The effect of hydrogen bond on the vibrational dynamics of genistein free and complexed with β-cyclodextrins

Article abstract of DOI:10.1002/jrs.2515

Inclusion, or host-guest, complexes are supramolecular assemblies in which two or more molecules hold together and organize by means of intermolecular noncovalent bonds. In the pharmaceutical field, inclusion complexation of drugs with unsubstituted and derivative β-cyclodextrins (β-CDs) has proven to be a successful method to improve the dissolution of water insoluble drugs. Genistein (Gen), an isoflavone constituent of Onodis spinosae radix, turned out to be a suitable guest molecule for the encapsulation into β-CD, resulting in a significant improvement of its aqueous solubility. In the present study, the modifications of the vibrational spectrum of Gen caused by its inclusion into β-CDs cavity have been characterized by means of Raman scattering experiments. These changes have been interpreted by comparing the experimental data with the vibrational wavenumbers and Raman intensities obtained by simulation for the free and complexed guest molecule. Following this strategy, we have obtained a deeper understanding of the host-guest interactions involved in the formation and stabilization of the complexes, with particular regard to the role played by the guest chemical groups, as well as to disentangle the effects directly related to the complexation process from those ascribed to other factors, such as formation of intra- and intermolecular hydrogen bonds.

Full text of DOI:10.1002/jrs.2515

Research Article
Received: 22 July 2009
Accepted: 26 August 2009
Published online in Wiley Interscience: 13 October 2009
(www.interscience.wiley.com) DOI 10.1002/jrs.2515
The effect of hydrogen bond on the vibrational
dynamics of genistein free and complexed
with β-cyclodextrins
Vincenza Crupi,a Domenico Majolino,a Alessandro Paciaroni,b
Barbara Rossi,c,d Rosanna Stancanelli,e Valentina Venutia∗
and Gabriele Vilianic,d
Inclusion, or host–guest, complexes are supramolecular assemblies in which two or more molecules hold together and
organize by means of intermolecular noncovalent bonds. In the pharmaceutical field, inclusion complexation of drugs with
unsubstituted and derivative β-cyclodextrins (β-CDs) has proven to be a successful method to improve the dissolution of water
insoluble drugs. Genistein (Gen), an isoflavone constituent of Onodis spinosae radix, turned out to be a suitable guest molecule
for the encapsulation into β-CD, resulting in a significant improvement of its aqueous solubility. In the present study, the
modifications of the vibrational spectrum of Gen caused by its inclusion into β-CDs cavity have been characterized by means of
Raman scattering experiments. These changes have been interpreted by comparing the experimental data with the vibrational
wavenumbers and Raman intensities obtained by simulation for the free and complexed guest molecule. Following this
strategy, we have obtained a deeper understanding of the host–guest interactions involved in the formation and stabilization
of the complexes, with particular regard to the role played by the guest chemical groups, as well as to disentangle the effects
directly related to the complexation process from those ascribed to other factors, such as formation of intra- and intermolecular
c
hydrogen bonds. Copyright ꢀ 2009 John Wiley & Sons, Ltd.
