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Chromium -VI Reagents: Synthetic Applications (SpringerBriefs in Molecular Science)

First, clothings coloured with natural dyes reduces the challenge of toxic runoff that could be suffered when synthetic dyes are used in textile and dyeing process. Secondly, using dyes from plants that grow in our environment eliminates the problems associated with production of synthetic dyes. Thirdly, natural dyes are non-toxic to work with. Fourthly, in working with natural dyes one gains enlightening experience from direct connection with nature.

Chromophores are groups with multiple bonds responsible for the colour of a compound. Auxochromes on the other hand are colour enhancers. They are groups that make coloured substances act as dyes. Many plants in Nigeria are sources of natural dyes Akpuaka, ; Nnabugwu and Okoro, , among which are R. Some of the local names by which R. Crude extracts from R. They are also used locally as a medication for the treatment of chicken-pox in children Akpuaka, Literature has shown that R.

Pterocarpus species is one of such plants which have been used for treatment of type 2 diabetes Mukherjee et al. The stem bark powder of Pterocarpus spp. The bark of the plant is used in treatment of some broncho-pulmorary ailments Akoegninou et al. Other useful properties exhibited by T. In addition to the aforementioned, the dyes from R.

These dyestuffs and many others Akpuaka, ; Nnabugwu and Okoro, ; Akpuaka et al. Considering the several applications of these dyes and the growing interest in the use of natural dyes, there is urgent need to elucidate the structures of these dyes. Moreover, the knowledge of the dye structures is useful for forensic investigations and the study of art history.

The present preliminary work investigated the basic chromophores and auxochromes present in dyestuffs from R. This is a vital step in the right direction towards understanding the makeup of some of these natural dyes. Ultraviolet and visible spectra were obtained on a Unico-Uv PC spectrophotometer using 1 cm quartz cells. The solvent used was ethanol or chloroform as the case may be. The absorption maxima were recorded in nanometers nm. The dyestuffs were extracted and isolated as reported in the literature Nnabugwu and Okoro, About g of R.

The soaked seeds were later agitated and there after filtered with a Buckner funnel. The light yellow filtrate was stirred vigorously with a magnetic stirrer for several hours until a dark blue solution was obtained. The solution was kept at room temperature for 2 weeks during which the dark blue dye precipitated out and collected at the bottom of the containing vessel as sediment. The supernatant liquid was poured out while the dark blue R.

About g of ground stem wood of P. After agitation, the steeped wood was filtered. The dark-red filtrate was concentrated via simple distillation to about one-third its original volume. The dark-red dye was dried at room temperature. About g of the T. The wood was agitated and filtered to obtain an orange-red filtrate. The filtrate was concentrated by simple distillation followed by evaporation through reduced pressure. The dyes being organic compounds were qualitatively analyzed by standard methods used for analysis of organic compounds as described in Vogel Furniss et al.

Specifically, the following tests were carried out on the dyestuffs, including melting point determination. About 50 mg of each of the dyestuffs was placed in an ignition tube and four pieces of metallic sodium 2 mm cube were added to each of the tubes. The tubes were heated gently at first in a Burnsen burner and then more vigorously until fumes have ceased to evolve. While the tubes were still red hot, each was dipped in a clean mortar containing about 10 ml of distilled water.

The tubes were then crushed inside the mortar and the solution was filtered. The filtrates were used to test for the presence of sulphur, nitrogen and halogens. Few crystals of sodium nitroprusside were added to 5 ml of each of the filtrates. Purple or deep blue violet colour indicates the presence of sulphur. A little quantity of ferric chloride was added to 5 ml of each filtrate and heated. The hot solution was cooled under tap and few drops of ferric chloride were added to it followed by acidification with dilute sulphuric acid. Blue or greenish blue colour indicates the presence of nitrogen.

Concentrated HNO 3 was added to 5 ml of each filtrate, heated and cooled under tap. Few drops of silver nitrate solution were then added. Precipitate indicates the presence of a halogen. Specific tests for functional groups were done on the dyestuffs to determine the functional groups present in them.

Dark red precipitate indicates the presence of aldehyde or ketone. Silver mirror indicates the presence of aldehyde while absence of silver mirror indicates the presence of ketone. Concentrated HCl was added to each of the dyestuffs and cooled. Then sodium nitrite was added to the solution. A brown precipitate indicates the presence of aromatic amines. Formation of a derivative gray colour confirms the presence of amines. Methanolic anhydrous iron III chloride solution was added to a solution of the dyestuff. A green solution indicates the presence of monohydric phenol. Neutral iron III chloride solution was added to a few crystals of the dyestuff.

Brick red solution indicates the presence of monohydric phenols. This method made it susceptible to sequence-specific bias or loss of specific sequences. Lynx Therapeutics merged with Solexa later acquired by Illumina in , leading to the development of sequencing-by-synthesis, a simpler approach acquired from Manteia Predictive Medicine , which rendered MPSS obsolete. However, the essential properties of the MPSS output were typical of later high-throughput data types, including hundreds of thousands of short DNA sequences. The Polony sequencing method, developed in the laboratory of George M.

