Novel dipeptide acylating agents

Abstract

Described are activated esters and oxazolone derivatives comprising the non-canonical amino acid, Aib; methods of making such compounds and methods of using such compounds in a process of making a target peptide comprising Aib. In addition, also the use of an activated dipeptide in a coupling reaction to obtain a target peptide, wherein the activated dipeptide comprises Aib, is described.

Description

[0001] NOVEL DIPEPTIDE ACYLATING AGENTS

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to activated dipeptides, such as oxazolone derivatives and ester derivatives, comprising a non-canonical amino acid, such as a-aminobutyric acid (Aib), methods of making such activated dipeptides and methods of using such activated dipeptides in a process of making a target peptide comprising one or more non-canonical amino acids.

[0004] BACKGROUND

[0005] Therapeutic peptides are a unique class of pharmaceutical agents composed of a series of well-ordered amino acids, usually with molecular weights of 500-10000 Da.

[0006] Chemical synthesis is the preferred method for the industrial preparation of peptides, because it can introduce versatile synthetic building blocks beyond the proteinogenic amino acids, such as unnatural amino acids, and biochemical or biophysical probes, allowing further modification or conjugation. Furthermore, the chemical synthesis process can be fully automated and easily scaled up. It provides a convenient and efficient approach for producing short- and mediumsized peptides, but often relies on the use of reagents and solvents which are classified as environmentally problematic substances by the European Chemicals Agency under the REACH (Registration, Evaluation, Authorisation, and Restriction of Chemicals) regulation. In addition, the chemical synthesis of long peptides remains challenging, and alternative strategies are therefore required.

[0007] Such an alternative approach is the recombinant synthesis of therapeutic peptides which is a powerful technique for the efficient production of peptides using modern biotechnology methods. The advantages of recombinant peptide synthesis include high yields, scalability greater purity, and the ability to introduce specific modifications for improved biological activity. However, if the target peptide comprises any modifications such as a side chain or a non- canonical amino acid (such as, e.g. semaglutide, pemvidutide (HXQFTFTSDYSKYLDEKAAKEFIQWLLQT.NH2 (Lactam: E-16, k-2), Tirzepatide,

[0008] Retatrutide, SAR441255, compound 0111 of WO 2022 / 129526 A1 , this technique may not be suitable due to the limitations of the expression system. A semi-synthetic or semi-recombinant approach can be a good alternative if recombinant peptide synthesis cannot be used for generating modified peptides. In a semi-synthetic approach, chemical synthesis is used to generate a partially synthetic peptide, which can then be combined with biological methods, such as recombinant expression of the modified portion. This method allows for the incorporation of non-natural amino acids or post-translational modifications that cannot be achieved through standard recombinant methods. A semirecombinant approach involves the use of both synthetic and recombinant techniques for peptide production. This technique can allow for greater control over peptide modification. For instance, semaglutide can be produced semi-recombinantly.

[0009] WO 2009 / 083549 (‘549) describes a method for the preparation of GLP-1 analogues and derivatives containing non-canonical amino acids (e.g. Aib). More specifically, ‘549 describes a ligation between a synthetically produced peptide fragment with a recombinantly produced larger fragment to obtain the desired backbone of semaglutide.

[0010] WO 2013 / 098191 describes the use of Fmoc-His-Aib-OH for the preparation of semaglutide.

[0011] There is however still a need to provide for alternative and more efficient processes for the preparation of therapeutic peptides comprising non-canonical amino acids, such as, e.g. a- aminoisobutyric acid (Aib).

[0012] SUMMARY

[0013] It is an objective of the present invention to overcome the above-mentioned drawbacks of the prior art.

[0014] It is another objective of the disclosure to provide a simple, cost-effective process for the production of peptides comprising one or more non-canonical amino acids, such as, e.g. Aib.

[0015] It is a further objective of the disclosure to provide activated but stable building blocks comprising a proteinogenic amino acid at the N-terminal and a non-canonical amino acid at the C-terminal. In particular, the present disclosure aims to provide dipeptide building blocks comprising His-Aib as activated ester or alternatively as an oxazolone.

[0016] It is another objective of the present disclosure to provide a simple, cost-effective, reproducible, commercially viable and / or industrially feasible process for the preparation of target peptides comprising at least one Aib, and more particularly comprising His-Aib. In particular embodiments such a target peptide is semaglutide, compound 0111 of WO 2022 / 129526 A1 , or tirzepatide. Compound 0111 of WO 2022 / 129526 A1 is

[0017] It is a further objective of the present disclosure to provide an efficient deprotection protocol for the deprotection of the ligated peptide. Preferably, the deprotection protocol is suitable in for a semi-recombinant approach.

[0018] According to a first aspect, the disclosure relates to an oxazolone of formula (6).

[0019] According to a second aspect, the disclosure relates to an activated ester of formula (5). According to a third aspect, the disclosure relates to a method of making an oxazolone of formula (6).

[0020] According to a fourth aspect, the disclosure relates to a method of making an activated ester of formula (5).

[0021] According to a fifth aspect, the disclosure relates to a process for the preparation of a target peptide, wherein the process comprises: a) providing a peptide to be coupled to an activated dipeptide, wherein the peptide is dissolved in water or an organic solvent or a mixture thereof, optionally wherein the organic solvent is miscible with water, and wherein the activated dipeptide comprises Aib; b) providing the activated dipeptide, wherein the activated dipeptide is dissolved in an organic solvent, optionally wherein the organic solvent is miscible with water; c) adding the activated dipeptide of step b to the peptide of step a; and optionally d) adding base or acid to adjust the pH.

[0022] The activated dipeptide may be an activated ester according to the second aspect of the disclosure or an ester obtainable by the method of the fourth aspect of the disclosure. Also, or alternatively, the activated dipeptide may be an oxazolone according to the first aspect of the disclosure or an oxazolone obtainable by the method according to the third aspect of the disclosure.

[0023] According to a sixth aspect, the disclosure relates the use of an activated dipeptide in a coupling reaction to obtain a target peptide, wherein the activated dipeptide comprises Aib. FIGURES

[0024] Fig. 1 shows Scheme 1 with additional details such as showing the various amino acids making up the backbone.

[0025] Fig. 2 shows Scheme 7 with additional details such as showing the various amino acids making up the backbone.

[0026] DESCRIPTION

[0027] In the following detailed description, numerous specific details are set forth to provide a full understanding of the subject technology. It will be apparent to one of ordinary skill in the art that the subject technology may be practised without some of these specific details. Well- known structures and techniques may not be shown in detail so as to not obscure the subject technology.

[0028] Documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer’s specifications, instructions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. In the event of a conflict between the definitions or teachings of such incorporated references and definitions or teachings recited in the present specification, the text of the present specification takes precedent.

[0029] In the following, the elements of the present disclosure will be described. These elements may be listed with specific elements, but it should be understood that they may be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed to limit the present disclosure to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0030] To facilitate an understanding of the present subject technology, a number of terms and phrases are defined below. Unless defined otherwise herein, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0031] In general, the present disclosure is directed to the provision of activated dipeptides. Examples of such activated dipeptides are oxazolones of formula (6) and activated esters of formula (5). The present disclosure also relates to methods of making such activated dipeptides, particularly wherein such dipeptides can be isolated. More particularly, the present disclosure is directed to activated dipeptides that can be used for large scale production and simplify process steps because they are stable enough to be isolated and / or to be stored at a temperature between -20 °C and 35 °C, preferably between -5 °C and 30 °C, without signs of significant degradation.

[0032] In addition, the activated dipeptides described herein or obtainable by the methods described herein can be used to produce a target peptide comprising a His-Aib moiety. More particularly, the activated dipeptides described herein or obtainable by the methods described herein can be used in peptide coupling reactions.

[0033] Definitions

[0034] Percentages, concentrations, amounts, and other numerical data may be expressed or presented herein in a "range” format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of "4% to 20%" should be interpreted to include not only the explicitly recited values of 4% to 20%, but to also include individual values and sub-ranges within the indicated range Thus, included in this numerical range are individual values such as 4, 5, 6, 7, 8, 9, 10, ... 18, 19, 20% and subranges such as from 4-10%, 5-15%, 10-20%, etc. This same principle applies to ranges reciting minimal or maximal values. Furthermore, such an interpretation should apply regardless of the breadth of the range, or the characteristics being described.

[0035] If no specific temperature is indicated, then it should be assumed that standard room temperature applies. As used herein, “standard room temperature” refers to a temperature interval of 19 °C to 30 °C.

[0036] It must be noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a filter" includes one or more of such different filters and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein. Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognise or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the present disclosure.

[0037] The term “and / or” wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term.

[0038] The term "comprise", and variations thereof such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or element or step or group of integer or element or step, but not the exclusion of any other integer or element or step or group of integer or element or step. When used herein the term “comprise” can be substituted with the terms “contain” or “include” or sometimes with “have”.

[0039] The term “obtainable” can be used interchangeably with “obtained”.

[0040] The term "about" is used herein to mean approximately, roughly, or around. When the term "about" is used in conjunction with a numerical range, it means within a statistically meaningful range of a value and modifies that range by extending the boundaries above and below the numerical values set forth. Such a range can be within an order of magnitude, typically within 10%, more typically within 5%, even more typically within 1% and most typically within 0.1% of the indicated value or range Sometimes, such a range can lie within the experimental error, typical of standard methods used for the measurement and / or determination of a given value or range.

[0041] As used herein, the term “ligated peptide” refers to a peptide obtained after a ligation reaction, sometimes also referred to as a coupling reaction. An example of such a ligation reaction can be found in Example 6 under the heading “Ligation Reaction”.

[0042] As used herein, the term “isolated” in reference to oxazolones according to the first aspect of the disclosure or activated esters according to the second aspect of the disclosure means that said oxazolones or esters are physically separated from the reaction mixture in which they were produced.

[0043] The amount of solvent employed in a chemical process, e.g., a reaction or crystallisation, may be referred to herein as a number of “volumes” or “vol” or “V”. For example, a material may be referred to as being suspended in 10 volumes (or 10 vol or 10 V) of a solvent. In this context, this expression would be understood to mean millilitres of the solvent per gram of the material being suspended, such as suspending 5 grams of a material in 10 volumes of a solvent means that the solvent is used in an amount of 10 millilitres of the solvent per gram of the material that is being suspended. In another context, the term “v / v” may be used to indicate the number of volumes of a solvent that are added to a liquid mixture based on the volume of that mixture. For example, adding solvent X (1.5 v / v) to a 100 ml reaction mixture would indicate that 150 mL of solvent X was added.

[0044] As used herein, the term “coupling” or “ligation” refers to a reaction where an amide bond is formed by reacting the C-terminal end of one peptide, e.g. a dipeptide, with the N-terminal end of another peptide. Conveniently, the coupling may be carried out in the presence of a coupling agent. Examples of coupling agents include but are not limited to benzotriazole- 1-yl- oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 3-

[0045] [Bis(dimethylamino)methyliumyl]-3H-benzotriazol-1-oxide hexafluorophosphate (HBTLI), 2- (1 H-Benzotriazole-1-yl)-1 ,1 ,3,3-tetranmethylaminium tetrafluoroborate (TBTLI), N-Ethyl-N’-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC*HCI), N,N’-dicyclohexylcarbodiimide (DCC) and N,N’-diisopropylcarbodiimide (DIC).

[0046] The wording “activation of carboxylic acid” refers to conversion of -OH of the carboxylic acid into a good leaving group by reacting with a carboxylic acid activator to result in an activated ester.

[0047] The term “activated ester” refers to an ester functional group that is highly susceptible toward nucleophilic attack. An activated ester undergoes the same reactions as their inactivated analogues but do so more rapidly. An activated ester can be prepared by a reaction of carboxylic acid function with a carboxylic acid activator, optionally in combination with an alcohol R3OH.

[0048] The term “protecting group” refers to a temporarily attached group to decrease reactivity of a functional group so that the protected functional group does not react under the reaction conditions to which the molecule is subjected in one or more subsequent steps.

[0049] The term “orthogonal set of protecting groups” as used herein refers to groups of protecting groups whose removal is accomplished in any order with reagents and conditions that do not affect protecting groups in any other orthogonal set. For instance, if a first protecting group can be removed by acid while a second protecting group remains unaffected by said acid, the first and the second protecting group are orthogonal protecting groups.

[0050] As used herein, an oxazolone or activated ester that is “stable in isolated form” means that said oxazolone or activated ester does not degrade via hydrolysation for several months (less than 0.5% hydrolysation during the indicated time frame), such as about 4 to 5 months, when stored at a temperature between about -20 °C and 25 °C. The amount of hydrolysation can be measured by qNMR using 1 ,3-benzodioxole as internal standard. Unstable oxazolones or activated esters hydrolyse by more than 1% w / w each day at room temperature.

[0051] Oxazolone derivatives

[0052] In a first aspect, the disclosure relates to an oxazolone derivative of formula (6),

[0053] Pg1and Pg2are both protecting groups. Conveniently, Pg1and Pg2can be different protecting groups such as e.g. Pg1is TFAc and Pg2being Ts. Preferably, Pg1and Pg2can be removed / cleaved under the same reaction conditions. Conveniently, Pg1and Pg2can be removed / cleaved in the same deprotection step. For example, Pg1is an acid-labile protecting group then it is preferable that Pg2is an acid-labile protecting group, too. Preferably, Pg1and Pg2are not an orthogonal set of protecting groups.

[0054] Pg1is a protecting group and can be selected from the group consisting of TFAc, Cbz, Dde or Moc. Preferably, Pg1is TFAc or Moc. Conveniently, TFAc and Moc protecting groups are suitable for use in semi-recombinant synthesis.

[0055] Pg2is a protecting group and can be selected from the group consisting of Ts, Bs, Mbs, Ths, Boc, Dnp, DCB, Bn, and Cbz.

[0056] The present inventors have surprisingly found that oxazolones according to the first aspect of the disclosure can be isolated and are surprisingly stable despite being activated. Conveniently, the oxazolones according to the first aspect of the disclosure are obtainable in isolated form. Advantageously, the oxazolones according to the first aspect of the disclosure can be generate in situ and used in a coupling reaction with another peptide directly or they can be isolated and stored. Conveniently, isolated oxazolones can be purified which can lead to a cleaner coupling reaction. In addition, less equivalents of the isolated oxazolone may be used in a coupling reaction compared to an oxazolone generated in situ and directly used in a coupling reaction. Advantageously, the use of isolated oxazolones allows for a convenient process because a bigger batch of oxazolones can be generated and be stored for later use. In some embodiments, the oxazolone is an isolated and / or purified oxazolone derivative. The oxazolone derivative may be purified by column chromatography. The oxazolone derivative may be purified by crystallisation.

[0057] Conveniently, by choosing protecting groups, such as e.g. Ts, oxazolones that are solid at ambient temperature can be generated. Typically, an oxazolone in solid form will correspond to a more stable oxazolone. The present inventors have, for instance, found, that the stability of the oxazolone typically increases from Pg2being Mbs to Ths to Ts / Bs when TFAc is used as Pg1.

[0058] In some embodiments, Pg1and Pg2are not orthogonal protecting groups. In some embodiments, each of Pg1and Pg2are acid labile. In some embodiments, each of Pg1and Pg2are cleavable by addition of a base and / or nucleophile. In some embodiments, each of Pg1and Pg2are cleavable by hydrogenation.

[0059] In some embodiments, Pg1is TFAc, Cbz, Dde, or Moc. In some embodiments, Pg2is Ts, Bs, Mbs, Ths, Boc, DCB, Dnp, Bn, or Cbz. In preferred embodiments, Pg2is Ts, Bs, Ths, or Boc.

[0060] In some particularly preferred embodiments, Pg1is TFAc, Dde, or Moc; and Pg2is Ts, Bs, or Ths. In some particular embodiments, the oxazolone is of formula (6E).

[0061] In some embodiments, Pg2is Ts, Bs, or Ths, and / or Pg1is Moc, wherein Rfis selected from the group consisting of

[0062] In some embodiments, Pg2is Ts, Bs, or Ths, and / or Pg1is Moc, wherein Rfis selected from the group consisting of

[0063] In some embodiments, Pg2is Ts, Bs, or Ths, and / or Pg1is Moc, wherein Rfis selected from the group consisting of

[0064] In some embodiments, the oxazolone derivative is selected from the group consisting of

[0065] In some particularly preferred embodiments, the oxazolone derivative is selected from the group consisting of Ester derivatives

[0066] In a second aspect, the disclosure relates to an activated ester of formula (5)

[0067] Pg1and Pg2are both protecting groups. Pg1and Pg2can be the same protecting group such as e.g. Pg1being Cbz and Pg2being Cbz. Conveniently, Pg1and Pg2can also be different protecting groups such as e.g. Pg1is TFAc and Pg2being Ts. Preferably, Pg1and Pg2can be removed / cleaved under the same reaction conditions. Preferably, Pg1and Pg2can be removed / cleaved in the same deprotection step. For example, Pg1is an acid-labile protecting group then it is preferable that Pg2is an acid-labile protecting group, too. Preferably, Pg1and Pg2are not an orthogonal set of protecting groups.

[0068] Pg1is a protecting group and can be selected from the group consisting of TFAc, Boc, Cbz, Dde or Moc. Preferably, Pg1is TFAc or Moc. Conveniently, TFAc and Moc protecting groups are suitable for use in semi-recombinant synthesis.

[0069] Pg2is a protecting group and can be selected from the group consisting of Ts, Bs, Mbs, Ths, Boc, Dnp, DCB, Bn, and Cbz.

[0070] The present inventors have surprisingly found that the activated esters according to the second aspect of the disclosure can be isolated and are surprisingly stable despite being activated. Conveniently, the activated esters according to the second aspect of the disclosure can be purified by chromatography and / or crystallisation. Conveniently, isolatable activated esters can be purified which leads to a cleaner coupling reaction. In addition, less equivalents of the isolated activated ester can be used in a coupling reaction compared to an activated ester generated in situ and directly used in a coupling reaction. Advantageously, the use of isolated activated esters allows for a convenient process because a bigger batch of activated esters can be generated and be stored for later use.

[0071] In some embodiments, Pg1and Pg2are cleavable under the same deprotection conditions. In some embodiments, Pg1and Pg2are not orthogonal protecting groups. In some embodiments, Pg2is selected from the group consisting of Ts, Bs, Mbs, Ths, Boc, DCB, Dnp, Bn, and Cbz. In particular embodiments, Pg2is Ts, Bs, Ths or Mbs. In some embodiments, Pg1is TFAc, Boc, Dde, Cbz or Moc. In some embodiments, Pg1is TFAc, Boc, Cbz or Moc, and Pg2is Ts. In some embodiments, Pg1is TFAc, Boc, Cbz, Dde, or Moc, and Pg2is Ths. In some embodiments, Pg1is TFAc, Boc, Cbz or Moc, and Pg2is Bs. In some embodiments, Pg1is TFAc, Boc, Cbz or Moc, and Pg2is Mbs or Ths.

[0072] In some embodiments, Pg1is TFAc. In some embodiments, Pg1is TFAc, and Pg2is Ts, Bs or Ths. In some embodiments, Pg1is Boc. In some embodiments, Pg1is Boc, and Pg2is Ts, Bs or Ths. In some embodiments, Pg1is Moc. In some embodiments, Pg1is Moc and Pg2is Ts, Bs or Ths. In some embodiments, Pg1is TFAc and Pg2is Cbz, and Ts, Bs or Ths.

[0073] In some embodiments, Pg1is Moc, and Rfis aryl or a linear or branched Ci-Ce alkyl group, wherein said Ci-Ce alkyl group is optionally substituted. In some embodiments, Pg2is Moc, and Rfis benzoyl, -CH3, -CH2CH3, -(CH2)3CH3, -(CH2)4CH3, -CH(CH3)(OCH3), or -CH2CH2CH2OCH3.

[0074] In some embodiments, Pg2is Ts, Bs, or Ths, and / or Pg1is Moc, wherein Rfis selected from the group consisting of

[0075] In some embodiments, R3is MNP or CSAP.

[0076] In some particular embodiments, the activated ester is selected from the group consisting of

[0077] Method of making an oxazolone derivative

[0078] In a third aspect, the disclosure relates to a method of making an oxazolone of formula (6) the method comprising reacting a compound of formula (4) or a salt thereof; coupling reagent to obtain the oxazolone of formula (6).

[0079] Pg1and Pg2are both protecting groups. Conveniently, Pg1and Pg2can be different protecting groups such as e.g. Pg1is TFAc and Pg2being Ts. Preferably, Pg1and Pg2can be removed / cleaved under the same reaction conditions. Preferably, Pg1and Pg2can be removed / cleaved in the same deprotection step. For example, Pg1is an acid-labile protecting group then it is preferable that Pg2is an acid-labile protecting group, too. Preferably, Pg1and Pg2are not an orthogonal set of protecting groups.

[0080] Pg1is a protecting group and can be selected from the group consisting of TFAc, Boc, Cbz, Dde or Moc. Preferably, Pg1is TFAc or Moc. Conveniently, TFAc and Moc protecting groups are suitable for use in semi-recombinant synthesis.

[0081] Pg2is a protecting group and can be selected from the group consisting of Ts, Bs, Mbs, Ths, Boc, Dnp, DCB, Bn, and Cbz.

[0082] In some embodiments, Pg1is a protecting group selected from the group consisting of TFAc, Boc, Dde, Cbz or Moc; and Pg2is a protecting group selected from the group consisting of Ts, Bs, Mbs, Ths, Boc, DCB, Dnp, Bn, and Cbz.

[0083] In some embodiments, Pg1and Pg2are both labile under acidic conditions. In some embodiments, Pg1and Pg2are both labile under nucleophilic conditions. In some embodiments, Pg1and Pg2are both cleavable by hydrogenation. In some embodiments, Pg1is TFAc. In some embodiments, Pg1is Boc. In some embodiments, Pg1is Moc.

[0084] In some embodiments, Pg1is Moc, and Rfis a linear or branched Ci-Ce alkyl group, wherein said Ci-Ce alkyl group is optionally substituted. In some embodiments, Pg1is Moc, and Rfis In some embodiments, Pg1is Dde.

[0085] In some embodiments, Pg1is Moc and is selected from the group consisting of

[0086] In some particular embodiments, Pg1is Moc and is selected from the group consisting of

[0087] In some embodiments, Pg2is Ts. In some embodiments, Pg2is Bs. In some embodiments, Pg2is Mbs. In some embodiments, Pg2is Ths.

[0088] In some embodiments, the coupling reagent is selected from the group consisting of 1- [Bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATLI), chloro-1 H-benzotriazol-1-yl)oxy](dimethylamino)- / \ / , / \ / - dimethylmethaniminium tetrafluoroborate (TCTLI), (Benzotriazol-1- yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), 4-(4,6-Dimethoxy-1 ,3,5- triazin-2-yl)-4-methylmorpholin-4-ium tetrafluoroborate (DMTMM BF4), T3P, and 2- Hydroxypyridine 1 -oxide (HOPO) in combination with diisopropylcarbodiimide (DIC), EDC, or N,N -Dicyclohexylcarbodiimide (DCC).

[0089] In some embodiments, the coupling reagent is PyBOP, DMT-MM*BF4, or DCC.

[0090] In some embodiments, the coupling reagent is DCC and the method also comprises adding a phenol derivative having a pKa below 6.0. In some embodiments, the coupling reagent is DCC and the method also comprises adding a phenol derivative having a pKa in the range of about 2 to about 6.

[0091] In some embodiments, the oxazolone derivative is generated in situ. Conveniently, PyBOP or DMT-MM*BF4 may be used to generate the oxazolone in situ.

[0092] In some embodiments, the stable oxazolone derivative is generated by using DCC as a coupling reagent.

[0093] In some embodiments, the method comprises an additional step of adding a peptide to generate a target peptide. In some embodiments, the target peptide is semaglutide. In some embodiments, the target peptide is a peptide comprising His-Aib.

[0094] In particular embodiments, the method comprises an additional step of isolating and optionally purifying the so obtained oxazolone. In particular embodiments, oxazolones obtainable by a method according to the third aspect of the disclosure are stable in isolated form.

[0095] Method of making an activated ester

[0096] In a fourth aspect, the disclosure relates to a method of making an activated ester of formula

[0097] (5) wherein the method comprises the steps of: a) providing a compound of formula (4), and b) reacting the compound of formula (4) with a coupling reagent or with a coupling reagent and an alcohol or thiol derivative to obtain the activated ester of formula (5).

[0098] Pg1and Pg2are both protecting groups. Pg1and Pg2can be the same protecting group such as e.g. Pg1being Cbz and Pg2being Cbz. Conveniently, Pg1and Pg2can also be different protecting groups such as e.g. Pg1is TFAc and Pg2being Ts. Preferably, Pg1and Pg2can be removed / cleaved under the same reaction conditions. Preferably, Pg1and Pg2can be removed / cleaved in the same deprotection step. For example, Pg1is an acid-labile protecting group then it is preferable that Pg2is an acid-labile protecting group, too. Preferably, Pg1and Pg2are not an orthogonal set of protecting groups.

[0099] Pg1is a protecting group and can be selected from the group consisting of TFAc, Boc, Cbz, Dde, or Moc. Preferably, Pg1is TFAc or Moc. Conveniently, TFAc and Moc protecting groups are suitable for use in semi-recombinant synthesis.

[0100] Pg2is a protecting group and can be selected from the group consisting of Ts, Bs, Mbs, Ths, Boc, Dnp, DCB, Bn, and Cbz.

[0101] X is sulfur or oxygen.

[0102] In some embodiments, Pg1and Pg2are both labile under acidic conditions. In some embodiments, Pg1and Pg2are both labile under nucleophilic conditions. In some embodiments, Pg1and Pg2are both cleavable by hydrogenation.

[0103] In some embodiments, Pg1is a protecting group selected from the group consisting of TFAc, Boc, Dde, Cbz, or Moc; and Pg2is a protecting group selected from the group consisting of Ts, Bs, Mbs, Ths, Boc, DCB, Dnp, Bn, and Cbz.

[0104] In some embodiments, Pg1and Pg2are both labile under acidic conditions. In some embodiments, Pg1and Pg2are both labile under nucleophilic conditions. In some embodiments, Pg1and Pg2are both cleavable by hydrogenation. In some embodiments, Pg1is TFAc. In some embodiments, Pg1is Boc. In some embodiments, Pg1is Moc.

[0105] In some embodiments, Pg1is Moc, and wherein Rfis a linear or branched Ci-Ce alkyl group, wherein said Ci-Ce alkyl group is optionally substituted. In some embodiments, Pg1is Moc,

[0106] In some embodiments, Pg1is Dde.

[0107] In some embodiments, Pg1is Moc and wherein Rfis selected from the group consisting of

[0108] In some particular embodiments, Pg1is Moc and wherein Rfis selected from the group consisting of

[0109] In some embodiments, Pg2is Ts. In some embodiments, Pg2is Bs. In some embodiments, Pg2is Mbs. In some embodiments, Pg2is Ths.

[0110] In some embodiments, R3is of formula (7),

[0111] Rais H, F, Cl, OCH3, NO2, SO2N(CH3)2, SO3H, C(O)Me, CN, or Ms;

[0112] Rbis H, F, Cl, NO2, SO2N(CH3)2, C(O)Me, CN, or Ms,

[0113] Rcis H, F CI, OCH3INO2, SO2N(CH3)2, SO3H, C(O)Me, CN, or Ms;

[0114] Rdis H, F, Cl, NO2, SO2N(CH3)2, C(O)Me, CN, or Ms; and Reis H, F, Cl, OCH3, NO2, SO2N(CH3)2, SO3H, C(O)Me, CN, or Ms; or wherein Raand Rbjointly form a 5 or 6 membered heterocycle.

[0115] In some embodiments, the alcohol derivative is a phenol derivative having a pKa < 7.5, such as a pKa in the range of about 6.5 to about 7.5.

