Design and synthesis of nucleolipids as possible activated precursors for oligomer formation via intramolecular catalysis: stability study and supramolecular organization

Background Fatty acid vesicles are an important part of protocell models currently studied. As protocells can be considered as pre-biological precursors of cells, the models try to contribute to a better understanding of the (cellular) origin of life and emphasize on 2 major aspects: compartmentalization and replication. It has been demonstrated that lipid-based membranes are amenable to growth and division (shell replication). Furthermore compartmentalization creates a unique micro-environment in which biomolecules can accumulate and reactions can occur. Pioneering research by Sugawara, Deamer, Luisi, Szostak and Rasmussen gave more insight in obtaining autocatalytic, self-replicating vesicles capable of containing and reproducing nucleic acid sequences (core replication). Linking both core and shell replication is a challenging feat requiring thorough understanding of membrane dynamics and (auto)catalytic systems. A possible solution may lie in a class of compounds called nucleolipids, who combine a nucleoside, nucleotide or nucleobase with a lipophilic moiety. Early contributions by the group of Yanagawa mentions the prebiotic significance (as a primitive helical template) arising from the supramolecular organization of these compounds. Further contributions, exploring the supramolecular scope regarding phospoliponucleosides (e.g. 5’-dioleylphosphatidyl derivatives of adenosine, uridine and cytidine) can be accounted to Baglioni, Luisi and Berti. This emerging field of amphiphiles is being investigated for surface behavior, supramolecular assembly and even drug ability. Results A series of α/β-hydroxy fatty acids and α-amino fatty acids, covalently bound to nucleoside-5′-monophosphates via a hydroxyl or amino group on the fatty acid was examined for spontaneous self-assembly in spherical aggregates and their stability towards intramolecular cleavage. Staining the resulting hydrophobic aggregates with BODIPY-dyes followed by fluorescent microscopy gave several distinct images of vesicles varying from small, isolated spheres to higher order aggregates and large, multimicrometer sized particles. Other observations include rod-like vesicle precursors. NMR was used to assess the stability of a representative sample of nucleolipids. 1D 31P NMR revealed that β-hydroxy fatty acids containing nucleotides were pH-stable while the α-analogs are acid labile. Degradation products identified by [1H-31P] heteroTOCSY revealed that phosphoesters are cleaved between sugar and phosphate, while phosphoramidates are also cleaved at the lipid-phosphate bond. For the latter compounds, the ratio between both degradation pathways is influenced by the nucleobase moiety. However no oligomerization of nucleotides was observed; nor the formation of 3′-5′-cyclic nucleotides, possible intermediates for oligonucleotide synthesis. Conclusions The nucleolipids with a deoxyribose sugar moiety form small or large vesicles, rod-like structures, vesicle aggregates or large vesicles. Some of these aggregates can be considered as intermediate forms in vesicle formation or division. However, we could not observe nucleotide polymerization or cyclic nucleotide function of these nucleolipids, regardless of the sugar moiety that is investigated (deoxyribose, ribose, xylose). To unravel this observation, the chemical stability of the constructs was studied. While the nucleolipids containing β-hydroxy fatty acids are stable as well in base as in acid circumstances, others degraded in acidic conditions. Phosphoramidate nucleolipids hydrolyzed by P-N as well as P-O bond cleavage where the ratio between both pathways depends on the nucleobase. Diester constructs with an α-hydroxy stearic acid degraded exclusively by hydrolysis of the 5′-O-nucleoside ester bond. As the compounds are too stable and harsh conditions would destruct the material itself, more reactive species such as lipid imidazolates of nucleotides need to be synthesized to further analyze the potential polymerization process. Graphical Abstract Vesicle information of a nucleolipid consisting of a nucleoside 5'-monophosphate and a α-hydroxy fatty acid.

