venerdì 28 settembre 2012

PREBIOTIC CHEMISTRY: rules or chaos, the prebiotic razor

It is told that a wise Rabin, was trying to resolve a quarrel between husband and wife. The husband complains to him of his wife. “You are right” answered the Rabin. His wife complained to him of her husband. “You are right” answered the Rabin. The Rabin’s wife comes out of behind a curtain, from where she had heard everything. “How can you say to both of them that they are right?”, she asked her husband. At this the wise Rabin answered: “You too are right”.
This little story, evoked by R. Panek in “L’universo oscuro” 2011, has been told by Vera Rubin, in a conference in 1996, to reassume the situation in which at this epoch cosmology found itself. But the story also illustrates in adequate way the state in which prebiotic chemistry finds itself today.
Who were the Alchemists? Chemists without rules.
Alchemy, at its origin had as its aim, the search for the philosopher’s stone and the origin of life. Such research led to the knowledge  

of the specific properties of many substances. However every alchemist had his own ideas, rules, methods, and the largest part of their work did not produce any result. When beginning at the 16th century rules were introduced, alchemy became science.

What were those rules? The fundamental laws of chemistry: the law of the conservation of mass by Lavoisier, the definition of compound by Proust, and the law of Dalton which introduced the atom.
Without laws, in whatever field of human knowledge, it is necessary to put rules or to establish conventions or reference points, otherwise we are in full chaos. Imagine how the street traffic would be without rules, that is without the highway code, without road signs and lights. In science also conventions are necessary. By convention hydrogen is indicated by the letter H, oxygen by the letter O, and water by H2O.
To measure the temperature of a body we need a thermometer. But to create a thermometer we must have reference points. These reference points are: the freezing point of water fixed at 0°C, and its boiling point at 100°C. We divide into a hundred parts this interval and we have the centigrade scale.
In astrophysics one wanted to understand what the universe would do: would it continue to expand or would it turn back? It was hence necessary to have a reference point, a source far away of light, and study its luminosity. For this purpose, it seems that the best sources of light are the supernovas of the group SNIa, called: standard candles. Studies of standard candles affirm that the universe is in continual expansion. Without reference points, without rules and conventions, one can go no way. Football also has its rules. Imagine football without rules: at the moment in which a penalty is about to be shot, the goalkeeper displaces the goal and puts it in the point of the corner. Rules whether agreeable or not must be everywhere. Only for artists, for their work, it is permitted not to have rules.
On the question of the origin of life, one starts with the ascertainment that life had its origin on our planet and that it could not have been a chance event. Prebiotic chemistry treats the formation of the fundamental substances for the synthesis of the macromolecules necessary to the origin of life in the prebiotic era. It is however difficult to establish what the physical-chemical conditions in prebiotic era were like. And so we cannot know with certainty if certain substances, the result of precise experiments, were or not present in the prebiotic era. There exists no rules, conventions, reference points; every researcher chooses his own, and so all are right. It seems absurd, but things are really like that, as confirmed by Orgel:
Leslie E. Orgel (Critical Reviews in Biochemistry and Molecular Biology, (39:99–123, 2004): «Prebiotic chemistry is concerned with molecules that are interesting to students of the origin of life which, they believe, could have been formed on the primitive Earth. Since we know very little about the availability of starting materials on the primitive Earth or about the physical conditions at the site where life began, it is often difficult to decide whether or not a synthesis is plausibly prebiotic. Not surprisingly, claims of the type, “My synthesis is more prebiotic than yours” are common. Nonetheless, there is fairly general agreement about the following restrictions on organic synthesis imposed by the requirement for prebioticity:
It must be plausible, at least to the proposers of a prebiotic synthesis, that the starting materials for a synthesis could have been present in adequate amounts at the site of synthesis.
Reactions must occur in water or in the absence of a solvent.
The yield of the product must be “significant,” at least in the view of the proposers of the synthesis.
Clearly “prebiotic” is a very elastic term, and it would not be wise to try to define it too closely».
Translated, the necessity for putting restrictions is recognized, but in fact the researcher is left free to choose his own restrictions. As every researcher is free to choose the prebiotic conditions, all the research is considered as linked with the origin of life and hence it is published. The result is a great number of publications, where every research worker obtains the substances fundamental for the origin of life in his own prebiotic ambience, often the fruit of fantasy and in contradiction with the prebiotic conditions of other scientists. Now it is evident that here there is a problem to be resolved; much the opposite of prebiotic chemistry, here we are in the presence of “prebiotic alchemy”.
Nucleotides are the constituents of the RNA. At the actual state of knowledge, the nucleotides in the prebiotic era could not exist and in fact they have never been obtained in experiments on prebiotic chemistry. Now, on the nucleotides and on the RNA, the absence of rules and of reference points, has led to the publication of some article which has no link with prebiotic chemistry. Some authoritative scientists, sustainers of the RNA world, have taken these articles as reference publishing the presence of nucleotides in prebiotic experiments.
It is sad to see authoritative scientists transmitting false information. And unfortunately, as all copy all, these information are even copied in test books for superior schools transferring false information to our students.
Now, coming from the above considerations it is evident how, to face the problem of the origin of life a new attitude is necessary. As suggested by Mario Ageno (Lezioni di Biofisica3,1984). «It is the same attitude that Urey inaugurated for research on the formation of the solar system: trying to gather, examining accurately, the actual state of things, all the indices and all the testimonies which can constitute conditions within limits for a future theory. On the basis of the indices and of the testimonies gathered, try to put together a sequence of possible events, that is which are not in contrast with our scientific knowledge,[…]».
Meanwhile the conditions within limits will impose restrictions which will have the function of “prebiotic razor”, eliminating all the elements which are in contradiction one with another. To reconstitute the sequence of the possible events, we can use: the simplest of the rules of logic,
1) The simplest of the rules of logic, that is hypothetic-deductive logic if…then, of the sort: if the petrol runs out, then the car is stationary.
2) Occam’s razor; William Ockham was a Franciscan Friar of the 14th century, to him is attributed the principle of Occam razor: one should always start from simple suppositions, obvious, and add afterwards complexity if necessary.
3) At length if this was not enough, we can follow the rule of common sense which as Descartes has written: common sense is the quality most frequently distributed among men.
In the end, as already said by Mario Ageno, condition within limits and possible events must not be in contrast with our scientific knowledge.
The primitive ambience: conditions within limits.

