WO2007064291A1 - Method and compounds for rna synthesis - Google Patents

Abstract

The 2-(4-tolylsulfonyl)ethoxymethyl (TEM) as a new 2'-OH protecting group is reported for the RNA synthesis on the solid support using the phosphoramidite chemistry. The usefulness of the 2'-0-TEM group is exemplified by the synthesis of 12 different oligo-RNAs of various sizes (14-38nt long). The stepwise coupling yield varied from 97 - 99 % with an optimized coupling time of 120s. Synthesis of all four pure phosphoramidite building blocks are also described. Two new reliable parameters, δc2' - δc3s and δn2' - δH3' have been suggested for the characterization of isomeric 2'-0-TEM and 3'-0-TEM as well as other isomeric mono 273 '-protected ribonucleoside derivatives. The most striking feature of this strategy is that the crude RNA prepared using our 2'-0-TEM strategy is sufficiently pure (>90 %) for molecular biology research without any additional purification step, thereby making oligo-RNAs easily available at a relatively low cost, saving both time and lab resources.

Description

METHOD AND COMPOUNDS FOR RNA SYNTHESIS

The present invention relates to RNA synthesis on solid supports.

The purpose of the invention is to improve yield and purity of the products form such synthesis.

This is achieved by using the compounds defined in claim 1 for the synthesis.

Furthermore, in a second aspect of the invention a chemical entity defined in claim 2 is used for 2'-OH protection of the compounds in claim 1.

Finally, in a third aspect, a method for solid support synthesis of RNA is defined in claim 5.

hi the Figures

Fig. 1 is a scheme (Scheme 1) showing synthesis of the 2'-O-Protecting groups used in oligo-RNA.

Fig. 2 is a scheme (Scheme 4) showing protection of ribonucleoside at 2'-OH by the TEM group.

Fig. 3 is PAGE pictures of crude products after deprotection.

Fig. 4 shows RNA cleavage efficiency of pure RNA.

Fig. 5 is a reversed-phase HPLC profile of products obtained by digestion.

Fig. 6 shows solid-phase synthesis of 12 oligoRNAs.

2-(4-Tolylsulfonyl)ethoxymethyl (TEM)

The present invention relates to a new 2'-OH Protecting Group for solid support RNA synthesis.

Introduction

Recent development of RNA interference (RNAi),¹ and evidence that short interferring RNA (siRNA)² effectively can silence gene expression have highlighted the need for dependable methodologies for the chemical synthesis of 20-25nt long oligo-RNA sequence. The most convenient way to obtain oligo-RNA is the solid support synthesis using the phorsphoramidite building blocks.³ For this, a correct choice of appropriate 2'-OH protecting group is necessary for the assembly of desired RNA sequence, because the nature of this 2'-0-protecting group can considerably influence the coupling reaction time⁴ and the coupling yield between the 3'-6>-phosphoramidite monomer block and the 5'-OH terminal of the ribonucleotide chain on the solid support, as well as the purity of the final product, hi order to circumvent this, stoically less hindered and more reactive

3'-(2-cyanoethyl-N-ethyl-iV-methylamino phosphoramidite) in conjunction with 2'-0-tBDMS group has been used without favorable results.⁴ Therefore, the design of an ideal 2'-OH protecting group for improving RNA synthesis has been a key issue during past ~3 decades.⁵ Presently available 2'-OH protecting groups can be classified to the following types depending upon the unblocking conditions: (1) Photosensitive groups such as Nbn (as shown in scheme I)⁶ and Nbom,⁷ (2) acid-labile acetal derivatives such as Mthp,⁸ MDMP,⁹' ¹⁰ Ctmp and Fpmp groups,¹¹ (3) base-labile groups such as Npes¹², Fnebe and Nebe,¹³ (4) reductively removable DTM group with labile S-S bond¹⁴, (5) the fluoride-labile groups such as tBDMS,^15"17 SEM,¹⁸ or CEE,¹⁹ which have been found to be useful in the solid-phase oligo-RNA synthesis.

An ideal 2'-OH protecting group should be²⁰ (1) easy to introduce, (2) achiral, (3) unable to migrate to vicinal 3'-OH, (4) completely stable under the conditions required for the assembly of the fully desired RNA sequence, as well as for its subsequent unblocking and release from the solid support. (5) It must be removable under conditions under which RNA is completely stable, and the 3' — >5' phosphodiester do not isomerize to 2' — »5' linkage. Though the 2'-protecting groups shown in Scheme 1 (Figure 1) are widely used, none of them however completely fulfills the above criteria. For example, tBDMS, the most widely used 2'-0-protecting group, is unstable²¹ in ammonia solution giving chain cleavage,²² migrates²³ to 3'-OH and gives relatively poor coupling yield with the required coupling time of ca 10 min.²⁴ The recent appearance of 2'-(9-ACE²⁵ (Scheme 1) is a considerable improvement in the synthesis of oligo-RNA compared to the use of 2'-<9-tBDMS group. But this strategy is not compatible with a conventional automated synthesizer, and also makes it impossible to monitor the coupling reactions in a standard solid-phase synthesizer with UV detector.²⁵

Scheme 1: The 2^f-0-Protecting groups used in oligo-RNA synthesis.

Hnebe: R=F Nebe :R=H

ACE

Recently, some new 2'-OH protecting group with formaldehyde acetal linker such as [(triisopropylsily)oxy]methyl (TOM)²⁶ and 2-cyanoethoxymethyl (CEM)²⁷ have been developed for the solid phase RNA synthesis. Using the 2'-0-TOM or 2'-0-CEM group, average coupling yield of 99% can be obtained in coupling time of 60-15Os. Presumably, the formaldehyde acetal linker poses relatively less steric hindrance toward the vicinal 3'-phosphoraniidite, thereby giving much higher coupling yield in a relatively short coupling time. As for the 2'-(9-CEM, it can be unblocked through a β -elimination process with fluoride ion as the base. As expected, it is also cleaved to some extent during the ammonia treatment, which increases the possibility of chain cleavage, and may limit its further use for larger oligo-RNA synthesis. Clearly, reducing the acidity of the α-proton to -CN group in 2'-0-CBM [2'-0-CH₂-O-CH₂-CH₂-CN]²⁷ will increase its stability in the ammonia deprotection step.

We report here our efforts to modulate the base-lability of α-proton by employing an appropriate substituent "X" in the aromatic moiety of 2-arylsulfonylethoxymethyl group as a potential 2'-OH protecting group (Scheme 2). Given the fact that the pK_a of the proton of RCH₂SO₂Ph (pK_Α = 27.9 in DMSO, R = PhO) is very similar to that of RCH₂CN (pK_a = 28.1 in DMSO, R = PhO),²⁸ introducing a pαrα-methyl substiruent (X = p-Ms in Scheme 2) to the phenyl, as in/?αrα-tolylsulfonylethoxymethyl (TEM) group, is expected to reduce the acidity of α-proton²⁹ and this electronic tuning may make the TEM, compared to CEM, a more stable 2'-OH protection group. This paper demonstrates solid support chemical synthesis of RNA using the TEM as a new 2'-OH protecting group. We also report the synthesis of all four native phosphoramidite building blocks, and their use in the automated RNA synthesis including the deblocking conditions to prepare pure oligo-RNA.

Scheme 2: Electronic nature of "X" dictates the fragmentation reactivity

Results and discussions

(A) Synthesis of the phosphoramidite building blocks. The reagent, TEM-Cl (5), was synthesized using a strategy shown in Scheme 3: 2-(4-tolylthio)ethanol (2) was synthesized from 4-methylbenzenethiol (I).³⁰ The thioether 2 was first oxidized to sulfone 3 in 97% yield by 35% hydrogen peroxide in aqueous acetic acid (H₂0/Ac0H, 1:1, reflux 20 min). Then sulfone 3 was treated with a mixture of DMSO, acetic acid and acetic anhydride at room temperature for two days, followed by purification by silica gel column chromatography to give O, ^-acetal 4 (74%). Finally, the reagent 5 was generated immediately before use by treatment of 4 with SO₂Cl₂ (1 eqv.) in CH₂Cl₂. This conversion of 4 — > 5 was a quantitative reaction, and no further purification step was necessary.

Scheme 3

Reagents and Conditions: (i) 2-Chloroethanol, 10 M NaOH aq. solution, ethanol, reflux 2 h; (ii) 35% hydrogen peroxide, HOAc, H₂O, reflux for 20 min; (iii) DMSO, HOAc, Ac₂O, r.t, 48 h; (iv) SO₂Cl₂, CH₂Cl₂, r.t., 1 h.

Acetyl (Ac), dimethylaminomethylene (Dmf) and phenoxyacetyl (Pac) were used to protect N⁴ of cytosine, N² of guanosine and N⁶ of adenosine, respectively. These exocyclic amino protected blocks and uridine were converted to their respective 5'-0-DMTr derivatives according to the published procedures.^31"33 The appropriately protected ribonucleoside was protected at 2'-OH by the TEM group through the procedure optimized by Pitsch and co works²⁶ to give a mixture of two 2'- and 3 '-isomeric ribonucleosides 7 and 8 (Scheme 4; Figure 2) by reacting 6a/6b/6c or 6d with Bu₂SnCl₂/ 'Pr₂NEt in 1, 2-dichloroethane at room temperature to form activated cyclic T-O, 3'-<9-dibutylstannylidene intermediate, followed by the treatment with 1.3 equivalent of TEM-Cl (5) at 80 ⁰C for 1 h. The two isomeric T-O and

3'-0-TEM can be isolated by silica gel column chromatography. The first isomer eluting from the column was the predominant product, 2'-0-TEM ribonucleosides 7a/7b/7c/7d in 26-38% yields. The second eluting isomer 8a/8b/8c/8d was isolated in 20-27% yields. The two isomers have been identified by NMR (see Section B). Compound 7a/7b/7c or 7d was further converted to the corresponding 5'-0-DMTr-2'-0-TEM-riobnucleoside 3'-(2-cyanoethyl ΛζiV-diisopropylphosphoramidite) 9a (79%) / 9b (68%) / 9c (60%) or 9d (54%) by using general procedure³⁴ (see experimental section).

Scheme 4 D

(20%) _(23%j

(27%)

(«) (iii)

Reagents and Conditions: (i) a, Bu₂SnCl₂, 'Pr₂NEt, ClCH₂CH₂Cl, r.t, 1 h; b, compound 5, 80 ⁰C, 1 h. (ii) 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, 'Pr₂NEt, CH₂Cl₂, r.t., 2 h. (iii) pimelic acid, EDAC, DMAP, pyridine, r.t., 4 h.

(B) Identification of isomeric 2 '-O-TEM and 3 '-0-TEM ribonucleosides by NMR

When ribonucleosides 6a/6b/6c or 6d was alkylated with TEM-Cl 5, two monoalkylated 2'/3'-0-TEM isomers were obtained. The isomer with lower Rf was converted to corresponding pimelates lOa/lOb/lOc or 1Od which showed the down-field shift of H-2' in ¹H NMR spectrum (data listed in Table 1), thereby showing that the lower Rf compound is 3'-0-TEM derivative.

