Probing the Role of Polyphenol Oxidation in Mediating Insect-Pathogen Interactions. Galloyl-Derived Electrophilic Traps for the Lymantria dispar Nuclear Polyhedrosis Virus Matrix Protein Polyhedrin

Article abstract of DOI:10.1021/jo982477n

Galloyl-derived orthoquinone probes have been designed, synthesized, and utilized in an ongoing study of insect-pathogen interactions. A stable galloyl-derived orthoquinone O-methyl ether modified with both acidic and fluorescent appendages was successful in trapping the model nucleophile cysteine, a test protein bearing a single cysteine residue, and the viral occlusion body matrix protein polyhedrin from Lymantria dispar nuclear polyhedrosis virus (LdNPV), a pathogen of the gypsy moth caterpillar (GMc). This latter observation may be related to the molecular mechanism by which gallotannins decrease LdNPV infectivity in GMc's. Sufficient site isolation was not achieved with a polymer-bound reactive galloyl hydroxyorthoquinone electrophile to permit similar nucleophile trapping to compete with oligomerization.

Full text of DOI:10.1021/jo982477n

5794
J . Org. Chem. 1999, 64, 5794-5803
P r obin g th e Role of P olyp h en ol Oxid a tion in Med ia tin g
In sect-P a th ogen In ter a ction s. Ga lloyl-Der ived Electr op h ilic Tr a p s
for th e Lym a n tr ia d ispa r Nu clea r P olyh ed r osis Vir u s Ma tr ix
P r otein P olyh ed r in
Ken S. Feldman,*,† Aruna Sambandam,† Katherine E. Bowers,† and Heidi M. Appel‡
Department of Chemistry and Pesticide Research Laboratory, The Pennsylvania State University,
University Park, Pennsylvania 16802
Received December 21, 1998
Galloyl-derived orthoquinone probes have been designed, synthesized, and utilized in an ongoing
study of insect-pathogen interactions. A stable galloyl-derived orthoquinone O-methyl ether
modified with both acidic and fluorescent appendages was successful in trapping the model
nucleophile cysteine, a test protein bearing a single cysteine residue, and the viral occlusion body
matrix protein polyhedrin from Lymantria dispar nuclear polyhedrosis virus (LdNPV), a pathogen
of the gypsy moth caterpillar (GMc). This latter observation may be related to the molecular
mechanism by which gallotannins decrease LdNPV infectivity in GMc’s. Sufficient site isolation
was not achieved with a polymer-bound reactive galloyl hydroxyorthoquinone electrophile to permit
similar nucleophile trapping to compete with oligomerization.
The role of polyphenolic compounds in mediating
by Felton, Duffey, and co-workers support the hy-
pothesis that the catechol chlorogenic acid, in the pres-
ence of the oxidant polyphenol oxidase (PPO), covalently
bonds to both the occlusion bodies (OB’s) and their
constituent protein polyhedrin of the tomato fruitworm
virus HzSNPV.3 The degree of covalent attachment
correlates strongly both with a decrease in rate of OB
dissolution, as is required for viral infection, and with a
decrease in host mortality. The presence of potentially
competing foliar proteins did not suppress inhibition of
infectivity. Similar observations attend Nakano’s work
with the catechol caffeic acid and a bacterial pathogen
of the silk moth caterpillar.4 At present, this inferential
evidence is consistent with a model in which the covalent
modification of a key viral protein(s) with polyphenol
thwarts viral infectivity. However, no direct chemical
characterization of the molecular-level details of these
presumed polyphenol-protein linkages has been forth-
coming.
Speculation on the nature of the conjugation process
commonly includes combination of a nucleophilic amino
acid residue (cysteine, lysine, histidine) with an electro-
philic orthoquinone derived from catechol oxidation.
Many model studies utilizing simple amino acid deriva-
tives and either in situ-generated or stable orthoquinones
undergird this speculation.5 The case for cysteine is
stronger than the nitrogen-based nucleophilic amino
acids, but the influence of the protein framework in
modulating the reactivity of these nucleophilic species
remains unknown.
insect-plant interactions remains an enduring issue in
chemical ecology. Numerous studies have demonstrated
negative consequences upon polyphenol consumption by
insects, including antidigestive/antiassimilatory effects,
enzyme inhibition, and feeding deterrence.1 More re-
cently, a beneficial impact of polyphenol ingestion, stem-
ming from a reduction in pathogen-induced mortality, has
been uncovered.2 This divergence of outcome serves to
underscore the complex and multifaceted nature of the
molecular-level interactions which might conceivably
underlie these phenomena.
Many of the deleterious effects of polyphenol consump-
tion have been attributed to noncovalent protein binding
via some favorable combination of hydrogen bonding and
hydrophobic association. However, polyphenol prophyl-
axis in insect-pathogen interactions may arise from
alternative modes of chemical interaction. Circumstantial
evidence in a few systems implicates covalent bond
formation between the phenol ring and a pathogen-
derived protein in diminishing mortality. Elegant studies
† Department of Chemistry.
‡ Pesticide Research Laboratory.
(1) (a) Schultz, J . C. In Chemistry and Significance of Condensed
Tannins; Hemingway, R. W., Karchesy, J . J ., Eds.; Plenum Press: New
York, 1989; p 417 and references therein. (b) Rhoades, D. F.; Cates, R.
G. Rec. Adv. Phytochem. 1976, 10, 168. (c) Bernays, E. A.; Driver, G.
C.; Bilgener, M. In Advances in Ecological Research; Bergon, M., Fitter,
A. H., Ford, E. D., MacFadyen, Eds.; Academic Press: London, 1989;
p 263. (d) Feeny, P. Ecology 1970, 51, 565. (e) Martin, J . S.; Martin,
M. M.; Bernays, E. A. J . Chem. Ecol. 1987, 13, 605. (f) Feeny, P.
Phytochemistry 1969, 8, 2119. (g) Mole, S.; Waterman, P. G. Phy-
tochemistry 1987, 26, 99. (h) Steinly, B. A.; Berenbaum, M. Ent. Exp.
Appl. 1985, 39, 3. (i) Bettolo, G. B. M.; Marta, M.; Pomponi, M.;
Bernays, E. A. Biochem. Syst. Ecol. 1986, 14, 249. (j) Veau de E. J . I.;
Schultz, J . C. J . Chem. Ecol. 1992, 18, 1437.
(2) (a) Schultz, J . C.; Hunter, M. D.; Appel, H. M. In Plant
Polyphenols: Synthesis, Properties, Significance; Hemingway, R. W.,
Laks, P. E., Eds.; Plenum Press: New York, 1992; p 621. (b) Schultz,
J . C.; Keating, S. T. In Microbial Mediation of Plant-Herbivore
Interactions; Barbosa, P., Krischik, V. A., J ones, C. G., Eds.; J ohn Wiley
& Sons: New York, 1991; p 489. (c) Hoover, K.; Yee, J . L.; Schultz, C.
M.; Rocke, D. M.; Hammock, B. D.; Duffey, S. S. J . Chem. Ecol. 1998,
24, 221.
The dynamics of another pathogen-herbivore system
consisting of the gypsy moth caterpillar (GMc) and
Lymantria dispar nuclear polyhedrosis virus (LdNPV)
appears to be mediated by polyphenolic compounds as
(3) (a) Felton, G. W.; Duffey, S. S. J . Chem. Ecol. 1990, 16, 1221.
(b) Felton, G. W.; Donato, K.; del Vecchio, R. J .; Duffey, S. S. J . Chem.
Ecol. 1989, 15, 2667.
(4) Nakano, H.; Tahara, S.; Iizuka, T.; Mizutani. Agric. Biol. Chem.
1987, 51, 549.
10.1021/jo982477n CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/20/1999

Mediating Insect-Pathogen Interactions
J . Org. Chem., Vol. 64, No. 16, 1999 5795
well.2a,2b,6 Gallotannins (polygalloylated glucose esters)
are the presumptive agents which attenuate the severity
of LdNPV infection in GMc’s. Gallotannins diminish GMc
fecundity, but that negative impact is more than com-
pensated by a significantly increased survivorship for
GMc’s infected with LdNPV.7 Development of a cellular-
or molecular-level rationale for these observations can
be guided by the tomato fruitworm study with the similar
HzSNPV and by the discovery of GMc midgut conditions
(pH ) 9-11, redox potential ) +47 to +304 mV)
conducive to catechol oxidation.8
F igu r e 1. Galloyl-derived probe molecules for polyhedrin
conjugation.
Therefore, it is possible that ingested gallotannins are
oxidized to reactive orthoquinones in the GMc midgut
and that these electrophiles bind covalently to the LdNPV
OB matrix protein polyhedrin (as well as other viral and/
or host proteins). This covalent linkage may disrupt any
number of recognition/disassembly processes required for
viral infection, although lack of any firm evidence on this
latter point renders speculation on these downstream
events untestable as yet.
A stable galloyl-derived model orthoquinone O-methyl
ether 1a , generated by oxidation of methyl 3-O-methyl-
gallate, efficiently traps cysteine and protected cysteine
derivatives as 1,6 conjugate adducts 3a /3b, eq 1.5i Con-
ate substructure characteristic of some ellagitannin
natural products.9 However, this putative oxidation
product of methyl gallate could be trapped by thiol
nucleophiles under conditions where dimerization is
suppressed (vide infra).
