William H. Nelson

ESR study of the guanine cation

It has been proposed that the primary direct radiation damage products in DNA are guanine cations and thymine anions. Experiments reported here characterize a guanine cation observed in a single crystal of guanine:HCl:H2O. ESR experiments were performed by x‐irradiating and observing the crystals at 15 K. Spectral parameters for the cation include N3 and N10 hyperfine couplings, a C8–Hα hyperfine coupling, and two small exchangeable couplings presumably from the N10 protons. The computed spin densities of ρ(N3)=0.283, ρ(N10)=0.168, and ρ(C8)=0.182 agree nicely with those observed for the guanine cation in DNA. In the single crystal the native molecule is protonated at N7. It is proposed that once the native molecule is oxidized it rapidly deprotonates at N7 to form the cation observed.

ESR and ENDOR study of the guanine cation: Secondary product in 5’‐dGMP

Previous ESR studies of x‐irradiated single crystals of 2’‐deoxyguanosine‐5’‐monophosphate have indicated the presence of a radical thought to be formed by deprotonation of a primary base cation at N1. In this communication are reported some results of detailed ESR and ENDOR experiments at 10 K conflicting with the above results. One of the radicals detected exhibited two α‐proton type couplings. The data analysis shows that one coupling is due to the exchangeable proton of the extra‐annular NH2 group, while the other is due to the proton bonded at C8. The experimental spin densities were ρ(N10)=0.33, and ρ(C8)=0.18. The results agree reasonably well with the INDO calculated spin density distribution of a radical formed by deprotonation at N10 of a primary cation radical. The radical is stable on warming to about 200 K where it anneals rapidly.

Estimation of errors in eigenvectors and eigenvalues from magnetic resonance results by use of linear data-fitting techniques

Abstract A technique for estimating errors in eigenvectors and eigenvalues resulting from magnetic resonance data is described. The procedure employs an error-propagation approach which permits use of linear data-fitting techniques. Also presented is a procedure for a simultaneous three-plane fit using the linear techniques.

Radical formation in X-irradiated single crystals of guanine hydrochloride monohydrate. II. ESR and ENDOR in the range 10-77 K.

In a study of guanine.HCl.H2O (Gm) single crystals X-irradiated at temperatures between 10 and 77 K, three radical species were found and characterized by ESR and ENDOR spectroscopy. All three are primary products in that they were present immediately following irradiation at T less than 10 K. Radical I, which apparently can exist in two slightly different conformations, was identified as the product of electron gain by the parent molecule and subsequent protonation at O6. Radical I decayed only after warming the crystals beyond 250 K. Radical II was the guanine cation previously reported (D. M. Close, E. Sagstuen, and W. H. Nelson, J. Chem. Phys. 82, 4386 (1985)); however, ENDOR data are reported here which confirm the previous results. The guanine cation in Gm resulted from electron loss from the parent and subsequent deprotonation at N7. It is proposed that Radical III results from OH attack at C8 of the parent molecule, followed by rupture of the C8-N9 bond and ring opening. The OH radicals thought to produce Radical III result from electron loss by the cocrystallized water molecules. The reaction leading to Radical III, unusual in solid-state radiation chemistry, is thought to be mediated by the specific hydrogen bonding network in this crystal.

ESR and ENDOR Study of Adenosine Single Crystals X-Irradiated at 10 K

Single crystals of adenosine were X-irradiated at 10 K and investigated between 10 and 300 K using K-band ESR and ENDOR spectroscopy. Two free radicals were analyzed. Radical I exhibits small hyperfine couplings to the C8-H, C2-H, and a N3-H protons, and was identified as the N3 protonated base anion radical. Radical II exhibits small hyperfine couplings to a C8-H and an exocyclic -N10-H proton. It is suggested that this is therefore the N10 deprotonated base cation radical. Enough data were not available to analyze a third primary radical believed to be located on the ribose moiety. Upon warming Radical I decays at ca. 40 K with no apparent successor. Likewise, no successor was identified for Radical II, which decays at ca. 100 K. At ca. 200 K there is ESR evidence for the C2 and C8 H-addition radicals. Their precursors have not been identified.

ESR/ENDOR Study of Guanine · HCl · 2H2O X-irradiated at 20K

Single crystals of guanine hydrochloride dihydrate, in which the guanine base is protonated at N7, were X-irradiated at 20K, 65K and 150K. Study with K-band ESR and ENDOR techniques indicated at least four radical species to appear in the temperature range 20-300K. Three of the radical species (Radicals 1, 2, and 3) were present immediately following irradiation at 20K. Radical 1 was identified as the molecular anion protonated at O6. Hyperfine couplings in Radical 1 to HC8, HN1, and HN7 were fully characterized with ENDOR spectroscopy. The data indicated this product to be trapped in four different conformations which coalesced into the most stable form as the sample temperature was raised to ca. 180K. Radical 2 was the C8 H-addition radical. For this radical, hyperfine couplings to HN7, HN9, and the two beta-methylene protons were fully characterized with ENDOR spectroscopy. Radical 3 was the result of hydroxyl addition to C8 of the guanine base. For Radical 3, full characterization by ENDOR was possible for couplings to HN7, HN9 and the proton at C8. Annealing the samples beyond 250K for several hours led to disappearance of Radical 1, and appearance of Radical 4. The evidence indicated that Radical 4 was the result of net hydrogen abstraction from N9 of the guanine base. The ENDOR results permitted full characterization of hyperfine couplings to HN7 and the two amino protons (the HN10s). From these results, and those from two other systems containing N7-protonated guanine bases, reaction mechanisms are proposed to account for formation of the products.

