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

Journal of Systems Chemistry volume 5, Article number: 5 (2014) Cite this article

Abstract

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.

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]-[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]).

Scheme 1
scheme1

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).

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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 self-assemble in spherical aggregates. Their stability towards intramolecular cleavage was examined and thus their ability to function as (activated) monomer for oligonucleotide synthesis was assessed.

Figure 1
figure1

General structure of nucleolipids studied in this manuscript: a: α-hydroxystearic acid (X = O), α-aminostearic acid (X = NH) and b: β-hydroxystearic acid.

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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 (±)-α-hydroxy stearic acid 3 is prepared in two steps from stearic acid 1 by bromination [24] using Hell-Volhard-Zelinsky condition giving compound 2 followed by hydrolysis in 88% overall yield (Scheme 2). The (±)-β-Hydroxy stearic acid 8 is synthesized by the procedure described by Masamune [25],[26], which involves homologation of palmitic acid 5[27] using in situ generated magnesium monomethylmalonate to a preformed acyl imidazole to produce the β-keto stearic acid methyl ester, which is reduced [28] with sodium borohydride in ethanol providing 7. Saponification with 1 N NaOH resulted in the formation of (±)-β-hydroxy stearic acid 8 in 77% overall yield (Scheme 3).

Scheme 2
scheme2

Synthesis of (±)-α hydroxy stearate. a) PBr3, Br2, 95°C, 6 h; b) 2 M NaOH, 85°C, 2 h; c) Et3N, BnBr, TBAI, toluene, reflux 12 h.

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Scheme 3
scheme3

Synthesis of (±)-β hydroxy stearate. a) 1,1′-carbonyldiimidazole, magnesium methylmalonate, THF, rt 24 h; b) NaBH4, EtOH, rt,15 min; c) 1 N NaOH, 15 min rt; d) Et3N, BnBr, TBAI, toluene, reflux 12 h.

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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 phosphoramidite approach is used for the synthesis of the lipid-nucleotide conjugates. The phosphoramidites of (±)-α- 12 and (±)-β-hydroxy benzyl stearate 13 (Scheme 4) were prepared by reaction of 4 and 9, respectively, with the phosphoramidite reagent 11 (Scheme 4), which was obtained by reaction of bis(diisopropylamino)chlorophosphine [30]10 with benzyl alcohol.

Scheme 4
scheme4

Synthesis of the phosphoramidite reagents. a) BnOH, Et3N, Et2O; b) 4, dry DCM, 1H-Tetrazole at 0°C, 15 min at rt; c) 9, dry DCM, 1H-Tetrazole at 0°C, 15 min at rt.

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The protected 2′-deoxyadenosine 14 and adenosine 16 were prepared in a similar manner as previously described [31],[32]. Likewise, protected thymidine 15 was prepared according to a previously reported procedure [31],[32] (Scheme 5).

Scheme 5
scheme5

Sugar-protected adenine and thymine nucleoside.

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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. Selective hydrolysis of the terminal isopropylidene group followed by oxidation with sodium periodate, and borohydride reduction, afforded the 1,2-acetonide 20, in 88% yield over three steps. After benzoylation of 20[33],[34] and treatment of 21 with Ac2O/H2SO4, a mixture of the anomers of 1,2-di-O-acetyl-3-O-benzyl-5-O-benzoyl-D-xylofuranose 22 was obtained. Condensation of 22 with N6-benzoyladenine [35],[36], provided the desired β-anomer 23 in 86% yield.

Scheme 6
scheme6

Synthesis of the protected xylofuranosyl nucleoside. a) NaH, DMF, BnBr, 0°C - rt 24 h; b) 1:1 methanol/1% aqueous sulfuric acid; c) NaIO4, H2O rt, NaBH4, EtOH 0°C - rt 2 h; d) benzoyl chloride, pyr; e) acetic acid, acetic anhydride, H2SO4; f) SnCl4, acetonitrile, rt 5 h; g) NH4OH, MeOH, rt, 2d; h) dry DMF, Imidazole, TBDMSCl. 24 h, rt; i) dry DCM, DMAP, CbzCl, 72 h, rt; j) HF in pyridine, pyridine, rt 4 h.

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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.

The synthesis of the phosphotriesters was accomplished in a one-pot method by reacting 12 and 13 with the protected nucleosides 14, 15, 16 and 27 in the presence of 1H-tetrazole followed by oxidation with hydrogen peroxide at -78°C, resulting in the phosphotriesters 29, 32, 35, 38, 41, 44, 46, and 48 (Scheme 7).

Scheme 7
scheme7

Synthesis of the ribose and deoxyribose nucleolipids. a) 12,1H-tetrazole, dry DCM, rt 4 h; b) 13, 1H-tetrazole, dry DCM, rt 4 h; c) H2O2, -78°C - rt, 30 min; d) H2, Pd/C, THF, K2CO3, H2O, rt 48 h, for 32 and 41, MeOH was used as co-solvent without adding K2CO3. 30, 36, 39 and 45 were isolated as 2 K+ salt.

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Deprotection of the benzyl and the carbobenzyloxy group of phosphotriesters 32 and 41 was performed by hydrogenolysis in the presence of Pd/C in THF/MeOH. Hydrogenolysis in THF/H2O as solvent in the presence of K2CO3, was carried out for the deprotection of the phosphotriesters of the adenine derivatives (29, 35, 38, 44, 46, and 48) to obtain phosphodiesters 30, 36, 39, 45, 47, 49.

α-(±)-Aminostearic acid 50 was synthesized based on the method reported by Toth [37] and Porter [38], starting from 1-bromohexadecane and diethylacetamido malonate. The crude material was directly used for esterification to obtain compound 50 (Schemes 8 and 9).

Scheme 8
scheme8

Synthesis of xylose nucleolipids. a) 12, dry DCM,1H-tetrazole, H2O2, rt, 4 h.; b) 13, dry DCM, 1H-tetrazole, H2O2, rt, 4 h.; c) H2, Pd/C, THF, rt, 48 h. Compounds 47 and 48 were isolated as 2Na+ salt.

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Scheme 9
scheme9

Synthesis of the phosphoramidate nucleolipids. a) dAMP, DCC, t- BuOH, H2O, reflux, 2 h; b) TMP, DCC, t- BuOH, H2O, reflux, 2 h; c) 0.5 N NaOH, MeOH, rt. Compounds 52 and 54 were isolated as 2Na + salt.

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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/H2O-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.

Figure 2
figure2

Structures of fluorescent dyes used for the visualization of vesicles.

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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) or a 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 3 shows microphotographs of compound 30 in H2O using dye 55 for visualization. Single vesicles are formed (Figure 3a) as well as several vesicle aggregates in which three (Figure 3b) or four (Figure 3c, d) water compartments are present. This could be intermediate stages in vesicle association or vesicle dissociation. Some of them (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 3
figure3

Representative fluorescent images of compound 30 with dye 55. Final concentrations of products for 3a-b, d =1.5 mM 30, 5.7 mM 55, 0.15 mM HCl, 7% DMSO in H2O; for 3c =1.1 mM 30, 4.4 mM 55, 0.11 mM HCl, 5% DMSO in H2O. (Scale bar 10 μm).

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Figure 4
figure4

Fluorescent microscopic images of the compound 33 with dye 56. Final concentrations of products for 4a-b =0.4 mM 33, 0.013 mM 56 in H2O/dioxane/THF (3: 47:50); for 4c-d =0.8 mM 33, 0.025 mM 56, 6% THF in H2O. (Scale bar 10 μm).

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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].

Figure 5
figure5

Fluorescent microscopic images of the compound 42 with dye 56. Final concentrations of products for 5a-c =0.3 mM 42, 0.009 mM 56, 5% THF in H2O. (Scale bar 10 μm).

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Figure 6
figure6

Fluorescent microscopic images of the compound 52 with dye 56. Final concentrations of products for 6a-b =0.39 mM 52, 0.013 mM 56 in H2O/dioxane/THF (3: 47:50); for 6c =0.66 mM 52, 0.022 mM 56, 5% THF in H2O. (Scale bar 10 μm).

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Figure 7
figure7

Fluorescent microscopic images of the compound 54 with dye 56. Final concentrations of products for 7a-c =0.3 mM 54, 0.009 mM 56, 2% THF in H2O. (Scale bar 10 μm).

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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 α- or 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).

31P NMR was used to study the stability of the nucleolipids in acidic (pH4) and in base (pH12) environment. One-dimensional 31P spectra were used as a fast screening experiment to monitor degradation of the conjugates. Two-dimensional 1H-31P correlation spectra were used to characterize the 31P 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 31P resonances with several 1H 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 31P and 1H NMR signals over time at different pH’s. All other compounds are stable in basic conditions (pH = 12 in D2O) 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 31P signals appear in 1D 31P 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 31P 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).

Figure 8
figure8

NMR study of 30 in acid conditions (pH-5.22 in DMSO). (a) time dependent degradation in acidic conditions monitored by 1D 31P NMR spectra. (b) degradation reaction of compound 30 in acidic conditions based on characterization of hydrolysis products by NMR. Arrows indicate cross peaks observed in the 2D spectrum depicted in C. (c)1H-31P-heteroTOCSY after 1 day in acidic conditions.

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Figure 9
figure9

NMR study of 52. (a) time dependent degradation in D2O. (b) HP-heteroTOCSY after 1 day in D2O. (c) HP-heteroTOCSY after 1 day in DMSO. (d) degradation reaction of nucleolipid 52 in acidic conditions. Arrows indicate cross peaks observed in the 2D spectra depicted in (b) and (c).

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Figure 10
figure10

Time dependent changes in 1D31P NMR spectrum of nucleolipid 33 in DMSO at acidic conditions. Emergence of 2 degradation products coming from phosphate (1) and Lip-OPO3 (2).

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Decomposition of the phosphoramidate 52 was first studied in aqueous, acidic conditions (Figure 10). We observed decrease of the original 31P signal (7 ppm) in D2O while to signals rose at 2 and 1 ppm. The latter were assigned to dAMP and inorganic phosphate respectively using [1H,31P]-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 (31P 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.

