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A study of the major gas-phase tautomer of adenine by microwave spectroscopy
Volume 156, number
1
CHEMICAL
A STUDY OF THE MAJOR GASPHASE BY MICROWAVE SPECTROSCOPY Ronald D. BROWN, Peter D. GODFREY,
24 March 1989
PHYSICS LETTERS
TAUTOMER
OF ADENINE
Donald McNAUGHTON
and Anthony
P. PIERLOT
Chemistry Department, Monash University, WellingtonRoad, Clayton, Victoria 3168, Australia Received I2 November
1988; in final form 17 January
I989
The microwave spectrum of the nucleic acid base adenine in a cw seeded supersonic beam has heen analyzed indicating that the amino, N (9)H tautomer is the most stable in an isolated environment. A comparison of the experimental rotational constants is made with those obtained using ab initio 3-2 1G basis SCF calculations for three ofthe lower- energy tautomers.
The occurrence of rare tautomeric forms of the nucleic acid bases has been suggested as a mechanism for spontaneous mutation [ 1 ] as well as being involved in chemical mutagenesis [2]. Thus the relative stability of the tautomers of these bases is of fundamental importance to the structure and functioning of the nucleic acids. Our previous studies of the pyrimidine bases, uracil [ 3 1, thymine [ 4 ] and cytosine [ 51, using our recently developed microwave spectrometer [ 6 1, established that for both uracil and thymine the diketo tautomer predominates in the gas phase. However, in the case of cytosine three distinct tautomers having similar abundances were observed. We now present data on the microwave spectrum of adenine, one of the two important purine bases. Microwave measurements on a seeded supersonic beam of adenine in argon expanded through a 550 urn diameter heated (280°C) nozzle were successful in detecting transitions of adequate S/N in the vicinity of 60 GHz. The spectroscopic parameters derived from 58 assigned, pa-type transitions are presented in table 1. If a very low frequency out-of-plane vibration is present the inertial defect can be larger than usual and negative even for planar molecules [ 71. Thus the value of the inertial defect of adenine is consistent with a planar cyclic molecule with a very low frequency out-of-plane vibration or a molecule with a slightly non-planar geometry. A number of weak lines were also observed in the spectrum that could not be assigned to this species but because of 0 009-2614/89/s ( North-Holland
03.50 0 Elsevier Science Publishers Physics Publishing Division )
Table 1 Derived spectroscopic
parameters
A (MHz) B (MHz) C (MHz) d (U.&Z) ‘) Transition request.
Fig.
.
frequencies
for adenine s) 2371.873(4) 1573.356518) 946.2576(4) -0.201
are available
from
the authors
on
I. Three possible low energy tautomers of adenine.
their low S/N we did not attempt an assignment. Recent semi-empirical calculations [ 8 ] employing the AM1 method predicted tautomer Al (lig. 1) to be the lowest energy tautomer with A2 predicted to be 28.0 kJ/mol higher in energy. All other amino tautomers were predicted to have relative energies greater than 48.5 kJ/mol. The lowest energy imine tautomer A3 was predicted to be 56.8 kJ/mol higher in energy than Al. By comparing the relative energies predicted using the AM1 method for the pyrimidine’ bases [ 81 with those obtained experimentally [ 3-51 the predicted relative energies are expected to be reliable to about 15 kJ/mol. Thus it is expected that B.V.
61
Volume 156, number
I
CHEMICAL PHYSICS LETTERS
Table 2 The 3-2 1G optimized parameters a) for three low energy tautomers Parameter
Al
C2NI N3C2 C4N3 csc4 C6C5 C6Nl N7C5 C8N7 N9C8 N9C4 HlOC2 Nl lC6 Hl2Nll Hl2Nl H13Nll H14C8 HlSN9 Hl5N7 N3C2N 1 C4N3C2 C5C4N3 C6C5C4 NlCK5 N7C5C4 CSN7C5 N9C8N7 C4N9C8 HIOC2C3 Nl IC6C5 Hl2Nl lC6 H12NlC6 Hl3NllC6 H14C8N7 H15N9C8 H15N7C8 H12NliC6C5 H13Nl lC6C5
1.3396 1.3269 1.3209 1.3913 1.3860 1.3249 1.3976 1.2932 1.3863 1.3692 1.0664 1.4131 1.0040
0.9961
1.0040 1.0637 0.9953
126.45 113.42 125.09 117.22 117.92 110.12 105.36 112.19 106.85 117.14 122.65 118.90
125.55 114.21 124.41 117.53 117.92 109.51 105.48 112.68 106.53
120.75 125.91 127.65
114.42 125.58 127.69
122.42 114.45
1.3434 1.3145 1.3359 1.3878 1.3963 1.3228 1.3874 1.3728 1.2965 1.3903
1.3587 1.2851 1.3719 1.3610 1.4408 1.4169 1.3845 1.2986 1.3844 1.3611
1.0670
1.0692
1.3538 0.9967
1.2554
0.9932 1.0641
0.9999 1.0098 1.0626 0.9956
0.9939 125.90 114.99 122.90 118.48 117.51 105.79 106.00 113.05 1OS.78 117.84 124.38 117.48
124.62 113.70 126.38 120.4 1 109.69 109.95 105.80 111.27 106.69 119.39 131.95
123.36 121.66
114.44 114.40 126.28 127.81
125.98
-461.90152 0.0 2.2 1.o 237 1 1571 948
-461.87233 76.6 4.5 0.0 2307 1578 945
-461.88406 45.8 0.3 7.5 2388 1543 941
-461.87667 65.2 1.5 3.4 2382 1547 942
A and angles in deg. constants
(A by 0.985, B and C by 0.995).
