Preparation of enantiopure methionine, arginine, tryptophan, and proline benzyl esters in green ethers by Fischer–Speier reaction

Article abstract of DOI:10.1007/s00726-018-2599-2

The simplest way to prepare the tosylate salts of amino acid benzyl esters, whose enantiomers are very important synthetic intermediates, is treatment of amino acid with benzyl alcohol and p-toluenesulfonic acid in a refluxing water-azeotroping solvent (Fischer–Speier esterification). However, to this day, the literature proposes only hazardous solvents, such as benzene, carbon tetrachloride, and chloroform, which must be absolutely avoided, or solvents, such as toluene and benzyl alcohol, which cause racemization because of too high boiling water azeotropes. On the other hand, the alternative successful use of cyclohexane, which we have recently reported for several amino acid benzyl esters, is inapplicable or not very efficient for ‘problematic’ amino acid such as tryptophan, arginine, and methionine, for which, indeed, the simple Fischer–Speier esterification is not described or poorly exemplified in the literature. Therefore, more polar solvents, in particular the green ethers CPME, TAME, and Me-THF, were selected and first considered for the preparation of methionine benzyl ester, previously accomplished in cyclohexane with modest yield. After discarding CPME and TAME, because causing racemization and decomposing under acidic conditions, respectively, we focused on Me-THF. In this ether, the benzyl esters of Met, Arg, and Trp could be obtained in good yield and, as proved by chiral HPLC or H NMR analysis, enantiomerically pure. The procedure was successfully extended to proline benzyl ester, which could be prepared enantiomerically pure and in quantitative yield both in cyclohexane and in Me-THF, thus avoiding the recently reported use of carbon tetrachloride.

Full text of DOI:10.1007/s00726-018-2599-2

Amino Acids
https://doi.org/10.1007/s00726-018-2599-2
ORIGINAL ARTICLE
Preparation of enantiopure methionine, arginine, tryptophan,
and proline benzyl esters in green ethers by Fischer–Speier reaction
Cristiano Bolchi1 · Francesco Bavo1 · Luca Regazzoni1 · Marco Pallavicini1
Received: 23 March 2018 / Accepted: 1 June 2018
© Springer-Verlag GmbH Austria, part of Springer Nature 2018
Abstract
The simplest way to prepare the tosylate salts of amino acid benzyl esters, whose enantiomers are very important synthetic
intermediates, is treatment of amino acid with benzyl alcohol and p-toluenesulfonic acid in a refuxing water-azeotroping
solvent (Fischer–Speier esterifcation). However, to this day, the literature proposes only hazardous solvents, such as benzene,
carbon tetrachloride, and chloroform, which must be absolutely avoided, or solvents, such as toluene and benzyl alcohol,
which cause racemization because of too high boiling water azeotropes. On the other hand, the alternative successful use of
cyclohexane, which we have recently reported for several amino acid benzyl esters, is inapplicable or not very efcient for
‘problematic’ amino acid such as tryptophan, arginine, and methionine, for which, indeed, the simple Fischer–Speier esteri-
fcation is not described or poorly exemplifed in the literature. Therefore, more polar solvents, in particular the green ethers
CPME, TAME, and Me-THF, were selected and frst considered for the preparation of methionine benzyl ester, previously
accomplished in cyclohexane with modest yield. After discarding CPME and TAME, because causing racemization and
decomposing under acidic conditions, respectively, we focused on Me-THF. In this ether, the benzyl esters of Met, Arg, and
Trp could be obtained in good yield and, as proved by chiral HPLC or H NMR analysis, enantiomerically pure. The procedure
was successfully extended to proline benzyl ester, which could be prepared enantiomerically pure and in quantitative yield
both in cyclohexane and in Me-THF, thus avoiding the recently reported use of carbon tetrachloride.
Keywords Amino acid benzyl ester · Water azeotrope · Racemization · Chiral HPLC · Mosher’s acid · TAME · CPME ·
Me-THF
Introduction
large majority of them as enantiomers of certifed purity and
through sustainable methods remains an open challenge, too
often neglected by both synthetic and analytical chemists.
Recently, we have reported the conversion of 11 l or d amino
acids (Ala, Phe, Phg, Tyr, Val, Leu, Met, Ser, Asp, Glu,
and Lys) into enantiopure benzyl esters p-toluenesulfonate
salts by treatment with benzyl alcohol and slightly more
than stoichiometric p-toluenesulfonic acid under refux in a
water-azeotroping solvent according to the Fischer–Speier
procedure (Bolchi et al. 2015a, 2017a) (Chart 1).
