Three-dimensional structure of anti-5,6-dimethylchrysene-1,2-dihydrodiol-3,4-epoxide: a diol epoxide with a bay region methyl group
Carol E. Afshar1,
Amy Kaufman Katz1,
H.L. Carrell1,
Shantu Amin2,
Dhimant Desai2 and
Jenny P. Glusker1,3
1 The Institute for Cancer Research, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111 and
2 American Health Foundation, 1 Dana Road, Valhalla, NY 10595, USA
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Abstract
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The three-dimensional structure of a dihydrodiol epoxide of 5,6-dimethylchrysene was elucidated by X-ray diffraction techniques. The effects of the steric overcrowding by the 5-methyl group in the bay region of this compound are described. The carbon atom of the 5-methyl group is found to lie out of the plane of the aromatic system, thereby avoiding the nearer C-H group of the epoxide ring; this C-H hydrogen atom is pushed in the opposite direction. As a result, the molecule is distorted so that the relative orientations of the epoxide group and the aromatic ring systems are very different for the diol epoxides of (nearly planar) benzo[a]pyrene (studied by Neidle and co-workers) and (distorted) 5,6-dimethylchrysene (described here). The main effect of the 5-methyl group is to change the relative angle between the epoxide-bearing ring (the site of attack when the diol epoxide acts as an alkylating agent) and the aromatic ring system (which is presumed to lie partially between the DNA bases in the DNA adduct that is about to be formed). This may favor some specific alkylation geometry.
Abbreviations: BaP, benzo[a]pyrene; 5,6-diMeC, 5,6-dimethylchrysene; PAH, polycyclic aromatic hydrocarbon
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Introduction
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When a polycyclic aromatic hydrocarbon (PAH) produces a neoplastic lesion, it has first been activated to a molecular species that can interact with informational macromolecules in the cell (1). A major mode of activation is the formation of a diol epoxide (1), which can then alkylate DNA. The carcinogenic potential of a PAH is often greatly increased when the PAH contains a bay region methyl group on the more aromatic side of the bay region (e.g. the 5 but not the 4 position in chrysene; see numbering in Figure 1
) (26). It has generally been assumed that the reason for this is that the bay region methyl group, by virtue of its bulk, causes significant molecular overcrowding in the diol epoxide and/or in the DNA adduct that is formed. This steric hindrance is presumed to favor a conformation that facilitates the progression of carcinogenesis (7,8). It has been found (for the parent PAHs) that 5-methylchrysene (with a bay region methyl group) is the only monomethylchrysene that is appreciably carcinogenic (2), but, surprisingly, its PAH derivative, 5,6-dimethylchrysene, is weaker as a tumor initiator (6).
Diol epoxides of fairly planar PAHs such as benzo[a]pyrene (BaP) react with DNA mainly at the amino groups of guanine residues (9). On the other hand, distorted PAHs (such as 7,12-dimethylbenz[a]anthracene) appear to attack the amino groups of both adenine and guanine in DNA (1014). This indicates that bay region distortions play a significant role in determining the course of PAHDNA adduct formation. We present here the crystal structure of the anti-diol epoxide of 5,6-dimethyl-chrysene (anti-5,6-dimethylchrysene-1,2-dihydrodiol-3,4-epoxide). Steric interactions between the bay region methyl group and the ring that bears the diol and epoxide groups are described for the first time. The formula of the compound studied here is presented in Figure 1
.
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Materials and methods
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Anti-5,6-dimethylchrysene-1,2-dihydrodiol-3,4-epoxide (5,6-diMeC diol epoxide) was synthesized as reported by Misra and co-workers (14). The crystal used for data collection was crystallized from tetrahydrofuran as a thin lath with dimensions 0.20x0.15x0.03 mm. It is monoclinic with unit cell dimensions a = 17.251(8) Å, b = 7.666(1) Å, c = 11.319(1) Å and ß = 99.91(7)°. The unit cell dimensions were determined using 250 measured Bragg reflections with
angles ranging from 1018° and having I > 25
(I). The space group was determined (15) to be P21/a with Z = 4. This space group is centrosymmetric, so that both the molecule and its mirror image are studied. This is an advantage to the precision of the structure determination because the phases of diffracted beams are well defined at 0° or 180° rather than any value between 0 and 360°.
