Identification of uric acid gluconucleoside-ascaroside conjugates in caenorhabditis elegans by combining synthesis and MicroED

Article abstract of DOI:10.1021/acs.orglett.0c02038

Few nucleoside-derived natural products have been identified from animals, despite the ubiquity of nucleosides in living organisms. Here, we use a combination of synthesis and the emerging electron microscopy technique microcrystal electron diffraction to determine the structures of several N³-(β-glucopyranosyl)uric acid derivatives in Caenorhabditis elegans. These noncanonical gluconucleosides further integrate an ascaroside moiety, for which we present a shortened synthetic route. The production of a phosphorylated gluconucleoside is influenced by evolutionarily conserved insulin signaling.

Full text of DOI:10.1021/acs.orglett.0c02038

pubs.acs.org/OrgLett
Letter
Identification of Uric Acid Gluconucleoside−Ascaroside Conjugates
in Caenorhabditis elegans by Combining Synthesis and MicroED
Brian J. Curtis, Lee Joon Kim, Chester J. J. Wrobel, James M. Eagan, Rubin A. Smith, Jessica E. Burch,
Henry H. Le, Alexander B. Artyukhin, Hosea M. Nelson,* and Frank C. Schroeder*
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ABSTRACT: Few nucleoside-derived natural products have been
identified from animals, despite the ubiquity of nucleosides in living
organisms. Here, we use a combination of synthesis and the emerging
electron microscopy technique microcrystal electron diffraction to
determine the structures of several N3-(β-glucopyranosyl)uric acid
derivatives in Caenorhabditis elegans. These noncanonical gluconu-
cleosides further integrate an ascaroside moiety, for which we present
a shortened synthetic route. The production of a phosphorylated
gluconucleoside is influenced by evolutionarily conserved insulin
signaling.
or nucleoside derivatives incorporating uric acid have been
he nematodes Caenorhabditis elegans and Pristionchus
Tpacificus are simple but powerful model systems for described. The only known uric acid incorporating nucleoside,
proposed to represent N3-(ribofuranosyl)uric acid (6), was
isolated in the 1960s from bovine blood and hypothesized to
function as a soluble carrier of uric acid.8−11 Uric acid serves as
a major antioxidant capturing reactive oxygen species that are
involved in diverse signaling pathways, regulating, e.g., immune
function and aging.12 Moderate levels of uric acid supple-
mentation were recently found to increase the life span in C.
elegans, possibly by activating DAF-16/FOXO downstream of
insulin/IGF-1 signaling.13 Analyzing the levels of uric acid-
derived metabolites in long-lived mutants of the insulin/IGF-1
receptor daf-2,14,15 we found that production of uglas#11 is
upregulated ∼10-fold specifically in the daf-2(e1368) allele, but
not in the daf-2(m577) allele, relative to wild-type worms,
whereas abundances of the putative building blocks glucosyl
uric acid and ascr#1 (7) as well as that of other ascarosides
were not significantly increased in daf-2 mutants (Figure 1b
and Figure S1).
human physiology and biochemistry.1−4 Over the past decade,
phenotypic screens of mutant collections and comparative
metabolomic studies have led to the discovery of an extensive
network of small molecule signals that plays a central role in
the life history of C. elegans, regulating, e.g., aging, behavior,
and development.5,6 These signaling molecules are derived
from combinatorial assembly of building blocks from all major
primary metabolic pathways, including nucleoside metabolism,
demonstrating its importance beyond the biosynthesis of the
canonical nucleosides in DNA and the different types of RNA.
The first example for this family of unusual nucleoside
derivatives is the signaling molecule npar#1 (1), which
incorporates a xyloadenosine and a glycoside of the
dideoxysugar paratose (Figure 1a).1 npar#1 (1) and other
ascaroside derivatives play a central role in regulating
development in P. pacificus and C. elegans, respectively.5,6
Comparative metabolomics of peroxisomal β-oxidation (pβo)
mutants and wild-type C. elegans7 revealed several additional
nucleoside derivatives, including nuclas#33 (2), incorporating
a 6-threonylcarbamoyl-adenosine (t6A) moiety esterified at its
3′-O-ribosyl position to ascaroside ascr#3, as well as
gluconucleoside, pugl#1 (3), featuring an N9 anomeric linkage
of 2-methylthio-N6-isopentenyladenine to glucose.
