Absolute configuration assignment of (+)-fluralaner using vibrational circular dichroism

Article abstract of DOI:10.1002/chir.22770

The absolute configurations of the separated enantiomers of fluralaner, a racemic animal health product used to prevent fleas and ticks, have been assigned using vibrational circular dichroism (VCD). The crystallographic structure of the active enantiomer (+)-fluralaner has previously been shown to have the (S) configuration using small molecule crystallography. We sought a faster analytical method to determine the absolute configuration of the separated enantiomers. When comparing the measured IR (infrared) and VCD spectra, it is apparent that the amide carbonyl groups appear in the IR but are nearly absent in the VCD. Computational work to calculate the VCD and IR using in vacuo models, implicit solvation, and explicitly solvated complexes has implicated conformational averaging of the carbonyl VCD intensities.

Full text of DOI:10.1002/chir.22770

Received: 10 April 2017
DOI: 10.1002/chir.22770
Revised: 25 August 2017
Accepted: 28 August 2017
R E G U L A R A R T I C L E
Absolute configuration assignment of (+)‐fluralaner using
vibrational circular dichroism
John Kong2 | Leo A. Joyce3
Edward C. Sherer1
| Jinchu Liu3 | Tiffany M. Jarrell2 | J. Chris Culberson1 |
1 Modeling & Informatics, MRL, Rahway,
New Jersey
Abstract
The absolute configurations of the separated enantiomers of fluralaner, a race-
mic animal health product used to prevent fleas and ticks, have been assigned
using vibrational circular dichroism (VCD). The crystallographic structure of
the active enantiomer (+)‐fluralaner has previously been shown to have the
(S) configuration using small molecule crystallography. We sought a faster ana-
lytical method to determine the absolute configuration of the separated enan-
tiomers. When comparing the measured IR (infrared) and VCD spectra, it is
apparent that the amide carbonyl groups appear in the IR but are nearly absent
in the VCD. Computational work to calculate the VCD and IR using in vacuo
models, implicit solvation, and explicitly solvated complexes has implicated
conformational averaging of the carbonyl VCD intensities.
2 Animal Health, Intervet, Inc., Rahway,
New Jersey
3 Process Research & Development, MRL,
Rahway, New Jersey
Correspondence
Edward Sherer, Modeling & Informatics,
MRL, Rahway, NJ 07065, USA.
Email: edward_sherer@merck.com
KEYWORDS
absolute configuration, Bravecto, DFT, fluralaner, VCD, vibrational circular dichroism
1 | INTRODUCTION
studied as individual entities or carefully controlled ana-
lytically unless proven differently.
Accurate assignment of the absolute configuration of a
chiral drug is an extremely important task in pharmaceu-
tical research and development. Approximately 50% of
marketed drugs are chiral with roughly half of those drugs
containing a mixture of enantiomers.1 When a chiral drug
is introduced in vivo, the enantiomers may display dra-
matically different biological activities. While one enan-
tiomer may be active in eliciting the desired medicinal
effect, the other enantiomer may be either inactive or con-
tribute to undesired side effects.1 The following are a few
pharmaceutical examples of enantiomers showing differ-
ent bioactivities: R‐thalidomide has been marketed as a
sedative while S‐thalidomide has been shown to cause
severe birth defects; R‐naproxen is used for arthritic pain
while S‐naproxen is teratogenic; and D‐ethambutol is an
antituberculosis drug while L‐ethambutol has been found
to cause blindness.2 For this reason, enantiomers must be
Fluralaner, an isoxazoline‐containing compound
shown in Scheme 1, is used to repel ticks for dogs and
fleas for cats. It is an insecticide/parasiticide that was first
synthesized by Nissan Chemical Industries, Ltd, targeting
γ‐aminobutyric acid (GABA) receptors and chloride‐gated
glutamate channels.3 Fluralaner is dosed as a racemic
mixture of R‐ and S‐enantiomers. Previously, single crys-
tal X‐ray crystallography (SCXRD) has been used to
assign the absolute configuration of fluralaner.3 Studies
of fluralaner have shown that the R‐enantiomer is inac-
tive while the S‐enantiomer is biologically active.3
While the absolute configuration of the active enantio-
mer, or (+)‐fluralaner, was assigned using SCXRD, grow-
ing a single crystal can be time‐consuming and hence
additional methods were explored to assign absolute con-
figuration without the need for crystallization. The current
study uses vibrational circular dichroism (VCD) as an
Chirality. 2017;1–11.
wileyonlinelibrary.com/journal/chir
© 2017 Wiley Periodicals, Inc.
1

