Method for the synthesis of a peptide with amino acids having basic side-chains

Abstract

Method for the solid-phase synthesis of a peptide comprising coupling cycles of amino acid building blocks to an amino acid chain, wherein at least one of said amino acid building blocks comprises one or more histidine residues with an unprotected side-chain, and at least one coupling cycle comprises using a coupling reagent selected from the group consisting of N-tert-Butyl-N'-ethylcarbodiimide and N-tert-Butyl-N'- isopropylcarbodiimide.

Description

[0001] Title: Method for the synthesis of a peptide with amino acids having basic side-chains.

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the general field of peptide synthesis. In particular, it relates to a method for the synthesis of a peptide comprising amino acids having basic side-chains.

[0004] BACKGROUND OF THE INVENTION

[0005] Amino acids having basic side-chains or peptide fragments containing such amino acids cannot be used as such in solid-phase peptide synthesis (SPPS) because, under coupling conditions, they undergo to undesired modification. As examples of undesired modification, the guanidine group of arginine (Arg) can be acylated during coupling reactions, while the imidazolyl group of histidine (His) reacts with the electrophilic coupling reagent DIC, both reactions result in impurities which lower the yield and the overall quality of the peptide.

[0006] To avoid this, such side-chains are traditionally protected with suitable protective groups before coupling these amino acids or peptide fragments containing these amino acids with an amino acid chain. This approach involves the formation of a covalent chemical bond between the basic side-chains and the protective groups. These protective groups are acid-labile and they are usually removed once the synthesis of the peptide on the solid support is completed. Very often the removal of these protective groups is done at the same time as the cleavage of the peptide from the solid support.

[0007] This strategy has some drawbacks.

[0008] Firstly, acylation of the basic side-chain is not always prevented by side-chain protection. In the case of N“- Fmoc-derivatives of arginine protected with Boc or Adoc, for example, acylation of the guanidine group occurs despite of the protecting group, leading to the formation of ornithine.

[0009] The introduction of side-chain protective groups can lower the overall quality of the peptide. In fact, in some cases, once removed from the peptide, they can reattach to the peptide itself, leading to impurities which can be also difficult to eliminate. Thus, not only the yield is impaired but also the purity of the peptide.

[0010] In addition, some protective groups can be very difficult to remove, for example Pbf is very difficult to be removed from arginine-rich peptides.

[0011] Further, while usually protective groups are removed during detaching of the peptide from the solid support, it can be necessary to have prolonged treatment and to use large amount of chemicals to detach the peptide from the solid support and at the same time remove the side-chain protective groups.

[0012] As an example, in the Fmoc-SPPS, where TFA at high concentration is used to obtain in the same step the removal of side-chain protective groups and the cleavage of the peptide from the resin, a very large amount of TFA is requested, especially with the protective groups that are difficult to be removed. However, it is universally recognised that TFA is a high impact solvent from an environmental point of view.

[0013] If a large amount of TFA is used, a greater volume of antisolvent is required to obtain the peptide as a precipitate.

[0014] Further, high concentration of TFA, addition of strong acids, high temperature and long time for cleavage are all factors causing degradation of the peptide.

[0015] For all these reasons, in the past scientists have tried to develop strategies for mild protection of basic sidechains of amino acids.

[0016] In "Side-chain unprotected Fmoc-Arg / His / Tyr-OH couplings and their application in solid-phase peptide synthesis through a minimal-protection / green chemistry strategy", Org. Process Res. Dev, 2022, 26, 1520- 1530, the authors proposed a method for side-chain mild protection of some amino acids. According to this method, when PyBOP is used instead of DIC as the coupling reagent for Fmoc-His-OH, no Nim-His modification is induced at the coupling step.

[0017] PyBOP however, is quite expensive if compared with carbodiimide coupling reagents and, during coupling, triazone is formed, which has explosive properties.

[0018] Further, the use of PyBOP is not compatible with other mild protection strategies of basic side-chain of amino acids. As an example, authors also observed that PyBOP is uncapable of driving the Fmoc-Arg(HCl)-OH coupling into completion and outlined the incompatibility of the use of PyBOP for His with unprotected sidechain and of DIC / Oxyma for Arg with unprotected side-chain in the case of peptides having both such amino acids.

[0019] Thus, there is still a need in the field for the development of new and efficient strategies for mild protection of basic side-chain of His residues in SPPS synthesis.

[0020] OBJECT OF THE INVENTION

[0021] Accordingly, the first object of the present disclosure is a method for the solid-phase synthesis of a peptide comprising amino acids having basic side-chain, in particular a method for the solid-phase synthesis of a peptide comprising one or more histidine residues, that does not show one or more of the above-mentioned drawbacks of the prior art.

[0022] Another object of the present invention is the use of selected coupling reagents in a coupling reaction of an amino acid building block to an amino acid chain.

[0023] SUMMARY OF THE INVENTION

[0024] In one aspect, the present disclosure relates to a method for the solid-phase synthesis of a peptide comprising a predetermined amino acid sequence, the method comprising coupling cycles of amino acid building blocks to an amino acid chain, wherein

[0025] - said amino acid building blocks comprise:

[0026] - an unprotected C-terminal carboxyl group and

[0027] - a protected N-terminal amino group, and

[0028] - said amino acid chain comprises an unprotected amino group, at least one of said amino acid building blocks comprises one or more His residues with unprotected sidechain, and at least one coupling cycle comprises using a coupling reagent selected from the group consisting of N-tert- Butyl-N'-ethylcarbodiimide and N-tert-Butyl-N'-isopropylcarbodiimide to couple said amino acid building block comprising one or more His residues with unprotected side-chain with said amino acid chain at the unprotected amino group of said amino acid chain, so that an amide bond is formed between said amino acid chain and said amino acid building block.

