A photocleavable masked nuclear-receptor ligand enables temporal control of C.elegans development

Article abstract of DOI:10.1002/anie.201307465

The development and lifespan of C.elegans are controlled by the nuclear hormone receptor DAF-12, an important model for the vertebrate vitaminD and liverX receptors. As with its mammalian homologues, DAF-12 function is regulated by bile acid-like steroidal ligands; however, tools for investigating their biosynthesis and function invivo are lacking. A flexible synthesis for DAF-12 ligands and masked ligand derivatives that enable precise temporal control of DAF-12 function was developed. For ligand masking, photocleavable amides of 5-methoxy-N-methyl-2-nitroaniline (MMNA) were introduced. MMNA-masked ligands are bioavailable and after incorporation into the worm, brief UV irradiation can be used to trigger the expression of DAF-12 target genes and initiate development from dauer larvae into adults. The invivo release of DAF-12 ligands and other small-molecule signals by using photocleavable MMNA-masked ligands will enable functional studies with precise spatial and temporal resolution. Copyright

Full text of DOI:10.1002/anie.201307465

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DOI: 10.1002/anie.201307465
Optogenetics
Hot Paper
A Photocleavable Masked Nuclear-Receptor Ligand Enables Temporal
Control of C. elegans Development**
Joshua C. Judkins, Parag Mahanti, Jacob B. Hoffman, Isaiah Yim, Adam Antebi, and
Frank C. Schroeder*
Abstract: The development and lifespan of C. elegans are
controlled by the nuclear hormone receptor DAF-12, an
important model for the vertebrate vitamin D and liver X
receptors. As with its mammalian homologues, DAF-12
function is regulated by bile acid-like steroidal ligands;
however, tools for investigating their biosynthesis and function
in vivo are lacking. A flexible synthesis for DAF-12 ligands
and masked ligand derivatives that enable precise temporal
control of DAF-12 function was developed. For ligand
masking, photocleavable amides of 5-methoxy-N-methyl-2-
nitroaniline (MMNA) were introduced. MMNA-masked
ligands are bioavailable and after incorporation into the
worm, brief UV irradiation can be used to trigger the
expression of DAF-12 target genes and initiate development
from dauer larvae into adults. The in vivo release of DAF-12
ligands and other small-molecule signals by using photo-
cleavable MMNA-masked ligands will enable functional
studies with precise spatial and temporal resolution.
gans, the NHR DAF-12 is a central regulator of life history; it
triggers reproductive development under favorable condi-
tions or developmental arrest at the long-lived dauer stage
when environmental conditions are unfavorable (Fig-
ure 1A).[5b,6] In addition, DAF-12 plays a major role in the
regulation of adult lifespan in response to signals from the
reproductive system.[7] DAF-12 is a homologue of the VDR,
farnesoid-X (FXR) and liver-X (LXR) receptors, and like its
vertebrate counterparts, DAF-12 function is regulated by
steroidal ligands.[5b] These DAF-12-activating steroids, collec-
tively called the dafachronic acids (DAs), feature a carboxyl-
ated side chain and varying functionalization of the steroidal
A- and B-rings.[8] We recently showed that previous hypoth-
eses concerning the endogenous ligands of DAF-12 and their
biosynthesis must be revised, and that the most prevalent
endogenous DAs include unexpected D1-desaturation and 3a-
OH hydroxylation (dafa#3 and hyda#1, respectively, see
Figure 1B).[8]
Several lines of evidence indicate that DAs serve different
functions at different time points in the wormsꢀ lifecycle[5,7f]
and that the biosynthesis of DAs occurs through different
routes in different tissues.[8b,9] These findings further increase
the significance of C. elegans as a model for vertebrate NHR
biology and associated small-molecule signaling pathways;
however, appropriate tools for investigating DA biosynthesis
and function in vivo are lacking. Further advancement of the
field will require the development of strategies that enable
the tissue-specific liberation of small molecules in live
C. elegans with precise temporal control. Herein, we intro-
duce 5-methoxy-N-methyl-2-nitroaniline (MMNA) amides as
photocleavable masking groups that are easy to attach,
biocompatible, and enable targeted release of different
DAF-12 ligands and putative biosynthetic precursors
in vivo. In addition, we report a short, flexible synthesis of
the new DAF-12 ligands (for syntheses of the previously
known dafa#1 and derivatives, see [10]).