It is now certain that the earth has a life of about 4,6 billions of years. The most ancient sedimentary rocks which conserve testimony of forms of primitive life have an age of about 3,5 billions of years. Hence life must have appeared on earth in the interval of time of about 1 billion years in which there does not exist testimony. To try to understand how life could have had its origin from inorganic matter, we must try to find out what  the ambience conditions were on the surface of the earth in the first billion of years.
Prof Mario Ageno has dedicated an entire volume to the reconstruction of time and ambiance of the appearance of life on earth: “Lezioni di biofisica 2, 1984”. The sources form which he gathered the information are more than 150 publications of various researchers which go from the beginning of the last century to almost 1980, that is the years of the most intense research on the argument. Such research extend from the formation of the earth to the epoch of the appearance of life and the primitive ambiance, in particular the origin and history of the ocean and the atmosphere. The conclusions to which Mario Ageno come and to which   agree the greater part of scientists are the following:
1) Our solar system and hence our planet had their origin
with the collapse of a gas nebula and dust 4,6 billion of years ago. It seems that the gravitational collapse lasted 1 million years. Hence in the sun was lit up a strong thermonuclear reaction which produced a solar wind, made up principally of protons and electrons, a millions times more intense than the actual one. The solar wind has blew away vapor and the gas residue of the nebula, which wrapped around the earth. Our planet hence remained without an atmosphere.

2) For at least another 100 millions of years the earth would have attracted comets and wandering bodies also of important dimensions, its surface was hence fused; catastrophical sporadic impacts are not excluded until almost 4 milliards of years ago. When this phase of accumulation has ended, the surface of the earth slowly begins to cool down and after another 100 millions of years about, it goes under the boiling point of water. The water carried by the comets or coming from the earth’s losing gas begins to condense.
3) The condensation of water has slowly given origin to a primitive ocean of about 1/10 of the actual one. With the exception of carbonates and silica the composition and the pH of the ocean were similar to the actual one. Phenomena of a local increase of the salinity due to evaporation of water, are not excluded.
With the presence of the ocean the water-cycle began, and it triggered a strong mechanism of feed-back. Such a mechanism made the average temperature of the planet, already about 4 milliards of years ago, equal to the actual one, and it maintained it more or less constant until our days. Phenomena of local overheating particularly close to volcanoes are not to be excluded.
5) It is very probable that the primitive atmosphere, restored by comets, meteorites and volcanism, was constituted by Methane (CH4), Ammonia (NH3), Water (H2O). and Hydrogen (H2). It has been evaluated that an atmosphere of such a composition maintained itself for about a half milliard of years; it is not excluded that there was the presence of other substances and compounds of sulphur. Oxygen was practically absent and hence the shield of Ozone (O3) was absent. The ultraviolet rays, in quantities much larger than today, attained, in the primitive ocean the depth of 10 m., destroying the organic substances and life which was forming. After about a half a million of years the primitive atmosphere was substituted by N2 (Nitrogen) CO (Carbon oxide) and CO2 (Carbon dioxide) of volcanic origin and H2O (Water vapor).
Further workers tried to put in crisis the conclusion of these studies, in particular for that which concerned the primitive atmosphere, in 1996 Miller defined this work as lacking in concrete data to sustain it.
6) Starting from these data, according to the majority of researchers, life had its origin on our planet about 4 milliards of years ago.
The medium composition of a cell puts in evidence the presence: (Escherichia coli):
Water                                                     70%       mol. Weight medium in D.     18
Inorganic ions
( K+, Mg++, Ca, PO43-,SO42-,…)        1%                                         "                  40
Carbohydrates                                    3%                                         "                  150
Amino acids
(precursors  of proteins)                     0,4%                                      "                 120
Lipids                                                  2%                                        "                 750
(precursors of  nucleic acids)              0,4%                                     "                 300
Proteins                                                15%                                      "                40.000
Nucleic acids                                       7%                                        "                 106-109
The proteins (like enzymes) control the metabolic reactions, they are made up of 20 different amino acids and except for glycine they are chiral, that is they exist in two forms D (Dexter) and its mirror image L (Left).
The abiotic synthesis of the amino acids (in laboratory, in industry, in prebiotic era) gives origin to 50% of molecules D e 50% of molecules L.   
 In living organisms only the form L is present.

