Solvent Effects on the UV-Vis Absorption Properties and Tautomerism of N-Confused Tetraphenylporphyrin.

The two tautomeric forms of N-confused tetraphenylporphyrin (NCTPP) show distinctly different absorption spectra. The existence of each tautomer in solution has been shown to be strongly solvent-dependent. In the present work, we have studied the tautomerization using absorption spectroscopy in 15 different solvents. While changes in the two tautomers are not large in the Soret band region, the distinct spectral changes between the two tautomers in the Q-band region provide a convenient way to measure the concentration of each tautomer. The resulting data shows a strong correlation between the tautomer and the H-bond accepting ability of the solvent. The anomaly in this data is for the alcoholic solvents ethanol and methanol, for which we observe evidence for H-bonding, presumably between the exterior N2 nitrogen of the NCTPP and the O-H proton of the solvent.

Single-Molecule Fluorescence Using Nucleotide Analogs: A Proof-of-Principle.

Fluorescent nucleotide analogues, such as 2-aminopurine (2AP) and pyrrolo-C (PyC), have been extensively used to study nucleic acid local conformational dynamics in bulk experiments. Here we present a proof-of-principle approach using 2AP and PyC fluorescence at the single-molecule level. Our data show that ssDNA, dsDNA, or RNA containing both 2AP and PyC can be monitored using single-molecule fluorescence and a click chemistry immobilization method. We demonstrate that this approach can be used to monitor DNA and RNA in real time. This is the first reported assay using fluorescent nucleotide analogs at the single-molecule level. We anticipate that single 2AP or PyC fluorescence will have numerous applications in studies of DNA and RNA, including protein-induced base-flipping dynamics in protein-nucleic acid complexes.

Spectroscopy of free-base N-confused tetraphenylporphyrin radical anion and radical cation.

The radical anions and radical cations of the two tautomers (1e and 1i) of 5,10,15,20-tetraphenyl N-confused free-base porphyrin have been studied using a combination of cyclic voltammetry, steady state absorption spectroscopy, and computational chemistry. N-Confused porphyrins (NCPs), alternatively called 2-aza-21-carba-5,10,15,20-tetraphenylporphyrins or inverted porphyrins, are of great interest for their potential as building blocks in assemblies designed for artificial photosynthesis, and understanding the absorption spectra of the corresponding radical ions is paramount to future studies in multicomponent arrays where electron-transfer reactions are involved. NCP 1e was shown to oxidize at a potential of E(ox) 0.65 V vs Fc(+)|Fc in DMF and reduce at E(red) -1.42 V, while the corresponding values for 1i in toluene were E(ox) 0.60 V and E(red) -1.64 V. The geometries of these radical ions were computed at the B3LYP/6-31+G(d)//B3LYP/6-31G(d) level in the gas phase and in solution using the polarizable continuum model (PCM). From these structures and that of H(2)TPP and its corresponding radical ions, the computed redox potentials for 1e and 1i were calculated using the Born-Haber cycle. While the computed reduction potentials and electron affinities were in excellent agreement with the experimental reduction potentials, the calculated oxidation potentials displayed a somewhat less ideal relationship with experiment. The absorption spectra of the four radical ions were also measured experimentally, with radical cations 1e(•+) and 1i(•+) displaying significant changes in the Soret and Q-band regions as well as new low energy absorption bands in the near-IR region. The changes in the absorption spectra of radical anions 1e(•-) and 1i(•-) were not as dramatic, with the changes occurring only in the Soret and Q-band regions. These results were favorably modeled using time-dependent density functional calculations at the TD-B3LYP/6-31+G(d)//B3LYP/6-31G(d) level. These results were also compared to the existing data of free base tetraphenylporphyrin and free base tetraphenylchlorin.

Laser-assisted single-molecule refolding (LASR).

To assemble into functional structures, biopolymers search for global minima through their folding potential energy surfaces to find the native conformation. However, this process can be hindered by the presence of kinetic traps. Here, we present a new single-molecule technique, termed laser-assisted single-molecule refolding (LASR), to characterize kinetic traps at the single-molecule level. LASR combines temperature-jump kinetics and single-molecule spectroscopy. We demonstrate the use of LASR to measure single-molecule DNA melting curves with ∼1°C accuracy and to determine the activation barrier of a model kinetic trap. We also show how LASR, in combination with mutagenesis, can be used to estimate the yields of competing pathways, as well as to generate and characterize transient, unstable complexes.

