Enzymatic and chromatographic resolution procedures applied to the synthesis of the phosphoproline enantiomers

Article abstract of DOI:10.1016/j.tetasy.2015.10.016

The preparation of enantiomerically pure pyrrolidine-2-phosphonic acid (phosphoproline, Pro^P) has been addressed through the synthesis of suitable racemates and subsequent resolution by independent enzyme-catalyzed and chiral HPLC methods. First, racemic phosphoproline derivatives bearing the necessary protecting groups have been synthesized in excellent global yields starting from inexpensive materials. Preparative HPLC resolution of the N-Cbz-protected aminophosphonate on a cellulose-based column allowed the isolation of enantiomerically pure enantiomers on a gram scale. Enzyme-catalyzed alkoxycarbonylation of the aminophosphonate was studied using different lipases, solvents, and carbonates. Candida antarctica lipase type A (CAL-A) provided the highest enantioselectivity when combined with benzyl 3-methoxyphenyl carbonate.

Full text of DOI:10.1016/j.tetasy.2015.10.016

Tetrahedron: Asymmetry 26 (2015) 1469–1477
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Enzymatic and chromatographic resolution procedures applied to
the synthesis of the phosphoproline enantiomers
Alicia Arizpe a, María Rodríguez-Mata b, Francisco J. Sayago a, María J. Pueyo a, Vicente Gotor b,
Ana I. Jiménez a, Vicente Gotor-Fernández b, , Carlos Cativiela a,
⇑
⇑
a Departamento de Química Orgánica, Instituto de Síntesis Química y Catálisis Homogénea (ISQCH), CSIC–Universidad de Zaragoza, 50009 Zaragoza, Spain
b Departamento de Química Orgánica e Inorgánica, Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, 33006 Oviedo, Spain
a r t i c l e i n f o
a b s t r a c t
The preparation of enantiomerically pure pyrrolidine-2-phosphonic acid (phosphoproline, ProP) has been
addressed through the synthesis of suitable racemates and subsequent resolution by independent
enzyme-catalyzed and chiral HPLC methods. First, racemic phosphoproline derivatives bearing the nec-
essary protecting groups have been synthesized in excellent global yields starting from inexpensive
materials. Preparative HPLC resolution of the N-Cbz-protected aminophosphonate on a cellulose-based
column allowed the isolation of enantiomerically pure enantiomers on a gram scale. Enzyme-catalyzed
alkoxycarbonylation of the aminophosphonate was studied using different lipases, solvents, and carbon-
ates. Candida antarctica lipase type A (CAL-A) provided the highest enantioselectivity when combined
with benzyl 3-methoxyphenyl carbonate.
Article history:
Received 12 October 2015
Accepted 21 October 2015
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
regulate essential physiological processes.6 As a consequence, inhi-
bitors of these dipeptidyl peptidases are being considered as novel
a
-Aminophosphonic acids, the phosphonic counterparts of
drug candidates for the treatment of prevalent diseases such as
a
-amino acids in which the carboxylic acid function is replaced
diabetes, immunological disorders, or cancer.1–3,5,7
by a phosphonic acid, display a wide variety of biological activities
that range from agrochemistry to medicine.1,2 Of particular
relevance in the medicinal field is the finding that small peptides
A feature common to the inhibition assays of DPP-IV and related
serine proteases by phosphoproline-containing dipeptides is the
use of diastereomeric mixtures.5 Thus, for these studies, racemic
containing a C-terminal
a
-aminophosphonate are inhibitors of
phosphoproline was coupled to the corresponding L-a-amino acid
serine proteases.1–3 A remarkable feature is that such peptide
phosphonates do not react with the active site of threonine, cys-
teine, or aspartyl proteases and are, therefore, specific inhibitors
of serine proteases.4 Moreover, modification of the terminal
and the mixture of diastereomeric dipeptides formed was used
as such for biological evaluation.5 Only exceptionally have the
peptide diastereomers been separated and tested as pure com-
pounds.5e,g,h This fact highlights the need for synthetic
methodologies that provide access to the phosphoproline
enantiomers in an efficient way and in sufficient quantities. The
ready availability of enantiopure phosphoproline derivatives is
also essential for the development of applications for this
a-aminophosphonate moiety and/or the preceding a-amino acid
residue(s) allows for considerable selectivity to target a particular
serine protease.2,3 Thus, dipeptides containing phosphoproline at
the C-terminus (Xaa-ProP, similar to those in Fig. 1) have been
shown to be selective irreversible inhibitors of dipeptidyl pepti-
dase IV (DPP-IV) and closely related serine proteases [DPP-II,
DPP8, DPP9, fibroblast activation protein (FAP)].2,3,5 These serine
proteases cleave dipeptide fragments from the N-terminus of
polypeptide chains with great specificity after a proline residue
and are involved in the metabolism of peptide hormones that
structurally singular
a-aminophosphonic acid (as is proline
within proteinogenic amino acids) in other fields such as
asymmetric organometallic catalysis8 and organocatalysis.9
Notwithstanding recent advances in the synthesis of
a
-aminophosphonic acids,1,2d,10 the development of efficient
methodologies that provide access to enantiomerically pure
phosphoproline remains a challenge. This is a subject of current
interest, as evidenced by the fact that about half of the papers
describing the isolation of the title compound in enantiopure or
enantioenriched form11 (through either stereoselective syntheses
⇑
Corresponding authors.
E-mail addresses: vicgotfer@uniovi.es (V. Gotor-Fernández), cativiela@unizar.es
(C. Cativiela).
http://dx.doi.org/10.1016/j.tetasy.2015.10.016
0957-4166/Ó 2015 Elsevier Ltd. All rights reserved.

1470
A. Arizpe et al. / Tetrahedron: Asymmetry 26 (2015) 1469–1477
O
O
P
O
N
N
N
O
OR
O
C
C
OR
P
P
R'
H
N
OAr
OAr
ArO
ArO
L-Pro-ProP
H2N
P(OR)3
L-Ala-ProP
+
Figure 1. Structure of dipeptides shown to be specific inhibitors of the serine
protease DPP-IV. Note that the phosphoproline residue is racemic.
