WO2024156694A1

WO2024156694A1 - Method for synthesizing polypeptides

Info

Publication number: WO2024156694A1
Application number: PCT/EP2024/051517
Authority: WO
Inventors: Markus GLAFFIG; Rabea HENNIG; Anna Mette HANSEN; Jens Fomsgaard; Jan Pawlas
Original assignee: Boehringer Ingelheim International Gmbh
Priority date: 2023-01-24
Filing date: 2024-01-23
Publication date: 2024-08-02

Abstract

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

Description

Method for Synthesizing Polypeptides

Field of the Invention

The present invention relates to a method for synthesizing a branched polypeptide based on solid phase peptide synthesis.

Background

Solid phase peptide synthesis (SPPS) of polypeptides pioneered by Bruce Merrifield (Merrifield, 1963) comprises synthesizing a polypeptide bound to an insoluble solid phase and subsequently cleaving of the polypeptide from the solid phase. SPPS comprise repetitive reaction cycles, in which amino acids are coupled to a nascent peptide bound to the solid phase by the formation of an amide bond (usually referred to as a peptide bond). The use of a solid phase enables the use of excess reagents and the removal of the reagents and other reaction facilitating agents by convenient filtration.

To achieve a high final (global) yield of the polypeptide it is desirable that the (local) yield of each coupling is as high as possible. It is preferred that the peptide bond formation in each reaction cycle is driven to completion. As the number of amino acids of a polypeptide increases the importance of achieving high local yield of each coupling becomes of ever greater importance.

The present peptide synthesis strategy relates to the preparation of peptides comprising at least an amino acid having an amino acid side chain with a functional group (branching inducing amino acid) such as lysine and lysine analogues which can serve as a locus for the formation of a peptide side chain.

An embodiment of the present invention relates to the synthesis of branched polypeptides, notably a polypeptide comprising a peptide side chain such as glucagon analogues. A peptide side chain can be formed by the application of several strategies. A peptide side chain may be formed while the polypeptide is attached to the resin and by the consecutive coupling of the relevant side chain amino acid moieties. Alternatively, the entire peptide side chain or part of the peptide side chain may be coupled to a relevant amino acid moiety of a polypeptide bound to the resin. Additionally, the peptide side chain may be assembled after the coupling of the branching amino acid but prior to the coupling of the next backbone amino acid. Or the peptide side chain is synthesized after the completion of the polypeptide backbone. Within the context of the present invention, after completion of the synthesis of a peptide side chain, either by the consecutive coupling of individual side chain amino acid moieties or by coupling part or the entire peptide side chain, the next amino acid of the polypeptide backbone is coupled. The amino acid serving as the locus for branching must at least comprise three reactive groups, including a group conducive for covalently linking a peptide side chain. Within the context of the present invention the locus for branching is an amino acid building block defined as PROT1-A(PROT2)-OH, where A is selected from diamino alkanoic acids comprising from 3 up to 10 carbon atoms, PROT1 constituting an a-amine protecting group and PROT2 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 coupling of the next amino acid of the polypeptide backbone, the amino acid side chain protecting group must be cleavable without cleaving the a-amine protecting group or any other amino acid side chain protecting groups present in the polypeptide attached to the resin (class (II) protecting groups). Thus, the degree of freedom for the selection of PROT1 and PROT2 is governed by the reaction scheme for synthesizing a polypeptide side chain.

The present invention seeks to optimize/increase the local (but also global) yield of the coupling of an amino acid serving as the locus for branching for the synthesis of a polypeptide comprising a backbone and a peptide side chain, such as an analogue of glucagon, using an SPPS scheme where the peptide side chain is assembled prior to the formation of the complete target polypeptide backbone, i.e. complete target polypeptide.

WO 2018069295 discloses a method for the preparation of a peptide comprising a lipophilically modified lysine side chain. More specifically, the method relates to a procedural scheme for the removal of trityl-based side chain protecting groups of lysine after formation of the target polypeptide backbone but prior to cleavage from the resin.

WO 2019120639 relates to the synthesis of a polypeptide containing an alloc N- terminal protected lysine or lysine derivative. The synthesis includes the acylation of the lysine side chain, removal of the alloc group and the coupling of an amino acid or peptide to the N-terminal of the lysine.

CN 106478806 presents a method for synthesizing semaglutide. Lys is provided in the form of Dde-Lys(Fmoc)-OH. CN 106478806 does not mention the temperature during coupling cycles.

of the Invention

One objective with the present invention is to increase the overall yield of a polypeptide synthesized using SPPS.

A further objective is to increase the overall yield of a branched polypeptide having more than 10 amino acids in the backbone, the polypeptide synthesized using SPPS.

Another objective is to increase the yield of the coupling of a lysine or lysine analogue, specifically to coupling of lysine to a backbone tryptophane.

An additional objective is to increase the local (but also global) yield of the coupling of an amino acid serving as the locus for branching for the synthesis of a polypeptide comprising a backbone and a peptide side chain, such as a glucagon analogue, using an SPPS scheme where a peptide side chain is assembled prior to the formation of the target polypeptide backbone.

A further objective is to provide improved conditions for coupling an amino acid (serving as locus for branching) to a tryptophan residue.

Another objective is to establish optimal conditions for coupling a side chain to a branching point amino acid such as lysine.

Summary of the Invention

The present invention relates to a method for synthesizing a polypeptide based on solid phase peptide synthesis, the polypeptide comprising a backbone, the backbone comprising one amino acid moiety A selected from diamino alkanoic acids comprising from 3 up to 10 carbon atoms and one peptide side chain covalently attached to the amino acid moiety A, the amino acid moiety A being coupled to an amino acid moiety W, the method comprising at least one reaction cycle comprising: (i) providing an amino acid building block PROT1-A(PROT2)-OH, PROT1 being an a- amine protection group and PROT2 being an amino acid side chain protection group;

(ii) performing activation of the amino acid building block PROT1-A(PROT2)-OH in absence of the solid phase for less than about 25 minutes at an elevated temperature of from about 20 °C up to about 50 °C, and;

(iii) coupling the activated amino acid building block PROT1-A(PROT2)-OH to the unprotected a-amine of amino acid moiety W of a peptide covalently attached to a solid phase thereby forming an amide bond.

Definitions

A solid phase peptide synthesis (SPPS) reaction cycle comprises procedures including de-protection of the protecting groups bound to the a-amine of a peptide or amino acid covalently attached to a solid phase and coupling of the carboxylic acid function of an a-amine protected amino acid moiety to the (free or deprotected) a- amine of a peptide or amino acid bound to the solid support thereby forming an amide bond. Prior to the coupling, an amino acid needs to be activated. In addition, a reaction cycle may also comprise the capping of (unreacted, deprotected) a-amines of peptides or amino acids bound to the solid phase which have not formed amide bonds, herein referred to as capping. Procedures may be repeated in a reaction cycle, e.g. a reaction cycle may comprise multiple coupling and capping procedures. Activities, during which compounds are removed from the reaction solution are collectively denote washings. A procedure of a reaction cycle can also be referred to as a step or stage. A procedure, step or stage may be characterized by any one of the following non-exhaustive procedures (steps, stages): usage of one or more significant compounds (examples of significant compound: activation agent, capping agent, de-protection agent, activated amino acid building block), activities between two washings, reaction procedure (e.g. activation of an amino acid building block, formation of an amide bond, cleavage of covalent bonds). Several procedures may also proceed in parallel, e.g. activation and coupling may occur simultaneously. Thus, within the context of the present invention, a reaction cycle may comprise a plurality of procedures (steps, stages), significant procedures (steps, stages) being de-protection, activation, coupling, and optionally capping.

