Hydroxypyridyl Imines: Enhancing Chromatographic Separation and Stereochemical Analysis of Chiral Amines via Circular Dichroism

Article abstract of DOI:10.1021/acs.joc.6b01162

Imine-bond formation between chiral amines and commercially available 3-hydroxypyridine-2-carboxaldehyde (HCA) was exploited for rapid determination of stereochemical composition. Chiral supercritical fluid chromatography (SFC) screening of the derivatized imine compounds led to the elucidation of multiple combinations of mobile and stationary phases that gave resolution of all members of a series of chiral amines. The first eluting enantiomer was generally the derivative of the (R)-amine enantiomer across the series that was studied, indicating that the imine formed from the (S)-amine has more favorable interaction with the chiral stationary phase of the column. These conditions were then applied to more challenging compounds, namely amino alcohols and diastereomers possessing more than one stereocenter. The approach was utilized to monitor stereoselective biocatalytic transamination and assign the absolute configuration of the enantiomeric products. Finally, hydrolysis of the imine bond of the derivative was shown to generate enantiopure amine starting materials without racemization. This further highlights the value of this approach for creating readily reversed derivatives that enhance chromatographic separation and aid in the determination of absolute configuration.

Full text of DOI:10.1021/acs.joc.6b01162

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Article
Hydroxypyridyl Imines: Enhancing Chromatographic Separation and
Stereochemical Analysis of Chiral Amines via Circular Dichroism
Leo A. Joyce, Erik L Regalado, and Christopher J. Welch
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b01162 • Publication Date (Web): 16 Aug 2016
Downloaded from http://pubs.acs.org on August 18, 2016
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Hydroxypyridyl
Separation and Stereochemical Analysis of Chiral Amines via
Circular Dichroism
Imines:
Enhancing
Chromatographic
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Leo A. Joyce*, Erik L. Regalado, Christopher J. Welch
Analytical Research & Development, Merck Research Laboratories, Rahway, New Jersey
07065, USA
*E-mail: leo.joyce@merck.com
ABSTRACT:
Imineꢀbond formation between chiral amines and commercially available 3ꢀ
hydroxypyridineꢀ2ꢀcarboxaldehyde (HCA) was exploited for rapid determination of
stereochemical composition. Chiral supercritical fluid chromatography (SFC) screening
of the derivatized imine compounds led to the elucidation of multiple combinations of
mobile and stationary phases that gave resolution of all members of a series of chiral
amines. The first eluting enantiomer was generally the derivative of the (R)ꢀamine
enantiomer across the series that was studied, indicating that the imine formed from the
(S)ꢀamine has more favorable interaction with the chiral stationary phase of the column.
These conditions were then applied to more challenging compounds, namely amino
alcohols and diastereomers possessing more than one stereocenter. The approach was
utilized to monitor stereoselective biocatalytic transamination and assign the absolute
configuration of the enantiomeric products. Finally, hydrolysis of the imine bond of the
derivative was shown to generate enantiopure amine starting material without
racemization. This further highlights the value of this approach for creating readily
reversed derivatives that enhance chromatographic separation and aid in the
determination of absolute configuration.
Keywords: chiral amines; derivatization; chiral separations; chiroptical sensing;
supercritical fluid chromatography; circular dichroism.
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GRAPHICAL ABSTRACT:
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INTRODUCTION:
The development and optimization of asymmetric reactions that selectively
produce a single stereoisomeric product has been an intense area of research focus for
several years.1ꢀ3 The chromatographic separation of enantiomers on chiral stationary
phases is a cornerstone capability that facilitates this area of modern chemical research.4ꢀ8
Today, chromatographic separation is almost always carried out directly on unmodified
compounds of interest. Indirect methods requiring chemical derivatization were
widespread in the past,9ꢀ10 and are still necessary in many cases where the compounds of
interest have poorly absorbing chromophores or their enantioseparation proves
challenging.11ꢀ13
Chiral primary amines, important to the synthesis of active
pharmaceutical ingredients,14ꢀ15 make up one such class of compound that can be difficult
to separate. The ideal derivatization reagents for chromatographic enantioseparation will
allow fast and convenient reaction, while enhancing detectability or separability. In
addition, the convenient removal of the derivatizing group is important, should access to
the original underivatized analyte be desired.
