Yu et al. BMC Chemistry(2024) 18:126
https://doi.org/10.1186/s13065-024-01232-0

BMC Chemistry

RESEARCH

Open AccessInteraction mechanism of oseltamivir
phosphate with bovine serum albumin:
multispectroscopic and molecular docking
study
Jing Yu1, Jian-Ming Liu1, Hui-Yi Chen1 and Wei-Ming Xiong2*
Abstract
Oseltamivir phosphate (OP) is an antiviral drug with potential risks to human health due to overuse, leading toserious consequences such as gastrointestinal disturbances, abnormal neuropsychiatric symptoms, and sudden
death. Therefore, gaining an in-depth understanding of its interaction with proteins is crucial. We investigated
the interaction between OP and bovine serum albumin (BSA) utilizing multispectral methods (i.e., fluorescence,
ultraviolet absorption, circular dichroism) combined with molecular docking techniques. Fluorescence spectroscopy
indicated that OP quenched BSA fluorescence by forming the OP-BSA complex. The Stern-Volmer constants (KSV )between OP and BSA were determined to be 3.06 × 103 L/mol, 2.36 × 103 L/mol, and 1.86 × 103 L/mol at 293 K,
298 K, and 303 K, respectively. OP occupies exclusively one binding site on BSA, and the fluorescent probe
displacement measurements revealed that this is BSA site I. Thermodynamic data (∆H, ∆S, and ∆G) obtained byfitting the van’t Hoff equation were − 77.49 kJ/mol, -176.54 J/(mol∙K), and − 24.88 kJ/mol, respectively, suggestingthat hydrogen bonding and van der Waals forces mainly participate in OP-BSA complex stabilization. Moreover, thereaction occurs spontaneously at room temperature. Synchronous fluorescence spectra indicated that OP interacts
with tryptophan residue of BSA. The results of ultraviolet (UV) and 3D fluorescence spectroscopy indicated that the
OP-BSA complex formation altered the microenvironment around amino acid residues. Circular dichroism spectra
revealed that the addition of OP decreased the α-helix content of BSA by 7.13%. Docking analysis confirmed that
OP binds to BSA site I through hydrogen bonding with amino acids VAL342, SER453, and ASP450. Finally, ADMET
studies were conducted to explore the pharmacokinetics of OP as an antiviral drug.
Keywords Oseltamivir phosphate, Bovine serum albumin, Multispectroscopic, Molecular docking*Correspondence:
Wei-Ming Xiong
xiongwm3@gxu.edu.cn
1School of Chemistry and Civil Engineering, Shaoguan University,
Shaoguan 512005, China
2
School of Physical Science & Technology, Guangxi University,
Nanning 530004, China© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Yu et al. BMC Chemistry(2024) 18:126

Introduction
The antiviral drug Oseltamivir phosphate (C16H31N2O8P,(3R,4R,5 S)-4-acetamido-5-amino-3-(1-ethyl epoxy)1-cyclohexene-1-carboxylic acid ethyl ester phosphate,OP, see Fig. 1) is a neuraminidase inhibitor that acts byblocking the release and transmission of viral progeny
from infected cells [1, 2]. In August 2016, the drug was
FDA-approved for the treatment and prevention of influenza A (including pandemic H1N1) and B virus infections [3]. It also inhibits tumor angiogenesis, growthand metastasis [4]. Its applications to ester-loaded drugdelivery systems, such as ionic complexes for palatability modulation, swellable hydrogel systems for modified
release, and liposome-encapsulated oseltamivir salts indry powder formulations for pulmonary delivery havebeen reported in the literatures [5, 6]. In addition, it canalso be used to limit the spread of infection in high-risk
populations, as recommended by the World Health Organization (WHO) [7]. However, researchers have demonstrated through numerous clinical trials that OP use did
not reduce the risk of hospitalization but increased theincidence of gastrointestinal adverse events [8, 9]. Studies
have shown that doses of OP may cause symptoms suchas nausea and vomiting in humans [10], and overdose of
OP may lead to serious consequences such as gastrointestinal disturbances [11], abnormal neuropsychiatric
symptoms [12], and sudden death [13, 14]. Therefore, it isnecessary to evaluate its toxicity and safety.
Drugs deliver their desired therapeutic effects by interacting with proteins in the targeted diseased tissue orsite of action [15]. The binding of drugs to proteins invivo significantly influences their therapeutic properties [16]. If a drug has a strong binding capacity, this may
prolong its residence time in the body, thereby increasing
the likelihood of adverse side effects. On the contrary, adrug with weak binding capacity may face issues related
to shorter half-life and early excretion from the body,making it less effective [17]. The therapeutic effectiveness of a drug molecule is impacted by its ability to bindto proteins, which can also influence its toxicity and stability during therapy [18]. This suggests that reversible

Fig. 1 Schematic diagram of OP molecular structure

Page 2 of 11

binding of drugs to proteins determines pharmacokinetic
parameters such as metabolism, distribution, transport,
and excretion of drugs [19–22]. Therefore, exploring thebinding mechanisms of drugs to proteins is crucial.
Among many proteins, bovine serum albumin (BSA)
has a three-dimensional structure similar to that ofhuman serum albumin (HSA), so it is often used as an
ideal model system for in vitro studies of interactions
with drugs [23, 24]. BSA is a versatile protein with multiple binding sites and several carboxylic and aminogroups that can interact with small molecules [25–27].
This makes it an effective transport protein that can create stable complexes with various external and internal
ligands, which may influence the effectiveness, distribution and storage of small molecule drugs [28–30]. Due tothe potential risks of OP, it is of vital importance to conduct explorations related to OP’s association with SA so
as to elucidate the relationship between OP and humanhealth.
In this study, we conducted a comprehensive analysisof the binding mode and interaction strength between
OP and BSA by employing various spectroscopic methods along with molecular docking. The experimental
techniques provided insights into the binding capacity,quenching type, force of action, binding site, secondary structure changes, and OP-BSA complex properties.
Meanwhile, molecular simulations rationalized the binding mode by highlighting the amino acid residues thatinteract with OP and estimated the binding free energy of
the OP-BSA complex. Additionally, OP was evaluated by
exploring its ADMET (absorption, distribution, metabolism, excretion and toxicity) properties in silico. Throughthese studies, we aim to understand the effect of OP on
BSA and provide valuable risk aversion data for the clinical application of this antiviral drug. Building on thisresearch, drug-protein interactions can be optimized toachieve the development of more effective and targeted
antiviral drugs.

