Przemysław J. Boratyński

Room-Temperature Distance Measurements of Immobilized Spin-Labeled Protein by DEER/PELDOR

Nitroxide spin labels are used for double electron-electron resonance (DEER) measurements of distances between sites in biomolecules. Rotation of gem-dimethyls in commonly used nitroxides causes spin echo dephasing times (Tm) to be too short to perform DEER measurements at temperatures between ∼80 and 295 K, even in immobilized samples. A spirocyclohexyl spin label has been prepared that has longer Tm between 80 and 295 K in immobilized samples than conventional labels. Two of the spirocyclohexyl labels were attached to sites on T4 lysozyme introduced by site-directed spin labeling. Interspin distances up to ∼4 nm were measured by DEER at temperatures up to 160 K in water/glycerol glasses. In a glassy trehalose matrix the Tm for the doubly labeled T4 lysozyme was long enough to measure an interspin distance of 3.2 nm at 295 K, which could not be measured for the same protein labeled with the conventional 1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-(methyl)methanethio-sulfonate label.

High-Spin S = 2 Ground State Aminyl Tetraradicals

Aminyl tetraradicals with planar tetraazanonacene backbones have quintet (S = 2) ground states and do not show any detectable thermal population of the low-spin excited states up to the highest temperature investigated (100 K) in the 2-methyltetrahydrofuran (2-MeTHF) matrix. This indicates that the nearest electronic excited state (triplet) is at least ~0.3 kcal mol(-1) higher in energy, that is, the triplet-quintet energy gap, ΔE(TQ) > 0.3 kcal mol(-1), which is consistent with the broken-symmetry-DFT-computed ΔE(TQ) of about 5 kcal mol(-1). In concentrated (ca. 1-10 mM) solutions of tetraradical 4 in 2-MeTHF at 133 K, a fraction of tetraradicals form a dimer (association constant, K(assoc) ≈ 60 M(-1)), with a weak, antiferromagnetic exchange coupling, J/k ≈ -0.1 K ~ 0.2 cal mol(-1), between the S = 2 tetraradicals. This weak intradimer exchange coupling is expected for two tetraradicals at the distance of about 6 Å. The most sterically shielded tetraradical 5 in 2-MeTHF has a half-life of 1 h at room temperature; the product of its decay is the corresponding tetraamine, suggesting that the hydrogen atom abstraction from the solvent is primarily responsible for the decomposition of the tetraradical.

