Synthesis and structure - Phytotoxicity relationships of acetylenic phenols and chromene metabolites, and their analogues, from the grapevine pathogen Eutypa lata

Article abstract of DOI:10.1021/np020415t

Eutypa lata, the fungus responsible for dying-arm disease in grapevines, produces a number of structurally related secondary metabolites, of which eutypine (1) has been implicated as the principal phytotoxin. However, analysis of an E. lata strain from California known to be pathogenic to grapevines showed that eutypine was not present, suggesting that other metabolites could be phytotoxic. Investigation of the relative phytotoxicities of individual metabolites has been limited by insufficient material and lack of a reliable bioassay. Metabolites of particular interest and their precursors were therefore synthesized, and a rapid, quantitative bioassay via topical application of individual compounds to disks of grape leaves and measurement of chlorophyll loss was developed to provide a relative measure of tissue damage. The recently reported metabolite eulatachromene (2) was found to have phytotoxicity greater than that of eutypine (1). The cyclization product, 5-formyl-2-methylvinyl[1]benzofuran (3), also showed significant activity, whereas the reduction product, eutypinol (4), was inactive, as was the quinol, siccayne (5). These results indicate that before strains of Eutypa are incriminated as pathogenic they must be analyzed for the presence or absence of specific constituents for which the phytotoxicity has been unequivocally established.

Full text of DOI:10.1021/np020415t

J . Nat. Prod. 2003, 66, 169-176
169
Syn th esis a n d Str u ctu r e-P h ytotoxicity Rela tion sh ip s of Acetylen ic P h en ols a n d
Ch r om en e Meta bolites, a n d Th eir An a logu es, fr om th e Gr a p evin e P a th ogen
Eu typa la ta
Leverett R. Smith,† Noreen Mahoney, and Russell J . Molyneux*
Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 800 Buchanan Street,
Albany, California 94710
Received August 30, 2002
Eutypa lata, the fungus responsible for dying-arm disease in grapevines, produces a number of structurally
related secondary metabolites, of which eutypine (1) has been implicated as the principal phytotoxin.
However, analysis of an E. lata strain from California known to be pathogenic to grapevines showed that
eutypine was not present, suggesting that other metabolites could be phytotoxic. Investigation of the
relative phytotoxicities of individual metabolites has been limited by insufficient material and lack of a
reliable bioassay. Metabolites of particular interest and their precursors were therefore synthesized, and
a rapid, quantitative bioassay via topical application of individual compounds to disks of grape leaves
and measurement of chlorophyll loss was developed to provide a relative measure of tissue damage. The
recently reported metabolite eulatachromene (2) was found to have phytotoxicity greater than that of
eutypine (1). The cyclization product, 5-formyl-2-methylvinyl[1]benzofuran (3), also showed significant
activity, whereas the reduction product, eutypinol (4), was inactive, as was the quinol, siccayne (5). These
results indicate that before strains of Eutypa are incriminated as pathogenic they must be analyzed for
the presence or absence of specific constituents for which the phytotoxicity has been unequivocally
established.
Eutyposis, or “dying-arm disease”, is a progressive fungal
disease that has resulted in worldwide economic losses in
the grape industry and also infects numerous types of fruit
trees.1-4 In California alone, losses of $260 million per
annum have been estimated as a consequence of reduced
yields, shortened life-span of the vines, and management
costs.5 The causative agent, the ascomycete Eutypa lata,
produces necrosis in the wood close to the point of infection,
stunting of new shoots, and small, deformed, chlorotic
leaves. The ultimate result is dieback of cordons and
reduced fruit production. Eutypine (1), a phenolic aldehyde
possessing an unusual five-carbon acetylenic side chain,
was isolated and identified by Renaud et al.6,7 from fungal
cultures of an unspecified strain of E. lata, together with
structurally related metabolites and reported to be the
principal phytotoxin on the basis of assays performed on
excised leaves and leaf protoplasts.8
quantity for evaluation, together with the chromene ana-
logues (6-8), and a quantitative bioassay for phytotoxicity
to grape leaves was developed.
A comparative study in our laboratory9 of metabolites
produced by two strains of E. lata obtained from grapevines
in California and Italy, respectively, showed that 1 was
not the primary constituent and led to the isolation and
identification of eulatachromene [6-hydroxymethyl-2,2-
dimethyl-2H-chromene] (2) from the Italian Eutypa culture.
In addition, 1 was found to be readily cyclized to the
benzofuran (3) in the presence of traces of acid, and the
corresponding alcohol, eutypinol (4), was the major me-
tabolite produced under most culture conditions. The
quinol, siccayne (5), initially isolated from Helminthos-
porium siccans,10 was also present in moderate amounts
in some strains of E. lata.9 These results suggested that
the phytotoxicity of E. lata might be due to a suite of
structurally related compounds each exhibiting a different
degree of activity, rather than 1 alone, and that 2, 3, and
5 could also contribute to phytotoxicity. To examine this
possibility, the metabolites were synthesized in sufficient
* To whom correspondence should be addressed. Tel: (510) 559-5812.
Fax: (510) 559-6129. E-mail: molyneux@pw.usda.gov.
† Mailing address: Department of Chemistry, Contra Costa College, 2600
Mission Bell Drive, San Pablo, CA 94806.
10.1021/np020415t This article not subject to U.S. Copyright. Published 2003 by the Am. Chem. Soc. and the Am. Soc. of Pharmacogn.
Published on Web 12/21/2002

170 J ournal of Natural Products, 2003, Vol. 66, No. 2
Smith et al.
F igu r e 1. Synthetic route to eutypine (1).
Resu lts a n d Discu ssion
A number of syntheses of eutypine (1) and certain of its
congeners have been developed.11-13 However, 1 was most
conveniently prepared in satisfactory yield by a modifica-
tion, summarized in Figure 1, of the method of Bates et
al.12 4-Hydroxy-3-iodobenzaldehyde (9) was prepared by the
method of Defranq et al.,11 then treated with acetic
anhydride and aqueous sodium hydroxide to give the
acetate (10).14 This was more convenient and gave better
yields than preparation by formylation of 2,4-diiodo-
acetoxybenzene, which gave rise to a mixture of regio-
isomers.12 The iodoaldehyde acetate (10) was coupled with
2-methyl-1-buten-3-yne in the presence of cuprous iodide
and tetrakis(triphenylphosphine)palladium(0), in triethyl-
amine/THF, to give 11,12,15 which was hydrolyzed by
lithium hydroxide in aqueous THF12 to give 1, identical to
the natural product in all respects, including GC-MS
retention time and fragmentation pattern.9 HPLC cleanup
of compound 11 was preferable, and HPLC purification of
1 was essential to prevent its cyclization to 3, which
occurred rapidly if impurities were not removed. This was
in contrast to the report12 that none of the cyclization
product was detectable on hydrolysis with LiOH, whereas
the only isolable product was the benzofuran under other
mildly basic conditions. Although 1 obtained after HPLC
cleanup showed no traces of impurity by spectroscopic
techniques and GC-MS, the melting point varied slightly
from batch to batch, and similar discrepancies were noted
in the literature values.11,12 It is possible that partial
thermal cyclization to the benzofuran occurs during the
melting point determination. The benzofuran (3) was
obtained in sufficient quantity for bioassays as a byproduct
of the eutypine HPLC purification and fully characterized
by spectroscopic methods, since earlier reports of its
occurrence in fungal filtrates7 and formation by cyclization
of eutypine6,12 had not provided details of its physical
properties or structural elucidation.
