Nanomole-scale assignment of configuration for primary amines using a kinetic resolution strategy

Article abstract of DOI:10.1021/ja310620c

The absolute configurations of primary amines were assigned using a kinetic resolution strategy with Mioskowski's enantioselective 1-(R,R) and 2-(S,S) acylating agents. A simple mnemonic was developed to determine the configuration. A pseudoenantiomeric pair of reagents, 1-(R,R) and 2-(S,S)-d ₃, was prepared and used to assay primary amines on a micromolar scale. The ESI-MS readout of the resulting acetamide products reproduced the selectivity factors from kinetic experiments. The method can be used on mixtures of amines and was validated with amine samples as small as 50 nmol.

Full text of DOI:10.1021/ja310620c

Communication
pubs.acs.org/JACS
Nanomole-Scale Assignment of Configuration for Primary Amines
Using a Kinetic Resolution Strategy
Shawn M. Miller,† Renzo A. Samame,† and Scott D. Rychnovsky*
Department of Chemistry, 1102 Natural Sciences II, University of CaliforniaIrvine, Irvine, California 92697, United States
S
* Supporting Information
ABSTRACT: The absolute configurations of primary
amines were assigned using a kinetic resolution strategy
with Mioskowski’s enantioselective 1-(R,R) and 2-(S,S)
acylating agents. A simple mnemonic was developed to
determine the configuration. A pseudoenantiomeric pair of
reagents, 1-(R,R) and 2-(S,S)-d3, was prepared and used to
assay primary amines on a micromolar scale. The ESI-MS
readout of the resulting acetamide products reproduced
the selectivity factors from kinetic experiments. The
method can be used on mixtures of amines and was
validated with amine samples as small as 50 nmol.
solation of natural products has reached the point where
Inanomole-scale structure assignments are becoming fea-
sible.1 Except for special cases (e.g., α-amino acids)2 the
assignment of absolute configuration on nanomole samples is
an unsolved problem. We report a new strategy for the
assignment of primary amines that is effective on micromole-
Figure 1. The rate of acetylation of amine 3 with the Mioskowski
reagents 1-(R,R) and 2-(S,S) was found to favor the 2-(S,S) reagent by
down to nanomole-scale samples and can be applied to
mixtures of amines.
a factor of 2.6. The ratio of starting material to product was monitored
Amines are often found in natural products, compounds of
1
by H NMR integration, and the experiments were done in triplicate.
medicinal interest, and synthetic intermediates.3 Direct
Each reaction used a 3-fold excess of the acylation reagent and was
methods to assign the absolute configuration of amines usually
involve derivatization and NMR analysis of the resulting
amides,4 but other methods have been developed.2,5 We have
developed a new method6 inspired by Horeau’s strategy for
determining the configuration of optically pure alcohols using a
strategy based on kinetic resolution.7 The method uses
Mioskowski’s enantioselective acetylating reagents 1-(R,R)
(Figure 1) and 2-(S,S).8 It is easy to implement, shows a
broad scope for different primary amines, and is extraordinarily
sensitive.
Initial studies to establish the feasibility of the method are
presented in Figure 1. (R)-1-(1-Naphthyl)ethylamine (3) was
treated with reagent 1-(R,R) or 2-(S,S) in an NMR tube in
CDCl3, and the conversion to acetamide 4 was followed by
integrating the NMR signal of the H atom adjacent to the
amine or amide. The rates of the reaction with the two
enantiomeric reagents were determined, and the 2-(S,S)
reagent was preferred by a factor of 2.6. This preference is
consistent with reports by Mioskowski using the reagents 1-
(R,R) and 2-(S,S) for kinetic resolutions with similar primary
amines in CHCl3.8 The reaction followed pseudo-first-order
kinetics under these conditions.
followed to ca. 50% conversion.