Results: A series of α/β-hydroxy fatty acids and α-amino fatty acids, covalently bound to nucleoside-5′-monophosphates via a hydroxyl or amino group on the fatty acid was examined for spontaneous self-assembly in spherical aggregates and their stability towards intramolecular cleavage. Staining the resulting hydrophobic aggregates with BODIPY-dyes followed by fluorescent microscopy gave several distinct images of vesicles varying from small, isolated spheres to higher order aggregates and large, multimicrometer sized particles. Other observations include rod-like vesicle precursors. NMR was used to assess the stability of a representative sample of nucleolipids. 1D 31 P NMR revealed that β-hydroxy fatty acids containing nucleotides were pH-stable while the α-analogs are acid labile. Degradation products identified by [ 1 H-31 P] heteroTOCSY revealed that phosphoesters are cleaved between sugar and phosphate, while phosphoramidates are also cleaved at the lipid-phosphate bond. For the latter compounds, the ratio between both degradation pathways is influenced by the nucleobase moiety. However no oligomerization of nucleotides was observed; nor the formation of 3′-5′-cyclic nucleotides, possible intermediates for oligonucleotide synthesis.
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Background
Fatty acid vesicles are an important part of protocell models currently studied [1,2]. As protocells can be considered as pre-biological precursors of cells [3], the models try to contribute to a better understanding of the (cellular) origin of life and emphasize on 2 major aspects: compartmentalization and replication [2,[4][5][6]. It has been demonstrated that lipid-based membranes are amenable to growth and division [1,7]. Small unilamellar vesicles divide after micelle addition [8]. Autocatalytic self-replicating micelles are formed from amphiphiles generated from the alkaline hydrolysis of ethyl caprylate (shell replication) [4]. Furthermore compartmentalization creates a unique micro-environment in which biomolecules can accumulate [9,10] and reactions can occur [11]. Pioneering research by Sugawara [12], Deamer [13], Luisi [4], Szostak [7,14,15] and Rasmussen [16] gave more insight in obtaining autocatalytic, self-replicating vesicles capable of containing and reproducing nucleic acid sequences (core replication).
Linking both core and shell replication is a challenging feat requiring thorough understanding of membrane dynamics [7] and (auto)catalytic systems [4,17]. A possible solution may lie in a class of compounds called nucleolipids, who combine a nucleoside, nucleotide or nucleobase with a lipophilic moiety. Early contributions by the group of Yanagawa [18] mentions the prebiotic significance (as a primitive helical template) arising from the supramolecular organization of these compounds. Further contributions, exploring the supramolecular scope regarding phospoliponucleosides (e.g. 5 -dioleylphosphatidyl derivatives of adenosine, uridine and cytidine) can be accounted to Baglioni, Luisi and Berti. This emerging field of amphiphiles is being investigated for surface behavior, supramolecular assembly and even drug ability [19,20]. Besides improving permeability, modifying medicinally active nucleosides or nucleotides with long alkyl chains has proven (also by our group) to be an adequate prodrug tactic [10,21]. Now we designed of a series of nucleolipids as possible activated precursors for obtaining oligonucleotides. Besides its role as supramolecular recognition element ensuring the vicinity of the nucleophilic 2′-and 3′-hydroxyl groups and the electrophilic (activated) phosphate, it is necessary that the lipid part of the conjugate is also a good leaving group. This may be achieved by intramolecular catalysis; as we have recently demonstrated that a carboxylic acid function introduced in α-position of a phosphoramidate or phosphodiester group may help in catalyzing the cleavage of the phosphoramidate or phosphodiester bonds. This occurs by means of a cyclic intermediate that forms under (mild) acidic conditions (Scheme 1b). One must also consider the competing acidic hydrolysis (Scheme 1a) and cleavage of the ester bond between nucleoside and phosphate (not depicted). Previous results have shown that the cleavage ratio of Nucleoside-O-P and P-X-leaving group depends on the nature of the latter bond, the leaving group (with or without nearby carboxyl group), nucleobase and pH (a more detailed discussion of these factors can be found in the work of Maiti et al [22]).
Depending on the cleaved bond, this might lead to oligonucleotide formation due to the leaving group properties of the lipid moiety or through the formation of cyclic nucleotides, (e.g. 3′-5′ cyclic GMP) which are able to polymerize in water to give short RNA fragments [23]. As the properties of the phospholipids with and without nucleoside are different, the potential of obtaining a dynamic system is present.
Here, we have investigated the potential of α/β-hydroxy fatty acids and α-amino fatty acids, covalently bound to nucleoside-5′-monophosphates via a hydroxyl or an amino group on the fatty acid ( Figure 1) to spontaneous selfassemble in spherical aggregates. Their stability towards intramolecular cleavage was examined and thus their ability to function as (activated) monomer for oligonucleotide synthesis was assessed.

