Comprehensive study of okaspirodiol: Characterization, total synthesis, and biosynthesis of a new metabolite from Streptomyces

Article abstract of DOI:10.1021/jo060149e

The new spiro[4.5]acetal okaspirodiol (4) was isolated from Streptomyces sp. Goe TS 19 as a secondary metabolite in yields up to 380 mg/L. The structure of this cryptic ketotetrol was elucidated by different methods including X-ray analysis, and its equil

Full text of DOI:10.1021/jo060149e

VOLUME 71, NUMBER 19
SEPTEMBER 15, 2006
© Copyright 2006 by the American Chemical Society
Comprehensive Study of Okaspirodiol: Characterization, Total
Synthesis, and Biosynthesis of a New Metabolite from Streptomyces
Tobias Bender,† Tim Schuhmann,† Jo¨rg Magull,‡ Stephanie Grond,*,† and
Paultheo von Zezschwitz*,†
Institut fu¨r Organische und Biomolekulare Chemie and Institut fu¨r Anorganische Chemie der
Georg-August-UniVersita¨t Go¨ttingen, Tammannstrasse 2-4, D-37077 Go¨ttingen, Germany
sgrond@gwdg.de; pzezsch@gwdg.de
ReceiVed January 23, 2006
The new spiro[4.5]acetal okaspirodiol (4) was isolated from Streptomyces sp. Go¨ TS 19 as a secondary
metabolite in yields up to 380 mg/L. The structure of this cryptic ketotetrol was elucidated by different
methods including X-ray analysis, and its equilibration under mildly acidic conditions furnishing three
additional isomers was thoroughly studied. Although metabolite 4 is not the thermodynamically favored
isomer, a high-yielding total synthesis was accomplished comprising a stereoselective spiro-
acetalization under equilibrium conditions. This approach benefits from the important influence of an
intramolecular hydrogen bond on the stabilization of the spiro[4.5]acetal moiety. The biosynthesis of 4
was investigated by feeding experiments with 13C-labeled precursors proving its origin from a new type
of the rare mixed acetate-glycerol biosynthetic pathway. All results are discussed on the basis of the
structural diversity of spiroacetals in nature and their chemical properties.
Introduction
tors. Moreover, the telomerase-inhibiting activity of griseorhodin
and rubromycin is attributed to the presence of a spiroacetal
moiety in these natural products.4 Whereas the latter are highly
functionalized, polycyclic compounds, various spiroacetals from
insects are volatile, simple molecules and act as pheromones.5
In contrast, the biological function of several other naturally
occurring spiroacetals has not been disclosed so far. Therefore,
Spiroacetals occur ubiquitously in nature as subunits of
miscellaneous natural products from different sources such as
microbes, fungi, plants, insects, and marine organisms, and some
of these compounds and/or their derivatives exhibit strong
biological activities.1 This comprises the reveromycins,2 inhibi-
tors of the mitogenic activity of epidermal growth factor, and
the cephalostatins,3 which are highly potent cell growth inhibi-
(2) (a) Koshino, H.; Takahashi, H.; Osada, H.; Isono, K. J. Antibiot. 1992,
45, 1420-1427. (b) Shimizu, T.; Usui, T.; Machida, K.; Furuya, K.; Osada,
H.; Nakata, T. Bioorg. Med. Chem. Lett. 2002, 12, 3363-3366. (c) Drouet,
K. E.; Ling, T.; Tran, H. V.; Theodorakis, E. A. Org. Lett. 2000, 2, 207-
210. (d) Shimizu, T.; Masuda, T.; Hiramoto, K.; Nakata, T. Org. Lett. 2000,
2, 2153-2156. (e) Cuzzupe, A. N.; Hutton, C. A.; Lilly, M. J.; Mann, R.
K.; McRae, K. J.; Zammit, S. C.; Rizzacasa, M. A. J. Org. Chem. 2001,
66, 2382-2393.
† Institut fu¨r Organische und Biomolekulare Chemie.
‡ Institut fu¨r Anorganische Chemie.
(1) Reviews: (a) Perron, F.; Albizati, K. F. Chem. ReV. 1989, 89, 1617-
1661. (b) Jacobs, M. F.; Kitching, W. Curr. Org. Chem. 1998, 2, 395-
436. (c) Mead, K. T.; Brewer, B. N. Curr. Org. Chem. 2003, 7, 227-256.
(d) Brimble, M. A.; Furkert, D. P. Curr. Org. Chem. 2003, 7, 1461-1484.
10.1021/jo060149e CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/27/2006
J. Org. Chem. 2006, 71, 7125-7132
7125

Bender et al.
the interest in the chemistry and pharmacology of this important
class of compounds is steadily growing.
of the enzymatic basis of microbial secondary metabolite
formation.
Regarding their synthesis, the major challenge frequently is
the stereoselective assembly of the spirocyclic structure with a
linking carbon atom which usually is a stereogenic center but
can easily isomerize under mildly acidic conditions. Luckily
though, in most cases, the natural products possess the ther-
modynamically favored configuration and conformation of the
spiro center,6 thus paving the way for a ring closure under
equilibrium conditions. The structural preferences are influenced
by steric and stereoelectronic (anomeric) effects7 which have
been most thoroughly studied in the case of simple spiro[5.5]
compounds8 such as (E)-2-methyl-1,7-dioxaspiro[5.5]undecane
(1). Yet, in the case of compounds bearing hydroxyl groups at
appropriate positions, intramolecular hydrogen bonds and
chelating effects can lead to an unexpected conformation as in
the monensin-water complex 2 with its axial arrangement of
the hydroxyl group.9 Therefore, the determination of the
stabilizing factors turns out to be essential for synthetic work
with spiroacetal natural products as has been shown for the
reveromycins2b-e and the talaromycins (3a,b), a group of spiro-
[5.5]acetals from the fungus Talaromyces stipitatus which
recently attracted extensive interest in synthesis.10 Herein, we
describe the isolation, chemical characterization, and enanti-
oselective total synthesis of the new natural product okaspirodiol
(4), a spiro[4.5]acetal bearing two hydroxyl groups which are
responsible for intramolecular hydrogen bonds and isomerization
processes.
Results and Discussion
Isolation and Chemical Characterization. Okaspirodiol (4)
was detected by detailed analysis of the metabolite pattern of
the Streptomyces sp. strain Go¨ TS 19 by methods of chemical
screening.12 Fermentation of this strain, which was isolated from
a Bavarian soil sample and found to be most closely related to
S. kanamyceticus (DSM 40500T),13 was carried out in a malt/
glucose/yeast medium (medium A). Okaspirodiol (4) was
isolated from the culture broth and purified by column chro-
matography on Sephadex LH-20 and silica gel yielding 60 mg/L
of a colorless solid. The production of 4 was significantly
enhanced by extraordinary high aeration (6.0 vvm) to afford
an improved yield of up to 275 mg/L. Feeding of glycerol to
the growing cultures raised the yield of 4 even further to 380
mg/L, a particularly high yield efficiently achieved with the
OSMAC method (one-strain-many-compounds).14 Staining with
orcin reagent led to an intense brown color, but okaspirodiol
(4) was not detectable under UV light on silica gel TLC plates.
