Synthesis of 5-epi-[6-2H2]valiolone and stereospecifically monodeuterated 5-epi-valiolones: Exploring the steric course of 5-epi-valiolone dehydratase in validamycin A biosynthesis

Article abstract of DOI:10.1021/jo0101003

In validamycin A biosynthesis, as well as that of acarbose, the valienamine and validamine moieties are ultimately derived from a C₇ sugar, sedoheptulose 7-phosphate, which is cyclized to 2-epi-5-epi-valiolone by a cyclase that operates via a dehydroquinate (DHQ) synthase-like mechanism. 2-epi-5-epi-Valiolone is first epimerized at C-2 to give 5-epi-valiolone and then dehydrated between C-5 and C-6 to yield valienone. To probe the dehydration mechanism of 5-epi-valiolone to valienone, stereospecifically 6α- and 6β-monodeuterated 5-epi-valiolones were synthesized. The key step in the synthesis was desulfurization of the tetrabenzyl-6,6-bis(methylthio)-5-epi-valiolone and introduction of the deuterium utilizing Zn, NiCl₂, ND₄Cl/D₂O, and THF. Extensive studies using various combinations of protio- and deuteroreagents and solvents probed the mechanism of the reductive desulfurization, which is crucial for the preparation of stereospecifically monodeuterated 5-epivaliolones. Incorporation experiments with the labeled precursors in the validamycin A producer strain, Streptomyces hygroscopicus var. limoneus, revealed that the dehydration of 5-epi-valiolone to valienone occurs by a syn elimination of water.

Full text of DOI:10.1021/jo0101003

5066
J . Org. Chem. 2001, 66, 5066-5073
Syn th esis of 5-epi-[6-2H2]Va liolon e a n d Ster eosp ecifica lly
Mon od eu ter a ted 5-epi-Va liolon es: Exp lor in g th e Ster ic Cou r se of
5-epi-Va liolon e Deh yd r a ta se in Va lid a m ycin A Biosyn th esis
Taifo Mahmud,*,† J un Xu, and Young Ung Choi
Department of Chemistry, Box 351700, University of Washington, Seattle, Washington 98195-1700
mahmud@chem.washington.edu
Received J anuary 24, 2001
In validamycin A biosynthesis, as well as that of acarbose, the valienamine and validamine moieties
are ultimately derived from a C7 sugar, sedoheptulose 7-phosphate, which is cyclized to 2-epi-5-
epi-valiolone by a cyclase that operates via a dehydroquinate (DHQ) synthase-like mechanism.
2-epi-5-epi-Valiolone is first epimerized at C-2 to give 5-epi-valiolone and then dehydrated between
C-5 and C-6 to yield valienone. To probe the dehydration mechanism of 5-epi-valiolone to valienone,
stereospecifically 6R- and 6â-monodeuterated 5-epi-valiolones were synthesized. The key step in
the synthesis was desulfurization of the tetrabenzyl-6,6-bis(methylthio)-5-epi-valiolone and introduc-
tion of the deuterium utilizing Zn, NiCl2, ND4Cl/D2O, and THF. Extensive studies using various
combinations of protio- and deuteroreagents and solvents probed the mechanism of the reductive
desulfurization, which is crucial for the preparation of stereospecifically monodeuterated 5-epi-
valiolones. Incorporation experiments with the labeled precursors in the validamycin A producer
strain, Streptomyces hygroscopicus var. limoneus, revealed that the dehydration of 5-epi-valiolone
to valienone occurs by a syn elimination of water.
In tr od u ction
the core units, valienamine and validamine, originally
derived from 2-epi-5-epi-valiolone, an intramolecular
aldol cyclization product of sedoheptulose 7-phosphate
(Scheme 1). This cyclization process was proposed to
occur via a dehydroquinate (DHQ) synthase-like cycliza-
tion mechanism.7 In further steps, in S. hygroscopicus
var limoneus 2-epi-5-epi-valiolone is epimerized at the
C-2 position to give 5-epi-valiolone and dehydrated
between C-5 and C-6 to yield valienone. The latter is a
proximate precursor of the cyclitol moieties in validamy-
cin A,5 giving rise to the unsaturated moiety directly and
to the saturated cyclitol unit indirectly via validone. The
conversion of 5-epi-valiolone to valienone resembles the
well-known dehydration of DHQ to dehydroshikimate,
which is catalyzed by two evolutionarily and mechanisti-
cally unrelated types of DHQ dehydratases. The type I
enzymes catalyze a syn elimination of the elements of
water, whereas the type II enzymes catalyze an anti
elimination. To determine what type of enzyme might
be catalyzing the dehydration of 5-epi-valiolone, it was
desirable to elucidate the steric course of this reaction.
To this end we developed a synthesis of stereospecifically
6R and 6â monodeuterated 5-epi-valiolones and explored
the fate of the deuterium in validamycin A biosynthesis.
Syntheses of various related cyclitol derivatives have
been reported by our group5-6,9 and other laboratories.10-13
Validamycin A, a major component of the validamycin
complex produced by Streptomyces hygroscopicus var.
limoneus,1 is an antifungal antibiotic widely used as a
crop protectant, especially to control rice sheath blight
disease caused by the phytopathogenic fungus, Rhizoc-
tonia solani.2 Validamycin A is composed of a pseudo-
disaccharide moiety, validoxylamine A,3 connected to
glucose. The pseudodisaccharide moiety consists of two
aliphatic C7 units, valienamine and validamine, which
are connected via a single nitrogen atom. The valien-
amine unit is also found in acarbose, an R-glucosidase
inhibitor and clinically useful antidiabetic drug.4
In our earlier work on the biosynthesis of validamycin
A in Streptomyces hygroscopicus var limoneus5 and
acarbose in Actinoplanes sp.,6 we had demonstrated that
† Phone: (206) 543-9950. Fax: (206) 543-8318.
(1) (a) Iwasa, T.; Yamamoto, H.; Shibata, M. J . Antibiot. 1970, 23,
595. (b) Horii, S.; Iwasa, T.; Kameda, Y. J . Antibiot. 1971, 24, 57. (c)
Horii, S.; Iwasa, T.; Mizuta, E.; Kameda, Y. J . Antibiot. 1971, 24, 59.
(d) Iwasa, T.; Kameda, Y.; Asai, M.; Horii, S.; Mizuno, K. J . Antibiot.
1971, 24, 119. (e) Horii, S.; Kameda, Y. J . Chem. Soc., Chem. Commun.
