Synthesis and biological activity of isopentenyl diphosphate analogues

Article abstract of DOI:10.1016/j.bmc.2003.11.033

A series of analogues of isopentenyl diphosphate (IPP) having a dicarboxylate moiety in place of the diphosphate were synthesized and investigated as inhibitors of undecaprenyl diphosphate (UPP) synthase and protein farnesyltransferase (PFTase). PFTase is involved in control of cell proliferation and is known to be inhibited by certain maleic acid derivatives bearing long alkyl substituents (≥12 carbons, e.g., chaetomellic acid). UPP synthase is a potential target for antimicrobial agents and utilizes isopentenyl diphosphate (IPP) as a substrate. A number of dicarboxylate- containing IPP analogues were prepared in 2-5 steps from commercially available starting materials with good overall yield (20-78%). These syntheses involved the conjugate addition of an organocuprate to dimethyl acetylenedicarboxylate (DMAD) followed by basic ester hydrolysis. The E-pentenylbutanedioic acid 32 showed inhibition of UPP synthase with an IC₅₀ of 135 μM. Compound 30 displays competitive inhibition of PFTase with a K_i of 287 μM.

Full text of DOI:10.1016/j.bmc.2003.11.033

Bioorganic & Medicinal Chemistry 12(2004) 763–770
Synthesis and biological activity of isopentenyl diphosphate
analogues
Andrew A. Scholte,a Lisa M. Eubanks,b C. Dale Poulterb and John C. Vederasa,*
aDepartment of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2
bDepartment of Chemistry, Henry Eyring Building, University of Utah, Salt Lake City, UT 84112, USA
Received 24 July 2003; accepted 5 November 2003
Abstract—A series of analogues of isopentenyl diphosphate (IPP) having a dicarboxylate moiety in place of the diphosphate were
synthesized and investigated as inhibitors of undecaprenyl diphosphate (UPP) synthase and protein farnesyltransferase (PFTase).
PFTase is involved in control of cell proliferation and is known to be inhibited by certain maleic acid derivatives bearing long alkyl
substituents (ꢀ12carbons, e.g., chaetomellic acid). UPP synthase is a potential target for antimicrobial agents and utilizes iso-
pentenyl diphosphate (IPP) as a substrate. A number of dicarboxylate-containing IPP analogues were prepared in 2–5 steps from
commercially available starting materials with good overall yield (20–78%). These syntheses involved the conjugate addition of an
organocuprate to dimethyl acetylenedicarboxylate (DMAD) followed by basic ester hydrolysis. The E-pentenylbutanedioic acid 32
showed inhibition of UPP synthase with an IC50 of 135 mM. Compound 30 displays competitive inhibition of PFTase with a Ki of
287 mM.
# 2003 Elsevier Ltd. All rights reserved.
The prevalence of bacterial resistance to current anti-
biotics has encouraged the search for new antimicrobial
compounds active against pathogenic microorganisms.
Enzymes involved in bacterial cell wall biosynthesis are
attractive targets for the development of new anti-
bacterial agents. Undecaprenyl diphosphate synthase
catalyzes the condensation of eight molecules of iso-
pentenyl diphosphate (IPP) with farnesyl diphosphate
(FPP) to produce undecaprenyl diphosphate (UPP),
whose function is to transport lipid II across the mem-
brane for bacterial cell wall biosynthesis.1 Inhibitors of
UPP synthase may give some insight on its specificity as
well as its molecular mechanism for the formation of
UPP. Recently, a crystal structure of UPP synthase and
a proposed substrate-enzyme binding model were
reported.2,3 Further understanding of this enzyme will
provide insight for the development of new antibacterial
agents. In the present study we targeted inhibition of
UPP synthase by dicarboxylate analogues of IPP
because certain alkyl-substituted dicarboxylates are
potent inhibitors of protein farnesyltransferase
(PFTase).4À6
PFTase catalyzes the transfer of the C15 farnesyl unit
from farnesyl diphosphate (FPP) to the cysteine residue
located in
a
tetrapeptide CAAX (A=aliphatic,
X=methionine or serine) sequence at the carboxyl ter-
minus of proteins.7 Prenylation of proteins is an impor-
tant post-translational modification of ribosomally
produced proteins and is important for cell prolifera-
tion.8 Examples of proteins that undergo prenylation
would include the family of small G proteins, in parti-
cular Ras, oncogenic proteins found in a variety of
tumor types including breast, ovarian and pancreatic.9
As prenylation of Ras is required for its activity, there
has been a considerable effort in discovering new
PFTase inhibitors as a potential treatment of cancer.
To date, most of the inhibitors developed are focused
on peptidomimetics that contain the CAAX recognition
motif. Much less attention has been paid to inhibitors
that resemble farnesyl diphosphate. For example, chae-
tomellic acid A (1) is a inhibitor of PFTase and com-
petes for the FPP binding site with an IC50 of 17 mM
(Fig. 1).10 Recently, two natural products, CJ-13,981 (2)
and CJ-13,982( 3), containing polyanionic functionality
were isolated and were shown to inhibit squalene syn-
thase, an enzyme that catalyzes the dimerization of two
FPP molecules to form squalene, an intermediate in
cholesterol biosynthesis.11 Inhibitors designed as prenyl
* Corresponding author at current address: Department of Chemistry,
University of Alberta, Edmonton, Alberta, Canada T6G 2G2. Tel.:
+1-780-492-5475; fax: +1-780-492-8231; e-mail: John.Vederas@
ualberta.ca
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.11.033

764
A.A. Scholte et al. / Bioorg. Med. Chem. 12 (2004) 763–770
Figure 1. Polyanionic natural compounds.
diphosphate analogues have been employed in substrate
specificity studies and may lead to new drugs.12 We now
report the synthesis of a series of new inhibitors that
contain a dicarboxylate moiety as inhibitors of enzymes
that utilize diphosphates. Analogues of isopentenyl di-
phosphate (IPP) containing a dicarboxylate moiety were
attractive because of IPP’s importance in the biosynth-
esis of a variety of isoprenoids, including FPP.
Scheme 1. Synthesis of IPP analogues. Reagents and conditions: (i)
NBS, PPh3, CH2Cl2, 16 h 40%; (ii) Mg, THF, Á, 2h; (iii) CuBr Me2S,
THF, À40 ꢁC, 2h; (iv) DMAD, THF, À78 ꢁC, 40% for 6 and 62% for
11 (over three steps); (v) LiOH, H2O/THF, quant for 7 and 12; (vi)
LiAlH4, THF, 88%; (vii) TsCl, NEt3, DMAP, CH2Cl2; (viii) LiBr,
acetone, 18 h, 60% (over two steps).
.
