View source for Chem321:Biocatalysts For Synthesis of Lipitor

Waste Reduction at Source: A Look at Biocatalysts For Synthesis of Lipitor®
Darla Davis (June 2011)
Chemistry Department-SUNY Potsdam-Potsdam, New York
Abstract: In the development of “green” pharmaceuticals, the application of biocatalysis has
revealed that fewer processing steps are involved, higher yields with less waste, and fewer costs
associated with materials and energy. Biocatalysts have been favored because of their ability to
catalyze almost any organic reaction in ambient conditions with minimal environmental costs
associated with large-scale production. The chemical industry is currently applying biocatalysts
in the design of pharmaceuticals, such as Lipitor®, to maximize efficacy while reducing toxicity.
Introduction: The chemical industry is both diverse and complex, as it is the chief developer and
producer of chemicals for commercial applications. The Chemical Manufacturers Association
reports that the $419 billion industry produces over 70,000 products and with these valuable
developments comes great concern about the repercussions involved with their synthesis, use,
and disposal (Derry, 2006). Reducing pollution and resolving environmental issues has been a
big concern for the industry, fortunately innovative chemists have embraced a new way of
thinking, introducing a whole new concept known as “green chemistry.” Green chemistry is
environmentally friendly, minimizes waste by favoring renewable resources, and reduces energy
consumption. Paul Anastas and John Warner were the first to coin a definition for green
chemistry: “Applying fundamental knowledge of chemical processes and products to achieve
elegant solutions with the ultimate goal of hazard-free, waste-free, energy efficient synthesis of
non-toxic products without sacrificing efficacy of function” (Anastas & Warner, 1998).
Likewise, in Green Chemistry: Theory and Practice (1998) Paul Anastas and John Warner
instituted twelve principles to be used as guidelines in its application:
• It is better to prevent waste than to treat or clean up waste after it is formed.
• Synthetic methods should be designed to maximize the incorporation of all materials used in
the process into the final product.
• Wherever practicable, synthetic methodologies should be designed to use and generate
substances that possess little or no toxicity to human health and the environment.
• Chemical products should be designed to preserve efficacy of function while reducing
toxicity.
• The use of auxiliary substances, e.g. solvents, separation agents, should be made unnecessary
wherever possible and innocuous when used.
• Energy requirements should be recognized for their environmental and economic impacts
and should be minimized. Synthetic methods should be conducted at ambient temperatures
and pressure.
• A raw material feedstock should be renewable rather than depleting, whenever technically
and economically practical.
• Unnecessary derivatization (blocking group, protection/deprotection, temporary modification
of physical/chemical processes) should be avoided wherever possible.
• Catalytic reagents (as selective as possible) are superior to stoichiometric reagents.
• Chemical products should be designed so that at the end of their function they do not persist
in the environment and break down into innocuous degradation products.
• Analytical methodologies need to be further developed to allow for real-time in-process
monitoring and control prior to the formation of hazardous substances.
• Substances and the form of a substance used in a chemical process should be chosen so as to
minimize the potential for chemical accidents, including releases, explosions and fires.
A major objective then, is to discover and develop products and processes that fulfill these very
goals.
All organic substances have a carbon backbone that binds to other essential elements
such as hydrogen and oxygen. Carbon atoms are capable of forming double bonds either
naturally or through organic reactions. A great step forward for green chemistry was Yves
Chauvin, Robert Grubbs and Richard Schrock’s discovery of a chemical process called
metathesis (Derry, 2006). In such reactions, catalyst molecules act to break and make double
bonds between carbon atoms, creating many new molecules. Catalyzed reactions refine the
nature and fate of synthetic reactions. Catalysts have incredible stability, can be used in small
amounts, and are regenerated after each reaction, making them a remarkable asset for
minimizing the amounts of chemicals involved in chemical processes. Metathesis applications
are used daily in the chemical industry, especially in the production of complex molecules for
pharmaceuticals because of their increased efficiency, are user friendly and more
environmentally benign (Nobelprize.org, 2011).
When it comes to greening up chemistry, the pharmaceutical industry has the most to
gain through improved chemical products and processes. Pharmaceutical production can be a
dirty business, often requiring multiple and costly steps that involve the use of toxic chemicals
and generation of waste. By reworking synthetic pathways, manufacturers can “improve the
atom economy of reactions and reduce the number of reaction steps to achieve the same
benefits” (ACS, 2011). As companies started to think about the triple bottom line of
environmental sustainability, economic performance, and social responsibility, the
pharmaceutical industry’s interest in green chemistry activity grew (Thayer & Houston, 2009).
Enzyme-mediated processes involving biocatalysts offer many advantages over
conventional, non-catalyzed methods and have become increasingly important as consumers
become more enviro-conscious. Biocatalysts come with a myriad of amenities in the context of
green chemistry: novelty, high yield, shorter process routes; increased selectivity; and lower
temperatures and pressures. They are completely renewable, cause no harm to animals or
humans, and generate drastically less waste per unit. With this in mind, there is no wonder why
biocatalytic processes are starting to dominate the fine chemical and pharmaceutical industry.