The ¹H and ¹³C NMR data of isomerically pure T- and 3'-0-TEM derivatives 7a - d/8a - d, T- and 3'-0-CEM derivatives 11a - c/12a - c, as well as T- and 3'-O-tBDMS derivatives of ribonucleosides 13a - 14c are listed in Table 1 for comparison. It can be seen from Table 1, regardless of alkylation or silylation at the 2'-OH or 3'-OH, the δnv do not follow any systematic upfield or downfield shift, and in fact the difference of δnv between the two isomers is negligible. On the other hand, ³Jy₁^ of all alkylated compounds 7a - 12c follow Reese's rule,³⁵ in that the ³

Jy^_' of 2'-isomers are smaller than those of the 3'-isomers, while it is not the case for the T, 3'-isomeric silylated compounds 13a - 14c. A comparison of δπr and δπy however showed that for all the mono 2V3'-O-alkylated compounds 7a - 12c, alkylation causes an upfield shift of the proton attached to the site of 2' or 3'-O-alkylation. The value of 5_H2' - δm¹ of the 2'-isomer is always smaller than that of the 3 '-isomer. On the contrary, an absolutely reversed rule can be drawn from the isomeric 273'-O-silylated compounds 13a - 14c. ¹³C NMR signals were also used by Ogilvie et al. to identify 2'-O- and 3'-O-silylated isomers.³⁶ They found silylation at a sugar hydroxyl leads to a downfield shift of the sugar carbon to which it is attached. Just as shown in Table 1, we can find that this rule also applies to all the isomeric 2'/3'-O-alkylated compounds 7a-12c. Alternatively, the value of δcr - δc_3' can be used as a more convenient identification parameter. The value of δcr - δcv of 2'-isomer is always larger than that of the corresponding 3 '-isomer.

Table 1. ¹H and ¹³C NMR chemical shifts of isomeric 2V3'-protected ribonucleoside derivatives.

7a 5.90(1.84) 4.21 4.48 -0.27 80.18 6 11.46

8a 5.90(3.70) 4.34 4.27 0.07 74.53 7 -1.37

7b 5.88* 4.18 4.44 -0.26 80.11 6 12.3

8b 5.93(2.10) 4.33 4.18 0.15 75.37 7 -0.24

(/>

C 7c 6.21(3.18) 4.86 4.56 0.30 80.46 7 10.14

CD C/> 8c 6.04(5.52) 4.91 4.44 0.47 74.35 7 , -2.90

7d 6.05(4.81) 4.66 4.47 0.19 80.03 7 9.62

8d 5.91(5.33) 4.80 4.36 0.44 74.04 7 -2.77 m lla 5.98(2.15) 4.29 4.50 -0.21 79.99 6 11.12

</> 12a 6.03(3.52) 4.47 4.38 0.09 74.18 7 -0.89 m 4.47 -0.17 79.73 6 11.8 m lib 5.95⁶ 4.30

12b 5.93(2.34) 4.39 4.28 0.11 75.16 7 0.06 lie⁸ 6.25(3.90) 4.92 4.58 0.34 80.14 7 9.76

C r 12c 6.06(5.71) 4.93 4.50 0.43 74.36 7 -2.54 m-

13a 5.95(2.65) 4.34 4.33 0.01 76.32 7 1.86 σ> 14a 5.96(4.17) 4.16 4.39 -0.23 71.30 7 -3.99

13b 6.11(5.34) 5.03 4.37 0.66 75.91 7 4.15

14b 6.07(4.93) 4.48 4.60 0.28 74.79 7 2.47

13c 5.74(7.42) 5.26 4.34 0.92 74.32 7 3.00

14c 5.71(5.97) 4.85 4.46 0.39 73.60 7 1.95

10« - 5.37 4.43 - 73.97 7 -

10b - 5.39 4.40 - 74.31 7 -

10c - 5.83 4.71 - 74.37 7 -

1Od 5.92 4.51 - 74.13 7 - ^a compounds lla-14c were synthesized according to literature procedure²⁷'³⁷ and their structures are shown in Scheme 5. ^b broad singlet.

Scheme 5. Structures of isomeric 273' mono protected ribonucleosides.

11c: B = _APac 13c: B = : G'^'-^BU

(C) Preparation of the solid support. Initially, we anchored the ribonucleoside to LCAA-CPG support through the succinate linker. Just as described by Pitsch,²⁶ the coupling yields for the first seven coupling steps were unsatisfactory. Pitsch and his coworkers have overcome this problem by employing a longer linker. Here we also adopted this strategy but a more convenient synthetic route was developed: Pimelic acid was reacted directly with the protected ribonucleosides 8a - 8d in presence 2 equivalents of N-(3-dimethylaminopropyl)-iV '-ethylcarbodiimide hydrochloride (EDAC) and DMAP in dry pyridine. This is a quick and clean reaction, completed in 4 h, worked up and purified by silica gel column chromatography to give the corresponding pimelates 10a - 1Od in 65-75% yield. The pimelates were subsequently immobilized to LCAA-CPG support by a modification of the general procedure of Pon and Yu?⁸ which involved shaking the mixture of pimelates, LCAA-CPG, benzotriazol-1- yloxytris (dimethylamino) phosphonium hexfluorophosphate (BOP), N-hydroxybenzotriazole (HOBt) and diisopropylethylamine (DIPEA) in acetonitrile for 2 h to give good loadings, 20-25 μmol/g. Prolonging the reaction time overnight gave however improved loading, 35 μmol/g.

(D) Automated RNA synthesis. We first synthesized U₁₂ under different conditions to optimize the synthesis cycle. This oligo was synthesized on an Applied Biosystems 392 DNA/RNA synthesizer with common DNA synthesis reagents. 2'-O-succinate-8a CPG was used as the solid support and 1.0 μmol RNA synthesis cycle was applied but with a modified coupling time. Average stepwise yield (ASWY), obtained by detritylation assay, was used to evaluate the synthesis efficiency. We first tried synthesis with 4, 5-dicarbonitrile-lH-imidazole (DCI) (p£_a = 5.2),³⁹ 5-(ethylthio)-lH-tetrazole (ETT) (pK_a = 4.28 ) and 5-(benzylthio)-lH-tetrazole (BTT) (ρK_a = 4.08)⁴⁰ as the activator with coupling time of 120 s. The ETT however gave the best result. Since an activator acts as an acid to protonate, and thereby activate the phosphoramidites, the more acidic the activator is, the faster the coupling reaction should be.⁴¹ On the other hand, as the activator becomes increasingly acidic, it can also lead to some concomitant loss of 5'-0-DMTr protecting group during coupling, which explains why the ETT, despite the fact it is less acidic than BTT, but gives a better coupling yield in the RNA synthesis. Next, with ETT as the activator, we tried different coupling time, 60 s, 90 s, 120 s, and found that the ASWY improved with longer coupling time. However, a coupling time of 150 s or 200 s did not improve the yields. Finally we settled for 120 s as the coupling time for the following synthesis. During the last experiments, we found the first 7 couplings always gave poor coupling yield, and after that the ASWY increased steadily. A three-methylene chain longer pimelated CPG instead of the conventional succinate (Scheme 4) improved the coupling yield however only slightly.

On the basis of the above experiments, 12 different oligo-RNAs (sequence shown in Table 2) were synthesized to test the efficacy of the TEM as the 2'-OH protecting group. The poly-U synthesis always gave perfect coupling yield, whereas the synthesis of the mixed RNA sequences, ON 3 - 12, particularly the one involving G-phosphoramidite, generally gave poorer yields. The average stepwise yield (ASWY) for each synthesis is listed in Table 2.

Table 2. Average stepwise and overall yields and mass of synthesized RNAs using the 2'-0-TEM protecting group.

OUgo [MH]⁺ [MH]⁺ Yield Crude product''

RNA sequence

RNAs (calcd) (found) (Overall yield)

ON F 5'-HO-U₂₀ -3'-OH 6062.42 6064.33 99.1⁶ 102 OD (53%) ^b 11683.37 11682.17

ON 2* 5'-HO-U₃₈ -3'-OH 98.4^δ 144 OD(40%)*

GACGUAAACGGCCACA

ON 3* 6719.16 6720.07 97.8* 112 OD(54%)⁴

AGUUC) ACUUGUGGCCGUUUAC ON 4^έ 6643.00 6643.92 98.0* 124 OD(64%)*

GUCGC)

5'-CGCGC ^u C

ON 5^C 3'-GCGCG G 5779.53 5777.13 97.5^C 21 OD(65%)^C

U _G A

ON 6^C 5'-CGCGC ^UU 3'-GCGCG₀C 4453.74 4451.56 97.0^c 17 OD(69 ι%θ/Λ)'^c

5'-CGCGC⁰ A

ON 7^e 3'-GCGCG _A A 4523.84 4521.69 97.0^c 20 OD(78% o/Λ)'^c

5'-CGCGC ^{A c}

ON 8^C 3'-GCGCG 5786.57 5784.14 97.6^C 18 OD(53%)^C U ^G

ON 9^C 5'-CGCGC ^{A U}A 3'-GCGCG _{A χj} G 5481.39 5479.18 97.2^C 21 OD(64%)^C

DN lO* 5'-CGCGC ^A° ^u 5152.18 5150.11 97.4^C 19 OD(63%)^{C f} A u⁽ 5'-CGCGC⁰U N lT t 3'^"-ΣG£CSG£CXG₁₁ nG 4493.76 4491.68 97.3^C 17 OD(68%)⁰

N 12⁰ OAAGAAAAAAUGAAG) 4918.137 4915.51 97.0^c 24 OD(68%)^C

^a average step-wise coupling yield; * oligos were synthesized in 1 μmol scale; ° oligos were synthesized in 0.2 μmol scale. 'Overall crude product yields were measured at 260 nm UV absorption.

(E) Post-synthesis deprotection treatment. Prior to deprotection of the oligo-RNAs, we tested the stability of 2'-(9-TEM in ammonia. Compound 7a was treated with 3% Cl₂CHCOOH in CH₂Cl₂ to give compound 15 (Scheme 6). Compound 15 was first treated with 33% MeNH₂ in EtOH at room temperature for 24 h. The reaction was monitored by TLC and found about 10% loss of 2'-0-TEM has taken place (the TLC is shown in the supporting imformation). This suggests that highly alkaline condition involving MeNH₂/EtOH is not a safe system for TEM. Then anhydrous methanolic ammonia (25% NH₃/Me0H)²² was tried. The reaction mixture was kept at 55 °C for 21 h and very small amount of TEM cleavage was observed. If the reaction was allowed to proceed at room temperature, the 2'-0-TEM cleavage was nearly negligible. So NH₃/Me0H was chosen for exocyclic amino deprotection. In view of the fact that about 5% cleavage of 2'-0-CEM is found to take place under the same solution, it is concluded that 2'-0-TEM is more stable in ammonia solution, just as we expected in the outset. Scheme 6.

is stable

7a 15

In order to find an optimal condition to remove the 2'-O-TEM, compound 15 was treated with neat Et₃N.-3HF and IM TBAF/THF separately. It was found only IM TBAF/THF is an efficient reagent for the deprotection of the 2'-O-TEM group in that the deprotection was found to be complete in 5 min at room temperature.

Thus the oligo anchored solid support were first treated with 25% NH₃/Me0H at room temperature for 20 h, then at 40 °C for 4 h to ensure the removal of the exocyclic amino protecting groups completely. After solvent removal and drying by coevaporation with dry THF, the samples were treated with IM TBAF/THF for 20 h at room temperature to make sure all 2'-0-TEM groups are deprotected. After desalting through a NAP-IO column and Sep-Pak column, the crude products were applied to PAGE or HPLC analysis. The PAGE pictures (see Figure 3) together with HPLC profiles show that the crude products of U₂₀ and U₃₈ are more than 80% pure.