Experimental

Benzyl 2-hydroxyoctadecanoate (4)

To a solution of α-hydroxy stearic acid 3 (10 g, 33.28 mmol), triethylamine (6.73 g, 9.25 ml, 66.56 mmol), TBAI (1.23 g, 3.33 mmol) in toluene (150 mL), benzyl bromide (5.7 g, 5.03 mL, 33.3 mmol) is added, and held at 90°C overnight. After the completion of the reaction, the reaction mixture is triturated with diethyl ether, the precipitate formed was washed with ether and dried to obtain the desired compound 4 as white solid (7.25 g, 55.7%). 1H NMR (300 MHz, CDCl3, ppm): δ 7.37 (m, 5H, Ar), 5.25 (q, 2H, CH2), 4.23 (m, 1H), 2.79 (br, 1H, -OH), 1.82-1.73 (m, 1H), 1.69 (m, 1H), 1.25 (s, 28H, -CH2), 0.90 (t, -CH2). 13C NMR (300 MHz, CDCl3, ppm): δ 175.41(C = O), 135.37(-C-), 128.75 (-C-Ar), 128.65 (-C-Ar), 128.44 (-C-Ar), 70.64 (-C-H), 67.36 (Ar-CH2), 34.52 (-CH2), 32.05 (-CH2), 29.82 (-CH2), 29.79 (-CH2), 29.66 (-CH2), 29.58 (-CH2), 29.49 (-CH2), 24.78 (-CH2), 22.81(-CH2), 14.24 (-CH3). HRMS (ESI+) m/z Calculated for C25H43O3 (MH+): 391.3212 found 391.2869. C25H42O3Na (MNa+): 413.3031 found 413.2918.

Benzyl 3-hydroxy stearate (9)

To a solution of β-hydroxy stearic acid 8 (2 g, 6.65 mmol), triethylamine (1.2 ml, 8.65 mmol), and TBAI (0.25 g, 0.66 mmol) in toluene, benzyl bromide (1.14 g, 0.8 mL, 6.65 mmol) was added, and stirred at 90°C overnight. After the completion of the reaction, the reaction mixture is triturated with diethyl ether, and the precipitate formed was washed with diethyl ether (2 × 50 mL). The residue obtained was purified on silica gel column chromatography using dichloromethane to obtain the desired compound 9[46] as white solid (1.47 g, 56%). 1H NMR (300 MHz, CDCl3): 7.34(m, 5H-Ph), 5.14 (s, 2H, CH2-Ph), 4.01 (m, 1H), 2.9 (br s, 1H, OH), 2.58-2.41 (m, 2H, CH2), 1.43(m, 2H, CH2), 1.41(m, 2H, CH2), 1.25 (s, 24H, CH2), 0.9 (t, 3H, CH3). 13C (300 MHz, CDCl3):172.50 (C = O), 135.32(C-Ph), 128.28(C-Ph), 128.02(C-Ph),127.92(C-Ph), 67.73(C, C-OH), 66.12(C, CH2-Ph), 41.08 (C, CH2),36.24 (C, CH2), 31.60 (C, CH2), 29.37 (C, CH2), 29.34 (C, CH2), 29.26 (C, CH2), 29.24 (C, CH2), 29.20 (C, CH2), 29.03 (C, CH2), 25.14 (C, CH2), 22.36 (C, CH2), 13.78 (C, CH3). HRMS(ES+) m/z Calculated for C25H42O3 (MH+): 391.3206, found 391.3199.

Benzyl, (benzyl stearate)-2-yl, N, N-diisopropyl phosphoramidite [(±)-12]

To a stirring mixture of benzyloxy bis(N, N’-diisopropylamino)phosphine (10 g, 29.55 mmol) and alpha-hydroxy benzyl stearate (5.77 g, 14.77 mmol) in dry DCM (100 mL), was added 0.5 M solution of 1H-Tetrazole (29.6 mL, 14.77 mmol) in ACN dropwise at 0°C. After the addition, cooling bath was removed and the reaction mixture was allowed to stir at rt for 15 min. The reaction mixture was diluted with DCM (200 mL), and the organic layer was washed with 1 M TEAB solution. The extracts were dried over Na2SO4 and concentrated in vacuum and purified by flash column chromatography using eluent 400:50:10-Hexane:EtOAc:TEA (Rf = 0.5) to obtain the phosphoramidite 12 (7.10 g, 71%). P31 NMR (CDCl3): δ 149.10, 148.69.

Benzyl, (benzyl stearate)-3-yl, N, N-diisopropyl phosphoramidite [(±)-13]

To a stirring mixture of benzyloxy Bis(N, N’-diisopropylamino) phosphine (11, 1.74 g, 5.12 mmol) and β-hydroxy stearic benzyl ester (9, 1 g, 2.56 mmol) in dry DCM (10 mL) was added 0.5 M solution of 1H-tetrazole (5.12 mL, 2.56 mmol) in ACN dropwise at 0°C. After the addition, cooling bath was removed and the reaction mixture was allowed to stir at room temperature for 15 min. The reaction mixture was diluted with DCM (150 mL), and was washed with 1 M TEAB (100 mL) solution. Dichloromethane layer was dried over Na2SO4 and concentrated in vacuo, and the obtained oil was purified by silica gel flash column chromatography using the eluent 400:50:10-Hexane:EtOAc:TEA (Rf = 0.5) to get 13 as an pale yellow oil (0.985 g, 61%). 31P NMR (300 MHz, CDCl3, ppm): δ 147.63, 147.07.

3-O-benzyl-1,2:5,6-Di-O-isopropylidene-α-D-glucofuranose (17)

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 (Na2SO4) 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. 1H NMR (300 MHz, CDCl3, ppm): 7.33-7.28 (m, 5H, Ar), 5.88 (d, 1H, 1′-H), 4.68-4.62 (m, 2H, CH2-Ar), 4.56 (d, 1H, 2′-H), 4.37-4.33 (m, 1H, 5′-H), 4.16-4.06 (m, 2H, 6′-H, 6″-H), 4.01-3.96 (m, 2H, 3′-H, 4′-H), 1.47 (s, 3H, CH3), 1.41(s, 3H, CH3), 1.35(s, 3H, CH3), 1.28(s, 3H, CH3). 13C NMR (300 MHz, CDCl3, ppm): δ 137.66, 128.33, 127.76, 127.58, 111.65, 108.86, 108.86, 105.27, 82.64, 81.68, 81.31, 72.50, 72.29, 67.35, 26.81, 26.75, 26.20, 25.40.

3-O-benzyl-1,2-O-isopropylidene-α-D-glucofuranose (18)

In a 250 mL round bottom flask 3-O-benzyl-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (7.00 g, 19.97 mmol) was dissolved in 1:1 methanol/1% aqueous sulfuric acid (110 mL), and the resulting solution stirred at rt. After completion of the reaction (approximately 5 h, monitored by TLC), the reaction mixture was quenched with triethylamine (pH7). The residue was concentrated in vacuo to afford the crude residue as syrup, which was purified by silica gel flash column chromatography using 5% MeOH in dichloromethane, affording the title compound 18[33] (5.05 g, 81%) as a colorless solid. 1H NMR (300 MHz, CDCl3, ppm): 7.31-7.28 (m, 5H, Ar), 5.91 (m, 1H, 1′-H), 4.71 (dd, 2H, CH2-Ar), 4.51 (s, 1H), 4.13 (m, 2H), 4.09 (m, 1H), 3.81 (dd, 2H), 3.03 (br, 1H), 2.02, (s, 1H), 1.47 (s, 3H), 1.29 (s, 3H). 13C NMR (300 MHz, CDCl3, ppm): δ171.29, 137.41, 128.70, 128.64, 128.11, 127.81, 111.80, 105.16, 82.21, 81.97, 80.01, 69.11, 64.33, 60.45, 26.73, 26.22, 21.03, 14.19. HRMS (ESI+) m/z Calculated for C16H22O6 (MH+): 311.1489, found 311.1486.

3-O-benzyl-1,2-O-isopropylidene-β-D-xylofuranose (20)

To a stirring solution of 3-O-benzyl-1,2- isopropylidene-α-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%). 1H NMR (300 MHz, CDCl3, ppm): δ 7.37-7.28 (m, 5H, Ar-H), 5.99 (d, 1H, 1′-H), 4.73 (dd, CH2-Ar), 4.64 (s, 1H, 4-′H), 4.30 (dd, 1H, 2′-H), 3.92 (d, 1H, 3′-H), 3.91-3.1 (m, 2H, 5′-H, 5″-H), 2.24 (dd, 1H), 1.48 (s, 3H, CH3), 1.33 (s, 3H, CH3). 13C NMR (300 MHz, CDCl3, ppm): δ 137.19 (-C-, Ar), 128.78(CH, Ar), 128.31 (CH, Ar), 127.85 (CH, Ar), 111.90 (C), 105.20(C-1′), 82.87 (C-4′), 82.60 (C-2′), 80.22 (C-3′), 72.04 (CH2-Ar), 61.09 (C-5′), 26.93 (CH3), 26.43(CH3). HRMS (ESI+) m/z Calculated for C15H20O5 [M + Na]+ : 303.1203, found 303.1207.

3-O-benzyl-1,2-O-isopropylidene-β-D-xylofuranose (21)

To a suspension of 3-O-Benzyl-1,2-O-isopropylidene-β-D-xylofuranose (4 g, 14.26 mmol) in benzoyl chloride (1.64 g, 2 mL,17.12 mmol), was added sulfamic acid (0.55 g, 5.70 mmol). The reaction mixture was stirred at 60°C for 3 h, allowed to come to room temperature and the reaction mixture was poured into ice cold saturated NaHCO3 solution, and extracted with minimum amount of diethyl ether. The organic solvents were dried on Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using eluents hexane:ethyl acetate (8:2) to obtain the compound 21[36] (4.8 g, 91%). 1H NMR (300 MHz, CDCl3, ppm): δ 8.01 (d, 2H), 7.54-7.51 (m, 1H), 7.40-7.37 (m, 2H), 7.30-7.26 (m, 4H), 7.24 (d, 1H), 5.99 (d, 1H), 4.70-4.60 (m, 3H), 4.57-4.48 (m, 3H), 4.04 (br s, 1H), 1.49 (s, 3H, CH3), 1.32 (s, 3H, CH3). 13C NMR (300 MHz, CDCl3, ppm): δ166.21, 137.16, 133.03, 129.88, 129.75, 128.53, 128.31, 128.07, 127.77, 111.82, 105.32, 82.13, 81.52, 78.14, 71.88, 26.83, 26.25.

