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Jose Manuel Seco was born in Dodro (La Coruna, Spain) in 1968. He Professor Ricardo Riguera was born in Lugo (Spain) in 1948. He received
received his B.Sc. (1991), M. Sc. (1992), and Ph.D. (1997) degrees in its M. S. degree in Chemistry from the University of Santiago de
Chemistry from the University of Santiago de Compostela, the latter under Compostela in 1971 and the Ph.D. degree in 1973 under the supervision
the supervision of Profs. Ricardo Riguera and Emilio Quinoa. He was of the late Prof. I. Ribas. From 1974 to 1976, he stayed at the University
awarded with the Ph.D. Extraordinary Prize in 1998. In 2001, he became College London as a postdoctoral fellow under the supervision of Prof.
Assistant Professor in Organic Chemistry. His current research interest Peter J. Garratt. After returning to the University of Santiago de
is focused in the development of new reagents and methods for the Compostela, he was appointed Lecturer of Organic Chemistry in 1978
determination of absolute stereochemistry of organic compounds by NMR. and became Full Professor in 1990. As academic, he has served the
university as Chairman of the Department, Dean of the Faculty of
Chemistry, and Vice-Chancellor. He also has authored textbooks for
students. His research interests have involved the fields of bioactive natural
products from terrestrial and marine organisms, in regard to new synthetic
methods and some aspects of medicinal chemistry. In the past few years,
he has focused on the development of methods for determination of the
absolute configuration by NMR.
Figure 1.
Figure 2.
of the chiral center of the auxiliary (CR; configuration ensure that the chemical shifts of L1 and L2 are
known) with that of the substrate (C(1), configura- different in the two species (diastereoisomers or
tion unknown). To this end, the changes in the conformers) that are used for the determination of
chemical shifts of the substituents of the asymmetric the conformation.
carbon of the substrate (L1 and L2) in the two For example, in the case of MPA, R1 is a methoxy
derivatives (or conformational species) are consid- group, Z is a carboxylic acid, and Y is a phenyl group
ered. (Figure 4).
These differences in the chemical shifts are repre-
sented by , and it is the sign of this parameter
(+ or -) that provides information about the config- 2.3. The NMR Spectra of the Derivatives
uration. For a particular substituent (e.g., L1), is As we have said, comparison of the NMR spectra
defined as the difference of chemical shifts of a given of the two derivatives and evaluation of the differ-
signal of the substituent (L1) in the two spectra ences in the chemical shifts of the signals for the
under consideration (diastereoisomers or conformers, substituents L1 and L2 (i.e., RS) must be performed.
depending on the routine chosen). As will be de- The signs of those parameters contain the informa-
scribed in this paper, there is a variety of ways to tion necessary for the configurational assignment,
express the differences in chemical shifts, their because they indicate the relative position of L1/L2,
significance, and their characteristics. For example, with respect to the anisotropic group Y; therefore, the
in Moshers method, a chiral secondary alcohol (i.e.,
observed differences must not be uncertain and must
2-pentanol; see Figure 3) is derivatized with the two
be clearly predictable.
enantiomers of an arylmethoxyacetic acid (AMAA)
i.e., (R)- and (S)-R-methoxy-R-phenylacetic acid (MPA), In the methods that are based on the formation of
the 1H NMR spectra of the two derivatives are two derivatives, it is important that both diastereo-
recorded, and the RS value is calculated for the isomers show a certain preference for a particular
substituents L1 (RSL1) and L2 (RSL2). In each conformation (NMR-significant conformer) indepen-
case, RS is the difference between the chemical dent of the particular substrate, and the Y group
shift in the (R)-MPA ester derivative ((R)) and in must project its magnetic anisotropy in a selective
the (S)-MPA derivative [(S)] (see Figure 3). way, i.e., on L1 in one derivative and on L2 in the
other.
2.2. Characteristics of the Auxiliary Reagents For example, in the MPA esters (Figure 3), it is
In general, the structure of the chiral auxiliary assumed that the representative conformer in terms
reagent (B) must incorporate groups with specific of NMR is the one in which the methoxy, carbonyl,
functions: and C(1)H groups are situated in the same plane.
(a) A polar or bulky group (R1) to fix a particular In this way, in the (R)-MPA derivative, the phenyl
conformation. group shields L1 (-CH2CH2CH3), whereas, in the (S)-
(b) A functional group (Z) (e.g., carboxylic acid) that MPA derivative, the shielded group is L2 (-CH3). The
provides a site for covalent attachment of the sub- comparison of both spectra leads to RSL1 < 0 and
strate. RSL2 > 0 (-0.14, -0.23, -0.09, and +0.013 ppm,
(c) A group (Y) that is able to produce an efficient respectively).
and space-oriented anisotropic effect (e.g., aromatic, If the absolute configuration of the alcohol were the
carbonyl) that selectively affects substituents L1 and opposite, the signs of RS will also be reversed (i.e.,
L2 of the substrate. This shielding/deshielding will RSL1 > 0, RSL2 < 0).
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 21
Figure 3.
Figure 5.
the way in which the CDAs work but also led to the ever since Mosher first reported7,8 its use in 1973.
development of new and more-efficient CDA reagents The procedure starts with the esterification of the
and methodologies giving a plus of reliability to the alcohol with the two enantiomers of MTPA. After the
absolute configuration assignment process that is two diastereomeric esters have been prepared, their
now based on both empirical and theoretical results. NMR spectra are acquired and compared. MTPA is
available both as the acid and the acid chloride, and
2.5. Substrates and Derivatives users must remember that, although derivatization
The ranges of substrates whose configuration can of the alcohol with the (R)-MTPA acid leads to the
be assigned by these methods have increased signifi- corresponding (R)-MTPA ester, the (R)-MTPA chlo-
cantly recently and include primary, secondary, and ride leads to the (S)-MTPA ester, which has led to
tertiary alcohols, diols, carboxylic acids, primary and confusion and some erroneous assignments.9
secondary amines, and sulfoxides. Appropriate chiral In his first study, Mosher used 19F NMR spectros-
auxiliary reagents have been developed for each case copysparticularly, the chemical shifts of the CF3
that produces derivatives such as esters, amides, groupssfor the assignment. In later studies, how-
hemiacetals, phosphonates, etc. This review covers ever, 1H and 13C NMR were utilized, and the chemical
all those chiral substrates, the reagents and methods shifts of the protons and C atoms of the chiral alcohol
most commonly used and attempts to offer a critical under examination were used, respectively. The
assessment of each method. absolute configuration of the substrate is deduced by
interpretation of the signs of values, using certain
empirical models. A description of those models,
3. Methods Based on Double Derivatization classified according to the NMR technique used,
follows.
3.1. Application to r-Chiral Secondary Alcohols 3.1.1.1. The Use of 19F NMR. 19F NMR was used
by Mosher to determine the absolute configuration
3.1.1. Methoxytrifluoromethylphenylacetic Acid (MTPA): of secondary alcohols.8a The procedure is based on the
Moshers Reagent anisotropic effect that the carbonyl group of the
auxiliary MTPA exerts on the CF3 group. For this
R-Methoxy-R-trifluoromethyl-R-phenylacetic acid purpose, Mosher assumed that the (R)- and (S)-
(MTPA; see Figure 6) has been the most commonly MTPA esters of secondary alcohols exist in a confor-
used derivatizing reagent for the determination of the
mation in which C(1)H, the carbonyl, and the CF3
absolute configuration of secondary alcohols by NMR
groups are situated in the same plane (Figure 7) in
the derivative where the phenyl ring is opposite to
the least bulky substituent of the alcohol. In the
derivative in which the phenyl ring is situated on the
same side as the most bulky substituent, the copla-
narity is lost and, as a consequence, the CF3 moves
from the deshielding zone to the shielding zone of the
carbonyl group.
When the bulkier substituent (e.g., L1) is on the
same side as the phenyl group, the CF3 resonates at
a higher fieldsbecause of greater shielding (Figure
7b)sthan if L1 were on the side of the methoxy group
Figure 6. (Figure 7a). These differences in chemical shifts are
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 23
Figure 7. Model for the correlation of configuration/NMR for 19F and NMR spectra of MTPA esters.
Figure 8. Models proposed by the Mosher model for (a, b) the assignment of configuration by 1H NMR and (c, d) the
expected sign of SR.
expressed in terms of the parameter SR(19F)CF3, made, regarding the spatial position of L1 and L2,
which is defined as the difference in the chemical with respect to the phenyl group of the MTPA moiety
shift of the trifluoromethyl signal in the (S)-MTPA on the basis of the signs of SR of the substituents.
ester (CF3(S)) and the chemical shift of the same In this case, Mosher assumed that the most repre-
signal in the (R)-MTPA ester (SR(19F)CF3 ) CF3- sentative conformation is that in which C(1)H, the
(S) - CF3(R)). carbonyl group, and the CF3 group are situated in
For example, an alcohol with the configuration the same plane (Figure 8a).
represented in Figure 7, in which L1 is more bulky Accordingly, the protons of substituent L2 are
than L2, gives a negative value of SR(19F)CF3 (<0). shielded by the phenyl ring in the (R)-MTPA ester,
If the configuration of the alcohol were the opposite, whereas those on L1 remain unaffected (Figure 8a).
or the size of the substituent L2 was much greater In the (S)-MTPA derivative, on the other hand, it is
than that of L1, the sign of SR would be positive. L1 and its protons that are shielded while L2 is
An advantage of this procedure lies in the clean- unaffected (Figure 8b). Therefore, the substituent L1
ness of the 19F NMR spectra, which generally only will be more shielded in the (S)-MTPA ester than in
contain signals that are due to the CF3 groups and the (R)-MTPA ester and the substituent L2 will be
eliminates the possibility of overlapping signals that more shielded in the (R)-MTPA than in the (S)-MTPA
often complicate proton spectra. Its application for ester derivative.
the configurational assignment will be discussed later These selective shieldings are expressed using the
in this review. parameter SR, which is defined as the difference
3.1.1.2. The Use of 1H NMR. The use of 1H NMR between the chemical shift of a certain proton in the
spectroscopy is undoubtedly more common for the (S)-MTPA ester and the chemical shift of the same
assignment of the configuration of secondary alco- proton in the (R)-MTPA derivative. All the protons
hols10 than the use of 19F NMR that has been shielded in the (R)-MTPA will present a positive SR
described previously. This approach is based on the value, whereas those shielded in the (S)-MTPA
anisotropic effect that the phenyl group of the chiral derivative will present a negative SR value.
auxiliary MTPA exerts on the substituents (L1/L2) of In the case of the alcohol shown in Figure 8c, for
the alcohol. This effect allows a correlation to be example, the signs for each proton in L1 and L2 are
24 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
as follows:
Figure 11.
Application of 13C NMR for the assignment of remember the following points that greatly limit its
configuration of alcohols presents some advantages application on a general basis:
but also some important limitations. (a) The number of alcohols of known configuration
Although the use of 13C NMR requires a greater (Figure 10) that have been used to test the validity
amount of sample than does 1H NMR, this technique of the correlations is rather small; therefore, the
could be especially interesting in cases where 1H accuracy of the configuration assigned to a certain
NMR is not useful because one of the substituents substrate has some uncertainty.
L1/L2 has no protons. Nevertheless, those wishing to (b) The SR values obtained are very small, in
use it as a general method for assignment should comparison to the 13C chemical shift scale.
26 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
Figure 18.
Figure 20.
Figure 21.
Figure 19.
most of the SR signs are in good agreement with
been described.26a 1 19
The H and F NMR spectra of the configuration, some compounds (21.3 and 21.6)
the two MTPA esters are recorded and the SR exhibit signals (underlined in the figure) that are
values calculated for the protons on one substituent inconsistent with their configuration and put a limit
and for the fluoro-substituents of the other substitu- on the validity of the method.
ent. The model proposed assumes that if the config- The purpose of the second alternative described26b
uration of the alcohol is that shown in Figure 20b, is to solve the difficulties presented by overlapping
then the protons in L1 must produce a negative SR signals for L1/L2 in aromatic and heteroaromatic
value, whereas the fluoro-substituents in L2 must alcohols. It is based on the combined use of the
have a positive SR value. When the configuration anisotropic effect caused by the aromatic ring of the
is the opposite, the signs of SR then will be reversed alcohol on the methoxy group of the auxiliary MTPA
(see Figure 20c). and that of the phenyl ring of the auxiliary on the
Figure 21 shows the alcohols of known absolute substituents L1/L2 of the alcohol.
configuration that have been used as support of the According to the author, in the (R)-MTPA ester of
mixed proton-fluorine NMR approach. Although the alcohol shown in Figure 22a, both aryl rings are
30 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
Figure 22.
Figure 24.
Figure 26. Moshers model8 and the revised model27 for the assignment of absolute configuration of MTPA esters.
sp conformer, whereas substituent L1 is slightly equilibrium, which is also the most important, from
shielded and substituent L2 is unaffected in the ap the standpoint of NMR, and is therefore suitable to
conformer (Figure 32b). predict the correct configuration. Figure 33 shows a
As a result of the characteristics discussed above, selection of compounds studied using that model.31
the aromatic group of the reagent will modify the Note that this is not the case for MTPA esters,
chemical shifts of substituents L1 and L2 in a very where the model used by Mosher is not truly repre-
selective way: In the (R)-MPA ester, it is expected sentative of the conformational composition.27
that strong shielding will be experienced by substitu- 3.1.2.3. MPA versus MTPA. In general, research-
ent L1 but only on the proportion of the molecules in ers have not been very critical when choosing be-
which substituent L1 is beside the phenyl group tween MPA and MTPA for the assignment of sec-
(conformer sp). On the other hand, in the (S)-MPA ondary alcohols. Quite frequently, they have over-
ester, substituent L1 will be slightly shielded, but only looked the anomalies detected with MTPA13 that
for those molecules in the ap conformation. Given were mentioned in the previous chapter.
that the sp conformer is more abundant than the ap In the case of MTPA esters of secondary alcohols,
conformer, substituent L1 is more shielded in the (R)- two factors contribute to these limitations:27,32 (a) the
MPA ester than in the (S)-MPA ester. Similar presence of three main conformers with similar
reasoning for substituent L2 leads to the conclusion populations and (b) the fact that some of these
that it will be more shielded in the (S)-MPA ester conformers produce shielding effects and others
than in the (R)-MPA ester. Thus, the differences in produce deshielding effects on substituents L1/L2 (see
chemical shifts (RS) will be positive in one case and Figure 25), the combination of which produces very
negative in the other (see Figure 32c). If the opposite small SR values and sometimes unexpected signs.
configuration is considered for the alcohol (i.e., sub- In contrast, the MPA esters present a simpler
stituent L1 in place of substituent L2), then the conformational composition (see Figure 32) with only
opposite distribution of signs is obtained (see Figure two conformers (sp/ap) and a clearer preference for
32d), thus demonstrating that the sign of RS is a one of those (sp), which, in turn, transmits a shielding
reliable indicator of the spatial arrangement of the effect to the substituents to give higher RS values
substituents. that are more reliable and homogeneous in terms of
distribution of the signs (Figure 34).
RSL1 ) L1(R) - L1(S) < 0 On the basis of the aforementioned discussion, the
choice between MTPA and MPA as a derivatizing
RSL2 ) L2(R) - L2(S) > 0 agent for secondary alcohols should be clearly in favor
of MPA.
The model proposed by Mosher8c,28a for the assign- 3.1.2.4. New MPA Analogues: The Effect of
ment of the configuration of MPA esters is, in fact, a Structural Modifications. In the search for more
representation of the most stable conformer of the efficient and reliable reagents, a variety of modifica-
36 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
Figure 35.
