Enzymatic preparation of cis and trans-3-amino-4-hydroxytetrahydrofurans and cis-3-amino-4-hydroxypyrrolidines

Article abstract of DOI:10.1016/j.bmc.2014.05.014

The lipase catalyzed resolution of cis and trans-3-amino-4-hydroxytetrahydrofurans and cis-3-amino-4-hydroxypyrrolidines have been studied. For all the heterocycles, the best enantioselectivity was obtained using Candida antarctica lipases A and B as cata

Full text of DOI:10.1016/j.bmc.2014.05.014

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Enzymatic preparation of cis and trans-3-amino-4-
hydroxytetrahydrofurans and cis-3-amino-4-hydroxypyrrolidines
⇑
⇑
Ángela Villar-Barro, Vicente Gotor , Rosario Brieva
Departamento de Química Orgánica e Inorgánica and Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, 33006 Oviedo (Asturias), Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The lipase catalyzed resolution of cis and trans-3-amino-4-hydroxytetrahydrofurans and cis-3-amino-4-
hydroxypyrrolidines have been studied. For all the heterocycles, the best enantioselectivity was obtained
using Candida antarctica lipases A and B as catalysts in hydrolytic processes. The absolute configuration of
the optically pure obtained heterocycles has been assigned.
Received 25 February 2014
Revised 6 May 2014
Accepted 9 May 2014
Available online xxxx
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
3-amino-4-hydroxytetrahydrofuran
3-amino-4-hydroxypyrrolidine
Heterocyclic amino alcohols
Enzymatic resolution
Biocatalysis
1. Introduction
O
OR
N
NH
H
N
NH2
O
N
The importance of optically active 3,4-disubstituted tetra-
hydrofurans and pyrrolidines is evident from their presence in a
wide variety of bioactive natural products and drugs, and their suc-
cessful applications as stereochemical control elements. In partic-
ular, the vicinal heterocyclic amino alcohols are used in
organocatalysis,1 as chiral auxiliaries,2 and building blocks for the
synthesis of complex molecules that exhibit interesting biological
activities.3
N
N
R
N
N
NH2
RO
F
X
R
Ar
O
P
OH
N
N
HO
HO
X = O, N-heteroaryl
F
1
3
O
RHN
2
Figure 1.
As a part of our research on the enzymatic preparation of
optically active functionalized heterocycles, we have reported the
synthesis and enzymatic resolution of trans-3-amino-4-hydroxy-
pyrrolidines.4 Now, we are interested in extending this biocatalytic
method to the synthesis of the cis-isomer derivatives, and also to
the cis- and trans-3-amino-4-hydroxytetrahydrofuran analogs,
since the presence of these moieties and their absolute (as well
as relative) stereochemistry are important for the biological
activity of the molecules that contain them.
Figure 1 shows three examples of these pharmacologically
active molecules: the pyrrolidine derivative of guanine, 1 is a
new class of bi-substrate inhibitor of human purine nucleoside,3a
the quinolone derivative, 2 shows antibacterial activity3f and
1,4-pyrazine derivatives, 3 are claimed as corticotropin releasing
factor, receptor antagonists.3e
Various synthetic approaches have been described for the
synthesis of these structures.5 Using biocatalytic methods, the
cis-relative configuration can be obtained by the SN2 displacement
reaction of the bromo functionality in the 3,4-heterocyclic bromo-
hydrins ( )-trans-5 and ( )-trans-12, whose resolution has been
recently reported.6 Another synthetic approach for the preparation
of the optically active 3-amino-4-hydroxypyrrolidines, is the enzy-
matic resolution of the 3-azido-4-hidroxypyrrolidines ( )-cis-15
and also the trans-isomer, described by Kamal et al.7
The method described herein is a new and convenient biocata-
lytic approach for the preparation of all the isomers of these
heterocyclic amino alcohols.
⇑
Corresponding authors. Tel.: +34 985 102 994; fax: +34 985 103 448.
E-mail addresses: vgs@uniovi.es (V. Gotor), rbrieva@uniovi.es (R. Brieva).
http://dx.doi.org/10.1016/j.bmc.2014.05.014
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
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2
Ángela Villar-Barro et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
HO
NHBoc
O
NHBoc
2. Results and discussion
O
NHBoc
Ph
Ph
Lipase
O
O
+
On the bases of our previous experience4,6 we choose as sub-
strates the N-Boc derivatives ( )-cis-8, ( )-trans-8 and ( )-cis-17a,
that have been synthesized in good yields by conventional meth-
ods, summarized in Scheme 1.
O
O
O
10 equiv. H2O
tBuOMe, 30 ºC
7
8
(3R,4R)-8
(3S,4S)-
( )-cis-
Scheme 2. Lipase catalysed hydrolysis of ( )-cis-8.