However, just as in the CEM strategy, deprotection of the mixed oligo-RNA sequence using the same deprotection procedure did not produce any satisfactory results. This was because of the formation of the adducts owing to the side-reaction of the purine bases as nucleophiles (at N7, N3, and Nl) with the α, β-unsaturated sulfone 16, generated by fluoride ion treatment, Scheme 7. The formation of these adducts have been evidenced by denatured PAGE as well as by MALDI-TOF MS analysis. In the CEM strategy²⁷, 10% n-propylamine and 1% bis (2-mercaptoethyl) ether was used as scavenger for the active α, β-unsaturated compound (acrylonitrile), and thereby adduct formation was suppressed efficiently. However, it should be kept in mind that n-propylamine also acts as a moderate base and can promote chain cleavage. So, with the hope to develop an improved scavenger, we tested several different deprotecting conditions for 2'-0-TEM with ON 3 and ON 4 as the substrates. The results are listed in Table 3. Comparison of these data suggests the following: (1) Deprotecting 2'-0-TEM with TBAF/THF is a fast process, 4 h and 24 h of reaction period nearly give the same yields. (2) Amine promoted chain cleavage is significant. When more basic amine such as DBU or piperidine was used, considerably more cleavage was observed. (3) n-Propylamine itself is an efficient scavenger, m the absence of bis(2-mercaptoethyl)ether, the deprotecting reaction is cleaner. (4) CH₃NO₂ was reported as a scavenger for acrylonitrile in the presence of amine such as NH₃ZMeCN, MeNH₂ZH₂O-EtOH-MeCN,⁴² but it is not the case in the presence of TBAF in THF. On the contrary, when CH₃NO₂ was used in conjunction with n-propylamine, it can inhibit the activity of n-propylamine. (5) The 2'-0-TEM group is found to be stable in ammonia. Treating the oligo with NH₃ZMeOH at room temperature for 20 h, then 40 ⁰C for 4 h

or 55 ⁰C for 3 h give similar results, which again proves the enhanced stability of 2'-0-TEM in

NH₃ZMeOH. (6) Up till now, the IM TBAF/THF with 10% n-propylamine and 1% bis (2-mercaptoethyl) ether remains to be the preferred reagent for deprotecting the 2'-O-TEM group fi'om the oligo-RNA. Recent unpublished data from this lab however suggests that the IM TBAF/THF with 10% morpholine at RT seems to be a more improved deprotection agent than IM TBAF/THF with 10% n-propylamine and 1% bis (2-mercaptoethyl) ether, which will be reported in a full paper.

ON 1 ON 2 ON 3 ON4 ON 12

mmø

*i^p8røp^

Jt-b

ON 5 ON6 ON7 ON8 ON9 ONlO ONIl

Figure 3.20% Denatured PAGE pictures of the crude products directly after deprotection. a: Xylene cyanol blue; b: Bromophenol blue. Scheme 7. The mechanism of the adduct formation.

ETSE_{h H} TE

14

Table 3. Conditions used for deblocking ON 3 and 4.

Entr Step I: NH₃ Step II: Isolated Yield (%) HPLC profile* y treatment 2'-Deprotection r Condition l^c Condition 4^f 53.7 Fig S3.1

T Condition 1 Condition 5^s 48.6 Fig S3.2

Condition 1 Condition 5 57.4 Fig S3.11

4^a Condition 1 Condition 6^h 50.4 Fig S3.3

5^b Condition 1 Condition f 43.7 Fig S3.4

6^a Condition 1 Condition 8^J 27.9 Fig S3.5

T Condition 1 Condition 9^k Fig S3.6

8^α Condition 1 Condition lθ' * Fig S3.7

</> 9^a Condition 1 Condition 11™ Fig S3.8

C CD 10^δ Condition 1 Condition 12" 49.1 Fig S3.9

(/>

11" Condition 2^d Condition 6 34.9 Fig S3.10

12* Condition 3^e Condition 6 52.1 Fig S3.12

71 m σ>

^a ON 4; ^b ON 3; ^c Condition 1: 28% NH₃MeOH, r.t, 20 h, 40 ⁰C, 4 h; ^d Condition 2: 28%

NH₃MeOH (with 1% CH₃NO₂), r.t, 20 h, 40 ⁰C, 4 h; ^e Condition 3: 28% NH₃MeOH, 55°C, 3 h; ^■'Condition 4: IM TBAF/THF [with 10% n-Propylamine, 1% bis (2-mecaptoethyl) ether], 24 h; ^s Condition 5: IM TBAF/THF [with 10% n-Propylamine, 1% bis (2-mecaptoethyl) ether] 4 h; ^h Condition 6: IM TBAF/THF (with 10% n-propylamine, 1% CH₃NO₂), 4 h; * Condition 7: IM TBAP/THF with 10% DBU, 1% CH₃NO₂), 4 h; ^j Condition 8: IM TBAF/THF (with 10% piperidine, 1% CH₃NO₂), 4 h; * Condition 9: IM TBAF/THF (with 10% CH₃NO₂), 4 h; ^l Condition 10: IM TBAF/THF (with 1% n-propylamine, 10% CH₃NO₂), 4 h; ^m Condition 11: IM TBAF/THF (with 10% n-propylamine, 10% CH₃NO₂), 4 h; " Condition 12: IM TBAF/THF (with 10% n-propylamine), 4 h; * no pure product obtained; ^φ HPLC conditions: KP-column, 0-40 min, buffer C→C/D 8/2. All of the HPLC profiles can be found in supporting information.

In this work, we have finally deprotected all of our oligo-Ranks, ON 3-12, in the following manner: (i) treatment with methanolic ammonia (RT, 20 h followed by 40⁰C for 4h)) as described above, and (ii) treatment with IM TBAF/THF containing 10% n-propylamine and 1% bis(2-mercaptoethyl) ether for 20 h at room temperature to give full length oligo-RNAs in good purity. The PAGE pictures of the crude products (>90% pure, which are acceptable for enzymological work, see below) are shown in Figure 1. These oligo-RNAs were subsequently examined by MALDI-TOF MS, which confirmed the chemical integrity of all the synthesized RNAs (see Table 2 and Figures S1.3 to Sl.13.).

(F) Enzymatic digestion of the crude RNA directly after deprotection without involving any purification step

The RNA synthesis with the TEM strategy described above can indeed give crude product with high purity. To address whether this crude product is pure enough for biology research, a

15nt long RNA (ON 12 in Table 2) was synthesized and, after deprotecting and desalting (see

Fig 1 for purity of the crude product), applied directly to RNase H digestion study. It may be noted that the synthesized crude RNA (compare its relative purity (-90 %) with that of the purified RNA) has nearly the same cleavage efficiency as the pure RNA (compare the RNase H promoted relative degradation rates in Figure 4). This suggests that the crude RNA prepared by our TEM strategy is satisfactory for most biological experiments. To our knowledge, it is the first approach that can provide crude RNA for biological studies directly without laborious, time-consuming purification step.

Figure 4 shows RNA cleavage efficiency of pure RNA and crude RNA (ON 12 in Fig 1) with RNase H (for the purity of pure RNA see the PAGE picture in Fig S5 in SI). Conditions of cleavage reactions: pure RNA (0.1 μM) or crude RNA (0.1 μM) and complementary DNA (1 μM) in buffer containing 20 mM Tris-HCl

(pH 8.0), 20 mM KCl, 10 mM MgCl₂ and 0.1 mM DTT at 21 ⁰C; 0.06 U of RNase H in a total reaction volume of 30 μL

The crude ON 12 was also subjected to digest by phosphodiesterase I (from Crotalus adamanteus Venom) and Shrimp Alkaline Phosphatase. The products were analyzed by RP-HPLC and the profile is shown in Figure 5. The crude products are completely digested to give pure nucleosides, which strong support that the oligo-RNA is in biologically active form and no modified base is present.

Figure 5 shows reversed-phase HPLC profile of the products obtained by digestion of crude ON 12 (R_t = 14.12 min) in Fig 1 with a mixture of phosphodiesterase I and Alkaline Phosphatase. The peaks correspond to uridine (3.83 min), guanosine (4.17 min) and adenosine (5.79 min). HPLC condition: C₁₈ RP column, 100*4.6mm, 1 ml/min, r.t, 0-10 min, buffer C→C/D 9/1, 10-35 min, buffer C/D 9/l→ C/D 2/8.

Conclusions

(1) A new set of NMR trends, involving δm_' - S_H3' and δc₂- - Sc₃-, was identified to characterize monomelic T-O- and 3'~O-alkylated or silylated ribonucleosides. Compared to earlier reported parameters such as δπr, J_Hi_', m; &cy and δc₂-, the proposed parameters are more regular and reliable.

(2) TEM has been developed as a new 2'-OH protecting group for solid support oligo-RNA synthesis. With this methodology, RNA synthesis can be carried out on standard solid support synthesizer with high average coupling yield and coupling time of only 120 second.

(3) The advantage of our 2'-0-TEM based strategy for RNA synthesis is its improved stability of the 2'-0-TEM group upon ammonia treatment, simpler post-synthesis deprotection procedure and high purity of the crude product than that of RNA prepared by 2'-O-cyanoethoxymethyl (2'-0-CEM) group.

(4) Furthermore, the crude RNA obtained by of our 2'-O-TEM based strategy is of high purity, which, after desalting, can be directly used in the biological research without further purification, which is an important advantage over other strategies based on either 2'-0-TBDMS,^15'17 2'-0-TOM,²⁶ 2'-0-CEM,²⁷ 2'-O-ACE,²⁵ or 2'-O-Fpmp.²⁰

Experimental section

Chromatographic separations were performed on Merck G60 silica gel. Thin layer chromatography (TLC) was performed on Merck pre-coated silica gel 60 F₂₅₄ glass-backed plates. ¹H NMR spectra were recorded at 270.1 MHz and 500 MHz respectively, using TMS (0.0 ppm) as internal standards. ¹³C NMR spectra were recorded at 67.9 MHz, 125.7 MHz and 150.9 MHz respectively, using the central peak of CDCl₃ (76.9 ppm) as an internal standard. ³¹P NMR spectra were recorded at 109.4 MHz using 85% phosphoric acid as external standard. Chemical shifts are reported in ppm (3 scale). MALDI-TOF mass spectra were recorded in positive ion mode for oligonucleotides and for other compounds as indicated. The mass spectrometer was externally calibrated with a peptide mixture using alpha-cyano-4-hydroxycinnamic acids as matrix. Anion exchange (AE) HPLC: Luna 5μ, NH₂, 10OA, 150*4.6 mm, 5 micron. Flow 1 ml/min at room tempreture, UV detector with detecting wavelength of 260 nm. Buffer A: 20 mM LiClO₄, 20 mM NaOAc in H₂O:CH₃CN (9: 1), pH 6.5 with AcOH. Buffer B: 600 mM LiClO₄, 20 mM NaOAc in H₂O:CH₃CN (9:1), pH 6.5 with AcOH. Reversed-phase (RP) HPLC: Kromasil 100, C18, 5μ, 100*4.6 mm. Fow 1 ml/min at room tempreture, UV detector with detecting wavelength of 260nm. Buffer C: 0.1M TEAA in H₂O:CH₃CN (95:5). Buffer D: 0.1M TEAA in H₂O:CH₃CN (50:50). Polyacrylamide gel electrophoresis (PAGE): 20% acrylamide (acrylamide/bisacrylamide 29:1), 7 M urea, TBE buffer.

