Biphasic Enantioselective Fluorescent Recognition of Amino Acids by a Fluorophilic Probe

Article abstract of DOI:10.1002/chem.201900880

A fluorophilic fluorescent probe based on a perfluoroalkyl-substituted bis(binaphthyl) compound was designed and synthesized. It displayed a highly enantioselective fluorescence response toward structurally diverse amino acids in a biphasic fluorous/aqueous system with enantiomeric fluorescent enhancement ratio (ef; ΔI_D/ΔI_L) values up to 45.2 (histidine). It can be used to determine the enantiomeric compositions of amino acids and also allows the amino acid enantiomers to be visually discriminated. NMR and mass-spectroscopic investigations provided insights into the observed high enantioselectivity. This biphasic fluorescent recognition was used to determine the enantiomeric composition of the crude phenylalanine products generated by an enzyme-catalyzed asymmetric hydrolysis under various reaction conditions. The fluorous-phase-based fluorescence measurement under the biphasic conditions was able to minimize the interference of other reaction components and thus has potential in asymmetric reaction screening.

Full text of DOI:10.1002/chem.201900880

A Journal of
Accepted Article
Title: Biphasic Enantioselective Fluorescent Recognition of Amino
Acids by a Fluorophilic Probe
Authors: Yuan-Yuan Zhu, Xue-Dan Wu, Mehdi Abed, Shuang-Xi Gu,
and Lin Pu
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To be cited as: Chem. Eur. J. 10.1002/chem.201900880
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10.1002/chem.201900880
Chemistry - A European Journal
Biphasic Enantioselective Fluorescent Recognition of Amino Acids by a
Fluorophilic Probe
Yuan-Yuan Zhu,[a,b] Xue-Dan Wu,[b] Mehdi Abed,[b] Shuang-Xi Gu,*[b,c] and Lin Pu*[b]
aSchool of Chemistry and Environmental Engineering, Wuhan Institute of Technology, Wuhan 430205, P. R. China
bDepartment of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA
cKey Laboratory for Green Chemical Process of Ministry of Education, School of Chemical Engineering and Pharmacy, Wuhan Institute
of Technology, Wuhan 430205, P. R. China
*Corresponding author e-mail: shuangxigu@163.com, lp6n@virginia.edu
Abstract. A fluorophilic fluorescent probe based on a perfluoroalkyl substituted bisbinaphthyl compound is designed and synthesized,
which displayed highly enantioselective fluorescence response toward structurally diverse amino acids in a biphasic fluorous/aqueous
system with ef (enantiomeric fluorescent enhancement ratios, ID/IL) values up to 45.2 (histidine). It can be used to determine the
enantiomeric compositions of amino acids and also allows the amino acid enantiomers to be visually discriminated. NMR and mass
spectroscopic investigations have provided insights into the observed high enantioselectivity. This biphasic fluorescent recognition has
been used to determine the enantiomeric composition of the crude phenylalanine products generated by an enzyme-catalyzed asymmetric
hydrolysis under various reaction conditions. The fluorous phase-based fluorescence measurement under the biphasic conditions can
minimize the interference of other reaction components and shows potential in asymmetric reaction screening.
fluorophilic probe (Scheme S1). Reaction of
binaphthyl-based aldehyde (
)-28 in the presence of base
followed by acidic deprotection gave the bisbinaphthyl
compound (R,R)- . This compound was found to be soluble
in fluorous solvent 1H,1H,2H,2H-perfluoro-1-octanol
1 with the 1,1’-
Introduction
Enantioselective fluorescent recognition of chiral organic
compounds are potentially useful for high throughput assay
of asymmetric reactions.1-3
One challenge for this
R
3
application is the possible interferences of various reaction
components on the fluorescence measurement when a
fluorescent probe is used to determine the enantiomeric
composition of the desired product in a reaction mixture. In
order to minimize such interferences, we have proposed a
strategy to develop the fluorous phase-based fluorescent
probes by utilizing the lipophobic, hydrophobic and
fluorophilic properties of the highly fluorinated materials.
Although the fluorous phase-based techniques have been
extensively applied to chemical reactions and separations,4-6
investigation of their use in fluorescent sensing has only just
started.7
a
(PFOH) and showed no fluorescence in this solvent.
Scheme 1. Synthesis of the Fluorophilic Probe (R,R)-3.
