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 Arginine residues with unprotected side-chain, and wherein at least one coupling cycle comprises the steps of: (a) contacting said amino acid building block comprising one or more Arginine residues with unprotected side-chain with a suitable coupling additive, where the ratio between said coupling additive and all the Arginine 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 a coupling reagent, (c) coupling the product obtained in step (b) at the unprotected amino group of said amino acid chain.

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 undesired modifications. As examples of undesired modification, the guanidine group of arginine (Arg) can be acylated during coupling reactions, and the imidazolyl group of histidine (His) may react with the electrophilic coupling reagent DIC (N,N'-Diisopropylcarbodiimide), both reactions result in the generation of 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 simultaneously with 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 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. A protective group that is difficult to remove may require harsh conditions such as a high amount of TFA, strong acids, high temperatures, prolonged time of treatment and / or the presence of carbocations to be removed.

[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 required, 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] Pbf is the most used protective group for Arg, even though it is very difficult to remove. It requires very high concentration of TFA and very long time of treatment. Removal of Pbf by TFA is a two step-reaction. The intermediate obtained after the first step is particularly difficult to be converted to the free Arg product, i.e. the product having Arg with unprotected side-chain. Very often it accompanies the final product as impurity, which is very difficult to be separated, thereby lowering the purity and the yield of the peptide.

[0016] For all these reasons, in the past scientists have tried to develop strategies for mild protection of basic sidechains of amino acids.

[0017] 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, Fmoc-Arg-OH was first preactivated with 1.5 eq DIC and 1.5 eq of Oxyma (AA / DIC / Oxyma 1 / 1 / 1 eq). Then, the activated amino acid was added to the substrate peptide resin to initiate the coupling.

[0018] The article clearly points out that subsequent addition of additional 1.5 eq of DIC is a necessary feature to result in high yield.

[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 Arg 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 arginine 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, and wherein at least one of said amino acid building blocks comprises one or more Arg residues with unprotected side-chain, and wherein at least one coupling cycle comprises the steps of:

[0029] (a) contacting said amino acid building block comprising one or more Arg residues with unprotected sidechain with a coupling additive selected from the group consisting of the following compounds:

[0030] Ri being CH or N,

[0031] R2being H, Cl, COCH3, CH2COCH3, or CH2OCH3,

[0032] R3being 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 equivalents;

[0033] (b) contacting the product obtained in step (a) with a coupling reagent,

[0034] (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.

[0035] Advantageously, this method does not require multiple additions of the coupling reagent to obtain the product with high yield.

[0036] The yield is also improved because the use of a covalently bound protective group for Arg, which can be difficult to be removed and results in impurities, is not required.

[0037] A further advantage is that the yield is improved without extending the reaction time when compared with at least some of the already known processes.

[0038] Also, a certain value of yield is reached in a shorter time when compared with at least some of the already known processes.

[0039] In addition, this method affords to increase the overall quality of the peptide, in terms of number and amount of impurities.

[0040] 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.

[0041] In addition, the overall quality of the peptide is also increased because there is no generation of impurities due to the reattachment of the protective groups of Arg residues or to protected groups of Arg residues not completely removed, as in the case of the use of Pbf. Further, the use of some chemicals, in some cases the less environmentally friendly chemicals, can be avoided or their amount can be reduced.

[0042] 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 Arg residues, such as Pbf.

[0043] In a further aspect, the present disclosure relates to the use of a coupling additive selected from the group consisting of the following compounds:

[0044] Ri being CH or N,

[0045] R2being H, Cl, COCH3, CH2COCH3, or CH2OCH3,

[0046] R3being CF3, Cl, or NO2, in a coupling reaction of an amino acid building block to an amino acid chain, wherein

[0047] - said amino acid building block comprise:

[0048] - an unprotected C-terminal carboxyl group,

[0049] - a protected N-terminal amino group,

[0050] - one or more Arg residues with unprotected side-chain, and

[0051] - said amino acid chain comprises an unprotected amino group, and 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Some features and advantages of the present invention will become apparent from the accompanying drawings, in which:

[0053] Figure 1 shows the HPLC-MS profile of etelcalcetide fragment of Example 1 (El), obtained with the method according to the present disclosure;

[0054] Figure 2 shows the HPLC-MS profile of etelcalcetide fragment of Comparative Example 1 (CE1), obtained with the method of the prior art;

[0055] Figure 3 shows the HPLC-MS profile of icatibant product of Example 2 (E2), obtained with the method according to the present disclosure;

[0056] Figure 4 shows the HPLC-MS profile of icatibant product of Comparative Example 2 (CE2), obtained with the method of the prior art;

[0057] Figure 5 shows the HPLC-MS profile of linear vasopressin fragment of Example 3 (E3), obtained with the method according to the present disclosure; and

[0058] Figure 6 shows the HPLC-MS profile of the peptide of Example 4 (E4), prepared according to the present disclosure.

[0059] DETAILED DESCRIPTION OF THE INVENTION

[0060] The method for the solid-phase synthesis of a peptide comprising a predetermined amino acid sequence according to the present invention comprises coupling cycles of amino acid building blocks to an amino acid chain, wherein

[0061] - said amino acid building blocks comprise:

[0062] - an unprotected C-terminal carboxyl group and

[0063] - a protected N-terminal amino group, and

[0064] - said amino acid chain comprises an unprotected amino group, and wherein at least one of said amino acid building blocks comprises one or more Arg residues with unprotected side-chain, and wherein at least one coupling cycle comprises the steps of:

[0065] (a) contacting said amino acid building block comprising one or more of Arg residues with unprotected sidechain with a coupling additive selected from the group consisting of the following compounds: (V) (VI) (VII) (VIII)

[0066] Ri being CH or N,

[0067] R2being H, Cl, COCH3, CH2COCH3, or CH2OCH3,

[0068] R3being 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;

[0069] (b) contacting the product obtained in step (a) with a coupling reagent,

[0070] (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.

[0071] 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-CI-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.

[0072] The terms "Oxyma" and "Oxyma Pure®" are used interchangeably in the present disclosure.

