A facile circular dichroism protocol for rapid determination of enantiomeric excess and concentration of chiral primary amines

Article abstract of DOI:10.1002/chem.200902650

A protocol for the rapid determination of the absolute configuration and enantiomeric excess (ee) of α-chiral primary amines with potential applications in asymmetric reaction discovery has been developed. The protocol requires derivatization of α-chiral primary amines through condensation with pyridine carboxaldehyde to quantitatively yield the corresponding imine. The Cu¹ complex with 2,2′-bis (diphenylphosphino)-l,l'- dinaphthyl (BINAP-Cu¹) with the imine yields a metal-to-ligand charge-transfer (MLCT) band in the visible region of the circular dichroism (CD) spectrum upon binding. Diastereomeric hostguest complexes give CD signals of the same signs but different amplitudes, allowing for differentiation of enantiomers. Processing the primary optical data from the CD spectrum with linear discriminant analysis (LDA) allows for the determination of the absolute configuration and identification of the amines, and processing with a super-vised multilayer perceptron artificial neural network (MLP-ANN) allows for the simultaneous determination of the ee and concentration. The primary optical data necessary to determine the ee of unknown samples is obtained in two minutes per sample. To demonstrate the utility of the protocol in asymmetric reaction discovery, the ee values and concentrations for an asymmetric metal-catalyzed reaction are determined. The potential of the application of this protocol in high-throughput screening (HTS) of ee is discussed.

Full text of DOI:10.1002/chem.200902650

FULL PAPER
DOI: 10.1002/chem.200902650
A Facile Circular Dichroism Protocol for Rapid Determination of
Enantiomeric Excess and Concentration of Chiral Primary Amines
[
a]
Sonia Nieto, Justin M. Dragna, and Eric V. Anslyn*
Abstract: A protocol for the rapid de-
termination of the absolute configura-
tion and enantiomeric excess (ee) of
a-chiral primary amines with potential
applications in asymmetric reaction dis-
covery has been developed. The proto-
col requires derivatization of a-chiral
primary amines through condensation
with pyridine carboxaldehyde to quan-
titatively yield the corresponding
upon binding. Diastereomeric host–
guest complexes give CD signals of the
same signs but different amplitudes, al-
lowing for differentiation of enantio-
mers. Processing the primary optical
data from the CD spectrum with linear
discriminant analysis (LDA) allows for
the determination of the absolute con-
figuration and identification of the
amines, and processing with a super-
vised multilayer perceptron artificial
neural network (MLP-ANN) allows for
the simultaneous determination of the
ee and concentration. The primary op-
tical data necessary to determine the ee
of unknown samples is obtained in two
minutes per sample. To demonstrate
the utility of the protocol in asymmet-
ric reaction discovery, the ee values
and concentrations for an asymmetric
metal-catalyzed reaction are deter-
mined. The potential of the application
of this protocol in high-throughput
screening (HTS) of ee is discussed.
I
imine. The Cu complex with 2,2’-bis
(
diphenylphosphino)-1,1’-dinaphthyl
Keywords: amines · artificial neural
network · circular dichroism · enan-
tiomeric excess · high-throughput
screening
I
(
BINAPÀCu ) with the imine yields a
metal-to-ligand
charge-transfer
(
MLCT) band in the visible region of
the circular dichroism (CD) spectrum
Introduction
For instance, optical techniques for the determination of
ee have emerged as a powerful approach to high-throughput
screening (HTS) because they are fast, simple, inexpensive,
Asymmetric catalysis is a commonly employed method for
the synthesis of enantiomerically enriched compounds.
[1]
[5]
and easily adaptable to determine concentration. For ex-
Traditionally, asymmetric catalysts are developed from a
combination of intuition, knowledge of the reaction mecha-
nism, molecular modeling, and trial and error, which are
time-consuming processes. Combining traditional techniques
with those used in the screening of large libraries of poten-
tial asymmetric catalysts could potentially accelerate the dis-
ample, our group and others have exploited the use of opti-
cal techniques based on enantioselective indicator-displace-
[6]
ment assays (eIDAs).
Recently, we reported a simple protocol based on the
[7]
combination of circular dichroism (CD) spectroscopy and
[8]
pattern-recognition-based techniques.