Experimental
Materials

BSA (M = 66.430 kDa, purity 96-99%, CAS# 9048-468) was acquired from Shanghai Aladdin Biochemical
Technology Co., Ltd. Phosphate buffer solution (PBS,
0.02 mol∙L− 1) with a pH of 7.43 was prepared using
NaH2PO4 and Na2HPO4 (p.a.). The BSA solution in PBS(1 µM) was then made and stored at 4 ℃. Prior to theexperiment, the OP (CAS# 204255-11-8) stock solution
in anhydrous ethanol (1 mM) was made. Ultrapure water
was used in all experiments.
The ZL10-250 A ultrasonic cleaner (Shanghai Zuo
Le Instrument Co., Ltd.) was utilized to facilitate OP
solubilization. UV-Vis spectra were measured using a
UV-670 device (Shanghai Meipuda Instrument Co., Ltd.).

Yu et al. BMC Chemistry(2024) 18:126

Fluorescence emission data were acquired on an F-380
fluorescence spectrophotometer (Tianjin Gangdong Science and Technology Co., Ltd.). Circular dichroism (CD)
spectra were recorded on a Chirascan Plus CD spectrometer (Applied Photophysics, Inc., U.K.). The solutions were thermostated using an SHZ-82 A water bathand a constant temperature oscillator (Changzhou Jintan
Jingda Instrument Manufacturing Co., Ltd.).
Experimental section
Fluorescence spectrometry

A 3 mL solution of BSA (1 µM) was added to a 1-cm
cuvette, and the solution was thermostated at 298 K. To
this solution, 30 µL of the OP stock solution (1 mM) was
added sequentially for 9 times, and the emission spectraof the OP-BSA adduct were collected after each addition of OP. The λex was configured at 280 nm, and fluorescence intensities were recorded within the range of
300–500 nm, employing a scan rate of 1200 nm/min. The
slit widths were adjusted to 5 nm. The same steps wererepeated at two additional temperatures, i.e., 293 K and
303 K.
Synchronous fluorescence spectroscopy (SFS)The SFS data of BSA and the OP-BSA complex at Δλ = 15
and 60 nm (Δλ = λem-λex) were measured at 298 K
between 250 nm and 350 nm. These experiments provided insights into the contribution Trp and Tyr residues
in the binding of OP to BSA.
3D fluorescence spectroscopy

The 3D fluorescence spectra were recorded at a 1:1 (OP:
BSA) mol ratio. Excitation wavelengths were scanned
from 200 to 500 nm, and emission intensities were monitored from 200 to 600 nm, applying 10-nm increment.
Fluorescent probe displacement

In a 1-cm cuvette containing 3 mL of BSA solution (1µM) was added along with 3.0 µL of ibuprofen stock solution (1.0 × 10− 3 M). Fluorescence spectra were collectedby sequentially adding 3.0 µL the OP solution (0.01 M)
eight times in total. The temperature was adjusted to
303 K, and the fluorescence spectra of the OP-BSA system were scanned in the range from 300 to 500 nm. The
same experiment was repeated using a 0.1 mM warfarinsolution. All other test parameters remained the same as
for fluorescence spectra acquisition.
Ultraviolet (UV) spectrometry

To a 1-cm quartz cuvette, 3.0 mL of BSA solution (1 µM)
was added, and 3.0 mL of PBS was used in the referencecell. The 3.0 µL of OP solution (0.1 mM) was added tothis cuvette and the reference cell three times, and the
UV absorption spectrum was scanned after each aliquot.

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The temperature was set to be 296 K, and the UV data
were collected in the range of 250–350 nm.
Circular dichroism (CD) spectra

A background scan of the PBS buffer solution was conducted using a circular dichroism (CD) spectrometerwithin a wavelength range from 200 to 260 nm. Subsequently, BSA solution in PBS (1 µM) was added to a
1-mm quartz cuvette, and the CD spectrum of BSA wasrecorded. Next, OP-BSA (concentration ratio equal to2:1) was added, and the CD spectrum was scanned threetimes. The final CD spectra of pure BSA and OP-BSA
adduct were obtained by subtracting the PBS backgroundsignal.
Molecular docking

The BSA coordinates were retrieved from the Protein
Data Bank (PDB ID 4F5S), while the initial conformationof OP was downloaded from PubChem. First, water molecules and irrelevant heteroatoms were removed using
Pymol, retaining only the protein A chain. Amino acidpKa values were calculated, and protonation states under
neutral conditions were assigned using the propka3
online tool. The structures of the ligand and receptor
were prepared for docking utilizing Autodock Tools1.5.7 software. Molecular docking experiments wereconducted using Watvina software (https://github.com/
biocheming/watvina), specifying a square box with side
lengths of 30 Å and a grid spacing step of 0.375 Å. The
maximum limit for searching for conformations was set
to 10,000, and conformational sampling and scoring were
performed using a genetic algorithm. Optimal conformations were selected by sorting the conformations according to the docking scores.
ADMET analysis

A computational ADME (absorption, distribution,
metabolism and excretion) profile was generated for
OP prior to its consideration for preclinical trials and invivo studies. The analysis was conducted using the SwissADME web platform (http://www.swissadme.ch/) [31].
The toxicity assessment of OP was performed utilizing
ADMETLAB 3.0 (https://admetlab3.scbdd.com/ ) [32].

