Stereoselective Separation of the Fungicide Bitertanol Stereoisomers by High-Performance Liquid Chromatography and Their Degradation in Cucumber

Article abstract of DOI:10.1021/acs.jafc.8b04594

Bitertanol is a widely used triazole fungicide and consists of four stereoisomers. A new high-performance liquid chromatography (HPLC) method was developed for simultaneous analysis of the four stereoisomers in apple, pear, tomato, cucumber, and soil. The mechanism of separation was explained with molecular docking and effects of thermodynamic parameters on the resolution. The absolute configuration and optical rotation of four stereoisomers were confirmed by X-ray diffraction and HPLC tandem circular dichroism, respectively. A good linearity (R² ≥ 0.999) was obtained for four stereoisomers in all matrix-matched calibration curves in the range of 0.02-10 mg/L. The mean recoveries of four stereoisomers in five matrices ranged from 74.6% to 101.0% with an intraday and interday relative standard deviation from 0.6% to 9.9%. Stereoselective degradation of bitertanol in cucumber was observed: (1R,2S)-bitertanol and (1R,2R)-bitertanol were preferentially degraded with enantiomeric fraction values from 0.5 to 0.43 at 7 d and 0.42 at 5 d, respectively. This research provides a useful tool for the analysis of bitertanol stereoisomers.

Full text of DOI:10.1021/acs.jafc.8b04594

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New Analytical Methods
Stereoselective separation of the fungicide bitertanol stereoisomers by high-
performance liquid chromatography and their degradation in cucumber
Lianshan Li, Beibei Gao, Zhaoxian Zhang, mailun Yang, Xin Li, Zongzhe He, and MingHua Wang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04594 • Publication Date (Web): 29 Nov 2018
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Stereoselective separation of the fungicide bitertanol stereoisomers
by high-performance liquid chromatography and their degradation
in cucumber
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Lianshan Li, Beibei Gao, Zhaoxian Zhang, Mailun Yang, Xin Li, Zongzhe He,
Minghua Wang*
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Department of Pesticide Science, College of Plant Protection, Nanjing Agricultural
University; State & Local Joint Engineering Research Center of Green Pesticide
Invention and Application, Nanjing, 210095, China
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∗Corresponding author. E-mail address: wangmha@njau.edu.cn (M. Wang)
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ABSTRACT
Bitertanol is a widely used triazole fungicide and consisted of four stereoisomers. A
new HPLC method was developed for simultaneous analysis of the four stereoisomers
in apple, pear, tomato, cucumber, and soil. The mechanism of separation was
explained with molecular docking and effects of thermodynamic parameters on the
resolution. The absolute configuration and optical rotation of four stereoisomers were
confirmed by X-ray diffraction and HPLC tandem circular dichroism detection,
respectively. A good linearity (R2  0.999) was obtained for four stereoisomers in all
matrix-matched calibration curves in the range of 0.02-10 mg/L. The mean recoveries
of four stereoisomers in five matrices ranged from 74.6% to 101.0% with an intra-day
and inter-day relative standard deviation of 0.6% to 9.9%. Stereoselective degradation
of bitertanol in cucumber was observed: (1R,2S)-bitertanol and (1R,2R)-bitertanol
were preferentially degraded with EF values from 0.5 to 0.43 at 7 d and 0.42 at 5 d,
respectively. This research provides a useful tool for the analysis of bitertanol
stereoisomers.
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Keywords: bitertanol, chiral separation, X-ray diffraction, stereoselective degradation
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INTRODUCTION
Chirality is a common property in pesticides.1 Approximately 25% of commercial
pesticides were chiral over the world in 1996, and more than 40% in China in 2006.2,3
Triazole fungicides play an essential role in preventing and controlling fungal disease
through inhibiting14α-demethylase involved in the biosynthesis of fungal sterols.
Triazole fungicides are becoming the most popular fungicides and have attracted an
increasing attention since the 1970s due to their high potency.4,5 Currently, there are
31 commercial triazole fungicides, and 26 of those have 1 or 2 asymmetric carbon
atoms containing 1 or 2 pairs of enantiomers.6 Most chiral triazole fungicides are
marketed in racemic forms, except for diniconazole and uniconazole.
