www.rsc.org/greenchem Green Chemistry A green-by-design biocatalytic process for atorvastatin intermediate
Steven K. Ma,a John Gruber,a Chris Davis,a Lisa Newman,a David Gray,a Alica Wang,a John Grate,a
Gjalt W. Huisman*a and Roger A. Sheldon*b
Received 18th June 2009, Accepted 10th September 2009
First published as an Advance Article on the web 23rd October 2009
DOI: 10.1039/b919115c
The development of a green-by-design, two-step, three-enzyme process for the synthesis of a keyintermediate in the manufacture of atorvastatin, the active ingredient of the cholesterol loweringdrug Lipitor R , is described. The first step involves the biocatalytic reduction of ethyl-4-chloroacetoacetate using a ketoreductase (KRED) in combination with glucose and aNADP-dependent glucose dehydrogenase (GDH) for cofactor regeneration. The (S)ethyl-4-chloro-3-hydroxybutyrate product is obtained in 96% isolated yield and >99.5% e.e. In thesecond step, a halohydrin dehalogenase (HHDH) is employed to catalyse the replacement of thechloro substituent with cyano by reaction with HCN at neutral pH and ambient temperature. Thenatural enzymes were highly selective but exhibited productivities that were insufficient for largescale application. Consequently, in vitro enzyme evolution using gene shuffling technologies wasemployed to optimise their performance according to predefined criteria and process parameters.
In the case of the HHDH reaction, this afforded a 2500-fold improvement in the volumetricproductivity per biocatalyst loading. This enabled the economical and environmentally attractiveproduction of the key hydroxynitrile intermediate. The overall process has an E factor (kg wasteper kg product) of 5.8 when process water is not included, and 18 if included.
in all commercialized syntheses of atorvastatin is ethyl (R)-4-
cyano-3-hydroxybutyrate 1, a.k.a. "hydroxynitrile" (see Fig. 2).
Atorvastatin calcium is the active ingredient of Lipitor R In the innovator's original commercial process for atorvastatin, first drug in the world with annual sales to exceed $10 billion.
the second stereogenic center in atorvastatin (at the 3-hydroxy Lipitor is a cholesterol-lowering drug, a member of the statin group) is set by diastereomeric induction, using cryogenic Downloaded by University of Calgary on 30 September 2011 family of so-called HMG-CoA reductase inhibitors that block borohydride reduction of a boronate derivative of the 5-hydroxy- Published on 23 October 2009 on http://pubs.rsc.org doi:10.1039/B919115C the synthesis of cholesterol in the liver.1 The structure of
3-keto intermediate 2 derived from 1.2
atorvastatin calcium is shown in Fig. 1. In common with other Previous routes that have been used for the industrial pro- statins, it comprises a chiral 3,5-dihydroxy carboxylate side chain duction of 1 are depicted in Fig. 3. Early routes involved
attached to a cyclic nucleus. Atorvastatin is unique in having the kinetic resolutions using microbes, and the use of (S)-hydroxy side chain attached to nitrogen in the nucleus.
butyrolactone produced from chiral pool raw materials, lactose
or malic acid. Later routes have involved asymmetric reduction
of ethyl 4-chloroacetoacetate, produced from diketene, using
an asymmetric hydrogenation catalyst, microbial cells, or an
enzyme. Alternative chemoenzymatic routes have been de-
scribed, using nitrilases3 or lipases4 but they have not, to
our knowledge, been commercialized. These processes tend
to require high enzyme loadings,1 making product recovery
difficult5 and adding significant costs to the process.
Structure of atorvastatin calcium.
The final step to 1 in all of the previous commercial processes
Atorvastatin's high volume demand coupled with the re- involves reaction of an ethyl 3-hydroxy-4-halobutyrate (a.k.a.
quirement for high chemical and optical purity has led to "halohydrin") with a cyanide ion in alkaline solution at elevated extensive efforts towards more economic production of its temperatures to form the hydroxynitrile 1. Alkaline conditions
chirality-setting intermediates.1 The key chiral building block
are necessary to form the nucleophilic cyanide anion (the pKaof HCN is about 9). However, the substrate and product arebase-sensitive compounds, resulting in extensive by-productformation. In one report, the reaction with the chlorohydrin aCodexis, Inc, 200 Penobscot Drive, Redwood City, CA, 94603, USA. was conducted at around 80 C and pH 10 with a reaction yield
of 85%.6 The product is a high-boiling oil and the by-products
bDelft University of Technology, Department of Biotechnology,Julianalaan 136, 2628, BL, Delft, Netherlands. include many which are close in boiling point.6 As a result,
a troublesome high-vacuum fractional distillation is required This journal is The Royal Society of Chemistry 2010 Green Chem., 2010, 12, 81–86 81
Synthetic route for atorvastatin calcium.