Keywords: inclusion complexes; Raman spectroscopy; normal mode analysis; DFT computation
Introduction
Cyclodextrins (CDs) are a group of structurally related macro-
cyclic oligosaccharides built up from 6, 7, or 8 glucopyranose
units called α, β, and γ -CD, respectively.[9] Their special molecular
structure, hydrophobic internal cavity, and hydrophilic external
surface give them the remarkable property of acting as molec-
ular hosts in the formation of inclusion complexes with various
guest molecules with suitable polarity and dimension via nonco-
valent host–guest interactions like Van der Waals, hydrophobic,
and hydrogen bonding.[10–12] The molecular encapsulation by
means of CDs has found extensive application in many fields,
including pharmaceutical technology, to improve the aqueous
solubility, dissolution rate, bioavailability, and stability of a vari-
ety of poorly water-soluble drugs.[13,14] The investigation of the
interactions between host and guest molecules is of interest in
view of the aforementioned technological applications, and is of
Genistein (5,7,4ꢁ-trihydroxyisoflavone, Gen, Fig. 1) is one of the
most known isoflavones. Isoflavones are found in a number
of plants, soybeans and soy products such as tofu and tex-
tured vegetable protein being the primary food sources. Gen
has been shown to interact with animal and human estrogen
receptors, causing effects in the body similar to those caused
by the hormone estrogen. It plays a relevant role in the treat-
ment and prevention of various cancers and other diseases, such
as cardiovascular ones and osteoporosis, among others.[1] Gen
protects against pro-inflammatory-factor-induced vascular en-
dothelialbarrierdysfunction, andinhibitsleukocyte–endothelium
interaction, thereby modulating vascular inflammation, which is
a major event in the pathogenesis of atherosclerosis.[2] It is a
potent tyrosine kynase inhibitor and also possesses antioxidant
activity.[3,4] Again, in vitro and animal studies proved that Gen
activity is involved in the inhibition of the cancerous cell growth.[5]
Gen makes some cells more sensitive to radiotherapy.[6] In addi-
tion, products containing Gen are now marketed as nutritional
supplements.
Nevertheless, Gen seems to have some disadvantages, which
considerably limit its potential clinical utility. These include
rapid in vivo metabolism and excretion, low serum level after
oral administration, and insufficient targeting of cancer cells. In
addition, Gen has rather poor water solubility. For nutraceutical
and pharmaceutical purposes, it needs to be solubilized to provide
sufficient concentration of available monomeric form of the
drug.[7,8]
∗
Correspondence to: Valentina Venuti, Department of Physics, University of
Messina, CNISM, UdRMessina, C.daPapardo, S.taSperone31, POBox55, 98166
S. Agata, Messina, Italy. E-mail: vvenuti@unime.it
a
Department of Physics, University of Messina, CNISM, Messina, Italy
b
DepartmentofPhysics,UniversityofPerugia,CEMINandINFMCRS-SOFT,06123
Perugia, Italy
c
d
e
Department of Physics, University of Trento, 38050 Povo, Trento, Italy
INFM CRS-SOFT, c/o University of Roma ‘La Sapienza’, 00185 Roma, Italy
Pharmaco-chemical Department, University of Messina, 98168 Messina, Italy
c
J. Raman Spectrosc. 2010, 41, 764–770
Copyright ꢀ 2009 John Wiley & Sons, Ltd.

The effect of hydrogen bond on the vibrational dynamics of genistein
≈290–300 ◦C), (2-hydroxypropyl)-β-cyclodextrin (HP-β-CD, FW
≈1170.0, mp≈278 ◦C, degreeofsubstitution≈0.6), andmethyl-β-
cyclodextrin (Me-β-CD, FW ≈1310.0, degree of substitution
≈1.7–1.9), were purchased from Fluka Chemie (Switzerland). They
were employed without any further purification.
Preparation of the solid inclusion complex
Inclusion complexes with 1 : 1 Gen/β-CD, Gen/HP-β-CD, and
Gen/Me-β-CD molar ratio were prepared by co-precipitation
method, by shaking the appropriate amounts of the drug and
macrocycle in water at 60 ◦C. The water used throughout the study
was deionized and double-distilled, then filtered trough 0.22-µm
Millipore GSWP filters (Bedford, MA, USA). Each mixture was stirred
for 1 h under controlled temperature (25.0 0.01 ◦C) using a
Telesystem stirring bath thermostat 15.40 with a Telemodul 40 ◦C
control unit which allowed an accuracy of 0.01 ◦C. The reaction
mixture was cooled in a refrigerator (4 ◦C) for about 24 h, and
the water was subsequently evaporated under vacuum (∼30 ◦C),
obtaining powders of Gen/β-CD, Gen/HP-β-CD, and Gen/Me-β-
CD inclusion complexes. The uncomplexed drug was filtered, prior
to solution evaporation, through Sartorius Minisart-SRP 15 PTFE
0.45-µm filters.
