A photoactive isoprenoid diphosphate analogue containing a stable phosphonate linkage: Synthesis and biochemical studies with prenyltransferases

Article abstract of DOI:10.1021/jo0623033

(Chemical Equation Presented) A number of biochemical processes rely on isoprenoids, including the post-translational modification of signaling proteins and the biosynthesis of a wide array of compounds. Photoactivatable analogues have been developed to study isoprenoid utilizing enzymes such as the isoprenoid synthases and prenyltransferases. While these initial analogues proved to be excellent structural analogues with good cross-linking capability, they lack the stability needed when the goals include isolation of cross-linked species, tryptic digestion, and subsequent peptide sequencing. Here, the synthesis of a benzophenone-based farnesyl diphosphate analogue containing a stable phosphonophosphate group is described. Inhibition kinetics, photolabeling experiments, as well as X-ray crystallographic analysis with a protein prenyltransferase are described, verifying this compound as a good isoprenoid mimetic. In addition, the utility of this new analogue was explored by using it to photoaffinity label crude protein extracts obtained from Hevea brasiliensis latex. Those experiments suggest that a small protein, rubber elongation factor, interacts directly with farnesyl diphosphate during rubber biosynthesis. These results indicate that this benzophenone-based isoprenoid analogue will be useful for identifying enzymes that utilize farnesyl diphosphate as a substrate.

Full text of DOI:10.1021/jo0623033

VOLUME 72, NUMBER 13
JUNE 22, 2007
© Copyright 2007 by the American Chemical Society
A Photoactive Isoprenoid Diphosphate Analogue Containing a
Stable Phosphonate Linkage: Synthesis and Biochemical Studies
with Prenyltransferases
Amanda J. DeGraw,† Zongbao Zhao,† Corey L. Strickland,‡ A. Huma Taban,§ John Hsieh,†
Michael Jefferies,† Wenshuang Xie,| David K. Shintani,§ Colleen M. McMahan,|
Katrina Cornish,¶ and Mark D. Distefano*,†
Department of Chemistry, UniVersity of Minnesota, Minneapolis, Minnesota 55455, Department of
Structural Chemistry, Schering-Plough Research Institute, Kenilworth, New Jersey 07033, Department of
Biochemistry, UniVersity of NeVada, Reno, NeVada 89557, Agricultural Research SerVice, Western
Regional Research Center, United States Department of Agriculture, Albany, California 94710, and
Yulex Corporation, Carlsbad, California 92008
distefan@chem.umn.edu
ReceiVed NoVember 7, 2006
A number of biochemical processes rely on isoprenoids, including the post-translational modification of
signaling proteins and the biosynthesis of a wide array of compounds. Photoactivatable analogues have
been developed to study isoprenoid utilizing enzymes such as the isoprenoid synthases and prenyltrans-
ferases. While these initial analogues proved to be excellent structural analogues with good cross-linking
capability, they lack the stability needed when the goals include isolation of cross-linked species, tryptic
digestion, and subsequent peptide sequencing. Here, the synthesis of a benzophenone-based farnesyl
diphosphate analogue containing a stable phosphonophosphate group is described. Inhibition kinetics,
photolabeling experiments, as well as X-ray crystallographic analysis with a protein prenyltransferase
are described, verifying this compound as a good isoprenoid mimetic. In addition, the utility of this new
analogue was explored by using it to photoaffinity label crude protein extracts obtained from HeVea
brasiliensis latex. Those experiments suggest that a small protein, rubber elongation factor, interacts
directly with farnesyl diphosphate during rubber biosynthesis. These results indicate that this benzophenone-
based isoprenoid analogue will be useful for identifying enzymes that utilize farnesyl diphosphate as a
substrate.
Introduction
of cholesterol,1 carotenoids,2 and terpenes,3 protein prenylation,4
and rubber biosynthesis.5 Protein prenylation is a post-
translational modification targeted by anti-cancer drugs6-9 given
that the farnesylation of mutant Ras proteins is required for
transformation.10 Substrate analogues that covalently modify
Farnesyl diphosphate (FPP) is an isoprenoid building block
utilized in a number of cellular processes including the synthesis
† University of Minnesota.
‡ Schering-Plough Research Institute.
§ University of Nevada, Reno.
¶ Yulex Corporation.
(1) Liscum, L. New Comp. Biochem. 2002, 36, 409-431.
| United States Department of Agriculture.
10.1021/jo0623033 CCC: $37.00 © 2007 American Chemical Society
Published on Web 05/04/2007
J. Org. Chem. 2007, 72, 4587-4595
4587

DeGraw et al.
their cognate targets are useful for both identifying previously
uncharacterized enzymes as well as for mapping the active sites
of known proteins. Photoaffinity labeling is one technique that
has proven useful in this regard. For that reason, we and others
have developed a number of analogues of farnesyl- and
geranylgeranyl diphosphates that can be used for this purpose.11-14
Initial photoactive isoprenoid analogues incorporated a di-
azotrifluoropropionate (DATFP) photophore into the isoprenoid
chain.12,14 Despite being accurate mimetics of FPP, they suffer
from low cross-linking efficiency and require prolonged short
wavelength UV irradiation for photoactivation. Molecules that
utilize benzophenone moieties proved to be an attractive
alternative for a number of reasons. Besides being more
chemically stable than diazo esters, they can be easily manipu-
lated in ambient light and require longer UV wavelengths for
activation, avoiding protein degradation.15 Given the significant
overlap in the benzophenone and isoprenoid structures, ana-
logues incorporating the benzophenone moiety as part of the
isoprenoid chain were developed.11,13,16-19 X-ray crystallo-
graphic analysis of these molecules revealed that they are nearly
superimposable with FPP in the active site of rat protein
farnesyltransferase, confirming they are structurally analogous
to FPP.13,20
analogue of cisplatin.22 The critical component in studies of this
type is the ability to detect cross-linked proteins in order to
isolate and identify them. Photoaffinity analogues of isoprenoid
diphosphates have often employed a 32P radiolabel for detection
given the ease of synthesis of these compounds.16,18,20 However,
because of the facile loss of the radiolabel due to the acid labile
nature of the allylic diphosphate linkage, the use of such
compounds has been impeded. The development of more stable
photoreactive analogues is needed to successfully accomplish
such studies.
