Title RNA創薬を目指した塩基部欠損ヌクレオシドを有する核 …...Title...

Title RNA創薬を目指した塩基部欠損ヌクレオシドを有する核酸オリゴマーの合成( 本文(Fulltext) )

Author(s) 長屋, 優貴

Report No.(DoctoralDegree) 博士(薬科学) 連創博甲第40号

Issue Date 2018-03-25

Type 博士論文

Version ETD

URL http://hdl.handle.net/20.500.12099/75253

※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

RNA

29

1

2

1 2 RNA (siRNA) 3 Argonaute 4 siRNA 5 67 (dRH)

3 dRH

1 dRH 2 dRH 3 4 5 6

4 2

1 2 (RHOBn) RNA 1 RHOBn 2 RHOBn RNA 3 4 5 6 7 Argonaute2-PAZ

2 2 (RHOPg) RNA

1 RHOPg 2 RHOPg RNA 3 RHOPg RNA CuAAC

5

6 7

2

9

21

26

30

24 22

1

35

15 19

11

31

12

41

43

36

8

25

32

29 28

33

37

62

44

1

1

DNA RNA

RNA (small interfering RNA, siRNA) RNA RNA (RNA interference, RNAi)

mRNAsiRNA i)

ii) iii) siRNA

siRNA 3′ 2

siRNA 3′

siRNA

3′siRNA

siRNA2

2siRNA

2

2

1

(Human Genome Project) 2% 2 2

1) 2 6 3 2

RNA non-coding RNA (ncRNA) (Figure 2-1) RNA

RNA ncRNARNA

(tRNA) RNA (rRNA) mRNAmicro RNA (miRNA) ncRNA 1

PIWI-interacting RNA (piRNA)

Figure 2-1. ncRNA

3

ncRNA

5 (Table 2-1) DNAmRNA miRNA siRNA

5

Table 2-1.

6 (Table 2-2) 2)

Table 2-2.

4

Vitravene ( Fomivirsen) Vitravene

21 Immediate early antigen 2 (IE2) mRNA

HIVIE2 Vitravene IE2 mRNA mRNA

IE21998 2011 6

2004 Macugen ( Pegaptanib) 27 (adenosine, guanosine)

2′ (cytidine, uridine) 2′5′ 40 kDa polyethylene glycol

2Macugen

165 (VEGF165)

2013 FDA Kynamro ( Mipomersen) Kynamro

20 5′ 3′ 5 2′ methoxyethoxy (MOE) Kynamro

lipoproteinn 1 apolipoprotein B (ApoB) mRNA ApoB mRNARNA-DNA RNA

RNase H ApoB mRNA ApoBlow density lipoprotein (LDL)

LDLKynamro

Exondys 51 ( Eteplirsen) 30 mer

Exondys 51 pre-mRNA 51 Exondys 51 pre-mRNA

()

5

Defitelio ( Defibrotide) 2013 EMA 2016 4 FDADefitelio

DNA 90% 210%

Spinraza ( Nusinersen) 182′ MOE

cytidine 5 (SMA) SMN2 Spinraza SMN2 mRNA

7 ISS-N1

2016 10 FDA

6

2 RNA (siRNA)

RNA (RNA interference, RNAi) 1998 Fire Mello3) RNAi

(Figure 2-2) 4)

Figure 2-2. RNAi

RNA (double-stranded RNA, dsRNA) RNase III

Dicer 3′ 2 ( ) 19-23 RNA (small interfering RNA, siRNA) siRNA RNA

(RNA induced silencing complex, RISC) Argonaute 2 (Ago2) Ago2 ATPHsp70/Hsc90 siRNA

( ) Ago2 Ago2Ago2 ( ) Ago2 RISC

Ago2 siRNA siRNA Dicer-2 RNAR2D2 RISC–loading complex (RLC) 5′

5′5′

RISC mRNA5′ 10

11 mRNA

7

3 Argonaute

RNAi Ago2 PIWI MIDPAZ N 4 L1 L2 2

(Figure 2-3) 5)

