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An exploratory ab initio conformational analysis of selected fragments of nicotinamide adenine dinucleotide (NAD+). Part II: adenosine
Journal of Molecular Structure (Theochem) 666–667 (2003) 431–437 www.elsevier.com/locate/theochem
An exploratory ab initio conformational analysis of selected fragments of nicotinamide adenine dinucleotide (NADþ). Part II: adenosine Suzanne K. Laua,b,*, Gregory A. Chassa,b,c,d, Botond Penkee,f, Imre G. Csizmadiaa,b,f a
b
Department of Chemistry, University of Toronto, Toronto, Ont., Canada M5S 3H6 Global Institute of Computational Molecular and Materials Science, 1422 Edenrose Street, Mississauga, Ont., Canada L5V 1H3 c Institut de Science et d’Inge´nierie Supramole´culaires, 8, alle´e Gaspard Monge, BP 70028, Strasbourg Cedex 67083, France d Department of Biomedical Sciences, Creighton University, 2500 California plaza, Omaha, NE 68178, USA e Protein Chemistry Research Group, Hungarian Academy of Sciences, University of Szeged, Do´m te´r 8, Szeged 6720, Hungary f Department of Medical Chemistry, University of Szeged, Do´m te´r 8, Szeged H-6720, Hungary
Abstract Ab initio molecular orbital computations were carried out on selected fragments of nicotinamide adenine dinucleotide (NADþ) at the RHF/3-21G level of theory. The definitions of the relative spatial orientation of all constituent atomic nuclei have been formulated in such a modular fashion so as to allow for portions of the whole to be studied independently. Key points of examination included the rotation of adjacent moieties and ring puckering. This work focuses on the possible conformations of the adenosine moiety. It is anticipated that the structural results from the truncated and modified fragments will closely resemble those of NAD as a whole. q 2003 Elsevier B.V. All rights reserved. Keywords: Ab initio; Coenzyme; Nicotinamide adenine dinucleotide; Adenine; Adenosine
1. Introduction The redox pair formed by the oxidized and reduced forms of nicotinamide adenine dinucleotide (NADþ/NADH) holds great biological importance, most notably in the energy producing process [1]. As depicted in Fig. 1, NADþ contains an adenylic acid and nicotinamide-50 -ribonucleotide combined by apyrophosphate bridge. Nicotinamide can carry * Corresponding author. Department of Chemistry, University of Toronto, Toronto, Ont., Canada M5S 3H6. E-mail addresses: [email protected] (S.K. Lau), [email protected] (G.A. Chass). 0166-1280/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.theochem.2003.08.118
a positive charge at the ring nitrogen, while the pyrophosphate link holds two negative charges. Adenosine is also a significant biological molecule in its own right. 20 -Deoxyadenosine 50 -phosphate forms an integral nucleotide in DNA, while its oxidated counterpart adenosine 50 -phosphate is present in RNA. It is involved in many signalling pathways as cyclic AMP, and has been shown to offer cardioprotection through the regulation of the adenosine receptor [2]. A quick perusal of the literature has shown that little ab initio structure work has been done on adenosine, although some have attempted to examine hydrogen bonding and hydration of the molecule using NMR spectra and molecular
432
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Fig. 1. Systematic modular numbering system of NADþ including pertinent dihedrals. The arrows indicate the approximate partitioning of the molecule. (1) Nicotinamide, (2) riboseN, (3) nicotinamide ribose (NmR), (4) adenine, (5) riboseA, (6) adenosine and (7) pyrophosphate bridge. This paper focuses primarily on (4) –(6).
modelling [3]. Much of the research interest has been focussed on base pair interactions in DNA and RNA. The structural characterization of NADþ presented a particular challenge, as it is a flexible molecule with over a dozen rotatable bonds that can adopt a wide variety of environmentally dependent conformations. Our first work characterized the possible conformations of the nicotinamide-50 -ribonucleotide portion of NADþ [4]. We now aim to study the adenosine portion in a similar manner. The study of
the adenosine moiety (Fig. 2a) can be considered a precursor for the future examination of adenosine monophosphate, diphosphate or triphosphate. Furthermore, a study of the interactions between the two aromatic ring groups would be suitable. Previous Xray data has suggested that a tryptophane derivative with similar aromatic rings exists at a perpendicular state [5,6], while molecular dynamic simulations of NADþ have indicated that ring interactions occur primarily in the stacked conformation [7]. Thus,
Fig. 2. (a) Fully numbered adenosine moiety, with dihedrals of interest labelled. (b) Fully numbered positively charged adenosine moiety, with dihedrals of interest labelled.