Different stereoisomers exhibit similar physicochemical properties in an achiral
environment, but different activities in the biological environment.7 Many papers
about enantioselective bioactivity, metabolism, degradation, and toxicity of chiral
triazole fungicides have been published in recent years.8-14 Bitertanol [(1RS,
2RS)-1-(biphenyl-4-yloxy)-3, 3-dimethyl-1-(1H-1, 2, 4-triazol-1 -yl) butan-2-ol] is an
efficient triazole fungicide consisting of four stereoisomers (Supporting Information
Figure S1) and was developed by Bayer in the 1970s.15,16 The ratio of four
stereoisomers in the current commercial bitertanol is 4:4:1:1. Bitertanol has been
frequently detected in food products and environmental samples due to frequent
applications over a long period of time.17,18 To date, the research about bitertanol was
mainly focused on residues and ecological toxicology. For instance, bitertanol
significantly inhibits the activity of CYP3A4 and has potential androgenic-disrupting
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effects.19,20 Bitertanol can induce and inhibit rat cytochrome P450-dependent
monooxygenases .21 The residue detection methods of bitertanol in baby food, food,
and atmospheric samples were established using various technologies.22-25 However,
the racemic bitertanol was used in the studies. The enantioselective bioactivity,
metabolism, degradation, and toxicity of each bitertanol stereoisomer have not been
reported.
Therefore, it is necessary to establish an effective method for the analysis of
bitertanol stereoisomers. The successful separation of racemate is fundamental to
realize bitertanol at the level of stereoisomers. Some remarkable works have been
accomplished regarding the separation of bitertanol by various techniques. For
example, Zhang et al. reported that 21 triazole fungicides, including bitertanol, were
separated with high-performance liquid chromatography (HPLC) -tandem mass
spectrometry.26 Chai et al. separated bitertanol by normal-phase HPLC with two
cellulose-based chiral columns.2 However, no publication has been found about the
absolute configuration and chiral analysis of bitertanol stereoisomers in the
environmental and food samples.
In this study, a reversed-phase HPLC method was established for simultaneous
determination of bitertanol stereoisomers in vegetables, fruits, and soil after the
solid-phase extraction (SPE) cleanup of the extracts. The absolute configuration and
optical rotation of bitertanol stereoisomers were confirmed. The stereoselective
degradation in cucumber was determined.
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MATERIALAND METHODS
Chemicals and reagents. The bitertanol standard (≥97% purity) was provided by
Jiangsu Sword Agrochemicals Co., Ltd. (Jiangsu, China). The optical isomers
(≥99.9%) were prepared by Chiralway Biotech Co., Ltd. (Shanghai, China).
HPLC-grade acetonitrile and methanol were purchased from TEDIA (Fairfield, USA).
Ultra-pure water was obtained using MUL-9000 water purification systems (Nanjing
Zongxin Water Equipment Co. Ltd., Nanjing, China). Analytical grade sodium
chloride, anhydrous sodium sulfate, and other reagents were acquired through
commercial sources. The Cleanert Florisil on a cartridge (500 mg, 6 mL) was
purchased from Agela Technologies (Tianjin, China).
Stock standards (1000 mg/L) of individual stereoisomers were prepared in
acetonitrile and a series of concentrations working standards 10, 5, 2, 0.5, 0.02 mg/L
were prepared by diluted in acetonitrile. Corresponding, the matrix-matched standard
solutions at 10, 5, 2, 0.5, and 0.02 mg/L were prepared by adding the standard
working solutions of four stereoisomers to the control matrices (apple, pear, cucumber,
tomato, and soil). All standard working solutions were placed in the dark at 4 ºC.
Chiral HPLC analysis. The separation was performed on an Agilent 1200 high
perform liquid chromatograph (Agilent, California, USA) equipped an UV-detector
and a chiral column Lux Cellulose-1 [cellulose tris (3,5- dime-thylphenylcarbamate)],
250 mm×4.6 mm(i.d.), 5 μm] (Phenomenex, California, USA). The separation
parameters were used to evaluate the effect of separation, including capacity factor k,
separation factor α, and resolution factor Rs, which were expressed as follows:
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k=(tR-t0)/t0
α=k1/k2
(1)
(2)
(3)
Rs=2(t2-t1)/(w2+w1)
where tR is the retention time of enantiomers, t0 is the void time, k is the retention
factor, and w represents the peak width at half height of stereoisomers.
