Proton irradiation: a key to the challenge of N-glycosidic bond formation in a prebiotic context

The formation of nucleosides in abiotic conditions is a major hurdle in origin-of-life studies. We have determined the pathway of a general reaction leading to the one-pot synthesis of ribo- and 2′-deoxy-ribonucleosides from sugars and purine nucleobases under proton irradiation in the presence of a chondrite meteorite. These conditions simulate the presumptive conditions in space or on an early Earth fluxed by slow protons from the solar wind, potentially mimicking a plausible prebiotic scenario. The reaction (i) requires neither pre-activated precursors nor intermediate purification/concentration steps, (ii) is based on a defined radical mechanism, and (iii) is characterized by stereoselectivity, regioselectivity and (poly)glycosylation. The yield is enhanced by formamide and meteorite relative to the control reaction.


Results
Irradiation experiments of nucleobases and carbohydrates were performed in three experimental conditions: 1, carbohydrate and adenine in solid film; 2, carbohydrate and adenine in formamide; 3, carbohydrate and adenine in formamide in the presence of powdered NWA 1465, selected as representative chondrite meteorite. Inorganic and organic composition, and cosmo-origin data of NWA 1465 are in SI # 2.
The solid film of adenine and carbohydrate (2-deoxyribose or ribose), or their solution in formamide (2.5 mL), with or without NWA 1465 powder (1.0% in weight, corresponding to 28 mg), was irradiated at 243 K with 170 MeV protons for 3 min. The uniform proton field was bounded to 10 × 10 cm 2 by the collimator system. The averaged linear energy transfer (LET) was 0.57 keV/μm and the calculated absorbed dose was 6 Gy. The presence of organics in the original sample of NWA 1465, observed in the ppb range 23 , was prevented by extracting the powder with NaOH (0.1 N), CHCl 3 -CH 3 OH (2:1 v/v) and sulfuric acid. The endogenous organics possibly present were removed by the extracting mixture during the treatment, and the remaining powder was used for the irradiation experiments. In agreement with previous data, the treated and untreated NWA 1465 powder performed as catalyst similarly during formamide irradiation (Table 1, note c) 13 . Materials and Methods are detailed in experimental methods and in SI # 3. The analysis was limited to products ≥1 ng/mL and the yield was calculated as percentage (%) of product per starting adenine.
We focused on the detection of nucleosides and nucleoside derivatives by ultrahigh performance liquid chromatography on-line to orbitrap-mass-spectrometry (UHPLC-MS/MS; Q Exactive-orbitrap mass analyzer). The structure of products was unambiguously defined by comparison of the mass fragmentation peaks with original commercial samples and with specifically laboratory-prepared samples as standards. The nucleosides showed all the expected molecular ions (M) and the specific fragment ions. When necessary, the assignment was further confirmed by the Addition Method 19 and by Matrix-assisted laser desorption/ionization mass spectrometry (MALDI) TOF analysis.
The complete set of UHPLC-MS/MS data, chromatographic profiles, and selected m/z fragmentation spectra are in SI #4. Chromatographic profiles also reports the magnification of the same reaction mixtures co-injected with standard nucleoside samples. The synthesis and analytical data of standard nucleosides are in SI #5.
Scientific RepoRts | 7: 14709 | https://doi.org/10.1038/s41598-017-15392-8 MALDI TOF analysis (Fig. 3) of the reaction in formamide and NWA 1465 confirmed the presence of products (3)(4)(5)(6) and (7a-b). The complete set of MALDI TOF analyses is SI#6 A-E. Higher molecular weight poly-glycosylated derivatives, corresponding to the addition of three (m/z = 484), four (m/z = 600), five (m/z = 716) and six (m/z = 832) sugar moieties, were also detected. In the irradiation of the solid film, the poly-glycosylation process progressed to a lesser extent, reaching the addition of up to four sugar moieties (SI #6-D). The m/z ions of higher molecular weight poly-glycosylated derivatives were characterized by the loss of a water molecule per novel glycosidic bond, as a consequence of the addition/elimination reaction. These data were further confirmed by the MS/MS analysis of the peak at 36.8 min in the HPLC chromatographic profile of the irradiation of adenine (1) and 2-deoxyribose (2) in formamide, corresponding to a tetra glycosylated derivative (Figure SI #4-I). The absence of the peaks of others poly-glycosylated compounds might be due to their low abundance. In this latter case, the possibility of the presence of non-covalent aggregates, rather than covalently-bound products, cannot be completely ruled out. In extant living systems, poly-glycosylated nucleosides bearing  oligosaccharide side chains have been detected in tRNA Met molecules involved in yeasts and plants replication processes 25 . They are products of post-translational modifications in humans and play key roles in cellular physiology and genotoxic stress response 26 .