Results and discussion
Fluorescence spectroscopy
Mechanism of fluorescence quenching by OP in BSA

Protein fluorescence quenching involves reducing the
intrinsic fluorescence intensity, of the of proteins, primarily derived from the phenylalanine, tyrosine andtryptophan residues, which frequently participate ininteractions with ligands [33]. Tryptophan contributes the most to the fluorescence, followed by tyrosine,
while phenylalanine has minimal fluorescence and isYu et al. BMC Chemistry(2024) 18:126

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Fig. 2 Fluorescence quenching spectra of BSA by OP at 293 K (A), 298 K (B) and 303 K (C) (CBSA = 1.0 × 10− 6 mol∙L− 1 decreases from a to k, the OP concentration increases from 0 to 1.0 × 10− 4 mol∙L− 1, with a step size of 1.0 × 10− 5 mol∙L− 1)

Table 1 Quenching constants of OP and BSA reactions at
different temperatures
T/K
293
298
303

R
0.99
0.99
0.98

Ksv(103L·mol− 1)
3.06
2.36
1.86

kq(1011L·mol− 1·s− 1)
3.06
2.36
1.86

SD
0.08
0.09
0.07

F0
= 1 + KSV [Q] = 1 + kq τ0 [Q] (1)F

Fig. 3 Stern-Volmer plots of the reactions of OP with BSA at different
temperaturesconsidered insignificant compared to the other two [34].
Different physicochemical mechanisms may lead to fluorescence quenching, including energy transfer, collisions,formation of complexes in the ground state, or reactionsin the excited state [35]. There are two primary typesof quenching: static and dynamic. Their differentiation
relies on their association with viscosity, temperature,and fluorescence lifetime. Static quenching occurs whena fluorescent substance and a quencher form a groundstate complex, while dynamic quenching occurs whenthe excited fluorophore relaxes to the ground state via
collisions with the quencher [36].
The influence of OP on the emission intensity (F) of
BSA is depicted in Fig. 2. With the addition of OP, thevalue of F decreased, and the maximum emission wavelength was blue-shifted, indicating that the microenvironment around BSA fluorophores became morehydrophobic upon OP binding. The changes in F values were further analyzed using the Stern-Volmer (S-V)
model [37]:where F and F0 represent emission intensity of the system in the presence or absence of OP, and KSV, kq, [Q]and τ0 represent the Stern–Volmer constant, bimolecularquenching rate constant, the molarity of OP, and the average fluorescence lifetime of biomolecules, respectively.
The S-V equation is a common tool for distinguishing
between the static and dynamic quenching mechanisms.
In the case of dynamic quenching, KSV is positively correlated with temperature since the diffusion coefficientsand the number of collisions increase with temperature.
On the other hand, KSV is negatively correlated with temperature in the case of static quenching, as temperature
promotes the dissociation of ground-state complexes[38].
Figure 3 shows the plot of F0/F against [Q] at threetemperatures. The corresponding KSV values, representing the slope of this trendline, are displayed in Table 1.
The observed decrease in KSV with rising temperatures
suggests the dominance of the static quenching mechanism, attributed to the formation of the OP-BSA adduct.
Furthermore, the kq values, exceeding the diffusion limit
for the dynamic quenching rate constant (2.0 × 1010
L·mol− 1·s− 1), provide additional confirmation for thisresult [39].
Double logarithmic plot of the S-V model

Additional insights into the impact of OP on BSA were
gained using the double logarithmic form of the S-V
equation (Eq. 2) [40], providing information on the binding constant (K) and the number of binding sites (n).

Yu et al. BMC Chemistrylg

(2024) 18:126

Page 5 of 11(F0 − F )= lg K + n lg [Q] (2)F

In this equation, [Q] represents the concentration of OP.
The double logarithmic plots are presented in Fig. 4, andthe corresponding values of slope (n) and intercept (logK)
are listed in Table 2. The findings indicate that OP binds
to a single binding site among many accessible sites on
BSA. Moreover, as the temperature increases, K values
decrease, suggesting the formation of the OP-BSA adductrather than collision quenching.
Probe displacement experiments

To determine the preferable binding sites for OP on BSA,
we performed probe displacement binding assays usingsite-specific probes for binding site I (warfarin) and binding site II (ibuprofen) [40]. Figure 5 shows the SternVolmer plots of the BSA-OP system without and withsite labeling at 303 K. The resulting values are listed inTable 3. We observed that the Ksv values of the BSA-OP
system all decreased in the presence of warfarin, suggesting that BSA-OP competes with warfarin for the same
binding i.e. for Site I, and that the Ksv values of the BSAOP binding to BSA Ksv values of site II did not changemuch in the presence of ibuprofen. This confirms that
BSA-OP binds near site I, which is located in BSA subdomain IIA.