Activation in the context of the present invention is initiated from the instance an a- amine-protected amino acid building block and at least one activating agent is present in a solution up to the instance of the formation of an amide bond.

Activation and activated amino acid building blocks: A carboxylic acid and an amine are not likely to engage in a condensation reaction forming an amide bond but instead will neutralize forming carboxylate and ammonium ion. In its widest sense activation (or the activation procedure) enables a carboxylic acid and an amine to form an amide bond. Activation may relate to the activation of a carboxylic acid function of an amino acid moiety by the engagement of at least one activation reagent (also referred to as coupling reagent). The carboxylic acid function is usually activated by the reaction with an electron withdrawing reagent. An activated carboxylic acid function 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 comprising a carboxylic acid/carboxylate bound to an activation reagent. The stability of the activated amino acid building block is partly dependent on the activation reagent(s) and the reaction conditions.

Within the context of the present invention an amino acid building block is an amino acid where the a-amine and optional reactive group of the amino acid side chain is protected by suitable protecting groups.

The term ‘amino acid moiety’ is used for denoting amino acids present in a peptide/polypeptide.

An activation agent is a molecule which activates an amino acid building block.

Capping is the activity of chemically modifying remaining unreacted a-amines after a coupling step into an entity which is not able to chemically react in downstream coupling cycles. Capping usually comprises the acylation or acetylation of an a- amine. Capping reagents can be acetic anhydride.

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 molecule comprising functional groups, notably an amine and a carboxylic acid, enabling the synthetic molecule to be used in SPPS and providing an amide bond.

A polypeptide comprises at least two amino acids up to any number of amino acids where the molecule is referred to as a protein. Human proteins have from about 200 amino acids.

In the context of the invention the term polypeptide is used to denote a branched polypeptide formed by the method disclosed herein. Also, the backbone of the branched polypeptide is referred to as a polypeptide.

The term peptide is used in conjunction with the side chain of the polypeptide. The peptide side chain has at least two amino acid moieties up to any number of amino acid moieties which is smaller than the number of amino acid moieties of the polypeptide backbone. Thus, a peptide side chain is the side chain of a branched polypeptide. The peptide side chain may also comprise a hydrocarbon moiety. Typically, the hydrocarbon moiety comprises from about 10 up to about 25 carbons, suitably from about 14 up to 20 carbon atoms.

Amino acid side chain is the side chain of an amino acid moiety. An amino acid side chain is attached to the a-carbon. Lysine is an example of an amino acid comprising a side chain.

Diamino propionic acid contains 3 carbon atoms whereas diamino decanoic acid contains 10 carbon atoms.

An acid-labile protecting group is a protecting group cleaved off under acid conditions.

A base-labile protecting group is a protecting group cleaved off under alkaline conditions.

Class (I) protecting groups denote protecting groups attached to reactive groups of amino acid building blocks not engaged in amide bond formation and excluding PROT2 protecting groups. Hence, class (I) protecting groups are preferably protecting groups covalently attached to reactive groups of the side chain of an amino acid building block. Examples of amino acid moiety side chain reactive groups are amine, carboxylic acid, alcohol, thiol, thioether. Class (II) protecting groups denote protecting groups attached to amines of amino acid building blocks engaged in amide formation (a-amines) and excluding PROT1 protecting groups.

The terminology of class (I) and class (II) protecting groups is introduced to distinguish such protecting groups from the groups PROT1 and PROT2 of the amino acid building blocks of formula PROT1-A(PROT2)-OH.

A is an amino acid moiety selected from diamino alkanoic acids comprising from 3 up to 10 carbon atoms

PROT1 is an a-amine protection group.

PROT2 is an amino acid side chain protection group.

By coupling is meant the formation of an amide bond (peptide bond).

WLESA denotes a peptide fragment Trp-Leu-Glu-Ser-Ala.

The term “about” is used for several parameters disclosed herein, such as temperature, time and yield. If not indicated otherwise the term “about” should be interpreted that the relevant parameter may divert by up to 5%.

An amino acid side chain (such as A side chain) is a side chain of an amino acid moiety/ amino acid building block. Lys is an example of an amino acid moiety/an 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 covalently attached to a backbone polypeptide. The side chain may comprise amino acid moieties and/or fatty acid moieties.

Brief description of figures

All figures 1 - 6 illustrate HPLC diagrams of the work-up solutions comprising the cleaved peptide (Dde-Lys(Fmoc)-Trp-Leu-Glu-Ser-Ala-NH2) of the synthesis described in example 3. Variables were the duration of pre-activation (5, 10, 15 min) and temperature 30°C and 35°C. Temperatures were kept constant during preactivation and coupling.

Figure 1 : 30°C, 5 min pre-activation, 45 min coupling

Figure 2: 30°C, 10 min pre-activation, 45 min coupling Figure 3: 30°C, 15 min pre-activation, 45 min coupling

Figure 4: 35°C, 5 min pre-activation, 45 min coupling

Figure 5: 35°C, 10 min pre-activation, 45 min coupling

Figure 6: 35°C, 15 min pre-activation, 45 min coupling

Disclosure of the invention

The method is based on SPPS comprising at least on reaction cycle (reaction cycle A), in which an amino acid building block selected from the group consisting of PROT1 -A(PROT2)-OH is coupled to an amino acid moiety W of a peptide covalently bound to a solid phase.

A is selected from diamino alkanoic acids comprising from 3 up to 10 carbon atoms.

PROT1 is an a-amine protection group.

PROT2 is an amino acid side chain protection group.

According to an aspect the diamino alkanoic acid is preferably an unbranched diamino alkanoic acid. Unbranched in the context of a diamino alkanoic acid refers to the side chain.

According to a further aspect the diamino alkanoic acid is selected from (a,w)- diamino alkanoic acids, preferably unbranched diamino alkanoic acid, wherein co denotes an amine at the distal end of the amino acid side chain, at the co carbon, and a second amine at the a carbon.

According to an aspect the diamino alkanoic acid comprises from 4 up to 8 carbon atoms, suitably from 5 up to 8, more preferably from 5 up to 7.

According to a further aspect the diamino alkanoic acid is selected from ornithine (2,5 diamino pentanoic acid) and lysine (2,6 diamino hexanoic acid), preferably lysine.

PROT1 and PROT2 are a-amine and amino acid side chain protection groups respectively and are preferably selected from protecting groups cleaved off under alkaline conditions with the proviso that only one of PROT1 and PROT2 is cleaved off under the same reaction cycle. PROT1 and PROT2 may be selected from Fmoc (Fluorenylmethyloxycarbonyl), Nsc (2-(4-Nitrophenylsulfonyl)ethoxycarbonyl), Bsmoc (1 ,1 -Dioxobenzo[b]thiophene-2- ylmethyloxycarbonyl), a-Nsmoc ((1 ,1 -Dioxonaphtho[1 ,2-b]thiophene-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)-3-methylbutyl, 2, 7-D i-tert-buty I- 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.

