Thiols are one of the most important classes of chemicals used broadly in organic synthesis, biological chemistry, and nanosciences. Thiol pKa values are key indicators of thiol reactivity and functionality. This study is an internally-referenced Raman-based pH titration method that enables reliable quantification of thiol pKa values for both mono- and di-thiols in water. The degree of thiol ionization is monitored directly using the peak intensity of the S-H stretching feature relative to an internal reference peak as a function of solution pH. The thiol pKa values and Raman activity relative to its internal reference were then determined by curveitting the experimental data with equations derived on the basis of the Henderson-Hasselbalch equation. Using this Raman titration method, first and second thiol pKa values for 1,2-benzenedithol in water were determined for the first time. This method is convenient to implement and its underlying theory is easy to follow.

We have synthesized dimethyl derivatives of the GFP chromophore (p-HOBDI) and several of its derivatives, and their photochemistry and photophysics were investigated using various steady-state and time-resolved techniques as follows. We first consider the effect of the £]-barrel on the optical properties of the GFP chromophore (p-HOBDI) experimentally by selective variation of the protonation state of chromophores and different solvents. Each of these forms shows a complex solvatochromic behavior and is governed by both polar and acid/base properties of the solvents. In contrast to their solution behavior, some O-alkyl GFP chromophore (alkoxy-BDI) derivatives exhibit large fluorescent enhancement in the solid state. The color of the crystalline BDI is tuned by substituent-mediated crystal packing, showing the potential applications in optoelectronic devices. Using femtosecond polarization-sensitive infrared (IR) spectrosceopy of the C=O stretching mode of the HOBDI, we have then discovered a near complete twisting around the ethylenic bridge between the phenolate and imidazolidinone groups upon electronic excitation. Cis/trans isomerization induced by the rotation around the bridge is thought to be responsible for the behavior of blinking in fluorescent protein; however, the mechanism of the thermal reverse isomerization is more problematic. Thus we synthesized BDI derivatives with decreasing para-donating ability, HO, CH3O, CH3, H, and Cl, and used a Hammett plot for the rate study. With a positive â value, we conceived, for the first time, a novel nucleophilic addition/elimination mechanism. Finally, the GFP chromophore falls into the general category of hydroxyarene photoacids, which exhibit high excited-state acidities but neutral ground states. A hydroxyl substituent at the meta position shows enhanced charge transfer and greater acidity in the excited state. As a result, we have demonstrated that the fast quenching of the excited state by internal conversion to the ground state is much slower in meta- than in para-HOBDI derivatives. This allows studies of this ultrafast intermolecular ESPT that competes with isomerization. The photoinduced dynamics of the meta isomer of GFP chromophore was further investigated using femtosecond transient absorption and fluorescence upconversion spectroscopies.

This thesis summarizes the results of studies on two aspects of nucleic acids. Chemically modified antisense oligonucleotides (AONs) have been evaluated with regards to their suitability for mRNA targeting in an antisense approach (Paper I – III). The chemically modified nucleotidic units 2'-O-Me-T, 2'-O-MOE-T, oxetane-T, LNA-T, azetidine-T, aza-ENA-T, carbocyclic-ENA-T and carbocyclic-LNA-T were incorporated into 15-mer AONs and targeted against a 15-mer RNA chosen from the coding region of SV-40 large T antigen. The comparative study showed that a single modified nucleotide in the AON with North-East locked sugar (oxetane-T and azetidine-T) lowered the affinity for the complementary RNA whereas North locked sugars (LNA-T, aza-ENA-T, carbocyclic-ENA-T, and carbocyclic-LNA-T) significantly improved the affinity. A comparative RNase H digestion study showed that modifications of the same type (North-East type or North type) in different sequences gave rise to similar cleavage patterns. Determination of the Michaelis-Menten parameters by kinetic experiments showed that the modified AONs recruit RNase H resulting in enhanced turnover numbers (kcat) although with weaker enzyme-substrate binding (1/Km) compared to the unmodified AON. The modified AONs were also evaluated with regards to resistance towards snake venom phosphodiesterase and human serum to estimate their stability toward exonucleases. The aza-ENA-T and carbocyclic-ENA-T modified AONs showed improved stability compared to all other modified AONs. In general, the modified AONs with North type nucleotides (except LNA-T) were found to be superior to the North-East type as they showed improved target affinity, comparable RNase H recruitment capability and improved exonuclease stability. The second aspect studied in this thesis is based on physicochemical studies of short RNA molecules utilizing NMR based pH titration and alkaline hydrolysis reactions (Paper IV – V). The NMR based (1H and 31P) pH titration studies revealed the effect of guaninyl ion formation, propagated electrostatically through a single stranded chain in a sequence dependent manner. The non-identical electronic character of the internucleotidic phosphodiesters was further verified by alkaline hydrolysis experiments. The internucleotidic phosphodiesters, which were influenced by guaninyl ion formation, were hydrolyzed at a faster rate than those sequences where such guaninyl ion formation was prevented by replacing G with N1-Me-G.