Mathieu Noël

Céline Clément-Blanc

Albert Meyer

Jean-Jacques Vasseur

François Morvan

Solid supports for the synthesis of 3'-aminooxy deoxy-or ribooligonucleotides and their 3'-conjugation by oxime ligation

Keywords: Oligonucleotides, Oxime, solid support, click, conjugates

Mono-and tri-ethyleneglycol aminooxy derivatives were reacted with levulinic acid, protected with dimethoxytrityl and immobilized on long-chain alkylamine controlled-pore glass (LCAA-CPG) solid support. The resulting solid supports were used to synthesize 3'aminooxy oligonucleotides having a shorter or longer spacer between the oligonucleotide and the aminooxy function. After completion of oligonucleotide synthesis, a first-step mild ammonia treatment was applied to remove the oligonucleotide protecting groups, followed by a second-step treatment with 50 mM methoxyamine at pH 4.2, releasing the 3'-aminooxy oligonucleotides from the solid support by an oxime exchange reaction. The resulting 3'aminooxy deoxy-or ribo-oligonucleotides were conjugated to various ketones (acetone, fluorenone, cyclohexanone) and aldehydes (pyrene, deoxycholic, biotin) with high efficiency by means of oxime ligation.

INTRODUCTION

Conjugation of oligonucleotides to diverse molecules has attracted substantial research interest as it offers an easy way to modulate their biological properties. Most of the conjugations are made either at the 5' or the 3' extremity of the oligonucleotides. The 3'modification has a great interest as it also increases the nuclease resistance. [START_REF] Juliano | Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides[END_REF] Different chemistries have been applied to conjugate oligonucleotides with different biomolecules or dyes including Diels-Alder ligation, [START_REF] Hill | Diels-Alder bioconjugation of diene-modified oligonucleotides[END_REF][START_REF] Marchan | Diels-Alder cycloadditions in water for the straightforward preparation of peptide-oligonucleotide conjugates[END_REF] Staudinger ligation [START_REF] Weisbrod | A nucleoside triphosphate for site-specific labelling of DNA by the Staudinger ligation[END_REF] copper catalyzed alkyne-azide cycloaddition, [START_REF] Bouillon | Microwave assisted "click" chemistry for the synthesis of multiple labeled-carbohydrate oligonucleotides on solid support[END_REF][START_REF] Gramlich | Postsynthetic DNA Modification through the Copper-Catalyzed Azide-Alkyne Cycloaddition Reaction[END_REF][START_REF] Pourceau | Azide Solid Support for 3 '-Conjugation of Oligonucleotides and Their Circularization by Click Chemistry[END_REF][START_REF] Pourceau | Synthesis of Mannose and Galactose Oligonucleotide Conjugates by Bi-click chemistry[END_REF][START_REF] El-Sagheer | Click chemistry with DNA[END_REF] copper free alkyne-azide cycloaddition, [START_REF] Jewett | Cu-free click cycloaddition reactions in chemical biology[END_REF][START_REF] Debets | Bioconjugation with Strained Alkenes and Alkynes[END_REF][START_REF] Jadhav | Synthesis and In Vivo PET Imaging of Hyaluronan Conjugates of Oligonucleotides[END_REF][START_REF] Massaad-Massade | New Formulation for the Delivery of Oligonucleotides Using "Clickable" siRNA-Polyisoprenoid-Conjugated Nanoparticles: Application to Cancers Harboring Fusion Oncogenes[END_REF] thiol Michael addition, [START_REF] Sanchez | Maleimide-Dimethylfuran exo Adducts: Effective Maleimide Protection in the Synthesis of Oligonucleotide Conjugates[END_REF][START_REF] Sanchez | Conjugation Reactions Involving Maleimides and Phosphorothioate Oligonucleotides[END_REF][START_REF] Sanchez | Easy introduction of maleimides at different positions of oligonucleotide chains for conjugation purposes[END_REF][START_REF] Meyer | Synthesis of Monoconjugated and Multiply Conjugated Oligonucleotides by "Click Thiol" Thiol-Michael-Type Additions and by Combination with CuAAC "Click Huisgen[END_REF][START_REF] Ligeour | Synthesis of galactoclusters by metal-free thiol "click chemistry" and their binding affinity for Pseudomonas aeruginosa lectin LecA[END_REF] thioether ligation, [START_REF] Meyer | Hetero-Click Conjugation of Oligonucleotides