Synthesis, binding, nuclease resistance and cellular uptake properties of 2′-O-acetalester-modified oligonucleotides containing cationic groups

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx

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Synthesis, binding, nuclease resistance and cellular uptake properties of 20 -O-acetalester-modified oligonucleotides containing cationic groups Annabelle Biscans a, Sonia Rouanet a, Jean-Rémi Bertrand b, Jean-Jacques Vasseur a, Christelle Dupouy a,⇑, Françoise Debart a,⇑ a b

Department of Nucleic Acids, IBMM UMR 5247, CNRS-Université Montpellier-ENSCM, UM Campus Triolet, Place E. Bataillon, 34095 Montpellier Cedex 05, France UMR 8203 CNRS, Université Paris-Saclay, Institut Gustave Roussy, 114 rue Edouard Vaillant, 94805 Villejuif Cedex, France

a r t i c l e

i n f o

Article history: Received 16 June 2015 Revised 24 July 2015 Accepted 25 July 2015 Available online xxxx Keywords: Oligonucleotides 20 -O-Acetalester Cationic groups Nuclease resistance Cell uptake

a b s t r a c t We report on the synthesis and properties of oligonucleotides (ONs) with 20 -O-acetalester modifications containing cationic side chains in a prodrug-like approach. In the aim to improve cell penetration and nuclease resistance, various different amino- or guanidino-acetalester were grafted to 20 -OH of uridine and the corresponding phosphoramidites were incorporated into ONs. Introduction of 20 -O-(2-aminomethyl-2-ethyl)butyryloxymethyl (AMEBuOM) modification into 20 -OMe ONs leads to high resistance towards enzymatic degradation and to destabilization of duplexes with complementary RNA strand. Spontaneous uptake experiments of a twelve-mer containing ten 20 -O-AMEBuOM-U units into A673 cells showed moderate internalization of ON within the cells whereas substantial internalization of the corresponding lipophilic 20 -O-pivaloyloxymethyl ON was observed for the first time. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Synthetic oligonucleotides (ONs) have been extensively used as potent molecules to knock down gene expression in various therapeutic strategies such as antisense or siRNA technologies.1 However, most oligonucleotide therapies are still limited by the inefficient in vivo delivery and the instability in extra- and intracellular media. The hydrophobic lipid-rich and negatively charged cell membrane obstructs cell penetration for ONs. Proposed solutions to overcome this hurdle have included the use of nanoparticle carriers or the attachment of various ligands such as peptides, cationic lipids, carbohydrates or small molecules designed to improve delivery.2–7 Despite significant advances, the stability and the intracellular delivery of synthetic ONs still remain the key point for therapeutic applications. Alternatively a large variety of chemical modified ONs have been explored and some of them were shown to improve cellular uptake.8 Conjugation or modification of ONs with lipophilic moieties is an attractive proposition to modify the pharmacokinetic and pharmacodynamic characteristics of ONs.6,9 In such a way, for two past decades our group has been ⇑ Corresponding authors. Tel.: +33 046 714 3837; fax: +33 046 704 2029 (C.D.); tel.: +33 046 714 3898; fax: +33 046 704 2029 (F.D.). E-mail addresses: [email protected] (C. Dupouy), francoise. [email protected] (F. Debart).

involved in the incorporation of hydrophobic groups into intrachain positions of ONs to provide lipophilicity to ONs. Thus a prodrug-like approach has been developed either masking the internucleoside phosphodiester bonds with S-acyl-2-thioethyl groups in ODNs10,11 or grafting the pivaloyloxymethyl (PivOM) group to 20 -OH in ORNs.12–14 These esterase-labile modifications were designed to enhance both nuclease resistance and cellular uptake,15 and once inside cells, modified ONs are converted by cytoplasmic esterases into native ONs. More recently, likewise efficient delivery of RNAi prodrugs containing phosphotriester backbone with esterase-labile groups was reported.16 Another means to improve cellular internalization properties is to introduce positive charges into ONs in order to reduce the overall negative charge of ONs and to favorably interact with the negatively charged cell membrane during cell penetration.17 Indeed, cationic or zwitterionic ONs have shown significant cell penetrating properties and stability towards hydrolytic enzymes.18–24 Particularly, the guanidinium group which has the advantage of remaining protonated over a wide pH range (pKa around 12.5) has been incorporated into ONs at selected positions on the 20 -OH ribose,25,26 nucleobases19,27 or phosphate backbone.20 In the same way, many 20 -O-tethered amino groups were reported to confer nuclease resistance properties and to promote cellular uptake of such cationic ON.24,28–30 Of particular interest 20 -O-(N-

http://dx.doi.org/10.1016/j.bmc.2015.07.054 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

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(aminoethyl)carbamoyl)methyl modified ONs demonstrated resistance to degradation in human serum and cell penetration, without any additives.24 In this context and in extension of the prodrug-like approach with 20 -O-acetalester modifications, we designed ONs modified with new 20 -O-acetalester groups bearing positive charges, and their properties have been studied. It is noteworthy that Damha’s group has been earlier interested in the synthesis of ONs containing amino acid based acetalesters particularly with lysine for its increased net positive charge.31 Unfortunately with lysine, alanine and phenylalanine as amino acids, 20 -O-acetalester ONs could not be obtained because they were partially hydrolyzed during HPLC purification/handling. In this work, in order to ensure the chemical stability of amino- or guanidino-containing acetalester groups we designed 20 -O-modifications with the carbon atom at a-position of the carbonyl moiety which was substituted with two electron donating methyl groups (2-amino-2-methylpropionyloxymethyl: AMPrOM) or ethyl groups (2-aminomethyl-2-ethyl-butyryloxymethyl: AMEBuOM and 2-guanidinomethyl-2-ethyl-butyryloxymethyl: GMEBuOM) by inductive effect (Fig. 1). While the pKa values of amino groups could not been determined experimentally, the amino groups are certainly positively charged at physiological pH as the pKa value of the free amino groups from amino acid derivatives is around 9. Herein we describe the synthesis of oligouridylates incorporating UGMEBuOM, UAMPrOM and UAMEBuOM, and the properties of 20 -O-methyl oligouridylates incorporating several UAMEBuOM units. 2. Results and discussion 2.1. Preparation of 20 -O-modified uridine phosphoramidites 5a-c and solid-phase synthesis of uridylates 6–18 To introduce the positively charged modifications into ONs, the phosphoramidite monomers 5a–c were synthesized (Scheme 1). The tert-butoxycarbonyl (Boc) group has been chosen to protect the amino or the guanidino functions during the phosphoramidites preparation and oligonucleotides assembly. This protection remains stable during the whole synthesis process and it is removed in acidic conditions which are not detrimental to the stability of acetalester groups. The synthetic approach was very similar to that previously described for 20 -O-modifications with PivOM or propionyloxymethyl groups (Scheme 1).14 First, the methylthiomethyl uridine derivative 1 was activated with sulfuryl chloride to give 20 -chloromethyl ether intermediate. Then reaction with the cesium salt of the desired Boc-protected amino or guanidino acid derivative afforded compounds 2a–c in high yields. For 2b and 2c, reactions were conducted with 90% and 83% yields, respectively from commercially available reagents: a-methylalanine or 2-aminomethyl-2-ethyl-butanoic acid which were Boc-protected before use. For BocGMEBuOM modification, the 2-N,N0 -di-Boc-

