Synthesis of photoaffinity-labeled daumone analogs

Full text of DOI:10.1002/bkcs.10453

Communication
DOI: 10.1002/bkcs.10453
BULLETIN OF THE
H. Kim and Y.-K. Paik
KOREAN CHEMICAL SOCIETY
Synthesis of Photoaffinity-Labeled Daumone Analogs
Heekyeong Kim† and Young-Ki Paik†,‡,*
†Department of Biomaterials Science and Engineering, Yonsei Proteome Research Center
‡Department of Integrated Omics for Biomedical Research, Yonsei University, Seoul 120-749, Korea.
*E-mail: paikyk@yonsei.ac.kr
Received May 1, 2015, Accepted May 27, 2015, Published online August 28, 2015
Keywords: Daumone, Dauer pheromone, Caenorhabditis elegans, Radioisotope, Photoaffinity labeling
The presence of dauer pheromone activity in Caenorhabditis
elegans1,2 was first reported by Golden and Riddle in 1982.3
However, the chemical structure and biological function of
these dauer pheromones remained unknown for more than
20 years. In 2005, Paik and coworkers identified the first dauer
pheromone,4 daumone, and determined its chemical structure
and biological activity. The basic structure of daumone con-
sists of ascarylose and a short-chain fatty acid, which is now
termed ascarosides (ascr). Further research on its biosynthesis
pathway5–7 has identified more than 160 daumone analogs or
ascarosides with diverse functions8 (e.g., dauer development,
mating attraction, aggregation, dispersal, and olfaction).
C. elegans use specified neurons to recognize a variety of
environmental cues (aggregation, mating behavior, and larval
development). The C. elegans genome contains a large num-
ber of G-protein-coupled receptors (GPCRs), which are
mainly expressed in chemosensory neurons. The five different
types of chemosensory neurons (ASI, ADF, ASG, ASJ, and
ASK) have been reported to be involved in dauer formation.8
The identification of the guanylyl cyclase/daf-11,9 and two
G-protein alpha subunits/gpa-2, gpa-3,10 suggested that the
GPCRs are potential candidates for dauer pheromone recep-
tors. For example, SRBC-64/6611 and SRG-36/37,12 which
are expressed in ASK and ASI neurons, respectively, were
identified as putative daumone receptors.
photoaffinity-labeled ascr#2 probe in an indirect receptor
assay system. The cell-specific overexpression showed that
DAF-37 regulates dauer formation when expressed in ASI
neurons and regulates adult behavior when expressed in
ASK neurons. However, the structures of these putative dau-
mone receptors and their modes of action have so far not been
well elucidated (i.e., SRBC-64/66, SRG-36, SRG-37, DAF-
37, and DAF-38), suggesting that there are more daumone
receptors that need to be identified and functionally validated.
To explore the range of GPCRs involved in daumone
metabolism and signaling (e.g., daumone 1-specific receptor,
daumone transporters, etc.), we designed and chemically
synthesized both radioactive and photoaffinity14-labeled dau-
mone analogs. First, a radiolabeled daumone 1, [14C]-1b, was
synthesized. Compound [14C]-2b was prepared by coupling
the first compound with the radiolabeled [14C]-phenyl azide
photoreactive group (Figure 1).
Before the radiolabeled [1’,2’-14C] daumone 1 [14C]-1b
was synthesized, we prepared unlabeled cold compounds to
verify the viability of the synthetic route (Scheme 1). The Hor-
ner–Wadsworth–Emmons (HWE) reaction of aldehyde 6 with
triethyl phosphonoacetate was carried out by the addition of
aldehyde 6 to a solution of triethyl phosphonoacetate and
NaH in THF at 0 ꢀC to give a selective E-form 8a in 79% yield.
The hydrogenation of 8a on Pd/C in EtOAc afforded 9a in
96% yield. The hydrolysis of 9a by 3M NaOH in THF/H2O
afforded daumone 1 (1a) in 75% yield. A hot reaction using
the radioactive isotope was performed in the same manner
as described above for the cold reaction. The HWE reaction
with [1,2-14C] triethyl phosphonoacetate provided the 14C-
labeled daumone 1 ([14C]-1b) (Scheme 1).
For the synthesis of the radioactive and photoaffinity-
labeled [14C]-2b (Scheme 2), azide 9 was coupled with an
N-Boc-ethylenediamine linker to afford amide 10 in 75%
yield. In the cold reaction, the resulting amide 10 was methy-
lated with CH3I to give methyl ether 11a in 80% yield.
The Boc group of phenyl azide probe 11a was deprotected
using 50% TFA/CH2Cl2, and coupled with daumone 1 (1a) in
the presence of EDC (1-Ethyl-3-(3-dimethylaminopropyl)-
carbodiimide) and HOBt to give 2a in 18% yield. A hot reac-
tion using a radioactive isotope was carried out in the same
manner as the cold reaction. Compound 10 was methylated
with [14C] CH3I (specific activity: 54 mCi/mmol) to give
Recently, two additional GPCR proteins, DAF-37 and
DAF-38, were identified by Park et al.13 using a
OH
OH
*
O
O
*
O
O
O
O
HO
HO
OH
OH
[
14C]-1b
1a (daumone 1)
N3
*
OCH3
H
N
O
N
O
H
O
O
HO
OH
[
14C]-2b
* denotes 14
C
Figure 1. Structures of daumone 1-based radioactive and photoaffi-
nity probe derivatives.
Correction added on 01 October 2015, after first online publication: ISSN (Print) has been corrected.
Bull. Korean Chem. Soc. 2015, Vol. 36, 2177–2178
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Wiley Online Library
2177

