Exploring the Interactions of Ferrocenylmethylnucleobase Derivatives With BSA and HHb: Insights From Electrochemical, Spectroscopic, ADMET, in Silico Docking, and MD Simulations

Abstract

The present study has investigated the binding interactions of four N‐ferrocenylmethyl‐nucleobase derivatives: N1‐ferrocenylmethyladenine (FcMeAd), N1‐ferrocenylmethyl‐cytosine (FcMeCy), N1‐ferrocenylmethylthymine (FcMeTh), and N6,9‐bis(ferrocenyl‐methyl)adenine ((FcMe)2Ad), with human hemoglobin (HHb) and bovine serum albumin (BSA). A combination of absorption spectroscopy, cyclic voltammetry, molecular docking, and molecular dynamics simulations is employed to investigate these interactions. The obtained results demonstrated that these derivatives can bind to the target proteins, inducing conformational changes with the binding affinity order: FcMeCy > FcMeTh > FcMeAd > (FcMe)2Ad. Molecular docking studies identified the preferred binding sites and modes, revealing that hydrogen and hydrophobic predominantly facilitate the binding to BSA and HHb, also exhibiting π–π stacking interactions with FcMeCy and FcMeAd. Dynamic simulations of the FcMeCy‐BSA and FcMeCy‐HHb complexes, selected based on both experimental and theoretical results, further confirmed their stability within the protein binding pockets. The RMSD, RMSF, rGyr, SASA, H‐bonds, MolSA, and PSA parameters consistently indicated that FcMeCy maintains stability in the receptor binding site throughout the 100 ns simulation.

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Exploring the Interactions of Ferrocenylmethylnucleobase
Derivatives With BSA and HHb: Insights From Electrochemical,
Spectroscopic, ADMET, in Silico Docking, and MD Simulations
Mohammed Larbi BEN AMOR,[a,b]Elhafnaoui LANEZ,[a,c]Yahia Bekkar,*[a]Aicha ADAIKA,[a]
Touhami LANEZ,[a]Kaouther NESBA,[d]and Lazhar BECHKI[e,f]
The present study has investigated the binding interac-
tions of four N-ferrocenylmethyl-nucleobase derivatives:
N-ferrocenylmethyladenine (FcMeAd), N-ferrocenylmethyl-
cytosine (FcMeCy), N-ferrocenylmethylthymine (FcMeTh), and
N,-bis(ferrocenyl-methyl)adenine ((FcMe)Ad), with human
hemoglobin (HHb) and bovine serum albumin (BSA). A combina-
tion of absorption spectroscopy, cyclic voltammetry, molecular
docking, and molecular dynamics simulations is employed to
investigate these interactions. The obtained results demon-
strated that these derivatives can bind to the target proteins,
inducing conformational changes with the binding anity order:
FcMeCy >FcMeTh >FcMeAd >(FcMe)Ad. Molecular dock-
ing studies identied the preferred binding sites and modes,
revealing that hydrogen and hydrophobic predominantly facili-
tate the binding to BSA and HHb, also exhibiting π–πstacking
interactions with FcMeCy and FcMeAd. Dynamic simulations of
the FcMeCy-BSA and FcMeCy-HHb complexes, selected based
on both experimental and theoretical results, further conrmed
their stability within the protein binding pockets. The RMSD,
RMSF, rGyr, SASA, H-bonds, MolSA, and PSA parameters consis-
tently indicated that FcMeCy maintains stability in the receptor
binding site throughout the  ns simulation.
1. Introduction
In recent decades, the exploration of molecular structures and
the conformational dynamics of proteins, driven by the develop-
ment of advanced analytical techniques, has become a central
focus in biophysical chemistry. The interaction of small organic
and organometallic compounds with blood-associated proteins
plays a critical role in shaping their ADMET (absorption, distri-
bution, metabolism, excretion, and toxicity) properties, which in
turn determines their bioavailability and overall toxicity. Among
[a] M. L. BE. AMOR, E. LANEZ, Y. Bekkar, A. ADAIKA, T. LANEZ
Faculty of exact Sciences, Department of Chemistry, VTRS Laboratory,
University of El Oued, B.P.789, El Oued 39000, Algeria
E-mail: yahia-bekkar@univ-eloued.dz
[b] M. L. BE. AMOR
Faculty of Technology, Department of Process Engineering and
Petrochemical, University of El Oued, P.O. Box 789, El Oued 39000, Algeria
[c] E. LANEZ
Faculty of Biology, Department of Cellular and Molecular Biology, University
of El Oued, El Oued 39000, Algeria
[d] K. NESBA
Faculty of Arts and Languages, Department of English Language, University
of El Oued, El Oued 39000, Algeria
[e] L. BECHKI
Faculty of Mathematics and Material Sciences, Department of Chemistry,
Kasdi Merbah University, Ouargla 30000, Algeria
[f] L. BECHKI
VPRS Laboratory, Department of Chemistry, Faculty of Mathematics and
Material Sciences, Kasdi Merbah University, Ouargla 30000, Algeria
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/slct.202404678
the various proteins circulating in the bloodstream, hemoglobin,
and serum albumins stand out as multifunctional and highly
soluble entities. In addition to their numerous physiological
functions, these proteins are particularly important for their
eciency in drug delivery systems, especially for biologically
relevant drugs.[,]
Human hemoglobin (HHb), is a globular iron-containing pro-
tein responsible for the red color of blood and serves as a carrier
for carbon dioxide, oxygen, and electrons across various tissues
and organs.[,]HHb’s ability to carry and release therapeutics in
a targeted manner has made it a subject of considerable inter-
est in the design of drug delivery systems, especially for the
treatment of various diseases. Bovine serum albumin (BSA), a
globular protein derived from bovine sources, is often utilized
as an alternative to human serum albumin (HSA) due to its
cost-eectiveness and ease of availability.[,]
Concurrently, the eld of organometallic chemistry, par-
ticularly the chemistry of ferrocene and its derivatives, has
expanded signicantly, leading to applications in electro-
chemical sensing,[–]chiral catalysis,[–]and medicinal
chemistry.[,]Despite these advances, the potential appli-
cations of ferrocenylmethyl-nucleobases (FcNB) remain
underexplored, especially when compared to the extensively
studied chemistry of purely organic nucleobase derivatives.[–]
The rst synthesis of FcNB was reported by Chen in ,[]who
successfully synthesized N-(ferrocenylmethyl)adenine through
the reaction of -chloropurine with ferrocenylmethylamine in
methoxyethanol. Since then, various ferrocene-modied nucleo-
sides have been described, with particular focus on their redox
properties and potential biological activities.[,]Moreover, the
anticancer activity of FcNB has been assessed in vitro through
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Figure 1. Structures of FcMeAd, FcMeCy, FcMeTh, and (FcMe)Ad.