[0029] Advantageously, this coupling reagent is milder if compared with others coupling reagents, which often are so reactive to generate both amide products and ester products.

[0030] Carbodiimides are, in fact, very selective toward the formation of the amide bond.

[0031] This method affords to increase the overall quality of the peptide, in terms of number and relative amount of impurities.

[0032] The yield is also improved because it is not required the use of covalently bound protective group for His residues, which can be difficult to remove and can originate impurities. In fact, this method does not necessarily require prolonged treatment or a large amount of chemicals to detach the peptide from the solid support, as compared to the method in the art, wherein large amount of chemicals is required to remove the side-chain protective groups of Arg residues simultaneously with the cleavage of the peptide from the resin.

[0033] In addition, there is no generation of impurities due to the reattachment of the protective groups of His residues, or to protective groups of His residues not completely removed.

[0034] Further, the use of some chemicals, in some cases the less environmentally friendly chemicals, can be avoided or their amount can be reduced.

[0035] An example of chemicals that are not required at all according to the present method, are carbocation scavengers, which are used to trap carbocations generated during the removal of covalently bound protective groups of His residues, such as Trt.

[0036] In a further aspect, the present disclosure relates to the use of a coupling reagent selected from the group consisting of N-tert-Butyl-N'-ethylcarbodiimide and N-tert-Butyl-N'-isopropylcarbodiimide in a coupling reaction of an amino acid building block to an amino acid chain, wherein:

[0037] - said amino acid building block comprises:

[0038] - an unprotected C-terminal carboxyl group,

[0039] - a protected N-terminal amino group,

[0040] - one or more His residues with unprotected side-chain, and

[0041] - said amino acid chain comprises an unprotected amino group.

[0042] BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Some features and advantages of the present invention will become apparent from the accompanying drawings, in which:

[0044] Figure 1 shows the UHPLC-MS profile of the peptide of Example 1 (El), prepared according to the present disclosure,

[0045] Figure 2 shows the UHPLC-MS profile of the peptide of Comparative Example 1 (CE1), prepared according to the prior art, and

[0046] Figure 3 shows the UHPLC-MS profile of the peptide of Example 10 (E10), prepared according to the present disclosure.

[0047] DETAILED DESCRIPTION OF THE INVENTION

[0048] The method for the solid-phase synthesis of a peptide comprising a predetermined amino acid sequence comprises coupling cycles of amino acid building blocks to an amino acid chain, wherein

[0049] - said amino acid building blocks comprise:

[0050] - an unprotected C-terminal carboxyl group and

[0051] - a protected N-terminal amino group, and

[0052] - said amino acid chain comprises an unprotected amino group, at least one of said amino acid building blocks comprises one or more His residues with unprotected sidechain, and at least one coupling cycle comprises using a coupling reagent selected from the group consisting of N-tert- Butyl-N'-ethylcarbodiimide and N-tert-Butyl-N'-isopropylcarbodiimide to couple said amino acid building block comprising one or more His residues with unprotected side-chain with said amino acid chain at the unprotected amino group of said amino acid chain, so that an amide bond is formed between said amino acid chain and said amino acid building block. The term "residue" refers to an amino-acid residue. Amino-acid residues are structures that lack a hydrogen atom of the amino group (-NH-CHR-COOH), or the hydroxyl moiety of the carboxyl group (NH2-CHR-CO-), or both (-NH-CHR-COO-); all units of a peptide chain are therefore amino acid residues.

[0053] A peptide is any compound produced by amide formation between a carboxyl group of one amino acid and an amino group of another. Peptides with fewer than about 10-20 residues are also called oligopeptides; those with more, polypeptides. Polypeptides of specific sequence of more than 50 residues are usually known as proteins. Within the present disclosure the term "peptide" refers to a peptide having a number of amino acid residues equal or less than 50.

[0054] With the wording "coupling the product obtained in step (b) with said amino acid chain at the unprotected amino group of said amino acid chain" it is meant that the product obtained in step (b) is C-terminally coupled with said amino acid chain at the unprotected amino group of said amino acid chain.

[0055] The term "protective group" refers to a chemical group temporarily attached to a functional group to generate a protected functional group and, consequently, decrease the reactivity of the functional group so that the protected functional group does not react under synthetic conditions to which the molecule is subjected in one or more subsequent steps.

[0056] The word "protected" is used to indicate a protected functional group, i.e. a functional group having a temporarily attached chemical group which decreases the reactivity of the functional group so that the protected functional group does not react under synthetic conditions to which the molecule is subjected in one or more subsequent steps.

[0057] The word "deprotecting" means removing the temporarily attached chemical group from the protected functional group to restore the functional group (free functional group).

[0058] The wording "coupling additive" refers to an additive used in coupling reactions to reduce side reactions and racemization.

[0059] The wording "coupling reagent", also known as "coupling activator", or "activator" refers to a chemical compound able to replace the hydroxyl group of the unprotected carboxylic acid of the amino acid building block with a leaving group. This replacement allows the formation of an activated intermediate that is able to be C-terminally coupled with the unprotected amino group of the amino acid chain, with the formation of an amide bond between the amino acid chain and the amino acid building block.

[0060] Advantageously, this method allows obtaining the coupling of amino acids building blocks containing one or more His residues to an amino acid chain without the need of protective groups covalently bound to the sidechain of these amino acid residues.