Figure 2-3. RISC Ago2

PIWI RNase H5) siRNA Ago2 RISC RISC

mRNA PIWImRNA Ago2 siRNA

5′ PIWI

MID PIWI 5′Ago2 6)

PAZ siRNA3′ 7) RISC

PAZ mRNAmRNA

PAZ 2

PAZ (Figure 2-4)

Figure 2-4. PAZ 2

8

N Ago2 8) Ago2 Hsp70/Hsc90siRNA siRNA

N siRNA

4 siRNA

1998 Fire Mello RNAi siRNAsiRNA i)

ii) iii)

siRNA siRNA

ATTR (TTR)

ATTRTTR mRNA

TTR mRNA siRNA9) Alnylam PharmaceuticalssiRNA siRNA

Phase 3

Alnylam N-siRNA N-

siRNA10) N- siRNA

Phase 2

9

5

3 (Figure 2-5)

11)

Figure 2-5.

Watson-CrickSpinraza

5′ 5-methylcytidine (Figure 2-6)Toll 9 (TLR9) 5′ CpG (cytidine- -

guanosine)

Spinraza 5′ CpG 5′ cytidine 5TLR9

Figure 2-6. cytidine 5

2′ Macugen 2′

(adenosine, guanosine) 2′ (adenosine, uridine) (Figure 2-7) Kynamro Spinraza 2′ MOE

2′Exondys 51

2

10

Figure 2-7. 2′

(O) (S) (Figure 2-8) Vitravene

Kynamro Spinraza

Figure 2-8.

11

6

siRNAsiRNA RNA

(Figure 2-9) 12) – 16)

PAZ

17) – 20)

Figure 2-9.

12

7 (dRH)

(dRH) [1]~[4]

[1]

2-Deoxy-D-ribose2 dRH (Scheme 2-1) 21), 22) A

2M ( 54%) B

p-A dRH ( 68%)

A B dRH

A

B

Scheme 2-1. dRH

[2]

2-Deoxy-D-ribose 1 (Scheme 2-2, 2-3) 23)

2-Deoxy-D-ribose 1 3 5 Tol1 Tol

dRH ( 4 61%) 1 1 ( 5 41%) Scheme 2-2 2-3

13

Scheme 2-2. 1 dRH

Scheme 2-3. 1 1 dRH

[3] 1

(Scheme 2-4) 24) 3 5 2 dRH

86%

Scheme 2-4. dRH

[4] dRH

(Scheme 2-5) 25)

100 ºC 1

1 5 41%

14

Figure 2-5. dRH

15

3 dRH

1 dRH

(dRH; 1) Cameron

26) (2) 3' 5' TBDPS3',5'-di-O-TBDPS- (3) HMDS

4 4 4

3 5 38% (Scheme 3-1)

Scheme 3-1. 3 4

3',5'-di-O-TBDPS- (3) 4 HMDS 3 TMS

4 35

(Scheme 3-2)

Scheme 3-2. 5

16

3 5' TBDPS5'-O-TBDPS- (6)

(Scheme 3-3) TLC 7

5 7

Scheme 3-3. 6 7

5'-O-TBDPS- (6) 3TMS

Cameron

7 5 HMDS

125 μM 1.5 6 7

3 8 8 7 5

(Scheme 3-4)

Scheme 3-4. 6 TMS 8

17

3 TMS 8dRH 9 5'-O-TBDPS- (6) 8

Pd/C9 36% Ferrier

1 10 31% (Scheme 3-5)

Scheme 3-5. Pd/C

(Scheme 3-6)

Scheme 3-6. Pd/C

3 TMS5'-O-TBDPS- (6) 3

TMS Pd/C- 5'-O-

TBDPS-dRH (9) 94% (Scheme 3-7)

18

Scheme 3-7. 6 5-O-TBDPS-dRH (9)

TBAF TBDPS dRH (1) 97% (Scheme 3-8)

Scheme 3-8. TBDPS

dRH (Scheme 3-9)

5

83%

Scheme 3-9. dRH

19

2 dRH

dRH dRH (1) (Scheme 3-10) 1 5 DMTr

5-O-DMTr-dRH (11) THF DIPEA3

12 11 3DMF EDC·HCl CPG

DMAP CPG

dRH 14

Scheme 3-10. dRH

(11 13) 3′ dRH

siRNA Dual-luciferase reporter assay system Renilla luciferase mRNA siRNA

5′

TBAFHPLC

MALDI-TOF/MS (Figure 3-1)

20

Figure 3-1.