N
Lewis acid
ref. 14
decarboxylation
ref. 13
R'
b
a
OMe
COOH
N
N
or resolution procedures) have been published in the last few
years.11a–d Moreover, it is only recently that the absolute
configuration of the phosphoproline enantiomers has been
established in an unambiguous manner.11c The most successful
diastereoselective procedures reported involve the addition of
R'
R'
electrochem.
ref. 14,16
this work
O
N
N
R'
R'
triethyl phosphite to
a chiral oxazolopyrrolidine bearing a
benzotriazol moiety11g or the addition of lithium diethyl
Scheme 1. Reported routes for the synthesis of racemic phosphoproline through an
intermediate iminium ion. The contribution of the present work is indicated.
phosphite to a chiral N-sulfinyl imine.11e An enantioselective
approach has been described involving
a Michael reaction
between a phosphoglycine imine and benzyl acrylate in the
presence of a chiral phase-transfer catalyst to furnish an (S)-
phosphoproline precursor with 85% ee.11b Chemical resolution
methods were applied in early studies to isolate (R)- and (S)-
addition of a Lewis acid to N-protected 2-methoxypyrrolidines14
(Scheme 1, path b). The latter compounds can be prepared by
electrochemical methoxylation (Scheme 1), as first reported by
Shono et al.14,16
c-lactams as alternative starting com-
pounds for the convenient preparation of the aforementioned
phosphoproline by coupling the racemate with
L-leucine and
We have proposed17
separation of the diastereomeric dipeptides by crystallization,
followed by hydrolysis and purification.11i In a recent report, the
phosphoproline enantiomers were isolated by acylation with
2-methoxylated pyrrolidines (Scheme 1). The use of lactams as
precursors of cyclic a-aminophosphonic acids offers several advan-
dibenzoyl-L-tartaric anhydride, separation of the diastereomeric
amides formed by column chromatography and hydrolysis.11a
Regarding the resolution by enzymatic methods, enantiopure (R)-
phosphoproline was obtained by kinetic resolution of a d-bromo-
tages. First, it avoids the use of electrochemical methods, which is
not straightforward in laboratories of synthetic organic chemistry.
Moreover, lactams can be easily transformed into the desired
a
-aminophosphonates without the isolation of any synthetic
a
-(chloroacetoxy)butylphosphonate with a protease and further
intermediate.17 Lactams of different ring size and bearing diverse
substituents are commercially or easily available, thus allowing
elaboration of the bromide and chloroacetoxy moieties and
cyclization.11c Herein, we have addressed the preparation of both
phosphoproline enantiomers by resolution of a cyclic precursor
using either enzymatic or chromatographic (chiral HPLC)
procedures.
access to a wide variety of cyclic
a-aminophosphonic acids. Most
importantly, the transformation of the lactam carbonyl into a
methoxy group ensures full regiochemical control when sub-
stituents are present, whereas electrochemical methoxylation
may afford mixtures of 2- and 5-methoxylated pyrrolidines. These
advantages have already been demonstrated in the synthesis of
substituted phosphoproline analogues.17
Accordingly, pyrrolidin-2-one (Scheme 2) was the substrate
of choice for the preparation of the racemic phosphoproline
derivatives to be subsequently resolved by enzymatic or chromato-
graphic methods. The presence of an N-acyl group is a prerequisite
2. Results and discussion
2.1. Synthesis of racemic phosphoproline derivatives
The success of resolution procedures applied to the production
of enantiomerically pure compounds relies, to a large extent,
on the stage of the synthetic route selected for enantiomer separa-
tion. At best, the transformations performed on the valuable
enantiomerically pure material obtained should be reduced to a
minimum and proceed with high yields under conditions that do
not compromise the chiral integrity of the stereogenic centers.
With this idea in mind, we envisaged the resolution of a racemic
phosphoproline precursor in which the pyrrolidine ring is already
formed and the amino and phosphonic acid functions are already
present and, if necessary, protected with groups that can be
cleaved smoothly at a final step. This would allow the isolation
DIBAL-H
CbzCl, LiHMDS
THF, -78 ºC
91%
O
O
N
H
N
THF, -78 ºC
Cbz
1
O
BF3·OEt2
P(OMe)3
+
OR
P
N
N
N
OMe
OMe
CH2Cl2, -20 ºC
Cbz
Cbz
Cbz
of the target enantiopure
transformation after the resolution step.
a-aminophosphonic acid with minimal
2
3
; R = H
; R = Me
4
MeOH, r.t.
PPTS
rac-
1
80% from
Accordingly, our first aim was the preparation of the appropri-
ate racemic phosphoproline precursor in an efficient manner.
Although racemic phosphoproline (and small peptides containing
it) has been obtained by cyclization of open-chain derivatives,5h,12
most of the reported routes make use of starting compounds with a
preformed pyrrolidine ring.13–15 In the majority of the latter cases,
the phosphonate moiety was introduced via reaction of an alkyl/
aryl phosphite with an iminium ion13,14 (Scheme 1), which in turn
was generated either by decarboxylation of N-substituted prolines
under a variety of conditions13 (Scheme 1, path a) or by the
H2, Pd/C
EtOH, rt
100%
O
P
N
H
OMe
OMe
5
rac-
Scheme 2. Synthesis of racemic phosphoproline derivatives to be used in resolu-
tion processes.

A. Arizpe et al. / Tetrahedron: Asymmetry 26 (2015) 1469–1477
1471
for the subsequent transformation into an
a
-aminophosphonate as
and ZWIX(ꢀ), reached baseline enantioseparation in both columns
for all compounds tested except for phosphoproline and its six-
membered ring homolog, which were not separated.
outlined in Scheme 1. An acyl substituent of the carbamate type,
namely the benzyloxycarbonyl (Z or Cbz) group, was selected for
this purpose because the final deprotection to liberate the amino
function can be accomplished under mild conditions. Moreover,
the aromatic unit in Cbz should facilitate UV detection in the HPLC
experiments.
