One-step preparation of enantiopure l- or d-amino acid benzyl esters avoiding the use of banned solvents

Article abstract of DOI:10.1007/s00726-017-2400-y

The enantiomers of amino acid benzyl esters are very important synthetic intermediates. Many of them are currently prepared by treatment with benzyl alcohol and p-toluenesulfonic acid in refluxing benzene or carbon tetrachloride, to azeotropically remove water, and then precipitated as tosylate salt by adding diethyl ether. Here, we report a very efficient preparation of eight l- or d-amino acid benzyl esters (Ala, Phe, Tyr, Phg, Val, Leu, Lys, Ser), in which these highly hazardous solvents are dismissed using cyclohexane as a water azeotroping solvent and ethyl acetate to precipitate the tosylate salt. With some work-up modifications and lower yield, the procedure can be applied also to methionine. Chiral HPLC analysis shows that all the benzyl esters, including the highly racemizable ones such as those of phenylglycine, tyrosine and methionine, are formed enantiomerically pure under these new reaction conditions thus validating the solvents replacement. Contrariwise, toluene cannot be used in place of benzene or carbon tetrachloride because leading to partially or totally racemized amino acid benzyl esters depending on the polar effect of the amino acid α-side chain as expressed by Taft’s substituent constant (σ*).

Full text of DOI:10.1007/s00726-017-2400-y

Amino Acids
DOI 10.1007/s00726-017-2400-y
ORIGINAL ARTICLE
One‑step preparation of enantiopure l‑ or d‑amino acid benzyl
esters avoiding the use of banned solvents
Cristiano Bolchi1 · Francesco Bavo1 · Marco Pallavicini1
Received: 25 January 2017 / Accepted: 22 February 2017
© Springer-Verlag Wien 2017
Abstract The enantiomers of amino acid benzyl esters are
very important synthetic intermediates. Many of them are
currently prepared by treatment with benzyl alcohol and
p-toluenesulfonic acid in refluxing benzene or carbon tet-
rachloride, to azeotropically remove water, and then pre-
cipitated as tosylate salt by adding diethyl ether. Here, we
report a very efficient preparation of eight l- or d-amino
acid benzyl esters (Ala, Phe, Tyr, Phg, Val, Leu, Lys, Ser),
in which these highly hazardous solvents are dismissed
using cyclohexane as a water azeotroping solvent and ethyl
acetate to precipitate the tosylate salt. With some work-up
modifications and lower yield, the procedure can be applied
also to methionine. Chiral HPLC analysis shows that all
the benzyl esters, including the highly racemizable ones
such as those of phenylglycine, tyrosine and methionine,
are formed enantiomerically pure under these new reaction
conditions thus validating the solvents replacement. Con-
trariwise, toluene cannot be used in place of benzene or
carbon tetrachloride because leading to partially or totally
racemized amino acid benzyl esters depending on the polar
effect of the amino acid α-side chain as expressed by Taft’s
substituent constant (σ*).
Keywords Amino acid benzyl ester · Water azeotrope ·
Racemization · Chiral HPLC · Cyclohexane · Toluene ·
Taft’s substituent constant
Introduction
The enantiomers of α-amino acid benzyl esters are very
important synthetic intermediates. Apart from their obvi-
ous wide use, documented by a great number of papers,
in peptides synthesis as C-protected amino acids deriva-
tives, easily deprotectable after peptide bond formation,
they are often employed to construct also more complex
chiral molecules of non-peptide nature. In fact, unlike the
carbohydrates derivatives, the α-amino acid esters and, in
particular, the benzyl esters are available in both the enan-
tiomeric forms from the ‘chiral pool’. Furthermore, they
do not suffer from that unneeded profusion of chirality,
carbon atoms and reactive functions, which is typical of
carbohydrates and which makes necessary multiple pro-
tecting groups and costly transformations to smaller, more
useful species. Recent examples can be cited to exemplify
the use of α-amino acid as benzyl esters for the synthesis
of non-peptide molecules in unichiral form: azetidinone-
isothiazolidinones from l-alanine and l-phenylglycine
(Cerić et al. 2010), 4-methylcarboxy-1,4-benzodiazepine-
2,5-diones from l-alanine and l-leucine (Verdié et al.
2008), α-methylene-β-hydroxy-γ-carboxy-γ-lactams from
l-phenylalanine, l-valine, l-leucine and l-threonine (Tek-
kam et al. 2013), isochaetominines from d- or l-valine, ala-
nine and tryptophan (Mao et al. 2016), differently substi-
tuted piperazine-2,5-diones (Liu et al. 2007) and imidazo
acetates (Liu et al. 2010).
Handling Editor: P. Meffre.
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-017-2400-y) contains supplementary
material, which is available to authorized users.
* Marco Pallavicini
marco.pallavicini@unimi.it
1
Dipartimento di Scienze Farmaceutiche, Sezione “Pietro
Our interest in α-amino acid esters has built on their
use as unichiral synthons (Bolchi et al. 2007; Bolchi et al.