We recently reported the development of a method for the study of chiral amines
by circular dichroism (CD) spectroscopy based around derivatization with 3ꢀhydroxy
pyridine–2ꢀcarboxaldehyde (HCA).16 Imineꢀbond formation between the HCA and the
amine to generate a CDꢀactive species occurs rapidly and in high yield. The resultant
imine has strong UV and CD signals at 317 nanometers, avoiding interference with most
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organic compounds. We also found that these imines were reasonably stable to high pH
liquid chromatography conditions, affording well shaped peaks in these studies. This
allowed us to couple the CD detector with an HPLC instrument to facilitate highꢀ
throughput analysis of the enantiopurity of synthetic chiral amines. Additionally, the sign
of the CD signal could be related to the absolute configuration of the original amine
within the suite of compounds that were tested. In this study we investigate the potential
for the use of HCA derivatization to provide an ‘easy on/easy off’ method for improving
chromatographic enantioseparation of amine analytes on chiral stationary phases while
enhancing UV absorption and providing a detectable CD signal that can be useful for
stereochemical analysis.
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Supercritical fluid chromatography (SFC), based on the application of
supercritical carbon dioxide as the main mobile phase component, was selected as a
separation technique for this work. Chiral SFC has recently emerged as the preferred tool
for analytical and preparative separation of chiral molecules,17ꢀ20 because it affords fast
separations, tolerates a large range of analyte polarities, and has an economical and green
carbon dioxide mobile phase.
RESULTS AND DISCUSSION
The first step of this study was to identify possible general SFC conditions for
enantioseparation of the imine derivatives. In order to do this, a diverse set of imines
were generated from the reaction of the corresponding chiral amines and HCA. The
resulting chiral imine mixtures were subjected to standard SFC screening using systems
routinely used for developing analytical methods in support of drug discovery and
development.21ꢀ22 A total of 12 chiral stationary phases (CSPs) in combination with
several polar modifiers were used in this study (see Supporting Information for full
screening chromatograms).
Selected examples from this evaluation are shown in Figure 1.
Cyclohexylethylamine (CEA) does not have any chromophoric elements, and thus is
invisible to UV detection even at 210 nm. As a result, no chiral separation methods can
be identified during direct screening of this amine with UV detection (Figure 1a).
However, when the imine derivative formed between CEA and HCA was screened
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baseline enantioseparation was found for multiple combinations of column and modifier.
In another example shown in Figure 1b, the low wavelength UV signal for 1ꢀmethylꢀ3ꢀ
phenylpropylamine (MPP) is sufficient for detection, but the poor chromatographic
performance (peak shape and incomplete resolution) on every CSPs is not suitable to
directly develop a chiral separation method. Similar to the CEAꢀimine example, multiple
chromatographic screening conditions delivered baseline separation of the MPPꢀimine
enantiomers with no need for subsequent optimization experiments.
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Figure 1. Chiral SFCꢀUV separation of amine and imine derivatives using standard screening conditions on
six different CSPs as detailed in the experimental section. a,b) Enantiomers of CEA, MPP and respective
imine derivative on the same columns using 25 mM iBuNH2 in MeOH as polar modifier. Y scale was kept
from imine to amine experiment to allow comparison. c) Map of best chiral SFC conditions for separation
of imineꢀderived products of seven different amines. Three different mobile phases (25 mM iBuNH2 in
MeOH, iPrOH, and EtOH/CO2) were screened. The scoring color system was developed based on baseline
(green), partial (yellow) or no separation (red) as illustrated in this figure.
