A simple quantitative chiral analysis of amino acid esters by fluorine-19 nuclear magnetic resonance using the modified James–Bull method

Article abstract of DOI:10.1002/chir.23028

A simple chiral analysis of amino acid esters by fluorine-19 nuclear magnetic resonance (¹⁹F?NMR) through the modified James–Bull method is described. Thus, amino acid ester acid salt was treated with 5-fluoro-2-formylphenylboronic acid and (S)-BINOL in the presence of triethylamine (TEA) and MS4A for 10?minutes. The reaction mixture was analysed by ¹⁹F?NMR directly to afford good quantifications.

Full text of DOI:10.1002/chir.23028

Received: 30 July 2018
DOI: 10.1002/chir.23028
Revised: 5 September 2018
Accepted: 18 September 2018
R E G U L A R A R T I C L E
A simple quantitative chiral analysis of amino acid esters by
fluorine‐19 nuclear magnetic resonance using the modified
James–Bull method
Naoto Hamaguchi | Yuta Okuno | Yohei Oe
| Tetsuo Ohta
Department of Biomedical Information,
Abstract
Faculty of Life and Medical Sciences,
A simple chiral analysis of amino acid esters by fluorine‐19 nuclear magnetic res-
onance (19F NMR) through the modified James–Bull method is described. Thus,
amino acid ester acid salt was treated with 5‐fluoro‐2‐formylphenylboronic
acid and (S)‐BINOL in the presence of triethylamine (TEA) and MS4A for
10 minutes. The reaction mixture was analysed by 19F NMR directly to afford
good quantifications.
Doshisha University, Kyoto, Japan
Correspondence
Yohei Oe, Department of Biomedical
Information, Faculty of Life and Medical
Sciences, Doshisha University, 1‐3
Miyakodani, Tatara, Kyotanabe, Kyoto
610‐0394, Japan.
Email: yoe@mail.doshisha.ac.jp
KEYWORDS
19F NMR, amino acids, James‐bull assembly, quantification
1 | INTRODUCTION
method to 19F NMR analysis would provide a highly effec-
tive analytical method by accompanying with the strong
α‐Amino acids form the proteins in living organisms.
Therefore, the natural forms (usually L‐amino acids) are
easily available and very useful as chiral building blocks.1
In addition, D‐amino acids, which are commonly known
as an unnatural form, are currently recognized as a useful
indicator of various changes such as ageing and diseases
in mammals.2 Therefore, quantitative chiral analysis of
amino acids and their derivatives is highly important.
Chiral analysis of biogenic compounds by use of
fluorine‐19 nuclear magnetic resonance (19F NMR) is cur-
rently attracting much attention because of the high sen-
sitivity of 19F NMR and very low background signals.3 For
example, Zhao et al reported the simultaneous analysis of
chiral amines using a pincer‐type palladium complex in
which various types of primary amines including amino
alcohols can be sensed. This method can be expanded to
amine sensing in foods and drinks.4 For discrimination
of chiral amines by hydrogen‐1 (1H) NMR, the method
for assembly of a chiral amine, a chiral diol and o‐
formylphenylboronic acid, so‐called the James–Bull
method, is very useful.5 Various types of chiral amines
can be analyzed qualitatively by 1H NMR analysis.
Although it is considered that the expansion of this
points of 19F NMR described above, the quantitative anal-
ysis by 19F NMR through the James–Bull method was
scarcely investigated in detail. In 2009, James and Bull's
group reported that ee of several chiral diols can be deter-
mined through the similar derivatization of the diol,
4‐fluoro‐2‐formylphenylboronic acid and (R)‐1‐phenyleth-
ylamine ((R)‐2a).6 However, only one amine (R)‐2a
was demonstrated; nevertheless, various chiral amines
1
were tested in their quantitative analysis by H NMR.
Chaudhari and Suryaprakash independently reported the
discrimination of chiral 1‐arylethylamines by 19F NMR
through a modified James–Bull method using (R,R)‐cyclo-
hexane‐1,2‐dicarboxylic acid as a chiral source and 3‐
fluoro‐2‐formylphenylboronic acid in 2012.7 However,
unequal intensity pattern of 19F NMR signals of the reac-
tion mixture with several racemic amines was observed
and the quantitative analysis failed. We investigated a
detailed investigation of stoichiometric chiral analysis of
α‐amino acid esters by 19F NMR through a modified
James–Bull method, in which triethylamine (TEA) was
found to be a very useful additive for successful assembly.
Here, we will present a simple chiral analysis of amino
acid esters by 19F NMR.
Chirality. 2018;1–7.
wileyonlinelibrary.com/journal/chir
© 2018 Wiley Periodicals, Inc.
1