[0073] 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.

[0074] 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 residues, polypeptides. Polypeptides of a 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] The word "deprotecting" means removing the temporarily attached chemical group from the protected functional group to restore the functional group (free functional group).

[0079] The wording "coupling additive" refers to an additive used in coupling reactions to reduce racemization.

[0080] 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.

[0081] Advantageously, this method allows coupling one or more amino acids building blocks containing one or more Arg residues to an amino acid chain without the need of protective groups covalently bound to the side-chain of Arg residues.

[0082] Further, this method allows obtaining good yields without the need of multiple additions of coupling reagent, or prolonged treatment to deprotect the Arg residues side-chain.

[0083] In addition, this method allows obtaining the peptide with high purity with respect to the one obtaining Pbf as protecting group for Arg residues.

[0084] In addition, no large amounts of chemicals are needed to detach the peptide from the solid support and, at the same time, remove the side-chain protective groups.

[0085] Further, there is no need to use carbocation scavengers for Arg protective groups, thus avoiding any secondary reaction associated to these chemicals. It is well known in the art that, when side-chain of Arg is protected with Pbf, DTT is a very effective carbocation scavenger that can be used during deprotection; anyway its use in TFA cleavage solutions has been limited. One reason is that DTT reacts with TFA to form a compound that can hinder successful HPLC purification of the peptide.

[0086] 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.

[0087] Further, this method is compatible with other mild protection strategies of basic side-chains, such the one of histidine.

[0088] 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.

[0089] 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.

[0090] The result of this interaction is that during coupling of the amino acid building block with the amino acid chain the basic side-chain does not react.

[0091] 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. If, as an example, the amino acid building block is Fmoc-Arg(HCI)-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.

[0092] At the same time, since each equivalent of Arg residues with unprotected basic side-chain of the amino acid building block interacts with one equivalent of the coupling additive, an excess of coupling additive is required so as to have coupling additive available for the coupling reaction.

[0093] 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.

[0094] Preferably, each molecule of coupling additive interacts with a single arginine residue and each arginine residue interacts with a single molecule of coupling additive.

[0095] The wording "excess of coupling additive" refers to the ratio between said coupling additive and all the Arg residues with unprotected side-chain of said amino acid building block being at least 1.0:1.0 equivalents and in particular at least 1.5:1.0 equivalents, so as to have each lateral chain of Arg residues interacting with a molecule of coupling additive and remaining coupling additive available for the coupling reaction.

[0096] Step (a) is preferably carried out at a temperature of 0 °C to 35 °C, more preferably at a temperature of 5 °C to 30 °C, even more preferably at temperature of 8 °C to 25 °C.

[0097] Step (a) is preferably carried out for 2 to 20 minutes, more preferably for 5 to 15 minutes.

[0098] The coupling reagent can be selected from the group consisting of N,N'-diisopropylcarbodiimide (DIC), N,N'- Dicyclohexylcarbodiimide (DCC), N-tertButyl-N'-Ethylcarbodiimide (tBEC), N-tertButyl-N'- isoPropylcarbodiimide (tBiPC), l-ethyl-3-(3'-dimethyl-amino-propyl)carbodiimide hydrochloride (EDC.HCI), l,3-bis(2,2-dimethyl-l,3,dioxolan-4-ylmethyl)carbodiimide (BDDC), l-cyano-2-ethoxy-2- oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), and their mixtures, preferably from the group consisting of N,N'-diisopropylcarbodiimide and l-tert-butyl-3- ethylcarbodiimide, more preferably the coupling reagent is N,N'-diisopropylcarbodiimide.

[0099] The ratio between said coupling additive and all the Arg residues with unprotected side-chain of said amino acid building block is preferably at least 1.7:1.0 equivalents, more preferably at least 2.0:1.0 equivalents, even more preferably at least 2.2:1.0 equivalents.

[0100] This ratio results in a strong reduction of undesired side reactions, and a reduced racemization, whilst not impairing the yield of the coupling reaction .

[0101] The ratio between said coupling additive and all the Arg residues with unprotected side-chain of said amino acid building block is preferably not more than 10.0:1.0 equivalents, more preferably not more than 8.0:1.0 equivalents, even more preferably not more than 5.0:1.0 equivalents.

[0102] This makes it possible to improve the efficiency of the method by avoiding unnecessary use of coupling additive and makes the method more sustainable and cost-effective.

[0103] In some embodiments said amino acid building block comprises one or more of Arg(HCI) residues with unprotected side-chain, preferably one Arg(HCI) residue with unprotected side-chain.

[0104] In some preferred embodiments said amino acid building block is Fmoc-Arg(HCI)-OH.

[0105] Advantageously, the guanidino side-chain of Fmoc-Arg(HCI)-OH is protonated as HCI salt, diminishing its nucleophilicity and suppressing the undesired lactam formation during Fmoc-Arg(HCI)-OH coupling. Also, this amino acid building block is particularly suitable since being commercially available at lower price if compared with other Arg building blocks.

[0106] Further, the side-chain of Fmoc-Arg(HCI)-OH is less basic with respect to the one of Fmoc-Arg-OH.

[0107] Preferably, the coupling additive is selected from the group consisting of compounds (I), (III) with Ri being CH, (IV) with R2 being H, (VII), (IX), (X) and (XI), more preferably from the group consisting of compounds (I), (VII), (IX), and (X), even more preferably the coupling additive is compound (VII).

[0108] Conditions for steps (b) and (c) are known by the person skilled in the art.

[0109] In some embodiments, said at least one coupling cycle comprises the following steps after said step (c):

[0110] (d) deprotecting the N-terminal amino group of the amino acid building block; and

[0111] (e) treating one or more times with a solvent, wherein at least one of the treatments is carried out with a mixture of solvent and said coupling additive.

[0112] Conditions for step (d) are known by the person skilled in the art.

[0113] The solvent is used to remove unreacted reagents and byproducts.

[0114] Preferably, the solvent used in the treatments is DMF or a mixture of NBP / DMC 8:2 vol.

