An Ugi-like Biosynthetic Pathway Encodes Bombesin Receptor Subtype-3 Agonists

Article abstract of DOI:10.1021/jacs.9b04183

Isocyanide functional groups can be found in a variety of natural products. Rhabduscin is one such isocyanide-functionalized immunosuppressant produced in Xenorhabdus and Photorhabdus gammaproteobacterial pathogens, and deletion of its biosynthetic gene cluster inhibits virulence in an invertebrate animal infection model. Here, we characterized the first "opine-glycopeptide" class of natural products termed rhabdoplanins, and strikingly, these molecules are spontaneously produced from rhabduscin via an unprecedented multicomponent "Ugi-like" reaction sequence in nature. The rhabdoplanins also represent new lead G protein-coupled receptor (GPCR) agonists, stimulating the bombesin receptor subtype-3 (BB3) GPCR.

Full text of DOI:10.1021/jacs.9b04183

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Article
An Ugi-like Biosynthetic Pathway Encodes
Bombesin Receptor Sub-type-3 Agonists
Joonseok Oh, Nam Yoon Kim, Haiwei Chen, Noah W. Palm, and Jason M. Crawford
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b04183 • Publication Date (Web): 19 Sep 2019
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An Ugi-like Biosynthetic Pathway Encodes Bombesin Receptor
Subtype-3 Agonists
Joonseok Oh,†,‡ Nam Y. Kim,†,‡ Haiwei Chen,§ Noah W. Palm,§ Jason M. Crawford*,†,‡,∥
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† Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States
‡ Chemical Biology Institute, Yale University, West Haven, Connecticut 06516, United States
§ Department of Immunobiology, Yale School of Medicine, New Haven, Connecticut 06520, United States
∥ Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, Connecticut 06536, United States
Supporting Information Placeholder
amino acid precursor.14,15 IsnB then catalyzes an oxidative
ABSTRACT: Isocyanide functional groups can be found in a
decarboxylation reaction on the isocyanide-functionalized amino
acid.14,15 Rhabduscin is produced by the IsnAB pathway followed
by glycosylation (Figure 1).7 For the second biosynthetic route,
ScoE-type non-heme iron (II)-dependent enzymes catalyze
isocyanide formation via an oxidative decarboxylation mechanism,
and this pathway has similarly been linked to virulence in
mycobacteria.16,17
variety of natural products. Rhabduscin is one such isocyanide-
functionalized immunosuppressant produced in Xenorhabdus and
Photorhabdus gammaproteobacterial pathogens, and deletion of its
biosynthetic gene cluster inhibits virulence in an invertebrate
animal infection model. Here, we characterized the first “opine-
glycopeptide” class of natural products termed rhabdoplanins, and
strikingly, these molecules are spontaneously produced from
rhabduscin via an unprecedented multicomponent “Ugi-like”
reaction sequence in nature. The rhabdoplanins also represent new
lead G protein-coupled receptor (GPCR) agonists, stimulating the
bombesin receptor subtype-3 (BB3) GPCR.
