C-Glycosyl Compounds in the Synthesis of the Phytotoxin Diplopyrone

I. C-Glycosides
C-glycosyl compounds, frequently referred to as “C-glycosides,” are compounds in
which the exo-glycosidic carbon-oxygen bond has been replaced with a carbon-carbon bond.1-3

Examples of naturally occurring C-glycosides are aloin, also known as barbaloin, which occurs
in various species of aloe, the antitumor aryl C-glycoside ravidomycin, and the nucleoside
antibiotic pyrazomydin (Figure 1). The chemistry and biological properties of C-glycosides have
long been of interest owing to their stability toward glycosidase enzymes; replacement of the
exocyclic carbon-oxygen bond with a carbon-carbon bond results in a hydrolytically stable
carbohydrate mimetic that may possess interesting applications in biological studies and in
medicine as therapeutics.4 Synthetic C-glycosides that have potent activity as enzyme inhibitors.5

The C-glycoside analog of GDP-L-fucose competitively inhibits fucosyltransferase.6 Iminosugar

C-glycosides inhibit several enzymes including glycosidases and they have been used clinically
in the treatment of lysosomal storage disorders. 7 In addition to their unique biological properties,
C-glycosyl compounds have also been utilized as intermediates in the asymmetric synthesis of
non-carbohydrate targets from carbohydrate starting materials, providing access to natural
products in enantiomerically pure form.8
Marine polyethers such as brevitoxin and the anticancer drug Halaven® have been
synthesized using pathways that rely on a pyranopyran core structure that is accessible from C-

1
glycosides.8 In this proposal, approaches to the synthesis of the fungal toxin diplopyrone are
described that involve a number of problems based on the chemistry of C-glycosides. Our main
objectives are the synthesis of the fungal toxin in both its natural and enantiomeric forms, along
with analogs for biological testing. Related objectives include the development of synthetic
methods based on glycosyl sulfones, new routes for pyranopyran synthesis, chain-extension, and
methods for C-vinyl glycoside synthesis.

II. (+)-Diplopyrone: Background and Significance

Diplopyrone is a phytotoxin isolated from the fungus Diplodia mutila and reported by
Evidente and coworkers in 2003.9,10 An endophytic fungus, meaning it can reside in the host
plant for a period of time before causing damage, D. mutila is considered to be responsible for
cork oak decline in parts of southern Europe where the disease has large and negative economic
and environmental impacts. Diplopyrone causes necrosis and wilting of cork oak (Quercus
suber) cuttings and is considered the main phytotoxin involved in the disease. Strains of this
fungus also infect other oak species as well as cypress; however, it is possible that the
diplopyrone from D. mutila may possess antimicrobial activity against these other strains.11 The
structure of diplopyrone was published on the basis of spectroscopic data and theoretical
calculations of its optical properties. Naturally occurring diplopyrone is dextrorotatory. To date,
there are no syntheses of diplopyrone, and natural material is scarce. As noted by the authors in
their paper on its structure: “the interesting potential applications of diplopyrone, produced in
very low amounts in the culture filtrates, require the availability of an efficient and
enantioselective synthesis.”10 Such a synthesis would not only provide confirmation of structure,
but it would also make material available for bioassays including those aimed at establishing
structure-activity relationships through the use of diplopyrone isomers and analogs.

III. Design and Retrosynthesis

Diplopyrone (1) is 6-[(1S)-1-hydroxyethyl]-2,4a(S),6(R),8a(S)-tetrahydropyrano[3,2-b]-
pyran-2-one. The “pyranopyran” bicyclic core of diplopyrone is found in many natural products
for which carbohydrates have proved to be well suited as starting materials in synthesis.8 The
first objective of this proposal is the synthesis of enantiomeric diplopyrone (2).

