Dewar Isomer Research Articles

Preparation and Kinetic Stabilization of Highly Strained Paracyclophanes

Abstract In this Account we describe the preparation, structures, and properties of the most highly strained paracyclophanes known to date, i.e. [4]- and [1.1]paracyclophanes and their derivatives. The preparation of these cyclophanes has been accomplished through the photochemical valence isomerization of corresponding 1,4-bridged Dewar benzene derivatives. These strained paracyclophanes are prone to polymerize and are not stable enough to isolate: [4]paracyclophane is rapidly consumed even below -100 °C in fluid solution, though it remains intact indefinitely in a frozen organic glass at 77 K, while [1.1]paracyclophane is stable only below -20 °C in solution. By introducing substituents which sterically hinder access to the bridgehead carbon atoms by other reagents, however, one can remarkably stabilize these species kinetically so as to allow the measurement of 1H NMR spectrum of the former and the isolation of the latter as crystals. The 1H NMR spectrum of the [4]paracyclophane strongly support, in conjunction with theoretical calculations, the sustenance of considerable aromaticity despite the extreme bending of its benzene ring. [4]Paracyclophane is so strained that it is thermodynamically less stable than the corresponding Dewar isomer and the thermal conversion of the benzene form to the Dewar form is observed for the first time. A fully unsaturated derivative of [4]paracyclophane is also generated and confirmed to prefer the structure of 1,2,3,4-tetradehydro[4]paracyclophane rather than that of π-bond isomer, bicyclo[4.2.2]deca-1,3,5,7,9-pentaene. The stabilized [1.1]paracyclophane is found to undergo efficient photochemical transformation into a benzene p,p′-dimer structure, from which the former is thermally regenerated upon mild heating. Structural characteristic features of the [1.1]paracyclophane and the benzene p,p′-dimer are revealed by X-ray crystallography. Limits for experimentally accessible strained paracyclophane are also discussed.

Ab initio study of the photochemical isomerization of thiophene derivatives

The photochemical isomerization reactions of thiophene, thiophene-2-carbonitrile, and 2-phenylthiophene were studied using ab initio methods. The results are in agreement with the previously reported data obtained through semiempirical methods. Triplet excited thiophene is a π,σ ∗ triplet with LSOMO at −9.94 eV and HSOMO at −9.51 eV and the biradical intermediate is a π,π ∗ species with LSOMO at −10.12 eV and HSOMO at −4.82 eV. In this case, the singlet excited state can evolve giving the Dewar thiophene, while the corresponding excited triplet state cannot be obtained. Furthermore, the triplet state cannot be converted into the biradical intermediate because this intermediate shows a higher energy than the triplet state, thus preventing the formation of the cyclopropenyl derivatives. Triplet excited thiophene-2-carbonitrile is a π,π ∗ species. It shows the LSOMO at −11.38 eV and the HSOMO at −7.36 eV. In this case, the direct irradiation involves the population of the excited singlet state, and then the formation of the Dewar isomer is possible. The intersystem crossing to the triplet state can occur; however, its interconversion into the corresponding biradicals cannot be efficient considering that the biradicals show a higher energy, even if for a little amount, than that of the triplet state. Triplet excited 2-phenylthiophene is a π,π ∗ species. It shows the LSOMO at −9.32 eV and the HSOMO at −6.24 eV. In this case, the direct irradiation involves the population of the excited singlet state, and then the formation of the Dewar isomer is possible. The energy of the excited singlet state was obtained from the UV absorption of the substrate. The intersystem crossing to the triplet state cannot occur; because it shows higher energy than the corresponding singlet state. Furthermore, its interconversion into the corresponding biradicals cannot be efficient considering that the biradicals show the same energy of the triplet state. The high efficiency of this reaction can be explained on the basis of low energy of the Dewar isomer.

Synthesis of an extremely crowded naphthalene via a stable norbornadienone.

