Synthesis of Deoxyradicinin, an Inhibitor of Xylella fastidiosa and Liberibacter crescens, a Culturable Surrogate for Candidatus Liberibacter asiaticus

Article abstract of DOI:10.1021/acs.jnatprod.9b01207

Pierce's disease of grapevine and citrus huanglongbing are caused by the bacterial pathogens Xylella fastidiosa and Candidatus Liberibacter asiaticus (CLas), respectively. Both pathogens reside within the plant vascular system, occluding water and nutrient transport, leading to a decrease in productivity and fruit marketability and ultimately death of their hosts. Field observations of apparently healthy plants in disease-affected vineyards and groves led to the hypothesis that natural products from endophytes may inhibit these bacterial pathogens. Previously, we showed that the natural product radicinin from Cochliobolus sp. inhibits X. fastidiosa. Herein we describe a chemical synthesis of deoxyradicinin and establish it as an inhibitor of both X. fastidiosa and Liberibacter crescens, a culturable surrogate for CLas. The key to this three-step route is a zinc-mediated enolate C-acylation, which allows for direct introduction of the propenyl side chain without extraneous redox manipulations.

Full text of DOI:10.1021/acs.jnatprod.9b01207

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Article
Synthesis of Deoxyradicinin, an Inhibitor of Xylella fastidiosa and
Liberibacter crescens, a Culturable Surrogate for Candidatus
Liberibacter asiaticus
Connor A. Brandenburg, Claudia A. Castro, Alex A. Blacutt, Elizabeth A. Costa, Kyler C. Brinton,
Diana W. Corral, Christopher L. Drozd, M. Caroline Roper, Philippe E. Rolshausen,
Katherine N. Maloney, and Jonathan W. Lockner*
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ABSTRACT: Pierce’s disease of grapevine and citrus huanglongbing are caused by the
bacterial pathogens Xylella fastidiosa and Candidatus Liberibacter asiaticus (CLas), respectively.
Both pathogens reside within the plant vascular system, occluding water and nutrient transport,
leading to a decrease in productivity and fruit marketability and ultimately death of their hosts.
Field observations of apparently healthy plants in disease-affected vineyards and groves led to
the hypothesis that natural products from endophytes may inhibit these bacterial pathogens.
Previously, we showed that the natural product radicinin from Cochliobolus sp. inhibits X.
fastidiosa. Herein we describe a chemical synthesis of deoxyradicinin and establish it as an inhibitor of both X. fastidiosa and
Liberibacter crescens, a culturable surrogate for CLas. The key to this three-step route is a zinc-mediated enolate C-acylation, which
allows for direct introduction of the propenyl side chain without extraneous redox manipulations.
ierce’s disease of grapevine and citrus huanglongbing (also
known as citrus greening) are devastating plant diseases
threatening important US agricultural industries. Both are
caused by Gram-negative bacterial pathogens, spread by way of
insect vectors. Pierce’s disease, known in California since the
was introduced to Florida in 2005, where it was able to spread
rapidly by way of its vector, the Asian citrus psyllid (in Florida
since 1998), infecting virtually all Florida citrus trees and
P
14,15
reducing production by nearly 75%.
CLas and Asian citrus
psyllids have been making their way west. The Asian citrus
psyllid was first detected in Southern California in 2008, and
the first trees tested huanglongbing positive in 2012. The
number of infected trees has been steadily increasing, and there
are now over 1700 trees that have tested positive for
1
800s, is caused by the bacterium Xylella fastidiosa, which
1
−3
inhabits the xylem of the plant.
As bacteria levels increase,
the xylem becomes blocked and the flow of water and nutrients
from the roots to the leaves is impeded, resulting in discolored
and dried leaves, shriveled fruit, and stunted vine growth,
16
huanglongbing. Notably, these huanglongbing finds have
been in backyard citrus trees, and huanglongbing has not yet
been found in commercial citrus groves.
4
−6
ultimately leading to vine death.
X. fastidiosa is transmitted
by xylem-feeding insects commonly known as leafhoppers.
Native leafhoppers are limited in their flight range and
transmission efficiency, and until recently, Pierce’s disease
outbreaks remained mostly isolated to small areas at a time.
However, in 1989, an especially effective insect vector, the
glassy-winged sharpshooter, was introduced to Southern
California. The glassy-winged sharpshooter has infested most
of Southern California, including the Malibu, Temecula Valley,
and San Diego wine regions, and has made its way north into
the Central Valley, posing an imminent threat to the California
wine, raisin, and table grape industries.
There is no known cure for either Pierce’s disease or
huanglongbing that targets the bacterium itself. A variety of
approaches are being evaluated to control infection, including
insecticide treatments to reduce insect vector populations,
developing disease-resistant vines and trees through either
breeding or genetic engineering, targeting the bacteria with
bacteriophages or other bacteria, or removing infected
17−19
plants.
Several small-molecule inhibitors of L. crescens (a
culturable surrogate for CLas) have been identified. Tolfe-
namic acid inhibits a transcriptional accessory protein in the
Huanglongbing is caused by the unculturable bacterium
7
−9
Candidatus Liberibacter asiaticus (CLas).
Unlike X.
Received: December 9, 2019
fastidiosa, CLas colonizes the phloem (food transport tissue)
of citrus trees. Huanglongbing-infected trees experience root
10,11
loss, dieback, starch buildup, and mottled yellow leaves.
Affected fruit are often green, stunted, and bitter and may drop
1
2,13
prematurely.
