CHEM-705 Biosynthesis and Isolation of Natural Products and Bioassay Screenings February – 2014

1. CHEM-705Biosynthesis and Isolation of Natural Products and Bioassay ScreeningsFebruary – 2014

2. CHEM-705Biosynthesis of Natural ProductsSet B  Fourth-Seventh LecturesProf. Dr. ShaheenFaiziFebruary - 2014

3. Success comes to those who make efforts vigorously, and strengthen their relationship with Allah, and proceed far afield with ease and outstrip their rivals, and manage well the affairs entrusted to them. (79:1-5)

4. Natural Products and their Biosynthesis Classification of Natural Products Classification based on chemical structure Classification based on physiological activity Classification based on taxonomy Classification based on biosynthesis (The best classification)

7. Classification based on taxonomy Citrus paradisi (Rutaceae) Citrus aurantium (Sour orange) b-Sitosterolglucoside Limonexic acid J. Agric. Food Chem., 2010, 58, 180-186 Phytother. Res., 2009, 23, 948-954 Aeglemarmelos (Rutaceae)

8. Biosynthetic classification of natural products  The carbon skeletons of the majority of natural products are assembled by characteristic sequences of enzyme-catalyzed reactions, starting from glucose, which is itself produced by reduction of CO2 in plants and autotrophic micro-organisms. These interrelated metabolic sequences provide the basis for a biosynthetic classification in which it is convenient to divide natural product, somewhat arbitrarily, into two groups, namely primary and secondary metabolites. The classification criteria are mainly biosynthetic and to a lesser extent functional. Thus the first group comprises those substances of widespread distribution (i.e. general metabolites), together with a variety of biopolymers such as the common polysaccharides, proteins, and nucleic acids.

9. Primary Metabolites • Products of general metabolism • Broad distribution in plants, animals and micro-organisms, e.g. amino-acids, acetyl-CoA, monosaccharides, mevalonic acid and nucleotides • Metabolically essential, e.g. sugars, amino acids, nucleotide bases, biopolymers such as common polysaccharides, proteins, and nucleic acids. Secondary Metabolites • Product of specialized pathways • Biosynthesized from primary metabolites • Restricted distribution, found mostly in plants and micro-organisms (often characteristic of individual genera, species or strains) e.g. alkaloids, terpenes, phenols, oligosaccharides, flavonoids, antibiotics. For secondary metabolites no clear biological function has been identified. These are called secondary metabolites because their roles in the metabolism of host organisms are not obvious. However, O.R. Gottlieb in his essay (Phytochemistry, 29, 1715, 1990 ) stated that contrary to a century old belief, the so called secondary (here designated special) metabolites are equally essential to plant life.

10. More recently it has been reported that whilst primary metabolites serve defined roles in the normal growth and development of living organisms, it seems likely that the secondary metabolites have evolved specifically to interact with and modulate the function of biological macromolecules. Most secondary metabolites are produced by bacteria, fungi, plants and marine organisms. Some of these molecules are known to provide a selective advantage against microbial attack or defence against infestation and disease,whilst others function as signalling molecules in quorum sensing,as pheromones or pigments facilitating reproduction. For over three billion years microorganisms have evolved to produce nucleic acids, proteins and other biological macromolecules alongside smaller metabolites.It is thus hardly surprising that there exists a small metabolite, or natural product, ligand for many of the distinct macromolecular targets that exist within today’s cells. Natural products are biosynthesised by protein catalysts, and as such natural product and protein structures must necessarily have co-evolved to bind to one another selectively.Indeed, it is likely that during evolution, there has existed a natural product ligand that could bind to all possible protein folds. In addition to this, many metabolites are known which can regulate their own biosynthesis or degradation through interaction with the genes, particularly mRNAs, which encode the enzymes responsible for their metabolism. (N. Dixon, L. S. Wong, T. H. Geerlings and J. Micklefield, Cellular targets of natural products, Nat. Prod. Rep., 2007, 24, 1288-1310).