The proposed structure of uglas#1 (4) includes uric acid,
glucose, and an ascaroside moiety; however, the connectivity of
the different moieties could not be elucidated on the basis of
MS2 spectra alone and thus remained undetermined as samples
that are large enough for NMR spectroscopic characterization
could not be isolated.7 To determine connectivity and obtain
Our attention was drawn to several other gluconucleoside
derivatives from the pβo mutant screen that appeared to
incorporate uric acid. These metabolites, uglas#1 (4), and a
phosphorylated analogue named uglas#11 (5) were proposed
to feature an N9-linked uric acid moiety, in analogy to
gluconucleoside pugl#1 (3). Although uric acid is ubiquitous
as a product of purine degradation, few natural small molecules
Received: June 19, 2020
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Figure 2. (a) Silyl Hilbert−Johnson glucosylation of uric acid (8)
favors N3 substitution (10) with the N1 (11) and N7 (12) isomers as
minor products. Arrows indicate relevant HMBC correlations; weak
correlations are shown as dashed lines (also see Figures S2−S4). (b)
Aminolysis of 10−12 and subsequent comparison with C. elegans exo-
metabolome by HPLC-HRMS revealed the N3-β-isomer (13,
gluric#1) as the major glucosyluric acid metabolite in C. elegans. (c)
Initial MicroED structural solution (left) and final refined structure
with overlaid density (right).
Figure 1. (a) Unusual purine nucleoside and gluconucleosides from
C. elegans and other nematodes. (b) Structures of uglas#1 and
uglas#11 proposed on the basis of MS2 analysis and analogy to other
glucosides and gluconucleosides in C. elegans,7 with dashed bonds
referring to tentative assignments. N3-(Ribofuranosyl)uric acid (6)
has been reported from bovine blood.8−11 Amounts of uglas#1 and
uglas#11 relative to ascr#1 (7) were quantified in the wild type and
mutants of the insulin/IGF-1 receptor daf-2 (also see Figure S1).
***p < 0.001.
linked isomer 13 (named gluric#1) is abundantly produced by
C. elegans (Figure 2b).
Given that not all regioisomers 10−12 provided complete
1
HMBC spectra and H line shapes of gluric#1 (13) were too
broad to obtain additional HMBC data, we sought to
unambiguously confirm our structural assignment for 13
using microcrystal electron diffraction (MicroED). MicroED
is an emerging cryo-electron microscopy (CryoEM) technique
that has recently been demonstrated to be a powerful tool for
small molecule structure elucidation but has remained largely
unproven in natural product applications.18,19 Synthetic
gluric#1 (13), isolated by reversed-phase flash chromatography
as a white powder, was deposited on a Quantifoil holey-carbon
TEM grid. Initial electron micrographs of the samples showed
numerous microcrystalline domains, including prisms 1−3 μm
long. Continuous rotation selected area diffraction data were
collected from 104 crystals. Merging of four data sets provided
a high-resolution (1.0 Å) and high-completeness direct method
solution (R1 = 14.0%). Importantly, without refinement or any
user input (besides the molecular formula), the initial
structural solution obtained from SHELXD20 clearly confirms
N3 glucosylation, despite lacking some atoms on the sugar
fragment (Figure 2c and Figure S5). Anisotropic refinement of
this structure allowed for placement of all atoms of gluric#1
(13), providing for unambiguous confirmation of the structure.
