3β-Acetoxy cholest-5-ene crystals: Catalytic synthesis, structural elucidation, contribution of intermolecular interactions and density functional theory calculations

Article abstract of DOI:10.1016/j.molstruc.2020.128833

In this work, we present the synthesis of 3β-acetoxy cholest-5-ene using zinc triflate Zn(OTf)₂ as a catalyst. The synthesized molecule has been characterized by single crystal XRD, spectroscopic techniques and density functional theory (DFT) calculations. The compound crystallizes in the monoclinic space group P2₁ with cell dimensions, a = 16.585(2) ?, b = 9.5175(12) ?, c = 17.656(2) ?, α = 90.00, β = 106.369(4), γ = 90.00. The systematic analysis of intermolecular interactions in crystal structure was accomplished by Hirshfeld surface analysis and fingerprint plot. Molecules are linked by a combination of C?H, C?C, H?H and O?H contacts, which have clear signatures in the fingerprint plots. The theoretical DFT/B3LYP calculations have been attempted to obtain the optimized geometry, IR/NMR spectra, natural bond orbitals, frontier molecular orbitals and various structure based molecular properties. We compared the conformations of the compound in solid state and in solution by calculation of dihedral angles and coupling constant values.

Full text of DOI:10.1016/j.molstruc.2020.128833

Journal Pre-proof
3β-acetoxy cholest-5-ene crystals: Catalytic synthesis, structural elucidation,
contribution of intermolecular interactions and density functional theory calculations
Ashraf Mashrai, Mohammad Jane Alam, Bushra Amer, Musheer Ahmed, Shabbir
Ahmad
PII:
S0022-2860(20)31158-3
DOI:
https://doi.org/10.1016/j.molstruc.2020.128833
MOLSTR 128833
Reference:
To appear in: Journal of Molecular Structure
Received Date: 20 May 2020
Revised Date: 27 June 2020
Accepted Date: 3 July 2020
Please cite this article as: A. Mashrai, M.J. Alam, B. Amer, M. Ahmed, S. Ahmad, 3β-acetoxy cholest-5-
ene crystals: Catalytic synthesis, structural elucidation, contribution of intermolecular interactions and
density functional theory calculations, Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/
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Credit Author Statement:
The contribution(s) of all authors is stated using relevant categories.
•
•
Ashraf Mashrai: Conceptualization, Synthesis of the compound, Crystallization, Writing-
Original draft preparation.
Mohammad Jane Alam: DFT calculations, theoretical data visualization, Writing-
Original draft preparation.
•
•
•
Bushra Amer: Characterization of the compound, Writing-Original draft preparation.
Musheer Ahmed: Single crystal XRD characterization and structure solution.
Shabbir Ahmad: Supervision, Software.

3β-Acetoxy cholest-5-ene crystals: Catalytic synthesis, structural elucidation,
contribution of intermolecular interactions and density functional theory
calculations
a,
b
c
d
b
Ashraf Mashrai , Mohammad Jane Alam* , Bushra Amer , Musheer Ahmed , Shabbir Ahmad
a
Department of Pharmacy, University of Science and Technology, Ibb Branch, Yemen
b
Department of Chemistry, Aligarh Muslim University, Aligarh 202002, India
b
Department of Physics, Aligarh Muslim University, Aligarh 202002, India
c
Faculty of Medicine and Health Sciences, Sana'a University, Yemen
d
Department of Applied Chemistry, Aligarh Muslim University, Aligarh 202002, India
ABSTRACT
In this work, we present the synthesis of 3β-acetoxy cholest-5-ene using zinc triflate Zn(OTf) as
2
a catalyst. The synthesized molecule has been characterized by single crystal XRD,
spectroscopic techniques and density functional theory (DFT) calculations. The compound
crystallizes in the monoclinic space group P2
1
with cell dimensions, a = 16.585(2) Ǻ, b =
9
.5175(12) Ǻ, c = 17.656(2) Ǻ, α = 90.00, β = 106.369(4), γ = 90.00. The systematic analysis of
intermolecular interactions in crystal structure was accomplished by Hirshfeld surface analysis
and fingerprint plot. Molecules are linked by a combination of C---H, C---C, H---H and O---H
contacts, which have clear signatures in the fingerprint plots. The theoretical DFT/B3LYP
calculations have been attempted to obtain the optimized geometry, IR/NMR spectra, natural
bond orbitals, frontier molecular orbitals and various structure based molecular properties. We
compared the conformations of the compound in solid state and in solution by calculation of
dihedral angles and coupling constant values.
Keywords: 3β-Acetoxy cholest-5-ene; Zinc triflate; Hirshfeld surface; DFT.
*
Corresponding author: janealam.ph@amu.ac.in , mjalamamu@gmail.com
1