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KONG ET AL.
of 1 hour and averaged those scans into one block. Each
of the runs involved averaging 5 blocks for each sample,
as well as the solvent. The solvent background average
was then subtracted from the sample average. Collection
times for samples and solvent were approximately 5 hours
each. The VCD spectra for each enantiomer were further
corrected using the respective enantiomer.
SCHEME 1 Chemical structure of fluralaner
alternative approach to identify the R‐ and S‐enantiomers
of fluralaner. The VCD chiroptical technique of combining
experimental VCD spectra with density functional theory
(DFT) calculated spectra has proven to be an accurate
way for determining the absolute configuration of chiral
molecules.4-12 At MRL (Merck Research Laboratories),
the absolute configurations of more than 350 diverse
compounds have been assigned, most of which remain
proprietary, but some of which have supported publica-
tions.5,13-18 The diversity in chemical structure has led to
an accumulation of experience in dealing with a vast array
of chemistry complexities and functionalities when apply-
ing VCD. Commonly, it is possible to identify all functional
groups of a given molecule in the VCD spectrum. During
assignment of the configuration of fluralaner, a compari-
son of measured and computed IR and VCD spectra
revealed a distinct lack of peaks (ie, nearly zero intensity)
corresponding to carbonyl stretches in the VCD spectrum.
A series of computational studies which explain these
observations are presented.
The general approach for VCD assignment at MRL,
including the detailed computational workflow, has been
published elsewhere.5 Based on the generation of a diverse
set of molecular mechanics conformations, we cluster con-
formations to a target representative set. These conformers
are then sent for refinement using density functional the-
ory. The vast majority of assignments can be accurately
madeusing only invacuo calculations; however, when mis-
matches in the spectra are observed, we pursue higher level
or implicitly (and explicitly) solvated calculations. Con-
formers of each test structure were geometry optimized at
the B3LYP/6‐31G** level, and stationary points were con-
firmed by performing frequency calculations.19-27 Calcu-
lated IR spectra saw improvement in quantitative peak
positioning when the compounds were further minimized
within the Solvation Model based on Density (SMD)
implicit solvent model of chloroform.28 All calculations
were performed using Gaussian 09.29 Output from the fre-
quency calculation is used to extract the IR and VCD spec-
tra.30 Frequencies were initially scaled by a value of 0.98
but are further scaled during comparison of experimental
and computational spectra.
2 | MATERIALS AND METHODS
Output conformers were ranked according to DFT
energy, and a clustering was performed to remove dupli-
cates. Two Boltzmann distributions were calculated based
on either the electronic energy (E) or the free energy (G).
The method for comparing spectra is based on a published
algorithm for calculating similarity between the experi-
mental and calculated IR or VCD spectra.31 The following
modifications were introduced to the algorithm: scale the
spectra (0 to 1 for IR, −1 to 1 for VCD) before comparing;
isolation of each peak for movement as opposed to
shifting groups of peaks; isolation of peaks independently
for IR and VCD spectra; peaks shifted by a default maxi-
mum shift of 25 cm−1; baseline correction of the experi-
mental spectrum when needed. When adjacent peaks
are shifted relative to each other, a gap in the spectrum
is introduced. The algorithm fits the disjoint ends of adja-
cent peaks to a parabola for a smooth transition by
employing a three‐point parabolic fit using the last 2 data
points of the left most peak and the first point of the next
peak. The method uses an overlap integral to estimate the
fit which translates to a range of possible fit values of −1.0
to 1.0 for the VCD curve and 0.0 to 1.0 for the IR. The
enantiomeric similarity index, or ESI, is the difference in
fit of one enantiomer minus the other.
In order to obtain pure enantiomeric samples of fluralaner,
chiral prep supercritical fluid chromatography (SFC) was
performed on the racemic mixture. Separation was achieved
using a Chiralpak IA column (30 × 250 mm I.D., with 5 μm)
with an isocratic mobile phase: CO2 /MeOH (60/40). In
order to separate sufficient quantity for VCD analysis, a
series of 30 stacked injections was used with the following
parameters: concentration of 50 mg/mL, injection vol of
0.5 mL, flow rate of 70 mL/min, back pressure of 100 bar,
temperature at 35°C, and detection at 254 nm. The final
product enantiomers after separation had ee values for A
and B measured at >99.9% and 97.9%, respectively.
Samples A (optical rotation: +56.2 at 589 nm, 25°C)
and B of fluralaner were dissolved in CDCl3 (50 mg/mL
for each sample). All experiments were performed using
a 0.10‐mm path length cell with BaF2 windows. The IR
and VCD spectra were recorded using a ChiralIR VCD
spectrometer equipped with the Dual PEM accessory
(BioTools, Jupiter, Florida), with 4 cm−1 resolution. A
dry N2 purge was used to eliminate water from the instru-
ment. Data were collected in blocks, where the instru-
ment recorded approximately 3000 scans over the course

