Golgi-modifying properties of macfarlandin E and the synthesis and evaluation of its 2,7-dioxabicyclo[3.2.1]octan-3-one core

Article abstract of DOI:10.1073/pnas.1001421107

Golgi-modifying properties of the spongian diterpene macfarlandin E (MacE) and a synthetic analog, t-Bu-MacE, containing its 2,7-dioxabicyclo[3.2.1]octan- 3-one moiety are reported. Natural product screening efforts identified MacE as inducing a novel morphological change in Golgi structure defined by ribbon fragmentation with maintenance of the resulting Golgi fragments in the pericentriolar region. t-Bu-MacE, which possesses the substituted 2,7-dioxabicyclo[3.2.1]octan-3-one but contains a tert-butyl group in place of the hydroazulene subunit of MacE, was prepared by chemical synthesis. Examination of the Golgi-modifying properties of MacE, t-Bu-MacE, and several related structures revealed that the entire oxygen-rich bridged-bicyclic fragment is required for induction of this unique Golgi organization phenotype. Further characterization of MacE-induced Golgi modification showed that protein secretion is inhibited, with no effect on the actin or microtubule cytoskeleton being observed. The conversion of t-Bu-MacE and a structurally related des-acetoxy congener to substituted pyrroles in the presence of primary amines in protic solvent at ambient temperatures suggests that covalent modification might be involved in the Golgi-altering activity of MacE.

Full text of DOI:10.1073/pnas.1001421107

Golgi-modifying properties of macfarlandin
E and the synthesis and evaluation of its
2,7-dioxabicyclo[3.2.1]octan-3-one core
Martin J. Schnermanna, Christopher M. Beaudrya, Anastasia V. Egorovab, Roman S. Polishchukb,c,
Christine Sütterlind,1, and Larry E. Overmana,1
aDepartment of Chemistry, 1102 Natural Sciences II, University of California, Irvine, CA 92697-2025; bTelethon Institute of Genetics and Medicine,
c
Via P. Castellino 111, Naples, 8013, Italy; Telethon Electron Microscopy Core Facility, Consorzio “Mario Negri Sud,” Santa Maria Imbaro (CH), 66030, Italy;
and dDepartment of Developmental and Cell Biology, 2011 Biological Sciences III, University of California, Irvine, CA 92697-2300
Contributed by Larry E. Overman, February 4, 2010 (sent for review January 18, 2010)
Golgi-modifying properties of the spongian diterpene macfar-
landin E (MacE) and a synthetic analog, t-Bu-MacE, containing its
2,7-dioxabicyclo[3.2.1]octan-3-one moiety are reported. Natural
product screening efforts identified MacE as inducing a novel
morphological change in Golgi structure defined by ribbon frag-
mentation with maintenance of the resulting Golgi fragments in
the pericentriolar region. t-Bu-MacE, which possesses the substi-
tuted 2,7-dioxabicyclo[3.2.1]octan-3-one but contains a tert-butyl
group in place of the hydroazulene subunit of MacE, was prepared
by chemical synthesis. Examination of the Golgi-modifying proper-
ties of MacE, t-Bu-MacE, and several related structures revealed
Fig. 1. Known Golgi-modifying agents.
that the entire oxygen-rich bridged-bicyclic fragment is required
for induction of this unique Golgi organization phenotype. Further
characterization of MacE-induced Golgi modification showed that
protein secretion is inhibited, with no effect on the actin or micro-
tubule cytoskeleton being observed. The conversion of t-Bu-MacE
and a structurally related des-acetoxy congener to substituted
pyrroles in the presence of primary amines in protic solvent at
ambient temperatures suggests that covalent modification might
be involved in the Golgi-altering activity of MacE.
specific localization of the Golgi is not required for general pro-
tein transport to the cell surface, but has recently been implicated
in directional protein transport in a polarized cell (18–20).
Screening of a library of small molecule marine natural products
revealed that the marine metabolite, macfarlandin E (MacE,
Fig. 2, 4), impacts Golgi structure in a unique way: The Golgi
ribbon is converted into small fragments that are maintained
in the pericentriolar position of the cell. This phenotype contrasts
with the effects of other small molecule natural products 1, 2, and
3 that cause Golgi fragmentation and concomitant loss of Golgi
positioning. A detailed study of MacE would offer significant in-
sight into the nature and mechanism of Golgi localization and
organization; however, access to this natural product is limited
to trace quantities (21). It was clear from the outset that further
study would require a collaborative approach incorporating
organic synthesis as a central element. We anticipated that a
synthetic program would ultimately provide not only MacE,
but also synthetic analogs whose evaluation would offer
additional insights.