electrochemical and spectroscopic assays.[]These nucleobases
have also emerged as valuable building blocks in supramolec-
ular chemistry, where they can engage in base-pair hydrogen
bonding while retaining their redox-active properties.[]
Motivated by these ndings and our ongoing interest in
the biological activity of ferrocene derivatives, the current
study focuses on the binding interactions, molecular docking,
and dynamic simulation studies of four N-ferrocenylmethyl-
nucleobase derivatives with BSA and HHb. By employing a
combination of electrochemical, spectroscopic, and computa-
tional approaches, we aim to further explore the potential of
these ferrocenyl compounds as therapeutic agents and enhance
our understanding of their interactions with key plasma proteins.
2. Experimental Section
2.1. Synthesis
The compounds N-ferrocenylmethyladenine (FcMeAd), N-
ferrocenylmethylcytosine (FcMeCy), N-ferrocenylmethylthymine
(FcMeTh), and N,-bis(ferrocenylmethyl)adenine ((FcMe)Ad),
illustrated in Figure , were synthesized through a coupling
reaction. This reaction involved the well-established (ferro-
cenylmethyl)trimethylammonium iodide[]and the respective
nucleobase, as previously described by the research group.[,]
The analytical data, including NMR and FT–IR spectra for these
compounds, as well as their structural characterization by single-
crystal X-ray diraction (XRD),[,]are provided in Figures S–S
(Supporting Information).
2.2. General Procedure to Synthesize
Ferrocenylmethylnucleobases
A solution of trimethylferrocenylmethylammonium iodide ( mg,
. mmol) in  mL of water was stirred thoroughly, and a  mmol
of nucleobase was gradually introduced. The reaction was main-
tained at a temperature of – K for – hours and then
cooled to room temperature. To remove residual quaternary ammo-
nium salt, the mixture was repeatedly extracted with distilled water.
Finally, the desired ferrocenylmethylnucleobase was isolated by
either chromatography or recrystallization.
2.3. Chemicals and Reagents
All reagents and solvents utilized in this study were of analytical
grade, sourced from various commercial suppliers, and employed
without further purication. Bovine serum albumin (BSA) was
sourced from Merck and used as received, with its concentration
determined by an extinction coecient of . M−cm−at
 nm.[]Stock solutions were stored at  °C and utilized within
 days of preparation. Human hemoglobin (HHb) was acquired
from Sigma–Aldrich and was also utilized without further purica-
tion. A phosphate buer solution (PBS) was prepared using sodium
dihydrogen phosphate and disodium hydrogen phosphate (both
from Sigma–Aldrich) in double-distilled water, and was employed
to maintain a physiological pH of .. Tetrabutylammonium tetrau-
oroborate (BuNBF) (electrochemical grade, %; Sigma–Aldrich)
served as the supporting electrolyte. Nitrogen gas (research grade,
.%) was provided by Linde Gaz Algeria.
3. Materials and Measurements
3.1. In Vitro Experiments
Voltametric assays were conducted with a PGZ voltammeter
utilizing Volta Master  V . software (Radiometer Analytical
SAS, France). Tetrabutylammonium tetrauoroborate (BuNBF)
served as the supporting electrolyte at a consistent concentra-
tion of . M. Nitrogen gas was bubbled through the solution to
remove air. The experiments employed a three-electrode electro-
chemical cell with a  ml capacity, featuring a glassy carbon (GC)
working electrode with a geometric area of . cm,aplat-
inum wire as the counter (auxiliary) electrode, and a Hg/HgCl
paste-covered wire as the reference electrode.
Absorption spectra were measured using a UV–Vis spectrom-
eter (Shimadzu , Japan) in a  ml cell with an optical path
length of  mm.
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Figure 2. Cyclic voltammograms of FcNB derivatives in . mM % ethanol/PBS were recorded in both the absence and presence of increasing
concentrations of BSA from  to  µMin  µMincrements, at a potential scan rate of . V s−. Inset: Plots of log(i/(i-i)) versus log(/[BSA]) used to
calculate the binding constants.
3.2. In Silico Experiments
3.2.1. Cheminformatics Prediction
For all compounds, the structures were drawn using ChemDraw
software.[]Each structure was veried and cleaned using the
structure icon, then saved as MDL Mol les (*.mol) for upload-
ing to the PASS prediction website.[]Subsequently, the “predict
activity” icon was selected to obtain the predicted activities
along with their corresponding Pa values.
In the SwissADME server,[]the import icon in the molecu-
lar sketcher was selected, opening a new window to choose the
previously prepared structure. The structure was displayed in the
molecular sketcher and then converted to the SMILES format.
Subsequently, the “Run” icon was then clicked to generate the
ADMET parameters and related values.
