De novo asymmetric synthesis and biological analysis of the daumone pheromones in Caenorhabditis elegans and in the soybean cyst nematode Heterodera glycines

Article abstract of DOI:10.1016/j.tet.2016.03.033

The de novo asymmetric total syntheses of daumone 1, daumone 3 along with 5 new analogs are described. The key steps of our approach are: the diastereoselective palladium catalyzed glycosylation reaction; the Noyori reduction of 2-acetylfuran and an ynone

Full text of DOI:10.1016/j.tet.2016.03.033

Accepted Manuscript
De Novo Asymmetric Synthesis and Biological Analysis of the Daumone Pheromones
in Caenorhabditis elegans and in the Soybean Cyst Nematode Heterodera glycines
Haibing Guo, James J. La Clair, Edward P. Masler, George O’Doherty, Yalan Xing
PII:
S0040-4020(16)30165-X
10.1016/j.tet.2016.03.033
TET 27573
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 23 January 2016
Revised Date: 4 March 2016
Accepted Date: 8 March 2016
Please cite this article as: Guo H, La Clair JJ, Masler EP, O’Doherty G, Xing Y, De Novo Asymmetric
Synthesis and Biological Analysis of the Daumone Pheromones in Caenorhabditis elegans and in the
Soybean Cyst Nematode Heterodera glycines, Tetrahedron (2016), doi: 10.1016/j.tet.2016.03.033.
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De Novo Asymmetric Synthesis and Biological Analysis
of the Daumone Pheromones in Caenorhabditis
elegans and in the Soybean Cyst Nematode
Heterodera glycines
Leave this area blank for abstract info.
Haibing Guo,a James J. La Clair, b Edward P. Masler,c George O’Doherty,*,d and Yalan Xing*,e
a
School of Nuclear Technology and Chemistry & Life Science, Hubei University of Science and Technology, Xianning 437100, China
Xenobe Research Institute, P. O. Box 3052, San Diego, CA 92163-1052, USA
Nematology Laboratory, USDA-ARS-NEA, Beltsville MD 20705, USA
b
c
d Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, USA
e Department of Chemistry, William Paterson University, 300 Pompton Rd, Wayne, NJ 07470, USA

1
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Tetrahedron
journal homepage: www.elsevier.com
De Novo Asymmetric Synthesis and Biological Analysis of the Daumone
Pheromones in Caenorhabditis elegans and in the Soybean Cyst Nematode
Heterodera glycines
Haibing Guo,a James J. La Clair, b Edward P. Masler,c George O’Doherty,*,d and Yalan Xing*,e
a
School of Nuclear Technology and Chemistry & Life Science, Hubei University of Science and Technology, Xianning 437100, China
Xenobe Research Institute, P. O. Box 3052, San Diego, CA 92163-1052, USA
Nematology Laboratory, USDA-ARS-NEA, Beltsville MD 20705, USA
b
c
d Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, USA
e Department of Chemistry, William Paterson University, 300 Pompton Rd, Wayne, NJ 07470, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The de novo asymmetric total syntheses of daumone 1, daumone 3 along with 5 new analogs are
described. The key steps of our approach are: the diastereoselective palladium catalyzed
glycosylation reaction; the Noyori reduction of 2-acetylfuran and an ynone, which introduce the
absolute stereochemistry of the sugar and aglycon portion of daumone; and an Achmatowicz
rearrangement, an epoxidation and a ring opening installing the remaining asymmetry of
daumone. The synthetic daumones 1 and 3 as well as related analogs were evaluated for dauer
activity in C. elegans and for effects on hatching of the related nematode H. glycines. This data
provides additional structure activity relationships (SAR) that further inform the study of
nematode signaling.
Available online
Keywords:
daumone pheromones
de novo asymmetric synthesis
dauer activity
2009 Elsevier Ltd. All rights reserved
.
hatching effect
Pd-glycosylation
1. Introduction
important pathogen of soybeans worldwide, and causes annual
losses of over $1 billion in the United States alone. Worldwide,
H. glycines and other plant-parasitic nematodes account for more
than $100 billion in crop losses annually.
Daumones comprise a family of small molecule pheromones that
can control multiple behaviors in the free-living nematode
Caenorhabditis elegans.1 Under unfavorable growth conditions, a
mixture of pheromones including 1-3 signal C. elegans to enter
the dauer stage, an enduring and non-ageing stage of the
nematode life cycle with distinctive adaptive features and
extended life.2 In 2005, Jeong isolated, characterized and
,
completed a total synthesis of daumone 1.3 4 Later that year we
reported a de novo asymmetric5 approach to 1.6 In 2007,
daumone pheromones 2 and 3 were isolated and characterized by
the Clardy group and were shown to have superior dauer
inducing properties.2a In 2009, Riddle reported that synthetic
daumone 1 did not have the same biological effects as the
isolated material.7 Specifically, synthetic daumone 1 required
near lethal doses to induce dauer effects, suggesting that other
pheromone impurities (e.g., 2 and 3) were present in the isolated
samples of daumone 1. More recently, other daumone-like
compounds have been isolated and reported to have pheromone
properties in C. elegans and related nematodes.8
Figure 1. Daumone Natural Products
With great pressure to discover novel agents that effectively
suppress plant-parasitic nematodes and are environmentally
responsible, biological targets such as egg hatching offer
promise.9 Although the genetics underlying the C. elegans and H.