Downloaded by University of Calgary on 30 September 2011 Published on 23 October 2009 on http://pubs.rsc.org doi:10.1039/B919115C Previous routes to the hydroxynitrile intermediate 1.
to recover product of acceptable quality, which results in still led us to propose the two-step, three-enzyme process for the further yield loss and waste.6
production of 1 shown in Fig. 4. The key step is the HHDH
Annual demand for 1 is estimated to be in excess of 100 mT,
catalysed reaction of the halohydrin 4 with HCN at neutral pH
making it highly desirable to reduce the wastes and hazards to produce 1.
involved in its manufacture, while reducing its cost and main- In addition to being highly efficient catalysts that typically taining or, preferably, improving its quality.
exhibit exquisite chemo-, regio-, and stereoselectivities, enzymes It is difficult to imagine a practical process for 1 that does
have many benefits from the viewpoint of developing green, not involve using cyanide, and we are not aware of any that has sustainable processes.13 They operate under mild conditions
been proposed. We reasoned that the key to providing a superior (ambient temperature and pressure as well as neutral pH), with process for 1 would be to find and develop a catalyst capable of
water as the preferred reaction medium. They are produced from accomplishing the cyanation reaction under mild conditions at renewable raw materials and are nontoxic and biodegradable.
neutral pH, so that by-product formation would be minimized.
Enzymes often provide the opportunity to reduce the number of Enzymes are known that catalyse the enantioselective ring- chemical steps in a synthetic process as there is no need for func- closing elimination of halohydrins to the corresponding epoxide tional group protection and deprotection. Finally, enzymatic in a reversible reaction.7 These enzymes are often referred to as
reactions are typically performed in standard multi-purpose halohydrin dehalogenases (HHDHs). It is also known, from the manufacturing plants. Notwithstanding all these advantages, the work of Nakamura and co-workers8,9 that these HHDHs accept
use of enzymes as catalysts in the large-scale production of fine cyanide as a non-natural nucleophile leading to the irreversible chemicals and pharmaceuticals had been hampered by perceived enantioselective formation of b-hydroxynitriles, under neutral, intrinsic limitations of natural enzymes.14,15 Such limitations
ambient conditions. More recently, this finding was further include insufficient activity towards non-natural substrates, elaborated and extended to other non-natural nucleophiles by insufficient activity at high substrate loadings due to substrate Janssen and co-workers.10–12 The availability of this enzyme
and/or product inhibition, and low operational stability under 82 Green Chem., 2010, 12, 81–86
This journal is The Royal Society of Chemistry 2010 Two-step, three-enzyme process for hydroxynitrile 1.
such economically viable conditions. While process engineering only 85%. To enable a practical large-scale process, the enzyme solutions might alleviate some of these problems,16 the optimal
loadings needed to be drastically reduced.
solution is to employ in vitro directed evolution technologies to DNA shuffling technology20 was used to improve the activity
generate enzymes that operate effectively with the desired sub- and stability of KRED and GDH while maintaining the nearly strate and under the desired, more practical process conditions;17
perfect enantioselectivity exhibited by the natural KRED. DNA instead of following the traditional path of compromising the shuffling involves the mutation of a gene encoding the enzyme process to fit the available catalyst, the catalyst is evolved to fit of interest to generate "libraries" of mutants. These libraries are the desired process. This enables the development of a process screened in high throughput, under conditions that approximate that is ‘green by design'.