The protocol is
[2]
covery process. However, this requires the rapid determi-
based on monitoring changes in the metal-to-ligand charge-
transfer (MLCT) bands of chiral and racemic metal com-
plexes upon the addition of chiral diamine analytes. This
technique allows for rapid screening of ee and the determi-
nation of concentration.
nation of enantiomeric excess (ee), which is difficult with
[3]
chromatographic techniques such as HPLC and GC.
A
ACHTUNGTRENNUNG
variety of methods are currently being developed to over-
[4]
ACHTUNGTRENNUNGc ome this problem.
a-Chiral primary amines are important building blocks in
[9]
chemical and pharmaceutical transformations.
Several
[
a] S. Nieto, J. M. Dragna, E. V. Anslyn
Department of Chemistry and Biochemistry
University of Texas at Austin, Austin, TX 78712 (USA)
Fax : (+1)512-471-8696
methods for the asymmetric synthesis of chiral amines are
under development. Rapid screening of asymmetric reac-
[10]
tions might hasten the discovery of an efficient route to
these versatile building blocks. Thus, a method for the rapid
E-mail: anslyn@austin.utexas.edu
[11]
screening of ee for a-chiral primary amines is desirable.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200902650.
Chem. Eur. J. 2010, 16, 227 – 232
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
227

Herein, we describe a protocol for the rapid screening of
to form the corresponding chiral Schiff base in situ
(Scheme 1). The overall reaction was fully complete in
under two hours and resulted in quantitative formation of
the imine. The imines were directly used without
ee of a-chiral primary amines. The protocol utilitizes the re-
I
ceptors (R)- and (S)-[Cu
A
H
U
G
R
N
U
G
A
H
U
G
R
N
U
G
2
6
which BINAP is 2,2’-bis (diphenylphosphino)-1,1’-dinaph-
thyl (See Figure 1a). Both (R)- and (S)-1 complexes show
[12]
AHCTUNGTRENNGNUp urification.
Scheme 1. Derivatization of the amines to form the corresponding Schiff
bases.
The addition of the chiral imines to a solution of receptor
(
R)-1 modulated the signal in the CD spectrum spanning
Figure 1. a) (R)- and (S)-1 complexes employed as receptors; b) Chiral
primary amines employed as analytes: a-methyl-benzylamine (MBA),
from approximately 320 nm to 470 nm (Figure 2). No CD
1
-cyclohexyl-ethylamine (CEA), 2-aminoheptane (AHP).
MLCT bands around 340 nm in the CD spectra, and the
changes in these MLCT bands after the addition of enantio-
merically enriched chiral guests give a method for enantio-
discrimination and concentration determination of the chiral
monoamines (shown in Figure 1b). To test the practicality
of the protocol, a sample from an asymmetric reaction was
prepared. The ee of the sample was determined by the CD
protocol and was compared with values obtained from the
1
well-accepted H NMR protocol developed by James and
[11a,b]
co-workers,
showing that the CD protocol allows for
rapid screening with comparable accuracy to standard itera-
tive protocols.
Results and Discussion
Design: For the protocol to be of use in rapid screening, a
system in which the analyte (chiral amine) does not have to
be derivatized is preferable. Derivatization is time consum-
ing and frequently requires purification before and after de-
rivatization. Additionally, either a commercially available
receptor or one that required only a one–two step synthesis
was sought because this would increase its usefulness to the
general chemistry community. Unfortunately, there was no
signal modulation through addition of an underivatized
chiral amine to (R)-1. Thus, we developed a method for de-
rivatization that is fast and does not require purification.
This minimizes the amount of time required for the derivati-
zation step and still allows the protocol to be amenable to
rapid ee determination.
Figure 2. a) CD spectrum for (R)-1
0.8 mm] (c=(R)-CPL, c=(S)-CPL);(b) Titration of (R)-1
with (R)- and (S)-CPI in acetonitrile at 354 nm. (*=(R)-CPL,
=(S)-CPL).
ACHTUNGTRENNUNG
[
ACHTUNGTRENNUNG
*
signals above 320 nm were observed for the chiral imines,
for copper alone with the imines, or for free BINAP. Thus,
the signal is the result of a MLCT in the coordination com-
plex of the imines with (R)-1.
Identification of amine and enantiodiscrimination: The ab-
solute configuration and the identity of the amines can be
determined by analyzing the primary optical data through
Detection scheme: As previously mentioned, signal modula-
tion of the receptor was not possible upon the addition of
underivatized chiral amines. To overcome this problem, the
chiral amine was condensed with 2-pyridine carboxaldehyde
[13]
linear discriminant analysis (LDA).
