Saturday, March 31, 2012

Development of an efficient, scalable, aldolase-catalyzed process for enantioselective synthesis of statin intermediates























Development of an efficient, scalable,
aldolase-catalyzed process for enantioselective synthesis of statin
intermediates


  1. William
    A. Greenberg
    *,
  2. Alexander
    Varvak
    ,
  3. Sarah
    R. Hanson
    ,
  4. Kelvin
    Wong
    ,
  5. Hongjun
    Huang
    ,
  6. Pei
    Chen
    , and
  7. Mark
    J. Burk
    *


- Author Affiliations


  1. Diversa Corporation, 4955 Directors Place, San Diego,
    CA 92121

  1. Edited by Barry M. Trost, Stanford University,
    Stanford, CA, and approved February 19, 2004 (received for review November
    14, 2003)

 


Next Section


Abstract


A process is reported for efficient,
enantioselective production of key intermediates for the common chiral side
chain of statin-type cholesterol-lowering drugs such as Lipitor (atorvastatin)
and Crestor (rosuvastatin). The process features a one-pot tandem aldol
reaction catalyzed by a deoxyribose-5-phosphate aldolase (DERA) to form a
6-carbon intermediate with installation of two stereogenic centers from
2-carbon starting materials. An improvement of almost 400-fold in volumetric
productivity relative to the published enzymatic reaction conditions has been
achieved, resulting in a commercially attractive process that has been run on
up to a 100-g scale in a single batch at a rate of 30.6 g/liter per h. Catalyst
load has been improved by 10-fold as well, from 20 to 2.0 wt % DERA. These
improvements were achieved by a combination of discovery from environmental DNA
of DERAs with improved activity and reaction optimization to overcome substrate
inhibition. The two stereogenic centers are set by DERA with enantiomeric
excess at >99.9% and diastereomeric excess at 96.6%. In addition,
down-stream chemical steps have been developed to convert the enzymatic product
efficiently to versatile intermediates applicable to preparation of
atorvastatin and rosuvastatin.


Biocatalysis has received attention
recently as a tool of enormous potential for the synthesis of pharmaceutical,
industrial, and agricultural chemicals and intermediates (15). To capitalize
further on this potential, enzymes must demonstrate not only superior
performance characteristics such as high enantioselectivity and low catalyst
loading but also cost-effectiveness and scalability. A successful industrial
biocatalytic process requires enzymes that can be produced cheaply in kilogram
quantities and are stable and active at very high substrate and product
concentrations, frequently in the presence of organic solvents. Although many
remarkable biocatalytic transformations have been reported on the
milligram-to-gram scale, especially in light of recent advances in directed
evolution and high-throughput screening for novel enzymes (68), the number of
processes that have been applied on an industrial scale remains limited (5, 9). This is
especially true for biocatalytic processes involving formation of carbon–carbon
bonds.


The aldol reaction is an extremely
useful transformation that allows the formation of new carbon–carbon bonds and
the introduction of up to two new stereocenters into the product. Controlling
the stereochemistry of aldol reactions can be challenging and has been an area
of intense research. Although several elegant asymmetric methods have been
developed (1012), there
remains a need for additional methods, and in recent years enzyme-catalyzed
approaches using aldolases have received attention as alternatives to chemical
methodologies (13).


Several years ago, Wong and
coworkers (14,
15) reported
unprecedented one-pot tandem aldol reactions catalyzed by a
deoxyribose-5-phosphate aldolase (DERA), in which 2 eq of acetaldehyde were
added in sequence to 2-carbon aldehyde acceptors to afford six-membered lactol
derivatives (Fig. 1).
Because the DERA-catalyzed reaction is an equilibrium process, the intermediate
4-carbon adduct (Fig.
1
) is reversibly formed under the reaction conditions. The second condensation
between this intermediate and a second equivalent of acetaldehyde drives the
equilibrium favorably because of the stability of the cyclized lactol form of
the product. The authors noted the structural similarity of the enzymatic
products to the lactone moiety (and its interchangeable 3,5-dihydroxy acid
form) of the cholesterol-lowering 3-hydroxy3-methylglutaryl (HMG)-CoA reductase
inhibitors mevastatin and lovastatin. Today, HMG-CoA reductase inhibitors
(collectively known as statins) have worldwide sales of approximately $20
billion, led by Pfizer's Lipitor (atorvastatin), the world's top-selling drug.
Atorvastatin and AstraZeneca's newly approved Crestor (rosuvastatin) (Fig. 2) are
completely synthetic molecules, unlike the earlier generation of statins that
were fungal metabolites or semisynthetic derivatives thereof. The synthetic
statins share the chiral 3,5-dihydroxy acid side chain also found in the
natural products, which is essential for activity and represents the greatest
challenge for preparation of these drugs.


