An indole-containing dauer pheromone component with unusual dauer inhibitory activity at higher concentrations

Article abstract of DOI:10.1021/ol901011c

In Caenorhabdltls elegans, the dauer pheromone, which consists of a number of derivatives of the 3,6-dideoxysugar ascarylose, is the primary cue for entry into the stress-resistant, "nonaging" dauer larval stage. Here, using activity-guided fractionation

Full text of DOI:10.1021/ol901011c

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2009
Vol. 11, No. 14
3100-3103
An Indole-Containing Dauer Pheromone
Component with Unusual Dauer
Inhibitory Activity at Higher
Concentrations
Rebecca A. Butcher, Justin R. Ragains, and Jon Clardy*
Department of Biological Chemistry and Molecular Pharmacology, HarVard Medical
School, 240 Longwood AVenue, Boston, Massachusetts 02115
jon_clardy@hms.harVard.edu
Received May 9, 2009
ABSTRACT
In Caenorhabditis elegans, the dauer pheromone, which consists of a number of derivatives of the 3,6-dideoxysugar ascarylose, is the primary
cue for entry into the stress-resistant, “nonaging” dauer larval stage. Here, using activity-guided fractionation and NMR-based structure elucidation,
a structurally novel, indole-3-carboxyl-modified ascaroside is identified that promotes dauer formation at low nanomolar concentrations but
inhibits dauer formation at higher concentrations.
Caenorhabditis elegans, a small nematode (round worm) that
has become an important model organism in development,
neurobiology, and aging research, senses the world primarily
through small molecules. In response to unfavorable growth
conditions, such as high population density, C. elegans enters
the dauer larval stage, which is specialized for survival under
harsh conditions1 (Figure 1a). Dauer formation is controlled
by the insulin/IGF-12 and TGFꢀ pathways,3 which also
control metabolism, stress resistance, and aging throughout
the lifecycle. To monitor its population density and trigger
entry into the dauer stage, C. elegans uses the dauer
pheromone that it secretes into its environment and senses
using chemosensory organs in its head.4 The dauer phero-
mone consists of a number of structurally related derivatives
of the 3,6-dideoxysugar ascarylose with fatty acid-like side
chains5 (Figure 1b). We have been naming these ascarosides
based on the carbon chain length of the fatty acid-like
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10.1021/ol901011c CCC: $40.75
Published on Web 06/22/2009
 2009 American Chemical Society