Huanglongbing can drastically impact the
yield productivity of a tree and can ultimately prove fatal. CLas
©
XXXX American Chemical Society and
American Society of Pharmacognosy
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2
0
bacterium. Through structure-based drug design, polycyclic
Scheme 1. Summary of Reported Syntheses of Radicinin
a
compounds (with IC values as low as 2.5 μM) were found to
(1), Deoxyradicinin (2), and Dihydrodeoxyradicinin (3)
50
21
inhibit the protein translocase ATPase subunit SecA. In a
drug repurposing approach, several tetracyclines (at 0.8−1.9
μM), cefotaxime (at 0.7 μM), and penicillin V (at 0.1 μM)
were found to provide nearly complete inhibition of L.
crescens, and recently streptomycin and oxytetracycline have
been approved for spraying on Florida citrus groves under
Section 18 emergency registration, although a recent study
indicates that oxytetracycline foliar sprays are ineffective at
2
2
17,18,23,24
controlling huanglongbing.
An interesting phenomenon observed in vineyards and citrus
groves infected with Pierce’s disease and huanglongbing,
respectively, suggested another possible avenue for fighting
these diseases. Occasionally, apparently healthy plants can be
found among the sick ones in areas of high disease pressure.
These “disease-escaped vines” (in the case of Pierce’s
a
Our approach permits access to a common intermediate in two steps
and obtention of 2 or 3 in one additional step.
2
5,26
disease)
ing)
or “survivor trees” (in the case of huanglongb-
show no or significantly reduced disease symptoms
2
7,28
Given the agricultural threats posed by X. fastidiosa and
CLas and the heavy reliance on insecticides to manage the
lethal diseases they cause, antibacterial tactics are desperately
needed. An ideal inhibitor of each bacterium (or both) would
be cost-effective to produce, nontoxic to both flora and fauna,
and easy to administer to infected plants. Secondary
metabolites derived from endophytic fungi hold great appeal,
because they are naturally endowed with many of these
properties. Here, we further promote the radicinin family of
natural products as useful antibacterials by demonstrating the
antibacterial potencies of both natural 1 and synthetic 2,
against X. fastidiosa and L. crescens (a culturable surrogate for
CLas).
and slower progression of disease relative to their neighbors.
Because both grapevines and citrus trees are clonally
propagated, the mechanisms behind these disease-escaped/
survivor phenotypes are likely not attributed to resistance/
tolerance encoded in the plant’s genome, leading us to explore
plant endophytesand their natural productsas a potential
26,29−31
source for suppressors of X. fastidiosa and CLas.
We previously reported the isolation of a Cochliobolus sp.
from disease-escaped Chardonnay grapevines in Temecula and
identified its major metabolite radicinin (1, Chart 1) as an
26
inhibitor of X. fastidiosa in vitro. To the best of our
knowledge, 1 has not yet been examined against CLas or its
culturable surrogate, L. crescens.
RESULTS AND DISCUSSION
■
Chart 1. Structures of Radicinin (1), Deoxyradicinin (2),
and Dihydrodeoxyradicinin (3)
Synthesis of Dihydrodeoxyradicinin (3), Deoxyradi-
cinin (2), and Related Compounds. In 2001, Bach
disclosed a means for securing 2-pyrones in three steps from
35
dioxinone 6. Very recently, this strategy was followed by
36
Marsico et al. for a synthesis of 2. However, this approach
requires the preparation of a trimethylsilyl-protected enol
ether, followed by Mukaiyama aldol addition and subsequent
alcohol oxidation using Dess-Martin periodinane. To minimize
step count, we preferred to avoid extraneous redox
37
manipulations. Furthermore, at the outset, our synthetic
plan emphasized dihydrodeoxyradicinin (3) as the initial target
(Scheme 2). We reasoned that late-stage oxidation of the
propyl side chain (i.e., conversion of 3 to 2) would permit us
easier passage through the earlier stage of the synthesis. Given
these considerations, we were drawn to direct enolate C-
acylation tactics, and Katritzky’s N-acyl benzotriazole tactic
In the search for antibacterials for use in agricultural settings,
synthetic approaches often complement isolation from natural
sources. The first chemical synthesis of 1 was reported in 1969.
Beginning with 3-oxoglutaric acid (4), racemic 1 was obtained
3
2
in 11 steps (Scheme 1).
Recently, a synthesis of
37−40
deoxyradicinin (2; biogenetic precursor to 1) was disclosed,
in which the target compound was obtained in five steps from
stood out among the array of options.
Among the virtues
of N-acylbenzotriazoles is their ease of preparation, bench
33
41
commercially available 5. A caveat of this approach is the
relatively high cost ($93.50 per gram) of this starting material.
Moreover, these syntheses employ expensive and/or toxic [e.g.,
SeO , Pb(OAc) ] reagents. Even the most recently reported
stability, and even commercial availability.
Our three-step synthesis of dihydrodeoxyradicinin (3)
commenced with C-acylation of the lithium enolate of
dioxinone 6 with N-acylbenzotriazole 11 (derived from butyric
acid) (Scheme 2). After column chromatography, the keto-
dioxinone 12 was refluxed in anhydrous toluene to elicit an
electrocyclization cascade, ultimately affording pyrone 10. This
intermediate precipitated out of the toluene solution, which
provided for convenient isolation via filtration. Pyrone 10 was
2
4
synthesis of 2 (which came to light while the present article
was in preparation) suffers from nonideal economies of
synthesis, including recourse to an extraneous redox
3
4
manipulation. In order for an antibacterial agent to achieve
widespread use in agricultural settings such as vineyards or
citrus groves, an economical synthesis is needed. One aim of
our work was to develop just such a synthesis of 1 and 2
then treated with crotonic acid in the presence of ZnCl and
2
42
POCl3 to afford the desired annulation product 3. Although
this sequence represents the most concise approach to 3, we
(Scheme 1).