21

3

5′ 3′ dRHdRH 3′→5′ (SVPD)

(Figure 3-2) 3′ (TT) (RHRH)

Conditions

Buffer: PBS (−)

Concentration: 3 μM

Nuclease: 1 unit/mL snake venom phosphodiesterase

Incubation temperature: 37 ºC

Figure 3-2.

dRHdRH 3′ TTRHRH

3′

22

4

260 nmπ-π

50 50% (Tm)

(dRH) siRNAsiRNA 3′ (TT)

(RHRH) siRNA

Conditions

Buffer: 10 mM NaH2PO4∙Na2HPO4 (pH 7.0) 100 mM NaCl Concentration: 3 μM

Figure 3-3.

Table 3-1. Tm

XX Tm (ºC) ΔTm (ºC)

TT 82.7

RHRH 81.8 -0.9

dRHdRH 81.2 -1.5

23

3′ dRH siRNATm Tm 82.7

ºC (TT) 81.8 ºC (RHRH) 81.2 ºC (dRHdRH) π-π

3′ dRH

siRNA siRNA

siRNA3′ siRNA

24

5

siRNAHeLa Dual-Luciferase reporter

assay system dRH siRNA3′ TT RHRH siRNA

Assay kit Dual-Glo Luciferase Assay System (Promega)

Vector: psiCHECK-2

Transfection reagent: Transfast Buffer: Opti-MEM

Conditions

Transfection time: 1 h

Incubation time: 24 h

Figure 3-4.

3′ dRHdRH siRNATT RHRH siRNA

siRNA 3′ dRH siRNA RNAi

25

6

50% siRNAdRHdRH siRNA 50% 37 ºCsiRNA PAGE GelRed

Conditions

Buffer: D-MEM (Phenol Red free) Concentration: 3 μM


Figure 3-5. 50%

3′ TT RHRH siRNA dRHdRH siRNATT RHRH siRNA 60

40%

26

4 2

1 2 (RHOBn) RNA

1 RHOBn

2 2 (RHOBn) 27) RHOBn

(RH) 3 5BEMP 2

(Scheme 4-1) 9 RHOBn

siRNARHOBn siRNA

Scheme 4-1. RHOBn

27

siRNAsiRNA

siRNA RHOBn

RHOBn (15) (RH, 16) RH 3 5

1,1,3,3- (TIPDS ) 3,5-O-TIPDS-RH (17) (Scheme 4-2)

Scheme 4-2. RH 3 5 TIPDS

173,5-O-TIPDS-RHOBn (18) TBAF TIPDS

27% RHOBn (15) (Scheme 4-3) 2TIPDS

(III)2 47% (Scheme 4-4)

Scheme 4-3. BnBr/NaH 2 RHOBn

Scheme 4-4. PhCHO-Et3SiH/FeCl3 2 RHOBn

28

2 RHOBn RNA

RHOBn (15) 5 DMTr5-O-DMTr-RHOBn (19) THF DIPEA 2

20 97% (Scheme 4-5)

Scheme 4-5. RHOBn

3 dRH 20 Renilla luciferase mRNA siRNA 3′RHOBnRHOBn (Figure 4-1)

5′

MALDI-TOF/MS

Figure 4-1.

29

3

5′ 3′ RHOBnRHOBn

(SVPD) (Figure 4-2)3′ TT RHRH

Conditions

Buffer: PBS (−) Concentration: 3 μM

Nuclease: 1 unit/mL snake venom phosphodiesterase


Figure 4-2.