16–18 days at 25 °C and by collecting, lyophilizing, and
extracting the growth medium.5b To summarize our frac-
tionation scheme, we fractionated the crude extract using C18
extraction, followed by silica gel chromatography and high
pressure liquid chromatography on a C18 column (see
Supporting Information). Activity was followed using the
dauer formation assay in which fractions are incorporated
into an agar plate, a small amount of food is added, and
adult worms are allowed to lay 50-100 eggs before removal.
The eggs are then allowed to develop, and the dauer/adult
ratio is determined. In addition to the primary dauer-inducing
components of the extract, ascarosides C6 and C9, a number
of minor components contributed to the overall activity. One
of these components was associated with a major UV peak
at 281 nm, suggesting that it might be structurally distinct
from the known ascarosides. This component was isolated
at approximately 7.5 µg/L of growth medium. Although
activity-guided fractionation suggested that this component
was less of a contributor to the overall activity than either
ascaroside C6 or C9, the fact that the component was present
at a very low concentration in the culture medium (∼19 nM
based on isolated yield) suggested that its specific activity
might be very high.
Figure 1. (a) Under favorable environmental conditions (low
population density, high food availability, low temperature), C.
elegans undergoes reproductive growth, progressing through four
larval stages (L1-L4) to the adult. Under unfavorable conditions
(high population density, low food availability, high temperature),
C. elegans will instead progress from the L2 larval stage to a stress-
resistant alternative L3 stage, the dauer. (b) C. elegans senses its
population density using dauer-inducing ascarosides (1-4) (para-
aminobenzoic acid, PABA).
HRESIMS analysis of the dauer pheromone component
(m/z 414.1513) indicated a molecular formula of C20H25NO7
(calcd. m/z for [M + Na]+, 414.1529). NMR analysis
included proton, dqf-COSY, gHSQC, gHMBC, and NOESY
experiments (Table 1 and Figure S1, Supporting Informa-
tion). Due to the small amount of compound isolated, NMR
experiments were performed using a 5 mm symmetrical
microtube (Shigemi) in a 600 MHz NMR instrument
equipped with a cryoprobe. The dqf-COSY spectrum indi-
portion. Minor modifications to the structures of the asca-
rosides dramatically affect their ability to induce dauer
formation.5a Synergism between some of the ascarosides
suggests that they may have different receptors.5b Further-
more, different ascarosides have differently shaped titration
curves and show different temperature dependencies.5b Here,
we report the structure of another dauer pheromone com-
ponent, indolecarboxyl-ascaroside C5 (5) (Figure 2), its total
synthesis, and characterization of its dauer-inducing activity.
1
Table 1. NMR Shifts Derived from H, dqf-COSY, gHSQC,
and gHMBC Spectra of Natural Indolecarboxyl-ascaroside C5
Salt in Methanol-d4
no.
δH mult. (J (Hz))
δC
HMBC
1
182.34
2a
2b
3a
3b
4
5
1′
2.27, m (J2a,2b ) 14.2)
2.41, m
1.85, m
1.91, m
3.88, m
1.19, d (J4,5 ) 6.0)
4.75, br s
35.48 C-1,3,4
C-1,3,4
35.21 C-1,2,4,5
C-1,2,4,5
72.41 C-2,1′
18.81 C-3,4
97.16 C-4,3′,5′
69.47 C-4′
2′
3.80, dt (J1′,2′ ) 2.7)
3′ax 2.05, ddd (J2′,3′ax ) 3.4, J3′ax,3′eq ) 12.7) 33.17 C-4′
3′eq 2.19, ddd (J2′,3′eq ) 2.7) C-1′,4′
4′
5′
5.11, ddd (J3′ax,4′ ) 11.0, J3′eq,4′ ) 4.7)
4.09, dq (J4′,5′ ) 9.7)
70.36 C-5′,6′,1′′
68.51 C-4′
Figure 2. Chemical structure of indolecarboxyl-ascaroside C5 (5).
6′
1.24, d (J5′,6′ ) 6.3)
18.06 C-4′,5′
166.36
108.11
133.26 C-2′′,4′′,5′′
138.26
127.22
121.64 C-4′′,8′′
122.32 C-5′′,6′′
123.42 C-4′′,9′′
112.75 C-5′′,7′′
1′′
2′′
3′′
4′′
5′′
6′′
7′′
8′′
9′′
7.99, s
As described previously, we generated a crude dauer
pheromone by cultivating C. elegans in liquid culture for
8.04, m
7.19, m
7.20, m
7.43, m
(5) (a) Butcher, R. A.; Fujita, M.; Schroeder, F. C.; Clardy, J. Nat. Chem.
Biol. 2007, 3, 420. (b) Butcher, R. A.; Ragains, J. R.; Kim, E.; Clardy, J.
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F. C. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 7708.
Org. Lett., Vol. 11, No. 14, 2009
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cated three distinct spin systemssa 3-substituted indole unit,
an ascarylose sugar, and a 4-carbon aliphatic chain (Figure
S2a, Supporting Information). The long-range HMBC cor-
relation between the ascarylose C-4′ proton and the C-1′′
carbonyl carbon, as well as the proton and carbon shifts of
the indole, suggested that the indole unit was connected via
an ester linkage to the ascarylose sugar at its C-4′ position.
Long-range HMBC correlations also established that the
aliphatic chain was in fact a pentanoic acid subunit that was
linked via an alcohol at the ω-1 position to the anomeric
carbon of the ascarylose sugar. Coupling constants and
NOESY correlations were used to assign tentatively the
relative stereochemistry of the sugar, as well as the ꢀ
configuration at the anomeric carbon and the relative
configuration of the pentanoic acid ω-1 alcohol (Figure S2b,
Supporting Information). The structure and absolute config-
uration of indolecarboxyl-ascaroside C5 (5) (Figure 2) were
then confirmed through total synthesis.
aldehyde intermediate, which was subsequently oxidized to
a carboxylic acid and then converted to methyl ester 9 via
treatment with TMSCHN2 (60%, 3 steps). Methanolytic
removal of the benzoyl protecting groups of 9 afforded diol
9a, which was then subjected to acylation conditions with
indole-3-carboxyl chloride. Surprisingly, attempts at the
monoacylation of 9a resulted primarily in the formation of
a product acylated at the 2 position of the ascarylose ring.
On the other hand, the doubly acylated product 10 could be
produced in good yield upon treatment with a large excess
of indole-3-carboxyl chloride and diisopropylethylamine.
Methanolysis of 10 produced the methyl ester precursor to
indolecarboxyl-ascaroside C5 as the only detectable monoa-
cylated product in 26% yield. Finally, the methyl ester was
converted to the final product with potassium trimethylsil-
anolate in refluxing THF. Since the natural indolecarboxyl-
ascaroside C5 was isolated as a salt, the synthetic molecule
was converted to a salt before obtaining NMR spectra,
including proton, dqf-COSY, gHSQC, gHMBC, and NOE-
SY, for comparison purposes (Figure S3, Supporting Infor-
mation). The NMR data of the natural and synthetic
indolecarboxyl-ascaroside C5 were virtually identical (see
Table 1 and Table S1 and Figures S1 and S3, Supporting
Information), and analysis of the natural and synthetic
molecules by CD spectroscopy indicated that they shared
the same absolute configuration (Supporting Information).
The synthetic indolecarboxyl-ascaroside C5 was titrated
in the dauer formation assay alongside ascarosides C3, C6,
and C9. Given that other ascarosides had previously shown
different temperature dependencies,5b all ascarosides were
titrated at both a lower (16 °C) and a higher (25 °C)
temperature (Figure 3). As was seen before, the EC50 of
ascaroside C3 is relatively temperature independent, whereas
the EC50’s of ascarosides C6 and C9 are smaller at higher
temperatures (indicating that C6 and C9 are much more
The synthesis of indolecarboxyl-ascaroside C5 proceeded
from dibenzoyl ascarylose6 (6) and commercially available
(R)-5-hexen-2-ol (7) (Scheme 1 and Supporting Information).
Scheme 1. Synthesis of Indolecarboxyl-ascaroside C5 (5)
In brief, glycosylation of 6 with 7 proceeded under the
conditions reported by Jeong et al. in good yield (73%) to
afford 8.6 Attempted cleavage of the olefin of 8 with KMnO4
resulted in an inseparable mixture of desired product car-
boxylic acid and unidentified impurities. A more selective
protocol employed the ozonolysis of 8 followed by treatment
with polymer-supported PPh3 (Janda Jel-PPh3) to afford the
Figure 3. Titration of ascaroside C6 (1), ascaroside C9 (2),
ascaroside C3 (3), and indolecarboxyl-ascaroside C5 (5) in the dauer
formation assay at either 16 or 25 °C.
(6) Jeong, P. Y.; Jung, M.; Yim, Y. H.; Kim, H.; Park, M.; Hong, E.;
Lee, W.; Kim, Y. H.; Kim, K.; Paik, Y. K. Nature 2005, 433, 541.
3102
Org. Lett., Vol. 11, No. 14, 2009