1972, 747. (f) Suami, T.; Ogawa, S.; Chida, N. J . Antibiot. 1980, 33,
98. (g) Kameda, Y.; Asano, N.; Yoshikawa, M.; Takeuchi, M.; Yamagu-
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(h) Ogawa, S.; Nose, T.; Ogawa, T.; Toyokuni, T.; Iwasawa, Y.; Suami,
T. J . Chem. Soc., Perkin Trans. 1 1985, 2369. (i) Kameda, Y.; Asano,
N.; Yamaguchi, T.; Matsui, K.; Horii, S.; Fukase, H. J . Antibiot. 1986,
34, 1491.
(2) Iwasa, T.; Higashide, E.; Yamamoto, H.; Shibata, M. J . Antibiot.
1971, 24, 107.
(3) (a) Kameda, Y.; Asano, N.; Yamaguchi, T.; Matsui, K. J . Antibiot.
1987, 40, 563. (b) Asano, N.; Takeuchi, M.; Kameda, Y.; Matsui, K.;
Kono, Y. J . Antibiot. 1990, 43, 722.
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Piepersberg, W. J . Biol. Chem. 1999, 274, 10889.
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Biochemistry 2000, 39, 2276. (b) Florova, G.; Lindsay, Y. M.; Brown,
M. S.; McArthur, H. A. I.; Denoya, C. D.; Reynolds, K. A. J . Org. Chem.
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(4) Truscheit, E.; Frommer, W.; J unge, B.; Muller, L.; Schmidt, D.
D.; Wingender, W. Angew. Chem., Int. Ed. Engl. 1981, 20, 744.
(5) Dong, H.; Mahmud, T.; Tornus, I.; Lee, S.; Floss, H. G. J . Am.
Chem. Soc. 2001, 123, 2733-2742.
(6) Mahmud, T.; Tornus, I.; Egelkrout, E.; Wolf, E.; Uy, C.; Floss,
H. G.; Lee, S. J . Am. Chem. Soc. 1999, 121, 6973.
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10.1021/jo0101003 CCC: $20.00 © 2001 American Chemical Society
Published on Web 06/28/2001

Synthesis of Deuterated 5-epi-Valiolones
J . Org. Chem., Vol. 66, No. 15, 2001 5067
Sch em e 1. Str u ctu r es of Va lid a m ycin A a n d Its Biosyn th etic In ter m ed ia tes a n d of Aca r bose
Sch em e 2. Syn th esis of Com p ou n d s 13a a n d 13b
It is known that an intramolecular aldol cyclization of
2,3,4,6-tetra-O-benzyl-1-C-[bis(methylthio)methyl]-D-xylo-
5-hexosulose (12) gives both C-5 stereoisomers 13a and
13b,13 while that of its dichloromethyl derivative, which
has been used in the preparation of valiolone,6 gives only
the 5S isomer. However, unlike the dichloromethyl
derivative which could be easily reduced to give the
dechlorinated analogue, desulfurization of 13a seemed
to be problematic. Reductive desulfurization of 13a using
Raney nickel was accompanied by elimination of the C-3
benzyloxyl group to give the 2-cyclohexenone derivative
14.13
In the present paper, we describe successful attempts
to selectively reduce the bis(methylthio) moiety of 13a
and introduce deuterium into the C-6 methylene group
of 5-epi-valiolone. Using combinations of protio- and
deuteroreagents and solvents, we probed the mechanism
and sequencial steps of the reductive desulfurization
process, which then allowed the preparation of stereospe-
cifically 6R and 6â deuterated 5-epi-valiolones. Results
on the utilization of the samples in validamycin A
biosynthesis are also presented.
t
Reagents: a . Ac2O, DMSO, rt., quant.; b. BuLi, CH2(SCH3)2,
-78 °C; c. LiAlH4, THF/Diglyme, rt.; d . TFAA, DMSO, Et3N,
CH2Cl2, -78 °C, 3 steps 70%; e. K2CO3, 18-crown-6, CH2Cl2, rt.,
90% (13a :13b ) 4:1).
ported by Fukase and Horii.13 Instead of using the
dichloroinosose derivative,6,13 bis(methylthio) derivatives
were employed to control the stereoselectivity of the
intramolecular aldol cyclization toward the desired 5R
stereochemistry of the final product (Scheme 2). Aldol
condensation of 12 with potassium carbonate in the
presence of 18-crown-6 gave an approximately 4:1 mix-
ture of 13a and 13b. The former was applied for the
synthesis of 5-epi-[6-2H2]valiolone reported here, whereas
the latter had originally been used in the synthesis of
N-substituted valiolamine analogues.13 In contrast to the
desulfurization of 13b with Raney nickel which gives
tetrabenzyl valiolone, reduction of 13a with Raney nickel
was accompanied by elimination of the 3-benzyloxy group
to give the 2-cyclohexenone derivative 14 (Scheme 3).
The second method of choice to reduce the bis(methyl-
thio) moiety in 13a was using tri-n-butyltin hydride (Bu3-
Resu lts
Syn th esis of 5-epi-[6-2H2]Va liolon e. 5-epi-[6-2H2]-
Valiolone was synthesized from 2,3,4,6-tetra-O-benzyl-
D-glucose by a route partly adopted from methods re-
(11) Fukase, H.; Horii, S. J . Org. Chem. 1992, 57, 3651.
(12) (a) Yoshikawa, M.; Cha, B. C.; Okaichi, Y.; Takinami, Y.;
Yokokawa, Y.; Kitagawa, I. Chem. Pharm. Bull. 1988, 36, 4236. (b)
Yoshikawa, M.; Murakami, N.; Yokokawa, Y.; Inoue, Y.; Kuroda, Y.;
Kitagawa, I. Tetrahedron 1994, 50, 9619. (c) Shing, T. K. M.; Tai, V.
W.-F. J . Org. Chem. 1995, 60, 5332. (d) Afarinkia, K.; Mahmood, F.
Tetrahedron Lett. 1998, 39, 7413. (e) Rassu, G.; Auzzas, L.; Pinna, L.;
Zanardi, F.; Battistini, L.; Casiraghi, G. Org. Lett. 1999, 1, 1213.
(13) Fukase, H.; Horii, S. J . Org. Chem. 1992, 57, 3642.

5068 J . Org. Chem., Vol. 66, No. 15, 2001
Sch em e 3. Syn th esis of 5-epi-[6-2H2]Va liolon e
Mahmud et al.