The invention relates to the synthesis of a polypeptide comprising a backbone and one peptide side chain which may be referred to as a branched polypeptide. The backbone contains amino acid moieties and one amino acid moiety A selected from diamino alkanoic acids comprising from 3 up to 10 carbon atoms which is coupled to W. The peptide side chain is covalently attached to the amino acid moiety A of the backbone. The backbone comprises only one amino acid moiety A formed from amino acid building blocks selected from PROT1 -A(PROT2)-OH serving as the locus for the construction/synthesis/formation of the peptide side chain. The backbone may comprise additional amino acids selected from diamino alkanoic acids comprising from 3 up to 10 carbon atoms, such as Lys.

W as well as WLESA and AWLESA form part of the peptide back-bone.

According to an aspect the polypeptide comprises one and only one peptide side chain, the peptide side chain covalently attached to an amino acid moiety as prescribed by the method of the invention, i.e. the amino acid moiety A.

According to an aspect PROT2 and class(l) protecting groups are selected from protecting groups simultaneously cleaved under the same reaction cycle. PROT2 and class(l) protecting groups may be selected from base-labile protecting groups, such as Fmoc. Protecting groups of class (II), of reactive groups of amino acid building blocks not to be engaged in amide bond formation (which may be referred to as amino acid side chain protecting groups such as amines), and protection groups of class (I) protecting amines of amino acid building blocks (a-amine protecting groups) to be engaged in amide formation (a-amines) are preferably selected such that only one class of protecting groups is cleaved off during the same reaction cycle. Class (I) protecting groups may be liable to cleavage under alkaline conditions (alkaline/base-labile protecting groups) while class (II) protecting groups may be liable to cleavage under acid conditions (acid-labile protecting groups).

PROT1 and PROT2 are suitably selected such that only one of PROT1 or PROT2 is cleaved off during the same reaction cycle. Furthermore, PROT2 is preferably selected from protecting groups which are cleaved off while no other protecting groups of class (II) are cleaved off during the same reaction cycle.

According to an aspect class (II) protecting groups are acid-labile while class (I) protecting groups are base-labile. This protection scheme is also referred to as an orthogonal protection scheme.

If the method relates to polypeptides formed from amino acid building blocks comprising reactive groups not engaged in amide bond formation protected by class (II) protecting groups, said class (II) protecting groups being acids-labile, then PROT1 and PROT2 are suitably base-labile signifying that said protection groups are cleaved off during alkaline conditions.

According to an aspect the method further comprises removing PROT2 prior to the assembly of the peptide side chain; and removing PROT1 after the assembly of the peptide side chain and attaching further amino acids to the backbone.

According to a further aspect, the amino acid moiety A is coupled the amino acid sequence WLESA.

According to yet a further aspect, the amino acid moiety A of the amino acid sequence WLESA is covalently bound to the solid phase.

When stating 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 by way of a suitable linker.

According to an aspect, the activation of step (ii) is performed at a temperature range 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. According to an aspect, the activation of step (ii) is performed less than about 22 minutes, preferably less than about 18 minutes.

According to an 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 -A(PROT2)-OH is activated.

According to an aspect, A is selected from unbranched (a,co)-diamino alkanoic acids, suitably ornithine and lysine, preferably lysine.

According to an aspect, PROT1 and PROT2 are selected from protection groups cleaved off under alkaline conditions, with the proviso that PROT1 and PROT2 are not cleaved off under the same reaction cycle.

According to an aspect, the method further comprises selective removal of PROT2.

According to an aspect, PROT1 is selected from Dde and ivDde, suitably Dde and PROT2 is Fmoc.

According to an aspect, the method further comprises at least one reaction cycle related to the formation of a peptide side chain attached to the amino acid moiety A, wherein the coupling is carried out at a temperature above about 30 °C, preferably from about 30 °C up to about 45 °C, preferably from about 30 °C up to about 35 °C.

According to yet a further aspect, the polypeptide is selected form:

His-X2-X3-GTFTSDYSKYL-X15-X16-X17-X18-A-X20-DFI-AWLESA wherein:

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

X3 is selected from Gin and His;

X15 is selected from Asp and Glu, preferably Asp;

X16 is selected from Glu and T, preferably Glu;

X17 is selected from Arg and T;

X18 is selected from Ala and Arg;

X20 is selected from Lys and His preferably Lys;

T is selected from Lys, Arg, or Orn; and the peptide side chain having the formula -Z²-Z¹;

Z¹ is a hydrocarbon chain having a polar group at one end of the chain and a connection to Z², -X-, positioned at the end of the chain distal from the polar group, 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;

-Z²- is a spacer of formula:

wherein: each Y is independently-NH, -NR, -S or -0, where R is alkyl, a protecting group or forms a linkage to another part of the spacer Z²; 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 bivalent organic moiety linking Y and X; and n is 1-10.

The method comprises at least one reaction cycle comprising the activation in absence of the solid phase of an a-amine protected amino acid building block thereby forming an activated a-amine protected amino acid building block and coupling of said activated a-amine protected amino acid building block to the unprotected a-amine of a peptide or amino acid covalently attached to a solid phase thereby forming an amide bond and wherein the duration of the activation is less than 25 minutes at an elevated temperature of from about 20 °C up to about 50 °C.

Activation may be accomplished in the presence of the solid phase, i.e. in the reaction vessel where the polypeptide is formed and attached to the solid phase (polymeric solid phase). An activation in the presence of the solid phase is frequently denoted as in-situ activation. An alternative is that activation is initiated in the absence of the solid phase. Activation in the absence of the solid phase and by inference in the absence of the nascent polypeptide linked to the solid phase is preferably conducted in an activation vessel separate from the reaction vessel where the polypeptide is formed. Activation of the a-amine protected amino acid building block is necessary for the establishment of an amide bond (peptide bond) between the a-amine-protected amino acid building block and the nascent peptide bound to the solid phase. Activation is initiated by the provision (addition) of at least one activating agent (an activating agent is in the field of SPPS also denoted as a coupling agent) and an a-amine-protected amino acid building block and normally commences until an amide bond is formed. The stability of an activated a-amine- protected amino acid building block is to an extend dependent on the activating agent and solvent composition. An activated a-amine-protected amino acid building block can be stable up to several hours, or up to days under convenient conditions, signifying that an activated a-amine-protected amino acid building block may be activated from the instance of activation until the formation of an amide bond. Thus, if the a-amine-protected amino acid building block is activated in the absence of the peptide-solid phase, it is present in its activated form after transfer to the peptide reaction vessel until formation of the amide bond. According to an aspect the activation is initiated in the absence of the solid phase preferably in a solution comprising at least one activation agent and preferably in an activation vessel (other than the reaction vessel comprising the solid phase). According to a further aspect the activation is performed by the application of two activating agents.