B
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a
Scheme 2. Details for Our Syntheses of 3, 2, and 15
Gratifyingly, this turned out to be the case, and we were able to
complete our three-step synthesis of deoxyradicinin (2) in a
manner akin to that of 3.
Having secured a three-step synthetic route from 6 to 2, we
next evaluated a few tactics for α-hydroxylation (i.e., 2 → 1).
In the pioneering 1969 synthesis, 2 was converted to 1 using
superstoichiometric Pb(OAc) followed by acetate hydrolysis.
4
Preferring to avoid the use of this toxic reagent, we screened
46−49
several iodine-based oxidation protocols.
We wish to
make mention of only the salient observations from that effort.
In all instances, unchanged 2 was recovered to some extent.
Exposure of 2 to phenyliodine(III) diacetate (PIDA) in
methanolic KOH afforded side-product 15, a consequence of
ring-opening of 2 (Scheme 2). Exposure of 2 to PIDA in
refluxing TFA/H O/CH CN afforded ca. 25% conversion of 2
2
3
to 3-epi-radicinin.
To complement our synthetic efforts, we evaluated
conditions for chiral supercritical fluid chromatographic
separation of the enantiomers of our synthetic racemic 3 as
well as our synthetic racemic 2. In lieu of synthetic radicinin
(
1), we made use of natural radicinin ((−)-1) isolated from
Alternaria radicina (vide infra). With these compounds in
hand, we were able to perform antibacterial assays.
Bioassay against X. fastidiosa and L. crescens.
Compound 1 is known to inhibit X. fastidiosa in a dose-
26
dependent fashion, and we tested it, deoxyradicinin (2), and
dihydrodeoxyradicinin (3) against X. fastidiosa and L. crescens.
Having observed high potency of racemic 2 against L. crescens
(
vide infra), we performed chiral supercritical fluid chromatog-
raphy (SFC) to obtain enantioenriched (−)-2 and (+)-2 for
follow-up evaluation.
a
Bt = benzotriazol-1-yl; LDA = lithium diisopropylamide; LiHMDS =
lithium hexamethyldisilazide; PIDA = phenyliodine(III) diacetate.
Against X. fastidiosa, 1 and 2 exhibit a comparable dose−
response relationship (Figure S1). While 2 (racemic, synthetic
in origin) is more potent than 1 (enantiopure, isolated from A.
radicina) at lower doses, 1 provided complete inhibition
(diameter = 10 cm) at the highest dose tested (2.12 μmol).
Notably, 3 shows activity only at doses greater than 0.53 μmol,
and even at the highest dose tested (2.12 μmol), it affords only
a modest (diameter = 3.4 cm) level of inhibition. Thus, it
appears that the propenyl side chain (serving as a conjugate
acceptor) is vital for maintaining potency.
Against L. crescens, 1 and 2 again exhibit a comparable dose−
response relationship, with both showing greater potency
against L. crescens than against X. fastidiosa (Figure S2).
Compared to 1 and 2, compound 3 exhibited attenuated
potency.
were unsuccessful in oxidizing 3 to 2 via the limited screening
effort undertaken. Thus, we opted to pursue a three-step
sequence for 2 that paralleled the synthesis of 3.
Initially, we encountered a pitfall in our first step toward
deoxyradicinin (2). Despite prior success in obtaining keto-
dioxinone 12 en route to 3, we were unsuccessful in isolating
desired keto-dioxinone 14 using the original protocol for C-
acylation. We reckoned that competing processes preclude the
formation of 14 from 6 and 13. The N-acylbenzotriazole 13
(
derived from crotonic acid) bears acidic hydrogens, and it
presents a further liability as a relatively unhindered α,β-
unsaturated carbonyl moiety. Meanwhile, the lithium enolate
of 6 is sufficiently basic to elicit undesired proton exchange
with 13. Making matters worse, the desired product 14 also
bears acidic α-hydrogens, further stifling the success of this
transformation. Fortunately for us, this is a problem that has
been encountered in other contexts, and we were delighted to
identify a solution that permitted us to proceed with our
planned synthesis of 2. In 1989, Morita introduced the notion
of using an organozinc aid in alkylation and acylation of
lithium enolates, demonstrating that “the presence of
dimethylzinc in the reaction of lithium enolates and electro-
philes effectively suppresses undesired α-proton exchange
reaction and enhances the efficiency of enolate alkylation
While 1 and its analogues have previously been reported to
26
possess antibacterial activity against X. fastidiosa, the capacity
of 1−3 to inhibit L. crescens has not been detailed until now. As
a note, the sample of 1 tested here was (−)-radicinin isolated
from Alternaria radicina, whereas compounds 2 and 3 were
synthesized as racemates. Racemic 2 as well as enantioenriched
(−)-2 and (+)-2 were found to potently inhibit L. crescens
(Figure S1).
We previously demonstrated the capacity of 1 and
derivatives with the conjugate acceptor intactbut not
derivatives lacking the conjugate acceptorto deactivate X.
fastidiosa proteases, consistent with enzymatic nucleophilic
4
3
and acylation.”