with Glycosides Using Bifunctional Phosphoramidites[END_REF] inverse-electron-demand Diels-Alder reaction [START_REF] Knall | Inverse electron demand Diels-Alder (iEDDA)-initiated conjugation: a (high) potential click chemistry scheme[END_REF][START_REF] Busskamp | Efficient labelling of enzymatically synthesized vinylmodified DNA by an inverse-electron-demand Diels-Alder reaction[END_REF] or Oxime ligation. [START_REF] Trevisiol | The oxyamino-aldehyde coupling reaction -an efficient method for the derivatization of oligonucleotides[END_REF][START_REF] Salo | Aminooxy functionalized oligonucleotides: Preparation, on-support derivatization, and postsynthetic attachment to polymer support[END_REF][START_REF] Defrancq | Use of an aminooxy linker for the functionalization of oligodeoxyribonucleotides[END_REF][START_REF] Forget | Highly efficient synthesis of peptide-oligonucleotide conjugates: Chemoselective oxime and thiazolidine formation[END_REF][START_REF] Katajisto | Solid-phase synthesis of multiantennary oligonucleotide glycoconjugates utilizing on-support oximation[END_REF][START_REF] Forget | 3'-Oligonucleotides conjugation via chemoselective oxime bond formation[END_REF][START_REF] Meyer | Oligonucleotide Sequential Bis-Conjugation via Click-Oxime and Click-Huisgen Procedures[END_REF][START_REF] Singh | Recent developments in oligonucleotide conjugation[END_REF][START_REF] Cieslak | Permanent or reversible conjugation of 2'-O-or 5'-O-aminooxymethylated nucleosides with functional groups as a convenient and efficient approach to the modification of RNA and DNA sequences[END_REF] With regards to oxime ligation, several phosphoramidite derivatives have been reported for the introduction of the aminooxy function at the 5'-end of oligonucleotides for their subsequent conjugation or cyclization. [START_REF] Salo | Aminooxy functionalized oligonucleotides: Preparation, on-support derivatization, and postsynthetic attachment to polymer support[END_REF][START_REF] Defrancq | Use of an aminooxy linker for the functionalization of oligodeoxyribonucleotides[END_REF][START_REF] Forget | Highly efficient synthesis of peptide-oligonucleotide conjugates: Chemoselective oxime and thiazolidine formation[END_REF][START_REF] Defrancq | Oxime bond formation for the covalent attachment of oligonucleotides on glass support[END_REF][START_REF] Edupuganti | Head-to-tail oxime cyclization of oligodeoxynucleotides for the efficient synthesis of circular DNA analogues[END_REF][START_REF] Lonnberg | Prompt site-selective DNA hydrolysis by Ce(IV)-EDTA using oligonucleotide multiphosphonate conjugates[END_REF][START_REF] Schmidt | Dialdehydes Lead to Exceptionally Fast Bioconjugations at Neutral pH by Virtue of a Cyclic Intermediate[END_REF][START_REF] Meyer | Phthalimide-Oxy Derivatives for 3-or 5-Conjugation of Oligonucleotides by Oxime Ligation and Circularization of DNA by "Bis-or Tris-Click" Oxime Ligation[END_REF] In contrast, the 3'-conjugation of oligonucleotides by oxime ligation has been poorly explored. The usual method reported in the literature to do so is to introduce a glycerol group at the 3'-end which is further oxidize to an aldehyde to eventually reacts with a aminooxy derivative. Thus, the modulation of the spacer between the oligonucleotide and the modification is not possible. Only one recent publication, from our group, reported the synthesis of a solid support allowing for the introduction of the aminooxy function at the 3'-end of oligonucleotides (Figure 1). [START_REF] Meyer | Phthalimide-Oxy Derivatives for 3-or 5-Conjugation of Oligonucleotides by Oxime Ligation and Circularization of DNA by "Bis-or Tris-Click" Oxime Ligation[END_REF] The resulting 3'-modified oligonucleotides were then conjugated with different aldehyde derivatives or circularized by means of bis-oxime ligation after the introduction of another aminooxy function at the 5'-end and reaction with a dialdehyde. [START_REF] Meyer | Phthalimide-Oxy Derivatives for 3-or 5-Conjugation of Oligonucleotides by Oxime Ligation and Circularization of DNA by "Bis-or Tris-Click" Oxime Ligation[END_REF]

