Observation of two N2-isobutyrylguanine tautomers by NMR spectroscopy

Article abstract of DOI:10.1002/mrc.3901

N₂-Isobutyrylguanine was prepared by treatment of guanine with isobutyryl chloride. Two tautomers, 1,7-dihydro-2-(isobutyroyl)amino-6H-purin-6- one and 1,9-dihydro-2-(isobutyroyl)amino-6H-purin-6-one, were identified in almost 1: 1 ratio in dichloromethane-dimethyl sulfoxide (1: 1 v/v) by NMR spectroscopy. By using the selective-inversion experiments, enthalpy, entropy, and free energy for activation were determined. This work represents the first report of guanine tautomers observed directly by NMR spectroscopy. Copyright

Full text of DOI:10.1002/mrc.3901

MRC Letters
Received: 27 August 2012
Revised: 17 October 2012
Accepted: 18 October 2012
Published online in Wiley Online Library: 20 November 2012
(wileyonlinelibrary.com) DOI 10.1002/mrc.3901
Observation of two N2-isobutyrylguanine
tautomers by NMR spectroscopy
Lijing Yang, Jia Li, Razvan Simionescu and Hongbin Yan*
N2-Isobutyrylguanine was prepared by treatment of guanine with isobutyryl chloride. Two tautomers, 1,7-dihydro-2-(isobutyroyl)
amino-6H-purin-6-one and 1,9-dihydro-2-(isobutyroyl)amino-6H-purin-6-one, were identified in almost 1 : 1 ratio in dichloromethane–
dimethyl sulfoxide (1 : 1 v/v) by NMR spectroscopy. By using the selective-inversion experiments, enthalpy, entropy, and free energy
for activation were determined. This work represents the first report of guanine tautomers observed directly by NMR spectroscopy.
Copyright © 2012 John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article.
1
Keywords: NMR; H; 13C; selective-inversion experiments; N2-isobutyrylguanine; tautomer; activation energy
NMR samples were prepared in a mixture of deuterated
dichloromethane and dimethyl sulfoxide (Cambridge Isotope
Introduction
Guanine is one of the nucleobases in nucleic acids. Guanine
nucleotides also serve critical roles in signal transductions, such
as in the G-proteins.[1] In medicinal chemistry, guanine analogues
have been developed as antiviral drugs.[2,3] Like the other
nucleobases, guanine is known to exist in a complex equilibrium
consisting of 36 possible tautomers/rotamers.[4] Some of these
guanine tautomers were suggested to be responsible for sponta-
neous point mutations in DNA.[5,6] Of these, eight tautomers and
rotamers are considered to be more stable in the gas phase
(Fig. 1).[7] Crystal structures of guanine monohydrate and
anhydrous guanine have been reported, representing the two
most stable tautomers, 1,7-dihydro-2-amino-6H-purin-6-one[8]
and 1,9-dihydro-2-(isobutyroyl)amino-6H-purin-6-one.[9] To date,
a number of computational studies have been carried out to
characterize these tautomers;[4,7,10–17] however, it is perhaps not
surprising that only a few experimental studies have been
reported in the gas phase,[18–25] whereas no observation of
tautomers has been made by NMR spectroscopy. This lack of
evidence in solution is most probably due to the extremely poor
solubility of guanine in both aqueous and organic solvents.
In our ongoing work with peptide nucleic acids (PNA),
N2-isobutyrylguanine was required for the construction of PNA
analogues. This compound has been previously synthesized
in several laboratories; however, evidence for purity of this
compound has been inconclusive.[26–29] In this work, we wish
to report the observation of 1,7-dihydro-2-(isobutyroyl)amino-
6H-purin-6-one and 1,9-dihydro-2-(isobutyroyl)amino-6H-purin-
6-one by NMR spectroscopy in a solution of dichloromethane
and dimethyl sulfoxide (DMSO).
Laboratories, Andover, MA, USA). Spectra were recorded on a
Bruker 600 MHz spectrometer at various temperatures. The 1D
selective-inversion experiments were performed using methods
reported in the literature.[30–32]
The tautomeric equilibrium is highly solvent dependent
When 1H NMR spectrum of N2-isobutyrylguanine was recorded in
DMSO-d6 at lower field (300 MHz), broad signals were generally
observed. At higher field (600 MHz), however, two apparent
structures start to emerge. Upon addition of deuterated
dichloromethane (CD2Cl2) and an appropriate amount of water
(H2O), two distinct isomers in the ratio of approximately 1 : 1 were
clearly resolved (Fig. 2).