Fig. 4 Double logarithmic curve of the reaction between OP and BSA

The thermodynamics of OP-BSA binding

The stabilization of the OP-BSA complex may result fromvarious non-covalent interactions, such as hydrophobic
forces, van der Waals forces, electrostatics, hydrogenbonding, and salt bridges [41]. According to Ross andSubramanian [42], the enthalpy change (ΔH) and entropychange (ΔS) determine the preferable type of interactions
between compounds and biomolecules. These thermodynamic data can be calculated from the Van’t Hoff model(Eqs. 3 and 4):

ln K =

Fig. 5 Stern-Volmer plot for the fluorescence of BSA-OP system in the absence and presence of site probes (warfarin and ibuprofen). CBSA = Cprobe=1 × 10− 6 mol∙L− 1bonding process, and R is universal gas constant. Fromthe linear plot of lnK vs. 1/T (Fig. 6), we obtained a ∆Hvalue of -77.49 kJ∙mol− 1 from the slope and a ∆S valueof -176.54 J∙mol− 1∙K− 1 from the intercept (see Table 2).
Moreover, Gibbs free energy was negative for all temperatures, indicating the spontaneity of OP binding to BSA.
These results imply that hydrogen bonding and van der
Waals interactions are the dominant forces governing thebinding process.

−∆H ∆S
+
(3)
(RT )
R

∆G = ∆H − T ∆S (4)In these equations, K represents the binding constant at
temperature T, ∆G is Gibbs free energy change of theTable 2 Binding constants and thermodynamic parameters for the reactions of OP with BSA at different temperatures
T(K)
293
298
303
a

K
(104 L∙mol− 1)
4.03
2.09
1.41

Ra

n

SDb

0.99
0.98
0.98

1.28
1.24
1.22

0.10
0.26
0.23

Linear correlation coefffcient for the K values. b Standard deviation for the K values∆G(KJ∙mol− 1)-25.76-24.88-23.99∆S(J∙mol− 1∙K− 1)-176.54∆H(KJ∙mol− 1)-77.49Yu et al. BMC Chemistry(2024) 18:126

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Table 3 Effect of site probes (warfarin and ibuprofen) on theinteraction of OP with BSA
T(K)
303 K

Ksv w/o site
probes3.06 × 103

Ksv with
ibuprofen
3.26 × 103

Ksv with
warfain7.15 × 102

R2
0.99/0.99/0.90

Fig. 6 Van’t Hoff plots for the reaction of OP with BSA

Synchronous fluorescence spectroscopy (SFS)The SFS provides insights into the changes in the polarity near Trp and Tyr, the main amino acid residues
contributing to the fluorescence emission of BSA [43].
According to Miller’s theory, setting Δλ = 15 nm andΔλ = 60 nm allows SFS to reveal changes in the microenvironment around Tyr and Trp caused by drug moleculebinding [44]. The SFS data of BSA after OP addition at
Δλ = 15 nm and Δλ = 60 nm are displayed in Fig. 7(A)and 7(B), respectively. The fluorescence intensity of
BSA decreased gradually with increasing OP and thequenching at Δλ = 60 nm was more pronounced thanthat at Δλ = 15 nm, suggesting that OP binds more tightly
to tryptophan residues than to tyrosine residues. In

addition, there was little change in λem for both (<1 nm),suggesting that the addition of OP had little effect on the
microenvironment of Trp and Tyr residues.
3D fluorescence spectroscopy (3D-FS)

The 3D-FS serves as a valuable tool for acquiring information about the microenvironment around Tyr andTrp, as well as the conformation of the peptide backbone [45]. The changes in 3D-FS data of BSA induced by
OP binding are presented in Fig. 8. In Fig. 8 (A-B), the3D emission spectra and contour plots of BSA and OPBSA are depicted. Figure 8A shows two major peaks inthe spectra of BSA and OP-BSA complex. The peak1 at
λex = 280 nm corresponds to the fluorescence emissionof Tyr and Trp [46]. Conversely, peak 2 at λex = 230 nm
provides structural information about the peptide backbone of BSA [47]. Upon the addition of an equimolar
amount of OP to BSA, the intensities of both peaks significantly decreased. The Stokes shift of peak 1 decrease
from 4500 to 3791. upon the addition of OP, suggests thatthe microenvironment of Tyr and Trp in BSA became
more hydrophobic upon ligand binding. Moreover, thedecrease from 2218 to 1750, along with a 10 nm blue shiftof peak(340 –330 nm), indicating conformational changesof the BSA peptide backbone and, consequently, alterations in the secondary structure of the entire BSA [48].
UV absorption spectroscopy

UV spectroscopy is a common technique to investigate protein-ligand interactions [49]. Figure 9 presents
the UV absorption spectra of BSA after the addition ofincreasing amounts of OP. The absorption maximum at
277 nm is attributed to the π-π* electronic transitionsin the aromatic residues of BSA, such as Tyr and Trp[50]. The results indicate that the peak shape and position remained unchanged upon the introduction of OP,
but the peak intensity slightly increased. This observation
further confirms the static quenching mechanism during

Fig. 7 Synchronous fluorescence spectra of the interaction between OP and BSA at Δλ = 15 nm (A) and Δλ = 60 nm (B). (CBSA = 1.0 × 10− 6 mol∙L− 1 decreases from a to j, the OP concentration increases from 0 to 9.0 × 10− 5 mol∙L− 1, with a step size of 1.0 × 10− 5 mol∙L− 1)

Yu et al. BMC Chemistry(2024) 18:126

Page 7 of 11

Fig. 8 3D fluorescence and contour maps of AC BSA, BD OP-BSA. CBSA = 1.0 × 10− 6 mol∙L− 1, COP = 1 × 10− 5 mol∙L− 1

Fig. 9 UV absorption spectra of OP and BSA (9 A) and Plot 1/(Aobs-A0) versus1/[OP] (9B). (CBSA = 1.0 × 10− 6 mol∙L− 1 decreases from a to e, the OP concentration increases from 0 to 4.0 × 10− 7 mol∙L− 1, with a step size of 1.0 × 10− 7 mol∙L− 1)

OP-BSA complex formation, influencing both the microenvironment around fluorophores and the backbone conformation. Additionally, based on the absorbance spectraof BSA binding to OP, the intrinsic binding constant (Kb)
was calculated as follows (Eq. 5).