Activation of the amino acid building block PROT1-A(PROT2)-OH in the absence of the 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 may be conducted from about 1 minute, from about 2 minutes, from 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 lower and upper time indications may be combined with each other. To an extent the duration of the activation is governed by the temperature during activation.

According to a further aspect the activation, suitably in the absence of the solid phase, 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 activated a-amine protected amino acid building block of (PROT1-A(PROT2)-OH) is formed.

Preferably the a-amine protected amino acid building block such as PROT1 - A(PROT2)-OH is charged at a molar ratio of above about 1 .5, suitably above about 2.0. The molar ratio is based on the loading of the solid phase, which is the theoretical number of amines available for forming amide/peptide bonds. Activating agents are preferably charged at molar amounts equal or similar to the molar amounts of the a-amine protected amino acid building block. Activating agents consumed during the process of activation, DIC is an example, may be charged at a total molar ratio above the ratio of the relevant a-amine protected amino acid building block.

Activation in the context of the present invention is the time from mixing the a-am ineprotected amino acid building block and at least one activating agent in the absence of a solid phase up to the instance of the formation of an amide bond corresponding to the addition of the solution comprising the activated amino acid building block to the solution comprising the solid phase.

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

Activation, the formation of an amino acid ester residue, is possible as long as relevant activation agents are present. Thus, activation may also take place after transferal of the solution comprising activated amino acid residues from an activation vessel to a peptide reaction vessel.

By coupling is meant the formation of an amide bond (peptide bond).

The rate of amide bond formation decreases with time and is partly correlated to the amount (molar ratio) of the activated a-amine-protected amino acid building block in relation to available amines of peptides bound to the solid phase.

Activating agents may be selected from the group of diisopropylcarbodiimide (DIC), Ethyl cyano(hydroxyimino)acetate (Oxyma, sometimes also referred to as OxymaPure), dicyclohexylcarbodiimide (DCC), 1 -Ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDCxHCI) (optionally in the presence of coupling additives such as 1 -Hydroxybenzotriazole (HOBt), 6-Chloro-1 -hydroxybenzotriazole (CI-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 tetramethyl uronium (HBTLI), O-(Benzotriazol-1 - yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate [O-[ N,N,N',N'-Tetramethyl-O-(1 H- benzotriazol-1 -yl)uronium hexafluorophosphate ] (TBTLI), 0- [(Ethoxycarbonyl)cyanomethylenamino]-N,N,N',N'-tetramethyluronium hexafluorophosphate (HOTII), O-[(Ethoxycarbonyl)cyanomethylenamino]-N,N,N',N'- tetramethyluronium tetrafluoroborate (TOTII), O-(1 H-6-chlorobenzotriazole-1 -yl)- 1 ,1 ,3,3-tetramethyluronium hexafluorophosphate (HCTLI), O-(6-Chlorobenzotriazol-1 - yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TCTLI), 1- [Bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyl uronium (HATLI), O-(7-Azabenzotriazole-1 -yl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate (TATLI), 1 -cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino- carbenium hexafluorophosphate (COMII), 1- [(Dimethylamino)(morpholino)methylene]-1 H-[1 ,2,3]triazolo[4,5-b]pyridine-1 -ium 3- oxide hexafluorophosphate (HDMA), HDMB, 6-chloro-1 - ((dimethylamino)(morpholino)-methylene)-1 H-benzotriazolium (HDMC), Benzotriazol- 1 -yloxytris(dimethylamino)phosphonium hexafluorophosphateC (BOP), (Benzotriazol- 1 -yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), [Ethyl cyano(hydroxyimino)acetato-O²]tri-1 -pyrrolidinylphosphonium hexafluorophosphate (PyOxim), 6-Chloro-benzotriazole-l -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), tetramethylammonium trifluoromethanethiolate ((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 (DMTMMCI), 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).

According to an aspect, activation is achieved by DIC and Oxyma e.g. by bringing together DIC and Oxyma and the relevant a-amine-protected amino acid building block in a suitable solvent. Suitable solvents in general for SPPS, applicable to any procedures of a 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 (DMPII), dimethylsulfoxide (DMSO), diethylsulfoxide (DESO), sulfolane, (1 R)-7,8-dioxabicyclo[3.2.1 ]octan-2-one (dihydrolevoglucosenone, cyrene®), N,N-dimethylacetamide (DMA), N,N,N',N'- tetraethyl sulfamide (TES), 1 -butyl-3-methylimidazolium chloride (BMIMCI), 1 -butyl-3- methylimidazolium bromide (BMIMBr), and 1 -butyl-3-methylimidazolium iodide (BMIMI).

Activation is implemented (carried out) at a temperature from about 20 °C up to about 50 °C. As elaborated above activation is the procedure initiated by bringing together at least one activation agent and an a-amine-protected amino acid building block. The act of activation provides an activated a-amine-protected amino acid building block which has a certain lifetime. Activated a-amine-protected amino acid building blocks react with amines forming amide (peptide) bonds. When activation is conducted in the absence of peptides/amino acids bound to a solid phase amide bond formation begins when the activated a-amine-protected amino acid building block is added to the suspension comprising the solid phase. If activation is commenced in the presence of the solid phase (in-situ activation) amide bond formation can start rapidly while still a-amine-protected amino acid building blocks are activated.

Within the context of the invention activation and for reasons elaborated herein, activation can also materialize while activated amino acid building blocks engage in amide bond formation (coupling). Even after transfer of the solution comprising an activated amino acid building block to the reaction vessel comprising the solid phase amino acid building blocks can be activated. The temperature of activation is preferably also the temperature of the coupling or at least under a period of coupling as long as a useful concentration of activated a-amine-protected amino acid building block is present in the reaction solution. According to a further aspect the activation is implemented (carried out) at a temperature of from about 20 °C up to about 45 °C, preferably from about 20 °C up to about 40 °C, from about 30 °C up to about 37 °C such as from about 30 °C up to about 35 °C.

The lower activation temperature can be 20 °C, 21 °C, 22 °C, 23°C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29°C, 30 °C while the upper activation temperature can 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 lower and upper temperatures may be combined to render a temperature range.

According to an aspect, activation and coupling is implemented (carried out) at the same temperature

Independent whether activation is initiated in the presence of the solid phase or in the absence of the solid phase it is preferred that activation, i.e. a period from the formation of activated a-amine-protected amino acid building blocks to amide bond formation, is preferably below about 50 minutes, below about 40 minutes, below about 35 minutes, below about 30 minutes, below about 25 minutes, preferably below about 20 minutes.

The duration of activation of the amino acid building block PROT1-A(PROT2)-OH in the absence of the 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 may be conducted from about 1 minute, from about 2 minutes, from about 5 minutes up to about 25 minutes, up to about 20 minutes, up to about 18 minutes, up to about 15 minutes.

According to an aspect activation of the amino acid building block PROT1- A(PROT2)-OH and coupling is performed at a temperature 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 temperatures of 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 being initiated in the absence of the solid phase suitably in an activation vessel being less than 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, from about 2 minutes, from about 5 minutes up to about 25 minutes, up to about 20 minutes, up to about 18 minutes, up to about 15 minutes..