Since then, others have turned to
26
alkylzincates (or zinc enolates) for C-acylations that were
addition to 1. Thus, we expected 2 to inhibit X. fastidiosa.
Our bioassay data confirm this in X. fastidiosa and expose a
similar vulnerability in L. crescens (Figure S1). Comparable
degrees of inhibition of L. crescens by enantioenriched samples
38,39,44,45
impossible with lithium enolates.
In our case, an
alkylzincate (or zinc enolate) species derived from 6 is
sufficiently less basic than the lithium enolate of 6, permitting
the formation and isolation of desired keto-dioxinone 14.
5
0
of (−)- and (+)-deoxyradicinin suggest that enzyme
C
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Figure 1. Minimum inhibitory concentration (MIC) for deoxyradicinin (2) against L. crescens. Theoretical dose refers to the applied concentration
of 2. Due to the poor aqueous solubility of 2, the actual concentration was significantly lower (see text and Figure S4). Kan, 1.5 μg/mL kanamycin
control; LCr, untreated L. crescens; mBM7, bBM7 + 1.0 mβc sterile growth media.
5
1
inactivation is not stereospecific, a finding that bodes well
from a developmental perspective.
Laboratories, Inc., Tewksbury, MA, USA) or DMSO-d (Sigma-
6
Aldrich) at 400 and 100 MHz, respectively, on a JEOL JNM-
ECZ400S NMR spectrometer (JEOL, Ltd., Akishima, Tokyo, Japan).
All spectra were referenced to residual solvent: 7.26 and 77.06 ppm
Our previously published work on radicinin suggested an
antibacterial mode of action involving enzyme (i.e., protease)
inhibition via conjugate addition to an unhindered α,β-
unsaturated carbonyl, and our bioassay results with 2 and
other analogues are consistent with this. Bioassay data on
additional related compounds further suggests that the rigid
bicyclic scaffold and bidentate 1,3-dicarbonyl are important
features influencing antibacterial potency (Chart S1 and
Figures S1 and S2).
Minimum Inhibitory Concentration of Deoxyradici-
nin. To further quantify the inhibitory effects of deoxyradi-
cinin against L. crescens, we performed an inhibition assay in
liquid bBM7 + 1.0 mβc medium and found the minimum
inhibitory concentration (MIC)the concentration at which
no measurable bacterial growth occursat a theoretical dose
of 8 μg/mL (Figure 1). However, the poor solubility of
deoxyradicinin in aqueous media resulted in a discrepancy
between the theoretical (applied) dose and the actual
1
13
1
13
for H and C NMR in CDCl ; 2.50 and 39.53 ppm for H and
C
3
26
NMR in DMSO-d . Analytical and preparative thin layer chromatog-
6
raphy (TLC) were performed on glass-backed silica gel plates. TLC
plates were visualized by exposure to ultraviolet light and
subsequently stained with p-anisaldehyde solution followed by
heating. Flash column chromatography was performed on silica gel
(0.040−0.063 mm, 230−400 mesh). Chiral supercritical fluid
chromatography was performed on an SFC-PICLab HT with Open
Bed fraction collection (PIC Solution SAS, Avignon, France).
Dimethyl sulfoxide (DMSO), ethyl acetate (EtOAc), and methanol
(
MeOH) were used as solvents for filter disc preparation.
Preparation of 11. To a solution of benzotriazole (28.6 g,
53
2
40.4 mmol) in CH Cl (300 mL) was added SOCl (7.1 g, 60.1
2
2
2
mmol) at 25 °C. After 30 min, butyric acid (5.3 g, 60.1 mmol) was
added in one portion. After 2 h, the white precipitate was removed via
filtration and rinsed with CH Cl (2 × 50 mL). The filtrate was
2
2
washed with 2 M NaOH (3 × 360 mL), dried over Na SO , filtered,
2
4
and concentrated in vacuo. The residue was purified by flash column
chromatography (silica gel; 4:1 hexanes−EtOAc) to yield 1-(1H-
benzo[d][1,2,3]triazol-1-yl)butan-1-one (11) as an off-white solid
(
measured) dose of soluble deoxyradicinin. As determined
by LC-MS, an average of 44.8% of deoxyradicinin was
solubilized in bBM7 + 1.0 mβc. Specifically, a dose of 8 μg/
mL yields a concentration of solubilized deoxyradicinin of 3.5
0.3 μg/mL (Figure S3). Thus, the MIC of soluble
deoxyradicinin is 3.5 ± 0.3 μg/mL.
(
10.01 g, 88%): mp 59−60 °C (2-propanol); R = 0.6 (4:1 hexanes−
f
1
EtOAc); H NMR (400 MHz, CDCl ) δ 8.30 (d, J = 8.2 Hz, 1 H),
3
8
.11 (d, J = 8.2 Hz, 1 H), 7.65 (t, J = 7.8 Hz, 1 H), 7.50 (t, J = 7.8 Hz,
±
1
H), 3.40 (t, J = 7.3 Hz, 2 H), 1.94 (m, 2 H), 1.10 (t, J = 7.5 Hz, 3
13
H); C NMR (100 MHz, CDCl ) δ 172.6, 146.2, 130.4, 126.1, 120.2,
3
1
13
114.5, 100.0, 37.4, 18.0, 13.7. H NMR and C NMR data are
54
EXPERIMENTAL SECTION
consistent with those found in the literature.
■
3
7,38
Preparation of 12.