active at higher temperatures). Indolecarboxyl-ascaroside C5
has no observable activity at 16 °C but is the most potent of
all the tested ascarosides at 25 °C (with an EC50 of
approximately 9.9 nM). Interestingly, at higher concentra-
tions, indolecarboxyl-ascaroside C5 seems to block its own
activity, resulting in a bell-shaped titration curve. To test
whether a high concentration of indolecarboxyl-ascaroside
C5 blocks the activity of the other ascarosides, we tested
the dauer-inducing activity of each ascaroside in combination
with a high concentration (6 µM) of indolecarboxyl-
ascaroside C5 (Figure S4, Supporting Information). These
results, however, suggest that a high concentration of indole-
carboxyl-ascaroside C5 does not block the activity of other
ascarosides. To test whether a low concentration of indole-
carboxyl-ascaroside C5 works additively or synergistically
with the other ascarosides, we tested the dauer-inducing
activity of the different ascarosides either alone or in
combination with other ascarosides (Figure S5, Supporting
Information). These results suggest that indolecarboxyl-
ascaroside C5 displays some synergism with ascaroside C3,
but not with other ascarosides.
Here, we report the structure elucidation, total synthesis,
and biological evaluation of indolecarboxyl-ascaroside C5.
The indole-3-carboxyl portion of the molecule is biosyn-
thetically and structurally unusual. Whereas the indole-3-
acetyl group, which is derived from tryptophan, is found in
many natural products (e.g., auxin),7 the indole-3-carboxyl
group, which is derived through an unknown mechanism,
has been observed only in a handful of natural products8
(that are otherwise structurally unrelated to indolecarboxyl-
ascaroside C5). The bell-shaped titration curve of indole-
carboxyl-ascaroside C5 suggests that high concentrations of
the molecule differentially modulate its receptor to block
dauer formation and suggest that it may be possible to
develop more potent molecules that block dauer formation.
The fact that low concentrations of indolecarboxyl-ascaroside
C5 show synergism with ascaroside C3, coupled with the
fact that high concentrations of indolecarboxyl-ascaroside
C5 cannot block the activities of other ascarosides, indicates
that indolecarboxyl-ascaroside C5 may target a different
receptor from the other ascarosides. Identification of new
ascarosides such as indolecarboxyl-ascaroside C5 suggests
that the chemical language that C. elegans uses to coordinate
population-wide activities like dauer formation is very
complex. Exploration of the biosynthesis of these molecules
will likely reveal equally complex control mechanisms for
their production.
Acknowledgment. We kindly thank Greg Heffron and
Gerhard Wagner (Harvard Medical School) for NMR time
and assistance. We thank Ralph Mazitschek (Massachusetts
General Hospital) for helpful conversations regarding syn-
thetic strategy and Edward Kim (Harvard College) for
assisting with synthetic steps. We thank Jesse Chen and Jack
Szostak (Harvard Medical School) for use of a CD spec-
tropolarimeter and Gary Ruvkun (Harvard Medical School)
for use of a microscope. Some worm strains used in this
work were provided by the Caenorhabditis Genetics Center,
which is funded by the NIH National Center for Research
Resources. This work was supported by a grant from NIH
CA24487 (J.C.). R.A.B. was the recipient of an NIH
Kirschstein National Service Research Award (GM077943)
and an NIH K99 Pathway to Independence Award
(GM087533).
Supporting Information Available: Supporting data and
NMR spectra of natural and synthetic indolecarboxyl-
ascaroside C5 (5), as well as synthetic intermediates to 5.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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Hahlbrock, K. Plant Physiol. 2005, 138, 1058. (b) Hu, J. F.; Wunderlich,
D.; Sattler, I.; Hartl, A.; Papastavrou, I.; Grond, S.; Grabley, S.; Feng, X. Z.;
Thiericke, R. J. Antibiot. (Tokyo) 2000, 53, 944.
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