Why synthesize the enantiomer first? There are two reasons for the choice of the enantiomer as
the initial target. First, as will be detailed in this section, viewing diplopyrone as a “C-glycoside
problem” opens up multiple synthetic routes that are based on common D sugars and some well
established protecting group chemistry that can be relied on to expedite the synthesis. The
second reason, less practical in nature but enticing nonetheless, stems from phytochemistry. It
is well known that stereoisomers of phytotoxins may possess different biological acitivity. 11,12
For example, the dihydroxynaphthaleneone (-)-regiolone is produced by Botrytis fabae, which is
a much more virulent pathogen than Botrytis cinerea, which produces the enantiomeric

2
phytotoxin (+)-isosclerone.13 Enantiomers of the phytotoxic lichen metabolite usnic acid possess
different activity, with (-)-usnic acid having the greatest phytotoxic activity. 14 Will enantiomeric
diplopyrone exhibit the same necrotic effects on Quercus suber as the natural material? Will it
exhibit inverse agonism by blocking the action of (+)-diplopyrone, or antimicrobial activity
against strains of D. mutila that infect other species? Synthetic derivatives of the phytotoxin
papyracillic acid revealed antibacterial and antifungal activity for this class of compounds and
allowed structure-activity relationships to be established.15 Fungal phytotoxins may as useful
lead compounds in the development of environmentally benign herbicides. or in the selection of
trees that are resistant to disease, prompting further interest in the synthesis of enantiomerically
pure phytotoxins.12,15
Part of the appeal to a carbohydrate-based strategy for the enantioselective synthesis of
diplopyrones lies in the mutltiple pathways can be developed from simple starting materials.
Viewing the target from the standpoint of C-6 Wittig chain-extension, defined here as a Type I
approach (Scheme 1), allows for formation of the pyranopyran core by intramolecular
esterification involving a chain-extended substrate 3, which is a “higher-carbon sugar.” Many
methods have been developed for the synthesis of higher-carbon sugars by chain extension. 16 In
this approach, the diplopyrone chirality centers at C-4a and C-8a are derived from C-5 and C-4
of the carbohydrate starting material, D-galactose. In an alternative approach, the pyranopyran
core of diplopyrone can potentially be formed from ring-closing metathesis (RCM) using a vinyl
glycoside such as 4 (Scheme 1, Type II approach). Using a Type II approach, the chirality
centers at C-6 and C-8a as numbered in diplopyrone can be seen to originate from C-6 and C-2
of either D-glucose or D-galactose. C-glycosidation is required at C-4a (diplopyrone numbering)
in the RCM-based synthesis.

Scheme 1. C-Glycoside-based approaches to diplopyrone

3
 The approaches outlined in Scheme 1 present opportunities for the development of new
synthesis methodology. An example is the preparation of the C-vinyl glycoside required in the
RCM (Type II) approach. The many applications of C-vinyl glycosides in organic chemistry
include their use as precursors to carbon-linked glycoconjugates such as analogs of ceramide
immunostimulants17 and nucleoside phosphonates,18 as well as higher-carbon cyclic carbohydrate
mimetics.19,20 Unlike their C-allyl glycoside counterparts, simple, unsubstituted C-vinyl
glycosides have proved to be more difficult to synthesize, as is well illustrated by their absence
in the examples cited in recent approaches to C-alkyl, C-aryl, and (substituted) C-vinyl
glycosides, some of which are based on modern, organocatalytic methods.21,22 Later in this
proposal, the synthesis of C-vinyl glycosides will be revisited as it applies to our approach to
diplopyrone. The synthesis of diplopyrone by the Wittig chain-extension approach (Type I) will
focus our attention on the development of glycosyl sulfones, nitriles, or alkynes as precursors to
hydroxyethyl C-glycosides, as outlined in the following section.