INTRODUCTION
Bacteria belonging to the genera Xenorhabdus and
Photorhabdus participate in multipartite interactions with insects,
nematodes, and in some cases humans.1,2 To mediate mutualistic
and pathogenic their interactions with differing animal hosts,
bacteria from these genera undergo phase variation,3 and during
their pathogenic phases, they produce an assortment of specialized
metabolites, including signaling molecules, antibiotics,
immunomodulators, protease inhibitors, cytotoxins, and
phenoloxidase inhibitors among others.4,5 Both Xenorhabdus and
Photorhabdus species produce the isocyanide-functionalized
metabolite rhabduscin (1), a potent phenoloxidase inhibitor, and
deletion of the gene cluster in Xenorhabdus nematophila inhibits
virulence in an invertebrate animal infection model when the
bacterium is inoculated at physiologically-relevant doses (Figure
1).6,7
Figure 1. Rhabduscin (1) biosynthesis and its degradation
product rhabduscin-formamide (2)
Isocyanide-containing compounds are a unique family of
compounds, as they are the only stable molecules with a formally
divalent carbon.8 Since the first terrestrial isocyanide xanthocillin
was identified in Penicillium notatum in 1950,9 ~400 natural
products bearing this functionality have been characterized
predominately from marine invertebrates, although they are
sometimes isolated as their N-formamide (e.g., 2) or isothiocyanate
degradation products.10-12 Natural isocyanides can be identified via
their chemoselective reaction with tetrazine probes in biological
media.13
Isocyanide substrates have also garnered broad synthetic utility
in multicomponent reactions, such as the Passerini three-
component reaction18 and the Ugi four-component reaction.19 The
former employs a carboxylic acid, a carbonyl compound, and an
isocyanide as the three components that lead to -acyloxyamides,
whereas the latter incorporates a fourth amine substrate to yield -
acylaminoamides. Both reactions generate a new stereogenic
center, often stereoselectively.20,21
Given the biological importance of the rhabduscin pathway
during pathogenesis, we collected metabolomic data in the current
study and evaluated it for the detection of potential rhabduscin-
derived metabolites. We focused on Xenorhabdus bovienii as a
There are two established proposals for isocyanide biosynthesis.
In the first route, IsnA-type enzymes are thought to excise the C2
carbon of ribulose-5-phosphate and transfer it onto the amine of an
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representative rhabduscin producer. Under electrospray ionization
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mass spectrometry conditions (ESI-MS), rhabduscin exhibits a key
daughter ion (m/z 188.0930), corresponding to its sugar moiety. We
identified and characterized a small family of new metabolites
termed rhabdoplanins A–D (3–6) in X. bovienii that share this
daughter ion, leading to the first examples of “opine-glycopeptide”
metabolites and identification of a new type of G protein-coupled
receptor (GPCR) agonist. Based on biomimetic synthetic studies,
we also propose an unprecedented natural “Ugi-like” pathway to
establish these new metabolites.
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RESULTS AND DISCUSSION
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The rhabdoplanins were isolated using
a mass-directed
purification strategy guided by the characteristic daughter ion
(Figure S1), i.e., focusing on molecules that shared the ESI-MS
rhabduscin daughter ion. The molecules also shared a distinctive
cinnamate-type UV-visible chromophore (Figure 3 and Figure
S38), facilitating analysis. The molecular formula of the most
abundant rhabdoplanin A (3) was assigned as C45H51N5O7 based
upon its protonated high-resolution (HR)-ESI-MS mass of m/z
774.3863 (calcd [M + H]+, m/z 774.3861, Figure S1). Initial
interpretation of the NMR data (1H) suggested that 3 could be an
Scheme 1. Synthetic route to model compounds 7 and 8 for
configurational assignment.
opine scaffold.22 This was further confirmed by direct (4'-N, CH3-
N via 1H,15N HMBC denoted in purple in Figure 2B) and indirect
detection of amide protons (8-NH, 10-NH) (Table S1). Other key
2D NMR correlations shown in Figure 2 supported the planar
structure of rhabdoplanin A as 3 (Figure 2).