RESULTS AND DISCUSSION

In order to introduce some versatility by easily modulating the length of the spacer between an oligonucleotide and its 3'-aminooxy function for subsequent oxime conjugation, we designed a new type of solid supports built from ethylene glycol derivatives (Figure 1). Herein, we report the synthesis of two solid supports allowing the 3'-conjugation of deoxyand ribo-oligonucleotides with different ketones and aldehydes. This novel approach allows for enhanced versatility in generating a variety of tailored oligonucleotide constructs, where the different solid supports can be utilized to efficiently modulate the target designs of 3'oligonucleotide conjugates.

Figure 1. Structure of previous phthalimide solid support [START_REF] Meyer | Phthalimide-Oxy Derivatives for 3-or 5-Conjugation of Oligonucleotides by Oxime Ligation and Circularization of DNA by "Bis-or Tris-Click" Oxime Ligation[END_REF] and new solid supports 7a-b.

To demonstrate the usefulness of this approach, we selected mono-and tri-ethyleneglycol as the starting compounds. However, it is worth noting that this synthetic strategy can be readily applied to ethyleneglycols of any length or to any diols. The synthesis of the solid supports 7a-b leading to 3'-aminooxy oligonucleotides with a mono-or a tri-ethylene glycol spacer proceeded in few steps (Scheme 1). Firstly, 2-bromo-ethanol, or a previously synthesized monotosyl-triethyleneglycol (1) [START_REF] Angeli | Glycoclusters with Additional Functionalities for Binding to the LecA Lectin from Pseudomonas aeruginosa[END_REF] were substituted with hydroxyl-phthalimide in the presence of K 2 CO 3 affording the phthalimide ethyleneglycol derivatives 2a-b. [START_REF] Salo | Aminooxy functionalized oligonucleotides: Preparation, on-support derivatization, and postsynthetic attachment to polymer support[END_REF][START_REF] Meyer | Phthalimide-Oxy Derivatives for 3-or 5-Conjugation of Oligonucleotides by Oxime Ligation and Circularization of DNA by "Bis-or Tris-Click" Oxime Ligation[END_REF] Secondly, the aminooxy function was deprotected with hydrazine, and after evaporation, levulinic acid was added to form the oxime derivatives 5a-b. After work-up, the crude was directly treated with dimethoxytrityl chloride to protect the hydroxyl function providing derivatives 6a-b after chromatography with an overall yield of 50% for the last three steps. Finally, 6a-b were immobilized by amide formation on long-chain alkylamino controlled-pore glass (LCAA-CPG 1000 Å), using EDC as the coupling reagent, affording the two solid supports 7a-b. The loading was measured by trityl assay (~40 µmol/g). Scheme 1. Synthesis of solid supports for the preparation of 3'-aminooxy oligonucleotides.

The elongation to full-length oligonucleotides (DNA or RNA) was performed on a DNA/RNA synthesizer, at the 1 mol scale, by means of solid phase oligonucleotide synthesis (SPOS) using standard 3'-phosphoramidite chemistry with fast-labile nucleobase protecting groups and 2'-O-pivaloyloxymethyl (PivOM) groups for the 2'-OH protection of the ribonucleotides, [START_REF] Lavergne | A Base-Labile Group for 2 '-OH Protection of Ribonucleosides: A Major Challenge for RNA Synthesis[END_REF] (Scheme 2). After elongation, the oligodeoxynucleotides 8a-b were first deprotected with concentrated aqueous ammonia for 1 h at room temperature to remove the cyanoethyl groups on the phosphates and the acyl groups on the nucleobases. For oligoribonucleotides a first-step treatment with 1 M DBU was applied to remove the cyanoethyl groups and then the second-step ammonia treatment for 3 h at 37 °C was performed to allow removal of the PivOM groups on the 2' position of ribose. [START_REF] Lavergne | A Base-Labile Group for 2 '-OH Protection of Ribonucleosides: A Major Challenge for RNA Synthesis[END_REF] The ammonia solution was collected, and after evaporation, the residue was analyzed confirming no undesired cleavage of the oligonucleotides from the solid supports. Finally, the 3'-aminooxyoligonucleotides 10a-b were released from the solid supports by treatment with a 0.4 M ammonium acetate buffer solution of 50 mM methoxyamine (pH 4.2) for 3 h at rt. The 3'aminooxy-oligonucleotides were purified by size exclusion chromatography on G25-sephadex (Nap-10) to remove the methoxyamine and the salts. The 3'-aminooxy-oligonucleotides were obtained in the range of 300 to 500 nmoles that is a similar amount in comparison with oligonucleotides synthesized on a standard succinyl solid support. HPLC and MALDI-TOF MS analyses showed the efficiency of the syntheses and confirmed the nature of the 3'-modified oligonucleotides (See SI). It has been reported that methoxyamine reacts with certain cytosines of tRNA. [START_REF] Piper | Selective Reaction of Methoxyamine with Cytidine Residues in Mammalian Initiator Transfer Ribonucleic-Acid[END_REF] Such degradation occurred after a 20 h treatment at 37 °C with a 3 M methoxyamine concentration and 10 mM MgCl 2 pH 5.5. In our case, no similar side reaction was observed, this could be explained by the short treatment (3 h) at room temperature with a low concentration of methoxyamine (50 mM) applied for the release of the aminooxy-oligonucleotides from the solid support.

