Chemoenzymatic synthesis of α-Hydroxy-β-methyl-γ-hydroxy esters: Role of the keto-enol equilibrium to control the stereoselective hydrogenation in a key step

Article abstract of DOI:10.1021/jo902227f

"Chemical Equation Presented" α-Hydroxy-β-methyl- γ-hydroxy esters not only are found in many natural products and potent drugs but also are useful intermediates in organic synthesis due to their highly functionalized skeleton that can be further manipula

Full text of DOI:10.1021/jo902227f

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Chemoenzymatic Synthesis of r-Hydroxy-β-methyl-γ-hydroxy Esters:
Role of the Keto-Enol Equilibrium To Control the Stereoselective
Hydrogenation in a Key Step
Cıntia D. F. Milagre,† Humberto M. S. Milagre,‡ Paulo J. S. Moran,† and
J. Augusto R. Rodrigues*,†
†Institute of Chemistry, University of Campinas-UNICAMP, PO Box 6154, 13084-970 Campinas, SP,
Brazil, and ‡Institute of Biosciences, UNESP-Univ. Estadual Paulista, 13506-900 Rio Claro, SP, Brazil
jaugusto@iqm.unicamp.br
Received October 20, 2009
R-Hydroxy-β-methyl-γ-hydroxy esters not only are found in many natural products and potent drugs
but also are useful intermediates in organic synthesis due to their highly functionalized skeleton that
can be further manipulated and applied in the synthesis of many compounds with remarkable
biological activities. This work was based on a chemoenzymatic approach to obtain these molecules
with three contiguous stereogenic centers in a highly enantio- and diastereoselective way. Two
distinct linear routes were proposed in which the key steps in both routes consisted of initial
stereocontrolled ketoester bioreduction followed by unsaturated carbonyl bioreduction or reduction
with Pd-C. Other key reactions in the synthesis include a Wasserman protocol for chain homo-
logation and a Mannich-type olefination with maintenance of enantiomeric excess for all inter-
mediates during the sequence. Whereas route A gave exclusively the skeleton with 3R,4R,5S
configuration (99% ee and 11.5% global yield after 7 steps), route B gave the skeleton with
3R,4R,5S and 3R,4S,5R configurations (dr 1:12, 98% ee and 20% global yield after 5 steps).
Introduction
the synthesis of a variety of bioactive molecules1 used as
pharmaceutical intermediates2 and are found in natural
products.3 In our continuing efforts toward the synthesis
Unsaturated carbonyl compounds are prochiral sub-
strates that can provide densely functionalized molecules
with two or more consecutive stereogenic centers. Asym-
metric reductive products such as optically pure R-hydroxy-
β-methyl-γ-hydroxy esters are important building blocks for
€
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Published on Web 02/09/2010
DOI: 10.1021/jo902227f
r
2010 American Chemical Society

Milagre et al.
JOCArticle
of such useful intermediates, we developed two novel chemo-
enzymatic routes for the preparation of enantio and
diastereomerically pure R-hydroxy-β-methyl-γ-hydroxy es-
ters.4 Our study has focused on a biocatalytical approach to
create the first chiral center from wild microbial whole cell
reduction of β-ketoesters and R,γ-ketoesters. The second
and third asymmetric centers were obtained by controlling
the absolute configuration of the adjacent stereogenic center
through a heterogeneous catalytic hydrogenation with
Pd-C of the double bond in the enol tautomer in route A
and by biocatalytic reduction of the exomethylene group
followed by reduction with nBu4NBH4 in route B (Schemes 1
and 5). Other key reactions in the synthesis include
such classical organic reactions as Claisen condensation, a
Wasserman chain homologation, a Mannich-type olefina-
tion with maintenance of stereo integrity of the involved
intermediates, and Pd-C catalyzed hydrogenation to obtain
a diastero- and enantioselective product by the reduction of
the appropriate enol formed in the keto-enol equilibrium.
99% ee after 2 h. Then, the chemical regio- and diastereo-
selective reduction of the R,β-unsaturated system 6 was
investigated in order to examine the substrate specificities
of the oxidoreductase yeasts. Initially a screening of bioca-
talysts to reduce 6 was carried out mediated by several yeasts
cells in growing conditions (Scheme 2).
Unfortunately, all of the yeasts evaluated (Saccharomyces
cerevisiae, Pichia kluyveri (CCT 3365), Pichia stipitis (CCT
2617), Pichia canadensis (CCT 2636), Rhodotorula minuta
(CCT 1751), Rhodotorula glutinis (CCT 2182)) were not able
to reduce 6 to 7, 8, or even 9 under several experimental
conditions. These results can be rationalized by the high
sterical hindrance caused by the TBS protecting group that
could reduce or hinder the interaction between substrate and
enzyme at the active site. To overcome these difficulties, we
exchanged biocatalysts for traditional chemical methods. A
catalytic hydrogenation of the CdC of (S)-6 mediated by
Pd-C in methanol under 1 atm gave after 16 h the methyl-
hydroxy product 7 in 45% yield. The reduction step of this
sequence proceed with modest diastereoselectivity giving a
mixture of syn: anti isomers (ca. 3:1). Surprisingly, when the
reaction was left more than 16 h, just one diasteroisomer, 8a,
was formed with excellent stereoselectivity (70% yield, >
99% de, 1H NMR spectroscopic analysis (300 MHz); >99%
ee). Alternatively, a one-pot, two-step tandem catalytic
hydrogenation in a round-bottom flask of (S)-6 with Pd-C
in methanol suspension under 1 atm after 15 h gave only one
diastereoisomer, 8a, with >99% ee. These unique high
diastereoselectivies could be attributed to the favorable
keto-enol equilibrium when methanol is used as a solvent
leading to epimerization of C-3 and subsequent reaction of 7
in the enolic form (Scheme 3).10 To our knowledge, there are
no reports of hydrogenation of an enol intermediate in a
keto-enol equilibrium using Pd-C (low pressure and room
temperature), although there are reports using hydrogena-
tion of enol acetates and silyl enol ethers from cyclic
ketones11 and with enolate ion.12
Results and Discussion
Our synthetic approach for route A is shown in Scheme 1.
