Chemoenzymatic synthesis of orthogonally protected (3R,4R)- and (3S,4S)-trans-3-amino-4-hydroxypyrrolidines

Article abstract of DOI:10.1016/j.tet.2013.04.112

Several orthogonally protected racemic trans-3-amino-4-hydroxypyrrolidines have been easily prepared from N-Cbz-3,4-epoxypyrrolidine. Resolution of each racemic compound was accomplished by means of lipase-catalyzed aminolysis, transesterification or hydr

Full text of DOI:10.1016/j.tet.2013.04.112

Tetrahedron 69 (2013) 5407e5412
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Chemoenzymatic synthesis of orthogonally protected (3R,4R)- and
(3S,4S)-trans-3-amino-4-hydroxypyrrolidines
*
ꢀ
Jesus A. Rodríguez-Rodríguez, F. Javier Quijada, Rosario Brieva, Francisca Rebolledo ,
*
Vicente Gotor
ꢀ
ꢀ
Departamento de Química Organica e Inorganica, Universidad de Oviedo, 33071 Oviedo, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Several orthogonally protected racemic trans-3-amino-4-hydroxypyrrolidines have been easily prepared
from N-Cbz-3,4-epoxypyrrolidine. Resolution of each racemic compound was accomplished by means of
lipase-catalyzed aminolysis, transesterification or hydrolysis reactions. In most cases, the corresponding
remaining substrates and the products were obtained with very high enantiomeric excesses.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 19 February 2013
Received in revised form 22 April 2013
Accepted 24 April 2013
Available online 30 April 2013
Keywords:
Pyrrolidine
Vicinal amino alcohol
Lipase
Transesterification
Hydrolysis
1. Introduction
The synthesis of optically active vicinal amino alcohols is a very
important task for organic chemists in view of the fact that these
compounds have been successfully used as chiral auxiliaries, li-
gands, and building blocks for complex molecules.1 Furthermore,
non-racemic pyrrolidines constitute key structural units found in
a variety of bioactive natural products and drugs.2 An excellent
representative example combining both a vicinal amino alcohol
moiety and a pyrrolidine ring is Voreloxin (1, Fig. 1), a first-in-class
anticancer quinolone derivative, that is, currently completing phase
two clinical trials in acute myeloid leukemia and platinum-resistant
ovarian cancer.3 Compound 1 is easily obtained by the coupling of
the selectively protected pyrrolidine (3S,4S)-2 (Fig. 1) with the
appropriate 7-chloro-1,8-naphthyridine derivative.3a
Fig. 1. Optically active pyrrolidine derivatives as precursors of Voreloxin.
As a part of our research on the chemoenzymatic synthesis of
optically active functionalized heterocycles,7 we planned the syn-
thesis and enzymatic resolution of different orthogonally protected
trans-3-amino-4-hydroxypyrrolidines8 that can be used as pre-
cursors of the optically active pyrrolidine trans-2. The synthesis
strategy reported here avoids the use of azide as a reagent and al-
lows us to study the influence of the protecting group on the exo-
cyclic nitrogen in the enzymatic resolution. Moreover, some of
these optically active derivatives are also precursors of di-
astereomeric cis-4-amino-3-hydroxypyrrolidine,9 which, in turn,
has been used in the synthesis of biologically active pyrrolidine
nucleotides.10
Some synthetic approaches aimed at preparing optically active
(3S,4S)-2 include the resolution of (ꢀ)-3 with mandelic acid,4 or the
enzymatic resolution of the azido derivative (ꢀ)-45 as key steps
(Fig. 1). In addition, the chiral pool synthesis of (3S,4S)-2 from D-
isoascorbic acid has also been described through
a 10-step
strategy.6
* Corresponding authors. Tel./fax: þ34 985103448; e-mailaddresses:frv@uniovi.es
(F. Rebolledo), vgs@uniovi.es (V. Gotor).
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.04.112

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J.A. Rodríguez-Rodríguez et al. / Tetrahedron 69 (2013) 5407e5412
2. Results and discussion
2.2. Enzymatic transesterification of pyrrolidines ( )-6, ( )-8,
and ( )-9
2.1. Synthesis of orthogonally protected racemic trans-4-
amino-3-hydroxypyrrolidine derivatives, and enzymatic ami-
nolysis of ( )-7
Resolution of the N,N-diallyl derivative (ꢀ)-6 via the trans-
esterification reaction was attempted using vinyl acetate as the acyl
donor and employing different lipaseesolvent combinations. Ini-
tially, these reactions were carried out on a 25 mg scale; a selection
of the results obtained is shown in Table 1. As can be seen, the
solvent has a strong influence on both the enantioselectivity and
the catalytic activity exhibited by CAL-B. This enzyme catalyzed the
acetylation of 6 with very high enantioselectivity in TBME and 1,4-
dioxane (Table 1, entries 2 and 8, respectively), though only the
reaction in TBME was found to be of practical utility. Furthermore,
PSL-IM showed very high enantioselectivity in all the tested sol-
vents, and was found to be a more efficient catalyst than CAL-B in
toluene and TBME. It is also of note the low catalytic activity
exhibited by the PSL toward 6 in comparison with that shown in the
transesterification, under analogues conditions, of (ꢀ)-trans-2-
(diallylamino)cyclopentanol (after 3 h of reaction at 28 ꢁC, the
conversion degree was 50%; E>200).11 This means that the ben-
zyloxycarbonyl group on the pyrrolidinic nitrogen has a dramatical
effect on the catalytic activity, but not on the enantioselectivity.