Three-dimensional X-ray diffraction data were measured with an Enraf-Nonius FAST area detector diffractometer using a rotating anode generator with a molybdenum anode [
(MoK
) = 0.71073 Å] employing a graphite monochromator. All measurements were carried out at low temperatures [120(2) K]. The data were measured as frames of 0.25° of rotation/frame for 40 s/frame at a crystal-to-detector distance of 50 mm with a
offset of 30°. The crystal-to-detector distance was calibrated with a crystal of basic beryllium acetate (cubic unit cell with a = 15.735 Å). The Munich Area Detector NE System program (15,16) was used for both collection and integration of the frames to yield integrated reflection intensity. A total of 12 522 data were measured in three crystal settings and were merged using XSCALE (17) to give 3367 unique reflections with Rmerge(I) = 0.069 to sin
/
max = 0.649/Å.
The crystal structure was determined by direct methods using the structure solution package MULTAN88 (18). The resulting structure was refined using the program ICRFMLS (19,20), which had been modified to refine on F2. After an initial isotropic refinement, all of the hydrogen atoms were located in an FoFc electron density map. The refinement was anisotropic for all non-hydrogen atoms and isotropic for hydrogen atoms. The quantity minimized in the least squares refinement was
w(Fo2Fc2) and the weighting scheme was w = 1/
2(Fo2). Scattering factors used were those published in International Tables for X-ray Crystallography (21). The agreement between the calculated and observed structures is R1 = 0.104 and wR1 = 0.043 for all data. The agreement with respect to F2 (the basis of the structure refinement) was R2 = 0.080 and wR2 = 0.089. R1(F > 4
F) = 0.042 (1795 reflections) with a goodness-of-fit of 1.059. The minimum and maximum electron densities in the final difference Fourier map are 0.46 e/Å3 and 0.58 e/Å3. The atomic numbering and bond lengths are given in Figure 2
. Molecular diagrams in the figures in this article were drawn with the in-house program ICRVIEW (22). Atomic coordinates are listed in Table I
; the atomic displacement parameters (anisotropic and isotropic) and calculated and observed structure factors are available from the authors. CH and OH bond lengths were extended along the bond vector to values found from neutron diffraction analyses of organic compounds in general (CH sp3 = 1.09 Å, CH sp2 = 1.07 Å, OH = 0.97 Å) (23) and the calculated atomic coordinates for hydrogen atoms are also listed in Table I
. The estimated standard deviations of atomic positions are also given in Table I
. They correspond to e.s.d. values of 0.002 Å for bonds involving C and O and 0.20.3° for angles.

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Fig. 2. Atomic numbering system used for 5,6-dimethylchrysene-1,2-diol-3,4-epoxide and bond lengths in Å. Oxygen atoms are stippled. Average estimated standard deviations are ~0.002 Å for CC and CO bonds and 0.02 Å for bonds involving hydrogen atoms.
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Results and discussion
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This structure analysis shows, as can be seen in Figure 2
, that the two hydroxyl groups in the epoxide-bearing ring are in the equatorial conformation (lying approximately in the plane of the aromatic ring system), as they also are in the anti-diol epoxide of BaP (24). All three bonds in the epoxide ring are similar in length (1.4611.466(2) Å), indicating that no appreciable distortions have occurred to alter the reactivity of this group (25,26). A comparison of the geometry of the PAH 5,6-dimethylchrysene (5,6-diMeC) (27) with that of its diol epoxide (this structure) shows that there is little additional distortion in the PAH ring system on diol epoxide formation, except in the epoxide-bearing ring, which has now become saturated. The conformation of this six-membered ring is similar to that found in other crystal structures containing analogous ring systems (24,2832); the only significant differences were that there are higher torsion angles around C13 in the structure described here. Thus the spatial relationship between the hydroxyl groups which form hydrogen bonds (thereby representing a biological binding mode of this molecule) and the epoxide ring itself (which represents the site of biochemical activity) is similar in both the diol epoxides of BaP and 5,6-diMeC.
