Bioconjugations Employing Unnatural Amino Acids

College Of William And Mary, Williamsburg VA

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Abstract

Project Abstract 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.