[0115] 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.

[0116] 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.

[0117] Advantageously, this avoids 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 an undesired coupling reaction at its unprotected basic side-chain during the subsequent coupling cycle.

[0118] Preferably, in step (e) the ratio between said coupling additive in said mixture and all the Arg residues with unprotected side-chain which are present in said amino acid chain is at least 1.0:1.0 equivalents, preferably at least 1.3:1.0 equivalents, more preferably at least 1.5:1.0 equivalents.

[0119] This allows to restore the interaction between the coupling additive and the basic side-chain of all the Arg residues of said amino acid chain.

[0120] Preferably, 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.

[0121] This guarantees that all the basic side-chain of all the Arg residues of said amino acid chain interact with the coupling additive and are thus protected against undesired side reaction during the subsequent coupling cycle.

[0122] Preferably, the solvent in step (e) is DMF.

[0123] In some embodiments, 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 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 1.3:1.0 equivalents, more preferably at least 1.5:1.0 equivalents.

[0124] 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 must be at least equal to the number of the arginine residues present in the amino acid building block.

[0125] In some preferred embodiments, all 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 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 1.3:1.0 equivalents, more preferably at least 1.5:1.0 equivalents.

[0126] In some alternative embodiments, all the coupling cycles following said at least one coupling cycle comprise using a coupling additive selected from the group consisting of the following compounds:

[0127] Ri being CH or N,

[0128] R2being H, Cl, COCH3, CH2COCH3, or CH2OCH3,

[0129] R3being CF3, Cl, or NO2, where the ratio between said coupling additive and all the Arg residues with unprotected side-chain of both said amino acid building block and 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. 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(s) of both said amino acid building block and 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.

[0130] This guarantees that not only the basic side-chain of all the Arg residues of said building block, but also that all the basic side-chain of all the Arg residues of said amino acid chain interact with the coupling additive and are thus protected against undesired side reaction during the subsequent coupling cycles.

[0131] In some embodiments, 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 said at least one of said amino acid building blocks comprises one or more Arg residues with unprotected sidechain and said amino acid chain comprises one or more His residues with unprotected side-chain, said at least one coupling cycle comprises the steps of:

[0132] (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:

[0133] Ri being CH or N,

[0134] R2being H, Cl, COCH3, CH2COCH3, or CH2OCH3,

[0135] R3being 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;

[0136] (b) contacting the product obtained in step (a) with a coupling reagent selected from the group consisting of N-tertButyl-N'-ethylcarbodiimide, N-tertButyl-N'-Ethylcarbodiimide, N-tertButyl-N'-isoPropylcarbodiimide, and their mixtures, preferably the coupling reagent is N-tertButyl-N'-Ethylcarbodiimide,

[0137] (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.

[0138] In case of amino acid building blocks comprising both one or more Arg residues with unprotected side-chain and one or more His residues with unprotected side-chain, scientists observed that the coupling additive is not effective in establishing an interaction in step (a) between the coupling additive itself and the unprotected basic side-chain of His residues, so that coupling additive does not protect the basic side-chain of His residue during coupling reaction of the amino acid building block with the amino acid chain.

[0139] As a consequence, unprotected imidazolyl group of His reacts e.g. with DIC, resulting in the formation of undesired by-products.

[0140] This side-reaction was also reported 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. In that case authors provided that PyBOP should be used as coupling reagent to avoid the side reaction between the imidazolyl group of His and DIC.

[0141] The authors also observed that PyBOP is uncapable of driving the Fmoc-Arg(HCI)-OH coupling into completion and outlined the incompatibility of the use of PyBOP for His residues with unprotected side-chain and of DIC / Oxyma for Arg residues with unprotected side-chain in the case of peptides having both such amino acids.

[0142] Inventors now have found that, in the case of one amino acid building block having at the same time one or more His residues with unprotected side-chain and one or more Arg residues with unprotected side-chain, by using in step (a) a coupling reagent selected from the group consisting of the following compounds:

[0143] Ri being CH or N,

[0144] R2being H, Cl, COCH3, CH2COCH3, or CH2OCH3,

[0145] R3being 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; and in step (b) a coupling reagent selected from the group consisting of N-tertButyl-N'-Ethylcarbodiimide, N- tertButyl-N'-isoPropylcarbodiimide, and their mixtures, preferably the coupling reagent is N-tertButyl-N'- Ethylcarbodiimide, the coupling is successfully achieved.

[0146] Further, the product of the coupling reaction is obtained with high yield.

[0147] In addition, no impurities due to the reaction of the coupling reagent with the imidazolyl group of His residues are observed.

[0148] This is due to the steric hindrance of the coupling reagent, which thus is not able to react with the His residues unprotected side-chain.

[0149] Exactly the same approach can be used when the amino acid building block comprises one or more Arg residues with unprotected side-chain and the amino acid chain comprises one or more His residues with unprotected side-chain.

[0150] So, if amino acid building blocks have one or more Arg residues with unprotected side-chain and the amino acid chain comprises one or more His residues with unprotected side-chain, by using in step (a) a coupling reagent selected from the group consisting of the following compounds:

[0151] Ri being CH or N,

[0152] R2being H, Cl, COCH3, CH2COCH3, or CH2OCH3,

[0153] R3being 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; and in step (b) a coupling reagent selected from the group consisting of N-tertButyl-N'-Ethylcarbodiimide, N- tertButyl-N'-isoPropylcarbodiimide, and their mixtures, preferably the coupling reagent is N-tertButyl-N'- Ethylcarbodiimide, the coupling is successfully achieved.

[0154] Also in this case, the coupling is achieved with high yield and without observing impurities due to the reaction of the coupling reagent with the imidazolyl group of His residues.

[0155] For these embodiments, preferably at least one coupling cycle after said at least one coupling cycle comprising steps (a)-(e) comprises the use of a coupling reagent selected from the group consisting of N- tertButyl-N'-Ethylcarbodiimide, N-tertButyl-N'-isoPropylcarbodiimide, and their mixtures, preferably the coupling reagent is N-tertButyl-N'-Ethylcarbodiimide.