We have proven that the presence of a bulky substituent adja-
cent to the secondary carbon bearing the hydroxyl group could
favour the enzymatic resolution of these compounds.4 Also, the
tert-butoxycarbonyl protecting group can be selectively removed
in the presence of the benzyloxycarbonyl group in the case of the
pyrrolidine ring. On the other hand, the presence of the
phenylacetyl ester is very convenient for a good light-absorbing
in UV-detector HPLC.
Initially, we studied the enzymatic hydrolysis of ( )-cis-3-tert-
butoxycarbonylamino-4-phenylacetoxytetrahydrofuran, ( )-cis-8.
The reactions were carried out at 30 °C in organic solvent (tBuOMe)
and using 10 equiv of water. Lipases from Candida antarctica (CAL-
A and CAL-B), Burkholderia cepacia (PSL-IM) and Thermomyces
lanuginosus (TL-IM) were tested. All the processes were carried
out on a 15 mg scale (Scheme 2).
The lipases CAL-A and CAL-B catalysed the hydrolysis of sub-
strate ( )-cis-8, (Table 1, entries 1 and 2) whereas, using TL IM or
PSL-IM the unaltered starting material was recovered after three
days of reaction. Both enzymes, CAL-A and CAL-B, showed very
high enantioselectivity but different reaction rate: using CAL-B, a
50% of conversion was achieved after 10 h of reaction, whereas
the process catalysed by CAL-A was slower, three days were neces-
sary to achieve the same conversion than with CAL-B. In both pro-
cesses, the product and the remaining substrate were obtained in
enantiopure form. On the other hand, the two enzymes showed
the same stereochemical preference. The assignation of absolute
configurations of the products and the remaining substrate is
described later.
In view of the excellent results obtained in the resolution
of the tetrahydrofuran derivatives, we tested the resolution
of substrate ( )-cis-1-benzyloxycarbonyl-3-tert-butoxycarbonyla-
mino-4-hydroxypyrrolydine, ( )-cis-17a under the same reaction
conditions (Scheme 4). The CAL-A catalysed hydrolysis showed
again a high enantioselectivity and a moderate reaction rate, both
enantiomers were obtained in enantiopure forms, after 48 h of
reaction (Table 1, entry 5). Surprisingly, the unaltered starting
material was recovered after two days of reaction when CAL-B
was used as biocatalyst.
In order to improve the reaction rate, we tested the methoxy-
methyl derivative ( )-cis-17b as substrate in the hydrolytic process
catalysed by lipases CAL-A or CAL-B, under the same reaction con-
ditions (Scheme 4).
Effectively, the reaction rate was improved. In the case of CAL-A
(Table 1, entry 7), only 23 h of reaction were necessary to achieved
a 50% of conversion, but more surprising was the strong influence
of the substrate structure in the process catalyzed by CAL-B since
in this case a 50% of conversion can be achieved after 10 h (Table 1,
entry 8). A very high enantioselectivity was observed for both
enzymes and, again, the substrate and the product were obtained
in their optically pure forms.
Finally, the enzymatic acylation processes of substrates ( )-cis-
7, ( )-trans-7 and ( )-cis-16 ware examined. For both enzymes,
very low or no conversions were observed when 2-phenyl acetate,
ethyl acetate or methoxymethyl acetate were used as acylating
agents. Also, using vinyl acetate, very low conversion was observed
in the acylation of ( )-cis-7 and ( )-trans-7. In the case of substrate
( )-cis-16, CAL-B showed a high enantioselectivity (E >200) and a
moderate reaction rate, after 48 h of a 50% of conversion. These
process are shown in Scheme 5, the results were very similar to
those already described for ( )-trans-16.4
The assignment of the absolute configurations to the optically
pure heterocycles obtained in the enzymatic processes was estab-
lished as follows.
The optically pure products of the lipase catalysed hydrolysis
(ꢀ)-cis-7 and (ꢀ)-trans-7 were treated with aq 3N HCl in methanol
Taking into account the results obtained in the resolution of the
substrate ( )-cis-8, we carried out the enzymatic hydrolysis of the
( )-trans-3-tert-butoxycarbonylamino-4-hydroxytetrahydrofuran,
( )-trans-8, using the same lipases and reaction conditions.
(Scheme 3, Table 1, entries 3 and 4).
The obtained results were very similar to those obtained in the
hydrolysis of the cis-isomer: very high enantioselectivity and dif-
ferent reaction rates were observed depending on the biocatalyst
used. The process catalysed by CAL-A was very slow, while in only
three hours of reaction a 50% conversion was achieved using CAL-B
as catalyst.