1
. Introduction
Cholesterol cell toxicity is well-known to be influenced by C-3 esterification [1]. The
esterification catalyzed through acyl coenzyme A, cholesterol acyl transferase intracellularly or
lecithin cholesterol acyl transferase in plasma. Cholesteryl esters have been observed in
lipoproteins and oxidised lipoproteins and it shows the way by which the cholesterol can be
accrued in cells and lipid droplets. Further, esters can potentially play a role as hydrophilic weak
acid transporters to target cancer cells [2]. In the last few decades, much attempt has been
devoted to developing transition metal catalyzed for the facile construction of the skeleton of
heterocycles [3]. Zinc triflate has been utilized as a catalyst in a comprehensive range of organic
syntheses in the past few years [4]. Hirshfeld surface analysis approach is a very convenient and
widely used tool in the field of crystallography for the investigation of different kinds of
intermolecular interactions and crystal packing. Hirshfeld surface [5-8] based tools seem to be
extremely appropriate for the visualization of variations in the intermolecular interactions as
crystal structure gives the most obvious insight of the intermolecular contacts and crystal
packing. The surfaces encode knowledge about all intermolecular interactions offers a facile way
for getting an idea on crystal packing. The associated fingerprint plot [9, 10] investigates
quantitatively the varieties of intermolecular contacts experienced by molecules and depicts this
valuable information in a suitable color plot. As part of our ongoing studies on crystal analysis of
steroidal compounds [11-14], catalytic synthesis, single crystal analysis of 3β-acetoxy cholest-5-
ene was carried out along with the Hirshfeld surface analysis to explore the close contacts and
crystal packing. DFT/B3LYP method is widely used in quantum chemical calculations of
molecules due to its low computational cost and good accuracy. This method has been employed
on various steroids molecules to obtain their structural information, spectra and physiochemical
2

properties [15-18]. In the present work, DFT/B3LYP calculation results are used not only to
support the experimental data like molecular geometry, FTIR and NMR spectra but also to
explore various structure based molecular properties. The optimized geometry and the simulated
spectra (IR and NMR) have been compared with the experimental one and they are found in
good agreement. Thus, structure based theoretical data like HOMO-LUMO energies, molecular
electrostatic potential (MEP), non-linear optical (NLO) parameters, thermodynamic quantities
and global reactivity indices of the present compound has been accurately estimated. In addition,
spectral, thermal and morphological properties were also investigated.
2
. Experimental and theoretical details
2
.1. General
All reagents and solvents were obtained commercially and utilized as received. Kofler apparatus
was used to detect the Melting point. The FTIR spectrum of present compound was recorded by
KBr pellets technique using Interspec 2020 FT-IR Spectrometer spectro Lab in the region 4000-
-1
4
00 cm . NMR spectra were measured in CDCl on a Bruker Avance II 400 NMR Spectrometer
3
1
13
at 400 MHz ( H-NMR) and 100 MHz ( C-NMR). Chemical shifts (δ in ppm) are reported
1
13
relative to the TMS ( H NMR) and to the solvent signal ( C NMR spectra). Elemental analyses
of the present compound were performed on Perkin Elmer 2400 CHN Elemental Analyzer.
Thermal study of the compound was performed using TGA/DTA-60H instrument (SHIMADZU)
-1
at a heating rate of 20 °C min from ambient temperature to 800°C. Powder X-ray diffraction
o
(
CuKα radiation, scan rate 3 /min, 293 K, λ= 1.54Å) was performed on a Bruker D8 Advance
Series 2 powder X-ray diffractometer. Mass spectra were measured on a JEOL D-300 Mass
Spectrometer. The surface morphology of the compound was monitored using JEOL JSM-
6
510LV scanning electron microscope (SEM), Thin layer chromatography (TLC) plates coated
3

with silica gel G were exposed to iodine vapors to see the homogeneity as well as the progress of
reaction. Sodium sulfate in anhydrous form was used as a drying agent.
2
.2. Synthesis 3β-acetoxy cholest-5-ene
To a mixture of cholesterol (1 mmol) and acetic anhydride (1 mmol) in methylene chloride (20
mL), Zn(OTf) (0.1 mol %) was added. This mixture was stirred at room temperature for 30 min.
2
Completion of reaction was monitored by TLC, after quenching with added sat. NaHCO3 aq the
products extracted into methylene chloride (3×20 mL). The organic layer after the washing with
water, dried over anhydrous Na SO and concentrated to obtain the desirable product [19]. The
2
4
compound was crystallized from acetone and obtained colorless crystals appropriate for X-ray
diffraction. Yield (91%); m.p. 112 °C; Anal. Calc. for C H O : C, 81.25; H, 11.29 (%). Found:
2
9
48
2
-1
C, 81.29; H, 11.26 (%); IR (KBr, cm ): 2948, 2870 (C-H, stretching), 1736 (C=O), 1465, 1368
1
(
C-H, bending), 1670 (C=C), 1246 (C-O); H NMR (CDCl
3
, 400 MHz): δ 5.4 (1H, t, C10H, J =
), 1.10 (3H, s, 8-
CH ) 0.67 (3H, s, 16-CH ), 0.90 and 0.85 (other methyl protons); C NMR (CDCl , 100 MHz):
2
.31, 2.78 Hz), 4.54 (1H, m, C3α-H, W1/2 = 14 Hz, axial), 2.01 (3H, s, OCOCH
3
1
3
3
3
3
δ 170.19 (C=O), 139.54 (C-5), 122.57 (C-10), 77.43 (C-O), 56.67, 56.17, 50.03, 42.28, 39.73,
3
2
9.52, 38.11, 37, 36.53, 36.21, 35.81, 31.88, 31.84, 28.24, 27.98, 27.76, 24.27, 23.89, 22.82,
+
.
2.58, 21.23, 21.03, 19.28, 18,72, 11.84; MS (ESI): m/z 428.37 [M ].
2
.3. X-ray diffraction
The intensity data (three dimensional) for the titled compound were generated at 100 K on
Bruker KAPPA APEXII DUO diffractometer using Cu Kα radiation (λ= 1.54178 Å). The
structure was solved by direct methods using SHELXS-97 software (SHELDRICK, 1990).
Isotropic refinement of the structure by least-squares methods was carried out by using
4

