Ion exchange resins are cross linked polymers of polystyrene, up on which a negatively charged or a positively charged functional group can be added to get an anion exchange resin (Resin- )or a cation exchange resin Resin +(Resin + Or Resin- ). When these resins are mixed with drug molecule which has a negative charge in its one or more functional group as a result of polarity, or as in salt form, or as a result of resonance, such a drug can form a complex with a resin as follow.
1.) Resin + Drug-
2.) Resin- Drug+
*The resulting resin drug complex stability depends on how strong are the acidic or basic functional groups on resin, stronger functional group result in to formation of very stable resin drug complex, and vice a versa , both has its applications in sustained release drug delivery systems, a resin with strong acidic or basic functional group tend to provide a much more delayed drug release, where drug release is bit faster with weak acidic and basic functional group resins.
When Resin- Drug+ or Resin + Drug- complex (drug resinate ) comes in contact with acid(in stomach) or base(in intestine), it start releasing drug molecule in exchange of similar charged ion for example if drug molecule is positively charged, then it is released from Resin- Drug+ complex when it come in contact with Hydrogen Ion( H+) , similarly a Resin + Drug- or basic drug resin complex will start releasing drug when it comes in contact with basic ions ( in intestine ) OH – NH3- .
Release of drug molecule from Resin +Drug complex takes place due to higher concentration of replacing ions (H+ , or OH – NH3- ).
Masking Taste of drug with ion exchange resin:
There are some drugs which are very bitter in taste like Bromhexin and Quinine, patients has very low acceptability for such drugs some time patients vomit and expelling all of the consumed dose which may result in to dosing error, therefore when such drugs are formulated with ion exchange resins which binds such drugs they do not release drug on taste buds over tongue as a result taste of drug is masked, and when it come in contact with gastric acid bromhexin is released in to gut.
Sustained-release drug delivery system:
Ion exchange resins may not alone give capability to formulate it in to a sustained release dosage form , to make it a good sustained release drug delivery system , proper selection of resin is important a resin with strong acidic or basic functional group provides strong complexation with drug therefore are good candidates for delayed drug release, likewise when early release is intended a weaker acidic or basic functional group resins are useful . Drug resinate is also required to be coated with semipermiable film forming polymers, as drug resinate complex can not be solely relied up on for the intended use. ( ethylcellulose ). In order to maintained the sustained release property of ion exchange resin drug complex it is pretreated with polyethylene glycol so that it do not swell and break open the film coating when it come in contact with gastric juice.
Ion exchange resins are required to be washed with, suitable organic solvent, to remove residual organic or chemical impurities in ion exchange resin, followed by washing with purified water, and regeneration if required, before using for actual process. Ion exchange resin are required to comply with requirements listed in 21 CFR 173.25 by US FDA.
Ion exchange resins have many other applications in pharmaceutical dosage form and drug delivery systems like , localized drug release, stabilization of drug molecule for chemical degradation .
Ion exchange resins are widely used in pharmaceutical industry for purification or raw water and in preparation of water for pharmaceutical use.
-Rajarshi Nareshkumar Patel
USFDA guidelines, GMP guidelines, WHO guidelines, Schedule M, FDA, European guidelines cleaning validation, process validation, water system validation, ICH guidelines, GMP audut compliance, equipment qualification, Installation Qualification, Operational Qualification, Performance Qualification, Calibration, Validation Protocol, SOPs etc.
Sunday, August 12, 2012
Novel Drugs: Cancer Chemotherapy Using Nanoparticles developed with Nanotechnology May Reduce Harmful Side Effects of Antineoplastic Agents.
Chemotherapy for cancer is most of the time associated with one or the other harmful side effect of antineoplastic drugs as these chemotherapeutic drugs themselves are very cytotoxic, i.e. they damage normal cells too.
Antineoplastic drugs bring about their anticancer action by inhibiting cancerour cells growth by virtue of alkylation of nucleotides in cancerous cells or by inhibition of folic acid uptake by cancerous cells or by inhibiting cell division by binding with tubulin and microtubulin in a cancerous cells, it is likely that these drug are also absorbed in to normal tissues, leading to untoward serious cytotoxic effects , like kidney damage and nerve damage in chemotherapy with cisplatin, a drug of choice in most of anticancer chemotherapies.