The ESI mass spectrum of 4 showed an ion peak at m/z ) 225
([M + Na]+), and the molecular formula was determined by
ESI-HRMS to be C10H18O4. The constitution of 4 was derived
from 1H NMR and 13C NMR experiments disclosing the scaffold
of a spiro[4.5]acetal with a signal of a quaternary carbon at
102.9 ppm. From 2D-COSY and HMBC experiments, the
structure of 4 was finally deduced to be a 3-hydroxymethyl-7-
methyl-1,6-dioxaspiro[4.5]decan-4-ol. Thus, okaspirodiol is a
new natural product with an unprecedented combination of rings
and substituents not found in nature or in synthetic products
before. It is distinguished from the spiro[4.5]acetals from insects
by its hydroxyl groups5 and only shows some similarities with
the talaromycins (3a,b) mentioned above in that they bear
comparable substituents but comprise just pyran rings.10
The absolute configuration of 4 was assigned on the basis of
the analysis of selected derivatives. Esterification with (R)- and
(S)-2-phenylbutyric acid and 1H NMR analysis of the thus
obtained diastereomeric products according to the method of
Helmchen15 indicated the (S)-configuration of C-4. Various
Focusing on the question of how nature synthesizes okaspirodi-
ol (4), some alternative biosynthetic pathways are likely to be
responsible for the formation of this new natural product. The
biosynthetic origin of such an unusually substituted spiroacetal
has not yet been investigated. At present, many microbial
spiroacetal metabolites are derived from acetate;5,11 however,
structure 4 also implies carbohydrate precursors such as glycerol
or even a heptose. We addressed these alternatives efficiently
by feeding experiments with 13C-labeled glycerol and acetate.
The putative biosynthetic mechanism is discussed in the light
(3) (a) Pettit, G. R.; Inoue, M.; Kamano, Y.; Herald, D. L.; Arm, C.;
Dufresne, C.; Christie, N. D.; Schmidt, J. M.; Doubek, D. L.; Krupa, T. S.
J. Am. Chem. Soc. 1998, 120, 2006-2007. (b) Mu¨ller, I. M.; Dirsch, V.
M.; Rudy, A.; Lopez-Anton, N.; Pettit, G. R.; Vollmar, A. M. Mol.
Pharmacol. 2005, 67, 1684-1689. (c) Lee, J. S.; Fuchs, P. L. J. Am. Chem.
Soc. 2005, 127, 13122-13123.
(4) (a) Ueno, T.; Takahashi, H.; Oda, M.; Mizunuma, M.; Yokoyama,
A.; Goto, Y.; Mizushina, Y.; Sakaguchi, K.; Hayashi, H. Biochemistry 2000,
39, 5995-6002. (b) Li, A.; Piel, J. Chem. Biol. 2002, 9, 1017-1026.
(5) Francke, W.; Kitching, W. Curr. Org. Chem. 2001, 5, 233-251.
(6) Exceptions were reported, e.g., in refs 1a, 5, and: Stok, J. E.; Lang,
C.-S.; Schwartz, B. D.; Fletcher, M. T.; Kitching, W.; de Voss, J. J. Org.
Lett. 2001, 3, 397-400.
(7) (a) Deslongchamps, P. Stereoelectronic Effects in Organic Chemistry;
Pergamon: Oxford, 1983. (b) Kirby, A. J. Stereoelectronic Effects; Oxford
University Press: New York, 1996.
(8) (a) Deslongchamps, P.; Rowan, D. D.; Pothier, N.; Sauve´, T.;
Saunders, J. K. Can. J. Chem. 1981, 59, 1105-1121. (b) Pothier, N.;
Goldstein, S.; Deslongchamps, P. HelV. Chim. Acta 1992, 75, 604-620.
(9) Ireland, R. E.; Ha¨bich, D.; Norbeck, D. W. J. Am. Chem. Soc. 1985,
107, 3271-3278.
(10) (a) Lynn, D. G.; Phillips, N. J.; Hutton, W. C.; Shabanowitz, J. J.
Am. Chem. Soc. 1982, 104, 7319-7322. (b) Iwata, C.; Maezaki, N.; Hattori,
K.; Fujita, M.; Moritani, Y.; Takemoto, Y.; Tanaka, T.; Imanishi, T. Chem.
Pharm. Bull. 1993, 41, 946-950. (c) Tietze, L. F.; Schneider, G.; Wo¨lfling,
J.; Fecher, A.; No¨bel, T.; Petersen, S.; Schuberth, I.; Wulff, C. Chem.-
Eur. J. 2000, 6, 3755-3760.
(12) Grabley, S.; Thiericke, R.; Zeeck, A. In Drug DiscoVery from
Nature; Grabley, S., Thiericke, R., Eds.; Springer: Berlin-Heidelberg, 1999;
pp 124-148.
(13) Phylogenetic and chemotaxonomic analyses were performed by R.
M. Kroppenstedt, DSMZ GmbH, Braunschweig, Germany.
(14) Bode, H. B.; Bethe, B.; Ho¨fs, R.; Zeeck, A. ChemBioChem 2002,
3, 619-627.
(11) Puder, C.; Loya, S.; Hizi, A.; Zeeck, A. Eur. J. Org. Chem. 2000,
729-735.
(15) Helmchen, G. Tetrahedron Lett. 1974, 15, 1527-1530.
7126 J. Org. Chem., Vol. 71, No. 19, 2006

Okaspirodiol: A New Metabolite from Streptomyces
SCHEME 1. Benzylation of Okaspirodiol (4) and
Isomerization of the Dibenzylated 7
substituents at the R-carbons of spiroacetals via the open-chain
ketodiol19 was not found for the hydroxyl group at C-4.
All isomers possess the chairlike conformation of the six-
membered ring together with the sterically favored equatorially
oriented methyl group. Okaspirodiol (4) and its isomer 4a with
their (S)-configuration at C-5 benefit from two anomeric effects
because of the axial-quasi-axial arrangement of the spiro C-O
bonds and therefore always outbalance the (R)-configured minor
isomers 4b and 4c. Interestingly, the natural product 4 is not
the thermodynamically most stable isomer which is 4a presum-
ably because of the steric preference of a trans relationship of
the substituents at C-3 and C-4. For evaluating the influence of
hydrogen bonding on the structural stabilization, okaspirodiol
(4) was benzylated to give a mixture of mono- and dibenzylated
products 6 and 7 (Scheme 1).20 Exposure of 7 to the equilibrating
conditions B and C, respectively, yielded only one additional
isomer 7a as expected because of suppression of the epimer-
ization of C-3. Yet, the ratio of isomer(s) with the (S)-
configuration at C-5 to the one(s) with the (R)-configuration
significantly dropped from 93:7 (condition B) or >95:5 (condi-
tion C) in the case of okaspirodiol (4) to just 60:40 (condition
B) or 71:29 (condition C) in the case of the dibenzylated 7.
Therefore, the (S)-configuration in 4 and 4a is not only
stereoelectronically favored but also must be extensively
stabilized by an intramolecular hydrogen bond between the C-4
hydroxyl group and O-6, especially in an aprotic medium. DFT
calculations on the B3LYP/6-31G* level of theory confirmed
this result revealing distances of 1.88 Å (isomer 4) and 2.10 Å
(isomer 4a) between O-6 and the relevant hydrogen atom,
although the calculated absolute energies of isomers 4-4c did
not reflect the observed equilibrium ratios (see Supporting
Information). It has been known for a long time that Lewis acids
can chelate and thus favor the cis orientation of the hydroxyl
group and O-6 in 4-hydroxy-spiro[4,5]acetals,18a yet this seems
to be the first evidence for a stabilization just by hydrogen
bonding.