In such a solvent mixture, the equilibrium between the two
tautomers is apparently affected by water content in the sample
(Fig. 3). In a titration experiment where water content varied, fast
exchange between the two tautomers was observed at low water
content. In this situation, the two tautomers coalesced. With the
increase of water in the mixture, the tautomeric exchange appears
to slow down to a point where the two tautomers were fully
resolved. Yet, the spectrum is dominated by exchange with water
upon further addition of water, resulting in broadening of the peaks.
Assignments of the 1H and 13C signals are listed in Tables 1 and 2.
These assignments are based largely on the HMBC correlations
(Fig. 4). Detailed correlations are annotated in Fig. 4. No correlation
involving C2 and C6 was detectable in all the experiments
performed, presumably because of the very small 2J(C–H) coupling
constants. Assignments of these two nuclei were based on
literature precedence of guanosine.[33]
Results and Discussion
*
Correspondence to: Hongbin Yan, Department of Chemistry, Brock University,
St. Catharines, Ontario L2S 3A1, Canada. E-mail: tyan@brocku.ca
N2-Isobutyrylguanine was readily synthesized by treating guanine
with isobutyryl chloride in dimethylformamide in the presence of
Hünig’s base (Scheme 1).
Department of Chemistry, Brock University, St. Catharines, Ontario, L2S 3A1,
Canada
Magn. Reson. Chem. 2013, 51, 60–64
Copyright © 2012 John Wiley & Sons, Ltd.

Observation of two N2-isobutyrylguanine tautomers by NMR spectroscopy
Figure 1. Eight most stable guanine tautomers and rotamers in the gas phase.
Scheme 1. Preparation of N2-isobutyrylguanine.
This allows for the Eyring plots, i.e. plot of the natural logarithm
of k/T against 1/T, which lead to the determination of enthalpy
and entropy for activation ΔH{ and ΔS{ following the relationship
shown in Eqn (1), where R is the gas constant, kB is the Boltzmann
constant, and h is the Planck’s constant.[31–33]
ꢀ
ꢁ
kh
ΔH{
R ln
¼ ΔS{ ꢀ
(1)
kBT
T
The ΔH{ and ΔS{ obtained previously allowed for the
calculation of ΔG{ at 300 K following Eqn (2) (Table 3).
ΔG{ ¼ ΔH{ ꢀ TΔS{
(2)
Conclusions
Figure 2. Resolution of the two tautomeric isomers in 1H NMR upon
addition of water. Bottom spectrum: N2-isobutyrylguanine in CD2Cl2–
DMSO-d6 containing only trace amount of water; top spectrum:
N2-isobutyrylguanine in CD2Cl2–DMSO-d6 containing approximately 2 mol.
equiv. of water.
Two major tautomers of N2-isobutyrylguanine were observed to
exist in a 1 : 1 ratio by NMR experiments. The equilibrium
between the two tautomers is highly solvent dependent. The free
energy for activation ΔG{ as determined by the selective-
inversion experiments is consistent with the ratio (1 : 1) observed
1
in the H NMR. It is likely that these two structures are involved
Thermodynamic parameters determined by the selective-
inversion experiments
in pre-organized assemblies through interactions with water.
This latter postulation has yet to be investigated.
Thermodynamic parameters (i.e. the free energy, enthalpy, and
entropy for activation ΔG{, ΔH{, and ΔS{) for the tautomeric
exchange in CD2Cl2–DMSO-d6 (1 : 1 v/v) were determined by the
selective-inversion experiments.
The H8a and H8b protons were excited at variable tempera-
tures with appropriate mixing times, and the ratio of the H8a
and H8b signals were taken that allows for the calculation of
exchange rate (k) at each individual temperature (Fig. 5).
Experimental
Synthesis
Guanine (Sigma-Aldrich) (6.00 g, 0.04 mol), which was dried
by heating at 60ꢁC in vacuo for 3 h, was suspended in dry
dimethylformamide (Sigma-Aldrich) (50ml), followed by addition
Magn. Reson. Chem. 2013, 51, 60–64
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L. Yang et al.
Figure 3. Stack of 1H NMR spectra with water titration. The numbers on the left of the spectra indicate molar ratios of water to N2-isobutyrylguanine.
1
Table 1. Assignments of H signals; spectrum was recorded at 255 K
Compound
CH3(i-Bu)
CH(i-Bu)a
H8
NH(i-Bu)
NH(2)
NH(7/9)
2a
2b
1.12 (d, J = 6.6)
2.73 (sep, J = 6.6) 2.74 (sep, J = 6.6)
7.84 (s)
8.03 (s)
11.65 (s)
12.09 (s)
12.24 (s)
13.01 (s)
13.48 (s)
aThe two resonance signals cannot be assigned to individual structure because of very small difference in their shifts.