A new drug delivery technique is being studied which uses Nanotechnology to deliver a cytotoxic drugs specifically directly in to the cancer cells , such drug delivery technique will be able to provide an efficient cancer chemotherapy that do not have much side effects as they pose today , it was observed that with nanoparticle drug delivery system the concentration of drug required to kill the cancerous cell is lesser than required in conventional chemotherapy therapy. As the drug is absorbed efficiently in to targeted cells and also drug is protected from degradation in blood stream , certain class of the anticancerdrugs are very unstable and stay in plasma for a very little time, therefor to achieve the required effect a higher concentration of drug may be required to be administrated.
Nanotechnology drug delivery system involves placing an anticancer drug in to a tiny particles known as nanoparticles which recognize cancerous cells and deliver the drug only to cancerous cells , as nanoparticles are very minute particles (1 nm to 100 nanometer) , the dose of drug required to kill the cancerous cells were also found to be very low as compared to conventional therapy . As the required effective dose it self gets reduced than conventional therapy , the harmful effect of anticancer drug are also likely to be reduced.
A team of scientists from the Massachusetts Institute of Technology and Brigham and Women's Hospital conducted study. They stored an prodrug of cisplatin (which is used in most of cancer chemotherapies) within nanoparticles which they developed to target a specific protein in cancerous cells in prostate gland.
After these prodrug loaded nanoparticles were absorbed by cancerous cells the prodrug was released in to the cancerous cells and was converted in to an active form . The team demonstrated that these prodrug carrying nanoparticles were able to kill cancer cells in culture more efficiently than the drug alone.
Study was conducted by researchers, led by Dr. Omid Farokhzad and Dr. Stephen Lippard, to study nanoparticle drug delivery system for an effective and safer option for chemotherapy in living animals. Their research work is published in Proceedings of the National Academy of Sciences, in Jan 2011 issue of the journal, the study was funded in part by NIH’s National Cancer Institute (NCI) and National Institute for Biomedical Imaging and Bioengineering (NIBIB).
By applying this drug delivery by nanoparticles they were able to shrink tumors in mice with smaller doses of the drug to reduce harmful side effects. Only 30% of the dose of prodrug of cisplatin was required to diminish the tumor by using the drug carrying nanoparticles, than that of standard dose of cisplatin as such.
Researchers initially studied different doses of nanoparticle bound drug in rats and mice, both the types of animals maintained their body weight and survived at higher doses of the drug when drug was delivered using nanoparticles than when injected without nanoparticles. It was also found that the kidney damage was less in rats which received the nanoparticle bound drug.
Also it was found that binding nanoparticles provided greater stability of cisplatin prodrug in blood stream than that of injected alone , after one hour about 77 % of prodrug was found in blood stream when it was delivered using nanoparticles compared to only 16% available drug in case of drug delivered without nanoparticles, cispaltin is very unstable drug and remains in blood for very short time , which calls for more dose to get the desired effect.
Rajarshi Patel
Antineoplastic drugs bring about their anticancer action by inhibiting cancerour cells growth by virtue of alkylation of nucleotides in cancerous cells or by inhibition of folic acid uptake by cancerous cells or by inhibiting cell division by binding with tubulin and microtubulin in a cancerous cells, it is likely that these drug are also absorbed in to normal tissues, leading to untoward serious cytotoxic effects , like kidney damage and nerve damage in chemotherapy with cisplatin, a drug of choice in most of anticancer chemotherapies.
A new drug delivery technique is being studied which uses Nanotechnology to deliver a cytotoxic drugs specifically directly in to the cancer cells , such drug delivery technique will be able to provide an efficient cancer chemotherapy that do not have much side effects as they pose today , it was observed that with nanoparticle drug delivery system the concentration of drug required to kill the cancerous cell is lesser than required in conventional chemotherapy therapy. As the drug is absorbed efficiently in to targeted cells and also drug is protected from degradation in blood stream , certain class of the anticancerdrugs are very unstable and stay in plasma for a very little time, therefor to achieve the required effect a higher concentration of drug may be required to be administrated.
Nanotechnology drug delivery system involves placing an anticancer drug in to a tiny particles known as nanoparticles which recognize cancerous cells and deliver the drug only to cancerous cells , as nanoparticles are very minute particles (1 nm to 100 nanometer) , the dose of drug required to kill the cancerous cells were also found to be very low as compared to conventional therapy . As the required effective dose it self gets reduced than conventional therapy , the harmful effect of anticancer drug are also likely to be reduced.
