Methods of analysis

This presentation of basic alkaloid synthesis pathways clearly reveals the diversity and complicity of this process in nature. Moreover, the large number of different pathways and synthesis routes proves the status of alkaloids as a phenomenon of the metabolic activity of organisms. Here, we have seen only the basic pathways and routes. In reality, each alkaloid has its own synthesis route. It is possible to find or to place it on one of the basic pathways. Certainly, experimental data is required for this, the obtaining of which necessitates deep research into molecular structure. Although the technical level of research in the leading laboratories is very high, deep structural and synthesis research is not easy. It is a very expensive and complex job. Pure chemical structure analysis does not suffice today to explain the nature of alkaloid behavioural synthesis in depth. Reactions require a lot of the energy derived from the Krebs cycle and, generally, enzymatic activity. Alkaloid studies use chemical methods of analysis to clarify the constructional and taxonomical nature of these compounds, together with biological and semi-biological methods to describe the role and behaviour of these molecules in life processes. It is well known that natural product molecules are biosynthesized by a chain of reactions which, with very few exceptions, are catalysed by enzymes32 221. This is especially important in the case of alkaloids - biologically very active secondary compounds, which have genetic background and environmental oscillators7. A discussion on the synthesis of alkaloids derived from different substracts in the metabolic system of a living organism should cover the form and construction blocks of substrate and the changes occurring in the synthesis. The most important step is to provide the answer to the question of origin and the link of the different compounds in the synthesis reactions. An alkaloid metabolism is concerned with the formation of new molecular substances or with the degradation of synthesized molecules. This metabolism is in reality connected with the active transport of metabolites, inorganic ions and organic atoms. Moreover, the converting of energy in biosynthetic and degradative processes is also a base for the reaction chains. The metabolic system of alkaloid pathways is clearly regulated. Preiss and Kosuge222 have emphasized that enzyme synthesis or degradation, and enzyme activity, are integrated to produce a more efficient modulation. Simpkins223 notes that there must be a positive correlation between the rate of overall physiological processes and the kinetic and regulatory properties of key enzymes. Moreover, he contends that the specific activity of key enzymes involved in a metabolic pathway would be expected to be high. Therefore, alkaloid synthesis is regulated by a mechanism linked to enzymatic strategy. This strategy seems to be one of the fundamental blocks in alkaloid analysis. It is generally known that enzymes are proteins with catalytic activity in the life system and metabolic processes. They were discovered by Sumner, who first isolated the enzyme urase in crystalline form from jack bean meal in 1926. This historic event was very important for both chemistry and biology. However, from today's perspective it is difficult to understand why the first isolation and crystallization of enzymes met with such strong criticism, and even derision, among large groups of leading scientists. Only the methodological development and subsequent works with crystallization, and the identification of many new enzymes, published more than 20 years later by Northrup et al.224, confirmed Sumner's discovery and finally established that the enzyme was a protein. The enzyme as a catalytic unit has a direct relation to alkaloid synthesis research. Even nowadays, not all metabolic enzymes have been isolated and named in alkaloid synthesis pathways. The development of enzymatic methods for the detection of the steps of the synthesis and degradation of alkaloid molecules seems to be very challenging for contemporary research. Mahler and Cordes212 have shown that enzymes catalyse reactions in four directions: (1) increasing reaction efficiency and speed; (2) increasing of utilization of substrate; (3) broadening of the spectrum of reactions; and (4) controlling cellular metabolism. All these catalyzing purposes are applicable to alkaloid synthesis and degradation reactions. Methods of alkaloid analysis are therefore focused on the molecular-level biology inside and outside the organism. On the other hand, any analysis of alkaloids is impossible without pure classical chemistry and bioorganic chemistry methods.

2.7.1. Methodological considerations

Alkaloids generally, and especially investigations into them throughout history, retain some kind of mystery. This is connected with their strong biological activity, which was observed and used even in ancient times, but without any explanation. Even in the middle of the 20th century, the scientific explanation of alkaloids was based on the opinion that these compounds were the products of waste organic metabolic processes. Only in 1960s and 1970s did alkaloids begin to be seen as evolutionary and biogenetic markers of living organisms225. Inspite of the fact that alkaloids are very different compounds, they can be classified and harmonized according to morphology and metabolism. As stated earlier, it is evident that an alkaloid pathway consists of a series of reactions and compounds as well as enzymes4-7 221. The sequence of all reactions leading to any alkaloid synthesis can be presented as follows:

1

1

A

A

A

t

t

t

P

P

P

t

t

t

a = precursor P = intermedia ^ = obligatory intermedia X = second obligatory intermedia m = final product of metabolism A = alkaloid synthesis P = postcursor of alkaloid.

The alkaloid is not the final product of a secondary metabolism. This new conception explains why alkaloids were traditionally considered to be unnecessary and undesirable compounds in organisms. Although the "waste" theory is no longer seriously entertained, there are many questions which remain unanswered. It is not quite clear why plants, animals and particularly micro-organisms produce alkaloids. Certainly, there are many hypotheses and theories regarding this problem, with compelling arguments but also with points open to criticism. A similar question is also related to biosynthesis: why does an alkaloid need its intermediate molecule in the synthesis process and why is it not derived directly from the precursor? In natural processes, there is a tendency to cut corners in developing links and pathways. However, in the alkaloid synthesis pathway just the opposite occurs, although this is also a process occurring in nature. Take, for example, the synthesis of quinolizidine alkaloids. From a theoretical point of view, the structural transformation of L-lysine into (—)-lupinine should be simpler; but according to empirical studies into ring and carbon spectra, a direct reaction does not exist32. This transformation occurs only by intermedia (cadaverine) reactions.

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