O

-O

O O O P O O

R=

U

H N

NH2

2'-O-GMEBuOM

NH2+ R

O

R=

NH3+

2'-O-AMPrOM

O R=

NH3+

2'-O-AMEBuOM

Figure 1. Chemical structures of 20 -O-amine- or guanidine-containing acetalester uridine used in this study. Abbreviations: GMEBuOM: 2-guanidinomethyl-2-ethyl butyryloxymethyl; AMPrOM: 2-amino-2-methyl propionyloxymethyl; AMEBuOM: 2-aminomethyl-2-ethyl butyryloxymethyl.

guanidinomethyl-2-ethyl-butyric acid derivative was prepared by guanidinylation of 2-aminomethyl-2-ethyl butanoic acid with N,N0 -di-Boc-N00 -triflylguanidine upon a described procedure.32 Introduction of the guanidine-containing acetalester group was carried out with similar yield (84%). After removal of the silyl protection upon treatment with Et3N.3HF, the 20 -O-modified uridine monomers 3a–c were further dimethoxytritylated in CH2Cl2 in the presence of DIEA to give 4a–c in similar 90% yield. 30 -phosphitylation was finally conducted in standard conditions and uridine phosphoramidites 5a–c were isolated with high purity in good yields. Before synthesis of 20 -O-modified ONs to study the properties of each modification, we prepared three model sequences URTTTTT-p (R = 20 -O-GMEBuOM 6, 20 -O-AMPrOM 7, 20 -O-AMEBuOM 8) incorporating each monomer building block 5a–c, respectively. The stability of the cationic 20 -O-acetalester groups was evaluated upon ON elongation on LCAA-CPG solid support and deprotection conditions. To maintain these modifications, ON deprotection could not be conducted in standard basic conditions. Consequently the normally used base-labile succinyl linker anchored to the support was replaced by an acid-labile phosphoramidate linker which after cleavage releases ON with a 30 -phosphoryl group.33 For these syntheses on a 1 lmol scale, elongation was performed upon the standard automated RNA synthetic procedure with a 180s coupling step. After ON elongation completion, a 1 M DBU solution in CH3CN was applied for 3 min to remove cyanoethyl groups then a treatment with 80% aqueous acetic acid was used for 4 h to cleave the monoester phosphoramidate linker as previously described.33 Unfortunately, in these acidic conditions the Boc protecting group was not removed therefore a TFA solution in dichloromethane (3:1) was applied for 30 min twice to deprotect the amino and guanidino functions while ONs were released from solid support. In the prospect of synthesizing G and A-containing ONs later, the stability of the hetero-sequence AGCTAGCU was evaluated in such acidic conditions. No depurination was observed after 30 min treatment with the TFA solution in dichloromethane. This data seems encouraging to use further a synthetic strategy based on acid-labile protections for nucleobases. Crude ONs 6, 7 and 8 were purified by RP-HPLC and characterized by MALDI-TOF mass spectrometry. RP-HPLC analyses of the purified ONs 6 and 7 showed the presence of an extra peak with a lower retention time than the peak of the desired ON corresponding to ON with 20 -OH-uridine. This result prompted us to study the stability of the 20 -O-acetalester modifications bearing amino or guanidino functions over 24 h in water at 25 °C. The fate of URTTTTT-p 6, 7 and 8 was monitored by RP-HPLC (Fig. 2). After 8 h in water, the GMEBuOM and AMPrOM groups (Fig. 2A and B, respectively) were totally hydrolyzed since the major peak corresponds to UTTTTT-p whereas the AMEBuOM group remained perfectly intact after 24 h (Fig. 2C). The electron withdrawing force for the guanidinium in ON 6 and for the ammonium on the carbon atom at a-position of the carbonyl function in ON 7 could explain the instability of the acetalester modification in both cases. Consequently, these too unstable modifications were not further evaluated. In contrast, the more stable 20 -O-AMEBuOM modification was investigated for hybridization properties, nuclease resistance and cellular penetration. For this purpose, the phosphoramidite 5c was incorporated into several 20 -O-methyl RNA oligomers (Table 1) at different positions within the sequence U10TT. Four sequences were designed to study the properties of these partially or fully modified RNA oligomers 11–14 with alternating 20 -O-AMEBuOM/20 -O-Me (11), five consecutive modifications at 50 -end of 20 -O-Me RNA 12, five consecutive modifications at 30 -end of 20 -O-Me RNA 13 and ten modifications for 14.

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O Si O

U

O

Si O

a) O

O

Si

HO

U

O

b)

O

S

Si

O

O

O

OH

R

U

O

c) O

2a 84% 2b 90% 2c 83% DMTrO

d) O iPr2N

P

O

O OCNE

a

BocGMEBuOM

R=

b

BocAMPrOM

R=

c

BocAMEBuOM

R=

H N

5a 85% 5b 72% 5c 83%

OH

O

O

R O

4a 89% 4b 92% 4c 90%

NHBoc NBoc

R O

R

3a 92% 3b 80% 3c 84%

U

O

O

U

O

O

O

1

DMTrO

NHBoc

NHBoc

Scheme 1. Synthesis of 20 -O-modified phosphoramidite uridine monomers. Reagents and conditions: (a) SO2Cl2 1 M in CH2Cl2, rt, 1.5 h then RCOOH, Cs2CO3, DMF, rt, 2 h; (b) Et3N, 3HF, THF, rt, 2 h; (c) DMTrCl, DIEA, CH2Cl2, rt, 2 h; (d) (iPr)2NP(Cl)OCNE, DIEA, DCM, rt, 2 h.

A

URTTTTT-3’P 0h

B

URTTTTT-3’P 0h

UTTTTT-3’P

8h

8h

24 h

24 h

10,0

URTTTTT-3’P 0h

UTTTTT-3’P

TTTTT-3’P

6,0 7,5

C

12,5

15,8

7,5

8h

24 h

10,0

min

12,5 min

15,0

17,5

7,5

10,0

12,5

15,0

17,5

min

Figure 2. RP-HPLC profiles of URTTTTT-p with (A) R = 20 -O-GMEBuOM 6, (B) 20 -O-AMPrOM 7, (C) 20 -O-AMEBuOM 8, incubated in water over 24 h at 25 °C. RP-HPLC analysis conditions: column Macherey-Nagel Nucleodur C18 8  125 mm, 100 Å; elution with a 20 min linear gradient of 0% to 30% of B (80% CH3CN in 0.05 M AcOAm) in eluent A (0.2% CH3CN in 0.05 M AcOAm). Flow rate: 2.0 mL min1, k 260 nm.

Comparison was performed with unmodified ON 9, fully modified 20 -O-Me ON 10 and fully modified 20 -O-PivOM ON 15. For the preparation of these modified ONs, the standard automated solid-phase synthesis on a 1 lmol scale was performed using the same conditions and solid support as described above for the model ONs 6–8. After elongation, ONs 9–15 were deprotected and released from solid support. The terminal 30 -phosphate of ONs 11–14 was enzymatically removed with calf intestinal phosphatase alkaline then crude ONs were purified by IEX-HPLC and were characterized by MALDI-TOF mass spectrometry (Table 1). 2.2. Hybridization studies The influence of 20 -O-AMEBuOM modified uridine on thermal stability of different RNA duplexes was examined (Table 1). Hybridization properties of ONs 11–14 with the complementary

strand rA12 were studied by UV-melting experiments at 260 nm and were compared to those of the unmodified duplex with ON 9, of the duplexes with the fully modified 20 -O-Me ON 10 and with the fully modified 20 -O-PivOM ON 15. Compared to the unmodified duplex (Tm 16.2 °C), the presence of ten consecutive AMEBuOM modifications in ON 14 destabilized the duplex (Tm 11.3 °C) whereas the fully PivOM modified ON 15 formed a more stable duplex (Tm 20.7 °C). Nevertheless the destabilization was not detrimental to the duplex formation (DTm/mod 0.5 °C). When five AMEBuOM modifications were introduced into 20 -O-Me ONs either alternatively in ON 11, or consecutively at 50 -end in ON 12, or consecutively at 30 -end in ON 13, the stability of duplexes decreased (DTm about 5–6 °C) relative to the duplex formed with fully 20 -O-Me ON 10 (Tm 27.7 °C). DTm values per modification range between 1.0 and 1.3 °C. This decrease confirms the destabilization induced by the 20 -O-acetalester modifications with positive

