domenica 5 maggio 2013

THE PROTEINS: THEIR CONSTITUENTS. Seven strokes of luck

ORIGIN OF LIFE. THE PROTEINS: AMINO ACIDS.


Post n.11 (English)

Hence the biogenic elements (C, N, O, H) are the only ones which, through their compounds, are adapted to carry out, in living organisms, the numerous biological functions. We know that between these compounds the most important  are the Nucleic acid, DNA and RNA, and the proteins.
The proteins constitute tissues and organs, they permit the cells to communicate, they control that which must enter and exit from the cell, they act as antibodies.
All living organisms have a complexity of independent functions which permit them: nourishment, growth, reproduction, evolution, reaction to stimuli, death. All these vital functions have in common the metabolism; that is that process of chemical reactions which co-operate with proteins (enzymes) which permit the living organisms to function. Inside the cell thousands of enzymes can be found which rule and program thousands of chemical reactions. No biological reaction and none of the functions mentioned above can happen without their intervention, not even the synthesis of the nucleic acids.
The proteins are macromolecules whose constituents are the amino acids.
But the amino acids, in the prebiotic era, were present in our planet?
In 1953 S. L. Miller, made the hypothesis of a primordial atmosphere made up CH4, NH3, H2O, H2, succeeded, applying electrical energy, in obtaining various organic substances and, among these, many amino acids of which components of our proteins. This experiment, carried out in plausible prebiotic conditions, marked the date of the birth of prebiotic chemistry.
In the years which followed various researchers have performed experiments both by varying the composition of the gas mixture and the sources of energy. All these studies have confirmed that in the prebiotic era, on our planet, the synthesis of a large number of organic substances was possible, and among these, amino acids were often present. Through those experiments was demonstrated the presence, in the prebiotic era, of about 60 different amino acids. Moreover also the presence of cyan hydric acid (HCN) precedent of purine, of formic aldehyde  (HCHO) precedent of ribose and of other important organic substances among which urea have been demonstrated.
It is remarkable that the same substances, in particular the amino acids, were found in the meteorites going back to the era of the formation of our solar system. The discovery of the amino acids in Miller’s experiments and their presence in the meteorites demonstrates, according to scientists, the ease of synthesis of these compounds. Even the sustainers of the “RNA World” have no doubts on the presence of amino acids in the prebiotic era. Manfred Eigen, referring to experiments like Miller’s, in “Gradini verso la vita” 1992, affirms: «That which renders significant these experiments is not so much the fact that in general amino acids form, but their relative frequency corresponds to those that one meets in nature, and in particular in organic compounds discovered in the meteorites».
At the beginning of the 90’s, some research workers expressed doubt about the presence of a primordial atmosphere made up of CH4, NH3, H2O, H2. These research workers have supposed a primordial atmosphere made up of CO2, N2 and H2O, and in such conditions the formation of the amino acids with application of energy does not take place. Miller has defined these studies hypotheses without sustaining data.
No serious research has doubted of the presence of amino acids in the prebiotic era.
We can conclude that numerous and strong indications demonstrate the presence of amino acids in the prebiotic era. From the synthesis of these molecules the proteins have their origin.
But why the amino acids? And again, are other solutions possible?
According to my knowledge, in 60 years of prebiotic chemistry, the only scientist who tried to give an answer to these questions was Mario Ageno in “Lezioni di biofisica 3” 1984.
Prof. Mario Ageno starts with the ascertainment  that the cells of living organisms do not contain molecules of intermediate dimensions, but they are made up of:
a) Little molecules as much possible simple, which can be found already in the ambiance, or are easy to synthesize.
b) Linear polymers (macromolecules) realized by repetitive operation as a limited number of small molecules (monomers).
As it is known the linear polymers can be obtained both by polyaddition  and by polycondensation. The polyaddition is obtained by the union
it.wikipedia.org/wiki/Polietilene
 
