Techniques for Biosynthesis

In the study of secondary metabolism the ongoing problem is twofold (i), to identify the source(s) in primary metabolism from which a secondary metabolite has its genesis and (ii), to identify by what mechanisms and manner of intermediates it is thus fashioned. It is structure that first hints at a solution made complete by a battery of special techniques. 

It is because, in the event, secondary metabolites derive from but a handful of primary metabolites along pathways involving generally the simplest reactions in organic chemistry and little rearrangement, that speculation about their biosynthesis can be so accurate. Also invaluable in this regard, is the identification of metabolites of similar structure in the same, or related, species. Thus benzyl-isoquinoline alkaloids [as (2.7)] and morphine (2.2) are both found in Papaver species. It follows reasonably that morphine derives from a compound type of (2.7) (Section 6.3.4). 

The wealth of intelligent speculation about the biosynthesis of secondary metabolites has provided a firm base from which to mount 

In addition to the results of feeding experiments with labelled precursors, clear information on the sequence of biosynthesis of several related metabolites produced by an organism may be obtained. This is done by feeding a very early precursor (usually C02) and noting the order in which the metabolites are labelled. This order gives the sequence of biosynthesis. 

Experiments with purified enzymes involved in biosynthesis, or even experiments with crude enzyme preparations, can provide important definition of a pathway. Studies with enzymes are briefly referred to, by way of introduction, in Section 2.3, as is the use of mutants. 

In addition, labeled precursors (used as tracers or markers) enriched in one or more positions with stable isotopes (e.g., H, N) play an important role in 

1 0) that allow rapid acquisition and interpretation of data with mass spectrometry (MS) and/or (only for isotopes with a nuclear spin 7 0) NMR, and (ii) radioactive isotopic labels (e.g., H, Na, U) which are very sensitively 

Biosynthesis of Heterocycles From Isolation to Gene Cluster, First Edition. Patrizia Diana and Girolamo Cirrincione. 

Structural determination using isotopes is often carried out using multiple ways. The information derived from the use of stable isotopes differs in many ways from that of radioisotopes, and the best demonstration of this relates to their distinctly different analytical measurements. Since isotopes have different masses, they can be determined through MS and NMR. Another consequence of the difference in mass is that molecules containing isotopes have different vibration modes these can be detected by infrared spectroscopy. Mass spectrometers are analytical instruments that allow the measurement of molecular masses of stable isotopes after their conversion into ions. Subsequently, the ion abundances are normalized as a percentage of the most abundant (unlabeled) species. The normalized crude ion abundances of an enriched molecule must be corrected for the measured natural abundance ( H, 0.02% C, 1.1% 0.4% 0,0.2%) of the stable isotopes present in the original molecule. 

---

Generally, the most powerful method for stmctural elucidation of steroids is nuclear magnetic resonance (nmr) spectroscopy. There are several classical reviews on the one-dimensional (1-D) proton H-nmr spectroscopy of steroids (267). C-nmr, a technique used to observe individual carbons, is used for stmcture elucidation of steroids. In addition, C-nmr is used for biosynthesis experiments with C-enriched precursors (268). The availability of higher magnetic field instmments coupled with the arrival of 1-D and two-dimensional (2-D) techniques such as DEPT, COSY, NOESY, 2-D J-resolved, HOHAHA, etc, have provided powerful new tools for the stmctural elucidation of complex natural products including steroids (269). 

Work on the biosynthesis of cholesterol began in earnest after Rudolf Schoenheimer and David Rittenberg, at Columbia University, developed isotopic tracer techniques for the analysis of biochemical pathways. In 1941, Rittenberg and Konrad Bloch were able to show that deuterium-labeled acetate (C2H, COO ) was a precursor of cholesterol in rats and mice. In 1949, James Bonner and Barbarin Arreguin postulated that three acetates could combine to form a single five-carbon unit called isoprene. 

In the previous chapters we have discussed the different classes of phenolic compounds, their chemical properties, and their biosynthesis. The characterization of phenolic compounds relied on the ability to isolate them from plant tissues. In this chapter we will discuss methods to isolate and characterize phenolic compounds, and methods to visualize them in planta. Chapter 5 focuses on techniques for the identification and characterization of some of these compounds using recently developed mass-spectrometry-based techniques. 

Marszalek PE, Oberhauser AF, Pang YP and Fernandez JM (1998) Polysaccharide elasticity governed by chair-boat transitions of the glucopyranose ring. Nature, 396 661-4 Marx-Figini M (1969) On the biosynthesis of cellulose in higher and lower plants. In Marchessault RH (ed), Proceedings of 6th cellulose conference. WUey, New York Mason DC (ed) (1975) Sawmill Techniques for Southeast Asia. Miller Freeman, San Francisco... 

Recently, Bale and Crout have described a double isotope technique for simultaneous measurement of incorporation of two precursors into a natural product. This method was utilized to demonstrate that ornithine is probably a slightly more efficient precursor than arginine for retronecine (127) biosynthesis. 

---