The present invention relates to adenosine or deoxyadenosine derivatives suitable for use in the synthesis of oligonucleotides, as well as a method for the synthesis of the above- mentioned derivatives.

Since the '80, the possibility of preparing synthetic oligonucleotides by using commercial equipment (synthesizers) and well-established protocols, such as for example the phos- phoramidite method, became widespread. The possibility of synthesizing oligonucleotides has, in turn, made a number of applications requiring the use of oligonucleotides possible, such as, for example, diagnostic tests, genetic inhibition experiments, the study of the molecular mechanism of drugs and, finally, supramolecular chemistry applications, with possible impacts in the electronic device production field. At the base of such applications there is the high selectivity of the hydrogen bond patterns, of the Watson and Crick type, or the Hoogsteen type, which allows linking in an expected manner the synthetic oligonucleotides together or with the natural ones, which in turn are depositaries of the genetic information. Through the chemical information contained in the synthetic oligonucleotides, therefore, it is possible to "dialogue" with the genetic information contained in the cells in the form of nucleic acids (DNA and RNA) in order to read the information thereof, i.e., to detect the presence of a given base sequence, or to change it.

The chemical synthesis also allows binding to the synthetic oligonucleotides a range of functional groups capable of adding new functionalities to the oligonucleotide, such as, for example, functional fluorescent groups, intercalating residues, or groups binding certain proteins or drugs.

The functionalization of the synthetic oligonucleotide is usually done through post- synthesis modifications, or directly on the synthesizer, using phosphoramidites that are modified compared to those normally used. The functionalization on the synthesizer has the advantage of a better chemical control and better guarantees for an efficient purification of the final compound. A range of modified phosphoramidites with about twenty functional substituents is commercially available, in order to obtain e.g. strands with a terminal amino group (-NH ₂ ) to address further post-synthesis modifications; with a terminal thiol group (-SH) to allow the anchorage of the oligonucleotide on gold surfaces; with a terminal dabcyl group to be used as a molecule quencher in combination with a fluorescent substituent on the other end of the strand to implement fluorescent probes of the "molecular beacon" type; with biotine to bond avidine; with cholesterol to modify the cellular uptake; with fluorescent substituents to implement probes; with psoralen for the intercalation in the double strand formed with the complementary strand and to bond it covalently after irradiation, etc.

In some of these amidites, the functional group is directly linked to phosphorous (in such a case, it does not contain the nucleoside base); in other cases, instead, it is linked at the position C8 of purines or the position C6 of pyrimidines. Modifications of the nitrogen bases in these positions generally allow maintaining unaltered the recognition of the complementary strands according to the Watson and Crick bonds. When the base is present, the modification can be inserted into a strand of the synthetic oligonucleotide and optionally incorporated several times in the same filament.

Among the numerous examples of modified purine and pyrimidine bases described in the literature, Pieles et al. Nucleic Acid Res. 1989, 17 8967-78 can be mentioned, which describe the preparation of a derivative of 8-deoxyadenosine linked to psoralen, which is capable, after its insertion in an oligonucleotide, of covalently binding the complementary strand after an appropriate light irradiation. According to the described synthesis method, the coupling between adenosine and psoralen takes place by exploiting the preformed 8- mercapto-adenosine with an alkyl derivative of psoralen according to the following scheme:

8HS-dA + Pso-OR-linker-Tos Pso-OR-linker-S-dA

Catglilialoglu et al. Tetrahedron Lett. 2006, 47, 711-14 discloses the derivatization of 8-Br deoxyadenosine with sulphur-containing alkylating agents. Such disclosure is limited to the formation of alkylated nucleosides with short alkyl chains that are free from further functionalizations. The modified nucleosides thus obtained are used only for structural studies, and there is no indication about the possibility of a use thereof in the synthesis of synthetic oligonucleotides.

Navacchia et al. Macromol. Rapid Commun. 2010, 31, 351-355 discloses the derivatization of 8-Br deoxyadenosine with a short chain dithioalkyl linker, to obtain a deoxyadenosine derivative to be polymerized in the presence of FeCl ₃ , thereby obtaining polymers capable of self-organizing that are suitable in applications in the electronics industry.