Macfarlandin E, a member of a family of rearranged diter-
penes, has been isolated from the dorid nudibranch Chromodoris
macfarlandi and from two marine sponge species (21–23).
Structurally, this diterpene is defined by a lipophilic cis-hydro-
azulene fragment joined to an oxygen-rich 2,7-dioxabicyclo
[3.2.1]octan-3-one moiety. Despite its presence in 13 related
natural products, including the closely related marine diterpene
aplyviolene (5), no studies related to the synthesis of the 2,
7-dioxabicyclo[3.2.1]octan-3-one subunit had been disclosed
organelle organization ∣ chemical biology ∣ natural product ∣ reactivity
he use of small molecule natural products has been particu-
T
larly fruitful in elucidating the dynamic features of Golgi
organization and the role of this organelle in protein modification
and transport. For example, brefeldin A (BFA, Fig. 1, 1) induces
loss of Golgi structure by enhancing retrograde transport of Golgi
proteins to the endoplasmic reticulum (ER) (1). Studies with this
natural product revealed key features of protein transport in the
early secretory pathway and the role of the small GTPase,
ADP-ribosylation factor (ARF) 1, in the regulation of membrane
dynamics (2, 3). The diterpene ilimaquinone (IQ, Fig. 1, 2) also
induces reversible fragmentation of Golgi membranes into uni-
sized vesicles (4), which might be the result of targeting compo-
nents of the activated methyl cycle (5). Studies with IQ identified
Gβγ and protein kinase D as previously undescribed regulators of
Golgi structure and function (6–8). The activities of BFA and IQ
contrast with those of the diterpene norrisolide (Fig. 1, 3) and
several synthetic analogs, which induce the irreversible conver-
sion of Golgi stacks into dispersed fragments in the cytosol
(9–12). Phospholipase A2 (PLA2) inhibitors also convert Golgi
membranes into large punctate juxtanuclear structures indicating
that PLA2 may control the tubulation of the Golgi complex (13,
14). In addition, screens of natural product-inspired chemical
libraries have identified compounds that block Golgi function
in protein transport to the cell surface without affecting overall
Golgi organization (15–17). Thus, identifying chemical entities
that modify Golgi structure and function continues to offer
fundamental insights into the function of this complex organelle.
The localization of the Golgi apparatus in close vicinity to the
centrosome is typical for interphase mammalian cells. The
Author contributions: M.J.S., C.M.B., C.S., and L.E.O. designed research; M.J.S., C.M.B.,
A.V.E., R.S.P., and C.S. performed research; M.J.S., C.M.B., R.S.P., C.S., and L.E.O. analyzed
data; M.J.S., C.S., and L.E.O. wrote the paper.
The authors declare no conflict of interest.
Data deposition: The atomic coordinates have been deposited in the Cambridge Structural
Database, Cambridge Crystallographic Data Centre, Cambridge CB2 1EZ, United Kingdom
[CSD reference nos. 762682 (7) and 762683 (17)].
1To whom correspondence may be addressed. E-mail: suetterc@uci.edu or leoverma@
uci.edu
This article contains supporting information online at www.pnas.org/cgi/content/full/
1001421107/DCSupplemental.
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MacE(4)
DMSO
BFA(1)
Fig. 3. MacE has a unique Golgi-modifying activity. NRK cells on coverslips
were treated with DMSO, BFA (3 μg∕mL), or MacE (20 μg∕mL) for 60 min at
37 °C, fixed, and processed for immunofluorescence analysis with an
antibody to the Golgi protein, giantin, and the DNA dye Hoechst 33342.
The scale bar corresponds to 10 μM.
Fig. 2. Structures of macfarlandin E, aplyviolene, and shahamin K.
prior to this report (24). Shahamin K (6), which has an identical
hydroazulene fragment, but a structurally simpler oxygenated
fragment, was the initial rearranged spongian diterpene to be
prepared by total synthesis (25).