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it would be of interest to complete the conformational analysis of the two ring groups, and then investigate the interaction between them. Insight into the conformation of NADþ in different environments will allow for a more complete understanding of the mechanism of its diverse functions at the molecular level, allowing for drug candidates with tailored ligand/receptor binding sites to be developed in the treatment of NAD-dependent pathologies. 2. Methods The GAUSSIAN 98 [8] program package has been used to perform preliminary ab initio calculations on the adenosine fragment of NADþ. The definitions of the relative spatial orientations of all constituent atomic nuclei have been formulated in a standardized and modular fashion so as to allow for portions of the whole to be studied independently (Fig. 1). The focus of this paper is on the adenosine group, which was first modelled by the semi-empirical AM1 method (data not shown). Previously obtained data for the optimization of the ribose molecule was used for the optimizations [4]. Few problems were encountered in the calculations of this moiety to date, primarily because the techniques developed while computing the conformations of the nicotinamide – ribose moiety were employed. It had been found that freezing the dihedrals of interest and sequentially unfreezing them with pre-optimized data generated the most accurate results. Each dihedral of interest was examined independently, and then incorporated into the larger study with multiple scans of up to three dihedrals. The dihedral xi2 was examined in conjunction with the hydroxy rotors ðd3i ; d4i Þ on the ribose portion of the molecule. Preliminary calculations were also made on a positively charged adenosine molecule found in the acidic environment created by crystallization solutions (Fig. 2b) [9,10]. These calculations would be of use in the event that a comparison between the X-ray crystallography and ab initio structures of NADþ was required. 3. Results and discussion As expected, bicyclic adenine rarely deviated from planarity due to the conjugated bonding pattern of
Fig. 3. Potential energy curve associated with the rotation about x2i of the adenosine moiety, computed at the RHF/3-21G level of theory.
the rings. A scan was performed on the link between the adenosine and ribose rings, labelled as xi2 . The minima were located at the gþ and g2 positions (Fig. 3). This differs from the nicotinamide –ribose dihedral, where conformers were found in all three positions (gþ ; a and g2 ). This is likely due to the fact that adenine contains a fused bicyclic ring structure rather than a single ring, which would increase the steric hindrance around the rotor. A point optimization of the fragment with xi2 set to the anti ðaÞ position was also made to verify that only two minima existed. Indeed, xi2 converged in the gþ conformation ðx2i ¼ 54:3959Þ; confirming the scan results. Although two virtually identical minima were found at gþ and g2 ; it appeared that x2i preferred the gþ conformation. The examination of all three dihedrals (x2i ; d3i ; d4i ) are summarized in Table 1. The fully optimized structure of adenosine is shown in Fig. 4 at its minima (x2i ; d3i ; d4i ) ¼ (gþ ; gþ ; a). The gþ structure must be preferred due to potential hydrogen bond stabilization between O6/H31 and N18/H17, with distances of 2.427 and ˚ , respectively. The most energetically favour2.089 A 2 able g conformer, (x2i ; d3i ; d4i ) ¼ (g2 ; a;gþ ), lacked ˚ ), while N18/H17 the O6/H31 hydrogen bond (3.736 A ˚ remained fairly stable at 2.050 A (Fig. 5). Thus, it appears that the O6/H31 bond between the ribose ring and adenine is critical in generating an energetically favourable conformation of adenosine. Minima obtained were further confirmed by frequency calculations (Table 2). It was expected that the scan of x2i of the charged adenosine moiety should closely resemble
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Table 1 Conformations obtained for the adenosine subgroup varying the torsional angles x2i ; d3i and d4i ; computed at the RHF/3-21G level of theory Conformer