For nucleic acids we take into consideration the RNA.
The constituents of RNA are:
The phosphate group: (H2PO4)-.
A sugar, the Ribose
 The nucleobases

A phosphate  group , a ribose molecule and one of the four nucleobases give origin to four different aggregations which take the name of nucleotides. As an example we report the Adenosin-5-phosphat; the nucleotides are the constituent of RNA.
In these  geometric representations, if a vertex is empty, the presence of an atom of carbon is understood. In the cytosine, for example, the position 3 there is Nitrogen, but in position 4 there is nothing. In the position 4, by convention carbon atom is omitted.
The formula is hence in reality the following:
The nucleotide Adenosin-5-phosphat,

A) The substances fundamental for the origin of life had their origin on our planet, or did they come for space.

What really happened 13,6 milliards of years ago, we do not know, yet we know that it happened and we called it the Big Bang. We also know that, after 380 thousands of years from the Big Bang, when the temperature of the universe at that time decreased by some thousands of degrees, the electrons (-) and the protons (+) linked, giving origin to hydrogen (H), Helium (He) and small quantities of Lithium (Li). The gravitational attraction between the atoms of these elements gave origin to the first stars. It was inside the massive stars, at a temperature
 of hundreds of millions of degrees through the nuclear fusion that, starting from hydrogen, other chemical elements were formed and among these Carbon (C), Nitrogen (N), and Oxygen (O), that is those elements that form the 99% of our body. The gravitational collapse of these stars, at thousands of millions of grades, completed the picture giving origin  to all the other natural elements. And so, for what concerns the elements, there is no doubt: we are “sons” of stars.
But are we also sons of space? That is the substance fundamental for the origin of life, amino acids, sugars, and nucleobases come from space?
The idea, expressed at first by Juan Orò in 1961 and taken up again in the 70’s by F. Hoyle and C. Wickramasinghe, has a certain fascination in itself. As it has been evident in the “conditions within limits”, the primordial atmosphere, brushed away by the solar wind, was reintroduced on our planet, probably by volcanism, by the impacts of meteorites of various dimensions and by the impacts of comets. With time the impacts became less frequent but they never ceased happening. Some of these meteorites, precipitated in the last two centuries, had been gathered and studied. A total of more than a thousand meteorites are conserved but only some, called “carbon condrite”, are interesting for our ends. These meteorites dated 4,5 milliards of years ago, had their origin during the formation of the solar system.
These chemical analyses, done at the beginning of the 70’s, have shown the presence of amino acids constituents of proteins. These amino acids present themselves in the two forms D and L and hence surely are  extraterrestrial and not biological. Hydrocarbons (made up of H and C) have been found, with a molecular weight even high.
In 1929 Haldane made the hypothesis that the primitive atmosphere, in our planet, was constituted by NH3, CO2, H2 H2O and traces of CH4. From these substances, with the input of energy, the substances fundamental for the origin of life would have been formed.
In 1953 S. Miller demonstrated that in atmosphere composed of CH4, NH3, H2O, e H2 in presence of liquid water and simulating lighting through electric discharges, many organic substances can be produced and among these a large number of different amino acids, these also in the two forms D and L. It is interesting to observe that the abundance of amino acids, in relations with their molecular weight, in the meteorites and those obtained in Miller’s experiments, present a certain coincidence. Some of these amino acids are equal to those present in our proteins. Moreover also in Miller’s experiments hydrocarbons of a composition like that of the hydrocarbons contained in meteorites have been found. Successive studies both on meteorites and on experiments of Miller’s type have demonstrated the presence of traces of purines  and pyrimidine. These substances have a molecular structure fairly close to that of