ordinary glycopeptide comprised of
rhabduscin, N-
We employed synthetic model standards to establish the
configuration of 3, as Marfey’s analysis23 was ineffective for this
molecule and optimization was not pursued due to the low quantity
of the purified natural product (~0.3 mg). Thus, arbitrarily selected
model compounds 7 and 8 representing diastereomers of the
aglycone of 3 were synthesized (Scheme 1) (See Supporting
Information). Upon installation of the opine core via reductive
amination using cyanoborohydride with L-Trp,24 two
diastereomeric products were separated and the configurations of
their stereogenic centers (denoted with * in Scheme 1) were
assigned as follows: each opine compound was subjected to
KMnO4 degradation to form enantiomeric 2-amino-N-phenethyl-3-
phenylpropanamide moieties and their configurations were
established as R and S via acid hydrolysis to generate respective
phenylalanine motifs followed by Marfey’s analysis (Figure
S37).23 The diastereomerically-pure compounds were used in the
subsequent synthetic steps to acquire 7 (SR) and 8 (SS). Having
acquired these two model compounds, the relative configuration of
3 was investigated by comparison of the 1H and 13C chemical shift
values of atoms comprising the flexible opine core (C-10, C-11, C-
20, C-21), using total absolute deviation (TAD) analysis (Table
S7). The analysis revealed that TAD between 3 and 8 (Σ|comp8
comp3|) showed a smaller value than that with its counterpart
-

(Σ|comp7 - comp3|)(1H 0.35 vs 1.04 ppm; 13C 3.7 vs 6.8) for both
NMR nuclei, corroborating that the relative configuration of 3 is
similar to 8. Ultimately, the absolute configuration of 3 was
established by comparison of its experimental electronic circular
dichroism (ECD) spectra with that of 8. As shown in Figure 3, the
ECD traces for both compounds support the opine-core as being
methyltryptophan, phenylalanine, and phenylethylamine as
components; however, multidimensional NMR (Figures S2–S13)
revealed a key HMBC cross-peak from H-8 to C-9, and their
corresponding chemical shift values indicated the presence of an
Figure 3. Comparison of UV (left) and ECD (right) of model
compound (8) with those of 3 for establishment of absolute
configuration.
Figure 2. (A) Full structures of 3–6; (B) Key 2D NMR
correlations for structural assignments; (C) Relative
configurational establishment of sugar motif. Dotted lines
indicate ROEs. Interproton distances are shown with
(experimental) or without parentheses (predicted). Numbering of
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enantiomeric, establishing the configuration of 3 as 10R,20R. The
precursors (Legend in Figure 4). These cellular precursors include
L- or D-N-methyl-Trp (L- or D-N-MeTrp) and phenylpyruvate (PP)
or its acyl-CoA mimic S-(2-acetamidoethyl) 2-oxo-3-
phenylpropanethioate (PP-SNAC) for formation of the opine core,
and phenylethylamine (PEA) for the installation of the terminal
motif of 3. Also, 2-oxo-N-phenethyl-3-phenylpropanamide (PPA),
the amide product of PP and PEA, was prepared to investigate the
likely coupling order for rhabdoplanin A generation. After 2d
incubation of rhabduscin in water or M9 medium with supplements
(5% casamino acids, 0.2% D-glucose, 1 mM MgSO4, 0.1 mM
CaCl2), reaction mixtures were extracted and analyzed.
Rhabdoplanin diastereomers were detected by HR-ESI-MS
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structures of the other rhabdoplanins were assigned employing
related approaches, excluding minor analogs 5 and 6, which were
supported by limited COSY/TOCSY NMR information, tandem
MS comparisons to 3, UV-visible spectra, and experimental ECD
spectra. Marfey’s analysis25 could also establish a D-allo-Ile
moietyin rhabdoplanin D (4) (See Supporting Information).
Because the isocyanide rhabduscin was a putative precursor of
the rhabdoplanins, which were formed in
a
largely
diastereoselective manner in cell culture, we proposed that the
rhabdoplanins were produced via a diastereoselective Ugi-like
multicomponent reaction sequence.8 To support this hypothesis, we
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Figure 4. Biomimetic synthesis of 3 using putative biosynthetic precursors.
conducted combinatorial biomimetic syntheses of rhabdoplanin A
(3) using four reaction groupings (groups a–d in Figure 4),
including isolated rhabduscin6 and other plausible cellular
analysis, observation of their characteristic daughter ions (m/z
188.0921 for the sugar and m/z 587.3027 for the aglycone, Figure
S1), and tandem MS comparisons to 3 (Figure S105). Their relative
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intensities are illustrated in Figure 4. These studies established
in regulating smooth-muscle contraction, exocrine and endocrine
processes, metabolism, and behavior.31 To confirm these
computational hits, we analyzed human BB3 activation using the
parallel receptor-ome expression and screening via transcriptional
output (PRESTO-Tango) assay in human HEK293 cells.32 As
illustrated in Figure 6A, rhabdoplanins A–D (3–6) activated the
receptor in a dose-dependent manner with significant stimulation
occurring between 1-10 M. QTOF-MS-based quantitative
analysis of culture supernatants shows that 3 is produced at a
physiologically relevant concentration of ∼25 μM in the
extracellular environment (Figure S110). However, the synthetic
model compounds 7 (SR) only weakly activated BB3 and 8 (SS)
partially inhibited baseline BB3 activity at 10 M (Figure 6B).