[0116] In some embodiments, the alcohol derivative is a phenol derivative having a pKa of about 7. In some embodiments, the alcohol derivative or thiol derivative is selected from the group consisting of

[0117] In particular embodiments, the alcohol derivative is 2-methoxy-4-nitrophenol or 5-chloro-2- hydroxy- / V, / \ / -dimethylbenzenesulfonamide.

[0118] In particular embodiments, the method comprises an additional step of isolating and optionally purifying the so obtained activated ester. In particular embodiments, activated esters obtainable by a method according to the third aspect of the disclosure are stable in isolated form.

[0119] Alternatively, in some embodiments, the activated ester is generated in situ.

[0120] In some embodiments, the method comprises an additional step of adding a peptide to generate a target peptide. In some embodiments, the target peptide is semaglutide, compound 0111 of WO 2022 / 129526 A1 , or tirzepatide.

[0121] Process for the preparation of a target peptide

[0122] In a fifth aspect, the disclosure relates to process for preparation of a target peptide, wherein the process comprises: a) providing a peptide to be coupled to an activated dipeptide, wherein the peptide is dissolved in water and / or an organic solvent, such as, e.g. an alcohol, NMP, DMSO, or a mixture thereof, and wherein the activated dipeptide comprises Aib; b) providing the activated dipeptide, wherein the activated dipeptide is dissolved in an organic solvent, optionally wherein the organic solvent is miscible with water; c) adding the activated dipeptide of step b to the peptide of step a; and optionally d) adding base or acid to adjust the pH.

[0123] The activated dipeptide can be an activated ester according to the second aspect of the disclosure or an activated ester obtainable by the method of the fourth aspect of the disclosure or an oxazolone according to the first aspect of the disclosure or an oxazolone obtainable by the method according to the third aspect of the disclosure. Conveniently, the activated dipeptide comprises Aib.

[0124] In particular embodiments, the target peptide comprises His-Aib.

[0125] The organic solvent may be selected from the group consisting of EtOH, / PrOH, NMP, DMSO, DMF, MeCN, THF, acetone, and a mixture thereof. In particular embodiments, the organic solvent is EtOH, iPrOH, DMSO, NMP, or a mixture thereof.

[0126] The process may be run at a pH in the range of 7.5 to 9. In some embodiments, the pH is in the range of 7.8 to 8.5.

[0127] The process may be run at a temperature in the range of 5 to 45 °C, preferably in a range between 19 °C to 30 °C.

[0128] In some embodiments, the ratio between the peptide to be coupled and the activated dipeptide is about 1 :1 to about 1 :3.5, such as 1 :2 to 1 :3.

[0129] In some embodiments, the target peptide is semaglutide, compound 0111 of WO 2022 / 129526 A1 , or tirzepatide.

[0130] In some embodiments, the process comprises an additional step, wherein the additional step is a deprotection step. In particular embodiments, the deprotection step comprises adding a nucleophile such as acetohydroxamic acid or hydroxylamine. In particular embodiments, the deprotection step comprises adding an acid, such as phosphor acid, hydrochloric acid or sulfonic acid. In further particular embodiments, the deprotection step comprises adding concentrate phosphoric acid. In particular embodiments, the deprotection step is a hydrogenation step.

[0131] Use of an activated dipeptide

[0132] In a sixth aspect, the disclosure relates to the use of an activated dipeptide in a coupling reaction to obtain a target peptide, wherein the activated dipeptide comprises Aib.

[0133] In some embodiments, the activated dipeptide is an oxazolone according to the first aspect of the disclosure.

[0134] In some embodiments, the activated dipeptide is an activated ester according to the second aspect of the disclosure.

[0135] In some embodiments, the activated dipeptide is an oxazolone obtainable according to the third aspect of the disclosure, and wherein the activated dipeptide is in isolated form.

[0136] In some embodiments, the activated dipeptide is an activated ester obtainable according to the fourth aspect of the disclosure, and wherein the activated dipeptide is in isolated form. Preferably, the activated dipeptide is suitable for use in semi-recombinant synthesis.

[0137] Overview of selected chemical moiety described in this disclosure

[0138] If there is discrepancy between the abbreviation / chemical name and the structure, then the structure takes precedence. Table 1 : Overview of chemical moieties, their chemical names, and abbreviations.

[0139]

[0140] List of embodiments

[0141] 1. An activated ester of formula (5), wherein

[0142] X is oxygen or sulfur; Pg1is a first protecting group;

[0143] Pg2is a second protective group; and

[0144] R3is MNP or CSAP.

[0145] 2. The activated ester according to embodiment 1 , wherein the ester is isolated or isolatable.

[0146] 3. The activated ester according to embodiment 1 or embodiment 2, wherein the ester is stable in isolated form.

[0147] 4. The activated ester according to any one of embodiments 1-3, wherein Pg1and Pg2are cleavable under the same deprotection conditions.

[0148] 5. The activated ester according to any one of embodiments 1-4, wherein Pg1and Pg2are not orthogonal protecting groups.

[0149] 6. The activated ester according to any one of embodiments 1-5, wherein R3is MNP, and optionally wherein X is oxygen.

[0150] 7. The activated ester according to any one of embodiments 1-6, wherein R3is CSAP and optionally wherein X is oxygen.

[0151] 8. The activated ester according to any one of embodiments 1-7, wherein Pg2is selected from the group consisting of Ts, Bs, Mbs, Ths, Dnp, Boc, DCB, Bn, and Cbz.

[0152] 9. The activated ester according to any one of embodiments 1-8, wherein Pg2is Ts, Bs, Mbs or Cbz.

[0153] 10. The activated ester according to any one of embodiments 1-9, wherein Pg2is Ts.

[0154] 11. The activated ester according to any one of embodiments 1-9, wherein Pg2is Bs.

[0155] 12. The activated ester according to any one of embodiments 1-8, wherein Pg2is Mbs or Ths.

[0156] 13. The activated ester according to any one of embodiments 1-12, wherein Pg1is TFAc, Boc,

[0157] Cbz, Dde or Moc.

[0158] 14. The activated ester according to any one of embodiments 1-12, wherein Pg1is TFAc.

[0159] 15. The activated ester according to any one of embodiments 1-12, wherein Pg1is Boc or Dde.

[0160] 16. The activated ester according to any one of embodiments 1-12, wherein Pg1is Cbz.

[0161] 17. The activated ester according to any one of embodiments 1-12, wherein Pg1is Moc.

[0162] 18. The activated ester according to embodiment 17, wherein Rfis a linear or branched Ci-Ce alkyl group, and wherein said Ci-Ce alkyl group is optionally substituted. 19. The activated ester according to embodiment 18, wherein the Ci-Ce alkyl group is linear.

[0163] 20. The activated ester according to embodiment 18 or embodiment 19, wherein the Ci-Ce alkyl group is substituted by an methoxy or ethoxy group.

[0164] 21. The activated ester according to embodiment 18 or embodiment 19, wherein the Ci-Ce alkyl group is substituted by a carbocyclic group or a heterocyclic group.

[0165] 22. The activated ester according to embodiment 17, wherein Rfis selected from the group consisting of 23. The activated ester according to any one of embodiments 1 to 5, wherein the activated ester is selected from the group consisting of

[0166] 24. An oxazolone derivative of formula (6), wherein

[0167] Pg1is a protecting group selected from the group consisting of TFAc, Boc, Cbz, Dde, and Moc, preferably wherein Pg1is TFAc, Moc, or Dde; and

[0168] Pg2is a protecting group selected from the group consisting of Ts, Bs, Mbs, Ths, Boc, Dnp, DCB, Bn, and Cbz.

[0169] 25. The oxazolone derivative according to embodiment 24, wherein the oxazolone derivative is isolated or isolatable.

[0170] 26. The oxazolone derivative according to embodiment 24 or embodiment 25, wherein the oxazolone derivative is stable in isolated form.

[0171] 27. The oxazolone derivative according to any one of embodiments 24-26, wherein Pg1is TFAc.

[0172] 28. The oxazolone derivative according to any one of embodiments 24-26, wherein Pg1is Boc or Dde.

[0173] 29. The oxazolone derivative according to any one of embodiments 24-28, wherein Pg2is Ts.

[0174] 30. The oxazolone derivative according to any one of embodiments 24-28, wherein Pg2is Bs.

[0175] 31. The oxazolone derivative according to any one of embodiments 24-28, wherein Pg2is Dnp.

[0176] 32. The oxazolone derivative according to any one of embodiments 24-28, wherein Pg2is Ths or Mbs.

[0177] 33. The oxazolone derivative according to any one of embodiments 24-28, wherein Pg2is Boc.

[0178] 34. The oxazolone derivative according to any one of embodiments 24-26, wherein the oxazolone derivative is selected from the group consisting of

[0179]

[0180] 35. The oxazolone derivative according to any one of embodiments 24-34, wherein the oxazolone derivative is obtainable in solid form.

[0181] 36. A method of making an oxazolone derivative (6) the method comprising reacting a compound of formula (4);

[0182] Pg1is a first protecting group;

[0183] Pg2is a second protecting group; and wherein

[0184] Pg1and Pg2are cleavable under the same deprotection conditions.

[0185] 37. The method according to embodiment 36, wherein Pg1is a protecting group selected from the group consisting of TFAc, Boc, Dde or Moc; and Pg2is a protecting group selected from the group consisting of Ts, Bs, Mbs, Ths, Boc, Dnp, DCB, Bn, and Cbz. 38. The method according to embodiment 36 or embodiment 37, wherein Pg1and Pg2are both labile under acidic conditions.

[0186] 39. The method according to embodiment 36 or embodiment 37, wherein Pg1and Pg2are both labile under nucleophilic conditions.

[0187] 40. The method according to embodiment 36 or embodiment 37, wherein Pg1and Pg2are both cleavable by hydrogenation.

[0188] 41. The method according to embodiments 36 or 37, wherein Pg1is TFAc.

[0189] 42. The method according to embodiments 36 or 37, wherein Pg1is Boc.

[0190] 43. The method according to embodiments 36 or 37, wherein Pg1is Moc, and optionally wherein Rfis a linear or branched Ci-Ce alkyl group, wherein said Ci-Ce alkyl group is optionally substituted.

[0191] 44. The method according to embodiment 43, wherein Rfis selected from the group consisting

[0192] 45. The method according to embodiments 36 or 37, wherein Pg1is Dde.

[0193] 46. The method according to any one of embodiments 36-37, or 41-45, wherein Pg2is Ts.

[0194] 47. The method according to any one of embodiments 36-37, or 41-45, wherein Pg2is Bs.

[0195] 48. The method according to any one of embodiments 36-37, or 41-45, wherein Pg2is Mbs.

[0196] 49. The method according to any one of embodiments 36-37, or 41-48, wherein Pg2is Ths or

[0197] Dnp.

[0198] 50. The method according to any one of embodiments 36-49, wherein the coupling reagent is selected from the group consisting of 1-[Bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5- b]pyridinium 3-oxide hexafluorophosphate (HATLI), chloro-1 H-benzotriazol-1- yl)oxy](dimethylamino)-N,N-dimethylmethaniminium tetrafluoroborate (TCTLI), (Benzotriazol- 1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), 4-(4,6-Dimethoxy-1 ,3,5- triazin-2-yl)-4-methylmorpholin-4-ium tetrafluoroborate (DMTMM BF4), T3P, and 2- Hydroxypyridine 1-oxide (HOPO) in combination with diisopropylcarbodiimide (DIC), EDC, orN,N’-Dicyclohexylcarbodiimide (DCC).

[0199] 51. The method according to any one of embodiments 36-50, wherein the coupling reagent is PyBOP.

[0200] 52. The method according to any one of embodiments 36-50, wherein the coupling reagent is DMT-MM*BF4, EDC, or T3P.

[0201] 53. The method according any one of embodiments 36-50, wherein the coupling reagent is DCC.

[0202] 54. The method according to embodiment 53, wherein DCC is added in combination with a phenol derivative having a pKa below 6, such as in the range of about 2 to about 6, is added.

[0203] 55. The method according any one of embodiments 36-54, wherein the method comprises an additional step of purifying the compound of formula (6).

[0204] 56. A method of making an activated ester of formula (5) wherein the method comprises the steps of: a) providing a compound of formula (4) or a salt thereof, and b) reacting the compound of formula (4) with a coupling reagent or with a coupling reagent and an alcohol derivative; and wherein

[0205] X is oxygen or sulfur;

[0206] Pg1is a first protecting group;

[0207] Pg2is a second protecting group; and wherein

[0208] Pg1and Pg2are cleavable under the same deprotection conditions. 57. The method according to embodiment 56, wherein Pg1is a protecting group selected from the group consisting of TFAc, Boc, Cbz, Dde or Moc and Pg2is a protecting group selected from the group consisting of Ts, Bs, Mbs, Ths, Boc, Dnp, DCB, Bn, and Cbz.

[0209] 58. The method according to embodiment 56 or embodiment 57, wherein Pg1and Pg2are both labile under acidic conditions.

[0210] 59. The method according to embodiment 56 or embodiment 57, wherein Pg1and Pg2are both labile under nucleophilic conditions.

[0211] 60. The method according to embodiment 56 or embodiment 57, wherein Pg1and Pg2are both cleavable by hydrogenation.

[0212] 61. The method according to embodiment 56 or embodiment 57, wherein Pg1is TFAc.

[0213] 62. The method according to embodiment 56 or embodiment 57, wherein Pg1is Boc.

[0214] 63. The method according to embodiment 56 or embodiment 57, wherein Pg1is Moc.

[0215] 64. The method according to embodiment 63, wherein Rfis a linear or branched Ci-Ce alkyl group, wherein said Ci-Ce alkyl group is optionally substituted.

[0216] 65. The method according to embodiment 63, wherein the Ci-Ce alkyl group is substituted by an methoxy or ethoxy group.

[0217] 66. The method according to embodiment 63, wherein the Ci-Ce alkyl group is substituted by a carbocyclic group or a heterocyclic group.

[0218] 67. The method according to embodiment 63, wherein Rfis selected from the group consisting of

[0219] 68. The method according to embodiment 56 or embodiment 57 or any one of embodiments 61 to 67, wherein Pg2is Bs.

[0220] 69. The method according to embodiment 56 or embodiment 57 or any one of embodiments 61 to 67, wherein Pg2is Ts. 70. The method according to embodiment 56 or embodiment 57 or any one of embodiments 61 to 67, wherein Pg2is Mbs.

[0221] 71. The method according to embodiment 56 or embodiment 57 or any one of embodiments 61 to 67, wherein Pg2is Ths.

[0222] 72. The method according to embodiment 56 or embodiment 57 or any one of embodiments 61 to 67, wherein Pg2is Cbz.

[0223] 73. The method according to embodiment 56 or embodiment 57 or any one of embodiments 61 to 67, wherein Pg2is Bn.

[0224] 74. The method according to any one of the embodiments 56 to 73, wherein R3is of formula (7), wherein

[0225] Rais H, F, Cl, OCH3, NO2, SO2N(CH3)2, SO3H, C(O)Me, CN, or Ms;

[0226] Rbis H, F, Cl, NO2, SO2N(CH3)2, C(O)Me, CN, or Ms;

[0227] Rcis H, F CI, OCH3, NO2, SO2N(CH3)2, SO3H, C(O)Me, CN, or Ms;

[0228] Rdis H, F, Cl, NO2, SO2N(CH3)2, C(O)Me, CN, or Ms; and

[0229] Reis H, F, Cl, OCH3, NO2, SO2N(CH3)2, SO3H, C(O)Me, CN, or Ms; or wherein Raand Rbjointly form a 5 or 6 membered heterocycle.

[0230] 75. The method according to any one of embodiments 56 to 75, wherein the alcohol derivative is a phenol derivative having a pKa < 7.5.

[0231] 76. The method according to any one of embodiments 56 to 76, wherein the alcohol derivative is a phenol derivative having a pKa of 6.5 to 7.5.

[0232] 77. The method according to any one of embodiments 56 to 77, wherein the alcohol derivative is a phenol derivative having a pKa of about 7.

[0233] 78. The method according to any one of the embodiments 56 to 74, wherein R3is 2-methoxy- 4-nitrophenol.

[0234] 79. The method according to any one of the embodiments 56 to 74, wherein R3is 5-chloro-2- hydroxy- / V, / \ / -dimethylbenzenesulfonamide. 80. The method according to any one of embodiments 56 to 79, wherein the coupling reagent is DOC, DIG, or EDC.

[0235] 81 . The method according to any one of embodiments 56 to 80, wherein the alcohol or thiol derivative is selected from the group consisting of d

[0236] 82. The method according to any one of embodiments 56 to 81 , wherein the alcohol derivative is 5-chloro-2-hydroxy- / V, / \ / -dimethylbenzenesulfonamide.

[0237] 83. The method according to any one of embodiments 56 to 81 , wherein the phenol derivative is 2-methoxy-4-nitrophenol.

[0238] 84. The method according to any one of embodiments 56 to 83, wherein the activated ester is generated in situ.

[0239] 85. The method according to any one of embodiments 36 to 55, wherein the oxazolone derivative is generated in situ.

[0240] 86. The method according to embodiment 84 or embodiment 85, wherein the method comprises an additional step of adding a peptide to generate a target peptide.

[0241] 87. The method according to embodiment 86, wherein the target peptide is semaglutide or Aib8, Arg34GLP- 1(7-37).

[0242] 88. The method according to embodiment 86, wherein the target peptide is compound 0111 of

[0243] WO 2022 / 129526 A1 or

[0244] HAibEGTFTSDVSSYLEEQAAREFIAWLVRGRKGGGGEASELSTAALGRLSAELHELATLPR TETGSGSP (SEQ ID NO: 1).

[0245] 89. The method according to embodiment 86, wherein the target peptide is tirzepatide or YAibEGTFTSDYSIAibLDKIAQKAFVQWLIAGGPSSGAPPPS (SEQ ID NO: 2).

[0246] 90. The method according to embodiment 86, wherein the target peptide is a peptide comprising Aib. 91. The method according to embodiment 86, wherein the target peptide is a peptide comprising His-Aib.

[0247] 92. A process for the preparation of a target peptide, wherein the process comprises: a) providing a peptide to be coupled to an activated dipeptide, wherein the peptide is dissolved in water, an organic solvent or a mixture thereof; b) providing the activated dipeptide, wherein the activated dipeptide is dissolved in an organic solvent, optionally wherein the organic solvent is miscible with water; c) addition the activated dipeptide of step b to the peptide of step a; d) adding base or acid to adjust the pH.

[0248] 93. The process according to embodiment 92, wherein the activated dipeptide is an activated ester according to any one of embodiments 1 to 23 or an oxazolone derivative according to any one of embodiments 24 to 35.

[0249] 94. The process according to embodiment 92 or embodiment 93, wherein the organic solvent is selected from the group consisting of EtOH, / PrOH, NMP, DMSO, MeOH, DMF, MeCN, THF, acetone, and a mixture thereof.

[0250] 95. The process according to embodiment 94, wherein the organic solvent is / PrOH, DMSO, NMP, or a mixture thereof.

[0251] 96. The process according to embodiment 94 or embodiment 95, wherein the organic solvent is NMP.

[0252] 97. The process according to any one of embodiments 92 to 96, wherein the pH is in a range of 7.5 to 9, preferably 7.8 to 8.5.

[0253] 98. The process according to any one of embodiments 92 to 97, wherein the process is run at a temperature in the range of 5 to 45 °C, preferably in a range between 20 to 30 °C.

[0254] 99. The process according to any one of embodiments 92 to 98, wherein the ratio between the peptide to be coupled and the activated dipeptide ranges between 1 :1.5 to 1 :3.5.

[0255] 100. The process according to any one of embodiments 92 to 98, wherein the ratio between the peptide to be coupled and the activated dipeptide is about 1 :2 to about 1 :3.

[0256] 101. The process according to any one of embodiments 92 to 100, wherein target peptide is semaglutide, and optionally the peptide is N{Epsilon-26}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4- (17- carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acet yl]-[Arg34]-GLP-1-(9-37)-peptide (8) or Arg38GLP-1 (9-37).

[0257] 102. The process according to any one of embodiments 92 to 100, wherein the target peptide is compound 0111 of WO 2022 / 129526 A1 or HAibEGTFTSDVSSYLEEQAAREFIAWLVRGRKGGGGEASELSTAALGRLSAELHELATLPR TETGSGSP (SEQ ID NO: 1).

[0258] 103. The process according to any one of embodiments 92 to 100, wherein the target peptide is tirzepatide or YAibEGTFTSDYSIAibLDKIAQKAFVQWLIAGGPSSGAPPPS (SEQ ID NO: 2).

[0259] 104. The process according to any one of embodiments 92 to 103, wherein the process further comprises a deprotecting step to obtain the target peptide.

[0260] 105. The process according to embodiment 104, wherein the deprotecting step comprises adding a nucleophile.

[0261] 106. The process according to embodiment 105, wherein the nucleophile is selected from the group consisting of acetohydroxamic acid, hydroxylamine, and / \ / ,2-dihydroxy benzamide or / V- hydroxy / V-methylacetamide.

[0262] 107. The process according to embodiment 104, wherein the deprotecting step comprises adding an acid.

[0263] 108. The process according to embodiment 107, wherein the acid is phosphor acid, hydrochloric acid or sulfonic acid.

[0264] 109. The process according to embodiment 104, wherein the deprotecting step is a hydrogenation step.

[0265] 110. The process according to embodiment 109, wherein the hydrogenation step comprises that addition of Pd / C.

[0266] 111. Use of an isolated activated ester derivative in a coupling reaction to obtain a target peptide, wherein the activated ester derivative is derivable from a dipeptide comprising Aib, preferably comprising His-Aib, preferably wherein the isolated activated ester derivative is suitable for use in semi-recombinant synthesis.

[0267] 112. Use according to embodiment 111 , wherein the isolated activated ester derivative is obtainable by a process according to any one of embodiments 56 to 84.

[0268] 113. Use according to embodiment 111 , wherein the isolated activated ester derivative is an activated ester according to any one of embodiments 1-23. 114. Use of an isolated oxazolone derivative in a coupling reaction to obtain a target peptide, wherein the oxazolone derivative is derivable from a dipeptide comprising Aib, preferably comprising His-Aib, preferably wherein the isolated activated ester derivative is suitable for use in semi-recombinant synthesis.

[0269] 115. Use according to embodiment 114, wherein the isolated oxazolone derivative is obtainable by a process according to any one of embodiments 36 to 55.

[0270] 116. Use according to embodiment 114, wherein the isolated oxazolone derivative is an activated ester according to any one of embodiments 24-35.

[0271] 117. Use of an activated dipeptide in a coupling reaction to obtain a target peptide, wherein the target peptide comprises Aib, preferably wherein the target peptide comprises His-Aib, preferably wherein the activated dipeptide is suitable for use in semi-recombinant synthesis.

[0272] 118. Use according to embodiment 117, wherein the activated dipeptide is an oxazolone according to the first aspect of the disclosure.

[0273] 119. Use according to embodiment 117, wherein the activated dipeptide is an activated ester according to the second aspect of the disclosure.

[0274] 120. The activated ester according to any one of the embodiments 1-23, wherein the activated ester is suitable for use in semi-recombinant synthesis.

[0275] 121. The activated oxazolone according to any one of the embodiments 24-35, wherein the activated oxazolone is suitable for use in semi-recombinant synthesis.

[0276] 122. The method according to any one of the embodiments 36-55, wherein the activated oxazolone is suitable for use in semi-recombinant synthesis.

[0277] 123. The method according to any one of the embodiments 56-83, wherein the activated oxazolone is suitable for use in semi-recombinant synthesis.

[0278] Examples

[0279] This experimental part starts with a list of abbreviations and is followed by a section including general methods for synthesizing intermediates and characterizing dipeptide analogues and activated derivatives of the invention. Then follows several examples which relate to the preparation of specific C-terminal Aib activated peptides, and at the end several examples have been included relating to the properties of these C-terminal Aib activated peptides and their use in semi-recombinant ligation of peptide or protein analogues to produce desired derivatives thereof. The examples serve to illustrate the invention.

[0280] List of Abbreviations

[0281] AcOH: Acetic acid

[0282] AHA: Acetohydroxamic acid

[0283] Aib: 2-Aminoisobutyric acid

[0284] Backbone: Peptide or peptide analogue

[0285] Boc: tert Butyloxycarbonyl

[0286] Bn: Benzyl

[0287] Bs Benzenesulfonyl

[0288] BSA / V,O-bis(trimethylsilyl)acetamide

[0289] Cbz Benzyloxycarbonyl

[0290] CH3CN: Acetonitrile

[0291] CSAP 4-Chloro-2-(N,N-dimethylsulfamoyl)phenyl

[0292] DCC: N,N’-Dicyclohexylcarbodiimide

[0293] DCM: Dichloromethane

[0294] DCU: Dicyclohexylurea

[0295] DHB: / V,2-dihydroxybenzamide

[0296] DIG: Diisopropylcarbodiimide

[0297] DI PEA: Diisopropylethylamine

[0298] DMAP: 4-Dimethylaminopyridine

[0299] DMF: / V, / V-Dimethylformamide

[0300] DMO: 4,4-dimethyloxazol-5(4H)-one

[0301] DMSO: Dimethyl sulfoxide

[0302] DMTMM BF4: 4-(4,6-Dimethoxy-1 ,3,5-triazin-2-yl)-4-methylmorpholin-4-ium tetrafluoroborate

[0303] EtOAc: Ethyl acetate

[0304] EtOH: Ethanol

[0305] Et2O: Diethyl ether

[0306] Eq.: Equivalent

[0307] FA: Formic acid

[0308] Fmoc: 9 H-fluoren-9-ylmethoxycarbonyl

[0309] GC: Gas chromatography HATLI 1-[Bis(dimethylamino)methylene]-1H-1 ,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

[0310] HexMoc: Hexanoyloxy(methoxy)carbonyl

[0311] HFIP: Hexafluoroisopropanol

[0312] HMA: / V-hydroxy- / V-methylacetamide

[0313] HOPO: 2-Hydroxypyridine 1 -oxide

[0314] HPLC: High-performance liquid chromatography

[0315] / -PrOH Isopropanol

[0316] IPC A: In process control sample A

[0317] LG: Leaving group

[0318] Mbs 4-Methoxybenzenesulfonyl

[0319] MeCN: Acetonitrile

[0320] MeOH Methanol

[0321] MeTHF Methyl tetrahydrofuran

[0322] MNP 2-methoxy-4-nitrophenyl

[0323] MS: Mass spectrometry

[0324] Ms Mesyl

[0325] MW: Molecular weight

[0326] NA: Normalized area

[0327] NHS: N-Hydroxysuccinimide

[0328] NMP: 1-Methyl-pyrrolidin-2-one

[0329] Pac Phenylacetyl

[0330] Pd / C: Palladium on carbon

[0331] PTFE: Polytetrafluoroethylene

[0332] PyBOP: (Benzotriazol-l-yloxy)tripyrrolidinophosphonium hexafluorophosphate qNMR: Quantitative nuclear magnetic resonance

[0333] Rf: Retention factor

[0334] RT: Room temperature

[0335] RP: Reverse phase

[0336] RPM Revolutions per minute

[0337] RX: Double jacketed reactor

[0338] TCTU: Chloro-1 H-benzotriazol-1-yl)oxy](dimethylamino)-N,N- dimethylmethaniminium tetrafluoroborate

[0339] TEA: Triethylamine

[0340] TFA: Trifluoroacetic acid THF: Tetrahydrofurane

[0341] Ths: Thiophene-2-sulfonyl

[0342] TIPS: Triisopropylsilane

[0343] Tj: Jacket temperature

[0344] TLC: Thin layer chromatography

[0345] Ts: Tosyl

[0346] TSTLI: / V, / V, / V’, / V-Tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate

[0347] Vol or V: Volume

[0348] Wt%: Weight percentage w / w: Weight / weight

[0349] LIPLC: Ultra-performance liquid chromatography

[0350] General Methods of Detection and Characterization

[0351] 1. LC-MS methods

[0352] Method: HPLC-MS_1 : RP-analysis was performed using Waters HPLC system and Waters evaporative light scattering (ELS) detector and Waters SQ Detector 2 mass spectrometer fitted with a Kinetix C18 column (26 pm, 4.6 x 50 mm, column own temperature 40°C). Eluent A: 0.10% (vol / vol) formic acid in water. Eluent B: 0.10% (vol / vol) formic acid in acetonitrile. The analysis was performed at RT by injecting an appropriate volume of the sample (preferably 0.5-5 pl) onto the column which was eluted with a gradient of A and B. The HPLC conditions, detector settings and mass spectrometer settings. Gradient: 5 to 100 % (vol / vol) B, 10 min., 1.2 ml / min. UV detection at 254 nm. MS ionization mode: API-ES+; Scan 100-1500 amu.