Computational studies at the HF/3-21G and B3LYP/6-31G(d) levels suggest that 5,6,8-tri(tert-butyl)-1,2,3,4-tetraphenylnaphthalene (2) is perhaps the most crowded naphthalene derivative that will show normal stability; more highly congested naphthalenes will prefer to exist as the corresponding Dewar isomers. Initial attempts to prepare 2 by reacting 3,4,5,6-tetraphenylbenzyne with 2,3,5-tri(tert-butyl)cyclopentadienone at 83 degrees C gave instead a stable norbornadienone, 1,2,4-tri(tert-butyl)-5,6,7,8-tetraphenyl-9-oxo-1,4-dihydro-1,4-methanonaphthalene (3), which was characterized by X-ray crystallography. The experimental and calculated (HF/3-21G) activation energies (E(a)) for the decarbonylation of 3 were quite high: 39 and 46 kcal/mol, respectively, a manifestation of the substantially increased strain in the transition state as the tert-butyl groups are forced together. The naphthalene 2 was obtained in good yield by heating 3 in refluxing toluene, and its X-ray structure showed exceptional distortions from a normal naphthalene geometry. However, 2 is not completely stable, and it decomposes upon prolonged heating in xylenes.

Quantitation and visualization of ultraviolet-induced DNA damage using specific antibodies: application to pigment cell biology.

The major types of DNA damage induced by sunlight in the skin are DNA photoproducts, such as cyclobutane pyrimidine dimers (CPDs), (6-4)photoproducts (6-4PPs) and Dewar isomers of 6-4PPs. A sensitive method for quantitating and visualizing each type of DNA photoproduct induced by biologically relevant doses of ultraviolet (UV) or sunlight is essential to characterize DNA photoproducts and their biological effects. We have established monoclonal antibodies specific for CPDs, 6-4PPs or Dewar isomers. Those antibodies allow one to quantitate photoproducts in DNA purified from cultured cells or from the skin epidermis using an enzyme-linked immunosorbent assay. One can also use those specific antibodies with in situ laser cytometry to visualize and measure DNA photoproducts in cultured cells or in the skin, using indirect immunofluorescence and a laser-scanning confocal microscope. This latter method allows us to reconstruct three-dimensional images of nuclei containing DNA photoproducts and to simultaneously examine DNA photoproducts and histology in multilayered epidermis. Using those techniques, one can determine the induction and repair of these three distinct types of DNA photoproducts in cultured cells and in the skin exposed to sublethal or suberythematous doses of UV or solar simulated radiation. As examples of the utility of these techniques and antibodies, we describe the DNA repair kinetics following irradiation of human cell nuclei and the photoprotective effect of melanin against DNA photoproducts in cultured pigmented cells and in human epidermis.

Infrared spectra of Dewar 4-picoline in low-temperature argon matrices and vibrational analysis by DFT calculation

Dewar 4-picoline produced by UV irradiation on 4-picoline in a low-temperature argon matrix was monitored by FT-IR spectroscopy. The observed infrared bands were assigned to an isomer which has a C–C bridging bond with the aid of DFT (density functional theory) calculation, where the 6-31++G ** basis set was used to optimize the geometrical structure. No other isomer bands were observed. A systematic comparison of the scaling factors derived from the observed and calculated wavenumbers for pyridine, 2-picoline, 4-picoline and their corresponding Dewar isomers indicates a linear relationship between the scaling factors and the vibrational wavenumbers.

Ab initio study on the photochemical isomerization of furan derivatives

The photochemical isomerization reactions of furan, 2-methylfuran, 2-trimethylsilylfuran, and furan-2-carbonitrile were studied using ab initio methods. The results are in agreement with the previously reported data obtained through semiempirical methods. In particular, the sensitized irradiation of furan derivatives populates the first excited triplet state of the furan, and this triplet state can evolve only through O-Ca cleavage. The selection of the bond to be broken can depend on energetic factors (furan-2-carbonitrile) or on kinetic factors (2-methylfuran, 2-trimethylsilylfuran). The direct irradiation of furan derivatives populates the singlet excited state and leads to a conversion into the Dewar isomer or into the corresponding triplet state through the usual intersystem crossing procedure. The efficiency of these processes determines the presence or the absence of isomerized furan derivatives in the reaction mixtures.