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A series of mobile phase compositions (Methanol/water and acetonitrile/water)
were used to explore the effect of mobile phase on the separation. Temperature
influences the rate of the analyte sorption and desorption with the stationary phase
and impacts the elution order of the stereoisomers.27,28 So, the impact of temperature
on separation was discussed from 15 ºC to 35 ºC. The thermodynamic parameters
were calculated according to the following equation of Van’t Hoff:
lnk = -ΔH0 / RT + ΔS0 / R + lnΦ
lnα = -ΔΔH0 / RT + ΔΔS0 / R
(4)
(5)
The ΔH0 and ΔS0 represent the changes molecular enthalpy and molecular entropy
of bitertanol stereoisomers between the mobile phase and stationary phase,
respectively. T, R, and Φ represent absolute temperature, gas constant 8.314 J/(mol・
K) and phase ratio, respectively. ΔΔH0 and ΔΔS0 were the values of ΔH2-ΔH1 and
ΔS2-ΔS1. If the Lnk and Lnα have a good linearity with 1/T, the value of -ΔH/R,
(ΔS/R+lnΦ), -ΔΔH0 / R, and ΔΔS0 / R could be calculated from the slop and intercept
based on equations (4) and (5).
Circular dichroism spectroscopy. The optical rotation and elution order of four
stereoisomers were determined using HPLC (JASCO LC2000) with ultraviolet
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detector (JASCO UV2075) tandem CD (JASCO CD2095) detector at 254 nm. The
mixed standard solution of four stereoisomers in acetonitrile was measured on Lux
Cellulose-1 with acetonitrile/water (55: 45, v/v) as the mobile phase at the flow of 0.8
mL/min.
X-ray crystallography. Single-crystal X-ray diffraction analysis of four
stereoisomers was carried out on a Bruker SMART APEX II CCD diffractometer with
graphite-monochromated Cu-Ka radiation (λ=1.54178 Å) at 150(2) K. Data reduction
and absorption corrections were performed with the SAINT and SADABS software
packages, respectively. The structures were resolved by direct methods and refined by
the full matrix least-squares based on F2 using the SHELXL-2016 program package.29
The non-hydrogen atoms were refined anisotropically. All hydrogen atoms were
placed at the calculated positions and refined as riding on the parent atoms.
Molecular docking. The three-dimensional structures of the stereoisomers of
bitertanol and the CSPs were developed, and the energy was minimized using Yasara
molecular docking software. The stereoisomers were docked in the structures of the
CSPs using Yasara molecular docking software. The results were sorted by binding
energy, and more positive energy indicates stronger binding. The optimal result was
chosen for further analysis.30-32
Sample preparation. Extraction. Approximately 10 g of samples (apple, pear,
cucumber, tomato, and soil) were weighed into a 250 mL conical flask, 5 mL water
and 30 mL acetonitrile was added and shaken 1 h in the ZQLY-180 thermostatic
oscillator (Shanghai, China) with 250 rpm/min at 25 ºC. The liquid phase was
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transferred into a 100 mL cylinder containing 2 g NaCl, then shaken for 1 min and
allowed to stand for 30 min. A half of acetonitrile portion was dehydrated through 5 g
of anhydrous Na2SO4 which followed by evaporating to dryness at 45 °C.
Cleaning up the soil samples. The residue was dissolved in 4 mL n-hexane/acetone
(98:2, v/v) and transferred into a Florisil SPE pretreated with 6 mL n-hexane. The
column was rinsed with 5 mL n-hexane/acetone (95:5, v/v) and eluted by 12 mL
n-hexane/acetone (88:12, v/v). The eluant was collected and evaporated at 45 ºC. The
extractive was dissolved with 1 mL acetonitrile and filtered through a 0.22-μm nylon
syringe filter for HPLC analysis.