3.1.2.8. The Effect of the Temperature on the alcohols, when their NMR spectra were recorded at
NMR of MPA Esters. A different and successful different temperatures, the changes in chemical
approach to obtain the best RS values without shifts consistently produced higher RS values38 of
modification of the structure of the auxiliary reagent practical interest.
consists of the controlled modification of the relative Thus, in the (R)-MPA ester of (-)-menthol, the
populations (conformer sp/ap) of the MPA esters by Me(8) and Me(9) protons are strongly shielded by
directly acting on the thermodynamic parameters the phenyl ring in the sp conformer (see Figure 39a,
governing the equilibrium,38 namely the polarity of resonances at 0.651 and 0.418 ppm, respectively),
the NMR solvent and the temperature of the NMR whereas Me(10) is slightly shielded in the ap con-
probe. former (0.888 ppm). A decrease of the probe temper-
Although changes in the NMR solvent30 did not ature should produce an increase in the number of
lead to any significant improvement of the RS molecules in the sp conformation (the most stable
values obtained for MPA esters of several secondary one) and a corresponding decrease in the ap popula-
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 39
Table 3. Chemical Shifts of MPA Esters of have their Me(8) and Me(9) groups shielded (in-
(-)-Menthol at Different Temperaturesa creased shielding in the average spectrum; reso-
chemical shift nances at 0.561 and 0.165 ppm) and fewer molecules
ester Me(8) Me(9) Me(10) have their Me(10) shielded by the phenyl group
T ) 296 K (increased shielding in the average spectrum; reso-
(R)-MPA 0.651 0.418 0.888 nance at 0.898 ppm).
(S)-MPA 0.846 0.642 0.822
RS -0.195 -0.224 0.066
Similarly, in the (S)-MPA ester, the Me(8) and
T ) 153 K
(R)-MPA 0.561 0.165 0.898
Me(9) groups become less shielded at lower temper-
(S)-MPA 0.895 0.681 0.785 atures and the signals are shifted from 0.846 and
RS -0.334 -0.516 0.113 0.642 ppm to 0.895 and 0.681 ppm, respectively,
a
CS2:CD2Cl2 ratio ) 4:1. whereas Me(10) is more intensely shielded at lower
temperatures, with a change in chemical shift from
tion (the least stable one). For this reason, as shown 0.822 to 0.785 ppm for the corresponding signal (see
in Table 3, at lower temperatures, more molecules Figure 39b).
Figure 40. Evolution with temperature of the RS values of MPA esters of secondary alcohols.
Table 4. RS Values for a Selection of MPA Esters at Different Temperaturesa
temp, RS (ppm)
T (K) H(1) H(3) H(4) H(5) H(6a) H(6e) H(6ax) H(7) H(8) Me(8) Me(9) Me(10)
(-)-Isopulegol
298 0.050 0.085 -0.210 -0.250 -0.216 0.039
213 0.100 0.175 -0.319 -0.373 -0.348 0.049
183 0.100 0.175 -0.346 -0.403 -0.40 0.049
(-)-Menthol
298 -0.45 -0.195 -0.224 0.066
153 -1.170 -0.334 -0.516 0.103
(-)-Borneol
298 0.050 0.255 0.080 -0.200
213 0.104 0.499 0.138 -0.312
183 0.125 0.596 0.162 -0.331
(R)-2-Butanol
298 0.101 -0.095 -0.161
263 0.120 -0.127 -0.207
230 0.138 -0.137 -0.259
203 0.156 -0.166 -0.316
a
CS2:CD2Cl2 ratio ) 4:1.
40 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
Figure 47. (a, b) NMR spectra of the (R)- and (S)-9-AMA esters of a marine metabolite and (c) comparison of the RS
values of the 9-AMA esters with those of the MPA esters.
in the NMR process. However, the difference in stituent L1 is shielded in conformer sp and substitu-
energy29 varies with the nature of the aryl ring and ent L2 remains unaffected, whereas in the ap con-
follows the order 9-AMA > 1-NMA > 2-NMA > MPA. former, substituent L2 is shielded and substituent L1
Thus, changing the aromatic ring plays a double is not affected (see Figure 46a). In the ester derived
role, because it affects both the intensity of the from the (S)-AMAA auxiliary, the opposite situation
aromatic magnetic current and the conformational occurs: L2 is shielded in the sp conformer and L1 is
orientation of the ring, with respect to the substrate. shielded in the ap conformer (see Figure 46b).
In fact, the larger the ring, the more intense the Comparison of the spectra of the (R)- and the (S)-
aromatic shielding effect and the greater the result- AMAA esters, and considering the greater population
ing difference of energy between the sp and ap of the sp conformer in comparison to that of the ap
conformers. The combined effect of these two factors conformer, leads to the following signs for RS (these
makes 9-AMA the most useful reagent of all, produc- are the same as those obtained in the MPA esters
ing RS values that are much greater than those with the same configuration):
caused by other reagents.
The substitution pattern of the ring is also impor- RSL1 ) L1(R) - L1(S) < 0
tant because the rotational barriers for the CR-Ar
and CR-CO bonds will be different. For example, the RSL2 ) L2(R) - L2(S) > 0
barrier in 1-NMA is greater than that for 2-NMA and
this, in turn, affects the NMR results. Naturally, if the absolute configuration of the alcohol
3.1.3.2. NMR Spectra of AMAA Esters. The is opposite to that shown above, then the signs should
NMR spectra of the AMAA esters are similar to those be reversed (see Figure 46c and d).
of the MPA esters, and the contribution of each Figure 47 illustrates the power of 9-AMA as a
conformer to the average NMR spectra also follows reagent for the assignment of the absolute configu-
the same trend. Thus, in the (R)-AMAA ester, sub- ration. The NMR spectra of the (R)- and (S)-9-AMA
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 43
Figure 49. RS values for selected 9-AMA, 1-NMA, 2-NMA, 2-ATMA, MPA, and MTPA esters of secondary alcohols.
signal distribution across the spectrum for the rel- The 500 MHz NMR spectrum of cis-androsterone30
evant protons and eliminates signal overlap. The is shown in Figure 52b and those of its (R)- and (S)-
reagents 2-NMA and 2-ATMA37 are less effective in 9-AMA ester derivatives in Figure 52a and c, respec-
this respect, because a large number of protons in tively. It is clear that an unambiguous assignment
the chain will be shielded to approximately the same of the signals in Figure 52b is impossible, because
extent and, therefore, considerable signal overlap is most of the signals are overlapped in a narrow range
likely. of 1 ppm. In contrast, the 9-AMA esters present an
This ability of 9-AMA to separate the signals of the extraordinary dispersion of these signals, and this
alcohol portion is essential for the correct assignment phenomenon allows the complete assignment of all
of the protons and calculation of the RS values and the protons in the molecule.
is not limited to linear substrates but can also be of As predicted, the maximum shielding is located in
use for cyclic alcohols.30 An illustrative example of the region near to the asymmetric center (rings A and
such a case is represented by cis-androsterone. B of the steroid) and is so intense that the chemical
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 45
Figure 50.
Figure 51. Variation of RS with the distance to the asymmetric C atom in 9-AMA, 2-NMA, and 2-ATMA esters of
lineal alcohols.
shifts of protons H(1), H(9), H(6), H(5), and H(7), sp conformer in its esters is greater than that in the
which are indistinguishable in cis-androsterone itself, other AMAAs.
are moved to the right of TMS and is enough to move
protons in the C ring. 3.1.4. Other Reagents
In conclusion, replacement of the phenyl ring of In addition to the reagents described thus far,
MPA by naphthyl or anthryl rings is shown to be a many other compounds have been proposed as CDAs
useful way to modify these reagents. Of all the for the assignment of configuration of secondary
AMAAs investigated, 9-AMA is the one that produces alcohols by NMR. Most of the methods reported in
the most marked changes in shifts and RS values, this section use 1H and 13C NMR, with only a fews
because (a) its aromatic system produces the most mostly those devoted to e.e. calculations rather than
intense magnetic field and (b) the population of the absolute configuration assignmentsinvolving 19F
46 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
Figure 52. 1H NMR spectra of cis-androsterone (b) and its (R)-9-AMA (a) and (S)-9-AMA esters (c).
Figure 55. (a) Structure of (R)-3-PBA and (b) model for the assignment of the configuration of secondary alcohols from
the NMR spectra of the 3-PBA esters.
Figure 64.
Figure 74.
Figure 73.
54 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
Figure 79.
56 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
Figure 83.
Figure 85. Empirical models for the determination of the absolute configuration of primary alcohols by NMR of their
MTPA esters in the presence of Eu(fod)3. Rm represents the medium-sized group and Rl represents the large-sized group.
58 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
Figure 87. Natural compounds studied with MTPA and steroidal side chains obtained by synthesis and used as models.
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 59
Table 10. Selected NMR Data (CD3OD) for Side-Chain Signals in Synthetic 24-Methyl-26-hydroxy Steroids and in
Natural Compounds 87.6 and 87.7
(R)-MTPA ester
compound 26-CHH2 27-CH3 28-CH3 C-24 C-27 C-28 (26-CH2)
22 Series
(87.8a) (24R,25S) 3.28 dd, 3.60 dd 0.90 d 0.95 d 39.7 13.8 17.1 4.19 dd, 4.31 dd
(87.9a) (24S,25R) 3.29 dd, 3.59 dd 0.90 d 0.97 d 39.5 13.8 16.8 4.13 dd, 4.38 dd
(87.9b) (24S,25S) 3.34 dd, 3.53 dd 0.87 d 1.02 d 39.2 13.6 19.0 4.21 br d
(87.8b) (24R,25R) 3.34 dd, 3.52 dd 0.88 d 1.02 d 39.3 13.6 19.2 4.16 dd, 4.21 dd
Saturated Series
(87.10a) (24R,25S) 3.38 dd, 3.55 dd 0.83 d 0.81 d 35.1 14.8 12.0 4.23 br d
(87.11a) (24S,25R) 3.38 dd, 3.49 dd 0.81 d 0.81 d 35.1 15.1 11.6 4.14 dd, 4.34 dd
(87.11b) (24S,25S) 3.36 dd, 3.58 dd 0.93 d 0.92 d 36.8 17.5 14.4 4.22 dd, 4.32 dd
(87.10b) (24R,25R) 3.37 dd, 3.57 dd 0.91 d 0.91 d 36.1 17.4 14.1 4.16 dd, 4.38 dd
Natural Compounds
(87.6) (24R,25S) 3.46 dd, 3.58 dd 0.91 d 0.97 d 40.4 14.5 17.5 (R)-MTPA: 4.17 dd, 4.33 dd
(S)-MTPA: 4.06 dd, 4.46 dd
(87.7) (24S,25S) 3.34 dd, 3.62 dd 0.92 d 0.92 d 36.7 17.4 14.3 (R)-MTPA: 4.24 dd, 4.32 dd
(S)-MTPA: 4.16 dd, 4.37 dd
Figure 88. Shape of the partial NMR spectra of the 26-methylene protons in (R)- and (S)-MTPA esters of (a) a 25S
steroid and (b) its 25R isomer.
used for secondary alcohols4a,62b,c and consisted of the esters of 2-phenylethanols with bulky Rm groups are
conversion of a mixture of the two enantiomers of a larger than those with smaller Rm groups is consis-
primary alcohol into the corresponding mixture of tent with the model. The procedure was tested on the
(R)-(+)-MTPA esters. The magnitude of the lan- 12 substrates shown in Figure 86, where the chiral
thanide-induced shift (LISOMe) produced by Eu(fod)3 center is not present as part of a ring in any of the
on the OMe group of the MTPA ester showed a cases.
correlation with the absolute configuration. The The application of this method to cyclic alcohols
diastereomeric (R)-(+)-MTPA esters with the larger was explored by Takahashi et al.63a with the terpenes
LISOMe value had configuration A and the diastere- 87.1 and 87.2 shown in Figure 87. The compounds
omer with the smaller LISOMe value had configuration were transformed to the corresponding (R)- and (S)-
B (see Figure 85). MTPA esters and the resulting LISOMe values
The results were rationalized in terms of the indicated configurations that were in agreement with
models shown in Figure 85, where the complexation the known configurations deduced by alternative
constant KA is larger than KB, because of the less- correlation procedures.
marked steric interaction between Eu(fod)3 and the The same method has also been applied to the
H atom at the chiral center in A than with Rm in B. determination of the stereochemistry of a variety of
The fact that the magnitudes of LISOMe for the steroidal side chains. Minale et al.63b,c assigned the
60 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
Figure 90. 19F NMR chemical shifts of MTPA esters of primary hydroxy-sulfonamides and empirical models.
In 1996, Seebach and co-workers67 proposed a has been shown29,30 to possess two very important
method based on 19F NMR analysis of the MTPA features: (a) a higher conformational preference (the
esters and, more specifically, directed toward the syn-periplanar form, sp), than other AMAA reagents,
determination of the absolute configuration of hy- and (b) the anthryl ring in the main conformer (sp)
droxy-sulfonamides. The study was comprised of six is particularly well-oriented, in terms of projecting
cyclic hydroxy-sulfonamides, one cyclic hydroxy-ester, its strong aromatic magnetic cone on substituent L1
and just one acyclic primary alcohol (2-methylbu- or L2.
tanol) of known configuration. A correlation between No such advantages are expected from MPA or
the relative configuration of the MTPA esters with MTPA, because they have lower conformational
the 19F NMR chemical shift of the CF3 group was preferences, less-favorable orientation of the phenyl
deduced. Figure 90 shows the structures, chemical ring (MTPA), and a weaker magnetic cone (phenyl
shifts, and the empirical model used to explain the versus anthryl). Indeed, when these three reagents
correlation. were tested using 2-methylpropan-1-ol as a model
compound, completely separate signals for the di-
3.2.2. 9-AMA astereotopic methyl groups were obtained only in the
The most general approach to the assignment of 9-AMA esters69 (RS ) 0.033 versus RS ) 0.002
the absolute configuration of primary alcohols is ppm for 9-AMA and MPA respectively, and no
based on the use of 9-AMA,68,69 because this reagent separation for MTPA).
62 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
Figure 91. Low-energy conformers (perspective and Newman projections) for (R)-9-AMA esters of primary alcohols. In
most cases, sp-a/a is the most populated and relevant for NMR studies.
Complex theoretical studies (including molecular Sixteen 9-AMA esters of primary alcohols were
mechanics, semiempirical, ab initio, and aromatic studied,69 and, in most cases (93.1-93.13; see Figure
shielding-effect calculations), complemented by D 93), the conformational preferences and the model
NMR, were performed69 with simple model com- previously described do, in fact, apply. This observa-
pounds to assess the viability of 9-AMA esters for this tion means that the NMR spectra of this set of
purpose. The results of these studies indicated that 9-AMA esters could be reliably interpreted on the
the main conformational processes involve rotation basis of the corresponding model, and NMR spec-
around 1 and 2 bonds (see Figure 91 a), generating troscopy can be used to correlate the absolute con-
three main low-energy conformations (syn-peripla- figuration at the asymmetric center of the 9-AMA and
nar-gauche+/anti (sp-g+/a), syn-periplanar-anti/ that of the alcohol.
anti (sp-a/a), and syn-periplanar-gauche-/anti (sp- Unfortunately, in three examples involving cyclic
g-/a); see Figure 91b), with one of these (sp-a/a) structures or bulky substituents (93.14-93.16), the
becoming the most significant, in terms of NMR calculations showed that the sp-a/a forms were not
spectroscopy in most cases. In all these conforma- the preferred forms and that the sp-g-/a and sp-g+/a
tions, the 9-AMA substructure maintains its most- forms make a very significant contribution. In these
stable sp form. cases, it is clear that the sp-a/a model is not valid
Because conformer sp-a/a is the more stable and for interpretation of the NMR spectra of the 9-AMA
representative, from the standpoint of NMR, for a esters.
primary alcohol with the stereochemistry shown in In conclusion, to assign the absolute configuration
Figure 92, substituent L1 will be more shielded in of a primary alcohol by this method, a preliminary
the (R)-9-AMA ester, whereas substituent L2 will be calculation must be performed (being the best force
in the (S)-9-AMA ester. Therefore, a negative RS field, PCff), especially when the chiral center is part
sign is expected for substituent L1 and a positive RS of a ring, or when strong steric interactions could be
sign is expected for substituent L2. in operation, to confirm that the anti form is the most
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 63
Figure 92. Conformational model for the determination of the absolute configuration of -chiral primary alcohols by
NMR of their 9-AMA esters.
stable one and, consequently, that the model shown number of substrates studied is small and their
in Figure 92 is representative and can be applied to structural variety is limited; therefore, the methods
the interpretation of the chemical shifts. are applicable only to the cases reported.
