Synthesis and quantitative analysis of diastereomeric linked ester conjugates with remote stereocenters using high-field nmr and chiral hplC

Article abstract of DOI:10.1002/chir.22217

A stereochemically safe high-yielding procedure for linking unprotected as well as protected hydroxycarboxylic acids to chiral secondary alcohols via glycolic acid linker is proposed. L-menthol has been linked with both enantiomers of mandelic, malic, and methoxyphenylacetic acid using bromo- or iodoacetyl group as a precursor of the glycolic acid linker. High-field nuclear magnetic resonance (NMR) and chiral high-performance liquid chromatography (HPLC) determination of high diastereomeric ratio (dr) (>99%) of the products bearing remote stereocenters was explored. Chiral HPLC allowed quantitation of the diastereomers up to dr 99.9/0.1. High-field NMR quantitation of the diastereomeric and parent alcoholic impurities in esters was demonstrated at the molar 0.3% and 0.03% levels, respectively. These analyses were done via comparison of integral intensities from major component ¹³C satellites in ¹H or even in ¹³C spectra to the ¹H or ¹³C signals of impurities. Despite lower sensitivity, the last option generally has much better selectivity. In this way the dynamic resolution is brought down by two orders. Chirality 25:793-798, 2013. 2013 Wiley Periodicals, Inc.

Full text of DOI:10.1002/chir.22217

CHIRALITY (2013)
Synthesis and Quantitative Analysis of Diastereomeric Linked Ester
Conjugates With Remote Stereocenters Using High-Field NMR and
Chiral HPLC
1
2
1
1
1
1
*
EVA DOYLE, JAAN PARVE, MARINA KUDRYASHOVA, SVEN TAMP, ALEKSANDER-MATI MÜÜRISEPP, LY VILLO,
LAURI VARES,2 TÕNIS PEHK,3 AND OMAR PARVE1
1Department of Chemistry, Tallinn University of Technology, Tallinn, Estonia
2Institute of Technology, University of Tartu, Tartu, Estonia
3National Institute of Chemical Physics and Biophysics, Tallinn, Estonia
ABSTRACT
A stereochemically safe high-yielding procedure for linking unprotected as well as
protected hydroxycarboxylic acids to chiral secondary alcohols via glycolic acid linker is proposed.
L-menthol has been linked with both enantiomers of mandelic, malic, and methoxyphenylacetic
acid using bromo- or iodoacetyl group as a precursor of the glycolic acid linker. High-field nuclear
magnetic resonance (NMR) and chiral high-performance liquid chromatography (HPLC) determi-
nation of high diastereomeric ratio (dr) (>99%) of the products bearing remote stereocenters was
explored. Chiral HPLC allowed quantitation of the diastereomers up to dr 99.9/0.1. High-field
NMR quantitation of the diastereomeric and parent alcoholic impurities in esters was demonstrated
at the molar 0.3% and 0.03% levels, respectively. These analyses were done via comparison of inte-
gral intensities from major component 13C satellites in 1H or even in 13C spectra to the 1H or 13C sig-
nals of impurities. Despite lower sensitivity, the last option generally has much better selectivity. In
this way the dynamic resolution is brought down by two orders. Chirality 00:000–000, 2013. © 2013
Wiley Periodicals, Inc.
KEY WORDS: glycolic acid linker; O-alkylation of carboxylic acid; prodrug homogeneity;
mandelic acid; lactic acid; malic acid; L-menthol
INTRODUCTION
stereoisomer.27 This value has to be at least as high as the limit
of quantitation (LOQ) for an assured analysis. Works where
high-performance liquid chromatography (HPLC)27–31 or
capillary electrophoresis32,33 (CE) have been used describe
particular efforts made in order to improve stereochemical
analysis. Chiral HPLC has been routinely used for the determi-
nation of er of enantiomeric compounds, but less often used for
the analysis of diastereomers with remote stereocenters.