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Cleaning up the vegetables and fruits samples. The residue was dissolved in 4 mL
n-hexane/acetone (98:2, v/v) and transferred into an SPE column pretreated with 6 mL
n-hexane/acetone (88:12, v/v). The column was washed with 5 mL n-hexane/acetone
(93:7, v/v) and eluted by 12 mL n-hexane/acetone (88:12, v/v). The eluant was
collected and evaporated to dryness at 45 ºC, then dissolved with 1 mL acetonitrile
and filtered through a 0.22 μm nylon syringe filter for HPLC analysis.
Method validation. The linearity, matrix effect, precision, accuracy, limit of
detection (LOD) and limit of quantitation (LOQ) were used to evaluate the feasibility
of the method for simultaneous determination of four bitertanol stereoisomers in fruits,
vegetables, and soil matrices. Linearity was assessed by the linear regression of
bitertanol stereoisomer peak areas versus the concentration. The matrix effect should
be ignored in the slope ratios of matrix/solvent ranging from 0.90 to 1.10. If the slope
ratios of the matrix/solvent were below 0.90 or higher than 1.10, a matrix suppression
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or enhancement effect should be concerned. The LOD and LOQ were defined as the
concentration that produced a signal-to-noise of (S/N) 3 and 10.
Accuracy and precision were estimated by recoveries, inter-day RSDs and intra-day
RSDs of five spiked samples replicated at three concentrations over a continuous
three days. The standard solutions were spiked to the control samples of apple, pear,
tomato, cucumber, and soil at 0.02, 0.05, and 0.2 mg/kg for each isomer. The spiked
samples were shaken for 1 min and overnight. The extraction and cleanup were
performed as described above. The racemization and transformed of the stereoisomers
in the sample processing was evaluated by separate recovery experiment for each
stereoisomer in five matrices.
Field experiment. The degradation test of bitertanol on cucumber was carried out
in Nanjing, China. The trial blocks were divided into 30 m2 and isolated by buffer
zone with three replicate plots. 40% WP of bitertanol was used as foliar spray at 800 g
(a.i.)/ha. The cucumber samples were collected in 2 h, 1, 2, 3, 5, 7, 10 days after
spraying. All samples were separately homogenized and stored at -20 °C. The
concentrations of four bitertanol stereoisomers were detected by HPLC.
The first-order kinetics equation was used to estimate the degradation kinetics of
the four stereoisomers in cucumber. The enantiomeric fraction (EF) was used to
evalute the enantioselectivity of the bitertanol stereoisomer. The half-lives of each
stereoisomer and EF were calculated using the following equations:
C=C0e-kt
(6)
(7)
T1/2=ln2/k
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EFA=C(1R,2S)/C(1R,2S) + C(1S,2R)
EFB=C(1R,2R)/C(1R,2R) + C(1S,2S)
(8)
(9)
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where C represents the concentrations of the analyte at time t, and the k is the
degradation rate constant. Cisomer is the concentration of the specified stereoisomer.
RESULT AND DISCUSSION
Chromatographic condition optimization. Mobile phase optimization Although,
both acetonitrile/water and methanol/water were used as the mobile phase in the
isocratic reversed-phase HPLC method, the baseline separation was obtained only
with acetonitrile/water as the mobile phase. Furthermore, a series of mobile phase
compositions were investigated by changing the proportion of acetonitrile from 55%
to 80%. When the acetonitrile percentage increased, the stereoisomers retention time
shortened, the peak width narrowed, and the resolutions decreased significantly.
Additionally, it was difficult to obtain the baseline separation when acetonitrile was
below 55% within an acceptable retention time. Baseline separation of four
stereoisomers was achieved with at the acetonitrile/water (55:45, v/v) as the mobile
phase with 0.8 mL/min at 25 °C, and all other parameters were constant. The
resolution of the first two enantiomers was 3.32, while the latter two enantiomers
were 9.28 with a retention time within 25 min (Figure S2 and Table S1). Bitertanol
had better solubility in acetonitrile than water and methanol. Therefore, acetonitrile
had a stronger power of eluting the analytes from the stationary phase. Zhang et al.