We initially carried out the synthesis of racemic trans-3-(dia-
llylamino)-4-hydroxypyrrolidine (ꢀ)-6 by opening the epoxide 57a
with diallylamine. The selection of diallylamine to incorporate the
amine function at the 3-position of the pyrrolidine was based on
the following reasons: (1) allyl groups are easily removed in the
presence of other protecting groups, (2) the opening reaction af-
fords a good yield, and (3) no side-products are formed.11 Moreover,
the presence of a bulky substituent adjacent to the secondary car-
bon bearing the hydroxyl group could favor the enzymatic resolu-
tion of this compound. As our aim in this study was also to
investigate the influence of the protecting group on the exocyclic
nitrogen, (ꢀ)-6 was converted into the unsubstituted amino alcohol
(ꢀ)-7 by removal of the allyl groups with N,N0-dimethylbarbituric
acid (NDMBA) in the presence of Pd(0)11,12 (Scheme 1). In addition,
(ꢀ)-7 was selectively N-acylated by treatment with vinyl acetate,
and also converted into the N-Boc derivative (ꢀ)-9 by reaction with
di-tert-butyl pyrocarbonate.
Table 1
Lipase-catalyzed transesterification of (ꢀ)-6a
Entry
Enzyme
Solvent
tb
eeS (%)c
eeP (%)c
cd
Ee
1
2
3
4
5
6
7
8
9
CAL-A
CAL-B
PSL-IM
CAL-B
PSL-IM
CAL-B
PSL-IM
CAL-B
PSL-IM
TBME
TBME
TBME
Toluene
Toluene
THF
THF
1,4-Dioxane
1,4-Dioxane
1
3
3
5
5
5
5
5
5
4
57
92
18
42
5
5
4
4
29
99
99
91
>99
90
>99
>99
>99
12
37
48
17
29
5
5
4
4
2
>200
>200
26
>200
19
>200
>200
>200
Scheme 1. Synthesis of some (ꢀ)-trans-3-amino-4-hydroxypyrrolidine derivatives.
Compound (ꢀ)-7 contains two groupsdamino and hydrox-
yldcapable of being transformed by a lipase in aminolysis and
transesterification reactions, respectively. Given that aminolysis
requires less activated esters than transesterification, we started off
by studying the aminolysis process. For this purpose, ethyl acetate
was used as the acyl donor, and several enzymes [lipases A and B
from Candida antarctica (CAL-A and CAL-B), lipase from Bur-
kholderia cepacia (PSL-IM)], and solvents [tert-butyl methyl ether
(TBME), toluene, and 1,4-dioxane] were tested. All the processes
were carried out on a 25 mg scale monitoring the progress of each
reaction by chiral-HPLC. Unfortunately, all the tested enzymes were
poor catalysts. The best result is the one shown in Scheme 2. After 3
days of reaction at 30 ꢁC, CAL-B catalyzed the selective acylation of
the amino group of 7 (the degree of conversion was 45%), though
with very low enantioselectivity (E¼5).13 In this process, the en-
zyme showed preference for the (3S,4S) enantiomer of the amino
alcohol 7, contrary to what might be expected on the basis of
Kazlauskas’ rule.14
a
Reactions were carried out using 25 mg (entries 1, 2, 4e9) or 1.0 mmol (entry 3)
of substrate. 5 equiv of vinyl acetate was employed in all cases.
b
Reaction time (days).
Determined by chiral-HPLC (see Section 4.12).
The degree of conversion (%) was calculated from the enantiomeric excesses of
c
d
the remaining substrate 6 (eeS) and the product 10 (eeP): c¼eeS/(eeSþeeP).
e
See Ref. 13.
In view of the results included in Table 1, the best option to
accomplish the resolution of (ꢀ)-6 is to use PSL-IM in TBME (Table
1, entry 3). In this case, both the remaining substrate (3S,4S)-6 and
the product (3R,4R)-10 were isolated with very high enantiomeric
excesses (eeꢂ92%) and yields (49 and 47%, respectively) after 3 days
of reaction.
Some attempts to achieve the enantioselective trans-
esterification of the acetamide derivative (ꢀ)-8 were unsuccessful.
A drawback of these processes was the low solubility of the sub-
strate in the usual organic solvents. After a screening of solvents, we
decided to employ vinyl acetate both as acyl donor and solvent.
Under these conditions, CAL-A and CAL-B catalyzed the reaction (a
conversion degree of 20 and 39%, respectively, was obtained after
48 h of reaction at 30 ꢁC), though with very low enantioselectivity
(E¼9). The reaction with PSL-IM was even less enantioselective. In
these processes, CAL-A and CAL-B showed opposing enantiopre-
ference,15 although CAL-B preferentially catalyzed the acetylation
of the (3R,4R) enantiomer (Scheme 3), as predicted by Kazlauskas’
rule.
Scheme 2. CAL-B-catalyzed aminolysis of AcOEt with (ꢀ)-7.

J.A. Rodríguez-Rodríguez et al. / Tetrahedron 69 (2013) 5407e5412
5409
hydrolysis. Reactions were carried out in different organic solvents
using 5 equiv of H2O. No satisfactory results were obtained in the
hydrolysis of the ester derivatives (ꢀ)-10 and (ꢀ)-12 (the reactions
were either very slow or only slightly enantioselective), in contrast
to the good results obtained in the transesterification of the cor-
responding hydroxyl derivative (see Tables 1 and 2).
However, CAL-B-catalyzed hydrolysis of (ꢀ)-11 in 1,4-dioxane
was more efficient than the transesterification of its precursor
(ꢀ)-8. After 2 days of reaction at 30 ꢁC, the degree of conversion was
49% and both the product (3R,4R)-8 and the remaining substrate
(3S,4S)-11 were obtained with 84% and 80% ee, respectively, (E¼28).
When the hydrolysis of the diacetyl derivative was attempted with
CAL-A in 1,4-dioxane, the reaction was very slow, though the
enantioselectivity was very high (Table 3, entry 1). Raising the
temperature to 45 ꢁC accelerated the process, but caused a drastic
fall in enantioselectivity (entry 2). Other solvents were tested and
the best conditions were obtained when the hydrolysis was carried
out in toluene at 30 ꢁC (Table 3, entry 3). In this process, both the
reaction rate and the enantioselectivity were moderate, which
allowed us to isolate the product (3S,4S)-8 with high ee (94%).