However, the methods that are available in the state of the art for the synthesis of modified adenosines or deoxyadenosines have the drawback that they need quite complex procedures, and that they fail to obtain high yields. Furthermore, the derivatives of the adenosine modified at position 8 described in the state of the art contain thioalkyl chains that are not longer than 3-5 carbon atoms, which not always allow binding a functional substituent with a sufficient adaptation capacity.

Object of the present invention is to overcome the drawbacks of the state of the art mentioned above.

These and other objects are achieved by the adenosine or deoxyadenosine derivatives modified at position 8, having the general formula (I) as defined in the annexed independent claim 1 and by the synthesis method of the above-mentioned derivatives as defined in the annexed independent claim 8. The dependant claims define further characteristics of the derivatives and the method for the synthesis thereof, and are an integral part of the description.

The synthesis method of the adenosine or deoxyadenosine derivatives modified at position 8 which is the object of the invention has the advantage of achieving high yields and requiring procedures that are less complex than those described in the state of the art to obtain other adenosine derivatives. Furthermore, the derivatives of adenosine modified at position 8 obtained by the method of the invention are advantageously suitable to be conjugated with a wide range of functional molecules, such as chromophores, fluorophores and intercalating residues. They are further suitable to be incorporated in the synthetic oligonucleotides, since they are compatible with the synthesis protocols that are employed in the commercial synthesizers of oligonucleotides, thus allowing the synthesis of modified DNA Dr RNA strands that are useful for adding potentialities to the interaction with the complementary strand, for the more disparate biochemical applications.

Further characteristics and advantages of the derivatives and the method of the invention will be apparent from the following detailed description, given by way of non-limiting example only, with reference to the reaction schemes 1, 2, and 3, which illustrate the synthesis of the phosphoramidite of 2'-deoxyadenosine modified at position 8 with a bis-thioalkyl linker and conjugated with psoralen (scheme 1) or with acridine (scheme 2) and the synthesis of the phosphoramidite of 2'-deoxyadenosine modified at position 8 with a thioal- kynyl linker (scheme 3), respectively.

[n particular, it has to be highlighted that with the synthesis reactions illustrated in the schemes 1, 2, and 3, the present inventors believe that bis-thioalkyl or thioalkynyl chains, which are longer than the linkers described in the state of the art, can be reacted with 8- bromo-deoxyadenosine, with minimal modifications to the reaction conditions, in order to directly introduce (scheme 3) or through further reactions (schemes 1 and 2) a wide variety of functional groups, such as chromophores, fluorophores, intercalating residues, alkylating agents, reactants for click-chemistry, etc., exemplified by the compounds described below. The obtainment of the derivatives set forth below is surprising, also because it had never been previously shown that it was possible to selectively protect the various functional groups present on the resulting modified adenosine, to obtain phosphoramidites usable for the synthesis of oligonucleotides with standard commercial automatic synthesizers.

Scheme 1

3r-deoxyadenosine (1) is reacted with hexane-l,6-dithiol, to give the deoxyadenosine de- ivative modified at position 8, indicated with (2) in scheme 1. The reaction takes place in in aqueous medium, preferably water and triethylamine (TEA preferably 10 eq.), at a temperature preferably ranging between 80-100° C, more preferably of about 100°C, and for a eaction time preferably ranging between 1.5 - 2.5 hours, more preferably of about 2 lours. rhe reaction proceeds with quantitative yields. By virtue of the fact that the reaction is carried out in an aqueous medium, the derivative (2) can be selectively isolated by extraction with an organic solvent without chromatographic processes, which provides an important synthesis advantage. rhe terminal thiol group of the derivative (2) is easily conjugated with a functional molecule, such as, for example, psoralen, in the form of Br-methyl psoralen (3), to give the derivative (4).