also found that acetonitrile presents a stronger eluting strength than methanol for
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bitertanol, baseline separation of bitertanol was obtained with acetonitrile and 0.1%
formic acid aqueous solution as mobile phase with 30 min. 30
Effect of temperature on chiral separation. The effects of temperature on bitertanol
enantiomers separation were carefully conducted from 15 ºC to 35 ºC on the Lux
Cellulose-1 chiral columns (Figure 1). When the temperature fell below 25 ºC, a more
efficient chiral separation was obtained, but the retention time increased. However,
increasing temperature shortened the retention time, but decreased the separation
efficiency. Finally, 25 ºC was used as the optimal temperature to separate bitertanol
stereoisomers. The results regarding k, Rs, and α are shown in the Table S2. The lnk′
and lnα had a good linearity with 1/T in the temperature range of 15 ºC to 35 ºC with
R2>0.989. The thermodynamic parameters indicated that (1R,2S)-(+)-bitertanol and
(1R,2R)-(+)-bitertanol had a weaker binding force with stationary than (1S,2R)-
(-)-bitertanol and (1S,2S)-(-)-bitertanol, respectively. The ΔΔH01 and ΔΔS10 were -1.24
kJ/mol and -3.53 J/(mol.K), showing that enthalpy drove the separation of
(1R,2S)-(+)-bitertanol and (1S,2R)-(-)-bitertanol. In contrast, ΔΔH02 and ΔΔS20 were
0.26 kJ/mol and 2.4 J/(mol.K), indicating that the separation of (1R,2R)-(+)-bitertanol
and (1S,2S)-(-)-bitertanol was driven by entropy (Table 1). The binding force between
stationary and stereoisomers will become weaker with the temperature increase, and
the stereoisomers are more easily eluted from the stationary phase. According to Chai
research, bitertanol stereoisomers were separated under normal-phase
chromatographic conditions. However, the result showed that the ∆∆H0 values were
negative and the ∆∆S0 values were positive, so, enthalpy and entropy contribute
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together to the separation of two enantiomers separation in normal-phase
chromatography with Lux-2 and Lux-3 chiral column.2
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Absolute configuration and optical rotation of stereoisomers. Crystallization
was utilized to obtain four single-crystals of bitertanol stereoisomers at a low
temperature, and single-crystal X-ray analysis is shown in Figure 2. The details of
data collection, structure refinement, and crystallography are summarized in Table 2.
Combining single-crystal X-ray analysis with the spectrogram of CD and UV (Figure
3) showed that the elution order of four enantiomers was (1R,2S)-(+)-bitertanol,
(1S,2R)-(-)-bitertanol, (1R,2R)-(+)-bitertanol and (1S,2S)- (-)-bitertanol, respectively.
This study has significance for confirming and oriented synthesis of high-efficient
stereoisomers for bitertanol in future research.
Molecular docking. The research of the interactions between the analytes and the
CSPs could explore the mechanisms of stereoisomer separation. The stereoisomers
were separately docked into the CSPs of the Lux-1 column. Binding energy for
(1R,2S)-(+)-bitertanol, (1S,2R)-(-)-bitertanol, (1R,2R)-(+)-bitertanol and (1S,2S)-(-)-
bitertanol were 5.3Cc, 5.4BCc, 5.6Bb, and 6.0Aa kcal/mol, respectively. In this research,
two kinds of forces were mentioned including π-π stacking and hydrogen bonds. The
π-π stacking was formed between the phenyl of bitertanol and the 3,5-dimethyl phenyl
of CSPs. Hydrogen bonds were formed between the 1,2,4-triazole in bitertanol and
the carbonyl of CSPs. The bond lengths and energies were also different. The higher
binding energy and shorter lengths may have contributed to easier combinations
between the CSPs and bitertanol stereoisomers, which resulted in the different elution
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orders. Beyond that, the steric hindrance, dipole-dipole interactions, and van der
Waals force may have contributed to the binding energy. The stereo matching between
the chiral cavity of CSPs and stereoisomers was also different, which is an important
factor to affect the chiral separation (Figure 4). Xie et al reported that hydrogen bonds
were the major factor in the separation of amide herbicides by AY-H
[amylasetris-(5-chloro-2-methylphenylcarbamate)],
AS-H
[amylasetris
((S)-1-phenylcarbamate)], OD-H [cellulose tris-(3, 5-dimethylphenylcarbamate)] and
OJ-H [cellulose tris-(4-methylbenzoate)] columns.32
Linearity, matrix effect, LOD, and LOQ. Four stereoisomers of bitertanol
showed a good linearity in solvent and five matrices in the range of 0.02-10 mg/kg
with the R2 > 0.999. The results indicated that no noticeable matrix effect was found
for four stereoisomers in soil, pear, apple, and tomato. However, a weak matrix
enhancement effect was found in cucumber for all stereoisomers in the range from
15.3% to 18.8%. The LOD and LOQ for four stereoisomers in five matrices were 2.2
-11.5 µg/kg and 7.2 -38 µg/kg, respectively (Table S3).