Church at Harvard, was among the first high-throughput sequencing systems and was used to sequence a full E. A parallelized version of pyrosequencing was developed by Life Sciences , which has since been acquired by Roche Diagnostics. The method amplifies DNA inside water droplets in an oil solution emulsion PCR , with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony. The sequencing machine contains many picoliter -volume wells each containing a single bead and sequencing enzymes.

Pyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs. Solexa , now part of Illumina , was founded by Shankar Balasubramanian and David Klenerman in , and developed a sequencing method based on reversible dye-terminators technology, and engineered polymerases. In , Solexa acquired the company Manteia Predictive Medicine in order to gain a massivelly parallel sequencing technology invented in by Pascal Mayer and Laurent Farinelli.

The cluster technology was co-acquired with Lynx Therapeutics of California. In this method, DNA molecules and primers are first attached on a slide or flow cell and amplified with polymerase so that local clonal DNA colonies, later coined "DNA clusters", are formed. To determine the sequence, four types of reversible terminator bases RT-bases are added and non-incorporated nucleotides are washed away. A camera takes images of the fluorescently labeled nucleotides. Then the dye, along with the terminal 3' blocker, is chemically removed from the DNA, allowing for the next cycle to begin.

Unlike pyrosequencing, the DNA chains are extended one nucleotide at a time and image acquisition can be performed at a delayed moment, allowing for very large arrays of DNA colonies to be captured by sequential images taken from a single camera. Decoupling the enzymatic reaction and the image capture allows for optimal throughput and theoretically unlimited sequencing capacity.

Applications

Here, a pool of all possible oligonucleotides of a fixed length are labeled according to the sequenced position. Oligonucleotides are annealed and ligated; the preferential ligation by DNA ligase for matching sequences results in a signal informative of the nucleotide at that position. The resulting beads, each containing single copies of the same DNA molecule, are deposited on a glass slide.

Ion Torrent Systems Inc. This method of sequencing is based on the detection of hydrogen ions that are released during the polymerisation of DNA , as opposed to the optical methods used in other sequencing systems. A microwell containing a template DNA strand to be sequenced is flooded with a single type of nucleotide. If the introduced nucleotide is complementary to the leading template nucleotide it is incorporated into the growing complementary strand.

This causes the release of a hydrogen ion that triggers a hypersensitive ion sensor, which indicates that a reaction has occurred. If homopolymer repeats are present in the template sequence multiple nucleotides will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal. DNA nanoball sequencing is a type of high throughput sequencing technology used to determine the entire genomic sequence of an organism.

The company Complete Genomics uses this technology to sequence samples submitted by independent researchers. Unchained sequencing by ligation is then used to determine the nucleotide sequence. Heliscope sequencing is a method of single-molecule sequencing developed by Helicos Biosciences. It uses DNA fragments with added poly-A tail adapters which are attached to the flow cell surface. The next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides one nucleotide type at a time, as with the Sanger method.

The reads are performed by the Heliscope sequencer. SMRT sequencing is based on the sequencing by synthesis approach. The sequencing is performed with use of unmodified polymerase attached to the ZMW bottom and fluorescently labelled nucleotides flowing freely in the solution. The wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected.

The fluorescent label is detached from the nucleotide upon its incorporation into the DNA strand, leaving an unmodified DNA strand. According to Pacific Biosciences PacBio , the SMRT technology developer, this methodology allows detection of nucleotide modifications such as cytosine methylation. This happens through the observation of polymerase kinetics. This approach allows reads of 20, nucleotides or more, with average read lengths of 5 kilobases.

The DNA passing through the nanopore changes its ion current. This change is dependent on the shape, size and length of the DNA sequence. Each type of the nucleotide blocks the ion flow through the pore for a different period of time. The method does not require modified nucleotides and is performed in real time.

Early industrial research into this method was based on a technique called 'Exonuclease sequencing', where the readout of electrical signals occurring at nucleotides passing by alpha- hemolysin pores covalently bound with cyclodextrin. Two main areas of nanopore sequencing in development are solid state nanopore sequencing, and protein based nanopore sequencing. The concept originated from the idea that single stranded DNA or RNA molecules can be electrophoretically driven in a strict linear sequence through a biological pore that can be less than eight nanometers, and can be detected given that the molecules release an ionic current while moving through the pore.

The pore contains a detection region capable of recognizing different bases, with each base generating various time specific signals corresponding to the sequence of bases as they cross the pore which are then evaluated. Another approach uses measurements of the electrical tunnelling currents across single-strand DNA as it moves through a channel. Depending on its electronic structure, each base affects the tunnelling current differently, allowing differentiation between different bases. The use of tunnelling currents has the potential to sequence orders of magnitude faster than ionic current methods and the sequencing of several DNA oligomers and micro-RNA has already been achieved.

Sequencing by hybridization is a non-enzymatic method that uses a DNA microarray. A single pool of DNA whose sequence is to be determined is fluorescently labeled and hybridized to an array containing known sequences. Strong hybridization signals from a given spot on the array identifies its sequence in the DNA being sequenced.

