NMR analysis of chiral alcohols and amines: Development of an environmentally benign "in tube" procedure with high efficiency and improved detection limit

Article abstract of DOI:10.1039/c1gc15191f

A fast and efficient approach was developed for the NMR analysis of chiral alcohols and amines using readily available enantiopure MPA (α-methoxy- α-phenylacetic acid) and MTPA (α-methoxy-α- trifluoromethylphenylacetic acid) as chiral derivatizing agents. The procedure requires less than 5 min (including sample preparation time) for analysis using routine NMR hardware and allows accurate measurements for <0.01 mg of the sample of chiral compounds. Direct "in tube" analysis can be performed with high efficiency to determine enantiomeric purity and absolute configuration, as well as to monitor reactions in asymmetric synthesis and catalysis. The developed procedure is superior in terms of waste-free analysis of chiral compounds for environmentally benign applications.

Full text of DOI:10.1039/c1gc15191f

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Green Chemistry
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PAPER
NMR analysis of chiral alcohols and amines: development of an
environmentally benign “in tube” procedure with high efficiency and
improved detection limit†
Nikolay V. Orlov and Valentine P. Ananikov*
Received 21st February 2011, Accepted 13th April 2011
DOI: 10.1039/c1gc15191f
A fast and efficient approach was developed for the NMR analysis of chiral alcohols and amines
using readily available enantiopure MPA (a-methoxy-a-phenylacetic acid) and MTPA
(a-methoxy-a-trifluoromethylphenylacetic acid) as chiral derivatizing agents. The procedure
requires less than 5 min (including sample preparation time) for analysis using routine NMR
hardware and allows accurate measurements for <0.01 mg of the sample of chiral compounds.
Direct “in tube” analysis can be performed with high efficiency to determine enantiomeric purity
and absolute configuration, as well as to monitor reactions in asymmetric synthesis and catalysis.
The developed procedure is superior in terms of waste-free analysis of chiral compounds for
environmentally benign applications.
imposes significant difficulties in a number of cases, particularly
Introduction
in those involving transition-metal-catalyzed transformations.
Outstanding progress in asymmetric synthesis and catalysis pro-
An alternative option is to utilize NMR spectroscopy, which
vided easy access to numerous classes of enantiomerically pure
became a popular and convenient tool for the determination
compounds andbecame oneof the leadingdrivingforces inmod-
of enantiomeric purity and absolute configuration of chiral
ern organic chemistry, biochemistry and drug discovery.1,2 With
compounds.3 The method is based on derivatization of chiral
recent development of the field, a demand has clearly emerged
compound to be analyzed with a suitable chiral derivatizing
for reliable methods for determination of absolute configuration
agent (CDA), which results in the discrimination of chemical
and enantiomeric purity of synthesized chiral products and
shifts of diastereomers in NMR spectra. The concept was proven
monitoring asymmetric transformations for mechanistic studies.
by successful application of several CDAs utilizing 1H, 13C,
Quite typically, numerous experiments of catalyst screening, op-
19F, 31P, 129Te and 195Pt NMR.4 Recently, we have reported the
timization of reactionconditions, substrate/selectivitytargeting,
first-principles development of a new 77Se CDA for chirality
etc. are performed repeatedly in an everyday laboratory practice.
Either manual or automatic optimization requires rapid analytic
scanning of a number of asymmetric synthesis experiments.
Thus, of particular importance are cost-efficient and eco-friendly
analytic methods where the analysis procedure itself is accurate
and fast with minimal production of wastes.
Chiral chromatography is currently most often used for
analysis with well known drawbacks in terms of: i) toxic
solvents; ii) expensive columns (to be replaced frequently due
to contamination in the case of analysis of crude mixtures); and
iii) inability of large numbers of rapid determinations. Another
important limit of chiral chromatography is the requirement
to remove traces of metals prior to analysis because chiral
stationary phases are very sensitive to such impurities. This
recognition within the structure of diastereomers.5 In addition
to detection of different NMR nuclei, several improvements of
the derivatization procedure were reported to enhance the NMR
analysis.6
To date the largest substrate scope for the determination
of enantiomeric purity and absolute configuration has been
established for a-methoxy-a-trifluoromethylphenylacetic acid
(MTPA or Mosher’s acid) and a-methoxy-a-phenylacetic acid
(MPA) as derivatizing agents.7,8 In a typical NMR measurement
with MTPA or MPA, derivatization is carried out as a separate
reaction and after the necessary workup a pure sample is
transferred into the NMR tube for spectral analysis (Scheme 1).
Some important limitations of this procedure are worth men-
tioning: i) workup and purification at the derivatization stage,
which produces waste; ii) kinetic resolution for some substrates
during the derivatization procedure, which results in incorrect
determination of enantiomeric purity; iii) a time consuming
procedure for chemical transformation; iv) a relatively large
amount of the chiral sample required for analysis.
Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences,
Leninsky Pr. 47, Moscow, 119991, Russia. E-mail: val@ioc.ac.ru
† Electronic supplementary information (ESI) available: Experimental
details, 1H, 13C, 19F NMR data. See DOI: 10.1039/c1gc15191f
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Scheme 1 A typical procedure for NMR recognition of chiral compounds.
In the present study we report a simple approach to carry
Reaction of the model mixture of R- and S-methylmandelates
out the derivatization procedure directly in the NMR tube
without any dedicated workup/purification being necessary. The
problem of kinetic resolution was solved in the present study and
a highly sensitive analytical protocol was developed to carry out
measurements on <0.01 mg of chiral sample on routine NMR
spectrometers. The scope of the NMR approach developed was
investigated for various chiral alcohols and amines. A valuable
Green Chemistry improvement was achieved for the developed
procedure, which at the same time became superior in terms of
higher efficiency.
with MPA and MTPA in various deuterated solvents
10 mg (4.3 ¥ 10-5 mol) of R-MTPA or 7.1 mg (4.3 ¥ 10-5 mol)
of S-MPA was placed in an NMR tube followed by addition of
0.6 mL of corresponding deuterated solvent. 3.8 ¥ 10-5 mol of
the mixture of R- and S-methylmandelates (molar ratio 70 : 30),
8.9 mg (4.3 ¥ 10-5 mol) of DCC and 1.0 mg (8.2 ¥ 10-6 mol) of
DMAP were subsequently added to the NMR tube. The mixture
was shaken for 1 min and kept at rt until solid particles precipi-
tated to the bottom or floated to the top of the solution (see Fig.