FIGURE 1. Isomerization studies of okaspirodiol (4).
attempts to obtain suitable crystals of 4 failed, but finally, X-ray
analysis of the p-bromobenzoate 5 of okaspirodiol was ac-
complished (see Supporting Information).16 These results alto-
gether showed the microbial metabolite 4 to possess the all-
(S)-configuration.
As expected for a spiroacetal, okaspirodiol (4) readily
isomerizes under mildly acidic conditions. When stored in
CDCl3 at room temperature for 48 h (condition A), three
additional isomers were formed which could readily be separated
by column chromatography on silica gel to yield a 29:47:5:19
ratio of 4-4c (Figure 1). To achieve a complete equilibration
under thermodynamic control, okaspirodiol was treated with
both methanolic HCl (condition B) and 10-camphorsulfonic acid
(CSA)/CD2Cl2 (condition C) at room temperature. Under the
former condition, the same four isomers were obtained, yet 4a
was by far the main component. In contrast, only 4 and 4a were
detected after isomerization under the aprotic condition C as
determined by 13C NMR analysis of the reaction mixtures. The
isomers were characterized by 1H NMR, 13C NMR, and NOESY
experiments. Most enlightening are the chemical shifts of 7-H
which show a downfield shift when 7-H is placed in a 1,3-
diaxial orientation with O-1 as in 4 and 4a.17 Additionally, the
cis relationship of O-6 with the oxygen substituent at C-4 exerts
a shielding effect on C-5 and 4-H.18 Finally, the respective
relationship of the C-4 hydroxyl and the C-3 hydroxymethyl
group was assigned on the basis of the experimental work and
NOE studies (see Supporting Information). Thus, an epimer-
ization of the spiro carbon C-5 and an unusual transacetalization
leading to the epimerization of C-3 create the four isomers 4-4c.
Quite remarkably, the frequently observed isomerization of
Enantioselective Total Synthesis. From the isomerization
studies, it was concluded that a selective total synthesis of 4
would be possible involving the spirocyclization of an acyclic
or monocyclic precursor under equilibrium conditions. Even
though the natural product is not the thermodynamically favored
isomer, it should indeed selectively be formed if the hydroxy-
methyl group ending up at C-3 is protected and the C-4 hydroxyl
group can exert its directing influence. A variety of methods
(16) CCDC 288701 contains the supplementary crystallographic data for
5. These data can be obtained free of charge via www.ccdc.cam.ac.uk/
conts/retrieving.html (or from the Cambridge Crystallographic Data Centre,
12 Union Road, Cambridge CB2 1EZ, U.K.; fax: (+44) 1223-336-033
or e-mail: deposit@ccdc.cam.ac.uk).
(17) (a) Francke, W.; Reith, W.; Sinnwell, V. Chem. Ber. 1980, 113,
2686-2693. (b) Ireland, R. E.; Daub, J. P. J. Org. Chem. 1983, 48, 1303-
1312. (c) Brimble, M. A.; Williams, G. M. J. Org. Chem. 1992, 57, 5818-
5822. (d) Zanatta, S. D.; White, J. M.; Rizzacasa, M. A. Org. Lett. 2004,
6, 1041-1044.
(18) (a) Kozluk, T.; Cottier, L.; Descotes, G. Tetrahedron 1981, 37,
1875-1880. (b) Cottier, L.; Descotes, G.; Grenier, M. F.; Metras, F.
Tetrahedron 1981, 37, 2515-2524.
(19) Evans, D. A.; Sacks, C. E.; Kleschik, W. A.; Taber, T. R. J. Am.
Chem. Soc. 1979, 101, 6789-6791.
(20) The formation of the monobenzylated 6 shows that the secondary
alcohol moiety in 4 is more reactive than the primary one, although a
procedure was used that is described as being selective for the protection
of primary alcohol moieties: Fukazawa, A.; Sato, H.; Masamune, T.
Tetrahedron Lett. 1987, 28, 4303-4306.
J. Org. Chem, Vol. 71, No. 19, 2006 7127

Bender et al.
SCHEME 2. Enantioselective Total Synthesis of
Okaspirodiol (4)
was obtained as a single diastereomer, most likely having an
(R)-configured anomeric carbon. The hydrogenation of this
substrate was still challenging,24 and thus different catalysts were
21a
investigated. The commonly employed Rh/Al2O3
led to
incomplete conversions as can be seen from the isolation of
the tricyclic diacetal 16 as a side product after cyclization. Better
results were achieved using the Adams catalyst PtO2 in ethyl
acetate, but the reaction had to be carefully monitored by TLC
to prevent a hydrogenation of the phenyl ring. After subsequent
cyclization, the desired compound 15 was obtained as a single
isomer in an excellent yield of 80%. Finally, hydrogenolysis of
the benzyl ether moiety was accomplished using Pd/C in MeOH
to furnish okaspirodiol (4). The analytical data of the synthetic
compound entirely matched those of the natural product, e.g.,
20
the optical rotation [R]D -53 (c 1.0, MeOH). Thus, a
completely stereoselective total synthesis was achieved furnish-
ing the novel spiro[4.5]acetal 4 in 52% yield over the five steps
from the known precursors 10 and 12. Resubjecting the
monobenzylated okaspirodiol 15 to CSA in CH2Cl2 yielded only
traces of a second isomer (20:1 ratio according to HPLC) which
must be the product of an epimerization of C-5.
Biosynthesis. Okaspirodiol (4) is the first small spiroacetal
isolated from actinomycetes. Only very few biosynthetic studies
on similar structures from nature have been carried out to date.
The biosynthetic pathway to low-boiling spiro compounds
isolated from fruit flies was investigated by De Voss et al. and
most likely involves several oxidation steps starting from fatty
acids.25 Another example is blazeispirol A isolated from the
fungus Agaricus blazei. In this case, the spiroacetal was found
to be of mevalonate origin.26
The carbon skeleton of okaspirodiol (4) indicates that the
biosynthesis possibly proceeds by the condensation of a glycerol
unit with an intermediate from the sugar pool like sedoheptulose
or from the carboxylic acid pool. Feeding experiments were
carried out by continuously adding sterile aqueous solutions of
[1-13C]acetate, [2-13C]acetate, or [U-13C3]glycerol, respectively,
to growing cultures of Streptomyces sp. Go¨ TS 19 during the
period of highest production of 4. NMR analysis of 4 isolated
from the [U-13C3]glycerol feeding experiment showed 13C-13C
couplings for all carbon atoms derived from intact incorporation
of C2- or C3-units of the precursor, but C-4 showed no coupling
(Table 1). The resulting labeling pattern does not match a
sedoheptulose-based biosynthetic pathway. In the case of a
sedoheptulose precursor, two C3- (C-12/C-7/C-8, C-2/C-3/C-
11) and two C2-units would form the carbon skeleton (see
Supporting Information). In addition, feeding 13C-labeled acetate
for the synthesis of spiroacetals have been elaborated over the
past decades,1 yet the concept of adding a lithiated terminal
alkyne bearing a protected hydroxyl group to a suitable lactone,
followed by hydrogenation of the triple bond and ring closure,
appeared to be the most attractive one.21 Thus, both fragments
of okaspirodiol can separately be prepared according to known
procedures and then combined in a very late stage of the
synthesis. A diastereoselective alkylation of (S)-diethyl malate
(8) according to Seebach et al.22a furnishes diester 9 which was
transformed to the desired lactone 10 in seven high-yielding
steps (53% overall yield from 9)22b (Scheme 2). Furthermore,
starting from (S)-propylene oxide (11), three straightforward
transformations yielded the desired THP-protected, (S)-config-
ured alkynol 12 (58% overall yield).23 This substrate was then
lithiated with ethereal MeLi and added to the lactone 10.