Reagents: a . Raney Ni, dioxane, reflux.; b. Bu3SnD, AIBN, toluene, reflux; c. Ni2BD2, EtOD; d . Zn, NH4Cl/H2O, THF; e. Zn, NiCl2,
ND4Cl/D2O, THF; f. wet 10% Pd/C, H2, 95% EtOH.
SnH) in the presence of AIBN.14 With this method, we
successfully converted 13a to tetrabenzyl-5-epi-valiolone
(15). However, replacement of Bu3SnH with Bu3SnD, to
provide the deuterium-labeled derivative, surprisingly,
failed. Attemps to reduce the bis(methylthio) moiety in
13a with Ni2BD2 or with LiAlD4/TiCl4 were also
unsuccessful, wherein Ni2BD2 mostly removed only one
of the methylthio moieties in 13a .
NH4Cl/H2O/THF at room temperature for 5 days yielded
almost 90% of 18, unlike treatment of 18 under the same
reaction conditions, which gave only a trace amount of
17. When a saturated solution of ND4Cl in D2O was used
in place of NH4Cl/H2O in the reaction of 17, about 50%
of [6-2H1]-18 was recovered together with 50% of [6-2H1]-
17 (Scheme 4). Interestingly, when purification of the
products was conducted using silica gel column chroma-
tography with toluene and ethyl acetate (20:1), almost
all the deuterium in [6-2H1]-17 was replaced by hydrogen.
To some degree, this replacement was also observed in
[6-2H1]-18, ranging from 0 atom % H in the earlier column
fractions to about 50 atom % H in the later ones.
However, this protonated species was probably not
generated from the existing [6-2H1]-18 but rather by the
in situ conversion of [6-2H1]-17 to 18 catalyzed by SiO2.
Stirring of compound 17 in a mixture of silica gel and
toluene:ethyl acetate (10:1) gave 18. Concurrent experi-
ments were carried out by stirring each of [6-2H1]-17 and
[6-2H1]-18 with a slurry of SiO2 in toluene/EtOAc (10:1)
at room temperature for 5 h, and the products were
analyzed by ESI-MS. The results indicated that most of
the deuterium in [6-2H1]-17 was replaced by hydrogen,
whereas that of [6-2H1]-18 was retained. Moreover,
exposure of 18 to Zn/ND4Cl/D2O/THF for 5 h did not yield
any deuterium incorporation into 18. This evidence
suggests that the 6-Hâ in 17 is much more acidic and
accessible to nucleophilic attack compared to the 6-HR
in 18. The complete replacement of the deuterium by
hydrogen in 17, but not in 18, during purifications also
suggests that 17 exists in a rapid equilibrium with its
enolate, some of which is converted more slowly to 18.
The second methylthio group can be reduced using a
mixture of activated zinc, NiCl2, saturated aqueous
ammonium chloride, and THF. Thin-layer chromatogra-
phy monitoring of simultaneous reactions of 17 and 18
with Zn, NiCl2, NH4Cl/H2O, and THF showed that 18 was
directly reduced to the final product 15, whereas 17 is
first epimerized to 18 prior to reduction to yield 15.
Syn th esis of 5-epi-[6r-2H1]Va liolon e a n d 5-epi-[6â-
2H1]Va liolon e. The synthesis of 5-epi-[6â-2H1]valiolone
([6â-2H1]-5) began with the reduction of the first meth-
ylthio group of 13a with Zn/NH4Cl/H2O/THF at room
temperature for 72 h to give 17 and its epimer 18 (4:5
ratio; total yield 83%). To provide additional product 18,
17 was isolated and re-treated with a fresh mixture of
15
16
Partial desulfurization was also achieved by vigorous
stirring of 13a with activated zinc, an aqueous solution
of saturated ammonium chloride, and THF at room
temperature.17 This reaction gave after 48 h of stirring
compounds 17 and 18 in roughly a 2:1 ratio which can
be separated by means of normal- or reversed-phase silica
gel column chromatography. The stereochemistry at C-6
of 17 and 18 was determined on the basis of NOE
experiments. Strong NOE correlations were observed
between 6â-H (3.45 ppm, d, J ) 1.3 Hz) and 2-H (4.15
ppm, dd, J ) 9.2, 1.3 Hz), 4-H (3.80 ppm, d, J ) 8.6 Hz),
6-SCH3 (2.20 ppm, s) in 17, whereas NOE correlation
between 6R-H (3.31 ppm, s) and 7-H2 (3.55 and 3.62 ppm,
ABq, J ) 9.0 Hz), 6-SCH3 (2.25 ppm, s) were observed in
18.
The complete reduction was eventually achieved by
utilizing a mixture of activated zinc and nickel chloride
in an aqueous (D2O) solution of saturated ammonium-d4
chloride and THF. After vigorous stirring overnight,
about 80% of the reactant was converted to [6-2H2]-15.
The synthesis was completed by catalytic hydrogenation
of [6-2H2]-15 with wet 10% Pd/C in a hydrogen atmo-
sphere for 16 h to give 5-epi-[6-2H2]valiolone ([6-2H2]-5)
in quantitative yield.
Mech a n istic Stu d y on th e Red u ctive Desu lfu r -
iza tion of th e Bis(m eth ylth io) Moieties by Zin c a n d
Nick el Ch lor id e. Treatment of 13a with activated zinc
in saturated aqueous NH4Cl and THF evidently only gave
17. However, under the same reaction conditions 17 is
slowly epimerized to 18. Vigorous stirring of 17 in Zn/
(14) Gutierrez, C. G.; Stringham, R. A.; Nitasaka, T.; Glasscock, K.
G. J . Org. Chem. 1980, 45, 3393.
(15) (a) Flippin, L. A.; Dombroski, M. A. Tetrahedron Lett. 1985,
26, 2977. (b) Kiyooka, S.; Abu Hena, M. Tetrahedron: Asymmetry 1996,
7, 2181.
(16) Mukaiyama, T.; Hayashi, M.; Narasaka, K. Chem. Lett. 1973,
291.
(17) Holton, R. A.; Crouse, D. J .; William, A. D.; Kennedy, R. M. J .
Org. Chem. 1987, 52, 2317.