2-(4-tolylthio)ethanol (2). To a solution of 4-methylbenzenethiol (1, 50 g, 0.394 mol) and 2-chloroethanol (47.6 ml, 0.71 mol) in ethanol (400 ml) was added dropwise aqueous 10 M NaOH (39.4 ml, 0.394 mol) within 1 h. The reaction mixture was refluxed for 2 h, cooled and concentrated on vacuum. The residue was diluted with AcOEt, washed with H₂O and dried over MgSO₄. After filtration, the filtrate evaporated on vacuum to give 76.6 g (97 %) of 2 as a colorless oil. ¹H NMR (270 MHz, CDCl₃): δ 2.32 (s, 3H), 3.06 (t, J= 5.9 Hz, 2H), 3.70 (t, J= 5.9 Hz, 2H), 7.11 (d, J= 9.1 Hz, 2H), 7.30 (d, J= 8.1 Hz, 2H). 2-(4-Tolysulfonyl)ethanol (3). To a solution of Compound 2 (20 g, 0.119 mol) in AcOH (50 ml) and H₂O (50 ml) on ice bath was added hydrogen peroxide (30%, 36.5 ml) dropwise over half an hour. Then the mixture was refluxed for 20 min and cooled. NaHCO₃ was added to the reaction mixture to neutralize this solution, followed by AcOEt. The organic layer was separated, washed with brine and water, concentrated on vacuum to give 22.9 g (96.3%) of 3 as a white solid. ¹H NMR (270 MHz, CDCl₃): δ 2.46 (s, 3H), 3.33 (t, J= 5.4 Hz, 2H), 3.99 (t, J= 5.4 Hz, 2H), 7.39 (d, J= 8.0 Hz, 2H ), 7.81 (d, J= 8.3 Hz, 2H). ¹³C NMR (67.9 MHz, CDCl₃): δ 21.7, 56.4, 58.3, 128.0, 130.1, 136.0, 145.2.

4-Tolysulfonylethyl methylthiomethyl ether (4). A solution of 4-tolysulfonyl ethanol (2 g, 10 mmol) in dimethyl sulfoxide (28 ml, 40 mmol) was treated with acetic anhydride (11.4 ml, 20 mmol) and acetic acid (19 ml, 20 mmol), the mixture was stirred at room temperature for 48 h and then added dropwise to a aqueous solution OfNaHCO₃. After a further hour of stirring, the reaction mixture was extracted with ethylacetate. The organic layer was separated, washed with saturated aqueous NaCl solution and dried over MgSO₄. The solvent was removed under reduced vacuum and the residual oil was applied to short column chromatography (ethyl acetate/cyclohexane, 1/5 to 2/5) to give the colorless oil 1.935 g, 74 %. ¹H NMR (270 MHz, CDCl₃): δ 2.08 (s, 3H), 2.46 (s, 3H), 3.40 (t, J= 6.3 Hz, 2H), 3.88 (t, J= 6.3 Hz, 2H), 4.54 (s, 3H), 7.36 (d, J= 7.9 Hz, 2H), 7.81 (d, J= 8.3 Hz, 2H). ¹³C NMR (67.9 MHz, CDCl₃): δ 14.1, 21.7, 56.2, 61.4, 75.6, 128.1, 129.9, 136.8, 144.9.

4-Tolysulfonylethoxymethyl chloride (5). 1.78 g (6.8 mmol) of 4-Tolysulfonylethyl methylthiomethyl ether (4) was dissolved in CH₂Cl₂ (20 ml). SO₂Cl₂ (0.95 g, 6.8 mmol) was added dropwise while the solution was kept on ice, and the reaction mixture was allowed to proceed for further two hours at room temperature. After evaporation of the solvent under reduced pressure, the product was obtained as colorless oil (1.69 g, 99%). It was used for next step synthesis without further purification. ¹H NMR (270 MHz, CDCl₃): δ 2.46 (s, 3H), 3.44 (t, J= 6.2 Hz, 2H ), 4.03 (t, J= 6.2 Hz, 2H), 5.35 (s, 3H), 7.36 (d, J= 8.0 Hz, 2H), 7.80 (d, J= 7.5 Hz, 2H). ¹³C NMR (67.9 MHz, CDCl₃): δ 21.7, 55.7, 63.7, 82.0, 128.1, 129.9, 136.5, 145.0.

5'-O-(Dimethoxytrityl)-2'-O-[2-(4-Tolylsulfonyl)ethoxy methyl] uridine (7a) and 5'-O-(Dimethoxytrityl)-3'-O-[2-(4-Tolylsulfonyl)ethoxy methyl] uridine (8a). To a solution of 5'-0-DMTr uridine (3.83 g, 7 mmol) in CH₂Cl₂ (30 ml) was added diisopropylethylamine (4.2 ml, 24.7 mmol) and dibutyltin dichloride (2.55 g, 8.4 mmol) and the reaction was allowed to proceed at room temperature for 1 hour. Then the mixture was heated to 80 ⁰C , 4-tolysulfonylethoxymethyl chloride (5) (2.5 g, 9.1 mmol) added dropwise, and the mixture left stirring at 80⁰C for 1 hour. The mixture was allowed to cool down and saturated NaHCO₃ was added. After shaking fiercely, the resulting turbid solution was filtered through Celit bar. CH₂Cl₂ was added to the filtrate and the organic layer was separated, dried over MgSO₄. The residue, after evaporation of the solvent, was applied to short column chromatography (CH₂Cl₂ with 1% Et₃N, ethyl acetate from 0 to 40%). The first eluted isomer was 7a (1.83 g, 34.4%). ¹H NMR (270 MHz, CDCl₃ + DABCO): δ 2.43 (s, 3H), 3.40 (t, J = 5.8 Hz, 2H), 3.54 (t, J = 2.7 Hz, 2H), 3.79 (s, 6H), 3.94 (m, IH), 4.05-4.16 (m, 2H), 4.21 (dd, J= 1.8, 2.0 Hz, IH, H-20, 4.48 (dd, J= 5.4, 5.5 Hz, IH, H-3'), 4.80 (d, J= 6.8, IH), 4.98 (d, J= 6.8, IH), 5.28 (d, J= 6.3, IH)₅ 5.90 (d, J = 1.8, IH, H-I'), 6.84 (d, J= 8.9, 4H ), 7.23-7.40 (m, HH), 7.78 (d, J= 8.3, 2H), 7.96 (d, J= 8.2, IH). ¹³C NMR (67.9 MHz, CDCl₃ + DABCO): δ 21.6,

55.3, 56.2, 61.4, 61.8, 68.7 (C-3')₃ 80.2 (C-2'), 83.2, 87.1, 88.1, 95.1, 102.2, 113.3, 127.2, 127.9, 128.0, 128.2, 130.0, 130.1, 130.2, 135.1, 135.3, 136.8, 140.0, 144.4, 145.0, 150.1, 158.8, 163.0.

MALDI-TOF MS: [M+Na]⁺ 781.20, calcd 781.25. The second eluted isomer was 8a (1.07 g, 20.2%). ¹H NMR (270 MHz, CDCl₃ + DABCO): δ 2.41 (s, 3H), 3.30-3.39(m, 3H), 3.57 (dd, J = 2.5, 2.3 Hz, IH), 3.79 (s, 6H), 3.87 (t, J= 5.2 Hz, IH), 3.99 (t, J= 6.1 Hz, IH), 4.22 (t, J = 5.3 Hz, IH), 4.27 (t, J= 5.0 Hz, IH, H-3'), 4.34 (d, J= 3.9, IH, H-2'), 4.72 (s, 2H), 5.35 (d, J= 8.1, IH), 5.90 (d, J= 3.7, IH, H-I'), 6.84 (d, J= 8.2, 4H ), 7.23-7.37 (m, HH), 7.75 (d, J= 8.3, 2H), 7.83 (d, J= 8.1, IH). ¹³C NMR (67.9 MHz, CDCl₃ + DABCO): δ 21.6, 55.3, 56.1, 61.9, 62.0, 74.5 (C-2'), 75.9 (C-3'), 81.7, 87.1, 89.8, 95.6, 102.4, 113.3, 127.2, 127.9, 128.0, 128.2, 129.9, 130.1, 130.2, 135.1, 135.2, 136.7, 140.0, 144.2, 145.0, 150.6, 158.8, 163.0. MALDI-TOF MS: [MfNa]⁺ 781.21, calcd 781.25. l^-Acetyl-5'-O-(Dimethoxytrityl)-2'-O-[2-(4-Tolylsulfonyl)ethoxy methyl] cytidine (7b) and l^-Acetyl-5'-O-(Dimethoxytrityl)-3'-O-[2-(4-Tolylsulfonyl)ethoxy methyl] I cytidine (8b). iV*-acetyl-5'-0-DMTr cytidine (3.95 g, 6.7 mmol) was treated as described for 7a and 8a to give 7b (2.04 g, yield 38.1%) and 8b (1.08 g, yield 20.1%). Compound 7b: ¹H NMR (270 MHz, CDCl₃ + DABCO): δ 2.20 (s, 3H), 2.41 (s, 3H), 2.96 (broad, IH), 3.43 (m, 2H), 3.54 (dd, J= 2.4 Hz, 2.4Hz, IH ), 3.61 (dd, J= 2.0 Hz, 1.6Hz, IH), 3.81 (s, 6H), 3.92 (q, J= 5.8 Hz, IH), 4.07-4.13 (m, 2H), 4.18 (d, J= 5.1 Hz, IH, H-20, 4.44 (t, IH, H-3'), 4.83 (d, J= 6.6 Hz, IH ), 5.13 (d, J= 6.6 Hz, IH), 5.88 (s, IH, H-I'), 6.86 (d, J= 8.9 Hz, 4H), 7.09 (d, J= 7.9 Hz, IH), 7.30-7.44 (m, 11H), 7.78 (d, J= 8.3 Hz, 2H), 8.47 (d, J= 7.5 Hz, IH), 9.17 (s, IH). ¹³C NMR (67.9 MHz, CDCl₃ + DABCO): δ 21.6, 24.9, 55.3, 56.3, 60.9, 61.7, 67.8 (C-3'), 80.1(C-2'), 82.9, 87.1, 89.8, 94.9, 96.5, 113.3, 127.2, 128.0, 128.1, 129.9, 130.1, 135.3, 135.5, 136.8, 144,3, 144.7, 144.9, 155.0, 158.7,162.6, 170.2. MALDI-TOF MS: [MH]⁺ 800.21, calcd 800.28. Compound 8^¹H NMR (270 MHz, CDCl₃ + DABCO): δ 2.21 (s, 3H), 2.42 (s, 3H), 3.35 (dd, J = 2.9 Hz, 2.2Hz, IH), 3.44 (q, J= 3.9 Hz, 2H ), 3.60 (dd, J= 2.0 Hz, 2.3Hz, IH), 3.81 (s, 7H), 4.03 (q, J= 4.7 Hz, IH), 4.18 (dd, J= 5.0, 5.4 Hz, IH, H-3'), 4.33 (m, 2H, H-2', 4'), 4.61 (d, J = 7.0 Hz, IH ), 5.68 (d, J= 7.0 Hz, IH), 5.93 (d, J= 2.1 Hz, IH, H-I'), 6.84 (d, J= 8.8 Hz, 4H), 7.17 (d, J= 7.5 Hz, IH), 7.23-7.34 (m, HH), 7.78 (d, J= 8.3 Hz, 2H), 8.35 (d, J= 7.5 Hz, IH), 8.99 (s, IH). ¹³C NMR (67.9 MHz, CDCl₃ + DABCO): δ 21.7, 25.0, 55.3, 56.1, 61.6, 61.9,

75.4, 75.6, 82.1, 87.1, 92.6, 95.7, 96.7, 113.4, 127.3, 128.0, 128.1, 128.2, 130.0, 130.2, 135.2, 135.3, 136.7, 144,2, 144.7, 144.9, 156.0, 158.8, 162.5, 170.2. MALDI-TOF MS: [MH]⁺ 800.23, calcd 800.28.