Herein, we disclose the chemical synthesis of an analog of
macfarlandin E (4), t-Bu-MacE (7, Scheme 1), that possesses
the fully elaborated oxygenated subunit of the natural product,
but has a lipophilic tert-butyl group in the place of the cis-hydro-
azulene subunit. We also describe the initial characterization of
the Golgi-modifying properties of natural MacE, analog 7, and
several additional synthetic analogs, as well as the facile conju-
gation of analog 7 and its desacetoxy congener with primary
amines to form pyrroles.
products; this prediction follows from an expected kinetic pre-
ference for initial formation of a five-membered rather than a
six-membered oxacyclic ring.
An alternative synthetic plan that addresses these concerns is
outlined in retrosynthetic format in Scheme 1. It was envisaged
that a late stage Baeyer–Villiger oxidation of the methyl ketone
side chain of compound 11 would install the C16 acetoxy group
(10, 26). This ketone was seen arising from acid-induced cycli-
zation of acetal 12, an intermediate in which oxocarbenium
ion formation is limited to the C15 position. Lactol 12 can be
simplified to acyclic precursor 13, which we anticipated forming
from enoxysilane 14 by oxidative cleavage of the cyclopentyl ring.
Such a plan would establish the cis relationship between the
tert-butyl and acetic acid side chains of intermediate 12 from a
readily accessible 3,4-trans-disposed cyclopentenyl precursor 14.
Guided by these considerations, we initiated the synthesis of
analogs 7 and 8 by Mukaiyama–Michael reaction of enantiomeri-
cally enriched silyl ketene acetal 15 and cyclopentenone 16
(Scheme 2, 27–29). Subsequent elimination of acetic acid was
induced by addition of water to the reaction mixture to produce
enone 17.‡ Reaction of 17 with aqueous trifluoroacetic acid
removed the auxiliary, and the resulting acid was methylated
to give cyclopentenone 18 (30). Coupling of this intermediate
with tert-butylcyanocuprate in the presence of tert-butyldimethyl-
silyl chloride provided the transsubstituted cyclopentene 19
(31, 32). Takai olefination cleanly afforded an intermediate
methyl enol ether, which underwent selective hydrolysis with
oxalic acid to deliver methyl ketone 14 (33, 34). Oxidative
cleavage of the double bond of 14 with osmium tetraoxide and
sodium periodate, followed by reaction of the crude product with
TMSCHN2 provided tricarbonyl product 13 in high yield. After
extensive screening, we discovered that reaction of 13 with
camphorsulfonic acid and trimethyl orthoformate in methanol
generated 2-methoxytetrahydrofuran dimethylacetal 20 as a 1∶1
mixture of C15 epimers in acceptable yield.
With access to tetrahydrofuran intermediate 20, we were able
to readily construct the 2,7-dioxabicyclo[3.2.1]octan-3-one ring
system (Scheme 3). Saponification of 20, followed by ketal
hydrolysis under acidic conditions, yielded carboxylic acid 12.
Cyclization of this intermediate with boron trifluoride etherate
(BF3 • OEt2) afforded 2,7-dioxabicyclo[3.2.1]octan-3-one 11,
which underwent Baeyer–Villiger oxidation with trifluoroper-
acetic acid to give analog 8 containing the dioxabicyclic fragment
found in the rearranged spongian diterpene aplyviolene (5) (35).
After failing in numerous attempts to directly introduce an
acetoxy substituent adjacent to the lactone carbonyl group of
analog 8, t-Bu-MacE (7) was prepared as summarized in the
lower half of Scheme 3. Using the readily separable β-epimer
of intermediate 20, α-hydroxylation with oxaziridine 21 generated
α-hydroxy ester 22 with high diastereoselectivity, albeit with the
Results and Discussion
MacE Has a Unique Golgi-Modifying Activity. We identified MacE in
a screen for compounds with Golgi-altering activity. MacE had a
unique effect on Golgi organization as incubation of normal rat
kidney (NRK) cells with this compound for 60 min at 37 °C
induced the conversion of the continuous Golgi ribbon into small
punctate structures in the pericentriolar region (Fig. 3 Right).
