Clarifying the structure of granadaene: Total synthesis of related analogue [2]-granadaene and confirmation of its absolute stereochemistry

Article abstract of DOI:10.1016/j.bmc.2012.09.017

Streptococcus agalactiae is an important agent in the infection of neonates in the first world. One of the most extended methods for its identification is based on the detection of a characteristic red pigment in the patient samples, named [12]-granadaene (1). In this article, we present a modular and flexible approach to simple analogues of this ornithine rhamno-polyene 1 and the elucidation of the most important features of its structure: the absolute configuration at C-27, the stereochemistry of the anomeric center and the link of the amino acid ornithine to the rest of the structure.

Full text of DOI:10.1016/j.bmc.2012.09.017

Bioorganic & Medicinal Chemistry 20 (2012) 6655–6661
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Bioorganic & Medicinal Chemistry
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Clarifying the structure of granadaene: Total synthesis of related analogue
[2]-granadaene and confirmation of its absolute stereochemistry
Miguel Paradas a, Rocío Jurado a, Ali Haidour a, Javier Rodríguez Granger b, Antonio Sampedro Martínez b,
Manuel de la Rosa Fraile b, Rafael Robles a, José Justicia a, Juan M. Cuerva a,
⇑
a Department of Organic Chemistry, Faculty of Sciences, University of Granada, C. U. Fuentenueva s/n, 18071 Granada, Spain
b Microbiology Service, University Hospital Virgen de las Nieves, La Caleta s/n, 18014 Granada, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 July 2012
Revised 6 September 2012
Accepted 11 September 2012
Available online 19 September 2012
Streptococcus agalactiae is an important agent in the infection of neonates in the first world. One of the
most extended methods for its identification is based on the detection of a characteristic red pigment
in the patient samples, named [12]-granadaene (1). In this article, we present a modular and flexible
approach to simple analogues of this ornithine rhamno-polyene 1 and the elucidation of the most impor-
tant features of its structure: the absolute configuration at C-27, the stereochemistry of the anomeric cen-
ter and the link of the amino acid ornithine to the rest of the structure.
Keywords:
Natural product
Granadaene
Ó 2012 Elsevier Ltd. All rights reserved.
Modular synthesis
Absolute stereochemistry
Structure
1. Introduction
in DMSO-d6/TFA-d mixtures (see Scheme 1) of scarce amounts of
compound 1, owing to the insolubility of the red pigment in any
Hemolytic Streptococcus agalactiae (group
B
streptococcus
other solvent mixture.7 Taking into account the relevance of this
red pigment in the worldwide detection of GBS in pregnant women
and the apparent biogenetic relationship with virulence factor
hemolysine, the clarification of some uncertainties in its structure
is mandatory. Thus, the absolute configuration at C-27, the config-
uration in the anomeric center of the sugar moiety, and the link of
the amino acid to the rest of the structure, remain unclear.8
Although the total synthesis of granadaene (1) would be essential
to determine all these structural unknowns, its biosynthetic path-
way and the structure of the hemolytic compound in GBS,5 its
structural complexity could hinder this goal. Thus, the preparation
of simple derivatives of granadaene (1), including different parts of
its structure, would be a good solution to clarify the structure of
the pigment. In this article, we have developed an easy, modular
approach to the synthesis of granadaene-type compounds (2–4)
(see Scheme 2), which has allowed the determination of the un-
known structural aspects for granadaene (1).
[GBS]) is the most important bacterium causing life-threatening
infections in neonates in the first world,1 constituting an important
cause of death in this population. Nowadays, its detection is man-
datory for all pregnant women between 35 and 37 weeks of gesta-
tion.2 In this context, one of the most extended method for GBS
identification is based on the detection of a characteristic red pig-
ment in the Granada medium.3 Despite its interest, little is known
about the nature of this key pigment and also unknown about the
nature of hemolysin GBS. The GBS hemolysin has yet to be isolated,
largely because its activity is unstable. Interestingly, GBS presents
the same genes for the synthesis of the red pigment and for the
GBS virulence factor hemolysine. The deduced proteins display
similarities to ABC (ATP binding cassette) transporters and
prokaryotic fatty acid biosynthesis enzymes.4,5 Until recently, a
carotene-type structure was assumed for GBS pigment,4 but
intringuingly, genes coding for carotene biosynthesis are not pres-
ent in GBS. In 2006, some of us isolated the main component of
that highly insoluble red pigment, which was named [12]-granad-
aene (1)6 and constituted the first example of a glycopolyene
isolated from gram-positive bacteria.7 A structure of ornithine
rhamno-polyene was tentatively proposed based on NMR studies
2. Results and discussion
Before to accomplish the total synthesis of granadaene (1), all
the structural unknowns of its structure have to be previously clar-
ified. To reach this objective, we decided to prepare simple ana-
logues of 1, as compounds 2, 3 and [2]-granadaene 4, a simple
derivative containing only two double bonds of the polyenic chain
⇑
Corresponding author.
E-mail address: jmcuerva@ugr.es (J.M. Cuerva).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.017

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M. Paradas et al. / Bioorg. Med. Chem. 20 (2012) 6655–6661
-bromorhamnose 1012 in the presence
anomer of 2.13 Subsequent acetoxy
OH
treatment of alcohol 9 with
of Hg(CN)2 gave exclusively the
L
1'
HO
OH
Me
a
HO2C
O
1
25
3
group saponification generated a mixture of two diastereoisomers.
The NMR analysis of that mixture showed that the 1H and 13C NMR
signals at C-27, C-28 and C-100 do not overlap and therefore and
unambiguous structural assignment can be carried out using the cor-
responding enantiopure starting materials 5. When the synthetic se-
quence was repeated using (R)-5 (see Scheme 3), similar yields were
obtained in all the reactions. Finally, the comparison of 1H and 13C
1'' 5''
O
H2N
N
H
27
O
2'
5'
26
5
[12]-Granadaene (1)
Scheme 1. Proposed structure of natural polyene pigment [12]-granadaene (1).