The ethyl benzoylacetate 1 was bioreduced, giving (S)-ethyl
3-hydroxyphenylpropanoate 2, which was produced on a
multigram scale5 (9.0 g) with 80% yield and 99% ee mediated
by the yeast Pichia kluyveri CCT 3365 immobilized on
alginate beads in a reactor (400 mL working volume) with
mechanical stirring and temperature control (30 °C). The
details were discussed in a recent publication.6
Protection of the β-hydroxy ester 2 as the TBS 3 ether
(TBSCl, imidazole, DMF, rt, 2 h, 90% yield and 99% ee),7
followed by alkaline ester hydrolysis of 3 (LiOH/H2O/EtOH
in 80% yield and 99% ee) gave the corresponding acid
4. Extension of 4 through the Wasserman protocol
([Ph3PCH2CN]Cl, EDCI and DMAP followed by ozonolysis
in methanol and dichloromethane)8 furnished the required
(S)-2-oxo ester 5 in 50% yield (after 2 steps) with preserva-
tion of the stereochemical integrity of the molecule. The
direct Mannich-type R-methylenation of 5 under optimized
conditions described previously9 yielded 6 in 85% yield and
Although enol 7 can exist as two geometric isomers, Z and
E, it is well-known that in Pd-catalyzed reactions, the overall
reaction is not controlled by the adsorption step but by the
step in which the hydrogen is transferred from the catalyst
surface to the molecule on the opposite side of the larger
substituent.13 In order to inspect these two isomeric struc-
tures, density functional theory calculations were performed
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H.; Rodrigues, J. A. R. Enzyme Microb. Technol. 2005, 37, 121–125. (b)
Milagre, H. M. S.; Milagre, C. D. F.; Moran, P. J. S.; Santana, M. H.;
Rodrigues, J. A. R. Org. Process Res. Dev. 2006, 10, 611–617. (c) Alonso, F.
O. M.; Antunes, O. A. C.; Oestreicher, E. G. J. Braz. Chem. Soc. 2007, 18,
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2005, 9, 174–180. (h) Zhao, H.; van der Donk, W. A. Curr. Opin. Biotechnol.
2003, 14, 583–589.
(5) The limitation of scale here is of practical use. We limited the reaction
scale to the use of a 400 mL stirred-tank reactor, 1.0 g of 1 per batch with no
loss in yield and ee. See ref 6 for schematic representation of the bioreactor
and a photo of the used platform.
(6) Milagre, C. D. F.; Milagre, H. M. S.; Moran, P. J. S.; Rodrigues,
J. A. R. J. Mol. Catal. B: Enzym. 2009, 56, 55–60.
(7) (a) Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis,
2nd ed.; John Wiley & Sons: New York, 1991. (b) Corey, E. J.; Venkateswarlu, A.
J. Am. Chem. Soc. 1972, 94, 6190–6191. (c) Nicolaou, K. C.; Webber, S. E.
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Chem. Soc., Perkin Trans. 1 2000, 901–910.
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M. L. A.; Moran, P. J. S.; Eberlin, M. N.; Rodrigues, J. A. R. J. Mass
Spectrom. 2007, 42, 1287–1293. (b) Rodrigues, J. A. R.; Siqueira, E. P. F.;
Mancilha, M.; Moran, P. J. S. Synth. Commum. 2003, 33, 331–340.
(10) (a) Makino, K.; Goto, T.; Hiroki, Y.; Hamada, Y. Tetrahedron:
Asymmetry 2008, 19, 2816–2828. (b) Flowers, B. J.; Gautreau-Service, R.;
Jessop, P. G. Adv. Synth. Catal. 2008, 350, 2947–2958. (c) Hamada, Y.;
Makino, K. J. Synth. Org. Chem. Jpn. 2008, 66, 1057–1065. (d) Diezi, S.;
Ferri, D.; Vargas, A.; Mallat, T.; Baiker, A. J. Am. Chem. Soc. 2006, 128,
4048–4057. (e) Singh, U. K.; Vannice, M. A. Appl. Catal., A 2001, 213, 1–24.
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Iwakura, C. Tetrahedron Lett. 2001, 42, 8323–8327. (h) Nitta, Y.; Kubota, T.;
Okamoto, Y. J. Mol. Catal. A: Chem. 2004, 212, 155–159. (i) Rodrigues, J. A.
R.; Moran, P. J. S.; Milagre, C. D. F.; Ursini, C. V. Tetrahedron Lett. 2004,
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SCHEME 1. Synthetic Route A
SCHEME 2. Possible Products from Yeast Reduction of (S)-6
with B3LYP/6-31Gþþ(d,p).14 Those calculations have
shown that tautomer (E)-7 (Scheme 3) is 1.55 Kcal/mol more
stable when compared with (Z)-7 and that both sides of the
CdC bond of enol (E)-7 should be blocked by the volumi-
nous -OTBS and -Ph groups (the dihedral angle between
C-OTBS groups and CdC bonds is -127.3°) leaving no room
for approximation of the hydrogen adsorbed on Pd-C to
either side of the CdC bond. Examination of the calculated
structure of enol (Z)-7 reveals that this enol is stabilized by an
(14) Frisch, M. J. et al. Gaussian 03, Revision A.1; Gaussian, Inc.:
Wallingford, CT, 2003. (See Supporting Information for the full citation.)
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SCHEME 3. Keto-Enol Equilibrium of 7 Leading to 8a with
High Stereoselectivity
FIGURE 1. Conformation of both enols E-7 and Z-7.
concomitant molecule lactonization to give 11. The data for
NOE are shown in Scheme 4. Chemical shifts and coupling
constants from 1H NMR experiments in addition to
GC-MS retention times and 13C NMR data are consistent
with the formation of lactone 11 from compound 8a. These
data were compared with the literature,15 and the absolute
configuration of 11 was established as 3R,4R,5S-11.16
The second strategy to obtain the desired R-hydroxy-
β-methyl-γ-hydroxy esters intermediates, route B, is shown
in Scheme 5. The first step was the Claisen condensation of
acetophenone with diethyloxalate, which proved to be
straightforward. A nearly quantitative yield of the 2,4-dioxo
ester 12 was obtained by addition of diethyl oxalate followed
by acetophenone to a freshly prepared NaOEt suspension in
THF in 92% yield.17
˚
internal hydrogen bond of 1.72 A between the OH and OTBS
groups. This internal hydrogen bond results in a distortion of
the dihedral angle between C-OTBS and CdC bonds to 26.1°,
bringing the OTBS nearer to the plane of the CdC bond and
therefore leaving room for the approximation of hydrogen
adsorbed on Pd-C to this side. This model justifies the
exclusive formation of 8a (see Figure 1). The high diasteros-
electivity obtained demonstrates that the OTBS group acts not
only as an effective protective group but also to assist the
vicinal chiral center of the substrate to stereoselectively drive
the reduction of CdC bond of the enol.