F
F
F
F
F
F
F
F
F
1. K2CO3,
O
18-crown-6
F
DMF, 50 o
C
F
F
F
F
F
F
F
HO
MOMO
+
2. HCl (conc.)
ethanol
CHCl3, reflux
Br
Br
N
OHC
1
(R)-2
F
F
F
F
F
F
F
F
F
F
F
F
MOM = CH3OCH2
F
Recently, we found that
a
bisbinaphthyl-based
O
N
F
F
F
F
fluorescent probe can carry out highly enantioselective
fluorescent recognition of amino acids in acetonitrile
solution.8,9 On the basis of this work, we have synthesized a
fluorophilic probe by incorporating a highly fluorinated alkyl
O
O
(R,R)-3
HO
OH
CHO
chain.
We have demonstrated that in a biphasic
OHC
aqueous/fluorous system, the probe can carry out highly
enantioselective fluorescent detection of amino acids by
selectively extracting the substrates from the aqueous phase
into the fluorous phase. This is the first example to conduct
enantioselective discrimination of amino acids by using a
fluorous phase-based separation process. It has been used to
determine the enantiomeric composition of crude chiral
amino acid samples generated from an enzyme-catalyzed
asymmetric reaction. Herein, these results are reported.
2. Fluorescence Response of (R,R)-3 toward D- and L-
Phenylalanine (Phe)
We studied the fluorescence response of (R,R)-
an amino acid D-/L-Phe in a biphasic system of H2O/PFOH
(2 mL/ 2 mL). When a solution of (R,R)-
(4.0 × 10-5 M, 2.0
mL) in PFOH was treated with Zn(OAc)2 (1.0 equiv, 4.0 mM
in H2O, 20 L), no change in fluorescence was observed
3 toward
3

(Figure 1a). Mixing this solution with a solution of D-Phe
(14.0 equiv, 2.0 mL) in water also did not cause any
fluorescence response. However, the fluorescence of (R,R)-
Results and Discussion
1. Synthesis of a Highly Fluorinated Bisbinaphthyl Probe
3
+Zn(II) in PFOH at  = 533 nm was turned on when it was
We prepared compound
1, a 2,6-dibromomethyl pyridine
mixed with the solution of D-Phe in aqueous
tetrabutylammonium hydroxide (TBAH) (2 mM) (Figure 1a).
with a highly fluorinated alkyl chain, as a precursor to a
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When L-Phe was used, under the same conditions, the
fluorescence enhancement was much smaller (Figure 1b). In
these experiments, the fluorescence of the bottom fluorous
solution of the two phases in a quartz cuvette was measured
directly with no need to remove the aqueous phase. Thus,
fluorescence intensity of (R,R)-3] was 11.4, representing a
high enantioselectivity.
(
R,R)-3 can discriminate the two enantiomers of D- and L-
Phe in the biphasic solution system in the presence of Zn(II)
and TBAH.
CO2H
NH2
D-/L-Phe
Figure 2. (a) Fluorescence intensity at533 nm (I533) of (R,R)-3 (1.0
equiv, 2.0 mL, 0.040 mM in PFOH) with (a) D- and (b) L-Phe (0 – 20
equiv, 2.0 mL, in 2 mM TBAH aqueous solution) in the presence of
Zn(OAc)2 (1.0 equiv, 20 μL, 4.0 mM in H2O). The error bars were
obtained from three independent experiments. (b) Fluorescence spectra
of (R,R)-3 with D- and L-Phe (14.0 equiv) in the presence of TBAH (2.0
mM) and Zn(II) (1.0 equiv). All the biphasic mixtures were stirred at rt
for 4 h, and fluorescence was measured 10 min after the mixing was
stopped (λexc = 435 nm. Slit: 2/2 nm).
We also prepared (S,S)-3, the enantiomer of (R,R)-3, and
examined its fluorescence response toward Phe under the same
conditions as above. It was found that L-Phe greatly enhanced the
fluorescence of (S,S)-3 but not D-Phe (Figure S2). The mirror
image relation between the fluorescence responses in Figure 2 and
Figure S2 confirmed the observed highly enantioselective
fluorescent recognition.
Figure 3 gives the photo images of the biphasic system after
(R,R)-3 (1.2 mM) was treated with D- or L-Phe (14.0 equiv, in
16.8 mM TBAH aqueous solution) for 4 h in the presence of
Zn(II) (1.0 equiv). It shows that upon UV lamp irradiation, the
fluorous phase with D-Phe in the lower layer gave bright yellow
emission but the one with L-Phe gave much weaker fluorescence
(Figure 3b). Thus, the enantiomers of this amino acid can be
visually discriminated.
