In-tube derivatization for determination of absolute configuration and enantiomeric purity of chiral compounds by NMR spectroscopy

Article abstract of DOI:10.1002/mrc.4419

We have developed an in-tube derivatization method using commercially available polymer-supported coupling agents to prepare derivatives of chiral compounds directly in NMR tube with high yield and purity. Because the method does not require any workup or purification, the configuration and enatiopurity can be quickly determined by NMR analysis for a small amount of chiral compounds, which is critical for today's fast-paced medicinal chemistry efforts in drug discovery. The application of the method was demonstrated for the derivatization of chiral amines, alcohols, diols, amino alcohols, thiols, and carboxylic acids using various chiral derivatizing agents and coupling agents. This article also serves as a practical guide for in-tube derivatization and selection of suitable chiral derivatizing agents and coupling agents for various types of chiral compounds. Copyright

Full text of DOI:10.1002/mrc.4419

Special issue research article
Received: 9 October 2015
Revised: 8 January 2016
Accepted: 27 January 2016
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/mrc.4419
In-tube derivatization for determination of
absolute configuration and enantiomeric purity
of chiral compounds by NMR spectroscopy
Jinhai Gao, Srinivasan Rajan and Bing Wang*
We have developed an in-tube derivatization method using commercially available polymer-supported coupling agents to pre-
pare derivatives of chiral compounds directly in NMR tube with high yield and purity. Because the method does not require
any workup or purification, the configuration and enatiopurity can be quickly determined by NMR analysis for a small amount
of chiral compounds, which is critical for today’s fast-paced medicinal chemistry efforts in drug discovery. The application of
the method was demonstrated for the derivatization of chiral amines, alcohols, diols, amino alcohols, thiols, and carboxylic acids
using various chiral derivatizing agents and coupling agents. This article also serves as a practical guide for in-tube derivatization
and selection of suitable chiral derivatizing agents and coupling agents for various types of chiral compounds. Copyright © 2016
John Wiley & Sons, Ltd.
Keywords: NMR; in-tube derivatization; polymer-supported coupling agents; absolute configuration; enantiomeric purity
polymer-supported CDAs for purification free derivatization of
Introduction
chiral substrates have been reported.[2,3] The chiral substrates react
More than half of the drugs in use and most of the drugs in
development nowadays are chiral molecules. Fast and efficient
methods for the determination of absolute configuration and enan-
tiomeric purity of chiral compounds have become increasingly im-
portant in supporting large number of asymmetric syntheses in
modern medicinal chemistry labs. While X-ray crystallography has
been a straightforward tool for the determination of absolute config-
uration, the requirement of a single crystal limits the routine applica-
tion of this method. Chiral chromatography is currently most often
used for chiral analysis. However, the application is impeded by
the amount of time and resources required to develop and optimize
the chiral separation method and the lack of reference compounds
for many new chemical scaffolds. Optical spectroscopy, such as cir-
cular dichroism, optical rotation, and vibrational circular dichroism,
has been also a useful tool for chiral analysis with some limitations.
Methods based on NMR spectroscopy for the analysis of chiral
molecules are very appealing because the instrument is readily
available and only a small amount of sample is needed for modern
NMR instruments. NMR analysis via chiral derivatization has the ad-
ditional benefit that it can provide accurate information for both
the absolute configuration and enantiomeric purity of chiral com-
pounds. In order to achieve the necessary chiral discrimination in
NMR spectra of enantiomers, chiral derivatizing agents (CDAs) have
been widely used to form diastereomeric derivatives for various
types of chiral molecules.[1] However, the synthesis of diastereo-
meric CDA derivatives requires a series of time-consuming steps
including reaction workup, separation, and purification. The applica-
tion of the CDA-based method has not been routinely used in
today’s fast-paced medicinal chemistry laboratories because of the
amount of material and time required to prepare the derivatives.
