Tracking the structural changes in a series of cholesterol solvates

Article abstract of DOI:10.1021/cg200971f

This article describes the isolation of seven new solvates of cholesterol using solvents of varying carbon chain length and overall size from propanol through to phenylethanol. The structural similarities that exist between these novel solid forms and also those cocrystals and solvates already observed are discussed.

Full text of DOI:10.1021/cg200971f

Article
pubs.acs.org/crystal
Tracking the Structural Changes in a Series of Cholesterol Solvates
Rhona J. Galloway,† Syed A. Raza,† Robert D. Young,† and Iain D. H. Oswald*,†
†Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, U.K.
S
* Supporting Information
ABSTRACT: This article describes the isolation of seven new
solvates of cholesterol using solvents of varying carbon chain
length and overall size from propanol through to phenyl-
ethanol. The structural similarities that exist between these
novel solid forms and also those cocrystals and solvates already
observed are discussed.
Despite the functional use of cholesterol in the body, it has
received notoriety in the press for being a contributing factor in
causing heart disease if present in high levels in the
bloodstream. Cholesterol is also a major component of
gallstones as the anhydrous form as well as in the form of
the monohydrate, and so there have been a number of studies
relating to the growth of this phase onto calcium carbonate, the
other main contributor to gallstones.4−7 Further theoretical
studies of cholesterol have been made investigating formation
of nanoparticles and also the applicability of the GROMOS
force field, designed primarily for biological systems, to model
the crystalline structures of smaller membrane sterols.8,9 At the
heart of all these studies, is the investigation of how cholesterol
molecules interact with one another and surrounding materials.
Further investigation of the solid-state forms of cholesterol and
how the molecules interact with one another in these systems
can contribute to our understanding of cholesterol in these
other environments.
INTRODUCTION
■
Cholesterol is one of the major steroids within mammalian cells
and forms an integral part of the cell membranes. The
molecular structure of cholesterol is ideally suited to these
surroundings, as it consists of a hydroxyl group, that interacts
with the polar headgroup of the phospholipid, and a tetracyclic
steroid body bonded to an eight carbon alkyl chain that
interacts with the hydrophobic fatty acid chains of the
phospholipid bilayer of the cell membrane (Scheme 1).
Scheme 1. Molecular Structure of Cholesterol
In the solid-state, cholesterol crystallizes in a number of
different forms in addition to the two anhydrous phases.10−14
In the Cambridge Structural Database (CSD),15,16 there is a
monohydrate (CSD refcode: CHOLES20),17 a hemimethanol
solvate (CHOLME02),18 two hemiethanol solvates (CHOLEU01,
CHOLEU10),19 an isobutylphosphocholine/tert-butanol coc-
rystal (MEQKAU),20 and finally a cocrystal with 4-iodophenol
(WOMHAI).21 The structures of all these compounds can be
broadly categorized into two distinct molecular architectures
depending on the size of the additional solvent or coformer. The
majority of these compounds crystallize in a bilayer struc-
ture with alternating hydrophobic and hydrophilic regions.
The molecules of cholesterol are positioned end-to-end such
that the isopropyl groups at the end of the alkyl chains interact
with one another. The second architecture is more compact,
Once orientated within the membrane, one of the roles of
cholesterol is to alter the physicochemical properties of the cell
membrane by subtly altering the interactions of the
surrounding fatty acid chains. It has been shown through
DSC and X-ray diffraction measurements that the addition of
cholesterol to the membrane structure causes changes in the
physical behavior of the phospholipid layers with respect to
temperature. Pure lipid layers possess a gel/liquid-crystal
transition at elevated temperatures whereby the chains “melt”
and become less ordered. The addition of >40 mol %
cholesterol causes the disappearance of this phase transition,
resulting in an “intermediary” structure that possesses a bilayer
thickness between that of the gel and liquid-crystal states.1,2
Furthermore, a study by Lund-Katz et al. demonstrated, using
surface pressure measurements, that the addition of cholesterol
to a membrane causes it to become more condensed and
hence harder which has an effect on the functioning of trans-
membrane proteins.3
Received: July 27, 2011
Revised: December 1, 2011
Published: December 5, 2011
© 2011 American Chemical Society
231
dx.doi.org/10.1021/cg200971f | Cryst. Growth Des. 2012, 12, 231−239

Crystal Growth & Design
Article
Figure 1. Crystal morphologies of the seven new cocrystals compared to the morphology of the methanol and ethanol solvates. Solvates of
cholesterol with (a) propanol, (b) butanol, (c) pentanol, (d) hexanol, (e) phenol, (f) benzyl alcohol·water, (g) phenylethanol, (h) methanol, or (i)
ethanol.