In this paper, we describe the synthesis of a benzophenone-
based farnesyl diphosphate analogue, 3b, containing a stable
phosphonophosphate group that replaces the labile allylic
diphosphate. A number of polyprenyl phosphonophosphates
have been reported as isoprenoid diphosphate mimics in the
design of inhibitors,23,24 immunoregulators,24,25 as well as affinity
purification reagents.26,27 The stability of 3b in aqueous acid
was investigated. Inhibition kinetics and photoaffinity labeling
were studied using protein prenyltransferases as model systems
to validate the utility of the new analogue. A co-crystal structure
of 3b with protein farnesyltransferase (PFTase) illustrates the
close complementarity between the new probe and FPP as well
as a previously synthesized photoactive FPP analogue. Finally,
3b was used in photolabeling experiments with protein mixtures
isolated from the latex of HeVea brasiliensis, the Brazilian
rubber tree. One of the cross-linked proteins previously desig-
nated as rubber elongation factor (REF) was identified by mass
spectral analysis. These experiments suggest that the REF
interacts directly with isoprenoid diphosphates and clearly
demonstrates the efficacy of this analogue for photoaffinity
labeling experiments involving the isolation and identification
of enzymes that employ isoprenoids as their substrates.
In addition to providing information on a specific, known
ligand or substrate, photoactivatable analogues of ligands can
be used to isolate and identify unknown binding partners. By
creating a photoaffinity labeling reagent structurally based on
suberoylanilide hydroxamic acid, Webb and co-workers were
able to identify a potential target for hybrid polar cytodiffer-
entiation agents through isolation of labeled proteins and mass
spectral analysis.21 Zhang and co-workers identified proteins
that interact with platinum-modified DNA using a photoreactive
(2) Fraser, P. D.; Bramley, P. M. Prog. Lipid Res. 2004, 43, 228-265.
(3) Christianson, D. W. Chem. ReV. 2006, 106, 3412-3442.
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(5) Puskas, J. E.; Gautriaud, E.; Deffieux, A.; Kennedy, J. P. Prog. Polym.
Sci. 2006, 31, 533-548.
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S.; Allen, C. M.; Gibbs, J. B.; Kohl, N. E. Biochemistry 1993, 32, 5167-
5176.
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Chem. 2001, 66, 3253-3264.
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1344-1354.
Results and Discussion
Synthesis and Stability of the Photoactive Analogue. In
our initial design of a more stable photoactive analogue, we
envisioned compound 3a, based on 2a, that would incorporate
a C-P bond at C-1 in lieu of the C-O bond present in 2a and
FPP (1, Figure 1).
It was considered desirable to retain the ether linkage between
the benzophenone moiety and the isoprenoid since that structural
feature is a minimal perturbation relative to the natural molecule
FPP; in earlier work, it had been shown that ether-containing
FPP analogues bound to protein farnesyltransferase (PFTase)
with higher affinity when compared to similar compounds
incorporating ester or amide linkages.13 Compound 4, an
intermediate from an earlier synthesis, was reacted under
Arbuzov conditions to yield the dimethylphosphonate (5) in
excellent yield (Scheme 1) in order to prepare compound 3a.
Unfortunately, efforts to convert the phosphonate diester to
the corresponding phosphonic acid (6) were not successful.
(15) Dorman, G.; Prestwich, G. D. Biochemistry 1994, 33, 5661-5673.
(16) Gaon, I.; Turek, T. C.; Weller, V. A.; Edelstein, R. L.; Singh, S.
K.; Distefano, M. D. J. Org. Chem. 1996, 61, 7738-7745.
(17) Marecak, D. M.; Horiuchi, Y.; Arai, H.; Shimonaga, M.; Maki, Y.;
Koyama, T.; Ogura, K.; Prestwich, G. D. Bioorg. Med. Chem. Lett. 1997,
7, 1973-1978.
(22) Zhang, C. X.; Chang, P. V.; Lippard, S. J. J. Am. Chem. Soc. 2004,
126, 6536-6537.
(23) Blau, N. F.; Wang, T. T. S.; Buess, C. M. J. Chem. Eng. Data 1970,
15, 206-208.
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J.; Liautard, J.-P.; Criton, M.; Montero, J.-L. J. Med. Chem. 2004, 47, 4600-
4612.
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149, 507-515.
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20566.
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Chem. Lett. 1997, 7, 2125-2130.
(19) Zhang, Y. W.; Koyama, T.; Marecak, D. M.; Prestwich, G. D.; Maki,
Y.; Ogura, K. Biochemistry 1998, 37, 13411-13420.
(20) Turek-Etienne, T. C.; Strickland, C. L.; Distefano, M. D. Biochem-
istry 2003, 42, 3716-3724.
(21) Webb, Y.; Zhou, X.; Ngo, L.; Cornish, V.; Stahl, J.; Erdjument-
Bromage, H.; Tempst, P.; Rifkind, R. A.; Marks, P. A.; Breslow, R.; Richon,
V. M. J. Biol. Chem. 1999, 274, 14280-14287.
4588 J. Org. Chem., Vol. 72, No. 13, 2007

Stable PhotoactiVe Isoprenoid Diphosphate Analogue
TABLE 1. IC50 Values for Inhibition of yPFTase by
Benzophenone-Based Isoprenoid Diphosphate Photoaffinity Labeling
Analogues
compound
IC50 (µM)
2aa
2bb
2cc
3b
0.85
2.3
3.7
0.96
a From ref 14. b From ref 13. c From ref 16.
Based on the above observations, it was decided to redesign
the target molecule to include an ester-linked benzophenone
(Compound 3b, Scheme 2) in place of the original ether linkage.
There was good evidence to believe that the ester-linked
analogue would effectively bind since a related analogue, 2b
(see Table 1), inhibited yeast PFTase with a KI of 380 nM.16
Compound 3b was prepared in six steps using the route shown
in Scheme 2.
FIGURE 1. Benzophenone-containing analogues of farnesyl diphos-
phate.
SCHEME 1. Original Strategy for the Synthesis of an
Analogue Containing a Stable Phosphonate Linkage
Geraniol (7) was first converted to the corresponding bromide
(8) using conditions described by Coates et al.,28 followed by
Arbuzov reaction with P(OCH3)3 which proceeded in good yield
to produce 9.23 The dimethyl phosphonate was then subjected
to oxidation with SeO2 according to the procedure described
by Umbreit and Sharpless29 to produce the allylic alcohol 10 in
78% yield. Apparently, the phosphonate moiety in 9 is suf-
ficiently deactivating such that oxidation by SeO2 occurs
predominantly at the alkene at the C-6 position and not at C-2.
Acylation of 10 to form the desired ester-linked benzophenone-
containing intermediate 11 proceeded in good yield (82%). In
contrast to our results obtained with the deprotection of
compound 5, where cleavage of the ether linkage was observed,
treatment of 11 with trimethylsilyl bromide and allyltrimeth-
ylsilane in CH2Cl2 resulted in clean deprotection of the
phosphonate esters to produce 12 in excellent yield (92%)
without any observable loss of the benzophenone moiety.30
Finally, phosphonate 12 was converted to the desired phospho-
nophosphate 3b (23% yield) by activation to the corresponding
imidazolide with carbonyl diimidazole followed by reaction with
anhydrous phosphoric acid.31 Purification of 3b was performed
by chromatographic separation using octyl-functionalized silica
gel. For photoaffinity labeling experiments [32P]-3b was pre-
Treatment of 5 with Me3SiCl or Me3SiBr resulted primarily in
cleavage of the benzyl ether linkage with only small amounts
of methyl ester cleavage occurring. Treatment of 5 under basic
conditions (LiOH) resulted in incomplete saponification to the
phosphonate monoester 6. Exposure of 5 to acidic conditions
produced decomposition.