1
1
1
=
+
(5)
Aobs − A0 Ac − A0 Kb (Ac − A0) [OP ]Aobs was the absorbance of BSA with varying concentrations of OP. A0 was the absorbance of BSA alone and Ac
the absorbance of OP-BSA complex. Based on the absorbance values, the relationship between 1/(Aobs-A0) and 1/[OP] was plotted (Fig. 9B). A graph of 1/(Aobs-A0) versus
1/[OP] yielded a linear plot with a slope equal to 1/Kb(AcA0) and an intercept equal to 1/(Ac-A0). It was found in
the plot that the value of Kb was 7.12 × 105 M− 1.

Yu et al. BMC Chemistry(2024) 18:126

Circular dichroism (CD)CD is another common tool for studying how the binding of small molecules affects protein conformation [51].
In this study, we employed CD to investigate the influence of OP on the structure of BSA. The CD spectrumof BSA (Fig. 10) displays two negative peaks at 208 nm
and 222 nm that originate from the π-π* and n-π* transitions in the α-helices. The intensity of these peaks reflects
the degree of helicity in BSA. The percentage of α-helicalsecondary structure can be calculated using the following
equation [52]:MRE =θobs (m deg)
(6)(10 × n × l × Cp)

Here, MRE is the ellipticity of the average residue, θobs isthe ellipticity, n is the number of amino acid residues, lrepresents the diameter of the cuvette, and Cp denotes
the molarity of BSA. The α-helical content is derived
from the ellipticity of the CD spectrum at 208 nm:( - MRE208 − 4000)
α% helix =× 100 (7)(33000 − 4000)
Calculated from Eqs. (6) and (7), the percentage of
α-helical structure in BSA was 48.08%, and after the addition of OP (Fig. 10, red curve), this content decreased to40.95%. This result confirms that OP binding inducedchanges in the secondary structure of BSA.
Molecular docking simulations

To gain deeper insights into the binding mode of OP to
BSA and identify crucial amino acids involved in ligand
binding, molecular docking calculations were performed [54]. Figure 11 shows the binding mode of OP
into site I of BSA. The ligand exhibits excellent shape

Page 8 of 11

complementarity with the binding site and a favorable
binding affinity of − 5.96 kcal/mol. Furthermore, theligand forms hydrophilic (electrostatic) interactions withthe receptor binding site.
As illustrated in Fig. 11D, OP interacts with 19 aminoacid residues of BSA. Among them, there are van der
Waals forces between OP and LEU346, VAL481, LEU480,
TYR451, LYS294, TYR340, GLN220, and VAL342,
SER453, and ASP450, and seven hydrogen bonds areformed between OP and VAL342, SER453, ASP450. Fiveof them were conventional H-bonds formed between H
atoms bound to highly electronegative atoms found at
distances of 2.75 Å, 2.76 Å, 1.82 Å, 1.97 Å, 3.0 Å respectively. Additionally, two weaker interactions involving
C-H bonds were found at distances of 2.98 Å and 2.08
Å. These results confirm the predominance of hydrogen bonding interactions in the stabilization of OP-BSA
complex, consistent with the data derived from the temperature dependence of fluorescence spectra. Moreover,
ARG194, LEU197, LEU454, VAL342 and TRP213 establish several hydrophobic interactions with OP. Since
TRP213 is the main fluorophore in BSA, the interaction
with this amino acid residue may explain the observed
decrease in BSA emission intensity. Additionally, molecular docking suggests the formation of alkyl-alkyl and
π-alkyl interactions between OP and TRP213, ARG194,
LEU454 and LEU197 residues of BSA, further contributing to the stabilization of the OP-BSA complex.
ADMET properties

The ADME properties of drugs were assessed using the
SwissADME server. The investigation involved a comprehensive evaluation of OP, with a specific focus on
its compliance with Lipinsk’s rule of five, encompassing
parameters such as molecular weight below 500 g/mol,less than 10 hydrogen bond acceptors, and fewer than 5

Fig. 10 Circular dichroism spectra of BSA and BSA-OP (1:2) systems CBSA = 1.0 × 10− 6 mol∙L− 1, COP = 2 × 10− 6 mol∙L− 1

Yu et al. BMC Chemistry(2024) 18:126

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Fig. 11 Analysis of BSA-site I and OP binding interface interactions (A and B are surface and ribbon structures, C and D are 3d and 2d plots of complex
interface interactions)hydrogen bond donors [53]. Findings from the supplementary materials in Table S1 revealed that OP did not
violate any of the rules, suggesting potential orally active.
Additionally, the consensus Log Po/w (the logarithm of
the n-octanol/water distribution) value of OP was determined to be -0.17, indicating moderate lipophilicity.
Moreover, SwissADME estimated the molar solubility of
OP in water to be 2.47 by calculating the Log S (SilicosIT) descriptor, indicating favorable water solubility [54].
Topological polar surface area (TPSA) of OP was foundto be 178.22 Å, exceeding 140 Å, suggesting suboptimal
permeability through various biological barriers [55]. OP
showed no inhibitory effects on CYPs (1A2, 2C19, 2C9,
2D6, and 3A4) but exhibited inhibition of the humanP-glycoprotein transporter (Pgp). Furthemore, the LogKp
value of the compound was calculated to be -11.13 cm/s,
indicating low skin permeability [56]. The Bio-availability
Score of 55% suggested favorable bioavailability.
The potential toxicity of OP was assessed using
ADMETLAB3, and the results were displayed in the Supplementary materils in Table S2. The findings revealed
that OP did not inhibit the hERG (human ether-a-go-gorelated gene) potassium channel, indicating it produced

no cardiac toxicity or side effects [57]. The Rat Oral Acute
Toxicity estimate for OP was 235 mg/kg, which can beclassified to Class 2 (50 mg/kg < LD50 ≤ 500 mg/kg), thus
it was non-toxic [60]. Additionally, the predicted Carcinogenicity Descriptor (CARC) value of 303 mg/kg bodyweight per day suggested that OP was unlikely to be carcinogenic. Consequently, OP was deemed non-carcinogenic and non-AMES toxic, enhancing its safety profile
for potential pharmaceutical applications. To conclude,
these results demonstrated that OP possessed favorable
pharmacokinetic properties, making it suitable for oral
administration and human consumption.