NMR data of (R,a)-2 with those previously described for the corre-
4''
sponding subunit in [12]-granadaene (1)7 (Tables 1 and 2) confirmed
the same absolute stereochemistry for compound 1.14
OH
O
2''
HO
HO
1''
Thus, 1H and 13C NMR signals corresponding to the anomeric
center C-100 are practically identical in both compounds. Addition-
ally, the NMR signals corresponding to the methyl group at C-27 in
2 are also very close to those previously reported for the natural
product 1. Therefore, the synthesis of compound 2 allowed us to
establish unambiguously two key structural characteristics of 1:
O
OH
O
O
O
1'
2'
25
25
NH2
N
H
HO
1
O
27
1
27
4
26
2
5'
2
26
4
2
3
4''
OH
O
HO
HO
1''
2''
the
a configuration for the anomeric center in the rhamnose moi-
O
OH
O
O
1'
ety and the R configuration at C-27.
25
3
NH2
Once the structural features of 1 involving the L-rhamnose moi-
27
1
N
H
2'
5'
ety were revealed, we turned our attention to the hemisphere con-
taining the amino acid subunit. Within this context, the link of the
amino acid fragment to the rest of the compound had to be deter-
mined. To this end, we accomplished the synthesis of the granad-
4
Scheme 2. Simple derivatives of [12]-granadaene (1).
(Scheme 2).9 The similarity between the chemical environments of
such fragments with those proposed in the original structure of 1
should ensure a simple comparison between the corresponding
spectroscopic data.
aene derivative 3, using the natural amino acid L-ornitine, which is
the only one available in Nature. In this case, we decided to try the
link between the amino acid and the polyenic chain using the ami-
no group located at C-20 (see Scheme 2). The synthesis of 3 (Scheme
4) started from protected ester 8, which had been previously used
in the synthesis of 2. After ester hydrolysis under strong basic con-
ditions, we performed the direct amidation of 11 with commercial
available hydrochloride 12 using a methodology recently devel-
oped by our group,15 yielding the corresponding amide 13 (50%
yield). After three consecutive cleavages of different protecting
groups, the derivative L-3 (67% yield) was isolated. Comparison
of 1H and 13C NMR observed for L-3 with those previously de-
scribed for [12]-granadaene (1)7 (see Tables 3 and 4) showed the
close relationship between these two compounds. The signals cor-
responding to the amino acid subunit and the polyenic chain
match almost perfectly (Tables 3 and 4). Therefore, we could con-
These compounds 2–4 contain all the unknown structural as-
pects in granadaene (1) and their synthesis can be accomplished
in a few steps. Thus, to determinate the absolute configuration at
C-27 and the configuration in the anomeric center of the rhamnose
including moiety, we decided to prepare compound 2, which has a
similar chemical environment that present in 1 for such hemi-
sphere of the molecule. The synthesis of 2 is depicted in Scheme 3.
We started from commercial available racemic 5, which was
subsequently transformed in aldehyde 610 in three steps.
Horner-Wadsworth-Emmons reaction11 between aldehyde 6 and
the commercial phosphonate 7 yielded diene 8 in good yield and
complete E,E stereoselectivity. Deprotection of THP group under
acidic conditions gave alcohol 9, which was subsequently used in
firm that [12]-granadaene (1) has a L-ornitinite amino acid in its
the glycosylation process. For it, we used the
which was synthesized in only two steps (acetylation and bromina-
tion with HBr under acidic conditions) from -rhamnose. Finally,
L-bromorhamnose 10,
structure, and the link of the amino acid to the rest of the molecule
occurs using the amino group located at C-20, ruling out the possi-
ble link using the amine at C-50.
L
O
P
COOEt
THPO
EtO
EtO
a) DHP, pTsOH
O
O
b) LiAlH4
7
COOEt
HO
OEt
THPO
H
R
c) PCC
DMPU,
n-BuLi
(75%)
E
E
8
5
6
(44%, three steps)
OH
HO
OH
a) Hg(CN)2
O
O
pTsOH
25
10
5" 1"
O
COOEt
HO
O
27
(97%)
b) K2CO3
MeOH
9
(R,α)- 2
(92%, two steps)
AcO
OH OH
AcO
OAc
Br
a) Ac2O, Py
O
b) HBr
OH OH
L-rhamnose
O
AcOH
10
(45%, two steps)
Scheme 3. Synthesis of sugar-containing derivative (R,a)-2 from (R)-5.

M. Paradas et al. / Bioorg. Med. Chem. 20 (2012) 6655–6661
6657
Table 1
Table 3
Key 1H NMR signals for 1 and 2
Key 1H NMR signals for 1 and L-3
a
a
H
1a
2a
H
1
L-3
1H NMR ppm (multiplicity, J)
1H NMR ppm (multiplicity, J)
1H NMR ppm (multiplicity, J)
1H NMR ppm (multiplicity, J)
100
4.63 (d, 1.5)
3.52 (dd, 3, 1.5)
3.39 (dd, 9, 3)
3.16 (t, 9)
3.43 (dd, 9, 6)
1.09 (d, 6)
6.1 (d, 15.1)
7.1 (dd, 15.1, 11)
6.5–6.2 (m)
5.72 (dt, 14.5, 7.2)
2.25 (m)
4.68 (bs)
3.55 (bs)
3.41 (m)
3.19 (t, 9)
2
3
6.1 (d, 15.1)
7.1 (dd, 15.1, 11)
6.5–6.2 (m)
5.72 (dt, 14.5, 7.2)
2.25 (m)
3.69 (sext, 6.1)
1.03 (d, 6.1)
4.32 (dd, 8.9, 5)
1.80 (m), 1.64 (m)
1.59 (m)
5.91 (d, 15.3)
7.16 (dd, 14.4, 10.8)
6.23 (m)b
6.05 (dt, 14.5, 7.2)
2.36 (m)
200
300
4, 6–24
25
26
27
28
20
400
500
3.45 (m)
600
1.12 (d, 6.1)
5.91 (d, 15.4)
7.22 (dd, 15.3, 10.6)
6.26 (m)b
6.34 (dd, 14.8, 11.2)
2.35 (m)
3.98 (m)
2
3
1.20 (d, 6.1)
4.33 (dd, 8.3, 6.7)
1.84 (m), 1.65 (m)
1.59 (m)
4, 6–24
25
26
27
28
30
40
50
2.77 (t, 6.7)
2.80 (bs)
3.69 (sext, 6.1)
1.03 (d, 6.1)
3.78 (dq, 12.4, 6.2)
1.09 (d, 6.1)
a
Spectra were recorded in DMSO-d6, 0.1% TFA-d.