F igu r e 2. Synthetic route to siccayne (5).
The conversion of eutypine (1) into siccayne (5) by two
five-step variants, with overall yields from 4-hydroxybenz-
aldeyde of 26% and 10%, respectively, has been reported
by Defranq et al.11 This appeared to be an inappropriate
approach because it involved 1, with its attendant lability,
as an intermediate. Siccayne was therefore prepared by
modification of the general approach of Pinault et al.,
reported as a six-step synthesis with an overall yield of
8.8%.16 The route reported here, summarized in Figure 2,
involved only five steps from the commercially available
3-iodophenol and approximately doubled the overall yield
to 16.7%. 3-Iodophenol (12) was subjected to diazo coupling
and reduced to the aminophenol (13), which was oxidized
to the iodoquinone (14).17 Reductive acetylation18 gave the
quinol diacetate (15), which was treated with 2-methyl-1-
buten-3-yne in the presence of tetrakis(triphenylphos-
phine)palladium and copper(I) iodide to yield siccayne
diacetate (16). Deacetylation to siccayne (5) was achieved
by base hydrolysis with lithium hydroxide. The basic
conditions gave a dark colored product which required
purification by HPLC prior to crystallization. Subsequently,
acidic hydrolysis conditions were found to give relatively
little discoloration of the crude product, but purification
was still necessary. Although both hydrolysis methods gave
comparable yields, hydrolysis with acid appears to be
preferable because residues bound to the HPLC column are

Grapevine Pathogen Eutypa lata
J ournal of Natural Products, 2003, Vol. 66, No. 2 171
less, and more easily removed, than with the dark colored
basic hydrolysate.
Eulatachromene (2) was isolated from an Italian culture
of Eutypa lata, and its structure established as 2,2-
dimethyl-6-hydroxymethylchromene by homo- and hetero-
nuclear shift correlation NMR experiments.9 Desert plants
of the Encelia genus (Asteraceae) have been reported to
produce a compound of this structure, but the 1H NMR
data19 showed significantly different shifts from those that
we observed for 2. In particular, the resonances observed
for the two aromatic ring protons adjacent to the hydroxy-
methyl group were approximately 0.5 ppm upfield relative
to the literature values. It therefore appeared probable that
the structure originally reported19 for the Encelia chromene
was incorrect and the regioisomeric 7-hydroxymethyl struc-
ture was more likely for this compound, since the structure
of eulatachromene (2) was rigorously established by cor-
relation experiments. The location of the hydroxymethyl
group para to the chromene ring oxygen functionality in 2
was also consistent with the structures of other fungal
metabolites occurring in E. lata, with 2 being an analogue
of 4 in which the pentenyne chain is reduced and cyclized
onto the phenolic group. Furthermore, the corresponding
6-hydroxymethylchromanone has recently been isolated
from submerged cultures of a Stereum species,20 and a
glycoside of 6-hydroxy-2,2-dimethylchromene has been
isolated from S. hirsutum,21 a fungus associated with
“esca”, a trunk disease of older grapevines. Nevertheless,
unequivocal confirmation of the structure required its
synthesis, which would also provide sufficient material for
evaluation of phytotoxicity.
F igu r e 3. Synthetic route to eulatachromene (2).
dimethylchromene, gave the aldehyde (7) in a yield of 40%,
and this was subsequently oxidized to the acid (8) in only
24% overall yield;27 another synthesis, via DDQ oxidation
of 2,2,6-trimethylchromene, was reported to give a 70%
yield.28 Both the acid and the aldehyde have previously
been isolated as natural products. The former compound
(8) was first obtained from the plant Anodendron affine
(Apocyanaceae) and named anofinic acid;29 subsequently
it has been found in the mushroom Lactarius deliciosus
(Russulaceae),30 the fungus Curvularia fallax (Hypho-
mycetes),31 the wood of the southeast Asian tree Bhesa
paniculata (Celastraceae),32 and the herb Gentiana algida
(Gentianaceae).33 The physical and spectroscopic data for
the synthetic material were completely consistent with
those reported for the natural product.29,30,32 Despite its
occurrence in two genera of fungi, it has not been identified
as a metabolite in the strains of Eutypa lata examined to
date, even though the corresponding chromene alcohol,
eulatachromene (2), has been isolated.9 The chromene
aldehyde (6) appears to be much less widely distributed in
nature, reported only in the steam distillate of liquid
cultures of the basidiomycete Lentinellus cochleatus.34
Eulatachromene (2) and the three chromenes (6-8),
together with eutypine (1), its cyclization product the
benzofuran (3), eutypinol (4), and siccayne (5) provided a
suite of compounds suitable for evaluation of their relative
phytotoxicity. All of these compounds were obtained by syn-
thesis, except for 4, which was available in sufficient quan-
tity from fungal cultures. The earliest signs of eutyposis
in grapevines are manifested in young leaves, which redden
and shrivel prior to complete dieback of the new shoots.
In view of this sensitivity, a bioassay was developed that
attempted to reproduce these symptoms without having to
treat whole plants, which would have involved unaccept-
ably large quantities of natural or synthetic compounds and
attendant problems with respect to uptake and transport
of the test materials through the root system. Tender leaves
of Cabernet Sauvignon, a susceptible variety, were selected,
and methanolic solutions of the metabolites or filtrate
The synthesis of 2,2-dimethyl-6-hydroxymethylchromene
(2) as an intermediate in the preparation of chroman
derivatives evaluated for their effects on platelet aggrega-
tion has been reported,22 but details of the method of
preparation and the physical properties of the compound
were not described, and no spectroscopic data were pro-
vided in support of this structure. The compound was
therefore synthesized de novo, based on methods used for
other chromene derivatives, as shown in Figure 3. Alkyl-
ation of methyl 4-hydroxybenzoate (17) with 3-chloro-3-
methyl-1-butyne23 smoothly gave propynyloxy benzoate
(18), which underwent rearrangement in refluxing diethyl-
aniline24,25 to give the 6-carbomethoxychromene (6) in
excellent yield and purity. This compound has been previ-
ously reported as a constituent of the bark of Piper
hostmannianum, and the spectroscopic properties were
consistent with this structure,26 although the natural
product was reported as an oil whereas the synthetic
compound was obtained as a low-melting solid. Finally, a
routine LAH reduction gave 2 in 72% overall yield.