Isolation of the Triplet Ground State Aminyl Diradical

Nitrogen-centered (aminyl) radicals are typically short-lived, and have been detected as intermediates in a variety of chemical and biological processes. 2] To the best of our knowledge, only two known stable aminyl radicals have been reported (more than 30 years ago), 4] while a few others have recently been isolated as aminyl metal complexes and as heteroatom (e.g., O, S) stabilized aminyls. Aminyl diradicals, in which the radical centers are connected through m-phenylene to form a planar crossconjugated p system devoid of stabilizing heteroatoms, are predicted by computational studies to possess a strong preference for a high-spin, triplet (S = 1) ground state, that is, a singlet–triplet energy gap (DEST) that is an order of magnitude greater than the thermal energy (RT) at room temperature. 9] Such a nitrogen-centered diradical with large DEST values and persistence at room temperature may be relevant to the design of strongly paramagnetic relaxation reagents as well as the development of magnetic materials and devices. 13] To date, the two known examples of aminyl diradicals have been examined only at very low temperature. Platz and coworkers first reported an aminyl diradical in a 2-methyltetrahydrofuran (2-MeTHF) matrix at about 195 8C. Recently, we prepared an aminyl diradical with an S = 1 ground state that was persistent in solution at about 100 8C. Herein we report the isolation of an S = 1 ground state aminyl diradical with a large DEST value and persistence at room temperature. The synthesis of the aminyl diradicals 1 and 2 demonstrates the balance between maintaining an effective 2pp–2pp overlap that enhances electron spin–spin interactions (exchange coupling), and the adequate protection of the carbon and nitrogen atoms with significant spin densities within the diazapentacene backbone in order to achieve the stability required for isolation. This result is significant as the 2pp–2pp overlap is generally severely perturbed by the increased steric bulk that is required for achieving radical stability, and thus a combination of a large DEST value and persistence at room temperature is not commonly accomplished. In addition, we detected an unexpected self-associated dimer of the S = 1 aminyl diradical 2 in 2-MeTHF solution; this species appears to resemble a p dimer. Notably, characterization of the dimer is facilitated by its unique zero-field splitting tensor (D). p Dimers of neutral organic radicals in solution are relatively rare and the reports of such phenomena are limited to those of S = =2 radicals. Diradicals 1 and 2 were prepared from the corresponding diamines 5 and 6, which were obtained from diamine 3 (Scheme 1). The structure of diamine 6 was confirmed by X-ray crystallography, and shows overcrowding of the bulky tert-undecyl groups so that the C C bond axes that connect the two outer pendant benzene rings to the backbone bend outward (Figure 1). This bending creates more space for the pendant benzene rings in order to attain moderate torsion angles of 52.0(4), 61.0(4), and 68.2(3)8 with respect to the diazapentacene backbone. Nevertheless, both nitrogen atoms are well-shielded and the diazapentacene backbone remains planar in spite of the steric congestion. The structure of diradical 2, which differs from its diamine precursor by the absence of two hydrogen atoms, is expected to be similar to that of diamine 6. Such a sterically shielded, planar structure should have enhanced stability and a strong preference for the S = 1 ground state. In spite of hindered access to the NH moieties in the diazapentacene backbone, double deprotonation of diamines 5 and 6 in THF/hexane at about 30 8C, followed by oxidation of the resultant dianions with iodine between 90 and 115 8C provided the corresponding diradicals 1 and 2, respectively. Generation of 1 is difficult because of its low solubility, and characterization of 1 is possible only by EPR spectroscopy, which gave spectra that are typical of the aminyl diradical in the S = 1 state (Figure S3 in the Supporting Information). In contrast, the generation of 2 is very efficient, and is facilitated by the good solubility of the diradical and its diamine precursor. Diradical 2 was prepared on a 1 mg (1 mmol) scale in custom-designed sample tubes, thus enabling sequential measurements by EPR spectroscopy and superconducting quantum interference device (SQUID) magnetometry at low temperatures. The EPR spectra of concentrated purple-blue solutions of 2 in 2-MeTHF at 132 K are strikingly complex, [*] Dr. P. J. Boratyński, Prof. Dr. S. Rajca, Prof. Dr. A. Rajca Department of Chemistry, University of Nebraska Lincoln, NE 68588-0304 (USA) Fax: (+ 1)402-472-9402 E-mail: arajca1@unl.edu Homepage: http://www.chem.unl.edu/rajca/rajcahome.html

Intramolecular Cyclization of Thiophene-Based [7]Helicenes to Quasi-[8]Circulenes

Intramolecular cyclization in a series of thiophene-based dibromo[7]helicenes (4-6) with different helix structures is investigated by vacuum pyrolysis, tin- and palladium-mediated C-C bond forming reactions. The product with the cyclic structure of the annelated aromatic rings, which resembles [8]circulene devoid of an atom linkage, is referred to as quasi-[8]circulene. Vacuum pyrolysis of 4 gives insoluble, unidentified products, while 5 and 6 yield the corresponding quasi-[8]circulenes under similar conditions. Thermogravimetry (TG) and differential scanning calorimetry (DSC) analyses for 4 indicate complex reaction pathways, while those for 5 and 6 show a single process corresponding to a loss of 1 equiv of Br(2) at about 330 degrees C. Pd-mediated reductive cyclization provides quasi-[8]circulenes for all three [7]helicenes, though only 4 gives a good isolated yield. Tributyltin hydride-mediated radical cyclization of 4-6 provides quasi-[8]circulenes in excellent yields, and it is practically insensitive to the helix structure. Experimental and calculated UV-vis absorption spectra for quasi-[8]circulenes and [8]circulenes are reported. The results suggest that the lack of atom linkage in quasi-[8]circulene does not significantly affect properties and conformation, compared to those for the corresponding [8]circulenes.

Antiproliferative Activity of Polyether Antibiotic – Cinchona Alkaloid Conjugates Obtained via Click Chemistry

A series of eight new conjugates of salinomycin or monensin and Cinchona alkaloids were obtained by the Cu(I)‐catalysed 1,3‐dipolar Huisgen cycloaddition (click chemistry) of respective N‐propargyl amides of salinomycin or monensin with four different Cinchona alkaloid derived azides. In vitro antiproliferative activity of these conjugates evaluated against three cancer cell lines (LoVo, LoVo/DX, HepG2) showed that four of the compounds exhibited high antiproliferative activity (IC50 below 3.00 μm) and appeared to be less toxic and more selective against normal cells than two standard anticancer drugs.