Acquisition of direct chemical evidence for gallotannin/
LdNPV polyhedrin covalent attachment under conditions
mimicking the GMc midgut would provide support for
this hypothesis. While it is possible that key interactions
with other viral (e.g., pp34, p25, pg41) or host proteins
play a critical role in reducing infectivity, polyhedrin is
a logical first target by analogy to the tomato fruitworm
work and by virtue of its overwhelming abundance in the
viral package. Two problems attend any attempts to
utilize galloyl-derived orthoquinones as protein traps
under physiological conditions: (1) The high reactivity
of 1b suggests that it will be difficult to maintain
adequate concentrations of a hydroxyorthoquinone dur-
ing exposure to viral protein, as would be required to
obtain sufficient quantities of covalent adduct for char-
acterization. In vivo, this problem may not be so acute,
as only a low steady-state concentration of electrophile
may be required to disrupt viral chemistry. (2) Reoxida-
tion of the first-formed adduct to furnish a new reactive
electrophilic orthoquinone, followed by trapping of this
species with a second protein nucleophile,5a,5b,5l,10 may
provide only cross-linked, intractable, uncharacterizable
materials. Polyphenol-based cross-linking of biological
macromolecules plays a central role in numerous meta-
bolic processes5a,j,11 and in fact may be important in
hampering virion release from the OB’s. Nevertheless,
these two observations pose great difficulties when the
goal is isolation and characterization of galloyl-polyhe-
drin monoadducts from a physiologically realistic reaction
medium.
jugate addition of histidine to this electrophile is prob-
lematic (∼16%), while lysine (or protected versions) is not
an effective coupling partner at all. Attempts to prepare
and isolate the parent unetherified hydroxyorthoquinone
1b were frustrated by its ready dimerization (below room
temperature) to furnish the dehydrohexahydroxydiphen-
(5) (a) Sugumaran, M.; Dali, H.; Semensi, V. Arch. Insect Biochem.
Physiol. 1989, 11, 127. (b) Salgues, M.; Cheynier, V.; Gunata, Z.; Wylde,
R. J . Food Sci. 1986, 51, 1191. (c) Dudley, E. D.; Hotchkiss, L. H. J .
Food Biochem. 1989, 13, 65. (d) Richard, F. C.; Goupy, P. M.; Nicolas,
J . J .; Lacombe, J .-M.; Pavia, A. A. J . Agric. Food Chem. 1991, 39, 841.
(e) Cilliers, J . J . L.; Singleton, V. L. J . Agric. Food Chem. 1990, 38,
1789. (f) Ito, S.; Palumbo, A.; Prota, G. Experientia 1985, 41, 960. (g)
Abul-Hajj, Y. J .; Tabakovic, K.; Gleason, W. B.; Ojala, W. H. Chem.
Res. Toxicol. 1996, 9, 434. (h) Iverson, S. L.; Shen, L.; Anlar, N.; Bolton,
J . L. Chem. Res. Toxicol. 1996, 9, 492. (i) Quideau, S.; Feldman, K. S.;
Appel, H. M. J . Org. Chem. 1996, 61, 6656. (j) Merritt, M. E.;
Christensen, A. M.; Kramer, K. J .; Hopkins, T. L.; Schaefer, J . J . Am.
Chem. Soc. 1996, 118, 11278. (k) Shen, X.-M.; Dryhurst, G. J . Med.
Chem. 1996, 39, 2018.
Two distinct experimental approaches were designed
to overcome these problems. The initial approach in-
volved anchoring an unetherified galloyl derivative to a
solid support. A reactive hydroxyorthoquinone 4 (Figure
1) derived by oxidation of the triphenolic precursor should
not be consumed by dimerization (or polymerization) to
the extent that site isolation is achieved. Removal of
excess oxidant, application of a basic protein solution to
trap the bound electrophile under biologically relevant
conditions, and eventual adduct cleavage from the resin
(6) (a) Keating, S. T.; Hunter, M. D.; Schultz, J . C. J . Chem. Ecol.
1990, 16, 1445. (b) Rossiter, M.; Schultz, J . C.; Baldwin, I. T. Ecology
1988, 69. 267.
(7) (a) Keating, S. T.; Yendol, W. G. Environ. Entomol. 1987, 16,
459. (b) Keating, S. T.; Yendol, W. G.; Schultz, J . C. Environ. Entomol.
1988, 17, 952. (c) Foster, M. A.; Schultz, J . C.; Hunter, M. D. J . Anim.
Ecol. 1992, 61, 509. (d) Keating, S. T.; Hunter, M. D.; Schultz, J . C. J .
Chem. Ecol. 1990, 16, 1445.
(9) Quideau, S.; Feldman, K. S., J . Org. Chem. 1997, 62, 8809.
(10) (a) Matheis, G.; Whitaker, J . R. J . Food Biochem. 1984, 137.
(b) Hanzlik, R. P.; Harriman, S. P.; Frauenhoff, M. M. Chem. Res.
Toxicol. 1994, 7, 177. (c) Xu, R.; Huang, X.; Hopkins, T. L.; Kramer,
K. Insect Biochem. Mol. Biol. 1997, 27, 101.
(11) (a) Kalyanaraman, B.; Premovic, P. I.; Sealy, R. C. J . Biol.
Chem. 1987, 262, 11080. (b) Brunet, P. C. J .; Coles, B. C. Proc. R. Soc.
London B. 1974, 187, 133. (c) Sugumaran, M. In Advances in Insect
Physiology; Evans, P. D., Wigglesworth, V. L., Eds.; Academic Press:
London: 1988; p 179.
(8) (a) Schultz, J . C.; Lechowicz, M. J . Oecologia 1986, 71, 133. (b)
Appel, H. M.; Maines, L. W. J . Insect Physiol. 1995, 41, 241. (c) Appel,
H. M.; Martin, M. M. J . Chem. Ecol. 1990, 16, 3277.

5796 J . Org. Chem., Vol. 64, No. 16, 1999
Feldman et al.
Sch em e 1
Sch em e 2
may provide the desired monoconjugation products. In
an alternative strategy, use of a stable galloyl-derived
orthoquinone monoether 5, which bears both a base-
solubilizing group (CO2H) and a fluorescent label, may
provide access to a polyhedrin monoadduct under condi-
tions similar to those in the GMc midgut. In this instance,
adduct overoxidation/cross-linking should be minimized
as excess oxidant is not present. The fluorescent tag will
facilitate reaction monitoring in these in vitro experi-
ments.
Sch em e 3
Resu lts a n d Discu ssion
Solid Su p p or t Stu d ies. The requirement for site
isolation of a reactive hydroxyorthoquinone like 1b was
reinforced by the feasibility study shown in Scheme 1.
Treatment of methyl gallate (6) with orthochloranil at
low temperature, followed by cannulation of this hydroxy-
orthoquinone solution into excess, chilled thiophenol, led
to a complex product mixture from which trace but
characterizable quantities of the mono- and bis(thio-
phenol) adducts 7 and 8, respectively, were isolated. No
signals characteristic of the orthoquinone dimer 9 could
be identified in the 1H NMR spectrum of the crude
product mixture. In fact, the 1H NMR data were not
inconsistent with the signature of predominantly oligo-
meric or polymeric material, perhaps resulting from a
nucleophile (PhSH)-initiated oligomerization of the puta-
tive intermediate 1b. Thus, the propensity for both
polymolecular condensation and overoxidation (cf. 8)
clearly renders 1b unsuitable as a solution phase thiol
trapping agent in vitro.
The initial attempts to prepare a galloyl hydroxyortho-
quinone-capped resin used H2O-compatible Tentagel-S
with a photolabile nitroveratrole linker, Schemes 2 and
3. A preliminary model study (Scheme 2) demonstrated
that this photochemical detachment protocol is tolerated
by the galloyl moiety. Thus, esterification of the protected
gallic acid 11 with the linker unit 1212 and desilylation
of the derived conjugate 13 afforded the phenolic species
14. Irradiation of this galloylated linker assembly under
standard conditions delivered the gallic acid 10 cleanly
and in good yield.
These encouraging results prompted studies with the
Tentagel-S system (Scheme 3). Synthesis of the requisite
galloyl-functionalized Tentagel resin 21 commenced with
the known veratraldehyde derivative 1513 and utilized
the trisilylated gallic acid 17 as the galloyl donor. Resin
loading was accomplished by combining the terminal
amine moieties of Tentagel-S with 10 equiv of activated
ester 18 in the presence of HOBt. An Ac2O chaser capped
off unreacted amine. The efficiency of galloyl loading was
assayed by irradiating a 138 mg sample of 19 for 9 h in
aqueous THF-EtOH, which furnished 16.3 mg of 17
following solvent evaporation (77% of the theoretical).
Desilylation of the resin-bound galloyl ester 19 and
(12) McMinn, D. L.; Greenberg, M. M. Tetrahedron 1996, 52, 3827.
(13) Venkatesan, H.; Greenberg, M. M. J . Org. Chem. 1996, 61, 525.

Mediating Insect-Pathogen Interactions
J . Org. Chem., Vol. 64, No. 16, 1999 5797
Sch em e 4
orthochloranil-mediated oxidation of the resulting tri-
phenol 20 furnished bright red beads after filtering and
extensive washing. Addition of an aqueous solution of
L-cysteine to these presumably electrophilically activated
beads instantaneously discharged the red color. Unfor-
tunately, irradiation of the resultant brown resin under
conditions which earlier met with success (e.g., 14 f 10,
19 f 17) did not provide any characterizable organic
material. It is possible that the long and flexible poly-
(ethylene glycol) chains which link the functionalized end
group to the polystyrene backbone of Tentagel-S could
not sustain the required site isolation. In this case, the
reactive galloyl hydroxyorthoquinone termini might have
been consumed through thiol-initiated oligomerization as
suggested by the observations with the model system 1b
(Scheme 1). Release of such a cross-linked oligomer from
the Tentagel matrix could be problematic.
The Wang resin presents an alternative support which
does offer better prospects for site isolation but at the
expense of H2O compatibility. Use of this resin requires
that the terminal cysteine-galloyl conjugate is stable to
the acidic cleavage conditions. To test this point, model
system 25 was prepared and subject to typical cleavage
conditions (50% TFA in CH2Cl2), eq 2. A good yield of
thesis of this deep red resin proceeded in an analogous
manner to the hydroxy analogue 29a (Scheme 4). Expo-
sure of these electrophile-terminated beads to cysteine
derivative 31 in THF or, independently, to 1,2-diamino-
benzene in HOAc, followed by TFA-mediated adduct
release and hydrogenolysis (with the cysteine adduct) or
esterification (with the diamine adduct), afforded small
quantities of the expected products 26 and 30, respec-
tively. Cysteine itself did not react with the Wang resin
29b in aqueous THF, plausibly because of solvent in-
compatibilities. These results demonstrate that a Wang
resin-bound galloyl orthoquinone is a competent partner
for combination with certain nucleophiles. Hence, the
failure to detect adduct formation with the related
hydroxyorthoquinone-capped resin 29a might speak to
the high and indiscriminate reactivity of this latter
species, even while presumably site-isolated on the Wang
resin. If the more tractable monoetherified galloyl ortho-
quinone species must be utilized in the planned protein
(cysteine) trapping experiments, the requirement for site
isolation, and hence solid-phase chemistry, is removed.