KONG ET AL.
3
Any choice of computational method when performing
Percentage contribution to the in vacuo Boltzmann distri-
butions are included in Figure 2 and total 78% for the elec-
tronic energy weighted distribution of the top four
conformers, but these same conformers contribute only
24% to the free energy distribution. This is indicative of a
conformationally diverse landscape. Free energy ranking
of the conformers provides 22 conformers which contribute
>1% to the distribution with nine conformers contributing
>5% to a total of 54%. Coordinates for dominant minima
are provided in Figure S1 Supporting Information.
VCD calculations may lead to artifacts in the overlay of
spectra owing to whether calculated modes are robust or
nonrobust.32,33 In the current study, all peaks noted in
the experimental spectrum were present in the computed
spectrum so this methodology was not applied in this
study. As will be discussed, carbonyl peaks were present
in the calculated spectrum only in the absence of a proper
Boltzmann population of conformations.
The major unifying feature of the dominant con-
formers, as ranked by electronic energy, is the presence
of an intramolecular hydrogen bond (see NH‐‐‐O═C in
conformer 1) between the two amide functional groups
(conformers 1‐4). The main structural difference between
conformer pairs 1, 2 and 3, 4 is a dihedral rotation of 180°
about the dihydroisoxazole‐phenyl bond. Though the
intramolecular hydrogen bond dominates the enthalpic
energy, there is an entropic penalty to forming this bond.
This entropic penalty is mostly due to the need to fold the
amide tail back upon itself. Further minimization of the in
vacuo conformers in implicit chloroform provides similar
energetic distributions with the four dominant electronic
energy minima equivalent to those shown in Figure 1,
however contributing only 63% to the Boltzmann weighted
spectrum compared to 78% for the calculations performed
in vacuo.
3 | RESULTS AND DISCUSSION
3.1 | Experimental spectra
Experimental determination of the VCD and IR spectra for
fluralaner was initially considered rather straightforward.
Fluralaner is sufficiently soluble in chloroform for the
spectra to be clean, and upon solvent subtraction, baseline
correction, and enantiomer correction, the spectra shown
in Figure 1 were obtained. From the IR panel, it is clear
that all fluralaner functional groups are present. However,
despite sharp peaks in the region from 1000 to 1500 cm−1 in
the measured VCD spectrum, the region 1600 to 1800 cm−1
where carbonyl signals are expected is conspicuously flat.
Of the assignments of absolute configuration which have
been performed at MRL, this represents the first example
of a compound containing amide bonds which does not
display carbonyl peaks in the measured VCD spectrum.
To better understand this observation, additional compu-
tational experiments were explored.
3.3 | In vacuo matching
Separated enantiomeric Samples A and B were individu-
ally compared to the in vacuo Boltzmann weighted
calculated IR and VCD spectra of the (R)‐configuration.
Experience indicates that an IR match of 0.7 to 0.9 repre-
sents a strong confirmation of the 2D functionality of the
molecule, and a VCD match in the range of 0.2 to 0.8 rep-
resents a good VCD match. A larger ESI provides more
confidence in the assignment of configuration. The initial
conformer search performed is not described in detail
3.2 | Conformational search
An initial conformational search of fluralaner was per-
formed in vacuo using B3LYP/6‐31G** and which identi-
fied four dominant conformations that are depicted in
Figure 2. The structure modeled was arbitrarily chosen
as the (R)‐configuration, since the spectra calculated for
enantiomers by definition will be equal and opposite.
FIGURE 1 Measured IR and enantiomer‐corrected VCD spectra for fluralaner (Sample A = black, Sample B = red). Curiously absent are
any VCD signals corresponding to the two carbonyl functional groups clearly evident in the IR