Pratesi”, Università degli Studi di Milano, Via Mangiagalli
25, 20133 Milan, Italy
1 3

C. Bolchi et al.
2015b) and on their potentially useful α,β-dehydrogenation
(Pallavicini et al. 2010) and then it has focused on some
important issues in the preparation and analytical char-
acterization of the dibenzyl esters of l-aspartic acid and
l-glutamic acid such as the esterification conditions, the
enantiomeric purity and the nature of the enantiomeric sys-
tems (Bolchi et al. 2015a).
Within this context, we have successfully proposed
the replacement of benzene and carbon tetrachloride with
cyclohexane, a solvent considered utilizable on an indus-
trial scale and surely much more acceptable on the basis
of overall health, environment, and safety criteria, and of
diethyl ether with 2-propanol and ethyl acetate, whose use
is recommended (Prat et al. 2016). In refluxing cyclohex-
ane, water is azeotropically removed at relatively low tem-
perature and the conversion of l-aspartic and l-glutamic
acid into the respective dibenzyl esters is complete without
racemization (Bolchi et al. 2015a). These results prompted
us to extend our investigation over other amino acids. To
comparatively evaluate our procedure using cyclohexane,
we considered those amino acid benzyl ester tosylates for
which literature long provides well-established examples of
preparation by treatment with benzyl alcohol and p-tolue-
nesulfonic acid in a water azeotroping solvent (see Table 1).
Apart from achiral glycine and the previously investigated
aspartic and glutamic acid, these are alanine (1), phenylala-
nine (2), tyrosine (3), phenylglycine (4), valine (5), leucine
(6), lysine (7) and serine (8). On the other hand, histidine,
asparagine, glutamine and threonine benzyl esters tosylates
are just cited as reactants (Kempf et al. 1991; Zhao et al.
2009; Abiko and Sekino 1983; Nishizawa and Saino 1977;
Bose et al. 1982) but, to the best of our knowledge, their
preparations have never been individually described, while
those of tryptophan and arginine are, in different respects,
problematic (Arai and Muramatsu 1983; Magnus et al.
1989; Biondini et al. 2010; Dorman and Cheng 1976) and
cysteine benzyl ester tosylate is unknown. Of the remaining
proline and methionine benzyl esters tosylates, for which
one and two preparations with only partial characterizations
are reported respectively (Dai and Etzkorn 2009; Kawa-
saki et al. 1980; Fayad et al. 2015), we decided to include
methionine (9). Therefore, we have considered the prepara-
tion of the benzyl esters tosylates 1a–8a and, in addition,
the methionine benzyl ester tosylate 9a, after verifying that
literature methods prescribe, for the nine esters, the use of
refluxing benzene, toluene, carbon tetrachloride or benzyl
alcohol itself regardless of safety and of racemization and
provide very few or no analytical data on the enantiomer
systems formed by the products and on their enantiomeric
composition (Fig. 2).
The simplest and most practiced way to obtain the enan-
tiomers of many α-amino acid benzyl esters is the treatment
of the unichiral amino acid, as such, with excessive benzyl
alcohol. The reaction is usually performed in the presence
of little more than 1 equiv of p-toluenesulfonic acid, both
to catalyze the reaction and to recover the α-amino-ester
as a crystalline and stable p-toluenesulfonate salt, and in a
solvent forming a heterogeneous azeotrope with water to
remove the water produced by the esterification and thus
to make the conversion quantitative (Zervas et al. 1957)
(Fig. 1). Unbelievably, in the literature procedures, ben-
zene, or even carbon tetrachloride, remains the most used
solvent and diethyl ether is widely employed to precipitate
the ester salt, although all these solvents are undesirable or
banned on any scale production. Furthermore, the optical
rotatory power and the melting point of the ester are usu-
ally the only analytical data provided besides the spectral
data, while the enantiomeric excess is almost never deter-
mined although amino acids and their derivatives are long
known to racemize with relative ease (Matsuo et al. 1967;
Manning 1970; Sato et al. 1970; Bada 1972; Smith and
Evans 1980; Dhaon et al. 1982; Smith and Sivakua 1983).
After analyzing the p-toluenesulfonates of l-aspartic
and l-glutamic acid dibenzyl esters and the respective race-
mates, prepared according to the above long-established
procedures (Bergmeier et al. 1993; Zervas et al. 1957), by
DSC and chiral HPLC effective in enantioseparation, we
have been able to critically revisit the literature prepara-
tive procedures based on treatment of the two acidic amino
acids with benzyl alcohol and on azeotropic water removal
under different conditions. To our surprise, we have found
that the enantiomeric purity of these diesters is almost
always and often wrongly taken for granted, even in works
in which the unichirality of the products is a central issue
of the investigation. Consequently, water azeotroping sol-
vents are chosen regardless not only of their environmental
hazard but also of the potential racemization of the ester.
Indeed, toxic or environmentally hazardous solvents, such
as benzene and carbon tetrachloride, in which racemization
does not take place probably due to the low water azeotrope
boiling point, are currently used in laboratory preparations.