Synthesis of Deuterated 5-epi-Valiolones
J . Org. Chem., Vol. 66, No. 15, 2001 5069
Sch em e 4. Sequ en cia l Red u ction s of Meth ylth io Moieties in 13a a n d Syn th esis of Ster eosp ecifica lly
La beled 5-epi-Va liolon e
Reagents: a . Zn, NH4Cl/H2O, THF; b. Zn, ND4Cl/D2O, THF; c. Zn, NiCl2, NH4Cl/H2O, THF; d . Zn, NiCl2, ND4Cl/D2O, THF; e. wet 10%
Pd/C, H2, 95% EtOH.
Zn/NH4Cl/H2O/THF, and the resulting 18 was pooled.
Treatment of 18 with Zn/ND4Cl/D2O/THF and NiCl2 at
room temperature for 16 h gave tetrabenzyl-5-epi-[6â-
2H1]valiolone ([6â-2H1]-15) with 93 atom % D and 81%
de, based on the integration of its 1H NMR signals
(Figure 1). The 6-Heq in [6â-2H1]-15 appeared as a singlet
(2.64 ppm), instead of a doublet (2.66 ppm) in 15, shifted
upfield around 0.02 ppm due to the deuterium isotope
effect.18 Purification of the synthesis product was carried
out using ODS C18 reversed-phase column chromatogra-
phy or HPLC to avoid epimerization and wash out of the
isotope. Deprotection of the benzyl moieties using wet
10% Pd-C in a hydrogen atmosphere gave 5-epi-[6â-2H1]-
valiolone ([6â-2H1]-5) in 66.4% yield from 18.
([6-2H2]-5) were fed to cultures (100 mL) of the valida-
1
mycin A producer, with /3 added at 24 h, 1/3 at 48 h, and
the rest at 55 h after inoculation. The cultures were
harvested on the seventh day of fermentation. The
fermentation broths were acidified to pH 3.0 with oxalic
acid, and the cells and other solids were removed by
centrifugation. The supernatants were directly subjected
to cation exchange column chromatography on Dowex
50W followed by rechromatography of the validamycin
complex on an anion exchange column (Dowex 1) to give,
typically, about 45 mg of validamycin A from a 100 mL
culture.
Selected ion monitoring (SIM) mass spectra ([M +
Na]+, [M + 1 + Na]+, [M + 2 + Na]+) of all samples
together with an authentic unlabeled sample of valida-
mycin A were recorded. Comparison of the intensities of
the isotope peak [M + 1 + Na]+ against the parent peak
[M + Na]+ of the samples and the reference sample
indicated the incorporation of about 19.2% of 5-epi-[6-
2H2]valiolone, 24.4% of 5-epi-[6R-2H1]valiolone, and 3.9%
of 5-epi-[6â-2H1]valiolone.
Conversely, preparation of 5-epi-[6R-2H1]valiolone was
carried out by treatment of 13a with Zn/ND4Cl/D2O/THF
to give [6-2H1]-17 and [6-2H1]-18. Typically, reactions
using Zn/ND4Cl/D2O/THF (deuterated reagents) gave
slightly lower yields (78%) with a 2:1 ratio of [6-2H1]-17
over [6-2H1]-18. [6-2H1]-17 was isolated and re-treated
with a fresh mixture of Zn/ND4Cl/D2O/THF to provide
additional [6-2H1]-18. Treatment of [6-2H1]-18 with Zn/
NH4Cl/H2O/THF and NiCl2 yielded tetrabenzyl-5-epi-[6R-
2H1]valiolone ([6R-2H1]-15) with 93 atom % D and 78%
de. The synthesis was completed by catalytic hydro-
genolysis of the benzyl moieties of [6R-2H1]-15 to give
5-epi-[6R-2H1]valiolone ([6R-2H1]-5) in 80% yield from
[6-2H1]-18.
2
The H NMR spectrum of the validamycin A sample
isolated from the feeding experiment with 5-epi-[6-2H2]-
valiolone (Figure 2B) had clearly indicated the incorpora-
tion of 5-epi-valiolone into validamycin A.5 The deuterium
is distributed in a 3:2 ratio between the valienamine and
the validamine moiety, because both derived from va-
lienone. In the saturated cyclitol moiety the majority of
the deuterium occupies the pro-6S position, reflecting the
anti stereochemistry of the reduction of valienone to
validone, with some scrambling to the pro-6R position
due to reversible enolization at the validone stage.5 The
In cor p or a tion Exp er im en ts w ith 5-epi-[6r-2H1]-
Va liolon e a n d 5-epi-[6â-2H1]Va liolon e to th e Va lid a -
m ycin A P r od u cer . Fifteen milligrams each of 5-epi-
[6R-2H1]valiolone ([6R-2H1]-5) and 5-epi-[6â-2H1]valiolone
([6â-2H1]-5) and, for comparison, 5-epi-[6-2H2]valiolone
2
identical signal was observed in the H NMR spectra of
validamycin A isolated from the feeding experiment with
(18) Bernheim, R. A.; Lavery, B. J . J . Chem. Phys. 1965, 42, 1462.
5-epi-[6R-2H1]valiolone (Figure 2C) and at much lower

5070 J . Org. Chem., Vol. 66, No. 15, 2001
Mahmud et al.
F igu r e 1. Partial 1H NMR spectra (300 MHz, CDCl3) of the compounds synthesized: (A) compound 17, (B) [6-2H1]-17, (C) compound
18, (D) [6-2H1]-18, (E) compound 15, (F) [6b-2H1]-15, (G) [6a-2H1]-15.
intensity from that with 5-epi-[6â-2H1]valiolone (Figure
2D). These results show that the dehydration of 5-epi-
valiolone to valienone occurs via a syn elimination of the
elements of water.
position and preventing epimerization of the mono-
methylthio product to a more reactive configuration.
Moreover, it was also reported that desulfurization of the
aliphatic derivative using Ni2BH2 is consistently more
difficult than the analogous procedure with aryl ring
containing substrates.15a
Discu ssion
A similar phenomenon has been observed with acti-
vated zinc/NH4Cl/H2O/THF which was reported effective
for the reductive desulfurization of R-phenylthio and
R-phenylsulfinyl carbonyl compounds.17 In the case of
13a , this reagent reductively eliminated only one of the
methylthio groups. This reaction proceeds by replacement
of the methylthio moiety with a hydrogen radical, which
evidently occurs solely toward the axial methylthio
moiety to give 17. The hydrogen radicals can be formed
at the Zn surface by the reaction of protons with electrons
released by the metal.20 Interestingly, under the same
reaction conditions 17 is slowly epimerized to 18, pre-
sumably catalyzed by a Lewis acid, [Zn(NH3)n]2+, via enol
formation. Treatment of 17 with mineral acid, HCl, or
saturated aqueous NH4Cl did not give 18. Since practi-
cally no epimerization has been observed from 18 to 17
in Zn/NH4Cl/H2O/THF or SiO2/toluene/EtOAc, the steri-
In the adaptation of the original Fukase and Horii
chemistry to the synthesis of 5, the reductive removal of
the methylthio moieties of 13a was surprisingly difficult.