2′-Deoxyribosides form more efficiently than ribosides, pyranosides prevail over furanosides, the β-isomer over the α-isomer, and the formation of nucleosides is always enhanced by formamide and by the meteorite. The synthesis of 2′-deoxyribosides and of ribosides progressed from acceptable to high yield. 2-D-deoxyribose (2) showed a reactivity higher than D-ribose (9). Irrespective of the experimental conditions, the highest yields were obtained in the presence of formamide and NWA 1465. The irradiation of adenine (1) and 2-deoxyribose (2) in dry state or in formamide afforded pyranosides (5)(6) in yield higher than that of the furanoside counterparts (3,4) (24.9% versus 12.3%, and 42.7% versus 24.4%, respectively. Table 1, entries 1 and 2). Similar results were observed for the reaction of D-ribose, where the pyranoside (12) prevailed over furanoside (11) [the ratio between (10) and (13) could not be determined due to overlapping peaks]. These data are in agreement with the prevalence of the pyranoside form for carbohydrates 2 and 9 when dissolved in formamide, as evaluated by 13 C-NMR analysis (Table 3. 83.3% versus 17.7%, and 81.8% versus 18.2%, respectively). Similar results were observed by 13 C-NMR analysis in water, where the furanose form accounts for 12% of all the ribose in solution 27 (SI#7D). Pyranosides have been suggested as plausible carbohydrate alternatives in preRNA molecules 28 instead     (Tables 1, 2). The tendency toward stabilizing (9) in its furanose form was previously reported on silica, associated with the prevalence of the β over the α stereo-isomer 29 . The formation of silicate five-membered diolate ring chelate 30 , characterized by the appropriate value of the O-C-C-O dihedral angle 31 , was reported to enhance the accumulation of the furanose form 32 . Furthermore, the elimination of water during the drying process increases the amount of the β-anomer 33 . The influence of zinc polydentate complexes on the formation of high proportion of β-furanose (50%) was also reported 29 . The formation of the latter is probably favored for geometrical reasons. From NMR results, the OH group at C5 interacts preferentially with the silica surface, so that the complexation with cations occurs with the OH at C2 and C3. These are more accessible because they are positioned on the opposite side of the molecular plane favoring the stabilization of the β-anomer.
We next evaluated the possible mechanism of the proton radiation-induced N-glycosylation. There is a consensus in the literature that the primary effect of ionizing radiation (like proton irradiation) on sugars is the abstraction of hydrogens from C-H bonds [34][35][36][37][38] . Hydrogen abstraction preferentially occurs on the carbons which are connected to an oxygen because of the electron withdrawing effect by the latter. For this reason, formation of C2-centered radicals of 2-deoxyribose is less likely as compared to that of radicals centered at other carbons 36 . The most stable product in the radiolysis of purine nucleobases is a C8-hydrogenated adduct 39,40 . M3 formed via the recombination of radiolysis products M1 and M2 (Fig. 4 and Figure SI #8-A) loses water in an exothermic reaction step (the computed reaction free energy change for this step is −13.9 kcal/mol). Based on computations using simplified models (for details see SI#8 and SI#10) this proceeds with the very low activation energy of 12.7 kcal/ mol if catalytic formamide molecules (in the formamidic acid tautomeric form) are included in the model. The transition state complex for the formamide-catalyzed water abstraction step is depicted in Figure SI#8-B. The product of the dehydration step, compound M4, then isomerizes to the nucleotide product in two consecutive  Table 3. 13 C-NMR determination of pyranoside and furanoside isomers of 2-deoxyribose (2) and ribose (9) in formamide a . a Anomeric ratios in formamide and in water calculated on the basis of the intensity of the C1 peaks. Adding to this selectivity, the amount of β-isomers increased in the presence of NWA 1465. β-Isomers usually prevailed in mineral catalysis conditions, presumably due to the preferred attack of the nucleobases from the less hindered side of the carbohydrate adsorbed on the mineral surface 42 . reaction steps with participation of two atomic hydrogens. In the first step the C8-position of the nucleobase loses a hydrogen, while in the second step the newly formed radical center at C1′ is saturated with another atomic hydrogen and leads to the nucleoside product M5. Since in these two steps the attacking atomic hydrogens form a covalent bond first with the C8 hydrogen and later with the C1′ carbon, both steps are markedly exothermic (the computed reaction free energies changes being −58.3 and −90.9 kcal/mol) and proceed essentially without an activation barrier. Based on the experimental data, 2-D-deoxyribose (2) is more active in the nucleoside formation reaction than D-ribose (9). Quantum chemical model calculations (SI #9, Table SI #9-A) suggest that a hydroxyl attached to the adjacent carbon has only a marginal effect on C-H bond dissociation energies. Because of this, there is no reason to assume that significant differences might occur in the rate of C1-radical formation from (9) and (2). On the other hand, there might be a difference in the degradation rate of these radicals, because of the absence of the 2-OH group in the 2-deoxy form. One of the main degradation channels of polyalcohol radicals is the loss of water from two vicinal hydroxyls located next to the radical center 41 . Because of the lack of the 2-OH, this degradation mechanism is irrelevant to 2-deoxyribose ( Figure SI #9-B). Thus, the higher activity of 2-deoxyribose in the glycosidic bond formation might be associated with its enhanced resistance towards degradation.