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To fully test the generality of this approach, imine derivatives of seven
commercially available amines were prepared and subjected to the same screening
protocol as described for CEA and MPP (Figure 1c). This set of compounds was
comprised of both aliphatic and aryl amines, and this representative set was used to help
identify general trends in enantioselectivity of the CSPs for the imine species. The results
were qualitatively sorted into three categories based on how well the enantiomers were
separated: full baseline resolution (green), partial resolution that could likely be
optimized (yellow), and no separation (red). This approach allowed us to identify two
CSPs (Chiralpak ADꢀ3 and Chiralcel OZꢀ3) that were general toward the chiral imine
derivatives, providing baseline enantioseparation for all of the compound mixtures with
any of the polar modifiers. Having identified suitable conditions for the enantioseparation
of chiral imineꢀderived amines, we next probed the elution order of the imine
enantiomers.
In order to track the absolute configuration of the eluting imine enantiomer, we
made use of a commercially available SFCꢀCD detector (Jasco, Inc). This is the same
detector used for HPLCꢀCD analysis, but makes use of a high pressure flow cell designed
to handle the high back pressure (~200 bar) required for SFC analysis.23 A single
isocratic method that gave the best resolution across the series was developed using a
Chiralpak ADꢀ3 column and iPrOH with iBuNH2 (25 mM). Though iBuNH2 itself is a
primary amine, virtually no reversibility of the desired imines were observed on the
experimental timescale. Each sample was made up of 75% (R)ꢀamine to ensure that the
absolute configuration could accurately be assigned to the eluting peak. Two additional
amines that had not been screened were included in this set, namely 1ꢀphenylpropylamine
(PPA) and 2ꢀaminoꢀ3ꢀmethylbutane (AMB), to demonstrate the applicability of these
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chromatography conditions beyond the set of compounds that was originally screened.
The UV and the CD signals at 317 nm were recorded for these separations. As illustrated
in Figure 2, the first eluting peak in each case is the larger 75% area peak, representing
the (R)ꢀenantiomer. This first peak has a negative CD signal at 317 nm, which is
consistent with what we reported previously for imines derived from a series of related
(R)ꢀamines.16 The enantiomeric elution order observed here is expected to apply only
among closely related compounds under study, and is not likely to be general across all
amines. These conditions delivered baseline resolution of both of the imines that had not
been screened previously, further demonstrating the generality of this approach.
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CEA (75% R)
2
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MBA (75% R)
MPP (75% R)
S
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PPA (75% R)
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Figure. 2. Chiral SFCꢀUVꢀCD analysis of six different imineꢀderivatives. Optimized chiral SFC method
from best screening conditions in fig. 1: Samples: 5 ꢁL injection of each mixture (75% (R)ꢀenantiomer and
25% (S)ꢀenantiomer) in ACN; column: Chiralpak ADꢀ3 (4.6 x 150 mm, 3 µm); temperature: 40˚C; flow
rate: 3 mL/min; isocratic mobile phase: 15% polar modifier (25 mM iBuNH2 in iPrOH) / 85% CO2, except
for AMB: 5% / 95%. Backpressure regulator (BPR): 150 bar. Detection: UV and CD at 317 nm.
In order to expand the utility of this method, we broadened the scope of our
analysis to incorporate amino alcohols (Figure 3a). The resultant imino alcohols formed
from the amino alcohols leucinol (Leuc) and 2ꢀphenylglycinol (2ꢀPG) were more polar
than those formed with the alkyl and aliphatic amines, and required an isocratic method
with a higher amount of iPrOH in order to elute the compounds from the column. These
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imino alcohols also appeared to be more prone to hydrolysis back to the amino alcohol
and HCA, and required a lower column temperature (20°C) in order to prevent onꢀ
column degradation. After making the required adjustments, baseline resolution was
observed for these two analytes. In both of these cases the 25% area peak corresponding
to the imino alcohol formed from the (S)ꢀenantiomer of the amino alcohol is the first to
elute, and has a negative signal. These seemingly contradictory results are consistent with
what has been observed previously for this type of substrate.15,24 In this case, a change in
CahnꢀIngoldꢀPrelog priority leads to a change in absolute configuration, while the largest
and smallest groups are consistent with the previous model. Hence, the imines formed
from the (S)ꢀamino alcohols give rise to a negative CD signal at 317 nanometers, and
elutes first under these chromatographic conditions.
