Improved Synthesis for Modular Ascarosides Uncovers Biological Activity

Article abstract of DOI:10.1021/acs.orglett.7b01009

A versatile synthesis of modular ascarosides, a family of signaling molecules from Caenorhabditis elegans and other nematodes, via hydrogenolysis of a cyclic sulfate derived from methyl-α-l-rhamnopyranoside is reported. The route enables selective introdu

Full text of DOI:10.1021/acs.orglett.7b01009

Letter
pubs.acs.org/OrgLett
Improved Synthesis for Modular Ascarosides Uncovers Biological
Activity
†
†
‡
§
Ying K. Zhang, Marco A. Sanchez-Ayala, Paul W. Sternberg, Jagan Srinivasan,
,†
and Frank C. Schroeder*
†Boyce Thompson Institute and Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853,
United States
‡
Howard Hughes Medical Institute and Division of Biology, California Institute of Technology, Pasadena, California 91125, United
States
§
Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, Massachusetts 01609, United States
*
S Supporting Information
ABSTRACT: A versatile synthesis of modular ascarosides, a
family of signaling molecules from Caenorhabditis elegans and
other nematodes, via hydrogenolysis of a cyclic sulfate derived
from methyl-α-L-rhamnopyranoside is reported. The route
enables selective introduction of different side chains at the 1,
2, and 4 positions of the sugar, as demonstrated for ascarosides
from C. elegans and Pristionchus pacificus. Bioassays with
synthetic samples of 4′-tigloyl ascaroside mbas#3 revealed its
role as an avoidance or dispersal signal.
ver the past decade, a group of specialized metabolites,
the nematode-derived modular metabolites (NDMMs),
expected to furnish ascarylose derivative 5 but instead produced
the dihydropyran 4 as the major product, likely as a result of
abstraction of the benzylic proton (Scheme 1). Inspired by the
O
have been identified from the model organism C. elegans and
1
,2
26
other nematodes.
These compounds are derived from
work of van Boom and co-workers, we then considered a
glycosides of the dideoxy sugars L-ascarylose and L-paratose,
which are decorated with diverse building blocks from amino
acid, nucleoside, carbohydrate, and fatty acid metabolism
strategy based on regioselective opening of cyclic sulfates,
whose utility is being increasingly recognized in carbohydrate
27
chemistry. Commercially available methyl L-rhamnopyrano-
side 6 was treated with 2,2-dimethoxypropane to afford 7,
followed by TBDPS protection. In the presence of excess
TBDPSCl, acetonide deprotection occurred during silica gel
chromatography to directly afford diol 8 in nearly quantitative
yield. Treatment of 8 with thionyl chloride led to cyclic sulfite
1
,3
(Figure 1). NDMMs function as signaling molecules that
regulate many aspects of nematode life history, including
4
−7
8,9
10
development,
tion,
lifespan, morphology, social communica-
6
,7,11−15
16−18
as well as interactions with other species.
Since even small changes in the chemical structures of NDMMs
can greatly affect biological activity and NDMMs can be active
9
, which was subsequently oxidized with sodium periodate in
12,19
at extremely low (femtomolar) concentrations,
require-
the presence of a catalytic amount of ruthenium trichloride to
give cyclic sulfate 10. In addition, the corresponding 4-
methoxybenzyl-protected derivative 14 was prepared as
ments for the purity of synthetic samples for bioassays are
particularly stringent. Previous syntheses of ascaroside- and
paratoside-based NDMMs employed 2,4-di-O-benzoylascary-
lose 2 as a key intermediate, which was obtained in six steps
26
described by van Boom and co-workers. Reduction of 14
4
,20−22
with tetrabutylammonium borohydride produced the two
from 1 (Figure 2).
An improved version of this route was
26
23
regioisomers 15 and 16 in a ratio of about 6:1, as reported.
reported by Knolker et al. However, 2 does not permit
̈
Better regioselectivity (15:1, easily separable) was achieved
when the corresponding 4′-TBDPS-protected derivative 10 was
used.