IV. Type I Approaches: Glycosyl Sulfones, Nitriles, and Alkynes

In 2008 our laboratory published a method for the preparation of glycosyl phenylsulfones
by the addition of benzenesulfinic acid to glycals. 23 Our method was based on a reaction
reported by Ley24 which we modified with the use of a different Lewis acid catalyst (Scheme 2).
The addition of benzenesulfinic acid to tri-O-acetyl-D-galactal by this method occurred in 93%
yield to give exclusively the -anomer of the sulfone. With the alkene properly located in the
product and the anomeric center functionalized for the next step, crystalline sulfone 5, prepared
in high yields by this method, seemed like a versatile precursor to the hydroxyethyl side-chain in
dipolopyrone using the C-glycosidation outlined in Scheme 1. Reductive coupling of the
glycosyl sulfone with acetaldehyde in the presence of samarium diioddie (samarium-Barbier
reaction) could in principle provide access not only to the hydroxyethyl side of diplopyrone but
also to a series of analogs through variation of the carbonyl acceptor in the coupling. 25,26 We
were unable to achieve this coupling to obtain 6, most likely due to reduction of the aldehyde
acceptor. It should be noted that the Beau method relies on anomeric 2-pyridyl sulfones, which
may be expected to behave differently than the phenyl sulfones we prepared. We would like to
revisit this approach in the course of these proposed studies. The synthesis of the 2-pyridyl
sulfone analog of 5 will be attempted by Ferrier rearrangement with 2-pyridyl sulfide, and
subsequent oxidation of the sulfide to the sulfone as described by Beau, or by the direct addition
of 2-pyridyl sulfinic acid. Alternatively, the phenyl sulfone may undergo reductive
desulfonation with lithium or sodium naphthenide and in situ alkylation with an aldehyde to give
a hydroxyalkyl derivative. There are two issues of stereochemistry in the synthesis of 8 by this
approach, one involving the anomeric center and the other the off-ring center. Based on our
experience with additions to galactose derivatives, we would expect the -anomer of 8 to be
favored; however, the off-ring center is not as easily predictable. We have been able to determine

4
the stereochemistry at a newly created off-ring chirality center of a higher-carbon galactose
derivative.27 If 2-pyridyl sulfones or phenyl sulfones are successful as precursors to the C-
hydroxyethyl side-chain, we would expand on this approach to synthesize analogs by using
different aldehyde or ketone acceptors in the samarium-Barbier coupling.

Scheme 2. Glycosyl sulfones and nitriles as precursors to C-hydroxyethyl glycosides

In other preliminary studies in our lab, alternative routes to the C-hydroxyethyl glycoside
were attempted based on glycosyl nitriles. The Ferrier rearrangement of tri-O-acetyl-D-galactal
in the presence of trimethylsilyl cyanide provided crystalline glycosyl nitrile 9 in 60% yield on a
20 gram scale (Scheme 2).28,29 Again, as in the case of the glycosyl sulfones, the nitriles seemed
suitable as precursors for the hydroxylethyl side-chain, by either of two sequences, one involving
reduction of the nitrile 10 to the corresponding aldehyde 11 followed by methylation, and the
other consisting of prior methylation of 10 to give a methyl ketone, followed by reduction to give
12. In spite of many attempts the outcomes of these nitrile-based approaches were not
promising. The addition of methyllithium to 10 followed by hydride reduction gave an alcohol
of undetermined configuration in low yield and poor reproducibility, while attempted DIBAL
reduction gave complex mixtures. Aside from the expected issues of epimerization at the
anomeric carbon of 11 and the methyl ketone leading to 12, there were likely also problems with
the double bond shifting into conjugation in these substrates. Compound 10 and the carbonyl
compounds derived from it such as 13 are also susceptible to elimination, as evidenced by the
formation of diene 14, which was detected in the reaction product of methyllithium addition to 8.

5
 Some success using the Type I approach to diplopyrone was realized in the construction
of the lactone ring while leaving the nitrile intact (Scheme 3). Selective removal of the tert-
butyldimethyl (TBDMS) protecting group from the C-6 hydroxyl in 8 was achieved with
methanol and an acid catalyst to give 15, which was oxidized to aldehyde 16 by the Parikh-
Doering method.30 The reaction of 16 with (carbethoxylmethylene)triphenylphosphorane under
conditions described by Valverde and coworkers gave ester 17 exclusively as the Z isomer.31
The cyclization of 17 to nitrile 18 proved more difficult than expected, perhaps because the
double bond in the pyranose ring of 17 places the C-4 hydroxyl group and ester group in poor
proximity for ring closure. We were able nonetheless to obtain 18 by this sequenc in 34%
overall yield from 8.32 Compound 18, termed diplopyrone nitrile, is an interesting material in
its own right and during the course of our research we plan to prepare additional quantities of 18
by this method for biological testing by the assay described later in this proposal. We will also
again examine transformations of the nitrile in 18 such as reduction to the aldehyde since 18 may
be less prone to the side-reactions we observed for 8.