Alternatively, someone replaces them, on both small and
large scale, by higher boiling toluene or benzyl alcohol
itself whilst ignoring that extensively racemized aspartic
and glutamic dibenzyl esters are formed under these more
severe azeotropic reflux conditions.
Here, we report the preparations of the enantiomerically
pure benzyl esters p-toluenesulfonates 1a–9a by treatment
with benzyl alcohol and p-toluenesulfonic acid in refluxing
cyclohexane together with their melting data and the ana-
lytical chiral HPLC resolutions of the respective racemates.
The same preparations were experimented in refluxing tol-
uene to demonstrate that more or less pronounced racemi-
zation instead occurs under these conditions. Benzene and
carbon tetrachloride were excluded from our experiments
for obvious safety reasons.
1 3

One-step preparation of enantiopure l- or d-amino acid benzyl esters avoiding the use of…
Table 1 Literature data for compounds 1a–9a
Cpda
Solventsb
mp °C
Yield %
References
1a
Benzene
BnOH
BnOHc
Toluene
Benzene
CCl4
113, 118
84,100
76
Gibian and Schroeder (1961); Tayama et al. (2012)
Winitz et al. (1956); Izumiya and Makisumi (1957)
Sato et al. (2004); Hirata (2009)
113,114
–
84–98
68
–
Poras and Kunesch (1998)
2a
165, 171
95
Zervas et al. (1957); Schwarz and Arakawa (1959); Deal and Welch (1997)
Xu et al. (2002)
188
96.5
79
Toluene
Benzene
Benzene
Toluene
Benzene
BnOH
–
Gray et al. (1991)
3a
4a
179
88
Zervas et al. (1957); Sato et al. (2008)
Akazome et al. (2000)
191
98
–
89
Llewellyn and Arndtsen (2005)
5a
6a
7a
157, 160
79
Zervas et al. (1957); Munegumi et al. (2012)
Izumiya and Makisumi (1957)
157
Toluene
Benzene
BnOH
156
82,91
Thaqi et al. (2008); Tagami et al. (2000)
Zervas et al. (1957); Gershon and Krull (1979); Winn et al. (1997)
Izumiya and Makisumi (1957)
153, 160
99,100
154
Toluene
Benzene
BnOH
–
70, 93
88
Larbig and Schmidt (2006); Harju et al. (2009); Fayad et al.(2015)
Takeoka et al. (2000)
–
148
13
Izumiya and Makisumi (1957); Hachisako et al. (1999)
Katsarava et al. (2007)
Toluene
CCl4
–
8a
9a
65,80, 94
Fölsch (1959); Boente et al. (1991); Holden et al. (2002)
Kawasaki et al. (1980)
Benzene
Toluene
133
–
56
75
Fayad et al. (2015)
a
In a few cases, data are of the other enantiomer
b
c
Used in the esterification reaction at reflux with water collecting Dean–Stark apparatus
In the presence of additives (hydrazines) or under argon or under reduced pressure
optical rotations. An exception is represented by two pat-
ents which denounce the risk of racemization in the prep-
aration of 1a and claim that it can be avoided, in toluene,
by adding a small quantity of phenylhydrazine or operat-
ing under inert atmosphere (Sato et al. 2004) and, in ben-
zyl alcohol, by applying a reduced pressure which allows
water to be distilled off at lower temperature (Hirata
2009).
Therefore, as the enantiomeric purity of the products
represents a central issue in these preparations, our inves-
tigation, aimed at developing a new procedure to prepare
enantiomerically pure 1a–9a without using hazardous
solvents, started from this last critical point. The race-
mates of the nine amino acid benzyl esters tosylates were
prepared in refluxing cyclohexane as below described
for 1a–9a and chiral HPLC methods were developed to
analytically resolve the enantiomers of the benzyl esters
possibly as such, namely without derivatization after
liberation from the tosylate by simple dichlorometh-
ane/aqueous carbonate extraction. Then, supported by
this indispensable analytical tool, we prepared 1a–9a.
Results and discussion
The state of the art in the preparation of the benzyl
esters 1a–9a is summarized in Table 1, where we exem-
plify what can be found for each aminoacid benzyl
ester, in unichiral l or d form, in the literature, namely
the solvents used in the esterification reaction, the melt-
ing points of the isolated p-toluenesulfonates and the
respective yields. Briefly, these data can be commented
as follows: (a) benzene is the most used solvent and its
use still persists, while toluene starts to be extensively
employed in the nineties, carbon tetrachloride seems to
be abandoned after 2000 and few examples are reported
for benzyl alcohol, mainly by some Japanese researchers;
(b) concordance between melting points is poor and often
these even lack, mostly in the preparations using toluene;
(c) the yields are generally very high with the significant
exception of 7a in benzyl alcohol and of 9a in benzene;
(d) last but the most important thing, the datum concern-
ing the enantiomeric composition is always absent, some-
times subrogated by a questionable comparison between
1 3

C. Bolchi et al.
Table 2 Comparison between the outcome of the esterification in
cyclohexane and in toluene
reaction was prolonged over night. After cooling to room
temperature, ethyl acetate was added to precipitate the
ester salt as a white solid. Exceptions to this general pro-
cedure were: (a) the use of 2.2 equivalents of p-toluene-
sulfonic acid for lysine esterification (7a), (b) the conver-
sion of the p-toluenesulfonate salt 8a, which separates as
an oil at the end of the reaction, into the high melting and
easy crystallisable hydrochloride and (c) the liberation
of l-methionine benzyl ester from the p-toluenesulfonate
salt, which separates as an oily phase at the end of the
reaction, and its precipitation as 9a by treatment with
TsOH in ethyl acetate. By the same procedure, we pre-
pared 1a–9a in refluxing toluene.