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HAMAGUCHI ET AL.
2 | MATERIALS AND METHODS
2.1 | General
3.2 | Optimization of analytical conditions
With 19F NMR reporter 1c, we then optimized the reac-
tion and measurement conditions for chiral quantitative
analysis of amino acid esters by 19F NMR. First, molar
ratio and several additives were tested, and the results
Amino acid esters acid salts were prepared according to
the literature procedure.8 Deuterated solvents were pur-
chased from the Cambridge Isotope Laboratories, Inc.
and used without further purifications. Unless noted oth-
erwise, all reagents and solvents were obtained from com-
mercial suppliers and used without further purifications.
NMR spectra were recorded with the Bruker Ascend
1
are summarized in Table 1. Thus, H and 19F NMR spec-
tra of the reaction mixture of ( )‐phenylalanine methyl
ester (2b) with 1c and (S)‐BINOL in CDCl3, with or with-
out additives, were measured. When 2b with an equal
molar amount of 1c and (S)‐BINOL were mixed and
reacted, two signals appeared on the 19F NMR spectrum
with an integral ratio of 1:1.40 (entry 1). These two sig-
nals were confirmed as the corresponding diastereomeric
imine (S,R)‐ and (S,S)‐3cb by the reaction using optically
pure (R)‐ and (S)‐2b. In this case, the starting amino acid
ester 2b remained and 3cb was obtained in only 77%
yield. The apparent disproportionation of the diastereo-
mer ratio of 3cb was considered to be due to the kinetic
resolution. Various reaction conditions were tested in
order to increase the chemical yield of 3cb. Although
the reaction was performed in toluene‐d8 and CD2Cl2 to
afford 3cb in 73% and 58% yield, respectively, the diaste-
reomer ratios of 3cb were not improved (entries 2 and 3).
While amino acid ester 2b still remained when the
reaction was carried out in toluene‐d8, an almost full con-
sumption of 2b was observed by use of CD2Cl2. However,
the imine that was generated by the reaction of 1c and 2b
without (S)‐BINOL was afforded as a by‐product, which
was presumably troublesome in the quantitative chiral
analysis. Increasing the amount of aldehyde 1c (1.1 eq)
and (S)‐BINOL (1.2 eq) affected these two values to afford
3cb in an 88% yield with a 1:1.14 diastereomer ratio
(entry 4). Use of a further excess amount of (S)‐BINOL
(1.5 eq) improved the diastereomer ratio up to 1:1.09
(entries 5 and 6). Because the present analysis is per-
formed on 19F NMR spectra, it was considered that the
addition of organic amines such as TEA would not pre-
vent the quantification so much. Indeed, the addition of
TEA effectively improved the conversion (entries 9 and
10), and the best diastereomer ratio (1;1.05) was obtained
when the reaction of 2b with 1.1 eq of 1c and 1.5 eq of
(S)‐BINOL was performed in CDCl3, in the presence of
MS4A and TEA (0.5 eq) (entry 10).
1
400 spectrometer (400 MHz for H, 100 MHz for 13C,
and 376 MHz for 19F) by using tetramethylsilane (TMS)
1
(δ = 0 ppm) as an internal standard for H NMR, and
CDCl3 (δ = 77 ppm) for carbon‐13 (13C) NMR.
2.2 | Quantitative analysis
Amino acid ester acid salt (0.03 mmol) and TEA (1.5 eq,
4.6 μL) were suspended in CDCl3 (0.3 mL). 5‐Fluoro‐2‐
formylphenylboronic acid (1c) (1.1 eq), (S)‐BINOL
(1.5 eq), 4A molecular sieves, and CDCl3 (0.7 mL) were
added. The solution was stirred for 10 minutes and then
analyzed by 19F NMR spectroscopy
3 | RESULTS AND DISCUSSION
3.1 | Effects of fluorinated Benzaldehydes
Initially, we tested several fluorinated benzaldehydes 1a‐c
as the 19F NMR chiral reporter in James' method
(Figure 1). Thus, a reaction of ( )‐1‐phenylethylamine
(2a), (S)‐BINOL, and aldehyde 1a‐c in CDCl3 was per-
formed at room temperature for 10 minutes, and then
1H and 19F NMR were measured. When 3‐fluoro‐2‐
formylphenylboronic acid (1a) was used, two signals were
obtained on the 19F NMR spectrum. The same reactions
by use of optically pure (S)‐ and (R)‐2a revealed that these
two peaks are (S,R)‐3aa and (S,S)‐3aa, and the difference
in these.
19F NMR shifts (Δδ) is 0.16 ppm (Figure 1a). A benzal-
dehyde bearing fluorine atom at C4‐position 1b, interest-
ingly afforded only one signal for the corresponding
diastereomer on the 19F NMR spectrum, although two
1
diastereomers were detected on its H NMR (Figure 1‐b).
Because amino acid esters were generally prepared
as acid salts by use of chlorination reagents, such as
SOCl2,8 it is preferred that the enantiomeric excesses
are determined directly by use of these salts. Thus, the
use of phenylalanine hydrochloride (2b·HCl) was exam-
ined (Table 2). In James' previous report,5b amino acid
ester hydrochloride was preliminarily treated with
Cs2CO3, and then the insoluble inorganic species were
removed by filtration. They also reported that the use of
5‐fluoro‐2‐formylphenylboronic acid (1c) was found as a
good chiral 19F NMR reporter as well as 1a from the view
point of peak separation (Figure 1c). Furthermore, the
assembly proceeded nicely to afford a 93% yield of 3ca,
whereas the yields of 3aa (28%) and 3ba (33%) were much
lower, presumably because of the steric hindrance and/or
electronic effects. Therefore, we selected 1c as a 19F NMR
chiral reporter in this work.