Substrate specificity of ultraviolet DNA endonuclease (UVDE/Uve1p) from Schizosaccharomyces pombe.

Schizosaccharomyces pombe ultraviolet DNA endonuclease (UVDE or Uve1p) has been shown to cleave 5' to UV light-induced cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PP). This endonuclease is believed to function in the initial step in an alternative excision repair pathway for the removal of DNA damage caused by exposure to UV light. An active truncated form of this protein, Delta228-Uve1p, has been successfully overexpressed, affinity purified and partially characterized. In the present study we present data from a detailed substrate specificity trial. We have determined that the substrate range of Uve1p is much greater than was originally believed. We demonstrate that this DNA damage repair protein is capable of recognizing an array of UV-induced DNA photoproducts (cis-syn-, trans-syn I- and trans-syn II CPDs, 6-4PP and Dewar isomers) that cause varying degrees of distortion in a duplex DNA molecule. We also demonstrate that Uve1p recognizes non-UV-induced DNA damage, such as platinum-DNA GG diadducts, uracil, dihydrouracil and abasic sites. This is the first time that a single DNA repair endonuclease with the ability to recognize such a diverse range of lesions has been described. This study suggests that Uve1p and the alternative excision repair pathway may participate broadly in the repair of DNA damage.

The solution structure of DNA decamer duplex containing the Dewar product of thymidylyl(3′→5′)thymidine by NMR and full relaxation matrix refinement

The (6-4) adducts and their Dewar isomers play an important role in cytotoxicity and mutation in skin cells exposed to sunlight. Structural study of the DNA duplex containing a site-specific photoproduct is an essential step toward understanding the molecular mechanism of the mutagenesis and the repair activity of UV-irradiated DNA. Here we use 1H NMR spectroscopy and full relaxation matrix refinement to investigate the solution structure of the duplex Dewar decamer. We find that the isomerization of the (6-4) adduct to its Dewar form induces a substantial change in overall structure of the oligonucleotide duplex. Contrasting base stacking of two lesion sites results in a large difference in the structural impacts induced by the two photoproducts, such as differential disruption of hydrogen bonding at the lesion sites and overall helical bending of 44° by the (6-4) lesion and 21° by the Dewar lesion.

Synthesis and characterisation of oligodeoxynucleotides containing thio analogues of (6-4) pyrimidine—pyrimidinone photo-dimers

A method for the preparation of an oligodeoxynucleotide, 20 bases in length, containing centrally located thio analogues of (6-4) pyrimidine-pyrimidinone thymine photo-dimers is reported. The approach is based on the selective irradiation, at 350 nm, of a Tp4ST (4ST=4-thiothymidine) step within a 20-mer having the sequence: d(ACTCGGACCT(4ST)CGCTGTGAT). Conversion of the S5-(6-4)/S5-thietane pyrimidine-pyrimidinone, initially formed, to its S5-Dewar isomer is by a subsequent irradiation at 300 nm. Both of the photo-dimer-containing oligonucleotides were purified by HPLC (ion exchange and reverse phase) and characterised by base composition analysis. The S5-(6-4)/S5-thietane pyrimidine-pyrimidinone containing 20-mer has a characteristic UV absorbance at 320 nm and exhibits strong fluorescence when excited at this wavelength. As expected, conversion to the S5-Dewar isomer abolished both the 320 nm absorbance and the fluorescence emission. The lengths of the oligonucleotides produced allowed the formation of stable double-stranded DNA, by hybridisation to a complementary sequence. Examination of these duplexes by circular dichroism spectroscopy showed that they formed B-DNA, with little changes to their gross structure as compared to the parent duplex. However, local structural perturbations in the region of the photo-dimer cannot be excluded. The S5-(6-4)/S5-thietane photoproduct lowered the tmby 10.5 deg. C and the Dewar isomer by 12 deg. C. The degree of curvature induced in the DNA sequence by the introduction of the photo-dimers was assessed by analysing the migration of modified and unmodified multimer ladders on polyacrylamide gels. Both photoproducts induced considerable bending into the DNA. A comparison with a six-base-pair T tract, a bending standard that has a known bend angle of 19°, gave values of around 47° for the S5-(6-4)/S5-thietane product and about 28° for the S5-Dewar isomer.

