Combined catalytic reactionsNatures way
Going from traditional step-by-step methods to one-pot coupled conversion saves raw materials and energy and reduces waste.
Organic synthesischemistry as it is done in the laboratory or manufacturing planttraditionally uses a step-by-step approach. In a typical sequence, a starting material A is converted into a final product D, and intermediate products B and C have to be isolated and purified in each conversion step.
Such multistep organic syntheses are still quite common in todays fine chemical industry, and they suffer from several disadvantages. They are often carried out noncatalytically using relatively large amounts of reagents that produce many kilograms of waste per kilo of final product. The separation and purification steps needed after each conversion step produce waste heat as energy is consumed. They require extra energy to overcome the thermodynamic hurdles to produce and isolate intermediates B and C if they lie in high-energy states.
On the other hand, biosynthesischemistry as Nature performs it in the cells of organismsgoes through a multistep cascade to convert starting material A to final product D without separation of intermediates B and C.
Such multistep combined syntheses are common in everyday life. They are carried out in a fully catalytic way by using enzymes with relatively limited amounts of reagents (cofactors) and thus produce much less waste. The mutual compatibility and high selectivity of the enzymatic conversions make it possible to proceed without intermediate recovery steps. They save energy by avoiding the separation and isolation of intermediates B and C.
The potential power of combined catalytic conversions to overcome thermodynamic hurdles in multistep syntheses is demonstrated here for the well-known glycolysis pathway.
The equilibria between fructose 1,6-diphosphate (A), dihydroxyacetone phosphate (B), and glyceraldehyde 3-phosphate (C) are quite unfavorable: [A]/[B]/[C] > 1000:20:1. Nevertheless, complete transformation of A to lactic acid (D) occurs through a coupled multienzymatic conversion of the more highly energetic C intermediate. In addition, the success of this overall conversion is brought about by the very fast aldolase- and isomerase-catalyzed equilibria, A B and B C, respectively.
Challenges in synthesis
The result of these efforts should drastically diminish the costs of energy consumption and waste treatment characteristic of current multistep processes in the fine chemical industry.
For the next generation of biosynthesis (microbial conversions), the challenge is to
These steps should free fermentation processes from excessively using renewables that end up in undesired, low-value biomass byproducts and minimize additional chemical modification steps to obtain the final product.
An industrial exampleBiotechnology
In the 1970s, cephalexin synthesis consisted of one fermentation reaction (step 3) together with seven organic synthesis steps (2):
1. Benzaldehyde → D-phenylglycine
2. D-Phenylglycine → D-phenylglycine chloride
3. Sugar + phenylacetic acid → penicillin G
4. Penicillin G → penicillin G sulfoxide
5. Penicillin G sulfoxide → penicillin G sulfoxide trimethylsilyl ester
6. Ring enlargement of penicillin G sulfoxide trimethylsilyl ester to fully protected 7-aminodeacetoxypenicillanic acid (7-ADCA)
7. Hydrolysis of fully protected 7-ADCA to 7-ADCA
8. D-Phenylglycine chloride + 7-ADCA → cephalexin
This multistep sequence used large amounts of energy (e.g., in low-temperature conversions in steps 57 and many recovery and solvent recycle steps), reagents and organic solvents (steps 2 and 48), and phenylacetic acid (no recycling was possible after the chemical hydrolysis step 7).
1. Benzaldehyde → D-phenylglycine amide
2. Sugar + adipic acid → 7-ADCA adipic acid amide
3. 7-ADCA adipic acid amide → 7-ADCA + adipic acid (recycled for use in step 2)
4. D-Phenylglycine amide + 7-ADCA → cephalexin
This sequence avoids low-temperature conversions, most of the previously used reagents and organic solvents, and consumption of adipic acid. The key improvements were
The following reagents (often used in more than stoichiometric amounts) and solvents (that could not be recycled completely) are no longer needed in the new cephalexin synthesis (2):
The two-step process reduced solvents by ~90%, auxiliary reagents by 100%, energy consumption by ~40%, and waste by ~15%. Highlights of the process are in the box, DSMs process for 7-ADCA.
In addition to the use of metabolic pathwayengineered microorganisms on a multiton scale, several promising multistep one-pot catalytic conversions have been described on the laboratory and industrial scale, using either a combination of enzymes or a combination of an enzyme and a chemical catalyst (see Table 1).