(9-(2′-O-acetyl-3′-O-benzyl-5′-O-benzoyl) β-D-xylofuranosyl) 6-N-benzoyladenine (23)

To a cooled solution of 5-O-benzoyl-3-O-benzyl-1,2-O-isopropylidene β-D-xylofuranose (4 g, 10.40 mmol) in AcOH (40 mL) and Ac2O (15 mL) was added drop wise sulfuric acid (1.5 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 CHCl3 (3 × 100mL), washed with saturated NaHCO3 (200 mL) and dried over Na2SO4. 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 N6-benzoyladenine (4.2 g, 17.50 mmol) in anhydrous acetonitrile (60 mL) was added drop wise 1 M SnCl4 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 NaHCO3 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 CHCl3 (3x100 mL). The combined filtrate was washed successively with saturated aq NaHCO3 (3 × 100 mL) and brine (2x100 mL), dried (Na2SO4). The filtrates were concentrated under reduced pressure. The residue obtained was purified by silica gel column chromatography using 3%MeOH in CHCl3 as eluent (Rf-0.5), affording nucleoside 23[36] (6.12 g, 86%) as a colorless solid. 1H NMR (500 MHz, DMSO- DMSO-d6, ppm): δ 9.36 (br s, 1H, -NH), 8.77 (s, 1H, H-2), 8.42 (s, 1H, H-8), 8.03 (dd, 5H, Ar), 7.59 (q, 2H, Ar), 7.50 (t, 2H, Ar), 7.43 (t, 2H, Ar), 7.27 (m, 5H, Ar), 6.44 (s, 1H, H-1′), 5.54 (s, 1H, H-2′), 4.76 (dd, 2H), 4.74 (dd, 1H), 4.65 (m, 1H), 4.16 (d, 1H),2.18 (s, 3H, CH3). 13C NMR (500 MHz, DMSO- d6, ppm): δ 169.69 (C = O, CH3), 166.38 (C = O, Ar), 164.63 (C = O, Ar-NH), 153.03 (C-2), 151.57 (C-6), 149.58 (C-4), 141.81 (C-8), 136.24 (CH-Ar), 133.84 (CH-Ar), 133.45 (CH-Ar), 132.92 (CH-Ar), 129.88 (CH-Ar), 129.68 (CH-Ar), 129.02 (CH-Ar), 128.84 (CH-Ar), 128.59 (CH-Ar), 128.39 (CH-Ar), 127.99 (CH-Ar), 122.92 (CH-Ar), 87.82(1′-C), 81.18 (4′-C), 79.98 (3′-C), 79.75 (C-2′), 72.36 (CH2-Ar), 62.43 (C-5′), 20.99 (CH3). HRMS (ESI+) m/z Calculated for C33H29N5O7 (MH+): 608.2139, found 608.2135.

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 CHCl3 to afford nucleoside 24 (0.8 g, 68%). 1H NMR (500 MHz, DMSO-d6, ppm): δ 8.17 (s, 1H, H-2), 8.14 (s, 1H, H-8), 7.35-7.27 (m, 7H, Ar, -NH2), 6.04 (d, 1H, 2′-OH), 5.95 (s, 1H, 1′-H), 4.94 (br, 2H, 5′-OH), 4.68 (s, 1H, 2′-H), 4.67 (dd, 2H, CH2), 4.34 (m, 1H, 4′-H), 4.05 (br s, 1H, 3′-H), 3.81-3.73 (m, 5′-H, 5″-H). 13C NMR (500 MHz, DMSO- DMSO-d6, ppm): δ 156.04 (C-6), 152.67(C-2), 149.21 (C-4), 139.01 (C-8), 138.04(-C-, Ar), 128.29(-CH, Ar), 127.56 (-CH, Ar), 127.40 (-CH, Ar), 118.67(C-5), 88.74 (1′-C), 82.68 (3′-C), 77.26 (2′-C), 71.29 (CH2-Ar), 59.44 (5′-C). HRMS (ESI+) m/z Calculated for C17H19N5O4 (MH+): 358.1509, found 358.1510.

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, N- dimethylformamide was added tert-butyldimethylchlorosilane (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 Na2SO4 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%). 1H NMR (500 MHz, CDCl3, ppm): δ 8.16 (s, 1H, H-2), 8.11 (s, 1H, H-8), 7.33 (m, 7H, Ar, -NH2), 6.05 (d, 1H, 2′-OH), 5.96 (d, 1H, 1′-H), 4.69 (s, 2H, 2′-H), 4.66-4.53 (m, Ar-CH2), 4.32 (m, 1H, 4′-H), 4.06 (m, 1H, 3′-H), 4.00-3.86 (m, 2H, 5′-H, 5″-H), 0.85 (s, 9H, -(CH3)3), 0.02 (s, 6H, -(CH3)2). 13C NMR (500 MHz, DMSO-d6, ppm): δ 155.85(C-6), 153.79 (-C = O, Cbz) 153.16(C-2), 149.52 (C-4), 139.04(C-8), 136.70 (-C-), 134.41 (-C-), 128.83 (-CH-, Ar), 128.68 (-CH-, Ar), 128.53 (-CH-, Ar), 128.50, (-CH-, Ar) 128.17 (-CH-, Ar), 127.91 (-CH-, Ar), 119.30 (C-5), 86.89 (C-1′), 83.31 (C-4′), 82.91 (C-3′), 79.92 (C-2′), 72.45 (-CH2, Bn), 70.57 (-CH2, Cbz), 60.37 (C-5′), 25.88 (-(CH3)3), 18.29 (-C-), -5.31 (CH3), -5.42 (CH3). HRMS (ESI+) m/z Calculated for C23H33N5O4Si1 (MH+): 472.2374, found: 472.2379.

(9-(5′-O-tert-butyldimethylsilyl-3′-O-benzyl-2′-O-benzyloxycarbonyl) β-D-xylofuranosyl) adenine (26)

To a solution of 9-(5′-O-tert-butyldimethylsilyl-3′-O-Benzyl-2′-O-benzyloxycarbonyl-β-D-xylofuranosyl)-adenine (2.5 g, 53 mmol) and DMAP (1.3 g, 10.60 mmol) in 50 mL of anhydrous dichloromethane was added benzyl chloroformate (CbzCl, 1.8 g, 1.78 mL, 10.60 mmol) at 0°C under argon atmosphere. After stirring for 72 hours at room temperature, the reaction mixture was diluted with DCM (200 mL) and washed with cold 1.0 M HCl aqueous solution (50 mL) and then with water (100 mL). The organic layer was dried over anhydrous NaSO4, filtered, and concentrated under reduced pressure. The residue obtained, was purified by silica gel column chromatography (hexane/EtOAc-10:1 to 1:1 v/v) to obtain 26 (2.85 g, 89%). 1H NMR (500 MHz, CDCl3, ppm): δ 8.25 (s, 1H, H-2), 8.05 (s, 1H, H-8), 7.28-7.17 (m, 10H, Ar), 6.38 (br, m, 2H, -NH2), 6.29 (s, 1H, 1′-H), 5.11 (s, 2H, CH2-Cbz) 4.64 (s, 2H, 2′-H), 4.61-4.52 (m, Ar-CH2), 4.27 (m, 1H, 4′-H), 4.10 (d, 1H, 3′-H), 3.97-3.87 (m, 2H, 5′-H, 5″-H), 0.82 (s, 9H, -(CH3)3), 0.01 (s, 6H, -(CH3)2). 13C NMR (500 MHz, CDCl3, ppm): 155.85, 153.79, 153.16, 149.52, 139.04, 136.70, 134.41, 128.83, 128.68, 128.53, 128.50, 128.17, 127.91, 119.30, 86.89, 83.31, 82.91, 79.92, 72.45, 70.57, 60.37, 25.88, 18.29, -5.31, -5.42. HRMS (ESI+) m/z Calculated for C31H39N5O6Si1 (MH+): 606.2742, found: 606.2729.

(9-(3′-O-benzyl-2′-O-benzyloxycarbonyl) β-D-xylofuranosyl) adenine (27)

To a solution of 26 (2 g, 33 mmol) in 30 mL of anhydrous THF was added 1 M TBAF in THF (8.63 g, 330 mmol) at 0°C under N2 atmosphere. The solution was stirred for 12 h at room temperature, and all volatiles were removed using a rotary evaporator. The residue was dissolved in EtOAc (100 mL) and washed with cold saturated NaHCO3 solution (30 mLx2), and brine (30 mL). The organic solvent was dried over Na2SO4 and filtered. The filtrate was concentrated and the obtained residue was purified by silica gel column chromatography (CH2Cl2/MeOH-9:1) to give 27 (1.41 g) as white solid in 86% yield. 1H NMR (500 MHz, CDCl3, ppm): δ 8.15 (s, 1H, H-2), 8.14 (s, 1H, H-8), 7.37-7.28 (m, 10H, 2xAr), 6.20 (s, 1H, 1′-H), 5.71 (t, 1H, 2′-H), 5.17 (s, 2H, -NH2), 5.03 (t, 1H, 5′-OH), 4.75 (dd, 2H, CH2-Ar), 4.38 (m, 1H, 4′-H), 4.30 (m, 1H, 3′-H), 3.78 (m, 2H, 5′-H, 5″-H). 13C NMR (500 MHz, CDCl3, ppm): 156.05, 153.24, 152.81, 149.14, 138.68, 137.57, 134.96, 128.53, 128.51, 128.32, 128.31, 127.70, 127.53, 118.52, 85.67, 82.58, 81.98, 79.90, 71.46, 69.75, 59.07. HRMS (ESI+) m/z Calculated for C25H25N5O6 (MH+):492.1877, found: 492.1881.