RHOBnRHOBn 3′ TT RHRH

RHOBnRHOBn TT 52

30

4

RHOBn siRNAsiRNA 3′

(TT) siRNA

Conditions

Buffer: 10 mM NaH2PO4∙Na2HPO4 (pH 7.0) 100 mM NaCl Concentration: 3 μM

Figure 4-3.

Table 4-1. Tm

XX Tm (ºC) ΔTm (ºC)

TT 77.0

RHOBnRHOBn 76.4 -0.6

3′ RHOBnRHOBn siRNATm Tm

77.0 ºC (TT) 76.4 ºC (RHOBnRHOBn) RHOBn

4 dRH

π-πRHOBn siRNA

31

5

HeLa Dual-Luciferase reporter assay system RHOBn siRNA3′ TT siRNA

Assay kit Dual-Glo Luciferase Assay System (Promega) Vector: psiCHECK-2

Transfection reagent: ScreenFect A Plus

Buffer: ScreenFect A Plus diluted transfection reagent

Conditions

Transfection time: 1 h

Incubation time: 24 h

Figure 4-4.

TT siRNARHOBnRHOBn siRNA

RHOBn

32

6

RHOBnRHOBn siRNA RHOBn

siRNA 50% 37 ºC siRNAPAGE GelRed

Conditions

Buffer: DMEM (Phenol Red free) Concentration: 3 μM


Figure 4-5. 50%

RHOBnRHOBn siRNA 12 20%TT RHRH

33

7 Argonaute2-PAZ

RNAi siRNA Ago2 PAZ(Ago2-PAZ) siRNA

RHOBn Ago2-PAZ

Ago2-PAZ pET15b (Figure 5-6)BL21 (DE3)

His-tag Ago2-PAZ 20)

Figure 4-6. Ago2-PAZ

BL21 (DE3) Ago2-PAZpET15b Talon Resin

Ago2-PAZSDS-PAGE MALDI-TOF/MS

Ago2-PAZ 1 L 0.2 mg

siRNA siRNA1 2Ago2-PAZ RHOBnRHOBn siRNA

19 21RNA (dsRNA) (Figure 5-7) Ago2-

PAZ 37 ºC 10 dsRNA

PAGE GelRed 3′ (TT) RNA

Figure 4-7. dsRNA

ColE1 origin

T7LacO

6xHis tag2Thrombin

hAgo2-PAZ.ape6146 bp

hAgo2/PAZ

AmpR

34

Figure 4-8. Ago2-PAZ dsRNA

TT RHOBnRHOBn dsRNA Ago2-PAZ

RHOBn Ago2-PAZRNAi

35

2 2 (RHOPg) RNA

1 RHOPg

siRNA

siRNA siRNAsiRNA

siRNA

2 (RHOPg) 3′ RHOPg Cu(I)

Cu-Catalyzed Azide Alkyne Cycloaddition (CuAAC) (Figure 4-9) siRNA Ago2-PAZ

RH 2 RHOBn 3′

CuAAC RHOBn

Figure 4-9. 3′ RHOPg CuAAC

2

3,5-O-TIPDS-RH (17) 3,5-O-TIPDS-RHOPg (20)

TBAF TIPDS RHOBn (21) 38% (Scheme 4-6)

36

Scheme 4-6. RHOPg

2 RHOPg RNA

RHOPg (21) 5 DMTr5-O-DMTr-RHOPg (22) THF DIPEA 2

23 (Scheme 4-7)

Scheme 4-7. RHOBn

3 dRH 23 Renilla luciferase mRNA siRNA 3′ RHOPg

(Figure 4-10)MALDI-TOF/MS

Figure 4-10.

37

3 RHOPg RNA CuAAC

(24) 4- (25) 1--1- -β- (26) 1- -1- -β- (27)1- (28) 5 3′ RHOPg

CuAAC (Figure 4-11) (24) (Scheme 4-8)

Figure 4-11.

Scheme 4-8.

