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Stretched Gelatin Gels as Chiral Alignment Media for the Discrimination of Enantiomers by NMR Spectroscopy.

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Angewandte
Chemie
NMR Spectroscopy
Stretched Gelatin Gels as Chiral Alignment
Media for the Discrimination of Enantiomers by
NMR Spectroscopy**
Kyryl Kobzar, Horst Kessler, and Burkhard Luy*
Verification of the enantiomeric purity of a product and
measurement of enantiomeric excess is an everyday problem
in modern organic chemistry. Therefore, the development of a
convenient method of measurement for this purpose is
desirable. Classical NMR spectroscopic techniques that discriminate enantiomers depend on chiral auxiliaries such as
chiral derivatizing agents, lanthanide chiral-shift reagents, and
chiral solvating agents.[1, 2] These methods work only for
functionalized chiral molecules of interest that can interact
with the auxiliaries to build detectable diastereomeric compounds or adducts. In contrast, chiral orienting media permit
enantiomer discrimination through the differential ordering
effect of enantiomers within the chiral phase.[3, 4] Therefore,
even compounds that lack polar groups, such as saturated
chiral hydrocarbons[5] and even prochiral elements in symmetrical molecules,[6, 7] can be distinguished by NMR spectroscopy in such media.
So far, only surfactant bilayers,[8–10] various chiral liquidcrystalline media,[11–15] and achiral liquid crystals with chiral
cages[16] have been reported to be suitable for enantiomeric
discrimination. Such media are usually difficult to prepare
and operate only within certain temperature ranges. Furthermore, their induced orientation depends on the strength of
the magnetic field. In contrast, covalently cross-linked,
stretched-polymer gels[17–19] are relatively easy to handle and
provide field-independent orientation.
The aim of the studies presented herein was to obtain
partial alignments in stretched chiral gels. Gelatin in the form
of gummibrchen (the famous German sweets) was used for
the initial proof of principle that alignment is possible with
this kind of polymer. In further experiments we were able to
show that stretched gelatin gels as chiral alignment media not
only make it possible to obtain structural information through
residual dipolar couplings,[20, 21] but also provide a way to
discriminate between enantiomers and to measure enantiomeric excess. Gelatin therefore represents a new subtype of
alignment media: polymer gels in which the spatial structure
is almost solely stabilized by hydrogen bonds.
Gummibrchen were swollen in deionized water to about
twice their original dimensions (Figure 1) and subsequently
[*] Dipl.-Chem. K. Kobzar, Prof. Dr. H. Kessler, Dr. B. Luy
Department Chemie
Lehrstuhl fr Organische Chemie II
Technische Universitt Mnchen
Lichtenbergstrasse 4, 85747 Garching (Germany)
Fax: (+ 49) 89-289-13210
E-mail: burkhard.luy@ch.tum.de
[**] B.L. and H.K. thank the Fonds der Chemischen Industrie and the
DFG (Emmy Noether fellowship LU 835/1-1; Ke 147/37-1) for
financial support. We also thank W. Rist and F. Rist for support.
Angew. Chem. Int. Ed. 2005, 44, 3145 –3147
Figure 1. Various stages of the preparation of stretched gelatin samples: A) gummibrchen; B) gummibrchen swollen in water; C) gelatin
gel (10 %) prepared in a pipette tip; D) gel after drying in the pipette
tip; E) equilibrated gelatin sample with a D2O deuterium splitting of
117 Hz (Figure 2 B); left: centimeter scale.
cut into roughly cylindrical shapes. They were then dried on a
glass capillary and inserted into NMR tubes with D2O. After
equilibration for two days, deuterium NMR spectra were
acquired with a clearly visible quadrupolar splitting in the
range of 20 Hz (Figure 2 A).
After this successful proof that partial alignment is
possible with gelatin-based gels, further experiments with
household gelatin were conducted. Heated gelatin solution
( 10 % w/v) was poured into a standard pipette tip with a
sealed end and dried for eight weeks under refrigeration
(Figure 1). From this, solid sticks with diameters of 1.9 mm
and uniform appearance were collected, put into NMR tubes,
and subjected to constrained swelling by direct addition of
D2O in a procedure similar to that described earlier for
polystyrene sticks.[19] After swelling for a couple of days and a
single solvent exchange to wash out unwanted substances, the
gel showed a quadrupolar splitting for D2O of 117 Hz at 25 8C
(Figure 2 B). A mixture of l-alanine (30 mg) and d-alanine
(25 mg; 9 % ee) was added to the sample and allowed to
diffuse into the gel. Although its spatial structure is largely
stabilized by hydrogen bonds, the gel, in the presence of polar
solutes over a two-month period, exhibited no change in the
deuterium quadrupolar splitting of the solvent. A specially
designed J-coupling experiment (Figure 3) was acquired that
allowed separation of the two enantiomers.
In principle, any order-dependent NMR interaction can
be used for the distinction of enantiomers. So far, mostly
1D[3, 4, 10, 11, 13, 14, 16, 24–30] and 2D[4, 5, 31–33] 2H NMR spectra of nonisotopically enriched samples have been reported in which
use was made of the difference in residual quadrupolar
couplings in chiral orienting media. However, the low natural
abundance of deuterium nuclei requires either long acquisition times or the use of deuterated compounds.
Recently, J-coupling spectroscopy on 1H[31] and 13C[34]
nuclei was proposed as an alternative technique in which
the different residual dipolar couplings (RDCs) of enantiomers lead to distinguishable NMR spectroscopic signals.
