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WO-2026093074-A1 - NEW PROTECTING GROUPS AS ANCHORS FOR LIQUID PHASE PEPTIDE SYNTHESIS

WO2026093074A1WO 2026093074 A1WO2026093074 A1WO 2026093074A1WO-2026093074-A1

Abstract

Object of the present invention are compounds of formula (1) as protecting groups to perform liquid phase peptide synthesis and a versatile, sustainable and cost-effective industrial process for liquid phase peptide synthesis.

Inventors

  • PAIO, ALFREDO
  • Nalin, Arnaldo
  • ROSSI, Emiliano
  • SACCO, Irene
  • MATTELLONE, Alexia
  • FEDERICO, Di Pietro
  • PROFETA, ROBERTO
  • MARCONI, GUIDO
  • MILLI, Lorenzo
  • BUZZETTI, Luca
  • GIRARDI, Giorgia
  • BAX, Pietro
  • TOSATO, FEDERICO

Assignees

  • F.I.S. FABBRICA ITALIANA SINTETICI S.P.A.

Dates

Publication Date
20260507
Application Date
20251021
Priority Date
20241029

Claims (12)

  1. 1) Use of a compound of formula (1): wherein nl, n2 and n3 are independently 0 or 1 and wherein X is OH, NH 2 or halogen, in liquid phase peptide synthesis.
  2. 2) Use of a compound of formula (1) according to claim 1, wherein nl, n2 and n3 are 0, or wherein nl and n3 are 0 and n2 is 1, or wherein nl and n2 are 0 and n3 is 1, or wherein nl and n2 are 1 and n3 is 0, or wherein n2 and n3 are 1 and nl is 0, or wherein nl, n2 and n3 are 1.
  3. 3) Use of a compound of formula (1) according to claim 1 or claim 2, wherein X is Br or NH 2 .
  4. 4) Use of a compound of formula (1) according to any one of the claims from 1 to 3, selected among the following compounds:
  5. 5) A method for protecting an amino acid or an amino acid derivative with a compound of formula (1) according to any one of the claims from 1 to 4, comprising the following steps: a) reacting the amino acid or amino acid derivative having a free carboxylic functional group, a protected N-terminus amino functional group and a side chain optionally protected at its functional groups, with a compound of formula (1) according to the claims from 1 to 4, in the presence of: i) a base, when X is a halogen; or ii) a coupling agent and optionally an additive, when X is OH; or iii) a base, a coupling agent and/or an activating agent, when X is NH 2 ; b) isolate the amino acid or amino acid derivative protected with compound of formula (1) obtained in step a).
  6. 6) A method according to claim 5, wherein in step a) case i) the base is potassium carbonate, in step a) case ii) the coupling agent is a carbodiimide selected from the group EDO, DOC, DIG, DSBC, DTBC or TBEC and the optional additive is DMAP and wherein in step a) case iii) the base is DIPEA and the coupling agent and/or the activating agent is selected from the group EDO, DOC, HOBt, HOAt, HATU, HBTU, TBTU.
  7. 7) A method for performing solution-phase peptide synthesis comprising a compound of formula (1) according to any one of the claims from 1 to 4 as C-terminus protecting group, comprising the following steps: al) protecting the carboxylic functional group of an amino acid or amino acid derivative with a compound of formula (1) according to anyone of the claims from 1 to 4, according to the method of claim 5 or claim 6; bl) deprotecting the amino functional group of the protected amino acid or amino acid derivative obtained in step al); cl) purifying the deprotected amino acid or amino acid derivative obtained in step bl); dl) reacting the deprotected amino acid or amino acid derivative obtained in step cl) with another amino acid or amino acid derivative protected at the amine function, to obtain a peptide or a peptide derivative; el) optionally purifying the peptide or peptide derivative obtained in step dl); fl) repeating steps bl), cl) dl) and el) to add other amino acids or amino acid derivatives to the peptide chain, until the complete peptide chain is obtained; gl) isolating the protected peptide obtained in step fl); hl) removing the C-terminus protecting group and any other protecting groups from the chain to obtain the peptide.
  8. 8) A method according to claim 7, wherein the amino function of the amino acids or amino acid derivatives that are added to the peptide chain is protected with a Fmoc group.
  9. 9) A method according to any one of the claims 7 or 8, wherein the reaction in step dl) is carried out using TBTU as coupling agent and DIPEA as base in DCM as solvent.
  10. 10) A method according to any one of the claims from 7 to 9, wherein in step el) and/or in step gl) the purification and/or isolation is performed by dosing a solution of the peptide in a solvent selected from the group ethyl acetate, isopropyl acetate, acetonitrile, DCM or CPME into a solvent selected from the group n-hexane, n-heptane, diethyl ether, di-isopropyl ether, MTBE or MIBE, to obtain peptide precipitation.