4
KONG ET AL.
FIGURE 2 Conformations contributing
to the Boltzmann conformational
distribution. Electronic energy
contribution (top) and free energy
contribution (bottom) percentages are
shown. Atom coloring: grey = carbon;
red = oxygen; blue = nitrogen;
green = chlorine; cyan = fluorine;
white = hydrogen
here but led to an undersampled conformer distribution.
Initial matching from this early conformational search
of the target compound indicated significant confidence
in the assignment; however, peaks in the calculated
VCD spectrum corresponding to carbonyl stretches were
not present in the experimental VCD spectrum. This lack
of agreement between calculated and experimental spec-
tra led to additional calculations.
directionality. Some peaks at the low end of the spectrum
are consistent across conformers and Boltzmann
weighting will not average them out. Peaks in the carbonyl
region may cancel each other out during Boltzmann
averaging but only if they are equally sampled. Poor con-
formational sampling can easily lead to unbalanced popu-
lations of the dominant conformer subsets leading to
residual peak signals in the 1600 to 1800 cm−1 range. Poor
sampling was apparent in the first conformational sam-
pling of fluralaner as the molecule exhibited a more
conformationally diverse landscape than expected. This is
evidenced by the difficulty of locating conformers 1 or 4
in the original search. Analysis of the distribution of con-
formers contributing less to the Boltzmann weighted spec-
trum indicated major drivers for conformational subsets
were rotamers of both the phenyl and amide dihedrals.
To resolve the expected undersampling, the conformer
search was extended to generate the Boltzmann weighted
spectra shown in Figure 4. For extended sampling, the tar-
get number of diverse conformers was increased from 50 to
150 conformations.
3.4 | Conformational dependence
Figure 3 provides an overlay of the calculated VCD spectra
for the four most dominant electronic energy conforma-
tions. These spectra show a high degree of variability in
the peak intensities, and more importantly in the peak
With a more experimentally relevant conformer set, it
was apparent that the lack of carbonyl signals in the mea-
sured VCD was due to conformational averaging of the
signal. This was only effectively matched computationally
with proper conformational sampling. Anytime the distri-
bution was unbalanced, residual peak signals were noted
in the computed VCD. Compounds containing carbonyls
are known to occasionally be problematic owing to the
robustness of modes. Nicu et al have shown that absolute
FIGURE 3 Conformational dependence on calculated in vacuo
VCD spectra (conformers 1‐4 = black, red, green, and blue,
respectively)