However, the lower limit of alignment in known liquid-
DOI: 10.1002/anie.200462736
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3145
Communications
Figure 2. NMR spectra acquired on stretched gelatin samples:
A) 2H NMR spectrum of a stretched gummibrchen sample swollen in
D2O; B) 2H NMR spectrum of a sample of household gelatin (see text
for details); C) 1H,13C-BIRDd,X-J-coupling spectrum of l-Ala/d-Ala
(1.2:1): the two enantiomers can be clearly distinguished. The spectrum was acquired with the sequence described in Figure 3 with 2048
t1 increments and processed in both dimensions in phase-sensitive
mode.
Figure 3. 1H,13C-BIRDd,X-J-coupling experiment for the phase-sensitive,
high-resolution detection of single-bond (DCH + 1JCH) couplings. 908
and 1808 pulses are indicated by black and white bars, respectively.
Unless otherwise indicated, the pulses are x-phase. Phase cycles are
f1 = y, y, y, y, y, y, y, y; f2 = x, x, x, x; f3 = x, x; frec = x, x,
x, x, x, x, x, x. Delays: D = 1/(1JCH+DCH), t and t’ are delays for
gradient application (1.2 ms). The ratio of gradient strengths are
G1:G2:G3 = 80:30:20.1 . The BIRDd,X element for suppression of longrange 1H,13C-couplings is shaded gray. Phase-sensitive absorption
(States-TPPI) is possible by cycling f1. Alternatively, simple t1-incrementation can be applied with the processing procedure described
previously in references [22, 23].
3146
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
crystalline media results in considerable RDCs that can easily
reach the size of 1JCH-coupling constants,[35] which therefore
limits one to very basic NMR spectroscopic experiments with
known disadvantages: 1H J-coupling spectra are not phasesensitive and broadened line widths from 1H,1H-RDCs lead to
spectra that in many cases are hardly interpretable. Directly
detected 13C J-coupling spectra, on the other hand, have the
disadvantage of a low signal-to-noise ratio.
In contrast, stretched gels with relatively weak induced
alignments permit the use of conventional heteronuclear
pulse-sequence building blocks, as the condition DCH ! 1JCH
can be easily fulfilled. We were therefore able to design a 1Hexcited and detected 13C,1H-correlated pulse sequence with
an additional BIRDd,X element[36] for the suppression of longrange 13C,1H scalar and dipolar couplings (Figure 3). This
experiment has acceptable sensitivity. Phase-sensitive detection and decreased cross-peak multiplicity decrease the signal
width in the indirect dimension sufficiently to separate very
small differences in single-bond DCH coupling constants.
The pulse sequence applied to the mixture of l-alanine/dalanine (1.2:1) diffused in a stretched gelatin gel gave two
multiplets for the b-CH3 groups with a well-resolved difference in outer J-multiplet components of 2.5 Hz (Figure 2 C).
A control experiment with a sample prepared with l-alanine
revealed only one signal at the same position as the stronger
component of the mixed sample. This is clear evidence that
the difference of the DCH RDCs of the enantiomers in chiral
stretched gelatin is the origin of the split signal (data not
shown). The spectrum of the enantiomeric mixture is so wellresolved that by cross-peak integration, an enantiomeric
excess of 7 5 % was obtained which is very close to the
expected value. Notably, the observed signal intensity is
dependent not only on the concentration of a single enantiomer, but also on the efficiency of coherence transfer via scalar
and dipolar couplings with the function: sin2(p[1JCH+DCH]D/
2) cos(p[1JCH+DCH]D). For the case shown in Figure 2 C this
leads to a very low systematic error of less than 0.3 % ee.
We have shown the usefulness of gelatin gels for the
partial alignment of molecules in aqueous environments.
They form three-dimensional networks of polypeptide chains
from the partial renaturation of native collagen,[37] which
eliminates the need for any added cross-linking agent to
connect the polymer chains, as is the case for other gels used
for the same purpose.[17–19, 38–40] Interestingly, this polymer
structure, which is held together almost solely through a
network of hydrogen bonds, is able to withstand the forces
present in the constrained swollen gels. However, household
gelatin gels are generally not stable at temperatures above
35 8C,[37] and the chemical stability of stretched gels with
respect to solutes must be studied in greater detail. At neutral
pH values and at room temperature, our samples did not
change alignment properties after more than two months.
Chiral alignment media lead to the differential orientation
of enantiomers and therefore allow discrimination of enantiomeric mixtures by NMR spectroscopy.[4] To our knowledge,
gelatin is the first chiral alignment medium that combines the
possibility of enantiomeric resolution with the advantages of
partial alignment through mechanical stretching. The easily
scalable alignment reported herein holds potential for the
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Angew. Chem. Int. Ed. 2005, 44, 3145 –3147
Angewandte
Chemie
application of more sophisticated pulse sequences with
greater sensitivity and decreased signal width due to reduced
multiplet patterns. The combination of chiral gel-based
alignment media, field-independent alignment, and fieldindependent J-coupling-based measurement techniques may
eventually allow these measurements to be taken on lowfield, low-cost NMR spectrometers, as long as chemical-shift
resolution is not necessary.
This approach toward enantiomeric resolution is not
limited to gelatin polymers and is probably transferable to
other chiral gels or achiral gels with chiral cages, which would
permit enantiomer discrimination in nonaqueous solutions as
well.
Received: November 26, 2004
Revised: January 21, 2005
Published online: April 18, 2005
.
Keywords: chirality · enantiomeric discrimination · gels ·
NMR spectroscopy · polymers
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