[0061] Further, this method allows obtaining good yields without the need of multiple coupling cycles, multiple additions of coupling reagent or prolonged treatment to deprotect the His residues side-chain.

[0062] In addition, this method allows obtaining the peptide with high purity with respect to the one obtaining Trt as protecting group for His residues.

[0063] In addition, a large amount of chemicals is not needed to detach the peptide from the solid support and, at the same time, remove the side-chain protective groups.

[0064] Further, there is no need to use carbocation scavengers for His residues protective groups, thus avoiding any secondary reaction associated to these chemicals. Also, degradation of the peptide is reduced with respect to covalently bound protective groups because the use of high concentration of TFA, the addition of strong acids, the use of high temperature and long time for cleavage can be avoided.

[0065] Further, this method is compatible with other mild protection strategies of basic side-chains, such the one of arginine.

[0066] The choice of the coupling reagent in the group consisting of N-tert-Butyl-N'-ethylcarbodiimide and N-tert- Butyl-N'-isopropylcarbodiimide is associated to their steric hindrance that prevent its reaction with the basic side chain of His residues.

[0067] The result of this steric hindrance is that the side-chain of His residues is prevented from reacting with the coupling reagent during coupling of the amino acid building block with the amino acid chain, as it is the case when DIC is used as coupling reagent.

[0068] The N-terminal amino group of said amino acids building blocks is preferably protected with a base-labile protecting group, more preferably Fmoc.

[0069] Preferably, said at least one coupling cycle comprises the steps of:

[0070] (a) contacting said amino acid building block comprising one or more His residues with unprotected sidechain with a coupling additive,

[0071] (b) contacting the product obtained in step (a) with said coupling reagent selected from the group consisting of N-tert-Butyl-N'-ethylcarbodiimide and N-tert-Butyl-N'-isopropylcarbodiimide, and

[0072] (c) coupling the product obtained in step (b) with said amino acid chain at the unprotected amino group of said amino acid chain, so that an amide bond is formed between said amino acid chain and said amino acid building block.

[0073] Preferably, the coupling reagent is N-tert-Butyl-N'-ethylcarbodiimide.

[0074] Preferably, at least one coupling cycle performed after said at least one coupling cycle comprising steps (a)- (c) comprises the use of a coupling reagent selected from the group consisting of N-tert-Butyl-N'- ethylcarbodiimide and N-tert-Butyl-N'-isopropylcarbodiimide, preferably N-tert-Butyl-N'-ethylcarbodiimide; more preferably, all the coupling cycles performed after said at least one coupling cycle comprising steps (a)- (c) comprise the use of a coupling reagent selected from the group consisting of N-tert-Butyl-N'- ethylcarbodiimide and N-tert-Butyl-N'-isopropylcarbodiimide, preferably N-tert-Butyl-N'-ethylcarbodiimide.

[0075] In one embodiment according to the present disclosure, if said at least one of said amino acid building blocks comprises one or more Arg residues with unprotected side-chain and one or more His residues with unprotected side-chain, said at least one coupling cycle comprises the steps of:

[0076] (a) contacting said amino acid building block comprising one or more Arg residues with unprotected sidechain and one or more His residues with unprotected side-chain with a coupling additive selected from the group of the following compounds:

[0077] Ri being CH or N,

[0078] R2being H, Cl, COCH3, CH2COCH3 or CH2OCH3,

[0079] R3 being CF3, Cl or NO2, where the ratio between said coupling additive and all the Arg residues with unprotected side-chain of said amino acid building block and is at least 1.5:1.0 equivalents;

[0080] (b) contacting the product obtained in step (a) with said coupling reagent selected from the group consisting of N-tert-Butyl-N'-ethylcarbodiimide and N-tert-Butyl-N'-isopropylcarbodiimide, preferably N-tert-Butyl-N'- ethylcarbodiimide;

[0081] (c) coupling the product obtained in step (b) with said amino acid chain at the unprotected amino group of said amino acid chain, so that an amide bond is formed between said amino acid chain and said amino acid building block.

[0082] It may be assumed that, due to the acidic nature of the coupling additive, in step (a) an interaction is established between the coupling additive itself and the unprotected basic side-chain of Arg, so that coupling additive protects the basic side-chain during the coupling reaction of the amino acid building block with the growing amino acid chain.

[0083] Thus, for each equivalent of unprotected basic side-chain of Arg residues of the amino acid building block, an equivalent of the coupling additive interacts in step (a) with said equivalent of unprotected basic side-chain.

[0084] The result of this interaction is that the basic side-chain does not react during coupling of the amino acid building block with the amino acid chain.

[0085] If the amino acid building block comprises two Arg residues having unprotected basic side-chain, in step (a) two equivalents of the coupling additive will interact with said basic side-chains.

[0086] If, as an example, the amino acid building block is Fmoc-Arg(HCl)-OH and the coupling additive is Oxyma, for each molecule of the amino acid building block, a molecule of Oxyma interacts in step (a) with the guanidinium lateral chain of an arginine residue.

[0087] Thus, if each molecule of coupling additive interacts with one single arginine residue, then the required ratio between said coupling additive and all the Arg residues with unprotected side-chain of said amino acid building block of at least 1.5:1.0 equivalents means that the number of molecules of the coupling additive must be at least 1.5 times the number of the arginine residues present in the amino acid building block. Preferably, each molecule of coupling additive interacts with a single arginine residue and each arginine residue interacts with a single molecule of coupling additive.

