Method for synthesizing polypeptide

Abstract

The present invention relates to a method for synthesizing a polypeptide based on a solid phase peptide synthesis process, said polypeptide comprising a main chain comprising an amino acid moiety Delta selected from diaminoalkanoic acids containing from 3 to up to 10 carbon atoms and a peptide side chain covalently linked to said amino acid moiety Delta, the amino acid moiety delta is coupled to the amino acid moiety W of the backbone, 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 specified temperature and time period, and coupling the amino acid building block to the backbone.

Description

Technical Field

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

[0002] Solid phase peptide synthesis (SPPS) of polypeptides, pioneered by Bruce Merrifield (Merrifield, 1963), involves synthesizing a polypeptide bound to an insoluble solid phase and subsequently isolating 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 via the formation of amide bonds (commonly known as peptide bonds). The use of a solid phase enables the use of excess reagents and the removal of reagents and other reaction promoters by convenient filtration.

[0003] To obtain a high final (overall) yield of a polypeptide, it is desirable that the (local) yield of each coupling be as high as possible. It is preferred that the peptide bond formation in each reaction cycle be driven to completion. As the number of amino acids in a polypeptide increases, the importance of achieving high local yields for each coupling becomes increasingly important.

[0004] The peptide synthesis strategy of the present invention involves preparing peptides including at least one amino acid having an amino acid side chain containing a functional group (branching-inducing amino acid), such as lysine and lysine analogs, which can serve as a site for peptide side chain formation.

[0005] One embodiment of the present invention relates to synthetic branched polypeptides, particularly polypeptides comprising peptide side chains (such as glucagon analogs). Peptide side chains can be formed by applying several strategies. When the polypeptide is attached to the resin and by continuous coupling of the relevant side chain amino acid moieties, peptide side chains can be formed. Alternatively, the entire peptide side chain or a portion of the peptide side chain can be coupled to the relevant amino acid moieties of the resin-bound polypeptide. In addition, the peptide side chains can be assembled after the branched amino acid coupling but before the next main chain amino acid coupling. Alternatively, the peptide side chains are synthesized after the polypeptide main chain is completed. In the context of the present invention, after completing the synthesis of the peptide side chains (either by continuous coupling of a single side chain amino acid moiety or by coupling part or the entire peptide side chain), the next amino acid coupling of the polypeptide main chain is performed. The amino acid as the branching site must include at least three reactive groups, which include groups for covalently linking the peptide side chains. In the context of the present invention, a branching site is an amino acid building block defined as PROT1-Δ(PROT2)-OH, wherein Δ is selected from a diaminoalkanoic acid comprising 3 to a maximum of 10 carbon atoms, PROT1 constitutes an α-amine protecting group, and PROT2 constitutes an amino acid side chain protecting group. If the peptide side chain is synthesized after the branching amino acid has been coupled but before the next amino acid in the polypeptide backbone is coupled, 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 group present in the polypeptide attached to the resin (class (II) protecting groups). Therefore, the degree of freedom in choosing PROT1 and PROT2 depends on the reaction scheme for synthesizing the polypeptide side chain.

[0006] The present invention seeks to optimize / increase the local (but also overall) yield of coupling of amino acids serving as branching sites for the synthesis of polypeptides comprising a backbone and peptide side chains (e.g., analogs of glucagon) using an SPPS protocol in which the peptide side chains are assembled prior to forming the complete target polypeptide backbone (i.e., the complete target polypeptide).

[0007] WO 2018069295 discloses a method for preparing peptides comprising lipophilically modified lysine side chains. More specifically, the method relates to a protocol for removing trityl-based side chain protecting groups of lysine after formation of the target polypeptide backbone but before cleavage from the resin.

[0008] WO 2019120639 relates to the synthesis of a polypeptide containing lysine or a lysine derivative with an allyloxycarbonyl N-terminal protection. The synthesis comprises acylation of the lysine side chain, removal of the allyloxycarbonyl group, and coupling of an amino acid or peptide to the N-terminus of lysine.

[0009] CN 106478806 proposes a method for synthesizing semaglutide. Lys is provided in the form of Dde-Lys(Fmoc)-OH. CN 106478806 does not mention the temperature during the coupling cycle.

[0010] Objectives of the present invention

[0011] It is an object of the present invention to increase the overall yield of polypeptides synthesized using SPPS.

[0012] An additional goal was to increase the overall yield of branched polypeptides having more than 10 amino acids in the backbone that were synthesized using SPPS.

[0013] Another goal was to increase the yield of couplings of lysine or lysine analogs, in particular the coupling of lysine to main-chain tryptophan.

[0014] A further goal was to increase the local (but also overall) yield of coupling of amino acids serving as branching sites for the synthesis of polypeptides comprising a backbone and peptide side chains (such as glucagon analogs) using an SPPS protocol in which the peptide side chains are assembled prior to forming the target polypeptide backbone.

[0015] A further aim was to provide improved conditions for the coupling of amino acids (as branching sites) to tryptophan residues.

[0016] Another goal was to establish optimal conditions for coupling side chains to branch point amino acids such as lysine. Summary of the Invention

[0017] The present invention relates to a method for synthesizing a polypeptide based on solid-phase peptide synthesis, wherein the polypeptide comprises a backbone comprising an amino acid moiety Δ selected from diaminoalkanoic acids containing 3 to a maximum of 10 carbon atoms and a peptide side chain covalently linked to the amino acid moiety Δ, wherein the amino acid moiety Δ is coupled to the amino acid moiety W, the method comprising at least one reaction cycle comprising:

[0018] (i) providing an amino acid building block PROT1-Δ(PROT2)-OH, PROT1 being an α-amine protecting group, and PROT2 being an amino acid side chain protecting group;

[0019] (ii) activating the amino acid building block PROT1-Δ(PROT2)-OH at an elevated temperature of from about 20° C. up to about 50° C. for less than about 25 minutes in the absence of said solid phase, and;

[0020] (iii) coupling the activated amino acid building block PROT1-Δ(PROT2)-OH to the unprotected α-amine of the amino acid part W of the peptide covalently linked to the solid phase, thereby forming an amide bond.

[0021] definition

[0022] The solid phase peptide synthesis (SPPS) reaction cycle includes the following procedures: deprotecting the protecting group bound to the α-amine of the peptide or amino acid covalently linked to the solid phase, and coupling the carboxylic acid functional group of the α-amine protected amino acid portion to the (free or deprotected) α-amine of the peptide or amino acid bound to the solid support, thereby forming an amide bond. Before coupling, the amino acid needs to be activated. In addition, the reaction cycle may also include blocking (referred to as blocking in this application) the (unreacted, deprotected) α-amine of the peptide or amino acid bound to the solid phase that has not formed an amide bond. The procedure can be repeated in one reaction cycle, for example, the reaction cycle may include multiple coupling and blocking procedures. The activities of removing compounds from the reaction solution are collectively referred to as washing. The procedures of the reaction cycle may also be referred to as steps or stages. The process, step or phase can be characterized by any of the following non-exhaustive processes (steps, phases): the use of one or more important compounds (examples of important compounds: activating agents, blocking agents, deprotecting agents, activated amino acid building blocks), activities between two washes, reaction processes (e.g., activation of amino acid building blocks, formation of amide bonds, cleavage of covalent bonds). Several processes can also be performed in parallel, for example, activation and coupling can occur simultaneously. Thus, in the context of the present invention, a reaction cycle can include multiple processes (steps, phases), wherein the important processes (steps, phases) are deprotection, activation, coupling and optional blocking.

[0023] Activation in the context of the present invention is the situation starting from the situation where the α-amine protected amino acid building block and at least one activating agent are present in solution until the formation of the amide bond.