Figure 1. Chemical structure of Gen. The labeling of the atoms is the one
adopted for the ab initio simulations.
great concern also from a general point of view as a prototype, for
example, of the enzyme–substrate interaction mechanism.[15,16]
In a previous study,[17] we investigated, in pure water, the effect
of complexation of unsubstituted and derivative β-CDs on the UV
absorption of Gen. A phase-solubility study allowed us to evaluate
the changes of isoflavone in the complexation state, suggesting
that it forms complexes with a stoichiometry of 1 : 1. Then, Fourier
transforminfraredspectroscopyintheattenuatedtotalreflectance
geometry (FTIR-ATR) has been successfully used to put into evi-
dence the host–guest interactions at the molecular level in solid
phase. Recently, based on molecular modeling investigations and
on the results of phase-solubility studies, a correlation between
the size of the CD cavity and the inclusion of Gen was found.[18]
Two-dimensional rotating-frame nuclear Overhauser effect spec-
troscopy experiments gave a clear explanation about the interact-
ing moieties. In particular, the 4-hydroxyphenyl ring penetrated
fromthewiderrimsideintotheβ-CDcavity. Thein vitrodissolution
of Gen entrapped in β-CD appeared to be definitely enhanced
withrespecttothatof theplainisoflavone. Finally, veryrecently, by
meansofanelasticincoherentneutronscatteringinvestigation,[19]
the specific host–guest interactions involved in the formation of
the Gen/β-CD inclusion complex in solid state were found to hin-
der its average dynamics in the picosecond timescale with respect
to the physical mixture, a blending of guest and host molecules
without complexation. In the same study, the improvement of the
thermal stability as a consequence of complexation has been hy-
pothesized by the observed increase of the rigidity of the system,
as quantified by the dynamical behavior versus temperature.
In the present work, we report on the results of a Raman
investigation aimed at getting deeper information on the
Raman measurements
Raman spectra of Gen and Gen/β-CD, Gen/HP-β-CD, and
Gen/Me-β-CD complexes were recorded on dried samples de-
posited on a glass slide, in air, by means of a microprobe setup
(Horiba-Jobin-Yvon, model Labram HR800), consisting of a He–Ne
laser, a narrow-band notch filter, an 80-cm focal length spectro-
graph using a 1800 grooves/mm grating, and a charge-coupled
device (CCD) detector with a red-extended response, cryogeni-
cally cooled by liquid nitrogen. Exciting radiation at 632.8 nm was
focused onto the sample surface with a spot size of about 1 µm2
through a 100× objective with NA = 0.9. To avoid unwanted
laser-induced transformations, neutral filters of different optical
densities were used, whenever necessary. Spectra were collected
in the wavenumber ranges 100–3500 cm−1. The resolution was
about 0.3 cm−1/pixel.
For the Raman spectra acquired in the wavenumber range
between
5 , a triple-monochromator spec-
and 100 cm−1
host–guest interactions involved in the formation of Gen/β-
CDs inclusion complex in solid phase. The comparison of
the Raman spectra of free molecule and inclusion complexes
evidenced significant wavenumber shifts, intensity variations, and
broadening as a consequence of complexation. These changes
have been interpreted on the basis of a vibrational analysis
performed by both ab initio quantum chemical calculations
and molecular dynamics, thereby relating such changes to the
geometry of the complex, disentangling single-molecule effects
from other factors, such as dimerization processes of the guest,
according to a reliable methodology already successfully applied
in the case of indomethacin[20,21] and ibuprofen.[22]
trometer (Horiba-Jobin-Yvon, model T64000) set in double-
subtractive/single configuration and equipped with 1800
grooves/mm grating was used. Micro-Raman spectra were excited
by the 514.5-nm wavelength of a argon/krypton ion laser and de-
tectedbyaCCDdetector. Theresolutionwasabout0.6 cm−1/pixel.