[0156] More preferably, all the coupling cycles after said at least one coupling cycle comprising steps (a)-(e) comprise the use of a coupling reagent selected from the group consisting of N-tertButyl-N'-Ethylcarbodiimide, N- tertButyl-N'-isoPropylcarbodiimide, and their mixtures, preferably the coupling reagent is N-tertButyl-N'- Ethylcarbodiimide.

[0157] Preferably, the peptide according to the present invention is selected from the group consisting of glucagon, etelcalcetide, icatibant, vasopressin, abaloparatide, semaglutide, salmon calcitonin, teriparatide, teduglutide, liraglutide, and their pharmaceutically acceptable salts.

[0158] The coupling additive selected from the group consisting of the following compounds: (V) (VI) (VII) (VIII)

[0159] Ri being CH or N,

[0160] R2being H, Cl, COCH3, CH2COCH3, or CH2OCH3,

[0161] R3being CF3, Cl, or NO2, can be used in a coupling reaction of an amino acid building block to an amino acid chain, wherein

[0162] - said amino acid building block comprises:

[0163] - an unprotected C-terminal carboxyl group,

[0164] - a protected N-terminal amino group,

[0165] - one or more Arg residues with unprotected side-chain, and

[0166] - said amino acid chain comprises an unprotected amino group, and 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.

[0167] Advantageously, the use of said coupling additives allows avoiding the use of protective groups that are covalently bound to the side chain of said Arg residues or multiple additions of coupling reagent.

[0168] EXPERIMENTAL

[0169] The term "room temperature" refers to a temperature ranging from about 15 °C to 35 °C.

[0170] Where not differently specified, all the operations are to be considered carried out at room temperature.

[0171] Example 1 - Etelcalcetide fragment synthesis

[0172] Etelcalcetide is a peptide having the following amino acid sequence:

[0173] Etelcalcetide contains 4 Arg residues in its sequence.

[0174] In Example 1 (El), etelcalcetide fragment DAIa-Darg-Darg-Darg-DAIa-DArg-NH2was synthesized according to the invention.

[0175] DArg and D-Arg mean the D-isomer of arginine.

[0176] Synthesis of etelcalcetide fragment was carried out by SPPS on rink amide MBHA resin. 6.8 gr of peptidyl resin Fmoc-DAIa-DArg(Pbf)-rink amide MBHA (100-200 mesh) corresponding to 5 g of starting resin with loading 0.65 mmol / g were swelled in DMF.

[0177] Fmoc deprotection was carried out with 2 cycles of 10 minutes each with 20% Piperidine:DMF followed by 2 treatments with DMF and 2 treatments with 0.5M Oxyma in DMF.

[0178] Fmoc-DArg(HCI)-OH (2.81g; 2eq) was suspended in 25 mL of DMF. Oxyma (2.3 g; 5 eq) was added to the solution.

[0179] Pre-activation was carried out with DIC (1.0 mL; 2 eq) for 5 minutes at room temperature. The coupling solution was then added to the resin and allowed to react for 2.5 h.

[0180] The solution was filtered away and the resin was washed 3 times with DMF.

[0181] Fmoc deprotection was carried out with 2 cycles of 10 minutes with 20% Piperidine / DMF followed by 2 treatments with DMF and 2 treatments with 0.5M Oxyma in DMF.

[0182] Fmoc-DArg(HCI)-OH (2.81g; 2 eq) was suspended in 25 mL of DMF. Oxyma (2.3 g; 5 eq) was then added to the solution.

[0183] Pre-activation was carried out with DIC (1.0 mL; 2 eq) for 5 minutes. The coupling solution was added to the resin and allowed to react for 2.5 h.

[0184] The solution was filtered away and the resin was washed 3 times with DMF.

[0185] Fmoc deprotection was carried out with 2 cycles of 10 minutes with 20% Piperidine:DMF followed by 2 treatments with DMF and 2 treatments with 0.5M Oxyma in DMF.

[0186] Fmoc-DArg(HCI)-OH (2.81g; 2 eq) was suspended in 25 mL of DMF. Oxyma (2.3 g; 5 eq) was added to the solution.

[0187] Pre-activation was carried out with DIC (1.0 mL; 2 eq) for 5 minutes. The coupling solution was added to the resin and allowed to react for 2.5 h.

[0188] The solution was then filtered away and the resin was washed 3 times with DMF.

[0189] Fmoc deprotection was carried out with 2 cycles of 10 minutes with 20% Piperidine:DMF followed by 2 treatments with DMF and 2 treatments with 0.5M Oxyma in DMF.

[0190] Fmoc-DAIa-OH (2.02g; 2 eq) was suspended in 25 mL of DMF. Oxyma (0.92 g; 2 eq) was added to the solution.

[0191] Pre-activation was carried out with DIC (1.0 mL; 2 eq) for 5 minutes. The coupling solution was added to the resin and allowed to react for 2.5 h.

[0192] The solution was filtered away and the resin was washed 3 times with DMF.

[0193] The peptide DAIa-Darg-Darg-Darg-DAIa-DArg-NH2was cleaved from the resin using mix solution TFA:TIS:H2O (90:5:5) for 4 h and the crude was precipitated in diisopropyl ether.

[0194] Comparative Example 1 - Etelcalcetide fragment synthesis

[0195] In Comparative Example 2 (CE1), etelcalcetide fragment DAIa-DArg-DArg-Darg-DAIa-DArg-NF was synthesized according to Org. Process Res. Dev, 2022, 26, 1520-1530.

[0196] Synthesis of etelcalcetide fragment was carried out by SPPS on rink amide MBHA resin. 6.8 gr of peptidyl resin Fmoc-DAIa-Arg(Pbf)-rink amide MBHA (100-200 mesh) corresponding to 5 g of starting resin with loading 0.65 mmol / g were swelled in DMF.

[0197] Fmoc deprotection was carried out with 2 cycles of 10 minutes with 20% Piperidine:DMF followed by 4 treatments with DMF.