Because the crude product 13 could not be hydrogenated, it
was treated with methanolic HCl to give the acetal 14 which
TABLE 1. 13C NMR Analyses (150.8 MHz, CD2Cl2) of Enriched 4
from Feeding Experiments
carbon
atom
δC
[U-13C3]glycerol,
(C,C) (Hz)
(ppm) [1-13C]acetatea [2-13C]acetatea
J
2
3
4
5
7
8
9
10
11
12
61.1
42.1
37.5
37.5; 32.5
(21) Examples: (a) Phillips, C.; Jacobson, R.; Abrahams, B.; Williams,
H. J.; Smith, L. R. J. Org. Chem. 1980, 45, 1920-1924. (b) Hayes, P.;
Fletcher, M. T.; Moore, C. J.; Kitching, W. J. Org. Chem. 2001, 66, 2530-
2533.
(22) (a) Hungerbu¨hler, E.; Naef, R.; Wasmuth, D.; Seebach, D.; Loosli,
H. R.; Wehrli, A. HelV. Chim. Acta 1980, 63, 1960-1970. (b) Ueki, T.;
Kinoshita, T. Org. Biomol. Chem. 2004, 2, 2777-2785.
(23) White, J. D.; Somers, T. C.; Reddy, G. N. J. Org. Chem. 1992, 57,
4991-4998.
(24) Similar problems were reported by other authors: (a) Horita, K.;
Oikawa, Y.; Nagato, S.; Yonemitsu, O. Chem. Pharm. Bull. 1989, 37, 1717-
1725. (b) Shimizu, Y.; Kiyota, H.; Oritani, T. Tetrahedron Lett. 2000, 41,
3141-3144.
77.9
102.9
67.6
32.6
20.0
30.9
66.9
21.7
1.66
2.96
2.34
45.5
40.5
32.5
32.5
45.5
32.5
40.5
1.17
1.16
1.42
1.83
a Specific incorporation.27
7128 J. Org. Chem., Vol. 71, No. 19, 2006

Okaspirodiol: A New Metabolite from Streptomyces
SCHEME 3. Labeling Pattern and Proposed Series of Biosynthetic Steps of Okaspirodiol (4)
gave okaspirodiol with a specific incorporation pattern27 and
thus confirmed that a polyketide chain and a glycerol unit form
the spiroacetal skeleton of okaspirodiol (4) (Scheme 3).
metabolites from actinomycetes (21-23)31 in addition to the
known autoregulators such as the A-factor (24) or virginiae
butanolide A29 (25) (Figure 2). Okaspirodiol (4) expands the
class of compounds from the mixed acetate-glycerol biosyn-
thetic pathway for the first time by the scaffold of a spiroacetal.
Prior to the present studies, no natural product has been known
whose biosynthesis comprises a similar set of reactions: an
unbranched tetraketide as a precursor for the aldol condensation
with a glycerol-derived C3-unit, a decarboxylation step, and an
acetalization, which occur within the mixed acetate-glycerol
pathway to yield the new hydroxylated spiro[4.5]acetal 4.
The incorporation pattern of 4 gives evidence that surprisingly
it is originated from the mixed acetate-glycerol biosynthetic
pathway which has recently gained new interest.28 From this,
two plausible biosynthetic routes (A, B) suggest a partially
reduced tetraketide 17 derived from a polyketide synthase (or
a fatty acid like pathway)28a which undergoes an aldol addition
of a dihydroxyacetone-derived 3-carbon unit (Scheme 3). The
proposed C11-product 18 is cyclized, dehydrated, and decar-
boxylated to form the hemiacetal 19. Rehydration with inverse
regioselectivity and intramolecular acetalization then lead to
okaspirodiol (4) concordantly with the observed incorporation
pattern (pathway A). Along the plausible alternative route
(pathway B), the five-membered ring is formed at first, so that
subsequent dehydration and decarboxylation give 20 which
finally forms 4 via rehydration and acetalization. Admittedly,
an alternative order of reactions and thus alternative structures
of the intermediates are also possible, e.g., thioester hydrolysis
or formation.
Some similarities for this biosynthetic pathway can be found
in the literature,29,30 such as for the well-known biosynthesis of
the virginiae butanolides. In addition, the mixed acetate-
glycerol biosynthetic pathway was recently found to be respon-
sible for the formation of a new diversity of furan and lactone
(25) (a) Stok, J. E.; Lang, C.-S.; Schwartz, B. D.; Fletcher, M. T.;
Kitching, W.; De Voss, J. J. Org. Lett. 2001, 3, 397-400. (b) Schwartz, B.
D.; McErlean, C. S. P.; Fletcher, M. T.; Mazomenos, B. E.; Konstanto-
poulou, M. A.; Kitching, W. M.; De Voss, J. J. Org. Lett. 2005, 7, 1173-
1176.
FIGURE 2. Microbial metabolites from other types of the mixed
acetate-glycerol biosynthetic pathway.
(26) Hirotani, M.; Kaneko, A.; Asada, Y.; Yoshikawa, T. Tetrahedron
Lett. 2000, 41, 6101-6104.
The biosynthetic and structural similarity of okaspirodiol (4)
with the “microbial hormones” 24 and 25 encouraged us to
screen for regulatory activity on growing actinomycetes strains.
No unambiguous results were obtained so far. No antibacterial
activity of 4 was found in plate diffusion assays with Escherichia
coli, Bacillus subtilis, Staphylococcus aureus, and Candida
albicans; however, growth inhibition was observed for some
(27) Specific incorporations were calculated according to: Scott, A. I.;
Townsend, C. A.; Okada, K.; Kajiwara, M.; Cushley, R. J.; Whitman, P. J.
J. Am. Chem. Soc. 1974, 96, 8069-8080.
(28) (a) Corre, C.; Challis, G. L. ChemBioChem 2005, 6, 2166-2170.
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2006, 33, 105-120.
(29) (a) Shikura, N.; Yamamura, J.; Nihira, T. J. Bacteriol. 2002, 184,
5151-5157. (b) Sakuda, S.; Higashi, A.; Tanaka, S.; Nihira, T.; Yamada,
Y. J. Am. Chem. Soc. 1992, 114, 663-668. (c) Yamada, Y.; Nihira, T.;
Sakuda, S. In Biotechnology of Antibiotics; Strohl, W. R., Ed.; Marcel
Dekker: New York-Basel-Hong Kong, 1997; pp 63-79.
(30) (a) Chang, Z.; Sitachitta, N.; Rossi, J. V.; Roberts, M. A.; Flatt, P.
M.; Jia, J.; Sherman, D. H.; Gerwick, W. H. J. Nat. Prod. 2004, 67, 1356-
1367. (b) Rohr, J.; Weissbach, U.; Beninga, C.; Ku¨nzel, E.; Siems, K.;
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A. Chem. Commun. 1998, 437-438.
