Trifunctional norrisolide probes for the study of Golgi vesiculation

Article abstract of DOI:10.1016/j.bmcl.2006.10.101

Inspired by the effect of norrisolide on the Golgi complex, we synthesized norrisolide probes that contain: the perhydroindane core of the parent natural product for Golgi localization, a crosslinking unit (aryl azide or epoxide) for covalent binding to t

Full text of DOI:10.1016/j.bmcl.2006.10.101

Bioorganic & Medicinal Chemistry Letters 17 (2007) 320–325
Trifunctional norrisolide probes for the study of Golgi vesiculation
Gianni Guizzunti,b Thomas P. Brady,a Vivek Malhotrab and Emmanuel A. Theodorakisa,*
aDepartment of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358, USA
bDepartment of Cell and Developmental Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla,
CA 92093-0358, USA
Received 17 August 2006; accepted 23 October 2006
Available online 6 November 2006
Abstract—Inspired by the effect of norrisolide on the Golgi complex, we synthesized norrisolide probes that contain: the perhydr-
oindane core of the parent natural product for Golgi localization, a crosslinking unit (aryl azide or epoxide) for covalent binding to
the target, and a tag (biotin or iodine) for subsequent target purification. We found that biotin-containing probes 14, 20 and
24 induced inefficient Golgi vesiculation. However, the iodinated probe 25 induced extensive and irreversible Golgi fragmentation.
This probe can be used for the isolation of the cellular target of norrisolide.
Ó 2006 Elsevier Ltd. All rights reserved.
The intracellular transport and sorting of biomolecules,
a process referred to as the secretory pathway, is essen-
tial to cell function and survival.1 Central to this process
is the Golgi complex, an assembly of membranes, whose
function involves the post-translational modification of
newly synthesized proteins and lipids, their sorting into
secretory vesicles, and their trafficking to the sites of
function.2 It has also been shown that during mitosis
the Golgi membranes undergo complete fragmentation
to small vesicles.3 However, the mechanisms governing
these vesiculation processes are currently poorly under-
stood.4 Small molecules that affect Golgi vesiculation
can be useful as probes for exploring this event and as
leads for controlling disease pathogenesis.5 For instance,
brefeldin A (1, Fig. 1) was found to cause fusion of Gol-
gi with endoplasmic reticulum (ER) and helped in
unraveling the Golgi to ER retrograde pathway.6 Secr-
amine (2) was shown to block protein transport from
Golgi to the plasma membrane,7 while CCL-19 (4)
blocks the exit of proteins from Golgi and induces Golgi
fragmentation.8 The marine sesquiterpene ilimaquinone
(3) was found to induce a reversible vesiculation of the
Golgi and led to the identification of Protein kinase D
as a component of the secretion machinery.9 More
recently, norrisolide (5)10 was shown to fragment the
Golgi complex in an irreversible manner.11
O
Me
Ph
O
N
HO
H
O
O
H
S
Br
HO
OMe
O
NH
OH
1: brefeldin A
2: secramine
MeO
O
OH
OH
O
OH
Me
H
H
O
N
H
O
O
Me
F
O
O
HN
Me
3: ilimaquinone
4: CLL-19
O
O
14
O
12
Me
O
O
O
O
O
Me
H
Me
O
9
9
O
O
H
Me Me
5: norrisolide
Me Me
6
Keywords: Natural products; Biotin; Crosslinking; Secretion; Golgi.
*
Corresponding author. Tel.: +1 858 822 0456; fax: +1 858 822
0386; e-mail: etheodor@chem.ucsd.edu
Figure 1. Structures of selected Golgi-disturbing agents.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.10.101

G. Guizzunti et al. / Bioorg. Med. Chem. Lett. 17 (2007) 320–325
321
Recent studies with norrisolide have suggested that the
OH
Me
OH
O
hydrocarbon-containing perhydroindane core of this
natural product binds to a receptor at the Golgi mem-
branes and induces reversible vesiculation.12 The vesicu-
lation becomes irreversible when this core is decorated
with electrophilic residues. Our efforts to explore this
concept led to identification of compound 6 (Fig. 1) that
was shown to induce an identical phenotype with 5. We
proposed that this effect is due to covalent reaction of
the epoxide units of 6 with its biological receptor.12
Based on these findings, we sought to synthesize and
study trifunctional norrisolide probes that would con-
tain the following functional groups: (a) the perhydroin-
dane core for Golgi localization, (b) a crosslinking
reagent for covalent binding to its putative target and,
(c) a tag for subsequent protein purification and
isolation.