Phase IdentificationX-ray Powder Diffraction. A small
where the cholesterol molecules from neighboring chains are
quantity (1−50 mg) of each recrystallized sample was analyzed
using transmission foil XRPD data collected on a Bruker AXS D8-
Advance transmission diffractometer equipped with a θ/θ geometry,
primary monochromated radiation (Cu Kα1, λ = 1.54056 Å), a Bruker
Vantec 1D position sensitive detector (PSD), and an automated
multiposition x−y sample stage.22 Samples were mounted on a 28
position sample plate supported on a polyimide (Kapton, 7.5 μm
thickness) film. Data were collected from each sample in the range
4−35° 2θ with a 0.015° 2θ step size and a 1 s·step−1 count time.
Figure ES2 of the Supporting Information contains the X-ray powder
diffraction patterns of all the bulk samples and confirms the identity of
the cocrystals. Slight differences in the diffraction patterns can be
attributed to the data collection temperature (293 K cf. 123 K for the
single crystal experiments) and preferred orientation.
intercalated with one another in a head-to-tail arrangement. In
this study we have attempted to systematically investigate the
effect of the size and rigidity of the solvent on the final crystal
structure so that a comparison can be made with those struc-
tures already found in the CSD.
EXPERIMENTAL SECTION
■
Sample Preparation. Cholesterol and solvents were purchased
from Sigma Aldrich and used as received.
Formation of Compounds 1−7. These compounds were
obtained from a 1:1 mixture of diethylether and the target solvent
(propanol, butanol, etc.). Cholesterol (103 mg, 0.266 mmol for 1; 100
mg, 0.259 mmol for 2; 98 mg for 3; 106 mg, 0.275 mmol for 4; 101
mg, 0.261 mmol for 5; 151 mg, 0.391 mmol for 6; and 256 mg, 0.663
mmol for 7) was dissolved in 3 cm3 of the 1:1 mixture of diethyl ether
and target solvent. The solvent was left to evaporate, allowing crystals
to form. In the case of propanol, after a few days, some small crystals
appeared on the side of the vial which were pushed into the mother
liquor in order to grow a suitable crystal for single crystal X-ray
diffraction. For compound 5, an excess of phenol was added (44 mg,
0.468 mmol) to 3 cm3 of dietheyl ether. The cocrystals from pentanol
and hexanol needed further addition of cholesterol in order to form
the precipitate on evaporation of the diethyl ether.
Crystal Morphology. Figure 1 shows the morphology of the
crystals from each of the crystallizations. The crystals from propanol,
butanol, and benzyl alcohol show a needle-like morphology whereas
the crystals from the other solvents possessed a lathe or plate
morphologythese are compared with the crystals of the methanol
and ethanol solvates.
Differential Scanning Calorimetry. DSC plots were obtained
using dynamic DSC (DSC 822e, Mettler Toledo, U.K.). Samples were
prepared by carefully weighing between 2.31 and 6.03 mg of each
sample into a 40 μL aluminum pan, which was then hermetically
sealed with a pinhole in the lid. An empty pin-holed 40 μL aluminum
pan was used as a reference. Both pans were subjected to a nitrogen
atmosphere. The pans were then heated at a rate of 10 °C/min from
293 to 463 K (well above the melting point of cholesterol). The
temperature and heat flow of the DSC instrument were calibrated with
indium and zinc. The results were analyzed using Mettler STAR
software. Figure ES1 of the Supporting Information shows the DSC
and TGA traces for each of the samples.
Thermal Gravimetric Analysis. TGA measurements were
performed on a Mettler Toledo TGA 751e. Each sample (7.86−
22.48 mg) was placed in a ceramic pan. An empty ceramic pan was
used as a reference, and both pans were subjected to a nitrogen atmo-
sphere. The pans were then heated from 303 to 463 K at 10°/min.
The results were analyzed using Mettler STAR software.
Crystal Structure Determination. X-ray diffraction intensities
were collected with Mo Kα radiation on a Bruker KAPPA Apex II
CCD diffractometer equipped with an Oxford Cryosystems Cryo-
stream-Plus variable-temperature device operating at 123 K.23
Absorption corrections were carried out using the multiscan procedure
SADABS (Sheldrick, 2004, based on the procedure described by
Blessing, 1995).24,25 The structures were solved by direct methods and
refined by full-matrix least-squares against F2 using all data
(SHELX).26 Due to the wavelength of the X-ray source (0.71073 Å),
the absolute configuration of the cholesterol molecule was not deter-
mined but the absolute structure was chosen to reflect the chirality
observed for the vast majority of structures in the literature in order to
make a direct comparison of the crystal structures. All hydrogen atoms
attached to carbon atoms were geometrically placed, and those partici-
pating in hydrogen bonding, i.e. hydroxyl hydrogens, were found in the
difference map. All non-H atoms were modeled with anisotropic dis-
placement parameters. Where disorder was present, the bond lengths
and angles were restrained to values found in the CSD.
Additional programs used included Materials Mercury 2.4,27
PLATON as incorporated in WINGX.28,29 Mercury, ChemBioDraw
12.0, and GIMP 2.630 were used in the production of the figures.
