TOF time of flight

List of abbreviations and symbols

°C

in some warm little pond with all sorts of ammonia and phosphoric salts, light, heat, electricity etc. present, that a protein compound was chemically formed, ready to undergo still more complex changes". A letter from Charles Darwin (1809-1882) to his friend Joseph D. Hooker in 1871. 1 Since the early days of Darwin's vision of his famous "warm little pond", scientists (alongside with the public) have been inspired by this fascinating idea. Charles Darwin was an exceptional scientist who was experienced in biology and geology. However, experimental evidences of the possibility of emergence of life from such a "warm little pond" have become mainly the duty of chemists. Since some decades, many researchers have taken the responsibility to proof that it would be possible to form the components of life starting from simple molecules which might have been present on the primitive Earth (Figure 1). [START_REF] Bada | Prebiotic Soup-Revisting the Miller Experiment[END_REF] In order to achieve this goal, researchers exploring the origin of life problem combine geological evidences, theoretical models, and laboratory experimentations that must be carried out under plausible prebiotic conditions.

Figure 1: Some main achievements and theories related to the study of origin of life

Pre-Darwin evolutionary ideas

Humans have been always wondering about their surrounding environments and have been trying to answer questions related to phenomena they see. Little by little, they were able to find out many reasonable answers to their inquiries. However, two concerns remained mysterious: life and death. Religion, philosophy and science tried to give answers to these two issues throughout history. Creationists believe that humans were created separately, in other words, they did not undergo evolution. In view of this, it is assumed in the modern scientific communities that evolutionary theories did not exist before Darwin. This belief might arise from the contradiction between the idea of evolution and religious thoughts that dominated the old eras. However, several scholars between the eighth and fourteenth century

proposed their ideas about evolution that have some similarities to Darwin's theory.

Al-Jahiz (776 -868) was an Iraqi zoologist who might be the first to propose an evolutionary biological theory. He observed the behaviour of animals and classified them according to their characteristics and similarities. He explained that adaptation of organisms is directed by the environmental conditions. In spite of similarities between Al-Jahiz's idea and Darwin's theory, the former believed that God guides the evolution of species and that organisms are developing for survival as a part of God's plans to preserve nature in order. [START_REF] Malik | An Untold Story in Biology: The Historical Continuity of Evolutionary Ideas of Muslim Scholars from the 8th Century to Darwin's Time[END_REF] Another example is the Persian architect, philosopher, physician and scientist, Nasir al-Din

Tusi (1201 -1274). Tusi proposed his elementary theory of evolution about 600 years before Darwin. He believed that elements have evolved into plants, then animals. He also supposed that humans have evolved from advanced animals. He believed in the ability of organisms to change and vary according to environmental conditions, which agrees with the Darwin's concept of natural selection. [START_REF] Alakbarli | A 13th-Century Darwin? Tusi's Views on Evolution[END_REF] An alternative example is Ibn Khaldun (1332 -1406), the writer of 'Al-Muqaddimah' (Prolegomena) where he presented similar ideas to modern theory of evolution and highlighted human evolution. Ibn Khaldun thought that humans evolved from an ape/monkey ancestor. He rejected the religious belief that dark skin is a sign of God's curse upon sinful human beings. Similarly to Al-Jahiz, he attributed the dark pigmentation of southern people to the hot southern climates. [START_REF] Malik | An Untold Story in Biology: The Historical Continuity of Evolutionary Ideas of Muslim Scholars from the 8th Century to Darwin's Time[END_REF] For centuries, it was believed that organic matter could only be derived from biological sources or living organisms. The first synthesis of an organic compound (Urea) from inorganic starting materials was accomplished by F. Wohler in 1828. Along the same line, many efforts have been carried out with the purpose of obtaining organic compounds.

Notably, A. Strecker found a way to synthesize amino acids starting from aldehydes, ammonia and cyanide. [START_REF] Strecker | Ueber Die Künstliche Bildung Der Milchsäure Und Einen Neuen, Dem Glycocoll Homologen Körper[END_REF] Oparin's idea went along with previous scenarios. The Russian biochemist first presented his ideas in 1924 in the first version of his booklet entitled "The Origin of Life" where he proposed that life must have arisen from a pool of organic compounds. Oparin claimed that life was the result of a smooth, gradual evolutionary developmental process. That is to say, it had come through chemical evolution. 6

Chemical evolution

The term "chemical evolution" stands for all chemical processes that took place in the primitive Earth and lead to the emergence of life. Thus, chemical evolution must have preceded the Darwinian biological evolution through which modifications of living things took place over time. Even though the mysterious line between chemistry and life is still not well understood, it is widely believed that the first living cell came into existence through long and accumulated processes of chemical reactions that occurred about 4.5 -3.5 billion years ago where complex molecules of life had been formed starting from simpler organic and inorganic molecules in the primitive oceans. 6 No one knows if such process occurred also elsewhere in the universe.

Life definition

Generally speaking, the origin of life could be considered as either the very beginning of biology, or the very last chemical steps which had led to the first living system. In fact, the definition of life is still disputable even with the huge knowledge in biology. One famous definition considers a living system as a combination of cells that can undergo metabolism, grow, adapt to their environment, respond to external effects, and reproduce. [START_REF] Benner | Defining Life[END_REF] This attempt to define life is indeed nothing but a way to show the characteristics and the observed properties of life. [START_REF] Lazcano | What Is Life?[END_REF] The absence of an exact definition of life indicates a difficulty to characterize it according to precise factors. Furthermore, some features of life can be observed in non-living systems, such as polymerization, self-assemblage, catalysis and exchange of matter and energy with surroundings. Thus, life cannot be identified by any individual characteristic.

However, as explained by Oparin 9 , life is a specific and extremely complicated combination of a large number of properties that can be found individually in non-living materials.

Considering the unidentified line between chemistry and life, nowadays definitions of life may be updated or reconsidered.

A common ancestor?

The story of life goes back to about 3.8 billion years ago, maybe more. [START_REF] Marchi | Widespread Mixing and Burial of Earth's Hadean Crust by Asteroid Impacts[END_REF] Despite that the real scenarios for the creation of the first living thing(s) is still indefinite, it is wildly accepted that it had been formed via series of chemical reactions that took place in parallel. These reactions might have existed in one place or might have started in different areas before they meet later to form the first living cell. [START_REF] Patel | Common Origins of RNA, Protein and Lipid Precursors in a Cyanosulfidic Protometabolism[END_REF] All past and present living things derive from one Last Universal Common Ancestor (LUCA). Being the first living thing in the tree of life does not mean that LUCA was a simple organism with narrow capacities; however, it may have been able to show an effective energetic metabolism and may have been also ready to face a wide range of environmental conditions. [START_REF] Castresana | Respiratory Chains in the Last Common Ancestor of Living Organisms[END_REF] It is probable that some kind of "chemical and biological" evolution had taken place before the appearance of LUCA. Being the last common ancestor of all forms of life that we know nowadays, LUCA had to contain membranes, proteins, RNA and DNA. It was able to make replication, transcription, translation and already possessed a complex metabolism. These kinds of perfect and complicated combinations might have been the result of a huge amount of evolutionary processes that might have taken place between the "true" (but lost) origin of life and the appearance of LUCA. [START_REF] Sutherland | Opinion: Studies on the Origin of Lifethe End of the Beginning[END_REF] It is possible that many attempts were made before the final form of life, as we know it, appeared.

Biological (Darwinian) evolution

In his book, on the origin of species, Darwin has presented his theory of Evolution. He supposed that all forms of life are derived from one common ancestor and proposed that evolution occurs by the means of natural selection, which he described as "preservation of favoured races in the struggle for life". [START_REF] Darwin | On the Origin of Species by Means of Natural Selection, or the Preservation of Favoured Races in the Struggle for Life[END_REF] Biological evolution stands for the "genetic" changes over time. These changes can be passed to next generations and this process is necessary for the appearance of new species on the long run.

The tree of life

As aforementioned, it is thought that all living organisms were derived from one unique common ancestor. Every living thing is then connected to every living thing on Earth. This connection is expressed by the tree of life. The tree of life shows the evolutionary relationships between all known types of life. It was originally designed to classify living organisms according to their physical and metabolic properties. Since Darwin, relevant drawings of the tree of life have been updated and modified based on the new discoveries.

Nowadays, the tree of life can be drawn in several ways that shows developing of each individual species as an independent branch. Nevertheless, a recent view of the tree of life has been presented. It shows total diversity, includes biological interactions between species and takes into account the linkage between various forms of life, such as the evolution of bacteria in relation to animals (Figure 2). 15

B. Conditions on Earth 4 billion years ago

Sir Arthur Conan Doyle once wrote: "When you have eliminated the impossible, whatever remains, however improbable, must be the truth". Sherlock Holmes in "The Sign of the Four", 1890

If the emergence of life on Earth was the case to be studied by Holmes, he would realize that the truth cannot be simply found after the elimination of the impossibilities. Nonetheless, several impossibilities can still be eliminated in this special case. For example, unreasonable starting materials or reactions that need conditions that would not be possible on Earth, must be ignored. Considering liquid water as the solvent of life, any organic solvent must be excluded. As a consequence, proposed reactions must be performed within a limited range of temperatures, from around zero (or a little less considering the presence of salts in the ocean)

and not more than 100 °C.

Answers to the questions "when, where and how" did life emerge should also be answered.

The debate weather life exists elsewhere in the universe or if "seeds" of life were brought to our Earth from space will not be discussed in this thesis. However, it is our belief that chemical evolution started on Earth.

It was assumed that microbial life might have appeared in the Hadean, the geological era starting from the formation of Earth and ending, more or less, at the end of the heavy meteoritic bombardment. However, it is widely accepted that the story of life started about 3.8 billion years ago, just after the late heavy bombardment [START_REF] Marchi | Widespread Mixing and Burial of Earth's Hadean Crust by Asteroid Impacts[END_REF] that lasted for few hundred million years during which huge number of meteorites struck the Earth. A new era, the Archean that witnessed the arising of life on Earth began near the end of this bombardment.

In order to achieve a reasonable answer to how life started on the early Earth, we must first have a better understanding of the early Earth conditions.

Unfortunately, most of the geological record during the Hadean eon had been erased due to the heavy tectonic activity of the young Earth. [START_REF] Pinti | The Origin and Evolution of the Oceans[END_REF] However, some information about that period has been captured thanks to zircons grains (ZrSiO4) found in Western Australia. [START_REF] Wilde | Evidence from Detrital Zircons for the Existence of Continental Crust and Oceans on the Earth 4.4 Gyr Ago[END_REF] These geological evidences demonstrate that the surface of the young primitive Earth was largely covered by liquid water that must have played a vital role in the synthesis of the first organic molecules and the appearance of life in the early Archean. As the moon was much closer to Earth than nowadays [START_REF] Walker | Lunar Nodal Tide and Distance to the Moon during the Precambrian[END_REF] , oceans were under vigorous motions and powerful tides that helped the surface of Earth to be mostly covered by water, or at least wet.

Despite the fact that sun was younger and weaker, Earth's temperature might be higher than nowadays due to the high concentrations of carbon dioxide that enhanced the greenhouse effect. [START_REF] Shaw | Earth's Atmosphere -Hadean to Early Proterozoic[END_REF] However, the temperature of Earth started to cool down before the birth of life, that is before 4 billion years ago, to be similar or just a little warmer than the present time. This could be explained by the declination of carbon dioxide in the atmosphere which helped in reducing the greenhouse effect. [START_REF] Pinti | The Origin and Evolution of the Oceans[END_REF] Today's ocean is slightly basic (pH 8.2). However, primitive ocean might have been slightly acidic due to the equilibrium with the large amount of carbon dioxide in the atmosphere. [START_REF] Pinti | The Origin and Evolution of the Oceans[END_REF] The composition of early Earth atmosphere has a degree of uncertainty; however, it can be estimated using thermodynamic modeling. [START_REF] Martin | Volcanic Emissions and the Early Earth Atmosphere[END_REF] Active volcanoes, that were major participants in the chemistry of the primitive atmosphere, liberated large amounts of gases, including hydrogen sulfide, H2S. [START_REF] Martin | Volcanic Emissions and the Early Earth Atmosphere[END_REF] Beside H2S and CO2, early Earth atmosphere contained CH4, H2, H2O, N2 and NH3. [START_REF] Zahnle | Earth's Earliest Atmospheres[END_REF] Each of these primeval atmospheric components was essential for the scenario of the synthesis of organic compounds in situ on Earth. Quick photochemical destruction of ammonia and methane in the atmosphere might appear as a serious problem against this scenario. However, this problem can be handled by presenting two issues. First, various works have shown that ammonia can be produced when introducing a spark into a mixture of CO2, N2 and water. Another argument is that the high altitude organic fog may have led to a shielding effect that protected both methane and ammonia from UV rays. [START_REF] Zahnle | Earth's Earliest Atmospheres[END_REF] Furthermore, significant amounts of ammonia could have dissolved in water as the pH of the early oceans was lower than the pKa of NH3 (about 9.2 at 25 °C). [START_REF] Cleaves | Prebiotic Chemistry: What We Know, What We Don[END_REF] It is wildly accepted that oxygen did not exist in diatomic form, O2. Conditions were globally reductive, thus iron was in its ferrous state, Fe +2 and was soluble. Free oxygen started to appear only in the mid of Archean, probably 2.7 billion years ago, and became significant in the Proterozoic era, which followed the Archean. [START_REF] Shaw | Earth's Atmosphere -Hadean to Early Proterozoic[END_REF] The arise of O2 during the Archean, and the following eras, was due to photosynthesis. That is to say, the appearance of oxygen was a consequence of life. A relative timeline of different eras that accompanied the appearance of life on Earth is presented in figure 3. Beside iron, elements that were present in the primordial continents must have been present also in the primitive ocean including sodium, calcium, magnesium, manganese, phosphorous (as phosphates), potassium and chlorine. [START_REF] Santosh | Hadean Earth and Primordial Continents: The Cradle of Prebiotic Life[END_REF] C. What molecules could come from space?

One major plausible source of organic molecules on the primitive Earth is their delivery by extraterrestrial objects. [START_REF] Anders | Abundances of the Elements: Meteoritic and Solar[END_REF] The organic matter in the present biosphere is estimated to be about 6 × 10 14 kg, while the delivery of organic carbon from interplanetary dust particles 4 billion years ago was estimated to be 10 8 kg per year. Such a rate indicates that the role of extraterrestrial organic matter could be important. [START_REF] Kitadai | Origins of Building Blocks of Life: A Review[END_REF] Extraterrestrial objects might have brought inorganic nutrients such as ammonia and phosphates, beside a wide range of organic molecules, including nitriles, aldehydes, may be amino acids, nucleobases, sugar-like compounds and sulfur-containing molecules. [START_REF] Kitadai | Origins of Building Blocks of Life: A Review[END_REF] On one hand, the extraterrestrial compounds can be identified by looking at nowadays meteorites' components. These meteorites are the remains of asteroids that have fallen to

Earth. Agencies such as the National Aeronautics and Space Administration (NASA) send expeditions to recover meteorites in Antarctica. These meteorites are less polluted than meteorites found in other environments. [START_REF] Crampton | Some Reactions of Ammonia and Primary Amines with Propanal, 2-Chloroethanal, 2, 2-Dichloroethanal and 2, 2, 2-Trichloroethanal in Acetonitrile[END_REF] They contain no terrestrial contaminants, and their composition can be precisely studied. [START_REF] Burton | Understanding Prebiotic Chemistry through the Analysis of Extraterrestrial Amino Acids and Nucleobases in Meteorites[END_REF] On the other hand, extraterrestrial molecules can be identified directly by observing space.

The detection of many organic molecules in solar nebula analogues indicates that solar nebula itself (during the formation of Earth) was rich of these compounds. It is broadly believed that these organics were brought to the surface of our young Earth from space by comets and asteroids bombardment. [START_REF] Öberg | The Comet-like Composition of a Protoplanetary Disk as Revealed by Complex Cyanides[END_REF] Furthermore, it has been suggested that Earth's water might have been captured from asteroids and comets that collided with the planet. In fact, water was one of the first molecules to be observed in the interstellar medium. Microwave radiation of water was observed from the direction of the Orion Nebula, Sagittarius B2 (Sgr B2), and W49. [START_REF] Cheung | Detection of Water in Interstellar Regions by Its Microwave Radiation[END_REF] In addition to H2O, singly deuterated water (HDO) was also reported toward the Kleinmann-Low nebula (KL) in Orion A. [START_REF] Turner | Microwave Detection of Interstellar HDO[END_REF] Moreover, emissions of waters containing heavier oxygen isotopes (H2 [START_REF] Wilde | Evidence from Detrital Zircons for the Existence of Continental Crust and Oceans on the Earth 4.4 Gyr Ago[END_REF] O and H2 18 O) were detected as well. [START_REF] Jacq | H2(O-18) in Hot Dense Molecular Cloud Cores[END_REF][START_REF] Hjalmarson | Recent Astronomy Highlights from the Odin Satellite[END_REF] Hydrogen sulfide is a common molecule in space; for example, it was detected in seven starforming regions. [START_REF] Thaddeus | Interstellar Hydrogen Sulfide[END_REF] The corresponding isotopes HDS and D2S were also detected. [START_REF] Vastel | First Detection of Doubly Deuterated Hydrogen Sulfide[END_REF] Microwave emission of ammonia was first observed in the interstellar medium toward Sgr B2 in 1968. [START_REF] Cheung | Detection of NH3 Molecules in the Interstellar Medium by Their Microwave Emission[END_REF] This discovery was followed by observations of other ammonia isotopes.

Microwaves of interstellar singly deuterated ammonia (NH2D) were detected in 1978. [START_REF] Turner | Microwave Detection of Interstellar Deuterated Ammonia[END_REF] Later on, doubly deuterated ammonia (NHD2) was detected in 2000. [START_REF] Roueff | Detection of Doubly Deuterated Ammonia in L134[END_REF] Two year later, the triply deuterated ammonia ND3 was also reported. [START_REF] Van Der Tak | Triply Deuterated Ammonia in NGC 1333[END_REF] Moreover, [START_REF] Hug | A New View of the Tree of Life[END_REF] NH3 was observed toward the Orion Molecular Cloud. [START_REF] Wilson | The Detection of Interstellar /N-15/H3[END_REF] Hydrogen cyanide is a simple volatile molecule that was observed in 1P/Halley comet in 1986. [START_REF] Despois | Observations of Hydrogen Cyanide in Comet Halley[END_REF] Before this observation, radio emission spectra of the two molecular isotopes of interstellar hydrogen cyanide (H [START_REF] Castresana | Respiratory Chains in the Last Common Ancestor of Living Organisms[END_REF] CN and H 13 CN) were detected. [START_REF] Snyder | Observations of Radio Emission from Interstellar Hydrogen Cyanide[END_REF] It has been found (using quantum chemical and kinetic techniques) that HCN molecules cannot dimerize under interstellar conditions, and thus it is impossible to form more complex molecules, for instance adenine, via successive neutral-neutral reactions between HCN units. [START_REF] Smith | The Production of HCN Dimer and More Complex Oligomers in Dense Interstellar Clouds[END_REF] Despite the stability of HCN toward the formation of complex organic molecules, HCN assemblies "reported as polymers" were detected in many bodies of our solar system including asteroids, moons, planets and comets. [START_REF] Matthews | Hydrogen Cyanide Polymers, Comets and the Origin of Life[END_REF] Besides HCN, more "complex" cyanides were detected as well. Spectral emission lines associated with the rotational states of acetonitrile (CH3CN) and cyanoacetylene (HC2CN)

were observed in the protoplanetary disk surrounding the young star MWC 480 (which has roughly twice the mass of our sun and is brighter). [START_REF] Öberg | The Comet-like Composition of a Protoplanetary Disk as Revealed by Complex Cyanides[END_REF] Longer HCnCN (n being an even number up to 10) have also been identified. [START_REF] Hudson | Reactions of Nitriles in Ices Relevant to Titan, Comets, and the Interstellar Medium: Formation of Cyanate Ion, Ketenimines, and Isonitriles[END_REF] In 2008, aminoacetonitrile (H2NCH2CN) was detected in the famous massive star-forming region Sagittarius B2(N). [START_REF] Belloche | Detection of Amino Acetonitrile in Sgr B2(N)[END_REF] Aminoacetonitrile is a possible precursor of the simplest amino acid, glycine. For this reason, its detection in the Interstellar Medium is of great interest. This interest is even greater for our team as aminonitriles are engaged directly in our research.

Another nitrile-containing molecule that has been recently detected with radio telescopes in the interstellar medium (in a cold molecular cloud of the Taurus region) is benzonitrile (C6H5CN). It may not have a big interest in the study of prebiotic chemistry; however, this nitrile is thought to be a precursor to more complex polycyclic aromatic hydrocarbons and polycyclic aromatic nitrogen heterocycles that are believed to have an extensive existence throughout the universe. [START_REF] Mcguire | Detection of the Aromatic Molecule Benzonitrile ( c -C 6 H 5 CN) in the Interstellar Medium[END_REF] Many aldehydes were also observed in the interstellar medium. Formaldehyde (H2CO) was first detected in 1969. [START_REF] Snyder | Microwave Detection of Interstellar Formaldehyde[END_REF] One of the first aldehydes to be detected in space was acetaldehyde (CH3CHO) in 1973. [START_REF] Fourikis | Microwave Emission of the 2-11/2-12 Rotational Transition in Interstellar Acetaldehyde[END_REF] Since 1980's, more aldehydes have been reported in the literature:

Propynal (HCCCHO) [START_REF] Irvine | A New Interstellar Polyatomic Molecule -Detection of Propynal in the Cold Cloud TMC-1[END_REF] , propenal (acrolein, H2CCHCHO), propanal (CH3CH2CHO) [START_REF] Hollis | Green Bank Telescope Detection of New Interstellar Aldehydes: Propenal and Propanal[END_REF] and glycolaldehyde (HOCH2CHO). [START_REF] Hollis | Interstellar Glycolaldehyde: The First Sugar[END_REF] Indeed, the detection of glycolaldehyde was of a big interest since it is structurally the simplest member of the monosaccharide sugars (monosaccharide sugars are carbohydrates with the formula CnH2nOn). [START_REF] Hollis | Interstellar Glycolaldehyde: The First Sugar[END_REF] Amino acids, which are the monomers of proteins, could have been also delivered to the primitive Earth through comets and meteorites. Several amino acids were first detected in the Murchison meteorite, which fell to Earth near Murchison village in southeastern Australia in September 1969. The Murchison meteorite contained more than 100 different amino acids (ranging from two to nine carbon atoms). Among these amino acids, 80 have been clearly identified. [START_REF] Pizzarello | The Organic Composition of Carbonaceous Meteorites: The Evolutionary Story Ahead of Biochemistry[END_REF] Not surprinsingly, the most abundant amino acid was glycine. Alanine, glutamic acid, valine and proline are other examples of proteinogenic amino acids that were detected.