R
O
NHBoc
HO
Br
O
N3
HO
NHBoc
b
Ph
a
b, c
d
O
O
O
O
O
O
6
O
8,
(
)-trans-5
7
( )-cis- R = benzyl
(
)-cis-
( )-cis-
4
O
R
(
O
NHBoc
HO
(
N3
HO
NHBoc
b
e
d
O
O
O
O
O
9
)-trans-10
(
)-trans-7
)-trans-8 R = benzyl
HO
NHBoc
HO
Br TBDMSO
Br
HO
N3
R
O
NHBoc
TBDMSO
c
N3
a
b
f
g
d
O
N
N
Cbz
16
N
N
N
N
N
Cbz
Cbz
Cbz
Cbz
)-trans-13
Cbz
)-cis-14
Cbz
17a,
R = benzyl
)-cis-17b, R = methoxymethyl
)-cis-17c, R = methyl
(
(
(
)-cis-
(
)-trans-12
(
(
(
)-cis-
11
15
( )-cis-
Scheme 1. Synthesis of substrates: (a) NBS, H2O, 4 h, rt.6 (b) RCOCl, K2CO3, CH3CN, 12 h, rt. (c) NaN3, DMSO, 110 °C, 12 h. (d) PMe3, Boc2O, NaOH 1 M, THF, rt, 3 h. (e) NaN3,
H2O/1,4-dioxane 1:4, 100 °C, 14 h. (f) TBDMSCl, imidazole, CH2Cl2, rt, 5 h. (g) CSA, anhydrous MeOH, rt, 3 h.
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Ángela Villar-Barro et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
Table 1
Lipase catalysed hydrolysis, in tBuOMe at 30 °C, using 10 equiv of water
b
a
Entry
Lipasea
Substrate
t (h)
ees (%)
Configuration
eep (%)
Configuration
cc (%)
Ec
1
2
3
4
5
6
7
8
CAL-A
CAL-B
CAL-A
CAL-B
CAL-A
CAL-B
CAL-A
CAL-B
( )-cis-8
( )-cis-8
75
10
7 (days)
3
48
48
23
10
>99
>99
>99
>99
>99
—
(3R,4R)
(3R,4R)
(3S,4R)
(3S,4R)
(3R,4S)
—
>99
>99
>99
>99
>99
—
(3S,4S)
(3S,4S)
(3R,4S)
(3R,4S)
(3S,4R)
—
50
50
50
50
50
—
>200
>200
>200
>200
>200
—
( )-trans-8
( )-trans-8
( )-cis-17a
( )-cis-17a
( )-cis-17b
( )-cis-17b
>99
>99
(3R,4S)
(3R,4S)
>99
>99
(3S,4R)
(3S,4R)
50
50
>200
>200
a
b
c
Weight ratio enzyme/substrate 1:1.
Determined by chiral HPLC.
Conversion, c = ees/(ees + eep), enantiomeric ratio, E ¼ ln½ð1 ꢀ cÞð1 ꢀ eesÞꢂ=ln½ð1 ꢀ cÞð1 þ eesÞꢂ.8
HO
NHBoc
In the case of the pyrrolidine derivatives, a better yield in the
selective deprotection of (+)-cis-16 was obtained when the reac-
tion was accomplished by saturating the solution of (+)-cis-16 in
ethanol with HCl and allowing the mixture to stir at room temper-
ature (Scheme 7).The specific rotation sign of the amino alcohol
(+)-cis-19 obtained, was opposite to that reported for the (3R,4S)-
enantiomer.3e
O
NHBoc
O
NHBoc
Ph
Ph
Lipase
10 equiv. H2O
tBuOMe, 30 ºC
+
O
O
O
O
O
8
(3R,4S)-7
( )-trans-8
(3S,4R)-
Scheme 3. Lipase catalysed hydrolysis of ( )-trans-8.
It should be noted that, for all the substrates, the lipases prefer-
entially catalyzed the hydrolysis of the expected enantiomer in
accordance with the Kazlauskas’ rule.10
HO
NHBoc
R
O
NHBoc
R
O
NHBoc
Lipase
+
O
O
10 equiv. H2O
tBuOMe, 30 ºC
N
N
N
3. Conclusion
Cbz
Cbz
Cbz
17a,b
(3R,4S)-
(3S,4R)-16
( )-cis-17a,b
a,
This paper describes a convenient approach to the preparation
of all the isomers of cis and trans-3-amino-4-hydroxytetrahydrofu-
ran and cis-3-amino-4-hydroxypyrrolidine in their optically pure
forms, via a CAL-A or CAL-B catalyzed hydrolysis. The pyrrolidine
derivative ( )-cis-16 that can also be resolved by a CAL-B catalysed
acylation of the hydroxyl group. In general, the hydrolysis cata-
lysed by CAL-B is faster than by CAL-A.
R = benzyl
b, R = metoxymethyl
Scheme 4. Lipase catalysed hydrolysis of ( )-cis-17a,b.
