Supporting Information

Estimating the potential toxicity of chiral diclofop-methyl: Mechanistic insight into

the enantioselective behavior

Fei Ding a, b, Wei Peng c, *, Yu-Kui Peng d, Bing-Qi Liu e

a Department of Environmental Engineering, Chang’an University, Xi’an 710054,

China

b Key Laboratory of Subsurface Hydrology and Ecological Effect in Arid Region of

Ministry of Education, Chang’an University, No. 126 Yanta Road, Yanta District,

Xi’an 710054, China

c State Key Laboratory of Physical Chemistry of Solid Surfaces and Fujian Provincial

Key Laboratory of Theoretical and Computational Chemistry, College of Chemistry

and Chemical Engineering, Xiamen University, Xiamen 361005, China

d Center for Food Quality Supervision, Inspection & Testing, Ministry of Agriculture

and Rural Affairs, Northwest A&F University, Yangling 712100, China

e Department of Agricultural Chemistry, Qingdao Agricultural University, Qingdao

266109, China

*Corresponding AuthorPhone/fax: +86-29-87092367E-mail: weipeng@cau.edu.cn

Supplementary Protocols:

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Time-Resolved Fluorescence Spectroscopy. Time-resolved fluorescence lifetime

was measured with a FLS1000 Photoluminescence Spectrometer (Edinburgh

Instruments, United Kingdom), utilizing the time-correlated single photon counting

mode with a hydrogen flash lamp excitation source, in air equilibrated solution at an

ambient temperature. The excitation wavelength was 280 nm and the number of

counts collected in the channel of maximum intensity was 4,000. The instrument

response function (IRF) was gauged exploiting Ludox to scatter light at the excitation

wavelength. The data were analyzed with a nonlinear least-squares iterative method

using the new Fluoracle® software package (Edinburgh Instruments, United

Kingdom), IRF was deconvoluted from the experimental data, and the resolution limit

after deconvolution was 0.2 ns. The value of χ2 (0.9～ 1.2), the Durbin-Watson

parameter (greater than 1.7), as well as a visual inspection of the residuals were

employed to judge how well the computed decay fit the data. Average fluorescence

lifetime (τ) for multiexponential function fittings were acquired from the following

relation (Patel and Datta, 2007; Henry et al., 2014; Zahid et al., 2019):

(1)

where τi are fluorescence lifetimes and Ai are their relative amplitudes, with i variable

from 1 to 2.

Site-Specific Competitive Assay. Enantioselective bioreaction domain studies

between target protein and diclofop-methyl enantiomers in the existence of three

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classic site markers (warfarin, diazepam, and digitoxin) were executed using the

fluorescence titration approach. The concentration of target protein and site markers

were held in equimolar (1.0 μM), then diclofop-methyl enantiomers were respectively

added to the protein-site markers mixtures. An excitation wavelength of 295 nm was

selected and the fluorescence emission wavelength was registered from 300 to 450

nm.

Circular Dichroism Spectra. Circular dichroism (CD) spectra were performed

with a J-1500 Circular Dichroism Spectrophotometer (Jasco, Japan) equipped with a

microcomputer, the apparatus was sufficiently purged with 99.9% dry nitrogen gas

before starting the instrument and then it was calibrate with d-10-camphorsulfonic

acid. All the CD spectra were conducted at 298 K with a single-position Peltier

thermostatted cell holders (PTC-517) attached to a water bath with an accuracy of

±0.1 oC. Each spectrum was recorded with use of a precision quartz cuvette of 10 mm

path length and taken at wavelengths between 200 and 260 nm range that provides a

signal extremely sensitive to small secondary conformational disturbances. Every

determination was the average of five continual scans encoded with 0.1 nm step

resolution and got at a speed of 50 nm min-1 and response time of 1.0 s. All observed

CD data were baseline subtracted for buffer and the evaluation of the secondary

structure components was gained by employing the Spectra ManagerTM Suite (Jasco,

Japan), which calculates the different designations of secondary structures by

comparison with CD spectra, determined from diverse proteins for which high-quality

X-ray diffraction data are available (Johnson, 1999; Goormaghtigh et al., 2009;

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Pescitelli et al., 2014).