2
SHELXL-97 (SHELDRICK, 1997) followed by anisotropic refinement on F of all the non-
hydrogen atoms. Crystallographic data (excluding structure factors) of 3β-acetoxy cholest-5-ene
reported in present article have been deposited with the Cambridge Crystallographic Data Centre
(CCDC) as deposition No. CCDC 1837266. Positions of all hydrogen atoms were obtained
geometrically with Uiso (H) = 1.2-1.5 Å Ueq (parent atom). A riding model was utilized in their
refinement (C-H = 0.98-1.00 Å).
2
.4. Hirshfeld surfaces calculations
Molecular Hirshfeld surfaces [20] in the crystal structure have been generated from electron
distribution which was computed as the sum of spherical atom electron densities [21]. Hirshfeld
surface is unique for a given crystal structure and set of spherical atomic electron densities [22].
This property suggests the possibility to know additional insight into the intermolecular
interaction of molecular crystals. In this work, the Hirshfeld surfaces as well as fingerprint plots
were produced using CrystalExplorer [23] with the results of X-ray studies. In the calculations,
bond lengths to hydrogen atoms were normalized to typical neutron values (C-H = 1.083 Å) [24]
in order to make sure the internal evenness and independence of results from the crystal structure
refinement method. The 2D fingerprint plots were presented by using the standard view with the
d and d distance scales (0.6-2.6 Å) displayed on the graph axes. The normalized contact
e
i
distance (dnorm) related to both d , d and the vdw radii of the atom, given in Eq. (1), enables to
e
i
identify the regions of particular importance for intermolecular interactions [25]
vdw
vdw
dnorm = di - ri
d – r
e
re
(1)
+
e
vdw
vdw
ri
5

Distances d
e i
and d in the expression of dnorm, where two Hirshfeld surfaces touch, display a red
spot identical in color intensity as well as size/shape because of the symmetry between d and d .
e
i
2
.5. Theoretical calculations
Present theoretical calculations were performed using hybrid functionals (B3LYP) within DFT
framework along with basis set 6-311G(d,p) by Gaussian 09 software [26-28]. The initial
coordinates of the present geometry were taken from the asymmetric unit of present crystal from
crystallographic information file. Potential energy surface scan was performed by varying two
dihedral angles i.e. C16-C6-O-C and C6-O-C5-C1 to check stable conformer of the 3β-acetoxy
cholest-5-ene. The optimized geometry and vibrational spectra of the title molecule were
computed in the ground electronic state at B3LYP/6-311G(d,p) level of theory (in vacuum). The
IR bands appeared in the FTIR were assigned with the great accuracy using animated modes
visualized by Gauss View 5 program [29]. The theoretical IR spectrum was presented by
-1
Lorentzian band shape with FWHM of 5 cm . In general, harmonic frequencies overestimate the
experimental IR frequencies mainly due to excluding anharmonic nature of molecular vibrations
in quantum chemical calculations. Therefore, a uniform scaling factor, 0.967, was used to scale
1
13
down the harmonic frequencies [30]. The NMR spectra ( H and C) were simulated in the
solution phase (CDCl ) using integral equation formalism of polarizable continuum model (IEF-
3
PCM) within GIAO-B3LYP/6-311G(d,p) method. The chemical shifts (in ppm) were calculated
with respect to TMS shielding (as reference) at same level of theory. Natural atomic charges,
hybridization, and hyper conjugative interactions energies were obtained by natural bond orbital
analysis (NBO) using NBO 3.1 program [31]. In addition, molecular energy, dipole moment,
frontier molecular orbitals (HOMO-LUMO), non-linear optical (NLO) parameters, molecular
6

electrostatic potential, thermodynamic data and various reactivity descriptors for the synthesized
compound have been also computed at same level of theory.
3
. Results and discussion
In this work, we synthesized 3β-acetoxy cholest-5-ene using Zn(OTf)
2
as a catalyst instead of
1
using pyridine [32]. Our approach offered the same product in less time and better yield. IR, H
1
3
NMR, C NMR, elemental analysis and single crystal XRD data agree with the supposed
structure depicted in scheme 1. The DFT/B3LYP results are used to support experimental data
for the presented compound.
Scheme 1: Synthesis of 3β-acetoxy cholest-5-ene from cholesterol.
3
.1. Description of crystal structure
48 2
The asymmetric unit of 3β-acetoxy cholest-5-ene, C29H O , consists of two crystallographically
independent molecules, A and B, while unit cell contains 4 molecules. In both molecules, the
three cyclohexane rings are in chair conformations while the cyclopentane rings show envelope
and twist conformations in molecules A and B, correspondingly. The synthesized compound
crystallizes in the monoclinic system with space group P2 due to the presence of 8 chiral centers
1
[24]. The molecules are arranged in head to tail manner and the crystal packing is stabilized by a
7

combination of intermolecular interactions (Fig. 1). The B/C and C/D ring systems are trans
fused about the C(12)-C(13) and C(16)-C(17) bonds, respectively, while the A/B ring junction is
quasi-trans. Side-chain is extended with a gauche-trans conformation of the terminal C28 and
C29 methyl groups (Fig. 1). The bond distances and bond angle in the compound are found to be
almost equivalent to earlier report and within normal ranges [33]. At position 3, the acetoxy
group is equatorial and antiperiplanar to the C3-C8 bond with torsion angle 177.95 and not
involved in any hydrogen bond. The absolute configurations of chiral centres were established
from the structure shown, these sites exhibit the following chiralities: C3 = S, C6 = R, C12 = S,
C13 = S, C16 = R, C17 = S, C21 = R and C22 = R. Due to the non-existence of the strong
hydrogen bond donor in the crystallize molecule, the cohesion of the crystal structure can only be
attributed to van der Waals interactions. Important crystallographic data along with refinement
details of the structural analyses are summarized in Table 1. It is important to mention that
previous report [34] did not provide any information concerning the intermolecular contacts,
however our, study demonstrates the existence of short intermolecular contacts.
8