4- (25) 4-

79% 28) 1- -1- -β- (26) 1- -1- -β- (27)

(30) 30 (III)β 26 29) 26

27 1- (28) 1-

2 78% 30)

38

3′ RHOPg

CuAAC 31) 3′ RHOPg

15 HPLCMALDI-TOF/MS

(24) (Retention time: 10 min, 12 min, 13 min) (Figure 5-12)

13

10 12

Figure 4-12. RHOPg 24 CuAAC

4- (25) (Retention time: 17 min, 25 min) (Figure 5-13)

25 17


39

1- -1- -β- (26) (Retention time: 11 min, 13.5 min, 15 min) (Figure 5-14)

1511 13.5


1- -1- -β- (27) 3′ RHOPg

(Figure 5-15)


40

1- (28) (Retention time: 11 min, 15 min, 17.5 min) (Figure 5-16)

17.511 15


3′ RHOPg

41

5

siRNA i) siRNAii) siRNA

iii) siRNA

RNAiRNAi

dRH siRNA

RH 2RHOBn siRNA

dRH

dTdRH

3′→5′

dT

siRNA HeLa dTRNAi

RH RHOBn

2RHOBn

RHOBn

50% RHOBn siRNARNAi dT siRNA

RHOBn siRNA

siRNA Ago2Ago2 PAZ (Ago2–PAZ)

Ago2–PAZsiRNA Ago2–PAZ dT RHOBn

42

CuAAC 2RH (RHOPg) 3′

CuAAC 3′

3′siRNA

RHOBn siRNA

siRNA Ago2-PAZ2 RH (RHOPg)

43

(

)

() (

)

( )

()

() ( )

44

1.

NMR JEOL JNM-ECS400 spectrometer JEOL JNM AL-400 spectrometer

DNA/RNA synthesizer Applied Biosystems Model 3400

NTS H-6

HITACHI U-2001 spectrophotometer SHIMADZU UV-2550 UV-VIS SPECTROPHOTOMETER

UV-2450 UV-VISIBLE SPECTROPHOTOMETER

WAKO SUNRISE TERMO

BERTHOLD TriStar LB941

MALDI/TOF-MS SHIMADZU AXIMA-CFR PLUS

GC/MS SHIMADZU GC-2010 GAS CHROMATOGRAPH

GCMS-QP2010 GAS CHROMATOGRAPH

MASS SPECTROMETER

HPLC SHIMADZU SPD-20A UV/VIS DETECTOR

CBM-20A COMMUNICATIONS BUS MODULE

CTO-20A COLUMN OVEN

DGU-20A3R DEGASSING UNIT

LC-20AT LIQUID CHGOMATOGRAPH

pH HORIBA F-52

FUJIFILM LAS-4000

2. NMR

1H NMR (CDCl3 : δ 7.26, DMSO-d6 : δ 2.49) ppm 13C NMR

(CDCl3 : δ 77.0, DMSO-d6 : δ 39.7) ppm 31P NMR H3PO4/CDCl3 (δ 0.00)

ppm

45

3.

3 1 Scheme 3-1

3′,5′-Di-O-(tert-butyldiphenylsilyl)thymidine (3)

Thymidine (2) (1.21 g, 5.00 mmol) imidazole (1.05 g, 15.4 mmol) DMF (20 mL) tert-buthyldiphenylsilyl chloride (3.9 mL, 15.0 mmol) 12

(n-hexane/EtOAc, 5:1~2:1) 3′,5′-O-di(tert-butyldiphenylsilyl)thymidine (3) (3.66 g, quant) 3,5-Di-O-(tert-butyldiphenylsilyl-1,2-didehydro-1,2-dideoxy-D-ribofuranose (4)

3′,5′-Di-O-(tert-butyldiphenylsilyl)thymidine (3) (7.73 g, 10.8 mmol) (NH4)2SO4 (2.27 g, 17.2 mmol) HMDS (78 mL) 150 °C 4

(n-hexane) 3,5-di-O-(tert-butyldiphenylsilyl-1,2-didehydro- 1,2-

dideoxy-D-ribofuranose (4) 5-O-(tert-butyldiphenylsilyl)methylfuran (5) (2.42 g, 38%)