Results and discussion
Chemistry Several types of phospholipid conjugates of nucleotides were synthesized as represented by structure A and B ( Figure 1). A stearic acid scaffold should provide an optimal balance between membrane fluidity and sufficient permeability [2]. A nucleoside moiety consists of either a (deoxy)ribofuranose or a xylofuranose linked to thymine or adenine; thus creating amphiphiles with large, polar head groups.
The benzyl esters of α-hydroxy stearate 4 was synthesized [29] by heating α-hydroxystearic acid 3, with benzyl bromide in presence of triethylamine, and catalytic amount of TBAI in toluene for 12 h in a yield of 56%. The benzyl ester of β-hydroxy stearate 9 is prepared in a similar way.
The 2′, 3′-O-protected xylofuranose derivative 27 with an adenine base moiety (Scheme 6) was prepared, starting from commercially available diacetone-D-glucose 17. Benzoylation of 17 was carried out using sodium hydride and benzyl bromide in dry dimethylformamide. Scheme 1 Rationale behind the design of a new series of nucleolipid containing hydroxy/amino fatty acid with a free α/β carboxyl group. Plain acid promoted nucleophilic cleavage (a) and by carboxyl group mediated, intramolecular catalyzed nucleophilic cleavage (b).
Deprotection of 23 was carried out by using saturated methanolic ammonia to give the nucleoside 24 in 68% yield. The selective silylation of the 5′-hydroxyl group of compound 24 was carried out with TBDMSCl, imidazole in dry DMF to obtain nucleoside 25. The 2′-hydroxyl group of compound 25 was protected with a carbobenzyloxy group. Finally, the 5′-O-TBDMS group is removed with 1 M TBAF in dry THF to obtain compound 27.
Deprotection of the benzyl and the carbobenzyloxy group of phosphotriesters 32 and 41 was performed by Scheme 5 Sugar-protected adenine and thymine nucleoside.
The synthetic protocol, used for the synthesis of the phosphoramidates of dAMP 51 and dTMP 53 was based on literature prescription [39,40] using dicyclohexylcarbodiimide (DCC) as coupling agent for the conjugation of nucleotides and amino acid. The phosphoramidates 51, 53 were obtained by refluxing the nucleoside monophosphates and the amino acid methyl ester in t-BuOH and water (5:1) in the presence of DCC (dicyclohexyldicarbodiimide). Deprotection of methyl ester was performed using 0.5 N NaOH in MeOH/H 2 O-5:1 at room temperature for 4 h result in the phosphoramidates 52 and 54.
The self-assembling properties of these nucleolipids were analyzed by fluorescent microscopy using water soluble, and organic soluble (chloroform) fluorescent dyes 55 and 56 respectively ( Figure 2). Synthesis of the new, water-soluble BODIPY dye 55 was carried out by conjugating 8-S-Methyl BODIPY [41] with taurine in presence of sodium hydrogen carbonate in DMSO:DCM (1:1) at room temperature. BODIPY 56 was prepared according to the procedure previously reported by Dehaen [42,43] at rt.
The nucleolipids, which have been analyzed, consist of an α-hydroxy fatty acid (30,33,36,47), a β-hydroxy fatty acid (39,42,45,49) or a α-amino fatty acid (52,54). The polar head group is a nucleoside monophosphate (dAMP or dTMP) connected to the lipid by a phosphodiester (30,33,36,39,42,45,47,49) or by a phosphoramidate bond (52,54). The sugar is either a deoxyribose (30,33,39,42,52,54), or a ribofuranose (36,45)  xylofuranose (47,49). The reason for this selection is that we would like to evaluate a) if the sugar moiety may influence the self-aggregation process, b) if oligomerization may lead to DNA and/or RNA sequences, c) if it would be possible to form 3′-O, 5′-O-cyclic phosphates in solution, d) if the properties of the leaving group may influence oligomerization or cyclic phosphate formation. For example, an α-hydroxy acid may lead to a 5-membered intermediate and a β-hydroxy acid to a 6-membered intermediate during activation of the phosphodiester bond (Scheme 1). A phosphoramidate may be activated as leaving group by acidification of the medium. A xylofuranose      may lead easier to cyclic nucleoside formation than a ribofuranose. The availability of as well A as T nucleolipids would allow us to study mixed vesicles, in which aggregation may be influenced by base pairing.
For studying the aggregation of the compounds, following 2′-deoxynucleolipids have been used: 30,33,39,42,52, and 54. The fluorescent Bodipy dyes (55, 56) were used to monitor self-assembly of the nucleolipids by visualization under fluorescent microscopy using a spin coat method on the surface of a microscopic glass plate. Vesicle formation of nucleolipids in water was facilitated by adding small amounts of organic solvents to solubilize respectively the dye (THF) or the nucleolipid (DMSO). Soon after dissolving the nucleolipid by vortexing, a structural transition towards thermodynamically more stable spherical structures is observed. Vesicular aggregations are formed ranging from about a few to 10 μm (large vesicles), depending on the dilution and solvent conditions. Also irregular (small and large tubular structures) aggregates are formed in some cases. A series of representative examples (most frequently occurring aggregates) for the phospholipids 30, 33, 42, 52, 54 are shown.  Figure 3d) are similar in morphology to the thread-like vesicles [7], which are formed before division in daughter vesicles. Figure 4 gives the images of compound 33 using dye 56. Here, single, spherical vesicles are formed using water-miscible organic solvents (Figure 4a, b), in water vesicles tend to associate (Figure 4c, d). Figure 5 is representative for the images observed using compound 42 and dye 56. As well the vesicles (5a, b, c) are observed as rod-like structures (5a, c) which may be precursor structures for vesicle formation. Finally, the aggregates formed by the phosphoramidate conjugates with an adenine (52) and thymine (54) base moiety were visualized using dye 56. The pictures of 52 ( Figure 6 in THF/dioxane) and 54 ( Figure 7b, c in water) shows the start of the formation of vesicle colonies [44].  For studying the potential of the nucleolipids to di (poly)merize and/or to form cyclic nucleotides, compounds with a deoxyribose (30,33,39,42,52 and 54), a ribose (36,45) or a xylose (47,49) sugar moiety were envisaged. Both basic and acid circumstances were considered. Oligomerization could occur by an intermolecular reaction in which the 2′-OH or 3′-OH group of the sugar moiety attacks the 5′-O-phosphoester function, using the hydroxy (amino) lipid as leaving group. The carboxylate group in the αor β-position may catalyze this reaction. Alternatively, a 3′-O, 5′-O-cyclic nucleotide may be formed (by intramolecular reaction) which could oligomerize in solution. During the synthesis of the compounds, we already observed that some of them are not stable in acidic medium. Therefore, all compounds were treated in acid and in base medium (pH4 and pH12) for a period of 48 h. However in none of the cases, polymerization products were detected using NMR spectroscopy. These negative results could be explained by the high chemical stability of the nucleolipids (only starting material present) or by hydrolysis of the compounds in acidic and/or basic medium. Therefore, we have evaluated this stability for representative examples (30,33,52,54). 31 P NMR was used to study the stability of the nucleolipids in acidic (pH4) and in base (pH12) environment. One-dimensional 31 P spectra were used as a fast screening experiment to monitor degradation of the conjugates. Two-dimensional 1 H-31 P correlation spectra were used to characterize the 31 P containing products formed by degradation of the nucleolipids. Correlations were established using a heteroTOCSY experiment with a DIPSI spinlock of 50 ms, allowing correlations of 31 P resonances with several 1 H resonances of adjacent spin systems.
The β-hydroxystearic acid containing nucleolipids (39,42,45,49) are stable in both acidic and basic conditions with no difference in the 31 P and 1 H NMR signals over time at different pH s. All other compounds are stable in basic conditions (pH = 12 in D 2 O) while degradation occurs in acidic conditions. For phosphate diesters, a gel is formed instantly upon lowering pH in water. Although (hydro)gelation is an interesting property and promising application of nucleolipids, this was not further investigated [45]. Due to hampered NMR measurements in aqueous conditions, sample degradation in acid medium was monitored in DMSO.
An example NMR study on an nucleolipid diester with α-hydroxy stearic acid is given in Figures 8 and 9 for compound 30. Original 31 P signals appear in 1D 31 P spectra between 0.1 and 0.0 ppm corresponding to Cα in R and S enantiomers in nucleolipid 30. Due to degradation in acidic conditions, a new 31 P signal rises slightly downfield (0.4 ppm) while the original signals decrease. In a 2D-heteroTOCSY the original signals close to 0 ppm correlate with protons in the spin systems of the ribose ring (5′/5″/4′) as well as the lipid (α, β, γ). The new signal at 0.4 ppm only correlates with protons of the lipid (α, β, γ), indicating that the covalent bond between lipid and phosphorus still exists after degradation. In acidic medium (pH4-5), the thymidine congener 33 is degraded in the same way as the adenine congener 30, which shows that the cleavage mechanism is not dependent on the nucleobase ( Figure 10).
Decomposition of the phosphoramidate 52 was first studied in aqueous, acidic conditions ( Figure 10). We observed decrease of the original 31 P signal (7 ppm) in D 2 O while to signals rose at 2 and 1 ppm. The latter were assigned to dAMP and inorganic phosphate respectively using [ 1 H, 31 P]-heteroTOCSY. Since the ratio of both new signals is constant over time, we suggest that initial cleavage occurs at both amide and ester bonds in the P linkage yielding dAMP and dA respectively. While dAMP is stable in the reaction medium, the phosphorylated lipid rapidly undergoes hydrolysis releasing inorganic phosphate. In DMSO, stability of the phosphorylated lipid is increased, making it observable as an intermediate in 1D and 2D NMR spectra ( 31 P signal at 8.8 ppm). The H8 signals from the nucleobases in 52, dAMP and dA are nicely resolved and allowed to determine a ratio of 1/4 for dAMP and dA formation:20% of adenosine monophosphate and lipid are formed via pathway A and 80% of deoxyadenosine, inorganic phosphate and lipid are formed in pathway B. The thymine containing congener of 52 (54) also follows both degradation pathways in acidic conditions: 53% pathway A with formation of dTMP and lipid and 47% pathway B with formation of thymidine, inorganic phosphate and the lipid. Indicating that the nucleobase influences the ratio between P-O and P-N bond cleavage. To summarize, among all investigated systems, only α-amino compounds have shown the desired, however nucleobase dependent, bond breakage upon acidifying; the only problem being that the nucleophile is water and not the 2 -or 3 -hydroxyl groups. The β-hydroxystearic acid containing nucleolipids are stable in both acidic and basic conditions. This difference between α and β derivatives is analogues to previous calculations (done on a model in which the nucleoside had been replaced by a methyl group), showing that the formation of a six-membered intermediate by the attack of the β-carboxyl group is higher in energy (5 kcalmol-1) than the five-membered ring formed by an α-carboxyl group [22]. The proposed mechanism, predicting acidic instability, in Scheme 1b is supported by the fact that only amino derivatives cleaved at the desired bond, due to the preferred protonation of a phosphoramidate over a phosphodiester.
To a stirring solution of diacetone glucose (10 g, 38.42 mmol) in anhydrous DMF at 0? C, was added sodium hydride (60% in mineral oil (w/w), total 2.3 g, 57.63 mmol) portion wise. Stirring was continued for 1 h at 0? C, and then benzyl bromide (5.51 g, 46.10 mmol) was added drop wise. After addition, ice bath was removed, and stirring continued overnight at room temperature. After completion of the reaction, excess of sodium hydride in the reaction mixture was quenched by the addition of ice cold water (25 mL). The reaction mixture was extracted with EtOAc (4 ? 150 mL) and the combined organic phase was dried (Na 2 SO 4 ) and concentrated under vacuum, and the residue was purified by silica gel column chromatography using eluents hexane:ethylacetate-8:2 to give the title compound 17 (13 g, 96%) as oil. 1

