Investigating Chiral Recognizability of Diastereomeric Crystallization of Mandelic Acid and L-Phenylalanine
Kim et al.
the use of chiral acids or bases to form diastereomeric
salts. Unlike the enantiomers from which they are formed,
diastereomers have different physical properties, including
crystal formation, solubility, a melting point, and pharma-
cological activities13 and thus can be easily separated from
each other by crystallization. Hence, optical resolution via
diastereomeric salt formation is the most practical method
for obtaining pure enantiomers from a racemate on both a
laboratory and industrial scale.12ꢀ14
model system composed of chiral mandelic acid and a
resolving agent (L-Phe) using AFM, NMR, and vibration
spectroscopy. In addition, the physical properties of the
two corresponding diastereomeric salts resulting from the
resolving agent L-phenylalanine and two mandelic acid
enantiomers are also examined using thermal analyses.
2. EXPERIMENTAL DETAILS
Even though diastereomeric crystallization was discov-
ered by Pasteur nearly 160 years ago,15 selecting the
appropriate resolving agent still remains a significant chal-
lenge. Thus, to utilize diastereomeric crystallization for
chiral separation, several recent studies have attempted to
clarify the important physical properties of diastereomeric
salts through the use of phase diagrams for solid–liquid
equilibriums16ꢀ17 and estimating the molecular structure of
less- and more-soluble diastereomeric salts.18ꢀ19
2.1. Chemicals
The enantiomers of mandelic acid and resolving agent
L-phenylalanine (99% purity, TCI Company, Japan)
were used as received. The 4-aminothiophenol (4-ATP),
p-Toluenesulfonyl chloride (TSC), pyridine anhydrous,
and 16-mercaptohexadecanoic acid (16-MHA), plus the
materials for the IR, Raman, and NMR spectroscopy were
all purchased from Sigma-Aldrich and used as received.
Deionized water was used to prepare the two diastere-
omeric salts by cooling crystallization.
Yet, despite such progress in chiral diasteromeric crys-
tallization, the chiral recognition mechanism between
enantiomers and the resolving agent has not been carefully
examined, as there is no direct technique for measuring the
interaction force between two enantiomers and the resolv-
ing agent. Several reports also showed the possibility of
a chiral selective crystallization process from a racemic
mandelic acid solution when applying L-phenylalanine
(L-Phe) as the resolving agent. The different intermolecu-
lar structures were revealed by the crystal structure of the
D-MA-L-Phe and L-MA-L-Phe salts.20 The X-ray diffrac-
tion pattern for both the crystals was measured to reveal
the role of the resolving agent by the function of the
molecular ratio.21 Both the papers indicated clearly the
structural difference between the two diasteromeric crys-
tals, and revealed the difference in the hydrogen bond
made an important role for the diasteromeric crystal for-
mation. On the other hand, Ichikawa et al. show a differ-
ent kind of the binding force can be also related to the
diasteromeric salt formation such as CH-ꢁ interaction and
ꢁ–ꢁ interaction using a different kind of diasteromeric
crystals.22 In this way, various kinds of the interaction
force can be involved in the chiral recognition techniques
performed by the crystallization process, but the mecha-
nism related to the selectiveness is unclear.23
2.2. Diastereomeric Crystallization
The diastereomeric crystals of L-mandelic acid-L-
phenylalanine and D-mandelic acid-L-phenylalaine were
prepared by cooling crystallization in a double-jacketed
Ruston reactor with a working volume of 50 mL. The
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individual enantiomers (D or L-form) of mandelic acid
IP: 60.51.239.205 On: Mon, 28 Mar 2016 21:25:29
(50 g/L) were first dissolved in distilled water, followed
Copyright: American Scientific Publishers
by the equivalent molar addition of L-phenylalanine as the
resolving agent. The solution was then heated to 80 C to
ꢀ
allow complete dissolution of the resolving agent. There-
after, to induce diastereomeric crystalꢀlization, the solution
was cooled to room temperature (25 C) at a cooling rate
ꢀ
of 10 C/hr with gentle agitation at 200 rpm. After reach-
ing room temperature, the solution was maintained at this
temperature for 2 hrs. The crystals were then filtered out
of the suspension using a 0.45 ꢂm filter membrane and
washed with ice water. The diastereomeric crystals were
re-crystallized at least 3 times to eliminate any impurities.
Finally, the retrieved crystals were dried in a vacuum oven
at room temperature for 1 day and stored in an esiccators
until analysis.
Recently, the highly sensitive technique of atomic force
microscopy (AFM) has been used to investigate mech-
anisms on a sub-molecular level, especially biological
interactions. For example, the interaction forces of biotin-
streptavidin,24–26 complementary DNA strands,27ꢀ28 and
antibody-antigen complexes29ꢀ31 have all been successfully
explored. In principle, AFM records the force based on the
deflection of the cantilever along the tip-surface distance.32
Therefore, AFM could feasibly be applied to detect the
interaction of a chiral enantiomer and a resolving-agent-
modified tip on a sub nN level.33
2.3. Modification of Tip and Sample for Chemical
Force Microscopy
The probe tip of the AFM was coated with
4-amino-L-phenylalanine using the method suggested by
Tsourkas.34 For this purpose, two types of micro cantilever
were selected for the tapping mode phase lag imaging
(RTESPA, f = 320 kHz, k = 40 N/m, Veeco Inc., U.S.A.)
and contact mode affinity force measurements (OMCL-
TR400PB-1, k = 0ꢃ09 N/m, Olympus). The tapping mode
probes were made of Si, and the probe tip coating with
gold film (thickness = 10 nm) after an undercoating of
Accordingly, this study demonstrates the potential for
understanding the chiral recognition mechanism of a
7140
J. Nanosci. Nanotechnol. 12, 7139–7147, 2012