Desulfurization using a common reducing agent, Raney
nickel, only gave an elimination product, 14, as reported
by Fukase and Horii.13 The success of the approach using
tri-n-butyltin hydride in the presence of AIBN, but its
failure with Bu3SnD, is also surprising, given that both
Bu3SnH and Bu3SnD have been effectively used to reduce
the dichloro analogues.5,6 Although this failure is not
clearly understood, kinetic isotope effects19 could be a
possible reason. The incomplete reduction of the methyl-
thio moieties in 13a by Ni2BD2, on the other hand, is
easily rationalized by the concomitant reduction of the
ketone function resulting in deactivation of the C-6
(19) Kreevoy, M. M. The Effect of Structure on Isotope Effects in
Proton-Transfer Reactions. In Isotopes in Organic Chemistry; Buncel,
E., Lee, C. C., Eds.; Elsevier, Amsterdam, 1976; Vol. 2, pp 1-29.
(20) Ahonen, M.; Sjo¨holm, R. Acta Chem. Scand. 1997, 51, 785.

Synthesis of Deuterated 5-epi-Valiolones
J . Org. Chem., Vol. 66, No. 15, 2001 5071
F igu r e 2. NMR spectra of validamycin A: (A) 500 MHz 1H NMR spectrum of unlabeled 1 in D2O, (B) 76.78 MHz 2H NMR
spectrum of 1 isolated from the feeding experiment with 5-epi-[6-2H2]valiolone ([6-2H2]-5) in H2O at 313 K,5 (C) 76.78 MHz 2H
NMR spectrum of 1 isolated from the feeding experiment with 5-epi-[6R-2H1]valiolone ([6R-2H1]-5) in H2O at 313 K, (D) 76.78
2
MHz H NMR spectrum of 1 isolated from the feeding experiment with 5-epi-[6â-2H1]valiolone ([6â-2H1]-5) in H2O at 313 K.
cally less accessible 6-HR (equatorial proton) must be
resistant to nucleophilic deprotonation. Therefore, once
the enolate intermediate has been converted to 18, the
difficult deprotonation of 6-HR traps this compound by
preventing its conversion back to the thermodynamically
more stable epimer 17.
Epimerization of the methylthio group from an equato-
rial position in 17 to an axial position in 18 makes this
group accessible to radical attack. The Zn/NH4Cl/H2O/
THF reagent, however, is lacking sufficient reducing
power to execute the second reduction. However, addition
of nickel chloride to the reaction mixture evidently
increases the reduction potential of the reagents. The first
step of the reaction probably involves the reduction of
the divalent nickel to a low-valent form adsorbed on the
zinc surface21 or to a free activated nickel precipitate.
Isolation of 14 as a side product when the reaction was
carried out at higher concentration, which resemble the
reaction of 13a with Raney nickel,13 supports the hy-
pothesis that nickel (Ni0) is directly involved in the
reduction reaction.
The success of the stereospecific synthesis of both 5-epi-
[6R-2H1]valiolone and 5-epi-[6â-2H1]valiolone has made
it possible to probe the steric course of the dehydration
of 5-epi-valiolone to valienone in validamycin biosynthe-
sis. The 24.4% incorporation rate of deuterium in val-
idamycin A isolated from a 5-epi-[6R-2H1]valiolone feeding
experiment compared to 3.9% of that from 5-epi-[6â-2H1]-
valiolone unambiguously revealed that the dehydration
reaction of 5-epi-valiolone to valienone occurs via a syn
elimination. The 3.9% incorporation of that from 5-epi-
[6â-2H1]valiolone could be rationalized by the fact that
an 80% de of 5-epi-[6â-2H1]valiolone was used in the
experiment.
The dehydration reaction of 5-epi-valiolone to valienone
is very similar to the interconversion of dehydroquinic
acid (DHQ) to dehydroshikimic acid (DHS), catalyzed by
3-dehydroquinate dehydratase (DHQase), in the shiki-
mate pathway.22 There are two distinct classes of DHQase
enzymes known, designated as type I and type II, whose
(21) Petrier, C.; Luche, J .-L. Tetrahedron Lett. 1987, 28, 2347.

5072 J . Org. Chem., Vol. 66, No. 15, 2001
Mahmud et al.
corn gluten meal which was obtained from Professor Higashide
and Takeda Chemical Co. Samples of validamycin A were
provided by Takeda Chemical Co. and Professor Kenneth L.
Rinehart J r., University of Illinois at Urbana.
F er m en ta tion a n d In cor p or a tion Exp er im en ts. Fer-
mentation and isolation of validamycin A, as well as the
incorporation experiments with labeled precursors, were car-
ried out as described previously.5
Syn th esis of La beled P r ecu r sor s. The synthesis of
compounds 13a and 13b from tetra-O-benzylglucose was
carried out following a method developed by Fukase and
Horii13 with some minor modifications.28
biophysical properties and reaction mechanisms are very
different.23-25 Type I DHQases catalyze a syn elimination
of a molecule of water from dehydroquinate (DHQ) using
an active-site lysine to form a covalent imine intermedi-
ate (Schiff’s base) as part of the reaction mechanism.26
Type II DHQases were originally identified as part of a
catabolic pathway for utilization of quinic acid but in
some cases are also involved in the shikimate pathway,
and they catalyze an anti elimination of water by an
unknown mechanism.27
In validamycin biosynthesis in Streptomyces hygro-
scopicus var. limoneus, the first reaction, cyclization of
sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone, is
catalyzed by a DHQ synthase-like enzyme.7 2-epi-5-epi-
Valiolone is epimerized at the C-2 position to give 5-epi-
valiolone. Dehydration of 5-epi-valiolone to valienone
takes place via a syn elimination, possibly involving a
type I DHQase-like mechanism. To shed more light on
the mechanism and origin of the enzymes involved in the
biosynthesis of validamycins, further studies at the
biochemical and genetic levels will be pursued.