Analogously to the transesterification of (ꢀ)-8, CAL-A preferentially
hydrolyzed the (3S,4S) enantiomer of 11, and CAL-B, its counterpart
(3R,4R).
Scheme 3. CAL-B catalyzed transesterification of (ꢀ)-8.
Similarly to the aforementioned pyrrolidine derivatives, reso-
lution of dicarbamate (ꢀ)-9 was evaluated using several lipases and
solvents. A selection of the obtained results is shown in Table 2. For
this substrate, CAL-B was found to be the most efficient enzyme in
all the tested solvents. Thus, the reaction in 1,4-dioxane, though
slow, occurred with high enantioselectivity, and both the remain-
ing substrate and the product were obtained with high ee after 5
days of reaction at 30 ꢁC (Table 2, entry 5). In spite of employing
anhydrous conditions in all the transesterification reactions, the
prolonged reaction time of this process favors the formation of
trace amount of acetic acid as a consequence of the lipase-catalyzed
hydrolysis of vinyl acetate. As the resulting acetic acid could neg-
atively affect enzymatic activity,16 we attempted the trans-
esterification in the presence of triethylamine. Effectively, under
these conditions, the enantioselectivity of the reaction continued to
be high, the E value being similar to that obtained in the absence of
Et3N (Table 2, entry 7). However, a moderate increase in the re-
action rate was observed, as the time required to achieve a 50%
conversion degree was reduced to 3 days.
Table 3
CAL-A-catalyzed hydrolysis of (ꢀ)-11a
Table 2
Lipase-catalyzed transesterification of (ꢀ)-9a
Solvent
T (ꢁC)
Time (days)
eeS (%)b
eeP (%)b
c
E
1
2
3
4
5
6
1,4-Dioxanec
1,4-Dioxanec
Toluened
Toluened
TBMEd
30
45
30
45
30
45
7
6
2
2
1
1
8
38
62
>99
21
>99
92
94
34
90
8
>200
35
63
16
23
29
40
75
19
84
Enzyme
Solvent
tb
eeS (%)c
eeP (%)c
c
E
1
2
3
4
5
6
7
8
CAL-B
PSL-IM
CAL-B
PSL-IM
CAL-B
PSL-IM
CAL-B
CAL-B
TBME
TBME
Toluene
Toluene
1,4-Dioxane
1,4-Dioxane
1,4-Dioxaned
1,4-Dioxaned
7
7
5
5
5
5
3
3.5
96
97
73
47
93
23
94
>99
58
36
50
78
94
92
94
91
62
73
59
38
50
20
50
52
13
8
6
TBMEd
>99
19
11
a
12
25 mg (entries 1, 2, 4e6) or 100 mg (entry 3) of substrate were employed. The
110
30
120
123
degree of conversion (c, %) and the E values were calculated as in Table 1.
b
Determined by chiral-HPLC (see Section 4.12).
5.0 equiv of water was used.
2.5 equiv of water was used.
c
d
a
Reactions were carried out with 5 equiv of vinyl acetate and 25 mg (entries 1e6)
or 200 mg (entries 7 and 8) of substrate. The degree of conversion (c, %) and the E
values were calculated as in Table 1.
2.4. Assignment of the absolute configuration to the enzy-
matically prepared compounds
b
Reaction time (days).
Determined by chiral-HPLC (see Section 4.12).
c
d
Triethylamine (10% v/v) was added.
The remaining substrate (ꢃ)-9 (isolated in the enzymatic
transesterification reaction with ee>99%: see Table 2, entry 8) was
treated with aq 3 N HCl at room temperature to selectively remove
the Boc group and isolate (þ)-7. The subsequent hydrogenolysis of
When comparing the results obtained in the transesterification
of the analogous racemic substrates (ꢀ)-6, (ꢀ)-8, and (ꢀ)-9, some
trends regarding the behavior of the different lipases can be
inferred. For instance, PSL-IM is very efficient for 6, the bearer of
a basic diallylamino group, whereas CAL-B was the most active
catalyst in the reactions with 9 and 8, both of which bear a non-
basic NHBoc or NHAc group. In addition, CAL-B showed higher
enantioselectivity with 9 than with 8, i.e., with the substrate bearer
of the bulkiest substituent.
the Cbz group of
7 afforded the unprotected 3-amino-4-
hydroxypyrrolidine, which was isolated as its dihydrochloride salt
(þ)-13 (Scheme 4, first row). Comparison of the sign of the specific
rotation of the obtained 13 with the reported value for (3R,4R)-
(ꢃ)-1317 establishes the absolute configuration (3S,4S) for its pre-
cursor (ꢃ)-9, and hence the (3R,4R) configuration for the enzy-
matically produced ester 12. Once the configuration of (ꢃ)-9 had
been assigned, the (3S,4S) configuration for the intermediate (þ)-7
was also established.
2.3. Enzymatic hydrolysis reactions of pyrrolidines ( )-10e12
Treatment of the racemic pyrrolidinols (ꢀ)-6, (ꢀ)-7, and (ꢀ)-9
with acetyl chloride yielded the acetyl derivatives (ꢀ)-10e12, re-
spectively, which were subsequently submitted to enzymatic
The optically active N,N-diallyl derivative 10 [produced in the
enzymatic transesterification reaction of (ꢀ)-6] was selectively
hydrolyzed using a methanolic solution of sodium methoxide

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J.A. Rodríguez-Rodríguez et al. / Tetrahedron 69 (2013) 5407e5412
Scheme 4. Assignment of the absolute configuration of the enzymatically prepared enantioenriched compounds.
