- Email: [email protected]
Biological process for preparing compounds labelled with stable isotopes
TIBTECH - JANUARY 1986
ability of products derived from transformed cells. In an effort to resolve the situation and to provide WHO with the best advice available, Petricciani announced that he will convene a special multidisciplinary group of the world's top biomedical experts to review the data, discuss the issues, and make recommendations. That meeting is expected to take place in mid-1986 and should provide the final green light for both
WHO and national regulatory authorities. This long awaited acceptance of continuous cell culture by two major regulatory organizations opens the way for the commercialization of a variety of specific products and procedures and was an optimistic introduction to the 1985 ESACT meeting. The next ESACT meeting will be held in Ness Ziona, Israel in April 1987.
Biological process for preparing compounds labelled with stable isotopes The combination of new techniques and instrumentation such as massspectrometry (MS) and nuclear magnetic resonance (NMR), and the development of innovative approaches for the preparing of biological compounds labelled with stable isotopes such as ~3C and 15N, is opening new research areas especially in human studies in biomedicine. New insight into metabolic mechanisms in humans is being gained without the exposure to hazardous radioactive materials. Study of problems such as the determination of maternal-fetal nutritional circulation or nutritional support in newborns 1'2 could not be envisioned without the use of stable isotopes. Numerous other metabolic studies in humans are being conducted using GC-MS (gas chromatographic-mass spectrometric) analysis of body fluids containing stable isotope tracers a. In vivo NMR techniques, still under development, offer new ways of observing the biochemical events of metabolism in living systems, from bacteria to man, in a non-invasive manner 4. In vivo NMR requires tracers labelled with nuclei such as those of ~3C and 25N. These emerging methodologies are
evolving into major tools for medical studies but the scarcity and prices of stable isotope labelled compounds are the bottleneck in this development. These methods depend on the availability of such compounds, in a wide variety of useful forms and in sufficient quantities. To illustrate the dimensions of the problem, amounts of 0.5-1 g, costing $100-1000 per gram depending on the amino acid in question, are required for metabolic determination of an 15N-labelled amino acid in one adult, using the sensitive GC-MS technique. Quantities of 13C-labelled compounds many times higher are required for in vivo NMR spectroscopy, which has lower sensitivity of detection. Currently most stable isotope compounds are prepared by long, isotopically inefficient, multi-step organic syntheses. Sometimes racemic products are obtained when only one isomer is of interest. The synthesis of radioactive compounds using organic methods is well established, but these procedures are not suitable for the synthesis of stable isotope labelled compounds. The former are made in miniscule amounts and are used with an unlabelled carrier, while the latter are prepared in gram
© 1986, Elsevier Science Publishers B.V., Amsterdam 0166- 9430/86/$02.00
The proceedings of the PHS meeting are almost ready for publication as a supplement to In vitro, whereas the proceedings of this year's ESACT meeting will be published in a volume of Developments in Biological Standardization published by S. Karger. R. BLIEM
Cambridge Life Sciences, Cambridge Science Park, Milton Road, Cambridge CB4 2QN, UK.