Figure 1. Fluorescence response of (R,R)-3 (1.0 equiv, 2.0 mL, 0.040
mM in PFOH) with (a) D- and (b) L-Phe (14.0 equiv, 2.0 mL, in 0 or 2
mM TBAH aqueous solution) in the presence of Zn(OAc)2 (1.0 equiv, 20
μL, 4 mM in H2O). (c) Fluorescence intensity at 533 nm (I533) versus the
concentration of TBAH. (d) I533 versus the equiv of Zn(OAc)2 (20 μL,
2~16 mM in H2O) with 2.0 mM TBAH. All the biphasic mixtures were
stirred at rt for 4 h. For (a)，(b) and (d), the fluorescence was measured
after the mixing was stopped for 10 min. For (c), the fluorescence was
recorded after the mixing was stopped for 10, 30, 60 min respectively.
(λexc = 435 nm. Slit: 2/2 nm)
We studied the fluorescence responses by varying the
concentration of TBAH as well as the time after the two
solution phases were separated while the mixing was
stopped.
As shown in Figure 1c, the fluorescence
enhancement of (R,R)-
3
+Zn at = 533 nm in the presence of

D-Phe greatly increased as the concentration of TBAH
increased from 1 mM to 2 mM which then slightly increased
at 3 mM TBAH. After that, the fluorescence started to
decrease slightly with increasing TBAH concentration. It
also shows that the time after the two phase mixing was
stopped had little influence on the fluorescence response.
We further studied the effect of the equivalency of Zn(II) on
the fluorescence response. As shown in Figure 1d, at
Zn(OAc)2 (1.0 equiv) the fluorescence enhancement of
Figure 3. (a) Photos of (R,R)-3 (1.0 mL, 1.2 mM, in PFOH) treated with
D- or L-Phe (14.0 equiv, 1.0 mL, in 16.8 mM TBAH aqueous solution) in
the presence of Zn(II) (1.0 equiv, 20 μL in H2O). (b) Under UV lamp
irradiation at 365 nm. Upper layer: aqueous phase; lower layer: fluorous
phase.
The fluorescence response of the two enantiomers of the
probe, (S,S)- and (R,R)-3, toward Phe at various ee values
[enantiomeric excess = ([D] - [L])/([D] + [L])] was investigated.
As shown in Figure 4, the plots of the fluorescence intensity at
533 nm versus the ee values of Phe present a mirror-image
relation between the two enantiomeric sensors. The linear
relations are obtained from the fluorescent response of (R,R)-3
and (S,S)-3 toward Phe with ee’s > 0 (D in excess) and < 0 (L in
(
R,R)-3 by D-Phe (14.0 equiv, in 2 mM TBAH) in the
biphasic system reached the maximum and no further
increase was observed at greater amount of Zn(OAc)2.
Fluorescence response of (R,R)-3 toward D- or L-Phe at
various concentrations in the presence of 2 mM TBAH and 1
equiv Zn(OAc)2 in the biphasic system was then
investigated. As shown in Figure 2a, the fluorescence
excess), respectively.
This plot demonstrates that the
intensity of (R,R)-3 at 533 nm increased continuously when
enantiomeric composition of the amino acid can be determined by
the biphasic enantioselective fluorescent recognition process.
the concentration of D-Phe increased from 2 to 20 equiv, but
when the concentration of L-Phe increased the fluorescence
intensity remained low. In Figure 2b, it shows that at 14.0
equiv of Phe, the enantioselective fluorescence enhancement
ratio [ef = (ID-I0)/(IL-I0) =
intensity in the presence of D- or L-amino acid. I0:
ID/IL. ID, IL: fluorescence
2
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The high enantioselectivity of (R,R)-3 in the fluorescent
recognition of His was further confirmed by the use of the
enantiomeric probe (S,S)-3 which gave the expected mirror image
relationship (Figure S8). We have used both (R,R)- and (S,S)-3 to
interact with His (12 equiv) under the biphasic conditions at
various enantiomeric compositions. The results in Figure 6
demonstrate that these probes can be used to determine the
enantiomeric composition of His. Visual discrimination of the
enantiomers of His similar to that shown for Phe in Figure 3 was
also observed (Figure S9).
Figure 4. Fluorescence intensity at 533 nm (I533) of (R,R)-3 (black curve)
and (S,S)-3 (red curve) (1.0 equiv, 2.0 mL, 0.04 mM in PFOH) with Phe
(14 equiv, 0.56 mM in 2 mM TBAH aqueous solution) at various ee in the
presence of Zn(II) (1.0 equiv, 4.0 mM in H2O). All the mixtures were
stirred at rt for 4 h, and fluorescence was measured 10 min after the
mixing was stopped. The error bars were obtained from three independent
experiments. λexc = 435 nm. Slit: 2/2 nm.