Preferably the reaction takes place in an organic solvent, dry dimethylformamide, in the presence of calcium carbonate at a temperature preferably ranging between 60-80° C, more preferably of about 80°C, and for a reaction time preferably ranging between 2.5-3.15 hours, more preferably of about 3 hours. rhe derivative (4) can be isolated as a solid product at the end of the work-up procedures, again without tedious and expensive chromatographic processes. rhe derivative (4) is then optionally transformed into the final product (7), which is a protected phosphoramidite, through a three-step process, the reaction conditions of which are described in detail in the following experimental section. Briefly, the three-step protection process provides firstly for the selective protection of the hydroxyl at the position 5' of the sugar radical with dimethoxytrityl chloride (DMT) to give the compound (5), which can be purified by chromatography. After that, the protection step of the ammino group at position 7 of the purine ring as the diphenylacetic acid amide to give the compound (6) follows, which compound can also be purified by chromatography. Finally, the hydroxyl at position 3' of the sugar radical of the compound (6) is phosphorylated to give the compound (7), which can be purified by chromatography. Scheme 2

The first synthesis step is identical to that described before with reference to scheme 1, leading to the obtainment of the derivative (2) of scheme 2. The terminal thiol group of the derivative (2) is easily conjugated with acridine, employed in the form of dichloromethoxy icridine (3), to give the derivative (4). ^P referably, the reaction takes place in an organic solvent, dry dimethylformamide, in the ^p resence of triethylamine (TEA) at a temperature preferably ranging between 60-80° C, nore preferably of about 80°C, and for a reaction time preferably ranging between 15-17 lours, more preferably of about 17 hours. rhe derivative (4) is easily isolated as a solid product at the end of the work-up procedures, without tedious and expensive chromatographic processes.

Dptionally, it is then followed by the three protection steps described above with reference o scheme 1.

Scheme 3

3r-deoxyadenosine (1) is reacted with hex-5-yne-l -thiol, to give the deoxyadenosine de- ivative modified at position 8, indicated with (2a) in scheme 3. The reaction takes place in in aqueous medium, preferably water and triethylamine (TEA), at a temperature preferably anging between 80-100° C, more preferably of about 100°C, and for a reaction time pref- ;rably ranging between 1.5-2.5, more preferably of about 2 hours. rhe reaction proceeds with quantitative yields. In this case also, the derivative (2a) of scheme 3 can advantageously be isolated without chromatographic processes. rhe reaction of scheme 3 is particularly advantageous, since it allows obtaining a deriva- ive suitable for use in click chemistry in very few passages that are easy to be carried out. such a derivative, having a terminal -C≡CH, can advantageously be conjugated with a nolecule containing an azide group, according to the click-chemistry methods. By way of jxample, molecules containing an azide group suitable to be conjugated with the derivative laving a terminal -C≡CH are 6-carboxy-fluorescein azide, 5-carboxytetramethyl rhoda- nine azide, biotine azide, ferrocene-azide (for reference, see the web site vww.baseclick.eu).

Generally, the synthesis process of the present invention, illustrated in detail by the three ;xemplary reaction schemes discussed above, allows attaching a linker with a thiol bond iirectly to the position C8 of the adenosine, deoxyadenosine, 2'-0-alkyl adenosine or 2'- 3-silyl adenosine. The specificity of this reaction is made possible by the synthesis in an iqueous environment, which ensures the proper solubility and reactivity of the reactants rnd allows the isolation of the product without the need of using expensive and tedious chromatographic methods. This type of synthesis is specific for the adenine ring, since the iifference in the chemical potential of guanine, under similar reactions conditions, would ead to the formation of guanosine.

EXPERIMENTAL SECTION

Equipment HPLC-MS analyses have been carried out with a HPLC Agilent 1 100 interfaced with a Bruker Esquire 3000+ spectrometer operating in ESI mode. For the chromatographic part, i Zorbax C8 column (4,6 x 150 mm, 5 5 μιη) was used, with a linear gradient from 70 to ?0% acetonitrile (ACN) in water in 20 minutes at a flow of 0,5 mL/minutes and UV detection at 260 nm.

NMR spectra were recorded with a Varian Mercury spectrometer operating a 400 mHz.

The oligonucleotides were synthesized with a Gene Assembler II + automatic synthesizer, by using the original protocols, unless otherwise indicated. For the preparative purification df RP-18, special Pharmacia FPLC columns were used, with a gradient generated by a peristaltic pump operating at 1.2 mL/minute, collecting 8 mL fractions.

Materials

8-Br-2'-deoxyadenosine was purchased from Berry & Associates, Dexeter, MI, U.S.A.

Synthesis of 4'-bromomethyl-4,5',8-trimethylpsoralen

The compound of the title was prepared following the procedure described by Pieles et al. [Nucleic Acids Res. 17, 8967-78, 1998).