Initial targets containing maleic acid units and structu-
rally rigid succinic acid units were designed as analogues
of IPP. Such units can achieve a spacing of negatively
charged oxygen atoms that is within 0.1 A of that in
the corresponding diphosphate moiety (Fig. 2).13
.
addition to a suspension of CuBr Me2S in THF pro-
duced the corresponding organocuprate, which was
subsequently added to DMAD to provide the desired
diester 6. Ester hydrolysis using lithium hydroxide
afforded the desired dicarboxylate 7. Compound 12 was
made in a similar manner, starting with ethyl-4-methyl-
4-pentenoate (8). Reduction of the ester with lithium
aluminum hydride gave the alcohol 9,15 which was then
converted to bromide 10 in two steps via the corre-
sponding tosylate.16 The protected diester 11 was made
as previously described by the conjugate addition to
DMAD. Subsequent base hydrolysis gave 12 in quanti-
tative yield.
1. Results and discussion
Previously, we reported a new synthetic route to chae-
tomellic acid A and its analogues.10 The key step in this
synthetic sequence involved the conjugate addition of
an organocuprate to dimethyl acetylenedicarboxylate
(DMAD). It was observed that use of copper (I) bro-
.
mide dimethyl sulfide complex (CuBr Me2S) gave the
preferential formation of product with the two esters
having a cis relationship.
To probe the importance of the external methylene
group for IPP binding to the enzyme active site, satu-
rated analogues of IPP were also synthesized as shown
in Scheme 2. Starting from 1-bromo-3-methyl-1-butane
(13), the corresponding organocuprate was formed and
added to DMAD. Conversion of the dimethyl ester to
the lithium dicarboxylate 15 was achieved using 1.0 M
lithium hydroxide. In a similar process, 18 was prepared
from 1-bromo-4-methyl-4-pentene (16).
1.1. Synthesis of diphosphate analogues
Compounds 7 and 12 were selected as initial synthetic
targets because of their similarity to IPP. Their synthesis
is shown in Scheme 1. Treatment of the commercially
available 3-methyl-3-buten-1-ol (4) with triphenylpho-
sphine and N-bromosuccinimide gave bromide 5.14
Formation of the Grignard reagent followed by its
Inhibitors based on succinic acid were also made to
determine if selective inhibition of cis- or trans-pre-
nyltransferases would be achieved. Compounds 21 and
24 were synthesized in seven steps using a Diels–Alder
reaction as the key transformation (Scheme 3). Conver-
sion of the dilithium salt 12 to its anhydride 19 with
dilute hydrochloric acid and subsequent reaction with
Figure 2. Comparison of diphosphate and maleates.

A.A. Scholte et al. / Bioorg. Med. Chem. 12 (2004) 763–770
765
ꢁ
.
Scheme 2. Synthesis of saturated analogues. Reagents and conditions: (i) Mg, THF, Á, 2h; (ii) CuBr Me2S, THF, À40 C, 2h; (iii) DMAD, THF,
À78 ꢁC, 60% for 14 and 82% for 17 (over three steps); (iv) LiOH, H2O/THF, quant for 15 and 18.
of the oxygen atoms in the dicarboxylate and this lack
of rotational freedom may not allow the molecule to
achieve the correct geometrical or spatial arrangement
for efficient binding to the active site of the enzyme. To
investigate the effect of increasing the rotational free-
dom within the two dicarboxylates, compounds 26 and
28 were prepared.
As shown in Scheme 4, syntheses of the desired com-
pounds 26 and 28 can be achieved via the conjugate
reduction of the corresponding substituted dimethyl
maleate. Initial attempts at this reduction utilizing either
dissolving metal reduction conditions (Li and NH3(l)) or
ruthenium-catalyzed reductions using triethylsilane did
not yield the desired product. Analysis of the reaction
mixtures with both sets of reagents indicated formation
of many side products. Recently, copper hydrides such
as hexa-m-hydrohexakis(triphenylphosphine) hexacopper
have been used as gentle reducing agents of a,b-unsatu-
rated esters, ketones and aldehydes.17 Therefore, treat-
ment of dimethyl (Z)-2-(2-methyl)butenylbutenedioate
(6) with hexa-m-hydrohexakis(triphenylphosphine) hex-
acopper gave the desired compound 25 in 61% yield.
Hydrolysis of the dimethyl esters with lithium hydroxide
generated the desired lithium salt 26. In a similar man-
Scheme 3. Synthesis of rigid IPP analogues. Reagents and conditions:
(i) HCl, Et2O, 80% for 19 and 76% for 22; (ii) cyclopentadiene, tolu-
ner compound 10 was reduced and subsequent base
mediated removal of the protective groups afforded 28
in quantiative yield.
ene, Á, 69% for 20 and 41% for 23; (iii) LiOH, H2O/THF, quant for
21 and 24.
cyclopentadiene gave the cycloadduct 20. NMR analysis
of the reaction mixture indicated preferential formation
of the endo adduct in a ratio of 2:1 to its exo product.
The stereochemical assignments of the cycloadduct
products were determined by using 2D TROESY NMR
experiments. In particular, interaction was observed for
the endo isomer between the bridged methylene and the
hydrogen a- to the carbonyl of the anhydride.
In addition to synthesizing analogues that contain
dicarboxylates with a cis relationship, two analogues
with trans dicarboxylates were also prepared (Scheme
Anhydride 20 was then hydrolyzed using lithium
hydroxide to afford 21. In a similar manner, treatment
of the lithium dicarboxylate 7 with dilute HCl gave
anhydride 22 in good yield. Conversion into the bicyclic
23 derivative was achieved by reaction with cyclopenta-
diene in toluene. Basic hydrolysis of anhydride 23 gave
the desired compound 24.
Scheme 4. Synthesis of flexible compounds. Reagents and conditions:
(i) [(C6H5)3PCu]6, toluene, 61% for 25 and 27; (b) LiOH, H2O/THF,
quant for 26 and 28.
The installment of a double bond between the two
dicarboxylates introduces restriction to the movement

766
A.A. Scholte et al. / Bioorg. Med. Chem. 12 (2004) 763–770
Scheme 5. Synthesis of trans IPP analogues. Reagents and conditions:
(i) Mg, THF, Á, 2h; (ii) DMAD, THF, À78 ꢁC, 8% for 29 and 3% for
31; (d) LiOH, H2O/THF, quantitative for 30 and 32.
Figure 3. Inhibition of PFTase by compound 30. Double-reciprocal
plot with FPP as the varied substrate at fixed concentrations of 30.
Concentrations of analogue 30 were 100 (*), 200 (&), 300 (&), and
400 (*) mM. Dansyl-Gly-Val-Ile-Ala was held constant at 2.4 mM.