The role of the 5-methyl group in the diol epoxide of 5,6-diMeC was the main focus of this study. The question is what is the effect of the additional methyl group on the three-dimensional structure of the diol epoxide, because 5-methylation greatly enhances the carcinogenicity of chrysene. The main steric interactions in the bay region are between hydrogen atoms (H19 and H19') on the 5-methyl group (C19) and the hydrogen atom (H4) on the bay region side of the epoxide group (Figure 3
). The molecule has assumed a conformation in which the minimum non-bonded HH distance is ~2 Å (H19H4 = 2.1 Å, H19'H4 = 2.3 Å). To ensure this, as shown in Figure 4
, C19 is pushed down below the plane of the aromatic system, while the epoxide group, including H4, is pushed up a large distance, ~2 Å. This avoids the steric clash that would occur if C19 and H4 were both to lie exactly in the plane of the aromatic system. The 5-methyl group is also constrained from free rotation by HH interactions with its adjacent 6-methyl group (H19"H20' = 2.1 Å) to the fixed orientation shown in Figure 3
. This might be part of the explanation for the greater carcinogenic activity of 5-methylchrysene versus 5,6-diMeC (6).

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Fig. 3. General view of the diol epoxide of 5,6-diMeC. The epoxide and methyl groups are indicated by filled bonds and some H . . . H interactions are shown.
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Fig. 4. A comparison of the relative orientations of the epoxide groups and the ring systems of the diol epoxides of 5,6-dimethylchrysene (this work) and BaP (24). (a) View of the diol epoxide of 5,6-diMeC along the plane through C5, C6 and C15 (heavier bonds), showing the relationship of the C19 methyl group (attached to C5) and the epoxide group (also shown with heavier bonds). (b) View of the diol epoxide of BaP (25) along the plane through the analogous plane to that in (a). Note how the epoxide group is pushed up in (a) relative to its position in (b). (c) View down the C3C4 bond of the epoxide group of 5,6-diMeC diol epoxide. The aromatic ring C7C8C9C10C17C18 is highlighted. (d) A similarly oriented view of the diol epoxide of BaP. Note the differences in the orientation of the aromatic rings furthest from the epoxide in (c) and (d).
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This structural analysis has shown the directions along which the C19 methyl group and the epoxide group adjust to steric overcrowding (Figure 1
). An alternative conformation that might be envisioned, in which the 5-methyl group moves in the opposite direction out of the plane of the aromatic ring system (`up' in the orientation in Figure 3
), is energetically much less likely. The reason for this is that H4 is already above the plane of the aromatic system to which it is attached and would require a much larger displacement to avoid a steric clash and therefore this second conformation is energetically disfavored.
Thus, the relief of steric overcrowding involves: (i) C19 being forced out of the plane of the aromatic system to which it is attached; (ii) increases in external CCC angles in the bay region; (iii) the epoxide group, including H4, being forced in the opposite direction from C19. These conformational effects bring the epoxide oxygen atom near to H19'; the C19H19' bond points almost directly towards this oxygen atom (H19'O3 = 2.7 Å), as shown in Figure 3
.
The relief of steric strain in the bay region distorts the aromatic system so that torsion angles about CC bonds deviate from the expected value of 0° for an aromatic system. This is especially evident in the bay region where torsion angles are as large as 23°, but it extends through the aromatic system, particularly along the C15C16 bond. The final result is that the relative angles between the planes of the aromatic system and the epoxide ring are different for the diol epoxides of 5,6-diMeC (115.5°) and of BaP (80.9°), a variation of ~35° (Figure 4
). Therefore, if intercalation of the flat portion of the PAH between the bases of DNA occurs, the epoxide-bearing ring (where the alkylating action occurs) will be in a different location for the two diol epoxides. In addition, the strain imposed on H4 may be transferred to C4, increasing its chemical reactivity.