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. 1H NMR (300 MHz, DMSO-d6, ppm): δ 8.33 (m, 1H, H-2), 8.18 (m, 1H, H-8), 7.38 -7.26 (m, 15H, Ar-H), 5.64 (br s, 1H), 5.34-5.04 (m, 6H, 3xCH2), 4.90-4.82 (m, 1H), 4.32 (m, 2H), 2.17 (s, 1H), 1.78 (m, 2H), 1.25 (m, 28H, CH2), 0.89 (t, 3H, CH3). 13C NMR (500 MHz, DMSO-d6, ppm): δ 170.16, 155.59, 154.37, 153.28, 139.08, 138.93, 134.83, 128.85, 128.72, 128.55, 128.18, 128.13, 128.01, 127.97, 120.04, 84.20, 83.03, 82.92, 70.31, 67.40, 37.55, 33.09, 32.06, 29.84, 29.79, 29.65, 29.49, 19.16, 24.66, 22.82, 14.24. HRMS (ESI+) m/z Calculated for C50H67N5O10P1 (MH+): 928.4619, found 928.4582.

2′-deoxyadenosine-5′-(O-stearic acid-3-yl) phosphate (30)

To the solution of 29 (0.2 g, 0.21 mmol) in THF, K2CO3 (60 mg, 4.31 mmol) and water (2 mL) is added, followed by Palladium (10%) on charcoal, and stirring continued at room temperature under Hydrogen for 72 h. After completion of the reaction, the mixture is filtered on celite 545, and the celite pad was washed with THF:Water-1:1, and the filtrate was evaporated and purified by column chromatography using eluent DCM:MeOH:H2O-17:7:1 to obtain the potassium salt of 30 as white solid. 1H NMR (500 MHz, DMSO-d6, ppm): δ 8.37 (d, 1H, H-2), 8.12 (d, 1H, H-8), 7.25 (t, 2H), 6.37 (m, 1H), 5.43 (br s, 1H), 4.44 (br, 1H) 4.29 (m, 1H), 3.96 (t, 1H), 3.90-3.85 (m, 1H), 3.78 (m, 1H), 2.74 (m, 1H), 2.28 (m, 1H), 1.66 (m, 1H), 1.511 (m, 1H), 1.23 (m, 28H), 0.86 (t, 3H). 13C NMR (500 MHz, DMSO-d6, ppm): 172.98, 172.89, 156.12, 152.02, 152.53, 152.39, 149.22, 149.17, 148.91, 139.57, 139.24, 139.11, 119.28, 119.00, 118.94, 88.02, 85.97, 83.96, 83.17, 73.49, 71.17, 71.14, 71.00, 65.44, 65.18, 61.92, 50.01, 31.31, 29.06, 28.91, 28.85, 28.83, 28.72, 24.65, 22.12, 13.99. 31P NMR (500 MHz, DMSO-d6, ppm): 1.31, 1.21. HRMS (ESI-) m/z Calculated for C28H47N5O8P1 (MH-): 612.3167, found 612.3171.

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%). H1 NMR (CDCl3, 300 MHz, ppm): δ 8.85 (br s, 1H, -NH), 7.38 (m, 15H, 3xAr), 6.38 (m, 1H), 5.20 (m, 7H), 4.89 (m, 1H), 4.30 (m, 3H), 1.87 (m, 5H), 1.25 (m, 29H), 0.89 (t, 3H). 13C NMR (CDCl3, 300 MHz, ppm): δ 171.15, 169.83, 169.73, 163.87, 163.78, 154.36, 154.33, 150.55, 150.51, 150.49, 150.47, 135.40, 135.36, 135.14, 135.08, 135.01, 134.93, 134.79, 134.72, 128.97, 128.93, 128.85, 128.76, 128.68, 128.51, 128.45, 128.14, 128.11, 127.99, 127.95, 111.86, 111.84, 111.63, 84.44, 82.60, 70.22, 70.17, 69.83, 67.41, 60.42, 37.26, 31.97, 29.75, 29.71, 29.67, 29.56, 29.40, 29.09, 24.67, 24.58, 22.74, 21.07, 21.05, 19.12, 14.24, 14.16, 12.40. 31P NMR (500 MHz, CDCl3, ppm): δ -1.00, -1.24, -1.80, -1.83. HRMS (ESI+) m/z Calculated for C50H67N2O12P1 (MH+): 919.4504, found 919.4512.

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:H2O-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%). 1H NMR (300 MHz, D2O, ppm): δ 7.82 (d, 1H, NH), 6.28 (d, 1H, H-5), 4.55 (br m, 2H), 4.14 (br m, 3H), 2.37 (m, 2H, 5′-H), 1.94 (m, 3H, CH3), 1.77 (br s, 2H, CH2), 1.42 (br, 2H), 1.18 (m, 26H, 13xCH2), 0.81 (t, 3H, CH3). 13C NMR (300 MHz, D2O, ppm): δ 173.02, 165.43, 150.90, 136.63, 110.88, 110.77, 85.53, 84.89, 74.38, 70.84, 70.60, 64.69, 39.23, 38.98, 33.09, 31.58, 29.53, 29.40, 29.27, 29.09, 24.74, 22.27, 13.53, 11.69, 11.61. 31P NMR (300 MHz, D2O, ppm): δ -0.80, -0.95. HRMS (ESI+) m/z Calculated for C28H49N2O10P1 (MH-): 603.3051, found 603.3054.

2′,3′-O-bisbenzyloxycarbonyl, adenosine-5′-((O-benzyl stearate)-2-yl, O-benzyl) phosphate (35)

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. 1H NMR (500 MHz, CDCl3, ppm): δ 8.28 (d, 1H, H-2), 8.07 (d, 1H, H-8), 7.34-7.29 (m, 20H, Ar), 6.21 (dd, 1H), 5.92 (m, 1H), 5.85 (br s, 2H), 5.65 (m, 1H), 5.18 (m, 1H), 5.12 (m, 6H), 5.87(m, 1H), 4.38 (m, 2H), 1.78 (m, 2H), 1.25 (m, 28H), 0.89 (t, 3H, CH3). 13C NMR (500 MHz, CDCl3, ppm): δ 170.13, 155.65, 154.09, 153.73, 153.47, 150.02, 139.38, 139.28, 135.27, 134.75, 134.59, 128.88, 128.85, 128.77, 128.71, 128.67, 128.600, 128.52, 128.11, 127.98, 127.92, 120.13, 85.63, 80.64, 76.17, 76.06, 73.98, 73.79, 70.63, 70.57, 69.83, 67.43, 66.2633.02, 32.97, 32.05, 29.83, 29.79, 29.66, 29.48, 29.45, 29.15, 24.64, 24.59, 22.81, 14.24. 31P NMR (500 MHz, CDCl3):-1.57,-1.61, -2.09. HRMS (ESI+) m/z Calculated for C58H72N5O13P1 (MH+): 1078.4936, found 1078.4946.

Adenosine-5′-(O-stearic acid-2-yl) phosphate (36)

To a solution of 35 (2 g, 1.85 mmol) in THF, K2CO3 (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. The filtrate was concentrated in vacuo and purified by silica gel column chromatography (DCM:MeOH:H2O-17:7:1) to obtain the potassium salt of 33 as white solid (0.62 g, 53%). 1H NMR (500 MHz, CDCl3, ppm): δ 8.4 (br s, 1H, H-2), 8.13 (s, 1H, H-8), 7.21 (br s, 2H, -NH2), 5.92 (d, 1H, H-1′), 5.32 (m, 1H), 4.61 (br, 1H), 4.21 (br s, 1H), 4.04 (s, 1H), 3.90 (br s, 1H), 2.08 (s, 1H), 1.68 (br, 1H), 1.52 (br, 1H), 1.22 (m, 28H), 0.86 (t, 3H, CH3).

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. 1H NMR (300 MHz, CDCl3, ppm): δ7.37-7.28 (m, 15H), 6.45 (m, 2H), 5.31 (d, 1H), 5.13-4.98 (m, 6H, 3 × CH2), 4.29-4.20 (m, 1H), 2.78-2.69 (m, 1H), 2.68-2.54 (m, 1H), 1.65 (m, 2H, CH2), 1.25 (m, 26H, 13xCH2), 0.89 (t, 3H, CH3). 13C NMR (300 MHz, CDCl3, ppm): δ 169.89, 155.84, 154.25, 153.16, 149.69, 138.63, 135.73, 134.76, 128.77, 128.69, 128.57, 128.52, 128.40, 128.35, 128.26, 128.24, 128.13, 127.99, 127.96, 127.88, 127.81, 119.89, 84.08, 83.97, 82.93, 82.82, 78.32, 78.25, 76.52, 76.45, 76.38, 70.12, 69.62, 69.54, 69.46, 66.63, 40.15, 37.46, 35.25, 31.91, 29.69, 29.65, 29.54, 29.43, 29.41, 29.35, 29.28, 24.79, 22.68, 14.12. 31 P NMR (500 MHz, CDCl3, ppm): δ -1.92, -1.98, -2.17, -2.31. HRMS (ESI+) m/z Calculated for C50H66N5O10P1 (MH+): 928.4619, found 928.4615.