1 for details). About 0.5–2 h was required without centrifugation
(see Fig. S4 in the ESI†) and 2–5 min using centrifugation (see
Fig. S1 in the ESI†). 1H and 19F NMR spectra were recorded for
the analysis (see Fig. S2 in the ESI† for NMR data).
Experimental part
NMR Spectroscopy
1H, 13C and 19F NMR measurements were performed using
Bruker 300, 500 and 600 MHz spectrometers. All 1D and
2D spectra were recorded at 303 K without spinning of the
Influence of water impurities on the reaction of the model mixture
of R- and S-methylmandelates with MPA and MTPA in CDCl3
10 mg (4.3 ¥ 10-5 mol) of R-MTPA or 7.1 mg (4.3 ¥ 10-5 mol)
of S-MPA was placed in an NMR tube followed by addition
of 0.6 mL of CDCl3. 2.8 ¥ 10-5 mol of mixture of R- and S-
methylmandelates (molar ratio 50 : 50) in case of R-MTPA or
3.8 ¥ 10-5 mol in case of S-MPA and corresponding amount of
water (see Table 2) were added to the NMR tube followed by
addition of 1.0 mg (8.2 ¥ 10-6 mol) of DMAP and 8.9 mg (4.3 ¥
10-5 mol) of DCC. Mixture was shaken for 1 min and kept at rt
1
NMR tube. H and 19F NMR measurements were carried out
with a single 90◦ pulse for the 18.0–0.9 mg quantities of the
analyzed samples. To obtain a good signal to noise ratio for the
0.09 mg and 0.009 mg of the analyzed samples, 128 and 1024
scans (with 30◦ pulses) were recorded, respectively. The spectra
were processed on a Linux workstation using the TopSpin 2.1
1
1
software package. 2D LR-COSY, H–13C HSQC and H–13C
HMBC NMR experiments were used for the full assignment of
signals of the studied diastereomers. 1H and 13C chemical shifts
are reported relative to the corresponding solvent signals used
as an internal reference; CFCl3 (0 ppm) was used for 19F as an
external reference. The detailed description of the NMR data is
given in the ESI.†
until solid particles floated to the top of the solution. 1H and 19
F
NMR spectra were recorded for the analysis (see Fig. S3 in the
ESI† for NMR data).
Developed “in tube” derivatization procedure of chiral alcohols
and amines using R-MTPA and S-MPA in CDCl3 (Method A)
10 mg (4.3 ¥ 10-5 mol) of R-MTPA was placed in an NMR tube
followed by addition of 0.6 mL of CDCl3. 2.8 ¥ 10-5 mol of the
corresponding alcohol or amine, 8.9 mg (4.3 ¥ 10-5 mol) of DCC
and 1.0 mg (8.2 ¥ 10-6 mol) of DMAP (here and later, DMAP
should be added in the case of chiral alcohols only, while DMAP
is not necessary for chiral amines) were subsequently added to
the NMR tube. The mixture was shaken for 1 min and kept at
rt until solid particles floated to the top of the solution (ca. 0.5–
2 h without centrifugation and ~5 min using centrifugation).
1H, 13C and 19F NMR spectra were recorded for the studied
General
For the experiments shown in Table 1 deuterated solvents were
used as received, in the other cases deuterated solvents were
dried over molecular sieves before use. The following tips may
be optionally used to improve the quality of the spectra. Samples
with precipitated or floating solids: an additional centrifugation
for 5 min at 1000 rpm directly in the NMR tube may be
applied (see Fig. S1 in the ESI†). Samples with floating solids:
an additional amount of solvent may be added to move the
floating solid out of the NMR detecting area (see Fig. 2 and
corresponding discussion in the text).
1
samples (typically either H or 19F NMR was enough for the
analysis).
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Fig. 1 Pictures of the “in tube” derivatization process: a) initial solution of MTPA or MPA and chiral analyzed alcohols; b) after addition of DCC
and DMAP, followed by shaking for 2 min; and reactions in various solvents after standing for 1 h: c) CD3CN; d) acetone-d6; e) toluene-d8; f)
benzene-d6; g) THF-d8; h) DMF-d7; i) DMSO-d6; j) CS2/CD2Cl2 (4/1); k) CDCl3.
Fig. 2 A routine NMR probe (a) and schematic representation of the tube alignment in the NMR probe showing analysis of the samples with
precipitated solids (b) and floating solids (c).
For the analysis involving S-MPA: 7.1 mg (4.3 ¥ 10-5 mol) of
S-MPA and 3.8 ¥ 10-5 mol of corresponding alcohol or amine
were taken using the same procedure.
5 min. 1H NMR spectra were recorded for the analysis (see Fig.
S5 in the ESI† for NMR data).
This procedure was successfully scaled for the analysis of 18.0–
0.9 mg of R- and S-methylmandelates (1.1 ¥ 10-4–5.4 ¥ 10-6 mol),
see Table 3.
Developed “in tube” derivatization procedure with better
tolerance to residual water impurity (Method C)
4.5 mg (2.7 ¥ 10-5 mol) of the analyzed sample – a model
mixture of R- and S-methylmandelates (molar ratio 70 : 30) and
12.4 mg (6 ¥ 10-5 mol) of DCC were placed in an NMR tube
followed by addition of 0.6 mL of deuterated solvent. 0.7 mg (6 ¥
10-6 mol) of DMAP and 5.0 mg (3 ¥ 10-5 mol) of S-MPA were
subsequently added. The mixture was shaken for 1 min and kept
at rt until solid particles floated to the top or precipitated to the
bottom of the solution (ca. 0.5–2 h without centrifugation and
Developed “in tube” derivatization procedure to analyze small
amounts of the chiral sample in CDCl3 and C6D6 (Method B)
The analyzed sample – a solution containing 0.09–0.009 mg of
the model mixture of R- and S-methylmandelates (molar ratio
70 : 30) and 0.6 mL of CDCl3 or C6D6 were placed into an NMR
tube, followed by addition of 1.0 mg (6.0 ¥ 10-6 mol) of S-
MPA and 1.3 mg (6.3 ¥ 10-6 mol) of DCC (in case of alcohols
0.1 mg (8.2 ¥ 10-7 mol) of DMAP should be added). Precipitate
did not form in chloroform solution and centrifugation was
not required, while the samples in benzene were centrifuged for
1
~5 min using centrifugation). H NMR spectra were recorded
for the analysis of diastereomers’ ratio (see Table S1 in the
ESI†).