Synthetic 2 was identical in all respects, including GC-
MS and HPLC retention times, UV spectroscopic data, and
1H and 13C NMR shifts, with those of the natural product,9
thus establishing the structure of the latter unequivocally;
the structure of the chromene isolated from Encelia spe-
cies19 therefore needs to be reexamined.
The success of this synthetic route permitted the prepa-
ration of sufficient quantities for biological evaluation of
additional chromenes with structural correspondence to
Eutypa metabolites. Thus, an identical route, starting with
4-hydroxybenzaldehyde, gave the chromene-6-carboxalde-
hyde (7), analogous to 1, in 74% overall yield. The chromene-
6-carboxylate (8), analogous to the minor metabolite,
eutypine carboxylic acid, was obtained in 69% overall yield
as a crystalline solid by hydrolysis of the ester (6). A
previous synthesis, involving formylation of the parent 2,2-

172 J ournal of Natural Products, 2003, Vol. 66, No. 2
Smith et al.
extracts were applied in a spot to the surface of excised
leaves with the stems immersed in water. Brown, circular
areas of necrosis surrounding the point of application
appeared, generally within 12 h, with some extracts and
metabolites but not with others. In the case of some of the
most active samples, signs of necrosis appeared within 1-2
h of their application. This qualitative assessment of
phytotoxicity suggested that a more quantitative assay
would be feasible and a method was devised to determine
the extent of necrosis by measuring the loss of chlorophyll
relative to control samples.
Leaf disks, 1 cm in diameter, were cut from Cabernet
Sauvignon grape leaves and placed on wet filter paper in
a Petri dish to maintain their viability. Aliquots of the
selected metabolites in MeOH, or plain MeOH as a control,
were applied to the center of the disks and the treated leaf
samples kept under ambient light and temperature for 64
h. Leaf disks from each treatment were collected, and the
chlorophyll was extracted with acetonitrile. The content of
chlorophyll a+b was determined by measuring the ab-
sorbance at 646.8 and 663.8 nm relative to that at 750 nm,
using the method of Porra et al.35 It was immediately
apparent that the extent of chlorophyll loss correlated
closely with the visual assessment of necrosis for each
metabolite and treatment; there was no visible necrosis in
leaf disks treated only with MeOH. Chlorophyll loss was
also concentration dependent, as shown in Figures 4A and
4B. Of the five metabolites tested, 4, a common constituent
of several Eutypa strains, and 59 showed essentially no
activity. Eutypine (1), the metabolite attributed as prima-
rily responsible for phytotoxicity,8 was only moderately
active, whereas its cyclization product, 3, showed signifi-
cant activity. In view of the reported facile conversion of 1
into 3,7,12 and our own experience of this transformation,
there has to be some concern as to whether such cyclization
was occurring under the bioassay conditions; if so, the
observed activity of 1 may in fact be due to the presence of
the cyclized product. In an exploratory experiment, pure 1
was applied to grapeleaf disks, which were then extracted
with acetone after 64 h. Analytical HPLC of the extract
showed that small amounts of 3 were present (data not
shown), indicating that a more detailed investigation of this
question is warranted. It is noteworthy that the dihydro
derivative of 3, (2S)-fomannoxin, has been isolated from
the wood rot fungus Heterobasidion annosum [Fomes
annosus] and shown to be toxic to germinating seeds and
young seedlings of Sitka spruce.36,37 Eulatachromene (2)
showed activity similar to that of 3 (Figure 4A), the
chlorophyll level decreasing to ca. 60% of control. It is
probable that further chlorophyll loss would occur with a
longer bioassay period, but the 64 h time limit was selected
as a convenient period to provide useful quantitative
results and beyond which chlorophyll loss due to normal
senescence of the leaf disks in the artificial Petri dish
environment was likely to occur.
F igu r e 4. Phytotoxicity of E. lata metabolites and synthetic analogues
measured as percent reduction in chlorophyll relative to control in the
grapeleaf disk bioassay. (A) E. lata metabolites eutypine (1), eulata-
chromene (2), 5-formyl-2-(methylvinyl)[1]benzofuran (3), eutypinol (4),
and siccayne (5). (B) Eulatachromene (2) and the synthetic analogues
6-carboxymethylchromene (6), 6-formylchromene (7), and 6-carboxy-
chromene (8).
together with the structurally related and co-occurring
benzofuran, euparin, affect photophosphorylation and elec-
tron transport in isolated spinach chloroplasts.40 The
syntheses of some halogenated chromenes as potential
herbicides have also been reported in the patent literature,
although specifics of the bioassay used were not de-
scribed.41,42 Our results show that some structurally simple
chromenes, including a metabolite of Eutypa lata, are
capable of producing necrosis in the grapeleaf disk bio-
assay. Moreover, the active chromenes 2 and 7, and the
corresponding benzofuran (3), exhibit greater activity than
the E. lata metabolites 1 and 4, with formyl and hydroxy-
methyl substituents, respectively, which possess an acet-
ylenic side chain. The conjugated acetylenic moiety is
therefore not an essential feature for bioactivity even
though many of the metabolites isolated to date possess
this functionality. As sufficient material becomes available,
additional metabolites or their synthetic counterparts will
be evaluated in an attempt to identify specific structural
features that are essential for phytotoxicity and possibly
establish the mode of action of such compounds. The
synthetic strategies developed in this investigation should
Comparison of eulatachromene (2) with three synthetic
analogues, the corresponding methyl ester (6), aldehyde (7),
and acid (8), respectively, showed that only 7 had analogous
activity; 6 and 8 were inactive in the bioassay (Figure 4B).
The reported biological activity of naturally occurring
chromenes has been confined almost entirely to toxicity
to insects and livestock and as photosensitizing antibiotics
to certain fungi and bacteria.38 However, it has recently
been shown that the chromenes present in Helianthella
quinquenervis, encecalin and demethylencecalin, inhibit
radicle growth of Amaranthus hypochondriacus (Prince’s
feather) and Echinochloa crusgalli (barnyard grass)39 and

Grapevine Pathogen Eutypa lata
J ournal of Natural Products, 2003, Vol. 66, No. 2 173
7.5% aqueous NaOH with magnetic stirring to give a clear,
light yellow solution. The solution was cooled in an ice-bath,
and Ac2O (7.5 mL, 79 mmol) was added to the rapidly stirred
solution. After 6 min, the mixture was diluted with Et2O (60
mL) and water (20 mL). The Et2O layer was separated and
washed with 5% aqueous NaHCO3 (3 × 50 mL), then dried
over Na2SO4. The solvent was removed on a rotary evaporator,
and the residue was subjected to a vacuum of 0.5 Torr, with
gentle warming, until Ac2O was no longer detectable by IR.
