Computational and Theoretical Chemistry 1217 (2022) 113891

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Computational and Theoretical Chemistry

journal homepage: www.elsevier.com/locate/comptc

Quantum chemical calculations of IR spectra of heparin

disaccharide subunits
Yulia B. Monakhova a, b, c, *, Polina M. Soboleva a, Elena S. Fedotova a, Kristina T. Musina a,
Natalia A. Burmistrova a
a
Institute of Chemistry, Saratov State University, Astrakhanskaya Street 83, 410012 Saratov, Russia
b
Spectral Service AG, Emil-Hoffmann-Straße 33, 50996 Cologne, Germany
c
University of Applied Sciences Aachen, Faculty of Chemistry and Biotechnology, Heinrich-Mußmann-Straße 1, 52428 Jülich, Deutschland

A R T I C L E I N F O A B S T R A C T

Keywords: Heparin is a natural polysaccharide, which plays essential role in many biological processes. Alterations in
IR spectroscopy building blocks can modify biological roles of commercial heparin products, due to significant changes in the
Heparin conformation of the polymer chain. The variability structure of heparin leads to difficulty in quality control using
Quality control
different analytical methods, including infrared (IR) spectroscopy. In this paper molecular modelling of heparin
Molecular modelling
disaccharide subunits was performed using quantum chemistry. The structural and spectral parameters of these
Quantum chemistry
Chemometrics disaccharides have been calculated using RHF/6-311G. In addition, over-sulphated chondroitin sulphate disac­
charide was studied as one of the most widespread contaminants of heparin. Calculated IR spectra were analyzed
with respect to specific structure parameters. IR spectroscopic fingerprint was found to be sensitive to substi­
tution pattern of disaccharide subunits. Vibrational assignments of calculated spectra were correlated with
experimental IR spectral bands of native heparin. Chemometrics was used to perform multivariate analysis of
simulated spectral data.

1. Introduction a role in inflammation, cell growth, angiogenesis, differentiation and

Due to its vitally important properties, such as anticoagulant activity,
heparin (HEP) has been widely used in clinical practice for many years
[1]. HEP is a natural polysaccharide that belongs to glycosaminoglycans
(GAG) and has a structural order with up to four levels of complexity
[2,3]. The structure of HEP is variable and depends both on the animal
origin and on the tissue it is extracted from. Considering the natural
sources of HEP, the numbers of potential contaminants are relatively
large compared to synthetic substances [4]. Moreover, HEP can include
structurally related contaminants that affect the quality of finished
product. It is known, that structural analogues of HEP do not exhibit any
anticoagulant activities typical for HEP caused by a pentasaccharide
sequence containing a glucosamine residue sulphated at position 3 [5].
In this regard, it has been found in 2007–8 that the presence of over-
sulphated chondroitin sulphate (OSCS) in pharmaceutical HEP results
in severe adverse health effects [6,7].
Besides the well-known anticoagulant role of heparin, GAG also play
invasion [8]. There is increasing evidence of their involvement in a wide
range of pathologies as disaccharides. Changes in GAG disaccharide
composition in pancreatic carcinoma or in human chondrodysplasia are
some of such examples [9–12]. Alterations in sulphation pattern may
well reflect modified biological roles of these molecules, because
different sulphation of the basic disaccharide subunits may significantly
affect the conformation of the polymer chain. On the other hand,
changes of important physicochemical properties (e.g. concentration,
pH, temperature, and the presence of cations) do not have any signifi­
cant effect on the secondary structure [13]. In this regard, reliable
methods of structural analysis of HEP in terms of sulphation pattern of
disaccharide subunits are critically needed.
Generally, characterization strategies for heparin can be divided into
analysis without any chemical or enzymatic pretreatment (e.g. size
exclusion chromatography, liquid chromatography-mass spectrometry
(LC-MS), MS-MS or NMR) and analysis based on controlled depoly­
merization of polysaccharide chains (e.g. high-performance liquid

* Corresponding author at: University of Applied Sciences Aachen, Faculty of Chemistry and Biotechnology, Heinrich-Mußmann-Straße 1, 52428 Jülich,
Deutschland.
E-mail address: monakhova@fh-aachen.de (Y.B. Monakhova).