PFTase (1.0 nM) was used to initiate reactions. Double-reciprocal plot
was generated by fitting the data to the appropriate form of the
Michaelis–Menten equation.
5). As was previously described, the addition of an
.
organocopper reagent, prepared from CuBr Me2S, to
DMAD gave primarily cis addition. The trans com-
pounds were accessed by the addition of the corre-
sponding organomagnesium reagent to DMAD,
followed by basic hydrolysis to give the desired com-
pounds 30 and 32. The yields for these compounds are
unoptimized.
Inhibition studies with E. coli UPP synthase involve a
radioactive assay. After incubation of [1-14C] IPP, FPP
and inhibitor with UPP synthase, the reaction products
are chromatographed on silica gel TLC. The plate with
radiolabeled products was exposed to a film and the
radioactivity distribution was determined by phosphor
autoradiography. The results show that the majority of
the dicarboxylates are poor inhibitors of UPP synthase.
Fortunately, compounds 30 and 32 with the trans
dicarboxylates show modest inhibition with an IC50 of
492and 135 mM, respectively.
1.2. Biological evaluation of the IPP analogues
Ten analogues of IPP (7, 12, 15, 18, 21, 24, 26, 28, 30,
32) were tested against PFTase and UPP synthase.
Recombinant yeast protein farnesyltransferase was
expressed in Escherichia coli and purified on a Ni(II)
column to homogeneity.18 The enzyme activity was
conveniently monitored using a continuous flourometric
assay using dansyl-Gly-Val-Ile-Ala at an enzyme con-
centration of 1.0 nM.19 The majority of compounds
tested, proved to be only poor inhibitors of PFTase with
IC50 values of greater than 1 mM. As shown in Table 1,
the bicyclic-based inhibitors 21 and 24 showed
improved inhibition with IC50’s of 812and 804 mM,
respectively. Analogue 30, with the dicarboxylates hav-
ing a trans relationship displayed the best inhibition of
PFTase with an IC50 of 384 mM. Compound 30 was also
shown to be a competitive inhibitor of PFTase (Fig. 3)
against FPP with a Ki=287Æ30 mM.
The lack of inhibition by these diphosphate mimics may
be due to poor binding of the substrate to the active site
of the enzyme. Recently, a co-crystal structure of pro-
tein farnesyltransferase indicated that the non-bridging
oxygen atoms of the two phosphates in diphosphate
moiety of the bound FPP molecule do not reflect in a
plane through the bridging oxygen.20,21 For binding to
the enzyme active site, specific geometric or spatial
arrangement of the diphosphate may be required, which
these dicarboxylates can not achieve in solution.
2. Conclusion
In summary, we have described the synthesis of a series
of ten analogues of diphosphates. Although these com-
pounds generally show only weak inhibition of PFTase
and UPP synthase, it was shown that 32 is a modest
inhibitor of UPP synthase. Synthesis of other mimics of
diphosphates and their interactions with enzymes that
utilize diphosphates is currently underway.
Table 1. IC50 values for IPP analogues
Compd
PFTase IC50 (mM)a
UPP synthase IC50 (mM)b
7
>1000
>1000
>1000
>1000
812Æ28
804Æ46
>1000
>1000
384Æ33
>1000
>1000
>1000
>1000
>1000
936Æ43
>1000
>1000
>1000
492Æ20
135Æ18
12
15
18
21
24
26
28
30
32
3. Experimental
3.1. General
a IC50 conditions: 1.0 nM PFTase, 50 mM Tris–HCl, 12mM MgCl
,
2
All reagents were purchased from Sigma or Aldrich and
were used without further purification unless otherwise
stated. All solvents were dried and distilled prior to use
according to standard procedures.22 Copper(I) bromide
12 mM ZnCl2, 5.8 mM DTT, 0.04% (w/v) n-dodecyl-b-d-maltoside,
pH 7.0 dansyl-Gly-Cys-Val-Ile-Ala was the peptide substrate.
b IC50 conditions: 0.3 mM UPPase, 100 mM Tris–HCl pH 8.5, 2mM
MgCl2, 5 mM FPP and 45 mM [1-14C] IPP (55 Ci/mol).

A.A. Scholte et al. / Bioorg. Med. Chem. 12 (2004) 763–770
767
dimethyl sulfide complex was purified as previously
reported by House et al.23 1H NMR data is reported to the
nearest 0.01 and 13C NMR data to the nearest 0.1 ppm.
temperature. After 30 min, the mixture is partitioned
between water and ether. The aqueous layer was
extracted with ether (3Â15 mL) and the combined
organic extracts were washed with an additional satu-
rated aqueous NH4Cl solution (25 mL), and brine (25
mL). Drying over MgSO4 and concentration in vacuo
gave an amber oil. Purification by flash column chro-
matography gave diester as a colorless oil.
3.2. Inhibition studies with protein farnesyl tranferase
Recombinant yeast PFTase was produced in E. coli and
purified by chromatography on a Ni(II) column as pre-
viously described.18 Catalytic rate constants were mea-
sured using a fluorescence assay that continuously
monitored farnesylation of dansylated pentapeptide
using a Spex Fluoromax model spectroflourimeter with
lex=340 (slit width=5.1 nm) and lem=486 nm (slit
width=5.1 nm) and 3 mm square cuvettes. For PFTase,
assays (250 mL) were conducted at 30 ꢁC in 50 mM Tris–
HCl, 12mM MgCl 2, 12 mM ZnCl2, 5.8 mM DTT, 0.04%
(w/v) n-dodecyl-b-d-maltoside, pH 7.0. Dansyl-Gly-
Cys-Val-Ile-Ala was the peptide substrate. Reaction
mixture were preincubated at 30 ꢁC for 5 min before the
reaction was initiated by PFTase previously diluted with
assay buffer containing 1 mg/mL bovine serum albu-
min. Initial rates were measured from the linear region
of each run, and all measurements were made in dupli-
cate. Rates were measured in counts/second per second
and converted to units of sÀ1 using a conversion factor
calculated from the slope of a line generated in a plot of
concentration of synthetic dansyl-Gly-((S)-farnesyl)Cys-
Val-Ile-Ala versus fluorescence intensity.
