The ability to prepare well-defined protein/small-molecule/solid-support bioconjugates 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, novel bioconjugations will be developed and optimized under physiological conditions, including the Glaser Hay coupling between terminal alkynes and the [2+2+2] cyclotrimerization between three alkynyl moieties. Secondly, a set of amino acids with reactive moieties (alkyne, azide, aminooxy, aldehyde etc) will be prepared to improve the efficiency of the bioconjugation reactions and prepare multivalent conjugates. 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. Utilizing optimized conditions, both model and medically relevant proteins will be immobilized onto solid-supports using the technologies to develop diagnostic protein chips. Finally, the developed technologies can be combined to produce multivalent bioconjugates that provide numerous therapeutic properties inaccessible to standard bivalent conjugates. This includes the linking of an antibody targeting agent to a small molecule drug and a fluorophore to be able to track drug delivery; however, there are numerous other applications of multivalent conjugates that can also be envisioned. Ultimately, the methodologies will be transitioned to biologically relevant systems including ubiquitin binding proteins, esterases, antibodies, and Cas9/dCas9. 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.
Bioconjugates provide a mechanism for the targeted diagnosis and treatment of cancer and other diseases. As such, improved methodologies for their preparation can further facilitate their therapeutic relevance. We aim to both develop novel bioconjugations, and to synthesize and incorporate unique unnatural amino acids into proteins in order to address current deficiencies associated with bioconjugate preparation.
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