12. Biosynthetic Classification of Secondary Metabolites C1 units (exformate and methionine)

17. ENZYMES: Enzymes are the catalysts for biological reactions. Their catalytic power is often quite prodigious and, coupled with their specificity for the substrate, this enables an organism to organize and select, for a given molecule, just one discrete metabolic pathway out of the many possible chemical reactions which that molecule and its progeny may undergo. The specificity of the enzyme for a particular substrate may be structural or stereochemical in origin. Enzymes are the most powerful and efficient catalysts known, yet at the same time the most subtle and most delicate. They catalyze almost all the reactions which go on in living systems, including many which the organic chemist finds difficult or impossible; and they do this under the mildest conditions of temperature and pH, in dilute solution in water. Enzymes are protein molecules, with molecular weights ranging from about ten thousand to several million. A given enzyme protein is made up of a unique, but apparaently random, sequence of all the 20 common amino-acids. Many enzymes contain, or work in conjunction with, coenzymes or metal ions which are essential for catalytic activity. Many, too, are aggregates of one, or sometimes two sorts of individual protein subunits, still others are organized in groups of small numbers of different enzymes, either in solution (multi-enzyme complex) or more or less firmly attached to specific sub cellular structures.These multi-enzyme systems can thus carry out sequences of several reactions, and contain one enzyme for each step of the sequence. The most important difference between enzyme catalysis and ordinary chemical catalysis is the binding step leading to the formation of the enzyme-substrate complex. This is the step most difficult to reproduce in attempts to approach enzyme rates and specificities in bimolecular reactions between simple compounds. The theory of substrate binding has been dominated by Fischer’s idea, expressed as long ago as 1894 that the substrate fits the enzyme much as a key fits a lock. Although Fischer’s lock-and-key hypothesis has proved immensely fruitful, and explains many of the general features of substrate specificity, its limitations have become more and more apparent as detailed information on enzyme-molecule interactions has accumulated in recent years. Taken literally the hypothesis presumes a rigid binding site, whereas there is a great deal of evidence for conformational mobility in proteins, both from X-ray structure determinations and from direct spectroscopic and kinetic measurement on enzymes in solution. Many enzyme-catalysed reactions require a substance to be present in addition to the enzyme and the substrate in order that the reaction may proceed. Such substances are called coenzymes or cofactors and form an essential part of the catalytic mechanism. The intact enzyme system, or holoenzyme, is thus formed from a protein portion called the apoenzyme and a non-protein component referred to variously as a prosthetic group, a cofactor, and more commonly a coenzyme. Apoenzyme + Coenzyme holoenzyme (Protein) (Intact enzyme system)

19. Experimental approaches for the development of biosynthetic studies have been predominantly based on the use of isotopically labeled intermediates. (Nat. Prod. Report, 23, 1046-1062, 2006). Fatty Acids Fatty acids are essential dietary factors, they have links with prostaglandin and they have involvement in structural membranes. Over 800 natural fatty acids have been identified. A few of these are very common, much studied, and biologically important. The following generalizations are based on the structure of the natural acids. To each statements, however, there are interesting and important exceptions. • Most natural acids, whether saturated or unsaturated, are straight –chain compounds with an even number of carbon atoms; odd acids, branched –chain acids, and cyclic acids also exist. • Although the range of chain length is great (C2 to >C80), the most common chain. length are C16, C18, C20 and C22. • Mono-unsaturated acids usually contain a cisolefinic bond in a limited number of preferred position in the carbon chain; trans-alkenoic and alkynoic acids are also known.

20. Most polyunsaturated acids have 2-6 cis double bonds in a methylene-interrupted pattern; acids with conjugated unsaturation also exist. • Substituted acids are uncommon but hydroxy-, oxo-, epoxy-, and fluoro-containing acids are known. Hydroxy –acids are found in brain lipids, woolware, milk lipids, cutins, plants and micro–organisms. • Lipids • Lipids are natural derivatives of fatty acids. In lipids fatty acids occur as esters usually, but not always, of glycerol (propane- 1, 2, 3triol). There are also lipids in which the fatty acids are present as amides of long chain amines. • J. Lipid Research, 46, 839-861 (2005).

41. Prostaglandins, Thromboxanes Linoleic acid is a dietary requirement for the healthy growth of animals and these type of acids are called ‘essential fatty acids’. These C18 acids and/or the higher polyene acids produced from them are involved in the production of the prostaglandins and thromboxanes. The prostaglandins are widely distributed in animal tissues and exhibit a wide range of pharmacological activities. A series of alkyl esters of PGE, and 20-methyl-PGE, were prepared in order to examine the effect of esterification on the inhibitory action of these compounds on platelet aggregation. Workers at the Upjohn Company have prepared many esters of PGE2 and PGE2 as potential pro-drugs.

43. Turner and Herz (Experientia, 33, 1133 1977), have presented a chemical model for these conversions which is based on known reactions of other endoperoxides with iron (II)- iron (III) redox system.