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a) from amino alcohols
2-PG (75% R)
Leuc (75% R)
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b) from diastereomeric amines
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Figure 3. a) Chiral SFCꢀUVꢀCD analysis of imineꢀderived amine alcohols using the method described in
fig. 2 at 40% modifier / 60% CO2. Samples: 5 ꢁL injection of each mixture (75% (R)ꢀenantiomers) in ACN.
b) Chiral SFCꢀUVꢀCD analysis of two different diastereomeric imineꢀderived amines. Chromatographic
conditions as described in Fig. 2 at 5% modifier / 95% CO2.
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To further demonstrate the utility of this method, we turned our attention to
amines with two stereocenters (Figure 3b). Developing a method for separating four
stereoisomers is more challenging than that for a pair of enantiomers. In this instance,
additional screening and optimization using the same column but with a different mobile
phase afforded superior resolution of the stereoisomeric products. It is clear from the UV
and CD signals above that the two enantiomers of a single diastereomer elute
consecutively, followed by both enantiomers of the other diastereomer. For example, the
first two imine peaks derived from 3ꢀmethylcyclohexylamine (MCHA) in Figure 3b,
between 2.5 and 3.5 minutes, have the same UV signal and CD signals with equal and
opposite magnitude. Similarly, the last set of peaks have the same UV signals, with equal
and opposite CD signals at 317 nm. The first eluting peak for each diastereomer, the first
and third overall eluting stereoisomeric peaks, have a positive CD signal at 317 nm. The
positive CD signal indicates that these peaks likely have the (S)ꢀconfiguration at the
carbon bearing the nitrogen. This switch in elution order may be due to the change in
solvent from iPrOH to MeOH. These compounds were only commercially available as
racemic mixtures of stereoisomers, and the relative configuration of each diastereomer
could not be assigned using this methodology. Caution must be taken in using this
method for determining the diastereomeric ratio (dr), since the diastereomeric imines
likely form to different extents and are prone to potential hydrolysis at different rates
while passing through the chromatographic stationary phase.
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To demonstrate the practical application of this system, we turned our attention to
the synthesis of Vernakalant. Vernakalant was discovered by Cardiome Pharma Corp,
and was developed to treat recentꢀonset atrial fibrillation through selective inhibition of
Naꢀ and Kꢀchannels.25 Merck recently reported an efficient asymmetric synthesis of this
compound, proceeding through chiral amine intermediate 2 shown in Figure 4a.26ꢀ27 The
stereochemistry for the two contiguous stereocenters on the cyclohexane ring was set in a
highly stereoselective fashion through an enzymeꢀcatalyzed dynamic asymmetric
transamination from the corresponding αꢀchiral ketone substrate 1. Three rounds of
evolution gave an enzyme that was selective in delivering the desired stereoisomer of
amine 2 with very high enantioꢀ and diastereoselectivity. The imine derivatization and
subsequent SFCꢀCD analysis was performed on amine intermediate 2. A mixture of all
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four stereoisomers was obtained by reductive amination with in the absence of any
enantioenriched influence. The crude reaction material could be used directly after
filtration through celite without further purification, since the absorbance of the HCAꢀ
imine is at a sufficiently long wavelength to avoid interference from any reaction
components.
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Figure 4. a) Stereoselective enzymatic transaminaton to afford Vernakalant intermediate 2. b) Chiral SFCꢀ
UVꢀCD analysis for the vernakalant amine as its imine derivative. c) Results of the transamination and
subsequent imineꢀformation, showing formation for an (S)ꢀselective (ATAꢀ234), or (R)ꢀselective (ATAꢀ
014) enzymes, as well as a variant evolved to give selectively (R,R) for this substrate (ATAꢀ303).
Chromatographic conditions; column: Chiralpak ADꢀ3 (4.6 x 150 mm, 3 µm); temperature: 40˚C; flow
rate: 3 mL/min; isocratic mobile phase: 15% polar modifier (25 mM iBuNH2 in 50% MeOH, 50% iPrOH) /
85% CO2. Backpressure regulator (BPR): 150 bar. Detection: UV and CD at 317 nm.