3.4.1. Dimethyl (Z)-2-(2-methyl)butenylbutenedioate (6).
The general procedure was followed. Thus 4-bromo-2-
methyl-1-butene (1.0 g, 6.71 mmol), magnesium turn-
.
ings (300 mg, 12.08 mmol), CuBr Me2S (1.38 g, 6.71
mmol), DMAD (800 mg, 5.6 mmol) were reacted as
before. Purification of the crude product by flash col-
umn chromatography (SiO2, petroleum ether/ether, 9:1)
gave 6 as an oil (490 mg, 41%). IR (film) 3077, 2953,
1
1732, 1651, 1436 cmÀ1; H NMR (CDCl3, 300 MHz) d
5.82(m, 1H), 4.76 (m, 1H), 4.70 (m, 1H), 3.82(s, 3H),
3.71 (s, 3H), 2.49 (m, 2H), 2.19 (m, 2H), 1.72 (s, 3H);
13C NMR (CDCl3, 75 MHz) d 169.5, 165.4, 150.0,
143.5, 119.6 111.2, 52.4, 51.9, 34.9, 32.5, 22.4; HRMS
(ES +ve) calcd [M+Na]+ for C11H16O4Na 235.0946,
found 235.0944.
3.4.2. Dimethyl (Z)-2-(2-methyl)pentenylbutenedioate
(11). The general procedure was followed. Thus 1-
bromo-4-methyl-4-pentene (1.0 g, 6.13 mmol), magne-
.
sium turnings (298 mg, 12.26 mmol), CuBr Me2S (1.63
g, 6.13 mmol), DMAD (0.63 mL, 4.85 mmol) were
reacted as before. Purification of the crude product by
flash column chromatography (SiO2, Petroleum ether/
ether, 9:1) gave 11 (680 mg, 62%) as an oil. IR (film)
3.3. Inhibition studies with undecaprenyl diphosphate
synthase
Recombinant his6-tagged UPP synthase from E. coli
was overproduced and purified by Ni (II) affinity chro-
matography essentially as described for PFTase.18 UPP
synthase activity was measured by incorporation of
radioactivity into UPP from [1-14C] IPP. Reaction mix-
tures (250 mL) contained 100 mM Tris–HCl (pH 8.5), 2
mM MgCl2, 5 mM FPP, 45 mM [1-14C] IPP (55 Ci/mol),
and inhibitor. UPP synthase (0.3 mM) and the samples
were incubated at 37 ꢁC for 10 min before being quen-
ched. The reaction mixtures were chromatographed on
silica gel TLC plates and the enzymatic product was
visualized by phosphor autoradiography.
1
3074, 2951, 1728, 1649, 1436 cmÀ1; H NMR (CDCl3,
300 MHz) d 5.8 (t, 1H, J=1.5 Hz), 4.71 (m, 1H), 4.65
(m, 1H), 3.80 (s, 3H), 3.70 (s, 3H), 2.32 (m, 2H), 2.03
(m, 2H), 1.67 (s, 3H), 1.58 (m, 2H); 13C NMR (CDCl3,
75 MHz) d 169.4, 165.4, 119.4, 110.8, 52.3, 51.8, 36.8,
33.5, 24.8, 22.2; HRMS (ES +ve) calcd for [M+Na]+
C12H18O4Na 249.1103, found 249.1108.
3.4.3. Dimethyl (Z)-2-(2-methyl)pentylbutenedioate (14).
The general procedure was followed. Thus 1-bromo-4-
methyl-4-pentene (1.0 g, 6.06 mmol), magnesium turn-
.
ings (300 mg, 12.12 mmol), CuBr Me2S (1.25 g, 6.06
mmol), DMAD (0.63 mL, 4.85 mmol) were reacted as
before. Purification of the crude product by flash col-
umn chromatography (SiO2, Petroleum ether/ether, 9:1)
gave 14 (830 mg, 60%) as an oil. IR (film) 2954, 2870,
3.4. General procedure for preparation of cis-maleyl
substituted dicarboxylates
In a flame dried, argon flushed round-bottom flask was
added freshly ground magnesium turnings (1.2equiv)
and THF (3 mL). To this was then added a solution of
bromide (1.0 equiv) in THF (5 mL) dropwise, and a
crystal of iodine. The reaction mixture was refluxed for
2h and cooled to room temperature. The prepared
Grignard was then added dropwise to a suspension of
cuprous bromide-dimethyl sulfide complex (1.2equiv)
in THF (10 mL) at À40 ꢁC. The resulting solution was
stirred at À40 ꢁC for 2h and then cooled to À78 ꢁC, and
freshly distilled DMAD (1 equiv) in THF (5 mL) was
added dropwise to give a dark red-brown mixture. After
1 h, the reaction mixture was quenched with a saturated
solution of ammonium chloride (5 mL, adjusted to pH 8
with 10% ammonia) and allowed to warm to room
1
1728, 1651, 1436 cmÀ1; H NMR (CDCl3, 300 MHz) d
5.79 (t, 1H, J=1.5 Hz), 3.81 (s, 3H), 3.70 (s, 3H), 2.32
(m, 2H), 1.50 (m, 3H), 1.19 (m, 2H), 0.85 (d, 6H, J=6.6
Hz); 13C NMR (CDCl3, 75 MHz) d 169.5, 165.5, 151.1,
119.0, 52.3, 51.8, 38.1, 34.6, 27.8, 24.8, 22.5; HRMS (ES
+ve) calcd for [M+Na]+ C12H20O4Na 251.1259, found
251.1261.
3.4.4. Dimethyl (Z)-2-(2-methyl)butylbutenedioate (17).
The general procedure was followed. Thus 1-bromo-3-
methyl-1-butane (1.0 g, 6.62mmol), magnesium turn-
.
ings (322 mg, 13.24 mmol), CuBr Me2S (1.76 g, 6.62
mmol), DMAD (784 mg, 5.52mmol) were reacted as
before. Purification of the crude product by flash col-

768
A.A. Scholte et al. / Bioorg. Med. Chem. 12 (2004) 763–770
umn chromatography (SiO2, petroleum ether/ether, 9:1)
gave 17 (970 mg, 82%) as an oil. IR (film) 2955, 2871,
3.6. General procedure for the formation of anhydrides
1
1731, 1635, 1436 cmÀ1; H NMR (CDCl3, 300 MHz) d
Acidification of a stirring solution of dilithium salts in
water (5 mL) with 1.0 N HCl at 0 ꢁC and extraction with
ether gave the anhydride. Purification by flash column
chromatography gave the corresponding anhydride as a
white solid.
5.79 (t, 1H, J=1.4 Hz), 3.81 (s, 3H), 3.69 (s, 3H), 2.33
(m, 2H), 1.56 (m, 3H), 1.35 (m, 2H) 0.87 (d, 6H, J=8.7
Hz); 13C NMR (CDCl3, 75 MHz) d 169.5, 165.5, 151.3,
118.9, 52.3, 51.8, 35.9, 32.4, 27.5 22.3; HRMS (ES +ve)
calcd for [M+Na]+ C11H18O4Na 237.1103, found
237.1096.
