carc$$0120
Carcinogenesis vol.18 no.2 pp.415–422, 1997
Benzo[a]pyrene and its analogues: structural studies of molecular
strain
Christopher J.Carrell, Thomas G.Carrell, H. L.Carrell,
Keith Prout1 and Jenny P.Glusker2
The Institute for Cancer Research, Fox Chase Cancer Center,
7701 Burholme Avenue, Philadelphia, PA 19111, USA and 1Chemical
Crystallography Laboratory, University of Oxford, 9 Parks Road, Oxford
OX1 3PD, UK
2To
whom correspondence should be addressed
The molecular geometry of benzo[a]pyrene, its 4-methyland 3,11-dimethyl derivatives, benzo[e]pyrene, and two
azabenzo[a]pyrenes are described. Results of these threedimensional crystal structure determinations, together with
those from previous studies in this laboratory of 11methylbenzo[a]pyrene, indicate the extent to which nonbonded interactions between hydrogen atoms contribute to
molecular distortions, particularly in the bay-region. This
strain is high if a bay-region methyl group is present. The
major effect is an increase in the C–C–C angles in that
area of the molecule, rather than torsion about bonds.
In addition, the effect of a nitrogen atom replacing one
of the C-H groups in the aromatic system is shown.
Molecules stack in planes ~3.5 Å apart. In benzo[a]pyrene, 5-azabenzo[a]pyrene and 3,11-dimethylbenzo[a]pyrene crystals the stacking is similar to that in graphite. 4Methylbenzo[a]pyrene molecules stack with less molecular
overlap. The packing in 4-aza-5-methylbenzo[a]pyrene consists of modules of four stacked molecules, packed in
a ‘tile-like’ arrangement. Nonbonded C····H interactions
between adjacent molecules lead to a herring-bone arrangement between these stacks. The types of C····H and π–π
interactions involving PAHs in the crystalline state,
described here, can also be expected to be found when the
PAHs bind to hydrophobic areas of biological macromolecules such as proteins, nucleic acids and membranes.
Introduction
Various polycyclic aromatic hydrocarbons (PAHs*) have been
shown to be carcinogenic and, of these, 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (BaP) are the
most highly investigated (1–7). The molecular structures of
chemical carcinogens contain information relative to their
biological fate, although we currently do not clearly understand
how to interpret them. Therefore detailed studies of chemical
bonding and of molecular strain (which may enhance chemical
reactivity at that point) are necessary for an understanding
of the molecular basis of chemical carcinogenesis. Threedimensional structural data are presented here on BaP, a potent
carcinogen, on benzo[e]pyrene (BeP) which is an inactive
isomer, and some analogues of BaP. These consist of PAHs,
methylated PAHs, and two aza analogues of BaP with a
nitrogen atom replacing one aromatic C-H group.
*Abbreviations: PAHs, polycyclic aromatic hydrocarbons; DMBA, 7,12dimethylbenz[a]anthracene; BaP, benzo[a]pyrene; BeP, benzo[e]pyrene.
© Oxford University Press
Crystals of BaP were originally reported by John Iball in
1936, and unit-cell dimensions were provided for both the
monoclinic and orthorhombic forms (8). A third polymorph,
also orthorhombic, has been reported more recently (9). The
two orthorhombic modifications have lower reported experimental densities than does the monoclinic form (8,9). A phase
change from monoclinic to orthorhombic has been observed
in the range 110–123°C by calorimetry (10). The crystal
structure of the monoclinic form was determined by Iball and
Young in 1956, based mainly on two-dimensional diffraction
data so that the molecular dimensions were not very precise
(11). Later Iball refined the structure with new diffraction data,
and obtained a more precise structure which included hydrogen
atom positions (12). Since there have been extensive improvements in the art of small-molecule crystallography since 1976,
and in order to provide structural data for comparison with
those of analogues, the crystal structure determination of the
monoclinic form of benzo[a]pyrene was repeated. Crystals
were studied by us at low temperatures (120 K) so that atomic
vibrations were decreased and, as a result, atomic positions
could be better determined. This low temperature (120K 5
–153°C) does not change the molecular geometry, although it
might have a slight effect on intermolecular distances. As a
result the molecular geometry is determined with higher
precision than before, a situation similar to that of our lowtemperature work on DMBA (13). The crystal structure of
BeP is also reported in a low-temperature study. This molecule,
unlike BaP, is not reported to be carcinogenic.