To avoid some of the disadvantages associated with the
preparation of the CDA-substrate derivatives, methods using
with the polymer-supported CDAs to produce CDA derivatives in
NMR solvent, and the NMR spectra can be collected without any
further manipulations. The drawbacks of this method include the
significant development time required to optimize the CDA-resin
properties (e.g. stability and swellability in NMR solvent, reactivity,
and regioselectivity with the chiral sustrate), and limited choices
of CDAs that can be applied. We have successfully implemented
the ‘mix and shake’ method with methoxyphenylacetic acid
(MPA) as the CDA,[3,4] but it did not always give satisfactory results
when we tried to expand the method to some other CDAs [e.g.
methoxytrifluoromethylphenylacetic acid (MTPA)]. Because of the
lack of commercial availabilty of polymer-supported CDAs, this
method does not offer the speed and flexibility needed in a drug
discovery enviroment.
Herein we report that by switching the polymer-supported re-
agent to the coupling reagent, rather than the CDA, a more versatile
and efficient method to carry out the derivatization procedure di-
rectly in NMR tube is achieved. This in-tube derivatization method
does not require the preparation of CDA-resins beforehand; as a re-
sult, we can screen many types of CDAs with a range of commer-
cially available polymer-supported coupling reagents in order to
obtain optimal chiral discrimination and accurate enatiopurity mea-
surements for various types of chiral compounds.
*
Correspondence to: Bing Wang, Global Discovery Chemistry, Novartis Institutes for
Biomedical Research, Inc., Cambridge, MA 02139, USA.
E-mail: bing.wang@novartis.com
Global Discovery Chemistry, Novartis Institutes for Biomedical Research, Inc.,
Cambridge, MA, 02139, USA
Magn. Reson. Chem. (2016)
Copyright © 2016 John Wiley & Sons, Ltd.

J. Gao, S. Rajan and B. Wang
main reason for incomplete conversion and long reaction time. This
can lead large errors in enantiomeric purity measurement because
of kinetic resolution. The water impurity could come from all the
components that are added in the NMR tube, and it is impractical
to keep all the materials completely dry before the derivatization
procedure. We found that adding some molecule sieve beads di-
rectly into the NMR tube is very effective to remove small amount
of water from the reaction mixture. The molecular sieves will either
stay at the bottom of NMR tube or float with the resin on CDCl3
without interfering with the NMR data collection.
Racemization of either CDA or the chiral substrate is another
important concern for the in-tube derivatization procedure. Fortu-
nately, many more efficient coupling agents have been developed
and become commercially available in polymer-supported format.
The flexibility in selection of coupling agents and CDAs for the in-
tube derivatization procedure allows the optimization of reaction
conditions so that racemization or other side reactions can be
avoided or minimized. Examples will be discussed in the application
sections to demonstrate how the racemization products were easily
identified and quantified by NMR, and the reaction conditions were
optimized to minimize the racemization.
Results and Discussion
General methodology and procedure of in-tube derivatization
method
Most commonly used CDAs have carboxylic acid groups, which will
be linked to the functional groups from chiral substrates. The syn-
thesis is usually initiated by an coupling reagent forming a reactive
intermediate with the CDA. The chiral substrate acts as a nucleo-
phile attacking the reactive intermediate to form the desired CDA
derivative. The development of in-tube derivatization method is
based on the following ideas: (i) the polymer-supported coupling
agent is used for the coupling of CDA and chiral substrates directly
in NMR tube in deuterated solvent; (ii) the polymer-supported cou-
pling agent and any by-products remain out of the solution (afloat
on top of the NMR solvent such as CDCl3) and the NMR detection
coil; and (ii) clean NMR spectra of the desired derivatives can be ob-
tained directly in the same NMR tube without any other
manipulations.