To emphasize the relevance of the keto-enol equilibrium,
we repeated the reaction using the less enolizable ketoester 10
under the same reaction conditions (Pd-C, H2, 1 atm,
MeOH, 32 h), as a model to verify the selectivity of carbonyl
reduction. The result showed no CdO reduction, and the
starting material 10 was quantitatively recovered. This result
shows the role of the enol tautomer for the reduction of the
ketone in the above conditions.
The bioreduction of 12 was performed according to the
Fadnavis protocol,10a and the desired product 13 was
achieved in 80% yield and 99% ee.18 The Mannich type
R-methylenation previously described was applied to R-13,
(15) (a) Matsubara, R.; Vital, P.; Nakamura, Y.; Kiyohara, H.; Kobayashi,
S. Tetrahedron 2004, 60, 9769–9784. (b) Matsubara, R.; Nakamura, Y.;
Kobayashi, S. Angew. Chem. Int. Ed. 2004, 43, 3258–3260.
(16) Data of relative and absolute configuration of compounds 8a and 11
15
were compared with data of their respective enantiomers shown in ref
.
These comparisons could be done because GC-MS retention times and 1H
NMR and 13C NMR data for the enantiomeric compounds are the same, and
the first chiral center therein was established accurately in the first step and
afterwards remained unchanged assisted by OTBS protecting group
throughout route. The same comparison procedure was used for compound
18.
(17) (a) Fadnavis, N. W.; Radhika, K. Tetrahedron: Asymmetry 2004, 15,
3443–3447. (b) Chang, C. Y.; Yang, T. K. Tetrahedron: Asymmetry 2003, 14,
2239–2245. (c) Jiang, X. H.; Song, L. D.; Long, Y. Q. J. Org. Chem. 2003, 68,
7555–7558. (d) Blaser, H. U.; Burkhardt, S.; Kirner, H. J.; Mossner, T.;
Studer, M. Synthesis 2003, 1679–1682. (e) Indolese, P. H.; Studer, M.; Jallet,
H. P.; Siegrist, U.; Blaser, H. U. Tetrahedron 2000, 56, 6497–6499. (f ) Studer,
M.; Herold, P.; Indolese, A. Burkhardt, S. Ciba Specialty Chemicals Holding, Inc.,
PCT Int. Appl. WO 9950223, 1999.
(18) Some difficulties were found in scaling up bioreduction of 12 due to
formation of aggregates of yeast and water in the highly volatile diethyl ether.
The vigorous stirring and a bath reactor coupled to a condenser was essential
to minimize these issues.
(19) (a) Siqueira Filho, E. P.; Rodrigues, J. A. R.; Moran, P. J. S.
Tetrahedron: Asymmetry 2001, 12, 847–852. (b) Hall, M.; Hauer, B.;
Stuermer, R.; Kroutil, W.; Faber, K. Tetrahedron: Asymmetry 2006, 17,
3058–3062. (c) Hall, M.; Stueckler, C.; Kroutil, W.; Macheroux, P.; Faber,
K. Angew. Chem., Int. Ed. 2007, 46, 3934–3937. (d) Stuermer, R.; Hauer,
B.; Hall, M.; Faber, K. Curr. Opin. Chem. Biol. 2007, 11, 203–213. (e)
Fryszkowaska, A.; Fischer, K.; Gardiner, J. M.; Stephens, G. M. J. Org.
Chem. 2008, 73, 4295–4298. (f) Hall, M.; Stueckler, C.; Ehammer, H.;
Pointner, E.; Oberdorfer, G.; Gruber, K.; Hauer, B.; Stuermer, R.; Kroutil,
W.; Macheroux, P.; Faber, K. Adv. Synth. Catal. 2008, 350, 411–418. (g)
Hall, M.; Stueckler, C.; Hauer, B.; Stuermer, Friedrich, T.; Breuer, M.;
Kroutil, W.; Macheroux, P.; Faber, K. Eur. J. Org. Chem. 2008, 1511–1516.
The relative stereochemistry of 8a was determined as 2,3-
1
syn-3,4-syn through H NMR chemical shift and coupling
constant analysis. As the absolute configuration of C-4 was
determined in the first step and was preserved since then and
knowing the relative stereochemistry of these three conti-
guous centers, the absolute configuration of 8a could be
established as 2R,3S,4S. To confirm these results the desily-
lation of 8a was performed with HF-pyridine in CH2Cl2 with
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SCHEME 4. Relative and Absolute Configuration of 11 Determined by NMR
SCHEME 5. Synthetic Route B
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TABLE 1. Microbial Reduction of (R)-14 Using Different Yeasts
yeasta
yield (%)
15 anti:syn (ee %)c
16 and/or 17
time (h)
Saccharomyces cerevisiaeb
Candida parapsilosis (CCT 3438)
Trichosporum cutaneum (CCT 1903)
Rhodotorula glutinis (CCT 2182)
-
73
75
76
-
-
-
-
-
48
24
30
24
77:23 (99)
74:26 (97)
85:15 (99)
aGrowing cells, YM medium, (R)-14 0.4 mmol (100 mg) in ethanol 1 mL, orbital stirring, 28 °C. bCommercial lyophilized yeast. Percentage of
enantiomeric excess of each diasteroisomer; enantiomeric and diastereoisomeric excess were determined by HPLC.
c
experiments as shown in Scheme 7, which are in agreement
with the literature.15
SCHEME 6. Cram Chelation Model of 15
Further determination of the absolute configuration of
18 was obtained using the chiral auxiliary reagent (S)-MPA
(R-methoxyphenylacetic acid)22 (Scheme 8) and comparison
of these data with the literature.15 The anisotropic effect of
the aromatic ring of (S)-MPA over the methyl group was
observed through the 0.61 ppm difference in the chemical
yielding R-14 in 80% and 99% ee with preserved stereo-
chemical integrity. The asymmetric biocatalytic reduction of
shift toward TMS, proving that both groups are synperipla-
nar. Thus, it has been possible to designate the absolute
14 could provide two chiral centers. Although the stereo-
configuration as (3R,4S,5R)-18.