(Scheme 4, second row). Removal of the allyl groups of the resulting
pyrrolidinol 6 afforded (ꢃ)-7, which has the (3R,4R) configuration,
as was established after comparison of the sign of its optical rota-
tion with that of the preceding compound 7 (Scheme 4, first row).
Thus, the absolute configuration (3R,4R) was assigned to the en-
zymatically produced 10. In addition, (3R,4R)-7 was transformed
into (3R,4R)-11 by conventional acetylation (Scheme 4, second
row). No racemization or epimerization was observed for com-
pounds showed in Scheme 4, such it was proven by the chiral-HPLC
and 1H NMR analyses.
Configuration of compound 11 arising from the enzymatic
processes shown in Scheme 3 and Table 3 was assigned by com-
parison of the corresponding chiral-HPLC chromatogram with that
obtained for (3R,4R)-11. Once the configuration for the diacetyl
derivative 11 had been established, the configuration of the acet-
amide 8 was also assigned. Lastly, configuration of both the
remaining substrate 7 and the product 8, isolated in the enzymatic
aminolysis of (ꢀ)-7 (Scheme 2), was also determined by chiral-
HPLC analysis.
101, 6.2 U/g) was purchased from Codexis. Immobilized lipase
from B. cepacia (PSL-IM, 783 U/g), which previously was classified
as Pseudomonas cepacia, was purchased from Amano Pharmaceu-
tical Co. For the enzymatic reactions, anhydrous solvents were
used. Melting points were taken on samples in open capillary tubes
and are uncorrected. IR spectra were recorded using KBr pellets (for
solids) or neat (for liquids). 1H NMR and proton-decoupled 13C NMR
spectra (CDCl3 solutions) were obtained using AC-300 or DPX-300
(1H, 300.13 MHz and 13C, 75.5 MHz) spectrometer using the
d scale
(parts per million) for chemical shifts; calibration was made on the
CDCl3 (13C; 76.95 ppm) or the residual CHCl3 (1H; 7.26 ppm) signals.
In almost all cases duplicity of some carbon signals (especially for
those of the ring) was observed due to both the presence of dif-
ferent conformers for the pyrrolidine ring and/or cisetrans isomers
around the N(1)e(C]O) bond.18 In Fig. 2 pyrrolidinic compounds
with the numbering used in the assignation of the NMR signals are
shown.
3. Conclusions
Different orthogonally protected racemic trans-3-amino-4-
hydroxypyrrolidine derivatives have been easily prepared starting
out from N-Cbz-3,4-epoxipyrrolidine. After testing different lipases,
solvents and reaction conditions, a successful enzymatic resolution
was obtained for each derivative. The transesterification catalyzed
by PSL-IM and CAL-B was found to be the most adequate via to
resolve the 3-diallylamino (ꢀ)-6 and 3-tert-butoxycarbonylamino
(ꢀ)-9 derivatives, respectively. The presence of a bulky substituent
in the neighboring position to the reactive OH group could be re-
sponsible for the high enantioselectivities obtained in these pro-
cesses. In contrast, these lipases were ineffective in the resolution
of the acetylamino derivative (ꢀ)-8, which bears a smaller sub-
stituent. Finally, CAL-A showed moderate enantioselectivity in the
hydrolysis of the diacetyl derivative (ꢀ)-11. Furthermore, the se-
lective deprotection of the different groups present in these com-
pounds has also been shown.
Fig. 2. Pyrrolidinic compounds.
4.2. Synthesis of ( )-trans-1-benzyloxycarbonyl-3-(dia-
llylamino)-4-hydroxypyrrolidine [( )-6]
Diallylamine (3.00 g, 30.6 mmol) was added to a solution of
epoxide 57a (3.20 g, 14.6 mmol) in ethanol (35 mL). After heating at
90 ꢁC for 48 h in a sealing tube, the solvent and the excess of amine
were evaporated in vacuo to give the crude product, which was
purified by flash chromatography (hexane/AcOEt 1:1). Compound
(ꢀ)-6 was obtained as a pale yellow oil (80% yield); nmax(neat) 3422,
1703 cmꢃ1
; dH (300.13 MHz, CDCl3) 7.41e7.27 (m, 5H, Ph),
5.92e5.73 (m, 2H, 2ꢄ CH]CH2), 5.29e5.09 (m, 6H, OeCH2, 2ꢄ
CH]CH2), 4.26 (q, 1H, 3J 6.9 Hz, H-4), 3.88e3.75 (m, 1H, H-5),
3.70e3.55 (m, 1H, H-2), 3.48e3.05 (m, 7H, H-20, H-3, H-50, and 2ꢄ
CH2eCH]CH2), 2.71 (br s, 1H, OH); dC (75.5 MHz, CDCl3) 154.8 (C]
O), 136.6 (C), [135.4 and 135.3 (CH]CH2)], 128.4 (CH), 127.9 (CH),
127.7 (CH), [117.8 and 117.7 (CH]CH2)], 71.2 and 70.6 (C-4), 66.8
(OeCH2), [66.6 and 66.0 (C-3)], 53.5 (NeCH2), [51.1, 50.7, 44.9, and
44.7 (C-2 and C-5)]; HRMS (ESIþ): MHþ, found 317.1868.
C18H25N2O3 requires 317.1860.
4. Experimental section
4.1. General
Lipase B from C. antarctica (CAL-B, Novozyme 435, available
immobilized on polyacrylamide, 7300 PLU/g) was gifted by Novo
Nordisk Co. Immobilized lipase A from C. antarctica (CAL-A, NZL-

J.A. Rodríguez-Rodríguez et al. / Tetrahedron 69 (2013) 5407e5412
5411
4.3. Synthesis of ( )-trans-3-amino-1-benzyloxycarbonyl-4-
hydroxypyrrolidine [( )-7]
CDCl3) 170.2 (C]O), 154.7 (C]O), 136.1 (C), 128.5 (CH), 128.1 (CH),
127.9 (CH), [75.4 and 74.4 (C-4)], 67.1 (OeCH2), [53.5 and 52.7
(C-3)], 49.6 (C-2, C-5)], 22.9 (CH3), 20.9 (CH3); HRMS (ESIþ): MNaþ,
found 343.1271. C16H20N2NaO5 requires 343.1264.