Briefly: 228 mg trimethoxy-psoralen was dissolved in 26 ml acetic acid (AcOH) and added with 1.89 mL bromomethyl ether (2.85 g, 22.8 mmol) and mixed at room temperature (RT). After 24 hours, a new aliquot of bromomethyl ether (2.85 g) was added, and the reaction was continued for additional 24 hours. The reaction mixture was then cooled in an ice bath for 2 hours, then filtered, and the solid obtained was washed with ethyl ether and concentrated in vacuum to yield 120 mg of the desired product. The stock solution, in combination with the washing fluid, was concentrated in vacuum and gave a new portion )f precipitate, which was washed and dried as before (136 mg). The two precipitated were assessed as having a comparable purity by NMR analysis, pooled, and subsequently used without further purifications. (80% yield).

Synthesis of 8-thiopentane-thiol-2'-deoxyadenosine

Γο a commercial suspension of 8-bromo-2'-deoxyadenosine (330 mg 1.0 mmol) in water ^625 mL) 1,5-pentane-dithiol (680 mg , 3 mmol) and triethylamine (1.5 mL, 10 mmol) were added. The mixture was heated at 100 °C for two hours (with coolant under reflux). A HPLC-MS check has allowed judging the conversion as satisfactorily, and the reaction was riot extracted with 2x100 mL ethyl acetate (EtOAc). The organic phase was concentrated in vacuum and the residue was used without further purifications (80% yield). It is appropriate to store the product under vacuum until the next reaction in order to prevent the formation of disulfides.

Synthesis of 8-thiopentane-tiomethylpsoralen-2'-deoxyadenosine

A solution of 8-thiopentane-thiol-2'-deoxyadenosine (400 mg 1.0 mmol), 3- bromomethylpsoralen (340 mg 1.0 mmol) Ca ₂ C0 ₃ (600 mg) in dry methylformamide (14 t L) was heated at 80 °C for 4 hours. The reaction product was extracted with EtOAc (2x 15 mL), and washed with water (4 x 10 mL). The product of the title was obtained by :vaporation under reduced pressure of the organic phase and used without further purifica- ions (85% yield).

Synthesis of 5 '-Q-dimethoxytrityl-8-thwpentane-trimethylpsoralen-2 '-deoxyadenosine

rhe hydroxyl 5' protection product of the derivative described above was prepared by dis- iolving the latter (625 mg, 1 mmol) in 8 mL dry pyridine, adding fresh dimethoxytrityl :hloride (400 mg, 1.2 mmol). The reaction is carried out under stirring at RT in an inert itmosphere for 1 hour. The raw reaction raw product is concentrated to a small volume by vacuum evaporation, diluted in EtOAc (40 mL) and washed with 10% p/v citric acid in wa- er (4 x 10 mL) and finally, with water (2 x 10 mL). The organic phase is purified on silica ;el by eluting with CH ₂ Cl ₂ /MeOH 98/2 v/v, thus obtaining the desired product in an 80% field. synthesis of 5 '-Q-dimethoxytrityl-4-diphenylacetamide-8-thiopentane-trime '- ieoxyadenosine

The compound of the title was obtained by dissolving the dimethoxytritylated derivative of the conjugated adenosine-psoralen (928 mg 1.0 mmol) in 5 mL dry pyridine. To this, trimethylsilyl chloride (0.635 mL 5 mmol) is added in an inert atmosphere, the mixture is left under stirring at RT for 30 minutes. Diphenylacetyl chloride (227 mg 1.2 mmol) is then added, and it is left under stirring for further 2 hours.

The excess of chlorinating agents is quenched with 1 mL MeOH for 15 minutes, after which 30% w/w ammonia in water (1 mL) is added and allowed to react for 30 minutes by running in the meantime control TLC (in CH2C12/MeOH 5% v/v) to control the successful removal of the SiMe ₃ groups from the OH at 2' and the possible bis-acetylated product. The raw reaction product is evaporated to dryness in vacuum, taken up with 100 mL CH ₂ C1 ₂ and washed with 10% w/v citric acid in water (2 x 25 mL), and finally with 2 x 25 mL water. The product is purified on silica gel by eluting with CH ₂ Cl ₂ /MeOH/Et ₃ N 98/2/1 v/v/v.