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Scheme 2 The derivatization procedure directly in the NMR tube (“in tube” procedure); DCC – N,N¢-dicyclohexylcarbodiimide, DMAP – 4-
dimethylaminopyridine, DCU – N,N¢-dicyclohexylurea.
solvents with density near or less than 1 g mL-1, precipitation of
DCU takes place (Fig. 1c–h), while in solvents with density more
Results and discussion
than 1.2 g mL-1, floating of insoluble DCU particles was found
to be the case (Fig. 1j and k). The experiments were repeated
several times and were found to be totally reproducible.
Modern NMR spectrometers utilize probes with the coils
located in the central part of the sample, in order to maintain
the highest possible homogeneity of magnetic field (Fig. 2).
To increase resolution in NMR spectra and simplify shims
adjustments the areas at the top and at the bottom of the
sample are shielded. Thus, these shielded areas do not influence
magnetic resonance measurements and give no signals to the
resulting spectrum as they are out of the NMR coil window. Such
a detection scheme is currently implemented in routine NMR
probes and it allowed direct analysis of the samples without iso-
lation and purification of diastereomers from insoluble particles
in all studied solvents (Fig. 1). In the case of precipitation, a
possible negative influence of the solids is diminished by the
bottom shield (Fig. 2b), while in case of floating solids, by
the top shield (Fig. 2c). We have tested both types of samples
on different NMR spectrometers operating at 300, 500 and
600 MHz and did not encounter any difficulties in recording 1H,
13C and 19F NMR spectra as well as 1H–1H and 1H–13C 2D NMR
experiments.
“In tube” derivatization procedure without wastes
The first necessary step was to improve the derivatization
procedure (Scheme 1) to avoid chemical wastes (generated in
the workup and purification steps) and to speed up the overall
time needed for analysis. An ideal derivatization procedure
should be carried out in the NMR tube with the resulting
solution being directly suitable for spectral analysis without
any additional manipulations. A promising approach in this
regard is DCC-promoted derivatization to prepare esters and
amides (Scheme 2). To the best of our knowledge such “in tube”
derivatization procedure was not explored for MPA and MTPA,
but an important prerequisite of successful implementation was
demonstrated with other CDAs9,10 and in our recent study.5
To optimize reaction conditions and develop a reliable “in
tube” protocol we have started with a model mixture of R- and
S-methylmandelates with a known composition of 70 : 30 molar
ratio. Derivatization was carried out by addition of 1.1 equiv. of
MTPA (or MPA), 1.1 equiv. of DCC and a catalytic amount of
DMAP (0.2 equiv.) to the analyzed mixture of alcohols in the
NMR tube. Indeed, we have observed the desired transformation
directly in the NMR tube, which resulted in the formation of
diastereomers at rt (Scheme 2).
Therefore, the derivatization procedure was successfully car-
ried out directly in the NMR tube in deuterated solvents
without the need for the separation/purification of diastere-
omers (Scheme 2). The procedure considerably decreases the
time needed for sample preparation and avoids chemical
wastes. NMR spectra were recorded without any additional
manipulations with the sample using standard hardware. These
experiments confirmed the possibility of the “in tube” NMR
procedure with MPA and MTPA for a large solvent scope
(Fig. 1).
To analyze the accuracy of the NMR measurements we have
determined the yield and the ratio of the diastereomers in
recorded 1H spectra. Both parameters are important to develop
an efficient procedure: 1) high yield is required to increase the
concentration of diastereomers and shorten NMR measurement
time; 2) the ratio is the key parameter, since it reflects the
accuracy of the measurements in determination of enantiomeric
composition of the analyzed sample. Analysis of all samples
revealed rather different pictures depending on the solvent used
(Table 1). High yields of diastereomers formed with MPA were
As the first necessary test we have evaluated this derivatiza-
tion procedure in various solvents commonly used in organic
synthesis and NMR spectroscopy (Fig. 1). Addition of DCC
to the solutions caused formation of white suspensions within
2–20 min in all cases except DMSO-d6 and DMF-d7. In DMF-
d7 the suspension was formed in 2 h, while DMSO solution
remained homogeneous after several days. Surprisingly, in all of
the studied cases it was possible to carry out the characterization
of chiral compounds directly in the NMR tube. Since NMR
analysis of heavily heterogeneous samples is not a common
practice we have addressed the possible scope of this approach
in more detail.
Of course, reliable NMR analysis of the initially formed
heterogeneous mixture (Fig. 1b) would hardly be possible.