tautomer observed is either tautomer Al or A2. Our previous studies of uracil, thymine and cytosine have shown that the rotational constants de62
117.44
A3
65.76 -65.85
B. (D) ~a (D) A (MHz) b, B (MHz) b1 C (MHz) b’
the
A2
1.3366 1.3244 1.3323 1.3811 1.3952 1.3338 1.3956 1.2927 1.3891 1.3673 1.0675 1.3390 0.9961
1.0634
energy (hartree) relative energy (k.l mol-‘)
a1 Bond lengths in b, Scaled rotational
of adenine Ala
0.995 1
24 March 1989
rived from geometries optimized using ab initio 32 1G basis SCF calculations, with subsequent scaling of the rotational constants by 0.985 for A and 0.995
Volume 156, number
1
CHEMICAL
PHYSICS LETTERS
for B and C, gives agreement with the experimentally derived rotational constants to within 15 MHz. The 3-2 1G optimized geometries calculated assuming planarity using the GAUSSIAN 82 suite of programs [ 91 with the Murtaugh-Sargent optimization algorithm for tautomers Al, A2 and A3 are given in table 2, The assumption of planarity for the amino group of Al was verified by using a non-planar configuration as an initial guess. A second minimum of much higher energy was found in the potential energy surface for Al where the amino hydrogens were located above and below the plane of the rings. The optimized parameters for this tautomer (A la) are also presented in table 2 as well as the scaled rotational constants, relative energies and dipole moment components for the optimized tautomers. Comparison of the rotational constants observed with the scaled rotational constants for the two lowest energy tautomers (A 1 and A2) and an imine tautomer A3 clearly indicates that it is tautomer Al that is being observed in the gas phase. Furthermore the observation of only a .&-type spectrum is consistent with tautomer Al as tautomers A2 and A3 are predicted to have their largest dipole moment components directed along the b axis and hence would be expected to exhibit primarily /&-type spectra. The observation of Al in the gas phase is in agree-
24 March 1989
ment with the conclusion obtained from gas phase ultraviolet photoelectron spectroscopy [ lo] and is also the tautomer observed in the crystal phase [ 111. This work was supported search Grants Scheme.
by the Australian
Re-
References [ 1 ] B. Pullman and A. Pullman, Advan. Heterccycl.
Chem. 13 (1971) 77. [2] R. Stolarski, B. Kierdaszuk, C.E. Hagberg and D. Shugar, Biochem. 23 ( 1984) 2906. [ 3 ] R.D. Brown, P.D. Godfrey, D. McNaughton and A.P. Pierlot, J. Am. Chem. Sot. 110 (1988) 2329. [4 ] R.D. Brown, P.D. Godfrey, D. McNaughton and A.P. Pierlot, J. Chem. Sot. Chem. Commun. (1988), in press. [ 5] R.D. Brown, P.D. Godfrey, D. McNaughton and A.P. Pierlot, J. Am. Chem. Sot. (1988), in press. [6 ] R.D. Brown, J.G. Crofts, P.D. Godfrey, D. McNaughton and A.P. Pierlot, J. Mol. Struct. ( 1988), in press. [7] W. Gordy and R.L. Cook, Microwave molecular spectra (Wiley-Interscience, New York, 1984) p. 688. [8] U. Norinder, J. Mol. Struct. THEOCHEM 15 1 (1987) 259. [9 ] AS. Binkley, M.J. Frisch, D.J. DeFrees, K. Ragavachari, R.A. Whiteside, H.B. Schlegel, E.M. Fluder and J.A. Pople, GAUSSIAN 82, QCPE ( 1984). [IO] J. Lin, C. Yu, S. Peng, I. Akiyama, K. Li, L. Kao Lee and P.R. LeBreton, J. Am. Chem. Sot. 102 ( 1980) 4627. [ 11 ] T.J. Kistenmacher and T. Shigematsu, Acta Cryst. B 30 (1974) 166.