3-O-benzyl-1,2-O-isopropylidene-β-D-xylofuranose (20)
To a stirring solution of 3-O-benzyl-1,2-isopropylideneα-D-glucofuranose (5 g, 16.11 mmol) in water (50 mL) was added sodium meta-periodate (4.13 g, 19.33) at room temperature. Stirring continued until consumption of starting material. The reaction mixture was diluted with ethanol (80 mL) and stirring continued for another 30 min. The reaction mixture is filtered on celite and the celite pad washed with ethanol. The filtrate was transferred to a 500 mL round bottom flask, and cooled to 0? C. Sodium borohydride (0.67 g, 17.72 mmol) was added in small portions to the filtrate. After completion of the addition, ice bath was removed and stirring continued for 2 h at room temperature. The reaction mixture was neutralized by drop wise addition of acetic acid, concentrated in vacuo and the residue is purified by silica gel column chromatography to get the title compound 20 [33,47] (4 g, 88.5%). 1 13 5 mL). Stirring was continued at room temperature until TLC analysis shows disappearance of the starting material, the mixture was poured into ice-water, extracted with CHCl 3 (3 ? 100mL), washed with saturated NaHCO 3 (200 mL) and dried over Na 2 SO 4 . The solvent was removed under reduced pressure and the residue is used directly in the next step. TLC-(7:3 hexanes EtOAc-Rf = 0.4).
To a suspension of 22 (5 g, 11.67 mmol) and N 6 -benzoyladenine (4.2 g, 17.50 mmol) in anhydrous acetonitrile (60 mL) was added drop wise 1 M SnCl 4 in dichloromethane (23.5 mL, 23.34 mmol) under argon. The resulting mixture was allowed to stir for 4 h at room temperature. After completion of the reaction, saturated aq NaHCO 3 was added slowly until the evolution of carbon dioxide ceased. Then the mixture was filtered through a pad of Celite 545, that was subsequently washed with CHCl 3 (3x100 mL). The combined filtrate was washed successively with saturated aq NaHCO 3 (3 ? 100 mL) and brine (2x100 mL), dried (Na2SO 4 ). The filtrates were concentrated under reduced pressure. The residue obtained was purified by silica gel column chromatography using 3% MeOH in CHCl 3 as eluent (Rf-0.5), affording nucleoside 23 [36] (6.12 g, 86%) as a colorless solid. 1