A team of scientists from the Massachusetts Institute of Technology and Brigham and Women's Hospital conducted study. They stored an prodrug of cisplatin (which is used in most of cancer chemotherapies) within nanoparticles which they developed to target a specific protein in cancerous cells in prostate gland.
After these prodrug loaded nanoparticles were absorbed by cancerous cells the prodrug was released in to the cancerous cells and was converted in to an active form . The team demonstrated that these prodrug carrying nanoparticles were able to kill cancer cells in culture more efficiently than the drug alone.
Study was conducted by researchers, led by Dr. Omid Farokhzad and Dr. Stephen Lippard, to study nanoparticle drug delivery system for an effective and safer option for chemotherapy in living animals. Their research work is published in Proceedings of the National Academy of Sciences, in Jan 2011 issue of the journal, the study was funded in part by NIH’s National Cancer Institute (NCI) and National Institute for Biomedical Imaging and Bioengineering (NIBIB).
By applying this drug delivery by nanoparticles they were able to shrink tumors in mice with smaller doses of the drug to reduce harmful side effects. Only 30% of the dose of prodrug of cisplatin was required to diminish the tumor by using the drug carrying nanoparticles, than that of standard dose of cisplatin as such.
Researchers initially studied different doses of nanoparticle bound drug in rats and mice, both the types of animals maintained their body weight and survived at higher doses of the drug when drug was delivered using nanoparticles than when injected without nanoparticles. It was also found that the kidney damage was less in rats which received the nanoparticle bound drug.
Also it was found that binding nanoparticles provided greater stability of cisplatin prodrug in blood stream than that of injected alone , after one hour about 77 % of prodrug was found in blood stream when it was delivered using nanoparticles compared to only 16% available drug in case of drug delivered without nanoparticles, cispaltin is very unstable drug and remains in blood for very short time , which calls for more dose to get the desired effect.
Rajarshi Patel
Kidney transplant will see more success rate than earlier.
Patients suffering from kidney failure are required to go for treatment like regular dialysis and kidney transplant, and that to be not all kidney transplants are successful , 1 out of every 3 kidney transplants are rejected by patients body (because of patients immune system) , it is because of the human body recognizes difference between its own body tissue and other persons body tissue and human body reject others persons body tissue by producing antibodies against the transplanted tissue , ultimately damaging and rejecting the transplanted kidney.
Dr. Robert A. Montgomery and his team from the Johns Hopkins University School of Medicine have found new technique , which has shown promising method which has greatly lowered the kidney rejection and has done kidney transplants successfully with their method.
They identified that there is an antigen on cells in human called as human leukocyte antigen, a person sensitized by human leukocyte antigen (HLA sensitized ) help human immune system to identify its own body tissue and incoming foreign tissue and trigger antibodies against transplanted kidney transplant and further leads to kidney rejection.
Therefore they developed a method to desensitize patients by filtering out anti-HLA antibodies they removed the anti-HLA antibodies from patient’s body by process of plasmapheresis. Patients were given
Low-dose intravenous immune globulin (antibody preparation made from pooled donor blood) as substitute for anti-HLA antibodies. The procedure of plasmapheresis was repeated several times before the actual kidney transplant was done , these patients were also given other immuno suppressant drugs which are normally given before kidney transplant.
More than 80% of the patients which received kidney transplant and plasmapheresis treatment were still alive after 8 years. Compared to observed lower life period for patients on either dialysis or HLA compatible kidney transplant ad dialysis.
The method is bit costly but is cost effective compared to the total other costs of regular dialysis are considered in kidney failure patients, and success rate.
What is plasmapheresis:
Plasmapheresis is a method of removing toxic or unwanted component from blood , by removing part of blood out of body and processing it in centrifuge to separate blood cells and plasma , the plasma containing toxic or unwanted antibodies is discarded and is replaced with donor plasma , albumin , or 70% albumin and 30% saline , a suitable anticoagulant drug is given to patient before the plasmapheresis procedure .
Dr. Robert A. Montgomery and his team from the Johns Hopkins University School of Medicine have found new technique , which has shown promising method which has greatly lowered the kidney rejection and has done kidney transplants successfully with their method.