KONG ET AL.
5
FIGURE 4 Overlay of in vacuo IR and VCD spectra for Samples A and B (calculated spectra in red, experimental spectra in black)
configuration assignment of pulegone is dependent on the
application of robust mode filtering due to the conforma-
tion of the carbonyl.32,33 In the case of fluralaner, the lack
of peak intensity in the experimental spectrum is
explained through cancellation of peaks via conforma-
tional averaging.
(nonintramolecular hydrogen bonded) conformers 5 to 9.
This difference is shown in Figure 5 by observing the over-
lay of two computed IR spectra for conformers 1 and 5.
Since the free energy IR is dominated by this NH stretch,
which is not represented as dominant in the experimental
IR, the electronic energy distribution dominated by four
conformers provides a better representation of the experi-
mentally relevant conformers.
While the statistics or overlays are not included, the
effect of assigning the absolute configuration of Samples
A and B using individual conformers from Figure 2 was
investigated. Since the low range of the VCD spectrum
dominates the match, even when there are residual peaks
in the carbonyl range, the assignments remain S for Sam-
ple A and R for Sample B.
From Figure 4, the absolute configuration for Sample A
((+)‐fluralaner) and Sample B ((−)‐fluralaner) can be
assigned S and R, respectively. Sample A is the active
(+)‐enantiomer, and thus the VCD analysis confirms ear-
lier crystallographic assignment of (S) as the active spe-
cies.3 Table 1 provides match statistics of the curve
overlays showing that the active enantiomer S for
Sample A has a final IR and VCD match of 0.78 and 0.74
with a very significant ESI of 0.79. For the in vacuo IR,
the curve fit is accurate across much of the range. However,
the carbonyl stretch at approximately 1780 cm−1 shows
evidence of a solvent shift. Since the default shifting algo-
rithm was limited to shifting individual peaks 25 cm−1, it
fails to shift that peak in the calculated spectrum to overlay
completely with the corresponding observed peak. The
algorithm can shift peaks by 0 to 100 cm−1, but we find
values >25 lead to problems with peaks crossing over each
other. The lower panels in Figure 4 provide the free energy
weighted overlays which have similar match statistics. The
main difference in the electronic energy and free energy
calculated IR spectra is the presence of a dominant IR
stretch approximately 1500 cm−1, corresponding to an
NH vibrational mode only present in the extended
3.5 | Implicit chloroform
While the statistical significance of the enantiomer assign-
ment was very high with the in vacuo modeling of
fluralaner, the observation of significant intramolecular
hydrogen bonding of the amides led us to consider the
possibility that, in chloroform, this interaction might be
interrupted through first shell solvation. Additionally,
even though the spectra matching algorithm shifts peaks
by up to 25 cm−1, the largest observed variation in exper-
imental IR peaks compared to the computed spectrum
was the carbonyl stretches. From Figure 4, the position
of the terminal carbonyl is at approximately 1760 cm−1

6
KONG ET AL.
TABLE 1 Matching statistics of the IR and VCD spectra (1000‐2000 cm−1 a
)
Initial IR
Final IR
Final VCD R
Final VCD S
ESI
In vacuo
Electronic energy
Sample A
0.63
0.63
0.78
0.77
−0.05
0.74
0.79
0.79
Sample B
0.74
−0.05
Free energy
Sample A
0.61
0.61
0.75
0.75
−0.01
0.78
0.79
0.79
Sample B
0.78
−0.01
Electronic energy (Supporting Information, 1000‐1500 cm−1
)
Sample A
0.68
0.89
−0.05
0.75
0.80
Implicit CHCl3
Electronic energy
Sample A
0.71
0.71
0.73
0.73
0.26
0.44
0.44
0.26
0.18
0.18
Sample B
In vacuo with 2 explicit CHCl3
Electronic energy
Sample A
0.61
0.61
0.71
0.71
−0.33
0.68
1.01
1.01
Sample B
0.68
−0.33
Free energy
Sample A
0.57
0.57
0.69
0.68
−0.10
0.67
0.77
0.77
Sample B
0.67
−0.10
Implicit CHCl3 with 2 explicit CHCl3
Electronic energy
Sample A
0.70
0.70
0.70
0.70
0.21
0.37
0.37
0.21
0.16
0.16
Sample B
In vacuo fragment (without bis‐amide tail)
Electronic energy
Sample A
0.47
0.61
−0.07
0.81
0.88
aDescription of the curve matching and derivation of ESI is provided in Materials and Methods
while the central carbonyl is at approximately 1690 cm−1
.
Table 1 indicate the ESI has decreased significantly
from 0.80 to 0.18. Absolute configuration of Sample A
would still be assigned as (S) and not (R) due to the bet-
ter fit. However, owing to initial solvent shifting of
selected regions of the IR and VCD spectra, the algo-
rithm is able to shift the improper computed enantio-
meric spectrum to overlay with the measured spectrum
to a much greater extent than with the in vacuo spectra.
The overlays of the incorrect enantiomers have not been
included but they are much worse than the overlays
shown in Figure 6 as they show evidence of improper
peak shifting done to force a match. For this reason,
even though modeling of the spectra in implicit solvent
improved the quantitative overlay of the carbonyl IR
stretches, it led to an overall less significant assignment
of absolute configuration.
When implicit solvation is included the carbonyl stretches
shift by 20 cm−1 in the calculated spectra to 1740 and
1670 cm−1. The experimental peaks are at 1700 and
1650 cm−1, which requires a shift from the in vacuo or
implicit IR peaks of 60 and 40 cm−1. This is too great a
shift for the algorithm, although these peaks correspond
to each other qualitatively. While the algorithmically
allowed shift could be set to have larger maximum shifts
for accommodation of solvent shifting, this causes artifac-
tual problems in the alignment of the rest of the spectrum
since peaks tend to cross over one another.
Figure 6 provides the overlays of the computed IR and
VCD spectra in implicit chloroform. This approach main-
tains the assignment of enantiomers that had been made
using in vacuo methodology. Statistics of the match in