Fig. 1.


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Fig. 1.


DERA-catalyzed tandem aldol
reaction.


Fig. 2.


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Fig. 2.


Structures of Lipitor (atorvastatin)
and Crestor (rosuvastatin).


Although the published
DERA-catalyzed reaction offered the potential to greatly simplify the process
for synthesizing the chiral side chain of statins, several issues would limit
its practicability for large-scale production. First, the catalyst load was
very high, ≈200 mg of DERA per gram of isolated product (RGraphicCl),
or 20% (wt/wt). Use of such a high concentration of enzyme would make the
process prohibitively expensive, in addition to making isolation of product
from the reaction mixture difficult. Second, the reaction time was on the order
of several days. Third, the concentration of the limiting reagent,
chloroacetaldehyde, was only 100 mM. Taken together, the volumetric
productivity of the published process was 2 g/liter per day. Hence, in this
process, the value of building an advanced statin intermediate, with
establishment of both stereogenic centers from achiral 2-carbon starting
materials in a single reaction, would be offset on process scale by a very
large reaction volume, long reaction time, high catalyst concentration, and
laborious product isolation. In addition to these points, the
enantioselectivity and diastereoselectivity of the DERA-catalyzed reaction was
unknown.


We addressed these limitations by (i)
discovering an improved DERA by screening genomic libraries prepared from
environmental DNA and (ii) developing a fed-batch reaction process to
overcome significant substrate inhibition. In addition, an inexpensive
oxidation method has been developed to convert the enzymatic lactol product
into (3R,5S)-6-chloro-2,4,6-trideoxy-erythro-hexonolactone
(1). The crude lactone was determined to have an enantiomeric excess
(ee) of >99.9% and diastereomeric excess (de) of 96.6%. After
crystallization, the lactone had an ee of >99.9% and de of 99.8%. Compound 1

was functionalized efficiently to provide side-chain intermediates for
synthesis of a variety of statins.


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Section
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Materials
and Methods


General. All reagents were purchased and used without additional
purification. Reactions were monitored by analytical TLC on silica gel 60 F254
plates and visualized by staining with acidic ceric ammonium molybdate or
potassium permanganate. 1H and 13C NMR spectra were
obtained on a Bruker (Billerica, MA) 500-MHz UltraShield spectrometer.
Fed-batch reactions were performed on a Dasgip (Juelich, Germany) Fedbatch-pro
system. GC was performed on an Agilent 6890 gas chromatograph.


Improved DERA. The improved DERA was discovered by high-throughput
screening of environmental DNA libraries, as described in Results and
Discussion
. The protein sequence of this aldolase is MNIAKMIDHTLLKPEATEQQIVQLCTEAKQYGFA
AVCVNP TWVKTA AR ELSGTDVRVCTVIGFPLGAT T PETKAFET TNA IENGAR EVDMVINIGA
LKSGQDELVERDIR AVVEA A AGR A LVKVI VETA LLTD EEKVR
ACQLAVKAGADYVKTSTGFSGGGATVEDVA LMRKTVGDR AGVKASGGVRDWKTAEA
MINAGATRIGTSSGVAIVTGGTGRADY.


Synthesis. The synthetic procedures described below are illustrated in
Figs. 3, 4, 5, 6.


Fig. 3.


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Fig. 3.


Synthesis of lactone (1).
Reagents and conditions: a, DERA, H2O, room temperature, 3 h; b,
NaOCl, HOAc, H2O, room temperature, 3 h.


Fig. 4.


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Fig. 4.


Reagents and conditions: a, NaCN,
DMF, 5% H2O, 40°C, 16 h; b, dimethoxypropane, DMF, catalytic H2SO4,
then trimethylsilyldiazomethane; c, NaOH, H2O, 40°C, 16 h.