Accuracy and precision. An excellent accuracy and precision of four
stereoisomers were obtained in five matrices. There was no racemization and
transformation of the stereoisomers in different matrices during the sample processing
step (Figure S3). The mean recoveries ranged from 74.6% to 101.0 % with 0.7–9.9 %
of intra-day (n=5) RSD and 0.6-7.1% of intre-day (n=15) RSD for bitertanol
stereoisomers (Table S4), which showed good accuracy and precision and could apply
to the simultaneous determination of four enantiomers of bitertanol in soil, vegetables,
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and fruits.
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Stereoselective degradation in cucumber. The dissipation of four stereoisomers
in cucumber followed first-order kinetics (R2, 0.9303-0.9954) and half-lives of
(1R,2S)-, (1S,2R)-, (1R,2R)-, (1S,2S)-bitertanol were 4.4, 5.1, 4.0, and 4.8 d (Figure
S4). The representative chromatogram is shown in Figure S5. The results indicated
that (1R,2S)-bitertanol and (1R,2R)-bitertanol were preferentially degraded in
cucumber and the half-lives of A-bitertanol (the first pair of enantiomers
((1R,2S)-bitertanol and (1S,2R)-bitertanol)) and B-bitertanol (the second pair of
enantiomers ((1R,2R)-bitertanol and (1S,2S)-bitertanol)) were all significantly
different (P<0.05). The values of EFA and EFB were decreased gradually with the time
increase from 0.5 at 2 h to 0.43 at 7 d and 0.42 at 5 d, and the EF was significantly
different compared to the initial EF (P <0.05) (Figure S6). Many chiral triazole
fungicides exist significant enantioselectivity degradation in many plants and soil. For
example, the remarkable stereoselective degradation of difenoconazole was found in
soil.7 Chiral pesticides in the different chiral environment may show different
stereoselective degradation. (R)-flutriafol was preferentially degraded in tomato,
whereas (S)-flutriafol degrades faster than (R)-flutriafol in cucumber.33 The enzyme
systems in the plant may play an important role to stereoselective degradation.34
Enantioselective uptake and translocation could also contribute to the enantioselective
enrichment for the chiral compounds.35
AUTHOR INFORMATION
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*Tel: +86 25 84395479. Fax: +86 25 84395479. E-mail: wangmha@njau.edu.cn.
Funding
This study was supported by the National Key Research and Development Program of
China (2016YFD0200207), the Top-notch Academic Programs Project of Jiangsu
Higher Education Institutions (PPZY2015B157) and the Program for Student
Innovation through Research and Training (1712A05).
Notes
The authors do not have any potential conflict of interest to declare.
ACKNOWLEDGMENT
We are grateful to Cuntao Dong (Hebei Agricultural University) for Molecular
Docking by Yasara software.
ASSOCIATED CONTENT
Supporting Information
The effects of acetonitrile and temperature on k, α, and Rs (Table S1, S2). Calibration
curves for bitertanol stereoisomers in solvent and matrix-matched standards (Table
S3). Accuracy and precision for four stereoisomers of bitertanol (Table S4). The
chemical structures of bitertanol stereoisomers (Figure S1). The influence of
proportion of acetonitrile to Rs for four stereoisomers (Figure S2). Typical
chromatograms of racemization and transformation of the stereoisomers in the sample
processing (Figure S3) Concentration of bitertanol stereoisomers in cucumber (Figure
S4). Typical chromatograms of bitertanol stereoisomers in cucumber samples (Figure
S5). EF value curve of bitertanol stereoisomers in cucumber (Figure S6).