Molecular Dynamics Simulation. Molecular dynamics (MD) simulation of the

protein-diclofop-methyl enantiomers was performed using a versatile GROMACS

package, version 2019, with the GROMOS96 54A7 force field (van der Spoel et al.,

2005; Huang et al., 2011; Kutzner et al., 2011; Schmid et al., 2011; Kutzner et al.,

2018). Simulation process was run under physiological conditions (pH＝7.4), and the

amino acid residues possessed acidity and basicity were adjusted to the protonation

states at neutrality condition. Initial conformations of protein and diclofop-methyl

enantiomers were, respectively, taken from the original X-ray diffraction crystal

structure that was solved at 2.5 Å resolution (entry codes 1H9Z) and the optimal

structure originated from molecular docking (Petitpas et al., 2001). The topology of

protein was yielded by GROMACS program directly, whereas diclofop-methyl

enantiomers by PRODRG2.5 Server (van Aalten et al., 1996; Schüttelkopf and van

Aalten, 2004). The simulation system was solvated with a periodic cubic box (the

volume is 8.771×5.061×8.664 nm3) filled with TIP3P water molecules and an

approximate number (14) of sodium counterion to neutralize the charge (Jorgensen et

al., 1983; Price and Brooks III, 2004). Totally, there are 43,648 crystallographic

solvent molecules, and the shortest distance between the adduct and the edge of the

box is set to 10 Å. Simulation was operated using the isothermal-isobaric (NPT)

ensemble with an isotropic pressure of 1 bar, and the temperature of the ligand,

protein and solvent (water and counterion) was separately coupled to an external bath

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held at 300 K, utilizing the Berendsen thermostat with 0.1 ps relaxation time

(Berendsen et al., 1984; van Gunsteren and Berendsen, 1984; Berendsen et al., 1986;

Combs, 1988; Rull et al., 1989). The LINCS algorithm was applied to constrain bond

lengths, and the long-range electrostatic interactions beyond 10 Å were modeled using

the Particle Mesh Ewald (PME) method with a grid point density of 0.1 nm and an

interpolation order of 4 (Darden et al., 1993; York et al., 1994; Hess et al., 1997). A

cutoff of 10 Å was utilized for van der Waals’ interactions. The MD integration time

step was 2.0 fs and covalent bonds were not constrained, and the system

configurations were saved every 2.0 ps. To decrease the atomic collisions with each

other, both gradient descent and conjugate gradient algorithm were adopted to

optimize the whole system (Hestenes and Stiefel, 1952; Gonze, 1996). First the

solvated starting structure was preceded by a 5,000-step gradient descent and then by

conjugate gradient energy minimization. Subsequently, 500 ps equilibration with

position restraints runs to remove possible unfavorable interactions between solute

and solvent, and after thorough equilibration, MD simulation was conducted for

50,000 ps. The result of MD simulation was ultimately appeared by Visual Molecular

Dynamics 1.9.4 (Humphrey et al., 1996; Hsin et al., 2008; Burgin et al., 2018), and

the program Discovery Studio Visualizer 4.0 (BIOVIA, San Diego, CA) was used to

exhibit the images of the MD simulation.

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Supplementary Results and Discussion:

Elaborate Interpretation of Enantiomeric Bioreaction Mechanism. It is visible

from the macroscopic behavior of the fluorescent biointeraction that the remarkable

enantioselectivity play a vital role in the biorecognition of the macromolecule-optical

isomers. In order to understand such photochemical phenomenon at the microscopic

scale, this story should dissect the inherent mechanism of the stereoselective

fluorescent reaction. As noted earlier, the shrinkage of fluorescence intensity has been

levigated both as a basic phenomenon, and as an origin of clue regarding molecular

biosystems (Žoldák et al., 2017; Carstea et al., 2019). These biological utilizations of

fluorescence evanishment are owing to the biomolecular reactions that lead to

extinguishing (Lakowicz, 2006). Typically both static and dynamic fluorescent

bioreactions request molecular touch between the fluorophore (Trp residue) and

ligand (diclofop-methyl enantiomers). Upon osculation, the fluorophore backs to the

ground-state without emission of a photon. This signifies fluorescence extinction

befalls without any eternal variation in the molecules, viz. without a photochemical

interaction (Lakowicz, 2000; Maity et al., 2019). We can think that the measurements

of fluorescence drop could be exploited to disclose the localization of fluorophores in

biological targets and their permeabilities to ligands, and the rate of collisional

reaction may likewise be utilized to assess the diffusion coefficient of the ligand.

Hence the appearance of fluorescence reduction relies on the chemical properties of

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the independent molecules, and exhaustive decryption of the mechanism of

fluorescent bioreaction might contribute to grasping the secret heart of

enantioselective biological recognition.

This essay used the classical Stern-Volmer equation to process the data of

emission spectra of the fluorophore, and a graphic of F0/F versus [Enantiomers]

produces an intercept of one on the y-axis and a slope equal to KSV (Fig. S3).