Because the NMR spectra are collected directly in the NMR
tube without any separation or purification, complete conversion
of the original chiral compounds is important in order to avoid
signal overlaps between any unreacted compounds and the de-
sired products. Peak overlapping makes it difficult to accurately
measure the chemical shifts and integrals, which are critical for
reliable determination of absolute configuration and enantio-
meric purity. The complete conversion is also critical for accurate
measurement of the enantiomeric purity because of the effect of
kinetic resolution. If the conversion is incomplete, the ratio of the
diastereomeric CDA derivatives will be affected by the relative
reaction rate of enantiomers with the CDA, potentially resulting
in the kinetic resolution of the products, leading to inaccurate
chiral analysis. The kinetic resolution will have no influence on
the final ratio of CDA derivatives provided that all chiral sub-
strates are completely converted. The real-time monitoring of
the in-tube derivatization procedure directly in NMR tube is
superior to the traditional synthesis approach, because it ensures
the complete conversion of the chiral substrate. The purification
procedure involved in the traditional synthesis also introduces
errors in the measurements because of the various degree of
sample loss.
In order to achieve complete transformation of the original chiral
samples, excess amount of CDA and coupling agent are used for
the in-tube derivatization procedure. The extra CDA reacts with
the excess amount of coupling agent on the resin and remain out
of the NMR detection coil. The typical procedure for in-tube deriva-
tization can be described as follows: (i) appropriate amount of
polymer-supported coupling agents (2 equiv relative to chiral sub-
strate) is weighed in NMR tube and NMR solvent (e.g. CDCl3) was
added to soak the resin. (ii) The CDA (1.5 equiv) is added and mixed
with the resin. (iii) The chiral substrate (1.0 equiv) is then added, and
the mixture is shaken either by hand or on a shaker if long reaction
time is required. (iv) NMR spectra are collected directly from the
same NMR tube to monitor the reaction until it is completed. With
the advantages of complete conversion and no sample loss during
the in-tube derivatization procedure, the amount of chiral com-
pound required is minimal as long as good quality 1H NMR spectra
can be obtained. On a modern high field NMR instrument with
cryo-probes, this can be achieved for as low as micrograms of
samples.
In-tube derivatization of chiral amines
Methoxytrifluoromethylphenylacetic acid (MTPA),[5,6] MPA,[7] and
Boc-phenylglycine (BPG)[8] (Scheme 1) are popular CDAs for chiral
NMR analysis of amines and alcohols. Polymer-supported
dicyclohexylcarbodiimide (PS-DCC) has been widely used as the
coupling agent for the preparation of CDA derivatives.[9] The in-
tube derivatization procedure was successfully carried out for the
transformation of amine 1 (Scheme 2) to (R)- and (S)-MPA amides
1
using PS-DCC. Monitoring the H NMR directly in the tube (Fig. 1
(a)), we observed full conversion in about 15 min. The absolute con-
figuration and enantiomeric purity (98% measured vs 99% re-
1
ported) of 1 can be determined from the H NMR spectra of (R)-
and (S)-MPA amides (Fig. 1(b)). Racemization and kinetic resolution
are rarely observed for derivatization of primary amines as the com-
plete conversion can be easily achieved in a very short period of
time. Excellent results were also obtained for the in-tube derivatiza-
tion of chiral amine 1 and 2 using MTPA and BPG as the CDA.
MTPA has been used as CDA for the configurational assignments
of cyclic secondary amines.[10,11] Because of the steric hindrance be-
tween secondary amine and MTPA, extended reaction times are re-
quired to reach full conversion. The transformation of secondary
amine 3 with (R)- and (S)-MTPA was completed within 2 h using
PS-DCC without any noticeable racemization. It is worth noting that
MTPA derivatives of secondary amines with very bulky subsitutions
are very difficult to make from our experience using the mix and
shake method. As an alternative to MTPA, we have used MPA suc-
cessfully to prepare the amide derivatives of a sterically hindered
During the optimization of the in-tube derivatization procedure,
we found that the presence of water in the reaction mixture is the
Scheme 1. Structures of CDAs.