In Table 2, the results of the preparations in the two sol-
vents are summarized and compared for each amino acid.
First, let’s consider 1a–8a and then, separately, 9a. The ester
salts 1a–8a are obtained in one-step and in very high yield.
The esterification of the amino acids 1–8 with benzyl alcohol
in refluxing cyclohexane takes place without evident racemi-
zation: no traces of the other enantiomer can be detected in
the HPLC chromatograms of the benzyl esters liberated from
the salts precipitated in very high or quantitative yield at the
end of the reaction. Only the benzyl esters of d-phenylgly-
cine and of l-lysine were found to contain a minimum quan-
tity of the opposite enantiomer (0.6 and 0.5% respectively).
The tosylates 1a–7a were precipitated by adding ethyl ace-
tate with yields ranging from 88 to 100%, while oily 8a was
converted into hydrochloride with an overall process yield
of 82%. On the contrary, with the exception of valine, the
esterification in toluene was accompanied by extensive race-
mization. Furthermore, in the case of amino acids with polar
alkyl side chain, such as lysine and serine, the chemical yield
of the benzylation in toluene was so modest that racemiza-
tion became a secondary issue and the quantification of the
enantiomeric excess not interesting in the isolated benzyl
ester salt and problematic in the complex reaction mixture.
Temperature near 100 °C is known to accelerate the
amino acid racemization (Smith and Sivakua 1983; Smith
and Reddy 1989) and indeed our chiral HPLC analyses
Cpd
Cyclohexane
mp °Ca
Toluene
Yield% ee%c ee%c
[α]bD
1a
2a
3a
4a
5a
6a
7a
109.95 (114.33) −3.78 92
169.03 (148.39) −7.48 94
178.20 (150.49) −14.10 92
192.59 (193.59) −35.21 95
161.89 (123.31) −3.36 88
159.26 (159.33) −2.09 100
162.89 (202.14) −5.93 85
100 80.8
100 75.3
100 21.5
98.8 ~0
100 100
100 89.7
99.0 –e
e
Hydrochloride 179.39
from 8a
(100.99d)
9a
−4.67 82
100
–
129.51 (132.05) +0.95 40
95.7 27.0
a
Melting peak maximum of the enantiomer in the DSC trace and, in
brackets, that of the racemate
b
In methanol (c 1) at 25 °C; in water (c 1) at 25 °C in the case of 7a
c
Determined on the liberated ester by chiral HPLC
d
Of the racemic p-toluenesulfonate
e
The ester yield is poor and byproducts are formed
.
NH2
NH2 TsOH
O
BnOH, TsOH
OH
*
R
R
Bn
*
refluxing water
azeotroping solvent
O
O
Fig. 1 General method to prepare amino acids benzyl esters and to
isolate them as p-toluenesulfonic acid salts
Cyclohexane was selected among usable solvents because
the physical properties of its water azeotrope (boiling
point, water content, and reciprocal solubility with water)
are very similar to those of benzene.
The amino acids were treated with 1.2 equivalents
of p-toluenesulfonic acid and 5 equivalents of benzyl
alcohol in refluxing cyclohexane for 4 h so as to azeo-
tropically remove water. In the case of methionine, the
HO
.
.
.
.
.
NH2
TsOH
TsOH
NH2
NH2 TsOH
NH2 TsOH
NH2
O
TsOH
O
O
O
O
O
Bn
Ph
Bn
Bn
Bn
Bn
O
O
O
O
Ph
2a
3a
5a
1a
4a
.
.
.
.
.
TsOH
NH2 TsOH
NH2 TsOH
S
NH2 TsOH
NH2
NH2 TsOH
O
HO
O
O
O
Bn
Bn
Bn
Bn
O
O
O
O
6a
7a
8a
9a
Fig. 2 l- or d-amino acid benzyl esters prepared as p-toluenesulfonates in cyclohexane
1 3

One-step preparation of enantiopure l- or d-amino acid benzyl esters avoiding the use of…
100
show how this parameter is important among the reaction
conditions: the only 20–30 °C enhancement for replace-
ment of cyclohexane with higher boiling toluene as a water
Val
Leu
Ala
80
Phe
azeotroping solvent promotes racemization with an extent
varying from amino acid to amino acid. In particular, we
60
observed that, in toluene, the racemization increases in the
40
Met
0,3
y = -125,09x + 78,811
2 = 0,8937
order valine < leucine < alanine < phenylalanine < tyros-
ine to become quantitative for phenylglycine (Table 2).