of thousands but also millions of molecules of the same type. It is the repetition of the same motive without any content of information, it causes polymers usually of the linear type, for example polyethylene, and it takes place through a chemical process fairly complex.
The polycondensation can happen between different molecules, through the elimination of water molecules. This type of polymerization cannot take place in the presence of water; however the chemical process is much more simple than the polyaddition. It can lead to plastic material, but it is used by living organisms for the construction of the macromolecules necessary to life. In fact, as they are not the monotonous repetition of the same motive, the polymers which result can contain information.
According to Ageno the choice of the linear polymers has a constructive logic of its own. Starting with a limited number of pieces, always with the same constructive operation, molecular structures different but all connected between them are realized. Probably there exists other solutions to accumulate informations and functions but they are all very complicated. The most convenient solution from an evolution point of view seems to be really that of the linear polymers. In the end chemical evolution has chosen the polymeric process the most simple, the polycondensation, and the simplest polymers that is linear polymers and Ageno adds: «Hence it seems very probable that, wherever they appear under the impulse of natural causes systems, in a way similar for their general characteristics to living organisms which we know, these systems are based for their reproduction, on some species of linear polymers. At the end, this solution can be attained more easily and before any other in the course of a chemical evolution which begins (this is inevitable) with small molecules made up of very few atoms. And hence, what monomers once accepted the solution of linear polymers?».
 Ageno examining the constituents of the proteins asks himself the question: «What can be imagined more simple than an amino acid, as structural  element of a linear polymer?». It is essentially a Carbon atom which establishes a bond with the most simple of elements, the H.
it.wikipedia.org/wiki/Amminoacido
 A second bond takes place with the most simple of the basic function, -NH2. A third link takes place with the simplest acid function, -COOH. In the end, the fourth link takes place with a side chain (R) of a  hydrocarbon simple  or with one of its derivates. Ageno concludes, that the ease with which they are found in nature, for example in meteorites, demonstrates that it is a question of simple molecules and which are easy to synthesize, and they suggest the conclusion that polypeptide chains represent the simplest solution.
Hence, to the question: why the amino acid? The answer: because they are simple and easy to synthesize.
Now the question is that the amino acids are yes simple and easy to synthesize, but they present a complex of properties without which life could not exist. Such complexity confronts us with, as we shall see, a difficult question on the problem of the origin of life, which normally does not fall in the ambiance of physical science.
When Ageno speaks of linear polymers, he refers to the primary structure that is to the disposition and to the structure of the amino acids in the polymer chain. The fact is that if the primary structure remained such, that is in all simplicity a long polymer, it would not perform any vital function. In reality the single proteins, over and above the primary structure, present a secondary structure and a tertiary structure. The proteins perform their vital functions only by having recourse to the secondary and tertiary structures.
To understand how such new structures are possible, it is necessary to examine the properties of amino acids.
1) The first characteristic of amino acids is exactly that of being simple molecules and easy to synthesize in a prebiotic ambiance.
2) A second property is that amino acids are soluble in water and stable. If they were not soluble in water, life would not exist, because they would have grouped together like piece of tar or like large oily stains at the surface of the water basins.
3) Miller’s experiments have shown the presence, in the prebiotic era, of about 60 different amino acids, but life uses only 20. If these could have reacted in water they would have given origin to an enormous number of polypeptides, of casual composition and length, but of no interest for the origin of life. Hence the amino acids must be soluble in water but they must not react in water. And in fact they form polymers through the polycondensation which in water is not permitted.
4) The peptide bond is a link which is realized between the group –COOH of an
biochimica.bio.uniroma1.it
 amino acid and the group –NH2 of another amino acid with the elimination of a molecule of H2O. The characteristic of this link is that it is a link in resonance. That is the  double link between the Carbon atom and Oxygen is not situated between the two atoms but it is distributed also on the link C-N. The consequence of this delocalization is that every zone on the peptide 
bond of the protein lies on a plane and the molecule of the protein cannot rotate around the peptide bonds. As we have said,  
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the proteins are not 
only long linear chains of amino acids.
This is only what is called a primary structure.

 The single proteins present a secondary structure and a tertiary structure through which they perform their vital functions. In the secondary structure they realize helical structures and pleated sheet structures, α-helix and β-pleated sheet. Well, so that these structures ca be realized, one necessary condition is that one prevents the rotation of the molecule around the peptide bond.
The delocalization  of the peptide bond prevents exactly such a rotation.
5) A second condition so that these secondary structures can realize themselves is that the constituent molecules be asymmetrical. We know that the amino acids, except for Glycine, are chiral symmetry, that is they exist in two forms asymmetrical one the mirror image of the other, called D and L.
Symmetrical molecules cannot realize either  α helix or β pleated sheet. The secondary structures of the proteins can be realized by choosing  one of the two asymmetrical forms. In the livings organisms the form L was chosen.
6) Another important characteristic of the peptide link is the presence of an atom of Hydrogen linked to azote. The link between these two
elements  gives origin to -Hδ+. As we can see in the image of the α helix, broken line, -Hδ+ links to the -CO δ- of another amino acid and  it is these links which stabilize  α helix. Without these links the secondary structure of the proteins could not exist.
7 ) To conclude, we have seen that in the amino acids Carbon is linked to a R side-chain.
These side-chains are not chosen by chance. Some of these are hydrophilic, mixable with water, others are hydrophobic not mixable with water. In the tertiary structure of the protein, that is the globular structure the R hydrophobic concentrates inside and the R hydrophilic in the external part. The final result is that the protein results very stable and it is defended from the      bios-project.blogspot.com
degradation of water.
It is known that the compounds in organic chemistry are about 1,5 million and grouped in families. There does not exist another family of organic compounds which presents likewise characteristics: the amino acids are unique, the passage was obliged.
One could conclude that the amino acids are versatile. But we have before us life and, if we wish to try to understand its origin, to the initial question: why the amino acids?
The answer that they are simple and easy to synthesize is not enough; because in reality they must possess the following characteristics:
1) Simple and easy to synthesize
2) Soluble and stable in water
3) They must not react in water
4) They must link between each other giving origin to a peptide bond in resonance.
5) They must be chiral
6) They must contain a -Hδ+ residue on the atom of azote
7) The R side-chain is not casual
If only one of these properties were missing, we do not know how life would have been or perhaps it would not exist. Amino acids are unique compounds.
And so the question is: but how is it possible for the amino acids to have all these properties, just there in these compounds unique, simple and easy to synthesize, exactly those properties necessary to life, when life is still becoming?
Seven strokes of luck?   

Giovanni Occhipinti





sabato 4 maggio 2013

RNA: its constituents. a colossal BLUFF



Post n. 11 (English)

Imagine that you want to construct a house. You charge architects paying a conspicuous advance. After a few months you go and control the state of the work and you find only the roof held up by an enormous scaffolding. The architects explain to you that there is no problem, one can easily start with the roof. Foundations and basic walls are already in the project.
You look again, amazed, at the state of the construction and you observe that the scaffolding is beginning to vacillate.
What are your feelings?
Hence, for the peculiarity of their atomic structures, the biogenic elements are the only ones which through their compounds are adapted to carry out, in living organisms, the numerous biological functions. We know that between these compounds the most important are the nucleic acids, DNA and RNA, and the proteins. The nucleic acids are the carriers of information for the synthesis of the proteins. Between the two nucleic acids the RNA has given origin to the theory of the “RNA World”. This postulate that life had its origin through the appearance of self-reproducing  molecules of RNA.

