[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO2019217116A1 - Méthode de synthèse en phase solution de peptides - Google Patents

Méthode de synthèse en phase solution de peptides Download PDF

Info

Publication number
WO2019217116A1
WO2019217116A1 PCT/US2019/029569 US2019029569W WO2019217116A1 WO 2019217116 A1 WO2019217116 A1 WO 2019217116A1 US 2019029569 W US2019029569 W US 2019029569W WO 2019217116 A1 WO2019217116 A1 WO 2019217116A1
Authority
WO
WIPO (PCT)
Prior art keywords
coupling
peptide
peptide synthesis
fmoc
solution
Prior art date
Application number
PCT/US2019/029569
Other languages
English (en)
Inventor
Cole SEIFERT
Original Assignee
Gap Peptides Llc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Gap Peptides Llc filed Critical Gap Peptides Llc
Publication of WO2019217116A1 publication Critical patent/WO2019217116A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups

Definitions

  • the present disclosure relates in general to the field of peptide synthesis.
  • the system provides for solution-phase peptide synthesis methods in organic solvents, some of which are immiscible with aqueous solutions, alkane solvents, or both, that allow for purification with minimal chromatography, recrystallization, or polymer supports, and allows for high overall yield and purity.
  • the disclosed systems and methods support a wide variety of scenarios and include various products and services.
  • SPPS is amenable to a wide range of protecting and coupling strategies, with the Fmoc//Bu strategy being among the most popular in the industry.
  • U.S. Patent No. 8,383,770 B2 teaches the use of the Fmoc and Boc N-terminus protecting groups in SPPS. Boc and Fmoc groups have been used for decades in all areas of peptide chemistry, and the preferred Fmoc group is almost entirely restricted to solid phase.
  • the key disadvantage of SPPS lies in the difficulty of scale-up: many polymer supports are expensive, and occupy the majority of the mass of the material to be worked with. Examples of economically feasible Fmoc protection schemes in solution are scarce, with few examples in the literature at all.
  • The‘891 patent teaches removal of this impurity by deprotecting with 4-aminomethylpiperidine (4AMP) instead of piperidine.
  • 4AMP 4-aminomethylpiperidine
  • This forms NFMP-CH2NH2 instead of NFMP, which due to the presence of the extra amino group, can be extracted into water.
  • the problem with this method lies in the high cost of using 4AMP.
  • Per Sigma Aldrich, 4AMP costs $3.80 per gram, while piperidine only costs $0.12 per gram. This is why this method is cost prohibitive, and why it has not been accepted by the industry.
  • Fmoc-based SolPPS can be seen in published patent application WO2017112809A1.
  • This patent teaches the use of a C-terminus group-assisted purification (GAP) protecting group to control the solubility of the target peptide to allow for selective precipitation after each successive coupling reaction.
  • GAP C-terminus group-assisted purification
  • this technology adapted Fmoc/tBu chemistry to solution-phase in a much more economically feasible manner, there are potential limitations inherent in the method. Namely, this method requires precipitation to remove the fulvene impurity (NFMP). While this method of removing NFMP is potentially more cost- effective than using 4AMP like in the‘891 patent, precipitations such as these can be problematic on a large scale, potentially limiting the overall scalability of the method.
  • the present disclosure addresses failings in the art by providing greener systems and methods for peptide synthesis utilizing reactions which occurs in solution phase, without the mass waste of polymer supports, but retains all of the purification benefits of SPPS as an alternative to both traditional SolPPS as well as SPPS, affording advantages of both methods.
  • a SolPPS strategy is presented that is economically feasible and useful for the commercial production of peptides.
  • Such green SolPPS may be achieved with a protection strategy that lends protected amino acids and potentially resulting peptides solubility in the reaction solvent and/or either aqueous solutions or alkane solvents.
  • a number of different protection strategies are possible, including, but not limited to, Fmoc//Bu, Boc/benzyl, Cbz, etc. Many of these strategies are currently used in both SPPS and SolPPS methods but are commonly restricted to toxic/non-green solvents in both phases.
  • a protecting group strategy such as Fmoc//Bu or GAP -PS as an example
  • Various amino acids protected in different ways have shown high solubility and coupling reaction efficiency in green solvents such as PC.
  • an adaptation of the group assisted purification peptide synthesis method to propylene carbonate is provided.
  • the GAP method allows for a high yield with high purity using the Fmoc//But strategy with solution-phase peptide synthesis (SolPPS).
  • SolPPS solution-phase peptide synthesis
  • the present invention enhances the scalability of the Group-Assisted Purification (GAP) method by adapting it to run in PC, avoiding the post coupling precipitation step and purifying the target peptide through a series of liquid-liquid extractions.
  • an adaptation of GAP peptide synthesis is presented wherein the chemistry is run in a DMF/DCM solvent mixture.
  • the DMF component of the solvent mixture prevents solubility in alkane solvents, while the DCM component of the mixture prevents solubility in aqueous systems. This allows for a similar tri-layer extraction method.
  • FIG. 1A depicts a prior art process of Solid Phase Peptide Synthesis (SPPS).
  • SPPS Solid Phase Peptide Synthesis
  • FIG. IB depicts a step of the GAP Peptide Synthesis (GAP -PS) process, specifically the use of a benzyl-type protecting group for C-terminus protection.
  • GAP -PS GAP Peptide Synthesis
  • FIG. 2 depicts a process for development of a protecting group utilized in FIG. IB.
  • FIG. 3 depicts a schematic for testing the orthogonality and GAP capability of the protecting group of FIG. 2.
  • FIGS. 4A-4B each depicts a schematic for the process of attaching the protecting group of FIG. 2 to various amino acids.
  • FIG. 5 depicts a schematic for the synthesis of a bivalirudin fragment using the protecting group of FIG. 2 for purposes of the exemplary non-limiting example of peptide synthesis of the present invention.
  • “or” if used to associate a list, such as A, B or C is intended to mean A, B, and C, here used in the inclusive sense, as well as A, B or C, here used in the exclusive sense.