HAMAGUCHI ET AL.
3
H
Ph
H
Ph
N
O
N
O
O
F
F
3
B
O
B
O
4
F
5
NH2
(S)-BINOL
H
2
+
CDCl3, r. t., 10 min.
Ph
B(OH)2
1
6
1a-c
2a
3-F (1a), 4-F (1b), 5-F (1c)
(S, S)-3aa-3ca
(S, R)-3aa-3ca
FIGURE 1 Effect of fluorinated benzaldehydes 1a‐c and nuclear magnetic resonance (NMR) spectra
a slightly excess amount of Cs2CO3 sometimes caused the
problematic racemization of amino acid esters and that
this racemization could be suppressed by using K2CO3
instead of Cs2CO3. Therefore, we started from the use of
these dicarbonates as a base. When 2b·HCl was treated
with 1c and (S)‐BINOL in the presence of MS4A and a
1.1 equivalent of K2CO3, an apparent disproportionation
of the diastereomer ratio was observed (entry 1). A simi-
lar result was obtained with Cs2CO3 (entry 2). The dispro-
portionation was considered to be caused by a low
chemical yield of 3cb (less than 63% yield). On the other
hand, the use of TEA was also effective in the quantita-
tive analysis of amino acid salt. Thus, the reaction was
carried out in the presence of a 1.1 equivalent of TEA to
afford 3cb in an 89% yield with a better diastereomer
ratio (1:1.18, entry 3). Increasing the amount of TEA
and (S)‐BINOL to 1.5 eq obtained 3cb in an almost quan-
titative yield with a sufficient diastereomer ratio (1:1.06,
entry 4).
3.3 | Scope of amino acid esters
Various amino acid ester salts (2b‐n·HX) were tested
under the optimized analytical conditions, and the results
are summarized in Table 3 (Experimental details and the
NMR spectra are provided in Supporting Information). As
shown in entries 2 and 3, ( )‐phenylalanine benzyl ester
p‐toluenesulfonic acid (PTSA) salt (2c·PTSA) and its allyl
ester (2d) also showed good separation of the 19F NMR
signals and a good diastereomer ratio (1:1.06 and 1:1.04,