Scheme 2: Synthesis of 3'-aminooxy-oligonucleotides. B* represents the protected nucleobase A Pac , G -tbu-Pac , C -Ac . Cne: cyanoethyl, Piv: pivaloyl. SPOS: Solid phase oligonucleotide synthesis.

To confirm the reactivity of the 3'-aminooxy-oligonucleotides, they were then engaged in derivatization reactions using different ketones and aldehydes, like acetone, cyclohexanone, fluorenone, pyrene carboxaldehyde, biotin or deoxycholic acetamide aldehydes (Scheme 4 and table 1). The aldehyde derivatives of biotin and deoxycholic acid 14 and 18 were synthesized in three steps, as follows: first, activation of the carboxylic function of biotin and deoxycholic acid as a N-hydroxysuccinimide derivative; second, conjugation with 3-amino-2hydroxy-propan-1-ol; and, third, oxidation of the diol by treatment with sodium periodate to generate the aldehyde function (Scheme 3). [START_REF]aldehydes species thanks to the acidic buffer used for the coupling by oxime ligation[END_REF] Scheme 3: Synthesis of aldehyde derivatives of biotin 14 and deoxycholic acid 18.

As already reported, [START_REF] Forget | Highly efficient synthesis of peptide-oligonucleotide conjugates: Chemoselective oxime and thiazolidine formation[END_REF] aminooxy function reacts easily with aldehydes and ketones, since such species could be in the air or glassware (e.g. acetone) or in some solvents (e.g. acetaldehyde).

Hence, it is strongly recommended to avoid a HPLC purification of the crude aminooxyoligonucleotides. It is safer to proceed rapidly to the oxime ligation between the crude 3'aminooxy-oligonucleotides and the desired aldehydes or ketones to introduce. After ligation, the conjugates can be easily purified by HPLC. The different 3'-aminooxy-deoxy-and ribooligonucleotides were dissolved in 0.4 M ammonium acetate buffer (pH 4.2) and ketones or aldehydes were added at rt. After 1 to 4 h of contact time, the conjugates were obtained and characterized by HPLC and MALDI-TOF MS (See SI). The HPLC profiles showed total conversion of the starting material and the formation of the conjugate as a single peak, except for the pyrene conjugate, where the two E and Z isomers were visualized.

CONCLUSION

Solid supports allowing the preparation of oligonucleotides with a 3'-aminooxy function were rapidly and easily synthesized in few steps from bromoethanol or triethyleglycol. According to this strategy, it is possible to modulate the length of the spacer between the oligonucleotide and the aminooxy function since any diol derivatives and ethyleneglycols of any length can be chosen as a spacer. After elongation by standard phosphoramidite chemistry, a two-step protocol was applied to first deprotect the oligonucleotides and second to release the 3'aminooxy oligonucleotides from the solid support. The corresponding 3'-modified deoxy-and ribo-oligonucleotides were conjugated to various ketones and aldehydes with high efficiency by oxime ligation.