Titration of (R)-1
with the imines showed that saturation was reached at two
equivalents (see Figure 2b). For this system, saturation gives
228
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 227 – 232

Facile Circular Dichroism Protocol
FULL PAPER
the maximum signal-to-noise ratio, and is thus best suited
for data collection and analysis; hence, all data was collected
at two equivalents. Each experiment was repeated five times
to ensure reproducibility. The ellipticities were recorded at
four different wavelengths (350, 355, 360, and 380 nm) and
analyzed with LDA. The selected wavelengths were chosen
around the positive maximum presented in the Cotton
effect to try to obtain all of the possible information from
the spectra. The use of more wavelengths does not give any
improvement to the resulting LDA plot. The analytes
showed good separation in the LDA plot (Figure 3). Positive
Figure 4. Two-dimensional PCA plot of CPI with three ee trainings sets
at three different [G] values: 0.2 mm, 0.8 mm and 1.4 mm.
t
from À1 to 1 in the eleven values listed above. The relative
weights of the PC1 and PC2 axes clearly show that this
system is much more responsive to concentration than ee
values. Yet, as described below, the errors found for the ee
values are still acceptable.
A MLP-ANN was trained with the ellipticities found for
the 33 samples of varying concentrations and ee values. The
number of hidden layers was varied until the MLP-ANN
could accurately generate the outputs from the optical
inputs. An MLP-ANN with 11 hidden layers gave the best
results
Figure 3. LDA plot of receptor (R)-1
0.8 mm] (&=(R)-CPI, *=(R)-HPI, ~=(R)-PPI, &=(S)-CPI, *=
S)-HPI, ~=(S)-PPI).
ACHTUNGTNERNUNG[ 0.4 mm] with all the analytes
[
(
scores along F2 were found for the R amines and negative
scores for the S amines. As expected, the opposite behavior
is observed for receptor (S)-1 (see the Supporting Informa-
tion). Jackknife analysis led to 100% identification and
enantioselective discrimination of the chiral primary
To assess the generality of the function developed by the
MLP-ANN, the ellipticities from [q]340 to [q]400 for six un-
known samples, independent of the training set, were col-
lected. These ellipticities were used as inputs in the MLP-
ANN, and its ability to generate the correct ee values and
concentrations was tested. The MLP-ANN calculates the
[14]
amines. Thus, this method permits the identification and
determination of handedness of chiral primary amines.
concentration [G] with an average error of 14.7% and cal-
t
culates the ee with an average error of 11.7%. The results
Quantitative determination of ee and concentration: The
next goal was the quantitative determination of ee and con-
centration for the chiral guest CEA by using the CD data
are summarized in Table 1.
Table 1. Determination of concentration and% ee of CEA test samples
by using ANN.
[15]
and an MLP-ANN.
All optical data was collected by
using a CD spectrometer and a robotically controlled 96-
well plate interface (ASU-605-JACSO, UK).
Because a supervised MLP-ANN was used, a training set
was necessary. The training set consisted of the ellipticities
from [q]340 to [q]400 at 1 nm intervals for 0.4 mm of receptor
Samples
G] [mM]
1
2
3
4
5
6
[16]
[
t
0.80
0.77
80
1.40
1.47
60
1.00
1.13
À40
À48
0.50
0.24
0
0.30
0.07
À20
10
1.20
1.36
À60
À72
[G] (ANN) [mM]
t
% ee
%
ee (ANN)
94
78
8
(
R)-1 in acetonitrile, charged with three different concentra-
tions (0.2 mm, 0.8 mm, and 1.4 mm) of CPI (the imine deriva-
tive of CEA), along with 11 ee values for each concentration
Analyzing an asymmetric reaction: The applicability of this
technique in the screening of asymmetric reactions was
demonstrated. To show the generality of the method, a dif-
ferent amine than CEA was analyzed. Asymmetric synthesis
of a-methylbenzylamine (MBA) was done by reacting ace-
tophenone and ammonium formate in the presence of a
chiral ruthenium catalyst according to a literature procedure
(
1, 0.8, 0.6, 0.4, 0.2, 0, À0.2, À0.4, À0.6, À0.8, and À1),
giving 33 solutions as the training set.