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The RNA is a large molecule whose   unities constitutive are four nucleotides.
These are the fundamental walls of RNA and they are constituted by a phosphate group, by D-Ribose (a pentose sugar) and one of the four nucleobases, Adenine, Guanine, Uracil, Cytosine.
The nucleotides, however, to be able to give origin to RNA must be activated, that is at only nucleotide it is necessary to link another two phosphate groups.
As we have already shown in the article concerning the “RNA World”, A. Graham Cairns-Smith has analysed the process through which, in laboratory,
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 activated nucleotides were obtained. He has counted 140 events (pouring, agitating, decanting, distilling, etc.) which should have been realized, in the prebiotic era, not by chance but in the appropriate way; that is like throwing a dice and obtaining number 6 for 140 times straight. Seeing these immense difficulties of procedure, following the principle of Occam’s razor, the most simple and obvious supposition is that the nucleotides, in the prebiotic era, never existed. And in fact Cairns-Smith in “Sette indizi sull’origine della vita” 1986 concludes: «[…]. These have the complete aspect of molecules programed for particular designs».
However, as we have seen, the nucleotides are constituted of the synthesis of three different molecules, which are hence the fundaments of the RNA.
The phosphate group which, even if in small quantities, is diffused on the whole surface of the planet.
The D-Ribose (a pentose sugar) and one of the four nucleobases : Adenine, Guanine, belonging to the family of the purine; Uracil and Cytosine, belonging to the family of the pyrimidine. Now, over and above the procedural difficulties to obtain activated nucleotides, Ribose and nucleobases were present in prebiotic era?       
  


Those who thinks that these substances were present in the prebiotic era, are principally the supporters of the RNA World. So, through the publications of their most influential scientists, Ciryl Ponnamperuma, Manfred Eigen and Leslie Orgel, we go and discover their cards. (It is useful to point out that for ammonium cyanide one intends hydrocyanic acid, HCN, and ammonia, NH3; and over more, hydrocyanic acid is called also hydrogen cyanide).
Ciryl Ponnamperuma “Origine della vita” 1984: «The purines were synthesized  for the first time in conditions which simulated those of the primitive earth by Orò(v. Studies in …1963), who demonstrated that one could obtain the synthesis of Adenine by a concentrated solution of ammonium cyanide. In its broad lines the reaction can be represented as 5 molecules of hydrogen cyanide that in the presence of ammonia give place to Adenine. This synthesis has be confirmed by Lowe […]. Afterwards, Orò succeeded in synthesizing Guanine and Xanthine by bringing to a temperature of 100-140°C a solution with water of amminoimidazolcarbossiammide. The rendering was of 1,5% for both the purines. It is possible that this was one of the ways in which the synthesis of purines on the primitive Earth took place, but the concentrations used by Orò were very much too high to correspond to a prebiotic situation. If the experimental concentrations had been really like those prebiotic, if for example lower concentrations had been used, then these reactions would be a great help to the understanding of the origin of the purines in the condition present in the prebiotic phase of the Earth. In spite of the efforts made, it was not possible to identify with certainty purines and pyrimidines among the final products in experiments which used electric charge. As a great quantity of hydrocyanic acid is formed, it is difficult to understand how the purines could be absent. Very little has been done in the region of the synthesis of the pyrimidines. Fox and Harada (v., 1961) have demonstrated that Uracil can be obtained by the heating of malic acid and urea. Whereas  urea is easily formed in the experiments which simulate the primitive Earth, there does not exist any indication of the presence of malic acid. […] and others (v., Formation of…,1963) have obtained also the synthesis of adenine through electrons flux on methane, ammonia and water […]. The greatest part of the radiations produced by radioactive sources is absorbed by solids, and as the earth’s crust has a thickness of about 30 km, this type of radiation could have had no role in the synthesis of organic material in the primitive oceans». Hence Ponnamperuma affirms, in 1984 that these reactions are not of great help to the understanding of the synthesis of the nucleobases in the prebiotic phase. These however do not offer any indication of the presence of nucleobases in the prebiotic era. On the other hand reactions do not respect the first and the second procedural rule and they are hence laboratory experiments of no interest for prebiotic chemistry.
 