The Cbz group was introduced by reaction of pyrrolidin-2-one
with lithium bis(trimethylsilyl)amide and benzyl chloroformate
(Scheme 2) to provide the N-Cbz protected pyrrolidinone 1. Com-
pound 1 is also available from commercial sources, albeit at a
higher price than inexpensive pyrrolidin-2-one. The carbonyl
group in 1 was reduced with diisobutylaluminium hydride to give
2. The unstable hemiaminal was immediately treated with cat-
alytic pyridinium p-toluenesulfonate (PPTS) in methanol to form
methoxyaminal 3, which was not isolated but directly reacted with
trimethyl phosphite in the presence of boron trifluoride-diethyl
ether. The latter step proceeded via an intermediate N-acylim-
The resolution of rac-4 was first tested at the analytical level
using 250 ꢁ 4.6 mm ChiralpakÒ IA, IB, and IC columns and eluting
with n-hexane/2-propanol mixtures. None or very poor resolution
was observed on the IA or IB stationary phases whereas ChiralpakÒ
IC showed excellent enantiodiscrimination ability. The effect pro-
duced by the addition of a third component to the elution mixture
was evaluated for the IC column. Among those tested, TBME
proved to be highly detrimental to the enantioseparation, whereas
acetone had the opposite effect. Moreover, elution with n-hexane/
acetone instead of ternary n-hexane/2-propanol/acetone mixtures
further improved the selectivity and resolution factors, and was
also beneficial to the sample solubility. The analytical conditions
finally selected for further extension to the preparative-scale reso-
lution of rac-4 were elution with a 72:28 mixture of n-hexane/
acetone on ChiralpakÒ IC. The chromatographic profile obtained
under these conditions is shown in Figure 2.
inium and provided the desired N-Cbz-protected
phonate rac-4 in 80% overall yield from 1 (Scheme 2). This result
highlights the utility of -lactams as precursors of the pyrro-
a-aminophos-
c
lidine-2-phosphonate moiety. In particular, the preparation of 3
from lactam 1 proceeds in a much better yield to that reported
for the synthesis of similar methoxylated N-carbamoyl pyrrolidi-
nes by electrochemical methods.14,16
The synthetic route in Scheme 2 allowed us to isolate the phos-
phoproline derivative rac-4 in high overall yield and multi-gram
quantities from inexpensive pyrrolidin-2-one and with minimal
purification of intermediate compounds (no purification step if
starting from commercial 1). Compound rac-4, with protected
amino and phosphonic acid functions, is a suitable candidate for
preparative HPLC resolution. For the enzymatic assays, rac-4 was
quantitatively transformed into rac-5 by catalytic hydrogenation
under standard conditions (Scheme 2). This aminophosphonate
should not be stored for long periods of time and was prepared
in small quantities.
Figure 2. HPLC analytical resolution of rac-4. Column: ChiralpakÒ IC
(250 ꢁ 4.6 mm). Eluent: n-hexane/acetone 72:28. Flow rate: 1.0 mL/min. UV
detection: 210 nm.
The optimal conditions established at the analytical level for the
resolution of rac-4 (Fig. 2) were then extended to the preparative
scale. For this purpose, a 250 ꢁ 20 mm ChiralpakÒ IC column was
used and the elution was performed with n-hexane/acetone
72:28 at 18 mL/min flow rate. The racemate (1.85 g) was dissolved
2.2. HPLC resolution
Based on our previous experience in the preparative-scale HPLC
in chloroform at a very high concentration (1 g/mL) and 150 lL
enantioseparation of protected non-natural
a
-amino acids,18 we
aliquots of this solution were injected successively. More than
900 mg of each enantiomer were thus isolated and both of them
were found to be enantiomerically pure (Fig. 3). This means that
98% of the starting racemic material was recovered in enantiomer-
ically pure form after a single passage through the column.
Next, the protecting groups in the resolved enantiomers of
4 were removed by treatment with a commercial solution of
hydrogen bromide in acetic acid at room temperature and the
resulting phosphoproline hydrobromides were neutralized by
ion-exchange chromatography (Scheme 3). Comparison of the
specific rotation measured for the free amino acids 6 with the
values reported in the literature11c allowed us to assign an
(R)configuration to the phosphoproline enantiomer derived from
the first eluted enantiomer of 4 (Fig. 3, Scheme 3). Accordingly,
an (S)stereochemistry was assigned to the more strongly retained
addressed the resolution of rac-4 by chromatographic methods
using the polysaccharide-based columns ChiralpakÒ IA,19a IB,19b
and IC.19c These stationary phases combine the excellent chiral
recognition ability typical of polysaccharide-derived phases with
high chemical stability.19–21 The latter feature stems from the
covalent bonding of the chiral selector to the silica matrix, which
makes these so-called immobilized ChiralpakÒ columns compati-
ble with all organic solvents19–21 at variance with their non-immo-
bilized counterparts, for which only hydrocarbon/alcohol mixtures
can be used. This distinctiveness may not be important for analyt-
ical assays, but is most often crucial for preparative purposes.18,21
In fact, the success of all our previously reported preparative
separations on immobilized polysaccharide-based columns18
relied on the use of chloroform or dichloromethane to dissolve
the sample at very high concentrations (typically, 500 mg of
sample per mL of solvent or above) and, in most cases, also on
the presence of chloroform, ethyl acetate, acetone, or tert-butyl
methyl ether (TBME) in the eluent. All preparative HPLC enan-
tioseparations previously carried out in our laboratories18 were
4
enantiomer. It is worth mentioning that the absolute
configuration of phosphoproline was an unresolved issue until
Hammerschmidt et al. assigned it unambiguously.11c,23
2.3. Enzymatic resolution
performed on
work being the first occasion on which the preparative HPLC
resolution of an -amino acid of phosphonic nature is undertaken.