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Hu, J.-F.; Wunderlich, D.; Sattler, I.; Ha¨rtl, A.; Papastavrou, I.; Grond, S.;
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J. Org. Chem, Vol. 71, No. 19, 2006 7129

Bender et al.
on Sephadex LH-20 (column: 100 × 2.5 cm, MeOH). The fractions
containing the spiroacetal 4 as detected by TLC were further
purified by column chromatography on silica gel (c-Hex/EtOAc
1:1) yielding pure okaspirodiol (4).
fungi. Further work to specify this antifungal activity is now in
progress with an expanded screening approach. The particular
ecological advantage of the high production (up to 380 mg/L)
of 4 and some other members of the mixed acetate-glycerol
biosynthetic pathway (e.g., 21-23) for the producing organism
needs further investigation.
(3S,4S,5S,7S)-3-Hydroxymethyl-7-methyl-1,6-dioxaspiro[4.5]-
decan-4-ol (4). The title compound was isolated as described above
as a colorless, crystalline solid: Rf ) 0.42 (CH2Cl2/MeOH 9:1);
20
1
mp 98 °C; [R]D -53 (c 1.0, MeOH); H NMR (600 MHz, CD3-
OD) δ 1.14 (d, 3J ) 6.1 Hz, 3 H, CH3), 1.21 (mc, 1 H, 8-H), 1.48
(mc, 1 H, 10-H), 1.58 (mc, 1 H, 8-H), 1.64-1.75 (m, 2 H, 9(10)-
Conclusion
The cryptic ketotetrol okaspirodiol (4) is a highly interesting
spiroacetal because of its two free hydroxyl groups which are
responsible for the unique isomerization pattern: the C-4
hydroxyl group permits a very effective stabilization of the
configuration and conformation of the spiro center through
hydrogen bonds. The C-3 hydroxymethyl group allows for a
transacetalization, a particular ansa isomerization, leading to
an epimerization at this carbon atom and the formation of the
thermodynamically more stable isomer 4a. The isomerization
studies also revealed the remarkable fact that the strain Strep-
tomyces sp. Go¨ TS 19 selectively forms the less stable isomer
4, and no isomerization products were isolated from the
fermentation broth. Additionally, the understanding of the
principles of stabilization strongly eased the total synthesis,
furnishing the new natural product in 52% yield over only five
highly stereoselective steps from known precursors.
Because okaspirodiol (4) is the first spiroacetal built from
an as yet unknown type of the rare mixed acetate-glycerol
biosynthetic pathway, it is an important example to emphasize
the variability of the secondary metabolite biosynthesis. Starting
from simple building blocks, a new structure is formed which
has not been found in nature before and has not yet been
invented by synthetic chemists’ ideas.
3
H), 1.80 (mc, 1 H, 9-H), 2.49 (mc, 1 H, 3-H), 3.69 (dd, J ) 7.8,
2J ) 11.0 Hz, 1 H, 2-H), 3.73-3.79 (m, 2 H, 2-H, CH2OH), 3.84
(d, 3J ) 9.0 Hz, 1 H, 4-H), 3.94 (ddq, 3J ) 2.0, 3J ) 6.1, 3J ) 12.5
1
Hz, 1 H, 7-H), 3.98 (t, J ) 8.5 Hz, 1 H, CH2OH); H NMR (600
3
MHz, CD2Cl2) δ 1.13 (d, J ) 6.1 Hz, 3 H, CH3), 1.16-1.27 (m,
1 H, 8-H), 1.52-1.62 (m, 5 H, 8(9,10)-H), 2.51 (mc, 1 H, 3-H),
2.83 (d, 3J ) 9.0 Hz, 1 H, 4-OH), 2.89 (dd, 3J ) 5.0, 3J ) 7.0 Hz,
1 H, CH2OH), 3.59-3.65 (m, 2 H, 2-H, CH2OH), 3.69-3.72 (m,
1 H, 2-H), 3.88-3.95 (m, 2 H, 4-H, CH2OH), 3.95 (ddq, 3J ) 2.5,
3
3J ) 6.1, J ) 12.5 Hz, 1 H, 7-H); 13C NMR (150.8 MHz, CD2-
Cl2) δ 20.0 (C-9), 21.7 (CH3), 30.9 (C-10), 32.6 (C-8), 42.1 (C-3),
61.1 (C-2), 66.9 (CH2OH), 67.6 (C-7), 77.9 (C-4), 102.9 (C-5); IR
(cm-1, KBr) 3445, 2934, 1441, 1386, 1128, 1078; MS (ESI) m/z
(%) 225.1 (100) [M + Na]+. Anal. Calcd for C10H18O4: 225.109746
[M + Na]+ (correct mass according to ESI-HRMS).
(3R,4S,5S,7S)-4-Hydroxy-7-methyl-1,6-dioxaspiro[4.5]dec-3-
yl-methyl 4-Bromobenzoate (5) and (3R,4S,5S,7S)-4-(4-Bro-
mophenylcarbonyloxy)-7-methyl-1,6-dioxaspiro[4.5]dec-3-yl-
methyl 4-Bromobenzoate (5a). To a solution of spiroacetal 4 (59.6
mg, 0.295 mmol) in dichloromethane (4 mL) were subsequently
added p-bromobenzoic acid (119 mg, 0.592 mmol), ethyl-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 116 mg,
0.605 mmol), and a catalytic amount of 4-(dimethylamino)pyridine
(3 mg). Because of an incomplete conversion, additional amounts
of p-bromobenzoic acid (60.0 mg, 0.301 mmol) and EDCI (60.0
mg, 0.313 mmol) were added after 4 h. The reaction mixture was
stirred overnight at room temperature and then diluted with
dichloromethane (20 mL) and water (10 mL). The phases were
separated, and the organic phase was washed with water (10 mL),
saturated NH4Cl solution (10 mL), and brine (10 mL), dried over
MgSO4, and concentrated in vacuo. The residue was purified by
flash column chromatography on silica gel (20 g, c-Hex/EtOAc
3:1). Two fractions were collected containing 26.8 mg (16%) of
the diester 5a (Rf ) 0.51) and 71.2 mg (63%) of the monoester 5
Experimental Section
Fermentation. Strain Go¨ TS 19 (Streptomyces sp.) was incubated
at 28 °C for 7 days and maintained on medium A agar plates. A
1 cm2 agar piece was used to inoculate 50 mL of preculture
(medium A, rotary shaker, 180 rpm, 48 h, 28 °C) in 300 mL
Erlenmeyer flasks (with three flow spoilers). A portion of 100 mL
of the preculture was used to inoculate a 2 L fermenter, containing
900 mL of medium A. Standard fermentation conditions: 72-
86 h, 500 rpm, aeration 6.0 vvm, 28 °C. Medium A: 10 g/L of
malt extract; 4 g/L of glucose; 4 g/L of yeast extract; culture plates:
additional 10 g/L of agar.
Feeding Experiments. Feeding experiments with [1-13C]acetate
and [U-13C3]glycerol were carried out under the conditions described
above. In general, precursors were administered to the fermentation
as sterile aqueous solutions. Continuous feeding with a low rate
pump was carried out with the 13C-labeled precursors between the
26th and 50th hour of fermentation. [1-13C]acetate: 560 mg/L in
100 mL of water (6.8 mmol/L culture broth), yielding 22 mg of
labeled 4. [U-13C3]glycerol: 400 mg of unlabeled glycerol was
added to 100 mg of 13C-labeled glycerol and dissolved in 100 mL
of water (1.05 mmol/L culture broth), yielding 380 mg of labeled
4. [2-13C]acetate: 250 mg of the 13C-labeled compound was
dissolved in 60 mL of water and added to a fermentation of
300 mL of medium A (1 L Erlenmeyer flasks, 250 rpm, 28 °C)
which was inoculated with 15 mL of preculture, yielding 42 mg of
labeled 4.