This solid supports could be used in combination with other building blocks like special phosphoramidites to introduce another orthogonal modification at the 5'-end of oligonucleotides allowing their bis-conjugation. Along this lane, we previously demonstrated that combination of oxime ligation and CuAAC conjugation are orthogonal. [START_REF] Meyer | Oligonucleotide Sequential Bis-Conjugation via Click-Oxime and Click-Huisgen Procedures[END_REF] The data reported in this manuscript increases the toolbox to synthesize oligonucleotide conjugates.

EXPERIMENTAL SECTION

General Methods: All commercial chemicals were reagent grade and were used without further purification. The DNA synthesis reagents and phosphoramidites were commercially available from Link Technologies or ChemGenes. Flash column chromatography was performed on silica gel 60 (40-63 μ ). [START_REF] Juliano | Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides[END_REF] H NMR (400 or 500 MHz) and [START_REF] Massaad-Massade | New Formulation for the Delivery of Oligonucleotides Using "Clickable" siRNA-Polyisoprenoid-Conjugated Nanoparticles: Application to Cancers Harboring Fusion Oncogenes[END_REF] C NMR spectra (100 or 126 MHz) were recorded in CDCl 3 or DMSO-d6 at room temperature. Compounds 1, 36 2a 33 and 2b [START_REF] Meyer | Phthalimide-Oxy Derivatives for 3-or 5-Conjugation of Oligonucleotides by Oxime Ligation and Circularization of DNA by "Bis-or Tris-Click" Oxime Ligation[END_REF] were prepared according to literature protocols. Deprotection affording 9a-b. For DNA: The CPG was withdrawn in a microtube and treated with aqueous conc. ammonia for 1 h at rt. The supernatant was removed and CPG was washed with water (3 × 1 mL). For RNA: The CPG was treated with a 1 M solution of DBU in dry acetonitrile for 3 min using two syringes, and washed with dry acetonitrile (3 × 5 mL).

General

Then the CPG was withdrawn in a microtube and treated with aqueous conc. ammonia for 3 h at 37 °C. The supernatant was removed and CPG was washed with water (3 × 1 mL).

Release

Biotin amide 1,2-propanediol 13

To a solution of biotin N-hydroxysuccinimide, [START_REF] Meyer | Synthesis of Monoconjugated and Multiply Conjugated Oligonucleotides by "Click Thiol" Thiol-Michael-Type Additions and by Combination with CuAAC "Click Huisgen[END_REF]

Scheme 4 :

 4 Scheme 4: Conjugation of 3'-aminooxy-(deoxy and ribo)-oligonucleotides with ketone or aldehyde derivatives.

  protocol of deprotection of aminooxy ethyleneglycol derivatives. To a solution of phthalimide derivative 2a-b (3.4 mmol) in 12 mL of absolute ethanol, hydrazine hydrate (900 L, 18 mmol) was added. A white precipitate appeared after 5 min and the reaction was stirred for 3 h. The precipitate was removed by filtration and the solution was evaporated followed by 3 coevaporations with ethanol affording 3a-b that were used without purification.General protocol for oxime formation. To a solution of aminooxy ethyleneglycolderivatives 3a-b (3.4 mmol) in 10 mL of methanol and 5 mL of DMF, 1 mL of 0.1M ammonium acetate aqueous buffer, 2 drops of acetic acid and levulinic acid (306 µL, 3.4 mmol) were added. The solution was stirred at rt for 5 h and the reaction was monitored by RP18 TLC. The solvents were evaporated. The residue was coevapored 3 times with dry pyridine and compounds 5a-b were used without purification. General protocol for dimethoxytritylation. To a solution of compounds 5a-b (3.4 mmol) in 20 mL of dry pyridine, dimethoxytrityl chloride (1.4g, 4.1 mmol) was added. After 2 h under magnetic stirring, 1 mL of methanol was added. After 5 min, saturated NaHCO 3 aqueous solution (50 mL) was added and the product was extracted with CH 2 Cl 2 (3 × 50 mL). The organic layers were dried over Na 2 SO 4 and evaporated under vacuum. The residue was purified by silica gel chromatography from CH 2 Cl 2 :Et 3 N, 98:2, (v/v) to CH 2 Cl 2 :Et 3 N:MeOH 83:2:15, (v/v/v), to afford compounds 6a-b. 4-((2-dimethoxytrityl-oxyethoxy)imino)-pentanoic acid 6a: Starting from 823 mg (4.0 mmol) of 2a gave 6a 1.15 g 50% (3 steps).