This phenotype is fundamentally distinct from that observed with
BFA (Fig. 3 Middle) and IQ, which caused Golgi relocalization
into the ER or complete Golgi fragmentation and dispersal, res-
pectively (1, 4). Additionally, MacE-induced Golgi fragments
appeared much smaller than those produced by PLA2 inhibitors
(13). Thus, this phenotype appears to be unique in its nature. To
complement this initial finding, we examined the effects of
compounds obtained during the course of the total synthesis of
shahamin K (6) (25), a collection that included both enantiomers
of 6, 14 compounds possessing the hydroazulene subunit, and the
structurally closely related natural product aplyviolene (5).* The
finding that none of these additional compounds were capable of
inducing a Golgi fragmentation phenotype similar to that seen
with MacE (4) suggests that the 2,7-dioxabicyclo[3.2.1]octan-3-
one fragment is critical for the Golgi activity of this natural
product.
Synthesis of Analogs Having the 2,7-Dioxabicyclo[3.2.1]octan-3-one
Ring System. Guided by the absence of Golgi-modifying activity
in structures possessing the hydroazulene motif, we selected as
preliminary synthetic targets analogs 7 (t-Bu-MacE) and 8 that
possess the intact oxygenated bridged-bicyclic subunit, yet incor-
porate a simple tert-butyl group in place of the hydroazulene
moiety. Initial consideration of the structure of analogs 7 and
8 pointed to their potential genesis from a much simpler di-
aldehyde intermediate 9 (Scheme 1).† However, further analysis
suggested that this concept likely would be unsuccessful in prac-
tice, because of unavoidable preferential formation of product 10
having the 2,8-dioxabicyclo[3.3.0]octan-3-one ring system found
in norissolide (3) and many other structurally related natural
*See SI Appendix for further information, including compounds that were screened and
selected immunofluorescent results (SI Appendix).
†To simplify comparisons between synthetic analogs and MacE, the atom numbering of the
natural product is used in Results and Discussion; proper atom numbering for synthetic
molecules can be found in SI Appendix.
‡The relative configuration of this product was confirmed by single crystal x-ray diffraction
analysis.
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Scheme 1 Analysis of 7 and 8.
opposite configuration at C12 of that found in MacE (36). The
α-epimer of 20 was converted to the β-epimer by equilibration,
allowing the overall efficiency of the conversion of 20 to 22 to
be improved to 66% (two recycles).§ Inverting the configuration
at C16 was achieved in two steps by Dess–Martin oxidation to
give the corresponding ketone, which was reduced with sodium
borohydride in high diastereoselectivity (>20∶1) to deliver 23
in 79% overall yield. Hydrolysis of the ester and the ketal,
followed by acylation yielded α-acetoxy acid 24, which, upon ex-
posure to BF3 • OEt2, converted to the bicyclic ketone 25 in high
yield. Baeyer–Villiger oxidation of this product delivered
t-Bu-MacE (7), whose structure was confirmed by single crystal
x-ray analysis.
Scheme 2 Synthesis of 20.
sections of DMSO-treated control cells contained usually five to
six quite thin cisternae and were frequently connected into a
ribbon-like structure.
The effect of MacE and t-Bu-MacE on the Golgi apparatus
was irreversible. Fragmented Golgi membranes in the pericen-
triolar region were still seen after incubating MacE or t-Bu-
MacE-treated cells in a fresh medium for 0.5 or 2 h (SI Appendix).
Whereas incubating MacE or t-Bu-MacE-treated cells in a fresh
medium for 5 h did not change the overall effect on Golgi
Effects of MacE and Its t-Butyl Analog on Golgi Organization and
Function. Incubation of NRK cells with t-Bu-MacE (7) at
40 μg∕mL produced a Golgi phenotype nearly identical to that
of MacE (Fig. 4A). The Golgi ribbon was converted into small
fragments that remained localized in the vicinity of the centro-
some, as seen by immunofluorescence analysis of the cis-Golgi
protein, giantin (Fig. 4A). Similar effects were also seen when
we analyzed the organization of additional Golgi markers, inclu-
ding GRASP65 and β-COP (cis-Golgi), mannosidase II (medial
Golgi), golgin-97 (trans-Golgi), or in HeLa cells (SI Appendix).
These Golgi-modifying activities of MacE and t-Bu-MacE
contrast to those of analogs 8, 11, and 25, which had no effect
on Golgi structure at concentrations of up to 80 μg∕mL
(SI Appendix). Intriguingly, t-Bu-MacE was less cytotoxic than
MacE, as we observed extensive cell death when incubating cells
with MacE (20 μg∕mL) for 6 or 12 h, but not with t-Bu-MacE
(40 μg∕mL). This biological activity of MacE and its tert-butyl
analog was specific for the Golgi apparatus, as the organization
of the endoplasmic reticulum appeared unaltered (SI Appendix).