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Table 1 Data on 20 -O-modified oligonucleotides ON

Sequences 50 ? 30 a

MALDI-TOF MSb

6 7 8 9 10 11 12 13 14 15 16 17 18

UGMEBuOMTTTTT-p UAMPrOMTTTTT-p UAMEBuOMTTTTT-p UUUUUUUUUUTT UOMeUOMeUOMeUOMeUOMeUOMeUOMeUOMeUOMeUOMeTT URUOMeURUOMeURUOMeURUOMeURUOMeTT URURURURURUOMeUOMeUOMeUOMeUOMeTT UOMeUOMeUOMeUOMeUOMeURURURURURTT URURURURURURURURURURTT UPivOMUPivOMUPivOMUPivOMUPivOMUPivOMUPivOMUPivOMUPivOMUPivOMTT Cy3-UUUUUUUUUUTT Cy3-UPivOMUPivOMUPivOMUPivOMUPivOMUPivOMUPivOMUPivOMUPivOMUPivOMTT Cy3-URURURURURURURURURURTT

Calcd

Found

2044.87 1961.07 2004.55 3608.26 3748.33 4463.03 4463.03 4463.03 5178.03 4748.67 4114.84 5254.32 5684.78

2045.52 1961.62 2004.07 3607.13 3747.36 4462.78 4460.86 4462.56 5175.89 4747.77 4113.54 5253.52 5683.95

Tmc (°C)

DTm/Modd (°C)

DTm/Mode (°C)

16.2 27.7 22.7 22.2 21.2 11.3 20.7

+1.2 +0.6 +0.6 +0.5 0.5 +0.5

1.0 1.1 1.3 1.6 0.7

MALDI-TOF MS data and duplex thermal stability (Tm °C) with rA12. Data is an average of three hybridization–melting cycles. Estimated error in Tm = ±0.5 °C. Complementary RNA strand: rA12 a UOMe = 20 -O-Me Uridine, UR = 20 -O-AMEBuOM Uridine, UPivOM = 20 -O-pivaloyloxymethyl Uridine. b Negative mode. c Tm values obtained from UV melting curves at 260 nm with 3 lM strand concentration in 10 mM sodium cacodylate, 100 mM NaCl, pH 7.0. d DTm/mod is the difference in Tm relative to the 20 -OH unmodified duplex per 20 -O-modification. e DTm/mod is the difference in Tm relative to the 20 -O-Me duplex per 20 -O-modification.

charges. These data are rather unexpected if we compare with reported hybridization properties of 20 -modified ONs containing cationic side chains which typically exhibit high binding affinity to complementary RNA.24,34 Nevertheless with aliphatic 20 -alkylamine modifications such as aminoethyl, aminopropyl, aminopentyl and aminohexyl a duplex destabilization was observed with increasing number of modified nucleotides compared to unmodified duplex.35,36 In addition, it is noteworthy that a decrease in affinity of ON 14 containing AMEBuOM sugars for RNA complement is obtained compared to simple tert-butyl side chain (ON 15). This result suggests that the positive charges do not compensate for the unfavorable effect of the steric hindrance of the alkyl substituents: ethyl and aminomethyl groups. Circular dichroism spectra of all duplexes formed with ONs 9–15 indicate the typical curve of a A-form helix geometry with a positive band near 262 nm and a strong negative band at 210 nm (Supplementary data). All the curves of modified duplexes are superimposable to the one of unmodified duplex. Therefore the AMEBuOM modification does not disturb the original A-form conformation of RNA duplex even though a lower duplex stability is noticed.

spleen phosphodiesterase (CSPD) (Fig. 3B). ONs 9–15 were treated with SVPD for 8 h and the percentage of intact ON was estimated by reverse-phase HPLC analysis after 1 h and 8 h incubation. It is noteworthy that modified ONs with AMEBuOM remained 80% intact (for ONs 11 and 13) or 65% intact (for ONs 12 and 14) while unmodified ON 9 and 20 -OMe ON 10 were degraded after 1 h incubation. Surprisingly, ON 14 was the least resistant to the nuclease after 8 h (50% intact) although it was fully modified with AMEBuOM groups. Nuclease stability in the presence of CSPD was investigated over a period of 24 h and all 20 -O-modified ONs were more resistant (>50% intact after 24 h) than 20 -OH ON 9 which was completely digested at 8 h incubation. These results suggest that 20 -O-AMEBuOM modifications confer RNA stability towards exonucleases as many 20 -O-modifications with alkylammonium substitutions.30,35–37 However, this assay demonstrates the superior 30 -exonuclease resistance of the 20 -O-PivOM modification (85% ON 15 intact after 8 h with SVPD) compared with the amine-containing 20 -O-acetalester modification (50% ON 14 intact after 8 h with SVPD). This finding argues against the superior stability of ONs with positively charged 20 -O-modifications over ONs with neutral 20 -O-alkyl substituents.24,29,38

2.3. Nuclease resistance

2.4. Cellular uptake

The 30 - and 50 -exonuclease resistance of ONs 11–14 containing five or ten 20 -O-AMEBuOM modifications have been determined using snake venom phosphodiesterase (SVPD) (Fig. 3A) and calf

One of the main objectives of this work was to evaluate the impact of positive charges in the 20 -O-acetalester modifications on the cell penetration properties. In order to do this, the Cy3-labeled 12-mer model (UAMEBuOM)10TT (ON 18) as well the Cy3-unmodified ON 16 and Cy3-(UPivOM)10TT ON 17 were synthesized. A673 cells (human Ewing’s sarcoma) were treated with ONs 16–18 (0.5 lM) in the absence of any transfection agents. After 3 h incubation in serum-free OptiMEM, cells were washed, fixed, mounted and analyzed by epi-fluorescence microscopy (Fig. 4).39 In these incubation conditions, unmodified ON 16 does not penetrate into the cells because no red fluorescence is detected as in untreated cells. This is in accordance with the fact that without modifications, ONs need to be vectorized to enter the cells. When PivOM or AMEBuOM modifications are introduced at 20 -position of the sugar ring, red fluorescence is detected into the cells. These results indicate that modified ONs 17 and 18 penetrated efficiently into the cells. Nevertheless the PivOM modification is more efficient than AMEBuOM one which was rather unexpected. Image

% total amount

A

B

100

100

80

80

60

60

40

40

20

20

0

9

10

11 12

13 14 15

Oligonucleotides

0

9

10

11 12 13 14 15

Oligonucleotides

Figure 3. Relative enzymatic stability of 20 -O-modified ONs 10–15 against SVPD (A) and CSPD (B) compared to the stability of unmodified ON 9. Incubation times for A: 0 h (black bar), 1 h (dark gray), 8 h (light gray); for B: 0 h (black bar), 8 h (dark gray), 24 h (light gray).