12
9
1
O
Me
H
(a)
EDC,
DMAP
14
N3
H
Me Me
HO
7
(77%)
Me Me
12
9
14
HO2C
N3
(b)
BocNH
I
(93%)
NHR
(10)
CsCO3, DMF
8
S
O
O
12
CO2H
O
Me
HN
O
NH
14
(13)
N3
(d)
EDC
H
11: R=Boc
12: R= H
The first probe that we evaluated was compound 14 in
which the perhydroindane motif is attached to an aryl
azide unit and biotin. It was expected that light-activa-
tion of the aryl azide would produce a reactive nitrene
intermediate that would react covalently with its recep-
tor and thus mimic the irreversible Golgi vesiculation
observed with the natural product.13 The biotin tag
could then allow the isolation and purification of the
target via affinity chromatography on resin-bound strep-
tavidin. The synthesis of 14 is summarized in Scheme
1.14 Coupling of 715 with 816 in the presence of EDC
and DMAP produced ester 9 in 77% yield. The phenol
functionality of 9 was then alkylated with N-Boc iodo-
propane (10) in the presence of CsCO3 in DMF to form
compound 11 (93% yield). TFA-induced Boc deprotec-
tion of 11, followed by coupling of the resulting amine
with biotin (13), gave rise to trifunctional probe 14.17
The bifunctional probe 15,17 containing the aryl azide
functionality and the biotin but not the perhydroindane
motif, was prepared in a similar manner and was used as
a control.
(89%)
HN
Me Me
(c)
DIEA
N3
14
N3
14
HN
12
12
O
O
O
O
MeO
O
O
O
Me
S
S
NH
H
HN
NH
HN
Me
Me
O
15
O
14
Scheme 1. Reagents and conditions: (a) 7 (1.0 equiv), 8 (1.1 equiv),
EDC (1.2 equiv), DMAP (1.2 equiv), CH2Cl2, 25 °C, 24 h, 77%; (b) 10
(1.3 equiv), CsCO3 (2.0 equiv), DMF, 25 °C, 4 h, 93%; (c) TFA
(5.0 equiv), CH2Cl2, 25 °C, 0.5 h, then; (d) 13 (1.3 equiv), DIEA
(3.0 equiv), EDC (1.4 equiv), DMF/CH2Cl2 1:1, 25 °C, 4 h, 89%.
The effect of compounds 14 and 15 in Golgi vesiculation
was evaluated using normal rat kidney (NRK) cells.
Cells grown on coverslips in complete growth medium
were treated with 14 and 15 (30 lM) for 60 min at
37 °C.18 The cells were then fixed and processed for
immunofluorescence microscopy.18 The Golgi complex
is shown in red and the cell nucleus is shown in blue.
Figure 2 highlights the results of this study. Comparison
of Figure. 2a and b indicates that compound 14 caused
Golgi fragmentation in 50% of the cell population. In
contrast, compound 15 did not lead to any fragmenta-
tion (data not shown), supporting the notion that the
perhydroindane motif is essential for norrisolide activi-
ty. As expected, the fragmentation induced by 14 was
completely reversible upon washing when the photoacti-
vation of the aryl azide unit did not take place (Fig. 2c).