It is worth mentioning that a recent HPLC analysis22 of almost 30
-aminophosphonic acids with different structures using two
chiral cinchona-based zwitterionic phases, ChiralpakÒ ZWIX(+)
a-amino acids of the carboxylic type, the present
Biocatalysis has received much attention over the last few
decades due to its potential in the production of enantiomerically
pure compounds under mild reaction conditions.24,25 However,
there are very few examples26 of biocatalytic procedures that
a
a
have been applied to the resolution of
a-aminophosphonic acid

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A. Arizpe et al. / Tetrahedron: Asymmetry 26 (2015) 1469–1477
did not proceed in any extension after 3 days at 30 °C using TBME
as the solvent. For this reason, the more reactive allyl 3-methoxy-
phenyl carbonate 7b31a was tested under similar conditions and
other lipases were included in the screening. Using carbonate 7b,
the highest activities were observed for Candida antarctica lipase
type
B (CAL-B) and Pseudomonas cepacia (PSL IM), which
promoted the reaction but in a non-selective manner, while
CAL-A was found to catalyze the allyloxycarbonylation of
aminophosphonate rac-5 to yield the unreacted substrate (S)-5
with 90% ee and the allyl carbamate (R)-8 with 20% ee
(Scheme 4) after 92 h of reaction. These ee values were
determined by HPLC analysis in an indirect manner since none of
these compounds can be easily detected by UV absorbance.
Moreover, the separation of these compounds by silica-gel
column chromatography was not possible due to the instability
observed for amine 5 under these conditions. For these reasons,
prior to HPLC assays, the crude obtained from the enzymatic
reaction was treated with benzyl chloroformate to give a mixture
of optically active carbamates (S)-4 and (R)-8 (Scheme 4).
Laborious column chromatography on silica gel was necessary to
separate these carbamates as they showed similar Rf values. The
Figure 3. HPLC analytical profile of the resolved enantiomers of 4 (conditions: see
Fig. 2). The configuration assigned to each enantiomer is shown.
O
P
N
OMe
OMe
Cbz
4
rac-
allyloxycarbonyl group in (R)-8 was then exchanged by
a
first
second
chiral HPLC
benzyloxycarbonyl moiety through a two-step procedure that
involved treatment with N,N-dimethylbarbituric acid (DMBA),
triphenylphosphine, and palladium acetate, and subsequent
protection with benzyl chloroformate (Scheme 4). The
enantioenriched Cbz-protected aminophosphonates (S)-4 and (R)-
4 obtained after these transformations and derived, respectively,
from the unreacted amine (S)-5 and the allyl carbamate (R)-8
were separately subjected to HPLC analysis for enantiomeric
purity determination. Their absolute stereochemistries were
assigned by comparison with enantiomerically pure samples of
(R)-4 and (S)-4 obtained in the preparative-scale HPLC resolution
process described above. The enantiomeric excesses determined
for the substrate (eeS) and product (eeP) were not consistent
with a conversion value around 50%. Similar discrepancies were
observed when the process was repeated and stopped at
different reaction times. This disagreement could be due to the
extensive chemical elaboration, including difficult purification,
required prior to enantioselectivity determination. In particular,
partial racemization of the product during the (R)-8-to-(R)-4
transformation cannot be discarded.
These difficulties prompted us to consider a change in the
approach. Thus, in order to avoid the inconveniences associated
with UV detection and purification of the allyl derivative, we envis-
aged the possibility of directly introducing a benzyloxycarbonyl
group in rac-5. For this purpose, two benzyl carbonates 9a–b were
prepared following reported procedures31b,c and their reactivity in
the benzyloxycarbonylation of rac-5 catalyzed by CAL-A was
studied (Scheme 5, Table 1). Carbonate 9a in TBME was highly
reactive and produced the complete transformation of the
starting material into the racemic Cbz-protected derivative rac-4.
On the other hand, the use of benzyl 3-methoxyphenyl carbonate
9b resulted, after 84 h, in the formation of (R)-4 with 82% ee and
the recovery of the starting material (S)-5 with 94% ee (Table 1,
entry 1). The enantiomeric excess of both compounds was
determined by HPLC after the transformation of the remaining
(S)-5 into the N-tosylated phosphonate (S)-10, which was
cleanly separated from the Cbz-derivative (R)-4 by column
chromatography on silica gel (Scheme 5). Enantiopure (R)-4 and
(S)-4 obtained by preparative HPLC resolution served as
references for the absolute configuration assignments. In an
attempt to optimize the enantioselectivity, other organic solvents
were tested in the CAL-A catalyzed benzyloxycarbonylation of
rac-5 with carbonate 9b, but none of them resulted in an
improved selectivity. Thus, no conversion was observed in the
enantiomer
enantiomer
O
O
P
P
OMe
OMe
OMe
N
N
OMe
Cbz
(R)-
Cbz
4
4
(S)-
1. HBr / AcOH, rt
2. Dowex 50WX8
100%
100%
O
O
P
P
OH
OH
OH
N
H
(R)-
N
H
OH
6
(S)-6
Scheme 3. Preparation of enantiopure phosphoproline by chromatographic (chiral
HPLC) resolution.
derivatives. In fact, most of the works reporting on the access to
these compounds by enzymatic methods are based on the
resolution of
(enzyme-catalyzed acylation or hydrolysis reactions, respectively)
followed by chemical transformations to replace the OH/OCOR
a
-hydroxy- or
a
-acyloxyphosphonates11c,27,28
a
substituent by an amino group and, if necessary, to elaborate
the side chain. This is the case of Hammerschmidt et al.’s
work mentioned in the Introduction,11c which describes the
highly enantioselective protease-catalyzed kinetic resolution of
a-(chloroacetoxy)phosphonate (99% ee) and subsequent
transformation into (R)-phosphoproline.
an
To the best of our knowledge, none of the few examples of bio-
catalytic procedures applied to the resolution of a-aminophospho-
nic acid derivatives26 involve a compound in which the amino
group is part of a cycle, as is the case in phosphoproline. This fact,
together with our experience in the enzymatic resolution of a- and
b-amino esters containing an azacycle,29 prompted us to explore
the kinetic resolution of the phosphoproline precursor rac-5 by
carbamoylation (alkoxycarbonylation) reactions. Hydrolases were
selected for this purpose, since they have been shown to provide
access to N-heterocyclic amines and amino acid derivatives with
high stereocontrol.29,30
As a starting point, and based on our previous work on the res-
olution of cyclic a
- and b-amino esters,29 the alkoxycarbonylation
of the amino function in rac-5 was attempted using Candida antarc-
tica lipase type A (CAL-A) and the allyl carbonates 7a–b (Scheme 4).