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

The reaction cycle may further comprise the capping of unreacted, unprotected a- amines of peptides or amino acids bound to the solid phase.

According to an aspect the capping comprises the addition of a capping composition comprising capping agent and a further activation agent such as DIC. Preferably, capping is initiated by the addition of the capping composition to the coupling solution.

Each reaction cycle comprises the cleavage of the a-amine protecting group of the peptide or amino acid attached to the solid phase prior to coupling.

It is contemplated that the method of the present invention can be implemented to the synthesis of a variety of polypeptides selected from analogues of glucagon or glucagon-like peptide-1 .

Any of the herein disclosed polypeptides, which can be synthesized by the disclosed method, are preferably synthesized using amino acid building blocks as herein defined, including naturally occurring amino acids and any chemically modified naturally occurring amino acids as well as any synthetic molecule comprising functional groups, notably an amine and a carboxylic acid, enabling the synthetic molecule to be used in SPPS and providing an amide bond. Thus, amino acid building blocks may be added to the polypeptide by way of a reaction cycle disclosed herein, be it the backbone of a target polypeptide or any peptide side chain attached to the backbone of a target polypeptide.

Polypeptides may be formed by a strategy including the formation of several peptide fragments which are subsequently coupled. Polypeptides comprising at least one peptide side chain may be formed by synthesizing at least part of the peptide side chain by SPPS and couple such a fragment to amino acid moieties of a backbone attached to a solid phase.

According to an aspect the method relates to a polypeptide selected from polypeptides comprising one peptide side chain, specifically peptides comprising one and only one peptide side chain.

According to a further aspect the entire peptide side chain of the polypeptide is synthesized by consecutive reaction cycles implemented after coupling of the amino acid building block serving as the locus for covalently attaching the peptide side chain (forming part of the backbone and typically amino acid moiety A) and before coupling of the next amino acid building block of the polypeptide backbone (main chain). If the peptide side chain is synthesized prior to the completion of the synthesis of the backbone PROT1 and PROT2 of the amino acid building block PROT1 -A(PROT2)-OH should preferably be selected such that PROT2 can be selectively deprotected with respect to PROT1 and any other protecting group (class (II) group protecting group) of amino acid side chains of amino acids present between the solid phase and the amino acid serving as the branching point. Thus, PROT2 is selected such that only PROT2 is deprotected in the deprotection procedure.

According to a further aspect, the method implements an SPPS strategy where class (II) protecting groups are acid-labile, signifying that the class (II) are cleaved under acidic conditions and that class (I) protecting are base-labile, are cleaved under alkaline conditions. If class (II) protecting groups are selected from the groups of acid-labile protecting groups, it is preferred that PROT1 and PROT2 are selected from the groups of alkaline(base)-labile protecting groups preferably with the proviso that PROT1 and PROT2 are selected such that only PROT2 is cleaved in a specific reaction cycle (which includes a de-protection procedure).

According to a further aspect class (I) protecting groups are Fmoc.

The peptide side chain may have the formula -Z²-Z¹; where -Z¹ is a fatty acid chain having a polar group at one end of the chain and a connection to Z², -X- at the end of the chain distal from the polar group; the polar group comprising a carboxylic acid or a carboxylic acid bioisostere, a phosphonic acid, or a sulfonic acid group; and -X- being a bond, -CO- -SO- or -SO2; -Z²- being a spacer of formula:

where each Y independently being -NH, -NR, -S or -0, where R is alkyl, a protecting group or forms a linkage to another part of the spacer Z²; each X independently being a bond, CO-, SO-, or SO2-; with the proviso that when Y is -S, X is a bond; each V independently being a bivalent organic moiety linking Y and X; and n is 1 -10.

According to an aspect -Z¹ may also be an acyl group of formula A-B-Alk-(CO)-; or a sulfonyl group of formula A-B-Alk-(SO2)-; A being a carboxylic acid or a carboxylic acid bioisostere preferably carboxylic acid; B being a bond, Ce arylene or Ce arylene- O-;

Aik being a saturated or unsaturated fatty acid chain comprising from 6 to 18 carbon atoms, preferably un-branched, optionally substituted with one or more of fluoro, C1-4 alkyl, trifluoromethyl, hydroxymethyl, amino, hydroxyl, C1-4 alkoxy, oxo and carboxyl; - Z² being -SA-, -SA-SB-, or -SB-SA-; -SA is a single amino acid residue selected from y- Glu, a-Glu, a-Asp, [3-Asp, Ala, [3-Ala (3-aminopropanoic acid (Dap)), and Gaba (4- aminobutanoic acid);

-SB- is a linker of general formula:

wherein n is 1 -10 and each Pu is independently selected from Pu' and Pu'"; each Pu' is independently a natural or unnatural amino acid moiety; and each Pu"' is independently a residue of general formula:

wherein m is 0-5 and p is 1 , 3, 4, or 5.

According to a further aspect, -Z²-Z¹ 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 an aspect, Z²-Z¹ is [17-carboxyheptadecanoyl]-isoGlu-PEG3-PEG3 or [17-carboxy-heptadecanoyl]-isoGlu-Gly-Ser-Gly-Ser-Gly-Gly.

PEG3 and PEG4 are oligomers of ethylene glycols containing 3 and 4 ethylene glycol units respectively.

Preferably, the carboxylic acid bioisostere has a proton having a pKa similar to the corresponding carboxylic acid. Examples of suitable bioisoteres may include, tetrazole, acylsulfomides, acylhydroxylamine, and squaric acid derivatives.

The fatty chain may be derived from a fatty acid, for example, it may be derived from a medium-chain fatty acid (MCFA) with an aliphatic tail of 6-12 carbon atoms, a long- chain fatty acid (LCFA) with an aliphatic tail of 13-21 carbon atoms, or a very long- chain fatty acid (LCFA) with an aliphatic tail of 22 carbon atoms or more. Examples of linear saturated fatty acids from which suitable fatty chains may be derived include tridecylic (tridecanoic) acid, myristic (tetradecanoic) acid, pentadecylic (pentadecanoic) acid, palmitic (hexadecanoic) acid, and margaric (heptadecanoic) acid. Examples of linear unsaturated fatty acids from which suitable fatty chains may be derived include myristoleic acid, palmitoleic acid, sapienic acid and oleic acid.

The fatty chain may be connected to Z² by an amide linkage, a sulfinamide linkage, a sulfonamide linkage, or by an ester linkage, or by an ether, thioether or amine linkage. Accordingly, the fatty chain may have at the co position, that is, the position distal to the polar group, a bond to Z² or an acyl (-CO-), sulfinyl (-SO-), or sulfonyl (- SO2-) group. Preferably, the fatty chain has an acyl (-CO-) group at the position distal to the polar group and is connected to Z² by an amide or ester linkage.

The method relates to a SPPS comprising a reaction cycle where an a-amine protected amino acid building block selected from the group consisting of PROT1- A(PROT2)-OH is coupled to a peptide or amino acid linked to a solid phase. The amino acid building block PROT1-A(PROT2)-OH c is used for the formation of the peptide side chain.