[0353] Method: UPLC-MS_2: RP-analysis was performed using Waters Acquity UPLC system and Waters Xevo G2-XS Q-tof mass spectrometer fitted with Waters BEH C18 column (1.7 pm, 2.1 x 150 mm, column own temperature 40°C). Eluent A: 0.10% (vol / vol) formic acid in water. Eluent B: 0.10% (vol / vol) formic acid in acetonitrile. The analysis was performed at RT by injecting an appropriate volume of the sample (preferably 0.5-5 pl) onto the column which was eluted with a gradient of A and B. The UPLC conditions, detector settings and mass spectrometer settings. Gradient: Linear 95% - 0% (vol / vol) A, 0% - 95% (vol / vol) B and 5% (vol / vol) D, 4 min., 0.4 ml / min. UV detection at 214 and 280 nm. MS ionization mode: API-ES+; Scan 50-4000 amu. Method: UPLC-MS_3: RP-analysis was performed using Waters Acquity LIPLC system and Waters Xevo G2-XS Q-tof mass spectrometer fitted with Waters BEH 04 column (1 .7 pm, 2.1 x 150 mm, column own temperature 40°C). Eluent A: 0.10% (vol / vol) formic acid in water. Eluent B: 0.10% (vol / vol) formic acid in acetonitrile. The analysis was performed at RT by injecting an appropriate volume of the sample (preferably 0.5-5 pl) onto the column which was eluted with a gradient of A and B. The LIPLC conditions, detector settings and mass spectrometer settings. Gradient: Linear 95% - 0% (vol / vol) A, 0% - 95% (vol / vol) B and 5% (vol / vol) D, 6 min., 0.4 ml / min. UV detection at 214 and 280 nm. MS ionization mode: API-ES+; Scan 50-4000 amu.

[0354] Method: UPLC-MS_4: RP-analysis was performed using Waters Acquity LIPLC system and Acquity QDa mass detector fitted with Waters BEH C18 column (1.7 pm, 2.1 x 50 mm, column own temperature 40°C). Eluent A: 0.05% (vol / vol) TFA in water. Eluent B: 0.05% (vol / vol) TFA in acetonitrile. The analysis was performed at RT by injecting an appropriate volume of the sample (preferably 0.5-10 pl) onto the column which was eluted with a gradient of A and B. The LIPLC conditions, detector settings and mass spectrometer settings. Gradient: Linear 5%

[0355] - 25% (vol / vol) B, 4 min, 0.45 ml / min. UV detection at 214 nm. MS ionization mode: API-ES+; Scan: 50-1250 amu.

[0356] Method: UPLC-MS_5: RP-analysis was performed using Waters Acquity UPLC system and Acquity QDa mass detector fitted with Waters BEH C18 column (1.7 pm, 2.1 x 50 mm, column own temperature 40°C). Eluent A: 0.05% (vol / vol) TFA in water. Eluent B: 0.05% (vol / vol) TFA in acetonitrile. The analysis was performed at RT by injecting an appropriate volume of the sample (preferably 0.5-10 pl) onto the column which was eluted with a gradient of A and B. The UPLC conditions, detector settings and mass spectrometer settings. Gradient: Linear 5%

[0357] - 60% (vol / vol) B, 4 min, 0.45 ml / min. UV detection at 214 nm. MS ionization mode: API-ES+; Scan: 100-1250 amu.

[0358] Method: UPLC-MS_6: RP-analysis was performed using Waters Acquity UPLC system and Acquity QDa mass detector fitted with Waters BEH C4 column (1.7 pm, 2.1 x 50 mm, column own temperature 40°C). Eluent A: 0.05% (vol / vol) TFA in water. Eluent B: 0.05% (vol / vol) TFA in acetonitrile. The analysis was performed at RT by injecting an appropriate volume of the sample (preferably 0.5-10 pl) onto the column which was eluted with a gradient of A and B. The UPLC conditions, detector settings and mass spectrometer settings. Gradient: Linear 5% - 95% (vol / vol) B, 4 min, 0.45 ml / min. UV detection at 214 nm. MS ionization mode: API-ES+;

[0359] Scan: 50-1250 amu.

[0360] Method: UPLC-MS_7: RP-analysis was performed using Waters Acquity LIPLC system and Acquity QDa mass detector fitted with Waters BEH C4 column (1.7 pm, 2.1 x 50 mm, column own temperature 40°C). Eluent A: 0.05% (vol / vol) TFA in water. Eluent B: 0.05% (vol / vol) TFA in acetonitrile. The analysis was performed at RT by injecting an appropriate volume of the sample (preferably 0.5-10 pl) onto the column which was eluted with a gradient of A and B. The LIPLC conditions, detector settings and mass spectrometer settings. Gradient: Linear 25%

[0361] - 60% (vol / vol) B, 3.5 min, 0.45 ml / min. UV detection at 214 nm. MS ionization mode: API-ES+; Scan: 50-1250 amu.

[0362] Method: UPLC-MS_8: RP-analysis was performed using Waters Acquity UPLC system and Acquity QDa mass detector fitted with Waters BEH C4 column (1.7 pm, 2.1 x 50 mm, column own temperature 40°C). Eluent A: 0.05% (vol / vol) TFA in water. Eluent B: 0.05% (vol / vol) TFA in acetonitrile. The analysis was performed at RT by injecting an appropriate volume of the sample (preferably 0.5-10 pl) onto the column which was eluted with a gradient of A and B. The UPLC conditions, detector settings and mass spectrometer settings. Gradient: Linear 25%

[0363] - 75% (vol / vol) B, 4 min, 0.40 ml / min. UV detection at 214 nm. MS ionization mode: API-ES+; Scan: 50-1250 amu.

[0364] Method: UPLC-MS_9: RP-analysis was performed using Waters Acquity UPLC system and Acquity QDa mass detector fitted with Waters BEH C4 column (1.7 pm, 2.1 x 50 mm, column own temperature 40°C). Eluent A: 0.05% (vol / vol) TFA in water. Eluent B: 0.05% (vol / vol) TFA in acetonitrile. The analysis was performed at RT by injecting an appropriate volume of the sample (preferably 0.5-10 pl) onto the column which was eluted with a gradient of A and B. The UPLC conditions, detector settings and mass spectrometer settings. Gradient: Linear 25%

[0365] - 60% (vol / vol) B, 7 min, 0.40 ml / min. UV detection at 214 nm. MS ionization mode: API-ES+; Scan: 50-1250 amu.

[0366] Method: UPLC-MS_10: RP-analysis was performed using Waters Acquity UPLC system and Acquity QDa mass detector fitted with Waters BEH C4 column (1.7 pm, 2.1 x 150 mm, column own temperature 40°C). Eluent A: 0.05% (vol / vol) TFA in water. Eluent B: 0.05% (vol / vol) TFA in acetonitrile. The analysis was performed at RT by injecting an appropriate volume of the sample (preferably 0.5-10 pl) onto the column which was eluted with a gradient of A and B. The LIPLC conditions, detector settings and mass spectrometer settings. Gradient: Linear 25% - 60% (vol / vol) B, 16 min, 0.40 ml / min. UV detection at 214 nm. MS ionization mode: API-ES+; Scan: 50-1250 amu.

[0367] 2. LC methods and GC methods

[0368] Method: HPLC_11 : RP-analysis was performed using Agilent 1260 infinity HPLC system fitted with Phenomenex EVO-C18 column (2.6 pm, 3.0 x 150 mm, column own temperature 40°C). Eluent A: 0.1% (vol / vol) TFA in water. Eluent B: 0.1% (vol / vol) TFA in acetonitrile. The analysis was performed at RT by injecting an appropriate volume of the sample (preferably 0.5-10 pl) onto the column which was eluted with a gradient of A and B. The LIPLC conditions, detector settings and mass spectrometer settings. Gradient: isocratic / linear 2% (vol / vol) B, 5 min, 2% - 30% (vol / vol) B, 2 min, 30% (vol / vol) B, 6.5 min, 30% - 98% (vol / vol) B, 4.5 min, 0.8 ml / min. Diode array detection at 210 nm.

[0369] Method: UPLC_12: RP-analysis was performed using Waters Acquity LIPLC system fitted with Waters BEH C4 column (1.7 pm, 2.1 x 150 mm, column own temperature 40°C). Eluent A: 10% (vol / vol) MeCN in water. Eluent B: 95% (vol / vol) MeCN in water. Eluent C: 1 % (vol / vol) TFA in 95% (vol / vol) MeCN in water. The analysis was performed at RT by injecting an appropriate volume of the sample (preferably 0.5-10 pl) onto the column which was eluted with a gradient of A and B and a constant flow of 10% C. Gradient: Linear 8% - 49% (vol / vol) B, 24 min, 0.40 ml / min. UV detection at 210 nm.

[0370] Method: UPLC_13: RP-analysis was performed using Thermo Scientific Vanquish UPLC system fitted with a Phenomenex Kinetix column (1.7 pm, 2.1 x 100 mm, C18, 100 A, column own temperature 40°C). Eluent A: 0.1 % (vol / vol) TFA in 10% (vol / vol) MeCN in water. Eluent B: 0.1 % (vol / vol) TFA in 90% (vol / vol) MeCN in water. The analysis was performed at RT by injecting an appropriate volume of the sample (preferably 0.5-10 pl) onto the column which was eluted with a gradient of A and B. Gradient: 0% - 25% - 75 % (vol / vol) B, 6 min, 0.40 ml / min. UV detection at 210 nm.

[0371] Method: HPLC_14: Normal phase analysis was performed using Agilent 1260 infinity HPLC system fitted with a Inertsil diol column (3.0 pm, 4.6 x 150 mm, LC-0003, column own temperature 40°C). Eluent A: 0.05 % (vol / vol) FA in 85% (vol / vol) n-heptane in I PA. Eluent B: 0.05 % (vol / vol) FA in 99.95% (vol / vol) I PA. The analysis was performed at RT by injecting an appropriate volume of the sample (preferably 0.5-10 pl) onto the column which was eluted with a gradient of A and B. Gradient: isocratic / linear 0% (vol / vol) B, 20 min, 0% - 35% (vol / vol) B, 1 min, 35% (vol / vol) B, 5 min, 1.2 ml / min. UV detection at 220 nm.

[0372] Method: HPLC_15: Normal phase analysis was performed using Agilent 1260 infinity HPLC system fitted with a Chiralpak IA-3 column (3.0 pm, 4.6 x 250 mm, LC-0009, column own temperature 40°C). Eluent: 0.05 % (vol / vol) FA in 80% (vol / vol) n-heptane in I PA. The analysis was performed at RT by injecting an appropriate volume of the sample (preferably 1-10 pl) onto the column, which was eluted isocratic over 25 min, 1.2 ml / min. UV detection at 220 nm.

[0373] Method: HPLC_16: Reverse phase analysis was performed using Agilent 1260 infinity HPLC system and UV detector fitted with an Agilent Poroshell 120 Chiral-V (Part N° 683975-604) (2.7 pm, 4.6 x 150 mm, column own temperature 40°C). Eluent: 20 mM ammonium formate, 0.2% Formic Acid in water / methanol 65 / 35. The analysis was performed at RT by injecting an appropriate volume of the sample (1 pl) onto the column, which was eluted isocratic over 50 min, 0.2 ml / min. UV detection at 230 nm.

[0374] Method: UPLC_17: RP-analysis was performed using Waters Acquity UPLC system fitted with Waters CSH C18 column (1.7 pm, 2.1 x 150 mm, column own temperature 60°C). Eluent A: 10% (vol / vol) MeCN in a pH 7.2 aqueous buffer solution of 20.0 mM Na2SC>4, 2.0 mM Na^PCL and 2.0 mM Na2HPO4. Eluent B: 70% (vol / vol) MeCN in water. The analysis was performed at RT by injecting an appropriate volume of the sample (preferably 0.5-10 pl) onto the column which was eluted with a gradient of A and B. Gradient: Linear 10% - 95% (vol / vol) B, 3.5 min, 0.45 ml / min. UV detection at 214 nm.

[0375] Method: GC_18: Gas chromatography analysis was performed using Agilent 6850 system fitted with Agilent CP-Volamine GC column (0.32 mm x 30 m). Nitrogen was used as a carrier gas (25 mL / min). The flow rates of zero air and hydrogen in flame ionized detector (FID) were 350 mL / min and 35 mL / min, respectively. The optimized HS injector parameters were oven start temperature, 40°C; isotherm time, 5 min; heating rate 10°C / min; oven end temperature, 230°C; isotherm time 3 min; inlet temperature, 225°C; flow 1.0 mL / min; injection volume, 2 mL; split ratio 5:1 or 20:1.

[0376] Method: KF_19: The water content is determined by Karl Fisher method using as solvent about

[0377] 40 mL of methanol. Titrate the solvent till neutralization.

[0378] Add about 10 mL of sample and titrate with Reactive for Karl Fischer factored. Calculation

[0379] VTx F x 0.1 Water content %w / w = -

[0380] Ws

[0381] Where:

[0382] Ws= Sample weight in g

[0383] VT= Volume of Karl Fischer reagent in mL

[0384] F=Factor of Karl Fischer reagent in mg / mL

[0385] Consider drift value when applying the above formula.

[0386] General Methods of Preparation

[0387] All commercial materials (Merck, Fluorochem, Nova, Combiblocks, Iris, ABCR, Johnson Matthey) were used without further purification. All solvents were reagent grade or HPLC grade. Yields refer to chromatographically pure compounds, %-conversions were obtained by comparison of high- or ultra-performance liquid chromatography (HPLC or LIPLC) peak areas of products and starting materials. Thin layer chromatography (TLC) or ultra-performance liquid chromatography with mass spectrometer (UPLC-MS) was used to monitor reaction progress. Thin layer chromatography was performed on aluminum plates, precoated with silica gel (Merck 25, 20 x 20 cm, 60 F254) using the indicated eluent systems. Visualization was carried out by illumination with a UV-lamp (254 nm) or by staining with a solution of potassium permanganate (dissolve 1.5g of KMnO4, 10g K2CO3, and 1.25mL 10% NaOH in 200mL water). High resolution mass spectrometry (HRMS) for characterizing the material and products was performed on Waters Xevo G2-XS Q-tof mass spectrometer. Peptides were quantified using charged aerosol detection (CAD) on a Thermo Scientific Vanquish LC-system.1H,13C and19F NMR spectra were recorded at 400 MHz, 100 MHz and 376 MHz, respectively, on a Bruker Aeon 400 instrument or 500.13 MHz (1H) and 125.758 MHz (13C), on a Bruker Ascend 500 instrument. For qNMR, 1 ,3-benzodioxole was used as the standard reference. Chemical shifts are reported in ppm on the 8 scale relative to the chemical shift of the deuterated solvent. Flash column chromatography (FC) was performed using silica gel 60 A (32-63 .m).

[0388] Compound 13 corresponds to compound 0111 of WO 2022 / 129526 A1.

[0389] / V';, / \ / T-bis(tert-butoxycarbonyl)-L-histidine (3Bg) Commercially available (Merck Cat. No. 853067)

[0390] A / T-benzyl- / \ / ';-(tert-butoxycarbonyl)-L-histidine (3Bh) Commercially available (Activate Scientific Cat. No. AS221982)

[0391] / V' tert-butoxycarbonyl)- / V osyl-L-histidine (3Bb) Commercially available (Aurum Pharmatech Cat. No M-1053)

[0392] To a stirred suspension of L-histidine (1) (40.0 g, 258.0 mmol) and triethylamine (TEA) (44.9 mL, 322.0 mmol) in methanol (MeOH) (300 mL) was added dropwise ethyl trifluoroacetate (38.3 mL, 322.0 mmol) over 20 minutes under mild cooling conditions at around 10°C (cold water bath). The resulting mixture was stirred at room temperature for 20 hours. A sample was taken out and diluted (1 :99) with MeCN / H2O (1 / 1). The sample was analyzed by method UPLC- MS_4. After complete conversion of the starting material the MeOH was evaporated, the residue was dispersed in tetra hydrofuran (THF) (300 mL) and evaporated to dryness (repeated three times). The residue was dried in vacuo, affording triethylammonium (2,2,2- trifluoroacetyl)-L-histidinate (2A) as a white solid (78.9 g, quantitative yield). Purity >99% (1H NMR).1H NMR (400 MHz, DMSO-d6) 6 ppm 1.13 (t, J=7.3 Hz, 5 H), 2.93-3.02 (m, 4 H), 3.09 (dd, J=14.4 Hz, J=4.2 Hz, 1 H), 4.31-4.39 (m, 1 H), 6.78 (s, 1 H), 7.63 (d, J = 1 .0 Hz, 1 H), 9.43 (bs, 1 H), 9.97 (bs, 1 H).13C NMR (100 MHz, DMSO-d6) 6 ppm 8.5, 28.1 , 45.1 , 53.9, 116.0 (q, J = 286.6 Hz), 117.0, 133.2, 134.6, 155.7 (q, J = 35.7 Hz), 172.1.19F NMR (376 MHz, DMSO- d6) 5 ppm -74.5. LCMS, m / z calcd. for C8H9F3N3O3 [M+H]+252.0591 , found 252.0889.

[0393] General Procedure A: Synthesis of TFAcHis(Pq2)-OH (3Aa-d)

[0394] Triethylammonium (2,2,2-trifluoroacetyl)-L-histidinate (2A) (10.0 mmol, 1.0 eq.) was dispersed in THF (16.0 mL) and 1.0 M aqueous sodium bicarbonate / sodium carbonate (NaHCC>3 / Na2CO3) buffer (pH=8.6, 13.0 mL) was added. The resulting suspension was cooled at 0°C under argon and aryl sulfonyl chloride (10.0 mmol, 1.0 eq.) was added dropwise. The cooling bath was removed, and the reaction mixture was stirred for 90-210 minutes, gradually becoming a clear pale-yellow solution. A sample was taken out and diluted (1 :99) with MeCN. The diluted sample was analyzed by method UPLC-MS_5. After complete conversion of the starting material the THF was removed under reduced pressure and the residue was adjusted to pH=3.0-5.5 using 1 M aqueous solution of hydrochloric acid (HCI) (approximate 8 mL). The resulting thick suspension was extracted with ethyl acetate (EtOAc) (3 x 30 mL). The combined organic phase was washed with 0.2 M aqueous solution of HCI (2 x 24 mL) and dried over anhydrous magnesium sulphate (MgSC>4). Evaporation of the solvent and drying in vacuo afforded compound 3.

[0395] General Procedure B: Synthesis of Pq1His(Pq2)Aib-OH

[0396]

[0397] 2-Aminoisobutyric acid (Aib-OH) (54,6 mmol, 1.2 eq.) was dispersed in anhydrous THF (85 mL) and / V,O-bis(trimethylsilyl)acetamide (BSA) (105.0 mmol, 2.4 eq.) was added. The resulting mixture was heated under reflux for 3.5-20 hours (until all the Aib was dissolved), then, still under argon, cooled to room temperature.

[0398] In another round-bottom flask protected L-histidine (3) (43.7 mmol, 1.0 eq.) was dissolved under argon in anhydrous THF (150 mL). 2-hydroxypyridine 1-oxide (HOPO) (48.0 mmol, 1.1 eq.) was added, followed in 10 minutes by / V, / V-dicyclohexylcarbodiimide (DCC) (50.2 mmol, 1.2 eq.). The resulting mixture was stirred for 3 hours. Anhydrous oxalic acid (6.6 mmol, 0.2 eq.) was added and stirring continued for 15-30 minutes. The resulting suspension was filtered directly into a 500 mL round-bottom flask and the solution of silyl protected Aib was added slowly. The resulting colorless suspension was stirred at 40°C to room temperature over 1-19 hours, gradually becoming a clear pale-yellow solution. The progress of the reaction was monitored using method HPLC-MS_1 , UPLC-MS_5 or UPLC-MS_6. The volume of the reaction mixture was reduced to approximate 100 mL and poured slowly into a vigorously stirred 5% aqueous solution of potassium bisulphate (KHSO4) (500 mL). The precipitate was filtered, washed with water (5 x 80 mL) and freeze-dried from MeCN. The crude product was dispersed in Et20 (150 mL), sonicated, filtered, and washed with Et20 (3 x 50 mL). This process was repeated once again. The collected solid was dried in vacuo.

[0399] General procedure C: Oxazolone formation = Bs, Ts, MBs, Ths, Dnp - , p: - , p:

[0400] = Boc, Pg2= Boc = Boc, Pg2= Boc

[0401] 4Bg: Pg1= Dde, Pg2= Ts 6Bg: Pg1= Dde, Pg2= Ts

[0402] 4Db: Pg12 6Db: Pg

[0403] = HexMoc, Pg = Ts1

[0404] = HexMoc, Pg2= Ts

[0405] 4Eb: Pg16Eb: Pg1

[0406] To protected dipeptide 4 (2.1 mmol, 1.0 eq.) suspended in MeCN or THF (15 mL) was added DCC (2.1 mmol, 1.0 eq.). The mixture was stirred at room temperature for 1-2 hours. Progress of the reaction was analyzed by1H NMR. The reaction mixture was filtered, and the filtrate was evaporated under reduced pressure. The solid was redissolved in MeCN (15 mL) and evaporated together with filter agent celite 545 (2-3 g). The solid was loaded to an empty column. Automatic, normal-phase flash column chromatography of the crude compound was carried out by gradient elution using EtOAc in heptane. The relevant fractions were pooled and concentrated under reduced pressure.

[0407] General procedure D: NHS ester formation of Pq1His(Pq2)Aib-OH

[0408] To protected dipeptide 4 (0.5 mmol, 1.0 eq.) dissolved in THF (3 mL) was added NHS (0.7 mmol, 1.3 eq.) followed by DCC (0.7 mmol, 1.3 eq.). The mixture was stirred at room temperature overnight. Progress of the reaction was analyzed by method UPLC-MS_7. The reaction mixture was filtered, and the filtrate was evaporated under reduced pressure. The crude was dissolved in DCM (2 mL) and filtered again using a PTFE filter before the solution was applied to a silica gel column. Automatic, normal-phase flash column chromatography of the crude compound was carried out by gradient elution using MeOH in DCM. The relevant fractions were pooled. MeCN (10 mL) was added as a co-solvent and concentrated under reduced pressure to give the NHS ester 5.

[0409] General procedure E: Phenol ester formation of Pq1 His(Pq2)Aib-OH.

[0410] To protected dipeptide 4 (0.6 mmol, 1.0 eq.) dissolved in either MeCN (7 mL) or a mixture of THF (5 mL) and DCM (2 mL) was added 2-methoxy-4-nitrophenol (0.9 mmol, 1.4 eq.) and 4- dimethylaminopyridine (DMAP) (0.1 mmol, 0.2 eq.) followed by DCC (0.8 mmol, 1.2 eq.). The mixture was stirred at room temperature from 1 hour to 18 hours. Progress of the reaction was analyzed by method UPLC-MS_7. The reaction mixture was filtered, and the filtrate was evaporated under reduced pressure. The crude was dissolved in DCM (2 mL) and filtered again using a PTFE filter before the solution was applied to a silica gel column. Automatic normalphase flash column chromatography of the crude compound was carried out by gradient elution using MeOH in DCM or EtOAc in heptane. The relevant fractions were pooled. MeCN (10 mL) was added as co-solvent and concentrated under reduced pressure. If DCU was present in the compound (seen in1H NMR) an extra trituration in MeCN (20 mL) followed by a filtration using a PTFE filter was performed. The filtrate was concentrated under reduced pressure to give phenol ester 5. Example 1 : Preparation of Pg1HisPg2-OH

[0411] Example 1a: Synthesis of / VT-(phenylsulfonyl)- / \ / a-(2,2,2-trifluoroacetyl)-L-histidine (3Aa) Small scale synthesis: Compound 3Aa was synthesized following general procedure A using triethylammonium (2,2,2-trifluoroacetyl)-L-histidinate (2A) (15.3 g, 50.0 mmol) and benzene sulfonyl chloride (6.4 mL, 50.0 mmol) After drying in vacuo, a white powder was obtained (17.9 g, 91.0% yield). Purity: >99.9% (ELSD, HPLC-MS_1), 97.0% (UV, 254 nm, HPLC-MS_1).1H NMR (300 MHz, DMSO-cfe) 6 ppm 2.94 (dd, J = 14.7, J = 10.3 Hz, 1 H), 3.05 (dd, J = 14.9 J = 4.4 Hz, 1 H), 4.53 (dd, J = 9.8, J = 4.4 Hz, 1 H), 7.42 (s, 1 H), 7.67 (t, J = 8.0 Hz, 2 H), 7.81 (t, J = 7.4 Hz, 1 H), 8.03 (d, J = 7.6 Hz, 2 H), 8.32 (s, 1 H), 9.64 (d, J=8.0 Hz, 2 H).13C NMR (125 MHz, DMSO-cfe) 6 ppm 28.5, 51.8, 115.0, 115.7 (q, J = 288 Hz), 127.0, 130.1 , 135.3, 137.1 , 140.3, 156.2 (q, J = 37 Hz), 171.0.19F NMR (376 MHz, DMSO-cfe) 6 ppm -74.4. LCMS, m / z calcd. for C14H13F3N3O5S [M+H]+392.0523, found 392.0470

[0412] Large scale synthesis: A slurry of L-histidine (100.0 g, 1.0 eq.) in MeOH (750 mL, 7.5 volumes (V)) was stirred at 20°C. TEA (81.5 g, 1.25 eq.) was added over 5 minutes followed by addition of ethyl trifluoroacetate (114.0 g, 1.25 eq.) over 30 minutes. The internal temperature during addition of ethyl trifluoroacetate was kept below 30°C (mild exotherm). The reaction mixture was stirred at 20°C for maximum 20 hours and then analyzed by method HPLC_11 (in process control sample A (IPC A): conversion > 98% NA1)).

[0413] After complete conversion, methyl tetrahydrofuran (MeTHF) was added (500 mL, 5 V) and the reaction mixture was concentrated at jacket temperature (Tj) = 40°C under vacuum to 4-5 weights (500-540 g). This operation was repeated twice. During the distillations the product began to precipitate. After two distillations, the slurry was sampled and analyzed by gas chromatography (method GC_18, split ratio 5:1) to monitor the residual MeOH whose limit was < 4% (IPC B). If the IPC was not satisfied, an additional distillation was necessary (addition of additional 5 volumes of MeTHF and concentrations under vacuum to 5 weights). The slurry was cooled at 0°C (potential hold point overnight 0 °C), then a solution of potassium hydrogen carbonate (KHCO3) was added (123.0 g, 1.9 eq. in 500 ml of water) over 30 minutes while maintaining the internal temperature below 0°C. Benzenesulfonyl chloride (114.0 g, 1.0 eq.) was added dropwise over 2 hours at 5°C. After complete addition, the reaction mixture was stirred at 5°C for 1 hour where some precipitation occurred. The solution was analyzed by method HPLC-11 (IPC C: conversion > 95% NA2potential hold point: overnight 5°C). MeTHF

[0414] •1

[0415] The conversion is calculated as normalized area between the product TFAcHis-OH and His-OH.