Unfortunately, the reaction with diallyl carbonate 7a (2.5 equiv)

A. Arizpe et al. / Tetrahedron: Asymmetry 26 (2015) 1469–1477
1473
O
P
O
P
N
H
OMe
N
OMe
OMe
OMe
Cbz
5
4
(S)-
(S)-
O
O
1) CbzCl, DIPEA
lipase
THF, rt
P
+
+
OMe
OMe
RO
O
N
H
2) chromatography
separation
TBME, 30 ºC
250 rpm
7a: R = H2C=CHCH2-
O
P
O
P
7b
: R = 3-OMe-C6H4-
5
rac-
OMe
OMe
N
N
OMe
OMe
O
O
O
O
(R)-8
(R)-8
1) DMBA, PPh3, Pd(OAc)2
THF, 40 ºC
2) CbzCl, DIPEA / THF, rt
O
P
N
OMe
OMe
Cbz
(R)-4
Scheme 4. Lipase-catalyzed kinetic resolution of rac-5 using allyl carbonates 7a–b.
O
P
O
P
OMe
N
N
OMe
OMe
OMe
H
Ts
O
(S)-5
(S)-10
O
1) TsCl, Et3N
CAL-A
THF, rt
P
RO
O
Ph
+
+
N
H
OMe
OMe
solvent
2) chromatography
separation
30 ºC, 250 rpm
O
P
O
P
9a
: R = Me2C=N-
5
rac-
9b: R = 3-OMe-C6H4-
N
OMe
OMe
OMe
N
OMe
Cbz
Cbz
4
4
(R)-
(R)-
Scheme 5. CAL-A catalyzed kinetic resolution of rac-5 using benzyl carbonates 9a–b.
Table 1
Kinetic resolution of rac-5 catalyzed by CAL-A using carbonate 9b in organic solventsa
commercially available N-protected pyrrolidin-2-one in a straight-
forward manner that provides the desired compound in high over-
all yield and multi-gram quantities without the isolation of any
intermediate species. The efficiency of the procedure highlights
b
b
Entry
Solvent
t (h)
eeP (%)
eeS (%)
cc (%)
Ed
1
2
3
TBME
Toluene
Et2O
84
96
96
82
71
29
94
30
13
54
30
31
35
8
2
the great value of
c-lactams as precursors of the pyrrolidine-
2-phosphonate moiety. The racemic phosphoproline derivative
has been subjected to HPLC resolution on a cellulose-based chiral
stationary phase. Specifically, a semi-preparative ChiralpakÒ IC
column has allowed the enantioseparation of approximately 2
grams of racemate eluting with a mixture of n-hexane/acetone.
Final removal of the protecting groups has provided both phospho-
proline enantiomers in enantiomerically pure form. In addition, the
resolution of the racemic phosphoprolinate has been explored
through enzyme-catalyzed alkoxycarbonylation reactions. From
the hydrolases toolbox, Candida antarctica lipase type A (CAL-A)
displayed the best activity and enantioselectivity. Different allyl
and benzyl carbonates were tested using a variety of organic sol-
vents. Under the optimal conditions, the unreacted aminophospho-
nate and the corresponding benzyl carbamate were obtained with
good enantiomeric excesses (94% and 82% ee, respectively). To the
best of our knowledge, this is the first biocatalytic resolution of a
a
Enzymatic reaction conditions: 2:1 ratio CAL-A:rac-5 in weight, carbonate 9b
(2.5 equiv), solvent (0.07 ), 30 °C, 250 rpm.
M
b
Determined by HPLC (see Section 4 for details).
Conversion, c = eeS/(eeS + eeP).
E = ln[(1ꢀc)ꢁ(1ꢀeeP)]/ln[(1ꢀc)ꢁ(1 + eeP)].
c
d
presence of tetrahydrofuran or 1,4-dioxane. Toluene and diethyl
ether led to lower activity than TBME and produced a loss of
selectivity in both substrate and product (Table 1, entries 2 and 3).
Even if the enantioselectivity reached did not allow the
isolation of enantiopure phosphoproline by these methods, it
should be emphasized that the present work is, to the best of our
knowledge, the first example of a biocatalytic resolution applied
to an a-aminophosphonic acid of cyclic structure.
cyclic a-aminophosphonic acid.
3. Conclusions
4. Experimental
4.1. General
An efficient methodology has been developed for the prepara-
tion of both phosphoproline enantiomers in enantiomerically pure
form. The procedure involves the synthesis of a racemic phospho-
proline derivative, with protected amino and phosphonic acid
functions, and its subsequent chromatographic resolution by
chiral HPLC. The racemic derivative has been synthesized from a
Candida antarctica lipase type A (CAL-A, immobilized NZL-101,
5.0 U/g) was purchased from Codexis. Candida antarctica lipase
type B (CAL-B, Novozyme 435, 7300 PLU/g) was a gift from Novo

1474
A. Arizpe et al. / Tetrahedron: Asymmetry 26 (2015) 1469–1477
Nordisk Co. Pseudomonas cepacia lipase immobilized on diatomite
(PSL IM, 943 U/g) was provided by Amano Europe. All reagents
were used as received from commercial suppliers without further
purification. DowexÒ 50WX8 (H+ form, 50–100 mesh) used for
ion-exchange chromatography was purchased from Sigma–
Aldrich. Thin-layer chromatography (TLC) was performed on
Macherey–Nagel PolygramÒ SIL G/UV254 precoated silica gel polye-
ster plates. The products were visualized by exposure to UV light
(254 nm), ninhydrin, iodine vapor or ethanolic solution of phos-
phomolybdic acid. Column chromatography was performed using
60 M (0.04–0.063 mm) silica gel from Macherey–Nagel. Melting
points were determined on a Gallenkamp apparatus. IR spectra
were registered on Nicolet Avatar 360 FTIR or Perkin-Elmer
1720-XFT spectrophotometers; mmax is given for the main absorp-
tion bands. 1H, 13C, and 31P NMR spectra were recorded on Bruker
AV-400 or DPX-300 instruments at room temperature using the
residual solvent signal as the internal standard; chemical shifts
(d) are expressed in ppm and coupling constants (J) in Hertz.