Hence, according to an aspect, the method relates to the synthesis of a polypeptide comprising a backbone and a peptide side chain covalently attached to the backbone. Reaction cycles related to the formation of a peptide side chain of a polypeptide are referred to as peptide side chain reaction cycles.

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 amino acid building block of formula PROT1-A(PROT2)-OH and as further characterized herein is formed by peptide side chain reaction cycles.

Reaction cycles related to the formation of a peptide side chain are referred to as peptide side chain reaction cycles or side chain reaction cycles.

Reaction cycles related to the formation of the polypeptide backbone are referred to as backbone reaction cycles.

Reaction cycles related to the formation of the polypeptide backbone are referred to as backbone reaction cycles. The reaction cycle comprising the coupling of an activated amino acid building block selected from PROT1-A(PROT2)-OH is hence a backbone reaction cycle. Any amino acid moiety of a backbone of a polypeptide, i.e. including the amino acid building block of formula PROT1-A(PROT2)-OH and as further characterized herein is formed by reaction cycles referred to as reaction cycles related to the formation of the backbone of the polypeptide or simply backbone reaction cycles.

Reaction cycles related to the formation (synthesis) of a peptide side chain attached to the amino acid moiety A is referred to as peptide side chain reaction cycles.

According to an aspect any one of activation, coupling, de-protection and optionally capping, preferably all of the procedures of one or more reaction cycles, preferably all reaction cycles, related to the formation a (peptide) side chain of a polypeptide (i.e. peptide side chain reaction cycles) is carried out at a temperature above about 30 °C, preferably from about 30 °C up to about 45 °C, preferably form about 30 °C up to about 35 °C.

According to a further aspect the de-protection procedure of reaction cycles related to the formation of the (peptide) side chain of a polypeptide (peptide side chain reaction cycles) is carried out in the presence of 1 ,8-Diazabicyclo[5.4.0]undec-7-ene (DBU). According to yet a further aspect, any one of activation, coupling, de-protection and optionally capping, preferably all of the procedures of one or more reaction cycles, preferably all reaction cycles, related to the formation of the backbone of a polypeptide (backbone reaction cycles) except the steps of the reaction cycle related to the amino acid moiety A is carried out at a temperature 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 up to about 55 °C, from about 40 °C up to about 50 °C, from about 45 °C up to about 50 °C.

Relevant to any reaction cycle, irrespective of the type of polypeptide disclosed herein, and relating to peptide side chain reaction cycles or backbone reaction cycles, the concentration of the relevant deprotection agent or deprotection agents, such as a base e.g. piperidine, or DBU may be variable under the de-protection procedure. Preferably the initial concentration of deprotection agent is lower than the final concentration. Accordingly, the deprotection agent is typically charged (added) multiple times, wherein the concentration of the deprotection agent in the reaction solution after the initial charge of deprotection agent is lower than the concentration after the final charge of deprotection agent.

An aspect relates to the application of a base, suitably piperidine, as the deprotection agent for the cleavage of the a-amine protecting groups (class (I)) related to backbone reaction cycles.

method

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 chain of the polypeptide typically 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.

The backbone of the polypeptide may comprise from about 10 up to about 50 amino acids.

According to a further aspect, the method relates to the synthesis of incretin analogues, for example incretin analogues selected from glucagon analogues, GLP-1 analogues, GIP analogues, oxyntomodulin analogues, exendin-4 analogues, and PYY analogues. Specifically, the method related to the synthesis of analogues of glucagon such as glucagon-like peptide-1 (GLP-1 ).

According to yet a further aspect, the method relates to the synthesis of a polypeptide selected from:

His-X2-X3-GTFTSDYSKYL-X15-X16-X17-X18-A-X20-DFI- A WLESA wherein:

X2 is selected from Aib, Ac3c, Ac4c and Ac5c; preferably Aib and Ac4c; X3 is selected from Gin and His; X15 is selected from Asp and Glu, preferably Asp; X16 is selected from Glu and T, preferably Glu ; X17 is selected from Arg and T; X18 is selected from Ala and Arg; X20 is selected from Lys and His preferably Lys;

Z¹ is a hydrocarbon chain, preferably a fatty chain, having a polar group at one end of the chain and a connection to Z², -X-, positioned at the end of the chain distal from the polar group, 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; -Z²- is a spacer of formula:

wherein: each Y is independently-NH, -NR, -S or -0, where R is alkyl, a protecting group or forms a linkage to another part of the spacer Z²; 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 bivalent organic moiety linking Y and X; and n is 1 -10.

According to an aspect X2 is selected from Aib and Ac4c; X3 is selected from Gin and His; X15 is Asp; X16 is Glu; X17 is selected from Arg and T; X18 is selected from Ala and Arg; X20 is Lys.

The peptide side chain -Z²-Z¹ is covalently bound to an amino acid moiety selected from diamino alkanoic acids comprising from 3 up to 10 carbon atoms, suitably diamino alkanoic acids comprises from 4 up to 8 carbon atoms, suitably from 5 up to 8, more preferably from 5 up to 7. Preferably, the diamino alkanoic acids are (a,w)- diamino alkanoic acids, preferably unbranched diamino alkanoic acid.

In some aspects Z1 is an acyl group of formula: A-B-Alk-(CO)- or a sulfonyl group of formula: A-B-Alk-(SO2)-; A is -COOH or a carboxylic acid bioisostere; B is a bond, Cearylene, or Cearylene-O-; Aik is a saturated or unsaturated hydrocarbon chain of 6 to 18 carbon atoms in length, optionally substituted with one or more substituents selected from fluoro, Ci-4alkyl, trifluoromethyl, hydroxymethyl, amino, hydroxyl, Ci-4alkoxy, oxo, and carboxyl;

-Z²- is -SA-, -SA-SB-, or -SB-SA-;

-SA- is a single amino acid moiety selected from y-Glu, a-Glu, a-Asp, [3-Asp, Ala, [3- Ala (3-aminopropanoic acid), and Gaba (4-aminobutanoic acid);

-SB- is a linker of general formula:

wherein m is 0-5 and p is 1 , 3, 4, or 5.

In some aspects m is 1 and p is 1 , that is, PU"' is a 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 herein as -PEG3-.

In some aspects, m is 2 and p is 1 , that is, Pll'" is a residue of 11 -amino-3,6,9- trioxaundecanoic acid (also known as H2N-PEG4-COOH). This residue is referred to herein as -PEG4-.

In some aspects, -Z¹ is an unbranched hydrocarbon chain comprising from 6 to 18 carbon atoms having a polar group at one end of the chain and a connection to Z², the polar group selected from carboxylic acid or a carboxylic acid bioisostere. In a further aspect, -Z²-Z¹ 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-GSGSGG; and

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

In a further aspect, -Z²-Z¹ is [17-carboxyheptadecanoyl]-isoGlu-PEG3-PEG3 or [17- carboxy-heptadecanoyl]-isoGlu-GSGSGG.

In an aspect, -Z²-Z¹ is [17-carboxyheptadecanoyl]-isoGlu-PEG3-PEG3 or [17- carboxy-heptadecanoyl]-isoGlu-GSGSGG.

In a further aspect, Z²-Z¹ is [17-carboxy-heptadecanoyl]-isoGlu-GSGSGG.