They identified that there is an antigen on cells in human called as human leukocyte antigen, a person sensitized by human leukocyte antigen (HLA sensitized ) help human immune system to identify its own body tissue and incoming foreign tissue and trigger antibodies against transplanted kidney transplant and further leads to kidney rejection.
Therefore they developed a method to desensitize patients by filtering out anti-HLA antibodies they removed the anti-HLA antibodies from patient’s body by process of plasmapheresis. Patients were given
Low-dose intravenous immune globulin (antibody preparation made from pooled donor blood) as substitute for anti-HLA antibodies. The procedure of plasmapheresis was repeated several times before the actual kidney transplant was done , these patients were also given other immuno suppressant drugs which are normally given before kidney transplant.
More than 80% of the patients which received kidney transplant and plasmapheresis treatment were still alive after 8 years. Compared to observed lower life period for patients on either dialysis or HLA compatible kidney transplant ad dialysis.
The method is bit costly but is cost effective compared to the total other costs of regular dialysis are considered in kidney failure patients, and success rate.
What is plasmapheresis:
Plasmapheresis is a method of removing toxic or unwanted component from blood , by removing part of blood out of body and processing it in centrifuge to separate blood cells and plasma , the plasma containing toxic or unwanted antibodies is discarded and is replaced with donor plasma , albumin , or 70% albumin and 30% saline , a suitable anticoagulant drug is given to patient before the plasmapheresis procedure .
Saturday, July 7, 2012
'India ready to make it big in outsourcing of medical devices'
'India ready to make it big in outsourcing of medical devices'
India is now in the limelight for its expertise in the medical devices space. The industry's inherent engineering strengths is being recognized by global majors who are looking to tap the emerging market opportunities to augment growth. This is where Bangalore-based MedVed sets up an advanced facility here, which has designed and developed high quality, reasonable priced implantable medical devices for cardiac rhythm management. The company's 'Stellar' brand for single and dual pacemakers, cardiac leads, pacing system analyzers will now revolutionize the heart surgery management. Dinesh Puri, chairman and managing director, MediVed Innovations Pvt Ltd provides an overview of the opportunities, business potential in the segment in a discussion with Nandita Vijay. Excerpts:
How do you see India emerging as a hub for contract design, manufacture of medical devices?
India is well placed in the outsourced contract design, development and manufacturing space because of its engineering capabilities in a wide spectrum of areas. Typically, there are two market opportunities in contract design and development of medical devices. One is that companies in the US and Europe can offload a whole range of existing medical device products to India to maximize the cost advantage. India has been proving to be a reliable and dependable source for this capability. The second is that global companies can look at India as a hub in the Asian region to undertake contract design, develop, manufacture and package the medical devices.
What according to you are the visible trends in this space?
A visible trend is that all international companies in the wake of the global economic slow down are looking to tap opportunities in the emerging markets. These are the markets of the future. Companies in the West are transforming business models to cater to the needs of the emerging markets which make up 4/5th of the global demand.
In the wake of these opportunities, what are the efforts taken by global cos to make the most of the situations?
The companies have begun to re-device the business strategy primarily because emerging markets are price sensitive. Therefore companies would look at alliances and joint ventures with Indian enterprises in these regions. In the US, the issue is 'performance over price' as against India where it is 'price over utility'. Therefore international companies need to look at the 'design-to-cost' factor to make medical devices available in India. This is where global majors will now have to look at design and manufacturing hubs in Asia and India, in particular, to tap the quality-cost advantage which will help improve gross margins. There is need for innovative thinking and India is now building its capability to be a platform for such prospects.
What are the challenges before medical devices cos to generate business?
The challenge for medical device companies is to raise funds and be able to repay within a realistic time frame. In this business, cycle time is longer. Therefore, both the investor and management have to be patient. There is also the issue of global sourcing of raw materials and dealing with imports because of inconsistent levy of import duty on raw materials which is higher than the finished products. Therefore, we need to be able to raise investments with a realistic time frame and calibrate expectations of time.
Could you tell us how MediVed has grown since its inception two years ago?
MediVed is in the league of the top six world-class medical devices companies. It has the advanced infrastructure and technical competence to offer services at a competitive cost. Our capability covers design, development of electro mechanical diagnostic and therapeutics devices besides implantable devices and active implantable like pacemakers. The company is currently doing five pilot projects for customers in the US. The assignments are in various phases of development. The projects are proving to test the capability in development of complex devices ranging from surgical to dental and diagnostics equipment apart from implantable devices. But, since pilots take a long time to convert into revenues, we are keen to tap the contract business space. By 2011, we hope to garner a turnover of Rs 150-crore which will be generated with contract assignments and the 'Stellar' brand pacemakers. The two opportunities could chip in a 50:50 revenue generation. The business opportunity in medical devices globally is close to $200 billion.