 With reference to Ribose, Ponnamperuma continues: «In contrast with the progress obtained in the field of primordial chemistry of the amino acids, the origin of another group of substances just as important biologically the monosaccharides (sugars), is even now fairly uncertain. Already in 1861 A. Butelow had shown that formaldehyde dissolved in water goes towards a process of condensation in an alkaline ambiance, giving place to a mixture of sugars. […]. There are for this reason various difficulties in accepting the hypothesis that formaldehyde could have been used as a precursor of the monosaccharides. Horowitz and Miller have made it clear that the high concentrations of formaldehyde used in some experiments do not reproduce in a realistic way the conditions of the primitive Earth. Objections have also been made to the use of very basic solutions. P. H. Abelson sustains that the concentration of ammonia free in the seas and in the atmosphere was not ever so elevated, and that a strong alkaline ocean never existed. Furthermore, formaldehyde polymerizes fairly rapidly giving paraformaldehyde, and so can go a certain distance from the ambiance […]. 
The fact of having reproduced in a satisfactory way the synthesis of the nucleobases and the sugars of the nucleic acids in conditions simulating those of the primitive Earth has inspired some researchers  to make searches on the abiotic formation of nucleosides». Sic!
It is not only one contradictions. Ponnamperuma falsifies the evidence of the facts by he himself listed. But why?
To understand such a conclusion, it is necessary to go back some years and refer to two affirmations by Manfred Eigen and others in “L’origine dell’informazione genetica” Le Scienze, 1981.  These quotations have already been amply introduced in the article concerning the “The RNA World”. With reference to the studies of Sol Spiegelman, Leslie Orgel and others Eigen affirms: «The fundamental conclusion of these studies is that the self-reproducing of the RNA in systems in vitro effectively takes place also without the intervention of sophisticated enzymes. It is possible to go ahead considering the evolutive consequence of self-reproducing of RNA without preoccupying oneself if this really happened in the prebiotic era: however it happened».
This affirmation was in reality the announcement of an announced disaster, because science does not proceed in this way.
And Manfred Eigen in the same article continues: «That which is important, here, is what these experiments reveal on Darwin’s processes. Natural selection and evolution, which are the consequences of self-reproducing, operates at the molecular level as at the level of the cells or the species».
But it was necessary to wait Ernst Mayr (L’unicità della biologia, 2005) to put into evidence, in a clear way, that the target of the selection is the phenotype and not the gene, and that this terminology is in flagrant conflict with Darwin’s basic thought?
At the end, in many researchers, sometime before, the idea had matured of extending Darwin’s theory also to molecules. In this way evolution through natural selection could be extended also to the origin of life. Everything should hence develop  according to this design. The idea slowly crystalized in their mind to the point of considering superfluous deepening the possibility of synthesis, in the prebiotic era, of nucleobases and ribose. And in fact Manfred Eigen in “Gradini verso la vita”.1992,with reference to the constitutive chemical unities necessary for life at the primordial era of our planet and in particular with reference to the nucleobases affirms: «To give a generic affirmative answer to this interrogation would mean, given the actual level of knowledge of organic chemistry, to deny the character of the basic question. […]. For the different constituents of the nucleic acids, methods of synthesis are known which in prebiotic conditions were fully realizable. The simplest to obtain is Alanine (the nucleobase A of the nucleic acids), for which different methods of synthesis exist, among which the most simple condensation of hydrocyanic acid […]. In ambiance without water that are, as Albert Eschenmoser has been able to demonstrate, other procedures of synthesis very elegant, efficient for all four nucleobases of the nucleic acids. With a degree of probability fairly close to certainty, the four nucleobases were, in the phase of their chemical synthesis, present in concentrations extremely different».
And so, after about 10 years, Eigen comes to the same false conclusion as Ponnamperuma. Moreover, as he could not give data to sustain the origin of nucleobases, he gives reactions in an ambiance without water that, in prebiotic chemistry, is not admitted.
In 1995 Christian De Duve, although a sustainer of the “RNA World” in “Polvere Vitale” affirms:« […] chemists have had a certain success in the production of the five organic components of the RNA, but with scarce rendering and in conditions at the same time very different from a prebiotic scenery and different for every substance. If one wants to combine the components in the right way, one comes across other problems, of such magnitude that no one has ever tried to do it in a prebiotic contest».
In 2004 Orgel, “Prebiotic Chemistry and the origin of the RNA World”, tries to unify the scene at least for the four nucleobases.
He shows how Adenine can be obtained from a eutectic solution, very concentrated, of NH3 and HCN at the temperature of -23,4 °C, that is the synthesis of Orò revisited. Orgel points out that among the products of this reaction, traces of Guanine were found.
Orgel observes how the Uracil can be obtained by the hydrolysis of Cytosine.
For the synthesis of Cytosine he takes first into consideration the reaction between cyan acetic aldehyde and urea concentrated, but he concludes that, in the prebiotic era, such a reaction is not plausible. However it seems to him more plausible the reaction of a eutectic solution of cyan acetylene with cyanide because it could proceed in parallel with the synthesis of adenine also in eutectic condition.
On paper under the word “eutectic” the synthesis of the four nucleobases were done with the same logic.  But temperature and concentrations indicated by Orgel are completely outside any prebiotic context. However, in which way all this could be translated in the prebiotic conditions of the primordial earth cannot be known. It is overmore necessary to keep in mind that these synthesis also cause the formation of many nucleobases without interest, even obstacles for the origin of life.
To be short, in 2004, that is after a half century of tentatives by the best chemists in the world, we can conclude that, in the prebiotic phase, no chemical process has produced the four nucleobases.
In 1994 in Le Scienze, “l’origine della vita sulla terra”, referring to Ribose L. Orgel writes: «Above all, in the lack of enzymes, it is a problem to synthesize Ribose in adequate quantities and with sufficient degree of purity».
In 2004, in “Prebiotic chemistry and the origin of the RNA World”, Orgel again takes up the now well-known reaction of Butlerow of 150 years ago. He writes that if this reaction could be addressed towards the synthesis of Ribose it would be ideal for the synthesis of sugar as a component of the nucleotides. Orgel quotes Zubey (1998, 2001) who repeated the reaction in the presence of lead. He also makes it clear how the calcium borate stabilises the pentoses (Ricardo and others, 2004). Orgel admits that all these reactions take place at pH to high, and with high concentrations of reagents. In the end he concludes that some progress has been mad, but there are still a series of obstacles to the synthesis which would produce significant and pure quantities of Ribose.  
Hence, after 150 years from Butlerow’s discovery, we can conclude that also Ribose in the prebiotic era never existed. And on nucleobases and Ribose, after 50 research, that which is left is only a handful of ashes.
Now it is clear how, to have tried to extend evolution by natural selection also to molecules, has extended the problem of the origin of life from mud to marsh. And yet, on nucleobases and sugars, that which was divulgated by the most respected scientific reviews, were the conclusion of these influential scientists. In truth there was some timid objection on the part of Freeman Dyson , Graham Cairns-Smith and some other  scientists.
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 However the greater part of the scientists (and unfortunately we modest observers), for some ten years , remained convinced that, although with some difficulties, nucleobases and Ribose, in the prebiotic era were present on our planet.
And so, a poker of aces was divulgated, but it was only a colossal Bluff.
You meanwhile, incredulous, go away from the building site accompanied by a deafening sound which confirms you in your presentiment.
And in 2013? Now it is enough, let us go out of this marsh.
In the prebiotic era, on our planet no chemical-physical process has given origin to Ribose and nucleobases. The origin of these substances, in such an epoch, stays only in the head of those who sustain the “RNA World”. And in fact that which remains of their theory, after more than half a century of research, is only an amass of ruins.
Now, if we wish to imagine eventual scenes which go over our planet, it is useful to go through the principal passage necessary to arrive at the RNA. I apologize to those in possession of different knowledge; they will find the description too synthetic, and hence not easy to understand, but I hope it is possible to have the intuition of a global judgement as a matter of fact.
Let us admit that in another planet, in prebiotic conditions different from ours, the synthesis of nucleobases and sugars really happened.  
In the pentoses  three carbon atoms are asymmetric and thus we have three chiral centres. This implies that the number of possible molecules (stereoisomers) is equal to 23 that is 8, of which four D (right) and four L (left) and among these the Ribose. It is true that every couple D-L has chemical-physic characteristics slightly different with respect to others, but from an energy point of view in a prebiotic phase, they all have the same probability to being synthesized. Now only for the spontaneous assemblage a nucleotide illustrated below,

