Synthesis of the rosette-inducing factor RIF-1 and analogs

Article abstract of DOI:10.1021/ja5046692

Studies on the origin of animal multicellularity have increasingly focused on one of the closest living relatives of animals, the choanoflagellate Salpingoeca rosetta. Single cells of S. rosetta can develop into multicellular rosette-shaped colonies through a process of incomplete cytokinesis. Unexpectedly, the initiation of rosette development requires bacterially produced small molecules. Previously, our laboratories reported the planar structure and femtomolar rosette-inducing activity of one rosette-inducing small molecule, dubbed rosette-inducing factor 1 (RIF-1), produced by the Gram-negative Bacteroidetes bacterium Algoriphagus machipongonensis. RIF-1 belongs to the small and poorly explored class of sulfonolipids. Here, we report a modular total synthesis of RIF-1 stereoisomers and structural analogs. Rosette-induction assays using synthetic RIF-1 stereoisomers and naturally occurring analogs defined the absolute stereochemistry of RIF-1 and revealed a remarkably restrictive set of structural requirements for inducing rosette development.

Full text of DOI:10.1021/ja5046692

Communication
pubs.acs.org/JACS
Open Access on 07/01/2015
Synthesis of the Rosette-Inducing Factor RIF‑1 and Analogs
Christine Beemelmanns,†,§ Arielle Woznica,‡ Rosanna A. Alegado,‡,∥ Alexandra M. Cantley,†
Nicole King,*,‡ and Jon Clardy*,†
†
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston,
Massachusetts 02115, United States
‡
Howard Hughes Medical Institute and Department of Molecular and Cell Biology, University of California, Berkeley, California
94720, United States
S
* Supporting Information
Undulation of the apical flagellum generates water currents
ABSTRACT: Studies on the origin of animal multi-
cellularity have increasingly focused on one of the closest
living relatives of animals, the choanoflagellate Salpingoeca
rosetta. Single cells of S. rosetta can develop into
multicellular rosette-shaped colonies through a process of
incomplete cytokinesis. Unexpectedly, the initiation of
rosette development requires bacterially produced small
molecules. Previously, our laboratories reported the planar
structure and femtomolar rosette-inducing activity of one
rosette-inducing small molecule, dubbed rosette-inducing
factor 1 (RIF-1), produced by the Gram-negative
Bacteroidetes bacterium Algoriphagus machipongonensis.
RIF-1 belongs to the small and poorly explored class of
sulfonolipids. Here, we report a modular total synthesis of
RIF-1 stereoisomers and structural analogs. Rosette-
induction assays using synthetic RIF-1 stereoisomers and
naturally occurring analogs defined the absolute stereo-
chemistry of RIF-1 and revealed a remarkably restrictive
set of structural requirements for inducing rosette
development.
that sweep bacteria against the microvillar collar, where they are
trapped and ultimately phagocytosed. One species of
choanoflagellate, Salpingoeca rosetta, exhibits both free-living
and multicellular colonial forms called rosettes; and this
transition provides the basis of our study (Figure 1).2
The rosette-shaped colonies formed by S. rosetta resemble
early stage morula embryos of diverse animals and develop
through a process of incomplete cytokinesis from a single
founding cell.4 The induction of rosette development requires a
bacterially produced signal from its prey Algoriphagus
machipongonensis.5 In a previous publication we identified the
planar structure of the first rosette-inducing factor (RIF-1, 1),
and we demonstrated its extraordinary femtomolar potency.6 In
this report, we describe a modular total synthesis that defines
the three-dimensional structure of RIF-1, the isolation of some
naturally occurring analogs, and a rosette-inducing assay to
establish initial structure−activity relations. In addition, we note
that synthetic RIF-1 by itself does not completely recapitulate
the activity of RIF-1 isolated from bacterial extract.