glycines developmental arrest behaviors appear not to be
identical,2 it is intriguing to consider what molecular mechanisms
the C. elegans dauer formation and H. glycines embryonic
developmental arrest might share, and how daumones 1-3 can be
used to discover such mechanisms. This biological hypothesis
involving daumones, along with their interesting structures,
inspired us to pursue their de novo synthesis. Herein we fully
Dauer formation in C. elegans is analogous to a protective
behavior of the soybean cyst nematode Heterodera glycines,
which is an obligate plant parasite. Embryos within H. glycines
eggs can undergo a developmental arrest that prevents hatching
of the infective stage until environmental conditions are optimal
and host plants are available. This nematode is the most

2
Tetrahedron
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describe the de novo asymmetric synthesis of daumones 1 and 3
and related analogs10 and their effects on C. elegans and H.
glycines biology.
OH
OH
HO
H3C
HO
H3C
O
O
O
O
O
O
OH
OH
CH3
CH3
Daumone 1
Daumone 3
O
H3C
O
O
OTBS
CH3
4
O
H3C
OTBS
+
OH
BocO
O
CH3
5
6
8
O
Scheme 3. Palladium Catalyzed Glycosylation
O
OH
CH3
H
Next, we turned our attention towards elaborating 9 into the
desired glycosylation precursor 5 (Scheme 3). Application of the
Achmatowicz reaction (NBS in buffered aq. THF) to 9 afforded
the ring-expanded pyranone product 12 in excellent yield
(91%).14 Hemiacetal 12 was then converted to its corresponding
Boc-protected pyranone 5. The second precursor 6 was then
completed by reduction (H2 10% Pd/C) of propargyl alcohol 11
in 82% yield. The palladium-catalyzed glycosylation of 5 with 6
occurred under Pd catalysis providing enone 4 as a single
diastereomer in 94% yield.15
7
Scheme 1. Retrosynthetic Analysis of Daumone 1 and 3
Our strategy for the synthesis of daumones 1 and 3 is
shown in Scheme 1. Retrosynthetically, both daumones 1 and 3
could be established from pyranone 4, which, in turn, could be
stereoselectively derived by a palladium-catalyzed coupling of
the pyranone 5 and aglycon alcohol 6.11 The synthesis of the
building blocks 5 and 6 was envisioned from ketone 7,12 and 4-
pentynol 8, respectively, using a Noyori reduction to install the
stereochemical features within both components.
Our next operation focused on the installation of the required
C-2 hydroxyl of the ascaroside ring.3 As shown in Scheme 4, we
began by using 13 as a model. Our initial studies indicated that
we were able to open epoxide 13 to its corresponding
bromohydrin 14 and subsequently reduce this material to the
desired ascaroside 15 under radical conditions using
tris(trimethylsilyl)silane (TTMSS).16
2. Results and Discussion
Scheme 2. Noyori Reduction to Install Stereochemistry
We began by exploring the Noyori reduction to install the
absolute stereochemistry in furan alcohol 9 and propargyl alcohol
11 respectively (Scheme 2).13 Using this approach, alcohol 9 was
obtained in 93% yield (>96% ee) from acylfuran 7 using the
Noyori (S,S) catalyst. Similar methods also allowed us to prepare
the fatty alcohol side chain. We began by preparing ynone 10
from 4-pentynol 8 by two approaches, either in one-step by
quenching with acetic anhydride in 75% yield, or metalation of
the TBS ether with n-BuLi and then quenching with acetaldehyde
followed by oxidation with MnO2 in 95% yield. Noyori reduction
of 10 provided alcohol 11 in 81% yield (> 96% ee).
Scheme 4. Redox Hydration Strategy to Install the Ascaroside
We also found a second solution through the regioselective
and diastereoselective reduction of 14 to 15 by LiAlH4.17 We
were then able to apply the latter procedure to the preparation of

3
18. First, enone 4 was diastereoselectively epoxidized to afford
16 in 81% yield by treatment with H2O2 in the presence of a
catalytic amount of NaOH.18 Fortunately, treatment of 16 with
LiAlH4 returned the correct isomer 18 in a manner that directly
mimicked the product obtained by reduction of the carbonyl first
with NaBH4 followed by opening of the epoxide 17 with LiAlH4.
(TCCA)/TEMPO oxidation of the primary alcohol in the
ACCEPTED MANUSCRIPT
suitably protected 20 to carboxylic acid 25 occurred in 93%
yield.19 Deprotection of the two acetate groups in 25 with LiOH
gave the desired product daumone 1 in 96% yield (Scheme 6).
This de novo approach enabled access to a series of daumone
analogues (Scheme 7) and fluorescent probes (Scheme 8), which
have been used in dauer inducing experiments to probe the
structure activity relationships as well as explore the
physiological mode of action.20 Both C-2 deoxy- and C3
hydroxy-daumone (27 and 29) were obtained from the palladium
glycosylation product 4. Reduction of 4 with NaBH4 gave allylic
alcohol 26 as a single diastereomer (78%). The deoxy-analogue
27 was established from 26 by diimide reduction of the pyran
double bond followed by TBS-ether deprotection and oxidation
to carboxylic acid 27 (53% overall yield from enone 4).