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In-tube derivatization for determination of absolute configuration and enantiomeric purity of chiral compounds by NMR spectroscopy
esters were achieved within 1–3 h without racemization, allowing
the accurate determination of chiral purity of the original chiral al-
cohols. Figure 2(a) showed the transformation of 4 with (R)-MPA
as monitored by NMR. The enantiomeric purity determined from
the ratio of diastereomeric (R)- and (S)-MPA esters of 4 was 90%
(Fig. 2(b)), which is in good agreement with the purity of 91% re-
ported from the supplier . The enantiomeric purity of 5 was 98%
from NMR analysis versus the reported purity of 99.5% .
O-Acetylmandelic acid (OAM) has been reported to be a more
reliable CDA for the configurational determination of some chiral al-
cohols, especially sterically crowded alcohols.[12] In-tube derivatiza-
tion of 4 and 5 with OAM was conducted using PS-DCC/DMAP.
However, enantiomeric purities measured from the OAM esters
prepared by PS-DCC were much lower (80% for 4; 86% for 5) than
those measured from MPA esters, suggesting that racemization of
OAM (supplied chiral purity 98%) occurred under the reaction con-
dition. In search for an alternative derivatization method, we tested
Scheme 2. Structures of compounds 1–12.
cyclic sencodary amine and established the conformational model
and correlation between the large shielding effects and the
configuration.[4]
the more activated polymer-supported N-alkyl-2-chloro pyridinium
triflate (PL-Mukaiyama).[13] For the derivatization of alcohols using
PL-Mukaiyama, an access amount of diisopropylethylamine (DIEA,
2 equiv) and a catalytic amoun of DMAP (0.2 equiv) were used to
facilitate the coupling reaction. We tested different reaction condi-
tions using either PS-DMAP with free DIEA or PL-DIEA with free
DMAP and found that the later combination gave better yield and
shorter reaction time with additional advantage that only small
NMR signals from free DMAP are present in the final 1H NMR spec-
tra. The enantiomeric purities determined from the OAM esters pre-
pared by PL-Mukaiyama were consistent with those reported for 4
(88% vs 91%) and 5 (96% vs 99.5%). It is recommended that PS-DCC
should be avoided when OAM is the preferred CDA for in-tube
derivatization.
In-tube derivatization of chiral alcohols
Derivatization of alcohols is more difficult than amines, so
4-(dimethylamino)pyridine (DMAP) is commonly used as a catalyst
in the coupling reaction. Although the NMR signals of free DMAP
are present in the NMR spectrum, these signals can be easily iden-
tified and usually do not interfere with the NMR data analysis. PS-
DCC resin with DMAP (0.2 equiv) was successfully employed for
the in-tube derivatization of 4 and 5 with (R)- and (S)-MPA. The
complete transformations of 4 and 5 to the corresponding MPA
Figure 1. (a) Transformation of 1 with (R)-MPA using PS-DCC monitored by
NMR. (b) 1H NMR spectra of (R)- and (S)-MPA amides of 1.
Figure 2. (a) Transformation of 4 with (R)-MPA using PS-DCC monitored by
NMR. (b) 1H NMR spectra of (R)- and (S)-MPA esters of 4.
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J. Gao, S. Rajan and B. Wang
Figure 3. Transformation of 6 with (R)-MPA using PS-DCC monitored by
NMR.
In-tube derivatization of chiral carboxylic acids
Phenylglycine methyl ester (PGME)[14,15] and 1-(9-anthryl)-2,2,2-
trifluoroethanol (TFAE)[16] have been used as the CDAs for NMR
analysis of chiral carboxylic acids. Different from the in-tube deriva-
tization procedure discussed in the preceding texts, the chiral car-
boxylic acid now first reacts with the polymer-supported coupling
agent to form the active intermediate, and the CDAs (PGME is an
amine and TFAE is an alcohol) act as nucleophiles during the cou-
pling reaction. The modified procedure was used for the prepara-
tion of PGME amide of 6 and TFAE ester of 7 using PS-DCC.