Intuitively, a linear Stern-Volmer plot is mostly suggestive of a lone type of

fluorophores, all equivalently approachable to chiral ligand (Eftink, 1991; Green et

al., 1993; Eftink, 1997). In the current situation, the target protein holds the

chromophore, the aromatic Trp-214 residue, which can be bioreacted with diclofop-

methyl enantiomers. In the meantime, the Stern-Volmer picture is linear, which

insinuates that just one class of bioreaction arises. However, it is essential to sense

that the emergence of a linear Stern-Volmer image does not attest that collisional

fluorescence reaction has transpired, for static interaction still conduces to linear

Stern-Volmer profiles. Interestingly, the order of magnitude of the bimolecular

reaction constant kq in the two chiral biosystems was found to be 1012

(1.892/2.838×1012 M-1 s-1), which implies efficient enantiomeric biointeraction

through static pattern, as a bimolecular reaction constant ～1.0×1010 M-1 s-1 could be

regarded as the highest data for the diffusion-governed bioreaction in aqueous phase

(Eftink and Ghiron, 1981; Eftink and Ghiron, 1987; Eftink and Ghiron, 1991). Thus

one may come to the rational verdict that the disappearance of target protein

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fluorescence is static under the circumstances, or rather, the stereoselective toxic

recognition of (R)-/(S)-stereoisomers by biomolecule is predominantly proceeded via

the generation of the biological target-chiral diclofop-methyl conjugate (Bozorgmehr

et al., 2015; Shakibapour et al., 2019; Aseman et al., 2019).

According to the quantitative Stern-Volmer enunciation of emission spectra of the

fluorophore, one might easily see that the enantiomeric biorecognition can occur as a

result of the emergence of a nonfluorescent ground-state adduct (static type) between

the biomacromolecule and chiral diclofop-methyl, and when this bioconjugate absorbs

light it goes back to the ground state without emission of a photon. It is popularly

accepted that static and dynamic fluorescent biointeraction could be discriminated

through their differing reliance on temperature and viscosity, but preferably by

fluorescence lifetime determinations (Choudhury et al., 2015; Tarif et al., 2019).

Ordinarily, in the case of static reaction, the ground-state bioconjugate is formed

between the ground-state fluorophore and the ligand, and the emission intensity will

be dribbled as a result of dropping the number of fluorophore. Regularly, the lifetime

of fluorophore maintains unaltered during fluorescent bioreaction process, since such

biochemical issue does not influence the excited-state of the fluorophore.

Simultaneously, neither the consistencies of fluorophore nor the inner filtering effects

meddle with lifetime measurements (Zhao et al., 2010; Hao et al., 2015; Arnett et al.,

2018). Therefore the examination of fluorescence lifetime of target protein may be

used to divulge the verificatory biointeraction characteristic if the Stern-Volmer map

is apparently linear, and today this physicochemical device has magnificently been

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hired by numbers of research groups to reveal the biorecognitions of functional

biomolecules with variously active substances (Mohan et al., 2019; Mos et al., 2019;

Patel et al., 2019; Sood et al., 2019). Nonetheless, the information of time-resolved

fluorescence lifetime might not only give precise cue about the time dependence of

the biological reaction, but uncloak the main feature of fluorophore environment

(Amaro et al., 2014; Benabou et al., 2019). Accordingly, to pierce the bioreaction

quality between macromolecule and chiral pesticide, and certify the results of

fluorescence emission spectra, the typical time-resolved fluorescence decay curves of

target protein at various molar ratios of diclofop-methyl enantiomers in Tris-HCl

buffer, pH＝ 7.4, are displayed in Fig. S4, and the corresponding time-resolved

fluorescence lifetimes and their amplitudes are captured in Table S1.

As has been argued, time-resolved fluorescence lifetime of the chromophore is

extremely perceptive with respect to its contiguous environmental alterations, and for

that reason, the detection of fluorescence lifetime variations of the chromophore can

redound to the inspection of numerous biophysical events during the biointeraction,

for instance, charge transfer, dipolar relaxation, molecular rotation, and extirpation of

the fluorophores (Wu et al., 2018; Lahiri et al., 2019; Valadan et al., 2019).