N⁶-Phenoj^acetyl-5'-O-(Dimethoxytrityl)-2 '-O-[2-(4-Tolylsulfonyl)etlιoxy methyl] adenosine (7c) and rf-Phenoxyacetyl -5'-O-(Dimethoxytrityl) -3'-0 -[2-(4-Tolylsulfonyl)ethoxy methyl] adenosine(Sc) . JV^-Phenoxyacetyl-S'-O-DMTr adenosine (0.9 g, 1.2 mmol) was treated as described for 7a and 8a to give 7c (0.31 g, 26.4%) and 8c (0.265 g, 22.8%). Compound 7c: ¹H NMR (270 MHz, CDCl₃ + DABCO): δ 2.41 (s, 3H), 3.28 (t, J= 3.0 Hz, 2H), 3.40-3.54 (m, 2H), 3.76-3.86 (m, 7H), 3.99-4.04 (m, IH), 4.25 (t, IH), 4,56 (t, IH, H-3'), 4.76-4.86 (m, 5H, H-2' included), 6.21 (d, J= 3.2 Hz, IH, H-I'), 6.78 (d, J= 8.9 Hz, 4H), 7.07 (m, 3H), 7.23-7.41 (m, 13H), 7.74 (d, J= 8.2 Hz, 2H), 8.23 (s, IH), 8.69 (s, IH). ¹³C NMR (67.9 MHz, CDCl₃ + DABCO): δ 21.6, 55.2, 56.1, 61.9, 63.0, 68.1, 70.3 (C-3'), 80.5 (C-2'), 84.1, 86.7, 87.3, 96.0, 113.2, 115.0, 122.5, 127.0, 127.8, 127.9, 128.2, 129.9, 130.1, 135.6, 136.7, 142.2, 144.5, 145.0,

148.4, 152.6, 157.0, 158.6, 166.6. MALDI-TOF MS: [MH]⁺ 916.18, calcd 916.01. Compound 8c: ¹H NMR (270 MHz, CDCl₃ + DABCO): δ 2.42 (s, 3H), 3.27-3.34 (m, 3H), 3.47 (dd, J = 4.0, 4.2 Hz, IH), 3.77 (s, 6H), 3.87-4.03 (m, 2H), 4.32 (t, J= 3.7 Hz, IH), 4.44 (t, J= 4.4 Hz, IH, H-3'), 4,72-4.86 (m, 4H), 4.91 (t, J= 5.2 Hz, IH, H-2') 6.04 (d, J= 5.5 Hz, IH, H-I'), 6.78 (d, J= 8.9 Hz, 4H), 7.03-7.37 (m, 16H), 7.75 (d, J= 8.2 Hz, 2H), 8.22 (s, IH), 8.72 (s, IH). ¹³C NMR (67.9 MHz, CDCl₃ + DABCO): δ 21.6, 55.2, 56.1, 61.7, 63.0, 68.1, 74.3 (C-2'), 77.2 (C-3'), 83.2, 86.7, 89.5, 95.5, 113.2, 115.0, 122.5, 127.0, 127.9, 128.0, 129.8, 130.0, 135.4, 135.5 , 136.7, 142.1, 144.4, 145.0, 148.4, 151.5, 152.4, 157.0, 158.6, 166.6. MALDI-TOF MS: [MH]⁺ 916.30, calcd 916.01. N² -(N, N-dimethylamino methylene)- 5'-O-(Dimethoxytrityl)-2'-O- [2-(4-Tolylsulfonyl)ethoxy methyl] guanosine (7d) and N² -(N, N-dimethylamino methylene)- 5'-O-(Dimethoxytrityl)-3'-O-[2-(4-Tolylsulfonyl)ethoxy methyl] guanosine (8d). N²-(N, iV-dimethylamino methylene)-5'-0-DMTr guanosine (4.5 g, 7 mxnol) was treated as described for 7a and 8a to give 7d (1.88 g, 31.4%) and 8d (1.60 g, 26.7%). 7d ¹HNMR (270 MHz, CDCl₃ + DABCO): δ 2.38 (s, 3H), 3.03(s, 3H), 3.07 (s, 3H), 3.27 (t, J= 5.2 Hz, 2H), 3.42 (broad, 2H), 3.71-3.76 (m, 7H), 4.0 (q, J= 5.1 Hz, IH), 4.23 (broad, IH), 4.47 (broad, IH, H-3'), 4.66 (t, J= 5.0 Hz, IH, H-2'), 4.82 (dd, J= 6.9, 7.0 Hz, 2H), 6.05 (d, J= 4.8 Hz, IH, H-I'), 6.80 (d, J= 8.6 Hz, 4H), 7.18-7.74 (m, 14H), 8.52 (s, IH), 9.25 (broad, IH). ¹³C NMR (67.9 MHz, CDCl₃ + DABCO): δ 21.6, 35.1, 41.3, 53.4, 55.2, 61.8, 63.6, 70.4 (C-3'), 80.0 (C-2'), 83.7, 85.6, 86.7, 95.3, 113.3, 120.6, 127.7, 127.8, 127.9, 128.1, 129.8, 130.0, 130.1,

135.5, 135.6, 136.0, 136.7, 144.5, 145.0, 150.4, 157.0, 157.9, 158.4, 158.6. MALDI-TOF: [MH]⁺ 853.33, calcd 853.32. 8d ¹H NMR (270 MHz, CDCl₃ + DABCO): δ 2.38 (s, 3H), 2.99 (s, 3H), 3.03 (s, 3H), 3.28-3.39 (m, 4H), 3.76 (s, 6H), 3.85-4.05 (m, 2H), 4.22 (t, J= 3.9 Hz, IH), 4.36 (t, J= 4.8 Hz, IH, H-3'), 4.70-4.82 (m, 3H, H-2', OCH₂O ), 5.91 (d, J= 5.3 Hz, IH, H-I'), 6.78 (d, J= 8.7 Hz, 4H), 7.17-7.75 (m, 14H), 8.44 (s, IH). ¹³C NMR (67.9 MHz, CDCl₃ + DABCO): δ 21.6, 35.1, 41.3, 55.2, 56.2, 61.7, 63.2, 74.0 (C-2'), 76.8 (C-3'), 82.2, 86.5, 88.2, 95.5, 113.2, 120.4, 127.8, 127.9, 128.1, 129.8, 129.9, 130.0, 135.5, 135.6, 136.5, 136.7, 144.5, 144.9, 150.2, 156.7, 157.9, 158.1, 158.6. MALDI-TOF: [MH]⁺ 853.30, calcd 853.32. 5'-O-(Dimethoxytrityl)-2'-O-[2-(4-Tolylsulfonyl)ethoxy methyl] uridine 3'-(2-cyanoehtyl N, N-diisopropyl phosphoramidite) (9a). A solution of Compound 7a (544 mg, 0.72 mmol) in dichloromethane (5 ml) was treated with ΛζiV-diisopropylethylamine (0.3 ml, 1.79 mmol) and 2-cyanoethyliV,iV-diisopropylchloropliosphor amidite (0.24 ml, 1.1 mmol) was added dropwise. The reaction was allowed to proceed for 1 h at room temperature. After quenched with MeOH (0.5 ml), the reaction was diluted with dichloromethane and washed with saturated NaHCO₃ solution, dried over MgSO₄, applied to short column chromatography (cyclohexane with 1% Et₃N, ethyl acetate from 10% to 50% ) to give the phosphoramidite 9a (0.54 g, 78.6%). ³¹P NMR (109.4 MHz, CDCl₃ + DABCO): 149.90, 151.00. MALDI-TOF: [MH]⁺ 959.16, calcd 959.36. rf-AcetylS'-O-ζDimethoxytrityl)^ '-O-[2-(4-Tolylsulfonyl)ethoxy methyl] cytidine 3'-(2-cyanoehtyl N, N-diisopropylphosphoramidite) (9b). Compound 7b (1.23 g, 1.54 mmol) was treated as described for 9a. Applying to short column chromatography (cyclohexane with 1% Et₃N, acetone from 20% to 50%) to give 9b (1.08 g, 67.9%). ³¹P NMR (109.4 MHz, CDCl₃ + DABCO): 150.16, 151.69. MALDI-TOF MS: [MH]⁺ 1000.29, calcd 1000.39. N⁵-Phenoxyacetyl-5'-O-(Dimethoxytrityl)-2'-O-[2-(4-Tolylsulfonyl)ethoxy methyl] adenosine 3'-(2-cyanoehtyl N, N-diisopropylphosphoramidite) (9c). 7c (0.246 g, 0.26 mmol) was treated as described for 9a. Applying to short column chromatography (petroleum ether with 1% Et₃N, acetone from 20% to 40%) to give 9c (0.18 g, 60.0%). ³¹P NMR (109.4 MHz, CDCl₃+ DABCO): 148.66, 148.79. MALDI-TOF: [MH]⁺ 1115.99, calcd 1116.42. N² -(N ,N-dimeihylamino methylene)- 5'-O-(Dimethoxytrityl) -2'-O- [2-(4-Tolylsulfonyl)ethoxy methyl] guanosine 3 '-(2-cyanoehtyl N N-diisopropylphosphoramidite) (9d). 7d (0.965 g, 1.13 mmol) was treated as described for 9a. Applying to short column chromatography (CH₂Cl₂ with 1% Et₃N, acetone from 10% to 30%) to give 9d (0.64 g, 53.9%). ³¹P NMR (109.4 MHz, CDCl₃ + DABCO): 148.67, 148.90. MALDI-TOF MS: [MH]⁺ 1053.38, calcd 1054.17. 5'-O-(Dimethoxytrityl)-3'-O-[2-(4-Tolylsulfonyl)ethoxy methyl] uridine 2'-O-pimelate (10a). To a solution of 8a (0.5 g, 0.66 mmol) in dry pyridine (10 ml) was added pimelic acid (258 mg, 1.61 mmol), N-(3-dimethylammopropyl)-iV'-ethylcarbodiirnide hydrochloride (EDAC, 284 mg, 1.48 mmol) and 4-dimethylaminopyridine (82 mg, 0.67 mmol). The reaction was allowed to proceed at room temperature for 4 h. After evaporation of solvent, the residue was diluted with CH₂Cl₂, washed with H₂O twice, saturated (TSnHU)₂CO₃ once, H₂O once, dried over MgSO₄ and applied to short column chromatography (dichloromethane with 1% Et₃N, methanol form 1% to 5%) to give 10a (448 mg, 75.4%). ¹B. NMR (270 MHz, CDCl₃ + DABCO): δ 1.32 (m, 2H), 1.63 (m, 4H), 2.21 (t, J= 6.9 Hz, 2H), 2.36 (t, J= 6.6 Hz, 2H), 2.43 (s, 3H), 3.19 (t, J= 3.8 Hz, 2H), 3.37 (dd, J= 1.9 Hz, IH), 3.58 (dd, J= 1.8 Hz, IH), 3.73 (t, J= 4.7 Hz, 2H), 3.79 (s, 6H), 4.14 (t, J= 2.4 Hz, IH), 4.43 (t, J= 5.1 Hz, IH, H3'), 4.54 (dd, J= 7.1 Hz, 2H), 5.29 (d, J = 8.1 Hz, IH), 5.37 (t, J= 4.8 Hz, IH, H2'), 6.11 (d, J= 4.7 Hz, IH), 6.84 (d, J= 8.0 Hz, 4H), 7.22-7.34 (m, HH), 7.72 (m, 3H). ¹³C NMR (67.9 MHz, CDCl₃ + DABCO): δ 21.7, 24.7, 25.7, 28.9, 33.8, 36.8, 55.3, 56.0, 61.6, 62.2, 73.5 (C3'), 74.0 (C2'), 82.3, 86.6, 87.2, 94.9, 102.7, 113.3, 127.3, 127.9, 128.0, 128.3, 129.9, 130.1, 130.2, 135.0, 139.5, 144.0, 144.8, 150.3, 158.8, 163.2, 172.6, 179.8. Maldi-Tof MS: [MH-Na]⁺ 1023.16, calcd 1023.31. rf-AcetylS'-O-φimethoxytrityl^'-O-ft-^-Tolylsulfonytyethoxy methyl] cytidine