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rhabduscin as a required precursor for spontaneous rhabdoplanin
production and, as expected, its isocyanide motif was required (See
group d, Figure 4). Group a clearly displayed that each
diastereomer and 3 were generated more efficiently in the cell
medium relative to water. Also, the production was not influenced
by the configuration of MeTrp; diastereomers I–III and 3 were
observed at a ratio of 34.2 (I):8.5 (II):20 (III):37.1 (3) with D-
MeTrp and 41.0 (I):5.1 (II):17.9 (III):35.9 (3) with L-MeTrp.
According to group b, the acyl-CoA mimic PP-SNAC, which was
used to facilitate formation of the amide bond (C28–NH–C29 in 3),
did not improve the yield. Instead, its addition led to the hydrolysis
of 1 to its inactive formamide degradation product (2), presumably
by decreasing the pH. The analysis of group c, utilizing 2-oxo-N-
phenethyl-3-phenylpropanamide (PPA), implied that the
diastereoselective formation becomes more evident given the
predominant production ratio of diastereomer III and 3, compared
to the other two. This suggests that stereoselective condensation,
often observed in an Ugi reaction with chiral amines,26 occurs in
spontaneous rhabdoplanin generation. The analysis of X. bovienii
Moldova extracts (denoted as Natural in Figure 4) exhibited a
similar production pattern to that of group c. This suggests that the
formation of the amide bond of PPA occurs first followed by a
sequential Ugi-like condensation to establish the rhabdoplanins in
cell culture (Figure 5). Utilizing identical synthetic conditions with
D- or L-Trp instead of MeTrp led to the generation of demethylated
derivatives of 3 (See Supporting Information, Figure S107 and
S108). However, these analogs were not detected in X. bovienii
cultures.
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CONCLUSION
The in vitro biomimetic syntheses support
a proposed
combinatorial biosynthetic pathway of rhabdoplanins via a
rhabduscin-derived and spontaneous Ugi-like reaction sequence
(Figure 5). As discussed above, the amide bond formation between
phenylpyruvate (PP) and phenylethylamine (PEA) occurs first,
followed by imine generation via condensation with N-MeTrp. This
facilitates decarboxylation, which could explain why rhabdoplanin
A (3) production was not influenced by the configuration of the
MeTrp amino acid building block. The nucleophilic carbanion of
rhabduscin then attacks C-8 to render the opine core
diastereoselectively, which is reminiscent of Ugi reactions utilizing
chiral amines.20,21,26 Finally, simple hydrolytic oxidation of the
condensation product leads to 3.
Figure 6. Biological activities of 3–6 (natural), and 7 and 8
(synthetic model compounds) against BB3 at various
concentrations (A) and 10 M (log M = -5) (B). **P < 0.01,
****P < 0.0001
A growing number of bioactive metabolites are now known to
be formed through spontaneous chemical reaction sequences in
cells, and the products of these pathways may regulate diverse
aspects of mammalian biology.33-37 From bacteria for example, this
includes a key step in the formation of the enterotoxin tilivalline
from Klebsiella oxytoca implicated in antibiotic-associated
hemorrhagic colitis in the host.38 Conversely, a human example
includes nitric oxide signaling in inflammation, which is largely
regulated by indirect products of spontaneous chemical reaction
sequences.39 Furthermore, many products of spontaneous chemical
reactions can potentially be sensed by host receptors, including
GPCRs.40 In these cases, the organisms regulate substrate supply of
the pathways. Indeed, X. bovienii likely accumulates MeTrp, which
feeds into spontaneous rhabdoplanin biosynthesis from rhabduscin,
whereas Trp-derived rhabdoplanins were undetected in cell culture.