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Scale Bar = 50 µm

Cy3

Cy3+DAPI

Cy3+DAPI+ Phase contrast

Untreated cells

Cy3-(U

) T

OH 10

2

16

Cy3-(U

) T

PivOM 10

2

17

) T

Cy3-(U

AMEBuOM 10

2

18

Figure 4. Fluorescence microscopy images of A673 cells treated with ONs 16–18 at 0.5 lM concentration for 3 h at 37 °C without any transfecting agent in OptiMEM medium. After fixation and mounting in DAPI containing anti-fading medium, cells were observed by fluorescence microscopy. Cell nuclei are colored in blue by DAPI and siRNA are in red due to Cy3 labeling. The scale bar is 50 lm.

analysis was performed to evaluate the fluorescence intensity in cells treated with ONs 16, 17 and 18 compared to untreated cells. The results (Fig. S21 in SI) confirmed that ON 17 with PivOM modifications penetrated into all the observed cells. In contrast, the AMEBuOM modified ON 18 and the unmodified ON 16 have a distribution profile closed to untreated cells indicating that cationic acetalester modification does not improve cell penetration as efficiently as expected. It is noteworthy that we do not observe any morphological modification in cell aspect during the incubation time indicating that cells well tolerate modified ONs. 2.5. Chemical Stability of 20 -O-AMEBuOM modifications in cell culture medium DMEM Unconvinced by the data obtained with cellular penetration of the 20 -O-AMEBuOM modified ON 18 in comparison with other reported ONs bearing 20 -cationic modifications22,24 we searched a reason for this surprising result. For this, we have assessed the chemical stability of AMEBuOM groups within the model ONs 11, 12 and 13 in comparison to PivOM groups (ON 15), in cell culture medium DMEM. The medium was not complemented by fetal calf serum as used for cell culture. ONs incubation was then performed in DMEM at 37 °C for time intervals of 0–72 h. The loss of acetalester groups was evaluated by RP-HPLC at different incubation times (Supplementary data, Figs. S20 and S21). After 24 h, the peaks corresponding to intact ONs 11–13 with five amino acetalester modifications were not noticeable in the chromatograms whereas 20 -O-PivOM ON 15 remained 70% intact. It appears that after the relatively short incubation time of 3 h, about 40% removal of AMEBuOM groups in ONs 11–13 occurred to give a mixture of several species with three, four or five cationic groups. In the same time, 97% of ON 15 remains intact. This result means that in the conditions of cellular uptake experiments 20 -OAMEBuOM modified ON 18 has lost some cationic groups which altered its properties unlike ON 17 with 20 -O-PivOM groups. The chemical instability of this cationic acetalester modification in DMEM might be the reason for lower internalization of ON 18 into A673 cells.

3. Conclusions In this work, we have presented a novel class of 20 -O-acetalester ONs containing a cationic side chain with the aim of improving the cellular uptake and nuclease resistance. Three uridine phosphoramidites with 20 -O-(amine or guanidine) acetalester 5a–c were prepared and were successfully incorporated into short ONs. The two modifications GMEBuOM and AMPrOM with a guanidinium or an ammonium side chain respectively were unstable during HPLC purification and handling therefore only the AMEBuOM modification was further investigated. Various ONs incorporating a mix of 20 -OMe and 20 -O-AMEBuOM groups and a fully modified ON were synthesized. Although this cationic acetalester modification leads to a moderate duplex destabilization, the A-form helix geometry was retained. This unexpected result is contrary to those found in the literature with 20 -O-cationic groups and could be explained by the unfavorable steric hindrance of the side chain not compensated by the positive charge. Greater nuclease stability was observed with all 20 -O-AMEBuOM modified ONs compared to 20 -OH or 20 -OMe ONs but 20 -O-PivOM ON is more resistant to 30 -exonuclease degradation. Spontaneous cellular uptake of ONs containing the cationic AMEBuOM or the lipophilic PivOM modification showed that the tBu side chain helped to penetrate into cells whereas moderate internalization of ON with the ammonium side chain was observed. We hypothesized that the reason for this result was due to the premature loss of some 20 -O-AMEBuOM groups from modified ONs, in cell culture medium. The cationic acetalester modifications were shown to be chemically less stable than neutral ones whereas lipophilic acetalester groups seem promising for an efficient spontaneous uptake of modified RNAs in a prodrug-like approach. 4. Experimental 4.1. General Pyridine and DIEA were distilled over calcium hydride. All reactions were performed in anhydrous conditions under argon. All

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compounds were purified by silica gel (particle size of 40–63 lm) column chromatography. NMR experiments were accomplished on a Bruker DRX 400 or 600 spectrometers at 20 °C. High resolution mass spectra were recorded with electrospray ionization (ESI) in positive mode on a Q-TOF Micromass spectrometer. 4.2. Chemistry 4.2.1. 20 -O-(2-N,N0 -Di-Boc-guanidinomethyl-2-ethyl-butyryloxymethyl)-30 ,50 -O-(tetraisopropyldisiloxane-1,3-diyl)uridine 2a To a solution of 20 -O-methylthiomethyl-30 ,50 -O-(tetraisopropyldisiloxane-1,3-diyl)uridine 1 (0.36 g, 0.67 mmol, 1.00 equiv) in anhydrous CH2Cl2 (7 mL) was added dropwise under argon a 1.0 M sulfuryl chloride solution in CH2Cl2 (1.00 mL, 1.00 mmol, 1.50 equiv). The mixture was stirred for 1.5 h at room temperature. After completion of the reaction, the chloromethyl ether derivative was obtained as brown foam after evaporation of the solvent and was directly used in the next step. 2-N,N’-di-Boc-guanidinomethyl-2-ethyl-butyric acid (see preparation in Supplementary data S19) (0.40 g, 1.03 mmol, 1.55 equiv) and cesium carbonate (0.17 g, 0.53 mmol, 0.80 equiv) were suspended in anhydrous DMF (5 mL). The mixture was stirred at room temperature for 2 h and the chloromethyl ether derivative in anhydrous CH2Cl2 (3 mL) was added dropwise. After stirring at room temperature for 1.5 h, the mixture was washed with a saturated aqueous NaHCO3 solution. The aqueous layer was then extracted with ethyl acetate. The organic layer was washed with water then brine and dried over Na2SO4. The solvent was concentrated under reduced pressure. The crude material was purified by silica gel column chromatography with a mixture of cyclohexane and ethyl acetate (70:30). The desired compound 2a was obtained as white foam (0.50 g, 0.56 mmol, 84%). 1H NMR (400 MHz, CDCl3) d 8.80 (s, 1H, NH); 8.58 (s, 1H, NH); 7.81 (d, J = 8.0 Hz, 1H, H-6); 5.72 (s, 1H, H-10 ); 5.64 (d, J = 8.8 Hz, 1H,H-5); 5.63, 5.48 (2dAB, JAB = 6.8 Hz, 1H+1H, OCH2O); 4.30 (m, 1H, H-20 ); 4.23 (m, 1H, H-30 ); 4.20 (m, 1H, H-50 ); 4.10 (m, 1H, H-40 ); 3.96 (m, 1H, H-500 ); 3.66 (m, 2H, CH2N); 1.67 (m, 4H, CH2CH3); 1.48, 1.46 (s+s, 9H+9H, CH3 Boc); 1.08-1.02 (m, 28H, iPr); 0.87 (t, J = 7.2 Hz, 6H, CH2CH3). 13C NMR (100 MHz, CDCl3) d 175.1 (C@O); 163.2 (C@O); 156.5 (C@O); 155.3 (C@O); 153.1 (C@N); 149.3 (C@O); 139.6 (C-6); 101.5 (C-5); 89.2 (C-10 ); 88.3 (OCH2O); 82.9 (Cq Boc); 81.6 (C-40 ); 81.5 (C-20 ); 79.1 (Cq Boc); 68.2 (C-30 ); 59.3 (C-50 ); 50.2 (Cq); 42.8 (CH2N); 28.3, 28.0 (CH3 Boc); 27.0, 26.3 (CH2CH3); 17.4–16.8 (CH3 iPr); 13.5–12.6 (CH iPr); 8.8, 8.5 (CH2CH3). HRMS (ESI+) m/z calcd for C40H71N5O13Si2 (M+H)+ 886.4665, Found 886.4661. 4.2.2. 20 -O-2-Boc-amino-2-methyl-propionyloxymethyl-30 ,50 -O(tetraisopropyldisiloxane-1,3-diyl)uridine 2b Using the same procedure as for synthesis of 2a, starting from 1 (3.83 g, 7.00 mmol, 1.00 equiv) and Boc-a-methylalanine (2.28 g, 11.20 mmol, 1.55 equiv) compound 2b was obtained as white foam (4.42 g, 6.30 mmol, 90%). 1H NMR (600 MHz, CDCl3) d 8.78 (s, 1H, NH); 7.83 (d, J = 8.4 Hz, 1H, H-6); 5.75 (s, 1H, H-10 ); 5.66 (dd, J = 7.8 Hz, J = 1.2 Hz, 1H, H-5); 5.60, 5.49 (2dAB, JAB = 6.6 Hz, 1H+1H, OCH2O); 4.28 (m, 1H, H-20 ); 4.25 (m, 1H, H-50 ); 4.22 (m, 1H, H-40 ); 4.10 (m, 1H, H-30 ); 3.97 (m, 1H, H-500 ); 1.54 (s, 6H, CH3); 1.40 (s, 18H, CH3 Boc); 1.10–1.03 (m, 28H, iPr). 13C NMR (150 MHz, CDCl3) d 174.6 (C@O); 163.3 (C@O); 154.9 (C@O); 149.8 (C@O); 139.6 (C-6); 101.8 (C-5); 89.3 (C-10 ); 89.0 (OCH2O); 81.7 (C-40 ); 81.4 (C-20 ); 79.8 (Cq Boc); 68.4 (C-30 ); 59.5 (C-50 ); 56.3 (Cq); 28.4 (CH3 Boc); 25.3 (CH3); 17.6–17.0 (CH3 iPr); 13.6– 12.8 (CH iPr). HRMS (ESI+) m/z calcd for C31H55N3O11Si2 (M+Na)+ 724.3273, Found 724.3278.