However, biocatalysts are nothing new to the chemical industry. According to the
American Chemical Society, the advantage of enzyme-catalyzed reactions has been well
understood, but with limited technology, they have not been practical for use in industry (2011).
Utilization of biocatalysts has been hampered by the “lack of consistent production and
formulation, limited scope of substrates compared with classical chemical methods, limited
stability and shelf life, and lack of easy separation and reuse” (ACS, 2011). Because biocatalysts
are highly selective, use on the industrial scale can be limited because availability is highly
restricted to those captured from nature (Thayer, & Houston, 2001). The speed of process
development is often slower for biocatalytic processes than their chemical counterparts. Modern
biotechnological tools now allow enzymes to be optimized for a desired reaction, but this
optimization is often too costly and time consuming to meet industrial timelines; therefore, broad
application remains elusive (Bio-Catalyst.com). Obstacles to overcome when using biocatalysts
is to increase both productivity and stability while lowering the cost of production, thus
improving yield of economically viable chemical processes (Thayer & Houston, 2001)
Application of Biocatalysts: Atorvastatin, commercially known as Lipitor®, is a statin drug
primarily indicated for use in patients at risk of heart attack, stroke, or heart disease by
suppressing biosynthesis of cholesterol (Pub Med Health, 2010). Currently, Lipitor® is the
largest selling pharmaceutical in the history of mankind, with annual sales of over $13 billion
worldwide. Being that cardiovascular disease is one of the leading causes of death today,
Lipitor® has succeeded in preventing countless cardiovascular events and saving the lives of
millions. To maintain its status as a world-class leading drug, Pfizer researchers have
reengineered the production process to develop a way to “reduce the use of chemicals by an
order of magnitude” (Pfizer, 2009).
Traditionally, commercial processes designed for the synthesis of Lipitor® had a
maximum yield of 95%. Production cost of product was as much as $85 per kilogram. An
eleven step process, reaction conditions had to be set at 100°C, using methanol solvent and
hydrogen gas, generating as much as 100 grams of waste per kilogram of product. Enantiomeric
purity <95% after purification by fractional distillation resulted in incomplete conversion and byproduct
formation that significantly reduced product yield. Hydroxynitrile (HN), the key chiral
building block for synthesis of the active ingredient, atorvastatin, found in Lipitor® required a
resolution step with 50% maximum yield. They also need hydrogen bromide to generate a
bromohydrin for cyanation. Substituting cyanide for halide under heated alkaline conditions,
formed extensive byproducts.
Biocatalysts have emerged as a versatile tool that offers a variety of enantioselective
routes to key chiral intermediates for the drug. Atorvastatin, the active ingredient in Lipitor, is a
completely synthetic molecule with a chiral 3,5- dihydroxy acid side chain that accounts for 25%
of the compounds molecular weight. This side chain also represents the greatest challenge for
preparation of the drug Lipitor®. Taking into account that biocatalysis is inherently green,
Presidential Green Chemistry Challenge Award winner, Codexis, developed a biocatalytic route
to the Lipitor® intermediate, hydroxynitrile (HN), (IUPAC name: ethyl (R)-4-cyano-3-
hydroxybutyrate). Biocatalysis of Lipitor® synthesis has shown to have excellent selectivity and
yields, purification only at the last step, reduced requirements, reduced byproducts, mild reaction
conditions, efficiency, and is green, safe, scalable and cost effective. The new HN synthesis
doesn’t require metal catalysts or chemical derivatization steps, and it is carried out at room
temperature and pressure at neutral pH in water in much less time. Reaction time is 8 hours and
because the product of the enzymatic process is enantiomerically and chemically pure, product
yield is at least 99%. The new E factor, a measure of process sustainability, is 5.8, significantly
lower than any of the previous routes to HN. The
two-step, three-enzyme mechanism can recover high-quality product by directly extracting the
reaction mixture because the enzymes are so active and stable. Codexis scientists used the
company's MolecularBreeding accelerated directed-evolution technology to engineer three
enzymes to accomplish the chemical reaction. The new HN synthesis starts with reduction of the
keto group of ethyl 4-chloroacetoacetate feedstock to form a chiral chlorohydrin. The reduction
is facilitated by two of the engineered enzymes, a ketoreductase and a glucose dehydrogenase.
The enzymes work in tandem with nicotinamide adenine dinucleotide to convert the
chloroketone to the chlorohydrin using glucose as a reductant, which avoids the need to use
hydrogen. More pivotal is the ensuing step in which the third enzyme, a halohydrin
dehalogenase, catalyzes substitution of chlorine with a cyano group to form hydroxynitrile. Both
enzymatic reaction steps take place under aqueous conditions at neutral pH and at atmospheric
pressure and just above room temperature, Grate pointed out. Overall, the Codexis process
provides more than 90% yield for each step and provides HN in high enantiomeric purity of
>99%. In addition, the low enzyme loading avoids emulsion formation and allows product
isolation by extraction rather than by more costly distillation (Ritter, 2006).