Optimized values (8)
½xi2 ; d3i d4i
xi2
gþ ½gþ gþ gþ ½gþ a gþ ½gþ g2 gþ ½agþ gþ ½aa gþ ½ag2 gþ ½g2 gþ gþ ½g2 a gþ ½g2 g2 g2 ½gþ gþ g2 ½gþ a g2 ½gþ g2 g2 ½agþ g2 ½aa g2 ½ag2 g2 ½g2 gþ g2 ½g2 a g2 ½g2 g2
45.5871 61.3466
52.5532
54.3876 261.0162 258.8578
259.1876
261.9397
d3i 88.5546 79.2342 Not found, migrated Not found, migrated Not found, migrated 2157.4757 Not found, migrated Not found, migrated 276.9195 92.5123 79.7864 Not found, migrated Not found, migrated Not found, migrated 2147.724 Not found, migrated Not found, migrated 283.1485
Energy (hartrees)
DEnergy (kcal mol21)
78.3654 -167.1856
2878.157847538 -878.162828733
þ3.1257 0.0000
235.9853
2878.159931011
þ1.8183
246.1692 71.8611 2161.6216
2878.159444534 2878.153416685 2878.157151647
þ2.1236 þ5.9062 þ3.5624
228.4122
2878.160705520
þ1.3323
236.4621
2878.160322927
þ1.5724
d4i
to gþ ½gþ a to gþ ½ag2 to gþ ½gþ a to gþ ½ag2 to gþ ½ag2
to g2 ½gþ a to g2 ½ag2 to g2 ½gþ a to g2 ½g2 g2 to g2 ½g2 g2
that of neutral adenosine. However, the potential energy curve produced had a very defined minimum at gþ (Fig. 5). Furthermore, there was an approximate 3.9117 kcal mol 21 difference in energy between the gþ and g2 conformers. A full examination of all three dihedrals (x2i ; d3i ; d4i )
demonstrated similar results, with eight minima found between the gþ and g2 conformers (Table 3). In addition, minima for the positively charged moiety were identical as those in neutral adenosine, although the global minima of each moiety were different (Fig. 6). The fully optimized structure of
Fig. 4. Optimized geometry of adenosine at its minima (x2i ; d3i ; d4i ) ¼ (gþ ; gþ ; a), computed at the RHF/3-21G level of theory. Potential hydrogen bonds are shown between O6/H31 and N18/H17, ˚ , respectively. with distances of 2.427 and 2.089 A
Fig. 5. Optimized geometry of an energetically favourable g2 conformer, (x2i ; d3i ; d4i ) ¼ (g2 ; a; gþ ) computed at the RHF/3-21G level of theory. Distances between O6/H31 and N18/H17 are 3.736 ˚ , respectively. and 2.050 A
S.K. Lau et al. / Journal of Molecular Structure (Theochem) 666–667 (2003) 431–437 Table 2 Confirmation of minima by frequency calculations, computed at the RHF/3-21G level of theory Conformer [xi2 ; d3i ; d4i ]
F1
F2
F3
gþ ½gþ gþ gþ½ gþ a gþ ½ag2 gþ ½g2 g2 g2 ½gþ gþ g2 ½gþ a g2 ½ag2 g2 ½g2 g2
26.1958 28.5352 28.0533 28.621 32.8401 21.0353 33.7452 36.9539
55.6107 50.9894 50.7084 50.9111 60.4951 53.273 55.399 58.0549
75.8169 75.4246 79.894 79.6078 86.456 83.8274 86.4975 89.2193
positively-charged adenosine is shown in Fig. 7 at its minima (xi2 ; d3i ; d4i ) ¼ (gþ ; g2 ; g2 ) with potential hydrogen bonds at O6/H31 and N18/H17 ˚ , respectively. Similar to of 2.354 and 2.10 A neutral adenosine, loss of hydrogen bond stabilization can be observed in the g2 ; where distances between O6/H 31 and N18/H 17 are 3.872 and ˚ , respectively (Fig. 8). Minima were 2.063 A confirmed by frequency calculations (Table 4).
435
The presence of the extra hydrogen on N22 in the adenine ring contributes to an approximate 245 kcal mol21 difference in energy between neutral and positively charged adenosine. The positive charge to N22 induced by the presence of hydrogen clearly stabilizes the heterocyclic ring system of adenosine, perhaps by increasing the electron-withdrawing effects of nitrogen and increasing a resonance effect. At some point in the future, it may be of interest to determine whether NAD þ in vivo prefers the conformation of adenosine induced by the acidic environment, as the positive charge may influence the interaction between the two ring moieties. Strong basis-set dependence with data from the 321G and 6-31G* calculations predicting differing minima has been shown by Wu and Houk, who studied NADþ model systems such as N-hydroxymethylpyridinium ion and N-ethylpyridinium ion [11]. Furthermore, this phenomenon has been previously reported for analogue allyl alcohols and ethers [12]. It would therefore be of interest to note the correlation between basis sets as calculations here are brought to increasing levels of theory.