This method of sequencing utilizes binding characteristics of a library of short single stranded DNA molecules oligonucleotides , also called DNA probes, to reconstruct a target DNA sequence. Non-specific hybrids are removed by washing and the target DNA is eluted. The benefit of this sequencing type is its ability to capture a large number of targets with a homogenous coverage. However, with the advent of solution-based hybridization, much less equipment and chemicals are necessary.

Mass spectrometry may be used to determine DNA sequences. With this method, DNA fragments generated by chain-termination sequencing reactions are compared by mass rather than by size. The mass of each nucleotide is different from the others and this difference is detectable by mass spectrometry. Single-nucleotide mutations in a fragment can be more easily detected with MS than by gel electrophoresis alone. The higher resolution of DNA fragments permitted by MS-based methods is of special interest to researchers in forensic science, as they may wish to find single-nucleotide polymorphisms in human DNA samples to identify individuals.

These samples may be highly degraded so forensic researchers often prefer mitochondrial DNA for its higher stability and applications for lineage studies. MS-based sequencing methods have been used to compare the sequences of human mitochondrial DNA from samples in a Federal Bureau of Investigation database [] and from bones found in mass graves of World War I soldiers. Even so, a recent study did use the short sequence reads and mass spectroscopy to compare single-nucleotide polymorphisms in pathogenic Streptococcus strains.

This approach directly visualizes the sequence of DNA molecules using electron microscopy. The first identification of DNA base pairs within intact DNA molecules by enzymatically incorporating modified bases, which contain atoms of increased atomic number, direct visualization and identification of individually labeled bases within a synthetic 3, base-pair DNA molecule and a 7, base-pair viral genome has been demonstrated.

One end of DNA to be sequenced is attached to another bead, with both beads being placed in optical traps. RNAP motion during transcription brings the beads in closer and their relative distance changes, which can then be recorded at a single nucleotide resolution. The sequence is deduced based on the four readouts with lowered concentrations of each of the four nucleotide types, similarly to the Sanger method.

A method has been developed to analyze full sets of protein interactions using a combination of pyrosequencing and an in vitro virus mRNA display method. The mRNA may then be amplified and sequenced. The combined method was titled IVV-HiTSeq and can be performed under cell-free conditions, though its results may not be representative of in vivo conditions. The success of a DNA sequencing protocol is dependent on the sample preparation.

As mentioned, several groups of process indicators could be used for measuring the sustainability or greenness of a process. There is no single set of universally accepted indicators. These indicators can take up different values if they are considered for the short, medium, or long term and also will depend on the type of stakeholders.

If that happens, there will be a steady increase in the total amount of anthropogenic material in that locality. Anthropogenic materials released into the environment should not affect global material cycles. Renewable resources should only be used at a rate not exceeding the natural local production rate of the renewable resource. If this principle is not followed, there will be a slow erosion of the renewable resource.

The natural variety of species and landscapes should be sustained maintain biodiversity. Five factors contribute to the total land area: Conclusions Green chemistry has become the important philosophy for the 21st century and beyond. Chemical and allied industries have taken this philosophy very seriously due to societal and governmental pressures with respect to environmental issues.

There are several levels in the green chemistry hierarchy, and each level is interested in certain indicators. Both developing a line of sight between the process and the environmental impact at a larger dimension and assigning a cost to this impact are probably impossible.

In addition, all the physical and chemical data, the short- and long-term impacts of chemicals on the environment, and the relationship between people and the ecology have not been fully studied.

On hindsight people realize the mistakes they have made, but by that time it may be too late. In addition to considering an ideal process and product, one needs to include an ideal user as well. The user could also help support the green chemistry initiative by being responsible in product selection, usage, and disposal. The user may be able to bring pressure on manufacturing organizations to adapt to sustainable development. This book deals with the latest developments in green chemistry and green process technologies, with relevant industrial examples.

Using principles of sustainability to determine what is green—a corporate perspective, Green Chem. Introduction 25 IChemE, The sustainability metrics. A new dimension in ecological evaluation, Ecol. Case studies for a novel VOC recovery technology, Environ. Drivers, metrics, and reduction to practice, Ann.

Adoption of green chemistry: An analysis based on US patents, Research Policy, Homogeneous catalysis leads the way, Angew. Methodology and Field Handbook. Environmental impacts, energy consumption, and engineering economics, Waste Management, Synthetic chemistry continues to develop various techniques for obtaining better products with less damaging environmental impacts. The control of reactivity and selectivity is always the central subject in the development of a new methodology of organic synthesis.

Novel, highly selective reagents appear every month. Periodic review articles and books appear in the literature on these newer reagents. The scope of this chapter is to focus on newer techniques experimental for improving the yield and reducing the duration of the reactions and also to discuss the need for a good synthetic design. In other words, newer methods of kinetic activation, which minimize the energy input by optimizing reaction conditions, will be discussed along with the need for an elegant synthetic design.

In most reactions, the reaction vessel provides three components as shown in Fig. Components of chemical reaction. The role of alternate reagents, solvents, and catalysts in greening chemical reactions is discussed in other chapters. In this chapter we shall see newer methods of kinetic activation of molecules in chemical reactions. Pressure and temperature are important parameters in reaction processes in chemical systems. However, it is a less well-known fact that other than thermally initiated reactions can also lead to sustainable results.

