Single-molecule fluorescence detection of a tricyclic nucleoside analogue.

Fluorescent nucleobase surrogates capable of Watson-Crick hydrogen bonding are essential probes of nucleic acid structure and dynamics, but their limited brightness and short absorption and emission wavelengths have rendered them unsuitable for single-molecule detection. Aiming to improve on these properties, we designed a new tricyclic pyrimidine nucleoside analogue with a push-pull conjugated system and synthesized it in seven sequential steps. The resulting C-linked 8-(diethylamino)benzo[b][1,8]naphthyridin-2(1H)-one nucleoside, which we name ABN, exhibits ε 442 = 20 000 M-1 cm-1 and Φ em,540 = 0.39 in water, increasing to Φ em = 0.50-0.53 when base paired with adenine in duplex DNA oligonucleotides. Single-molecule fluorescence measurements of ABN using both one-photon and two-photon excitation demonstrate its excellent photostability and indicate that the nucleoside is present to > 95% in a bright state with count rates of at least 15 kHz per molecule. This new fluorescent nucleobase analogue, which, in duplex DNA, is the brightest and most red-shifted known, is the first to offer robust and accessible single-molecule fluorescence detection capabilities.

Structure-based design of guanosine analogue inhibitors targeting GTP cyclohydrolase IB towards a new class of antibiotics.

GTP cyclohydrolase (GCYH-I) is an enzyme in the folate biosynthesis pathway that has not been previously exploited as an antibiotic target, although several pathogens including N. gonorrhoeae use a form of the enzyme GCYH-IB that is structurally distinct from the human homologue GCYH-IA. A comparison of the crystal structures of GCYH-IA and -IB with the nM inhibitor 8-oxo-GTP bound shows that the active site of GCYH-IB is larger and differently shaped. Based on this structural information, we designed and synthesized a small set of 8-oxo-G derivatives with ether linkages at O6 and O8 expected to displace water molecules from the expanded active site of GCYH-IB. The most potent of these compounds, G3, is selective for GCYH-IB, supporting the premise that potent and selective inhibitors of GCYH-IB could constitute a new class of small molecule antibiotics.

2H NMR Reveals Liquid State-Like Dynamics of Arene Guests Inside Hexameric Pyrogallol[4]arene Capsules in the Solid State.

The dynamics of guests in molecular encapsulation complexes have been studied extensively in solution, but the corresponding behavior of those guests when the capsules are present in the solid state is not as well understood. Here we report on comparative solution 1H and solid-state 2H NMR measurements of encapsulation complexes of fluorene(-d 2), fluoranthene(-d 10), and pyrene-(-d 10) in pyrogallol[4]arene hexamers assembled in the solid state by ball milling. In solution, the 1H spectra show that these rigid guests tumble and exchange positions quickly within the capsules' interiors, with the exception of pyrene, which has slower tumbling and positional exchange. Static solid-state 2H NMR using the deuterated guests shows that, when the capsules are in the solid state, their guests retain the liquid state-like dynamics observed for the capsules in solution. When the pyrogallol[4]arene hexamers' pendant decyl groups were substituted with propyl groups, guest dynamics in the solid state were slowed. We propose that these pendant alkyl groups form an interdigitated and dynamic waxy domain surrounding the capsules in the solid state, and that the greater mobility of the decyl groups is translated across the walls of the host, resulting in more rapid guest dynamics in the capsules' interiors.

Synthesis of Fluorescence Turn-On DNA Hybridization Probe Using the DEA tC 2'-Deoxycytidine Analog.

DEA tC is a tricyclic 2'-deoxycytidine analog that can be incorporated into oligonucleotides by solid-phase synthesis and that exhibits a large fluorescence enhancement when correctly base-paired with a guanine base in a DNA-DNA duplex. The synthesis of DEA tC begins with 5-amino-2-methylbenzothiazole and provides the DEA tC nucleobase analog over five synthetic steps. This nucleobase analog is then silylated using N,O-bis(trimethylsilyl)acetamide and conjugated to Hoffer's chlorosugar to provide the protected DEA tC nucleoside in good yield. Following protective-group removal and chromatographic isolation of the ß-anomer, dimethoxytritylation and phosphoramidite synthesis offer the monomer for solid-phase DNA synthesis. Solid-phase DNA synthesis conditions using extended coupling of the DEA tC amidite and a short deprotection time are employed to maximize efficiency. By following the protocols described in this unit, the DEA tC fluorescent probe can be synthesized and can be incorporated into any desired synthetic DNA oligonucleotide. © 2018 by John Wiley & Sons, Inc.