3.6.1. (Z)-2-(2-Methyl)pentenylbutenedioic acid, di-car-
bonic anhydride (19). The acidification of di-lithium salt
12 (100mg 0.471 mmol) gave salt 19 (90 mg, 99%) as a
white powder: IR 2939, 1843, 1772, 1700, 1652, 1436,
3.5. General procedure for preparation of trans-maleyl
substituted dicarboxylates
1
1376 cmÀ1; H NMR (CDCl3, 500 MHz) d 5.88 (s, 1H),
In a flame dried, argon flushed round-bottom flask was
added freshly ground magnesium turnings (1.2equiv)
and THF (3 mL). To this was then added a solution of
bromide (1.0 equiv) in THF (5 mL) dropwise, and a
crystal of iodine. The reaction mixture was refluxed for
2h and cooled to room temperature. The prepared
Grignard was then added dropwise to a stirring solution
of freshly distilled DMAD (1 equiv) at À78 ꢁC to give
dark brown-red mxture. After 1 h, the reaction mixture
was quenched with a saturated solution of ammonium
chloride (5 mL, adjusted to pH 8 with 10% ammonia)
and allowed to warm to room temperature. After 30
min, the mixture is partitioned between water and ether.
The aqueous layer was extracted with ether (3Â15 mL)
and the combined organic extracts were washed with an
additional saturated aqueous NH4Cl solution (25 mL),
and brine (25 mL). Drying over MgSO4 and concen-
tration in vacuo gave an amber oil. Purification by flash
column chromatography gave diester as a colorless oil.
4.74 (s, 1H), 4.68 (s, 1H), 2.40 (t, 2H, J=7.0 Hz), 2.06
(t, 2H, J=7.5 Hz), 1.70 (m, 5H); 13C NMR (CDCl3,
125 MHz) d 174.3, 170.5, 150.9, 144.3, 119.9, 110.9,
36.8, 33.8, 24.8, 22.2; HRMS (EI) calcd for [M+.
C10H13O3 180.0786, found 180.0781.
]
3.6.2. (Z)-2-(2-Methyl)butenylbutenedioic acid, di-car-
bonic anhydride (22). The acidification of di-lithium salt
7 (100 mg 0.471 mmol) gave anhydride 22 (90 mg, 99%)
as a colorless oil: IR 2916, 1697, 1648, 1424, 1380 cmÀ1
;
1H NMR (CDCl3, 300 MHz) d 5.90 (s, 1H), 4.78 (s, 1H),
4.71 (s, 1H), 2.54 (m, 2H), 2.40 (t, 2H, J=6.9 Hz), 1.73
(s, 3H); 13C NMR (CDCl3, 75 MHz) d 173.9, 170.5,
150.4, 143.2, 120.4, 111.5, 34.9, 32.5, 22.4; HRMS (EI)
calcd for [M+.] C9H10O3 166.0630, found 166.0628.
3.7. General procedure for the basic hydrolysis of dimethyl
esters to lithium salts
To the di-ester (50 mg) in THF–H2O (2mL, 1:1) was
added 1.0 N LiOH (3 equiv) and the mixture was stirred
at 50 ꢁC and monitored for the consumption of starting
material by TLC. The solvent was removed in vacuo
and the remaining residue was dissolved in H2O (4 mL).
Freeze-drying of the aqueous layer gave the respective
lithium salt.
3.5.1. Dimethyl (E)-2-(2-methyl)butenylbutenedioate
(29). The general procedure was followed. Thus 4-
bromo-2-methyl-1-butene (750 mg, 5.03 mmol), magne-
sium turnings (245 mg, 10.07 mmol), and DMAD (0.56
mL, 4.57 mmol) were reacted as before. Purification of
the crude product by flash column chromatography
(SiO2, hexanes/ethyl acetate, 10:1) gave 29 as an oil (78
mg, 8%). IR (film) 3075, 2953, 1725, 1645, 1436 cmÀ1
;
3.7.1. (Z)-2-(2-Methyl)butenylbutenedioic acid, di-lithium
salt (7). The hydrolysis of ester 6 (100 mg, 0.471 mmol)
gave salt 7 (90 mg, 99%) as a white powder: IR 3379,
;
3079, 2968, 2937, 1648, 1555, 1419 cmÀ1 1H NMR
(CD3OD, 300 MHz) d 5.51 (m, 1H), 4.70 (m, 2H), 2.39
(m, 2H), 2.23 (m, 2H), 1.73 (s, 3H); 13C NMR (CD3OD,
75 MHz) d 179.0, 174.8, 151.3, 147.2, 120.3, 109.9, 35.5,
33.1, 21.9; HRMS (ES–ve) calcd for [MÀH]À C9H11O4
183.0657, found 183.0663.
1H NMR (CDCl3, 300 MHz) d 6.73 (s, 1H), 4.70 (m,
1H), 4.67 (m, 1H), 3.78 (s, 3H), 3.74 (s, 3H), 2.91 (m,
2H), 2.13 (m, 2H), 1.74 (s, 3H); 13C NMR (CDCl3,
100 MHz) d 167.2, 165.9, 147.8, 144.8, 126.5, 110.7,
52.6, 51.8, 37.2, 26.7, 22.3; HRMS (ES +ve) calcd for
[M+H]+ C11H17O4 213.1121, found 213.1122.
3.5.2. Dimethyl (E)-2-(2-methyl)pentenylbutenedioate
(31). The general procedure was followed. Thus 1-
bromo-4-methyl-4-pentene (750 mg 4.60 mmol), mag-
nesium turnings (223 mg, 9.20 mmol) and DMAD (0.38
mL, 2.80 mmol) were reacted as before. Purification of
the crude product by flash column chromatography
(SiO2, hexanes/ethyl acetate, 10:1) gave 31 as an oil (30
mg, 3%). IR (film) 3073, 2929, 1725, 1649, 1436, 1373
3.7.2. (Z)-2-(2-Methyl)pentenylbutenedioic acid, di-lithium
salt (12). The hydrolysis of ester 11 (100 mg 0.471
mmol) gave salt 12 (90 mg, 99%) as a white powder: IR
;
3379, 3079, 2968, 2937, 1648, 1555, 1419 cmÀ1 1H
NMR (D2O, 300 MHz) d 5.48 (m, 1H), 4.70 (m, 2H),
2.21 (t, 2H, J=8.0 Hz), 2.08 (t, 2H, J=7.4 Hz), 1.72(s,
3H), 1.59 (dpent, 2H, J=1.4 Hz, 7.4 Hz); 13C NMR
(D2O, 75 MHz) d 179.9, 175.4, 152.6, 148.6, 120.7,
110.4, 37.4, 34.9, 25.8, 22.3; HRMS (ES–ve) calcd for
[MÀH]À C9H11O4 197.0814, found 197.0819.