2′-deoxyadenosine-5′-(O-stearic acid-3-yl) phosphate (39)

To a stirring solution of 38 (0.2 g, 0.21 mmol) in THF, K2CO3 (60 mg, 4.31 mmol) and water (2 mL) is added. To this Pd (10%) on charcoal is added and kept for stirring at rt under hydrogen for 12 h. After completion, the reaction mixture is filtered on celite-545 and the solvent is evaporated and purified by column chromatography (DCM:MeOH:H2O-17:7:1) to obtain the potassium salt of 39 as white solid. 1H NMR (500 MHz, D2O, ppm): δ 8.48 (d, 1H), 8.09 (d, 1H), 6.42 (d,1H), 4.61 (br s, 1H), 4.26 (br s, 1H), 4.16 (br m, 2H, -NH2), 2.64 (m, 3H), 1.68 (m, 2H), 1.25 (m, 2H), 1.31-1.22 (m,2H), 1.01 (m, 18H, CH2), 0.66 (t, 3H, CH3). 13C NMR(500 MHz, D2O, ppm): 176.53, 154.04, 150.78, 147.64, 139.41, 117.66, 85.66, 83.89, 74.11, 70.77, 70.49, 64.73, 61.14, 58.56, 41.62, 40.05, 39.94, 35.24, 35.05, 33.61, 31.40, 29.34, 29.23, 28.91, 28.02, 24.66, 22.09, 13.37. 31P NMR(500 MHz, D2O, ppm): δ -1.04, -1.12. HRMS (ESI-) m/z Calculated for C28H48N5O8P1 (MH-): 612.3167, found: 612.3173.

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. 1H NMR (500 MHz, CDCl3, ppm): δ 9.27 (br s, 1H, NH), 7.43-7.31 (m, 15H, 3 × Ar-H), 6.37 (m, 1H), 5.16 (s, 2H, Ar-CH2), 5.12-5.03 (m, 4H, Ar-CH2), 4.86 (m, 1H), 4.18 (m, 2H), 2.74-2.64 (m, 2H), 2.36 (m, 1H),2.03 (m, 1H), 1.89 (d, 2H), 1.69 (m, 2H), 1.25 (m, 26H, -CH2), 0.89 (t, 3H, CH3). 1H NMR (300 MHz, CDCl3, ppm): δ 169.87, 163.75, 154.36, 150.49, 135.63, 135.59, 135.11, 135.02, 134.76, 134.73, 128.85, 128.76, 128.68, 128.63, 128.50, 128.46, 128.36, 128.28, 128.09, 128.01, 127.95, 111.81, 111.73, 84.52, 84.44, 82.59, 82.48, 78.07, 78.02, 70.22, 70.20, 69.87, 69.80, 69.70, 69.63, 67.13, 67.06, 67.00, 66.72, 66.68, 40.25, 40.18, 40.12, 37.28, 37.20, 35.39, 31.97, 29.75, 29.71, 29.69, 29.60, 29.51, 29.41, 29.38, 24.91, 24.85, 22.71, 14.18, 12.44. 31P NMR(300 MHz, CDCl3): δ -1.68, -1.86, -2.06, -2.25. HRMS (ES+) m/z Calculated for C50H67N2O12P1 (MH+): 919.4504, found 919.4498.

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:H2O-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%). 1H NMR (500 MHz, D2O, ppm): δ 7.84 (br m, 1H), 6.32 (br m, 1H), 4.60 (br m, 2H), 4.16 (m, 3H), 2.61 (m, 2H), 2.39 (m, 2H), 1.94 (s, 3H), 1.72 (m, 2H), 1.21 (m, 26H, CH2), 0.83 (s, 3H, CH3). 13C NMR (500 MHz, D2O, ppm): δ 174.93, 165.58, 151.16, 136.86, 111.04, 85.69, 85.20, 84.89, 73.60, 71.08, 70.80, 65.06, 64.67, 40.54, 39.33, 35.21, 31.78, 29.70, 29.60, 29.28, 24.80, 22.46, 13.74, 11.86. 31P NMR(500 MHz, CDCl3): δ -1.13, -1.15. HRMS (ES+) m/z Calculated for C28H49N2O10P1 (MH-): 603.3051, found 603.3048.

2′,3′-O-dibenzyloxycarbonyl, adenosine-5′-(O-(benzyl stearate)-3-yl, O-benzyl) phosphate (44)

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. 1H NMR (500 MHz, CDCl3, ppm) : δ 8.27 (s, 1H, H-2), 8.01-7.97 (s, 1H, H-8), 7.33-7.25 (m, 20H, 4xAr-H), 6.19 (d, 1H), 5.93-5.91 (m, 3H), 5.62 (m, 1H), 5.12-5.01 (m, 8H, CH2-Ar), 4.81 (m, 1H), 4.38 (m, 1H), 4.30-4.28 (m, 1H), 4.24-4.22 (m, 1H), 2.7-2.70 (m, 1H), 2.61-2.57 (m, 1H), 2.21 (br s, 1H), 1.67-1.60 (m, 2H), 1.25 (m, 26H), 0.89 (t, 3H, CH3).13C NMR (500 MHz, CDCl3, ppm): 170.05, 155.70, 154.00, 153.72, 153.48, 149.90, 139.26, 135.78, 134.69, 134.54, 128.85, 128.75, 128.70, 128.64, 128.54, 128.52, 128.40, 128.06, 127.98, 120.16, 85.83, 85.79, 85.63, 80.65, 80.59, 76.66, 73.79, 70.65, 70.53, 69.67, 66.64, 65.94, 40.22, 35.31, 32.03, 29.81, 29.77, 29.68, 29.61, 29.54, 29.47, 29.40, 24.86, 22.79, 14.23. 31P NMR (500 MHz, CDCl3, ppm): δ (q -1.96, -2.07, -2.25, -2.39). HRMS(ES+) m/z Calculated for C58H72N5O13P1 (MH+): 1078.4937, found 1078.4946.

Adenosine-5′-(O-stearic acid-3-yl)-phosphate (45)

To the solution of 44 (0.3 g, 0.27 mmol) in THF, K2CO3 (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: H2O-17:7:1) to obtain the potassium salt of 45 as white solid. 1H NMR (500 MHz, D2O, ppm): δ 8.42 (d, 1H, H-2), 8.12 (s, 1H, H-8), 7.24 (br s, 2H, -NH2), 5.91 (d, 1H), 4.53 (m, 1H), 4.19 (m, 2H), 4.01 (br s, 1H), 3.93 (m, 2H), 2.68 (m, 1H), 2.40 (ddd, 1H), 1.91 (m, 1H), 1.60 (m, 1H), 1.45 (m, 1H), 1.23 (m, 26H, CH2), 0.86 (t, 3H, CH3). 13C NMR (500 MHz, D2O, ppm): δ 172.50, 165.45, 155.98, 152.55, 149.58, 139.32, 139.19, 127.96, 126.94, 118.83, 86.93, 83.74, 74.01, 73.88, 71.17, 70.82, 70.64, 67.48, 64.73, 64.41, 60.48, 53.30, 47.85, 43.37, 42.56, 35.52, 31.28, 29.04, 28.69, 24.89, 22.08, 13.95. 31P NMR (500 MHz, D2O): δ -0.69. HRMS(ES-) m/z Calculated for C28H48N5O9P1 (MH-): 628.3116, found 594.3061.

9-((O-benzyl, O-(O-benzyl stearate)-2-yl-5′ phospho), 3′-O-benzyl, 2′-O-benzyloxycarbonyl xylofuranosyl) adenine (46)

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. 1H NMR (300 MHz, CDCl3, ppm): δ 8.34 (s, 1H, H-2), 8.09 (m, 1H, H-8), 7.38-7.21 (m, 20H, Ar-H), 6.33 (m, 1H), 5.93 (br s, 1H), 5.41 (br s, 1H), 5.20-5.02 (m, 6H, CH2-Ar), 4.83 (m, 1H), 4.67 (m, 1H), 4.54 (m, 1H), 4.44 (m, 3H), 4.09 (br m, 1H), 1.80 (m, 2H), 1.25 (m, 26H, CH2), 0.87 (m, 3H, CH3). 13C NMR (300 MHz, CDCl3, ppm): δ 170.03, 155.58, 153.84, 153.37, 149.69, 139.24, 139.15, 136.38, 136.27, 135.73, 135.65, 135.27, 134.46, 129.04, 128.85, 128.67, 128.57, 128.47, 128.42, 128.19, 128.12, 128.06, 127.92, 119.54, 87.38, 87.33, 82.60, 82.52, 81.34, 81.23, 81.16, 80.02, 79.94, 76.04, 75.97, 72.40, 70.80, 69.87, 69.82, 69.72, 69.65, 67.25, 65.37, 65.30, 65.21, 65.10, 65.03, 33.06, 32.97, 32.02, 29.80, 29.75, 29.71, 29.60, 29.45, 29.41, 29.11, 24.60, 22.78, 14.22. 31P NMR (300 MHz, CDCl3, ppm): δ -1.58, -1.68, -1.82, -1.91. HRMS (ESI+) m/z Calculated for C57H72N5O11P1 (MH+):1034.5038, found: 1034.5048.

9-xylofuranosyl adenine-5′-(O-stearic acid-2-yl) phosphate (47)

To a solution of 46 (100 mg, 0.09 mmol) in THF, K2CO3 (27 mg, 0.18 mmol) and water (2 mL) is added. To this Palladium (10%) on charcoal (10 mg) is added and held for stirring at rt under hydrogen for 12 h. After the completion of the reaction, the mixture is filtered on celite and the solvent is removed in vacuo. The obtained residue was purified by column chromatography with the eluent (DCM:MeOH:H2O-17:7:1) to obtain the potassium salt of 47 as white solid. 1H NMR (300 MHz, CDCl3, ppm): δ 8.35 (m, 1H, H-2), 8.13 (s, 1H, H-8), 7.31 (d, 2H), 5.91 (br s, 1H), 4.31 (m, 2H), 4.12 (m, 2H), 4.05 (m, 1H), 3.87 (m, 1H), 3.80-3.61 (m, 1H), 1.63 (m, 2H), 1.19 (m, 24H, CH2), 0.85 (t, CH3). 13C NMR (300 MHz, CDCl3, ppm): δ 156.06, 155.96, 152.43, 149.02, 148.78, 139.79, 118.44, 89.42, 83.62, 82.01, 80.93, 75.05, 74.87, 60.64, 59.27, 54.96, 31.34, 29.20, 28.78, 28.28, 22.12, 13.92. 31P NMR (300 MHz, CDCl3, ppm): δ 1.67, 1.36. HRMS (ESI-) m/z Calculated for C28H48N5O9P1 (MH-): 628.3116, found: 628.3123.