Nonchiral HPLC of such conjugates is complicated due to the
low resolution of the isomers.
The use of prodrugs for optimal introduction of a drug
molecule into the target site,1–5 development of their synthesis,
and more suitable constituent combinations,1 especially within
frames of mutual prodrug concept,2 have been of continuous
interest.1–7 The compounds linked into prodrugs have a certain
bioactivity by themselves. They are bound together by linker
molecules in order to obtain better target properties, such as
improved bioavailability8 and fewer side effects.7 A variety of
moieties to be linked and linkers can be used to achieve the
desired construct, ranging from ethers and esters9,10 to
amides.11 Several prodrugs and biopolymers have been synthe-
sized by linking together chiral hydroxycarboxylic acids via
glycolic acid linker.12–14 The use of chloroacetate as a precursor
of glycolic acid linker has been reported.15 Although linking of
bile acids reported is undoubtedly a complex task, the yield of
such conjugates (27%) could still be further improved by modi-
fying the process.
Developing methods in three fields: 1) for linking together
chiral compounds, 2) for the analysis of diastereomeric ratio
(dr) of the conjugates gained, and 3) for the determination of
enantiomeric ratio (er) of chiral starting compounds forms a
symbiotic process. For instance, a number of methods based
on nuclear magnetic resonance (NMR)15–25 spectroscopy of
diastereomers are used for the determination of er and the
absolute configuration of chiral compounds.
The possibility of diastereomer differentiation by NMR
spectroscopy generally depends on the number of bonds
between the chiral centers.34,35 The selectivity for 13C nuclei
1
is usually much higher than for H. The sensitivity of both
1H and 13C NMR in the analysis of different impurities has
often been underestimated. However, recently an interesting
work has been published reporting a highly efficient and
environmentally benign procedure for fast NMR analysis of
small samples of chiral alcohols and amines.36 The authors
derivatize samples directly in the NMR tube. This technique
gives to the proposed analytical approach an additional,
highly advantageous economical dimension. Furthermore,
Additional Supporting Information may be found in the online version of this article.
Contract grant sponsor: Estonian Ministry of Education; Contract grant num-
ber: SF0140133s08, SF0690034s09, SF0180073s08, SF0140060s12, ETF8289,
ETF8880, TAR8103.
Contract grant sponsor: Archimedes Foundation; Contract grant number:
3.2.0501.10-0004 and AR12171.
* Correspondence to: L. Villo, Department of Chemistry, Tallinn University of
Technology, Ehitajate tee 5, 19086 Tallinn, Estonia. E-mail: lee@chemnet.ee
Received for publication 21 May 2013; Accepted 17 June 2013
DOI: 10.1002/chir.22217
Published online in Wiley Online Library
(wileyonlinelibrary.com).
Diastereomeric homogeneity of the target conjugates
depends on both the er of the starting compounds as well as
on the stability of the constituents that could racemize26
throughout the linking procedure. The requirements to
homogeneity of the target prodrug are stringent; the desired
level is usually set at molar 0.1% content of the undesired
© 2013 Wiley Periodicals, Inc.

DOYLE ET AL.
rate, 1 ml/min; detection, UV 254nm; the retention time of the diastereo-
mers: (14S)-9 11.06 min, (14R)-9 13.51 min.
regarding sensitivity of the NMR method, the authors suc-
cessfully demonstrated measurements of as small as <10-5 g
of a chiral sample. This significant achievement confirms that,
actually, for the quantitative analysis of trace impurities in many
systems high-field 1H NMR could be considered more suitable
than HPLC or CE, affording comparable or higher sensitivity
along with more reliable identification. The use of high-field
NMR also results in better selectivity for analysis. In the current
work, in order to enhance the reliability of the quantitation,
certain products obtained were analyzed using both chiral
HPLC and 800/200 MHz 1H/13C NMR. However, the success
of analysis of minor impurities using either high field NMR or
HPLC always depends on the structure of the diastereomers
as well as on the presence of different other trace impurities
that may cause overlapping of signals.