Amino acid esters are very important synthetic intermedi-
ates (Bolchi et al. 2007; Liu et al. 2007; Verdié et al. 2008;
Bolchi et al. 2009; Cerić et al. 2010; Liu et al. 2010; Palla-
vicini et al. 2010; Tekkam et al. 2013; Straniero et al. 2014;
Bolchi et al. 2015b; Mao et al. 2016). However, despite
widespread and long documented use, the attainment of the
Handling Editor: P. Mefre.
For the frst time, we have successfully used cyclohexane
in such amino acid esterifcations instead of banned ben-
zene, carbon tetrachloride, and chloroform or of toluene,
which unexpectedly causes complete or partial racemization
of all the prepared benzyl esters, with the exception of that
of valine, because of its higher boiling point. We have also
demonstrated that even moderate racemizations, resulting
in relatively high enantiomeric excesses (> 75%), can be
deleterious for the many amino acid benzyl esters tosylates
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00726-018-2599-2) contains
supplementary material, which is available to authorized users.
* Cristiano Bolchi
cristiano.bolchi@unimi.it
1
Dipartimento di Scienze Farmaceutiche, Sezione “Pietro
Pratesi”, Università degli Studi di Milano, via Mangiagalli
25, 20133 Milan, Italy
Vol.:(0123456789)
1 3

C. Bolchi et al.
NH3+ TsO-
O
•
Proline, a unique amino acid for its rigid architecture,
and proline derivatives, such as the benzyl ester, have
been extensively used as chiral synthons and auxiliaries
and widely investigated for the attractive conformational
properties of diproline, oligoprolines, polyprolines, and
proline-rich peptides (Dai and Etzkorn 2009; Martin
et al. 2013; Bräuer et al. 2016; Zanna et al. 2016; Liu
and Wang 2017). However, although enantiomeric purity
is an indispensable requisite for such uses and studies,
we found no chiral HPLC determinations of the enantio-
meric excess, for instance, of starting l- or d-proline ben-
zyl ester. On the other hand, the only reported Fischer–
Speier esterifcation with benzyl alcohol was recently
accomplished in carbon tetrachloride (Dai and Etzkorn
2009).
NH2
BnOH, TsOH
+ H2O
OH
refluxing water
azeotroping solvent
(cyclohexane)
*
R
R
Bn
*
O
O
Chart 1 General method to prepare amino acids benzyl esters and to
isolate them as p-toluenesulfonic acid salts
forming racemic compounds. In fact, the enantiomeric
excesses corresponding to the eutectics in the respective
binary melting point and ternary solubility phase diagrams
are often higher than the enantiomeric excesses of the these
partially racemized benzyl esters, and thus, their successive
enantioenrichment by crystallization/trituration processes is
precluded (Bolchi et al. 2017b).
•
In a continuation of our efort to prepare enantiopure
amino acid benzyl esters under acceptable conditions, we
have extended our investigation over further amino acids.
Again, our intent was to develop efcient Fischer–Speier
esterifcation procedures avoiding racemization and using
safer solvents. We focused on l-proline, l-tryptophan, l-argi-
nine, and l-methionine benzyl esters salts 1–4 (Chart 2). The
direct conversion of these four amino acids into benzyl esters
salts by simple treatment with benzyl alcohol was a prob-
lematic challenge in itself, as stated already 60 years ago in
a systematic study on the synthesis of amino acid benzyl
ester p-toluenesulfonates by Izumiya and Makisumi (1957).
Unlike for the previously studied amino acids, the litera-
ture provides not only a very poor and inadequate analytical
characterization for both chemical and enantiomeric purity
of these benzyl esters, but also very few and often inadvis-
able or little reliable procedures. Therefore, we frst veri-
fed whether Fischer–Speier esterifcation is applicable to
these amino acids by screening acceptable water-azeotroping
solvents and then whether enantiomeric purity is preserved
under the selected esterifcation conditions. Here, we report
the results of our double-focused research.
Tryptophan is the most degradable of the proteinogenic
amino acids and literature on its direct conversion into
benzyl ester is very elusive. Only one and two dated pro-
cedures are reported to obtain the benzyl ester p-toluene-
sulfonate and hydrochloride, respectively, under condi-
tions unsuitable for scaling-up (Wilchek and Patchornik
1962; Otsuka and Inouye 1964; Arai and Muramatsu
1983). Analytical characterization is limited to melting
point and elemental analysis and, only for the hydrochlo-
ride, also to the optical rotatory power. No information
is available on the enantiomeric purity. Few more recent
reports refer to old preparations or propose very small-
scale procedures which are not ameliorative and do not
provide new or updated analytical data (Magnus et al.
