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

Abstract

The preparation of native S‐palmitoylated (S‐palm) membrane proteins is one of the unsolved challenges in chemical protein synthesis. Herein, we report the first chemical synthesis of S‐palm membrane proteins by removable‐backbone‐modification‐assisted Ser/Thr ligation (RBMGABA‐assisted STL). This method involves two critical steps: 1) synthesis of S‐palm peptides by a new γ‐aminobutyric acid based RBM (RBMGABA) strategy, and 2) ligation of the S‐palm RBM‐modified peptides to give the desired S‐palm product by the STL method. The utility of the RBMGABA‐assisted STL method was demonstrated by the synthesis of rabbit S‐palm sarcolipin (SLN) and S‐palm matrix‐2 (M2) ion channel. The synthesis of S‐palm membrane proteins highlights the importance of developing non‐NCL methods for chemical protein synthesis.

Enhanced native chemical ligation by peptide conjugation in trifluoroacetic acid

“…First, 12 (2 mM) and 13 (2.1 mM) were dissolved in TFA, and the peptide dithioacetal thioester 15a was formed within 5 min at 30°C, as confirmed by ESI-MS (observed: 10,459.90 Da; calculated: 10,460.30 Da). Solid 15a was isolated after concentration and precipitation and then treated with weakly acidic H 2 O/CH 3 CN (v/v = 1/1) buffer (0.1 mM, Py/acetic acid, pH 5.6) to the backbone amide and TFA cocktails (TFA/TIPS/H 2 O, 95/2.5/2.5, v/v/v) to remove the auxiliary group ( 46 ). The resulting peptide underwent free-radical desulfurization by treatment with TECP/VA-044 to give the product 15d , which was purified by HPLC (42% isolated yield starting from peptide 12 ), and its molecular mass verified by ESI-MS ( Fig.…”

Chemical Synthesis of Proteins with Base‐Labile Posttranslational Modifications Enabled by a Boc‐SPPS Based General Strategy Towards Peptide C‐Terminal Salicylaldehyde Esters

“…The lability of S ‐palmitoyl thioester renders its synthesis rather difficult and challenging. Access to the S ‐palmitoylated peptide has been achieved by direct Fmoc‐SPPS [32a] or post‐Fmoc‐SPPS‐modification; [10a, 32b,c] however, the length of the peptide was limited, or a complicated protocol was required. Additionally, the use of NCL for peptides containing S ‐palmitoylation modification was precluded, due to the reactivity of the S ‐palmitoyl thioester towards the thiol species in the NCL system.…”

“…Thus, the non‐NCL ligation methods have become the appropriate choices to tackle this challenging task. In 2020, Zheng's group reported the synthesis of the S ‐palmitoylated sarcolipin and M2 ion channels, taking advantage of STL [10a] . In their report, synthesis of the peptide SAL ester with S ‐palmitoylation was very difficult.…”

Recent Advances in Glycopeptide and Glycoprotein Synthesis: A Refined Synthetic Probe towards the Biological World

“…Apparently, the preparation of glycoproteins to mimic natural complexity is a fascinating target, and also an attractive, arduous and problem‐driven journey, calling for continuous efforts and wisdom to come. While some challenges in protein synthesis ( e.g ., poor solubility, difficult folding) can be facilitated via the introduction of glycosylation, it can be anticipated that the ongoing development of protein chemical synthesis and semisynthesis tools, [ 16,116‐123 ] exemplified with various chemical ligation, conjugation and purification technologies, will contribute to the progress of glycoprotein synthesis. Meanwhile, a primary concern under development is the construction of diverse forms of glycosidic linkage, between each glycan unit and also between the glycan chain and protein backbone, where stereoselectivity and regioselectivity issues are frequently interrogated.…”