The basic requirement is to capture the energy required by a reaction. The energy required for synthesis as well as that required for cooling are of interest here. Approaches are being taken and possibilities investigated to use until now scarcely used forms of energy, so-called nonclassical energy forms, in order to optimize the duration and product yield and avoid undesired side products.

Teams working in this area are also interested in the energetic aspects of the preparation of starting substances and Newer Synthetic Methods 29 products and the conditioning of reaction systems e. We now have six well-documented methods of activating molecules in chemical reactions, which can be grouped as follows: Each of these methods has its advantages and niche areas of applications, alongside its inherent limitations. A comparative study of these techniques is given in Table 2. What do we mean by classical and nonclassical energy forms?

In classical processes, energy is added to the system by heat transfer; by electromagnetic radiation in the ultraviolet UV , visible, or infrared IR range; or in the form of electrical energy. On the other hand, microwave radiation, ultrasound, and the direct application of mechanical energy are among the nonclassical forms. Not only can this high-energy input enhance mechanical effects in heterogeneous processes, but it is also known to induce new reactions, leading to the formation of unexpected chemical species.

What makes sonochemistry unique is the remarkable phenomenon of cavitation, currently the subject of intense research, which has already yielded thought-provoking results. With highfrequency ultrasound, the chemistry produced displays characteristics similar to high-energy radiation more radicals are created. One of the most striking features in sonochemistry is that there is often an optimum value for the reaction temperature.

In contrast to classical chemistry, most of the time it is not necessary to go to higher temperatures to accelerate a process. In fact, heterogeneous reactions are those in which ultrasound is likely to play the most important role by selective accelerations between potentially competitive pathways. It was reported that the sonochemical decomposition of volatile organometallic precursors was shown to produce nanostructured materials in various forms with high catalytic activities.

Ultrasound is known to enhance the reaction rate, thus minimizing the duration of a reaction. A large number of published examples, which highlight this observation, are shown in Appendix 2. Use of Microwaves for Synthesis In synthetic chemistry, was an important year for the use of microwave devices. Since that year, countless syntheses initiated by microwaves have been carried out on a laboratory scale. The result is often a drastic reduction in the reaction time with comparable product yields, if microwaves are used instead of classical methods of energy input.

Unwanted side reactions can often be suppressed and solvents dispensed with. Reactions listed in Appendix 2. Apart from the obvious advantages of the use of microwaves in chemical syntheses, microwave technologies are being tested as energy- and cost-saving alternatives.

Newer Synthetic Methods 33 Electro-Organic Methods Over the past 25 to 30 years, the use of electrochemistry as a synthetic tool in organic chemistry has increased remarkably. According to Pletcher and Walsh , more than electro-organic synthetic processes have been piloted at levels ranging from a few tons up to tons. Many excellent reviews and publications highlight the synthetic utility of electro-organic methods Lund and Baizer, These cover a broad spectrum of applications of electrochemical methods in organic synthesis, including their use in the pharmaceutical industry.

Mild reaction conditions, ease of control of solvent and counter-ions, high yields, high selectivities, as well as the use of readily available equipment, simply designed cells, and regular organic glassware make the electrochemical syntheses very competitive to the conventional methods in organic synthesis. This approach was very successful for synthesis of the organosilicon compounds Fry and Touster, Elegant and Cost-Effective Synthetic Design The heart of synthesis is in the design of the synthetic scheme for the given target molecule.

All the technological advances discussed above can only supplement the synthetic scheme. Structures of atropine, tropinone, and cocaine. Thus, synthesis was often a matter of utilitarian necessity rather than the creative, elegant art form illustrated by the work of many of the great synthetic chemists such as Woodward and Corey. In , Robinson approached the synthesis in a totally radical way. Tropinone was obtained by condensation of succinaldehyde with acetone and methylamine in aqueous solution see Fig. In fact, we can view this synthesis of tropinone as one of the earliest examples of multicomponent reactions MCR.

MCRs are convergent reactions in which three or more starting materials react to form a product, where basically all or most of the atoms contribute to the newly formed product. Carbonyl compounds played a crucial role in the early discovery of multicomponent reactions. One example is the Mannich reaction see Fig. Thus, the chemistry development time, which can typically take up to 6 months for a linear six-step synthesis, is considerably shortened.

With only a limited number of chemists and technicians, more scaffold synthesis programs can be achieved within a shorter time. Conclusions The various reaction types most commonly used in synthesis can have different degrees of impact on human health and the environment. Substitution reactions, on the other hand, necessarily generate stoichiometric quantities of substances as byproducts and 38 Green Chemistry and Processes waste. As such, elimination reactions are among the least atom-economical transformations.

For any synthetic transformation, it is important to evaluate the hazardous properties of all substances necessarily being generated from the transformation, just as it is important to evaluate the hazardous properties of all starting materials and reagents that are added in a synthetic transformation. The atom-economy of various reaction types is shown in Fig. The most atom-economy—suited reactions are condensations, multicomponent reactions, and rearrangements. Atom-economy of various reaction types. Maximize yield per step. Maximize atom-economy per step. In multistep syntheses, perform the following: Minimize frequency of substitutions protecting group strategies and redox reactions.

