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\begin{document}

\title{Fluorescent Probe for Simultaneous Detection of Human Serum Albumin and
Sulfur Dioxide: A Theoretical Analysis}



\author[1]{ang jia}%
\author[2]{he huang}%
\author[2]{zhong-fu zuo}%
\author[2]{xuezheng liu}%
\author[2]{Yongjin Peng}%
\affil[1]{First Affiliated Hospital of Jinzhou Medical University}%
\affil[2]{Jinzhou Medical University}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
A small molecule probe for simultaneous detection of HSA and SO2 via
their distinct fluorescent signals was designed recently. This effective
tool provided a significant boost in understand underlying mechanism of
synergistic action between SO2 and HSA in disease. The structure and
fluorescent character of this probe molecule were studied under density
functional theory in this work. The different stable conformations of
probe C23 were found through theoretical method which explained the no
experimental fluorescent character of the probe itself. The electron
excitation analysis indicated the charge transfer process in the
restricted C23 (binding to the hydrophobic cavity of HSA) and CS (C23
reaction with SO2) when the molecules were under optical excitation. The
theoretical results could be helpful for understanding the electronical
properties in the probe and providing the insights for designing new
probe molecules.%
\end{abstract}%



\sloppy


\textbf{1 Introduction}

Studies have shown that SO\textsubscript{2}-induced HSA free radicals
could cause tissue damage during allergic reactions, eosinophilic
inflammation, and lung disease.\textsuperscript{1-6} An effective tool
of simultaneous detection of HSA and SO\textsubscript{2} is important
for understanding the underlying mechanism of synergistic action between
SO\textsubscript{2} and HSA in disease. Recently Liang et al designed a
fluorescent small molecule probe for simultaneous detection of HSA and
SO\textsubscript{2} via their distinct fluorescent
signals.\textsuperscript{7} The design concept was as showed in Scheme
1.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Scheme 1 probe C23

The probe itself does not fluoresce due to intramolecular rotation. In
the presence of HSA, the probe molecules specifically bind to the
hydrophobic cavity of HSA, causing the probe molecules to be unable to
rotate, resulting in strong red fluorescence. When the probe coexists
with SO\textsubscript{2} alone, a specific Michael addition reaction
occurs (as shown in Scheme 2), which reduces the \selectlanguage{greek}π-\selectlanguage{english}conjugated structure
of the probe and produces obvious blue fluorescence.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Scheme 2 CS--Michael addition reaction of probe C23 and
SO\textsubscript{2} (HSO\textsubscript{3}\textsuperscript{-1} in the
solution)

When the probe is combined with HSA, the Michael addition reaction
between the probe and SO\textsubscript{2} is rapidly catalyzed by HSA,
and this catalytic effect of HSA greatly improves the detection
sensitivity of SO\textsubscript{2}. Studies have shown that the amino
group of Lys199 in HSA contributes most of the catalytic
activity.\textsuperscript{7} The results of liang et al showed good
performance of this probe molecule for the simultaneous determination of
HSA and SO\textsubscript{2} molecules in urine and cells. And this probe
was expected to be used in the diagnosis of liver cancer, drug
evaluation, and the study of physiological and pathological functions of
HSA and SO\textsubscript{2}. In this work, the structure and fluorescent
character of this probe molecule were studied under density functional
theory. The rotation of the unsaturated C-C bonds in the probe molecule
led to several stable structures coexisted. The transfer between
different stable structures provided the non-radiative energy loss
pathway when the probe molecule recovered from the excited state to the
ground state which led to no fluorescence in this process. The
calculated results indicated the red fluorescence would be generated if
the rotation of the unsaturated C-C bonds in the probe molecule was
prohibited such as the probe molecule was stabilized in the hydrophobic
cavity of HSA. The calculation also indicated that the Michael reaction
between the probe molecule and SO\textsubscript{2}would be another
effective way for the stabilization of the probe molecule and generating
blue fluorescence which made this probe molecule is able of simultaneous
determination of HSA and SO\textsubscript{2}molecules. All the figures
were rendered by means of VMD 1.9.3 software\textsuperscript{8} and the
analyses were finished by using the Multiwfn 3.7
code\textsuperscript{9}.

\textbf{2 Method}

The processes of the calculation were as follows:

(1) Generate initial conformations of probes with
Confab;\textsuperscript{10}

(2) Use Crest to call xtb to do batch structural optimization under the
GFN2-xTB method,\textsuperscript{11} and then call isostat in
Molclus\textsuperscript{12} program to screen out several conformations
with the lowest energy for next step;

(3) Use Molclus to call ORCA\textsuperscript{13} to perform optimization
and vibrational frequency analysis on the conformations generated from
step (2) under PBE0/def2-TZVP,\textsuperscript{14, 15} and then single
point energy and TDDFT calculation under wB2GP-PLYP/def2-TZVP so as to
obtain the free energy with high precision.\textsuperscript{16, 17}

\textbf{3 Results and Discussion}

The four most stable conformations of probe C23 were found as shown in
Figure1. The difference among the four conformations were clearly
depicted in the front and side view of the molecular structure of the
probes due to the rotational bonds. The transfer among the different
conformations provided the non-radiation energy loss when the probe C23
recovered back to the ground state from excited states, led to no
fluorescence generation with the probe C23 itself. When the probe was
incorporated into a hydrophobic cavity of HSA, the bonding interaction
between the probe and HSA resulted in the inhibition of non-irradiative
energy loss pathways and an increase in fluorescence within the red
channel.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