SCHEME 2. Synthesis of an Analogue Containing a Stable Phosphonate Linkage
J. Org. Chem, Vol. 72, No. 13, 2007 4589

DeGraw et al.
previously observed reduction in binding when a carbonyl group
is involved in linkage between the benzophenone moiety and
the isoprenoid.
X-ray Crystallographic Studies. In contrast to our earlier
work that always employed analogues containing an allylic
diphosphate, compound 3b possesses a phosphonate linkage that
is one carbon out of register relative to FPP. Crystals of 3b
bound to rPFTase were obtained, and the structure of the
resulting complex was determined via X-ray diffraction experi-
ments to investigate the effect of this modification. The structure
of 3b bound to rPFTase is shown in Figure 3; for comparison,
the structure of FPP bound to rPFTase is shown in a superposi-
tion (panel A). Figure 4 highlights the key amino acid residues
involved in the binding.
Electron density maps clearly show that 3b is bound in the
rPFTase active site cavity. The presence of the higher molecular
weight phosphate atoms in the molecule provides helpful
information in defining the binding mode. Comparison of the
protein in the structure of the rPFTase:3b complex and the
rPFTase:FPP:SCH66336 ternary complex shows a 0.2 Å rms
deviation for all protein atoms, indicating that no significant
movement of the protein side chains has occurred.32,33 Interest-
ingly, the two phosphate P atoms from FPP and those from 3b
are essentially superimposable, suggesting that the phosphono-
phosphate substitution present in the analogue is not deleterious.
Due to the absence of the intervening oxygen atom in 3b (the
oxygen attached to C-1 in FPP) and the presence of the bulky
benzophenone group, there are small perturbations in the
structure of the isoprenoidal portion of the analogue; the average
rms deviation for all atoms in the diphosphate unit and the first
two isoprene units is 0.8 Å. However, these changes are in
general small, indicating close complementarity between the
natural substrate, FPP, and 3b. A superposition of 3b and 2a,
a related diphosphate-based analogue with an ether-linked
benzophenone, is shown in Figure 3, panel B. As was observed
above with FPP, there is substantial overlap between these two
structures particularly in the diphosphate unit and the first two
isoprene units. However, there is a significant difference in the
position of the benzophenone group in 3b relative to 2a. Clearly,
the presence of the more conformationally restricted ester
linkage in 3b alters the orientation of the benzophenone
compared to that of the more flexible ether-linked analogue 2a.
This structural difference may account for the greater inhibitory
potency observed with 2a compared with 3b. Nevertheless, it
can be concluded that beyond the greater size of the benzophe-
none groups that causes them to protrude beyond the FPP
binding site, analogues such as 3b are good mimics of FPP and
that the use of a phosphonophosphate group does not cause any
significant changes in the mode of binding to PFTase.
FIGURE 2. Stability of phosphonophosphate 3b compared to geranyl
diphosphate (GPP).
pared from phosphonate 12 using a similar procedure employing
[32P]-H3PO4 followed by purification via a small reversed-phase
column.
In earlier work, it was noted that the ester linkage between
the benzophenone and isoprenoid moieties present in compounds
similar to 3b is stable to the acidic conditions typically used in
reversed-phase chromatography to separate and identify cross-
linked peptides obtained from photoaffinity labeling experiments
with proteins. Unfortunately, this is not the case for the C-O
bond at C-1 between the isoprenoid and phosphate moieties in
allylic diphosphates such as 2a and 2b. In such molecules, while
the phosphate anhydride linkage between the R and â phosphates
is relatively stable to acid, the bond between C-1 and O-1 is
labile. As illustrated in Figure 2, treatment of geranyl diphos-
phate (GPP), which contains a C-O bond at C-1 like 2a, with
a mixture of H2O and CH3CN containing 0.2% TFA (common
conditions for peptide analysis) results in hydrolysis of the allylic
diphosphate with a half-life of approximately 2 h.
This significant rate of degradation via phosphate loss makes
it impractical to use 32P-labeled allylic diphosphates for pho-
toaffinity labeling applications when tryptic digestion and
subsequent peptide sequencing are desired. In contrast, treatment
of phosphonophosphate 3b under the same conditions results
in less than 5% hydrolysis even after 50 h of reaction (Figure
2). This enhanced stability significantly increases the utility of
this benzophenone-containing analogue.
Inhibition Kinetics. Given the inherent stability of the C-P
bond at position C-1, phosphonophosphate 3b was tested as a
competitive inhibitor of yPFTase. The rate of yPFTase-catalyzed
farnesylation of N-dansyl-GCVIA was measured in the presence
of a fixed concentration of FPP and varying concentrations of
3b. An IC50 value of 960 nM was calculated for 3b, which is
comparable to previously synthesized benzophenone-based FPP
analogues (see Table 1). The similarity in values between the
ether-linked analogue 2a and 3b was unexpected, given the
Photolabeling of Protein Prenyltransferases with [32P]-
3b. To verify the ability of phosphonophosphate 3b to act as a
photoaffinity label of protein prenyltransferases, the radiolabeled
analogue [32P]-3b was synthesized using a method developed
by Bartlett and co-workers,34 adapted to incorporation of a
(32) Strickland, C. L.; Weber, P. C.; Windsor, W. T.; Wu, Z.; Le, H.
V.; Albanese, M. M.; Alvarez, C. S.; Cesarz, D.; del Rosario, J.; Deskus,
J.; Mallams, A. K.; Njoroge, F. G.; Piwinski, J. J.; Remiszewski, S.;
Rossman, R. R.; Taveras, A. G.; Vibulbhan, B.; Doll, R. J.; Girijavallabhan,
V. M.; Ganguly, A. K. J. Med. Chem. 1999, 42, 2125-2135.
(33) Strickland, C. L.; Windsor, W. T.; Syto, R.; Wang, L.; Bond, R.;
Wu, Z.; Schwartz, J.; Le, H. V.; Beese, L. S.; Weber, P. C. Biochemistry
1998, 37, 16601-16611.
(28) Coates, R. M.; Ley, D. A.; Cavender, P. L. J. Org. Chem. 1978,
43, 4915-4922.