O
O
NHBoc
4
HO
NHBoc
HO
NHBoc
3
O
3
4
3
4
CAL-B
+
+
tBuOMe
48 h, 30 ºC
O
N
N
N
4. Experimental
E > 200, c = 50
Cbz
(3R,4S)-17c
Scheme 5. CAL-B catalysed acetylation of ( )-cis-16.
Cbz
Cbz
General Remarks. Enzymatic reactions were carried out in a
Gallenkamp incubatory orbital shaker. Immobilized Candida ant-
arctica lipase B, CAL-B (Novozym 435, 7300 PLU/g), was a gift from
Novo Nordisk co., immobilized CAL-A (lipase NZL-101, 6,2 U/g) is
commercialized by Codexis, immobilized Burkholderia cepacia
lipase (PS-SD, 23,000 U/g) is commercialized by Amano
Pharmaceuticals and Thermomyces lanuginosus (TL-IM, 560 TBU/
g) is commercialized by Novo Nordisk. Chemical reagents were
commercialized by Aldrich, Merck, ACROS organics or Alfa Aesar.
Solvents were distiled over an appropriate desiccant under nitro-
gen. Flash chromatography was performed using Merck silica gel
60 (230–400 mesh). Optical rotations were measured using a
Perkin–Elmer 343 polarimeter and are quoted in units of 10ꢀ1
16
(
)-cis-
16
(3R,4S)-
NHBoc
HO
NH2
HO
∗
∗
O
O
HCl 3N / MeOH
7
18
cis-(3S,4S)-
cis-(3S,4S)-
7
18
trans-(3R,4S)-
trans-(3R,4S)-
Scheme 6. Deprotection of Boc group in aq 3N HCl.
deg cm2 gꢀ1 1H NMR, 13C NMR and DEPT spectra were recorded
.
in a Bruker AC-300, Bruker AC-300 DPX or Bruker NAV-400 spec-
trometer using CDCl3 or MeOH-d4, as solvent. The chemical shift
values (d) are given in ppm. For pyrrolidins, duplicity of some car-
bon signals (especially for those of the ring) and broad signals in 1H
NMR were observed due to the presence of different conformers
around the N(1) (C@O) bond. APCI+ and ESI+ using a Hewlett–
HO
NHBoc
HO
NH2
HCl gas
EtOH
N
N
Cbz
Cbz
cis-(3S,4R)-16
cis-(3S,4R)-19
Packard 1100 chromatograph mass detector or EI+ with
a
Scheme 7. Deprotection of Boc group in HCl saturated ethanol.
Hewlett–Packard 5973 mass spectrometer were used to record
mass spectra (MS). IR spectra were recorded in a UNICAM Mattson
3000 FT. The enantiomeric excesses were determined by chiral
HPLC analysis on a Hewlett–Packard 1100, LC liquid chromato-
graph, using a CHIRALPAK OJ-H column (4.5 ꢁ 250 mm) and
CHIRALPAK IA column (4.6 ꢁ 250 mm).
at room temperature to remove the Boc group and isolate (ꢀ)-cis-
18 and (ꢀ)-trans-18, whose specific rotation signs were in agree-
ment with those reported for the (3S,4S) and (3R,4S)-enantiomers
respectively9 (Scheme 6).
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4
Ángela Villar-Barro et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
The starting materials ( )-cis-3-azido-4-phenylacetoxytetra-
4.3. General procedure for the acylation of ( )-cis-7, ( )-trans-7
and ( )-cis-16
hydrofuran, ( )-cis-6, and ( )-cis-1-benzyloxycarbonyl-3-azido-4-
hydroxypyrrolidine, ( )-cis-15, were prepared following the
methodology described in Ref. 6
To a solution of the corresponding N-tert-butoxycarbonylami-
noalcohol (3.0 mmol) in acetonitrile (30 mL), the corresponding
acyl chloride (4.6 mmol) and K2CO3 (623 mg, 4.5 mmol) were
added. The mixture was stirred at room temperature for 12 h.
Then, the solvent was removed under reduced pressure and ethyl
acetate (25 mL) was added to the crude residue; the mixture was
washed with water (3 ꢁ 15 mL) and the organic phase was dried
over Na2SO4. The solvent was removed under reduced pressure
and the crude residue was purified by flash chromatography on sil-
ica gel.