To a solution of N,N-diisopropylamine
General Experimental Procedures. All reactions were carried
out in flame-dried glassware with magnetic stirring under an argon
atmosphere, unless noted otherwise. ACS reagent grade or anhydrous
dichloromethane (CH Cl ), tetrahydrofuran (THF), and toluene
were used without further purification. Compound 6 was obtained
from Acros Organics (Morris Plains, NJ, USA). Compound 16 was
obtained from Sigma-Aldrich (St. Louis, MO, USA). Compound 5
(3.2 mL, 22.5 mmol) in anhydrous THF (60 mL) was added n-
butyllithium (2.5 M in hexanes, 9.9 mL, 24.8 mmol) dropwise over 20
min at −78 °C. To this lithium diisopropylamide (LDA) solution was
added a solution of 6 (2.4 mL, 17 mmol) in THF (60 mL) dropwise
over 15 min at −78 °C. After 1.5 h, a solution of 11 (2.9 g, 15 mmol)
in THF (60 mL) was added, and the reaction was warmed to room
temperature overnight. The reaction was quenched with saturated
2
2
52
was prepared in two steps from 16 according to published methods.
Melting points were measured on an Electrothermal 1101D Mel-
Temp digital melting point apparatus. Optical rotations were
measured on an Atago Polax-2L polarimeter. Infrared spectra were
aqueous NH Cl (6 mL) and concentrated in vacuo to afford a golden-
4
brown syrup. Water (300 mL) was added to the syrup, and it was
transferred to a separatory funnel for extraction with EtOAc (3 × 150
mL). The organic layers were combined, washed with saturated
aqueous Na CO (300 mL), dried over MgSO , filtered, and
1
recorded on a Thermo Scientific Nicolet iS10 FTIR spectrometer. H
and 13C NMR spectra were recorded in CDCl (Cambridge Isotope
3
2
3
4
D
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1
concentrated in vacuo. The residue was purified by flash column
chromatography (silica gel; 1:1 hexanes−EtOAc) to yield 2,2-
EtOAc); H NMR (400 MHz, CDCl ) δ 6.92 (dq, J = 15.6, 6.9 Hz, 1
3
H), 6.16 (m, 1 H), 5.36 (s, 1 H), 3.44 (s, 2 H), 1.94 (m, 3 H), 1.69 (s,
6 H); C NMR (100 MHz, CDCl ) δ 192.5, 165.2, 161.0, 145.6,
1
3
dimethyl-6-(2-oxopentyl)-4H-1,3-dioxin-4-one (12) as a colorless oil
3
1
1
13
(
1.21 g, 38%): R = 0.5 (1:1 hexanes−EtOAc); H NMR (400 MHz,
130.9, 107.4, 96.8, 44.7, 25.1, 18.5; H NMR and C NMR data are
35
f
CDCl ) δ 5.34 (s, 1 H), 3.31 (s, 2 H), 2.48 (t, J = 7.2 Hz, 2 H), 1.71
s, 6 H), 1.67−1.61 (m, 2 H), 0.93 (t, J = 7.4 Hz, 3 H); C NMR
100 MHz, CDCl ) δ 203.3, 164.7, 160.8, 107.2, 96.6, 47.1, 45.0, 25.0,
consistent with those found in the literature.
32
3
1
3
(
(
Preparation of 9. A 0.1 M solution of 14 (0.228 g, 1.09 mmol)
in anhydrous toluene (10.9 mL) was stirred under reflux for 10 min.
The white precipitate was collected via filtration and washed with
toluene (2 × 10 mL), yielding (E)-4-hydroxy-6-(prop-1-en-1-yl)-2H-
pyran-2-one (9) as an off-white solid (0.124 g, 75%): mp 183−185
3
1
6.9, 13.5.
Preparation of 10. A 0.1 M solution of 12 (1.11 g, 5.23 mmol)
3
2
in anhydrous toluene (52.3 mL) was stirred under reflux for 1 h. The
white precipitate was collected via filtration and washed with toluene
1
°C; R = 0.3 (10:1 CH Cl −MeOH); H NMR (400 MHz, DMSO-
d ) δ 11.63 (s, 1 H), 6.46 (dq, J = 15.6, 6.9 Hz, 1 H), 6.15 (dd, J =
15.6, 1.4 Hz, 1 H), 5.98 (d, J = 1.8 Hz, 1 H), 5.22 (d, J = 1.8 Hz, 1
H), 1.82 (dd, J = 6.9, 1.4 Hz, 3 H); C NMR (100 MHz, DMSO-d )
δ 170.4, 163.1, 159.0, 133.8, 123.4, 99.8, 89.4, 18.1; H NMR and
NMR data are consistent with those found in the literature.
Preparation of Deoxyradicinin (2).
(0.4 mL, 4.4 mmol) and ZnCl (0.3 g, 2.5 mmol) were added crotonic
f
2
2
(
2 × 20 mL), yielding 4-hydroxy-6-propyl-2H-pyran-2-one (10) as an
6
1
off-white solid (0.637 g, 79%): R = 0.3 (10:1 CH Cl −MeOH); H
NMR (400 MHz, CDCl ) δ 11.33 (brs, 1 H), 6.01 (d, J = 2.1 Hz, 1
f
2
2
1
3
3
6
C
1
13
H), 5.59 (d, J = 2.1 Hz, 1 H), 2.45 (t, J = 7.5 Hz, 2 H), 1.70−1.61 (m,
1
3
35,58
2
1
H), 0.94 (t, J = 7.4 Hz, 3 H); C NMR (100 MHz, CDCl ) δ
3
1
13
42,56
72.9, 168.7, 167.2, 101.6, 89.9, 35.6, 20.2, 13.5; H NMR and
NMR data are consistent with those found in the literature.