Refer to H-4.
b
a
Spectra were recorded in DMSO-d6, 0.1% TFA-d.
Refer to H-4.
b
Table 2
13C NMR signals for 1 and 2
Table 4
C
1a
2a
13C NMR signals for 1 and L-3
13C NMR ppm
13C NMR ppm
C
1a
L-3a
100
97.4
70.8
70.5
71.8
68.8
97.6
70.6
70.5
71.9
68.7
13C NMR ppm
13C NMR ppm
200
300
2
3
124.0
138.8
130–136
131.7
39.9
124.3
138.6
133.2b
130.4
40.0
71.0
18.7
51.4
28.0
400
500
4, 6–24
25
26
27
28
20
600
17.7
17.8
2
3
124.0
138.8
130–136
131.7
39.9
119.4
144.6
141.0b
130.0
39.7
71.0
18.7
51.1
27.9
23.5
38.1
4, 6–24
25
26
27
28
30
71.0
18.7
71.0
18.8
40
23.7
38.1
50
a
a
Spectra were recorded in DMSO-d6, 0.1% TFA-d.
Refer to C-4.
Spectra were recorded in DMSO-d6, 0.1% TFA-d.
Refer to C-4.
b
b
Although the synthesis depicted before were useful to determi-
introduction of different sugars and/or amino acids in the final
molecule, which could allow to carry out further biological studies
in more details.18 To check the feasibility of our approach, we fo-
cussed in the synthesis of the shortest member of the family,
[2]-granadaene (4),6 which posses all the structural motifs present
in natural [12]-granadaene (1) (Schemes 5 and 6).
nate each of the unknown structural features of granadaene (1), a
new synthetic approach was necessary to obtain a compound con-
taining all the important structural aspects of 1. In this sense, the
synthesis of granadaene-type compounds could be accomplished
taking into account a modular and convergent approach, ensuring
the stereochemistry of the polyprenic chain (Scheme 5). Within
this context, Suzuki–Miyaura coupling has been extensively used
in the synthesis of stereocontrolled polyenic chains.16,17 The chem-
ical compatibility of this methodology also allows the presence of
In this case, a direct Suzuki–Miyaura coupling between frag-
ments 15 and 16 would yield the desired compound 4 (Scheme 6).
Amino acid derivative 15 was prepared from commercially avail-
able protected L
-ornithine 12 and 3-bromoacrylic acid 14,19 as
L
-rhamnose (16) and
L
-ornithine (15) fragments in the correspond-
previously described in the synthesis of L-3.15 On the other hand,
fragment 16 was readily prepared from epoxide 17 in a few steps,
ing subunits. Moreover, this approach would also allow the
MeO
H3N
O
H
N
Boc
Cl
12
KOH
COOEt
COOH
THPO
THPO
THPO
MeOH
(58%)
I2, Ph3P
Imidazole
8
11
(50%)
O
OMe
O
OH
O
O
1'
H
25
a) pTsOH
NH2
N
N
H
Boc
HO
1
N
H
2'
27
4
5'
26
2
b) NaOH 1M
c) CF3CO2H
13
L-3
(67%, three steps)
Scheme 4. Synthesis of amino acid-containing derivative L-3.

6658
M. Paradas et al. / Bioorg. Med. Chem. 20 (2012) 6655–6661
Table 5
O
Key 1H NMR signals for 1 and 4
MeO
O
MeO
O
HO
Br
O
H
1a
4a
H
H
14
N
N
Boc
N
H
Br
1H NMR ppm (multiplicity, J)
1H NMR ppm (multiplicity, J)
+
Boc
NH3
Cl-
I2, Ph3P
Imidazole
(53%)
100
4.63 (d, 1.5)
3.52 (dd, 3, 1.5)
3.39 (dd, 9, 3)
3.16 (t, 9)
3.43 (dd, 9, 6)
1.09 (d, 6)
6.1 (d, 15.1)
7.1 (dd, 15.1, 11)
6.5–6.2 (m)
5.72 (dt, 14.5, 7.2)
2.25 (m)
3.69 (sext, 6.1)
1.03 (d, 6.1)
4.32 (dd, 8.9, 5)
1.80 (m), 1.64 (m)
1.59 (m)
15
12
4.66 (bs)
200
3.53 (bs)
3.39 (dd, 9.4, 3.1)
3.18 (t, 9)
300
400
OAc
500
3.42 (dd, 9.2, 6.3)
1.10 (d, 6.1)
6.04 (d, 15.3)
7.01 (dd, 15, 11)
6.25 (m)b
AcO
Br
OAc
Me
600
OAc
O
2
3
Li
H
O
10
AcO
O
OAc
Me
HO
O
EDTA Complex
4, 6–24
25
26
27
28
20
R
(42%)
Hg(CN)2
(88%)
6.09 (m)
2.32 (m)
17
18
19
3.74 (dt, 12.2, 6.1)
1.06 (d, 6)
OAc
O
4.30 (dt, 8.3, 6.7)
1.80 (m,), 1.63 (m)
1.57 (m,)
30
AcO
O
OAc
Me
O
catechol-BH
40
B
50
2.77 (t, 6.7)
2.79 (bs)
O
16
a
Spectra were recorded in DMSO-d6, 0.1% TFA-d.
Refer to H-4.
b
Scheme 5. Preparation of fragments 15 and 16.
with complete stereochemical control (Scheme 5). As we estab-
lished before, it is known that the glycosylation reaction of rham-
nose derivative 10 mediated by Hg(CN)2 yields the corresponding
Table 6
13C NMR signals for 1 and 4
a
anomer, and the absolute configuration at C-27 was determined
a
a
C
1
4
as R. Thus, the preparation of 16 had to include all these structural
requirements. In this case, the stereochemical control on C-27 was
obtained by nucleophylic opening of commercial available enantio-
pure epoxide (R)-17 by lithium acetylide-EDTA complex, to yield
alcohol 1820 in modest yield (42%). Then, alcohol 18 was efficiently
glycosylated with 10, under the conditions indicated in Scheme 3.