The packing of molecules will give some indication of the charge distribution in the molecule. The two hydroxyl groups act as hydrogen bond donors; while the two hydrogen bond acceptors are the oxygen of the epoxide and the hydroxyl group nearest to the epoxide, group, as shown in Figure 5
. The arrangement of charges around the epoxide oxygen atom may affect the charge distribution around C4, the site of alkylation of DNA (33). This could enhance the alkylating function of this diol epoxide, because a hydrogen bond to the epoxide group can be considered to be an incipient protonation (an initial step in epoxide ring opening). The manner in which two hydroxyl groups approach and form intermolecular hydrogen bonds to the epoxide and its adjacent hydroxyl group oxygen atoms is reminiscent of the same intermolecular stereochemistry in crystals of cis-2,3-epoxycyclooctanol (34). In the anti- and syn-diol epoxides of BaP there are additional interactions that are not found in the structure examined here. In the anti-diol epoxide of BaP there is a C-HO interaction to the epoxide oxygen atom from a hydrogen atom on a hydroxyl-bearing carbon atom (24). In the syn-diol epoxide of BaP there is a somewhat short interaction between the epoxide oxygen atom and one of the carbon atoms of the epoxide group (32).

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Fig. 5. Packing of molecules showing the O-HH hydrogen bonds that are formed. Note the hydrogen bonding to the epoxide oxygen atom.
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Thus, the introduction of a bay region methyl group has forced the aromatic ring system and the epoxide group to distort considerably so that the spatial relationship between the epoxide group and the aromatic portion of the ring system is appreciably altered. Since the amino groups of guanine lie in the minor groove of DNA, whereas those of adenine lie in the wider major groove, it is possible that the bulkiness of bay region-substituted PAH diol epoxides and the spatial relationships of certain functional groups influence the adenine:guanine ratio of attacked bases. An overall evaluation of the significance of these findings to the process of carcinogenesis must await further structural studies of diol epoxides.
Anisotropic and isotropic vibrational parameters and observed and calculated structure factors are available from the authors.
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Acknowledgments
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This study was supported by Research Grant CA-10925 (to J.P.G.), Cancer Center Core Grant CA-06927 (to F.C.C.C.) and Cancer Center Support Grant CA-17613 (to S.A.) from the National Institutes of Health and an appropriation from the Commonwealth of Pennsylvania (to F.C.C.C.). The results and interpretation are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute.
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Notes
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3 To whom correspondence should be addressed Email: jp_glusker{at}fccc.edu 
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References
|
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-
Sims,P., Grover,P.L., Swaisland,A., Pal,K. and Hewer,A. (1974) Metabolic activation of benzo[a]pyrene proceeds by a diol-epoxide. Nature, 256, 326328.
-
Hecht,S.S., Bondinell,W.E. and Hoffmann,D. (1974) Chrysene and methylchrysenes: presence in tobacco smoke and carcinogenicity. J. Natl Cancer Inst., 52, 11211133.
-
Dipple,A. (1976) In Searle,C.E. (ed.), Chemical Carcinogens, ACS Monograph 173. American Chemical Society, Washington, DC, pp. 245314.
