WO2018141146A1 - Synthèse chimique totale de peptide lasso - Google Patents

Synthèse chimique totale de peptide lasso Download PDF

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Publication number
WO2018141146A1
WO2018141146A1 PCT/CN2017/087855 CN2017087855W WO2018141146A1 WO 2018141146 A1 WO2018141146 A1 WO 2018141146A1 CN 2017087855 W CN2017087855 W CN 2017087855W WO 2018141146 A1 WO2018141146 A1 WO 2018141146A1
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Prior art keywords
cryptand
ionic liquid
peptide
linker
lasso
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PCT/CN2017/087855
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English (en)
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Ming Chen
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Ming Chen
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Publication of WO2018141146A1 publication Critical patent/WO2018141146A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
    • 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

Definitions

  • the present invention relates to the new strategy using cryptand-ionic liquid complex as a multi-linker support for the chemical synthesis of lasso peptides BI-32169 and its analogs.
  • Lasso peptides are an important class of natural products that belong to the family of ribosomally biosynthesized and post-translationally modified peptides (RiPPs) .
  • Various interesting biological activities of these peptides have been reported, such as enzyme inhibition, receptor antagonism and/or antimicrobial activities.
  • the sequence of lasso peptide exhibits a knotted topology involving an N-terminal lactam-bridged ring (7-9 amino acids) , which is threaded by a exocyclic C-terminal tail (7-15 amino acids) .
  • the antimicrobial lasso peptides e.g. Microcin J25 and lassomycin, have been found to lost their activities when their conformations were unthreaded.
  • the unique knotted feature was proposed to be a key factor for the activity of lasso peptide.
  • ionic liquids that are organic salts in liquid states at low temperature or even at room temperature, have been used as an efficient reaction medium in peptide synthesis.
  • Miao et al. described a new method called ionic-liquid-supported peptide synthesis (ILSPS) , in which the imidazolium-based ionic liquid acted as a high-loading and recoverable support for synthesizing oligopeptides.
  • ILSPS ionic-liquid-supported peptide synthesis
  • imidazolium-based ionic liquid can form a stable inclusion complex with hollow circular compounds via hydrogen bonds or ⁇ - ⁇ interactions.
  • These findings give us inspiration to develop a cryptand-ionic liquid complex as support for lasso peptide synthesis.
  • the present invention relates to a multi-linker strategy for the chemical synthesis of lasso peptide.
  • the multi-linker synthesis strategy is given in Figure 1B, in which three anchors or linkers are introduced for constructing a lasso peptide with knotted structure.
  • the C-terminal tail of lasso peptide (AA hollow circles in Figure 1B) is prolonged after first amino acid is anchored to an initial linker (middle linker in Figure 1B) .
  • the N-terminal ring of lasso peptide (AA solid circles in Figure 1B) is then formed by taking the amino acid anchored to the second linker (left linker in Figure 1B) as starting point.
  • the direction of the ring formation around the C-terminal tail could be controlled by the third linker (right linker in Figure 1B) , chirality of the peptidyl support and the distribution of rigid residues like L-proline in the sequence.
  • the tail sequence then threads through the ring after cleavage of the linkages.
  • the multi-linker strategy required a special support which should provide the following features: containing multiple linkers, no steric hindrance between adjacent linkers and increasing solubility of peptide.
  • a cryptand-ionic liquid complex as support, in which the imidazolium cation of ionic liquid contains the first linker for C-terminal tail formation of lasso peptide and the cryptand furnishes the second and third linkers for N-terminal ring formation for the multi-linker strategy of lasso peptide synthesis.
  • BI-32169 contains a 9-mer N-terminal ring (G 1 LPWGCPSD 9 ) established via an isopeptide linkage and a 10-mer C-terminal tail (I 10 PGWNTPWAC 19 ) threaded through the ring.
  • a disulfide bridge is then formed between the C-terminal cysteine residue of the tail (C 19 ) and the cysteine residue in the N-terminal ring (C 6 ) ( Figure 2B, 2C) .
  • the cryptand-ionic liquid supported approach is also a useful synthetic tool for producing and studying the D-retro enantiomers of lasso peptides ( Figure 2D) , which has not been reported before.
  • Figure 1A-2B are (A) conventional single-linker strategy of lasso peptide synthesis and (B) multi-linker strategy of lasso peptide synthesis.
  • AA amino acid
  • Figure 2A-2D are (A) primary structure of BI-32169 composed of L-amino acids, (B) crystal structure of BI-32169 composed of L-amino acids, (C) secondary structure diagram of BI-32169 composed of L-amino acids and (D) secondary structure diagram of D-retro-inverso BI-32169, which is composed of D-amino acids in a reversed sequence (in italics) .
  • Figure 3 (left side) is, according to certain embodiments, the complexation between cryptand 2 and ionic liquid 1.
  • the interactions (hydrogen bonds and ⁇ - ⁇ stacking) are shown in dotted line.
  • Figure 3A-3C (right side) are, according to certain embodiments, 1 H NMR spectra of (A) ionic liquid 1, (B) equimolar mixture of 2 and 1 and (C) cryptand 2.
  • Figure 4A-4C are, according to certain embodiments, (A) attempt at coupling of amino acid with cryptand-ionic liquid complex support, (B) attempt at complexation of peptidyl ionic liquid with cryptand and (C) cryptand-ionic liquid supported total synthesis of BI-32169.