  • the term“one or more” as used herein, depending at least in part upon context may be used to describe any feature, structure, or characteristic in a singular sense or may be used to describe combinations of features, structures or characteristics in a plural sense.
  • terms, such as“a,”“an,” or“the,” again, may be understood to convey a singular usage or to convey a plural usage, depending at least in part upon context.
  • the term“based on” may be understood as not necessarily intended to convey an exclusive set of factors and may, instead, allow for existence of additional factors not necessarily expressly described, again, depending at least in part on context.
  • SolPPS solution-phase peptide synthesis
  • the present invention discloses a method of peptide synthesis wherein the protecting strategy is selected from a group consisting of: Fmoc/tBu, Boc/benzyl, Cbz, GAP, Nvoc, Nitrobenzyl, or azide.
  • the present invention discloses a method of peptide synthesis wherein the coupling and deprotection reactions are performed such that the peptide is synthesized in the C to N direction.
  • the present invention discloses a method of peptide synthesis wherein the coupling and deprotection reactions are performed such that the peptide is synthesized in the N to C direction.
  • the present invention discloses a method of peptide synthesis wherein the peptide remains in the reaction solvent, and the aqueous and alkane solvent washes remove impurities generated during the coupling and deprotection reactions from the reaction solvent.
  • a novel SolPPS method begins with the choice of a green reaction solvent that is immiscible with both aqueous solutions and alkane solvents: for this example, propylene carbonate (PC).
  • PC propylene carbonate
  • This characteristic is vital to avoiding distillations and other purification strategies because it allows for a wider range of wash protocols to take advantage of different solubility properties of different impurities in the reaction mixture.
  • Fmoc-protected amino acids show a high solubility in PC with the addition of 1 eq. of mild organic base. Boc protected amino acids and deprotected amino acids show similar solubility as well.
  • Benzyl diphenylphosphine oxide (BnDppOH) as seen in the GAP -PS method also shows an affinity for the solvent, thereby providing a reaction solvent for attaching the GAP protecting group to an Fmoc-protected amino acid.
  • BnDppOH can be attached to the amino acid in PC via a number of different coupling reagents, including EDCI and TFFH. Subsequently, deprotection of the Fmoc group can be carried out with a myriad of deprotection reagents, including diethylamine, DBU, piperidine, and tertbutylamine, and additional coupling reactions can be performed.
  • quenching agents can be used to nullify excess activated amino acids and lend desired solubility characteristics to the resulting molecule.
  • decylamine could be used to quench activated amino acid and increase the resulting molecule’s solubility in alkane solvents.
  • deprotected excess amino acid is often rendered water- soluble and can be removed from the PC via aqueous washes.
  • fulvene impurities such as NFMP created by piperidine Fmoc deprotection, can be removed from the PC by alkane solvent washes. Boc deprotection, as well as /Bu type global deprotection, can be performed with TFA as well.
  • FIGS. 4A- 4B depict a schematic for the process of attaching the GAP protecting group of FIG. 2 to the amino acid leucine, and synthesis of the bivalirudin fragment is illustrated in FIG. 5.
  • Compound 6 is first treated with 30% tertbutylamine (TBA) in PC for 15 minutes, with 7 eq.
  • octane thiol added as a scavenging agent, to remove the Fmoc group, followed by multiple alkane solvent washes to remove the excess TBA, and then multiple (about three) saturated ammonium chloride washes to remove amino acid impurities while simultaneously concentrating the PC solution.
  • the next Fmoc amino acid (3 eq. relative to peptide) is dissolved in PC, 3 eq. of organic base such as DIPEA or 2,4,6-TMP is added, followed by 3 eq. of coupling reagent such as TFFH or TBTU to activate the Fmoc amino acid.
  • the activated amino acid solution is then added to the dried peptide solution in PC to effect the coupling.
  • reaction mixture is washed with a quenching agent, such as long-chain (C10-C18) aliphatic thiols, long-chain (C10-C18) aliphatic alcohols, long-chain (C 10-08) aliphatic amines, long-chain (C10-C18) aliphatic selenols, aliphatic polyamines, aliphatic polyalcohols, primary or secondary amines, or primary or secondary alcohols for an hour.
  • a quenching agent such as long-chain (C10-C18) aliphatic thiols, long-chain (C10-C18) aliphatic alcohols, long-chain (C 10-08) aliphatic amines, long-chain (C10-C18) aliphatic selenols, aliphatic polyamines, aliphatic polyalcohols, primary or secondary amines, or primary or secondary alcohols for an hour.
  • the reaction mixture is washed about
  • the crude product after coupling has a high degree of purity, with only trace amounts (if any) of impurities from the deprotection and coupling reactions. These same steps are repeated with different Fmoc amino acids to grow the peptide chain.
  • the novel SolPPS method can easily remove any impurities simply by washing the reaction mixture with combinations of aqueous solutions and alkane solvents. This allows for the elimination of the precipitation step from the traditional GAP -PS method. Additionally, because PC is somewhat soluble in aqueous solutions, the reaction mixture volume can be concentrated during the same liquid-liquid extraction protocols, completely avoiding evaporation, distillation, or other more arduous means of concentration.
  • FIGS. 4A-4B each depicts a schematic for the process of attaching the protecting group of FIG. 2 to various amino acids.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne d'une manière générale le domaine de la synthèse de peptides. En général, le système fournit des procédés de synthèse en phase solution de peptides dans des solvants organiques, dont certains sont non miscibles avec des solutions aqueuses, des solvants alcanes, ou les deux, qui permettent une purification avec une chromatographie, une recristallisation ou des supports polymères minimaux, et permet un rendement global élevé et une pureté élevée. Les systèmes et les procédés de l'invention prennent en charge une grande variété de scénarios et comprennent divers produits et services.
PCT/US2019/029569 2018-05-06 2019-04-29 Méthode de synthèse en phase solution de peptides WO2019217116A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201862667591P 2018-05-06 2018-05-06
US62/667,591 2018-05-06