9-(3′-O-benzyl) β-D-xylofuranosyl)-adenine (24)
To a solution of nucleoside 23 (2 g, 3.29 mmol) in MeOH (20 mL) was added saturated methanolic ammonia (100 mL) in a sealed tube and the mixture was stirred at 85? C for 3 h. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure and the residue was coevaporated with toluene (5x10 mL). The residue obtained was purified by column chromatography using 5% MeOH in CHCl 3 to afford nucleoside 24 (0.8 g, 68%). 1

9-(5′-O-tert-butyldimethylsilyl-3′-O-benzyl) β-D-xylofuranosyl) adenine (25)
To a cooled suspension of 9-(5′-O-tert-butyldimethylsilyl-3-O-Benzyl-β-D-xylofuranosyl)-adenine (6 g, 16.78 mmol) and imidazole (2.85 g, 41.97 mmol) in anhydrous N, Ndimethylformamide was added tert-butyldimethylchloro silane (3.03 g, 16.78 mmol) in anhydrous DMF under argon. The reaction mixture was allowed to stir for 20 h at room temperature. After completion of the reaction, the organic solvent was removed under high vacuum. The residue was dissolved in 150 mL of ethyl acetate, the solution was washed with two times 80 mL of water, and one time with 30 mL of brine and the extract was dried on Na 2 SO 4 and the organic solvent was concentrated under reduced pressure. The residue obtained was purified on silica gel column chromatography using 2% MeOH in DCM to obtain the compound 25 as colorless solid (7.56 g, 95%). 1 13