Specificities and Rates of Binding of Anti-(6-4) Photoproduct Antibody Fragments to Synthetic Thymine Photoproducts

Pyrimidine (6-4) pyrimidone photoproducts are some of the major DNA photolesions induced by ultraviolet (UV) light. A monoclonal antibody (64M5) specific to a (6-4) photoproduct has been established and the corresponding single-chain antibody (64M5scFv) has been prepared. In this study, we characterized the ligand selectivities of 64M5 and 64M5scFv using synthetic octadeoxynucleotides containing either a central cis-syn cyclobutane thymine dimer (T[c,s]T), the (6-4) photoproduct of TpT (T[6-4]T), or its Dewar isomer (T[Dewar]T) by means of enzyme-linked immunosorbent assays (ELISA). Both 64M5 and 64M5scFv recognized T[6-4]T, but not the other photoproducts. We synthesized several biotinylated oligonucleotides of different lengths containing (T[6-4]T) to analyze the effects of the antigen size on the binding rates of an antigen binding fragment (64M5Fab) and 64M5scFv by means of surface plasmon resonance. The association rate constants for oligonucleotides of different sizes containing T[6-4]T as to 64M5Fab were found to be almost the same (1.9-5.6 x 10(5) M(-1) x s(-1)), while the dissociation rate constant for the largest oligonucleotide (d8mer, 8.0 x 10(-5) s(-1)) was significantly smaller than that for the d2mer (4.2 x 10(-2) s(-1)). These results indicate that 64M5Fab recognized the d2mer as the epitope and that the binding affinity for T[6-4]T depended on the flanking oligonucleotides. The dissociation rate constants for 64M5scFv as to the antigen analogs were almost the same as those for the various T[6-4]T-oligonucleotides as to 64M5Fab, suggesting that the conformations of these antibody binding regions are pretty similar to each other.

Reaction Mechanism of (6-4) Photolyase

The (6-4) photolyase catalyzes the photoreversal of the (6-4) dipyrimidine photoproducts induced in DNA by ultraviolet light. Using the cloned Drosophila melanogaster (6-4) photolyase gene, we overproduced and purified the recombinant enzyme. The binding and catalytic properties of the enzyme were investigated using natural substrates, T[6-4]T and T[6-4]C, and the Dewar isomer of (6-4) photoproduct and substrate analogs s5T[6-4]T/thietane, mes5T[6-4]T, and the N-methyl-3'T thietane analog of the oxetane intermediate. The enzyme binds to the natural substrates and to mes5T[6-4]T with high affinity (KD approximately 10(-9)-10(-10) M) and produces a DNase I footprint of about 20 base pairs around the photolesion. Several lines of evidence suggest that upon binding by the enzyme, the photoproduct flips out of the duplex. Of the four substrates that bind with high affinity to the enzyme, T[6-4]T and T[6-4]C are repaired with relatively high quantum yields compared with the Dewar isomer and the mes5T[6-4]T which are repaired with 300-400-fold lower quantum yield than the former two photoproducts. Reduction of the FAD cofactor with dithionite increases the quantum yield of repair. Taken together, the data are consistent with photoinduced electron transfer from reduced FAD to substrate, in a manner analogous to the cyclobutane pyrimidine dimer photolyase.