3. Fluorescence Response of (R,R)-3 Toward Other Amino
Acids
We studied the fluorescent response of (R,R)-3 toward 18
common chiral amino acids (including Phe) in the
fluorous/aqueous phases. As shown in figure S3, all 17 amino
acids could lead to fluorescence enhancement except proline.
Among the amino acids enhancing the fluorescence of the probe,
14 of the amino acids generated enantioselective fluorescent
response except alanine and tyrosine. The D enantiomers of 9
amino acids including histidine, tryptophan, glutamine, serine,
methionine, arginine, cysteine, threonine, and asparagine
enhanced the fluorescence of (R,R)-3 greater than their L-
enantiomers, whereas the L-enantiomers of 5 amino acids
including lysine, glutamic acid, leucine, aspartic acid and valine
generated greater fluorescence enhancement than the
corresponding D-enantiomers. Figure S4 shows that after the two
phases of (R,R)-3 (1.0 equiv, 0.040 mM in 2 mL PFOH), D/L-
amino acids (10.0 equiv, 2.0 mL in 3 mM TBAH aqueous
solutions), Zn(II) (2.0 equiv, 20 L in H2O) were mixed for 3 h,
high ef values were observed for 6 amino acids: histidine (33.5),
tryptophan (5.7), glutamine (4.8), serine (4.6), methionine (4.1)
and lysine (6.3, IL/ID).
We have explored various conditions for the biphasic
recognition of histidine (His) by (R,R)-3 (Figures S5, S6 and S7).
As shown in Figure 5a, under the optimized conditions, the
fluorescence of (R,R)-3 in PFOH at  = 540 nm was greatly
enhanced in the presence of Zn(II) (2.0 equiv) by D-His (in 3 mM
TBAH) from 1 to 12 equiv but not by L-His. Figure 5b gives the
fluorescence spectra of (R,R)-3 upon treatment with 12.0 equiv D-
and L-His which gave an ef of 45.2, an excellent
enantioselectivity.
Figure 6. Fluorescence intensity at 540 nm (I540) of (R,R)-3 (black curve)
and (S,S)-3 (red curve) (1.0 equiv, 2.0 mL, 0.040 mM in FTOH) toward
histidine (12 equiv, 0.48 mM in 3.0 mM TBAH water solution) at various
ee in the present of Zn(II) (2.0 equiv, 8.0 mM in H2O). All the mixtures
were stirred at rt for 4 h, and fluorescence was measured 10 min after the
mixing was stopped. The error bars were obtained from three independent
experiments. λexc = 435 nm. Slit: 4/4 nm.
4. NMR and mass spectroscopic studies for the interaction of
(R,R)-3 with D-/L-Phe and D-/L-His
In order to gain a better understanding on the fluorescence
response of (R,R)-3 toward the amino acids, we have conducted
1H NMR and mass spectroscopic investigations.
1
The H NMR spectra of (R,R)-3 in PFOH after being treated
with an aqueous solution of D- or L-Phe was obtained (Figure 7a).
A capillary tube filled with acetone-d6 was added as an external
standard. As shown in Figure 7a (the full spectra were displayed
in Figure S10), when (R,R)-3 was treated with D- or L-Phe, the
aldehyde signal at  8.87 of the probe completely disappeared and
new and broad peaks appeared between 6.6 to 7.2 (Figures 7a5
1
and 7a6). The H NMR spectra for the reactions of (R,R)-3 with
D- and L-Phe in the homogenous solution of DMSO-d6 were also
obtained. As shown in Figures 7b4 and 7b5, both D- and L-Phe
reacted with (R,R)-3+Zn(II) completely to give the corresponding
1
products with well-defined sharp H NMR signals. Singlets at 
8.32 and 8.38 in Figures 7b4 and 7b5, can be assigned to the imine
protons in the proposed products 4 from the condensation of the
aldehyde groups of (R,R)-3 with D- and L-Phe respectively in
DMSO-d6 in the presence of Zn(OAc)2.
Figure 5. (a) Fluorescence intensity at 540 nm of (R,R)-3 (2.0 mL, 0.040
mM in PFOH, 1.0 equiv.) with. D- and L-His (2.0 mL, in 3.0 mM TBAH
aqueous solution, 1.0 – 14.0 equiv) in the presence of Zn(OAc)2 (20 μL, 8
mM in H2O, 2.0 equiv). (b) Fluorescence spectra of (R,R)-3 with D- and
L-His (12.0 equiv) in the presence of TBAH (3.0 mM) and Zn(II) (2.0
equiv). All the mixtures were stirred at rt for 4 h, and fluorescence was
measured 10 min after the mixing was stopped. The error bars were
obtained from three independent experiments. λexc = 435 nm. Slit: 4/4 nm.