Finally, hydroboration of alkyne moiety with catecholborane in
THF gave intermediate 16, which was subsequently used in the next
step. Suzuki–Miyaura coupling of both fragments 15 and 16 gener-
ated diene (E,E)-20, which possesses the main framework of 1. Fi-
nally, deprotection in two steps (saponification of acetate groups,
and removing of Boc group and ester hydrolysis under acidic condi-
tions) yielded analogue 4 in 65% yield (two steps). Again the chem-
ical shifts for compound 4 are in agreement with those reported for
compound 1 confirming again its fundamental structural features
(see Tables 5 and 6).
13C NMR ppm
13C NMR ppm
100
97.4
70.8
70.5
71.8
68.8
97.6
70.7
70.6
200
300
400
71.9
68.8
17.9
123.1
138.4
130.3b
139.7
40.0
500
600
17.7
2
3
124.0
138.8
130–136
131.7
39.9
4, 6–24
25
26
27
28
20
71.0
18.7
51.1
27.9
23.5
38.1
71.0
18.8
51.3
28.2
23.8
38.5
30
40
50
a
Spectra were recorded in DMSO-d6, 0.1% TFA-d.
b
Refer to C-4.
3. Conclusions
In conclusion, a modular and flexible approach to compounds of
granadaene family has been described. The absolute configuration
at C-27 and the stereochemistry of the anomeric center were also
determined, as well as the position of the link of the amino acid
fragment to the rest of the compound. We are now working in
the total synthesis of [12]-granadaene (1) and some other deriva-
tives including other different sugars and amino acids to check
their biological activities.
4. Experimental Section
4.1. General details
Deoxygenated solvents and reagents were used for all reactions.
THF was freshly distilled from Na. CH2Cl2 was freshly distilled from
P2O5. Products were purified by flash chromatography on Merck
OAc
Pd(Ph3P)4
AcO
O
OAc
Me
MeO
O
O
15 16
+
BocHN
H2N
K3PO4
70 ºC
O
N
H
20
(50%)
OH
HO
OH
Me
HO
O
a) NaOH/MeOH
O
2'' 4''
1'' O
1'
25
3
b) CF3CO2H
(65%)
1
O
27
N
H
2'
5'
4
Scheme 6. Synthesis of [2]-granadaene (4).

M. Paradas et al. / Bioorg. Med. Chem. 20 (2012) 6655–6661
6659
silica gel 50. Yields refer to analytically pure samples. NMR spectra
were recorded in NMR 400 MHz and 500 MHz spectrometers. The
following known compounds were isolated as pure samples and
showed NMR spectra matching those of the reported compounds:
6,10 10,12 14,19 and 18.20
1H), 3.68 (s, 3H), 3.65–3.49 (m, 1H), 3.47–3.43 (m, 1H),
3.43–3.38 (m, 1H), 3.19 (t, J = 9.2 Hz, 1H), 2.38–2.32 (m, 2H),
1.12 (d, J = 6.1 Hz, 3H), 1.09 (d, J = 6.1 Hz, 3H). 13C NMR
(125 MHz, DMSO-d6) d 166.2 (C), 144.6 (CH), 141.0 (CH), 130.0
(CH), 119.4 (CH), 97.6 (CH), 71.9 (CH), 71.0 (CH), 70.6 (CH), 70.5
(CH), 68.7 (CH), 59.7 (CH2), 51.2 (CH3), 18.8 (CH3), 17.8 (CH3);
HRMS-ESI(+) for C15H24O7Na Calcd 339.1414, Found 339.1422
[M+Na]+.
4.2. Synthesis of (R,a)-2
4.2.1. Preparation of compound 8
To a solution of phosphonate 7 (916 mg, 3.66 mmol) in dry THF
(10 mL) at 0 °C, DMPU (913 mg, 7.12 mmol) and n-BuLi
(4.07 mmol, 1.63 mL, 2.5 M in hexane) were added and the new
mixture was stirred for 30 min. Then, the mixture was cooled to
ꢀ78 °C and a solution of aldehyde 610 (350 mg, 2.03 mmol) in
dry THF (10 mL) was slowly added. The mixture was stirred for
1 h at ꢀ78 °C, warmed until 0 °C and stirred for additional 3 h.