KONG ET AL.
7
calculated IR were equivalent to the nonexplicit model of
fluralaner in implicit solvent. This indicates that implicit
solvation is successfully able to approximate the solvent
shift effect on the carbonyls from a nonexplicitly solvated
in vacuo model. Table 1 provides an overview of the
match statistics for overlays shown in Figure 8 for the
in vacuo explicit complex matching. The ESI for the elec-
tronic energy Boltzmann weighted spectra improves
from 0.80 to 1.01, though the overall IR match is slightly
worse.
3.7 | Explicit solvation of fluralaner in
implicit chloroform
FIGURE 5 Overlay of calculated in vacuo IR showing effect of
intramolecular hydrogen bonding (conformer 1 in black,
Short of modeling fluralaner in a chloroform solvent box,
the best approximation of the physical environment from
the experimental VCD determination is the first shell sol-
vation (even if this is limited to only 2 solvent molecules
in this case) in an implicit model of chloroform. From
Table 1, it is clear that the same decrease in ESI is calcu-
lated when considering implicit solvent estimates for
enantiomeric fit. From the overlay of the IR in Figure 9,
it is apparent that the carbonyl peaks in the IR have
shifted further to occupy positions at 1720 and
1650 cm−1, positions which are now closely in‐line with
the experimental positions. This supports the notion that
the in vacuo treatment of nonsolvated fluralaner can be
used to fit the IR and VCD with the assumption that dis-
crepancies in the position of the IR carbonyl peaks are arti-
facts due to the lack of proper solvation. Upon significant
additional computational expense to model the solvation
effects on the carbonyls, those peaks come into better
alignment but offer no better statistical match when
assigning absolute configuration. Owing to the same
undersampling (since conformers are exactly those
depicted in Figure 7) the VCD spectrum calculated with
implicit treatment of the explicit complex also shows arti-
factual peaks in the carbonyl region.
conformer 5 in red). Large variation in computed IR observed in
moving from an intramolecular hydrogen‐bond‐driven‐folded
conformation and the extended conformation. Orange box indicates
the main peak associated with extended conformation amide NHs
3.6 | Explicit solvation of fluralaner
Attempts at quantitatively improving the overlay by
modeling the conformational distribution and spectros-
copy in implicit chloroform were extended to include
modeling of explicit solvation using two molecules of
CHCl3. Since a hydrogen bond is most likely to form
between the solvent and the carbonyl groups of fluralaner,
we performed a conformational scan of a solvated complex
beginning from the four dominant in vacuo conformations
with added explicit solvent. The resultant conformational
distribution is provided in Figure 7. Many complexes of rel-
atively equivalent energy were identified, and it was clear
that conformational sampling of the explicit complexes
would be problematic since the available space was not
exhaustively sampling. Additionally, sampling of solvated
extended conformers was not pursued. Interestingly, the
positions of the explicitly solvated carbonyls from the
FIGURE 6 Overlay of implicit CHCl3 IR and VCD spectra for Samples A and B (calculated spectra in red, experimental spectra in black)

8
KONG ET AL.
FIGURE 7 Conformations contributing
to the Boltzmann distribution for explicitly
solvated fluralaner using two molecules of
CHCl3. Electronic energy contribution
(top) and free energy contribution
(bottom) percentages are shown. Atom
coloring: grey = carbon; red = oxygen;
blue = nitrogen; green = chlorine;
cyan = fluorine; white = hydrogen
FIGURE 8 Overlay of explicit CHCl3 IR and VCD spectra for Samples A and B (calculated spectra in red, experimental spectra in black)
structures.5 A VCD and IR match will generally always
be better when modeling the entire chemical species,
3.8 | Assignment of absolute
configuration using a fragment
and in turn, there will be more confidence in the assign-
A fragment‐based approach can be pursued if the mole-
cule of interest is significantly complex or flexible, or if
computational resources are limited. Owing to advances
in conformational sampling and computational resources,
we have largely relied only on modeling complete
ment if approximations in structure are not made. How-
ever, we report our investigation of this approach since
truncation of the amide tail led to a highly confident
assignment of absolute configuration. Observations made
here concerning the ability to truncate the assigned