[0024] Activation and activated amino acid building blocks: Carboxylic acids and amines are less likely to participate in condensation reactions to form amide bonds, but rather neutralize to form carboxylate groups and ammonium ions. Broadly speaking, activation (or activation procedure) allows carboxylic acids and amines to form amide bonds. Activation can involve activating the carboxylic acid functional group of the amino acid moiety through the participation of at least one activating reagent (also known as a coupling reagent). The carboxylic acid functional group is typically activated by reaction with an electron-withdrawing reagent. The activated carboxylic acid functional group of the 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 that includes a carboxylic acid / carboxylate group 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.

[0025] In the context of the present invention, amino acid building blocks are amino acids wherein the α-amine as well as optionally reactive groups of the amino acid side chain are protected by suitable protecting groups.

[0026] The term "amino acid moiety" is used to denote the amino acids present in a peptide / polypeptide.

[0027] Activators are molecules that activate amino acid building blocks.

[0028] Capping is the chemical modification of any remaining unreacted α-amine after the coupling step into an entity that is incapable of chemically reacting in the downstream coupling cycle. Capping typically involves acylation or acetylation of the α-amine. Acetic anhydride can be used as the capping agent.

[0029] Amino acid moieties and amino acid building blocks include naturally occurring amino acids and any chemically modified naturally occurring amino acids as well as any synthetic molecules that include functional groups, particularly amine and carboxylic acid functional groups, that enable their use in SPPS and provide amide bonds.

[0030] A polypeptide comprises at least two amino acids up to any number of amino acids, wherein the molecule is called a protein. Human proteins have approximately 200 amino acids.

[0031] In the context of the present invention, the term polypeptide is used to refer to the branched polypeptide formed by the method disclosed in the present application. In addition, the main chain of the branched polypeptide is called polypeptide.

[0032] The term peptide is used in conjunction with the side chains of polypeptides. The peptide side chains have at least two amino acid moieties up to any number of amino acid moieties less than the number of amino acid moieties of the polypeptide backbone. Thus, peptide side chains are side chains of branched polypeptides. The peptide side chains may also include hydrocarbon moieties. Typically, the hydrocarbon moieties include from about 10 to up to about 25 carbon atoms, suitably from about 14 to up to 20 carbon atoms.

[0033] An amino acid side chain is the side chain of an amino acid moiety. The amino acid side chain is attached to the α-carbon. Lysine is an example of an amino acid that includes a side chain.

[0034] Diaminopropionic acid contains 3 carbon atoms, while diaminodecanoic acid contains 10 carbon atoms.

[0035] Acid labile protecting groups are protecting groups that are cleaved under acid conditions.

[0036] A base labile protecting group is a protecting group that is cleaved under basic conditions.

[0037] Class (I) protecting groups refer to protecting groups that are attached to reactive groups in the amino acid building blocks that do not participate in amide bond formation, and do not include PROT2 protecting groups. Thus, Class (I) protecting groups are preferably protecting groups that are covalently attached to reactive groups in the side chains of the amino acid building blocks. Examples of reactive groups in the side chains of the amino acid moiety are amines, carboxylic acids, alcohols, thiols, and thioethers.

[0038] Class (II) protecting groups refer to protecting groups attached to amines (α-amines) in amino acid building blocks that do not participate in amide formation and do not include PROT1 protecting groups.

[0039] The terminology of class (I) and class (II) protecting groups is introduced to distinguish these protecting groups from the groups PROT1 and PROT2 of the amino acid building block of the formula PROT1-Δ(PROT2)-OH.

[0040] Δ is an amino acid moiety selected from diaminoalkanoic acids containing 3 up to 10 carbon atoms.

[0041] PROT1 is an α-amine protecting group.

[0042] PROT2 is an amino acid side chain protecting group.

[0043] Coupling refers to the formation of an amide bond (peptide bond).

[0044] WLESA represents the peptide fragment Trp-Leu-Glu-Ser-Ala.

[0045] The term "about" is used for several parameters disclosed in this application, such as temperature, time and yield. If not otherwise indicated, the term "about" should be interpreted as the relevant parameter can deviate by up to 5%.

[0046] Amino acid side chains (e.g., delta side chains) are side chains of amino acid moieties / amino acid building blocks. Lysine is an example of an amino acid moiety / amino acid building block having an amino acid side chain. Peptide side chains (i.e., side chains of polypeptides (peptide side chains)) are side chains covalently attached to a backbone polypeptide. Such side chains may include amino acid moieties and / or fatty acid moieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] all Figures 1 to 6 Each depicts an HPLC profile of a work-up solution comprising the cleaved peptide (Dde-Lys(Fmoc)-Trp-Leu-Glu-Ser-Ala-NH2) synthesized as described in Example 3. The variables are the duration of preactivation (5, 10, 15 min) and the temperature (30°C and 35°C). The temperature was kept constant during preactivation and coupling.

[0048] Figure 1: 30°C, 5 min preactivation, 45 min coupling.

[0049] Figure 2 : 30°C, 10 min preactivation, 45 min coupling.

[0050] Figure 3 : 30°C, 15 min preactivation, 45 min coupling.

[0051] Figure 4 : 35°C, 5 min preactivation, 45 min coupling.

[0052] Figure 5 : 35°C, 10 min preactivation, 45 min coupling.

[0053] Figure 6 : 35°C, 15 min preactivation, 45 min coupling. DETAILED DESCRIPTION

[0054] The method is based on SPPS comprising at least one reaction cycle (reaction cycle A) wherein an amino acid building block selected from PROT1-Δ(PROT2)-OH is coupled to an amino acid moiety W of a peptide covalently bound to a solid phase.

[0055] Δ is selected from diaminoalkanoic acids containing 3 up to 10 carbon atoms.

[0056] PROT1 is an α-amine protecting group.

[0057] PROT2 is an amino acid side chain protecting group.

[0058] According to one aspect, the diaminoalkanoic acid is preferably an unbranched diaminoalkanoic acid. Unbranched in the context of diaminoalkanoic acid refers to a side chain.

[0059] According to another aspect, the diaminoalkanoic acid is selected from (α,ω)-diaminoalkanoic acids, preferably unbranched diaminoalkanoic acids, wherein ω represents the amine on the ω carbon distal to the amino acid side chain and is the second amine counted from the α carbon.

[0060] According to one aspect, the diaminoalkanoic acid comprises from 4 up to 8 carbon atoms, suitably from 5 up to 8 carbon atoms, more preferably from 5 up to 7 carbon atoms.

[0061] According to another aspect, the diaminoalkanoic acid is selected from ornithine (2,5-diaminopentanoic acid) and lysine (2,6-diaminohexanoic acid), preferably lysine.

[0062] PROT1 and PROT2 are α-amine and amino acid side chain protecting groups, respectively, and are preferably selected from protecting groups that are cleaved under alkaline conditions, with the proviso that only one of PROT1 and PROT2 is cleaved in the same reaction cycle.

[0063] PROT1 and PROT2 can be selected from: Fmoc (fluorenylmethoxycarbonyl), Nsc (2-(4-nitrophenylsulfonyl)ethoxycarbonyl), Bsmoc (1,1-dioxobenzo[b]thiophen-2-ylmethoxycarbonyl), α-Nsmoc ((1,1-dioxonaphtho[1,2-b]thiophen-2-yl)methoxycarbonyl), Dde (N-[1-(4,4-dimethyl-2,6-dioxocyclohexylene-1-yl)ethyl]), ivDde ( 1-(4,4-dimethyl-2,6-dioxocyclohexan-1-ylidene)-3-methylbutyl, 2,7-di-tert-butyl-Fmoc, 2-fluoro-Fmoc, 2-monoisooctyl-Fmoc, 2,7-diisooctyl-Fmoc, TCP (tetrachlorophthaloyl), Pms (2-phenyl(methyl)sulfonium)ethoxycarbonyl tetrafluoroborate), Esc (ethanesulfonylethoxycarbonyl), and Sps (2-(4-sulfophenylsulfonyl)ethoxycarbonyl).

[0064] PROT1 is preferably selected from Dde and ivDde. PROT1 is suitably Dde. PROT2 is preferably Fmoc.