It was prepared from (ꢀ)-6 following the methodology de-
scribed in Ref. 19. The crude material was purified by flash chro-
matography (ethyl acetate/methanol 4:1) to yield (ꢀ)-7 (85%) as
4.7. General procedure for the enzymatic aminolysis and
transesterification reactions on a 25 mg scale
a colorless oil; nmax(neat) 3351, 3292, 1696 cmꢃ1
;
dH (300.13 MHz,
CDCl3) 7.40e7.27 (m, 5H, Ph), 5.11 (s, 2H, OeCH2), 3.98e3.88 (m,1H,
H-4), 3.79e3.67 (m, 2H, H-2, H-5), 3.38e3.25 (m, 2H, H-20, H-50),
3.21e3.08 (m, 1H, H-3), 2.40e2.08 (br s, 3H, OH, NH2); dC
(75.5 MHz, CDCl3) 155.1 (C]O), 136.6 (C), 128.4 (CH), 127.9 (CH),
127.7 (CH), [76.1 and 75.5 (C-4)], 66.8 (OeCH2), [57.4 and 56.8 (C-
3)], [53.1, 51.9, 51.7, and 51.4 (C-2, C-5)]; HRMS (ESIþ): MNaþ, found
259.1043. C12H16N2NaO3 requires 259.1053.
To a mixture of racemic substrate (25 mg) and lipase (25 mg),
under a nitrogen atmosphere, the corresponding anhydrous or-
ganic solvent (0.8 mL) and the acyl donor (5 equiv) were added. The
mixture was stirred at 30 ꢁC and 250 rpm, and the progress of the
reaction was analyzed by chiral-HPLC. After the time indicated in
Scheme 2, and Tables 1 and 2, the enzyme was filtered and washed
with methanol. Solvents were eliminated under reduced pressure
and the crude material analyzed by chiral-HPLC.
4.4. Synthesis of ( )-trans-3-(acetamido)-1-
benzyloxycarbonyl-4-hydroxypyrrolidine [( )-8]
4.8. Enzymatic transesterification of ( )-6 on a 1 mmol scale
A solution of (ꢀ)-7 (0.227 g, 0.961 mmol) in vinyl acetate
(5.0 mL) was stirred at 30 ꢁC for 24 h. After this time, solvent was
evaporated under reduced pressure and the resulting crude was
purified by flash chromatography (ethyl acetate as eluent) to yield
(ꢀ)-8 (69%) as a white solid; mp 159e160 ꢁC; nmax(KBr) 3244, 1702,
The mixture containing substrate (ꢀ)-6 (0.316 g,1.00 mmol), PSL-
IM (0.316 g), TBME (10 mL), and vinyl acetate (0.461 mL, 5.00 mmol)
was allowed to react until achieve a degree of conversion nearly 50%
(3 days at 30 ꢁC; Table 1, entry 3). After this time, the enzyme was
filtered and washed with methanol. Solvents were eliminated under
reduced pressure and the resulting crude material, which was
formed by the optically active (3S,4S)-6 and the ester (3R,4R)-10, was
submitted to flash chromatography (hexane/ethyl acetate 3:1).
1647 cmꢃ1
; dH (300.13 MHz, CD3OD) 7.42e7.26 (m, 5H, Ph), 5.13 (s,
2H, OeCH2), 4.18e4.07 (m, 2H, H-3, H-4), 3.80e3.70 (m, 1H, H-2 or
H-5), 3.65e3.53 (m, 1H, H-5 or H-2), 3.42e3.30 (m, H-20, H-50
overlapped with the signal of the solvent), 1.93 (s, 3H, CH3); dC
(75.5 MHz, CDCl3) 173.3 (C]O), 156.8 (C]O), 138.1 (C), 129.5 (CH),
129.1 (CH), 129.0 (CH), [74.7 and 73.9 (C-4)], 68.2 (OeCH2), [57.3
and 56.5 (C-3)], [53.0, 52.7, 50.2, and 50.1 (C-2, C-5)], 22.4 (CH3);
HRMS (ESIþ): MHþ, found 279.1333. C14H19N2O4 requires 279.1339.
4.8.1. (3S,4S)-1-Benzyloxycarbonyl-3-(diallylamino)-4-hydro-
20
xypyrrolidine [(3S,4S)-6]. Yield 49% (0.155 g, 0.49 mmol); [
þ47.8 (c 1.0, CHCl3), ee 92%.
a]
D
4.5. Synthesis of ( )-trans-1-benzyloxycarbonyl-3-(tert-bu-
4.8.2. (3R,4R)-3-Acetoxy-1-(benzyloxycarbonyl)-4-(diallylamino)
toxycarbonyl)amino-4-hydroxypyrrolidine [( )-9]
pyrrolidine [(3R,4R)-10]. Colorless oil; yield 47% (0.168 g,
20
0.47 mmol); [
a
]
ꢃ17.2 (c 1.0, CH2Cl2), ee 99%; nmax(neat) 1742,
D
To a solution of (ꢀ)-7 (0.300 g, 1.27 mmol) in anhydrous
dichloromethane (12 mL), di-tert-butyl pyrocarbonate (0.360 g,
1.64 mmol) was added. After 24 h of reaction at room temperature,
solvents were eliminated and the residue was subjected to flash
chromatography (hexane/ethyl acetate 1:1). Compound (ꢀ)-9 was
isolated as a white solid; yield 87%; mp 140e142 ꢁC; nmax(KBr) 3323,
1711 cmꢃ1
; dH (300.13 MHz, CDCl3) 7.42e7.27 (m, 5H, Ph),
5.87e5.69 (m, 2H, 2ꢄ CH]CH2), 5.29e5.07 (m, 7H, OeCH2, H-3, 2ꢄ
CH]CH2), 3.87 (dd, 1H, 3J 6.4, j2Jj 12.0 Hz, H-2), 3.72e3.58 (m, 1H,
H-5), 3.52e3.26 (m, 3H, H-20, H-4, H-50), 3.24e3.08 (m, 4H, 2ꢄ
CH2eCH]CH2), 2.06 (s, 3H, CH3); dC (75.5 MHz, CDCl3) 170.2 (C]
O), 154.5 (C]O), 136.5 (C), [135.3 and 135.1 (CH]CH2)], 128.4 (CH),
127.9 (CH), 127.8 (CH), 117.5 (CH]CH2), [74.1 and 73.1 (C-3)], 66.9
(OeCH2), [64.2 and 63.3 (C-4)], 53.2 (NeCH2), [50.4 and 50.0 (C-2)],
46.5 (C-5), 20.9 (CH3); HRMS (ESIþ): MNaþ, found 381.1787.