However, for the studied suspensions we have observed either
precipitation (CD3CN, C6D6, acetone-d6, toluene-d8, THF-d8,
DMF-d7) or floating (CDCl3, CS2/CD2Cl2) of the insoluble
DCU particles within a short period of time (Fig. 1c–h and 1j–k,
respectively). The main factor, which plays an important role in
the studied reaction, is the density of the solvent. In the case of
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Table 1 Derivatization and 1H NMR analysis of the mixture of R- and S-methylmandelates with a known composition (70 : 30 molar ratio) using
MPA and MTPA derivatizing agents directly in the NMR tube in different solvents
Yield, % (R/S ratio)b
Entry
Solvent
Density/g mL-1
Solidsa
MPA
MTPA
1
2
3
4
5
6
7
8
9
CD3CN
0.84
0.87
0.94
0.95
0.99
1.04
1.19
1.26/1.36
1.50
precipitated
precipitated
precipitated
precipitated
precipitated
precipitated
floated
94 (73 : 27)
93 (75 : 25)
100 (71 : 29)
100 (71 : 29)
66 (77 : 23)
24 (77 : 23)
26 (71 : 29)
75 (69 : 31)
98 (71 : 29)
70 (72 : 28)
67 (73 : 27)
100 (70 : 30)
100 (71 : 29)
23 (80 : 20)
0c
Acetone-d6
Toluene-d8
Benzene-d6
THF-d8
DMF-d7
DMSO-d6
CS2/CD2Cl2 (4/1)
CDCl3
0c
floated
floated
77 (69 : 31)
73 (78 : 22)
a See Fig. 1 for the nature of the system with precipitated or floating solids (note also, that DCU is partially soluble in some solvents). b The ratio of
diastereomers determined by NMR analysis is shown in parentheses. c The reaction did not take place.
Table 2 Influence of moisture on the yield and R/S ratio of derivati-
zation of racemic mixture of R- and S-methylmandelates with S-MPA
and R-MTPA (in CDCl3)
observed in toluene-d8, benzene-d6 and CDCl3 (Table 1; entries
3, 4 and 9), good yields were found in CD3CN, acetone-d6 and
CS2/CD2Cl2 (Table 1; entries 1, 2 and 8), moderate yield was
detected in THF-d8 (Table 1, entry 5) and poor yields were
observed in DMF-d7, DMSO-d6 (Table 1; entries 6, 7). Except
for toluene-d8, benzene-d6 and CS2/CD2Cl2, lower yields of
diastereomers were observed in the case of MTPA (Table 1).
What is more crucial, a rather large error (up to 10%) in the
determined ratio of diastereomers was observed in some cases
(Table 1).11
The results suggested that enantiomers reacted with different
rates (Scheme 2), thus resulting in kinetic resolution and leading
to incorrect spectral analysis. Although the results were in
qualitative agreement with the expected amounts of R and S
enantiomers in the analyzed sample, the problem of kinetic
resolution should be solved in order to develop a quantitative
analytical procedure.
Conversion, % (R/S ratio)
Entry Amount of added water/equiv.a S-MPAb
R-MTPA
1
0
100 (51 : 49)
86 (55 : 45)b
93 (52 : 48)c
79 (62 : 38)c
49 (62 : 38)c
16 (67 : 33)c
12 (64 : 36)c
11 (67 : 33)c
2
3
4
5
6
0.1
0.5
1.0
3.0
5.0
92 (52 : 48)
52 (55 : 45)
49 (55 : 45)
43 (53 : 47)
42 (53 : 47)
a Based on the initial amount of the alcohols. b 1.1 equiv. of CDA was
used. c 1.5 equiv. of CDA was used.
Comparing MTPA and MPA, we have observed that the
former is less tolerant to moisture. Higher sensitivity of MTPA
to moisture effects (resulting in lower yields and larger error)
was observed in all experiments of this series with added water
(Table 2, entries 2–6).
The origin of incorrect measurements: tremendous effect of water
Surprisingly, we have found that the incomplete conversion and
kinetic resolution were caused by water impurities. To illustrate
the influence of water, a special set of experiments was performed
with water added to the analyzed sample in the NMR tube.
The effect was observed for derivatization of 70 : 30 mixture
of enantiomers discussed above, as well as for 50 : 50 mixture
(racemate) of R- and S-methylmandelates (Table 2).
To reduce the amount of water, the well-known technique of
drying CDCl3 by molecular sieves was applied. Usage of 1.1
equiv. of MPA as CDA resulted in complete conversion of the
alcohols and their ratio was quite satisfactorily determined by
1H NMR spectroscopy within an expected error of the method
of ~1% (Table 2, entry 1). Even trace amounts of water caused a
noticeable decrease of the yield to 92% and a twofold increase of
the error in the determined ratio of diastereomers (Table 2, entry
2). Addition of as small as 0.5 equiv. of water (which corresponds
to only 0.25 mg) resulted in a dramatic decrease of conversion
(52%) and an increase of the error of the measurements up to
5% (Table 2, entry 3). Incomplete conversions and noticeable
kinetic resolution were observed in all other cases as well (Table
2, entries 4–6).
The use of 1.1 equiv. of MTPA in CDCl3 dried over molecular
sieves resulted in 86% conversion of the alcohols and showed
noticeable kinetic resolution, while the same level of solvent
purification was enough for MPA (Table 2, entry 1). Since
rigorous purification of the solvent could be impractical, the
results with MTPA were further improved simply by increasing
the amount of the CDA to 1.5 equiv.: 93% vs. 86% conversion
and a satisfactory ratio of diastereomers with ~2% error (Table
2, entry 1).
The study clearly revealed the influence of water as a main
reason for kinetic resolution and the origin of the relatively
large error observed in the analysis. Utilization of an excess
of the CDAs allowed tolerance to water traces to some extent.12
Thus, an optimal procedure was developed using dry CDCl3 and
MPA (1.1 equiv.) or MTPA (1.5 equiv.) directly in the NMR
tube without isolation and purification of diastereomers. The
formation of solid particles had no influence on the quality of
1H, 13C and 19F NMR measurements.
The experiments highlighted the reaction yield of the deriva-
tization step to be a key-importance criterion for correct deter-
mination of the enantiomeric content of the sample (not only
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1
Scheme 3 The difference in NMR chemical shifts (in ppm) of the baseline resolved H (red), 13C (blue) and 19F (violet) signals of diastereomers
prepared from the corresponding chiral alcohols and amines with R-MTPA (1–8) and S-MPA (9–18) directly in the NMR tube (Method A, see
Experimental part).
1
for sensitivity reasons, but also for correct diastereomers ratio).
If the reaction is complete, kinetic resolution has no influence
on the determined ratio of the diastereomers. Therefore, the
“in tube” procedure (Scheme 2) which allows real-time NMR
monitoring of the derivatization procedure with detection of
the amounts of reacted (and unreacted) reagents is superior
compared to the standard method. In the case of the standard
method unreacted reagents are separated on the purification step
(Scheme 1) which makes it difficult to establish the yield. The
accuracy of NMR measurements carried out with the mixture of
diastereomers at incomplete conversion of the analyzed sample
may substantially suffer from kinetic resolution.