Finally, we studied the nucleobase regioselectivity of the glycosylation. The glycosylation process under irradiation conditions selectively afforded N9 isomers, which are the isomers that molecular evolution has selected for the formation of nucleic acids. If electron delocalization enables the appearance of the radical character on nitrogens other than the N9 of the aromatic ring (which is the case of adenine), then the water-elimination from the adduct formed between the nucleobase and carbohydrate radicals (similar to the conversion of M3 to M4 in Fig. 4) drives the reaction towards the stabilization of the glycosidic bond. This however assumes that one H is available on the nitrogen carrying the radical character (the OH is coming from the carbohydrate radical). If no H is available on the nitrogen (which is for instance the case of N7), then the high-energy adduct cannot be stabilized and will most likely fall apart (Fig. 5). This mechanism is probably responsible for the absence of glycosylation on N1 and N7 of adenine, justifying the observed formation of N 6 -glycosylated adducts.

Discussion and Conclusions
The major interest of a prebiotically plausible synthesis of nucleosides resides in the hurdles that this topic has met. In its general purport, the term prebiotic refers to compounds or processes that are robust, energetically not excessively demanding, based on commonly available starting materials. Nucleosides can be obtained through de novo synthesis by the oxazoline chemistry [15][16][17] or by the formamido pyrimidine chemistry 19 . Both pathways are ingenuous, afford nucleosides in good yields, and provide important data on nucleoside chemistry. The strengths of the oxazoline-based syntheses are their high regio-and stereoselectivity, the weaknesses are the multi-step procedures involved and the fact that they only apply to ribonucleosides. The formamido pyrimidine-based syntheses are high regioselective, moderately stereoselective, multi-step, only apply to purines and afford a mixture of furanosides and pyranosides. The prebiotic worth of these syntheses is inversely proportional to the procedural complexities involved, requiring numerous concentration, purification and supplementation steps, designed to specifically overcome intermediate reactions bottlenecks. How do other approaches, and the results presented here compare with these previous studies? The difficulty of formation of the β-glycosidic bond between preformed moieties 14 may be overcome considering alternative routes, as reaction in dry phase or radical chemistry. Considering that this reaction is substantially a dehydration process, Orgel and coworkers 21 studied it by treating adenine and guanine with D-ribose by heating in dry phase. The reaction yielded 6-ribosylamino adenine and 2-ribosylamino guanine as the only recovered products. This result showed that the reaction in dry phase does not allow for an adequate regioselectivity, since the exocyclic nitrogen atoms are more reactive as nucleophiles than the endocyclic N9-or N1-atoms. Better regioselectivity was observed when performing the reaction in the presence of magnesium salts and inorganic phosphates, affording riboadenosine and riboguanosine in low yield (4% and 9%, respectively) as a mixture of αand β-isomers.
As for the radical chemistry approach, the data presented in this paper define the pathway of the reaction and the characterization of the products. The reaction is one-pot, high-yield, stereo-and regioselective, and applies both to ribo-and deoxyribonucleosides. In addition, in the presence of one representative part of the meteorite tested, the carbonaceous chondrite NWA 1465, it favors furanosides over pyranosides, which are the anomeric forms present in extant nucleic acids.
Irradiation experiments. General procedure. The solid film (12 mg) of adenine and carbohydrates, or the solution of adenine (0.04 mmol) and carbohydrate (2-deoxyribose or ribose; 0.008 mmol) in formamide (2.0 mL), with or without NWA 1465 meteorite powder (1.0% in weight with respect to formamide, corresponding to mg) were irradiated at 243 K with 170 MeV protons generated by the Phasotron facility of the Joint International Nuclear Institute (JINR; Dubna, Russia) for 3 min. The uniform proton field was bounded to 10×10 cm 2 by the collimator system. The averaged linear energy transfer (LET) was about 0.57 keV/μm and the calculated absorbed dose was 6 Gy.
Further details of the experiments and computations are given in the Supplementary Material.