4.2.3. 20 -O-2-Boc-aminomethyl-2-ethyl-butyryloxymethyl-30 ,50 O-(tetraisopropyldisiloxane-1,3-diyl)uridine 2c Using the same procedure as for synthesis of 2a, starting from 1 (0.64 g, 1.20 mmol, 1.00 equiv) and 2-Boc-aminomethyl-2-ethylbutanoic acid (0.46 g, 1.86 mmol, 1.55 equiv) compound 2c was obtained as white foam (0.74 g, 0.99 mmol, 83%).1H NMR (600 MHz, CDCl3) d 10.42 (s, 1H, NH); 8.42 (s, 1H, NH); 7.82 (d, J = 8.4 Hz, 1H, H-6); 5.72 (s, 1H, H-10 ); 5.66 (d, J = 8.4 Hz, 1H, H5); 5.54, 5.51 (2dAB, JAB = 6.6 Hz, 2H, OCH2O); 4.34 (m, 1H, H-20 ); 4.25–4.20 (m, 2H, H-30 +H-50 ); 4.10 (m, 1H, H-40 ); 3.96 (m, 1H, H500 ); 3.35 (m, 2H, CH2NH); 1.67 (q, J = 7.2 Hz, 2H, CH2CH3); 1.63 (q, J = 7.8 Hz, 2H, CH2CH3); 1.45 (s, 9H, CH3 Boc); 1.10–1.03 (m, 28H, iPr); 0.86 (m, 6H, CH2CH3). 13C NMR (150 MHz, CDCl3) d 176.0 (C@O); 162.6 (C@O); 156.1 (C@O); 149.8 (C@O); 138.9 (C6); 101.8 (C-5); 89.0 (C-10 ); 88.2 (OCH2O); 81.7 (C-40 ); 81.0 (C20 ); 79.4 (Cq Boc); 67.9 (C-30 ); 59.2 (C-50 ); 50.6 (Cq); 43.5 (CH2NH); 28.4 (CH3 Boc); 25.6, 25.1 (CH2CH3); 17.4–16.8 (CH3 iPr); 13.4–12.6 (CH iPr); 8.3, 8.1 (CH2CH3). HRMS (ESI+) m/z calcd for C34H61N3O11Si2 (M+H)+ 744.3923, Found 744.3925. 4.2.4. 20 -O-(2-N,N0 -di-Boc-guanidinomethyl-2-ethyl-butyryloxymethyl)-uridine 3a To a solution of 2a (0.47 g, 0.53 mmol, 1.00 equiv) in anhydrous THF (8 mL) was added Et3N-3HF solution (220 lL, 1.32 mmol, 2.50 equiv). After stirring for 2 h at room temperature, the deprotection was complete and the reaction mixture was treated with triethylammoniumacetate buffer (2 M, pH 7). The aqueous layer was extracted with ethyl acetate. The organic layer was washed with water then brine and dried over Na2SO4. The solvent was concentrated under reduced pressure. The crude material was purified by silica gel column chromatography with a step gradient of CH2Cl2 and methanol (0–3%). The desired compound 3a was obtained as white foam (0.32 g, 0.49 mmol, 92%). 1H NMR (400 MHz, CDCl3) d 11.44 (s, 1H, NH); 9.68 (s, 1H, NH); 8.43 (t, J = 5.6 Hz, 1H, NH); 7.85 (d, J = 8.0 Hz, 1H, H-6); 5.82 (d, J = 4.0 Hz, 1H, H-10 ); 5.72 (d, J = 8.0 Hz, 1H, H-5); 5.51, 5.41 (2dAB, JAB = 6.0 Hz, 2H, OCH2O); 4.49 (m, 1H, H-20 ); 4.31 (m, 1H, H-30 ); 4.06 (m, 1H, H-40 ); 3.97 (m, 1H, H-50 ); 3.81 (m, 1H, H-500 ); 3.60 (m, 2H, CH2N); 1.62 (m, 4H, CH2CH3); 1.47 (s, 18H, CH3 Boc); 0.83 (m, 6H, CH2CH3). 13C NMR (100 MHz, CDCl3) d 172.6 (C@O); 161.2 (C@O); 160.9 (C@O); 154.0 (C@O); 150.8 (C@N); 148.0 (C@O); 139.0 (C-6); 100.0 (C-5); 87.2 (C-10 ); 86.1 (OCH2O); 82.5 (C-40 ); 80.9 (Cq Boc); 78.8 (C-20 ); 77.1 (Cq Boc); 66.7 (C-30 ); 58.7 (C-50 ); 51.0 ((CH2CH3)2CCH2); 40.2 (CH2N); 25.8, 25.6 (CH3 Boc); 23.8 (CH2CH3); 5.9 (CH2CH3). HRMS (ESI+) m/z calcd for C28H45N5O12 (M+H)+ 644.3143, Found 644.3149. 4.2.5. 20 -O-2-Boc-amino-2-methyl-propionyloxymethyl uridine 3b Using the same procedure as for synthesis of 3a, starting from 2b (4.40 g, 6.28 mmol, 1.00 equiv) compound 3b was obtained as white foam (2.31 g, 5.00 mmol, 80%). 1H NMR (600 MHz, CDCl3) d 9.37 (s, 1H, NH); 7.81 (d, J = 8.4 Hz, 1H, H-6); 5.80 (d, J = 4.2 Hz, 1H, H-10 ); 5.73 (d, J = 7.8 Hz, 1H, H-5); 5.53, 5.40 (2dAB, JAB = 6.0 Hz, 2H, OCH2O); 4.49 (m, 1H, H-20 ); 4.34 (m, 1H, H-30 ); 4.11 (m, 1H, H-40 ); 3.94 (dd, J = 12.0 Hz, J = 1.8 Hz, 1H, H-50 ); 3.82 (dd, J = 12.0 Hz, J = 1.2 Hz, 1H, H-500 ); 1.48, 1.47 (s+s, 3H+3H, CH3); 1.41 (s, 18H, CH3 Boc). 13C NMR (150 MHz, CDCl3) d 174.3 (C@O); 163.5 (C@O); 155.1 (C@O); 155.0 (C@O); 141.7 (C-6); 102.4 (C-5); 90.0 (C-10 ); 89.4 (OCH2O); 85.1 (C-40 ); 82.4 (C-20 ); 80.5 (Cq Boc); 69.2 (C-30 ); 61.4 (C-50 ); 56.0 (Cq); 28.3 (CH3 Boc); 25.3 (CH3). HRMS (ESI+) m/z calcd for C19H29N3O10 (M+H)+ 460.1931, Found 460.1927.