Alternatively, OsO4 was used to diastereselectively oxidize the
double bond of 26 to the rhamnose isomer 28 in excellent yield
(96%). The deprotection of the TBS-group with TBAF followed
by oxidation with TEMPO provided the C-3 hydroxy daumone
analogue 29 (42% overall yield from enone 4).
Three fluorescent daumone analogs 31, 33 and 34 (Scheme 9)
were synthesized in parallel by a peptide coupling of green and
blue fluorescent amines 30 and 32 with daumone 1, and amine 32
with daumone 3, in the presence of HBTU and Et3N respectively,
in good yield (31: 72%, 33: 78%, 34: 69%). Previously we
showed that fluorescent compound 34 can serve as a probe to
track the uptake and localization of dauer-inducing pheromones
in C. elegans.20
Scheme 5. Synthesis of Daumone 3
With diol 18 in hand, we were able to complete the synthesis
of 3 using a six-step procedure. As shown in Scheme 5, this
began by protection using acetic anhydride to provide the bis-
acetate 19. At this stage, removal of the TBS protection with aq.
HCl set the stage for installation of the terminal unsaturation.
Alcohol 20 was oxidized to aldehyde 21 followed by treatment
with Horner–Wadsworth–Emmons (HWE) reaction that afforded
ethyl ester 22. Completion of the synthesis of 3 arose through
tandem acetate deprotection and trans-esterification to methyl
ester 23 followed by hydrolysis with LiOH in THF/H2O. Overall
this six step process delivered 3 in 69 % overall yield from the
central intermediate 18.
OH
OH
HO
H3C
HO
H3C
O
O
OTBS
OH
TBAF
98%
O
O
CH3
CH3
18
24
OH
TEMPO
NaClO
KBr
HO
H3C
Scheme 7. Synthesis of Daumone Analogues.
O
O
O
NaHCO3
OH
Next, we applied an in vivo assay to gain an understanding of
the structure-activity relationships (SAR) associated with dauer
induction. In order to visualize the spacial and temporal effect
using the fluorescent analogues, we developed a specific dosing
assay that allowed the washing away of excess drug.20 While
many of the published assays use treatment during multiple
stages or long-term growth,1,2,4 our studies have found that the
uptake of these materials is rapid and does not require constant
exposure. We have found that the C. elegans treated with
daumones 1 or 3 remain in the dauer state long after the worms
have been washed with fresh media. In fact, time course
imagining studies have shown the fluorescent analogues 33 and
34 are retained in C. elegans for greater than 24 h.
61%
CH3
LiOH, MeOH
Daumone 1
Then HCl
96%
OAc
OAc
AcO
H3C
AcO
H3C
TCCA, TEMPO,
O
O
OH
O
NaHCO3,Acetone
93%
O
O
OH
CH3
CH3
25
20
Scheme 6. Synthesis of Daumone 1
We previously reported the preparation of daumone 1 from
triol 24, via a two-step TBS-group deprotection (18, 98% yield)
and chemoselective oxidation (61% yield).6 Herein, we report an
improved synthesis of daumone 1 from bis-acetate 20 (89% yield
Following this liquid based assay,20 C. elegans worms were
exposed to a 1.0 µM solution of 1, 3, 23, 29, 31, 33 and 34 and
the percent of dauer formation was ascertained after transfer to
over two steps). Thus
a
trichloroisocyanuric acid

4
Tetrahedron
ACCEPTED MANUSCRIPT
agar media (Fig. 2). This assay allowed us to test the effects of
the carboxylic acid terminus either via an ester in 23 or amide in
the compounds at different stages of worm development. For
comparative purposes,20 the activity is reported as compared to
compound 3 (positive control) and solvent alone (negative
control).
34 were tolerated.8 Combined these observations provide an
important next step in establishing the development of
fluorescent probes for the further study of nematode biology. In
addition, the design of this assay allowed us to specifically
identify that 3, 23, and 34 are active when presented not only
during L1 but also at the egg stage or at entry to L1. While the
current convention,2a,4,7 screen over multiple stages, this assay
provides the additional confirmation as to the stage and timing of
the dauer induction.
OH
H
HO
H3C
N
O
NH3
Cl
N
B
F
O
+
O
F
O
N
O
OH
CH3
30
1
OH
Daumones 1 and 3 were also evaluated in a H. glycines hatch
assay.21 Eggs were continuously exposed to aqueous preparations
(1, 2, 6, 10, 1000 µM) of either daumone in vitro. Each treatment
was replicated 16-24 times over 4 experiments. Percent hatch
means were compared by 1-way ANOVA. After 6 days,
daumone 3 stimulated hatching from treated eggs by a mean of
21 ± 1% above control (P < 0.05) at all doses between 1 µM and
10 µM. Notably, higher doses (e.g., 1 mM) had no effect.