63
1
CHEMICAL
A STUDY OF THE MAJOR GASPHASE BY MICROWAVE SPECTROSCOPY Ronald D. BROWN, Peter D. GODFREY,
24 March 1989
PHYSICS LETTERS
TAUTOMER
OF ADENINE
Donald McNAUGHTON
and Anthony
P. PIERLOT
Chemistry Department, Monash University, WellingtonRoad, Clayton, Victoria 3168, Australia Received I2 November
1988; in final form 17 January
I989
The microwave spectrum of the nucleic acid base adenine in a cw seeded supersonic beam has heen analyzed indicating that the amino, N (9)H tautomer is the most stable in an isolated environment. A comparison of the experimental rotational constants is made with those obtained using ab initio 3-2 1G basis SCF calculations for three ofthe lower- energy tautomers.
The occurrence of rare tautomeric forms of the nucleic acid bases has been suggested as a mechanism for spontaneous mutation [ 1 ] as well as being involved in chemical mutagenesis [2]. Thus the relative stability of the tautomers of these bases is of fundamental importance to the structure and functioning of the nucleic acids. Our previous studies of the pyrimidine bases, uracil [ 3 1, thymine [ 4 ] and cytosine [ 51, using our recently developed microwave spectrometer [ 6 1, established that for both uracil and thymine the diketo tautomer predominates in the gas phase. However, in the case of cytosine three distinct tautomers having similar abundances were observed. We now present data on the microwave spectrum of adenine, one of the two important purine bases. Microwave measurements on a seeded supersonic beam of adenine in argon expanded through a 550 urn diameter heated (280°C) nozzle were successful in detecting transitions of adequate S/N in the vicinity of 60 GHz. The spectroscopic parameters derived from 58 assigned, pa-type transitions are presented in table 1. If a very low frequency out-of-plane vibration is present the inertial defect can be larger than usual and negative even for planar molecules [ 71. Thus the value of the inertial defect of adenine is consistent with a planar cyclic molecule with a very low frequency out-of-plane vibration or a molecule with a slightly non-planar geometry. A number of weak lines were also observed in the spectrum that could not be assigned to this species but because of 0 009-2614/89/s ( North-Holland
03.50 0 Elsevier Science Publishers Physics Publishing Division )
Table 1 Derived spectroscopic
parameters
A (MHz) B (MHz) C (MHz) d (U.&Z) ‘) Transition request.
Fig.
.
frequencies
for adenine s) 2371.873(4) 1573.356518) 946.2576(4) -0.201
are available
from
the authors
on
I. Three possible low energy tautomers of adenine.
their low S/N we did not attempt an assignment. Recent semi-empirical calculations [ 8 ] employing the AM1 method predicted tautomer Al (lig. 1) to be the lowest energy tautomer with A2 predicted to be 28.0 kJ/mol higher in energy. All other amino tautomers were predicted to have relative energies greater than 48.5 kJ/mol. The lowest energy imine tautomer A3 was predicted to be 56.8 kJ/mol higher in energy than Al. By comparing the relative energies predicted using the AM1 method for the pyrimidine’ bases [ 81 with those obtained experimentally [ 3-51 the predicted relative energies are expected to be reliable to about 15 kJ/mol. Thus it is expected that B.V.
61
Volume 156, number
I
CHEMICAL PHYSICS LETTERS
Table 2 The 3-2 1G optimized parameters a) for three low energy tautomers Parameter
Al
C2NI N3C2 C4N3 csc4 C6C5 C6Nl N7C5 C8N7 N9C8 N9C4 HlOC2 Nl lC6 Hl2Nll Hl2Nl H13Nll H14C8 HlSN9 Hl5N7 N3C2N 1 C4N3C2 C5C4N3 C6C5C4 NlCK5 N7C5C4 CSN7C5 N9C8N7 C4N9C8 HIOC2C3 Nl IC6C5 Hl2Nl lC6 H12NlC6 Hl3NllC6 H14C8N7 H15N9C8 H15N7C8 H12NliC6C5 H13Nl lC6C5
1.3396 1.3269 1.3209 1.3913 1.3860 1.3249 1.3976 1.2932 1.3863 1.3692 1.0664 1.4131 1.0040
0.9961
1.0040 1.0637 0.9953
126.45 113.42 125.09 117.22 117.92 110.12 105.36 112.19 106.85 117.14 122.65 118.90
125.55 114.21 124.41 117.53 117.92 109.51 105.48 112.68 106.53
120.75 125.91 127.65
114.42 125.58 127.69
122.42 114.45
1.3434 1.3145 1.3359 1.3878 1.3963 1.3228 1.3874 1.3728 1.2965 1.3903
1.3587 1.2851 1.3719 1.3610 1.4408 1.4169 1.3845 1.2986 1.3844 1.3611
1.0670
1.0692
1.3538 0.9967
1.2554
0.9932 1.0641
0.9999 1.0098 1.0626 0.9956
0.9939 125.90 114.99 122.90 118.48 117.51 105.79 106.00 113.05 1OS.78 117.84 124.38 117.48
124.62 113.70 126.38 120.4 1 109.69 109.95 105.80 111.27 106.69 119.39 131.95
123.36 121.66
114.44 114.40 126.28 127.81
125.98
-461.90152 0.0 2.2 1.o 237 1 1571 948
-461.87233 76.6 4.5 0.0 2307 1578 945
-461.88406 45.8 0.3 7.5 2388 1543 941
-461.87667 65.2 1.5 3.4 2382 1547 942
A and angles in deg. constants
(A by 0.985, B and C by 0.995).