2'-O-pimelate (10b). 8b (150 mg, 0.19 mmol) was treated as described for 10a to give 10b (123 mg, 70.0%). ¹H NMR (270 MHz, CDCl₃ + DABCO): δ 1.37 (m, IH), 1.64 (m, 2H), 2.21 (s, 3H), 2.31 (t, J= 6.6 Hz, IH), 2.37 (t, J= 3.4 Hz, IH), 2.42 (s, 3H), 3.16 (t, J= 3.7 Hz, 2H), 3.39 (dd, J= 2.0 Hz, IH), 3.67 (m, 3H), 3.81 (s, 6H), 4.19 (t, J= 6.9 Hz, IH), 4.40 (m, 2H), 4.53 (d, J= 7.1 Hz, IH), 5.39 (t, J= 4.3 Hz, IH, H2'), 6.1 (d, J= 2.1 Hz, IH, Hl'), 6.85 (d, J= 7.4 Hz, 4H), 7.02 (d, J= 7.5, IH), 7.71 (d, J= 8.2, 2H), 8.27 (d, J= 7.6, IH). ¹³C NMR (67.9 MHz, CDCl₃ + DABCO): δ 21.7, 24.7, 24.8, 24.9, 28.6, 33.8, 34.8, 55.3, 56.1, 61.4, 61.7, 72.4 (C3'), 74.3 (C2')_> 81.7, 87.3, 88.8, 94.9, 96.8, 113.4, 123.8, 127.5, 128.0, 128.1, 128.4, 130,0, 130.2, 130.3, 135.1, 136.1, 136.9, 143.9, 144.7, 144.9, 149.8, 158.9, 162.8, 170.6, 172.2, 178.4. MALDI-TOF MS: [M+Na]⁺ 964.31, calcd 965.04.

N⁵ -Phenoxyacetyl-5 '-O-(Dimethoxytrityl)-3 '-O-[2-(4-Tolylsulfonyl)ethoxy methyl] adenosine 2'-O-pimelate (10c). 8c (214 mg, 0.23 mmol) was treated as described for 10a to give 10c (160 mg, 64.8%). ¹H NMR (270 MHz, CDCl₃ + DABCO): δ 1.32 (m, IH), 1.61 (m, 2H), 2.29-2.39 (m, 2H), 2.42 (s, 3H), 3.22 (t, J= 5.8 Hz, 2H), 3.33 (dd, J= 3.8 Hz, IH), 3.56 (dd, J= 3.1 Hz, IH), 3.77 (m, 8H), 4.27 (m, IH), 4.57 (s, 2H), 4.71 (t, J= 5.5 Hz, IH, H3'), 4.86 (s, 2H), 5.83 (t, J= 4.4 Hz, IH, H2'), 6.26 (d, , J= 4.1 Hz, IH, Hl'), 6,79 (d, J = 8.8 Hz, IH), 7.04 (m, 3H), 7.15-7.39 (m, 13H), 7.73 (d, J= 8.3 Hz, 2H), 8.27 (s, IH), 8.74 (s, IH). ¹³C NMR (67.9 MHz, CDCl₃ + DABCO): δ 21.6, 24.3, 24.4, 28.2, 33.6, 33.7, 55.4, 56.0, 61.7, 62.5, 68.2, 73.9 (C3'), 74.4 (C2'X 82.7, 86.6, 86.8, 95.2, 113.2, 115.2, 123.0, 127.8, 128.1, 129.8, 130.1, 130.3, 135.4, 135.5, 136.8, 141.8, 144.3, 145.0, 148.5, 151.4, 153.0, 157.1, 158.7, 166.8, 172.4, 176.6. MALDI-TOF MS: [M]⁺ 1058.36, calcd 1058.38

N²-(N,N-dimethylamino methylene)- 5'-O-(Dimethoxytrityl)-3'-O- [2-(4-Tolylsulfonyl)ethoxy methyl] guanosine 2'-O-pimelate (1Od). 8d (165 mg, 0.19 mmol) was treated as described for 10a to give 1Od (126 mg, 65.6%). ¹H NMR (270 MHz, CDCl₃ + DABCO): δ 1.35 (m, IH), 1.61 (m, 2H), 2.20 (t, J= 7.3 Hz, IH), 2.38 (m, 4H), 3.07 (s, 3H), 3.11 (s, 3H), 3.15 (t, J= 2.4 Hz, 2H), 3.26 (dd, J= 2.6 Hz, IH), 3.43 (dd, J= 2.2 Hz, IH), 3.71-3.77 (m, 8H), 4.21 (m, IH), 4.51 (m, 3H), 5.92 (t, J = 3.2 Hz, IH, H2'), 5.98 (d, J = 2.3 Hz, IH, Hl'), 6.79 (m, 4H), 7.21-7.38 (m, 11 H), 7.71 (m, 3H), 8.58 (s, IH). ¹³C NMR (67.9 MHz, CDCl₃ + DABCO): δ 21.6, 24.8, 25.8, 29.0, 33.9, 35.2, 36.9, 41.3, 55.3, 55.9, 61.5, 62.7, 73.5 (C3'), 74.1 (C2'), 81.7, 86.5, 86.6, 95.0, 113.2, 127.0, 127.8, 127.9, 128.1, 129.9, 130.0, 135.5, 136.5, 136.8, 144.3, 145.5, 152.2, 156.9, 157.6, 158.5, 158.6, 172.6, 179.6. MALDI-TOF MS: [M]⁺ 995.37, calcd 995.10.

2'-0~[2-(4-Tolylsulfonyl)ethoxy methyl] uridine (15). Compound 7a (115 mg, 0.15 mmol) was treated with 3% DCA / DCM (5 ml) and the reaction was continued at room temperature for 5 minutes. Then the mixture was diluted with ethyl acetate and washed with saturated NaHCO₃, dried over MgSO₄. After short chromatography column (CH₂Cl₂/Me0H, 9/1, v/v), compound 15 (23 mg, 33%) was given. ¹H NMR (270 MHz, CD₃OD): δ 2.42 (s, 3H), 3.50 (t, J= 5.7 Hz, 2H), 3.73 (dd, J= 2.6 Hz, IH), 3.83-3.97 (m, 4H), 4.09 (t, J= 4.2 Hz, IH), 4.19 (t, J= 5.4 Hz, IH), 4.71 (dd, J= 7.0 Hz, IH), 5.67 (d, J= 8.1 Hz, IH), 5.88 (d, J= 3.7 Hz, IH), 7.40 (d, J= 7.8 Hz, 2H), 7.77 (d, J= 8.0 Hz, 2H), 8.02 (d, J= 8.0 Hz, IH). ¹³C NMR (67.9 MHz, CD₃OD): δ 20.28, 55.76, 60.38, 61.87, 68.85, 79.20, 84.72, 88.01, 94.72, 101.30, 127.90, 129.57, 137.09, 141.11, 144.95, 150.82, 164.83. MALDI-TOF MS: [MH]⁺ 457.22, calcd 457.12. Preparation of the solid support. To a solution of 1Oa-IOd (0.1 mmol) in dry acetonitrile (20 ml) was added LCAA-CPG (1 g, Biotech company), and then diisopropylethylamine (1.74 ml, 10 mmol), benzotriazol-1-yloxytris (dimethylamino) phosphonium hexfluorophosphate (BOP, 84 mg, 0.2 mmol) and N-hydroxybenzotriazole (27 mg, 0.2 mmol). After shaking at room temperature for 2 h, this reaction mixture was filtered, washed in turn with acetonitrile, CH₂Cl₂, methanol, CH₂Cl₂ and diethyl ether. Then the solid was suspended in dry pyridine (20 ml) with acetic anhydride (2.25 ml) and 4-dimethylaminopyridine (DMAP, 465 mg), shaken for 2 h at room temperature. After filtration, the solids were washed with pyridine, toluene, CH₂Cl₂, methanol, CH₂Cl₂, diethyl ether in turn and dried over P₂O₅ under high vacuum. The loadings, determined by detritylation assay, are 20-25 μmol/g.

RNA synthesis and purification. All the RNAs are assembled on Applied Biosystems 392 DNA/RNA synthesizer. All syntheses were carried out in trityl off mode. The synthesis cycle and reagents can be found in supplementary information. After the assembly, the solid supports were removed form the cartridges and treated with 25% NH₃/MeOH (4ml) at room temperature for 20 h, then at 40⁰C for 4 h. Then the supernatant solutions were separated from the solid supports, evaporated to dryness. After coevaporation with dry THF twice, they are treated with IM tetrabutylammonium fluoride in THF (this solutions was dried with 4 A molecular sieve overnight before use) containing 10% n-propylamine and 1% bis(2-mercaptoethyl) ether at room temperature for 20 h. The reactions were quenched by addition of an equal volume of dd water and applied to NAP-IO column according to manufacturer's instruction with dd water as the eluting buffer. To ensure remove the fluoride completely, the collected fragments were again applied to Sep-Pak cartridge.