3.2.2. Molecular Docking
Ligand Preparation: The chemical structures of FcMeAd,
FcMeCy, FcMeTh, and (FcMe)Ad were optimized using the
Gaussian  software package[ ]with density functional theory
(DFT) at the BLYP level and the –++G(d,p) basis set.[,]
Receptor Preparation: The bovine serum albumin (BSA) recep-
tor (PDB ID V)[ ]and the human hemoglobin (HHb) receptor
(D)[]were selected as the targets for this investigation. The
D structures of the target receptors were retrieved from the
RCSB Protein Data Bank.[]Initial preparation of the receptor
involved using AutoDock Tools (ADT),[ ]which involved remov-
ing all water molecules and co-factors to obtain the raw protein
structure. Subsequently, the active site was dened using Mole-
gro Virtual Docker software based on the initial position of
the co-crystallized ligand. Polar hydrogens were then added,
followed by the assignment of Kollman charges. All docking
studies were conducted on a computer equipped with a Pentium
. GHz processor and . GB of RAM running the Windows 
operating system.
3.2.3. Molecular Dynamic Simulation
Molecular dynamics (MD) simulations were performed using the
Desmond software from Schrödinger LLC,[]employing an NPT
ensemble at  K and  bar pressure. Each simulation spanned
 ns with a  ps relaxation period for the ligands, utilizing
the OPLS force eld.[]Long-range electrostatics were calculated
using the particle mesh Ewald approach,[]with a Coulomb
interaction cut-o set at . Å. Water molecules were explicitly
represented by the simple point charge (SPC) model.[]Pressure
and temperature were managed using the Martyna-Tuckerman-
Klein method (. ps coupling constant) and the Nose-Hoover
chain algorithm, respectively.[]Nonbonded interactions were
computed with an r-RESPA integrator, which updated short-
range interactions at each step while long-range interactions
were updated every three steps. Trajectories were recorded
at . ps intervals, and ligand-receptor interactions were ana-
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Tab le 1 . The linear equations of log(i/(i-i)) versus log(/[BSA]), binding
constant, and binding free energy values of FcMeAd-BSA, FcMeCy- BSA,
FcMeTh- BSA, and (FcMe)Ad- BSA obtained from CV data at pH =. and
T= K.
Adduct Equation RK×
[M−]
−G
[kJ mol−]
FcMeAd-BSA y =.x +. . . .
FcMeCy-BSA y =.x +. . . .
FcMeTh-BSA y =.x +. . . .
(FcMe)Ad-
BSA
y=.x +. . . .
lyzed utilizing the Simulation Interaction Diagram tool within
Desmond.
4. Results and Discussion
4.1. Cyclic Voltammetry Study
Figure depicts the cyclic voltammetric behavior of . mM
FcNB derivatives under two conditions: in the absence and pres-
ence of BSA at a bare GC electrode. The introduction of BSA
led to a signicant negative shift in the anodic peak potential
and a considerable reduction in the anodic peak current density.
This observed reduction in current density can be attributed to
the formation of slowly diusing adducts,[]which diminish the
concentration of free compounds necessary for charge transfer
reactions.[]The physical interaction between the FcNB deriva-
tives and BSA accounts for the negative shift in anodic peak
potential.[,]
To determine the binding free energies and binding con-
stants of the compounds, Equation () was employed. This
equation correlates the reduction in the anodic peak current
density of the adducts to the free FcNB derivatives. In this
equation, i represents the anodic peak current density in the
presence of BSA, idenotes the anodic peak current density in
the absence of BSA, and [BSA] indicates the concentration of
BSA.
log 1
[BSA]=log i
i0−i+log K()
The binding number, which indicates the stoichiometric ratio
of the interaction, was determined to be , indicating the for-
mation of a : association complex between the inclusion
complexes and BSA.[]The value was derived from the slope of
the linear plot of log(i/(i-i)) versus log(/[BSA]), as shown in the
inset of Figure .
The Binding constant values for FcMeAd, FcMeCy, FcMeTh,
and (FcMe)Ad with BSA were derived from the y-intercept of
theplotandwerefoundtobe.×,.×,.
×and . ×M−, respectively. The binding energies
(G, in kJ mol−), as listed in Table , were calculated using
Figure 3. Cyclic voltammograms of the free compounds (. mM) and their
BSA adducts ( µM), Scan rate  mV s−.
Figure 4. Redox process of the studied compounds with BSA: FcNB
represents FcMeAd, FcMeCy, FcMeTh, and (FcMe)Ad.
Equation ():[]
G=−RT ln K ()
where T is the absolute temperature equal to  K, R is the gas
constant, . J mol−K−.
4.1.1. Ratio of Binding Constants
The behavior of . mM solutions of FcMeAd both in the absence
and presence of  µMBSA, is illustrated by the cyclic voltammo-
grams presented in Figure (for FcMeCy, FcMeTh, and (FcMe)Ad
see Figure S, Supporting Information). These voltammograms
enable the calculation of the binding constant ratios between
the reduced form of FcNB derivatives and BSA, and the oxi-
dizedform[FcNB]
+and BSA. The binding constant ratios can be
determined by analyzing the shifts in anodic and cathodic peak
potentials induced by the addition of BSA.
When BSA induces shifts in both anodic and cathodic peak
potentials, the equilibria depicted in Figure can be utilized.
Equation (), derived from the Nernst equation applied to these
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Tab le 2. Electrochemical data of the free and BSA-bound FcMeAd,
FcMeCy, FcMeTh, and (FcMe)Ad were used to calculate the ratio of the
binding constants.
Sample code Epa Epc E[V] E[V] Kox/Kred
FcMeAd . . . . .
FcMeAd-BSA . . .
FcMeCy . . . . .
FcMeCy- BSA . . .
FcMeTh . . . . .
FcMeTh-BSA . . .
(FcMe)Ad . . . . .
(FcMe)Ad-
BSA
. . .
equilibria, describes the redox interactions of the compounds
under investigation with BSA, as shown in Figure .Theterm
FcNB includes FcMeAd, FcMeCy, FcMeTh, and (FcMe)Ad. These
ndings elucidate the interaction mechanisms between the
ferrocenylmethylnucleobase derivatives and BSA.