Then, EtOAc was added and the organic layer was washed with sat-
urated solution of NH4Cl. The organic layer was dried (anhyd
Na2SO4) and the solvent was removed. The residue was submitted
to flash chromatography on silicagel (hexane/EtOAc 8/2) to yield
ester 8 (393 mg, 72%). Colorless oil; 1H NMR (400 MHz, CDCl3) d
7.32–7.19 (m, 1H), 6.28–6.04 (m, 2H), 5.80 (d, J = 15.4 Hz, 1H),
4.72–4.69 (m, 1H), 4.18 (q, J = 7.2 Hz, 2H), 3.95–3.81 (m, 2H),
3.50–3.42 (m, 2H), 2.48–2.26 (m, 2H), 1.84–1.76 (m, 1H), 1.72–
1.64 (m, 2H), 1.46–1.58 (m, 2H), 1.28 (t, J = 7.2 Hz, 3H), 1.12 (d,
J = 6.2, 3H); 13C NMR (100 MHz, CDCl3) d 167.3 (C), 144.7 (CH),
140.1 (CH), 130.7 (CH), 119.9 (CH), 98.3 (CH), 72.6 (CH), 62.8
(CH2), 60.3 (CH2), 40.0 (CH2), 31.1 (CH2), 25.6 (CH2), 21.6 (CH3),
19.9 (CH2), 14.4 (CH3); HRMS-ESI(+) for C15H24O4Na Calcd
291.1566. Found 291.1573 [M+Na]+.
4.3. Synthesis of (L)-3
4.3.1. Saponification of ester 8
Ester 8 (155 mg, 0.58 mmol) was treated with 5% solution of
KOH in MeOH (15 mL) for 30 h. Then, 2 N HCl was added until
pH 6, the mixture was diluted with EtOAc and the organic layer
was washed with water. The organic layer was dried (anhyd
Na2SO4) and the solvent was removed to yield acid 11 (126 mg,
91%) without further purification. Colorless oil; 1H NMR
(400 MHz, CDCl3) d 7.42–7.28 (m, 1H), 6.31–6.09 (m, 2H), 5.86–
5.77 (m, 1H), 4.78–4.62 (m, 1H), 3.97–3.80 (m, 2H), 3.54–3.43
(m, 1H), 2.52–2.28 (m, 2H); 1.96–1.44 (m, 6H), 1.13 (d, J = 6.2 Hz,
3H); 13C NMR (100 MHz, CDCl3) d 172.2 (C), 146.7 (CH), 141.1
(CH), 130.3 (CH), 118.9 (CH), 96.1 (CH), 70.4 (CH), 62.5 (CH2),
41.0 (CH2), 31.1 (CH2), 25.5 (CH2), 19.8 (CH2), 18.6 (CH3).
4.3.2. Preparation of amide 13
To a solution of I2 (94 mg, 0.37 mmol) in dry CH2Cl2 (10 mL),
Ph3P (97 mg, 0.37 mmol) was added, giving the solution
a
brown–yellow color. Then, imidazole (89 mg, 1.28 mmol) was
added, changing the color to light yellow. Subsequently, acid 11
(69 mg, 0.29 mmol) was added and the solution was stirred for
5 min at room temperature, and then commercial amine 12
(86 mg, 0.35 mmol) was added. The mixture was stirred until
completely consumption of the starting material (checked by
TLC, around 24 h). Then, CH2Cl2 was added, and the solution was
washed with 2 N HCl and water before being dried with
anhyd Na2SO4, and the solvent removed. The residue was
submitted to flash chromatography on silicagel (hexane/EtOAc
4.2.2. Synthesis of alcohol 9
To a solution of ester 8 (60 mg, 0.22 mmol) in EtOH (10 mL) at
rt, pTsOH (3 mg, 0.011 mmol) were added and the mixture was
stirred for 16 h. Then, EtOAc was added and the organic layer
was washed with brine. The organic layer was dried (anhyd
Na2SO4) and the solvent was removed. The residue was submitted
to flash chromatography on silicagel (hexane/EtOAc 7/3) to yield
alcohol 9 (40 mg, 97%). Colorless oil; 1H NMR (400 MHz, CDCl3) d
7.31–7.21 (m, 1H), 6.26 (dd, J = 15.2, 10.8 Hz, 1H), 6.17–6.08 (m,
2H), 5.83 (d, J = 15.4 Hz, 1H), 4.20 (q, J = 7.2 Hz, 2H), 3.92 (dd,
J = 12.0, 6.3 Hz, 1H), 2.34 (dd, J = 12.8, 6.4 Hz, 2H), 1.31–1.27 (m,
3H), 1.23 (d, J = 6.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) d 167.2
(C), 144.5 (CH), 139.8 (CH), 131.2 (CH), 120.4 (CH), 67.2 (CH),
60.4 (CH2), 42.8 (CH2), 23.2 (CH3), 14.4 (CH3); HRMS-ESI(+) for
1/1) to yield amide 13 (68 mg, 50%). Colorless oil; ½a D25
ꢁ
+ 17.3
(c = 0.75, CHCl3); 1H NMR (400 MHz, CDCl3) d 7.25–7.16 (m, 1H),
6.52–5.96 (m, 2H), 5.94–5.81 (m, 1H), 4.75–4.57 (m, 1H), 4.22–
4.15 (m, 1H), 3.81–3.75 (m, 2H), 3.74 (s, 3H), 3.55–3.44 (m, 1H),
3.20–3.07 (m, 1H), 2.75–2.58 (m, 4H), 1.97–1.75 (m, 6H), 1.77–
1.47 (m, 4H), 1.43 (s, 9H), 1.22 (d, J = 6.9 Hz, 3H); 13C NMR
(100 MHz, CDCl3) d 173.0 (C), 166.1 (C), 156.2 (C), 142.1 (CH),
133.3 (CH), 131.4 (CH), 127.8 (CH), 113.1 (CH),79.2, 70.4 (CH),
62.5 (CH2), 52.5 (CH3), 45.8 (CH), 39.9 (CH2), 33.1 (CH2), 29.8
(CH2), 28.4 (CH3), 26.4 (CH2), 22.9 (CH2), 19.9 (CH2), 18.6 (CH3);
HRMS-ESI(+) for C24H40O7N2Na Calcd 491.2727. Found 491.2737
[M+Na]+.
C
10H17O3 Calcd 185.1172. Found 185.1188 [M+H]+.
4.2.3. Preparation of (R, )-2
a
To a solution of alcohol 9 (40 mg, 0.22 mmol) and Hg(CN)2
(50 mg, 0.2 mmol) in dry CH3CN (10 mL), bromorhamnose 10
(115 mg, 0.33 mmol) was added in three portions during 3 h, and
the mixture was stirred for additional 4 h. Then, the solvent was
removed. The residue was solved in CH2Cl2 and washed with 1 M
solution of KBr, saturated solution of NaHCO3 and water. The or-
ganic layer was dried (anhyd Na2SO4) and the solvent was re-
moved. The residue was submitted to flash chromatography on
silicagel (hexane/EtOAc 7/3) to yield the corresponding glycosyla-
tion product (90 mg, 91%). This compound was solved in MeOH
(10 mL) and K2CO3 (136 mg, 1 mmol) was added, and the mixture
was stirred for 10 min. Then, brine was added and the mixture was
extracted with EtOAc, dried (anhyd Na2SO4) and the solvent re-
4.3.3. Synthesis of model compound (L)-3
To a solution of amide 13 (65 mg, 0.14 mmol) in EtOH (10 mL) at
rt, pTsOH (1.3 mg, 0.007 mmol) was added and the mixture was
stirred for 24 h. Then, EtOAc was added and the organic layer was
washed with brine. The organic layer was dried (anhyd Na2SO4)
and the solvent was removed. The residue was submitted to flash
chromatography on silicagel (EtOAc) to yield the corresponding
alcohol (54 mg, 97%). Colorless oil; 1H NMR (400 MHz, CDCl3) d
7.18 (dd, J = 14.8, 10.8 Hz, 1H), 6.22 (dd, J = 15.2, 10.8 Hz, 1H),
6.08 (dd, J = 14.4, 7.2 Hz, 1H), 5.86 (d, J = 15.2 Hz, 1H), 4.60 (m,
2H), 3.80 (q, J = 6.4 Hz, 2H), 3.68 (s, 3H), 3.10 (m, 1H), 2.45–2.20
(m, 2H), 1.60–1.84 (m, 8H), 1.38 (s, 9H), 1.21 (d, J = 6.2 Hz, 3H). This
alcohol was dissolved in MeOH (10 mL) and NaOH 1 M (0.28 mL,
0.28 mmol) were added and the mixture was stirred for 16 h.