C20H26N2NaO4 requires 381.1785.
1682 cmꢃ1
; dH (300.13 MHz, CDCl3) 7.40e7.30 (m, 5H, Ph), 5.13 (s, 2H,
OeCH2), [4.86 and 4.77 (br s,1H, NH)], 4.30e4.18 (m,1H, H-4 or H-3),
4.03e3.89 (m,1H, H-3 or H-4), 3.84 (dd,1H, 3J 6.8, j2Jj 11.0 Hz, H-2 or
H-5), 3.72 (dd, 1H, 3J 5.7, j2Jj 11.8 Hz, H-5 or H-2), 3.48e3.23 (m, 2H,
H-20, H-50), 2.90 (br s, 1H, OH),1.44 (s, 9H, tBu); dC (75.5 MHz, CDCl3)
[156.2 and 155.7 (C]O)], 154.8 (C]O), 136.4 (C), 128.4 (CH), 128.0
(CH), 127.8 (CH), 80.4 (C), [74.8 and 73.7 (C-4)], 67.0 (OeCH2), [57.1
and 56.6 (C-3)], [51.5, 51.2, and 49.2 (C-2, C-5)], 28.2 (CH3); HRMS
(ESIþ): MNaþ, found 359.1589. C17H24N2NaO5 requires 359.1577.
4.9. Enzymatic transesterification of ( )-9 on a 200 mg scale
The mixture containing substrate (ꢀ)-9 (0.200 g, 0.595 mmol),
CAL-B (0.200 g), 1,4-dioxane (6.0 mL), triethylamine (0.60 mL), and
vinyl acetate (0.274 mL, 2.98 mmol) was allowed to react at 30 ꢁC.
The reaction was monitorized by chiral-HPLC and stopped when
the enantiomeric excess for the substrate was >99% (3.5 days; Table
2, entry 8). After this time, the enzyme was filtered and washed
with methanol. Solvents were eliminated under reduced pressure
and the resulting crude material, which was formed by the optically
active (3S,4S)-9 and the ester (3R,4R)-12, was submitted to flash
chromatography (hexane/ethyl acetate 1:1).
4.6. Synthesis of ( )-trans-3-(acetamido)-4-acetoxy-1-(ben-
zyloxycarbonyl)pyrrolidine [( )-11]
Anhydrous pyridine (1.6 mL, 20 mmol) and acetyl chloride
(1.4 mL, 20 mmol) were added to a solution of (ꢀ)-7 (1.0 g,
4.2 mmol) in anhydrous dichloromethane (50 mL). After stirring at
room temperature for 12 h, the reaction mixture was successively
washed with aq 1 N HCl (3ꢄ50 mL) and brine (40 mL). The organic
phase was dried with Na2SO4 and evaporated to reduced pressure
to give a residue, which was subjected to flash chromatography
(ethyl acetate as eluent). White solid; yield 79%; mp 119e121 ꢁC;
4.9.1. (3S,4S)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-
hydroxypyrrolidine [(3S,4S)-9]. Yield 45% (90 mg, 0.268 mmol);
[a
]
20 ꢃ18.1 (c 1.0, CH2Cl2), ee>99%.
D
nmax(neat) 1743, 1707, 1683 cmꢃ1
; dH (300.13 MHz, CDCl3) 7.41e7.28
(m, 5H, Ph), 6.45 (br s, 1H, NH), 5.19e5.02 (m, 3H, OeCH2, H-4),
4.44e4.33 (m, 1H, H-3), 3.90e3.69 (m, 2H, H-2, H-5), 3.53e3.32 (m,
2H, H-20, H-50), 2.06 (s, 3H, CH3), 1.97 (s, 3H, CH3); dC (75.5 MHz,
4.9.2. (3R,4R)-3-Acetoxy-1-(benzyloxycarbonyl)-4-(tert-butox-
ycarbonylamino)pyrrolidine [(3R,4R)-12]. Yield 46% (0.103 g,
20
0.274 mmol); [
a
]
ꢃ0.7 (c 1.0, CH2Cl2), ee 91%.; nmax(KBr) 1744,
D

5412
J.A. Rodríguez-Rodríguez et al. / Tetrahedron 69 (2013) 5407e5412
1719,1706 cmꢃ1
; dH (300.13 MHz, CDCl3) 7.41e7.28 (m, 5H, Ph), 6.45
(ꢀ)-6: Chiralcel OJ-H; hexane/propan-2-ol 94:6, 0.8 mL/min, 30 ꢁC;
tR¼14.9 (3R,4R) and 16.5 (3S,4S) min; RS¼1.7.