  11. 11) A method according to any one of the claims from 7 to 10, further comprising a step hl) of regeneration of the C-terminus protecting group obtained after deprotection in step gl) into a compound of formula (1) according to any one of the claims from (1) to (4) to be employed in step al). 12) A compound of formula (1): (1), wherein nl, n2 and n3 are independently 0 or 1, X is OH, NH 2 or halogen, and wherein, when n2 is 1 and nl and n3 are 0, X is not OH. 13) A compound of formula (1) according to claim 12, wherein nl, n2 and n3 are 0, or wherein nl and n3 are 0 and n2 is 1, or wherein nl and n2 are 0 and n3 is 1, or wherein nl and n2 are 1 and n3 is 0, or wherein n2 and n3 are 1 and nl is 0, or wherein nl, n2 and n3 are 1. 14) A compound of formula (1) according to claim 12 or claim 13, wherein X is Br or NH 2 . 15) A compound of formula (1) according to any one of the claims from 12 to 14 selected among the following compounds: (12) (13) (36). 16) An amino acid or amino acid derivative protected with at least a compound of formula (1) according to any one of the claims from 1 to 4 as protecting group, or a peptide or a peptide derivative comprising at least a compound of formula (1) according to any one of the claims from 1 to 4 as protecting group. 17) A kit for peptide synthesis comprising at least an amino acid or amino acid derivative protected with a compound of formula (1) according to anyone of the claims from 1 to 4, and at least a peptide synthesis coupling agent. 18) A screening kit for peptide synthesis comprising at least two or a plurality of compound of formula (1) according to anyone of the claims from 1 to 4. 19) Use of compound of formula (1) according to claims from 1 to 4 for the preparation of the pentapeptide Thymopentin, having formula H-Arg-Lys- Asp-Val-Tyr-OH. 20) Use according to claim 19, wherein the compound of formula (1) has the formula of compound (8). 21) A method according to any one of the claims from 7 to 11, wherein in step el) and/or in step gl) the purification and/or isolation is performed by dosing a solution of the peptide in a solvent selected from the group ethyl acetate, isopropyl acetate, acetonitrile, DCM or CPME into n-heptane, to obtain peptide precipitation. 22) A method according to any one of the claims from 7 to 11, wherein in step el) and/or in step gl) the purification and/or isolation is performed by dosing a solution of the peptide in a solvent selected from the group ethyl acetate, isopropyl acetate, acetonitrile, DCM or CPME into MTBE, to obtain peptide precipitation. 23) A method according to any one of the claims from 7 to 11, wherein in step el) and/or in step gl) the purification and/or isolation is performed by dosing a solution of the peptide in DCM into a solvent selected from the group n-hexane, n-heptane, diethyl ether, di-isopropyl ether, MTBE or MIBE, to obtain peptide precipitation. 24) A method according to any one of the claims from 7 to 11, wherein in step el) and/or in step gl) the purification and/or isolation is performed by dosing a solution of the peptide in DCM into n-heptane or MTBE, to obtain peptide precipitation. 25) A method for performing liquid phase peptide synthesis, comprising the following steps: a2) protecting the carboxylic functional group of an amino acid or amino acid derivative with a tag, wherein said tag is a compound of formula (1) according to anyone of the claims from 1 to 4, and wherein said amino acid or amino acid derivative is protected at the amino functional group with a Fmoc protecting group, to obtain a tag-protected amino acid or amino acid derivative (anchoring step); b2) deprotecting the amino functional group of the protected amino acid or amino acid derivative obtained in step a2) with DBU in a solvent or solvent mixture comprising acetonitrile (Fmoc cleavage step); c2) adjusting the pH of the mixture obtained in step b2) to a pH comprised in the range from 7 to 1 (quenching of DBU); d2) proceeding with the following steps: a3) treating the mixture obtained in step c2) with n-heptane or limonene, separating the phases and discarding the n- heptane or limonene phase (DBF removal); b3) reacting the mixture obtained in step a3) with an amino acid or amino acid derivative protected with a Fmoc protecting group at the amine function, to obtain a peptide or a peptide derivative (coupling); c3) optionally treating the mixture containing the peptide or peptide derivative obtained in step b3) with propylamine (capping); d3) treating the mixture containing the peptide or peptide derivative obtained in step b3) or step c3) with Me-THF and an aqueous solution, separating the phases and discarding the aqueous phase (aqueous work up); e3) switching the solvent of the mixture containing the peptide or peptide derivative obtained in step d3) to a solvent mixture comprising acetonitrile (solvent switch); or, alternatively, with the following steps: a4) reacting the mixture obtained in step c2) with an amino acid or amino acid derivative protected with a Fmoc protecting group at the amine function, to obtain a peptide or a peptide derivative (coupling); b4) optionally treating the mixture containing the peptide or peptide derivative obtained in step a4) with propylamine (capping); c4) treating the mixture containing the peptide or peptide derivative obtained in step a4) or step b4) with n-heptane or limonene, separating the phases and discarding the n- heptane or limonene phase (DBF removal); d4) treating the mixture containing the peptide or peptide derivative obtained in step c4) with Me-THF and an aqueous solution, separating the phases and discarding the aqueous phase (aqueous work up); e4) switching the solvent of the mixture containing the peptide or peptide derivative obtained in step d4) to a solvent mixture comprising acetonitrile (solvent switch); or, alternatively, with the following steps: a5) reacting the mixture obtained in step c2) with an amino acid or amino acid derivative protected with a Fmoc protecting group at the amine function, to obtain a peptide or a peptide derivative (coupling); b5) precipitating the peptide or peptide derivative obtained in step a5), isolating the peptide or peptide derivative from the mixture and washing the resulting peptide or peptide derivative; e2) repeating the steps from b2) to d2) to add other amino acids or amino acid derivatives protected at the amino functional group with a Fmoc protecting group to the peptide or peptide derivative chain, until the complete peptide or peptide derivative chain is obtained; f2) removing the tag protecting group from the C-terminus of the peptide or peptide derivative obtained in step e2) (tag cleavage) and, optionally, any other protecting groups from the chain (side chain protecting group deprotection); g2) isolating the deprotected peptide or peptide derivative obtained in step f2). 26) A method according to claim 25, wherein in step a2) the tag is selected among the following compounds:
  12. (12) (13) (36). 27) A method according to claim 25 or claim 26, wherein in step a2) the tag is selected among the following compounds: (6) (8). 28) A method according to anyone of the claims from 25 to 27, wherein in step b2) the DBU is used between 0.3 equivalents and 1.0 equivalent with respect to the equivalents of the amino acid or amino acid derivative, at a temperature comprised in the range from -10° C to 25° C. 29) A method according to anyone of the claims from 25 to 28, wherein in step b2) the DBU is used as 1.0 equivalent with respect to the equivalents of the amino acid or amino acid derivative, at a temperature of -10° C. 30) A method according to anyone of the claims from 25 to 28, wherein in step b2) the DBU is used as 0.3 equivalents with respect to the equivalents of the amino acid or amino acid derivative, at a temperature of 25° C. 31) A method according to any one of the claims from 25 to 30, wherein in step c2) the pH is adjusted by adding to the mixture MSA or a solution of HCI in CPME. 32) A method according to any one of the claims from 25 to 31, wherein in step b3), a4) or a5), the reaction is carried out using TBTU as coupling agent and DIPEA as base in acetonitrile as solvent. 33) A method according to any one of the claims from 25 to 32, wherein in step b5) the precipitation of the peptide or peptide derivative is performed by dosing the mixture obtained in step a5) into a solvent selected from the group n-hexane, n-heptane, diethyl ether, di-isopropyl ether, di-phenyl ether, MTBE or MIBE, to obtain peptide precipitation. 34) A method according to any one of the claims from 25 to 33, wherein in step b5) the precipitation of the peptide or peptide derivative is performed by dosing the mixture obtained in step a5) into di-isopropyl ether, to obtain peptide precipitation. 35) A method according to any one of the claims from 25 to 32, wherein in step b5) the precipitation of the peptide or peptide derivative is performed by cooling the mixture to a temperature comprised in the range from -15° C to 15° C. 36) A method according to any one of the claims from 25 to 35, wherein in step f2) the cleavage of the tag is carried out by means of catalytic hydrogenation or by hydrolysis. 37) A method according to any one of the claims from 25 to 36, further comprising a step h2) wherein the compound of formula (1) used as tag in step a2) is recovered starting from the mixture of step f2) or step g2) to be recycled in the same method. 38) Use of a compound of formula (1) according to anyone of the claims from 1 to 4 in liquid phase peptide synthesis, according to a method according to any one of the claims from 25 to 37. 39) Use according to claim 38, wherein the compound of formula (1) is selected among the following compounds:

Description

Description New protecting groups as anchors for liquid phase peptide synthesis. Technical Field [001] The present invention refers to protecting groups for liquid phase peptide synthesis. Background Art [002] Peptide chemistry has experienced a significant grown in recent decades mainly due to the employment of peptides as therapeutics. Since the introduction of insulin almost a century ago, in fact, a large number of peptide drugs have reached the market for a wide range of diseases, including diabetes, cardiovascular diseases, gastrointestinal diseases, cancer, infectious diseases, vaccine development, HIV infection and chronic pain. [003] Therapeutic peptides are composed of a series of well-ordered amino acids, usually with molecular weights of 500-5000 Da. The high demand for this class of pharmaceutical agents has prompted great advances in peptide synthesis development. [004] The first approach used to prepare peptides is known as classical solution peptide synthesis (CSPS). This strategy, however, is tedious, highly timeconsuming and requires a great human effort. In practice, this technique is almost inapplicable for the preparation of medium-sized and long peptides. [005] A second approach, referred to as solid-phase peptide synthesis (SPPS), greatly simplified the workup and removed the need to purify intermediates (J. Am. Chem. Soc., 1963, 85, 2149-2154). This method shows poorer performance with respect to the classical solution peptide synthesis and requires the use of a large excess of solvents and reagents to compensate the poorer solubility and reactivity of the compound attached to the insoluble resin. Despite these disadvantages, however, SPPS significantly shorten the time required for the peptide preparation and can easily be scaled up. Some limits arise when SPPS is used to produce long peptides (>20 amino acids), as this strategy often results in the accumulation of undesired byproducts and erroneous sequences that need to be removed through pricey chromatographic methods. [006] For these reasons, a third synthetic approach was developed, where the best attributes of SPPS and CSPS were combined. In this third approach, called liquid-phase peptide synthesis (LPPS), all the reactions are carried out in solution like in CSPS, allowing to reduce the excess of reagents and the huge amount of solvent required by SPPS. In LPPS the growing peptide chain is supported on a soluble tag or anchor, which confers the chain special properties that facilitate solution chemistry, while, at the same time, greatly simplifying the separation of peptide from excess reagents and byproducts, which is the advantage of SPPS chemistry. In practice, LPPS consists of the elongation of the peptide chain in solution thanks to a peculiar protecting group attached to one of its terminus that, after coupling reaction, is also the driver for the purification of each intermediate before the following amino acid addition. These tags/anchors attached to the peptide chain terminus, in fact, act as the polystyrene-based solid supports employed in standard SPPS, thus simplify the workup after each step of the synthesis. At the same time, however, maintain the peptide in solution and therefore realize the conditions of CSPS. At the end of the chain elongation, the tag/anchor is removed as any other protecting group used in peptide synthesis. [007] LPPS potential for scalability as well as its reduced use of reagents and solvents makes this technique more sustainable and environmentally friendly than any other chemical method developed up to date. [008] In order to be suitable as tags/anchors for LPPS, these peculiar protecting groups need to show simultaneously the property of easily dissolving in aqueous or organic solvents and a feature capable of significantly distinct the peptide from the other reagents and byproducts found in the mixture, so to simplify their removal by precipitation, filtration, or extraction at the end of each step. [009] It follows that the selection of adequate protecting groups suitable to be employed as tags/anchors is the primary criterion for a successful and effective LPPS strategy. [010] The first protecting groups used as LPPS tag were soluble polyethylene glycol polymers (PEGs). The application of these compounds for the preparation of peptides is reported for example in J. Am. Chem. Soc. 1974, 96, 7333-7336 or in J. Org. Chem. 1980, 45, 5364-5370. [011] Later, the so-called Membrane-enhanced peptide synthesis (MEPS) was developed as a variant of PEG tag-based technology, where the reagents are removed by organic solvent nanofiltration (Org. Process Res. Dev. 2010, 14, 1313-1325 and Macromolecules, 2017, 50, 1626-1634). [012] Another kind of protecting group developed for LPPS includes Perfluoroalkyl (fluorous) tags, which are simultaneously fluorophilic, lipophobic, and hydrophobic. This technology is described, for example, in Tetrahedron Letters, 2002, 43, 7809-7812, in Tetrahedron