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And Guanosine Monophosphate (GMP) Have Long-Lived VOL. 53, 1965 CHEMISTRY: RAHN, SHULMAN, AND LONGWORTH 893 6 Caglioti, L., and P. Grasselli, Chem. Ind. (London), 1964, 153 (1964). 6Eliel, E. L., Stereochemistry of Carbon Compounds (New York: McGraw-Hill Book Co., Inc., 1962), p. 236. THE UV-INDUCED TRIPLET STATE IN DNA BY R. 0. RAHN, R. G. SHULMAN, AND J. W. LONGWORTH BELL TELEPHONE LABORATORIES, MURRAY HILL, NEW JERSEY Communicated by W. 0. Baker, March 30, 1965 Previous investigations have shown that the phosphorescence from native DNA is not the sum of the emission from the individual nucleotides.1 . 2 In this note we shall present evidence from electron spin resonance (ESR) and optical emission studies which shows that the observed emission from UV-irradiated DNA is from the thymine moieties. Because thymine only phosphoresces when it loses its N1 proton (pK = 9.8), these results are consistent with an effective transfer of this proton from thymine to adenine during emission. All measurements reported were made at 770K on samples dissolved in a 1:1 ethylene glycol-water glass. Concentrations used for making optical measure- ments were in the range 10-3-10-4 M. For ESR measurements the concentrations ranged from 3 X 10-4 M to 3 X 10-2 M. Optical emission measurements were made using Bausch and Lomb 0.5-meter grating monochromators for both excita- tion and emission. The light was modulated at 150 cps and phase-sensitive de- tection was used to observe the signal. A Varian Associates X-band spectrometer with 100 kc/s modulation was used for the ESR measurements. For excitation in the microwave cavity an Osram 200-watt high-pressure Hg lamp was used with an aqueous NiSO4 filter. With the exception of poly rAU and poly dAT, at least three different samples from different sources were used for all of these measure- ments. At 770K, neutral pH, the purine nucleotides adenosine monophosphate (AMP) and guanosine monophosphate (GMP) have long-lived triplet states which phos- phoresce.3 4 The pyrimidine nucleotides thymidine monophosphate (TMP) and cytidine monophosphate (CMP) show no phosphorescence at neutral pH. In DNA, the observed phosphorescence bears little resemblance to either of the two purine nucleotides for, as shown in Figure 1, the DNA emission is less intense, red- shifted, and less structured than the purines. Measurements of the triplet-state lifetimes show that native DNA has a 0.3-sec decay time compared with 2.3 sec for AMP and 1.3 sec for GMP. From Figure 1 we estimate the quantum yield of phosphorescence of DNA to be --0.2 per cent compared with -2 per cent for GMP. As part of an attempt to understand these observations, measurements were carried out on synthetic polynucleotides which serve as models for DNA and RNA. Among the systems studied was poly rAU which is a double-stranded polymer with Watson-Crick hydrogen bonding between the adenine and uracil bases. In this system, where the adenine and uracil bases alternate in a single strand, the observed emission resembled that of uridine at pH > 10 where the N1 proton is removed Downloaded by guest on September 30, 2021 894 CHEMISTRY: RAHN, SHULMAN, AND LONGWORTH PROC. N. A. S. GUANOSINE U, I- 0-J 0 0 ADENOSINE DN 3500 4000 4500 5000 WAVELENGTH (A) FIG. 1.-Relative phosphorescence of DNA and its purine nucleotides. U) H ,'OLY dAT N 3500 4000 4500 5000 5500 WAVELENGTH (A) FIG. 2.-Phosphorescence of T-, poly-dAT, and DNA. (U-). This suggested the examination of the thymine analogue poly-dAT where the adenine-thymine base pairing is the same as that found in DNA and where ade- nine and thymine bases alternate in a single strand. Phosphorescence measure- ments revealed a striking similarity between DNA and poly-dAT as shown in Figure 2, with both molecules having a decay time of -0.3 sec (see Table 1). Since both poly-dAT and DNA have less than a 15 per cent contribution from adenine as judged from the structure observed, this result suggests that in both poly-dAT and DNA the emission is from thymine in some perturbed state. A study of the phosphorescence of thymidine as a function of pH showed that thymidine which has lost a proton at the N1 position (pK = 9.8) has an excited triplet state which phosphoresces with a decay time of 0.50 sec. Because the N1 pro- ton of thymidine is involved in hydrogen bonding at the N1 position of adenine, both DNA and poly-dAT act as if thymine shifts its proton to adenine and emits as the deprotonated base (T-). Adenosine and AMP, on the other hand, have been shown' to have their triplet states quenched by protonation at their N1 posi- tion (pKa - 4). Hence, this mechanism is consistent with the failure to observe extensive phosphorescence from adenine in either poly-dAT or DNA. Further- Downloaded by guest on September 30, 2021 VOL. 53, 1965 CHEMISTRY: RAHN, SHULMAN, AND LONGWORTH 895 TABLE 1 ESR PROPERTIES OF TRIPLETS (D2 + 3E2)'/2 cm 1 T (see) ESR T (sec) Optical DNA 0.200 0.29 J+ 0.03 0.27 4+ 0.02 dAT 0.201 0.32 4± 0.03 0.34 ± 0.03 T- 0.198 0.50 + 0.1 0.50 + 0.03 G 0.145 1.26 1.3 A 0.126 2.6 2.6 more, preliminary measurements indicate that guanine phosphorescence in a poly G-poly C copolymer is quenched, which is also consistent with the thymine phos- phorescence being the main component of the DNA emission. Stronger evidence that the DNA triplet is the same as T- came from the ESR studies of the triplets. Previously, we had observed the ESR signals from AMP (pH 7) and GMP (pH 7, pH 1). The resonances observed were the so-called Am = 2 transitions of the metastable triplet of the sort first observed by Van der Waals and de Groot.5 The resonance position defines a field Hmin where (D2 + 3E2) 1/2 = [3/4 (h V) 2-3(gHmin))2]1/2 in which D and E describe the dipole-dipole interaction between the two spins in the triplet through the spin Hamiltonian. H = OH*9*S + D(S2 - 2/3) + E(S2 _ S2). When it became clear from the emission studies that T- had a reasonably long-lived triplet state, we searched for the T- resonance although a previous search had not shown any ESR triplet in thymidine at pH 7. The resonance in T- was found at Hmin = 1067 gauss at a microwave frequency of 9105 Mc/sec. Subsequent obser- vations showed almost identical resonances in poly-dAT and DNA with Hmin = 1046 and 1054 gauss, respectively. In a similar fashion, U- also showed a reso- nance, although neutral uridine did not. The values of (D2 + 3E2) /' and of r, the triplet decay time measured by turning off the light, for DNA, dAT, and T- are listed in Table 1. The T- resonance in native DNA had a signal:noise ratio of 35: 1 (with a 1-sec time constant), and no other resonances were observed. The values of r measured from the emission are seen to be in excellent agreement with those measured by the ESR. The agreement between the value of (D2 + 3E2) 1/2 in these three compounds is very close when compared with the range of values observed in other pyrimidines and purines shown in Table 2. The values of Hmin can be measured with an ac- curacy of better than one gauss. Although there are interesting small differences TABLE 2 ESR PROPERTIES OF DIFFERENT BASES (D2 + 3E2)1/2 T (see) ESR Thymidine, pH 11 (T-) 0.198 0.50 Uridine, pH 11 (U-) 0.163 0.40 Orotic acid 0.184 0.30 2. 4-Dimethoxypyrimidine 0.144 1.63 4. 6-Dimethyl pyrimidine-2-one 0.132 0.60 2-Aminopyrimidine 0.111 0.25 Adenosine 0.126 2.6 Guanosine 0.145 1.26 Downloaded by guest on September 30, 2021 896 CHEMISTRY: RAHN, SHULMAN, AND LONGWORTH PROC. N. A. S. of Hmin for T-, dAT, and DNA of several gauss, their similarities to each other and their differences from the other resonances are the points to be noticed. In T-, U-, and orotic acid where the canonical diketo structures have only one ring double bond, (D2 + 3E2)l/2 is larger than the other compounds with more than one double band in their canonical structures. In orotic acid it was possible to measure three Am = 1 edge absorptions6 and to determine that D = 0.18 cm-' and E = 0.016 cm-' while in GMP all six edge absorptions were observed and D = 0.141 cm-' and E = 0.0172 cm-'. The range of D values observed in these molecules agrees with the values calculated for 7r-7r* triplets.7 In addition, the small values of E are characteristic' of the small departures from cylindrical sym- metry found in 7r-7r* rather than no7r* triplets. The larger values of D in T- and orotic acid indicate a more localized triplet. This suggests that the T- triplet is a 7r-T* excitation somewhat localized in the 4-5 double bnod, since this is where the thymine dimer8 and the thymine free radical9 are found. Paramagnetic metal ions have been shown9 to reduce considerably the yield of the thymine free radical in UV-irradiated DNA. It is also known that the phosphorescence of DNA is quenched4 by paramagnetic Mn2+ ions. We added Mn2+ to the DNA samples in the ratio Mn2+/PO4 = 0.20 and found the triplet ESR signal to be quenched by more than a factor of 20.
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