Electronic Modifications of Fluorescent Cytidine Analogues Control Photophysics and Fluorescent Responses to Base Stacking and Pairing.

The rational design of fluorescent nucleoside analogues is greatly hampered by the lack of a general method to predict their photophysics, a problem that is especially acute when base pairing and stacking change fluorescence. To better understand these effects, a series of tricyclic cytidine (tC and tCO ) analogues ranging from electron-rich to electron-deficient was designed and synthesized. They were then incorporated into oligonucleotides, and photophysical responses to base pairing and stacking were studied. When inserted into double-stranded DNA oligonucleotides, electron-rich analogues exhibit a fluorescence turn-on effect, in contrast with the electron-deficient compounds, which show diminished fluorescence. The magnitude of these fluorescence changes is correlated with the oxidation potential of nearest neighbor nucleobases. Moreover, matched base pairing enhances fluorescence turn-on for the electron-rich compounds, and it causes a fluorescence decrease for the electron-deficient compounds. For the tCO compounds, the emergence of vibrational fine structure in the fluorescence spectra in response to base pairing and stacking was observed, offering a potential new tool for studying nucleic acid structure and dynamics. These results, supported by DFT calculations, help to rationalize fluorescence changes in the base stack and will be useful for selecting the best fluorescent nucleoside analogues for a desired application.

Fluorescence Turn-On Sensing of DNA Duplex Formation by a Tricyclic Cytidine Analogue.

Most fluorescent nucleoside analogues are quenched when base stacked and some maintain their brightness, but there has been little progress toward developing nucleoside analogues that markedly increase their fluorescence upon duplex formation. Here, we report on the design and synthesis of a new tricyclic cytidine analogue, 8-diethylamino-tC (8-DEA-tC), that responds to DNA duplex formation with up to a 20-fold increase in fluorescent quantum yield as compared with the free nucleoside, depending on neighboring bases. This turn-on response to duplex formation is the greatest of any reported nucleoside analogue that can participate in Watson-Crick base pairing. Measurements of the quantum yield of 8-DEA-tC mispaired with adenosine and, separately, opposite an abasic site show that there is almost no fluorescence increase without the formation of correct Watson-Crick hydrogen bonds. Kinetic isotope effects from the use of deuterated buffer show that the duplex protects 8-DEA-tC against quenching by excited state proton transfer. These results, supported by DFT calculations, suggest a rationale for the observed photophysical properties that is dependent on duplex integrity and the electronic structure of the analogue.

Exploiting Atropisomerism to Increase the Target Selectivity of Kinase Inhibitors.

Many biologically active molecules exist as rapidly interconverting atropisomeric mixtures. Whereas one atropisomer inhibits the desired target, the other can lead to off-target effects. Herein, we study atropisomerism as a possibility to improve the selectivities of kinase inhibitors through the synthesis of conformationally stable pyrrolopyrimidines. Each atropisomer was isolated by HPLC on a chiral stationary phase and subjected to inhibitor profiling across a panel of 18 tyrosine kinases. Notably different selectivity patterns between atropisomers were observed, as well as improved selectivity compared to a rapidly interconverting parent molecule. Computational docking studies then provided insights into the structure-based origins of these effects. This study is one of the first examples of the intentional preorganization of a promiscuous scaffold along an atropisomeric axis to increase target selectivity, and provides fundamental insights that may be applied to other atropisomeric target scaffolds.

Characterizing substrate selectivity of ubiquitin C-terminal hydrolase-L3 using engineered α-linked ubiquitin substrates.

The ubiquitin-proteasome system (UPS) is highly complex and entails the concerted actions of many enzymes that function to ubiquitinate proteins targeted to the proteasome as well as enzymes that remove and recycle ubiquitin for additional rounds of proteolysis. Ubiquitin C-terminal hydrolase-L3 (UCH-L3) is a human cytosolic deubiquitinase whose precise biological function is not known. It is believed to hydrolyze small peptides or chemical adducts from the C-terminus of ubiquitin that may be remnant from proteasomal processing. In addition, UCH-L3 is a highly effective biotechnological tool that is used to produce small or unstable peptides/proteins recalcitrant to production in Escherichia coli expression systems. Previous research, which explored the substrate selectivity of UCH-L3, demonstrated a substrate size limitation for proteins/peptides expressed as α-linked C-terminal fusions to ubiquitin and also suggested that an additional substrate property may affect UCH-L3 hydrolysis [ Larsen , C. N. et al. (1998) Biochemistry 37 , 3358 - 3368 ]. Using a series of engineered protein substrates, which are similar in size yet differ in secondary structure, we demonstrate that thermal stability is a key factor that significantly affects UCH-L3 hydrolysis. In addition, we show that the thermal stabilities of the engineered substrates are not altered by fusion to ubiquitin and offer a possible mechanism as to how ubiquitin affects the structural and unfolding properties of natural in vivo targets.