3
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10.1002/chem.201900880
Chemistry - A European Journal
their different mass spectra indicate very different stability of
these products. The NMR and mass spectroscopic studies suggest
that both D- and L-Phe could react with (R,R)-3 to generate the
corresponding diastereomeric complexes like
4 and these
diastereomers should have different stability and structural
rigidity leading to the observed enantioselective fluorescence
responses.
The reactions of (R,R)-3 with D- and L-His in PFOH and
1
DMSO-d6 gave H NMR spectra very similar to those shown in
Figures 7a and 7b respectively. In both solvents, the aldehyde
signal of (R,R)-3 completely disappeared to give broad signals in
PFOH and sharp and well-defined signals in DMSO-d6 (Figure
S12). In the mass spectrum for the reaction of (R,R)-3 with D-His
in PFOH, a weak signal at m/z 1542.4 was observed for the
proposed double condensation product 5 (calcd for [M-H]-:
1542.3) (Figure S13a). This signal was not observable for the
reaction of (R,R)-3 with D-His in DMSO and with L-His in both
PFOH and DMSO (Figure S13b-d). A peak at m/z = 1406.3 was
observed for the reaction of D-His with (R,R)-3 in both PFOH and
DMSO solutions (Figure S13a and S13c) which can be attributed
to the mono condensation product 6 (Calcd for [M-H]-: 1406.2).
This peak was not observed in the mass spectra for the reaction of
(R,R)-3 with L-His in PFOH and DMSO (Figure S13b and S13d).
1
Although the H NMR spectra show complete reaction of the
aldehyde groups of (R,R)-3 with the amino acid, the
corresponding double condensation products might not be stable
under the conditions of the mass spectroscopic experiments and
give only very weak or nonobservable signal.
H
H
O
F
F
NH
NH
O
O
O
N
O
O
N
O
F
F
F
F
F
F
N
N
H
H
N
N
O
O
F
F
O
O
O
O
Zn
Zn
1
F
F
F
F
O
F
F
Figure 7. (a) H NMR spectra of (R,R)-3 (1.0 equiv, 1.2 mM in PFOH)
N
F
F
H
after being treated with D- and L-Phe (14.0 equiv, 1.0 mL, 16.8 mM, in
16.8 mM TBAH aqueous solution) in the presence of Zn(II) (1.0 equiv, 20
μL, 60 mM in H2O). (b) 1H NMR spectra of (R,R)-3 (1.0 equiv, 0.40
mM) in DMSO-d6 after being treated with D- and L-Phe (14.0 equiv, 100
μL, 56.0 mM, in 56.0 mM TBAH D2O solution) in the presence of Zn(II)
(1.0 equiv, 20 μL, 20 mM in D2O). The mixtures were all stirred at rt for
4 h. The intense signals from the solvent PFOH in the upfield were
removed for clarity. The full spectra are given in figure S10.
N
F
F
F
F
F
F
F
F
F
F
O
CHO
HO
NH
F
F
F
F
H
F
F
5
6
5. Determination of the enantiomeric composition of the
crude Phe products prepared from an enzyme-catalyzed
asymmetric reaction
We have applied the probes (R,R)- and (S,S)-3 to evaluate the
enantiomeric composition of an amino acid product generated in
an enzyme-catalyzed asymmetric hydrolysis of a racemic amino
acid ester. Previously, Kise and coworkers reported that racemic
amino acid esters such as D-/L-Phe-O-Me can undergo highly
enantioselective hydrolysis in the presence of the enzyme α-
chymotrypsin (CT) to generate L-Phe (Scheme 2).10 We carried
out this asymmetric hydrolysis in various solvent systems and
used (R,R)- and (S,S)-3 to determine the ee of the crude product
Phe under the biphasic conditions according to Figure 4. On the
basis of the fluorescence spectra of the crude products in Figure
S14, we estimated the ee’s of these products obtained from
various conditions as shown in Table 1. All the samples greatly
enhanced the fluorescence of (S,S)-3 in PFOH at 533 nm, but
caused much weaker fluorescence enhancement on (R,R)-3. This
shows that the hydrolysis product is predominately L-Phe rather
than D-Phe. The ee’s obtained by the fluorescence measurement
under the biphasic recognition are close to those determined by
HPLC as shown in Table 1. This demonstrates that the fluorous
phase-based fluorescent probe can be used to screen the reaction
conditions for the asymmetric synthesis of the amino acid.