Fig. 5.


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Fig. 5.


Preparation of chiral standards for
analysis of 1. Reagents and conditions: a, NaBH4, MeOH; b,
15% trifluoroacetic acid, CH2Cl2, 1% triethylsilane.


Fig. 6.


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Fig. 6.


Synthesis and retro-aldol reaction
of fluorogenic DERA substrate. a, 4-methylumbelliferone, K2CO3,
DMF, 75°C, 16 h; b, H2O/20% CH3CN, Dowex 50WX8-100, 2
days.


(3R,5S)-6-Chloro-2,4,6-trideoxy-erythro-hexose
(2).
Lyophilized
crude DERA lysate (1.3 g of powder, 220 mg of pure DERA) was dissolved in 70 ml
of water. An aqueous solution containing 1.5 M chloroacetaldehyde and 3.1 M
acetaldehyde was fed into the stirred enzyme solution at a rate of 16.66 ml/h
for 3 h, resulting in a final volume of 120 ml. Acetone (200 ml) was added to
precipitate protein, and the mixture was filtered through Celite. The filtrate
was concentrated under reduced pressure to a volume of 50 ml and extracted
three times with 75 ml of ethyl acetate. The organic fraction was dried over
anhydrous sodium sulfate and concentrated under reduced pressure to provide
crude lactol (11.12 g) as a light-yellow oil. The crude product was carried on
to the oxidation step without additional purification.


(3R,5S)-6-Chloro-2,4,6-trideoxy-erythro-hexonolactone
(1).
Crude
lactol (2) (75.0 g, ≈0.45 mol) was dissolved in glacial acetic
acid (600 ml). Aqueous sodium hypochlorite (265 ml of a 13% solution) was added
dropwise to the solution over 3 h. The reaction mixture was concentrated under
reduced pressure to a volume of 200 ml, neutralized by addition of solid sodium
bicarbonate, and extracted twice with 500 ml of ethyl acetate. The organic fraction
was dried over sodium sulfate, concentrated to dryness under reduced pressure,
and dissolved in 350 ml of chloroform. The solution was cooled slowly to –20°C,
and after 12 h, crystalline product was collected, washed with cold chloroform,
and dried under vacuum. Lactone 1 was obtained as white crystals (44.0
g, 0.267 mol, 45% over two steps): 1H NMR (500 MHz) CDCl3
δ 5.01 (1H, m), 4.47 (1H, m), 3.80 (1H, m), 3.68 (1H, m), 2.69 (2H, d, J

= 3.6 Hz), 2.09 (1H, m), 1.97 (1H, m). 13C NMR (125 MHz) CDCl3
δ 170.15, 74.81, 62.50, 46.55, 38.54, 32.77.


(4R,6R)-[6-Cyanomethyl-2,2-dimethyl(1,3)dioxan-4-yl]acetic
acid methyl ester (3).

Lactone 1 (10.0 g, 60.7 mmol)
was dissolved in 200 ml of dimethylformamide (DMF). Sodium cyanide (8.93 g, 182
mmol) was dissolved in 10 ml of water. The aqueous cyanide solution was added
to the DMF solution of chlorolactone. The mixture was stirred at 40°C for 16 h.
At the 4-h time point, NMR analysis indicated the presence of chloro diol
carboxylate, an epoxide intermediate, and cyano diol carboxylate. After 16 h,
the nitrile was the major product. The crude product mixture was partially
concentrated under reduced pressure to remove the water (to a final volume of
≈100 ml). It then was acidified carefully to pH ≈ 3 with
concentrated sulfuric acid (≈3 ml) and purged for 1 h with nitrogen gas.
Dimethoxypropane (21.2 g, 203.3 mmol) was added, and the mixture was stirred at
room temperature for 2 h. The solution then was titrated with
trimethylsilyldiazomethane (2 M solution in hexane) until bubbling was no
longer observed and the final pH was ≈7. Excess
trimethylsilyldiazomethane was quenched with 0.1 ml of acetic acid. Water (200
ml) was added, and the product was extracted with 200 ml of ethyl acetate. The
organic layer was back-extracted with 100 ml of water and then dried over
sodium sulfate and concentrated under reduced pressure. The residue was
purified on silica, eluting with 4:1 hexane/ethyl acetate, yielding 6.52 g of
light-yellow oil (48% over 3 steps from chlorolactone 1). Epoxide
intermediate: 1H NMR (sodium salt) (500 MHz) 2H2O