Table 2. Assignments of 13C signals; spectrum was recorded at 255 K
Compound
CH3 (i-Bu)
CH (i-Bu)
C5
C8
C2
C4
C6 (C O)
153.0, 155.6
C O (i-Bu)
180.41, 180.44
2a
2b
19.22, 19.23
35.3
120.3
111.9
137.9
142.3
147.4, 148.2
149.5
157.3
of dry N,N-diisopropylethylamine (Sigma-Aldrich) (6.90 ml, 0.04 mol)
and isobutyryl chloride (Sigma-Aldrich) (4.16 ml). The mixture was
heated at 100ꢁC under stirring for 3 h. Upon cooling, methanol
(2ml) was added, and the solvents were subsequently distilled off
under reduced pressure. The residue was stirred with isopropanol
(Sigma-Aldrich) (60 ml) at 80ꢁC and filtered while hot. The solid
was again treated with isopropanol (60 ml) at 80ꢁC and filtered
while hot. The solid residue was dried in vacuo and purified by
column chromatography on silica gel. The appropriate fractions,
which were eluted with dichloromethane–methanol (92 : 8 v/v),
were combined and evaporated under reduced pressure to give
the title compound as a white solid (3.54 g, 40%). Rf = 0.66
(n-butanol/acetic acid/water 3 : 1 : 1). HRMS (EI): 221.0901 [M+],
C9H11N5O2 requires 221.09128.
content, Cambridge Isotope Laboratories) and dry DMSO-d6
(0.30 ml, 99.96% deuterium content, Cambridge Isotope Laboratories)
in a dry NMR tube followed by addition of H2O (1.6 ml, 89 mmol)
using a 10 ml Hamilton syringe.
NMR experiments were carried out on a Bruker AV600
spectrometer running Topspin 2.1 PL 6 on a Windows 7 PC.
1H NMR and 13C spectra were measured at 600 and 150.9 MHz,
respectively. Chemical shifts were given in parts per million.
Typical acquisition parameters for 2D experiments were as
follows: 2048 points and 256 increments processed to
1024 ꢂ 1024 points, two transients for COSY, two for HSQC, four
for NOESY, and eight for HMBC and TOCSY. NOESY spectrum was
acquired with 1000 ms mixing time. The H–C coupling constant
in the HSQC and HMBC was set to 145 Hz, and long-range coupling
constant in the HMBC was set to 6 Hz. 1D selective-inversion
experiments were carried out using a selective excitation pulse
sequence at four different temperatures (254.6, 262, 271.2, and
285.4K). At each temperature, at least four selective-inversion
experiments were run with mixing time ranging from 4 to 100 ms.
NMR measurements
To prepare the NMR sample, N2-isobutyrylguanine (7.3 mg,
33 mmol) was dissolved in dry CD2Cl2 (0.30 ml, 99.9% deuterium
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Magn. Reson. Chem. 2013, 51, 60–64

Observation of two N2-isobutyrylguanine tautomers by NMR spectroscopy
Figure 4. COSY and HMBC spectra of N2-isobutyrylguanine in CD2Cl2–DMSO-d6 (1 : 1, v/v).
Figure 5. Examples of the selective-inversion spectra. The numbers in milliseconds given in the figure are mixing times. (a) Excitation at H8a; (b)
excitation at H8b.
1H, 13C, COSY, HSQC, HMBC, NOSEY, and TOCSY spectra of
compound 2 are included in the Supporting Information.
Table 3. The free energy, enthalpy, and entropy for activation (ΔG{,
ΔH{, and ΔS{) for the tautomeric exchange in CD2Cl2–DMSO-d6
(1 : 1 v/v)
ΔH{ (kJ/mol) ΔS{ (J/mol) ΔG{ (300 K) (kJ/mol)
Acknowledgements
Excitation at H8b
Excitation at H8a
35.1
34.7
70.3
69.5
14.0
14.0
This work was supported by the Natural Science and Engineering
Research Council of Canada. The authors wish to thank Professor
Alex Bain for the helpful discussions.
Magn. Reson. Chem. 2013, 51, 60–64
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L. Yang et al.
[18] M. Y. Choi, R. E. Miller. J. Am. Chem. Soc. 2006, 128, 7320–7328.
References
[19] W. Caminati. Angew. Chem. Int. Ed. 2009, 48, 9030–9033.
