Synthesis of Stable Cholesteryl–Polyethylene Glycol–Peptide Conjugates with Non-Disperse Polyethylene Glycol Lengths

Abstract

A method for conjugating cholesterol to peptide ligands through non-disperse polyethylene glycol (ND-PEG) through a non-hydrolysable linkage is described. The iterative addition of tetraethylene glycol macrocyclic sulfate to cholesterol (Chol) renders a family of highly pure well-defined Chol-PEG compounds with different PEG lengths from 4 up to 20 ethylene oxide units, stably linked through an ether bond. The conjugation of these Chol-PEG compounds to the cyclic (RGDfK) peptide though Lys5 side chains generates different lengths of Chol-PEG-RGD conjugates that retain the oligomer purity of the precursors, as analysis by HRMS and NMR has shown. Other derivatives were synthesized with similar results, such as Chol-PEG-OCH 3 and Chol-PEG conjugated to glutathione and Tf1 peptides through maleimide−thiol chemoselective ligation. This method allows the systematic synthesis of highly pure uniform stable Chol-PEGs, circumventing the use of activation groups on each elongation step and thus reducing the number of synthesis steps.

Application of Quality by Design to the robust preparation of a liposomal GLA formulation by DELOS-susp method

“…1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was purchased from Corden Pharma (Liestal, Switzerland), cholesterol was purchased from Panreac Química (Castellar del Vallès, Spain), and myristalkonium chloride (MKC) from United States Biological (Salem, US). Cholesterol pegylated with arginyl–glycyl–aspartic acid peptide (Chol-PEG 400 -RGD) was synthetized as described by E. Cristóbal-Lecina et al [14] . Ethanol (EtOH) was purchased from Scharlab (Sentmenat, Spain), dimethyl sulfoxide (DMSO) from Sigma-Aldrich (St. Louis, US), carbon dioxide (CO 2 ) and nitrogen (N 2 ) from Carburos Metálicos (Cornellà de Llobregat, Spain).…”

Hierarchical Quatsome-RGD Nanoarchitectonic Surfaces for Enhanced Integrin-Mediated Cell Adhesion

“…This molecular building block was synthesized as previously described. 43,53 The other one was a commercially available cholesterol molecule, functionalized with a long thiolterminated poly(ethylene glycol) chain (chol-PEG 3000 -SH) with a contour length of ca. 21 nm, 54 providing the nanovesicles with a flexible Au-anchoring moiety.…”

“…One of them was cholesterol bound to a cyclic RGD peptide through a poly­(ethylene glycol) spacer (chol-PEG 200 -c­(RGDfK)), which provides a ligand for integrins to the nanovesicles. This molecular building block was synthesized as previously described. , The other one was a commercially available cholesterol molecule, functionalized with a long thiol-terminated poly­(ethylene glycol) chain (chol-PEG 3000 -SH) with a contour length of ca. 21 nm, providing the nanovesicles with a flexible Au-anchoring moiety.…”

“…The equipment used to produce the nanovesicle formulations through the DELOS-susp method is a small-scale reactor that has been previously described . Chol-PEG 200 -c­(RGDfK) (RGD-PEG) was prepared as described previously: , 9.28 μmol were dissolved in 800 μL of DMSO before adding 2.08 mL of an ethanolic solution dropwise with 69.6 μmol of cholesterol (Panreac, Barcelona, Spain) and 0.27 μmol of chol-PEG 3000 -SH (Nanocs, New York, USA). The resulting mixture of the three cholesterol derivatives with a molar ratio of chol/chol-PEG 200 -c­(RGDfK)/chol-PEG 3000 -SH of 260/30/1 was loaded into a 7.5 mL high-pressure vessel and volumetrically expanded with compressed CO 2 to reach a working pressure of 11.5 MPa.…”

Impact of Chemical Composition on the Nanostructure and Biological Activity of α-Galactosidase-Loaded Nanovesicles for Fabry Disease Treatment

“…Miristalkonium chloride (MKC) was purchased from US Biological (Salem, United States). Cholesterol-PEG200-RGD was prepared as described previously in Cabrera et al 39 or from Cristóbal-Lecina et al 52 Agalsidase alfa (GLA, 1 mg mL −1 , Replagal ® ) was purchased from Shire-Takeda. Ethanol HPLC grade (Teknocroma Sant Cugat del Vallès, Spain) was purchased with high purity and used without further purifications.…”