The product was a very viscous, almost colorless, clear oil,
which slowly crystallized to a white mass, mp 47-49 °C. Yield
was 4.36 g (15.0 mmol, 93%). GC-EIMS: tR 11.80 min; m/z
290 (M+, 23), 248 (100), 247 (60), 219 (4), 191 (5), 127 (4), 119
(5), 92 (13). Spectroscopic data agreed with that reported in
the literature,11 except that the latter reference did not include
the 13C NMR signal at δ 123.7, corresponding to one of the
aromatic carbon atoms.
prove adaptable to the synthesis of other E. lata metabo-
lites and their structural analogues.
The fairly rapid bioassay provides a convenient system
whereby filtrate extracts from various strains of pathogenic
and nonpathogenic E. lata can be screened for the presence
of phytotoxins and used for bioassay-directed fractionation
of the most toxic metabolites. It is anticipated that with a
sufficiently large selection of fungal strains, not only from
the United States but also from other grape-growing areas
of the world, a specific metabolite or group of metabolites
can be selected as diagnostic for pathogenic strains and
used to predict and control the spread of dying-arm disease
in vineyards. Attempts are currently underway to correlate
the presence of individual metabolites with DNA probes
specific for pathogenic and nonpathogenic E. lata strains
developed using PCR techniques.43
4-Acet oxy-3-(3′-m et h ylb u t -3-en -1-yn yl)b en za ld eh yd e
(11). This compound was prepared by a modification of the
method of Bates et al.,12 based also on a related procedure
described by Brisbois et al.15 (see Supporting Information). The
product (11) was obtained in 63% yield as a yellow, crystalline
material, mp 47-49 °C. Spectroscopic data agreed with that
of Bates et al.;12 complete assignments of the 1H NMR
spectrum not previously reported are as follows: 1H NMR
(CDCl3) δ 1.99 (3 H, dd, J ) 0.8, 1.6 Hz, -CH3), 2.36 (3H, s,
-OCOCH3), 5.37 (1H, appearing as a “pentuplet”, J ) 1.6 Hz,
dCH2), 5.43 (1H, dq, appearing as a “sextet”, J ) 0.8 and 1.6
Hz, dCH2), 7.26 (1H, d, J ) 8.4 Hz, H-5), 7.83 (1H, dd, J )
8.4 Hz, 2 Hz, H-6), 7.98 (1H, dd, J ) 2 Hz, 0.4 Hz, H-2), 9.95
(1H, d, J ) 0.4 Hz, -CHO); 13C NMR (CDCl3) δ 20.7
(-OCOCH3), 21.2 (CH3), 82.1 (C-1′), 96.9 (C-2′), 118.8 (C-3),
123.2 (C-5), 123.4 (dCH2), 126.2 (C-3′), 130.1 (C-6), 134.2 (C-
1), 134.6 (C-2), 155.9 (C-4), 168.0 (-OCOCH3), 190.2 (-CHO).
Eu typ in e (1). This compound was prepared from 4-acetoxy-
3-(3′-methylbut-3-en-1-ynyl)benzaldehyde (11) by deacetylation
with LiOH in aqueous THF using the method of Bates et al.12
Crude material rapidly (<1 day) isomerized to 3 if not promptly
processed to remove impurities. HPLC purification gave a
stable, cream-colored, crystalline solid. Despite consistently
clean NMR spectra, melting ranges varied, for example, 70.5-
72.5 °C for one batch, 67.5-71 °C for another (lit. 76-77 °C;11
67-69 °C12). Spectroscopic properties were in agreement with
those reported by Defranq et al.11 and Bates et al.:12 GC-EIMS
(monoTMS derivative) tR 13.75 min; m/z 258 (M+, 100), 243
(82), 227 (9), 203 (22), 199 (43), 189 (15), 185 (28), 141 (12),
128 (12), 115 (15), consistent with that of the natural product;9
1H NMR (CDCl3) δ 2.03 (3H, dd, J ) 1.6 Hz, 0.8 Hz), 5.41 (1H,
p, J ) 1.6 Hz), 5.49 (1H, dq, appearing as a “sextet”, J ) 1.6
Hz, 0.8 Hz), 6.37 (1H, br s), 7.07 (1H, d, J ) 8.4 Hz), 7.79 (1H,
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
determined on a Mel-Temp apparatus and are uncorrected.
IR spectra were obtained with a Nicolet Magna-IR 550 Series
II spectrometer. High-resolution MS results were determined
by the Mass Spectrometry Facility, College of Chemistry,
University of California, Berkeley. NMR spectra were obtained
at 298 K from samples dissolved in CDCl3 with TMS as an
internal standard on
a Bruker ARX400 spectrometer at
frequencies of 400.13 MHz (1H) and 100.62 MHz (13C). A 90°
pulse at a 7-8 s repetition rate was used for 1H experiments,
and a 30° pulse at a 2.3 s repetition rate was used for 13C
experiments. The number of attached protons for 13C signals
was determined from DEPT90 and DEPT135 assays.
Methyl 4-hydroxybenzoate, 3-chloro-3-methyl-1-butyne, 3-
iodophenol, copper(I) iodide, tetrakis(triphenylphosphine)-
palladium, and 2-methyl-1-but-3-yne were obtained from
Sigma-Aldrich (Milwaukee, WI). Zinc powder (-100 mesh) was
provided by Alfa Products (Danvers, MA).
When necessary, synthetic products were purified by pre-
parative HPLC using a 250 × 21.4 mm i.d., 8 µm, C18
Dynamax column, and 50 mm guard column (Rainin), with
isocratic elution by CH3CN/H2O (1:1) pumped (Beckman110B)
at a flow rate of 10 mL/min and UV detection at 254 nm
(Gilson 115). Purity was checked and compounds established
as being identical to natural metabolites by GC-MS analysis
of their TMS derivatives, prepared by suspension of the sample
(ca. 0.5 mg) in dry pyridine (100 µL) in a 1.0 mL Reacti-Vial
(Pierce, Rockford, IL), to which was added N-methyl-N-
(trimethylsilyl)trifluoroacetamide (MSTFA) (100 µL) (Pierce).
The reaction mixture was then heated at ca. 60 °C for 1 h.
Analyses were performed on a Hewlett-Packard 5890 Series
II instrument equipped with a 5971 mass-selective detector
(MSD) and a 60 m × 0.32 mm i.d., 0.25 µm, SE-30 fused Si
capillary column (J &W Scientific). The column was held at an
initial temperature of 105 °C for 0.2 min, ramped at 30 °C/
min for 0.5 min, programmed from 120 °C to 300 °C at 10 °C/
min, and held at the final temperature for 10 min. Helium
was used as carrier gas with a head pressure of 60 psi.