The documented stability of the monoether galloyl ortho-
quinones to dimerization/oligomerization and/or subse-
quent adduct reoxidation suggests that soluble versions
of the trapping electrophile, such as 5, can be explored
profitably to probe galloyl-protein interactions relevant
to the LdNPV-GMc system.
the cysteine conjugate 26, free of decomposition products,
was isolated. With these promising results in hand,
synthesis of Wang resins terminated in both galloyl
hydroxyorthoquinone and galloyl methoxyorthoquinone,
29a ,b, respectively, proceeded as shown in Scheme 4. The
hydroxyl functionality of the resin was acylated with
either acid 11 or acid 17 in two repetitive cycles of
diisopropylcarbodiimide-mediated esterification. Post-
esterification treatment of the crude galloylated resins
with Ac2O capped off any unreacted alcohol. Resin
loading was assayed, in the case of 28b, by treatment of
14.7 mg of material with 50% TFA/CH2Cl2 for 15 min.
Evaporation of the supernatant furnished 2.5 mg of 3-O-
methyl gallic acid (92% of the theoretical). Oxidation of
28a with excess orthochloranil provided bright orange
beads (29a ?) following extensive washing. Unfortunately,
the thiol trapping/resin release experiments did not
proceed any more satisfactorily with the Wang resin than
they did with the Tentagel system. Treatment of these
orange beads with either L-cysteine, the cysteine deriva-
tive 31, or PhSH all led to immediate color discharge.
However, TFA-mediated cleavage of the reaction product-
(s) did not afford even trace amounts of the expected
thiol-gallate adducts whose spectral data were in hand
from previous studies.
Whether the Wang resin itself contributed to the
failure of the desired chemistry was probed by examina-
tion of the nucleophile addition/resin release chemistry
of the galloyl methoxyorthoquinone 29b, a stable ana-
logue of the hydroxy species 29a whose solution coun-
terpart shows no tendency toward polymerization. Syn-
Solu ble P r obe Stu d ies. Synthesis of the base-soluble
fluorescently labeled galloyl orthoquinone 5 proceeded as
outlined in Scheme 5. The dansyl amide of serine benzyl
ester, 32, was condensed with the O-methyl dibenzyl

5798 J . Org. Chem., Vol. 64, No. 16, 1999
Feldman et al.
Sch em e 5
F igu r e 2. Fluorescence data for the interaction of ortho-
quinone 5 with 39 and 40.
ensure that the cysteine thiol of 39 was in the reduced
form. The DTE was removed just prior to addition of the
orthoquinone 5 through exhaustive dialysis against
phosphate buffer.
gallic acid derivative 33 to furnish ester 34 en route to
the unprotected galloyl-serine-dansyl construct 35 (H2/
Pd). Orthochloranil-mediated oxidation of 35 in 1:2
Et2O-THF then afforded the pure orthoquinone 5 in
moderate yield as a pink solid. The solvent composition
noted above was critical in preserving solubility on the
starting catechol 35 while at the same time promoting
precipitation of the pure product 5 from the reaction
mixture. Failure to isolate 5 in this manner led to
contaminated material whose purification was problem-
atic. Orthoquinone 5 was insoluble in pure H2O but did
dissolve in pH 7.8 phosphate buffer. The electrophilicity
of this galloyl orthoquinone was similar to the previously
studied 1a , as indicated by the moderate yields of adduct
formation (37 and 38) upon reaction with either cysteine
(36) or the cysteine derivative 31, respectively (Scheme
5). The regiochemical assignment of these cysteine ad-
Treatment of 40 in pH 7.8 phosphate buffer with a 6.5-
fold molar excess of 5 resulted in a modest increase in
fluorescence intensity (ca. 10%) and a slight blue shift
(∼6 nm), Figure 2. Similar exposure of 39 to 6.5 equiv of
5 in pH 7.8 buffer led to an 80% increase in fluorescence
output and a pronounced (19 nm) blue shift, Figure 2.
Both observations are consistent with a reaction between
39 and 5 in which the dansyl moiety becomes imbedded
in a hydrophobic microenvironment as might accompany
attachment to the protein. While the lack of a similar
fluorescence response with the thiol-less but otherwise
identical protein 40 is suggestive of chemistry occurring
at the cysteine residue of 39, these measurements do not
address the issue of covalent vs noncovalent attachment
per se.
1
dition products rested upon comparisons of H and 13C
Evidence which implicates the cysteine thiol of 39 in
covalent bond formation can be gleaned from the results
of some simple chemical tests. Direct assay of solvent
accessible thiol groups in proteins can be accomplished
through use of Ellman’s reagent, 5,5′-dithiobis(2-nitro-
benzoic acid (DTNB, 41),16 eq 3. Titration of 39 with
NMR data with those of 3a /3b.
The utility of orthoquinone 5 as a thiol trap within the
more demanding context of a protein target was probed
with a well-characterized cysteine-containing model pro-
tein prior to examining components of the relatively
unexplored LdNPV system. The structurally described
dihydrofolate reductase (DHFR) mutant N37C/C85A/
C152S 3914 was chosen for its manageable size (for
eventual electrospray MS analysis), availability in ser-
viceable quantities and high purity, and solubility/
stability in pH 7.8 phosphate buffer. This particular
mutant has a single cysteine residue engineered into a
solvent-exposed loop. A C-terminus hexahistidine puri-
fication tag raises an issue of competition for electrophile
5 (vide supra), but subsequent studies (vide infra) allay
these concerns. A second DHFR double mutant C85A/
C152S 40,15 which lacks cysteine entirely, was used as a
control in the labeling studies with 5. Neither 39 nor 40
exhibited fluorescence at 560 nm, the wavelength of
interest for the dansyl chromophore in 5. Both proteins
were treated with dithioerythritol (DTE) prior to use to
DTNB followed by spectrophotometric quantitation of the
generated 5-mercapto-2-nitrobenzoate (MNB, 42) re-
vealed the expected result: almost all of the single thiol
group (0.84 ( 0.04) is modified by reagent (cf. 43). A
similar experiment with the thiol-less protein 40 led to
no measurable production of 42, either before or after
treatment with 5. In the trials of interest, treatment of
(14) Gegg, C. V.; Bowers, K. E.; Matthews, C. R. In Techniques in
Protein Chemistry VII; Academic Press: Orlando, FL, 1996; p 439.
(15) Iwakura, M.; J ones, B. J .; Luo, J .; Matthews, C. R. J . Biochem.
1995, 117, 480.
(16) Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70.

Mediating Insect-Pathogen Interactions
J . Org. Chem., Vol. 64, No. 16, 1999 5799
protein)17d,e were bonding to 5. No structural information
on polyhedrin is available, and so the accessibility of 5
to these three nucleophilic residues cannot be ascer-
tained. Similar mobility of native polyhedrin and 2-mer-
captoethanol-treated polyhedrin upon SDS-PAGE was
observed, a result consistent with an absence of disulfide
bridges.
The DTNB assays for cysteine modification in polyhe-
drin were more encouraging. In the absence of ortho-
quinone 5, 2.08 ( 0.10 of polyhedrin’s three cysteine
residues were modified by DTNB. However, only ca. 1.30
( 0.06 cysteines, on average, were modified by DTNB
after exposure of polyhedrin to a 6.4 molar excess of 5.
Thus, these assays implicate ca. 0.8 cysteine residues per
polyhedrin, on average, as forming covalent attachments
to 5.
Companion experiments on a partially denatured
sample of polyhedrin18 engendered similar results. DTNB
assays conducted on a sample of polyhedrin in 6 M urea,
and on polyhedrin pretreated with a 2.5 molar excess of
orthoquinone 5 in 6 M urea, revealed that ∼0.6 equiv of
cysteine per protein, on average, were modified by 5.
Unfortunately, further attempts to characterize the puta-
tive polyhedrin-5 adduct via electrospray MS were not
fruitful. Neither the native protein nor its 5 conjugate
gave interpretable ESMS signals. The higher molecular
weight of polyhedrin (∼31 kDa) and its tendency to
aggregate compared with 39 may have contributed to the
failure of this technique to provide useful information.
In summary, the development of two distinct galloyl
orthoquinone-based strategies for protein thiol covalent
trapping was pursued. The initial approach, which relied
on achieving site-isolation of a very reactive hydroxy-
orthoquinone gallate on a solid support, did not provide
a useful thiol-trapping reagent. It is likely that sufficient
site isolation was not achieved. A second approach
utilized a soluble, stable methoxyorthoquinone construct
which did react productively with cysteine and a small
cysteine-containing model protein. Unambiguous evi-
dence for similar reaction with the larger and less stable
protein of interest in the LdNPV system, polyhedrin, was
not forthcoming. However, the results of DTNB free thiol
assays were consistent with covalent modification of one
of the three polyhedrin cysteines by orthoquinone reagent
5. Application of this probe reagent to the GMc/LdNPV
system is planned, and results will be reported in due
course.
F igu r e 3. Fluorescence data for the interaction of ortho-
quinone 5 with polyhedrin.
39 with 1.0 molar equiv of 5 and then DTNB released
ca. 0.5 equiv of MNB. Thus, approximately half of the
original thiol in 39 is consumed by 5 when admixed in
equimolar ratio. When 6.5 molar equiv of 5 to 39 was
used, a similar DTNB assay produced no detectable
MNB. In this instance, the excess of electrophile 5 was
sufficient to modify all of the protein’s free thiol.