Next, we explored the effect of different protective groups in
position 2′ on the outcome of subsequent glycosylation
reactions. Although the use of the 2′-benzyl derivative would
offer advantages for the synthesis of some modular ascarosides,
we expected 2′-benzoyl protection to be preferable, as
regioselective modification or introduction of other substituents
at the 2′- and 4′-positions and, thus, is ill-suited for the selective
synthesis of 2′- or 4′-substituted NDMMs.
We aimed to develop a synthesis for a key intermediate that
would enable straightforward differentiation of the 2′ and 4′
positions. Recognizing that the crucial 3′ deoxygenation would
be facilitated by using an activating group that could double as a
protecting group for the 2′ hydroxy group, we turned our
attention to Klemer−Rodemeyer-like transformations. Based
2
4,25
on literature precedent,
inverse Klemer−Rodemeyer
Received: April 3, 2017
fragmentation of 2,3-O-benzylidinerhamnose derivative 3 was
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.7b01009
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Organic Letters
Letter
Scheme 1. Inverse Klemer−Rodemeyer Fragmentation of
2
,3-O-Benzylidinerhamnose Derivative 8 (a) and
Preparation of Ascaroside Derivatives via
Methoxyrhamnose-Derived Cyclic Sulfates (b, c)
Figure 1. Modular ascarosides incorporating building blocks from
neurotransmitter (osas#9), folate (ascr#8), tryptophan (icas#3), and
short-chain fatty acid (mbas#3) metabolism from C. elegans and, from
the satellite model organism P. pacificus, dimeric ascarosides ubas#1
and dasc#1.
19
biological activity of mbas#3, in part because previous
syntheses yielded insufficient material for bioassays.
Starting from benzoyl derivative 13, icas#9 was synthesized
in nine steps with an overall yield of 15% (Scheme 2). After
removal of the anomeric methyl using BBr and glycosylation of
3
(
2R)-5-hexen-2-ol, the terminal double bond was oxidized to
the corresponding carboxylic acid 24. Following debenzoylation
and dibenzylation, the TBDPS group was removed to yield
intermediate 27. Finally, attachment of the indole carboxy
moiety and subsequent debenzylation afforded icas#9. Using an
analogous sequence, dasc#1 was synthesized from key
intermediate 13 in eight steps with an overall yield of 7%
(Scheme 2). For the synthesis of mbas#3, we had to take into
account that the presence of the tigloyl and unsaturated side
chain preclude a hydrogenation step. Therefore, we here
employed 4-methoxybenzyl for 2′-protection, which could be
easily removed using trifluoroacetic acid. In this way, mbas#3
was synthesized from key intermediate 13 in eight steps with an
overall yield of 22% (Scheme 2).
Figure 2. Previous approach to ascaroside synthesis and the desired
key intermediate.
anchimeric assistance of the benzoyl carbonyl would provide
better stereoselectivity of the glycosylation step. In test
reactions with (2R)-5-hexen-2-ol, we found that the 2′-benzyl
derivative produced acceptable yields of the desired α-anomer
(
2
ratio α:β = 7:1); however, we ultimately proceeded using the
′-benzoyl derivative 13 because of better yields in subsequent
steps, in addition to better stereoselectivity of the glycosylation
(
ratio α:β > 50:1).
To demonstrate the synthetic utility of 13, we selected three
Next, we explored the biological properties of mbas#3 in
bioassays with C. elegans. Since it is structurally related to icas#3
(Figure 1), differing only in the nature of the 4′-substituent, we
tested mbas#3 in an assay that had previously been used to
modular ascarosides: icas#9 and mbas#3 from C. elegans and
dasc#1 from P. pacificus. The dimeric ascaroside dasc#1 has
recently been shown to regulate mouth form development in P.
1
0
12
pacificus, and synthetic dasc#1 is urgently needed for the
study of the underlying signaling pathways, which are of great
interest for understanding development of morphology in
characterize icas#3 as an attractive pheromone (Figure 3).