1989; Biondini et al. 2010). Sufce it to say that the
frst and unique NMR spectra of the unsalifed benzyl
ester date to 2015 (Taglang et al. 2015). Poor and costly
commercial availability confrms that tryptophan ben-
zyl ester, free, or salifed is a tricky compound to work
around.
•
•
Arginine is another amino acid sui generis, because of
the highly basic distal guanidine residue, and the mono-
and di-p-toluenesulfonate of its benzyl ester are not rou-
tine derivatives. We found only one example of Fischer–
Speier esterifcation, accomplished in 1/1 benzyl alcohol/
benzene, without NMR characterization and analytical
data on enantiomeric purity (Dorman and Cheng 1976).
Methionine had been already investigated by us. As pre-
viously said, we have recently reported its conversion to
Results and discussion
For the four amino acids, results of literature survey are
briefy detailed hereafter.
.
.
NH TsOH
NH2 TsOH
S
NH2
O
.
NH2 HCl
H
N
O
O
HN
HN
O
O
Bn
Bn
Bn
Bn
.
O
O
TsOH
NH2
O
3
2
4
1
Chart 2 l-Amino acid benzyl esters salts prepared by Fischer–Speier esterifcation with benzyl alcohol
1 3

Preparation of enantiopure methionine, arginine, tryptophan, and proline benzyl esters in…
enantiopure benzyl ester by treatment with benzyl alco-
hol in refuxing cyclohexane. Our procedure avoided the
use of benzene and the almost complete racemization
observed in toluene. However, because of degradation
of the substrate, its yield was only 40%, lower than that
reported in benzene (56%) (Kawasaki et al. 1980) and far
from that claimed in toluene (75%) (Fayad et al. 2015).
we frst prepared l-phenylalanine benzyl ester in refuxing
CPME according to the reported procedures in cyclohexane
and in toluene. Chiral HPLC analysis showed that the ester
product, obtained in high yield, was extensively racemized.
We thus abandoned CPME and considered 20 degrees lower
boiling TAME, whose water azeotrope boils at 74 °C, a few
degrees higher than the water azeotropes of cyclohexane and
benzene. TAME is ranked as a ‘recommended’ solvent by
CHEM21 selection guide (Prat et al. 2016). Unfortunately,
attempts at preparing amino acid benzyl esters by treatment
with benzyl alcohol and p-toluenesulfonic acid in this water-
azeotroping solvent were unsuccessful. The most reliable
cause was the instability of tertiary ethers to acids: boiling
TAME in the presence of p-toluenesulfonic acid over-night
resulted in at least 40% degradation of the ether to 2-methyl-
2-butene, 2-methyl-1-butene, and methanol. On paper, the
third option, Me-THF, a green alternative to THF for better
health and environmental score (Byrne et al. 2016; Aul and
Comanita 2007; Aycock 2007), looked very well: low boil-
ing point (71 °C) and stability to acids so as to avoid product
racemization and solvent degradation, respectively. Indeed,
the treatment of l-methionine with a large excess of benzyl
alcohol and a slight excess of p-toluenesulfonic acid in boil-
ing Me-THF over-night and the subsequent work-up accord-
ing to the previously reported procedure in cyclohexane
aforded l-methionine benzyl ester p-toluenesulfonate with
95.7% e.e. and in 62% yield, one and a half relative to the
yield obtained in cyclohexane. In Me-THF, maximum yield
coincided with 10:1 benzyl alcohol/methionine molar ratio.
Lower ratios led to some percentage points lower yields,
while higher ratios were avoided, because the consequent
boiling point enhancement of the reaction mixture could
cause degradation and racemization.
l‑Methionine benzyl ester p‑toluenesulfonate (4)
Within this context, we started our investigation just from
methionine to improve the yield of its transformation into
benzyl ester p-toluenesulfonate by the Fischer–Speier
method while maintaining product enantiopurity high.