 the following passages are necessary:
1) The separation of the pentoses from all the other sugars.
2) The separation of the four pentoses D (right) from the L to avoid crossed reactions.
3) The separation of the D-Ribose  from the other three sugars D to avoid superposed reactions.
4) The separation of the four nucleobases necessary for the RNA from all the others.
5) Presence of a consistent source of phosphate (H2PO4-) in solution.
6) A particular orientation, to obtain the four nucleotides, of all the nucleobases on OH of C-1, and to avoid the reaction with OH from C-2, C-3, C-5, (as illustrated above).
7) A particular orientation of the phosphate group on OH of C-5 to the end of avoiding the reaction with the OH from C-2, C-3 (as illustrated above)
8) The orientation of all the nucleobases in the position β (that in upwards as in the figure).
9) The reaction of phosphate of the nucleotides with OH in the position C-3 of other nucleotides, with the exclusion of the position C-2, to give origin to the RNA.
In over 50 years of research, there have not been and there is not even now a chemist who has succeeded in corroding points 1, 2, 3, 4, 5. And there has not been and there is not even now a biologist who can give an explanation to points 6, 7, 8, 9 without the intervention of specific enzymes.
To conclude:
On our planet.
In the prebiotic era, on our planet, no chemical-physic process has given origin to Ribose and nucleobases; the laws of chemistry and physics stopped their formation. And if we rewind the film of the origin of life and we reproject it, in all probability we would see again the same story.
In the universe:
We cannot exclude that in different prebiotic conditions, on some other planet, sugars and nucleobases are formed. But life without water in a liquid state cannot exist. In such conditions, the laws of physics and chemistry being universal, in no planet, except for a miracle, can the 9 points, above mentioned, be realized spontaneously. Hence, in no part of the universe ever existed or will exist a “RNA World”.
And if there is still who wants to use the usual passe-partout phrase: one cannot exclude such a possibility, what to say: dreams cannot be prohibited.

                                                                                     Giovanni Occhipinti

Translated by Silvia Occhipinti  (04.10.2014)



venerdì 3 maggio 2013

L'RNA: I SUOI COSTITUENTI. Un bluff





Post n.10
NOI, GLI ALIENI, LA MATERIA. Ma un'altra vita è possibile (le macromolecole fondamentali)
Immaginatevi di voler costruire una casa. Incaricate degli architetti versando un cospicuo anticipo. Dopo alcuni mesi andate a controllare lo stato dei lavori e trovate solo il tetto sorretto da una enorme ponteggio. Gli architetti vi spiegano che non c’è problema, si può benissimo iniziare dal tetto. Fondamenta e muri portanti, nel progetto, ci sono già. Riguardate ancora, sbigottiti, lo stato dei lavori e notate che l’impalcatura inizia a vacillare.
Che sensazioni provate?
Dunque, per le peculiarità delle loro strutture atomiche, gli elementi biogeni sono gli unici che, attraverso i loro composti, sono adatti a svolgere, negli organismi viventi, le numerose funzioni biologiche. Sappiamo che tra questi composti i più importanti sono gli acidi nucleici, DNA e RNA, e le proteine. Gli acidi nucleici sono portatori dell’informazione per la sintesi delle proteine.
Tra i due acidi nucleici l’RNA ha dato origine alla teoria del “mondo a RNA”. Essa postula che la vita ebbe origine attraverso la comparsa di molecole autoreplicanti di RNA.
immag. orig.
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Ma i costituenti dell’RNA erano presenti in epoca prebiotica?
L'RNA è una grande molecola le cui unità costitutive sono quattro nucleotidi.
Essi sono i muri portanti dell’RNA e sono costituiti da un gruppo fosfato, dal D-Ribosio (uno zucchero pentoso) e una delle quattro basi azotate, Adenina, Guanina, Uracile, Citosina.
I nucleotidi però, per poter dare origine all’RNA devono essere attivati, cioè ad ogni nucleotide bisogna legare altri due gruppi fosfato.
Come abbiamo già evidenziato nell’articolo riguardante il mondo a RNA,A. Graham Cairns-Smith ha analizzato la procedura attraverso la quale, in laboratorio, si sono ottenuti nucleotidi attivati.

immag.orig.
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Egli ha contato 140 eventi (versare, agitare, decantare, distillare, ecc.) che avrebbero dovuto succedersi, in epoca prebiotica, non a caso ma in maniera appropriata; cioè, come lanciare un dado e ottenere il numero 6 per 140 volte di seguito. Viste queste enormi difficolta procedurali, seguendo il principio del rasoio di Occam, la supposizione più semplice e ovvia è che i nucleotidi in epoca prebiotica non sono mai esistiti. E infatti Graham Cairns-Smith in “Sette indizi sull’origine della vita”, 1986, conclude: «[…]. Essi hanno tutto l’aspetto di molecole congegnate per scopi particolari».
Comunque, come abbiamo visto, i nucleotidi sono costituiti dalla sintesi di tre molecole diverse, che sono poi le fondamenta dell’RNA. Il gruppo fosfato che, anche se in piccole quantità, si trova diffuso su tutta la superficie del pianeta.
Il D-Ribosio(uno zucchero pentoso) e una delle quattro basi: Adenina e Guanina, appartenenti alla famiglia delle Purine; Uracile e Citosina appartenenti alla famiglia delle Pirimidine.
Ora, al di là delle difficoltà procedurali per ottenere nucleotidi attivati, ma Ribosio e Basi azotate erano presenti in epoca prebiotica?