In addition, the actin and microtubule cytoskeleton were normal
after treatment of cells with these compounds for 60 min
(SI Appendix).
On an ultrastructural level, MacE and t-Bu-MacE treatment
induced significant alterations of Golgi morphology with discon-
nected and shorter Golgi stacks and extensively swollen cisternae
being produced (Fig. 4B and C). In contrast, Golgi stacks in thin
§The high level of stereochemical control achieved in the hydroxylation reaction is readily
rationalized by preferential orientation of the enolate away from the tetrahydrofuran
ring and delivery of the oxygen electrophile to the face opposite the tert-butyl group.
Scheme 3 Synthesis of 7 and 8.
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Schnermann et al.

MacE(4)
A
C
t-Bu-MacE(7)
DMSO
1250
1000
750
500
250
0
t-Bu-
MacE
DMSO
MacE
B
D
t-Bu-MacE(7)
MacE(4)
DMSO
c
b
a
E
100
80
60
40
20
0
100
80
60
40
20
0
BFA
MacE
DMSO BFA 0.2
1
2
5
1.6
8
16 40
t-Bu-
MacE
t-Bu-MacE (µg/mL)
MacE (µg/mL)
Fig. 4. Characterization of the Golgi-modifying activity of MacE and t-Bu-MacE (A) NRK cells on coverslips were treated with DMSO, MacE (20 μg∕mL),
t-Bu-MacE (40 μg∕mL), fixed, and analyzed by immunofluorescence with an antibody to giantin. Magnifications of the boxed areas are shown in the insets.
(B) NRK cells treated for 60 min with DMSO, 20 μg∕mL MacE, or 40 μg∕mL t-Bu-MacE were analyzed by electron microscopy. Arrows indicate the organization
of Golgi membranes under these conditions. The scale bar for DMSO and t-Bu-MacE-treated cells corresponds to 300 nm, for MacE-treated cells to 510 nm.
(C) Morphometric analysis was performed to determine the average length of Golgi stacks for each experimental condition. The values for MacE or t-Bu-MacE-
treated cells are statistically distinct from the DMSO control (t test: p < 0.01). (D) Cells transfected with VSV-GtsO45-GFP were incubated for 60 min with DMSO
BFA (5 μg∕mL) and indicated concentrations of MacE and t-Bu-MacE prior to fixation or incubation for 60 min to 32 °C. The fraction of cells with VSV-GtsO45-GFP
at the plasma membrane was determined for >100 cells per data point (see Fig. S4 for representative images, n ¼ 4). (E) For BFA (5 μg∕mL), MacE (20 μg∕mL),
t-Bu-MacE (40 μg∕mL), the fraction of cells in which VSV-GtsO45-GFP had reached the Golgi was determined in >100 cells (n ¼ 4).
organization, we observed significant cell death in the population
of MacE-treated cells. The irreversibility of this phenotype con-
trasts with that of other well-studied Golgi disrupting agents, such
as brefeldin A, and suggests that the putative biological target of
MacE or t-Bu-MacE may be modified covalently.
protein transport from the Golgi to the ER by treating cells first
with these compounds for 60 min, followed by treatment with
BFA for 45 min. We found that Golgi enzymes were able to un-
dergo BFA-induced relocalization to the ER in the presence of
both MacE and t-Bu-MacE, although relocalization was less com-
plete than in control cells (SI Appendix). Together, these findings
suggest that the effect of MacE and t-Bu-MacE on protein
secretion is predominantly a result of a block in Golgi to plasma
membrane transport.
