Tatiana I. Troitskaya

Physiological-Temperature Distance Measurement in Nucleic Acid using Triarylmethyl-Based Spin Labels and Pulsed Dipolar EPR Spectroscopy

Resolving the nanometer-scale structure of biomolecules in natural conditions still remains a challenging task. We report the first distance measurement in nucleic acid at physiological temperature using electron paramagnetic resonance (EPR). The model 10-mer DNA duplex has been labeled with reactive forms of triarylmethyl radicals and then immobilized on a sorbent in water solution and investigated by double quantum coherence EPR. We succeeded in development of optimal triarylmethyl-based labels, approach for site-directed spin labeling and efficient immobilization procedure that, working together, allowed us to measure as long distances as ~4.6 nm with high accuracy at 310 K (37 °C).

Room-Temperature Electron Spin Relaxation of Triarylmethyl Radicals at the X- and Q-Bands.

Triarylmethyl radicals (trityls, TAMs) represent a relatively new class of spin labels. The long relaxation of trityls at room temperature in liquid solutions makes them a promising alternative for traditional nitroxides. In this work we have synthesized a series of TAMs including perdeuterated Finland trityl (D36 form), mono-, di-, and triester derivatives of Finland-D36 trityl, the deuterated form of OX63, the dodeca-n-butyl homologue of Finland trityl, and triamide derivatives of Finland trityl with primary and secondary amines attached. We have studied room-temperature relaxation properties of these TAMs in liquids using pulsed electron paramagnetic resonance (EPR) at two microwave frequency bands. We have found the clear dependence of phase memory time (Tm ∼ T2) on the magnetic field: room-temperature Tm values are ∼1.5-2.5 times smaller at the Q-band (34 GHz, 1.2 T) than at the X-band (9 GHz, 0.3 T). This trend is ascribed to the contribution from g-anisotropy that is negligible at lower magnetic fields but comes into play at the Q-band. In agreement with this, the difference between T1 and Tm becomes more pronounced at the Q-band than at the X-band due to increased contributions from incomplete motional averaging of g-anisotropy. Linear dependence of (1/Tm - 1/T1) on viscosity implies that g-anisotropy is modulated by rotational motion of the trityl radical. On the basis of the analysis of previous data and results of the present work, we conclude that, in the general situation where the spin label is at least partly mobile, the X-band is most suitable for application of trityls for room-temperature pulsed EPR distance measurements.

Triarylmethanols with Bulky Aryl Groups and the NOESY/EXSY Experimental Observation of a Two-Ring-Flip Mechanism for the Helicity Reversal of Molecular Propellers

Triarylmethanols - the direct precursors of persistent trityl radicals - are racemic mixtures of chiral three-bladed molecular propellers. Depending on bulkiness of aryl groups they exhibit various liabilities to interconversion, the half- life time of room temperature racemization varying in a range between 8.4 hours and 1.32 years. NOESY/EXSY experiment performed on two representative models strongly supports the two-ring flip mechanism for the configurational interchange.

Triarylmethyl Radicals: An EPR Study of 13C Hyperfine Coupling Constants

Abstract Triarylmethyl (TAM) radicals are widely used in electron paramagnetic resonance (EPR) spectroscopy as spin labels and in EPR imaging as spin probes for in vivo oxymetry. One of the key advantages of TAMs is the extremely narrow EPR line, especially in case of deuterated analogs (~2.5 μT). Another advantage is their slow spin relaxation even at physiological temperatures; in particular, this characteristic enables the use of pulsed dipolar EPR methods for distance measurements in biomolecules. In this study, a large series of TAM radicals and their deuterated analogs was synthesized, and the corresponding spectroscopic parameters including 13C hyperfine constants were determined for the first time. We observed negligible dependence of 13C hyperfine constants on the solvent or on the structure or number of substituents at para-C atoms of the aromatic rings. In addition, we demonstrated that 13C signals at natural abundance can be used for successful distance measurements at room temperature by pulsed electron double resonance (PELDOR or DEER).

A combinatorially convenient version of synthesis of 5-substituted oxazole-4-carboxylic acid ethyl esters

Reaction of ethyl isocyanoacetic acid with sodium hydride in anhydrous benzene, followed by treatment with carboxylic acid chlorides or N-(acyloxy)pyrrolidine-2,5-diones, gives 5-substituted oxazole-4-carboxylic acid esters. The procedure is applicable to derivatives of various carboxylic acids, including saturated aliphatic, α,β-unsaturated, alicyclic, aromatic, heterocyclic, and N-Boc-protected amino acids.

A straightforward and convenient synthesis of Cbz-protected 2-(1-aminoalkyl)oxazole-5-carboxylates

Cbz-protected 2-(l-aminoalkyl)oxazole-5-carboxylates are readily obtainable by a two-step enantioselective sequence involving reaction of benzyloxycarbonyl protected amino acids with α-chloro-β-ketoesters and subsequent cyclization of the resulting acyloxyketones to required oxazoles upon treatment with neat ammonium trifluoacetate.