We reasoned that, in the case of the easily degradable
methionine, refuxing reaction in an apolar solvent such as
cyclohexane, in which the amino acid is highly insoluble,
could advantage substrate degradation over its conversion
into ester, thus lessening yield of the desired ester prod-
uct. Therefore, we searched for alternative solvents form-
ing heterogeneous water azeotropes, similar to benzene and
cyclohexane for relatively low boiling point, water-rich azeo-
tropic composition, and modest reciprocal solubility with
water, but more polar than hydrocarbons. Ethers were the
natural candidates to satisfy such requirements. The most
recent solvent selection guides and green chemistry litera-
ture, which classify solvents on the basis of EHS (environ-
mental, health, and safety) properties, unanimously rank the
classical ethers as ‘hazardous’ or ‘highly hazardous’, but
provide discordant indications on the new ethers (Prat et al.
2013, 2016; Byrne et al. 2016). These, developed to circum-
vent the issues of the previous ones, are ranked as ‘recom-
mended’ or ‘problematic’ depending on the weight given to
each of the selected solvent properties (fash point, autoigni-
tion temperature, volatility, explosive limits, freezing point,
solubility in water, persistence in the environment, peroxide
formation, stability to acids and bases, synthetic accessi-
bility, derivation from renewable resources, toxicity, etc.).
Nevertheless, rankings based on the most stringent criteria
converge to identify TAME (tert-amyl-methyl ether), CPME
(cyclopentyl-methyl ether), and Me-THF (2-methyl-tetrahy-
drofuran) as acceptable green alternatives to hazardous tra-
ditional ethers, such as diisopropyl ether, methyl-tert-butyl
ether, and THF, and also to banned or hazardous halogenated
hydrocarbons. Therefore, we considered the replacement of
cyclohexane with these ethers. First, we tested CPME, which
is particularly recommended for its high fash point, high
stability to acids, low peroxide formation, low miscibility
with water, and low toxicity (Azzena et al. 2015; Byrne et al.
2016). However, its relatively high boiling point (106 °C)
raised concerns of possible racemization of the benzyl ester,
as we had previously observed esterifying other amino acids
in boiling toluene (Bolchi et al. 2015a, 2017a). Therefore,
l‑Arginine benzyl ester mono‑p‑toluenesulfonate
(3)
Prompted by this improvement, we decided to address argi-
nine and tryptophan, two ‘difcult’ amino acids, which
we had previously tried to transform into benzyl ester in
cyclohexane or in toluene, but to no avail. To our knowl-
edge, the only detailed and reliable example of preparation
of l-arginine benzyl ester can be found in a patent (Dorman
and Cheng 1976), where the ester is isolated in 85% yield as
di-p-toluenesulfonate hydrate and characterized for melting
point, optical activity, and elemental analysis. Unfortunately,
the reaction is accomplished in benzene. We developed a
Fischer–Speier procedure in boiling Me-THF and worked up
the reaction to isolate the mono-p-toluenesulfonate, which
was found to have a higher melting point than di-p-tolue-
nesulfonate (99 vs 67 °C) and to be not hygroscopic. We
maintained the 10:1 benzyl alcohol/arginine ratio, identical
to that adopted for methionine and similar to that reported
1 3

C. Bolchi et al.
in the above patent (11:1). At the end of the reaction, after
removing the upper Me-THF phase, the downer phase was
washed with ethyl acetate, dried under vacuum, and poured
into DCM/aqueous Na2CO3 to remove p-toluenesulfonic
acid. Concentration of the DCM phase aforded pure argi-
nine benzyl ester salifed only at the more basic guanidine
residue with p-toluenesulfonic acid as a white solid in 58%
yield. A similar yield was also obtained using 5:1 benzyl
alcohol/arginine molar ratio. Attempts at removing both
p-toluenesulfonic acid molecules to recover free arginine
benzyl ester were unsuccessful: treatment with an organic
solvent and water containing stronger bases than carbonate
caused ester hydrolysis and no arginine benzyl ester could
be recovered from the organic phase. This behavior seriously
hampered our ability to determine the enantiomeric excess
of the arginine benzyl ester by chiral HPLC according to the
analytical procedures applied to the other amino acid benzyl
esters previously synthesized. Difculty in desalifying the
guanidine moiety without ester hydrolysis suggested that
we desist and rather salify also the liberated alpha-amino
group, but with the enantiomer of a chiral acid so as to
diastereomerize the two arginine benzyl ester enantiomers.