High-resolution mass spectra were obtained on a Bruker Micro-
tof-Q spectrometer. Optical rotations were measured on JASCO
P-1020 or Perkin-Elmer 241 polarimeters. The HPLC systems used
for the chromatographic and enzymatic resolution studies are
described in the following sections. Carbonates 7b,31a 9a,31b and
9b31c were prepared following reported procedures. Racemic
samples of the allyl derivative rac-8 and the tosyl derivative rac-
10 were synthesized to be used as references for HPLC and/or
TLC analysis in the enzymatic studies.
p-toluenesulfonate (514 mg, 2.04 mmol). After stirring at room
temperature for 2 h, triethylamine (884 mg, 1.22 mL, 8.74 mmol)
was added. The solvent was evaporated and the crude methoxyam-
inal 3 thus obtained was dissolved in anhydrous dichloromethane
(80 mL) and kept under argon. Trimethyl phosphite (2.53 g,
2.41 mL, 20.43 mmol) was added and the resulting solution was
cooled to ꢀ20 °C. Boron trifluoride-diethyl ether (2.90 g, 2.59 mL,
20.43 mmol) was added dropwise and the reaction mixture was
slowly warmed to room temperature and stirred for 12 h. After
quenching with water (20 mL), the two layers were separated
and the aqueous phase was extracted with dichloromethane
(2 ꢁ 30 mL). The combined organic extracts were dried, filtered,
and concentrated. Purification by column chromatography (eluent:
ethyl acetate/hexanes 9:1) afforded rac-4 as a colorless oil (5.11 g,
16.31 mmol, 80% yield). IR (neat)
(CDCl3, 400 MHz) d 1.81–1.94 (m, 1H, H4), 1.97–2.40 (m, 3H,
m
1705, 1248, 1028 cmꢀ1. 1H NMR
0
H3+H4 ), 3.37–3.86 (m, 8H, H5+OMe), 4.18–4.36 (m, 1H, H2),
5.06–5.21 (m, 2H, CH2Ph), 7.21–7.44 (m, 5H, Ph). 13C NMR (CDCl3,
100 MHz) d (duplicate signals are observed for most carbons, an
⁄
asterisk
indicates the minor rotamer) 23.5⁄, 24.5 (C4); 26.9,
27.7⁄ (C3); 46.8, 47.1⁄ (C5); 53.1 (d, J = 6.9 Hz, OMe); 52.9 (d,
J = 6.5 Hz, OMe); 53.2 (d, J = 162.3 Hz, C2); 67.3, 67.6⁄ (CH2Ph);
127.9 (Ph); 128.1 (Ph); 128.3 (Ph); 128.5 (Ph); 136.6 (Ph); 155.1⁄,
155.2 (C⁄O). 31P NMR (CDCl3, 162 MHz) d (duplicate signal, an
asterisk indicates the minor rotamer) 27.3⁄, 27.6. HRMS (ESI)
C
14H20NNaO5P [M+Na]+: calcd 336.0971, found 336.0987.
4.4. HPLC resolution of rac-4: isolation of (R)-dimethyl N-
(benzyloxycarbonyl)pyrrolidine-2-phosphonate and (S)-dimethyl
N-(benzyloxycarbonyl)pyrrolidine-2-phosphonate, (R)-4 and (S)-4
4.2. Synthesis of N-(benzyloxycarbonyl)pyrrolidin-2-one, 1
A 1 M solution of lithium bis(trimethylsilyl)amide in tetrahy-
drofuran (23.71 mL, 23.71 mmol) was slowly added to a solution
of pyrrolidin-2-one (2.02 g, 23.71 mmol) in anhydrous tetrahydro-
furan (60 mL) kept at ꢀ78 °C under argon. After stirring at this
temperature for 30 min, benzyl chloroformate (4.05 g, 3.38 mL,
23.71 mmol) was added dropwise. The reaction mixture was
slowly warmed to ꢀ40 °C and stirred at this temperature for 3 h.
Next, saturated aqueous ammonium chloride (30 mL) was added
and the resulting mixture was allowed to warm to room tempera-
ture. The two layers were separated and the aqueous phase was
further extracted with dichloromethane (2 ꢁ 30 mL). The com-
bined organic extracts were dried, filtered, and concentrated.
Purification by column chromatography (eluent: hexanes/ethyl
acetate 2:1) afforded 1 as an oil, which crystallized upon cooling;
white solid (4.74 g, 21.63 mmol, 91% yield). Mp 36–37 °C. Spectro-
scopic data were in agreement with reported values.32 This com-
pound is also available from commercial sources.
The HPLC resolution of rac-4 was carried out on a Waters 600
HPLC system equipped with a 2996 photodiode array detector
(used at the analytical level) and a 2487 dual wavelength absor-
bance detector (used for the preparative-scale resolution). Analyt-
ical assays were performed on 250 ꢁ 4.6 mm ChiralpakÒ IA, IB, and
IC columns eluting with different binary or ternary mixtures (IA,
IB: n-hexane/2-propanol; IC: n-hexane/2-propanol, n-hexane/
2-propanol/chloroform, n-hexane/2-propanol/TBME, n-hexane/
2-propanol/acetone, n-hexane/acetone) at flow rates ranging from
0.8 to 1.0 mL/min. The preparative resolution was carried out on a
250 ꢁ 20 mm ChiralpakÒ IC column eluting with n-hexane/acetone
72:28 at a flow rate of 18 mL/min. The process was monitored by
UV absorbance at 215 nm. The racemate rac-4 (1.850 g) was
dissolved in chloroform (1.85 mL) and 150 lL aliquots of this solu-
tion were injected consecutively every 20 min. Each injection was
collected into three separate fractions, with equivalent fractions of
successive injections being combined. Evaporation of the first and
third fractions provided, respectively, enantiomerically pure (R)-4
(909 mg) and (S)-4 (905 mg). The second fraction (17 mg) was
found to contain a 33:67 mixture of (R)-4/(S)-4 and was discarded.