According to several investigators, loss of a proton from the
α-methine is the initial necessary step in amino acid race-
mization and, consequently, factors favoring the methine
dissociation and stabilizing the incipient carbanion facili-
tate racemization (Smith and Evans 1980). Among these
factors, several studies agree that the most important ones
are the protonation of the α-amine function, the formation
of small amounts of the conjugate acid (COOH2+) promot-
ing enolization, the esterification of the carboxyl group pre-
cluding its conversion to –COO− and, discriminant among
the various amino acids, the electron-with-drawing capacity
of the side chain, better if not α-branched (Manning 1970;
Bada 1972; Smith and Sivakua 1983). The reported race-
mizability rankings put phenylglycine first; phenylalanine,
alanine and leucine follow in sequence at a great distance,
whilst valine is last with practically null racemization (Sato
et al. 1970; Smith and Evans 1980). Such racemization
susceptibility is well linearly correlated to the electron-
egativity of the side chain, expressed by the Taft’s polar
constant (σ*), decreasing from phenyl (0.60, Phg) to ben-
zyl (0.225, Phe), methyl (0.00, Ala), i-butyl (−0.125, Leu)
and i-propyl (−0.20, Val) (Sato et al. 1970). Indeed, i-pro-
pyl appears to be a trade-off between inductive and steric
effects (α-branching) and, compared to i-butyl, it depresses
racemization more than expected from its σ* value, only
slightly smaller than that of i-butyl (Sato et al. 1970). As
shown in Table 2 and in Fig. 3, the extent of racemization
of our corresponding benzyl esters, prepared in toluene,
parallels the above ranking and is acceptably correlated to
side chain σ* with complete racemization of phenylglycine
benzyl ester and null racemization of valine benzyl ester at
the opposite extremities and intermediate racemizations for
phenylalanine, alanine and leucine benzyl esters.
R
20
0
Phg
0,6
-0,3 -0,2 -0,1
0
0,1
0,2
0,4
0,5
0,7
Amino acid side-chain σ∗
Fig. 3 Plot of the enantiomeric excesses of the benzyl esters of
valine, leucine, alanine, phenylalanine, methionine and phenylglycine
prepared in toluene against the σ* values of the amino acids side-
chain
In contrast, under the acidic conditions of our esterifica-
tion, the electron-with-drawing capacity of unionized
p-hydroxybenzyl exceeds that of benzyl and results in the
higher racemization of tyrosine. The higher electron-with-
drawing capacity of p-hydroxybenzyl compared to benzyl
can be inferred, for instance, from the lower basicity of
tyrosine and of tyramine in comparison with phenylalanine
(Nagai et al. 2008) and phenylethylamine (Martin 1971;
Brubacher and Bender 1966) respectively.
Coming to the methionine ester 9a, it is to be noted
that the older preparation in refluxing benzene has a 56%
yield and is supported by melting point and optical rota-
tion as analytical data (Kawasaki et al. 1980), while the
more recent one in refluxing toluene has a 75% yield but
provides neither melting point nor optical rotation (Fayad
et al. 2015). In both cases, the enantiomeric excess is an
unknown issue. Our results using cyclohexane are in line
with those of the older preparation in benzene. The yield
is modest and 9a, which separates as an oil at the end of
the reaction, cannot be precipitated as a solid directly from
the reaction mixture. First, benzyl alcohol and p-toluene-
sulfonic are removed by extractions, and then 9a is precip-
itated as a pure solid from ethyl acetate by adding p-tol-
uenesulfonic acid. Anyway, chiral HPLC analyses show
that both the crude ester and the crystallized salt have high
enantiomeric excess (95.6 and 95.7% respectively), when
the preparation is accomplished in refluxing cyclohexane,
while a wide racemization (27.0% e.e.) occurs when tolu-
ene is used. A sample of 9a, prepared in refluxing benzene
according to the literature procedure, exhibited the same
enantiomeric excess (95.6%) as that obtained in cyclohex-
ane. The wide extent of racemization in toluene, near to
that of tyrosine and much higher than those of amino acids
with alkyl side chains, is consistent with previous observa-
tions on the ready racemization of methionine under both
With regard to tyrosine, the literature studies (Liardon
and Ledermann 1986) indicate a racemizability slightly
lower than that of phenylalanine under alkaline conditions,
while we found that its benzyl ester, prepared in toluene, is
much more racemized (21.5% e.e.) than that of phenylala-
nine (75.3%). The p-hydroxybenzyl side chain has an ion-
izable phenol function and its electron-with-drawing capac-
ity, unlike the unionizable side chains of the above amino
acids, is pH-dependent. Reasonably, at the alkaline pHs of
the racemization studies, it is less electronegative than ben-
zyl, because negatively charged, and consequently the race-
mization of tyrosine is lower than that of phenylalanine.