9–16 and amines 6–8, 17, 18 (Scheme 3). High quality H, 13C
and 19F NMR spectra were recorded using routine technique
without any additional manipulations for all studied compounds
with both MTPA (1–8) and MPA (9–18). A good accuracy was
observed in the NMR analysis with the average error <1%.
Determination of the enantiomeric composition of the sample
was done simply by integration of the corresponding baseline
resolved NMR signals. Chemical shift differences between the
baseline resolved NMR signals of diastereomers were in a range
of 0.011–0.207 ppm for 1H, 0.016–0.443 ppm for 13C and 0.025–
0.289 ppm for 19F (Scheme 3). Signals of the MeO-group of
MTPA in H or 13C NMR spectra were baseline resolved in
1
many cases so they can be used for accurate measurements of
the diastereomer ratio. In the case of MPA, the signals of the CH-
group of MPA can be involved in the measurements. Baseline
resolved 19F NMR signals were observed in all studied cases
with MTPA (see ESI†).
Scope and application of the developed procedure
The scope of the developed “in tube” derivatization procedure
was investigated for variety of chiral samples of alcohols 1–5,
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The developed “in tube” derivatization protocol can also
be applied for determination of the absolute configuration
of chiral alcohols and amines. It should be mentioned that
even partially resolved signals of diastereomers can be taken
into account because only the difference of chemical shifts is
important in such a case.13 The well-known double derivatization
method using two enantiomers of CDA,14 as well as a single
derivatization method developed by Riguera and co-workers15
based on registration of NMR spectra of diastereomers at
different temperatures were successfully applied. As an example,
assignment of the absolute configuration of menthol by a single
derivatization protocol is shown in Fig. 3 using the developed “in
compounds, since there are no losses of the sample in the
separation/purification stages. The pictures of the NMR tubes
containing 18–0.9 mg of the analyzed sample of a 70 : 30 mixture
of R- and S-methylmandelates in CDCl3 (floating of insoluble
particles) and C6D6 (precipitation) are shown in Fig. 4.
As one would obviously expect, the lower the quantity of
reagents used, the smaller the amount of insoluble particles
formed (Fig. 4), thus leading to faster precipitation (or floating)
and significantly decreasing the overall time needed for analysis.
Excellent yield of the derivatization reaction and very good
accuracy in the NMR measurements were observed in benzene-
d6 (Table 3, entries 1–4). Even with a tiny amount of analyzed
chiral sample of 0.9 mg, a suitable 1H NMR spectrum was
recorded with a single scan (Table 3, entry 4) and the time
needed for NMR measurements was less than 1 min. Taking
into account that the “in tube” procedure needs ca. 2–3 min
for NMR sample preparation, less than 5 min of overall time is
required for a complete procedure.
1
tube” derivatization procedure. High quality H NMR spectra
of the S-MPA ester were recorded both at rt (Fig. 3a) and at
low temperature (Fig. 3b). The presence of insoluble particles
(floated to the top of the sample) did not affect the results, which
were in total agreement with the literature data.14
To determine the detection limit of the developed “in tube”
procedure the series of experiments under optimized conditions
(Method B) were further carried out until as little as 0.009 mg
of the sample was reached (Table 3; entries 5, 6). H NMR
spectra of high quality suitable for accurate measurements of
Improved detection limit and reliable procedure for fast screening
of chiral samples
1
We have found that an “in tube” derivatization procedure
can be efficiently used to detect even small amounts of chiral
Fig. 3 Assignment of the absolute configuration of menthol by a single derivatization coupled with the “in tube” procedure: fragments of 1H NMR
spectra recorded at 303 K (a) and 223 K (b) in CDCl3 (see ref. 14a for details of assignment procedure).
Fig. 4 Scaling of the “in tube” procedure in CDCl3 (a–d) and C6D6 (e–h). The amount of the analyzed sample: a and h – 18.0 mg; b and g – 9.0 mg;
c and f – 4.5 mg; d and e – 0.9 mg.
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Table 3 Minimal detection limit and accuracy of “in tube” derivatization and NMR analysis of the mixture of R- and S-methylmandelates (70 : 30)
using S-MPA in CDCl3 and benzene-d6
Conversion, % (R/S ratio)c
Entry
Analyzed sample/mg
Methoda
Concentration/mol L-1b
Benzene-d6
CDCl3
1
2
3
4
5
6
18.0
9.0
4.5
0.9
0.09
0.009
A
A
A
A
B
1.8 ¥ 10-1
9.0 ¥ 10-2
4.5 ¥ 10-2
9.0 ¥ 10-3
9.0 ¥ 10-4
9.0 ¥ 10-5
100 (69.7 : 30.3)
100 (69.9 : 30.1)
100 (70.4 : 29.6)
100 (69.0 : 31.0)
100 (70.3 : 29.7)d
100 (69.3 : 30.7)d
100 (69.4 : 30.6)
98 (69.6 : 30.4)
94 (67.1 : 32.9)
78 (73.4 : 26.6)
82 (71.0 : 29.0)d
87 (70.5 : 29.5)d
B
a See Experimental part. b Initial concentration of the analyzed sample in the NMR tube. c The ratio of diastereomers determined by 1H NMR
analysis is shown in parenthesis. d 10 equivs of CDA and DCC were used.
the diastereomer ratio (Fig. 5) were obtained on a 600 MHz
instrument with 128 scans (ca. 5 min) and 1024 scans (ca. 1h) for
0.09 and 0.009 mg of analyzed sample, respectively. To enhance
the performance with such small amounts of the chiral samples
fixed amounts of S-MPA and DCC were used. Clearly, the
developed “in tube” protocol allows accurate measurements,
even for trace amounts of the analyzed chiral samples.