Derivatized samples (0.1-0.2 µL) were introduced through an
SGE model OC1-3 on-column injector held at ambient tem-
perature. The MSD was operated at 70 eV in the EI mode with
scanning from 75 to 600 amu at a sampling rate of 1.5 scans/
s. A postinjection delay of 7.0 min was set in order to avoid
MS data acquisition during elution of the solvent and deriva-
tization reagent.
dd, J ) 8.4 Hz, 2 Hz), 7.89 (1H, d, J ) 2 Hz), 9.84 (1H, s); 13
C
NMR (CDCl3) δ 23.3, 80.5, 98.9, 110.7, 115.5, 123.9, 125.8,
129.9, 131.8, 134.4, 161.2, 190.1.
5-F or m yl-2-(m eth ylvin yl)-1-ben zofu r a n (3). This com-
pound was isolated by HPLC in varying yields as a crystalline
byproduct, mp 58-59 °C, from preparations of 1: GC-EIMS
tR 11.94 min; m/z 186 (M+, 100), 185 (79), 157 (26), 128 (12),
115 (5), 92 (5); IR νmax (NaCl, thin film) cm-1 3125, 3096, 2955,
2914, 2725, 1694, 1606, 1560, 1444, 1336, 1277, 1115, 887, 807;
UV (MeOH) λmax nm (log ꢀ) 204 (3.84), 256 (4.62); 1H NMR
(CDCl3) δ 2.14 (3H, dd, J ) 1.4, 1.0 Hz, -CH3), 5.25 (1H,
appearing as a “pentuplet”, J ) 1.4 Hz, dCH2), 5.85 (1H, m,
dCH2), 6.72 (1H, s, H-3), 7.54 (1H, d, J ) 8.8 Hz, H-7), 7.83
(1H, dd, J ) 8.6, 1.8 Hz, H-6), 8.06 (1H, d, J ) 1.6 Hz, H-4),
10.04 (1H, -CHO); 13C NMR (CDCl3) δ 19.2 (-CH3), 103.0 (C-
3), 111.7 (C-7), 114.7 (dCH2), 124.0 (C-4), 126.3 (C-6), 129.7,
132.3, 132.4 (C-3a, C-5, CdCH2) 158.2, 158.8 (C-2, C-7a), 191.7
(-CHO); HREIMS m/z 186.0681 (calcd for C12H10O2, 186.0681).
4-Am in o-3-iod op h en ol (13). This compound was prepared
by the method of Kvalnes,17 except that the extraction with
ether was omitted; the product (13) was recrystallized directly
from the reaction solvent as a light-colored, fibrous solid, in
50% yield, mp 134.5-136.5 °C (dec); lit. darkening at 135 °C,
melting at 140 °C with charring:17 1H NMR (DMSO-d6) δ 4.51
(2H, br s), 6.58 (1H, dd, J ) 8.6, 2.6 Hz), 6.38 (1H, d, J ) 8.6
4-Hyd r oxy-3-iod oben za ld eh yd e (9). This compound was
prepared by the method of Defranq et al.,12 except that the
product was purified by recrystallization from H2O (ca. 75 mL
of H2O per gram of crude product) rather than by column
chromatography: GC-EIMS (monoTMS derivative) tR 12.50
min; m/z 320 (M+, 41), 305 (100), 185 (10), 178 (13), 177 (11),
150 (7), 149 (11).
4-Acetoxy-3-iod oben za ld eh yd e (10). 4-Hydroxy-3-iodo-
benzaldehyde (4.02 g, 16.2 mmol) was dissolved in 15 mL of

174 J ournal of Natural Products, 2003, Vol. 66, No. 2
Smith et al.
Hz), 7.01 (1H, d, J ) 2.4 Hz), 8.70 (1H, s); 13C NMR (DMSO-
d6) δ 83.6, 115.2, 116.6, 124.1, 140.9, 149.0.
[C-4′]), 123.0 (C-5), 125.7 (C-2), 126.4 (C-3′); 147.9, 149.0 (C-1
and C-4); 168.6 (-OCOCH3), 169.0 (-OCOCH3); HREIMS m/z
258.0894 (calcd for C15H14O4, 258.0892).
2-Iod oqu in on e (14). This compound was prepared by the
method of Kvalnes17 using saturated aqueous K2Cr2O7, instead
of Na2Cr2O7 solution. 4-Amino-3-iodophenol (5.12 g; 21.8 mmol)
was dissolved in concentrated H2SO4 (38 mL) and H2O (110
mL) at 50 °C. The mixture was cooled in an ice bath to 10 °C,
and saturated aqueous K2Cr2O7 (165 mL) added in portions
over 15 min at 14-17 °C. After stirring for an additional 5
min, the ice bath was removed and stirring continued for a
further 10 min. The slurry was filtered and the solid washed
with H2O (4 × 10 mL) to give 3.73 g (15.9 mmol, 73%) of 14:
mp 61-63 °C; lit. 62 °C, after sublimation;17 IR νmax (NaCl,
thin film) cm-1 3308, 3073, 3046, 1667, 1651, 1619, 1570, 1271,
Sicca yn e (5). (a) Via base hydrolysis. LiOH‚H2O (0.154 g;
3.67 mmol) in a 60 mL flask was cooled in an ice bath and
H2O (2.0 mL) added to dissolve the solid. A solution of siccayne
diacetate (16) (0.233 g; 0.903 mmol) in THF (1.5 mL) was
added with rapid stirring. After 30 min, the dark mixture was
diluted with Et2O (50 mL) and acidified by addition of 10%
aqueous HCl (5 mL). After mixing and allowing to settle, the
aqueous layer was removed by pipet. The red-amber Et2O layer
was washed with saturated aqueous NaCl (2 × 5 mL), then
dried over anhydrous Na2SO4. Solvent removal (rotary evapo-
rator, then ca. 1 Torr) gave a molasses-colored, very viscous
oil, from which siccayne was isolated by HPLC (two passes).
The yield of purified siccayne was 0.106 g (0.609 mmol, 67%):
white solid, mp 114.5-116 °C; lit.114-116 °C;16 GC-EIMS (as
di-TMS derivative) tR 13.60 min; m/z 318 (M+, 100), 304 (13),
303 (42), 287 (8), 263 (8); IR νmax (NaCl, thin film) cm-1 3500-
3100, 2955, 2920, 2206, 1616, 1450, 1232; 1H NMR (CDCl3) δ
2.00 (3H, dd, J ) 1.6, 1.2 Hz, -CH3), 4.69 (1H, br s, -OH),
5.35 (1H, “pentuplet”, J ) 1.6 Hz, dCH2), 5.42 (1H, br s, -OH),
5.43 (1H, dq, appearing as a “sextet”, J ) 1.6, 1 Hz, dCH2),
6.74 (1H, dd, J ) 8.8, 3 Hz, H-6), 6.80-6.83 (2H, m, H-2 and
H-5); 13C NMR (CDCl3) δ 23.4 (-CH3); 81.9 (-Ct), 97.6 (-Ct),
110.0 (C-3); 115.5, 117.4 (C-2, C-6); 118.0 (C-5), 123.1 (dCH2
[C-4′]), 126.1 (C-3′); 148.7, 150.8 (C-1, C-4). (b) Via acid
hydrolysis. Siccayne diacetate (16) (0.051 g; 0.198 mmol) in
THF (5.0 mL) was treated with 6 M aqueous HCl (1.0 mL)
and the clear, almost colorless solution stirred in the closed
flask at room temperature for 51 h. The mixture was diluted
with Et2O (50 mL) and the small lower layer which separated
removed by pipet. The Et2O solution was then washed with
saturated aqueous NaCl (4 × 5 mL) and dried over Na2SO4.