RESULTS AND DISCUSSION
■
Cholesterol was crystallized with seven alcoholic solvents of increas-
ing size: propanol, butanol, pentanol, hexanol, phenol, benzyl
alcohol, and phenylethanol. The descriptions of the crystal
structures follow, and the crystallographic parameters for the
compounds under study and also those of previous studies
can be found in Tables 1 and 2.
Thermal Analysis. Figure ES1 of the Supporting Informa-
tion shows the thermal analysis plots for each of the cocrystals.
It can be observed that each of the cocrystals 1−4 shows a
desolvation event followed by the melting of the cholesterol at
∼423 K. The desolvations of the propanol and butanol solvates
232
dx.doi.org/10.1021/cg200971f | Cryst. Growth Des. 2012, 12, 231−239

Crystal Growth & Design
Article
Table 1. Crystal Structure Refinement Details for Compounds 1−7
1
2
3
4
5
6
7
chemical formula
C57H100O3
833.37
C58H102O3
847.40
C59H104O3
861.62
C60H106O3
875.45
C33H52O2
480.75
C61H102O4
899.43
C62H102O3
895.44
Mr
crystal system, space group monoclinic, P21 monoclinic, P21 monoclinic, C2 monoclinic, C2 monoclinic, P21 monoclinic, P21 monoclinic, C2
a, b, c (Å)
15.0249(12)
6.1107(5)
28.663(2)
97.386(4)
2609.8(4)
2
15.0634(9)
6.0931(4)
29.1765(18)
96.583(3)
2660.2(3)
2
42.9792(15)
10.3021 (4)
6.2807 (2)
96.225(2)
2764.55(17)
2
42.982(2)
10.3695(7)
6.2684(4)
96.166(3)
2777.7(3)
2
11.5935(9)
6.1697(5)
21.1002(16)
105.446(4)
1454.8(2)
2
15.699(2)
7.5133(9)
23.492(3)
94.643(9)
2761.8(6)
2
42.676(4)
10.2044(11)
6.3396(6)
97.501(6)
2737.1(5)
2
β (deg)
V (Å3)
Z
Dx (mg m−3
)
1.061
1.058
1.108
1.047
1.098
1.082
1.086
temp (K)
123(2)
7248
123(2)
9921
123(2)
9902
123(2)
6988
123(2)
5735
123(2)
4652
123(2)
6021
no. of reflns for cell
2θmax (deg)
48.90
53.06
50.52
49.84
52.64
48.70
52.64
μ (mm−1
)
0.062
0.062
0.065
0.061
0.066
0.065
0.064
reflns collected
unique [Rint]
no. I > 2u(I)
20182
26928
30565
19989
12342
15071
11188
8364[0.0230]
6666
10339 [0.0248]
7994
5162[0.0364]
4468
4924[0.0600]
3915
5789[0.0214]
5222
8204 [0.0319]
6354
5354[0.0254]
4755
T
min, Tmax
0.69, 0.75
603
0.64, 0.75
583
0.66, 0.75
310
0.63, 0.75
351
0.65, 0.75
329
0.65, 0.75
616
0.53, 0.75
327
params
R1 [F > 4u(F)]
wR2 (F2, all data)
S
0.0428
0.039
0.0642
0.0676
0.0774
0.2357
1.092
0.0466
0.1290
1.027
0.0391
0.0886
1.016
0.0475
0.1335
1.045
0.1829
0.2146
0.998
1.044
1.061
ρmax (e Å−3
ρmin (e Å−3
)
0.189
0.672
0.833
0.623
0.585
0.146
0.281
)
−0.292
−0.752
−0.278
−0.286
−0.230
−0.153
−0.205
Table 2. Unit Cell Parameters for the Crystal Structures of Cholesterol in the Cambridge Structural Database15,16
cholesterol
cholesterol
cholesterol
hemiethanol
solvate form I
cholesterol
hemiethanol
solvate form II
cholesterol isobutyl-
phosphocholine
isobutanol solvate
cholesterol
4-iodophenol
cocrystal
cholesterol
form I
cholesterol
form II
monohydrate hemimethanol
form III
solvate
CSD
Refcode
CHOEST20
CHOEST21
CHOLES20
CHOLME02
CHOLEU01
CHOLEU10
MEQKAU
WOMHAI
crystal
system,
triclinic, P1
triclinic, P1
triclinic, P1
triclinic, P1
triclinic, P1
monoclinic, P21
monoclinic, C2
monoclinic,
P21
space
group
a, b, c (Å)
14.172(7)
34.209(18)
10.481(5)
27.565(10)
38.624(16)
10.748(4)
93.49(3)
90.90(3)
117.15(3)
10151(7)
16
12.390(30)
12.410(30)
34.360(60)
91.90(10)
98.10(10)
100.80(10)
5128
12.2735(17)
34.237(7)
6.2739(8)
90.224(14)
93.705(10)
91.576(14)
2629.8(7)
4
12.787(2)
35.310(11)
12.225(1)
97.80(2)
100.40(2)
99.06(2)
5284(2)
8
12.775(2)
68.668(15)
12.213(2)
16.994(10)
11.314(7)
28.164(15)
6.302(<1)
10.295(<1)
41.964(3)
α, β, γ (deg) 94.64(4)
90.67(4)
100.43(1)
104.07(3)
91.03(<1)
96.32(4)
V (Å3)
5033(4)
8
105367(3)
16
5252
2
2722.3(3)
2
Z
8
are quite clean events; the desolvations of the pentanol and
hexanol show other events surrounding the desolvation which
may be due to an increase in disorder of the solvent molecule in
the crystal lattice before it leaves the lattice. The TGA plots
show that compounds 1, 2, and 4 (Figures ES1a, ES1b, and
ES1d) all lose a mass that is equivalent to the stoichiometry
that is found in the crystal structure analysis (7, 12, and 9%).