N
uclear Hormone Receptors (NHRs) play a central role in
metazoan development and metabolism.[1] Many NHRs are
regulated by small-molecule ligands, and extensive studies of
mammalian vitamin-D receptor (VDR),[2] peroxisome pro-
liferator-activated receptors (PPARs),[3] and estrogen recep-
tors (ERs)[4] have shown that the binding of a variety of
natural and synthetic ligands can lead to different gene
expression profiles.[2,4]
The nematode Caenorhabditis elegans is a particularly
useful model organism for the study of NHR biology because
of its short lifecycle and the close homology of many of its
signaling pathways to those of higher organisms.[5] In C. ele-
[*] M. Sc. J. C. Judkins, Dr. P. Mahanti, J. B. Hoffman, I. Yim,
Prof. Dr. F. C. Schroeder
Boyce Thompson Institute and Department of Chemistry and
Chemical Biology, Cornell University
Analysis of the substitution patterns of the identified
DAF-12 ligands led us to choose lithocholic acid (1) and
chenodeoxycholic acid (3) as inexpensive starting materials
(Figure 1B). LiAlH4 reduction of 1 followed by Ag2CO3
oxidation and side-chain extension through Horner–Wads-
worth–Emmons (HWE) reaction produced intermediate 2,
which after basic hydrolysis, diastereoselective hydrogenation
with (S)-[Ru(OAc)2(H8-BINAP)],[10b] and dehydrogenation
with IBX·NMO yielded dafa#2 in only 6 steps and 32%
overall yield. Subsequent dissolving metal reduction of the
D4-double bond in dafa#2 by using Li/NH3 produced the
putative biosynthetic precursor dafa#4 (see the Supporting
Information). The synthesis of the D7-unsaturated dafa#1 and
1 Tower Road, Ithaca, New York 14853 (USA)
E-mail: fs31@cornell.edu
Homepage: http://bti.cornell.edu/schroeder
Prof. Dr. A. Antebi
Max Planck Institute for Biology of Ageing
Joseph Stelzmann Str. 9b, 50931 Cologne (Germany)
[**] We thank Maciej Kukula (BTI Mass Spectrometry Facility) for
assistance with HRMS. This work was supported in part by the
National Institutes of Health (R01 GM088290 and T32 GM008500).
Some strains were provided by the CGC, which is funded by the NIH
Office of Research Infrastructure Programs (P40 OD010440).
Supporting information for this article, including experimental
details, is available on the WWW under http://dx.doi.org/10.1002/
anie.201307465.
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12% overall yield. Subsequent treatment with IBX and TFA
introduced the D1-double bond with high regioselectivity to
yield dafa#3, whereas reduction with K-selectride produced
the 3a-OH-substituted hyda#1. A prior synthesis[10b] of dafa#1
used the same approach for the introduction of the chiral side
chain, but at a much earlier stage.
To develop inactive, photocleavable derivatives of DAF-
12 ligands or biosynthetic precursors that could be incorpo-
rated in vivo, the properties of 5-methoxy-N-methyl-2-nitro-
aniline (MMNA) amides were investigated (Figure 2). Com-
pared to the nitroindole and nitrodehydroquinoline deriva-
tives previously used for masking carboxylic acids,[12] MMNA
offers similar quantum efficiency (Table S1 in the Supporting
Information) but is commercially available and less prone to
undesired oxidative degradation. MMNA-masked variants of
dafa#1, dafa#4 (a likely intermediate in DAF-12 ligand
biosynthesis),[8b] and a simple 2-methyl-branched carboxylic
acid, (RS)-2-methylundecanoic acid, were prepared. Irradi-
ation at 365 nm resulted in nearly quantitative conversion into
the free acids, as indicated by NMR spectroscopic analysis of
Figure 1. A) Under favorable conditions, cholesterol is converted into
ligands of the nuclear hormone receptor DAF-12, thereby triggering
development into adult worms. Under unfavorable conditions, ligand
biosynthesis is abolished, DAF-12 binds to its corepressor DIN-1, and
larvae are arrested at the long-lived dauer stage. B) Synthesis of DAF-
12 ligands (dafa#1-dafa#3 and hyda#1, see www.smid-db.org for
nomenclature) and derived photocleavable MMNA-masked ligands.