General Procedure A for linking
A
carboxylic acid was dissolved in acetonitrile (1 vol). N,N-
Diisopropylethylamine (DIPEA) (1.5 eq per one carboxyl group) was
added dropwise on stirring. After that, L-(–)-menthyl haloacetate (2 or
3) dissolved in EtOAc (1 vol) was added dropwise to the solution of
trialkylammonium carboxylate. The reaction progress was monitored by
thin-layer chromatography (TLC). After the reaction was complete, the
reaction mixture was diluted with EtOAc (50 mL) and the product was
washed with 5% solution of NaHCO3 (2 x 15 mL) (the product was also
washed with a 5% solution of sodium sulfite (1 x 15 mL) if iodoacetate
was linked) and with brine (2 x 15 mL). The solution of the reaction
product was then dried on anhydrous Na2SO4, filtered, evaporated, and
purified by column flash chromatography over silica gel.
General Procedure B for linking
carboxylic acid was dissolved in acetonitrile (1 vol). N,N-
MATERIALS AND METHODS
General Experimental Procedures
A
1H and 13C NMR spectra were recorded in CDCl3 solutions on Bruker
Avance 800 and 400MHz spectrometers. All signals were referenced rela-
tive to solvent signal (7.27 ppm for 1H and 77.00ppm for 13C). 2D FT
methods were used for the full assignment of NMR spectra.
Diisopropylethylamine (DIPEA) (1.5 eq per one carboxyl group) was
added dropwise on stirring. After that, L-(–)-menthyl bromoacetate 2
dissolved in acetonitrile (1 vol) was added dropwise to the solution of
trialkylammonium carboxylate. The reaction progress was monitored by
TLC. After the reaction was complete the reaction mixture was evaporated
on a rotary evaporator and the residue was taken up in EtOAc (50 mL).
The product was washed with 5% solution of NaHCO3 (2 x 15mL) and with
brine (2 x 15 mL). The solution of the crude reaction product was then dried
on anhydrous Na2SO4, filtered, evaporated, and purified by column flash
chromatography over silica gel.
Quantitation of trace impurities by 1H spectra. Analytical test procedure:
regular 99.9% quality deuterated CDCl3 and a regular quality 5 mm sample
tube was used; 128 scans were collected into 32 K data points (acquisition
time 2.5sec) with 40° pulse and 20sec relaxation delay. Free induction
decay (FID) was transformed into128K data points without any window
functions.
Quantitation of trace impurities by 13C spectra. Analytical test procedure:
on using 13C satellites for the quantitative determination of impurities, signal
from about 10mg of mixture in CDCl3 solution was collected overnight
(10,000 scans) into 128K data points (acquisition time 2.0 sec) with 30°
pulse and 2 sec relaxation delay. In order to obtain better resolution at base-
line, Gaussian multiplication of FID with LB= -0.2 Hz and GB = 0.7 were used
before FID transformation into 256K data points. For HPLC analysis a
Shimadzu Prominence HPLC set was used.
HPLC determination of the enantiomeric ratio of (S)-4/(R)-4 was
performed using a Phenomenex Lux column, eluent: 10% isopropanol/
n-hexane (isopropanol contains 0.5% TFA); flow rate, 1.0 ml/min; detec-
tion, UV 254 nm; the retention time of the enantiomers: (S)-4 8.60 min,
(R)-4 10.48 min.
RESULTS AND DISCUSSION
Herein we have focused on the base-catalyzed linking of
unprotected and protected chiral hydroxycarboxylic acids37,38
to menthol as a model compound via glycolic acid linker
(Scheme 1). A haloacetyl39,40 group attached to menthol was
used as the precursor of the linker. The synthetic goal of the
work is the development of a stereochemically safe and techni-
cally simple high-yielding protocol for the linking. The analyti-
cal goals of the work are the exploration of methods that
would allow to determine dr of the target conjugates at the
content of 0.1–0.3% of the minor diastereomer and the develop-
ment of a 1H NMR method for quantitation of traces of parent
alcohol or other impurities at molar ratio equal to or less than
0.1% in the ester.