Then, amberlyst was added until to reach pH 7, the mixture was fil-
move. Compound (R,a)-2 was obtained (65 mg, 98%) without fur-
ther purification. Colorless oil; ½a D25
ꢁ
+ 18.5 (c = 0.85, CHCl3); 1H
NMR (500 MHz, DMSO-d6) d 7.22 (dd, J = 15.3, 10.6 Hz, 1H), 6.34
(dd, J = 14.8, 11.2 Hz, 1H), 6.26 (ddd, J = 12.1, 7.4, 4.0 Hz, 1H),
5.91 (d, J = 15.4 Hz, 1H), 4.68 (bs, 1H), 3.78 (dq, J = 12.4, 6.2 Hz,

6660
M. Paradas et al. / Bioorg. Med. Chem. 20 (2012) 6655–6661
tered and the solvent removed. The residue was submitted to
flash chromatography on silicagel (EtOAc/MeOH 8/2) to yield the
corresponding acid (38 mg, 74%). This compound was immediately
used in the next step. Colorless oil; 1H NMR (400 MHz, CD3OD) d
7.11 (dd, J = 15.0, 10.8 Hz, 1H), 6.29–6.20 (m, 1H), 6.10 (dd,
J = 14.8, 7.3 Hz, 1H), 6.02 (d, J = 15.2 Hz, 1H), 3.87–3.78 (m, 1H),
3.72 (s, 1H), 3.03 (t, J = 6.3 Hz, 2H), 2.36 (dd, J = 12.9, 6.5 Hz, 2H),
1.86 (dd, J = 12.3, 4.9 Hz, 2H), 1.72–1.61 (m, 2H), 1.40 (s, 9H), 1.13
(d, J = 6.0 Hz, 3H). This acid (38 mg, 0.1 mmol) was subsequently
treated with trifluoroacetic acid (0.2 mL, 2.6 mmol) for 15 min.
Then, the excess of acid was removed, to yield amine (L)-3
(25 mg, 93%). Colorless oil; 1H NMR (400 MHz, DMSO-d6 + 0.1%
CF3CO2D) d 7.16 (dd, J = 14.4, 10.8 Hz, 1H), 6.23 (dd, J = 15.0,
10.6 Hz, 1H), 6.05 (dd, J = 14.5, 7.2 Hz, 1H), 5.91 (d, J = 15.3 Hz,
1H), 4.33 (dt, J = 8.3, 6.7 Hz, 1H), 3.98 (td, J = 13.2, 5.9 Hz, 1H),
2.80 (bs, 2H), 2.20–2.45 (m, 2H), 1.89–1.83 (m, 1H), 1.68–1.63 (m,
1H), 1.62–1.56 (m, 2H), 1.20 (d, J = 6.1 Hz, 3H). 13C NMR
(150 MHz, DMSO-d6 + 0.1% CF3CO2D) d 175.4 (C), 164.9 (C), 138.6
(CH), 133.2 (CH), 130.4 (CH), 124.3 (CH), 71.1 (CH), 51.4 (CH),
40.0 (CH2), 38.1 (CH2), 28.0 (CH2), 23.7 (CH2), 18.7 (CH3).
4.4.3. Preparation of intermediate 20
To flask containing alkyne 19 (307 mg, 0.88 mmol),
a
catecholborane (105 mg, 0.88 mmol) was added under Ar
atmosphere and the mixture was stirred at 70 °C for 3 h. Then,
the mixture was solved in dry and strictly deoxygenated THF
(5 mL) and a mixture of 15 ((120 mg, 0.33 mmol), Pd[PPh3]4
(19 mg, 0.017 mmol) and K3PO4 (162 mg, 0.99 mmol) in THF
(5 mL) was added. This new mixture was stirred at 70 °C for
48 h. Then, brine was added and the mixture was extracted with
EtOAc. The organic layer was dried (anhyd Na2SO4) and the
solvent was removed. The residue was submitted to flash chroma-
tography on silicagel (hexane/EtOAc 7/3) to yield 20 (110 mg, 55%).
Colorless oil; ½a D25
ꢁ
ꢀ 30.4 (c = 0.8, CHCl3); 1H NMR (500 MHz,
CDCl3)
d 7.19 (dd, J = 14.9, 10.9 Hz, 1H), 6.22 (dd, J = 14.9,
11.0 Hz, 1H), 6.09–5.99 (m, 1H), 5.87 (d, J = 15.0 Hz, 1H), 5.24
(dd, J = 10.1, 3.3 Hz, 1H), 5.16 (dd, J = 3.4, 1.8 Hz, 1H), 5.03 (t,
J = 9.9 Hz, 1H), 4.82 (s, 1H), 4.65 (dd, J = 12.4, 5.2 Hz, 1H), 3.89–
3.78 (m, 1H), 3.73 (s, 3H), 3.12 (d, J = 6.1 Hz, 2H), 2.47–2.29 (m,
2H), 2.13 (s, 3H), 2.02 (s, 3H), 1.97 (s, 3H), 1.87 (dd, J = 14.2,
6.1 Hz, 2H), 1.75–1.65 (m, 3H), 1.42 (s, 9H), 1.17 (d, J = 6.3 Hz,
3H), 1.14 (d, J = 6.1 Hz, 3H); 13C NMR (125 MHz, CDCl3) d 173.0
(C), 170.3 (C), 170.2 (C), 170.1 (C), 166.0 (C), 156.2 (C), 141.5
(CH), 138.4 (CH), 131.1 (CH), 122.3 (CH), 95.1 (CH), 72.6 (CH),
71.2 (CH), 70.6 (CH), 69.3 (CH), 66.8 (CH), 52.6 (CH), 52.1 (CH),
40.5 (CH2), 40.1 (CH2), 32.3 (C), 29.8 (CH2), 28.5 (CH3), 26.4
(CH2), 21.1 (CH3), 21.0 (CH3), 20.9 (CH3), 19.0 (CH3), 17.5 (CH3);
HRMS-ESI(+) for C31H48O13N2Na Calcd 679.3048. Found 679.3075
[M+Na]+.