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A. Biscans et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

4.2.6. 20 -O-2-Boc-aminomethyl-2-ethyl-butyryloxymethyl uridine 3c Using the same procedure as for synthesis of 3a, starting from 2c (0.71 g, 0.95 mmol, 1.00 equiv) compound 3c was obtained as white foam (0.40 g, 0.80 mmol, 84%). 1H NMR (600 MHz, CDCl3) d 9.58 (s, 1H, NH); 9.15 (s, 1H, NH); 7.87 (d, J = 7.8 Hz, 1H, H-6); 5.86 (d, J = 3.6 Hz, 1H, H-10 ); 5.73 (d, J = 7.8 Hz, 1H, H-5); 5.53, 5.36 (2dAB, JAB = 6.6 Hz, 2H, OCH2O); 4.44 (m, 1H, H-20 ); 4.38 (m, 1H, H-30 ); 4.10 (m, 1H, H-40 ); 4.00 (m, 1H, H-50 ); 3.86 (dd, J = 12.6 Hz, J = 1.8 Hz, 1H, H-500 ); 3.29 (m, 2H, CH2NH); 1.61 (m, 4H, CH2CH3); 1.41 (s, 9H, CH3 Boc); 0.83 (t, J = 7.8 Hz, 6H, CH2CH3). 13C NMR (150 MHz, CDCl3) d 175.4 (C@O); 163.3 (C@O); 156.1 (C@O); 154.4 (C@O); 141.1 (C-6); 102.5 (C-5); 89.5 (C-10 ); 89.3 (OCH2O); 84.9 (C-40 ); 81.9 (C-20 ); 79.8 (Cq Boc); 69.3 (C-30 ); 61.3 (C-50 ); 50.8 (Cq); 43.7 (CH2NH); 28.3 (CH3 Boc); 24.7 (CH2CH3); 8.1 (CH2CH3). HRMS (ESI+) m/z calcd for C22H35N3O10 (M+H)+ 502.2401, Found 502.2405. 4.2.7. 20 -O-(2-N,N0 -di-Boc-guanidinomethyl-2-ethylbutyryloxymethyl)-50 -O-(4,40 -dimethoxytrityl)uridine 4a A solution of 3a (0.65 g, 1.00 mmol, 1.00 equiv) in anhydrous CH2Cl2 (5 mL) was treated under argon with DIEA (192 lL, 1.10 mmol, 1.10 equiv) and dimethoxytrityl chloride (0.41 g, 1.20 mmol, 1.20 equiv) was added in small portions over 15 min. The mixture was stirred for 1.5 h at room temperature. A saturated aqueous NaHCO3 solution was added. The aqueous layer was then extracted with CH2Cl2. The organic layer was washed with water then brine and dried over Na2SO4. The solvent was concentrated under reduced pressure. The crude material was purified by silica gel column chromatography with a step gradient of CH2Cl2 and methanol (0–2%) with 1% pyridine. The desired compound 4a was obtained as yellow foam (0.84 g, 0.89 mmol, 89%). 1H NMR (400 MHz, CDCl3) d 11.49 (s, 1H, NH); 9.80 (s, 1H, NH); 8.55 (t, J = 5.6 Hz, 1H, NH); 8.01 (d, J = 8.4 Hz, 1H, H-6); 7.38–6.81 (m, 13H, HAr); 5.91 (d, J = 1.2 Hz, 1H, H-10 ); 5.65, 5.51 (2dAB, JAB = 6.0 Hz, 2H, OCH2O); 5.27 (d, J = 8.0 Hz, 1H, H-5); 4.45 (m, 1H, H-30 ); 4.33 (m, 1H, H-20 ); 4.02 (m, 1H, H-40 ); 3.78, 3.77 (s+s, 3H+3H, 2OCH3); 3.65 (m, 2H, CH2CH2CH2N); 3.50 (m, 2H, H-50 ); 1.66 (m, 4H, CH2CH3); 1.47, 1.42 (s+s, 9H+9H, CH3 Boc); 0.86 (m, 6H, CH2CH3). 13C NMR (100 MHz, CDCl3) d 175.3 (C@O); 163.5 (C@O); 163.4 (C@O); 158.7, 158.6, 144.3, 135.2, 135.0 (Cq, DMTr); 156.3 (C@O); 153.2 (C@N); 150.2 (C@O); 149.6 (C-6); 139.7, 136.0, 130.1, 130.0, 128.1, 128.0, 127.1, 123.7, 113.2 (CH, Car); 102.1 (C-5); 88.4 (C-10 ); 88.3 (OCH2O); 87.0 (OCq, DMTr); 83.1 (Cq Boc); 83.0 (C-40 ); 82.4 (C-20 ); 79.1 (Cq Boc); 68.4 (C-30 ); 61.0 (C-50 ); 55.2 (OCH3, DMTr); 50.3 ((CH2CH3)2CCH2); 42.9 (CH2N); 28.2, 28.0 (CH3); 26.0, 25.9 (CH2CH3); 8.4, 8.3 (CH2CH3). HRMS (ESI+) m/z calcd for C49H63N5O14 (M+H)+ 946.4450, Found 946.4457. 4.2.8. 20 -O-2-Boc-amino-2-methyl-propionyloxymethyl-50 -O(4,40 -dimethoxytrityl)uridine 4b Using the same procedure as for synthesis of 4a, starting from 3b (0.39 g, 0.76 mmol, 1.00 equiv) compound 4b was obtained as yellow foam (0.56 g, 0.70 mmol, 92%). 1H NMR (600 MHz, CDCl3) d 8.68 (s, 1H, NH); 8.01 (d, J = 8.4 Hz, 1H, H-6); 7.39–6.84 (m, 13H, HAr); 5.92 (s, 1H, H-10 ); 5.61, 5.50 (2dAB, JAB = 6.0 Hz, 2H, OCH2O); 5.29 (d, J = 7.8 Hz, 1H, H-5); 4.49 (m, 1H, H-30 ); 4.32 (m, 1H, H-20 ); 4.06 (m, 1H, H-40 ); 3.80, 3.79 (s+s, 3H+3H, 2OCH3); 3.57 (m, 1H, H-50 ); 3.53 (dd, J = 10.8 Hz, J = 2.4 Hz, 1H, H-500 ); 3.42–3.27 (m, 2H, CH2NH); 1.64 (m, 4H, CH2CH3); 1.41 (s, 9H, CH3 Boc); 0.85 (m, 6H, CH2CH3). 13C NMR (150 MHz, CDCl3) d 175.7 (C@O); 163.9 (C@O); 158.9, 158.8, 144.5, 135.4, 135.2 (Cq, DMTr); 155.0 (C@O); 156.2; 150.3 (C@O); 138.0 (C-6); 130.3, 130.2, 128.4, 128.2, 127.3, 113.5 (CH, Car); 102.3 (C-5); 88.6