Scheme 3. Synthesis of diplopyrone nitrile

As a precursor to the hydroxylethyl side-chain of diplopyrone, an alkyne may also serve

to be useful provided that hydration and reduction can be carried out. (Scheme 4). Using a
method described by Isobe, tri-O-acetyl-D-galactal will be converted to TMS-alkyne derivative
19 in one pot by a Ferrier-type reaction.32-34 Hydration of 19 followed by reduction of the
resulting methyl ketone by classical methods would provide the target C-glycosyl product;
however, it is possible that the methyl ketone may be problematic for the reasons noted earlier.
An attractive alternative is the catalytic reductive-hydration described by Herzon. 35 Alkenes are
compatible with the reaction conditions but acetate esters are not, so deacylation will likely occur
(by transesterification with propanol which is one of the reagents). Since the TMS-protected
alkynes are acceptable substrates for the procedure, it may be possible to obtain 20 from 19
directly. Benzylidenation and protection of the side-chain hydroxyl in 20 gives 21, which should
be suitable for further elaboration to introduce the lactone ring.

6
Scheme 4. Side-chain introduction by Alkyne Reductive Hydration

V. Type II Approaches: Allyl, Alkynyl, and Vinyl Glycosides

Synthesis of diplopyrone based on a Type II approach opens up the possibility to use a
ring-closing methathesis (RCM) to form the bicylic pyranopyran core. Recent results from our
laboratory on the synthesis of an RCM precursor are described below. The main challenges in
this approach are the introduction of the hydroxyl ethyl side-chain with the appropriate
stereochemistry and the synthesis of a vinyl glycoside for building the lactone ring by RCM. We
have solved the first of these problems through the use of an organotitanium reagent, as shown in
Scheme 5 and described below.
We decided to introduce the hydroxyethyl side-chain early in the synthesis for two
reasons. In terms of design, we considered that a non-chelation controlled methylation of
aldehyde 22 would provide 24 stereoselectively. Our previous studies of the addition of
organometallic reagents to pendodialdo-1,4-furanoses revealed high levels of Felkin-Anh
stereoselectivity when methyl(triisopropoxy)titanium was used instead of methyllithium or
Grignard reagents.36 Additionally, NMR data published by Lemieux for diastereomers 23 and 24
would allow for structural confirmation. 37 In our study, commercially available 1,2:3,4-di-O-
isopropylidene--D-glalactopyranose was oxidized with catalytic TEMPO in the presence of
bis(acetxoy)iodo-benzene (BAIB) on a five-gram scale to give unstable aldehyde 22 which was
used immediately in the next step.38 This oxidation was also be carried out using a polymer-
bound reagent,39 which gave improved yields on smaller (1-2 mmol) scales, but turned out to be
less desirable for scaled-up preparations of 22. Straightforward chromatographic purification
gave material of high purity for use in the addition of organometallic reagents.

It should be noted that the addition of organometallic reagents to aldehyde 22 has

provided products that have found use as building blocks for trisaccharides used to study
monoclonal antibodies,37 enzyme inhibitors,40 and higher-carbon sugars.16 However, most of the
addition reactions of organometallic reagents to 22 provide mixtures of diastereomers. In need of
a highly stereoselective route to 24, we decided to examine the addition of
methyl(triisopropoxy)titanium to 22 and were able to achieve a d.r. of 19:1 in favor of desired
24.27 Confirmation of the structure of 24 was provided by x-ray analysis of the p-bromobenzoate
25. Previously, the highest 24:23 ratios reported were on the order of 2.5:1 to 3:1. 41 Multi-gram
quantities of 24 were prepared by the route shown in Scheme 5.