[0088] Preferably, the ratio between said coupling additive and all the Arg residues with unprotected side-chain of said amino acid building block is at least 1.7:1.0 equivalents, preferably at least 2.0:1.0 equivalents, more preferably at least 2.2:1.0 equivalents.

[0089] Compound (I) is also known as HOSu, compound (II) as HODhbt, compound (III) as HOBt when Ri is CH and as HOAt when Ri is N, compound (V) as HOt, compound (VI) as HOCt, compound (VII) as Oxyma or Oxyma Pure®, compound (VIII) as 6-CF3-HOBt when R3 is CF3, as 6-Cl-HOBt when R3 is CF3, and as 6-NO2-HOBt when R3 is NO2, compound (IX) as Oxyma-B®, compound (X) as HONB, and compound (XI) as HOPy.

[0090] The terms "Oxyma" and "OxymaPure®" are used interchangeably in the following disclosure.

[0091] Said coupling additive is preferably selected from the group consisting of compounds (I), (III) with Ri being CH, (IV) with R2 being H, (VII), (IX), (X) and (XI), preferably from the group consisting of compounds (I), (VII), (IX), and (X), more preferably the coupling additive is compound (VII).

[0092] Preferably, said at least one coupling cycle comprises the following steps after said step (c):

[0093] (d) deprotecting the N-terminal amino group of the amino acid building block; and

[0094] (e) treating one or more times with a solvent, wherein at least one of the treatments, preferably at least the last treatment, more preferably the last and the second to last treatments are carried out with a mixture of said solvent and said coupling additive.

[0095] The solvent is used to remove unreacted reagents and byproducts.

[0096] Step (e) of treating one or more times with a solvent, wherein at least one of the treatments is carried out with a mixture of said solvent and said coupling additive, is required if said coupling cycle comprising steps (a), (b), and (c) is not the last coupling cycle for the solid-phase synthesis of the peptide.

[0097] In fact, during step (d) of deprotecting the N-terminal amino group of the amino acid building block, e.g. by using piperidine, the interaction between the coupling additive and the basic side-chain of Arg residues of the amino acid building block coupled in step (c) with the amino acid chain is lost and it has to be restored before starting a new coupling cycle in order to avoid any side reaction of these side-chains occurring with the new coupling cycle.

[0098] Advantageously, this avoid that the Arg residues with unprotected side-chain that has become part of the amino acid chain in one coupling cycle comprising said steps (a)-(c) or already present in the amino acid chain undergo to undesired coupling reaction at its unprotected basic side-chain during the subsequent coupling cycle.

[0099] In step (e) the ratio between said coupling additive in the mixture and all the Arg residues with unprotected side-chain present in said amino acid chain is preferably at least 1.0:1.0 equivalents, more preferably at least 1.1:1.0, even more preferably at least 1.3:1.0, most preferably at least 1.5:1.0.

[0100] Thus, if each molecule of coupling additive interacts with one single arginine residue, then the required ratio between said coupling additive and all the Arg residues with unprotected side-chain of said amino acid chain of at least 1.0:1.0 equivalents means that the number of molecules of the coupling additive in the mixture must be at least equal to the number of the arginine residues present in the amino acid building block.

[0101] Step (e) of treating one or more times, wherein at least one of the treatments is carried out with a mixture of said solvent and said coupling additive, is required if said coupling cycle comprising steps (a), (b), and (c) is not the last coupling cycle for the solid-phase synthesis of the peptide. If there are one or more coupling cycles occurring after said coupling cycle comprising said steps from (a) to (e), said one or more coupling cycles occurring after said coupling cycle comprising said steps from (a) to (e) comprise treating with a mixture of said solvent and said coupling additive, wherein, preferably, the ratio between said coupling additive in said mixture and all the Arg residues with unprotected side-chain present in said amino acid chain is at least 1.0:1.0 equivalents, more preferably at least 1.3:1.0, even more preferably at least 1.5:1.0.

[0102] If there are one or more coupling cycles occurring after said coupling cycle comprising said steps from (a) to (e), preferably all the coupling cycles except the last one occurring after said coupling cycle comprising said steps from (a) to (e) comprise treating with a mixture of said solvent and said coupling additive, wherein, preferably, the ratio between said coupling additive in said mixture and all the Arg residues with unprotected side-chain present in said amino acid chain is at least 1.0:1.0 equivalents, more preferably at least 1.3:1.0, even more preferably at least 1.5:1.0.

[0103] If said at least one of said amino acid building block comprises one or more His residues with unprotected side-chain and said amino acid chain comprises one or more Arg residues with unprotected side-chain, said amino acid chain is treated one or more times before being used in said coupling cycle with a mixture of solvent and a coupling additive selected from the group consisting of:

[0104] Ri being CH or N,

[0105] R2being H, Cl, COCH3, CH2COCH3 or CH2OCH3, R3 being CF3, Cl or NO2. In this case, the ratio between said coupling additive in the mixture and all the Arg residues with unprotected side-chain present in said amino acid chain is preferably at least 1.0:1.0 equivalents, more preferably at least 1.1:1.0, even more preferably at least 1.3:1.0, most preferably at least 1.5:1.0.

[0106] Conditions for steps (a), (c), (d) and (e) are known by the person skilled in the art.

[0107] Preferably, the solvent used in the treatments is DMF or a mixture of NBP / DMC 8:2 vol.