  • Figure 5 is, according to certain embodiments, synthesis of unthreaded topoisomer of BI-32169 (ESI-MS calculated for C 95 H 125 N 23 O 24 S 2 [M+2H] 2+ : 1019.6595; Found 1019.6560) .
  • Figure 6 are, according to certain embodiments, HPLC profiles of (a) crude synthesized BI-32169, (b) purified synthesized BI-32169, (c) native BI-32169 and (d) unthreaded topoisomer of BI-32169 synthesized by SPPS method.
  • Figure 7 is, according to certain embodiments, MS 2 spectrum of oxidized BI-32169. Four series of fragment ions (singly and doubly protonated fragments) and their corresponding peaks (marked with italic serial numbers) are showed respectively.
  • Figure 8 is, according to certain embodiments, MS 2 spectrum of reduced BI-32169. Three series of fragment ions (singly and doubly protonated fragments) and their corresponding peaks (marked with italic serial numbers) are showed respectively.
  • Figure 9 is, according to certain embodiments, MS 2 spectrum of reduced unthreaded topoisomer of BI-32169. Three series of fragment ions (singly and doubly protonated fragments) and their corresponding peaks (marked with italic serial numbers) are showed respectively.
  • Figure 10 is, according to certain embodiments, the protected cryptand assemblies that are axial enantiomers of each other.
  • Ph i Pr phenylisopropyl
  • TDPS tert-butyldiphenylsilyl
  • Linear peptide GLPWGCPSDIPGWNTPWAC was prepared by stepwise Fmoc-SPPS on an Advanced ChemTech (ACT-396) automated peptide synthesizer on 2-chlorotrityl chloride resin (100-200 mesh, 1.06 mmol/g) in situ activation protocols to couple Fmoc-protected amino acid (4.0 eq. to resin loading) to the resin using PyBOP (4.0 eq. ) as coupling reagent in the presence of N-methylmorpholine (8.0 eq. ) .
  • the Fmoc group was deprotected with 20%piperidine/DMF.
  • the side chain ODmab of residue D 9 was selectively removed using 2%hydrazine in DMF.
  • the cyclization via isopeptide bond was carried out using PyBOP (4.0 eq. ) and N-methylmorpholine (4.0 eq. ) .
  • Cyclized peptide was cleaved from the resin at room temperature in TFA/phenol/water/TIPS (88: 5: 5: 2) for 3h.
  • Cold diethyl ether was then added to the filtered cleavage mixture and the peptide precipitated out.
  • Pure peptide (0.2 mM) were oxidized by stirring at room temperature in 0.1M NH 4 OAc/DMF for 12h after washing with further cold diethyl ether.
  • the oxidized peptide was purified by semipreparative reversed-phase HPLC equipped with a Waters XBridgeTM BEH3000 C18 column (4.6 ⁇ 150 mm) at a flow rate of 10.0 ml/min in 0–50%acetonitrile/0.1%TFA gradient and then lyophilized overnight.
  • the solvent was then washed with 20% (m/v) NaCl solution and deionized water.
  • the cysteine ( t Bu) -loaded ionic liquid (6) was obtained via an Fmoc-deprotection with tris (2-aminoethyl) amine (TAEA) .
  • Fmoc-alanine-OH protected A 18
  • the anchoring (esterification) and coupling reactions were both performed efficiently under conditions using the reagent combination of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDCI) and ethyl (hydroxyimino) cyanoacetate (Oxyma ) without pre-drying treatment.
  • the cryptand (2) was introduced to constitute a cryptand-ionic liquid complex support loaded with Fmoc-alanine-cysteine ( t Bu) (8) .
  • the protected amino acid moieties tryptophan (W 17 ) , proline (P 16 ) , threonine (T 15 ) , asparagine (N 14 ) , tryptophan (W 13 ) , glycine (G 12 ) , proline (P 11 ) , isoleucine (I 10 ) , aspartic acid (D 9 ) and serine (S 8 ) were sequentially coupled to 8, to form 9, in which the hydroxyl side chain of the residue S 8 was chosen as a suitable anchor group.
  • Tryptophan (W 4 ) and proline (P 3 ) could be appropriate alternatives in the light of their positions (opposite to S 8 ) in the ring sequence of BI-32169. However, neither of them has a chemically linkable side chain for anchoring to the support.
  • tryptophan (W 4 ) with its derivative, 2-amino-3- (1-carboxyl-indolin-3-yl) propanoic acid (namely 2H, 3H-1-carboxytryptophan, W′ 4 ) .
  • the moiety W′ 4 was preferred since it could be converted to tryptophan residue in the final cleavage stage.
  • the second and third linkages were first broken in minutes through a simple photolytic cleavage under mild heating conditions (30°C) . After vacuum evaporation, the concentrated solution was transferred into a cleavage cocktail which consists of TFA, phenol, water and triisopropylsilane (TIPS) with a ratio of 88: 5: 5: 2, to remove all the remaining protecting groups in the peptide chain.
  • TIPS triisopropylsilane
  • the reaction mixture was added dropwise in ice-cold hexane and the complex support loaded with unprotected peptide (15) precipitated immediately. It should be noted that cold ether is not applicable for separation due to its high dissolving capability for the compounds with [BAr F 4 ] anion.
  • the lasso peptide was then released from the complex support by 0.1 M sodium hydroxide in water/tetrahydrofuran (1: 3) with argon protection for 6 hours, and the solution was afterwards exposed to air for 12 hours to generate the disulfide bridge between the residues C 1 and C 6 .
  • utilizing a higher concentration (1.0 M) solution of sodium hydroxide could obviously shorten the liberation time, yet led to an undesirable opening of the N-terminal ring and thus destroyed the lasso conformation of BI-32169.
  • the solution was neutralized with 0.1 M aqueous citric acid and the solvent was distilled in vacuum.
  • BI-32169 D-retro-inverso BI-32169 was prepared via cryptand-ionic liquid supported approach using an (R a ) -enantiomer of 2 as cryptand assembly (16, Figure 10) and corresponding D-amino acids as building units. Finally, the lasso peptide BI-32169 was obtained in a 98.35%pure form, and in an overall yield of 1.97%by preparative scale HPLC.