The use of the auxiliary reagents MTPA and MPA
did not generate either the required RS values or 3.3.1. MTPA
homogeneity in the sign distribution, and this situ-
ation confirmed that these compounds could not be The use of MTPA with tertiary alcohols71 was
used as auxiliary reagents with chiral primary alco- explored for just two examples: pinan-2-ols 95.1 and
hols. 95.2 (see Figure 95).
A good example of the application of this procedure A combination of MM2 calculations and NOE and
for the determination of the configuration of a anisotropic effects (measured as SR of the methyl
complex substance is shown in Figure 94, which groups) was used to predict the configuration of these
depicts oxazinin-1, a cytotoxic compound that has substrates. The authors state that MTPA can be used
been isolated from the digestive glands of an edible in combination with conformational analysis; how-
mussel (Mytilus galloprovincialis). That was recently ever, the method was not expanded to include other
published by Fattorusso et al.70 In that paper, MM alcohols.
calculations were initially performed to check the
reliability of the NMR method with that particular 3.3.2. Methoxy-(2-naphthyl)acetic Acid (2-NMA)
substrate, and the stereochemistry was determined A method for the prediction of the absolute config-
using the 9-AMA esters only after a positive result uration of acyclic R-methyl-substituted tertiary al-
had been obtained. cohols has been published and involves the use of
methoxy-(2-naphthyl)acetic acid (2-NMA) as an aux-
3.3. Application to r-Chiral Tertiary Alcohols iliary reagent.72
Although the determination of the configuration of The method is based on an analysis of the NMR
chiral secondary alcohols by NMR has been satisfac- spectra of the 11 compounds of known configuration
torily accomplished through the use of the various shown in Figure 96. According to the authors, the
alternatives already discussed (see Section 3.1), a RS signs obtained are systematically distributed as
general methodology for chiral tertiary alcohols has positive and negative for substituents L1 and L2.
not yet been established. This fact is mainly due to This arrangement can be represented by the model
intrinsic problems associated with these substrates, shown in Figure 97, and it was used by the authors
such as difficulties in making derivatives (i.e., esters) for the assignment of configuration. The -methylene
in high yields and their unsuitable conformational and methyl protons directly bonded to the tertiary
characteristics. A few attempts have been made to hydroxyl group should not be considered for the
find solutions to these problems but usually the assignment.
64 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
Figure 100. (a, b) Moshers model for the correlation of the configuration with the 1H NMR shifts and (c and d) expected
signs of SR.
66 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
Figure 101. Generation of Z-conformers in MTPA amides and the most significant characteristics of each one.
The combination of these three orientations of the of the aromatic ring to the chemical shifts (aromatic
phenyl group with the two sp and ap conformers anisotropy increments) of each conformer.
generates six possible situations (as in the case of In the conformational equilibrium, each conformer
esters).27 These six possibilities can be reduced to just plays a different role (shielding or deshielding) and
three (sp1, ap3, and ap1; see Figure 101)sas in the overall result is the weighted average among the
MTPA esters27son the basis of theoretical calcula- conformer populations and their shielding/deshield-
tions (MM and AM1). The most stable conformer is ing effects. From the NMR standpoint, the L1 sub-
sp1, with the other two conformers (ap3 and ap1) stituent of an (S)-MTPA amide is shielded in con-
being energetically less stable (see Figure 102). former sp1 (see Figure 103a), while substituent L2
In the sp1 conformer, the CF3 group is syn- is virtually unaffected because it is deshielded in
periplanar to the carbonyl and the phenyl ring is conformer ap1 and shielded in conformer ap3 (Figure
coplanar with the CR-OMe bond. Overall, this ar- 103a). The magnitude of the shielding/deshielding
rangement leads to selective shielding on one of the effects on substituent L2 depends on the difference
substituents (L1/L2) of the amine (see Figure 102a). in the populations of the two conformers. Similarly,
In the other two conformers (ap1 and ap3), the CF3 in the (R)-MTPA amide, substituent L2 is shielded
is anti-periplanar to the carbonyl group. However, in conformer sp1 and substituent L1 is almost un-
in conformer ap1, the phenyl ring is coplanar with affected (deshielded in conformer ap1 and shielded
the CR-OMe bond, causing deshielding on one sub- in conformer ap3) (Figure 103b).
stituent of the amine (see Figure 102b), whereas in The overall result is that the L1 substituent is more
conformer ap3, the phenyl ring is coplanar to the shielded in the (S)-MTPA than in the (R)-MTPA
CR-CF3 bond, which produces shielding (see Figure amide. Conversely, substituent L2 is more shielded
102c). in the (R)-MTPA than in the (S)-MTPA amide. When
These shielding/deshielding effects were quantita- the differences in the chemical shifts are calculated
tively demonstrated by calculating the contributions for an amine with the configuration shown in Figure
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 67
Figure 103. Low-energy conformations of (a) (S)-MTPA and (b) (R)-MTPA, as obtained by MM and AM1 calculations.
68 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
Figure 104. (a) Low-energy conformations of the (R)-MTPA amide of (+)-bornylamine. Solid arrows show shielding, and
dashed arrows show deshielding effects. (b) Simplified models for (R)- and (S)-MTPA amides.
in shift obtained and the uneven distribution of SR Z and E rotamers result from rotation around the
signs;13,27 such problems for MTPA amides have not CO-NH bond, with the Z arrangement being the
been reported. predominant one. The CR-CO rotation generates the
two usual conformers for this type of system: sp
3.4.2. MPA (OMe and carbonyl in a syn-periplanar disposition)
and ap (OMe and carbonyl in an anti-periplanar
The application of MPA to the determination of the disposition) (Figure 106). In both the sp and ap forms,
configuration of R-substituted primary amines is a the OMe is anti, with respect to the CR-Ar bond,
very recent development.76,77 At present, there are and the phenyl group is coplanar to the CR-H bond.
two methods available for this reagent. The method
Theoretical calculations showed that the ap con-
described in this chapter involves the use of the two
enantiomers of MPA,76a whereas only one enantiomer former has a lower energy than the sp conformer (this
is necessary in the procedure described in section 4.2 is opposite to the case for MPA esters, where sp is
of this review.77 the most stable conformer). The evolution of the NMR
spectra of the MPA amide of (+)-bornylamine at
The method consists of the derivatization of the
different temperatures confirmed these conforma-
amine with the two enantiomers of MPA, acquisition
tional characteristics.
of the 1H NMR spectra of the corresponding (R)- and
(S)-MPA amides (preferably in a nonpolar solvent76a), The results of all these studies indicate that, in
and analysis of the RS parameters. solution, the MPA amides exist in a conformational
The RS signs for substituents L1/L2 are correlated equilibrium composed of two main conformers: ap-Z
to the absolute configuration of the amine through (MeO and carbonyl, anti-periplanar) and sp-Z (MeO
out a nonempirical model, which has been established and carbonyl, syn-periplanar; carbonyl and C(1)H
from a detailed study of aromatic shielding effects bond, syn-periplanar). In both cases, the methoxy
and structural calculations76a (molecular mechanics group, the carbonyl group, the N-H bond, and the
(MM), semiempirical (AM1, PM3) and ab initio), as CR-H bond are in the same plane and the phenyl
well as by NMR experiments76a (low-temperature and ring is almost coplanar to the CR-H bond (Figure
dynamic NMR). 106).
The conformational analysis shows that the main Accordingly, in (R)-MPA amides, the L2 substituent
conformational processes involve rotations around is shielded by the phenyl ring in the ap conformer
the CO-NH and CR-CO bonds (see Figure 106). Two whereas substituent L1 is shielded in the sp con-
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 69
Figure 105. SR values (ppm, CDCl3) of the (R)- and (S)-MTPA amides of a selection of amines of known configuration.
former (Figure 107a). In the (S)-MPA amides, how- The reliability of the assignment obtained from the
ever, substituent L1 is shielded in the ap conformer model shown in Figure 107c and d has been demon-
and substituent L2 is shielded in the sp conformer strated experimentally with a series of amines of
(see Figure 107b). Given that the ap conformer is known absolute configuration (see Figure 108); in all
more populated than the sp conformer, the L2 sub- cases, the signs of the RS values are consistent with
stituent is more shielded in the (R)-MPA than in the the predictions.
(S)-MPA amides and, conversely, substituent L1 is 3.4.2.1. MPA versus MTPA. When determining
more shielded in the (S)-MPA than in the (R)-MPA the configuration of a substrate, it is important to
amides. Comparison of the spectra of the two deriva- select the most suitable CDA available. In the case
tives of an amine with the configuration shown in of amines, note that there are no significant differ-
Figure 107c shows that the RS value is positive for ences in the magnitude of obtained for MPA
substituent L1 and negative for substituent L2 (with (RS) and MTPA (SR) amides, indicating that
opposite signs for the enantiomeric amine) (see neither of these reagents has a clear advantage over
Figure 107d). In practice, this method effectively the other75 (see Figure 108). This situation may seem
considers the ap conformer as a simplified model for surprising initially, because the population of the
the assignment of configuration of the amine by predominant conformer in MTPA amides is smaller
correlation of the RS signs with the configuration. than that in MPA amides. However, in the former
70 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
case, the phenyl ring is better orientated for shielding 3.4.3. AMAAs
than in MPA amides and the combination of the two
factors leads to similar shift changes. Other than MPA and MTPA, other arylmethoxy-
3.4.2.2. The Effect of the Polarity of the Sol- acetic acids with different aryl rings (9-AMA and
vent on the NMR of MPA Amides. MM calcula- 1-NMA) have been explored as CDAs for amines.76a
tions on MPA amides revealed that the dipolar The predominant conformer in the amides derived
moment of the predominant ap-Z conformer is smaller from both reagents was ap-Z (as in MPA amides).
than that of the sp-Z conformer.76a Thus, the popula- Despite the presence of an anthryl or naphthyl ring
tion of ap-Z (and, therefore, the RS values) should in these reagents, NMR experiments show that the
increase if the polarity of the NMR solvent is reduced. RS values obtained for 9-AMA and 1-NMA amides
This proposal has been experimentally proven76a are no higher than those obtained with MPA or
by acquiring NMR spectra in several solvents, which MTPA. This is probably due to a lower population of
all showed the expected trend for RS values: RS- the shielding conformer or to an unfavorable orienta-
CS2 > RSCDCl3 > RS(CD3)2CO (see Figure 109). tion of the aryl ring. In either case, the practical
Therefore, one way to increase the efficacy of MPA result is small RS values; therefore, the use of
as a CDA for amines (larger RS values) involves 9-AMA and 1-NMA as CDAs for amines does not
recording the spectra of the corresponding amides in provide any advantages over MTPA or MPA.
nonpolar solvents such as CS2.
3.4.4. Boc-phenylglycine (BPG)
A similar improvement in the RS data for MPA
esters was obtained by reducing the temperature of The use of Boc-phenylglycine (BPG; see Figure 110)
the NMR probe. This technique was described in as an auxiliary reagent78 fills a gap that neither
Section 3.1.2.8. MTPA, MPA, nor the other AMAA reagents were able
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 71
Figure 107. (a, b) Conformational equilibrium for MPA amides. (c, d) Prediction of RS signs.
Figure 108. Selected RS values (ppm) obtained from the 1H NMR spectra of (R)- and (S)-MPA amides (in CDCl3) of the
illustrated amines. For the sake of comparison, SR values obtained with MTPA are shown in parentheses.
to cover due to the small magnitude of the differences The procedure consists of the derivatization of the
between the chemical shifts (RS or SR) they amine of unknown configuration with the two enan-
generate. tiomers of BPG, recording of the 1H NMR spectra of
72 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
Figure 112. Main conformers of BPG amides: (a) sp Figure 114. Selected RS values for (R)- and (S)-BPG
conformer and (b) ap conformer. amides.
The ap conformer is the major component in the For the sake of comparison, Figure 116 shows the
mixture and, therefore, is the most significant, from values observed in a selection of BPG (RS), MPA
the NMR standpoint. As such, it can be used as a (RS), and MTPA (SR) amides derived from amines
simplified model to correlate the RS signs with the of known configuration. The data show beyond doubt
absolute configuration of the amines. This simplified that BPG produces the largest values and, hence, the
model is shown in Figure 115 and can be used to most-accurate assignments.
place substituents L1 and L2 on the amine by simply The reason for the advantage that is inherent in
considering the relevant RS signs. BPG systems, in comparison to the other reagents,
Figure 115. Simplified model for the assignment of the configuration of BPG amides.
Figure 117. Main orientation of the phenyl ring and direction of maximum shielding in (a) BPG, (b) MPA, and (c) MTPA
amides.
Figure 119. Model used to correlate the configuration with the 1H NMR shifts and RS signs.
Table 11. Comparison of Values between FDPEA and MTPA Amides
L-isoleucinol L-phenylalaninol
D-phenylalaninol,
position FDPEA MTPA FDPEA MTPA FDPEA
R-CH 0.0045 -0.0420 -0.0725 0.0400 0.0240
-CH 0.3120 0.0000
-CH2 0.2410 -0.0270 -0.2880
0.2855 -0.0010 -0.3410
-CH3 0.2260 -0.0420
-CH2 -0.43750 0.0500 -0.4825 0.0570 0.4355
-0.5085 0.0700 -0.5360 0.0540 0.4880
-CH2 0.3910 -0.0840
0.3095 -0.0900
-CH3 0.2830 -0.0560
-NH 0.0020 -0.1420 -0.0230 0.1210 -0.0695
Figure 127. (a, b) Syn and (c, d) anti rotamers for the (S)- and (R)-MTPA amides of (2S,6S)-(2-carbomethoxy-
methyl)-6-methylpiperidine.
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 79
Figure 132. Conformational equilibrium in the esters of (R)- and (S)-9-AHA with (a, b) an R-chiral carboxylic acid and
(c) predicted RS signs. Energies calculated by AM1 for (S)-2-methylbutiric acid.
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 81
Figure 134. Conformational model for the determination of the absolute configuration of R-chiral carboxylic acids by
NMR of their 9-AHA esters.
(S)-2-methylbutyric acid as the model substrate. configuration of carboxylic acids: MM and AM1/PM3
Their NMR showed that the higher RS values were methods were used to calculate the geometries and
obtained with ethyl 2-hydroxy-2-(9-anthryl) acetate relative stabilities of the conformers of the 9-AHA
(9-AHA; see Figure 131) as the auxiliary, and, esters; D NMR data were used to provide experimen-
therefore, 9-AHA was chosen for further studies as tal evidence of the theoretical findings; and aromatic
a CDA. shielding effect.87
Theoretical and experimental studies showed the The studies previously described all established
suitability of this reagent for the assignment of the that the conformational composition of any ester of
82 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
Figure 137. Representation of the direction of the maximum shielding in the esters of (a) 9-AHA and (b) 135.1. Models
for the determination of the absolute configuration on R-chiral carboxylic acids by NMR of their trans-2-phenyl-1-cyclohexanol
esters (c, d and e).
separate the resonances of the enantiomeric protons of its esters almost never overlap with those of the
of the substrates (see Figure 136). substrate, whereas the presence in 135.1 of numer-
Aromatic shielding-effect calculations corroborated ous different protons is a clear limitation in this
the shieldings obtained with this reagent. From a respect.
practical standpoint, 135.1 has an advantage over To analyze the role of the aryl ring in 135.1, the
9-AHA in that it is commercially available; on the possibility of increasing the RS values by changing
other hand, the signals of 9-AHA in the NMR spectra the aromatic ring was explored and new reagents
84 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
Figure 138. SR values obtained for enantiomeric mixtures of the acids shown, using (S)-methyl mandelate as the reagent
(chemical shift of the (S)-acid-(S)-methyl mandelate minus that of the (R)-acid-(S)-methyl mandelate).
Figure 140. (a) Structure of PGDA and PGME amides and (b) empirical model for the assignment of the absolute
configuration of carboxylic acids.
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 85
Figure 141. SR values for the PGDA and PGME amides of the carboxylic acids shown.
Figure 144. SR values for the amides of the carboxylic acids shown with PGME (obtained from the (S)-PGME amides
of the racemic acids).
Figure 148. (a) Structure of Pectenotoxin-6, (b) SR values in C5D5N, (c) SR values in CDCl3, and (d) structure of
Tanikolide lactone.