[0416] 9

[0417] The conversion was calculated considering the ratio between the A% of TFAcHis(Bs)-OH and the sum of A% of TFAcHis-OH and TFAcHis(Bs)-OH (1200 mL, 12 V) was added at 0 °C, then an aqueous solution of sulfonic acid 6% (839.0 g, 0.8 eq.) was added to decrease the pH from 7.7 to 2.5-3.0. The mixture was heated to 20°C. The biphasic mixture was stirred for 30 minutes at 20 °C, then the phases were allowed to settle for 30 minutes followed by separation of the phases. To the organic layer was added brine (500 mL, 5V). The biphasic mixture was stirred for 30 minutes at 20 °C, then the phases were allowed to settle for 30 minutes followed by phase separation. Finally, the organic phase (1590.0 g, whose weight was between 15.9 weights (w) ± 0.2) was concentrated at Tj = 20°C under vacuum to about 6 weights of L-histidine (610.0 g, 6.4 weight ±0.3), then diluted with MeTHF (1050 mL, 10.5 V) to have the same initial volume and concentrated under vacuum at 20°C to a final weight of about 6 weights (647.0 g, 6.4 weight ±0.3). During the distillation, the product precipitated. A slurry sample was taken to check residual water in the supernatant by Karl Fisher titration (I PC: residual water 0.6-1.1 w / w, method KF_19). When the slurry was determined anhydrous, the mixture was cooled at 0-5°C and aged for 1 hour at 0-5°C, followed by filtration. The wet filter was washed with cold MeTHF (2 x 100 ml, 1 V) and dried at 20°C under vacuum for 20 hours to afford 3Aa (188.0 g). Purity 98.8% (HPLC_11), 99.3 w / w% (qNMR).

[0418] Typically, the compound 3Aa was achieved in 74-75% yield and purity > 98.4%, 98-100 w / w% (qNMR).

[0419] Example 1 b: Synthesis of / VT-tosyl- / \ / a-(2,2,2-trifluoroacetyl)-L-histidine (3Ab)

[0420] Compound 3Ab was synthesized following general procedure A using triethylammonium (2,2,2-trifluoroacetyl)-L-histidinate (2A) (15.3 g, 50.0 mmol) and tosyl chloride (10.0 g, 50.0 mmol) After drying in vacuo a white powder was obtained (19.6 g, 96.0% yield). Purity: 95.0% (UV, 254 nm, HPLC-MS_1).1H NMR (400 MHz, DMSO-cfe) 6 ppm 2.39 (s, 3 H), 2.92 (dd, J=14.8 Hz, J=10.2 Hz, 1 H), 3.03 (dd, J=14.8 Hz, J=4.4 Hz, 1 H), 4.48-4.58 (m, 1 H), 7.38 (s, 1 H), 7.48 (d, J = 8.2 Hz, 2 H), 7.90 (d, J = 8.3 Hz, 2 H), 8.29 (d, J = 0.9 Hz, 1 H), 9.63 (d, J = 8.0 Hz, 1 H), 13.26 (bs, 1 H).13C NMR (100 MHz, DMSO-cfe) 6 ppm 21.1 , 28.4, 51.8, 114.9, 115.6 (q, J = 290 Hz), 127.1 , 130.5, 134.3, 137.0, 140.2, 146.3, 156.1 (q, J = 36 Hz), 171.0.19F NMR (376 MHz, DMSO-cfe) 6 ppm -74.5. LCMS, m / z calcd. for C15H15F3N3O5S [M+H]+406.0679, found 406.0611. Example 1c: Synthesis of / VT-4-(methoxyphenyl)sulfonyl- / \ / ';-(2,2,2-trifluoroacetyl)-L-histidine

[0421] Compound 3Ac was synthesized following general procedure A using triethylammonium (2,2,2-trifluoroacetyl)-L-histidinate (2A) (30.8 g, 100.0 mmol) and 4-methoxybenzenesulfonyl chloride (20.7 g, 100 mmol). After THF removal, the solution was adjusted to pH 3.0 using 1 M aqueous solution of HCI (approximately 180 mL). The resulting thick suspension was filtered; the precipitate was washed with water (3 x 100 mL) and air dried over three days. The finely powdered material was dispersed in diethyl ether (Et2O) (200 mL) and sonicated for 5 minutes. The product was filtered and washed with Et2O (2 x 100 mL). Drying in vacuo at 60 °C afforded a white powder (34.3 g, 81% yield). Purity >99.9% (ELSD, HPLC-MS_1), >99.9% (UV, 254 nm, HPLC-MS_1).1H NMR (400 MHz, DMSO-cfe) 6 ppm 2.92 (dd, J = 15.6 Hz, J = 10.2 Hz, 1 H), 3.03 (dd, J = 14.8, J = 3.9, 1 H), 3.85 (s, 3 H), 4.48-4.56 (m, 1 H), 7.17 (dt, J = 9.1 , J = 2.1 , 2 H), 7.37 (s, 1 H), 7.97 (dt, J = 9.0, J = 2.1 , 2 H), 8.27 (d, J = 1.4, 1 H), 9.63 (d, J = 8.0, 1 H), 13.1 (bs, 1 H).13C NMR (125 MHz, DMSO-cfe) 6 ppm 28.5, 51.9, 56.0, 114.3, 115.3, 115.7 (q, J = 286.4), 136.9, 140.1 , 156.2 (q, J = 36.4), 164.4, 171.1.19F NMR (376 MHz, DMSO-cfe) 6 ppm -74.4. LCMS, m / z calcd. for C15H15F3N3O6S [M+H]+422.0628, found 422.0771.

[0422] Example 1d: Preparation of / VT-(thiophen-2-ylsulfonyl)- / \ / a-(2,2,2-trifluoroacetyl)-L-histidine

[0423] Compound 3Ad was synthesized following general procedure A using triethylammonium (2,2,2-trifluoroacetyl)-L-histidinate (2A) (15.3 g, 50.0 mmol) and thiophen-2-ylsulfonyl chloride (9.1 g, 50.0 mmol). After workup and evaporation of the solvent, the solid residue was homogenized and triturated with diethyl ether (2 x 50 mL) with the aid of sonication. The resulting white solid was dried in vacuo affording a white powder (17.8 g, 81.0% yield. Purity 99.0% (ELSD, HPLC-MS_1), >98.0% (UV, 254 nm, HPLC-MS_1).1H NMR (400 MHz, DMSO- cfe) 6 ppm 2.95 (dd, J=15.0 Hz, J=10.2 Hz, 1 H), 3.06 (dd, J=15.0 Hz, J=3.8 Hz, 1 H), 4.49- 4.59 (m, 1 H), 7.29 (dd, J=5.1 Hz J=3.9 Hz, 1 H), 7.41 (d, J=1.0 Hz, 1 H), 8.03 (dd, J=3.9 Hz, J=1.4 Hz, 1 H), 8.23 (dd, J=5.0 Hz, J=1.4 Hz, 1 H), 8.31 (d, J=1.4 Hz, 1 H), 9.65 (d, J=8.0 Hz, 1 H), 13.12 (bs, 1 H).13C NMR (100 MHz, DMSO-cfe) 6 ppm 28.5, 51.9, 114.9, 115.7 (q, J=286.4 Hz), 128.9, 136.1 , 136.6, 137.0, 137.8, 140.4, 156.2 (q, J=36.4 Hz), 171.1.19F NMR (376 MHz, DMSO-cfe) 6 ppm -74.4. LCMS, m / z calcd. for C^H FsNsC^Na [M+Na]+419.9906, found 419.9858.

[0424] Example 1e: Preparation of / VT-(2,4-dinitrophenyl)- / \ / a-(2,2,2-trifluoroacetyl)-L-histidine (3Ap)

[0425] Boc-L-His(Dnp)-OH (2.0 g, 4.7 mmol) was dissolved in 4 M HCI in 1 ,4-dioxane (30 mL). The solution was stirred at rt. for 1.5 hours. Next, the solution was concentrated under reduced pressure. The resulting solid was redissolved in MeCN (30 mL) and evaporated. This was repeated two times in total. The resulting white solid was then dissolved in MeOH (30 mL). Ethyl trifluoroacetate (1.7 mL, 14.2 mmol) was added and the mixture was heated to 40 °C. Triethylamine (2.0 mL, 14.2 mmol) was added dropwise, and the mixture was stirred for 2 hours. Afterwards, the mixture was concentrated under reduced pressure. MeCN (30 mL) was added, and the mixture was concentrated again. The resulting solid was redissolved in EtOAc (30 mL) and washed with a 5% aqueous solution of KHSO4. The organic phase was collected, dried over MgSCL, filtered, and concentrated giving the product as a yellow solid (1.7 g, 85.8% yield). Purity 96.0% (UPLC-MS_5).1H NMR (400 MHz, DMSO-cfe) 6 ppm 3.05 (dd, J=15.2 Hz, J=9.9 Hz, 1 H), 3.15 (dd, J=15.4 Hz, J=4.0 Hz, 1 H), 4.53-4.68, m, 1 H), 7.27 (s, 1 H), 7.95 (d, J=8.7 Hz, 1 H), 8.11 (s, 1 H), 8.67 (d, J=7.2 Hz, 1 H), 8.93 (s, 1 H), 9.70 (s, 1 H), 13.1 (bs, 1 H).13C NMR (100 MHz, DMSO-cfe) 6 ppm 28.3, 52.3, 115.8 (q, J=286.3 Hz), 117.7, 121.3, 128.8, 129.7, 134.4, 137.3, 138.3, 143.7, 146.5, 156.4 (q, J=36.4 Hz), 171.2.19F NMR (376 MHz, DMSO-cfe) 6 ppm -74.4 LCMS, m / z calcd. for C14H11F3N5O7 [M+H]+418.0605, found 418.0670.

[0426] L-Histidine (1) (2.0 g, 12.9 mmol) was dissolved in 1 M NaHCCh (30 mL, pH 10.0). The solution was cooled in an ice bath and benzyl chloroformate (3.7 mL, 25.8 mmol) was added dropwise to the solution. Precipitation was observed during addition of the benzyl chloroformate. Therefore, THF (20 mL) was added to the solution. The pH was kept at 9.8-10.0 with 2 M NaOH during the addition of the benzyl chloroformate. After the addition, a sample was taken out and diluted (1 :99) with MeCN. The diluted sample was analyzed with method UPLC-MS_5. After complete conversion of the starting material, the THF was removed under reduced pressure. The pH was then decreased to 4.0 under stirring using 1 M HCI. The product was extracted with EtOAc. The organic phase was collected, dried over MgSC>4, filtered, and concentrated leading to a solid. The solids were washed with water and Et2O. The solids were collected by filtration and dried under vacuum. The product was purified by column chromatography eluting with a gradient from DCM to 8% MeOH in DCM (DCM containing 0.25% AcOH). After drying in vacuo overnight a white powder was obtained (3.8 g, 70.0% yield). Purity 88.5% (UPLC-MS_5).1H NMR (400 MHz, DMSO-cfe) 6 ppm 2.77-2.87 (m, 1 H), 2.88-2.98 (m, 1 H), 4.21-4.32 (m, 1 H), 4.99 (s, 1 H), 5.42 (s, 1 H), 7.21-7.45 (m, 9 H), 7.47- 7.58 (m, 3 H), 8.22 (d, J = 1.3 Hz, 1 H), 12.47 (bs, 1 H).13C NMR (100 MHz, DMSO-cfe) 6 ppm 29.5, 53.5, 65.4, 69.2, 114.6, 127.7, 127.8, 128.3, 128.4, 128.6, 128.7, 134.7, 136.9, 137.0, 139.6, 148.2, 156.0, 173.2. LCMS, m / z calcd. for C22H22N3O6 [M+H]+424.1503, found 424.1567.

[0427] Example 1q: Preparation of (1-(4,4-dimethyl-2,6-dioxocvclohexylidene)ethyl)- / \ / T-tosyl-L- histidine (3Db)

[0428] To a solution of L-Histidine (1) (1.0 g, 6.4 mmol) in ethanol (30 mL) was added triethylamine (1 .3 mL, 9.7 mmol) and 2-acetyldimedone (1.3 g, 7.1 mmol). The suspension was heated under reflux under an atmosphere of nitrogen for 18 h. Complete conversion was seen with method UPLC-MS_4. EtOH was removed under reduced pressure. The solid was dissolved in NaHCOs buffer pH 8.7 1 M (9 mL). THF (10 mL) was added, and the solution was cooled in an ice batch. Tosyl chloride (1.4 g, 7.4 mmol, 1.2 eq.) was added to the ice-cold solution. The cooling bath was removed, and the reaction mixture was stirred for 45 min where it became a clear mixture. A sample was taken out, diluted (2:98) with MeCN / Water (1 :1) and analyzed using UPLC- MS_4. Some starting material (~3%) was still present. The THF in the reaction mixture was evaporated under reduced pressure and when the product precipitated the evaporation was stopped. The stirred mixture was acidified using 1 M HCI to pH 3. The white precipitate was filtered and washed with water (2 x 5 mL) and diethyl ether (2 x 5 mL). The precipitate was dried for several hours under vacuum at 40°C. After drying in vacuo, a white powder was obtained (2.8 g, 90.7% yield). Purity 98.4% (HPLC-MS_7).1H NMR (500 MHz, DMSO-cfe) 6 ppm 0.93 (s, 6 H), 2.24 (s, 3 H), 2.29 (s 3 H), 2.40 (s, 3 H), 2.98-3.12 (m, 2 H), 4.80-4.88 (m, 1 H), 7.45-7.52 (m, 3 H), 7.91 (d, J=8.4 Hz, 2 H), 8.30 (d, J=1.4 Hz, 1 H), 13.41 (d, J=8.0 Hz, 1 H), 13.41 (bs, 1 H).13C NMR (100 MHz, DMSO-cfe) 6 ppm 17.5, 21.2, 27.8, 29.6, 30.7, 52.4, 54.8, 107.2, 115.8, 125.5, 127.2, 128.1 , 130.5, 134.3, 137.0, 138.9, 146.3, 171.0, 172.2. LCMS, m / z calcd. for C23H28N3O6S [M+H]+474.1693, found 474.1825. (((hexanoyloxy)methoxy)carbonyl)- / \ / T-tosyl-L-histidine (3Edb)

[0429] Boc-L-His(Ts)-OH (3Bb) (10.0 g, 24.4 mmol) was dissolved in TFA (20 mL) and anisole (6 mL) at 0 °C. After one hour the deprotection was finished. A white precipitate was formed. TFA was evaporated under reduced pressure. The solid was treated with diethyl ether (50 mL) at 0 °C and stirred for several minutes. Filtration did not work due to very fine powder. The solid (divided in two with 2x 50 ml diisopropyl ether in each 250 mL centrifuge plastic flask) was centrifugated at 4000g for 6 min and the solvent was decanted. The process was repeated one more time. The solids in the two flasks were dried under vacuum overnight. After drying in vacuo, a white powder was obtained (10.1 g, 97.7% yield, TFA salt). Purity 97.0% (UV, 214 nm, UPLC-MS_5).

[0430] The L-His(Ts)-OH TFA salt (2.3 g, 5.2 mmol) was dispersed in anhydrous DMF (8 mL) followed by addition of triethylamine (2.2 mL, 15.8 mmol). A solution of (((4- nitrophenoxy)carbonyl)oxy)methyl hexanoate (1.6 g, 5.2 mmol) in anhydrous DMF (2 mL) was added dropwise. The resulting yellow suspension was stirred under nitrogen for 0.5 hours. The reaction mixture was diluted with EtOAc (40 mL) and washed with 0.1 M aqueous solution of citric acid (2 x 40 mL), and saturated aqueous solution of sodium chloride (2 x 30 mL). The organic phase was dried over anhydrous sodium sulphate, filtered, and evaporated. The residue was dissolved in DCM and purified by column chromatography with gradient elution from DCM to MeOH / DCM 1 :9 was performed. The pure fractions were pooled and MeCN (10 mL) was added as a co solvent during the evaporation of the solvents. After drying in vacuo, a white powder was obtained (2.0 g, 78.8% yield). Purity 94.2% (HPLC-MS_5).1H NMR (400 MHz, DMSO-cfe) 6 ppm 0.82 (t, J=7.0 Hz, 3 H), 1.18-1.30 (m, 4 H), 1.50 (p, J=7.2 Hz, 2 H), 2.29 (t, J=7.4 Hz, 2 H), 2.40 (s, 3 H), 2.77 (dd, J=14.2 Hz, J=9.6 Hz, 1 H), 2.89 (dd, =17.1 Hz, J=4.5 Hz, 1 H), 4.18-4.26 (m, 1 H), 5.57 (s, 2 H), 7.38 (s, 1 H), 7.49 (d, J=8.2 Hz, 2 H), 7.82 (d, J=8.1 Hz, 2 H), 8.27 (d, J=1.3 Hz, 1 H), 12.8 (s, 1 H).13C NMR (100 MHz, DMSO-cfe) 6 ppm 13.7, 21.1 , 21.7, 23.8, 29.4, 30.5, 33.2, 53.2, 79.4, 115.0, 127.2, 130.6, 134.5, 136.8, 140.4, 154.2, 172.1 , 172.6. LCMS, m / z calcd. for C21H28N3O8S [M+H]+482.1592, found 482.1573.

[0431] Example 2: Synthesis of Pg1His(Pg2)Aib-OH

[0432] Example 2a: Preparation of (S)-2-methyl-2-(3-(1-(phenylsulfonyl)-1 H-imidazol-4-yl)-2-(2,2,2- trifluoroacetamido)propanamido) propanoic acid (4Aa)

[0433] Small scale: Compound 4Aa was synthesized following general procedure B using NT- (phenylsulfonyl)- / V“-(2,2,2-trifluoroacetyl)-L-histidine (3Aa) (17.1 g, 43.7 mmol). After drying in vacuo, a white powder was obtained (16.6 g, 79% yield). Purity >99.9% (ELSD, HPLC-MS_1), 97% (UV, 254 nm, HPLC-MS_1). Method HPLC-MS_1 : Retention time 6.42 min.1H NMR (400 MHz, DMSO-cfe) 6 ppm 1.24 (s, 3 H) 1.3 (s, 3 H), 2.84 (dd, J=15.3 Hz, J=9.8 Hz, 1 H), 2.94 (dd, J=14.8 Hz, J=4.8 Hz, 1 H), 4.51-4.64 (m, 1 H) 7.35 (s, 1 H) 7.68 (t, J = 7.72 Hz, 2 H) 7.81 (t, J = 7.48 Hz, 1 H) 8.02 (d, J = 7.64 Hz, 2 H) 8.34 (d, J = 5.12 Hz. 2 H) 9.44 (d, J = 8.32 Hz, 1 H) 12.30 (bs, 1 H).13C NMR (100 MHz, DMSO-cfe) 6 ppm 24.6, 24.7, 29.9, 52.0, 55.1 , 114.9, 115.7 (q, J = 286.4 Hz), 127.0, 130.1 , 135.3, 136.9, 137.2, 140.2, 155.9 (q, J = 36.3 Hz), 168.5, 175.1.19F NMR (376 MHz, DMSO-cfe) 6 ppm -74.2. LCMS, m / z calcd. for CisH Fs^OeSNa [M+Na]+499.0870, found 499.0926.

[0434] Large scale: BSA (99.0 g, 486.0 mmol, 1.6 eq.) was added to a MeTHF (208 ml, 1.7 V) slurry of Aib (40.0 g, 388 mmol, 1.3 eq.). After addition, the mixture was heated under reflux (range temperature: 85-90 °C) and stirred under these conditions for at least 24 hours. No I PC is foreseen but the conversion was assumed complete when the reaction mixture was a clear solution. The reaction mixture was cooled at 20 °C and used directly in the next coupling.

[0435] Activation of3Aa with DCC / HOPO: A slurry of 3Aa (120.0 g, 298.0 mmol, 1.0 eq.) and HOPO (34.8 g, 313.0 mmol, 1.05 eq.) in MeTHF (1080 ml, 9 V) was cooled at 10 °C. Then a solution of DCC (73.9 g, 358.0 mmol, 1.2 eq.) in MeTHF (1320 ml, 11 V) was added over 1 hour by maintaining the internal temperature between 5°C and 10°C. After addition, the reaction mixture was stirred for 1.5 hour at 10°C and then analyzed by method HPLC_11 to confirm the complete conversion (IPC H: conversion > 98% NA3). Then, the suspension was filtered to remove the solid dicyclohexylurea (DCU) (about 0.6-0.7 weights). The wet DCU cake was washed with MeTHF (120 ml, 1 V) and the filtered solution, containing the HOPO ester of 3Aa, was cooled at 10°C and used directly in the next step.

[0436] Coupling-. T o the reactor containing the solution of HOPO ester of 3Aa at 10 °C, under nitrogen atmosphere, a solution of silyl protected Aib was added dropwise over 30 minutes under stirring. After addition, the reaction was stirred for 1.5 hours at 10 °C and then analyzed by method HPLC_11 (IPC I: residual starting material < 5% NA4). If, the conversion was below 95%, then the reaction was left an additional 2 h at 10°C. (Potential holding points overnight, or weekend at 0°C).

[0437] Workup-. The reaction mixture was quenched by adding, over 30 minutes at 10 °C, a solution of magnesium acetate tetrahydrate (141.0 g, 657.0 mmol, 2.2 eq.) in water (1200 ml, 10 V). The biphasic mixture was stirred for 30 minutes at 20 °C, then the phases were allowed to settle for 30 minutes and then separated.

[0438] The organic phase was extracted twice with a solution of NaHCOs (55.2 g, 657.0 mmol, 2.2 eq.) in water (1200 ml, 10 V). After the separation, the two aqueous phases were combined and diluted with MeTHF (2400 ml, 20 V), cooled at 5°C and acidified at pH 2.5 by adding sulfuric acid 25% (292.0 g, 746.0 mmol, 2.5 eq.). Then the biphasic mixture was heated at 20°C and the phases were allowed to settle for 30 minutes and then separated. Finally, the organic phase was washed with water (600 ml, 5 V). After separation of two phases, the organic layer (whose weight is between 22 weights ± 0.3) was concentrated at Tj = 20°C under

[0439] 3The conversion during the acid activation is calculated as normalized area (NA%), considering the ratio between the A% of HOPO ester of 3Aa and the sum of A% of HOPO ester of 3Aa and 3Aa starting material.

[0440] 4The conversion during the coupling is calculated as normalized area (NA%), considering the ratio between the A% of 4Aa and the sum of A% of the 4Aa, the 3Aa and the HOPO ester of 3Aa. vacuum to 5 weights (about 600 g), then diluted with MeTHF (2000 ml, 16.6 V) and concentrated under vacuum at 20°C to a final weight of 5 weights (about 600 g). During the distillation, the product precipitated out of solution. A sample of the slurry was taken to check residual water in the supernatant by Karl Fisher (I PC L: residual water <1.2% w / w, method KF_19), to conclude the distillation. Then, the slurry was cooled at 20°C and the slurry stirred for 30 minutes (not more than 3 hours) before being filtered. The wet cake was washed with MeTHF (120 ml, 1 V) and dried at 20°C under vacuum for 20 hours to afford 4Aa (113.4 g, 289.0 mmol, HPLC Purity: 99.4%, HPLC Assay: 92.9% w / w, D-enantiomer: <0.25% w / w HPLC_16). Typically, the dipeptide 4Aa was achieved in 72-76% yield and purity > 99%, HPLC assay 91.1-92.9% w / w since the product was an hemi solvate of MeTHF. In fact, the solvent amount in the most representative dipeptides was around 7.6 to 8.5% w / w by GC analysis (method CG_18, split ratio 20:1), in line with the theorical amount of solvent to be hemi solvate 8.3% w / w.

[0441] Example 2b: Preparation of (S)-2-methyl-2-(3-(1-tosyl-1 H-imidazol-4-yl)-2-(2,2,2- trifluoroacetamido)propanamido) propanoic acid (4Ab)

[0442] Compound 4Ab was synthesized following general procedure B using / VT-(tosyl)- / Va-(2,2,2- trifluoroacetyl)-L-histidine (3Ab) (21.1 g, 52.0 mmol). After drying in vacuo, a white powder was obtained (24.1 g, 94.0% yield). Purity >99.9% (ELSD, HPLC-MS_1), 96% (UV, 265 nm, HPLC-MS_1). Method HPLC-MS-1 : Retention time 5.31 min.1H NMR (400 MHz, DMSO-cfe) 6 ppm 1.24 (s, 3 H), 1.30 (s, 3 H), 2.39 (s, 3 H), 2,83 (dd, J = 15.0 Hz, J = 9.7 Hz, 1 H), 2.93 (dd, J = 15.0 Hz, J = 4.8 Hz, 1 H), 4.52-4.62 (m, 1 H), 7.32 (s, 1 H), 7.47 (d, J = 8.3, 2 H), 7.89 (d, J = 8.3 Hz, 2 H), 8.29 (d, J = 1.2 Hz. 1 H), 9.92 (s, 1 H), 9.44 (d, J = 8.3 Hz, 1 H), 12.27 (bs, 1 H).13C NMR (100 MHz, DMSO-cfe) 6 ppm 21.1 , 24.6, 24.7, 29.9, 52.1 , 55.1 , 114.8, 115.7 (q, J = 288.2 Hz), 127.1 , 130.5, 134.3, 136.8, 140.2, 146.3, 155.9 (q, J = 36.5 Hz), 168.5, 175.2.19F NMR (376 MHz, DMSO-cfe) 6 ppm -74.2. LCMS, m / z calcd. for C19H22F3N4O6S [M+H]+491.1207, found 491.1244.

[0443] Example 2c: Preparation of (S)-2-(3-(1-((4-methoxyphenyl)sulfonyl)-1 H-imidazol-4-yl)-2-

[0444] (2,2,2-trifluoroacetamido)propanamido)-2-methylpropanoic acid (4Ac)

[0445] Compound 4Ac was synthesized following general procedure B using NT-4-

[0446] (methoxyphenyl)sulfonyl- / Va-(2,2,2-trifluoroacetyl)-L-histidine (3Ac) (22.6 g, 53.6 mmol). After drying in vacuo, a white powder was obtained (25.2 g, 92.0% yield). Purity 99.0% (ELSD, HPLC-MS_1), 97.0% (UV, 254 nm, HPLC-MS_1). Method HPLC-MS-1: Retention time 5.13 min.1H NMR (400 MHz, DMSO-cfe) 6 ppm 1.24 (s, 3 H), 1.31 (s, 3 H), 2.83 (dd, J = 14.9 Hz, J = 9.7 Hz, 1 H), 2.93 (dd, J = 15.0 Hz, J = 4.8 Hz, 1 H), 2.39 (s, 3 H), 4.52-4.62 (m, 1 H), 7.16 (d, J = 9.0 Hz, 2 H), 7.30 (s, 1 H), 7.95 (d, J = 9.0 Hz, 2 H), 8.27 (s, 1 H), 8.34 (s, 1 H), 9.44 (d, J = 8.3 Hz, 1 H), 12.26 (bs, 1 H).13C NMR (100 MHz, DMSO-cfe) 6 ppm 24.6, 24.7, 29.9, 52.1 , 55.1 , 56.0, 114.7, 115.3, 115.7 (q, J = 288.2 Hz), 128.3, 129.7, 136.7, 140.1 , 155.9 (q, J = 36.5 Hz), 164.3, 168.5, 175.2.19F NMR (376 MHz, DMSO-cfe) 6 ppm -74.2. LCMS, m / z calcd. For C19H22F3N4O7S [M+H]+507.1156, found 507.1075.