https://doi.org/10.1016/j.comptc.2022.113891
Received 2 June 2022; Received in revised form 13 September 2022; Accepted 21 September 2022
Available online 25 September 2022
2210-271X/© 2022 Elsevier B.V. All rights reserved.
Y.B. Monakhova et al.
chromatography (HPLC), capillary electrophoresis (CE) and LC-MS
disaccharide compositional analysis or oligosaccharide sequence anal­
ysis) [14]. Obviously, single analytical methods such as NMR, HPLC-MS
etc. are high-quality analytical techniques, however, researchers
encounter some difficulties during screening stage because data coming
from these instruments is very complex and difficult to interpret.
Simpler alternative as IR spectroscopy also suitable for quality con­
trol of heparin due to chemometrical calculations that improve the ef­
ficiency of instrumental methods [3,15]. Although IR spectrum of HEP is
well-known, the majority of studies describing quality control of HEP
did not completely assign all IR bands to corresponding structural fea­
tures [2,16,17]. Often vibrational assignment is performed based on
features arising from the higher order structure. Therefore, the differ­
ences in spectral characteristics and reasons for classifying a sample as
pure or contaminated are not always clear. To find structural features
related to variability of IR spectra it is necessary to compare the spectra
to structure of the HEP building blocks. Disaccharide compositional
analysis by IR spectroscopy remains a challenge due to the need the
detection of relatively subtle structural features (e.g. sulpho group po­
sition or differences in linkage position), so theoretical study of struc­
tural and spectral characteristics is of great importance to improve the
quality of interpretation of spectral data.
Currently, there are some papers dedicated to theoretical de­
scriptions of the major disaccharide sequences of HEP and its structural
analogs [4]. Quantum chemical calculations have been successfully used
for modelling of GAGs fragments and were effective for evaluation of
their properties. The approaches for simulation of HEP and other
sulphated GAGs were based mostly on Hartree–Fock (HF) ab initio and
B3LYP Density Functional Theory (DFT) methods (using Pople basis sets
6-31G*, 6–31 + G* and 6–311 + G**) [18]. For example, M. Hricovíni
[19] used DFT to make a detailed study of the molecular structure and
spin–spin coupling constants of HEP tetrasaccharides representing the
predominant HEP repeating-sequence, while M. Remko et al. [13] used
HF/6–31G(d), B3LYP/6–31G(d), and B3LYP/6–311 + G(d, p) methods
to study the acidic dimeric structural units and the pentamer of HEP, as
well as their anionic form. The molecular structure of trimeric units and
the pentamer of HEP (including their anionic forms and sodium salts)
was also studied by B3LYP/6-31G(d) method [20], while authors of
[21–23] used DFT modelling to explain the sulphate patterns in GAGs
(mono-, di-, and oligosaccharides) and found strong correlations be­
tween spectral and structural characteristics found via cryogenic
infrared spectroscopy and InfraRed Multiple Photon Dissociation
(IRMPD). It should be mentioned that previous works were mainly
aimed directly at investigating the structure of various GAG. At the same
time, the molecular structure is responsible for the unique vibrational
spectral characteristics of HEP. Moreover, all levels of a HEP structure
can and do affect spectral and activity data [3]. According to our liter­
ature search, spectral characteristics of HEP were previously determined
only in several studies [19,23,24] that focused on laborious instru­
mental techniques.
In the present study we were focused on theoretical calculation and
studying of IR spectral characteristics of twelve HEP and OSCS disac­
charide subunits with different sulphation. Investigated structures were
optimized by the restricted Hartree-Fock (RHF) method with the 6-311G
basis set [25]. HOMO and LUMO energies, the ionization energy, the
absolute hardness and dipole moment were calculated and discussed. In
addition, we thoroughly investigated the normal vibration frequencies
and intensities of bands in the IR spectra of studied compounds. Vibra­
tional assignments were performed to characterize and clearly describe
IR bands. In addition, we performed multivariate analysis of calculated
IR spectra using principal component analysis (PCA) method that
allowed us to evaluate the inherent similarities and differences. It ap­
pears that substitution pattern of the HEP disaccharides causes well-
