Research Article
A Conformational Model for MTPA Esters of
Chiral N-(2-Hydroxyalkyl)acrylamides
Eduardo M. Rustoy,1 Alicia Baldessari,1 and Leandro N. Monsalve1,2
Laboratorio de Biocata´lisis, Departamento de Quı´mica Orga´nica y UMYMFOR, Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, Pabello´n , Piso , Ciudad Universitaria, CEGA Buenos Aires, Argentina
INTI-CONICET, Avenida Gral. Paz , Ed. , San Mart´ın, BJKA Buenos Aires, Argentina
Correspondence should be addressed to Leandro N. Monsalve; monsalve@inti.gob.ar
Received May ; Revised July ; Accepted July ; Published August
Academic Editor: Daniel Glossman-Mitnik
Copyright © Eduardo M. Rustoy et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
e absolute stereochemistry of novel chiral N-(-hydroxylalkyl)acrylamides prepared by a lipase-catalyzed resolution was
successfully determined by 1H NMR of their MTPA esters. e method was validated for this particular case by computational
experiments.
1. Introduction
It is well known that chiral acrylamides are useful compounds
in organic synthesis [–].
e absolute stereochemistry of chiral compounds is
determined by using several methods []. Among the meth-
ods available, NMR spectroscopy using chiral derivatizing
agents such as -methoxy--(triuoromethyl)phenylacetic
acid (MTPA) has been widely used in the determination of
con guration of stereogenic centers bearing either hydroxyl
or amine groups. More recently, this methodology has been
applied for the derivatization of chiral primary alcohols thus
determining the absolute con guration of stereogenic centers
at a two- and three-bond distance from the hydroxyl group
[–]. Moreover, the modi ed Mosher’s method has been
used to determine the absolute con guration of primary
alcohols with chiral methyl groups at C- [].
However, it should be noted that this modi cation of
Mosher’s method seemed to be unsuccessful for determining
their stereochemistry of MTPA esters of some simple C-
branched primary alcohols with conjugated groups or a con-
secutive chiral centre at C-. According to Tsuda et al. the dif-
ference in chemical shi between diastereotopic oxymethy-
lene protons is similar for both diastereomers [].
We have previously performed a lipase-catalyzed synthe-
sis of nonchiral and chiral N-(-hydroxyalkyl)acrylamides
(Scheme ) [, ].
We employed the modi ed Mosher’s method for the
determination of %ee. e absolute con guration of products
was found to be (S). However, a report by Puertas et al. []
describes the enantioselective behavior of Candida antarctica
lipase B (CAL B) aording the (R)-acrylamides instead of (S)-
acrylamides as products, using racemic amines as starting
materials.
Considering the absence of a conformational model for
MTPA esters of -chiral primary alcohols and the results
of our experiments showing opposite selectivity to previous
experience, we decided to test the reliability of the method for
our case. e aim of this work was to validate the results by
conformational analysis of MTPA esters and thus obtain a
reasonable explanation for NMR data on a molecular basis.
2. Results and Discussion
We performed the synthesis of chiral N-(-hydroxya-
lkyl)acrylamides a–d and their corresponding MTPA esters
a–d and a–d as described in order to assign their absolute
stereochemistry and the degree of stereoselectivity achieved
in each case (Scheme ).
A er puri cation, the MTPA esters were analyzed by
1H NMR spectroscopy in CDCl3. e results observed for
compounds (S)-b and (S)-b were taken as example and
showed signi cant dierences in the chemical shi for many
Hindawi Publishing Corporation
Advances in Chemistry
Volume 2014, Article ID 736417, 6 pages
http://dx.doi.org/10.1155/2014/736417

Advances in Chemistry
O
O
Et
HO
R
NH2 HN
O
R OH
+
2
CAL B
2? = R = –CH3; 2? = R = –CH2CH3; 2? = R = –(CH2)2CH3;
2? = R = –(CH2)3CH3
S : Lipase-catalyzed synthesis of a–d.