[0447] Example 2d: Preparation of (S)-2-methyl-2-(3-(1-(thiophen-2-ylsulfonyl)-1 H-imidazol-4-yl)-2-

[0448] (2,2,2-trifluoroacetamido)propanamido)propanoic acid (4Ad)

[0449] Compound 4Ad was synthesized following general procedure B using / \ / T-(thiophen-2- ylsulfonyl)- / Va-(2,2,2-trifluoroacetyl)-L-histidine (3Ad) (17.8 g, 44.8 mmol). After the last Et20 wash the resulting pale-yellow solid was dried in vacuo, dispersed in 2-propanol (90 mL), sonicated, filtered, and washed with 2-propanol (2 x 10 mL). The collected precipitate was lyophilized from acetonitrile, affording an off-white powder (15.0 g, 69.0% yield). Purity >99.9% (ELSD, HPLC-MS_1), 99.0% (UV, 254 nm, HPLC-MS_1). Method HPLC-MS-1 : Retention time 3.97 min.1H NMR (400 MHz, DMSO-cfe) 6 ppm 1.27 (s, 3 H), 1.32 (s, 3 H), 2.80-3.00 (m, 2 H), 4.55-4.66 (m, 1 H), 7.29 (t, J = 4.4 Hz, 1 H), 7.35 (s, 1 H), 8.01 (d, J = 2.9 Hz, 1 H), 8.23 (d, J = 4.3 Hz, 1 H), 8.31 (s, 1 H), 8.37 (s, 1 H), 9.46 (d, J = 8.3 Hz, 1 H), 12.26 (bs, 1 H).13C NMR (100 MHz, DMSO-cfe) 6 ppm 24.66, 24.70, 29.9, 52.0, 55.1 , 115.7 (q, J= 288 Hz) 114.8, 128.9, 136.0, 136.6, 136.7, 140.4, 155.9 (q, J = 36.5 Hz) 168.5, 175.2.19F NMR (376 MHz, DMSO- cfe) 6 ppm -74.1. LCMS, m / z calcd. For C16H18F3N4O6S [M+H]+483.0614, found 483.0745. Example 2e: Preparation of (S)-2-(3-(1-(2,4-dinitrophenyl)-1 H-imidazol-4-yl)-2-(2,2,2- trifluoroacetamido)propanamido)-2-methylpropanoic acid (4Ap)

[0450] Compound 4Ap was synthesized following general procedure B using A / T-(2,4-dinitrophenyl)- / V“-(2,2,2-trifluoroacetyl)-L-histidine (3Ap) (1.5 g, 3.6 mmol). The product was purified by flash column chromatography eluting with a gradient from DCM to 10% MeOH in DCM affording the title compound as a yellow solid (1.1 g, 61% yield). Purity 97.0% (UPLC-MS_5).1H NMR (400 MHz, DMSO-cfe) 6 ppm 1.33 (s, 3 H), 1.36 (s, 3 H), 2.95 (dd, J=14.9 Hz, J=9.4 Hz, 1 H), 3.03 (dd, J=14.9 Hz, J=4.9 Hz, 1 H), 4.58-4.68 (m, 1 H), 7.20 (s, 1 H), 7.91 (d, J=8.8 Hz, 1 H), 8.04 (s, 1 H), 8.39 (s, 1 H), 8.66 (dd, J=8.9 Hz, J=2.Q Hz, 1 H), 8.92 (d, J=2.Q Hz, 1 H), 9.49 (d, J=8.2 Hz, 1 H), 12.3 (bs, 1 H).13C NMR (100 MHz, DMSO-cfe) 6 ppm 24.7, 24.8, 29.8, 52.6, 55.2, 115.8 (q, J=286.5 Hz), 117.4, 121.3, 128.8, 129.5, 134.5, 137.1 , 138.7, 143.7, 146.4, 156.0 (q, J=36.3 Hz), 168.8, 175.3.19F NMR (376 MHz, DMSO-cfe) 6 ppm -74.1. LCMS, m / z calcd. for CI8HI8F3N6O8 [M+H]+503.1133, found 503.1150.

[0451] Example 2f: Preparation of (S)-2-(3-(1-(tert-butoxycarbonyl)-1 H-imidazol-4-yl)-2-((tert- butoxycarbonyl)amino)propanamido)-2-methylpropanoic acid (4Bg)

[0452] Compound 4Bg was synthesized following general procedure B using / V“, / Vt-bis(tert- butoxycarbonyl)-L-histidine (3Bg) (17.8 g, 44.8 mmol). After the last Et20 wash the resulting pale-yellow solid was dried in vacuo, dispersed in 2-propanol (90 mL), sonicated, filtered, and washed with 2-propanol (2 x 10 mL). The collected precipitate was lyophilized from acetonitrile, affording an off-white powder (15.0 g, 69.0% yield). Purity 96.4% (UPLC_17).1H NMR (500 MHz, DMSO-cfe, 80 °C) 5 ppm 1.35 (s, 9 H), 1.36 (s, 3 H), 1.38 (s, 3 H), 1.57 (s, 9 H), 2.71 (dd,

[0453] J=14.8, J=9.1 Hz, 1 H), 2.84 (dd, J=14.6, J=4.6 Hz, 1 H), 4.18 (m, 1 H), 6.62 (bs, 1 H), 7.23 (s, 1 H), 7.81 (s, 1 H), 8.07 (s, 1 H).13C NMR (100 MHz, DMSO-cfe) 6 ppm 24.6, 24.7, 27.4, 28.1 , 53.5, 55.0, 78.1 , 85.0, 114.2, 136.5, 139.5, 146.7, 155.1 , 170.5, 175.5. LCMS, m / z calcd. for C20H33N4O7 [M+H]+441.2344, found 441.2269. Example 2q: Preparation of (S)-2-(3-(1-benzyl-1 H-imidazol-4-yl)-2-(((tert-butyloxy) carbonyl)amino)propanamido)-2-methylpropanoic acid (4Bh)

[0454] Procedure: Preparation of silyl protected 2-amino isobutyric acid:

[0455] A 350 mL screw-cap pressure vessel was charged with Aib (3.07 g, 29.8 mmol) and the vessel was flushed with argon. Then, dry THF (200 mL) and BSA (9.0 mL, 36.4 mmol) were added, the vial was closed with screw cap and the stirring mixture was heated (heating bath temperature 80°C) for 7 hours after which the closed pressure vessel was cooled to room temperature in water bath. The resulting mixture was transferred to a dropping funnel under argon.

[0456] Activation of3Bh with DCC / HOPO A 250 mL round-bottom flask was charged with 3Bh (7.48 g, 21.7 mmol) and HOPO (2.65 g, 23.9 mmol) and the flask was flushed with argon. Then dry THF (100 mL) was added, and the mixture was stirred at room temperature for 5 minutes under argon. The mixture was cooled to 0 °C and DCC (5.84 g, 26.3 mmol) dissolved in dry THF (initially 10 mL, then rinse with 3 x 5 mL) was added via septum / needle and syringe under argon over 10 minutes. After addition, the stirring at 0 °C continued for another 10 minutes and then the cooling bath was removed. The stirring continued for 3.5 hours. Then, anhydrous oxalic acid (330 mg, 3.67 mmol) was added as a solid and the mixture was stirred at room temperature for 1 hour. Then, the mixture was very quickly filtered through sinter (S4) and the solids were washed with dry tetrahydrofuran (1 x 10 mL, 1 x 24 mL). To minimize decomposition of the activated acid with moisture, all the operations during filtration were performed as quickly as possible and the air was not allowed to pass through the solids on the sinter to avoid cooling (due to solvent evaporation) and moisture condensation. The filtrate was collected in a 500 mL round-bottom flask and directly after filtration, the solution was flushed with argon. The solution was cooled to 0 °C under argon and the mixture with the silyl protected Aib solution was added dropwise from dropping funnel over 15 minutes and the funnel was then washed with dry THF (2 x 10 mL). After addition, cooling continued for another 10 minutes, then the cooling bath was removed, and the stirring continued for 16 hours at room temperature. The rection was monitored using HPLC-MS_1. Analysis showed conversion to 4Bh (estimated 5-10% of residual starting material 3Bh according to HPLC-MS). Thin layer chromatography (TLC) analysis of the reaction mixture also showed 4Bh formation (SiC>2, MeCN / H2O 10:1 + 0.5% acetic acid (AcOH); 4Bh: Rf=0.20, starting material: Rf=0.10). The reaction mixture was cooled to 0 °C and added to a 5% aqueous solution of sodium dihydrogen phosphate (500 mL) pre-cooled to 0 °C. The mixture was transferred to separatory funnel, then more of 5% aqueous solution of sodium dihydrogen phosphate (300 mL) was added and the mixture was extracted with DCM (1 x 500 mL, 1 x 200 mL, 1 x 200 mL). The organic extracts were dried over anhydrous sodium sulfate (Na2SO4). After decantation, the volatiles were removed on rotary evaporator to give crude product (11.23 g), which was purified by column chromatography (silica gel 60, 0.040-0.063 mm, 430 g; eluent: MeCN / H2O 20:1 to 10:1 + 0.5% AcOH). Pure fractions were pooled, concentrated in vacuo, and water (200 mL) was added. Freeze-drying gave pure 4Bh as an off-white powder (6.6 g, 71.0% yield). Purity >99.9% (ELSD, HPLC-MS_1). Rf(SiO2, MeCN / H2O 15:1 + 0.5% AcOH): 0.10.1H NMR spectrum (500 MHz, DMSO-cfe) 6 ppm 1.26 (s, 3 H), 1.39-1.28 (m, 12 H), 2.83-2.57 (m, 2 H), 4.18-4.01 (m, 1 H), 5.12 (s, 2 H), 6.80-6.65 (m, 1 H), 6.90 (s, 1 H), 7.26-7.19 (m, 2 H), 7.39-7.26 (m, 3 H), 7.72 (s, 1 H), 7.92 (s, 1 H).13C NMR (125 MHz, DMSO-cfe) 6 ppm 24.5, 24.7, 28.1 , 30.7, 49.6, 54.2, 55.2, 78.1 , 117.0, 127.5, 127.7, 128.6, 136.8, 137.6, 155.0, 170.5, 175.7. LCMS, m / z calcd. for C22H3IN4O5[M+H]+431 .2289, found 431.2290.

[0457] Example 2h: Preparation of (S)-2-(3-(1-benzyl-1 H-imidazol-4-yl)-2-

[0458] (((benzyloxy)carbonyl)amino)propanamido)-2-methylpropanoic acid (4Ch)

[0459] To a 2L round-bottom flask containing (S)-2-(3-(1 -benzyl- 1 H-imidazol-4-yl)-2-((tert- butoxycarbonyl)amino)-propanamido)-2-methylpropanoic acid (4Bh) (6.5 g, 15.1 mmol) was added dry DCM (60 mL). To the resulting solution TFA (200 mL) was added. The mixture was stirred at room temperature for 1 .5 hours. LC-MS showed complete tert-butyloxycarbonyl (Boc) deprotection. The volatiles from the reaction mixture were removed on rotary evaporator placed in a fume hood (bath temperature maximum 30 °C). Residues of TFA were removed by multiple co-evaporation with dry DCM (8 x 100 mL) to give (S)-2-(2-amino-3-(1-benzyl-1 H- imidazol-4-yl)propanamido)-2-methylpropanoic acid bistrifluoroacetate salt as an off-white amorphous solid (8.4 g, 15.0 mmol, quantitative yield). The compound was transferred to a 2 L round-bottom flask and dissolved with 1 ,4-dioxane (40 mL), water (100 mL), and KHCO3 (11.7 g, 117.0 mmol) (gas evolution). Then, 1 ,4-dioxane (20 mL) was added followed by solid / V-(benzyloxycarbonyloxy)succinimide (3.8 g, 15.3 mmol) and 1 ,4-dioxane (40 mL) to wash the walls of the flask. The mixture was stirred for 17 hours at room temperature. Method HPLC- MS_1 showed complete conversion of H-His(Bn)Aib-OH to product (4Ch). The reaction mixture was cooled to 0 °C and 10% aqueous solution of sodium dihydrogen phosphate (300 mL) was added. The mixture was transferred to separatory funnel and the mixture was extracted with EtOAc (1 x 400 mL, 1 x 200 mL, 1 x 200 mL). The organic extracts were dried over anhydrous Na2SO4. After decantation, the volatiles were removed on rotary evaporator to give crude product (6.88 g), which was purified by column chromatography (silica gel 60, 0.040-0.063 mm, 200 g; eluent: MeCN / H2O 15:1 to 10:1). Pure fractions were pooled, concentrated in vacuo, and 5% aqueous MeCN (600 mL) was added. Lyophilization gave a fine light white powder (5.64 g, 80.0% yield). Purity: >99.9% (ELSD, HPLC-MS_1). Rf (SiO2, MeCN / H2O 10:1): 0.20.1H NMR (500 MHz, DMSO-cfe) 6 ppm 1.26 (s, 3 H), 1.31 (s, 3 H), 2.88- 2.64 (m, 2 H), 4.28-4.13 (m, 1 H), 4.98 (s, 2 H), 5.12 (s, 2 H), 6.92 (s, 1 H), 7.44-7.17 (m, 11 H), 7.73 (s, 1 H), 8.04 (s, 1 H).13C NMR (125 MHz, DMSO-cfe) 6 ppm 24.7, 24.8, 30.9, 49.6, 54.5, 55.2, 65.3, 117.1 , 127.4, 127.6, 127.7, 127.7, 128.3, 128.6, 136.8, 137.0, 137.4, 137.6, 155.6, 170.5, 175.5. LCMS, m / z calcd. for C25H29N4O5 [M+H]+465.2132, found 465.2053.

[0460] Example 2i: Preparation of (S)-2-(3-(1-((benzyloxy)carbonyl)-1 H-imidazol-4-yl)-2-

[0461] (((benzyloxy)carbonyl)amino)propanamido)-2-methylpropanoic acid (4Ci)

[0462] Compound 4Ci was synthesized following general procedure B using / V^ / V1- bis((benzyloxy)carbonyl)-L-histidine (3Ci) (1.0 g, 2.4 mmol). After the reaction was completed the THF was removed under reduced pressure. Water was added and the pH adjusted to around 3. The product was extracted with EtOAc. The organic phase was collected, dried over MgSO4, filtered, and concentrated. The product was purified by flash column chromatography eluting with a gradient from MeCN to 2% water in MeCN. Pure fractions were collected and after lyophilization an off-white solid was obtained (0.8 g, 67.0%). Purity 98.4% (UPLC-MS_6).1H NMR (400 MHz, DMSO-d6) 6 ppm 1.30 (s, 3 H), 1.34 (s, 3H H), 2.65-2.77 (m, 1 H), 2.79- 2.89 (m, 1 H), 4.22-4.35 (m, 1 H), 4.91-5.03 (m, 2 H), 5.42 (s, 2 H), 7.23-7.45 (m, 10 H), 7.46- 7.52 (m, 2 H), 8.13 (s, 1 H), 8.22 (s, 1 H), 12.26 (bs, 1 H).13C NMR (100 MHz, DMSO-cfe) 6 ppm 24.7, 24.8, 53.8, 55.0, 65.3, 69.1 , 114.5, 127.5, 127.7, 128.2, 128.3, 128.6, 128.6, 134.7136.7, 137.0, 139.8, 148.2, 155.7, 170.4, 175.4. LCMS, m / z calcd. for C26H28N4O7Na [M+Na]+531.1850, found 531.1860. Example 2j: Preparation of (S)-2-(2-((1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl)amino)-

[0463] 3-(1-tosyl-1 H-imidazol-4-yl)propanamido)-2-methylpropanoic acid (4Db)

[0464] Compound 4Db was synthesized following general procedure B using / V“-(1-(4,4-dimethyl-2,6- dioxocyclohexylidene)ethyl)- / VT-tosyl-L-histidine (3Db) (2.7 g, 5.7 mmol). After the reaction was completed. The THF was reduced under reduced pressure (~10 mL removed). MeTHF was added (30 mL) to the aqueous solution followed by Zn(OAc)2 (2 eq) dissolved in water (50 mL). After extraction the phases were separated, and the organic phase was washed with water (2 x 30 mL) and brine (1 x 30 mL). The organic phase was dried over MgSCL, filtered, and concentrated. The product was purified by flash column chromatography eluting with a gradient from DCM to 5% MeOH in MeCN. The pure fractions were combined and evaporated under reduced pressure. After drying in vacuo overnight, a white solid material was obtained (1025.0 mg, 32.2% yield). Purity 95.7% (UPLC-MS_10).

[0465] 1H NMR (400 MHz, DMSO-cfe) 6 ppm 0.92 (s, 6 H), 1.18(s, 3 H ), 1.25 (s, 3 H ), 2.24 (s, 4 H), 2.25 (s, 3 H), 2.40 (s, 3 H), 2.84 (dd, J = 14.9 Hz, J = 7.9 Hz , 1 H), 2.93 (dd, J = 14.8 Hz, J = 7.9 Hz , 1 H), 4.68 (q, J = 7.6 Hz, 1 H), 7.46 (s, 1 H), 7.47, (d, J = 8.6 Hz, 2 H), 7.91 (d, J = 8.4 Hz, 2 H), 8.32 (d, J = 1.0 Hz, 1 H), 8.48 (s, 1 H), 12.34 (bs, 1 H), 13.35 (d, J = 7.6 Hz, 1 H).13C NMR (100 MHz, DMSO-cfe) 6 ppm 17.5, 21.1 , 24.0, 24.9, 27.8, 29.6, 31.8, 52.4 (bs, 2 C) 55.0, 55.4, 107.1 , 115.7, 127.2, 130.5, 134.3, 136.9, 138.9, 146.3, 168.1 , 171.9, 174.9, 190- 200 (2 x carbonyl from the Dde ring, broad and weak). LCMS, m / z calcd. for C27H35N4O7S [M+H]+559.2221 , found 559.2055.

[0466] Example 2k: Preparation of (S)-2,2-dimethyl-4,7,11-trioxo-5-((1-tosyl-1 H-imidazol-4- yl)methyl)-8,10-dioxa-3,6-diazahexadecanoic acid (4Edb)

[0467] Compound 4Edb was synthesized following general procedure B using A / “- (((hexanoyloxy)methoxy)carbonyl)- / \ / T-tosyl-L-histidine (3Edb) (0.5 g, 1.0 mmol). After the reaction was completed, the reaction mixture was poured slowly into a vigorously stirred 5% aqueous solution of potassium bisulphate (KHSO4) (100 mL). The precipitate was filtered, washed with water (5 x 20 mL) and freeze-dried from MeCN to obtain a white material (338.2 mg, 76.0% yield). Purity 91.0% (UPLC-MS_2).1H NMR (400 MHz, DMSO-cfe) 6 ppm 8.84 (t, J=6.8 Hz, 3 H), 1.18-1.31 (m, 10 H), 1.50 (p, J=7.2 Hz, 2 H), 2.31 (t, J=7.4 Hz, 2 H), 2.40 (s, 3 H), 2.67 (dd, J=15.1 Hz, J=9.0 Hz, 1 H), 2.80 (dd, J=14.9 Hz, J=5.1 Hz, 1 H), 4.22-4.29 (m, 1 H), 5.56 (s, 2 H), 7.34 (s, 1 H), 7.49 (d, J=8.1 Hz, 2 H), 7.62 (d, J=8.6 Hz, 1 H), 7.91 (d, J=8.4 Hz, 2 H), 8.27 (d, J=1.3 Hz, 1 H).13C NMR (100 MHz, DMSO-cfe) 6 ppm 13.7, 21.1 , 21.7, 23.8, 24.6, 30.4, 30.7, 33.2, 53.4, 54.9, 79.4, 114.8, 127.2, 130.5, 134.4, 136.6, 140.5, 146.3, 153.9, 169.7., 172.0, 175.2. LCMS, m / z calcd. for C25H34N4O9SNa [M+Na]+589.1939, found 589.1964.

[0468] Example 3: Oxazolone formation.

[0469] Example 3a: Preparation of (S)- / V-(1-(4,4-dimethyl-5-oxo-4,5-dihydrooxazol-2-yl)-2-(1-

[0470] (phenylsulfonyl)-1 H-imidazol-4-yl)ethyl)-2,2,2-trifluoroacetamide (6Aa)

[0471] Small scale synthesis: Compound 6Aa was synthesized following general procedure C using (S)-2-methyl-2-(3-(1-(phenylsulfonyl)-1 H-imidazol-4-yl)-2-(2,2,2- trifluoroacetamido)propanamido) propanoic acid (4Aa) (1.0 g, 2.1 mmol). Purification by flash column chromatography with gradient elution EtOAc / heptane 1 :5 to pure EtOAc was performed. After drying in vacuo, a white powder was obtained (0.7 g, 73.0% yield). Purity >99.0% (1H NMR). Active content of material from1H qNMR was 92.0% w / w.1H NMR (400 MHz, DMSO-cfe) 6 ppm 1.23 (s, 3 H), 1.26 (s, 3 H), 3.00 (dd, J = 14.9 Hz, J = 8.7 HZ, 1 H), 3.12 (dd, J = 15.0 Hz, J = 5.8 HZ, 1 H), 4.92-4.97 (m, 1 H), 7.51 (d, J = 1.2 Hz, 1 H), 7.65-7.72 (m, 2 H), 7.81 (tt, J = 7.5, J= 1.8 Hz, 1 H), 8.01-8.06 (m, 2 H), 8.34 (d, J = 1.4 Hz, 1 H), 9.92 (d, J = 7.9 Hz, 1 H).13C NMR (100 MHz, DMSO-cfe) 6 ppm 23.6, 23.7, 28.4, 47.6, 65.2, 115.5, 115.5 (q, J= 288.2 Hz), 127.1 , 130.1 , 135.3, 137.1 , 137.2, 139.3, 156.6 (q, J = 36.9 Hz), 159.9, 180.7.19F NMR (376 MHz, DMSO-d6) 5 ppm -74.3. LCMS, m / z calcd. for C18H18F3N4O5S [M+H]+459.0944, found 459.0986.

[0472] Large scale synthesis: Equipment (Reactor): 0.5-liter double jacketed reactor (RX) with thermostat, distillation setup, N2headspace. All intakes correlated from the assay of dipeptide. The reactor was charged with dipeptide 4Aa (25.0 g, 90.0% w / w, calc. 22.5 g, 47.2 mmol) and EtOAc (9 vol, 179.7 g -205 mL). The resulting white slurry was stirred at 30°C for 20 minutes. Subsequently, a solution of DCC (10.9 g, 53.0 mmol, 1.1 eq) in EtOAc (20.4 g, 23 mL, 1 vol) was added. The slurry was stirred at Tj 30±5°C for 1 h and then at 20 - 25°C for 19h. The reaction mixture was sampled and analyzed with quantitative HPLC. Result: 98.8% conversion, 97.0% assay yield 6Aa. The batch was collected from the reactor in a glass beaker and filtered through a silica-gel plug (22.5 g silica gel 60, conditioned with EtOAc, height 1.8 cm, width 6 cm) in a glass filter (p3) in 12 minutes applying slight vacuum in the receiving vessel. The DCU cake / silica gel plug was washed with EtOAc (220 mL, 10 vol) and pulled dry. Meanwhile the reactor was cleaned with EtOAc and dried with nitrogen (N2). The DCU / silica plug filtrate (clear, near colorless to very pale-yellow solution) was returned to the reactor. The batch (450 mL, 20 vol) was heated to 59°C and 350.0 mL (15.5 vol) EtOAc was distilled off under vacuum at 50-44°C (pressure -0.30 bar) until a batch volume of 100 mL (4.5 vol). Subsequently toluene was added, and 280.0 mL (8 vol) EtOAc / toluene were distilled of at SOSO °C until a batch volume of 100 mL (4.5 vol). Total distillation time 1.5h. After the distillations toluene (110 mL, 5 vol) was added to the slurry of 6Aa at 51 °C. The batch volume was 210 mL (9.5 vol). The batch was subsequently cooled slowly to 20 °C in 2-3h and then stirred overnight at 20 ± 2°C. The batch was then filtered. The product filter was washed with toluene (45 mL, 2 vol), and then with n-heptane (45 mL, 2 vol). The wet cake was harvested and dried in a porcelain tray in a vacuum stove (<40°C) for 4h (no weight loss compared to 3h) affording 18.77 g of a white solid.

[0473] Analyses: Quantitative HPLC (58.94 mg in 50.00 mL THF): assay 98.0% w / w, purity >99.9% no impurities observed. qNMR (34.47 mg product, 18.65 mg 1 ,3,5-trimethoxybenzene) in -1.2 mL DMSO: Assay 99.0% w / w, DCU 0.13% w / w, toluene 0.08% w / w. Chiral HPLC: enantiomeric excess >99.9%. Isolated yield corrected for assay of 99.0%: 86.0%.

[0474] Example 3b: (S)- / V-(1-(4,4-dimethyl-5-oxo-4,5-dihydrooxazol-2-yl)-2-(1-tosyl-1 H-imidazol-4- yl)ethyl)-2,2,2-trifluoroacetamide (6Ab)

[0475] The title compound 6Ab was synthesized following general procedure C using ((S)-2-methyl- 2-(3-(1-tosyl-1 H-imidazol-4-yl)-2-(2,2,2-trifluoroacetamido)propanamido) propanoic acid 4Ab (2.0 g, 4.1 mmol). Purification with gradient elution EtOAc / heptane 1 :5 to 1 :1 was performed. After drying in vacuo overnight, a white powder was obtained (2.0 g, 99.0% yield). Purity >98.0% (1H NMR). Active content of material from1H qNMR was 99.7% w / w.1H NMR (400 MHz, DMSO-cfe) 6 ppm 1.23 (s, 3 H), 1.26 (s, 3 H), 2.39 (s, 3 H), 2.99 (dd, J = 14.9 Hz, J = 8.7 Hz, 1 H), 3.11 (dd, J = 14.9 Hz, J = 5.9 Hz, 1 H), 4.87-4.98 (m, 1 H), 7.45-7.52 (m, 3 H), 7.91 (d, J = 8.4 Hz, 2 H), 8.30 (d, J = 1.3 Hz, 1 H), 9.92 (d, J = 7.4 Hz, 1 H).

[0476] 13C NMR (100 MHz, DMSO-cfe) 6 ppm 21.1 , 23.6, 23.7, 28.4, 47.6, 65.2, 115.4, 115.5 (q, J = 288.2 Hz), 127.2, 130.5, 134.2, 137.0, 139.2, 146.4, 156.2 (q, J = 37.0 Hz), 159.9, 180.7.19F NMR (376 MHz, DMSO-cfe) 6 ppm -74.3. LCMS, m / z calcd. For C19H20F3N4O5S [M+H]+473.1101 found, 473.1224.

[0477] Example 3c: Preparation of (S)- / V-(1-(4,4-dimethyl-5-oxo-4,5-dihydrooxazol-2-yl)-2-(1-((4- methoxyphenyl) sulfonyl)-1 H-imidazol-4-yl) ethyl)-2,2,2-trifluoroacetamide (6Ac)

[0478] Compound 6Ac was synthesized following general procedure C using (S)-2-(3-(1-((4- methoxyphenyl) sulfonyl)-1 H-imidazol-4-yl)-2-(2,2,2-trifluoroacetamido) propanamido)-2- methylpropanoic acid (4Ac) (2.5 g, 4.9 mmol). Purification with gradient elution EtOAc / heptane 1 :5 to 1 :1 was performed. After drying in vacuo overnight, a white powder was obtained (1.25 g, 52.0% yield). Purity >98.0% (1H NMR). Active content of material from1H qNMR was 96.0% w / w.1H NMR (400 MHz, DMSO-cfe) 6 ppm 1.24 (s, 3 H), 1.26 (s, 3 H), 2.39 (s, 3 H), 3.00 (dd, J = 14.8 Hz, J = 8.7 Hz, 1 H), 3.11 (dd, J = 14.8 Hz, J = 6.0 Hz, 1 H), 3.85 (s, 3 H), 4.88-4.98 (m, 1 H), 7.17 (d, J = 9.0 Hz, 2 H), 7.46 (s, 1 H), 7.97 (d, J = 9.0 Hz, 2 H), 8.30 (d, J = 1.4 Hz, 1 H), 9.92 (d, J = 7.9 Hz, 1 H).13C NMR (100 MHz, DMSO-cfe) 5 ppm 23.6, 23.7, 28.4, 47.6, 56.0, 65.2, 115.3, 115.5 (q, J = 288.2 Hz), 128.3, 129.7, 136.9, 139.1 , 156.1 (q, J = 36.9 Hz), 159.9, 164.4, 180.7.19F NMR (376 MHz, DMSO-cfe) 5 ppm -74.3. LCMS, m / z calcd. for C19H20F3N4O6S [M+H]+489.1050 found, 489.1118.