(br s, 1H, NH), 5.19e5.02 (m, 3H, H-4, OeCH2), 4.44e4.33 (m, 1H, H-
3), 3.90e3.69 (m, 2H, H-2, H-5), 3.53e3.32 (m, 2H, H-20, H-50), 2.06
(s, 3H, CH3), 1.97 (s, 3H, CH3); dC (75.5 MHz, CDCl3) 170.2 (C]O),
154.7 (C]O), 136.1 (C), 128.5 (CH), 128.1 (CH), 127.9 (CH), [75.4 and
74.4 (C-4)], 67.1 (OeCH2), [53.5 and 52.7 (C-3)], 49.6 (C-2, C-5)],
22.9 (CH3), 20.9 (CH3); HRMS (ESIþ): MNaþ, found 401.1684.
C19H26N2NaO6 requires 401.1683.
(ꢀ)-10: Chiralcel OJ-H; hexane/propan-2-ol 94:6, 0.8 mL/min,
ꢁ
30 C; tR¼12.1 (3R,4R) and 13.5 (3S,4S) min; RS¼1.7.
(ꢀ)-7: Chiralcel OJ-H; hexane/ethanol 93:7, 0.8 mL/min, 40 ꢁC;
tR¼25.4 (3S,4S) and 28.0 (3R,4R) min; RS¼2.1.
(ꢀ)-8: Chiralcel OJ-H; hexane/ethanol 93:7, 0.8 mL/min, 40 ꢁC;
tR¼19.7 (3S,4S) and 22.5 (3R,4R) min; RS¼3.0. This compound
was also analyzed using: Chiralcel OJ-H; hexane/propan-2-ol
92:8, 0.8 mL/min, 40 ꢁC; tR¼17.2 (3S,4S) and 30.9 (3R,4R) min;
RS¼10.4.
4.10. Enzymatic hydrolysis of ( )-11
(ꢀ)-11: Chiralcel OJ-H; hexane/propan-2-ol 92:8, 0.8 mL/min,
40 ꢁC; tR¼35.9 (3S,4S) and 40.5 (3R,4R) min; RS¼2.1.
(ꢀ)-9: Chiralpack IA; hexane/ethanol 91:9, 0.8 mL/min, 30 ꢁC;
tR¼19.1 (3R,4R) and 22.1 (3S,4S) min; RS¼3.1.
To a solution of (ꢀ)-11 (100 mg, 0.313 mmol) in toluene (3.2 mL),
CAL-A (200 mg), and H2O (15 mL) were added. The mixture was
allowed to react during 2 days at 30 ꢁC (Table 3, entry 3) as in-
dicated in the general procedure (Section 4.7). The resulting crude
material, which was formed by the optically active (3S,4S)-8 and
(3R,4R)-11, was submitted to flash chromatography (ethyl acetate
as the eluent).
(ꢀ)-12: Chiralpack IA; hexane:ethanol 91:9, 0.8 mL/min, 30 ꢁC;
tR¼15.0 (3R,4R) and 20.3 (3S,4S) min; RS¼6.4.
Acknowledgements
4.10.1. (3S,4S)-3-(Acetamido)-1-benzyloxycarbonyl-4-hydroxyp-
yrrolidine [(3S,4S)-8]. Yield 36% (31 mg, 0.113 mmol); [
a]
20 ꢃ5.9 (c
D
Financial support from the Spanish MICINN (CTQ2011-24237) is
gratefully acknowledged.
1.0, MeOH), ee 94%.
4.10.2. (3R,4R)-3-(Acetamido)-4-acetoxy-1-(benzyloxycarbonyl)pyr-
rolidine [(3R,4R)-11]. Yield 55% (55 mg, 0.172 mmol); ee 62%.
References and notes
4.11. Optically active compounds involved in the de-
termination of the absolute configuration
1. (a) Lee, H.-S.; Kang, S. H. Synlett 2004, 1673e1685; (b) Bergmeier, S. C. Tetra-
hedron 2000, 56, 2561e2576; (c) Ager, D. J.; Prakash, I.; Schaad, D. R. Chem. Rev.
1996, 96, 835e875.
2. (a) O’Hagan, D. Nat. Prod. Rep. 2000, 17, 435e446 and references therein;
(b) Daly, J. W.; Spende, T. F. In Alkaloids: Chemical and Biological
Perspectives; Pelletier, S. W., Ed.; Wiley: New York, NY, 1986; Vol. 4,
Chapter 1.
3. (a) Tsuzuki, Y.; Tomita, K.; Shibamori, K.; Sato, Y.; Kashimoto, S.; Chiba, K. J. Med.
Chem. 2004, 47, 2097e2109; (b) Scatena, C. D.; Kumer, J. L.; Arbitrario, J. P.;
Howlett, A. R.; Hawtin, R. E.; Fox, J. A.; Silverman, J. A. Cancer Chemother.
Pharmacol. 2010, 66, 881e888 and references therein.
4.11.1. (3S,4S)-7. A mixture of (3S,4S)-9 (100 mg, 0.297 mmol;
ee>99%) and aq 3 N HCl (4.0 mL) was stirred at room temperature
during 24 h. Aqueous solution was washed with CH2Cl2 (2ꢄ5 mL),
and then, aq 4 N NaOH was added until pH was strongly basic. The
aqueous phase was extracted with CH2Cl2 (3ꢄ10 mL). Organic phase
was dried with Na2SO4, and the organic solvents were eliminated to
4. Tsuzuki, Y.; Chiba, K.; Mizuno, K.; Tomita, K.; Suzuki, K. Tetrahedron: Asymmetry
2001, 12, 2989e2997.
5. Kamal, A.; Shaik, A. A.; Sandbhor, M.; Malik, M. S.; Kaga, H. Tetrahedron Lett.
yield enantiopure (3S,4S)-7 (yield 70%). [
a
]
20 þ7.4 (c 1.0, CH2Cl2).
D
2004, 45, 8057e8059.