1 3

C. Bolchi et al.
acidic (Manning 1970) and alkaline conditions (Smith and
Reddy 1989; Liardon and Ledermann 1986). It is imput-
able to the thioether group, namely to the electron-with-
drawing capacity of the methylthioethyl side-chain, which
is sensibly higher than that of alkyl chains. We have not
found the σ* value of the methionine side-chain in the lit-
erature. Therefore, we have considered the reported σ* of
the following two series of alkyloxy and alkylthio residues:
(a) propyloxy (1.57), ethoxymethyl (0.50) and methoxy-
ethyl (0.24) and (b) propylthio (1.38) and ethylthiomethyl
(0.56) (Hansch and Leo 1979; Hansch et al. 1995). Based
on these values and on their parallel disposition, for which
σ* decreases by shifting both O and S towards the end of
the three carbon chain, we have extrapolated a near 0.3 σ*
value for the lacking methylthioethyl residue and we have
used it to insert methionine into the enantiomeric excess/
side-chain plot. As shown in Fig. 3, the position of methio-
nine benzyl ester results acceptably in line with those of the
other amino acids esters thus confirming the correlation of
the racemization with the electron-with-drawing capacity
of the amino acid side-chain.
We were also interested in gaining information about
the nature of the enantiomeric systems formed by the nine
amino acid benzyl ester p-toluenesulfonates in the solid
state, because they are isolated by precipitation/crystalliza-
tion. The DSC analyses show that the enantiomers of the
p-toluenesulfonates of the benzyl ester of phenylalanine,
tyrosine and valine melt at temperatures sensibly higher
than the respective racemates thus suggesting that their
enantiomeric systems are conglomerates. However, such a
suggestion is confirmed only for 3a, whose IR spectrum is
superimposable to that of the racemate, and not for 2a and
5a, which have IR spectra different from those of the race-
mates. On the other hand, it can be excluded that conglom-
erates are formed by the enantiomers of the other p-tolue-
nesulfonates, which melt about at the same temperature as
the corresponding racemates (alanine, phenylglycine, leu-
cine and methionine benzyl ester) or lower (lysine and ser-
ine benzyl ester).
diethyl ether. The enantiopurity of the amino acids benzyl
esters prepared in refluxing cyclohexane and the racemiza-
tion of the same esters prepared in refluxing toluene were
demonstrated by chiral HPLC analysis. The racemization
is favored by the high boiling temperature of toluene and
increases with the electronegativity of the amino acid side
chain, as indicated by the good correlation found between
enantiomeric excess and Taft’s polar substituent constant of
the amino acid side chain. Methionine, tyrosine and phe-
nylglycine benzyl esters showed the highest susceptibility
to racemize.
Materials and methods
1H NMR spectra were recorded on a Varian Gemini 300
operating at 300 MHz. Chemical shifts are reported in ppm
relative to residual solvent (CHCl3, DMSO and metha-
nol) as internal standard. The melting points were deter-
mined by DSC analysis and the melting peak maximum
is reported for each compound. The DSC curves were
recorded and integrated with the aid of a TA Instruments
DSC 2010 apparatus. Optical rotations were determined
in a 1 dm cell of 5 mL capacity using a Perkin-Elmer 241
polarimeter. Enantiomeric excesses were determined by
chiral HPLC on 150 mm × 4.6 mm i.d. columns at 210 or
220 nm.
General procedure for the preparation of 1a–9a
The esterifications were carried out on l amino acids with
the exception of phenylglycine, the d enantiomer of which
was used. A mixture of amino acid (0.05 mol), p-toluene-
sulfonic acid (0.06 mol), benzyl alcohol (0.25 mol) and
cyclohexane (30 mL) was refluxed for 4 h using a Dean-
Stark apparatus to separate water that was azeotroped out
as it formed. The reaction mixture was cooled to room
temperature and ethyl acetate (80 mL) was added. After
stirring for 1 h, the precipitate was collected by filtration
and dried to give the corresponding benzyl ester p-tolue-
nesulfonate as a white solid. According to this procedure,
the amino acids 1–6 were converted into the correspond-
ing benzyl ester p-toluenesulfonates 1a–6a. The benzyla-
tion of 7 was accomplished in the same manner but in the
presence of more p-toluenesulfonic acid (0.11 mol) to give
the di-p-toluenesulfonate 7a as a white solid. The p-tolue-
nesulfonate 8a separated at the end of the reaction as an
oil; instead of adding ethyl acetate, the supernatant was
removed, the oily phase was washed with cyclohexane and
then poured into dichloromethane/aqueous Na2CO3. After
removing the water layer and evaporating dichlorometh-
ane, the residue was treated with hydrochloric methanol to
Conclusions
In conclusion, we report a new straightforward procedure to
obtain a series of enantiomerically pure amino acid benzyl
esters in excellent yield by treatment of the amino acid with
benzyl alcohol without using water azeotroping solvents
that are highly hazardous, such as benzene and carbon
tetrachloride, or that cause racemization, such as toluene.