The hexanol solvate shows a change in the gradient of the TGA
which signifies a change from the loss of residual solvent to the
loss of solvent from the crystalline lattice. The second loss is
equivalent to the quantity of solvent in the crystal structure.
The proportion of solvent that is lost from compound 3
(Figures ES1c) is a little lower than would be expected (7% cf.
10%), but refining the crystal structure with a lower occupancy
of solvent resulted in extra electron density surrounding the
solvent molecules; that is, the stoichiometry is correct at 2:1
cholesterol/pentanol. A possible explanation could be that
during the prepartion of the sample and subsequent equili-
bration of the pan inside the TGA instrument, some solvent
was lost from the crytal structure; for crystal structure analysis,
the crystals were placed under oil before putting them onto the
instrument under a nitrogen cold stream which would preserve
the solvent in the crystal.
Compounds 5 and 7 (Figures ES1e and ES1g) show an
endothermic event at ∼373 K with a constant loss of mass
throughout the experiment. There was no separate endother-
mic event that could be assigned to the melting of cholesterol.
Intriguingly, hot-stage microscopy showed that the samples
remained beyond 373 K and melted at the melting point of
cholesterol with indications that solvent was being lost on
heating. Figures ES1e and ES1g show that, at the melting point
of cholesterol, compound 5 has lost 18% of its mass and
compound 7 has lost 14% of its mass, which are consistent with
the weights of solvent in the crystal structures identified by
233
dx.doi.org/10.1021/cg200971f | Cryst. Growth Des. 2012, 12, 231−239

Crystal Growth & Design
Article
single crystal diffraction (20% and 14%, respectively). Beyond
the melting point, the mass reduced in each of these solids
indicated the sublimation of cholesterol.
The three molecules interact through hydrogen bonding
between the alcohol moieties on each of the molecules to form
chains along the b-direction (O1···O1S, 2.650(3) Å; O2···O1,
2.676(2) Å; O1S···O2, 2.683(3) Å). Neighboring chains are
packed through the application of the 21-screw axis so that the
cholesterol molecules interact in a head-to-tail manner. Due to
the paucity of hydrogen-bonding groups in the alkyl chain, the
cholesterol molecules only interact through van der Waals
contacts, and so one observes different molecular confomations
of the two independent molecules.
Compound 6 (Figure ES1f) showed two endothermic peaks
at ∼330 K which could be attributed to the loss of benzyl
alcohol and water. Increasing the end point of the experiment
to 230 K showed no further events that could be attributed to
the melting of another compound. The mass lost at the melting
point of cholesterol was 21%, which is greater than the mass
of benzyl alcohol and water, which indicates that during heat-
ing it is likely that some of the cholesterol was lost through
sublimation.
This structure is not extensively layered into hydrogen
bonded and hydrophobic regions, in contrast to the cases of the
methanol and ethanol solvates. Instead, the propanol molecules
are in discrete locations in the crystal structure (Figure 3).
Crystal Structure Analysis. 2:1 Cholesterol/Propanol
Solvate (1). In 2008, Uskokovic investigated the effects of
́
varying the crystallization conditions on the morphology of
crystals of cholesterol.12 As part of this study, Uskokovic
crystallized cholesterol from a mixture of propanol and water
and analyzed these crystals using both DSC and X-ray powder
diffraction. Despite the use of propanol in the reaction mixture,
the author did not observe any changes in the diffraction
pattern or the thermal analysis trace that would suggest the
presence of another phase. Nevertheless, the formation of a
potential propanol solvate was investigated due to the previous
successful isolation of methanol and ethanol solvates by various
groups.18,19
́
A new compound was observed and found to crystallize in
space group P21 with two molecules of cholesterol and one
propanol molecule in the asymmetric unit (Figure 2). All three
Figure 3. Packing diagram of compound 1 viewed down the b-
direction. The molecules are colored by symmetry equivalent.
Molecule 1 is green; molecule 2 is blue; propanol is red; and the
disordered components are yellow. The inset shows the close
interaction between C27 of molecule 1 and O2, where the position
of C27 in both components does not alter significantly.
While molecule 2 and the solvent show a great deal of disorder,
molecule 1 shows relatively little disorder, which could be
attributed to a weak CH···O interaction between the hydrogen
attached to C27 and the hydroxyl group (O2) of a neighboring
molecule (Figure 3, inset).