Fig. 1. Crystallographic structure of 3β-acetoxy cholest-5-ene: (a) asymmetric unit (b) 2D-view
c) central projection view and (d) geometry with labelling.
(
Table 1
Crystal data and structure refinement for 3β-acetoxy cholest-5-ene.
Empirical formula
Formula mass
Wavelength (λ)
Crystal system
Space group
Unit cell dimensions
a, Ǻ
b, Ǻ
c, Ǻ
α (deg)
29 48 2
C H O
428.67
0.71073 Å
Monoclinic
P21
16.585(2)
9.5175(12)
17.656(2)
90.000
β (deg)
γ (deg)
106.369(4)
90.000
2674.0(6)
4
1.065
0.064
3
Volume, Ǻ
No. of molecules per unit cell (Z)
Calculated density, Mg m
Absorption coefficient (μ, mm )
-3
-1
F(000)
952
Crystal size
0.32× 0.21× 0.15 mm
9

θ
range for data collection
2.38 to 25.50˚
Limiting indices
Reflections collected
Data/restraints/parameters
-20<=h<=20, -11<=k<=11, -18<=l<=21
19658
9607/178/567
2
Goodness-of-fit on F
0.956
Final R indices [I>2
R indices (all data)
σ
(I)]
R1 = 0.0686, wR2 = 0.1363
R1 = 0.1757, wR2 = 0.1786
2
o
2
2
2
2
2 2 1/2
R = Σ ||F | – |F ||/Σ|F | with F >2σ(F ). wR = [Σw(|F |–|F |) /Σ|F | ]
1
o
c
o
o
2
o
c
o
3
.2. Molecular Hirshfeld surfaces
Hirshfeld surface is a valuable tool for understanding the surface characteristics of the molecules.
It shows the area where molecules come into contact and therefore its analysis provides the
additional insight into the intermolecular interactions within the crystal. The Hirshfeld surface is
described by regions where the part of the electron density from the molecule of interest is equal
to the contribution from the other molecules. The two distances are defined for each point on the
e i
surface: d (the distance from the point to the nearest nucleus external to the surface), and d (the
distance to the nearest nucleus internal to the surface). The molecular Hirshfeld surfaces of 3β-
acetoxy cholest-5-ene were generated using a standard (high) surface resolution with the 3D
dnorm surfaces. The surfaces were mapped over a fixed color scale of -0.23 (red) to 1.2 Å (blue).
The shape index and curvedness were mapped in the color range of -0.99 to 1 and of -4.0 to 0.4
respectively. The molecular Hirshfeld surface (dnorm, shape index and curvedness) of 3β-acetoxy
cholest-5-ene are presented in Fig. 2. The normalized contact distances, dnorm were mapped into
the Hirshfeld surfaces for comparison of intermolecular interactions in crystal structures.
Negative values of dnorm as shown by the red color indicate contacts shorter than the sum of van
der Waals radii. The white color visualizes intermolecular distances close to van der Waals
contacts with dnorm equal to zero. In turn, positive dnorm values indicated by blue color define
contacts longer than the sum of van der Waals radii. The bright red regions in the Hirshfeld
1
0

surface are appeared due to the O---H and C---H interaction in the compound. The H---H and C--
C contacts can be seen by other visible spots on the surface correspond. Shape index describe
-
the surface shape, and it is sensitive to very fine changes in surface shape, mainly in areas where
the total curvature (or the curvedness) is very low. The information expressed by shape index is
given with the 2D fingerprint plot. The flat areas of the surface have correspondence to low
values of curvedness, while sharp curvature areas are related to high values of curvedness. This
usually tends to divide the surface into patches, representing interactions between neighboring
molecules. The 2D fingerprint plots can be decomposed to highlight close contacts of particular
atom pair [11]. The 2D fingerprint plots, analyses all the intermolecular contacts simultaneously,
revealed that the main intermolecular interactions in the present compound were C---H, H---H,
O---H and C---C intermolecular interactions (Fig. 3). H---H interactions, which cover most of
regions in the 2D fingerprint plots, have a major contribution to the total Hirshfeld surfaces
(
85.9%). The O---H contacts are appeared as distinct spikes pointing in the direction of the lower
left of the plot. In these complementary regions one molecule acts as donor ( e di) and the
other as an acceptor ( ). The minimum value of ( ) i.e. the shortest contact is around
.9Å which point out the significance of these interactions. The proportion of H---O interactions
d
>
d
e
<
d
i
e i
d + d
1
comprising 8.2% of the total Hirshfeld surfaces for the molecule. The top left and bottom right
regions of the fingerprint plot are the characteristic ‘‘wings’’ which are identified as a result of
C---H interactions. The decomposition of the fingerprint plot for H---C contacts comprised 5.4%
of the total Hirshfeld surface area. Finally, C---C contacts comprised 0.5% of the total Hirshfeld
surface area. Thus the nature of the interaction for the title compound is straightforwardly
understood using Hirshfeld surface, with the results further highlighting the importance of the
technique in mapping out the interactions within the crystal and this tool has very important
1
1

promise in crystal engineering. The Hirshfeld surface allows a much more detailed scrutiny by
exhibiting all the intermolecular interactions and also by quantifying them in two dimensional
fingerprint plot within the crystal.
Fig. 2. Hirshfeld surfaces mapped with (a) dnorm, (b) surface index and (c) curvedness of 3β-
acetoxy cholest-5-ene. The surfaces are shown as transparent to allow visualization of the
orientation and conformation of the functional groups in the molecule.
1
2