Chi ritiene che queste sostanze fossero presenti in epoca prebiotica, sono principalmente i sostenitori del mondo a RNA. Allora, attraverso le pubblicazioni dei loro più autorevoli scienziati, Ciryl Ponnamperuma, Manfred Eigen e Leslie Orgel, andiamo a scoprire le loro carte. (è utile precisare che per cianuro di ammonio si intende acido cianidrico, HCN e ammoniaca, NH3; inoltre, l’acido cianidrico vien chiamato anche cianuro di idrogeno)
C. Ponnamperuma “Origine della vita”, 1984: «Le purine sono state sintetizzate per la prima volta in condizioni che simulavano quelle della Terra primitiva da Orò (v., Studies in..., 1963), il quale dimostrò che si poteva ottenere la sintesi di adenina da una soluzione concentrata di cianuro d'ammonio. Nelle sue linee essenziali la reazione può essere raffigurata come 5 molecole di cianuro di idrogeno che, in presenza di ammoniaca, danno luogo ad adenina. Questa sintesi è stata confermata da Lowe e collaboratori […]. In seguito, Orò riuscì a sintetizzare guanina e xantina portando a una temperatura di 100-140°C una soluzione acquosa di amminoimidazolocarbossimmide. Il rendimento è stato dell'1,5% per ambedue le purine. È possibile che questo sia uno dei modi in cui è avvenuta la sintesi delle purine sulla Terra primitiva, ma le concentrazioni usate da Orò erano di gran lunga troppo alte per corrispondere a una situazione prebiotica. Se le condizioni sperimentali fossero state davvero simili a quelle prebiotiche, se, per esempio, si fossero usate concentrazioni più basse, allora queste reazioni sarebbero di grande aiuto alla comprensione dell'origine delle purine nelle condizioni presenti nella fase prebiotica della Terra.. Nonostante gli sforzi fatti, non è stato possibile identificare con sicurezza purine e pirimidine tra i prodotti finali in esperimenti che impiegavano scariche elettriche. Poiché si forma una gran quantità di cianuro di idrogeno, è difficile capire come le purine potrebbero essere assenti. Ben poco è stato fatto nel campo della sintesi delle pirimidine. Fox e Harada (v., 1961) hanno dimostrato che uracile può essere ottenuto per riscaldamento di acido malico e urea. Mentre l'urea si forma facilmente negli esperimenti che simulano la Terra primitiva, non esiste alcuna indicazione della presenza di acido malico. […] e altri (v., Formation of.., 1963) hanno ottenuto anche la sintesi di adenina mediante irraggiamento con elettroni di metano, ammoniaca e acqua.[…]La maggior parte delle radiazioni prodotte da sorgenti radioattive viene assorbita dai solidi, e poiché la crosta terrestre ha uno spessore di circa 30 km, questo tipo di radiazione potrebbe non aver avuto alcun ruolo nella sintesi di materiale organico negli oceani primitivi».
Quindi Ponnamperuma afferma, nel 1984, che queste reazioni non sono di grande aiuto alla comprensione delle sintesi delle basi azotate nella fase prebiotica.
Esse quindi non offrono alcun indizio della presenza delle basi azotate in epoca prebiotica. D’altra parte reazioni non rispettano la prima e la seconda regola procedurale e sono, quindi, esperimenti di laboratorio di nessun interesse per la chimica prebiotica.
In riferimento al ribosio, Ponnamperuma continua: «In contrasto con i progressi compiuti nel campo della chimica primordiale degli amminoacidi, l'origine di un altro gruppo di sostanze altrettanto importanti biologicamente, i monosaccaridi (zuccheri), è tuttora assai incerta. Già nel 1861 A. Butlerow aveva mostrato che formaldeide (HCHO) sciolta in acqua va incontro a un processo di condensazione, in ambiente alcalino, dando luogo a una miscela di zuccheri. […] Ci sono peraltro varie difficoltà nell'accettare l'ipotesi che la formaldeide possa essere stata utilizzata come precursore dei monosaccaridi. Horowitz e Miller hanno sottolineato che le elevate concentrazioni di formaldeide usate in alcuni esperimenti non riproducono in modo realistico le condizioni della Terra primitiva. Sono state sollevate obiezioni anche all'uso di soluzioni molto basiche. P. H. Abelson sostiene che la concentrazione di ammoniaca libera nei mari e nell'atmosfera non fu mai molto elevata, e che un oceano fortemente alcalino non è mai esistito. Inoltre, la formaldeide polimerizza assai rapidamente dando paraformaldeide, e può pertanto allontanarsi dall'ambiente. […].Il fatto di avere riprodotto in modo soddisfacente la sintesi delle basi e degli zuccheri degli acidi nucleici in condizioni simulanti quelle della Terra primitiva ha spinto alcuni ricercatori a compiere indagini sulla formazione abiogena di nucleosidi».  Sic!
Non è una solo una contraddizione, Ponnamperuma falsifica l’evidenza dei fatti da lui stesso elencati. Ma perché?
Per comprendere tale conclusione, bisogna tornare indietro di qualche anno e fare riferimento a due affermazioni di Manfred Eigen e altri in “L’origine dell’informazione genetica”, (Le Scienze, 1981). Queste citazioni sono state già ampiamente introdotte nel post riguardante il “Mondo a RNA”. In riferimento agli studi di Sol Spiegelman, Leslie Orgel e altri egli afferma: «La conclusione fondamentale di questi studi è che l’auto replicazione dell’RNA in sistemi in vitro avviene effettivamente anche senza l’intervento di enzimi sofisticati. È possibile andare avanti a considerare le conseguenze evolutive dell’auto replicazione dell’RNA senza doversi preoccupare se questa realmente avvenne in tempi prebiotici: essa comunque avvenne».
Questa affermazione era in realtà l’ annuncio di un disastro annunciato, perché la scienza non procede in questo modo.
E Manfred Eigen nello stesso articolo continua:
«Ciò che è importante, qui, è quello che questi esperimenti rivelano sui processi darwiniani. La selezione naturale e l’evoluzione, che sono conseguenze dell’auto replicazione, opera a livello delle molecole così come a livello delle cellule o delle specie».
Ma bisognava aspettare Ernst Mayr (L’unicità della biologia,2005) per mettere in evidenza, in modo chiaro, che il bersaglio della selezione è il fenotipo e non il gene, e che questa terminologia è in netto conflitto con il pensiero Darwiniano di base?
In definitiva, in parecchi ricercatori, era sorta da tempo l’idea di estendere la teoria di Darwin anche alle molecole. In questo modo l’evoluzione per selezione naturale poteva essere estesa anche all’origine della vita. Tutto doveva quindi scorrere secondo questo disegno. L’idea lentamente si cristallizzò nella loro mente tanto da considerare superfluo approfondire la possibilità di sintesi, in epoca prebiotica, di basi azotate e ribosio. E infatti Manfred Eigen in “Gradini verso la vita”, 1992, in riferimento alle unità costitutive chimiche necessarie per la vita ai primordi del nostro pianeta e in particolare alle basi azotati afferma: «Dare una risposta affermativa generica a questo interrogativo significherebbe, dato l’attuale livello conoscitivo della chimica organica, negarne il carattere di questione di fondo.