20
(Rf ) 0.26) as colorless solids. 5: mp 91 °C; [R]D -12 (c 1.0,
1
3
MeOH); H NMR (300 MHz, CD2Cl2) δ 1.14 (d, J ) 6.1 Hz,
3 H, CH3), 1.16-1.28 (m, 1 H, 8-H), 1.50-1.90 (m, 5 H, 8(9,10)-
H), 2.66-2.80 (m, 2 H, 3-H, OH), 3.77 (dd, 3J ) 7.5, 2J ) 9.0 Hz,
3
1 H, 2-H), 3.91 (t, J ) 8.5 Hz, 1 H, 2-H), 3.93 (ddq, J ) 2.1,
3J ) 6.1, 3J ) 12.5 Hz, 1 H, 7-H), 4.02 (dd, J ) 8.0, J ) 9.0 Hz,
1 H, CH2OBz), 4.28 (dd, J ) 8.0, J ) 11.0 Hz, 1 H, CH2OBz),
3
3
4.56 (dd, J ) 5.5, J ) 7.0 Hz, 1 H, 4-H), 7.63-7.66 (m, 2 H,
Ar-H), 7.82-7.86 (m, 2 H, Ar-H); 13C NMR (75.5 MHz, CD2-
Cl2) δ 20.0 (C-9), 21.9 (CH3), 31.2 (C-10), 32.7 (C-8), 40.4 (C-3),
64.7 (C-2), 67.7 (C-7), 68.7 (CH2OBz), 76.9 (C-4), 103.2 (C-5),
128.1 (CH, C-Ar), 129.6 (CH, C-Ar), 131.3 (Cquat, C-Ar), 132.0
(Cquat, C-Ar), 165.9 (CO2); IR (cm-1, KBr) 3417, 2964, 2930,
2867, 1716, 1591, 1387, 1331, 1272, 1082; MS (ESI) m/z (%) 407.1
(100) [M + Na]+, 792.7 (40) [2M + Na]+, 1340.0 (34) [4M-C7H4-
BrO2]+. 5a: mp 55 °C; [R]D20 -134 (c 1.0, MeOH); 1H NMR (300
3
MHz, CD2Cl2) δ 1.16 (d, J ) 6.1 Hz, 3 H, CH3), 1.20-1.32 (m,
1 H, 8-H), 1.50-1.90 (m, 5 H, 8(9,10)-H), 3.08 (mc, 1 H, 3-H),
3
2
3
3.82 (dd, J ) 6.1, J ) 9.0 Hz, 1 H, 2-H), 3.93 (ddq, J ) 2.1,
3J ) 6.1, 3J ) 12.5 Hz, 1 H, 7-H), 4.20 (t, J ) 9.0 Hz, 1 H, 2-H),
4.37 (dd, 3J ) 7.0, 2J ) 11.0 Hz, 1 H, CH2OBz), 4.58 (dd,
3J ) 7.5, 2J ) 11.0 Hz, 1 H, CH2OBz), 5.12 (d, 3J ) 9.0 Hz, 1 H,
4-H), 7.46-7.56 (m, 4 H, Ar-H), 7.63-7.66 (m, 2 H, Ar-H),
7.82-7.86 (m, 2 H, Ar-H); 13C NMR (75.5 MHz, CD2Cl2) δ 20.2
(C-9), 22.0 (CH3), 30.7 (C-10), 32.5 (C-8), 37.8 (C-3), 64.9 (C-2),
Isolation and Purification. The culture broth was separated from
the mycelium by filtration, and the mycelium was discarded. The
culture filtrate was passed through an Amberlite XAD-2 column,
and impurities were washed out with deionized water. The
metabolites were eluted with MeOH, and evaporation yielded crude
extracts (ca. 1.5 g/L) which were applied to gel chromatography
7130 J. Org. Chem., Vol. 71, No. 19, 2006

Okaspirodiol: A New Metabolite from Streptomyces
66.9 (C-7), 67.8 (CH2OBz), 77.2 (C-4), 103.3 (C-5), 128.0 (CH,
C-Ar), 128.4 (CH, C-Ar), 129.1 (CH, C-Ar), 129.2 (CH, C-Ar),
131.1 (Cquat, C-Ar), 131.5 (Cquat, C-Ar), 131.7 (Cquat, C-Ar),
131.9 (Cquat, C-Ar), 165.6 (2 C, CO2); IR (cm-1, KBr) 3424, 2931,
2875, 1723, 1592, 1396, 1291, 1127; MS (ESI) m/z (%) 591 (65)
[M + Na]+, 1158.5 (100) [2M + Na]+; CD (MeOH) λextr ([Θ]) )
254 (-126 044), 238 (45 741), 224 (16 486), 211 (39 208) nm.
(3S,4S,5S,7S)-4-Benzyloxy-3-hydroxymethyl-7-methyl-1,6-
dioxaspiro[4.5]decane (6) and (3S,4S,5S,7S)-4-Benzyloxy-3-ben-
zyloxymethyl-7-methyl-1,6-dioxaspiro[4.5]decane (7). The spiro-
acetal 4 (33.6 mg, 0.166 mmol) was dissolved in DMF (4 mL) and
cooled to -60 °C, and sodium hydride (60% in mineral oil, 16.7
mg, 0.42 mmol) was added. After being stirred for 1 h at -60 °C,
the mixture was allowed to warm to -10 °C and kept at this
temperature for another 30 min. The reaction mixture was again
cooled to -60 °C, and a solution of benzyl bromide (49 µL, 71
mg, 0.41 mmol) in DMF (1 mL) was added. The resulting mixture
was stirred for 20 h at room temperature, cooled to 0 °C, quenched
with water (2 mL), and stirred for 5 min at room temperature. The
reaction mixture was diluted with water (20 mL) and dichlo-
romethane (20 mL). The aqueous phase was extracted with
dichloromethane (2 × 25 mL), and the combined organic layers
were washed with water (2 × 20 mL), dried over Na2SO4, and
concentrated in vacuo. The crude product was purified by flash
column chromatography on silica gel (10 g, c-Hex/EtOAc 5:1). Two
fractions were collected containing 8.6 mg (14%) of the dibenzyl-
ated derivative 7 (Rf ) 0.20) and 33 mg (68%) of the monoben-
zylated compound 6 (Rf ) 0.11) as colorless oils. 6: [R]D20 -13 (c
1.0, MeOH); 1H NMR (600 MHz, CD2Cl2) δ 1.09 (d, 3J ) 6.1 Hz,
3 H, CH3), 1.11-1.21 (m, 1 H, 8-H), 1.36-1.39 (m, 1 H, 10-H),
1.48-1.52 (m, 1 H, 8-H), 1.54-1.60 (m, 1 H, 9-H), 1.64-1.75
organic phase was washed with saturated NaHCO3 solution (10
mL) and brine (10 mL) and dried over Na2SO4. Concentration in
vacuo yielded 150 mg of a yellow oil, which was dissolved in
MeOH (15 mL). Concentrated hydrochloric acid (20 µL) was added
to this solution, and the mixture was stirred for 60 min at room
temperature. When the reaction had ceased, saturated NaHCO3
solution (10 mL) was added and the mixture was diluted with
diethyl ether (100 mL). The phases were separated, and the aqueous
phase was extracted with diethyl ether (2 × 50 mL). The combined
organic layers were washed with saturated NaHCO3 solution (10
mL) and brine (10 mL) and dried over Na2SO4. Concentration in
vacuo yielded 88 mg (81%) of the title compound 14 as a colorless
20
oil, whose purity was >95%: Rf ) 0.20 (c-Hex/EtOAc 1:2); [R]D
+21 (c 1.0, MeOH); 1H NMR (250 MHz, CD2Cl2) δ 1.21 (d, 3J )
3
2
8.3 Hz, 3 H, 5′-H), 2.38 (dd, J ) 6.5, J ) 16.5 Hz, 1 H, 3′-H),
2.48 (dd, 3J ) 4.6, 2J ) 16.5 Hz, 1 H, 3′-H), 2.85 (mc, 1 H, 4-H),
3
2
3.38 (s, 3 H, OCH3), 3.58 (dd, J ) 6.5, J ) 9.3 Hz, 1 H, CH2-
OBn), 3.78 (dd, 3J ) 7.0, 2J ) 9.3 Hz, 1 H, CH2OBn), 3.80-4.02
3
(m, 3 H, 5(4′)-H), 4.25 (d, J ) 4.6 Hz, 1 H, 3-H), 4.51 (s, 2 H,
CH2Ph), 7.21-7.40 (m, 5 H, Ar-H); 13C NMR (62.9 MHz, CD2-
Cl2) δ 22.3 (C-5′), 28.8 (C-3′), 41.6 (C-4), 50.4 (OCH3), 65.9 (C-
4′), 67.8 (CH2OBn), 69.7 (C-5), 73.1 (CH2Ph), 76.2 (C-2′), 77.5
(C-3), 85.5 (C-1′), 106.2 (C-2), 127.6 (CH, C-Ar), 128.3 (CH,
C-Ar), 138.3 (Cquat, C-Ar); IR (cm-1, film) 3444, 2937, 2268,
1634, 1453, 1370, 1282, 1222, 1110. Anal. Calcd for C18H24O5:
343.15147 [M + Na]+ (correct mass according to ESI-HRMS).