[0479] Example 3d: Preparation of (S)- / V-(1-(4,4-dimethyl-5-oxo-4,5-dihydrooxazol-2-yl)-2-(1-

[0480] (thiophen-2-ylsulfonyl)-1 H-imidazol-4-yl)ethyl)-2,2,2-trifluoroacetamide (6Ad) The title compound 6Ad was synthesized following general procedure C using (S)-2-methyl-2- (3-(1-(thiophen-2-ylsulfonyl)-1 H-imidazol-4-yl)-2-(2,2,2-trifluoroacetamido)- propanamido)propanoic acid (4Ad) (0.5 g, 1.0 mmol). Purification with gradient elution EtOAc / heptane 1 :19 to 1 :1 was performed. The pure fractions were combined and evaporated under reduced pressure. The solid was redissolved in MeCN and evaporated under reduced pressure. After drying in vacuo overnight, a white crystallin powder was obtained (325.0 mg, 66.0% yield). Purity >98% (1H NMR). Active content of material from1H qNMR was 95.7% w / w.1H NMR (400 MHz, DMSO-cfe) 6 ppm 1.21 (s, 3 H), 1.32 (s, 3 H), 3.03 (dd, J = 14.8 Hz, J = 8.8 Hz, 1 H), 3.14 (dd, J = 14.8 Hz, J= 5.9 Hz, 1 H), 4.96 (m, 1 H), 7.29 (dd, J = 4.9 Hz, J= 4.0 Hz, 1 H), 7.52 (d, J = 0.9 Hz, 1 H), 8.03 (dd, J = 3.9 Hz, J= 1.4 Hz, 1 H), 8.23 (dd, J = 4.7 Hz, J= 1.3 Hz, 1 H), 8.32 (d, J = 1.4, 1 H), 9.93 (d, J = 7.9 Hz, 1 H).13C NMR (100 MHz, DMSO-cfe) 6 ppm 23.6, 23.8, 47.6, 65.2, 115.35, 115.54 (q, J= 288 Hz), 136.1 , 136.5, 137.8, 139.4, 156.2 (q, J = 37.0 Hz ) 159.9, 180.7.19F NMR (376 MHz, DMSO-cfe) 6 ppm -74.3. LCMS, m / z calcd. for C16H16F3N4O5S2 [M+H]+465.0509, found 465.0552.

[0481] Example 3e: Preparation of (S)-N-(1-(4,4-dimethyl-5-oxo-4,5-dihydrooxazol-2-yl)-2-(1-(2,4- dinitrophenyl)-1 H-imidazol-4-yl)ethyl)-2,2,2-trifluoroacetamide (6Ap)

[0482] The title compound 6Ap was synthesized following general procedure C using (S)-2-(3-(1-(2,4- dinitrophenyl)-1 H-imidazol-4-yl)-2-(2,2,2-trifluoroacetamido)propanamido)-2-methylpropanoic acid (4Ap) (0.3 g, 0.6 mmol). Purification with gradient elution EtOAc / heptane 1 :1 to EtOAc was performed. The pure fractions were combined and evaporated under reduced pressure. After drying in vacuo overnight, a yellow oil was obtained (181.0 mg, 63.0% yield). Purity >99.0% (1H NMR). Active content of material from1H qNMR was 90.0 % w / w.1H NMR (400 MHz, CDCh) 6 ppm 1.32 (s, 3 H), 1.39 (s, 3 H), 3.24 (dd, J=15.3 Hz, J=4.5 Hz, 1 H), 3.31 (dd, J=15.3 Hz, J=4.5 Hz, 1 H), 5.16-5.22 (m, 1 H), 6.93 (s, 1 H), 7.61 (d, J=1.3 Hz, 1 H), 7.65 (d, J=8.7 Hz, 1 H), 8.55 (d, J=7.44 Hz, 1 H), 8.59 (dd, J=8.7 Hz, J=2.5 Hz, 1 H), 8.86 (d, J=2.5 Hz, 1 H).13C NMR (100 MHz, CDCh) 6 ppm 24.3, 24.4, 48.1 , 65.8, 115.8 (q, J=285.8 Hz), 118.0, 121.6, 128.5, 129.4, 134.9, 136.9,138.7, 144.7, 147.3, 157.2 (q, J=37.6 Hz), 160.3, 180.3.19F NMR (376 MHz, CDCh) 6 ppm -75.9. LCMS, m / z calcd. for CisHieFsNeCh [M+H]+485.1027, found 485.1008. Example 3f: Preparation of tert-butyl (S)-4-(2-((tert-butoxycarbonyl)amino)-2-(4,4-dimethyl-5- oxo-4, 5-dihvdrooxazol-2-yl)ethyl)-1 H-imidazole-1-carboxylate (6Bg)

[0483] The title compound 6Bg was synthesized following general procedure C using (S)-2-(3-(1-(tert- butoxycarbonyl)-1 H-imidazol-4-yl)-2-((tert-butoxy-carbonyl)amino)propanamido)-2- methylpropanoic acid (4Bg) (0.3 g, 0.7 mmol). Purification with gradient elution EtOAc / heptane 1 :1 to 3:1 was performed. The pure fractions were combined and evaporated under reduced pressure. After drying in vacuo overnight, a white semi-solid material was obtained (208.0 mg, 72.0% yield). Purity 85.0% (1H NMR). Active content of material from1H qNMR was 83.7% w / w.1H NMR (400 MHz, DMSO-cfe) 6 ppm 1.28-1.39 (m, 15 H), 1.56 (s, 9 H), 2.89 (dd, J=14.7 Hz, J=8.4 Hz, 1 H), 2.98 (dd, J=14.7 Hz, J=5.6 Hz, 1 H), 4.55 (q, J=8.0 Hz, 1 H), 7.30 (s, 1 H), 7.34 (d, J=8.0 Hz, 1 H), 8.12 (s, 1 H).13C NMR (100 MHz, DMSO-cfe) 6 ppm 23.6, 23.7, 27.2, 27.9, 29.4, 48.7, 65.0, 78.4, 85.0, 114.6, 136.6, 138.4, 146.5, 154.9, 161.8, 181.1. LCMS, m / z calcd. for C2oH3oN406Na [M+Na]+445.2058, found 445.2080.

[0484] Example 3q: Preparation of (S)-2-(1-((1-(4,4-dimethyl-5-oxo-4,5-dihydrooxazol-2-yl)-2-(1- tosyl-1 H-imidazol-4-yl)ethyl)amino)ethylidene)-5,5-dimethylcyclohexane-1 ,3-dione (6Db)

[0485] The title compound 6Db was synthesized following general procedure C using (S)-2-(2-((1- (4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl)-amino)-3-(1-tosyl-1 H-imidazol-4- yl)propanamido)-2-methylpropanoic acid (4Db) (1.0 g, 1.8 mmol). Purification with gradient elution EtOAc / heptane 1 :19 to 9:1 was performed. The pure fractions were combined and evaporated under reduced pressure. After drying in vacuo overnight, a white semi-solid material was obtained (565.0 mg, 85.0% yield). Purity >98.0% (1H NMR). Active content of material from1H qNMR was 96.0% w / w.1H NMR (400 MHz, DMSO-cfe) 6 ppm 0.94 (s, 6 H), 1.23 (s, 3 H), 1.25 (s, 3 H), 2.26 (bs, 4 H), 2.40 ( s, 3 H), 2.43 (s, 3 H), 3.11 (d, J = 5.4 Hz, 2 H), 5.22-5.28 (m, 1 H), 7.48 (d, J = 8.2 Hz, 2 H), 7.57 (s, 1 H), 7.92 (d, J = 8.4 Hz, 2 H), 8.30 (d, J = 1.1 Hz, 1 H), 13.46 (d, J = 7.8 Hz, 1 H).13C NMR (100 MHz, DMSO-cfe) 6 ppm 17.5, 21.1 , 23.3, 23.6, 27.8 (bs), 29.6, 29.9, 50.5, 51.6 (bs), 52.9 (bs), 65.2, 107.5, 116.4, 127.2, 130.5, 134.2, 137.1 , 146.3, 159.9, 172.7, 180.5, 190-200 (2 x carbonyl from the Dde ring, broad and weak). LCMS, m / z calcd. for C27H32N4O6S [M+H]+541.2115, found 541.2136.

[0486] Example 3h: Preparation of (S)-(((1-(4,4-dimethyl-5-oxo-4,5-dihvdrooxazol-2-yl)-2-(1-tosyl-

[0487] 1 H-imidazol-4- hexanoate

[0488] The title compound 6Edb was synthesized following general procedure C using (S)-2,2- dimethyl-4,7,11-trioxo-5-((1-tosyl-1 H-imidazol-4-yl)methyl)-8,10-dioxa-3,6- diazahexadecanoic acid (4Edb) (300.0 mg, 0.53 mmol). Purification with gradient elution EtOAc / heptane 1 :1 to pure EtOAc was performed. The pure fractions were combined and evaporated under reduced pressure. After drying in vacuo overnight, a white semi-solid material was obtained (240.0 mg, 83.0% yield). Purity >98.0% (1H NMR). Active content of material from1H qNMR was 96.0% w / w.1H NMR (400 MHz, DMSO-cfe) 5 ppm 0.88 (t, J=7.0 Hz, 3 H), 1.09 (s, 3 H), 1.23-1.35 (m, 7 H), 1.63 (p, J=7.4 Hz, 2 H), 2.35 (t, J=7.Q Hz, 2 H), 2.44 (s, 3 H), 3.03-3.14 (m, 2 H), 4.88 (dt, J=8.2 Hz, J=4.6 Hz, 1 H), 5.69-5.78 (m, 2 H), 6.37 (d, J=8.2 Hz, 1 H), 7.05 (d, J=1.1 Hz, 1 H), 7.35 (d, J=8.0 Hz, 2 H), 7.79 (d, J=8.4 Hz, 2 H), 7.89 (d, J=1.3 Hz, 1 H).13C NMR (100 MHz, DMSO-cfe) 6 ppm 13.9, 21.8, 22.3, 24.1 , 24.2, 24.3, 29.5, 31.2, 34.0, 49.1 , 65.4, 80.0, 115.2, 127.5, 130.6, 134.8, 136.6, 139.2, 146.6, 154.2, 161.4, 172.7, 180.4. LCMS, m / z calcd. for C25H32N4O8SNa [M+Na]+571.1833, found 571.1841.

[0489] Example 4: NHS ester formation of Pg1His(Pg2)Aib-OH. imidazol-4-yl)-2-(2,2,2-trifluoroacetamido) propanamido) propanoate (5Aac)

[0490] Compound 5Aac was synthesized following general procedure D using (S)-2-methyl-2-(3-(1- (phenylsulfonyl)-1 H-imidazol-4-yl)-2-(2,2,2-trifluoroacetamido) propenamide) propanoic acid (4Aa) (238.0 mg, 0.5 mmol). Purification with gradient elution DCM to MeOH / DCM 1 :9 was performed. After drying in vacuo overnight, a white powder was obtained (243.0 mg, 86.0% yield). Purity >92.0% (UPLC-MS_7). Active content of material from1H qNMR was 95.0% w / w (mixture of oxazolone and NHS ester).1H NMR (400 MHz, DMSO-cfe) 5 ppm 1.42 (s, 3 H), 1.49 (s, 3 H), 2.76 (bs, 4 H), 2.86-2.96 (m, 2 H), 4.55-4.65 (m, 1 H), 7.35 (s, 1 H), 7.77 (t, J = 7.6 Hz, 2 H), 7.81 (t, J = 7.5 Hz, 1 H), 8.02 (d, J = 7.44 Hz, 2 H), 8.32-8.35 (m, 1 H), 8.86 (s, 1 H), 9.55 (d, J = 8.2 Hz, 1 H).13C NMR not reported as the compound was a mix of oxazolone and activated NHS ester.19F NMR (376 MHz, DMSO-cfe) 6 ppm -70.1. LCMS, m / z calcd. for C22H22F3N5NaO8S [M+Na]+596.1033, found 596.1118.

[0491] Example 4b: Preparation of 2,5-dioxopyrrolidin-1-yl (S)-2-methyl-2-(3-(1-tosyl-1 H-imidazol-4- yl)-2-(2,2,2-trifluoroacetamido) propanamido) propanoate (5Abc)

[0492] Compound 5Abc was synthesized following general procedure D using (S)-2-methyl-2-(3-(1- tosyl-1 H-imidazol-4-yl)-2-(2,2,2-trifluoroacetamido) propenamide) propanoic acid (4Ab) (238.0 mg, 0.5 mmol). Purification with gradient elution DCM to MeOH / DCM 1 :9 was performed. After drying in vacuo overnight, a white powder was obtained (138.0 mg, 52.0% yield). Purity 91% (UPLC-MS_7). Active content of material from1H qNMR was 90.0% w / w (mixture of oxazole and NHS ester).1H NMR (400 MHz, DMSO-cfe) 6 ppm 1.42 (s, 3 H), 1.48 (s, 3 H), 2.39 (s, 3 H), 2.76 (bs, 4 H), 2.86-2.97 (m, 2 H), 4.54-4.66 (m, 1 H), 7.31 (s, 1 H), 7.47 (d, J = 7.7 Hz, 2 H), 7.89 (d, J = 7.9 Hz, 2 H), 8.29 (s, 1 H), 8.86 (s, 1 H), 9.54 (d, J = 8.0 Hz, 1 H).13C NMR not reported as the compound was a mix of oxazolone and activated NHS ester.19F NMR (376 MHz, DMSO-cfe) 6 ppm -74.1. LCMS, m / z calcd. for C23H24F3N5NaO8S [M+Na]+596.1033, found 596.1118.

[0493] Example 5: Phenol ester formation of Pg1His(Pg2)Aib-OH

[0494] 1 H-imidazol-4-yl)-2-(2,2,2-trifluoroacetamido) propanamido) propanoate (5Aaa)

[0495] Compound 5Aaa was synthesized following general procedure E using (S)-2-methyl-2-(3-(1- (phenylsulfonyl)-1 H-imidazol-4-yl)-2-(2,2,2-trifluoroacetamido) propenamide) propanoic acid (4Aa) (300.0 mg, 0.6 mmol). Purification with gradient elution DCM to MeOH / DCM 1 :13 was performed. After drying in vacuo overnight, a white powder was obtained (277.0 mg, 70.0% yield). Purity 98.5% (UPLC-MS_7). Active content of material from1H qNMR is 95.0% w / w.1H NMR (400 MHz, DMSO-cfe) 6 ppm 1.38 (s, 3 H) 1.46 (s, 3 H), 2.90 (dd, J=16.0 Hz, J=9.4 Hz, 1 H), 2.98 (dd, J=14.7 Hz, 1 H), 3.89 (s, 3 H) 4.61-4.69 (m, 1 H) 7.10 (d, J = 8.6 Hz, 1 H) 7.38 (d, J = 0.9 Hz, 1 H) 7.63-7.70 (m, 2 H) 7.80 (tt, J = 7.5 Hz, J = 1.0 Hz, 1 H) 7.85-7.92 (m, 2 H) 7.98-8.04 (m, 2 H) 8.32 (d, J = 1.4 Hz, 1 H) 8.91 (s, 1 H) 9.58 (d, J = 8.1 Hz, 1 H).13C NMR (100 MHz, DMSO-cfe) 5 ppm 24.3, 24.4, 51.9, 55.3, 56.6, 107.9, 114.9, 115.6 (q, J= 286.4 Hz), 116.2, 117.1 , 119.9, 123.3, 127.0, 130.1 , 135.2, 136.9, 137.2, 140.0, 144.9, 145.8, 151.5, 155.9 (q, J = 36.4 Hz), 169.1 , 171.2.19F NMR (376 MHz, DMSO-cfe) 6 ppm -74.1. LCMS, m / z calcd. for C25H24F3N5O9SNa [M+Na]+650.1139, found 650.1082. 2-(3-(1-

[0496] Compound 5Abb was synthesized following general procedure E using ((S)-2-methyl-2-(3-(1- tosyl-1 H-imidazol-4-yl)-2-(2,2,2-trifluoroacetamido) propanamido) propanoic acid (4Ab) (150.0 mg, 0.3 mmol). Purification with gradient elution DCM to MeOH / DCM 1 :9 was performed. After drying in vacuo overnight, a white powder was obtained (170.0 mg, 78.0% yield). Purity 93.0% (UPLC-MS_7). Active content of material from1H qNMR is 93.0% w / w.1H NMR (400 MHz, DMSO-cfe) 6 ppm 1.35 (s, 3 H) 1.44 (s, 3 H) 2.38 (s, 3 H) 2.69 (s, 6 H), 2.85 (dd, J=15.3 Hz, J=9.2 Hz, 1 H), 2.92 (dd, J=14.7 Hz, J=5.4 Hz, 1 H), 4.54-4.65 (m, 1 H) 7.20 (d, J = 8.7 Hz, 1 H) 7.33 (s, 1 H) 7.47 (d, J = 8.1 Hz, 2 H) 7.75 (d, J = 2.6 Hz, 1 H) 7.81 (dd, J = 8.7 Hz, J = 2.6 Hz, 1 H) 7.89 (d, J = 8.4 Hz, 1 H) 8.28 (d, J = 1.3 Hz, 1 H) 9.0 (s, 1 H) 9.56 (d, J = 8.0 Hz, 1 H).13C NMR (100 MHz, DMSO-cfe) 6 ppm 21.0, 23.9, 24.1 , 24.4, 37.2, 51.9, 55.1 , 114.8, 115.5 (q, J = 286.3 Hz), 126.8, 127.1 , 129.1 , 129.4, 130.2, 130.4, 134.1 , 134.2, 136.7, 139.7, 146.3, 147.2, 155.9 (q, J= 36.4 Hz), 169.4, 172.0.19F NMR (376 MHz, DMSO-cfe) 5 ppm -74.2. LCMS, m / z calcd. for C25H25F3N5O9S [M+H]+708.1171 , found 708.1107.

[0497] Compound 5Aca was synthesized following general procedure E using (S)-2-(3-(1-((4- methoxyphenyl)sulfonyl)-1 H-imidazol-4-yl)-2-(2,2,2-trifluoroacetamido)propanamido)-2- methylpropanoic acid (4Ac) (400.0 mg, 0.8 mmol). Purification with gradient elution EtOAc / heptane 4:6 to pure EtOAc was performed. The pure fractions were combined and evaporated under reduced pressure. After drying in vacuo overnight, an off-white solid material was obtained (148.0 mg, 28.4% yield). Purity >95.0% (1H NMR). Active content of material from1H qNMR is 90.5% w / w.1H NMR (400 MHz, CDCI3) 6 ppm 1.60 (s, 3 H), 1.63 (s, 3 H), 2.93 (dd, J=15.1 Hz, J=7.5 Hz, 1 H), 3.09 (dd, J=15.3 Hz, J = 3.8 Hz, 1 H), 3.88 (s, 3 H), 3.89 (s, 3 H), 4.62-4.69 (m, 1 H), 7.00 (d, J=9.0 Hz, 1 H), 7.10 (s, 1 H), 7.16 (d, 1 H, J = 8.7), 7.81 (d, J=2.5 Hz, 1 H), 7.86 (m, 3 H), 7.96 (d, J=1.1 Hz, 1 H), 8.12 (s, 1 H), 8.15 (d, J = 6.4 Hz, 1 H).13C NMR (100 MHz, CDCI3) 6 ppm 24.7, 24.9, 30.1 , 52.5, 56.0, 56.5, 57.0, 107.8, Hz), 115.2, 115.3, 115.7 (q, J=288), 116.6, 123.5, 128.6, 130.1 , 136.1 , 139.7, 145.1 , 146.5, 151.7, 157.0 (q, J=37.7 Hz), 165.0, 168.8, 171.3.19F NMR (376 MHz, CDCI3) 5 ppm -75.9. LCMS, m / z calcd. for C26H27F3N5O10S [M+H]+658.1425, found 658.1270.

[0498] Example 5d: Preparation of benzyl (S)-4-(2-(((benzyloxy)carbonyl)amino)-3-((1-(4-chloro-2-

[0499] ( / V, / V-dimethylsulfamoyl)phenoxy)-2-methyl-1-oxopropan-2-yl)amino)-3-oxopropyl)-1 H- imidazole-1 -carboxylate (5Cib)

[0500] Compound 5ACib was synthesized following general procedure E using (S)-2-(3-(1- ((benzyloxy)carbonyl)-1 H-imidazol-4-yl)-2-(((benzyloxy)carbonyl)amino)propanamido)-2- methylpropanoic acid (4Ci) (150.0 mg, 0.3 mmol). Purification with gradient elution EtOAc / heptane 1 :1 to pure EtOAc was performed. The pure fractions were combined and evaporated under reduced pressure. After drying in vacuo overnight, an off-white solid material was obtained (151.0 mg, 71.0% yield). Purity 98.2% (UPLC-MS_6). Active content of material from1H qNMR is 97.2% w / w.1H NMR (400 MHz, DMSO-cfe) 6 ppm 1.44 (s, 3 H), 1.47 (s, 3 H), 2.68 (s, 6 H), 3.33 (s, 2 H), 4.36 (m, 1 H), 4.91 (s, 2 H), 5.38 (s, 2 H), 7.16-7.34 (m, 7 H), 7.35- 7.53 (m, 6 H), 7.70-7.78 (m, 2 H), 8.17 (2, J=1.1 Hz), 8.84 (s, 1 H).13C NMR (100 MHz, DMSO- d6) 6 ppm 24.19, 24.2, 30.3, 37.3, 53.4, 55.0, 65.2, 69.1 , 114.5, 127.0, 127.4, 127.7, 128.2, 128.3, 128.5, 128.6, 129.3, 129.4, 130.1 , 134.1 , 134.6, 136.6, 136.9, 139.4, 147.4, 148.0, 155.6, 171.3, 172.3. LCMS, m / z calcd. for C34H37CIN5O9S [M+H]+726.1995, found 726.1634 Table 2. Overview of synthesized protected dipeptides.

[0501] Example 6: Method for activation of dipeptide and ligation to GLP-1

[0502] The purpose of this example is to study the use of different activators in combination with dipeptide 4Aa or4Ab from example 2a and 2b of the invention in a ligations reaction to produce a protected GLP-1 API analogue. Focus is to find stable activated dipeptides which can ligate in an aqueous environment. The GLP-1 analogue being ligated in this example is monoacylated GLP-1 analogue N{Epsilon-26}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17- carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acet yl]-[Arg34]-GLP-1-(9-37)-peptide (8), which may be prepared, e.g., as described in Example 10 of WO 2018 / 083335. The protected dipeptide (5) is attached to the / V-terminus at position 9 of this GLP-1 analogue in the ligation reaction to give 9 (see Scheme 1). Compound 9 is diprotected semaglutide, i.e. semaglutide having two protecting groups, Pg1and Pg2.

[0503] Scheme 1. Dipeptide activation and ligation to GLP-1 backbone (8) to obtain di-protected semaglutide (9).

[0504] The activation of the Aib motif in the dipeptide fragment gives rise to some complexity. With highly reactive leaving groups (LG) such as e.g., HOAt, HOBt, HOPO and phenols with pKa <6.5 the Aib motif can ring close upon itself to make an activated oxazolone moiety 4,4- dimethyloxazol-5(4H)-one (DMO) as seen in

[0505] Scheme 2. For less reactive phenols with pKa >7.5 the ester form will be the most predominate compound of the activated dipeptide. Activated compounds which are stable and have a well- balanced reactivity for aminolysis compared to hydrolysis is preferred in this example. However, in situ activation can be used for the more reactive leaving groups as the DMO will be formed as the most stable compound. Certain oxazolones / DMOs are stable and can be isolated and purified on silica gel (see example 3). In the end of this example the isolated DMO is compared to the esters to see the difference in the ligation reaction (see Table 3).

[0506] • Leaving group R3OH with pKa < 6.5 predominantly gives 6Ab (DMO activation)

[0507] • E.g. HOAt, HOBt, HOPO, and electron deficient phenols

[0508] • Leaving group R3OH with pKa > 7.5 gives more of the dipeptide-ester

[0509] • See e.g. some of the LG in Table 3 with pKa between 6.5 to 7.5.

[0510] Scheme 2. The ratio between DMO and ester activation as a function of pKa of the corresponding leaving group.

[0511] Procedure.

[0512] Purified activated dipeptides in Scheme 1 :

[0513] The activated dipeptides in this screening example were synthesized from dipeptide 4Aa or 4Ab and the different LG’s shown in Scheme 1. General procedures C, D and E were used to synthesise the activated dipeptides. All activated dipeptides were isolated before a ligation reaction between activated dipeptide and GPL-1 backbone was performed. The ratio between ester and DMO form was followed using UPLC-MS. Samples from the reaction were withdrawn at different times points, diluted (1 / 99) with MeCN / H2O (1 / 1) and analyzed using method UPLC-MS_7. The ratio in the activation process was estimated by area comparison with UV at 214 nm after overnight reaction time. For clarity in comparison, the combined area of ester and DMO was normalized to 100% (see Table 3).

[0514] Ligation reaction with activated dipeptide: Charge a titrator-chamber (75 mL) with the GPL-1 backbone 8 solution (12.8 mM, 1.0 mL, 1.0 eq., mono acylated BB). Ethanol (EtOH) (950.0 pl) was added. pH was measured to 7.1. The pH of the solution was titrated with 0.5 M NaOH to reach pH 8.0. The different activated dipeptides in Scheme 1 (19.2 pmol, 1.5 eq.) was dissolved in NMP (150 pL). The activated dipeptide solution was added over 9-10 min in 5-10 pL portions. pH was adjusted with 0.5 M NaOH in the pH interval 8.0-8.2. The progress of the reaction was followed using UPLC-MS. Samples from the reaction were withdrawn at different time points, diluted (2 / 98) with MeCN / H2O (1 / 1) and analyzed using method UPLC-MS_7 or UPLC-MS_8. The conversion in the ligation process was estimated by area comparison with UV at 214 nm. For clarity in comparison, the combined area of remaining backbone (SM), product, and over ligated product was normalized to 100%. The product peak and over ligated product are combined as “Product Conversion” in Table 3. The over ligated product and product will, during the cleavage step, be cleaved to API in example 8.

[0515] Table 3. Ligation with isolated and activated dipeptides of 4Aa or 4Ab.

[0516] 1) pKa values calculated in Scifinder".

[0517] 2) pKa for this class should not be compared to the pKa of the phenol class.

[0518] Results:

[0519] Table 3 lists the results of the ratio of the ester / DMO in the synthesis of the activated dipeptide. The time for the ligation reaction and the conversion to protected GLP-1 analogue 9Aa or 9Ab. Entry 1 and 2 gives the most efficient phenol ester as they are stable as esters and perform the ligation to full conversion in 2-3 hours. Several other LG’s also work in the ligation reaction but may be less attractive due to fast hydrolysis and lower product formation (entry 4, 7 and 8). Mixture of ester and DMO in the activation give rise to problems if the activated dipeptide ester should be isolated. The compound is not well-defined, and a purification step would be necessary (entries 3, 4, 6, 7 and 8). Activation via e.g. DCC to give a DMO activated dipeptide gives both a well-defined activated dipeptide and good conversion to protected API (entry 9). Typically, if the LG has a pKa < 6.5, the oxazolone derivative will predominantly be formed. If the LG has a pKa in the range of about 6.5 to about 7.5, a mixture of activated ester and oxazolone may be observed; a LG having a pKa above 7.5 typically results predominantly in the activated ester. As can be seen from Table 3, steric and electronic factors have an influence on the ratio between oxazolone and ester in the pKa interval from 7.0-7.5. For instance, it is believed that a phenol having a substituent in the ortho position favours the formation of the activated ester over the oxazolone in this pKa interval.