We accomplished the esterification in refluxing cyclohex-
ane, which is not hazardous and forms an aqueous azeo-
trope similar to that of benzene, and precipitated the esters
as p-toluenesulfonates by adding ethyl acetate instead of
1 3

One-step preparation of enantiopure l- or d-amino acid benzyl esters avoiding the use of…
give the corresponding hydrochloride as a white solid. The
benzylation of 9 was prolonged over night and, at the end
of the reaction, 9a separated as an oil, which was poured
into dichloromethane/water. After removing the organic
layer, the water phase was made alkaline with NaHCO3 and
extracted with ethyl acetate. The organic extract was con-
centrated to a small volume and a slight excess of p-tolue-
nesulfonic acid was added to precipitate 9a as a white crys-
talline solid.
The racemic benzyl esters were prepared in the same
manner as the enantiomeric forms and all the p-toluenesul-
fonates precipitated as white solids by adding ethyl acetate,
excluding only that of methionine benzyl ester, which was
recovered as described for 9a.
1H NMR (300 MHz, DMSO-d6) δ 2.27 (s, 3 H), 2.98 (m, 2
H), 4.26 (m, 1 H), 5.14 (s, 2 H), 6.66 (d, J = 7.6 Hz, 2 H),
6.93 (d, J = 7.6 Hz, 2 H), 7.09 (s, 2 H), 7.26 (s, 2 H), 7.34 (s,
3 H), 7.48 (d, J = 7.6 Hz, 2 H), 8.37 (br s, 3 H), 9.38 (br s, 1
H).
rac‑Tyrosine benzyl ester p‑toluenesulfonate
Mp 150.49 °C; HPLC analysis as for 3a: the d enantiomer
corresponds to the second eluted peak (tR = 20.0 min).
d‑Phenylglycine benzyl ester p‑toluenesulfonate (4a)
Obtained as a white solid in 95% yield: mp 192.59 °C;
[α]2D5 = −35.21 (c 1, MeOH); 98.8% e.e. (determined by
HPLC analysis on a Phenomenex Lux 3 μ Cellulose-1 col-
umn; hexane/MeOH/EtOH 79/13/7, 1 mL/min; 220 nm,
l‑Alanine benzyl ester p‑toluenesulfonate (1a)
1
Obtained as a white solid in 92% yield: mp 109.95 °C;
[α]2D5 = −3.78 (c 1, MeOH); 100% e.e. (determined by
HPLC analysis on a Phenomenex Lux 3 μ Cellulose-1 col-
umn; hexane/iPrOH 9/1, 1 mL/min; 220 nm, tR = 8.0 min);
1H NMR (300 MHz, CDCl3) δ 1.46 (d, J = 7.6 Hz, 3H),
2.30 (s, 3H), 4.04 (m, 1H), 4.99 (d, J = 12.3 Hz, 1H), 5.08
(d, J = 12.3 Hz, 1H), 7.06 (d, J = 7.6 Hz, 2H), 7.22–7.29
(m, 5H), 7.72 (d, J = 7.6 Hz, 2H), 8.24 (br s, 3H).
tR = 7.25 min); H NMR (300 MHz, CDCl3) δ 2.30 (s, 3
H), 4.94 (d, J = 12.3 Hz, 1 H), 5.00 (d, J = 12.3 Hz, 1 H),
5.13 (m, 1 H), 7.02 (m, 2 H), 7.14–7.30 (m, 10 H), 7.51 (d,
J = 8.2 Hz, 2 H), 8.70 (br s, 3 H).
rac‑Phenylglycine benzyl ester p‑toluenesulfonate
Mp 193.59 °C; HPLC analysis as for 4a: the l enantiomer
corresponds to the first eluted peak (tR = 6.6 min).
rac‑Alanine benzyl ester p‑toluenesulfonate
l‑Valine benzyl ester p‑toluenesulfonate (5a)
Mp 114.33 °C; HPLC analysis as for 1a: the d enantiomer
corresponds to the second eluted peak (tR = 8.7 min).
Obtained as a white solid in 88% yield: mp 161.89 °C;
[α]2D5 = −3.36 (c 1, MeOH); 100% e.e. (determined by
HPLC analysis on a Phenomenex Lux 3 μ Cellulose-1 col-
umn; hexane/iPrOH 9/1, 1 mL/min; 220 nm, tR = 5.3 min);
1H NMR (300 MHz, CDCl3) δ 0.88 (pt, J = 6.4 Hz, 6
H), 2.18 (m, 1 H), 2.31 (s, 3 H), 3.90 (m, 1 H), 5.01 (d,
J = 12.3 Hz, 1 H), 5.12 (d, J = 12.3 Hz, 1 H), 7.09 (d,
J = 8.2 Hz, 2 H), 7.28 (s, 5 H), 7.75 (d, J = 8.2 Hz, 2 H),
8.23 (br s, 3 H).