[0198] Fmoc-DArg(HCI)-OH (2.81g; 2 eq) was suspended in 25 mL of DMF and Oxyma (0.92 g; 2 eq) was added to the solution.

[0199] Pre-activation was carried out with DIC (1.0 mL; 2 eq) for 5 minutes. The coupling solution was added to the resin and stirred at room temperature. After 1.5 h DIC (1.0 mL; 2 eq) was added in the reactor and allowed to react for lh.

[0200] The solution was filtered away and the resin was washed 3 times with DMF.

[0201] Fmoc deprotection was carried out with 2 cycles of 10 minutes with 20% Piperidine:DMF followed by 4 treatments with DMF.

[0202] Fmoc-DArg(HCI)-OH (2.81g; 2 eq) was suspended in 25 mL of DMF and Oxyma (0.92 g; 2 eq) was added to the solution.

[0203] Pre-activation was carried out with DIC (1.0 mL; 2 eq) for 5 minutes. The coupling solution was added to the resin and stirred at room temperature. After 1.5 h DIC (1.0 mL; 2 eq) was added in the reactor and allowed to react for 1 h.

[0204] The solution was filtered away and the resin was washed 3 times with DMF.

[0205] Fmoc deprotection was carried out with 2 cycles of 10 minutes with 20% Piperidine:DMF followed by 4 treatments with DMF.

[0206] Fmoc-DArg(HCI)-OH (2.81g; 2 eq) was suspended in 25 mL of DMF and Oxyma (0.92 g; 2 eq) was added to the solution.

[0207] Pre-activation was carried out with DIC (1.0 mL; 2 eq) for 5 minutes. The coupling solution was added to the resin and stirred at room temperature. After 1.5 h DIC (1.0 mL; 2 eq) was added in the reactor and allowed to react for 1 h.

[0208] The solution was filtered away and the resin was washed 3 times with DMF.

[0209] Fmoc deprotection was carried out with 2 cycles of 10 minutes with 20% Piperidine:DMF followed by 4 treatments with DMF.

[0210] Fmoc-DAIa-OH (2.02 g; 2 eq) was suspended in 25 mL of DMF and Oxyma (0.92 g; 2 eq) was added to the solution.

[0211] Pre-activation was carried out with DIC (1.0 mL; 2 eq) for 5 minutes. The coupling solution was added to the resin and stirred at room temperature. After 1.5 h DIC (1.0 mL; 2eq) was added in the reactor and allowed to react for 1 h.

[0212] The solution was filtered away and the resin was washed 3 times with DMF.

[0213] The peptide DAIa-Darg-Darg-Darg-DAIa-DArg-NH2was then cleaved from the resin using a mix solution TFA:TIS:H2O (90:5:5) for 4 h and the crude was precipitated in diisopropyl ether.

[0214] In Figure 1 the HPLC chromatogram of Fmoc-A-R-R-R-A-R-NH2obtained according to Example 1 (El) and Comparative example 1 (CE1) is reported.

[0215] In Table 1 the assignment of the peaks is reported. Table 1

[0216] From the comparison between the two approaches, it clearly emerges from the data of Table 1 that higher yield and a lower amount of impurities are obtained for etelcalcetide with the approach of the present invention (El) in comparison with the approach disclosed in the reference Org. Process Res. Dev, 2022, 26, 1520-1530 (CE1).

[0217] In fact, the area of the target peptide is 73.87% for El while only 33.15% for CE1, demonstrating that, for the same reaction time, etelcalcetide (Fmoc-A-R-R-R-A-R-NH2) is obtained in higher quantity with the approach according to the present disclosure.

[0218] Further, for the same reaction time, a lower amount of impurities is obtained with the approach according to the present disclosure (26.14% vs. 66.85%, where these values are the sum, for each of El and CE1, of the Area% of Fmoc-A-R-R-R-A-NH2, Fmoc-A-R-R-A-R-NH2, Fmoc-A-R-R-A-NH2, Fmoc-A-R-A-R-NH2, Fmoc-A-R-A-NH2and Fmoc-A-A-R-NH2.

[0219] Example 2 - Icatibant synthesis

[0220] Icatibant is a peptide having the following amino acid sequence:

[0221] H-DArg-Arg-Pro-Hyp-Gly-2Thi-Ser-DTic-Oic-Arg-OH

[0222] In Example 2 (E2), icatibant was synthesized according to the invention.

[0223] The equivalent of 5 gr of Fmoc-Pro-Hyp(tBu)-Gly-Thi-Ser-D-Tic-Oic-Arg(Pbf)-2CTC resin with loading of 1 mmol / g were swelled with 50 mL of DMF for 20 minutes.

[0224] Fmoc-deprotection was carried out with two cycles of 10 minutes each with 20% Piperidine / DMF followed by 2 treatments with DMF and 2 treatments with 0.5M Oxyma in DMF.

[0225] Fmoc-Arg(HCI)-OH (2 eq; 4.3 gr) and Oxyma (5 eq; 3.55 gr) were dissolved in 30 mL DMF.

[0226] Pre-activation was carried out with DIC (2 eq; 1.55 mL) for 5 minutes at room temperature.

[0227] The coupling solution was added to the resin and allowed to react for 2.5 h at room temperature, followed by 3 treatments with 30 mL DMF.

[0228] Fmoc-deprotection was carried out with two cycles of 10 minutes with 10% Piperidine / DMF followed by 2 treatments with DMF and 2 treatments with 0.5M Oxyma in DMF.

[0229] Fmoc-D-Arg(HCI)-OH (2 eq; 4.3 gr) and Oxyma (5 eq; 3.55 gr) were dissolved in 30 mL DMF.

[0230] Pre-activation was carried out with DIC (2 eq; 1.55 mL) for 5 minutes. The coupling solution was added to the resin and allowed to react for 2.5 h at room temperature, followed by 3 treatments with 30 mL DMF.

[0231] Fmoc-deprotection was carried out with two cycles of 10 minutes with 20% Piperidine / DMF followed by 2 treatments with DMF and 2 treatments with 0.5M Oxyma in DMF.