a) LiAlH4, reflux; b) Ag2CO3-Celite, reflux; c) triethyl-2-phosphonopropi-
onate; LiCl, DIEA; d) LiOH; e) (S)-[Ru(OAc)2(H8-BINAP)], H2;
f) IBX·NMO; g) TMSCHN2; h) BOM–Cl, DIEA; i) IBX, TFA; j) Li, NH3;
k) Burgess reagent, reflux; l) K-selectride; m) 1: C2O2Cl2, DMF; 2: 5-
methoxy-N-methyl-2-nitroaniline, pyridine. A NOESY spectrum was
used to confirm the trans configuration of the double bond in 2 (see
Figure S1). DIEA=N,N-diisopropylethylamine, BINAP=2,2’-bis(di-
phenylphosphanyl)-1,1’-dinaphthyl, IBX=2-iodoxybenzoic acid,
NMO=N-methylmorpholine N-oxide, TMSCHN2 =trimethyl-
silyldiazomethane, TFA=trifluoroacetic acid, DMF=N,N-dimethyl-
formamide.
Figure 2. A) Irradiation of MMNA–dafa#4 at 365 nm yielded dafa#4
and byproducts 7 and 8. B) UV/Vis spectra of MMNA-masked (RS)-2-
methylundecanoic acid with increasing irradiation at 365 nm in
dafa#3 followed a similar sequence but required protection of
the 7a-hydroxy group as a benzyloxymethyl ether (BOM)
during the initial part of the synthesis. Deprotection was
concomitant with the dissolving metal reduction of the D4-
unsaturated intermediate 4 to achieve a trans-decalin config-
uration of the steroid core (Figure 1B). Oxidation with
Ag2CO3 adsorbed on Celite (Ag2CO3-Celite) yielded ketoal-
dehyde 5, which after HWE reaction, Burgess elimination,[11]
and late-stage diastereoselective reduction with (S)-[Ru-
(OAc)2(H8-BINAP)] yielded (25S)-dafa#1 in 10 steps and
1
CH3CN/H2O (3:1). C) Aromatic region of the H NMR spectra
(600 MHz, CDCl3) of MMNA–2-methylundecanoic acid before (top)
and after (bottom) irradiation, showing signals for MMNA–2-methyl-
undecanoic acid (blue arrows), 7 (red arrows), and 8 (green arrows).
Note that MMNA–2-methylundecanoic acid exists as a mixture of
three rotamers in a ratio of 1:0.83:0.32, thus resulting in three sets of
1
1H NMR signals (see Figure S3). D) Aliphatic regions of the H NMR
spectra (600 MHz, CDCl3) of dafa#4, MMNA–dafa#4 (mixture of
rotamers), and irradiated MMNA–dafa#4, indicating quantitative con-
version of MMNA–dafa#4 into dafa#4.
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the reaction mixtures (Figure 2 and Figure S2 in the
Supporting Information). We then investigated whether the
MMNA-protected DAF-12 ligands are biocompatible and
stable under physiological conditions, and whether these
derivatives could be used to liberate DAF-12 ligands or
precursors in vivo. For these studies, daf-9(dh6) mutant
worms were used, which are defective in the CYP450
enzyme that catalyzes the last step in DAF-12 ligand
biosynthesis.[6b,8a,13] As a result, daf-9(dh6) mutant worms
lack endogenous DAF-12 ligands and constitutively arrest
development as long-lived dauer larvae unless synthetic
ligands are added that trigger resumption of development
into normal adult worms (“dauer rescue”).[8a]
confirm that the irradiation of MMNA–dafa#1-treated worms
in fact triggers development through activation of DAF-12,
we used daf-9(dh6) animals that express green fluorescent
protein (GFP) under the control of the promoter of a highly
conserved microRNA, mir-84, a known DAF-12 target
involved in lifespan regulation (Figure 3B).[7c,9a] mir-84 is
strongly expressed in two rows of cells along the sides of the
worm body (the seam cells), and thus ligand-based activation
of DAF-12 in pmir-84:GFP worms leads to green fluores-
cence in the seam cells.[7c,9a] As shown in Figure 3, irradiation
of daf-9(dh6)(pmir-84:GFP) worms treated with MMNA–
dafa#1 produced strong fluorescence in the seam cells similar
to that observed following treatment with unmodified dafa#1
(also see Figures S5 and S6).