Preparation of Dihydrodeoxyradicinin (3).
C
To a solution of POCl3
55
2
4
2,56
To a solution
acid (0.05 g, 0.6 mmol) and 9 (0.097 g, 0.6 mmol) at 85 °C with
stirring for 4 h. The reaction was quenched with a small handful of ice
and Na CO (10% aq w/v %, 15 mL). The product was extracted
of POCl (2.4 g, 15.3 mmol) and ZnCl (1.2 g, 8.8 mmol) were added
3
2
crotonic acid (0.2 g, 2.19 mmol) and 10 (0.3 g, 2.2 mmol) at 85 °C
with stirring for 4 h. The reaction was quenched with a small handful
of ice and Na CO (10% aq w/v %, 50 mL). The product was
2
3
with CH Cl (2 × 40 mL). The organic layers were combined, dried
2
2
over Na SO , filtered, and concentrated in vacuo. The residue was
2
3
2
4
extracted with CH Cl (2 × 100 mL). The organic layers were
purified by flash column chromatography (silica gel; CH Cl followed
2
2
2 2
combined, dried over Na SO , filtered, and concentrated in vacuo.
by 10:1 CH Cl −MeOH) to afford (E)-2-methyl-7-(prop-1-en-1-yl)-
2
4
2
2
The residue was purified by flash column chromatography (silica gel;
CH Cl followed by 10:1 CH Cl −MeOH) to afford 2-methyl-7-
2,3-dihydropyrano[4,3-b]pyran-4,5-dione (2) as a dark brown solid
1
(0.091 g, 63%): R = 0.4 (10:1 CH Cl −MeOH); H NMR (400
2
2
2
2
f
2
2
propyl-2,3-dihydropyrano[4,3-b]pyran-4,5-dione (3) as a light brown
MHz, CDCl ) δ 6.99−6.87 (m, 1 H), 6.02 (dd, J = 15.4, 1.6 Hz, 1 H),
3
1
solid (0.253 g, 52%): R = 0.4 (10:1 CH Cl −MeOH); H NMR (400
5.83 (s, 1 H), 4.80−4.69 (m, 1 H), 2.70−2.58 (m, 2 H), 1.95 (dd, J =
f
2
2
1
3
MHz, CDCl ) δ 5.88 (s, 1 H), 4.79−4.72 (m, 1 H), 2.71−2.58 (m, 2
H), 2.47 (t, J = 7.5 Hz, 2 H), 1.71 (sextet, J = 7.2 Hz, 2 H), 1.54 (d, J
6.3 Hz, 3 H), 0.98 (t, J = 7.3 Hz, 3 H); C NMR (100 MHz,
7.0, 1.6 Hz, 3 H), 1.53 (d, J = 6.4 Hz, 3 H); C NMR (100 MHz,
3
CDCl )δ 187.5, 177.1, 164.5, 155.5, 141.2, 118.2, 99.9, 93.1, 76.7,
3
1
3
1
13
=
39.1, 20.9, 17.8; H NMR and C NMR data are consistent with
32,59
CDCl ) δ 186.6, 176.0, 172.3, 158.1, 100.0, 99.1, 77.4, 43.8, 36.4,
those found in the literature.
Preparative chiral SFC furnished
3
1
13
2
0.4, 19.9, 13.5; H NMR and C NMR data are consistent with
those found in the literature.
separate samples of enantioenriched (−)-2 and (+)-2. Method
conditions: Chiralpak AS-3, 4.6 × 100 mm, 3 μm; 20% isocratic in 2.0
min; 20 mM ammonium formate in MeOH; 3.5 mL/min; 160 bar; 25
°C; APCI (+); 3.0 μL injection.
3
2,57
Analytical chiral SFC method
screening identified a suitable means for separating (−)-3 and (+)-3.
Method conditions: Chiralpak AD-3, 4.6 × 100 mm, 3 μm; 10%
isocratic in 5 min; 20 mM ammonium formate in MeOH; 3.5 mL/
min; 160 bar; 25 °C; APCI (+); 1.0 μL injection. Preparative-scale
separation was not done for 3.