4.1. Synthesis of ( )-trans-3-azido-4-hydroxytetrahydrofuran,
( )-trans-10
To a solution of 3,4-epoxytetrahydrofuran 9 (1.0 g, 11.6 mmol)
in a mixture of 1,4-dioxane/H2O 4:1 (50 mL), sodium azide (2.2 g,
33.8 mmol) was added. The mixture was stirred at 100 °C, for
12 h, in a sealed tube. After this time, the resulting mixture was
extracted with EtOAc (3 ꢁ 10 mL), the organic phase was dried
over Na2SO4 and the crude residue was purified by flash chroma-
tography on silica gel (hexane/EtOAc 7:3) to afford the product
( )-trans-10 as a colourless oil in a 80% yield. The spectroscopic
data were consistent with those previously described.5d Better
overall yield can be achieved using the crude residue in the next
step of the synthesis without further purification.
4.3.1. ( )-cis-3-tert-Butoxycarbonylamino-4-phenylacetoxytetrahy-
drofuran, ( )-cis-8
Flash chromatography eluent: hexane/EtOAc 6:4 Yellow oil,
yield 80%. 1H NMR (CDCl3, 300.13 MHz): d 7.42–7.27 (m, 5H),
5.25 (m, 1H, H-4), 4.45–4.43 (m, 2H, NH and, H-2 or H-5), 4.14–
4.01 (m, 2H, H-2 and H-5), 3.82 (m, 1H, H-2 or H-5), 3.72 (s, 2H),
3.43 (m, 1H, H-3), 1.45 (s, 9H); 13C NMR (CDCl3, 75.5 MHz): d
170.5 (CO), 155.7 (CO), 133.4 (C), 129.1 (CH), 128.8 (CH), 127.4
(CH), 80.0 (C), 73.3 (CH), 72.6 (CH2), 70.0 (CH2), 52.1 (CH), 41.5
4.2. General procedure for the synthesis of ( )-cis- and ( )-
trans-3-tert-butoxycarbonylamino-4-hydroxytetrahydrofuran
and ( )-cis-1-benzyloxycarbonyl-3-tert-butoxycarbonylamino-
4-hydroxypyrrolidine, ( )-cis-7, ( )-trans-7 and ( )-cis-16
(CH2), 28.3 (CH3); IR (neat, NaCl):
m ,
3341, 1730, 1711 cmꢀ1
HRMS-ESI+ calcd for [C17H23NO5Na]+ (M+Na)+ 344.1468 m/z, found
To a solution of ( )-cis-6, ( )-trans-10 or ( )-cis-15 (4 mmol) in
THF (40 mL), NaOH 1M (8 mL), PMe3 (12 mmol) and di-tert-butyl-
pyrocarbonate (12 mmol) was added. The resulting mixture was
stirred at room temperature for 3 h. After this time, the solvent
was removed under reduced pressure to afford the corresponding
crude residue that was purified by flash chromatography on silica
gel (hexane/EtOAc 3:7).
344.1466.
4.3.2. ( )-trans-3-tert-Butoxycarbonylamino-4-phenylacetoxytetra-
hydrofuran, ( )-trans-8
Flash chromatography eluent: hexane/EtOAc 6:4 Yellow oil,
yield 80%. 1H NMR (CDCl3, 300.13 MHz): d 7.40–7.25 (m, 5H),
5.11 (m, 1H, H-4), 4.85 (br s, 1H, NH), 4.12–4.02 (m, 3H, H-2, H-3
and H-5), 3.74–3.68 (m, 2H, H-2 and H-5), 3.66 (s, 2H), 1.45 (s,
9H); 13C NMR (CDCl3, 75.5 MHz): d 170.9 (CO), 155.9 (CO), 133.4
(C), 129.2 (CH), 128.8 (CH), 127.2 (CH), 79.0 (CH), 77.6 (C), 72.1
(CH2), 71.9 (CH2), 56.6 (CH), 41.10 (CH2), 28.3 (CH3); IR (neat,
4.2.1. ( )-cis-3-tert-Butoxycarbonylamino-4-hydroxytetrahydro-
furan, ( )-cis-7
White solid, yield 85%; mp 148–154 °C. 1H NMR (CDCl3,
300.13 MHz): d 5.22 (br s, 1H), 4.32 (br s, 1H, H-4), 4.16 (br s,
1H, H-3), 4.07–3.96 (m, 2H, H-2 and H-5), 3.78 (dd, J 10.1, J 2.3
Hz, 1H, H-5), 3.53 (t, J 8.0 Hz, 1H, H-2), 1.45 (s, 9H); 13C NMR
(CDCl3, 75.5 MHz): d 155.9 (CO), 80.0 (C), 74.4 (CH), 70.6 (CH2),
NaCl):
m
3341, 1730, 1711 cmꢀ1; HRMS-ESI+ calcd for [C17H23NO5-
Na]+ (M+Na)+ 344.1468 m/z, found 344.1478.