the conversion at fixed times would simplify this procedure,
although the inherent sensitivity of NMR spectroscopy still
limits this method.6 In contrast, mass spectrometry (MS) is
exquisitely sensitive and allows for the rapid detection of
species with different masses. Therefore, we envisioned a more
powerful MS-based approach using deuterium-labeled pseu-
doenantiomers to identify the relative rates of acetylation with
reagents 1 and 2 (Figure 2). Amine 5 would react with reagent
1-(R,R) to produce unlabeled acetamide 6-(M), whereas
reagent 2-(S,S)-d3 would afford deuterated acetamide 6-(M
+3). To maintain pseudo-first-order kinetics, the reagents 1 and
2 should be used in excess. This kinetic regime would allow a
direct measurement of the selectivity factor by comparison of
the relative ratios of 6-(M)+ and 6-(M+3)+ peaks in the MS
analysis.9 MS methods have been used previously to estimate
the enantiomeric excess of compounds,10 but rarely have they
been used to assign absolute configurations.7d Unlike the NMR
kinetics in Figure 1, this MS method uses a single rapid
experiment with a simple analysis.
The NMR analysis is straightforward but requires milligram
Received: October 28, 2012
samples of substrate and significant instrument time. Analyzing
© XXXX American Chemical Society
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Table 1. Determination of the Absolute Configurations of
Chiral Primary Amines by Enantioselective Protio/
a
Deuteroacetylation and ESI-MS Analysis
Figure 2. The selectivity for the enantioselective acylation reaction can
be determined by using the pseudoenantiomeric Mioskowski reagents
1-(R,R) and 2-(S,S)-d3 and analyzing the acylated product 6 by ESI-
MS.
The deuterated reagent 2-(S,S)-d3 was prepared using a
modified version11 of Mioskowski’s procedure.8a A 1:3:3 ratio
of amine, 1-(R,R), and 2-(S,S)-d3 gave reproducible kinetics and
was used in the experiments. The two acylation reagents are
stable crystalline solids and do not exchange acetyl groups. A
mixture of the solid reagents was stable indefinitely, and it was
dissolved in chloroform to produce the stock solutions used in
these experiments. In the standard procedure, a solution
containing 0.5 μmol of amine was combined with the mixture
of reagents 1-(R,R) and 2-(S,S)-d3 in a total volume of 50 μL of
CHCl3 and allowed to stand for 60 min, at which point the
reaction was quenched with methanol. The mixture was then
analyzed directly by electrospray ionization (ESI) MS.12 The
results are shown in Tables 1 and 2.
Despite the modest selectivities of the reagents, clear
differences in the MS peak intensities, coupled with small
standard deviations, allowed for the confident assignment of the
absolute configurations of a variety of amines (Table 1). The
selectivity factors ranged from 1.12 to 6.6 for the examples
shown. Multiple trials were run for each amine, and the number
of trials and the standard deviation are listed for each entry in
the table. In most cases, the standard deviation was very small
(<0.1). In a few cases, the standard deviation was larger (entries
6 and 8), but these examples corresponded to unusually high
selectivity and did not present any ambiguity in the analysis.
Enantiomeric pairs showed the expected complementary
reactivities (entries 1 and 2 and entries 3 and 4).13 Perhaps
the largest source of error was deviation of the 1:2 ratio in the
stock solutions.14 The results for amino alcohol substrates are
gathered in Table 2. The range of selectivities was similar to
that for the nonprotic substrates in Table 1.
A mnemonic for assigning the absolute configuration of the
amine from the observed selectivity with the reagents 1-(R,R)
and 2-(S,S)-d3 is presented in Figure 3. In each case, the “large”
group is drawn to the left and the “small” group is drawn to the
right. When 1-(R,R) reacts faster, the amine is forward, and
when 2-(S,S)-d3 reacts faster, the amine is back. The amines in
Table 1 are oriented to match the mnemonic in Figure 3 (with
the large group to the left and the small group to the right).