[0108] If said at least one of said amino acid building blocks comprises one or more Arg residues with unprotected side-chain and one or more His residues with unprotected side-chain and / or at least one of said amino acid building block comprises one or more His residues with unprotected side-chain and said amino acid chain comprises one or more Arg residues with unprotected side-chain, in all the coupling cycles following said at least one coupling cycle the ratio between said coupling additive and all the Arg residues with unprotected side-chain of said amino acid building block and of said amino acid chain is at least 1.5:1.0 equivalents, preferably at least 2.0:1.0 equivalents, more preferably at least 2.2:1.0 equivalents.

[0109] Thus, if each molecule of coupling additive interacts with one single arginine residue, then the required ratio between said coupling additive and all the Arg residues with unprotected side-chain of both said amino acid chain of at least 1.5:1.0 equivalents means that the number of molecules of the coupling additive must be at least 1.5 times the total number of the arginine residues present in the amino acid building block and in the amino acid chain, i.e. the number of the arginine residues present in the amino acid building block plus the number of the arginine residues present in the amino acid chain.

[0110] So, if said at least one of said amino acid building blocks comprises one or more Arg residues with unprotected side-chain and one or more His residues with unprotected side-chain, alternatively to the use of a mixture of said solvent and said coupling additive in step e), it can be used in all the coupling cycles following said at least one coupling cycle a ratio between said coupling additive and all the Arg residues with unprotected side-chain of said amino acid building block and of at least 1.5:1.0 equivalents, preferably at least 2.0:1.0 equivalents, more preferably at least 2.2:1.0 equivalents.

[0111] On the other hand, if said at least one of said amino acid building block comprises one or more His residues with unprotected side-chain and said amino acid chain comprises one or more Arg residues with unprotected side-chain, alternatively to the treatment of said amino acid chain one or more times before being used in said coupling cycle with a mixture of said solvent and said coupling additive, it can be used in all the coupling cycles following said at least one coupling cycle a ratio between said coupling additive and all the Arg residues with unprotected side-chain of said amino acid chain and of at least 1.5:1.0 equivalents, preferably at least 2.0:1.0 equivalents, more preferably at least 2.2:1.0 equivalents.

[0112] Preferably, the peptide is selected from the group consisting of glucagon, abaloparatide, semaglutide, salmon calcitonin, teriparatide, teduglutide, liraglutide, and their pharmaceutically acceptable salts.

[0113] The coupling reagent selected from the group consisting of N-tert-Butyl-N'-ethylcarbodiimide and N-tert- Butyl-N'-isopropylcarbodiimide, preferably N-tert-Butyl-N'-ethylcarbodiimide, can be used in a coupling reaction of an amino acid building block to an amino acid chain, wherein:

[0114] - said amino acid building block comprise:

[0115] - an unprotected C-terminal carboxyl group,

[0116] - a protected N-terminal amino group,

[0117] - one or more His residues with unprotected side-chain, and

[0118] - said amino acid chain comprises an unprotected amino group. Advantageously, the use of said coupling reagents avoids using protective groups that are covalently bound to the side-chain of said His residues and at the same time do not form explosive by-products, while being compatible with mild protection strategies of other amino acids.

[0119] EXPERIMENTAL

[0120] Where not otherwise specified, all the operations are to be considered carried out at room temperature.

[0121] The term "room temperature" refers to a temperature ranging from about 15 °C to 35 °C.

[0122] Example 1 - Synthesis of esapeptide F-H-C-P-R-G

[0123] In Example 1 (El), esapeptide Phe-His-Cys-Pro-Arg-Gly was synthesized according to the invention.

[0124] The esapeptide was obtained starting from the tetrapeptide Gly-Arg-Pro-Cys(Trt)-Fmoc preloaded Rink amide MBHA resin.

[0125] The resin was treated two times with DMF, then other two times with 0.5 M Oxyma in DMF.

[0126] Unprotected histidine was coupled to the tetrapeptide as follows.

[0127] Fmoc-His-OH (2 eq with respect to the loading of the resin) was suspended in 25 mL of DMF and Oxyma (5 eq respect to the loading of the resin) was added to the solution. Pre-activation was carried out with tBEC (2 eq respect to the loading of the resin) for 5 minutes. The coupling solution was then added to the Fmoc- deprotected amino acid chain attached to the resin and left to react for 2 h.

[0128] Fmoc deprotection was obtained with 20 vol% piperidine in DMF for 10 minutes.

[0129] The resin was then treated two times with DMF, then other two times with 0.5 M Oxyma in DMF.

[0130] Then, phenylalanine has been coupled as follows:

[0131] Fmoc-Phe-OH (2 eq with respect to the loading of the resin) was suspended in 25 mL of DMF and Oxyma (2 eq with respect to the loading of the resin) was added to the solution. Pre-activation was carried out with tBEC (2 eq with respect to the loading of the resin) for 5 minutes. The coupling solution was then added to the resin and left to react for 1 h.

[0132] Fmoc deprotection was obtained with 20 vol% piperidine in DMF for 10 minutes.

[0133] The resin was then treated two times with DMF, then other two times with 0.5 M Oxyma in DMF.

[0134] Cleavage of the esapeptide from the resin and removal of protecting groups has been obtained with a mixture of TFAS / TIS / H2O (90 / 5 / 5 vol) for 2 h.

[0135] After cleavage, solution was analysed by UHPLC equipped with quaternary pump, UV detector and ESI-Q-TOF mass using as mobile phase A water +0.1% of TFA and acetonitrile as mobile phase B.

[0136] The UHPLC-MS profile of Example 1 is reported in Figure 1.

[0137] The peak at 9.809 min is attributable to an impurity present at 8.8 wt%.

[0138] The peak at 10.570 min is attributable to the desired esapeptide% (M / Z: 715), present at 91.2 wt%.