Removal of solvent (rotary evaporator, then ca. 1 Torr) gave a
yellow oil, from which 5 was isolated by HPLC (one pass).
Duplicate experiments gave 20 mg (0.115 mmol; 58%) and 23
mg (0.132 mmol; 67%), respectively, of 5, mp 113-115 °C. IR
and NMR spectra matched those of material obtained from
base hydrolysis.
1
1096, 961, 917, 822; H NMR (CDCl3) δ 6.84 (1H, dd, J ) 10,
2.4 Hz), 7.00 (1H, d, J ) 10 Hz), 7.68 (1H, d, J ) 2.4 Hz); 13
NMR (CDCl3) δ 119.5, 134.6, 136.7, 146.2, 180.2, 184.0.
C
2-Iod oh yd r oqu in on e Dia ceta te (15). This compound was
prepared by reductive acetylation18 of 2-iodoquinone. A mixture
of 2-iodoquinone (0.946 g; 4.04 mmol) and Zn powder (1.91 g;
29.2 mmol) was chilled in an ice/water bath and then treated
with an ice-cold solution of concentrated aqueous HCl (3 mL)
in Ac2O (20 mL) with vigorous stirring. The dark quinone color
disappeared completely in less than 90 s; after 3 min, the ice
bath was removed, and stirring continued for 7 min. The
mixture was diluted with Et2O (60 mL), filtered through cotton
wool to remove unreacted Zn, and then washed with H2O (2
× 20 mL) followed by saturated aqueous NaCl (2 × 20 mL).
The ethereal solution was dried over Na2SO4 and concentrated
(rotary evaporator, then ca. 1 Torr) to give a pale amber oil,
which crystallized under vacuum (1.24 g; 3.88 mmol, 96%):
mp 82-84.5 °C; lit. 86-87 °C recrystallized from aqueous
EtOH;17 IR νmax (NaCl, thin film) cm-1 3090, 2936, 1763, 1586,
1476, 1370, 1203, 1170, 1011, 923; 1H NMR (CDCl3) δ 2.27
(3H, s), 2.35 (3H, s), 7.08 (1H, dd, J ) 8.4 and 0.4 Hz), 7.11
(1H, dd, J ) 8.8, 2.4 Hz), 7.57 (1H, dd, J ) 2.4, 0.4 Hz); 13C
NMR (CDCl3) δ 21.0, 21.1, 90.0, 122.6, 123.0, 132.2, 148.4,
148.9, 168.4, 168.8.
Sicca yn e Dia ceta te (16). To a 100 mL three-necked flask,
flushed with N2, was added 2-iodohydroquinone diacetate
(1.833 g; 5.72 mmol), CuI (0.208 g; 1.09 mmol), and tetrakis-
(triphenylphosphine)palladium (0.373 g; 0.323 mmol). Triethyl-
amine (17 mL) was added with stirring, followed by anhydrous
THF (10 mL). After a few minutes a clear, light amber solution
was produced, to which 2-methyl-1-buten-3-yne (4.5 mL; 47
mmol) was added. A momentary lightening in color was
followed by a gradual (1-2 min) darkening, and within a few
minutes, turbidity was followed by formation of a granular
precipitate. The flask was covered with aluminum foil and
stirring continued at room temperature for 25 h. The mixture
was then diluted with hexane/ether (9:1) (70 mL). After stirring
and settling, the supernatant was filtered through cotton wool
into a separatory funnel and the cream-colored solid remaining
in the flask rinsed with 4 × 15 mL portions of the solvent
mixture. The combined organic phase was washed with
saturated aqueous NH4Cl (60 mL), 5% aqueous NaHCO3 (60
mL), and saturated aqueous NaCl (60 mL). After drying over
anhydrous Na2SO4, the solution was passed through a short
column of silica gel (5.0 g), eluted with an additional 100 mL
of solvent. Concentration on a rotary evaporator gave a pale
crystalline solid admixed with an orange oil. The solid was
washed with hexane (3 × 5 mL), then dried at ca. 1 Torr to
give a pale yellow solid (0.998 g), mp 66-67.5 °C. Concentra-
tion of the hexane washes gave additional product (0.053 g),
mp 64-67 °C. Total yield: 1.051 g (4.07 mmol, 71%); GC-EIMS
tR 14.33 min; m/z 258 (M+, 6), 216 (20), 174 (100), 159 (9), 147
(5), 127 (5); IR νmax (NaCl, thin film) cm-1 3072, 2978, 2925,
1768, 1759, 1614, 1577, 1486, 1371, 1218, 1170, 910; 1H NMR
(CDCl3) δ 1.96 (3H, dd, J ) 1.6, 1 Hz, -CH3), 2.28 (3H, s,
-OCOCH3), 2.32 (3H, s, -OCOCH3), 5.32 (1H, m, J ) 1.8 Hz,
dCH2), 5.38 (1H, dq, appearing as a “sextet”, J ) 2, 1 Hz,
dCH2), 7.05 (1H, dd, J ) 8.8, 2.4 Hz, H-5), 7.08 (1H, dd, J )
8.8, 0.8 Hz, H-6), 7.22 (1H, dd, J ) 2.4, 0.8 Hz, H-2); 13C NMR
(CDCl3) δ 20.7 (-OCOCH3), 21.0 (-OCOCH3), 23.2 (-CH3),
82.5 (-Ct), 96.2 (-Ct), 118.5 (C-3), 122.5 (C-6), 122.9 (dCH2
Meth yl 4-(1,1-Dim eth yl-2-p r op yn yloxy)ben zoa te (18).