Figure 2. Asymmetric unit of compound 1. The main component of
the disorder is colored by atom and the minor component colored
blue. The color coding for this and subsequent figures is as follows:
carbon, gray; oxygen, red; hydrogen, white.
2:1 Cholesterol/Butanol Solvate (2). Compound 2 is
isostructural with compound 1 (see Table 1), but only one
cholesterol molecule (molecule 2) shows any disorder in the
alkyl chain while the solvent remains disordered. As one might
expect, the hydrogen bonding interactions are a similar length
to those observed in compound 1 (O1···O1S, 2.670(4) Å;
O2···O1, 2.704(3) Å; O1S···O2, 2.703(4) Å). The weak
hydrogen bond that was observed in compound 1 is retained in
this structure at a slightly shorter distance (H27A···O2S, 2.63 Å;
cf. 2.73 Å).
2:1 Cholesterol/Pentanol Solvate (3). Extending the alkyl
chain length of the solvent molecule further to pentanol
produces a new crystalline structure that crystallizes in C2
(compound 3). There is one molecule of cholesterol and half a
molecule of pentanol that lies perpendicular to the cholesterol
molecule in the asymmetric unit; the solvent has been modeled
molecules show some degree of disorder, with molecule 2
showing the greatest proportion. Molecule 1 has small
differences in orientation of the isopropyl end group, whereas
molecule 2 shows disorder along the whole length of the alkyl
chain. The propanol molecule is disordered over two positions
with the oxygen and primary carbon residing in the same
position. These latter two places of disorder reside close to one
another in the crystal structure, and so it is probable that the
disorder in each region is related to one another. However,
assigning the same occupancies to the cholesterol and propanol
disorder, one observed a slight increase in the R-factor
indicating a poorer fit to the data. The disorder was better
modeled as 65:35 and 54:46 for the cholesterol and propanol,
respectively.
234
dx.doi.org/10.1021/cg200971f | Cryst. Growth Des. 2012, 12, 231−239

Crystal Growth & Design
Article
as disordered over two sites with occupancy 0.35:0.15, but in
reality the solvent is likely to be disordered over many other
sites with occupancies too low for the refinement of the atomic
positions. The cholesterol hydrogen bonds to a symmetry
equivalent molecule as well as the disordered solvent (O1···O1
2.623(4) Å; O1···O1S 2.644(9) and 2.712(9) Å; O1···O1SA
2.717(6) Å). There are two values for the distance between O1
and O1S due to the two positions the oxygen atom of the
solvent atom adopts.
As far as the packing is concerned, the cholesterol molecules
are arranged in a head-to-tail manner but there is a clear distinc-
tion in the packing of the molecules in this structure compared
to the packing in compounds 1 and 2 (Figure 4, cf. Figure 3).
Figure 5. Disordered solvent molecules in compound 3 (left) and
compound 4 (right). The solvent is disordered over the bc-plane, as
depicted in Figure 4. The solvent molecules are disordered over the 2-
fold rotation (green line). The second molecule of pentanol (O1SA)
will have another site of occupation that can be generated via the 2-
fold rotation but has been omitted for clarity.
compound 3, with the solvent molecules arranged perpendic-
ular to the direction of the cholesterol molecules. Table 1
shows that the unit cell parameters for compound 4 are very
similar in the a-direction, as the contributions to this direction
from the solvent molecules in both structures are disordered in
the bc-plane. The b- and c-directions show some differences in
lengths that can be attributed to the change in solvent length
and orientation. The cholesterol molecule hydrogen bonds to a
symmetry equivalent molecule and also to the two components
of the disordered solvent molecule (O1···O1 2.608(5) Å;
O1···O1S 2.70(3) Å; O1···O1S 2.63(3) Å) (Figure 5).
1:1 Cholesterol/Phenol Cocrystal (5). The attempted
crystallization of phenol with cholesterol was to investigate
whether the iodine substituent on iodophenol was an important
factor in the crystallization of the WOMHAI in the intercalated
structure rather than the bilayer structure observed for the
other solvates.
Compound 5 crystallizes with one molecule of cholesterol
and one molecule of phenol in space group P21 and with cell
parameters that are different from those of the previous com-
pounds (Table 1). However, if one looks closely at the unit cell
parameters, one can actually convert the P-centered cell into a
distorted C-centered cell through the application of the following
matrix transformation to the P-centered cell (−1 0 −2 1 0 0 0
−1 0). The resulting unit cell lengths from this transformation are
similar to those found for compounds 3 and 4, with the angles
necessarily different from 90° (a = 43.979 Å, b = 6.305 Å, c =
10.305 Å, α = 89.853°, β = 103.371°, γ = 92.248°). Therefore,
there is a similarity between the structures despite the large
differences in the cell parameters.