4.3. Synthesis of dimethyl N-(benzyloxycarbonyl)pyrrolidine-2-
phosphonate, rac-4
A 1 M solution of diisobutylaluminium hydride in hexanes
(30.70 mL, 30.70 mmol) was slowly added to a solution of 1
(4.48 g, 20.43 mmol) in anhydrous tetrahydrofuran (100 mL) kept
at ꢀ78 °C under argon. After stirring at this temperature for 2 h,
the reaction was treated with saturated aqueous sodium acetate
(30 mL) and allowed to warm to room temperature. A 3:1 mixture
of diethyl ether and saturated aqueous ammonium chloride
(48 mL) was then added and the resulting mixture was stirred until
a suspension was formed. The solid was filtered off under reduced
pressure and washed with diethyl ether (2 ꢁ 20 mL). The organic
layer was separated and the aqueous phase was further extracted
with diethyl ether (2 ꢁ 30 mL). The combined organic extracts
were washed with water (20 mL) and brine (20 mL), dried, filtered,
and evaporated to provide the hemiaminal 2 as oil. It was then
dissolved in methanol (100 mL) and treated with pyridinium
(R)-4: colorless oil, [
a]
D
22 = ꢀ60.0 (c 0.53, CHCl3). (S)-4: colorless oil,
[a]
22 = +61.3 (c 0.52, CHCl3). Spectroscopic data were identical to
D
those obtained for rac-4.
4.5. Synthesis of (R)-pyrrolidine-2-phosphonic acid, (R)-
phosphoproline, (R)-6
A 33% solution of hydrogen bromide in acetic acid (8 mL) was
added to (R)-4 (400 mg, 1.28 mmol) and the reaction mixture was
stirred at room temperature for 2 h. The solvent was evaporated
and the residue was submitted to ion-exchange chromatography
on DowexÒ 50WX8 (H+ form, 50–100 mesh) eluting with water.
Lyophilization of the aqueous solution afforded (R)-6 as a white
solid (193 mg, 1.28 mmol, 100% yield); mp 272–274 °C (lit. 11c
265–270 °C). [
a]
21 = ꢀ49.8 (c 1.1, 1 M NaOH) {lit. 11c [
a]
20 = ꢀ49.1
D
D

A. Arizpe et al. / Tetrahedron: Asymmetry 26 (2015) 1469–1477
1475
(c 1.1, 1 N NaOH)}. Spectroscopic data were in agreement with
210 nm. Samples of rac-4, rac-10, and enantiopure (R)-4 and
(S)-4 (from the preparative HPLC resolution, see above) were used
as references for retention times and absolute configuration
assignment.
reported values.11c
4.6. Synthesis of (S)-pyrrolidine-2-phosphonic acid, (S)-
phosphoproline, (S)-6
(S)-10: [a]
20 = +76.7 (c 1.0, CHCl3) for 94% ee; spectroscopic data
D
were identical to those obtained for rac-10.
An identical procedure to that described above was applied to
transform (S)-4 (400 mg, 1.28 mmol) into (S)-6 (193 mg,
4.9. Synthesis of dimethyl N-(allyloxycarbonyl)pyrrolidine-2-
phosphonate, rac-8
1.28 mmol, 100% yield); mp 272–274 °C. [a]
21 = +51.1 (c 1.1, 1 M
D
NaOH). Spectroscopic data were identical to those obtained for
(R)-6.
N,N-Diisopropylethylamine (74 mg, 99 lL, 0.57 mmol) was
added to a solution of rac-5 (82 mg, 0.46 mmol) in anhydrous
tetrahydrofuran (3 mL) kept at room temperature under an argon
atmosphere. The mixture was cooled to 0 °C and allyl chloroformate
4.7. Synthesis of dimethyl pyrrolidine-2-phosphonate, rac-5
A mixture of rac-4 (200 mg, 0.64 mmol) and 10% Pd/C (20 mg)
in ethanol (10 mL) was stirred overnight at room temperature
under an atmospheric pressure of hydrogen gas. Filtration of the
catalyst and evaporation of the solvent provided rac-5 as colorless
(69 mg, 60 lL, 0.57 mmol) was added. The reaction mixture was
allowed to warm to room temperature and stirred overnight. The
solvent was evaporated and the crude was purified by column chro-
matography (eluent: ethyl acetate) to afford rac-8 as colorless oil
oil (115 mg, 0.64 mmol, 100% yield). IR (neat)
m
3315, 1247,
(57 mg, 0.22 mmol, 47% yield). IR (neat) m 1704, 1648, 1248,
1031 cmꢀ1 1H NMR (CDCl3, 400 MHz) d 1.61–2.24 (m, 5H, NH
.
1029 cmꢀ1
.
1H NMR (CDCl3, 400 MHz) d 1.80–1.93 (m, 1H, H4),
0
0
+H3+H4), 2.81–2.89 (m, 1H, H5), 2.92–3.00 (m, 1H, H5 ), 3.25–3.32
(m, 1H, H2), 3.72 (d, J = 10.4 Hz, 3H, OMe), 3.73 (d, J = 10.4 Hz,
3H, OMe). 13C NMR (CDCl3, 100 MHz) d 26.0 (d, J = 8.1 Hz, C4),
26.7 (C3), 47.6 (d, J = 11.6 Hz, C5), 53.0 (d, J = 4.0 Hz, OMe), 53.0
(d, J = 4.1 Hz, OMe), 53.6 (d, J = 164.7 Hz, C2). 31P NMR (CDCl3,
1.95–2.30 (m, 3H, H3+H4 ), 3.35–3.59 (m, 2H, H5), 3.64–3.80 (m,
6H, OMe), 4.17–4.30 (m, 1H, H2), 4.51–4.62 (m, 2H, CH2CH@),
5.13–5.20 (m, 1H, CH@CH2), 5.22–5.34 (m, 1H, CH@CH2), 5.82–
5.96 (m, 1H, CH@CH2). 13C NMR (CDCl3, 100 MHz) d (duplicate
⁄
signals are observed for most carbons; an asterisk indicates the
162 MHz)
d
31.1. HRMS (ESI) C6H14NNaO3P [M+Na]+: calcd
minor rotamer) 23.4⁄, 24.5 (C4); 26.8, 27.6⁄ (C3); 46.7, 46.9⁄ (C5);
52.9 (d, J = 6.6 Hz, OMe); 53.1 (d, J = 7.2 Hz, OMe); 53.1 (d,
J = 162.3 Hz, C2); 66.1, 66.4⁄ (CH2CH@); 117.5, 117.9⁄ (CH@CH2);
132.8 (CH@CH2); 155.0 (CO). 31P NMR (CDCl3, 162 MHz) d (duplicate
signal, an asterisk ⁄ indicates the minor rotamer) 27.3⁄, 27.7. HRMS
(ESI) C10H18NNaO5P [M+Na]+: calcd 286.0815, found 286.0833.