2 ,

 2 and dried. The trityl assay indicated a loading of 40 mol/g for 7a and 41 mol/g for 7b. Synthesis of oligonucleotides 8a-b. The oligonucleotides were elongated from solid support protocols. Detritylation was performed for 65 s using 3 % trichloroacetic acid (TCA) in CH 2 Cl 2 . For the coupling step: benzylmercaptotetrazole (0.3 M in anhydrous CH 3 CN) was used as the activator along with y v b 2′-deoxyribonucleoside-3′-O-2-cyanoethyl, N,N-diisopropylphosphoramidites phenoxyacetyl or tertbutylphenoxyacetyl (0.1 M in CH 3 CN, 30 s coupling time) or 2′-O-pivaloyloximethylyribonucleoside-3′-O-2-cyanoethyl, N,N-diisopropylphosphoramidites (0.1 M in CH 3 CN, 180 s coupling time). The capping step was performed using commercially available solutions (Cap A: phenoxyacetic anhydride:pyridine:THF 10:10:80 v/v/v, and Cap B: 10 % Nmethylimidazole in THF) for 60 s. The oxidation step was performed with a standard, diluted iodine solution (0.1 M I 2 , THF:pyridine:water 90:5:5, v/v/v) for 15 s.

  from solid support affording 10a-b. The CPG with the solid-supported oligonucleotides 9a-b were treated with a solution of 50 mM CH 3 ONH 2 ,HCl in 0.4 M ammonium acetate pH 4.2 (2 mL) for 3 h at rt under stirring. The supernatant was applied on a Sephadex G25 column for desalting. The solution was lyophilized affording the 3'aminooxy-(deoxy or ribo)-oligonucleotides 10a-b. Warning: Since aminooxy is a very reactive function, 10a-b should be used rapidly. The glassware must be washed with ethanol to avoided any trace of acetone. Conjugation with ketones or aldehyde derivatives. Fluorenone 50 mM in MeOH, Pyrene carboxyaldehyde 50 mM in DMF, Deoxycholic amide ethanal 50 mM in MeOH, biotin amide ethanal 50 mM in MeOH. To 3'-aminooxy-EG/EG 3 -deoxyoligonucleotides 10a-b 15 nmol in 25 µL of water were added 225 µM of 0.4M NH 4 + AcO -pH 4.2 and 10 eq of ketone or aldehyde derivatives. To 3'-aminooxy-EG 3 -ribooligonucleotides 10b 5 nmol in 10 µL of water were added 20 µM of 0.4M NH 4 + AcO -pH 4.2 and 10 eq of ketone or aldehyde derivatives. After 1 h to 4 h the conjugate was purified by size exclusion chromatography on Sephadex G25. The conversion of 3'-aminooxy-oligonucleotide into its conjugate was better than 90% (See SI). HPLC purification gave between 19 to 48% of pure conjugate.

Table 1 :

 1 3-aminooxy-deoxy or ribo-oligonucleotides and their conjugates.