Unmistakably, the fluorescence lifetime decay curves fitted daintily to a biexponential

function, and this phenomenon advocate the existence of rotational isomers which

could be associated with the single electronic transition of fluorophore in equilibrium

state in the tight structure of biomolecule (Beechem and Brand, 1985; Ross and

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Jameson, 2008; Otosu et al., 2010). It may be known from Table S1 that the relative

fluorescence lifetimes of biopolymer are τ1＝ 3.29 ns and τ2＝7.43 ns (χ2＝ 1.06),

respectively, while in the maximal amount of (R)-/(S)-enantiomers, the relative

fluorescence lifetimes of biomacromolecule are τ1＝3.03/2.86 ns and τ2＝7.14/7.03 ns

(χ2＝1.04/1.01), respectively. Undeniably, the biexponential decay of the aromatic Trp

residue stems from the dual emission from the 1La and 1Lb excited-states, and the

presence of disparate rotamers, concerning the Cα-Cβ or the Cβ-Cγ bond of Trp residue

was endorsed by Szabo and Rayner (1980) to afford the groundwork for unraveling

the biexponential decay. Specifically, a fluorophore in a homogeneous

microenvironment is anticipated to expose monoexponential fluorescence lifetime

decay, while for the zwitterion of Trp residue, it is unearthed frequently that a

fluorophore own a biexponential decay outline, and the molecular clarification might

embrace either ground-state heterogeneity or excited-state bioreactions (Alcala et al.,

1987; McLaughlin and Barkley, 1997; Rolinski et al., 2007). More importantly, there

is a lone style of fluorophore in the current biopolymer, ground-state heterogeneity

can be evoked by the emergence of manifold conformational states of the biological

target, which arouses the fluorophore to experience a different microenvironment and

possess a dissimilar decay time in each conformation (Schneckenburger et al., 1988;

Moors et al., 2008; Ariga et al., 2015). In fact, due to steric effects between the side

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chain of the Trp residue and the backbone of the polypeptide chain, all rotamers are

not absolutely equal. After the formation of the target protein-chiral pesticide

bioconjugates, the biointeraction group closest to the indole segment is the small

amino group, thereby such conformer own the greatest population and the lifetime is

7.43 ns (Periasamy, 2008; Praus et al., 2008; Xie et al., 2011). In contrast, if the amino

and carbonyl groups near the indole ring, this rotamer could hold the brief lifetime of

3.29 ns (Flora et al., 1998; Millar, 2000; Barakat and Patra, 2013). And the elucidation

of conformers in functional macromolecule is circumscribed to the aqueous solution,

and the presence of various Trp residue rotamers has scrupulously been authenticated

by means of other experimental methods such as nuclear magnetic resonance (Yang

and Zhang, 2010; Julien et al., 2012; Williams et al., 2013). So the current job would

not try to assign the individual ingredient of the lifetime of Trp-214 residue, and

conversely the average fluorescence lifetime has been rented to qualitatively probe the

enantioselective biorecognition essence between the biomolecule and optical isomers.

It is so obvious that the mean fluorescence lifetime of target protein is 6.11 ns,

which square with the previous value released by Abou-Zied and Al-Shihi (2008a,

2008b) in a more recent consideration. At various amounts of (R)-/(S)-diclofop-

methyl, the mean fluorescence lifetimes of biopolymer vary from 6.11 ns to 6.10 ns,

τ0/τ≈1, expounding that the contraction of fluorescence intensity of the Trp residue is

largely dominated by static reaction. At the same time, we may palpably heed that the

mean fluorescence lifetime state a slight fluctuation, but the amplitude is in the

acceptable scope. Perhaps such fact originates from the charge transfer from the

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indole ring in the aromatic Trp-214 residue to the neighboring substituent group,

which gives rise to the weak augmentation on biointeraction intensity of the target

protein-chiral diclofop-methyl and then result in the slight reduction in the average

fluorescence lifetime (Pan et al., 2011; Stella, 2011; Pahari et al., 2015). It is

indicative of a teeny dedication of dynamic trait as just the (R)-/(S)-enantiomers that

undergo dynamic bioreaction have been exclusive donation in the diminution of the

fluorescence lifetime. For this reason the time-resolved fluorescence energy transfer

efficiency (E) reckoned from fluorescence lifetimes determinations solely represents

the energy transfer during the dynamic behavior and is harvested based on the

relationship: E＝1－τ/τ0, where τ and τ0 are the fluorescence lifetime of Trp residue in

the presence and absence of diclofop-methyl enantiomers, respectively. The sizes of E

computed from time-resolved fluorescence data are calculated to be 0.1637%,

respectively, in the macromolecule-chiral pesticide complexes at a molar ratio of

target protein to (R)-/(S)-stereoisomers of 1︰4. Patently, this value is exceedingly

small, thus one might believe that the phenomenon of energy transfer is bechanced in

the procedure of biological recognition, but the transfer efficiency is terrifically tiny,

nearly negligible. These minute analyses on the basis of the data of time-resolved

fluorescence conform to the thoughtful clarification of fluorescence emission spectra,

site-specific competitive assay, circular dichroism spectra, and computational

toxicology, that is, biorecognition of chiral pesticide with biomacromolecule is

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substantially progressed via static reaction, or the generation of the noncovalent

bioconjugates between the target protein and chiral diclofop-methyl, and the

bioreaction domain of optical isomers is situated in the proximity of the Trp-214

residue (subdomain IIA), accordingly provoking some conspicuous alterations in the

spatial conformation of biopolymer. This tale should surely prompt that although the

conformational transition of the biological target can be actuated by (R)-/(S)-isomers,

multiexponential fluorescence decay manner could be ascribed to the dissimilar

conformations of target protein rather than an apportionment to different Trp residues

in a biomolecule of one conformation at neutral pH (Chen et al., 1991; Stayton and

Sligar, 1991; Siemiarczuk et al., 2004; Albani, 2009; Amiri et al., 2010a, 2010b).

