The ability to prepare well-defined protein/small molecule/solid-support conjugates possesses significant advantages in the treatment and diagnosis of a variety of diseases. However, several issues associated with the preparation of the bioconjugates preclude their widespread application. This proposal aims to address these issues via the use of unnatural amino acid technologies to rapidly synthesize highly active and well- defined conjugates. Specifically, a variety of unnatural amino acids will be synthetically prepared and assessed for their utility in bioconjugations. First, utilizing pre-existing UAAs, a novel bioconjugation will be developed through the demonstration and optimization of a novel Glaser Hay coupling between terminal alkynes in a physiological setting. Secondly, a set of amino acids with reactive moieties (alkyne, azide, aminooxy, etc) will be prepared with a variable methylene tether to provide a degree of separation between the protein and the reactive functional group, to improve efficiency of the bioconjugation. Moreover, the increased reactivity of a aminooxy group (which has not previously been investigated as an unnatural amino acid) will provide improved conjugation conditions at mild pH and temperature. Additional unnatural amino acids will be prepared that can be activated by light irradiation. The installation of a photolabile protecting group on an aldehyde functionality will allow for the successful incorporation of a reactive aldehyde into the protein due to the ability of the photolabile group to block its innate reactivity. Brief light irradiation will restore the aldehyde and afford optimized coupling conditions. Following the synthesis and in vitro assessment of these novel amino acids, they will be evaluated for site-specific incorporation into model proteins using evolved aminoacyl tRNA synthetase/tRNA pairs. Ultimately, the technology will be examined for optimized coupling conditions and biological efficacy. Finally, utilizing optimized conditions both model and medically relevant proteins will be immobilized onto solid-supports using the technologies to develop diagnostic protein chips. The tools developed within the proposed research will not only significantly advance the fields of therapeutics and diagnostics, but also be extremely useful in the training of undergraduate researchers towards their future careers within the scientific arena.

Public Health Relevance

Bioconjugates provide a mechanism for the targeted diagnosis and treatment of cancer. As such, improved methodologies for their preparation can further facilitate their therapeutic relevance. We aim to develop a novel Glaser Hay bioconjugation, and synthesize and incorporate novel unnatural amino acids into proteins in order to address current deficiencies associated with bioconjugate production. Moreover, this technology will afford a means to access well-defined conjugates in a rapid and efficient fashion.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
1R15GM113203-01A1
Application #
9018635
Study Section
Synthetic and Biological Chemistry B Study Section (SBCB)
Program Officer
Fabian, Miles
Project Start
2016-04-01
Project End
2019-03-31
Budget Start
2016-04-01
Budget End
2019-03-31
Support Year
1
Fiscal Year
2016
Total Cost
Indirect Cost
Name
College of William and Mary
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
074762238
City
Williamsburg
State
VA
Country
United States
Zip Code
23187
Nimmo, Zachary M; Halonski, John F; Chatkewitz, Lindsay E et al. (2018) Development of optimized conditions for Glaser-Hay bioconjugations. Bioorg Chem 76:326-331
Young, Douglas D; Schultz, Peter G (2018) Playing with the Molecules of Life. ACS Chem Biol 13:854-870
Chatkewitz, Lindsay E; Halonski, John F; Padilla, Marshall S et al. (2018) Investigation of copper-free alkyne/azide 1,3-dipolar cycloadditions using microwave irradiation. Bioorg Med Chem Lett 28:81-84
Maza, Johnathan C; Howard, Christina A; Vipani, Megha A et al. (2017) Utilization of alkyne bioconjugations to modulate protein function. Bioorg Med Chem Lett 27:30-33
Villa, Jordan K; Tran, Hong-Anh; Vipani, Megha et al. (2017) Fluorescence Modulation of Green Fluorescent Protein Using Fluorinated Unnatural Amino Acids. Molecules 22:
Maza, Johnathan C; Nimmo, Zachary M; Young, Douglas D (2016) Expanding the scope of alkyne-mediated bioconjugations utilizing unnatural amino acids. Chem Commun (Camb) 52:88-91
Padilla, Marshall S; Farley, Christopher A; Chatkewitz, Lindsay E et al. (2016) Synthesis and incorporation of a caged tyrosine amino acid possessing a bioorthogonal handle. Tetrahedron Lett 57:4709-4712
Lampkowski, Jessica S; Uthappa, Diya M; Halonski, John F et al. (2016) Application of the Solid-Supported Glaser-Hay Reaction to Natural Product Synthesis. J Org Chem 81:12520-12524
Maza, Johnathan C; McKenna, Jaclyn R; Raliski, Benjamin K et al. (2015) Synthesis and Incorporation of Unnatural Amino Acids To Probe and Optimize Protein Bioconjugations. Bioconjug Chem 26:1884-9