selectivity achieved in this kind of reduction is excellent, the
chemoselectivity of whole cell bioreductions with respect to
CdC versus CdO bond reduction is often poor as a result of
Conclusions
In summary, we have achieved the syntheses of R-hydroxy-
β-methyl-γ-hydroxy esters, which are a versatile chiral synthon
possessing three stereogenic centers, through two distinct
chemoenzymatic routes with excellent stereoselectivity. Nota-
ble features of these approaches include biocatalytic reductions
and an outstanding stereo- and regioselective Pd-C hydro-
genation due to the keto-enol equilibrium, in accordance
with a model obtained by theoretical calculations. The syn-
thetic strategies used to obtain these skeletons were efficient
and provide the desired products in good overall yield.
Furthermore, the biocatalytic pathways were performed on a
multigram scale, and both syntheses are amenable to scale-up
to produce important chiral synthons. In principle, these
approaches are readily applicable for the preparation of more
complex organic structures of industrial interest.
the presence of competing alcohol dehydrogenases, since the
enoate reductases and the alcohol dehydrogenases depend
on the same nicotinamide cofactor.19 Thus, several yeast
strains were evaluated for the bioreduction of 14 in order to
find the most reactive ones (Table 1).
In all cases, the bioreduction of R-14 afforded exclusively
15 in good yields, with diastereoselectivity and excellent
enantioselectivity.20 It is well-known that in R,β-unsaturated
ketone systems, the double bond is reduced preferably.21
Only in rare cases, when R,β-unsaturated ketone resonance is
destabilized by electron-withdrawing groups bonded to
double bonds, the CdO is reduced first, which justifies the
absence of 17. The 14 γ-carbonyl is not electrophilic enough
to favor a NADH/NADPH type hydride attack. Thus for-
mation of 16 is not observed. The bioreduction of R-14 was
carried out on a multigram scale by the yeast Rhodotorula
glutinis CCT 2182, giving 15 in 90:10 diastereoisomeric ratio
favoring the isomer anti. As the γ-carbonyl of 15 was not
reduced by any of the employed yeasts, a chemical reduction
with nBu4NBH4 was carried out. Reduction of 15 gave
lactones 18 (white solid) and 11 (yellow oil) in 92:8 (trans:
cis) selectivity (anti-Felkin products) as a result of favored
five-membered ring formation. The high 1,3-anti selectivity
can be explained by the Cram chelation model due to the OH
group in the β position (Scheme 6).
Experimental Section
Ethyl (S)-(-)-3-hydroxy-3-phenylpropanoate, (S)-2. A solu-
tion of sodium alginate (150 mL, 2%) was added to a suspension
of Pichia kluyveri CCT 3365 (23 g wet biomass) in distilled water
(50 mL, pH 6.5). This mixture was extruded using syringe
nozzles with inner diameters of 0.8 mm into a solution of CaCl2
(0.2 mol/L) to produce beads with 2 mm diameters. After 20
min, the beads were filtered and washed with water to remove
the excess of CaCl2. The beads were suspended in distilled water
(300 mL, pH 6.5) containing glucose (6 g) and R-chloroaceto-
phenone (40 mg) in a 400 mL working volume bioreactor and
stirred at 300 rpm at 30 °C. After activation of the yeast for 2 h,
substrate 1 (5.2 mmol in 3.0 mL of ethanol) was added. The
reaction was monitored by GC-MS, and after 24 h the beads
were filtered and washed with ethyl acetate. The bead-free
The configuration of 11 was already established in route
A. The relative configuration of 18 was determined as 3,4-cis-
1
4,5-cis which is supported by J constants from H NMR
experiments and increments in NOE signals of NOESY1D
(20) This bioreduction is easily scaled up, and as described previously, the
limitation of scale is of practical use (a 400 mL stirred-tank reactor).
(21) Faber, K. Biotransformations in Organic Chemistry, 4th ed.; Springer-
Verlag: Berlin, 2000.
(22) For a revision about absolute configuration assembly through
~ ꢀ
NMR, see: Riguera, R.; Seco, J. M.; Quinoa, E. Chem. Rev. 2004, 104,
17–117.
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SCHEME 7. Assignment of Relative Configuration of 18
SCHEME 8. Absolute Configuration Assembly of 18 by 1H
NMR Employing (S)-MPA
99% ee. (S)-4 was pure enough for further manipulation. Color-
less oil;25 [R]D20 = -16.8 (c 0.90, MeOH); IR (neat) ν max (cm-1
)
3300-2500, 1713, 1436; 1H NMR (300.1 MHz, CDCl3)
δ -0.87 (s, 3H), 0.02 (s, 3H), 1.01 (s, 9H), 2.81 (dd, 1H, J =
4.0 and J = 10.9 Hz), 2.99 (dd, 1H, J = 5.8 and J = 9.1 Hz), 5.50
(dd, 1H, J = 4.0 Hz and J = 4.0 Hz), 7.51 (m, 5H); 13C NMR
(75.0 MHz, CDCl3) δ -4.5 (CH3), -5.2 (CH3), 18.2 (C0), 25.6
(CH3), 46.0 (CH2), 72.0 (CH), 125.7 (CH), 127.5 (CH), 128.3
(CH), 143.5 (C0),176.7 (C0).
Methyl (S)-(-)-4-(tert-butyldimethylsilyloxy)-2-oxo-4-phenyl-
butanoate, (S)-5. (Cyanomethylene)triphenylphosphonium chlor-
ide (4.17 g, 12 mmol) was dissolved in water (25 mL) and
dichloromethane (25 mL). Sodium hydroxide (1.26 g, 32 mmol)
in water (6 mL) was slowly added. After 20 min, the two layers were
separated. The organic layer was dried (MgSO4) and added to a
solution of (S)-4 (1.94 g, 7 mmol), DMAP (0.2 g) and EDCI (2.13 g,
11 mmol) in dichloromethane (50 mL) at 0 °C under a nitrogen
atmosphere. After 0.5 h, the reaction mixture was warmed to room
temperature and left stirring for 15 h. Water (50 mL) was added,
and the two layers separated. The organic layer was dried (MgSO4)
and concentrated in vacuum, resulting in a yellow paste. The yellow
paste was dissolved in dichloromethane (10 mL) and methanol
(20 mL) and cooled to -78 °C. Ozone was then bubbled through
the solution until the reaction mixture turned colorless-gray
(around 2 h). The reaction was followed by TLC. Nitrogen was
then passed through the solution to remove ozone excess. The
reaction mixture was warmed to room temperature and concen-
trated in vacuum. Purification by DSC, eluting with hexane/EtOAc
25%, gave (S)-5 as a pale yellow oil8a (0.87 g, 50% over two steps).