[…]. Per i diversi costituenti degli acidi nucleici sono noti metodi di sintesi che in condizioni prebiotiche erano pienamente realizzabili. La più semplice da ottenere è l’adenina (la base A degli acidi nucleici), per la quale esistono diversi metodi di sintesi, tra cui la più semplice condensazione di acido cianidrico. […]. In ambiente non acquoso vi sono, come ha potuto mostrare Albert Eschenmoser, altri procedimenti di sintesi molto eleganti, efficaci per tutti e quattro le basi degli acidi nucleici.
Con un grado di probabilità assai prossimo alla certezza, le quattro basi erano, nella fase della loro sintesi chimiche, presenti in concentrazioni estremamente diverse».
E così, dopo circa 10 anni, Eigen arriva alla stessa falsa conclusione di Ponnamperuma. Inoltre non potendo portare dati a sostegno dell’origine delle basi azotate, riporta reazioni in ambiente non acquoso che, in chimica prebiotica, non sono ammesse.
Nel 1995 C. de Duve, pur essendo un sostenitore dell’RNA, in “Polvere vitale”, afferma: «[…] i chimici hanno avuto un certo successo nella produzione dei cinque componenti organici dell'RNA, ma con scarso rendimento ed in condizioni ad un tempo molto diverse da uno scenario prebiotico e diverse per ogni sostanza. Volendo combinare i componenti nel modo giusto ci si imbatte in altri problemi, di tale grandezza che nessuno ha mai tentato di farlo in un contesto prebiotico».
Nel 2004 Orgel, “ Prebiotic Chemistry and the Origin of the RNA World”,2004, tenta di unificare lo scenario almeno per le quattro basi azotate.
Egli mette in evidenza come l’Adenina si può ottenere da una miscela eutettica, molto concentrata, di HCN e NH3 (acido cianidrico e ammoniaca) alla temperatura di  -23,4°C, cioè la sintesi di Orò rivisitata. Sottolinea che tra i prodotti di questa reazione si sono trovate tracce di guanina.
Orgel fa notare come l’uracile si possa ottenere dall’idrolisi della citosina.
Per la sintesi della citosina prende prima in considerazione la reazione tra ciano-acetaldeide e urea concentrata ma conclude che, in epoca prebiotica, una simile reazione non è plausibile. Le sembra però più plausibile la reazione di una soluzione eutettica di ciano acetilene con cianato perché potrebbe procedere in parallelo con la sintesi dell’adenina anch’essa in condizione eutettica.
Sulla carta, sotto la parola “eutettica”, la sintesi delle quattro basi azotate sono state condotte sotto la stessa logica. Ma temperatura e concentrazioni indicate da Orgel son completamente fuori da ogni contesto prebiotico. Comunque, in che modo tutto questo si sarebbe tradotto nelle condizioni prebiotiche della terra primordiale non è dato sapere. Bisogna inoltre tenere presente che queste sintesi portano anche alla formazione di tante altre basi azotate di nessun interesse anzi di ostacolo per l’origine della vita.
In definitiva, nel 2004, cioè dopo oltre mezzo secolo di tentativi da parte dei migliori chimici del mondo, possiamo concludere che, nella fase prebiotica, nessun processo chimico ha prodotto le quattro basi azotate.
Nel 1994 in Le scienze, “L’origine della vita sulla terra”, in riferimento al ribosio L. Orgel scrive: «Innanzitutto, in mancanza di enzimi, è problematico sintetizzare ribosio in quantità adeguate e con un sufficiente grado di purezza».
Nel 2004, in Prebiotic Chemistry and the Origin of the RNA World egli riprende però l’ormai nota reazione di Butlerow di 150 anni fa. Scrive che se questa reazione potesse essere indirizzata verso la sintesi del ribosio sarebbe ideale per la sintesi dello zucchero componente dei nucleotidi. Cita Zubey(1998, 2001) che ha ripetuto la reazione in presenza di piombo. Mette anche in evidenza come il borato di calcio stabilizza i pentosi (Ricardo e altri, 2004). Ammette che tutte queste reazioni avvengono a pH troppo elevato e con elevate concentrazioni di reagenti. Infine conclude che alcuni progressi sono stati fatti, ma che vi sono ancora una serie di ostacoli alla sintesi che produca quantità significative e pure di ribosio.
Così, dopo 150 anni dalla scoperta di Butlerow, possiamo concludere che anche il ribosio in epoca prebiotica non è mai esistito. E su basi azotate e ribosio, dopo cinquant’anni di ricerche, ciò che rimane è solo un pugno di cenere.
Ora è chiaro come, aver cercato di estendere l’evoluzione per selezione naturale anche alle molecole, ha spinto il problema dell’origine della vita dal pantano alla palude. Intanto, su basi azotate e zuccheri, ciò che veniva divulgato dalle più blasonate riviste scientifiche erano le conclusioni di questi autorevoli scienziati. Vi fu in verità qualche timida obiezione da parte di Freeman Dyson, Graham Cairns-Smith e qualche altro scienziato. Comunque la maggior parte degli scienziati (e purtroppo anche noi modesti osservatori), per qualche decennio, rimase convinta che, se pur con qualche difficoltà, basi azotate e ribosio, in epoca prebiotica erano presenti sul nostro pianeta.
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In epoca prebiotica, sul nostro pianeta nessun processo chimico fisico ha dato origine a ribosio e basi azotate. L’origine di queste sostanze, in tale epoca, risiede solo nella testa dei sostenitori del “mondo a RNA”. E infatti ciò che rimane della loro teoria, dopo oltre mezzo secolo di ricerche, è solo un cumulo di macerie.
Ora, se vogliamo immaginare eventuali scenari che vanno oltre il nostro pianeta è utile ripercorrere i principali passaggi necessari per arrivare all’RNA. Mi scuso con quanti in possesso di conoscenze diverse troveranno la descrizione troppo sintetica e quindi di non facile comprensione, ma spero si possa intuire un giudizio globale di fattibilità.
Ammettiamo che in un altro pianeta, in condizioni prebiotiche diverse dal nostro, la sintesi delle basi azotate e degli zuccheri sia realmente avvenuta.
Nei pentosi sono asimmetrici tre atomi di carbonio e quindi si hanno tre centri chirali. Ciò comporta che il numero di molecole possibili (stereoisomeri) sia pari a 23, ossia 8, di cui quattro D (Destro) e quattro L (Levo) e tra questi il D Ribosio. È vero che ogni coppia DL presenta caratteristiche chimico-fisiche leggermente diverse rispetto alle altre, ma da un punto di vista energetico in una fase prebiotica, hanno tutti la stessa probabilità di essere sintetizzate. Ora, per l’assemblaggio spontaneo di un solo nucleotide qui  illustrato,