Table 3 Conformations obtained for the positively charged adenosine subgroup varying the torsional angles x2i ; d3i and d4i computed at the RHF/3-21G level of theory Conformer
Optimized values (8)
[xi2 ; d3i ; d4i
xi2
gþ ½gþ gþ gþ ½gþ a gþ ½gþ g2 gþ ½agþ gþ ½aa gþ ½ag2 gþ ½g2 gþ gþ ½g2 a gþ ½g2 g2 g2 ½gþ gþ g2 ½gþ a g2 ½gþ g2 g2 ½agþ g2 ½aa g2 ½ag2 g2 ½g2 gþ g2 ½g2 a g2 ½g2 g2
55.1857 62.6176
66.7451
65.3539 23.4387 272.4606
270.2056
270.8383
Energy (hartrees)
DEnergy (kcal mol21)
d3i
d4i
87.0527 74.9216 Not found, migrated to Not found, migrated to Not found, migrated to 2143.0699 Not found, migrated to Not found, migrated to 275.9683 95.4318 76.2185 Not found, migrated to Not found, migrated to Not found, migrated to 2143.1771 Not found, migrated to Not found, migrated to 276.8192
76.4222 2164.2027
2878.544926258 2878.551080792
þ5.0444 þ1.1824
235.5169
2878.552157445
þ0.5067
243.206 67.4728 2156.1714
2878.552965137 2878.552965002 2878.539101223 2878.545247615
0.0000 þ8.7000 þ4.8427
230.5331
2878.548669705
þ2.6953
238.718
2878.549121389
þ2.4119
gþ ½gþ a gþ ½ag2 gþ ½gþ a gþ ½g2 g2 gþ ½g2 g2
g2 ½gþ a g2 ½ag2 g2 ½gþ a g2 ½g2 g2 g2 ½g2 g2
436
S.K. Lau et al. / Journal of Molecular Structure (Theochem) 666–667 (2003) 431–437 Table 4 Confirmation of minima by frequency calculations, computed at the RHF/3-21G level of theory
Fig. 6. Potential energy curve associated with the rotation about x2i of the positively-charged adenosine moiety, computed at the RHF/3-21G level of theory.
Fig. 7. Optimized geometry of positively-charged adenosine at its minima (x2i ; d3i ; d4i ) ¼ (gþ ; g2 ; g2 ), computed at the RHF/3-21G level of theory. Potential hydrogen bonds are shown between O6/H31 and ˚ , respectively. N18/H17, with distances of 2.354 and 2.10 A
Conformer [x2i ; d3i ; d4i ]
F1
F2
F3
gþ ½gþ gþ gþ ½gþ a gþ ½ag2 gþ ½g2 g2 g2 ½gþ gþ g2 ½gþ a g2 ½ag2 g2 ½g2 g2
26.1996 28.9924 26.9605 27.7951 33.198 32.0525 32.9831 32.0124
55.0869 53.1113 50.9243 51.3511 63.2129 62.0061 59.6958 60.1255
76.6393 77.3137 74.1477 75.0869 77.9338 79.7404 76.5724 76.6985
4. Conclusion The minima for the three dihedrals x2i ; d3i and d4i in adenosine was found at gþ ; gþ and a using the RHF/321G level of theory. The positively charged adenosine moiety was also optimized to (x2i ; d3i ; d4i ) ¼ (gþ ; g2 ; g2 ) at the RHF/3-21G level of theory. All minima were confirmed by frequency calculations. The next step would be to bring the converged conformations to higher levels of theory, and begin combining this fragment with a structurally optimized pyrophosphate bridge. In this way, we hope to completely characterize each portion of NADþ so that we may integrate the segments and fully develop an ab initio model for the molecule. With a better understanding of the oxidized form of NAD, ab initio studies of NADH and its analogues can be easily conducted, allowing for further research encompassing the computational, biochemical and pharmacological fields.
Acknowledgements
Fig. 8. Optimized geometry of an energetically favourable g2 conformer, (x2i ; d3i ; d4i ) ¼ (g2 ; g2 ; g2 ) computed at the RHF/3-21G level of theory. Distances between O6/H31 and N18/H17 are 3.872 ˚ , respectively. and 2.063 A
This work was supported with grants from the Global Institute Of COmputational Molecular and Materials Sciences (GIOCOMMS), Toronto, Ont., Canada. Thanks are extended to Christopher N.J. Marai, Jeremy H. Keller and David H. Setiadi for helpful discussion. One of the authors (IGC) wishes to thank the Ministry of Education for a Szent-Gyo¨rgyi Visiting Professorship.