(29) Umbreit, M. A.; Sharpless, K. B. J. Am. Chem. Soc. 1977, 99, 5526-
5528.
(30) Hammerschmidt, F. Monatsh. Chem. 1991, 122, 389-398.
(31) Freeman, G. A.; Rideout, J. L.; Miller, W. H.; Reardon, J. E. J.
Med. Chem. 1992, 35, 3192-3196.
(34) Bartlett, D. L.; King, C. H.; Poulter, C. D. Methods Enzymol. 1985,
110, 171-184.
4590 J. Org. Chem., Vol. 72, No. 13, 2007

Stable PhotoactiVe Isoprenoid Diphosphate Analogue
FIGURE 3. Crystallographic analysis of analogue 3b bound to rPFTase. (A, left image): Superposition of 3b and FPP bound to PFTase. (B,
center and right images): Stereoview of a superposition of 3b and a related diphosphate analogue 2a. Analogue 3b is shown in green (carbon), red
(oxygen), and purple (phosphorus). FPP is shown in blue. Analogue 2a is shown in yellow (carbon), red (oxygen), and purple (phosphorus). The
solvent-accessible protein surface is white with the Zn atom shown in red.
FIGURE 4. Stereoview of 3b bound to rPFTase. Residues highlighted in red are from the R subunit; black are from the â subunit. The catalytic
zinc is shown as a red sphere.
radiolabel, and purified using a method previously described.35
Photolysis of the prenyltransferases with [32P]-3b at a concen-
tration 10-fold higher than the IC50 value determined for
yPFTase was performed with 300-400 nm light15 for 2 h.
Preferential labeling of the â subunit was observed for both
yeast protein farnesyltransferase (yPFTase) and human protein
geranylgeranyltransferase type I (hPGGTAse-I) (Figure 5:
yPFTase and [32P]-3b and Figure 6: hPGGTase-I and [32P]-
3b; lane 2′ in each). Upon addition of the natural substrate (FPP
for yPFTase, GGPP for hPGGTase-I) in the photolysis reactions
containing [32P]-3, protection from labeling was observed
(Figure 5: yPFTase and [32P]-3b and Figure 6: hPGGTase-I
and [32P]-3b; lane 3′ in each). Phosphorimaging analysis was
used to quantify the relative labeling efficiencies of each subunit
for each enzyme. For yPFTase, the â subunit was labeled 4.8-
fold over the R subunit. Inclusion of the natural substrate FPP,
1, in the photolysis reaction resulted in a substantial (2.3-fold)
decrease in â subunit labeling. In the case of hPGGTase-I
photolabeling by [32P]-3b, the â subunit was labeled 24-fold
over the R subunit, while inclusion of the physiologically
relevant substrate GGPP resulted in a substantial decrease in â
subunit labeling (8.7-fold). The observed labeling specificity is
in agreement with results obtained from other photoactive
analogues with yPFTase and hPGGTase-I, all implicating â
subunit involvement in prenyl group recognition.13,16
The applicability of this probe for labeling specific targets
in a mixture of proteins was demonstrated by the photolysis of
a partially purified sample of hPGGTase-I with [32P]-3b. Of
the crude mixture of proteins, the â subunit is the major labeled
species, while no labeling of the R subunit is observed (Figure
7). In this crude mixture, a large number of proteins are clearly
visible by Sypro-orange staining. Interestingly, the phospho-
rimage (Figure 7, lane 1′) reveals three major labeled species
with the â subunit of hPGGTase-I as the most intensely labeled
protein. Thus, although 3b is not absolutely specific for
hPGGTase-I, it obviously manifests substantial selectivity in
this complex mixture. Based on the promising results obtained
with 3b labeling in crude extracts, we decided to explore the
ability of this probe to identify an unknown target.
Photolabeling of HeWea brasiliensis Washed Rubber Par-
(35) Turek, T. C.; Gaon, I.; Distefano, M. D. J. Labelled Compd.
Radiopharm. 1997, 39, 139-146.
ticles with [32P]-3b. H. brasiliensis, the Brazilian rubber tree,
J. Org. Chem, Vol. 72, No. 13, 2007 4591

DeGraw et al.
FIGURE 7. Analysis of crude hPGGTase-I with [32P]-3b by SDS-
PAGE. Lanes 1 and 1′ contain samples of crude cell lysate after ion-
exchange chromatography, irradiated at 350 nm in the presence of [32P]-
6. Lane 1 shows protein identified with Sypro-orange staining. Lane
1′ shows radiolabeled protein identified with phosphorimaging.
FIGURE 5. Analysis of photolabeling of pure yPFTase with [32P]-3
by SDS-PAGE. Lanes 1 and 1′ contain samples of yPFTase and [32P]-
3b that were not irradiated. Lanes 2 and 2′ contain samples of yPFTase
and [32P]-3 irradiated at 350 nm. Lanes 3 and 3′ contain samples of
yPFTase and [32P]-3b irradiated in the presence of FPP. Lanes 1, 2,
and 3 show proteins identified with Sypro-orange staining. Lanes 1′,
2′, and 3′ show the radiolabeled proteins identified with phosphor-
imaging.
FIGURE 8. Analysis of photolabeling of H. brasiliensis washed rubber
particles with [32P]-3b by SDS-PAGE. Lanes 1 and 1′ contain samples
of WRPs and [32P]-3b irradiated at 350 nm. Lanes 2 and 2′ contain
samples of WRPs and [32P]-3b irradiated in the presence of FPP. Lanes
1 and 2 show proteins identified with Sypro-orange staining. Lanes
1′and 2′ show radiolabeled proteins identified with phosphorimaging.
FIGURE 6. Analysis of photolabeling of pure hPGGTase-I with [32P]-
3b by SDS-PAGE. Lanes 1 and 1′ contain samples of hPGGTase-I
and [32P]-3b that were not irradiated. Lanes 2 and 2′ contain samples
of hPGGTase-I and [32P]-3b irradiated at 350 nm. Lanes 3 and 3′ contain
samples of hPGGTase-I and [32P]-3 irradiated in the presence of GGPP.
Lanes 1, 2, and 3 show proteins identified with Sypro-orange staining.
Lanes 1′, 2′, and 3′ show the radiolabeled proteins identified with
phosphorimaging.
biosynthesis have yet to be unequivocally identified and
reported. Based on the observation that FPP can act as an
initiator of rubber biosynthesis in washed rubber particles
(WRPs), it was decided to utilize 3b to identify FPP binding
components in the complex mixture of proteins present in H.
brasiliensis WRPs. Photolysis of suspensions of WRPs contain-
ing 10 µM [32P]-3b was performed for 6 h. Electrophoretic
separation of the reaction mixture and visualization of the
proteins indicated the presence of a wide range of species. The
WRPs from H. brasiliensis are clearly a complex mixture.