Ab initio quantum chemical calculations
QuantumchemicalcalculationsonthemoleculeofGen(monomer
and dimer) were performed using the GAUSSIAN 03 program
suite,[23] using unrestricted density functional theory (DFT);[24]
the non-local B3LYP functional hybrid method was employed.[25]
The standard 6-31G(d) basis set[26] was used for the geometry
optimizationandwavenumberanalysis. Fortheplotsoftheoretical
Raman and Infrared spectra (this latter used for comparison with
the experimental FTIR spectrum of uncomplexed Gen as obtained
in a previous study[17] and here reported), a Lorentzian lineshape
with a line width of 4.0 cm−1 was used.
Materials and Methods
Samples
The isoflavone Gen (C15H10O5, FW ≈270.24) was purchased
from Sigma Aldrich Chemie (USA). β-CD (FW ≈1135.0, mp
c
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V. Crupi et al.
(a)
(a)
(b)
(b)
(c)
(d)
1550
1600
1650
Wavenumber/cm-1
1700
Figure 2. Chemical structure of simulated dimeric structures of Gen: dim1
(a) and dim2 (b).
Figure 4. Experimental Raman spectrum (1550–1730 cm−1 range) of
uncomplexed Gen (a), and computed Raman spectra of dim1 (b), dim2 (c),
and intrabonded single Gen molecule (d).
(a)
(a)
(b)
(c)
(b)
(d)
1500 1550 1600 1650 1700 1750
Wavenumber/cm-1
Figure 5. Experimental FTIR-ATR spectrum (1465–1770 cm−1 range) of
uncomplexed Gen (a), and computed IR spectra of dim1 (b), dim2 (c), and
intrabonded single Gen molecule (d).
Figure 3. Chemical structure of simulated Gen molecule with (a) and
without (b) H-bond.
We focused our attention on the Raman spectrum of uncom-
plexed Gen in the wavenumber range 1500–1800 cm−1. The
choice of this range is justified by the fact that, when using
Raman spectroscopy, no free β-CD peaks are present, so that
complexation-induced changes in the Gen spectrum can be easily
revealed.[20–22]
Figure 4(a) reports the experimental Raman spectrum of
uncomplexed Gen. As can be seen, six characteristic peaks are
clearly distinguishable in the experimental spectrum, centred at
Molecular dynamics simulations
The complexation process of Gen with β-CD was investigated by
classical molecular dynamics simulations, by using the PCMODEL
8.0 package (Serena Software, Bloomington, IN, USA).[27] The
force field employed is Molecular Mechanics MMX.[28] The starting
structure for the molecular dynamics simulation of β-CD was
obtained from the Protein Data Bank.[29] The Gen structure was
set up by GaussView 3.0.
∼1569, ∼1584, ∼1590, ∼1615, ∼1618, and ∼1647 cm−1
.
In Figs 4 and 5 we compare the experimental Raman and
FTIR-ATR spectra, respectively, of uncomplexed Gen (the latter
as obtained in Ref. [17]), with those computed for the two
investigated dimeric structures and for a single Gen molecule
Results and Discussion
Quantumchemicalcalculationswereperformedontwotypologies with intramolecular H-bond.
of dimeric structures of Gen, differently oriented, labeled dim1 and
We observe that for both Raman and FTIR-ATR spectra in
dim2, both of them having intra- and intermolecular hydrogen the investigated range, the spectra computed for dimeric and
bond (Fig. 2(a) and (b), respectively), and on a single molecule of single intrabonded Gen are almost identical. A comparison with
Gen with and without intramolecular hydrogen bond involving the experimental spectrum allowed us to tentatively assign the
C
O and O–H groups (Fig. 3(a) and (b), respectively).
characteristic peaks of uncomplexed Gen in this zone, but did
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The effect of hydrogen bond on the vibrational dynamics of genistein
(a)
(b)
(a)
(c)
(b)
(c)
(d)
(e)
(f)
(d)
12
24
36
48
60
72
84
0
50
100
150
i (1,3N)
Wavenumber/cm-1
Figure 7. Normalized displacements ri as a function of the atomic degree
of freedom i for each atom of Gen under the effect of the normal modes
at ∼1567 cm−1 (a), ∼1630 cm−1 (b), ∼1652 cm−1 (c), ∼1673 cm−1 (d),
∼1675 cm−1 (e), and ∼1713 cm−1 (f).