The preference for N3 glucosylation in our synthesis is
consistent with calculated π-electron densities at purine
nitrogen atoms21 and previously reported N3 glucosylation
and ribosylation of uric acid using a silver-based silyl Hilbert−
samples for exploring biological function, we pursued a
synthetic approach that would provide access to different
structural isomers. Previous work showed that under
thermodynamic control, guanine glucosylation using the silyl
Hilbert−Johnson method favors N9 attachment, as in RNA
and DNA, for 2′-O-benzoylated substrates.16 However, when
applied to the case of uric acid (8) and protected glucose (9),
1
we obtained a mixture of three regioisomers. H−13C HMBC
spectra of the three isomers in conjunction with chemical shift
data of uric acid17 suggested that the major product
represented the N3 regioisomer (10, 39%), whereas the two
minor products were assigned as the N1-substituted (11, 9%)
and likely N7-substituted (12, 4%) regioisomers. N9
glucosylation was not observed (Figure 2a). However, not all
expected HMBC correlations could be observed in the spectra
of the N1 and N7 regioisomers, due to the broad line shapes of
some of the NH protons (Figure 2a and Figures S2−S4).
Aminolysis of the major N3 isomer (10) as well as combined
N1 and N7 isomers (11 and 12) produced samples of the three
glucosyl uric acid isomers. Comparison with C. elegans exo-
metabolome samples using HPLC-HRMS indicated that N3-
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Johnson approach.22 These results further suggested that
uglas#1 and uglas#11, like gluric#1 (13), also represent N3-β-
linked gluconucleosides, in contrast to the original proposal7
and canonical N9-linked purine nucleosides in DNA and RNA,
and in agreement with the connectivity in the previously
reported N3-(ribofuranosyl)uric acid (6).8−11
For the synthesis of the proposed 2,4-dideoxyglycoside
(ascaroside) moiety in uglas#1 and uglas#11, we employed a
new, shorter route to access the key intermediate, dibenzoy-
lated ascarylose (16).23 Methylation of α-L-rhamnose (14)
followed by tosylation using p-TsCl and Bu2SnCl2 under basic
conditions regioselectively afforded a 3-O-tosylated intermedi-
ate24,25 which was reduced upon treatment with NaH and
LiAlH4, affording 1-O-methyl ascarylose, which was sub-
sequently benzoylated with BzCl, furnishing 15. Optimized
reaction conditions prevented the formation of a branched-
chain deoxyfuranose side product derived from ring con-
traction, which had been reported for similar deoxygenation
reactions (see Methods).26 Demethylation of 15 afforded
target precursor 16, which was converted into dibenzylated
ascaroside 17 as previously described.27
To determine the position of attachment of the ascaroside
side chain to N3-(β-glucopyranosyl)uric acid (13), we first
pursued a nonselective approach. Steglich esterification of
dibenzylated ascr#1 (17)27 and gluric#1 (13) provided a
mixture of three dibenzylated uglas#1 isomers, containing the
6′-O-substituted isomer (18, 40%) as the major component,
which was separated from two minor isomers derived from
acylation of secondary alcohols (Figure 3a).
The 1H NMR spectroscopic signals of one of the two minor
isomers exhibited extreme line broadening of the glucose-
attached protons, likely due to slow interconversion of different
conformers, which prevented full NMR spectroscopic charac-
terization of this compound (Figure S6). However, dqfCOSY
spectra processed to emphasize quickly decaying signals
demonstrated 2′-O-acylation (19, 15%). The other minor
isomer was identified as the 3′-O isomer (20, 8%), whereas a
4′-O-acylated isomer was not observed. Subsequent Pd/C-
catalyzed hydrogenation of the 6′-O-substituted isomer (18)
and a 2:1 mixture of the 2′-O (19) and 3′-O (20) isomers
provided three uglas#1 isomers whose retention times and MS2
spectra were compared to that of natural uglas#1. We found
that the synthetic 2′-O isomer 21 co-eluted with C. elegans
metabolite uglas#17 and produced virtually identical MS2
spectra (Figure S7). In addition, we detected small amounts
of the 3′-O (22) and 6′-O (23) isomers, named uglas#12 and
uglas#14, respectively, in C. elegans endo-metabolome samples
(Figure 3b and Figure S8).