quantities with almost all of the molecules enriched. Compared to 13C and 15N, deuterated compounds are easier to make by organic synthesis, but their use is restricted by the strong isotope effects associated with deuterium. Furthermore, some deuterated sites are susceptible to loss of label by exchange with water, or are easily scrambled enzymatically, unlike most carbons and nitrogens. However, specifically deuterated drugs are used in pharmacological studies and the isotopic effect may be used to advantage if it can be designed to slow certain metabolic steps 5. Biotechnology offers attractive alternatives to organic methods of preparation, avoiding the use of complicated precursors, the isolation of intermediates, the intricate purification and optical resolution of products. Therefore, they result in high yields and in efficient isotope incorporation. Several approaches are used in biotechnological preparation of labelled compounds. One is to label whole organisms by growing them on labelled precursors and subsequently fractionating the biomass into various biochemicals. It is most commonly used with plants and algae growing o n 13C02. Although the incorporation of 13C02 by photosynthesis is very efficient, the technique is wasteful because of (a) the enormous diversion of 13C into unwanted biomass and (b) the elaborate purification schemes needed. Yet photosynthetic systems are still attractive for the uniform labelling of several storage carbohydrates from the relatively cheap 13CO2, especially if specific production can be regulated by the organism. For instance, the
TIBTECH - JANUARY
1986
osmoregulated alga Dunalliela salina can be used to accumulate 13C carbohydrates, with control exerted by changing the salts concentration 6. Unfortunately, uniformly labelled compounds are not much favoured in NMR work. The adjacency of two or more 13C nuclei causes multiple splitting of their resonances, resulting in complicated spectral patterns. The trend in stable isotope labelling leads clearly towards site-specific labelled species. If organic chemical methods are used, 13C must be laboriously introduced into predetermined sites. Indeed the most common method of preparing ~3C compound is to substitute the terminal carbon (such as the carboxyl in amino and organic acids) by a [13C]cyano moiety. Nevertheless, the scarcity of other site-specific labelled biological compounds, especially those with ~3C introduced at internal backbone sites (which are important because the label is less susceptible to removal during catabolism), is a major obstacle for using these compounds in certain biomedical investigations with GC-MS and NMR. In recent years fermentation and immobilized biocatalyst techniques have become the major alternatives to chemical synthesis of organic and amino acids, growth factors and antibiotics. The outstanding achievements of these approaches on a large scale has induced their use for the preparation of biomolecules labelled with ~3C and 15N. The process must conserve the expensive labelled precursors (15NH4C1 costs $50 per gram, and [xaC]glucose $600-1200 per gram) and their design is dominated by considerations of (a) the limiting quantity of isotopic precursors, (b) the danger of label dilution from external and internal pools and (c) the need to recover residual labeled starting materials. Batch sizes are usually small, not exceeding several litres, and processes, therefore, are labour intensive. However, since the economics are completely dominated by the cost of the labelled precursors, concern over prices of raw materials and energy or capital is secondary. Overproducing fermentations Fermentation by overproducing
bacteria is suitable for the production of compounds requiring multi-step enzymatic synthesis. It was used for the preparation of several ~5Nlabelled amino acids and antibiotics such as L-[15N]glutamate, L - [ 1 5 N ] lysine and [15N]actinomycin D 7-1°. As in conventional fermentation, the selected bacteria are grown under optimal conditions for production, which must be modified so as to maximise the product in relation to the isotopic precursor. For 15N, 6 0 80% of the initial precursor is incorporated into the desired product and only about 10-25% into the biomass, which may be used as the source of various other labelled biomolecules. For laC labelling, the metabolic centrality of glycolysis reduces isotopic incorporation could be achieved by separating microbial glucose or [laC]acetate as the sole carbon source. More efficient isotopic incorporation could be achieved by separating microbial growth from amino acid production. The internal amino acid pools must be depleted before the introduction of the isotope. The production process for the labelled compounds can, in effect, be used for its own analysis. NMR spectroscopy is the only method for non-destructive monitoring of the intracellular assimilation of carbon and nitrogen and is particularly suitable for biotechnological processes where high concentration of metabolites are involved. It has been applied to various problems of microbial fermentation of natural products ~m2 where growth parameters and changes in nutrient and product transport could be monitored simultaneously in real time, and may be developed for on-line analysis. Thus, in vivo NMR has resulted in the optimization of the uptake of isotopic labelled precursors. It also lends itself for screening of overproducing bacterial strains, thereby opening up the possibility of producing additional labelled compounds. The use of exotic bacteria, such as anaerobic strains capable of growth on a range of substrates including 13CO2 and simple onecarbon compounds, which may be subsequently induced to over-