Conclusion
In this study, we systematically assessed the interactions between OP and BSA using a combination ofmultiple spectroscopic methods and molecular docking simulations. The fluorescence spectroscopy results
demonstrated that the addition of OP leads to thequenching of intrinsic fluorescence in BSA, primarily via
a static quenching mechanism. Our investigation further
revealed that OP exclusively binds to a specific bindingsite on BSA, and the displacement of the warfarin probeYu et al. BMC Chemistry(2024) 18:126

confirmed this site to be BSA site I. The thermodynamic
analysis of the OP-BSA binding process indicated negative values for ∆H, ∆S and ∆G. These findings strongly
suggested a spontaneous process driven by hydrogen bonding and van der Waals forces. Synchronous
fluorescence spectra indicated that OP interacts withtryptophan residue of BSA. Furthermore, UV and CD
spectroscopy indicated that the formation of OP-BSA
complex induced changes in the protein secondary structure. Molecular docking calculations complemented ourexperimental results by validating that site I is the energetically most favorable binding site for OP on BSA. The
docking also highlighted amino acid residues essential
for OP-BSA interaction, and confirmed the dominanceof hydrogen bonding and hydrophobic forces. Subsequently, an ADMET study forecasted favorable physicochemical and pharmacokinetic proffles for OP. This work
contributes to a thorough understanding of the OP-BSA
bonding mechanism, combining experimental and theoretical insights. The elucidation of these interactions is of
considerable reference value for advancing research on
the impact of OP on human health and furthering ourunderstanding of the mechanisms underlying the actionof antiviral drugs.
Abbreviations
UV	Ultraviolet
OP	Oseltamivir phosphate
BSA	Bovine serum albumin
HSA	Human serum albumin
ADMET	Absorption, distribution, metabolism, excretion and toxicity
SFS	Synchronous fluorescence spectroscopy
3D-FS	3D fluorescence spectroscopy
CD	Circular dichroism
Log Po/w	The logarithm of the n-octanol/water distribution
TPSA	Topological polar surface area
Pgp	P-glycoprotein transporter
CARC	Carcinogenicity descriptor
hERG	Human ether-a-go-go-related gene

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s13065-024-01232-0.
Supplementary Material 1
Acknowledgements
Not applicable to this study.
Author contributions
Experiments was accomplished by JY, JML and YHC. The molecular docking
study was accomplished by WMX. JY and WMX accomplished the writingof the article. JY and WMX were the study designers. All authors read andapproved the final manuscript.
Funding
This research was funded by Shaoguan Science and Technology Bureau(Project No.210726114532129,220607144531375), Shaoguan University(SY2020KJ14, 408-9900045509).

Page 10 of 11

Data availability
The datasets used and/or analyzed during the current study are available fromthe corresponding author on reasonable request.

Declarations
Ethics approval and consent to participate
Not applicable to this study.
Consent for publication
Not applicable to this study.
Competing interests
The authors declare no competing interests.
Received: 5 February 2024 / Accepted: 24 June 2024

References
1. Yurtdaş-Kırımlıoğlu G, Görgülü Ş, Güleç K, et al. Nanoarchitectonics of PLGA
based polymeric nanoparticles with oseltamivir phosphate for lung cancer
therapy: in vitro-in vivo evaluation. J Drug Deliv Sci Tec. 2022;67:102996.
2. Huang MF, Lin YR, Chang YT, et al. Reductive amination assistance for quantification of oseltamivir phosphate and oseltamivir carboxylate by HPLC-MS/MS. J Chromatogr B. 2018;1087:23–8.
3. El-Shorbagy HI, Mohamed MA, El-Gindy A, et al. Development of UPLC
method for simultaneous assay of some COVID-19 drugs utilizing novel
instrumental standard addition and factorial design. Sci Rep-uk. 2023;13:5466.
4. Yurtdaş-Kırımlıoğlu G, Güleç K, Görgülü Ş, et al. Oseltamivir phosphate loaded
pegylated-eudragit nanoparticles for lung cancer therapy: characterization,
prolonged release, cytotoxicity profile, apoptosis pathways and in vivo antiangiogenic effect by using CAM assay. Microvasc Res. 2022;139:104251.
5. Qorri B, Mokhtari RB, Harless WW, et al. Repositioning of old drugs for Novel
Cancer therapies: continuous therapeutic perfusion of aspirin and Oseltamivir Phosphate with Gemcitabine Treatment disables Tumor Progression,
Chemoresistance, and metastases. Cancers. 2022;14(15):3595.
6. Kapishon V, Allison S, Whitney RA, et al. Oseltamivir-conjugated polymeric
micelles prepared by RAFT living radical polymerization as a new active
tumor targeting drug delivery platform. Biomater Sci-uk. 2016;4:511–521.
7. Aziz S, Scherlieβ R, Steckel H. Development of high dose oseltamivir phosphate dry powder for inhalation therapy in viral pneumonia. Pharmaceutics.
2020;12(12):1154.
8. Hanula R, Bortolussi-Courval E, Mendel A, et al. Evaluation of Oseltamivir used
to prevent hospitalization in outpatients with influenza. Jama Intern Med.
2024;184(1):18–27.
9. Han NY, Mi Oh J, Kim IW. Assessment of adverse events related to antiinfluenza neuraminidase inhibitors using the FDA adverse event reporting
system and online patient reviews. Sci Rep-uk. 2020;10:3116.
10. Doll MK, Winters N, Boikos C, et al. Safety and effectiveness of neuraminidase inhibitors for influenza treatment, prophylaxis, and outbreak control: asystematic review of systematic reviews and/or meta-analyses. J Antimicrob
Chemoth. 2017;72(11):2990–3007.
11. Huh K, Kang M, Shin DH, et al. Oseltamivir and the risk of neuropsychiatricevents: a National, Population-based study. Clin Infect Dis. 2020;71(9):406–14.
12. Shin JS, Ku KB, Jang Y, et al. Comparison of anti-influenza virus activity andpharmacokinetics of oseltamivir free base and oseltamivir phosphate. J
Microbiol. 2017;55:979–83.
13. Hama R. Fifty sudden deaths may be related to central suppression. Brit Med
J. 2007;335:59.
14. Hama R, Bennett CL. The mechanisms of sudden-onset type adverse reactions to oseltamivir. Acta Neurol Scand. 2017;135(2):148–60.
15. Xiang H, Zhou M, Li Y, et al. Drug discovery by targeting the protein–protein
interactions involved in autophagy. Acta Pharm Sin B. 2023;13(11):4373–90.
16. Rogóż W, Pożycka J, Owczarzy A, et al. Comparison of Losartan and Furosemide Interaction with HSA and their influence on HSA antioxidant potential.
Pharmaceuticals. 2022;15(5):499.
17. Di L. An update on the importance of plasma protein binding in drug discovery and development. Expert Opin Drug Dis. 2021;16(12):1453–65.