Salifcation with α-methoxy-α-trifuoromethylphenylacetic
acid (Mosher acid) ofered the promising chance of deter-
mining the enantiomeric excess of arginine benzyl ester
mono-p-toluenesulfonate by H NMR. Therefore, we sali-
fed racemic arginine benzyl ester mono-p-toluenesulfonate
with the R enantiomer of Mosher’s acid. The resulting 1:1
diastereomeric mixture of l-arginine benzyl ester tosylate
(R)-Mosher carboxylate and d-arginine benzyl ester tosylate
(R)-Mosher carboxylate was analyzed by H NMR in CD3OD
at 60 mM concentration to check whether salifcation with
a single Mosher acid enantiomer induced anisochrony (∆δ)
between the signals of the two arginine ester enantiomers.
Spectral non-equivalence (0.07 ppm ∆δ) was detected for
the proton linked to the stereogenic arginine carbon: the
chemical shift of its triplet (J = 6.4 Hz) was 4.00 ppm in
the lR stereoisomer and 4.07 ppm in the dR stereoisomer
(Fig. 1, spectrum E). The Δδ and J values were such as
to avoid any overlapping between the two triplets. NMR
spectra at 600 MHz of prepared non-equimolar lR/dR mix-
tures containing decreasing percentages of dR diastereomer
showed that the presence of the CH downfeld triplet of the
minor diastereomer was clearly detectable when higher or
equal to 1%. Based on these results and on the inspection of
the H NMR spectrum of the (R)-Mosher carboxylate of the
l-arginine benzyl ester mono-p-toluenesulfonate prepared
in Me-THF, we concluded that conversion of l-arginine into
benzyl ester in refuxing Me-THF occurs without racemiza-
tion and providing the ester with>98% e.e. (Fig. 1, spectra,
B, C and D).
l‑Tryptophan benzyl ester hydrochloride (2)
The successive challenge was the preparation of trypto-
phan benzyl ester, which has never been obtained by the
Fig. 1 H NMR signal of the proton linked to the stereogenic carbon
ester p-toluenesulfonate (R)-Mosher carboxylate (B–D) and of rac-
arginine benzyl ester p-toluenesulfonate (R)-Mosher carboxylate (E)
of
latel(-Aar)g,inoifniets bmeinxztyulreesswteirthpi-ntoclrueeansiensgulafmonoautents(Ro)f-Md-oarsghienrinceabrbeonxzyy-l
1 3

Preparation of enantiopure methionine, arginine, tryptophan, and proline benzyl esters in…
Fischer–Speier procedure. Considering the instability of
tryptophan at acidity and high temperature, some research-
ers have proposed a procedure based on the conversion
into N-carboxy-α-amino acid anhydride by treatment with
phosgene in dioxane and subsequent reaction with benzyl
alcohol and diethyl ether saturated with hydrogen chloride
at 0 °C (Wilchek and Patchornik 1962) or, alternatively, the
transesterifcation with equimolar benzyl p-toluenesulfonate,
formed from TsCl and benzyl alcohol in situ, in benzyl alco-
hol at 80 °C and in the presence of equimolar p-toluenesul-
fonic acid (Arai and Muramatsu 1983). In both cases, the
benzyl ester was isolated as hydrochloride. More recently, a
very small-scale preparation has been reported, which uti-
lizes benzyl chloride in ionic liquids afording the product
in a modest 34% yield (Biondini et al. 2010). Our attempts
at esterifying l-tryptophan with benzyl alcohol in the pres-
ence of p-toluenesulfonic acid by azeotropical removal of
water were completely unsuccessful in both boiling toluene
and cyclohexane, whereas they succeeded when we used
Me-THF and 10:1 or 5:1 benzyl alcohol/amino acid molar
ratio, as in the case of arginine. After refuxing for 24 h and
removing Me-THF under vacuum, the residue was poured
into EtOAc/aqueous Na2CO3. Concentration of the organic
layer aforded crude tryptophan benzyl ester, which was
isolated as a hydrochloride in 62% by treatment with hydro-
chloric dioxane in TAME at 0 °C. Chiral HPLC analysis
under conditions efective in enantioseparation did not show
any trace of d-tryptophan benzyl ester.