1
cmÀ1; H NMR (CDCl3, 400 MHz) d 6.74 (s, 1H), 4.71
(m, 1H), 4.68 (m, 1H), 3.79 (s, 3H), 3.76 (s, 3H), 2.74
(m, 2H), 2.07 (t, 2H, J=7.6 Hz), 1.70 (s, 3H), 1.58 (m,
2H); 13C NMR (CDCl3, 100 MHz) d 167.3, 166.0, 148.1,
145.3, 126.3, 110.1, 52.4, 51.6, 37.7, 27.6, 26.9, 22.2;
HRMS (EI) calcd for [M+.] C12H18O4 226.1205, found
226.1200.
3.7.3. (Z)-2-(2-Methyl)pentylbutenedioic acid, di-lithium
salt (15). The hydrolysis of ester 14 (100 mg 0.438

A.A. Scholte et al. / Bioorg. Med. Chem. 12 (2004) 763–770
769
1
mmol) gave salt 15 (93 mg, 99%) as a white powder: IR
3433, 2954, 2870, 2845, 1640, 1586, 1428 cmÀ1 1H
1573, 1427 cmÀ1; H NMR (D2O, 400 MHz) d 4.72(m,
;
2H), 2.54 (m, 1H), 2,41 (dd, 1H, J=5.4 Hz, 14.3 Hz),
2.14 (dd, 1H, J=9.5 Hz, 14.3 Hz), 2.04 (m, 2H), 1.70 (s,
3H), 1.42(m, 4H) 13C NMR (D2O, 100 MHz) d 185.6,
182.6, 148.9, 110.1, 46.9, 42.0, 37.9, 32.5, 25.8, 24.1,
22.3; HRMS (ES–ve) calcd for [MÀH]À C10H15O4
199.0963, found 199.0964.
NMR (D2O, 300 MHz) d 5.48 (m, 1H), 2.21 (m, 2H),
1.45 (m, 3H), 1.23 (m, 2H), 0.88 (d, 6H, J=6.6 Hz); 13C
NMR (D2O, 125 MHz) d 179.4, 174.8, 152.6, 119.8,
38.4, 35.3, 37.6, 25.3, 22.5, 22.4; HRMS (ES–ve) calcd
for [MÀH]À C10H15O4 199.0970, found 199.0976.
3.7.4. (Z)-2-(2-Methyl)butylbutenedioic acid, di-lithium
salt (18). The hydrolysis of ester 17 (99 mg 0.462
mmol) gave salt 18 (102mg, 94%) as a white powder:
3.7.9. (E)-2-(2-Methyl)butenylbutenedioic acid, di-lithium
salt (30). The hydrolysis of ester 29 (14 mg 0.066 mmol)
gave salt 30 (14 mg, 99%) as a white powder: IR 3362,
2938, 1648, 1567, 1390 cmÀ1; 1H NMR (D2O, 300 MHz)
d 6.34 (s, 1H), 4.71 (s, 1H), 4.69 (s, 1H), 2.59 (t, 2H,
J=8.1 Hz), 2.09 (t, 2H, J=7.5 Hz), 1.69 (s, 3H); 13C
NMR (D2O, 75 MHz) d 178.1, 177.2, 147.8, 144.4,
129.4, 110.7, 37.3, 28.0, 22.3; HRMS (ES–ve) calcd for
[MÀH]À C9H11O4 183.0652, found 183.0653.
1
IR 3409, 2954, 2871, 1638, 1574, 1428 cmÀ1; H NMR
(D2O, 300 MHz) d 5.48 (m, 1H), 2.23 (m, 2H), 1.58 (m,
1H), 1.32(m, 2H), 0.87 (d, 6H, J=6.6 Hz); 13C NMR
(D2O, 75 MHz) d 180.0, 175.5, 153.4, 120.2, 37.1, 33.4,
27.9, 22.5; HRMS (ES–ve) calcd for [MÀH]À C9H13O4
185.0814, found 185.0819.
3.7.5. 2-(4-Methyl-pent-4-enyl)-bicyclo[2.2.1]hept-5-ene-
2,3-dioic acid, dilithium salt (21). The hydrolysis of the
anhydride 20 (16 mg, 0.069 mmol) gave salt 21 (25 mg,
99%) as a white powder. IR 3373, 3074, 2967, 1704,
3.7.10. (E)-2-(2-Methyl)pentenylbutenedioic acid, di-li-
thium salt (32). The hydrolysis of ester 31 (15 mg 0.066
mmol) gave salt 32 (15 mg, 99%) as a white powder: IR
1
3328, 2935, 1566, 1502, 1428 cmÀ1; H NMR (D2O,
1
1649, 1551, 1416 cmÀ1; H NMR (D2O, 400 MHz) d
400 MHz) d 6.29 (s, 1H), 4.69 (m, 2H), 2.40 (t, 2H,
J=7.82Hz), 1.98 (t, 2H, J=7.5 Hz), 1.66 (s, 3H), 1.47
(p, 2H, J=7.7 Hz); 13C NMR (CD3OD, 75 MHz) d
178.4, 177.3, 148.7, 144.8, 129.0, 110.3, 37.7, 29.1, 27.1,
22.3; HRMS (ES–ve) calcd for [MÀH]À C10H13O4
197.0808, found 197.0806.
6.29 (dd, 1H, J=2.99 Hz, 5.55 Hz), 6.18 (dd, 1H,
J=2.97 Hz, 5.45 Hz), 4.59 (m, 2H), 2.83 (b, 1H) 2.79 (b,
1H), 2.64 (d, 1H. J=3.05 Hz), 2.07 (m, 3H), 1.73 (4H,
m), 1.57 (d, 1H, J=9.35 Hz), 1.28 (td, 1H, J=1.76 Hz,
8.52Hz); 13C NMR (D2O, 125 MHz) d 184.3, 183.3,
149.0, 138.0, 136.6, 110.1, 64.5, 51.9, 49.5, 47.7, 47.2,
42.8, 38.5, 24.5, 22.5; HRMS (ES-ve) calcd for [MÀH]À
C15H19O4 263.1278, found 263.1276.
3.8. General procedure for Diels–Alder reaction
To a stirring solution of butenedioic acid anhydride (1
equiv) in toluene (2mL) was added freshly prepared
cyclopentadiene (5 equiv). The resulting reaction mix-
ture was stirred at reflux for 5 h. Analysis of the reac-
tion mixture by TLC showed disappearance of starting
material. Solution was cooled to room temperature and
solvent was removed in vacuo. Purification of the crude
product by flash column chromatography gave the
desired cycloadduct.