Fig. 3. 2D fingerprint plots of 3β-acetoxy cholest-5-ene. (a) full (b) resolved into C-H (c) C-C
d) H-H (e) O-H contacts showing the percentages of contacts contributed to the total Hirshfeld
surface area molecule.
(
3
3
.3. DFT calculation results
.3.1. Conformational search and molecular geometry
Potential energy surface (PES) scan has been performed to obtain the most stable structure by
rotating acetoxy group of the present compound, Fig. 4. The dihedral angles i.e. C-C6-O-C5 and
o
o
C6-O-C5-C1 for the most stable geometry on PES are found to be 153.8 (Exp. 156.1 ) and
o
o
1
79.9 (Exp. 177.9 ) respectively. The molecular geometry with β-acetoxy, obtained by PES
1
3

scan, is in good agreement with the XRD geometry. The optimized molecular structure (most
stable geometry) of 3β-acetoxy cholest-5-ene molecule along with numbering of atoms is shown
in Fig. 5. The numbering scheme is used as per Fig. 5 to discuss DFT results. The minimum self-
consistent field (SCF) energy and dipole moment are found to be -806233.7902 kcal/mol and
2
.016 Debye, respectively. The comparison of some selected optimized geometrical parameters
with the X-ray diffraction data is shown in Table 2. They are in a reasonable agreement as low
values of RMSE and MAD (1-3° for angles) are found. However, any discrepancies noted may
be due to fact that the XRD data belongs to molecule with many surrounding interactions while
the DFT results were obtained for molecule in isolated form. The molecular geometries obtained
from DFT calculation and XRD experiment have been globally compared by superimposing
atom by atom (Fig. 6) and RMSE value is found to be 0.477 Å at B3LYP/6-311G(d,p) level of
theory.
1
4

Fig. 4. PES scan of acetoxy group of 3β-Acetoxy cholest-5-ene molecular geometry.
Fig. 5. Optimized molecular geometry of 3β-acetoxy cholest-5-ene.
Fig. 6. Superimposed molecular structure of 3β-acetoxy cholest-5-ene obtained by XRD and
DFT.
Table 2
Some selected structural parameters of 3β-acetoxy cholest-5-ene.
a
a
Bond lengths (Å)
Exp.
B3LYP/6-
11G(d,p)
Bond angles (
⁰
)
Exp.
B3LYP/6-
311G(d,p)
3
C1-C5
C5=O
1.462
1.214
1.510
1.205
C1-C5-O
C1-C5=O
113.6
124.9
110.6
125.2
1
5

C5-O
O-C6
C6-C8
C6-C16
C11-C12
C11=C23
C8-C11
C50-C52
MAD
1.322
1.464
1.510
1.491
1.514
1.312
1.506
1.539
1.352
1.455
1.530
1.520
1.533
1.335
1.516
1.544
0.020
0.024
O=C5-O
C5-O-C6
121.4
119.1
107.4
108.8
112.25
112.3
119.3
114.2
124.2
117.5
107.1
110.2
111.1
112.5
119.7
110.5
1.5
O-C6-C16
O-C6-C8
C8-C6-C16
C47-C50-C52
C37-C50-C52
C69-C67-C73
RMSE
1.9
Dihedral angles (
C1-C5-O78-C6
C16-C6-C8-C11
O78-C6-C16-C13
C16-C6-O78-C5
C37-C50-C52-C58
MAD
⁰
)
Dihedral angles (⁰ )
177.9
50.3
172.0
156.1
177.0
179.9
53.9
176.6
153.8
179.6
O77=C5-O78-C6
O78-C6-C8-C11
C8-C6-O78-C5
C37-C50-C52-C54 58.7
C47-C50-C52-C54 179.6
0.5
169.0
82.2
0.2
172.5
85.2
55.6
177.2
2.7
RMSE
2.9
a
Obtained from single crystal XRD data; numbering scheme is adopted as per Fig. 5
RMSE: root mean square error, MAD: mean absolute deviation
3
.3.2. Vibrational analysis
The present compound consists of 79 atoms consequently it has 231 normal vibrations. Out of
these, some important vibrational modes have been discussed. Present compound consists of four
rings (six member R1/R2/R3 and five member R4, Fig. 5) with CH/CH /CH groups, carbon
2
3
chain with CH/CH /CH groups and one acetoxy group. Some important vibrational assignments
2
3
and comparison of FTIR band frequencies and intensities with the theoretical one are presented
in Table 3. Only the major contributions of the vibrations are mentioned in the Table 3. This
table is self-explanatory for the vibrational assignments however some vibrations need
discussions. The low values of deviations RMSE and MAD show the reasonable agreement
between theoretical (scaled) and experimental IR frequencies. The observed FTIR and simulated
IR spectra of the compound are shown in Fig. 7. In general, aliphatic CH and acyclic CH
3
2
-1
-1
stretching vibrations occur in the regions 2975-2865 cm and 2940-2880 cm respectively [34].
The IR bands due to CH symmetric bending (Umbrella) vibration appear often in the region
3
1
6