The group assignments are empirical. Aromatic rings and
carbonyls, both of which contain sp2 carbons, behave as large
groups. Alcohols (Table 2) behave as small groups, even when
they carry substituents (Table 2, entry 6). Protected alcohols
(Table 1, entries 9, 11, and 14) behave as bulky substituents
and show a small preference for the large side. Aliphatic groups
show only very modest preferences (Table 1, entry 13).
a
The amine (0.01 M) and the acylating agents 1 and 2 were combined
in a 1:3:3 molar ratio in 50 μL of CHCl3. After 60 min, the mixture
was diluted with MeOH and analyzed by ESI-MS. The sodium peaks
(M+23) were analyzed in all cases. The standard deviation (σ) for the
multiple runs is included.
b
c
Remote substituents influence the selectivity in a modest but
predictable fashion (Table 1, entries 10 and 12). These
examples all follow the mnemonic in Figure 3, but caution
should be exercised in interpreting very small selectivities; we
consider any selectivity less than 1.2:1 to be inconclusive. In
most cases, the assignments of the small and large groups are
obvious and, combined with the relative reactivity observed
with the reagents 1-(R,R) and 2-(S,S)-d3, lead to direct and
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a
Table 2. Absolute Configurations of α-Amino Alcohols
approach, a mixture containing 50 nmol (<10 μg) of each of
the three amines 10−12 was analyzed using the standard
protocol with 1-(R,R) and 2-(S,S)-d3 (1.5 μmol each) in 50 μL
of solution. ESI-MS analysis allowed the selectivity and thus the
absolute configuration to be defined for all three amines in a
single experiment.
a−cSee the corresponding footnotes in Table 1.
We have developed a new method for assigning the absolute
configurations of primary amines using kinetic resolution
reagents. The method can be completed in less than 2 h,
uses no exotic equipment beyond a standard ESI-MS
instrument, and is sensitive enough to analyze nanomole
samples. The method can be applied to simple mixtures of
amines without prior separation. It will be useful in natural
product assignments, medicinal chemistry, and synthetic
chemistry. The development of kinetic resolution strategies
for the determination of absolute configurations remains an
active area of research in our laboratory.
Figure 3. Mnemonic for assigning the absolute configuration of a
primary amine on the basis of its relative reactivity with the acylating
agents 1-(R,R) and 2-(S,S)-d3. Adjacent alcohols behave as small
groups, and aryls and esters behave as large groups.
unambiguous assignments of the absolute configuration for
primary amines.
The origins of the selectivity in the acylation reaction have
not been defined, and the analysis is complicated by a large
solvent effect observed for these reagents.8 The rate-
determining step is likely the formation of the tetrahedral
intermediate, but there may be exceptions to this proposal.8b
The structure of the favored neutral intermediate formed by the
reaction of (R)-α-methylbenzylamine with 2-(S,S) is repro-
duced in Figure 4.15 The faster-reacting diastereomeric pair, the
ASSOCIATED CONTENT
* Supporting Information
■
S
Preparation of deuterated reagent 2-(S,S)-d3, the standard
procedure for the configuration assignment of primary amines,
representative data and analysis, and coordinates for
intermediate in Figure 4. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
srychnov@uci.edu
■
Author Contributions
†S.M.M and R.A.S contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Figure 4. The most stable tetrahedral intermediate for the acylation of
(R)-α-methylbenzylamine with 1-(R,R) or 2-(S,S) is shown. The 2-
(S,S) reagent and the R amine are the observed faster-reacting pair.
Calculations were performed at the B3LYP/6-31G(d) level.15
Support was provided by NSF Award CHE 1152449. Input
from Dr. Viengkham Malathong and Dr. Noel Powell is
gratefully acknowledged.
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The standard experiment uses 0.5 μmol (ca. 75 μg) of amine
for each determination, but the method itself can be much
more sensitive (eq 1). To demonstrate the potential of this
■
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D
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