4.4. Synthesis of model compound [2]-granadaene (4)
4.4.1. Preparation of amide 15
To a solution of I2 (404 mg, 1.59 mmol) in dry CH2Cl2 (10 mL),
Ph3P (417 mg, 1.59 mmol) was added, giving the solution a
brown–yellow color. Then, imidazole (324 mg, 4.66 mmol) was
added, changing the color to light yellow. Subsequently, acid
1419 (176 mg, 1.17 mmol) was added and the solution was stirred
for 5 min at room temperature, and then commercial amine 12
(300 mg, 1.06 mmol) was added. The mixture was stirred until
completely consumption of the starting material (checked by
TLC, around 24 h). Then, CH2Cl2 was added, and the solution was
washed with 2 N HCl and water before being dried with anhyd
Na2SO4, and the solvent removed. The residue was submitted to
flash chromatography on silicagel (hexane/EtOAc 6/4) to yield
4.4.4. Synthesis of [2]-granadaene (4)
To a solution of amide 20 (110 mg, 0.18 mmol) in MeOH
(10 mL), NaOH 1 M (1.26 mL, 1.26 mmol) was added and the
mixture was stirred for 16 h. Then, amberlyst was added until
pH 7, the mixture was filtered and the solvent was removed.
The residue was submitted to flash chromatography on silicagel
(EtOAc/MeOH 9/1) to yield the corresponding alcohol (51 mg,
amide 15 (214 mg, 53%). Colorless oil; ½a D25
ꢁ
+ 12.6 (c = 0.52, CHCl3);
55%). Colorless oil;
½
a 2D5
ꢁ
ꢀ 3.9 (c = 0.8, CHCl3); 1H NMR
1H NMR (400 MHz, CDCl3) d 7.50 (d, J = 13.5 Hz, 1H), 6.58 (d,
J = 13.4 Hz, 1H), 4.64 (dd, J = 12.3, 7.3 Hz, 1H), 3.75 (s, 3H), 3.21–
3.06 (m, 2H), 1.96–1.84 (m, 2H), 1.79–1.66 (m, 2H), 1.44 (s, 9H);
13C NMR (100 MHz, CDCl3) d 172.7 (C), 163.6 (C), 156.4 (C), 130.6
(CH), 123.3 (CH), 95.5 (CH), 79.4 (C), 52.6 (CH3), 39.9 (CH2), 29.0
(CH2), 28.4 (CH3), 26.4 (CH2); HRMS-ESI(+) for C14H23O5N2Br Calcd
401.0682. Found 401.0699 [M+Na]+.
(400 MHz, CD3OD) d 7.19 (dd, J = 15.0, 10.8 Hz, 1H), 6.39–6.28
(m, 1H), 6.18 (dd, J = 14.8, 7.3 Hz, 1H), 6.11 (d, J = 15.2 Hz, 1H),
4.40 (s, 1H), 3.96–3.86 (m, 1H), 3.80 (s, 3H), 3.70 (dd, J = 9.3,
3.3 Hz, 1H), 3.46–3.40 (m, 1H), 3.11 (t, J = 6.3 Hz, 2H), 2.52–
2.37 (m, 2H), 1.99–1.88 (m, 2H), 1.81–1.69 (m, 2H), 1.63–1.54
(m, 3H), 1.48 (s, 9H), 1.29 (d, J = 6.1 Hz, 3H), 1.21 (d, J = 6.0 Hz,
3H); 13C NMR (125 MHz, CDCl3) d 176.1 (C), 167.8 (C), 158.5
(C), 144.0 (CH), 141.7 (CH), 139.5 (CH), 132.1 (CH), 99.3 (CH),
79.8 (CH), 73.9 (CH), 73.0 (CH), 72.8 (CH), 70.1 (CH), 48.4 (CH),
41.6 (CH2), 41.1 (CH2), 37.9 (CH2), 32.3 (C), 28.8 (CH3), 25.9
(CH2), 19.2 (CH3), 18.0 (CH3); HRMS-ESI(+) for C25H42O10N2Na
Calcd 553.2737. Found 553.2746 [M+Na]+. This alcohol (51 mg,
0.1 mmol) was treated with trifluoroacetic acid (0.3 mL,
3.8 mmol) for 5 min. Then, the excess of acid was removed, to
yield 4 (37 mg, 91%). Colorless oil; 1H NMR (600 MHz, DMSO-d6
+ 0.1% CF3CO2D) d 7.01 (dd, J = 15.0, 11.0 Hz, 1H), 6.25 (dd,
J = 14.9, 11.2 Hz, 1H), 6.09 (m, 1H), 6.04 (d, J = 15.3 Hz, 1H),
4.66 (s, 1H), 4.30 (dt, J = 8.3, 6.7 Hz, 1H), 3.74 (td, J = 12.2,
6.1 Hz, 1H), 3.53 (s, 1H), 3.42 (dd, J = 9.2, 6.3 Hz, 1H), 3.39 (dd,
J = 9.4, 3.1 Hz, 1H), 3.18 (t, J = 9.3 Hz, 1H), 2.79 (s, 2H), 2.32 (m,
2H), 1.80 (dt, J = 11.5, 7.3 Hz, 1H), 1.63 (dd, J = 14.1, 6.1 Hz, 1H),
1.61–1.54 (m, 2H), 1.10 (d, J = 6.1 Hz, 3H), 1.06 (d, J = 6.0 Hz,
3H). 13C NMR (150 MHz, DMSO-d6 + 0.1% CF3CO2D) d 173.3 (C),
165.3 (C), 139.7 (CH), 138.4 (CH), 130.3 (CH), 123.1 (CH), 97.6
(CH), 71.9 (CH), 71.0 (CH), 70.7 (CH), 70.6 (CH), 68.8 (CH), 51.3
(CH), 40.1 (CH2), 38.5 (CH2), 28.2 (CH2), 23.8 (CH2), 18.8 (CH3),
17.9 (CH3).