4.3.3. ( )-cis-1-Benzyloxycarbonyl-3-tert-butoxycarbonylamino--
4-phenylacetoxypyrrolidine, ( )-cis-17a
70.4 (CH2), 53.5 (CH), 28.3 (CH3); IR (neat, NaCl):
m 3400–3200,
1690, cmꢀ1 HRMS-ESI+ calcd for [C9H17NO4Na]+ (M+Na)+
;
Flash chromatography eluent: hexane/EtOAc 8:2 Yellow oil,
yield 95%. 1H NMR (CDCl3, 300.13 MHz): d 7.45–7.25 (m, 10H),
5.15 (s, 2H), 4.32 (m, 1H, H-4), 3.87–3.43 (m, 4H, H-3 and, H-2 or
H-5), 3.36 (s, 2H), 3.2 (m, 1H, H-2 or H-5), 1.46 (s, 9H); 13C NMR
(CDCl3, 75.5 MHz): d 175.8 (CO), 170.4 (CO), 154.7 (CO), 136.5
(C), 133.6 (C), 129.4 (CH), 129.1 (CH), 128.8 (CH), 128.6 (CH),
128.1 (CH), 127.5 (CH), 127.2 (CH), 80.0 (C), 73.1 and 72.9 (CH),
67.1 and 65.9 (CH2), 50.9 and 50.6 (CH2), 49.4 (CH2), 48.1 (CH),
226.1050 m/z, found 226.1030.
4.2.2. ( )-trans-3-tert-Butoxycarbonylamino-4-hydroxytetrahy-
drofuran, ( )-trans-7
White solid, yield 85%; mp 144–146 °C. 1H NMR (CDCl3,
300.13 MHz): d 4.79 (br s, 1H), 4.30 (m, 1H, H-4), 4.09 (m, 2H, H-
2 and H-5), 3.97 (m, 1H, H-3), 3.71 (dd, J 9.9 Hz, J 2.9, 1H, H-5),
3.64 (dd, J 9.4 Hz, J 2.9, 1H, H-2), 1.47 (s, 9H); 13C NMR (CDCl3,
75.5 MHz): d 155.9 (CO), 80.3 (C), 77.3 (CH), 73.6 (CH2), 71.0
41.6 and 41.0 (CH2), 28.3 and 27.0 (CH3). IR (neat, NaCl):
m 3053,
1715, 1705 cmꢀ1; HRMS-ESI+ calcd for [C25H30N2O6Na]+ (M+Na)+
(CH2), 59.8 (CH), 28.3 (CH3); IR (neat, NaCl):
m 3400–3200, 1689,
cmꢀ1; HRMS-ESI+ calcd for [C9H17NO4Na]+ (M+Na)+ 226.1050 m/z,
477.1996 m/z, found 477.1949.
found 226.1061.
4.3.4. ( )-cis-1-Benzyloxycarbonyl-3-tert-butoxycarbonylamino--
4-(2-methoxyacetoxy)pyrrolidine, ( )-cis-17b
4.2.3. ( )-cis-1-Benzyloxycarbonyl-3-tert-butoxycarbonylamino-
4-hydroxypyrrolidine, ( )-cis-16
Yellow oil, yield 85%. 1H NMR (CDCl3, 300.13 MHz): d 7.35 (s,
5H), 5.34 (br s, 1H, H-4), 5.13 (s, 2H), 4.82 (m, 1H, H-3), 4.44 (br
s, 1H, NH) 4.08 (s, 2H), 3.89 (m, 1H, H-2 or H-5), 3.65 (m, 2H, H-
2 and H-5), 3.44 (s, 3H), 3.19 (m, 1H, H-2 or H-5), 1.45 (s, 9H);
13C NMR (CDCl3, 75.5 MHz): d 169.9 (CO), 155.3 (CO), 155.1 (CO),
136.8 (C), 128.9 (CH), 128.5 (CH), 128.4 (CH), 80.7 (C), 73.8 and
73.1 (CH), 70.0 (CH2), 67.5 (CH2), 59.8 (CH3), 51.7 (CH), 51.2 and
Colourless oil, yield 87%. 1H NMR (CDCl3, 300.13 MHz): d 7.35
(m, 5H), 5,26 (m, 1H), 5.12 (s, 2H), 4.41–4.05 (m, 2H, H-3 and H-
4), 3.71 (m, 1H, H-2 or H-5), 3.54 (m, 2H, H-2 and H-5), 3.35–
3.17 (m, 1H, H-2 or H-5); 1.45 (s, 9H); 13C NMR (CDCl3,
75.5 MHz): d 155.6 (CO), 155.1 (CO), 136.5 (C), 128.5 (CH), 128.1
(CH), 127.9 (CH), 80.0 (C), 70.4 and 69.6 (CH), 67.1 (CH2), 53.1
50.9 (CH2), 48.4 (CH2), 28.7 (CH3); IR (neat, NaCl):
1710 cmꢀ1 HRMS-ESI+ calcd for [C25H30N2O6Na]+ (M+Na)+
431.1794 m/z, found 431.1789.
m 3345, 1730,
(CH2), 52.6 (CH), 48.0 (CH2), 28.3 (CH3); IR (neat, NaCl):
m 3400–
3200, 1689, cmꢀ1; HRMS-ESI+ calcd for [C17H24N2O5Na]+ (M+Na)+
;
359.1583 m/z, found 359.1577.