Baseline resolution of the four HCAꢀimines was not achieved using either pure
MeOH or iPrOH as a polar modifier, but rather a 1:1 mixture of the two solvents was
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used to give the best resolution (Figure 4). The first two eluting peaks showed negative
CD signals at 317 nm, indicating that they likely have the (R)ꢀconfiguration at the carbon
bearing the nitrogen. This is different from what we observed previously, where the two
enantiomers of each diastereomer eluted together as a pair. This could be due to the
mixture of the solvents, or possibly because of the size and additional interactions
available to the aromatic side chain attached to the other stereocenter on the cyclohexane
ring. The second eluting peak was confirmed to be the desired (R,R)ꢀstereoisomer by
injection of the authentic sample, confirming the tentative assignment made by sign of
the CD signal.28 Based on this information, the assignment of each stereoisomer is made
according to the label in Figure 4a. After optimizing the separation method for the four
stereoisomers, we focused on analyzing the formation of the product by enzymeꢀ
mediated transamination. Three enzymes were selected for the synthesis: ATAꢀ234,
ATAꢀ014, and ATAꢀ303. This panel of enzymes was selected to contain diversity in
selectivity, with one general Sꢀselective (ATAꢀ234) component, one general Rꢀselective
(ATAꢀ014) enzyme, and one that had been evolved for ketone substrate 1 (ATAꢀ303).
The reactions were allowed to stir for one day, and the products subsequently analyzed
after extraction into ethyl acetate and subsequent imine formation. The results of these
reactions are shown in Figure 4. The product generated by ATAꢀ234 was comprised of
only two stereoisomers, each with approximately equal area by UV. These stereoisomers
gave positive CD signals at 317 nm, which supports formation of the (S)ꢀconfiguration at
the carbon attached to nitrogen. In contrast, the other two stereoisomers were formed in
approximately the same concentration with ATAꢀ014. These stereoisomers had negative
signals at 317 nm, indicating that the stereoisomeric products formed both have the (R)ꢀ
configuration at the carbon bearing nitrogen. Finally, ATAꢀ303 gave rise to the same two
diastereomers as ATAꢀ014. As anticipated, this enzyme afforded selective formation of
the desired Vernakalant intermediate stereoisomer.
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This approach allows relatively straightforward differentiation of enantiomeric
imines formed from corresponding chiral amines. The imine formation occurs rapidly,
and is stable to high pH chromatographic conditions. A large CD signal is generated in a
portion of the spectrum that previously had no discernible signals, and the sign of the
output signal allows the assignment of absolute configuration to be made with some
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degree of confidence. The ability to remove the HCA derivatizing reagent to afford the
pure enantiomer, desirable in preparative enantioseparations or enantiopurity upgrades,
was studied further. In order to test that HCA met this criterion as a derivatizing reagent,
we studied the hydrolysis of the imine formed between HCA and MBA. Imines are
known to be susceptible to acidꢀcatalyzed hydrolysis, and hence we began our study
focusing on hydrochloric acid (1N HCl). Addition of five equivalents of HCl relative to
the imine gave complete hydrolysis of the imine bond and regeneration of the starting
amine and HCA (Figure 5). There was concern that the addition of acid could lead to
undesired racemization of the stereocenter during hydroloysis. A chiral SFC method was
developed for the MBA analyte alone (Figure 1c), and this method showed that no
observable racemization occurred for either enantiomer during the process of hydrolysis
(see supporting information for additional details). Hence, this group indeed performs as
an “easy on easy off” protecting group while maintaining stereochemical integrity during
its incorporation and removal.
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HCA
amine integration
1845 ± 4 µV*sec
MBA
*
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*imine derivative
+
HCA
imine
hydrolysis
amine integration
1839 ± 8 µV*sec
imine mix
+
0
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min
1N HCl
Figure 5. Monitoring amine recovery by comparison of achiral reversed phase HPLC profiles before and
after imine hydrolysis. 2 mM 3ꢀhydroxypyridineꢀ2ꢀcarboxaldehyde (HCA) + 2 mM methylbenzylamine
(MBA). 10 ꢁL 1N HCl (5 eq) was added to the respective imine derivative mixture and then analyzed. All
experiments were performed in triplicate (n = 3). Detailed chromatographic conditions are described in the
experimental section.