3′-O-benzyloxycarbonyl-2′-deoxyadenosine-5′-(O-(benzyl stearate)-2-yl, O-benzyl) phosphate (29)
To a stirred solution of 14 (1 g, 25.94 mmol) and 12 (2.45 g, 38.92 mmol) in 5 mL dry DCM was added 0.5 M solution of 1H-Tetrazole (25.94 mL, 129.74 mmol) drop wise at 0? C. The reaction mixture was allowed to stir at room temperature for 4 h. Then the reaction mixture was cooled down to -78? C and hydrogen peroxide 35% (W/V, 10 mL) was added. After stirring for 5 min at -78? C, cooling bath was removed and the reaction mixture was allowed to stir at room temperature for further 30 min. The reaction mixture is diluted with DCM (150 mL) and washed with 1 M phosphoric acid (70 mL), 5% aq. sodium bicarbonate (70 mL) and with brine (60 mL), dried over sodium sulfate, filtered and concentrated in vacuo. The obtained residue was purified by column chromatography using EtOAc to obtain the title compound 29 as oil. 1

3′-O-benzyloxycarbonyl-thymidine-5′-(O-(benzyl stearate)-2-yl, O-benzyl) phosphate (32)
To the stirring solution of 15 (2 g, 5.31 mmol) and 12 (5 g, 8 mmol) in dry DCM (5 mL), was added 0.5 M solution of 1H-Tetrazole (53 mL, 26.57 mmol) drop wise at 0? C and the reaction mixture was stirred at rt for 4 h. Then the reaction mixture was cooled down to -78? C and hydrogen peroxide 35% (W/V, 15 mL) was added. After stirring for 5 min at -78? C, cooling bath was removed and the reaction mixture was allowed to stir at rt for 30 min. The reaction mixture is diluted with DCM (200 mL) and washed with 1 M phosphoric acid (100 mL), dilute sodium bicarbonate (100 mL) and with brine (100 mL), dried over sodium sulfate, filtered, concentrated in vacuo. The obtained pale yellow oil was purified by column chromatography using EtOAc as eluent to give the desired compound 32 as oil (4.35 g, 89%

Thymidine-5′-(O-stearic acid-2-yl) phosphate (33)
To a stirring solutions of 32 (2 g, 2.17 mmol) in THF: MeOH-1:1 (30 mL) was added Palladium on Charcoal (10%), and kept for stirring under hydrogen for 6 h at room temperature. After completion of the reaction, the reaction mixture is passed through celite pad and the celite pad is washed with THF-MeOH mixture (200 mL). The organic solvents were removed under vacuum, and the obtained white residue was purified by silica gel chromatography using DCM:MeOH:H 2 O-17:7:1. The organic solvents were removed with a rotavapor and the aqueous solvent was removed with a lyophilizer to get the desired product 33 as white solid (0.81 g, 61%). 1   To the stirring solution of 16 (2.5 g, 4.668 mmol) and 0.5 M solution of 1H-Tetrazole (46.7 mL, 23.34 mmol) in anhydrous dichloromethane, was added 12 (0.88 g, 7 mmol) in dry dichloromethane, drop wise at 0? C and the reaction mixture was stirred at rt for 4 h. Then the reaction mixture was cooled down to -78? C and hydrogen peroxide 35% (W/V) (15 mL) was added. After stirring for 5 min at -78? C, cooling bath was removed and the reaction mixture was allowed to stir at room temperature for 30 min. The reaction mixture is diluted with DCM (200 mL) and washed with 1 M phosphoric acid (100 mL), 5% aqueous sodium bicarbonate (100 mL) and with brine (100 mL). Organic layer was separated and dried over sodium sulfate, filtered and concentrated in vacuo. The obtained pale yellow oil was purified by silica gel column chromatography with eluent EtOAc to give 35 (4.55 g, 90%) as oil. 1 13  Adenosine-5′-(O-stearic acid-2-yl) phosphate (36) To a solution of 35 (2 g, 1.85 mmol) in THF, K 2 CO 3 (0.52 g, 37 mmol) and water (2 mL) was added. To this, Pd (10%) on Charcoal is added and kept for stirring at room temperature under hydrogen for 72 h. After the completion of the reaction, the mixture is filtered on celite 545, filtrate was washed with THF:Water-1:1.

3′-O-benzyloxycarbonyl, deoxyadenosine-5′-(O-benzyl, O-(benzyl stearate)-2-yl) phosphate (38)
To the stirring solution of 14 (0.3 g, 0.77 mmol) and 13 (0.734 g, 1.16 mmol) in 5 mL dry DCM was added 0.5 M solution of 1H-tetrazole (12.5 mL, 6.22 mmol) drop wise at 0? C, and the reaction mixture was stirred at room temperature for 12 h. Then the reaction mixture was cooled down to -78? C and hydrogen peroxide 35% (W/V) was added. After stirring for 5 min at -78? C, cooling bath was removed and the reaction mixture was allowed to stir at rt for 30 min. The reaction mixture is diluted with DCM and washed with 1 M phosphoric acid, 5% aq. sodium bicarbonate and with brine, dried over sodium sulfate, filtered and concentrated on vacuo, and purified by column chromatography (EtOAc) which gives 38 as an oil. 1