Concerning the choice of solvent, CDCl3 solutions are suitable
for analysis of 18.0–9.0 mg of the sample with high yield and
accuracy (Table 3; entries 1, 2), the error in determination of
enantiomeric content of the analyzed sample was less than
1%. However, further decrease of the sample amount is not
recommended using Methods A and B (Table 3, entries 3–6).
A modified Method C was developed to increase the accuracy
of the measurements in CDCl3 with small amounts of chiral
samples. Method C has shown 100% conversion and very good
accuracy 69.3 : 30.7 using commercially available CDCl3 (see
Experimental part and Table S1 in the ESI†; cf. entry 3 in Table
3 for Method B).
Excellent results were observed in benzene-d6 for the whole
range of the studied samples 18.0–0.009 mg (Table 3). As
shown in Table 3, in all cases the error in determination of
the enantiomeric content of the analyzed sample was less
than 1%. We have found that tolulene-d8 can be also used to
replace benzene-d6 for NMR measurements using the same
experimental procedure. Complete conversion of the reagents
and a 69.3 : 30.7 R/S ratio was observed in toluene-d8 (cf. entry
3, Table 3 for benzene-d6).
Finally, in most cases, the error in the measurements did not
exceed 0.3%, which confirms the high accuracy of the developed
procedure.
Conclusions
We have shown that MTPA and MPA can be used for the
derivatization of chiral alcohols and amines directly in the NMR
tube, followed by spectral analysis of enantiomeric purity and
determination of absolute configuration without any isolation
1
Fig. 5 Fragments of H NMR spectra of diastereomers in CDCl3 and C6D6 with small sample amounts: a) 0.09 mg; and b) 0.009 mg. Signals
assigned to diastereomers of R- and S-methylmandelate are marked in red ( ) and blue ( ), respectively; signals of unreacted alcohol are shown in
green ( ).
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or purification steps. The crucial role of the residual amount
of water was revealed for the first time and it was shown that
the accuracy of the measurements can be greatly improved by
a simple modification of the experimental procedure (solvent
drying over molecular sieves and applying an excess of the
CDA or DCC). Using standard NMR spectrometers, a highly
efficient and environmentally benign procedure was developed
for fast analysis of the chiral samples (<5 min), which can be
suitable for routine usage for asymmetric synthesis and catalysis
applications. Successful measurements of as small as <10-5 g of
chiral sample were demonstrated.
Russ. Chem. Rev., 2009, 78, 457; (u) S. Luo, L. Zhang and J.-P. Cheng,
Chem.–Asian J., 2009, 4, 1184.
3 (a) J. M. Seco, E. Quinoa and R. Riguera, Chem. Rev., 2004, 104, 17;
(b) G. Uccello-Barretta, F. Balzano and P. Salvadori, Curr. Pharm.
Des., 2006, 12, 4023; (c) D. Parker, Chem. Rev., 1991, 91, 1441; (d) R.
Hulst, R. M. Kellogg and B. L. Feringa, Recl. Trav. Chim. Pays-
Bas, 1995, 114, 115; (e) K. M. Błazewska and T. Gajda, Tetrahedron:
Asymmetry, 2009, 20, 1337; (f) A. P. Kumar, D. Jin and Y.-I. Lee, Appl.
Spectrosc. Rev., 2009, 44, 267; (g) T. J. Wenzel, Discrimination of chiral
compounds using NMR spectroscopy, New York,Wiley, 2007; (h) T. J.
Wenzel and C. D. Chisholm, Chirality, 2011, 23, 190; (i) R. Rothchild,
Enantiomer, 2000, 5, 457; (j) T. J. Wenzel and C. D. Chisholm, Prog.
Nucl. Magn. Reson. Spectrosc., 2011, 59, 1.
4 Examples of CDAs: (a) Y. Takeuchi, M. Segawa, H. Fujisawa, K.
Omata, S. N. Lodwig and C. J. Unkefer, Angew. Chem., Int. Ed.,
2006, 45, 4617; (b) T. Fujiwara, M. Segawa, H. Fujisawa, T. Murai,
T. Takahashi, K. Omata, K. Kabuto, S. N. Lodwig, C. J. Unkeferc and
Y. Takeuchi, Tetrahedron: Asymmetry, 2008, 19, 847; (c) Y. Takeuchi,
H. Fujisawa and R. Noyori, Org. Lett., 2004, 6, 4607; (d) J. M.
Seco, E. Quinoa and R. Riguera, Tetrahedron: Asymmetry, 2000,
11, 2781; (e) J. M. Seco, E. Quinoa and R. Riguera, Tetrahedron:
Asymmetry, 2002, 13, 919; (f) J. M. Seco, E. Quinoa and R. Riguera,
Tetrahedron, 1999, 55, 569; (g) R. Garcıa, J. M. Seco, S. A. Vazquez,
E. Quinoa and R. Riguera, J. Org. Chem., 2006, 71, 1119; (h) R.
Garcıa, J. M. Seco, S. A. Vazquez, E. Quinoa and R. Riguera, J. Org.
Chem., 2002, 67, 4579; (i) B. Lopez, E. Quinoa and R. Riguera, J.
Am. Chem. Soc., 1999, 121, 9724; (j) K. M. Sureshan, T. Miyasou, S.
Miyamori and Y. Watanabe, Tetrahedron: Asymmetry, 2004, 15, 3357;
(k) Habel, D. Spiteller and W. Boland, J. Chromatogr., A, 2007, 1165,
182; (l) N. Harada, M. Watanabe, S. Kuwahara, A. Sugio, Y. Kasaia
and A. Ichikawa, Tetrahedron: Asymmetry, 2000, 11, 1249; (m) B. E.
Dominguez, P. E. Garcia and J. Cardenas, Tetrahedron: Asymmetry,
2005, 16, 3976; (n) O. T. du Boullay, A. Alba, F. Oukhatar, B. Martin-
Vaca and D. Bourissou, Org. Lett., 2008, 10, 4669; (o) T. Murai, H.