7
Scheme 5. Stereoselective chain-extension with methyltitanium

The stereoselective synthesis of 24 solved one of the two problems of our approach to (-)-
diplopyrone using RCM for the lactone formation, the other being the required C-glycosidation
at C-4a in the diplopyrone structure. For this part of the synthesis, we decided to rely on the use
of allyl glycosides because of the successful synthesis of ladder ethers and other complex targets
with a pyranopyran core in which C-allyl glycosides were key intermediates. 8 Protection of the
alcohol in 24 was straightforward and protecting group manipulations leading to 27, although
inefficient, did provide material to probe the key C-glycosidation step (Scheme 6). Treatment of
27 with allyltrimethylsilane and a Lewis acid gave exclusively the -anomer of 28, along with
unreacted starting material.42 We encountered a serious problem in the attempted alkene
isomerization of 28 to give 29, in that a significant amount of propenylidene glycoside 30 was
obtained, using methods which were expected to produce only minor amounts of this material. 43
While we were able to solve this issue through the use of Grubbs II catalyst for the
isomerization,44 the poor yields in the preceding steps remain problematic and need to be
addressed. Other conditions for the cleavage of isopropylidene groups in compounds similar to
26 have been reported and will be attempted here. We can also attempt to improve the C-
allylation using a modification Kishi which demonstrated that higher yields of C-allylation can
be obtained if the acetate is first converted to a p-nitrobenzoate.45 Provided that selective
deacylation C-1 of 27 can be achieved under mild conditions,46 it will be possible to obtain a
more reactive substrate for C-allylation.
Remaining steps from the C-propenyl glycoside 29 to both a dihydroxylated analog of (-)-
diplopyrone and (-)-diplopyrone itself are outlined in Scheme 7. Precedent exists for alkene
methathesis using a C-(1-propenyl)glycoside from the work of Franck on the synthesis of C-
glycosyl ceramides.17 Conversion of the propenyl to a vinyl glycoside was also possible by cross
metathesis with Grubbs II catalyst in an atmosphere of ethylene. The end-game from this
approach provides access to (-)-diplopyrone as well as dihydroxylated analog 34 from 33. The
steps leading from 33 to (-)-diplopyrone are removal of the isopropylidene protecting group and

8
reductive elimination of the 3,4-di-O-methanesulfonate derivative of the resulting diol. 47 This
stereoselective synthesis of enantiomeric (-)-diplopyrone would confirm the proposed structure
for the phytotoxin and provide material for biological testing. Synthesis of the natural, (+)-
diplopyrone is addressed at the end of this proposal.

Scheme 6. C-glycosidation with allyltrimethylsilane

Scheme 7. Proposed synthesis of (-)-dihydroxydiplopyrone and (-)-diplopyrone

9
 The synthesis of (-)-diplopyrone outlined in Schemes 6 and 7 would be greatly improved
if a more efficient route to an RCM precursor could be developed. A C-vinyl analog of 28
would be more desirable for this purpose than C-allyl, but there are few methods available for
the synthesis of simple unsubstitued vinyl glycosides. Scheme 8 illustrates some of the methods
that have been described for the synthesis of C-vinyl and related C-glycosyl pyranosides,17,20-22,32-
34,48-49
and additional examples are included in a review by Linhardt. 50 Most of these methods are
indirect, requiring several steps, and in some cases appear to work with narrow choices of
protecting groups. Also noteworthy is the apparent lack of success of the catalytic methods in
entries 2 and 3 as a means to synthesize unsubstituted vinyl glycosides, which was specifically
noted by the authors of the method in entry 2. Entry 3 is intriguing in the sense that previous
studies by Ley showed that 2-benzenesulfonyl cyclic ethers could be directly substituted with
vinylmagnesium bromide in the presence of zinc bromide to give C-vinyl compounds directly.24
Adapting this approach to the glycosyl sulfone 5 can be investigated as an approach to a C-vinyl
glycoside. If successful, the resulting 1,4-diene 35 would be subjected to regioselective oxidation
at the less-substituted olefin to give 36. Selective epoxidation51 of the less-substituted double
bond followed by reductive ring opening of the epoxide in 36 would provide the required
hydroxyethyl side-chain of diplopyrone.