Given that 4 and 7 induced a significant change to the orga-
nization of the Golgi apparatus, we investigated if there was a
concomitant block in protein transport. We performed vesicular
stomatitus virus G protein (VSV-G) transport assays in which we
first accumulated a GFP-tagged temperature sensitive form of
VSV-G (VSV-GtsO45-GFP) in the ER by incubating cells at a non-
permissive temperature of 40.5 °C to prevent proper folding of
this protein (37). During the last hour of this incubation, we
treated the cells with MacE or t-Bu-MacE to induce Golgi frag-
mentation or with DMSO as a control. Then, we either fixed cells
or shifted them to the permissive temperature of 32 °C, allowing
VSV-GtsO45-GFP to fold and be transported first to the Golgi and
then to the cell surface (SI Appendix). With both 4 and 7, we ob-
served a concentration-dependent block of protein transport
to the cell surface so that at the highest concentration,
VSV-GtsO45-GFP was no longer detected at the plasma mem-
brane (Fig. 4D). It is of interest that at these concentrations of
MacE and t-Bu-MacE, VSV-GtsO45-GFP was able to reach the
Golgi in most cells, although a substantial portion of this protein
remained in the ER. This result suggests that protein transport
between the ER and the Golgi is affected to a lesser extent than
transport between the Golgi and the plasma membrane (Fig. 4E).
We also examined effects of MacE and t-Bu-MacE on retrograde
Conjugation of t-Bu-MacE (7) and Analog 8 with Primary Amines. The
plausibility of the 6-acetoxy-2,7-dioxabicyclo[3.2.1]octan-3-one
moiety readily reacting with nucleophiles led us to examine
the reactivity of t-Bu-MacE (7) and des-acetoxy analog 8 with bio-
logically relevant nucleophiles. NMR and mass spectroscopic
observation of the reaction of perdeutero-benzylamine and 7
in THF-d8 at room temperature identified pyrrole 26¶ as the prin-
cipal product (Scheme 4, 38). Notably, no intermediate species
could be observed prior to the formation of 26 and equimolar
quantities of acetic acid and benzyl acetamide. The pyrrole
product 27, which presumably derives through the intermediacy
of 26, can be isolated in moderate yield by sequential addition of
benzylamine and camphorsulfonic acid (CSA) to a methanolic
solution of 7. As would be expected from these results, exposure
of 8 to benzylamine generates the pyrrole product 28 in high yield
¶Efforts to isolate 26 have been complicated by the formation of additional products upon
concentration.
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conjugate formation are under way. The synthetic methods de-
scribed herein will serve to generate additional chemical struc-
tures that will facilitate these studies, as well as provide
synthetic access to MacE.
Materials and Methods
Cell Culture. NRK and HeLa cells were grown in Advanced DMEM (Invitrogen),
supplemented with 2% FBS and 2 mM glutaMAX-I (GIBCO) in a 5% CO2
incubator.
Antibodies and Reagents Used in This Study. Anti-giantin, anti-β-COP and anti-
GRASP65 (Sütterlin Lab), anti-Golgin-97 [kindly provided by Ed Chan (Univer-
sity of Florida)], anti-mannosidase II [kindly provided by Kelley Moreman
(University of Georgia)], anti-calreticulin and anti-α-tubulin (Sigma) were
used. BFA was from MP Biochemicals; phalloidin-Rhodamine was from Sigma.
Fluorochrome-conjugated secondary antibodies were from Invitrogen.
Scheme 4 In situ observation of 26 and synthesis of 27 and 28.
(Scheme 4). A feasible mechanism to explain the conversion of
the oxygenated core of t-Bu-MacE and analog 8 to pyrrole pro-
ducts consists of nucleophilic addition to the C16 acetoxy group
and breakdown of the ensuing tetrahedral intermediate to
generate a transient dialdehyde (9, Scheme 2) that subsequently
undergoes Paal–Knorr-type pyrrole formation (39). Elimination
of the C12 acetoxy group and benzylamine addition to the resul-
tant α,β-unsaturated iminium leads to the formation of 26.
Subsequent addition of CSA promotes amine exchange with
methanol and Fisher esterification to give 27.
These findings highlight a potential functional role for the oxy-
genated dioxabicyclo[3.2.1]octanone as a masked 1,4-dialdehyde
progenitor leading to a protein bound pyrrole species. The reac-
tion of 1,4-dicarbonyl compounds with lysine residues to form
pyrrole-protein conjugates has been proposed as the origin of cel-
lular toxicity of n hexane via in vivo formation of 2,5-hexadione
and levuglandin E2, a δ-keto aldehyde derived from the pro-
staglandin precursor endoperoxide H2 (40, 41). An additional
feature, apparent from the observed formation of 26 and 27, is
the role of the C12 acetoxy group as a latent leaving group
enabling the formation of structures that possess two sites of
covalent modifications via pyrrole formation and subsequent
C12 reactivity. The requirement of acetoxy groups at C12 and
C16 to induce the unique Golgi phenotype reported herein
suggests that pyrrole formation and potentially cross-linking
might be involved in mediating the interaction of MacE and
t-Bu-MacE with the putative biological target.