The intriguing isocyanide functionality in rhabduscin was critical
Figure 5. Proposed biosynthesis of the rhabdoplanins via an
Ugi-like reaction sequence. *Often stereoselectively generated
in the Ugi reaction.
To initiate biological analysis of the new opine-glycopeptide
scaffolds, we first employed a high-throughput computational
screening strategy using the established similarity ensemble
approach (SEA).27 The SEA results suggested that the
rhabdoplanins shared structural similarities with agonists of the
bombesin receptor subtype-3 (BB3) GPCR,28-30 which is involved
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to mediate the multicomponent reaction sequence to establish the
target UV trace and a higher mass range (>m/z 700), leading these
fractions to be further purified employing repetitive semi-prep
HPLC (Phenomenex Luna C18(2); isocratic elution of 15% MeCN
for 3 and 4, and 18% for 5 and 6) to purify rhabdoplanins A–D (3–
6).
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rhabdoplanins. While the mechanism proposed is distinct from the
classical Ugi reaction, rhabdoplanin formation is reminiscent of the
Ugi reaction (i.e., Ugi-like). To the best of our knowledge,
analogous multicomponent reactions have not been described
previously in natural product biosynthesis. These new metabolites
also activate the BB3 GPCR in the single-digit M-level range.
Based on these studies, we expect that functional isocyanide-
mediated multicomponent reactions may be more widespread in
nature.
In vitro Synthesis of Rhabdoplanin A (3). To implement in vitro
synthesis of 3, rhabduscin was obtained from a large cultivation of
BL21(DE3) E. coli harboring pEIsnAB-GT1760, which encodes
the rhabduscin biosynthetic gene cluster and glycosyltransferase
from P. luminescens.6,7 A 5 mL LB liquid culture with 0.1 mg/mL
of ampicillin was initiated by inoculation of a single colony of the
E. coli strain. The cultivation was incubated overnight (37 °C and
250 rpm) and 250 L of the cultivation was used to inoculate 6 × 5
mL fresh LB cultures with 0.1 mg/mL of ampicillin. These seed
cultures were similarly incubated overnight. The M9 minimal
medium (M9) and supplements (6 L) were prepared with reference
to a previous study6 and the seed cultures were utilized to inoculate
1 × 6 L M9 with 0.1 mg/mL of ampicillin. These cultures were
grown (37 °C and 250 rpm) to allow them to reach O.D. of 0.5 and
IPTG (1 mM) was added to each culture. The temperature was
dropped to 25 °C and further cultivated for 2 d. The cultivation was
concentrated and crude extract was processed utilizing the method
elaborated in section “Identification of 3–6” and rhabduscin was
purified using a previous strategy.6 In vitro synthesis was
conducted at pH 7 using commercially available precursors except
for synthetic PP-SNAC and isolated rhabduscin in Figure 3. Each
experimental set (50 L) with combination of precursors displayed
in Figure 3 was prepared in triplicates and incubated at 30 °C and
250 rpm for 2 d. After incubation, the resulting mixtures were
concentrated and analyzed to measure relative production of
rhabdoplanin A. Diastereomers I–III in Figure 4 were confirmed as
described in the main text and their ion counts were analyzed for
comparative production levels. The experiment set “Natural” was
prepared from a 2 d cultivation of X. bovienii Moldova in LB (100
mL, duplicate), which was extracted using XAD-7 HP resins in a
similar manner described in section “Identification of 3–6”. PP-
SNAC was prepared as follows41: phenylpyruvic acid (0.50 g) was
dissolved in a mixture of anhydrous THF (10 mL) and N,N-
dimethylformamide (20 uL) under N2 at 0 °C. Oxalyl chloride (0.40
mL) was added slowly and stirred for 30 min and the reaction was
warmed to r.t. Solvent and excessive oxalyl chloride were removed
to produce 0.50 g of product mixture, which was extracted with
dichloromethane three times. Without purification, acid chloride
with 10% DMAP was dissolved in anhydrous dichloromethane at
0 °C and N-acetyl cysteamine (SNAC, 0.2 g) was added. The
product mixture was extracted with EtOAc three times and the
resulting extract was subjected to RP-HPLC to purify PP-SNAC.