δ 3.87 (1H, m), 2.88 (1H, m), 2.58 (1H, app. t, J = 4.2 Hz), 2.34
(1H, dd, J 1 = 3.1 Hz, J 2 = 2.8 Hz), 2.09
(2H, d, J = 6.7 Hz), 1.47 (1H, dt, J 1 = 9.5 Hz, J

2 = 5.0 Hz), 1.39 (1H, m). Compound 3: 1H NMR (500
MHz) CDCl3 δ 4.35 (1H, m), 4.17 (1H, m), 3.70 (3H, s), 2.59
(1H, dd, J 1 = 15.8 Hz, J 2 = 6.9 Hz), 2.51
(2H, m), 2.43 (1H, dd, J 1 = 15.8 Hz, J 2 =
6.1 Hz), 1.78 (1H, dt, J 1 = 12.6 Hz, J 2 =
2.5 Hz), 1.48 (3H, s), 1.39 (3H, s), 1.33 (1H, dd, J 1 = 18.3
Hz, J 2 = 11.6 Hz); 13C NMR (125 MHz) CDCl3

δ 171.22, 116.92, 99.76, 65.56, 65.16, 51.97, 41.04, 35.58, 29.90, 25.12,
19.79.


(3R,5S)-3,5,6-Trihydroxyhexanoic
acid (4).
Lactone
1 (330 mg, 2 mmol) was dissolved in 5 ml of water. Sodium hydroxide (176 mg,
4.4 mmol) was added, and the mixture was stirred at 40°C for 16 h. The solvent
was removed under reduced pressure to provide a white solid, which was
dissolved in 2H2O for NMR analysis: 1H NMR
(sodium salt) (500 MHz) 2H2O δ 4.08 (1H, m), 3.79
(1H, m), 3.50 (1H, m), 3.41 (1H, m), 2.31 (2H, m), 1.52 (1H, m), 1.48 (1H, m); 13C
NMR (125 MHz) 2H2O δ 179.93, 70.41, 67.92, 67.19,
45.80, 41.52.


Chiral Analysis. Enantio- and diastereoselectivity of crude and
recrystallized lactone 1 were determined by chiral GC using β-Dex
225 + β-Dex 120 columns in series. A representative GC trace is shown in Fig. 7. A mixture of
all four possible diastereomers was prepared as illustrated in Fig. 5 and described
below.


Fig. 7.


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Fig. 7.


GC conditions: columns, β-Dex
225 (30 m × 0.25 mm × 0.25 μm) plus β-Dex 120 (30 m × 0.25 mm × 0.25
μm); oven temperature, 180°C; carrier gas He flow rate, 1.0 ml/min. (A)
Chromatogram of lactone 1 standard with an approximate ratio of (3R,5S)/(3S,5R)/(3R,5R)/(3S,5S)
= 1:1:3:3. (B) Chromatogram of actual sample from the chemoenzymatic
process with an analysis result of >99.94% ee and 96.60% de using the
indicated GC method. Peak 1, (3R,5R) or (3S,5S)
stereoisomer; peak 2, (3R,5R) or (3S,5S)
stereoisomer; peak 3, (3S,5R) stereoisomer; peak 4, (3R,5S)
stereoisomer.