Figure 151. (a) Structure of (aR)-BNDO. (b) Ideal conformation of an (aR)-BNDO monoester. (c) Position of the acid
substituents in the diasteromeric esters. (d) Model for the assignment of configuration from the values.
Figure 152. (a) 1H aSaR values for the BNDO esters of the carboxylic acids shown. (b) 13C aSaR values.
Table 14. Values of the -Methyl and Other confirm the preponderance of the ideal conformation
Diagnostic Resonances in Diastereomeric Syn and in solution.
Anti 1-Arylethylamides from 3-Methylcarboxylic
Acids The 1H values for a very limited number (four)
of chiral acids are shown in Figure 152a. 13C
values for three of those compounds were also re-
ported (Figure 152b).
Figure 154. Conformational model for the determination of the sign of ) (syn) - (anti) of protons in R1 and R2.
Figure 155. ) (syn) - (anti) values for amides from -chiral acids with known relative configurations Ar ) 1-Np
data given in boldface type.
90 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
Figure 158.
Figure 157. RS values for the amides of the -chiral carboxylic acids tested with PGME.
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 91
Figure 164. Analysis of the diastereomeric nonequivalence in the (R)-MPA esters of the enantiomers of tanshindiol (164.1).
The shielding on the methylene protons is different in each case.
retical calculations were not performed to confirm 3.9. Application to Chiral Polyhydroxylated
this hypothesis. Compounds
This reagent/model was used to establish the In many fields of chemistrysnatural products,
stereochemistry of several natural products such as organic synthesissresearchers often must determine
158.1, which is a degradation product from a diter- the absolute configuration of substrates that have
penoid isolated from an insect wax99a (see Figure 158) several chiral centers and functional groups, such as
and the gambieric acids, which are polyethers iso- polyols. Usually, when facing these situations through
lated from marine dinoflagellates.99b In the latter the use of the NMR methods, the direct application
case, the authors found it necessary to ensure that to polyalcohols of the models originally developed for
the proposed PGME model was acceptable for these monoalcohols was the solution that was used, without
compounds by performing a conformational analysis considering that such a solution could be highly
(NOE, HOHAHA). The resulting modified conforma- inappropriate. This situation was exposed in a work13
tion is shown in Figure 159. where several examples taken from the literature
illustrate the magnitude and characteristics of the
3.8. Application to Chiral Sulfoxides: MPA problem.
In those reports, attempts were made to assign the
A procedure for the determination of the absolute absolute configuration of all the asymmetric centers
configuration of chiral sulfoxides has been devel- of a polyalcohol by application of the NMR method
oped100 and requires the introduction of a chiral to the fully esterified derivatives with (R)- and (S)-
anisotropic moiety by transformation of the sul- MTPA; however, the SR data obtained generally
foxides into N-(methoxyphenylacetyl)sulfoximines. did not allow a completely unambiguous assignment
The method consists of the amination of the sulfoxide of configuration.
with O-mesitylsulfonylhydroxylamine, which pro-
ceeds with complete retention of chirality at the S
atom, followed by introduction of (R)- and (S)-MPA
at the N atom (Figure 160).
Subsequent application of the empirical model
shown in Figure 161 allows the configuration to be
deduced on the basis of the SR values (SR ) (S)
- (R)).
The utility of this model and the reliability of the
configurational assignments are based on the coher-
ence of the NMR data obtained for the MPA-derived
sulfoximides shown in Figure 162, which are derived
from eleven chiral sulfoxides of known absolute
configuration (162.5-162.15). In addition, four ra-
cemic sulfoxides were derivatized with racemic MPA
and the NMR data of the corresponding MPA-derived
sulfoximides 162.1-162.4 were shown to produce
shifts that apparently confirmed the model: the Figure 165. Polypropionate metabolites from the marine
signs are systematically arranged on both sides of mollusk Onchidium sp. (165.1 and 165.2) and 1H NMR
chemical shifts of chiral bis-MPA esters of (2R,4R)-(-)-
the MPA plane, but the absolute configuration of each pentanediol, indicating the different shielding caused by
derivative was not available for proper validation. the auxiliary reagents.
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 93
Figure 166. (R)- and (S)-MPA diester of an anti-1,2-diol and expected RS signs. Arrows show the shielding produced
by the phenyl groups.
Figure 163 shows how the hexa-MTPA ester of In practice, the assignment of the configuration of
5-desacetylaltohyrtin A101 (163.1) generates val- compounds that have several functional groups with
ues around C-5, C-35, C-38, C-42, and C-47 that do the same reactivity (i.e. 2,3-pentanediol) can be
not present the necessary alternation of signs around addressed in two possible ways: either the appropri-
substituents L1/L2 and the same occurs with C-16, ate protection/deprotection protocols are used to
C-19, and C-24 of squamostatin D25b (19.5; see Figure achieve selective derivatization of each hydroxyl
19). Other examples of this type include annonaceous group or, more simply, the two alcohol groups are
acetogenins such as bullatacin12a,102a (rolliniastatin- derivatized together in a single reaction.
2, 163.2; see Figure 163), sootepensin A25a (19.4; see In the first case, the configuration at each asym-
Figure 19), 4-deoxyannomontacin102b (163.3); a don- metric C atom is examined separately, using the
naienin derivative102c (163.4); and sponge metabolites models developed for monofunctional compounds;
penaresidins23 (19.1 and 19.2; see Figure 19). however, the protection/deprotection operations will
This lack of homogeneity of the SR signs involves be time- and sample-consuming and this is a signifi-
a clear risk of misassignment and means that, to cant drawback.
some extent, the considerations known to be valid for There could be special cases where the reactivity
monoesters are not fulfilled in those bis- or tris- of the functional groups with the CDA is sufficiently
MTPA esters. The aforementioned article13 reminds different to achieve selective derivatization of one of
us that the fundamentals of the NMR method are the groups without protecting the other. One par-
related to aromatic shielding, which is a through- ticularly noteworthy example of this situation using
space phenomenon that affects protons several bonds MPA as the reagent is represented by tanshindiol
away from the hydroxylic C atom that has the (164.1; see Figure 164), which is a plant metabolite
auxiliary reagent. Therefore, the SR values ob- that possesses vicinal primary and tertiary hydroxyl
served in those polyderivatized compounds are, in groups.103 When both enantiomers of 164.1 were
fact, the result of the combined effects (shielding/ derivatized with (R)-MPA, single esters on the pri-
deshielding) of all the MTPA phenyl rings and cannot mary hydroxyl group were obtained and their NMR
be interpreted as if they were produced by just one were used for the assignment on the basis of the
phenyl group as in monofuntional compounds. models shown in Figure 164. Note that MPA is not a
94 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
Figure 167. (R)- and (S)-MPA diester of a syn-1,2-diol, and expected RS signs. Arrows show the shielding produced by
the phenyl groups.
reliable reagent for assignment of configuration of Years later, the correspondence between the abso-
primary alcohols (see section 3.2 of this review) and lute configuration of a series of diols of known
that its application in the case of tanshindiol (164.1) configuration and the NMR spectra of their bis-
is determined by the chelating ability of the tertiary AMAA derivatives was studied by the same group,
hydroxyl group. and a general methodology to determine the config-
In the second and more general approach, the two uration of 1,n-diols (1,2-diols, 1,3-diols etc.) from the
functional groups are made to react with the auxil- NMR spectra of the bis esters was presented.105a In
iary at the same time and the preparation of the bis this procedure, the diol is fully esterified with the R
derivatives is straightforward. However, as men- and the S auxiliary (MPA or 9-AMA) and the
tioned previously, the shifts observed in the NMR signs are interpreted as being the result of the
spectra of the fully derivatized compound (i.e., the combined action of the two auxiliary units.
bis-ester of the diol) cannot be interpreted using the
models for monofunctional compounds. New and This approach is based on the fact that the aro-
specific models that correlate the absolute stereo- matic shielding is a through-space phenomenon; each
chemistry of diols with the NMR spectra of their bis- reagent unit contributes to the chemical shift of a
AMAA esters have recently been described and will given proton in a different way (shielding or deshield-
be presented in the next section of this review. ing) and with a different strength, depending on the
actual structure of the diol. As a consequence, the
3.9.1. MPA and 9-AMA
The use of MPA on substrates that have several chemical shifts (and RS values) observed are the
chiral hydroxyl groups was first described by Riguera result of the additivity of the shielding/deshielding
et al.104 in a study on polypropionate metabolites from effects produced by all the auxiliary reagents present
the marine mollusk Onchidium sp. The configura- in the bis ester. This overall effect can be predicted
tions of the onchitriols 165.1 and 165.2 were estab- for each stereochemical combination if the conforma-
lished by comparison of the RS values of the bis- tional composition of the AMAA esters is known. In
MPA esters with those of selected diols of known fact, the predicted RS signs are fully in agreement
absolute configuration, such as (2R,4R)-(-)-pen- with the experimental results for many diols of
tanediol (Figure 165). known absolute configuration.
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 95
Figure 168. Diagnostic RS (9-AMA, MPA) and SR (MTPA) signs for the four stereoisomers of (a) 1,2-diols, (b) 1,3-
diols and (c) 1,4-diols. R3 represents the substructure between the chiral centers in diols other than 1,2 systems.
Clearly, if the experimental values were inter- diester, which makes prediction of the sign of their
preted as if they were caused by just one reagent unit RS values impossible. For its part, the CRH and
(and neglecting the effect of the other), using the CRH are shielded only in the bis-(S)-MPA-diesters;
models for monofunctional compounds, the resulting thus, a positive RS should be expected for both
assignment may be incorrect. protons CRH and CRH. Once again, if the config-
If we consider the conformational characteristics uration of the diol were the opposite, the signs also
of the AMAA esters of secondary alcohols previously would be the opposite.
discussed, and apply them to the case of a vicinal diol, The same reasoning can be used to predict the
the schematic diagrams that are presented in Figure distribution of the RS signs in other diols different
166 result. than 1,2-diols. Figure 168 shows the distinctive RS
In Figure 166a, the shielding/deshielding zones patterns of the four stereoisomers of 1,2-, 1,3-, and
originated by the aryl group of an anti-1,2-diol 1,4-diols and constitutes a guide for the assignment
derivatized as bis-MPA ester are presented: in the of the configurations.
bis-(R)-MPA ester, the R2 group and CRH are Note that, although for monofunctional compounds,
shielded, whereas in the bis-(S)-MPA ester, only the the RS signs provide sufficient information to
R1 group and CRH are shielded. Thus, negative RS distinguish between the two enantiomers (R or S),
(R,R - S,S) values should be expected for R2, and in diols with two asymmetric centers, four stereo-
CRH, and positive RS values are expected for R1, chemical situations are possible (the syn and anti
and CRH. If the configuration of the diol were the pairs: RR, SS, RS, or SR). In fact, four different
opposite, the signs also would be opposite. combinations of shielding/deshielding effectssand,
When a syn-1,2-diol is considered (Figure 167), consequently, four different sets of RS signssare
caution must be taken into consideration, because predicted and experimentally obtained for those four
only the signals due to the protons in R positions are stereochemical arrangements: one for each stereo-
useful for assignment. Figure 167 shows that R1 and isomer. Thus, the absolute configurations of the two
R2 are both shielded in the bis-(R)- and (S)-MPA chiral centers in any of the four stereochemical
96 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
Figure 170. (a) Diols used in this study. Models for the
determination of configuration of (b) syn- and (c) anti-1,2-
diols with MTPA. Only protons inside the box fit the rule
for syn diols.107
Figure 174. (a) Signs of the SR obtained for the bis-MTPA esters of syn- and anti-cyclohexane-1,3-diols used in this
study, and (b) predicted SR values.
central part). They also state that it should not be In the field of natural products, numerous ex-
applied to anti-1,3-diols, where substituents L1/L2 amples can be found of the assignment of configura-
cannot be considered and the central part is, in their tion by NMR of the bis- or tris-MTPA esters. Only in
opinion, not surely diagnostic. However, in all the a few cases have the authors based their assignment
cases, the SR signs obtained experimentally (Figure on the use of simpler synthetic diols of known
173) are in full agreement with those predicted by configuration as models for comparison. For example,
Rigueras model for those configurations as shown in Minale et al. determined the configuration of the side
Figure 168. chains of marine polyhydroxysteroids110a such as
In the same work, six-membered cyclic systems 175.1 and 175.2, using synthetic 2,3-dimethylpen-
were examined and four zones with distinct tane-1,2-diols and 2-methyl-3-ethylheptane-1,2-diols,
values, depending on the position around the ring, respectively, as models.110b
were proposed (see Figure 174). Five cyclohexane- The bis-MTPA derivatives of C27- and C29-alkane-
1,3-diols derived from dihydroxyvitamin D3 and with 6,8-diols (175.3; see Figure 175) were prepared in
known absolute configuration were examined as bis- another report;110c however, their NMR data were
MTPA esters. Figure 174a shows the experimental used simply to discriminate among stereoisomers (in
SR sign distributions and Figure 174b shows the fact, only the small shifts at Me-1 were considered!)
empirical models that have been used. and the absolute configuration was not proposed.
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 99
Figure 176. Partial 1H NMR spectra (300 MHz) of (a) the (R)-9-anthrylmethoxyacetic acid ester of alcohol 176.1; (b)
alcohol 176.1; (c) the (S)-9-anthrylmethoxyacetic acid ester of alcohol 176.1.
100 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
Figure 177. (a) Esterification shifts (AR values) measured for the (R)-9-AMA ester of 176.1. (b) Esterification shifts
(AS values) measured for the (S)-9-AMA ester. Larger shifts are highlighted.
Figure 178. AR and AS values for (R) and (S)-9-AMA esters of alcohols 178.1-178.5. (Larger values are given in
boldface type.)
If the sp/ap conformer ratio is modified in a certain stituents L1/L2, with respect to the phenyl group of
way (i.e., an increase of the sp conformer), the NMR the auxiliary (absolute configuration known), can be
spectra should translate such a change in ratio into determined by the NMR response to the disturbance
a predictable modification in the chemical shifts in the equilibrium.
associated with the substituents (i.e., greater popula- Two different procedures have been described to
tion of the sp conformer in the (R)-MPA ester leads induce the conformational change:
to greater shielding of substituent L1 in the average (a) Modification of the equilibrium by reducing the
spectrum). In this way, the spatial position of sub- temperature of the NMR probe. This approach has
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 101
Figure 179. Diagram for the assignment of the absolute configuration of a secondary alcohol from AR or AS values.
Figure 180. Esterification shifts for the (R)- and (S)-9-AMA esters of alcohols 176.1 and 178.1-178.6, expressed as mean
values (mL1 and mL2).
found application in the assignment of the absolute preparation of only one derivative has been recently
configuration of secondary alcohols using MPA as an presented by Riguera et al.35 and consists of a
auxiliary reagent (see Section 4.1.3). comparison of the NMR spectrum of the alcohol with
(b) Modification of the equilibrium by selective that of one of its esters prepared with either the (R)-
chelation of one of the conformers. This procedure has or the (S)-9-AMA chiral auxiliary.29,30 When the two
been described for the assignment of secondary resulting spectra are compared, the signals of sub-
alcohols and primary amines using MPA as an stituents L1 and L2 can be easily distinguished,
auxiliary reagent and barium(II) salts as chelators because the protons of one of those substituents (the
(Sections 4.1.4 and 4.2, respectively). one that is under the shielding cone of the anthryl
4.1. Application to r-Chiral Secondary Alcohols ring in the derivative) move upfield in the 9-AMA
ester, with respect to the free alcohol, whereas the
4.1.1. 9-AMA Esters: Esterification Shifts protons of the other substituents (unaffected by the
A procedure for the assignment of the absolute aromatic shielding) resonate at practically the same
configuration of secondary alcohols involving the field in both the 9-AMA ester and in the free alcohol.
102 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
Figure 185. (a) Correlation model for secondary alcohols as -D-glucopyranosides. (b) D - ROH values for dimethyl D-
and L-malate (in ppm, at room temperature, CDCl3).