Daumone 1 had no effect on the hatching at any dose. Given the
mechanical barriers to chemical penetration of nematode eggs,
hatch stimulation by daumone 3 is significant. Whether hatch
stimulation is the result of the daumone penetrating the egg and
directly affecting the embryo, or the effect of a change in
eggshell permeability related to the daumone molecule, are open
and important questions22 relating to mode of action. Daumone 3
is likely effective at less than 1 µM in H. glycines, with an
effective concentration range of ~ 10-fold. Daumone 3 was
effective at similar doses in inducing dauer formation in C.
elegans.[20] The transition from hatch stimulation occurs at > 10
µM. This loss of effect may not involve general toxicity, since
hatched worms in the presence of 1 mM daumone 3 exhibited the
same behavior as control groups.
HO
H3C
O
HBTU/Et3N
72%
H
N
O
N
N
B
O
CH3
F
H
N
F
31
N
OH
H
HO
H3C
N
O
O
NH3
Cl
+
O
O
O
OH
CH3
O
32
1
OH
HO
H3C
N
O
H
N
O
HBTU/Et3N
78%
O
N
CH3
O
H
O
33
O
N
OH
H
N
HO
H3C
O
NH3
Cl
O
+
O
O
O
OH
O
CH3
32
3
OH
3. Conclusion
HO
H3C
N
O
HBTU/Et3N
69%
H
O
N
O
In conclusion, de novo asymmetric total syntheses of daumones
1, 3, and five analogs were accomplished. This includes the
development of new synthetic routes to 3, 29, 31, 33, and 34.
These syntheses take advantage of our highly enantio- and
diastereo-controlled palladium catalyzed glycosylation reaction
and Noyori reduction of two ketones. Daumones 1 and 3 and the
fluorescent analogs were evaluated in dauer inducing and egg
hatching assays. Daumone 3 is an effective inducer of dauer
formation in C. elegans and of hatch stimulation in H. glycines.
Conversely, daumone 1 was less effective than daumone 3 in
each of the two nematode species.
N
H
O
O
CH3
O
34
Scheme 8. Synthesis of Fluorescent Daumones.
From this screen, we found that three compounds, 3, 23, and 34,
were identified as having significant effects on dauer induction.
Data from these studies revealed two key SAR features. First, the
inclusion of a terminal-unsaturation within the olefinic side chain
was key to improving the dauer activity. Second, these studies
provided further confirmation that the addition of functionality at
Figure 2. Dauer induction assays. Compounds were screened by treating 200-300 worms as egg, during hatching, or in L1 stage with 1 µM of the ascribed
compound in a volume of 200 µL of M9 media for 20 min, as previously reported.20 The worms were then washed with fresh media, transferred to blank NG
agar plates, and incubated for 72 h at 23 °C. The percentage of dauer worms was determined from the average of five repetitions of this assay. Each screen
was conducted with a solvent control at 0-2% dauer formation (negative control) and established daumone 3 (positive control). Dauer states were counted in
agar based upon their characteristic morphology and verified by counting the worms after treatment with 1% (w/v) aqueous SDS solution. The average and
deviation of both plate and SDS-treated larvae are presented.

5
3.82 (dq, J = 12.6, 6 Hz, 1H), 3.76 (ddq, J = 6.0, 6.0, 6.0 Hz,
4. Experimental section
ACCEPTED MANUSCRIPT
1H), 2.44 (dt, J = 7.2, 1.8 Hz, 1H), 2.09 (m, 3H), 2.04 (m, 3H),
1.91 (ddd, J = 13.8, 11.4, 3 Hz, 1H), 1.68-1.46 (m, 8H), 1.33 (d,
J = 6.6 Hz, 6H); 13C NMR (150 MHz, CDCl3) δ 202.6, 170.6,
170.3, 93.8, 72.3, 70.8, 70.0, 67.0, 44.0, 37.0, 29.6, 25.4, 22.3,
21.4, 21.3, 19.2, 17.9. HRMS: Calculated for [C17H28O7Na+]:
367.1227, Found: 367.1733.
1
General Information: H and 13C NMR spectra were recorded
on 270 MHz and 600 MHz spectrometers. Chemical shifts are
1
reported relative to CDCl3 (δ 7.26 ppm) for H NMR and CDCl3
(δ 77.0 ppm) for 13C NMR. Infrared (IR) spectra were obtained
on FT-IR spectrometer. Optical rotations were measured with a
digital polarimeter in the solvent specified. Only new procedures
and compounds are reported in this experimental section. For
compounds not included in this section, please see the supporting
information.
(8R,E)-ethyl8-(3,5-diacetoxy-6-methyl-tetrahydro-2H-pyran-
2-yloxy)non-2-enoate
(22).
Ethyl
(triphenylphosphoranylidene)acetate (396.7 mg, 1.14 mmol) was
dissolved in 0.6 mL of CH2Cl2, into which aldehyde 21 (190 mg,
0.55 mmol) was added. The reaction was stirred vigorously at
room temperature for 12 h before solvent was removed in
vacuum. Chromatography on silica gel (30% EtOAc/Hexane)
gave ethyl ester 22 as colorless oil (220 mg, 0.53 mmol, 96.5%).
(2R,3R,5R,6S)-2-((R)-7-(tert-butyldimethylsilyloxy)heptan-2-
yloxy)-6-methyltetrahydro-2H-pyran-3,5-diyl diacetate (19).