UV-irradiation for 15 min caused an irreversible Golgi
fragmentation in a small percent of the cells (Fig. 2d).18
As a negative control, we used probe 15, containing bio-
tin but not the norrisolide core. The results, shown in
Figure 3, indicate that both compounds 14 and 15 led
to isolation of the same protein bands. This suggests
that these protein bands are due to non-specific interac-
tions of biotin with cellular proteins.19
In parallel with the above studies, we considered an
alternative design of trifunctional probes in which the
photoactivated aryl azide unit would be replaced by an
epoxide functionality. This design was inspired by the
observation of compound 6 inducing an identical phe-
notype to that of norrisolide in Golgi membranes.12
We hypothesized that a probe that combines the per-
hydroindane motif with an epoxide unit and biotin
would ensure the irreversible Golgi vesiculation and al-
low the isolation of its target protein. This hypothesis
led us to synthesize and evaluate probes 20 and 24.
Despite the low extent of irreversibility, we attempted
isolation of the protein target of compound 14. After
UV irradiation, the cells were lysed and analyzed by
Western blot to identify proteins containing biotin.18
The synthesis of compound 20 is shown in Scheme 2.
Alkylation of 16 with 1,3-dibromopropane (1.1 equiv)
in refluxing acetone using K2CO3 as the base produced

322
G. Guizzunti et al. / Bioorg. Med. Chem. Lett. 17 (2007) 320–325
a)
b)
a)
b)
180
130
100
73
54
48
35
c)
d)
24
Figure 3. Immunoblot of total protein lysate of cells treated with 14 (a)
and 15 (b), and subjected to photoactivation. Biotinylated proteins are
visualized by Streptavidin–HRP.
of biotin tag. It is possible that the steric hindrance
caused by biotin in combination with its intrinsic affinity
for biotin receptors leaves insufficient amounts of these
probes on the Golgi membranes thus dramatically
reducing their effect on the Golgi complex. In other
Figure 2. Effect of 14 on Golgi membranes. NRK cells were treated
with 14 for 0 min (a) and 60 min (b). Cells were then exposed to
photoactivation (d) and subsequently washed and incubated for
60 min. Control cells not subjected to UV irradiation are shown in
(c). Golgi is shown in red; nuclei in blue.
OH
O
selectively the C14-monoalkylated adduct that, after
treatment with NaN3, gave rise to azide 17 (83% yield
over two steps). The C-12 phenolic oxygen was then
benzylated and the adjacent methyl ester was saponified
to afford carboxylic acid 18 (94% yield over two steps).
Coupling of 18 with alcohol 7, followed by Staudinger
reduction20 of the pendant azide group, produced amine
19 (75% combined yield) that was coupled with biotin
(13). Hydrogenolysis of the C12 benzyl ether of 19, fol-
lowed by alkylation of the resulting alcohol with epi-
bromohydrin using CsCO3 as the base, produced
probe 20 (59% yield over three steps).21
14
(a) Br(CH2)3Br
(b) NaN3, DMF
14
HO12
N3
HO 12
(83%)
MeO2C
MeO2C
17
16
(c) BnBr, CsCO3
(d) NaOH, THF
(94%)
O
14
NH2
BnO
O
12
O
14
(e) EDC, 7
Me
N3
BnO 12
O
(f) Ph3P
A similar strategy was devised for the construction of
probe 24 (Scheme 3).21 Compound 21 was selectively
alkylated first at the C14 phenol (epibromohydrin,
K2CO3) and then at the C12 center using EZ-link
PEO-iodoacetylbiotin (23)22 and CsCO3, to form 24 in
55% yield (over two steps).