	Sequences

  calcd. for C 12 H 20 N 3 O 3 S, 286.1220, found 286.1232. OH, J= 1.7 and 4.8 Hz); 7.76 (br, 1H, NH).[START_REF] Massaad-Massade | New Formulation for the Delivery of Oligonucleotides Using "Clickable" siRNA-Polyisoprenoid-Conjugated Nanoparticles: Application to Cancers Harboring Fusion Oncogenes[END_REF] C{ 1 H}-NMR (126 MHz, DMSO-d6): ppm 12.4, 17.0, 23.0,23.4, 26.0, 26.9, 27.1, 28.5, 30.1, 31.6, 32.3, 32.8, 33.7, 35.0, 35.6, 36.2, 41.5, 41.9, 45.9, 46.1, 47.4, 48.5, 63.5, 69.9, 70.5, 70.9, 173.1. HRMS (ESI/Q-TOF): [M+H] + calcd. for C 27 H 48 NO 5 , Calcd 466.3532, found 466.3537.To a solution of deoxycholamide 1,2-propanediol (280 mg, 0.6 mmol) in CH 3 OH:H 2 O:THF, 4:2:2, (v/v/v) (8 mL), sodium periodate (155 mg , 0.73 mmol) was added. After 2 h, the solution was evaporated and the residue was dissolved in methanol (10 mL). After filtration and evaporation of filtrate the crude was purified by silica chromatography using a gradient of methanol (5 to 15%) in CH 2 Cl 2 affording 18 as a clear foam, 240 mg, 92% Rf: 0.75 (20% MeOH/CH 2 Cl 2 ), Mixture of ethanal (20%) and dimethylacetal (80%). 1 H-NMR (400 MHz, DMSO-d6): ppm 0.59 (s, 3H, CH 3 ); 0.84 (s, 3H, CH 3 ); 0.91 (d, 3H, CH 3 , J = 6.5 Hz); 0.94-1.84 (m, 24H, CH 2 , CH); 1.92-2.13 (m, 2H, C23, CH 2 ); 3.04 (t, 2H, CH 2 , J = 5.5 Hz); 3.21 (s, 2H, OMe) 3.34-3.42 (m, 3H, C3 CH, CH 2 ); 3.78 (br, 1H, C12 CH); 4.16 (d, 1H, OH, J =4.1 Hz); 4.37-4.43 (m, 0.8H, CH(OMe) 2 ); 4.45 (d, 1H, OH, J = 4.3 Hz); 7.76 and 8.22(2 m, 1H, NH); 9.41 (s, 0.2H, CHO).[START_REF] Massaad-Massade | New Formulation for the Delivery of Oligonucleotides Using "Clickable" siRNA-Polyisoprenoid-Conjugated Nanoparticles: Application to Cancers Harboring Fusion Oncogenes[END_REF] C{ 1 H}-NMR (100 MHz, DMSO-d6): ppm 12.3, 16.9, 23.0, 23.4, 26.0, 26.9, 27.1, 28.5, 30.1, 31.6, 32.2, 32.8, 33.72, 35.0, 35.6, 36.2, 41.5, 43.9, 45.9, 46.1, 47.4, 49.1, 53.4, 54.8, 69.8, 70.9, 95.7, 172.7, 200.1. HRMS (ESI/Q-TOF): [M+H] + calcd. for C 26 H 44 NO 4 , 434.3270, found 434.3279.