This circumstantial evidence implicates cysteine as the
reactive amino acid with orthoquinone 5, but it does not
address the possibility that other nucleophilic amino
acids in 39 (e.g., lysine, histidine) may also trap 5 to a
minor extent. However, localization of probe 5 to C37 in
39 can be deduced from comparison of the mass spectral
data for 39 before and after treatment with orthoquinone.
Electrospray MS was capable of detecting a parent ion
for this small and relatively well-behaved protein (obsd
m/e 18 761, calcd m/e 18 764). Similar analysis of the
5-labeled version of 39 provided evidence for incorpora-
tion of one molecule of 5 only (obsd m/e 19 266, calcd m/e
19 266, ) 39 + 5). Since the DTNB studies indicated that
cysteine was modified, and the MS data were consistent
with attachment of only a single molecule of 5, it appears
that the galloyl-derived orthoquinone remains quite
selective for thiol nucleophiles even within the highly
complex microenvironment of a protein.
These promising model system results provided en-
couragement for investigating the interaction of LdNPV
polyhedrin with orthoquinone 5. Polyhedrin17 in pH 8.5
phosphate buffer was incubated with 6.4 equiv of 5 and
then examined by fluorescence spectroscopy, Figure 3.
The response was much less pronounced than with 39
and more closely resembled that of the control protein
40. Only a slight increase in fluorescence intensity (ca.
5%) and a modest blue shift (∼6 nm) accompanied
polyhedrin/5 interaction, which raised concerns about
whether any of the protein’s three cysteine residues
(positions 133, 144, and 178 in the 245 amino acid
Exp er im en ta l Section
Fluorescence spectra were obtained on a titrating dif-
ferential/ratio spectrofluorometer. High-resolution fast atom
bombardment mass spectra were run at the University of
Texas at Austin. Electrospray mass spectra were run at the
Protein Chemistry Laboratory, Department of Molecular Biol-
ogy and Pharmacology, Washington University School of
Medicine, St. Louis, MO. Combustion analyses were performed
by Midwest Microlabs, Indianapolis, IN, or by Galbraith
Laboratories, Knoxville, TN. Liquid (flash) chromatography19
was carried out using 32-63 mm silica gel and the indicated
solvent. Et2O and THF were purified by distillation from
sodium/benzophenone under nitrogen, while CH2Cl2 was dis-
tilled from CaH2 under nitrogen. Moisture sensitive reactions
(17) (a) Bilimoria, S. L. In Viruses of Invertebrates; Kurstak, E., Ed.;
Marcel Dekker: New York, 1991; p 1. (b) Vlak, J . M.; Rohrmann, G.
F. In Viral Insecticides for Biological Control; Maramorosch, K.,
Sherman, K. E., Eds.; Academic Press: Orlando, FL, 1985; p 489. (c)
Kozlov, E. A.; Levitina, T. L.; Gusak, N. M. In The Molecular Biology
of Baculoviruses; Doerfler, W., Bihm, P., Eds.; Springer-Verlag Ber-
lin: Heidelberg, Germany, 1986; Vol. 131, p 135. (d) Smith, I. R. L.;
van Beek, N. A. M.; Podgwaite, J . D.; Wood, H. A. Gene 1988, 71, 97.
(e) Chang, M. T.; Lanner-Herrera, C.; Fikes, M. J . Invert. Path. 1989,
53, 241. (f) McCarthy, W. J .; Liu, S. J . Invert. Path. 1976, 28, 57.
(18) Circular dichroism measurements of native polyhedrin and
polyhedrin in 6 M urea revealed that the native protein possessed a
number of elements of secondary structure which were altered upon
urea treatment.
(19) Still, W. C.; Kahn, M.; Mitra, A. J . Org. Chem. 1978, 43, 2923.

5800 J . Org. Chem., Vol. 64, No. 16, 1999
Feldman et al.
were carried out in predried glassware under an inert atmo-
sphere of argon (Ar). Copies of 13C NMR spectra are provided
in the Supporting Information to establish purity for those
compounds that were not subject to combustion analyses.
Oxid a tion of Meth yl 3,4,5-Tr ih yd r oxyben zoa te (6).
Methyl 3,4,5-trihydroxybenzoate (6) (0.50 g, 2.7 mmol) in 60
mL of dry Et2O was added dropwise over 3 h to a stirring
solution of orthochloranil (0.74 g, 3.0 mmol) in 12 mL of dry
Et2O at -30 °C. The reaction mixture was transferred to a
dry ice-CH3CN-cooled (-42 °C) addition funnel and was added
to a solution of thiophenol (0.34 mL, 3.3 mmol) in 19 mL of
dry CH2Cl2 over 1 h. The solvents were removed under reduced
pressure, and a mixture of the two products 7 and 8 was
isolated by silica gel chromatography, using sequential elution
with CH2Cl2, 2% CH3OH in CH2Cl2, and finally 5% CH3OH
and 0.25% HOAc in CH2Cl2. Monoadduct 7 and bisadduct 8
were separated by preparative TLC, using 5% CH3OH and
0.25% HOAc in CH2Cl2, to obtain 33 mg (3%) of 7 and 11 mg
(1%) of 8.
0.25 (s, 6 H), 0.18 (s, 6 H); 13C NMR (75 MHz, CD3COCD3) δ
173.7, 166.0, 154.8, 152.8, 148.7, 148.2, 142.1, 141.3, 127.6,
122.8, 116.2, 112.3, 110.4, 107.0, 69.1, 64.3, 56.8, 55.8, 51.8,
30.8, 26.5, 26.4, 25.3, 19.4, 19.2, -3.5, -3.6; FABMS m/z
(relative intensity) 694 (MH+, 5).
4-(3-(Ca r bom eth oxy)p r op oxy)-5-m eth oxy-2-n itr oben -
zyl 4,5-Dih yd r oxy-3-m eth oxyben zoa te (14). A solution of
tetrabutylammonium fluoride (TBAF) (1.0 M in THF, 0.63 mL,
0.63 mmol) was syringed into a solution of galloylated ni-
trobenzyl derivative 13 (0.20 g, 0.29 mmol) in 10 mL of THF
under Ar. The reaction proceeded to completion within 5 min
(TLC), at which point the reaction mixture was added to ice-
cold 1 M H3PO4. The product was extracted into Et2O, washed
with brine, dried over Na2SO4, filtered, and concentrated, and
the residue was purified by silica gel chromatography using
30% EtOAc in hexanes as the eluent. The product was obtained
as an off-white solid (0.13 g, 99%): IR (CH2Cl2) 1733 cm-1; 1H
NMR (300 MHz, CD3COCD3) δ 7.73 (s, 1 H), 7.32 (s, 1 H), 7.27
(d, J ) 1.9 Hz, 1 H), 7.22 (d, J ) 1.9 Hz, 1 H), 5.62 (s, 2 H),
4.19 (t, J ) 6.3 Hz, 2 H), 3.97 (s, 3 H), 3.87 (s, 3 H), 3.63 (s, 3
H), 2.55 (t, J ) 7.3 Hz, 2 H), 2.17-2.08 (m, 2 H); 13C NMR (75
MHz, CD3COCD3) δ 173.7, 166.2, 154.8, 148.64, 148.60, 146.0,
141.1, 140.0, 127.8, 121.2, 112.1, 111.7, 110.4, 105.7, 69.1, 63.9,
56.7, 56.5, 51.7, 30.7, 25.2; FABMS m/z (relative intensity) 465
(MH+, 8).
7: IR (CH2Cl2) 1725 cm-1; 1H NMR (300 MHz, CD3COCD3)
δ 7.23-6.97 (m, 6 H), 3.69 (s, 3 H); 13C NMR (75 MHz,
CD3COCD3) δ 167.7, 148.6, 147.6, 138.9, 136.6, 129.7, 129.5,
127.3, 126.0, 110.8, 107.7, 52.0; FABMS m/z (relative inten-
sity) 293 (MH+, 30); HRMS (FABMS) calcd for C14H12O5S1
292.040 545, found 292.040 028.
Alcoh ol 16. DCC (2.18 g, 10.6 mmol) in 20 mL of dry CH2Cl2
was added dropwise over 20 min to a stirring solution of
nitrated vanillin derivative 1513 (2.0 g, 7.1 mmol) and 2,4,5-
trichlorophenol (2.09 g, 10.6 mmol) in 39 mL of dry CH2Cl2
under Ar at room temperature. The reaction mixture was
filtered at 4 h after the completion of addition to remove
dicyclohexylurea, and the filtrate was diluted with CH2Cl2 and
H2O. The organic layer was separated out, and the aqueous
layer was reextracted with CH2Cl2. The organic layers were
combined, washed with brine, dried over Na2SO4, filtered, and
concentrated to yield the crude trichlorophenyl ester of 15.
Purification of this ester was achieved via silica gel chroma-
tography, eluting with 40% EtOAc in hexanes, to yield 2.95 g
(90%) of the ester as a bright yellow solid: IR (CH2Cl2) 1772,
8: IR (CH2Cl2) 1712 cm-1; 1H NMR (300 MHz, CD3COCD3)
δ 7.27-7.11 (m, 10 H), 3.66 (s, 3 H); 13C NMR (75 MHz,
CD3COCD3) δ 167.7, 150.3, 141.0, 137.9, 134.9, 129.6, 127.9,
126.5, 106.6, 52.1; FABMS m/z (relative intensity) 401 (MH+,
16); HRMS (FABMS) Calcd for C20H16O5S2 400.043 917, found
400.043 872.