The overall aim in these studies was to determine the amount
of distortion due to steric overcrowding in these molecules.
Such distortions are much less evident in BaP and its analogues
than in DMBA (13). Finally the effect of a bay-region methyl
group in 3,11-dimethyl-BaP (which has carcinogenic activity)
is assessed. Structural data have already been reported by us
for 11-MeBaP (disordered) (14) and can now be analysed in
detail. The numbering in each BaP analogue has been chosen
to be as near as possible the same as that for 11-MeBaP (14).
Materials and methods
X-ray diffraction analysis
X-ray diffraction intensities were measured at low temperatures (120 6 2 K)
for each crystal, except for 5-azaBaP which was measured at room temperature.
Diffraction data were obtained from each crystal on an Enraf-Nonius FAST
diffractometer with MoKα radiation (λ 5 0.71073 Å) from a rotating anode,
and with a graphite monochromator. Unit-cell dimensions were measured
from 250 intense Bragg reflections in the θ range 11–18.8°. Intensity data
were measured by the rotation method with box integration. Frame sizes were
0.20°–0.25°, and the time of measurement varied between 20 and 60 s. Further
information is provided in Table I. Intensities of duplicate and equivalent
reflections were averaged. Crystals of 4-aza-5-methyl BaP had a large mosaic
spread, of the order of 1.8°. Therefore, the weakest data with intensity I less
than 1.0 σ(I) were not used. For the other crystals, all of the unique intensity
data were used. Intensity data were measured to the edge of the copper sphere
for 4-MeBaP and 3,11-diMeBaP, and well beyond for BeP. Intensity data were
considered to be ‘observed’ when I . 2σ(I). The program MADNES (15,16)
was used during the data collection and for the integration of data frames.
The program XSCALE and in-house computer programs (17,18) were used
415
C.J.Carrell et al.
Table I. Unit cell dimensions (in Å and degrees), details of data measurement and structure refinement
Space group
a
b
c
α
β
γ
Number of molecules
per unit cell
Unit cell volume (Å3)
1
BaP
2
BeP
3
5-azaBaP
4
4-aza-5-MeBaP
5
4-MeBaP
6
3,11-diMeBaP
P21/c
4.489(1)
20.309(6)
13.372(5)
90.0
96.59(2)
90.0
4
P1
11.307(5)
11.537(4)
11.006(5)
106.44(2)
114.70(2)
74.30(3)
4
P212121
4.608(2)
13.122(3)
20.095(5)
90.0
90.0
90.0
4
P21/n
9.118(4)
19.493(9)
29.017(7)
90.0
91.05(2)
90.0
16
Pca21
21.649(7)
11.453(2)
5.255(2)
90.0
90.0
90.0
4
P21/n
4.703(2)
16.739(6)
17.501(5)
90.0
90.18(2)
90.0
4
1211.0(6)
1231.8(9)
1215.1(7)
5154.4(6)
1303.0(7)
1377.7(9)
Scheme I
for the reduction of the raw diffraction data. The internal R value (on the
intensity) was in the range of 0.085 for all of the crystals. No absorption
correction was applied because crystals were small and their absorption
coefficients µ were low, ~0.08 mm–1.
Structure determination and refinement
Crystal structures, except for those of BeP and 4-aza-5MeBaP, were determined
by direct methods with MULTAN88 (19). The structure of BeP, space group
PO(1,–), was solved in the space group P1 and the center of symmetry was
located in the model so obtained (20). The structure of 4-aza-5MeBaP, space
group P21/n, with 4 molecules in the asymmetric unit, presented problems in
structure determination because of its inherent hypersymmetry. Ultimately
this crystal structure was elucidated by use of the program SIR92 (20) in
space group P21, and the crystallographic symmetry center was subsequently
identified from the P21 model with 8 molecules in the asymmetric unit. The
in-house program ICRFMLS (21,22) was used for a full-matrix least-squares
refinement of the structures (based on F2). Scattering factors used were
those published in International Tables for X-ray Crystallography (23). The
weighting scheme was w 5 1/[σ2(Fo2)]. Hydrogen atoms were found using
difference electron-density maps. After the final anisotropic refinement (isotropic for hydrogen atoms), the R values listed in Table I were obtained.