Environmental Effects on Primary Radical Formation in Guanine: Solid-State ESR and ENDOR of Guanine Hydrobromide Monohydrate

Single crystals of guanine hydrobromide monohydrate, in which the guanine base is protonated at N7, were X-irradiated at 8 and 65 K. Using K-band ESR, ENDOR, and field-swept-ENDOR (FSE) techniques, the crystals were studied between 8 K and room temperature. There was evidence for five different radicals, RI-RV, immediately following irradiation at 8 or 65 K. RI was identified as the O6-protonated anion. It decayed near room temperature with no detectable successor. RII was identified as the N7-deprotonated cation, and decayed near 130 K. RIII is thought to be a ring-opened product formed by C8-N9 bond rupture; upon warming, it decayed at 150 K. RIV is the well-known C8 H-addition radical. These four radicals have been observed previously in the hydrochloride salt of guanine monohydrate. RV is novel, however, with magnetic characteristics consistent with those of the product formed by net OH addition to C5 of the unsaturated C4-C5 bond. It is characterized by four alpha-proton couplings indicating pi-electron spin as follows: 13% at C8; 11% at N7; and 12% at N10. RV decayed between 240 and 255 K with no detectable successor. Upon further warming, very weak resonance lines due to additional, unidentified radicals were observed. A comparison of these results with those obtained from other systems containing N7-protonated guanine bases demonstrates the effect of the environment on the primary radical formation.

Electron paramagnetic resonance and electron nuclear double resonance studies of X-irradiated crystals of cytosine hydrochloride. Part I : Free radical formation at 10 K after high radiation doses

Anhydrous single crystals of cytosine hydrochloride (protonated at N3) have been X-irradiated at 10 K and studied using K-band EPR, ENDOR and FSE spectroscopy. At least seven radicals were present at 10 K after X irradiation with a dose of about 150 kGy. Two different protonation states of the one-electron reduced cytosine cation were observed: an amino-protonated species (R1) and the pristine one-electron reduced species (R2) with zero net charge. Apparently three deprotonated versions of the one-electron oxidized cytosine cation were formed: the amino-deprotonated cation (R3), an N3-deprotonated cation (R4) and an N1-deprotonated cation (R5). Finally, two products formed by net hydrogen addition to the cytosine base were observed: a C5 hydrogen-addition radical (R6) and a C6 hydrogen-addition radical (R7). The crystalline lattice of cytosine hydrochloride is characterized in part by a cytosine base initially protonated at the N3-position, thus forming a cytosine base cation, and in part by an extended network of hydrogen bonding involving the chlorine anions. Proton transfer properties of pristine one-electron oxidation and reduction base products in this lattice are discussed and are suggested as explanations of the unusual multitude of positions for deprotonation of the one-electron oxidized species as well as for the two protonation states of the reduction product observed. The magnetic parameters for the amino-protonated species R1 agree well with those extracted from previous studies of cytosine derivatives in frozen solutions and in various glasses.

Radiation damage to DNA base pairs. II. Paramagnetic resonance studies of 1-methyluracil-9-ethyladenine complex crystals X-irradiated at 10 K

Single crystals of the co-crystalline complex of 1-methyluracil and 9-ethyladenine were X-irradiated and studied using EPR, ENDOR and FSE spectroscopic techniques at 10 K. All together seven radicals were identified, and experimental evidence for at least one more species, as well as for a very low population of radical pairs, is available. Oxidation and reduction products appear to be stabilized at both base constituents of the pair. Of the 1-methyluracil moiety, the product formed by net hydrogen abstraction from the methyl group was observed, together with the 1-methyluracil anion and the 1-methyluracil-5-yl radical. From the 9-ethyladenine moiety, the N3-protonated 9-ethyladenine anion is stabilized. In addition, the 9-ethyladenine cation as well as traces of the amino-deprotonated cation were observed, together with the C8-H hydrogen adduct. The presence of oxidation and reduction products in each of the two bases may indicate that negligible energy transfer takes place between them. This behavior is different from that observed in the similar pair of 1-methylthymine-9-methyladenine. There also seems to be minor proton exchange between the two stacks of molecules: Interbase protonation-deprotonation channeled through the hydrogen-bonding scheme seems to be almost completely suppressed.

Electronic g-factor measurement from ENDOR-induced EPR patterns: malonic acid and guanine hydrochloride dihydrate

Measurement of electronic g-factors (g) from radicals in irradiated organic crystals is generally difficult because the overall EPR pattern is usually the composite of several components, e.g., from multiple radicals and from multiple magnetic sites. However, when an ENDOR line is fully resolved, the method of ENDOR-induced EPR (EI-EPR, or EIE) in principle permits identification of the EPR pattern from the individual component yielding the line. To examine this method as an approach useful for measuring g, we used it to measure those of known radicals in two different crystal systems. First, to verify correspondence of the EIE and EPR sufficient for using EIE patterns to extract g, we used both EIE and EPR to measure g of (*CH(COOH)(2) from irradiated crystals of malonic acid. Then, to illustrate the procedure applied to a system giving a more complex EPR pattern, we used EIE to measure g of the O6-protonated anion radical of guanine in irradiated guanine.HCl.2H(2)O crystals. EPR results from the malonic acid radical are g(max)=2.00374(2), g(mid)=2.00331(2), and g(min)=2.00234(3); EIE results from the same radical are g(max)=2.00375(2), g(mid)=2.00334(2), and g(min)=2.00238(2), where numbers in parentheses indicate statistical uncertainties in the respective least significant digits. In addition, eigenvectors from the two sets of measurements agree to approximately 1 degrees. Results from the guanine radical are g(max)=2.00490(2), g(mid)=2.00318(4), and g(min)=2.00218(4). (The uncertainties should reliably indicate relative accuracy, while absolute accuracy is within +/-0.0002 as indicated by simultaneous measurement of Cr(3+) in MgO.)