[R]D20 = -48.0 (c 1.50, MeOH); IR (neat) ν max (cm-1) 1755, 1732,
1254; MS m/z (%) 159 (100), 131 (31), 89 (50), 75 (85); 1H NMR
(300.1 MHz, CDCl3) δ -0.85 (s, 3H), 0.02 (s, 3H), 1.11 (s, 9H),
3.10 (dd, 1H, J = 4.0 and J = 11.0 Hz), 3.59 (dd, 1H, J = 5.3 and J
= 9.1 Hz), 4.11 (s, 3H), 5.40 (dd, 1H, J = 4.0 Hz and J = 4.0 Hz),
7.51 (m, 5H); 13C NMR (75.0 MHz, CDCl3) δ -4.1 (CH3), -4.6
(CH3), 18.6 (C0), 26.3 (CH3), 50.6 (CH2), 53.3 (CH3), 72.0
(CH), 126.3 (CH), 128.1 (CH), 128.8 (CH), 144.1 (C0), 161.5
(C0), 192.3 (C0).
mixture was extracted with ethyl acetate, dried over anhydrous
MgSO4, and filtered, and the solvent was evaporated. The crude
material was purified by flash chromatography, eluting with
10% ethyl acetate in hexane (to recover acetophenone side
product) and 20% ethyl acetate in hexane (to recover 2 with 80%
yield). Colorless oil;23 ee = 99%, [R]2D0 = -20.0 (c 2.05, MeOH);
IR (neat) ν max (cm-1) 3420, 1727, 1452; MS m/z (%) 194 (Mþ),
3
120 (14), 107 (100) and 77 (59); 1H NMR (300.1 MHz, CDCl3) δ
1.25 (t, 3H, J = 7.3 Hz), 2.70 (dd, 2H, J = 4.4 and J = 8.4 Hz),
4.16 (q, 2H, J = 7.1 Hz), 5.11 (t, 1H, J = 4.4 Hz), 7.31 (m, 5H);
13C NMR (75.0 MHz, CDCl3) δ 14,1 (CH3), 43.4 (CH2), 60.7
(CH2), 70.2 (CH), 125.5 (CH), 127.5 (CH), 128.2 (CH), 142.3
(C0), 172.1 (C0).
Ethyl (S)-(-)-3-(tert-butyldimethylsilyloxy)-3-phenylpropanoate,
(S)-3. A solution containing (S)-2 (2.7 g, 14 mmol) previously dried
in a vacuum for 30 min, tert-butyldimethylsilyl chloride (3.7 g, 24
mmol), imidazole (2.9 g, 42 mmol) and anhydrous DMF (15 mL)
was stirred for 3 h under Ar atmosphere at room temperature. Then
this mixture was dissolved in hexane (50 mL) and washed with
water (3 ꢀ 50 mL). The combined organic phases were dried over
MgSO4 and evaporated. The residue was purified by DSC (hexane/
EtOAc 20%) affording (S)-3 with 90% yield and 99% ee as a
colorless oil.24 [R]D20 -26.7 (c 1.02, MeOH); IR (neat) ν max (cm-1
)
1738, 1258, 1090; MS m/z (%) 293 (4), 103 (35) and 75 (100); 1H
NMR (300.1 MHz, CDCl3) δ -0.09 (s, 3H), 0.02 (s, 3H), 0,86 (s,
9H), 1.25 (t, 3H, J = 7.3 Hz), 2.54 (dd, 1H, J = 4.0 and J = 10.2
Hz), 2.73 (dd, 1H, J = 4.9 and J = 9.1 Hz), 4.15 (m, 2H), 5.15 (dd,
1H, J = 4.3 Hz), 7.30 (m, 5H); 13C NMR (75.0 MHz, CDCl3) δ
-4.6 (CH3), -5.1 (CH3), 14.3 (CH3), 18.1 (C0), 25.7 (CH3), 46.6
(CH2), 60.5 (CH2), 72.2 (CH), 125.7 (CH), 127.4 (CH), 128.1 (CH),
144.0 (C0), 171.0 (C0).
Methyl (S)-(-)-4-(tert-butyldimethylsilyloxy)-2-oxo-3-methylene-
4-phenylbutanoate, (S)-6. The reaction was carried out under a
nitrogen atmosphere. The ketoester 5 (1.0 mmol) and a freshly
prepared solution of morpholine (0.0264 g, 0.3 mmol) in glacial
acetic acid (5.0 mL) were mixed in a 25 mL double-necked round-
bottomed flask with a coiled reflux condenser flask. Molecular sieves
(S)-(-)-3-(tert-Butyldimethylsilyloxy)-3-phenylpropanoicAcid,
(S)-4. Compound (S)-3 (2.43 g, 8 mmol) was dissolved in ethanol
(10 mL) and cooled to 0 °C. Lithium hydroxide monohydrate
(1.37 g, 32 mmol) was dissolved in water (5 mL) and slowly added
to the (S)-3 solution. This mixture was magnetically stirring with
gradual warming. After 15 h, the solvent was evaporated. The
residue was dissolved in acidified water (pH 4.0) and washed with
hexane (3 ꢀ 10 mL). The combined organic phases were dried
over MgSO4 and evaporated to afford (S)-4 with 80% yield and
˚
(4 A) and p-formaldehyde (0.29 g, 9.0 mmol) were then added, and
the mixture was stirred and heated (reflux). After 2 h, the reaction
mixture was cooled to room temperature and neutralized with solid
NaHCO3 followed by repeated (3 times) extractions with equal
volumes of ethyl acetate. The combined organic extracts were washed
with brine and water and dried over anhydrous MgSO4 followed by
evaporation under reduced pressure. The product was filtered
through a chromatographic column filled with silica gel. Pale yellow
oil;9a [R]D20 = -56.0 (c 0.92, MeOH); IR (neat) ν max (cm-1) 1742,
1684, 1472, 1255; MS m/z (%) 319 (Mþ), 277 (17), 171 (100), 115
(23) He, C.; Chang, D.; Zhang, J. Tetrahedron: Asymmetry 2008, 19,
1347–1351.