HN
O
(S)
R OH
H
N
(S) O
R
(R)
O
MeO
Ph
HbHa
O
H
N
(S) O
R
(S)
O
CF3CF3 MeO
Ph
HbHa
O
(R)-MTPA, TEA (S)-MTPA, TEA
or
2
22 2 2
(S)-2? R = CH3
(S)-2? R = CH2CH3
(S)-2? R = (CH2)2CH3
(S)-2? R = (CH2)3CH3
(S)- ? R = CH3
(S)- ? R = CH2CH3
(S)- ? R = (CH2)2CH3
(S)- ? R = (CH2)3CH3
(S)- ? R = CH3
(S)- ? R = CH2CH3
(S)- ? R = (CH2)2CH3
(S)- ? R = (CH2)3CH3
S : MTPA esters (S)-a–d and (S)-a–d.
signals. Particularly, signals for diastereotopic protons Ha
and Hb of the (R)-MTPA amido ester (S)-b were for the
major isomer at . and . ppm and for the minor isomer
at . and . ppm (Figure , Spectrum A). For the (S)-
MTPA ester (S)-b these signals are reversed, so that those
corresponding to the major isomer are observed at . and
. ppm and the signals for the minor isomer are observed at
. and . ppm (Figure , Spectrum B). e integration of
the above-mentioned signals in both cases gave the same %
ee value for b.
e same procedure was applied to study the enan-
tiomeric purity of the other three products a, c, and d.
Table (columns and ) shows these results.
is pattern for the oxymethylene protons of chiral
primary alcohols esteri ed with MTPA is according to previ-
ously published results on the determination of the absolute
stereochemistry of a series of chiral primary alcohols with
a methyl group at C- position [] and chiral ,-dihidro-
xyketones []. In these works the absolute con guration
of stereogenic centers was assigned by considering the
between oxymethylene protons. Larger values of (R)-
MTPA derivative were diagnosis for R stereochemistry on the
carbon vicinal to oxymethylene whereas smaller values
of (R)-MTPA derivative were diagnosis for the opposite
stereochemistry (S). Accordingly, (S)-MTPA derivatives of
(R)-primary alcohols showed smaller values for their
oxymethylene proton signals than those prepared from (S)-
primary alcohols.
For (R)-MTPA derivatives of chiral N-(-hydroxya-
lkyl)acrylamides here reported, we found = 0.01 ppm for
the major isomer and = 0.11 ppm for the minor isomer.
is fact should indicate that the absolute stereochemistry of
the major isomer is (R,S) and therefore (S) is the absolute
con guration of the products a–d.
On the other hand the stereoselective behavior of lipases
towards hydrolysis of esters of chiral secondary alcohols has
been extensively studied. e Kazlauskas rule is intended to
predict the behavior of lipases in such cases and, according to
this rule, lipases tend to hydrolyze esters of chiral secondary
alcohols having absolute con guration (R) faster than their
enantiomer []. Lipases also showed the same stereochemi-
cal preference in transesteri cation and aminolysis reactions
[, ].
is model takes into consideration that the substituent
seniority can be correlated with substituent size, which is not
always the case. e enormous diversity of substrates accep-
ted by lipases and reaction conditions applied showed that
this rule is not met in some circumstances [, ].

Advances in Chemistry
4.324.344.364.384.404.424.444.464.48
(2R,2S)
(a)
(2S,2 S) (2S,2 S)
4.324.344.364.384.404.424.444.464.48

(b)
F : 1H NMR signals for diastereotopic protons Ha and Hb in the (R)-MTPA amido ester (S)-b (Spectrum A) and (S)-MTPA amido
ester (S)-b (Spectrum B).
T : Conformers of both diastereomers of b.
Compound Diastereomer Tor () Tor () (Kcal/mol) %P
(R)-b (R,R)
. .
− . .
− . .
. .
(S)-b (R,S)
− . .
− − . .
. .
− . .
− . .
. .