the nucleobases. In neither of the two cases mentioned above was however discovered the slightest trace of sugar and nucleobases, that is the constituents of the nucleic acid, whereas it is now certain that amino acids are present. Hence from the depth of space, arrives evidence of the chemical processes like those that happened on our planet according to the “conditions within limits” already exposed. But it still remains to be cleared if the organic molecules of the meteorites already made part of the gas cloud and dust which gave origin to the solar system or if these were formed during the condensation phase of the cloud itself through inputs of energy.
Miller’s experiment has inspired many other experiments, in which were used sources of energy and mixtures of gas of different types. These experiments have demonstrated that, in prebiotic conditions, one can obtain a large variety of organic substances with a biological interest and, among these, formaldehyde (HCHO) and hydrogen cyanide (HCN). One must not under valuate the importance of these little molecules because they could have been the precursors of sugar, and nucleobases and amino acids. In fact ribose, a fundamental molecule for RNA, is a pentamer of formaldehyde (HCHO) in the sense that five molecules of formaldehyde could give origin to a ribose molecule. And adenine, nucleobase fundamental for nucleic acid,   is a pentamer of hydrogen cyanide (HCN), that is five molecules of hydrogen cyanide could give origin to a adenine molecule. All this leads us to conclude that, on our planet, the natural processes can produce the fundamental substances necessary to the origin of life.
Around the end of the ‘70’s, the idea of an extraterrestrial origin of the substances fundamental for the origin of life, received a new impulse. In the interstellar clouds of gas (more or less ionized) and dust, radio astronomers
have discovered various simple organic substances and among these formic aldehyde (HCHO) and hydrogen cyanide (HCN). A complete list of all the organic substances (about 40) found in space until the beginnings of the 80’s is contained in an article by Leo Blitz: Complessi giganti di nubi molecolari nella galassia, Le Scienze 1982.
However no trace of molecules a bit more complex, important for the origin of life.
It was thought that one could find them in the near future, but it was not clear in what way these molecules could have arrived from space onto our planet. As single molecules they would have been destroyed by the solarultraviolet, mortal not only for living organisms but also for the molecules fundamental for the origin of life. If the molecules had been contained inside asteroids fallen on our planet, they would have been destroyed by the enormous heat given off by the impact, or remained prisoners inside the fragments.
However, around the middle of the 80’s, the majority of scientists implied in research on prebiotic chemistry, was of the opinion that the extraterrestrial origin of organic substances only demonstrated the ease with which these molecules can be synthesized in the presence of carbon, hydrogen, azote and oxygen.
Such an opinion was also well synthesized by Mario Ageno who concludes (Lezioni di biofisica 3, 1984): «Even if a minimum fraction of it succeeded in the end in surviving and in joining in some way the hydrosphere of the planet, it would be difficult for the chemical evolution to go further, up to the appearance of living organisms, without a continuous supply and that of really different magnitude, of substances of new synthesis and local origin».
The discovery of organic molecules in space, implied however a reflection. If the interstellar clouds contain prebiotic organic molecules, these must have been present also in the cloud which gave origin to the solar system. It was hence thought that, during the formation of the solar system, in the planets close to the sun, because of the enormous heat, these molecules were destroyed. They were however saved in the colder zones, that is at the limits of the solar system, where they were incorporated in the comets.
Hence was diffused, among scientists, the conviction that comets were a residue of the nebulous which gave origin to the solar system.
The hunt after the comets begins. 
Around the middle of the 90’s in the coma of the comets