A mixture of KI (6.0 g), K2CO3 (6.0 g), methyl 4-hydroxy-
benzoate (3.05 g, 25.0 mmol), acetone (30 mL), and 3-chloro-
3-methyl-1-butyne (6.8 mL, 60.5 mmol) was heated with
stirring and exclusion of moisture at 49-53 °C. After 48 h,
the mixture was allowed to cool to room temperature, then
partitioned between Et2O (100 mL) and 1.0 M aqueous NaOH
(100 mL) in a separatory funnel. The organic fraction was
washed with additional 1.0 M NaOH (2 × 100 mL) and dried
over anhydrous MgSO4. Removal of solvent (rotary evaporator,
then ca. 1 Torr) gave a clear yellow oil (yield 4.29 g, 19.7 mmol,
79%), which rapidly crystallized: mp 49-53 °C; GC-EIMS m/z
218 (M+, 6), 203 (66), 152 (67), 121 (100); IR νmax (NaCl, thin
film) cm-1 3292, 2991, 2951, 2111, 1720, 1604, 1507, 1435,
1
1280, 1249, 1139, 1114; H NMR (CDCl3) δ 1.69 (6H, s), 2.62
(1H, s), 3.88 (3H, s), 7.24 (2H, d, J ) 9 Hz), 7.97 (2H, d, J )
9 Hz); 13C NMR (CDCl3) δ 29.6, 51.9, 72.4, 74.7, 85.3, 119.5,
123.9, 131.0, 159.8, 166.9; HREIMS m/z 218.0941 (calcd for
C
13H14O3, 218.0943).
6-Met h oxyca r b on yl-2,2-d im et h yl-2H -ch r om en e (6).
Methyl 4-(1,1-dimethyl-2-propynyloxy)benzoate (18) (3.22 g,
14.8 mmol) and N,N-diethylaniline (15 mL) were stirred
magnetically in a 25 mL flask, until almost all of the solid
dissolved, giving a yellow solution. The mixture was brought
to reflux (210-220 °C), at which point the remaining solid had
dissolved. After 75 min the reaction was allowed to cool to room
temperature. The mixture was diluted with Et2O (75 mL),
washed (caution; exothermic) with 6 M aqueous HCl (4 × 30
mL) followed by saturated aqueous NaCl (2 × 10 mL), and
dried over anhydrous Na2SO4. Solvent removal (rotary evapo-
rator, then 0.25 Torr) gave an amber oil, which soon crystal-
lized into prisms and rosettes; yield 3.09 g (14.2 mmol, 96%).
The product could be further purified by sublimation under
vacuum, which gave ca. 96% recovery of an almost white
crystalline solid: mp 43-45 °C; GC-EIMS tR 12.16 min; m/z

Grapevine Pathogen Eutypa lata
J ournal of Natural Products, 2003, Vol. 66, No. 2 175
218 (M+, 8), 203 (100), 144 (11), 115 (8); IR νmeltmax cm-1 3044,
2977, 2951, 1720, 1641, 1609, 1576, 1490, 1441, 1367, 1291,
1274, 1196, 1166, 1096, 960, 766; UV (MeOH) λmax nm (log ꢀ)
240 (4.60), 282 (3.64); 1H NMR (CDCl3) δ 1.44 (6H, s), 3.86
(3H, s), 5.63 (1H, d, J ) 10 Hz), 6.33 (1H, d, J ) 10), 6.77 (1H,
dd, J ) 8.4 hz, 0.4 Hz), 7.67 (1H, dd, J ) 2 Hz, 0.4 Hz), 7.80
(1H, dd, J ) 8.4 Hz, 2 Hz); 13C NMR (CDCl3) δ 28.3, 51.8, 77.4,
116.2, 120.7, 121.7, 122.6, 128.1, 131.0, 131.1, 157.2, 166.8;
HREIMS m/z 218.0946 (calcd for C13H14O3, 218.0943).
2,2-Dim eth yl-2H-ch r om en e-6-car boxaldeh yde (7). 4-(1,1-
Dimethyl-2-propynyloxy)benzaldehyde (0.40 g, 2.13 mmol) in
N,N-diethylaniline (4.0 mL) was heated under reflux (205-
210 °C) in a 5 mL flask under anhydrous conditions, with
stirring. The initially yellow solution rapidly became dark
amber in color. After 5 h, IR of a sample of the reaction mixture
showed no alkyne C-H band. The reaction mixture was
diluted with Et2O (25 mL), then washed with 6 M aqueous
HCl (4 × 10 mL), followed by H2O (10 mL). Drying over
anhydrous Na2SO4, followed by solvent removal, gave an oil,
which was dissolved in hexane/ether (9:1) and passed through
5 g of silica gel, eluting with 150 mL of the same solvent.
Removal of the solvent gave the chromene carboxaldehyde (7)
as an oil: yield 0.33 g (1.76 mmol, 82%); GC-EIMS tR 10.65
min; m/z 188 (M+, 9), 174 (12), 173 (100), 144 (7), 115 (13); IR
6-Hyd r oxym eth yl-2,2-d im eth yl-2H-ch r om en e; Eu la ta -
ch r om en e (2). 6-Methoxycarbonyl-2,2-dimethyl-2H-chromene
(5) (1.247 g, 5.72 mmol) was reduced by stirring with LiAlH4
(0.722 g, 19 mmol) in anhydrous Et2O (50 mL) with gentle
reflux, under an atmosphere of N2. After 30 min, the reaction
mixture was chilled in an ice bath, and 15% aqueous NaOH
(5 mL) was added dropwise over a 10 min period with vigorous
stirring. Partway through the quenching process, Et2O (25 mL)
was added. After all the NaOH solution had been added, a
clear colorless supernatant was obtained, over white semisolid
clumps. The supernatant was decanted, the solid was washed
with Et2O (3 × 10 mL), and the combined Et2O phase was
dried over anhydrous Na2SO4. Solvent removal gave a clear,
colorless, viscous oil: yield 1.066 g (5.61 mmol, 98%); GC-EIMS
(mono-TMS derivative) tR 12.05 min; m/z 262 (M+, 8), 247
(100), 173 (13), 158 (5), consistent with that of the natural
product;9 IR νmax (NaCl, thin film) cm-1 3600-3150, 3039,
2975, 2933, 2872, 1638, 1614, 1490, 1361, 1261, 1212, 1151;
UV (MeOH) λmax nm (log ꢀ) 224 (4.51), 264 (3.56), 312 (3.44);
1H NMR (CDCl3) δ 1.42 (6H, s), 1.80 (1H, s), 4.54 (2H, s), 5.60
(1H, d, J ) 9.6 Hz), 6.30 (1H, d, J ) 9.6 Hz), 6.74 (1H, d, J )
8 Hz), 6.96 (1H, d, J ) 2 Hz), 7.07 (1H, dd, J ) 8, 2 Hz); 13C
NMR (CDCl3) δ 28.0, 65.1, 76.3, 116.4, 121.3, 122.2, 125.4,
128.1, 131.1, 133.2, 152.6; HREIMS m/z 190.0993 (calcd for
ν
max (NaCl, thin film) cm-1 3046, 2977, 2934, 2820, 2734, 1690,
1639, 1601, 1572, 1486, 1364, 1278, 1146, 1119, 963; UV
(MeOH) λmax nm (log ꢀ) 252 (4.46), 298 (3.75); 1H NMR (CDCl3)
δ 1.47 (6H, s), 5.68 (1H, d, J ) 10 Hz), 6.36 (1H, d, J ) 10
Hz), 6.85 (1H, dm, J ) 8.4 Hz), 7.50 (1H, dd, J ) 2, 0.4 Hz),
7.63 (1H, dd, J ) 8.4, 2 Hz), 9.82 (1H, s); 13C NMR (CDCl3) δ
28.5, 77.9, 116.8, 121.2, 121.4, 127.8, 130.0, 131.5, 131.9, 158.7,
190.6; HREIMS m/z 188.0834 (calcd for C12H12O2, 188.0837).