H
F
O
O
N
O
F
N
H
F
O
F
F
O
O
O
Zn
F
O
F
F
F
H
N
F
F
F
F
O
F
H
F
F
F
4
The mass spectra (MALDI-TOF, DCTB negative mode) of the
reaction mixtures of (R,R)-3 with D- and L-Phe in PFOH and
DMSO were obtained (Figure S11). In PFOH, the reaction of
(R,R)-3 with D-Phe gave a peak at m/z 1562.6 as shown in Figure
S11a, consistent with the proposed complex 4 (calcd for [M-H]-:
1562.3), but a much weaker peak at m/z 1562.4 was observed for
the reaction of (R,R)-3 with L-Phe in PFOH (Figure S11b). In
DMSO, the reaction of (R,R)-3 with D-Phe also gave an intense
peak at m/z 1562.3 (Figure S11c), but this peak was not observed
for the reaction of (R,R)-3 with L-Phe (Figure S11d). Although
1
Scheme 2. An Enzyme-Catalyzed Enantioselective Hydrolysis
the H NMR spectra in Figures 7b4 and 7b5 show that (R,R)-3
for the Asymmetric Preparation of L-Phe.
reacted with D- and L-Phe in DMSO to give similar products,
4
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Chemistry - A European Journal
CO2Me
CO2H
Synthesis and Characterization of (R,R)-3. (a) A mixture of 1
(263 mg, 0.355 mmol, 1.0 equiv), (R)-2'-hydroxy-2-
(methoxymethoxy)-[1,1'-binaphthalene]-3-carbaldehyde [(R)-2,
318 mg, 0.887 mmol, 2.5 equiv], potassium carbonate (196 mg,
1.42 mmol, 4.0 equiv), 18-crown-6 (9 mg, 0.036 mmol, 0.1 equiv)
and N,N-dimethylformamide (5.0 mL) was heated at 50 oC for 21
h at which TLC showed the completion of the reaction. The
mixture was poured into cold water (30 mL), and then extracted
with ethyl acetate (2×25 mL). The combined organic layer was
washed with water (1×30 mL) and brine (2×30 mL), and dried
-chymotrypsin (CT)
+
H2O
NH3Cl
NH2
L-Phe
Et3N
various solvents
D-/L-Phe-O-Me
racemic mixture
Table 1. Comparison of ee values of the crude Phe productsa
from the enzymatic hydrolysis of D/L-Phe-O-Me determined by
fluorescence (FL) and HPLC analyses
Yield ee/%
ee/%
Deviation
/%
Product
Phe-1
Phe-2
Phe-3
Phe-4
Phe-5
Phe-6
Phe-7
Solvent
/%
(FL) (HPLC)b
with anhydrous Na2SO4.
After filtration, the filtrate was
Acetonitrile
(10% H2O)
Acetonitrile
(20% H2O)
Acetonitrile
(30% H2O)
2-Propanol
(10% H2O)
1-Propanol
(10% H2O)
Ethanol
concentrated under reduced pressure to give a crude product as a
yellow oil, which was purified by column chromatography on
silica gel by gradient elution with 15-30% ethyl acetate in hexane
to afford the dimethoxymethyl protected product diMOM-(R,R)-3
as a light yellow solid (296 mg) in 64% yield. 1H NMR (600
MHz, CDCl3) δ 10.55 (s, 2H), 8.54 (s, 2H), 8.01 (dd, J = 8.7, 5.6
Hz, 4H), 7.89 (d, J = 8.1 Hz, 2H), 7.44 – 7.19 (m, 16H), 6.01 (s,
2H), 5.10 (d, J = 3.8 Hz, 4H), 4.71 (d, J = 6.0 Hz, 2H), 4.61 (d, J
= 6.1 Hz, 2H) , 2.93 (s, 6H), 2.14-2.17 (m, 2H), 1.92-1.95 (m,
2H). 13C NMR (150 MHz, CDCl3) δ 191.06, 166.03, 162.64,
158.31, 154.04, 153.71, 137.08, 133.88, 130.97, 130.68, 130.28,
130.15, 129.35, 129.29, 128.25, 127.31, 127.09, 126.14, 126.09,
126.08, 125.12, 124.30, 118.52, 114.19, 105.64, 100.37, 65.80,
57.20, 36.80-14.23 (m). 19F NMR (564 MHz, CDCl3) δ -80.74 (t,
J = 9.7 Hz, 3F), -114.25 (m, 2F), -121.58 – -121.59 (m, 2F), -
121.78– -121.88 (m, 4F), -122.61 – -122.68 (m, 2F), -123.06 – -
78
90.1
87.4
82.3
90.3
83.7
81.8
91.5
99.8
96.2
94.2
99.5
94.5
94.8
99.8
-9.7
-8.8
64
75
34
50
36
78
-11.9
-9.2
-10.8
-13.0
-8.3
(10% H2O)
Acetone
(10% H2O)
aDL-phenylalanine methyl ester hydrochloride: 45 mM; Et3N: 45 mM;
o
CT: 12 μΜ; Solvent: 20 mL; 25 C，24 h. ee = ([L] - [D])/([L] + [D].