6-Chloro-3,5-dioxohexanoic acid
tert-butyl ester (16)
(2.0 g, 8.5 mmol) was dissolved in 40 ml of methanol at 0°C. Sodium borohydride
(750 mg, 20 mmol) was added, and the mixture was stirred for 30 min at 0°C. The
solvent was removed under reduced pressure, and the residue was extracted
between 75 ml of ethyl acetate and 50 ml of water. The organic layer was dried
over anhydrous sodium sulfate and concentrated under reduced pressure. The
crude diol product (300 mg) was dissolved in 5 ml of 15% trifluoroacetic acid
in dichloromethane with 1% triethylsilane. The mixture was stirred at room
temperature for 1.5 h and then concentrated under reduced pressure. The residue
was extracted between 30 ml of ethyl acetate and 30 ml of concentrated aqueous
sodium bicarbonate. The organic layer was dried over sodium sulfate, concentrated,
and purified by silica gel chromatography, eluting with 3:1 ethyl
acetate/hexane, with partial separation of diastereomeric pairs of lactones.
Fraction A consisted of 52 mg of a 1:1 mixture, and fraction B consisted of 27
mg of a 1:3 mixture in favor of the 3R,5R/3S,5S
pair. Spectral data for the 3R,5S/3S,5R racemic
mixture: 1H NMR (500 MHz) CDCl3 δ 5.01 (1H, m), 4.47
(1H, m), 3.80 (1H, m), 3.68 (1H, m), 2.69 (2H, d, J = 3.6 Hz), 2.09 (1H,
m), 1.97 (1H, m); 13C NMR (125 MHz) CDCl3 δ 170.15,
74.81, 62.50, 46.55, 38.54, 32.77. Spectral data for the 3S,5S/3R,5R

racemic mixture: 1H NMR (500 MHz) CDCl3 δ 4.50 (1H,
m), 4.32 (1H, m), 3.72 (2H, dd, J 1 = 5.1 Hz, J 2
= 1.1 Hz), 2.92(1H, m), 2.53 (1H, m), 2.39 (1H, m), 1.83 (1H, m); 13C
NMR (125 MHz) CDCl3 δ 170.01, 76.24, 63.47, 45.81, 39.49,
34.95.


Fluorogenic DERA Substrate. The substrate was prepared from known methyl
5-toluenesulfonyl-2-deoxyriboside (17). The
toluenesulfonate (12.75 g, 42.2 mmol) was dissolved in 75 ml of DMF. To the
solution was added potassium carbonate (11.68 g, 84.5 mmol) and
4-methylumbelliferone (9.29 g, 52.8 mmol). The mixture was stirred at 75°C for
16 h. Water (300 ml) was added, and the mixture was extracted twice with 200 ml
of ethyl acetate. The organic layer was back-extracted with 100 ml of 0.1 M
aqueous sodium hydroxide, dried over sodium sulfate, and concentrated. The
crude product (9.05 g) was dissolved in 25 ml of acetonitrile and 100 ml of
water. Dowex 50WX8-100 (2.5 g) was added, and the mixture was stirred at room
temperature. After 1.5 h, the mixture was exposed to reduced pressure to remove
methanol and then returned to atmospheric pressure. After 2 days, the mixture was
filtered, concentrated under reduced pressure, and purified by silica gel
chromatography, eluting with a gradient from 100% ethyl acetate to 10%
acetone/ethyl acetate. The product was obtained as a white foam (5.31 g, 62%)
as a 1:1 mixture of anomers: 1H NMR (500 MHz) DMSO-d6
δ 7.70 (1H, m), 6.97 (2H, m), 6.32 (1H, m, 1α), 6.20 (1H, m,
1β), 6.19 (1H, m), 5.40 (1H, br), 5.17 (1H, br), 4.0–4.3 (4H, m), 2.38
(3H, s), 1.65–2.0 (2H, m); 13C NMR (125 MHz) CDCl3 δ

161.63, 160.12, 154.68, 153.35, 126.43, 113.18, 112.38, 111.17, 101.28, 97.86,
97.05, 82.93, 80.89, 70.93, 70.69, 70.56, 68.94, 42.11, 18.09.


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Section


Results
and Discussion


Discovery of an Improved DERA. To improve the commercial viability of the process, we
sought to discover and develop a DERA that was improved over the enzyme from Escherichia
coli
in two specific parameters: tolerance to high substrate concentrations
and reduced catalyst load. Thus, we desired an enzyme that would produce more
product per liter while at the same time using less enzyme than would be
required with E. coli DERA. In an effort to access the most diverse
range of enzymes that occur in nature, we create large genomic libraries by
extracting DNA directly from environmental samples collected around the world
from a variety of habitats (18). These
environmental genomic libraries are screened through a variety of
high-throughput methods including both fluorogenic activity-based screens and
sequence-based screens (19). Our
fluorescent DERA assay, based on technology developed by Reymond and coworkers
(20), uses a
fluorogenic substrate analog that undergoes a retro-aldol reaction in the
presence of a DERA (Fig.
6
). The resultant fragment spontaneously eliminates to produce fluorescent
4-methylumbelliferone. Using these methods, we discovered >15 DERAs that
exhibited activity in the title reaction. All these enzymes were overexpressed
and compared directly to the E. coli DERA in the commercially relevant
reaction (Fig. 1).
One enzyme was found to be superior, and results for this DERA are described in
the following section. Because it was discovered from large, mixed genomic
libraries created from environmental DNA, the source organism for the improved
DERA is unknown. The protein sequence has <30% identity to E. coli