P h ytotoxicity Bioa ssa y w ith Gr a p elea f Disk s. Leaf
disks (1 cm diameter) were cut from greenhouse grown
Cabernet Sauvignon grape leaves (4-5 cm at the widest point)
with a cork borer with the major veins being avoided. Ten
leaves were used for each concentration such that each of the
tested compounds and its associated control had one leaf disk
from each of the same 10 leaves. The 10 leaf disks for each
sample were arranged around the perimeter of filter paper (7.0
cm Whatman #3) saturated with H2O (2 mL) and placed in a
Petri dish (100 × 15 mm). Compounds to be tested were
dissolved in MeOH, and a volume of 5 µL was applied as a
single spot at the center of the surface of each leaf disk; the
control disks were similarly treated with 5 µL of MeOH. The
Petri dishes were covered with lids and the test samples
exposed at room temperature to ambient lighting on a labora-
tory bench; this consisted of approximately 12 h of mixed
natural and artificial light and 12 h of darkness. An additional
volume of H2O (1.5 mL) was added to each Petri dish after 24
h. The degree of leaf disk browning was measured after 64 h
by comparing the chlorophyll content of the treated leaf disks
to the control disks. All 10 leaf disks for a given compound
and concentration were placed in DMF (10 mL) and shaken
in the dark for 4 h at 250 rpm. Chlorophyll concentrations were
calculated according to the method of Porra et al.35 using the
formula
C
12H14O2, 190.0994).
2,2-Dim et h yl-2H -ch r om en e-6-ca r b oxylic Acid ; An o-
fin ic Acid (8). 6-Methoxycarbonyl-2,2-dimethyl-2H-chromene
(5) (0.102 g, 0.468 mmol) was hydrolyzed by stirring with
NaOH (0.34 g, 8.5 mmol) dissolved in absolute EtOH (2.5 mL)
and H2O (0.5 mL) at room temperature for 4.5 h. The mixture
was diluted with Et2O (15 mL), then acidified with 6 M
aqueous HCl (5 mL). After separation, the Et2O layer was
dried over anhydrous Na2SO4, then evaporated to give the
carboxylic acid (7): mp 159-162 °C; lit. 158.5-160 °C,29 150-
156 °C;30 yield 0.083 g (0.407 mmol, 87%); GC-EIMS (mono-
TMS derivative) tR 13.71 min; m/z 276 (M+, 6), 262 (20), 261
(100), 189 (5), 144 (8), 115 (7); IR νmax (NaCl, thin film) cm-1
3300-2400 (br), 2975, 1674, 1606, 1575, 1446, 1412, 1299,
1276, 1200, 1123, 954, 768; UV (MeOH) λmax nm (log ꢀ) 240
(4.58), 282 (3.60); 1H NMR (CDCl3) δ 1.46 (6H, s), 5.65 (1H, d,
J ) 10 Hz), 6.36 (1H, d, J ) 10 Hz), 6.80 (1H, d, J ) 8.4 Hz),
7.74 (1H, d, J ) 2 Hz), 7.88 (1H, dd, J ) 8.4, 2 Hz), 10.5 (1H,
v br); 13C NMR (CDCl3) δ 28.4, 77.6, 116.3, 120.7, 121.61,
121.63, 128.8, 131.1, 131.9, 158.0, 171.8; HREIMS m/z 204.0783
(calcd for C12H12O3, 204.0786).
total chlorophyll a +b (µg/mL) ) 17.67A646.8 + 7.12A663.8
where A646.8 ) absorbance at 646.8 nm - 750 nm and A663.8
)
absorbance at 663.8 nm - 750 nm. Results were plotted as
remaining chlorophyll (percent of control) versus amount of
applied sample (µg) and are shown in Figure 4.
4-(1,1-Dim eth yl-2-p r op yn yloxy)ben za ld eh yd e. A mix-
ture of 4-hydroxybenzaldehyde (1.26 g, 10.3 mmol) and 3-chloro-
3-methyl-1-butyne (5.0 mL, 44.5 mmol) was heated under
reflux over KI (3.0 g) and K2CO3 (3.0 g) in dry acetone (15
mL) for 47 h, with stirring and exclusion of moisture. The
reaction was cooled to room temperature, diluted with ether
(50 mL), washed with 1 M aqueous NaOH (3 × 50 mL), and
dried over anhydrous MgSO4. After solvent removal, the
sample was dissolved in hexane/ether (9:1) (ca. 20 mL) then
passed through silica gel (5 g), using 150 mL more solvent to
complete elution. This gave the 4-(1,1-dimethyl-2-propynyl-
oxy)benzaldehyde as a light amber oil: yield 1.74 g (9.26 mmol;
90%); GC-EIMS tR 9.48 min; EIMS m/z 188 (M+, 5), 173 (31),
159 (11), 122 (78), 121 (100), 115 (10), 93 (11); IR νmax (NaCl,
thin film) cm-1 3290, 2991, 2939, 2828, 2738, 2111, 1698, 1601,
1577, 1505, 1248, 1220, 1138; 1H NMR (CDCl3) δ 1.72 (6H, s),
2.66 (1H, s), 7.33 and 7.81 (each 2H, AB multiplets), 9.90 (1H,
s); 13C NMR (CDCl3) δ 29.6, 72.5, 75.0, 85.0, 119.6, 130.7, 131.3,
161.2, 190.9; HREIMS m/z 188.0834 (calcd for C12H12O2,
188.0837).
Ack n ow led gm en t. This research was conducted under a
Cooperative Agreement between USDA-ARS (No. 58-5325-0-
158) and the American Vineyard Foundation (Project V200);
we thank the AVF for financial support and Mr. Patrick
Gleeson, Executive Director, for his encouragement. The
granting of a sabbatical leave to L.R.S. by Contra Costa College
is gratefully acknowledged.
Su p p or tin g In for m a tion Ava ila ble: Procedure for synthesis of
compound 11. This material is available free of charge via the Internet
at http://pubs.acs.org.
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(4) Munkvold, G. P.; Marois, J . J . Plant Dis. 1994, 78, 200-207.

176 J ournal of Natural Products, 2003, Vol. 66, No. 2
Smith et al.
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