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Title: Biological and chemical approaches toward site-directed protein modifications and a novel synthetic method to rhodamine B derivatives for protein labeling
Authors: CHEN XI
Keywords: protein modification, site-specific, ribonuclease A, lysozyme, somatostatin, rhodamine
Issue Date: 18-Jun-2010
Citation: CHEN XI (2010-06-18). Biological and chemical approaches toward site-directed protein modifications and a novel synthetic method to rhodamine B derivatives for protein labeling. ScholarBank@NUS Repository.
Abstract: The modification of proteins and the further decoration of them using reactive chromophores represent a recent topic of scientific interest. In this thesis, novel approaches toward site-directed protein modifications will be explored via either biology or chemistry means. In conjunction with those novel protein modification approaches, reactive rhodamine B dyes were prepared as useful chromophores to decorate those functionalized proteins and peptide. First of all, a biology method termed selective pressure incorporation (SPI) was developed to incorporate non-canonical amino acid (NCAA) into proteins in a site-directed fashion as exemplified by the incorporation of azidohomoalanine (AHA) into ribonuclease A (RNase A) in this thesis. AHA which is an analogue of methionine was synthesized with a considerably enhanced yield compared to recently reported protocols. This NCAA was efficiently incorporated into the protein RNase A via SPI, thus giving rise to a site-directed bi-azido functionalized RNase variant with two surface AHA located at AHA1 and AHA41 respectively. Two chemistry approaches toward site-directed protein modifications were also established that target cysteine (Cys) and lysine (Lys) respectively. The first cysteine-targeting approach requires the protein substrates to bare a single accessible cysteine residue on its surface, such as serum albumin proteins, antibody monomers and etc.. Compared to this method, the second chemistry approach which addresses lysine residue displays a wider application scope as lysine typically exists on most protein surfaces. The solvent accessibility of each lysine residue as well as the interaction between lysine and other residues in close vicinity play an important role to achieve the site-directionality. The success of this work was demonstrated by the mono-biotinlylation of RNase A, lysozyme C and somatostatin (SST) using a novel bioconjugation reagent?biotin-PEG-NHS. The last part of this thesis is dedicated to the preparation of functionalized rhodamine B dyes as reactive chromophore to label functional protein/peptides, such as azido-antibody and SST. This is a one step approach toward functionalized rhodamine B dyes via Steglich esterification. Compared to the previously reported protocol, this route is more practical, concise and avoids the usage of highly reactive reagents. Aside from rhodamine B ethyl ester, three reactive rhodamine B derivatives were prepared. They are propargyl rhodamine B ester for targeting azido group, azidoethyl rhodamine B ester for targeting ethynyl group and monosulfone-OH rhodamine B ester for targeting accessible disulfide bonds. Finally, their applicability for labeling corresponding functionalized protein/peptide is demonstrated.
Appears in Collections:Master's Theses (Open)

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