Values for R are based on F, while those for weighted R (wR) are based on
F2; R values based on F are approximately half of those for F2.
Atomic coordinates of the refined structures are listed in Table II. Atomic
displacement parameters and observed and calculated structure factors have
been deposited (see note at the end of this article for information on how to
obtain these data). Molecular diagrams were drawn with the in-house program
ICRVIEW (24). The overall numbering used in this article is shown in Scheme I.
Results and discussion
The structures presented here, for which coordinates are
listed in Table II, have bond lengths with estimated standard
deviations (e.s.d. values) of the order of 0.002 Å, and e.s.d.
values for angles of ~0.2° (2° if a hydrogen atom is involved);
416
values are lower for benzo[e]pyrene. These values indicate
that differences .0.005 Å in distances or 0.5° in bond
angles (excluding hydrogen atoms) are probably significant.
No disorder was evident in any of the crystal structures.
Averaged bond lengths are shown for each in Figure 1. In
these PAHs the average benzene C–C is ~1.39 Å; longer
bonds have more single-bond character, shorter bonds have
more double-bond character. Short, almost pure double bonds
(1.34 Å) are found in 4-MeBaP (C4–C5 5 1.328 Å), 1.345 Å
for BaP, and 1.348 Å for 3,11-diMeBaP. Note the decrease in
K-region double-bond lengths in 4-MeBaP.
The unit-cell dimensions reported here for BaP are in line
with those reported earlier by Iball for the monoclinic form
(8,11,12). We failed to obtain any crystals of the reported
orthorhombic forms of BaP, unit-cell dimensions 7.59, 7.69,
and 22.38 Å, space group P212121, density 1.274 g/cc and
9.96, 13.90 and 9.22 Å, density 1.310 g/cc. These unit-cell
volumes are more in line with value expected for a methylated
BaP, although the reported densities are lower than any of the
values we find (1.35–1.38 g/cc) for BaP and its methylated
and dimethylated derivatives.
The molecular strain shown by these BaP derivatives is the
result of a steric interaction between H10 and H11. The
minimum nonbonded H····H distance (when C–H distances
have been ‘normalized’ to 1.09 Å) appears to be ~2.0 Å, even
in the strained bay-region area. Major geometric indicators of
molecular strain in BaP and its analogues are the C–C–C
angles and the C–C–C–C torsion angles in the bay region.
Thus the molecule overcomes steric hindrance by increasing
the angles and twisting some bonds in the bay region. The
extent to which it does each of these depends to some extent
on the environment; in other words, the molecules show some
flexibility. The average bay-region C–C–C angle of 122.4(3)°
in BaP and its analogues with no bay-region methyl group is
extended to 124.7(3)° when a bay-region methyl group is
present. Bay-region torsion angles in BaP and its analogues
studied here are of the order of 0–3° if no methyl group is
present, and is slightly increased to 3–15° in the presence of
a methyl group. In 11-MeBaP one of the two molecules in the
asymmetric unit of the crystal has a torsion angle of 15.2°
while the other has a torsion angle of 3.8°, possibly indicating
the extent to which the molecule can flex or is disordered
(14). The value for this torsion angle in 3,11-diMeBaP (with
only one molecule in the asymmetric unit and more precisely
measured) is 3.8°. The high torsion angles (up to 23°) reported
for DMBA are not observed for BaP (13). The expansion of
angles in the bay region of DMBA would cause steric problems,
[a]pyrene and its analogues
Fig. 1. Bond distances in Å for (a) BaP, (b) BeP, (c) 5-azaBaP, (d) 4-aza-5-MeBaP, (e) 4-MeBaP, (f) 3,11-diMeBaP, and (g) 11-MeBaP (14). Carbon atoms
are unfilled circles, nitrogen atoms are filled circles, and hydrogen atoms are smaller circles. The bonds in the bay region of each molecule are filled.
and therefore torsion angles, rather than interbond angles, are
increased in this molecule (13). The effects of steric hindrance
are evident in the structures of the methylated derivatives of
BaP. They are inherent properties of the molecules, but,
as described, show some slight variability in response to
molecular packing.