If forced to use oxidations, opt for hydrogen peroxide as oxidant. If forced to use reductions, opt for hydrogen as reductant. Devise catalytic methods where catalysts are recycled and reused. Opt for solventless reactions, recycle solvents, or use benign solvents ionic liquids. EtOH, ZnBr2, , r. Lie Ken Jie, M. Newer Synthetic Methods 43 NaOH, MW, 25sec a: Newer Synthetic Methods 51 In the last decade, green chemistry has been widely recognized and accepted as a new means for sustainable development. Industries are often forced to pay heavy prices to meet with the standards set by the pollution regulatory boards while using the traditional methods of treating or recycling waste.

Also, with growing environmental problems, law-making boards are now looking more critically at the possible hazardous effects of a larger number of chemical substances. This can be quite obviously attributed to three general characteristics of catalysts: Catalytic reagents reduce the energy of the transition state, thereby reducing the energy input required for a process. Catalysts are required in small quantities.

In the case of biocatalysts, the number of catalysts generally enzymes needed compared to the quantity of reactants is very low. The regeneration and reversibility of catalysts are good for green processes. As much as it is a key in achieving economic objectives, catalysis is also a powerful tool in realizing the goals of green chemistry. It is calculated by dividing the molecular weight of the desired product by the sum total of the molecular weight of all substances produced in the stoichiometric equation for the reactions involved.

Some authors also describe it as the number of atoms of all the reactants that are converted into atoms of the desired product in a reaction. For instance, when replaced with cleaner catalyzed oxidation, traditional oxidations using oxidants such as permanganate or chromium reagent as shown in Fig. Jones oxidation of secondary alcohol. Atom-economical oxidation of secondary alcohol.


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The oxidation contained in Fig. Trost and co-workers Trost, used a variety of palladium catalysts to effect allylic alkylation reaction. The reaction, as it occurs at room temperature, is also an example of catalysis reducing energy usage. Though the usage of HF, a toxic substance, is a drawback of the process, the recovery of HF is effected with The process shown in Fig. This leaves a need for truly catalytic procedures: Use of zeolites in an acid-catalyzed rearrangement of epoxides to carbonyl compounds Elings et al.

Traditionally, Lewis acids such as ZnCl2 were used in stoichiometric amounts for the type of reaction displayed in Fig. The following examples are two commercially relevant processes. The products are precursors of chemicals used for their fragrance see Fig. Zeolites and clay-catalyzed, high-AE reactions.

The use of zeolites in the manufacture of cumene is of immense importance. About 7 million metric tons of cumene are produced annually worldwide. The earlier-used process involved alkylation of benzene over a solid phosphoric acid or an aluminum chloride catalyst. Both catalysts are toxic in nature. In addition, it also generates less waste and requires less energy than the earlier catalysts, thus simultaneously satisfying various conditions of green chemistry. The use of zeolites in making industrial processes ecocompatible is growing with the widespread research on using these as catalysts.

One such example of zeolite being used to better the existing process is that of the Meerwin—Ponndorf—Verly MPV reduction. The MPV reduction process is an extensively used technology for reducing aldehydes and ketones to their corresponding alcohols. MPV reduction using zeolite. The stoichiometric requirement of aluminum alkoxide, due to the slow exchange of the alkoxy group, was an inherent drawback in the method.

The example in Fig. In this reaction, the trans-alcohol was the preferred product in the traditional MPV reduction. The zeolitecatalyzed reaction forms the thermally less stable cis-isomer, which is an important fragrance chemical intermediate. Catalysis offers an edge over stoichiometric reactions in achieving selectivity in production, when mono substitution is preferred over disubstitution, when one stereo-isomer is preferred over another or one regioisomer over another.

Hence, by driving the reaction to a preferred product, catalyzed reactions decrease the amount of waste generated while reducing the energy requirements, as mentioned earlier. The contribution of Spiney and Gogate Spivey and Gogate, in developing heterogeneous catalysts for the condensation of acetone to methyl isobutyl ketone MIBK is commendable. The reaction typically requires stoichiometric amounts of base and could also result in considerably overcondensed products. In the production of biologically active molecules pharmaceuticals and pesticides , there is often a need to produce chiral molecules as the pure enantiomer.

CO , electrolysis 2 2. This need has directed the focus onto asymmetric catalysis using chiral metal complexes and enzymes. The Novartis process for the synthesis of the optically active herbicide s -metachlor Blaser and Spindler, involves a chiral metal complex as a catalyst see Fig. An iridium I complex of a chiral ferrocenyldiphosphine catalyzes the asymmetric hydrogenation of a prochiral imine, a key step in the process.