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CONCLUSIONS
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We have demonstrated that 3ꢀhydroxypyridineꢀ2ꢀcarboxaldehyde (HCA) can function as
an efficient derivatizing agent for subsequent chiral SFC analysis. Optimal conditions
were found to give good resolution of a large suite of imineꢀderived chiral amines, with
the (R)ꢀenantiomer generally eluting first for this suite of compounds under the optimized
conditions. These conditions were then expanded to encompass a wider substrate scope,
namely amino alcohols and compounds with multiple stereocenters. Separation across all
of the substrates was achieved using the same chromatographic stationary phase, namely
Chiralpak ADꢀ3. To demonstrate the practical application of this sensing ensemble, a
biocatalytic transamination forming a chiral amine intermediate in the synthesis of
Vernakalant was monitored. Conditions were identified that formed predominantly the
(R)ꢀ or (S)ꢀ enantiomer, with respect to the stereocenter bound to nitrogen. Finally, the
imine bond was hydrolyzed to generate enantiopure amine starting material and HCA
with no corresponding racemization seen.
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EXPERIMENTAL
Chiral SFC Screening Conditions
Chiral SFC screening experiments were carried out on 4.6 mm × 150 mm, 3 µm columns
by gradient elution at a flow rate of 3 mL/ min as described previously in the literature.21
The SFC eluents were solvent A (CO2) and solvent B (25 mM iBuNH2 in
MeOH/iPrOH/EtOH or ACN). The mobile phases were programmed as follows: linear
gradient from 1% to 40% B in 5, hold at 40% B for 1 min. The column and samples were
maintained at a temperature of 40 °C and 20 °C, respectively.
Derivatization of chiral amine analytes
Derivatization of chiral amines was carried out using the protocol described previously.16
Briefly, 10 mM acetonitrile stock solutions were made of HCA and the desired amine
analyte. Next, equimolar amounts of the amine analytes and HCA (200 uL) were mixed
to afford the respective imine products, followed by dilution with 600 uL acetonitrile
This analysis was done in situ, with no need to purify the product or remove water from
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the mixture. The formation of chiral imineꢀderived amines leads to a strong CD signal at
317 nm which has been used in this study for the analysis.
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Vernakalant Amine Synthesis
9
Synthesis of Vernakalant ketone intermediate 1, as well as subsequent transamination
reactions, were carried out as described in the literature.26 To form the mixture of
stereoisomers, ketone intermediate 1 (0.2 mmol) was stirred in methanol (5 mL).
Ammonium hydroxide (10 mmol) and sodium cyanoborohydride (10 mmol) were then
added, and the mixture was allowed to stir overnight at room temperature. The mixture
was filtered through celite, concentrated in vacuo, and used without further purification.
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Imine Hydrolysis
The imine formation was carried out as described previously, thorough equimolar
addition of the desired amine and HCA. To this solution was added 1N HCl (10 uL), and
the product was briefly mixed before HPLC analysis to determine extent of imine
hydrolysis.
SUPPORTING INFORMATION:
Supporting information is available online: SFC chromatograms of chiral screening;
monitoring racemization in imine hydrolysis.
AUTHOR INFORMATION:
Corresponding author: leo.joyce@merck.com
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
We are grateful to MRL Postdoctoral Research Fellows Program for financial support
provided by a fellowship (E.L.R), and to the MRL Process and Analytical Chemistry
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Screening and High Throughput Analysis Laboratory for making available the use of the
instrumentation used in this study.
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REFERENCES
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23 For detailed instrumental specifications, please see jascoinc.com
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W.; Anslyn, E. V. J. Am. Chem. Soc. 2011, 133, 13746ꢀ13752.
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28 Characterization of authentic standards was undertaken in Ref. 26.
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