We can sum up the current state of this field as follows: Most of the multistep one-pot conversions have been reported in carbohydrate synthesis, using combinations of enzymatic conversions. Combinations of metal-catalyzed and biocatalytic conversions are not yet common. After some early examples in the 1980s, more than a decade of inactivity ensued, followed by an increasing interest during the past few years for combined catalytic conversions.
The very first example of the combined action of an enzyme and a metal catalyst was the direct one-pot conversion of D-glucose to D-mannitol. In this process, the enzyme glucose isomerase converts glucose to a ~1:1 glucosefructose mixture and ensures that this mixture remains in equilibrium. At the same time, the copper catalyst preferentially hydrogenates the fructose to mannitol (Figure 2).
The kinetic picture is quite complicated (Table 2). The three types of kinetics are expressed in TONs (turnover numbers). In the enzymatic isomerization (MichaelisMenten kinetics), only two of the six sugar forms are substrates for the enzyme. In the heterogeneous hydrogenation on copper, the adsorption constants vary from 3 to 10 L/mol. Only ~25% of the copper surface is covered with D-fructose, but that sugar reacts much faster than the adsorbed glucose. In the acidbase homogeneous catalysis of mutarotation, the rate for D-glucose is 50 times slower than that for D-fructose.
This is a good example of the selective conversion of one of two enantiomers in equilibrium. The fine tuning of simultaneous catalytic conversion is important for the synthesis of enantiomerically pure compounds in 100% yield from racemic starting materials.
Some recent examples on the laboratory scale have been reported (1115) for the concomitant action of a chemical and a biocatalyst for the required racemization and enantioselective conversions, respectively.
A multienzyme one-pot example
The four consecutive enzymatic conversion steps in one reactor without any separation of intermediates consist of
Combined with the broad substrate specificity of DHAP aldolases, this sequence constitutes a simple procedure for the synthesis of a wide variety of carbohydrates from readily available glycerol and pyrophosphate (5, 6).
Investigations of such multistep synthesis methods without isolation of intermediate products require appropriate in situ analytical methods so that the investigator knows what is happening during the consecutive conversions. The combination of NMR spectroscopy with starting materials selectively enriched in isotopes such as 13C, 15N, and 17O provides a powerful window (18, 19). In this way, a sequence of conversions can be well characterized, even in complicated matrices of catalysts, reagents, and mixed solvent systems.
This approach is illustrated in Figure 5 for the cross-linking of in situgenerated galactohexodialdose from lactose protein mixtures, as studied by using selective 13C labeling (20). The undesired background from 13C NMR signals of the other components of the reaction mixture is suppressed, allowing straightforward detection of the reaction species of interest.
A synthesis renaissance
In this respect, future clean synthesis methods should be inspired by the achievements in the field of modern detergent formulations that have up to six different enzymes in them (22): an advanced multicatalytic one-pot conversion of dirty laundry to clean laundry plus dirt, with the washing machine as the in-house catalytic reactor that simultaneously separates the product (clean laundry) from waste (dirt).
Economic and environmental goals will cause chemical and biotechnological conversion methods to merge into integrated biologychemistry routes, based on optimum feedstocks. Conservation of matter and energy from starting material to end product is required to achieve sustainable conversion processes. Neither chemistry nor biotechnology, neither fossil fuels nor renewable feedstocks will be the ultimate winneronly the combination of these disciplines and resources.
Rob Schoevaart is a postdoctoral fellow at the Leiden Institute of Chemistry (Department of Industrial Fermentative Chemistry, Leiden University, Einsteinweg 55, PO Box 9502, Leiden, 2300 RA, The Netherlands; firstname.lastname@example.org). He received his M.Sc. in organic chemistry and microbiology from Nij megen University, The Netherlands, and his Ph.D. in organic chemistry from the Delft University of Technology, The Netherlands. At Leiden, he works on combined biological and chemical catalytic conversion procedures. His experience includes the application of multienzyme systems in organic synthesis and with in situ NMR analysis, and he has published six scientific papers.
Tom Kieboom is general science manager at DSM Food Specialties R&D (Postbus 1, Delft, 2600 MA, The Netherlands; email@example.com) and part-time professor of organic chemistry in the Department of Industrial Fermentative Chemistry, Leiden Institute of Chemistry (firstname.lastname@example.org). He received his M.Sc. and Ph.D. in organic chemistry from Delft University of Technology, where he later was appointed as full professor in bioorganic chemistry. His main research interests and expertise are in the application of chemical and biocatalysis in organic chemistry, with special emphasis toward carbohydrate conversions in combination with in situ NMR. He has published more than 160 scientific papers.