-1
1
420-1375 cm [34]. These vibrations are observed in the present FTIR band within the said
region as shown in Table 3. Scaled IR frequencies of these vibrations are found close with the
-1
experimental one. The strong IR band observed at 1736 cm is assigned to C=O stretching
vibration in acetoxy group of the compound. The scaled frequency of C=O stretching vibration is
-1
1
746 cm for the present molecule. The C-O stretching vibration is assigned to the strong band
-1
-1
observed at 1246 cm . The calculated harmonic frequency of this vibration is 1257 cm . These
vibrations of acetoxy group are found in agreement with those obtained theoretically as well as
-1
of reported data in literature [35]. Vibrational modes found below 1000 cm are mixed with
skelton vibrations.
Table 3
-1
Important vibrational frequencies (cm ), integrated intensity (km/mol) and assignments.
a
b
FTIR
harmonic
3078
3071
3016
2972
1805
1726
1515
1494
1416
1358
1257
1177
1123
1047
1038
scaled
2976
2969
2917
2874
1746
1669
1465
1444
1369
1313
1216
1138
1086
1013
1004
956
IR intensity Assignment
2
2
2
2
1
1
1
1
1
1
1
1
1
1
9
948vs
936vs
906sh
870sh
736vs
670w
465s
82.7
86.9
56.6
28.9
209.7
2.7
14.9
4.4
10.3
1.3
ν
ν
ν
ν
ν
ν
β
β
β
β
ν
β
β
CH (C-chain)
C-H (R1, R3)
CH (C-chain)
3
2
C-H (R2)
C5=O
C=C (R2)
C-H
442sh
368s
C-H (R3,R4,C-chain)
CH (umbrella) +
β
C-H
3
312w
246vs
137m
085w
032s
C-H
C5-O +
twist CH
491.2
7.6
2.2
β
+
C-H
C-C-C +
C-C-C
C6-O] (acetoxy group)
CH (acetoxy group) + C-C +
CH (acetoxy group, R2) + C6-O
2
β
γ CH
3
C-H + ν C-C + β
173.8
40.6
3.6
[
γ CH + ν
3
80sh
γ
γ
+
β
γ
ν
β C-C-C
3
9
88
ν
3
9
56m
ν
C-C
C-C-C +
C23-H + βrock CH (rings)
9
8
02m
00m
937
854
906
826
722
4.9
4.3
2.7
2 3
γ [CH + CH ] (rings, C-chain)
2
7
47
[
ν
C-C +
β C-C-C] (rings) +
βrock CH (R4)
7
38m
2
6
12m
625
66
55
604
16
11
7.0
β C-C-C (rings) + γ C-H
RMSE
MAD
Abbreviation used: vs-very strong, s-strong, m-medium intensity, sh-shoulder, w-weak, ν-
1
7

stretching, β- bending motion, γ -out-plane deformation vibrations, RMSE: root mean square error
-
1
-1
(
in cm ), MAD: mean absolute deviation (in cm )
a
harmonic wavenumbers were scaled by factor 0.967 [30].
vibrations having major contribution; numbering scheme is adopted as per Fig. 5
b
Fig. 7. Simulated and experiment IR spectra 3β-acetoxy cholest-5-ene.
3
.3.3. NMR spectra
1
13
The H NMR, C NMR and Distortionless Enhancement by Polarization Transfer (DEPT)
1
13
studies confirmed the molecular structure of 3β-acetoxy cholest-5-ene. The recorded H and C-
NMR spectra of the molecule in CDCl are compared with the theoretical one obtained at GIAO-
3
B3LYP/6-311G(d,p)/IEF-PCM. They are found in good agreement. Experimental and simulated
1
8

1
13
NMR spectra ( H, C) are presented in Fig. S1- S4 (supplementary files). The numbering of
atoms was used as per Fig. 5. Most of the proton NMR signals are observed within the narrow
region 0-3 ppm may be due to relatively high electron density over the nuclei (shielding effect).
1
Two H-NMR signals observed in the region 4-6 ppm are assigned to olefinic proton at δ 5.4 and
4
.5 δ for C3
ppm and 4.5 ppm respectively for olefinic proton (H79) and C3
identified relatively at high ppm value because of low electron density over these nuclei. In the
α
-proton. The corresponding theoretical chemical shift values are found to be 5.7
α-proton (H7). These are
1
3
C-NMR spectrum, the signals are appeared in the region 0-180 ppm. Most of the signals are
occurred in the region 0-80 ppm while three signals are appeared at high ppm values (120-180).
These signals observed within range 120-180 ppm are due to C=O of acetoxy group and C=C.
These carbon atoms have shown their presence at high ppm because these found with
respectively large positive and almost zero charges (Fig. S5). It is often extra difficult to obtain a
1
3
full aspect from a simple C spectrum, particularly if there is overlap in the spectral like our
case. An alternative approach would be to acquire a DEPT spectrum (Fig. S6). The DEPT
sequence produces signals only for protonated carbons by changing the flip angle of the final 1H
pulse (45°, 90°, or 135°). The DEPT with flip angle 45° detects signals for CH, CH
2 3
and CH ,
with the same sign. The DEPT-90° provides only CH peaks. The DEPT-135° gives signals with
1
3
positive sign for CH and CH while negative peaks for CH . In the DEPT-135 the C signal
3
2
1
13
enhancement is achieved by polarization transfer from H to C. Other advantage of DEPT
spectra over simple carbon NMR spectra is its ability to identify protonated carbon peaks that
overlap one of the deuterated solvent peaks [36]. The nature of each carbon in the compound was
deduced through DEPT spectrum (Fig. S6) by polarization pulses of 135˚, obtaining negative
2 3
signals (down) for CH groups while positive signals (up) for CH, CH . The DEPT-135 showed
1
9