To a solution of diol 18 (752 mg, 2 mmol) in pyridine (2 mL),
was added Ac2O (1 mL) and DMAP (35 mg). The reaction
mixture was stirred for 12 h. Water was added to destroy the
excess acetic anhydride, extracted (3 x 50 mL) with Et2O washed
with 40 mL of saturated CuSO4 solution for three times, dried
(Na2SO4), and concentrated under reduced pressure. The crude
product was purified using silica gel flash chromatography
eluting with 5% ether/hexane to give acetate 19 (912 mg, 1.98
mmol, 99%); a colorless oil; Rf = 0.49 (10% EtOAc/hexane);
Rf = 0.57 (40% EtOAc/Hexane); [α]25 = -22 (c = 1.0, MeOH);
D
IR (thin film, cm-1), 2936, 1743, 1233, 1038; 1H NMR (600
MHz, CDCl3) δ 6.96 (dt, J = 13.8, 7.2 Hz, 1H), 5.82 (dt, J = 15.6,
1.8 Hz, 1H), 4.83 (m, 1H), 4.79 (ddd, J = 10.8, 9.6, 4.2 Hz, 1H),
4.73 (s, 1H), 4.18 ( q, J = 7.2 Hz, 2H), 3.84 (dq, J = 12, 6 Hz,
1H), 3.76 (ddq, J = 6.0, 6.0, 6.0 Hz, 1H), 2.22 (dt, J = 7.2, 1.2
Hz, 1H), 2.11 (m, 3H), 2.06 (m, 3H), 1.93 (ddd, J = 13.8, 11.4, 3
Hz, 1H), 1.60-1.35 (m, 8H), 1.28 (t, J = 7.2 Hz, 3H); 1.17 (d, J =
6.6 Hz, 3H), 1.13 (d, J = 6 Hz, 3H); 13C NMR (150 MHz,
CDCl3) δ 170.3, 170.1, 166.7, 148.9, 121.5, 93.6, 72.2, 70.6,
69.8, 66.8, 60.4, 60.2, 36.8, 32.2, 29.3, 27.9, 25.3, 21.2, 19.0,
17.6, 14.3; HRMS: Calculated for [C21H34O8Na+]: 437.2146,
Found: 437.2151.
[α]25 = -66 (c = 1.1, CH2Cl2); IR (thin film, cm-1), 2958, 1743,
D
1371, 1309, 1037, 835; 1H NMR (600 MHz, CDCl3) 4.74 (m,
1H), 4.70 (ddd, J = 11.4, 10.2, 4.2 Hz, 1H), 4.65 (s, 1H), 3.85
(dq, J = 9.6, 6.6 Hz, 1H), 3.76 (m, 1H), 3.60 (t, J = 6.6 Hz, 2H),
2.11 (ddd, J = 13.2, 3.6, 3.6 Hz, 1H), 2.10 (s, 3H), 2.05 (s, 3H),
1.93 (ddd, J = 13.2, 11.4, 3 Hz, 1H), 1.58 (m, 2H), 1.54 (m, 2H),
1.43 (m, 2H), 1.34 (m, 2H), 1.17 (d, J = 6.6 Hz, 3H), 1.12 (d, J =
6.0 Hz, 3H), 0.89 (s, 9H), 0.04 (s, 6H); 13C NMR (150 MHz,
CDCl3) 170.4, 170.1, 93.7, 72.4, 70.7, 70.0, 68.8, 63.3, 37.2,
32.9, 29.5, 26.1, 26.0, 25.9, 25.6, 21.3, 21.2, 19.1, 18.4, 17.7, --
5.2; HRMS: Calculated for [C23H44O7SiNa+]: 483.2749, found:
483.2755.
(8R,E)-methyl-8-(3,5-dihydroxy-6-methyl-tetrahydro-2H-
pyran-2-yloxy)non-2-enoate (23). To a 20 mL flask was added
ethyl ester 22 (136 mg, 0.328 mmol), MeOH (3 mL), Lithium
hydroxide (30 mg, 1.25 mmol). The resulting solution was stirred
at room temperature for 24 h. Aqueous HCl (0.5 M, 3 mL) was
added before solvent was removed in vacuum. Chromatography
on silica gel (80% EtOAc/Hexane) gave methyl ester 23 as
colorless oil (103 mg, 0.309 mmol, 99%). Rf = 0.58 (EtOAc);
2-((R)-7-hydroxyheptan-2-yloxy)-tetrahydro-6-methyl-2H-
pyran-3, 5-diacetate (20). To a solution of TBS-ether 19 (460.6
mg, 1 mmol) in dry MeOH (2 mL), 6 N HCl (2 mL) was added at
room temperature under the argon atmosphere. After 4 h, the
reaction mixture was diluted with ether (20 mL) and quenched
with saturated NaHCO3, extracted (3 x 5 mL) with Et2O, dried
(Na2SO4), and concentrated under reduced pressure. The crude
product was purified by passing a silica gel pad eluting with pure
ether to give alcohol 20 (350 mg, 0.97 mmol, 97%); a colorless
[α]25 = -81 (c = 1.0, MeOH); IR (thin film, cm-1), 3404, 2934,
D
2180, 1991, 1723, 1655, 1438, 1273, 1203, 1101, 1128, 1029,
984, 854; 1H NMR (600 MHz, CDCl3) δ 6.96 (dt, J = 15.6, 6.6
Hz, 1H), 5.83 (dt, J = 15.6, 1.2 Hz, 1H), 4.69 (s, 1H), 3.80-3.77
( m, 1H), 3.72 (s, 3H), 3.65 (dt, J = 6.6, 3.0 Hz, 1H), 3.59-3.57
(m, 1H), 2.22 (ddd, J = 9.0, 7.2, 1.8Hz, 1H), 1.88-1.72 (m, 2H),
1.61(m, 2H), 1.55-1.35 (m, 6H), 1.27 (d, J = 6. Hz, 3H), 1.22 (d,
J = 6 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 167.3, 149.5,
121.7, 96.1, 71.3, 70.0, 69.3, 69.1, 51.5, 36.9, 35.2, 32.0, 27.8,
25.0, 20.0, 17.7; HRMS: Calculated for [C16H28O6Na+]:
339.1778, Found: 339.1784.