Novartis process for the synthesis of the optically active herbicide. Production of phenol from benzene. Solutia USA , in joint work with the Boreskov Institute of Catalysis, Russia, developed a one-step process to manufacture phenol from benzene using nitrous oxide as the oxidant see Fig. Production of cumene from benzene. Production of p-methoxyacetophenone from methoxybenzene. Manufacture of methylethyl ketone MEK from ethylene and butylenes. The Rhodia process for the production of p-hydroxyacetophenone from methoxybenzene using clay as the catalyst eliminates the use of toxic chemicals such as AlCl3 and BF3 and also eliminates toxic waste see Fig.

The Catalytic process for the manufacture of methylethyl ketone MEK from ethylene and butylenes uses a mixture of palladium, vanadium, and molybdenum oxides as catalyst see Fig. The original process used chlorinated chemicals, which led to a large amount of chlorinated waste that posed several problems during disposal.

The Enichem process for the preparation of propene oxide from propylene involves using H2O2 as the oxidizing agent using titanium silicate catalyst see Fig. The Avetis process for preparing halo benzaldehyde is to oxidize corresponding halo toluene using air and a mixture of iron, vanadium, and molybdenum oxide catalyst see Fig. The catalytic process eliminates the formation of chlorinated byproducts. Preparation of propene oxide from propylene. Microbial mediated aromatic ring hydroxylation.

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Biocatalysis is the other option when selectivity sterio or regio is a priority in a reaction. The various aspects of biocatalysis are discussed elsewhere in the book; the following are some examples of biocatalysts that have been used in important synthesis. Kirner conducted microbial ring hydroxylation and side chain oxidation of hetero-aromatics see Fig. As the example in Fig. The classical method calls for the protection of the carboxy group of Penicillin-G, making it a four-step process. Enzymatically, this conversion can be achieved in a single step Sheldon, Genetic engineering also comes in handy when dealing with chemical reactants that are not biological substrates.

It involves the conversion of a ketone into a lactone commonly using the reagent m-chloroperoxybenzoic acid m-CPBA. This reagent is both sensitive to shocks and explosive. This is a classic example of biocatalysis making a reaction eco-compatible. Synthesis of cephalexin through the use of CLECs. The reaction is also run in an aqueous medium.

The industrial scope of the reaction is under study. Enzymes do have their disadvantages. Their solvent incompatibility and instability restrict their industrial use. Altus Biologics has developed cross-linked enzyme crystals CLECs to increase the versatility of enzymes in organic reactions. CLECs exhibit a high level of stability in extreme conditions of temperature and pH and in exposure to both aqueous and organic solvents. The N-protection step of methyl phenyl glycinate in the classical synthesis was eliminated.

Genetically engineered microbes have been used by Draths and Frost a, b to synthesize common but important chemicals such as adipic acid and catechol see Fig. The noteworthy aspect of this work is that the starting materials were renewable feedstock.

This reaction addresses this principle and more, as it can be seen. Classical catechol synthesis beginning with benzene obtained from petroleum, a nonrenewable feedstock involves a multistep process see Fig. Classical synthesis of catechol. Biocatalysis for the synthesis of catechol from a renewable source.

The biocatalyzed reaction is a far better process than the classical one, as it replaces the hazardous starting chemical, benzene, with D-glucose and tremendously decreases the energy demands apart from replacing a nonrenewable feedstock with a renewable one. In a similar effort, Ho and colleagues have succeeded in creating recombinant Saccharomyces yeast that can ferment glucose and xylose simultaneously to ethanol see Fig.

Cellulose biomass made of materials such as grasses, woody plants, etc. Recombinant yeast for fermentation of both glucose and xylose. The central role these catalysts play in directing the course of a reaction, thereby minimizing or eliminating the formation of side products, cannot be disputed. Hence, catalysis—or rather, designed catalysis—is the mainstay of green chemical practices. References Altus Biologics, Inc.

Catalysis and Green Chemistry 67 Draths, K. Oxford University Press, New York, Biotransformations have been known since the early stages of human civilization and have been used since then to make fermented foods and beverages. These numbers suggest the important role of biocatalysis to the chemical industry.

DNA sequencing

Chemical reactions performed by microorganisms or catalyzed by enzymes are essentially the same as those carried out in 69 70 Green Chemistry and Processes TABLE 4. The most striking differences between enzymes and chemical catalysts are summarized in Table 4. Advantages Within Industrial Applications Microorganisms are undoubtedly the most superior enzyme sources among living organisms.

They show high adaptability to new environments and high growth rates. These characteristics are especially useful for easy handling and large-scale cultivation without a high cost. At present, several new techniques such as extractive biocatalysis, immobilization, biocatalysis in organic solvents, and recombinant DNA technology for enzyme engineering are rapidly being developed in order to make biocatalysis industrially viable. Furthermore, protein engineering and cell technology, such as cell fusion, will become useful techniques for microbial transfor- Biocatalysis: A large number of biologically and chemically useful compounds are prepared through microbial transformations.

Challenges to Make Biocatalysis Industrially Viable Many of the unique features of the enzymatic reactions prove to be limitations for their commercial use. Approaches to overcome the limitations of biocatalysis. Many of these problems have been addressed by a large variety of approaches, all of which can be summarized, as shown both in Fig. Enzyme engineering helps in designing the enzyme for a given transformation. Green Chemistry 73 concentration , use of organic co-solvent or micelles and carrying out the catalysis in organic solvent have been attempted with considerable success.