[0232] Cleavage was carried out with 100 mL of cleavage mix TFA / TIS / DODT (80:10:10) for lh at 5 °C and 3 h at room temperature.

[0233] The resin was then filtered out and the solution was precipitated with 380 mL of DIPE.

[0234] The precipitate was filtered, washed 2 times with 50 mL DIPE and dried.

[0235] Comparative Example 2 - Icatibant synthesis

[0236] In Comparative Example 2 (CE2), icatibant was synthesized according to Org. Process Res. Dev, 2022, 26, 1520-1530.

[0237] The equivalent of 5 gr of Fmoc-Pro-Hyp(tBu)-Gly-2Thi-Ser-D-Tic-Oic-Arg-2CTC resin with loading of 1 mmol / g were swelled with 50 mL of DMF for 20 minutes.

[0238] Fmoc-deprotection was carried out with two cycles of 10 minutes with 1.5% and 11.5% Piperidine / DMF followed by 2 treatments with DMF and 2 treatments with 0.5M Oxyma in DMF.

[0239] Fmoc-Arg(HCI)-OH (2 eq; 4.33 gr) and Oxyma (2 eq; 1.42 gr) are dissolved in 30 mL DMF.

[0240] Pre-activation with DIC (2 eq; 1.55 mL) for 5 minutes.

[0241] The coupling solution was added to the resin and stirred at room temperature. After 1.5 h DIC (2 eq; 1.55 mL) was added in the reactor and allowed to react for lh (total reaction time 2.5 h), followed by 3 treatments with 30 mL DMF.

[0242] Fmoc-deprotection was carried out with two cycles of 10 minutes with 20% Piperidine / DMF followed by 4 treatments with DMF (30 mL).

[0243] Fmoc-D-Arg(HCI)-OH (2 eq; 4.33 gr) and Oxyma (2 eq; 1.42 gr) were dissolved in 30 mL DMF.

[0244] Pre-activation was carried out with DIC (2 eq; 1.55 mL) for 5 minutes.

[0245] The coupling solution was added to the resin and stirred at room temperature. After 1.5 h DIC (2 eq; 1.55 mL) was added in the reactor and allowed to react for lh (total reaction time 2.5 h), followed by 3 treatments with 30 mL DMF.

[0246] Fmoc-deprotection was carried out with two cycles of 10 minutes with 20% Piperidine / DMF followed by 4 treatments with DMF.

[0247] Cleavage was carried out with 100 mL of cleavage mixTFA / TIS / DODT (80:10:10) for lh at 5 °C and 3h at room temperature.

[0248] The resin was then filtered out and the solution was precipitated with 380 mL of DIPE.

[0249] The precipitate was filtered, washed 2 times with 50 mL DIPE and dried.

[0250] In Figure 2 the HPLC chromatogram of icatibant obtained according to Example 2 (E2) and Comparative example 2 (CE2) is reported.

[0251] In Table 2 the assignment of the peaks is reported. Table 2

[0252] The wording "Des-AA peptide" is commonly used in the field for describing the peptide where the amino acid "AA" was not incorporated. Thus, with "Icatibant Des-Arg" it is meant icatibant where the last Arg is missing and with "Icatibant Des-Arg-Arg" it is meant icatibant where the last two Arg are missing.

[0253] Also in this case, from the comparison between the two approaches, it clearly emerges from the data reported in Table 2, that higher yield and lower amount of impurities are obtained for icatibant synthesis with the approach of the present invention (E2) in comparison with the approach disclosed in the reference Org. Process Res. Dev, 2022, 26, 1520-1530 (CE2).

[0254] In fact, the area of the target peptide is 89.9% for E2 while only 27.7% for CE2, demonstrating that, for the same reaction time, icatibant is obtained in higher quantity with the approach according to the present disclosure.

[0255] Further, for the same reaction time, a lower amount of impurities is obtained with the approach according to the present disclosure (10.1% vs. 72.9%, where these values are the sum, for each of E2 and CE2, of the Area% of Icatibant Des-Arg and Icatibant Des-Arg-Arg.

[0256] Example 3 - Linear Vasopressin synthesis

[0257] Linear vasopressin is a peptide having the following amino acid sequence:

[0258] H-Cys-Tyr-Phe-GIn-Asn-Cys-Pro-Arg-Gly-NHz

[0259] In Example 1 (El), vasopressin was synthesized according to the invention.

[0260] 5 gr of rink amide MBHA resin with loading of 0.7 mmol / g were swelled with 50 mL of DMF.

[0261] Fmoc-deprotection was carried out with two cycles of 10 minutes with 10 % Piperidine / DMF followed by 4 treatments with DMF.

[0262] Fmoc-Gly-OH (2 eq; 2.1 gr) and Oxyma (2 eq; 1.0 gr) were dissolved in 30 mL DMF.

[0263] Pre-activation was carried out with DIC (2 eq; 1.1 mL) for 5 minutes.

[0264] The coupling solution was added to the resin and allowed to react for 90 minutes at room temperature, followed by 3 treatments with DMF.

[0265] Fmoc-deprotection was carried out with two cycles of 10 minutes with 10% Piperidine / DMF followed by 2 treatments with DMF and 2 treatments with 0.5M Oxyma in DMF.

[0266] Fmoc-Arg(HCI)-OH (2.5 eq; 3.8 gr) and Oxyma (7.5 eq; 3.7 gr) were dissolved in 30 mL DMF.

[0267] Pre-activation was carried out with with DIC (2.5 eq; 1.4 mL) for 5 minutes.

[0268] The coupling solution was added to the resin and allowed to react for 2.5 h at room temperature, followed by 3 treatments with 30 mL DMF. Fmoc-deprotection was carried out with two cycles of 10 minutes with 10% Piperidine / DMF followed by 2 treatments with DMF and 2 treatments with 0.5M Oxyma in DMF.

[0269] Fmoc-Pro-OH (2 eq; 2.4 gr) and Oxyma (2 eq; 1.0 gr) were dissolved in 30 mL DMF.