(24) Manzocchi, A.; Casati, R.; Fiecchi, A.; Santaniello, E. J. Chem. Soc.,
Perkin Trans. 1 1987, 12, 2753–2757.
(25) Moussa, M. M.; Le Quesne, P. W. Tetrahedron Lett. 1996, 37, 6479–
6482.
3
(59),89(76),75(79);1H NMR (300.1 MHz, CDCl3) δ-0.87 (s, 3H),
0.03 (s, 3H), 0.98 (s, 9H), 3.98 (s, 3H), 5.70 (s,1H), 6.39 (s, 1H), 6.61
(s, 1H), 7.49 (m, 5H); 13C NMR (75.0 MHz, CDCl3) δ -4.6 (CH3),
-4.9 (CH3), 18.4 (C0), 25.9 (CH3), 52.8 (CH3), 71.5 (CH), 126.1
1416 J. Org. Chem. Vol. 75, No. 5, 2010

Milagre et al.
JOCArticle
(CH), 127.1 (CH), 128.0 (CH), 131.5 (CH2), 142.3 (C0), 148.3 (C0),
163.7 (C0), 186.7 (C0).
pH 4.5, 50 mL) containing glucose (5 g). The cells were allowed
to activate for 3 h, and diethyl ether was added (200 mL),
followed by the addition of R-chloroacetophenone (150 mg)
dissolved in diethyl ether (10 mL). The contents were vigorously
stirred on a magnetic stirrer at 28 °C for 3 h in a bath reactor
coupled to a condenser. Substrate 12 (1.1 g, 45 mmol) dissolved in
diethyl ether (50 mL) was added, and the contents were stirred.
After 48 h, the cells were filtered with Celite. The aqueous phase
was acidified with 6 mol/L HCl (10 mL) and extracted with EtOAc
(3 ꢀ 50 mL). The combined organic layer was washed with
brine, dried over anhydrous MgSO4, and concentrated by
rotatory evaporation to give a viscous yellow oil. This oil was
dissolved in ethanolic HCl (50 mL) and stirred at room tem-
perature overnight. Ethanol was then evaporated, and the
residual oil was purified by column chromatography using
hexane/EtOAc 25%, affording 13, a white solid17a,d with low
melting point in 80% yield and 99% ee. Mp 36 to 38 °C; [R]2D0=
-5.5 (c 1.0, CHCl3); HPLC (Chiralcel OJ, hexane/iPrOH = 9:1,
0.1% trifluoroacetic acid, flow rate = 0.7 mL/min, λ = 254 nm)
Methyl 4-(tert-butyldimethylsilyloxy)-2-oxo-3-methyl-4-phenyl-
butanoate (syn:anti mixture), 7. Pd-C (5%, 0.005 mmol) was
dissolved in anhydrous methanol (2 mL) and stirred under a H2
atmosphere (1 atm). After 30 min, 0.01 mmol of (S)-6 dissolved in
anhydrous methanol was added to the mixture. The reaction was
maintained under magnetic stirring, at room temperature and under
a H2 atmosphere (1 atm) for 16 h to obtain (S)-7 and 15 h more to
obtain (S)-8a. The mixture was filtered with filter paper, and the
solvent was evaporated. The product were purified by column
chromatography (hex/EtOAc 20%) affording a colorless oil in
70% yield; HPLC (Chiralcel OJ, hexane/iPrOH = 9:1, 0.1%
trifluoroacetic acid, flow rate = 0.4 mL/min, λ = 254 nm) tR
=
)
44.2 min (3S,4S)-7; tR = 45.1 min (3R,4S)-7; IR (neat) ν max (cm-1
1730, 1283, 1090; MS m/z (%) 173 (100), 117 (16), 105 (14), 89 (59),
73 (67), 59 (40); 1H NMR (300.1 MHz, CDCl3) 7-syn δ 0.20 (s, 9H),
0.91 (s, 6H), 1.10 (d, 3H, J=6.6Hz), 3.68(m, 1H), 3.87(s, 3H), 5.03
(d, 1H, J = 5.1 Hz), 7.27 (m, 5H); 7-anti δ 4.11 (s, 3H) and 4.72 (d,
1H, J = 9.2 Hz); 13C NMR (75.0 MHz, CDCl3) 7-syn δ -4.5 (2
CH3), 12.9 (CH3), 26.2 (3 CH3), 53.3 (CH3), 50.5 (CH), 75.5 (CH),
142.5 (C0), 162.0 (C0), 195.9 (C0); 7-anti δ -4.7 (2 CH3), 13.8 (CH3),
26.2 (3 CH3), 51.3 (CH3), 53.3 (CH), 79.3 (CH), 143.2 (C0), 164.9
(C0), 197.6 (C0). HRMS 336.17569. C18H28O4Si requires 336.17572.
Methyl (2R,3S,4S)-4-(tert-butyldimethylsilyloxy)-2-hydroxy-
3-methyl-4-phenylbutanoate, 8a. Colorless oil; [R]2D0 -32.4 (c
tR = 27.4min (S)-13; tR = 29.6 min (R)-13; IR (neat) ν max (cm-1
)
3489, 1738, 1685; MS m/z (%) 222 (Mþ), 149 (19), 120 (20), 105
(100), 77 (48); 1H NMR (300.1 MHz, CDCl3) δ 1.28 (t, 3H, J =
7.1 Hz), 3.50 (dd, 2H, J = 7.0), 4.30 (q, 2H, J = 7.1 Hz),
4.64-4.62 (m, 1H), 7.40-7.60 (m, 3H), 7.95 (d, 2H, J = 7.1
Hz); 13C NMR (75.0 MHz, CDCl3) δ 14.1 (CH3), 42.1 (CH2), 61.8
(CH2), 67.2 (CH), 128.1 (CH), 128.7 (CH), 128.8 (CH), 133.5 (C0),
173.8 (C0), 197.2 (C0).