Numerous reviews on conventional approaches, such as immobilization techniques, genetic engineering, and extractive biocatalysis, have appeared in the literature at regular intervals. The subsequent isolation of DNA polymerases that can function at high temperatures has revolutionized the biotechnology industry. Bioremediation is often the most cost-effective means of cleaning up contaminated soil and water. However, bioremediation may not always be viewed as an appropriate treatment option due to the chemical nature of the contaminant or a mixture of contaminants present at a site.

Yet microbial biodiversity is so immense that it is usually possible to either isolate from nature, or evolve in the laboratory, a microbial culture capable of treating almost any type or mixture of environmental contaminants. Applications for Enrichment Cultures One example of the use of enrichment culture techniques was the isolation of a microbial culture that could degrade a chemical warfare agent. Resolution of CPA by soil-enriched Pseudomonas putida. Still, researchers were able to use enrichment culture techniques to isolate a bacterial culture that cannot only survive exposure to this deadly compound and its derivatives but can also use the chemical as a food source for growth.

Besides their role in degrading unwanted chemicals and pollutants, enrichment culture techniques can also be used to isolate microbial cultures that possess biochemical pathways that are useful for making chemicals by biocatalysis. For example, consider the conversion of racemic 2-chloropropanoic acid CPA to L-CPA, by the dehalogenase from Pseudomonas putida—the necessary strain AJ1 was isolated from the environment with high-chlorine-containing compounds—the road tanker off-loading point see Fig. Enrichment culture techniques can also be used for bioremediation to detoxify xenobiotic pollutants such as polycyclic aromatic hydrocarbons PAHs , heterocyclic polyaromatics, and halogenated aromatics in soils and sediments through microbial degradation.

An effective way to do this is by isolating microbes through enrichment cultures with the substrate one wants to detoxify as a limiting compound. Approach Enrichment culture techniques rely on creating a condition in which the survival and growth of bacterial cultures, with whatever Biocatalysis: Green Chemistry 75 traits are desired, are favored. The nutritional composition of the microbial growth media can be adjusted so that an environmental contaminant serves as the only available source of food and energy or the growth conditions favor the growth of only those bacteria that can grow at a certain temperature or in the presence of other chemicals.

In these ways, the conditions can be controlled in the laboratory to allow for the selection of those bacteria that can provide solutions to various problems. In addition to selecting naturally occurring microbial cultures that possess a desired metabolic trait, it is also possible to use enrichment culture techniques to develop microbial cultures with unique biochemical traits. The substrate range of enzymes catalyzing a certain reaction can be expanded through the use of enrichment culture techniques. This process of evolving new biochemical traits in the laboratory can also be accelerated by the use of directed evolution.

The similar shape and polarity within a series of substrates of different reactivity bioisosteres eliminate effects due to differences between enzyme-substrate binding ES , which is hence a good method of extending the range of substrates that can be chosen for the transformation. A number of instances can be cited from the literature wherein the isosteres had similar transformations. Bacterial dioxygenase-catalyzed cis-dihydroxylation of the tetracyclic arene benzo[c]phenanthrene was found to occur exclusively at fjord region cavity region bonds.

The isosteric compounds benzo[b]naphthol [1,2-d]furan and benzo[b]naphthol[1,2-d]thiophene were also similarly cis-dihydroxylated at the fjord region bonds by bacterial dioxygenases Boyd et al. The isosteres 1,2dihydronaphthalene, 2,3-dihydrobenzothiophene, and 2,3-dihydrobenzofuran gave similar corresponding diol products on incubation with Pseudomonas putida UV4.

Often times in nonaqueous media enzymes exhibit properties drastically different from those displayed in aqueous buffers. These novel properties are given in Table 4. In addition to those mentioned in Table 4. Once organic solvent becomes a reaction medium, there cannot be contamination, which thus precludes release of proteolytic enzymes by microbes and favors the direct application of the process in an industrial setting.

Most proteins enzymes inherently function in an aqueous environment, and hence their behavior in nonaqueous solvents is completely different due to the loss in the three-dimensional structure. Thus, only polar solvents Biocatalysis: Effects 1 Enhances the reaction rates. In many cases maximal rate of the reaction in water—organic mixture is higher than the rate of the same reaction in aqueous buffers Khmelnitsky et al. Changes the reaction pathway by promoting change in substrate cleavage and product synthesis Pal and Gertler, ; Blankeney and Stone, There are cases when stability of enzymes drastically improved in water—organic solvent mixtures as compared to aqueous media Guagliardi et al.

Shift in the direction of the biocatalyzed reaction Deschrevel et al. Homogenous biocatalysis in organic solvents requires the solubility of enzymes in nonaqueous media. Since proteins inherently function in aqueous environments, initial efforts were to study biocatalysis in water—organic mixtures. Biocatalysis in nonaqueous systems using water—miscible organic solvents was studied in detail and has been reviewed previously Butler, ; Blinkovsky et al.