defined changes of IR spectral profile.
2
Computational and Theoretical Chemistry 1217 (2022) 113891
2. Methods and computational details
Quantum chemical calculations were performed using the Firefly QC
package [26], which is partially based on the GAMESS (US) [27] source
code using the Saratov State University cluster. The geometry of HEP
and OSCS disaccharide subunits were optimized using the RHF/6-311G.
The basis set was chosen according to the required calculation accuracy.
Visualization of obtained results was carried out using the ChemCraft v.
1.8 graphical software for visualization of quantum chemistry compu­
tations (https://www.chemcraftprog.com).
Chemometrics Add-in for Microsoft Excel MS was used for chemo­
metric modelling by PCA as described in [28]. PCA is multivariate
decomposition method, whose idea is to reduce the dimensionality of a
data set consisting of a large number of interrelated variables, while
retaining as much as possible of the variation present in the data set. This
is achieved by transforming to a new set of variables (principle com­
ponents), which are uncorrelated and ordered so that the first few
components retain most of the variation in all the original variables
[29]. The rows of a data matrix X correspond to samples while columns
correspond to variables. PCA decomposes the data matrix X as the sum
of the outer product of vectors score t (i.e. contain information regarding
the interaction of the samples to each other) and loading p (i.e. eigen­
vectors of the covariance matrix) plus a residual matrix E.
The PCA model was performed on calculated IR spectral data of HEP
disaccharides in 2000–900 cm− 1 range. IR spectral data with Gaussian
broadening were exported and organized in the form of a matrix for the
chemometric data processing. The order of resulting matrix was 12 ×
291.
We chose porcine HEP powder as an example of native HEP for
experimental IR spectrum. The sample was supplied by Spectral Ser­
vice’s quality control laboratory (Cologne, Germany). IR measurements
were performed on IRAffinity-1S FTIR spectrometer (Shimadzu, Kyōto,
Japan) in the spectral region of 4000–400 cm− 1 using KBr pellet. The
spectrum was obtained by averaging 20 scans at a resolution of 2 cm− 1.
For sample preparation, 3 mg of a HEP sample was mixed with 297 mg of
KBr, and sample was evacuated for 15 min and pressed under vacuum on
a laboratory press PGR-400 (Monitoring LLC, Russia).
3. Results and discussion
3.1. Geometric and energetic parameters of HEP and OSCS disaccharide
subunits
The structure of HEP has increasing levels of complexity starting
from monosaccharide composition to higher orders of structure [3]. In
this work, disaccharides were chosen as our primary object of structural
theoretical analysis. HEP is usually considered a mixture of variously
sulphated linear polysaccharides consisting of 1–4 linked uronic acid
and D-glucosamine [3,30]; while OSCS consists of 1,3-linked glucuronic
acid and N-acetylgalactosamine residues containing four sulphate
groups per building block [3]. The disaccharides composition of the
underlying structure of HEP is known to be different for various animal
species. Typically, about 70 % of constituent disaccharides of native HEP
are trisulphated [3,4]. The flexibility of the pyranose rings and their
ability to rotate around glycosidic linkage regions result in existence of
several HEP conformations that depend both on the structure of residue
and on the sulphation degree [3,31]. In this regard, we optimized the
geometry of twelve disaccharide subunits of HEP and OSCS (Fig. 1,
Fig. S1, Table 1). Using the RHF/6-311G method, the obtained struc­
tures did not have any negative values in the Hessian matrix, thus,
confirming the attribution of optimized structures to corresponding
conformers. We have observed that the β-glycosidic linkage between
monosaccharides has been formed. It was found that the bond angle
between the sulphated residues of iduronic acid and glucosamine in HEP
is almost independent of the substituents position, while the main
changes in geometry are associated with the values of torsion angles
Y.B. Monakhova et al.
Fig. 1. Repeating disaccharide subunits of HEP (A) and OSCS (B) (R1 = H, SO−3 ; R2 = H, SO−3 ; R3 = H, acetyl (Ac), SO−3 ); optimized structure of HEP disaccharide
6 (C).
Table 1
Substituents of disaccharide subunits of HEP.
1 2 3 4 5 6
R1 H H H H SO3H
R2 H H SO3H SO3H SO3H
R3 H SO3H H SO3H H SO3H
(Fig. 2, Table S1).
Comparison of HEP disaccharide subunits geometry has shown that
