate preactivation reactors. Preactivation was carried out in the presence of DIC and Oxyma in the presence of DMF for 5, 10, 20, and 30 minutes, respectively, before addition to the SPPS reactor for the coupling reaction with Trp-Leu-Glu-Ser-Ala. This reaction cycle, including preactivation and coupling to WLESA, was carried out at room temperature (RT), 35°C, and 55°C. The results are shown in Tables 3, 4, and 5 below.

[0079] [Table 3] The data in Table 3 show the purity of the cleaved samples as determined by HPLC analysis (UV detection at 290 nm). These data indicate that the purity, i.e., the yield, after the coupling of Dde-Lys(Fmoc)-OH is in fact less than 100%. However, the impurities present in the samples are the accumulation of impurities from six couplings of peptide fragments after the coupling of Dde-Lys(Fmoc)-OH. More important than the actual purity of the samples is the fact that only trace amounts (<0.1%) of unreacted peptide (in the form of acetylated peptide or peptide with a free amino group) were detected from this coupling at room temperature.

[0080] A further series of equivalent experiments was performed using 20 and 30 minutes of preactivation at RT, 35°C, and 55°C. The results are shown in Tables 4 and 5 below. At 55°C, the purity (i.e., yield) of the sample rapidly decreased, clearly indicating the appearance of acetylated peptides and peptides with free amino groups. Furthermore, because only tryptophan residues and Fmoc and Dde groups are visible at 290 nm, acetylated peptides and peptides with free amino groups have much lower UV absorption at this wavelength. In contrast to the product containing all three, only tryptophan residues are present in the uncoupled species. The UV absorption of the product was estimated to be four times that of the uncoupled species (acetylated peptides and peptides with free amines), which corresponds to the molar ratios shown in Table 5 below. Therefore, the majority of the amino groups did not react during the coupling of Dde-Lys(Fmoc)-OH. At 35°C and room temperature, not even a trace of unreacted peptide was observed.

[0081] [Table 4]

[0082] [Table 5]

[0083] [Table 6]

Claims

1. 1. A method for synthesizing a polypeptide based on solid phase peptide synthesis, wherein the polypeptide comprises a backbone, the backbone comprising one amino acid moiety Δ selected from diaminoalkanoic acids containing 3 to 10 carbon atoms and one peptide side chain covalently attached to the amino acid moiety Δ, wherein the amino acid moiety Δ is coupled to an amino acid moiety W of the backbone, the method comprising: (i) providing an amino acid building block PROT1-Δ(PROT2)-OH, where PROT1 is an α-amine protecting group and PROT2 is an amino acid side chain protecting group; (ii) performing activation of the amino acid building block PROT1-Δ(PROT2)-OH in the absence of a solid phase at an elevated temperature of about 20° C. to about 50° C. for less than about 25 minutes; (iii) coupling the activated amino acid building block PROT1-Δ(PROT2)-OH to the unprotected α-amine of the amino acid moiety W of the amino acid sequence covalently attached to the solid phase, thereby forming an amide bond; The method comprises at least one reaction cycle comprising:

2. removing PROT2 prior to association of peptide side chains; removing PROT1 after assembly of the peptide side chains and attaching additional amino acids to the backbone; The method of claim 1 further comprising:

3. 3. The method of claim 1 or 2, wherein the amino acid moiety Δ is coupled to the amino acid sequence WLESA.

4. 4. The method of claim 3, wherein A of the amino acid sequence WLESA is covalently attached to a solid phase.

5. 5. The method according to any one of claims 1 to 4, wherein the activation in step (ii) is carried out at a temperature in the range of from about 20°C to about 45°C, preferably from about 20°C to about 40°C, preferably from about 20°C to about 35°C, preferably from about 30°C to about 35°C.

6. The method of any one of claims 1 to 5, wherein the activation in step (ii) is carried out for less than about 22 minutes, preferably less than about 18 minutes.

7. 7. The method of any one of claims 1 to 6, wherein activation is maintained until at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75% of the amino acid building blocks PROT1-Δ(PROT2)-OH are activated.

8. 8. The method according to any one of claims 1 to 7, wherein Δ is selected from unbranched (α,ω)-diaminoalkanoic acids, preferably lysine.

9. 9. The method of any one of claims 1 to 8, wherein PROT1 and PROT2 are selected from protecting groups that are cleaved under alkaline conditions, with the proviso that PROT1 and PROT2 are not cleaved under the same reaction cycle.

10. The method of any one of claims 1 to 9, further comprising selective removal of PROT2.

11. The method of any one of claims 1 to 10, wherein PROT1 is selected from Dde and ivDde, and PROT2 is Fmoc.

12. 11. The method of claim 10, further comprising at least one reaction cycle involving the formation of a peptide side chain attached to the amino acid moiety Δ, wherein the coupling is carried out at a temperature above about 30°C, preferably between about 30°C and about 45°C, preferably between about 30°C and about 35°C.

13. The polypeptide is His-X2-X3-GTFTSDYSKYL-X15-X16-X17-X18-A-X20-DFI-ΔWLESA is selected from: X2 is selected from Aib, Ac3c, Ac4c, and Ac5c, preferably Aib and Ac4c; X3 is selected from Gln and His; X15 is selected from Asp and Glu, preferably Asp; X16 is selected from Glu and Ψ, preferably Glu; X17 is selected from Arg and Ψ; X18 is selected from Ala and Arg; X20 is selected from Lys and His, preferably Lys; Ψ is selected from Lys, Arg, or Orn; The peptide side chain has the formula -Z 2 -Z 1 and Z 1 However, a polar group and Z 2 a hydrocarbon chain having a bond to, -X- located at the end of the chain distal from the polar group; The polar group comprises a carboxylic acid or carboxylic acid bioisostere, a phosphonic acid, or a sulfonic acid group, and -X- is a bond, -CO-, -SO-, or -SO 2 and -Z 2 - is the spacer in the following formula 【Chemistry 1】 wherein each Y is independently —NH, —NR, —S, or —0, and R is an alkyl, a protecting group, or a spacer Z 2 and each X independently represents a bond, CO—, SO—, or SO 2 - with the proviso that when Y is -S, X is a bond, each V is independently a divalent organic moiety attached to Y and X, and n is 1 to 10.

14. The polypeptide is His-AibQGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA, His-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA, and His-Ac4c-QGTFTSDYSKYLDERRAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA The method according to any one of claims 1 to 13, wherein the compound is selected from the group consisting of:

15. The method of any one of claims 1 to 13, wherein the polypeptide is His-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA.

16. The polypeptide is H-His-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH 2 16. The method of claim 15, wherein:

17. The method of any one of claims 1 to 16, wherein the polypeptide is a glucagon analogue.

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