Equation () denes Ef
0and Eb
0as the formal potentials
for the FcNB+/FcNB redox couple in their free and BSA-bound
states, respectively. The shifts in these formal potentials, derived
from the cyclic voltammograms shown in Figure , are detailed
in Table .UsingtheseEvalues from Table , the binding
constant ratios were calculated according to Equation (). The
results, summarized in Table , indicate that the oxidized forms
of the FcNB derivatives bind to BSA with a slightly higher anity
than their reduced forms.
E0=E0
f−E0
b=E0(FcNB)−E0(FcNB −BSA)=0.06 log Kox
Kred
()
4.1.2. Diusion Coecients
Figure illustrates the electrochemical behavior of FcMeAd, (for
FcMeCy, FcMeTh, and (FcMe)Ad check Figure S, Supporting
Information) at varying scan rates, revealing distinct and stable
Figure 6. iversus v/plots of FcMeAd (. mM) in the absence and
presence of BSA at scan rates ranging from . to . V s−under the
experimental conditions of Figure .
anodic peaks. The diusion coecients for both the free and
BSA-bound forms of these compounds were calculated using the
Randles–Sevcik Equation ():[]
i=2.69 ×105n3/2SCD1/2v1/2()
where i represents the peak current (A), n is the number of elec-
trons transferred during oxidation, S is the electrode surface area
(cm), C is the bulk concentration (mol cm−) of the electroactive
species, D is the diusion coecient (cms−), and v is the scan
rate (V s−).
The linear relationship between the square root of the scan
rates and the anodic peak current density (Figure ) indicates
a diusion-controlled redox process.[]Diusion coecients for
both the free and BSA-bound FcMeAd (The rest of the lig-
ands in Figure S, Supporting Information) were derived from
the slopes of the linear regressions based on Equation (). The
lower diusion coecients observed for the BSA-bound ligands,
in comparison to their free counterparts, further support the
formation of adducts[](Table ).
Figure 5. Cyclic voltammetric behavior of . mM FcMeAd on GC electrode in the absence and the presence of  µMBSA in % ethanol/BPS at pH =.
and scans rates ., ., ., . and . V s−.
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Tab le 3 . Diusion constants values of the free and BSA-bound forms of
FcMeAd, FcMeCy, FcMeTh, and (FcMe)Ad.
Adduct Equation RD[cm
s−]
FcMeAd y =.x +. . . ×−
FcMeAd-BSA y =.x +. . . ×−
FcMeCy y =.x +. . . ×−
FcMeCy-BSA y =.x +. . . ×−
FcMeTh y =.x +. . . ×−
FcMeTh-BSA y =.x +. . . ×−
(FcMe)Ad y =.x +. . . ×−
(FcMe)Ad-
BSA
y=.x +. . . ×−
4.2. Absorption Spectral Study
4.2.1. Bovine Serum Albumin Interaction Analysis
The study on the binding interactions between BSA and the
compounds FcMeAd, FcMeCy, FcMeTh, and (FcMe)Ad was con-
Tab le 4 . Binding free energy and binding constant values for FcMeAd,
FcMeCy, FcMeTh, and (FcMe)Ad compounds with BSA from UV–Vis data
at pH =. and T = K.
Adduct Equation RK×
[M−]
−G
[kJ mol−]
BSA-FcMeAd y =−.x–. . . .
BSA-FcMeCy y =−.x–. . . .
BSA-FcMeTh y =−.x–. . . .
BSA-(FcMe)Ad y =−.x–. . . .
ducted using a . MPBS (pH .) containing % ethanol. To
each . mMsolution of the compounds, incremental portions
of the BSA solution were added. The resulting mixtures were
scanned within the – nm wavelength range. Since BSA
does not absorb within this wavelength range, a notable peak at
 nm, attributed to the π→π∗transition in the conjugated
ring of the ferrocene moiety, showed a decrease in intensity with
the continuous addition of BSA (Figure ).
Figure 7. Absorbance spectra of BSA–FcNB complexes. Inset: Plots of /[BSA] versus A/(A–A) are used to calculate the binding constants.
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Figure 8. Absorption spectral change of HHb (in . mMPBSatpH.)in
thepresenceofFcNBatK.
The binding constants (K) were calculated from the absorp-
tion data using the Benesi–Hildebrand Equation ().[]
A0
A−A0
=ε0
ε−ε01+1
K[BSA]()
In Equation (), A0and Arepresent the absorbances of the
studied ligands and their complexes with BSA, respectively, while
εand εare respectively their extinction coecients. A plot of
/[BSA] versus A/(A–A) gave a slope of ε/(ε−ε)Kband a ‘ y’
intercept equal to ε/(ε−ε), where Kis the ratio of the slope to
the y-intercept. The binding constants obtained and their corre-
sponding free binding energies calculated using Equation ()[]
are summarized in Table :
4.2.2. Human Hemoglobin Interaction Analysis
Figure illustrates the UV-Vis spectral changes resulting from
increasing concentrations of FcNB. The normal hemoglobin spec-
trum can be divided into three regions.[]The rst region, at
 nm, corresponds to globin absorption due to π–π*transi-
tions of the carbonyl (C═O) groups in amino acids, specically
tryptophan and tyrosine, resulting in a peak at . nm.[]The
second region, known as the B or Soret band, is observed at
 nm and arises from π–π* transitions of the heme iron por-
phyrin complex within the hydrophobic pocket.[]The third
region, the Q band, spans – nm and is associated with the
oxy- and deoxy-forms of heme.[]
Upon the addition and gradual increase of FcNB concen-
tration to the hemoglobin solution, a systematic increase in
the globin absorption peak at  nm was observed, while
the absorbance peaks in the B and Q bands at  nm and
– nm, respectively, decreased. The hyperchromic shift at
 nm is attributed to the increased concentration of the FcNB
molecule, which shows its λmax at  nm.[]Conversely, the
hypochromic shift in the B and Q bands indicates the interaction
between hemoglobin and the ferrocene derivatives.[]The FcNB
molecules access the heme group within the hemoglobin, alter-
Figure 9. Absorbance spectra of  mMHHb in . MPBS at pH =. in the absence and presence of increasing concentration of FcNB derivatives.