Moreover, we also discerned that the impact of diclofop-methyl enantiomers on time-

resolved fluorescence lifetime decay of the chromophore on target protein has some

distinction, further substantiating the biointeraction of the macromolecule-chiral

pesticide conserve prominent enantioselectivity. And such chiral selectivity retains

diverse influence on the biomolecular conformational variations triggered by unequal

(R)-/(S)-diclofop-methyl and the free energy of the biomacromolecule-optical

isomers. These analytical outcomes matches the elaborate interpretation on the ground

of circular dichroism and molecular modeling, and a comparable narration has been

specified very recently by Abou-Zied et al. (2011, 2012, 2013, 2015) for the

commentary of the biorecognition of hydroxypyridines, hydroxyquinolines, and

hydroxyphenyl benzazoles with available biopolymers by engaging some modern

biophysical experimental techniques such as fluorescence spectroscopy and UV/vis

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absorption spectra.

Stereoselective Biorecognition Domain. From the findings of the fluorescent

bioreaction between biological macromolecule and chiral pesticide, we may observe

clearly that the biointeraction position of diclofop-methyl enantiomers is ascertained

to be located at the propinquity of the fluorophore on target protein, i.e. subdomain

IIA. In order to validate the experimental results, this attempt uses site-specific

competitive assay to concretely illustrate the biorecognition region of (R)-/(S)-

stereoisomers on biological target. With regard to the target protein utilized in the

present affair, the pioneering effort of Sudlow et al. (1973, 1975a, 1975b, 1976) of

competitive binding surveys defined site I and site II as an independent place for some

drugs, with 5-dimethylaminonaphthalene-1-sulfonamide and dansylsarcosine as two

specific markers, but did not allot they to the cavity of the protein molecule. Luckily,

the X-ray diffraction crystallographic consequences of Carter’s (1989, 1990, 1994a,

1994b) laboratory unwrapped the bioreaction patch corresponding to site I and site II

to stand in subdomains IIA and IIIA, respectively. Structurally, site I is known as the

warfarin-azapropazone site, and formed as a placket in subdomain IIA, the solitary

Trp-214 residue of target protein in this domain. The internal wall of the region is

shaped by hydrophobic side chains, whereas the gate to the hole is encompassed by

positively charged residues (He and Carter, 1992; Dockal et al., 1999; Wang et al.,

2013). And analogously, site II corresponds to the hollow of subdomain IIIA, and is

known as the indole-benzodiazepine site, which is approximately the same size as site

I, the inner of the cave is comprised by hydrophobic residues and the outside area

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posed two essential residues (Arg-410 and Tyr-411). In general, site I ligands are

bulky heterocyclic anions with the charge localized in a fairly central position in the

ligand. This discriminates them from the molecules typical of site II, situated in

subdomain IIIA, which are chiefly aromatic and might be neutral; a charge, if subsist,

is anionic and lied more marginally on the ligand (Dockal et al., 2000; Dockal et al.,

2000; Curry, 2009). Nowadays a lot of diverse substances are trusted to situate in the

district labeled site I and site II by Sudlow et al. (1973, 1975a, 1975b, 1976) that they

would be regarded here together in spite of their gigantic variety. A number of them

are therapeutic pharmaceuticals, e.g. azapropazone, indomethacin, phenylbutazone

and warfarin are among the site I agents (Montero et al., 1986; Kragh-Hansen, 1988;

Pinkerton and Koeplinger, 1990; Ryan et al., 2011), while site II compounds contains

clofibrate, diazepam, flufenamic acid and naproxen (Sollenne and Means, 1979;

Zunszain et al., 2008; Ryan et al., 2011). Afterwards, encouraging biochemical proofs

of Kragh-Hansen, Brodersen’s and Tillement’s teams deemed that digitoxin is

different from both of the two Sudlow’s regions, and perch on what was designated as

site III (Brodersen et al., 1977; Tillement et al., 1980; Kragh-Hansen, 1985).

Consequently, for the current discussion, the competitors utilized involved warfarin, a

typical marker for site I, diazepam for site II, and digitoxin for site III.