Figure 187. (a) Structure of the sp and ap conformers of an (R)-MPA ester. (b) Chemical shifts of substituents L1 and L2
in the sp and ap conformers. (c) Average chemical shifts of L1 and L2 at room temperature (T1). (d) Average chemical shifts
of L1 and L2 at low temperature (T2).
Figure 188. Distribution of shielded and deshielded areas in the (R)- and (S)-MPA esters of (-)-menthol and selected
T1T2 values.
Figure 190. Diagram to deduce the absolute configuration of an alcohol from the experimental T1T2 signs of either its
(R)- or (S)-MPA ester.
106 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
Figure 191. Partial spectra of the (R)-MPA ester of diacetone-D-glucose at (a) 303, (b) 223, and (c) 193 K in CS2/CD2Cl2.
Figure 192. Plot of the proportional increase of RS with decreasing probe temperature T (in K) for the Me(9) group of
(-)-menthyl esters of arylmethoxyacetic acids.
of MPA esters, this is the sp conformer and, therefore, parameter is positive for substituent L1 (the group
greater shielding of the substituent located on the located on the same side as the Ph ring) and negative
same side as the Ph group in the (R)-MPA ester is for substituent L2 (the group located on the other
expected. The practical application of this approach side) and provides the configurational information.
requires the preparation of a single MPA ester (either Several alcohols of known absolute stereochemistry
the R or the S derivative) and comparison of two 1H have been used to demonstrate the general ap-
NMR spectra taken at two sufficiently different plicability of this phenomenon. Figure 188 shows the
temperatures.112 The differences in chemical shifts shielded and deshielded areas in the MPA esters of
are now measured as T1T2 (the chemical shift at the (-)-menthol. Other alcohols that have been tested are
higher temperature T1 minus that at the lower shown in Figure 189. From a practical standpoint, it
temperature T2) and obey the scheme shown in seems that temperatures lower than -70 C are not
Figure 187, where, at lower temperature, the signal necessary, and, therefore, the commonly used CDCl3
for substituent L2 moves to lower field and that of or a CS2/CD2Cl2 (4:1) mixture can be used as the
substituent L1 to higher field. The sign of this NMR solvent. The scheme shown in Figure 190
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 107
Figure 194. HF optimized geometries for the (a) sp-Ba2+ and (b) ap-Ba2+ conformers of the (R)-MPA ester of 2-propanol.
The sp-Ba2+ geometry is more stable than that of ap-Ba2+, by 13.9 kcal/mol.
108 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
Figure 196. Diagram to deduce the absolute configuration of a chiral secondary alcohol from the Ba experimental
values of one ester (either the (R)- or the (S)-MPA derivative).
amine by this method. Figure 201 shows the changes evaluation of their enantiomeric excess, and the
produced in the spectra of the (R)-MPA amide of assignment of their absolute configuration at the
L-valine methyl ester upon the addition of Ba(ClO4)2. microscale level and in a single operation.
As discussed previously for MPA esters, amines Two different cases can be distinguished:
that have functional groups in substituents L1/L2 that (a) If the absolute configuration of each enantiomer
are able to complex with the Ba2+ ion should not be in a mixture (including racemic ones) is to be deter-
assigned by this method. 4-Oxo-R-amino acids, such mined, the mixture is derivatized with either (R)- or
as 202.1 (see Figure 202), are good examples that (S)-9-AMA, according to standard procedures. The
show the effect of such competitive chelation.114 mixture of 9-AMA esters is submitted to HPLC NMR
and comparison of the 1H NMR spectra of the two
5. New Trends HPLC peaks allows the assignment of the configu-
5.1. Hyphenated High-Pressure Liquid ration based on the shielding/deshielding effects
Chromatography (HPLC) NMRMS Techniques caused in substituents L1/L2 by the auxiliary reagent
for Automatization and Microscale Analysis of known configuration (see Figure 203 and Section
The development of hyphenated techniques for 3.1.3).
rapid separation of mixtures and spectroscopic analy- (b) If assignment of the absolute configuration of
sis of its components is a topic of great interest, a single enantiomer is the objective, then the alcohol
particularly when the amount of sample is limited is derivatized with a 3:1 mixture of (R)- and (S)-9-
and the need for automatization is high. This is the AMA. The unequal mixture of 9-AMA esters is
case for high-pressure liquid chromatography nuclear subjected to HPLC NMR and the spectra of both
magnetic resonance-mass spectroscopy (HPLC NMR- peaks are compared, taking into consideration that
MS), which has been shown to be extremely useful the major component of the mixture corresponds to
in certain pharmaceutical and medical applications. the (R)-ester and the minor component corresponds
Quite recently, Riguera et al. reported115 that the to the (S)-ester (see Figure 204).
derivatization of mixtures of enantiomeric secondary In both cases, ,1 mg of the alcohol is needed for
alcohols with 9-AMA, followed by tandem HPLC derivatization and no more than 10-15 g of the
NMR, allows the separation of the enantiomers, the mixture of 9-AMA esters, dissolved in acetonitrile-
110 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
Figure 197. Partial 1H NMR spectra (250 MHz) for the (R)-MPA ester of diacetone-D-glucose (a) before and (b) after the
addition of Ba2+.
Figure 198. Main conformers present in the equilibrium of (R)-MPA amides in the absence and presence of a barium(II)
salt.
d3, are injected onto a standard reverse phase column experimental conditions and many examples of such
eluted with acetonitrile-d3/deuterium oxide/formic separation.
acid mixtures. The number and structural variety of the alcohols
The generality of this approach, including the that have been used to validate the procedure indi-
reliability of the NMR data obtained in acetonitrile- cate beyond doubt that this methodology is quite
d3/deuterium oxide/formic acid mixtures instead of general for secondary alcohols and that it can be used
deuteriochloroform, was demonstrated by its success- in the analytical and semipreparative scales. The
ful application to the secondary alcohols of known simplification involved in the use of the same auxil-
absolute configuration (205.1-205.18, shown in Fig- iary reagent for both the HPLC separation and the
ure 205, including saturated, unsaturated, aromatic, NMR configurational assignment, and its implemen-
acyclic, and cyclic compounds). tation in directly coupled HPLC NMR should be
The authors also mention that if actual isolation extremely useful for the automatization of many
of the 9-AMA esters of the enantiomeric alcohols is processes involving optically active compounds. HPLC
needed for preparative purposes or else, higher NMR has been recently used to purify and analyze
loading can be obtained when the HPLC separation the two MTPA esters of a very small sample of a
is performed with normal-phase HPLC and provides natural product.116
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 111
Figure 200. Diagram to deduce the absolute configuration of a chiral primary amine from the experimental Ba signs,
from either the (R)- or (S)-MPA amide.
112 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.
Figure 201. Partial 1H NMR spectra of the (R)-MPA amide of L-valine methyl ester in the presence (upper) and absence
(bottom) of barium perchlorate. The observed shifts are highlighted.
Figure 203. Enantioresolution and assignment of the absolute configuration of the enantiomers of a chiral secondary
alcohol by tamden HPLC NMR of its (R)-9-AMA esters (the assignment is the opposite if (S)-9-AMA is chosen as the
reagent).
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 113
Figure 204. Assignment of the absolute configuration of a chiral secondary alcohol by derivatization with a (R)-9-AMA/
(S)-9-AMA 3:1 mixture followed by tamden HPLC NMR of the diasteromeric esters.
Figure 206. MPA, MTPA, and BPG resins used in this mix and shake methodology.
volved; however, these calculations also showed that of substrate sample is available. In a paper recently
the differences in energy between the derivatives are published, the enantioresolution of secondary alco-
frequently so small that calculations can in no way hols (either an unequal or racemic mixture of enan-
replace the use of a collection of compounds of known tiomers) and the assignment of the absolute config-
absolute configuration and representative structures uration of the enantiomers is achieved in a single
as a test compounds. operation and with just a few micrograms of sub-
In regard to the substrates investigated, most of strate by tandem HPLC NMR of the 9-AMA deriva-
the efforts have been directed to monofunctional tives. In another publication by the same group, the
compounds. Secondary alcohols and primary amines simplification of the methodology is attained using
have been the most frequently investigated, and solid matrix-bound auxiliaries that allow the deriva-
although other functional groups were also studied, tization to happen in the NMR tube and within a few
those remain the most amenable to assignment by minutes. In this way, the NMR spectra can be
NMR procedures. Quite recently, an extension of the obtained after just mixing the resin and the substrate
procedure to diols has been described, which will without other operations being necessary.
surely open the way to the assignment by NMR of In summary, NMR procedures for assignment of
other polyfunctional compounds. the absolute configuration have evolved in a few
Although many procedures that are based on the decades from the original Moshers method to a
use of NMR of nucleus different from 1H (i.e., 19F, mature field, where a menu containing a large
13
C) have been described and recommended on the number of CDAs is available for the study of different
basis of the simplified NMR spectra obtained, the low substrates using a variety of approaches. New excit-
sensitivity of those nuclei to the anisotropic effects ing advances will undoubtedly emerge in the future.
produced by the CDA originate very small shifts that
overcome that advantage. Nevertheless, the simpli- 7. Acknowledgment
fication of the spectra renders those reagents very
The authors are grateful to Ministerio de Ciencia
useful when the enantiomeric excess of a mixture
y Tecnologa and Xunta de Galicia for financial
(but not the configurational assignment of each
support (currently through Grant Nos. BQU2002-
enantiomer), is the objective.
01195 and PGIDT02BTF20902PR) and to their co-
Other than papers directed to widen the scope of
workers, who have contributed to many of the articles
functional groups amenable to be studied by the
described herein.
NMR procedure, in the past few years, some efforts
have been described to simplify the method and
reduce the amount of sample. In the classical ap- 8. References
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CHIRALITY 00:000000 (2009)
Wiley-Liss, Inc.
TABLE 1. Chemical shift nonequivalence (DDd, ppm) for protons of compounds 1, 3, 4, and 5 in presence of (S)-BINOL in
different deuterated solvents at 228C (signals from protons H4 and H7 not reported due to their broadness by effect of the
benzimidazole tautomerism)
Compound Host/guest ratio H6 H5 H8A H8B H12 H13 H15 H16 H14 H17 H18
1 (CD2Cl2) 1:1 0.000 0.042 0.018 0.084 0.033 0.005 0.017 0.004
1 (CDCl3) 1:1 0.000 0.000 0.014 0.055 0.017 0.000 0.007 0.000
1 (tol-d8) 1.5:1 bd 0.007 0.000 0.040 0.020 0.014 0.022 0.004
3 (CD2Cl2) 1:1 bd bd 0.008 0.031 0.027 0.054 0.000 0.009
3 (CDCl3) 1:1 bd bd 0.000 0.025 0.016 0.050 0.000 0.005
3 (tol-d8) 4:1 bd bd 0.032 0.000 0.000 0.016 bd 0.006
4 (CD2Cl2) 1:1 bd 0.011 0.019 0.028 0.072 0.003 0.005 0.000
4 (CDCl3) 1:1 0.022 0.014 0.020 0.025 0.075 0.000 0.008 0.000
4 (tol-d8) 1.5:1 bd 0.000 0.000 0.015 0.054 0.006 0.009 0.000
5 (CD2Cl2) 1:1 bd bd 0.000 0.021 0.035 0.060 0.007 0.000 0.012 0.000 0.000
5 (CDCl3) 1:1 bd bd 0.015 0.005 0.028 0.060 0.005 0.000 0.008 0.000 0.000
5 (tol-d8) 1:1 bd bd 0.000 0.007 0.015 0.015 bd 0.000 0.015 0.000 0.000
In all cases, H8A refers to as the downfield H8 proton and H8B to the upfield H8 proton.bd, broad signal.
lack of splitting in the signals from the side chain protons In addition to the induced proton nonequivalence, the
H16, H17, and H18 suggests that this part of the molecule is formation of the host-guest complex produced changes in
far away from the binding sites involved in the enantiore- the chemical shifts of the involved molecular species. In
cognition process (in fact, only H15, which are the closest
chain protons to the pyridine ring, manifest some degree
of splitting).
The effect of the enantiorecognition was also evident in
the 19F-NMR spectrum of the CHF2 group of compound 4,
whose original doublet (JF-H: 74 Hz) slightly split in the
presence of (S)-BINOL (Fig. 5). Interestingly, no extra
splitting was observed in the 19F-NMR signal of the tri-
fluoromethyl group of compound 3 (a triplet with JF-H: 7.5
Hz), probably due to the high mobility of the OCH2CF3
chain and its relatively far distance from the sites of inter-
action with (S)-BINOL.
Clearly, among the solvents studied, toluene-d8 was the
less favorable due to the relatively low solubility of com-
pounds 3 and 4 (for those compounds, the host/guest
molar ratios achieved in toluene solution were 5.4:1 and
1.3:1, respectively, as determined by integration of the cor-
responding 1H signals).
Fig. 9. Intermolecular NOE on H03 of (S)-BINOL after irradiation of H8AB protons of esomeprazole (intramolecular NOEs on esomeprazole H13 and
H14 protons are shown).
Chirality DOI 10.1002/chir
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Method development and validation for the separation and de-
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Steve Lee
School of Science and Mathematics, Roosevelt University, Chicago and Schaumburg, IL, splee@roosevelt.edu
Received August 4, 2004. Accepted September 16, 2004.
Abstract: Moshers established technique of measuring the enantiomeric ratio of a chiral compound has been
adapted to be suitable for an undergraduate organic chemistry experiment. In this technique a chiral alcohol or
amine reacts with the Mosher acyl chloride, -methoxy--(trifluoromethyl) phenylacetyl chloride (MTPA-Cl), to
yield the corresponding diastereomeric ester or amide. The ester or amide products are analyzed by 1H and/or 19F
NMR spectroscopy, and signals for diastereotopic groups on the products are integrated to determine the ratio of
the diastereomeric products and consequently the ratio of enantiomeric substrates. This experiment has the
benefits of helping students (1) apply advanced topics in stereochemistry, such as stereoheterotopic ligands and
kinetic resolution; (2) learn a quantitative application of NMR spectroscopy, including 19F NMR; and (3) be
introduced to microscale techniques. Furthermore, even though this is a microscale NMR technique, it can be
conducted on a low-field (60- or 90-MHz) NMR spectrometer with satisfactory results.
2004 The Chemical Educator, S1430-4171(04)06843-X, Published on Web 11/16/2004, 10.1333/s00897040843a, 960359sl.pdf
360 Chem. Educator, Vol. 9, No. 6, 2004 Steve Lee
Control Test for the Possibility of Kinetic Resolution amines because of the slower rates with sterically bulky
substrates; however to help minimize the effect of the different
In Moshers method the principle reaction might undergo a rates upon the product ratio, the reaction conditions are
kinetic resolution and so must be investigated for this designed to include an excess of the Mosher acyl chloride and
possibility in a control test. Because the reaction of the two a sufficient reaction time to ensure a complete reaction for both
enantiomeric substrates with the Mosher acyl chloride leads to diastereomeric reactions.
diastereomeric products, it must be confirmed that the ratio of
diastereomeric products accurately reflects the ratio of the The Laboratory Experiment
original enantiomers. A kinetic resolution would invalidate the
Mosher method for that particular substrate. A kinetic The chemical reagents in this experiment are all commercially
resolution involves resolving a mixture of enantiomers by available, for example, from the Sigma-Aldrich Company; however,
reacting them with a chiral, nonracemic agent and not allowing because the Mosher acyl chloride is expensive, the corresponding acid
the two reactions to go to completion. The different reaction can instead be purchased at a lower cost and converted with thionyl
chloride. The procedure involves a 50-hour reflux, and so
rates of the two enantiomers lead to one diastereomeric
unfortunately it is not conducive to most student laboratory formats.
product being formed faster than the other, thereby resolving This would have to be conducted by the instructor or laboratory
the two enantiomers. A kinetic resolution is not desired in the personnel [2].