Two resonance forms were purified as a mixture based upon NMR
traces (Figure S99) and the in vitro synthesis was implemented as
a mixture.
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EXPERIMENTAL SECTION
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General Experimental Procedures. UV/Vis spectra were
acquired on an Agilent 1260 Infinity system equipped with a photo
diode array (PDA) detector (Agilent Technologies, CA, USA). Full
1D and 2D NMR data sets were recorded on an Agilent 600 MHz
NMR spectrometer (DD2) equipped with an inverse cold probe (3
1
mm), utilizing standard NMR pulse libraries. For H,15N-HMBC,
f1 chemical shift range was adjusted to 500 ppm and the gain value
was adjusted to 60. Flash column chromatography was performed
on LiChroprep reverse phase (RP)-18 (40–63 mm, Merck, NJ,
USA) or a Biotage-Isolera One (Biotage, Charlotte, NC, USA)
utilizing a Biotage SNAP cartridge (KP-C18-HS 120 g). LC-MS
analysis was implemented on an Agilent 1260 Infinity system using
a Phenomenex Luna C18(2) (100 Å) 5 m (4.6 × 150 mm)
(Phenomenex, CA, USA) column and a PDA detector coupled with
a single quadrupole electrospray ionization mass spectrometry
instrument (Agilent 6120). Separation and isolation of
rhabdoplanins (3–6) and synthetic analogues (7 and 8) were carried
out with an Agilent Prepstar HPLC system with an Agilent Polaris
C18-A 5 m (21 × 250 mm) column, a Phenomenex Luna C18(2)
(100 Å) 10 m (10 × 250 mm) column, and an Agilent phenyl-
hexyl 5 m (10.0 × 250 mm) column. High-resolution ESI-MS
(HR-ESI-MS) data were recorded on an Agilent iFunnel 6550
quadrupole time-of-flight (QTOF) MS instrument fitted with an
electrospray ionization (ESI) source coupled to an Agilent 1290
Infinity HPLC system. Experimental ECD spectra were recorded
on a Chirascan CD spectrometer (Applied Photophysics, Inc., MA,
USA). XAD-7 HP resins were also obtained from Fisher Scientific
(Waltham, MA, USA).
Identification of 3–6. A 5 mL Luria Bertani (LB) liquid culture
was initiated by inoculation of a single colony of Xenorhabdus
bovienii Moldova. Upon overnight growth under aerobic
conditions (30 °C and 250 rpm), the culture (250 L) was used to
inoculate 18 × 5 mL fresh LB cultures, and incubated using the
identical cultivation conditions overnight. These starter cultures
were used to inoculate 1 × 18 L of M9 medium with supplements
(5% casamino acids, 0.2% D-glucose, 1 mM MgSO4, 0.1 mM
CaCl2). These cultures were grown for two days (30 °C and 250
rpm) and then centrifuged at 14,000  g (r.t.) for 30 min. XAD-7
HP resins (20 g/L) were added to the supernatants and the mixtures
were incubated for 2 h at 37 °C and 180 rpm. The pooled filtered
resins were extracted with MeOH (18 L each) and the extract was
filtered and evaporated under reduced pressure to yield the crude
material. The crude extract (~100 g) was chromatographed over a
Biotage SNAP cartridge (KP-C18-HS 120 g) with a programmed
gradient elution method (0→100% MeOH in water for 1 h) to
generate 10 fractions (Fr.1–Fr.10). Each fraction was analyzed
using LC-MS to search for rhabduscin-based compounds based
upon the distinctive UV trace for the cinnamate motif (Figure S38)
and corresponding MS data. Potential rhabdoplanins were detected
in Fr.8 based upon LC-MS analysis and they were further purified
using prep RP HPLC equipped with an Agilent Polaris C18-A
column (5→50% MeCN in water with 0.01% TFA for 60 min, 8
mL/min, 1 min automated fraction collection). Among 60 fractions
(Fr.8.1–Fr.8.60), Frs.8.23–28 contained compounds showing the
SEA and PRESTO-Tango Assay.27,32 SEA was implemented on
the Shoichet lab server (http://sea.bkslab.org/). HTLA cells, a
