Conjugation of Amphiphilic Proteins to Hydrophobic Ligands in Organic Solvent

Lillo, Antonietta M.; Lopez, Ciana L.; Rajale, Trideep; Yen, Hung-Ju; Magurudeniya, Harsha D.; Phipps, Mary Lisa; Balog, Eva Rose M.; Sanchez, Timothy C.; Iyer, Srinivas; Wang, Hsing-Lin; Michalczyk, Ryszard; Rocha, Reginaldo C.; Martinez, Jennifer S.

doi:10.1021/acs.bioconjchem.8b00354

Title: Conjugation of Amphiphilic Proteins to Hydrophobic Ligands in Organic Solvent

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Abstract

Protein–ligand conjugations are usually carried out in aqueous media in order to mimic the environment within which the conjugates will be used. Here, 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 ofmore »« less

Authors:

^[1]; Lopez, Ciana L. ^[1];

^[1]; Yen, Hung-Ju ^[2];

^[1]; Phipps, Mary Lisa ^[1]; Balog, Eva Rose M. ^[3];

^[1]; Iyer, Srinivas ^[1]; Wang, Hsing-Lin ^[4];

^[1];

^[1]

Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Academia Sinica, Taipei (Taiwan). Inst. of Chemistry
Univ. of New England, Biddeford, MA (United States). Dept. of Chemistry and Physics
Southern Univ. of Science and Technology, Shenzhen (China). Dept. of Materials Science and Engineering

Publication Date:: 2018-07-06

Research Org.:: Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

Sponsoring Org.:: USDOE Laboratory Directed Research and Development (LDRD) Program; USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities Division; USDOE National Nuclear Security Administration (NNSA)

OSTI Identifier:: 1461400

Report Number(s):: LA-UR-18-21399
Journal ID: ISSN 1043-1802

Grant/Contract Number:: AC52-06NA25396

Resource Type:: Accepted Manuscript

Journal Name:: Bioconjugate Chemistry

Additional Journal Information:: Journal Volume: 29; Journal Issue: 8; Journal ID: ISSN 1043-1802

Publisher:: American Chemical Society

Country of Publication:: United States

Language:: English

Subject:: 59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Biological Science

Citation Formats


                    Lillo, Antonietta M., Lopez, Ciana L., Rajale, Trideep, Yen, Hung-Ju, Magurudeniya, Harsha D., Phipps, Mary Lisa, Balog, Eva Rose M., Sanchez, Timothy C., Iyer, Srinivas, Wang, Hsing-Lin, Michalczyk, Ryszard, Rocha, Reginaldo C., and Martinez, Jennifer S.. Conjugation of Amphiphilic Proteins to Hydrophobic Ligands in Organic Solvent.  United States: N. p., 2018. 
Web.  doi:10.1021/acs.bioconjchem.8b00354.

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                    Lillo, Antonietta M., Lopez, Ciana L., Rajale, Trideep, Yen, Hung-Ju, Magurudeniya, Harsha D., Phipps, Mary Lisa, Balog, Eva Rose M., Sanchez, Timothy C., Iyer, Srinivas, Wang, Hsing-Lin, Michalczyk, Ryszard, Rocha, Reginaldo C., & Martinez, Jennifer S.. Conjugation of Amphiphilic Proteins to Hydrophobic Ligands in Organic Solvent.  United States.  https://doi.org/10.1021/acs.bioconjchem.8b00354

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                    Lillo, Antonietta M., Lopez, Ciana L., Rajale, Trideep, Yen, Hung-Ju, Magurudeniya, Harsha D., Phipps, Mary Lisa, Balog, Eva Rose M., Sanchez, Timothy C., Iyer, Srinivas, Wang, Hsing-Lin, Michalczyk, Ryszard, Rocha, Reginaldo C., and Martinez, Jennifer S.. Fri .  
"Conjugation of Amphiphilic Proteins to Hydrophobic Ligands in Organic Solvent".  United States.  https://doi.org/10.1021/acs.bioconjchem.8b00354.  https://www.osti.gov/servlets/purl/1461400.

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@article{osti_1461400,

  title        = {Conjugation of Amphiphilic Proteins to Hydrophobic Ligands in Organic Solvent},

  author       = {Lillo, Antonietta M. and Lopez, Ciana L. and Rajale, Trideep and Yen, Hung-Ju and Magurudeniya, Harsha D. and Phipps, Mary Lisa and Balog, Eva Rose M. and Sanchez, Timothy C. and Iyer, Srinivas and Wang, Hsing-Lin and Michalczyk, Ryszard and Rocha, Reginaldo C. and Martinez, Jennifer S.},

  abstractNote = {Protein–ligand conjugations are usually carried out in aqueous media in order to mimic the environment within which the conjugates will be used. Here, 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.},

  doi          = {10.1021/acs.bioconjchem.8b00354},

  journal      = {Bioconjugate Chemistry},

  number       = 8,

  volume       = 29,

  place        = {United States},

  year         = {2018},

  month        = {7}

}

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