the desired manufacturing process, and improved mutants are Herein, we describe a novel, economically viable and envi- selected for further evolution. Their genes are recombined ronmentally attractive process for the large-scale synthesis of 1
in vitro to create the next generation of libraries to be screened that includes the laboratory evolution of three enzymes to meet for further improved progeny. The gene libraries are transformed pre-defined process parameters. Details of the enzyme evolution and expressed in a host organism, such as Escherichia coli, and aspects of the HHDH enzyme have been reported elsewhere.18
screened in high throughput for enzyme variants that exhibitthe desired improved properties. DNA shuffling is becoming Results and discussion
a proven method to efficiently improve properties of enzymes,such as specific activity, stereoselectivity, optimum pH, organic In the process,19 a ketoreductase (KRED) and glucose dehy-
solvent tolerance, and stability.21
drogenase (GDH) are used for the enantioselective reduction Through several generations of such DNA shuffling, GDH of ethyl 4-chloroacetoacetate (3), using glucose as reductant,
activity was improved by a factor of 13 and KRED activity by to ethyl (S)-4-chloro-3-hydroxybutyrate (4). Glucose is oxidized
a factor of 7. The enantioselectivity of the improved KRED to gluconic acid, which is neutralized by sodium hydroxide via remained >99.5%. With the improved enzymes, the reaction a pH-stat. Subsequent to its recovery, 4 is converted to 1 by
was complete in 8 h with an increased loading of 3 to 160 g L-1,
Downloaded by University of Calgary on 30 September 2011 reaction with HCN (pK 9) at neutral pH, catalyzed by a reduced KRED loading to 0.57 g L-1, and lowered GDH loading Published on 23 October 2009 on http://pubs.rsc.org doi:10.1039/B919115C halohydrin dehalogenase (HHDH). This reaction releases the to 0.38 g L-1 (Table 1). With a 9.5¥ lowering of the enzyme H+ and Cl-, and must also be pH-statted, which is achieved loading there were no emulsion problems. Phase separation using aqueous NaCN as the base and source of CN-.
required less than one minute and provided 4 in >95% recovered
The activities of natural KRED and GDH for the reduction yield of >99.9% e.e.
of 2 to 3 were low (Table 1). With 100 g L-1 of 3, a total of 9 g L-1
The activity improvement for the ketone reduction biocata- natural, recombinantly produced KRED (6 g L-1) and GDH lysts over the course of evolution compared with the wild type (3 g L-1) were required to complete the reaction in 15 h. However, enzyme is shown in Fig. 5.
due to emulsion formation, recovery of 4 was problematic.
Similarly, the activity of natural HHDH for cyanation of 4
Although the analytical yield of 4 was >99%, the recovered
to 1 (Fig. 6) was extremely low and the enzyme showed poor
yield, after an hour to allow for some emulsion separation, was stability in the presence of the substrate and product. With
20 g L-1 of 4 and 30 g L-1 of recombinantly-produced natural
Evolution of a KRED/GDH biocatalyst for reduction of 3
HHDH, the rate of reaction had virtually approached 0 by 72 h.
Product recovery was difficult due to work-up challenges inthe filtration and phase separation steps caused by the large amount of enzyme. In addition to the extremely low activityand poor stability of the natural HHDH for the cyanation Substrate loading/g L-1 of 4, the enzyme was also strongly inhibited by the product.
Since 1 is more water-soluble than the substrate 4, attempts to
Biocatalyst loading/g L-1 overcome the inhibition by extraction of product into an organic Chemical purity/% >98 (GC) solvent such as EtOAc, n-BuOAc, or MTBE were unsuccessful.
E.e. of 4/%
However, after many iterative rounds of DNA shuffling, with Phase separation of organic product phase from aqueous screening in the presence of iteratively higher concentrations of phase containing enzyme/min product, the inhibition was largely overcome and the HHDH Space–time yield/gproduct L-1 d-1 activity was increased >2500-fold compared to the wild-type Catalyst yield (gproduct/gcat) This journal is The Royal Society of Chemistry 2010 Green Chem., 2010, 12, 81–86 83

Improvements in the biocatalysts' performances for the reduction of 3. The numbers identifying the enzymes represent the number of
generations of evolution. The KREDs were exposed to thermal challenges before their assay as indicated in the figure.
The green features of the new process
As noted above, previous commercial processes involved kinetic
resolution (50% maximum yield), syntheses from chiral pool
precursors involving bromine chemistry or asymmetric hydro-
genation of 3. All of these processes ultimately involve substitu-
tion of halide by cyanide under warm, alkaline conditions (e.g.