In the light of the present protocol, this scenario inspect the enantioselective

biointeraction between macromolecule and chiral diclofop-methyl in the existence of

various competitive chemicals, and the relative fluorescence emission intensity (F/F0)

versus (R)-/(S)-diclofop-methyl concentration ([Enantiomers]) figures is manifested

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in Fig. S5. As can be viewed from Fig. S5, both optical isomers could diminish the

emission intensity of the fluorophore when the three site-specific agents present

respectively in the enantiomeric biorecognition systems, but the amplitude of

fluorescence decline has giant divergence. This chemical phenomenon explicated

crystally that (R)-/(S)-enantiomers and site-specific substance may compete for the

bioreaction patch on target protein, and the notable discrepancies in the fluorescence

decrease extent might be noticed in the presence of different competitive ligands.

Evidently, the emission intensity of the fluorophore is respectively rejected

6.874%/10.65% when the (R)-/(S)-diclofop-methyl add to the biomacromolecule-

warfarin conjugate; while the emission strength of the fluorophore will respectively

drop 20.25%/29.04% and 17.87%/26.25% if the (R)-/(S)-isomers emerge in the

biomolecule-diazepam/digitoxin adducts. Doubtlessly, warfarin has the highest

inhibitory degree for the stereoselective recognition of the target protein-chiral

pesticide, or rather, the strongly competitive bioreaction relationship between chiral

pesticide and warfarin is occurred in biopolymer, and further enable the

macromolecule-diclofop-methyl enantiomers to own the smallest biorecognition

intensity. Such event testified that (R)-/(S)-stereoisomers and warfarin indeed act on

the same biointeraction domain, that is they have the uniform ligand reaction position,

subdomain IIA (Sudlow’s site I). It is noteworthy that (R)-/(S)-diclofop-methyl can

still shorten ～ 20% and 25% emission intensity of the fluorophore, respectively,

clarifying that the emersion of diazepam/digitoxin did not have significant

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interference with the enantioselective bioreaction of biomolecule with chiral diclofop-

methyl, or, more exactly, the capital biorecognition area of chiral pesticide and

diazepam/digitoxin is not overlapped on target protein, and the biointeraction region

of the optical isomers is fixed to be located within the Sudlow’s site I, where the

aromatic Trp residue situate in the structural domain on biomacromolecule (Fasano et

al., 2005; Ghuman et al., 2005; Simard et al., 2006). Certainly, the conclusion of the

site-specific competitive assay is not only compatible greatly with the experimental

outcomes of fluorescence spectroscopy, but also in consonance with the upshots of

enantiomeric biological reaction based upon the approach of computational

toxicology.

Conformational Alteration. As previously mentioned, the results of fluorescence

spectroscopy implicated that the enantioselective biorecognition of chiral diclofop-

methyl by target protein could cause remarkable decrease in the emission intensity of

the fluorophore, and such issue alluded that the orderly spatial conformation of

biomolecule has changed to a certain extent, because a conformational alteration of a

biopolymer engendered by ligand bioreaction may commonly bring about a variation

of the fluorescence intensity (Hovius et al., 2000; Akbar et al., 2016; Samokhvalov et

al., 2018). Further, the experimental consequences of site-specific competitive assay

evinced that both optical isomers are centered in the Sudlow’s site I (subdomain IIA)

on biomacromolecule, and this is another smoking gun for macromolecular

conformational changes, as conformational alterations in the present globular protein

are frequently distinct with many site I substances (Kragh-Hansen, 1990; Peters Jr.,

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1995; Yamasaki et al., 1996; Kragh-Hansen et al., 2013). This event was taken to

mean that the “configurational adaptability” embraces more than the adjacent

neighborhood of a ligand and might impact the compactness or decompaction of

structure of the entire protein molecule.

To scrutinize the quantitatively conformational changes of biological target during

the enantiomeric biointeraction, the far-UV CD spectra of biomolecule with different

amounts of (R)-/(S)-stereoisomers were collected in Fig. S6, and secondary structure

contents evaluated based upon raw CD data were gathered in Table S2. It was obvious

that the CD curve of the lone biopolymer presented two negative peaks in the far-UV

region at 208 nm and 222 nm (negative Cotton effect), which are the representative

earmark of the α-helical structure of globular protein (Johnson Jr., 1988; Besley and

Hirst, 1999; Wallace and Janes, 2001). A logical explication is that the negative peaks

between 208 nm and 209 nm and 222 nm and 223 nm are governed via both n→π*

and π→π* transitions of amide groups and are also impressed by the geometries of the

polypeptide backbones (Sreerama and Woody, 1994; Kelly and Price, 2000; Corrêa

and Ramos, 2009). It can be stated in Table S2 that free target protein includes 58.4%