3,4-Bis(ter t-b u t yld im et h ylsiloxy)-5-m et h oxyb en zoic
Acid (11). Diisopropylethylamine (8.5 mL, 49 mmol) was
syringed into a solution of methyl 3-O-methyl gallate (10)20
(2.0 g, 11 mmol) and tert-butyldimethylsilyl chloride (6.2 g,
41 mmol) in 37 mL of dry DMF under Ar. The reaction mixture
was stirred for 16 h at room temperature and was poured into
ice-cold 1 M H3PO4. The product was extracted into hexanes
and then 1:1 Et2O-hexanes. The combined organic layers were
washed with brine, dried over Na2SO4, filtered, and concen-
trated to provide an orange oil. The trisilylated product (and
some disilylated material) was treated with 35 mL of 1:1:3
H2O-THF-AcOH (an additional amount of THF may be
required to obtain a homogeneous solution) for 90 min, and
the reaction mixture was poured into ice-cold water. The
product was extracted into 1:1 Et2O-hexanes, washed with
copious quantities of water and then brine, dried over Na2SO4,
filtered, and concentrated to yield 3.36 g (75%) of an off-white
powder. The acid 11 was used without further purification:
1
1690 cm-1; H NMR (300 MHz, CDCl3) δ 10.44 (s, 1 H), 7.62
(s, 1 H), 7.55 (s, 1 H), 7.41 (s, 1 H), 7.30 (s, 1 H), 4.29 (t, J )
6.1 Hz, 2 H), 4.01 (s, 3 H), 2.90 (t, J ) 7.1 Hz, 2 H), 2.42-2.32
(m, 2 H); 13C NMR (75 MHz, CDCl3) δ 187.6, 169.8, 153.4,
151.5, 145.6, 143.6, 131.4, 131.0, 130.6, 126.0, 125.6, 125.2,
109.9, 108.2, 68.2, 56.6, 30.1, 24.0; CIMS m/z (relative inten-
sity) 462 (MH+, 8); HRMS (FABMS) calcd for C18H14Cl3N1O7
461.991 410, found 461.991 464. The trichlorophenyl ester (0.1
g, 0.2 mmol) was dissolved in 3.4 mL of 6:1 THF-H2O at 0
°C. NaBH3CN (0.16 g, 2.60 mmol) was added in one portion
to the reaction mixture, followed by 1 M HCl dropwise, until
the pH of the solution ranged between 3 and 4. After 20 min
at 0 °C, the reaction mixture was diluted with H2O, and the
product was extracted into EtOAc. The organic layer was
washed with brine, dried over Na2SO4, filtered, and concen-
trated. The crude benzylic alcohol 16 was chromatographed
on silica gel, using 40% EtOAc in hexanes, to yield 74 mg (73%)
1
IR (CH2Cl2) 1718 cm-1; H NMR (200 MHz, CDCl3) δ 7.31 (d,
J ) 2.0 Hz, 1 H), 7.27-7.26 (m, 1 H), 3.83 (s, 3 H), 1.00 (s, 9
H), 0.98 (s, 9 H), 0.23 (s, 6 H), 0.16 (s, 6 H); 13C NMR (50 MHz,
CDCl3) δ 171.9, 151.7, 147.4, 142.0, 120.9, 116.3, 106.5, 55.2,
26.0, 25.8, 18.8, 18.6, -3.9, -4.0; FABMS m/z (relative
intensity) 413 (MH+, 100). Anal. Calcd for C20H36O5Si2: C,
58.21; H, 8.79. Found: C, 58.56; H, 8.81.
of a yellow solid. 16: IR (CH2Cl2) 1772 cm-1 1H NMR (300
;
4-(3-(Ca r bom eth oxy)p r op oxy)-5-m eth oxy-2-n itr oben -
zyl 3,4-Bis(ter t-bu tyld im eth ylsiloxy)-5-m eth oxyben zoa te
(13). Nitrobenzyl alcohol 1212 (0.16 g, 0.55 mmol), gallic acid
derivative 11 (0.15 g, 0.36 mmol), and Ph3P (0.11 g, 0.40 mmol)
were suspended in 5.5 mL of dry Et2O under Ar. Diethyl-
azodicarboxylate (DEAD) (63 µL, 0.40 mmol) was added
dropwise to the reaction mixture via syringe over 20 min. The
clear yellow solution obtained turned cloudy on stirring
overnight under Ar. The reaction mixture was concentrated
to dryness, and the crude product obtained was purified by
silica gel chromatography using 20% EtOAc in hexanes as the
eluent, to yield a pale yellow oil (0.20 g, 81%): IR (CH2Cl2)
MHz, CDCl3) δ 7.72 (s, 1 H), 7.55 (s, 1 H), 7.30 (s, 1 H), 7.20
(s, 1 H), 4.97 (s, 2 H), 4.20 (t, J ) 6.0 Hz, 2 H), 3.99 (s, 3 H),
2.89 (t, J ) 7.2 Hz, 2 h), 2.38-2.29 (m, 2 H); 13C NMR (75
MHz, CDCl3) δ 170.0, 154.3, 147.0, 145.7, 139.6, 132.7, 131.4,
131.0, 130.6, 126.0, 125.3, 111.2, 109.7, 68.0, 62.7, 56.4, 30.4,
24.2; CIMS m/z (relative intensity) 463 (M+, 3); HRMS (EIMS)
calcd for C18H14Cl3N1O7 462.9992, found 462.9980.
3,4,5-Tr is(ter t-bu tyldim eth ylsilyloxy)ben zoic Acid (17).
Diisopropylethylamine (12.2 mL, 35.3 mmol) was syringed into
a suspension of 3,4,5-trihydroxybenzoic acid (2.0 g, 12 mmol)
and tert-butyldimethylsilyl chloride (8.88 g, 29.4 mmol) in 30
mL of dry DMF under Ar at room temperature. The suspension
turned into a clear solution after 30 min and was stirred for
an additional 90 min. The reaction mixture was added to cold
1 M H3PO4, and the product was extracted into hexanes. The
organic extract was washed with a saturated solution of
NaHCO3 followed by brine, dried over Na2SO4, filtered, and
concentrated to a clear oil. The crude tetrasilylated derivative
1
1732 cm-1; H NMR (300 MHz, CD3COCD3) δ 7.73 (s, 1 H),
7.31 (s, 1 H), 7.30-7.29 (m, 2 H), 5.64 (s, 2 H), 4.19 (t, J ) 6.3
Hz, 2 H), 3.98 (s, 3 H), 3.88 (s, 3 H), 3.63 (s, 3 H), 2.55 (t, J )
7.3 Hz, 2 H), 2.17-2.08 (m, 2 H), 1.00 (s, 9 H), 0.99 (s, 9 H),
(20) Scheline, R. R. Acta Chem. Scand. 1966, 20, 1182.

Mediating Insect-Pathogen Interactions
J . Org. Chem., Vol. 64, No. 16, 1999 5801
was treated with 1:1:3 H2O-THF-AcOH (35 mL; additional
THF may be required to obtain a homogeneous solution) for 1
h at room temperature. The reaction mixture was poured into
ice-cold H2O, and the product was extracted into 1:1 Et2O-
hexanes, washed with H2O and brine, dried over Na2SO4,
filtered, and concentrated to a white solid (5 g, 83%), which
was used without further purification: IR (CH2Cl2) 3000-
2400, 1724 cm-1; 1H NMR (300 MHz, CD3COCD3) δ 7.28 (s, 2
H), 1.02 (s, 9 H), 0.96 (s, 18 H), 0.27 (s, 12 H), 0.18 (s, 6 H);
13C NMR (50 MHz, CDCl3) δ 171.7, 148.5, 144.4, 121.0, 116.1,
26.2, 26.1, 18.8, 18.5, -3.6, -3.9; CIMS m/z (relative intensity)
513 (MH+, 100). Anal. Calcd for C25H48O5Si3: C, 58.54; H, 9.43.
Found: C, 58.28; H, 9.29.
Activa ted Ester 18. DEAD (141 µL, 0.90 mmol) was added
dropwise via syringe pump over 25 min to a stirred suspension
of activated benzyl alcohol 16 (0.56 g, 1.2 mmol), 3,4,5-tris-
(tert-butyldimethylsiloxy)benzoic acid (17) (0.42 g, 0.81 mmol),
and Ph3P (0.23 g, 0.89 mmol) at room temperature under Ar.
The reaction mixture was stirred for 16 h and then concen-
trated to dryness. The crude product was purified by silica gel
chromatography, using 20% EtOAc in hexanes as the eluent,
to obtain 0.72 g (92%) of 18: IR (CH2Cl2) 1772, 1717 cm-1; 1H
NMR (300 MHz, CDCl3) δ 7.75 (s, 1 H), 7.54 (s, 1 H), 7.30 (s,
1 H), 7.27 (s, 2 H), 7.04 (s, 1 H), 5.72 (s, 2 H), 4.21 (t, J ) 6.0
Hz, 2 H), 3.88 (s, 3 H), 2.88 (t, J ) 7.2 Hz, 2 H), 2.38-2.29 (m,
2 H), 0.99 (s, 9 H), 0.94 (s, 18 H), 0.24 (s, 12 H), 0.14 (s, 6 H);
13C NMR (75 MHz, CDCl3) δ 169.9, 165.7, 153.9, 148.6, 147.2,
145.7, 143.7, 139.8, 131.5, 131.0, 130.6, 128.0, 126.1, 125.3,
121.3, 115.6, 110.5, 109.8, 68.0, 63.4, 56.2, 30.4, 26.12, 26.10,
24.2, 18.8, 18.5, -3.7, -3.9; FABMS m/z (relative intensity)
959 (M + 2, 1); HRMS (FABMS) calcd for C43H62Cl3N1O11Si3
957.269 631, found 957.268 942.
Resin Loa d in g w ith 18. Tentagel-S NH2 (0.25 g) was
allowed to swell in dry DMF under Ar for 10 min. Ester 18
(0.72 g, 0.75 mmol) and HOBt (0.10 g, 0.75 mmol) were added
to the swollen resin, and the reaction was allowed to proceed
for 12 h. The resin beads were filtered out, washed well with
EtOAc and acetone, and dried under vacuum. The unfunc-
tionalized NH2 termini were capped off by treating the dried
resin beads with 0.28 g of DMAP, 2.80 mL of Ac2O, and 22.5
mL of pyridine for 2 h. The resin beads 19 were washed with
EtOAc, acetone, H2O, and then acetone and then dried under
vacuum.