Interestingly, phosphorimaging analysis of the photolysis mix-
ture gave a very different picture. Two major labeled species
were observed as well as several minor bands (Figure 8, lane
is among the many species of angiosperms known to produce
rubber of high molecular weight and is the predominant source
of commercially available natural rubber. The latex obtained
from these trees has been shown to contain enzymatically active
rubber particles that, when purified and incubated with an allylic
diphosphate initiator and isopentenyl diphosphate as the mono-
mer, are able to carry out biosynthesis of cis-1,4-polyisoprene.36
Despite intense research, the proteins involved in rubber
(36) McMullen, A. I.; McSweeney, G. P. Biochem. J. 1966, 101, 42-
47.
4592 J. Org. Chem., Vol. 72, No. 13, 2007

Stable PhotoactiVe Isoprenoid Diphosphate Analogue
TABLE 2. Tryptic Peptides Determined by MS/MS Analysis
amino acid
residues
ion
score
confidence
interval %
calcd mass
obsd mass
sequence
17-40
41-58
2689.3547
1849.0065
1605.8846
1109.599
1621.8795
753.4869
1158.6477
1088.5946
2689.3516
1849.0023
1605.8828
1109.6146
1621.8759
753.4849
YLGFVQDAATYAVTTFSNVYLFAK
DKSGPLQPGVDIIEGPVK
SGPLQPGVDIIEGPVK
FSYIPNGALK
FVDSTVVASVTIIDR
SLPPIVK
120
135
111
70
24
41
100.000
100.000
100.000
99.999
82.268
99.591
100.000
97.974
43-58
68-77
78-92
93-99
100-110
118-128
1158.6460
1088.5912
DASIQVVSAIR
SLASSLPGQTK
82
34
1′). The presence of FPP during the labeling reaction reduced
the amount of radiolabel throughout the gel (Figure 8, lane 2′).
Mass Spectral Analysis of Photolabeled Proteins from
HeWea brasiliensis Washed Rubber Particles. The identifica-
tion of the cross-linked proteins from the photolabeling of H.
brasiliensis WRPs with [32P]-3b was determined by performing
a second set of photolysis reactions. Samples were fractionated
by isoelectric focusing followed by SDS-PAGE. Surprisingly,
the higher molecular weight proteins shown to be labeled
previously were not observed. Instead, a series of proteins (10-
20 kDa) appeared to be labeled (see Supporting Information).
These labeled protein bands were extracted from the gel,
subjected to tryptic digestion, and analyzed by MALDI-MS/
MS. A peak list of the data was then generated and searched
using the MASCOT database. A 14.6 kDa protein, rubber
elongation factor, was identified (Table 2) based on the masses
of eight different peptides. Together these represent ap-
proximately 69% of the complete primary sequence. A number
of as yet unidentified peptides were also observed at low
abundance.
Given the apparent contradiction in the observed labeled
proteins, the originally observed 35 and 70 kDa labeled proteins
(as shown in Figure 7) were isolated, subjected to tryptic
digestion, and analyzed by MALDI-MS/MS. Upon searching
of the MASCOT database, it was found that both the 35 and
the 70 kDa proteins could be attributed to REF. The appearance
of the 14.6 kDa REF protein at these higher molecular weights
appears to be due to oligomerization. Czuppon et al. have
previously found that REF exists as a homotetramer of 58 kDa
when isolated from raw latex.37 A study done by Yeang and
co-workers showed that another rubber particle protein, Hev
b3, is prone to aggregation upon prolonged storage.38
a photoactivatable analogue of FPP suggests that REF can
interact with isoprenoid diphosphates. This interaction may be
key for rubber biosynthesis. However, even if this is the case,
it is unlikely that REF is the sole protein responsible for rubber
synthesis. We are currently using compound 3b and related
analogues to identify other components, including the unidenti-
fied peptides noted above, present in WRPs that contribute to
the catalytic machinery of rubber biosynthesis.
Conclusion
Compound 3b is a photoactive phosphonophosphate-contain-
ing analogue of FPP with enhanced stability that can be
efficiently prepared in six steps from geraniol. Kinetic and
structural studies with 3b and PFTase demonstrate that this probe
is a good mimic of isoprenoid diphosphates. The greater stability
of 3b has allowed us to use it to label and identify a specific
protein that may be involved in rubber biosynthesis. These
findings suggest that 3b and analogues thereof will be useful
tools for probing the structure and function of FPP utilizing
enzymes.
Experimental Section
General Methods and Materials. See the Supporting Informa-
tion.
Dimethyl Geranylphosphonate (9). A mixture of 8 (35 g, 0.16
mol) and trimethyl phosphite (22 g, 0.18 mol) was heated to reflux
for 4 h. After cooling to rt, the mixture was chromatographed on
silica gel (hexanes/ethyl acetate, 5:1 to 1:2) to give the product as
a colorless oil (36 g, 91%): 1H NMR (CDCl3, 300 MHz) δ 5.15
(1H, m), 5.03 (1H, m), 3.72 (3H, s), 3.69 (3H, s), 2.55 (2H, dd, J
) 7.8, 21.9 Hz), 2.03 (4H, m), 1.64 (3H, s), 1.63 (3H, d, J ) 5.7
Hz), 1.57 (3H, s); 13C NMR (CDCl3, 75 MHz) δ 140.4 (d, J )
14.3 Hz), 131.6, 123.8, 112.0 (d, J ) 11.2 Hz), 52.6, 52.5, 39.6 (d,
J ) 2.9 Hz), 25.6, 25.4 (d, J ) 142.0 Hz), 17.6, 16.2, 16.1; 31P
NMR (CDCl3, 121 MHz) δ 31.7. HRMS (ESI) calcd for C12H24O3P
[M + H]+ 247.1463; found 247.1453.