they can be divided into three groups based on the values of the torsion
angles: disaccharides 1, 2, and 9 (group I), disaccharides 3, 4, 5, 6, 10
and 11 (group II) and disaccharides 7, 8 and 12 (group III). Group I
contain disaccharides with H atoms as R1 and R2, group II has SO3H
group as R2, and group III has SO3H group as R1 and H atoms as R2. R2
has the strongest influence on the values of torsion angles; the influence
of R3 is insignificant. In addition, when the R2 is changed from -SO3H
group to H atom, the influence of R1 increases. The values of torsion
angles for OSCS and HEP disaccharide subunits is significantly different
(Fig. 2, Table S1); visual comparison between trisulphated HEP and
OSCS disaccharides is shown in Fig. 3.
Calculated parameters, such as HOMO and LUMO energies, the
ionization energy, the absolute hardness and dipole moment of the
studied disaccharides are summarized in Table S2. Comparison of
electronic characteristics shows that disaccharides 3, 11 and 13 has the
least absolute hardness and the largest reactivity; the opposite was
observed for disaccharides 2, 4, 6 and 8. HEP disaccharide containing H
atoms as R1 and R2 acts as the best electron donor, while HEP disac­
charide with three -SO3H groups is the worst electron donor. Di­
saccharides 3, 5, 7 and 13 have the largest values of dipole moment,
while disaccharides 2 and 12 have the least values of dipole moment.
Representative UV–vis spectrum of native HEP are shown in Fig. S2
[32].
Computational and Theoretical Chemistry 1217 (2022) 113891
7 8 9 10 11 12
SO3H SO3H SO3H H H SO3H SO3H
SO3H H H H SO3H SO3H H
H SO3H Ac Ac Ac Ac
3.2. IR spectral analysis of HEP and OSCS disaccharides subunits
Calculated IR spectra of HEP and OSCS disaccharide subunits are
presented in Fig. 4 and Table S3. The primary IR spectral bands of HEP
were attributed to absorbance of N-sulfonate groups, N-acetyl groups
and iduronate residues rings [33,34]. IR spectroscopic profiles and as­
signments of absorption bands of native HEP samples have already been
described previously [15]. The main difficulty in analysis of IR spectrum
of native HEP lies in variability of analytical signal due to the structure
complexity. Besides, IR spectroscopic bands are broad and usually
overlap, so differences in spectral profiles of samples is difficult to
associate with vibrations of certain groups. Here, we compared IR
spectra of HEP and OSCS disaccharide subunits computed via RHF/6-
311G method with the experimental IR spectrum of native HEP
(Fig. 4, Table S3). Before that, both the computed and the corresponding
experimental spectra were normalized to the intensity of the strongest
band.
Calculated frequencies of HEP disaccharide subunits were found to
be quite similar to the frequencies in the experimental spectra of native
HEP. The only exception were asymmetrical stretching vibrations of
–COOH group where frequencies differed by more than 200 cm− 1. Di­
saccharides forming the backbone of HEP have similar structure, how­
ever, various substitution patterns affect their IR spectral profiles in
different ways (Fig. 4, Table S3). The band corresponding to –COOH
asymmetrical stretching at around 1900 cm− 1 is very similar for all
calculated disaccharides. This resemblance in terms of the peak position
and intensity without any significant effect from various substituents is
observed for all studied structures. N-acetylated HEP has changes in the
3
Y.B. Monakhova et al.
Fig. 2. Torsion angles deviation for calculated HEP and OSCS disaccharide subunits.
Fig. 3. Arrangement of atoms of trisulphated HEP (blue) and OSCS (violet)
disaccharides in a three-dimensional space. (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of
this article.)
IR spectral profile associated with amide group vibrations at around
1850, 1650, 1570 and 1490 cm− 1. Moreover, changes in substitution
pattern lead to changes in spectral profiles associated with non-specific
vibrations in the ring and vibrations of the –COH side groups and the
glycosidic bonds. Sulphated disaccharides show more complex IR profile
in the range between 1300 and 1000 cm− 1. In addition, the presence of
4
Computational and Theoretical Chemistry 1217 (2022) 113891
two -SO3H groups leads to a decrease in resolution for this spectral
range. The IR spectrum of OSCS disaccharide is more resolved.
Calculated and experimental IR spectra were analyzed via chemo­
metric (PCA) modelling to find any inherent similarities and differences.
All HEP disaccharides can be projected onto the first four principal