Desilyla tion of Resin 19. A 100 mg amount of resin 19
was swollen in 1 mL of dry THF for 20 min. A 1.0 M solution
of TBAF (0.01 mL, 0.10 mmol) was syringed into the reaction
mixture, causing the beads to turn brown in color. The reaction
mixture was shaken manually for 5 min, and the beads were
then filtered out. The resin 20 was washed well with THF and
dried under vacuum.
butyldimethylsiloxy)-5-methoxybenzoic acid (11) (1.0 g, 2.4
mmol) and Ph3P (0.70 g, 2.7 mmol) in 37 mL of dry Et2O at
room temperature under Ar. DEAD (0.42 mL, 2.7 mmol) was
added dropwise to the reaction mixture via syringe pump over
1 h, and the resultant clear yellow solution was stirred
overnight at room temperature. The reaction mixture was
concentrated, and the residue was chromatographed on silica
gel, eluting with 20% EtOAc in hexanes, to obtain 23 (0.91 g,
70%) as a pale yellow solid: IR (CH2Cl2) 1710 cm-1; 1H NMR
(200 MHz, CDCl3) δ 7.38 (d, J ) 8.4 Hz, 2 H), 7.27 (d, J ) 1.9
Hz, 1 H), 7.21 (d, J ) 1.8 Hz, 1 H), 6.91 (d, J ) 8.5 Hz, 2 H),
5.26 (s, 2 H), 3.82 (s, 3 H), 3.80 (s, 3 H), 0.98 (s, 9 H), 0.97 (s,
9 H), 0.21 (s, 6 H), 0.20 (s, 6 H); 13C NMR (75 MHz, CDCl3) δ
166.3, 159.5, 151.6, 147.3, 141.1, 129.7, 128.5, 122.0, 115.7,
113.9, 106.0, 66.2, 55.2, 26.0, 25.8, 18.7, 18.5, -3.9, -4.1; CIMS
m/z (relative intensity) 533 (MH+, 55). Anal. Calcd for C28H4O6-
Si2: C, 63.12; H, 8.32. Found: C, 63.03; H, 8.44.
4-Meth oxyben zyl 3-Meth oxy-1,2-d ioxocycloh exa -3,5-
d ien e-5-ca r boh yd r a te (24). A 1.0 M solution of TBAF in
THF (3.3 mL, 3.3 mmol) was syringed into a pale yellow
solution of 23 (0.80 g, 1.5 mmol) in 53 mL of dry THF under
Ar. The reaction mixture turned pale green instantaneously
and was poured into ice-cold 1 M H3PO4, resulting in a
brownish-yellow solution. The product was extracted into Et2O,
and the organic layer was washed with brine, dried over
Na2SO4, filtered, and concentrated to a brown oil. The crude
product was purified by chromatography on silica gel using
50% EtOAc in hexanes as eluent to yield 0.41 g (90%) of the
catechol derived from 23 as a reddish-brown oil: IR (CH2Cl2)
1710 cm-1; 1H NMR (300 MHz, CD3COCD3) δ 7.40 (d, J ) 8.8
Hz, 2 H), 7.28 (d, J ) 1.9 Hz, 1 H), 7.20 (d, J ) 1.9 Hz, 1 H),
6.92 (d, J ) 8.7 Hz, 2 H), 5.24 (s, 2 H), 3.83 (s, 3 H), 3.77 (s, 3
H); 13C NMR (75 MHz, CD3COCD3) δ 166.6, 160.5, 148.5,
145.7, 139.7, 130.7, 129.5, 121.7, 114.6, 111.6, 105.7, 66.6, 56.5,
55.4; CIMS m/z (relative intensity) 304 (M+, 4); HRMS (EIMS)
Calcd for C16H16O6 304.0947, found 304.0923.
A solution of this catechol (0.12 g, 0.40 mmol) in 6 mL of
dry Et2O was added via syringe pump to a solution of
orthochloranil (0.11 g, 0.43 mmol) in 4 mL of dry Et2O under
Ar at -30 °C over 3.5 h. The reaction mixture was stirred for
an additional 14 h at -15 °C. The product was obtained as a
fine red precipitate, which was filtered out, washed with cold
Et2O, and dried under reduced pressure to yield 84 mg (70%)
of orthoquinone 24: IR (CH2Cl2) 1723, 1672, 1626 cm-1 1H
;
NMR (300 MHz, CDCl3) δ 7.34 (d, J ) 8.3 Hz, 2 H), 6.92 (d, J
) 8.0 Hz, 2 H), 6.74 (s, 1 H), 6.48 (s, 1 H), 5.27 (s, 2 H), 3.84
(s. 3 H), 3.83 (s, 3 H); 13C NMR (75 MHz, CDCl3) δ 179.9, 174.2,
164.2, 160.1, 153.2, 141.5, 130.4, 126.6, 124.2, 114.1, 106.4,
68.3, 56.3, 55.3; FABMS m/z (relative intensity) 304 (M + 2,
10). Anal. Calcd for C16H14O6: C, 63.35; H, 4.69. Found: C,
63.57; H, 4.67.
Or th och lor a n il Oxid a tion of Resin 20 a n d Attem p ted
Cystein e Tr a p p in g. The deprotected phenolic resin 20
obtained as described above was allowed to swell for 20 min
in 5 mL of dry Et2O under Ar. The swollen resin suspension
in Et2O was then cooled to -30 °C. A solution of orthochloranil
(8 mg, 0.03 mmol) in 1 mL of dry Et2O was added to the
reaction flask via syringe. The reaction flask was maintained
at -30 °C for 2 h, and then the deep-red resin beads 21 were
filtered out, washed well with Et2O, and dried under vacuum.
A solution of L-cysteine (36) (11 mg, 0.09 mmol) in 1 mL of
2:1 H2O-THF was added to this deep-red resin suspended in
5 mL of THF under Ar, resulting in a discharge of the deep-
red color of the beads. The reaction was allowed to proceed
for an additional 2 h, at which point the beads were filtered
out and washed sequentially with THF, 1:1 H2O-THF, and,
finally, THF. The brown-colored resin was dried under vacuum,
swollen in 4 mL 1:1 THF-EtOH, and then irradiated with a
450 W mercury arc lamp whose output was filtered through a
40% aqueous solution of CuSO4 for 2 days under Ar, while
monitoring the supernatant by TLC. No evidence for cysteine-
4-Meth oxyben zyl 5-Meth oxy-2-((S)-cystein yl)-3,4-d ih y-
d r oxyben zoa te (25). Orthoquinone 24 (80 mg, 0.26 mmol)
in 4.3 mL of THF was added dropwise via syringe pump over
3.5 h to a stirring solution of L-cysteine (36) (30 mg, 0.25 mmol)
in 2.2 mL of 2:1 H2O-THF under Ar. The reaction mixture
was stirred for an additional 20 min and then diluted with
H2O and EtOAc. The two layers were separated, and the
aqueous layer was lyophilized to obtain 61 mg (55%) of the
adduct 25 as a pinkish-white solid: IR (CH2Cl2) 1718, 1611
cm-1; 1H NMR (300 MHz, CD3COCD3-0.1 M DCl, 9:1) δ 7.42
(d, J ) 8.6 Hz, 2 H), 6.93 (s, 1 H), 6.92 (d, J ) 8.4 Hz, 2 H),
5.26 (s, 2 H), 4.09 (dd, J ) 8.9 Hz, 4.1 Hz, 1 H), 3.81 (s, 3 H),
3.76 (s, 3 H), 3.60 (dd, J ) 14.7 Hz, 4.1 Hz, 1 H), 3.34 (dd, J
) 14.7 Hz, 9.0 Hz, 1 H); 13C NMR (75 MHz, CD3COCD3: 0.1
M DCl, 9:1) δ 169.3, 168.7, 160.5, 149.4, 148.7, 137.9, 131.0,
128.7, 128.5, 114.6, 109.5, 106.6, 67.6, 56.5, 55.5, 52.8, 35.7;
FABMS m/z (relative intensity) 424 (MH+, 100); HRMS
(FABMS) calcd for C19H22NO8S 424.106 614, found 424.107 533.
5-Meth oxy-2-((S)-cystein yl)-3,4-d ih yd r oxyben zoic Acid
(26). Adduct 25 (0.04 g, 0.09 mmol) was dissolved in 2.3 mL
of dry CH2Cl2 under Ar, and then 2.3 mL of TFA was added
to the solution. The reaction mixture turned pink gradually.
The reaction was allowed to proceed for 15 min, and then the
solvents were removed under reduced pressure to afford a
1
gallic acid adduct 22, either by TLC or by H NMR, could be
discerned.
4-Meth oxyben zyl 3,4-Bis(ter t-bu tyld im eth ylsiloxy)-5-
m eth oxyben zoa te (23). p-Methoxybenzyl alcohol (0.45 mL,
3.6 mmol) was syringed into a suspension of 3,4-bis(tert-

5802 J . Org. Chem., Vol. 64, No. 16, 1999
Feldman et al.
brown solid. The crude product was dissolved in H2O and was
washed with EtOAc repeatedly until the wash came out
colorless. The aqueous layer was lyophilized to obtain 17 mg
(59%) of 26 as a pinkish-white solid: 1H NMR (300 MHz,
CD3OD) δ 6.98 (s, 1 H), 3.88 (s, 3 H), 3.72 (dd, J ) 14.5 Hz,
3.2 Hz, 1 H), 3.59-3.55 (m, 1 H), 2.97 (dd, J ) 14.4 Hz, 10.4
Hz, 1 H); 13C NMR (75 MHz, CD3COCD3-2 M DCl, 9:1) δ
170.6, 169.4, 149.2, 148.7, 137.8, 128.3, 109.3, 106.7, 56.5, 52.8,
34.9; FABMS m/z (relative intensity) 304 (MH+, 100).
