Traceless native chemical ligation of lipid-modified peptide surfactants by mixed micelle formation

Abstract

Biology utilizes multiple strategies, including sequestration in lipid vesicles, to raise the rate and specificity of chemical reactions through increases in effective molarity of reactants. We show that micelle-assisted reaction can facilitate native chemical ligations (NCLs) between a peptide-thioester - in which the thioester leaving group contains a lipid-like alkyl chain - and a Cys-peptide modified by a lipid-like moiety. Hydrophobic lipid modification of each peptide segment promotes the formation of mixed micelles, bringing the reacting peptides into close proximity and increasing the reaction rate. The approach enables the rapid synthesis of polypeptides using low concentrations of reactants without the need for thiol catalysts. After NCL, the lipid moiety is removed to yield an unmodified ligation product. This micelle-based methodology facilitates the generation of natural peptides, like Magainin 2, and the derivatization of the protein Ubiquitin. Formation of mixed micelles from lipid-modified reactants shows promise for accelerating chemical reactions in a traceless manner.

Fig. 1. Traceless lipid-facilitated acceleration of NCL.

a Schematic representation of traceless lipid-facilitated NCL via micelle mixing. b NCL reaction between aliphatic alkyl peptide thioesters (1a–c, 5a or 6a) and photocaged cysteine-based peptides (2a,b), which yields the desired polypeptides (3a,b, 7 and 8, respectively). Control reactions were performed with peptides containing short alkyl chains (1d, 2c, 5b, and 6b). Subsequent photocleavage reaction of the ligated products (3a,b, 7 or 8) leads to the formation of the uncaged polypeptides (4, 9, and 10, respectively).

Fig. 2. Kinetic measurements of NCL between LYRMG-Z (1a–d) and CDap(PhCn)ANK (2a–c).

a NCL reaction between 1a–d (1 mM) and 2b (1 mM). b NCL reaction between 1a (1 mM) and 2a–c (1 mM). c NCL reaction between 1a (1 mM–50 µM) and 2b (1 mM–50 µM). Peptides were ligated at room temperature and pH 7.0 in the presence of TCEP·HCl (10 mM). Decapeptide LYRMGCDap(PhCn)ANK (3a,b) formation was monitored over time using combined liquid chromatography (LC), mass spectrometry (MS), and evaporative light-scattering detection (ELSD) measurements. At each time point, the fraction ligated was determined by integration of the ligated product with detection at 210 nm as a fraction of the sum of {starting material cysteine peptide 2 + ligated product}. Error bars represent standard deviations (SD) (n = 3).

Fig. 3. Various ligations (Asn-Cys, Leu-Cys) and photocleavage reaction.

a Kinetic measurements of NCL between the peptide thioesters LYRMN-αCOSC8 (5a) (1 mM) or LYRML-αCOSC8 (6a) (1 mM) and the peptide CDap(PhC16)ANK (2b) (1 mM). Decapeptide (7 and 8, respectively) formation was monitored over time using combined HPLC-ELSD-MS measurements. Error bars represent standard deviations (SD) (n = 3). b Photocleavage reaction of the auxiliary on NCL product LYRMGCDap(PhC16)ANK (3b) to generate the decapeptide LYRMGCDapANK (4). The photocleavage reaction was monitored using HPLC-ELSD-MS (raw ELSD data shown).

Fig. 4. One-pot strategy for the total synthesis of Magainin 2 (15).

HPLC (210 nm) traces corresponding to the precursors, intermediates, and final product. Retention times were verified by MS.

Fig. 5. Derivatization of the natural protein Ubiquitin by lipid-facilitated NCL.

a HPLC (210 nm) traces corresponding to the precursors Ubi-αCOSC8 (16) and CK(PhC16)ANK (S4) (top) and the ligated product Ubi-CK(PhC16)ANK 17 (after 5 h of NCL reaction; bottom). Retention times were verified by MS. b ESI-TOF MS spectrum of the ligated product 17. Deconvoluted spectrum calculated from this ESI-TOF spectrum is also shown in the top left. The deconvolution value (9485.0) corresponds with the molecular weight of the product.