Photoinduced electron-transfer within osmium(II) and ruthenium(II) bis-terpyridine donor acceptor dyads.

The synthesis and photophysical characterization of two different series of electron donor-acceptor dyads containing Ru(II) and Os(II) bis-terpyridines (M(tpy)(2)(2+)) were prepared and studied in order to compare the oft-studied Ru(tpy)(2)(2+) chromophore with the less studied Os(tpy)(2)(2+) chromophore. The first series of dyads incorporates a benzoquinone (BQ) group as the electron acceptor, whereas the second contains a substituted pyromellitimide (PI) group as the electron acceptor. Steady-state emission experiments indicated efficient quenching of the 3MLCT emission of the electronically excited Os(II)-BQ complexes (7-8) compared to both model complexes (3-4) and the Os(II)-PI complex 10. Femtosecond pump-probe absorption experiments on 7-8 yielded ultrafast electron transfer rate constants (kET) of approximately 2.0 x 10(11) s(-1) (7) and 1.3 x 10(10) s(-1) (8) that were in good agreement with the low emission quantum yield results. Charge-recombination (kCR) in these complexes was also quite rapid, with rate constants of approximately 6.7 x 10(10) s(-1) (7) and 1.2 x 10(10) s(-1) (8). The analogous Ru(II) complexes underwent charge separation with rate constants of 7.6 x 10(10) s(-1) (5) and approximately 2.3 x 10(10) s(-1) (6), while charge recombination in these complexes occurred with rate constants of approximately 2.1 x 10(10) s(-1) (5) and approximately 5.3 x 10(10) s(-1) (6). Electron transfer in the pyromellitimide-containing complexes occurred only for Os(II)-PI (10), which exhibited significantly slower electron transfer (approximately 4.3 x 10(6) s(-1)) and charge recombination (approximately 7.7 x 10(6) s(-1)) rate constants. The nearly thermoneutral free energy of electron transfer and short excited state lifetime in the case of Ru(II)-PI (9) presumably prevents electron transfer in this compound.

Exploring RNA folding one molecule at a time.

RNA molecules fold into stable native structures to perform their biological function. RNA folding can be influenced by ions, co-factors, and proteins through numerous mechanisms. Understanding these mechanisms at the molecular level is important for elucidating the structure-function relationship in biologically important RNAs. Recent developments in single molecule spectroscopy have provided new approaches to investigate RNA folding and have allowed identification of kinetic intermediates that would otherwise remain hidden in ensemble-averaged experiments. Here we summarize some of these developments, which provide new insight into the effect of Mg(2+) ions in RNA folding landscapes, the role of cooperativity in RNA tertiary folding, the stepwise folding of RNA during transcription, and the hierarchical assembly of RNA-protein complexes.

Focus on function: single molecule RNA enzymology.

The ability of RNA to catalyze chemical reactions was first demonstrated 25 years ago with the discovery that group I introns and RNase P function as RNA enzymes (ribozymes). Several additional ribozymes were subsequently identified, most notably the ribosome, followed by intense mechanistic studies. More recently, the introduction of single molecule tools has dissected the kinetic steps of several ribozymes in unprecedented detail and has revealed surprising heterogeneity not evident from ensemble approaches. Still, many fundamental questions of how RNA enzymes work at the molecular level remain unanswered. This review surveys the current status of our understanding of RNA catalysis at the single molecule level and discusses the existing challenges and opportunities in developing suitable assays.

Ultrafast spectroscopy of free-base N-confused tetraphenylporphyrins.