4
6
Preparation of 15. To a solution of KOH (0.056 g, 1.0 mmol)
in MeOH (2 mL) at 0 °C was added 2 (0.022 g, 0.10 mmol) over 10
min. Phenyliodine(III) diacetate (0.064 g, 0.20 mmol) was added
over 10 min, and the reaction allowed to warm to room temperature
overnight. The reaction was quenched with 0.1 M HCl (0.2 mL). The
product was extracted with CH Cl (2 × 10 mL). The organic layers
5
3
Preparation of 13. To a solution of benzotriazole (28.6 g,
2
40.4 mmol) in CH Cl (300 mL) was added SOCl (7.1 g, 60.1
2
2
2
mmol) at 25 °C. After 30 min, crotonic acid (5.2 g, 60.1 mmol) was
added in one portion. After 2 h, the white precipitate was removed via
filtration and rinsed with CH Cl (2 × 50 mL). The filtrate was
2
2
were combined, dried over Na SO , filtered, and concentrated in
2
4
vacuo. The residue was purified by flash column chromatography (1:1
hexanes−acetone) to afford 3-((E)-but-2-enoyl)-4-hydroxy-6-((E)-
prop-1-en-1-yl)-2H-pyran-2-one (15) as an off-white solid (11 mg,
2
2
washed with 2 M NaOH (3 × 360 mL), dried over Na SO , filtered,
2
4
and concentrated in vacuo. The residue was purified by flash column
chromatography (silica gel; 4:1 hexanes−EtOAc) to yield (E)-1-(1H-
benzo[d][1,2,3]triazol-1-yl)but-2-en-1-one (13) as an off-white solid
1
50%): R = 0.4 (1:1 hexanes−acetone); H NMR (400 MHz, CDCl )
f
3
δ 7.64 (d, J = 15.3 Hz, 1 H), 7.34−7.23 (m, 1 H), 6.92 (dq, J = 15.2,
7.0 Hz, 1 H), 6.03 (d, J = 15.5 Hz, 1 H), 5.88 (s, 1 H), 2.02 (d, J = 6.9
Hz, 3 H), 1.96 (d, J = 6.9 Hz, 3 H); C NMR (100 MHz, CDCl ) δ
(
9.96 g, 89%): mp 91−93 °C (2-propanol); R = 0.6 (4:1 hexanes−
f
1
13
EtOAc); H NMR (400 MHz, CDCl ) δ 8.35 (d, J = 8.2 Hz, 1 H),
3
3
8
2
1
.12 (d, J = 8.5 Hz, 1 H), 7.73−7.59 (m, 1 H), 7.57−7.43 (m, 3 H),
193.0, 183.2, 163.4, 160.8, 147.4, 140.2, 128.3, 122.9, 100.8, 99.6,
19.1, 18.9.
1
3
.12 (d, J = 5.5 Hz, 3 H); C NMR (100 MHz, CDCl ) δ 163.6,
3
1
50.0, 146.3, 131.4, 130.2, 126.2, 121.4, 120.1, 114.8, 19.9; H NMR
Isolation of (−)-Radicinin (1). (−)-Radicinin was obtained from
13
53
and C NMR data are consistent with those found in the literature.
the fermentation of Alternaria radicina (ATCC 96831) shaken at 190
3
7,38
1
Preparation of 14.
To anhydrous THF (84 mL) was added
rpm in potato dextrose broth for 24 days. H NMR data are consistent
26,32,59
hexamethyldisilazane (8.8 mL, 42 mmol) at −78 °C. n-Butyllithium
with those found in the literature.
Analytical chiral SFC
(
16.8 mL, 42 mmol, 2.5 M in hexanes) was added dropwise over 10
confirmed the enantiopurity of (−)-1 (see Supporting Information).
Method conditions: Chiralpak AD-3, 4.6 × 100 mm, 3 μm; 40%
isocratic in 5 min; 20 mM ammonium formate in MeOH; 3.5 mL/
min; 160 bar; 25 °C; APCI (+); 1.0 μL injection.
Antibacterial Assays. Compounds were evaluated using an in
vitro assay of their ability to inhibit X. fastidiosa or L. crescens growth.
Compounds were dissolved in DMSO, EtOAc, or MeOH and applied
to sterile filter discs (Difco) to achieve desired doses. Culture
techniques for each bacterium are described below. After incubation
with compound-loaded filter discs at 28 °C for 5 to 7 days, the
diameters of clear zones of inhibition were measured and recorded.
Each assay was performed in triplicate, and the average diameter of
inhibition reported. Compounds that afforded significant inhibition at
min. After 20 min, a solution of 6 (4 mL, 30 mmol) in THF (12 mL)
was added dropwise over 10 min. After 1 h, diethylzinc (42 mL, 42
mmol, 1.0 M in hexanes) was slowly added. After 20 min, the mixture
was warmed to −20 °C. A solution of 13 (6.75 g, 36 mmol) in THF
(
18 mL) was added, and stirring was continued for 2 h. The reaction
was quenched with 1 M HCl (240 mL), and the aqueous layer was
acidified to pH 1−2 using 1 M HCl. The product was extracted with
EtOAc (2 × 300 mL). The organic layers were combined, dried over
MgSO , filtered, and concentrated in vacuo. The residue was purified
by flash column chromatography (silica gel; 3:1 hexanes−EtOAc) to
yield 2,2-dimethyl-6-[(3E)-2-oxopent-3-en-1-yl]-2,4-dihydro-1,3-diox-
4
in-4-one (14) as a colorless oil (1.016 g, 16%): R = 0.5 (1:1 hexanes−
f
E
https://dx.doi.org/10.1021/acs.jnatprod.9b01207
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
higher doses were tested at lower doses to further characterize dose
response.
Authors
Connor A. Brandenburg − Department of Chemistry, Point
Loma Nazarene University, San Diego, California 92106,
United States
Claudia A. Castro − Department of Microbiology and Plant
Pathology, University of California, Riverside, California 92521,
United States
Alex A. Blacutt − Department of Microbiology and Plant
Pathology, University of California, Riverside, California 92521,
United States
Xylella fastidiosa Inhibition Assay. Pierce’s disease medium
60
(
PD3) top agar (0.8% agar) was prepared, cooled to 60 °C, and
amended 10% v/v with 6-day-old X. fastidiosa liquid culture (PD3, 28
C, 180 rpm shaking), and the OD600 was adjusted to 0.1. This
°
amended top agar was then dispensed to evenly coat previously
poured PD3 agar plates. After 2 days of incubation at 28 °C,
compound-loaded filter discs were placed in the center of the plates.