DERA and has closest homology to putative DERAs from Gram-positive organisms.


Aldolase Reaction. As noted previously, the reported volumetric productivity
levels observed for the reaction between acetaldehyde and chloroacetaldehyde
using E. coli DERA were exceedingly low (2 g/liter per day). To address
this point, we further investigated the cause for limited volumetric
productivity as well as the high catalyst load requirement in this
transformation. Both the E. coli enzyme and our DERA were examined for
inhibition, and no effect was observed when acetaldehyde concentration was
increased when holding chloroacetaldehyde concentration constant. By contrast,
when attempts were made to increase the chloroacetaldehyde concentration, the
reaction was halted completely. Inhibition by chloroacetaldehyde, a potent
electrophile, was overcome by process improvements that entailed slow feeding
of the substrates over 3 h, at a constant ratio of 2:1
acetaldehyde/chloroacetaldehyde, into the reaction at a rate such that they
were consumed as fast as they were added. Thus, chloroacetaldehyde did not
reach inhibitory concentrations, and the reaction proceeded to completion. The
improved DERA was expressed on a scale of hundreds of grams, and directly
compared (with respect to catalyst load and volumetric productivity) with the E.
coli
enzyme under the same substrate feeding conditions.


We examined lactol productivity
levels that could be achieved under the improved process conditions. The E.
coli
DERA performed significantly better, producing a maximum of 76 g/liter
(456 mM) product over 3 h, with a minimum catalyst load of 4.8% (wt/wt). When
lower concentrations of E. coli DERA were used, much lower product
yields were achieved. Even greater performance was observed with our improved
DERA, which reached 93 g/liter (558 mM) product over 3 h, with a catalyst load
of only 2.0% (wt/wt). By discovering a DERA with improved activity and
substrate tolerance and converting from a batch reaction to a fed-batch
process, we were able to reach ≈7-fold higher product concentrations
(>0.5 M) while at the same time using less enzyme (<2 g DERA per liter,
or ≈75 μM), compared with the published process. Tolerance to even
higher aldehyde concentrations may be engineered into the aldolase by directed
evolution. Thus, higher concentrations of product could be achieved in the same
time while expending less than half as much enzyme, rendering the process
significantly more cost-effective.


Oxidation to Lactone ( 1

). The published procedure for oxidation of the enzymatic lactol
product to the lactone entailed reaction with bromine and barium carbonate (14, 21). Although
this process was effective on a laboratory scale, the expense and toxicity of
bromine would prohibit its use in a large-scale process. We identified a very
inexpensive method for the selective oxidation to lactone 1 by using
aqueous sodium hypochlorite and acetic acid. Lactone 1 is crystalline,
providing a readily scalable method for purification by recrystallization. This
process yielded a product of exceptional enantiomeric and diastereomeric
purity, as described in the following section.


Enantio- and Diastereoselectivity. Because the enzymatic product 2 exists as an
equilibrium mixture of α and β anomers, chiral analysis of the
enzymatic reaction was performed after oxidation to compound 1 to
simplify the process. Because two stereogenic centers are set in a single pot,
four isomers (two enantiomeric pairs of diastereomers) can be formed. Relative
to the desired (3R,5S) product, improper setting of the first, or
second, center leads to the diastereomeric (3R,5R) and (3S,5S)
isomers, respectively. Improper setting of both centers leads to the
enantiomeric (3S,5R) isomer, as illustrated in Fig. 8. Because both
centers are set in the same reaction, exquisite enantioselectivity is observed.
Each of the two individual centers is set with 98–99% ee, thus the likelihood
that the undesired stereochemistry is formed at both centers on the same
molecule is vanishingly small. For instance, if the first center is set
improperly, it is most likely that the second center will be set properly,
leading to the diastereomeric (3S,5S) product. The two
diastereomeric products were observed as a combined 1.7% of the total crude
lactone concentration, and the enantiomeric (3S,5R) isomer was
observed in <0.05%, giving a de of 96.6% and ee of >99.9%. The
diastereomeric impurities were separated effectively from the desired product
by recrystallization, providing 1 with an ee of >99.9% and de of
99.8%. Diastereoselectivity may be improved further by performing directed
evolution on the aldolase.