Yield based on the amount of L-Phe formed in theory.
bDetermined by Daicel Chiral Technologies (China) Co., LTD (Figure
S18).
123.12 (m, 2F), -126.03 – -126.09 (m, 2F). [α]2D3 = +7.8 (c = 1.0,
CHCl3).
(b) A mixture of diMOM-(R,R)-3 (239 mg, 0.182 mmol, 1.0
equiv), conc. HCl (2.0 mL), ethanol (1.0 mL) and chloroform (1.0
mL) was heated at reflux for 9 h. The resulting mixture was
cooled to rt, and a saturated NaHCO3 solution was added
dropwise into the mixture until no gas evolved. The mixture was
poured into H2O (30 mL) and extracted with ethyl acetate (1×30
mL). The organic layer was washed with H2O (1×20 mL) and
brine (1×20 mL), and then dried with anhydrous Na2SO4. After
filtration, the filtrate was concentrated under reduced pressure to
give a crude product as a yellow solid, which was purified by
column chromatography on silica gel by gradient elution with 15-
25% ethyl acetate in hexane to afford (R,R)-3 as a yellow solid
(181 mg) in 83% yield. 1H NMR (600 MHz, CDCl3) δ 10.53 (s,
2H), 10.16 (s, 2H), 8.27 (s, 2H), 7.96 (d, J = 9.0 Hz, 4H), 7.90 (d,
J = 8.4 Hz, 2H), 7.41 (d, J = 9.0 Hz, 2H), 7.36-7.39 (m, 6H), 7.30
(t, J = 8.4 Hz, 2H), 7.26-7.25 (m, 2H), 7.22 (d, J = 8.4 Hz, 2H),
6.21 (s, 2H), 5.15 (m, 4H), 3.37-3.32 (m, 2H), 2.24-2.18 (m, 2H),
1.99-1.94 (m, 2H). 13C NMR (150 MHz, CDCl3) δ 196.72,
165.93, 158.66, 153.70, 153.62, 137.97, 137.61, 133.75, 130.50,
130.39, 129.73, 129.57, 128.36, 127.66, 126.96, 125.57, 124.91,
124.41, 124.11, 122.32, 118.81, 117.80, 114.71, 105.72, 70.95,
65.64, 32.05-14.22 (m). 19F NMR (564 MHz, CDCl3) δ -80.72 (t,
J = 9.9 Hz, 3F), -114.19 (m, 2F), -121.82 – -121.86 (m, 6F), -
122.60 – -122.67 (m, 2H), -123.05 – -123.10 (m, 2H), -126.02 – -
126.08 (m, 2H). HRMS Calcd for C60H39F17NO7 (MH+):
6. Conclusion
We have demonstrated that a fluorophilic probe (R,R)-3 in
combination with Zn(II) can carry out biphasic fluorescent
recognition of structurally diverse amino acids with fair to
excellent enantioselectivity. This study represents the first
example for the enantioselective fluorescent recognition of amino
1
acids in the fluorous phase. The H NMR and mass analyses of
the reaction of (R,R)-3 with D- and L-amino acids indicate the
formation of imine-Zn(II) complexes from the condensation of the
aldehyde groups of the probe with the amino acid enantiomers
and the differences in stability and structural rigidity of these
complexes should have contributed to the observed
enantioselective fluorescent responses. The biphasic amino acid
recognition has been used to determine the enantiomeric
composition of the crude products generated by an enzyme-
catalyzed asymmetric preparation of a chiral amino acid under
various conditions which shows potential of this method in chiral
catalyst screening.