3
3
3
2
the existence of six sp methyl, eleven sp methylene and eight methine (seven sp and one sp )
carbons.
3
.3.4. Other molecular properties
Quantum mechanical parameters describing various molecular properties obtained at B3LYP/6-
11G(d,p) level are tabulated in Table 4. The donor-acceptor NBO hyperconjugative interaction
3
(
2)
energies, E , were estimated at same level of theory to investigate charge delocalization within
the molecule. The strong hyperconjugative interactions for the present compound are predicted
for LP (2) O78→ π*(C5=O77), LP (2) O77→σ*(C1 - C5) and LP (2) O77→ σ*(C5 - O78) with
(
2)
E values 47, 18 and 32 kcal/mol respectively. The hybridization of the atoms and the electron
density (ED, in %) of atom in localized electron pair bond for acetoxy group of present
compound is presented in Table 5. A bonding orbital for
σ
C5=O77 with occupancy 1.99652 has
1
.91
1.35
3
4.09% C5 character in a sp
hybrid and 65.91% O77 character in a sp
hybrid while for π
1
.00
C5=O77 with occupancy 1.99031 has 30.40% C5 character in a sp
hybrid and 69.60% O77
9
9.99
1.91
1.35
character in a sp
hybrid. The sp hybrid on carbon and sp hybrid on oxygen atom have
1
.00
respectively 65.50% and 57.38% p-character for
σ
C5=O77, while for
π C5=O77 the sp hybrid
9
9.99
on carbon and sp
hybrid on oxygen atom have respectively 99.48% and 99.87% p-character.
In Table 5, oxygen (O78) has larger polarization coefficient (0.8359) because of higher
electronegativity. Natural atomic charges obtained by NBO analysis are shown in Fig. S5. The
-30
mean first hyperpolarizability of present compound is found to be 0.6481 x 10 esu, which is
-30
about 2 times greater than of urea (0.3728 x 10 esu) [37]. This value shows that the 3β-acetoxy
cholest-5-ene may be used for NLO application. The MEP plot is obtained by mapping
electrostatic potential onto total electron density of the molecule to know its binding sites i.e.
nucleophilic/electrophilic (Fig. 8). The electrostatic potentials are shown over the surface in
2
0

terms of colour scheme. The regions with blue, green and red/ yellow colours define the positive,
zero and negative values of the potential respectively. In the MEP plot, most of the regions over
the molecule shown by green colour are characterized as neutral part of the molecule. The
regions over the O77 and O7 atoms (acetoxy group) represent the negative potential (dark
red/yellow coloured/ high electron density). The light blue colour regions spotted over the H-
atoms of molecule are characterized by positive potential. The frontier molecular orbitals i.e.
HOMO and LUMO play important role in chemical reactions. The HOMO, LUMO energy Eigen
values and HOMO-LUMO energy gap help to determine the chemical reactivity of molecules.
The spatial plot of HOMO and LUMO of the present molecule is shown in Fig. 9. The HOMO
and LUMO energy Eigen values, HOMO-LUMO energy gap and various global reactivity
descriptors computed at B3LYP/6-311G(d,p) level of theory are presented in Table 4. In Fig. 9,
the HOMO is delocalized mainly over the ring (R1 and R2), while the LUMO is delocalized
mainly over the acetoxy group. Thus, HOMO-LUMO plot shows that these frontier molecular
orbitals are localized over some part of the molecule. HOMO and LUMO are found completely
absent over the other rings and side carbon chain. Therefore, these regions may not participate
much in chemical reactions as its molecular orbitals are of too large in energy for the reactions.
A large value of HOMO-LUMO gap (~6 eV) exhibits good thermodynamic stability and low
chemical reactivity. The global reactivity descriptors based on HOMO and LUMO energy Eigen
values are presented in Table 4. These chemical descriptors were estimated using relations given
in literatures [38-44]. These global descriptors are important parameters for molecule in
quantitative structure – activity relationship (QSAR) modeling [45].
Table 4
Some quantum chemical parameters for 3β-acetoxy cholest-5-ene.
2
1

Parameters
Minimum SCF energy
B3LYP/6-311G(d,p)
-806233.7902
Parameters
EHOMO (eV)
B3LYP/6-311G(d,p)
-6.460
(
kcal/mol)
Dipole Moment (Debye)
Polarizability ( ) in a.u.
2.0159
328.7104
ELUMO (eV)
HOMO-LUMO
Gap (eV)
Global reactivity
descriptors (eV)
Hardness
0.270
6.190
α
-30
HyperPolarizability (
in e.s.u
β
)
0.6481 x 10
451.6656
472.435
Zero point vibrational
energy (kcal/mol)
Total thermal energy
3.365
-3.095
-0.270
1.423
Chemical potential
Electron affinity
Electrophilicity
(
kcal/mol)
Heat Capacity (Cv)
cal/mol-kelvin
Entropy (S)
130.317
209.564
cal/mol-kelvin
Electronegativity
3.095
6.460
0.148
6.201
0.011
Ionization potential
-1
Softness (eV )
Electrofugality
Nucleofugality
Table 5
NBO analysis of acetoxy group in 3β-acetoxy cholest-5-ene at B3LYP/6-311G(d,p) level of theory
a
Bond (A-B)
Occupancy
1.97660
ED %
ED %
NBO (% p character)
0.7797sp (76.26) +
A
B
3
.22
σ
σ
σ
σ
σ
π
σ
σ
C1 - H2
C1 - H3
C1 - H4
C1 - C5
C5 - O77
C5 - O77
C5 - O78
C6 - O78
60.79
60.80
60.78
51.55
34.09
30.40
30.78
30.13
39.21
39.20
39.22
48.45
65.91
69.60
69.22
69.87
C
0
.00
0
.6262sp3 (0.04)H
.10
1.98734
1.97389
1.98119
1.99652
1.99031
1.99197
1.98513
0.7797sp (75.55) +
C
0
.00
0
.6261sp3 (0.04)
H
.26
C
0.7797sp (76.49) +
.6262sp 0(0.04)H
0.7180 sp 0 (71.41) +
.6961 sp1 (62.32)C
0.5838 sp (65.50)
0
.0
0
2.5
C
1
.66
0
.91
C
+ 0.8119
1.35
sp (57.38)
O
1
.00
0.5514 sp (99.48) + 0.8342
C
99.99
sp
(99.87)
O
2.57
0.5548 sp (71.82) + 0.8320
C
2.11
sp (67.84)
O
4
.48
C
0.5489 sp (81.53) + 0.8359
2.29
sp (69.54)O
sp (42.51)
sp (99.92)
sp (62.48)
0.74
LP (1) O77
LP (2) O77
LP (1) O78
LP (2) O78
1.97657
1.84856
1.96488
1.79556
1.00
1.67
1.00
sp (99.95)
Abbreviation used: ED: electron density
a
numbering scheme is adopted as per Fig. 5
2
2