417
C.J.Carrell et al.
Table II. Atomic coordinates expressed as fractions of unit-cell edges
(see Table I for unit-cell dimensions). Estimated standard deviations are
listed in parentheses and refer to the last quoted digit, i.e. 0.3425(2) 5
0.3425 6 0.0002
The geometries of intermolecular interactions of BaP
analogues provide a clue to their mode of interaction with
hydrophobic aromatic regions of biological macromolecules.
The unit-cell dimensions reported in Table I show that there
is a short axis of 4.5–4.7 Å for BaP, 5-azaBaP and 3,11diMeBaP, and a slightly longer one of 5.255 Å in 4-MeBaP.
Molecules of the BaP, 5-azaBaP, and 3,11-diMeBaP stack
up this short axis as shown in Figures 2(a)–(c), with a
distance of ~3.5 Å between molecular planes. The molecules
are stacked at an angle to the unit-cell axis of ~50° 5 sin–1
418
Table II. continued
(3.5/4.6). The distances of 3.5 Å between molecular planes
are, however, too large to indicate any transfer of charge such
as is found in molecular complexes of polycyclic aromatic
hydrocarbons with, for example, trinitrobenzene; in these
complexes the interplanar separation is often nearer to 3.2 Å
rather than 3.5 Å (25,26). When viewed perpendicular to the
molecular planes, as shown in Figures 2(d)–(f), these three
compounds stack in a similar manner to the hexagonal sheets
of carbon atoms in graphite (27,28).
The unexpected finding was the overall molecular packing
in 4-aza-5-MeBaP, illustrated in Figure 3. Here groups of four
molecules associate to give the tiling-like effect shown in this
diagram. The width of four molecules appears to match the
length of a molecule and indicates that non-bonded C····H as
well as C····C interactions are important in packing.
Thus the compounds BaP, 5-azaBaP, and 3,11-diMeBaP
form infinite stacks of molecules extending through the crystal,
so that each molecule is sandwiched between two others.
This indicates a strong preference for such stacking between
aromatic molecules. In 4-aza-5-MeBaP stacks of four
molecules are formed as a unit and these then pack perpendicular to each other in the ‘tiling pattern’ shown in Figure 4.
[a]pyrene and its analogues
Table II. continued
Table II. continued
BeP molecules stack, but only in pairs which form units that
lie at an angle to other pairs. Molecules of 4-MeBaP stack in
planes 3.5 Å apart, but with much less overlap than is seen in
the other compounds studied.
The stacking of molecules is one component of the crystal
structures. Another is the non-bonded C····H distances so that
the hydrogen atoms on the periphery of the molecule determine
the side-by-side packing. Some H····H distances in BaP are
illustrated in Figure 4. In crystals of the dimethyl compound,
molecules related by a center of symmetry are coplanar, but
in BaP crystals this is not the case.
This analysis has also shown that replacement of a CH
group by nitrogen to give an aza-BaP does not have a
profound effect on intermolecular interactions. The packing
in BaP and 5-azaBaP is similar when viewed down the
419
C.J.Carrell et al.
Fig. 2. View along the molecular planes for (a) BaP, (b) 5-azaBaP, and (c) 3,11-diMeBaP, and views onto the molecular planes for (d) BaP, (e) 5-azaBaP, and
(f) 3,11-diMeBaP. The upper molecule has been drawn with heavier bonds.
short axis even though the space groups of these two
compounds are different. Apparently the tendency of
molecules to stack has not been affected by the substitution.
On the other hand, in 4-aza-5-MeBaP an almost linear
N····H–C interaction is formed to another molecule. The
N····H–C angle is 170–180° and the N····H distances are
2.4–2.6 Å. This results in the association of two molecules
in a planar arrangement, as shown in Figure 5. A similar
interaction is formed by 5-aza-BaP but only one N····H–C
interaction occurs between pairs of molecules.