oil, [α]25 = –86 (c = 1.12, CH2Cl2); IR (thin film, cm-1) 2975,
D
1
2935, 2861, 1739, 1230, 1105, 1032, 983; H NMR (600 MHz,
CDCl3) 4.81 (m, 1H), 4.77 (ddd, J = 11.4, 10.2, 4.2 Hz, 1H), 4.72
(s, 1H), 3.84 (dq, J = 9.6, 6.6 Hz, 1H), 3.74 (m, 1H), 3.62 (t, J =
6.6 Hz, 2H), 2.10 (ddd, J = 13.2, 3.6, 3.6 Hz, 1H), 2.09 (s, 3H),
2.03 (s, 3H), 1.92 (ddd, J = 13.2, 11.4, 3 Hz, 1H), 1.57 (m, 2H),
1.54 (m, 2H), 1.43 (m, 2H), 1.36 (m, 2H), 1.15 (d, J = 6.6 Hz,
3H), 1.11 (d, J = 6.0 Hz, 3H); 13C NMR (150 MHz, CDCl3)
170.4, 170.1, 93.7, 72.3, 70.7, 70.0, 66.8, 62.7, 37.1, 32.7, 29.4,
25.8, 25.5, 21.2, 21.1, 19.1, 17.7; HRMS: Calculated for
[C17H30O7Na+]: 369.1884, found: 369.1891
(8R,E)-8-(3,5-dihydroxy-6-methyl-tetrahydro-2H-pyran-2-
yloxy)non-2-enoic acid (daumone 3). To a 20 mL flask was
added methyl ester 23 (13 mg, 0.04 mmol), THF/H2O (4:1, 0.4
mL), Lithium hydroxide (1.44 mg, 0.06 mmol). The resulting
solution was stirred at room temperature for 24 h. Aqueous HCl
(0.5 M, 0.4mL) was added to adjust the pH to 7, and then the
solvent was removed in vacuum. Chromatography on silica gel
(5% MeOH/EtOAc) gave acid 1 as colorless oil (12 mg, 0.0397
2-methyl-6-((R)-7-oxoheptan-2-yloxy)-tetrahydro-2H-pyran-
3, 5-yl diacetate (21). A solution of alcohol 20 (240 mg, 0.693
mmol) and trichloroisocyanuric acid (171.2 mg, 1.386 mmol) in
ethyl ether (30 mL) was stirred at –30 oC, and then 2,2,6,6-
tetramethylpeperidinooxy (5 mg, 0.034 mmol) was added in one
portion. The mixture was stirred for 20 min and then quenched
with 30 mL saturated sodium carbonate. The organic layer was
separated and the aqueous layer was extracted with ether.
Combined the organic layer and washed with brine, dried
(Na2SO4), filtered and concentrated. Chromatography on silica
gel (20% EtOAc/Hexane) gave aldehyde 21 (210 mg, 0.61 mmol,
mmol, 99%). Rf = 0.28(EtOAc) ; [α]25 = -62 (c = 1.0, MeOH);
D
IR (thin film, cm-1), 3703, 3364, 2930, 2357, 2164, 2084, 2010,
1
1954, 1696, 1272, 1127, 981, 667; H NMR (600 MHz, CD3OD)
6.93 (dt, J = 15.6, 7.2 Hz, 1H), 5.79 (dt, J = 15.6, 1.2 Hz, 1H),
4.62 (s, 1H), 3.78-3.75 ( m, 2H), 3.69 (s, 1H), 3.63-3.58 (m, 1H),
3.49 (ddd, J = 10.8, 9.6, 4.8Hz, 1H), 2.23-2.21 (m, 2H), 2.05-
1.91 (m, 2H), 1.74 (ddd, J = 12.6, 11.4, 2.4Hz, 1H), 1.58-1.23
(m, 4H), 1.19 (d, J = 6.6 Hz, 3H), 1.11 (d, J = 6 Hz, 3H); 13C
NMR (150 MHz, CD3OD) 169.9, 148.8, 122.4, 96.4, 71.3, 70.0,
68.8, 67.2, 36.9, 34.8, 31.9, 28.0, 25.2, 18.2, 16.9; HRMS:
Calculated for [C15H26O6Na+]: 325.1622, Found: 325.1627.