Tetr a ben zyl-5-epi-va liolon e (15). (a ) Usin g Tr i-n -bu tyl-
tin Hyd r id e/AIBN. To a solution of 13a (100 mg, 0.155 mmol)
in toluene (1 mL) were added tributyltin hydride (1.66 µL, 0.62
mmol) and AIBN (10 mg, 0.062 mmol), and the mixture was
refluxed for 2 h and then cooled to room temperature. The
products were extracted with EtOAc (5 mL), and the organic
solution was washed with 2 N HCl, saturated aqueous
NaHCO3, and brine. The organic solvent was evaporated under
reduced pressure, and the extract was purified over a silica
gel column (toluene/EtOAc ) 10:1) to give 15 (60 mg, 70%).
(b) Usin g Zn /NiCl2/NH4Cl/H2O/THF . To a solution of 13a
(480 mg, 0.745 mmol) in THF (50 mL) were added activated
zinc (5 g), a saturated aqueous solution of NH4Cl (10 mL), and
anhydrous NiCl2 (1 g), and the mixture was stirred vigorously
at room temperature for 20 h. Twenty milliliters of EtOAc was
added, and the mixture was filtered through a filter paper to
remove the solid materials. The filtrate was washed with 2 N
HCl, saturated aqueous NaHCO3, and brine. The organic
solvent was dried over Na2SO4 and evaporated under reduced
pressure. The extract was purified over a silica gel column
(toluene/EtOAc ) 20:1-10:1) to give 15 (340 mg, 85%) and 17
(34 mg, 7.6%).
Exp er im en ta l Section
Gen er a l. 1H, 2H, and 13C NMR spectra were recorded on
Bruker AF-300 or AM500 NMR spectrometers with MacNMR
5.5 PCI as the instrument controller and data processor.
Optical rotations were measured on a J ASCO DIP-370 digital
polarimeter. Low-resolution electrospray mass spectra were
recorded on a Bruker-Esquire liquid chromatography-ion trap
mass spectrometer with electrospray, APCI, and nanospray
ionization sources. A Fison VG Quattro II electrospray ioniza-
tion mass spectrometer was used to measure the high-
resolution mass spectroscopy. A ISF-4-V shaker, Adolf Kuhner
AG, was used for the fermentations. All synthetic reactions
were carried out under an atmosphere of dry argon at room
temperature in oven-dried glasswares unless otherwise noted.
Reactions were monitored by TLC (silica gel 60 F254, Merck)
with detection by UV light or by alkaline permanganate spray
or Ce(SO4)2/H2SO4 solution. Column chromatography was
performed on 230-400 mesh silica gel (Aldrich) or YMC*Gel
120 Å ODS 63-210 µm. Ion exchange chromatography was
carried out on 100 mesh Dowex-50W and 100 mesh Dowex 1
(Sigma).
Ma ter ia ls. All chemicals and solvents were of reagent or
HPLC grade and were used without further purification unless
otherwise noted. Activated zinc was prepared by washing a
20 g portion of well-ground zinc dust in a fritted glass funnel
with three 50 mL portions each of 4% aqueous HCl, water,
methanol, and ether.17 The active zinc was ground again to
remove lumps and then dried first at 110 °C for 15 min and
then at room temperature under reduced pressure for several
hours. Cultures of Streptomyces hygroscopicus var. limoneus
were purchased from the American Type Culture Collection
(ATCC 21431 and 21432) or obtained from Professor Eiji
Higashide, Okayama University, J apan (No. T-7545). Fermen-
tation ingredients were purchased from Difco or Sigma except
15: yellowish syrup, [R]D +25.5° (c ) 0.96, CHCl3, 25 °C).
ESI-MS: m/z 575 (M + Na)+. 1H NMR (500 MHz, CDCl3) δ:
2.46 (1H, d, J ) 16.0 Hz), 2.66 (1H, d, J ) 16.0 Hz), 2.77 (1H,
s), 3.38 (1H, d, J ) 9.3 Hz), 3.49 (1H, d, J ) 9.3 Hz), 3.86 (1H,
d, J ) 6.2 Hz), 3.91 (1H, dd, J ) 6.2, 8.0 Hz), 4.41 (1H, d, J )
8.0 Hz), 4.47-4.95 (8H), 7.16-7.42 (10H).
Tetr a ben zyl-5-epi-[6-2H2]va liolon e ([6-2H2]-15). The pro-
cedure was identical with that in b for 15, except ND4Cl and
D2O were used in place of NH4Cl/H2O.
[6-2H2]-15: yellowish syrup. ESI-MS: m/z 577 (M + Na)+.
HR ESI-MS calcd for C35H34D2O6Na [M + Na]+: 577.2533,
found 577.2529. 1H NMR (500 MHz, CDCl3) δ: 2.77 (1H, s),
3.38 (1H, d, J ) 9.3 Hz), 3.49 (1H, d, J ) 9.3 Hz), 3.86 (1H, d,
J ) 6.2 Hz), 3.91 (1H, dd, J ) 6.2, 8.0 Hz), 4.41 (1H, d, J )
8.0 Hz), 4.47-4.95 (8H), 7.16-7.42 (10H).
5-epi-[6-2H2]Va liolon e ([6-2H2]-5). To a solution of 110 mg
of [6-2H2]-15 in 95% aqueous ethanol (8 mL) was added wet
10% Pd/C (200 mg), and the mixture was stirred at room
temperature under an H2 atmosphere for 16 h. The suspension
was passed through a Celite column to remove the catalyst
and then filtered through a membrane filter. The solvent was
evaporated in vacuo to give [6-2H2]-5 in quantitative yield.
[6-2H2]-5: yellowish syrup, [R]D +6.5° (c ) 0.86, MeOH, 25
°C). ESI-MS: m/z 217 (M + Na)+. HR ESI-MS calcd for
1
C7H10D2O6Na [M + Na]+: 217.0655, found 217.0655. H NMR
(500 MHz, CD3OD) δ: 3.42 (1H, d, J ) 11 Hz), 3.63 (1H, d, J
) 11 Hz), 3.71 (1H, t, J ) 9.3 Hz), 3.85 (1H, d, J ) 9.3 Hz),
4.06 (1H, d, J ) 9.3 Hz). 13C NMR (125 MHz, CDCl3): δ 64.7
(t), 74.7 (s), 75.9 (d), 79.1 (d), 79.9 (d), 206.3 (s).
(22) Haslam, E. Shikimic Acid: Metabolism and Metabolites; J ohn
Wiley and Sons: Chichester, 1993.