The in vivo stability of the MMNA-protected DAF-12
ligands was tested by placing arrested daf-9(dh6) worms in
growth media containing 1 mm MMNA–dafa#1 or MMNA–
dafa#4. All treated worms remained arrested for the entire
duration of the experiment (2 days), thus indicating that the
MMNA-protected dafachronic acids do not act as DAF-12
ligands and are not hydrolyzed to form free DAF-12 ligands.
Worms treated with MMNA–dafa#1 remained viable as
demonstrated by resumption of development upon UV
irradiation of the plates (Figure S4). To test whether the
MMNA derivatives are taken up by the worms and can be
used to generate active DAF-12 ligand inside the worm, we
treated arrested daf-9(dh6) worms with MMNA-masked
dafa#1, washed them extensively, and transferred them to
untreated agar plates (Figure 3A). Treated worms did not
develop and remained arrested during the entire experiment
(up to 6 days), even when using high concentrations of
MMNA-masked ligand. However, brief irradiation (365 nm,
90 sec) of arrested daf-9(dh6) worms up to 4 days after
treatment with MMNA–dafa#1 consistently triggered
resumption of development to the adult stage. These results
show that 1) MMNA-masked steroids are readily taken up by
C. elegans, 2) the MMNA derivatives are nontoxic and are
retained in the worm body for several days, and 3) brief,
innocuous irradiation is sufficient to unmask biologically
relevant quantities of active ligand inside the worm. To
These results demonstrate that MMNA-masked deriva-
tives can be used to deliver functional NHR ligands inside the
worm body with precise temporal control, and present a first
example for the light-triggered in vivo release of endogenous
small molecule signals in C. elegans. MMNA-protected DAF-
12 ligands and ligand precursors provide new tools for the
study of the signaling cascades upstream and downstream of
DAF-12,[5a,7f,14] which are of great interest for further devel-
oping C. elegans as a model for aging in higher animals.[7c,15]
Steroids that are structurally related to the DAs, for example
common bile acids,[16] may play a role in mammalian lifespan
regulation and should be similarly amenable to MMNA
derivatization. In combination with tissue-specific gene
knockouts, localized irradiation of animals treated with
MMNA-masked signaling molecules will enable the study of
tissue-specific biosyntheses and functions, one of the major
challenges in understanding small-molecule signaling in
C. elegans and other metazoans.[5b] Lastly, we report herein
an improved synthesis that provides more direct access to
newly identified and known DAF-12 ligands than previously
reported routes.[10]
Received: August 25, 2013
Revised: November 2, 2013
Published online: January 21, 2014
Figure 3. The in vivo release of dafa#1 activates DAF-12 and triggers development in ligand-deficient daf-9(dh6) mutant worms. A) Simplified
Scheme for assay. B) Left, positive control: addition of synthetic dafa#1 to arrested daf-9(dh6)(pmir-84:GFP) worms triggers seam cell
fluorescence (white arrows) and development. Center: worms treated with MMNA–dafa#1 remain arrested, even after several days, and no seam
cell fluorescence is observed. Right: worms treated with MMNA–dafa#1 initiated development upon irradiation up to 4 days after treatment and
show strong GFP expression in the seam cells (white arrows).
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A. Bethke, J. C. Judkins, J. Wollam, K. J. Dumas, A. M. Zimmer-
Keywords: amides · gene expression · photolysis ·
small-molecule signaling · steroid hormones
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