Immunofluorescence. NRK or HeLa cells grown on coverslips were fixed for
10 min in 4% formaldehyde in PBS and incubated in blocking buffer
(2.5% FBS, 0.1% Triton-X 100). Primary and secondary antibodies were di-
luted into blocking buffer. Cells were imaged with a Zeiss Axiovert 200M
microscope and analyzed with linear adjustments with the Zeiss Axiovision
software.
Compound Treatment. NRK cells on coverslips were treated with MacE,
t-Bu-MacE, and BFA at the indicated concentration in complete medium
supplemented with 25 mM Hepes pH 7.4 for the indicated periods of time.
Control incubations were done with DMSO.
Electron Microscopy. NRK cells were grown in 6 well dishes and treated with
DMSO, MacE, or t-Bu-MacE for 60 min. After their fixation with 1% glutar-
aldehyde in Hepes buffer, they were harvested by scraping, washed in PBS,
embedded in Epon 812, and cut into thin sections. Analysis of thin sections
was performed under a Tecnai-12 electron microscope (FEI/Philips). Images
were taken using an ULTRA VIEW CCD digital camera. Morphometric analysis
of Golgi stacks was performed in 20 cells (1–4 stacks per cell) for each experi-
mental condition (overall; 41 stacks in DMSO, 38 in MacE, and 43 in
t-Bu-MacE) using the ANALYSIS software.
Transport of VSV-GtsO45-GFP. HeLa cells grown on coverslips were transfected
with the plasmid VSV-GtsO45-GFP (37) and left at 37 °C for 24 h. Cells were
then shifted to 40.5 °C for 5 h and an additional 1 h in the presence of DMSO,
MacE, or t-Bu-MacE. The cells were then fixed or shifted to 32 °C for 60 min
to allow protein transport to the cell surface. Arrival of VSV-GtsO45-GFP at the
cell surface was quantified in at least 100 cells per each experimental
condition.
Conclusions. A unique Golgi phenotype has been identified for the
spongian diterpene MacE (4) defined by fragmentation of the
Golgi ribbon with localization of the Golgi fragments in the peri-
centriolar region and concurrent block of protein transport out of
the Golgi. Limited access to significant quantities of the natural
product prompted the development of a synthesis of the substi-
tuted dioxabicyclo[3.2.1]octanone ring system characteristic of
MacE and related natural products. These studies included the
synthesis of the truncated analog t-Bu-MacE, which induces
the MacE Golgi phenotype, and other analogs whose biological
studies outlined key elements of the structure activity relation-
ships in this series. In addition, t-Bu-MacE was found to be less
cytotoxic than MacE. The conversion of the oxygenated core of
these MacE analogs to pyrrole products has suggested a potential
mechanism of action for these compounds. Further studies direc-
ted at elucidating the target and the relevance of pyrrole-protein
Compounds. See SI Appendix for experimental details, 1H and 13C NMR spec-
tra of previously undescribed compounds, and x-ray structures of t-Bu-MacE
(7) and compound 17.
ACKNOWLEDGMENTS. We thank Dr. Joe Ziller, University of California, Irvine,
for the single-crystal x-ray analyses and Dr. John Greaves, University of
California, Irvine, for mass spectrometric analyses. We gratefully acknowl-
edge the late Professor John Faulkner, Scripps Institution of Oceanography
(SIO), for encouragement and authentic macfarlandin E. We thank Professor
Gerwick, SIO, and Dr. Hyukjae Choi, SIO, for purification and access to
authentic aplyviolene. This research was supported by the NIH Neurological
Disorders & Stroke Institute (Grant NS-12389) and by NIH postdoctoral
fellowship support for M.J.S. (CA-138084) and C.M.B. (GM-079937). NMR,
mass spectra, and the x-ray analyses were obtained at UC Irvine using
instrumentation acquired with the assistance of NSF and NIH Shared Instru-
mentation grants. Unrestricted funds from Amgen, Merck, and Roche
Biosciences are also gratefully acknowledged.
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