HEK293 cell line that stably expresses -arrestin-TEV and tTA-
Luciferase (a kind gift from Gilad Barnea, Brown University), were
seeded in 96-well tissue culture plates in DMEM containing 10%
FBS and 1% penicillin/streptomycin. One day after plating (after
reaching approximately 90% confluence), 200 ng per well GPCR-
Tango plasmids in 20 L DMEM were mixed with 400 ng
polyethylenimine in an equal volume of DMEM and incubated for
20 minutes at room temperature before adding the transfection
mixture to the HTLA cells. 16-24 hrs after transfection, medium
was replaced with 180 L fresh DMEM containing 1%
penicillin/streptomycin and 10 mM HEPES, and 20 L indicated
concentration of compounds 3–8 were added into the supernatants.
Supernatants were aspirated 16-24 hr after stimulation and 50 µL
per well of Bright-Glo solution diluted 20-fold with PBS containing
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20 mM HEPES was added to each well. After 20 min incubation at
room temperature, luminescence was quantified using
Spectramax i3x (Molecular Devices, San Jose, CA). Activation
fold for each sample was calculated by dividing relative
luminescence units (RLU) for each condition by RLUs from media
alone controls.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
9
Publications
website.
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Experimental details including structural elucidation of 4–6 and
synthesis procedures, and NMR, and MS experimental spectra
(PDF)
AUTHOR INFORMATION
Corresponding Author
*jason.crawford@yale.edu
ORCID
Author Contributions
J.O. and J.M.C. conceived and designed the study. J.O.
characterized structure and function of metabolites by NMR,
synthesis, and computation. N.Y.K. conducted heterologous
expression studies. H.C. conducted Presto-Tango studies. N.W.P.
oversaw Presto-Tango studies. J.M.C. oversaw the project. J.O. and
J.M.C. wrote the manuscript, and all authors reviewed and edited
the final manuscript.
(21) Bock, H.; Ugi, I. Multicomponent reactions. II. Stereoselective
Synthesis of 1(S)-Camphor-2-Cis-Methylidene-Isocyanide and Its
Application in Passerini-and Ugi-reaction. J. Prakt. Chem. 1997, 339, 385-
389.
Notes
The authors declare no competing financial interests.
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Opines, Chemical Mediators of Parasitism. Phytochemistry 1993, 34, 31-
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Separation of Synthetic Nopaline and Isonopaline and Their Absolute
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Containing Major Advanced Glycation End-Products of Lysine and
Arginine. J. Pept. Res. 2005, 66, 111-124.
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Nonempirical Method Using LC/MS for Determination of the Absolute
Configuration of Constituent Amino Acids in a Peptide: Elucidation of
Limitations of Marfey's Method and of Its Separation Mechanism. Anal.
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Asymmetric Multicomponent Reactions. Chem. Soc. Rev. 2012, 41, 3969-
4009.
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ACKNOWLEDGMENT
We thank Alexandra Gatsios, Dr. Yick Chong Lam (Yale University), and
Prof. Alan Healy (NYU, Abu Dhabi) for helpful discussions. We also
recognize Dr. Daneel Ferreira (University of Mississippi) and Wesley
Yoshida (University of Hawaii) for practical advice on the study. The
authors acknowledge financial support from the National Institutes of
Health (1DP2-CA186575), the Burroughs Wellcome Foundation
(1016720), the Camille and Henry Dreyfus Foundation (TC-17-011), and
Yale University.
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