Dimethyl (8-Hydroxy-3,7-dimethylocta-2,6-dienyl)phospho-
nate (10). To a suspension of SeO2 (31 mg, 0.27 mmol), salicylic
acid (190 mg, 1.3 mmol), and 90% t-Bu-OOH (4.5 mL) in CH2-
Cl2 (20 mL) was added 9 (2.5 g, 10 mmol) in CH2Cl2 (5.0 mL) at
rt. The mixture was diluted with Et2O after being stirred for 21 h,
washed with dilute Na2SO3 solution and brine, then dried over Na2-
SO4. The solvents were evaporated, and the residue was chromato-
graphed on silica gel (hexanes/ethyl acetate, 1:1 to 0:1) to give the
alcohol as a colorless oil (1.3 g, 78% yield based on the converted
starting material), together with the recycled starting material (0.95
g): 1H NMR (CDCl3, 300 MHz) δ 5.26 (1H, m), 5.08 (1H, m),
3.91 (2H, s), 3.71 (3H, s), 3.67 (3H, s), 2.98 (1H, br s), 2.51 (2H,
dd, J ) 8.1, 21.9 Hz), 2.11 (4H, m), 1.60 (3H, s), 1.59 (3H, m);
13C NMR (CDCl3, 75 MHz) δ 139.9 (d, J ) 14.6 Hz), 135.5, 124.6,
112.4 (d, J ) 11.4 Hz), 68.6, 52.6, 52.5, 39.1 (d, J ) 3.1 Hz), 25.2
(d, J ) 3.9 Hz), 25.1 (d, J ) 140.3 Hz), 15.9 (d, J ) 2.9 Hz),
Rubber elongation factor (REF), also known as the latex
allergen Hev b1,38 is a known constituent in H. brasiliensis latex
compromising 10-60% of the total protein.39 While it is
possible that REF can exist as carrier or binding partner in rubber
biosynthesis, it has been directly implicated in playing a larger
role. A study done by Dennis and co-workers showed that
treatments of WRPs to either hydrolyze or remove REF
completely abolishes prenyltransferase activity. However, at-
tempts to reconstitute rubber biosynthesis in REF depleted
WRPs with solubilized REF were unsuccessful, making the
exact role of REF in rubber biosynthesis uncertain.40 In our
experiments, the cross-linking of REF in WRP suspensions with
(37) Czuppon, A. B.; Chen, Z.; Rennert, S.; Engelke, T.; Meyer, H. E.;
Heber, M.; Baur, X. J. Allergy Clin. Immunol. 1993, 92, 690-697.
(38) Yeang, H. Y.; Cheong, K. F.; Sunderasan, E.; Hamzah, S.; Chew,
N. P.; Hamid, S.; Hamilton, R. G.; Cardosa, M. J. J. Allergy Clin. Immunol.
1996, 98, 628-639.
(39) Dennis, M. S.; Light, D. R. J. Biol. Chem. 1989, 264, 18608-18617.
(40) Durauer, A.; Csaszar, E.; Mechtler, K.; Jungbauer, A.; Schmid, E.
J. Chromatogr., A 2000, 890, 145-158.
J. Org. Chem, Vol. 72, No. 13, 2007 4593

DeGraw et al.
13.6; 31P NMR (CDCl3, 121 MHz) δ 31.8; IR (NaCl, cm-1) ν 3400
(br), 2955 (m), 1719 (m), 1683 (m). HRMS (ESI) calcd for
C12H24O4P [M + H]+ 263.1412; found 263.1419.
2925 (w), 1715 (m), 1655 (m), 1101 (m). HRMS (ESI) calcd for
C24H27O9P2 [M - H]- 521.1136; found 521.1174. The ammonium
counterions within this compound are not observed during mass
spectral analysis.
Dimethyl (E,E)-((8-(3-Benzoyl)benzoyloxy)-3,7-dimethylocta-
2,6-dienyl)phosphonate (11). To a solution of 10 (2.2 g, 8.3 mmol)
and 3-benzoyl benzoic acid (2.3 g, 10 mmol) in anhydrous CH2Cl2
(100 mL) were added DCC (2.1 g, 10 mmol) and DMAP (50 mg,
0.40 mmol) at rt. The resulting suspension was stirred for 20 h at
room temperature and then filtered through Celite. The filtrate was
concentrated and chromatographed on silica gel (hexanes/ethyl
acetate, 2:1 to 1:2) to give the ester as a viscous oil (3.3 g, 82%):
1H NMR (CDCl3, 300 MHz) δ 8.43 (1H, m), 8.24 (1H, dt, J )
7.8, 1.8 Hz), 7.97 (1H, dt, J ) 7.8, 1.5 Hz), 7.79 (1H, m), 7.77
(1H, m), 7.45-7.62 (4H, m), 5.50 (1H, m), 5.17 (1H, m), 4.70
(2H, s), 3.72 (3H, s), 3.69 (3H, s), 2.55 (2H, dd, J ) 7.8, 21.9 Hz),
2.16 (2H, m), 2.09 (2H, m), 1.70 (3H, s), 1.65 (3H, d, J ) 3.3 Hz);
13C NMR (CDCl3, 75 MHz) δ 195.8, 165.6, 139.9 (d, J ) 14.3
Hz), 137.9, 137.0, 134.0, 133.2, 132.8, 131.0, 130.6, 130.1, 130.0,
129.4, 128.5, 128.4, 112.5 (d, J ) 11.1 Hz), 70.9, 52.6, 52.5, 38.9
(d, J ) 2.6 Hz), 26.1 (d, J ) 3.4 Hz), 25.4 (d, J ) 140.0 Hz), 16.2
(d, J ) 2.4 Hz), 14.0; 31P NMR (CDCl3, 121 MHz) δ 31.6; IR
(NaCl, cm-1) ν 3065 (w), 2955 (s), 2854 (m), 1731 (s), 16675 (s)
1602 (s), 1579 (m). HRMS (ESI) calcd for C26H32O6P [M + H]+
471.1936; found 471.1945.
Ammonium (E,E)-8-(3-Benzoylbenzoyloxy)-3,7-dimethylocta-
2,6-dienylphosphonate (12). To a solution of 11 (1.0 g, 2.1 mmol)
in anhydrous CH2Cl2 (30 mL) were added allyltrimethylsilane (0.50
mL, 3.1 mmol) and bromotrimethylsilane (0.90 mL, 6.8 mmol) at
rt. The mixture was stirred for 38 h and concentrated in vacuo.
The residue was dissolved in H2O (4.0 mL), neutralized with NH4-
HCO3, then lyophilized to afford the phosphonic acid in its
ammonium form as a white powder (0.87 g, 92%): 1H NMR (25
mM NaHCO3 in D2O, 500 MHz) δ 7.80 (1H, s), 7.60 (1H, s), 7.27
(1H, d, J ) 6.0 Hz), 7.15 (2H, m), 7.00 (2H, m), 6.89 (2H, m),
5.06 (1H, s), 5.01 (1H, d, J ) 5.5 Hz), 4.15 (2H, s), 2.10 (2H, d,
J ) 15.0 Hz), 1.67 (4H, m), 1.28 (3H, s), 1.17 (3H, s); 13C NMR
(25 mM NaHCO3 in D2O, 75 MHz) δ 195.1, 165.3, 136.8, 136.4
(d, J ) 13.0 Hz), 136.0, 133.8, 132.7, 129.9, 129.8, 129.6, 129.2,
128.1, 117.1, 70.9, 38.6, 28.9 (d, J ) 131.2 Hz), 25.9, 15.3, 13.2;
31P NMR (25 mM NaHCO3 in D2O, 121 MHz) δ 22.8. IR (NaCl,
cm-1) ν 2971 (w), 2922 (w), 1720 (s), 1655 (s), 1602 (w). HRMS
(ESI) calcd for C24H26O6P [M - H]- 441.1473; found 441.1481.