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Tab le 5 . The linear equations of /[FcNB] versus A/(A-A), binding con-
stant and binding free energy values of FcMeAd-HHb, FcMeCy- HHb,
FcMeTh- HHb, and (FcMe)Ad- HHb obtained from ES data at pH =.
and T = K.
Adduct Equation RK×
[M−]
−G
[kJ mol−]
FcMeAd-HHb y =−.x–. . . .
FcMeCy-HHb y =−.x–. . . .
FcMeTh-HHb y =−.x–. . . .
(FcMe)Ad-HHb y =−.x–. . . .
ing its environment and exposing it to the aqueous medium.[]
The isosbestic point at  nm suggests an equilibrium binding
of a two-state system comprising protein-bound and free drug
molecules in the FcNB-hemoglobin complex.[]The observed
spectral changes in the hemoglobin spectrum, induced by the
FcNB drug, are attributed to non-covalent molecular interac-
tions between hemoglobin and FcNB, resulting in alterations
to the protein’s microenvironment.[]Figure presents a
zoomed view of the Q region, highlighting the interactions
between FcMeAd, FcMeCy, FcMeTh, and (FcMe)Ad with human
hemoglobin (Table ).
The binding constants of the FcNB-HHb adducts were deter-
mined using the Benesi-Hildebrand Equation (), which is based
on the decrease in absorbance upon the addition of FcNB com-
pounds to an HHb.[]The plots of /[FcMeNb] versus A/(A–A)
are presented in Figure .
Tab le 6 . Prediction of BSA and HHb inhibition activity of FcMeAd, FcMeCy,
FcMeTh, and (FcMe)Ad.
Compound Activity/Pa
BSA Inhibition HHb Inhibition
FcMeAd . .
FcMeCy . .
FcMeTh . .
(FcMe)Ad . .
4.3. Cheminformatics Prediction
4.3.1. PASS Online
The online tool PASS, standing for Prediction of Activity Spectra
for Substances, is readily accessible at no cost and is specically
designed to predict the potential biological activities of drug-like
molecules.[]Its functionality hinges on an intricate analysis of
structure-activity relationships (SAR) within an extensive repos-
itory comprising over  organic compounds.[]As such,
PASS serves as a pivotal resource for rening chemical synthesis
strategies and guiding the execution of biological assays.[]In
the present investigation, PASS[ ]was employed to forecast the
BSA and HHb inhibition activities of FcMeAd, FcMeCy, FcMeTh,
and (FcMe)Ad, with the ndings summarized in Table .
In evaluating BSA inhibition activity, both FcMeCy and
FcMeTh exhibit relatively comparable values, with FcMeCy show-
Figure 10 . Plots of A/(A−A) versus /[FcNB] were used to calculate the binding constants of ligands FcMeAd, FcMeCy, FcMeTh, and (FcMe)Ad with HHb.
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Tab le 7 . Physicochemical, pharmacokinetics, and medicinal chemistry properties of FcMeAd, FcMeCy, FcMeTh, and (FcMe)Ad using SwissADME server.
Compound MW
[g mol−]
HBA HBD TPSA
[Å]
Consensus
Log Po/w*)
MR GI
Absorp-
tion
BBB Per-
meant
P-gp
Sub-
strate
Lipinski Bioavailability
Score
PAI NS
(alert)
Brenk
(alert)
FcMeAd .   . . . High Yes Yes Yes .  
FcMeCy .   . . . High Yes No Yes .  
FcMeTh .   . . . High Yes No Yes .  
(FcMe)2Ad .   . . . High No Yes Yes**).  
MW, Molecular Weight; HBA, Num. H-Bond Acceptors; HBD, Num. H-Bond Donors; MR, Molar Refractivity; TPSA, Topological Polar Surface Area; GI,
Gastrointestinal; BBB, Blood-Brain Barrier; P-gp, P Glycoprotein
*) Average of ve predictions
**)  MW >, PAINS: Pan-assay Interference Compounds.
ing slightly higher inhibition at ., compared to FcMeTh’s
.. In contrast, FcMeAd and (FcMe)Ad display somewhat
lower BSA inhibition activities than FcMeCy and FcMeTh.
When examining HHb inhibition, FcMeTh demonstrates the
highest inhibitory activity with a value of ., indicating its sig-
nicant potential in forming complexes with the target receptor.
In comparison, FcMeAd and (FcMe)Ad show lower HHb inhi-
bition values, suggesting a diminished impact on HHb activity
relative to the other two compounds.
4.3.2. Drug-Likeness Prediction
Drug likeness is conceptualized as a delicate balance of
molecular properties and structural characteristics, indicating
the degree of similarity between specic compounds and
established drugs.[]This assessment is guided by various
molecular properties, including hydrogen bonding, electronic
distribution, hydrophobicity, molecular weight, bioavailability,
pharmacophore constituents, reactivity, toxicity, and metabolic
stability.[]One of the widely employed tools for estimating
solubility and permeability, thereby predicting the potential of
compounds as drug candidates, is Lipinski’s Rule.[]Accord-
ing to Lipinski’s Rule of , compounds with more than ve
hydrogen bond donors (the sum of NH and OH groups), a
molecular weight exceeding , a Log P value above , and
more than ten hydrogen bond acceptors (the sum of nitrogen
and oxygen atoms) are more likely to experience poor absorp-
tion or permeation. The drug-likeness properties of FcMeAd,
FcMeCy, FcMeTh, and (FcMe)Ad, as assessed using the Swis-
sADME server,[]are depicted in Table . All tested compounds
(FcMeAd, FcMeCy, FcMeTh, and (FcMe)Ad) comply with Lipin-
ski’s rule of ve, indicating good drug-like properties, and exhibit
high gastrointestinal absorption, suggesting suitability for oral
administration. They all have a bioavailability score of ., indi-
cating moderate bioavailability. Notably, FcMeAd and (FcMe)Ad
can cross the blood-brain barrier, unlike FcMeCy and FcMeTh.