[0139] Comparative Example 1

[0140] In Comparative Example 1 (CE1), esapeptide Phe-His-Cys-Pro-Arg-Gly was synthesized exactly in the same way as Example 1, except that DIC has been used as coupling reagent instead of tBEC. After cleavage, solution was analysed by UHPLC equipped with quaternary pump, UV detector and ESI-Q-TOF mass using as mobile phase A water +0.1% of TFA and acetonitrile as mobile phase B.

[0141] The UHPLC-MS profile of Comparative Example 1 is reported in Figure 2.

[0142] The peak at 10.173 min is attributable to an impurity present at 18.1 wt%.

[0143] The peak at 10.966 min is attributable to the desired esapeptide (M / Z: 715), present at 72.1 wt%.

[0144] The peak at 10.173 min is attributable to the adduct formed by the reaction of the esapeptide with DIC (M / Z: 841, +126 mAU with respect to the esapeptide), present at 9.8 wt%.

[0145] The peak ratio between the target esapeptide and the adduct formed by the reaction of the esapeptide with DIC was 89:11.

[0146] Examples 2-9 - Synthesis of pentapeptides and tetrapeptides

[0147] Five different pentapeptides (E2-E6) and three different tetrapeptides (E7-E9) where synthesized according to the invention; details are reported in Table 1.

[0148] Synthesis was carried out at room temperature in glass syringes fitted with a polyethylene porous disc and connected to a vacuum source to remove excess reagents and solvents, by using 0.3 g of preloaded Fmoc- Gly resin (E2, E7-E9) and Fmoc-Gly-Phe-Leu resin (E3-6). Specifically, preloaded MBH resin (loading 0.5 mmol g-1) was used in both cases.

[0149] After the swelling of the resin in 2 mL of the selected solvent (DMF or NBP / DMC 8:2 vol), the Fmoc protective group was removed by 20 vol% piperidine in the selected solvent by treating 2 times x 2 mL, 15 min each, and then the resin was treated with the selected solvent for 3 times x 1.5 mL, 1 min each.

[0150] The amino acids were pre-activated by Oxyma and tBEC (2.0 eq of each of them with respect to the loading of the resin, except for E3 and E4, where 20 eq of tBEC were used) for 3 minutes and coupled to the resin for 90 minutes.

[0151] After each coupling step, the Fmoc protective group was removed by treating the peptide attached to the resin with 20 vol% piperidine in the selected solvent (2 times x 2 mL, 15 min each) and then by treating with the selected solvent (3 times x 1.5 mL, 1 min each).

[0152] After the last Fmoc deprotection, the peptide attached to the resin was further treated with DCM (3 times x 2 mL, 1 min each) and dried under vacuum for 12 hours. The dry peptide resin was suspended in 3 mL of TFA / TIS / H2O (95.0 / 2.5 / 2.5 v / v / v) mixture and stirred for 2 h to obtain cleavage of the peptide from the resin. The resin was then filtered off, treated with TFA (1 time x 1 mL, 1 min) and diisopropylether (25 mL) was added to the solution cooled to 4 °C dropwise. The peptide was finally filtered and dried under vacuum to obtain the crude peptide.

[0153] After cleavage, solution was analysed by UHPLC equipped with quaternary pump, UV detector and ESI-Q-TOF mass using as mobile phase A water +0.1% of TFA and acetonitrile as mobile phase B.

[0154] As said, the adduct of of tBEC or DIC with the His residues of the target peptide should have +126 mAU with respect to the target peptide.

[0155] In Table 1 is reported that no +126 mAU adduct has been detected in case of the use of tBEC; thus, no adduct due to the reaction of imidazolyl group of His residue with tBEC was detected (n.d.: not detectable).

[0156] Table 1 - Examples 2-9

[0157] Comparative Examples 2 and 3 - Synthesis of pentapeptides

[0158] Two pentapeptides (CE2, CE3) where synthesized as comparative examples with the same procedure used for E2-E3 but using DIC instead of tBEC; details are reported in Table 2.

[0159] It was found that, when using DIC, the +126 mAU adduct, due to the reaction of imidazolyl group of His residue with the coupling reagent, is clearly detectable and its amount is 7-10 wt% with respect to the target pentapeptide.

[0160] Table 2 - Comparative Examples 2-3

[0161] Example 10 - Synthesis of heptapeptide Y-H-A-R-G-F-L

[0162] In Example 10 (E10), heptapeptide Tyr-His-Ala-Arg-Gly-Phe-Leu was synthesized according to the invention.

[0163] Synthesis of the heptapeptide was carried out at room temperature in glass syringes fitted with a polyethylene porous disc and connected to a vacuum source to remove excess reagents and solvents, by using 0.3 g of preloaded NH2-Gly-Phe-Leu-MBH resin (loading 0.7 mmol g-1). The resin was swelled in 1.5 mL of NBP / DMC 8:2.

[0164] Fmoc-Arg(HCl)-OH, Fmoc-Ala-OH, Fmoc-His-OH and Fmoc-Tyr-OH were subsequently coupled to the NH2- Gly-Phe-Leu-MBH resin to obtain the target heptapeptide as follows: Fmoc-Arg(HCl)-OH, Fmoc-Ala-OH, Fmoc-His-OH and Fmoc-Tyr-OH (2.0 eq, 0.42 mmol) were pre-activated by OxymaPure® (5.0 eq, 1.05 mmol) and tBEC (2.0 eq, 65.0 uq, 0.42 mmol) for 5 minutes and coupled to the amino acid chain anchored to the resin for 2 h minutes.