Experiment Section
General Data. All synthetic reactions were carried out under
nitrogen atmosphere unless otherwise noted. DL-phenylalanine
methyl ester hydrochloride was purchase from Chem-Impex
International, Inc. Other chemicals were purchased from Sigma
Aldrich Chemical Co. or Alfa Aesar. The fluorous solvent PFOH
was purchased from SynQuest Laboratories. Other solvents used
in the fluorescent measurement were HPLC grades. Optical
rotations were measured on a Jasco P-2000 digital polarimeter.
NMR spectra were recorded on a Varian-600 MHz spectrometer.
1208.2455, found: 1208.2439. [α]2D3 = +143.1 (c = 2.0, CHCl3).
Synthesis and Characterization of (S,S)-3. Compound (S,S)-3,
the enantiomer of (R,R)-3, was obtained as a yellow solid in a
two-step yield of 75% by using the same procedure described
above but starting with (S)-2. The 1H/13C/19F NMR data of (S,S)-
3 were consistent with those of (R,R)-3. [α]2D3 = – 142.3 (c = 2.0,
CHCl3).
1
Chemical shifts for H NMR spectra were reported in parts per
million relative to solvent signals at 7.26 ppm for CDCl3, 2.05
ppm for acetone-d6, 2.50 ppm for DMSO-d6. Chemical shifts for
13C NMR were reported relative to the centerline of a triplet at
77.16 ppm for CDCl3. Steady-state fluorescence emission spectra
were recorded on Horiba FluoroMax-4 spectrofluorometer. Mass
spectra were obtained from the University of Illinois at Urbana-
Champaign (UIUC) Mass Spectrometry Facility.
Enzymatic Hydrolysis of DL-Phenylalanine Methyl Ester
Hydrochloride.
Typically, DL-phenylalanine methyl ester
hydrochloride (194 mg, 0.9 mmol, 1.0 equiv) was dispersed in an
5
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6
10.1002/chem.201900880
Chemistry - A European Journal
organic solvent (18 mL), and then an equimolar amount of
triethylamine (125 μL, 0.9 mmol, 1.0 equiv) and an aqueous
solution (2.0 mL) of enzyme CT (6.0 mg) were successively
added to the organic suspension. The final concentration of DL-
phenylalanine methyl ester hydrochloride was 45 mM, and the
total volume of solvent was 20 mL, containing 10% (in volume)
H2O. For the conditions with the use of acetonitrile containing
20% and 30% H2O, the ester salt (194 mg) was first dispersed in
16 and 14 mL acetonitrile respectively, and then an equimolar
amount of triethylamine and an aqueous solution (4.0 and 6.0 mL)
of the enzyme CT (6.0 mg) were successively added to the
organic suspension. The reaction mixture was stirred at rt for 24
h. At the end of the 3 reactions in acetonitrile (10% H2O), 2-
propanol (10% H2O) and acetone (10% H2O), precipitates were
collected by filtration and dried under vacuum to give crude
products Phe-1 (58.1 mg, 78% yield), Phe-4 (25.5 mg, 34%
yield), Phe-7 (57.9 mg, 78% yield). The reactions in the other 4
solvents including acetonitrile (20% H2O), acetonitrile (30%
H2O), 1-propanol (10% H2O) and ethanol (10% H2O) gave only
small amount of precipitate because of better solubility of Phe in
these solvents. The solvents were first removed under vacuum,
and then 9.0 mL acetonitrile and 1.0 mL H2O were added to the
mixture. The precipitates were collected by filtration and dried
under vacuum to give crude products Phe-2 (47.7 mg, 64% yield),
Phe-3 (55.7 mg, 75% yield), Phe-5 (37.3 mg, 50% yield) and Phe-
6 (26.9 mg, 36% yield). The crude products Phe-1 ~ Phe-7 were
directly used for ee measurement by using the fluorescence probe
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solutions of 1.2 mM (R,R)-3 or (S,S)-3 in PFOH, 2.0 or 3.0 M
TBAH aqueous solutions and 4.0 or 8.0 M Zn(OAc)2 aqueous
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Acknowledgement: This work was partially supported by the US
National Science Foundation (CHE-1565627), National Natural
Science Foundation of China (Nos. 21877087, 21602164,
21402148), Wuhan International Scientific and Technological
Cooperation Project (No. 2017030209020257), and Wuhan
Institute of Technology Scientific Research Fund (No. K201716).
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Supplementary Materials Available: Additional experimental
description and spectroscopic data.
Keywords:
Fluorous phase, enantioselective recognition,
fluorescent probe, amino acid, BINOL
6
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10.1002/chem.201900880
Chemistry - A European Journal
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7
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