Additionally, FcMeAd, FcMeTh, and FcMeCy are P-glycoprotein
substrates, while (FcMe)Ad is not. FcMeAd and (FcMe)Ad have
higher molar refractivity values, which could inuence their
pharmacokinetics and interactions with biological targets. None
of the compounds show PAINS alerts, indicating low nonspecic
biological activity. However, Brenk alerts, which predict poten-
tial instability or toxicity, were present in all compounds to
varying degrees. Despite these alerts, the compounds, particu-
larly FcMeAd and (FcMe)Ad show promise as drug candidates,
although they may require further optimization to enhance
stability and reduce toxicity.[]
4.3.3. ADMET Prediction
The ADMET properties of the compounds FcMeAd, FcMeCy,
FcMeTh, and (FcMe)Ad were predicted using the ADMETlab
server and the DataWarrior program, with the results docu-
mented in Tables and . Regarding absorption, FcMeCy shows
the highest membrane permeability among the tested com-
pounds, while FcMeTh and FcMeAd are lower. Both FcMeCy
and FcMeTh can inhibit P-gp, potentially enhancing bioavail-
ability, while (FcMe)Ad is a P-gp substrate.[]All compounds
exhibit high human intestinal absorption (HIA), with (FcMe)Ad
having the highest value. In distribution, FcMeCy has the high-
est plasma protein binding, whereas FcMeAd and (FcMe)Ad
show signicant blood-brain barrier (BBB) permeability, indi-
cating potential CNS eects.[]For metabolism, FcMeAd and
(FcMe)Ad are metabolized by P CYPA and P CYP,
while FcMeAd and FcMeTh are substrates for P CYP, aect-
ing metabolic stability.[]In terms of elimination, FcMeTh has
the longest half-life and highest clearance rate, indicating rapid
elimination, whereas FcMeAd and (FcMe)Ad have moderate
values, suggesting longer retention in the body.
The domain of drug toxicology is pivotal in preclinical
research due to its signicant impact on drug attrition rates
during both the discovery and development stages.[]Conse-
quently, accurate in silico predictions of compound toxicity are
crucial for minimizing costs and expediting the drug discovery
and development process.[]The toxicity proles of FcMeAd,
FcMeCy, FcMeTh, and (FcMe)Ad were rigorously evaluated.
Quantitative assessments were conducted using the Pro-tox-III
webserver (https://comptox.charite.de/protox/),[]and qual-
itative evaluations were performed via the ADMETlab server
(ADMETlab . (scbdd.com)).[]The ndings are detailed in
Table .
The safety proles of the compounds were assessed through
four key toxicity descriptors: cardiotoxicity, cytotoxicity, immuno-
toxicity, and mutagenicity as determined by the Ames test.
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Tab le 8 . ADME properties of the compounds FcMeAd, FcMeCy, FcMeTh, and (FcMe)Ad using ADMETlab server.
Compound Absorption Distribution Metabolism; P CYP Elimination
Caco- p
[cm s−]
Pgp [I] Pgp [S] HIA PPB% VD
[L Kg−]
BBB A   T/ hCLml
min−
kg−
ISI SIS
FcMeAd −. . . . . . . . . . . . . . .
FcMeCy −. . . . . . . . . . . . . . .
FcMeTh −. . . . . . . . . . . . . . .
(FcMe)2Ad −. . . . . . . . . . . . . . .
PPB, Plasma Protein Binding; VD, Volume Distribution; BBB, Blood–Brain Barrier; T / , Half-Life Time; CL, Clearance Rate; I, Inhibitor; S, Substrate.
Tab le 9 . Toxicity risk assessment of the compounds FcMeAd, FcMeCy, FcMeTh, and (FcMe)Ad using Pro-tox-III and ADMETlab servers.
Compound Mutagenicity Cytotoxicity Cardiotoxicity Immunotoxicity Toxicity*)
hERG H-HT AMES
FcMeAd None None None None . . .
FcMeCy None None None None . . .
FcMeTh None None None None . . .
(FcMe)2Ad None None None None . . .
hERG: Human ether-à-go-go related gene; H-HT: Human Hepatotoxicity; AMES: Ames Mutagenicity
*) calculated by ADMETlab server.
Cardiotoxicity is a crucial consideration due to its potential
impact on cardiac ion channel function, particularly the human
ether-a-go-go-related gene (hERG) potassium channel, which is
essential for regulating cardiac action potentials.[]The data
indicates that none of the tested compounds demonstrated
signicant cardiotoxic eects. Specically, the hERG values for
FcMeAd, FcMeCy, FcMeTh, and (FcMe)Ad were ., .,
., and ., respectively, suggesting that these compounds
pose minimal risk of cardiotoxicity compared to established
cardiotoxic agents.[]Cytotoxicity assesses the potential of
compounds to cause cellular damage or death.[]The results
revealed that all compounds exhibited no signicant cytotoxic-
ity, implying that they do not impair cell viability at the tested
concentrations. This nding is indicative of a low propensity for
these compounds to induce cellular damage or promote cell
death. The evaluation of immunotoxicity determines whether
compounds adversely aect immune system function.[]The
results indicated no signicant immunotoxin eects for any
of the compounds, suggesting that they do not compromise
immune system activity. The Ames test, which evaluates the
potential of compounds to cause genetic mutations, revealed
that FcMeAd, FcMeCy, FcMeTh, and (FcMe)Ad exhibited no
mutagenic activity, with AMES values of ., ., ., and
., respectively.