[0520] Example 7: Screening of coupling reagents

[0521] The purpose of this example is to study and compare the use of different coupling reagents in combination with dipeptide 4Aa or 4Ab of the invention in ligation reactions to produce protected GLP-1 analogue. Process robustness is in focus in this example, examples of alternative ways of producing the activated dipeptide 5Aaa, 5Abc, 6Aa and 6Ab in situ activated dipeptide via protocol A versus purified activated dipeptide via protocol B) (see Scheme 3) and how efficient the activated dipeptides perform in the ligation to the / V-terminal of the GLP-1 analogue are presented. This is shown as a combined conversion which consist of product 1 (P1) and product 2 (bis ligated, P2). The surplus that is needed of the activated dipeptide for the reaction to proceed as desired is also determined. The GLP-1 backbone analogue being ligated at the / V-terminal in this example is N{Epsilon-26}-[2-[2-[2-[[2-[2-[2- [[(4S)-4-carboxy-4-(17- carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acet yl]-[Arg34]-GLP-1-(9-37)-peptide (8), which may be prepared, e.g., as described in example 10 of WO 2018 / 083335.

[0522] Protocol A:

[0523] Scheme 3. In situ versus isolated activated dipeptide in the ligation of GLP-1 backbone 8.

[0524] Procedure.

[0525] In situ formation of DMO activated dipeptide (protocol A):

[0526] Dipeptide 4Aa or 4Ab (1.2-1.5 eq.) was dissolved in either MeCN or NMP (115 pL). To the suspension was added an organic base (for T3P: 1-methylimidazole is used, 5.0 eq.), (for DMTMM BF4: NMM is used, 2.0 eq.), (for PyBOP: TEA is used, 2.1 eq.) (for DIG no base was used). The coupling reagent (1.2-1.7 eq.) was added as a DMF or NMP solution (8.0-29.0 pL) to the dipeptide solution and the reaction was stirred for 35-60 min at 20-40°C before being added to the GLP-1 backbone analogue 8 (15.0-26.0 pmol, 1.0 eq.).

[0527] Purified and activated dipeptide (protocol B):

[0528] The dipeptides activated as esters in this example were 5Aaa and 5Abc. The DMO activated dipeptide was 6Ab. General procedures C, D and E were used to synthesise and purify the activated dipeptides.

[0529] Ligation Reaction:

[0530] A titrator-chamber (75 mL) was charged with an aqueous solution of the mono acylated GLP- 1 backbone 8 (12.8 mM, 1.0 mL, 1.0 eq.). Isopropanol ( / -PrOH), EtOH or NMP (950.0 pl) was added to the solution. pH was measured to 7.1 . The pH of the solution was titrated with 0.5 M NaOH to reach pH 8.0 using an auto titrator (Titrando / dosino®). The activated dipeptide (19.2 pmol, 1.5 eq.) was dissolved in NMP (120-150 mL). The activated dipeptide solution was added over 9-10 min in 5-10 iL portions. pH was adjusted with 0.5 M NaOH in the pH interval 8.0-8.2. The progress of the reaction was followed using UPLC-MS. Samples from the reaction were withdrawn at different times points, diluted (1 / 20) with MeCN / H2O (1 / 1) and analysed using method UPLC-MS_6 or UPLC-MS_7. The conversion in the ligation process was estimated by area comparison with UV at 214 nm. For clarity in comparison, the combined area of remaining starting material (SM), product, and over ligated product was normalized to 100%. The product peak (P1) and over ligated product (P2) are combined as “Conversion” in Table 4. The over ligated product and product will be cleaved to API during the deprotection step in example 8.

[0531] Table 4. Process variations, surplus needed of activated dipeptide and conversion.

[0532] Results:

[0533] The in situ generated 6Aa, entries 1 and 2 in Table 4, do not give full conversion to protected product. Entries 3-5 shows it is slightly beneficial to apply a solid well defined and purified activated dipeptide in the ligation reaction. The purified dipeptide 6Ab in entry 3 convert the GLP-1 backbone 8 to protected product 9 with 1.5 equivalents of the dipeptide 6Ab. In entries 4 and 5 the effect on the conversion can be seen when lowering the equivalents of 6Ab. More backbone starting material is used in entry 5 than 4 due to competing hydrolysis of 6Ab when the concentration of backbone starting material is below 20 mg / mL. The conversion in entry 5 serve as comparison for the last entries 6-9 where four different coupling reagents are used. The purified esters 5Abc and 5Aaa in entries 7-8 showed almost similar conversion as entry 5. However, the NHS activated dipeptide e.g. 5Abc is not well defined, as it is a mixture of NHS ester and the DMO specie, when activated as described in Table 3, entry 3. The NHS ester is better suited for in-situ pre-activation in the ligation reaction. The in situ pre-activated 6Ab from PyBOP and DMTMM BF4 in entries 6 and 9 gave less conversion to product compared to entry 5. The PyBOP reagent has been the standard reagent of choice for the activation of the protected dipeptide (see patent WO 2013 / 098191). Use of DMTMM BF4 salt as activator gave less bis ligation (P2) but more equivalents are needed to reach completion of the reaction. The most beneficial activated dipeptide to use in the ligation reaction, is the purified DMO activated dipeptide e.g., 6Aa-d and 6Db or the purified esters 5 which have been obtained from 4 in combination with e.g., the phenols a-b from these results.

[0534] Example 8: Deprotection step

[0535] The purpose of this example is to study the use of different nucleophilic cleavage reagents in the deprotection reaction to produce the final GLP-1 analogue (10). Process robustness and how efficient the reagents are to cleave the protection groups of the GLP-1 analogue are in focus in this example. The surplus, that is needed of the reagents for the reaction to proceed as desired are also determined. The GLP-1 analogue being deprotected at the His-Aib moiety in this example is protected GLP-1 analogue (9Aa) which may be prepared, e.g., as described in Example 7. The deprotection reaction is shown in Scheme 4.

[0536] Scheme 4. Screening of different deprotecting reagents.

[0537] Procedure.

[0538] Deprotection:

[0539] A titrator-chamber (75 mL) was charged with the protected GLP-1 analogue 9Aa from the ligation reaction (11.5 mM, 1 .0 mL, 1.0 eq.). The solution consists of approximate 30% organic solvent (NMP, / -PrOH or EtOH) and 70% Milli-Q water from the previous ligation reaction. Reaction at pH 10.5:

[0540] The buffer (Na2HPO4 / NaH2PO4, 0.4 M, 0.8 mL) was added to the nucleophilic deprotection reagent (2.3 mmol, 200.0 eq.). If the pH was measured to approximately 10.5, HCI (1.0 M) was added to the buffer solution to lower pH to 10.3. If the deprotection reagent lowered the pH to 5-8, NaOH (4.0 M) was added to the solution to increase the pH to 10.5. The deprotection reagent solution was added to the peptide solution. MilliQ water was added to dilute the peptide solution (5.8 mM, 2.0 mL). The titrator titrated the solution to pH 10.5 and kept the pH at this value over time by addition of NaOH (1.0 M).

[0541] Reaction at pH 11.0: The buffer (Na2HPO4 / NaH2PO4, 0.4 M, 0.8 mL) was added to the deprotection reagent (2.3 mmol, 200.0 eq.). If the deprotection reagent lowered the pH to 5-8, NaOH (4.0 M) was added to the solution to raise the pH to 10.9. The deprotection reagent solution was added to the peptide solution. MilliQ water was added to dilute the peptide (5.8 mM, 2.0 mL). The titrator titrated the solution to pH 11.0 and kept the pH at this value over time by addition of NaOH (1.0 M).

[0542] The progress of the reaction was determined using UPLC-MS. Samples from the reaction were withdrawn at different times points, diluted (2 / 98) with MeCN / H2O (1 / 1) and analyzed using method UPLC-MS_7 or UPLC-MS_8. The conversion efficiency in the deprotection process was estimated by area comparison with UV at 214 nm. For clarity in comparison, the combined area of remaining starting material and product was normalized to 100%. When the reaction was finished based on the UPLC-MS analysis, the pH of the reaction was lowered with AcOH to pH 8 and the reaction mixture was stored at 5°C. The results are shown in Table 5.

[0543] Table 5. Use of different deprotection reagents to obtain deprotected product.

[0544] Results:

[0545] Entries 1 and 2 in Table 5 shows that the conversion to product is faster at pH 11 .0 compared to 10.5. However, pH 10.5 may be better for the peptide stability over time. Leaving out the nucleophilic reagent results in lower cleavage rate to product (see entry 3). Several nucleophilic reagents have been tested as cleavage reagent in the deprotection reaction (entries 4-12). The best reagents regarding fast conversion are found in entries 5- 7 and ID- 12.

[0546] Example 9: Combined ligation and deprotection to obtain semaglutide.

[0547] Example 9a - small scale

[0548] The purpose of this example is to show a combined ligation and deprotection on small-scale to produce a GLP-1 analogue followed by a UF / DF purification step. The activated dipeptide (6Aa) was used in this example and coupled to mono acylated GLP-1 analogue (8).

[0549] Deprotection was performed with acetohydroxamic acid (AHA) under alkaline conditions to produce semaglutide 10 (see Scheme 5).

[0550] Scheme 5. Combined ligation and deprotection of an GLP-1 analogue on small scale.

[0551] Procedure

[0552] Ligation:

[0553] A titrator-chamber (75 mL) was charged with an aqueous solution (20 mL) of the mono acylated GLP-1 backbone 8 (1100.0 mg, 55.0 mg / ml, 14.1 mM, 1.0 eq., MW = 3891.3 g / mol). The solution was diluted with EtOH (10 mL). pH was measured to 7.6. The pH of the solution was titrated with 1.0 M NaOH to reach pH 8.0 using an auto titrator (Titrando / dosino®). The activated dipeptide 6Aa (207.3 mg, 452.2 mol, 1.6 eq.) was dissolved in NMP (1000 iL, 200 mg / mL). The activated dipeptide solution was added over 20 min in 50 piL portions. The vial with the dipeptide solution was washed with NMP (50 piL) and added as well. The pH was adjusted with 1.0 M NaOH in the pH interval 8.0-8.2. The progress of the reaction was followed using UPLC-MS or LIPLC. Samples from the reaction were withdrawn at different time points and diluted (2:98) with MeCN / H2O (1 / 1) and analyzed using method UPLC-MS_7 or UPLC- MS_10. The conversion was estimated by area comparison with UV at 214 nm. For clarity in comparison, the combined area of remaining backbone, product, and bis ligated product was normalized to 100%. The product peak (P1) and bis ligated product (P2) were combined as “Product Conversion” in Table 6. After 160 min. the ligation was finished. The result is shown in Table 6, example 9a.

[0554] Deprotection:

[0555] To the ligation solution (1230.0 mg, 38.0 mg / mL, 8.8 mM, 1.0 eq.) was added acetohydroxamic acid (4.2 g, 56.5 mmol, 200.0 eq.) and K3PO4 3xH2O (1.5 g, 5.5 mmol). After addition of EtOH (6 mL) and adjustment of pH with 4.0 M KOH to 11 .3 (approx. 11.0 mL) the reaction was stirred for 3.5 hours. The pH of the reaction was adjusted to 8.2 with 4 M HCI (10.5 mL). A small sample was withdrawn and diluted (2:98) with MeCN / H2O (1 / 1) and analyzed using UPLC- MS_7 or UPLC-MS_10. The conversion in the deprotection step was estimated by area comparison with UV at 214 nm. For clarity in comparison, the combined area of remaining backbone, product, and by-product / s were normalized to 100%. The result is shown in Table 7, example 9a.

[0556] UF / DF:

[0557] The deprotected peptide solution (1162.8 mg, 17.8 mg / mL, 4.3 mM) was reduced to 50 mL and the solvent exchanged with 10 % EtOH (5x 50 mL) using a peristaltic pump equipped with a membrane filter (Hydrosart 5000 MW from Sartorius). The volume was reduced further to 20 mL and the system washed with 10% EtOH (2 x 10 mL). The solution was stored in a Nunc tube at -20°C. Yield: 924.0 mg (79.0%, CAD determination). Purity 86.0% (UPLC-MS_10). UPLC-MS_3 calc. 4111.12 m / z; found [M+3H]3+: 1371.35; [M+4H]4+: 1028.76.

[0558] Example 9b - large scale

[0559] The purpose of this example is to show a combined ligation and deprotection on large scale to produce a GLP-1 analogue 10. The dipeptide 4Aa was pre-activated using DMTMM BF4in this example and coupled to mono acylated GLP-1 backbone 8. Deprotection was performed with acetohydroxamic acid under alkaline conditions to produce 10 as shown in Scheme 6.

[0560] Scheme 6. Combined ligation and deprotection of an GLP-1 analogue on large scale.

[0561] Procedure.

[0562] Ligation:

[0563] A 1 L flask was charged with an aqueous solution (265.0 mL) of the mono acylated GLP-1 backbone 8 (24.2 g, 6.2 mmol, 1.0 eq., MW = 3891.3 g / mol). A 50 mL beaker was charged with dipeptide 4Aa (7.7 g, 14.9 mmol, 2.4 eq., assay corrected, assay is 92.3%) and 20 mL NMP was added to give a 0.7M solution of dipeptide. A second beaker was charged with DMTMM BF4(4.5 g, 13.7 mmol, 2.2 eq.) and 20 mL NMP was added. When the dipeptide was dissolved, the dipeptide solution was added into the DMTMM BF4suspension, followed by Et3N (4.16 mL, 29.8 mmol, 4.8 eq.). The mixture was stirred for 25 minutes, during which it became a clear solution. The activated dipeptide solution was added over 7 minutes using a syringe pump to the solution of the mono acylated GLP-1 backbone 8. The solution was stirred at room temperature for 30 minutes. pH was adjusted to 8.0 with Et3N during the reaction. The progress of the reaction was followed using UPLC_12. Samples from the reaction were withdrawn at different time points and diluted (1 :99) with AcOH / MeCN / H2O (2 / 1 / 1) and analysed using method UPLC_12. The conversion in the ligation process was estimated by area comparison with UV at 210 nm. For clarity in comparison, the combined area of remaining backbone, product, and over ligated product was normalized to 100%. The product peak (P1) and bis ligated product (P2) are combined as “Product Conversion” in Table 6. After 30 min. the ligation was finished. The result is shown in Table 6, example 9b.

[0564] Deprotection:

[0565] The ligation solution was diluted with water (190 mL) to decrease concentration to 50 g / L for the deprotection. Acetohydroxamic acid (11.7 g, 155.0 mmol, 25 eq.) and Na3PO410xH2O (19.0 g, 50 mmol, resulting in a 0.1 M solution) was added to the solution followed by adjustment of the pH with 4.0 M NaOH to 10.5 (27 mL). The reaction was stirred at room temperature for 7 hours followed by adjustment of pH to 8.5 with glacial AcOH (6.5 mL). A small sample was withdrawn and diluted (2:98) with AcOH / MeCN / H2O (2 / 1 / 1) and analyzed using method UPLC_12. The conversion in the deprotection step was estimated by area comparison with UV at 210 nm. For clarity in comparison, the combined area of remaining backbone, product, and by-product / s were normalized to 100%. The result is shown in Table 7, example 9b.

[0566] UF / DF:

[0567] The deprotected peptide solution (25.6 g, 47.4 mg / mL, 11.5 mM) was reduced to 500 mL and the solvent exchanged with 25% EtOH (3 x 500 mL) using a peristaltic pump equipped with a membrane filter (Hydrosart 5000 MW from Sartorius). The system was washed with 10% EtOH (2 x 500 mL). The solution was stored in a glass vial at -20°C. Yield: 21.3 g (68.0%, correlated with an internal standard). Purity 82.0% (UPLC_12). UPLC-MS_3 calc, mass (mono isotope): 4111.12 m / z; Found [M+3H]3+: 1371.35; [M+4H]4+: 1028.77.

[0568] Example 10: Combined ligation and deprotection to obtain compound 13

[0569] The purpose of this example is to show a combined ligation and deprotection on a large scale to produce compound 13 (compound 0111 of WO 2022 / 129526 A1) in this example. The dipeptide 4Aa was pre-activated using DMTMM BF4and coupled to mono acylated peptide 11 (precursor for compound 0111 of WO 2022 / 129526 A1). Deprotection was performed with acetohydroxamic acid under alkaline conditions to produce 13 (see Scheme 7).

[0570] Scheme 7. Combined ligation and deprotection to obtain compound 13 by reacting peptide 11 with a dipeptide.

[0571] Procedure.

[0572] Ligation:

[0573] A 1 L flask was charged with an aqueous solution (677.1 mL) of the mono acylated backbone 11 (47.4 g, 6.22 mmol, 1.0 eq., MW= 7624.4 g / mol). A beaker was charged with dipeptide 4Aa (10.4 g, 20.2 mmol, 3.25 eq., assay corrected, assay is 92.3%) and NMP (99 mL) was added (105 mg / mL dipeptide). To this solution was added DMTMM BF4(6.2 g, 18.7 mmol, 3.0 eq.), followed by NMM (4.4 mL, 40.4 mmol, 6.5 eq.). The mixture was stirred for 15 minutes, during which time it became a clear solution. The activated dipeptide solution was added over 15 minutes, using a syringe pump, to the mono acylated backbone 11 solution. The solution was stirred at room temperature for 30 minutes. If pH decreased below 8.3, 1 M NaOH was added to keep pH at 8.3 or above (39 mL were required). The progress of the reaction was analyzed using LIPLC. Samples from the reaction were withdrawn at different time points, diluted (1 :99) with MeCN / H2O (1 / 1) and analyzed using method UPLC_13. The conversion in the ligation reaction was estimated by area comparison with UV at 210 nm. For clarity in comparison, the combined area of remaining backbone, product, and bis ligated product were normalized to 100%. The product peak (P1) and bis ligated product (P2) were combined as “Product Conversion” in Table 6. After 30 min. the ligation was finished. The result is shown in Table 6, example 10.

[0574] Deprotection:

[0575] A 0.77 M K2HPO4 buffer solution (120 mL, pH 10.5) was added to the ligation solution above. The final concentration of K2HPO4 was 0.08 M. pH was adjusted to 10.5 with 1 M KOH. Acetohydroxamic acid (11.7 g, 155.0 mmol, 25 eq.) was added and pH adjusted to 10.5 with 1 M KOH (21 mL). The solution was stirred at room temperature for 16 h. Then pH adjustment with 2.0 M AcOH (13.3 mL) to pH 8.5. See Table 7, example 10. Yield: 34.1 g (70.0%, correlated with an internal standard). Purity 80.0% (UPLC_13). UPLC-MS_3 calc, mass (mono isotope): 7842.00 m / z; Found [M+7H]7+: 1121.25; [M+8H]8+: 981.23.

[0576] Example 11

[0577] The purpose of this example is to show a combined ligation and deprotection on small-scale to produce a GLP-1 analogue 10. The activated dipeptide (5Aca) was used in this example and coupled to mono acylated GLP-1 backbone 8. Deprotection was performed with acetohydroxamic acid under alkaline conditions to produce 10 (see Scheme 8).

[0578] Scheme 8. Enablement of different protection groups in the GLP-1 analogue synthesis process.

[0579] Procedure.

[0580] Ligation:

[0581] A titrator-chamber (75 mL) was charged with an aqueous solution (1 mL) of the mono acylated GLP-1 backbone 8 (100.0 mg, 100.0 mg / ml, 25.7 mM, 1.0 eq., MW = 3891.3 g / mol). The solution was diluted with EtOH (1 mL). pH was measured to be 8.3. The pH of the solution was titrated with 1 M HCI to reach pH 7.8. The pH of the solution was titrated with 1.0 M NaOH to reach pH 8.0 using an auto titrator (Titrando / dosino®). The activated dipeptide 5Aca (37.3 mg, 51.0 mol, 2.0 eq. 90.5% active) was dissolved in NMP (330 .L, 100 mg / mL). The activated dipeptide solution was added over 20 min with a Hamilton syringe pump to the GLP-1 backbone solution. The vial with the dipeptide solution was washed with NMP (20 iL) and this was added as well. During the addition of dipeptide, the pH of the reaction mixture was adjusted with 1.0 M NaOH in the pH interval 8.0-8.2. The progress of the reaction was followed using UPLC-MS. Samples from the reaction were withdrawn at different time points, diluted (2:98) with MeCN / H2O (1 / 1) and analyzed using method UPLC-MS_7. The conversion in the ligation process was estimated by area comparison with UV at 214 nm. For clarity in comparison, the combined area of remaining backbone, product, and bis ligated product was normalized to 100%. The product peak (P1) and bis ligated product (P2) are combined as “Product Conversion” in Table 6. After 180 min the ligation was finished. The result is shown in Table 6, example 11 . The solution was stored overnight at -20°C.

[0582] Deprotection:

[0583] To half of the ligation solution (55.5 mg, 47.5 mg / mL, 1.15 mL, 11.3 mM, 1.0 eq.) was added acetohydroxamic acid (48.2 mg, 643.0 mol, 50.0 eq.) and 1.0 M aqueous K3PO4 solution (127 |j.L) to give a phosphate concentration of 0.1 M. After adjustment of pH with 4.0 M KOH to 10.5 (27 jiL) the reaction was stirred for 22 hours under pH control. The pH of the reaction was adjusted to 8.2 with 4 M HCI. A small sample was withdrawn, diluted (2:98) with MeCN / H2O (1 / 1) and analyzed using method UPLC-MS_7. The conversion in the deprotection stepwas estimated by area comparison with UV at 214 nm. For clarity in comparison, the combined area of remaining backbone, product, and by-product / s were normalized to 100%. The result is shown in Table 7, example 11. The remaining solution was stored in a Nunc tube at -20°C. Yield: 29.8 mg (56.0%, CAD determination). Purity 95.7% (UPLC-MS_7). UPLC-MS_3 calc, mass (mono isotope): 4111.12 m / z; Found [M+3H]3+: 1371.38; [M+4H]4+: 1028.79.

[0584] Example 12

[0585] The purpose of this example is to show a combined ligation and deprotection on a small-scale to produce GLP-1 analogue 10. The activated dipeptide (6Ac) was used in this example and coupled to mono acylated GLP-1 backbone 8. Deprotection was performed with acetohydroxamic acid under alkaline conditions to produce 10 (see Scheme 9).

[0586] Scheme 9. Enablement of different protection groups in the GLP-1 analogue process.

[0587] Procedure.

[0588] Ligation:

[0589] Ligation was performed following the protocol in example 11 using dipeptide 6Ac (26.7 mg, 51.4 mol, 2.0 eq. 94.0% active). 6Ac was dissolved in NMP (260 iL, 100 mg / mL) and added over 20 min to the mono acylated GLP-1 backbone 8 solution (100.0 mg, 50.0 mg / ml, 12.9 mM, 1.0 eq., MW = 3891.3 g / mol). The progress of the reaction was followed using method UPLC-MS_7. After 180 min the ligation was finished. The result is shown in Table 6, example 12.

[0590] Deprotection:

[0591] To half of the ligation solution (56.3 mg, 56.3 mg / mL, 1.0 mL, 12.9 mM, 1.0 eq.) was added acetohydroxamic acid (48.5 mg, 642.8 mol, 50.0 eq.) and 1.0 M aqueous K3PO4 solution (110 |j.L) to give a phosphate concentration of 0.1 M. After adjustment of pH with 4.0 M KOH to 10.5 (75 jiL) the reaction was stirred for 22 hours under pH control. The pH of the reaction was adjusted to 7.5 with 4 M HCI. Samples were withdrawn during the reaction and analyzed as in example 11. The result is shown in Table 7, example 12. The remaining solution was stored in a Nunc tube at -20°C. Yield: 39.4 mg (74.0%, CAD determination of all peaks x 85.9%). Purity 85.9% (UPLC-MS_7). UPLC-MS_3 calc, mass (mono isotope): 4111.12 m / z; Found [M+3H]3+: 1371.37; [M+4H]4+: 1028.79.

[0592] Example 13

[0593] The purpose of this example is to show a combined ligation and deprotection on small-scale to produce a GLP-1 analogue 10. The activated dipeptide (6Ad) was used in this example and coupled to mono acylated GLP-1 backbone 8. Deprotection was performed with acetohydroxamic acid under alkaline conditions to produce 10 (see Scheme 10).

[0594] Scheme 10. Enablement of different protection groups in the GLP-1 analogue synthesis process.

[0595] Procedure

[0596] Ligation:

[0597] Ligation was performed following the protocol in example 11 using dipeptide 6Ad (25.9 mg, 51.4 mol, 2.0 eq. 95.7% active). The 6Ad was dissolved in NMP (260 iL, 100 mg / mL) and added over 20 min to the mono acylated GLP-1 backbone 8 solution (100.0 mg, 50.0 mg / ml, 12.9 mM, 1.0 eq., MW = 3891.3 g / mol). The progress of the reaction was followed using method UPLC-MS_7. After 180 min the ligation was finished. The result is shown in Table 6, example 13.

[0598] Deprotection:

[0599] To half of the ligation solution (60.0 mg, 54.5 mg / mL, 1.1 mL, 12.7 mM, 1.0 eq.) was added acetohydroxamic acid (52.5 mg, 700.0 mol, 50.0 eq.) and 1.0 M aqueous K3PO4 solution (125 |j.L) to give a phosphate concentration of 0.1 M. After adjustment of pH with 4.0 M KOH to 10.5 (80 jiL) the reaction was stirred for 22 hours under pH control. The pH of the reaction was adjusted to 8.2 with 4 M HCI. Samples were withdrawn during the reaction and analyzed as in example 11. The result is shown in T able 7, example 13. The remaining solution was stored in a Nunc tube at -20°C. Yield: 34.0 mg (60.0%, CAD determination). Purity 94.0% (UPLC- MS_7). UPLC-MS_3 calc, mass (mono isotope): 4111.12 m / z; Found [M+3H]3+: 1371.39; [M+4H]4+: 1028.79.

[0600] Example 14

[0601] The purpose of this example is to show a combined ligation and deprotection on a small-scale to produce a GLP-1 analogue 10. The activated dipeptide (6Ap) was used in this example and coupled to mono acylated GLP-1 analogue 8. Deprotection was performed with acetohydroxamic acid (AHA) under alkaline conditions to produce 10 (see Scheme 11).

[0602] Scheme 11 . Enablement of different protection groups in the GLP-1 analogue synthesis process.

[0603] Procedure

[0604] Ligation:

[0605] Ligation was performed following the protocol in example 11 using dipeptide 6Ap (27.7 mg, 51.4 .mol, 2.0 eq. 90.3% active). 6Ad was dissolved in NMP (280 iL, 100 mg / mL) and was added over 20 min to the mono acylated GLP-1 backbone 8 solution (100.0 mg, 50.0 mg / ml, 12.9 mM, 1.0 eq., MW = 3891.3 g / mol). The progress of the reaction was followed using method UPLC-MS_7. After 180 min the ligation was finished. The result is shown in Table 6, example 14.

[0606] Deprotection:

[0607] To half of the ligation solution (56.2 mg, 56.2 mg / mL, 1.0 mL, 12.8 mM, 1.0 eq.) was added acetohydroxamic acid (192.7 mg, 2562.0 mol, 200.0 eq.) and 1.0 M aqueous K3PO4 solution (111 |iL) to give a phosphate concentration of 45.3 mM. After adjustment of pH with 4.0 M KOH to 10.5 (1278.0 iL) the reaction was stirred for 22 hours under pH control. The pH of the reaction was adjusted to 8.2 with 4 M HCI. Samples were withdrawn during the reaction and analyzed as in example 11 . The result is shown in Table 7, example 14. The remaining solution was stored in a Nunc tube at -20°C. Yield: 36.7 mg (70%, CAD determination). Purity 94.0% (UPLC-MS_7). UPLC-MS_3 calc, mass (mono isotope): 4111.12 m / z; Found [M+3H]3+: 1371.39; [M+4H]4+: 1028.79.

[0608] Example 15

[0609] The purpose of this example is to show a combined ligation and deprotection on small-scale to produce GLP-1 analogue 10 followed by UF / DF purification step. Activation of dipeptide 4Ch was performed in situ with DCC and coupled to mono acylated GLP-1 backbone 8. The deprotection of Cbz and Bn was performed with palladium on carbon with 1 atmosphere hydrogen gas (see Scheme 12). (

[0610] Scheme 12. Enablement of different protection groups in the GLP-1 analogue synthesis process.

[0611] Procedure.