3.7.6. 2-(3-Methyl-but-3-enyl)-bicyclo[2.2.1]hept-5-ene-
2,3-dicarbonic anhydride (24). The hydrolysis of the
anhydride 23 (16 mg, 0.065 mmol) gave salt 24 (24 mg,
99%) as a white powder.: IR 3417, 3375, 3078, 2988,
2967, 2884, 1646, 1576, 1469 cmÀ1 1H NMR (D2O,
;
500 MHz) d 6.31 (d,d, 1H, J=5.50 Hz, 2.93 Hz), 6.18
(d,d, 1H, J=5.50 Hz, 2.93 Hz), 4.76 (m, 2H), 2.81 (m,
2H), 2.63 (d, 2H, J=3.05 Hz), 2.04 (dt, 2H, J=3.30 Hz,
7.33 Hz), 1.95 (dt, 1H, J=4.76 Hz, 12.46 Hz), 1.72 (s,
3H), 1.51 (m, 4H), 1.27 (dt, 1H, J=1.70 Hz, 8.71 Hz);
13C NMR (D2O, 125 MHz) d 184.3, 183.1, 149.5, 137.8,
136.8, 109.7, 64.4, 61.7, 49.7, 17.6, 47.1, 41.6, 34.9, 22.7;
HRMS (ES +ve) calcd for [M+Na]+ C14H18O4Na
273.1097 found 273.1094.
3.8.1. 2-(4-Methyl-pent-4-enyl)-bicyclo[2.2.1]hept-5-ene-
2,3-dicarbonic anhydride (20). The general procedure
was used. Thus, (Z)-2-(3-methyl-pent-3-enyl)-butene-
dioic acid, anhydride 19 (50 mg, 0.277 mmol) and
cyclopentadiene (0.11 mL, 1.385 mmol) were reacted as
before. Purification of the crude product by flash col-
umn chromatography (SiO2, hexanes/ethyl acetate,
10:1) gave 20 as an oil (40 mg, 59%). IR 3073, 2943,
3.7.7. (Z)-2-(2-Methyl)butenylbutane dioic acid, dilithium
salt (26). The general procedure was followed. The
hydrolysis of ester 25 (21 mg, 0.098 mmol) gave salt 26
(24 mg, quantitative) as a white powder. IR 3372, 2937,
1
1771, 1650, 1459; H NMR (CD2Cl2, 500 MHz) d 6.34
(d,d, 1H, J=5.65 Hz, J=2.90 Hz), 6.26 (d,d, 1H,
J=5.65, 2.90 Hz), 4.72(m, 1H), 4.66 (m, 1H), 3.42(m,
1H), 3.19 (d, 1H, J=4.58 Hz), 3.03 (m, 1H), 2.07 (m,
3H), 1.78 (m, 2H), 1.69 (s, 3H), 1.64 (t, d J=13.13 Hz,
J=4.12 Hz), 1.56 (m, 2H), 1.45 (m, 2H); 3C NMR
(CD2Cl2, 75 MHz) d 174.8, 171.4, 145.1, 137.8, 135.9,
110.9, 59.7, 51.9, 51.3 (2C), 47.2, 38.0, 35.5, 24.2, 22.2;
HRMS (EI) calcd for [M+.] C15H18O3 246.1256 found
246.1254.
1
1580, 1433 cmÀ1; H NMR (D2O, 500 MHz) d 4.75 (m,
2H), 2.56 (m, 1H), 2.45 (dd, 1H, J=5.2Hz, 14.4 Hz),
2.18 (dd, 1H, J=4 Hz, 14 Hz), 2.03 (m, 2H), 1.72 (s,
3H), 1.59 (m, 2H); 13C NMR (D2O, 100 MHz) d 185.5,
182.6, 148.6, 110.4, 46.8, 42.1, 36.2, 31.1, 22.5; HRMS
(ESÀve) calcd for [MÀH]À C9H13O4 185.0808, found
185.0809.
3.7.8. (Z)-2-(2-Methyl)pentenylbutane dioic acid, dilithium
salt (28). The general procedure was followed. The
hydrolysis of ester 27 (21 mg, 0.098 mmol) gave salt 28
(24 mg, quantitative) as a white powder. IR 3356, 2936,
3.8.2. 2-(3-Methyl-but-3-enyl)-bicyclo[2.2.1]hept-5-ene-
2,3-dicarbonic anhydride (23). The general procedure
was used. Thus (Z)-2-(3-methyl-but-3-enyl)-butenedioic

770
A.A. Scholte et al. / Bioorg. Med. Chem. 12 (2004) 763–770
acid, anhydride 22 (80 mg, 0.484 mmol) and cyclo-
pentadiene (0.175 mL, 2.42 mmol) were reacted as
before. Purification of the crude product by flash col-
umn chromatography (SiO2, hexanes/ethyl acetate,
15:1) gave 23 (60 mg, 54%) as an amber oil. IR 3073,
Acknowledgements
These investigations were supported by the Natural
Sciences and Engineeering Research Council of Canada,
the Canada Research Chair in Bioorganic and Medic-
inal Chemistry, the Alberta Heritage Foundation for
Medical Research (scholarship to A.A.S.), and National
Institutes of Health Grant GM 21328.
2952, 1779, 1649, 1457 cmÀ1 1H NMR (CD2Cl2,
;
500 MHz) d 6.36 (d,d, 1H, J=5.71 Hz, 2.99 Hz), 6.29
(d,d, 1H, J=5.85 Hz, 2.86 Hz), 4.77 (m, 1H), 4.72 (m,
1H), 3.44 (m, 1H), 3.24 (d, 1H, J=4.75 Hz), 3.06 (m,
1H), 2.30 (m, 1H), 2.16 (m, 1H), 2.07 (m, 1H), 1.81 (m,
3H), 1.74 (s, 3H). 13C NMR (CD2Cl2, 125 MHz) d
174.6, 171.3, 144.4, 137.5, 135.9, 111.3, 59.5, 52.3, 51.4,
51.1, 47.4, 34.6, 34.0, 22.4; HRMS (EI) calcd for [M+.
C14H16O3 232.1099, found 232.1096.
]
References and notes
1. Robyt, J. In Essentials of Carbohydrate Chemistry;
Springer: New York, 1998; p 305.
2. Fujihashi, M. Z. Y-W., Higuchi, Y., Li, X-Y., Koyama, T.
and Miki, K., Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 4337.
3. Fujikura, K.; Zhang, Y. W.; Fujihashi, M.; Miki, K.;
Koyama, T. Biochemistry 2003, 42, 4035.