The ammonium counterions within this compound are not observed
during mass spectral analysis.
(E,E)-â-P-(3-Benzoylbenzoyloxy)-3,7-dimethylocta-2,6-di-
enylphosphonophosphate ([32P]-3b). A mixture of phosphonate
1 (4.0 mg, 8.0 µmol) and 1,1′-carbonyldiimidazole (4.0 mg, 24
µmol) in anhydrous DMF (250 µL) was stirred for 12 h at rt. 32P-
Labeled phosphoric acid was then prepared by lyophilizing 250
µL of 1% (v/v) phosphoric acid and 250 µL of 10 mCi/mL 32P-
phosphoric acid over P2O5 overnight. Activated phosphonate in
DMF was then added via syringe to the flask containing the dry
32P-labeled phosphoric acid (4.0 mg, 40 µmol), left to stir for 24 h
at rt, and monitored by TLC (6:3:1 2-propanol/NH4OH/H2O).
Solvent was then removed under a stream of N2(g). The residue
was dissolved in 25 mM NH4HCO3 (3 mL) and applied to a Sep-
Pak column equilibrated in the same solution. The column was
washed with a step gradient of 25 mM NH4HCO3 and increasing
CH3CN (10% increase per step, 2 mL per step). Fractions were
collected and analyzed by TLC (6:3:1 2-propanol/NH4OH/H2O).
Under these conditions, the desired product 2 eluted at 30-40%
CH3CN/25 mM NH4HCO3. Product fractions were pooled, lyoph-
ilized, and dissolved in 1.0 mL of 25 mM NH4HCO3. UV analysis
(ꢀ258 24 100 mol-1 cm-1) and liquid scintillation counting were used
to determine the solution concentration and specific activity (0.55
mM and 334 Ci/mol). The solution was used for protein cross-
linking experiments without further purification.
Acid Stability Study of GPP. Five hundred microliters of a 1.0
mM solution of GPP in 50:50 25 mM NH4HCO3/CH3CN containing
0.2% TFA was allowed to sit at rt. Fifty microliter aliquots were
removed at prescribed intervals (h) and analyzed by reverse-phase
HPLC. A gradient of solvent A (25 mM NH4HCO3) and solvent B
(CH3CN) and detecting at 214 nm (flow rate, 0.7 mL/min) was
employed. Elution was performed by a 40 min linear gradient of
20-80% B. Integration of the peak corresponding to GPP was
performed using HPLC software. Hydrolysis half-life was deter-
mined from a plot of remaining starting material (%) as a function
of time.
Acid Stability Study of 3b. The same procedure as described
for GPP was performed with a 0.30 mM solution of 3b in 50:50
25 mM NH4HCO3/CH3CN containing 0.2% TFA.
Purification of yPFTase and hPGGTase-I. yPFTase was
purified as described by Mayer et al.41 and published in our earlier
work.16 hPGGTase-I was purified using modifications of published
procedures.12,42,43
Ammonium (E,E)-P-8-(3-Benzoylbenzoyloxy)-3,7-dimethyl-
octa-2,6-dienylphosphonophosphate (3b). A mixture of the phos-
phonate 12 (100 mg, 230 µmol) and 1,1′-carbonyldiimidazole (60
mg, 370 µmol) in anhydrous DMF (4.0 mL) was stirred for 5 h at
rt. To this clear solution was added directly 98% phosphoric acid
solid (∼50 mg, 510 µmol). The solvent was removed under high
vacuum after being stirred for 27 h. The residue was dissolved in
H2O (1.0 mL) and loaded on an octyl-functionalized silica gel
column (18 cm × 1 cm). The column was washed with a step
gradient of H2O and MeOH (10 mL per step, 10% MeOH per step)
from 0 to 70% MeOH. Fractions were collected, and those
containing product as determined by TLC were pooled and
lyophilized. The lyophilized powder was dissolved in H2O, passed
through a short, strong acidic ion exchange column, then washed
with H2O. The resulting solution was neutralized with NH4HCO3
and lyophilized to give the product in its ammonium form as a
white powder (28 mg, 23%): 1H NMR (D2O, 300 MHz) δ 7.77
(1H, s), 7.75 (1H, d, J ) 7.5 Hz), 7.50 (1H, d, J ) 7.5 Hz), 7.09-
7.31 (6H, m), 5.16 (1H, m), 5.03 (1H, m), 4.26 (2H, s), 2.33 (2H,
dd, J ) 7.5, 21.5 Hz), 1.78 (4H, m), 1.39 (3H, d, J ) 3.3 Hz), 1.31
(3H, s); 13C NMR (D2O, 75 MHz) δ 197.5, 166.4, 138.2 (d, J )
14.0 Hz), 136.6, 135.7, 134.3, 133.4, 130.6, 129.8, 129.5, 129.4,
128.7, 128.4, 115.5 (d, J ) 9.7 Hz), 71.1, 38.5, 28.4 (d, J ) 135.2
Hz), 25.7, 15.4, 13.1; 31P NMR (D2O, 121 MHz, pD 7.0, NH4+) δ
16.4 (d, J ) 26.4 Hz), -9.6 (d, J ) 26.4 Hz); IR (NaCl, cm-1) ν
Enzyme Assays. A continuous fluorescence assay was employed
to monitor yPFTase activity as described by Gaon et al.16 modified
from the original published procedures.44,45
Enzyme Inhibition Experiments. The concentration of 3b was
varied (0, 0.4, 0.6, 1.5, 3, 4.5, 6 µM), while the natural substrate,
1, and yPFTase were maintained at a fixed concentrations (10 µM
and 11 nM). Enzymatic rates were obtained from linear regression
analysis of the time-dependent fluorescence emission data using
the fluorimeter software, and the IC50 was calculated from a plot
of the enzymatic rate versus concentration of 3b.
X-ray Crystallography Studies. Crystals of the rPFTase:3b
complex were prepared by soaking 3b into preformed crystals using
methods previously described.32 X-ray diffraction data for the
PFTase:3b complex were collected on a Rigaku rotating anode
(41) Mayer, M. P.; Prestwich, G. D.; Dolence, J. M.; Bond, P. D.; Wu,
H. Y.; Poulter, C. D. Gene 1993, 132, 41-47.
(42) Stirtan, W. G.; Poulter, C. D. Arch. Biochem. Biophys. 1995, 321,
182-190.
(43) Zhang, F. L.; Diehl, R. E.; Kohl, N. E.; Gibbs, J. B.; Giros, B.;
Casey, P. J.; Omer, C. A. J. Biol. Chem. 1994, 269, 3175-3180.