[0612] In situ activation of dipeptide:

[0613] Dipeptide 4Ch (216.0 mol, 100.0 mg) was dissolved in THF (1200 mL). To the solution was added DCC (204.0 mol, 42.4 mg) and the reaction was stirred for 60 min at 20°C. The precipitate was filtered with PTFE filter (0.45 mm). The THF was evaporated under reduced pressure. The crude was redissolved in DCM (2 mL) and evaporated under reduced pressure to give a white solid. Active content of material from1H qNMR is 72.5% w / w.

[0614] Ligation:

[0615] A titrator-chamber (75 mL) was charged with an aqueous solution (1 mL) of the mono acylated GLP-1 backbone 8 (100.0 mg, 100.0 mg / ml, 25.7 mM, 1.0 eq., MW = 3891.3 g / mol). The solution was diluted with EtOH (1 mL). pH was measured to 8.3. The pH of the solution was titrated with 1 M HCI to reach pH 8.0. The pre-activated dipeptide 4Ch (31.6 mg, 51.0 mol, 2.0 eq. 72.5% active) was dissolved in NMP (230 .L, 100 mg / mL). The oxazolone solution was added over 20 min to the reaction mixture. The pH was adjusted with 1 M NaOH in the pH interval 8.0-8.1. The progress of the reaction was followed using UPLC-MS. Samples from the reaction were withdrawn at different time points, diluted (2:98) with MeCN / H2O (1 / 1) and analyzed using method UPLC-MS_7. The conversion in the ligation process was estimated by area comparison with UV at 214 nm. For clarity in comparison, the combined area of remaining backbone, product, and bis ligated product was normalized to 100%. The product peak (P1) and bis ligated product (P2) are combined as “Product Conversion” in Table 6. After 60 min. the ligation was finished. The result is shown in Table 6, example 15.

[0616] Deprotection:

[0617] The ligation mixture was transferred to a 10 mL glass vial. 2 mL 50% EtOH / H2O was added. Concentration of the protected backbone 9 was 25 mg / mL. The vial was closed with a septum. The solvent was degassed with nitrogen for 15 minutes. Pd / C type 5R39 (20% w / w) was added, and the flask was degassed with nitrogen. 1 Atmosphere of hydrogen gas was applied, and the nitrogen was flushed out of the flask for 45 sec. The reaction was stirred at RT for 18 hours.

[0618] A sample was taken out from the mixture after 2 hours with a syringe and filtered through a small PTFE filter before analysis by method UPLC-MS_7. The Cbz protection group was cleaved but not the benzyl protection group. The reaction was stirred for another 16 hours then filtered through a small PTFE filter before a sample was analyzed by method UPLC-MS_7. 29.6 % conversion to GLP-1 10 was obtained from protected GLP-1 9. See Table 7, example 15. The remaining solution was stored in a Nunc tube at -20°C. Yield: 29.2 mg (27.6%, CAD determination of all peaks x 27.6%). Purity 27.6% (UPLC-MS_9). UPLC-MS_3 calc, mass (mono isotope): 4111.12 m / z; Found [M+3H]3+: 1371.33; [M+4H]4+: 1028.76.

[0619] Example 16

[0620] The purpose of this example is to show a combined ligation and deprotection on small-scale to produce a GLP-1 analogue 10. Pre-activation of the dipeptide 4Ci with DCC was used in this example and coupled to mono acylated GLP-1 backbone 8. The deprotection of Cbz was performed with palladium on carbon with 1 atmosphere hydrogen gas (see Scheme 13).

[0621] Scheme 13. Enablement of different protection groups in the GLP-1 analogue synthesis process.

[0622] Procedure.

[0623] The pre-activation and ligation were performed following the protocols in example 15 using dipeptide 4Ci (35.1 mg, 51.0 mol, 2.0 eq. 71.8% active). The in-situ formed oxazolone of 4Ci was dissolved in NMP (230 .L, 100 mg / mL). The activated dipeptide solution was added over 20 min to the solution of the mono acylated GLP-1 backbone 8 (100.0 mg, 50.0 mg / ml in EtOH / hhO 1 :1 , 12.9 mM, 1.0 eq., MW = 3891.3 g / mol). The progress of the reaction was followed using method UPLC-MS_7. After 60 min no more backbone starting material 8 was converted to product 9. The result is shown in Table 6, example 16.

[0624] Deprotection: The ligation mixture was transferred to a 10 mL glass vial. 50% EtOH / hhO (2 mL) was added. Peptide 9 concentration was 25 mg / mL. The vial was closed with a septum. The solvent was degassed with nitrogen for 15 minutes. Pd / C type 5R39 (20% w / w) was added, and the flask was degassed with nitrogen. 1 atmosphere of hydrogen was applied, and the nitrogen was flushed out of the flask for 45 sec. The reaction was stirred at rt for 20 hours.

[0625] A sample was taken out from the mixture after 2 hours with a syringe and filtered through a small PTFE filter before analysis using method UPLC-MS_7. The reaction was not completed. The reaction was stirred for 16 hours before a second sample was analyzed. The reaction was completed, and the solution was filtered through a small PTFE. The result is shown in Table 7, example 16. The remaining solution was stored in a Nunc tube at -20°C. Yield: 85.9 mg (81.2%, CAD determination of all peaks x 78.8%). Purity 78.8% (UPLC-MS_9). UPLC-MS_3 calc, mass (mono isotope): 4111.12 m / z; Found [M+3H]3+: 1371.35; [M+4H]4+: 1028.76.

[0626] Example 17

[0627] The purpose of this example is to show a combined ligation and deprotection on a small-scale to produce GLP-1 analogue 10. Activated dipeptide 5Cib was used in this example and coupled to mono acylated GLP-1 backbone 8. The deprotection of Cbz was performed with palladium on carbon with 1 atmosphere hydrogen (see Scheme 14).

[0628] Scheme 14. Enablement of different protection groups in the GLP-1 analogue process.

[0629] Procedure.

[0630] Ligation was performed following the protocol in example 14 using dipeptide 5Cib (35.1 mg, 51.0 mol, 2.0 eq. 97.0% active). The 5Cib was dissolved in NMP (230 .L, 100 mg / mL) and was added over 20 min to the mono acylated GLP-1 backbone 8 solution (100.0 mg, 50.0 mg / ml in EtOH / hhO 1 :1 , 12.9 mM, 1.0 eq., MW = 3891.3 g / mol). The progress of the reaction was followed using method UPLC-MS_9. The result is shown in Table 6, example 17.

[0631] Deprotection:

[0632] The ligation mixture was transferred to a 10 mL glass vial. 50% EtOH / H2O (2 mL) was added. Peptide 9 concentration was 25 mg / mL. The vial was closed with a septum. The solvent was degassed with nitrogen for 15 minutes. Pd / C type 5R39 (20% w / w) was added, and the flask was degassed with nitrogen. 1 atmosphere of hydrogen was applied, and the nitrogen was flushed out of the flask for 45 sec. The reaction was stirred at rt for 20 hours.

[0633] A sample was taken out from the mixture after 2 hours with a syringe and filtered through a small PTFE filter before analysis by method UPLC-MS_9. The reaction was not completed. The reaction was stirred for 20 hours before a second sample was analyzed. The reaction was completed, and the solution filtered through a small PTFE filter. The result is shown in Table 7, example 17. The remaining solution was stored in a Nunc tube at -20°C. Yield: 79.0 mg (75.2%, CAD determination of all peaks x 73.0%). Purity 73.0% (UPLC-MS_9). UPLC-MS_3 calc, mass (mono isotope): 4111.12 m / z; Found [M+3H]3+: 1371.33; [M+4H]4+: 1028.76.

[0634] Example 18

[0635] The purpose of this example is to show a combined ligation and deprotection on small-scale to produce a GLP-1 analogue 10. Activated dipeptide 6Bg was used in this example and coupled to mono acylated GLP-1 backbone 8. The deprotection of Boc was performed with

[0636] Scheme 15. Enablement of different protection groups in the GLP-1 analogue synthesis process.

[0637] Procedure.

[0638] Ligation was performed following the protocol in example 15 using dipeptide 6bg (25.9 mg, 51.0 mol, 2.0 eq. 83.7% active). 6Bg was dissolved in NMP (220 .L, 100 mg / mL) and added over 20 min to the mono acylated GLP-1 backbone 8 solution (100.0 mg, 50.0 mg / ml in EtOH / hhO 1 :1 , 12.9 mM, 1.0 eq., MW = 3891.3 g / mol). The progress of the reaction was followed using method UPLC-MS_7. The result is shown in Table 6, example 18.

[0639] The solvent mixture was exchanged (5 x 2 mL) with MiliQ water using spin filtration (Amicon Ultra-15 centrifugal filters with 3000 MW cutoff from Merck Millipore).

[0640] Deprotection:

[0641] The spin filtered ligation mixture (2.25 mL, 36.8 mg / mL, MW = 4313.8 mol / g) was transferred to a 10 mL glass vial. Concentrated phosphoric acid (85%) (1.1 mL) was added. The progress of the reaction was followed using UPLC-MS_9. The reaction was completed in 5 hours. The solution was neutralized with 4M NaOH to pH 7. The solution was stored in a Nunc tube at - 20°C. The result is shown in Table 7, example 18. Yield: 48.0 mg (65.4%, CAD determination of all peaks x 84.5%). Purity 84.5% (UPLC-MS_9). UPLC-MS_3 calc, mass (mono isotope): 4111.12 m / z; Found [M+3H]3+: 1371.32; [M+4H]4+: 1028.76.

[0642] Example 19

[0643] The purpose of this example is to show a combined ligation and deprotection on small-scale to produce GLP-1 analogue 10. Activated dipeptide 6Db was used in this example and coupled to mono acylated GLP-1 backbone 8. The deprotection of Dde and Ts were performed with hydroxyl amine (see Scheme 16).

[0644] (

[0645] Scheme 16. Enablement of different protection groups in the GLP-1 analogue synthesis process.

[0646] Procedure.

[0647] Ligation was performed following the protocol in example 11 using dipeptide 6Db (28.9 mg, 51.0 mol, 2.0 eq. 96.2% active). 6Db was dissolved in NMP (290 .L, 100 mg / mL) and added over 20 min to the mono acylated GLP-1 backbone 8 solution (100.0 mg, 50.0 mg / ml in EtOH / H2O 1 :1 , 12.9 mM, 1.0 eq., MW = 3891.3 g / mol). The progress of the reaction was followed using method UPLC-MS_7. The result is shown in Table 6, example 19.

[0648] Deprotection:

[0649] Approximately half of the ligation mixture was used (1.1 mL, 65.0 mg, 14.7 mol). Hydroxyl amine (50% w / v in water) (37 .l, 214 mg, 643.0 mol) was added followed by pH adjustment with 1 M potassium phosphate buffer (110 .L) to give a total concentration of 0.1 M. Manual pH control at 10.5 was achieved over time by addition of 1 M potassium hydroxide in 2-4 .L portions. The progress of the reaction was followed using UPLC-MS_7. The reaction was completed in 4.5 hours. The solution was neutralized with 1 M HCI to pH 7.5 and was stored in a Nunc tube at -20°C. The result is shown in Table 7, example 19. Yield: 54.6 mg (91.0%, CAD determination of all peak x 73.0%). Purity 73.0% (UPLC-MS_9). UPLC-MS_3 calc, mass (mono isotope): 4111.12 m / z; Found [M+3H]3+: 1371.39; [M+4H]4+: 1028.79.

[0650] Example 20

[0651] The purpose of this example is to show a combined ligation and deprotection on small-scale to produce compound 13 (compound 0111 of WO 2022 / 129526 A1). Activated dipeptide 6Db was used in this example and coupled to mono acylated backbone 11. The deprotection of

[0652] Dde and Ts were performed with hydroxyl amine (see Scheme 17). (

[0653] Scheme 17. Enablement of different protecting groups for synthesis compound 0111.

[0654] Procedure.

[0655] The ligation in this example deviated from the ligation protocols in example 11 by an addition of 1.0 M aqueous phosphate solution (100 .L) to the backbone 11 solution to give a phosphate concentration of 100.0 mM with pH 8.05 making it easier to control pH during addition of the dipeptide. The dipeptide 6Db (8.4 mg, 13.1 mol, 2.0 eq. 84.0% active) was dissolved in NMP (84.0 jiL, 100 mg / mL) and added over 20 min to the mono acylated backbone 11 solution (50.0 mg, 6.5 mol, 45.5 mg / ml, in 1.1 mL EtOH / H2O / phosphate buffer 1 :1 :0.1 , 1.0 eq., MW = 7624.4 g / mol). The progress of the reaction was followed using method UPLC-MS_9. The result is shown in Table 6, example 20.

[0656] Deprotection:

[0657] Half of the ligation mixture was used (505.0 .L, 26.8 mg, 3.3 .mol). Hydroxyl amine (50% w / v in water) (10.9 .l, 5.4 mg, 165.0 mol, 50.0 eq.) was added followed by pH adjustment with 1 M potassium hydroxide (10.0 .L) to give a total concentration of 100 mM potassium phosphate with a pH of 10.5 in the reaction mixture. Manual pH control at 10.5 was achieved over time by addition of 1 M potassium hydroxide in portions of 2-4 .L (total 16.0 .L used). The progress of the reaction was followed using UPLC-MS_9. The reaction was followed over 24 hours where 7 hours was at RT and the remaining time at -20°C. The result is shown in Table 7, example 20. The solution was neutralized with 1 M HCI to pH 7.5 and the remaining solution was stored in a Nunc tube at -20°C. Yield: 12.7 mg (49.0%, CAD determination of all peak x 65.9%). Purity 65.9% (UPLC-MS_9). UPLC-MS_3 calc, mass (mono isotope): 7842.00 m / z; Found [M+6H]6+: 1308.01 ; [M+7H]7+: 1121.29.

[0658] Example 21 :

[0659] The purpose of this example is to show a combined ligation and deprotection on small-scale to produce a GLP-1 analogue 10. Activated dipeptide 6Edb was used in this example and coupled to mono acylated GLP-1 backbone 8. The deprotection of Hexmoc and Ts were performed with AHA (see Scheme 18). (

[0660] Scheme 18. Enablement of different protection groups in the GLP-1 analogue process.

[0661] Procedure.

[0662] Ligation was performed following the protocol in example 11 using dipeptide 6Edb (36.7 mg, 52.0 mol, 2.0 eq. 78.0% active). 6Edb was dissolved in NMP (360.0 .L, 100 mg / mL) and added over 20 min to the mono acylated GLP-1 backbone 8 solution (100.0 mg, 50.0 mg / ml in EtOH / H2O 1 :1 , 12.9 mM, 1.0 eq., MW = 3891.3 g / mol). The progress of the reaction was followed using method UPLC-MS_7. The reaction was finished in less than 3 hours. The result is shown in Table 6, example 21.

[0663] Deprotection:

[0664] Half of the ligation mixture was used in the deprotection step (1150.0 .L, 57.7 mg, 13.0 mol). Acetohydroxamic acid (48.7 mg, 649.8 mol, 50.0 eq.) and 1.0 M aqueous K3PO4 solution (130.0 jiL) were added to give a phosphate concentration of 100.0 mM. After adjustment of pH with 4.0 M KOH to 10.5 (70.0 .L) the reaction was stirred for 2.5 hours under pH control. The progress of the reaction was followed using UPLC-MS_7. The solution was neutralized with 6 M HCI (50.0 jiL) to pH 7.5. The remaining solution was stored in a Nunc tube at -20°C. The result is shown in Table 7, example 21. Yield: 40.0 mg (74.8%, CAD determination of all peak x 79.8%). Purity 79.8% (UPLC-MS_7). UPLC-MS_3 calc, mass (mono isotope): 4111.12 m / z; Found [M+3H]3+: 1371.39; [M+4H]4+: 1028.79. Example 22:

[0665] The purpose of this example is to show a combined ligation and deprotection on a small-scale to produce a GLP-1 analogue 10. Activated dipeptide 6Aa was used in this example and coupled to mono acylated GLP-1 backbone 8. The deprotection of TFAc and Bs were performed with two different cleavage reagents / V-hydroxy- / V-methylacetamide (HMA) or N,2- dihydroxybenzamide (DHB) (see scheme 19).

[0666] Scheme 19. Enablement of different cleavage reagents in the GLP-1 analogue synthesis process.

[0667] Procedure.

[0668] Ligation:

[0669] A titrator-chamber (75 mL) was charged with an aqueous solution (2 mL) of the mono acylated GLP-1 backbone 8 (200.0 mg, 100.0 mg / ml, 25.7 mM, 1.0 eq., MW = 3891.3 g / mol). The solution was diluted with milliQ water (1.6 mL) and EtOH (1.8 mL). pH was measured to be 8.2. The pH of the solution was titrated with 1 M HCI to reach pH 7.9. The pH of the solution was titrated with 1.0 M NaOH to reach pH 8.1 using an auto titrator (Titrando / dosino®). The activated dipeptide 6Aa (38.5 mg, 82.0 .mol, 1.6 eq. 98.2% active) was dissolved in NMP (197 |j.L, 197 mg / mL). The activated dipeptide solution was added over 22 min with a Hamilton syringe pump to the GLP-1 backbone solution. The vial with the dipeptide solution was washed with NMP (10 jiL) and this was added as well. During the addition of dipeptide, the pH of the reaction mixture was adjusted with 1.0 M NaOH in the pH interval 8.0-8.2. The progress of the reaction was followed using UPLC-MS. Samples from the reaction were withdrawn at different times and diluted (2:98) with MeCN / H2O (1 / 1) and analyzed using method UPLC-MS_7. The ratio efficiency in the ligation process was estimated by area comparison in UV at 214 nm. For clarity in comparison, the combined area of remaining backbone, product, and over ligated product was normalized to 100%. The product peak (P1) and over ligated product (P2) are combined as “Product Conversion” in table 6. After 180 min the ligation was finished. The result is shown in table 6, example 22. The solution was stored overnight at -20°C.

[0670] Deprotection with HMA:

[0671] To half of the ligation solution (111.8 mg, 48.6 mg / mL, 2.3 mL, 11.2 mM, 1.0 eq.) was added aqueous ethanol (30% v / v,1 mL), / V-hydroxy- / V-methylacetamide (HMA) (458.0 mg, 5.2 mmol, 200.0 eq.) and 1.0 M aqueous K3PO4 solution (450.0 iL) to give a phosphate concentration of 0.1 M. After adjustment of pH with 4.0 M KOH (1.0 mL) to 11 and addition of EtOH (400.0 .L) the reaction was stirred for 5 hours under pH control followed by storage in the refrigerator overnight. The pH of the reaction was adjusted to 8.3 with 4 M HCI. During the reaction small samples were withdrawn, diluted (2:98) with MeCN / H2O (1 / 1) and analyzed using the same method for the ligation step. The ratio efficiency in the ligation process was estimated by area comparison in UV at 214 nm. For clarity in comparison, the combined area of remaining backbone, product, and by-product / s were normalized to 100%. The result is shown in table 7, example 22A. The remaining solution was stored in a Nunc tube at -20°C. Yield: 94.0 mg (89.0%, CAD determination). Purity 81.7% (UPLC-MS_7). UPLC-MS_3 calc, mass (mono isotope): 4111.12 m / z; Found [M+3H]3+: 1371.41 ; [M+4H]4+: 1028.81.

[0672] Deprotection with DHB:

[0673] To half of the ligation solution (111.8 mg, 48.6 mg / mL, 2.3 mL, 11.2 mM, 1.0 eq.) was added / V,2-dihydroxybenzamide (DHB) (788.0 mg, 5.2 mmol, 200.0 eq.) and 1.0 M aqueous K3PO4 solution (450.0 iL) to give a phosphate concentration of 0.1 M. After adjustment of pH with 4.0 M KOH (1.9 mL) to 11 and addition of EtOH (600.0 iL) the reaction was stirred for 5 hours under pH control followed by storage in the refrigerator overnight. The pH of the reaction was adjusted to 8.2 with 4 M HCI. The precipitated material was removed by filtration. The solid was washed with aqueous ethanol (30% v / v, 2 x 0.5 mL). During the reaction small samples were withdrawn, diluted (2:98) with MeCN / H2O (1 / 1) and analyzed using the same method as for the ligation step. The ratio efficiency in the ligation process was estimated by area comparison in UV at 214 nm. For clarity in comparison, the combined area of remaining backbone, product, and by-product / s were normalized to 100%. The result is shown in table 7, example 22B. The remaining solution was stored in a Nunc tube at -20°C. Yield: 83.0 mg (79.0%, CAD determination). Purity 82.7% (UPLC-MS_7). UPLC-MS_3 calc, mass (mono isotope): 4111.12 m / z; Found [M+3H]3+: 1371.41 ; [M+4H]4+: 1028.81.

[0674] Table 6. Use of different protocols and dipeptide reagents to obtain protected product.

[0675] 1) GLP-1 as backbone

[0676] 2) Compound 0111 as backbone

[0677] 3) UV signals at 210 or 214 nm. 4) 28.5% of other small peaks formed during ligation.

[0678] 5) ~7% of the Cbz was cleaved during the ligation.

[0679] 6) An over acylation impurity (~5%) was include in the peak.

[0680] Results: The examples in Table 6 shows that the ligation with different protected dipeptides to either GLP-1 backbone 8 or backbone 11 generally performs very well for both activated dipeptides (examples 9a, 11 , 12, 13, 14, 17, 18, 19, 20 and 21) and in situ activated dipeptides (examples 9b, 10 and 16). Only example 15 gives a low conversion in the ligation reaction due to a benzyl protected imidazole on the histidine. There could be a small difference in ligation conversion between a purified dipeptide activated as an oxazolone (example 9a) compared to an in situ generated oxazolone (example 9b). In the later example more equivalents should be used to reach completion. There may also be a very small difference in ligation conversion in Table 6 when using a dipeptide activated as an oxazolone compared to a dipeptide activated as an ester (example 12 versus 11 and 17), which was also found in Table 4.

[0681] Table 7. Deprotection of peptides from Table 6.

[0682] 1) CAD measurement of the crude peaks and multiplied with the UPLC purity or CAD on the separated product peak.

[0683] 2) Yield based on area under curve of the product UV signal and multiplied with a UV standard.

[0684] 3) Removal of Cbz was finished after 2 hours whereas, the benzyl was cleaved ~29% after 18 hours.

[0685] 4) Starting material from ligation combined with partial Bn cleavage.

[0686] 5) Gel formation was observed. Yield expected to be higher if the solution was homogenic.

[0687] 6) 20% w / w of 5% palladium on charcoal was used.

[0688] 7) Compound 0111 as backbone

[0689] Results: The examples in Table 7 shows that the deprotection of the different protected GLP-1 backbone 9 or protected compound 12 (protected compound 0111 of WO 2022 / 129526 A1) generally performs very well, except for example 15 (dipeptide with Cbz / Bn protection), which is very difficult to deprotect and to some degrees example 14 (dipeptide with TFAc / Dnp protection). The yields are generally around 70% with some variation to both sides. Both nucleophiles at high pH (examples 9a, 9b, 10, 11 , 12, 13, 14, 19, 20 and 21) and hydrogenation at neutral pH (examples 16 and 17) would work to deprotect the protected peptides. An acidic deprotection was also carried out with success (example 18). Finally, it can also be seen in Table 7 that both small and large scale deprotection works equally well (examples 9a and 9b).

[0690] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skills in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

CLAIMS1. A method of making an oxazolone derivative of formula (6) or an activated ester of formulathe method comprising: reacting a compound of formula (4);with (i) a coupling reagent, (ii) with a coupling reagent and / V-hydroxysuccinimide, or (iii) with a coupling reagent and R3-XH to obtain the oxazolone derivative of formula (6) or the activated ester of formula (5); whereinPg1is TFAc, Dde or Moc;Pg2is selected from the group consisting of tosyl (Ts), benzenesulfonyl (Bs), 4- methoxybenzenesulfonyl (Mbs), thiophenylsulfonyl (Ths), 2,5-dichlorobenzoyl (DCB), benzyl (Bn), and benzyloxycarbonyl (Cbz);X is oxygen or sulfur;R3is a of formula (7), whereinRais H, F, Cl, OCH3, NO2, SO2N(CH3)2, SO3H, C(O)Me, CN, or Ms;Rbis H, F, Cl, NO2, SO2N(CH3)2, C(O)Me, CN, or Ms;Rcis H, F CI, OCH3, NO2, SO2N(CH3)2, SO3H, C(O)Me, CN, or Ms;Rdis H, F, Cl, NO2, SO2N(CH3)2, C(O)Me, CN, or Ms; andReis H, F, Cl, OCH3, NO2, SO2N(CH3)2, SO3H, C(O)Me, CN, Ms; or wherein Raand Rbjointly form a 5 or 6 membered heterocycle; and whereinPg1and Pg2are cleavable under the same deprotection conditions.

2. The method according to claim 1 , wherein the coupling reagent is DIC, EDC, or DCC, preferably DCC.

3. The method according to claim 1 or 2, wherein R3-XH is selected from the group consisting of4. An activated ester of formula (5), whereinX is oxygen or sulfur;Pg1is a protecting group selected from the group consisting of TFAc, or Moc;Pg2is a protecting group selected from the group consisting of tosyl (Ts), benzenesulfonyl (Bs), 4-methoxybenzenesulfonyl (Mbs), thiophenylsulfonyl (Ths), 2,4- dinitrophenyl (Dnp), terf-butyloxycarbonyl (Boc), 2,5-dichlorobenzoyl (DCB), benzyl (Bn), and benzyloxycarbonyl (Cbz); andR3is 2-methoxy-4-nitrophenyl (MNP), 4-chloro-2-(N,N-dimethylsulfamoyl)phenyl (CSAP) or / V-succinimidyl, preferably 2-methoxy-4-nitrophenyl (MNP), 4-chloro-2-(N,N- dimethylsulfamoyl)phenyl (CSAP).

5. The ester according to claim 4, wherein the ester is selected from the group consisting of6. An oxazolone of formula (6), whereinPg1is a protecting group selected from the group consisting of TFAc, Dde, or Moc; and Pg2is a protecting group selected from the group consisting of tosyl (Ts), benzenesulfonyl (Bs), 4-methoxybenzenesulfonyl (Mbs), thiophenylsulfonyl (Ths), tert-butyloxycarbonyl (Boc), 2,4-dinitrophenyl (Dnp), 2,5-dichlorobenzoyl (DCB), benzyl (Bn), and benzyloxycarbonyl (Cbz).

7. The oxazolone according to claim 6, wherein the oxazolone is selected from the group consisting of8. The activated ester according to claim 4 or claim 5 or the oxazolone according to claim 6 or 7, wherein the ester or oxazolone is isolated or isolatable.

9. The activated ester according to claim 4 or claim 5 or the oxazolone according to claim 6 or claim 7, wherein the ester or oxazolone is stable in isolated form.

10. A process for producing a target peptide comprising Aib, wherein the process comprises: a) providing a peptide to be coupled to an activated dipeptide is obtainable by a method according to any one of claims 1 to 3, wherein the peptide is dissolved in water or an alcohol or a mixture thereof, and wherein the activated dipeptide comprises Aib; b) providing the activated dipeptide, wherein the activated dipeptide is dissolved in an organic solvent, optionally wherein the organic solvent is miscible with water; c) addition the activated dipeptide of step b to the peptide of step a; and optionally d) adding base or acid to adjust the pH.

11. The process according to claim 10, wherein the target peptide is semaglutide or a precursor thereof.

12. The process according to claim 10, wherein the target peptide is tirzepatide or a precursor thereof.

13. The process according to claim 10, wherein the activated dipeptide is an activated ester according to claim 4 or claim 5 or an oxazole according to claim 6 or claim 7.

14. Use of an isolatable activated ester or an isolatable oxazolone in a coupling reaction to obtain a target peptide, wherein the activated ester or oxazolone is a dipeptide comprising an Aib moiety, optionally wherein the dipeptide comprises His-Aib.

15. Use of an activated ester according to claim 4 or claim 5 or an oxazole according to claim6 or claim 8 in a coupling reaction to obtain a target peptide.

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