4
HAMAGUCHI ET AL.
TABLE 1 Optimization of analytical conditions for phenylalanine methyl ester (2b)a
Entry
1c (eq)
(S)‐BINOL (eq)
Solvent
Additive
Dr (S,R: S,S)
Yield (%)
1
2
3
4
5
6
7
8
9
1.0
1.0
1.0
1.1
1.1
1.1
1.1
1.1
1.1
1.0
1.0
1.0
1.2
1.5
2.0
1.2
1.5
1.2
CDCl3
Toluene‐d8
CD2Cl2
CDCl3
CDCl3
CDCl3
CDCl3
CDCl3
CDCl3
–
1:1.40
1:1.95
1:1.14
1:1.18
1:1.24
1:1.09
1:1.13
1:1.16
1:1.13
77
73
–
–
58
–
88
–
92
–
88
MS4A
MS4A
88
72
MS4A
>99
TEA (0.5 eq)
10
1.1
1.5
CDCl3
MS4A
1:1.05
>99
TEA (0.5 eq)
aReaction conditions: phenylalanine methyl ester (2b) was treated with 1c and (S)‐BINOL in the presence of given additives in solvent at room temperature for
10 min. The reaction mixture was analyzed by 1H and 19F NMR.
TABLE 2 Optimization of analytical conditions for phenylalanine methyl ester hydrochloride (2b•HCl)a
Entry
(S)‐BINOL (eq)
Additive (eq)
Dr (S,R: S,S)
Yield (%)
1
2
3
4
1.2
1.2
1.2
1.5
K2CO3 (1.1)
Cs2CO3 (1.1)
TEA (1.1)
1:1.79
1:1.55
1:1.18
1:1.06
45
63
89
TEA (1.5)
>99
aReaction conditions: phenylalanine methyl ester hydrochloride (2b·HCl) was treated with 1c (1.1 eq) and (S)‐BINOL in the presence of additive and MS4A in
CDCl3 at room temperature for 10 min. The reaction mixture was analyzed by 1H and 19F NMR.
respectively). Tyrosine methyl ester hydrochloride
(2e·HCl) and tryptophan methyl ester hydrochloride
(2f·HCl) were also successfully separated to afford the
corresponding imines 3ce and 3cf with a 1:1.07 and
1:1.07 diastereomer ratio, respectively (entries 4 and 5).
Compared with these arylalanine derivatives, small
Δδ values were obtained with the other amino acid
esters. For example, a Δδ value of the corresponding
diastereomers 3cg derived from aspartic acid methyl ester
hydrochloride (2g·HCl) was only 0.12 ppm (entry 6).