(3S,4S,5S,7S)-3-Benzyloxymethyl-7-methyl-1,6-dioxaspiro[4.5]-
decan-4-ol (15), Method A. The alkyne 14 (107 mg, 334 µmol)
was dissolved in MeOH (10 mL), and Rh on activated Al2O3 (5%,
40.2 mg) was added. The mixture was vigorously stirred for 4.5 h
at room temperature under a hydrogen atmosphere (1 atm) and then
filtered through 2 cm Celite. The solids were washed with MeOH
(20 mL), and the filtrate was concentrated in vacuo to a volume of
approximately 15 mL. Then, concentrated hydrochloric acid was
added (10 µL), and the mixture was stirred for 15 h at room
temperature. The solution was diluted with diethyl ether (200 mL)
and water (20 mL). The layers were separated, and the organic
phase was washed with saturated NaHCO3 solution (10 mL) and
brine (10 mL) and dried over Na2SO4. The solvent was removed
in vacuo, and the residue was separated by flash column chroma-
tography on silica gel (10 g, c-Hex/EtOAc 3:1) to yield 6.0 mg
(6%) of (1S,4S,5S,7R)-4-benzyloxymethyl-7-methyl-2,6,11-trioxa-
tricyclo[5.3.1.01,5]undecane (16) (Rf ) 0.58, c-Hex/EtOAc 1:1) and
9.8 mg (10%) of the title compound 15 (Rf ) 0.40, c-Hex/EtOAc
3
(m, 2 H, 9(10)-H), 2.52 (mc, 1 H, 3-H), 3.29 (d, br, J ) 7.5 Hz,
1 H, OH), 3.40 (dt, 3J ) 4.5, 2J ) 11.0 Hz, 1 H, CH2OH), 3.64 (d,
3
2
3J ) 9.5 Hz, 1 H, 4-H), 3.83 (dd, J ) 2.5, J ) 11.0 Hz, 1 H,
CH2OH), 3.83-3.92 (m, 2 H, 2-H), 3.87 (ddq, 3J ) 2.1, 3J ) 6.1,
3J ) 12.5 Hz, 1 H, 7-H), 4.54 (d, 2J ) 11.8 Hz, 1 H, CH2Ph), 4.57
(d, 2J ) 11.8 Hz, 1 H, CH2Ph), 7.21-7.32 (m, 5 H, Ar-H);
13C NMR (75.5 MHz, CD2Cl2) δ 20.3 (C-9), 21.5 (CH3), 31.3 (C-
10), 32.4 (C-8), 40.0 (C-3), 59.6 (C-2), 66.0 (C-7), 66.9 (CH2OBn),
73.5 (C-4), 84.0 (CH2Ph), 102.0 (C-5), 128.1 (CH, C-Ar), 128.2
(CH, C-Ar), 128.7 (CH, C-Ar), 138.3 (Cquat, C-Ar); MS (ESI)
20
m/z (%) 293.1 (100) [M + H]+, 606.9 (40) [2M + Na]+. 7: [R]D
1
-147 (c 1.0, MeOH); H NMR (300 MHz, CD3COCD3) δ 1.02
3
(d, J ) 6.1 Hz, 3 H, CH3), 1.05-1.18 (m, 1 H, 8-H), 1.35-1.42
20
1
(m, 1 H, 10-H), 1.48-1.82 (m, 4 H, 8(9,10)-H), 2.71 (mc, 1 H,
1:1) as colorless oils. 15: [R]D -17 (c 1.0, MeOH); H NMR
(250 MHz, CD2Cl2) δ 1.15 (d, 3J ) 6.1 Hz, 3 H, CH3), 1.14-1.25
(m, 1 H, 8-H), 1.48-1.82 (m, 5 H, 8(9,10)-H), 2.58 (mc, 1 H, 3-H),
3
3-H), 3.63-3.78 (m, 4 H, 2(4)-H, CH2OBn), 3.83 (ddq, J ) 2.1,
3J ) 6.1, 3J ) 12.5 Hz, 1 H, 7-H), 3.95 (t, J ) 8.5 Hz, 1 H, 2-H),
2
2
3
4.42 (d, J ) 11.8 Hz, 1 H, CH2Ph), 4.50 (d, J ) 11.8 Hz, 1 H,
CH2Ph), 4.60 (s, 2 H, CH2Ph), 7.22-7.40 (m, 10 H, Ar-H);
13C NMR (75.5 MHz, CD3COCD3) δ 20.7 (C-9), 22.2 (CH3), 31.8
(C-10), 33.1 (C-8), 39.5 (C-3), 66.7 (C-7), 69.6 (C-2), 71.2 (CH2-
OBn), 73.4 (CH2Ph), 73.5 (CH2Ph), 84.3 (C-4), 103.3 (C-5), 128.0
(CH, C-Ar), 128.3 (CH, C-Ar), 128.3 (CH, C-Ar), 128.7 (CH,
2.68 (d, J ) 8.5 Hz, 1 H, 4-OH), 3.46 (t, J ) 9.1 Hz, 1 H, CH2-
3
OBn), 3.70 (t, J ) 9.1 Hz, 1 H, CH2OBn), 3.75 (dd, J ) 7.5,
2J ) 8.8 Hz, 1 H, 2-H), 3.81 (t, J ) 8.8 Hz, 1 H, 2-H), 3.93 (ddq,
3
3
3
3J ) 2.0, J ) 6.1, J ) 12.5 Hz, 1 H, 7-H), 3.98 (dd, J ) 7.9,
3J ) 8.5 Hz, 1 H, 4-H), 4.45 (d, 2J ) 14.0 Hz, 1 H, CH2Ph), 4.51
(d, J ) 14.0 Hz, 1 H, CH2Ph), 7.20-7.40 (m, 5 H, Ar-H); 13C
2
C-Ar), 129.0 (CH, C-Ar), 139.6 (Cquat, C-Ar), 139.9 (Cquat
,
NMR (62.9 MHz, CD2Cl2) δ 19.8 (C-9), 21.6 (CH3), 31.1 (C-10),
32.5 (C-8), 40.9 (C-3), 67.2 (C-7), 69.1 (C-2), 69.6 (CH2OBn),
73.1 (CH2Ph), 76.8 (C-4), 102.9 (C-5), 127.5 (CH, C-Ar), 127.6
C-Ar); MS (ESI) m/z (%) 405.3 (100) [M + Na]+, 383.3 (5)