 Hyakutake and Hale-Bopp, organic compounds are discovered and among these formic aldehyde (HCHO) and hydrogen cyanide (HCN).
However still no trace of molecules a bit more complex, important for the origin of life. Yet it has been observed that, when in the interstellar space water molecules, methanol, ammonia and hydrocarbons are deposed on silica dust, frozen grain are formed. According to some scientists, inside these grains, organic substances could have been accumulated, complex ones included, and the layer of ice would have protected them from the ultraviolet. Other researchers hold that, if the layer of ice had not been sufficiently thick, the organic substances would have been broken by the ultraviolet rays. The residues, as they could not disperse into space, could have reacted early or late giving origin to more complex molecules. When they enter into the atmosphere, the organic substances contained in the cavities of the granules, are protected from overheating. Frozen granules have been reproduced in laboratory, and named analogous to frozen granules. In these analogous granules have been individuated simple organic substances like ketones, ethers and alcohols.
However when we reread the article of M. Bernstein, A. Sandford and J. Allamandola: Dallo spazio le molecule della vita (Le Scienze, 1999), apart from the emphasis, understandable for one who works at the NASA, There is no trace of molecules important for the origin of life.
In 2002, twenty years after the publication of the article by Leo Blitz and hence after another twenty years of research in radio astronomy, is published by P. Ehrenfreund and others: “Astrophysical and astrochemical insight into the origin of life, (Rep. Prog. Phys. 65 (2002) 1427-1487, a list up to date (about 70) of organic substances found by radio astronomy in space. Among these substances Ehrenfreund includes Glycine, a very simple amino acid that makes part of our proteins, but he adds an interrogation.
Also in this new list there is not yet the trace of complex molecules important for the origin of life. In the same article Ehrenfreund publishes the list of the substances found in the comets Hyakutake and Hale-Bopp. All these  substances were already found in the interstellar space. Hence it does not seem that inside the comets particular reactions of synthesis take place. In the Ehrenfreund table, Glycine is still included but this time without any question mark. Reading through his comment on the table, he writes: «Glycine, the simplest amino acid, has not yet been detected, […]». In 2006 was brought to earth the dust of the comet Wild 2, taken by the  Stardust mission . Analysis has shown the presence of amine and molecules made of long chains of carbon atoms. In 2009, after three years of the publication of the first data, NASA announces with much emphasis, that in the dust of Wild 2, when the analysis had been redone, also Glycine was discovered.
What can one conclude? Finally they did it.
It is opportune, to conclude, to add some points, starting with some parameters.
The massive stars, that form inside the immense clouds of gas and dust, have an average life of about 3 million years and the greater part of their energy they give it out under form of ultraviolet rays. Also our sun gives out ultraviolet rays which would destroy amino acids and nucleobases up to the threshold of the solar system. When the sun had its origin, the intensity of radiation was a thousand  times superior to the actual situation. Space is hence permeated by rays lethal not only for the living organisms, but also for molecules important for the origin of life. In the interstellar clouds matter is extremely rarefied. At the temperature of -260°C the average density is 100 molecules of hydrogen per cm3 and small fractions of azote, oxygen, and carbon. These gas, atomized or ionized by the ultraviolet rays, through casual impacts have given origin to very simple substances like methane, ammonia, water and formaldehyde, hydrogen cyanide, ammines too. A part of these substances is destroyed by ultraviolet rays to reform later in other places. Now the molecules fundamental for the origin of life like amino acids, sugars, and nucleobases, even if they are molecules with a molecular weight relatively low, they are not so simple, but have their own complexity. What is the probability that molecules of this type could have formed in space, from atoms and ions extremely rarefied through casual impacts and at such a low temperature and what is the probability that they could have resisted against the ultraviolet rays? Descartes’ good sense suggests that such a probability is practically zero. And then, even if  some molecule useful to the origin of life should form, what could be its usefulness? The quantity of molecules necessary to the origin of life is of such an order of magnitude that space should be permeated, and instead space is permeated with rays which destroy these molecules. And so for what concerns substances fundamental for the origin of life, we are not “sons” of space.
Space with its ultraviolet rays is the dwelling of death.
The first living organisms on earth knew something about that, being obliged to live in mud or in water deeper than 10 meters. Only after two milliard years, when microorganisms had learned to produce oxygen, did they succeed in strapping from death some kilometer of space and they could occupy the surface and the atmosphere of our planet.
However, living organisms exist thanks to the fact that space is the kingdom of death. Life was possible on earth thanks to the fact that space was sterilized by the ultraviolet rays.
The organic substances known by us are now, on the earth, more than 1,5 million. Imagine if in space, through casual impacts and in the absence of ultraviolet rays, the substances fundamental for the origin of life had originated. But then by casual impact also all the sugar, that is tens of sugars, and all the nucleobases possible, perhaps hundreds, and then hundreds of thousands of  organic substances, Having arrived on earth, in an oceanic melting pot, from these substances only a miracle could have given origin to life. Life needs regulating principles. Without regulating principles there is no life. And these regulating principles do not exist in space but they can exist only on earth or on similar planets. And then, organic molecules which radio astronomy finds in space are nothing but ashes of an over lasting cremation thanks to which life has been able to originate on earth.
That which has been exposed above makes one conclude that space is not the seat of the origin of the substances fundamental for the origin of life.
The comet impacts on our planet have restored in the prebiotic epoch the primitive atmosphere; they have added water to our planet and probably also some simple organic substances like formic aldehyde, hydrocyanic acid, etc.
Probably during the phase of contraction of the primitive cloud, from compounds present in the cloud like methane, Ammonia, Hydrogen and water, through addition of energy, many organic substances were produced, and among these the amino acids. Some of these amino acids were imprisoned in the meteorites, other have been destroyed by the solar ultraviolet and the high temperature of contraction of the cloud.
Therefore, the conclusion to which scientists had come to in the 80’s is still valid: natural processes, in prebiotic conditions, on our planet, are able to produce the substances fundamental for the origin of life, and the contribution of simple organic substances, coming from space, if ever it existed, is to be considered without influence.
We are “sons” of the earth.