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ISSN (Print) 0253-2964 | (Online) 1229-5949
BULLETIN OF THE
KOREAN CHEMICAL SOCIETY
N3
N3
NH
O
O
CCl3
O
O
b
R1O
R1O
a
a
b or d
O
O
O
OH
OH
BzO
BzO
BzO
BocHN
HO
O
N
H
O
OBz
OBz
OBz
5
4
6
9
10
N3
N3
OCH3
b
H
N
c
O
OR2
O
O
O
N
H
O
Cold rxn
BocHN
OCH3
O
O
d
c
O
O
O
BzO
O
HO
N
O
H
OBz
OR1
Cold rxn
OH
2a
11a
7a
8a, R1 = Bz, R2 = Et
1a, R1 = H, R2 = H
e
N3
N3
*
OCH3
d
e
H
N
O
OR2
*
*
*
O
O
*
*
Hot rxn
OCH3
O
N
H
O
O
O
O
g
BocHN
f
O
O
O
BzO
N
H
O
HO
OBz
OR1
Hot rxn
OH
[
14C]-11b
[
14C]-2b
* denotes 14
C
[
14C]-7b
[
[
14C]-8b, R1 = Bz, R2 = Et
14C]-1b, R1 = H, R2 = H
h
Scheme 2. Synthesis of radioactive and photoaffinity labeled
daumone 1 [14C]-2b. Reagents and conditions: (a) N-Boc-
ethylenediamine, EDC, HOBt, CH2Cl2, rt, 18 h, 75%; (b) CH3I,
K2CO3, acetone, reflux, 4 h, 80%; (c) i) 50% TFA, CH2Cl2, rt, 1 h;
ii) 1a (daumone 1), EDC, HOBt, DIEA, CH2Cl2, rt, 6 h, 18%;
(d) [14C] CH3I, K2CO3, acetone, reflux, 4 h; (e) i) 50% TFA, CH2Cl2,
rt, 1 h; ii) 1a (daumone 1), EDC, HOBt, DIEA, CH2Cl2, rt, 6 h.
* denotes 14
C
Scheme 1. Synthesis of radioactive labeled [10,20-14C] daumone
1 [14C]-1b. Reagents and conditions: (a) (R) - (-) -5- hexen-2-ol,
TMSOTf, 4 Å MS, CH2Cl2, -20ꢀC, 2 h, 85%; (b) i) O3, CH2Cl2,
-78ꢀC, ii) DMS, 24 h, 68%; (c) triethyl phosphonoacetate, NaH,
THF, 0ꢀC, 30 min, 79%; (d) H2, Pd/C, EtOAc, rt, 1 h, 96%;
(e) 3M-NaOH, H+, THF/H2O, rt, 48 h, 75%; (f) [1,2-14C] triethyl
phosphonoacetate, NaH, THF, 0ꢀC, 30 min; (g) H2, Pd/C, EtOAc,
rt, 1 h; (h) 3M-NaOH, H+, THF/H2O, rt, 48 h.
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the radioactive, photoaffinity-labeled daumone 1 ([14C]-2b)
(Scheme 2).
In summary, we successfully prepared radiolabeled dau-
mone 1 [14C]-1b and photoaffinity-labeled phenyl azide
[14C]-2b. The synthesized radioactive, photoaffinity-labeled
derivatives may be useful for identifying various daumone-
1 binding proteins in C. elegans. We also anticipate that the
labeled compounds will provide tools for a systematic screen-
ing of the proteins potentially involved in daumone metabo-
lism (e.g., transport, storage), which may also be broadly
present in other species (e.g., fungi, insects, and mammals).
Acknowledgment. This work was supported by a grant from
the National Research Foundation of Korea (2011-0028112
to YKP).
SupportingInformation. Additionalsupportinginformation
is available in the online version of this article.
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Bull. Korean Chem. Soc. 2015, Vol. 36, 2177–2178
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