. .
− . .
− . .
− . .
− − . .
For this reason, we considered that this primary conclu-
sion should be submitted to further analysis, since this result
seems dicult to be interpreted in terms of the Kazlauskas
rule that predicted (R)-con guration. erefore the stereoch-
emical outcome of the enzymatic aminolysis of ethyl acrylate
was not con rmed. Previous results on the enzyme-catalyzed
transesteri cation of acrylates [] derivatives of such com-
pounds showed enantioselectivity towards (R)-enantiomer.
Furthermore, some authors reported that enzyme-
catalyzed hydrolysis and transesteri cation of alcohols []
and hydrolysis of amide [] derivatives of such compounds
showed enantioselectivity towards (R)- or (S)-enantiomer
depending on the catalyst and the reaction conditions.
Moreover, we could not establish precisely if the reason-
ing from previously published results could also be applied
in the determination of absolute stereochemistry of the chiral
primary alcohols belonging to products a–d, by means of 1H
NMR analysis of their MTPA derivatives. Besides the experi-
mental fact that the con guration of the chiral products was
known, no clear evidence was found that could be inter-
preted or generalized on a molecular basis (i.e., evidence of
conformational restrictions). For this reason, we decided to
perform some experiments in silico in order to provide an
independent explanation for experimental results.
Conformational search experiments were performed
with MTPA derivatives (S)-b (S,S) and (R)-b (R,S). In
these experiments the torsion angles O–C–C–C (Tor)
and MeO–C–C=O (Tor) (Figure ) were varied in order to
obtain the most stable conformers of both compounds and
thus interpret the chemical shi dierences observed bet-
ween their oxymethylene protons and H in 1H NMR
experiments.
As it can be observed in Table , up to nine stable con-
formers were found for (S)-b within Kcal/mol above the
global minimum. Most conformers had their methoxyl group
synperiplanar to their carbonyl ester, which is according
to previous calculations on other MTPA esters by DFT
methods. However, many rotamers along O–C and C–C
bond were found to be stable enough to be important
contributions to conformer population. is fact explains the
broader signals and lower values between oxymethylene
protons for (S)-b isomer. It was observed that signals at
. and . ppm are broad (more than Hz wide) and not
completely resolved.
On the other hand, only four stable conformers were
found for the diastereomer (R)-b. All of them had their
methoxyl group synperiplanar to their carbonyl ester. Major
contributions to the conformational population were the H

Advances in Chemistry
OMe
F3C Ph
O
CH2CH3
H
N
O
Tor1
Tor2
O
F : Torsion angles Tor (red and bold) and Tor (blue and bold) employed for conformational search of compound (S)-b.
Ph
OMe
CF3
Ha
OHb
NH(C=O)–CH=CH2
H3 C H2C
H
O
0
0.54
0.72
1.54
–
−70
5
E (kcal/mol)
(a)
2.3 A˚
(b)
F : (a) Newman projections along the axes C–C (up) and C–C (down) for the most stable conformer of (R)-b. Torsion angles
Tor and Tor are indicated. Major contributions to this type of conformation are highlighted in the energy level diagram on the right. (b)
Picture of a D molecular render of the same conformer showing intramolecular hydrogen bonding between amide hydrogen and a uorine
atom (H–F distance: . angstrom).
antiperiplanar to oxygen attached to C (Figure ). Two
stable conformers have this conformation and they con-
tribute to .% of the total conformer population. Moreover,
these conformers show one oxymethylene hydrogen (Hb)
synperiplanar to the aromatic ring and the other (Ha)
synperiplanar to the C=O bond. ese observations could
explain the large dierence of chemical shi s between both
oxymethylene protons (. ppm) and the coupling con-
stants observed for Ha-H and Hb-H (. Hz). e
occurrence of intramolecular hydrogen bonding between the
amide hydrogen and a uorine atom was also con rmed for
both conformers. We assume that this hydrogen bond plays a
key role in conformer stability.
ese results are dicult to assimilate to those predicted
in Kazlauskas rule based on the size of the substituents in the
stereocenter and designed to predict which enantiomer of a
secondary alcohol reacts faster in enzyme catalyzed reactions.
e rule is reliable with substrates having substituents which
dier signi cantly in size.