Syn th esis of Wa n g Resin 29a . Wang resin (2.09 g, ca. 2.09
mmol -OH) in 20 mL of dry CH2Cl2 was treated with 17 (1.17
g, 2.30 mmol), diisopropylcarbodiimide (DIC) (0.29 g, 2.3
mmol), and DMAP (51 mg, 0.42 mmol) at room temperature
under Ar for 14 h. The beads were filtered out, washed with
CH2Cl2 and THF, and dried under vacuum. The cycle was
repeated once more to obtain resin 27a . Resin 27a was treated
with Ac2O (1.9 mL, 20 mmol), pyridine (1.62 mL, 20 mmol),
and DMAP (12 mg, 0.1 mmol) to cap off the unreacted -OH
groups. The resin was washed well with THF and CH2Cl2 and
then dried under vacuum. This galloylated resin was then
swollen in 18 mL of dry THF for 10 min and treated with a
1.0 M solution of TBAF in THF (4.4 mL, 4.4 mmol). Filtration
and washing of the resulting resin with CH2Cl2 afforded resin
28a as pale brown beads. The deprotected resin 28a (1 g, ca.
1 mmol) in 20 mL of CH2Cl2 was then treated with a solution
of orthochloranil (0.49 g, 2.0 mmol) in 4 mL of CH2Cl2 at -30
°C for 12 h. This orthoquinone-containing resin was resubmit-
ted to a second cycle of oxidant after initial filtration and
washing with CH2Cl2 to furnish the electrophilic orange colored
resin beads 29a . The resin 29a obtained was stored at -10
°C.
Syn th esis of Wa n g Resin 29b. Wang resin (4.8 g) in 35
mL of dry CH2Cl2 was treated with 11 (1.44 g, 3.85 mmol),
DIC (0.60 mL, 3.85 mmol), and DMAP (0.09 g, 0.70 mmol) at
room temperature under Ar for 14 h. The beads were filtered
out, washed with CH2Cl2 and THF, and dried under vacuum.
The cycle was repeated once more to obtain resin 27b. Resin
27b was treated with 3.3 mL of Ac2O, 10 mL of pyridine, and
0.02 g of DMAP to cap off the unreacted -OH groups. The
resin was washed well with THF and CH2Cl2 and then dried
under vacuum. This galloylated resin was then swollen in 100
mL of dry THF for 10 min and treated with a 1.0 M solution
of TBAF in THF (7.7 mL, 7.7 mmol). Filtration and washing
of the resulting resin with THF, 1:1 H2O-THF, and then THF
again afforded resin 28b. The deprotected resin 28b in 58 mL
of CH2Cl2 was then treated with a solution of orthochloranil
(0.95 g, 3.85 mmol) in 35 mL of CH2Cl2 at -30 °C for 12 h.
This orthoquinone-containing resin was resubmitted to oxidant
after initial filtration and washing with CH2Cl2 to furnish the
electrophilic deep-red resin beads 29b. The resin 29b obtained
was stored at -10 °C.
Cystein e Ad d u ct 26. Wang resin 29b (0.2 g) in THF was
added in small portions to a solution of cysteine derivative 315i
(0.14 g, 0.40 mmol) in 3 mL of THF under Ar over 45 min.
The resin beads were allowed to stand in the reaction mixture
overnight and were then filtered out and washed well with
THF. The cysteine adduct was cleaved from the resin by
treating the beads with 2 mL of 1:1 TFA-CH2Cl2 for 35 min.
The resin beads were filtered off, and the filtrate was concen-
trated to yield a brown residue. The crude product was purified
by silica gel chromatography using 5% CH3OH and 0.25%
acetic acid in CH2Cl2 as the eluent. Final purification was
effected by preparative TLC, using 5% CH3OH and 0.25%
acetic acid in CH2Cl2 as the solvent, to obtain 9 mg (12%,
assuming 100% resin loading) of the desired cysteine product.
This cysteine derivative was converted into, and characterized
as, the known compound 26 by hydrogenolysis over Pd black.
P h en a zin e 30. Resin 29b (0.30 g) in 5 mL of dry CH2Cl2
under Ar was treated with a solution of 1,2-diaminobenzene
(0.12 g, 1.10 mmol, 5 equiv) in 5 mL of glacial HOAc. The beads
turned dark brown instantly and were allowed to stand in the
reaction mixture for 20 h. The resin was filtered out, washed
well with CH2Cl2, and dried under vacuum. The brown resin
was allowed to swell in 2 mL of CH2Cl2, and then 2 mL of
TFA was added to the reaction mixture. The beads turned pink
upon standing and were filtered off after 30 min. The filtrate
was concentrated to yield the crude phenazine derivative. The
crude product was purified by preparative TLC, using 10%
CH3OH and 0.25% acetic acid in CH2Cl2, to obtain 8 mg (14%,
assuming 100% resin loading) of product. A portion of the
phenazine product (6 mg, 0.02 mmol) in 1 mL of dry CH3OH
was converted to the corresponding methyl ester 30 by reaction
with a 2.0 M solution of trimethylsilyldiazomethane in hexanes
(0.11 mL, 0.22 mmol). Removal of solvents afforded pure 30
in quantitative yield. IR (CDCl3) 1711 cm-1 1H NMR (360
;
MHz, CDCl3) δ 8.62 (d, J ) 1.5 Hz, 1 H), 8.42 (m, 1 H), 8.27
(m, 1 H), 7.90 (m, 2 H), 7.66 (d, J ) 1.3 Hz, 1 H), 4.25 (s, 1 H),
4.05 (s, 3 H); 13C NMR (90 MHz, CDCl3) δ 166.5, 155.2, 144.2,
143.4, 143.1, 138.1, 131.7, 131.4, 131.3, 130.2, 129.7, 124.9,
105.4, 56.8, 52.8; EIMS m/z (relative intensity) 268 (M+, 19);
HRMS (EIMS) calcd for C15H12N2O3 268.0844, found 268.0848.
Ben zyl (S)-2-(5-(Dim et h yla m in o)n a p h t h ylen e-1-su l-
fon a m id yl)-3-h yd r oxyp r op ion a te (32). A solution of dansyl
chloride (0.54 g, 2.0 mmol) in 24 mL of 2:1 acetone-H2O was
added to a stirred solution of L-serine benzyl ester hydrochlo-
ride (0.39 g, 2.0 mmol) in 6 mL of saturated aqueous NaHCO3.
The reaction mixture was stirred in the dark for 14 h, followed
by evaporation of acetone and acidification of the reaction
mixture with 1 M H3PO4. The product was extracted into
EtOAc, and the organic extract was dried over Na2SO4, filtered,
and concentrated to a fluorescent green oil (0.68 g, 88%). The
crude product 32 was used without further purification: IR
(CH2Cl2) 1743 cm-1; 1H NMR (300 MHz, CDCl3) δ 8.55 (d, J )
8.3 Hz, 1 H), 8.30 (d, J ) 8.6 Hz, 1 H), 8.21 (dd, J ) 7.3 Hz,
1.1 Hz, 1 H), 7.56 (t, J ) 8.1 Hz, 1 H), 7.47 (t, J ) 7.9 Hz, 1
H), 7.29-7.26 (m, 3 H), 7.19 (d, J ) 7.6 Hz, 1 H), 7.11-7.08
(m, 2 H), 5.87 (br s, 1 H), 4.89 (s, 1 H), 4.87 (s, 1 H), 4.00 (ddd,
J ) 7.3 Hz, 4.0 Hz, 4.0 Hz, 1 H), 3.85-3.74 (m, 2 H), 2.88 (s,
6 H); 13C NMR (75 MHz, CDCl3) δ 169.4, 151.5, 134.6, 134.5,
130.5, 129.6, 129.4, 129.3, 128.4, 128.3, 128.1, 127.7, 122.0,
118.8, 115.2, 67.2, 63.4, 57.8, 45.2; FABMS m/z (relative
intensity) 429 (MH+, 76). Anal. Calcd for C22H24N2O5S: C,
61.68; H, 5.61; N, 6.54. Found: C, 61.06; H, 5.81; N, 6.33.
Ben zyl (S)-2-(5-(Dim eth yla m in o)n a ph th ylen e-1-su lfon -
a m id yl)-3-((3,4-bis(ben zyloxy))-5-m eth oxyben zoyl)oxy)-
p r op ion a te (34). Dansyl amide 32 (0.42 g, 0.99 mmol), 3,4-
bis(benzyloxy)-5-methoxybenzoic acid (33)21 (0.40 g, 1.1 mmol),
and DMAP (0.02 g, 0.2 mmol) were dissolved in 12 mL of dry
CH2Cl2 under Ar. DIC (0.17 mL, 1.1 mmol) was syringed into
the reaction mixture, which was then stirred at room temper-
ature overnight. The reaction mixture was diluted with CH2Cl2
and poured into 1 M H3PO4. The organic layer was washed
with brine, dried over Na2SO4, filtered, and concentrated. The
crude product was purified by chromatography on silica gel,
eluting with 30% and then 40% EtOAc in hexanes. Compound
34 was obtained as a fluorescent green foam (0.53 g, 70%):
1
IR (CH2Cl2) 1723 cm-1; H NMR (300 MHz, CDCl3) δ 8.50 (d,
J ) 8.5 Hz, 1 H), 8.24-8.18 (m, 2 H), 7.48-7.01 (m, 20 H),
5.72 (d, J ) 8.0 Hz, 1 H), 5.12 (s, 2 H), 5.04 (s, 1 H), 5.03 (s, 1
H), 4.92 (s, 2 H), 4.46-4.37 (m, 2 H), 4.28-4.25 (m, 1 H), 3.79
(s, 3 H), 2.83 (s, 6 H); 13C NMR (75 MHz, CDCl3) δ 168.4, 164.9,
153.0, 151.8, 151.6, 141.6, 137.2, 136.4, 134.6, 134.3, 130.6,
129.5, 129.2, 129.0, 128.3, 128.24, 128.21, 128.0, 127.83,
127.80, 127.7, 127.3, 123.7, 122.8, 118.4, 114.9, 108.4, 106.9,
74.7, 70.7, 67.5, 64.3, 55.9, 55.0, 45.1; FABMS m/z (relative
intensity) 775 (MH+, 50). Anal. Calcd for C44H42N2O9S: C,
68.22; H, 5.43; N, 3.62. Found: C, 68.05; H, 5.60; N, 3.65.
(S)-2-(5-(Dim eth ylam in o)n aph th ylen e-1-su lfon am idyl)-
3-((3,4-d ih yd r oxy-5-m eth oxyben zoyl)oxy)p r op ion ic Acid
(35). Debenzylation of 34 (0.53 g, 0.69 mmol) was carried out
in the presence of 10% Pd/C (250 mg) in 50 mL of dry THF.