tautomer observed is either tautomer Al or A2. Our previous studies of uracil, thymine and cytosine have shown that the rotational constants de62
117.44
A3
65.76 -65.85
B. (D) ~a (D) A (MHz) b, B (MHz) b1 C (MHz) b’
the
A2
1.3366 1.3244 1.3323 1.3811 1.3952 1.3338 1.3956 1.2927 1.3891 1.3673 1.0675 1.3390 0.9961
1.0634
energy (hartree) relative energy (k.l mol-‘)
a1 Bond lengths in b, Scaled rotational
of adenine Ala
0.995 1
24 March 1989
rived from geometries optimized using ab initio 32 1G basis SCF calculations, with subsequent scaling of the rotational constants by 0.985 for A and 0.995
Volume 156, number
1
CHEMICAL
PHYSICS LETTERS
for B and C, gives agreement with the experimentally derived rotational constants to within 15 MHz. The 3-2 1G optimized geometries calculated assuming planarity using the GAUSSIAN 82 suite of programs [ 91 with the Murtaugh-Sargent optimization algorithm for tautomers Al, A2 and A3 are given in table 2, The assumption of planarity for the amino group of Al was verified by using a non-planar configuration as an initial guess. A second minimum of much higher energy was found in the potential energy surface for Al where the amino hydrogens were located above and below the plane of the rings. The optimized parameters for this tautomer (A la) are also presented in table 2 as well as the scaled rotational constants, relative energies and dipole moment components for the optimized tautomers. Comparison of the rotational constants observed with the scaled rotational constants for the two lowest energy tautomers (A 1 and A2) and an imine tautomer A3 clearly indicates that it is tautomer Al that is being observed in the gas phase. Furthermore the observation of only a .&-type spectrum is consistent with tautomer Al as tautomers A2 and A3 are predicted to have their largest dipole moment components directed along the b axis and hence would be expected to exhibit primarily /&-type spectra. The observation of Al in the gas phase is in agree-
24 March 1989
ment with the conclusion obtained from gas phase ultraviolet photoelectron spectroscopy [ lo] and is also the tautomer observed in the crystal phase [ 111. This work was supported search Grants Scheme.
by the Australian
Re-
References [ 1 ] B. Pullman and A. Pullman, Advan. Heterccycl.
Chem. 13 (1971) 77. [2] R. Stolarski, B. Kierdaszuk, C.E. Hagberg and D. Shugar, Biochem. 23 ( 1984) 2906. [ 3 ] R.D. Brown, P.D. Godfrey, D. McNaughton and A.P. Pierlot, J. Am. Chem. Sot. 110 (1988) 2329. [4 ] R.D. Brown, P.D. Godfrey, D. McNaughton and A.P. Pierlot, J. Chem. Sot. Chem. Commun. (1988), in press. [ 5] R.D. Brown, P.D. Godfrey, D. McNaughton and A.P. Pierlot, J. Am. Chem. Sot. (1988), in press. [6 ] R.D. Brown, J.G. Crofts, P.D. Godfrey, D. McNaughton and A.P. Pierlot, J. Mol. Struct. ( 1988), in press. [7] W. Gordy and R.L. Cook, Microwave molecular spectra (Wiley-Interscience, New York, 1984) p. 688. [8] U. Norinder, J. Mol. Struct. THEOCHEM 15 1 (1987) 259. [9 ] AS. Binkley, M.J. Frisch, D.J. DeFrees, K. Ragavachari, R.A. Whiteside, H.B. Schlegel, E.M. Fluder and J.A. Pople, GAUSSIAN 82, QCPE ( 1984). [IO] J. Lin, C. Yu, S. Peng, I. Akiyama, K. Li, L. Kao Lee and P.R. LeBreton, J. Am. Chem. Sot. 102 ( 1980) 4627. [ 11 ] T.J. Kistenmacher and T. Shigematsu, Acta Cryst. B 30 (1974) 166.
63