Conjugation of Amphiphilic Proteins to Hydrophobic Ligands in Organic Solvent

Abstract

Protein-ligand conjugations are usually carried out in aqueous media in order to mimic the environment within which the conjugates will be used. In this work, we focus on the conjugation of amphiphilic variants of elastin-like polypeptide (ELP), short elastin (sEL), to poorly water-soluble compounds like OPPVs ( p-phenylenevinylene oligomers), triarylamines, and polypyridine-metal complexes. These conjugations are problematic when carried out in aqueous phase because hydrophobic ligands tend to avoid exposure to water, which in turn causes the ligand to self-aggregate and/or interact noncovalently with hydrophobic regions of the amphiphile. Ultimately, this behavior leads to low conjugation efficiency and contamination with strong noncovalent "conjugates". After exploring the solubility of sEL in various organic solvents, we have established an efficient conjugation methodology for obtaining covalent conjugates virtually free of contaminating noncovalent complexes. When conjugating carboxylated ligands to the amphiphile amines, we demonstrate that even when only one amine (the N-terminus) is present, its derivatization is 98% efficient. When conjugating amine moieties to the amphiphile carboxyls (a problematic configuration), protein multimerization is avoided, 98-100% of the protein is conjugated, and the unreacted ligand is recovered in pure form. Our syntheses occur in "one pot", and our purification procedure is a simple workup utilizing a combination of water and organic solvent extractions. This conjugation methodology might provide a solution to problems arising from solubility mismatch of protein and ligand, and it is likely to be widely applied for modification of recombinant amphiphiles used for drug delivery (PEG-antibodies, polymer-enzymes, food proteins), cell adhesion (collagen, hydrophobins), synthesis of nanostructures (peptides), and engineering of biocompatible optoelectronics (biological polymers), to cite a few.

Atom Transfer Radical Polymerization for Biorelated Hybrid Materials

“…Russell et al have investigated chymotrypsin-poly­(2-(dimethylamino)­ethyl methacrylate) (CT-pDMAEMA) solubility and activity in acetonitrile . Protein–polymer conjugate synthesis has also been performed in organic solvent in order to increase the conjugation yield of an amphiphilic protein to a hydrophobic ligand (or polymer) . Moreover, Thayumanavan et al found that polymeric supramolecular assemblies could help efficiently transport proteins from an aqueous phase to an organic phase .…”

“…103 Protein−polymer conjugate synthesis has also been performed in organic solvent in order to increase the conjugation yield of an amphiphilic protein to a hydrophobic ligand (or polymer). 104 Moreover, Thayumanavan et al found that polymeric supramolecular assemblies could help efficiently transport proteins from an aqueous phase to an organic phase. 105 Ionic liquids are another class of nonaqueous media that are attractive because they are nonvolatile, more environmentally friendly, and have tunable properties.…”

Synthesis and Characterization of Cholesteryl Conjugated Lysozyme (CHLysozyme)

“…The chemical stability (e.g., against thermal stress) of proteins has also been improved by modification with polysaccharides, which are hydrophilic groups [ 9 , 10 , 11 ]. The chemical modification of proteins has mostly been via conjugation with hydrophilic groups, but there are a few reports on chemical modification using hydrophobic groups [ 12 , 13 ]. Chemical modification of hydrophobic groups to water soluble polymers has been reported.…”

“…Nuclear-magnetic resonance (NMR) and13 C NMR spectra of CHLysozyme in DMSO-d6 at 25 °C. (a) 1 H NMR spectrum, and (b)13 C NMR spectrum, CHLysozyme concentration: 21.4 mg/mL.…”

Cholesteryl-Conjugated Ribonuclease A Exhibits Enzyme Activity in Aqueous Solution and Resistance to Dimethyl Sulfoxide

“…10−12 Although most proteins are modified with hydrophilic groups, some studies have introduced modifications with hydrophobic groups. 13,14 In natively structured proteins, hydrophobic residues are burried; 15,16 however, protein denaturation, induced by physical stress, heat, or chemical exposure, allows these residues to become exposed. 13,17−19 Aggregation of proteins is mediated by hydrophobic interactions between the newly exposed hydrophobic residues of denatured proteins.…”