HO2C
(75%)
18
H
Me Me
O
14
O
19
O
HN
O
(g) EDC, 13
(h) H2, Pd/C
12
(i) epibromohydrin
S
O
Me
H
Evaluation of probes 20 and 24 in NRK cells was per-
formed using the conditions discussed above. Incuba-
tion of NRK cells with 20 induced less than 10% of
Golgi fragmentation in the entire cell population
(Fig. 4a). Moreover, this effect was fully reversible after
cell washing (Fig. 4b). Similar results were obtained with
compound 24 (data not shown). The Golgi fragmenta-
tion induced by probes 20 and 24 was clearly less effec-
tive than that observed using norrisolide and compound
6 that, under identical conditions, led to complete and
irreversible vesiculation in more than 90% of the cell
population.12
O
(59%)
HN
NH
Me Me
O
20
Scheme 2. Reagents and conditions: (a) 1,3-dibromopropane
(1.1 equiv), K2CO3 (2.0 equiv), acetone, 60 °C, 4 h, 87%; (b) NaN3
(1.5 equiv), DMF, 25 °C, 16 h, 95%; (c) BnBr (1.6 equiv), CsCO3 (2.0
equiv), DMF, 25 °C, 16 h, 95%; (d) NaOH (1 N) in THF (1:1) 50 °C,
4 h, 99%; (e) 7 (1.0 equiv), 18 (2.0 equiv), EDC (2.0 equiv), DMAP
(2.0 equiv), CH2Cl2, 25 °C, 24 h, 83%; (f) PPh3 (1.3 equiv), H2O
(1.3 equiv), THF, 25 °C, 24 h, 90%; (g) 13 (1.3 equiv), EDC (1.3 equiv),
CH2Cl2/DMF 1:1, 25 °C, 12 h, 92%; (h) H2 (1 atm), 10% Pd/C
(0.2 equiv), AcOEt, 25 °C, 6 h, 79%; (i) epibromohydrin (1.3 equiv),
CsCO3 (1.3 equiv), DMF, 25 °C, 12 h, 81%.
We hypothesized that the low extent of Golgi vesicula-
tion induced by probes 20 and 24 is due to the presence

G. Guizzunti et al. / Bioorg. Med. Chem. Lett. 17 (2007) 320–325
323
words, the biotin tag could interfere with the ability of
the perhydroindane motif to localize these probes on
the Golgi. This hypothesis is also supported by the re-
sults obtained with probe 14 that again led to a de-
creased extent of Golgi vesiculation. This analysis led
us to reconsider our overall strategy and design an alter-
native probe in which the biotin unit would be replaced
by a radioisotope. Isolation of the norrisolide target(s)
could then be pursued via radiolabeling.
(a)
O
epibromohydrin
K2CO3
O
OH
Me
H
12
O
(83%)
14
14
O
OH
HO
12
Me Me
21
O
Me
H
S
O
The structure of epoxide 22 offers the unique advantage
of facile incorporation of radioactive iodine at the
aromatic ring at the C15 center. The synthesis of the
non-radioactive probe 2521 is highlighted in Scheme 4
and involves iodination of 22 with NaI and chloramine
T,23 followed by alkylation of the C12 phenol with epi-
bromohydrin (74% yield over two steps).
O
NHR
NH
HN
Me Me
O
23
22
(b) CsCO3
(70%)
O
O
NH
O
14
O
Incubation of NRK cells with compound 25 induced
Golgi vesiculation at concentrations similar to those
used for norrisolide and 6. Importantly, this fragmenta-
tion was found to occur in more than 80% of cells and
was irreversible after standard washing (Fig. 5). In fact,
probe 25 induced comparable Golgi vesiculation to that
obtained with analogue 6. This result suggests that a
probe 25 armed with a radioactive isotope 125I could
be used to tag the protein target and lead to its isolation
by radiochromatography.24
O
O
12
NH
S
O
Me
O
O
HN
NH
O
H
24
Me Me
Scheme 3. Reagents and conditions: (a) epibromohydrin (1.3 equiv),
K2CO3 (1.3 equiv), acetone, 60 °C, 2 h, 83%; (b) 22 (1.0 equiv), 23
(R@(CH2CH2O)2CH2CH2NHCOCH2I)
(1.5 equiv), DMF, 25 °C, 16 h, 70%.