4.4. Molecular Docking Results
To elucidate the specic binding sites and interactions between
FcMeAd, FcMeCy, FcMeTh, and (FcMe)Ad with BSA and HHb, and
to validate the cyclic voltammetry and absorption spectroscopy
results, semi-exible docking was performed using AutoDock
. and AutoDock Vina.[,]The crystal structure of BSA (PDB
ID: V)[]and HHb (PDB ID: D)[ ]with a resolution of .
and . Å was retrieved from the Protein Data Bank.[]For the
docking calculations involving FcNB derivatives, the grid size
was set to  × × Å in the x, y, and z directions, with a
grid point spacing of . Å for the two target proteins. The
conformation with the lowest binding energy was selected for
further analysis.[]All the visualizations of the interactions were
generated using the Protein-Ligand Interaction Proler (PLIP)
web service.[]
4.4.1. Bovine Serum Albumin Docking Analysis
The energy scoring parameters, including binding energy, inhi-
bition constant, intermolecular energy, van der Waals (vdW), and
electrostatic energies,[]are presented in Table .
The negative binding energies for all studied ligands indi-
cate that the interactions between BSA and FcNB derivatives are
spontaneous.[]Molecular docking results indicate that hydro-
gen bonding, hydrophobic interactions, and π-πstacking play
signicant roles in the binding process. Figure  depicts the
interaction of FcMeAd, FcMeCy, FcMeTh, and (FcMe)Ad with
residues in the active site of BSA, with oxygen, nitrogen, and
iron atoms colored red, blue, and brown, respectively. Hydro-
gen bonds are shown in blue, hydrophobic interactions in short
dashes, and π–πstacking interactions in dashes. Detailed inter-
action residues, distances, and interaction types are summarized
in Table S (Supporting Information).
Table S (Supporting Information) reveals that all compounds
interact with BSA through hydrogen bonding and hydrophobic
interactions. Specic residues and amino acids involved in these
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Tab le 1 0. Docked results with the BSA interacting residues after  runs of docking.
Adduct Lowest Binding
Energy
[kJ mol−]
Inhibition
Constant
(. K),
µM
Final Inter-
molecular
Energy
[kJ mol−]
vdW +H-Bond +
Dissolve Energy
[kJ mol−]
Electrostatic
Energy
[kJ mol−]
Final Total
Internal
Energy
[kJ mol−]
FcMeAd-BSA −. . −. −. −. −.
FcMeCy-BSA −. . −. −. −. −.
FcMeTh-BSA −. . −. −. −. −.
(FcMe)Ad-BSA −. . −. −. −. −.
Figure 11. Best docking poses for BSA-FcMeAd, BSA-FcMeCy, FcMeTh, and BSA-(FcMe)Ad generated with PLIP web service illustrating the H-bonds,
hydrophobic and π-πstacking interactions.
interactions are listed in Table S (Supporting Information), which
also shows the distances between hydrogen and receptor atoms
(H-A). Notably, for the BSA-FcMeAd and BSA-FcMeCy complexes,
molecular docking results indicate a π–πstacking interaction
between the positively charged amino acid residues Tyr- and
Phe-, and the benzene ring, with distances of . and . Å,
respectively.
4.4.2. Human Hemoglobin Docking Analysis
A molecular docking study was conducted to predict the con-
formational structures and binding anities of FcMeAd, FcMeCy,
FcMeTh, and (FcMe)Ad with HHb. Among the  docked
poses generated, the pose with the lowest binding energy
was selected as the best representative for each compound.[]
The docking results indicate that all compounds bind within
the central cavity of HHb, specically near the αandα
chains.
The lowest energy docked poses were found to have binding
anities of −., −., −., and −. kcal mol−for FcMeTh,
(FcMe)Ad, FcMeAd, and FcMeCy, respectively. Figure  illus-
trates the lowest energy conformation of FcMeCy, with the other
compounds’ lowest energy conformations presented in Figure
S (Supporting Information).
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Figure 12 . D and D view of the best docking pose for the interaction of FcMeCy with HHb.
The free binding energy values, ranging from −. to
−. kcal mol−, suggest moderate binding anities between
the FcNB derivatives and HHb. Also, the negative values indi-
cate spontaneous binding, with FcMeCy showing the highest
anity among the studied compounds.[]The interactions
between HHb and the studied compounds are primarily driven
by van der Waals forces, hydrogen bonding, and π–πstack-
ing interactions, as depicted in the D interaction diagrams.
These ndings are consistent with the results of spectroscopic
interaction studies, further validating the observed binding
mechanisms.
4.5. Molecular Dynamics Simulation
The most promising compounds for each target were identi-
ed based on docking scores and predicted biological activi-
ties. These selected protein-ligand complexes underwent  ns
molecular dynamics (MD) simulations to assess their stability.[]
Structural and dynamic properties were analysed through sev-
eral key metrics: root mean square deviation (RMSD), root
mean square uctuation (RMSF), radius of gyration (rGyr), and
solvent-accessible surface area (SASA). RMSD was employed
to quantify overall structural stability,[]while RMSF provided
insights into local uctuations within the protein chain and
ligand.[]The rGyr metric assessed the compactness of the
protein structure,[]and SASA evaluated the exposure of the
complex to the solvent, oering additional information on its
stability.[]A detailed analysis of these receptor-ligand interac-
tions is presented below:
4.5.1. Bovine Serum Albumin and FcMeCy
The stability of the receptor-ligand complex was evaluated by
comparing RMSD and RMSF values relative to the unbound
protein structure. The RMSD prole for the complex is illus-
trated in Figure a. The unbound BSA exhibited slightly greater
deviations than its ligand-bound counterpart. Notably, the BSA-
FcMeCy complex achieved equilibrium more swiftly, as indicated
by average RMSD values of . ±. nm for the com-
plex and . ±. nm for the unbound BSA (Figure a).
These ndings suggest that FcMeCy reduces structural devi-
ations in BSA, thereby enhancing the stability of the BSA-
FcMeCy complex.[]Protein uctuations during the simulation,
depicted in Figure a, revealed notable deviations (∼. Å) in
residues Trp-, Trp-, Gln-, Asp-, Glu-, Asp-, and
Leu-, which are involved in ligand interactions. The Lig-
andRMSF,showninFigurea, exhibited uctuations below
. Å, which are within acceptable limits.[]The secondary
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(a) (b)
Figure 13 . RMSD plots for BSA-FcMeCy a) and HHb-FcMeCy b) complexes.