[0065] The present invention relates to the synthesis of a polypeptide comprising a main chain and a peptide side chain, which can be referred to as a branched polypeptide. The main chain comprises an amino acid moiety and an amino acid moiety Δ selected from diaminoalkanoic acids containing 3 to up to 10 carbon atoms, Δ coupled to W. The peptide side chain is covalently linked to the amino acid moiety Δ of the main chain. The main chain comprises only one amino acid moiety Δ, which is formed by an amino acid building block selected from PROT1-Δ(PROT2)-OH, which serves as a site for the construction / synthesis / formation of the peptide side chain. The main chain can include additional amino acids selected from diaminoalkanoic acids containing 3 to up to 10 carbon atoms, such as Lys.

[0066] W as well as WLESA and ΔWLESA form part of the peptide backbone.

[0067] According to one aspect, the polypeptide comprises one and only one peptide side chain covalently linked to an amino acid moiety as defined by the method of the invention (ie amino acid moiety Δ).

[0068] According to one aspect, PROT2 and class (I) protecting groups are selected from protecting groups that are cleaved simultaneously under the same reaction cycle. PROT2 and class (I) protecting groups can be selected from base-labile protecting groups, such as Fmoc. Preferably, class (II) protecting groups (which can be referred to as amino acid side chain protecting groups, such as amines) are selected for reactive groups in the amino acid building blocks that do not participate in amide bond formation, and class (I) protecting groups (α-amine protecting groups) are selected for protecting amines (α-amines) that participate in amide formation in the amino acid building blocks, such that only one class of protecting groups is cleaved during the same reaction cycle. Class (I) protecting groups can be easily cleaved under alkaline conditions (alkaline / base-labile protecting groups), while class (II) protecting groups can be easily cleaved under acidic conditions (acid-labile protecting groups).

[0069] PROT1 and PROT2 are suitably selected so that during the same reaction cycle, only one of PROT1 or PROT2 is cleaved. In addition, PROT2 is preferably selected from the following protecting groups, which are cleaved while no other type (II) protecting groups are cleaved during the same reaction cycle.

[0070] According to one aspect, the class (II) protecting groups are acid labile, whereas the class (I) protecting groups are base labile. This protection scheme is also known as an orthogonal protection scheme.

[0071] If the method involves a polypeptide formed from amino acid building blocks comprising a reactive group not participating in amide bond formation protected by a Class (II) protecting group, said Class (II) protecting group being acid labile, PROT1 and PROT2 are suitably base labile, indicating that the protecting group is cleaved under basic conditions.

[0072] According to one aspect, the method further comprises removing PROT2 before peptide side chain assembly; and removing PROT1 after peptide side chain assembly and attaching additional amino acids to the backbone.

[0073] According to another aspect, the amino acid portion Δ is coupled to the amino acid sequence WLESA.

[0074] According to yet another aspect, the amino acid portion A of the amino acid sequence WLESA is covalently bound to a solid phase.

[0075] When it is stated that an amino acid sequence is covalently linked to a solid phase, it is to be understood that the amino acid sequence may be linked to the solid phase via a suitable linker.

[0076] According to one aspect, the activation of step (ii) is carried out at a temperature ranging from about 20°C up to about 45°C, preferably from about 20°C up to about 40°C, preferably from about 30°C up to about 35°C.

[0077] According to one aspect, the activation of step (ii) is performed for less than about 22 minutes, preferably less than about 18 minutes.

[0078] According to one aspect, the 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 block PROT1-Δ(PROT2)-OH is activated.

[0079] According to one aspect, Δ is selected from unbranched (α,ω)-diaminoalkanoic acids, suitably ornithine and lysine, preferably lysine.

[0080] According to one aspect, PROT1 and PROT2 are selected from protecting groups that are cleaved under alkaline conditions, with the proviso that PROT1 and PROT2 are not cleaved in the same reaction cycle.

[0081] According to one aspect, the method further comprises selectively removing PROT2.

[0082] According to one aspect, PROT1 is selected from Dde and ivDde, suitably, Dde and PROT2 are Fmoc.

[0083] According to one aspect, the method further comprises at least one reaction cycle for the formation of a peptide side chain Δ-linked to the amino acid moiety, wherein the coupling is carried out at a temperature of above about 30°C, preferably from about 30°C to a maximum of about 45°C, preferably from about 30°C to a maximum of about 35°C.

[0084] According to yet another aspect, the polypeptide is selected from:

[0085] His-X2-X3-GTFTSDYSKYL-X15-X16-X17-X18-A-X20-DFI-ΔWLESA

[0086] in:

[0087] X2 is selected from Aib, Ac3c, Ac4c and Ac5c; preferably Aib and Ac4c;

[0088] X3 is selected from Gln and His;

[0089] X15 is selected from Asp and Glu, preferably Asp;

[0090] X16 is selected from Glu and Ψ, preferably Glu;

[0091] X17 is selected from Arg and Ψ;

[0092] X18 is selected from Ala and Arg;

[0093] X20 is selected from Lys and His, preferably Lys;

[0094] Ψ is selected from Lys, Arg or Orn;

[0095] And the peptide side chain has the formula -Z 2 -Z 1 ;

[0096] Z 1 is a hydrocarbon chain having a polar group at one end of the chain and a Z-containing group at the end of the chain away from the polar group. 2 The connecting part -X-.

[0097] wherein the polar group comprises a carboxylic acid or a carboxylic acid bioisostere, a phosphonic acid or a sulfonic acid group; and -X- is a bond, -CO-, -SO- or -SO2;

[0098] -Z 2 - is a spacer having the formula:

[0099]

[0100] Wherein: each Y is independently -NH, -NR, -S or -O, wherein R is an alkyl group, a protecting group or a spacer Z 2 each X is independently a bond, CO-, SO- or SO2-; with the proviso that when Y is -S, X is a bond; each V is independently a divalent organic portion connecting Y and X; and n is 1 to 10.

[0101] 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, thereby forming an activated α-amine protected amino acid building block, and coupling the activated α-amine protected amino acid building block to an unprotected α-amine of a peptide or amino acid covalently attached to a solid phase, thereby forming an amide bond, wherein the activation is at an elevated temperature of about 20° C. up to about 50° C. for less than 25 minutes.

[0102] Activation can be accomplished in the presence of a solid phase, i.e., in the reaction vessel in which the polypeptide is formed and attached to the solid phase (polymer solid phase). Activation in the presence of the solid phase is generally referred to as in situ activation. An alternative is to initiate activation in the absence of the solid phase. Activation in the absence of the solid phase and, presumably, the absence of the nascent polypeptide attached to the solid phase is preferably performed in an activation vessel that is different from the reaction vessel in which the polypeptide is formed. In order to establish an amide bond (peptide bond) between the α-amine-protected amino acid building block and the nascent peptide bound to the solid phase, the α-amine-protected amino acid building block must be activated. Activation is initiated by providing (adding) at least one activating agent (also referred to as a coupling agent in the field of SPPS) and the α-amine-protected amino acid building block, and is typically continued 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 agent and solvent composition. Activated α-amine-protected amino acid building blocks can be stable for up to several hours, or, under appropriate conditions, for up to several days, indicating that the activated α-amine-protected amino acid building block can be activated from the activation state until the amide bond is formed. Thus, if the α-amine protected amino acid building block is activated in the absence of a peptide solid phase, it remains in its activated form after transfer to the peptide reaction vessel until the amide bond is formed. According to one aspect, the activation is initiated in the absence of a solid phase, preferably in a solution comprising at least one activating agent, and preferably in an activation vessel (rather than a reaction vessel comprising a solid phase). According to another aspect, the activation is performed by applying two activating agents.

[0103] The activation of the amino acid building block PROT1-Δ(PROT2)-OH in the absence of a solid phase is less than about 25 minutes, less than 22 minutes, less than 21 minutes, less than about 20 minutes, less than about 18 minutes, less than about 15 minutes. Activation can be performed for about 1 minute, about 2 minutes, about 5 minutes up to about 25 minutes, up to about 20 minutes, up to about 18 minutes, up to about 15 minutes. Any of the indicated upper and lower time limits are combinable with each other. The duration of the activation is determined, to some extent, by the temperature during activation.