Publications (1)

Publication Number Publication Date
WO2019217116A1 true WO2019217116A1 (fr) 2019-11-14

Family

ID=68467467

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2019/029569 WO2019217116A1 (fr) 2018-05-06 2019-04-29 Méthode de synthèse en phase solution de peptides

Country Status (1)

Country Link
WO (1) WO2019217116A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11827660B2 (en) 2019-02-01 2023-11-28 Sederma Synthesis strategy for gap protecting group
US12024537B2 (en) 2018-05-31 2024-07-02 Sederma Compositions and methods for chemical synthesis

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6753409B1 (en) * 1996-08-21 2004-06-22 Aventis Pharmaceuticals Inc. STABLE NON-HYGROSCOPIC CRYSTALLINE FORM OF N-[N-[N-(4-(PIPERIDIN-4-YL)BUTANOYL)-N-ETHYLGLYCYL]ASPARTYL]-L-β-CYCLOHEXYL ALANINE AMIDE, INTERMEDIATES THEREOF, AND PREPARATION THEREOF AND OF ANTITHROMBOTIC AZACYCLOALKYLALKANOYL PEPTIDES AND PSEUDOPEPTIDES
US20140100355A1 (en) * 2012-10-09 2014-04-10 Novartis Ag Solution Phase Processes for the Manufacture of Macrocyclic Depsipeptides and New Intermediates
WO2017112809A1 (fr) * 2015-12-21 2017-06-29 Taxas Tech University System Système et procédé de synthèse de peptides gap en phase solution