9-((O-benzyl, O-(O-benzyl stearate)-3-yl-5′ phospho), 3′-O-benzyl, 2′-O-benzyloxycarbonyl xylofuranosyl) adenine (48)

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. 1H NMR (500 MHz, CDCl3, ppm): δ 8.33 (s, 1H, H-2), 8.10 (m, 1H, H-8), 7.38-7.19 (m, 20H, 4 × Ar), 6.33 (d, 1H, H-1′), 6.14 (br s, 2H, -NH2), 5.42 (br s, 1H), 5.19 (m, 2H), 5.08-5.00 (m, 4H), 4.83 (m, 1H), 4.66 (dd, 1H), 4.54 (dd, 1H), 4.38 (m, 1H), 4.35 (m, 2H), 4.10 (m, 1H), 3.55-3.46 (m, 1H), 2.77-2.72 (m, 1H, H-5′), 2.63-58 (m, 1H, H-5″), 1.66 (m, 2H, CH2), 1.25 (m, 28H, CH2), 0.89 (t, 3H, CH3). 13C NMR (500 MHz, CDCl3, ppm): δ 169.91, 155.68, 153.75, 153.22, 149.53, 139.02, 136.24, 135.82, 135.62, 134.36, 128.95, 128.75, 128.63, 128.58, 128.57, 128.54, 128.36, 128.27, 128.12, 128.10, 128.00, 127.92, 128.86, 119.41, 87.25, 82.44, 81.24, 79.91, 76.28, 72.27, 70.69, 69.47, 66.54, 64.92, 40.33, 35.26, 31.94, 29.71, 29.71, 29.68, 29.64, 29.56, 29.45, 29.37, 29.31, 24.76, 22.98, 22.71, 14.16. HRMS (ES-) m/z Calculated for C57H72N5O11P1 (MH+): 1034.5038, found: 1034.5026.

β-D-xylofuranosyladenine-5′-(O-stearic acid-3-yl)-phosphate (49)

To the solution of 48 (0.2 g, 0.21 mmol) in THF, K2CO3 (60 mg, 4.31 mmol) and water (2 mL) is added. To this Pd (10%) on charcoal is added and held for stirring at rt under hydrogen for 12 h. After the completion of the reaction, the mixture is filtered on celite-545 and the solvent is evaporated and the residue is purified on column chromatography (DCM:MeOH:H2O-17:7:1) to obtain the potassium salt of 49 as white solid. H1 NMR (500 MHz, DMSO-d6, ppm): δ 8.26 (m, 1H), 8.11 (d, 1H, H-8), 5.98 (br s, 1H), 4.63 (m, 2H), 4.46 (m, 2H), 4.25 (m, 2H), 3.60 (t, 1H), 2.44 (m, 2H), 1.78 (t, 1H), 1.55 (m, 2H), 1.06 (m, 26H, CH2), 0.74 (t, 3H, CH3). 13C NMR (600 MHz, DMSO-d6, ppm): δ 179.31, 162.09, 154.98, 152.10, 152.03, 148.07, 147.75, 139.95, 139.86, 118.24, 118.09, 89.68, 88.46, 81.43, 81.39, 80.54, 80.11, 79.59, 75.66, 75.53, 74.57, 62.98, 62.75, 61.24, 48.57, 44.01, 35.17, 33.73, 31.45, 29.33, 29.24, 28.93, 28.12, 24.69, 24.63, 22.97, 22.17, 13.51. 31P NMR (500 MHz, D2O, ppm): -0.94, -1.09. HRMS (ES-) m/z Calculated for C28H48N5O9P (MH+): 628.3117, found: 628.3119.

2′-deoxyadenosine-5′-(N-(methyl stearate)-2-yl)-phosphoramidate (51)

In a 100 mL two neck flask, 2′-deoxyadenosine 5′-O-monophosphate (0.3 g, 0.9 mmol) and methyl α-aminostearate 50 (1.42 g, 4.52 mmol) were dissolved in a mixture of t- butanol and water (5:1). Triethylamine (0.5 mL), and a freshly prepared solution of N, N’-dicyclohexylcarbodiimide (DCC) in t-butanol (0.5 g/mL) was added to the reaction mixture under argon atmosphere, and the reaction mixture was allowed to reflux for 2 h. The progress of the reaction was monitored by TLC. Another 5 eq of DCC was added to the reaction mixture, and was continued refluxed under argon for 1 h. Upon completion, the reaction mixture was cooled down to room temperature and the solvent was concentrated under vacuo. The residue obtained was purified by silica gel column chromatography (DCM:MeOH:H2O-17:7:1) to obtain 51 (0.44 g, 77%) as white solid. 1H NMR (500 MHz, DMSO, ppm): δ 8.38 (d, 1H, H-2), 8.12 (s, 1H, H-8), 7.22 (br s, 2H), 6.35 (t, 1H), 4.41 (s, 1H), 3.92 (br s, 1H), 3.78-3.74 (m, 2H), 3.67 (m, 1H), 3.55 (s, 2H), 3.52 (s, 1H), 3.02 (dd, 3H), 2.69-2.63 (m, 1H), 1.47 (m, 2H), 1.22-1.14 (m, 28H, 14xCH2), 0.85 (t, 3H, CH3). 13C NMR (500 MHz, DMSO, ppm): δ 175.11, 155.95, 152.50, 149.18, 139.20, 118.86, 117.45, 82.97, 71.34, 64.14, 54.47, 51.28, 45.20, 31.26, 29.01, 28.87, 28.73, 28.67, 24.94, 22.07, 13.93, 8.46. 31P NMR (500 MHz, DMSO): δ 4.51. HRMS (ESI+) m/z Calculated for C29H50N6O7P1(MH-): 625.3483 found 625.3453.

2′-deoxyadenosine-5′-(N-stearic acid-2-yl)-phosphoramidate (52)

A solution of 51 (0.4 g, 0.63 mmol) in MeOH/H2O (4:1 v/v, 3 mL, containing 0.4 M of NaOH (0.051 g, 1.27 mmol) was stirred at room temperature under nitrogen for 2 h. After completion of the reaction, the solvents were removed under reduced pressure. The resulted crude reaction mixture was purified by silica gel chromatography (DCM:MeOH:H2O-17:7:1) to obtain 52 (0.28 g, 71%) as white solid. 1H NMR (500 MHz, DMSO, ppm): δ 8.42 (d, 1H, H-2), 8.11 (d, 1H, H-8), 7.23 (br s, 2H, -NH2), 6.37 (m, 1H), 4.44 (d, 1H), 3.97 (m, 1H), 3.82-3.71 (m, 3H), 2.69 (m, 1H), 2.27 (m, 1H), 1.65 (br s, 1H), 1.21 (m, 28H), 0.85 (t, 3H). 13C NMR (500 MHz, DMSO, ppm): δ 176.18, 156.08, 152.59, 149.31, 139.31, 119.01, 86.45, 83.21, 71.62, 64.31, 64.09, 55.13, 54.78, 31.87, 31.39, 29.14, 25.90, 25.87, 22.20, 14.06. 31P NMR (500 MHz, DMSO): δ 7.08, 6.96. HRMS (ES-) m/z Calculated for C28H49N6O7P1 (MH-): 611.3327, found: 611.3323.

Thymidine-5′-(N-(methyl stearate)-2-yl)-phosphoramidate (53)

In a 100 mL two neck flask, 2′-deoxythymidine 5′-O-monophosphate (0.5 g, 1.55 mmol) and methyl α-aminostearate 50 (2 g, 6.20 mmol) were dissolved in a mixture of t- butanol and water (5:1). Triethylamine (0.5 mL), and a freshly prepared solution of N, N’-dicyclohexylcarbodiimide (DCC, 1.6 g, 7.75 mmol) in t- butanol (0.5 g/mL) was added to the reaction mixture under argon atmosphere, and the reaction was allowed to reflux for 2 h. Another 5 eq of DCC was added to the reaction mixture which was refluxed for 1 h. Upon completion, the reaction mixture was cooled down to room temperature and the reaction mixture was concentrated in vacuo. The residue obtained was purified by silica gel column chromatography (DCM:MeOH:H2O-17:7:1) to obtain 53 (0.69 g, 72%) as white solid. 1H NMR (500 MHz, DMSO): δ 10.82 (br s, 1H, -NH), 7.28 (d, 1H), 5.70 (m, 1H), 5.24 (s, 1H), 3.76 (br s, 1H), 2.67 (s, 2H), 1.56 (m, 2H), 1.32 (br s, 2H), 0.71 (m, 28H, 14xCH2), 0.34 (m, 3H, CH3).

13C NMR (300 MHz, DMSO, ppm): δ 175.26, 163.79, 150.50, 136.26, 109.62, 86.03, 85.92, 83.74, 70.97, 63.82, 54.54, 54.44, 51.19, 49.19, 45.10, 34.30, 31.28, 30.21, 29.03, 28.75, 28.70, 25.01, 24.62, 23.83, 22.07, 13.87, 12.06, 8.36. 31P NMR (300 MHz, DMSO, ppm): δ 3.70. HRMS (ES-) m/z Calculated for C29H52N3O9P1 (MH-): 616.3368, found: 616.3371.

Thymidine-5′-(N-stearic acid-2-yl)-phosphoramidate (54)

A solution of 53 (0.5 g, 0.8 mmol) in MeOH/H2O (4:1-v/v), was added 0.4 M NaOH (0.065 g, 1.61 mmol), and the mixture was stirred at room temperature under nitrogen for 2 h. The solvent was removed under reduced pressure. The resulting crude material was purified by chromatography (DCM:MeOH:H2O-17:7:1) to obtain 54 (0.34 g, 69%) as white solid. 1H NMR (500 MHz, DMSO): δ11.24 (s, 1H), 7.84 (s, 1H), 6.22 (q, 1H), 5.36 (br s, 1H), 4.31 (d, 1H), 3.86-3.72 (m, 1H), 2.12-2.01 (m, 2H), 1.80 (m, 1H), 1.76 (), 1.23 (m, 28H), 0.86 (t, 3H, CH3). 13C NMR (500 MHz, DMSO): 176.13, 163.89, 150.58, 136.39, 109.79, 86.12, 83.84, 71.35, 71.00, 31.38, 29.12, 26.03, 25.85, 22.18, 14.05, 12.16, 12.12. 31P NMR (500 MHz, DMSO): δ 7.29, 7.00. HRMS (ES-) m/z Calculated for C28H49N3O9P1 (MH-): 602.3211, found 602.3210.