sono necessari i seguenti passaggi:
1) Separazione dei pentosi da tutti gli altri zuccheri
2) Separazione dei quattro pentosi Destro dai Levo per evitare reazioni incrociate.
3) Separazione del D-Ribosio dagli altri tre zuccheri Destro per evitare reazioni sovrapposte.
4) Separazione delle quattro basi necessarie per l'RNA da tutte le altre.
5) Presenza di una fonte consistente di fosfato (H2PO4)- in soluzione.
6)Particolare orientamento, per ottenere i quattro nucleotidi, di tutte e quattro basi sull’OH del C-1, ed evitare la reazione con l'OH del C-2, C-3, C-5. (come illustrato sopra)
7) Particolare orientamento del gruppo fosfato sull'OH del C-5 al fine di evitare la reazione con l'OH del C-2, C-3. (come illustrato sopra)
8) Orientamento di tutte le basi nella posizione β (cioè verso l’alto come in figura)
E infine per l’RNA:
9) Reazione del fosfato dei nucleotidi con l'OH in posizione C-3 di altri nucleotide, con l’esclusione della posizione C-2, per dare origine all’RNA.
In oltre 50 anni di ricerche, non c’è stato e non c’è a tutt’oggi un chimico che sia riuscito a scalfire i punti 1,2,3,4,5. E non c’è stato e non c’è a tutt’oggi un biologo che possa dare una spiegazione ai punti 6,7,8,e 9 senza l’apporto di enzimi specifici.
Concludendo:
Sul nostro pianeta.
In epoca prebiotica, sul nostro pianeta nessun processo chimico fisico ha dato origine a ribosio e basi organiche; le leggi della chimica e della fisica ne impedirono la loro formazione. Esse riavvolgiamo il film dell’origine della vita e lo riproiettiamo, con ogni probabilità, rivedremmo la stessa storia.
Nell’universo.
Non possiamo escludere che in condizioni prebiotiche diverse, in qualche altro pianeta, si siano formati zuccheri e basi azotate. Però la vita senza acqua allo stato liquido non può esistere. In tali condizioni, essendo le leggi della fisica e della chimica universali, in nessun pianeta, a meno di un miracolo, possono realizzarsi spontaneamente i 9 punti sopra elencati. Quindi, in nessuna parte dell’universo e mai esistito e mai esisterà un “Mondo a RNA”.
E se c’è ancora chi vuole utilizzare la solita frase passe-partout: non si può escludere tale possibilità; che dire, i sogni non li si può proibire.

                                                                                   Giovanni Occhipinti
(Di eventuali errori o informazioni errate contenuti in questa articolo sono responsabile solo io G.O.).
Prossimo articolo, leggere pagina info