However, this meteorite also contained a number of non-proteinogenic amino acids, for instance α-aminoisobutyric acid. [START_REF] Kvenvolden | Evidence for Extraterrestrial Amino-Acids and Hydrocarbons in the Murchison Meteorite[END_REF] Many amino acids (despite that most of them are not engaged in today's proteins formation)

were also observed in other meteorites. These observations are a solid argument for the extraterrestrial origin of at least a part of amino acids. What is more interesting about few of meteoritic amino acids is the slight enantiomeric excess of the L-over the R-amino acids. [START_REF] Burton | Understanding Prebiotic Chemistry through the Analysis of Extraterrestrial Amino Acids and Nucleobases in Meteorites[END_REF] These meteoritic amino acids might have contributed to the origin of homochirality in life on Earth. [START_REF] Pizzarello | Prebiotic Amino Acids as Asymmetric Catalysts[END_REF] In carbonaceous chondrites (a rare class of meteorites) more than 80 amino acids were detected, 12 of them were protein-amino acids: alanine, aspartic acid, glutamic acid, glycine, isoleucine, leucine, phenylalanine, proline, serine, threonine, tyrosine, and valine. [START_REF] Kitadai | Origins of Building Blocks of Life: A Review[END_REF] Up to now, cysteine has not yet been observed in space, however, it has been proposed that cysteine precursor CysCN (HSCH2CH(NH2)CN) may be synthesized in interstellar dust clouds by radical combination reactions. [START_REF] Knowles | Radical Formation of Amino Acid Precursors in Interstellar Regions? Ser, Cys and Asp[END_REF] All organisms depend on nucleic acids to encode genetic information. Nucleobases are substituted one-or two-ring nitrogen heterocyclic compounds that are essential for this process. For this reason, it is strongly believed that nucleobases might have played an essential role in the early evolution of life. In fact, researches concerning the detection of nucleobases in meteorites are much less developed in comparison to those that were carried out for amino acids. However, the detection of some nucleobases in meteorites were reported in the literature. [START_REF] Callahan | Carbonaceous Meteorites Contain a Wide Range of Extraterrestrial Nucleobases[END_REF] Purines (Adenine, guanine, hypoxanthine, and xanthine) and one pyrimidine (uracil) have been identified in the Murchison, Murray and/or Orgueil meteorites. [START_REF] Stoks | Uracil in Carbonaceous Meteorites[END_REF][START_REF] Martins | Extraterrestrial Nucleobases in the Murchison Meteorite[END_REF] Other unusual nucleobase analogues that are not found in Earth's biology (purine, 2,6-diaminopurine, and 6,8-diaminopurine) were also identified in these meteorites. [START_REF] Callahan | Carbonaceous Meteorites Contain a Wide Range of Extraterrestrial Nucleobases[END_REF] The detection of various nucleobases in meteorites, support their extraterrestrial origin (Figure 4). Miller ran his experiment in simulated primitive conditions by applying an electric discharge to a mixture of gases in a reducing atmosphere (CH4, H2, H2O and NH3). [START_REF] Miller | A Production of Amino Acids Under Possible Primitive Earth Conditions[END_REF] Miller's work has been continued by his student, J. Bada. Bada analysed/re-analysed archived samples of Miller's original experiments found in his laboratory using more sensitive techniques. While a relatively small number of amino acids had been detected by Miller, Bada found that more than 40 amino acids and amines were synthesized. [START_REF] Bada | New Insights into Prebiotic Chemistry from Stanley Miller's Spark Discharge Experiments[END_REF] Furthermore, seven organosulfur compounds in unreported 1958 Miller's samples, in which H2S gas was considered in the primitive atmosphere, were identified using HPLC and TOF mass spectroscopy. [START_REF] Parker | Prebiotic Synthesis of Methionine and Other Sulfur-Containing Organic Compounds on the Primitive Earth: A Contemporary Reassessment Based on an Unpublished 1958 Stanley Miller Experiment[END_REF] All of the amino acids that were identified in the Murchison meteorite have been also found in Miller experiment's products. [START_REF] Miller | The Atmosphere of the Primitive Earth and the Prebiotic Synthesis of Amino Acids[END_REF] In fact, Bada was not the first to detect sulphur containing amino acids. Miller himself have reported that methionine was a product of the action of an electric discharge on a simulated primitive reduced atmosphere containing H2S or CH3SH. [START_REF] Parker | Prebiotic Synthesis of Methionine and Other Sulfur-Containing Organic Compounds on the Primitive Earth: A Contemporary Reassessment Based on an Unpublished 1958 Stanley Miller Experiment[END_REF][START_REF] Van Trump | Prebiotic Synthesis of Methionine[END_REF][START_REF] Parker | Primordial Synthesis of Amines and Amino Acids in a 1958 Miller H 2 S -Rich Spark Discharge Experiment[END_REF] Beside racemic methionine, other sulphur-containing organic compounds were detected again in unpublished 1958

Miller's experiment samples. [START_REF] Parker | Prebiotic Synthesis of Methionine and Other Sulfur-Containing Organic Compounds on the Primitive Earth: A Contemporary Reassessment Based on an Unpublished 1958 Stanley Miller Experiment[END_REF] Miller experiments are not the only prebiotic process to yield amino acids. Plausible prebiotic synthesis of amino acids from HCN reactions in water has been reported. [START_REF] Matthews | The Origin of Proteins: Heteropolypeptides from Hydrogen Cyanide and Water[END_REF] It is strongly believed that amino acids are not the direct products of the electric discharge in Miller experiment. They probably arise from a Strecker synthesis (Figure 5), followed by the hydrolysis of transiently formed amino nitriles in aqueous phase. [START_REF] Cleaves | Prebiotic Chemistry: What We Know, What We Don[END_REF][START_REF] Miller | Production of Some Organic Compounds under Possible Primitive Earth Conditions 1[END_REF] In 1990, a Strecker reaction was proposed to be the last step in a plausible prebiotic synthesis of histidine starting from erythrose and formamidine. In this case, the first product was Imidazole-4-acetaldehyde, which was then converted to histidine in the presence of hydrogen cyanide and ammonia. 67

Nucleobase

Miller's experiment have led to the modern era of prebiotic chemistry. From that time on, prebiotic chemists have been trying to synthesize biomolecules under plausible prebiotic conditions. J. Oro was able to report the first prebiotic synthesis of the nucleobase adenine, a basic constituent of nucleic acid, starting from hydrogen cyanide and ammonia in 1961. [START_REF] Oró | Synthesis of Purines under Possible Primitive Earth Conditions. I. Adenine from Hydrogen Cyanide[END_REF][START_REF] Orgel | The Origin of Life-a Review of Facts and Speculations[END_REF] Adenine can be regarded as a pentamer of HCN. The initial step in a possible pathway to adenine is the dimerization of HCN followed by further reactions to give HCN trimer (aminomalononitrile) and tetramer (diaminomaleonitrile). Then the tetramer undergoes photochemical isomerization followed by a ring closure reaction, and finally the ring reacts with another HCN molecule to provide adenine (Figure 6). 70 Furthermore, the photochemical rearrangement of diaminomaleonitrile in sunlight to give amino imidazole carbonitrile was demonstrated by Ferris and Orgel in 1966. [START_REF] Ferris | Studies in Prebiotic Synthesis. I. Aminomalononitrile and 4-Amino-5-Cyanoimidazole 1,2[END_REF] The Imidazole ring can also give other purines in this mixture. [START_REF] Cleaves | Prebiotic Chemistry: What We Know, What We Don[END_REF] Beside purines, the prebiotic synthesis of Pyrimidines was also investigated. Uracil was produced from a mixture of malic acid and urea. [START_REF] Fox | Synthesis of Uracil under Conditions of a Thermal Model of Prebiological Chemistry[END_REF] Few years later, the synthesis of cytosine from cyanoacetylene (HCCCN) and cyanate (NCO -) was reported (Figure 7). [START_REF] Cleaves | Prebiotic Chemistry: What We Know, What We Don[END_REF][START_REF] Sanchez | Cyanoacetylene in Prebiotic Synthesis[END_REF]

Figure 7: Proposed mechanisms for the prebiotic synthesis of pyrimidines

Thymine was obtained in a good yield from the reaction of uracil with formaldehyde and formate. 74

Sugars

The formose reaction (also known as Butlerov synthesis) has been considered as a prebiotic source of sugars. This reaction was discovered by Butlerov's in 1861. [START_REF] Butlerow | Bildung Einer Zuckerartigen Substanz Durch Synthese[END_REF] It has a great importance concerning the study of the origin of life, as sugars (ribose and deoxyribose) are key molecules to construct RNA and DNA skeletons.

In the formose reaction, formaldehyde oligomerizes in the presence of alkaline earth hydroxides (eg. Ca(OH)2 or CaCO3). It is a simple reaction in which sugar elongation is achieved via repetitive additions of formaldehyde molecules. The importance of alkaline earth hydroxides as a catalyst for this reaction is not a problem from a prebiotic point of view as these alkalines can be found in (or replaced by) clays. [START_REF] Cleaves | Prebiotic Chemistry: What We Know, What We Don[END_REF] The presence of Kaolin, a white clay composed mainly of kaolinite (Al2Si2O5(OH)4), in the reaction mixture can catalyse the formation of sugars, including ribose. [START_REF] Cleaves | Prebiotic Chemistry: What We Know, What We Don[END_REF][START_REF] Gabel | Model for Origin of Monosaccharides[END_REF] Pentoses were formed from mixtures of formaldehyde, glyceraldehyde, and borate minerals such as colemanite (Ca2B6O115H2O) or kernite (Na2B4O7). [START_REF] Ricardo | Borate Minerals Stabilize Ribose[END_REF] The formose reaction is favoured by the presence of glycolaldehyde, which is the first product of the reaction. [START_REF] Cleaves | Prebiotic Chemistry: What We Know, What We Don[END_REF] The observation of glycoaldehyde in space endorses the importance that the formose reaction might have had as a major pathway in the synthesis of sugars on the early Earth.

Figure 8 shows a simple pathway to some of the common sugars, however, more complex mixtures of sugars would be obtained from the formose reaction. For example, Decker et al.

reported more than 40 sugars in one reaction mixture. 78

Figure 8: A simple pathway for sugar synthesis via the formose reaction

On the other hand, the formose reaction is not universally accepted for the prebiotic synthesis of sugars because of some weaknesses in this proposal. The difficulty to control the reaction (i.e. nothing would stop the elongation process under alkaline media) is one reason for this rejection. Another problem is that the conditions of the reaction may lead to the degradation of sugars. [START_REF] Reid | Synthesis of Sugars in Potentially Prebiotic Conditions[END_REF] For example, at pH seven, ribose and other sugars are found to be labile and are able to decompose. [START_REF] Larralde | Rates of Decomposition of Ribose and Other Sugars: Implications for Chemical Evolution[END_REF] Furthermore, sugars are able to undergo several reactions in a rate that would be faster than their accumulation in the environment. [START_REF] Cleaves | Prebiotic Chemistry: What We Know, What We Don[END_REF] As an alternative prebiotic synthesis, sugar synthesis was achieved by photoredox systems. It has been reported that simple sugars can be formed from hydrogen cyanide by ultraviolet irradiation in the presence of cyanometallates. 81

Nucleosides and nucleotides

As prebiotic synthesis of both sugars and nucleotides are possible, it was logical to go further in the construction of more complex biomolecules. In this regard, we will discuss the attempts to obtain nucleosides and nucleotides as crucial examples of "more complex" biomolecules. Nucleosides are essential components in DNA and RNA, and understanding their development on Earth is fundamental in the study of the origin of life.

Of course, in today's biology enzymes facilitate the synthesis of nucleotides, but it is not realistic to imagine that such evolved bio-catalysts were involved in the early stages of chemical evolution. [START_REF] Zubay | Prebiotic Synthesis of Nucleotides[END_REF] Therefore, "nonenzymatic" prebiotic synthesis should be proposed. [START_REF] Fahrenbach | Common and Potentially Prebiotic Origin for Precursors of Nucleotide Synthesis and Activation[END_REF] Here, a lot of questions arise, for instance: why was ribose preferred over other sugars? Why not another sugar? It is also still unclear why the present RNA bases (but not other possible alternatives that were probably present on early Earth) would have been selected by prebiotic reactions. [START_REF] Fialho | Glycosylation of a Model Proto-RNA Nucleobase with Non-Ribose Sugars: Implications for the Prebiotic Synthesis of Nucleosides[END_REF] The earliest attempts to obtain nucleosides in a prebiotic way was simply to heat a dry mixture of ribose and nucleobases (purines or pyrimidines). About 8 % of β-D-inosine was obtained when hypoxanthine was used in the presence of a mixture of some seawater salts.

Mixtures of α-and β-isomers were obtained in low yields when adenine and guanine were used. [START_REF] Fuller | Studies in Prebiotic Synthesis[END_REF] However, heating a mixture of ribose and uracil did not give the expected nucleosides, uridine. Similarly, cytidine was not produced when cytosine was used (Figure 9). [START_REF] Cleaves | Prebiotic Chemistry: What We Know, What We Don[END_REF] The synthesis of pyrimidine nucleoside was achieved using a similar methodology. 86 Phosphates are believed to be an essential compound in primitive Earth. Small amounts of condensed phosphates are emitted in volcanic fumaroles. [START_REF] Yamagata | Volcanic Production of Polyphosphates and Its Relevance to Prebiotic Evolution[END_REF] Prebiotic formation of nucleotides were attempted through nucleosides phosphorylation. It was found that nucleosides can be phosphorylated upon dry heating with acidic phosphates. [START_REF] Beck | Phosphorylation with Inorganic Phosphates at Moderate Temperatures[END_REF] Trimetaphosphate (TMP) has been found to be an active phosphorylating agent for nucleosides. [START_REF] Cleaves | Prebiotic Chemistry: What We Know, What We Don[END_REF] Diiminosuccinonitrile (DISN) was considered as a potential prebiotic phosphorylating promoter. [START_REF] Ferris | Prebiotic Synthesis and Reactions of Nucleosides and Nucleotides[END_REF] Very interestingly, significant yields of ribonucleotides have been obtained in stepwise reactions, in which products were isolated after each reaction and submitted to next step. [START_REF] Powner | On the Prebiotic Synthesis of Ribonucleotides: Photoanomerisation of Cytosine Nucleosides and Nucleotides Revisited[END_REF] This methodology was able to produce pyrimidine and purine ribonucleotides in good yields. [START_REF] Powner | Potentially Prebiotic Synthesis of Pyrimidine β-D-Ribonucleotides by Photoanomerization/Hydrolysis of α-D-Cytidine-2′-Phosphate[END_REF] Moreover, a prebiotic transformation of a pyrimidine nucleoside into a purine nucleoside has been reported. [START_REF] Becker | Origin of Life: A High-Yielding, Strictly Regioselective Prebiotic Purine Nucleoside Formation Pathway[END_REF] A recent study has shown how a possible RNA ancestor (2,4,6-triaminopyrimidine (TAP)) can be glycosylated in water without the presence of ribose. This study supposed that ribose might not have been essential for prebiotic nucleoside formation at the very beginning of life development, as ancient RNA (reported as proto-RNA) might have used TAP (or other similar models). [START_REF] Fialho | Glycosylation of a Model Proto-RNA Nucleobase with Non-Ribose Sugars: Implications for the Prebiotic Synthesis of Nucleosides[END_REF] It has also been reported that 2-aminoimidazole, that was found to be a plausible prebiotic molecule, can act as a nucleotide activating group for nonenzymatic RNA copying. 83

E. Prebiotic synthesis of peptides

The big role proteins play in the present life suggests that amino acids and peptides might have had a huge contribution in the emergence and development of life. [START_REF] Danger | Pathways for the Formation and Evolution of Peptides in Prebiotic Environments[END_REF] It is very likely that amino acids were present in the primitive ocean. As we have previously reported it, amino acids are the most interesting products from Miller's experiment. [START_REF] Miller | A Production of Amino Acids Under Possible Primitive Earth Conditions[END_REF] Strecker synthesis is a reasonable prebiotic reaction that leads to the formation of these compounds. [START_REF] Strecker | Ueber Die Künstliche Bildung Der Milchsäure Und Einen Neuen, Dem Glycocoll Homologen Körper[END_REF] Moreover, many amino acids have been detected among the organic contents of meteorites. [START_REF] Cleaves | Prebiotic Chemistry: What We Know, What We Don[END_REF] Peptide bond formation in water starting from α-amino acids is thermodynamically unfavorable, but not impossible. Indeed, spontaneous peptidization of several amino acids in aqueous medium, eg. Cysteine 94 and Serine [START_REF] Godziek | Dynamics of Spontaneous Peptidization of L-, d-and Dl-Serine in an Abiotic Solution as Investigated with Use of TLC-Densitometry and the Auxiliary Chromatographic Techniques[END_REF] , has been demonstrated. Considering a primitive soup full of different amino acid units, every single addition to the peptidic sequence would give the newly formed peptide a different reactivity toward the hydrolysis of some parts of it or promoting the synthesis of more complex peptidic sequence. Several attempts to obtain peptides under prebiotic conditions (eg. drying/wetting cycle experements) ended up with diketopiperazines as major undesired byproducts. [START_REF] Danger | Pathways for the Formation and Evolution of Peptides in Prebiotic Environments[END_REF] However, it is assumed that these cyclic dipeptides can still be considered as precursors of dipeptides upon hydrolysis, and they can be opened by aminolysis, and thus lead to peptide elongation. This scenario would be a game of "trial and failure" processes toward self-organization and polymerization (Figure 10). However, resulting peptides from spontaneous polymerization of amino acids would be obtained only with very low yields. Moreover, considerable amount of hydrolysis of peptide bonds under the postulated conditions in the primitive ocean must be taken into account.

Warming up the reaction media will increase the equilibrium constants for the formation of peptides; however, very high temperatures are unreasonable from a prebiotic point of view.

Therefore, it would be hard to form significant amounts of peptides without the existence of catalysts in order to fill the energy gap for peptide formation. 96 Adenosine triphosphate (ATP) is used as a catalyst and provides the required energy for peptide bond formation in nowadays living systems.

In a primitive model, activating agents simpler than ATP and/or activated forms of amino acids are required to promote peptide elongation. Among models that have been proposed the formation of N-carboxyanhydrides of amino acids seems reasonable. These compounds could have been formed in the presence of bicarbonate in high concentration. 96 It has been reported

that this process can be achieved in the presence of cyanate by converting first amino acids into N-carbamoylamino acids which then can form N-carboxyanhydrides in the presence of carbonate (in equilibrium with saturated CO2 in the aqueous medium) (Figure 11-A). 96 Under similar conditions, peptide bonds were also obtained from dicarboxylic acids, including aspartic acid, derivatives in which the extra carboxylic acid in these types of molecules would undergo an intramolecular cyclization in order to form succinic anhydrides that can be reacted with nucleophiles in following steps (Figure 11-B). 96 It has been proposed that amino acids can be activated and converted into peptides under hot, anaerobic, aqueous conditions. Indeed, in the presence of NiS, FeS, CO and H2S (or CH3SH) peptide bonds were formed under "geothermal conditions". [START_REF] Huber | Peptides by Activation of Amino Acids with CO on ( Ni , Fe ) S Surfaces : Implications for the Origin of Life[END_REF] These results endorse the scenario that first reactions that lead to the formation of life had been catalysed on the surfaces of metal sulfide minerals.

Indeed, some scientists who study the origin of life went farther with minerals. They proposed that they had the crucial role in the appearance of life.

Iron-sulfur world

Iron-sulfur world was one of the pioneering hypothesis in this domain. Iron-sulfur clusters have a vital role in metabolism and they are involved in a broad range of essential functions in all living cells such as DNA maintenance, gene expression regulation, protein translation, energy production, and antiviral response. Mitochondrion, endoplasmic reticulum, cytosol, and the nucleus contain Fe-S Proteins. Fe-S enzymes participate in the metabolism of amino acids in fungi. [START_REF] Bonfio | UV-Light-Driven Prebiotic Synthesis of Iron-Sulfur Clusters[END_REF][START_REF] Braymer | Iron-sulfur Cluster Biogenesis and Trafficking in Mitochondria[END_REF] As a result of this essential role of Fe-S clusters as cofactors of proteins involved in biological cells, humans with a deficiency in the production of any these related protein suffer from serious diseases. 100

The fundamental role of Fe-S clusters (see p.34, Figure 19) of nowadays life can be considered as a biological heritage that is still kept for the earliest days of life. 101 Wächtershäuser proposed his idea of an "iron-sulfur world" in 1988. He suggested that early life might have formed on the surface of iron sulfide minerals. It has been later demonstrated that iron-sulfur clusters may have formed on early in the Earth ocean, with the help of UV light that drives their synthesis. These clusters may have coordinated and had been stabilized by a wide range of thiol-containing peptide. [START_REF] Bonfio | UV-Light-Driven Prebiotic Synthesis of Iron-Sulfur Clusters[END_REF] Wächtershäuser and Huber have found that an activated thioester can be formed starting from CO and CH3SH in the presence of FeS. 102 Furthermore, thioacetic acid, which can be regarded as a simple acetyl-CoA analogue, was also formed in the presence of H2S. The authors suggested that this reaction can be considered as the leading reaction for a chemoautotrophic origin of life in which, for example, amino acids would have been converted into simple peptides on FeS surfaces.

Unlike photosynthesis, the term "chemoautotrophic" was presented by Wächtershäuser to describe the system where simple forms of lifes obtain their nutrition via the oxidation of non-organic compounds, in this case FeS. He postuleted a pioneer organism with a merged composition between an organic superstructure and an inorganic substructure. In this scenario, the growth of the organic superstructure would have been achieved thanks to the Fe-S surface that could afford a reproductive metabolism by an autocatalytic process. Hence, the theory of a chemoautotrophic origin of life proposed that a common origin of Bacteria, Archaea and Eukarya had aroused through a volcanic iron-sulfur world. 103 Iron and sulphide may be arranged in cage structures ranging from [Fe2-S2], [Fe3-S4], [Fe4-S4], up to [Fe8-S8] clusters. 101 A familiar example of iron-sulfur structures is pyrite (FeS2).

Wächtershäuser proposed that life emerged on pyrite surfaces in volcanic or hydrothermal vents. On pyrite surfaces, peptides, nucleic acids and lipids would have been formed. 104 Rusell and Hall have gone farther and proposed that life had emerged from iron monosulphide bubbles that could have worked as primitive membranes in the Hadean ocean. 105 On the other hand, the iron-sulfer world theory was rejected by some scientists who believed that sequenced cellular genomes of nowadays living systems does not support Wächtershäuser proposal. 106

Zinc world

Alternatevely, the zinc world hypothesis proposes that the emergence of life started on zinc sulfide (ZnS, sphalerite) surfaces of hydrothermal origin and was derived by UV-rich solar radiation. ZnS could precipitate at the surface of continents under the exposure of solar light. 107 These mineral structures submitted to UV radiations would have facilitate the formation of the first biomolecules. These mineral sulfides might have also absorbed the excess radiation and thus protect biomolecules from photo-dissociation. Zn 2+ ions are expected byproducts in the photosynthesis of ZnS structures. As a result, life might have been evolved under high levels of Zn 2+ ions. Indeed, this suggestion agrees with the fact that Zn 2+

ions have important and specific roles in modern organisms. 108 Zinc world and iron-sulfur world scenarios promote the "metabolism-first" concept. This concept proposes that replication processes were preceded by many other chemical reactions that had been catalysed by minerals and had yielded a mixture of organic molecules. These compounds were able to react, polymerize, and undergo self-assemblage in order to form proteins, RNA and DNA later.

Thioester world

Another hypothesis that is related to metabolism first concept is de Duve's thioester world. 109 Christian de Duve suggested that a thioester world might have preceded the RNA world (that we will discuss later) in the prebiotic evolution. He suggested that peptides might have been synthesized with the help of thioester bond energy, establishing a growing protometabolism. 110,111 Thioester bond has a high-energy character and thus is highly reactive toward amino acids.

The thioester world hypothesis has gained a strong support since it has been first proposed. 111

The most famous example of biological thioesters is CoA (see p.33, Figure 16). This wellknown coenzyme contributes in many biological processes in all domains of life. Indeed, CoA can be regarded as a molecular fossil from an early metabolic state and that has been kept in life's memory. 112 An ancient CoA-thioester model might have appeared in the very early form of life. It might have been even participated in the metabolism of LUCA itself! 111,113

RNA world

Alexander Rich first proposed the concept of the RNA world in 1962. 114 He assumed that RNA might have played the key role in the early history of life after discovering that ribozymes, a type of RNA molecules, catalyse the formation of proteins beside being able to replicate itself. The "replication-first" concept thus suggests that the appearance of RNA, which is able to carry and transfer information, preceded the formation of other metabolisms.

In order to form simple RNA strands, nucleotides had to form and polymerize, which might be possible in hydrothermal vents in the ocean or in "warm little ponds" in the primitive Earth. 115 Even though, it is the nowadays most consensual theory, RNA world hypothesis has faced several objections due to some accompanying difficulties such as the very limited catalytic properties, the instability, and the high complexity of the RNA molecules, complexity that makes their prebiotic synthesis difficult to imagine. 116

RNA world / protein world: A partnership?

In nowadays biochemistry, proteins catalyse chemical reactions and DNA stores the genetic information (with RNA transmitting the information from DNA, and allowing the synthesis of proteins). The problem weather proteins or nucleotides came first presents a famous chicken-or-egg dilemma.

Still believing in the RNA world, some scientists do not believe in an independent RNA world and they argue that the RNA world alone could have never existed without amino acids and peptides that "shaped" it. 117 On one hand, RNA world concept relies on the fact that RNA is able to carry and transfer information. On the other hand, proteins-first concept depends on the catalytic properties of proteins for a wide range of chemical reactions. However, it has been proposed that both peptides and oligonucleotides might have contributed in the emergence of life in a complementary partnership. 118 With this hypothesis, it is reasonable to assume that both metabolism and genetics shared a common origin.

It has also been assumed that RNA, protein and even lipid precursors might have aroused from common origins starting from hydrogen cyanide and hydrogen sulfide in the presence of UV light. [START_REF] Patel | Common Origins of RNA, Protein and Lipid Precursors in a Cyanosulfidic Protometabolism[END_REF] This scenario suggested that reactions had been developed separately in different streams and that the resulting products had been mixed together later in pools, in a way very similar to what is now done in laboratories with flow chemistry.

Another scenario has nominated formamide as the possible simple starting material for the synthesis of a wide range of primordial biomolecules that are necessary for both genetic and metabolic processes such as nucleic bases, nucleotides, sugars, and amino acids (Figure 12). 119

F. Today's role of thiols in biology

Sulfur and oxygen share many chemical properties as they belong to the same group of elements in the periodic table. However, sulfur atom has a higher radius, which leads to a less electronegativity. Also, sulfur containing biomolecules are more hydrophobic than their oxygen counterparts. 120 The fact that SH is more nucleophilic than OH makes the thiol function one of the most reactive functional groups in biology. Moreover, thiols often occur as thiolates under physiological pH conditions, which make them even more reactive. Thiolates can easily chelate with different metals such as iron [START_REF] Bonfio | UV-Light-Driven Prebiotic Synthesis of Iron-Sulfur Clusters[END_REF] , zinc 121 , nickel 122 , copper 123 , and cobalt 124 . This chelating behaviour of thiols, especially in cysteine residues, might have participated in framing of the metalloproteins models that we know nowadays.

The presence of H2S on primitive Earth support the fact that many sulfur-containing molecules were probably present in the Hadean/Archean ocean. Indeed, sulfur biomolecules have vital importance in all living organisms. They are involved, inter alia, in free radicals scavenging, functionality of enzymes, as well as in DNA methylation and repair. 125,126 . The diversity and the significance of active organosulfur molecules strongly suggests that sulfurcontaining compounds, including cysteine and methionine residues, had an important role in the early evolutionary history of life. 127 Methionine is of crucial importance for mRNA translation. It is the initiating amino acid in the synthesis of eukaryotic and archaeal proteins, while N-formyl-Methionine plays this role in prokaryotes. 120 Cysteine-rich proteins are found in all living organisms and often play fundamental roles as hormones, growth factors, ion channel modulators and enzyme inhibitors in various biological pathways. 128 The similar functions that sulfur containing molecules play in plant and animal cells strongly support the hypothesis of a shared ancestor of these two kingdoms. 129 It has been proposed that primitive living organisms used elemental sulfur itself, or simple sulfur molecules, for energy metabolism (i.e. breathing) under oxygen-free atmosphere. This belief is supported by the highly conserved sulfur metabolism in nowadays bacteria that can be regarded as "survivals" of ancient living organisms. 130

Sulfur metabolism in living cells

In humans, sulfur inorganic species and sulfur containing biomolecules are obtained from a normal equilibrated diet. The main sulfur-containing biomolecules active in human metabolism and their metabolism are presented in Figure 13. 120

The methionine cycle

In the methionine cycle, methionine is converted to SAM (S-Adenosyl Methionine). SAM is a methyl donor, and it is converted to SAH (S-Adenosyl Homocysteine) after losing its methyl group. S-adenosylhomocysteine hydrolase converts SAH into homocysteine and adenosine (Figure 14). 120,131

Figure 14: Summary of the methionine cycle

Homocysteine can either recycled back to methionine through this cycle, or it can be used to form other sulfur-containing amino acids (Figure 13).