In general, enzyme activity in a homogenous mixture of water—organic solvent is extremely sensitive to the nature and amount of organic solvent Budde and Khmelnitsky, It is interesting to note that in many cases the maximal rate of the reaction in a water—organic mixture is higher than the rate of the same reaction in aqueous buffers Khmelnitsky et al. The most obvious reason for shifting to water—organic mixtures as a reaction medium is to enable bioconversion of substrates poorly soluble in water.

Application of water—organic mixtures often enables a shift in the direction of a biocatalyzed reaction due to a decrease in the content of water, a reaction substrate. For example, synthesis of dipeptides using chymotrypsin and procine pancreatic lipase present good examples of reverse reaction becoming predominant while moving from aqueous media to water—organic mixtures Deschrevel et al. The authors reported an increase in the dipeptide concentration in reaction medium concurrent with the decrease in the water content.

It is worth mentioning that bioconversions in water—organic solvent mixtures are not limited to monomeric enzymes. Aspartate transcarbamylase ATCase from E. Ionic liquids in mixtures with water display a potential to modify properties of biocatalyst. Green Chemistry 79 Malhorta, Conclusions Biocatalysis in nonaqueous systems has proven itself as a powerful tool.

A combination of directed evolution and rational enzyme design is likely to result in many more exciting developments in the near future. Use of Cyclodextrins The use of enzymes as valuable catalysts in organic solvents has been well documented. However, some of their features limit their application in organic synthesis, especially the frequently lowerenzyme activity under nonaqueous conditions, which constitutes a major drawback in the application of enzymes in organic solvents.

In addition, many enzymatic reactions are subject to substrate or product inhibition, leading to a decrease in the reaction rate and enantioselectivity. To overcome these drawbacks and to make enzymes more appealing to synthesis, cyclodextrins are used. The effects of the cyclodextrins range from increasing the availability of insoluble substrates to reducing substrate inhibition to limiting product inhibition.

In each case, the effects of the cyclodextrins are interpreted in terms of the formation of inclusion complexes. It is thus demonstrated that cyclodextrins can be used rationally to increase the utility of enzymes in organic synthesis. In an interesting study, cyclodextrins were used as regulators for the Pseudomonas cepacia lipase PSL and macrocyclic additives to enhance the reaction rate and enantioselectivity E in TABLE 4.

This maintains activity, but is not suited for pure organic solvents. Very helpful in resolution of racemic compounds with high enantioselectivity.

Preliminary characterization of some natural dyes

HIP results in highly active and stable preparations. Sodium bis 2Activity depends on water ethylhexyl content and organic solvent. Molecular biology Involves techniques of techniques molecular biology, and success in this area depends on screening methods. An innovative method of forming an inclusion complex with the product was reported by Easton et al. Use of Crown Ethers Today it is well established that enzymes can be catalytically active in organic solvents. Compared to aqueous solutions, the use of an organic reaction medium can have some interesting advantages, such as the enhanced thermal stability of the enzyme, the easy separation of the suspended enzyme from the reaction medium, the increased solubility of substrates, the favorable equilibrium shift to synthesis over hydrolysis, the suppression of water-dependent side reactions, and possibly the new stereo- selectivity properties of the enzyme.

An important drawback of the use of organic solvents for enzyme reactions is that the activity of the enzyme is generally several orders of magnitude lower than in aqueous solution. Prior lyophilization of the enzyme from an aqueous solution, which is buffered at the pH of optimal aqueous enzyme activity, if necessary in the presence of an inhibitor, improves the activity in organic solvent. In recent years the effects of crown ethers on enzyme reactions in organic solvents have been investigated. Depending on their ring size and structure, crown ethers can form complexes with metal ions, ammonium groups, guanidinium groups, and water, species that are all common in enzymatic reactions.

Therefore, these results show that pretreatment of enzymes by lyophilization with crown ethers or by simply adding C-6 to the organic solution can enhance the enzyme activity to a level where they are suitable for practical applications. Moreover, it was reported that for relatively reactive substrates the enantioselectivity of proteases in organic solvent is very sensitive for small changes in solvent composition.

This offers the possibility to tune the enantioselectivity and to apply these enzymes as catalysts for conversions of both the L- and the D-enantiomers Johan et al. The acceleration of the initial rate, V 0 , ranged from less than fold to more than fold. This suggests that molecular imprinting is likely the primary cause of subtilisin activation by crown ethers, as recently suggested Santos et al.

The PCL co-lyophilized with each additive showed simultaneous enhanced enzyme activity and enantioselectivity when compared to the native lipase lyophilized from buffer alone; in contrast, such enhancement was not observed for the colyophilized CRL. Hence, this method seems to be of practical use for the large-scale production of optically active compounds Mine et al.

Process Design Now that the various methodologies to overcome the limitations of biocatalysis have been discussed, a brief account of process design will give the necessary information to make these processes Biocatalysis: Green Chemistry 83 industrially viable. Three categories of questions need to be answered for any process design: How much material do you need to supply to customer— g, 1 kg, 10 kg? How long does one batch take to run in process per unit of reaction volume? How long do you have to make your delivery? How large is the reaction vessel?

How long can the vessel be practically and safely operated? Establish a baseline process. Determine how to run the process to meet a target delivery.