0.60, MeOH); IR (neat) ν
(cm-1) 3530, 1738, 1410, 1253;
max
1
MS m/z (%) 163 (34), 115 (21), 107 (31), 91 (31), 75 (100); H
NMR (300.1 MHz, CDCl3) δ 0.29 (s, 9H), 1.12 (s, 6H), 1.26 (d,
3H, J = 7.0 Hz), 2.45 (m, 1H), 3.05 (d, 1H, J = 4.4 Hz), 4.08 (s,
3H), 4.86 (d, 1H, J = 8.4 Hz), 7.59 (m, 5H); 13C NMR (75.0
MHz, CDCl3) δ -4.5 (CH3), -3.9 (CH3), 10.7 (CH3), 26.4 (3
CH3), 53.0 (CH3), 71.8 (CH), 77.1 (CH), 127.4 (2 CH), 127.9
(CH), 128.5 (2 CH), 143.8 (C0), 175.7 (C0). HRMS 338.19134.
C18H30O4Si requires 338.19129.
Ethyl (R)-(-)-2-Hydroxy-3-methylene-4-oxophenylbutano-
ate, 14. The reaction was carried out under a nitrogen atmo-
sphere. The ketoester 13 (1.0 mmol) and a freshly prepared
solution of morpholine (0.0264 g, 0.3 mmol) in glacial acetic acid
(5.0 mL) were mixed in a 25 mL double-necked round-bottomed
˚
flask with a coiled reflux condenser flask. Molecular sieves (4A)
and p-formaldehyde (0.29 g, 9.0 mmol) were then added, and the
mixture was stirred and heated (reflux). After 2 h, the reaction
mixture was cooled to room temperature and quenched with
solid NaHCO3 followed by repeated (3 times) extractions with
equal volumes of ethyl acetate. The combined organic extracts
were washed with brine and water and dried over anhydrous
MgSO4 followed by evaporation under reduced pressure. The
product was purified by DSC chromatography (hexane/EtOac
(3R,4R,5S)-3-Hydroxy-4-methyl-5-phenyl-dihydrofuran-2-one,
11. A HF-pyridine solution (0.8 mL) was added to 8a (15 mg, 0.04
mmol) dissolved in anhydrous methylene chloride (5 mL), under
magnetic stirring, Ar atmosphere at 0 °C with gradual warming.
The reaction was monitored by GC-MS. The reaction was
neutralized with solid NaHCO3. The solvent was evaporated
and the residue purified by column chromatography (hex/EtOAc
15%) affording 11 in 65% yield as a colorless oil.15 IR (neat) ν max
70%) affording 14, a pale yellow oil,9a in 85% yield. [R]2D0
=
-21.0 (c 2.4, MeOH); IR (neat) ν max (cm-1) 3489, 1739, 1659,
(cm-1) 3362, 1776, 1455, 1096; MS m/z (%) 192 (Mþ), 148 (80),
1597; MS m/z (%) 234 (Mþ), 205 (6), 132 (15), 149 (16), 120 (5),
3
3
115 (10), 105 (30), 91 (100), 77 (25); 1H NMR (300.1 MHz,
CDCl3) δ 0.87 (d, 3H, J = 7.0 Hz), 2.75-2.96 (m, 1H), 4.22 (d,
1H, J = 9.9 Hz), 5.63 (d, 1H, J = 8.1 Hz), 7.13 (m, 2H),
7.31-7.43 (m, 3H), 7.38-7.45 (m, 2H); 13C NMR (75.0 MHz,
CDCl3) δ 13.3 (CH3); 42.1 (CH); 72.2 (CH); 82.4 (CH); 125.7
(CH); 128.5 (CH); 128.6 (CH); 135.5 (C0); 177.5 (C0).
105 (100), 77 (34); 1H NMR (300.1 MHz, CDCl3) δ 1.26 (t, 3H,
J = 8.0 Hz), 4.18 (q, 2H, J = 7.0), 5.34 (s, 1H), 5.82 (s, 1H), 6.19
(s, 1H), 7.40-7.60 (m, 3H), 7.79 (d, 2H, J = 7.0 Hz); 13C NMR
(75.0 MHz, CDCl3) δ 15.2 (CH3), 62.3 (CH2), 71.7 (CH), 127.3
(CH2), 128.4 (CH), 128.5 (2 CH), 129.6 (2 CH), 132.9 (C0), 144.9
(C0), 172.5 (C0), 197.2 (C0). HRMS 234.04671. C13H14O4
requires 234.08921.
Ethyl 2,4-Dioxo-4-phenylbutanoate, 12. A freshly prepared
sodium ethoxide solution (Na 0.02 mol in 10 mL of ethanol at
0 °C) was added to acetophenone (1.0 g, 0.009 mol) and
diethyloxalate (2.7 g, 0.02 mol) dissolved in anhydrous THF,
under a Ar atmosphere. This solution was stirred at 0 °C for 2 h
and quenched by addition of cooled 2 mol/L HCl (120 mL, 0 °C)
and extracted with EtOAc. The combined organic layers were
dried over MgSO4 and filtered, and the solvent was evaporated.
The residue, a yellow paste,17c was pure enough for further
Ethyl (2R)-2-Hydroxy-3-methyl-4-oxo-4-phenylbutanoate, 15.