(1.3 equiv),
CsCO3
In conclusion, we present herein the results of a study
aiming to develop norrisolide-based trifunctional probes
that could be used to identify the cellular target of this
unusual natural product. Inspired by the finding that
norrisolide and its analogue 6 induce an irreversible
fragmentation of the Golgi membranes, we attempted
to conjugate this structure with biotin and isolate its tar-
a)
b)
Figure 4. NRK cells treated with 20 for 60 min (a); then washed and
incubated in fresh medium for 60 min (b). Golgi is shown in red; nuclei
in blue. Similar results are obtained with probe 24.
O
O
O
O
O
14
14
15
HO 12
O 12
I
15
O
O
O
O
Me
(a) NaI, chloramine T
(b) epibromohydrin
Me
(74%)
H
H
Me Me
Me Me
22
25
Figure 5. Effect of 6 and 25 on Golgi membranes. Untreated NRK
cells (a) and NRK cells treated for 60 min with 6 (b) and 25 (c). Cells
treated as in (c) are then washed and allowed to recover for 60 min in
fresh medium (d).
Scheme 4. Reagents and conditions: (a) NaI (1.2 equiv), chloramine T
(1.1 equiv), CH3CN/H2O 10:1, 25 °C, 0.5 h, 89%; (b) epibromohydrin
(2.0 equiv), CsCO3 (1.5 equiv), DMF, 25 °C, 12 h, 83%.

324
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get by affinity chromatography. None of the biotin-
based probes induced significant Golgi fragmentation
to justify further studies. The only proteins isolated with
these probes were non-specific biotin targets. However,
iodinated probe 25 induces sufficient Golgi vesiculation
at comparable concentrations to that of the natural
product. Thus, this probe represents an appropriate re-
agent for further biological studies aiming to isolate
the cellular target of norrisolide.
Acknowledgment
Financial support by the NIH (CA 086079 to EAT, and
GM 46224 and GM 53747 to V.M.) is gratefully
acknowledged.
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17. Spectroscopic and analytical data for compounds 14–15.
25
Compound 14: ½aꢀD +48.7 (c = 0.45, CH2Cl2); Rf = 0.2
(3% MeOH in CH2Cl2); 1H NMR (400 MHz, CDCl3) d
7.99 (m, 1H), 7.95 (d, J = 8.4 Hz, 1H), 6.67 (d, J = 8.4 Hz,
1H), 6.54 (s, 1H), 5.92 (br s, 1H), 4.68 (t, J = 8 Hz, 1H),
4.84 (m, 1H), 4.29 (m, 1H), 4.12 (m, 2H), 3.51 (m, 2H),
3.12 (m, 1H), 2.89 (m,1H), 2.86 (m, 1H), 2.72 (d,
J = 12.4 Hz, 1H), 2.38 (t, J = 7.2 Hz, 2H), 2.25–2.20 (m,
2H), 2.05 (t, J = 4.8 Hz, 2H), 1.78–1.42 (m, 11H), 1.25–
1.03 (m, 4H) 0.99 (s, 3H), 0.90 (s, 3H), 0.87 (s, 3H); 13C
NMR (100 MHz, CDCl3) d 173.9, 164.4, 160.5, 146.0,
133.7, 115.2, 110.4, 103.2, 83.7, 69.4, 61.7, 55.4, 52.8, 42.9,
41.4, 40.5, 38.4, 37.9, 35.3, 33.1, 32.9, 28.5, 28.2, 28.1, 27.0,
26.7, 25.9, 20.8, 20.4, 19.4, 13.1; HRMS, calcd for
C32H46N6O5S [M+Na+]: 649.31478, found: 649.31469.