HAMAGUCHI ET AL.
5
TABLE 3 Scope of amino acid estersa
19F NMR shift
Dr
Entry
Amino acid ester 2
HX
Δδ
(S,R: S,S)
1
2
3b
R' = me (2b)
R' = Bn (2c)
R' = Allyl (2d)
HCl
PTSA
–
0.36
0.41
0.39
1:1.06
1:1.06
1:1.04
4
R' = me (2e)
HCl
0.35
1:1.07
5
6
7
8
R' = me (2f)
R' = me (2g)
R' = me (2h)
R' = me (2i)
HCl
HCl
HCl
HCl
0.34
0.12
0.04
0.08
1:1.07
1:1.08
1:1.07
1:1.08
9
R' = me (2j)
R' = me (2j)
R' = Bn (2k)
HCl
HCl
PTSA
0.01
0.07
0.06
n.d.d
1:1.04
1:1.05
10c
11
12
R' = me (2l)
R' = me (2l)
R' = Bn (2m)
R' = Allyl (2n)
HCl
HCl
PTSA
–
0.03
0.00
0.03
n.d.e
n.d.d
n.d.d
1:1.01
n.d.d
13c
14
15b
aReaction conditions: amino acid derivatives 2·HX was treated with 1c (1.1 eq.) and (S)‐BINOL (1.5 eq) in the presence of TEA (1.5 eq) and MS4A in CDCl3 at
room temperature for 10 min. The reaction mixture was analyzed by 1H and 19F NMR.
bTEA (0.5 eq) was used.
cCD2Cl2 was used instead of CDCl3.
dNot determined.
eNot distinguished.
Further decreasing Δδ was obtained with valine
(2h·HCl) and with leucine (2i·HCl) derivatives (entries
7 and 8). Although, only 0.01 ppm differences in 19F
chemical shifts of the corresponding diastereomeric
imines derived from ( )‐alanine methyl ester hydrochlo-
ride (2j·HCl) were observed when CDCl3 was used as a
solvent (entry 9), the use of CD2Cl2 instead of CDCl3
slightly improved the Δδ value (0.07 ppm) and enabled
us to determine the diastereomer ratio (1:1.06, entry
10). Fortunately, 19F NMR signals of the corresponding
diastereomers of alanine benzyl ester PTSA salt
(2k·PTSA) appeared with Δδ = 0.06 and dr = 1:1.05
even in CDCl3 (entry 11). Phenylglycine methyl ester
hydrochloride (2l·HCl) did not obtain a good Δδ value
in both CDCl3 and in CD2Cl2, and the diastereomer
ratios were not determined (entries 12 and 13). Use of
its benzyl ester PTSA salt 2m·PTSA afforded almost
the same Δδ as in CDCl3 (0.03 ppm); however, the dia-
stereomer ratio could be determined because of the
sharpness of the signals (entry 14). 19F shifts of the

6
HAMAGUCHI ET AL.
diastereomers derived from phenylglycine allyl ester (2n)
were not discriminated at all (entry 15).
(Experimental details and the NMR spectra are provided
in Supporting Information), suggesting that the present
19F NMR method resulted in the highly quantitative
chiral analysis.
A mixed solution of D‐ and L‐amino acid methyl
esters was analyzed. Thus, a mixed solution of 20% ee of
L‐phenylalanine methyl ester hydrochloride (2b·HCl),
80% ee of D‐2f·HCl, and racemic 2k·PTSA was prepared
and treated with 1c and (S)‐BINOL under the optimized
conditions. As a result of the 19F NMR analysis, ee
values of L‐2b, D‐2f, and 2k were determined in
17.9%, 82.2%, and almost racemate (1.0% ee), respectively
(Figure 3; Experimental details and the NMR spectra
are provided in Supporting Information). This result
suggested that enantio‐purities of multiple amino acid
esters could be analyzed at one time.
3.4 | Quantitative chiral analysis of amino
acid esters
Various ee of 2e·HCl (75, 50, 20, 0, −20, −50, and −75%
ee) were prepared. The ee values were determined by
both chiral HPLC and the present 19F NMR method and
the correlation of these ee values are shown in Figure 2
4 | CONCLUSION
We have investigated the quantitative chiral analysis of
amino acid derivatives through the James–Bull method,
in which TEA was found to be effective for the reaction
conversion without significant loss of enantiomeric
excess. The operation is very easy, and the accuracy of
the quantification is sufficient. ee values of a mixture of
alanine benzyl ester, tryptophan methyl ester, and phe-
nylalanine methyl ester could be simultaneously deter-
mined through our present method.
FIGURE 2 Comparison of ee values of tyeosine methyl ester
hydrochloride (2e·HCl) between HPLC and the present method
FIGURE 3 19F NMR spectrum of a mixed solution of L‐phenylalanine methyl ester hydrochloride (2b·HCl, 20% ee), D‐tryptophan methyl
ester hydrochloride (2f·HCl, 80% ee), and DL‐alanine benzyl ester PTSA salt (2k·PTSA)

HAMAGUCHI ET AL.
7
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ORCID
Yohei Oe http://orcid.org/0000-0001-5298-3852
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How to cite this article: Hamaguchi N, Okuno
Y, Oe Y, Ohta T. A simple quantitative chiral
analysis of amino acid esters by fluorine‐19 nuclear
magnetic resonance using the modified James–Bull
method. Chirality. 2018;1–7. https://doi.org/
10.1002/chir.23028