RIF-1 belongs to the small and poorly explored class of
sulfonolipids.7 Sulfonolipids have been reported as constituents
of the cell envelopes of Bacteroidetes bacteria and are thought
to contribute to the gliding motility frequently found in this
group.8 Sulfonolipids (2−4) closely resemble sphingolipids,
such as (dihydro)ceramides (Figure 2, 5), that are important
membrane components in eukaryotes and act as both structural
components and signaling molecules for cell death, survival,
differentiation, and migration.9 Sphingolipids and sulfonolipids
have been reported rarely in bacteria and so far have only been
isolated from the Bacteroidetes phylum and Sphingomonas
genus, where their biological functions are poorly understood.10
Both are amides of a fatty acid and an amine base called either
sphingosine (for sphingolipids) or capnine (for sulfonolipids).
Whereas sphingosine originates in the condensation of serine
with a fatty acid followed by reduction and dehydrogenation,
labeling studies with deuterated amino acids suggest that the
capnine base is biosynthesized via the condensation of a fatty
acyl-CoA with cysteic acid.11
ulticellularity, the transition from a unicellular to a
Mmulticellular organism, evolved at least 25 times within
eukaryotes, but it evolved only once in the animal lineage.1
Choanoflagellates, the closest living relatives of animals, have
emerged as important model organisms for reconstructing the
transition to multicellularity.2 Choanoflagellate cells have a
spherical to prolate spheroid cell body and an apical collar of
microvilli surrounding a single flagellum (Figure 1)3 that
resembles the feeding cells (choanocytes) of sponges.
To elucidate the stereochemistry of RIF-1 (Figure 2, 1), we
designed a flexible synthetic approach so that multiple
derivatives of RIF-1 could be produced without changing the
Figure 1. Morphogenesis of the choanoflagellate S. rosetta upon
exposure to the prey bacterium A. machipongonensis: (A) unicellular
slow swimmer and (B) multicellular colonial rosette form (drawing:
courtesy of Mark Dayel).
Received: May 9, 2014
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ja5046692 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
Scheme 1. Representative Synthesis of (A) α-Hydroxy Acid
a
and (B) Precursor of Capnine Base
Figure 2. (A) RIF-1 and known sulfonolipids; (B) retrosynthesis of
RIF-1.
main synthetic route. The absolute stereochemistry of RIF-1
was unknown, but we assumed it to be homologous to reported
sulfonolipids (2−4).7,12 Therefore, we first focused on the two
completely unknown stereocenters at C-2′ and C-5, which
generates four possible diastereoisomeric targets.
The synthesis of the α-hydroxy fatty acid commenced with
the addition of cuprate reagent to benzyl-protected R(−) or
S(+)-glycidol (Scheme 1).13 After TBS protection, chain
elongation was pursued by metathesis reaction with a second
generation Hoveyda-Grubbs catalyst. A metathesis reaction at
this point allowed access to other fatty acid precursors with
different chain length and substitution pattern. The newly
generated double bond and the Bn-protecting group were
removed with Pd/C under hydrogen atmosphere in one step.
The primary alcohol was then treated with Dess-Martin reagent
and directly oxidized under standard reaction conditions with
NaClO2 in the presence of 2-methyl-2-butene yielding the
desired α-hydroxy fatty acid 8. The alternative fatty acid
precursor 10 could be obtained by addition of alkyne 9 to
glycidol ether 6, subsequent TBS protection of the secondary
alcohol, and reduction of the triple bond with PtO2. Alkynes
like 9 were synthesized according to literature procedures.14 To
assemble the sphingosine/capnine moiety, alkyne 12 was
treated with nBuLi and reacted with Garner’s aldehyde 11 in
the presence of HMPA to yield compound 13 in acceptable
75% (syn:anti 20:80) yield (Scheme 1).15 Over the course of
the synthesis, it became apparent that a late stage Mitsunobu
reaction for the introduction of the sulfonic acid group was a
more attractive synthetic approach than using a cysteine-
derived Garner’s aldehyde as described by Takikawa et al.12d
The next step involved a hydrosilylation reaction of 13 with
benzyldimethylsilane (BDMS-H) as reported by Trost.16 The
reaction proceeded with excellent regiocontrol (>95:5) and
afforded the desired silylated compound 14 in 90% yield.