[0104] According to another aspect, activation is suitably maintained in the absence of a solid phase 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) is formed.

[0105] Preferably, the α-amine protected amino acid building blocks, e.g., PROT1-Δ(PROT2)-OH, are loaded at a molar ratio of greater than about 1.5, suitably greater than about 2.0, based on the loading of the solid phase, which is the theoretical number of amines available for amide / peptide bond formation.

[0106] The activator is preferably loaded in a molar amount equal to or similar to the molar amount of the α-amine protected amino acid building block. The activator consumed during the activation process (e.g., DIC) can be loaded at a ratio higher than the total molar ratio of the relevant α-amine protected amino acid building block.

[0107] Activation in the context of the present invention is the time from mixing the α-amine protected amino acid building block with at least one activating agent in the absence of a solid phase until the point at which the amide bond is formed, corresponding to adding the solution comprising the activated amino acid building block to the solution comprising the solid phase.

[0108] The α-amine protected amino acid building block activated in the absence of a solid phase is transferred from the activation vessel to the polypeptide reaction vessel. Once the activated α-amine protected amino acid building block is transferred to the peptide reaction vessel containing the peptide or amino acid on the solid phase, coupling begins.

[0109] Activation (ie, formation of amino acid ester residues) can proceed as long as the relevant activating agent is present. Thus, activation can also occur after transferring the solution comprising the activated amino acid residues from the activation vessel to the peptide reaction vessel.

[0110] Coupling refers to the formation of an amide bond (peptide bond).

[0111] The rate of amide bond formation decreases over time and is related in part to the amount (molar ratio) of activated α-amine protected amino acid building blocks compared to the available amines of the peptide bound to the solid phase.

[0112] The activating agent may be selected from the group consisting of diisopropylcarbodiimide (DIC), ethyl cyano(hydroxyimino)acetate (Oxyma, sometimes also referred to as OxymaPure), dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCxHCl) (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-dicarboximide (HONB), tetramethylbenzotriazole hexafluorophosphate (HBTU), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyl tetrafluoroborate [O-[N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)hexafluorophosphate] ](TBTU), O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyl (HOTU), O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyl tetrafluoroborate (TOTU), O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyl hexafluorophosphate (HCTU), O-(6-chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyl tetrafluoroborate (TCTU), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, tetramethyl azabenzotriazole hexafluorophosphate (HATU), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyl tetrafluoroborate (TATU), 1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholinyl-carbonium hexafluorophosphate (COMU), 1-[(dimethylamino)(morpholinyl)methylene]-1H-[1,2,3]triazolo[4,5-b]pyridin-1-ium 3-oxide hexafluorophosphate (HDMA), HDMB, 6-chloro-1-((dimethylamino)(morpholinyl)-methylene)-1H-benzotriazolium (HDMC), benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), (benzotriazol-1-yloxy)tripyrrolidinylphosphonium hexafluorophosphate (PyBOP), [ethylcyano(hydroxyimino)acetate-O] 2 ]tris-1-pyrrolidinylphosphonium (PyOxim), 6-chloro-benzotriazol-1-yloxy-tris-pyrrolidinylphosphonium hexafluorophosphate (PyClock), N,N,N',N'-tetramethylchloroformamidinium hexafluorophosphate (TCFH), N-(chloro(morpholinyl)methylene)-N-methylmethylammonium hexafluorophosphate (DMCH), chlorotripyrrolidinylphosphonium hexafluorophosphate (Pyclop), tetramethylfluoroformamidinium hexafluorophosphate (TFFH), tetramethylammonium trifluoromethanethiol ((Me4N)SCF3), 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 (DMTMMBF4) and 2-(4,6-dimethoxy-1,3,5-triazinyl)trialkylammonium salts (DMT-Ams).

[0113] According to one aspect, activation is achieved by DIC and Oxyma, for example by placing DIC and Oxyma together with the relevant α-amine protected amino acid building block in a suitable solvent.

[0114] Suitable solvents for SPPS in general for any sequence of the reaction cycle are 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-dimethyl-2-imidazolidinone (DMI), 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 (dihydro-levulinone, ), N,N-dimethylacetamide (DMA), N,N,N',N'-tetraethylsulfonamide (TES), 1-butyl-3-methylimidazolium chloride (BMIMCl), 1-butyl-3-methylimidazolium bromide (BMIMBr) and 1-butyl-3-methylimidazolium iodide (BMIMI).

[0115] Activation is carried out (conducted) at a temperature of about 20°C to a maximum of about 50°C. As described above, activation is a procedure that begins by bringing together at least one activating agent and an α-amine protected amino acid building block. The activation process provides an activated α-amine protected amino acid building block with a certain lifetime. The activated α-amine protected amino acid building block reacts with an amine to form an amide (peptide) bond. When activation is carried out in the absence of a peptide / amino acid bound to a solid phase, amide bond formation begins when the activated α-amine protected amino acid building block is added to a suspension comprising a solid phase. If activation is initiated in the presence of a solid phase (in situ activation), amide bond formation can begin rapidly while the α-amine protected amino acid building block is still being activated.

[0116] In the context of activation according to the present invention and for the reasons explained herein, activation can also be achieved when the activated amino acid building block participates in amide bond formation (coupling). The amino acid building block can be activated even after the solution comprising the activated amino acid building block is transferred to the reaction vessel comprising the solid phase. The temperature of activation is preferably also the temperature of coupling or at least the temperature during coupling, as long as an effective concentration of the activated α-amine protected amino acid building block is present in the reaction solution.

[0117] According to another aspect, the activation is carried out at a temperature of about 20°C up to about 45°C, preferably about 20°C up to about 40°C, about 30°C up to about 37°C, such as about 30°C up to about 35°C.

[0118] The lower limit of the activation temperature may be 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., or 30° C., and the upper limit of the activation temperature 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. The lower limit temperature and the upper limit temperature may each be combined to present a certain temperature range.

[0119] According to one aspect, activation and coupling are performed at the same temperature.

[0120] Regardless of whether activation is initiated in the presence or absence of a solid phase, it is preferred that the activation (i.e., the time period 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.

[0121] The duration of activation of 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, less than 15 minutes. Activation can be performed from about 1 minute, about 2 minutes, about 5 minutes up to about 25 minutes, up to about 20 minutes, up to about 18 minutes, up to about 15 minutes.

[0122] According to one aspect, the activation of the amino acid building block PROT1-Δ(PROT2)-OH and the coupling are carried out at a temperature of from about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C up 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, the activation suitably being initiated in the absence of a solid phase in the activation vessel, the activation being carried out 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 in a temperature range of from about 1 minute, about 2 minutes, about 5 minutes up to about 25 minutes, up to about 20 minutes, up to about 18 minutes, up to about 15 minutes.

[0123] If activation is initiated in an activation vessel comprising a solution comprising at least one activating agent and an α-amine protected amino acid building block (in the absence of a solid phase), said solution is subsequently transferred to a solution comprising a solid phase, i.e., to a polypeptide reaction vessel comprising a solid phase.

[0124] The reaction cycle may further comprise capping of unreacted, unprotected α-amines of the peptide or amino acid bound to the solid phase.

[0125] According to one aspect, the capping comprises adding a capping composition comprising a capping agent and an additional activating agent (such as DIC). Preferably, the capping is initiated by adding the capping composition to the coupling solution.

[0126] Each reaction cycle involves cleavage of the α-amine protecting group of the peptide or amino acid attached to the solid phase prior to coupling.

[0127] It is contemplated that the methods of the present invention can be practiced to synthesize a variety of polypeptides selected from analogs of glucagon or glucagon-like peptide-1.