80 C, pH 10), causing extensive by-product formation and the
concomitant need for high-vacuum fractional distillation. The
process described here is greener than previous processes when
HHDH-catalyzed cyanation of 4.
assessed according to the twelve principles of green chemistry.22
Principle 1: waste prevention. The highly selective biocatalytic Fig. 7 shows the progress of cyanation reactions, using HHDH reactions afforded a substantial reduction in waste. In the final catalysts spanning multiple generations of directed evolution.
process, raw material is converted to product with >90% isolated With the improved HHDH, the reaction of 140 g L-1 4 using
yield affording product that is more than 98% chemically pure 1.2 g L-1 enzyme was complete in 5 h (Table 2). The product was with an enantiomeric excess of > 99.9%. Furthermore, the Downloaded by University of Calgary on 30 September 2011 isolated in 92% yield.
avoidance of alkaline-induced by-products obviates the needfor further yield-sacrificing fractional distillation. The highly Published on 23 October 2009 on http://pubs.rsc.org doi:10.1039/B919115C active evolved biocatalysts are used at such low loadings that
countercurrent extraction can be used to minimize solvent
volumes. Moreover, the butyl acetate solvent is recycled with an
efficiency of 85%. The E factor (kg waste per kg product)23 for
the overall process is 5.8 if process water is excluded (2.3 for the
reduction and 3.5 for the cyanation). If process water is included,
the E factor for the whole process is 18 (6.6 for the reduction and
11.4 for the cyanation). The main contributors to the E Factor,
as shown in Table 3, are solvent (EtOAc and BuOAc) losses
which constitute 51% of the waste, sodium gluconate (25%),
NaCl and Na SO (combined ca. 22%). The three enzymes and
the NADP cofactor accounted for < 1% of the waste. The mainwaste streams are aqueous and directly biodegradable.
Progress of cyanation reactions using HHDH catalysts. Sub- Principle 2: atom economy. The atom economy24 is only 45%.
strate : biocatalyst = 100 : 1 (w/w). Rd 1 = expression mutant of wild The use of glucose as the reductant for cofactor regeneration type enzyme. Rd 22 gave 130 g L-1 product.
is cost effective but not particularly atom efficient. However,glucose is a renewable resource and the gluconate co-product is The dramatic improvement in the activities of three different enzymes demonstrates that DNA shuffling technologies can Principle 3: less hazardous chemical syntheses. The reduction enable large-scale enzymatic processes that otherwise would reaction uses starting materials that pose no toxicity to human not have been economically viable. Our completely enzymatic health or the environment. It avoids the use of potentially process has been scaled-up to 2000 L reactors. In addition to hazardous hydrogen and heavy metal catalysts throughout the this two-step process, running the reactions as a one-pot process, process thus obviating concern for their removal from waste sequentially or concertedly, was demonstrated on a laboratory streams and/or contamination of the product. While cyanide must be used in the second step, as in all practical routes to HN, 84 Green Chem., 2010, 12, 81–86
This journal is The Royal Society of Chemistry 2010 Evolution of the enzymatic cyanation catalyst Principle 8: reduce derivatization. The process avoids deriva- tization steps, i.e. it is step-economic25 and involves fewer unit
operations than earlier processes, most notably by obviating the trouble-prone product distillation or the bisulfite-mediated Substrate loading/g L-1 separation of dehydrated by-products.26
Principles 11 and 12: real-time analysis for pollution preven- Biocatalyst loading/g L-1 tion, and inherently safer chemistry. The reactions are run in Chemical purity/% pH-stat mode at neutral pH by computer-controlled addition of E.e. of 1/%
base. Gluconic acid generated in the first reaction is neutralized Phase separation of organic with aq. NaOH and HCl generated in the second step is product phase from aqueousphase containing enzyme/min neutralized with aq. NaCN, regenerating HCN (pK 9) in situ.
Space–time yield/g The pH and the cumulative volume of added base are recorded Catalyst yield (gproduct/gcat) in real time. Feeding NaCN on demand minimizes the overallconcentration of HCN affording an inherently safer process.