7

(OCq, DMTr); 88.5 (C-10 ); 87.3 (OCH2O); 83.3 (C-40 ); 82.6 (C-20 ); 79.7 (Cq Boc); 68.5 (C-30 ); 61.1 (C-50 ); 55.4 (OCH3, DMTr); 51.1 (Cq); 44.9 (CH2NH); 28.5 (CH3 Boc); 25.0 (CH2CH3); 8.3 (CH3). HRMS (ESI+) m/z calcd for C43H53N3O12 (M+Na)+ 826.3527, Found 826.3518. 4.2.9. 20 -O-2-Boc-aminomethyl-2-ethyl-butyryloxymethyl-50 -O(4,40 -dimethoxytrityl)uridine 4c Using the same procedure as for synthesis of 4a, starting from 3c (2.29 g, 4.97 mmol, 1.00 equiv) compound 4c was obtained as yellow foam (3.41 g, 4.48 mmol, 90%). 1H NMR (600 MHz, CDCl3) d 8.84 (s, 1H, NH); 8.00 (d, J = 8.4 Hz, 1H, H-6); 7.41–7.14 (m, 13H, HAr); 5.92 (d, J = 2.4 Hz, 1H, H-10 ); 5.58, 5.51 (2dAB, JAB = 6.6 Hz, 2H, OCH2O); 5.26 (d, J = 8.4 Hz, 1H, H-5); 4.53 (m, 1H, H-40 ); 4.38 (m, 1H, H-20 ); 4.10 (m, 1H, H-30 ); 3.80, 3.79 (s+s, 3H+3H, 2OCH3); 3.51 (m, 2H, H-50 +H-500 ); 1.52, 1.50 (s+s, 3H+3H, CH3); 1.43 (s, 9H, CH3 Boc). 13C NMR (150 MHz, CDCl3) d 174.4 (C@O); 163.1 (C@O); 162.7 (C@O); 158.7, 158.6, 144.4, 135.4, 135.1 (Cq, DMTr); 155.0 (C@O); 150.1 (C@O); 140.0 (C-6); 130.2, 130.1, 128.1, 128.0, 127.1, 123.7, 113.3 (CH, Car); 102.0 (C-5); 88.8 (C-10 ); 88.5 (OCH2O); 87.0 (OCq, DMTr); 83.3 (C-40 ); 83.2 (C20 ); 80.4 (Cq Boc); 68.3 (C-30 ); 61.3 (C-50 ); 56.0 (OCH3, DMTr); 28.3 (CH3 Boc); 25.3 (CH3). HRMS (ESI+) m/z calcd for C40H47N3O12 (M+Na)+ 784.3057, Found 784.3056. 4.2.10. 20 -O-(2-N,N0 -Di-Boc-guanidinomethyl-2-ethyl-butyryloxymethyl)-30 -O-(2-cyanoethyl-N,N-diisopropyl phosphoramidite)-50 -O-(4,40 -dimethoxytrityl)uridine 5a To a solution of 4a (0.78 g, 0.81 mmol, 1.00 equiv) in anhydrous CH2Cl2 (8.2 mL) previously passed through an alumina column was added dropwise a mixture of N,N-diisopropylethylamine (354 lL, 2.03 mmol, 2.50 equiv) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (397 lL, 1.78 mmol, 2.20 equiv) in CH2Cl2. The mixture was stirred for 2 h at room temperature under argon. After reaction completion, ethyl acetate previously washed with a saturated aqueous NaHCO3 solution was added and the reaction mixture was poured into saturated NaCl/NaHCO3 solution (1:1 v/v). The aqueous layer was extracted with ethyl acetate and organic layers were dried over Na2SO4. The solvent was concentrated under reduced pressure. The crude material was purified by silica gel column chromatography with an isocratic elution with a mixture of CH2Cl2 and ethyl acetate (6:4, v/v) containing 1% pyridine. The desired phosphoramidite 5a was obtained as white foam (0.79 g, 0.69 mmol, 85%). 31P NMR (121 MHz, CD3CN): d 155.8, 154.2. HRMS (ESI+) m/z calcd for C58H80N7O15P (M+H)+ 1146.5528, Found 1146.5540. 4.2.11. 20 -O-2-Boc-amino-2-methyl-propionyloxymethyl-30 -O(2-cyanoethyl-N,N-diisopropylphosphoramidite)-50 -O-(4,40 dimethoxytrityl)uridine 5b Using the same procedure as for synthesis of 5a, starting from 4b (3.75 g, 4.93 mmol, 1.00 equiv) compound 5b was obtained as white foam (3.42 g, 3.55 mmol, 72%). 31P NMR (121 MHz, CD3CN): d 150.5, 149.3. HRMS (ESI+) m/z calcd for C49H64N5O13P (M+H)+ 962.4316, Found 962.4323. 4.2.12. 20 -O-2-Boc-aminomethyl-2-ethyl-butyryloxymethyl-30 O-(2-cyanoethyl-N,N-diisopropylphosphoramidite)-50 -O-(4,40 dimethoxytrityl)uridine 5c Using the same procedure as for synthesis of 5a, starting from 4c (1.50 g, 1.86 mmol, 1.00 equiv) compound 5c was obtained as white foam (1.56 g, 1.55 mmol, 83%). 31P NMR (121 MHz, CD3CN): d 150.4, 149.5. HRMS (ESI+) m/z calcd for C52H70N5O13P (M+Na)+ 1026.4605, Found 1026.4606.

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A. Biscans et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