3′-O-benzyloxycarbonyl thymidine 5′-(O-(benzyl stearate)-3-yl, O-benzyl)-phosphate (41)
To a stirring solution of 15 (0.3 g, 0.77 mmol) and 13 (0.734 g, 1.16 mmol) in 5 mL dry DCM was added 0.5 M solution of 1H-tetrazole (12.5 mL, 6.22 mmol) drop wise at 0? C, and the reaction mixture was stirred at room temperature for 12 h. Then the reaction mixture was cooled down to -78? C and hydrogen peroxide 35% (W/V) was added. After stirring for 5 min at -78? C, cooling bath was removed and the reaction mixture was allowed to stir at rt for 30 min. The reaction mixture is diluted with DCM and washed with 1 M phosphoric acid, 5% aq. sodium bicarbonate and with brine, dried over sodium sulfate, filtered and concentrated on vacuo, purified by column chromatography (EtOAc) which gives 41 as an oil. 1

Thymidine-5′-(O-stearic acid-3-yl)-phosphate (42)
To the stirring solutions of 41 (2 g, 2.17 mmol) in THF: MeOH-1:1 (30 mL) was added Pd (10%) on charcoal, and kept for stirring under hydrogen for 6 h at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture is passed through celite pad and the celite pad is washed with THF-MeOH mixture (200 mL). The organic solvents were removed under vacuum, and the obtained white residue was purified by silica gel chromatography using DCM:MeOH:H 2 O-17:7:1. The organic solvents were removed in the rotavapor and the aqueous solvent was removed by lyophilization to obtain the desired product 42 as white solid (0.94 g, 71%). 1   To a stirring solution of 16 (2 g, 3.73 mmol) and 13 (2.81 g, 4.48 mmol) in dry DCM was added 0.5 M solution of 1H-tetrazole (0.5 M in dry acetonitrile (75 ml, 37.34 mmol) dropwise at 0? C and the reaction mixture was stirred at rt for 12 h. Then the reaction mixture was cooled down to -78? C and hydrogen peroxide 35% was added. After stirring for 5 min at -78? C, cooling bath was removed and the reaction mixture was allowed to stir at rt for 30 min. The reaction mixture is diluted with DCM and washed with 1 M phosphoric acid, dilute sodium bicarbonate and with brine, dried over sodium sulfate, filtered and concentrated in vacuo, purified by column chromatography with eluent EtOAC to give 44 as oil. 1

Adenosine-5′-(O-stearic acid-3-yl)-phosphate (45)
To the solution of 44 (0.3 g, 0.27 mmol) in THF, K 2 CO 3 (77 mg, 0.55 mmol) and water 2 mL was added. To this Pd (10%) on charcoal is added and held for stirring at rt under hydrogen for 12 h. After completion of the reaction, the reaction mixture is filtered on celite and the solvent is evaporated and purified by column chromatography (DCM: MeOH: H 2 O-17:7:1) to obtain the potassium salt of 45 as white solid. 1   To a stirred solution of 27 (1.2 g, 24.41 mmol) and 12 (2.3 g, 36.62 mmol) in 12 mL dry DCM was added 0.5 M solution of 1H-Tetrazole (24.41 mL, 122.07 mmol) drop wise at 0? C, and the reaction mixture was stirred at room temperature for 12 h. Then the reaction mixture was cooled down to -78? C and hydrogen peroxide 35% (W/V) was added. After stirring for 5 min at -78? C, cooling bath was removed and the reaction mixture was allowed to stir at room temperature for further 30 min. The reaction mixture is diluted with DCM (150 mL) and washed with 1 M phosphoric acid (100 mL), 5% aq. sodium bicarbonate (100 mL) and with brine (80 mL), dried over sodium sulfate, filtered and concentrated in vacuo. The obtained residue was purified by column chromatography (EtOAc) giving 46 (100 mg, 4%) as an oil. 1   To a stirred solution of 27 (1.2 g, 2.44 mmol) and 13 (2.3 g, 3.66 mmol) in 20 mL dry DCM was added 0.5 M solution of 1H-Tetrazole (54 mL, 24.4 mmol) drop wise at 0? C, and the reaction mixture was stirred at room temperature for 12 h. Then the reaction mixture was cooled down to -78? C and hydrogen peroxide 35% (W/V) was added. After stirring for 5 min at -78? C, cooling bath was removed and the reaction mixture was allowed to stir at rt for 30 min. The reaction mixture is diluted with DCM and washed with 1 M phosphoric acid, 5% aq. sodium bicarbonate and with brine, dried over sodium sulfate, filtered and concentrated in vacuo, purified by column chromatography (EtOAc) which gives 48 as an oil.