88%) as colorless oil; Rf = 0.66 (40% EtOAc/Hexane); [α]25 = -
D
41 (c = 1.0, MeOH); IR (thin film, cm-1), 2929, 1741, 1234,
1040; 1H NMR (600 MHz, CDCl3) δ 9.76 (t, J = 1.8 Hz, 1H),
4.82 (m, 1H), 4.78 (ddd, J = 11.4, 10.2, 4.8 Hz, 1H), 4.72 (s, 1H),
(R)-N-(2-(3-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-
indacene-3-propionyl) propanamido)ethyl)-6-(tetrahydro-3,5-

6
Tetrahedron
dihydroxy-6-methyl-2H-pyran-2-yloxy) heptanamide (31).
coli (OP50) as a food source under standard uncrowded and well-
ACCEPTED MANUSCRIPT
To a solution of amine 30 (5 mg, 0.013 mmol) in THF (0.1 mL),
was added Et3N (3.9 µl), HBTU (10.2 mg, 0.027 mmol) and
daumone 2 (7.4 mg, 0.027 mmol). The reaction mixture was
stirred overnight, concentrated under reduced pressure, purified
using silica gel flash chromatography eluting with 12 %
MeOH/Et2O to give amide 31 (5.5 mg, 0.009 mmol, 72%); Rf =
0.54 (20% MeOH/EtOAc); IR (thin film, cm-1) 3397, 2967, 2930,
2861, 1607, 1469, 1140, 1043, 850; 1H NMR (600 MHz,
CD3OD) δ 7.50 (s, 1H), 7.08 (d, J = 3.6 Hz, 1H), 6.40 (d, J = 3.6
Hz, 1H), 6.28 (s, 1H), 4.70 (s, 1H), 3.84 (ddq, J = 6.0, 6.0, 6.0
Hz, 1H), 3.77 (m, 1H), 3.68 (dq, J =9.6, 6.0 Hz 1H), 3.58 (ddd, J
= 10.2, 10.2, 4.2 Hz, 1H), 3.35 (t, J = 6.0 Hz, 2H), 3.34 (t, J =
6.0 Hz, 2H), 3.29 (t, J = 7.8 Hz, 2H), 2.68 (t, J = 7.8 Hz, 2H),
2.58 (s, 3H), 2.35 (s, 3H), 2.24 (t, J = 7.8 Hz, 2H), 2.02 (ddd, J
=13.2, 3.6, 3.6 Hz, 1H), 1.83 (ddd, J = 13.2, 11.4, 3.0 Hz, 1H),
1.67-1.58 (m, 2H), 1.57-1.50 (m, 2H), 1.46-1.33 (m, 2H), 1.28 (d,
J = 6.0 Hz, 3H), 1.18 (d, J = 6.0 Hz, 3H); HRMS: Calculated for
[C29H43BF2N4O6Na+]: 615.3136, found: 615.3140.
fed conditions at 20 °C unless otherwise stated.
Dauer Formation Assay. Adult worms were placed on a plate
containing 3 mL of NGM agar and incubated at 20 °C for 48-96
h. After incubation, adult worms were removed, and the eggs or
entry to-L1, L1-staged progeny were collected by centrifugation
after suspension of the agar in 5 mL of M9 media. Eggs, entry to-
L1 stage or L1 stage worms were then resuspended in 190 µL of
M9 media in a 96 well plate and treated with a 10 µL solution of
the desired compound suspended in 50% (v/v) aqueous DMSO or
50% (v/v) aqueous ethanol. After incubation for 4 h at 23 °C, the
solution was transferred to a 1 mL NGM agar plate, and the entry
to-L1 stage or L1 stage larvae were then incubated for 52-72 h at
23 °C. The number of eggs or larvae was counted before and
after incubation. A 1% (w/v) aqueous SDS solution (1.0 mL) was
added to the plate, and the surviving worms were counted as
dauer larvae.2a,4,7
5. Acknowledgements
6. Supplementary data
(R)-N-(2-(2-(7-(dimethylamino)-2-oxo-2H-chromen-4-
yl)acetamido)ethyl)-6-(tetrahydro-3, 5-dihydroxy-6-methyl-
2H-pyran-2-yloxy) heptanamide (33) To a solution of amine 32
(20 mg, 0.05 mmol) in THF (0.5 mL), was added Et3N (8.4 µl),
HBTU (22.7 mg, 0.06 mmol) and daumone 2 (16.4 mg, 0.06
mmol). The reaction mixture was stirred overnight, concentrated
under reduced pressure, purified using silica gel flash
chromatography eluting with 20% MeOH/Et2O to give amide 33
(21 mg, 0.039 mmol, 78%); Rf = 0.47 (10% MeOH/EtOAc);
Supplementary data
(experimental
procedures
and
characterization for the synthesis of all compounds, and copies of
1H NMR, 13C NMR for all new compounds can be found in the
supplementary data) associated with this article can be found, in
the online version, at…
[α]25 = -40 (c = 1.6, MeOH); IR (thin film, cm-1) 3352, 2931,
We are grateful to NIH (GM09025901 to G.A.O.), NSF (CHE-
0749451 to G.A.O.), and National Nature Science Foundation of
China (21202136) for the support of our research programs.