Compound 15: Rf = 0.2 (3% MeOH in CH2Cl2); 1H
NMR (400 MHz, CDCl3) d 7.92 (d, J = 8.4 Hz, 1H),
7.82 (t, J = 4.8 Hz, 1H), 6.65 (dd, J = 8.4, 2 Hz, 1H), 6.53
(d, J = 1.6 Hz, 1H), 6.10 (br s, 1H), 5.57 (br s, 1H), 4.56
(m, 1H), 4.27 (m, 1H), 4.11 (t, J = 5.2 Hz, 2H), 3.85 (s,
3H), 3.51 (q, J = 5.2 Hz, 2H), 3.12 (m, 1H), 2.86 (dd,
J = 12.8, 5.2 Hz, 1H), 2.72 (d, J = 12.8 Hz, 1H), 2.35 (t,
J = 7.6 Hz, 2H), 2.04 (m, 2H), 1.77–1.60 (m, 4H), 1.44 (m,
2H); 13C NMR (100 MHz, CDCl3) d 173.8, 165.1, 163.7,
160.4, 146.1, 133.7, 114.7, 110.4, 103.2, 69.2, 61.7, 60.1,
55.4, 51.9, 40.5, 38.2, 35.4, 28.5, 28.2, 28.1, 25.9; HRMS,

G. Guizzunti et al. / Bioorg. Med. Chem. Lett. 17 (2007) 320–325
325
calcd for C21H28N6O5S [M+Na+]: 499.17396, found:
499.1742.
6.50 (m, 2H), 6.11 (br s, 1H), 5.78 (s, 1H), 5.10 (s, 2H),
4.86 (d, J = 8.8 Hz, 2H), 4.75 (t, J = 6.8 Hz, 1H), 4.50 (m,
2H), 4.31 (m, 2H), 4.29 (m, 2H), 4.04 (m, 2H), 3.42 (m,
2H), 3.14 (m, 1H), 2.91 (m, 3H), 2.71 (m, 1H), 2.34 (t,
J = 6.8 Hz, 2H), 2.20 (m, 2H), 2.01 (m, 2H), 1.70–1.01 (m,
11H), 0.99 (s, 3H), 0.89 (s, 3H), 0.86 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 173.1, 165.4, 163.2, 162.9, 160.1,
133.7, 107.5, 106.0, 100.7, 83.4, 69.6, 61.8, 61.7, 60.0, 55.3,
55.2, 52.7, 50.2, 41.4, 40.5, 37.7, 36.8, 35.9, 33.6, 31.4, 29.7,
28.9, 28.3, 27.9, 26.5, 25.5, 20.8, 20.3, 19.4, 13.1; HRMS,
calcd for C35H51N3O7S [M+Na+]: 680.33454, found:
18. Reagents and cell: NRK cells were grown on coverslips as
described in Ref. 11. Stock solutions (5 mg/ml) of
norrisolide analogues were made in DMSO and stored
at ꢁ20 °C. The working concentration of the compounds
was 30 lM for each coverslip and cells were incubated at
37 °C for 60 min. An equal volume of DMSO was used as
a negative control for each compound. For the crosslink-
ing reaction, cells were irradiated for 15 min with UV light
using a UVL-21 hand lamp (1 mW/cm2 at 366 nm) placed
at 2 cm distance from the cells. To test the irreversibility of
Golgi fragmentation, cells were washed 4 times with
phosphate-buffered saline (PBS) (150 mM NaCl, 1.8 mM
NaH2PO4, and 8.4 mM Na2HPO4) and then incubated in
fresh complete medium at 37 °C for 90 min.
25
680.2249. Compound 24: ½aꢀD +17.9 (c = 0.3, CH2Cl2);
Rf = 0.3 (70% ether in hexanes); 1H NMR (400 MHz,
CDCl3) d 8.54 (br s, 1H), 7.88 (d, J = 8.8 Hz, 1H), 6.82 (m,
1H), 6.56 (dd, J = 8.8, 2.4 Hz, 1H), 6.48 (d, J = 2.4 Hz,
1H), 6.36 (s, 1H), 5.37 (s, 1H), 4.69 (t, J = 8 Hz, 1H), 4.57
(s, 2H), 4.48 (m, 1H), 4.32 (dd, J = 11.2, 2.8 Hz, 1H), 4.29
(m, 1H), 3.95 (dd, J = 10.8, 5.6 Hz, 1H), 3.67–3.53 (m,
12H), 3.43–3.34 (m, 2H), 3.11 (m, 1H), 2.95–2.86 (m, 3H),
2.78–2.68 (m, 2H) 2.21 (m, 2H), 2.01 (br s, 2H) 1.75–
1.01(m, 13H), 0.99 (s, 3H), 0.90 (s, 3H), 0.87 (s, 3H); 13C
NMR (100 MHz, CDCl3) d 173.3, 168.5, 164.6, 163.2,
159.6, 133.6, 112.5, 106.9, 100.8, 83.2, 70.2, 70.1, 69.9,
69.4, 69.1, 67.9, 61.7, 60.1, 55.2, 52.8, 49.9, 44.5, 43.8, 41.4,
40.5, 39.1, 38.9, 37.8, 35.9, 33.1, 32.9, 28.2, 28.0, 26.6, 25.6,
20.7, 20.3, 20.2, 19.4, 13.1; HRMS, calcd for
C40H60N4O10S [M+H+]: 789.41084, found: 789.4107.