[0165] After each coupling step, the Fmoc protective group was removed by 20% Piperidine solution in NBP / DMC 8:2 (2 times x 1.5 mL, 15 min each) and then the amino acid chain anchored to the resin was treated with NBP / DMC 8:2 (3 times x 1.5 mL, 2 min each).

[0166] After the last Fmoc deprotection, the peptide attached to the resin was further treated with NBP / DMC 8:2 (3 times x 1.5 mL, 2 min each), DCM (3 times x 1.5 mL,21 min each) and dried under a vacuum for 12 hours. The dry peptide resin was suspended in 3 mL of the TFA / TIS / H2O (95 / 2.5 / 2.5 v / v / v) mixture and stirred for 2 h. The resin was filtered off, washed with TFA (1 time x 1 mL, 1 min) and diisopropylether (10 mL) cooled to 4 °C was added to the solution dropwise. The peptide was filtered and dried in vacuo to obtain the crude product that was directly analyzed by UHPLC-MS.

[0167] The UHPLC-MS profile of Example 10 is reported in Figure 3.

[0168] The peak at 14.001 min is attributable to the target peptide.

[0169] The small peak before the one of the target peptide is attributable to an impurity of the column.

[0170] No adduct due to the reaction of imidazolyl group of His residue with tBEC was observed.

[0171] No adduct due to the reaction of guanidine group of Arg residue was observed.

[0172] ABBREVIATIONS

[0173] AA amino acid

[0174] Adoc Adamantyloxycarbonyl

[0175] Boc tert-Butyloxycarbonyl

[0176] DIC N,N'-Diisopropylcarbodiimide

[0177] DMC dimentylcarbonate

[0178] DMF N,N-dimethylformamide

[0179] Fmoc 9-Fluorenylmethoxycarbonyl

[0180] HOAt l-Hydroxy-7-azabenzotriazole

[0181] HOBt N-hydroxybenzotriazole

[0182] HOCt ethyl l-hydroxy-lH-l,2,3-triazole-4-carboxylate

[0183] HODhbt 3-hydroxy-3,4-dihydro-4-oxo-l,2,3-benzotriazine

[0184] HONB N-hydroxy-5-norbornene-endo-2,3-dicarboxymide

[0185] HOSu N-hydroxysuccinimide

[0186] HOt N-hydroxytetrazole

[0187] NBP N-butylpirrolidone

[0188] OxymaPure®, Oxyma 2-cyano-2-(hydroxyimino)acetate

[0189] Oxyma-B® 5-(hydroxyimino)-l,3-dimethylpyrimidine-2,4,6(lH,3H,5H)-trione

[0190] Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl

[0191] PyBOP (Benzotriazol-l-yloxy)tri(l-pyrrolidinyl)phosphonium hexafluorophosphate

[0192] RT room temperature

[0193] SPPS Solid Phase Peptide Synthesis tBEC N-tert-Butyl-N'-ethylcarbodiimide tBIC N-tert-Butyl-N'-isopropylcarbodiimide

[0194] TFA Trifluoroacetic acid

[0195] TIS Triisopropylsilane

[0196] Trt Trytil

[0197] UHPLC High Performance Liquid Chromatography

[0198] 6-CF3-HOBt 6-trifluoromethyl-l-hydroxybenzotrialzole

[0199] 6-Cl-HOBt l-hydroxy-6-chlorobenzotriazole

[0200] 6-NO2-HOBt l-hydroxy-6-nitrobenzotrialzole

Claims

Claims1. Method for the solid-phase synthesis of a peptide comprising a predetermined amino acid sequence, the method comprising coupling cycles of amino acid building blocks to an amino acid chain, wherein- said amino acid building blocks comprise:- an unprotected C-terminal carboxyl group and- a protected N-terminal amino group, and- said amino acid chain comprises an unprotected amino group, at least one of said amino acid building blocks comprises one or more His residues with unprotected sidechain, and at least one coupling cycle comprises using a coupling reagent selected from the group consisting of N-tert- Butyl-N'-ethylcarbodiimide and N-tert-Butyl-N'-isopropylcarbodiimide to couple said amino acid building block comprising one or more His residues with unprotected side-chain with said amino acid chain at the unprotected amino group of said amino acid chain, so that an amide bond is formed between said amino acid chain and said amino acid building block.

2. Method according to claim 1, wherein said at least one coupling cycle comprises the steps of:(a) contacting said amino acid building block comprising one or more His residues with unprotected sidechain with a coupling additive,(b) contacting the product obtained in step (a) with said coupling reagent selected from the group consisting of N-tert-Butyl-N'-ethylcarbodiimide and N-tert-Butyl-N'-isopropylcarbodiimide, and(c) coupling the product obtained in step (b) with said amino acid chain at the unprotected amino group of said amino acid chain, so that an amide bond is formed between said amino acid chain and said amino acid building block.

3. Method according to claim 1 or claim 2, wherein the coupling reagent is N-tert-Butyl-N'-ethylcarbodiimide.

4. Method according to any of claims 1 to 3, wherein at least one coupling cycle performed after said at least one coupling cycle comprising steps (a)-(c) comprises the use of a coupling reagent selected from the group consisting of N-tert-Butyl-N'-ethylcarbodiimide and N-tert-Butyl-N'-isopropylcarbodiimide, preferably N- tert-Butyl-N'-ethylcarbodiimide.