(23) Kleanthous, C.; Deka, R.; Davis, K.; Kelly, S. M.; Cooper, A.;
Harding, S. E.; Price, N. C.; Hawkins, A. R.; Coggins, J . R. Biochem.
J . 1992, 282, 687.
(24) Harris, J .; Kleanthous, C.; Coggins, J . R.; Hawkins, A. R.; Abell,
C. A. J . Chem. Soc., Chem. Commun. 1993, 1080.
(25) (a) Shneier, A.; Harris, J .; Kleantous, C.; Coggins, J . R.;
Hawkins, A. R.; Abell, C. A. Bioorg. Med. Chem. Lett. 1993, 3, 1399.
(b) Harris, J .; Gonzales-Bello, C.; Kleanthous, C.; Hawkins, A. R.;
Coggins, J . R.; Abell, C. Biochem. J . 1996, 319, 333.
(26) (a) Chaudhuri, S.; Duncan, K.; Graham, L. D.; Coggins, J . R.
Biochem. J . 1991, 275, 1. (b) Sheiner, A.; Kleanthous, C.; Deka, R.;
Coggins, J . R.; Abell, C. J . Am. Chem. Soc. 1991, 113, 9416.
(27) Bottomley, J . R.; Clayton, C. L.; Chalk, P. A.; Kleanthous, C.
Biochem. J . 1996, 319, 559.
Tetr a ben zyl-6(S)-m eth ylth io-5-epi-va liolon e (17) a n d
Tetr a ben zyl-6(R)-m eth ylth io-5-epi-va liolon e (18). To a
solution of 13a (500 mg, 0.776 mmol) in THF (12.5 mL) were
added activated zinc (2.75 g) and a saturated aqueous solution
of NH4Cl (12.5 mL), and the mixture was stirred vigorously
at room temperature for 72 h. Twenty milliliters of EtOAc was
t
(28) An application of BuLi in place of n-BuLi in the reaction of 9
to 10 and using a silica gel column with n-hexane/EtOAc as solvent
system to purify compound 12, to overcome the difficulty encountered
by Fukase and Horii in isolating compound 12, which was reportedly
too unstable to be isolated.

Synthesis of Deuterated 5-epi-Valiolones
J . Org. Chem., Vol. 66, No. 15, 2001 5073
added, and the mixture was filtered through a filter paper to
remove the solid materials. The filtrate was washed with 2 N
HCl, saturated aqueous NaHCO3, and brine. The organic
solvent was dried over Na2SO4 and evaporated under reduced
pressure. The extract was purified over a silica gel column
(toluene/EtOAc ) 20:1-10:1) to give 17 (214 mg, 46%) and 18
(172 mg, 37%).
17: yellowish syrup, [R]D +1.85° (c ) 0.76, CHCl3, 25 °C).
ESI-MS: m/z 621 (M + Na)+. HR ESI-MS calcd for C36H38O6-
NaS [M + Na]+: 621.2287, found 621.2257. 1H NMR (500 MHz,
CDCl3) δ: 2.20 (3H, s), 2.74 (1H, s), 3.36 (1H, d, J ) 9 Hz),
3.45 (1H, d, J ) 1.3 Hz), 3.80 (1H, d, J ) 9.0 Hz), 3.90 (1H, d,
J ) 9.3 Hz), 4.15 (1H, dd, J ) 1.3, 9.3 Hz), 4.25 (1H, t, J ) 9.3
Hz), 4.37-5.00 (8H), 7.20-7.44 (10H). 13C NMR (125 MHz,
CDCl3): δ 16.5 (q), 60.2 (t), 62.4 (d), 69.1, 73.2, 73.3, 75.3 (all
t), 75.5 (s), 81.1 (d), 85.1 (d), 85.5 (d), 127.3-128.2 (all d), 137.0,
137.8 (s), 138.2 (s), 138.3 (s), 197.4 (s).
activated zinc (2.1 g), a saturated D2O solution of ND4Cl (10
mL), and anhydrous NiCl2 (415 mg), and the mixture was
stirred vigorously at room temperature for 15 h. Twenty
milliliters of EtOAc was added, and the mixture was filtered
through a filter paper to remove the solid materials. The
filtrate was washed with brine. The organic solvent was dried
over Na2SO4 and evaporated under reduced pressure. The
extract was purified over an ODS column (MeOH-H2O ) 80:
20) to give ([6â-2H]-15 (110 mg, 43%) with recovery of 18 (130
mg, 47%).
[6â-2H1]-15: yellowish syrup. ESI-MS: m/z 576 (M + Na)+.
HR ESI-MS calcd for C35H35DO6Na [M + Na]+: 576.2471, found
576.2462. 1H NMR (300 MHz, CDCl3) δ: 2.64 (1H, s), 2.76 (1H,
s), 3.39 (1H, d, J ) 9.3 Hz), 3.50 (1H, d, J ) 9.3 Hz), 3.86 (1H,
d, J ) 6.2 Hz), 3.91 (1H, dd, J ) 6.2, 7.8 Hz), 4.40 (1H, d, J )
7.8 Hz), 4.48-4.95 (8H), 7.18-7.42 (10H).
5-epi-[6r-2H1]Va liolon e ([6r-2H1]-5). To a solution of 128
mg (0.23 mmol) of [6R-2H1]-15 in 95% aqueous ethanol (12.8
mL) was added wet 10% Pd/C (128 mg), and the mixture was
stirred at room temperature under an H2 atmosphere for 16
h. The suspension was passed through a Celite column to
remove the catalyst and then filtered through a membrane
filter. The solvent was evaporated in vacuo, and the sample
was passed through a Sephadex LH-20 (MeOH) to give [6R-
2H1]-5 (35.6 mg, 80%).
18: yellowish syrup, [R]D -44.1° (c ) 0.83, MeOH, 25 °C).
ESI-MS: m/z 621 (M + Na)+. HR ESI-MS calcd for C36H38O6-
NaS [M + Na]+: 621.2287, found 621.2298. 1H NMR (500 MHz,
CDCl3) δ: 2.09 (3H, s), 2.90 (1H, s), 3.31 (1H, brs), 3.55 (1H,
d, J ) 9.0 Hz), 3.62 (1H, d, J ) 9.0 Hz), 3.88 (1H, dd, J ) 6.2,
8.0 Hz), 3.98 (1H, d, J ) 6.2 Hz), 5.04 (1H, d, J ) 8.0 Hz),
4.44-4.91 (8H), 7.09-7.44 (10H). 13C NMR (125 MHz,
CDCl3): δ 15.3 (q), 57.7 (t), 71.2 (t), 73.5 (2C), 74.4, 74.5 (all
t), 75.0 (s), 81.2 (d), 81.8 (d), 127.7-128.3 (all d), 137.2 (s),
137.6 (s), 137.8 (s), 138.0 (s, C6H5- × 4), 201.3 (s).