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6753409B1 (en) * 1996-08-21 2004-06-22 Aventis Pharmaceuticals Inc. STABLE NON-HYGROSCOPIC CRYSTALLINE FORM OF N-[N-[N-(4-(PIPERIDIN-4-YL)BUTANOYL)-N-ETHYLGLYCYL]ASPARTYL]-L-β-CYCLOHEXYL ALANINE AMIDE, INTERMEDIATES THEREOF, AND PREPARATION THEREOF AND OF ANTITHROMBOTIC AZACYCLOALKYLALKANOYL PEPTIDES AND PSEUDOPEPTIDES
US20140100355A1 (en) * 2012-10-09 2014-04-10 Novartis Ag Solution Phase Processes for the Manufacture of Macrocyclic Depsipeptides and New Intermediates
WO2017112809A1 (fr) * 2015-12-21 2017-06-29 Taxas Tech University System Système et procédé de synthèse de peptides gap en phase solution

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DE MARCO ET AL.: "C -. N and N -. C Solution Phase Peptide Synthesis Using the N-acyl 4-Nitrobenzenesulfonamide as Protection of the Carboxylic Function", ORGANIC & BIOMOLECULAR CHEMISTRY, vol. 11, no. 23, 21 July 2013 (2013-07-21), pages 3786 - 96, XP055257059, DOI: 10.1039/c3ob40169c *
HOU ET AL.: "Progress in Chemical Synthesis of Peptides and Proteins", TRANSACTIONS OF TIANJIN UNIVERSITY, vol. 23, no. 5, 23 June 2017 (2017-06-23), pages 401 - 419, XP036310733, DOI: 10.1007/s12209-017-0068-8 *
LAWRENSON ET AL.: "The Greening of Peptide Synthesis", GREEN CHEMISTRY, vol. 19, no. 7, 2 March 2017 (2017-03-02), pages 1685 - 1691, XP055651343, DOI: 10.1039/C7GC00247E *
SEIFERT ET AL.: "GAP Peptide Synthesis via Design of New GAP Protecting Group: An Fmoc/tBu Synthesis of Thymopentin Free from Polymers, Chromatography and Recrystallization", EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, vol. 2016, no. 9, 8 March 2016 (2016-03-08), pages 1714 - 1719, XP055651402, DOI: 10.1002/ejoc.201600026 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12024537B2 (en) 2018-05-31 2024-07-02 Sederma Compositions and methods for chemical synthesis
US11827660B2 (en) 2019-02-01 2023-11-28 Sederma Synthesis strategy for gap protecting group

Similar Documents

Publication Publication Date Title
CN108697770B (zh) 用于溶液相gap肽合成的系统和方法
EP3266792B1 (fr) Procédé de synthèse peptidique
EP3802554B1 (fr) Procédé de synthèse peptidique en phase solution et stratégies de protection associées
Goodman et al. New reagents, reactions, and peptidomimetics for drug design
WO2019217116A1 (fr) Méthode de synthèse en phase solution de peptides
CN108440654B (zh) 一种抗菌活性环六肽Thermoactinoamide A的合成方法
Denoël et al. Progress in lanthionine and protected lanthionine synthesis
US11827660B2 (en) Synthesis strategy for gap protecting group
JP2003055396A (ja) ペプチドの高速溶液合成方法
KR20230175279A (ko) 펩티드의 화학적 합성을 위한 조성물
Oualid et al. Synthesis of atypical diubiquitin chains
Kobayashi et al. Toward the total synthesis of onchidin, a cytotoxic cyclic depsipeptide from a mollusc
CN105622424A (zh) 化合物及其制备方法和应用
US8981049B2 (en) Aziridine mediated native chemical ligation
CN114276411A (zh) 含有三硫醚键的环肽化合物的固相合成方法
Vathsala et al. Peptide Bond Formation via N α-Protected Diacyldiselenides
Noguchi et al. Development of a Mirror-Image Screening System for Chiral Natural Products
CN109912696A (zh) 一种环孢菌素a及其类似物的液相合成方法
DE20023789U1 (de) Markierte Peptide und Zwischenprodukte, die sich zu deren Herstellung eignen

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19800715

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19800715

Country of ref document: EP

Kind code of ref document: A1