components (PCs), which described 92.67 % of the total variance (with
39.30 % described by the PC1, 26.31 % by the PC2, 14.38 % by the PC3,
and 12.68 % by the PC4). The PCA loadings were in accordance with the
main differences in IR spectra (Fig. S3). The PCA model showed that
samples formed four isolated clusters in the PC1-PC2-PC4 space
(Fig. S3). HEP disaccharides from Group I and Group III had the negative
score values along PC2, that can be attributed to their lower absorbance
at 1390 cm− 1, 1270 cm− 1 and 1100–1000 cm− 1. According to the
loadings, the highest contribution to PC4 came from the signals pro­
vided by bands located at 1400–1300 cm− 1 range, 1820 cm− 1, 1500
cm− 1 and 965 cm− 1 (positive) and those at 1237 cm− 1, 1100 cm− 1 and
1420 cm− 1 (negative). HEP disaccharides from Group I and Group II had
higher positive scores along PC4 compared to HEP disaccharides from
Group III and Group IV. Moreover, HEP disaccharides from Group I and
Group II were characterized by both higher intensity at 1400–1300 cm− 1
and lower intensity at around 1237 cm− 1. The opposite was observed for
HEP disaccharides from Group III and Group IV.
In summary, the differentiation of HEP disaccharides was observed
according to their substitution patterns. Each cluster contains di­
saccharides with the same R1 and R2 substituents, thus, confirming that
the influence of R3 is insignificant to both torsion angles and IR spectra.
Native HEP and OSCS disaccharides have been used as test structures to
Y.B. Monakhova et al.
Fig. 4. IR spectra of native HEP, HEP and OSCS disaccharide subunits plotted with a Gaussian broadening. Group differentiation of HEP disaccharides made in
accordance with PCA. The legend reflects the disaccharide numbers.
evaluate similarity with HEP disaccharides. It was found that native HEP
and HEP disaccharides from Group II had similar scores that indicates
the similarity of their spectral characteristics. Moreover, Group II con­
tains trisulphated HEP disaccharides that confirms the actual predomi­
nance of these disaccharides in native HEP. OSCS disaccharide has closer
characteristics with HEP disaccharides from Group IV. Their spectral
similarities seem to make it difficult to detect OSCS in native HEP.
4. Conclusions
The vibrational infrared spectra of HEP and OSCS disaccharide
subunits were investigated using quantum mechanics methods. RHF/6-
311G level of theory provided good performance for the calculations of
relative intensities and frequencies for vibrational bands in the range of
1800–1000 cm− 1. Comparing the results of quantum–mechanical
approach with experimental IR spectrum of native HEP revealed that the
treatment at the RHF/6-311G level of theory offers reliable modelling of
the IR spectra. RHF modelling demonstrated that geometric parameters
as well as the features of the IR spectrum of HEP disaccharides depended
on the substitution pattern. We have shown that N-sulphation doesn’t
5
Computational and Theoretical Chemistry 1217 (2022) 113891
affect the conformation of the β-glycosidic linkage, while O-sulphation
appears to have a measurable effect on the conformation of the
β-glycosidic linkage.
In addition, multivariate approach for analysis of calculated IR
spectra allowed to evaluate the inherent similarities and differences in
the set of calculated and experimental spectra. PCA modelling of
calculated IR spectral data differentiated the disaccharides according to
their structures. PCA model also confirmed the plausibility of the
calculated IR spectra of disaccharides. In this regard, an important
practical result is that theoretical IR spectral profiles combined with
appropriate multivariate modelling can be used to assess the disaccha­
ride composition of native HEP that may facilitate HEP authentication.
Therefore, we are proceeding to further studies of the larger fragments
of HEP that ultimately can provide more detailed information about
HEP.
CRediT authorship contribution statement
Yulia B. Monakhova: Writing – review & editing, Supervision,
Project administration. Polina M. Soboleva: Formal analysis,
Y.B. Monakhova et al.
Investigation, Writing – original draft, Visualization. Elena S. Fedo­
tova: Formal analysis, Investigation. Kristina T. Musina: Formal
analysis, Investigation. Natalia A. Burmistrova: Conceptualization,
Investigation, Writing – original draft, Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgements
This work was supported by the Russian Science Foundation, Russia
(project 18-73-10009).
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