[0270] Pre-activation was carried out with DIC (2 eq; 1.1 mL) for 5 minutes.

[0271] The coupling solution was added to the resin and allowed to react for 1.5 h at room temperature, followed by 3 treatments with 30 mL DMF.

[0272] Fmoc-deprotection was carried out using two cycles of 10 minutes with 10% Piperidine / DMF followed by 2 treatments with DMF and 2 treatments with 0.5M Oxyma in DMF.

[0273] Fmoc-Cys(Trt)-OH (2 eq; 4.1 gr) and Oxyma (2 eq; 1.0 gr) were dissolved in 30 mL DMF.

[0274] Pre-activation was carried out with DIC (2 eq; 1.1 mL) for 5 minutes.

[0275] The coupling solution was added to the resin and allowed to react for 1.5 h at room temperature, followed by 3 treatments with 30 mL DMF.

[0276] Fmoc-deprotection was carried out with two cycles of 10 minutes with 10% Piperidine / DMF followed by 2 treatments with DMF and 2 treatments with 0.5M Oxyma in DMF.

[0277] Fmoc-Asn-OH (2 eq; 2.5 gr) and Oxyma (2 eq; 1.0 gr) were dissolved in 30 mL DMF.

[0278] Pre-activation was carried out with DIC (2 eq; 1.1 mL) for 5 minutes.

[0279] The coupling solution was added to the resin and allowed to react for 1.5 h at room temperature, followed by 3 treatments with 30 mL DMF.

[0280] Fmoc-deprotection was carried out with two cycles of 10 minutes with 10% Piperidine / DMF followed by 2 treatments with DMF and 2 treatments with 0.5M Oxyma in DMF.

[0281] Fmoc-GIn-OH (2 eq; 2.6 gr) and Oxyma (2 eq; 1.0 gr) were dissolved in 30 mL DMF.

[0282] Pre-activation was carried out with DIC (2 eq; 1.1 mL) for 5 minutes.

[0283] The coupling solution was added to the resin and allowed to react for 1.5 h at room temperature, followed by 3 treatments with 30 mL DMF.

[0284] Fmoc-deprotection was carried out with two cycles of 10 minutes with 10% Piperidine / DMF followed by 2 treatments with DMF and 2 treatments with 0.5M Oxyma in DMF.

[0285] Fmoc-Phe-OH (2 eq; 2.7 gr) and Oxyma (2 eq; 1.0 gr) were dissolved in 30 mL DMF.

[0286] Pre-activation was carried out with DIC (2 eq; 1.1 mL) for 5 minutes.

[0287] The coupling solution was added to the resin and allowed to react for 1.5 h at room temperature, followed by 3 treatments with 30 mL DMF.

[0288] Fmoc-deprotection was carried out with two cycles of 10 minutes with 10% Piperidine / DMF followed by 2 treatments with DMF and 2 treatments with 0.5M Oxyma in DMF.

[0289] Fmoc-Tyr-OH (2 eq; 2.8 gr) and Oxyma (2 eq; 1.0 gr) were dissolved in 30 mL DMF.

[0290] Pre-activation was carried out with DIC (2 eq; 1.1 mL) for 5 minutes.

[0291] The coupling solution was added to the resin and allowed to react for 1.5 h at room temperature, followed by 3 treatments with 30 mL DMF. Fmoc-deprotection was carried out with two cycles of 10 minutes with 10% Piperidine / DMF followed by 2 treatments with DMF and 2 treatments with 0.5M Oxyma in DMF.

[0292] Fmoc-Cys(Trt)-OH (2 eq; 4.1 gr) and Oxyma (2 eq; 1.0 gr) were dissolved in 30 mL DMF.

[0293] Pre-activation was carried out with DIC (2 eq; 1.1 mL) for 5 minutes.

[0294] The coupling solution was added to the resin and allowed to react for 1.5 h at room temperature, followed by 3 treatments with 30 mL DMF.

[0295] Fmoc-deprotection was carried out with two cycles of 10 minutes with 10% Piperidine / DMF followed by 2 treatments with DMF and 2 treatments with 0.5M Oxyma in DMF.

[0296] Cleavage was carried out for 2 h at room temperature with 75 mL of cleavage mix TFA / TIS / DTT (87:8:5) prepared at 10 °C.

[0297] The resin was then filtered out and the solution was precipitated with 120mL of DIPE.

[0298] The precipitate was filtered, washed 2 times with DIPE and dried.

[0299] In Figure 3 the HPLC chromatogram of linear vasopressin obtained according to Example 3 (E3) is reported. No deletion impurities were observed.

[0300] Example 4- Synthesis of heptapeptide Y-H-A-R-G-F-L

[0301] In Example 4 (E4), heptapeptide Tyr-His-Ala-Arg-Gly-Phe-Leu was synthesized according to the invention.

[0302] 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 NHj-Gly-Phe-Leu-MBH resin (loading 0.7 mmol g1). The resin was swelled in 1.5 mL of NBP / DMC 8:2.

[0303] Fmoc-Arg(HCI)-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(HCI)-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.

[0304] 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).

[0305] 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.

[0306] 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 HPLC-MS.

[0307] The HPLC-MS profile of Example 10 is reported in Figure 3.

[0308] The peak at 14.001 min is attributable to the target peptide. The small peak before the one of the target peptide is attributable to an impurity of the column.

[0309] No adduct due to the reaction of imidazolyl group of His residue with tBEC, which should have +126 mAU with respect to the target peptide, was observed.

[0310] No adduct due to the reaction of guanidine group of Arg residue was observed.