ChemInform Abstract: The First Observation of Thermal Transformation of Strained Paracyclophane into the Dewar Isomer.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform Abstract: X‐Ray Crystal Structures and ab initio Calculations on Photochemically Formed Dewar Isomers of Type (I) of 4(3H)‐Pyrimidinone Derivatives.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

X-ray Crystal Structures and ab Initio Calculations on the Photochemically Formed Dewar Isomers of the 4(3H)-Pyrimidinone Derivatives

The structures of the 5-oxo-2,6-diazabicyclo[2.2.0]hex-2-enes (imine Dewar pyrimidinones) 2a-h have been studied by X-ray diffraction analysis and ab initio calculations at the HF/6-31G(d,p) and MP2/6-31G(d,p) levels of theory. The crystal structures of 1-alkyl-3-tert-butyl-6-methyl imine Dewar pyrimidinones 2a-c have been determined at low temperature. The X-ray diffraction studies revealed that both the 2-azetidinone and dihydroazete rings are almost planar, and their eight bond angles are nearly 90 degrees (81-100 degrees ). The C1-N2(=C3) bond distance in the dihydroazete ring is longer due to the bond angle strain by ca. 0.06 Å than that of a typical straight-chain imine molecule. Full geometry optimizations on the imine Dewar isomers (2a and 2d-h) show deviations between the HF and MP2 geometries. The MP2 structure of the 3-tert-butyl-1,6-dimethyl imine Dewar pyrimidinone 2a is compared with those of the X-ray diffraction. The results of the calculations are found to be in good agreement with the X-ray diffraction data. The full geometry optimizations are necessary with the inclusion of electron correlation for the highly strained molecules. The ab initio calculations of the 2-azetidinone 9, 3,4-dihydroazete 11, and N-ethylidenemethylamine 12 have been carried out at the HF and MP2/6-31G(d,p) levels of theory to compare with the structures, orbital hybridization, bond orders, and charge distributions of the Dewar pyrimidinones 2. The theoretical results reveal that the MP2 C1-N2 bond distance of the Dewar pyrimidinone 2a is consistent with the abnormally elongated X-ray C1-N2 bond distance. The electronegative N2 and N6 atoms in the Dewar pyrimidinones 2 give the great positive charge on the C1 atom. The smaller bond angles (ca. 90 degrees ) in the Dewars 2 than typical sp(2) and sp(3) bond angles (ca. 120 and 109 degrees ) increase p character in the endocyclic bonds and decrease p character in the exocyclic bonds.

Thermodynamic studies of the hybridization properties of photolesions in DNA.

In order to gain insight into the mechanism of mutations induced by photolesions in DNA, the thermal melting curves of oligonucleotide duplexes containing the (6-4) photoproducts of TT and TC and the Dewar isomer were measured at varying concentrations, and the thermodynamic parameters were determined. The duplexes were designed to be used as models for template-primer systems. In the case of d(CAXYAGCACGAC) x d(GTCGTGCTN), in which XY = T(6-4)T, T(6-4)C, or T(Dewar)T and N = A, G, C, or T, the -deltaG degrees values were the largest when N = G, and the difference between N = G and N = C or T was 1.1, 0.8, and 0.5 kcal/mol at 25 degrees C for XY = T(6-4)T, T(6-4)C, and T(Dewar)T, respectively. These results provide evidence of base pair formation between the 3'-pyrimidone and the opposite guanine, which was proposed previously to explain the T --> C transition induced at the 3' side of the T(6-4)T photoproduct. This interaction was weakened by isomerization to the Dewar photoproduct, which would explain the lower mutation specificity. For d(CAXYAGCACGAC) x d(GTCGTGCTGN), no obvious differences were observed regarding the base opposite the 5'-pyrimidine of the photoproducts. In contrast to the tertiary structure, determined by NMR, of a duplex containing T(6-4)T in its center [Kim, J.-K., & Choi, B.-S. (1995) Eur. J. Biochem. 228, 849-854], base pair formation at the 5'-pyrimidine was not detected for any photoproduct used in this study, even when the base opposite the 3'-pyrimidone of T(6-4)T was changed to adenine.