KONG ET AL.
9
FIGURE 9 Overlay of explicit CHCl3 in implicit CHCl3 IR and VCD spectra for Samples A and B (calculated spectra in red, experimental
spectra in black)
molecule and factors complicating assignment of flexible
molecules have been made for some natural product lin-
ear diterpenes.34
The amide tail of fluralaner was truncated to provide
the fragment compound displayed in Figure 10. There
are only two in vacuo conformational minima for this spe-
cies. This serves to greatly reduce the conformational
search space, removes any ability for the formation of
intramolecular hydrogen bonds, and deletes both car-
bonyls. As demonstrated earlier, the truncated carbonyls
are not relevant due to their lack of presence in the exper-
imental VCD spectrum. If the analysis of the full species is
limited to the range of 1000 to 1500 cm−1, the match
remains consistent (Figure S1 and Table 1).
Figure 11 provides the overlays of the IR and VCD for
the (S)‐enantiomer of the fragment onto the experimental
spectra of Sample A over the full and truncated spectral
ranges. While the VCD and IR match are very good over
most of the range, it is clear that matching the fragment
FIGURE 11 Overlay of in vacuo IR and VCD spectra for Sample
A onto the fragment (calculated spectra in red, experimental spectra
of fluralaner in black)
fails to include IR content of the amides so the match in
this region is quite poor. Statistics of the fragment match
provided in Table 1 indicate that the correct absolute con-
figuration for Samples A and B could have been made
with IR overlap of 0.61 to 0.74 and ESI of approximately
0.90. In this case, completely ignoring the carbonyls in
the calculated structure still provides enough chemical
information in the IR and VCD to properly assign abso-
lute configuration.
4 | CONCLUSION
Absolute configuration assignment of fluralaner using
VCD indicated that the active (+)‐enantiomer of the race-
mic mixture has the (S) configuration. This assignment
supports the previous crystallographic determination that
the active enantiomer or (+)‐fluralaner is (S). The absence
of carbonyl peaks in the experimental VCD was shown,
through a set of computational studies, to be caused by
averaging of the conformationally dependent positive
and negative peaks in the VCD spectrum. Reducing the
range of the match to ignore the carbonyls or truncating
the chemical structure provided equivalent matching
FIGURE 10 Conformations contributing to the Boltzmann
conformational distribution of the (R) configuration of the
fragment. Electronic energy contribution percentages are shown.
Atom coloring: grey = carbon; red = oxygen; blue = nitrogen;
green = chlorine; cyan = fluorine; white = hydrogen

10
KONG ET AL.
8. Freedman TB, Cao XL, Dukor RK, Nafie LA. Absolute configu-
ration determination of chiral molecules in the solution state
using vibrational circular dichroism. Chirality. 2003;15:743‐758.
and proper assignment of the (S) configuration. When
VCD and IR spectra are compared and all functional
groups are not apparent in both spectra, it can be indicative
of conformational averaging of some structural features.
When conformational ensembles provide a calculated
Boltzmann weighted spectrum which contains peaks in
the regions with no experimental peak intensity, it may
be a sign of undersampling in the conformational search.
While calculation of spectra using implicit and explicit
solvation would be expected to better approximate the
spectroscopic environment, it was determined that a confi-
dent assignment of absolute configuration could be made
using only in vacuo calculations.
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ACKNOWLEDGMENTS
12. Joseph‐Nathan P, Gordillo‐Roman B. Vibrational circular
dichroism absolute configuration determination of natural prod-
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Springer; 2015;100:311‐452.
We thank the MRL modeling tools development group,
especially Joe Shpungin for work on implementing a con-
formational sampling routine.
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disulfide‐based prodrugs for cytosol‐specific drug delivery.
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ORCID
Leo A. Joyce http://orcid.org/0000-0002-8649-4894
14. Li H, Chen CY, Nguyen H, et al. Asymmetric synthesis of cyclic
Edward C. Sherer http://orcid.org/0000-0001-8178-9186
indole aminals via 1,3‐stereoinduction. The
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