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 NaHCO3 (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. 1H NMR(300 MHz, DMSO, ppm): δ 7.47 (br s, 1H, -CH), 7.36 (d, 1H, -CH), 7.24 (br s, 1H, -CH), 7.11 (d, 1H, -CH), 6.49-6.47 (m, 1H, -CH), 6.31-6.29 (m, 1H, -CH), 3.99 (t, 2H, -CH2), 2.92 (t, 2H, -CH2). 13C NMR (500 MHz, DMSO, ppm): δ 147.79, 132.15, 129.05, 122.45, 122.18, 114.57, 113.77, 112.42, 40.42.

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 D2O 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 D2O (0.1 M). 31P NMR was used to study the stability of the nucleolipids in acidic (pH4) and in base (pH12) environment. One-dimensional 31P spectra were used as a fast screening experiment to monitor degradation of the conjugates. Two-dimensional 1H-31P correlation spectra were used to characterize the 31P 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 31P resonances with several 1H 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 α- or β-hydroxyl 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 α-hydroxy 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.

Methods

All reactions were performed in an inert atmosphere (argon). Chemicals and solvents were purchased from Sigma-Aldrich, TCI Europe, and Alfa Aesar, and were used without further purification. Unless otherwise mentioned, each reaction vessel was oven dried prior to use. Column chromatography was performed using silica gel (63-200 mesh) obtained from the Sigma-Aldrich Company. Analytical thin-layer chromatography (TLC) was performed on Merck pre-coated aluminum plates (silica gel 60, F254) and visualized under 254-nm UV light. 1H, 31P and 13C NMR spectra were obtained on a Bruker 300 MHz and Bruker 500 MHz instrument at ambient temperature in CDCl3-d3, MeOD-d4, D2O and DMSO-d6. Chemical shifts are reported as values in parts per million (ppm) downfield from internal standard tetramethylsilane (δ = 0.0 ppm) from the residual solvent signal. Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; m, multiplet; dd, doublet of doublet; br, broad peak. NMR signal assignment of sugar protons and carbons are numbered with a prime. 31P NMR chemical shifts are referenced to an external 85% H3PO4 standard (d =0.000 ppm). Mass spectra were performed on a Hewlett Packard MALDI-TOF spectrometer. Fluorescent microscopy images were recorded by Olympus Fluoview FV 1000 spectrometry.

References

  1. 1.

    Del Bianco C, Mansy SS: Nonreplicating protocells. Acc Chem Res 2012, 45: 2125–2130. 10.1021/ar300097w

    CAS  Article  Google Scholar 

  2. 2.

    Mann S: Systems of creation: the emergence of life from nonliving matter. Acc Chem Res 2012, 45: 2131–2141. 10.1021/ar200281t

    CAS  Article  Google Scholar 

  3. 3.

    Walde P: Building artificial cells and protocell models: experimental approaches with lipid vesicles. Bioessays 2010, 32: 296–303. 10.1002/bies.200900141

    CAS  Article  Google Scholar 

  4. 4.

    Bachmann PA, Luisi PL, Lang J: Autocatalytic self-replicating micelles as models for prebiotic structures. Nature 1992, 357: 57–59. 10.1038/357057a0

    CAS  Article  Google Scholar 

  5. 5.

    Andes-Koback M, Keating CD: Complete budding and asymmetric division of primitive model cells to produce daughter vesicles with different interior and membrane compositions. J Am Chem Soc 2011, 133: 9545–9555. 10.1021/ja202406v

    CAS  Article  Google Scholar 

  6. 6.

    Chen IA, Roberts RW, Szostak JW: The emergence of competition between model protocells. Science 2004, 305: 1474–1476. 10.1126/science.1100757

    CAS  Article  Google Scholar 

  7. 7.

    Zhu TF, Szostak JW: Coupled growth and division of model protocell membranes. J Am Chem Soc 2009, 131: 5705–5713. 10.1021/ja900919c

    CAS  Article  Google Scholar 

  8. 8.

    Luisi PL, Rasi PS, Mavelli F: A possible route to prebiotic vesicle reproduction. Artif Life 2004, 10: 297–308. 10.1162/1064546041255601

    Article  Google Scholar 

  9. 9.

    Minshall RD, Malik AB: Transport across the endothelium: regulation of endothelial permeability. In Handbook of Experimental Pharmacology. Edited by: Moncada S, Higgs A. Springer-Verlag, Heidelberg, Germany; 2006:107–144.

    Google Scholar 

  10. 10.

    Hostetler KY, Richman DD, Sridhar CN, Felgner PL, Felgner J, Ricci J, Gardner MF, Selleseth DW, Ellis MN: Phosphatidylazidothymidine and phosphatidyl-ddC: assessment of uptake in mouse lymphoid tissues and antiviral activities in human immunodeficiency virus-infected cells and in rauscher leukemia virus-infected mice. Antimicrob Agents Chemother 1994, 38: 2792–2797. 10.1128/AAC.38.12.2792

    CAS  Article  Google Scholar 

  11. 11.

    Sunami T, Matsuura T, Suzuki H, Yomo T: Synthesis of functional proteins within liposomes. Methods Mol Biol 2010, 607: 243–256. 10.1007/978-1-60327-331-2_20

    CAS  Article  Google Scholar 

  12. 12.

    Kurihara K, Tamura M, Shohda K, Toyota T, Suzuki K, Sugawara T: Self-reproduction of supramolecular giant vesicles combined with the amplification of encapsulated DNA. Nat Chem 2011, 3: 775–781. 10.1038/nchem.1127

    CAS  Article  Google Scholar 

  13. 13.

    Chakrabarti AC, Breaker RR, Joyce GF, Deamer DW: Production of RNA by a polymerase protein encapsulated within phospholipid vesicles. J Mol Evol 1994, 39: 555–559. 10.1007/BF00160400

    CAS  Article  Google Scholar 

  14. 14.

    Mansy SS, Schrum JP, Krishnamurthy M, Tobe S, Treco DA, Szostak JW: Template-directed synthesis of a genetic polymer in a model protocell. Nature 2008, 454: 122–125. 10.1038/nature07018

    CAS  Article  Google Scholar 

  15. 15.

    Mansy SS, Szostak JW: Thermostability of model protocell membranes. Proc Natl Acad Sci U S A 2008, 105: 13351–13355. 10.1073/pnas.0805086105

    CAS  Article  Google Scholar 

  16. 16.

    Rasmussen S, Chen L, Nilsson M, Abe S: Bridging nonliving and living matter. Artificial Life 2003, 9: 269–316. 10.1162/106454603322392479

    Article  Google Scholar 

  17. 17.

    Bissette AJ, Fletcher SP: Mechanisms of autocatalysis. Angewandte Chemie-Int Edition 2013, 52: 12800–12826. 10.1002/anie.201303822

    CAS  Article  Google Scholar 

  18. 18.

    Yanagawa H, Ogawa Y, Furuta H, Tsuno K: Spontaneous formation of superhelical strands. J Am Chem Soc 1989, 111: 4567–4570. 10.1021/ja00195a006

    CAS  Article  Google Scholar 

  19. 19.

    Rosemeyer H: Nucleolipids: natural occurrence, synthesis, molecular recognition, and supramolecular assemblies as potential precursors of life and bioorganic materials. Chem Biodiversity 2005, 2: 977–1062. 10.1002/cbdv.200590082

    CAS  Article  Google Scholar 

  20. 20.

    Allain V, Bourgaux C, Couvreur P: Self-assembled nucleolipids: from supramolecular structure to soft nucleic acid and drug delivery devices. Nucleic Acids Res 2012, 40: 1891–1903. 10.1093/nar/gkr681

    CAS  Article  Google Scholar 

  21. 21.

    Gangadhara KL, Lescrinier E, Pannecouque C, Herdewijn P: Hydroxy fatty acids for the delivery of dideoxynucleosides as anti-HIV agents. Bioorg Med Chem Lett 2014, 24: 817–820. 10.1016/j.bmcl.2013.12.093

    CAS  Article  Google Scholar 

  22. 22.

    Maiti M, Michielssens S, Dyubankova N, Maiti M, Lescrinier E, Ceulemans A, Herdewijn P: Influence of the nucleobase and anchimeric assistance of the carboxyl acid groups in the hydrolysis of amino acid nucleoside phosphoramidates. Chemistry 2012, 18: 857–868. 10.1002/chem.201102279

    CAS  Article  Google Scholar 

  23. 23.

    Morasch M, Mast CB, Langer JK, Schilcher P, Braun D: Dry polymerization of 3′, 5′-cyclic GMP to long strands of RNA. Chembiochem 2014, 15: 879–883. 10.1002/cbic.201300773

    CAS  Article  Google Scholar 

  24. 24.

    Polyakova SM, Belov VN, Yan SF, Eggeling C, Ringemann C, Schwarzmann G, de Meijere A, Hell SW: New GM1 Ganglioside Derivatives for Selective Single and Double Labelling of the Natural Glycosphingolipid Skeleton. European Journal of Organic Chemistry 2009, 5162–5177.

  25. 25.

    Brooks DW, Lu LDL, Masamune S: C-acylation under virtually neutral conditions. Angew Chem Int Ed Engl 1979, 18: 72–74. 10.1002/anie.197900722

    Article  Google Scholar 

  26. 26.