Mosher method because this would invalidate the method for Regarding alcohol and amine substrates, four compounds have
that particular substrate; however, the effect of the different been tested and found to be suitable for this technique. Their NMR
rates is minimized by using an excess of the Mosher acyl data and configurations have been reported previously [2] and are
chloride and a sufficiently long reaction time to allow both updated here. These compounds have the added benefit of having both
reactions to proceed to completion. their racemic and enantiopure forms commercially available so that
The possibility for a kinetic resolution can be considered in samples of varying enantioenrichments can conveniently be prepared.
an example with sec-butanol (4) as shown in Scheme 2. The The compounds are sec-butanol (4), -trifluoromethylbenzyl alcohol
reactions of (S)-1 with (R)-4 and (S)-4 lead to diastereomeric (5), methyl mandelate (6), and -methylbenzylamine (7). Obviously,
countless other alcohols or amines can be tested with this technique.
products. Because a pair of enantiomers is reacting with a
chiral reagent to form diastereomeric products, it is likely that H CH3 H CF3 H CO2CH3 H CH3
the two reactions will proceed with different rates (i.e., kRR will H2N Ph
HO HO Ph HO Ph
likely be different than kRS). This difference in rates could
potentially be significant enough to alter the product ratio in 4 5 6 7
which case the enantiomeric substrates would be kinetically
resolved; however, for this method to be valid, this difference The overall procedure for the experiment is logistically simple. In
in rates must not be significant enough to alter the product the first laboratory period students start their reactions by adding their
ratio. In other words, the product ratio of (R,R)-8 to (R,S)-8 reagents into a conical vial, which is left in their laboratory drawers at
needs to accurately reflect the ratio of (R)-4 to (S)-4 in order ambient temperature [5]. In the following laboratory period students
for this method to validly determine the enantiomeric ratio of 4 carry out the work-up, with no purification required, and analyze their
in the original sample. product samples by NMR spectroscopy [6]. The recommended
reaction time is 12 hours at ambient temperature, but the reaction
H O H mixture can be kept at ambient temperature for one week without a
kRR F 3C
HO O (R,R)-8 noticeable degradation of the product.
O Ph OCH3 Students can be organized into pairs or groups. One student could
F3C (R)-4 carry out the control test using a racemic sample of the substrate while
Cl + kRR kRS
the other student(s) in the group use an enantioenriched sample of the
Ph OCH3 O
H H same substrate. Students then compare each others NMR spectra with
kRS
(S)-1 F 3C
O (R,S)-8 the literature NMR data to fully assign their spectra, determine the
HO
Ph OCH3 enantiomeric ratio, and determine the configuration of the major
(S)-4 enantiomer.
A sample experiment is shown in Scheme 3. In this case, (S)-1
Scheme 2 reacts with (R)-5 and (S)-5 to yield the corresponding esters (R,R)-9
and (R,S)-9. The 1H NMR spectra in Figure 1 show the signals for the
It must, therefore, be tested whether the difference in the methoxy hydrogens in 9. (These signals are split into quartets due to
rates of the two reactions (i.e., kRR and kRS) is significant long distance 1H-19F coupling.) In the control reaction, a racemic
enough to change the product ratio of (R,R)-8 to (R,S)-8. This sample of 5 was used. The 1H NMR spectrum of its product sample
possibility of kinetic resolution can be tested by running a (Figure 1a) shows that the diastereomeric products are in nearly equal
control experiment with a racemic sample of the enantiomeric amounts (51.9% to 48.1%), which indicates that the enantiomeric
substrate. If a racemic sample of the substrate reacts with the substrates are not kinetically resolved. The slight deviation from the
expected ratio of 50:50 can be explained by the use of a sample of (S)-
Mosher acyl chloride to yield a 50:50 mixture of the two
1 that was not completely enantiopure. It was labeled with 98% e.e. as
diastereomeric products, this would confirm that our method is determined by GLC according to the commercial source. From the
valid so that the ratio of diastereomeric products accurately second reaction starting with enantioenriched 5, the relative
reflects the initial ratio of enantiomeric substrates. On the other integration in its NMR spectrum (Figure 1b) indicates a
hand, if the racemic sample of the substrate reacts to yield an diastereomeric ratio of 64.2% to 35.8%, or 28.4% d.e. for (R,R)-9 and
unequal mixture of diastereomeric products, this would consequently 28.4% e.e. (R) for 5 (d.e. = diastereomeric excess = %
indicate that the substrate is not suitable for this method. For major diastereomer % minor diastereomer; e.e. = enantiomeric
this reason, the Mosher method has generally been limited to excess = % major enantiomer % minor enantiomer). This value is
sterically-unhindered, primary and secondary alcohols and close to that (25% e.e.) based upon a mass analysis of racemic and
enantiopure samples of 5 used to prepare the enantioenriched sample.
2004 The Chemical Educator, S1430-4171(04)06843-X, Published on Web 11/16/2004, 10.1333/s00897040843a, 960359sl.pdf
A Microscale NMR Experiment for Organic Chemistry Chem. Educator, Vol. 9, No. 6, 2004 361
Table 1. Summary of NMR data for the diastereomeric products from reaction with (S)-1
Diastereomeric products Configuration of substrate Chemical shifts (ppm)
H3C H O CH3 H OCH3 CF3a
OCH3 8 R 1.255 5.096 3.564 72.00
O S 1.335 5.096 3.566 72.00
Ph CF3
F 3C H O R 6.263 3.601 76.17
OCH3 S 6.342 3.479 76.40
Ph O 9
Ph CF3
H3CO2C H O R 3.769 6.094 3.697 72.20
OCH3 10 S 3.740 6.115 3.552 72.20
Ph O
Ph CF3
H 3C H O R 1.499 5.175 3.361 69.25
OCH3 S 1.539 5.175 3.403 69.44
Ph N 11
H Ph CF3
a 19
F NMR for the trifluoromethyl group in bold with CFCl3 as the internal reference set to 0 ppm.
Figure 1. Expanded regions of the 1H NMR (60 MHz) spectra for (R,R)-9 and (R,S)-9 for the methoxy hydrogens.
The assignment of the signals for the methoxy hydrogens in the a microscale with typical product yields of 20 to 50 mg, it is still
diastereomeric products ( 3.601 ppm for (R,R)-9 and 3.479 ppm for suitable for a low-field NMR spectrometer. The product samples have
(R,S)-9) are based upon the chemical shifts reported by Mosher [2] also been analyzed with a higher-field 300-MHz instrument, which
and based upon the use of enantiopure (R)-5 (which was purchased provides much better separations of the signals. In this example, the
from the Sigma-Aldrich Company) to provide the major enantiomer in two signals are completely separated at 300 MHz.
the enantioenriched sample of 5. The consistency of these two A summary of the NMR data for all four diastereomeric products is
independent factors also helps to confirm the accuracy of the given in Table 1. The reactions were carried out with the S enantiomer
assignment. of 1. The table shows the chemical shifts for hydrogen or fluorine
atoms in the diastereomeric products. With only a few exceptions,
H CF3 O H CF3
diastereotopic groups led to different chemical shifts. This data is an
F3C (R,R)-9
HO Ph O Ph update and is consistent with that reported by Mosher [2]; however, in
O Ph OCH3 one case, Mosher reported the signals for the methine hydrogens in 10
(R)-5
F3C
Cl +
to be identical for both diastereomers. Using a 300-MHz NMR
Ph OCH3 spectrometer, however, these signals appear separately at 6.094 and
O F3C H
F3C H 6.115 ppm. Complete 1H and 19F NMR data for these compounds are
(S)-1 F3C (R,S)-9
HO Ph O Ph included in the experimental section.
Ph OCH3 This technique can be adapted into a laboratory experiment in a
(S)-5 variety of ways. In a straightforward approach, students can be given
a known substrate with an unknown enantiomeric ratio that is
Scheme 3 determined by the instructor. The students would then be required to
carry out the reaction, analyze the product sample by 1H and/or 19F
The NMR spectra shown in Figure 1 are from an upgraded Anasazi
NMR, and determine the enantiomeric ratio and the major enantiomer.
EFT 60-MHz NMR spectrometer. Although the signals are not
Alternatively, the technique can be attached to another experiment
completely separated at the baseline, the spectrum is still acceptable
involving an asymmetric synthesis or resolution. For example, the
for determining the ratio of diastereomeric products for educational
resolution of -methylbenzylamine with tartaric acid is commonly
purposes. This shows that even though the technique is carried out on
2004 The Chemical Educator, S1430-4171(04)06843-X, Published on Web 11/16/2004, 10.1333/s00897040843a, 960359sl.pdf
362 Chem. Educator, Vol. 9, No. 6, 2004 Steve Lee
used in organic chemistry courses [4]. The determination of the filtered, and concentratedmaking sure that all traces of diethyl ether
enantiomeric ratio of the resolved -methylbenzylamine has been was removed [14]. Typical yields are approximately 70 to 90% with
reported to be carried out by optical rotation [4a], chiral stationary 20 to 50 mg of product. The entire residue was dissolved in 0.5 mL
phase HPLC [7], and chiral shift reagents [8]. This Mosher technique CDCl3 with TMS and filtered directly into an NMR tube. For 19F
provides another means of determining the success of the students NMR analysis, a drop of CCl3F was added as the internal standard.
resolution of -methylbenzylamine, which has not been previously Filtration was performed with a disposable glass pipette and a
reported. The technique works particularly well with - Kimwipe plug.
methylbenzylamine because the 19F NMR signals for the NMR Data. 3,3,3-Trifluoro-2-methoxy-2-phenylpropionic acid
diastereotopic trifluoromethyl groups are very far apart providing a sec-butyl ester. [(R,R)-8]: 1H NMR (CDCl3, 300 MHz, ) 7.544
pedagogically interesting NMR experiment for observing another 7.261 (m, 5H), 5.1325.056 (m, 1H), 3.564 (s, 1H), 1.5511.737 (m,
nucleus besides 1H or 13C [9]. 2H), 1.255 (d, J = 6.2 Hz, 3H), 0.939 (t, J = 7.4 Hz, 3H). 19F NMR
(CDCl3, 56.5 MHz, ) 72.0 (m, 3F). [(R,S)-8]: 1H NMR (CDCl3, 300
Summary MHz, ) 7.5447.261 (m, 5H), 5.1325.056 (m, 1H), 3.566 (s, 1H),
1.7371.551 (m, 2H), 1.335 (d, J = 6.2 Hz, 3H), 0.828 (t, J = 7.4 Hz,
The Mosher technique provides an alternative means of 3H). 19F NMR (CDCl3, 56.5 MHz, ) 72.0 (m, 3F).
determining the enantiomeric ratio of chiral alcohols and 3,3,3-Trifluoro-2-methoxy-2-phenylpropionic acid 2,2,2-
trifluoro-1-phenylethyl ester. [(R,R)-9]: 1H NMR (CDCl3, 300
amines, which can conveniently be used as a stand-alone
MHz, ) 7.5277.250 (m, 10H), 6.263 (qt, J = 6.6 Hz, 1H), 3.601 (qt,
organic experiment or attached to another experiment
J = 1.2 Hz, 3H). 19F NMR (CDCl3, 56.5 MHz, ) 72.3 (m, 3F),
involving an asymmetric synthesis or resolution. If the
76.17 (d, J = 6.6 Hz, 3F). [(R,S)-9] 1H NMR (CDCl3, 300 MHz, )
configurations of the enantiomers have been reported, students 7.5277.250 (m, 10H), 6.342 (qt, J = 6.7 Hz, 1H), 3.479 (qt, J = 1.2
can also determine the configuration of the major enantiomer. Hz, 3H). 19F NMR (CDCl3, 56.5 MHz, ) 72.3 (m, 3F), 76.40 (d, J
This technique is pedagogically beneficial as it introduces = 6.6 Hz, 3F).
microscale techniques and integrates 1H and 19F NMR 3,3,3-Trifluoro-2-methoxy-2-phenylpropionic acid methoxycar-
spectroscopy with advanced stereochemical topics that are not bonylphenylmethyl ester. [(R,R)-10]: 1H NMR (CDCl3, 300 MHz,
always sufficiently applied in organic chemistry courses. ) 7.4357.370 (m, 10H), 6.094 (s, 1H), 3.769 (s, 3H), 3.697 (qt, J =
1.2 Hz, 3H). 19F NMR (CDCl3, 56.5 MHz, ) 72.2 (m, 3F). [(R,S)-
Experimental Section 10]: 1H NMR (CDCl3, 300 MHz, ) 7.435-7.370 (m, 10H), 6.115 (s,
1H), 3.740 (s, 3H), 3.552 (qt, J = 1.1 Hz, 3H). 19F NMR (CDCl3, 56.5
General Remarks. All chemicals were purchased from the MHz, ) 72.2 (m, 3F).
Aldrich-Sigma Company. Chloroform was dried over calcium 3,3,3-Trifluoro-2-methoxy-2-phenyl-N-(1-phenylethyl)-
hydride, or its anhydrous form was purchased and used directly. propanamide. [(R,R)-11]: 1H NMR (CDCl3, 300 MHz, ) 7.533
Pyridine was dried over KOH and distilled over BaO, or its anhydrous 7.254 (m, 10H), 7.2287.014 (m, 1H), 5.2015.151 (m, 1H), 3.361
form was purchased and used directly [10]. All other chemicals were (qt, J = 1.5 Hz, 3H), 1.499 (d, J = 6.9 Hz, 3H). 19F NMR (CDCl3, 56.5
purchased and used without further purification. The sample of - MHz, ) 69.25 (m, 3F). [(R,S)-11]: 1H NMR (CDCl3, 300 MHz, )
methoxy--(trifluoromethyl)phenylacetyl chloride purchased from the 7.5337.254 (m, 10H), 7.2287.014 (m, 1H), 5.2015.151 (m, 1H),
Aldrich-Sigma Company is labeled as having 98% e.e. as determined 3.403 (qt, J = 1.5 Hz, 3H), 1.539 (d, J = 6.9 Hz, 3H). 19F NMR
by GLC [11]. 1H NMR spectra were taken on a Bruker Avance DPX (CDCl3, 56.5 MHz, ) 69.44 (m, 3F).
300-MHz (16 scans, data size = 32K) or Anasazi-upgraded Varian
360A (60 MHz, 4 scans, data size = 16K) FT-NMR spectrometer in Acknowledgments. The author would like to acknowledge
deuterochloroform (CDCl3) with tetramethylsilane (TMS) as the
the National Science Foundation (DUE 0310353), the Max
internal standard. 19F NMR spectra (56.5 MHz, 4 scans, data size =
16K) were taken on an Anasazi-upgraded Varian 360A FT-NMR Goldenberg Foundation, and Roosevelt University for funding
spectrometer in deuterochloroform (CDCl3) with the Anasazi EFT system. He would also like to thank Professor
trichlorofluoromethane (CCl3F) as the internal standard set to 0 ppm. Jeffrey Bjorklund and North Central College for the use of
Safety and Hazards. All experiments should be performed in their Bruker Avance DPX 300-MHz NMR spectrometer.
fumehoods with suitable eye and skin protection and with proper
expert supervision. Any eye and skin contact, inhalation, or ingestion Supporting Materials. A student handout, assignment, and
should be avoided with all reagents. Chloroform is a cancer suspect experimental notes for the instructor are available in a Zip file
agent. Calcium hydride is a flammable solid and moisture-sensitive.
(http://dx.doi.org/10.1333/s00897040843a).