produce, could become another economical way of obtaining uniformly labelled ~3C products. Biotransformations Single step transformations by immobilized or free biocatalysts such as enzymes and bacteria, will give superior yields of labelled biochemicals because bacterial growth is segregated from the production step. In the growth phase the desired enzymatic activity is induced in the microorganism. The unlabelled precursors are then removed to avoid dilution of label before the isotope is introduced. Such reactions have been used for preparing gram quantities of l~N-labelled alanine, aspartate, tyrosine, tyramine, DOPA and dopamine in very high (90-100%) isotopic yields 13-16. Synthesis using isolated enzymes could not compete with these economical and efficient whole-cell biotransformations where the induced cells contain other enzymes which exhaust cofactors or substrates. The limited number of nitrogenous groups in biomolecules means that biotransformation is very useful for 15N labelling: the 15N label is almost always introduced into specific sites. Single step biocatalysis is also suitable for 13C site specific labelling but it depends on the organic synthesis of some specifically labelled precursors (for instance, 1- or 2,4-(13C)phenol for the making of the corresponding labelled L-tyrosine17. Another way of site-specific labelling with 13C is by quasi-fermentative condensation of an intermediate with specifically labelled acetate or pyruvate, in a reaction of minimal metabolic recycline. Regulatory phenomena are the key to success of such approaches, demonstrated by the preparation of L-(3- or 3'-13C)isoleucine TM. The exploitation of pyruvate carboxylase, which fixes 13CO2, may be another approach for introducing ~3C into specific positions in amino acids. Developments of biotechnological methods such as the introduction of genetically engineered organisms with increased enzymatic capabilities and higher labelling efficiency, the improvement of bound biocatalysis for multi-enzymatic synthesis, and the establishment of
TIBTECH - JANUARY 1986
t i s s u e c u l t u r e s for e u k a r y o t i c products, s h o u l d b e f u r t h e r a p p l i e d to stable i s o t o p e t e c h n o l o g y . S u c h progress will p r o v i d e the i m p e t u s for the future of in vivo N M R a n d G C - M S i n medicine.
AVIVALAPIDOTAND ZVI E. KAHANA
Department o f Isotope Research, W e i z m a n n Institute o f Science, Rehovot, Israel. ~"
References 1 Gilfillan, C. A., Tserng, K-Y. and Kalhan, S. C. (1985) Biol. Neonate 47, 141-147 2 Amir, J., Reisner, S. H. and Lapidot, A. (1980) Pediatr. Res. 14, 1238-1244 3 Holliday, D. and Rennie, M. J. (1982) Clin. Sci. 63, 485-496 4 Radda, G. K. and Shulman, R.G. (1984) in Biomedical Magnetic Res-
5
6
7 8
9 10 11
onance (James, T.L. and Margulis, A.L. eds), pp. 201-207, Radiology Research and Education Foundation Darland, G., Kropp, H., Kahan, F. M., Hajdu, R., Walker, R. and VandenHeuvel, W. J. A. (1985) International Symposium on the Synthesis and Applications of lsotopically Labelled Compounds (Kansas City), p. 79 Gopher, A. and Lapidot, A. (1985) 2nd International Symposium on the Synthesis and Applications of Isotopically Labelled Compounds (Kansas City), p. PB14 Kahana, Z. E. and Lapidot, A. (1983) Anal. Biochem. 132, 160-164 Irving, C. S., Cooney, C.L., Brown, L. T., Gold, D., Gordon, J. and Klein, P. D. (1983) Anal. Biochem. 131, 9 3 98 Shaefer, R. H., Formica, J. L., Delfini, C., Brown, S.C. and Miroue, P.A. (1982) Biochemistry 21, 6491-6503 Juretschke, H-P. and Lapidot, A. (1985) Eur. ]. Biochem. 147, 313-324 Haran, N., Kahana, Z. E. and Lapidot, A. (1983)]. Biol. Chem. 258, 12929-
12933 12 Inbar, L., Kahana, Z. E. and Lapidot, A. (1985) Eur. ]. Biochem. 149, 601-607 13 Kahana, Z. E. and Lapidot, A. (1982) Anal. Biochem. 126, 389-393 14 Mocanu, A., Niac, G., Ivanof, A., Gorun, V., Palibroda, N., Vargha, E., Bologa, M, and Barzu, O. (1982) FEBS Lett. 143, 153-156 15 Kahana, Z. E. and Lapidot, A. (1984) Third European Congress of Biotechnology, Vol. I, pp. 439-443, Verlag-Chemie 16 Kahana, Z. E. and Lapidot, A. (1985) Anal. Biochem. 149, 549-554 17 Matwiyoff, N. A., Unkefer, C.J. and Walker, T, E. (1983) in Synthesis and Application of Isotopically Labelled Compounds (Duncan, N.P. and Susan, A.B., eds), pp. 467-475, Elsevier 18 Kahana, Z. E. and Lapidot, A. (1985) 2nd International Symposium on the Synthesis and Applications of Isotopically Labelled Compounds (Kansas City) p. 123
ability of products derived from transformed cells. In an effort to resolve the situation and to provide WHO with the best advice available, Petricciani announced that he will convene a special multidisciplinary group of the world's top biomedical experts to review the data, discuss the issues, and make recommendations. That meeting is expected to take place in mid-1986 and should provide the final green light for both
WHO and national regulatory authorities. This long awaited acceptance of continuous cell culture by two major regulatory organizations opens the way for the commercialization of a variety of specific products and procedures and was an optimistic introduction to the 1985 ESACT meeting. The next ESACT meeting will be held in Ness Ziona, Israel in April 1987.