Rapid-scan EPR of immobilized nitroxides

X-band electron paramagnetic resonance spectra of immobilized nitroxides were obtained by rapid scan at 293 K. Scan widths were 155 G with 13.4 kHz scan frequency for (14)N-perdeuterated tempone and for T4 lysozyme doubly spin labeled with an iodoacetamide spirocyclohexyl nitroxide and 100 G with 20.9 kHz scan frequency for (15)N-perdeuterated tempone. These wide scans were made possible by modifications to our rapid-scan driver, scan coils made of Litz wire, and the placement of highly conducting aluminum plates on the poles of a Bruker 10″ magnet to reduce resistive losses in the magnet pole faces. For the same data acquisition time, the signal-to-noise for the rapid-scan absorption spectra was about an order of magnitude higher than for continuous wave first-derivative spectra recorded with modulation amplitudes that do not broaden the lineshapes.

Diastereoselective Corey–Chaykovsky 9-Epoxymethylation of Cinchona Alkaloids: Access to Chiral Scaffolds with Diverse Functionalities

Reaction of dimethylsulfonium methylide with Cinchona alkaloid ketones proceeds with complete diastereoselectivity to give epoxides of 8,9-like configuration. The reaction of dimethylsulfoxonium methylide gives different isomers, albeit with lower (4:1) selectivity. α-Epimerization of the alkaloid ketones resulted in formation of two separable diasteromeric products. The configurations of the epoxides were elucidated on the basis of NMR data combined with DFT calculations. Models explaining observed selectivity are discussed. The epoxides were efficiently transformed to a number of derivatives through selective S(N)2-type ring-opening reactions with various nucleophiles, often without the need of additional purification steps.

Oxidation of Annelated Diarylamines: Analysis of Reaction Pathways to Nitroxide Diradical and Spirocyclic Products

Oxidation of diaryldiamine 2, a tetrahydrodiazapentacene derivative, provides diarylnitroxide diradical 1 accompanied by an intermediate nitroxide monoradical and a multitude of isolable diamagnetic products. DFT-computed tensors for EPR spectra and paramagnetic (1)H NMR isotropic shifts for nitroxide diradical 1 show good agreement with the experimental EPR spectra in rigid matrices and paramagnetic (1)H NMR spectra in solution, respectively. Examination of the diamagnetic products elucidates their formation via distinct pathways involving C-O bond-forming reactions, including Baeyer-Villiger-type oxidations. An unusual diiminoketone structure and two spirocyclic structures of the predominant diamagnetic products are confirmed by either X-ray crystallography or correlations between DFT-computed and experimental spectroscopic data such as (1)H, (13)C, and (15)N NMR chemical shifts and electronic absorption spectra.

Site and stereoselectivity in sulfa-Michael addition to equivocally activated conjugated dienes

The regiochemical course of sulfa-Michael addition of thiols to terminally divergently activated dienes partially incorporated in a cyclic system, as in substituted 2-(3-oxocyclohexenyl)-vinyls, can be to some extent altered by the choice of activation method. The ratio of products of competing 1,4- and 1,6-addition reactions is dependent on the potency of the electron withdrawing group. The iminium ion catalysis favors 1,6-addition to the cyclic ketone group, changing the product preference for dienes terminated with ketone and aryl ester groups. Application of 9-epi-aminoquinine analogues and an acid allowed for both regioselective and enantioselective (up to 90% ee) addition at the distant δ position.

Stereoselective C9 Arylation and Vinylation of Cinchona Alkaloids

A simple and efficient method for the highly stereoselective C-9 arylation and vinylation of Cinchona alkaloids was developed. Both 9S- and 9R-chloroquinine with PhMgBr yielded 9S-phenylquinine (X-ray structure). The reactions with various aryl and vinyl Grignard reagents resulted in the series of 9S-aryl and vinyl alkaloid derivatives. The stereochemical outcome was rationalized by coordination of the magnesium atom to the quinuclidine nitrogen, thus directing the nucleophilic attack at the C-9 stereogenic center.