Figure 6. Experimental Raman spectrum (0–150 cm−1 range) of uncom-
plexed Gen (a), and computed Raman spectra of dim1 (b), dim2 (c), and
intrabonded single Gen molecule (d).
Table 1. Experimental and computed wavenumbers (in cm−1) for
uncomplexed Gen
Experimental
Computed
System
wavenumber (cm−1
)
wavenumber (cm−1
)
(a)
(b)
1550
1650
1750
Wavenumber/cm-1
1569
1584
1590
1615
1618
1647
1567
1630
1652
1673
1675
1713
Uncomplexed Gen
(c)
(d)
1500
1550
1600
1650
1700
1750
Wavenumber/cm-1
not allow a distinction between dimeric and monomeric (with
intramolecular H-bond) structures, since these vibrations are
practicallydegenerate.Inordertoclarifythispoint,weanalyzedthe
low-wavenumber region of the Raman spectrum of uncomplexed
Geninthe5–150 cm−1 range, andcompareditwiththecomputed
spectra of the two Gen dimers and of the single intrabonded Gen
molecule, see Fig. 6.
Figure 8. Experimental Raman spectrum (1550–1775 cm−1 range) of
uncomplexed Gen (a), Gen/β-CD (b), Gen/HP-β-CD (c), and Gen/Me-β-
CD (d) inclusion complexes. In the inset, the computed Raman spectrum
for a single Gen molecule without intramolecular H-bond is reported.
scaling factors, we prefer to report the uncorrected values, since
the choice of these factors is somewhat arbitrary.
Relevant differences emerge in this range among the computed
spectra, especially between the two dimers (which are very
similar to each other) and the single Gen. For both dimeric
structures we observe a very low wavenumber peak, centered
at about 10–13 cm−1, which, from the analysis of the normal
modes, has been assigned to the bending of the hydrogen bond
connecting the two Gen molecules. This peak is obviously not
present in the computed spectrum of the single molecule of
Gen. Therefore, comparison between computed and experimental
low-wavenumber Raman spectra suggests the coexistence in
uncomplexed Gen of single and dimeric forms, intra- and
intermolecularly hydrogen-bonded, respectively.
On the basis of these considerations and of the comparison
between experimental and computed Raman wavenumbers and
intensities, we make the assignments reported in Table 1.
A small shift to higher wavenumbers of the computed modes
is generally observed in Table 1, but it is thought to derive
from anharmonicity effects and from the general tendency of
quantumchemicalmethodstooverestimatetheforceconstantsof
bonding among lightatoms in the equilibrium configuration. Even
though the difference between the experimental and calculated
vibrational wavenumbers can be minimized by using suitable
In Fig. 7 we report the normalized displacements ri for each
atom of Gen, labeled as in Fig. 1, under the effect of the normal
modes computed at ∼1567 (a), ∼1630 (b), ∼1652 (c), ∼1673
(d), ∼1675 (e), and ∼1713 cm−1 (f). On the x-axis, i = 1, 2, 3 . . .
correspond to the displacements x1, y1, z1, respectively, and so on.
From the analysis of the normal modes reported in the figure, it
turns out that the vibration falling at ∼1567 cm−1 predominantly
involves the hydrogens and carbons of the Gen phenolic ring
(atoms 23–30 and 11–16, i = 67–90 and i = 31–48, respectively).
The modes computed at ∼1630 and ∼1652 cm−1 are mainly
localized on the atoms of the O(22)–H(23) group, on carbons 9
and 10, and on hydrogens 20, 21, 25, 26. The modes at ∼1673
and ∼1675 cm−1 are mainly attributable to vibrations involving
the C and H atoms of the phenolic and condensed rings of Gen
(atoms 1–16, atom 20, atoms 23–30), while the computed mode
at ∼1713 cm−1 corresponds to a vibration mainly localized on the
O(22)–H(23) group of condensed rings and on hydrogen 25.