4.3. Oligonucleotides synthesis and purification RNA syntheses were performed on an ABI model 394 DNA/RNA synthesizer (Applied Biosystems) on a 1 lmol scale. Phosphoramidites were vacuum dried prior to their dissolution in extra dry acetonitrile (Biosolve) at 0.1 M. Coupling was performed with 5-benzylmercaptotetrazole (BMT, 0.3 M) as activator. The oxidizing solution was 0.1 M iodine in THF/pyridine/H2O (78:20:2; v/v/v) (Link Technologies). The capping step was performed with a mixture of 5% phenoxyacetic anhydride (Pac2O) in THF and 10% N-methylimidazole in THF (Link Technologies). Detritylation was performed with 3% TCA in CH2Cl2. ONs deprotection and ONs releasing from solid support were carried out with different solutions according the ON. Cy3-50 -labeled ONs were synthesized by using Cy3-phosphoramidite (GE Healthcare). Crude ONs were analyzed and purified by IEX-HPLC (Dionex DNAPacÒ PA100, 4  250 mm for analysis or 9  250 for semi-preparative purpose, Buffer A: 20% CH3CN in 25 mM Tris–HCl pH 8, Buffer B: 20% CH3CN containing 200 mM NaClO4 in 25 mM Tris–HCl pH 8, flow rate: 1.5 mL/min for analysis or 5 mL/min for semi-preparative purpose, detection at 260 nm) or RP-HPLC (Macherey-Nagel Nucleodur C-18, 100 Å, 8  125 mm, Buffer A: 0.05 M AcOAm in 0.2% CH3CN, Buffer B: 0.05 M AcOAm in 80% CH3CN, 2 mL/min flow rate, detection at 260 nm). Purified ONs were characterized by MALDI-TOF MS (Perseptive Biosystems, spectra recorded on a Voyager-DE spectrometer equipped with a N2 laser 337 nm). ONs were desalted using a C18 cartridge (Sep-PakÒ Classic) and lyophilized. 4.3.1. Synthesis and purification of URTTTTT models 6–8 and 20 -O-modified ONs 11–14 LCAA-CPG 500Å (Glen Research) was used as solid support. URTTTTT 6–8 and ONs 11–14 were assembled in TWIST™ synthesis columns (Glen Research) with 20 -O-acetalester phosphoramidites 5a-c, dT phosphoramidites and 20 -OMe phosphoramidites (Chemgenes). Coupling step was performed for 180 s except for the first T phosphoramidite coupling on the solid support extended to 360 s. Capping step was performed for 160 s extended to 300 s for the first T phosphoramidite coupling. ONs deprotection and release from the solid support were carried out with 1.0 M DBU solution in anhydrous CH3CN for 3 min and then with a dry solution of TFA/CH2Cl2 3:1 (v/v) for 30 min (2). The TFA/CH2Cl2 solution was removed, neutralized by an aqueous solution of 0.05 M sodium acetate (pH 8) and lyophilized. Crude ONs 6–8 were analyzed and purified by RP-HPLC and characterized by MALDI-TOF MS. ONs were desalted using a C18 cartridge (Sep-PakÒ Classic) equilibrated with water and they were eluted with water/CH3CN (50/50). Crude ONs 11–14 were desalted using a C18 cartridge (Sep-PakÒ Classic) equilibrated with 100 mM TEAAc and eluted with 12.5 mM TEAAc/CH3CN (50:50). The desalted material was treated with phosphatase alkaline (CARN = 0.4 mM, 104 U/mL, CIP buffer, 2 h, 37 °C), analyzed by IEX-HPLC, MALDI-TOF MS and finally IEXHPLC purified. ONs were desalted using a C18 cartridge (Sep-PakÒ Classic) equilibrated with 100 mM TEAAc and they were eluted with 12.5 mM TEAAc/CH3CN (50:50). 4.3.2. Synthesis and purification of ONs 9–10 dT Q-Linker (Glen Research) was used as solid support. ON were assembled in TWIST™ synthesis columns (Glen Research) with 20 -O-PivOM phosphoramidites, dT phosphoramidites and 20 -OMe phosphoramidites (Chemgenes). Coupling step was performed for 180 s and capping step for 160 s. ONs deprotection and release from the solid support were carried out with 1.0 M DBU solution in anhydrous CH3CN for 3 min and then with a 30% aqueous ammonia solution for 3 h. The deprotection solution was

evaporated in the presence of isopropylamine (13% of total volume) under reduced pressure, analyzed by IEX-HPLC, MALDI-TOF MS and finally IEX-HPLC purified. ONs were desalted using a C18 cartridge (Sep-PakÒ Classic) equilibrated with 100 mM TEAAc and they were eluted with 12.5 mM TEAAc/CH3CN (50:50). 4.3.3. Synthesis and purification of ON 15 dT Q-Linker (Glen Research) was used as solid support. ON 15 was assembled in a TWIST™ synthesis column (Glen Research) with 20 -O-PivOM phosphoramidites and dT phosphoramidites (Chemgenes). Coupling step was performed for 180 s and capping step for 160 s. ON deprotection and cleavage from the solid support were carried out with 1.0 M DBU solution in anhydrous CH3CN for 3 min and then with a BuNH2/THF (2:1) solution for 2 h. The deprotection solution was evaporated with water under reduced pressure, analyzed by IEX-HPLC, MALDI-TOF MS and finally IEX-HPLC purified. ON 15 was desalted using a C18 cartridge (Sep-PakÒ Classic) equilibrated with 100 mM TEAAc and was eluted with 12.5 mM TEAAc/CH3CN (50:50). 4.4. Thermal denaturation studies Tm experiments were performed on a CARY 300 UV Spectrophotometer (Varian Inc.) equipped with a Peltier temperature controller and thermal analysis software. The samples were prepared by mixing oligonucleotide solutions of ONs 9–15 with complementary rA12 together to give 3 lM final concentration in 10 mM sodium cacodylate, 100 mM NaCl, pH 7. A heating–cooling–heating cycle in the 5–90 °C temperature range with a gradient of 0.5 °C min1 was applied. Tm values were determined from the maxima of the first derivative plots of absorbance versus temperature. 4.5. CD Spectroscopy studies CD spectra were recorded on a Jasco J-815 spectropolarimeter. Duplexes formed with ONs 9–15 and rA12 were diluted to a concentration of 3 lM in a 10 mM sodium cacodylate buffer, 100 mM sodium chloride, pH 7 in a total volume of 1000 lL. Measurements were performed in a 1.0 cm path length quartz cuvette at 1 °C. The wavelength range was set to 340–200 nm with a scanning speed of 100 nm min1. Raw data were acquired over 3 scans. 4.6. Enzymatic stability studies with snake venom phosphodiesterase (SVPD) and calf spleen phosphodiesterase (CSPD) Aqueous solutions of ONs 9–15 were prepared at a concentration of 0.4 mM. Each ON (10.5 lL, 4.2 nmol) was mixed with 4.2 lL of ammonium citrate solution at 450 mM and 23.1 lL of water. SVPD (4.2 lL, 0.2 U/mL) was added and the mixture was incubated at 37 °C for the required time then frozen. Aqueous solutions of ONs 9–15 were prepared at a concentration of 0.8 mM. Each ON (5.25 lL, 4.2 nmol) was incubated with 4.2 lL of CSPD (5 U/mL) and 32.55 lL of 50 g/L THAP: 450 mM ammonium citrate buffer (22:1) mixture at 37 °C for the required time then frozen. 4.7. Cellular uptake A673 human Ewing sarcoma cell line was a generous gift from Dr. Elizabeth R. Lawlor (University of Michigan, USA). Cells were grown in Opti-MEM medium (Gibco, USA) and 1% penicillin–streptomycin antibiotics (Gibco).

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A. Biscans et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

To evaluate capacity of modified ONs to penetrate into the cells, 50 -Cy3-labeled ONs 16–18 were used. One day before treatment, A673 cells were seeded on 12-wells plate containing a cover slide rinsed once in ethanol bath and once in sterile water. Then medium will be discarded and ONs (at 500 nM final concentration) were added to cells in 500 lL OptiMEM medium and incubated for 3 h at 37 °C, 5% CO2 in moist atmosphere. Cells were then washed with PBS, and 4% formal solution in PBS (1 mL) was added for 20 min at room temperature. Cells were washed three times with PBS and mounted on slide with DAPI fluoromount G (SouthernBiotech) before being observed with an epifluorescence microscope (Observer; Zeiss) to confirm the presence of intracytoplasmic siRNA. Untreated cells present only a blue fluorescence due to nucleus coloration. Acknowledgment Annabelle Biscans acknowledges University of Montpellier for financial support. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2015.07.054. References and notes 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

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Please cite this article in press as: Biscans, A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.054