Tsuji, S. Imaizumi and T. Maruyama, Chem. Lett., 2010, 39, 524;
(p) J.-L. Gras, T. Soto and A. Heumann, Eur. J. Org. Chem., 2000,
837; (q) S. Rodrıguez-Escrich, D. Popa, C. Jimeno, A. Vidal-Ferran
and M. A. Pericas, Org. Lett., 2005, 7, 3829; (r) I. Louzao, J. M.
Seco, E. Quinoa and R. Riguera, Chem. Commun., 2010, 46, 7903;
(s) M. Kobayashi, Tetrahedron, 1998, 54, 10987; (t) D. B. Weibel,
T. R. Walker, F. C. Schroeder and J. Meinwald, Org. Lett., 2000, 2,
2381; (u) F. C. Schroeder, D. B. Weibel and J. Meinwald, Org. Lett.,
2004, 6, 3019; (v) R. T. Williamson, A. C. B. Sosa, A. Mitra, P. J.
Seaton, D. B. Weibel, F. C. Schroeder, J. Meinwald and F. E. Koehn,
Org. Lett., 2003, 5, 1745; (w) M. Campagna, M. Trzoss and S. Bienz,
Org. Lett., 2007, 9, 3793; (x) R. A. Gariani, F. Simonelli, A. R. M.
Oliveira, A. Barison and J. V. Comasseto, Tetrahedron: Asymmetry,
2006, 17, 2930; (y) G. Uccello-Barretta, R. Bernardini, F. Balzano
and P. Salvadori, Chirality, 2002, 14, 484.
5 N. V. Orlov and V. P. Ananikov, Chem. Commun., 2010, 46, 3212.
6 Optimization of the derivatization procedure: (a) S. Porto, J. M. Seco,
J. F. Espinosa, E. Quinoa and R. Riguera, J. Org. Chem., 2008, 73,
5714; (b) S. Porto, J. Duran, J. M. Seco, E. Quinoa and R. Riguera,
Org. Lett., 2003, 5, 2979; (c) T. Arnauld, A. G. M. Barrett, B. T.
Hopkins and F. J. Zecri, Tetrahedron Lett., 2001, 42, 8215; (d) M.
Adamczyk and J. R. Fishpaugh, Tetrahedron Lett., 1996, 37, 7171;
(e) J. M. Seco, L.-H. Tseng, M. Godejohann, E. Quinoa and R.
Riguera, Tetrahedron: Asymmetry, 2002, 13, 2149.
7 Development of MTPA and MPA: (a) J. A. Dale and H. S. Mosher,
J. Am. Chem. Soc., 1973, 95, 512; (b) J. A. Dale, D. L. Dull and H. S.
Mosher, J. Org. Chem., 1969, 34, 2543; (c) B. M. Trost, J. L. Belletire,
S. Godleski, P. G. McDougal and J. M. Balkovec, J. Org. Chem.,
1986, 51, 2370; (d) B. M. Trost, R. C. Bunt and S. R. Pulley, J. Org.
Chem., 1994, 59, 4202.
Acknowledgements
The research work was supported by Research Grant MK-
1434.2010.3 and RFBR grant 11-03-01055.
References
1 Representative examples: (a) C. Grondal, M. Jeanty and D. Enders,
Nat. Chem., 2010, 2, 167; (b) B. M. Trost and C. S. Brindle, Chem. Soc.
Rev., 2010, 39, 1600; (c) K. C. Nicolaou, J. S. Chen and S. M. Dalby,
Bioorg. Med. Chem., 2009, 17, 2290; (d) D. Enders, C. Wang and J. X.
Liebich, Chem.–Eur. J., 2009, 15, 11058; (e) K. C. Nicolaou and J. S.
Chen, Chem. Soc. Rev., 2009, 38, 2993; (f) S. Mukherjee, J. W. Yang,
S. Hoffmann and B. List, Chem. Rev., 2007, 107, 5471; (g) D. Enders,
C. Grondal and M. R. M. Huttl, Angew. Chem., Int. Ed., 2007, 46,
1570; (h) J. Seayad and B. List, Org. Biomol. Chem., 2005, 3, 719;
(i) D. Enders and T. Balensiefer, Acc. Chem. Res., 2004, 37, 534; (j) T.
Hayashi and K. Yamasaki, Chem. Rev., 2003, 103, 2829; (k) M. C.
Perry and K. Burgess, Tetrahedron: Asymmetry, 2003, 14, 951; (l) B.
M. Trost and M. L. Crawley, Chem. Rev., 2003, 103, 2921; (m) Q.-H.
Fan, Y.-M. Li and A. S. C. Chan, Chem. Rev., 2002, 102, 3385; (n) M.
Shibasaki and N. Yoshikawa, Chem. Rev., 2002, 102, 2187; (o) R.
Noyori, Angew. Chem., Int. Ed., 2002, 41, 2008; (p) R. Noyori and
T. Ohkuma, Angew. Chem., Int. Ed., 2001, 40, 40; (q) G. Helmchen
and A. Pfaltz, Acc. Chem. Res., 2000, 33, 336; (r) M. J. Palmer and
M. Wills, Tetrahedron: Asymmetry, 1999, 10, 2045; (s) R. Noyori and
S. Hashiguchi, Acc. Chem. Res., 1997, 30, 97; (t) B. M. Trost and
D. L. VanVranken, Chem. Rev., 1996, 96, 395; (u) H. C. Kolb, M.
S. VanNieuwenhze and K. B. Sharpless, Chem. Rev., 1994, 94, 2483;
(v) K. B. Sharpless, W. Amberg, Y. L. Bennani, G. A. Crispino, J.
Hartung, K. S. Jeong, H. L. Kwong, K. Morikawa, Z.-M. Wang, D.
Xu and X.-L. Zhang, J. Org. Chem., 1992, 57, 2768; (w) R. Noyori
and M. Kitamura, Angew. Chem., Int. Ed. Engl., 1991, 30, 49; (x) S.
Masamune, W. Choy, J. S. Petersen and L. R. Sita, Angew. Chem.,
Int. Ed. Engl., 1985, 24, 1.