Composition of the waste per kg of 1 product in the new
% Contribution % Contribution We have developed and deployed a two-step, three-enzyme, and Quantity (kg to E (excluding green-by-design process for the manufacture of the key hydrox- ynitrile intermediate for atorvastatin. We used directed evolution technologies to optimize the performance of all three enzymes to meet pre-defined process criteria. The overall volumetric productivity per mass catalyst load of the cyanation process was improved 2500-fold, comprising a 14-fold reduction in BuOAc (85% recycle) 0.46 EtOAc (85% recycle) 2.50 reaction time, a 7-fold increase in substrate loading, a 25-fold reduction in enzyme use, and a 50% improvement in isolated yield. Concomitant significant process simplification resulted in an overall reduction in energy use and waste production. These results demonstrate the power of state-of-the-art enzyme opti-mization technologies in enabling economically viable, green-by- it is used more efficiently (higher yield) and under less harsh design biocatalytic processes that would otherwise remain mere conditions compared to previous processes.
Principle 4: design safer chemicals. This principle is not Downloaded by University of Calgary on 30 September 2011 applicable as the hydroxynitrile product is the commercial Published on 23 October 2009 on http://pubs.rsc.org doi:10.1039/B919115C starting material for atorvastatin.
Principle 5: safer solvents and auxiliaries. Safe and environ- mentally acceptable butyl acetate is used, together with water, as the solvent in the biocatalytic reduction reaction and extraction butyrate (3), and ethyl 4-(R)-cyano-3-hydroxybutyrate (1)
of the hydroxynitrile product; no auxiliaries are used. Solvent were purchased from Sigma-Aldrich. Ketoreductase (KRED; use is minimized by employing countercurrent extraction.
EC1.1.1.184) glucose dehydrogenase (GDH; EC1.1.1.47) and Principles 6 and 9: design for energy efficiency, and catalysis.
halohydrin dehalogenase (HHDH; EC3.8.1.5) were produced as In contrast with previous processes, which employ elevated tem- semi-purified lyophilisates by fermentation using recombinant peratures for the cyanation step and high pressure hydrogenation E. coli strains carrying the corresponding genes and subsequent for the reduction step, both steps in our process are very efficient work-up as described. The desired enzymes constitute 30–50% biocatalytic transformations. The reactions are run at or close to of this material by weight.
ambient temperature and pressure and pH 7 and the very highenergy demands of high vacuum distillation are dispensed with Determination of (S)-ECHB (4) and (R)-HN (1) by chiral GC
altogether, resulting in substantial energy savings. The turnovernumbers for the different enzymes are >105 for KRED and Sample preparation and derivatization.
A sample of 4 or 1
GDH and >5 ¥ 104 for HHDH. Because of the low enzyme was converted to its trifluoroacetyl derivative by reaction with concentration used, immobilization of the biocatalyst to make trifluoroacetic anhydride (TFAA). One drop of 4 in a GC sample
it recyclable is neither practical, nor economic.
vial was treated with approximately 0.3 mL of TFAA. The vial Principles 7 and 10: use of renewable feedstocks, and design for was capped and held at room temperature for approximately degradation. The enzyme catalysts and the glucose co-substrate 30 min. Excess TFAA was removed under a stream of nitrogen.
are derived from renewable raw materials and are completely A few drops of methanol were added and blown off. The sample biodegradable. The by-products of the reaction are gluconate, was diluted with hexane (1 mL) and injected (1 mL) into the NADP (the cofactor that shuttles reducing equivalents from GC (Agilent 6890 GC and Agilent 19091s-433 GC/MS with GDH to KRED) and residual glucose, enzyme, and minerals a b-cyclodextrin dimethyl (B-DM) column (30 m ¥ 0.25 mm) and the waste water is directly suitable for biotreatment.
[Chiraldex (Astec)]). Instrumental parameters: inlet—250 C,
This journal is The Royal Society of Chemistry 2010 Green Chem., 2010, 12, 81–86 85
flow—88 mL min-1, split—60 : 1; temperature program: 80 C
acetate phases were concentrated in vacuo to give 61.6 g (93%) (for 4) or 100 C (for 1) for 30 min, 15 C min-1, final T = 180 C,
of 1. The chemical purity as determined by GC was 99.5%
held for 5 min; detector: 300 C; 30 mL min-1 H , 400 mL min-1
and the chiral purity was >99.5% e.e. for the R-enantiomer air; retention times for enantiomers of 4: S at 24 min, R at 25 min;
(no S-enantiomer was detected).
retention times for enantiomers of 1: R at 22 min, S at 23 min.