The molecules interact through hydrogen bonds between the
hydroxyl groups to form chains along the b-axis (O1···O1S
2.754(2) Å; O1S···O1 2.6193(19) Å). The neighboring chains
are intercalated so that the cholesterol molecules are arranged
head-to-tail with only short contacts between hydrogen atoms
on each of the molecules on different chains (Figure 6). The
depth of intercalation is not as great as that found in compound
3; therefore, there is not a definitive layer of solvent molecules
(cf. Figure 4).
2:1:1 Cholesterol/Benzyl Alcohol/Water Solvate (6). While
the products of the previous crystallizations could be isolated
over the course of a day, the product from the crystallization of
cholesterol from benzyl alcohol took over a week to appear. X-ray
analysis of the crystals showed that water had been incorporated
into the structure of cholesterol along with a targeted benzyl
alcohol molecule. Compound 6 crystallizes with two molecules
of cholesterol, one molecule of benzyl alcohol, and one of water
in space group P21. The a- and b-parameters for this compound
Figure 4. Packing diagram of compound 3 viewed down the b-direction.
The disordered solvent molecules are colored blue and red, depicting
the major and minor components, respectively.
The solvent molecules in 1 and 2 were observed to be in
discrete locations with some interaction between the alkyl chain
of the solvent and the steroid moeity of cholesterol. This occurs
due to the pseudoparallel orientation of the solvent with respect
to the cholesterol molecule. In compound 3, the perpendicular
orientation of the solvent allows it to form a layer at a = 0, 1/2,
etc. with the cholesterol molecules sandwiched between these.
This change to a more layered structure is most likely due to
the increase in disorder of the solvent compared to the
propanol and butanol solvates. The solvent molecules are
disordered such that the oxygen atoms are in a position to
interact with the hydroxyl groups of cholesterol molecules that
are equivalent by translation along the b-direction (Figure 5).
2:1 Cholesterol/Hexanol Solvate (4). The solvate with
hexanol, compound 4, is isostructural with compound 3. The
cholesterol molecule in this structure shows slight disorder
around the isopropyl group, which equates to a 70:30 ratio of
components. This solvate shows the same layered structure as
235
dx.doi.org/10.1021/cg200971f | Cryst. Growth Des. 2012, 12, 231−239

Crystal Growth & Design
Article
are similar to those of the first two compounds; however, the
c-parameter is significantly shorter. Despite the similarity of the
cell parameters, the packing of the molecules is significantly
different, which is due to the incorporation of the water molecule.
The water allows for further hydrogen bonding, which enables the
dimerization of the formally isolated hydrogen bonded chains.
The cholesterol molecules in the chain interact favorably so
that the flat sides of the ring system (α-side) are adjacent to
one another, as opposed to the case of compound 1, where the
ring systems of neighboring molecules are perpendicular to one
another (Figure 7).8 Chains of molecules are formed along the
b-direction using all three components (O1S···O1 2.663(2) Å;
O1···O2 2.771(2) Å; O2···O2S 2.670(2) Å; O2S···O1S 2.770(3) Å)
with an additional hydrogen bond between the water molecule
and a benzyl alcohol of another chain (O2S···O2 2.760(2) Å)
(Figure 8).
2:1 Cholesterol/Phenylethanol Solvate (7). The last solvate
studied in this series was that of cholesterol with phenylethanol.
These molecules crystallize together in a 2:1 ratio in monoclinic
space group C2. The cholesterol molecule is fully ordered apart
from the hydroxyl hydrogen atom, which is disordered over two
sites due to the disorder in the solvent. The phenylethanol
resides on the 2-fold axis and is found to be disordered over this
site (Figure 9). The hydrogen bonding in this crystal is very
simple, with chains of molecules running along the c-direction.
Due to the disorder of the solvent oxygen atom, there are two
distances quoted for the interaction between cholesterol and
the solvent: one which is slightly below average for this type of
Figure 6. Packing diagram of compound 5 viewed along the b-direction.
The phenol molecules are colored blue. The layers are not as well
differentiated as those found in compounds 3 and 4.
Figure 7. Comparison of the packing diagrams of compounds 1 (lower) and 6 (upper) viewed along the b-direction. The cholesterol molecules in
compound 6 interact via their α-sides, which has previously been calculated as being a favorable motif.8
236
dx.doi.org/10.1021/cg200971f | Cryst. Growth Des. 2012, 12, 231−239

Crystal Growth & Design
Article
Figure 8. Single hydrogen bonded chain (left) equivalent to other
compounds. The water molecules allow further hydrogen bonding to
neighboring chains to form “dimers” (right).
Figure 9. Disordered phenylethanol molecule. The different com-
ponents that are related by the 2-fold rotation (green) are highlighted
in blue and green.
interaction and one which is slightly above (O1···O1S 2.494(4) Å;
O1···O1S 2.824(3) Å; O1···O1 2.604(3) Å).
Figure 10. Packing diagram of cholesterol monohydrate showing the
bilayer structure (left). A molecular overlay of the independent
molecules and their conformational variability (right).
The crystal structure is isostructural to both compounds 3
and 4. The cholesterol molecules are intercalated with those
from neighboring chains so that a layered structure is formed.