Figure 3 shows the transformations of 6 to the corresponding (R)-
PGME amide using PS-DCC. We used the same equivalence of
CDA in this example to evaluate the extent of conversion while min-
imizing the NMR signals from unreacted PGME. The conversion of
chiral carboxylic acids 6 was estimated to be >90%, which is suffi-
cient for configuration determination but may cause error for
enatiopurity measurement because of potential kinetic resolution.
The real-time NMR monitoring during the in-tube derivatization
procedure not only provide quantitative measurement of reaction
components but also provide detailed structural information and
understanding of the reaction mechanism to guide the optimization
of reaction conditions. This was demonstrated by the in-tube
derivatiozation of chiral carboxylic acid 8, which is prone to
epimerization and isomerization. Not surprisingly, when PS-DCC
was first used for the in-tube derivatiozation of 8 with (S)-PGME, a
mixture of three components with ratio of 4 : 3 : 3 were observed
in 1H NMR spectrum (Fig. 4(a)). Detailed analysis of the NMR data re-
vealed that the mixture is the (S)-PGME esters of the following prod-
ucts: the original (S)-8, racemization product (R)-8, and the olefin
isomer 13 (Scheme 3). The racemization was believed to occur dur-
ing the activation step of the carboxylic acid with DCC. 2-Isobutoxy-
1-isobutoxycarbonyl-1,2-dihydroquinoline (IIDQ) has the advantage
over conventional carbodiimide-based coupling reagents that no
pre-activation step is required, and therefore, the acid can be added
at the last step in order to minimize the chance of racemization.
When PL-IIDQ[17] was used as the coupling agent for the in-tube
Figure 4. (a) 1H NMR spectrum of in-tube derivatization products of 8 and
(S)-PGME using PS-DCC. (b) 1H NMR spectra of (R)- and (S)-PGME amides of 8.
derivatization of 8, a single product was obtained for each (R)- and
(S)-PGME amides of 8 (Fig. 4(b)), allowing unambiguous determina-
tion of the configuration and chiral purity of 8. PL-IIDQ proved to be
a very efficient coupling agent with minimal racemization of the chi-
ral carboxylic acid. The drawback is the release of isobutanol, which
will show NMR signals when it is used for in-tube derivatization.
In-tube derivatization of other chiral functional groups
The CDA method has been also applied to other chiral functional
groups such as thiol, diol, and amino alcohol. The in-tube derivati-
zation of thiol (9), cyanohydrin (10), and diol (11) with MPA using
PS-DCC was carried out successfully with excellent yield. Interest-
ingly, we found that the derivatization of amino and hydroxyl
groups in 12 with MPA can be achieved in two separate steps. First,
the amine group was transformed to MPA amide using PS-DCC by
following the derivatization procedure for primary amines. The
amidation of amine group was selective, and no transformation of
hydroxyl group was detected in 1H NMR spectrum. The configura-
tion of the chiral amine can be determined by comparison of the
NMR spectra of (R)- and (S)-MPA amides. Second, the same NMR
sample with either (R)- or (S)-MPA amide was further transformed
to MPA esters of the hydroxyl group when additional MPA and
PS-DCC/DMAP were added to the NMR tube. The configuration of
the hydroxyl group can be determined by comparison of the
NMR spectra of (R)- and (S)-MPA esters. This step-by-step derivatiza-
tion method avoid the complication of analyzing the bis-MPA deriv-
atives for which the chemical shift changes are complicated by the
‘combined shielding effects’ of MPA amide and ester.[18]
Conclusion
A fast and efficient in-tube derivatization method has been devel-
oped for the determination of absolute configurations and enantio-
meric purity of chiral compounds. Common problems such as
Scheme 3. Racemization and olefin isomerization of 8 during amide
coupling with (S)-PGME using PS-DCC.