Preparative enzymatic reduction of ECAA (3) to (S)-ECHB (4)
A 3 L jacketed three-neck flask equipped with a pH electrode 1 M. M ¨uler, Angew. Chem., Int. Ed., 2005, 44, 362.
2 D. E. Butler, T. V. Le, A. Millar and T. N. Nanninga, US5155251
connected to a pH stat (Schott system) was charged with 570 ml of 100 mM triethanolamine. The pH was adjusted to 7 using 3 G. DeSantis, Z. Zhu, W. A. Greenberg, K. Wong, J. Chaplin, S. R.
Hanson, B. Farwell, L. W. Nicholson, C. L. Rand, D. P. Weiner, D. E.
D-glucose (298 g; 1.64 M) was added.
Robertson and M. J. Burk, J. Am. Chem. Soc., 2002, 124, 9024; G.
The temperature was raised to 25 C. KRED (854 mg) and
DeSantis, K. Wong, B. Farwell, K. Chatman, Z. Zhu, G. Tomlinson, GDH (578 mg) were charged as lyophilized powders. Then, Na- H. Huang, X. Tan, L. Bibbs, P. Chen, K. Kretz and M. J. Burk, J. Am. NADP (98 mg) was added followed by butyl acetate (370 ml).
Chem. Soc., 2003, 125, 11476.
The reaction was started by the addition of 3 (240 g; 1.46 moles)
4 F. H. Hoff and T. Anthonsen, Tetrahedron: Asymmetry, 1999, 10,
from an addition funnel while maintaining the pH at 6.9 ± 0.05 5 D. R. Yazbek, C. A. Martinez, S. Hu and J. Tao, Tetrahedron: using the pH stat with 4 N NaOH feeding. The reaction was Asymmetry, 2004, 15, 2757.
completed in 8 h at 25 C at which point 357 ml 4 N NaOH
6 H. Matsuda, T. Shibata, H. Hashimoto and M. Kitai, U.S. Patent, 5, 908,953, 1999 to Mitsubishi Chemical Corporation.
was consumed. A sample was analyzed by GC to check for 7 M. M. Elenkov, B. Hauer and D. B. Janssen, Adv. Synth. Catal., 2006, completion. The reaction mixture was heated to 50 C for 30 min
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and 5 g of Celite was added to facilitate filtration. The reaction 8 T. Nakamura, T. Nagasawa, F. Yu, I. Watanabe and H. Yamada, mixture was cooled to 25 C and filtered through a Celite pad.
Biochem. Biophys. Res. Commun., 1991, 180, 124.
9 T. Nakamura, T. Nagasawa, F. Yu, I. Watanabe and H. Yamada, The two layers were separated and the aqueous solution was Tetrahedron, 1994, 50, 11821.
extracted with 350 mL butyl acetate. The two organic extracts 10 J. H. Lutje Spelberg, L. Tang, M. van Gelder, R. M. Kellogg and were combined and the solvents were removed to obtain 232.61 g D. B. Janssen, Tetrahedron: Asymmetry, 2002, 13, 1083.
11 J. H. Lutje Spelberg, J. E. T. van Hylckama Vlieg, L. Tang, D. B.
4, as a light yellow-colored liquid. The enantiomeric
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excess of 4 was >99.5% (the R-enantiomer was not detected).
12 G. Hasnaoui, J. H. Lutje Spelberg, E. de Vries, L. Tang, B. Hauer and D. B. Janssen, Tetrahedron: Asymmetry, 2005, 16, 1685.
Preparative enzymatic cyanation of (S)-ECHB (4) to HN (1)
13 R. A. Sheldon, Chirotechnology; Industrial Synthesis of Optically Active Compounds, Marcel Dekker, New York, 1993; C. H. Wong and A 1 L jacketed three-neck round bottom flask equipped with G. M. Whitesides, Enzymes in Synthetic Organic Chemistry, Elsevier,Amsterdam, 1994.
a rubber septum, a pH electrode connected to a pH stat, 14 U. T. Strauss, U. Felfer and K. Faber, Tetrahedron: Asymmetry, 1999, and a mechanical stirrer was charged with 400 ml 500 mM Downloaded by University of Calgary on 30 September 2011 NaCN. The whole system was airtight since gaseous HCN is 15 A. Bommarius and B. Bommarius-Riebel, Fundamentals of Biocatal- Published on 23 October 2009 on http://pubs.rsc.org doi:10.1039/B919115C generated. The pH of the solution was 11.2. The pH electrode ysis, Wiley-VCH, Weinheim, 2005.