α-helix, 9.3% β-sheet, 10.1% turn and 22.2% random coil; upon biorecognition with

(R)-/(S)-enantiomers, a diminution of α-helix was viewed from 58.4% (free target

protein) to 49.9%/48.5% (target protein-(R)-/(S)-isomers), and then there was a rise in

β-sheet, turn and random coil from 9.3%, 10.1% and 22.2% (free target protein) to

12.1%/12.4%, 12.7%/13.2% and 25.3%/25.9 (target protein-(R)-/(S)-stereoisomers) at

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a molar ratio of target protein to chiral diclofop-methyl of 1︰ 6. Apparently, the

abasement of α-helical constituent with an amplification in the β-sheet, turn and

random coil structures confirmed that diclofop-methyl enantiomers fashions

noncovalent bonds with the residues in the polypeptide chain on target protein, and

then sparking off the destabilization of the regularly spatial conformation in protein

molecule, e.g. some degree of structural protension of target protein after it recognize

optical isomers (Woody, 1995; Greenfield, 2006; Iranfar et al., 2012; Chanphai and

Tajmir-Riahi, 2019). Meanwhile, we could also mark from Table S2 that the influence

of (R)-stereoisomer on the secondary structure of target protein is less than (S)-

stereoisomer, and this is probably because the bioreaction intensity of (S)-

stereoisomer with target protein outweigh (R)-enantiomer, namely, the noncovalent

bonds yielded between (S)-enantiomer and the crucial residues are relatively strong,

and thereby having somewhat large perturbation on the regularly spatial conformation

of target protein. Such biochemical phenomenon further demonstrated that the toxic

biointeraction is guaranteed to possess enantioselectivity when the intrinsic

macromolecule face chiral pesticide in living organisms, and the (R)-/(S)-isomers of

chiral toxicant would produce different effects on biomacromolecular conformation,

whereas the conformation of biopolymer is related inseparably to its biological

function, and so that these optical isomers shall ultimately exhibit various

toxicological effects in the body (Lashuel et al., 2013; Nwamba and Ibrahim, 2014;

Moarefi, 2016).

Besides, it should be noted that the biomolecular conformational alterations

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provoked by (R)-/(S)-diclofop-methyl in the current experimental conditions does not

mean the serious damage in the orderly spatial conformation of target protein, but the

self-regulation of the macromolecular spatial structure so as to qualify diclofop-

methyl enantiomers to act on the bioreaction domain of biological target more perfect.

Thus, strictly speaking, the compact α-helical structure of target protein is still

predominated in the stereoselective biorecognition processes. Practically, such

biological macromolecule is not in a stationary, “platonic” state at physiological pH;

on the contrary, it is imagined as the limber and quickly altering in shape, a “kicking

and screaming, stochastic” biopolymer in the biological organism. As narrated by

Weber (1972, 1975), the overall biomacromolecule welters in ～ 40 ns. The main

reason is that the loop-link structure in biomolecule approves fast distention,

constringency, and flexure, some of it inherent, some of it associated with

biointeraction with ligands. Usually, the cooperative motions of the target protein

molecule to adapt ligand bioreaction arise in 0.1～ 0.3 s (Choi et al., 1990). For

example, Gurd and Rothgeb (1979) deliberated that molecular oxygen, a ligand of Trp

residue fluorescence, may enter the 10 Å chink between helices h1 and h2, underneath

which inheres the single Trp residue of the biopolymer, within 6.0 ns. The outcomes

of previous discussions also learned that much of the flexibility of target protein is the

result of separate integrant movements of segments of the biological molecule

(Benson and Hallaway, 1970; Hvidt and Wallevik, 1972). Meantime, high

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interchangeability of hydrogens is a singular schtick of the macromolecule among

nonenzymatic proteins, and is possibly a fruit of its slack structure and its proclivity

for biorecognition with many substances (Willumsen, 1971; Volynskaya et al., 1994).

Nevertheless, elements of protein are incessantly moving on more swift time ranges

(Hansen et al., 2013; Chattopadhyay and Haldar, 2014), for instance, the sole Trp

residue side chain of target protein, attained by time-resolved fluorescence

spectroscopy, revolves individually at a speedy rate, nearly 10-10 s rotational

correlation time (Munro et al., 1979; Meadows et al., 2014). As a result, the biological

target leased in this task in liquid solution might be viewed as having a unitary frame

whole, but it is likely more authentic to consider it as an assemblage of wriggly,

flexible fragments, often converting in three-dimensional spatial conformation by

unclosing and occluding of primary cranny. Adopting such respiratory mode, and with

plenty of its amino acid side chains continually in motion on a microscale, the

multifunctional biopolymer is well fitted to snatch or emancipate the various bioactive

compounds, e.g. (R)-/(S)-enantiomers of a chiral pesticide that it assimilate, catalyzes,

degrades, distributes, excretes, metabolizes, transports…in the organisms (Abboud et

al., 2017; Horváthy et al., 2017; Neelofar and Ahmad, 2017; Litus et al., 2018;

Rabbani and Ahn, 2019).