4.11.2. (3S,4S)-3-Amino-4-hydroxypyrrolidine
dihydrochloride
6. Kumar, A. R.; Reddy, J. S.; Rao, B. V. Tetrahedron Lett. 2003, 44, 5687e5689.
7. (a) Rodríguez-Rodríguez, J.; Brieva, R.; Gotor, V. Tetrahedron 2010, 66,
6789e6796; (b) Recuero, V.; Brieva, R.; Gotor, V. Tetrahedron: Asymmetry 2008,
19, 1684e1688.
[(3S,4S)-13]. To a solution of (3S,4S)-7 (50 mg, 0.21 mmol) in de-
oxygenated methanol (2.0 mL), 10% PdeC (10 mg) was added. The
mixture was stirred under a hydrogen atmosphere at room tem-
perature for 7 h. After this time, the catalyst was filtered through
a pad of CeliteÒ and washed with methanol. Conc. HCl (several
drops) was added to the solution and solvents were evaporated
8. This unit is also a structural part of other drugs: (a) Hong, J.; Zhang, Z.; Lei, H.;
Cheng, H.; Hu, Y.; Yang, W.; Liang, Y.; Das, D.; Chen, S.-H.; Li, G. Tetrahedron Lett.
2009, 50, 2525e2528; (b) Zbinden, K. G.; Anselm, L.; Banner, D. W.; Benz, J.;
ꢀ
Blasco, F.; Decoret, G.; Himber, J.; Kuhn, B.; Panday, N.; Ricklin, F.; Risch, P.;
giving (3S,4S)-13$2HCl. Yield 58%; [
a
]
20 þ12.4 (c 0.5, H2O); ee>99%.
Schlatter, D.; Stahl, M.; Thomi, S.; Unger, R.; Haap, W. Eur. J. Med. Chem. 2009,
44, 2787e2795, Some optically active trans-3-amino-4-hydroxypyrrolidine
D
20
Ref. 17 for (3R,4R)-13: [
a
]
D
ꢃ14.6 (c 1.08, H2O).
ꢀ
derivatives have shown to be efficient organocatalysts: (c) Martín-Rapun, R.;
ꢁ
Fan, X.; Sayalero, S.; Bahramnejad, M.; Cuevas, F.; Pericas, M. A. Chem.dEur. J.
2011, 17, 8780e8783.
9. Schaus, S. E.; Larrow, J. F.; Jacobsen, E. N. J. Org. Chem. 1997, 62,
4197e4199.
4.11.3. (3R,4R)-6. Compound (3R,4R)-10 (145 mg, 0.405 mmol) was
dissolved in a 0.50 M methanolic solution of sodium methoxide
(4.0 mL, 2.0 mmol) and the mixture was stirred at room tempera-
ture for 4 h. Elimination of the solvent yielded a residue, which was
purified by flash chromatography (hexane/ethyl acetate 1:1) as
10. (a) Rejman, D.; Panova, N.; Klener, P.; Maswabi, B.; Pohl, R.; Rosenberg, I. J. Med.
ꢂ
ꢂ
ꢀ
ꢀ
Chem. 2012, 55, 1612e1621; (b) Kocalka, P.; Rejman, D.; Vanek, V.; Rinnova, M.;
ꢂ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢂꢂ
Tomeckova, I.; Kralíkova, S.; Petrova, M.; Pav, O.; Pohl, R.; Budesínsky, M.;
ꢂ
Liboska, R.; Tocík, Z.; Panova, N.; Votruba, I.; Rosenberg, I. Bioorg. Med. Chem.
eluent. Yield 93%; [
a
]
20 ꢃ52.6 (c 1.0, CH2Cl2), ee 99%.
Lett. 2010, 20, 862e865.
D
ꢀ
~
11. Gonzalez-Sabín, J.; Morís-Varas, F.; Pena, C.; Rebolledo, F.; Gotor, V. J. Mol. Catal.
B: Enzym. 2009, 59, 111e115.
4.11.4. (3R,4R)-7. It was prepared from (3R,4R)-6 as indicated for
ꢀ
12. Garro-Helion, F.; Merzouk, A.; Guibe, F. J. Org. Chem. 1993, 58, 6109e6113.
the racemic compound (ꢀ)-7. [
a
]
20 ꢃ6.8 (c 1.0, CH2Cl2), ee 99%.
13. (a) Chen, C. S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. Am. Chem. Soc. 1982, 104,
7294e7299; (b) Faber, K.; Hoenig, H. Program ‘Selectivity’. http://borgc185.
kfunigraz.ac.at/sites/research/model.htm.
D
4.11.5. (3R,4R)-11. It was obtained from (3R,4R)-7 following the
14. Kazlauskas, R. J.; Weissfloch, A. N. E.; Rappaport, A. T.; Cuccia, L. A. J. Org. Chem.
procedure described for the racemic compound (ꢀ)-11. [
a]
20 þ2.7 (c
D
1991, 56, 2656e2665.
15. This behaviour has also been observed with other substrates, see: (a) Ref. 7a.
(b) Recuero, V.; de Gonzalo, G.; Brieva, R.; Gotor, V. Eur. J. Org. Chem. 2006,
4224e4230.
1.0, CH2Cl2), ee 99%.
4.12. HPLC determination of the enantiomeric excesses
16. Theil, F. Tetrahedron 2000, 56, 2905e2919.
17. Limberg, G.; Lundt, I.; Zavilla, J. Synthesis 1999, 178e183.
18. Koskinen, A. M. P.; Helaja, J.; Kumpulainen, E. T. T.; Koivisto, J.; Mansikkamaki,
€
The enantiomeric excess for each compound obtained in the
enzymatic reactions was determined by chiral-HPLC. Results ob-
tained in the analysis of racemic samples are as follows:
H.; Rissanen, K. J. Org. Chem. 2005, 70, 6447e6453.
19. Pena, C.; Gonzalez-Sabín, J.; Rebolledo, F.; Gotor, V. Tetrahedron: Asymmetry
2008, 19, 751e755.
~
ꢀ