Subsequent Tamao-Fleming oxidation using TBAF and H2O2
(2 h) yielded ketone 15. Gratifyingly, a one-pot procedure as
reported by Trost starting from compound 13 could be
accomplished affording ketone 15 in slightly higher overall yield
(82%). Finally, β-hydroxy ketone was stereoselectively reduced
using either Et2BOMe as chelating reagent to give almost
a
Conditions: (a) Mg, CuI, THF, −20 °C, 84%; (b) TBSCl, TEA,
DMAP, DMF, quant.; (c) 5-methyl-1-hexene, 5 mol % Hoveyda-
Grubbs II catalyst, CH2Cl2, 40 °C, then; (d) Pd/C, H2, EtOAc:EtOH
1:1, 2 d, 79% over 2 steps; (e) DMP, 30 mol % NaHCO3, CH2Cl2, 0
°C → RT; then (f) NaClO2, 2-methyl-butene, THF:BuOH:H2O
(3:1:1), RT, 3 h, 65% over 2 steps; (g) nBuLi, HMPA, THF, −78 °C,
82%; (h) TBSCl, TEA, DMAP, DMF, quant.; (i) PtO2, H2, EtOAc, 1
d, quant.; (j) nBuLi, HMPA, THF, −78 °C, 75% (syn:anti 20:80); (k)
−
[Cp*Ru(NCCH3)3]+PF6 , BDMS-H, acetone, 0 °C → RT, 1 h, 90%;
(l) TBAF, THF, 15 min, 0 °C; then H2O2, MeOH, K2CO3, 12 h, RT,
87%; (m) Et2BOMe, NaBH4, THF:MeOH 4:1, 77% (syn:anti >
90:10); (n) Me4NB(OAc)3H, MeOH:AcOH, −40 °C, 91% (syn:anti
20:80); (o) PtO2, H2, EtOAc, 1 d, quant.
exclusively syn-diol 16 in 77% (dr, syn:anti > 90:10),17 or
Me4NB(OAc)3H to furnish anti-diol 17 with lower but
satisfactory diastereoselectivity.18 In addition, the alkyne moiety
of 13 was hydrogenated using PtO2 in nearly quantitative yield.
Since the stereochemistries of RIF-1’s hydroxy groups at C-2′
and C-5 were unknown, we first continued our synthetic
approach with α-hydroxy fatty acid 8 and syn-diol 16. The
cyclic isopropylaminal and Boc protecting groups were
removed in 6 N HCl at 60 °C yielding free sphingoid base
19, which was suitable for condensation with a fatty acid
(Scheme 2).
The sphingolipid core structure 20 was assembled by
treatment of 19 and fatty acid 8 with peptide coupling reagent
EDAC (Scheme 2). Subsequent protection with TBSOTf and
selective deprotection with TFA of the primary alcohol yielded
the key precursor for RIF-1. Finally, a Mitsunobu reaction of 20
with thioacetic acid, one-pot deprotection and oxidation of the
primary thiol with H2O2 afforded sulfonolipid 1 in an overall
yield of 8% (9 steps) starting from Garner’s aldehyde 11. The
spectroscopic data of 1 were in full agreement with the reported
B
dx.doi.org/10.1021/ja5046692 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
a
Scheme 2. (A) Completion of the Total Synthesis and (B) Synthesis of Structurally Related Sulfonolipids
a
Conditions: (a) 6 N HCl, MeOH, 60 °C, 6 h; then (b) EDAC, compound 8, CH2Cl2, 2 h, 58% over 2 steps; (c) TBSOTf, 2,6-lutidine, DMAP,
CH2Cl2, 89%; (d) 10% TFA in H2O, THF, 0 °C → RT, 6 h, 58% + 30% sm; (e) PPh3, DIAD, CH2Cl2, 1 h, 0 °C, then CH3COSH, 81%; (f) TFA,
H2O2, 4 h, RT, 65%.
data on natural RIF-1.6 For sulfonolipids 21−28 an analogous
synthetic route was performed.19
In a complementary approach we also investigated the
diversity of sulfonolipids produced by A. machipongonensis.5,6
The already reported sulfonolipids flavochristamide A and B (2,
3) were only detected in negligible amounts by LC-MS.
However, sulfobacin B (4) turned out to be one of the major
sulfonolipid products under our standard growth condition.19
By detailed analysis of the lipid extracts we were able to
isolate and characterize four unknown sulfonolipids 24, 29−31,
which we named accordingly (sulfobacins C−F, Figure 3A).