Biological metabolism of cysteine and homocysteine

In animals, the biosynthesis of cysteine starts with serine and homocysteine. Cystathionine βsynthase joins these two amino acids to form cystathionine. An enzyme, cystathionase, breaks down cystathionine into cysteine by releasing α-ketobutyrate and NH3. This process is called transsulfuration (Figure 15). 120,132

Figure 15: Transsulfuration pathway in mammals to obtain cysteine

The obtained cysteine can be used to produce other sulfur containing molecules such as CoA, taurine (Tau), glutathione (GSH, an intra-cell free radical scavenger), and iron-sulfur clusters (Figure 13). 132 The structure of Coenzyme A (CoA) was first revealed in 1953. CoA biosynthesis requires cysteine, vitamin B5, and adenosine triphosphate (ATP) (Figure 16). Taurine is an amino sulfonic acid highly concentrated and widely distributed in animal and human tissues such as brain, retina, muscle, liver and kidney. Taurine has antioxidant activities in biological systems, besides regulation of Ca 2+ levels (Figure 18). 135

Glutathione is the most abundant thiol compound in cells. It has important antioxidant activities in plants, animals, fungi, and bacteria. It binds to several toxic compounds for detoxification purposes. It also helps to maintain the suitable cell redox status (Figure 18). 136 clusters) (Figure 19). Their importance in the living cells arises from the fact that they are able to deliver electrons via the Fe 2+ to Fe 3+ oxidation reaction. 101,137,138

Figure 19: Iron clusters redox behaviour

The SMM cycle of flowering plants

Sulfur metabolism in plants presents some specificities. In this regard, we believe that the Smethylmethionine (SMM) cycle deserves to be considered. It was proposed that the SMM cycle contributes in the synthesis of methionine. SMM acts as a storage of methionine in plant tissues. SMM is first produced from methionine in non-seed tissues, and then transported through the phloem from leaves into seeds, where it is reconverted to methionine thanks to the SMM cycle (Figure 20). 139 In fact, methionine is a key metabolite in plant cells.

It plays an essential role in the initiation of mRNA translation and the synthesis of proteins. 139,140 As mentioned before, many simple aldehydes were probably present in the primitive ocean 142 and they are plausible precursors for α-amino acids. Reacting with ammonia and hydrogen cyanide, they would have first give α-aminonitriles which upon hydrolysis would have delivered amino acids (Figure 21). 143 However, it has been reported that the reaction of nitriles with water is a slow process 144 , so slow that, once formed in the ocean, aminonitriles would have had sufficient time to react with species more nucleophilic than water.

Figure 21: Strecker reaction

Nitriles are known to react with aminothiols to give thiazolines, which in turn can be hydrolysed to mercaptoamides. 145,146 Starting from α-aminonitriles and cysteine, the expected products of this two-step process are dipeptides (Figure 22).

Figure 22: The reaction of aminonitriles with cysteine to obtain dipeptides

Compared to any activation process starting from acids, this nitrile scenario has the advantage of not necessitating any strong energy source. The acid does not need to be activated as it is delivered directly in an activated form by the Strecker reaction.

This chapter focuses on the synthesis and reactivity of different types of aminonitriles toward aminothiols, under plausible prebiotic conditions. In the first part of this chapter, we discuss the synthesis of aminonitriles that we used in reaction with aminothiols (cysteine, homocysteine and cysteine amide). The second part of the chapter studies the reactivity of these aminonitriles toward thiazoline formation. It includes some competitive reactions between different starting materials. The formation of active thioesters, nitrogen heteroaromatics, and other by-products of our reactions are also discussed. Some of these results are supported by theoretical calculations.

I.2 Synthesis of aminonitriles

Some nitriles that we used are commercially available, some are not. Their synthesis is described here.

I.2.1 Synthesis of racemic α-aminonitriles

We used Strecker synthesis to obtain racemic aminonitriles. In 1988, Denis and Guillemin used this reaction to obtain several aminonitriles starting from the related aldehydes. 147 We followed their procedure in order to obtain racemic 2-aminobutanenitrile 1, Val-CN 2 and Met-CN 3 (Scheme 1).

Scheme 1

The common by-products of this method were cyanohydrins. Denis and Guillemin have separated their aminonitriles by distillation. We used the same method. However, for reactions with smaller quantities we used column chromatography as another efficient method to recover the products.

I.2.2 Synthesis of L-aminonitriles

L-aminonitriles were obtained starting from the corresponding Fmoc protected L-amino acids. We activated the acids as acid chlorides using thionyl chloride, SOCl2. Then, we introduced ammonia gas to provide the corresponding amides. By using cyanuric chloride, we were able to dehydrate amides into nitriles. Fmoc group was finally removed using piperidine to obtain the required L-aminonitriles (Scheme 2).

Scheme 2

In the last step, L-Methionine nitrile 11 was obtained upon deprotection. However, when we tried to deprotect Fmoc-L-Valine 8, the resulting L-Valine nitrile 10 was found to be unstable and mixture of products has been obtained. As a consequence, we used only racemic Val-CN 2 in our reactions.

I.2.1.a Synthesis of L-Phe-CN 15

On the other hand, we did not succeed to obtain the acid chloride of phenylalanine. When we started from Fmoc-Phe-OH, we found that the corresponding acid chloride 12 was not stable.

We think that Friedel-Crafts by-products were formed. Similar results were obtained when we used oxalyl chloride, (COCl)2, as an alternative to thionyl chloride (Scheme 3).

Scheme 3

Strangely, when Fmoc protecting group was replaced by Boc, we obtained the good product.

In this case, we succeeded to obtain Boc-L-Phe-NH2 13 and Boc-L-Phe-CN 14 successively.

However, this nitrile 15 was not stable under acidic conditions and it hydrolysed in the last deprotection step to form the corresponding acid, L-Phe-OH 16 (Scheme 4).

Scheme 4

Still trying to obtain the L-Phe-CN 15, Cbz protected L-Phe was attempted. The acid was activated as a mixed anhydride, ammonia was introduced to form the amide 17, and cyanuric chloride was used to provide the nitrile 18. However, the last step was not successful and a mixture of products was produced in this case (Scheme 5).

Scheme 5

I.2.1.b Synthesis of L-Leu-CN 21

The observed stability of the phenylalanine nitrile under acidic conditions, in the previous example, makes the stability of similar simple aminonitriles questionable. In order to study the stability of these nitriles in acidic media, we decided to synthesize some of them in a similar way starting from Boc protected L-amino acids. As an example, L-Leu-CN 21 was obtained. We found that it is only poorly stable under similar conditions. The last step was followed by extraction in D2O. The 1 H-NMR showed a mixture of the nitrile 21, the acid 22 and the amide (Scheme 6).

Scheme 6

I.2.1.c Synthesis of L-Pro-CN 26

We also tried to synthesize the aminonitrile of proline, an example to study the stability of an amino nitrile with a secondary amine. We started this synthesis form Boc-L-Proline, we activated the acid as mixed anhydride, introduced ammonia to provide the amide 24, converted the amide into nitrile 25 using cyanuric chloride, and finally deprotected the amine with TFA (Scheme 7). However, this nitrile 26 could not be isolated and a mixture of unidentified products was produced in this case.

Scheme 7

The instability of leucine and phenylalanine nitriles demonstrates that at least some simple aminonitriles (aa-CN) are poorly stable in strong acidic conditions. As we will see later, longer peptidic chains ending with nitriles (aa-(aa)n-CN) are more stable under similar acidic conditions.

Despite that we could not obtain the nitrile starting from the Boc protected proline, we thought to attempt Fmoc as a protecting group of proline's amine as this protecting group can be removed under basic conditions to give the required nitrile 26. The acid was activated as an acid chloride 27, followed by amide formation 28. The corresponding nitrile 29 was obtained using the cyanuric chloride. However, we were not able to isolate the product 26 in pure form when the protected nitrile was treated with piperidine (Scheme 8).

Scheme 8

The deprotection step was repeated using Et3N, and by introducing ammonia gas in DCM.

However, none of these methods was successful. Finally, the last step worked when we used methanol as a solvent and introduced ammonia to the solution to obtain Pro-CN 26. However, this nitrile was not stable when passing through a silica column and thus it was difficult to purify it. Another idea for purification was to obtain the ammonium chloride salt by introducing HCl gas, but we found that the nitrile 26 did not survive such acidic condition and hydrolysed into the acid.

I.2.2 Synthesis of (S)-2-amino-1,4-butanedinitrile 32

Scheme 9

In order to study the relative reactivity of α and β-aminonitriles, we decided to synthesize an α,β-aminonitrile, a compound having both an α-aminonitrile and a β-aminonitrile. The bisnitrile 32 has been obtained starting from a protected asparagine (AsnCbz). We first tried the transformation of the carboxylic acid into amide. However, in this case, the obtained product was the amido-nitrile 30. This unexpected nitrile formation in the first step can be explained by the following mechanism (Figure 23). 148 Indeed, this was lucky, as it shortens our synthesis.

Figure 23: A reported mechanism for this unexpected nitrile formation

Cyanuric chloride was used to form the second nitrile group 31. Finally, Cbz group was removed by hydrogenation (Scheme 9).

I.2.3 Synthesis of 2-amino-4-cyanobutanoic acid 35

Scheme 10 γ-aminonitrile 35 was synthesized starting from protected glutamine, MeOGlnCbz. The amide was first transformed into the corresponding nitrile 33 using cyanuric chloride. Then, LiOH was used for the saponification of the ester into the carboxylic acid 34, and finally, Cbz group was deprotected by hydrogenation to obtain the required nitrile 35 (Scheme 10).

I.2.4 Synthesis of (S)-2-aminopentanedinitrile 38

Scheme 11

An α,γ-amino bisnitrile, a bisnitrile which have both an α-aminonitrile and a γ-aminonitrile, was synthesized in order to study the competition between α and γ-amino nitriles. We have followed a previously described method to obtain the required bisnitrile 38. 149 It was synthesized in 3 steps. Starting from GlnCbz, the acid was activated by SOCl2 and then ammonia was introduced to form the amide 36. Then, cyanuric chloride was used to form the bisnitrile 37 and the product 38 was obtained in the last step upon the deprotection of the Cbz group (Scheme 11).

I.2.5 Synthesis of N-acetyl-α-aminonitrile 40

Scheme 12

N-acetyl-α-aminonitrile is a good candidate as a simple model which will allow us to check the reactivity of a peptide chain ending with a nitrile. The required amino nitrile 40 was simply obtained starting from acetic anhydride and aminoacetonitrile 39 in pyridine at 0 °C for one day (Scheme 12). 150

I.2.6 Synthesis of Gly-Gly-CN 42

Scheme 13

We followed a previously described method to obtain Cbz-Gly-CN 41 starting from Cbz protected glycine in a good yield. 151 Then, the Cbz group was removed by hydrogenation to give product 42 (Scheme 13).

I.2.7 Synthesis of Gly-Gly-Gly-CN 45

Scheme 14

We started the synthesis from the commercially available diglycine. We protected this dipeptide with Boc group to give 43. 152 Then we activated the acid like in previous synthesis and added aminoacetonitrile 39 to give the nitrile 44. 151 Finally, the Boc group was removed in acidic condition to give Gly-Gly-Gly-CN 45, as its trifluoroacetate salt (Scheme 14).

I.3 Reactions between aminonitriles and aminothiols I.3.1 Reaction of aminoacetonitrile with cysteine

We first studied the reaction of aminoacetonitrile (Gly-CN 39) with cysteine. 153 The probable slight acidity of primitive oceans supports our work because acidity can favour Pinner reaction (Figure 26). 154 In this reaction, nitrile reacts with an alcohol (or a thiol) to

give an imino-ether (or an imino-thioether).

Figure 26: Pinner reaction

In the case of an aminonitrile reacting with cysteine, the mechanism starts with thiol attack on the nitrile (i.e. Pinner reaction). Then, the amine adds onto the obtained iminium double bond in 50 to form a non-observed thiazolidine 51, which upon NH3 loss gives the more stable thiazoline cycle 46. This thiazoline is thus slowly hydrolysed to give the dipeptide 47 (Figure 27).

Figure 27: Proposed mechanism of the formation of Gly-Cys dipeptide 47 in our mixture

In addition, we performed this reaction in H2O and followed it by mass spectrometry. This technique confirmed the formation of both the thiazoline ring 46 and Gly-Cys 47 (Figure 28). We observed the formation of the thiazoline 46 and Gly-Cys 47 at concentrations as low as 5

x 10 -4 mol. L -1 . We have repeated the experiment in different temperatures and pH's and we have found that the process is quicker at higher temperatures but goes well also at room temperature. The ring formation is quicker under basic conditions. We believe that under such conditions the thiol function is deprotonated giving the more nucleophilic thiolate species. Under acidic condition the nucleophilic species is probably the thiol itself. We also noticed that the hydrolysis step is quicker under acidic conditions. This probably implies that the thiazoline ring is activated through protonation step before H2O addition.

Although, these reactions were applicable under a wide range of pH's (4 -9) and temperatures (20 -70 °C), we (in most cases) choose 45 °C and a pH at ca. 5.5 -6.5 as standard conditions for the following reactions, taking into account the primitive conditions that have been discussed in the introduction. The evolution of Gly-Cys formation at 45 °C and different pH's were followed by 1 H-NMR and is presented in (Figure 29). The figure

shows that the formation of the cycle 46 is faster under basic conditions. These conditions gives more stability to the cycle 46. The hydrolysis of this cycle is faster under more acidic conditions. We did not observe reactions between aminoacetonitrile and any other proteinogenic amino acid that we tested, including serine. In this case, it appears that the alcohol function of serine is not nucleophilic enough to attack the CN triple bond. Therefore, the presence of a thiol function is mandatory in our reactions.

Finally, in order to study the effect that minerals might have on these reactions, we perform this reaction in the presence of Zn (II), which is mandatory for the formation of zinc fingers.

We found that addition of ZnCl2 had no effect on the formation of the thiazoline ring 46 or the Gly-Cys dipeptide 47.

I.3.2 Reaction of aminoacetonitrile with homocysteine

As it contains both thiol and amine functions, we expected homocysteine to react similarly to cysteine. Indeed, aminoacetonitrile 39 reacted with homocysteine in a similar way to that observed with cysteine. In this case, the intermediate is a six-membered ring 52. The hydrolysis of this dihydrothiazine leads to the final product, Gly-Hcy dipeptide 53 (scheme 16).

Scheme 16

We performed the reaction in D2O and followed it with 1 H-NMR (Figure 30). We found that homocysteine reacts slightly slower than cysteine. We believe that this is due to the fact that the formation of a five-membered ring, in the case of cysteine, is kinetically more favoured than the formation of a six-membered ring. Despite this remark, the difference was not much and it can be considered that both cysteine and homocysteine reacts in similar rates.

Scheme 19

However, the obtained dipeptides in the last two examples 58 and 61 are not composed of completely "proteinogenic" amino acids, and we thought that trying to obtain peptides consisting of proteinogenic amino acids would be more interesting to us. In order to obtain a wider library of "proteinogenic amino acid"-containing dipeptides, we introduced relevant aminonitriles to react with cysteine, and by this way, several aa-Cys examples were obtained.

We followed these reactions by 1 H-NMR without purification. Furthermore, the expected products were detected in mass spectrometry of the mixtures when H2O was used as a solvent. We chose to use the nitriles derived from methionine and valine. Both of these nitriles reacted with cysteine, and the expected thiazolines and the dipeptides were identified (Scheme 20).

Scheme 20

Valine and methionine nitriles reacted in a similar way with homocysteine. In this case, the expected six-membered rings were obtained. Similarly to thiazolines, these six-membered rings are more stable in basic conditions (pH 7 -8) and they hydrolyse faster in lower pH's, and/or upon heating, to give the corresponding dipeptides (Scheme 21).

Scheme 21

As we have previously discussed in the first part of this chapter, some of our nitriles were not stable under acidic conditions. One of these nitriles is L-Leu-CN 21 that was obtained in a mixture with the acid and amide. However, we tried to add cysteine to this mixture, despite it was not pure, as another example of our reactions. In this case, the expected thiazoline was observed (Scheme 22). Similarly to other thiazolines, this one was identified by the appearance of a nearly triplet peak at 4.97 ppm for CH on the cycle and an ABX system from 3.5 to 3.7 ppm corresponding to the CH2.

Scheme 22

I.3.4 N-Acetyl aminoacetonitrile with cysteine

N-Acetyl aminoacetonitrile, which could be considered as a model for any other N-acyl acetonitrile, including peptidic nitriles, also reacted with cysteine in good rate (Scheme 23).

Scheme 23

The formation of the expected thiazoline ring 71 was confirmed by the appearance of a small triplet signal (4.9 ppm) corresponding to CH, and the ABX system (from 3.4 to 3.6 ppm) corresponding to CH2 on the cycle. (Figure 31). In this example however, the hydrolysis step was quick, and the major signals observed on the 1 H-NMR were that of the dipeptide 72.

Figure 31: Reaction of cysteine with N-acetyl aminoacetonitrile

I.3.5 Reaction of β-aminopropionitrile with cysteine and homocysteine

On the contrary, the reaction of cysteine was much slower when α-aminoacetonitrile 39 was replaced by β-aminopropionitrile 73. In this case, the reaction took few days to complete but eventually yield the expected amide 75 (Scheme 24). This result agrees with our theoretical calculations that will be discussed at the end of this chapter. It is noteworthy that the corresponding thiazoline 74 in this example hydrolysed quickly and was not observed on 1 H-NMR spectra.

Scheme 24

As in the case of cysteine, β-aminopropionitrile 73 reacted with homocysteine much slower than aminoacetonitrile 39 (Scheme 25).

Scheme 25

Trying to test the effect of moving the nitrile farther from the amine, we used the -nitrile of glutamic acid 35 155 and added it to a cysteine solution in D2O. No reaction was observed in this case (Scheme 26).

Scheme 26

Thus, according to our findings, if a mixture of various α, β and -aminonitriles was present in the primitive ocean, α-aminonitriles would have had the best chance to react with aminothiols and would have led to the formation of "regular" peptides, consisting only of αamino acid residues. Fortunately, these results agree with the living systems that we know nowadays, in which only α-amino acids take part in the formation of proteins.

I.3.6 Competition between α and β-aminonitrile

We used to preserve an acceptable range of pH's (5.5 -6.5) in our reactions, and to maintain the temperatures at the selected values (45 °C or room temperature). However, our comparisons of the reactivity of two nitriles in separate containers are not very accurate as the pH (or even the temperature) may vary a little bit, and thus the conditions will not be exactly the same. In order to achieve error-free results, we performed competition reactions where both nitriles in the same container and react freely with a chosen aminothiols.

I.3.6.a Competition between α and β-aminonitrile with cysteine

We used an equimolar amounts of cysteine, aminoacetonitrile 39 and β-aminopropionitrile 73 in D2O and followed the reactions by 1 H-NMR (Figure 32). Clearly, cysteine reacted with the α-aminonitrile much faster than with its β-analogue. The reaction with α-aminonitrile took few hours to complete. On the other hand, it took several days in the case of β-aminonitrile.

I.3.6.b Competition between α and β-aminonitrile with homocysteine

Similarly to cysteine, homocysteine reacted with aminoacetonitrile 39 much faster than with β-aminopropionitrile 73. Here also, while the reaction of α-aminonitrile took few hours to complete, it took several days in the case of β-aminonitrile (Figure 33).

I.3.7 Reaction with 2-amino-1,4-butanedinitrile

In the aforementioned examples, we have tested the chemoselectivity of aminothiols toward α-and β-aminonitriles (as separate molecules) in competition reactions and we found that reactions with α-aminonitriles are always much faster than with β-aminonitriles. As a complement, we investigated what happens when both nitriles were located on the same molecule, that is to say, when chemoselectivity can be also regarded as regioselectivity.

Kieu DUNG LY (during her M2R internship in our group) synthesized the bis-nitrile derived from aspartic acid 32 156 and examined its reaction toward cysteine. In this case, only the α-aminonitrile reacted with cysteine giving the corresponding thiazoline 78 which was stable under our conditions (Scheme 27).

Scheme 27

1D ( 1 H and [START_REF] Sutherland | Opinion: Studies on the Origin of Lifethe End of the Beginning[END_REF] C-NMR) spectra demonstrated that only one nitrile reacted with cysteine to give a thiazoline cycle. The use of 2D NMR spectroscopy (HMBC) showed that the reaction occurred on the α-aminonitrile side, while the β-aminonitrile remained unreacted (an equimolar cysteine/bisnitrile mixture was used), once again demonstrating the selectivity toward α-aminonitriles (Figure 34).

Reaction of cysteine with GlyGly-CN and GlyGlyGly-CN

In order to obtain a simple example of a tripeptide, we mixed Gly-Gly-CN  with cysteine.

The corresponding thiazoline 79 for this tripeptide was observed in this case and it was characterized in 1 H-NMR by its triplet (4.91 ppm) and its ABX system (3.38 to 3.6 ppm) relative to the CH2 in the cycle (Scheme 28).  We will demonstrate later that this nitrile may be regarded as a plausible prebiotic molecule.

Scheme 28

Similarly, when we mixed Gly-Gly-Gly-CN 45 with cysteine, we observed the formation of the thiazoline ring 80. This formation was demonstrated by the appearance of a triplet (4.9 ppm) and the ABX system (3.3 to 3.6 ppm) in the 1 H-NMR. This reaction was relatively rapid at pH 8 and 45 °C. Under these conditions, 15 % of the thiazoline was obtained after 40 min and this conversion increased to 40 % after 2 hours (Scheme 29).

Scheme 29

In these cases, we used slightly basic conditions to be sure to observe thiazoline rings. Under acidic conditions, hydrolysis of thiazolines 79 and 80 would be quicker leading to the corresponding peptides Gly-Gly-Cys 81 and Gly-Gly-Gly-Cys 82.

I.4.2 Reaction of aminoacetonitrile with Cys-Gly

When Cys-Gly 83 was used, the tripeptide Gly-Cys-Gly 85 was obtained with a very good conversion (Scheme 30).

Scheme 30

I.4.3 Competition between cysteine and Cys-Gly

We have also studied the competition between Cys-Gly dipeptide and cysteine itself. The competition between these two aminothiols was followed by 1 H-NMR (Figure 35). It appeared that the difference between reaction rates toward dipeptide formation was small. Indeed, the figure shows that the reaction with Cys-Gly was slightly faster than that one with cysteine.

Figure 35: (Conversion = consumption of starting materials, forming thiazoline and the amide)

This shows that the acid function of cysteine can be replaced by a secondary amide. In other words, any longer peptide with an available aminothiol function at one of its NH2-ends would react with Gly-CN (or any other α-aminonitrile).

These results confirm our suggestion that longer peptides ending with an aminonitrile would react with cysteine (or any peptidic chain starting with aminothiol). Furthermore, this reaction would link two peptidic chains together if they meet the needed requirements (i.e. NH2-end of one chain is an aminothiols, the CO2H-end of the other chain is replaced by CN) (Figure 36).

Figure 36: Connecting two peptidic chains together via thiazoline ring formation

I.5 Reactions with cysteine amide

In common with other amino acids, cysteine is a polar water-soluble molecule. Consequently, reactions between cysteine and aminonitriles lead to highly polar products, which are also soluble in water. This makes it difficult to purify our products by the common chromatography column method (in normal phase). For this reason, we decided to test reactions with cysteine amide. In this case, we expected the isolation of products to be an easy process.

We used two methods to synthesize cysteine amide 86. The first (and simplest) method was a one-step method starting from cysteine ethyl ester. It was enough to mix the ester with a 26 % ammonia solution for one day to obtain the amide with a good "yield" (Scheme 31).

Scheme 31

In spite of the apparent good "yield", the product was not pure enough and contained some remaining starting material that hydrolysed to give cysteine, and cystine. From this mixture we were not able to obtain a "good-quality" cysteine amide. For this reason, we used an alternative three-step method to obtain it. Starting from Fmoc-Cys(Trt)-OH, we obtained the amide 87. Then, we cleaved the Fmoc group using piperidine to provide 88 and finally, the trityl group was removed by TFA to provide the desired amide as a TFA salt 86 (Scheme 32).

Scheme 32

The second "multistep" method provided the amide in high purity, and thus it was used to obtain cysteine amide that we used in our reactions.

I.5.1 Reaction of cysteine amide with aminoacetonitrile

As an aminothiol, cysteine amide reacted with aminoacetonitrile in the same manner as cysteine.

First, with the aim of studying the selectivity between these two aminothiols, we added aminoacetonitrile, as an example of α-aminonitrile, to an equimolar mixture of cysteine and cysteine amide 86.

This competition reaction was performed in D2O at 45 °C and followed by 1 H-NMR. The aminonitrile was totally consumed after 26 hours. We found that both candidates reacted in similar rates, indicating that the presence of the amide group, in place of the acid, has no significant effect on the reaction (Figure 37).

I.5.2 Reaction of cysteine amide with aminoacetonitrile

As reported before, both methionine and valine nitriles reacted with cysteine and the expected thiazolines and dipeptides were identified. However, none of these products was isolated.

From this perspective, we repeated these reactions with cysteine amide. In this case, we perform the reactions in slightly basic water solutions (pH 8) in order to guarantee the formation and stability of heterocyclic rings. Using these conditions, starting with Lmethionine nitrile used in an enantiopure form, we were able to isolate the corresponding thiazoline 90, but only in 16 % yield (Scheme 33).

Scheme 33

We also used Val-CN 2 (racemic mixture). In this case, a one to one mixture of diastereoisomers of the thiazoline 92 was isolated (30 % yield) (Scheme 34).

Scheme 34

Both isolated yields are deceptive  : we believed that this is due to some degradation of products on silica.

It should be noticed that the hydrolysis of the valine derived thiazoline 92 to obtain the dipeptide 93 was found to be very slow process, indeed much slower than the hydrolysis of the methionine derivative 90 (and of the simplest glycine derivative 46). This is probably due to the presence of the bulky isopropyl group in 92.

 90 and 92 were isolated as free amines.

I.6 Reaction of aminoacetonitrile with cysteamine

We also tested the reaction of α-aminoacetonitrile 39 with cysteamine 94, the simplest stable aminothiol. In this case, the corresponding thiazoline ring 95 should be formed and be hydrolysed to give the amide 96 (scheme 35).