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References [1] L. Stryer, Biochemistry, Freeman, New York, 1988, pp. 320 – 321. [2] V. Shneyvays, L.K. Mamedora, D. Leshem, Exp. Clin. Cardiol. 7 (2002) 138– 145. [3] P. Acharya, J. Chattopadhyaya, J. Org. Chem. 67 (2002) 1852. [4] S.K. Lau, G.A. Chass, S. Lovas, B. Penke. Submitted for publication. [5] T. Ishida, M. Tarui, Y. In, M. Ogiyama, M. Doi, M. Inoue, FEBS Lett. 333 (1993) 214. [6] Y. Shimohigashi, I. Maeda, T. Nose, K. Ikesue, H. Sakamoto, T. Ogawa, Y. Ide, M. Kawahara, T. Nezu, Y. Terada, K. Kawano, M. Ohno, J. Chem. Soc., Perkin Lett. 1 2479 (1996). [7] P.E. Smith, J.J. Tanner, J. Mol. Recognit. 13 (2000) 27. [8] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, V.G. Zakrzewski, J.A. Montgomery,
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Jr., R.E. Stratmann, J.C. Burant, S. Dapprich, J.M. Millam, ¨ . Farkas, J. Tomasi, V. A.D. Daniels, K.N. Kudin, M.C. Strain, O Barone, M. Cossi, R. Cammi, B. Mennucci, C. Pomelli, C. Adamo, S. Clifford, J. Ochterski, G.A. Petersson, P.Y. Ayala, Q. Cui, K. Morokuma, D.K. Malick, A.D. Rabuck, K. Raghavachari, J.B. Foresman, J. Cioslowski, J.V. Ortiz, A.G. Baboul, B.B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R. Gomperts, R.L. Martin, D.J. Fox, T. Keith, M.A. Al-Laham, C.Y. Peng, A. Nanayakkara, C. Gonzalez, M. Challacombe, P.M.W. Gill, B.G. Johnson, W. Chen, M.W. Wong, J.L. Andres, M. Head-Gordon, E.S. Replogle, J.A. Pople, Gaussian 98 (Revision A.9), Gaussian, Inc., Pittsburgh PA, 1998. B. Guillot, C. Jelsch, C. Lecomte, Acta Cryst. C56 (2000) 726. B. Guillot, C. Lecomte, A. Cousson, C. Scherf, C. Jelsch, Acta Cryst. D57 (2001) 981. Y-D. Wu, K.N. Houk, J. Am. Chem. Soc. 113 (1991) 58. S.D. Kahn, W.J. Hehre, J. Am. Chem. Soc. 109 (1987) 666.
An exploratory ab initio conformational analysis of selected fragments of nicotinamide adenine dinucleotide (NADþ). Part II: adenosine Suzanne K. Laua,b,*, Gregory A. Chassa,b,c,d, Botond Penkee,f, Imre G. Csizmadiaa,b,f a
b
Department of Chemistry, University of Toronto, Toronto, Ont., Canada M5S 3H6 Global Institute of Computational Molecular and Materials Science, 1422 Edenrose Street, Mississauga, Ont., Canada L5V 1H3 c Institut de Science et d’Inge´nierie Supramole´culaires, 8, alle´e Gaspard Monge, BP 70028, Strasbourg Cedex 67083, France d Department of Biomedical Sciences, Creighton University, 2500 California plaza, Omaha, NE 68178, USA e Protein Chemistry Research Group, Hungarian Academy of Sciences, University of Szeged, Do´m te´r 8, Szeged 6720, Hungary f Department of Medical Chemistry, University of Szeged, Do´m te´r 8, Szeged H-6720, Hungary
Abstract Ab initio molecular orbital computations were carried out on selected fragments of nicotinamide adenine dinucleotide (NADþ) at the RHF/3-21G level of theory. The definitions of the relative spatial orientation of all constituent atomic nuclei have been formulated in such a modular fashion so as to allow for portions of the whole to be studied independently. Key points of examination included the rotation of adjacent moieties and ring puckering. This work focuses on the possible conformations of the adenosine moiety. It is anticipated that the structural results from the truncated and modified fragments will closely resemble those of NAD as a whole. q 2003 Elsevier B.V. All rights reserved. Keywords: Ab initio; Coenzyme; Nicotinamide adenine dinucleotide; Adenine; Adenosine
1. Introduction The redox pair formed by the oxidized and reduced forms of nicotinamide adenine dinucleotide (NADþ/NADH) holds great biological importance, most notably in the energy producing process [1]. As depicted in Fig. 1, NADþ contains an adenylic acid and nicotinamide-50 -ribonucleotide combined by apyrophosphate bridge. Nicotinamide can carry * Corresponding author. Department of Chemistry, University of Toronto, Toronto, Ont., Canada M5S 3H6. E-mail addresses: [email protected] (S.K. Lau), [email protected] (G.A. Chass). 0166-1280/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.theochem.2003.08.118
a positive charge at the ring nitrogen, while the pyrophosphate link holds two negative charges. Adenosine is also a significant biological molecule in its own right. 20 -Deoxyadenosine 50 -phosphate forms an integral nucleotide in DNA, while its oxidated counterpart adenosine 50 -phosphate is present in RNA. It is involved in many signalling pathways as cyclic AMP, and has been shown to offer cardioprotection through the regulation of the adenosine receptor [2]. A quick perusal of the literature has shown that little ab initio structure work has been done on adenosine, although some have attempted to examine hydrogen bonding and hydration of the molecule using NMR spectra and molecular
432
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Fig. 1. Systematic modular numbering system of NADþ including pertinent dihedrals. The arrows indicate the approximate partitioning of the molecule. (1) Nicotinamide, (2) riboseN, (3) nicotinamide ribose (NmR), (4) adenine, (5) riboseA, (6) adenosine and (7) pyrophosphate bridge. This paper focuses primarily on (4) –(6).