In a further aspect, the polypeptide has a sequence selected form:

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;

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-/V/-/2.

In a further aspect, the invention relates to polypeptides obtained by the method as defined above.

Herein is disclosed the SPPS of the polypeptide with the sequence:

/-/-His-Ac4c-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Ala-Ala- Lys-Asp-Phe-lle-Lys[N-(17-carboxy-heptadecanoyl-y-Glu-Gly-Ser-Gly-Ser-Gly-Gly-)]- Trp-Leu-Glu-Ser-Ala-/V/-/2. The building blocks of the peptide side chain are within the squared bracket. The polypeptide backbone contains 29 building blocks which are consecutively numbered from 1 to 29, His being number 1 and Ala number 29. The polypeptide has a mol-weight of 4231 ,6 g/mol.

The synthesis was performed in a 600 liter SPPS reactor and a 332 liter preactivation reactor. The synthesis was executed at a 12.61 mol scale resulting in 53,4 kg target polypeptide at 100% yield.

All amino acids were coupled individually apart from the consecutive Gly-Gly of the side chain which was coupled as a di-peptide. The peptide was synthesized using an Fmoc-based strategy implying that all amino acid building block, i.e. individual natural amino acids, the synthetic Ac4c amino acid and the Gly-Gly di-peptide, were provided having the a-amine protected with the Fmoc group. One exception was the Lys at position 24, which was provided in the form of Dde-Lys(Fmoc)-OH signifying that Dde was the a-amine protecting group while Fmoc was the amino acid side chain protecting group.

The dicarboxylic acid fatty hydrocarbon at the distal end of the peptide side chain (distal with respect to the polypeptide backbone) was provided with the distal carboxylic acid residue protected by tBu (tert-butyl), tBuOOC-CieH32-COOH.

The peptide side chain was assembled prior to the coupling of lie.

All reactive amino acid side chain amines were protected by acid-labile protecting groups selected from Boc (fe/t-butyloxycarbonyl), Trt (trityl), tBu, OtBu, and Pbf (2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl). Hence, an ‘orthogonal’ scheme was applied where Fmoc and Dde were removed under alkaline conditions whereas all amino acid side chain protection groups were removed under acid conditions simultaneously with cleavage of the polypeptide from the resin forming a crude polypeptide. List of building blocks:

Backbone:

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-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-lle-OH; Dde-Lys(Fmoc)-OH; Fmoc- Trp(Boc)-OH; Fmoc-Leu-OH; Fmoc-Glu-OtBu; Fmoc-Ser(tBu)-OH; Fmoc-Ala-OH

Peptide side chain:

Fmoc-Gly-Gly-OH; Fmoc-Ser(tBu)-OH; Fmoc-Gly-OH; Fmoc-Ser(tBu)-OH; Fmoc- Gly-OH; Fmoc-Glu-OtBu; Octadecane dioic acid mono tert-butyl ester

In general, each reaction cycle comprised the procedures of de-protection of the a- amine of amino acid or peptide bond to resin, coupling of a-amine protected amino acid, and acetylation of un-reacted a-amines (capping).

Prior to the coupling of amino acid 29, Ala, a Ramage linker (Fmoc-RMG-OH) was coupled to a polystyrene-based resin modified with aminoethyl (AM resin) implementing the following scheme:

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 agent in the presence of DIC

6) Wash with DMF

The amino acids 1-22 and 24-29 (except amino acid lle(23)) of the backbone were attached to the amino acid or peptide bound to the resin by the cycle of the following scheme:

1) 2,5 vol% piperidine in DMF (/V,/V-Dimethylformamide)

2) 12,5 vol% piperidine in DMF 3) Wash with DMF

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

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

6) Wash with DMF

The building blocks of the peptide side chain were attached by a cycle of 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) Coupling of Fmoc-AA-OH and tBuOOC-CieH32-COOH in the presence of DIC, Oxyma and DMF

6) Wash with DMF

7) Capping agent, acetic acid in DMF

8) Wash with DMF

The coupling of lie (23) was executed according to the following scheme:

1 ) 5 vol% piperidine in DMF

2) Wash with DMF

3) NH2OH x HCI in the presence of DIPEA (/V,/V-Diisopropylethylamine) and DMF

4) Wash with DMF

5) Repetition of steps 1 to 4

6) 10 vol% piperidine in DMF

7) Step 6 is repeated twice

8) Fmoc-lle-OH coupling in the presence of DIC Oxyma and DMF

9) Step 8 is repeated

10) Capping agent in the presence of DIC

11 ) Wash with DMF

Following the 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 pre-activated in the separate pre-activation reactor. Pre-activation was generally conducted for less than 10 minutes in the presence of DIC and Oxyma in the presence of DMF prior to addition to the SPPS reactor.

All de-protection, pre-activation, coupling and capping procedures related to the backbone polypeptide were executed under a temperature ranging from 45 °C to 50°C. The exception was the de-protection of the Fmoc a-amine protecting group of Trp preceding the coupling of Dde-Lys(Fmoc)-OH and the deprotection, activation and coupling of Dde-Lys(Fmoc)-OH which were conducted at a temperature of 30 to 35°C.

All de-protection, pre-activation, coupling and capping procedures related to the peptide side chain were executed under a temperature of 30 to 35°C.

Generally, the coupling was conducted during a period of from 30 to 50 minutes. Capping was carried out for 15 to 30 minutes.

The polypeptide was cleaved from the resin using a TFA (trifluoroacetic acid)/scavenger and precipitated by MTBE (ethyl-Te/t-Butyl Ether). The crude polypeptide was dissolved in an aqueous buffer and IPA. Subsequently, the polypeptide was purified in three different Reverse Phase Chromatographic (RPC) steps, subsequently concentrated by ultrafiltration prior to isopropanol removal by diaf iltration. The final product was isolated by lyophilization.

The SPPS yield was 93.9 kg peptide resin (prior to cleavage). Based on the resin 208 g of target polypeptide was generated per 1 kg of resin. The quantified yield was given by 93.9 kg peptide resin multiplied by 0.208 giving 19.5 kg target polypeptide on resin, i.e. 4.6 mol. The quantified yield (target polypeptide on resin) prior to cleavage was 4.6/12.61 around 36.5%. After cleavage 21 .2 kg target polypeptide was obtained, i.e. around 5.0 mol and 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 net target polypeptide was obtained (2.86 mol). The total overall yield after concentration and lyophilization (based on the theoretical amount of 53.4 kg target polypeptide for 12.61 mol synthesis scale) was 22.7% (12.1 kg divided by 53.4 kg) net target polypeptide corresponding to 2.86 mol).

In this example the activation of the model amino acid Dde-Lys(Fmoc)-OH in the presence of Oxyma and DIC, as activation agents, and benzylamine was examined.

Without activation of an amino acid the carboxylic acid and the amine of two amino acids will not condense, to form an amide bond/peptide bond, but neutralize.

Activation is the process of transforming an amino acid into a species which readily reacts with an amine thereby forming an amide bond. Also, the reactivity of the activated amino acid must be balanced such that the activated amino acid predominantly reacts with the intended amine.