Fig. 8. MEP plot of 3β-acetoxy cholest-5-ene.
Fig. 9. HOMO and LUMO spatial plots.
2
3

3
.4. Elemental analysis
To determining the constituents and purity compositions of the crystallized compound, analysis
of carbon and hydrogen was carried out. The calculated elemental analysis of C H O is C,
2
9
48
2
8
1.25 and H, 11.29 (%) and the experimental; C, 81.29; H, 11.26 (%). As we can see the
experimental and calculated values within ±0.4%, confirming the purity of compound >95%.
3
.5. Conformations of the compound in solution and solid state
We examined the conformations of crystallized compound in solution via NMR and solid state
through X-ray. The angles were measured from CIF files (solid state data). Nuclear Overhauser
Effect Spectroscopy (NOESY) spectrum of 3β-Acetoxy cholest-5-ene has been also reported.
This spectrum exhibits signature due to interactions within the molecule through space and also
shows the correlation between protons which are close enough in space. In spectrum, the cross
3
peaks signals are appeared due to protons that are close in space. The coupling constants ( JH-H
)
were obtained by analysis of NOESY NMR (Fig. S7) of the molecule in CDCl , and angles were
3
3
determined using the on-line interactive data table. When this J corresponded to two or more
H-H
possible angles, the nearby one (between -90 and +90° from the solid state angle) was chosen
[46]. The estimate of the dihedral angles deduced from the measured coupling constants are
presented in Table 6. Remarkably, we analyzed less difference in the conformations of the
compound in the solid state and in solution phase, showing that the bending of rings at the A, B
junction was also less in solution phase than in the solid phase.
Table 6
Comparison of torsion angles of 3
β-acetoxy cholest-5-ene.
a
3
Torsion angle
Measured angle
J
H-H (Hz)
Calculated
2
4

(
solid state)
127.27
176.90
174.93
156.70
43.59
(solution)
C10-C5-C4-C3
C4-C5-C10-C11
C8-C7-C6-C13
C7-C6-C13-C12
H10-C10-C11-H11
H10-C10-C11-H11
β
α
2.75
53.34
-74.14
167.16
49.37
H11
α
-C11-C12-H12
- C11-C12-H12
Fig. 1(d)
8.7
4.4
166.46
42.09
H11
β
a
3
.6. Scanning electron microscopy
The surface morphology of the crystal was studied through scanning electron microscopy
SEM). The SEM micrograph (Fig. 10) indicated presence of brick-shaped, some irregular
shaped particles and it shows that the crystals grown in big size.
(
2
5

Fig. 10. SEM micrograph of 3β-acetoxy cholest-5-ene.
3
.7. Density measurement
48 2
The calculated density of C29H O was determined by taking the ratio of the cell mass to cell
volume. The equation for the density is defined as ρcalc = MZ/NV, where M is the chemical
formula weight of crystals, Z is the number of formula units in one cell, N is Avogadro's number
and V is the volume of the unit cell. According to the crystallographic data, it is observed that a
3
=
16.585(2) Å, b = 9.5175(12), c = 17.656(2), M = 428.67, Z = 4, and V = 2674.0(6) Å . So the
-3
calculated density of the crystal was found to be 1.065 Mg m . Experimentally, the density of
the 3β-acetoxy cholest-5-ene crystal was measured using the buoyancy method at temperature 22
2
6

°
C in silicon oil. This experimental density of the as-grown crystal is found to be 1.069 ± 0.003
-3
Mg m , which is almost equal to the calculated density.
3
.8. Deliquescence test
To verify the deliquescence of the 3β-acetoxy cholest-5-ene crystal, at the room temperature the
crystal sample was subjected to air atmosphere without any protection [47]. At the starting, it
was transparent, and the weight was about 0.7872 g. After five days, we could not observe any
change in the weight (0.7875 g). Still, after 15 days we could not notice changes in the weight
(0.7880 g). These observations undoubtedly indicate that 3β-acetoxy cholest-5-ene has good
deliquescence resistance and is stable in air.
3
.9. Thermogravimetric analysis (TGA)
TGA was performed to know the purity and thermal stability of the present crystal. The TG
curve of crystal revealed that it is stable up to 280 °C and does not undergo any phase transition
Fig. S8. The disintegration process started after 280 °C and continued till complete
decomposition and at 450 °C, all the mass was lost and nothing was left as residue. Thus, no
weight loss or phase transition around or before its melting point was observed which confirmed
the nonexistence of any lattice entrapped solvent or moisture on the grown material.
3
.8. Powder XRD
The powder XRD pattern of 3β-acetoxy cholest-5-ene is shown in Fig. S9. The XRD profiles
showed that grown crystal was of single phase without detectable impurity. The appearances of
sharp and strong peaks confirmed the good crystallinity of the grown crystals. The lattice
2
7