The solvent molecule is orientated so that the phenyl group lies
1
in the bc-plane at a = 0, /2, etc. The solvent molecules in this
solvate are all orientated in the same way, mimicking the
behavior in compound 4, as opposed to compound 3,
where the disordered components are orientated in opposite
directions.
Comparison with Known Structures. There are eight
structures present in the database for which the structure has
been fully characterized, and their cell parameters can be found
in Table 2. While there are subtle differences in the structures
leading to differences in cell parameters, the overall molecular
architectures show two distinct variations. The bilayer
architecture is exhibited by both of the anhydrous forms, the
monohydrate, and the methanol and ethanol solvates. A good
example of this is the monohydrate (Figure 10, left). All of
these structures except for the high temperature polymorph
of the hemiethanol solvate crystallize in P1 with multiple
molecules in the unit cell, where, in many of the cases, dif-
ferent molecules are related by pseudosymmetry. In the
monohydrate, there are four different molecular conformations
(despite being Z = 8) (Figure 10, right), which indicates the
pseudosymmetry in the crystal structure as discussed by Hsu et al.
in their paper.14
The second type of architecture is exhibited by the two
cocrystals MEQKAU and WOMHAI. In these structures, the
cholesterol molecules interact in a head-to-tail manner with
other chains that are related by a 21-screw axis (Figure 11). This
type of architecture seems to reduce the disorder and thermal
motion exhibited by the cholesterol molecules as the tail groups
are in close proximity to the strong hydrogen bonds. The
Figure 11. Head-to-tail packing in MEQKAU. Colors depict the
different orientations of cholesterol molecules. Atom colors: carbon,
gray; oxygen, red; nitrogen, blue; phophorus, orange.
question that was posed at the outset of this investigation was
how big does the coformer have to be before a change from the
bilayer to the intercalated structure was invoked?
From the outset, a novel molecular packing was observed for
the simplest alcohols studied: propanol and butanol. In these
structures the packing of the cholesterol molecules is in the
head-to-tail arrangement, which is the distinctive feature of the
second type of molecular architecture, but instead of observing
the layers of alternating hydrophilic and hydrophobic regions,
one observes that the solvent molecules are located in discrete
locations in the crystal structure. The crystal structures of
compounds 5 and 6 show no isostructurality with any of the
five other solvates isolated as part of this study, or between
themselves, but the two structures show a stepwise change from
one architecture (compounds 1 and 2) to the other (3, 4, and
7). If one considers compound 6, the dimer unit in this
237
dx.doi.org/10.1021/cg200971f | Cryst. Growth Des. 2012, 12, 231−239

Crystal Growth & Design
Article
Figure 12. Crystal structures of compound 6 (left) and compound 5 (right) with an overlay depicting the similarity of the dimer chain of compound
6 and the single hydrogen bonded chain in compound 5.
structure resembles the single hydrogen bonded chains found
in the phenol solvate (compound 5), where the phenol
molecules reside at both sides of the cholesterol chain when
viewed along the b-axis (Figure 12 (right)). Figure 12 shows a
block representation of the hydrogen bonded chains in order to
show the similarity in the packing of these chains more clearly.
When depicted like this, it is clear that the chains are “tilted” to
a different extent in each of the structures. The lower the
degree of “tilt”, the closer the structure is to the layered
structure observed in the iodophenol cocrystal (WOMHAI).
Figure 13 shows a similar overlay of the block representation
over the structure of compound 7. There is a change in the
hydrogen bonded chain to only include the solvent molecule on
one side of the cholesterol molecules that allows neighboring
chains to pack closely so that the layered hydrophilic and
hydrophobic structures can be formed. The disorder of the
pentanol and hexanol molecules mimics the role of the bulkier
additives rather than following the packing arrangement
observed in the smaller alcohols: propanol and butanol.
CONCLUSIONS
■
In this paper we have described the formation of seven new
solvates of cholesterol. All of these novel forms adopt crystal
structures that are removed from the bilayer structure observed
for the simple solvates presently in the CSD. In each of these
structures, the cholesterol molecules interact in a head-to-tail
manner as opposed to tail-to-tail structure of the bilayer. The
solvates of the simplest two alcohols and benzyl alcohol form a
new molecular architecture where the solvent molecules are
found in discrete regions in the crystal structure. Phenol forms
an intermediary structure with more extended hydrophobic and
hydrophilic regions than the previous solvates. The solvates
with pentanol, hexanol, and phenylethanol are all isostructural
with each other and are essentially isostructural with an
Figure 13. Crystal structure of compound 7. Note that the
phenylethanol participating in the hydrogen bonded chain lies only
on one side of the cholesterol chain (cf. compound 5).
238
dx.doi.org/10.1021/cg200971f | Cryst. Growth Des. 2012, 12, 231−239

Crystal Growth & Design
Article
(26) Sheldrick, G. M. Acta Crystallogr., Sect. A: Fundam. Crystallogr.