Biological process for preparing compounds labelled with stable isotopes The combination of new techniques and instrumentation such as massspectrometry (MS) and nuclear magnetic resonance (NMR), and the development of innovative approaches for the preparing of biological compounds labelled with stable isotopes such as ~3C and 15N, is opening new research areas especially in human studies in biomedicine. New insight into metabolic mechanisms in humans is being gained without the exposure to hazardous radioactive materials. Study of problems such as the determination of maternal-fetal nutritional circulation or nutritional support in newborns 1'2 could not be envisioned without the use of stable isotopes. Numerous other metabolic studies in humans are being conducted using GC-MS (gas chromatographic-mass spectrometric) analysis of body fluids containing stable isotope tracers a. In vivo NMR techniques, still under development, offer new ways of observing the biochemical events of metabolism in living systems, from bacteria to man, in a non-invasive manner 4. In vivo NMR requires tracers labelled with nuclei such as those of ~3C and 25N. These emerging methodologies are
evolving into major tools for medical studies but the scarcity and prices of stable isotope labelled compounds are the bottleneck in this development. These methods depend on the availability of such compounds, in a wide variety of useful forms and in sufficient quantities. To illustrate the dimensions of the problem, amounts of 0.5-1 g, costing $100-1000 per gram depending on the amino acid in question, are required for metabolic determination of an 15N-labelled amino acid in one adult, using the sensitive GC-MS technique. Quantities of 13C-labelled compounds many times higher are required for in vivo NMR spectroscopy, which has lower sensitivity of detection. Currently most stable isotope compounds are prepared by long, isotopically inefficient, multi-step organic syntheses. Sometimes racemic products are obtained when only one isomer is of interest. The synthesis of radioactive compounds using organic methods is well established, but these procedures are not suitable for the synthesis of stable isotope labelled compounds. The former are made in miniscule amounts and are used with an unlabelled carrier, while the latter are prepared in gram
© 1986, Elsevier Science Publishers B.V., Amsterdam 0166- 9430/86/$02.00
The proceedings of the PHS meeting are almost ready for publication as a supplement to In vitro, whereas the proceedings of this year's ESACT meeting will be published in a volume of Developments in Biological Standardization published by S. Karger. R. BLIEM
Cambridge Life Sciences, Cambridge Science Park, Milton Road, Cambridge CB4 2QN, UK.