Synthesis of 5 '-0-dimethoxytrityl-4-diphenylacetamide-8-thiopentane-trimet hylpsoralen-2 - deoxyadenosine-3 '-O-cyanoethyl-diisopropyl-phosphoramidite (synthesis of phosphor a- midite)

The product derivative from the previous reaction (1.122 g 1.0 mmol) is dissolved in dry CH ₂ C1 ₂ (5 mL) and added with ethyldiisopropylamine (0.85 mL, 5 mmol), cyanoethyl- diisopropyl chlorophosphite (0.36 mL, 1.6 mmol). The reaction is kept stirred in an inert atmosphere at RT for 30 minutes. The reaction is diluted with 30 mL CH ₂ C1 ₂ and washed with NaHC0 ₃ (8% p/v in water) and subsequently with brine (10% p/v NaCl in water). The product is purified by silica chromatography in a gradient of 20/80/10 ethyl ace- tate/cyclohexane/triethylamine and 90/10/10 yielding a spongy solid in a 50% yield.

Synthesis of 8-thiopentane-thio-acridine-2 '-deoxyadenosine

8-thiopentane-thiol-2-deoxyadenosine (400 mg 1.0 mmol) is dissolved in dry dimethylfor- mamide (DMF) (10 mL) and triethylamine (1.3 mL, 10 mmol) together with 2,9-dichloro- 6-methoxy acridine (600 mg, 2 mmol). The mixture is heated at 80 °C in a closed tube for 17 hours. The product is pulled dry and chromatographed on silica gel by eluting with CH ₂ Cl ₂ /MeOH 90/10 to yield the product of the title as a fluorescent yellow/greed solid, in a 70% yield.

Synthesis of 5 '-0-dimethoxytrityl-8-thiopentane-thio-acridine-2 '-deoxyadenosine

8-thiopentane-thioacridine-2'-deoxyadenosine (630 mg, 1.0 mmol) is dissolved in 8 mL dry pyridine and added with fresh dimethoxytrityl chloride (400 mg, 1.2 mmol). The mixture is allowed to react for 1 hour at RT in an anhydrous atmosphere, after that it is concentrated to a small volume, re-dissolved in ethyl acetate (40 mL) and washed with 10% w/v citric acid in water (4 x 10 mL), and finally with water (2 x 10 mL). The organic phase is purified on silica gel by eluting with CH ₂ Cl ₂ /MeOH/triethylamine 97/2/1 v/v, thus obtaining the desired product in a 65% yield.

Synthesis of 5 '-0-dimethoxytrityl-4-diphenylacetamide-8-thiopentam-thio-ac ridine-2 - deoxyadenosine

The previous compound (930 mg 1.0 mmol) is protected on the amino group of the adenosine by dissolving it in dry pyridine (5 mL) and reacting it in an anhydrous atmosphere for 30 minutes with trimethylsilyl chloride (0.635 mL 5.0 mmol) under stirring at RT. The mixture of diphenylacetyl chloride (277 mg 1.2 mmol) is added, and it is left to react for 2 hours. MeOH (1 mL) is added, and after 15 minutes NH ₄ OH (1 mL al 30%) p/v) is added. A.s in the corresponding psoralen derivative, the removal of the silyl-protecting group and the possible bis-protected amide is followed. The above-described work-up is repeated, followed by a purification on silica gel with 99/1 CH ₂ Cl ₂ /triethylamine to obtain a 70%) ield.

Synthesis of 5 '-0-dimethoxytrityl-4-diphenylacetamide-8-thiopentane-thio-a cridine-2 - ieoxyadenosine-3'-OR-cyanoethyl-diisopropyl-phosphoramidite

The previous compound (1.120 g, 1 mmol) is dissolved in dry dichloromethane (5 mL) and ethyldiisopropylamine (0.85 mL, 5 mmol) and, finally, added with cyanoethyl-diisopropyl chlorophosphite (0.36 mL, 1.6 mmol). The reaction is kept stirred in an inert atmosphere at RT for 30 minutes. The reaction is diluted with 30 mL CH ₂ C1 ₂ and washed with NaHC0 ₃ (8% p/v in water) and subsequently with brine (10% p/v NaCl in water). The product is purified by chromatography on silica in a gradient of 20/80/10 and 90/10/10 ethyl ace- tate/cyclohexane/triethylamine, yielding a spongy solid in a 50% yield.