Synthetic compound 24, missing the C-5 hydroxy group, had
identical spectroscopic data as isolated sulfonolipid sulfobacin
D, suggesting the depicted absolute stereochemistry. These
synthetic and isolated materials allowed a preliminary
structure−activity analysis for rosette induction. All synthesized
(1, 21−28) and isolated sulfonolipids (1, 4, 24, 29−31) as well
as sphingolipid intermediates of type 20 were tested over a
broad concentration range (μM to fM) in a robust rosette
colony-induction assay with the S. rosetta RCA cell line.19 We
also tested the corresponding capnine bases (32−34), which
were obtained by hydrolysis with methanolic HCl. However,
RIF-1 diastereomers (21−23), RIF-1 analogs (24−31), and
capnine bases (32−34) did not induce rosette formation in S.
rosetta. Small amounts of isomers of sulfonolipids 24 and 31
were also tested, but they too showed no rosette-inducing
activity.19 Only synthetic and natural RIF-1 (1) stimulated the
development of solitary slow swimmers into rosette colonies.
This unexpectedly restricted set of structural requirements
indicates a highly specific substrate−receptor interaction.
Synthetic RIF-1 does not completely replicate the biological
activity of RIF-1 isolated from A. machipongonensis as shown by
quantitative comparison (Figure 3B). We are currently
exploring the reasons for this discrepancy by investigating
additional bacterially produced molecules with rosette-inducing
activity, molecules that synergize with RIF-1, and methods of
delivering these highly hydrophobic signals.
In summary, we have defined the three-dimensional structure
of RIF-1 through a modular total synthesis, characterized four
new naturally occurring sulfonolipids, established the tight
structural requirements for RIF-1’s biological activity, and
discovered that signals beyond RIF-1 may be needed for full
activity.
Figure 3. (A) Isolated sulfonolipids from A. machipongonensis, and
corresponding capnine bases; and (B) dose−response curve of S.
rosetta (fM concentration range) after treatment with natural isolate
RIF-1 (black) and synthetic RIF-1 (red); error bars indicate standard
deviation.
ASSOCIATED CONTENT
* Supporting Information
Syntheses, isolation procedures, compound characterization,
and assay data. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
jon_clardy@hms.harvard.edu; nking@berkeley.edu
■
C
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Journal of the American Chemical Society
Communication
Mol. Cell Biol. 2008, 9, 139. (c) Futerman, A. H.; Hannun, Y. A. EMBO
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(b) An, D.; Na, C.; Bielawski, J.; Hannun, Y. A.; Kasper, D. L. Proc.
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Present Addresses
§Leibniz Institute for Natural Product Research and Infection
Biology e.V., Hans-Knoll-Institute (HKI), Beutenbergstrasse
̈
11a, 07745 Jena, Germany.
∥Center for Microbial Oceanography Research and Education,
1950 East-west Rd., Honolulu, HI 96822.
Notes
(11) (a) Raman, M. C.; Johnson, K. A.; Clarke, D. J.; Naismith, J. H.;
Campopiano, D. J. Biopolymers 2010, 93, 811. (b) White, R. H. J.
Bacteriol. 1984, 159, 42. Analogous results were obtained by feeding
2H- and 13C-labeled amino acids to a culture of A. machipongonensis
and will be reported in due course.
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(b) Gupta, P.; Naidu, S. V.; Kumar, P. Tetrahedron Lett. 2004, 45,
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the NIH GM099533 (N.K.) and
GM086258 (J.C.). C.B. was supported by a Leopoldina
Research Fellowship (LPDS 2011-2) from the German
Academy of Sciences Leopoldina. A.W. is supported by the
National Science Foundation Graduate Research Fellowship
Program under grant no. DGE 1106400. N.K. is an Investigator
in the Howard Hughes Medical Institute and a Senior Scholar
in the Integrated Microbial Biodiversity Program of the
Canadian Institute for Advanced Research. The authors thank
Dr. P. Stallforth for insightful discussions on synthetic strategy,
and Mark Dayel (http://www.dayel.com) for choanoflagellate
illustrations.
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