Future of Biocatalysts: Codexis’ evolved enzymes have “improved the volumetric productivity
of the reduction reaction by approximately 100-fold and that of the cyanation reaction by
approximately 4,000-fold” (Ritter, 2006). This reengineered process involves many
environmental and health benefits by reducing waste, solvent usage, energy consumption, and
need for specialized manufacturing equipment, and increasing worker safety (EPA, 2011). Goals
for industrial processing such as “sustainable development”, “green chemistry,” or
“environmentally benign manufacturing,” would be difficult to attain without the availability of
biocatalysts that perform optimally in ambient conditions (Bommarius & Riebel, 2004).
Increasing emphasis on becoming “green” through the use of environmentally benign products
and processes means that pharmaceutical companies will embrace the evolution of biocatalysts,
particularly in the production of atorvastatin. In a report by Dr. Nicholas Derry (2007), he
discusses the viability of many green chemistry processes that were once frowned upon, but
records indicate that industrial-scale biocatalytic processes more than doubled between 1992 and
2002. (Straathof, 2002) and have proved to be profitable as consumers yearn to be more
environmental friendly. Moreover, there are significant economic benefits from utilizing the
revised process of HN production for the synthesis of atorvastatin, as the pharmaceutical industry
is based on maximum efficacy with minimum toxicity.
The future looks bright for Lipitor®, as it continues to grow steadily and dominate the
market in cardiovascular medications. The latest generation of Lipitor® that involves green by
design processes enables access to global markets that have expectations for cleaner and efficient
products.
References
American Chemical Society (ACS). (2011).
• Chemical society to honor “Heroes of Chemistry” during national meeting.
http://portal.acs.org:80/portal/acs/corg/content?_nfpb=true&_pageLabel=PP_ARTICLE
MAIN&node_id=222&content_id=WPCP_010675&use_sec=true&sec_url_var=region1
&__uuid=3b11aeb5-b955-4d8c-aa63-cf11f04d71e8 (accessed 29 June 2011).
• Greening up process chemistry.
http://portal.acs.org:80/portal/acs/corg/content?_nfpb=true&_pageLabel=PP_ARTICLE
MAIN&node_id=445&content_id=CNBP_026112&use_sec=true&sec_url_var=region1
&__uuid=d35f770f-f99c-4429-9866-f2f4fcf07299 (accessed 29 June 2011).
Affholter, J., Apel, W. A., Arnold, F., et. Al. (1999). New Biocatalysts: Essential tools
for a sustainable 21st century chemical industry. Council for Chemical Research.
Bio-catalyst.com Time for biocatalysis and bio-based processes?
Biocatalysis:How enzymes, biofuels, and sustainable chemistry are changing
the world. http://www.bio-catalyst.com/hot-and-new-inbiocatalysis/
biocatalysis/time-for-biocatalysis/ (accessed 30 June 2011).
Bommarius, A. S. & Riebel, B. R. (2004). Inroduction to Biocatalysts.
http://media.wiley.com/product_data/excerpt/48/35273034/3
527303448.pdf (accessed 22 June 2011).
Derry, N. (2007). Green chemistry. Victorian Curriculum and Assessment Authority, Units 1-2.
Environmental Protection Agency. (2011). Green Chemistry.
http://www.epa.gov/gcc/pubs/pgcc/winners/grca06.html (accessed 16 June 2011).
Greenberg, W. A., Varvak, A., Hanson, S. R., Wong, K., Huang, H., Chen, P., and Burk, M. A.
(2004). Development of an efficient, scalable, aldolase-catalyzed process for
enantioselective synthesis of statin intermediates.
http://www.pnas.org/content/101/16/5788.full.pdf (accessed 30 June 2011).
Nobelprize.org. (2011). Press Release: The Nobel Prize in chemistry 2005.
http://nobelprize.org/nobel_prizes/chemistry/laureates/2005/press.html (accessed 30 June
2011).
Pfizer. (2009). Pfizer 2008 Annual Report.
http://media.pfizer.com/files/annualreport/2008/annual/revie
w2008.pdf (accessed 16 June 2011).
PubMedHealth. (2010). Atorvastatin.
http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0000009/
(accessed 16 June 2011).
Ritter, S. K. (2006). Going green keeps getting easier. Science & Technology, 84, 24-27.
Roumeliotis, G. (2006). Green Lipitor building block synthesis wins kudos for Codexis.
Pharma Technologist.com. http://www.in- pharmatechnologist.com/Processing-
QC/Green-Lipitor-building-block- synthesis-wins-kudos-for-Codexis
(accessed 1 July 2011).
Straathof, A. J., Panke, S., & Schmid, A. (2002). The production of fine chemicals
by biotransformations. Current Opinion of Biotechnology, 13, 548–556.
Thayer, A. M. & Houston, E.
• (2001). Biocatalysis. Chemical and Engineering News, 79, 27-34.
http://pubs.acs.org/cen/coverstory/7921/7921biocatalysis.html (accessed 30 June 2011).
• (2009). Sustainable syntheses. Chemical and Engineering News.
http://portal.acs.org/portal/fileFetch/C/CNBP_024675/pdf/CNBP_024675.pdf (accessed
30 June 2011).

[[Category:Chemistry 321]]