l‑Phenylalanine benzyl ester p‑toluenesulfonate (2a)
Obtained as a white solid in 94% yield: mp 169.03 °C;
[α]2D5 = −7.48 (c 1, MeOH); 100% e.e. (determined by
HPLC analysis on a Phenomenex Lux 3 μ Cellulose-1 col-
umn; hexane/iPrOH 9/1, 1 mL/min; 220 nm, tR = 9.2 min);
1H NMR (300 MHz, CDCl3) δ 2.30 (s, 3 H), 3.12 (m, 1 H),
3.23 (m, 1 H), 4.32 (m, 1 H), 4.88 (d, J = 12.3 Hz, 1 H),
4.99 (d, J = 12.3 Hz, 1 H), 6.95-7.26 (m, 12 H), 7.70 (d,
J = 7.6 Hz, 2 H), 8.27 (br s, 3 H).
rac‑Valine benzyl ester p‑toluenesulfonate
Mp 123.31 °C; HPLC analysis as for 5a: the d enantiomer
corresponds to the second eluted peak (tR = 5.9 min).
rac‑Phenylalanine benzyl ester p‑toluenesulfonate
Mp 148.39 °C; HPLC analysis as for 2a: the d enantiomer
corresponds to the second eluted peak (tR = 10.9 min).
l‑Leucine benzyl ester p‑toluenesulfonate (6a)
Obtained as a white solid in quantitative yield: mp 159.26
°C; [α]2D5 = −2.09 (c 1, MeOH); 100% e.e. (determined
by HPLC analysis on a Phenomenex Lux 3 μ Amyl-
ose-2 column; hexane/iPrOH 95/5, 1 mL/min; 220 nm,
l‑Tyrosine benzyl ester p‑toluenesulfonate (3a)
Obtained as a white solid in 92% yield: mp 178.20 °C;
[α]2D5 = −14.10 (c 1, MeOH); 100% e.e. (determined by
HPLC analysis on a Phenomenex Lux 3 μ Amylose-2 col-
umn; hexane/iPrOH 95/5, 1 mL/min; 220 nm, tR = 18.5 min);
1
tR = 8.3 min); H NMR (300 MHz, CDCl3) δ 0.74 (pt,
J = 5.3 Hz, 6 H), 1.66 (m, 3 H), 2.30 (s, 3 H), 3.96 (m, 1
H), 5.02 (d, J = 12.3 Hz, 1 H), 5.11 (d, J = 12.3 Hz, 1 H),
1 3

C. Bolchi et al.
1
7.07 (d, J = 8.2 Hz, 2 H), 7.27 (s, 5 H), 7.74 (d, J = 8.2 Hz,
1 mL/min; 210 nm, tR = 11.3 min); H NMR (300 MHz,
CDCl3) δ 1.84 (s, 3H), 2.10 (m, 2H), 2.30 (s, 3H), 2.43
(m, 2H), 4.14 (m, 1H), 5.00 (d, J = 12.3 Hz, 1H), 5.12 (d,
J = 12.3 Hz, 1H), 7.08 (d, J = 7.0 Hz, 2H), 7.24–7.30 (m,
5H), 7.73 (d, J = 7.0 Hz, 2H), 8.35 (br s, 3H).
2 H), 8.26 (br s, 3 H).
rac‑Leucine benzyl ester p‑toluenesulfonate
Mp 159.33 °C; HPLC analysis as for 6a: the d enantiomer
corresponds to the first eluted peak (tR = 7.70 min).
rac‑Methionine benzyl ester p‑toluenesulfonate
l‑Lysine benzyl ester di‑p‑toluenesulfonate (7a)
Mp 132.05 °C; HPLC analysis as for 9a: the d enantiomer
corresponds to the first eluted peak (tR = 8.9 min).
Obtained as a light yellow solid in 85% yield: mp 162.89
°C; [α]2D5 = −5.93 (c 1, H2O); 99.0% e.e. (determined by
HPLC analysis of the liberated and N,N’-dibenzoylated
amino acid on a Phenomenex Lux 3 μ Cellulose-1 column;
Compliance with ethical standards
1
hexane/iPrOH 8/2, 2 mL/min; 220 nm, tR = 14.1 min); H
Conflict of interest The authors declare that they have no conflict of
interest.
NMR (300 MHz, CD3OD) δ 1.34–1.58 (m, 2 H), 1.66 (m,
2 H), 1.92 (m, 2 H), 2.36 (s, 6 H), 2.86 (t, J = 7.6 Hz, 2 H),
4.09 (t, J = 6.4 Hz, 1 H), 5.24 (d, J = 12.3 Hz, 1 H), 5.31
(d, J = 12.3 Hz, 1 H), 7.22 (d, J = 8.2 Hz, 4 H), 7.37 (m, 5
H), 7.70 (d, J = 8.2 Hz, 4 H).
Ethical approval This article does not contain any studies with
human participants or animals performed by any of the authors.
Informed consent Informed consent was obtained from all individual
participants included in the study.
rac‑Lysine benzyl ester di‑p‑toluenesulfonate
Mp 202.14 °C; HPLC analysis as for 7a: the N,N’-diben-
zoylated d enantiomer corresponds to the first eluted peak
(tR = 5.0 min).
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