RNase H digestion assays. Escherichia coli RJNase H (5 units/μL, specific activity 420000 units mg ^-1, molecular weight 21000 g moF¹), T4 polynucleotide Kinase (30 units/μL) and [7-³²P]ATP were purchased from Amersham Pharmacia Biotech (Sweden). The pure 15 mer RNA (ON 12) was from IBABioTAGnology (received as a crude form and purified by PAGE, the purity is shown in Figure S5). Synthesis of the complementary DNA was carried out as previously described.⁴³ The ON 12 was synthesized, deprotected, desalted by NAP-10 column and Sep-Pak cartridge just as upper description to give the crude ON 12, which was used directly for RNase H digestion. The RNA was 5'-end labeled with ³²P using T4 polynucleotide kinase, [7-³²P]ATP by standard procedure. The RNase H digestion of synthesized crude RNA and pure RNA was carried out according to the following procedure: target pure RNA (0.1 μM) or crude RNA (0.1 μM) (specific activity 70000 cpm) and 10-fold excess of complementary DNA (1 μM) were incubated in a buffer, containing 20 mM Tris-HCl (pH 8.0), 20 mM KCl, 10 mM MgCl₂, 0.1 mM EDTA and 0.1 mM

DTT at 21 ⁰C in the presence of 0.06 U E. coli RNase H. Prior to the addition of the enzyme

reaction components were pre-annealed in the reaction buffer by heating at 80 ⁰C for 4 min

followed by 1.5 h equilibration at 21 ⁰C. Total reaction volume was 30 μL. Aliquots of 3 μL were taken after 2, 5, 10, 15, 25, 40 and 60 min and the reactions were terminated by mixing with stop solution (7 μL), containing 0.05 M EDTA, 0.05% (w/v) bromophenol blue and 0.05% (w/v) xylene cyanole in 80% formamide. The samples were subjected to 20% 7 M urea PAGE and visualized by autoradiography. Quantitation of cleavage products was performed using a Molecular Dynamics Phosphorlmager.

Phosphodiesterase digestion assays. Shrimp Alkaline Phosphatase and Phosphodiesterase I (Crotalus adamanteus Venom) are from Amersham Pharmacia Biotech, Sweden. A reaction mixture containing 20 mM Tris-HCl (pH 8.0), 10 ml MgCl₂, Shrimp Alkaline Phosphatase (0.5 unit) and Phosphodiesterase I (Crotalus adamanteus Venom) (0.4 unit) was added to Rude ON 12 (1 OD unit at 260 nm) with total reaction volume of 30 μL. The reaction mixture was incubated at 37 D for 24 h and subjected to HPLC analysis directly (0.2 OD/injection).

References 1. Fire, A.; Xu, S.; Montgomery, M. K.; Kostas, S. A.; Driver, S. E.; Mello, C. C, Nature 1998, 391, 808-811.

2. Elbashir, S. M.; Harborth, J.; Lendechel, W.; Yalcin, A.; Weber, K.; Tuschl, T., Nature 2001, 411, 494-498.

3. Reese, C. B., Org. Biomol. Chem. 2005, 3, 3851-3868. 4. Gasparutto, D.; Molko, D.; Teoule, R., Nucleosides & Nucleotides 1990, 9, 1087-1098.

5. Reese, C. B., Protection of 2'-Hydroxy Functions of Ribonucleosides. In Current Protocols in Nucleic Acid Chemistiy, Beaucage, S. L.; Bergstrom, D. E.; Glick, G. D.; Jones, R. A., Eds. John Wiley & Sons: New York, 2000.

6. Chaulk, S. G; MacMillan, A. M., Nucl. Acids Res. 1998, 26, 3173-3178.

7. Schwartz, M. E.; Breaker, R. R.; Asteriadis, G. T.; deBear, J. S.; Gough, G. R., Bioorg. Med. Chem. Lett. 1992, 2, 1019-1024. 8. Reese, C. B.; SafQiill, R.; Sulston, J. E., J. Am. Chem. Soc. 1967, 89, 3366-3368.

9. Sandstroem, A.; Kwiatkowski, M.; Chattopadhyaya, J., J. Acta Chem. Scad. 1985, B39, 273-290.

10. Sandstroem, A.; Kwiatkowski, M.; Chattopadhyaya, J., Nucleosides & Nucleotides 1985, 4, 177-181. 11. Lloyd, W.; Reese, C. B.; Song, Q.; Vandersteen, A. M.; Vistina, C; Zhang, P. Z., J. Chem. Soc. Perkin Trans. 1 2000, 165-176.

12. Pfister, M.; Schirmeister, H.; Mohr, M.; Farkas, S.; Stengele, K. P.; Reiner, T.; Dunkel, M.; Gokhale, S.; Charubala, R.; Pfleiderer, W., HeIv. Chim. Acta 1995, 78, 1705-1737.

13. Matysiak, S.; Pfleiderer, W., HeIv. Chim. Acta 2001, 84, 1066-1085. 14. Semenyuk, A.; Foldesi, A.; Johansson, T.; Estmer-Nilsson, C; Blomgren, P.; Brannvall, M.; Kirsebom, L. A.; Kwiatkowski, M., J. Am. Chem. Soc. 2006, 128, 12356-12357.

15. Ogilvie, K. K.; Sadana, K. L.; Thompson, E. A.; Quilliam, M. A.; Westmore, J. B., Tetrahedron Lett. 1974, 33, 2861-2863.

16. Ogilvie, K. K.; Theriault, N.; Sadana, K. L., J. Am. Chem. Soc. 1977, 99, 7741-7743. 17. Usman, N.; Ogilvie, K. K.; Jiang, M. Y; Cedergren, R. J., J. Am. Chem. Soc. 1987, 109,

7845-7854.

18. Wada, T.; Tobe, M.; Nagayama, T.; Furusawa, K.; Sekine, M., Tetrahedron Lett. 1995, 36, 1683-1684.

19. Umemoto, T.; Wada, T., Tetrahedron Lett. 2004, 45, 9529-9531. 20. Rao, M. V.; Reese, C. B.; Schehlmann, V.; Yu, P. S., J. Chem. Soc. Perkin Trans. 1 1993, 43-55.

21. Stawinski, J.; Strδmberg, R.; Thelin, M.; Westman, E., Nucl. Acids Res. 1988, 16, 9285-9308.

22. Wu, T.; Ogilvie, K. K.; Pon, R. T., Nucl. Acids Res. 1989, 17, 3501-3517. 23. Jones, S. S.; Reese, C. B., J. Chem. Soc. Perkin 1 1979, 2762-2764.

24. Scaringe, S. A.; Francklyn, C; Usman, N., Nucl. Acids Res. 1990, 18, 5433-5441.

25. Scaringe, S. A.; Wincott, F. E.; Caruthers, M. H., J. Am. Chem. Soc. 1998, 120, 11820-11821.

26. Pitsch, S.; Weiss, P. A.; Jenny, L.; Stutz, A.; Wu, X., HeIv. Chim. Acta 2001, 84, 3773-3795.

27. Ohgi, T.; Masutomi, Y; Ishiyama, K.; Kitagawa, H.; Shiba, Y; Yano, J., Org. Lett. 2005, 7, 3477-3480.

28. Bordwell, F. G, Ace. Chem. Res. 1988, 21, 456-463.

29. Josephson, S.; Balgobin, N.; Chattopadhyaya, J., Tetrahedron Lett. 1981, 22, 4537-4540.

30. Jain, M.; Fan, J.; Baturay, N. Z.; Kwon, C. H., J. Med. Chem. 2004, 47, 3843-3852.

31. Watanabe, K. A.; Fox, J. J., Angew. Chem. Internal Edit. 1966, 5, 579-580.

32. Sheppard, T. L.; Rosenblatt, A. T.; Breslow, R., J. Org. Chem. 1994, 59, 7243-7248.

33. Chaix, C; Duplaa, A. M.; Molko, D.; Teoule, R., Nucl. Acids Res. 1989, 17, 7381-7393. 34. McBride, L. J.; Caruthers, M. H., Tetrahedron Lett. 1983, 24, 245-248. 35. Fromageot, H. P. M.; Griffin, B. E.; Reese, C. B.; Sulston, J. E.; Trentham, D. R., Tetrahedron Lett. 22, 705-710.

36. Ogilvie, K. K.; Schifman, A. L.; Penney, C. L., Can. J. Chem. 1979, 57, 2230-2238.

37. Milecki, J.; Zamaratski, E.; Maltseva, T. V.; Foldesi, A.; Adamiak, R.; Chattopadhyaya, J., Tetrahedron Lett. 1999, 55, 6603-6622.

38. Pon, R. T.; Yu, S., Tetrahedron Lett. 1997, 38, 3331-3334.

39. Vargeese, C; Carter, J.; Yegge, J.; Krivjansky, S.; Settle, A.; Kropp, E.; Peterson, K.; Pieken, W., Nucl. Acids Res. 1998, 26, 1046-1050.

40. Lieber, E.; Enkoji, T., J. Org. Chem. 1961, 4472-4479. 41. Dahl, B. H.; Nielsen, J.; Dahl, O., Nucl. Acids Res. 1987, 15, 1729-1743.

42. Umemoto, T.; Wada, T., Tetrahedron Lett. 2005, 46, 4251-4253.

43. Pradeepkumar, P. L; Zamaratski, E.; Foldesi, A.; Chattopadhyaya, J., J. Chem. Soc, Perkin Trans. 2 2001, 402-408.

Solid-phase synthesis of 12 oligoRNAs (14-38mer, crude purity >90%) have ; been exemplified using this new 2'-0-TEM group

A New Method for RNA Synthesis Using a New 2'-O-(4-tolylsulfonylethoxymethyl) (TEM) Protecting Group

For RNA synthesis on the solid-support, the common synthetic strategy is to use a 4,4'-dimethoxytrityl (DMTr) group at the 5'-OH and a t-butyldimethylsilyl (TBDMS) group for 2'-OH protections. Several other 2'-protecting groups such as photolabile 4-nitro- or 2-nitrobenzyl group, or an

NHR₃

1a 1c 1d

1b

2a 2b 2c 2d

R₁ = R₂ = Acetyl; R₂ = R₃ = Pac;

-CH₂-O-2' R₄ = -CH₃ or -CH₂CH₂CN

Q = (Q₂₉) = TEM (see below for Q₂₃)

Where, Q = (Q₁) CF₃- ; (Q₂) CCI₃- ; (Q₃) CBr₃- ; (Q₄) MeS- ; (Q₃) CF₃S- ; (Q₆) CF₃S(O)- ; (Q₇) CF₃SO₂-; (Q₈) C₆H₅-S(O)- ; (Q₉) 0-Me-C₆H₄-S(O)- ; (Q₁₀) P-Me-C₆H₄-S(O)- ; (Q₁₁) /H-Me-C₆H₄-S(O)- ; (Q₁₂) 0-CI-C₆H₄-S(O)- ; (Q₁₃) P-Cl-C₆H₄-S(O)- ; (Q₁₄) /H-CI-C₆H₄-S(O)- ; (Q₁₅) 0-F-C₆H₄-S(O)- ; (Q₁₆) P-F-C₆H₄-S(O)- ; (Q₁₇) /H-F-C₆H₄-S(O)- ; (Q₁₈) 0-Br-C₆H₄-S(O)- ; (Q₁₉) P-Br-C₆H₄-S(O)- ; (Q₂₀) Zn-Br-C₆H₄-S(O)- ; (Q₂₁) 0-1-C₆H₄-S(O)- ; (Q₂₂) P-I-C₆H₄-S(O)- ; (Q₂₃) /H-I-C₆H₄-S(O)- ; (Q₂₄) o-NO₂- C₆H₄-S(O)- ; (Q₂₅) P-NO₂-C₆H₄-S(O)- ; (Q₂₆) /H-NO₂-C₆H₄-S(O)- ; (Q₂₇) C₆H₅-SO₂- ; (Q₂₈) (MWe- C₆H₄-SO₂; (Q₂₉) P-Me-C₆H₄-SO₂- (TEM); (Q₃₀) /H-Me-C₆H₄-SO₂- ; (Q₃₁) 0-CI-C₆H₄-SO₂- ; (Q₃₂) p-CI- C₆H₄-S(O₂)- ; (Q₃₃) /H-CI-C₆H₄-S(O₂)- ; (Q₃₄) 0-F-C₆H₄-SO₂- ; (Q₃₅) P-F-C₆H₄-SO₂- ; (Q₃₆) /H-F-C₆H₄- SO₂-; (Q₃₇) 0-Br-C₆H₄-SO₂- ; (Q₃₈) P-Br-C₆H₄-S(O₂)- ; (Q₃₉) /H-Br-C₆H₄-SO₂- ; (Q₄₀) 0-!-C₆H₄-SO₂- ; (Q₄₁) P-I-C₆H₄-SO₂- ; (Q₄₂) /H-I-C₆H₄-SO₂- ; (Q₄₃) 0-NO₂-C₆H₄-SO₂- ; (Q₄₄) P-NO₂-C₆H₄-SO₂- ; (Q₄₅) /H-NO₂-C₆H₄-SO₂-; (Q₄₆) MeS(O)- ; (Q₄₇) MeS(O₂)- ;