Scheme 35

As we did with cysteine amide, we studied the competition between cysteamine 94 and cysteine. We introduced both aminothiols in a D2O solution containing aminoacetonitrile 39, and followed the reaction by 1 H-NMR (Figure 38). The results did not show a big difference between both competitors toward thiazoline and amide formation. Even though cysteine reacted slightly faster than cysteamine, the result indicates once again (as with cysteine amide) that the presence of the carboxyl group in aminothiols is not compulsory for the formation of thiol-containing dipeptides. 36). In this case, the reaction was very slow, probably because of the bulkiness of the two methyl substituents.

Scheme 36

Furthermore, the thiazoline was not seen on the 1 H-NMR and the only detected product was the final dipeptide (Figure 39). This might be due to the electron donating property of the methyl groups, making the nitrogen atom of the intermediate thiazoline ring more basic.

Protonation of this nitrogen atom would thus be easier, hence the hydrolysis step quicker.

I.8 Theoretical calculations

In order to explain the observed selectivity, Anne-Milet (from theoretical chemistry team in our lab) calculated the level of the π* orbital of a series of nitriles (Table 1).

As shown in the table, the lowest calculated orbital was that of the protonated form of aminoacetonitrile 39. Such level would explain its greater reactivity compared to other nitriles. The non-protonated form of aminoacetonitrile 39 is predicted to be much less reactive and so is probably not involved in the reaction mechanism.

The π* orbital of β-aminopropionitrile 73 is higher (it is less reactive) and the simplest aminonitrile 102 is predicted to be even less reactive (no reaction from glutamic nitrile 35). In contrast, α-substitution of aminoacetonitrile, as in α-aminopropionitrile 101, should not alter significantly its reactivity (Table 1

I.9 Observation of a catalytic behaviour of aa-Cys and aa-Hcy dipeptides

On the basis of our experiments, we propose that aa-Cys and aa-Hcy dipeptides were overrepresented in the primitive ocean (compared to non-thiol containing dipeptides). These dipeptides are thiols and as such could be major players in a "thioester world". 109 Indeed, when we mixed Gly-Cys 47 with an excess of aminoacetonitrile 39 in D2O solution at 45°C, a peak was observed at 194.16 ppm in the [START_REF] Sutherland | Opinion: Studies on the Origin of Lifethe End of the Beginning[END_REF] C NMR of the reaction mixture. Such chemical shift is characteristic of the thioester function (Figure 41). Thioester 104 was observed also in 1 H-NMR (Figure 40). We believe that it belongs to compound 104. We also noticed the formation of glycine amide 105. These products would derive from the first formed C=N double-bonded addition product 106. The thioester was partly hydrolysed to give glycine 107. We were also able to characterize among the reaction products the amidonitrile Gly-GlyCN 42 ( 13 C NMR: 27.58,40.34,116.74,167.58 ppm), meaning that the thioester reacted with the non-protonated amino group of GlyCN 39 (which is possible because of the low pKa of GlyCN: 5.55). 157 For instance, in an experiment in which we used globally 4 eq. of GlyCN 39 (relative to cysteine), after 2 days at 45°C, the observed GlyOH 107/GlyNH2 105/GlyGlyCN 42 ratio was found to be 21/37/42. This demonstrates that Gly-Cys 47 is able to promote the formation of a peptide bond from a nitrile (Figure 40). Similar results were obtained from Gly-Hcy 53. Peaks corresponding to at least two products were detected near 135-140 ppm. They might correspond to two different imidazoles (named Im1 and Im2). 115: the homocysteine thioester analogue of 104. Big peaks at 166. 65 (166.66) and 176.07 (178.33) belong to 47 (and 53).

One peak of both 104 and 115 sticks to the foot of the 166.6 ppm peak of 47 and 53

The formation of other products was also evidenced in the reaction of cysteine with excess GlyCN 39. In the [START_REF] Sutherland | Opinion: Studies on the Origin of Lifethe End of the Beginning[END_REF] C NMR spectra peaks were observed at 130-140 ppm (Figure 41).

The mass spectrum of a reaction mixture in water also showed the formation of various products (Figure 42). This mass spectrum first confirmed the presence of Gly-GlyCN 42 (protonated, found 114.0656, calcd 114.0667). Another mass was detected at 113.0817. It could be attributed to the amidine 108 (calcd 113.0817). However, in accordance with the observed [START_REF] Sutherland | Opinion: Studies on the Origin of Lifethe End of the Beginning[END_REF] C NMR of the mixture, we propose than this amidine 108 cyclized and that this mass peak should be attributed to the imidazole 110. Indeed, the cyclization of an amidinonitrile similar to 108 into an aminoimidazole (5-amino-2-methyl-1H-imidazole) has already been reported. 158 At least some of the other products observed in the mass spectrum would be evolution products of 110. For instance, this imidazole could loss ammonia to give the stabilized cation 111 that would in turn react with 110 (in its free amine form) to yield the bis-imidazole 112 (M+H, found 208.1300, calcd 208.1310). 110 itself could react with thioester 104 to give the amide 113 (or 114) (Figure 43). Similar results were obtained in case of addition of 39 to 53. In this case, the thioester 115 was identified. In order to further ascertain the cyclisation of the imino-compound 108 Anne-Milet, once again, calculated its stability in comparison to cyclized forms. We used the strategy described previously for table 1 results, with the 6-31+G(d,p) basis set (Fig. 11). Not surprisingly 110 was calculated to have free enthalpy 12.7 kcal/mol lower than 108 and 6.9 kcal/mol more stable than 109.

Interestingly, it was found that the dissociation of 110 into 111 + NH3 cost only around 7 kcal/mol. The ∆H of dissociation is nearly 19 kcal/mol (18.67 kcal/mol) but due to the dissociative character of the process, the ∆G value drops to 7.01 kcal/mol. This process does not show a well-defined TS. Thus we think that the formation of cation 111 proposed in (Figure 44) is a plausible event.

We studied more precisely the cyclisation step from 108 to 109 (Figure 45). According to our hypothesis, aa-Hcy dipeptides could have been easily formed in the primitive ocean. The presence of homocysteine was highly probable due to the large amounts of H2S blown up from volcanoes and therefore the needed mercapto-aldehyde precursor 116 could be formed from acrolein and would lead to homocysteine in the presence of ammonia and hydrogen cyanide via Strecker reaction. Furthermore, acrolein is a product of an electric spark on a mixture of methane and water (Scheme 37). 61

Scheme 37

However, 3-mercaptopropanal (3-sulfanylpropanal) is known to be very difficult to isolate because of autocondensation reactions. Thus, we synthesized homocysteine nitrile (Hcy-CN), in a non-prebiotic way in order to study the behavior that it might have had under primitive conditions.

II.2 Synthesis of homocysteine nitrile

We started the synthesis from acrolein and triphenylmethanethiol to obtain 3tritylsulfanylpropanal 117. 159 The aldehyde 117 was isolated and submitted to a modified Strecker condensation. 160 The last step was deprotection of the Trt group under acidic conditions to obtain Hcy-CN 120, which was finally recovered in D2O solution as a salt with trifluoroacetic acid (Scheme 38).

Scheme 38

Traditional Strecker reaction has been also tried to obtain the product 119. However, a considerable amount of cyanohydrin 121 was obtained in this case (Scheme 39).

Scheme 39

II.3 Homocysteine nitrile behaviour in water

Hcy-CN 120 was stable under strong acidic conditions. No hydrolysis was observed. It could be expected that it would be able to polymerize in a similar way to Cys-CN under neutral pH (see chapter 3). However, Hcy-CN 120 showed a completely different behaviour. Instead of polymerization, it cyclized to form homocysteine thiolactone 54 (Figure 46). primitive ocean, and it can then be engaged in our scenario.

II.4 Prebiotic behaviour of Hcy-thiolactone

It has been reported in the literature that, under slightly basic conditions, Hcy-thiolactone undergoes a dimerization reaction to form Hcy-Hcy cyclic dipeptide 122, the diketopiperazine of homocysteine, or disulfide polymers 123 depending on the used conditions (Figure 48). 161 We found that Hcy-thiolactone 54 is stable in slightly acidic water at 45 °C for a long time.

However, we wanted to make sure that no hydrolysis happened during this reaction. If this was the case, homocysteine, which is the product of the hydrolysis of thiolactone 54, is expected to react with aminoacetonitrile to give dihydrothiazine cycle 52 (see chapter 1). We wanted to confirm that the observed cycle in the previous NMR is not the cycle 52. According to these results, it can be postulated that Hcy-thiolactone 54 can be considered as a molecular "staple" which would permit the connection of two peptide chains (one containing a nitrile and the other containing an amine) together (Figure 52).

II.5 Aminoacyl transfer via a thioester

The reactivity of the obtained tripeptide 126 has been studied. Similarly to Gly-Cys 47 and Gly-Hcy 53, Gly-Hcy-Gly-CN 126 is a thiol-containing small peptide and can be expected to give thioesters when reacted with nitriles. From the obtained [START_REF] Sutherland | Opinion: Studies on the Origin of Lifethe End of the Beginning[END_REF] C-NMR of the mixture, we were able to detect the formation of a thioester, most probably compound 132.

Similarly to the previous examples, we studied the reactivity of this thioester 132. In this case, thioester 132 reacted with the amino group of the starting thiolactone 54 to give Gly-Hcy-thiolactone 133 which was the dominant product resulting from the thioester (Figure 53). This is our second example of an "aminoacyl" transfer via a thioester (after Gly-Gly-CN 42 formation). Indeed, Gly-CN 39 and Hcy-thiolactone 54 have similar pKa's (near 6), so that a significant amount of free amines are present in both cases under our conditions.

However, the expected Gly-GlyCN 42 was not observed in this reaction mixture but only the thiolactone 133.

Figure 53: "Aminoacyl" transfer via a thioester

This result gives extra evidence that our thiol-containing peptides are able to promote the formation of peptide bonds via thioester bond formation and thus might have played a role in a "thioester world".

We synthesized an authentic sample of Gly-Hcy-thiolactone 133 in order to compare its NMR spectra with the spectra of our prebiotic mixtures (Scheme 41).

Scheme 41

The reactivity of the obtained Gly-Hcy-thiolactone 133 has also been studied. As the amine of Hcy thiolactone is poorly basic, we postulated that this amine could act as leaving group

(which is of course unusual for amines). If so, 133 would be an activated form of Glycine and therefore, for instance, Gly-Cys would be formed from it in the presence of cysteine. In order to test this idea, cysteine was added to a pure sample of this thiolactone in D2O. However, no

Gly-Cys was formed.

II.6 Formation of bis-thiol tetrapeptides

The reactivity of the obtained nitriles 125 and 126 towards aminothiols group has been studied. It has been found that upon the addition of cysteine to a mixture of these nitriles, the bis-thiol tetrapeptide Gly-Hcy-Gly-Cys 136 was formed. It has been characterized by 1 H-NMR (Figure 54). When both nitriles were present, the thiazine-containing tripeptide 125 was found to be more reactive toward the formation of tetrapeptide 136 than the hydrolysed one 126.

Figure 54: Prebiotic formation of bis-thiol tetrapeptide Gly-Hcy-Gly-Cys 136

Similarly, Gly-Hcy-Gly-Hcy 138 was formed when Hcy was added (Figure 55).

Figure 55: Prebiotic formation of bis-thiol tetrapeptide Gly-Hcy-Gly-Hcy 138

After some days, these bis-thiols were oxidized to form intramolecular sulfur bridges (Figure 56) However, when we analysed our disulfide samples by high resolution mass spectrometry (HMRS), we found that the detected exact masses were not those of the expected acids but those of the corresponding amides (Figure 56).

Figure 56: Intramolecular (S-S) bond formation

We have no definitive explanation for this unexpected result. Indeed, the acid to amide transformation necessitate an activation of the acid. A very tentative mechanism is presented in the following scheme (given from Gly-Hcy-Gly-Hcy as an example) (Figure 57). Even more cysteine residues are found in proteins. For instance, metallothioneins are small proteins containing about 60 residues of which up to 20 are cysteine units units. 164 They can complex zinc cations, and other metals such as copper and cadmium. 165,166 The presence of cysteine in antioxidant glutathione and its use in the synthesis of coenzyme A are other examples of the widespread scope of this sulfur amino acid. 132 Prebiotic synthesis of cysteine is not so obvious. Still, two plausible pathways starting from glycine might have participated in this synthesis (Figure 58). We have previously argued that cysteine residues in peptides may have participated in important catalytic sites when life appeared on Earth. The peptide richest in cysteine is polycysteine, Cysn. 167 In this chapter, we present our results about synthetic ways to form this polymer. Of course, it is hard to believe that long chain polycysteines were formed in the prebiotic ocean. However, the presence of many cysteine residues in more diversified proteins are plausible, and the regular cysteine polymer can be regarded as a simplified (even though longer) model for such proteins.

To form polycysteine under plausible prebiotic conditions, in water, we needed activated cysteine derivatives. We choose to synthesize nitrile 144, thioacid 145 and three thioesters 146, 147 and 148 as plausible models that might be presented in the ocean 4 billion years ago (Figure 60).

III.2.1 Synthesis of cysteine nitrile 144

We first proposed a pathway toward the synthesis of cysteine nitrile starting from Boc-Cys(Trt)-OH as described in (Scheme 42). The advantage of starting from this protected cysteine is that the removal of both Boc group on the amine and the Trt group on the thiol would be performed in a single step. We successfully obtained the amide 149, then the nitrile 150, with good yields. TFA was added to 150 and this deprotection process was followed by extraction in D2O. However, the last step did not provide cysteine nitrile 144 as we expected.

Instead, a mixture was obtained. The 13 C-NMR spectra of this mixture allowed us to identify the cyclic nitrile 151 beside its hydrolysis product 152 (Scheme 42). Upon time, the nitrile 151 disappeared and the acid 152 became dominant in the mixture.

Scheme 42

This result was attributed to the removal of the trityl group before Boc leading to molecule 153. The free thiol in 153 was able to react on the carbamate group and cyclized rapidly to give thiocarbamate 151, which hydrolysed fast to obtain the corresponding acid 152 (Figure 61).

Scheme 43

III.2.2 Synthesis of cysteine thioacid 145

We were also interested in the study of the behaviour of the thioacid of cysteine 145. To synthesize it, we started from Boc,Trt-protected cysteine. The acid was first activated with DCC, and then triphenylmethanethiol was introduced to provide the thioester 157. Finally, the product 145 containing the free amine, thiol, and thioacid functions was obtained in a single deprotection step (Scheme 44). In this case, no hydrolysis to thiocarbamate cycle was noticed.

Scheme 44

III.2.3 Synthesis of cysteine ethyl thioester 146

In order to obtain cysteine ethyl thioester, Boc-Cys(Trt)-OH was activated as a mixed anhydride and ethanethiol was then introduced to provide Boc-Cys(Trt)-SEt 158. Then, the product 146 was formed in a single deprotection step in which protecting groups on cysteine were successfully removed without damaging the thioester group (Scheme 45).

Scheme 45

However, in this case a by-product was formed: the ester 159, whose formation is explained in figure 62. The Rf, rates of flow on TLC paper, for product 158 was very close to that of this ester. For this reason, we were not able to obtain the product 158 in high purity. Indeed, having such an impurity in our "prebiotic soup" should not be a problem, as the ester 159 will not polymerize. However, we tried to overcome this difficulty by repeating the reaction with the use of excess amount of ethanethiol for a short duration (15 minutes); however, similar results were obtained, that is to say, the alcohol always reacted with the thioester.

Figure 62: Formation of the undesired ester 159

In order to obtain the thioester 158 in pure form, we intended to avoid the formation of isobutyl alcohol. To do so, we activated the acid using DCC with catalytic amount of DMAP.

This way, the thioester 158 was obtained in a good yield and a good purity (Scheme 46).

Scheme 46

III.2.4 Synthesis of cysteine propyl thioester 147, and cysteine benzyl thioester 148

The same method was used to synthesize propyl thioester 147 and benzyl thioester 148 in a high yield. Despite the fact that cysteine benzyl thioester 148 can hardly be regarded as a primitive component, we considered it as an additional example that might lead to polycysteine formation in water. Both protecting groups (Boc and Trt) were removed in one step to provide the final cysteine thioesters 147 and 148 (Scheme 47).

Scheme 47

III.3 Formation of polycysteine

III.3.1 Starting from cysteine nitrile

In the first chapter, we have discussed the possibility to form a dipeptide between two amino acid precursors in which one holds a nitrile group and the other has an aminothiol moiety.

Cysteine nitrile contains both nitrile and aminothiol functions, it should be able to polymerize. The nitrile precursor of cysteine is also interesting as it is a plausible interstellar molecule. [START_REF] Knowles | Radical Formation of Amino Acid Precursors in Interstellar Regions? Ser, Cys and Asp[END_REF] Indeed, if Cys-CN 144 was found to be stable when it was kept in acidic water (pH 1), the formation of its dimer 162 was observed as soon as pH was fixed at 5 -6. Another product was observed to which we tentatively assigned a trimeric structure 163, and after few hours, a non-soluble white polymer precipitated out of the solution (Scheme 48).

Scheme 48

These results were first observed by 1 In this experiment, we were not able to characterize the dimer 165 with two adjacent thiazolines. We believe that it was not formed. The results indicated that the thiazoline of the dimer 162 was first hydrolysed to give the dimer 164 before it reacted with another Cys-CN unit (at least in this experiment) (Figure 65). The reaction did not stop at this level of oligomerization. The monomer, dimer, and trimer, all contain nitrile and aminothiol moieties and thus reacted further to give longer chains.

These longer chains were not soluble in D2O and thus started to precipitate.

We wanted to study this polymer using MALDI-TOF mass spectrometry. So, we repeated the reaction in degassed (to avoid as much as possible any disulfide bridge formation) H2O (to avoid any deuteration). In order to prepare the sample for mass analysis, the precipitate was washed several times with water to remove traces of TFA, before drying. The obtained solid was then dissolved in DMSO and a MALDI analysis was undertaken.

The expected polycysteine masses should give values that are separated by 103, the mass of a cysteine residue. However, the observed results did not agree we our expectation, the difference between the observed masses being 192, so much higher than 103 (without being 206, which would correspond to 2 Cys residues)

Believing that the polymer consisted of polycysteine assemblies, we tried to interpret our results by proposing some plausible structures, the question being: what is this the mass 192 related to? This value could be explained starting with two thiazole rings, hence two cysteine units, with mass equal to 170. These "dimers" could lose a proton and each formed anion would neutralized by a sodium cation: 170 -1 + 23 = 192.

Therefore, we propose that the observed masses may correspond to the chelation of polythiazoline rings with sodium cations in which each ion chelates with two cycles (Figure 66). The presence of conjugated thiazoline rings would help the formation of this repetitive Indeed, some ring hydrolysis might have occurred, then some thiols could have been oxidized

to disufides, facts that would explain some other masses that we observed.

Polythiazoline structures have already been described in the literature. For example, linear polythiazolines were obtained form polythioureas. 168 Cyclic oligothiazolines were formed by the head-to-tail cyclooligomerization of doubly deprotected linear fragments. 169 Moreover, poly(thiazole) amino acids , which are not far from our rings, are found in nature. The biosynthesis of thiazole itself can be achieved through cyclo-dehydration of cysteine moiety of cysteine-containing peptides followed by an oxidation step, hence via thiazoline structures (Figure 67). 170

III.3.2 Starting from thioacid

Like any other proteinogenic amino acid, cysteine alone does not significantly oligomerize in water. This can be explain by the fact that the hydroxyl group is a bad leaving group. In contrast, the sulfhydryl group is a good leaving group. Thus, we expected that replacing cysteine by its thioacid analogue would allow an easy polycysteine formation.

However, in contrast with what was observed for cysteine nitrile, we found that cysteine thioacid 145 is not stable in strong acidic conditions (pH 1). Under these conditions the thioacid was completely hydrolysed into cysteine within few hours (Scheme 49).

Scheme 49

To avoid this hydrolysis we extracted a newly formed thioacid 145 in D2O and quickly added sodium carbonate to reach pH 7. Under these neutral conditions, the hydrolysis process was slower, but another product was formed to which we assigned the cyclic structure 152 (Figure 68). Indeed, we did not expect to obtain such structure which is not formed when carbonate and cysteine are mixed. Thus, we believe that the presence of hydrosulfide ions, which were released in the solution upon thioacid hydrolysis, promoted the observed process (Figure 69 a). A plausible mechanism is presented in (Figure 69-b). We assume that some thiocarbonate and/or dithiocarbonate ions were formed via the reaction of carbonate ions with HS -. These thio-and dithio-carbonic acid ions (or some O=C=S resulting from their decomposition) then reacted with the thiol and amine functions of cysteine to give the cyclic thiocarbamate 152.

III.3.3 Starting from cysteine thioesters

Thioesters are well-known activated forms of amino acids. A thioester function would react with an aminothiol nucleophile to give a peptide bond. Most probably, the thiol group would start the nucleophilic substitution process on the thioester, and then the amine reacts to form the peptide bond (Figure 70). This process is used in the native ligation procedure to synthesize polypeptides. 171 Starting from any thioester of cysteine, polycysteine should be formed. For this reason, we synthesized ethyl, propyl and benzyl thioesters. Unlike cysteine nitriles, no thiazoline rings are expected in this case. The predicted masses for the polymer should be separated by one cysteine unit (103). Similarly to cysteine nitrile, cysteine ethyl thioester was stable in acidic D2O. When we changed the pH to slightly acidic (around 6), we noticed on [START_REF] Sutherland | Opinion: Studies on the Origin of Lifethe End of the Beginning[END_REF] C-NMR the disappearance of the starting thioester 146, and the appearance of new C=O peaks, both amides and thioesters.

After few hours, the solution in the NMR tube became turbid, indicating the formation of an insoluble polymer, most probably polycysteine. We repeated this process in water in which the precipitated solid was recovered after 24 hours for mass spectrometry analysis (Scheme 50).

Scheme 50

In accordance with our expectations, MALDI analysis showed nice series of masses corresponding to cysteine polymer, clearly separated by 103 (Figure 71). This time, only single peaks were observed due to the absence of thiazoline rings.

Figure 71: MALDI MS-spectrometry of polycysteine

The obtained masses may correspond to cyclic polycysteine structures. For example, the observed peak 2493.134 matches with 169, (Cys)24 (calculated exact mass = 2495.210). The observed difference between these values can be attributed to the presence of one or two disulfide (S-S) bonds (Figure 72). There is no doubt that the formation of small cyclic structures is possible. However, we believe that the presence of "big" macrocyclic polycysteine structures is uncertain. Therefore, we presented an alternative possible structure for the observed "bigger" masses. We proposed that small cyclic structures, considering that they have free thiols, might form thioester bonds.

For instance, the previous value (2493.134) may correspond to 170, a cyclo(Cys)5 in which each thiol holds various cysteine chains via a thioester bond (Figure 73).

Of course, each mass may correspond to several possible (but similar) structures. That it so say, one cyclic polycysteine, or a cycle that link with other cysteine residues.

III.3.3.b Cysteine propyl and cysteine benzyl thioesters

Cysteine propyl thioester 147 was also stable in acidic water. Polycysteine started to precipitate out of the solution around neutral pH at room temperature (Scheme 51). However, MALDI analysis did not give sufficient results in this case. This might be because of very low concentrations for example.

Scheme 51

When benzyl thioester 148 was synthesized and kept in water under our prebiotic conditions, some turbidity was observed in the mixture indicating the formation of insoluble products (Scheme 52). However, the precipitation process was slow in this case. This might be due to the low solubility of benzyl containing thioester in water. Finally, we observed a milky emulsion-like product which was hard to recover. As a result, MALDI analysis were not sufficient in this case. However, benzyl thiol "by-product" was detected when the mixture was extracted with DCM. This confirms that some polymers were certainly formed.

Scheme 52

Chapter IV: Prebiotic formation of thiol-rich polypeptides starting from aminothiolcontaining amidonitriles

IV.1 Introduction

In the previous chapter, we have reported that cysteine nitrile was stable under strong acidic conditions, and that it started to polymerize in slightly acidic water (pH 5 -6). After complete hydrolysis of thiazoline rings, the final polymer in this case would consist exclusively of cysteine residues. As we discussed before, the formation of this polymer, (Cys)n cannot be regarded as probable in the primitive ocean, as it would have required a pool with very high cysteine nitrile concentrations. However, it would be more reasonable to imagine that thiol rich (but not the richest) peptides might have formed in the primitive ocean and, if we are true, might have engaged in the process of appearance of life. Our idea was to synthesize aminonitrile models that would polymerize and lead to the formation of polycysteine units separated by one (or more) amino acids, that is to say, poly(Cys-aa) or poly(Cys-aa1-aa2). If such polymerizations happen to work, then it will be reasonable to propose that thiazoline formation from longer Cys-terminated amidonitriles permitted the synthesis of long thiol-containing peptides, with a "reasonable" number of cysteine residues.

For this project, we needed to synthesize Cys-aa-CN (and Cys-aa1-aa2-CN) samples. We expected these nitriles to have a behaviour similar to that found with cysteine nitrile, so that we predicted that linear (and/or cyclic) peptides would form from them in slightly acidic or neutral water (Figure 74).

Figure 74: Aminothiol-containing amidonitriles are plausible precursors for thiol-rich peptides

We chose Cys-Gly-CN, Cys-Ala-CN, Cys-Val-CN and Cys-Met-CN as models to obtain peptidic chains. Furthermore, as we aimed to study the possibility to obtain polycysteine units that would be separated by two amino acids, we also decided to synthesize the simplest candidate, Cys-Gly-Gly-CN.

In this work, we will use amidonitriles produced from classical chemical synthesis. However, we believe that it is reasonable to propose that they may have been present in the primitive ocean, a prerequisite. As an example, a possible prebiotic synthesis of Cys-Gly-CN is presented in figure 75. First, cysteine could be activated as a thioester, which would condense with GlyCN to provide Cys-Gly-CN 171.

Scheme 54

IV.2.3 Synthesis of Cys-Gly-Gly-CN

We first obtained Gly-Gly-OMe 191 by treating diglycine with thionyl chloride in methanol (Scheme 55). 172 This ester was used to introduce the Gly-Gly moiety onto cysteine.