B) When can a chemical reaction, realized in laboratory, be defined prebiotic?
One of the scopes of prebiotic chemistry, is that of understanding through what processes were formed the constituents of the proteins and of the nucleic acids in the prebiotic era. A corollary of this scope is the question: how does one establish if a reaction, realized in a laboratory equipped for such a scope, can be considered plausible also in the prebiotic era? It results that without a minimum of rules, there is no answer to this question. To establish these rules, it is however necessary to start with concrete examples. These examples are the first publications which concern the origin of the constituents of the nucleic acids. Such examples, for the time being, are exposed in a synthetic way, but in the next article they will be analyzed in detail.
In 1965 is published by C. Ponnamperuma e R. Mack: Nucleotide synthesis under possible primitive earth conditions (Science (1965), 148,3674): «[…]An aqueous solution of nucleoside and phosphate was lyophilized in a Pyrex tube and the tube then sealed off and heated to 160° for 2 hrs.». The experiment consisted in causing a reaction between a nucleoside (that is a ribose linked to a nucleobase) with a phosphate to obtain the nucleotides. The experiment gave origin to nucleotides and created much interest, so much so that it was quoted by many researchers as a probable way of synthesis of nucleotides in the prebiotic era. From one quotation to another, it was at length inserted on a text book for students where, the way of synthesis, from probable became certain. As ten years and more passed, doubt was born on the prebiotic significant of the experiment. Tens of researchers fixed themselves in experiments to render it still valid; but finally in 2004 L. Orgel, (Critical Reviews in Biochemistry and Molecular Biology, 39:99–123, 2004), with extreme fair play, concluded that the synthesis of the nucleotides still proposed today are not convincing.
But why Ponnamperuma’s experiment is not convincing?
Because Ponnamperuma and all the other researchers who followed him had bought the nucleosides in specialized laboratories, but no one knows how the nucleosides
were formed in the prebiotic era. The different sorts of nucleosides are perhaps hundreds; if in the prebiotic era they had spontaneously originated, there would have been a mixture of hundreds of different nucleosides. But the experiments above mentioned did not use a probable mixture of this type, no, the researchers acquired the right ones.
In the prebiotic era, the indication of the existence of the nucleosides and the phosphate used in the experiment, are zero.
And so for forty years discussions were made about nothing.
But why, how was it possible? Because of the lack of the “rules of procedure”.
From this experience we can however extract the first procedural rule.

 1) A chemical reaction, realized in laboratory, can be defined prebiotic, if for the reacting substances, there exist strong indications of their presence in the prebiotic era.