In the acylation reaction of every chiral alkanolamine
used in this work the enzyme showed an enantioselective
behavior opposite to that described by Kazlauskas rule,
preferably getting the product by reaction of alkanolamine
with (S) con guration instead of the (R) predicted by the rule.
In a the fact could be explained considering that the hydrox-
ymethyl group is clearly larger than the methyl group, but
in b ethyl and hydroxymethyl substituents are about the
same size and compounds c and d have their alkyl sub-
stituent larger than their hydroxyalkyl substituent. In these
two compounds the Kazlauskas rule could be applied in terms
of substituent size.
e opposite con guration observed between the exper-
imental results and that predicted by the rule could be
attributed more likely to electronic eects than to steric hin-
drance. For instance, Maraite et al. showed that Pseudomonas
stutzeri lipase stereoselectivity for benzoin acylation could
be explained by hydrogen bonding between Tyr- hydroxyl
group and carbonyl oxygen of the substrate []. A similar
eect could be attained by hydrogen bonding between
hydroxyl moiety of alkanolamine and the threonine-rich loop
of residues – of CAL B. erefore it could be suggested
that the chemo- and enantioselectivity of the lipase-catalyzed
N-acylation of chiral alkanolamines must be driven by the
presence of the hydroxyl group in one substituent rather than
the dierence in the substituent size.
3. Conclusions
In this work we have proposed a conformational model for
MTPA esters of chiral N-(-hydroxyalkyl)acrylamides. e in
silico conformational analysis of the corresponding diastere-
omers (R)-b and (S)-b showed that the dierences in
conformational restrictions, and consequently 1H NMR sig-
nal splitting for oxymethylene protons, arose from speci c
intramolecular interactions rather than nonspeci c steric
hindrance. is analysis was also useful for explaining the
chemical shi dierences for both diastereomers and served

Advances in Chemistry
for the validation of the stereochemical determination of
chiral N-(-hydroxyalkyl)acrylamides obtained by lipase-
catalyzed resolution.
4. Experimental
.. General Remarks. 1H NMR spectra were recorded in
CDCl3 as solvent using a Bruker Avance II spectrometer
operating at MHz. Chemical shi s are reported in units
(ppm) relative to tetramethylsilane (TMS) set at ppm, and
coupling constants are given in hertz. e synthesis of N-
(-hydroxyalkyl)acrylamides a–d and their corresponding
MTPA derivatives a–d and a–d were performed as des-
cribed previously [].
.. Molecular Modeling. e structures of (R,R)--(acr-
yloylamino)butyl ,,-triuoro--methoxy--phenylpropa-
noate (R)-b and (R,S)--(acryloylamino)butyl ,,-
triuoro--methoxy--phenylpropanoate (S)-b were subm-
itted to conformational search using the molecular mechan-
ics method MM+ and a conformational search algorithm
integrated on HyperChem .. e selected torsions to vary
were O–C–C–C (Tor) and MeO–C–C=O (Tor)
and conformations of energies below Kcal/mol over the
global minimum were submitted to geometry optimization
using Gamess []. Energies were minimized employing DFT
method BLYP using – g basis function. Solvent eect
was simulated using the PCM method with the standard
parameters for chloroform included in the so ware package.
Optimized structures for each isomer were visualized using
ChemBioD Ultra . and overlaid in order to discard
repeated structures.
Conflict of Interests
e authors declare that there is no conict of interests
regarding the publication of this paper.
Acknowledgments
e authors thank UBA X, CONICET PIP --
/, and ANPCyT PICT - for partial nancial
support. Eduardo M. Rustoy, Alicia Baldessari, and Leandro
N. Monsalve are research members of CONICET.
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