HPLC determination of the diastereomeric ratio of (14S)-9/(14R)-9
was performed using an IA column with chiral stationary phase ”amylose
tris(3,5-dimethylphenyl-carbamate immobilized on 5μm silica gel“ (Daicel
Chiralpak IA; 0.46cm Ø / 25cm); eluent, 5% isopropanol/n-hexane; flow
Scheme 1. The synthesis of the chiral carboxylic acid conjugates.
Chirality DOI 10.1002/chir

HIGH FIELD NMR QUANTITATION OF IMPURITIES
Chirality DOI 10.1002/chir

DOYLE ET AL.
In order to estimate the scope and limitations of the
commercial (S)-MA analyzed in the form of diastereomers 9
was found to be a bit less than 0.1% (see Table 1, run 10).
Thus, the LOQ could be estimated to be between 0.1–0.3%
of the minor diastereomer (14R)-9 in the mixture of diastereo-
mers determined by chiral HPLC.
synthetic method proposed, both enantiomers of mandelic
acid (MA, 4), malic acid (8), and methoxyphenylacetic acid
(MPA, 5) were linked separately to the menthyl bromoacetate
(2) (Table 1). In addition, MA was linked in the form of
artificial mixtures of enantiomers (Table 1; runs 11–13). Also,
lactic acid (6) and 2-methoxy-2-benzylacetic acid (MBA, 7)
were linked successfully to bromoacetate 2. Depending on
the structure of the carboxylic acid, one significant difference
was observed: for the O-alkylation of dicarboxylic malic acid
(8) a higher temperature and longer reaction time were
needed to afford products in satisfactory yield. Neither
racemization nor stereochemical discrimination was noted
for the compounds in the linking process. Reaction medium
CH3CN/EtOAc (1/1; v/v) is preferable, since it affords
higher yields of the products in comparison with neat acetoni-
trile (Table 1). This could be related to better solubility of
menthyl haloacetates 2 and 3 or other components in the
mixture of the solvents. The isolated yield of the products
gained in the solvent mixture ranges from 74% to 92%
(Table 1), while for malic acid (8) the conditions should be
optimized.
For the NMR determination of the dr the use of 1H and 13
C
NMR spectra was explored. Comparison of differential 13C
and 1H shielding effects in some diastereomers in which
chiral centers are separated by six (2 C-C and 4 C-O) bonds
are presented in Table 2. Much higher selectivity of 13C
NMR for the differentiation of diastereomers was observed
and can be used for the determination of dr in cases with
not very different concentrations of diastereomers (e.g., run
7 in Table 1). For the quantitative determination of minute
diastereomeric impurities we propose to use the comparison
of 13C satellite integrals of H or even 13C NMR spectra and
1
1H or 13C signals from the impurities, respectively. In this
way the dynamic resolution is brought down by about two
orders! The 13C satellite intensity on one side of the main
1
signal is equal to 0.535% in H spectrum and (1–4)× 0.535% in
13C spectrum, the latter depending on the number of carbon
atoms directly bonded to the carbon atom under analysis.
The suitability of menthyl iodoacetate (3) for linking
(Table 1, run 3) was demonstrated. This is important because
it extends the scope of the method to chloroacetates as
precursors to glycolic acid linker that can be synthesized also
by selective enzyme-catalyzed acylation of complex mole-
cules (followed by halogen exchange).