for leucine (90%) (Bolchi et al. 2017a). On the contrary,
reaction in boiling cyclohexane or Me-THF quantitatively
aforded the benzyl ester p-toluenesulfonate without any
racemization. Comparison of our preparations with literature
Fischer–Speier esterifcation in carbon tetrachloride cannot
be made, because optical rotation is not provided by the
authors and the oily consistency of the p-toluenesulfonate
hampers evaluation of melting points (Dai and Etzkorn
2009). It is, nevertheless, presumable that also the esterif-
cation in carbon tetrachloride, a, however, banned solvent,
yields enantiomerically pure proline benzyl ester because of
the low refuxing temperature. On the other hand, l-proline
benzyl ester hydrochloride, which is a high-melting solid,
is somewhat better characterized than p-toluenesulfonate in
the literature, although only for optical rotation and melting
point (Neuman and Smith 1951; Adam and Fleš 1959; Hwu
et al2002). However, in the absence of any enantiomeric
composition data, its enantiomeric purity is also question-
able depending on the preparation conditions. Our results
indicate that temperatures little higher than cyclohexane and
Me-THF boiling point can be deleterious for the l-proline
benzyl ester enantiomeric purity, which cannot thus be taken
for granted in the case of its hydrochloride either.
Conclusions
Preparing enantiopure benzyl ester salts of methionine, argi-
nine, tryptophan, and proline by the simple Fischer–Speier
procedure is, for several reasons, a non-trivial challenge, as
suggested by the very few examples in the literature. Poor
solubility and instability of the substrates add to pronounced
tendency to racemization common to almost all the amino
acids, here further documented by proline benzyl ester in
boiling toluene and phenylalanine benzyl ester in CPME at
refux. Reference analytical characterization is largely faulty
and, for enantiomeric purity, completely absent. Moreover,
cyclohexane, which can successfully replace banned or
unsuitable solvents to azeotropically remove water in the
esterifcation of several amino acids, is totally inefective
or little efcient for the esterifcation of these amino acids,
except for proline. Therefore, we searched for an alternative
solvent and developed a straightforward procedure to obtain
enantiopure methionine, arginine, tryptophan, and proline
benzyl esters in good-to-excellent yield by treatment of the
amino acid with benzyl alcohol and p-toluenesulfonic acid
in refuxing Me-THF, a green ether whose low boiling water
azeotrope allows any racemization to be avoided. Other two
green ethers were considered, CPME and TAME. However,
the former was discarded, because it causes racemization for
its high boiling point and the latter was not used for its insta-
bility to acidity. The enantiomeric excesses were determined
by chiral HPLC and, in the case of arginine benzyl ester, by
l‑Proline benzyl ester p‑toluenesulfonate (1)
We have previously reported that the benzyl esters tosylates
of neutral amino acids with unfunctionalized alkyl side
chains, such as leucine and valine, undergo limited or no
racemization when prepared in boiling toluene, whereas
those of amino acids with electron-with-drawing side
chains, aromatic or not, such as phenylglycine, tyrosine,
and methionine, extensively racemize under the same
conditions (Bolchi et al. 2017a). Beside the electron-with-
drawing capacity of the side chain, the protonation of the
α-amine function is the other important factor that facilitates
racemization favoring methine dissociation and stabilizing
the incipient carbanion (Smith and Sivakua 1983). Com-
pared to the amino acids with unfunctionalized alkyl side
chain, proline stands out for the higher basicity of its amine
function (pKa 10.60), secondary because alkylated by the
side chain (Dwyer 2005). Therefore, higher susceptibility
to racemization was expected for proline than for leucine.
Indeed, Fischer–Speier esterifcation of l-proline with ben-
zyl alcohol in boiling toluene provided the benzyl ester
p-toluenesulfonate in quantitative yield but with only 85%
enantiomeric excess, a value determined by chiral HPLC
that was signifcantly lower than that previously registered
1 3

C. Bolchi et al.
H NMR analysis of the p-toluenesulfonate (R)-mosherate.
The present procedures widen the application feld of the
Fischer–Speier esterifcation from amino acids containing
OH, NH2, and COOH functions in the side chain to amino
acids, such as tryptophan, methionine, and arginine, whose
susceptibility to degradation, due to 3-indolyl, methylthio,
and guanidyl residue, respectively, is well documented
(Unger and Holzgrabe 2018).
J=12.1 Hz, 1H) 7.14 (d, J=8.2 Hz, 2H), 7.22–7.32 (m, 5H),
7.72 (d, J =8.2 Hz, 2H), 8.75 (bs, 1H), 9.58 (bs, 1H); 13C
NMR (75 MHz,CD3OD) δ 168.54, 141.99, 140.44, 134.99,
128.51, 128.35, 128.32, 128.22, 125.55, 67.91, 59.41, 45.84,
+
27.88, 23.04, 19.97. HRMS (ESI) m/z calcd for C12H16NO2
(MH+) 206.11756, found 206.11741.