quantities with almost all of the molecules enriched. Compared to 13C and 15N, deuterated compounds are easier to make by organic synthesis, but their use is restricted by the strong isotope effects associated with deuterium. Furthermore, some deuterated sites are susceptible to loss of label by exchange with water, or are easily scrambled enzymatically, unlike most carbons and nitrogens. However, specifically deuterated drugs are used in pharmacological studies and the isotopic effect may be used to advantage if it can be designed to slow certain metabolic steps 5. Biotechnology offers attractive alternatives to organic methods of preparation, avoiding the use of complicated precursors, the isolation of intermediates, the intricate purification and optical resolution of products. Therefore, they result in high yields and in efficient isotope incorporation. Several approaches are used in biotechnological preparation of labelled compounds. One is to label whole organisms by growing them on labelled precursors and subsequently fractionating the biomass into various biochemicals. It is most commonly used with plants and algae growing o n 13C02. Although the incorporation of 13C02 by photosynthesis is very efficient, the technique is wasteful because of (a) the enormous diversion of 13C into unwanted biomass and (b) the elaborate purification schemes needed. Yet photosynthetic systems are still attractive for the uniform labelling of several storage carbohydrates from the relatively cheap 13CO2, especially if specific production can be regulated by the organism. For instance, the
TIBTECH - JANUARY
1986
osmoregulated alga Dunalliela salina can be used to accumulate 13C carbohydrates, with control exerted by changing the salts concentration 6. Unfortunately, uniformly labelled compounds are not much favoured in NMR work. The adjacency of two or more 13C nuclei causes multiple splitting of their resonances, resulting in complicated spectral patterns. The trend in stable isotope labelling leads clearly towards site-specific labelled species. If organic chemical methods are used, 13C must be laboriously introduced into predetermined sites. Indeed the most common method of preparing ~3C compound is to substitute the terminal carbon (such as the carboxyl in amino and organic acids) by a [13C]cyano moiety. Nevertheless, the scarcity of other site-specific labelled biological compounds, especially those with ~3C introduced at internal backbone sites (which are important because the label is less susceptible to removal during catabolism), is a major obstacle for using these compounds in certain biomedical investigations with GC-MS and NMR. In recent years fermentation and immobilized biocatalyst techniques have become the major alternatives to chemical synthesis of organic and amino acids, growth factors and antibiotics. The outstanding achievements of these approaches on a large scale has induced their use for the preparation of biomolecules labelled with ~3C and 15N. The process must conserve the expensive labelled precursors (15NH4C1 costs $50 per gram, and [xaC]glucose $600-1200 per gram) and their design is dominated by considerations of (a) the limiting quantity of isotopic precursors, (b) the danger of label dilution from external and internal pools and (c) the need to recover residual labeled starting materials. Batch sizes are usually small, not exceeding several litres, and processes, therefore, are labour intensive. However, since the economics are completely dominated by the cost of the labelled precursors, concern over prices of raw materials and energy or capital is secondary. Overproducing fermentations Fermentation by overproducing
bacteria is suitable for the production of compounds requiring multi-step enzymatic synthesis. It was used for the preparation of several ~5Nlabelled amino acids and antibiotics such as L-[15N]glutamate, L - [ 1 5 N ] lysine and [15N]actinomycin D 7-1°. As in conventional fermentation, the selected bacteria are grown under optimal conditions for production, which must be modified so as to maximise the product in relation to the isotopic precursor. For 15N, 6 0 80% of the initial precursor is incorporated into the desired product and only about 10-25% into the biomass, which may be used as the source of various other labelled biomolecules. For laC labelling, the metabolic centrality of glycolysis reduces isotopic incorporation could be achieved by separating microbial glucose or [laC]acetate as the sole carbon source. More efficient isotopic incorporation could be achieved by separating microbial growth from amino acid production. The internal amino acid pools must be depleted before the introduction of the isotope. The production process for the labelled compounds can, in effect, be used for its own analysis. NMR spectroscopy is the only method for non-destructive monitoring of the intracellular assimilation of carbon and nitrogen and is particularly suitable for biotechnological processes where high concentration of metabolites are involved. It has been applied to various problems of microbial fermentation of natural products ~m2 where growth parameters and changes in nutrient and product transport could be monitored simultaneously in real time, and may be developed for on-line analysis. Thus, in vivo NMR has resulted in the optimization of the uptake of isotopic labelled precursors. It also lends itself for screening of overproducing bacterial strains, thereby opening up the possibility of producing additional labelled compounds. The use of exotic bacteria, such as anaerobic strains capable of growth on a range of substrates including 13CO2 and simple onecarbon compounds, which may be subsequently induced to over-
produce, could become another economical way of obtaining uniformly labelled ~3C products. Biotransformations Single step transformations by immobilized or free biocatalysts such as enzymes and bacteria, will give superior yields of labelled biochemicals because bacterial growth is segregated from the production step. In the growth phase the desired enzymatic activity is induced in the microorganism. The unlabelled precursors are then removed to avoid dilution of label before the isotope is introduced. Such reactions have been used for preparing gram quantities of l~N-labelled alanine, aspartate, tyrosine, tyramine, DOPA and dopamine in very high (90-100%) isotopic yields 13-16. Synthesis using isolated enzymes could not compete with these economical and efficient whole-cell biotransformations where the induced cells contain other enzymes which exhaust cofactors or substrates. The limited number of nitrogenous groups in biomolecules means that biotransformation is very useful for 15N labelling: the 15N label is almost always introduced into specific sites. Single step biocatalysis is also suitable for 13C site specific labelling but it depends on the organic synthesis of some specifically labelled precursors (for instance, 1- or 2,4-(13C)phenol for the making of the corresponding labelled L-tyrosine17. Another way of site-specific labelling with 13C is by quasi-fermentative condensation of an intermediate with specifically labelled acetate or pyruvate, in a reaction of minimal metabolic recycline. Regulatory phenomena are the key to success of such approaches, demonstrated by the preparation of L-(3- or 3'-13C)isoleucine TM. The exploitation of pyruvate carboxylase, which fixes 13CO2, may be another approach for introducing ~3C into specific positions in amino acids. Developments of biotechnological methods such as the introduction of genetically engineered organisms with increased enzymatic capabilities and higher labelling efficiency, the improvement of bound biocatalysis for multi-enzymatic synthesis, and the establishment of
TIBTECH - JANUARY 1986
t i s s u e c u l t u r e s for e u k a r y o t i c products, s h o u l d b e f u r t h e r a p p l i e d to stable i s o t o p e t e c h n o l o g y . S u c h progress will p r o v i d e the i m p e t u s for the future of in vivo N M R a n d G C - M S i n medicine.