C23-1 C23-2\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

C23-3 C23-4

Figure1 Four most stable conformations of probe C23

To illustrate the reaction sites within the probe molecule, the
electrostatic potential of C23-1 was analyzed using Multiwfn 3.7 from
the wave function generated under wB2GP-PLYP/def2-TZVP combination from
ORCA program. The electrostatic potential of C23-1 was as shown in
Figure2. Several local maximum and minimum values of electrostatic
potential except those within the
N-CH\textsubscript{2}-CH\textsubscript{3} groups were depicted in the
Figure2 which showed the potential reaction sites with the HSA. The
results were agreed with the previous work.\textsuperscript{7}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

Figure2 Electrostatic potential of C23-1

The electron excitation process within the probe C23 was analyzed under
TDDFT method within the ORCA program. The structure difference between
the ground state S\textsubscript{0} and lowest excited state
S\textsubscript{1} were small for all four conformations. The electron
density difference between the S\textsubscript{0} and
S\textsubscript{1}as shown in Figure3a and Figure3b for C23-1 and C23-4
respectively could show the electron transfer in the excitation process.
The electron density difference between the S\textsubscript{0} and
S\textsubscript{1}in C23-2 and C23-3 were similar to C23-1 and C23-4
respectively. The electron transfer in the excitation process could be
indicated from hole region (h+, orange) to electron region (e-, green)
in the figures. As can be seen from Figure3, the electron excitation in
the probe C23 should be charge transfer process.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image9/image9}

\end{center}
\end{figure}

Figure3 Electron density difference between the S\textsubscript{0} and
S\textsubscript{1}

(a)C23-1 and (b)C23-4

The probe C23 could detect HSA and SO\textsubscript{2} simultaneously
due to the different fluorescence signal while binding with them.
Michael addition reaction between probe C23 and
SO\textsubscript{2}(HSO\textsubscript{3}\textsuperscript{-1}) could
saturate the rotational bonds in the C23 and stabilize the conformation
which led to the strong fluorescence signal within the blue channel. To
understand this mechanism, the conformation and electron excitation
process of CS (C23 combined with
HSO\textsubscript{3}\textsuperscript{-1}) were analyzed using the same
methods which were used for C23 before. The stable conformations of CS
structure were reduced to two (CS-1 and CS-2 shown in Figure4). It can
be clearly seen that due to the steric hindrance effect of
HSO\textsubscript{3} group, the rotational C-C bonds between the two
rings in the probe were saturated and no longer rotated. This Michael
addition reaction cut off the non-radiative pathway and led to the blue
fluorescence signal within the electron excitation process of CS.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image10/image10}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image11/image11}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image12/image12}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image13/image13}

\end{center}
\end{figure}

CS-1 CS-2

Figure4 Two stable conformations of CS

The electron density difference between the S\textsubscript{0} and
S\textsubscript{1} of CS-1 was shown in Figure 5. The electron
excitation in the CS-1 should be taken as charge transfer process but
with different direction from C23.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image14/image14}

\end{center}
\end{figure}

Figure 5 Electron density difference between the S\textsubscript{0} and
S\textsubscript{1} of CS-1

The excitation and emitting fluorescence wavelength within probe C23 and
CS were analyzed with ORCA program under wB2GP-PLYP/def2-TZVP and
CAM-B3LYP/ def2-TZVP method. The results were summarized in Table 1. The
calculated \selectlanguage{greek}λ\selectlanguage{english}\textsubscript{ex} (wB2GP-PLYP in dmso) values in the table
1 were closed to the experimental values which indicated the red and
blue fluorescence channel with probe C23 and CS respectively. Due to
unrealized optimization function of first excited state
S\textsubscript{1} under wB2GP-PLYP function in ORCA program, the
CAM-B3LYP functional was utilized for the emitting fluorescence
wavelength calculation. Although a clear deviation from the experimental
wavelength, a similar trend of wavelength changing with the experimental
value was found in the calculation.

Table1 The calculated excitation and emitting fluorescence wavelength
(nm) within probe C23 and CS\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{Probe}\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\selectlanguage{greek}\textbf{λ\textsubscript{ex}(wB2GP-PLYP}\selectlanguage{english} \textbf{in gas)}\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\selectlanguage{greek}\textbf{λ\textsubscript{ex}(wB2GP-PLYP}\selectlanguage{english} \textbf{in dmso)}\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\selectlanguage{greek}\textbf{λ\textsubscript{ex}(CAM-B3LYP}\selectlanguage{english} \textbf{in gas)}\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\selectlanguage{greek}\textbf{λ\textsubscript{em}(CAM-B3LYP}\selectlanguage{english} \textbf{in gas)}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{C23-1}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{731.2}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{552.4}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{981.8}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{1186.6}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{C23-2}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{733.0}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{562.4}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{988.8}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{1202.7}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{C23-3}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{737.9}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{575.8}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{994.4}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{1206.2}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{C23-4}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{738.8}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{585.2}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{1029.9}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{1281.0}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{CS-1}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{455.9}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{466.1}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{649.2}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{745.2}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{CS-2}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{454.1}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{465.8}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{645.8}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{743.0}\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{4 Conclusions}

The different stable conformations of probe C23 were found through
theoretical method which explained the no fluorescent character of the
probe itself. The extreme points of electrostatic potential of C23
showed the potential reaction sites with the HSA and
SO\textsubscript{2}. After the Michael addition reaction with
SO\textsubscript{2} (HSO\textsubscript{3}\textsuperscript{-1}), some
rotated C-C bonds in C23 were saturated in CS structure which reduced
the number of the stable conformations and led to the blue fluorescence
generation. The electron excitation analysis indicated the charge
transfer process in the C23 and CS when the molecules were under optical
excitation. These results provided the insights in preparing a wide
range of fluorescent probes and great potential for widespread
industrial applications.

\textbf{Funding Information}

This work was supported by the Natural Science Foundation of Liaoning
Province (JYTQN201923), National Natural Science Foundation of China
(81374051)

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