4. Lingham, R. B.; Silverman, K. C.; Bills, G. F.; Cascales,
C.; Sanchez, M.; Jenkins, R. G.; Gartner, S. E.; Martin,
I.; Diez, M. T.; Pelaez, F.; Mochales, S.; Kong, Y. L.;
Burg, R. W.; Meinz, M. S.; Huang, L.; Nallinomstead,
M.; Mosser, S. D.; Schaber, M. D.; Omer, C. A.; Pom-
pliano, D. L.; Gibbs, J. B.; Singh, S. B. Appl. Microbiol.
Biot. 1993, 40, 370.
5. Gibbs, J.; Pompliano, D.; Mosser, S.; Rands, E.; Ling-
ham, R.; Singh, S.; Scolnick, E.; Kohl, N.; Oliff, A. J.
Biol. Chem. 1993, 268, 7617.
3.9. General procedure for the conjugate reduction of
maleyl substituted diester
In a flame-dried, argon-flushed round-bottom flask was
added maleyl substituted diester. In an inert atmo-
sphere, [(Ph3P)CuH]6 was weighed and added to round
bottom flask under positive N2 pressure. Deoxygenated
toluene (2mL) containing water (50 mL) was added, and
the resultant red solution was stirred at room tempera-
ture until starting material had been consumed by TLC
analysis. The cloudy red-brown reaction mixture was
opened to air, and stirring was continued for 1 h. Fil-
tration of mixture through Celite and resulting filtrate
was concentrated in vacuo gave crude product which
was purified by flash chromatography.
6. Singh, S. B.; Liesch, J. M.; Lingham, R. B.; Silverman,
K. C.; Sigmund, J. M.; Goetz, M. A. J. Org. Chem. 1995,
60, 7896.
3.9.1. Dimethyl (Z)-2-(2-methyl)butenylbutanedioate
(25). The general procedure was followed. Thus dime-
thyl (Z)-2-(2-methyl)butenylbutanedioate 6 (25 mg,
0.118 mmol) and [(Ph3P)CuH]6 (115 mg, 0.06 mol) were
reacted as before. Purification of the crude product by
flash column chromatography (SiO2, hexanes/ethyl ace-
tate, 10:1) gave 25 (17 mg, 67%) as an oil. IR 3075,
7. Adjei, A. A. Drugs Fut. 2000, 25, 1069.
8. Leonard, D. M. J. Med. Chem. 1997, 40, 2971.
9. Bis, J. L. Cancer Res. 1989, 49, 4682.
10. Ratemi, E. S.; Dolence, J. M.; Poulter, D.; Vederas, J. C.
J. Org. Chem. 1996, 61, 6296.
11. Watanabe, S.; Hirai, H.; Kambara, T.; Kojima, Y.; Nish-
ida, H.; Sugiura, A.; Yamauchi, Y.; Yoshikawa, N.; Har-
wood, H. J., Jr.; Huang, L. H.; Kojima, N. J. Antibiot.
(Tokyo) 2001, 54, 1025.
2952, 1738, 1650, 1437 cmÀ1
;
1H NMR (CDCl3,
300 MHz) d 4.71 (m, 1H), 4.65 (m, 1H), 3.68 (s, 3H),
3.65 (3s, 3H), 2.82 (m, 1H), 2.70 (dd, 1H, J=9.1 Hz,
16.3 Hz), 2.44 (dd, 1H, J=5.1 Hz, 16.2 Hz), 2.0 (t, 2H,
J=7.8 Hz), 1.79 (m, 1H), 1.68 (s, 3H), 1.63 (m, 1H); 13C
NMR (CDCl3, 75 MHz) d 175.3, 172.3, 144.5, 110.7,
51.9, 51.7, 40.7, 35.8, 35.0, 19.8, 22.3; HRMS (EI) calcd
for [M+.] C11H18O4 214.1205, found 214.1204.
12. Turek-Etienne, T. C.; Strickland, C. L.; Distefano, M. D.
Biochemistry 2003, 42, 3716.
13. The distances were calculated based on the smallest
separation between un-minimized conformations.
14. Pattenden, G. A. T.; Simon, J. J. Chem. Soc., Perkin
Trans. 1 1988, 1077.
15. Snider, B. B.; Merrit, J. E. Tetrahedron 1991, 47, 8663.
16. van der Gen, A.; Wiedhaup, K.; Swoboda, J. J.; Duna-
than, H. C.; Johnson, W. S. J. Am. Chem. Soc. 1973, 95,
2656.
17. Mahoney, W. S. B. D.M.; Stryker, J. M. J. Am. Chem.
Soc. 1988, 110, 291.
18. Harris, C. M.; Derdowski, A. M.; Poulter, C. D. Bio-
chemistry 2002, 41, 10554.
19. Cassidy, P. B. D. J.M.; Poulter, C. D. Methods Enzymol.
1995, 250, 30.
20. Long, S. B.; Casey, P. J.; Beese, L. S. Biochemistry 1998,
37, 9612.
21. Strickland, C. L. W. W.T.; Syto, R.; Wang, L.; Bond, R.;
Wu, R.; Schwartz, J.; Le, H. V.; Beese, L. S.; Weber, P. C.
Biochemistry 1998, 37, 16601.
3.9.2. Dimethyl (Z)-2-(2-methyl)pentenylbutanedioate
(27). The general procedure was followed. Thus, dime-
thyl (Z)-2-(2-methyl)pentenylbutenedioate 27 (100 mg,
0.442mmol) and [(Ph 3P)CuH]6 (350 mg, 0.18 mol) were
reacted as before. Purification of the crude product by
flash column chromatography (SiO2, hexanes/ethyl ace-
tate, 10:1) gave 25 (62mg, 61%) as an oil. IR 3074,
2951, 1738, 1650, 1437 cmÀ1
;
1H NMR (CDCl3,
400 MHz) d 4.68 (m, 1H), 4.63 (m, 1H), 3.67 (s, 3H),
3.64 (s, 3H), 2.84 (m, 1H), 2.70 (ddd, 1H, J=2.3 Hz, 9.2
Hz, 16.5 Hz), 2.41 (ddd, 1H, J=2.1 Hz, 5.2 Hz, 16.4
Hz), 1.98 (t, 2H, J=7.4 Hz), 1.66 (s, 3H), 1.60 (m, 1H),
1.42(m, 3H); 13C NMR (CDCl3, 100 MHz) d 175.3,
172.3, 145.0, 110.2, 51.7 (2C), 41.0, 37.3, 35.8, 31.4,
24.7, 22.2; HRMS (ES+ve) calcd for [M+H]+
C12H20O4Na 251.1254, found 251.1256.
22. Perrin, D. D. A. W.L.F.; Perrin, D. R. Purification of
Laboaratory Chemicals, 2nd ed.; Pergamon: New York,
1980.
23. House, H. O. C. C.-Y.; Wilkins, J. M.; Umenm, M. J.
Org. Chem. 1975, 40, 1460.