First, this training set was analyzed by using principle
[13]
component analysis (PCA) to demonstrate that simultane-
ous ee and concentration determination is feasible
(
Figure 4). A slightly rotated axis shows concentration along
[
10b]
PC1’ and ee along PC2’. This PCA plot was performed to
display the ANN training data simply in two dimensional
space, and not to show reproducibility with multiple repli-
cates. The three strips parallel to PC2’ span the ee values
(see Scheme 2).
methylbenzylamine by H NMR and C NMR spectroscopy.
The enantiopurity of the sample was first determined by the
The crude sample was identified as a-
1
13
[11a,b]
NMR protocol used by James and co-workers,
giving a
Chem. Eur. J. 2010, 16, 227 – 232
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
229

E. V. Anslyn et al.
[1]
ing of a few thousand samples per day , which is not possi-
ble with this protocol. However, the primary optical data in
this protocol is collected in only two minutes per sample.
More traditional techniques such as chiral HPLC takes 10–
[1]
Scheme 2. Asymmetric reaction used to synthesize a sample of MBA of
20 minutes per sample. Thus, this protocol outperforms
traditional techniques and can reasonably be described as a
rapid-screening method. Additionally, HPLC and GC meth-
ods usually require purification of the sample prior to analy-
sis. Whereas the reported protocol requires a two hour deri-
vatization, it can be done in situ and in parallel in 96-well
plates. Thus, for the large sample sizes this protocol is de-
signed for, the two hour time period is a relatively short
amount of time when compared with the screening time.
The speed at which the optical data is collected could be
improved allowing for HTS. Previously, our group has dem-
onstrated that a 96-well plate reader can collect optical data
for 84 samples in two minutes. However, in the case of the
CD spectrometer, the commercially available automated
plate interface uses a pump to inject samples from a 96-well
plate into a cuvette. Injection of the samples is the most
time-consuming part of the process, with the collection of
the primary optical data occupying a relatively small portion
of the time. Thus, with improvements to the automated
plate interface, the protocol could approach sample sizes
comparable to those previously reported for UV/Vis spec-
troscopy, which would allow for true high-throughput
screening.
unknown ee.
value of 72% of the R enantiomer (44% ee, see the Sup-
porting Information).
Next, the ee was also determined by using the protocol re-
ported here. A training set was loaded onto a 96-well plate
consisting of PPI (the imine derivative of MBA) at three dif-
ferent concentrations (0.2, 0.8, and 1.4 mm) and eleven ee
values at each concentration (1, 0.8, 0.6, 0.4, 0.2, 0, À0.2,
À0.4, À0.6, À0.8, and À1). An MLP-ANN was trained by
using the ellipticities from [q] to [q] at 2 nm intervals as
inputs. A network containing twenty hidden layers gave the
best results.
The imine (PPI) was synthesized in situ by direct addition
of 2-pyridine carboxaldehyde to the crude reaction mixture
[6i]
3
40
400
(
Scheme 2). Without purification, the imine was loaded onto
the same 96-well plate that was used for the training set.
There were three sets at two different concentrations (0.7
and 1.1 mm). The samples were run in triplicate at two dif-
ferent concentrations to demonstrate both that this protocol
works at different concentrations, and that even with impur-
ities from the reaction mixture, the average errors would be
comparable to those previously obtained by using prepared
solutions of pure imines.
Conclusion
The ee and concentration values calculated by using the
MLP-ANN are summarized in Table 2. The average value
Our previous eIDA and CD methods analyzed chiral biden-
tate analytes, namely, diols, amino acids, and diamines. As
described above, the monodentate primary amines did not
show an optical response, whereas conversion to bidentate
imines gave enantioselective discrimination. Although the
scope here was the determination of ee and concentration of
chiral monoamines, the approach of conversion to bidentate
chelating ligands may prove to be a general strategy for
analysis of simple functional groups, allowing the extension
of this protocol to many functional groups by using a single-
step in situ derivatization.
In summary, a new protocol that allows for the rapid and
simultaneous determination of ee and concentration of
chiral primary amines has been developed. Furthermore, the
ee of a sample from an asymmetric reaction was determined
by using this CD technique and displayed close agreement
with a literature protocol. The speed, accuracy, and simplici-
ty of this method, as well as the possibility of simultaneous
concentration determination, make this method clearly ame-
nable to rapid screening. Additionally, improvements to the
automated plate reader on the CD spectrometer may allow
for this protocol to be applied in true HTS. We are currently
employing this technique in the analysis of a wide variety of
asymmetric reactions and other organic functional groups.
t
Table 2. Determination of% ee and [G] of MBA for unknown samples
from the asymmetric reaction.