2 Selected recent reviews: (a) A. M. Palmer and A. Zanotti-Gerosa,
Curr. Opin. Drug Discovery Dev., 2010, 13, 698; (b) R. Wohlgemuth,
Curr. Opin. Microbiol., 2010, 13, 283; (c) S. Sengupta and X. Shi,
ChemCatChem, 2010, 2, 609; (d) M. Node, T. Kajimoto and M.
Ozeki, Heterocycles, 2010, 81, 1061; (e) Y. Takemoto, Chem. Pharm.
Bull., 2010, 58, 593; (f) L. Albrecht, A. Albrecht, H. Krawczyk and
K. A. Jorgensen, Chem.–Eur. J., 2010, 16, 28; (g) Y. Sohtome and K.
Nagasawa, Synlett, 2010, 1; (h) M. Bartok, Chem. Rev., 2010, 110,
1663; (i) S. G. Zlotin and N. N. Makhova, Russ. Chem. Rev., 2010, 79,
543; (j) B. Weiner, W. Szymanski, D. B. Janssen, A. J. Minnaard and
B. L. Feringa, Chem. Soc. Rev., 2010, 39, 1656; (k) S. J. Zuend, M.
P. Coughlin, M. P. Lalonde and E. N. Jacobsen, Nature, 2009, 461,
968; (l) S. Park and H. Sugiyama, Angew. Chem., Int. Ed., 2010, 49,
3870; (m) A. Moyano, N. El-Hamdouni and A. Atlamsani, Chem.–
Eur. J., 2010, 16, 5260; (n) E. Marques-Lopez, R. P. Herrera and M.
Christmann, Nat. Prod. Rep., 2010, 27, 1138; (o) A. J. Boersma, R.
P. Megens, B. L. Feringa and G. Roelfes, Chem. Soc. Rev., 2010, 39,
2083; (p) B. M. Trost and M. K. Brennan, Synthesis, 2009, 3003;
(q) S. Bertelsen and K. A. Jorgensen, Chem. Soc. Rev., 2009, 38,
2178; (r) J. E. D. Martins and M. Wills, Tetrahedron, 2009, 65,
5782; (s) T. Matsuda, R. Yamanaka and K. Nakamura, Tetrahedron:
Asymmetry, 2009, 20, 513; (t) K. P. Volcho and N. F. Salakhutdinov,
8 Examples of applications of MTPA and MPA: (a) T. Kusumi, T. Ooi,
Y. Ohkubo and T. Yabuuchi, Bull. Chem. Soc. Jpn., 2006, 79, 965;
(b) J. Gao, H. Haas, K. Y. Wang, Z. Chen, W. Breitenstein and S.
Rajan, Magn. Reson. Chem., 2008, 46, 17; (c) Y. Yang, M. M. Kayser
and D. Hooper, Chirality, 2005, 17, 131; (d) A. V. Malkov, M. R.
Gordon, S. Stoncius, J. Hussain and P. Kocˇovsky, Org. Lett., 2009,
11, 5390.
9 Examples of improved derivatization procedure: (a) F. Freire, E.
Quinoa and R. Riguera, Chem. Commun., 2008, 4147; (b) M.
Amberg, U. Bergstra¨sser, G. Stapf and J. Hartung, J. Org. Chem.,
2008, 73, 3907; (c) A.-S. Chauvin, G. Bernardinelli and A. Alexakis,
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The Royal Society of Chemistry 2011
Green Chem., 2011, 13, 1735–1744 | 1743
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View Article Online
Tetrahedron: Asymmetry, 2006, 17, 2203; (d) A.-S. Chauvin, G.
Bernardinelli and A. Alexakis, Tetrahedron: Asymmetry, 2004, 15,
1857; (e) G. Uccello-Barretta, R. Bernardini, R. Lazzaroni and P.
Salvadori, J. Organomet. Chem., 2000, 598, 174.
12 Extra dry purification of solvent and all reagents is a time consuming
procedure, which is accompanied by significant waste production.
In addition, some amounts of water may be present in the analyzed
samples taken from crude reaction mixtures. Therefore, the analytical
procedure with some tolerance to water residues is preferred.
13 Complete conversion is not required for determination of absolute
configuration, thus a number of different solvents may be used
(Table 1). However, complete conversion is required for accurate
measurements of diastereomer ratio (see text).
14 (a) J. M. Seco, E. Quinoa and R. Riguera, Tetrahedron: Asymmetry,
2001, 12, 2915; (b) S. K. Latypov, J. M. Seco, E. Quinoa and R.
Riguera, J. Org. Chem., 1996, 61, 8569; (c) V. Leiro, J. M. Seco, E.
Quinoa and R. Riguera, Chem.–Asian J., 2010, 5, 2106; (d) F. Freire,
E. Lallana, E. Quinoa and R. Riguera, Chem.–Eur. J., 2009, 15,
11963.
10 Examples of direct derivatization: (a) C. Sabot, M. Mosser, C.
Antheaume, C. Mioskowski, R. Baati and A. Wagner, Chem.
Commun., 2009, 3410; (b) T. Reiner, F. N. Naraschewski and J.
Eppinger, Tetrahedron: Asymmetry, 2009, 20, 362; (c) M. E. Powell,
A. M. Kelly, S. D. Bull and T. D. James, Tetrahedron Lett., 2009, 50,
876; (d) Y. Perez-Fuertes, A. M. Kelly, J. S. Fossey, M. E. Powell, S.
D. Bull and T. D. James, Nat. Protoc., 2008, 3, 210; (e) A. M. Kelly,
Y. Perez-Fuertes, J. S. Fossey, S. L. Yeste, S. D. Bull and T. D. James,
Nat. Protoc., 2008, 3, 215.
11 The sample with known composition of the R- and S-
methylmandelates (70 : 30 molar ratio) was used to determine the
accuracy, thus the largest error of 10% was found for MTPA in
THF-d8 with the 80 : 20 ratio determined in the analysis (Table 1,
entry 5).
15 (a) S. K. Latypov, J. M. Seco, E. Quinoa and R. Riguera, J. Am.
Chem. Soc., 1998, 120, 877; (b) V. Leiro, J. M. Seco, E. Quinoa and
R. Riguera, Org. Lett., 2008, 10, 2733.
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