[6r-2H1]-5: colorless syrup. ESI-MS: m/z 216 (M + Na)+.
HR ESI-MS calcd for C7H11D1O6Na [M + Na]+: 216.0593, found
Tetr a ben zyl-6(S)-m eth ylth io-5-epi-[6-2H1]va liolon e ([6-
2H1]-17) a n d Tetr a ben zyl-6(R)-m eth ylth io-5-epi-[6-2H1]-
va liolon e ([6-2H1]-18). The procedure was identical with that
for 17 and 18, except ND4Cl and D2O were used in place of
NH4Cl/H2O and the purification of the products was conducted
using an ODS column (MeOH-H2O ) 80:20).
1
216.0585. H NMR (300 MHz, CD3OD) δ: 2.58 (1H, brs), 3.42
(1H, d, J ) 11 Hz), 3.64 (1H, d, J ) 11 Hz), 3.72 (1H, t, J ) 9
Hz), 3.85 (1H, d, J ) 9 Hz), 4.07 (1H, d, J ) 9 Hz).
5-epi-[6â-2H1]Va liolon e ([6â-2H1]-5). To a solution of 100
mg (0.18 mmol) of [6â-2H1]-15 in 95% aqueous ethanol (10 mL)
was added wet 10% Pd/C (100 mg), and the mixture was
stirred at room temperature under an H2 atmosphere for 16
h. The suspension was passed through a Celite column to
remove the catalyst and then filtered through a membrane
filter. The solvent was evaporated in vacuo, and the sample
was passed through a Sephadex LH-20 column (MeOH) to give
[6â-2H1]-5 (28.7 mg, 82%).
[6-2H1]-17: yellowish syrup. ESI-MS: m/z 622 (M + Na)+.
HR ESI-MS calcd for C36H37DO6NaS [M + Na]+: 622.2348,
found 622.2364. 1H NMR (500 MHz, CDCl3) δ: 2.20 (3H, s),
2.74 (1H, s), 3.36 (1H, d, J ) 9 Hz), 3.80 (1H, d, J ) 9.0 Hz),
3.90 (1H, d, J ) 9.3 Hz), 4.15 (1H, d, J ) 9.3 Hz), 4.25 (1H, t,
J ) 9.3 Hz), 4.37-5.00 (8H), 7.20-7.44 (10H).
[6-2H1]-18: yellowish syrup. ESI-MS: m/z 622 (M + Na)+.
HR ESI-MS calcd for C36H37DO6NaS [M + Na]+: 622.2348,
found 622.2347. 1H NMR (500 MHz, CDCl3) δ: 2.10 (3H, s),
2.90 (1H, s), 3.55 (1H, d, J ) 9.0 Hz), 3.62 (1H, d, J ) 9.0 Hz),
3.88 (1H, dd, J ) 6.2, 8.0 Hz), 3.98 (1H, d, J ) 6.2 Hz), 5.04
(1H, d, J ) 8.0 Hz), 4.44-4.91 (8H), 7.09-7.44 (10H).
Tetr a ben zyl-5-epi-[6r-2H1]va liolon e ([6r-2H1]-15). To a
solution of [6-2H1]-18 (240 mg, 0.40 mmol) in THF (30 mL)
were added activated zinc (2 g), a saturated aqueous solution
of NH4Cl (10 mL), and anhydrous NiCl2 (400 mg), and the
mixture was stirred vigorously at room temperature for 16 h.
Twenty milliliters of EtOAc was added, and the mixture was
filtered through a filter paper to remove the solid materials.
The filtrate was washed with brine. The organic solvent was
dried over Na2SO4 and evaporated under reduced pressure.
The extract was purified by ODS column chromatography
(MeOH-H2O ) 80:20) to give ([6R-2H1]-15 (143 mg, 65%) with
a recovery of [6-2H1]-18 (83.2 mg, 35%).
[6â-2H1]-5: colorless syrup. ESI-MS: m/z 216 (M + Na)+.
HR ESI-MS calcd for C7H11D1O6Na [M + Na]+: 216.0593, found
1
216.0584. H NMR (300 MHz, CD3OD) δ: 2.65 (1H, brs), 3.41
(1H, d, J ) 11 Hz), 3.64 (1H, d, J ) 11 Hz), 3.72 (1H, t, J ) 9
Hz), 3.85 (1H, d, J ) 9 Hz), 4.07 (1H, d, J ) 9 Hz).
Ack n ow led gm en t. The authors are deeply indebted
to Professor Heinz G. Floss for his guidance and helpful
discussions, to Professor Eiji Higashide of Okayama
University, J apan, for providing a strain of Streptomyces
hygroscopicus var. limoneus (No. T-7545), to Professor
Kenneth L. Rinehart J r., University of Illinois at Ur-
bana, and the Takeda Company, Osaka, J apan, for
generous gifts of validamycin A and corn gluten meal.
This work was supported by the National Institutes of
Health through Grant AI20264.
[6r-2H1]-15: yellowish syrup. ESI-MS: m/z 576 (M + Na)+.
HR ESI-MS calcd for C35H35DO6Na [M + Na]+: 576.2471, found
576.2453. 1H NMR (300 MHz, CDCl3) δ: 2.46 (1H, s), 2.76 (1H,
s), 3.38 (1H, d, J ) 9.3 Hz), 3.49 (1H, d, J ) 9.3 Hz), 3.86 (1H,
d, J ) 6.2 Hz), 3.91 (1H, dd, J ) 6.2, 8.0 Hz), 4.41 (1H, d, J )
8.0 Hz), 4.48-4.95 (8H), 7.18-7.42 (10H).
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra of
compounds 15, [6-2H2]-15, [6R-2H1]-15, [6â-2H1]-15, 17, [6-2H1]-
17, 18, [6-2H1]-18, [6R-2H1]-5, and [6â-2H1]-5. This material is
available free of charge via the Internet at http://pubs.acs.org.
Tetr a ben zyl-5-epi-[6â-2H1]va liolon e ([6â-2H1]-15). To a
solution of 18 (275 mg, 0.46 mmol) in THF (50 mL) was added
J O0101003