[0128] Any polypeptide disclosed herein that can be synthesized by the disclosed methods is preferably synthesized using amino acid building blocks as defined herein, including naturally occurring amino acids and any chemically modified naturally occurring amino acids, as well as any synthetic molecules that include functional groups (particularly amines and carboxylic acids) that enable the synthetic molecules to be used in SPPS and provide amide bonds. Thus, amino acid building blocks can be added to a polypeptide, whether the backbone of the polypeptide of interest or any peptide side chain attached to the backbone of the polypeptide of interest, by the reaction cycles disclosed herein.

[0129] Polypeptides can be formed by a strategy involving the formation of several peptide fragments which are subsequently coupled. Polypeptides comprising at least one peptide side chain can be formed by synthesizing at least a portion of the peptide side chain by SPPS and coupling the fragment to the amino acid portion of the backbone attached to a solid phase.

[0130] According to one aspect, the method involves a polypeptide selected from polypeptides comprising one peptide side chain, in particular peptides comprising one and only one peptide side chain.

[0131] According to another aspect, the entire peptide side chain of the polypeptide is synthesized by a continuous reaction cycle that is performed after coupling of the amino acid building block that serves as the site for covalent attachment of the peptide side chain (forming part of the backbone and typically the amino acid portion Δ) and before coupling of the next amino acid building block of the polypeptide backbone (backbone / main chain). If the peptide side chain is synthesized before the synthesis of the backbone is complete, PROT1 and PROT2 of the amino acid building block PROT1-Δ(PROT2)-OH should preferably be selected so that PROT2 can be selectively deprotected relative to PROT1 and any other protecting groups (class (II) protecting groups) present on the amino acid side chains of the amino acids between the solid phase and the amino acid serving as the branch point. Thus, PROT2 is selected so that only PROT2 is deprotected during the deprotection procedure.

[0132] According to another aspect, the method implements an SPPS strategy, wherein the class (II) protecting group is acid labile, meaning that the class (II) protecting group will cleave under acidic conditions, and the class (I) protecting group is base labile, meaning that it will cleave under alkaline conditions. If the class (II) protecting group is selected from acid labile protecting groups, it is preferred that PROT1 and PROT2 are selected from alkaline / base labile protecting groups, preferably with the constraint that PROT1 and PROT2 are selected such that only PROT2 is cleaved in a particular reaction cycle (which includes a deprotection procedure).

[0133] According to one other aspect, the class (I) protecting group is Fmoc.

[0134] The peptide side chain may have the formula -Z 2 -Z 1 ; where -Z 1 is a fatty acid chain having a polar group at one end of the chain and a Z at the end of the chain remote from the polar group. 2 The polar group comprises a carboxylic acid or a carboxylic acid bioisostere, a phosphonic acid or a sulfonic acid group; and -X- is a bond, -CO-, -SO- or -SO2; -Z 2 - is a spacer having the formula:

[0135]

[0136] wherein each Y is independently -NH, -NR, -S or -O, wherein R is an alkyl group, a protecting group or a spacer Z 2 each X is independently a bond, CO-, SO- or SO2-; with the proviso that when Y is -S, X is a bond; each V is independently a divalent organic portion connecting Y and X; and n is 1 to 10.

[0137] According to one aspect, -Z 1 It can also be an acyl group of the formula AB-Alk-(CO)-; or a sulfonyl group of the formula AB-Alk-(SO2)-; A is a carboxylic acid or a carboxylic acid bioisostere, preferably a carboxylic acid; B is a bond, a C6 arylene group, or a C6 arylene-O-;

[0138] Alk is a saturated or unsaturated fatty acid chain comprising 6 to 18 carbon atoms, preferably unbranched, optionally substituted with fluorine, C 1-4 Alkyl, trifluoromethyl, hydroxymethyl, amino, hydroxy, C 1-4 One or more of alkoxy, oxo and carboxyl; -Z 2 Yes-S A -、-S A -S B -or-S B -S A -;-S A is a single amino acid residue selected from the group consisting of γ-Glu, α-Glu, α-Asp, β-Asp, Ala, β-Ala (3-aminopropionic acid (Dap)) and Gaba (4-aminobutyric acid);

[0139] -S B - is a linker having the following general formula:

[0140]

[0141] wherein n is 1 to 10, and each Pu is independently selected from P U i and P U iii ;

[0142] Each P U i is independently a natural or unnatural amino acid moiety; and each P U iii are independently residues of the general formula:

[0143]

[0144] wherein m is 0 to 5, and p is 1, 3, 4 or 5.

[0145] According to one other aspect, -Z 2 -Z 1 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.

[0146] According to one aspect, Z 2 -Z 1 It is [17-carboxyheptadecanoyl]-isoGlu-PEG3-PEG3 or [17-carboxy-heptadecanoyl]-isoGlu-Gly-Ser-Gly-Ser-Gly-Gly.

[0147] PEG3 and PEG4 are ethylene glycol oligomers containing 3 and 4 ethylene glycol units, respectively.

[0148] Preferably, the pKa of the proton of the carboxylic acid bioisostere is similar to the pKa of the corresponding carboxylic acid.Examples of suitable bioisosteres may include tetrazoles, acylsulfonamides, acylhydroxylamines, and squaric acid derivatives.

[0149] The fatty chain can 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 straight chain saturated fatty acids from which suitable fatty chains can be derived include tridecanoic acid / tridecanoic acid, myristic acid / tetradecanoic acid, pentadecanoic acid / pentadecanoic acid, palmitic acid / hexadecanoic acid, and heptadecanoic acid / heptadecanoic acid. Examples of straight chain unsaturated fatty acids from which suitable fatty chains can be derived include myristoleic acid, palmitoleic acid, sapienic acid, and oleic acid.

[0150] The fatty chain may be linked to Z via an amide linkage, a sulfenamide linkage, a sulfonamide linkage, or via an ester linkage, or via an ether, thioether or amine linkage. 2Therefore, the fatty chain may have a Z at the ω position (ie, away from the polar group) 2 Preferably, the fatty chain has an acyl group (-CO-) at a position away from the polar group and is connected to Z via an amide or ester linkage. 2 .

[0151] The method relates to a SPPS comprising a reaction cycle in which an α-amine protected amino acid building block selected from PROT1-Δ(PROT2)-OH is coupled to a peptide or amino acid attached to a solid phase. The amino acid building block PROT1-Δ(PROT2)-OH is used to form a peptide side chain.

[0152] Thus, according to one aspect, the method involves the synthesis of a polypeptide comprising a backbone and peptide side chains covalently linked to the backbone.

[0153] The reaction cycle involved in forming the peptide side chains of a polypeptide is called the peptide side chain reaction cycle.

[0154] In a more specific aspect, any amino acid moiety of a peptide side chain, including the amino acid moiety attached to the side chain of an amino acid building block of the formula PROT1-Δ(PROT2)-OH and other features as described herein, is formed by peptide side chain reaction cycles.

[0155] The reaction cycle involving the formation of peptide side chains is called the peptide side chain reaction cycle or side chain reaction cycle.

[0156] The reaction cycle involved in forming the polypeptide backbone is called the backbone reaction cycle.

[0157] The reaction cycle involved in forming the polypeptide backbone is called a backbone reaction cycle. A reaction cycle comprising the coupling of activated amino acid building blocks selected from PROT1-Δ(PROT2)-OH is therefore a backbone reaction cycle. Any amino acid portion of the backbone of a polypeptide (i.e., comprising an amino acid building block of the formula PROT1-Δ(PROT2)-OH and other features as described herein) is formed by a reaction cycle involved in forming the polypeptide backbone, or simply a backbone reaction cycle.

[0158] The reaction cycle involving the formation (synthesis) of a peptide side chain attached to the amino acid portion Δ is called a peptide side chain reaction cycle.

[0159] According to one aspect, all, preferably all, reaction cycles of any one, preferably one or more, of activation, coupling, deprotection and optional capping involving the formation of (peptide) side chains of the polypeptide (i.e., peptide side chain reaction cycles) are carried out at a temperature of above about 30°C, preferably from about 30°C to a maximum of about 45°C, preferably from about 30°C to a maximum of about 35°C.