Yu et al. BMC Chemistry(2024) 18:126

18. Zhao C, Han SY, Li PP. Pharmacokinetics of Gefitinib: roles of drug metabolizing enzymes and transporters. Curr Drug Deliv. 2017;14(2):282–8.
19. Griffin BT, Guo JF, Presas E, et al. Pharmacokinetic, pharmacodynamic and biodistribution following oral administration of nanocarriers containing peptide
and protein drugs. Adv Drug Deliver Rev. 2016;106:367–80.
20. Wani TA, Zargar S, Hussain A, Spectroscopic. Thermodynamic and molecularDocking studies on Molecular mechanisms of drug binding to proteins.
Molecules. 2022;27(23):8405.
21. Reddy DS, Kongot M, Singh V, et al. Biscoumarin–pyrimidine conjugates aspotent anticancer agents and binding mechanism of hit candidate withhuman serum albumin. Arch Pharm. 2020;354(1):2000181.
22. Ali MS, Singh E, Muthukumaran J, et al. Non-steroidal anti-inflammatory drug
Effect on the binding of plasma protein with antibiotic drug Ceftazidime:
Spectroscopic and in Silico Investigation. Int J Mol Sci. 2023;24(19):14811.
23. Banu A, Khan RH, Mohssen TA, et al. Multispectroscopic and computational
studies of interaction of bovine serum albumin, human serum albumin andbovine hemoglobin with bisacodyl. J Mol Struct. 2022;1249:131550.
24. Gu Jl, Liu HR, Huang XY, et al. Investigation of the separate and simultaneous bindings of warfarin and fenofibrate to bovine serum albumin. Int J Biol
Macromol. 2023;236:123978.
25. Lv Y, Liang Q, Li Y, et al. Study of the binding mechanism between hydroxytyrosol and bovine serum albumin using multispectral and molecular docking.
Food Hydrocolloids. 2022;122:107072.
26. Gu J, Yang G, Li X, et al. Difference in the binding mechanism of distinct
antimony forms in bovine serum albumin. Biometals. 2021;34(1):1–18.
27. Das S, Pahari S, Sarmah S, et al. Lysozyme-luteolin binding: molecular insights
into the complexation process and the inhibitory effects of luteolin towards
protein modification. PCCP. 2019;21(23):12649–66.
28. Yessenkyzy A, Saliev T, Zhanaliyeva M, et al. Polyphenols as caloricrestriction mimetics and Autophagy Inducers in Aging Research. Nutrients.
2020;12(5):1344.
29. Das S, Sarmah S, Hazarika Z, et al. Targeting the heme protein hemoglobin by (–)-epigallocatechin gallate and the study of polyphenol–protein
association using multi-spectroscopic and computational methods. PCCP.
2020;22(4):2212–28.
30. Zhao J, Huang L, Li RJ, et al. Multispectroscopic and computational evaluation of the binding of flavonoids with bovine serum albumin in the presenceof Cu2+. Food Chem. 2022;385:132656.
31. Daina A, Michielin O, Zoete V. SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small
molecules. Sci Rep-uk. 2017;7:42717.
32. Allah AE, Guerrab W, Maatallah M, et al. Synthesis, characterization, crystal
structure, DFT, HSA, ADMET prediction and antibacterial activity of thiohydantoin analogues. J Mol Struct. 2024;1310:138324.
33. Raghav D, Mahanty S, Rathinasamy K. Characterizing the interactions of the
antipsychotic drug trifluoperazine with bovine serum albumin: probing
the drug-protein and drug-drug interactions using multi-spectroscopic
approaches. Spectrochim Acta A. 2020;226:117584.
34. Ali MS, Hamad A, Al-Lohedan, et al. Interaction of the lysozyme with anticoagulant drug warfarin: Spectroscopic and computational analyses. Heliyon.
2024;10:30818.
35. Bai J, Sun XK, Geng B, et al. Interaction mechanism of Cu+/Cu2+ on bovine
serum albumin: Vitro simulation experiments by spectroscopic methods.
Spectrochim Acta A. 2023;293:122491.
36. Javaheri-Ghezeldizaj F, Soleymani J, Kashaniand S, et al. Multi-spectroscopic,
thermodynamic and molecular dockimg insights into interaction of bovine
serum albumin with calcium lactate. Microchem J. 2020;154:104580.
37. Vakili S, Baher E, Vaezi Z, et al. In Vitro Study of the Interaction between
Ochratoxin A and Human serum albumin by Spectroscopic and MolecularDocking methods. ChemistrySelect. 2023;8:1–10.
38. Rahman N, Khalil N. Characterization of sulfasalazine-bovine serum albumin
and human serum albumin interaction by spectroscopic and theoretical
approach. Spectrochim Acta A. 2023;300:122865.
39. Ali MS, Rehman MT, Al-Lohedan HA, et al. Study of the binding of Cuminaldehyde with bovine serum albumin by Spectroscopic and Molecular modelingmethods. J Spectrosc. 2023;2023:4191046.
40. Esazadeh K, Azimirad M, Yekta R, et al. Multi-spectroscopies and molecularsimulation insights into the binding of bovine serum albumin and sodium
tripolyphosphate. J Photoch photobio A. 2023; 444:114999.

