Alanine Radicals:  Structure Determination by EPR and ENDOR of Single Crystals X-Irradiated at 295 K

Abstract

Single crystals of the amino acid l-α-alanine have been X-irradiated at room temperature, and the free radical formation has been studied using X-band and K-band EPR, ENDOR, and EIE (ENDOR-induced EPR) spectroscopy in the temperature interval 220−295 K. Aided by the results from EIE, as well as ENDOR from selected magnetic field positions, nine hyperfine coupling tensors were obtained and assigned to three different radicals. Room-temperature relaxation behaviors characterized by efficient W1 x and W1e and by slow W1 n relaxation rates allowed for determination of the signs of the various hyperfine couplings from the ENDOR spectra obtained at room temperature. The temperature dependence of the W1 x relaxation is qualitatively discussed. The EPR spectra from alanine are dominated by the well-known resonance of the “stable alanine radical”, SAR, formed by a net deamination of the protonated alanine anion. Precise hyperfine coupling tensors due to the α-proton coupling, the methyl group coupling, and a dipolar coupling to a methyl group of a neighboring molecule, as well as the g tensor, are given for this radical. Spectral simulations show that these parameters in a satisfactory manner reproduce all observable features of the resonance from this radical. Radical R2, apparently formed in roughly the same amounts as SAR, exhibits the structure H3N+−•C(CH3)C(O)O-. It is formed from alanine by a net H-abstraction from the Cα position. The hyperfine coupling tensor to the freely rotating methyl group was obtained from both X-band and K-band data. K-band spectra obtained at several temperatures between 220 and 290 K revealed that the amino group is not freely rotating; that is, the three protons of the amino group are locked in their hydrogen bonds also after radical formation. A significant increase in ENDOR line widths upon increasing temperature made the ENDOR lines due to the amino protons practically nonobservable at 295 K. However, the three corresponding hyperfine coupling tensors were easily obtained from K-band ENDOR data at 220 K. The B 0 and B 2 values for β-coupling to N+−H fragments were determined to be −4.3 and 117.6 MHz, respectively. Due to partly unresolved nitrogen hyperfine interaction leading to larger line widths, the individual EPR lines from radical R2 are of far less intensity as compared to those of the SAR. However, simulations strongly indicate that there is an almost equal relative distribution (60%:40%) of the two radicals. Two hyperfine coupling tensors were assigned to two conformations of a third minority radical species (radical R3) which tentatively is suggested to be the species H2N−•C(CH3)C(OH)O. Possible mechanisms for the formation of the radicals are discussed in light of the basic radiation chemistry of the amino acids. The simultaneous presence of two stable radicals of similar relative amounts in alanine may have consequences for the use of alanine as a radiation dosimeter.

Density functional theory as a tool for the structure determination of radiation-induced bioradicals

“…It can therefore be concluded that the amino group is slightly 'skewed' from the radical plane in the radical R2 conformation. This is in line with the conclusions of Sagstuen et al, 64 who suggested a value of 7.3°, assuming a perfectly planar radical backbone. In their work, 8.3, 8.2 and 5.5 MHz were proposed for the principal values of the N4 hyperfine interaction.…”

“…At the UB3LYP/6-311G(d) level, the total spin density on the central carbon atom C2 amounts to 0.93 (0.96 with the IGLO III basis set), which is substantially higher than the previously estimated value of 0.72 reported by Sagstuen et al 64 It can therefore be expected that the hyperfine interaction will be mainly due to the methyl and amino protons. For the methyl protons, our calculations suggest comparable couplings to those obtained for radical R1, as can be seen from Table 1.…”

“…Sagstuen et al assigned two hyperfine coupling tensors for the methyl protons corresponding to two conformations of the radical R3 in the crystal lattice. 64 Apart from the H8, H9, H10 values present in Table 1, they also reported a value of 33.1 MHz for the isotropic hfcc and 37.7, 30.9 and 30.8 as the principal values for a second radical R3 conformation in the crystal lattice. Again, the agreement between the theoretical and experimental values is excellent.…”

“…2. 64 Recently however, Sagstuen and co-workers presented experimental evidence for the existence of R2 in irradiated alanine. 64 -66 In these contributions, evidence for the existence of a third radical R3 was put forward.…”

Solved? The reductive radiation chemistry of alanine

“…However, it puts the detected nitrogen on an alanine molecule neighboring the radical and not on the detached ammonium, as can be seen in Figure SI-2 of the supplementary information. The H(C) dipolar coupling detected by Sagstuen et al 17 at room temperature was attributed to a nearby methyl group. Our calculations confirm this analysis: the measurements agree very well with the calculated hyperfine tensor obtained by averaging the individual proton tensors of the methyl group located just below the SAR radical (see Figure SI-2).…”