Scheme 55

In this synthesis, the treatment of Boc-Cys(Trt)-OH with IBCF and NMM was followed by Cys-Gly-CN 171 was found to be stable for a long period in acidic water (pH 1). When the pH was nearly neutralized (pH 6.5) by adding sodium bicarbonate, the solution started to become turbid indicating the formation of polymers (Scheme 57)

Scheme 57

Few hours later, a white solid started to precipitate at the bottom of the flask. After one night, the white thin layer that precipitated was recovered and dried. Figure 76 shows the observed solution during the polymerization process. Unfortunately, the expected masses were not observed in MALDI analysis. This might be attributed to the poor solubility of the solid in DMSO. This is one reason for which we decided to introduce amino acids other than glycine. These amino acids contain other "more organic" substituents that we expected to increase the solubility of our polymers in organic solvents and would lead to better results.

IV.3.2 Formation of poly(Cys-Ala)

After deprotection of the nitrile 185, Cys-Ala-CN 188 wat extracted as TFA salt in water. We found that it was stable in acidic water for hours. When the pH was neutralized (6.5 -7) using sodium bicarbonate, the clear water solution started to become turbid and within few hours, a very thin layer of a white solid started to precipitate indicating the formation of poly(Cys-Ala) (Scheme 58).

Scheme 58

We first run this reaction in D2O that was neutralized and left unstirred. The solution was left unstirred for overnight, then the solid was recovered and subjected to MALDI-TOF analysis. In order to prepare the matrix for MALDI analysis, 10 % of acetonitrile was added to DHB solution in DMSO. This solution was mixed with our polymer (dissolved in DMSO) and this mixture was dropped on the plate and introduced to MALDI.

The obtained spectrum was rather complex. It did not show the expected peptides as a series of single masses (a mass for each (Cys-Ala)n), but for each given peptide length, a set of CH's thiazolines CH 2 's thiazolines H 2 O CH 188 CH masses was observed (Figure 78). These masses were attributed to the presence of a varied number of hydrolysed and non-hydrolysed thiazoline rings in each chain.

Figure 78: MALDI MS-spectrometry of poly(Cys-Ala)

For example, the masses around 1152 correspond to seven "monomers" (= Cys-Ala) ending with a nitrile (observed as [M+Na] + ). Focusing on this region, seven single masses were observed. The highest number of possible thiazoline rings in the case of (Cys-Met)7-CN is six rings, which corresponded to a calculated mass 1134.4598 (observed 1134.965). With less rings, higher masses are expected, the difference between two peaks being 18.

Figure 79: Peaks corresponding to (Cys-Met)7-CN on MALDI spectrometry

For instance, taking the peak of 1184.979, this mass corresponds to a (Cys-Met)7-CN in which three thiazoline rings were hydrolysed (and thus three rings still present). An example of such structure is presented in (Figure 79). Of course, we cannot determine the precise place of thiazoline rings in the chain. Other structures are possible, so that the observed mass must correspond to a mixture of oligomers. What is sure is that all these structures are heptamers of CysGly, i.e. chains containing 14 amino acids 197 (Figure 80).

IV.3.3 Formation of poly(Cys-Met)

Cys-Met-CN 190 solution started to become turbid at neutral pH, indicating the formation of insoluble polymers (Scheme 59). After leaving the solution at room temperature without stirring for overnight, a thin layer of white solid was observed at the bottom of the container.

This solid was recovered and subjected to MALDI spectrometry (in DMSO solution)

Scheme 59

Several polypeptide chains of various length (up to 25 chains, i.e. 50 amino acids) were observed. Just like in the case of (Cys-Ala)n, we observed a set of masses for each peptide length. Here also, various numbers of thiazoline rings remained in the formed chain (Figure 81).

Figure 81: MALDI MS-Spectrometry for poly(Cys-Met)

Taking (Cys-Met)14-CN as an example, several masses were detected (Figure 81). The observed masses started with 3068.278; this value indicates the presence of 13 thiazoline rings (the maximum number of rings possible for 14 "monomeric" Cys-Met). This mass is followed by 3091.303 (12 thiazoline rings) and so on.

It should be noticed that, contrarily to the previous example for which [M+Na] + masses were observed, in this case [M+H] + masses were detected. However, some odd single peaks were also observed on the MALDI. As an example, the peak at 1081.959 shown in figure 84 does not seem to fit with the observed series of masses.

We were not able to relate this mass a peptidic chain consisting of thiazoline rings and amides. Thus, another structure should be proposed in this case. A list of macrocycles that we observed is presented in table 4. Masses presented in table 4 are average masses. To confirm the proposed structures, we recorded their high resolution mass spectra (HRMS-SEI). As shown in figure 86, the exact masses agree with the proposed thiazoline-containing cyclopeptides. However, the quantities of cyclo(CysMet)7 and cyclo(CysMet)8 in the mixture of polymers were not sufficient to allow the measurement of their exact mass.

No

IV.3.4 Formation of poly(Cys-Val)

Cys-Val-CN 189 was found to be stable under acidic conditions and its solution started to become turbid at neutral pH, indicating the formation of insoluble polymers (Scheme 60).

Scheme 60

We first tried the reaction in D2O and followed it by NMR spectroscopy. After one hour, the spectra showed the formation of the early stages of the first oligomers (dimers, trimers,…) (Figure 87). After few hours, the polymerization process continued and eventually led to a polymer that was not soluble in D2O and precipitated at the bottom of the NMR tube.

Figure 87: 1 H-NMR showing the beginning of Cys-Val-CN polymerization

We repeated this reaction in H2O and left the solution at room temperature without stirring.

The obtained solid was subjected to MALDI spectrometry.

Several polypeptide chains of various length (up to 30 chains, i.e. 60 amino acids) were observed. Sets of peaks were also observed in the mass spectrometry analysis indicating the presence of various numbers thiazoline rings (Figure 88).

-CN

CH's thiazolines CH2's thiazolines

CHVal CHCys

Figure 88: MALDI MS-Spectrometry of poly(Cys-Val)

We noticed that the farther we go in MALDI spectra, the less precise values we observed.

That is to say, in higher masses, the values had some little deviations from the expected ones.

This observation can be attributed to the formation of disulfide bridges (inter-or intramolecular). If so, it is logic to have more disulfides in the case of higher masses.

Examples of some observed values are given in table 5. Similarly to what we observed in the case of poly(Cys-Met), cyclic structures were observed.

No. of monomers

These cycles containing thiazolines were identified precisely by means of high resolution mass spectrometry (HRMS-SEI) (Figure 89). In this example, we decided to try MS/MS experiments, that were performed by Maxime Bridoux (CEA -Centre DAM -Île de France), in order to detect the expected fragmentations of these cyclic structures. For example, when the peak of the cycle 205 (553) was separated and submitted to further energy (collision energy 15), the expected fragments were observed (Figure 90). Once again, confirming the proposed structures. Similar spectra were obtained with other macrocycles. Similarly to Cys-Gly-CN 171, Cys-Gly-Gly-CN 196 was found to be stable in very acidic water (pH 1). After few hours at pH 6.5, poly(Cys-Gly-Gly) chains started to precipitate (Scheme 61). The recovered solid was dried and finally subjected to MALDI-TOF analysis

Scheme 61

However, the mass resulted from MALDI was not satisfactory and did not show the expected masses. This might be due to the very poor solubility of the obtained polymers in DMSO.

IV.4 First attempts to observe a catalytic behaviour of the obtained polymers

To study the catalytic effect of our polymers in the formation of peptide bonds, poly(Cys-Val) was used as an example. A small amount of poly(Cys-Val) was put in D2O. DCl was added to the obtained suspension in order to promote the hydrolysis of thiazoline rings in peptide chains and the mixture was heated at 45 °C for 24 hours, before it was divided in six NMR tubes. In each tube, we added a "monomer". We used Glycine, glycine amide, aminoacetonitrile, serine, cysteine and homocysteine. The six tubes were left at room temperature and the evolution of each mixture was followed by NMR spectroscopy.

Expected products were oligopeptides (at least dipeptides), but we did not observe any evolution product even after two months.

In these first experiments, the complete lack of catalytic effect (especially with aminoacetonitrile 39, that we know that it forms GlyGlyCN 42 in the presence of thiols) might be due to the low solubility of long thiol-containing peptides. Another reason could be that thiazoline derived from valine were too stable, so that we had only a small amount of "free" thiol functions. Such high stability was observed in the case of the thiazoline of Val-Cys-NH2 92, in which the hydrolysis step was very slow. Thus, poly(Cys-Val) was probably not the best polymer to test. Such experiments should be repeated with other polymers.

Conclusion

Our research focuses on the prebiotic synthesis of peptides, especially thiol-rich ones. This research support the metabolism first hypothesis, an alternative proposal to the RNA world hypothesis. It is related to de Duve's thioester world, and to Sutherland's cyanosulfidic protometabolism. We proposed that this world, that we proposed to name "Thiol Rich Peptide (TRP) world", had appeared in primitive ocean thanks to the presence of significant amounts of HCN that permitted the synthesis of aminonitriles from aldehydes. The chemistry of this world is based on thiols and nitriles. Thiol-containing peptides would have been the most important molecules in this world.

In the first chapter, we study the reaction of aminoacetonitrile (Gly-CN) with cysteine. This reaction leads to the formation of a thiazoline cycle, which is then hydrolysed to yield the dipeptide Gly-Cys. Similarly, homocysteine gives Gly-Hcy via a 6-membered cycle. These reactions were found to work under a wide range of pH's and temperatures. However, we chose to run most of our reactions in slightly acidic water (pH 6 at 45 °C). In this way, we were able to obtain several dipeptides (aa-Cys or aa-Hcy) by introducing different aminonitriles (the first products of the Strecker reaction) (Figure 91).

Figure 91

We found that these thiol-containing dipeptides were able to form active thioester bonds that promote the formation of longer peptide chains. For instance, any dipeptide nitrile aa1-aa2-CN (the simplest example being Gly-GlyCN), produced from the reaction of H2N-aa2-CN with a thioester of aa1, would react with cysteine to give the tripeptide aa1-aa2-Cys, and with homocysteine to give aa1-aa2-Hcy. We have demonstrated that dipeptides like GlyCys are able to promote the formation of heterocycles that play an important role in today's biochemistry. This remarkable catalytic behaviour might have had a crucial role to promote chemical evolution processes.

In the second chapter, we study the behaviour of homocysteine nitrile (Hcy-CN) in water. We found that it cyclized to form homocysteine thiolactone (Hcy-thiolactone). This argues for the existence of this thiolactone in the primitive ocean. Hcy-thiolactone showed a double reactivity, thiolactone opening by amines followed by aminothiol condensation reaction with nitriles. For example, when Hcy-thiolactone reacted with aminoacetonitrile, Hcy-Gly-CN was formed. The obtained thiol-containing dipeptide reacted again with aminoacetonitrile to form Gly-Hcy-Gly-CN (Figure 92).

Figure 92

In the third chapter, we discuss the plausible prebiotic formations of polycysteine. We found that cysteine nitrile (Cys-CN) in water lead to the formation of a cysteine dimer, a trimer, and finally to the formation of a non-soluble polymer, polycysteine. This was also the case when

Cys-CN was replaced by cysteine ethyl ester, another plausible prebiotic precursor of poly(Cys) (Figure 93).

Figure 93

Finally, we discuss the behaviour of Cys-aa-CN model molecules under our prebiotic conditions in the last chapter. In this case, the corresponding polypeptides, containing up to 50 amino acid units, have been formed (Figure 94).

Figure 94

In addition to the linear polypeptides, macrocyclic polythiazolines have also been detected, that would lead to macrocyclic polypeptides after hydrolysis of thiazoline rings. We believe that such thiol-rich cycles, especially if formed from various Cys-aa-CN (an example is presented in figure 95), could have a catalytic role in the prebiotic ocean.

Figure 95

Perspectives

In this study, we have used the corresponding nitriles of glycine, methionine and valine (beside other aminonitriles) to react with aminothiols. However, using other aminonitriles (which may correspond to proteinogenic or non-proteinogenic amino acids) will lead to the formation of a wide range of thiol-containing dipeptides.

Similarly, introducing new amidonitriles (Cys-aa-CN) in water will give different polypeptides. In this part of our study, we have used one type of amidonitriles for each attempt. These attempts have led to the formation of polypeptides that were always separated by one type of repeating units. However, mixing two or more different amidonitriles together in water will probably lead to the formation of polypeptide sequences containing different amino acid residues with random (or slightly selective) arrangement. These random assemblies of amino acid residues would be closer to the real scenarios that gave rise to life.

Peptides with longer repeating units were obtained when CysGlyGly-CN monomers were used to obtain cysteine-rich peptides in which Cys residues were separated by two Gly residues. Further steps of our research will focus on the synthesis of various Cys-aa1-aa2-CN or Cys-aa1-aa2-…-aan-CN in order to obtain more complex polymers.

Even though we did not notice an obvious effect of zinc ions in our first attempts (p. 50 and p. 79), the effect of metal cations that were present in the primitive ocean cannot be totally ignored. For instance, it has been found that sodium and potassium ions have different effects on cyclic and linear peptide hydrolysis. 173 In our polymer formation experiments, replacing sodium with another metal may lead to different structures or, at least, may enhance the probability to form a given structure over the others. Beside the presence of metal cations, concentration factor of our prebiotic models in water should be taken into consideration. For instance, concentration would affect the reaction between aminothiols and aminonitriles as well as possible S-S bond forming reactions, either intra-or intermolecular. Such factor would affect, for instance, the size of the obtained cyclopeptides.

Last but not least, the reaction of aminothiols with aminonitriles can be generally used to couple any two peptides. Therefore, this chemoselective reaction can be considered as an alternative approach to chemical ligation technique in peptide synthesis.

Experimental Part

General experimental methods

The standard handling techniques under an inert atmosphere were used for sensitive air and moisture reagents. The reactions were monitored by thin layer chromatography (TLC) using commercial aluminum-backed silica gel plates (Merck, Kieselgel 60 F254). TLC spots were viewed under ultraviolet light, by heating the plate after treatment with Phosphomolybdic acid (PMA) solution or by heating the plate after treatment with Ninhydrin solution (1.5

g/100 mL n-butanol and 3 mL acetic acid). Product purifications by gravity column chromatography were performed using Macherey-Nagel silica gel 60 (70-230 mesh).

1 H-NMR and 13 C-NMR spectra were recorded on a Bruker Avance 500 ( 1 H: 500 MHz, [START_REF] Sutherland | Opinion: Studies on the Origin of Lifethe End of the Beginning[END_REF] C:

125 MHz) and 400 ( 1 H: 400 MHz, [START_REF] Sutherland | Opinion: Studies on the Origin of Lifethe End of the Beginning[END_REF] C: 100 MHz) spectrometers.

Chemical shifts for 1 H spectra are values from tetramethylsilane in CDCl3 (δ 0.00 ppm),

MeOH peak in CD3OD (δ 3.31 ppm) or relative to H2O peak in D2O (calibrated at 4.7 ppm).

Chemical shifts for [START_REF] Sutherland | Opinion: Studies on the Origin of Lifethe End of the Beginning[END_REF] C spectra are values from CDCl3 (δ 77.16 ppm), or CD3OD (δ 49.00 ppm). For MALDI analysis, DMSO was used as a solvent to dissolve the obtained polymers and DHB (2,5-Dihydroxybenzoic acid) was used as a matrix.

(S)-(9H-fluoren-9-yl)methyl (1-chloro-4-(methylthio)-1-oxobutan-2-yl)carbamate (5)

Following to the general procedure, Fmoc-L-Met-OH (540 mg, 1.45 mmol) reacted with SOCl2 (1.73 g, 14.5 mmol) to give a yellowish solid (quantitative). 75, 156.29, 143.80, 143.64, 141.43, 127.91, 127.22, 125.09, 125.09, 120.16, 68.23, 67.46, 47.20, 29.95, 19.34, 17.22.

General procedure for the preparation of the amides of Fmoc-L amino acids:

Fmoc amino acid chloride was dissolved in DCM (15 mL). The mixture was cooled to 0 °C then ammonia gas was bubbled for 1-2 min. Excess ammonia and DCM were evaporated gently under vacuum. The mixture was then quenched with water and the product was extracted in EA (3 X 20 mL), dried over Na2SO4 and concentrated under vacuum. Following to the general procedure, Fmoc-L-Val-Cl 4 (1.05 g, 2.59 mmol) reacted with ammonia. Crude product was concentrated and purified by silica column chromatography (95-50 % pentane/EA), and the product was collected, concentrated under vacuum to give 6 as a white solid (0.86 g, yield = 86 %). 143.76, 141.35, 127.75, 127.12, 125.03, 120.01, 66.97, 47.25, 30.80, 29.72, 19.19, 17.76.

(S)-(9H-fluoren-9-yl)methyl (1-amino-4-(methylthio)-1-oxobutan-2-yl)carbamate (7) 176

Following to the general procedure, Fmoc-L-Met-Cl 5 (1 g, 2.56 mmol) reacted with ammonia. Crude product was concentrated and purified by silica column chromatography (95-50 % pentane/EA), and the product was collected, concentrated under vacuum to give 7 as a white solid (0.83 g, yield = 95 %). 26, 143.82, 14150, 127.93, 127.26, 120.19, 67.12, 53.6, 47.37, 31.42, 30.24, 15.38 General procedure for the preparation of the nitriles of Fmoc-L amino acids:

Amide 6 or 7 (1 equiv.) was dissolved in DMF (6 mL). To this solution was added cyanuric chloride (1 equiv.). The mixture was stirred for 2 h at rt. Then, 20 mL of water was added and the aqueous phase was extracted with EA three times. The organic layer was then recovered and washed with water 5 times to get rid of remaining DMF. The resulted solution was concentrated under reduced pressure and purified by silica column chromatography.

(S)-(9H-fluoren-9-yl)methyl (1-cyano-2-methylpropyl)carbamate (8) 177

Following to the general procedure, Fmoc-L-Val-NH2 6 (860 mg, 2.5 mmol) reacted with cyanuric chloride (940 mg, 5 mmol). The product was purified by silica column chromatography (95-75 % pentane/EA), collected and concentrated under vacuum to give 8 as a white solid (651 mg, 80 %). 55, 143.52, 141.38, 127.90, 127.20, 125.00, 120.12, 117.91, 67.47, 49.08, 47.10, 31.82, 18.58, 18.07. 42, 141.38, 127.90, 127.18, 124.91, 118.05, 67.46, 47.07, 41.86, 32.17, 29.64, 15.47.

(S)-2-amino-4-(methylthio)butanenitrile (11)

Fmoc-L-Met-CN 9 (474 mg, 1.35 mmol) was dissolved in 6 mL of 2/1 mixture of DCM /Piperidine. The reaction was followed by TLC at rt. After 15 min, the mixture was concentrated and was purified by silica column chromatography (98-10 % pentane/EA), collected and concentrated under vacuum to give 11 as a yellowish oil (140 mg, 80 %).

[α] 20 D -24.02 (c 0.97, CHCl3); 85, 41.98, 34.40, 29.88, 29.88, 15.44.

(S)-tert-butyl (1-amino-1-oxo-3-phenylpropan-2-yl)carbamate (13) 179

Boc-Phe-OH (1 g, 3.77 mmol) was dissolved in 40 mL of THF at -20 °C under argon. NMM (622 µL, 5.65 mmol) and IBCF (733 µL, 5.65 mmol) were added respectively and the reaction left to stir for 15 min. Ammonia gas was then introduced to the solution for 4 minutes and then left stirring for overnight at rt. The mixture was gently evaporated, quenched with water and extracted with DCM. Organic layer was collected, dried over Na2SO4 and evaporated under vacuum. The crude was submitted to silica column chromatography (95-20 % pentane/EA). The solvent was collected and concentrated under vacuum to give 13 as a white solid (quantitative). (S)-tert-butyl (1-cyano-2-phenylethyl)carbamate ( 14) 180 Boc-Phe-NH2 13 (345 mg, 1.3 mmol) was dissolved in 6 mL DMF. Cyanuric chloride (184.4 mg, 1.3 mmol) was added. The mixture was stirred for 2 h at rt. Then it was quenched with water, and extracted with EA (3 x 20 mL). Organic phase was washed with water (5 x 20 mL), dried over NaSO4 and evaporated under vacuum to yield a white solid (95 %). 89, 129.51, 129.02, 127.94, 121.74, 118.34, 81.45, 45.06, 39.20, 28.20.

(S)-benzyl (1-amino-1-oxo-3-phenylpropan-2-yl)carbamate (17) 176

Cbz-Phe-OH (250 mg, 0.84 mmol) was dissolved in 30 mL THF at -20 °C under argon.

NMM (138 µL, 1.25 mmol) and IBCF (162 µL, 1.25 mmol) were added respectively and the reaction left to stir for 15 min. Ammonia gas was then introduced to the solution for 5 minutes and then left stirring for overnight at rt. The mixture was gently evaporated, quenched with water and extracted with DCM. Organic layer was collected, dried over Na2SO4 and evaporated under vacuum. The crude was submitted to silica column chromatography (0.2-1.5 % MeOH/DCM). The solvent was collected and concentrated under vacuum to give 17 as a white solid (91 %). Cbz-Phe-NH2 17 (220 mg, 0.74 mmol) was dissolved in 7 mL DMF. Cyanuric chloride (136 mg, 0.74 mmol) was added. The mixture was stirred for 2 h at rt. Then it was quenched with water, and extracted three times with EA. Organic phase was washed five times with water, dried over NaSO4 and evaporated under vacuum. The crude was submitted to silica column chromatography (95-70 % pentane/EA). The solvent was collected and concentrated under vacuum to give 18 as a white solid (78 %).

[α] 20 D -20.9 (c 1.00, CHCl3); 50, 133.54, 129.49, 129.11, 128.66, 128.55, 128.33, 128.07, 117.99, 67.81, 43.75, 39.03, 30.93.

(S)-tert-butyl (1-amino-4-methyl-1-oxopentan-2-yl)carbamate (19) 181

Boc-Leu--OH (1 g, 4.32 mmol) was dissolved in 40 mL THF at -20 °C under argon. NMM (713 µL, 6.49 mmol) and IBCF (841 µL, 6.49 mmol) were added respectively and the reaction left to stir for 15 min. Ammonia gas was then introduced to the solution for 5 minutes and then left stirring for overnight at rt. The mixture was gently evaporated, quenched with water and extracted with EA. Organic layer was collected, dried over Na2SO4

and evaporated under vacuum. The crude was submitted to silica column chromatography (95-15 % Pentane/EA). The solvent was collected and concentrated under vacuum to give 19 as a white solid (65 %). 81, 80.22, 52.58, 41.08, 28.32, 24.76, 22.96, 21.93.

(S)-tert-butyl (1-cyano-3-methylbutyl)carbamate (20) 180

Cbz-Phe-NH2 19 (510 mg, 2.22 mmol) was dissolved in 7 mL DMF. Cyanuric chloride (408 mg, 2.22 mmol) was added. The mixture was stirred for 2 h at rt. Then it was quenched with water, and extracted three times with EA. Organic phase was washed five times with water, dried over NaSO4 and evaporated under vacuum. The crude was submitted to silica column chromatography (95-80 % pentane/EA). The solvent was collected and concentrated under vacuum to give 20 as a pale yellow oil (92 %). 27, 119.13, 81.20, 42.20, 40.87, 28.24, 24.80, 22.21, 21.85.

(S)-1-cyano-3-methylbutan-1-aminium trifluoroacetate (21)

The nitrile 20 (225 mg, 1.06 mmol) was dissolved in DCM (4 mL), iPr3SiH (870 µL, 4.24 mmol) and TFA (817 µL, 10.6 mmol) were added respectively. The mixture was stirred for 1 h and then was extracted in D2O (1 mL) as a TFA salt. Finally, the aqueous layer was left under vacuum at room temperature to remove the remaining DCM. 38.72, 24.54, 21.54, 20.41.

(S)-(9H-fluoren-9-yl)methyl 2-(chlorocarbonyl)pyrrolidine-1-carboxylate (27) 175

To a solution of Fmoc-Pro-OH (1 g, 2.96 mmol) in dry DCM (10 mL) was added oxalyl chloride (COCl)2 (500 µL, 5.93 mmol) gently at 0 °C, 1 drop of DMF was added. The solution was stirred for 2 h at rt. The mixture was evaporated gently at 35 °C to give a pale green oil that was taken to the next step without further purification. 41, 174.38, 163.74, 154.74, 153.85, 143.88, 143.81, 143.68, 143.53, 141.46, 141.35, 141.32, 141.27, 127.78, 127.76, 127.74, 127.17, 127.10, 127.09, 127.06, 125.07, 124.99, 124.79, 124.72, 120.09, 120.01, 67.93, 67.79, 67.57, 67.31, 47.20, 47.18, 47.12, 46.65, 30.91, 30.49, 29.34, 24.06, 22.96.

(S)-(9H-fluoren-9-yl)methyl 2-cyanopyrrolidine-1-carboxylate (28)

The obtained Fmoc-Pro-Cl in the previous step 27 was dissolved in DCM (15 mL). The mixture was cooled to 0 °C then ammonia gas was bubbled for 1 min. Excess ammonia and DCM were evaporated gently under vacuum. The mixture was then quenched with water and the product was extracted in EA (3 X 20 mL), dried over Na2SO4 and concentrated under vacuum. The crude was submitted to silica column chromatography (80-10 % pentane/EA, then with 1 % MeOH/DCM). The product was collected and concentrated under vacuum to give 28 as a pale yellow oil (84 %). 7.71-7.63 (m, 2H), 7.59 (t, J = 7.3 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.38-7.28 (m, 2H), 4.65-4.17 (m, 4H), 3.65-3.23 (m, 2H), 2.34-1.92 (m, 4H); 13 C-NMR (125 MHz, CDCl3, both conformers were observed) (δ, ppm): 154. 33, 153.78, 143.92, 143.83, 143.71, 143.62, 141.36, 127.88, 127.26, 127.19, 125.17, 125.07, 125.03, 120.10, 119.01, 118.80, 68.21, 67.86, 47.56, 47.18, 47.04, 46.45, 45.99, 31.88, 30.76, 24.72, 23.73.