The photophysical characterization of the two tautomers (1e and 1i) of 5,10,15,20-tetraphenyl N-confused free-base porphyrin, as well as the tautomer-locked 2-methyl 5,10,15,20-tetraphenyl N-confused free-base porphyrin, was carried out using a combination of steady state and time-resolved optical techniques. N-Confused porphyrins, alternatively called 2-aza-21-carba-porphyrins or inverted porphyrins, are of great interest for their potential as building blocks in assemblies designed for artificial photosynthesis, and understanding their excited-state properties is paramount to future studies in multicomponent arrays. Femtosecond resolved transient absorption experiments reveal spectra that are similar to those of tetraphenylporphyrin (H2TPP) with either Soret or Q-band excitation, with an extinction coefficient for the major absorbing band of 1e that was about a factor of 5 larger than that of H2TPP. The lifetime of the S1 state was determined at a variety of absorption wavelengths for each compound and was found to be consistent with time-resolved fluorescence experiments. These experiments reveal that the externally protonated tautomer (1e) is longer lived (tau = 1.84 ns) than the internally protonated form (1i, tau = 1.47 ns) by approximately 369 ps and that the N-methyl N-confused porphyrin was shorter lived than the tautomeric forms by approximately 317 ps (DMAc) and approximately 396 ps (benzene). Steady-state fluorescence experiments on tautomers 1e and 1i and the N-methyl analogues corroborate these results, with fluorescence quantum yields (Phi(Fl)) of 0.046 (1e, DMAc) and 0.023 (1i, benzene), and 0.025 (DMAc) and 0.018 (benzene) for the N-methyl N-confused porphyrin. The lifetime and quantum yield data was interpreted in terms of structural changes that influence the rate of internal conversion. The absorption and transient absorption spectra of these porphyrins were also examined in the context of DFT calculations at the B3LYP/6-31G(d)//B3LYP/3-21G(d) level of theory and compared to the spectra/electronic structure of H2TPP and tetraphenyl chlorin.

Photophysical properties of a series of free-base corroles.

Four free-base corroles with electron-donating or electron-withdrawing groups on the para or 2 through 6-positons of the meso phenyl rings were prepared via either Paolesse or Gross conditions and investigated for their absorption and emission properties. The triaryl corroles 5,10,15-triphenylcorrole, 5,10,15-tris(pentafluorophenyl)corrole, 5,10,15-tris(p-nitrophenyl)corrole, and 5,10,15-tris(p-methoxyphenyl)corrole were examined. Absorption, steady-state, and time-resolved fluorescence measurements were performed on all compounds in both nonpolar (dichloromethane) and polar (dimethylacetamide) solvents. The experimental evidence points to hydrogen bonding with an internal N-H group as the most likely factor in the solvent-dependent photophysical behavior of these corroles, that is also highly dependent upon substitution.

Synthesis and spectroscopy of a series of substituted N-confused tetraphenylporphyrins.

A series of N-confused tetraphenylporphyrins (H(2)NCTPPs) with substituents on either the para- or the 3,5-positions of the meso phenyl rings were prepared using Lindsey conditions. Both electron-withdrawing and electron-donating groups were chosen in order to probe the effects of peripheral substitution on the properties of the macrocycles. The series includes 5,10,15,20-tetra-(4-R-phenyl) N-confused porphyrins (where R = bromo (1), iodo (2), cyano (3), methoxy (4), 2',5'-dimethoxyphenyl (5), or ethynyl (6)) and 5,10,15,20-(3,5-di-tert-butylphenyl) N-confused porphyrin (7). Absorption and steady-state fluorescence measurements were carried out, and quantum yields were measured for all compounds in both dichloromethane (CH(2)Cl(2)) and dimethylacetamide (DMAc).

One-step synthesis and characterization of difunctionalized N-confused tetraphenylporphyrins.

Three disubstituted N-confused porphyrins (2-4) were prepared in ca. 4% yield using a one-pot synthesis. These porphyrins bear 3,5-di-tert-butylphenyl groups substituted at the C(5) and C(20) meso positions and para-substituted (Br, NO(2), ethynyl) phenyl groups at the C(10) and C(15) meso positions. The specific orientation of the aryl rings around the macrocycle in porphyrin 2 was definitively determined using a combination of 1D ((1)H and (13)C) and 2D (gHMQC and gHMBC) NMR spectroscopy. The absorption spectra of 2-4 in CH(2)Cl(2) and dimethylacetamide are similar to those of N-confused tetraphenylporphyrin in the same solvents but have Soret and Q-bands that are shifted to lower energies. Steady-state fluorescence measurements revealed Q(x)(0,0) and Q(x)(0,1) bands similar in energy to the unsubstituted NCPs 1i and 1e. The fluorescence quantum yield results for two of these NCPs (2, 4) are atypical of porphyrin behavior and are being further investigated by time-resolved spectroscopy.