All the reactions that do not adapt themselves to this first rule, can be interesting laboratory experiments, but they have no prebiotic value.
In 1961 Juan Orò, one of the most engaged in research on prebiotic chemistry, publishes: Synthesis of purines under possible primitive earth conditions. I. Adenine from hydrogen cyanide. The Adenine was obtained heating at 70°C a high concentration of cyanide acid (HCN) in the presence of ammonia (NH3). In this experiment were obtained many organic substances and, among these, Adenine with a concentration of the order of 0,5%. Also this experiment was the object of a great interest and was widely quoted. Now, in the prebiotic era, there are strong indications of the presence of NH3 and of HCN and hence the experiment follows the first rule of procedure. But HCN  is a gas, as also NH3. And then, how could one obtained such a high concentration of cyanide acid, in an ammonia ambience and at the temperature of 70°C, in prebiotic era? Orò does not explain it but, after the publication of his article he expresses the idea that the organic substances necessary for the origin of life would have come from space.
The suspicion comes that he himself was not filly convinced by his experiment.
Those who believed, on the other hand, were many other researchers who sought how it was possible to obtain high concentrations of HCN in the prebiotic era.
In laboratory experiments it was discovered that at the temperature of -23,4°C and at a high concentration, 74,5 mole in % of HCN (about 800g/L), after a certain time a polymer of HCN forms. This compound, extracted and treated with water, gives origin  to small percentages of adenine. One must be careful though: if the temperature is some degree higher the polymer does not form end goodbye to adenine. Moreover, a solution of this concentration takes the name of an “eutectic solution”. We must be careful also here, because this means that if the temperature lowers another degree, the solution freezes entirely, immediately, the polymer can no longer form and goodbye adenine.
How it would have been possible for such restrictive conditions to be realized on our planet in the prebiotic era, the researchers do not say.
And all of us imagine a planet for long periods, perhaps for tens of thousands of years, at the temperature of -23°C, not a degree more not a degree less.
Or else to imagine a primitive Artic where under thick layer of ice the useful concentration and the temperature of -23°C were attained, neither a grade higher nor a grade lower.
But in this case, what should have been the temperature of the atmosphere, -150°C or -200°C .
Also in the precedent example, Ponnamperuma should have made clear what, in prebiotic era, could have to lyophilize the solution, by what process one could substitute the Pyrex tube hermetically closed and how would take place the heating at 160°C.
But Ponnamperuma does not say it.
And at the end we conclude that this is not a serious way of proceeding.
From this experience we can however extract the second rule of procedure.
2) A chemical reaction, realized in laboratory, can be defined prebiotic if it takes place inside the ambient conditions of the primitive earth. If the laboratory experiment takes place in ambient conditions and concentration of the reagents improbable in the primitive earth, then the author must explain, in a convincing way, how his experiment in the laboratory can be translated in the prebiotic era.
All the reactions which do not adapt also to this second rule can be interesting laboratory experiments but they are not of prebiotic value.
These minimum rules to define prebiotic a reaction carried out in a laboratory, can certainly be criticized and improved or changed but the rules are certainly necessary.
Without procedural rules, one risks to reason in emptiness for another 50 years. And above all, as shown by the above examples, young researchers risk working for years on research whose result is already known: zero
C) In a chemical reaction, realized in a laboratory, what must be the concentration of the reacting substances, and what must be its rendering so that it be defined prebiotic
As we have already observed at the precedent point, prebiotic chemistry has the scope of understanding through what processes were formed the constituents of the proteins and the constituents of the nucleic acids in the prebiotic era. The problem is that in all the laboratory experiments which simulate these processes, likely or  unlikely, the quantity of the products obtained is very small. As we now know, Miller has demonstrated that the amino acids form easily through an abiotic way. Now, let us imagine that these amino acids were formed in the primitive atmosphere. These, transported in a primordial ocean, would have given origin to a “prebiotic mixture” very diluted. In such conditions, how would the amino acid molecules meet to react and give origin to the first peptides? Christian De Duve has exposed clearly the problem in “Polvere vitale” 1998: «[…] Let us imagine a brief sequence of 3 steps from A to B, from B to C, and from C to D each with a rendering (high for a prebiotic reaction) of 1%. In terms of A the rendering of B will be 0,01%, that of C 0,0001% and that of D 0,000001%, that is of one for a million. Even in the best conditions, chemists must fight against this sort of tendency to evanescence». The fact is that we need at least thirty steps for the proteins, and for the nucleic acids some hundred, evanescence would already be a good result. And so, how does one resolve the problem and what do scientists think of it?
On this question we can divide scientists in three groups.
For the first group the problem does not exist.
To this group belong all those who follow the line of thought of J. B. Haldane and Jacques Monod. According to these scientists, life would have had its origin by chance. Some insist by chance of a probabilistic sort, others think of a chance as almost miraculous. And if life had its origin by chance, why should we preoccupy ourselves with the concentration of its constituents?  
The second group feigns that the problem does not exist.
To this group belong the evolutionists fascinated by the “self-reproducing” of Dawkins and some of the researchers who believe in the “RNAWorld”. For these researchers the primordial mixture is a fortress to be defended. Where can exist populations of self-reproducing molecules of RNA in competition for the availability of food, if not in the primordial mixture? Without the primordial mixture the theory collapses. The problem hence is avoided. Manfred Eigen in fact speaks of hydric basins, that is of a variety of ambiences and where at least one of these would have to be adapted for its concentration and temperature to the origin of life.
For the third group, the problem, on the other hand, the problem exists.
But then, if the problem exists, how shall we solve it?
To suggest a solution, J. D. Bernal was the first, in 1951. As it is known argil are formed of various crystalline strata superposed. Every stratus is made up of two under strata, one of  silica tetrahedron (Si2O52-)n and the other of hydrated alumina octahedron [Al2(OH)42+]n. Without going in too much detail, for the presence of electric charges, the various strata or under strata neutralize at a distance. Between  one under strata and another their remain hence empty spaces where water molecules or molecules that have electric dipoles can lodge themselves. 