(b) In Me-THF. A mixture of l-proline (0.05 mol), p-tol-
uenesulfonic acid (0.06 mol), benzyl alcohol (0.25 mol), and
Me-THF (30 mL) was heated at refux with a Dean–Stark
trap under vigorous stirring for 4 h. The reaction mixture was
cooled to rt and concentrated under vacuum. The obtained
residue was washed twice with cyclohexane (60 ml). After
vacuum pump drying, 1 was obtained as a pure oil in quan-
titative yield.
Materials and methods
1H NMR spectra were recorded on a Varian Gemini 300
operating at 300 MHz and 13C NMR at 75 MHz. Chemi-
cal shifts are reported in ppm relative to residual solvent
(CHCl3 and methanol) as internal standard. HRMS: stock
solution was prepared by dissolution in 200 µL of CH3OH;
prior analysis, an aliquot of stock solution was diluted in
water containing 0.1% HCOOH. Sample analysis: sample
was introduced by fow injection directly into the ESI inter-
face (Finnigan IonMax). Nebulization was achieved by 1
unit of nitrogen and applying 5 kV spray voltage. Spectra
were acquired using the orbitrap analyzer of a LTQ Orbit-
rap XL instrument. Resolution was set at 100,000 (FWHM
at 400 m/z) and scan range was 150–700 m/z. The melting
points were determined 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. The enantiomeric excess of the ben-
zyl esters of l-proline, l-tryptophan, and l-methionine was
determined by chiral HPLC, while that of l-arginine by H
NMR analysis of the p-toluenesulfonate (R)-Mosher carbox-
ylate salt. Reference racemic samples of the four amino acid
benzyl esters were prepared from the corresponding racemic
amino acids as described for the l enantiomers.
l‑Tryptophan benzyl ester hydrochloride (2)
A mixture of l-tryptophan (0.05 mol), p-toluenesulfonic
acid (0.06 mol), benzyl alcohol (0.50 mol), and Me-THF
(120 mL) was heated at refux with a Dean–Stark trap under
vigorously stirring for 24 h. The reaction mixture was cooled
to rt and the solvent was evaporated under vacuum. The
obtained residue was poured into EtOAc/aqueous Na2CO3
and, after removing the water layer, the organic phase was
evaporated under vacuum. The residue was diluted with
TAME and cooled to 0 °C, and hydrochloric dioxane was
added to the solution. The precipitated hydrochloride was
collected by fltration and washed twice with ethyl acetate
to give 2 in 62% yield. M.p. = 207.20; [α]2D5 = + 4.56 (c 2,
MeOH); 100% e.e. (determined by HPLC analysis on a Phe-
nomenex Lux 3 μ Cellulose-2 column, 150 mm× 4.6 mm
i.d.; hexane/2-PrOH 9/1; 1 mL/min; 280 nm, tR =13.57 min);
1H NMR (300 MHz, CD3OD) δ 3.34 (dd, J=7.0, 15.2, Hz,
1H), 3.43 (dd, J=6.4, 15.2, Hz, 1H),4.35 (dd, J =6.4, 7.0,
Hz, 1H), 5.16 (d, J = 12.3 Hz, 1H), 5.21 (d, J = 12.3 Hz,
1H) 7.05-7.41 (m, 8H), 7.52 (d, J=8.2 Hz, 1H); 13C NMR
(75 MHz, CD3OD) δ 168.90, 136.86, 134.74, 128.29,
128.21, 126.86, 124.26, 121.50, 118.93, 117.49, 111.31,
106.02, 67.80, 53.37, 26.27. HRMS (ESI) m/z calcd for
C18H19N2O2+ (MH+) 295.14410, found 295.14426.