Please cite this article in press as: Villar-Barro, Á;. et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.014

Ángela Villar-Barro et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
4.3.5. ( )-cis-1-Benzyloxycarbonyl-3-acetoxy-4-tert-butoxycar-
4.6. General procedure for the enzymatic acylation
bonylaminopyrrolidine, ( )-cis-17c
Colourless oil, yield 80%. 1H NMR (CDCl3, 300.13 MHz): d 7.52–
7.31 (m, 5H), 5.23 (m, 1H, H-3), 5.13 (s, 2H), 4.75 (m, 1H, H-4), 4.41
(m, 1H, NH), 3.89 (m, 1H, H-2 or H-5), 3.62 (m, 2H, H-2 and H-5),
3.19 (m, 1H, H-2 or H-5), 2.13 (s, 3H), 1.59 (s, 9H); 13C NMR (CDCl3,
75.5 MHz): d 170.7 (CO), 154.93 (CO), 130.7 (C), 128.5 (CH), 128.1
(CH), 128.0 (CH), 80.3 (C), 77.3 (CH), 67.2 (CH2), 50.9 (CH), 48.26
The reaction mixture containing the corresponding N-Boc-
amino alcohol (15 mg), the lipase (15 mg) and the acylating agent
(5 equiv) in tBuOMe (1 mL), was shaken at 30 °C and 250 rpm in an
orbital shaker. The progress of the reaction was monitored by TLC
until achievement of the required conversion. The enzyme was
t
then removed by filtration and washed with BuOMe. The solvent
(CH2), 28.3 (CH3), 22.65 (CH3); IR (neat, NaCl):
m
1744, 1719,
was evaporated under reduced pressure and the crude residue
was purified by flash chromatography on silica gel using a gradient
hexane/EtOAc from 8:2 to 3:7.
1706 cmꢀ1 HRMS-ESI+ calcd for [C19H26N2O6Na]+ (M+Na)+
;
401.1683 m/z, found 401.1660.
4.4. General procedure for the enzymatic hydrolysis
4.6.1. (3R,4S)-1-Benzyloxycarbonyl-3-acetoxy-4-tert-butoxycar-
bonylaminopyrrolidine. (3R,4S)-17c
20
The reaction mixture, containing the corresponding hydroxya-
cylated-N-Boc-aminoalcohol (15 mg), the lipase (15 mg) and H2O
(10 equiv) in BuOMe (2,5 mL), was shaken at 30 °C and 250 rpm
Yellow oil, yield 18%; [
a
]
D
ꢀ2.0 (c 0.07, MeOH), ee >99%.
t
4.7. Assignment of the absolute configuration of
tetrahydrofuran derivatives
in an orbital shaker. The progress of the reaction was analyzed
by TLC until the achievement of the required conversion (approx-
imately). Then, the enzyme was removed by filtration and washed
t
The CAL-B catalyzed hydrolysis of ( )-cis-8, ( )-trans-8 and ( )-
cis-17b were carried out on a 50 mg scale in order to obtain the
optically pure 3-tert-butoxycarbonylamino-4-hydroxyheterocy-
cles, that were deprotected to the corresponding amino alcohols.
with BuOMe. The crude residue was purified by flash chromatog-
raphy on silica gel using a gradient hexane/EtOAc from 8:2 to 3:7.
4.4.1. (3S,4S)-3-tert-Butoxycarbonylamino-4-hydroxytetrahydro-
furan (3S,4S)-7
20
4.7.1. Synthesis of (3S,4S)-3-amino-4-hydroxytetrahydrofuran,
(3S,4S)-(ꢀ)-18
White solid, yield 48%; [
a
]
ꢀ3.6 (c 0.10, MeOH), ee >99%.
D
The optically pure product (ꢀ)-cis-7 or (ꢀ)-trans-7 (10 mg,
0.05 mmol) obtained from the CAL-B catalyzed hydrolysis, was
4.4.2. (3R,4R)-3-tert-Butoxycarbonylamino-4-phenylacetoxytetra-
hydrofuran, (3R,4R)-8
20
dissolved in MeOH (1 mL) and treated with aq 3N HCl (20 lL).
Yellow oil, yield 49%; [
a
]
D
+16.3 (c 0.10, EtOH), ee >99%.