Scheme 3-3 5′-O-(tert-Butyldiphenylsilyl)thymidine (6)

Thymidine (2) (486 mg, 2.01 mmol) DMAP (49.2 mg, 403 μmol) pyridine (10 mL) tert-buthyldiphenylsilyl chloride (600 μL, 2.34 mmol) 15

(n-hexane/EtOAc, 3:1~0:1)

5′-O-(tert-butyldiphenylsilyl)thymidine (6) (882 mg, 91%) 1H NMR (CDCl3) δ 9.25 (s, 1H, NH), 7.67–7.64 (m, 4H, H-Ph), 7.50 (d, J = 0.9 Hz, 1H, H-6), 7.47–7.37 (m, 6H, H-Ph), 6.43 (dd, J = 8.4 Hz, 5.8 Hz, 1H, H-1′), 4.58–4.56 (m, 1H, H-3′), 4.05–4.03 (m, 1H, H-4′), 3.98 (dd, J = 11.8 Hz, 2.7 Hz, 1H, H-5′α), 3.85 (dd, J = 11.8 Hz, 2.7 Hz, 1H, H-5β), 2.43 (ddd, J = 13.8 Hz, 5.8 Hz, 2.3 Hz, 1H, H-2′α), 2.20 (ddd, J = 13.8 Hz, 8.4 Hz, 5.8 Hz,1H, H-2′β), 1.62 (d, J = 0.9 Hz, 3H, CH3 of thymine), 1.08 (s, 9H, C(CH3)3); 13CNMR (CDCl3) δ 163.8, 150.5, 135.5, 135.3, 132.8, 132.3, 130.1, 130.0, 128.0, 127.9, 111.2, 87.1,

46

84.7, 72.3, 64.1, 41.0, 27.0, 19.3, 12.1; MS (DART) m/z 504 [M+Na]+, HRMS(DART) Calcd. for C26H32N2NaO5Si [M+Na]+: 503.6180. Found: 503.6163.

5-O-(tert-butyldiphenyl)-1,2-didehydro-1,2-dideoxy-3-O-trimethylsilyl-D- ribofuranose (7)

5′-O-(tert-Butyldiphenylsilyl)thymidine (6) (969 mg, 2.02 mmol) (NH4)2SO4 (536 mg, 4.06 mmol) HMDS (29 mL) 150 °C 2

(n-hexane) 5-O-(tert-butyldiphenylsilyl)methylfuran (5) 5-O-tert-butyldiphenylsilyl-1,2-didehydro-1,2-dideoxy-3-O- trimethylsilyl-D-

ribofuranose (7)

Scheme 3-4 5-O-(tert-butyldiphenyl)-1,2-didehydro-1,2-dideoxy-D-ribofuranose (8)

5′-O-(tert-Butyldiphenylsilyl)thymidine (6) (2.41 g, 5.01 mmol) (NH4)2SO4 (1.33 g, 10.0 mmol) HMDS (37 mL) 150 °C 1.5

K2CO3 (141 mg, 1.02 mmol) MeOH (40 mL)

12

(n-hexane/EtOAc, 5:1~2:1) 5-O-(tert-butyldiphenylsilyl)methylfuran (5) 5-O-tert-

butyldiphenylsilyl-1,2-didehydro-1,2-dideoxy-D-ribofuranose (8)

Scheme 3-5 5-O-(tert-butyldiphenyl)-1,2-dideoxy-D-ribofuranose (9)

5′-O-(tert-Butyldiphenylsilyl)thymidine (6) (969 mg, 2.02 mmol) (NH4)2SO4 (535 mg, 4.05 mmol) HMDS (14 mL) 150 °C 1.5

K2CO3 (8.0 mg, 57.9 μmol) MeOH (4 mL) 12

(n-hexane:EtOAc=50:1~20:1) 5-O-tert-butyldiphenyl-1,2-didehydro-1,2-dideoxy-D-ribofuranose (8) (621 mg, crude) 10% Pd/C (62.7 mg, 10 wt%) MeOH (4 mL)