Fig. 8.


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Fig. 8.


Enantio- and diastereoselectivity of
the DERA-catalyzed process.


Conversion of 1 to Statin
Intermediates.
Although the DERA-catalyzed process
economically provided highly pure 1 in only two steps, it remained to
convert this compound to desired statin intermediates by converting the
chloride to the relevant functionality (e.g., a nitrile for atorvastatin and
hydroxyl group for rosuvastatin). This process entailed opening the lactone
ring of 1 with appropriate nucleophiles and introducing suitable protecting
groups. For example, triol acid 4 was formed cleanly and quantitatively
after treatment of lactone 1 with aqueous NaOH at 45°C over 16 h (Fig. 4).
Furthermore, when 1 was stirred at 45°C in wet DMF with 3 eq of NaCN (Fig. 9), the lactone
was hydrolyzed within minutes to the chloro diol intermediate 5. This
intermediate was consumed over several hours, and epoxide intermediate 6

concomitantly increased in concentration, as revealed by 1H and 13C
NMR spectroscopy. The epoxide subsequently was opened regioselectively by
cyanide, with complete conversion in 16 h. At the 4-h time point, the chloro
diol, epoxide, and cyano diol were present in approximately equimolar
quantities. It should be noted that epoxide 6 also was observed as an
intermediate during the basic hydrolysis of lactone 1 to afford the
triol acid 4.


Fig. 9.


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Fig. 9.


Cyanide displacement through an
epoxide intermediate.


Protection of the cyano diol 7
in a two-step, single-pot process provided intermediate 3 in 48% overall
yield from 1. It should be noted that these are preliminary results, and
there is potential for additional optimization of the chemical steps that follow
the DERA-catalyzed process. Likewise, it should be possible to further improve
the enzyme's specific activity and tolerance of high aldehyde concentrations in
the DERA process by directed evolution (8). The complete
process to the protected, fully elaborated, enantiomerically pure atorvastatin
side chain intermediate 3 entailed only five steps from
chloroacetaldehyde and acetaldehyde.


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Conclusions


By a combination of discovery of
superior DERAs from environmental DNA libraries and process improvements to
overcome substrate inhibition, we developed a highly efficient, cost-effective,
scalable, and enantioselective biocatalytic process. The aldolase-catalyzed
step proceeded with an ee of >99.9% and de of 96.6%. After oxidation,
crystalline lactone 1 was obtained with an ee of >99.9% and de of
99.8%. The volumetric productivity of the enzymatic process, which has been
performed on a 100-g scale, was improved to 30.6 g/liter per h (from 0.08
g/liter per h for a published process). Downstream chemical transformations
have been developed to convert the enzymatic product to key statin
intermediates. The complete process, integrating a key biocatalytic step
forming two carbon–carbon bonds stereoselectively with subsequent chemical
transformations, represents a general, practical, and economical route to
statins.


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Acknowledgments


We thank C. Abulencia, D. Wyborski,
A. Milan, and M. Miller for enzyme discovery; J. Verruto and X. Tan for
subcloning; T. Kaneko and J. Patel for fermentation; T. Richardson and M. Podar
for bioinformatics support; and J. Gemsch and L. Bibbs for sequencing.


Previous SectionNext Section


Footnotes




  • * To whom correspondence may be addressed. E-mail: wgreenberg@diversa.com or mburk@diversa.com.
  • This paper was submitted directly (Track II) to the
    PNAS office.
  • Abbreviations: DERA, deoxyribose-5-phosphate aldolase;
    ee, enantiomeric excess; de, diastereomeric excess; DMF,
    dimethylformamide.
  • Copyright © 2004, The National Academy of Sciences

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