It has been calculated that in 1 cm3 of argil, the surface of these empty spaces is almost equal to the surface of a football field. Because most molecules fundamental for the origin of life have electric dipoles, Bernal proposed that these molecules could have accumulated in the argil.
Other researchers took into consideration pools of water at margins of continents and subject to continual evaporation which could have concentrated the fundamental molecules (Chemical evolution and the origin of life, R. E. Dickerson, Le Scienze, 1978). Miller and Orgel proposed the cryogen methods, that is eliminating water under the form of ice crystals. In such a way that the solution became always more concentrated (article already quoted). However, those who affirm that the problem exists, are convinced that some process of accumulation of such substances must have happened. Attention though, because some of the propositions contain a trap.
Let us take the example of the pools of water at the margins of continents. And if the evaporation does not reach the right point? What does one do? One waits for another round? We shall say! In the arc of thousands or millions of years, everything is possible. All right, but there always remains local and isolated processes which can take place or not take place, or happen without attaining the right point. Moreover the pools surely contained Miller’s amino acids, that is D and L. In what way would they have been separated? The researchers do not say. And if the pools of water contained the amino acids of Miller, these were about 60.The amino acids which constituted the first proteins were perhaps no more than 15. In what way proteins could have been formed excluding all the others? The researchers don’t say.
And hence, here the trap hides itself. Behind the appearance
of a natural process, evaporation or crystallization, in reality resurges like the Phoenix, another time by chance. Hence, if we do not want to fall once more in the trap of chance, the process of accumulation could not have been sporadic and local. It must have been a process
which had to realize itself at any moment, in the sense that one could not wait for heat or cold, for the sun or the stars. This because during the waiting the important substances could surely have participated in secondary reactions or could have been destroyed. Moreover it must have been a fairly diffused process on the whole surface of the planet. This because a local event at that epoch was exposed with so many vicissitudes to render it casual. A local and sporadic process, in fact casual, would reduce also the possibility of life in other planets.    
Now, if a process of accumulation is all the same necessary, then what the sense of speaking of the rendering of a reaction? A constituent can be obtained with a low rendering (or to use a chemical term a trace), through some process it is accumulated and can reach also elevated concentration.
On the question of concentration of the substances fundamental for the origin of life, it is impossible not to mention that which in biophysics is called: the problem of phosphorous.
This element is found in nature under the form of minerals of volcanic origin, the apatite (calcium phosphate Ca5(PO4)3[F, OH, Cl]) which, under the form of very small crystals are disseminated on all the surface of the planet. We have no element which can indicate that the situation in prebiotic era was different from now. The apatite however are not very soluble and their concentration in the water is 30 µg/L (µg/L millionth  of grams for litre). To give origin to chemical reactions a concentration of this type is for chemists really insignificant. And yet phosphorous, under the form of phosphate, enters to make part of the structure of the nucleic acids, it is fundamental in the transfer of energy participating in many metabolic reactions. Some researchers have shown how the characteristics of phosphorous are unique and it does not seem that some other element can substitute it in living organisms. One hence concludes that it is a substance indispensable to life, even if at low concentration but diffused on all the surface of the planet, it becomes all the same co-opted. It hence seems that for life and for its origin the extreme diffusion of a substance is irrelevant. To be clear, here one does not mean that phosphorous was accumulated, but simply, as we shall see in the next article, that perhaps a problem of phosphorous does not exist.
Hence a fundamental substance for the origin of life can also be at a concentration very low and undergo accumulation processes, or also as in the case of phosphorous, as it is indispensable to life, it is anyway co-opted.
From what is written above we can conclude with the third and last rule of procedure.
3) A chemical reaction, realized in laboratory, is defined prebiotic even if the concentration of the reagents and the rendering of the products are very low. That because in any way a process of accumulation is necessary. Reaction and process of accumulation however must be realized, in prebiotic era, at any moment and in a diffuse way on the surface of the planet.
Once the primitive ambiance has been identified, we can recapitulate
 The substances fundamental for the origin of life had their origin on Earth.
1st  procedural rule
A chemical reaction, realized in laboratory, can be defined prebiotic, if for the reacting substances, there exist strong indications of their presence in the prebiotic era.
2nd  procedural rule
A chemical reaction, realized in laboratory, can be defined prebiotic if it takes place inside the ambient conditions of the primitive earth. If the laboratory experiment takes place in ambient conditions and concentration of the reagents improbable in the primitive earth, then the author must explain, in a convincing way, how his experiment in the laboratory can be translated in the prebiotic era.
3rd  procedural rule
A chemical reaction, realized in laboratory, is defined prebiotic even if the concentration of the reagents and the rendering of the products are very low. That because in any way a process of accumulation is necessary. Reaction and process of accumulation however must be realized, in prebiotic era, at any moment and in a diffuse way on the surface of the planet.
                                                                                      Giovanni Occhipinti
  Translated by Silvia Occhipinti (30.09.2013)