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Supplementary Tables:

Table S1Time-resolved fluorescence lifetime of target protein (10 μM) as a function of amounts of diclofop-methyl enantiomers

Enantiomeric biosystems τ1 (ns) τ2 (ns) A1 A2 τ (ns) χ2

Free protein 3.29 7.43 0.32 0.68 6.11 1.06Protein-(R)-diclofop-methyl (1︰2) 3.37 7.22 0.29 0.71 6.10 1.09Protein-(R)-diclofop-methyl (1︰4) 3.03 7.14 0.25 0.75 6.11 1.04Protein-(S)-diclofop-methyl (1︰2) 3.24 7.16 0.27 0.73 6.10 1.03Protein-(S)-diclofop-methyl (1︰4) 2.86 7.03 0.22 0.78 6.11 1.01

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Table S2Secondary structure ingredients of target protein (5.0 μM) stereoselective

biorecognition with diclofop-methyl enantiomers at pH＝7.4 computed by the Spectra

ManagerTM Suite

Enantiomeric biosystemsSecondary structure components (%)

α-Helix β-Sheet Turn Random Free protein 58.4 9.3 10.1 22.2

Protein-(R)-diclofop-methyl (1︰6) 49.9 12.1 12.7 25.3Protein-(S)-diclofop-methyl (1︰6) 48.5 12.4 13.2 25.9

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Supplementary Figures:

Fig. S1. Molecular structures of (R)-diclofop-methyl (A) and (S)-diclofop-methyl (B).

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Fig. S2. Calculated Root-Mean-Square Deviation (RMSD) for the diclofop-methyl enantiomers and the backbone Cα atoms of target protein from MD simulation at

temperature of 300 K with respect to their docking results as a function of the simulation time. The blue and red trajectories portray RMSD data for the backbone Cα

atoms of target protein and the diclofop-methyl enantiomers, respectively. Panel (A): target protein-(R)-diclofop-methyl bioconjugate; and panel (B): target protein-(S)-

diclofop-methyl complex.

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Fig. S3. Stern-Volmer plot representing fluorescence bioreaction of target protein (1.0

μM) at pH＝7.4 and T＝298 K in the existence of different amounts of (R)-diclofop-

methyl (black square, ■) and (S)-diclofop-methyl (red circle, ●). Fluorescence

emission intensity was registered at λex＝295 nm, and the λem maximum emerged at

345 nm. All data were corrected for diclofop-methyl enantiomers fluorescence and

each point was the mean of three independent observations±S.D. ranging 0.49%－3.95%.

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Fig. S4. Time-resolved fluorescence lifetime decays of target protein in Tris-HCl

buffer (pH＝7.4) as a function of the concentrations of (R)-diclofop-methyl (panel

(A)) and (S)-diclofop-methyl (panel (B)), respectively. c(target protein)＝10 μM,

c(diclofop-methyl enantiomers)＝0 (red circle, ●), 20 (green equilateral triangle, ▲),

and 40 (blue inverted equilateral triangle, ▼) μM. The sharp profile on the left (black square, ■) is the lamp pattern.

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Fig. S5. Fluorescence inhibitory effects of target protein and target protein-site

markers biosystems at pH＝7.4 and T＝298 K. Enantioselective biointeraction

isotherm of (R)-diclofop-methyl (panel (A)) and (S)-diclofop-methyl (panel (B)) induced reduction of target protein fluorescence and decrease of target protein-site

markers complexes fluorescence. (Black square, ■): blank; (red circle, ●): warfarin; (green equilateral triangle, ▲): diazepam; and (blue inverted equilateral triangle, ▼): digitoxin. All data were corrected for diclofop-methyl enantiomers fluorescence and

295758

each value was the average of three separate determinations±S.D. ranging 0.21%－2.17%.

Fig. S6. Circular dichroism spectra of the target protein-diclofop-methyl enantiomers

adducts at pH＝7.4 and T＝298 K, 5.0 μM target protein in the presence of 0 (black

solid line) and 30 μM (red solid line) diclofop-methyl enantiomers. Panel (A): target protein-(R)-diclofop-methyl conjugates; and panel (B): target protein-(S)-diclofop-

methyl systems.

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