Bodipy (55)
8-S-Methyl Bodipy (120 mg, 0.5 mmol, prepared according to ref. [41]) is dissolved in DMSO/DCM (1/1; v/v, 5 ml) and mixed with taurine (60 mg, 0.5 mmol) and NaHCO 3 (42 mg, 0.5 mmol). The resulting mixture is stirred at room temperature until TLC indicates complete consumption of the starting material, and the formation of a highly polar compound displaying blue fluorescence. The reaction mixture is diluted with water (10 ml) and dichloromethane (10 ml) and extracted. The aqueous layer is collected and lyophilized to yield the desired product 55 as a pale yellow solid. 1

Vesicles preparation and visualization
Nucleolipid aggregate/vesicle formation was performed by dissolving the nucleolipids in water or DMSO and dilute with water or THF/dioxane (1:1) in a glass vial. To this solution Bodipy fluorescent dye either in water, THF or chloroform was added, vortexed for 10 seconds to stimulate vesicle formation and set aside for 5 min. The pH of the nucleolipids emulsion is found to be 6.82. If the pH was lowered further, the emulsion appeared opalescent. An aliquot of the mixture (100 μL) was pipetted out from this reaction mixture and spin coated at 3000 rpm on a microscope glass plate for 2 min, resulting in a thin layer adequate for optical microscopy.
Vesicles formed in presence of Bodipy fluorescent dye were monitored under fluorescent microscopy and the images were recorded with Olympus Fluoview FV1000 by carrying out excitation wavelength readings at 532 nm with 100 ? zoom. Two fluorescent dyes were used for the vesicle encapsulation: aqueous soluble dye 55 (soluble in both water) and organic soluble dye 56 (chloroform/THF soluble), which were used at a concentration of 0.5 μM.

Stability study by NMR
Samples were prepared in D 2 O or DMSO and the pD of the sample was adjusted by the addition of a small volume (a few μL) of HCl or NaOH solutions in D 2 O (0.1 M). 31 P NMR was used to study the stability of the nucleolipids in acidic (pH4) and in base (pH12) environment. One-dimensional 31 P spectra were used as a fast screening experiment to monitor degradation of the conjugates. Two-dimensional 1 H-31 P correlation spectra were used to characterize the 31 P containing products formed by degradation of the nucleolipids. Correlations were established using a Proton-detected hetero-TOCSY experiment [24] with a DIPSI spinlock of 50 ms, allowing correlations of 31 P resonances with several 1 H resonances of adjacent spin systems.

Conclusions
We have synthesized a series of amphiphiles in which the polar group consists of an adenine or thymine nucleotide and the lipid moiety is based on stearic acid. The nucleolipids are constructed from a phosphodiester or phosphoramidate bond formed between αor βhydroxyl group or the α-amino group present on the lipid moiety and the 5′-phosphate group of the nucleotide. These molecules have been analyzed for their potential to form vesicles in water. This can be considered as a model of a protocell with a shell containing covalently bond nucleotides, which may be used to establish an information system in the vesicle by polymerization. The functionalized lipid may function as leaving group for the polymerization reaction and the α (or β) carboxylic acid may catalyze phosphodiester cleavage.
The nucleolipids with a deoxyribose sugar moiety may form small or large vesicles, rod-like structures or vesicle aggregates. Some of these aggregates can be considered as intermediate forms in vesicle formation or division. It seems that a diversity of communication systems, by diffusion (in/out the vesicle) or exchange of material between vesicles (via vesicle fusion), between such vesicles exist. Suggesting that a protocell could stay out of equilibrium by diverse forms of material exchange.
However, we could not observe nucleotide polymerization or cyclic nucleotide formation of these nucleolipids, regardless of the sugar moiety that is investigated (deoxyribose, ribose, xylose). To unravel this observation, the chemical stability of the constructs was studied. While the nucleolipids containing β-hydroxy fatty acids are stable as well in base as in acid circumstances, others degraded in acidic conditions. Phosphoramidate nucleolipids hydrolyzed by P-N as well as P-O bond cleavage where the ratio between both pathways depends on the nucleobase. Diester constructs with a αhydroxy stearic acid degraded exclusively by hydrolysis of the phosphorus to 5′-O-nucleoside ester. To summarize, among all investigated systems, only α-amino compounds have shown the desired bond breakage, the only problem being that the nucleophile is water and not the 2 -or 3hydroxyl groups. Favoring the intramolecular mechanism in order to promote polymerization, acyclic sugar moieties such as in GNA could be considered. Also their prebiotic relevance makes them ideal candidates to explore protocells capable of simultaneous core and shell replication [48]. As the compounds are too stable and harsh conditions would destruct the material itself, more reactive species (such as lipid imidazolates of nucleotides) need to be synthesized to further analyze the potential polymerization process. This research could be based on the original work of L. Orgel [49], in which he used phosphorimidazolate for nucleotide polymerization. Furthermore, quantitative investigation is in order to address the interesting (hydro) gelating properties of these new phosphodiester nucleolipids in acidic aqueous environment.