16 A. Liese, K. Seelbach, and C. Wandrey, Industrial Biotransforma- was calibrated at room temperature 20–23 C before use. The
tions, Wiley-VCH, Weinheim, 2000; G. J. Lye, P. A. Dalby and J. M.
vessel was sealed and the pH was adjusted to 7.5 using conc.
Woodley, Org. Process Res. Dev., 2002, 6, 434.
H SO ( 7 mL). HHDH was charged as an aqueous solution 17 K. A. Powell, S. W. Ramer, S.B. del Cardayre, W. P. C. Stemmer, M. B.
(1.05 g in 20 mL de-ionized water) and the reaction mixture was Tobin, P. F. Longchamp and G. W. Huisman, Angew. Chem., Int. Ed.,
2001, 40, 3948; M. T. Reetz and K.-E. Jaeger, Chem.–Eur. J., 2000,
heated to 40 C. Then, the pH stat control was started and 70 g
6, 407; S. B. Rubin-Pitel and H. Zhao, Combinatorial Chemistry &
(0.42 moles) of 4 was added with a syringe over 10 min. The
High Throughput Screening, 2006, 9, 247.
pH stat maintained the pH at 7.3 ± 0.05 with the addition of 18 R. J. Fox, S. C. Davis, E. C. Mundorff, L. M. Newman, V. Gavrilovic, S. K. Ma, L. M. Chung, C. Ching, S. Tam, S. Muley, J. Grate, J.
a 25% NaCN solution containing 0.25% NaOH. After 18 h, Gruber, J. C. Whitman, R. A. Sheldon and G. W. Huisman, Nat. 73 mL of NaCN/NaOH solution was consumed. At that point, Biotechnol., 2007, 25, 338.
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with conc. H SO ( 2.5 mL). For safety reasons, the reaction was Giver, M. Bueno, J. R. Cherry, T. V. Borchert, W. P. Stemmer and J.
cooled to 25 C. The pH electrode and the rubber septum were
Minshull, Nat. Biotechnol., 1999, 17, 893–6.
removed [caution: HCN gas present]. The flask was equipped 21 J. F. Chaparro-Riggers, K. M. Polizzi and A. S. Bommarius, Biotechnol. J. with a condenser and nitrogen dip tube extending into the liquid.
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HCN in the off gas). The reaction mixture was heated to 40 C. A
23 R. A. Sheldon, Chem. Ind. (London, UK), 1992, 903; R. A. Sheldon, Chem. Commun., 2008, 3352; R. A. Sheldon, Green Chem., 2007, 9,
nitrogen bleed was used to facilitate the removal of HCN. After 2.5 h, HCN removal was complete (<5 ppm in the off gas). The 24 B. M. Trost, Science, 1991, 254, 1471; B. M. Trost, Angew. Chem.,
mixture was cooled and treated with 3.5 g of Celite and 3.5 mL Int. Ed. Engl., 1995, 34, 259.
bleach (containing 6.1% sodium hypochlorite). This mixture 25 P. A. Wender, M. P. Croatt and B. Witulski, Tetrahedron, 2006, 62,
7505; P. A. Wender, V. A. Verma, T. J. Paxton and T. H. Pillow, Acc. was filtered through a Celite pad. The filtrate was extracted Chem. Res., 2008, 41, 40.
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86 Green Chem., 2010, 12, 81–86
This journal is The Royal Society of Chemistry 2010

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The World Journal of Biological Psychiatry, 2008; 9(1): 623 World Federation of Societies of Biological Psychiatry (WFSBP)Guidelines for Biological Treatment of Substance Use and RelatedDisorders, Part 1: Alcoholism MICHAEL SOYKA1, HENRY R. KRANZLER2, MATS BERGLUND3, DAVID GORELICK4,VICTOR HESSELBROCK2, BANKOLE A. JOHNSON5, HANS-JU THE WFSBP TASK FORCE ON TREATMENT GUIDELINES FOR SUBSTANCE USEDISORDERS*