modelling [3]. Much of the research interest has been focussed on base pair interactions in DNA and RNA. The structural characterization of NADþ presented a particular challenge, as it is a flexible molecule with over a dozen rotatable bonds that can adopt a wide variety of environmentally dependent conformations. Our first work characterized the possible conformations of the nicotinamide-50 -ribonucleotide portion of NADþ [4]. We now aim to study the adenosine portion in a similar manner. The study of
the adenosine moiety (Fig. 2a) can be considered a precursor for the future examination of adenosine monophosphate, diphosphate or triphosphate. Furthermore, a study of the interactions between the two aromatic ring groups would be suitable. Previous Xray data has suggested that a tryptophane derivative with similar aromatic rings exists at a perpendicular state [5,6], while molecular dynamic simulations of NADþ have indicated that ring interactions occur primarily in the stacked conformation [7]. Thus,
Fig. 2. (a) Fully numbered adenosine moiety, with dihedrals of interest labelled. (b) Fully numbered positively charged adenosine moiety, with dihedrals of interest labelled.
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433
it would be of interest to complete the conformational analysis of the two ring groups, and then investigate the interaction between them. Insight into the conformation of NADþ in different environments will allow for a more complete understanding of the mechanism of its diverse functions at the molecular level, allowing for drug candidates with tailored ligand/receptor binding sites to be developed in the treatment of NAD-dependent pathologies. 2. Methods The GAUSSIAN 98 [8] program package has been used to perform preliminary ab initio calculations on the adenosine fragment of NADþ. The definitions of the relative spatial orientations of all constituent atomic nuclei have been formulated in a standardized and modular fashion so as to allow for portions of the whole to be studied independently (Fig. 1). The focus of this paper is on the adenosine group, which was first modelled by the semi-empirical AM1 method (data not shown). Previously obtained data for the optimization of the ribose molecule was used for the optimizations [4]. Few problems were encountered in the calculations of this moiety to date, primarily because the techniques developed while computing the conformations of the nicotinamide – ribose moiety were employed. It had been found that freezing the dihedrals of interest and sequentially unfreezing them with pre-optimized data generated the most accurate results. Each dihedral of interest was examined independently, and then incorporated into the larger study with multiple scans of up to three dihedrals. The dihedral xi2 was examined in conjunction with the hydroxy rotors ðd3i ; d4i Þ on the ribose portion of the molecule. Preliminary calculations were also made on a positively charged adenosine molecule found in the acidic environment created by crystallization solutions (Fig. 2b) [9,10]. These calculations would be of use in the event that a comparison between the X-ray crystallography and ab initio structures of NADþ was required. 3. Results and discussion As expected, bicyclic adenine rarely deviated from planarity due to the conjugated bonding pattern of
Fig. 3. Potential energy curve associated with the rotation about x2i of the adenosine moiety, computed at the RHF/3-21G level of theory.
the rings. A scan was performed on the link between the adenosine and ribose rings, labelled as xi2 . The minima were located at the gþ and g2 positions (Fig. 3). This differs from the nicotinamide –ribose dihedral, where conformers were found in all three positions (gþ ; a and g2 ). This is likely due to the fact that adenine contains a fused bicyclic ring structure rather than a single ring, which would increase the steric hindrance around the rotor. A point optimization of the fragment with xi2 set to the anti ðaÞ position was also made to verify that only two minima existed. Indeed, xi2 converged in the gþ conformation ðx2i ¼ 54:3959Þ; confirming the scan results. Although two virtually identical minima were found at gþ and g2 ; it appeared that x2i preferred the gþ conformation. The examination of all three dihedrals (x2i ; d3i ; d4i ) are summarized in Table 1. The fully optimized structure of adenosine is shown in Fig. 4 at its minima (x2i ; d3i ; d4i ) ¼ (gþ ; gþ ; a). The gþ structure must be preferred due to potential hydrogen bond stabilization between O6/H31 and N18/H17, with distances of 2.427 and ˚ , respectively. The most energetically favour2.089 A 2 able g conformer, (x2i ; d3i ; d4i ) ¼ (g2 ; a;gþ ), lacked ˚ ), while N18/H17 the O6/H31 hydrogen bond (3.736 A ˚ remained fairly stable at 2.050 A (Fig. 5). Thus, it appears that the O6/H31 bond between the ribose ring and adenine is critical in generating an energetically favourable conformation of adenosine. Minima obtained were further confirmed by frequency calculations (Table 2). It was expected that the scan of x2i of the charged adenosine moiety should closely resemble