Liberibacter crescens Inhibition Assay. bBM7 + 1.0 mβc top
61
agar (0.8% agar) was prepared, cooled to 60 °C, and amended 10%
v/v with 4-day-old L. crescens liquid culture (mBM7, 28 °C, 180 rpm
shaking). This amended top agar was then dispensed to evenly coat
previously poured bBM7 + 1.0 mβc agar plates, and compound-
loaded filter discs were placed in the center of the plates. See Jain et
Elizabeth A. Costa − Pfizer Inc., La Jolla, California 92121,
United States
Kyler C. Brinton − Department of Chemistry, Point Loma
Nazarene University, San Diego, California 92106, United
States
Diana W. Corral − Department of Chemistry, Point Loma
Nazarene University, San Diego, California 92106, United
States
Christopher L. Drozd − Department of Microbiology and Plant
Pathology, University of California, Riverside, California 92521,
United States
M. Caroline Roper − Department of Microbiology and Plant
Pathology, University of California, Riverside, California 92521,
United States
62
al. for justification for the use of L. crescens as a culturable surrogate
for CLas.
Minimum Inhibitory Concentration Assay for Deoxyradici-
nin against L. crescens. The minimum inhibitory concentration was
calculated for deoxyradicinin in liquid bBM7 + 1.0 mβc as described
31
previously. In brief, deoxyradicinin was dissolved in methanol, filter
sterilized, and added to a 96-well plate to give concentrations from 0.5
to 10 μg/mL. Methanol was evaporated off in a biosafety cabinet
overnight. The following day, 150 μL of sterile bBM7 + 1.0 mβc was
added to the wells, along with 50 μL of 4-day-old liquid culture
(
1
OD600 nm ∼0.27) of L. crescens. The plate was incubated at 28 °C at
50 rpm. Absorbance was read at 600 nm using an Infinite 200 Pro
plate reader (Tecan Group Ltd., Switzerland) daily for 11 days. The
lowest dose at which no growth was observed was recorded as the
MIC. Each treatment had four technical replicates.
In Vitro Solubility Assay for Deoxyradicinin. For quantifica-
tion of solubilized deoxyradicinin, 96-well plates containing bBM7 +
Philippe E. Rolshausen − Department of Botany and Plant
Sciences, University of California, Riverside, California 92521,
United States
Katherine N. Maloney − Department of Chemistry, Point Loma
Nazarene University, San Diego, California 92106, United
States; orcid.org/0000-0003-3472-7593
1
.0 mβc media were prepared as described above, but with no
bacteria. Three concentrations were tested for solubility: 7.5, 8, and
8
.5 μg/mL. Plates were incubated at 28 °C at 150 rpm for 48 h and
50 μL aliquots were analyzed via LC-MS. Prior to LC-MS analysis,
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.9b01207
1
protein was precipitated with methanol. After centrifugation at
1
6000g for 15 min at 4 °C, supernatant was analyzed by LC-MS. LC-
Notes
MS was performed at the UC Riverside Metabolomics Core Facility
on a Synapt G2-Si quadrupole time-of-flight mass spectrometer
The authors declare no competing financial interest.
(
Waters) coupled to an I-class UPLC system (Waters). Separations
ACKNOWLEDGMENTS
were carried out on a CSH phenyl-hexyl column (2.1 × 100 mm, 1.7
μM) (Waters). The mobile phases were (A) water with 0.1% formic
acid and (B) acetonitrile with 0.1% formic acid. The flow rate was 250
μL/min, and the column was held at 40 °C. The injection volume was
1
4
was operated in positive ion mode (50 to 1200 m/z) with a 100 ms
scan time. Source and desolvation temperatures were 150 and 600 °C,
respectively. Desolvation gas was set to 1100 L/h and cone gas to 150
L/h. All gases were nitrogen except the collision gas, which was argon.
Capillary voltage was 1 kV. Samples were analyzed in random order.
Leucine enkephalin was infused and used for mass correction. Data
were analyzed using QuanLynx software (Waters).
■
We gratefully acknowledge Point Loma Nazarene University
PLNU) for supporting this work through research grants from
(
the Department of Chemistry and Research Associates. This
research was also funded by the California Department of
Food and Agriculture (CDFA) Pierce’s Disease/Glassy-
Winged Sharpshooter (PD/GWSS) Board (Grant #1687),
and the United States Department of Agriculture (USDA
Project No. CA-R-PPA-5139-CG). We also gratefully acknowl-
edge Lia Lozano and Jennifer Cordoza of PLNU for providing
−)-radicinin (1) for bioassay and Dr. Chambers Hughes and
Trevor Purdy of the Scripps Institution of Oceanography for
assistance in acquiring mass spectrometry data.
μL. The gradient was as follows: 0 min, 1% B; 1 min, 1% B; 8 min,
0% B; 8.5 min, 100% B; 10 min, 100% B; 10.5 min, 1% B. The MS
(
ASSOCIATED CONTENT
sı Supporting Information
■
*
REFERENCES
■
The Supporting Information is available free of charge at
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Supplemental figures, H and 13C NMR spectra, and
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Corresponding Author
Jonathan W. Lockner − Department of Chemistry, Point Loma
Nazarene University, San Diego, California 92106, United
States; orcid.org/0000-0001-6148-0579;
Email: jlockner@pointloma.edu
F
https://dx.doi.org/10.1021/acs.jnatprod.9b01207
J. Nat. Prod. XXXX, XXX, XXX−XXX

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G
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