The mixture was stirred at room temperature for 24 h. Then,
the solvent was removed under reduced pressure. The crude
residue was purified by flash chromatography on silica gel
(dichloromethane/methanol 8:2). The specific rotation signs of
the pure products were in accordance with that reported for
the (3S,4S)-(ꢀ)-3-amino-4-hydroxytetrahydrofuran and the (3R,4S)-
(ꢀ)-3-amino-4-hydroxytetrahydrofuran.9
4.4.3. (3R,4S)-3-tert-Butoxycarbonylamino-4-hydroxytetrahydro-
furan, (3R,4S)-7
20
White solid, 48%; [
a
]
ꢀ4.2 (c 0.15, MeOH), ee >99%.
D
4.4.4. (3S,4R)-3-tert-Butoxycarbonylamino-4-phenylacetoxytetra-
hydrofuran, (3S,4R)-8
20
Yellow oil, yield 49%; [
a
]
+9.0 (c 0.10, MeOH), ee >99%.
D
4.7.2. (3S,4S)-(ꢀ)-18
25
Yield 50%. [
a]
ꢀ6.0 (c 0.01, MeOH), ee >99%.
D
4.4.5. (3S,4R)-1-Benzyloxycarbonyl-3-tert-butoxycarbonylamino-
4-hydroxypyrrolidine, (3S,4R)-16
20
4.7.3. (3R,4S)-(ꢀ)-18
Colourless oil, yield 47%; [
a]
+7.2 (c 0.07, MeOH), ee >99%.
D
25
Yield 50%. [
a]
ꢀ14.0 (c 0.01, MeOH), ee >99%.
D
4.4.6. (3R,4S)-1-Benzyloxycarbonyl-3-tert-
4.8. Assignment of the absolute configuration of pyrrolidine
derivative
butoxycarbonylamino-4-phenylacetoxypyrrolidine, (3R,4S)-17a
20
Yellow oil, yield 49%; [
a
]
+35.0 (c 0.10, CHCl3), ee >99%.
D
4.8.1. Synthesis of (3S,4R)-1-Benzyloxycarbonyl-3-amino-4-
hydroxypyrrolidine, (3S,4R)-19
4.4.7. (3R,4S)-1-Benzyloxycarbonyl-3-tert-butoxycarbonylami-
no-4-(2-methoxyacetoxy)pyrrolidine, (3R,4S)-17b
20
To a solution of the optically pure product (+)-cis-16 (20 mg,
0.059 mmol) obtained from the CAL-B catalyzed hydrolysis of
( )-cis-17b in ethanol (1.2 mL), HCl-saturated ethanol (10 mL)
was added and the mixture was stirred at room temperature
for 2 h. Then, the solvent was removed under reduced pressure.
The crude residue was purified by flash chromatography on silica
gel (dichloromethane/methanol 8:2). The specific rotation
signs of the pure products were in accordance with that
reported for the (3S,4R)-(+)-1-benzyloxycarbonyl-3-amino-4-
hydroxypyrrolidine.3e
Yellow oil, yield 49%; [
a
]
+10.9 (c 0.10, MeOH), ee >99%.
D
4.5. Determination of the ee by HPLC analysis
Furan derivatives: previous to HPLC analysis, the hydroxyl
group in cis or trans-8, was acylated to the phenyl acetyl ester;
Chiralpak OJ-H, 30 °C, hexane/2-propanol (90:10), UV 210 nm,
0.8 mL minꢀ1, tR 17.25 min (3R,4R)-8, tR 20.20 min (3S,4S)-8, tR
21.47 min (3S,4R)-8, 29.43 min (3R,4S)-8. Pyrrolidine derivatives:
Chiralpak IA, 30 °C, hexane/2-propanol (75:25), UV 210 nm,
0.8 mL minꢀ1, tR 11.12 min (3S,4R)-16, tR 14.79 min (3R,4S)-16; tR
8.32 min (3R,4S)-17a; tR 10.53 min (3S,4R)-17a; tR 8.58 min
(3R,4S)-17b; tR 9.21 min (3S,4R)-17b; tR 28.88 min (3R,4S)-17c;
tR 13.74 (3S,4R)-17c.
4.8.2. (3S,4R)-(+)-19
25
Yield 80%. [
a]
+14.0 (c 0.03, CHCl3), ee >99%.
D
Please cite this article in press as: Villar-Barro, Á;. et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.014

6
Ángela Villar-Barro et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
O’Donnell, C. J.; Schwarz, J. B.; Segelstein, B. E. WO 2010150192 A1, 2010, CAN,
Acknowledgments
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This work was supported by the Ministerio de Ciencia e Innova-
tion of Spain (Project CTQ 2011-24237). A. V.-B. thanks Principado
de Asturias for a predoctoral fellowship.
Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.05.014.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
References and notes
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Please cite this article in press as: Villar-Barro, Á;. et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.014