In view of the problems experienced with the use of C-allyl glycosides in our (-)-
diplopyrone synthesis, the last two methods (entries 5 and 6) based on alkynes are more
attractive than the method in entry 4. Recent developments in the addition of TMS acetylene
derivatives to glycals and glycosyl acetates34,52 suggest that a sequence consisting of C-
alkynylation followed by Lindlar reduction would provide a suitable RCM precursor. One
approach would be to convert glycosyl acetate 27 to the alkynyl glycoside 37 (Scheme 9).
Chemoselective reduction of the alkyne in the presence of the less reactive benzyl ether,
followed by acylation of the C-2 hydroxyl group leads to RCM precursor 38. Successful RCM
will provide 33, for transformations to (-)-diplopyrone and dihydroxy analog 34, as outlined in
Scheme 6.
An alternative approach based on glycals is proposed based on a recent report by Isobe
on the C-alkynylation of glycals with bis(trimethylsilyl)acetylene. 34 If successful, this synthesis
(Scheme 10) will provide an efficient route to desmethyldiplopyrone, beginning with glycal 39
which is readily available from acetobromoglucose or acetobromogalactose by the reported
large-scale prep.53 The glycosidation of 39 with bis(trimethylsilyl)acetylene as described,
followed by sodium borohydride reduction as described gives exclusively alkynyl alcohol 40.
Selective deprotection with DBU54 gave diol 41, which was converted to RCM precursor 43, as
shown. Cyclization of 43 occurred smoothly to give desmethyldiplpyrone acetate 44. An
immediate objective of this research will be to scale up the preparation of 44 and pursue the
remaining steps leading to (-)-diplopyrone. These consist of deacylation, oxidation, and
methylation of the resulting aldehyde.

10
Scheme 8. Methods for the synthesis of C-vinyl glycosides

11
Scheme 9. Synthesis of (-)-diplopyrone based on a C-alkynyl glycoside

Scheme 10. Synthesis of desmethyldiplopyrone

12
VI. (+)-Diplopyrone
Carbohydrates are attractive starting materials for the proposed syntheses of (-)-
diplopyrone and its analogs in enantiomerically pure form, since two of the four required
chirality centers are incorporated from the starting sugars. The starting carbohydrates for these
syntheses are the common D sugars galactose and glucose and the diplopyrones obtained would
be enantiomeric to the natural product that was isolated by Evidente. What about the synthesis
of natural, (+)-diplopyrone? As outlined in this section, the synthesis of (+)-diplopyrone can be
accomplished from L-sugars, which have become more readily available recently through
improved syntheses from D-sugars. Owing to the symmetry of galactose, interchange of groups
at C-1 and C-6 results in enantiomeric structures (Scheme 11). Several syntheses of L-galactose
from D-galactose have been based on this concept, including the preparation of di-O-
isopropylidene derivative 46.55 The recent 6-step synthesis reported by Okamoto utilizes
straightforward reactions and requires no chromatographic purification of any intermediates. 56 L-
Galactose can be used in either of our proposed Type 1 or Type approaches to diplopyrone.

Scheme 11. Aldose interchange for galactose

The synthesis of L-glucose can be carried out from sodium -D-glucoheptonate, using the
method of Jenkinson. This method, title “short and sweet” by the authors, produces L-glucose in
five steps in multi-gram quantities with no purifications of intermediates required.57 Suitable for
undergraduates, this method could also be adapted to explore a method for C-vinyl glycoside
synthesis as shown in Scheme 12. Conversion of the known aldehyde (R=H or protecting group
if necessary) 47 to the acetylenic alcohol 48, followed by tosylation and deprotection should give
the C-alkynyl pyranoside 50. This type of sequence has been used previously for the synthesis of
an alkynyl -C-riboside.58,59

Scheme 12. Synthesis of C-alkynyl L-glucose

13
VII. Summary of Targets and New Synthesis Methodology
The proposed syntheses of diplopyrone and its structural analogs from carbohydrates rely
on the further development of synthetic methods for natural products that contain a pyranopyran
core. Briefly, these methods can be grouped into the following categories:

 Glycosyl sulfones as precursors to C-glycosides

 Stereoselective alkylation of carbohydrates using alkyl titanium reagents
 Synthesis of the pyranopyran core of diplopyrone using cis-selective Wittig chain
extension/lactonization and ring-closing metathesis
 Synthesis of C-vinyl glycosides

While described in the context of a specific natural product synthesis, we anticipate that these
methods will find application in the solution of other problems in which carbohydrates may be
viewed as suitable intermediates for access to novel structures. A summary of the targets that
will be pursued in this research is given in Table 1.