Abstract

L'invention concerne un procédé supporté par un liquide cryptand-ionique pour la première synthèse totalement chimique du peptide lasso BI-32169 et de ses analogues. La technique supportée par un liquide cryptand-ionique s'est avérée être un outil synthétique puissant et contrôlable pour produire ou modifier des peptides en forme de lasso modifiés chimiquement et aussi pour étudier les relations structure-activité des peptides lasso. Ainsi, il est très souhaitable que cette stratégie de synthèse avec un support multi-lieur de conception puisse bénéficier de l'expansion de diverses banques de peptides lasso pour la découverte de nouveaux peptides bioactifs.
PCT/CN2017/087855 2017-02-06 2017-06-12 Synthèse chimique totale de peptide lasso WO2018141146A1 (fr)

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CN106749497B (zh) * 2017-02-06 2020-10-30 陈铭 套索多肽的化学全合成
CN112321699B (zh) * 2020-11-05 2021-09-14 深圳深创生物药业有限公司 一种司美格鲁肽的合成方法

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CN101180307A (zh) * 2005-03-15 2008-05-14 麦吉尔大学 离子液体支载的合成
WO2012146729A1 (fr) * 2011-04-29 2012-11-01 Philipps-Universität Marburg Peptides lasso utilisés en tant qu'échafaudages pour la greffe de peptides
WO2014036213A1 (fr) * 2012-08-31 2014-03-06 The Trustees Of Princeton University Peptides d'astexine
CN105392480A (zh) * 2013-05-17 2016-03-09 马凯特大学 由离子液体组合物形成的含结构性多糖和大环化合物的复合材料
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