(S)-pyrrolidine-2-carbonitrile (26)

Fmoc-L-Pro-CN 29 (582 mg, 1.83 mmol) was dissolved in 10 mL of 3/1 mixture of DCM /Piperidine. The reaction was followed by TLC rt. After 15 min, the mixture was concentrated and was purified by silica column chromatography (98-10 % pentane/EA), collected and concentrated under vacuum to give 26 (quantitative). 78, 118.09, 51.95, 51.74, 49.92, 49.48, 29.60, 29.54, 22.38, 21.91.

(S)-benzyl (1-amino-3-cyano-1-oxopropan-2-yl)carbamate ( 30)

Cbz-Asn-OH (2 g, 7.54 mmol) was dissolved in 100 mL THF. NMM (1.65 mL, 15.03 mmol) and IBCF (1.95 mL, 15.03 mmol) were added at 0 °C. NH3 (gas) was bubbled in the mixture during 30 minutes at 0 °C. The mixture was stirred overnight at RT. The mixture was concentrated under reduced pressure, then water was added and the organic layer was extracted 3 times with EA (3 x 30 mL). The organic phase was evaporated under pressure to obtain a white solid. The product was purified using silica column with (90-25 % Pentane/EA. White solid (49 %). 2, 156.9, 136.5, 128.1, 127.7, 127.5, 116.9, 66.7, 51.0, 20.0.

(S)-benzyl (1,2-dicyanoethyl)carbamate ( 31) 30 (900 mg, 3.64 mmol) was dissolved in DMF (7 mL). To this solution was added cyanuric chloride (336 mg, 1.82 mmol). The mixture was stirred for 3 h at rt. The 20 mL of water was added and the aqueous phase was extracted with EA (3 x 25 mL). The organic layer was separated, concentrated under reduced pressure to obtain 31 as a white solid (91 %). 9, 136.2, 128.2, 127.9, 127.6, 116.4, 115.2, 66.9, 39.4, 21.5.

(S)-2-aminosuccinonitrile (32)

Bis-nitrile 31 (680 mg, 2.97 mmol) was dissolved in 20 mL acetonitrile. Catalytic amount of Pd/C was added, and the solution was stirred under hydrogen atmosphere for 6 h at rt. Then, the mixture was filtered on celite and washed with methanol. The filtrate was concentrated under reduced pressure to obtain 32 as a yellow oil (60 %) 6, 115.9, 40.1, 23.6.

methyl 2-(((benzyloxy)carbonyl)amino)-4-cyanobutanoate (33)

Cbz-Gln-OMe (1g, 3.41 mmol) was dissolved in 6 mL DMF. Cyanuric chloride (328 mg, 1.71 mmol) was added. After stirred for 1.5 h at rt, 10 mL water was added and the mixture was extracted 4 timed with EA. The organic layer was washed 5 times with water and evaporated under reduced pressure. The crude product was purified using silica column with (90-50 % Pentane/EA to give product 33 as a white solid (64 %). 2, 155.9, 135.9, 128.6, 128.4, 128.2, 118.6, 67.4, 52.9, 52.8, 28.7, 13.6.

2-(((benzyloxy)carbonyl)amino)-4-cyanobutanoic acid (34) 182 33 (598 mg, 2.17 mmol) was dissolved in 3 mL tBuOH/H2O (2 : 1). Then, LiOH (182 mg, 4.33 mmol) was added and the mixture was stirred for 1 h at 0 °C. The mixture was acidified with HCl (1 N) and extracted with EA (4 x 15 mL). The organic layer was evaporated under reduced pressure to give a yellowish oil (73 %). 174. 1, 156.1, 135.7, 128.6, 128.4, 128.2, 118.5, 67.6, 52.6, 28.3, 13.6.

2-amino-4-cyanobutanoic acid (35)

The product 34 (411 mg, 3.21 mmol) was dissolved in 10 mL EA. A catalytic amount of Pd/C was added and the mixture was stirred under hydrogen atmosphere for 4 h at rt. Finally, the mixture was filtered by celite with EA to give the product 35 as white solid (60 %). 53.3, 26.3, 13.5.

(S)-benzyl (1,5-diamino-1,5-dioxopentan-2-yl)carbamate ( 36) SOCl2 (0.52 mL, 7.14 mmol) was dissolved in 15 mL dry CH3OH at 0 °C under argon.

CbzGln (1 g, 3.57 mmol) was added to the solution, and stirred for overnight at rt. The mixture was evaporated, dry methanol was added at -10 °C and ammonia gas was bubbled during 30 min. The mixture was stirred for 48 h at rt. The white solid was separated from the obtained red solution by filtration to give 36 (50 %). 174. 2, 174.1, 154.4, 137.5, 128.8, 128.3, 128.2, 65.9, 54.8, 32.0, 28.2.

(S)-benzyl (1,3-dicyanopropyl)carbamate ( 37)

The product 36 (422 mg, 1.58 mmol) was dissolved in 12 mL DMF. Cyanuric chloride (292 mg, 1.58 mmol) was added to the mixture and left stirring for 3 h at rt. 10 mL water was added and the mixture was extracted with EA (5 x 10 mL). The organic phase was washed with water (5 x 10 mL), evaporated under reduced pressure to obtain 37 as a yellowish oil (40 %). 8, 135.2, 128.7, 128.4, 117.4, 116.8, 68.2, 41.8, 29.4, 13.8.

(S)-2-aminopentanedinitrile (38)

Product 37 (158 mg, 1.45 mmol) was dissolved in 6 mL EA. Catalytic amount of Pd/C was added to the mixture under hydrogen atmosphere and the mixture was stirred for 4 h at rt. The mixture was filtered by celite with EA to afford the product 38 as an orange oil (quantitative).

[α] 9, 118.4, 41.8, 30.8, 13.7.

N-(cyanomethyl)acetamide (40) 150

Aminoacetonitrile 39 (500 mg, 5.4 mmol) was dissolved in 10 mL pyridine at 0 °C. acetic anhydride (0.76 mL, 8.1 mmol) was added to the solution and the mixture was stirred overnight at rt. Pyridine was evaporated, water was added, pH was increased to 8 by adding NaOH solution (1 N), and the mixture was extracted with EA (5 x 10 ml). The organic phase was evaporated under reduced pressure to give the product as a white solid (32 %). 12, 116.82, 41.28, 27.52.

2-(2-((tert-butoxycarbonyl)amino)acetamido)acetic acid (43) 152

To a solution of diglycine (500 mg, 3.79 mmol) in aquous NaOH (1 N) at 0 °C was added a solution of (Boc)2O (910 mg, 4.17 mmol), in dioxane. The solution was left stirring for overnight at rt. The mixture was then concentrated and 7.5 mL NaOH (1 N) was added to the residue. Then, HCl (1 N) was added slowly to reach pH 2. The mixture was extracted three times with EA. The organic layer was collected, dried over NaSO4, concentrated under reduced vacuum to give 43 as a white solid (40 %). 65, 170.20, 156.24, 78.54, 43.50, 41.03, 28.66.

2-(ammoniomethyl)-4,5-dihydrothiazole-4-carboxylate (46)

Cysteine (3.9 mg, 0.032 mmol) and aminoacetonitrile hydrochloride (3 mg, 0.032 mmol) were dissolved in D2O (0.7 mL) in a NMR tube under Argon.

The product was identified in the reaction mixture. Cysteine (3.9 mg, 0.032 mmol) and aminoacetonitrile hydrochloride (3 mg, 0.032 mmol)

were dissolved in D2O (0.7 mL) in a NMR tube under Argon.

The product was identified in the reaction mixture.

2-(2-((tert-butoxycarbonyl)amino)acetamido)-3-Sulfanylpropanoic acid (49)

The ester 48 (85 mg, 0.28 mmol) was dissolved in 9 mL (THF/H2O 2/1 solution). LiOH (29.1 mg, 0.69 mmol) was added at 0 °C. The mixture was stirred for 2 h at 0 °C. The mixture was extracted three times with EA. The organic layer was collected, dried over Na2SO4, concentrated under reduced vacuum to give 49 as a white solid (65 %). 22, 170.45, 156.60, 80.81, 53.76, 44.02, 28.30, 26.55. 71, 156.74, 60.62, 44.57, 24.37, 22.29; HRMS (ESI): calcd for C6H11N2O2S [M + H] + : 175.0541, found 175.0542.

2-(ammoniomethyl

2-(2-ammonioacetamido)-4-Sulfanylbutanoate (53)

The product was identified in the reaction mixture. Boc-Gly-OH (500 mg, 2.85 mmol) was dissolved in 30 mL THF. NMM (0.85 mL, 7.7 mmol)

and IBCF (0.55 mL, 4.28 mmol) were added at -20 °C. After 15 min, Hcy-thiolactone hydrochloride (526 mg, 3.48 mmol) was added to the mixture. The mixture was stirred overnight at rt. The mixture was concentrated under reduced pressure, then water was added and the organic layer was extracted 3 times with EA. The organic phase was evaporated under pressure. The crude product was purified using silica column with (90-50 % Pentane/EA to provide 55 as a white solid (88 %). 39, 170.42, 156.18, 80.20, 59.03, 44.12, 31.26, 28.30, 27.43 33, 170.84, 156.61, 80.64, 51.26, 44.01, 36.12, 28.31, 20.77.

2-(2-acetamidoacetamido)-4-Sulfanylbutanoic acid (72)

In an NMR, N-acetyl-α-aminonitrile (3.2 mg, 0.033 mmol) was dissolved in 0.7 mL D2O.

Cysteine (3.96 1, 174.9, 170.9, 56.4, 42.6, 26.3, 21.7.

2-(3-ammoniopropanamido)-3-Sulfanylpropanoate (75)

In an NMR tube, the β-aminonitrile 73 (2 mg, 0.029 mmol) was dissolved in 0.7 mL D2O.

DCl was added to achieve pH 5.5. Cysteine (3.47 mg, 0.029 mmol) was added. The solution was left at 45 °C and the product was identified by NMR without purification.

1 H-NMR (500 MHz, D2O) (δ, ppm): 4.24 (t, J = 5.9 Hz, 1H), 3.13 (s, 4H,), 2.77 (m, 2H);

13 C-NMR (125 MHz, D2O) (δ, ppm): 176. 5, 171.9, 56.9, 35.6, 26.1.

2-(3-ammoniopropanamido)-4-Sulfanylbutanoate (77)

77 was not isolated, but characterized by NMR. 1 H-NMR (500 MHz, D2O) (δ, ppm): 4.15 (dd, J = 9.4, 4.7 Hz, 1H), 3.12 (t, 2H), 2.58 (m, 2H), 2.46-2.38 (m, 2H), 1.89-1.82 (m, 2H).

2-(1-ammonio-2-cyanoethyl)-4,5-dihydrothiazole-4-carboxylate (78)

In an NMR tube, the bis-nitrile (32) (7.67 mg, 0.08 mmol) was dissolved in 0.7 mL D2O. DCl was added to achieve the pH 5.5. Then, cysteine (9.68 mg, 0.08 mmol) was added and the solution was left at 45 °C. 78 was characterized by NMR without purification. 2, 177.3, 118.6, 79.6, 50.3, 36.3, 24.1.

2-((2-ammonioacetamido)methyl)-4,5-dihydrothiazole-4-carboxylate (79)

In an NMR tube, GlyGly-CN (6 mg, 0.053 mmol) and cysteine (6.4 mg, 0.053 mmol) were mixed in 0.7 mL D2O. NaHCO3 was used to afford the pH 8. The product was identified by NMR without purification.

1 H-NMR (500 MHz, D2O) (δ, ppm): 4.91 (dd, J = 9.3, 8.6 Hz, 1H), 4.26-4.16 (m, 2H), 3.61 (dd, J = 11.2, 9.8 Hz, 1H), 3.50 (s, 2H), 3.38 (dd, J = 11.2, 8.1 Hz, 1H). (0.4mL, 22.1 mmol) was added at 0 °C. After stirring for 1 h at rt, the solution was evaporated and the obtained solid was dissolved in 20 mL DCM. NH3 (gas) was bubbled in the solution for 2 min. the mixture is then extracted three times in DCM. The organic layer was collected, dried and evaporated to give 87 (quantitative).

2 nd method: Fmoc-L-Cys(Trt)-OH (2 g, 3.42 mmol) was dissolved in 50 mL DCM at 0 °C under argon. NMM (0.48 mL, 3.76 mmol) and IBCF (0.53 mL, 4.1 mmol) were added respectively and the reaction left to stir for 1 hour at 0 °C. 10 mL of ammonoia solution ( 26%) were added and the reaction was stirred for 15 min at 0 °C. Solvent was gently evaporated and then water was added and the product was extracted with DCM. Solvent was dried using

Na2SO4 and evaporated under vacuum to obtain the desired product as a white solid (quantitative). 5, 156.0, 144.3, 143.6, 141.30, 129.5, 128.1, 127.7, 127.1, 127.0, 125.0, 120.0, 67.3, 66.8, 53.6, 47.1, 33.6; HRMS (ESI): calcd for C37H32N2O3SNa [M + Na] + : 607.2026, found 607.2005.

2-amino-3-(tritylthio)propanamide (88)

87 (895 mg, 1.53 mmol) was dissolved in 15 mL DCM, 3 mL of piperidine was added. After 15 min at rt, the mixture was evaporated and purified by silica gel chromatography with 3 % MeOH/DCM to give the product as a white solid (63 %). 74, 144.59, 129.62, 128.00, 126.84, 67.08, 53.98, 37.27.

1-amino-3-Sulfanyl-1-oxopropan-2-aminium trifluoroacetate (86)

88 (1.41 g, 3.98 mmol) was dissolved in 5 mL DCM, iPr3SiH (3.2 mL, 15.7mmol) was added to the solution, then, TFA (3 mL, 38.6 mmol) was added. The mixture was stirred for 1 h at rt, then evaporated. The crude product was washed several times with DCM and finally evaporated to give a white solid (quantitative). 52.93, 34.89, 34.43, 29.18, 14.12 Homocysteine thiolactone (10 mg, 0.065 mmol) and aminoacetonitrile hydrochloride (12 mg, 0.13 mmol) were dissolved in D2O (0.7 mL) in a NMR tube under inert atmosphere, pH = 6.

The product was identified in the reaction mixture. 55 (80 mg, 0.29 mmol) was dissolved in DCM (2 mL). TIPS (0.5 mL) and TFA (0.5 mL)

were added to this mixture. The mixture was left stirring for 30 min at rt. The solution was evaporated and the crude product was washed 3 times with DCM. The product was then evaporated under reduced pressure to afford 133 as TFA salt (quantitative). 167.22, 59.33, 40.38, 29.79, 27.47.

(R)-(9H-fluoren-9-yl)methyl (1-cyano-2-(tritylthio)ethyl)carbamate (155)

Fmoc-L-Cys(Trt)-NH2 (2.13 g, 3.65 mmol) was dissolved in 6 mL DMF. Cyanuric chloride (0.67 g, 3.65 mmol) was added. The mixture was stirred for 30 min at rt. Then it was quenched with water, and organic layer was extracted with DCM (4 x 50 mL). Organic phase was washed with water (4 x 50 mL), dried, evaporated under vacuum to yield a white solid (88 %).

[𝛼] 7, 143.8, 143.4, 141.3, 129.4, 128.2, 127.9, 127.2, 127.1, 124.9, 120.1, 117.2, 67.9, 67.3, 47.0, 41.9, 34.3; HRMS (ESI): calcd for C37H30N2O2SNa [M + Na] + : 589.1920, found 589.1902.

(R)-2-amino-3-(tritylthio)propanenitrile (156)

Fmoc-L-Cys(Trt)-CN (1.5 g, 2.65 mmol) was dissolved in 8 mL DCM. Piperidine (4 mL) was added. The mixture was stirred for 15 min at rt and finally the solution was evaporated to obtain a yellow solid which was then purified by column (90-40 % pentane/EA) to obtain the product as a white solid (55 %).

[𝛼] 20 D +24.5 (c 1.00, CHCl3); 1, 129.5, 128.2, 127.1, 120.6, 67.5, 42.8, 37.2.

(R)-1-cyano-2-Sulfanylethanaminium trifluoroacetate (144)

L-Cys(Trt)-CN (18 mg, 0.052 mmol) was dissolved in 1.5 mL DCM. TIPS (43 μL, 0.21 mmol) and TFA (40 μL, 0.52 mmol) were added respectively and the solution was stirred for 1 h at rt. The product was extracted with 1.5 mL D2O. 196.88, 154.84, 144.25, 143.45, 129.91, 129.52, 128.04, 127.71, 127.07, 126.90, 115.30, 80.26, 70.43, 67.11, 59.26, 34.27, 28.30 28, 129.56, 128.07, 126.93, 200.19, 154.89, 59.16, 34.20, 28.36, 23.46, 14.31 65, 154.87, 144.26, 136.93, 126.93, 129.56, 80.40, 59.14, 34.22, 33.48; HRMS (ESI): calcd for C34H35O3NS2Na 1, 115.2, 63.4, 53.8, 44.1, 37.5, 25.6, 24.5. 27, 155.67, 144.35, 129.57, 128.15, 126.99, 115.98, 80.62, 67.24, 60.49, 33.64, 28.36, 27.42. 169.93, 155.32, 144.41, 129.60, 128.09, 126.92, 80.27, 67.22, 53.49, 52.37, 48.13, 33.65, 28.22, 18.94. 171. 82, 170.40, 144.42, 129.57, 128.08, 126.88, 67.21, 57.12, 52.05, 31.35, 28.25, 18.89, 17.66 81, 170.44, 155.29, 144.45, 129.57, 128.09, 126.91, 80.09, 67.14, 52.39, 51.51, 31.63, 29.74, 28.32, 15.34 50, 155.85, 144.19, 129.49, 128.19, 127.07, 80.91, 67.47, 54.00, 48.57, 33.28, 28.34, 17.54. 174. 85, 170.95, 155.59, 129.57, 128.10, 126.90, 80.47, 67.24, 57.08, 53.62, 33.09, 31.22, 28.26, 18.93, 17.50 174. 72, 170.74, 155.59, 144.36, 129.56, 128.13, 126.95, 80.75, 67.28, 53.60, 51.81, 33.37, 31.42, 29.75, 28.29, 15.34 170.29, 155.89, 144.19, 129.46, 128.19, 127.07, 80.91, 67.47, 53.98, 48.57, 33.28, 28.34, 28.23, 17.55. 29, 170.65, 155.82, 144.26, 129.49, 128.18, 127. 76, 144.26, 129.55, 128.17, 127.01, 118.73, 80.88, 77.23, 67.49, 35.75, 29.71, 28.22, 19.36. 7.43 (d, J = 7.5 Hz, 6H), 7.30 (t, J = 7.4 Hz, 6H), 7.27-7.17 (m, J = 13.2, 6.1 Hz, 3H), 6.75 (s, 1H), 4.91-4.36 (m, 2H), 3.66 (d, J = 5.3 Hz, 1H), 2.84-2.65 (m, 1H), 2.54 (dd, J = 13.3, 4.8 Hz, 1H), 2.04-1.82 (m, 1H), 1.41 (s, 9H), 1.01 (t, J = 6.0 Hz, 9H); 13 C-NMR (125 MHz, CDCl3) (δ, ppm): 170. 30, 155.90, 144.29, 129.55, 128.18, 127.00, 117.40, 80.85, 67.48, 53.35, 46.37, 32.35, 31.66, 28.23, 18.36, 17.89 25, 144.23, 129.53, 128.18, 127.03, 117.67, 80.92, 67.50, 53.50, 39.48, 32.76, 32.10, 29.50, 28.23, 15.41 65, 119.41, 54.20, 36.78, 24.62, 16.92. 81, 117.98, 54.22, 47.56, 30.40, 24.69, 24.69, 17.75, 17.45. 79, 118.17, 54.19, 39.92, 30.13, 28.58, 24.52, 13.86.

(R)-tert-butyl (1-((cyanomethyl)amino)-

2-((2-methoxy-2-oxoethyl)amino)-2-oxoethanaminium chloride (191) 172

Diglycine (3.3 g, 24.98 mmol) was dissolved in MeOH (75 mL). SOCl2 (3 mL, 37.47 mmol) was added dropwise at 0 °C and the reaction was left to stir for 3 h at rt. The mixture was concentrated, washed three times with Et2O ether and evaporated under reduced pressure to afford the 191 as a white solid (quantitative). 17, 166.54, 51.39, 40.44, 40.11. 89, 169.83, 168.95, 155.49, 144.19, 129.49, 128.17, 127.08, 80.92, 67.42, 52.46, 52.23, 42.96, 40.86, 28.24, 18.99. 28, 172.25, 170.39, 156.30, 144.55, 129.36, 127.76, 126.66, 79.82, 66.73, 54.13, 42.49, 42.39, 33.62, 27.52. 14, 171.85, 169.86, 156.00, 144.24, 129.50, 128.15, 127.04, 80.78, 67.27, 54.08, 50.52, 43.19, 42.55, 33.61, 28.31. 12, 169.39, 156.16, 144.11, 129.48, 128.26, 127.20, 115.92, 81.23, 67.52, 54.39, 42.82, 33.32, 28.34, 27.18. 168.46, 116.65, 54.24, 42.00, 27.37, 24.68. Life emerged on Earth probably about 3.8 billion years ago, on a planet that was largely covered by water. This work focuses on the prebiotic synthesis of peptides, especially thiolrich ones. We studied the reactions of aminonitriles (the first products of the Strecker reaction) with cysteine and homocysteine. These reactions lead to the formation of 5-or 6membered rings which are then hydrolysed to give the corresponding dipeptides (aa-Cys or aa-Hcy). The obtained thiol-containing dipeptides are able to promote the formation of longer peptide chains via thioesters bonds, and to promote the formation of some heterocycles. Homocysteine nitrile cyclizes in water to form homocysteine thiolactone, which shows a double reactivity, thiolactone opening by amines followed by aminothiol condensation reaction with nitriles. Cysteine nitrile and the S-ethyl thioester of cysteine lead to the formation of polycysteine, while Cys-aa-CN molecules gives linear and cyclic polypeptides.

Our results support the hypothesis that thiol-containing peptides would have been important molecules in the early stages of life development.

Keywords: origin of life, prebiotic chemistry, metabolism first hypothesis, thiol-rich peptides, aminonitriles, amidonitriles, cysteine, homocysteine, imidazoles, polycysteine La vie a émergé sur Terre il y a probablement 3,8 milliards d'années, sur une planète largement recouverte d'eau. Ce travail porte sur la synthèse prébiotique de peptides, en particulier de peptides riches en thiol. Nous avons étudié les réactions des aminonitriles (les premiers produits de la réaction de Strecker) avec la cystéine et l'homocystéine. Elles conduisent à la formation de cycles à 5 ou 6 chaînons qui sont ensuite hydrolysés pour donner les dipeptides correspondants (aa-Cys ou aa-Hcy). Les dipeptides contenant un thiol obtenus sont capables de favoriser la formation de chaînes peptidiques plus longues via des liaisons thioesters et de favoriser la formation de certains hétérocycles. L'homocystéine nitrile se cyclise dans l'eau pour former l'homocystéine thiolactone, qui présente une double réactivité, la thiolactone est ouverte par des amines puis on observe une condensation de l'aminothiol ainsi formé avec les nitriles. Le nitrile de cystéine et le thioester de S-éthyle de la cystéine conduisent à la formation de polycystéine, tandis que les molécules de type Cys-aa-CN donnent des polypeptides linéaires et cycliques. Nos résultats soutiennent l'hypothèse que des peptides contenant des thiols auraient joué un rôle important dans les premiers stades du développement de la vie.

Mots-clés : origine de la vie, chimie prébiotique, hypothèse « métabolisme d'abord », peptides riches en thiols, aminonitriles, amidonitriles, cystéine, homocystéine, imidazoles, polycystéine

Figure 2 :

 2 Figure 2: An example of the tree of life: Ivica Letunic, Mariana Ruiz Villarreal. The image was generated using iTOL: Interactive Tree Of Life, an online phylogenetic tree viewer and Tree Of Life resource

Figure 3 :

 3 Figure 3: A relative timeline showing the different eras that accompanied the appearance of life on Earth

27

 27

Figure 4 :

 4 Figure 4: Some nucleobases that were detected in meteorites

Figure 5 :

 5 Figure 5: The Strecker mechanisms for the formation of amino acids from NH3, HCN, and aldehydes (or ketones)

Figure 6 :

 6 Figure 6: Proposed pathway for the formation of adenine in aqueous NH4CN solution

Figure 9 :

 9 Figure 9: Heating ribose with uracil (and cytosine) did not give the corresponding nucleotides

93

 93

Figure 10 :

 10 Figure 10: A plausible simple example; a dipeptide surrounded by different amino acids (x, y and z). Preferred routs depend on equilibrium constants for the formation of peptide bonds (a). Every resulting peptide (b) has a specific reactivity that control the following steps (c) (i.e., hydrolysis, elongation, or cyclization).

Figure 11 :

 11 Figure 11: Elongation of peptides from either c-terminal (A) or N-terminal (B) activation of amino acids in the presence of cyanate

Figure 12 :

 12 Figure 12: Syntheses of some biomolecules from formamide

Figure 13 :

 13 Figure 13: A simplified pathways of the main active sulfur-containing molecules in human metabolism. Met: methionine, SAM: S-adenosylmethionine, SAH: S-adenosylhomocysteine, Hcy: homocysteine, Cyst: cystathionine, Cys: cysteine, Tau: taurine, CoA: coenzyme A, GSH: glutathione, Fe-S: iron-sulfur clusters. a) methionine adenosyl transferase, b) x-methyl transferases, c) S-adenosylhomocysteine hydrolase, d) betaine:Hcy methyltransferase, e) cystathionine β-synthase, f) cystathionine γ-lyase

  133

Figure 16 :

 16 Figure 16: CoA structure

Figure 17 :

 17 Figure 17: Simplified pathway showing the importance of CoA in Krebs cycle

Figure 18 :

 18 Figure 18: Taurine and glutathione structures

Figure 20 :

 20 Figure 20: The SMM cycle of flowering plants

  This reaction was conducted in D2O solution and followed by NMR spectroscopy. Formation of the expected thiazoline ring 46 was evidenced by the apparition of signals at ca. 5 ppm (a tripletlike dd, H α to CO2), and from 3.4 to 3.7 ppm (2 dd, SCH2). After some time, new signals grew, including a triplet at 4.4 ppm (H α to C(O)N) and a thin doublet like signal at ca. 2.9 ppm (SCH2), both characteristic of Gly-Cys 47 (Figure 24).