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Figure caption
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Fig 1. Effects of temperature on bitertanol stereoisomers separation.
Fig 2. X-ray diffraction diagrams of bitertanol stereoisomers.
Fig 3. UV and CD spectrograms of bitertanol stereoisomers on HPLC.
Fig 4. Binding mode of the stereoisomers with CSP of the Lux-1.
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Table 1 The Van’t Hoff equations and thermodynamic parameters.
ΔΔH01
kJ/mol
ΔΔS01
J/(mol·k)
ΔΔH02
kJ/mol
ΔΔS02
J/(mol·k)
Stereoisomers
lnk
lnα
lnk=347.1/T+0.082
(1S, 2R)-bitertanol
(R2=0.991)
lnk=577.9/T-0.592
lnα1=149.5/T-0.424
(1R, 2S)-bitertanol
(1R, 2R)-bitertanol
-1.24
-3.53
(R2=0.994)
(R2=0.967)
lnk=524.8/T-0.243
(R2=0.995)
lnk=580.7/T-0.120
Lnα2=-30.63/T+0.288
(1S, 2S)-bitertanol
0.26
2.4
(R2=0.989)
(R2=0.933)
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Table 2 Crystal data and structure refinement for bitertanol stereoisomers
Crystal data and structure
refinement
Peak 1a
Peak 2
Peak 3
Peak 4
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
a (Å)
b (Å)
c (Å)
α (deg)
β (deg)
C20H23N3O2
337.41
150(2)
1.54178
Triclinic
P1
12.1896(19)
12.615(2)
13.147(2)
75.858(10)
75.858(10)
78.953(10)
1868.7(5)
4
C20H23N3O2
337.41
150(2)
1.54178
Triclinic
P1
12.129(5)
12.599(5)
13.150(5)
75.979(8)
74.315(14)
78.851(13)
1859.4(13)
4
C20H23N3O2
337.41
150(2)
1.54178
Orthorhombic Orthorhombic
P212121
11.3683(16)
11.4327(16)
14.1772(19)
90
90
90
1842.6(4)
4
1.216
C20H23N3O2
337.41
150(2)
1.54178
P212121
11.385(2)
11.427(2)
14.135
90
90
90
1839.0(6)
4
1.219
γ (deg)
Volume (Å 3)
Z
D calc(Mg/m3)
Absorptioncoefficient
(mm-1)
1.199
1.205
0.629
0.632
720
0.638
720
0.639
F (000)
720
720
3.646
68.606
to 3.564
59.697
to 4.969
68.471
to
4.977 to 68.567
 range n()
-14<=h<=14, -12<=h<=10, -8<=h<=13,
-15<=k<=14, -8<=k<=13, -11<=k<=13,
-11<=h<=13,
-13<=k<=13,
-17<=l<=15
Index ranges
-15<=l<=15
-11<=l<=13
-16<=l<=17
Reflections collected
11575
4896
6470
16979
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Independent reflections
8766
3907
3083
3341
Data/restraints/parameters
Unique reflns (Rint)
8766/3/917
0.0482
3907/51/917 3083/0/231
3341/0/231
0.0512
0.0483
0.0427
R1=0.0514,
wR2=0.1323 wR2=0.1820 wR2=0.1170
R1=0.0759,
R1=0.0425,
R1=0.0277,
wR2=0.0722
1.069
Final R indices [I>2σ(I)]
2
1.019
-0.1(2)
0.260
1.026
1.017
Goodness-of-fit on F
Absolute
structure
-0.4(6)
0.0077(11)
0.0076(7)
parameter
and 0.591
and 0.214
and 0.178
-0.143 e.Å-3
and
Largest diff. peak and hole
-0.232e.Å-3
-0.300 e.Å-3 -0.190e.Å-3
Noe: Peak 1-4 represents the elution order of bitertanol stereoisomers on Lux-1.
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Fig 1. Effects of temperature on bitertanol stereoisomers separation.
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Fig 2. X-ray diffraction diagrams of bitertanol stereoisomers
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Fig 3. UV and CD spectrograms of bitertanol stereoisomers on HPLC.
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