202.0604, found 202.0613.
4.8. General procedure for the enzymatic kinetic resolution of
rac-5
To a suspension of rac-5 and the lipase (1:2 in weight) in dry
solvent (0.07
M
) kept under a nitrogen atmosphere was added car-
4.10. Synthesis of dimethyl N-tosylpyrrolidine-2-phosphonate,
rac-10
bonate 7a–b or 9a–b (2.5 equiv). The system was shaken at 30 °C
and 250 rpm until the conversion was approximately 50% (TLC,
eluent: ethyl acetate). The enzyme was filtered and washed with
tetrahydrofuran (5 ꢁ 2 mL). The solvent was evaporated and the
crude obtained was elaborated in one of the following ways:
Triethylamine (12 mg, 17 lL, 0.12 mmol) was added to a solu-
tion of rac-5 (17 mg, 0.09 mmol) in anhydrous tetrahydrofuran
(1 mL) kept at room temperature under an argon atmosphere.
The mixture was cooled to 0 °C and tosyl chloride (23 mg,
0.12 mmol) was added. The reaction mixture was allowed to warm
to room temperature and stirred for 6 h. After evaporation of the
solvent, the residue was purified by column chromatography (elu-
ent: ethyl acetate) to afford rac-10 as a white solid (16 mg,
(A) Carbonates 7a–b (see Scheme 4): The crude was treated
with benzyl chloroformate in the presence of N,N-diiso-
propylethylamine (similarly to that described in Section 4.9)
to give a mixture of enantioenriched (S)-4 and (R)-8. The two
carbamates were separated by column chromatography on
silica gel (eluent: ethyl acetate). Enantioenriched (R)-8 was
transformed into (R)-4 by treatment with N,N-dimethylbar-
bituric acid, triphenylphosphine, and palladium acetate (as
described in Ref. 33; no purification was performed) and
subsequent reaction with benzyl chloroformate in the
presence of N,N-diisopropylethylamine (similarly to that
described in Section 4.9). The enantioenriched N-Cbz deriva-
tives (S)-4 and (R)-4 obtained were subjected to HPLC anal-
ysis for ee determination.
(B) Carbonates 9a–b (see Scheme 5): The crude was treated
with tosyl chloride in the presence of triethylamine (as
described for the synthesis of rac-10) to give a mixture of
enantioenriched N-tosyl and N-Cbz derivatives (S)-10 and
(R)-4. The two compounds were separated by column
chromatography on silica gel (eluent: ethyl acetate) and sub-
jected to HPLC analysis for ee determination.
0.05 mmol, 51% yield). Mp 119–120 °C. IR (KBr)
m 1348, 1309,
1263, 1189, 1158, 1037 cmꢀ1 1H NMR (CDCl3, 300 MHz) d 1.35–
.
1.49 (m, 1H, H4), 1.52–1.72 (m, 1H, H4’), 1.89–2.11 (m, 2H, H3),
2.39 (s, 3H, Me), 3.34–3.41 (m, 2H, H5), 3.77 (d, J = 10.5 Hz, 3H,
OMe), 3.84 (d, J = 10.5 Hz, 3H, OMe), 4.07–4.16 (m, 1H, H2), 7.28
(d, J = 8.2 Hz, 2H, Ph), 7.69 (d, J = 8.2 Hz, 2H, Ph). 13C NMR (CDCl3,
75 MHz) d 21.6 (Me), 24.5 (C4), 27.0 (C3), 49.3 (C5), 53.0 (d,
J = 6.9 Hz, OMe), 54.2 (d, J = 7.2 Hz, OMe), 55.3 (d, J = 171.2 Hz,
C2), 127.6 (Ph), 129.9 (Ph), 134.8 (Ph), 144.0 (Ph). 31P NMR (CDCl3,
121 MHz) d 26.1. HRMS (ESI) C13H20NNaO5PS [M+Na]+: calcd
356.0692, found: 356.0721.
Acknowledgments
Financial support to this work was provided by Ministerio de
Economía
y Competitividad–FEDER (grants CTQ2011-24237,
CTQ2013-44153-P, and CTQ2013-40855-R; FPU fellowship to M.
R.-M.), Consejo Superior de Investigaciones Científicas (JAE predoc-
toral fellowship to A.A.) and Gobierno de Aragón–Fondo Social
Europeo (research group E40). The authors thank Novo Nordisk
Co. for the generous gift of CAL-B (Novozyme 435).
HPLC elution times and conditions: (R)-4 9.5 min, (S)-4
11.8 min; (S)-10 18.0 min, (R)-10 30.9 min; Hewlett Packard 1100
chromatograph, 250 ꢁ 4.6 mm ChiralcelÒ OJ-H column, 30 °C, n-
hexane/ethanol 85:15, 0.8 mL/min flow rate, UV monitoring at

1476
A. Arizpe et al. / Tetrahedron: Asymmetry 26 (2015) 1469–1477
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3,5-dimethylphenylcarbamate
of
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-ProP, -ProP) in TLC
and ion-exchange chromatography. This assignment was suggested to be
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configuration of the X-ray structures was established considering the Flack
parameter value. The specific rotation obtained in the present work for (R)-6,
[
a
]
21 = ꢀ49.8 (c 1.1, 1 M NaOH), nicely agrees with that reported by
D
Hammerschmidt et al. [
a
]
D
20 = ꢀ49.1 (c 1.1, 1 M NaOH) in Ref. 11c. Moreover,
to double-check our assignment, we prepared one of the crystallized ureas
mentioned above, by hydrogenation of (R)-6 and further reaction with (S)-1-
phenylethyl isocyanate, and the compound obtained showed spectroscopic and
optical properties in agreement with those described (Ref. 11c). A recent report
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a carbon during this process cannot be discarded; details on the
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