Scheme 8

appropriate acetal protecting group, such as FPMP, are also used^1"10. Two new 2'-OH protecting groups, such as bis(2-acetoxyethyloxy)methyl (ACE) and triisopropylsilyloxymethyl (TOM), have also been recently developed. 2'-Acetal derivatives with electron-withdrawing substituents, such as l-(2-cyanoethoxy) ethyl, were also identified (cleaved by fluoride anion under aprotic conditions). Gough et al. have introduced the fluoride-cleavable 4-nitrobenzyloxymethyl protecting group. Recently, electron-withdrawing substituents into formaldehyde acetal type protecting groups has also been introduced¹¹. This approach has led to the development of a novel protecting group, 2-cyanoethoxymethyl (CEM), which has allowed synthesis of rather large oligo-RNA¹¹.

We here report that the nature of the electron- withdrawing group into formaldehyde acetal type protecting groups for the protection of the 2'-OH has been varied in a facile manner to produce a battery of appropriately 2'-protected derivatives, [Q-(CBb)₂-O-CH₂- in la-d, where Q = Q₁ to Q₄₆ in Figure 1], which has been successfully used to produce pure oligo-RNAs in very good yields and purity on the solid support using either phosphiteamidite, H-phosphonate or by phosphotriester approach. We here show, as an example, the use of 4-tolylsulfonylethoxymethyl (TEM) group for 2'-OH protection for the preparation of building blocks 2a-d for the solid-phase RNA synthesis [R = p-Me-C₆H₄-SO₂-(CH₂)₂-O-CH₂- in 2a-d in Figure 1] using the phosphoramidite approach.

The 2',3'-O-dibutylstannylidene derivatives of the nucleobase-protected 5'-0-DMTr ribonucleosides were treated with 4-tolylsulfonylethoxymethyl (TEM) chloride to give a mixture of the T-O- and 3'-0-TEM derivatives with the latter being the dominant product (30-50%). They were subsequently converted to the corresponding base-protected 5'-O-DMTr-2'-O-TEM phosphoramidites 2a-d, yields varying from 50 -80%. Compounds 2a-d were characterized by NMR (for Characterizations and purities of 2a-d, see Figs 2-9) and mass-spectroscopy. We have subsequently employed the 2'-O-TEM-protected building blocks 2a-d (as in Fig 1) for the synthesis U₂₀, U₃₈ as well as several mixed RNA sequences containing all four ribonucleotides as proof of the principle: Seq #1: 5'-GAC GUAAAC GGC CAC AAG UUC-3' and Seq #2: 5'-ACT UGT GGC CGU UTA CGT CGC-3'. They were deprotected first by the treatment of methanolic ammonia at RT for 24 hours, then at 50⁰C for 4 h, followed by treatment with 1 M TBAF/THF for 24 hours at RT (see below for latest procedure for deprotection). The HpIc purities of the crude U₂₀ and U₃₈ in Figs 10 and 11, whereas the PAGE pictures of the crude Seq# 1 and #2 are shown in Figs 12, which also show that the coupling yield in each step was very satisfactory. The structural integrities of all oligo-RNAs were proven by the Maldi-Tof mass spectrometry. We have subsequently synthesized several oligo-RNA using our 2'-0-TEM methodology which has been reported in a full paper: "2-(4-Tolylsulfonyl)ethoxymethyl (TEM) - A New 2'-OH Protecting Group For Solid Support RNA Synthesis", Org. Biomol. Chem. (2007) (DOI: 10.1039/B614210A). (manuscript enclosed). For a smoother deprotection of the 2'-0-TEM group following comments are useful: the IM TBAF/THF with 10% n-propylamine and 1% bis (2-mercaptoethyl) ether remains to be the preferred reagent for deprotecting the 2'-0-TEM group from the oligo-RNA. Recent unpublished data from this lab however suggests that the IM TBAF/THF with 10% morpholine at RT seems to be a more improved deprotection agent than IM TBAF/THF with 10% n-propylamine and 1% bis (2-mercaptoethyl) ether, which will be reported in a full paper.

References (1) Scaringe, S. A.; Wincott, F. K; Caruthers, M. H. J. Am. Chem. Soc.1998, 120, 11820-11821.

(2)(a) Ogilvie, K. K.; Beaucage, S. L.; Schifman, A. L.; Theriault, N.Y.; Sadana, K. L. Can. J.

Chem. 1978, 56, 2768-2780. (b) Usman, N.;Ogilvie, K. K.; Jiang, M.-Y.; Cedergren, R. J. J. Am.

Chem. Soc. 1987, 109, 7845-7854.

(3) (a) Tanaka, T.; Tamatsukuri, S.; Ikehara, M. Nucleic Acids Res. 1986,i4, 6265-6279. (b) Hayes, J. A.; Brunden, M. J.; Gilham, P. T.; Gough, G. R. Tetrahedron Lett. 1985, 26, 2407-2410.

Pitsch, S.; Weiss, P. A.; Wu, X.; Ackermann, D.; Honegger, T. HeIv. CHm. Acta 1999, 52,

1753-1761.

(4)(a) Griffin, B. E.; Reese, C. B. Tetrahedron Lett. 1964, 5, 2925-2931. (b) Rao, M. V.; Reese, C.

B.; Schehlmann, V.; Yu, P. S. J. Chem.Soc, Perkin Trans. 1 1993,43-55. (5) (a) Gough, G R.; Miller, T. J.; Mantick, N. A. Tetrahedron Lett.1996, 31, 981-982. (b) Welz, R.; Muller, S. Tetrahedron Lett. 2002, 43,795-797.

(6) Pitsch, S.; Weiss, P. A.; Jenny, L.; Stutz, A.; Wu, X. HeW. ChimActa 2001, 54, 3773-3795.

(7) Micura, R. Angew. Chem. Int. Ed. 2002, 41, 2265-2269.

(8) Matysiak, S.; Fitznar, H.-P.; Schnell, R.; Pfleiderer, W. HeW. CUm. Acta 1998, 51, 1545-1566.

(9) Umemoto, T.; Wada, T. Tetrahedron Lett. 2004, 45, 9529-9531.

(10) (a) Ti, G. S.; Gaffney, B. L.; Jones, R. A. J. Am. Chem. Soc. 1982, 104, 1316-1319. (b) Chaix, C; Molko, D.; Teoule, R. Tetrahedron Lett.1989, 30,71-74.

11. Ohgi, T, Masutomi, Y, Ishiyama, K, Kitagawa, H, Shiba, Y, and Yano, J. Organic Letters, 7(6), 3477-3480 (2005).

Claims

CLAIMS:

1. A compound, usable in RNA synthesis, according to any of the formulae la-Id or 2a-2d below:

1a 1b 1c 1d

2a 2b 2c 2d

R₁ = R₂ = Acetyl; R₂ = R₃ = Pac;

R = Qs _^CH₂-O-2" R₄ = -CH₃ or -CH₂CH₂CN

Q = (Q₂₉) = TEM (see below for Q₂₉J

Where, Q = (Q₁) CF₃- ; (Q₂) CCI₃- ; (Q₃) CBr₃- ; (Q₄) MeS- ; (Q₅) CF₃S- ; (Q₆) CF₃S(O)- ; (Q₇) CF₃SO₂-; (Q₈) C₆H₅-S(O)- ; (Q₉) D-Me-C₆H₄-S(O)- ; (Q₁₀) P-Me-C₆H₄-S(O)- ; (Q₁₁) WT-Me-C₆H₄-S(O)- ; (Q₁₂) OCI-C₆H₄-S(O)- ; (Q₁₃) P-CI-C₆H₄-S(O)- ; (Q₁₄) /W-CI-C₆H₄-S(O)- ; (Q₁₅) 0-F-C₆H₄-S(O)- ; (Q₁₆) P-F-C₆H₄-S(O)- ; (Q₁₇) m-F-C₆H₄-S(O)- ; (Q₁₈) 0-Br-C₆H₄-S(O)- ; (Q₁₉) P-Br-C₆H₄-S(O)- ; (Q₂₀) /W-Br-C₆H₄-S(O)- ; (Q₂₁) 0-1-C₆H₄-S(O)- ; (Q₂₂) P-I-C₆H₄-S(O)- ; (Q₂₃) /W-I-C₆H₄-S(O)- ; (Q₂₄) 0-NO₂- C₆H₄-S(O)- ; (Q₂₅) P-NO₂-C₆H₄-S(O)- ; (Q₂₆) /W-NO₂-C₆H₄-S(O)- ; (Q₂₇) C₆H₅-SO₂- ; (Q₂₈) o-Me- C₆H₄-SO₂; (Q₂₉) P-Me-C₆H₄-SO₂- (TEM); (Q₃₀) /W-Me-C₆H₄-SO₂- ; (Q₃₁) 0-CI-C₆H₄-SO₂- ; (Q₃₂) p-CI- C₆H₄-S(O₂)- ; (Q₃₃) /W-CI-C₆H₄-S(O₂)- ; (Q₃₄) 0-F-C₆H₄-SO₂- ; (Q₃₅) P-F-C₆H₄-SO₂- ; (Q₃₆) /W-F-C₆H₄- SO₂-; (Q₃₇) 0-Br-C₆H₄-SO₂- ; (Q₃₈) P-Br-C₆H₄-S(O₂)- ; (Q₃₉) /W-Br-C₆H₄-SO₂- ; (Q₄₀) 0-1-C₆H₄-SO₂- ; (Q₄₁) P-I-C₆H₄-SQ₂- ; (Q₄₂) /W-I-C₆H₄-SO₂- ; (Q₄₃) 0-NO₂-C₆H₄-SO₂- ; (Q₄₄) P-NO₂-C₆H₄-SO₂- ; (Q₄₅) /W-NQ₂-C₆H₄-SO₂-; (Q₄₆) MeS(O)- ; (Q₄₇) MeS(O₂)- ;

2. A chemical entity, usable for the protection of the 2'-OH in RNA synthesis, represented by the formula:

wherein Q is defined as in claim 1.

3. The chemical entity as claimed in claim 2 for RNA synthesis on solid support.

4. A method for RNA synthesis on solid support comprising using any combination of compounds according to claim 1.

5. The method as claimed in claim 4, comprising 2'-OH protection with a chemical entity as defined in any of claims 2-4.