[M + H]+. Anal. Calcd (%) for C24H30O4 (382.50): C, 75.36; H,
7.91. Found: C, 75.05; H, 7.61.
(CH, C-Ar), 128.3 (CH, C-Ar), 138.7 (Cquat, C-Ar); IR (cm-1
,
(2R,3S,4S)-4-Benzyloxymethyl-2-[(S)-4-hydroxypent-1-ynyl]-
2-methoxytetrahydrofuran-3-ol (14). A solution of (S)-2-[(tet-
rahydro-2H-pyran-2-yl)oxy]-4-pentyne (12, 61 mg, 0.36 mmol) in
diethyl ether (20 mL) was cooled to 0 °C, and methyllithium (0.35
mL, 0.34 mmol, 0.97 M in pentane) was added. After being stirred
for 5 min at 0 °C, the solution was transferred by cannula to second
flask containing a precooled solution (0 °C) of (2S,3S)-3-benzyl-
oxymethyl-2-[(tetrahydro-2H-pyran-2-yl)oxy]-4-butanolide (10, 111
mg, 0.362 mmol) in diethyl ether (20 mL). The resulting mixture
was stirred for 20 min at 0 °C and then for 3 h at room temperature.
An aqueous solution of NH4Cl (20%, 10 mL) was added, and the
mixture was stirred for 10 min. The layers were separated, and the
KBr) 2934, 1735, 1617, 1457, 1385, 1229, 1087; MS (ESI) m/z
(%) 293.1 (100) [M + H]+, 315.2 (40) [M + Na]+. Anal. Calcd
(%) for C17H24O4 (292.37): C, 69.84; H, 8.27. Found: C, 69.68;
H, 8.22. 16: 1H NMR (250 MHz, CD2Cl2) δ 1.38 (s, 3 H, CH3),
1.52-2.00 (m, 6 H, 8(9,10)-H), 2.45 (mc, 1 H, 4-H), 3.39 (t, J )
9.1 Hz, 1 H, CH2OBn), 3.67 (dd, 3J ) 6.2, 2J ) 9.1 Hz, 1 H, CH2-
OBn), 3.98 (t, J ) 8.5 Hz, 1 H, 3-H), 4.18 (t, J ) 8.5 Hz, 1 H,
3-H), 4.47-4.53 (m, 3 H, 5-H, CH2Ph), 7.20-7.40 (m, 5 H, Ar-
H); 13C NMR (62.9 MHz, CD2Cl2) δ 19.4 (C-9), 23.5 (CH3), 28.7
(C-10), 34.2 (C-8), 40.5 (C-4), 69.3 (CH2OBn), 73.3 (CH2Ph), 73.4
(C-3), 80.2 (C-5), 110.0 (C-7), 113.7 (C-1), 127.5 (CH, C-Ar),
127.7 (CH, C-Ar), 128.3 (CH, C-Ar), 138.6 (Cquat, C-Ar); MS
J. Org. Chem, Vol. 71, No. 19, 2006 7131

Bender et al.
(DCI, 70 eV) m/z (%) 291.2 (10) [M + H]+, 308.2 (100) [M +
NH4]+, 598.5 (65) [2M + NH4]+. Anal. Calcd for C17H22O4:
291.15894 [M + H]+ (correct mass according to ESI-HRMS).
Method B. The alkyne 14 (10.0 mg, 31.2 µmol) was dissolved
in ethyl acetate (2 mL). PtO2 (3.0 mg, 13 µmol) was added, and
the resulting suspension was stirred for 20 min at room temperature
under a hydrogen atmosphere (1 atm). The reaction mixture was
filtered through 1.5 cm Celite and washed with 20 mL of MeOH,
and the filtrate was concentrated in vacuo. Dichloromethane (1.5
mL) and (+)-10-camphorsulfonic acid (3.5 mg, 15 µmol) were
added, and the mixture was stirred for 5 h at room temperature
under TLC control. When the reaction had ceased, the solution was
diluted with diethyl ether (50 mL), washed with saturated NaHCO3
solution (5 mL), water (2 × 10 mL), and brine (10 mL), and dried
over Na2SO4. The solvent was removed in vacuo, and the residue
was purified by flash column chromatography on silica gel (10 g,
c-Hex/EtOAc 4:1) to yield 7.3 mg (80%) of the title compound
15.
Streptomyces sp. Go¨ TS 19. Anal. Calcd (%) for C10H18O4
(202.25): C, 59.39; H, 8.97. Found: C, 59.68; H, 8.92.
Computations. Ab initio DFT calculations were performed using
Spartan 04 for Windows (Wavefunction Inc., Irvine, CA). All
isomers of 4 were geometry minimized using the B3LYP method
with a 6-31G* basis set.
Acknowledgment. This work was financially supported by
the Deutsche Forschungsgemeinschaft (SFB 416, Project B14).
S.G. and P.v.Z. thank Prof. Dr. Axel Zeeck and Prof. Dr. Armin
de Meijere, respectively, for their support. The authors are
grateful to Dr. Heiko Schill for his assistance in performing
the computational studies.
Supporting Information Available: General experimental
methods; procedures for the isomerization of okaspirodiol (4) and
analytical data of isomers 4a-4c; 1H NMR and 13C NMR spectra
of all new compounds including the 13C NMR spectrum of the
natural product from the [U-13C3]glycerol feeding experiment; a
short graphical explanation for the exclusion of a sedoheptulose-
based biosynthesis; tabularly summary of HMBC and COSY
correlations for okaspirodiol 4; NOESY spectra and a graphical
summary of observed NOE cross-peaks for compounds 4-4c;
structure, total energies, and Cartesian coordinates for calculated
structures 4-4c; and ORTEP drawing, X-ray data, and details for
the X-ray data acquisition for 5 (CIF format). This material is
available free of charge via the Internet at http://pubs.acs.org.
Synthesis of Okaspirodiol (4). The benzylated precursor 15 (15
mg, 51 µmol) was dissolved in MeOH (3 mL), and Pd/C (10%,
3 mg) was added. After being stirred for 4 h at room temperature
under a hydrogen atmosphere (1 atm), the reaction mixture was
filtered through 1.5 cm of Celite and washed with 20 mL of MeOH.
The solvent was removed in vacuo, and the residue was purified
by flash column chromatography on silica gel (5 g, c-Hex/EtOAc
4:1) to furnish 8.5 mg (82%) of the title compound 4. The optical
20
rotation {[R]D -53 (c 1.0, MeOH)} and all spectroscopic data
were consistent with the data of okaspirodiol (4) isolated from
JO060149E
7132 J. Org. Chem., Vol. 71, No. 19, 2006