Samples
1_1
1_2
1_3
2_1
2_2
2_3
%ee (ANN)
60
62
62
52
62
68
[
G]
t
t
(ANN) [mM]
[mM]
0.84
0.70
0.86
0.70
0.86
0.70
1.23
1.10
1.21
1.10
1.21
1.10
[
G]
obtained was 61% ee. Assuming that the James procedure
yields the real value for the ee, the average error for ee is
17%, which is higher than but comparable to that of 11.7%,
calculated by using solutions of the pure imine CPI. With re-
spect to concentration, the average error for the asymmetric
reaction is 13.5%, which is also similar for the error of
14.7% that was calculated by using solutions of a pure
imine. The error found represents the accuracy of our
method, which we note consistently gives a low ee value
compared with the accepted method. In contrast, the preci-
sion, which is represented by a standard deviation of the ee
values, is much better (5.2%).
Applications in rapid and high-throughput screening of ee:
The term rapid screening is used throughout this paper in-
stead of HTS. This is because true HTS requires the screen-
230
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 227 – 232

Facile Circular Dichroism Protocol
FULL PAPER
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Experimental Section
Preparation of the imines: A stoichiometric amount of the corresponding
chiral amines was added to a stirred solution of 2-pyridine carboxalde-
hyde (9.5 mL, 0.1 mmol) in acetonitrile (2 mL) with 4 ꢁ molecular sieves.
After stirring for 2 h at room temperature, the solvent was removed in
vacuo yielding the corresponding crude imines as pale-yellow oils. These
imines were directly used in the analysis.
[
(
R)- and (S)-N-(1-Phenylethyl)-2-pyridylmethanimine, (R)- and (S)-PPI:
1
6
2
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3
): d=8.64–8.63 (m, 1H),
.48 (s, 1H), 8.06–8.04 (m, 1H), 7.85–7.81 (m, 1H), 7.49–7.26 (m, 6H),
.77 (q, J=6.7Hz, 1H), 1.56 ppm (d, J=6.6Hz, 3H).
8
4
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(
R)- and (S)-N-(1-Cyclohexylethyl)-2-pyridylmethanimine, (R)- and (S)-
1
CPI: Yield: 19.7 mg, 91%; H NMR (400 MHz, CDCl
.8Hz, 1H), 8.3 (s, 1H), 7.98–7.96 (m, 1H), 7.84–7.79 (m, 1H), 7.40–7.37
m, 1H), 3.18–3.15 (m, 1H), 1.84–1.68 (m, 4H), 1.48–1.46 (m, 2H), 1.32–
.24 (m, 2H), 1.21 (d, J=6.5Hz, 3H), 1.19–0.96 ppm (m, 2H).
R)- and (S)-N-(2-hepthyl)-2-pyridylmethanimine, (R)- and (S)-HPI:
3
): d=8.62 (d, J=
4
(
1
(
1
Yield: 19.2 mg, 94%; H NMR (400 MHz, CDCl
3
): d=8.63–8.62 (m, 1H),
8
3
3
.34 (s, 1H), 7.97–7.95 (m, 1H), 7.83–7.82 (m, 1H), 7.39–7.37 (m, 1H),
.42 (m, 1H), 1.58–1.57 (m, 2H), 1.29–1.22 (m, 9H), 1.11–1.10 ppm (m,
H).
James procedure: In a dry vial with 4 ꢁ molecular sieves 2-formylphenyl-
boronic acid (14.9 mg, 0.1 mmol) and (S)-1,1’-bi-2-naphthol (28.6 mg,
[
0
(
.1 mmol) were dissolved in CDCl
3
(1 mL). a-Methylbenzylamine
3
4
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12.7 mL, 0.1 mmol) obtained from the asymmetric reaction was added
onto the solution (see Scheme S1 in the Supporting Information). The
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sulting solution is shown in Figure S2 in the Supporting Information.
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Linear discriminant analysis (LDA): The XLSTAT program was used to
carry out the LDA studies. LDA studies allow for differentiation and
classification of the analytes. The generalization error of this classifica-
tion method was measured by using Jackknife analysis.
Principle component analysis (PCA): XLSTAT was also used for PCA
analysis. PCA allows multivariate data to be represented in lower-dimen-
sional space. The first two principal components are invoked to visualize
the objects in two-dimensional space. The first principal component
PC1) is directed along the maximum variance. The second principal
component (PC2) is orthogonal to PC1 and carries the second maximum
extent of variance.
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