Figure 24 :

 24 Figure 24: Reaction of cysteine with aminoacetonitrile, Gly-CN 39 at rt. a) mixture of starting materials, b) mostly thiazoline ring 46, c) Gly-Cys dipeptide 47. Conditions: room temperature, pH 6.5, concentration 10 -2 mol/L

Figure 28 :

 28 Figure 28: Reaction of cysteine with Gly-CN in H2O; ESI spectrometry of the reaction mixture confirms the thiazoline and dipeptide formation

Figure 29 :

 29 Figure 29: Evolution of a mixture of GlyCN 39 and cysteine in D2O at 45 °C followed by 1 H-NMR, at various pH's: a) pH 4, b) pH 6, c) pH 8. SM: starting materials. Concentration 4 × 10 -2 mol/L

Figure 30 :

 30 Figure 30: Reaction of homocysteine with Gly-CN 39 at 45 °C, pH = 6.5, Conc. = 10 -2 mol/L, followed by 1 H-NMR; a) starting mixture, b) reaction after 6 hours contains the cycle 52 and the dipeptide 53, and c) Gly-Hcy 53 at the end of the reaction

Figure 32 :

 32 Figure 32: Competition between α and β-aminonitrile with cysteine. (Conversion = consumption of starting materials, forming thiazoline and the amide)

Figure 33 :

 33 Figure 33: Competition between α and β-aminonitrile with homocysteine. (Conversion = consumption of starting materials, forming thiazoline and the amide)

Figure 34 :

 34 Figure 34: Reaction of aspartic acid bis-nitrile with cysteine, 1D NMR (left), 2D (HMBC) experiment (right) demonstrating the selectivity toward the α-nitrile

Figure 37 :

 37 Figure 37: Competition between cysteine and cysteine amide 86 with aminoacetonitrile. (Conversion = consumption of starting materials)

Figure 38 :

 38 Figure 38: Competition between Cysteine and cysteamine with aminoacetonitrile. (Conversion = consumption of starting materials)

Figure 39 :

 39 Figure 39: Reaction of aminoacetonitrile with penicilamine

Figure 40 :Figure 41 :

 4041 Figure 40: 1 H-NMR showing the formation of thioester xx upon addition of excess GlyCN to Gly-Cys

Figure 42 :Figure 43 :

 4243 Figure 42 : Mass spectrum of a reaction of an excess GlyCN 39 with GlyCys 47

Figure 44 :Figure 45 :

 4445 Figure 44: Minimized conformation for compounds 108 to 111. B3LYP/6-31+G** with a continuum to mimic the solvent effect of water. ∆G's relative to 110 in kcal/mol

Figure 46 :

 46 Figure 46: Homocysteine nitrile cyclization

Figure 47 : 3 h

 473 Figure 47: 1 H-NMR monitoring of Hcy-CN cyclization at rt. a) just after deprotection, b) after 3 h

Figure 48 : 40 Figure 49 :

 484049 Figure 48: Opening of Hcy-thiolactone ring by another molecule

  To do this, we prepared two solutions in which the first contained the cycle 52 (by mixing homocysteine and aminoacetonitrile 39 in D2O at 45 °C for 2 hours) and the cycle 125 (by mixing Hcy-thiolactone 54 with aminoacetonitrile 39 in D2O at 45 °C for 3 hours). Mixing the two solutions together confirmed that the observed SCH2 signals did not belong to the same molecule (Figure50).

Figure 50 :Figure 51 :

 5051 Figure 50: 1 H-NMR of (CH2-S) protons: a) dihydrothiazine 125, b) dihydrothiazine 52, c) a mixture of 125 and 52

Figure 52 :

 52 Figure 52: Suggested example for homocysteine thiolactone as a molecular "staple"

Figure 57 : 1 Introduction

 571 Figure 57: Tentative explanation of amide formation

61

 61

Figure 58 :

 58 Figure 58: Two proposed mechanisms for prebiotic synthesis of cysteine starting from Glycine

Figure 59 :

 59 Figure 59: Cysteine Strecker synthesis

Figure 60 :

 60 Figure 60: Activated cysteine derivatives

Figure 61 :

 61 Figure 61: Explanation of the unexpected cyclisation

  H-NMR spectrometry. Formation of the expected thiazoline ring 162 with its Cys-Cys dimer structure was demonstrated by the apparition of signals at ca. 5.38 ppm (a triplet-like dd, H α to CN), and from 3.66 to 3.78 ppm (2 dd, SCH2) (Figure 63).

Figure 63 :

 63 Figure 63: 1 H-NMR spectrum of the monomer, Cys-CN (blue) and dimer formation (red) after 30 min

Figure 64 :

 64 Figure 64: 1 H-NMR spectrum showing the dimer and the trimer of cysteine residues

Figure 65 :

 65 Figure 65: Formation of the trimer 163 via the dimer 164

  structure. If we are true, the polymer was thus mainly composed of these rings.Two examples of these structures 166 (with 10 thiazoline rings) and 167 (with 12 thiazoline rings) are shown in figure 66. (Indeed our proposed structure for trimer 163 implies thee hydrolysis of the ring).

Figure 66 :

 66 Figure 66: Proposed structures of the observed masses (166 for M = 985.021 and 167 for M= 1177.126)

Figure 67 :

 67 Figure 67 : Biosynthesis of thiazole peptides

Figure 68 :

 68 Figure 68: 1 H-NMR demonstrated the formation of the cyclic molecule 152 resulted from cysteine thioacid 145 at pH 7

Figure 69 :

 69 Figure 69: Formation of the cyclic thiocarbamate 152 starting from cysteine thioacid 145

Figure 70 :

 70 Figure 70: Cysteine thioester is a way to obtain polycysteine

Figure 72 :

 72 Figure 72: A proposed polycysteine structure for M = 2493.134

Figure 73 :

 73 Figure 73: A proposed polycysteine structure for M = 2493.134

Figure 75 : 2 . 1 53 IV. 2 . 2

 75215322 Figure 75: A possible prebiotic synthesis of Cys-Gly-CN

  Scheme 56

Figure 76 :

 76 Figure 76: Polymerization process of Cys-Gly-CN under argon. a) Cys-Gly-CN in degassed water, b) turbid solution after few hours, c) the recovered solid , (Cys-Gly)n, stored under argon

  After few hours the NMR tube showed a precipitate out of the solution indicating the polymerization of Cys-Ala-CN units. The formation of thiazoline rings was characterized via 1 H-NMR spectroscopy by the appearance of broad signals around 5 ppm (CH of thiazolines), and in the region from 3.3 to 3.6 (CH2 of thiazolines) (Figure 77).

Figure 77 :

 77 Figure 77: Observation of thiazoline rings of poly(Cys-Ala) in 1 H-NMR

Figure 80 :

 80 Figure 80: A possible structure of M = 1184.979

Figure 82 :

 82 Figure 82: Peaks correspond to (Cys-Met)14-CN on MALDI

Figure 83 :

 83 Figure 83: A possible structure for M = 1374.3

Figure 84 :

 84 Figure 84: Unusual peak corresponds to the cyclic structure 203

Figure 85 :

 85 Figure 85: Formation of the cyclic structure 200

Figure 86 :

 86 Figure 86: Some detected cyclic structures of poly(Cys-Met)

Figure 89 :

 89 Figure 89: Some detected cyclic structures of poly(Cys-Val)

Figure 90 :

 90 Figure 90: MS/MS of 553 (m/z), observation of the expected fragments

1 H

 1 -NMR spectra are reported as following: chemical shift (ppm), multiplicity (s: singlet; d: doublet; dd: doublet of doublets; t: triplet; sex: sextet; sep: septet; m: multiplet), coupling constants (Hz) and integration. Proton and carbon signal assignments were established using COSY, HSQC, and HMBC experiments.High-resolution mass spectra (HRMS) were recorded on a Waters G2-S Q-TOF mass spectrometer and LTQ Orbitrap XL (Thermo Scientific). HPLC analysis of compounds was performed on a Thermo Scientific Dionex Ultimate 3000 apparatus equipped with a Varian 380-LC DEDL detector. MS/MS experiments: Low resolution ESI analysis was performed on amazon speed (Bruker Daltonics) -IonTrap Spectrometer.

  (t, J = 7.5 Hz, 2H), 7.31 (t, J = 7.5 Hz, 2H), 5.86 (s, 1H), 5.59 (s, 1H), 5.39 (d, J = 7.0 Hz, 1H), 4.42 (s, 2H), 4.21 (t, J = 6.7 Hz, 1H), 4.03 (br, 1H), 2.07-2.21 (m, 1H), 0.98 (d, J = 6.4 Hz, 3H), 0.95 (d, J = 6.4 Hz, 3H); 13 C-NMR (100 MHz, CDCl3) (δ, ppm): 173.45,

  ppm):7.71 (d, J = 7.5 Hz, 2H), 7.53 (d, J = 7.4 Hz, 2H), 7.35 (t, J = 7.4 Hz, 2H), 7.26 (t, J = 7.4 Hz, 2H), 5.50 (d, J = 8.9 Hz, 1H), 4.34-4.58 (m, 3H), 4.14 (t, J = 6.2 Hz, 1H), 1.98 (m, 1H), 1.04 (d, J = 6.4 Hz, 3H), 1.00 (d, J = 6.5 Hz, 3H);

21 (

 21 general procedure, Fmoc-L-Met-NH2 7 (0.86 g, 2.32 mmol) reacted with cyanuric chloride (0.48 g, 2.32 mmol). The product was purified by silica column chromatography (95-70 % pentane/EA), collected and concentrated under vacuum to give 9 as a white solid (0.69 g, 84 %). m.p. 136.5-137.5 °C; [α] 20 D -33.33 (c 1.00, CHCl3); 1 H-NMR (500 MHz, CDCl3) (δ, ppm): 7.76 (d, J = 7.28 Hz, 2H), 7.57ppm (d, J = 6.24 Hz, 2H), 7.41 (t, J = 7.81 Hz, 2H), 7.33 (t, J = 7.29 Hz, 2H), 5.33 (d, J = 7.5 Hz, 1H), 4.84 (d, J = 7.5, 1H), 4.50 (d, J = 6.9 Hz, 2H), 4.

  (s, 1H), 5.30 (s, 1H), 5.02 (s, 1H), 4.35 (s, 1H), 3.07 (d, J = 6.6 Hz, 2H),1.41 (s, 9H).

  ); 13 C-NMR (100 MHz, CDCl3) (δ, ppm): 135.

1 H

 1 -NMR (500 MHz, CDCl3) (δ, ppm): 6.19 (s, 1H), 5.55 (s, 1H), 4.91 (d, J = 7.9 Hz, 1H), 4.14 (s, 1H), 1.78-1.61 (m, 2H), 1.50 (dt, J = 13.7, 7.2 Hz, 1H), 1.44 (s, 9H), 0.94 (t, J = 6.1 Hz, 6H); 13 C-NMR (125 MHz, CDCl3) (δ, ppm): 175.06, 155.

  1.75-1.57 (m, 2H), 1.47 (s, 9H), 0.98 (d, J = 6.6 Hz, 6H); 13 C-NMR (125 MHz, CDCl3) (δ, ppm): 154.

1 H

 1 -NMR (500 MHz, D2O) (δ, ppm): 4.48 (q, J = 5.3 Hz, 1H), 1.90-1.96 (m, 1H), 1.54-1.85 (m, 2H), 0.92 (d, J = 6.2 Hz, 6H); 13 C-NMR (125 MHz, D2O) (δ, ppm): 116.10, 39.81,

  conformers were observed) (δ, ppm): 174.

1 H

 1 -NMR (500 MHz, CDCl3) (δ, ppm):7.77 (d, J = 7.5 Hz, 2H), 7.59 (d, J = 7.3 Hz, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 6.57 (s, 1H), 5.30 (s, 1H), 4-NH2 28 (515 mg, 1.53 mmol) was dissolved in 10 mL DMF. Cyanuric chloride (282.3 mg, 1.53 mmol) was added. The mixture was stirred for 1 h at rt. Then it was quenched with water, and extracted three times with EA. Organic phase was washed five times with water, dried over NaSO4 and evaporated under vacuum. The crude was submitted to silica column chromatography (95-70 % pentane/EA). The solvent was collected and concentrated under vacuum to give 29 as a colourless oil (81 %).

  , JBX = 8.2 Hz, 1H), 3.02-2.84 (2 dd, AB of ABX, JAX = 5.4 Hz, JBX = 8.2 Hz, JAB = 17 Hz, 2H); 13 C-NMR (CD3OD, 125 MHz) (δ, ppm): 172.

  ppm): 7.22 (m, 5H), 5.05 (s, 2H), 4.90 (t, J = 7.0 Hz, 1H), 3.00 (d, J = 7.0 Hz, 2H); 13 C-NMR (CD3OD, 125 MHz) (δ, ppm): 155.

1 H

 1 -NMR (CD3OD, 400 MHz) (δ, ppm): 4.04 (t, J = 5.2 Hz, 1H), 2.84 (d, J = 5.2 Hz, 2H); 13 C-NMR (CD3OD, 100 MHz) (δ, ppm): 119.

1 H

 1 -NMR (500 MHz, DMSO) (δ, ppm): 7.31 (m, 5H), 7.28, 7.01, 6.75 (s, 5H), 5.03 (s, 2H), 3.90 (m, 1H), 2.11 (m, 2H), 1.87-1.70 (m, 2H); 13 C-NMR (125 MHz, DMSO) (δ, ppm):

1 H

 1 -NMR (500 MHz, CDCl3) (δ, ppm): 4.19 (d, J = 5.9 Hz, 2H), 2.07 (s, 3H); -OH (1 g, 4.78 mmol) was dissolved in dry THF at-20 °C under argon. NMM (525 µL, 4.78 mmol) and IBCF (620 µL, 4.78 mmol) were added respectively and the reaction left to stir for 20 min. Then, aminoacetonitrile hydrochloride 39 (442.3 mg, 5.74 mmol) was added and the mixture was left stirring under argon for overnight at rt. the mixture was concentrated, quenched with water and extracted three times with EA. The organic phase was dried over Na2SO4 and evaporated. The crude product was submitted to silica column chromatography (95-30 % pentane/EA). The solvent was collected and concentrated under vacuum to give 41 as a white solid (93 %).

  mg, 3.54 mmol) was dissolved in 30 mL acetonitrile. Catalytic amount of Pd/C was added, and the solution was stirred under hydrogen atmosphere for 6 h at rt. Then, the mixture was filtered on celite and washed with methanol. The filtrate was concentrated under reduced pressure to obtain 42 as pale yellow oil (92 %).

1 H

 1 -NMR (500 MHz, DMSO) (δ, ppm): 8.04 (t, J = 5.8 Hz, 1H), 6.98 (t, J = 6.0 Hz, 1H), 3.76 (d, J = 5.8 Hz, 2H), 3.57 (d, J = 6.1 Hz, 2H), 1.39 (s, 9H); 13 C-NMR (125 MHz, DMSO) (δ, ppm): 171.

  , 0.032 mmol) and aminoacetonitrile hydrochloride (3 mg, 0.032 mmol) were dissolved in D2O (0.7 mL) in a NMR tube under Argon. The product was identified in the reaction mixture.

  : Fmoc-L-Cys(Trt)-OH (1.21 g, 2.07 mmol) was dissolved in 25 mL DCM, SOCl2

1 H 8 (

 18 -NMR (500 MHz, D2O) (δ, ppm): 4.17(t, J = 5.6 Hz, 1H), 3.02 (t, J = 6.0 Hz, 2H); 13 C-NMR (125 MHz, D2O) (δ, ppm): 170.0,54.2, 24.(168 mg, 1.29 mmol) was dissolved in 15 mL H2O. Cys-NH2.TFA (280 mg; 1.29 mmol) was added. The pH of the solution was adjusted to 8 by adding Na2CO3. The solution was stirred at 45 °C during 2.5 h. The aqueous phase was extracted three times with EA. The organic layer was dried over Na2SO4, filtered, and concentrated under vacuum. After purification by silica gel chromatography (1-10 % MeOH/DCM), the thiazoline 90 was obtained as an orange oil(16 % yield).

  ppm): 4.59 (dd, X of ABX, JAX = 5.6 Hz, JBX = 6.7 Hz, 1H), 3.02 (2 dd, AB of ABX, JAB = 14.8 Hz, JAX = 5.6 Hz, JBX = 6.7 Hz, 2H); 13 C-NMR (125 MHz, D2O) (δ, ppm): 114.6, 43.8, 24.1.General procedure for the synthesis of 157, 158, 160 and 161 esters:L-Boc-Cys(Trt)-OH (1 equiv.) was dissolved in DCM (15 mL), the solution was cooled to 0 °C, then DMAP (0.1 equiv.) and thiol (Triphenylmethanethiol, ethanethiol, propanethiol and benzylthiol) (1.2 equiv.) were added respectively. To this mixture, a solution of DCC(1.1 equiv.) in DCM (4 mL) was added dropwise and a white precipitate started to form. The temperature was raised from 0 °C to r.t. and the mixture was stirred for 3 hours. The mixture was filtered and the filtrate was evaporated. Flash chromatography afforded the pure esters

13 C

 13 -NMR (125 MHz, CDCl3) (δ, ppm):

[

  or 161(1 equiv.) was dissolved in DCM (2 mL), iPr3SiH(8 equiv.) and TFA(20 equiv.) were added respectively. The mixture was stirred for 1 h and then it was extracted with D2O (1.5 mL). Finally, the aqueous layer was left under vacuum at room temperature to remove the remaining DCM to provide the required products 145, 146, 147 and 148 as TFA salts.Following the general procedure, 157 (174 mg, 0.24 mmol), iPr3SiH (502 µL, 1.92 mmol) and TFA (471 µL, 4.8 mmol) were used to give 145 as TFA salt.

  mg, 0.24 mmol) was dissolved in DCM(5 mL), iPr3SiH (393 µL, 1.92 mmol) andTFA (370 µL, 4.78 mmol) were added respectively. The mixture was stirred for 1 h and then it was extracted with H2O (1.5 mL). Finally, the aqueous layer was left under vacuum at room temperature to remove the remaining DCM to provide the required products 171 as a TFA salt (quantitative). The solution was then neutralized with NaHCO3 and the resulted polymers were identified by MALDI.General method for the preparation of methyl esters Boc-Cys(Trt)-aa-OMe:Boc-L-Cys(Trt)-OH (1 equiv.) was dissolved in dry THF (~ 40 mL) at -20 °C under argon.NMM(3 equiv.) and IBCF (1.5 equiv.) were added respectively and the reaction left to stir for 1 h. Then, ester 173, 174 or 175(1.1 equiv.) was added to the mixture and was left stirring under argon for overnight at rt. The mixture was concentrated, quenched with water and extracted three times with DCM. The organic phase was dried over Na2SO4 and evaporated under reduced pressure. Flash chromatography afforded the pure esters 176, 177 and 178.Following the general procedure, Boc-L-Cys(Trt)-OH(1.5 g, 3.24 mmol) was dissolved in

  Following the general procedure, Boc-L-Cys(Trt)-OH (719 mg, 1.55 mmol) was dissolved. NMM (342 µL, 3.1 mmol) and IBCF (241 µL, 1.86 mmol) were added respectively to give the mixed anhydride. Val-OMe (260 mg, 1.55 mmol) was added. The crude product was submitted to silica column chromatography (95-60 % pentane/EA). The solvent was collected and concentrated under vacuum to give 177 as a white solid (96 %).

  Following the general procedure, To 177 (200 mg, 0.69 mmol) solution, LiOH(73 mg, 1.73 mmol) was added. The crude product was submitted to silica column chromatography (90-10 % pentane/EA). The solvent was collected and concentrated under vacuum to give 180 as a white solid (55 %).

  J = 5.5 Hz, 1H), 2.84-2.62 (m, 1H), 2.54 (dd, J = 13.1, 5.1 Hz, 1H), 2.29-2.12 (m, 1H), 1.41 (s, 9H), 0.91 (dd, J = 12.7, 6.9 Hz, 6H); 13 C-NMR (125 MHz, CDCl3) (δ, ppm):

  ; HRMS (ESI): calcd for C32H38N2O5SNa [M + Na] + : 585.2394Following the general procedure, to 178 (2.4 g, 3.80 mmol) solution, LiOH (399 mg, 9.5 mmol) was added. The crude product was submitted to silica column chromatography (90-10 % pentane/EA). The solvent was collected and concentrated under vacuum to give 181 as a white solid (74 %). m.p. 56.2-58.2 °C; [α] 20 D +6.474 (c 1.00, CHCl3); 1 H-NMR (400 MHz, CDCl3) (δ, ppm): 10.39 (br, 1H), 7.41 (d, J = 7.7 Hz, 6H), 7.29 (t, J = 7.7 Hz, 6H), 7.21(t, J =7.3 Hz, 3H ), 6.85 (s, 1H,), 4.86 (s, 1H), 4.62 (d, J = 4.4 Hz, 1H), 3.87 (dd, J = 12.3, 7.1 Hz, 1H), 2.77 (dd , J = 13.0, 7.1 Hz, 1H), 2.51 (dd, J = 13.0, 5.0 Hz, 1H), 2.49 (t, J = 7.3 Hz, 2 H), 2.16-2.23 (m, 1H), 2.03 (s, 3H), 1.94-2.01 (m, 1H), 1.41 (s, 9H); 13 C-NMR (125 MHz, CDCl3) (δ, ppm):

  ; HRMS (ESI): calcd for C32H38N2O5S2Na [M + Na] + : 617.2120, found 617.2120.General method for the preparation of amides Boc-Cys(Trt)-aa-NH2:Acid 179, 180 or 181 (1 equiv.) was dissolved in dry THF at -20 °C under argon. NMM (2 equiv.) and IBCF(1.5 equiv.) were added respectively and the reaction left to stir for 30 min.Then, ammonia gas was introduced to the mixture for 10 minutes and was left stirring under argon for overnight at rt. The mixture was concentrated, quenched with water and extracted three times with EA. The organic phase was dried over Na2SO4 and evaporated under reduced pressure. Flash chromatography afforded the pure amides 182, 183 and 184Following the general procedure, 179 (595 mg, 1.11 mmol), NMM (0.19 mL, 1.7 mmol) and IBCF (0.29 mL, 2.23 mmol) were used. The crude product was submitted to silica column chromatography (90-50 % pentane/EA). The solvent was collected and concentrated under vacuum to give 182 as a white solid (88 %). m.p. 97.3-101.2 °C; [α] 20 D -8.0 (c 0.65, CHCl3); 1 H-NMR (400 MHz, CDCl3) (δ, ppm): 7.42 (d, J = 7.6 Hz, 6H), 7.30 (t, J = 7.5 Hz, 6H), 7.24 (t, J = 7.2 Hz, 3H), 6.47 (s, 1H), 6.31 (d, J =7.7 Hz, 1H), 5.21 (s, 1H), 4.74 (d, J = 3.7 Hz, 1H), 4.43 (quin, J = 7.3 Hz, 1H), 3.75 (d, J = 4.9 Hz, 1H), 2.71 (dd, J = 12.8, 7.4 Hz, 1H), 2.61 (dd, J = 12.8, 5.3 Hz, 1H), 1.42 (s, 9 H) 1.36 (d, J = 7.2 Hz, 3H); 13 C-NMR (100 MHz, CDCl3) (δ, ppm): 174.06,

  Following the general procedure, 180 (300 mg, 0.53 mmol), NMM(88 µL, 0.8 mmol) and IBCF (138 µL, 1.1 mmol) were used. The crude product was submitted to silica column chromatography (90-80 % pentane/EA). The solvent was collected and concentrated under vacuum to give 183 as a white solid (73 %).

  ),3.78 (d, J = 4.7 Hz, 1H), 2.71 (dd, J = 12.3, 7.0 Hz, 1H), 2.60 (dd, J = 13.0, 5.3 Hz, 1H), 2.40-2.19 (m, 1H), 1.41 (s, 9H), 0.92 (d, J = 6.9 Hz, 3H), 0.86 (d, J = 6.9 Hz, 3H); 13 C-NMR (125 MHz, CDCl3) (δ, ppm): 173.

  Following the general procedure, 182 (440 mg, 0.8 mmol), cyanuric chloride (152 mg, 0.8 mmol) were used. The crude product was submitted to silica column chromatography(90-50 % pentane/EA). The solvent was collected and concentrated under vacuum to give 182 as a white solid (91 %). m.p. 202.4-203.4 °C; [α] 20 D -8.5 (c 1.00, CHCl3); 1 H-NMR (400 MHz, CDCl3) (δ, ppm): 7.36 (d, J = 7.7 Hz, 6H), 7.23 (t, J = 7.5 Hz, 6H), 7.16 (t, J = 7.2 Hz, 3H), 6.48 (s, 1H), 4.72 (quin, J = 7.4 Hz, 1H), 4.61 (d, J = 5.4 Hz, 1H), 3.60 -3.65 (m, 1H), 2.67 (dd, J = 13.3, 7.5 Hz, 1H), 2.46 (dd, J = 13.3, 5.0 Hz, 1H), 1.40 (d, J =7.2 Hz, 3H), 1.34 (s, 9H); 13 C-NMR (100 MHz, CDCl3) (δ, ppm): 170.08, 155.