Strikingly, we found that, in contrast to icas#3, which was
tested in parallel, mbas#3 is not attractive and instead triggers
avoidance behavior. Avoidance behavior was observed in assays
with the common laboratory strain N2 Bristol as well as in
CB4856, a C. elegans strain originally collected in Hawaii that
differs from the laboratory strain N2 in that it has a higher
2
8,29
animals.
The indole ascaroside icas#9 is one of the most
abundant indole ascarosides, acts as a strong hermaphrodite
attractant in C. elegans, and contributes to induction of larval
12,30
diapause (dauer).
In contrast, nothing is known about the
B
DOI: 10.1021/acs.orglett.7b01009
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Organic Letters
Letter
Scheme 2. Synthesis of mbas#3, dasc#1, and icas#9 from 13
Figure 3. Quadrant assay used to measure attractive and repulsive
properties of icas#3 and mbas#3. (a) Agar plates were divided into
quadrants, with opposite quadrants containing icas#3 at 1 pM or
mbas#3 at 10 nM. Worms were placed into the center of the plate and
their location tracked over time. (b) Chemotaxis indices measured for
1
2
icas#3 and mbas#3. See Srinivasan et al. for methods.
on reductive opening of a rhamnose-derived cyclic sulfate. The
new approach enabled facile access to synthetic samples of very
high purity of three modular ascarosides, icas#3, dasc#1, and
mbas#3. Testing of synthetic mbas#3 revealed that this
ascaroside mediates a novel, unexpected behavioral phenotype.
Availability of synthetic mbas#3 and other ascarosides of high
purity will enable further, more detailed studies of their
biological roles.
ASSOCIATED CONTENT
Supporting Information
■
31
*
S
propensity to aggregate on food. In order to exclude the
possibility that the observed behavioral effects of mbas#3 are
due to synergy with other ascarosides produced by the worms
during the assays, we also tested the effect of mbas#3 on daf-22
mutant worms that do not produce any ascaroside-based
signaling molecules because of a defect in peroxisomal β-
oxidation. Similar to the results we obtained for N2 Bristol
and CB4856, mbas#3 triggered avoidance behavior in daf-22
mutant worms.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.7b01009.
Detailed experimental procedures and full spectroscopic
data for all major compounds (PDF)
1
9
AUTHOR INFORMATION
Corresponding Author
■
These results suggest that mbas#3 serves as a dispersal signal
in C. elegans that may antagonize the attractive properties of
indole ascarosides such as icas#3 and icas#9 in a manner similar
to the antagonistic effects of high concentrations of ascr#3 on
*
E-mail: fs31@cornell.edu.
ORCID
Frank C. Schroeder: 0000-0002-4420-0237
Notes
12
icas#3-mediated attraction. Avoidance behavior induced by
mbas#3 also differs from previously described avoidance
behavior triggered by another ascaroside, osas#9. osas#9, an
ascaroside specifically produced in large quantities by starved
L1 larvae, triggers avoidance behavior only in starved worms,
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
11
but not in worms that have access to food, whereas mbas#3,
which is produced by C. elegans larvae and adults, triggers
avoidance even in the presence of food.
This work was supported in part by the NIH (GM113692 and
GM088290 to FCS) and the Howard Hughes Medical Institute.
We thank Robert J. Micikas (University of Pennsylvania) for
helpful comments and Alexander Artyukhin (Boyce Thompson
Institute) for assistance with mass spectrometry.
In summary, we report a synthetic strategy that enables
selective preparation of 2′- or 4′-substituted ascarosides, based
C
DOI: 10.1021/acs.orglett.7b01009
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Organic Letters
Letter
(
27) Megia-Fernandez, A.; Morales-Sanfrutos, J.; Hernandez-Mateo,
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DOI: 10.1021/acs.orglett.7b01009
Org. Lett. XXXX, XXX, XXX−XXX