The mutagenicities of methylated benzo[a]pyrenes indicate
that 11-MeBaP is a very potent mutagen and that 1-MeBaP,
3-MeBaP, BaP, 4-MeBaP and 12-MeBaP are also potent
420
mutagens (2,29). Tumor-initiating activity of monomethyl
BaP derivatives is also high for this group of mutagens
(30). The methyl groups presumably assist in the fitting of
the molecule in a metabolizing enzyme and in interacting
with DNA bases when covalently bound. Thus, the molecular
stacking in 3,11-diMeBaP, which is similar to that in BaP
(see Figure 2), may indicate likely interactions with biological
macromolecules containing flat aromatic or heterocyclic ring
systems. Environmental forces that affect these PAHs in the
body would most likely be π–π and C–H····C interactions,
because such molecules aggregate in hydrophobic areas.
These are also the types of interactions described here in
the crystalline state.
[a]pyrene and its analogues
Fig. 3. Molecular packing in crystals of 4-aza-5-MeBaP showing the ‘tiling pattern’ described in the text. The view is along the molecular planes.
Note added in proof
Deposited material includes details of data measurement and structure refinement, anisotropic displacement parameters, and observed and calculated
structure factors. Order NAPS document No. 05351 from NAPS c/o Microfiche
Publications, PO Box 3513, Grand Central Station, New York, NY 101633513, USA.
Acknowledgements
We thank Guido Daub (deceased) who provided crystalline specimens of
some of the compounds studied. This work was supported by Grants CN-10
from the American Cancer Society (to JPG), CA-10925 and CA-06927
from the National Institutes of Health and by an appropriation from the
Commonwealth of Pennsylvania (to FCCC). Its contents are solely the
responsibility of the authors and do not necessarily represent the official views
of the American Cancer Society or the National Cancer Institute.
References
1. Huggins,C.B., Pataki,J. and Harvey,R.G. (1967) Geometry of carcinogenic
aromatic hydrocarbons. Proc. Natl Acad. Sci. USA, 58, 2253–2260.
2. Utesch,D., Glatt,H. and Oesch,F. (1987) Rat hepatocyte-mediated bacterial
mutagenicity in relation to the carcinogenic potency of benz[a]anthracene,
benzo[a]pyrene, and twenty-five methylated derivatives. Cancer Res., 47,
1509–1515.
3. Wislocki,P.G., Fiorentini,K.M., Fu,P.P., Yang,S.K. and Lu,A.Y.H. (1982)
Tumor-initiating ability of the 12 monomethylbenz[a]anthracenes.
Carcinogenesis, 3, 215–217.
4. Harvey,R.G. (1991) Polycyclic Aromatic Hydrocarbons: Chemistry and
Carcinogenicity, Cambridge University Press, Cambridge, UK, pp. 26–49.
5. Glusker,J. (1985) X-ray analyses of PAH metabolite structures.
In Harvey,R.G. (ed.) Polycyclic Aromatic Hydrocarbons and Cancer. ACS
Monograph No. 283. Washington, DC, pp. 125–185.
6. DiGiovanni,J., Diamond,L., Harvey,R.G. and Slaga,T.J. (1983)
Enhancement of the skin tumor-initiating activity of polycyclic aromatic
hydrocarbons by methyl-substitution at non-benzo ‘bay-region’ positions.
Carcinogenesis, 4, 403–407.
7. Santella,R., Kinoshita,T. and Jeffrey,A.M. (1982) Mutagenicity of some
methylated benzo[a]pyrene derivatives. Mutat. Res., 104, 209–213.
8. Iball,J. (1936) The crystal structure of condensed ring compounds. III.
Three carcinogenic hydrocarbons, 1:2-benzpyrene, methylcholanthrene
and 5:6-cyclopenteno-1:2-benzanthracene. Z. Krist., 94, 7–21.
421
C.J.Carrell et al.
Fig. 4. H····H distances (in Å) and side-by-side packing in BaP.
9. Contag,B. (1978) Benzo[a]pyrene polymorphism. Naturwissenschaften,
65, 108–109.