Synthesis of S-hexa-5-vnyl-ethanethioate

6-chloro-l-hexine (0.60 mL, 5 mmol) is dissolved in dry DMF (5 mL). To the solution, potassium ethanethioate is added, then it is left at 50°C under stirring for 3 hours. The reaction mixture is extracted with Et ₂ 0 (2 x 15 mL) and washed with H ₂ 0 (5 x 30 mL). The solvent is brought to dryness in a rotavapor and the product is used without further purification (quantitative yield).

Synthesis of hex-5-vnyl-l -thiol

S H S-hex-5-ynyl-ethanethioate (780 mg, 5 mmol) is dropped in a LiAlH ₄ solution (500 mg, 10 urnol) in dry THF (15 mL). It is reacted for 5 minutes, then H ₂ 0 is added, until when there is no more gas release. It is acidified with citric acid, then it is extracted with Et ₂ 0 (2 x 10 nL). The solvent is brought to dryness in a rotavapor and the product is used without further purification (60% yield).

Synthesis of 8-thiohex-5-ynyl-2 '-deoxy adenosine

Γο a commercial suspension of 8-bromo-2'-deoxyadenosine (330 mg 1.0 mmol) in ^' water ^' 625 mL) hex-5-yne-l -thiol (342 mg, 3 mmol) and triethylamine (1.5 mL, 10 mmol) were idded. The mixture was heated to 100 °C for two hours (with coolant under reflux). The -eaction product was hot extracted with ethyl acetate (EtOAc) (2 x 100 mL). The organic jhase was concentrated in vacuum and the residue was purified on silica gel by eluting with CH ₂ Cl ₂ /MeOH/triethylamine 89/10/1, to yield the desired product in a 30% yield.

Synthesis of 5 '-dimethoxytrityl-8-thiohex-5-ynyl-2 '-deox adenosine

?-thiohex-5-yne-2'-deoxyadenosine (363 mg 1.0 mmol) is dissolved in 8 mL dry pyridine, ind added with fresh dimethoxytrityl chloride (400 mg, 1.2 mmol). The reaction is carried 3ut under stirring at RT in an inert atmosphere for 1 hour. The raw reaction product is concentrated to a small volume by evaporation in vacuum, diluted in EtOAc (40 mL) and washed with 10% p/v citric acid in water (4 x 10 mL) and, finally, with water (2 x 10 mL). rhe organic phase is purified on silica gel by eluting with CH ₂ Cl ₂ /MeOH 95/5 v/v, thus obtaining the desired product in a 78% yield.

Synthesis of 5 '-dimethoxytrityl-8-thiohex-5-ynyl-2 '-deoxyadenosine-4-N-diphenylacetamide

rhe compound obtained above (557 mg, 1.0 mmol) is dissolved in dry pyridine (5 mL) and ^• eacted, under a nitrogen atmosphere under stirring, with trimethylsilyl chloride (0.635 TiL, 5.0 mmol). After 30 minutes, the diphenylacetyl chloride (277 mg 1.2 mmol) is added, md the reaction is continued for 2 hours. MeOH (1 mL) is added, and after 15 minutes ^Η ₄ ΟΗ (1 mL al 30% p/v). As in the corresponding psoralen derivative, the removal of :he silyl protecting group and the possible bis-protected amide is followed. The already described work-up is followed, with successive purification on silica gel with H ₂ Cl ₂ /MeOH/triethylamine 89/10/1, thus obtaining a 90% yield.

Synthesis of 8-thiohex-5-ynyl-5'-Q-dimethoxytrityl -4-diphenylacetamide-2'- ieoxyadenosine 3 '-O '-cyanoethyl-diisopropyl phosphoramidite

A solution is prepared, which contains the compound described above (557 mg, 1 mmol) in dry dichloromethane (5 mL) and ethyldiisopropylamine (0.85 mL, 5 mmol). To this solution, cyanoethyl-diisopropyl chlorophosphite (0.36 mL, 1.6 mmol) is added. The reaction is kept stirred in an inert atmosphere at RT for 30 minutes. The reaction is diluted with 30 mL CH ₂ C1 ₂ and washed with NaHC0 ₃ (8% p/v in water) and subsequently with brine (10% p/v NaCl in water).