47

12 (n-hexane/EtOAc, 50:1~10:1)

5-O-tert-butyldiphenyl-1,2-dideoxy-D-ribofuranose (9) (224 mg, 36%) 5-O-tert-butyldiphenylsilyl-2,3-dideoxy-1-O-methyl-D-

ribofuranose (10) (148 mg, 31%)


5′-O-(tert-Butyldiphenylsilyl)thymidine (6) (962 mg, 2.00 mmol) (NH4)2SO4 (530 mg,

4.01 mmol) HMDS (15 mL) 150 °C 1.5

K2CO3 (55.3 mg, 400 μmol) MeOH (17 mL) 12

10% Pd/C (32.0 mg, 5 wt%) i-PrOH (4 mL) 12

(n-hexane/EtOAc, 10:1~3:1) 5-O-tert-butyldiphenyl-1,2-dideoxy-D-ribofuranose (9) (499 mg, 70%) 1H NMR (CDCl3) δ 7.70–7.66 (m, 4H, H-Ph), 7.46–7.37 (m, 6H, H-Ph), 4.43–4.40 (m, 1H, H-3),

3.96 (dd, J = 8.4 Hz, 5.7 Hz, 2H, H-5), 3.85–3.82 (m, 1H, H-4), 3.78 (dd, J = 10.7 Hz, 4.3 Hz, 1H, H-1α), 3.60 (dd, J = 10.7 Hz, 6.4 Hz, 1H, H-1β), 2.20–2.10 (m, 1H, H-2α), 1.93–1.86 (m, 1H, H-2β), 1.07 (s, 9H, C(CH3)3); 13C NMR (CDCl3) δ 135.6, 135.5, 133.2, 133.1, 129.8, 129.8, 127.7, 86.1, 77.2, 74.3, 67.1, 64.8, 34.8, 26.8, 19.2; MS (DART) m/z 379 [M+Na]+, HRMS (DART) Calcd. for C21H28NaO3Si [M+Na]+: 379.1705. Found: 379.1750.


5′-O-(tert-Butyldiphenylsilyl)thymidine (6) (482 mg, 1.00 mmol) ammonium sulfate (265 mg, 2.01 mmol) HMDS (8 mL) 150 °C 1.5

K2CO3 (28.5 mg, 206 μmol) MeOH (15 mL) 2

(n-hexane/Et2O, 3:1~2:1) 5-O-tert-butyldiphenyl-1,2-didehydro-1,2-dideoxy-D-ribofuranose (8) (340 mg, mixture) 10% Pd/C (53.6 mg, 15 wt%) i-PrOH (10 mL)

48

24 Pd/C (n-hexane/EtOAc,

7:1~2:1) 5-O-(tert-butyldiphenyl)-1,2-dideoxy-D-ribofuranose (9) (338 mg, 94%)

Scheme 3-8 1,2-Dideoxy-D-ribofuranose (1)

5-O-(tert-butyldiphenyl)-1,2-dideoxy-D-ribofuranose (9) (215 mg, 603 μmol) THF (2.7 mL) 1 M TBAF in THF (660 μL, 660 μmol) 12

(CH2Cl2/acetone, 5:1~2:1) 1,2-dideoxy-D-ribofuranose (1) (68.8 mg, 97%)

1H NMR (CD3OD) δ 4.21–4.18 (m, 1H, H-3), 3.97–3.87 (m, 2H, H-1), 3.75–3.72 (m, 1H, H-4), 3.57–3.49 (m, 2H, H-5), 2.18–2.03 (m, 1H, H-2α), 1.88–1.81 (m, 1H, H-2β); 13C NMR (CD3OD) δ 88.1, 73.8, 68.0, 63.6, 35.9; MS (DART) m/z 141 [M+Na]+, HRMS (DART) Calcd. for C5H10NaO3 [M+Na]+: 141.1209. Found: 141.1250.