Pyridine is flammable and an irritant. Potassium hydroxide is
corrosive. Barium oxide is corrosive and moisture-sensitive. Diethyl
ether is flammable and toxic and can form explosive peroxides. sec- References and Notes
Butanol (4) is a flammable liquid and irritant. -
1. For reviews of methods of determining enantiomeric ratios, see (a)
Trifluoromethylbenzyl alcohol (5) is an irritant. -Methylbenzylamine
Eliel, E. L.; Wilen, S. H. Stereochemistry of Organic Compounds;
(7) is corrosive and toxic. -Methoxy--(trifluoromethyl)phenylacetyl Wiley & Sons: New York, 1994; pp 214274; (b) Rothchild, R.
chloride (1) is corrosive and moisture-sensitive. Chem. Educator [Online], 1996, S1430-4171(96)03036-11(3); DOI
General Procedure for MTPA Derivatives. Into a cleaned and 10.1333/s00897960036a; (c) Rothchild, R. Enantiomer 2000, 5 (5),
dried 3-mL conical vial were added in the following order: anhydrous 457471; (d) Shah, R. D.; Nafie, L. A. Curr. Opin. Drug Discov.
pyridine (300 L), enantiopure Mosher acyl chloride (35.4 mg, 26.2 Devel. 2001, 4, 764775; (e) Finn, M. G. Chirality 2002, 14, 534
L, 0.140 mmol) [12], anhydrous chloroform [13] (300 L), and the 540; (f) Wenzel, T. J.; Wilcox, J. D. Chirality 2003, 15, 256270.
substrate (0.100 mmol). This was capped with a silicone-rubber seal, 2. Dale, J. A.; Dull, D. L.; Mosher, H. S. J. Org. Chem. 1969, 34, 2543
stirred gently, and then left at ambient temperature in the students 2549.
drawers until the following laboratory period, usually one week. The
3. Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973, 95, 512519.
reaction solution was quenched with 1 mL H2O and taken up in 20
mL diethyl ether. The layers were separated, and the organic layer 4. (a) Ault, A. J. Chem. Educ. 1965, 42, 269; (b) Krause, J. G. J. Chem.
was washed with 5 mL each of 1 M HCl (aq), saturated Na2CO3 (aq), Educ. 1994, 71, 596.
and saturated NaCl (aq). The organic layer was then dried (Na2SO4),
2004 The Chemical Educator, S1430-4171(04)06843-X, Published on Web 11/16/2004, 10.1333/s00897040843a, 960359sl.pdf
A Microscale NMR Experiment for Organic Chemistry Chem. Educator, Vol. 9, No. 6, 2004 363
5. This first part of the experiment where students simply combine the the Mosher method. Mosher reports the use of this substrate with
reagents is brief; so, the instructor may wish to arrange the success [2].
laboratories so that students are finishing another experiment during 10. The chloroform and pyridine were dried by the instructor after which
the beginning of this laboratory period or use the time to introduce the drying apparatus was shown and the drying technique was
the Mosher method. explained to the students. Other instructors may wish to have
6. At Roosevelt University, the students analyze their samples by NMR laboratory personnel dry these reagents or simply purchase
on an Anasazi EFT upgraded 60-MHz spectrometer. Because they anhydrous samples.
have used the instrument in previous courses and/or laboratories, 11. Two different samples of the -methoxy--
they are able to obtain a spectrum quickly. The students use the (trifluoromethyl)phenylacetyl chloride are commercially available
spectrometer only to obtain a spectrum and then save and transfer the from the Aldrich-Sigma Company with 98% e.e. or 99+% e.e., as
spectra files via disk or email for processing (i.e., to zoom, integrate, determined by GLC.
print, etc) later on our science or university computers using the
NUTS software. This provides a rapid rotation of student use on the 12. Mosher [3] provides this additional note, which requires attention:
spectrometer while permitting students to access and analyze their The formation of a precipitate at this point indicates that the
spectra at a later time at their convenience. The author has also on pyridine was wet. A slight opalescence can be tolerated but a definite
occasion taken the class on a field trip to a nearby institution (North precipitate requires that the reaction can be abandoned and repeated
Central College) to use their 300-MHz FT-NMR. with dry reagents.
7. Krumpolc, Miroslav. J. Chem. Educ. 1991, 68, A176A178. 13. Mosher reports using CCl4 as the solvent, but due to its high costs,
CHCl3 has been used as the solvent with acceptable results.
8. Viswanathan, Tito; Toland, Alan. J. Chem. Educ. 1995, 72, 945946.
14. Even with thorough concentration on a high vacuum, diethyl ether
9. Another possible application involves the enzymatic resolution of - consistently appeared in the 1H NMR spectrum; thus, after initial
methylbenzylalcohol, which was recently reported as an organic concentration, the residue was dissolved in some CHCl3 and
chemistry experiment (Stetca, D.; Arends, I. W. C. E.; Hanefeld, U., concentrated again to ensure that all of the diethyl ether was
J. Chem. Educ. 2002, 79, 13511352.). Hanefield and coworkers removed. The NMR signals for diethyl ether appear adjacent to the
reported the determination of the enantiomeric excess by optical signals for the products.
rotation; however, the enantiomeric excess could be determined with
2004 The Chemical Educator, S1430-4171(04)06843-X, Published on Web 11/16/2004, 10.1333/s00897040843a, 960359sl.pdf
PROTOCOL
This protocol details the most commonly used nuclear magnetic resonance (NMR)-based method for deducing the configuration of
otherwise unknown stereogenic, secondary carbinol (alcohol) centers (R1R2CHOH (or the analogous amines where OH is replaced by
NH2)). This Mosher ester analysis relies on the fact that the protons in diastereomeric a-methoxy-a-trifluoromethylphenylacetic acid
(MTPA) esters (i.e., those derived from conjugation of the carbinol under interrogation with MTPA) display different arrays of
chemical shifts (ds) in their 1H NMR spectra. The protocol consists of the following: (i) preparation of each of the diastereomeric
S- and R-MTPA esters and (ii) comparative (DdSR) analysis of the 1H NMR spectral data of these two esters. By analyzing the sign of
the difference in chemical shifts for a number of analogous pairs of protons (the set of DdSR values) in the diastereomeric esters
(or amides), the absolute configuration of the original carbinol (or amino) stereocenter can be reliably deduced. A typical Mosher
ester analysis requires approximately 46 h of active effort over a 1- to 2-d period.
INTRODUCTION
The absolute configuration of a non-racemic sample of a chiral HO2C CF3 HO2C CF3
molecule is a fundamental and essential property of that com- H O Ph OMe MeO Ph H O
H
pound. This is especially so when the compound is of biological R2 O
CF3 2R 2S R2 O
CF3
R1 R2 OH R1
relevance (e.g., a natural product or a synthetic compound under Ph OMe CIOC CF3 R1 CIOC CF3 MeO Ph
method.
Figure 2 | Commonly encountered representations of the conformations (rotational isomers) used for the
In addition to absolute configuration, analysis of each of the diastereomeric MTPA esters 4S and 4R. The phenyl group shielding effect is
Mosher ester derivatives can be used to indicated by the gray arrows in each representation.
gain other types of stereochemical informa-
tion. Mosher showed that one can deduce
the ratio of two enantiomers in a given sample (i.e., enantiomeric dominant conformer for 4S versus that for 4R reveals a key distin-
ratio (er [major]:[minor]), sometimes expressed as the enantio- guishing feature. Protons residing within the R2 substructure of 4S are
meric excess (%ee %major %minor)) by measuring the relatively more shielded (and upfield in its spectrum) and, conversely,
relative intensities of analogous resonances (1H and/or 19F) in protons within the R1 substructure of 4R are relatively more shielded;
each of the diastereomers 4R and 4S5. (Complementary measure- this is the crux of the Mosher method3,7. Moreover, the reach of this
ment of this ratio is sometimes performed by gas or liquid anisotropic shielding effect extends a considerable distance within the
chromatographic analysis.) While valuable11, this variant is not molecule so that a fairly large number of (even remote) protons within
further discussed in this protocol, but it is mentioned here since each R group are differentially shielded in the two diastereomers; this is
one encounters language like Mosher ester analysis was used to the crux of the modified (or advanced) Mosher method10. A con-
determine the enantiomeric purity of this mixture in the literature. sequence of all of the above is that the signs of the DdSR (defined, by
The success of the Mosher ester method for deducing the absolute convention, to be dS dR8,10) values for protons residing in R1 will be
configuration of a secondary alcohol relies upon the empirically positive and those in R2 will be negative. Thus, by knowing which
based (and validated) conformational picture that is shown in protons have a positive versus a negative DdSR value, one can deduce
Figure 2. Briefly, the important conformation for each diastereomer the (sub)structures of R1 versus R2, which translates directly to the
is taken to be the one in which the ester adopts the usual s-trans absolute configuration of the secondary carbinol center in 1the
arrangement about its OCO bond (analogous to the major con- original objective of the exercise.
formation for acyclic, secondary amide CN bonds), and both the Mosher ester (and amide) analysis, especially the modified version,
trifluoromethyl (CF3) substituent of the MTPA moiety and the is remarkably powerful and general. The most complementary alter-
methine proton of the secondary alcohol moiety are syn-coplanar native, predicated on the same concepts, is the use of O-methylman-
(01 dihedral angle) with the carbonyl group. Thus, all of the under- delate esters (or methoxyphenyl acetates), which was introduced by
lined atoms in the R1R2C(H)OC(O)CCF3 substructure of Trost et al.12. Although there are instances where the Mosher method
each Mosher ester are coplanar. Although this is not the only must be applied with caution (e.g., for substrates with more than one
conformation populated in the ensemble of (rapidly interconverting) carbinol and/or amine functional groups), discussion of such subtle-
rotamers available to 4, it is the one that dominates the spectroscopic ties is beyond the scope of this protocol. Interested readers are
features that distinguish the diastereomeric MTPA esters. Or, as encouraged to consult the comprehensive review by the Riguera
Mosher and coworkers stated so clearly, these conformations are group2. Finally, note that some of the developers of more recent
intended to represent a model which successfully correlates the NMR-based methods similar to the Mosher analysis have elected to
known results. These are not intended to represent the preferred use the convention DdRS dR dS. This is opposite to the DdSR
ground state conformation of the molecules under consideration. convention used universally in Mosher analyses. One will get to the
They may in fact measure an effective average of many conforma- correct answer either way, but it is essential that users unambiguously
tions or may represent a minor conformation which, however, exerts state which way the data are being interpreted and reported.
a proportionately large differential shielding of the [R1] and [R2] The protocol detailed below consists of two main components:
groups. Admittedly the success of the correlation tends to reinforce (i) individually prepare (synthesize) each of the two diastereomeric
the belief that these do indeed represent major conformations of the MTPA esters and then (ii) comparatively analyze the 1H NMR
molecules in question, but it must still be borne in mind that the spectral data of each of the two diastereomeric MTPA esters. We
possibility exists that this is a fortuitous array which happens to serve have chosen ()-menthol (5; Fig. 3) as a representative example of
as an empirical correlation of the results7. the universe of secondary carbinols 1. Of course, the absolute
An aryl group (e.g., the phenyl substituent in each of the MTPA configuration of ()-menthol is known, but the synthesis and
esters 4) is known to impose an anisotropic, magnetic shielding effect analysis methodologies are unchanged by that fact. Given the
on protons residing above (or below) the plane of the aryl ring. This sensitivity of 1H NMR spectroscopy, it is virtually never necessary
shielding results in a more upfield chemical shift for the affected to carry out the preparation of the MTPA esters for a Mosher
(spatially proximal) proton(s) in the NMR spectrum. Inspection of the analysis on a very large scale, even when the supply of carbinol 1 is
Cl (3R) on a 50 mg scale and in option C, 6R from R-(+)-MTPA-OH performed for each of the diastereomeric MTPA esters serially
(2R) on a 10 mg scale. The interpretation of the data (described in rather than in parallel. Although this may seem like an obvious
Steps 27) is performed in an identical manner, regardless of how the point (as an interchange of the two samples will lead to the
pair of diastereomeric esters was prepared. assignment of precisely the wrong absolute configuration!), experi-
Throughout the protocol, it is essential that the individual ence teaches that it is an easy issue over which uncertainty can arise
samples, spectra and data for each of the two diastereomers never in the mind of the experimenter.
MATERIALS
REAGENTS . Silica gel thin-layer chromatography (TLC) plates (SIL G/UV254, 0.25 mm
. (R)-(+)-a-Methoxy-a-trifluoromethylphenylacetic acid (2R, R-(+)-MTPA- layer thickness; Machery Nagel, M805024)
OH; e.g., Acros) . Ceric ammonium molybdate, phosphomolybdic acid, anisaldehyde
. (S)-()-a-Methoxy-a-trifluoromethylphenylacetic acid (2S, S-()-MTPA- or potassium permanganate (e.g., TCI America, Fisher Scientific or
OH; e.g., Aldrich) Aldrich)
. (S)-(+)-a-Methoxy-a-trifluoromethylphenylacetyl chloride (3S, S-(+)- EQUIPMENT
MTPA-Cl; prepared from R-(+)-MTPA-OH5,6,13, but this reagent is also . Screw-capped glass vial or other similar reaction vessel with a
commercially available; e.g., Fluka) relatively small headspace with respect to the volume of reaction
. (R)-()-a-Methoxy-a-trifluoromethylphenylacetyl chloride (3R, R-()- solvent
MTPA-Cl; prepared from S-()-MTPA-OH5,6,13, but this reagent is also . Glass microliter syringes (Hamilton) or Wiretrols (Drummond) for
commercially available; e.g., Lancaster) measuring small volumes of liquid
. ()-Menthol (5, (1R,2S,5R)-()-2-isopropyl-5-methylcyclohexanol; e.g., Aldrich) . Common glassware (round-bottomed flask, Erlenmeyer flask, disposable
. N,N-DCC (e.g., Acros) pipets, etc.)
. Pyridine (e.g., Aldrich) . Magnetic stirrer and stir bar small enough to fit the reaction vial
. 4-Dimethylaminopyridine (e.g., Aldrich) . Rotary evaporator (Buchi)
. Chloroform-d (e.g., Cambridge Isotope Lab) . Balance (Mettler)
. Hexanes, ethyl acetate, diethyl ether, dichloromethane (CH2Cl2; standard . Handheld UV lamp (UVP)
reagent-grade laboratory solvents) . Small chromatography column
. Silica gel, bulk (typically 40- to 63-mm-diameter particle size, with nominal . NMR sample tube (5 mm diameter; New Era)
60 A pore size) . 1H NMR spectrometer (Z200 MHz; Varian, Bruker)
PROCEDURE
Individually prepare (synthesize) and collect the 1H NMR spectrum for each of the two diastereomeric MTPA esters.
1| Examples of three methods are provided as follows: (A) preparation of S-MTPA-menthyl ester 6S from ()-menthol (5)
and the R-acid chloride (3R, R-()-MTPA-Cl) on a 10 mg scale; (B) preparation of S-MTPA-menthyl ester 6S from ()-menthol
(5) and the R-acid chloride (3R, R-()-MTPA-Cl) on a 50 mg scale; and (C) preparation of R-MTPA-menthyl ester 6R from
()-menthol (5), the R-Mosher acid (2R, R-(+)-MTPA-OH) and DCC on a 10 mg scale.
(A) S-MTPA-menthyl ester 6S from ()-menthol (5) and the R-acid chloride (3R, R-()-MTPA-Cl) on a 10 mg scale
(i) Fit a screw-capped 4 ml glass vial with a Teflon-coated magnetic stir bar.
(ii) Transfer ()-menthol (5, 10.0 mg, 64 mmol) and dry pyridine (16 ml, 0.20 mmol, 3.1 equiv.) to the vial.
(iii) Dissolve the contents of the vial in anhydrous CH2Cl2 or chloroform (CHCl3) (1 ml, [5] 0.064 M). If a source of one of
these anhydrous solvents is not readily available, either can be dried using the same procedure described in the following
critical step.
m CRITICAL STEP If CHCl3 is selected as the solvent, it should first be passed through a column of fresh silica gel or
alumina immediately before use to remove the ethanol (B0.75% (vol/vol)) that is present as a stabilizer in most commercial
sources. For example, insert a small cotton plug into the neck of a disposable pipet and load the pipet with fresh silica
gel (or alumina) to a height of approximately 5 cm. Pass several milliliters of CHCl3 through this column using pressure
applied from a pipet bulb.
(iv) Add the R-()-MTPA-Cl (3R, 23 ml, 0.12 mmol, 1.9 equiv.) to the mixture. This transfer can be performed using a
microliter syringe or Wiretrol.
(v) Cap the vial and stir at ambient temperature.
(vi) Monitor the progress of the reaction by TLC analysis, by eluting with a solvent composed of hexanes and ethyl acetate in
a 4:1 ratio, and visualizing the plate by UV monitoring and/or staining with an appropriate TLC stain (e.g., ceric ammo-
nium molybdate, phosphomolybdic acid, anisaldehyde or potassium permanganate). Because they are less polar, the MTPA
esters will elute on silica gel more rapidly than the precursor carbinol (i.e., with a higher retention factor (Rf) on the TLC
plate). For the case of menthol (5) to 6R, the former has an Rf of 0.46 and the latter 0.82. The choice of TLC elution
solvent will vary depending on the polarity of the starting substrate under analysis.