(syn/anti mixture) (General procedure) Yeast medium (125 mL)
was autoclaved for 15 min at 121 °C and 1.5 atm. After cooling, 3
mL of yeast inoculum was added, and the reaction was kept at 30
°C in an orbital shaker for 48 h. Then, 14 (1 mmol) was dissolved
in ethanol (1 mL) and added to the growing medium. The
reaction was monitored by GC-MS. At the end of reaction,
the cells were centrifuged and the aqueous phase extracted with
EtOAc. The combined organic layers were dried with MgSO4,
and the solvent was evaporated affording a pale yellow oil.15 The
residue was purified by TLC (hexane/EtOAc 65%). HPLC
(Chiralcel OJ, hexane/iPrOH = 4:1, flow rate = 0.4 mL/min,
λ =254nm) tR=19.9min(2R,3R)-15; tR =21.4 min (2R,3S)-15;
IR (neat) ν max (cm-1) 3484, 1736, 1683, 1596; MS m/z (%) 236
manipulation, 92% yield. Mp 34-36 °C; IR (neat) ν max (cm-1
)
3125, 1742, 1600, 1615, 1451; MS m/z (%) 220 (Mþ), 147 (100),
3
105 (38), 120 (5), 77 (20); 1H NMR (300.1 MHz, CDCl3) δ 1.42
(t, 3H, J = 7.3 Hz), 4.40 (q, 2H, J = 6.9); 7.05 (s, 1H); 7.45-7.62
(m, 3H), 8.01 (d, 2H, J = 8.0 Hz); 13C NMR (75.0 MHz, CDCl3)
δ 14.4 (CH3), 63.1 (CH2), 98.4 (CH), 128.0 (CH), 129.0 (CH),
134.2 (CH), 135.3 (C0), 162.6 (C0), 170.3 (C0), 191.1 (C0).
Ethyl (R)-(-)-2-Hydroxy-4-oxo-4-phenylbutanoate, 13. Yeast
cells (20 g) were suspended in sodium citrate buffer (0.1 mol/L,
1
(Mþ), 163 (16), 105 (100), 77 (32); H NMR syn (300.1 MHz,
3
CDCl3) δ 1.26 (t, 3H, J = 7.0 Hz), 1.29 (d, 3H, J = 7.0 Hz), 3.28
(s, 1H), 3.93 (dq, 1H, J = 4.2 and 7.0 Hz,), 4.25 (q, 2H, J = 7.0
J. Org. Chem. Vol. 75, No. 5, 2010 1417

Milagre et al.
JOCArticle
Hz), 4.58 (m, 1H), 7.40-7.65 (m, 3H), 7.90-8.05 (m, 2H); anti δ
1.20 (t, 3H, J = 7.1 Hz), 1.36 (d, 3H, J = 7.3 Hz), 3.61 (d, 1H,
OH, J = 8.3 Hz), 3.98 (dq, 1H, J = 4.6 and 7.3 Hz,), 4.10-4.25
(m, 2H), 4.39 (dd, 1H, J = 4.6 and 8.3 Hz), 7.40-7.65 (m, 3H),
7.90-8.00 (m, 2H); 13C NMR syn (75.0 MHz, CDCl3) δ 12.1
(CH3), 14.0 (CH3), 44.3 (CH2), 61.9 (CH), 71.6 (CH), 128.4 (CH),
128.7 (CH), 133.3 (CH), 135.7 (C0), 173.1 (C0), 201.6 (C0); anti δ
14.0 (CH3), 14.1 (CH3), 44.0 (CH2), 61.5 (CH), 73.1 (CH), 128.3
(CH), 128.7 (CH), 133.4 (CH), 135.9 (C0), 173.1 (C0), 201.6 (C0).
(3R,4S,5R)-3-Hydroxy-4-methyl-5-phenyl-dihydrofuran-2-one, 18.
(cis/trans mixture) Compound 15 (1.0 mmol) was dissolved in
anhydrous ethanol (2 mL) under magnetic stirring and cooled to
-78 °C. Then, nBu4NBH4 (1.0 mmol) was slowly added, and the
temperature was increased to -23 °C. After 1 h, acetone (5 mL)
and saturated NaHCO3 solution were added. The aqueous
phase was extracted with EtOAc (3 ꢀ 10 mL), and the com-
bined organic layers dried over MgSO4 and concentrated under
vacuum. The residue was purified by TLC (45% hexane/
EtOAc) affording the diastereoisomeric mixture (3R,4S,5R)-
18 (white solid) (3R,4R,5R)-11 (colorless oil) in 98:2 ratio. The
relative stereochemistries of 11 and 18 were determined by 1H
NMR, NOESY1D and literature comparison.15 White solid,
MP 150-151 °C; IR (neat) ν max (cm-1) 3443, 1758, 1414, 1294,;
MHz, CDCl3) δ 7.4 (CH3), 41.1 (CH); 72.1 (CH); 80.2 (CH); 125.2
(CH); 128.2 (CH); 128.6 (CH); 135.1 (C0); 177.0 (C0).
(3R,4S,5R,20S)-4-Methyl-2-oxo-5-phenyl-tetrahydrofuran-3-yl
(20-methoxy-20-phenylacetate), 19. Anhydrous methylene chlor-
ide (1.5 mL) was added to 18 (10 mg, 0.05 mmol), and then
DMAP and (S)-MPA (4 mg, 0.05 mmol) were added to this
solution. The mixture was cooled to 0 °C, and DCC (10 mg, 0.005
mmol) was added. The reaction was maintained under magnetic
stirringwithgradual warming. After 28 h the mixturewas filtered,
and the solvent evaporated. The residue was purified by column
chromatography (20% hexane/EtOAc) and yielded a white
solid.15 IR (neat) ν
(cm-1) 3445, 1798, 1756, 1455; MS m/z
max
(%) 121 (100), 122 (9), 105 (9), 91 (12), 77 (19). 1H NMR (300.1
MHz, CDCl3) δ 0.10 (d, 3H, J = 7.3 Hz), 2.96-3.02 (m, 1H), 3.45
(s, 3H), 4.96 (s, 1H), 5.55 (d, 1H, J = 5.1 Hz), 5.77 (d, 1H, J = 6.9
Hz), 7.25-7.45 (m, 10H).
Acknowledgment. The authors thank the Brazilian science
foundations FAPESP, CAPES, CNPq and FUNCAMP-
UNICAMP for their financial support. We also thank Prof.
Carol Collins for reviewing the manuscript.
MS m/z (%) 192 (Mþ), 148 (80) 133 (15), 115 (10), 105 (30), 91
3
(100), 77 (25); 1H NMR (300.1 MHz, CDCl3) δ 0.65 (d, 3H, J =
7.3 Hz), 2.75 (s, 1H), 2.98-3.08 (m, 1H), 4.79 (d, 1H, J = 6.8 Hz),
5.57 (d, 1H, J = 4.6 Hz), 7.25-7.38 (m, 5H); 13C NMR (75.0
Supporting Information Available: General experimental
and compound characterization data. This material is available
free of charge via the Internet at http://pubs.acs.org.
1418 J. Org. Chem. Vol. 75, No. 5, 2010