D
1
2865, 1611, 1404, 1131, 1026, 980; H NMR (600 MHz, D2O) δ
7.36 (d, J = 9.0 Hz, 1H), 6.66 (dd, J = 9.0, 2.4 Hz, 1H), 6.33 (d, J
= 2.4 Hz, 1H), 6.01 (s, 1H), 4.77 (s, 1H), 3.89 (m, 1H), 3.80 (ddq,
J = 6.0, 6.0, 6.0 Hz, 1H), 3.68 (dq, J =9.6, 6.0 Hz 1H), 3.67 (s,
2H), 3.65 (ddd, J = 10.2, 10.2, 4.2 Hz, 1H), 3.41 (t, J = 6.0 Hz,
2H), 3.35 (t, J = 6.0 Hz, 2H), 3.01 (s, 6H), 2.09 (ddd, J =13.2,
3.6, 3.6 Hz, 1H), 2.07 (t, J = 7.8 Hz, 2H), 1.79 (ddd, J = 13.2,
11.4, 3.0 Hz, 1H), 1.51-1.44 (m, 1H), 1.43-1.36 (m, 2H), 1.35-
1.29 (m, 1H), 1.29-1.17 (m, 2H), 1.28 (d, J = 6.0 Hz, 3H), 1.16
(d, J = 6.0 Hz, 3H); 13C NMR (67.5 MHz, CD3OD) δ 176.6,
171.6, 164.4, 157.3, 154.9, 152.8, 127.0, 110.7, 110.6, 109.9,
98.9, 97.5, 72.4, 71.3, 70.0, 68.5, 40.5, 40.4, 40.3, 40.0, 38.1,
37.2, 36.2, 27.0, 26.7, 19.5, 19.4, 18.3; HRMS: Calculated for
[C28H41N3O8Na+]: 570.2789, found: 570.2788.
7. References and notes
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(8R,E)-8-(3,5-dihydroxy-6-methyl-tetrahydro-2H-pyran-2-
yloxy)-N-(2-(2-(7-(dimethylamino)-2-oxo-2H-chromen-4-
yl)acetamido)ethyl)non-2-enamide (34). To a solution of amine
32 (6 mg, 0.015 mmol) in THF (0.125 mL), was added Et3N (3
ul), HBTU (4.7 mg, 0.0125 mmol) and acid 3 (5 mg, 0.0125
mmol). The reaction mixture was stirred overnight, concentrated
under reduced pressure, purified using silica gel flash
chromatography eluting with 10% MeOH/EtOAc to give amide
34 (5 mg, 0.00872 mmol, 69%); Rf = 0.55 (10% MeOH/EtOAc);
[α]25D = – 30 (c = 0.5, MeOH); IR (thin film, cm-1), 3781, 3358,
2933, 2252, 2149, 1997, 1699, 1615, 1531, 1453, 1403, 1252,
1128, 1030, 983, 834; 1H NMR (600 MHz, CDCl3) δ 7.32 (d , J =
9 Hz, 1H), 6.92 (dt, J = 15.6, 7.2 Hz, 1H), 6.77-6.71 (m, 1H),
6.54 (d, J = 3Hz, 1H), 6.03 (s, 1H), 5.81 (dt, J = 15.6, 1.2 Hz,
1H), 4.63 (s, 1H), 3.79-3.78 (m, 1H), 3.70 (s, 1H), 3.67-3.60 (m,
2H), 3.41(t, J = 6.0 Hz, 2H), 3.34 (t, J = 6.0 Hz, 2H), 3.06 (s,
6H), 2.26-2.22 (m, 1H), 1.97-1.88 (m, 2H), 1.78-1.73 (m, 1H),
1.58-1.54 (m, 2H), 1.50-1.45 (m, 2H), 1.43-1.38 (m, 2H), 1.28
(m, 2H), 1.20 (d, J = 6.6 Hz, 3H), 1.11 (d, J = 6 Hz, 3H); 13C
NMR (150 MHz, CD3OD) δ 170.3, 167.9, 163.1, 156.0, 153.6,
151.4, 149.2, 144.6, 125.7, 123.5, 121.9, 109.5, 108.7, 97.6, 96.4,
71.2, 70.0, 68.8, 67.2, 39.3, 39.0, 38.7, 36.9, 34.8, 31.8, 28.1,
28.0, 25.2, 18.1, 16.9; HRMS: Calculated for [C30H43O8N3Na+]:
596.2942, Found: 596.2948.
3
In this manuscript we refer to the naturally occurring
ascarosides with dauer inducing properties as daumones. There is
an effort to change the natural product class name from
“daumones” to “ascarosides”. This new naming convention
derives it name from ascarylose, the unique sugar associated with
this class of natural products. Unfortunately, the name
ascarosides is already assigned by carbohydrate nomenclature
conventions to any natural and unnatural ascarylose with a group
attached to the anomeric oxygen (i.e., ascaroside is akin to
glucoside).
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