Page 11 of 11

41. Gokavi NM, Nandibewoor ST, Gowda JI. Investigations of the interaction mechanism between orphenadrine hydrochloride and bovine
serum albumin by spectroscopic and voltammetric techniques. J Fluorsc.
2023;33:2061–2073.
42. Tian ZY, Tian LY, Shi Man, et al. Investigation of the interaction of a polyaminemodified flavonoid with bovine serum albumin (BSA) by spectroscopic
methods and molecular simulation. J Photoch photobio B. 2020;209:111917.
43. Ross PD, Subramanian S. Thermodynamics of protein association reactions:forces contributing to stability. Biochem. 1981;20(11):3096–3102.
44. El Gammal RN, Elmansi H, El-Emam AA, et al. Insights on the in-vitro binding
interaction between donepezil and bovine serum albumin. BMC Chem.
2023;17(31):1–11.
45. Tian ZY, Ding TL, Niu HJ, et al. 2-Phenylquinoline-polyamine conjugate(QPC): Interaction with bovine serum albumin (BSA). Spectrochim Acta A.
2023;300:122875.
46. Zhou H, Xiong ZY, Ma XL, et al. A multispectral study and computer simulation on the interaction of pomalidomide with human serum albumin. J Mol
Liq. 2023;382:121947.
47. Wani TA, Bakheit AH, Abounassif MA. Study of interactions of an anticancer
drug neratinib with bovine serum albumin: spectroscopic and moleculardocking approach. Front Chem. 2018;6:00047.
48. Mohapatra P, Chandrasekaran N. Effects of black cumin-based antimalarial
drug loaded with nano-emulsion of bovine and human serum albumins byspectroscopic and molecular docking studies. Heliyon. 2023;9(1):12677.
49. Duman B, Erkmen C, Kabir MZ. In vitro interactions of two pesticides, propazine and quinoxyfen with bovine serum albumin: spectrofluorometric andmolecular docking investigations. Spectrochim Acta A. 2023;300:122907.
50. Shahabadi N, Hadidi S, Abdoli Z, et al. Interaction of a cobalt(III) complex containing β-amino alcohol with human serum albumin (HSA): spectroscopic
and molecular docking methods. J Mol Liq. 2023;384:122187.
51. Gu Jl, Huang XY, Ma YX, et al. Spectroscopic study on the separate and simultaneous interaction of nicotinic and its metabolite to bovine serum albumin.
J Mol Liq. 2022;358:119106.
52. Rostamnezhad F, Fatemi MH. Comprehensive investigation of binding ofsome polycyclic aromatic hydrocarbons with bovine serum albumin: spectroscopic and molecular docking studies. Bioorg Chem. 2022;120:105656.
53. Tian ZY, Tian LY, Shi M, et al. Investigation of the interaction of a polyaminemodified flavonoid with bovine serum albumin (BSA) by spectroscopic
methods and molecular simulation. J Photoch Photobio B. 2020;209:111917.
54. Zaheri M, Azimirad M, Yekta R, et al. Kinetic and thermodynamic aspects on
the interaction of serum albumin with sodium hydrosulffte: spectroscopic
and molecular docking methods. J Photoch Photobio A. 2023;442:114804.
55. Chen X, Li H, Tian L, et al. Analysis of the physicochemical properties of acaricides based on lipinski’s rule of five. J Comput Biol. 2020;27(9):1–10.
56. Guerraoui A, Goudjil M, Direm A, et al. A rhodanine derivative as a potential
antibacterial and anticancer agent: crystal structure, spectral characterization,
DFT calculations, Hirshfeld surface analysis, in silico molecular docking and
ADMET studies. J Mol Struct. 2023;1280:135025.
57. Shafreen RB, Lubinska M, Rózańska A, et al. Human serum interactions withphenolic and aroma substances of Kaffir (Citrus hystrix) and Key lime (Citrus
aurantifolia) juices. J Lumin. 2018;201:115–122.
58. Potts, RO, Guy, RH. Predicting skin permeability. Pharm Res.
1992;9(5):663–669.
59. Adedotun IO, Abdul-Hammed M, Hamzat BA, et al. Molecular docking,
ADMET analysis, and bioactivity studies of phytochemicals from phyllanthus
niruri as potential inhibitors of hepatitis C virus NSB5 polymerase. J Indian
Chem Soc. 2022;99:100321.
60. Li X, Chen L, Cheng F, et al. In silico prediction of chemical acute oral toxicity
using multi-classification methods. J Chem Inf Model. 2014;54(4):1061–1069.

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