In Fig. 8 we compare the experimental Raman spectrum of
uncomplexed Gen with that of the Gen/β-CD, Gen/HP-β-CD, and
Gen/Me-β-CD inclusion complexes.
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V. Crupi et al.
As can be seen, the Raman bands of the inclusion complexes
generally appear slightly broader and shifted with respect to the
corresponding bands of uncomplexed Gen. These features reveal
a decrease of the vibrational relaxation time of the corresponding
vibration (broadening) and a change in the strength of the
bonds among the atoms involved in that vibration (shift), and
are indicative of the existence of host–guest interactions.
In the inset of the same figure we report the computed Raman
spectrum for a single Gen molecule for which the formation of
intramolecular hydrogen bond was prevented in the simulation
procedure.
Asgeneralresult,weobservethatthepeakat∼1738 cm−1 inthe
inset has a higher wavenumber with respect to the corresponding
one in the computed spectrum of intrabonded Gen (Fig. 4(d))
revealed at ∼1713 cm−1. Again, an overall high wavenumber shift
of the peaks of the Raman spectra of the inclusion complexes
is revealed with respect to uncomplexed Gen. This allows us
to hypothesize that, as a consequence of complexation, the
intramolecular H-bond between the C O group and the nearest
OH group is broken, thereby ‘releasing’ the C O group that can
vibrate at a higher wavenumber (normal mode at ∼1738 cm−1
in the computed Raman spectrum reported in the inset). This is
experimentally confirmed by the high wavenumber shift revealed,
upon complexation, for the C O stretching peak, which from
∼1647 cm−1 in the experimental spectrum of uncomplexed
Gen moves to ∼1664 cm−1 for Gen/β-CD, to ∼1664 cm−1 for
Gen/HP-β-CD, and to ∼1664 cm−1 for Gen/Me-β-CD. This result
is in agreement with many other studies on a variety of inclusion
complexes with β-CDs,[30–32] where this high wavenumber shift,
indicative of a strengthening of the carbonyl radical double
bond, has been associated to the breakdown of intra- and/or
intermolecular hydrogen bonding and the establishment of less
intense associations, i.e. a monomeric dispersion of the drug
within the hydrophobic cavity of β-CDs, and relative interaction
with primary or secondary OH groups and/or with interstitial and
intracavity crystallization water molecules.
and correspond to vibrations involving the atoms of the phenolic
ring (computed mode at ∼1673 cm−1) and of the condensed rings
(computed modes at ∼1675 and 1681 cm−1), confirming that the
inclusion of Gen in the β-CD cavity induces the breakdown of the
intramolecular H-bond between C O group and the nearest OH
group.
Other relevant spectral changes have been revealed in the
region below 1500 cm−1 but they are difficult to interpret since
that range contains also characteristic bands of pure β-CDs, and
for this reason they not discussed here.
As already mentioned, the inclusion of Gen into β-CD, HP-β-CD,
andMe-β-CDhasalreadybeenobservedbyFTIR-ATRspectroscopy
by investigating the 400–4000 cm−1 range.[17] This reveals for all
these host molecules a similar basic complexation mechanism
as for Gen. This general result is confirmed by the present data.
In Ref. [17], the O–H stretching region (3000–3800 cm−1) was
analyzed in detail, and differences in the degree of association via
H-bonding during the complexation process were postulated to
explain the spectral modifications revealed upon complexation.
In addition, the present data provide new insight into the
understanding of the host–guest interactions of Gen/β-CD
inclusion complexes by a vibrational analysis that explores
experimentalregionspreviouslynotinvestigated(5–150 cm−1),or
where (1500–1800 cm−1) in any case FTIR spectroscopy becomes
less significant for the structural characterization of the inclusion
compounds, since the bands of β-CDs cover a large part of the
spectrum.