434
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Table 1 Conformations obtained for the adenosine subgroup varying the torsional angles x2i ; d3i and d4i ; computed at the RHF/3-21G level of theory Conformer
Optimized values (8)
½xi2 ; d3i d4i
xi2
gþ ½gþ gþ gþ ½gþ a gþ ½gþ g2 gþ ½agþ gþ ½aa gþ ½ag2 gþ ½g2 gþ gþ ½g2 a gþ ½g2 g2 g2 ½gþ gþ g2 ½gþ a g2 ½gþ g2 g2 ½agþ g2 ½aa g2 ½ag2 g2 ½g2 gþ g2 ½g2 a g2 ½g2 g2
45.5871 61.3466
52.5532
54.3876 261.0162 258.8578
259.1876
261.9397
d3i 88.5546 79.2342 Not found, migrated Not found, migrated Not found, migrated 2157.4757 Not found, migrated Not found, migrated 276.9195 92.5123 79.7864 Not found, migrated Not found, migrated Not found, migrated 2147.724 Not found, migrated Not found, migrated 283.1485
Energy (hartrees)
DEnergy (kcal mol21)
78.3654 -167.1856
2878.157847538 -878.162828733
þ3.1257 0.0000
235.9853
2878.159931011
þ1.8183
246.1692 71.8611 2161.6216
2878.159444534 2878.153416685 2878.157151647
þ2.1236 þ5.9062 þ3.5624
228.4122
2878.160705520
þ1.3323
236.4621
2878.160322927
þ1.5724
d4i
to gþ ½gþ a to gþ ½ag2 to gþ ½gþ a to gþ ½ag2 to gþ ½ag2
to g2 ½gþ a to g2 ½ag2 to g2 ½gþ a to g2 ½g2 g2 to g2 ½g2 g2
that of neutral adenosine. However, the potential energy curve produced had a very defined minimum at gþ (Fig. 5). Furthermore, there was an approximate 3.9117 kcal mol 21 difference in energy between the gþ and g2 conformers. A full examination of all three dihedrals (x2i ; d3i ; d4i )
demonstrated similar results, with eight minima found between the gþ and g2 conformers (Table 3). In addition, minima for the positively charged moiety were identical as those in neutral adenosine, although the global minima of each moiety were different (Fig. 6). The fully optimized structure of
Fig. 4. Optimized geometry of adenosine at its minima (x2i ; d3i ; d4i ) ¼ (gþ ; gþ ; a), computed at the RHF/3-21G level of theory. Potential hydrogen bonds are shown between O6/H31 and N18/H17, ˚ , respectively. with distances of 2.427 and 2.089 A
Fig. 5. Optimized geometry of an energetically favourable g2 conformer, (x2i ; d3i ; d4i ) ¼ (g2 ; a; gþ ) computed at the RHF/3-21G level of theory. Distances between O6/H31 and N18/H17 are 3.736 ˚ , respectively. and 2.050 A
S.K. Lau et al. / Journal of Molecular Structure (Theochem) 666–667 (2003) 431–437 Table 2 Confirmation of minima by frequency calculations, computed at the RHF/3-21G level of theory Conformer [xi2 ; d3i ; d4i ]
F1
F2
F3
gþ ½gþ gþ gþ½ gþ a gþ ½ag2 gþ ½g2 g2 g2 ½gþ gþ g2 ½gþ a g2 ½ag2 g2 ½g2 g2
26.1958 28.5352 28.0533 28.621 32.8401 21.0353 33.7452 36.9539
55.6107 50.9894 50.7084 50.9111 60.4951 53.273 55.399 58.0549
75.8169 75.4246 79.894 79.6078 86.456 83.8274 86.4975 89.2193
positively-charged adenosine is shown in Fig. 7 at its minima (xi2 ; d3i ; d4i ) ¼ (gþ ; g2 ; g2 ) with potential hydrogen bonds at O6/H31 and N18/H17 ˚ , respectively. Similar to of 2.354 and 2.10 A neutral adenosine, loss of hydrogen bond stabilization can be observed in the g2 ; where distances between O6/H 31 and N18/H 17 are 3.872 and ˚ , respectively (Fig. 8). Minima were 2.063 A confirmed by frequency calculations (Table 4).
435
The presence of the extra hydrogen on N22 in the adenine ring contributes to an approximate 245 kcal mol21 difference in energy between neutral and positively charged adenosine. The positive charge to N22 induced by the presence of hydrogen clearly stabilizes the heterocyclic ring system of adenosine, perhaps by increasing the electron-withdrawing effects of nitrogen and increasing a resonance effect. At some point in the future, it may be of interest to determine whether NAD þ in vivo prefers the conformation of adenosine induced by the acidic environment, as the positive charge may influence the interaction between the two ring moieties. Strong basis-set dependence with data from the 321G and 6-31G* calculations predicting differing minima has been shown by Wu and Houk, who studied NADþ model systems such as N-hydroxymethylpyridinium ion and N-ethylpyridinium ion [11]. Furthermore, this phenomenon has been previously reported for analogue allyl alcohols and ethers [12]. It would therefore be of interest to note the correlation between basis sets as calculations here are brought to increasing levels of theory.