How is MediVed positioned to tap the opportunities?
MediVed has the expertise in medical device engineering, design and prototype. It can provide the proof of concept in the lab study and later scale up manufacture.
We are gearing up to offer our expertise across all segments in medical devices from electrical-electronics-mechanical engineering to software. These include high precision machining of components, sub assemblies to printed circuit boards assembling, sourcing of implantable grade materials and sterile packaging, global sourcing of bio gradable materials which are implantable grade materials steel, noble metals, novel biomaterials like nitinol, polymers, silicones, epoxy of medical grade. This is possible because of our state-of-the-art ISO 13485 compliant in-house multi disciplinary engineering and manufacture infrastructure that includes micro electronics, polymer sciences, bio mechanical, laser welding-hermetic sealing which is supported by 100,000 class clean room and complex device assembly areas. We have the expertise to provide the required regulatory compliance documentation for medical devices manufacturing quality management systems.
What is the future of medical devices industry?
The future holds immense potential for the medical devices industry. This is because of the strong growth of the healthcare space. India can score over China in the medical devices space and grab the contract design and development orders. The country has a sound record in adherence to IPR which is vital for medical devices because it follows English law which is also referred to as the contract law of the Commonwealth countries. International Courts uphold the English law.
There is multi-disciplinary R&D efforts and engineering capability in multiple sciences of electronics, chip design, software mechanical and medical engineering which are complex. The country has already made a mark in pharmaceuticals, development of new chemical entities and clinical research. The time has now come for India to take its place in the medical devices space. This medical device engineering opportunity is bigger than the Information Technology (IT) boom and we need to capitalise
India is now in the limelight for its expertise in the medical devices space. The industry's inherent engineering strengths is being recognized by global majors who are looking to tap the emerging market opportunities to augment growth. This is where Bangalore-based MedVed sets up an advanced facility here, which has designed and developed high quality, reasonable priced implantable medical devices for cardiac rhythm management. The company's 'Stellar' brand for single and dual pacemakers, cardiac leads, pacing system analyzers will now revolutionize the heart surgery management. Dinesh Puri, chairman and managing director, MediVed Innovations Pvt Ltd provides an overview of the opportunities, business potential in the segment in a discussion with Nandita Vijay. Excerpts:
How do you see India emerging as a hub for contract design, manufacture of medical devices?
India is well placed in the outsourced contract design, development and manufacturing space because of its engineering capabilities in a wide spectrum of areas. Typically, there are two market opportunities in contract design and development of medical devices. One is that companies in the US and Europe can offload a whole range of existing medical device products to India to maximize the cost advantage. India has been proving to be a reliable and dependable source for this capability. The second is that global companies can look at India as a hub in the Asian region to undertake contract design, develop, manufacture and package the medical devices.
What according to you are the visible trends in this space?
A visible trend is that all international companies in the wake of the global economic slow down are looking to tap opportunities in the emerging markets. These are the markets of the future. Companies in the West are transforming business models to cater to the needs of the emerging markets which make up 4/5th of the global demand.
In the wake of these opportunities, what are the efforts taken by global cos to make the most of the situations?
The companies have begun to re-device the business strategy primarily because emerging markets are price sensitive. Therefore companies would look at alliances and joint ventures with Indian enterprises in these regions. In the US, the issue is 'performance over price' as against India where it is 'price over utility'. Therefore international companies need to look at the 'design-to-cost' factor to make medical devices available in India. This is where global majors will now have to look at design and manufacturing hubs in Asia and India, in particular, to tap the quality-cost advantage which will help improve gross margins. There is need for innovative thinking and India is now building its capability to be a platform for such prospects.
What are the challenges before medical devices cos to generate business?
The challenge for medical device companies is to raise funds and be able to repay within a realistic time frame. In this business, cycle time is longer. Therefore, both the investor and management have to be patient. There is also the issue of global sourcing of raw materials and dealing with imports because of inconsistent levy of import duty on raw materials which is higher than the finished products. Therefore, we need to be able to raise investments with a realistic time frame and calibrate expectations of time.