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In-tube derivatization for determination of absolute configuration and enantiomeric purity of chiral compounds by NMR spectroscopy
racemization and incomplete conversion during the coupling reac-
tion were overcome by optimization of the reaction conditions and
selection of optimal coupling agents. The application and optimiza-
tion of the in-tube derivatization method were successfully
demomstrated for various types of chiral functional groups and
coupling agents in CDCl3. Other NMR solvents such as
dicholoromethene-d2 and aceton nitrile-d4 have been commonly
used in resin-based synthesis and are also good choices for the in-
tube derivatization. The procedure is easy to implement and can
be conducted quickly in any chemistry or analytical NMR laboratories.
With the availability of today’s highly sensitive NMR instruments, the
method can be used to determine the absolute configuration and
enantiomeric purity of chiral compounds at microscale level.
until it is completed. For the derivatization of alcohol, DMAP
(0.002 mmol, 0.2 equiv) was added to catalyze the reaction.
In-tube derivatization using PL-Mukaiyama
Nineteen milligrams of PL-Mukaiyama (0.02 mmol, 1.1 mmol/g
loading, 2 equiv) with PL-DIEA (0.02 mmol, 6 mg, 3.5mmol/g load-
ing, 2 equiv) was added in a 5 mm NMR tube, and 0.6ml of CDCl3
was added to soak the resin. DMAP (0.002 mmol, 0.2 equiv) was
used to catalyze the derivatization of alcohol (0.01 mmol, 1 equiv)
in CDCl3, following the same procedure as described in the preced-
ing texts.
In-tube derivatization using PL-IIDQ
Twelve milligrams of PL-IIDQ (0.02 mmol, 1.7mmol/g loading, 2
equiv) was weighted in a 5 mm NMR tube, and 0.6ml of CDCl3
was added to soak the resin for 10 min. PGME (0.01 mmol, 1 equiv)
was added followed by carboxylic acid 8 (0.01 mmol, 1 equiv). The
reaction was monitored by 1H NMR spectroscopy until it’s
completed.
Experimental
Materials
All chiral amines, alcohols, carboxylic acids, and chiral derivative
agents were ordered from Sigma-Aldrich or Fluka (Milwaukee, WI).
PGME was supplied as HCl salt, which was removed by using PL-
HCO3 MP cartridge. Polymer-supported coupling agents PS-DCC
and PS-DMAP were purchased from Biotage (Biotage AB, Sweden).
PL-Mukaiyama, PL-IIDQ, PL-DIEA were purchased from Polymer
Laboratories (Amherst, MA). CDCl3 was purchased from Cambridge
Isotope Laboratories (Andover, MA).
Acknowledgement
We greatly appreciate Dr Kian Tan for his insightful advices and
careful review of the manuscript.
NMR spectroscopy
References
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TCI probehead with z-gradient. All spectra were recorded at 300 K,
and the chemical shifts for 1H NMR spectra were referenced to in-
ternal tetramethylsilane at 0 ppm.
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In-tube derivatization using PS-DCC
Seventeen milligrams of PS-DCC (0.02 mmol, 1.2mmol/g loading, 2
equiv) was weighted in a 5 mm NMR tube, and 0.6ml of CDCl3 was
added to soak the resin for 10 min. The cleanness of the resins can
1
be checked by H NMR spectrum, and the resins can be washed
with additional CDCl3 to remove any impurities from the resin if
necessary. The carboxylic acid CDA (0.015 mmol, 1.5 equiv) in CDCl3
solution was added and mixed with the resin for 10 min. Chiral
amine or alcohol substrate ( 0.01 mmol, 1 equiv) in CDCl3 solution
was added, and the NMR tube is shaken either by hand or on a
shaker if long reaction time is necessary. NMR spectra were
collected directly from the same NMR tube to monitor the reaction
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