[20] A. Gahlmann, S. T. Park, A. H. Zewail. J. Am. Chem. Soc. 2009, 131,
2806–2808.
[21] J. L. Alonso, I. Peña, J. C. López, V. Vaquero. Angew. Chem. Int. Ed.
2009, 48, 6141–6143.
[1] A. M. Spiegel. Mol. Cell. Endocrinol. 1987, 49, 1–16.
[2] F. P. Clausen, J. Juhl-Christensen. Org. Prep. Proc. Int. 1993, 25, 373–401.
[3] C. K. Chu, Recent Advances in Nucleosides: Chemistry and Chemotherapy,
Elsevier, Amsterdam, 2002.
[4] W. Liang, H. Li, X. Hu, S. Han. Chem. Phys. 2006, 328, 93–102.
[5] P. Strazewski. Nucleic Acids Res. 1988, 16, 9377–9398.
[6] M. B. Topal, J. R. Fresco. Nature 1976, 263, 285–289.
[7] C. M. Marian. J. Phys. Chem. A 2007, 111, 1545–1553.
[8] K. Guille, W. Clegg. Acta Cryst. 2006, C62, o515–o517.
[9] U. Thewalt, C. E. Bugg, R. E. Marsh. Acta Cryst. 1971, B27, 2358–2363.
[10] D. Kosenkov, Y. Kholod, L. Gorb, O. Shishkin, D. M. Hovorun, M. Mons,
J. Leszczynski. J. Phys. Chem. B 2009, 113, 6140–6150.
[11] M. Sabio, S. Topiol, W. C. Lumma Jr.. J. Phys. Chem. 1990, 94,
1366–1372.
[22] E. Nir, C. Plützer, K. Kleinermanns, M. de Vries. Eur. Phys. J. D 2002, 20,
317–329.
[23] M. Mons, I. Dimicoli, F. Piuzzi, B. Tardivel, M. Elhanine. J. Phys. Chem. A
2002, 106, 5088–5094.
[24] A. Abo-Riziq, B. Crews, L. Grace, M. S. de Vries. J. Am. Chem. Soc.
2005, 127, 2374–2375.
[25] F. Piuzzi, M. Mons, I. Dimicoli, B. Tardivel, Q. Zhao. Chem. Phys. 2001,
270, 205–214.
[26] T. Nasr, Y. Taniguchi, D. Sasaki. Heterocycles 2007, 71, 2659–2668.
[27] A. Saleh, J. G. D’Angelo, M. D. Morton, J. Quinn, K. Redden,
R. W. Mielguz, C. Pavlik, M. B. Smith. J. Org. Chem. 2011, 76,
5574–5583.
ꢀ
ꢀ
ꢀ
[12] M. Hanus, F. Ryjácek, M. Kabelác, T. Kubar, T. V. Bogdan, S. A. Trygubenko,
P. Hobza. J. Am. Chem. Soc. 2003, 125, 7678–7688.
[13] H. Langer, N. L. Doltsinis. J. Chem. Phys. 2003, 118, 5400–5407.
[14] L. Gorb, J. Leszczynski. J. Am. Chem. Soc. 1998, 120, 5024–5032.
[15] M. Monajjemi, B. Honarparvar, S. M. Nasseri, M. Khaleghian. J. Struct.
Chem. 2009, 50, 67–77.
[28] Z. Timár, L. Kovács, G. Kovács, Z. Schmél. J. Chem. Soc., Perkin Trans. 1
2000, 19–26.
[29] D. W. Will, G. Breipohl, D. Langner, J. Knolle, E. Uhlmann. Tetrahedron
1995, 51, 12069–12082.
[16] C. Colominas, F. J. Luque, M. Orozco. J. Am. Chem. Soc. 1996, 118,
6811–6821.
[30] A. D. Bain, G. J. Duns, F. Rathgeb, J. Vanderkloet. J. Phys. Chem. 1995,
99, 17338–17343.
[17] O. Plekan, V. Feyer, R. Richter, M. Coreno, G. Vall-llosera, K. C. Prince,
A. B. Trofimov, I. L. Zaytseva, T. E. Moskovskaya, E. V. Gromov, J. Schirmer.
J. Phys. Chem. A 2009, 113, 9376–9385.
[31] A. D. Bain. Prog. Nucl. Magn. Reson. Spectroc. 2003, 43, 63–103.
[32] A. D. Bain. Ann. Rep. NMR Spec. 2008, 63, 23–48.
[33] F. Hansske, D. Madej, M. J. Robins. Tetrahedron 1984, 40, 125–135.
wileyonlinelibrary.com/journal/mrc
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