[0160] According to another aspect, the deprotection procedure of the reaction cycle involving the formation of the (peptide) side chain of the polypeptide (peptide side chain reaction cycle) is carried out in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

[0161] According to yet another aspect, all procedures, preferably all reaction cycles, involving any, preferably one or more, reaction cycles of activation, coupling, deprotection and optional capping to form the backbone of the polypeptide (backbone reaction cycle), except the steps of the reaction cycle involving the amino acid moiety Δ, are carried out at a temperature of above about 30°C, preferably above about 35°C, preferably above about 40°C, preferably above about 45°C, such as from about 35°C to a maximum of about 55°C, from about 40°C to a maximum of about 50°C, from about 45°C to a maximum of about 50°C.

[0162] For any reaction cycle, regardless of the type of polypeptide disclosed herein, and whether it involves a peptide side chain reaction cycle or a main chain reaction cycle, the concentration of the relevant one or more deprotecting agents (e.g., a base such as piperidine or DBU) can be varied under the deprotection procedure. Preferably, the initial concentration of the deprotecting agent is lower than the final concentration. Therefore, the deprotecting agent is typically loaded (added) multiple times, wherein after the initial loading of the deprotecting agent, the concentration of the deprotecting agent in the reaction solution is lower than the concentration after the final loading of the deprotecting agent.

[0163] One aspect involves the use of a base, suitably piperidine, as a deprotecting agent for the cleavage of α-amine protecting groups (type (I)) involved in the backbone reaction cycle.

[0164] Polypeptides synthesized by the method of the present invention

[0165] According to another aspect, the method involves the synthesis of a polypeptide comprising a backbone and a peptide side chain covalently linked to the backbone.

[0166] The peptide side chain of the polypeptide generally comprises amino acids and a hydrophobic entity at the distal region of the side chain. The hydrophobic entity may be a hydrocarbon chain having a polar group at the distal end.

[0167] The backbone of the polypeptide may include from about 10 up to about 50 amino acids.

[0168] According to another aspect, the method relates to the synthesis of incretin analogs (e.g., incretin analogs selected from glucagon analogs, GLP-1 analogs, GIP analogs, oxyntomodulin analogs, exendin-4 analogs, and PYY analogs). Specifically, the method relates to the synthesis of glucagon analogs such as glucagon-like peptide-1 (GLP-1).

[0169] According to yet another aspect, the method involves synthesizing a polypeptide selected from the group consisting of: His-X2-X3-GTFTSDYSKYL-X15-X16-X17-X18-A-X20-DFI-ΔWLESA

[0170] in:

[0171] 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;

[0172] Ψ is selected from Lys, Arg or Orn;

[0173] And the peptide side chain has the formula -Z 2 -Z 1 ;

[0174] Z 1 is a hydrocarbon chain (preferably an aliphatic chain) having a polar group at one end of the chain and a Z at the end of the chain located away from the polar group. 2 The connecting part -X-.

[0175] wherein the polar group comprises a carboxylic acid or a carboxylic acid bioisostere, a phosphonic acid or a sulfonic acid group; and -X- is a bond, -CO-, -SO- or -SO2;

[0176] -Z 2 - is a spacer having the formula:

[0177]

[0178] Wherein: each Y is independently -NH, -NR, -S or -O, wherein R is an alkyl group, a protecting group or a spacer Z 2 each X is independently a bond, CO-, SO- or SO2-; with the proviso that when Y is -S, X is a bond; each V is independently a divalent organic portion connecting Y and X; and n is 1 to 10.

[0179] 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.

[0180] Peptide side chain-Z 2 -Z 1Covalently bonded to an amino acid moiety selected from diaminoalkanoic acids containing 3 to a maximum of 10 carbon atoms, suitably diaminoalkanoic acids containing 4 to a maximum of 8 carbon atoms, suitably diaminoalkanoic acids containing 5 to a maximum of 8, more preferably diaminoalkanoic acids containing 5 to a maximum of 7 carbon atoms. Preferably, the diaminoalkanoic acid is an (α,ω)-diaminoalkanoic acid, preferably an unbranched diaminoalkanoic acid.

[0181] In some aspects, Z1 is an acyl group having the formula AB-Alk-(CO)- or a sulfonyl group having the formula AB-Alk-(SO2)-; A is -COOH or a carboxylic acid bioisostere; B is a bond, C6 arylene or C6 arylene-O-; Alk is a saturated or unsaturated hydrocarbon chain having a length of 6 to 18 carbon atoms, which is optionally substituted with fluorine, C 1-4 Alkyl, trifluoromethyl, hydroxymethyl, amino, hydroxy, C 1-4 Alkoxy, oxo and carboxyl are substituted with one or more substituents;

[0182] -Z 2 -Yes-S A -、-S A -S B -or-S B -S A -;

[0183] -S A - is a single amino acid moiety selected from the group consisting of γ-Glu, α-Glu, α-Asp, β-Asp, Ala, β-Ala (3-aminopropionic acid) and Gaba (4-aminobutyric acid);

[0184] -S B - is a linker having the following general formula:

[0185]

[0186] wherein n is 1 to 10, and each Pu is independently selected from P U i and P U iii ; Each P U i is independently a natural or unnatural amino acid moiety; and each P U iii are independently residues of the general formula:

[0187]

[0188] wherein m is 0 to 5, and p is 1, 3, 4 or 5.

[0189] In some aspects, m is 1 and p is 1, ie, PU iiiIt is the residue of 8-amino-3,6-dioxaoctanoic acid (also known as {2-[2-aminoethoxy]ethoxy}acetic acid and H2N-PEG3-COOH). This residue is referred to as -PEG3- in this application.

[0190] In some aspects, m is 2 and p is 1, ie, PU iii It is the residue of 11-amino-3,6,9-trioxaundecanoic acid (also known as H2N-PEG4-COOH). This residue is referred to as -PEG4- in this application.

[0191] In some aspects, -Z 1 is an unbranched hydrocarbon chain comprising 6 to 18 carbon atoms having a polar group at one end of the chain and Z 2 The connecting portion is selected from carboxylic acid or carboxylic acid bioisostere.

[0192] In another aspect, -Z 2 -Z 1 Selected from

[0193] (i) [17-Carboxy-heptadecanoyl]-isoGlu-PEG3-PEG3;

[0194] (ii) [17-carboxy-heptadecanoyl]-isoGlu;

[0195] (iii) [13-Carboxy-tridecanoyl]-isoGlu-PEG3-PEG3;

[0196] (iv) [Carboxyphenoxynonanoyl]-isoGlu-PEG3-PEG3;

[0197] (v) [13-Carboxy-tridecanoyl]-isoGlu-PEG4-PEG4;

[0198] (vi) [17-carboxy-heptadecanoyl]-PEG3-PEG3-isoGlu;

[0199] (vii) [17-carboxy-heptadecanoyl]-isoGlu-GSGSGG; and

[0200] (viii) [17-Carboxy-heptadecanoyl]-Ala-Ala-PEG3-PEG3.

[0201] In another aspect, -Z 2 -Z 1 It is [17-carboxyheptadecanoyl]-isoGlu-PEG3-PEG3 or [17-carboxy-heptadecanoyl]-isoGlu-GSGSGG.

[0202] In one aspect, -Z2 -Z 1 It is [17-carboxyheptadecanoyl]-isoGlu-PEG3-PEG3 or [17-carboxy-heptadecanoyl]-isoGlu-GSGSGG.

[0203] In another aspect, Z 2 -Z 1 It is [17-carboxy-heptadecanoyl]-isoGlu-GSGSGG.

[0204] In another aspect, the polypeptide has a sequence selected from the group consisting of:

[0205] His-AibQGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA;

[0206] His-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA;

[0207] His-Ac4c-QGTFTSDYSKYLDERRAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA;

[0208] According to another aspect, the polypeptide has the following sequence: His-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA or H-His-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2.