Could you tell us how MediVed has grown since its inception two years ago?
MediVed is in the league of the top six world-class medical devices companies. It has the advanced infrastructure and technical competence to offer services at a competitive cost. Our capability covers design, development of electro mechanical diagnostic and therapeutics devices besides implantable devices and active implantable like pacemakers. The company is currently doing five pilot projects for customers in the US. The assignments are in various phases of development. The projects are proving to test the capability in development of complex devices ranging from surgical to dental and diagnostics equipment apart from implantable devices. But, since pilots take a long time to convert into revenues, we are keen to tap the contract business space. By 2011, we hope to garner a turnover of Rs 150-crore which will be generated with contract assignments and the 'Stellar' brand pacemakers. The two opportunities could chip in a 50:50 revenue generation. The business opportunity in medical devices globally is close to $200 billion.
How is MediVed positioned to tap the opportunities?
MediVed has the expertise in medical device engineering, design and prototype. It can provide the proof of concept in the lab study and later scale up manufacture.
We are gearing up to offer our expertise across all segments in medical devices from electrical-electronics-mechanical engineering to software. These include high precision machining of components, sub assemblies to printed circuit boards assembling, sourcing of implantable grade materials and sterile packaging, global sourcing of bio gradable materials which are implantable grade materials steel, noble metals, novel biomaterials like nitinol, polymers, silicones, epoxy of medical grade. This is possible because of our state-of-the-art ISO 13485 compliant in-house multi disciplinary engineering and manufacture infrastructure that includes micro electronics, polymer sciences, bio mechanical, laser welding-hermetic sealing which is supported by 100,000 class clean room and complex device assembly areas. We have the expertise to provide the required regulatory compliance documentation for medical devices manufacturing quality management systems.
What is the future of medical devices industry?
The future holds immense potential for the medical devices industry. This is because of the strong growth of the healthcare space. India can score over China in the medical devices space and grab the contract design and development orders. The country has a sound record in adherence to IPR which is vital for medical devices because it follows English law which is also referred to as the contract law of the Commonwealth countries. International Courts uphold the English law.
There is multi-disciplinary R&D efforts and engineering capability in multiple sciences of electronics, chip design, software mechanical and medical engineering which are complex. The country has already made a mark in pharmaceuticals, development of new chemical entities and clinical research. The time has now come for India to take its place in the medical devices space. This medical device engineering opportunity is bigger than the Information Technology (IT) boom and we need to capitalise
India-Canada to foster joint R&D projects in biotech, health research, medical devices
India-Canada to foster joint R&D projects in biotech, health research, medical devices
Under the Canada-India agreement for scientific and technological cooperation, India and Canada will soon foster joint research and development (R&D) projects in areas of biotechnology, health research, pharmaceuticals, medical devices and nanotechnology as it applies to these sectors.
The key objectives of this joint programme are to encourage domestic competitiveness through the transfer of technology and knowledge resulting from international S&T partnerships; to foster international S&T partnerships and collaborative research with an emphasis on industrial outcomes; to accelerate the commercialization of R&D that would benefit Canada and the partner country, through international partnerships, with a focus on small and medium-sized enterprises; to access international technologies for Canadian enterprises; to promote Canadian R&D capacity and Canada as a destination for foreign technology-based investments; to encourage the mobility of researchers and to promote Canada as a career destination for foreign researchers and highly qualified personnel; and to strengthen overall bilateral S&T relations.
The Department of Biotechnology (DBT) will be the implementing organization on the Indian side, while the International Science and Technology Partnerships Canada (ISTPCanada), a non-governmental organization will be implementing the project on the Canadian side.
This Canada-India programme aims to foster and support the development of collaborative R&D projects that bring together companies, research organizations, academics and other collaborators from both countries for the joint development of innovative products or processes. It aims to stimulate innovative R&D projects (engaging small-to-medium-sized companies and/or larger, well established firms) that address a specific market need or challenge; demonstrate high industrial relevance and commercial potential; and aim to deliver benefit to all participants, and more broadly, to both nations. These projects help participants to become more competitive by developing global research-based alliances with the potential to foster increased or expanded international R&D collaboration.
Eligible Indian applicants for this programme include researchers and managers of Indian companies, academic institutions, research hospitals or other R&D institutions (including not-for-profit research institutes recognized by DBT) that are headquartered and operate in India.