(44) Cassidy, P. B.; Dolence, J. M.; Poulter, C. D. Methods Enzymol.
1995, 250, 30-43.
(45) Pompliano, D. L.; Gomez, R. P.; Anthony, N. J. J. Am. Chem. Soc.
1992, 114, 7945-7946.
4594 J. Org. Chem., Vol. 72, No. 13, 2007

Stable PhotoactiVe Isoprenoid Diphosphate Analogue
generator equipped with osmic mirrors and a Raxis-IV image plate
detector. With the detector set at 150 mm, data were collected in
323 contiguous 0.30° oscillation images each exposed for 8 min.
The data extend to 2.2 Å resolution and have a Rmerge of 4.6% with
a 5.8-fold multiplicity. The structure was refined using CNX2002
(Accelrys Inc.) to an Rfactor of 18.3% and an Rfree of 21.9%.
Photolysis Reaction of Protein Prenyltransferases with [32P]-
3b. All reactions (100 µL) were performed in silinized quartz test
tubes (10 × 75 mm) and contained 52 mM Tris-HCl (pH 7.5), 5.8
mM DTT, 12 mM MgCl2, 12 µM ZnCl2, 25 mM NH4HCO3, 10
µM [32P]-3b (∼10-fold above IC50 value), and 400 nM pure enzyme
(yPFTase, hPGGTase-I) or 21.2 µg of partially purified (after ion-
exchange FPLC) protein sample containing hPGGTase-I. For
substrate protection experiments, FPP or GGPP was added to a
final concentration of 100 µM. Reactions were photolyzed for 2 h
at 4 °C using a Rayonet Mini-Reactor fitted with 6-350 nm bulbs
and a spinning platform. Samples were spun to ensure an even
exposure to the light. Loading buffer (100 µL) was then added to
each sample, and samples were heated to 100 °C for 3 min followed
by analysis with 12% Tris-glycine SDS-polyacrylamide gel elec-
trophoresis. Gels were stained with Sypro-orange and subjected to
32P-phosphorimaging at rt.
Preparation of HeWea brasiliensis Washed Rubber Particles.
H. brasiliensis WRPs used in this study were prepared as previously
described by R. Krishnakumar et al.46 and Cornish et al.47
Photolysis Reaction of HeWea brasiliensis Washed Rubber
Particles with [32P]-3b. All reactions (∼100 µL) were performed
in silinized quartz test tubes (10 × 75 mm) and contained 0.1 M
Tris-HCl (pH 7.5), 1.25 mM MgSO4, 5 mM DTT, 10 µM [32P]-
3b, and a suspension of H. brasiliensis WRP (40 µL, 4.51 µg
protein/µL). For substrate protection experiments, FPP was added
to a final concentration of 100 µM. Reactions were photolyzed for
6 h using the apparatus described above. Loading buffer (50 µL)
was then added to each sample, and samples were heated to 100
°C for 3 min followed by gel analysis as described above.
Tryptic Digest of Labeled Proteins from Photolysis Reaction
of HeWea brasiliensis Washed Rubber Particles with [32P]-3b.
Proteins from previously described photolysis reactions (no SDS-
PAGE analysis) were extracted with 5 mL of sequential extraction
reagent (5.0 M urea, 2.0 M thiourea, 2.0% CHAPS, 2% SB-3-10,
40 mM Tris, and 0.2% Bio-Lyte 3 to 10 ampholyte). Samples were
incubated in this buffer for 1 h at rt with constant rocking and then
centrifuged at max speed in a microcentrifuge for 15 min. The
supernatant was then transferred to a 50 mL conical tube, and 4
volumes of cold acetone were added. Acetone-precipitated protein
pellets were washed twice with 80% acetone in water. Pellets were
dried under N2 and prepared for fractionation using the ZOOM
IEF fractionator according to manufacturer’s instructions. After IEF
fractionation, samples from the different pH ranges were acetone-
precipitated as described above. Samples were then dissolved in
45 µL of Laemmli sample buffer (0.06 M Tris-HCl, pH 6.8, 0.01%
bromophenol blue, 1.5% SDS, 10% glycerol) and analyzed by SDS-
PAGE using 4-20% gradient gel. Gels were stained with Coo-
massie and subjected to 32P-phosphorimaging at rt. Radiolabeled
protein bands were excised using a pipet tip (three spots from each
band). For tryptic digestion of proteins, samples were reduced and
alkylated using 10 mM dithiothreitol and 100 mM iodoacetamide
in water, then digested with 100 ng of trypsin in 25 mM NH4-
HCO3.
Mass Spectral Analysis of Labeled Proteins. Samples from
tryptic digestion were spotted onto a MALDI target and eluted with
70% CH3CN, 0.2% formic acid containing 5 mg/mL MALDI matrix
(R-cyano-4-hydroxycinnamic acid), and 0.5 µL was then spotted
onto the MALDI target. Analysis was performed using an Applied
Biosystems 4700 Proteomics Analyzer with TOF/TOF Optics.
MALDI-MS data were acquired in reflector mode from a mass
range of 700-4000 Da, and 1250 laser shots were averaged for
each mass spectrum. Each sample was internally calibrated using
the trypsin autolysis products (m/z of 842.51 and 2211.10) as
internal standards. The eight most intense ions from the MS
spectrum were then subjected to MS/MS analysis. The mass range
was 70 to precursor ion with a precursor window of -1 to 3 Da
with an average 5000 laser shots for each spectrum. A peak list
was created by GPS Explorer software (Applied Biosystems) from
the raw data based on signal-to-noise filtering and included de-
isotoping. The resulting file was then searched by MASCOT
(Matrix Science) using user specified databases. A tolerance of 20
ppm was used if the sample was internally calibrated and 200 ppm
tolerance if the default calibration was applied. Protein identification
was validated by the following criteria: greater than 20 ppm mass
accuracy on all MS ions and all ions in at least two MS/MS spectra,
which were not modified, had to be accounted for.
Acknowledgment. This research is supported by the Na-
tional Institutes of Health Grant GM58442 and National
Institutes of Health Predoctoral Training Grant T32-GM08700
(to A.J.D.).
Supporting Information Available: Image of the SDS-PAGE
used for the first mass spectral analysis of the photolysis reaction
of HeVea brasiliensis washed rubber particles with [32P]-3b. General
1
methods and materials. Copies of H NMR, 31P NMR, and mass
spectral data for 3b as well as the intermediates in its synthesis.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(46) Krishnakumar, R.; Cornish, K.; Jacob, J. J. Rubber Res. (Kuala
Lumpur, Malaysia) 2001, 4, 131-139.
(47) Cornish, K.; Siler, D. J.; Grosjean, O.-K.; Goodman, N. J. Nat.
Rubber Res. 1993, 8, 275-285.
JO0623033
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