  Following the general procedure, 183 (150 mg, 0.27 mmol), cyanuric chloride (49 mg, 0.27 mmol) were used. The crude product was submitted to silica column chromatography (90-80 % pentane/EA). The solvent was collected and concentrated under vacuum to give 186

  Following the general procedure, 184 (880 mg, 1.4 mmol), cyanuric chloride (263 mg, 1.4 mmol) were used. The crude product was submitted to silica column chromatography (90-70 % pentane/EA). The solvent was collected and concentrated under vacuum to give 187 as a white solid (72 %). m.p. 126.6-128.2 °C; 1 H-NMR (400 MHz, CDCl3) (δ, ppm): 7.42 (d, J = 7.7 Hz, 6H), 7.31 (t, J = 7.6 Hz, 6H), 7.23 (d, J = 7.2 Hz, 3H), 6.73 (d, J = 7.5 Hz, 1H), 4.97 (d, J = 7.4 Hz, 1H), 4.65 (s, 1H), 3.70 (d, J = 5.0 Hz, 1H), 2.59 (t, J = 7.1 Hz, 1H), 2.53 (dd, J = 13.3

  ; HRMS (ESI): calcd for C32H37N3O3S2Na [M + Na] + : 598.2174, found 598.2180.General method for the preparation of nitriles (Cys-aa-CN):Nitrile (185, 186 or 187) (1 equiv.) was dissolved in DCM (2 mL), iPr3SiH (8 equiv.) and TFA (20 equiv.) were added respectively. The mixture was stirred for 1 h and then it was extracted with D2O (1.5 mL). Finally, the aqueous layer was left under vacuum at room temperature to remove the remaining DCM to provide the required products 188, 189 and 190 as TFA salts.Following the general procedure, 185 (30 mg, 0.058 mmol), iPr3SiH (85 µL, 0.46 mmol) and TFA(89 µL, 1.16 mmol) were used to give 188 as TFA salt.

  2H), 1.52 (d, J = 7.3 Hz, 3H); 13 C-NMR (125 MHz, D2O) (δ, ppm): 167.

  Following the general procedure, 186 (42 mg, 0.08 mmol), iPr3SiH (127 µL, 0.62 mmol) and TFA (120 µL, 1.54 mmol) were used to give 189 as TFA salt.

1 H

 1 -NMR (500 MHz, D2O) (δ, ppm): 4.53 (d, J = 7.1 Hz, 1H), 4.10 (t, J = 5.8 Hz, 1H), 3.04-2.85 (m, 2H), 2.11-1.97 (m, 1H), 0.95 (d, J = 6.7 Hz, 3H), 0.89 (d, J = 6.8 Hz, 3H);

  Following the general procedure, 187 (30 mg, 0.05 mmol), iPr3SiH (82 µL, 0.40 mmol) and TFA (77 µL, 1.00 mmol) were used to give 190 as TFA salt.

  (Trt)-OH (500 mg, 1.08 mmol) was dissolved in dry THF(30 mL) at -20 °C under argon. NMM (261 µL, 2.37 mmol) and IBCF (168 µL, 1.29 mmol) were added respectively and the reaction left to stir for 20 min. Then, 191 (217 mg, 1.18 mmol) was added and the mixture was left stirring under argon for overnight at rt. The mixture was concentrated, quenched with water and extracted three times with EA. The organic phase was dried over Na2SO4 and evaporated. The crude product was submitted to silica column chromatography (0.5-10 % MeOH/DCM). The solvent was collected and concentrated under vacuum to give 192 as a white solid (50 %).

  t, J = 12.2, 4.9 Hz, 3H),6.97 (s, 1H), 6.50 (t, J = 5.8 Hz, 1H), 4.79 (d, J = 5.1 Hz, 1H), 3.94 (dd, J = 9.4, 4.3 Hz, 2H), 3.89 (d, J = 5.8 Hz, 2H), 3.69 (s, 3H), 2.75 (dd, J = 12.9

  mg, 0.79 mmol) was dissolved in 30 mL THF/water solution (2/1). LiOH(83.3 mg, 1.99 mmol) was added to the mixture at 0 °C. The mixture was left to stir for 2 h at rt. The solution was concentrated and water was added. Then, NaHSO4 (sat.) was added to afford pH 2-3. The mixture was extracted three times with EA. The organic layer was collected, dried over Na2SO4 and concentrated under reduced pressure. The crude product was submitted to silica column chromatography (0.5-10 % MeOH/DCM). The solvent was collected and concentrated under vacuum to give 193 as a white solid (41 %).

  mg, 0.69 mmol) was dissolved in dry THF(30 mL) at -20 °C under argon. NMM (114 µL, 1.04 mmol) and IBCF (108 µL, 0.83 mmol) were added respectively and the reaction left to stir for 25 min. Then, ammonia gas was bubbled in the mixture for 5 minutes.The mixture was left stirring under argon for overnight at rt. The excess ammonia was evaporated gently, and the mixture was quenched with water and extracted three times with EA. The organic phase was dried over Na2SO4 and evaporated. The crude product was submitted to silica column chromatography (0.5-10 % MeOH/DCM). The solvent was collected and concentrated under vacuum to give 194 as a white solid (40 %).

  mg, 0.23 mmol) was dissolved in 9 mL DMF. Cyanuric chloride(41.5 mg, 0.23 mmol) was added. The mixture was stirred for 3 h at rt. Then, the mixture was quenched with water, and was extracted three times with EA. The organic phase was washed with five times with water, dried, evaporated under vacuum. The crude product was submitted to silica column chromatography (90-10 % pentane/EA). The solvent was collected and concentrated under vacuum to give 195 as a white solid (72 %).[α] 20 D +16.4 (c 3.00, CHCl3); 1 H-NMR (500 MHz, CDCl3) (δ, ppm): 7.61 (s, 1H), 7.40 (d, J = 7.8 Hz, 6H), 7.30(t, J = 10.3, 4.9 Hz, 6H), 7.27-7.18 (m, 3H), 6.70 (s, 1H), 4.96 (d, J = 4.4 Hz, 1H), 4.02-3.74 (m, 4H), 3.58 (s, 1H), 2.71 (dd, J = 13.1, 7.6 Hz, 1H), 2.64 (dd, J = 13.1, 5.3 Hz, 1H), 1.42 (s, 9H); 13 C-NMR (125 MHz, CDCl3) (δ, ppm): 171.

  mg, 0.11 mmol) was dissolved in DCM (2 mL), iPr3SiH (182 µL, 0.88 mmol) and TFA (171 µL, 2.2 mmol) were added respectively. The mixture was stirred for 1 h and then it was extracted with D2O (1 mL). Finally, the aqueous layer was left under vacuum at room temperature to remove the remaining DCM to provide the required products 196 as a TFA salt (quantitative).

Table 1

 1 ).

	Nitrile	π* value	reaction rate
		-0.03632	quick
		0.01698	no reaction (?)
		-0.03010	quick
		0.00504	slower
		0.00353	quick
		-0.00547	quick
		0.00616	slower
		0.01556	no reaction
		0.03491	no reaction
	α-CN	-0.03566
	β-CN	-0.00500	reacts at α-CN

3

  Indeed, the observed masses belonged to peptides that are mainly ended with a nitrile,

	No. of monomers	No. of thiazolines	Calculated molecular wt. (M+Na) +	Observed average mass (M+Na) +
	(Cys-Met)13-Cys-Met-CN	13	3068.5522	3068.278
	(Cys-Met)13-Cys-Met-CN	10	3122.5981	3122.250
	(Cys-Met)13-Cys-Met-CN	9	3140.6134	3140.684
	(Cys-Met)14-Cys-Met-CN	9	3374.9524	3375.55
	(Cys-Met)15-Cys-Met-CN	11	3573.2608	3573.852
	(Cys-Met)15-Cys-Met-CN	8	3627.3067	3626.564

However, chains that ends with COOH were observed sometimes. Other examples on some observed masses on MALDI are given in table

3.

Table 3

 3

. of monomers No. of thiazolines Chemical formula Calculated molecular wt. (M+H) + Observed average mass (M+H) +

	Cyclo(Cys-Met)3	3	C24H37N6O3S6 +	649.978	649.074
	Cyclo(Cys-Met)4	4	C32H49N8O4S8 +	866.3027	865.745
	Cyclo(Cys-Met)5	5	C40H61N10O5S10 +	1082.6264	1081.959
	Cyclo(Cys-Met)6	6	C48H73N12O6S12 +	1298.9501	1298.332
	Cyclo(Cys-Met)7	7	C56H85N14O7S14 +	1515.2738	1515.213
	Cyclo(Cys-Met)8	8	C64H97N16O8S16 +	1731.5975	1731.996

Table 4

 4

No. of thiazolines Calculated molecular wt. (M+Na) + Observed average mass (M+Na) + (Cys-Val)13-Cys-Val-CN

	8	2708.907	2709.719
	(

Cys-Val)15-Cys-Val-CN

	9	3096.251	3095.221
	(

Cys-Val)17-Cys-Val-CN

		12	3446.754	3447.252
	(Cys-Val)21-Cys-Val-CN	17	4165.773	4162.424
	(Cys-Val)24-Cys-Val-CN	19	4736.564	4734.501

Table 5

 5

1 H-NMR (500 MHz D2O) (δ, ppm):

	2.84-2.93 (~d, 2 H), 3.84 (s, 2 H), 4.38 (t, J = 5.3 Hz, 1 H);

13 C-NMR (125 MHz D2O) (δ, ppm): 26

	.09, 40.51, 56.83, 166.6, 176.06; HRMS (ESI): calcd for C5H11N2O3S [M + H] + :
	179.0490, found 179.0492.

1 H-NMR (500 MHz D2O) (δ, ppm):

	4.28
	(q, J = 4.6, 1 H), 3.75 (s, 2 H), 2.43-2.57 (m, 2 H), 1.88-2.03 (m, 2 H); 13 C-NMR (500 MHz
	D2O) (δ, ppm): 178.36, 167.02, 54.09, 40.63, 35.58, 20.43; HRMS (ESI): calcd for
	C6H12N2O3S [M + H] + : 193.0647, found 193.0650.
	tert-butyl (2-oxo-2-((2-oxotetrahydrothiophen-3-yl)amino)ethyl)carbamate (55)

-NMR (500 MHz, CDCl3) (δ, ppm): 11

	; HRMS (ESI): calcd for C11H18N2O4SNa [M + Na] + : 297.0885, found
	297.0890.
	2-(2-((tert-butoxycarbonyl)amino)acetamido)-4-Sulfanylbutanoic acid (56)
	Thiolactone 55 (382 mg, 1.4 mmol) was dissolved in 4 mL THF, 4.2 mL of NaOH (1 N) was
	added stepwise, and the mixture was stirred under argon for overnight at rt. The mixture was
	concentrated and then acidified to pH 6 with NaHSO4 (saturated solution). The mixture was
	extracted four times with EA. The organic layer was recovered, dried over NaSO4 and
	concentrated under reduced vacuum to afford 56 (83 %)
	.51 (br, 1H), 7.38 (s, 1H), 5.81 (s, 1H), 4.72 (s,
	1H), 3.70-3.98 (m, 2H), 2.58 (q, J = 7.4 Hz, 2H), 2.18 (s, 2H), 1.60 (t, J = 8.1 Hz, 1H), 1.45
	(s, 9 H); 13 C-NMR (125 MHz, CDCl3) (δ, ppm): 174.

1 H

  mg, 0.033 mmol) was added. The mixture was kept at 45 °C. 72 was characterized by NMR without isolation.

	1 H-NMR (500 MHz, D2O) (δ, ppm): 4.35 (t, J = 5.4 Hz, 1H, CH), 3.89 (s, 2H, CH2), 2.86
	(dd, J = 2.6, 5.7 Hz, 2H), 2.00 (s, 3H); 13 C-NMR (125 MHz, D2O) (δ, ppm): 176.

;

  HRMS(ESI): calcd for C46H43O3NS2Na [M + Na] + : 744.2577, found 744.2579.

	(S)-S-ethyl 2-((tert-butoxycarbonyl)amino)-3-(tritylthio)propanethioate (158) 183
	Following the general procedure, L-Boc-Cys(Trt)-OH (1 g, 2.16 mmol), DMAP (270 mg,
	0.216 mmol), ethanethiol (170 mg, 2.6 mmol) and DCC (570 mg, 2.37 mmol) were used. The
	crude product was purified by silica column chromatography (1-10 % EA/pentane) to obtain
	158 as a white solid (98 %).

m.p. 45-50 °C; [𝛼] 20 D -1.63 (c 1.00, CHCl3); 1 H-NMR (500 MHz, CDCl3) (δ, ppm): 7.4 (m, 15H), 5.1 (dd, 1H), 4.2 (s, 1H), 2.8 (q, 2H), 2.5 (dd, 2H), 1.5 (s, 9H), 1.2 (t, 3H); 13 C-NMR (125 MHz, CDCl3) (δ, ppm): 144.

1-oxo-3-(tritylthio)propan-2-yl)carbamate (172)

  Hz, 1H), 2.75 (dd, J = 13.0, 7.2 Hz, 1H),2.58 (dd, J = 13.3, 5.3 Hz, 1H), 1.42 (s, 9H);

	m.p. 81.8-82.5 °C; 1 H-NMR (500 MHz, CDCl3) (δ, ppm): 7.48-7.39 (m, 6H), 7.31 (t, J =
	10.3, 4.9 Hz, 6H), 7.27-7.17 (m, 3H), 6.61 (s, 1H), 4.65 (s, 1H), 4.28-3.94 (m, 2H), 3.76 (d, J
	= 5.9
	Boc-Cys(Trt)-OH (500 mg, 1.08 mmol) was dissolved in dry THF (40 mL) at -20 °C under
	argon. NMM (261 µL, 2.37 mmol) and IBCF (168 µL, 1.29 mmol) were added respectively
	and the reaction left to stir for 20 min. Then, aminoacetonitrile (110 mg, 1.18 mmol) was
	added and the mixture was left stirring under argon for overnight at rt. The mixture was
	concentrated, quenched with water and extracted three times with EA. The organic phase was
	dried over Na2SO4 and evaporated. The crude product was submitted to silica column

chromatography

(95-40

% pentane/EA. The solvent was collected and concentrated under vacuum to give 172 as a white solid (67 %). 13 C-NMR (125 MHz, CDCl3) (δ, ppm): 171.

  THF. NMM(1.07 mL, 9.72 mmol) and IBCF (0.63 mL, 4.6 mmol) were added respectively to give the mixed anhydride. Ala-OMe (0.452 g, 3.24 mmol) was added. The crude product was submitted to silica column chromatography (95-80 % pentane/EA). The solvent was collected and concentrated under vacuum to give 176 as a white solid (80 %).

	m.p. 195.4-196.5 °C; 1 H-NMR (400 MHz, CDCl3) (δ, ppm): 7.35 (d, J = 7.5 Hz, 6H), 7.23
	(t, J = 7,5 Hz, 6H), 7.16 (dd, J = 13.2, 7.2 Hz, 3H), 6.44 (br, 1H), 4.69 (br, 1H), 4.42 (quin, J

= 7.2 Hz, 1H),

3.75 (br, 1H), 3.63 (s, 3H)

, 2.60-2.73 (b ,1 H), 2.45 (dd, J = 12.9, 4.9 Hz, 1H),

1.35 (s, 9 H)

, 1.28 (d, J = 7.1 Hz, 3H); 13 C-NMR (100 MHz, CDCl3) (δ, ppm): 172.84,

13 C-NMR (125 MHz, CDCl3) (δ, ppm):

  1H),1.42 (s, 9H), 0.88 (dd, J = 12.1, 6.9 Hz, 6H);

1 H-NMR (500 MHz, CDCl3) (δ, ppm): 7.43 (d, J = 7.4 Hz, 6H), 7.29 (t, J = 7.6 Hz, 6H), 7.22 (t, J = 7.3 Hz, 3H), 6.58 (s, 1H), 4.74 (s, 1H), 4.45 (dd, J = 8.8, 5.0 Hz, 1H), 3.80 (s, 1H), 3.69 (s, 3H), 2.76 (d, J = 4.8 Hz, 1H), 2.52 (dd, J = 13.0, 5.1 Hz, 1H), 2.19-2.06 (m,

13 C-NMR (100 MHz, CDCl3) (δ, ppm): 171.

  ; HRMS (ESI): calcd for C33H41N2O5S [M + H] + : 577.2731, found 577.2722.

	(S)-methyl 2-((R)-2-((tert-butoxycarbonyl)amino)-3-(tritylthio)propanamido)-4-
	(methylthio)butanoate (178)
	Following the general procedure, Boc-L-Cys(Trt)-OH (3 g, 6.48 mmol) was dissolved. NMM
	(2.14 mL, 19.44 mmol) and IBCF (1.26 mL, 9.72 mmol) were added respectively to give the
	mixed anhydride. Met-OMe (1.294 g, 6.48 mmol) was added. The crude product was
	m.p. 121.9-123.2 °C; [α] 20 D +25.7 (c 1.03, CHCl3); 1 H-NMR (400 MHz, CDCl3) (δ, ppm):
	7.41 (d, J = 7.7 Hz, 6H), 7.25 (t, J = 7.6 Hz, 6H), 7.17(t, J =7.2 Hz, 3H), 6.85 (d, J = 7.0 Hz,
	1H), 5.07 (d, J = 6.0 Hz, 1H), 4.63 (dd, J =12.6, 7.4 Hz, 1H), 3.86 (s ,1H), 3.64 (s, 3H), 2.78
	(dd, J = 12.6, 7.2 Hz, 1H), 2.53 (dd, J = 12.6, 4.9 Hz, 1H), 2.46 (t, J = 7.4 Hz, 2H), 2.09-2.18
	(m, 1H), 1.99 (s, 3H), 1.88-1.97 (m, 1H), 1.41 (s, 9H);

submitted to silica column chromatography (90-80 % pentane/EA). The solvent was collected and concentrated under vacuum to give 178 as a white solid (59 %).

General method for the preparation of acids Boc-Cys(Trt)-aa-OH:

	(S)-2-((R)-2-((tert-butoxycarbonyl)amino)-3-(tritylthio)propanamido)propanoic acid
	(179)
	Following the general procedure, To 176 (930 mg, 1.69 mmol) solution, LiOH (178 mg, 4.24
	mmol) was added. The crude product was submitted to silica column chromatography (90-30
	% pentane/EA). The solvent was collected and concentrated under vacuum to give 179 as a
	white solid (85 %).
	m.p. 80.7-81.2 °C; [α] 20 D +10.38 (c 1.05, CHCl3): 1 H-NMR (400 MHz, CDCl3) (δ, ppm):
	7.35 (d, J = 7.7 Hz, 6H), 7.22 (t, J = 7.7 Hz, 6H), 7.16 (t, J = 7.3, 3H), 6.59 (d, J = 5.5 Hz,
	1H), 4.75 (br, 1H), 4.41 (quin, J = 6.8 Hz, 1H), 3.76 (br, 1H), 2.66 (dd, J = 12.8, 7.2 Hz, 1H),
	An ester 176, 177 or 178 (1 equiv.) was dissolved in THF/water solution (2/1). LiOH (2.5
	equiv.) was added to the mixture at 0 °C. The mixture was left to stir for 2 h at rt. The
	solution was concentrated and water was added. Then, NaHSO4 (sat.) was added to afford pH
	2-3. The mixture was extracted three times with DCM. The organic layer was collected, dried
	over Na2SO4 and concentrated under reduced pressure. Flash chromatography afforded the
	pure acids 179, 180 and 181.

; HRMS (ESI): calcd for C33H40N2O5S2Na [M + Na] + : 631.2276, found 631.2278. 2.47 (dd, J = 12.8, 4.7 Hz, 1H), 1.34 (s, 9 H) 1.32 (d, J = 7.5 Hz, 3H); 13 C-NMR (100 MHz, CDCl3) (δ, ppm): 174.01, 170.

  .81-4.77 (m, 1H), 4.03 (t, J = 5.7 Hz, 1H), 2.96-2.81 (m, 2H), 2.53-2.36 (m, 2H), 2.13-1.95 (m, 2H), 1.90 (s, 3H);

1 H-NMR (500 MHz, D2O) (δ, ppm): 413 C-NMR (125 MHz, D2O) (δ, ppm): 167.

1 H-NMR (500 MHz, CDCl3) (δ, ppm): 7

  .60 (s, 1H),7.44 (s, 1H), 7.38 (d, J = 7.7 Hz, 6H), 7.27 (t, J = 9.3, 5.9 Hz, 6H), 7.20 (t, J = 7.3 Hz, 3H), 6.78 (s, 1H),6.16 (s, 1H), 5.43-5.18 (m, 1H), 4.00-3.58 (m, 4H), 2.69-2.56 (m, 2H), 2.19 (s, 1H), 1.38 (s, 9H);

13 C-NMR (125 MHz, CDCl3) (δ, ppm): 172.

H-NMR (500 MHz, D2O) (δ, ppm):3.76 (dd, J = 5.7, 4.5 Hz, 1H),

2.62 (dd, J = 15.1, 5.8

Scheme 15: Synthesis of an authentic sample of Gly-Cys dipeptide 47

The evolution of this reaction was followed by 1 H-NMR for 15 hours in which we observe the declination of the starting materials and the evolution of the products (Figure 25). 138 at 24 °C (pH 6) followed by 1

H-NMR

Scheme 17

Adding this sample to our "prebiotic" mixture confirmed the formation of Gly-Hcy 53.

I.3.3 Reaction of alpha substituted aminonitriles with cysteine and homocysteine

We chose to study the substitution effect of aminoacetonitrile (in the alpha position) on our reactions. We first tried a reaction between α-aminobutanenitrile 1 and cysteine and we observed the formation of the thiazoline ring 57 on the 1 H-NMR spectrum (Scheme 18).

Scheme 18

We also tried the α-aminonitrile 59 in which two methyl groups are located on the aminoacetonitrile's methylene, and observed the formation of the corresponding thiazoline 60 (Scheme 19).

General procedure for the preparation of racemic amino nitrile:

NH4Cl (3 equiv.) and NaCN (1 equiv.) were dissolved in distilled water (20 mL). The mixture was cooled to 0 °C for 5 minutes and then the aldehyde (propionaldehyde, isobutyraldehyde or 3-(Methylthio)propionaldehyde) (1 equiv.) was added. The mixture was left at room temperature for 4 hours. Organic layer was extracted with DCM (3 × 20 mL), dried over MgSO4, concentrated under vacuum. Flash chromatography afforded the amino nitriles.

2-aminobutanenitrile (1)

Following the general procedure, NH4Cl (2.21 g, 41.31 mmol), NaCN (675 mg, 13.77 mmol) and propionaldehyde (800 mg, 13.77 mmol). Crude product was concentrated and purified by silica column chromatography (95-50 % pentane/EA). The product was collected, and concentrated under vacuum to give 1 (580 mg, 50 %).

2-amino-3-methylbutanenitrile (2) 174

Following the general procedure, NH4Cl (8 g, 133.3 mmol), NaCN (2.18 g, 44.4 mmol) and isobutyraldehyde (3.2 g, 44.4 mmol). Crude product was concentrated and purified by silica column chromatography (95-50 % pentane/EA), and the product was collected, concentrated under vacuum to give 2 (1.5 g, 35 %). 49.78, 32.87, 18.78, 17.56.

2-amino-4-(methylthio)butanenitrile (3)

Following the general procedure, NH4Cl (1.60 g, 3 mmol), NaCN (0.47 g, 9.6 mmol) and 3-(Methylthio)propionaldehyde (1 g, 9.6 mmol). Crude product was concentrated and purified by silica column chromatography (90-20 % pentane/EA), and the product was collected, concentrated under vacuum to give 3 (0.53 g, 45 %). 97, 42.12, 34.55, 30.02, 15.59.

General procedure for the preparation of the acid chlorides of Fmoc-L amino acids:

To a solution of Fmoc amino acid (1 equiv.) in dry DCM (5 mL) was added SOCl2 (10 equiv.) gently at 0 °C. The solution was stirred for 15 minutes at rt. The mixture was concentrated under vacuum to give the product that was taken to the next step without further purification.

(S)-(9H-fluoren-9-yl)methyl (1-chloro-3-methyl-1-oxobutan-2-yl)carbamate (4) 175

Following to the general procedure, Fmoc-L-Val-OH (500 mg, 1.47 mmol) reacted with SOCl2 (1.75 g, 14.7 mmol) to give a yellowish solid (quantitative). (t, J = 7.3 Hz, 2 H), 5.56 (d, J = 8.8 Hz, 1 H), 4.34-4.49 (m, 2H), 4.13 (t, J = 6.7 Hz, 1 H), 2.30 (q, J = 6.2 Hz, 1H), 0.97 (d, J = 6.5 Hz, 3H), 0.87 (d, J = 6.7 Hz, 3H); 13 C-NMR (100 MHz, CDCl3) (δ, ppm): 174. 75, 156.29, 143.80, 143.64, 141.43, 127.91, 127.22, 125.09, 125.09, 120.16, 68.23, 67.46, 47.20, 29.95, 19.34, 17.22.

(4R)-2-(1-amino-2-methylpropyl)-4,5-dihydrothiazole-4-carboxamide (92)

Val-CN.HCl (35 mg, 0.26 mmol) was dissolved in 5 mL H2O. Cys-NH2.TFA (57 mg; 0.26 mmol) was added. The solution was adjusted to pH = 7 by adding Na2CO3. The solution was stirred at 45 °C during 24 h. The aqueous phase was extracted three times with EA. The organic layer was dried over Na2SO4, filtered, and concentrated under vacuum. After purification by silica gel chromatography (1-10 % MeOH/DCM), the thiazoline 92 was obtained as a yellow oil (30 % yield). 74, 169.70-169.43, 58.66-58.38, 55.65-55.63, 30.01-29.94, 25.30-25.02, 17.79-17.62, 16.85-16.65 In an NMR tube, cysteamine 94 (3.1 mg, 0.027 mmol) was dissolved in 0.7 mL D2O. Then, aminoacetonitrile 39 (2.5 mg, 0.027 mmol) was added. The mixture was left at 45 °C and followed by NMR. 196.51, 59.95, 24.93, 23.66, 13.33.

(S)-3-Sulfanyl-1-oxo-1-(propylthio)propan-2-aminium trifluoroacetate (147)

Following the general procedure, 160 (30 mg, 0.058 mmol), iPr3SiH (94 µL, 0.46 mmol) and TFA (88 µL, 1.15 mmol) were used to give 147 as TFA salt.

(S)-1-(benzylthio)-3-Sulfanyl-1-oxopropan-2-aminium trifluoroacetate (148)

Following the general procedure, 161 (30 mg, 0.053 mmol), iPr3SiH (89 µL, 0.43 mmol) and TFA (82 µL, 1.06 mmol) were used to give 148 as TFA salt.

1 H-NMR (400 MHz, D2O) (δ, ppm): 6.90 (m, 5H), 4.07 (t, 1H), 3.48 (m, 2H), 2.72-2.83 (m, 2H), 2.69 (m, 2H).