Eligible Canadian applicants must be researchers or managers of for-profit companies that operate and are headquartered in Canada. Canadian subsidiaries of companies headquartered outside of Canada are typically not eligible for support. However, as ‘benefit to Canada’ is a key objective and among the most important selection criteria, ISTPCanada may grant an exception to such subsidiaries if they have R&D facilities in Canada and can demonstrate that Canada will accrue clear economic benefit from the bilateral R&D project. Academic institutions, research hospitals, other institutes or research associations are strongly encouraged to participate in the projects as co-investigators.
Each proposal must include an eligible lead from Canada and India. Although it is not mandatory, projects that engage a technology developer and a technology end-user/first customer are strongly encouraged.
Under the Canada-India agreement for scientific and technological cooperation, India and Canada will soon foster joint research and development (R&D) projects in areas of biotechnology, health research, pharmaceuticals, medical devices and nanotechnology as it applies to these sectors.
The key objectives of this joint programme are to encourage domestic competitiveness through the transfer of technology and knowledge resulting from international S&T partnerships; to foster international S&T partnerships and collaborative research with an emphasis on industrial outcomes; to accelerate the commercialization of R&D that would benefit Canada and the partner country, through international partnerships, with a focus on small and medium-sized enterprises; to access international technologies for Canadian enterprises; to promote Canadian R&D capacity and Canada as a destination for foreign technology-based investments; to encourage the mobility of researchers and to promote Canada as a career destination for foreign researchers and highly qualified personnel; and to strengthen overall bilateral S&T relations.
The Department of Biotechnology (DBT) will be the implementing organization on the Indian side, while the International Science and Technology Partnerships Canada (ISTPCanada), a non-governmental organization will be implementing the project on the Canadian side.
This Canada-India programme aims to foster and support the development of collaborative R&D projects that bring together companies, research organizations, academics and other collaborators from both countries for the joint development of innovative products or processes. It aims to stimulate innovative R&D projects (engaging small-to-medium-sized companies and/or larger, well established firms) that address a specific market need or challenge; demonstrate high industrial relevance and commercial potential; and aim to deliver benefit to all participants, and more broadly, to both nations. These projects help participants to become more competitive by developing global research-based alliances with the potential to foster increased or expanded international R&D collaboration.
Eligible Indian applicants for this programme include researchers and managers of Indian companies, academic institutions, research hospitals or other R&D institutions (including not-for-profit research institutes recognized by DBT) that are headquartered and operate in India.
Eligible Canadian applicants must be researchers or managers of for-profit companies that operate and are headquartered in Canada. Canadian subsidiaries of companies headquartered outside of Canada are typically not eligible for support. However, as ‘benefit to Canada’ is a key objective and among the most important selection criteria, ISTPCanada may grant an exception to such subsidiaries if they have R&D facilities in Canada and can demonstrate that Canada will accrue clear economic benefit from the bilateral R&D project. Academic institutions, research hospitals, other institutes or research associations are strongly encouraged to participate in the projects as co-investigators.
Each proposal must include an eligible lead from Canada and India. Although it is not mandatory, projects that engage a technology developer and a technology end-user/first customer are strongly encouraged.
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
- William
A. Greenberg *, - Alexander
Varvak, - Sarah
R. Hanson, - Kelvin
Wong, - Hongjun
Huang, - Pei
Chen, and - Mark
J. Burk *
- Author Affiliations
- Edited by Barry M. Trost, Stanford University,
Stanford, CA, and approved February 19, 2004 (received for review November
14, 2003)
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 (1–5). 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 (6–8), 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 (10–12), 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.
View larger version:
Fig. 1.
DERA-catalyzed tandem aldol
reaction.
View larger version:
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 (R
Cl),
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.
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.
View larger version:
Fig. 3.
Synthesis of lactone (1).
Reagents and conditions: a, DERA, H2O, room temperature, 3 h; b,
NaOCl, HOAc, H2O, room temperature, 3 h.
View larger version:
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.
View larger version:
Fig. 5.
Preparation of chiral standards for
analysis of 1. Reagents and conditions: a, NaBH4, MeOH; b,
15% trifluoroacetic acid, CH2Cl2, 1% triethylsilane.
View larger version:
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.
View larger version:
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.
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.
View larger version:
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.
View larger version:
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.
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.
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.
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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