	162.6, 172.5. HRMS (ESI/Q-TOF): [M+H] + calcd. for C 13 H 24 N 3 O 4 S, Calcd 318.1482, found
	318.1490.
	Biotin amide ethanal 14
	To a solution of biotin amide 1,2-propanediol (120 mg, 0.37 mmol) in CH 3 OH:H 2 O, 4:2, (v/v)
	(6 mL), sodium periodate (88 mg , 0.41 mmol) was added. After 2 h, the solution was
	evaporated and the residue was dissolved in methanol (10 mL). After filtration and
	evaporation of filtrate the crude was purified by silica chromatography using a gradient of
	methanol (5 to 15%) in CH 2 Cl 2 affording 14, 80 mg, 76%. Rf: 0.65 (20% MeOH/CH 2 Cl 2 ).
	Mixture of ethanal (70%) and dimethylacetal (30%), 1 H-NMR (400 MHz, DMSO-d6): ppm
	1.21-1.67 (m, 6H, (g,f,h), CH 2 ); 2.07 (t, 1.3H, (e) CH 2 , J = 7.2 Hz); 2.17 (t, 0.7H, (e) CH 2 , J =
	7.2 Hz); 2.57 (d, 1H, (l) CH 2 , J = 12.5 Hz); 2.82 (dd, 1H, (l) CH 2 , J = 5.1 Hz J = 12.5 Hz);
	3.08-3.13 (m, 1.3H, (c) CH 2 (i) CH); 3.22 (s, 1.8H, OCH 3 ); 3.85 (d, 0.7H, (c) CH 2 , J = 5.4
	Hz); 4.09-4.16 (m, 1H, (k) CH); 4.27-4.33 (m, 1H, (j) CH); 4.38-4.44 (m, 0.7H, CH(OMe) 2 );
	6.15, 6.18 and 6.35, 6.40 (2 x 2s, 2H, NHCONH); 7.78 and 8.24 (t and bs, 1H, CONH, J =
	5.52 Hz); 9.42 (s, 0.7H, CHO). Deoxycholamide 1,2-propanediol 17 Associated content
	To a solution of deoxycholic acid N-hydroxysuccinimide, 17 (490 mg, 1.0 mmol) and Et 3 N
	(140 µL, 1.1 mmol) in DMF (10 mL), 3-amino-2-hydroxy-propan-1-ol (100 mg, 1.1 mmol)
	(150 mg 0.44 mmol) and Et 3 N (148 µL, 1.06 dissolved in 1 mL of DMF was added and stirred at 50 °C for 3 h. After evaporation the
	mmol) in dry CH 3 CN (10 mL), 3-amino-2-hydroxy-propan-1-ol (48 mg, 0.53 mmol) residue was purified by silica gel chromatography from 5% to 20% of MeOH in CH 2 Cl 2
	dissolved in 3 mL of DMF was added and stirred at rt for 2 h. After evaporation, the residue affording the diol derivative 17 as a clear oil, 300 mg, 64%. Rf: 0.5 (20% MeOH/CH 2 Cl 2 ),
	was purified by silica gel chromatography from 5% to 20% MeOH in CH 2 Cl 2 affording the 1 H-NMR (500 MHz, DMSO-d6): ppm 0.59 (s, 3H, CH 3 ); 0.84 (s, 3H, CH 3 ); 0.91 (d, 3H, CH 3 ,
	diol derivative 13, 120 mg, 86%. Rf : 0.5 (MeOH/CH 2 Cl 2 , 2:8, v/v). 1 H-NMR (400 MHz, J = 6.5 Hz); 0.95-1.83 (m, 24H, CH 2 , CH); 1.94-2.14 (m, 2H, C23, CH 2 ); 2.93-3.39 (m, 5H,
	DMSO-d6): ppm 1.23-1.37 (m, 2H, (g) CH 2 ); 1.39-1.66 (m, 4H, (f,h) 2CH 2 ); 2.09 (t, 2H, (e) propyl CH 2 , CH); 3.45 (qd, 1H, C 3 , CH, J = 5.4 Hz, J = 10.8 Hz); 3.78 (br, 1H, C12, CH);
	CH 2 , J = 7.5 Hz); 2.57 (d, 1H, (l) CH 2 , J = 12.4 Hz); 2.82 (dd, 1H, (l) CH 2 , J = 5.2 Hz J = 12.5 4.19 (d, 1H, OH, J = 4.1 Hz); 4.46 (d, 1H, OH, J = 4.3 Hz); 4.50 (t, 1H, OH, J= 5.9 Hz); 4.71
	Hz); 2.93-3.29 (m, 5H, (a,c) CH 2 , (i) CH); 3.43-3.49 (m, 1H, (b) CH); 4.1-4.16 (m, 1H, (k) CH); 4.27-4.33 (m, 1H, (j) CH); 4.51 (t, 1H, OH, J =5.8 Hz); 4.72 (d, 1H, OH, J=4.9 Hz); 6.35 and 6.41 (2s, 2H, (m,n) NH); 7.76 (t, 1H, (d) CONH, J = 5.7 Hz). 13 C{ 1 H}-NMR (100 MHz, DMSO-d6): ppm 25.2, 27.9, 28.1, 35.0, 39.4, 39.7, 41.9, 55.3, 59.1, 60.9, 63.5, 70.5, (dd, 1H, Deoxycholamide ethanal 18

[START_REF] Massaad-Massade | New Formulation for the Delivery of Oligonucleotides Using "Clickable" siRNA-Polyisoprenoid-Conjugated Nanoparticles: Application to Cancers Harboring Fusion Oncogenes[END_REF]

C{ 1 H}-NMR (400 MHz, DMSO-d6): ppm 25.2, 27.9, 28.0, 34.9, 39.7, 49.1, 55.3, 59.1, 60.9, 95.7, 162.6, 172.1, 200.1. HRMS (ESI/Q-TOF): [M+H] + *S Supporting Information. NMR data of 6a-b, 13, 14, 17, 18; HPLC and MALDI-TOF spectra of 3'-aminooxy-(deoxy or ribo)-oligonucleotides and their conjugates.

ACKNOWLEDGMENTS

We thank the Institut des biomolécules Max Mousseron (IBMM) for financial support through the AAP-IBMM2017 funding program. F. M. is a member of Inserm.

AUTHOR INFORMATION

Corresponding Authors E-mail francois.morvan@umontpellier.fr ORCID Dr François Morvan https://orcid.org/0000-0001-5077-0028 Dr Jean-Jacques Vasseur https://orcid.org/0000-0002-4379-6139

Notes

The authors declare no competing financial interest.