Immunofluorescence microscopy was performed as
described in Ref. 11.
Immunoblot: Cells treated with 14 and 15 for 60 min and
irradiated with UV light were collected and lysed with
loading buffer (60 mM Tris, pH 6.8, 5% 2-mercap-
toethanol, 2% SDS, 0.01% Bromophenol blue, and 10%
glycerol). Proteins in the lysate were separated by SDS–
PAGE using a 10% running gel. Proteins were transferred
on a nitrocellulose membrane (Western blot) (60 min,
350 mA) that was then kept in blocking buffer (50 mN,
Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20, and 1%
BSA) for 30 min. The membrane was incubated for 1 h at
room temperature with Streptavidin–HRP (BD Pharmin-
gen) diluted in blocking buffer. After washing 3 times with
PBS, it was added the reagent for ECL (Perkin-Elmer).
Kodak Biomax films were used for exposure.
25
Compound 25: ½aꢀD +22.2 (c = 0.1, CH2Cl2); Rf = 0.3
(70% ether in hexanes); 1H NMR (400 MHz, CDCl3) d
8.24 (s, 1H), 6.60 (d, J = 6.4 Hz, 1H), 4.73 (t, J = 7.6 Hz,
1H), 4.44–4.33 (m, 2H), 4.09–3.99 (m, 2H), 3.38 (m, 2H),
2.95–2.87 (m, 4H), 2.83 (m, 1H), 2.22 (m, 1H), 1.75–1.01
(m, 11H), 0.99 (s, 3H), 0.90 (s, 3H), 0.86 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 164.2, 160.8, 160.5, 142.3, 115.7,
99.3, 83.7, 70.3, 69.8, 52.7, 50.1, 50.0, 49.9, 44.7, 44.5, 41.4,
37.7, 33.0, 32.8, 29.6, 26.5, 20.7, 20.3,19.4, 13.1; HRMS,
calcd for C25H33IO6 [M+H+]: 557.1400, found: 557.1412.
23. Kometani, T.; Watt, D. S.; Ji, T. Tetrahedron Lett. 1985,
26, 2043.
19. Chapman-Smith, A.; Cronan, J. E., Jr. Biomol. Eng. 1999,
16, 119.
20. (a) Liu, L.; Hong, Z.-Y.; Wong, C.-H. ChemBioChem
2006, 7, 429; (b) Soellner, M. B.; Nillson, B. L.; Raines, R.
T. J. Am. Chem. Soc. 2006, 128, 8820.
21. Compound 23 is commercially available from Pierce
Biotechnology, Inc. Rockford, IL.
22. Spectroscopic and analytical data for compounds 20, 24,
and 25. Compound 20: Rf = 0.4 (5% MeOH in CH2Cl2);
1H NMR (400 MHz, CDCl3) d 7.84 (d, J = 8.8 Hz, 1H),
24. (a) Sun, P.; Wang, G. X.; Furuta, K.; Suzuki, M. Bioorg.
Med. Chem. Lett. 2006, 16, 2433; (b) Hashimoto, M.;
Kato, Y.; Hatanaka, Y. Tetrahedron Lett. 2006, 47, 3391.