There exists a multitude of activation agents. Here, DIC and Oxyma were used for transforming the amino acid into an activated state, an ester.

The activated ester of Dde-Lys(Fmoc)-OH readily reacts with the benzylamine according to the scheme below while Oxyma is liberated:

The activated ester of Dde-Lys(Fmoc)-OH is not stable in aqueous mixtures and degrades during HPLC analysis, but readily reacts with benzyl amine. Thus, the benzylamide of Dde-Lys(Fmoc)-OH serves as the proxy for the activated ester of Dde-Lys(Fmoc)-OH.

Activation of Dde-Lys(Fmoc)-OH in the presence of DIC and Oxyma was performed at 30 °C, 35 °C, 40 °C, 45 °C, 50 °C and 55 °C, respectively, for 0.5 min, 5 min, 10 min, 15 min, 20 min, 25 min and 30 minutes and then reacted with benzylamine In table 1 , unreacted starting material (SM) refers to Dde-Lys(Fmoc)-OH while activated ester (AE) refers to the benzylamide of Dde-Lys(Fmoc)-OH.

Table 1 : Results related to the activation of Dde-Lys(Fmoc)-OH with DIC, Oxyma and benzylamine as a function of activation temperatures and time.

From table 1 one can derive the following:

• As temperature increases the amount of AE increases to an optimum and then decreases at a given time

• As temperature increases the amount of impurities increases from a given time (indicated by shading in gray)

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%.

Example 3

In this example the peptide Dde-Lys(Fmoc)-Trp-Leu-Glu-Ser-Ala-NH2 was synthesized using the protocols presented in example 1.

Lys was provided as Dde-Lys(Fmoc)-OH and was activated in a separate preactivation reactor at 30 °C or at 35 °C for a period of 5, 10 and 15 min, respectively. After activation the solution containing the activated ester of Dde-Lys(Fmoc)-OH was transferred to the SPPS reactor. Activated esters of Dde-Lys(Fmoc)-OH reacted with the a-amine of tryptophan (Trp) under formation of an amide bond at a temperature equal to the temperature during the activation, i.e. 30 °C or 35 °C for a duration of 45 min. Subsequently, after 45 min, unreacted a-amines of Trp were capped (acetylated) by the addition of a capping agent (2 molar equivalents) in the presence of DIC (2 molar equivalents). Subsequent the capping, the peptides were cleaved from the resin with TFA, neutralized (NH4OAc:IPA:H2O) and diluted with DMF. After the work-up the solution comprising the cleaved peptides was analyzed by HPLC.

Figures 1- 6 present the HPLC chromatograms of the work-up solutions comprising

5 the cleaved peptide (Dde-Lys(Fmoc)-Trp-Leu-Glu-Ser-Ala-NH2). 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% to 10% and 10% to 90% at the end-point flow was 1 ,0 ml/min.

Table 2 below presents ‘significant peaks’ in the diagrams of figures 1-6. No. 6 is the o peptide Dde-Lys(Fmoc)-Trp-Leu-Glu-Ser-Ala-NH2.

Example 4

In Example 4, the conditions of the coupling of Dde-Lys(Fmoc)-OH were further investigated. The peptide Trp-Leu-Glu-Ser-Ala was synthesized using the protocols 5 presented in Example 1 and 3. For this purpose, pre-activation of Dde-Lys(Fmoc)- OH was performed in a separate pre-activation reactor. Pre-activation was performed for 5, 10, 20 and 30 minutes, respectively, in the presence of DIC and Oxyma in the presence of DMF prior to addition to the SPPS reactor for the coupling reaction with Trp-Leu-Glu-Ser-Ala, while the temperatures room temperature (RT), 35°C and 55°C were used for this reaction cycle including pre-activation and coupling to WLESA.

The results are shown in Tables 3, 4 and 5 below.

Table 3

The data from Table 3 show the purity in an HPLC analysis of the cleaved samples (UV detection at 290 nm). and they do show that the purity, and hence the yield, after the coupling of Dde-Lys(Fmoc)-OH is less than 100%. But the impurities present in the samples are accumulated impurities from the six couplings of the peptide fragment after the coupling of Dde-Lys(Fmoc)-OH. What is more important than the actual purity of the samples is the fact that we could only detect traces (< 0.1 %) of unreacted peptide from this coupling at room temperature (in form of acetylated peptide or peptide with free amino groups).

A further set of equivalent experiments were performed using 20 and 30 min preactivation at RT, 35 °C and 55 °C. The results are shown in Table 4 and Table 5, below. It is clear that at 55 °C, the purities of the samples (and hence the yields) are rapidly decreasing, and acetylated peptide and the peptide with free amino groups appear. Moreover, the acetylated peptide and the peptide with free amino groups have a much lower UV absorption at 290 nm, because only the tryptophan residue and the Fmoc and Dde groups are visible at this wavelength. Only the tryptophane residue is present in the un-coupled species in contrast to the product which contains all three. It is estimated that the product’s UV absorption is 4 times that of the uncoupled species (acetylated and free amine containing peptides), which corresponds to the molar ratios shown below in table 5. A large fraction of the amino groups has thus not reacted during the coupling of Dde-Lys(Fmoc)-OH. No traces of unreacted peptide were seen at 35 °C and at room temperature.

Table 4

Table 5

55 °C, Pre-activation 20 min

Table 5 cont.

55 °C, Pre-activation 30 min

Claims

1 . A method for synthesizing a polypeptide based on solid phase peptide synthesis, the polypeptide comprising a backbone, the backbone comprising one amino acid moiety A selected from diamino alkanoic acids comprising from 3 up to 10 carbon atoms and one peptide side chain covalently attached to the amino acid moiety A, the amino acid moiety A being coupled to an amino acid moiety W of the backbone, the method comprising at least one reaction cycle comprising:

(i) providing an amino acid building block PROT1-A(PROT2)-OH, PROT1 being an a- amine protection group and PROT2 being an amino acid side chain protection group;

(iii) coupling the activated amino acid building block PROT1-A(PROT2)-OH to the unprotected a-amine of amino acid moiety W of an amino acid sequence covalently attached to a solid phase thereby forming an amide bond.

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

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

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

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

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

7. The method according to anyone 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-A(PROT2)- OH is activated.

8. The method according to any one of the preceding claims, wherein A is selected from unbranched (a,co)-diamino alkanoic acids, preferably lysine.

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

10. The method according to any one of the preceding claims, wherein the method further comprises selective removal of 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 related to the formation of a peptide side chain attached to the amino acid moiety A, wherein the coupling is carried out at a temperature above about 30 °C, preferably from about 30 °C up to about 45 °C, preferably from about 30 °C up to 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-AWLESA wherein:

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

X3 is selected from Gin and His;

X15 is selected from Asp and Glu, preferably Asp;

X16 is selected from Glu and T, preferably Glu;

X17 is selected from Arg and T;

X18 is selected from Ala and Arg; X20 is selected from Lys and His preferably Lys;

-Z²- is a spacer of formula:

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

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

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

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

16. The method according to claim 15, wherein the polypeptide is /-/-His-Ac4c- QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)- WLESA-/VH2.

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