The complexation process of Gen and β-CD was also in-
vestigated by classical molecular dynamics simulation, thereby
obtaining information on the geometrical arrangement of the
guest molecule in the cavity of the host. A starting system consti-
tuted by one molecule of Gen without intramolecular hydrogen
bond and one molecule of β-CD in vacuum was allowed to evolve
at room temperature. Ten different starting systems were built,
assigning a different initial conformation of Gen with respect to
β-CD in each of them. During the molecular dynamics runs, we
observed that all the starting systems resulted in a complexation
event. A tight-fit inclusion complex between Gen and β-CD was
in a sense expected, considering that the molecular dimensions
of isoflavones are approximately in the 0.5–1.5 nm range, thereby
having a good correlation with the typical size of the central cavity
of β-CD. By analysing the complexes resulting from the ten differ-
ent initial configurations, we verified that the structure in Fig. 9 is
the favoured one.
Again we notice that, when Gen is entrapped in the analysed
macrocycles, the characteristic peak of pure Gen at ∼1569 cm−1
,
assigned to a vibration involving the carbon and hydrogen atoms
of phenolic ring, is not revealed in the spectrum of Gen/β-CD
complex, whereas it is still present as shoulder at ∼1576 cm−1
in Gen/HP-β-CD complex and at ∼1576 cm−1 in Gen/Me-β-
CD complex. The diminishing of the band intensity indicates a
decreaseandhinderingofthisvibration,whereastheshifttohigher
wavenumber is due to the strengthening of the corresponding
bond. Both these spectral changes can be justified considering a
restriction of these groups within the β-CDs cavities.
The complex geometry, with the whole aromatic ring in the
β-CD cavity with a mode of penetration from the wider rim side,
appears in agreement with that proposed by other authors.[18]
From the classical simulation, it seems that the structure of the
complex is stabilized by the presence of H-bonds involving the
phenolic OH and the C O groups of Gen and some OH groups of
β-CD (Fig. 9).
This scenario is consistent with the changes induced on the
variousH-bondedenvironmentsduringthecomplexationprocess,
as revealed by FTIR-ATR spectroscopy,[17] which suggested a
redistribution of water molecules of crystallization (interstitial
and intracavity) of β-CD among the different hydrogen bonds
sites, due to the formation of H-bonds between Gen and β-CD.
Further, the peak at ∼1584 cm−1 in uncomplexed Gen does not
suffer any wavenumber shift as a consequence of complexation.
On the other hand, the peak at ∼1590 cm−1 slightly shifts to
higher wavenumbers for all analyzed complexes: in particular, it
shifts to ∼1592 cm−1 for Gen/β-CD system, to ∼1592 cm−1 for
Gen/HP-β-CD, and to ∼1591 cm−1 for Gen/Me-β-CD.
Finally, clearly observable effects in all the investigated
complexes concern the appearance in the experimental spectral
window between 1600 and 1630 cm−1 three distinct peaks
at ∼1606, ∼1615, and ∼1626 cm−1 for Gen/β-CD; at ∼1606,
∼1615, and ∼1626 cm−1 for Gen/HP-β-CD; and at ∼1606, ∼1615,
and ∼1625 cm−1 for Gen/Me-β-CD inclusion complex. These
experimental peaks are well reproduced from the computed
Raman spectrum of a single Gen molecule for which the formation
of intramolecular hydrogen bond is prevented (see inset in Fig. 8)
Conclusion
Raman spectroscopy and numerical simulations have been used
to investigate the interactions at the molecular level driving the
c
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Copyright ꢀ 2009 John Wiley & Sons, Ltd.
J. Raman Spectrosc. 2010, 41, 764–770

The effect of hydrogen bond on the vibrational dynamics of genistein
a successful methodology for the elucidation of host–guest
interactions driving the formation of supramolecular assemblies
in the solid phase.
Acknowledgements
We would like to thank A. Defant and G. Guella for useful
discussions, G. Mariotto for making micro-Raman apparatus
available, and PAT (Provincia Autonoma di Trento) for financial
support.
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Figure 9. Structure of the Gen/β-CD inclusion complex, resulting from the
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in agreement with a previous FTIR-ATR study on the same
systems.
As a main result, a large high wavenumber shift (∼17 cm−1
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agreement with the rearrangement of the H-bond environments
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presence of H-bonds involving the phenolic OH and the C
O
groups of Gen and some OH groups of β-CD.
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