AVIVALAPIDOTAND ZVI E. KAHANA
Department o f Isotope Research, W e i z m a n n Institute o f Science, Rehovot, Israel. ~"
References 1 Gilfillan, C. A., Tserng, K-Y. and Kalhan, S. C. (1985) Biol. Neonate 47, 141-147 2 Amir, J., Reisner, S. H. and Lapidot, A. (1980) Pediatr. Res. 14, 1238-1244 3 Holliday, D. and Rennie, M. J. (1982) Clin. Sci. 63, 485-496 4 Radda, G. K. and Shulman, R.G. (1984) in Biomedical Magnetic Res-
5
6
7 8
9 10 11
onance (James, T.L. and Margulis, A.L. eds), pp. 201-207, Radiology Research and Education Foundation Darland, G., Kropp, H., Kahan, F. M., Hajdu, R., Walker, R. and VandenHeuvel, W. J. A. (1985) International Symposium on the Synthesis and Applications of lsotopically Labelled Compounds (Kansas City), p. 79 Gopher, A. and Lapidot, A. (1985) 2nd International Symposium on the Synthesis and Applications of Isotopically Labelled Compounds (Kansas City), p. PB14 Kahana, Z. E. and Lapidot, A. (1983) Anal. Biochem. 132, 160-164 Irving, C. S., Cooney, C.L., Brown, L. T., Gold, D., Gordon, J. and Klein, P. D. (1983) Anal. Biochem. 131, 9 3 98 Shaefer, R. H., Formica, J. L., Delfini, C., Brown, S.C. and Miroue, P.A. (1982) Biochemistry 21, 6491-6503 Juretschke, H-P. and Lapidot, A. (1985) Eur. ]. Biochem. 147, 313-324 Haran, N., Kahana, Z. E. and Lapidot, A. (1983)]. Biol. Chem. 258, 12929-
12933 12 Inbar, L., Kahana, Z. E. and Lapidot, A. (1985) Eur. ]. Biochem. 149, 601-607 13 Kahana, Z. E. and Lapidot, A. (1982) Anal. Biochem. 126, 389-393 14 Mocanu, A., Niac, G., Ivanof, A., Gorun, V., Palibroda, N., Vargha, E., Bologa, M, and Barzu, O. (1982) FEBS Lett. 143, 153-156 15 Kahana, Z. E. and Lapidot, A. (1984) Third European Congress of Biotechnology, Vol. I, pp. 439-443, Verlag-Chemie 16 Kahana, Z. E. and Lapidot, A. (1985) Anal. Biochem. 149, 549-554 17 Matwiyoff, N. A., Unkefer, C.J. and Walker, T, E. (1983) in Synthesis and Application of Isotopically Labelled Compounds (Duncan, N.P. and Susan, A.B., eds), pp. 467-475, Elsevier 18 Kahana, Z. E. and Lapidot, A. (1985) 2nd International Symposium on the Synthesis and Applications of Isotopically Labelled Compounds (Kansas City) p. 123