l‑Proline benzyl ester p‑toluenesulfonate (1)
(a) In cyclohexane. A mixture of l-Proline (0.05 mol), p-tol-
uenesulfonic acid (0.06 mol), benzyl alcohol (0.25 mol), and
cyclohexane (30 mL) was heated at refux with a Dean–Stark
trap under vigorous stirring for 4 h. The reaction mixture
was cooled to rt and the upper layer was removed. The
obtained residue was washed twice with cyclohexane
(60 ml). After vacuum pump drying, 1 was obtained as a
pure oil in quantitative yield. [α]2D5 = − 22.5 (c 1, MeOH);
100% e.e. (determined by HPLC analysis on a Phenomenex
Lux 3 μ Amyloae-2 column, 150 ×4.6 mm i.d.; hexane/2-
l‑Arginine benzyl ester mono‑p‑toluenesulfonate (3)
A mixture of l-arginine (0.05 mol), p-toluenesulfonic acid
(0.11 mol), benzyl alcohol (0.50 mol), and 2-Me-THF
(100 mL) was heated at refux with a Dean–Stark trap under
vigorously stirring for 24 h. The reaction mixture was cooled
to rt and the upper layer was removed. The lower layer was
washed with ethyl acetate twice and, after vacuum pump dry-
ing, poured into DCM/aqueous Na2CO3. Water was removed
and DCM was evaporated to give 3 as a white solid in 58%
yield. M.p. = 98.94 °C; [α]2D5 = - 2.26 (c 1, MeOH);> 98%
e.e. (determined by H NMR analysis); 1H NMR (300 MHz,
1
PrOH 8/2; 1 mL/min; 220 nm, tR = 8.91 min); H NMR
(300 MHz, CDCl3) δ 1.82–2.12 (m, 3H), 2.35 (s, 4H), 3.52
(m, 2H), 4.52 (m, 1H), 5.09 (d, J =12.1 Hz, 1H), 5.17 (d,
1 3

Preparation of enantiopure methionine, arginine, tryptophan, and proline benzyl esters in…
CD3OD) δ 1.53-1.81 (m, 4H), 2.35 (s, 3H), 3.13 (t,
J=6.4 Hz, 2H), 3.49 (t, J=5.8 Hz, 1H), 5.14 (d, J=12.2 Hz,
1H), 5.19 (d, J=12.2 Hz, 1H) 7.22 (d, J=8.2 Hz, 2H), 7.31-
7.42 (m, 5H), 7.70 (d, J=8.2 Hz, 2H); 13C NMR (75 MHz,
CDCl3) δ 175.33, 157.25, 141.55, 140.48, 135.60, 128.97,
128.56, 128.30, 128.15, 125.68, 66.68, 53.79, 40.85, 30.57,
19.95, 13.58. HRMS (ESI) m/z calcd for C12H18NO2S+
(MH+) 240.10527, found 240.10531.
Compliance with ethical standards
Conflict of interest The authors declare that they have no confict of
interest.
+
25.01, 21.25. HRMS (ESI) m/z calcd for C13H21N4O2
(MH+) 265.16590, found 265.16592. The (R)-Mosher car-
boxylates were prepared by adding an equimolar amount
of (R)-α-methoxy-α-trifluoromethylphenylacetic acid to
a methanolic solution of l-arginine benzyl ester mono-p-
toluenesulfonate (3) or of rac-arginine benzyl ester mono-p-
toluenesulfonate and evaporating the solvent under vacuum.
lR-Salt 1H NMR (600 MHz, CD3OD) δ 1.51-1.80 (m, 2H),
1.83-2.07 (m, 2H), 2.39 (s, 3H), 3.16 (m, 2H), 3.56 (s, 3H),
4.00 (t, J=6.4 Hz, 1H), 5.27 (d, J=12.0 Hz, 1H), 5.31 (d,
J = 12.0 Hz, 1H), 7.26 (d, J = 8.2 Hz, 2H), 7.29-7.46 (m,
8H), 7.66 (m, 2H), 7.72 (d, J=8.2 Hz, 2H). lR/dR-Salt: 1H
NMR (600 MHz, CD3OD) δ 1.52–1.82 (m, 2H), 1.83–2.07
(m, 2H), 2.39 (s, 3H), 3.16 (dt, J=6.4, 2.0 Hz, 2H), 3.56 (s,
3H), 4.00 (t, J = 6.4 Hz, 0.5H), 4.07 (t, J = 6.4 Hz, 0.5H),
5.27 (d, J = 12.0 Hz, 1H), 5.31 (d, J = 12.0 Hz, 1H), 7.26
(d, J=8.2 Hz, 2H), 7.29–7.46 (m, 8H), 7.66 (m, 2H), 7.72
(d, J = 8.2 Hz, 2H). lR-Salt 13C NMR (75 MHz, CD3OD)
δ 170.61, 169.02, 157.29, 141.90, 140.56, 135.75, 135.01,
128.58, 128.40, 128.36, 128.26, 127.49, 125.55, 124.87 (q,
J=288.0 Hz), 85.30 (q, J=24.8 Hz), 67.75, 54.07, 52.17,
40.13, 27.28, 23.92, 19.99; 19F NMR (282 MHz, CD3OD)
δ -67.45.
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.
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