The first observation of thermal transformation of strained paracyclophane into the Dewar isomer

M. Okuyama, M. Ohkita and T. Tsuji, Chem. Commun., 1997, 1277 DOI: 10.1039/A702663C

Synthesis of Medium and Large Ring Compounds, XLIII. Synthesis and Isomerization of 8,9‐Dimethyl[6]paracyclophane and Its Bridged Dewar Isomer

AbstractAn improved sonochemical procedure for the synthesis of dimethyl [6]paracyclophane‐8,9‐dicarboxylate (2a) on a 10‐g scale is described. The title compound 2d was synthesized from 2a in three steps. Irradiation of 2d yielded the Dewar isomer 3. In solution, the reaction enthalpy for the thermal isomerization of 3 to 2d was determined to be ‐162 ± 16 kJ · mol‐1, while the activation energy for this reaction was found to be 123 ± 12 kJ · mol‐1.

Photochemical fluorescence probes: rate distributions in solid polymers

Non-exponential fluorescence decays due to the photochemical reaction of 10-cyano-9-tert-butyl-anthracene towards the Dewar isomer are analyzed to gain information on the free volume of a polymer glass (poly- n-butyl-methacrylate) at different temperatures. The simple model of a multiexponential fitting procedure is well suited to following the temperature dependence in the envelope of this distribution qualitatively, but it fails to detect finer structures. More demanding methods (maximum entropy and exponential series) result in at least trimodal distributions indicating preferred polymer free volume voids within a broad distribution. Increasing temperature favours the decay time range associated with the largest free volume void.

Detection of Ultraviolet Photoproducts in Mouse Skin Exposed to Natural Sunlight

In the present study, we for the first time investigated the formation of ultraviolet (UV) photoproducts, cyclobutane pyrimidine dimers (CPDs), pyrimidine‐pyrimidone (6–4) photoproducts (64PPs) and Dewar isomers, in vivo in shaved and depilated C3H/HeN mouse skin exposed to natural sunlight (NSL) at noon for 5 min to 1 h in mid‐summer, using a highly sensitive immunohistochemical method. This method permits the quantitative analysis of UV‐photoproducts in formalin‐fixed, paraffin‐embedded sections with specific antibodies against CPDs, 64PPs and Dewar isomers. We demonstrated that the induction of CPDs in vivo in mouse skin by NSL was exposure time‐dependent, but the accumulation of 64PPs or Dewar isomers was comparatively low in the skin sections from mice exposed to NSL in vivo. The results indicate that CPDs are the main photoproducts in vivo induced by sunlight and that their formation and repair may be important in connection with carcinogenesis in sun‐exposed areas of human skin.

Inhibition of RNA and DNA synthesis in UV-irradiated normal human fibroblasts is correlated with pyrimidine (6-4) pyrimidone photoproduct formation

UV-irradiation of living cells results in an inhibition of RNA and DNA synthesis. The purpose of this study was to determine whether specific photoproducts or the total combined yield of lesions were responsible for these effects. Asynchronously dividing human fibroblasts from normal donors were irradiated with UVC (254 nm), broad spectrum UVB (290–320 + nm, Westinghouse FS20 lamp) or narrow spectrum UVB (310–315 nm, Philips TL01 lamp) at fluences which induce known yields of cyclobutane pyrimidine dimers, pyrimidine (6-4) pyrimidone photoproducts or Dewar isomers. DNA synthesis was approximately 3–4 times more sensitive to both UVC and UVB irradiation than RNA synthesis. The immediate inhibition of RNA and DNA synthesis was correlated with (6-4) rather than overall photoproduct formation suggesting that the (6-4) photoproduct is the mediator of these inhibitory effects. In support of this suggestion we found that photoreactivation of cells cultured from the marsupial, mouse Sminthopsis crassicaudata, resulted in removal of 70% of pyrimidine dimers from the overall genome, but had only a slight effect on the recovery of RNA synthesis.