(vii) After the reaction is complete (typically less than 2 h), partition the reaction mixture between diethyl ether (3 ml) and
water (1 ml).
(viii) Mix the layers thoroughly.
2007 Nature Publishing Group http://www.nature.com/natureprotocols
(ix) Separate the layers and place the ether layer into a small Erlenmeyer flask.
(x) Add 3 ml of ether to the aqueous layer that remains in the reaction vial and repeat Steps (viii) and (ix), adding this
ether layer to the original one in the Erlenmeyer flask.
(xi) Repeat Step (x).
(xii) Dry the combined ether extracts with a suitable solid drying agent (anhydrous solid Na2SO4 or MgSO4) in a 25- to 50-ml
Erlenmeyer flask. Typically, approximately 1 g of drying agent is used for the approximately 10 ml of combined organic
extracts. Swirl the suspension occasionally over a period of approximately 5 (for MgSO4) or 30 (for Na2SO4) min. In
instances where the substrate might contain acid-sensitive functionality, the use of Na2SO4 is recommended.
(xiii) Filter the ether, containing the Mosher ester, from this suspension into a round-bottomed flask (B25 ml).
(xiv) Evaporate the volatiles using a rotary evaporator under reduced pressure using a 2025 1C water bath.
(xv) Purify the product by chromatography on silica gel (any of flash14, medium pressure liquid chromatography (MPLC), HPLC,
gravity column or prep-TLC plate methods can be used), eluting with hexanes/ethyl acetate (40:1) and monitoring the
fractions using TLC (4:1, hexanes/ethyl acetate) (or another commonly available detection method like UV absorbance or
differential refractive index).
(xvi) Combine the fractions containing the purified ester product 6S and remove the solvents under reduced pressure using a
2035 1C water bath.
(xvii) If necessary or desired, remove the remaining traces of ethyl acetate and hexane elution solvents from the purified product
by attaching the sample flask to a high-vacuum source (e.g., 0.11 torr) at ambient temperature for approximately 1 h.
(xviii) Record the 1H NMR spectrum of 6S in deuterochloroform (CDCl3).
(xix) Prepare the R-MTPA-menthyl ester 6R by repeating Steps (ixvii) using S-(+)-MTPA-Cl in place of R-()-MTPA-Cl.
(xx) Record the 1H NMR spectrum of 6R in CDCl3.
(B) S-MTPA-menthyl ester 6S from ()-menthol (5) and the R-acid chloride (3R, R-()-MTPA-Cl) on a 50 lg scale
(i) Fit a screw-capped 2 ml glass vial with a Teflon-coated magnetic stir bar.
(ii) Transfer ()-menthol (5, 50 mg, 0.32 mmol) and dry pyridine (1 ml, 12.5 mmol, 39 equiv.) to the vial.
(iii) Dissolve the contents of the vial in anhydrous CDCl3 (100 ml, [5] 3.2 mM). Use of CDCl3 as the solvent allows one to
assay the reaction mixture directly by 1H NMR spectroscopy.
(iv) Add the R-()-MTPA-Cl (3R, 1 ml, 5.2 mmol, 16 equiv.) to the vial, cap the vial and stir the contents at room temperature.
(v) Perform Step 1A(vi).
(vi) After the reaction is complete (typically less than 1 h), dilute the reaction mixture with dry CDCl3 (0.6 ml).
(vii) Transfer the entire CDCl3 solution to a standard (5 mm diameter) NMR sample tube.
(viii) Record a 1H NMR spectrum.
(ix) Prepare the R-MTPA-menthyl ester 6R by repeating Steps 1B(ivii), using S-(+)-MTPA-Cl in place of R-()-MTPA-Cl.
(x) Record the 1H NMR spectrum of 6R in CDCl3.
(C) R-MTPA-menthyl ester 6R from ()-menthol (5), the R-Mosher acid (2R, R-(+)-MTPA-OH) and DCC on a 10 mg scale
(i) Fit a screw-capped 4 ml glass vial with a Teflon-coated magnetic stir bar.
(ii) Transfer ()-menthol (5, 10.0 mg, 64 mmol) and R-(+)-MTPA-OH acid (2R, 32 ml, 0.2 mmol, 3.1 equiv.) to the vial.
(iii) Dissolve the contents of the vial in anhydrous CH2Cl2 or CHCl3 (1 ml, [5] 0.064 M). If a source of one of these anhydrous
solvents is not readily available, either can be dried using the same procedure described in the critical step above,
following Step 1A(iii).
(iv) Add DCC (42 mg, 0.2 mmol, 3.1 equiv.) to the vial.
! CAUTION DCC is an irritant and can lead to sensitization. Avoid any direct contact with the skin or inhalation of particulates.
(v) Add 4-dimethylaminopyridine (25 mg, 0.20 mmol, 3.1 equiv.), tightly cap the vial and stir the contents at room
temperature.
(vi) Perform Step 1A(vi).
(vii) After the reaction is complete (typically 1224 h), filter TABLE 1 | Dd (dS dR) data for the S- and R-MTPA-menthyl Mosher
the white precipitate through a cotton plug. This solid is esters 6S and 6R.
largely the unwanted by-product, N,N-dicyclohexylurea. DdSR (dS dR)
(viii) Purify the product as described in Steps 1A(xivxvii). d S-ester d R-ester
(ix) Record the 1H NMR spectrum of 6R in CDCl3. (6S) (ppm) (6R) (ppm) ppm Hz (500 MHz)
(x) Prepare the S-MTPA-menthyl ester 6S by repeating
6ax 1.12 0.98 +0.14 +70
Steps (iviii), using S-()-MTPA-OH (2S) in place of 6eq 2.13 2.08 +0.05 +25
R-(+)-MTPA-OH (2R). 5Me 0.94 0.91 +0.03 +15
(xi) Record the 1H NMR spectrum of 6S in CDCl3. 5ax 1.54 1.52 +0.02 +10
1ax 4.90 4.88 +0.02 +10
Comparatively analyze the 1H NMR spectral data of each of 4ax 0.87 0.86 +0.01 +5
the two diastereomeric MTPA esters 4eq 1.70 1.70 0.00 0
2007 Nature Publishing Group http://www.nature.com/natureprotocols
2| Unambiguously assign as many proton resonances as 3ax 1.04 1.06 0.02 10
possible in the 1H NMR spectrum for each of the 3eq 1.67 1.69 0.02 10
diastereomeric esters 4S and 4R. 2ax 1.42 1.45 0.03 15
2Mea 0.67 0.77 0.10 50
3| Determine the difference in chemical shifts (DdSR) for 2Meb 0.74 0.87 0.13 65
each of the assignable analogous pairs of protons for the 2 1.56 1.88 0.32 160
S- and R-MTPA esters according to the following convention: For the menthyl MTPA esters 6S and 6R, all proton resonances are uniquely identifiable. However, this
need not be the case, and, for many (most?) complex structures, not every proton in the compound is
SR
Dd d(S-MTPA ester)d(R-MTPA ester) (or Dd dS SR
uniquely identifiable in its 1H NMR spectrum. In such cases, it is prudent to use only the subset of
analogous proton pairs for which unambiguous assignments are known.
dR). Gather all of the resonances of positive and, then,
negative DdSR values together. These DdSR data for the menthyl MTPA esters 6S versus 6R are presented in Table 1, listed in the
order from most positive to most negative.
4| Using the conformations shown in Figure 2 for the MTPA esters of generic secondary alcohols, decide which protons of the
esters under study are part of the R1 substituent and which part of R2. More specifically, those protons that have positive DdSR
values reside within R1, whereas those with negative values belong to R2. The structures shown in Figure 4 for the menthyl
esters 6S and 6R clearly show that the protons with positive DdSR values (i.e., those in R1) are all on one side (front) of the
plane of the MTPA moiety, whereas those with negative values are all on the opposite (back) side of that plane.
! CAUTION Occasionally, one of the proton resonances proves to be an exception to this trend. If so, it is usually for a proton
with a DdSR value that is relatively small in magnitude because this exceptional proton resides either near the plane of the
MTPA ester moiety or quite remotely. For this reason, the secondary carbinol proton itself (i.e., H1 in 6) is typically ignored in
the analysis; an important corollary is that protons having the largest DdSR values are, qualitatively, weighted very heavily in
the analysis.
5| Use the Cahn Ingold Prelog convention15 to assign the configuration of the original carbinol center as R or S. C1 of
()-menthol has the R configuration, as the substituent priority sequence is 1, OH; 2, C2; 3, C6; and 4, H.
6| Report the data. A typical chemist-friendly format for reporting the synthesis and spectral properties of a Mosher ester is
presented in Box 1. Specifically, it describes the preparation of both the S- and R-MTPA esters of ()-menthol (6S and 6R,
respectively).
7| Finally, one particularly insidious aspect of the Mosher method relates to the Cahn Ingold Prelog convention nomenclature
mentioned above and represents a potential pitfall. This problem is presented as a caveat in Box 2.
? TROUBLESHOOTING
50/65 TIMING
10
Me Steps 1A(iiv): 1520 min
H Steps 1A(v) and (vi): 13 h
Me +10 160 Me
H O H Me H H O Steps 1A(viixiv): 2040 min
Me H CF3 10 Me H CF3
O H 3 1 OMTPA O Steps 1A(xv) and (xvi): 2060 min
H 2 H
Me OMe 5
H
+25 Me MeO Step 1A(xvii): 530 min
4 6
H
Me
H
H
Step 1A(xviii): B3.5 h
15
6S
+15 H 6R Step 1A(xix): 530 min
+70
Steps 1B(iiv): 1520 min
SR = S R
Step 1B(v): 13 h
Figure 4 | Conformations used for the analysis of the menthyl esters 6S and 6R and the resulting DdSR Steps 1B(vi) and (vii): 25 min
(dS dR) values (in Hz) for each of the protons on the front and back sides of the menthyl moiety. Step 1B(viii): 540 min
J 7.0 Hz). In an entirely analogous fashion, the R-MTPA-menthyl ester 6R was prepared using S-(+)-MTPA-Cl (3S). 6R: 1H NMR (CDCl3) d 7.52
(m, 2H), 7.40 (m, 3H), 4.88 (ddd, 1H, J 4.5, 11.0, 11.0 Hz), 3.53 (q, 3H, J 1.0 Hz), 2.08 (dddd, 1H, J 2.0, 3.5, 4.0, 12 Hz), 1.88 (dsept,
1H, J 3.0, 7.0), 1.70 (dddd, 1H, J 3.5, 3.5, 3.5, 13 Hz), 1.69 (dddd, 1H, J 3, 3, 3, 13 Hz), 1.52 (ddddq, 1H, J 3.5, 3.5, 12, 12, 6.5 Hz),
1.45 (dddd, 1H, J 3.0, 3.0, 11.0, 12.5 Hz), 1.06 (dddd, 1H, J 3.5, 13, 13, 13 Hz), 0.98 (ddd, 1H, J 12, 12, 12 Hz), 0.91 (d, 3H, J 6.5
Hz), 0.87 (d, 3H, J 7.0 Hz), 0.86 (dddd, 1H, J 3.5, 12.5, 12.5, 12.5 Hz), and 0.77 (d, 3H, J 7.0 Hz).
BOX 2 | THE DIRTY LITTLE SECRET ABOUT THE ABSOLUTE CONFIGURATION OF THE
MOSHER ACID CHLORIDE
Upon conversion of the MTPA acid (MTPA-OH) to the MTPA acid chloride (MTPA-Cl), there is a switch in the relative priority of two of the groups.
Specifically, the trifluoromethyl group (CF3) is higher in priority than the carboxyl group (COOH) in the acid, but lower than the chlorocarbonyl
group (COCl) in the acid chloride. Thus, the R enantiomer of the Mosher acid (R-(+)-MTPA-OH, 2R) gives rise to the S enantiomer of the Mosher
acid chloride (S-(+)-MTPA-Cl, 3S) (and vice versa for the S acid 2S to the R acid chloride 3R). This represents a somewhat rare instance in which
the absolute configuration of a stereocenter changes as a result of a chemical reaction, even though none of the four bonds to the stereogenic
carbon was involved in the reaction.
3 2 2 3 R1 O 2
3
HO2C CF3 ClOC CF3 2 CF3
R O
Ph OMe Ph OMe Ph OMe
4 1 4 1 4 1
2R 3S 4R
R-(+)-MTPA-OH S-(+)-MTPA-Cl R Mosher ester
R Mosher acid S Mosher acid chloride
As was shown but not otherwise emphasized earlier in Figure 1, it follows that the R Mosher acid gives rise to the R Mosher ester, but that it is
the S Mosher acid chloride that gives rise to the R Mosher ester (since there is another change in relative priority: CF3 is lower than COCl but
higher than COOR). It is, of course, essential that every user of Mosher ester analysis considers this fact when performing the analysis.
Otherwise, again, precisely the opposite (and wrong) absolute configuration will be assigned16. Somewhat ironically, this potential problem has
been exacerbated by the fact that the acid chloride became commercially available in the early 1990s. That is, earlier workers would often
prepare the acid chloride from purchased, say, R acid, convert the derived acid chloride to the R Mosher ester and properly deduce the correct
absolute configuration, without even realizing that they were passing by way of the S acid chloride.
Finally, it is also important that users report the method by which they made their Mosher esters with absolute clarity, so that no doubt is
left in readers minds about the configuration of each MTPA ester. The experimental description in Box 1 represents one such unambiguous
presentation. If the publication venue does not lend itself to full experimental description, then an unambiguous statement like esterification
of # with (S)- and (R) MTPA-Cl occurred only at the C-8 hydroxy group to give the (R)- and (S)-MTPA esters #a and #b, respectively17 is to be
highly commended and recommended. All too often and in fact typically, authors are ambiguous on the issue of the precise origin of their R- and
S-esters. For example, simple statements like Mosher ester analysis led to the assignment of the R configurationy and R- and S-MTPA esters
were prepared from reaction of carbinol X with the MTPA acid chlorides abound and leave doubt. This does not mean that the method was
misused (i.e., that the user was unaware of the CIP switch), but it does (and should) erode the confidence that readers can have in the
conclusions reached. The only instances in which it is clear that an error was madeand we have found a number of published examples of
thisis where it is unambiguously stated that the R-Mosher ester was prepared using R-MTPA-Cl. Ironically, these cases are at least somewhat
self-correcting, since the informed reader will know to reverse whatever conclusions were drawn from these analyses.
The bottom line is that Mosher ester analysis is an extremely reliable method for determining absolute configuration, provided that users
be aware of the above pitfall and that they demonstrate that awareness by reporting the method of synthesis (origin) of each Mosher ester in
an unambiguously stated manner so that others will have the full measure of confidence in the conclusions.
Step 5: 5 min
Step 6: 12 h
? TROUBLESHOOTING
Troubleshooting advice regarding potential issues with the preparation, purification, and/or NMR data collection for esters 4R
and 4S can be found in Table 2.
Slow reaction Reaction solution too dilute Make sure substrate alcohol and MTPA-OH or
MTPA-Cl are present at Z0.05 M, and monitor
reaction conversion carefully by TLC analysis
Product contamination, especially for Leaching of impurities (e.g., plasticizers) Use only glass reaction vessels, pipets, etc., and
microscale from plastic labware by organic solvents Teflon rather than plastic tubing
Product contamination by excess MTPA- Need to use Z10 equiv. of MTPA-Cl because Quench the reaction mixture Me2N(CH2)3NH2 before
Cl, again, especially for microscale of limited availability of carbinol adding dilute HCl during workup3
Use stock solutions to accurately measure reagent
quantities18
Overlapping resonances complicating Overall molecular complexity and/or coinci- Carry out 2D-NMR analysis (e.g., COSY or HMQC) to
NMR interpretation dental chemical shift similarity unambiguously assign overlapping resonances
ANTICIPATED RESULTS
Each purified diastereomeric Mosher ester (4R and 4S) will be prepared and isolated in 5080% yield from the starting
carbinol 1. 1H NMR spectroscopic data of sufficient quality for subsequent Dd analysis will be obtained for each ester. After
the prescribed DdSR analysis is performed, the absolute configuration of the carbinol 1 will have been deduced.
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