    Qin J, Rao A, Chen XA, Zhu XH, Liu ZD, Huang XH, Degrado S, Huang Y, Xiao D, Aslanian R, Cheewatrakoolpong B, Zhang HT, Greenfeder S, Farley C, Cook J, Kurowski S, Li Q, van Heek M, Chintala M, Wang GF, Hsieh YS, Li FB, Palani A: Discovery of a potent nicotinic acid receptor agonist for the treatment of dyslipidemia. ACS Med Chem Lett 2011, 2: 171–176. 10.1021/ml100251u

    CAS  Article  Google Scholar 

  27. 27.

    Ratovelomanana-Vidal V, Girard C, Touati R, Tranchier JP, Hassine BB, Genêt JP: Enantioselective hydrogenation of β-keto esters using chiral diphosphine-ruthenium complexes: optimization for academic and industrial purposes and synthetic applications. Adv Synth Catal 2003, 345: 261–274. 10.1002/adsc.200390021

    CAS  Article  Google Scholar 

  28. 28.

    Yang D, Gao Q, Zheng BF, Zhu NY: Et2AlCl-promoted asymmetric phenylseleno group transfer radical cyclization reactions of unsaturated beta-hydroxy esters. J Org Chem 2004, 69: 8821–8828. 10.1021/jo048322z

    CAS  Article  Google Scholar 

  29. 29.

    Horneff T, Herdtweck E, Randoll S, Bach T: Stereoselective allyl transfer to chiral alpha-methoxycarbaldehydes: a model study related to the C-9/C-15 fragment of geldanamycin. Bioorg Med Chem 2006, 14: 6223–6234. 10.1016/j.bmc.2006.05.056

    CAS  Article  Google Scholar 

  30. 30.

    Iwashita M, Makide K, Nonomura T, Misumi Y, Otani Y, Ishida M, Taguchi R, Tsujimoto M, Aoki J, Arai H, Ohwada T: Synthesis and evaluation of lysophosphatidylserine analogues as inducers of mast cell degranulation. Potent activities of lysophosphatidylthreonine and its 2-deoxy derivative. J Med Chem 2009, 52: 5837–5863. 10.1021/jm900598m

    CAS  Article  Google Scholar 

  31. 31.

    Abbas S, Bertram RD, Hayes CJ: Commercially available 5′-DMT phosphoramidites as reagents for the synthesis of vinylphosphonate-linked oligonucleic acids. Org Lett 2001, 3: 3365–3367. 10.1021/ol0166045

    CAS  Article  Google Scholar 

  32. 32.

    Johnson DC 2nd, Widlanski TS: Facile deprotection of O-Cbz-protected nucleosides by hydrogenolysis: an alternative to O-benzyl ether-protected nucleosides. Org Lett 2004, 6: 4643–4646. 10.1021/ol048426w

    CAS  Article  Google Scholar 

  33. 33.

    Saito Y, Zevaco TA, Agrofoglio LA: Chemical synthesis of C-13 labeled anti-HIV nucleosides as mass-internal standards. Tetrahedron 2002, 58: 9593–9603. 10.1016/S0040-4020(02)01246-2

    CAS  Article  Google Scholar 

  34. 34.

    Santra A, Guchhait G, Misra AK: Efficient acylation and sulfation of carbohydrates using sulfamic acid, a mild, eco-friendly catalyst under organic solvent-free conditions. Green Chem 2011, 13: 1345–1351. 10.1039/c1gc15122c

    CAS  Article  Google Scholar 

  35. 35.

    Saneyoshi M, Satoh E: Synthetic nucleosides and nucleotides .13. Stannic chloride catalyzed ribosylation of several 6-substituted purines. Chem Pharm Bull 1979, 27: 2518–2521. 10.1248/cpb.27.2518

    CAS  Article  Google Scholar 

  36. 36.

    Gaubert G, Babu BR, Vogel S, Bryld T, Vester B, Wengel J: Synthesis and RNA-selective hybridization of alpha-L-ribo- and beta-D-lyxo-configured oligonucleotides. Tetrahedron 2006, 62: 2278–2294. 10.1016/j.tet.2005.12.007

    CAS  Article  Google Scholar 

  37. 37.

    Gibbons WA, Hughes RA, Charalambous M, Christodoulou M, Szeto A, Aulabaugh AE, Mascagni P, Toth I: Lipidic Peptides .1. Synthesis, Resolution and Structural Elucidation of Lipidic Amino-Acids and Their Homo-Oligomers and Heterooligomers. Liebigs Annalen Der Chemie 1990, 1175–1183.

  38. 38.

    Culbertson SM, Porter NA: Unsymmetrical azo initiators increase efficiency of radical generation in aqueous dispersions, liposomal membranes, and lipoproteins. J Am Chem Soc 2000, 122: 4032–4038. 10.1021/ja9934605

    CAS  Article  Google Scholar 

  39. 39.

    Moffatt JG, Khorana HG: Nucleoside polyphosphates .10. Synthesis and some reactions of nucleoside-5′ phosphoromorpholidates and related compounds - improved methods for preparation of nucleoside-5′ polyphosphates. J Am Chem Soc 1961, 83: 649–658. 10.1021/ja01464a034

    CAS  Article  Google Scholar 

  40. 40.

    Adelfinskaya O, Herdewijn P: Amino acid phosphoramidate nucleotides as alternative substrates for HIV-1 reverse transcriptase. Angew Chem Int Ed Engl 2007, 46: 4356–4358. 10.1002/anie.200605016

    CAS  Article  Google Scholar 

  41. 41.

    Goud T, Tutar A, Biellmann J: Synthesis of 8-heteroatom-substituted 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene dyes (BODIPY). Tetrahedron 2006, 62: 5084–5091. 10.1016/j.tet.2006.03.036

    CAS  Article  Google Scholar 

  42. 42.

    Verbelen B, Leen V, Wang L, Boens N, Dehaen W: Direct palladium-catalysed C-H arylation of BODIPY dyes at the 3- and 3,5-positions. Chem Commun 2012, 48: 9129–9131. 10.1039/c2cc34549h

    CAS  Article  Google Scholar 

  43. 43.

    Leen V, Van der Auweraer M, Boens N, Dehaen W: Vicarious nucleophilic substitution of alpha-hydrogen of BODIPY and its extension to direct ethenylation. Org Lett 2011, 13: 1470–1473. 10.1021/ol200148u

    CAS  Article  Google Scholar 

  44. 44.

    Carrara P, Stano P, Luisi PL: Giant vesicles “colonies”: a model for primitive cell communities. Chembiochem 2012, 13: 1497–1502. 10.1002/cbic.201200133

    CAS  Article  Google Scholar 

  45. 45.

    Iwaura R, Yoshida K, Masuda M, Yase K, Shimizu T: Spontaneous fiber formation and hydrogelation of nucleotide bolaamphiphiles. Chem Mater 2002, 14: 3047–3053. 10.1021/cm020263n

    CAS  Article  Google Scholar 

  46. 46.

    Martin OR, Zhou W, Wu X, Front-Deschamps S, Moutel S, Schindl K, Jeandet P, Zbaeren C, Bauer JA: Synthesis and immunobiological activity of an original series of acyclic lipid a mimics based on a pseudodipeptide backbone. J Med Chem 2006, 49: 6000–6014. 10.1021/jm060482a

    CAS  Article  Google Scholar 

  47. 47.

    Lenagh-Snow GM, Araujo N, Jenkinson SF, Rutherford C, Nakagawa S, Kato A, Yu CY, Weymouth-Wilson AC, Fleet GW: Inhibition of nonmammalian glycosidases by azetidine iminosugars derived from stable 3,5-di-O-triflates of pentoses. Org Lett 2011, 13: 5834–5837. 10.1021/ol2024482

    CAS  Article  Google Scholar 

  48. 48.

    Zhang LL, Peritz A, Meggers E: A simple glycol nucleic acid. J Am Chem Soc 2005, 127: 4174–4175. 10.1021/ja042564z

    CAS  Article  Google Scholar 

  49. 49.

    Inoue T, Orgel L: Oligomerization of (guanosine 5′-phosphor)-2-methylimidazolide on poly(C) - an RNA-polymerase model. J Mol Biol 1982, 162: 201–217. 10.1016/0022-2836(82)90169-3

    CAS  Article  Google Scholar 

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Acknowledgements

We thank FWO (G0780.14 N) and KU Leuven for financial support and C. Biernaux for editorial help.

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Affiliations

  1. Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Leuven, Minderbroederstraat-10, Leuven, 3000, Belgium

    Kishore Lingam Gangadhara, Puneet Srivastava, Jef Rozenski, Henri-Philippe Mattelaer, Eveline Lescrinier & Piet Herdewijn

  2. Department of Chemistry, Molecular Design and Synthesis, KU Leuven, Leuven, Belgium

    Henri-Philippe Mattelaer, Volker Leen & Wim Dehaen

  3. Department of Chemistry, Molecular Imaging and Photonics, KU Leuven, Leuven, Belgium

    Johan Hofkens

Authors
  1. Kishore Lingam Gangadhara
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  2. Puneet Srivastava
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  3. Jef Rozenski
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  4. Henri-Philippe Mattelaer
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  5. Volker Leen
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  8. Eveline Lescrinier
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  9. Piet Herdewijn
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Correspondence to Piet Herdewijn.

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Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

KLG performed synthesis and experiments for vesicle formation and assistance in fluorescence microscopy. PS assisted with organic synthesis and JR performed mass spectrometry determination; HPM wrote the introduction and revised the manuscript. VL and WD performed the synthesis of bodipy dyes. JH performed fluorescence microscopy. EL did the NMR experiments and wrote the NMR section. PH was responsible for the ideation, follow up of the research groups, coordination and writing the manuscript. All authors read and approved the final manuscript.

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Gangadhara, K.L., Srivastava, P., Rozenski, J. et al. Design and synthesis of nucleolipids as possible activated precursors for oligomer formation via intramolecular catalysis: stability study and supramolecular organization. J Syst Chem 5, 5 (2014). https://doi.org/10.1186/s13322-014-0005-3

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Keywords

  • Nucleolipid
  • Vesicles
  • Hydroxy fatty acids
  • Protocell
  • Chemical stability
  • Supramolecular assembly
  • Intramolecular catalysis
  • Fluorescence microscopy
  • BODIPY
  • NMR stability study