4.4.2. Synthesis of alkyne 19
To a solution of alcohol 1820 (330 mg, 3.57 mmol) and Hg(CN)2
(812 mg, 2.21 mmol) in dry CH3CN (6 mL), bromorhamnose 10
(1.89 g, 5.36 mmol) was added in three portions during 3 h, and
the mixture was stirred for additional 4 h. Then, the solvent was
removed. The residue was solved in CH2Cl2 and washed with 1 M
solution of KBr, saturated solution of NaHCO3 and water. The or-
ganic layer was dried (anhyd Na2SO4) and the solvent was re-
moved. The residue was submitted to flash chromatography on
silicagel (hexane/EtOAc 7/3) to yield alkyne 19 (1.23 g, 97%). Color-
less oil; ½a 2D5
ꢁ
ꢀ 76.5 (c = 0.33, CHCl3); 1H NMR (400 MHz, CDCl3) d
5.32 (d, J = 3.4 Hz, 1H), 5.30 (d, J = 3.3 Hz, 1H), 5.19 (d, J = 1.6 Hz,
1H), 5.07 (t, J = 10.0 Hz, 1H), 4.10 (dt, J = 11.8, 8.2 Hz, 1H), 3.91
(dq, J = 12.3, 6.3 Hz, 1H), 2.44 (qdd, J = 16.8, 6.1, 2.6 Hz, 2H), 2.18
(s, 1H), 2.15 (s, 3H), 2.05 (s, 3H), 1.98 (s, 3H), 1.26 (d, J = 6.1 Hz,
3H), 1.21 (d, J = 6.3 Hz, 3H); 13C NMR (100 MHz, CDCl3) d 170.3
(C), 170.2 (C), 170.1 (C), 95.7 (CH), 81.0 (C), 72.4 (CH), 71.3 (CH),
70.5 (CH), 70.2 (CH), 69.3 (CH), 66.7 (CH), 26.9 (CH2), 21.1 (CH3),
21.0 (CH3), 20.9 (CH3), 19.0 (CH3), 17.4 (CH3); HRMS-ESI(+) for
C
17H24O8Na Calcd 379.1363. Found 379.1370 [M+Na]+.

M. Paradas et al. / Bioorg. Med. Chem. 20 (2012) 6655–6661
6661
6. For the nomenclature of granadaene-type polyenes we selected the general
name granadaene preceded by the number of double bonds in brackets.
Therefore, natural granadaene 1 is [12]-granadaene.
7. Rosa, M.; Rodríguez-Granger, J.; Haidour-Benamin, A.; Cuerva, J. M.; Sampedro,
A. Appl. Environ. Microbiol. 2006, 72, 6367.
Acknowledgments
We thank the Regional Government of Andalucía (project P09-
FQM-4571), and MICINN (project CTQ-2011.22455). MP thanks Re-
gional Government of Andalucía for his fellowship.
8. The enantiomeric series of the subunit of rhamnose and ornithine amino acid
are supposed to be the natural ones.
9. For
a better comparison of the model structures with the original [12]-
granadaene, we have use the same numeration for all the analogues positions.
10. Kobayashi, Y.; Matsuumi, M. J. Org. Chem. 2000, 65, 7221.
References and notes
11. (a) Horner, L.; Hoffmann, H.; Wippel, H. G.; Klahre, G. Chem. Ber. 1959, 92,
2499; (b) Wadsworth, W. S., Jr.; Emmons, W. D. J. Am. Chem. Soc. 1961, 83,
1733; (c) Maryanoff, B. E.; Reitz, A. B. Chem. Rev. 1989, 89, 863.
1. Rodríguez-Granger, J.; Alvargonzalez, J. C.; Berardi, A.; Berner, R.; Kunce, M.;
Hufnagel, M.; Melin, P.; Decheva, A.; Orefici, G.; Poyart, C.; Telford, J.; Efstration,
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Fraile, M. Eur. J. Clin. Microbiol. Infect. Dis. 2012. http://dx.doi.org/10.1007/
s10096-012-1559-0.
2. Verani, J. R.; McGee, L.; Schrag, S. J. Division of Bacterial Diseases, National Center
for Immunization and Respiratory Diseases, Centers for Disease Control and
Prevention (CDC). 2010, 59, 1–36.
3. Rosa-Fraile, M.; Rodríguez Granger, J.; Cueto Lopez, M.; Sampedro Martinez, A.;
Biel Gaye, E.; Haro, J. M.; Andreu, A. J. Clin. Microbiol. 1999, 37, 2674.
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14. The corresponding 1H signals for (S,
a)-2 at C-27 (3.50 ppm), C-28 (1.14 ppm)
and C-100 (4.75 ppm) does not match with those reported for 1.
15. Morcillo, S. P.; Álvarez de Cienfuegos, L.; Mota, A. J.; Justicia, J.; Robles, R. J. Org.
Chem. 2011, 76, 2277.
16. Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457.
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18. In this sense, this methodology has allowed us to prepare granadaene
derivatives containing simple amines (as N-octylamine) and amino acids (as
L-alanine) in excellent yields.
19. Just, G.; Ouellet, R. Can. J. Chem. 1976, 54, 2925.
20. Dimitriadis, C.; Gill, M.; Harte, M. F. Tetrahedron: Asymmetry 1997, 8, 2153.
5. (a) Doran, K. S.; Nizet, V. Mol. Microbiol. 2004, 54, 23; (b) Liu, G. Y.; Nizet, V.
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