Figure 3. (a) Nonselective synthesis of uglas#1 isomers. (b) HPLC-
HRMS ion chromatograms showing that synthetic 2′-O regioisomer
21 co-eluted with the major natural uglas#1 isomer in C. elegans
metabolome extracts. In addition, the natural extracts contain small
amounts of the 3′-O and 6′-O isomers, 22 and 23, respectively.
Scheme 1. Selective Synthesis of uglas#1 and uglas#11
Next, we pursued selective synthesis of the major natural
isomer, 2′-O-acylated 21. gluric#1 (13) was protected as the
4′,6′-O-benzylidene derivative (Scheme 1) and then coupled
with dibenzylated ascr#1 (17) via Steglich esterification, which
produced primarily 2′-O-acylated isomer 24, in addition to a
trace of 3′-O isomer 25 (>25:1), in overall 64% yield.
Subsequent hydrogenation gave uglas#1 (21) in 83% yield.
1
The much better line shapes of the H NMR spectra of the
benzylidene derivative 24 allowed to further confirm our
earlier assignment of uglas#1 as the 2′-O-acylated isomer
(Figure S9).
To determine the position of the phosphate moiety in
uglas#11 (26) (Scheme 1), we considered the structure of
iglu#2 (27), a glucosylated indole derivative from C. elegans,
for which 3′-O-phosphorylation had been demonstrated
(Figure 4).28 Phosphitylation of 24 using dibenzyl N,N-
diethylphosphoramidite and 1H-tetrazole, followed by oxida-
tion with t-butyl hydroperoxide selectively yielded the
protected 3′-O-phosphate in 70% yield over two steps.
Subsequent deprotection of benzyl and benzylidene moieties
via Pd/C-catalyzed hydrogenation furnished uglas#11 (26),
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of uglas#11 (26) production was observed only in the daf-
2(e1368) mutant, whereas this was not observed for daf-
2(m577), which carries a mutation in a different location of the
DAF-2 ligand binding domain. Ongoing studies investigate
potential roles of uglas#1 and uglas#11 and related metabolites
for phenotypes associated with perturbation of insulin
signaling.14
ASSOCIATED CONTENT
■
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* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.0c02038.
Experimental procedures, supporting figures, supporting
tables, and NMR spectra (PDF)
Accession Codes
Figure 4. Examples of modular glucosyl metabolites/ascaroside O-
glycoside esters produced by C. elegans with the indicated variation in
substitution specificity.
CCDC 2020283 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
data_request@ccdc.cam.ac.uk, or by contacting The Cam-
bridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
whose HPLC retention times and MS2 spectra were
indistinguishable from those of natural uglas#11 in the C.
elegans metabolome (Figures S10 and S11). Using synthetic
uglas#11 as a standard, we determined that uglas#11
concentrations range from ∼25 nM in the wild type to ∼250
nM in daf-2(e1368) worms (Figures S1 and S12).
Taken together, our results show that connectivity of the
building blocks in the uglas family of metabolites deviates from
that found in canonical nucleosides and previously identified C.
elegans metabolites. Furthermore, we report a much-simplified
route for the synthesis of protected ascarylose (16), which will
greatly facilitate synthesis of diverse ascaroside-derived signal-
ing molecules.
Although NMR spectroscopic techniques provide sufficient
proof for the structures of a majority of newly discovered
natural products, the case presented here highlights the
challenges that can arise from broad line shapes or the absence
of non-exchangeable protons near heteroaromatic cores.
Moreover, growing crystals large enough for single-crystal X-
ray crystallography is frequently not feasible. MicroED
addresses these challenges, here providing unambiguous
corroboration of N3 substitution of 13, which otherwise
could only be inferred indirectly from solution-state NMR
spectra of a precursor, 10. The ability to obtain crystal
structures from microcrystals of natural products (provided as
simple powders without crystallization) will likely facilitate
identification of a wide range of novel metabolites.