[0311] ABBREVIATIONS

[0312] AA amino acid

[0313] Adoc Adamantyloxycarbonyl

[0314] Arg Arginine

[0315] BDDC l,3-bis(2,2-dimethyl-l,3,dioxolan-4-ylmethyl)carbodiimide

[0316] Boc tert-Butyloxycarbonyl

[0317] COMU l-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate

[0318] DCC N,N'-Dicyclohexylcarbodiimide

[0319] DIVIC dimentylcarbonate

[0320] DTT Dithiothreitol

[0321] DIC N,N'-Diisopropylcarbodiimide

[0322] DMF N,N-dimethylformamide

[0323] EDC.HCI l-ethyl-3-(3'-dimethyl-amino-propyl)carbodiimide hydrochloride

[0324] Fmoc 9-Fluorenylmethoxycarbonyl

[0325] HOAt l-hydroxy-7-azabenzotriazole

[0326] HOBt N-hydroxybenzotriazole

[0327] HOCt ethyl l-hydroxy-lH-l,2,3-triazole-4-carboxylate

[0328] HODhbt 3-hydroxy-3,4-dihydro-4-oxo-l,2,3-benzotriazine

[0329] HONB N-hydroxy-5-norbornene-endo-2,3-dicarboxymide

[0330] HOPy 1-hydroxy -2-pyridione

[0331] HOSu N-hydroxysuccinimide

[0332] HOt N-hydroxytetrazole

[0333] HPLC High Performance Liquid Chromatography

[0334] NBP N-butylpirrolidone

[0335] Oxyma Pure®, Oxyma 2-cyano-2-(hydroxyimino)acetate

[0336] Oxyma-B® 5-(hydroxyimino)-l,3-dimethylpyrimidine-2,4,6(lH,3H,5H)-trione Pbf 2,2A6,7-pentamethyldihydrobenzofuran-5-sulfonyl

[0337] PyBOP (Benzotriazol-l-yloxy)tri(l-pyrrolidinyl)phosphonium hexafluorophosphate

[0338] SPPS Solid Phase Peptide Synthesis tBEC N-tertButyl-N'-Ethylcarbodiimide tBiPC N-tertButyl-N'-isoPropylcarbodiimide

[0339] Trt Trytil

[0340] TFA Trifluoroacetic acid

[0341] TIS Triisopropylsilane

[0342] 6-CF3-HOBt 6-trifluoromethyl-l-hydroxybenzotriazole

[0343] 6-CI-HOBt l-hydroxy-6-chlorobenzotriazole

[0344] 6-NO2-HOBt l-hydroxy-6-nitrobenzotriazole

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, and wherein at least one of said amino acid building blocks comprises one or more Arg residues with unprotected side-chain, and wherein 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 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 a coupling reagent,(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.

2. Method according to claim 1 wherein the coupling reagent is selected from the group consisting of N,N'- diisopropylcarbodiimide, N,N'-Dicyclohexylcarbodiimide, N-tertButyl-N'-Ethylcarbodiimide, N-tertButyl-N'- isoPropylcarbodiimide, l-ethyl-3-(3'-dimethyl-amino-propyl)carbodiimide hydrochloride, l,3-bis(2,2- dimethyl-l,3,dioxolan-4-ylmethyl)carbodiimide, l-cyano-2-ethoxy-2-oxoethylidenaminooxy) dimethylamino-morpholino-carbenium hexafluorophosphate, and their mixtures, preferably from the group consisting of N,N'-diisopropylcarbodiimide, N-tertButyl-N'-Ethylcarbodiimide, and N-tertButyl-N'- isoPropylcarbodiimide, more preferably from the group consisting of N,N'-diisopropylcarbodiimide and N- tertButyl-N'-Ethylcarbodiimide, even more preferably the coupling reagent is N,N'- diisopropylcarbodiimide.

3. Method according to claim 1 or claim 2 wherein 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.

4. Method according to any of claims 1 to 3 wherein the ratio between said coupling additive and all the Arg residues with unprotected side-chain of said amino acid building block is not more than 10.0:1.0 equivalents, preferably not more than 8.0:1.0 equivalents, more preferably not more than 5.0:1.0 equivalents.

5. Method according to claim 5 wherein said amino acid building block comprises one or more of Arg(HCl) residues with unprotected side-chain, preferably one Arg(HCl) residue with unprotected side-chain.

6. Method according to claim 6 wherein said amino acid building block is Fmoc-Arg(HCl)-OH.

7. Method according to any of claims 1 to 6 wherein the coupling additive is 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).

8. Method according to any of claims 1 to 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 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, preferably at least 1.1:1.0 equivalents, more preferably at least 1.3:1.0 equivalents, even more preferably at least 1.5:1.0 equivalents.

11. Method according to any of claims from 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 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 1.3:1.0 equivalents, more preferably at least 1.5:1.0 equivalents.

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 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 1.3:1.0, more preferably at least 1.5:1.0.

13. Method according to any of claims 1 to 7 , wherein all the coupling cycles following said at least one coupling cycle comprise using 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 both said amino acid building block and 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.

14. Method according to any of claims from 1 to 13, 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 said at least one of said amino acid building blocks comprises one or more Arg residues with unprotected side-chain and said amino acid chain comprises 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:Ri being CH or N,R2being H, Cl, COCH3, CH2COCH3, or CH2OCH3,R3 being CF3, Cl, or NO2,(b) contacting the product obtained in step (a) with a coupling reagent selected from the group consisting of N-tertButyl-N'-Ethylcarbodiimide, N-tertButyl-N'-isoPropylcarbodiimide, and their mixtures, preferably the coupling reagent is N-tertButyl-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.

15. Method according to claim 14, wherein at least one coupling cycles after said at least one coupling cycle comprising steps (a)-(e) comprises the use of a coupling reagent selected from the group consisting of N- tertButyl-N'-Ethylcarbodiimide, N-tertButyl-N'-isoButylcarbodiimide, and their mixtures.

16. Method according to claim 15, wherein all the coupling cycles after said at least one coupling cycle comprising steps (a)-(e) comprise the use of a coupling reagent selected from the group consisting of N- tertButyl-N'-Ethylcarbodiimide, N-tertButyl-N'-isoButylcarbodiimide, and their mixtures.

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

18. Use of a coupling additive selected from the group consisting ofRi being CH or N,R2being H, Cl, COCH3, CH2COCH3, or CH2OCH3,R3 being CF3, Cl, or NO2, 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 Arg residues with unprotected side-chain, and- said amino acid chain comprises an unprotected amino group, and 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.

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