-
Kashino,S., Zacharias,D.E., Prout,C.K., Carrell,H.L., Glusker,J.P., Hecht,S.S. and Harvey,R.G. (1984) Structure of 5-methylchrysene, C19H14. Acta Crystallogr., C40, 536540.[ISI]
-
Afshar,C.E., Carrell,H.L., Harvey,R.G., Kiselyov,S., Amin,S. and Glusker,J.P. (1994) Bay-region distortions in a methanol adduct of a bay-region diol epoxide of the carcinogen 5-methylchrysene. Carcinogenesis, 17, 25072511.[Abstract]
-
Amin,S., Desai,D. and Hecht,S.S. (1993) Tumor initiating activity on mouse skin of bay region diol epoxides of 5,6-dimethylchrysene and benzo[c]phenanthrene. Carcinogenesis, 14, 20332037.[Abstract]
-
Misra,B.J., Amin,S. and Hecht,S.S. (1992) Metabolism and DNA binding of 5,6-dimethylchrysene in mouse skin. Chem. Res. Toxicol., 5, 242247.[ISI][Medline]
-
Szeliga,J., Hilton,B.D., Chmurny,G.N., Krzeminski,J., Amin,S. and Dipple,A. (1997) Characterization of DNA adducts formed by the four configurationally isomeric 5,6-dimethylchrysene 1,2-dihydrodiol 3,4-epoxides. Chem. Res. Toxicol., 10, 378385.[ISI][Medline]
-
Jeffrey,A.M., Jennette,K.W., Blobstein,H., Weinstein,J.B., Beland,F.A., Harvey,R.G., Kasai,H., Miura,I. and Nakanishi,K. (1976) Benzo[a]pyrenenucleic acid derivative found in vivo: structure of a benzo[a]pyrene tetrahydrodiol epoxideguanosine adduct. J. Am. Chem. Soc., 98, 57145715.[ISI][Medline]
-
Dipple,A., Pigott,M.A., Agarwal,S.K., Yagi,H., Sayer,J.M. and Jerina,D.M. (1987) Optically active benzo[c]phenanthrene diol epoxides bind extensively to adenine in DNA. Nature, 327, 535536.[ISI][Medline]
-
Agarwal,R., Yagi,H., Jerina,D.M. and Dipple,A. (1996) Benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxide adducts in native and denatured DNA. Carcinogenesis, 17, 17731776.[Abstract]
-
Bigger,C.A.H., Flickinger,D.J., Strandberg,J., Pataki,J., Harvey,R.G. and Dipple,A. (1990) Mutational specificity of the anti-1,2-dihydrodiol 3,4-epoxide of 5-methylchrysene. Carcinogenesis, 11, 22632265.[Abstract]
-
Szeliga,J., Amin,S., Zhang,F.-J. and Harvey,R.G. (1999) Reactions of dihydrodiol epoxides of 5-methylchrysene and 5,6-dimethylchysene with DNA and deoxyribonucleotides. Chem. Res. Toxicol., 12, 347352.[ISI][Medline]
-
Misra,B., Amin,S. and Hecht,S.S. (1992) Dimethylchrysene diol epoxides: mutagenicity in Salmonella typhimurium, tumorigenicity in newborn mice, and reactivity with deoxyadenosine in DNA. Chem. Res. Toxicol., 5, 248254.[ISI][Medline]
-
Karaulov,C. (1991) ABSURDProgram to Aid in Processing Data from MADNES, as Modified for Space Group Determination. (see ref. 16).
-
Pflugrath,J. and Messerschmidt,A. (1989) MADNES. Munich Area Detector (New EEC) System, version EEC 11/09/89, with Enhancements. Enraf-Nonius Corporation, Delft, The Netherlands.
-
Kabsch,W. (1988) Evaluation of single crystal X-ray diffraction data from a position-sensitive detector. J. Appl. Crystallogr., 21, 916924.[ISI]
-
Debaerdemaeker,Y., Germain,G., Main,P., Refaat,L.S., Tate,C. and Woolfson,M.M. (1988) MULTAN88Computer Programs for the Automatic Solution of Crystal Structures from X-ray Diffraction Data. University of York, York, UK.
-
Gantzel,P.K., Sparks,R.A., Long,R.E. and Trueblood,K.N. (1969) UCLALS4, Program in Fortran IV. Department of Chemistry, UCLA, Los Angeles, CA, USA.
-
Carrell,H.L. (1975) ICRFMLS, Modification of UCLALS4. The Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA.
