Solid‐Phase Thiol–Ene Lipidation of Peptides for the Synthesis of a Potent CGRP Receptor Antagonist

Abstract

We report a new method herein coined SP‐CLipPA (solid‐phase cysteine lipidation of a peptide or amino acid) for the synthesis of mono‐S‐lipidated peptides. This technique utilizes thiol–ene chemistry for conjugation of a vinyl ester to a free thiol of a semiprotected, resin‐bound peptide. Advantages of SP‐CLipPA include: ease of handling, conversions of up to 91 %, by‐product removal by simple filtration, and a single purification step. Additionally, the desired lipidated products show high chromatographic separation from impurities, thus facilitating RP‐HPLC purification. To showcase the utility of SP‐CLipPA, we synthesized a potent calcitonin gene‐related peptide (CGRP) receptor antagonist peptide in excellent yield and purity. This peptide, selected from a series of lipidated analogues of CGRP8–37 and CGRP7–37, has potential for the treatment of migraine.

Chemical Synthesis of Native S‐Palmitoylated Membrane Proteins through Removable‐Backbone‐Modification‐Assisted Ser/Thr Ligation

“…During the SPPS,the 4-monomethoxytrityl (Mmt)-protected Cys9 was installed into the peptide.A fter peptide assembly, the Mmt group in the resin-bound peptide precursor was selectively removed by treatment with 2% trifluoroacetic acid (TFA) cocktails to give afree thiol. [19] Thereleased thiol then reacted with palmitic anhydride to generate S-palm SLN 1.U nfortunately,t he crude peptide 1 was found to be insoluble,and no target product was detected in the reversedphase high-performance liquid chromatography (RP-HPLC) profile ( Figure 1b). We also examined other previously developed strategies [20] (such as the use of NMP/DMF cosolvents,microwave irradiation, pseudoproline,and O-acyl isopeptide,and the attachment of solubilizing tags on peptide side chains) to improve the synthetic efficiency.However,no significant improvement was observed by RP-HPLC (see Figure S1 in the Supporting Information), presumably owing to the strong hydrophobicity of 1.…”

Replacement of the Acrid tert‐Butylthiol and an Improved Isolation Protocol for Cysteine Lipidation on a Peptide or Amino Acid (CLipPA)

“…[15] We have previously used this peptide to demonstrate a solid-phase variant of CLipPA (SP-CLipPA). [16] S-lipidation with either vinyl palmitate or vinyl benzoate in the presence of either t-NonylSH or tBuSH gave excellent conversions (>95 %) to the desired lipopeptides 4b and 4c. On a multi-milligram scale, S-palmitoylated lipopeptide 4b was isolated in a 47 % yield, a substantial improvement to that reported previously.…”

“…Peptide sequence 4a is a potent antagonist of the calcitonin gene‐related peptide (CGRP) receptor used in the development of new migraine treatments . We have previously used this peptide to demonstrate a solid‐phase variant of CLipPA (SP‐CLipPA) . S ‐lipidation with either vinyl palmitate or vinyl benzoate in the presence of either t‐NonylSH or t BuSH gave excellent conversions (>95 %) to the desired lipopeptides 4b and 4c .…”

“…On a multi-milligram scale, S-palmitoylated lipopeptide 4b was isolated in a 47 % yield, a substantial improvement to that reported previously. [16] Optimization of the lipopeptide isolation procedure. The large excesses of the radical quenchers, TIPS and tBuSH/ t-Non-ylSH as well as vinyl ester required for optimal conversion of peptides, and the complex degradation profile of DMPA upon UV irradiation can severely complicate the analysis of the reaction profile by HPLC and hamper subsequent HPLC purification.…”

Synthesis, Antibacterial Activity, and Nephrotoxicity of Polymyxin B Analogues Modified at Leu-7, d-Phe-6, and the N-Terminus Enabled by S-Lipidation

“…Encouraged by these reports and the recent progression of several polymyxins, including Spero’s SPR206, Qpex BioPharma’s QPX9003, , and MicuRx’s MRX8 to clinical trials, we focused on preparing polymyxin B analogues that contained unique lipids anchored by a novel 2-thioethyl ester linkage via the peptide backbone at the N-terminus, position 7, position 6, or the combination of several of these sites. The 2-thioethyl ester linkage can be introduced using thiol–ene-promoted S -lipidation (which we termed CLipPA: C ysteine Lip idation on a P eptide or A mino acid). − CLipPA S-lipidation takes place via a radical-initiated thiol–ene reaction between the free thiol of a cysteine residue (or surrogate) and the terminal sp 2 carbon atom on a fatty acid vinyl ester. We chose to preserve the five cationic Dab residues based on the widely accepted mechanism of action studies; these cationic side-chain amino groups are essential to initiate disabling of lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria through electrostatic interactions, prior to the subsequent lipid tail insertion into the membrane. , …”