The product is purified by chromatography on silica in a gradient of 20/80/10 and 90/10/10 ethyl acetate/cyclohexane/triethylamine, yielding a spongy solid in a 60% yield.

Synthesis of oligonucleotides

The amidites corresponding to the three conjugates (with psoralen, acridine, alkyne) are diluted in acetonitrile at a 0.1 mM concentration (0.08 mM in the case of the slightly less soluble acridine derivative), as any common phosphoramidite, and loaded on the Gene Assembler 11+ synthesizer in the position corresponding to that of the modified base X. Syntheses of oligonucleotides having the following sequence: 5'CGTGCXTCCTAGC3' are carried out by using the standard protocols for the 1.3 μη ο^Γ synthesis scale, taking care of lengthening the coupling times by two minutes for the X base.

The readings carried out on the dimethoxytrityl cation removed after the coupling reaction give the following results:

PI -norm (non-modified oligo X = A):

coupling efficiency of 96.9 %

Pl-pso (X = A conjugated with psoralen):

coupling efficiency of 99.9 %

Pl-acr (X = A conjugated with acridine):

coupling efficiency of 97.6%

Pl-yne (X = A conjugated with alkyne):

coupling efficiency of 98.5%

As it can be seen from the results, the standard coupling protocols (although with the times elongated by two minutes) provide a normal incorporation of the modified adenosines in the oligonucleotide synthesized with efficiency totally comparable, if not superior, to that of the standard amidite.

The efficiency is also comparable with the next base, cytidine at position 5, where:

PI -norm:

coupling efficiency of 99.0%

Pl-pso:

coupling efficiency of 102.8%(*)

Pl-acr:

coupling efficiency of 98.6%

Pl-yne:

coupling efficiency of 100.8%

(*) a 2-3% error in the reading of the cation absorption is the norm for this type of equipment.

At the end of the synthesis, the cartridges are dried with an air jet and placed inside of a 1.6 mL plastic vial supplied with a watertight cap. The cartridge is filled with 30% w/v aqueous ammonia, closed and left overnight (16 hours) at 50 °C to allow the release from the CPG and the removal of the protecting groups. In the morning, the cartridge is cooled and opened, and the liquid is transferred into a 12 mL tube. The cartridge is washed thrice with 1 mL mil-li-Q water and the aqueous phases are combined to the previous ammonia solution. The contents of the tube is dried in a vacuum centrifuge, and the resulting precipitate is dissolved in 500 μΐ, triethylammonium acetate (TEA) 0.1 M in Milli-Q water and purified on a preparative 1.6x 12 cm RP-18 column in 0.1 M TEA in milli-Q water in a gradient from 0 to 20% acetonitrile.

The appropriate fractions are controlled by HPLC before being combined and lyophilized again. There are recovered, respectively:

PI -norm: 68.8 OD, equal to 0.59 μηιοΐ with a 36% whole yield in relation to the used CPG;

Pl-pso: 64.15 OD, equal to 0.55 μπιοΐ with a 28% whole yield in relation to the used CPG; Pl-acr: 63.32 OD, equal to 0.54 μηιοΐ with a 31% whole yield in relation to the used CPG; Pl-yne: 104.64 OD, equal to 0.89 μηιοΐ with a 45% whole yield in relation to the used CPG.

The UV absorption spectra of the four oligonucleotides synthesized are those expected: λη _ΐ 3χ 270 nm, Pl-pso shows a second absorption at 340 nm, Pl-acr shows a second absorption extending from 320 to 450 nm, while the absorption of the alkynyl group in Pl- yne is not measured in the visible range.

Pl -pso and Pl-Acr fluoresce by excitation at 350 nm and 370 nm, respectively, showing emission curves similar to those reported in the derivatives of this type.

The purified oligonucleotides show the expected mass value by ESI spectrometry:

PI -norm:

exact mass calculated 3908.7 measured 3908.8

Pl-pso:

exact mass calculated 4282.8 measured 4283.0

Pl-acr:

exact mass calculated 4283.7 measured 4284.3

(contains 1 CI atom)

Pl-yne:

exact mass calculated 4020.7 measured 4020.7