Table 1. Summary of Synthetic Targets

VIII. Biological Testing of Synthetic Targets

            Diplopyrone isolated from Diplodia mutila was assayed for phytotoxic activity
using cuttings from cork oak and tomato seedlings.10  A leaf puncture assay method with tomato
and cork oak leaves was used to test afritoxinones obtained from Diplodia africana and showed
that changes in the stereochemistry of the phytotoxins and other structural changes produced
different levels of necrosis.60 These assays are straightforward to carry out and will be conducted
with assistance from the Biology Department at Villanova using the synthetic targets prepared in
our laboratory. Specifically, the work will be examining the effect of our compounds on the
generation of leaf necrosis in tomato seedlings. Villanova University has a large greenhouse as
well as growth chambers that control temperature, humidity, light and photoperiod. Tomato
leaves from seedlings (~1 month old) will be micro-punctured with a needle and then exposed to
20 l droplets of various concentrations of compounds dissolved in sterile water (4% methanol).

14
The size of lesions will be photographed and measured regularly via ImageJ software over the
next two weeks and compared to application of control solutions. The assay is similar to the one
described by Evidente.60 Dr. Dennis Wykoff, Professor of Biology, will assist with the
development of these assays, and he has a background in plant biology. Initially, the assay will
be developed with commonly available tomato seedlings (Marmande variety); however, it is
likely that a few genetically diverse varieties will be tested to optimize the genetic background of
the Lycopersicon esculentum for the given compounds. Depending on the results, the assay will
be developed for the host plant (cork oak) as well; however, tomato seedlings appear to be the
easiest first step for developing this necrosis assay.

IX. Broader Impacts

The pyranopyran core structure addressed in this proposal occurs in the natural product
diplopyrone, the causative agent in cork oak decline, and also in numerous natural products,
many with biological activity including antimicrobial activity. The methods developed for the
synthesis of diplopyrone will impact a broad range of interesting natural products that contain
this common scaffold, not only in terms of synthetic methodology, but also through the
availability of materials, both natural and non-natural, for further study.
In terms of student participation in this research, it is noted that the P.I. has a record of
mentoring undergraduates in research that was recognized by the Villanova University
Outstanding Mentor Award he received in 2015. Through the support of this proposal,
undergraduates will continue to benefit in terms of their personal and professional growth in their
academic careers. The postdoctoral researcher will gain valuable experience and hopefully
pursue a university faculty position, as has been the case for previous Mendel Science
Experience postdoctoral fellows who have been successful.
In terms of outreach, we will partner with the Agnes Irwin School, an all girls school
located within walking distance to Villanova. This past spring, I hosted an event here at
Villanova with participants from an inner-city school (Friends Select School) and Agnes Irwin
that included a presentation on organic chemistry and a hands-on session in which the
participants carried out analysis of analgesic drugs using thin-layer chromatography. While this
type of activity is useful and we are willing to continue to offer it, we are most excited about
expanding our collaboration in science education with the Agnes Irwin School to include student
research. Based on our planning meeting held during the past summer with AIS faculty and
administration, our proposed programming will include:
(a) Independent Science Research (ISR): The existing program at Agnes Irwin will be expanded
to include two students for summer research at Villanova in the laboratory of the P.I. for periods
of 4 – 8 weeks. Student participants would be required to submit a written report on their
activities to be included in the AIS Advancements in Science journal. Faculty at Agnes Irwin
have noted first hand that when their students engage in independent, novel research in a
university setting, they return to AIS more confident in their lab skills, and excited with their
decision to pursue a STEM career.
(b) AP Chem Support: The AP chemistry class at Agnes Irwin will be brought to Villanova to
carry out experiments in the organic chemistry instructional laboratory in the spring semester.
(c) STEM Club/After Session: Villanova chemistry faculty will visit the AIS after session
program to meet with students, carry out demonstrations, and supervise students in hands-on
activities.

15
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