2008, 64, 112.
iodophenol cocrystal in the CSD but for the change in space
group symmetry.
(27) Macrae, C. F.; Bruno, I. J.; Chisholm, J. A.; Edgington, P. R.;
McCabe, P.; Pidcock, E.; Rodriguez-Monge, L.; Taylor, R.; van de
Streek, J.; Wood, P. A. J. Appl. Crystallogr. 2008, 41, 466.
(28) Spek, A. L. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2009, 65,
148.
(29) Farrugia, L. J. J. Appl. Crystallogr. 1999, 32, 837.
(30) Mattis, P.; Kimball, S. GNU Image Manipulation Program (www.
gimp.org); Copyright 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009,
2010.
ASSOCIATED CONTENT
* Supporting Information
■
S
DSC and TGA analysis of the solvates along with the powder
X-ray diffraction results of the solvates formed. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: iain.oswald@strath.ac.uk. Fax: +44 141 552 2562.
Telephone: +44 141 548 2157.
■
ACKNOWLEDGMENTS
■
The authors would like to thank Alan R. Kennedy and Alastair
J. Florence for the use of the X-ray diffractometers and Naomi
Watts for help with PXRD. The authors would like to thank the
reviewers for their useful comments. The authors would also
like to thank the Nuffield Trust for a summer scholarship for
R.J.G. and the EPSRC for provision of X-ray equipment under
the Glasgow Centre for Physical Organic Chemistry.
REFERENCES
■
(1) Maulik, P. R.; Shipley, G. G. Biophys. J. 1996, 70, 2256.
(2) Calhoun, W. I.; Shipley, G. G. Biochemistry 1979, 18, 1717.
(3) Lund-Katz, S.; Laboda, H. M.; McLean, L. R.; Phillips, M. C.
Biochemistry 1988, 27 (9), 3416.
(4) D’Ascenzo, G.; Cardarelli, E.; Domenico Magri, A.; Bica, T.;
Sabbatini, M. Clin. Chem. 1978, 24, 119.
(5) Kumar, S.; Burns, S. J.; Blanton, T. N. J. Mater. Res. 1995, 10, 216.
(6) Frincu, M. C.; Sharpe, R. E.; Swift, J. A. Cryst. Growth Des. 2004,
4, 223.
(7) Frincu, M. C.; Fleming, S. D.; Rohl, A. L.; Swift, J. A. J. Am. Chem.
Soc. 2004, 126, 7915.
(8) Cromie, S. R. T.; Ballone, P. J. Chem. Phys. 2009, 131 (3),
034906.
(9) Baran, M.; Mazerski, J. Chem. Phys. Lipids 2002, 120, 21.
(10) Sheih, H. S.; Hoard, L. G.; Nordman, C. E. Nature 1977, 267,
287.
(11) Sheih, H. S.; Hoard, L. G.; Nordman, C. E. Acta Crystallogr.,
Sect. B: Struct. Sci. 1981, 37, 1538.
́
(12) Uskokovic, V. Steroids 2008, 73, 356.
(13) Hsu, L. Y.; Nordman, C. E. Science 1983, 220, 604.
(14) Hsu, L. Y.; Kampf, J. W.; Nordman, C. E. Acta Crystallogr., Sect.
B: Struct. Sci. 2002, 58, 260.
(15) Allen, F. H. Acta Crystallogr., Sect. B: Struct. Sci. 2002, 58, 380.
(16) Allen, F. H.; Motherwell, W. D. S. Acta Crystallogr., Sect. B:
Struct. Sci. 2002, 58, 407.
(17) Craven, B. M. Acta Crystallogr., Sect. B: Struct. Sci. 1979, 35,
1123.
(18) Sheih, H. S.; Nordman, C. E. Acta Crystallogr., Sect. E: Struct.
Rep. Online 2002, 58, o79.
(19) Sheih, H. S.; Hoard, L. G.; Nordman, C. E. Acta Crystallogr.,
Sect. B: Struct. Sci. 1982, 38, 2411.
(20) Karasev, V. A.; Fundamensky, V. S.; Bannova, I. I.; Franke, V.
D.; Stefanov, V. E. Biochim. Biophys. Acta 2000, 1466, 26.
(21) Bhatt, P. M.; Desiraju, G. R. CrystEngComm 2008, 10, 1747.
(22) Florence, A. J.; Baumgartner, B.; Weston, C.; Shankland, N.;
Kennedy, A. R.; Shankland, K.; David, W. I. F. J. Pharm. Sci. 2003,
92 (9), 1930.
(23) Cosier, J.; Glazer, A. M. J. Appl. Crystallogr. 1986, 19, 105.
(24) Sheldrick, G. M. SADABS, Version 2004-1; Bruker-AXS:
Madison, WI, 2004.
(25) Blessing, R. H. Acta Crystallogr. Sect. A: Fundam. Crystallogr.
1995, 51, 33.
239
dx.doi.org/10.1021/cg200971f | Cryst. Growth Des. 2012, 12, 231−239