The artificial mixtures of MA enantiomers prepared were
analyzed prior to linking, using chiral HPLC. The analysis
afforded results (up to 99.8/0.2 er) in accordance with the
amounts of (R)-MA added. The mixtures of MA were linked
with bromoacetate 2 and dr of the products was analyzed by
using chiral HPLC and 800/200 MHz NMR (Table 1, runs
11–13). The change along the dr-s determined by chiral
HPLC was found to be in line with the differences in the er
of MA starting samples. The systematic nearby constant
difference between the er values determined by chiral HPLC
for the starting MA samples and the corresponding dr values
of the products, measured by both chiral HPLC and NMR
could be explained by different elution properties of carbox-
ylic acids vs. corresponding esters. This evidently causes an
underestimation of the content of the minor R-enantiomer of
MA in the samples. The contamination of (R)-MA in the
Fig. 1. The use of 13C satellites in 200 MHz 13C NMR spectra for the deter-
mination of low concentrations of diastereomers in which chiral centers are
separated by six bonds.
TABLE 2. The differential shielding effects in the NMR spectra
δ differences (R-S) between compounds *
C atom no.
(14R)-9 (14S)-9
(14R)-10 (14S)-10
(14R)-11 (14S)-11
(14R)-12 (14S)-12
+0.009
2
3
6
7
8
9
10
11
12
13
13C
+0.018
-0.013
+0.037
+0.017
-0.009
+0.026
-0.031 n.d. n.d.
+0.042 n.d.
-0.021 -0.002
+0.018 n.d.
n.d. n.d.
13C
-0.004
13C 1H
13C 1H
13C 1H
13C 1H
13C 1H
13C
-0.021 n.d. n.d.
-0.050 +0.04
n.d. +0.005
+0.021 +0.018
n.d. -0.007
-0.031
-0.017 -0.02(eq) n.d.
-0.007 +0.04
+0.024 +0.009
+0.028 +0.027
-0.006 -0.010
-0.004
-0.011 0.004(eq) n.d.
+0.027 n.d.
n.d. +0.003
-0.012 -0.006
n.d. +0.001
n.d.
n.d.
13C 1H
13C
-0.020 +0.017 -0.002
-0.059
0.015 +0.009 -0.002
-0.117
n.d. +0.002 -0.003
+0.012
-0.024 +0.003 -0.001
-0.012
*n.d., difference was not detected, for 1H only rows, where
differences were observed, are indicated.
Chirality DOI 10.1002/chir

HIGH FIELD NMR QUANTITATION OF IMPURITIES
Fig. 2. A precise quantitation of parent menthol at molar 0.03% level in glycolic acid ester by using 800 MHz 1H NMR.
1
The use of 13C satellites in 13C spectra for quantitative
analysis seems to be an unreasonable idea, but it is an option
4. A sensitive high-field H NMR technique for quantita-
tion of parent alcoholic impurity in ester at LOQ = molar
0.03% has been demonstrated.
1
in cases where 13C satellites in H NMR spectrum cannot be
used. In modern high-field instruments about 10 mg is
enough to perform such an analysis for medium-sized
molecules. In Figure 1 quantitative analysis of diastereomeric
impurity via 13C satellites in 13C NMR spectra of (14R)-9/
ACKNOWLDGMENTS
The authors thank the Estonian Ministry of Education and Re-
search (grants: SF0140133s08, SF0690034s09, SF0180073s08,
SF0140060s12, ETF8289, ETF8880, TAR8103) and the Archime-
des Foundation (project no. 3.2.0501.10-0004 and AR12171).
1
(14S)-9 is illustrated. In H NMR spectra of diastereomers
with remote stereogenic centers it is often hard to find a good
integration region for the minor isomer. (For (14R)-9/(14S)-9
mixture the signal from H14 could be used; but in the current
case this position was overlapped by a signal from a minute
other impurity.) However, the low concentration of more
differing compounds, for instance, parent menthol in menthyl
glycolate, can be precisely quantitated at molar 0.03% level by
1H high-field NMR even despite partially overlapping signals
from H1 of free menthol and from an additional trace impurity
(Fig. 2).
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Chirality DOI 10.1002/chir