10. Casellato,F., Vecchi,C. and Girelli,A. (1973) Heat-induced crystal transition
of BaP. Chem. & Ind., 479–480.
11. Iball,J. and Young,D.W. (1956) Structure of 3:4-benzpyrene. Nature
(London), 177, 985–986.
12. Iball,J., Scrimgeour,S.N. and Young,D.W. (1976) 3,4-Benzopyrene (a new
refinement). Acta Cryst., B32, 328–330.
13. Klein,C.L., Stevens,E.D., Zacharias,D.E. and Glusker,J.P. (1987) 7,12Dimethylbenz[a]anthracene: refined structure, electron density distribution,
and endoperoxide structure. Carcinogenesis, 8, 5–18.
14. Prout,K., Smith,A.D., Daub,G.H., Zacharias,D.E. and Glusker,J.P. (1992)
11-Methylbenzo[a]pyrene: bay region distortions. Carcinogenesis, 13,
1775–1782.
15. Pflugrath,J. and Messerschmitt,A. (1989) MADNES, Munich Area Detector
(New EEC) System, version EEC 11/09/89, with enhancements by EnrafNonius Corporation, Delft, The Netherlands.
16. ABSURD—Program to aid in processing data from MADNES, as modified
by C. Karaulov (1991) for space group determination.
17. Kabsch,W. (1988) Evaluation of single-crystal X-ray diffraction data from
a position-sensitive detector. J. Appl. Cryst., 21, 916–924.
18. Carrell,H.L., Shieh,H.-S. and Takusagawa,F. (1981) The Crystallographic
Program Library of the Institute for Cancer Research, Fox Chase Cancer
Center, Philadelphia, PA, USA.
19. Debaerdemaeker,Y., Germain,G., Main,P., Refaat,L.S., Tate,C. and
Woolfson,M.M. (1988) MULTAN88—Computer programs for the
automatic solution of crystal structures from X-ray diffraction data. The
University of York, York, UK.
20. Altomare,A., Cascarano,G., Giacovazzo,C. Guagliardi,A., Burla,M.C.,
Polidori,G. and Camalli,M. (1994) SIR92—a program for automatic
solution of crystal structures by direct methods. J. Appl. Cryst., 27, 435.
21. Gantzel,P.K., Sparks,R.A., Long,R.E. and Trueblood,K.N. (1969)
UCLALS4, Program in Fortran IV.
22. Carrell,H.L. (1975) ICRFMLS, modification of UCLALS4, The Institute
for Cancer Research, Fox Chase Cancer Center.
23. International Tables for X-ray Crystallography (1974) Vol. IV. Kynoch
Press: Birmingham, UK (Present distributor Kluwer Academic Publishers:
Dordrecht, Germany).
24. Erlebacher,J. and Carrell,H.L. (1992) ICRVIEW. Program from The
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia,
PA, USA.
25. Mayoh,B. and Prout,C.K. (1972) Molecular complexes. Part 13. Influence
of charge transfer interactions on the structure of π–π* electron donoracceptor molecular complexes. J. Chem. Soc., Faraday Trans., 1072–1082.
26. Herbstein,F.H. (1971) Crystalline π-molecular compounds: chemistry,
spectroscopy, and crystallography. In Dunitz,J.D. and Ibers,J.A (eds)
Perspectives in Structural Chemistry. Vol. IV. Wiley, New York, pp. 166–395.
27. Bernal,J.D. (1924) The structure of graphite. Proc. Roy. Soc. (London)
A106, 749.
28. Hassel,O. and Mark,H. (1924) Über die Kristallstruktur des graphits. [The
crystal structure of graphite.] 25, 317–337.
29. Osborne,M.R. and Crosby,N.T. (1987) Benzopyrenes. Cambridge
University Press, Cambridge.
422
Fig. 5. Dimerization of 4-aza-5-MeBaP, which may contribute to the
packing motif in Figure 3.
30. Sax, N.I. (1981) Cancer Causing Chemicals. Van Nostrand Reinhold
Company, New York.
Received on May 7, 1996; revised on October 2, 1996; accepted on October
17, 1996