The reaction mixture was stirred for 14 h at room temperature
under 1 atm of H2. The Pd/C was removed by filtration through
Celite, and the THF was evaporated under reduced pressure
to afford crude 35. Silica gel chromatography, using 10%
methanol in CH2Cl2, and then 100:1 EtOAc-HOAc, yielded
(21) Chhabra, S. C.; Gupta, S. R.; Sharma, N. D. Ind. J . Chem. Sect.
B 1978, 16, 1079.

Mediating Insect-Pathogen Interactions
J . Org. Chem., Vol. 64, No. 16, 1999 5803
35 as a fluorescent green foam (0.34 g, 98%): IR (CH2Cl2) 1716
cm-1; 1H NMR (300 MHz, CD3COCD3) δ 8.50 (dd, J ) 8.5 Hz,
0.9 Hz, 1 H), 8.39 (d, J ) 8.7 Hz, 1 H), 8.27 (dd, J ) 7.3 Hz,
1.3 Hz, 1 H), 7.17 (dd, J ) 7.5 Hz, 0.6 Hz, 1 H), 7.08 (d, J )
1.9 Hz, 1 H), 7.05 (d, J ) 1.9 Hz, 1 H), 4.45-4.32 (m, 3 H),
3.81 (s, 3 H), 2.83 (s, 6 H); 13C NMR (75 MHz, CD3COCD3) δ
170.5, 166.0, 152.6, 148.3, 137.4, 130.9, 130.6, 130.5, 129.4,
128.7, 124.0, 120.8, 120.3, 115.9, 111.8, 106.0, 65.3, 56.5, 56.0,
45.5; FABMS m/z (relative intensity) 505 (MH+, 82); HRMS
(FABMS) calcd for C23H24N2O9S 504.120 253, found 504.120 037.
Or th oqu in on e 5. A solution of 35 (200 mg, 0.40 mmol) in
9 mL of 2:1 THF-Et2O (sufficient THF for dissolving 35,
making up the remaining volume with Et2O) was added
dropwise via an addition funnel to a solution of orthochloranil
(0.11 g, 0.44 mmol) in 2 mL of dry Et2O at -42 °C over 30
min. The deep red solution was stirred for an additional 30
min at -42 °C and then stored in a -20 °C freezer for 12 h.
The pale pink precipitate obtained was filtered out, washed
with copious volumes of cold Et2O, and dried under reduced
pressure to yield 88 mg (44%) of orthoquinone 5. Orthoquinone
5 was characterized as the corresponding phenazine derivative,
prepared in a 26% yield as described previously for 30: IR
(CH2Cl2) 1724 cm-1; 1H NMR (200 MHz, CD3OD) δ 8.36-8.20
(m, 5 H), 8.11 9d, J ) 1.5 Hz, 1 H), 8.02-7.97 (m, 2 H), 7.47
(dd, J ) 8.6 Hz, 7.3 Hz, 1 H), 7.41-7.33 (m, 1 H), 7.39 (d, J )
1.4 Hz, 1 H), 6.76 (d, J ) 7.4 Hz, 1 H), 4.50-4.47 (m, 2 H),
4.19-4.15 (m, 1 H), 4.15 (s, 3 H), 2.51 (s, 6 H); 13C NMR (90
MHz, CD3OD) δ 166.1, 155.8, 152.7, 145.0, 143.9, 143.8, 138.5,
137.5, 133.1, 132.9, 132.3, 131.2, 130.81, 130.80, 130.4, 130.3,
129.8, 129.0, 125.0, 124.1, 120.4, 115.6, 106.5, 66.2, 57.0, 45.4;
FABMS m/z (relative intensity) 575 (MH+, 3); HRMS (FABMS)
calcd for C29H27N4O7S 575.160 046, found 575.159 712.
Cystein e Ad d u ct 37. Dropwise addition of a solution of
orthoquinone 5 (0.03 g, 0.07 mmol) in 2 mL of THF containing
100 µL of water to a stirred solution of L-cysteine in 1 mL of
2:1 H2O-THF at room temperature over 30 min afforded a
clear pale yellow solution. The reaction mixture was diluted
with H2O and EtOAc, and the aqueous layer was lyophilized
to yield crude 37 as a white solid. The white solid was then
dissolved in CH3OH and filtered through a membrane filter.
The filtrate was collected and dried under vacuum to afford
37 (17 mg, 46%) as a white solid: IR (KBr) 1718 cm-1; 1H NMR
(200 MHz, CD3OD) δ 8.46 (d, J ) 8.7 Hz, 1 H), 8.32 (d, J )
8.7 Hz, 1 H), 7.55-7.45 (m, 2 H), 7.18 (d, J ) 7.4 Hz, 1 H),
6.95 (s, 1 H), 4.57-4.53 (m, 1 H), 4.49-4.32 (m, 2 H), 4.09 (t,
J ) 5.1 Hz, 2 H), 3.84 (s, 1 H), 3.80-3.73 (m, 1 H), 3.66-3.58
(m, 1 H), 3.07-3.05 (m, 1 H), 2.83 (s, 6 H); 13C NMR (90 MHz,
CD3COCD3-2 M DCl, 9:1) δ 170.6, 169.3, 167.4, 149.1, 148.4,
139.7, 138.1, 130.6, 129.9, 128.3, 128.0, 127.7, 127.3, 126.4,
120.7, 107.5, 65.0, 56.7, 55.9, 52.9, 47.7, 35.6; FABMS m/z
(relative intensity) 624 (MH+, 75); HRMS (FABMS) calcd for
CH3OH and 0.25% HOAc in CH2Cl2 as the eluent. Adduct 38
was obtained as a yellow solid (30 mg, 42%): IR (CH2Cl2) 1714
1
cm-1; H NMR (360 MHz, CD3COCD3) δ 8.46 (d, J ) 8.6 Hz,
1 H), 8.38 (d, J ) 8.7 Hz, 1 H), 8.25(d, J ) 7.3 Hz, 1 H), 7.52-
7.46 (m 2 H), 7.33-7.30 (m, 10 H), 7.15 (d, J ) 7.6 Hz, 1 H),
5.11-4.99 (m, 4 H), 4.45-4.37 (m, 4 H), 3.77 (s, 3 H), 3.34-
3.24 (m, 2 H), 2.81 (s, 6 H); 13C NMR (90 MHz, CD3COCD3) δ
171.3, 166.7, 157.0, 152.6, 148.6, 147.8, 138.0, 137.5, 136.8,
130.8, 130.7, 130.6, 129.26, 129.20, 128.86, 128.81, 128.7,
128.5, 127.1, 124.0, 120.4, 115.9, 112.6, 107.7, 67.4, 67.0, 65.8,
56.6, 55.25, 55.20, 45.6, 38.6; FABMS m/z (relative intensity)
848 (MH+, 15); HRMS (FABMS) calcd for C41H42N3O13S2
848.215 908, found 848.216 732.
P r otein Con ju ga tion Stu d ies. DHFR mutants 39 and 40
were provided by Dr. C. R. Matthews, Department of Chem-
istry and Center for Biomolecular Structure and Function, The
Pennsylvania State University, University Park, PA 16802.14,15
Polyhedrin was isolated from LdNPV-infected Lymantria
dispar larvae.17f The purity of polyhedrin was judged by SDS-
polyacrylamide gel electrophoresis. The alkaline isolate of
polyhedrin in 0.1 M Na2CO3 and 0.17 M NaCl (pH 10.6) was
dialyzed into 50 mM and then 10 mM phosphate buffer (pH
8.5), stored at -20 °C, and used without further purification.
DHFR mutants 39 and 40 were dialyzed into 4 L of fresh,
degassed 10 mM phosphate buffer (pH 7.8), while polyhedrin
was dialyzed into degassed 10 mM phosphate buffer (pH 8.5)
prior to use. Both buffers contain 1 mmol DTE in the first 1 L
volume; subsequent volumes do not contain the reducing
agent. The concentrations of 39 and 40 were determined
spectrophotometrically by the absorbance at 280 nm using the
extinction coefficient for wild-type DHFR (ꢀ280 ) 3.11 × 104
M-1 cm-1). Polyhedrin concentrations were determined by the
absorbance at 276 nm, using the extinction coefficient deter-
mined by the method of Gill and von Hippel22 (ꢀ276 ) 4.28 ×
104 M-1 cm-1).
DTNB assays were performed by incubating orthoquinone
reagent 5 with protein for 1 h at 4 °C, followed by the addition
of DTNB 41 and a second period of incubation at 4 °C for 1 h.
MNB 43 concentrations were measured at 412 nm, using the
molar extinction coefficient ꢀ412 ) 1.37 × 104 M-1 cm-1. All
spectroscopic determinations were carried out at 25 °C.
Fluorescence assays were carried out by incubating the
protein of interest and orthoquinone reagent 5 in the indicated
buffer for 1 h at 4 °C prior to spectrofluorometric analysis.
Fluorescence spectra were recorded at 15 °C for 39 and 40 and
at 25 °C for polyhedrin.
Ack n ow led gm en t. We thank the NSF (Grant IBN-
9407308) for support of this work.
Su p p or tin g In for m a tion Ava ila ble: Copies of 13C NMR
spectra for compounds 7, 8, 13, 14, 16, 18, 25, 26, 30, 32, 35,
37, and 38. This material is available free of charge via the
Internet at http://pubs.acs.org.
C
26H30N3O11S2 624.132 178, found 624.132 041.
Cystein e Ad d u ct 38. Orthoquinone 5 (48 mg, 0.10 mmol)
in 2.2 mL of THF containing 100 µL of water was added
dropwise via an addition funnel to a solution of cysteine
derivative 315i (31 mg, 0.09 mmol) at room temperature over
10 min. Removal of solvents under reduced pressure afforded
crude 38, which was purified by preparative TLC using 5%
J O982477N
(22) Gill, S. C.; von Hippel, P. H. Anal. Biochem. 1989, 182, 319.