Table 3 Conformations obtained for the positively charged adenosine subgroup varying the torsional angles x2i ; d3i and d4i computed at the RHF/3-21G level of theory Conformer
Optimized values (8)
[xi2 ; d3i ; d4i
xi2
gþ ½gþ gþ gþ ½gþ a gþ ½gþ g2 gþ ½agþ gþ ½aa gþ ½ag2 gþ ½g2 gþ gþ ½g2 a gþ ½g2 g2 g2 ½gþ gþ g2 ½gþ a g2 ½gþ g2 g2 ½agþ g2 ½aa g2 ½ag2 g2 ½g2 gþ g2 ½g2 a g2 ½g2 g2
55.1857 62.6176
66.7451
65.3539 23.4387 272.4606
270.2056
270.8383
Energy (hartrees)
DEnergy (kcal mol21)
d3i
d4i
87.0527 74.9216 Not found, migrated to Not found, migrated to Not found, migrated to 2143.0699 Not found, migrated to Not found, migrated to 275.9683 95.4318 76.2185 Not found, migrated to Not found, migrated to Not found, migrated to 2143.1771 Not found, migrated to Not found, migrated to 276.8192
76.4222 2164.2027
2878.544926258 2878.551080792
þ5.0444 þ1.1824
235.5169
2878.552157445
þ0.5067
243.206 67.4728 2156.1714
2878.552965137 2878.552965002 2878.539101223 2878.545247615
0.0000 þ8.7000 þ4.8427
230.5331
2878.548669705
þ2.6953
238.718
2878.549121389
þ2.4119
gþ ½gþ a gþ ½ag2 gþ ½gþ a gþ ½g2 g2 gþ ½g2 g2
g2 ½gþ a g2 ½ag2 g2 ½gþ a g2 ½g2 g2 g2 ½g2 g2
436
S.K. Lau et al. / Journal of Molecular Structure (Theochem) 666–667 (2003) 431–437 Table 4 Confirmation of minima by frequency calculations, computed at the RHF/3-21G level of theory
Fig. 6. Potential energy curve associated with the rotation about x2i of the positively-charged adenosine moiety, computed at the RHF/3-21G level of theory.
Fig. 7. Optimized geometry of positively-charged adenosine at its minima (x2i ; d3i ; d4i ) ¼ (gþ ; g2 ; g2 ), computed at the RHF/3-21G level of theory. Potential hydrogen bonds are shown between O6/H31 and ˚ , respectively. N18/H17, with distances of 2.354 and 2.10 A
Conformer [x2i ; d3i ; d4i ]
F1
F2
F3
gþ ½gþ gþ gþ ½gþ a gþ ½ag2 gþ ½g2 g2 g2 ½gþ gþ g2 ½gþ a g2 ½ag2 g2 ½g2 g2
26.1996 28.9924 26.9605 27.7951 33.198 32.0525 32.9831 32.0124
55.0869 53.1113 50.9243 51.3511 63.2129 62.0061 59.6958 60.1255
76.6393 77.3137 74.1477 75.0869 77.9338 79.7404 76.5724 76.6985
4. Conclusion The minima for the three dihedrals x2i ; d3i and d4i in adenosine was found at gþ ; gþ and a using the RHF/321G level of theory. The positively charged adenosine moiety was also optimized to (x2i ; d3i ; d4i ) ¼ (gþ ; g2 ; g2 ) at the RHF/3-21G level of theory. All minima were confirmed by frequency calculations. The next step would be to bring the converged conformations to higher levels of theory, and begin combining this fragment with a structurally optimized pyrophosphate bridge. In this way, we hope to completely characterize each portion of NADþ so that we may integrate the segments and fully develop an ab initio model for the molecule. With a better understanding of the oxidized form of NAD, ab initio studies of NADH and its analogues can be easily conducted, allowing for further research encompassing the computational, biochemical and pharmacological fields.
Acknowledgements
Fig. 8. Optimized geometry of an energetically favourable g2 conformer, (x2i ; d3i ; d4i ) ¼ (g2 ; g2 ; g2 ) computed at the RHF/3-21G level of theory. Distances between O6/H31 and N18/H17 are 3.872 ˚ , respectively. and 2.063 A
This work was supported with grants from the Global Institute Of COmputational Molecular and Materials Sciences (GIOCOMMS), Toronto, Ont., Canada. Thanks are extended to Christopher N.J. Marai, Jeremy H. Keller and David H. Setiadi for helpful discussion. One of the authors (IGC) wishes to thank the Ministry of Education for a Szent-Gyo¨rgyi Visiting Professorship.
S.K. Lau et al. / Journal of Molecular Structure (Theochem) 666–667 (2003) 431–437
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