(a) (b)
Figure 14. RMSF plots of bovine serum albumin a) and human hemoglobin b) with FcMeCy in MD simulations study.
(a) (b)
Figure 15 . RMSF plots of FcMeCy when run with bovine serum albumin a) and human hemoglobin b) in MD simulations study.
structure elements (SSE) distribution analysis (Figure S, Sup-
porting Information) indicated that the SSE percentage for the
FcMeCy-bound and unbound protein were .% and .%,
respectively, reecting negligible changes over the  ns sim-
ulation period.[]Ionic interactions in the FcMeCy-BSA complex
and the formation of water bridges with His-, Arg-, and
Asp- were observed (Figure a). Protein-ligand contacts
over the trajectory are presented in Figure S (Supporting Infor-
mation), with residues exhibiting multiple specic interactions
with FcMeCy, indicated by darker orange shades. Overall, ve key
parameters were analyzed to elucidate the stability of FcMeCy
within the BSA protein during the  ns simulation (Figure a).
The radius of gyration showed signicant uctuations at  ns,
stabilizing thereafter with uctuations ranging from . to
. Å. No intramolecular interactions were detected through-
out the simulation period. The SASA plot demonstrated initial
uctuations up to  ns, followed by stabilization for the remain-
der of the simulation. MolSA and PSA plots further supported
the stability of FcMeCy during the simulation.
4.5.2. Human Hemoglobin and FcMeCy
Figure b presents the RMSD plot of FcMeCy in complex with
HHb. The RMSD data for both the free and FcMeCy-bound HHb
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Figure 16 . Histogram showing the interaction fraction of FcMeCy with BSA a) and HHb b) during  ns simulation.
Figure 17 . Ligand properties during  ns simulations for compound FcMeCy.
reveal an initial rise in deviation, peaking around . nm at
 ns, followed by a stabilization phase from  ns onwards.
This indicates that the ligand actively engaged in stabilizing
interactions with HHb early in the simulation. The slightly lower
RMSD values observed for the HHb-FcMeCy complex com-
pared to the free protein suggest that the complex achieved
marginally greater stability. Among the protein residues, only
Leu-, Val-, and Ser- exhibited uctuations near . Å,
while Tyr- displayed a higher uctuation of approximately . Å
(Figure b). Notably, these uctuations occurred in residues that
are not directly involved in ligand binding. The RMSF analysis of
FcMeCy (Figure b) demonstrated stability, with all uctuations
remaining below . Å.
The SSE percentage (Figure S, Supporting Information)
showed minimal change throughout the  ns simulation, fur-
ther indicating the structural integrity of the complex. The
protein-ligand interaction analysis (Figure b) revealed a pre-
ponderance of hydrophobic interactions and water bridges, with
Asp- emerging as the most frequent interaction partner as
indicated by the dark orange shades in Figure S (Support-
ing Information). Additionally, the radius of gyration exhibited
uctuations between . and . Å (Figure b), while other
parameters, including SASA, MolSA, and PSA, consistently sup-
ported the stable behavior of FcMeCy within the HHb complex
over  ns of the simulation.
5. Conclusion
This study oers a detailed characterization of the binding inter-
actions between four N-ferrocenylmethylnucleobase and the
proteins bovine serum albumin (BSA) and human hemoglobin
(HHb), utilizing cyclic voltammetry, spectroscopic technique, and
in silico approaches. The binding free energies derived from
these analyses indicate that the interactions are thermody-
namically favorable and spontaneous, primarily facilitated by
hydrogen bonding and hydrophobic interactions. The binding
free energies, as determined through absorption spectroscopy,
ranged from −. to −. kJ mol−1, with binding constants
(K) in the order of M−for both BSA and HHb. These ndings
suggest a moderate binding anity, indicating that although the
ferrocenyl derivatives exhibit modest interaction with the pro-
teins, they are nonetheless capable of facilitating transport and
delivery within biological contexts. The order of binding anity,
observed as FcMeCy >FcMeTh >FcMeAd >(FcMe)Ad, reects
the capacity of these compounds to induce conformational
alterations in the protein structures. Cyclic voltammetry results
further suggest that electrostatic interactions predominantly
govern the binding of these compounds to BSA. Molecular dock-
ing identied the primary binding sites of the ligands within
the cavity of BSA and in proximity to the αandα subunits
of HHb, with hydrophobic forces and hydrogen bonds serving
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as the principal stabilizing interactions. Moreover, the molecular
dynamics simulations highlighted the complexation process and
consequent conformational changes, particularly with FcMeCy,
as evidenced by alterations in the compactness of the receptor
structures. Collectively, this research advances our understand-
ing of the binding mechanisms of organometallic compounds
with heme proteins and holds potential implications for the
design of future therapeutic agents.
Author Contributions
M.L.B.A. did software, formal analysis, investigation, methodol-
ogy, writing reviewing, and editing. E.L. performed supervision,
validation, reviewing and editing. Y.B. performed validation,
writing, reviewing, and editing. A.A. did software, writing, and
editing. T.L. performed supervision, validation. K.N. did reviewing
and editing. L.B. performed supervision, validation.
Acknowledgements
This work was supported by the directorate-general of scien-
tic research and technological development (DGRSDT) and the
Laboratory of valorisation and Technology of Saharan Resources
(VTRS) (project code: BLUN).
Conict of Interests
The authors declare no conict of interest.
Data Availability Statement
The data that support the ndings of this study are available
from the corresponding author upon reasonable request.
Keywords: Bovine Serum Albumin •Ferrocenylmethyl-
Nucleobases •Human Hemoglobin •Molecular Docking •
Molecular Dynamic Simulation
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