5. Method according to claim 4, wherein all the coupling cycles performed after said at least one coupling cycle comprising steps (a)-(c) comprise the use of a coupling reagent selected from the group consisting of N-tert-Butyl-N'-ethylcarbodiimide and N-tert-Butyl-N'-isopropylcarbodiimide, preferably N-tert-Butyl-N'- ethylcarbodiimide.6 Method according to any of claims 2 to 5, wherein, if said at least one of said amino acid building blocks comprises one or more Arg residues with unprotected side-chain and one or more His residues with unprotected side-chain, said at least one coupling cycle comprises the steps of:(a) contacting said amino acid building block comprising one or more Arg residues with unprotected sidechain and one or more His residues with unprotected side-chain with a coupling additive selected from the group consisting of the following compounds:Ri being CH or N,R2being H, Cl, COCH3, CH2COCH3 or CH2OCH3,R3 being CF3, Cl or NO2, where the ratio between said coupling additive and all the Arg residues with unprotected side-chain of said amino acid building block is at least 1.5:1.0 equivalents;(b) contacting the product obtained in step (a) with said coupling reagent selected from the group consisting of N-tert-Butyl-N'-ethylcarbodiimide and N-tert-Butyl-N'-isopropylcarbodiimide, preferably N-tert-Butyl-N'- ethylcarbodiimide;(c) coupling the product obtained in step (b) with said amino acid chain at the unprotected amino group of said amino acid chain, so that an amide bond is formed between said amino acid chain and said amino acid building block.

7. Method according to claim 6, wherein said coupling additive is selected from the group consisting of compounds (I), (III) with Ri being CH, (IV) with R2being H, (VII), (IX), (X) and (XI), preferably from the group consisting of compounds (I), (VII), (IX), and (X), more preferably the coupling additive is compound (VII).

8. Method according to claim 6 or claim 7, wherein said at least one coupling cycle comprises the following steps after said step (c):(d) deprotecting the N-terminal amino group of the amino acid building block; and(e) treating one or more times with a solvent, wherein at least one of the treatments is carried out with a mixture of said solvent and said coupling additive.

9. Method according to claim 8, wherein in step (e) at least the last treatment is carried out with a mixture of said solvent and said coupling additive, preferably the last and the second to last treatments are carried out with a mixture of said solvent and said coupling additive.

10. Method according to claim 8 or claim 9, wherein in step (e) the ratio between said coupling additive in the mixture and all the Arg residues with unprotected side-chain present in said amino acid chain is at least 1.0:1.0 equivalents, preferably at least 1.1:1.0, more preferably at least 1.3:1.0, even more preferably at least 1.5:1.0.

11. Method according to any of claims 8 to 10, wherein one or more coupling cycles occurring after said coupling cycle comprising said steps from (a) to (e) comprise treating with a mixture of said solvent and said coupling additive, preferably wherein the ratio between said coupling additive in said mixture and all the Arg residues with unprotected side-chain present in said amino acid chain is at least 1.0:1.0 equivalents, more preferably at least 1.3:1.0, even more preferably at least 1.5:1.0.

12. Method according to claim 11, wherein all the coupling cycles except the last one occurring after said coupling cycle comprising said steps from (a) to (e) comprise treating with a mixture of said solvent and said coupling additive, preferably wherein the ratio between said coupling additive in said mixture and all the Arg residues with unprotected side-chain present in said amino acid chain is at least 1.0:1.0 equivalents, more preferably at least 1.3:1.0, even more preferably at least 1.5:1.0.

13. Method according to any of claims 1 to 12, wherein if said at least one of said amino acid building block comprises one or more His residues with unprotected side-chain and said amino acid chain comprises one or more Arg residues with unprotected side-chain, said amino acid chain is treated one or more times before being used in said coupling cycle with a mixture of solvent and a coupling additive selected from the group consisting of:Ri being CH or N,R2being H, Cl, COCH3, CH2COCH3 or CH2OCH3, R3 being CF3, Cl or NO2.

14. Method according to claim 13, wherein the ratio between said coupling additive in the mixture and all the Arg residues with unprotected side-chain present in said amino acid chain is at least 1.0:1.0 equivalents, preferably at least 1.1:1.0, more preferably at least 1.3:1.0, even more preferably at least 1.5:1.0.

15. Method according to any of claims 1 to 7, wherein, if said at least one of said amino acid building blocks comprises one or more Arg residues with unprotected side-chain and one or more His residues with unprotected side-chain and / or at least one of said amino acid building block comprises one or more His residues with unprotected side-chain and said amino acid chain comprises one or more Arg residues with unprotected side-chain, in all the coupling cycles following said at least one coupling cycle the ratio between said coupling additive and all the Arg residues with unprotected side-chain of said amino acid building block and of said amino acid chain is at least 1.5:1.0 equivalents, preferably at least 2.0:1.0 equivalents, more preferably at least 2.2:1.0 equivalents.

16. Method according to any of claims 1 to 15, wherein the peptide is selected from the group consisting of glucagon, abaloparatide, semaglutide, salmon calcitonin, teriparatide, teduglutide, liraglutide, and their pharmaceutically acceptable salts.

17. Use of a coupling reagent selected from the group consisting of N-tert-Butyl-N'-ethylcarbodiimide and N- tert-Butyl-N'-isopropylcarbodiimide, preferably N-tert-Butyl-N'-ethylcarbodiimide, in a coupling reaction of an amino acid building block to an amino acid chain, wherein:- said amino acid building block comprises:- an unprotected C-terminal carboxyl group,- a protected N-terminal amino group,- one or more His residues with unprotected side-chain, and- said amino acid chain comprises an unprotected amino group.