-
Ibers,J.A. and Hamilton,W.C. (eds) (1974) International Tables for X-ray Crystallography, Vol. IV. Kynoch Press, Birmingham, UK (present distributor Kluwer Academic Publishers: Dordrecht, Germany).
-
Erlebacher,J. and Carrell,H.L. (1992) ICRVIEW. The Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA.
-
Allen,F.H., Kennard,O., Watson,D.G., Brammer,L., Orpen,A.G. and Taylor,R. (1987) Tables of bond lengths determined by X-ray and neutron diffraction. Part 1. Bond lengths in organic compounds. J. Chem. Soc. Perkin Trans. II, S1S19.
-
Neidle,S., Subbiah,A., Cooper,C.S. and Ribeiro,O. (1980) Molecular structure of (±)7
,8ß-dihydroxy-9ß,10ß-epoxy-7,8,9,10-tetrahydrobenz[a]pyrene: an X-ray crystallographic study. Carcinogenesis, 1, 249254.[ISI]
-
Glusker,J.P., Carrell,H.L., Zacharias,D.E. and Harvey,R.G. (1974) Crystallographic studies of K-region arene oxides: 7,12-dimethylbenz[a]anthracene 5,6-oxide and phenanthrene 9,10-oxide. Cancer Biochem. Biophys., 1, 4352.[ISI]
-
Glusker,J.P., Zacharias,D.E., Carrell,H.L., Fu,P.P. and Harvey,R.G. (1976) Molecular structure of benzo(a)pyrene 4,5-oxide. Cancer Res., 36, 39513957.[Abstract]
-
Zacharias,D.E., Kashino,S., Glusker,J.P., Harvey,R.G., Amin,S. and Hecht,S.S. (1984) The bay-region geometry of some 5-methylchrysenes: steric effects in 5,6- and 5,12-dimethylchrysenes. Carcinogenesis, 5, 14211430.[Abstract]
-
Glusker,J.P., Zacharias,D.E., Whalen,D.L., Friedman,S. and Pohl,T.M. (1982) Internal hydrogen bond formation in a syn-hydroxyepoxide. Science, 215, 695696.[ISI]
-
Zacharias,D.E., Glusker,J.P., Whalen,D.L., Friedman,S. and Pohl,T.M. (1995) Ring pucker in dihydroaromatic epoxides: the molecular structure of r-1-hydroxy-t-2-methyl-t,t-3-epoxy-1,2,3,4-tetrahydronaphthalene. Struct. Chem., 6, 8997.[ISI]
-
Klein,C.L. and Stevens,E.D. (1984) Molecular structure of anti-3,4-dihydroxy-1,2,3,4-tetrahydronaphthalene 1,2-oxide. Cancer Res., 44, 15231526.[Abstract]
-
Klein,C.L. and Stevens,E.D. (1988) Experimental electron density distribution of (1R*,2S*,3R*,4R*)-3,4-epoxy-1,2,3,4-tetrahydro-1,2-naphthalenediol. Acta Crystallogr., B44, 5055.[ISI]
-
Neidle,S. and Cutbush,S.D. (1983) X-ray crystallographic analysis of (±)7ß,8
-dihydroxy-9ß,10ß-epoxy-7,8,9,10-tetrahydrobenz[a]pyrene: molecular structure of a `syn' diol epoxide. Carcinogenesis, 4, 415418.[ISI][Medline]
-
Fetzer,S.M., Huang,C.-R., Harvey,R.G. and LeBreton,P.R. (1993) Photoelectron and ab initio molecular orbital investigations of genotoxic benz[a]anthracene metabolites: electronic influences on DNA binding. J. Phys. Chem., 97, 23852394.[ISI]
-
Carrell,H.L., Whalen,D.L. and Glusker,J.P. (1997) Hydrogen bonding in cis-2,3-epoxycyclooctanol. Struct. Chem., 8, 149154.[ISI]
Received November 4, 1998;
revised March 30, 1999;
accepted April 9, 1999.