[0209] In another aspect, the present invention relates to a polypeptide obtained by a method as defined herein above.

[0210] Example

[0211] Example 1

[0212] The present application discloses a SPPS having a polypeptide with the following sequence:

[0213] 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 building blocks of the peptide side chains are enclosed in square brackets. The polypeptide backbone contains 29 building blocks, which are numbered consecutively from 1 to 29, with His being numbered 1 and Ala being numbered 29. The polypeptide has a molar weight of 4231.6 g / mol.

[0214] 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 and produced 53.4 kg of the target polypeptide in 100% yield.

[0215] Except for the Gly-Gly on the side chain, which was coupled continuously as a dipeptide, all amino acids were coupled individually. The peptides were synthesized using an Fmoc-based strategy, which means that all amino acid building blocks provided (i.e., individual natural amino acids, synthetic Ac4c amino acids, and Gly-Gly dipeptides) had α-amines protected by Fmoc groups. The only exception is the Lys at position 24, which is provided as Dde-Lys(Fmoc)-OH, indicating that Dde is an α-amine protecting group, while Fmoc is an amino acid side chain protecting group.

[0216] Provide a dicarboxylic acid aliphatic hydrocarbon at the distal end of the peptide side chain (relative to the distal end of the polypeptide main chain), the distal carboxylic acid residue is protected by tBu (tert-butyl), tBuOOC-C 16 H 32 -COOH.

[0217] The peptide side chains were assembled prior to coupling of Ile.

[0218] All reactive amino acid side chain amines were protected by 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). Therefore, an "orthogonal" protocol was employed, wherein Fmoc and Dde were removed under basic conditions, while all amino acid side chain protecting groups were removed under acidic conditions, and the polypeptide was simultaneously cleaved from the resin to form a crude polypeptide.

[0219] List of building blocks:

[0220] Main chain:

[0221] 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-Leu-O H; 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

[0222] Peptide side chains:

[0223] Fmoc-Gly-Gly-OH; Fmoc-Ser(tBu)-OH; Fmoc-Gly-OH; Fmoc-Ser(tBu)-OH; Fmoc-Gly-OH; Fmoc-Glu-OtBu; octadecane dicarboxylic acid mono-tert-butyl ester.

[0224] Typically, each reaction cycle includes a procedure for deprotecting the α-amine of a resin-bound amino acid or peptide, coupling of an α-amine-protected amino acid, and acetylation (capping) of unreacted α-amine.

[0225] Prior to the coupling of amino acid 29 (i.e., Ala), a Ramage linker (Fmoc-RMG-OH) was coupled to a polystyrene-based resin modified with aminoethyl (AM resin) using the following protocol:

[0226] 1) 2.5 vol% piperidine in DMF

[0227] 2) 12.5 vol% piperidine in DMF

[0228] 3) Wash with DMF

[0229] 4) Fmoc-RMG-OH in DMF in the presence of DIC and Oxyma

[0230] 5) Capping agent in the presence of DIC

[0231] 6) Wash with DMF.

[0232] Amino acids 1 to 22 and 24 to 29 of the backbone (except amino acid Ile (23)) are linked to the resin-bound amino acids or peptides by cycles of the following scheme:

[0233] 1) 2.5% by volume of piperidine in DMF (N,N-dimethylformamide)

[0234] 2) 12.5 vol% piperidine in DMF

[0235] 3) Wash with DMF

[0236] 4) Fmoc-AA-OH in DMF in the presence of DIC and Oxyma (coupling)

[0237] 5) Capping agent, acetic acid (capping) in the presence of DMF

[0238] 6) Wash with DMF.

[0239] The building blocks of the peptide side chains are connected through cycles according to the following scheme:

[0240] 1) 0.1 vol% DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) in DMF

[0241] 2) 0.1 vol% DBU in DMF

[0242] 3) 0.7 vol% DBU in DMF

[0243] 4) Wash with DMF

[0244] 5) In the presence of DIC, Oxyma and DMF, Fmoc-AA-OH reacted with tBuOOC-C 16 H 32 Coupling of -COOH

[0245] 6) Wash with DMF

[0246] 7) Capping agent in DMF, acetic acid

[0247] 8) Wash with DMF.

[0248] The coupling of Ile(23) was performed according to the following scheme:

[0249] 1) 5% by volume piperidine in DMF

[0250] 2) Wash with DMF

[0251] 3) NH2OH xHCl in the presence of DIPEA (N,N-diisopropylethylamine) and DMF

[0252] 4) Wash with DMF

[0253] 5) Repeat steps 1 to 4

[0254] 6) 10% by volume piperidine in DMF

[0255] 7) Repeat step 6 twice

[0256] 8) Fmoc-Ile-OH coupling in the presence of DIC, Oxyma and DMF

[0257] 9) Repeat step 8

[0258] 10) Capping agent in the presence of DIC

[0259] 11) Wash with DMF.

[0260] After His coupling, the peptide-resin was subjected to the following protocol:

[0261] 1) 2.5 vol% piperidine in DMF

[0262] 2) 12.5 vol% piperidine in DMF

[0263] 3) Wash with DMF

[0264] 4) Wash with IPA (isopropyl alcohol)

[0265] 5) Dry.

[0266] All Fmoc protected amino acids were preactivated in a separate preactivation reactor. Prior to addition to the SPPS reactor, preactivation was typically performed in the presence of DIC and Oxyma in the presence of DMF for less than 10 minutes.

[0267] All deprotection, preactivation, coupling, and capping procedures involving the backbone polypeptide were performed at a temperature range of 45°C to 50°C. The exception was the deprotection of the Fmoc α-amine protecting group of Trp prior to coupling with Dde-Lys(Fmoc)-OH, where deprotection, activation, and coupling were performed at a temperature of 30 to 35°C.

[0268] All deprotection, preactivation, coupling and capping procedures involving peptide side chains were performed at a temperature between 30 and 35 °C.

[0269] Typically, coupling is performed over a period of 30 to 50 minutes. Endcapping is performed for 15 to 30 minutes.

[0270] 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. Subsequently, the polypeptide was purified in three different reverse phase chromatography (RPC) steps, then concentrated by ultrafiltration and then the isopropanol was removed by diafiltration. The final product was isolated by lyophilization.

[0271] The SPPS yield was 93.9 kg peptide resin (before cracking). Based on the resin, 208 g of target polypeptide was generated per 1 kg of resin. The quantitative yield was given as 93.9 kg peptide resin multiplied by 0.208, which gave 19.5 kg of target polypeptide on the resin, i.e. 4.6 mol. The quantitative yield (target polypeptide on the resin) before cracking was 4.6 / 12.61, which was approximately 36.5%. After cracking, 21.2 kg of target polypeptide was obtained, i.e. approximately 5.0 mol, with a yield of 109%. The RPC yield (yield of all three RPC steps) was 65% (13.6 kg / 21.2 kg). After concentration and lyophilization, 12.1 kg of net target polypeptide (2.86 mol) was obtained. The total overall yield after concentration and lyophilization (based on the theoretical amount of 53.4 kg of target polypeptide for a 12.61 mol synthesis scale) was 22.7% (12.1 kg divided by 53.4 kg) of net target polypeptide, corresponding to 2.86 mol).

[0272] Example 2

[0273] 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 examined.

[0274] If the amino acid is not activated, the carboxylic acid and amine of the two amino acids will not condense to form an amide bond / peptide bond, but will be neutralized.

[0275] Activation is the process of converting an amino acid into a species that readily reacts with an amine to form an amide bond. Furthermore, the reactivity of the activated amino acid must be balanced so that the activated amino acid reacts primarily with the intended amine.

[0276] There are a variety of activating agents. Here, DIC and Oxyma are used to convert amino acids into their activated state, i.e., esters.