Our data are in agreement with earlier reports that natural
glycosylated uric acid derivatives represent the N3-linked
isomers (e.g., in bovine blood),8−11 in contrast to many other
nucleoside derivatives described from animals, and also
contrasting with the structure of pugl#1 (3), an N9-linked
gluconucleoside from C. elegans. Notably, the N3-glucosyluric
acid moiety has not been previously characterized from natural
sources, despite the ubiquity of the building blocks uric acid
and glucose. The most abundant members of the uglas family,
uglas#1 (21) and uglas#11 (26), feature 2′-O attachment of
the ascaroside side chain, whereas all previously identified
ascarosyl glucosides are attached at position 1′ (e.g., glas#10,
28) or 6′ (e.g., iglas#1, 29).7,29 These findings suggest that
uglas#1 (21) and related modular metabolites are the products
of highly specific biosynthetic pathways. Notably, upregulation
AUTHOR INFORMATION
■
Corresponding Authors
Hosea M. Nelson − Department of Chemistry and Biochemistry,
University of California, Los Angeles, California 90095, United
States; orcid.org/0000-0002-4666-2793; Email: hosea@
chem.ucla.edu
Frank C. Schroeder − Boyce Thompson Institute and
Department of Chemistry and Chemical Biology, Cornell
University, Ithaca, New York 14853, United States;
orcid.org/0000-0002-4420-0237; Email: fs31@
cornell.edu
Authors
Brian J. Curtis − Boyce Thompson Institute and Department of
Chemistry and Chemical Biology, Cornell University, Ithaca,
New York 14853, United States
Lee Joon Kim − Department of Chemistry and Biochemistry,
University of California, Los Angeles, California 90095, United
States
Chester J. J. Wrobel − Boyce Thompson Institute and
Department of Chemistry and Chemical Biology, Cornell
University, Ithaca, New York 14853, United States
James M. Eagan − Ascribe Bioscience, Ithaca, New York 14853,
United States; orcid.org/0000-0003-4010-9353
Rubin A. Smith − Boyce Thompson Institute and Department of
Chemistry and Chemical Biology, Cornell University, Ithaca,
New York 14853, United States
Jessica E. Burch − Department of Chemistry and Biochemistry,
University of California, Los Angeles, California 90095, United
States
Henry H. Le − Boyce Thompson Institute and Department of
Chemistry and Chemical Biology, Cornell University, Ithaca,
New York 14853, United States
Alexander B. Artyukhin − Chemistry Department, College of
Environmental Science and Forestry, State University of New
York, Syracuse, New York 13210, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.0c02038
D
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Notes
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Letter
(27) Zhang, Y. K.; Sanchez-Ayala, M. A.; Sternberg, P. W.;
Srinivasan, J.; Schroeder, F. C. Org. Lett. 2017, 19, 2837−2840.
(28) Stupp, G. S.; Von Reuss, S. H.; Izrayelit, Y.; Ajredini, R.;
Schroeder, F. C.; Edison, A. S. ACS Chem. Biol. 2013, 8, 309−313.
(29) Von Reuss, S. H.; Bose, N.; Srinivasan, J.; Yim, J. J.; Judkins, J.
C.; Sternberg, P. W.; Schroeder, F. C. J. Am. Chem. Soc. 2012, 134,
1817−1824.
The authors declare the following competing financial
interest(s): F.C.S. is a founder of Ascribe Bioscience Inc.
ACKNOWLEDGMENTS
■
The authors thank David Kiemle (ESF Syracuse) for help with
the acquisition of NMR spectra and Russell N. Burkhardt and
Bennett W. Fox (Boyce Thompson Institute) for assistance
with mass spectrometry. This work was partly supported by the
National Institutes of Health (R35 GM131877 to F.C.S. and
T32GM008500 to B.J.C.) and the Howard Hughes Medical
Institute. The authors also thank the Packard Foundation
(fellowship to H.M.N.) and Bristol Myers Squibb (Unre-
stricted Grant in Synthetic Organic Chemistry to H.M.N.) for
generous support.
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