[0277] The activated ester of Dde-Lys(Fmoc)-OH readily reacts with benzylamine with the release of oxime according to the following scheme:

[0278]

[0279] The activated ester of Dde-Lys(Fmoc)-OH is unstable in aqueous mixtures and degrades during HPLC analysis, but readily reacts with benzylamine. Therefore, the benzylamide of Dde-Lys(Fmoc)-OH was used as an alternative to the activated ester of Dde-Lys(Fmoc)-OH.

[0280] The 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 min, 5 min, 10 min, 15 min, 20 min, 25 min and 30 min, respectively, and then reacted with benzylamine.

[0281] 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.

[0282] Table 1: Results involving activation of Dde-Lys(Fmoc)-OH with DIC, Oxyma and benzylamine as a function of activation temperature and time.

[0283]

[0284] The following conclusions can be drawn from Table 1:

[0285] ●As the temperature increases, the amount of AE increases to an optimum value and then decreases within a given time.

[0286] As the temperature increases, the amount of impurities increases from a given time (indicated by gray shading)

[0287] The percentage of impurities at 30°C and 30 min is 1-(0.7416+0.2406)=1.78%.The percentage of impurities at 50°C and 30 min is 1-(0.4392+0.1223)=43.85%.

[0288] Example 3

[0289] In this example, the peptide Dde-Lys(Fmoc)-Trp-Leu-Glu-Ser-Ala-NH2 was synthesized using the protocol set forth in Example 1.

[0290] Lys was provided in the form of Dde-Lys(Fmoc)-OH and activated in a separate pre-activation reactor at 30°C or 35°C for periods of 5, 10, and 15 minutes, respectively. After activation, the solution containing the activated ester of Dde-Lys(Fmoc)-OH was transferred to the SPPS reactor. The activated ester of Dde-Lys(Fmoc)-OH reacted with the α-amine of tryptophan (Trp) under conditions of amide bond formation at a temperature equivalent to that during activation (i.e., 30°C or 35°C) for 45 minutes. Subsequently, after 45 minutes, the unreacted α-amine of Trp was capped (acetylated) by adding a capping agent (2 molar equivalents) in the presence of DIC (2 molar equivalents).

[0291] After capping, the peptide was cleaved from the resin with TFA, neutralized (NH4OAc:IPA:H2O) and diluted with DMF. After workup, the solution including the cleaved peptide was analyzed by HPLC.

[0292] Figures 1 to 6 An HPLC chromatogram of a workup solution containing the cleaved peptide (Dde-Lys(Fmoc)-Trp-Leu-Glu-Ser-Ala-NH2) is presented. 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 initially 90%:10% and reached 10%:90% at an endpoint flow rate of 1.0 ml / min.

[0293] The following Table 2 presents Figures 1 to 6 Number 6 is the peptide Dde-Lys(Fmoc)-Trp-Leu-Glu-Ser-Ala-NH2.

[0294]

[0295] Example 4

[0296] In Example 4, the conditions for the coupling of Dde-Lys (Fmoc) -OH were additionally investigated. The peptide Trp-Leu-Glu-Ser-Ala was synthesized using the protocol described in Examples 1 and 3. For this purpose, pre-activation of Dde-Lys (Fmoc) -OH was performed in a separate pre-activation reactor. Prior to addition to the SPPS reactor for the coupling reaction with Trp-Leu-Glu-Ser-Ala, pre-activation was performed in the presence of DIC and Oxyma in the presence of DMF for 5 minutes, 10 minutes, 20 minutes, and 30 minutes, respectively, while room temperature (RT), 35°C, and 55°C were used for the reaction cycle (including pre-activation and coupling with WLESA). The results are shown in Tables 3, 4, and 5 below.

[0297] Table 3

[0298]

[0299]

[0300] The data in Table 3 show the purity of the cleaved sample in HPLC analysis (UV detection at 290 nm). And it does show that the purity and therefore the yield of Dde-Lys(Fmoc)-OH after coupling is less than 100%. However, the impurities present in the sample are impurities accumulated from the six couplings of the peptide fragment after coupling of Dde-Lys(Fmoc)-OH. More important than the actual purity of the sample is the fact that only trace amounts (<0.1%) of unreacted peptide (in the form of acetylated peptides or peptides with free amino groups) can be detected from this coupling at room temperature.

[0301] Another set of equivalent experiments was performed at RT, 35°C and 55°C using 20 and 30 min pre-activation. The results are shown in Tables 4 and 5 below. It is clear that at 55°C, the purity (and therefore the yield) of the samples dropped rapidly, and acetylated peptides and peptides with free amino groups appeared. In addition, the acetylated peptides and peptides with free amino groups had much lower UV absorption at 290nm because only tryptophan residues and Fmoc and Dde groups were visible at this wavelength. Only tryptophan residues were present in the uncoupled material, in contrast to the product containing all three groups. It is estimated that the UV absorption of the product was 4 times that of the uncoupled material (acetylated peptide and peptide containing free amine), corresponding to the molar ratios shown in Table 5 below. Therefore, most of the amino groups did not react during the Dde-Lys(Fmoc)-OH coupling. No trace of unreacted peptide was seen at 35°C or at room temperature.

[0302] Table 4

[0303] Pre-activation 20min (%) Ac / free amine (%) Pre-activation 30min (%) Ac / free amine (%) 55℃ / area% 59.55 2.34 / 0.28 48.90 6.55 / 0.36 35℃ / area% 91.04 - 90.71 - RT / area% 90.43 - 91.74 -

[0304] Table 555℃, pre-activation 20min

[0305]

[0306]

[0307] Table 5 continued 55℃, pre-activation 30min

[0308] product Acetylation Free amines molar ratio 48.9 26.2(6.55*4) 1.44(0.36*4) Normalization 64％ 34％ 1.9％

Claims

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

2. The method according to claim 1, further comprising: removing PROT2 prior to assembly of the peptide side chains; and Following assembly of the peptide side chains PROT1 is removed and the other amino acids are attached to the backbone.

3. The method according to claim 1 or 2, wherein the amino acid portion Δ is coupled to the amino acid sequence WLESA. The method according to claim 3 , wherein A of the amino acid sequence WLESA is covalently bound to the solid phase.

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

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

7. The method of any one of the preceding claims, 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 block PROT1-Δ(PROT2)-OH is activated.

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

9. The method according to any one of the preceding claims, 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 in the same reaction cycle.

10. The method according to any one of the preceding claims, wherein the method further comprises selectively removing PROT2.

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

12. The method according to claim 10, further comprising at least one reaction cycle associated with the formation of a peptide side chain Δ-linked to the amino acid moiety, wherein the coupling is performed at a temperature of greater than about 30°C, preferably from about 30°C to a maximum of about 45°C, preferably from about 30°C to a maximum of about 35°C.

13. The method according to any one of the preceding claims, wherein the polypeptide is selected from: His-X2-X3-GTFTSDYSKYL-X15-X16-X17-X18-A-X20-DFI-ΔWLESA in: 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; And the peptide side chain has the formula -Z 2 -Z 1 ; Z 1 is a hydrocarbon chain having a polar group at one end of the chain and a Z at the end of the chain away from the polar group. 2 The connecting part -X-. wherein the polar group comprises a carboxylic acid or a carboxylic acid bioisostere, a phosphonic acid or a sulfonic acid group; and -X- is a bond, -CO-, -SO- or -SO2; -Z 2 - is a spacer having the formula: Wherein: Y is independently -NH, -NR, -S or -O, wherein R is an alkyl group, a protecting group or a spacer Z 2 another part of the formation of a connection; X is each independently a bond, CO-, SO- or SO2-; with the proviso that when Y is -S, X is a bond; V is each independently a divalent organic portion connecting Y and X; and n is 1 to 10.

14. The method according to any one of the preceding claims, wherein the polypeptide is selected from: 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.

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 method of claim 15, wherein the polypeptide is H-His-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2.

17. The method of any one of the preceding claims, wherein the polypeptide is an analog of glucagon.

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