3 2 Scheme 3-10

1,2-Dideoxy-5-O-(4,4′-dimethoxytrityl)-D-ribofuranose (11) 1,2-Dideoxy-D-ribofuranose (1) (101 mg, 853 μmol) DMTrCl (376 mg, 1.11 mmol)

pyridine (3.3 mL) 12

(CH2Cl2/acetone, 20:1~10:1) 1,2-dideoxy-5-O-(4,4′-dimethoxytrityl)-D-ribofranose (11) (265 mg, quant) 1H NMR (400 MHz, CDCl3) δ 7.43 (d, J = 7.8 Hz, 2H, H-Ph), 7.36–7.16 (m, 7H, H-Ph), 6.82 (d, J = 9.2 Hz, 4H, H-Ph), 4.30–4.27 (m, 1H, H-3), 3.99–3.96 (m, 2H, H-1), 3.90–3.86 (m, 1H, H-4), 3.79 (s, 6H, OCH3), 3.23 (dd, J = 9.8 Hz, 4.8 Hz, 1H, H-5α), 3.07 (dd, J = 9.8 Hz, 6.2 Hz, 1H, H-5β), 2.19–2.10 (m,

1H, H-2α), 1.92–1.84 (m, 1H, H-2β); 13CNMR (CDCl3) δ 158.4, 144.8, 136.0, 136.0, 130.0, 128.1, 127.8, 126.8, 113.1, 86.2, 85.0, 74.6, 67.0, 64.5, 55.2, 34.8; MS (EI) m/z 420 (M+)

3-O-[(2-Cyanoethoxy)(N,N-diisopropylamino)phosphanyl]-1,2-dideoxy-5-O-(4,4′- dimethoxytrityl)-D-ribofuranose (12)

1,2-Dideoxy-5-O-(4,4′-dimethoxytrityl)-D-ribofuranose (11) (292 mg, 694 μmol) THFDIPEA (730 μL, 4.18 mmol) 2-cyanoethyldiisopropylchloro

49

phosphoamidite (250 μL, 1.12 mmol) 2

(n-hexane/EtOAc, 4:1) 3-O-[(2-Cyanoethoxy)(N,N-diisopropylamino)phosphanyl]-1,2-dideoxy-5-O-(4,4′-dimethoxytrityl)-D-ribofuranose (12) (451 mg, quant) 31P NMR (160 MHz, CDCl3) δ 148.3, 148.6.

dRH (14)

1,2-Dideoxy-5-O-(4,4′-dimethoxytrityl)-D-ribofuranose (11) (102 mg, 243 μmol) pyridine

(2.4 mL) DMAP (30.4 mg, 249 μmol) succinic anhydride (112 mg, 1.12 mmol) 23

DMF (5.3 mL) CPG (546 mg, 53.5 μmol) EDC·HCl (41.6 mg, 217 μmol) 72

(20 mL) 100 mM DMAP [DMAP (370 mg, 3.03 mmol), acetic anhydride (3 mL), pyridine (27 mL)] 72

(20 mL) (20 mL) (20 mL) dRH

(14) (479 mg) 14 (6.4 mg) / (3:1)

25 mL 25 mL ( :54.5 μmol/g)

(μmol/g) = Abs498 ∙ Vol (HClO4) ∙ ε498 ∙ Weight (mg)-1

dRH dRH

(Applied Biosystems Model 3400) 1 μmol 100 mM dRH 12 150 mM

MeCN dRH (14) 1 μmol (18.3 mg)

/ (3:1) (1.2 mL) 12

1.5 mL / (3:1) TBAF (1 M solution in THF, 1 mL) 12 100 mM TEAA *1

buffer 30 mL (15 mL) 50% CH3CN in

50

(4 mL) loading solution *2 (180 μL) 20% PAGE*3 (500 V, 20 mA)

*4 (20 mL) Sep-Pak (15 mL)

50% CH3CN in (3 mL)

*1 2 M TEAA buffer [ (114.38 mL) (277.6 mL) (Milli-Q ) 1 L mess up pH 7.0 ] 20 *2 Formamide (9 mL) 1 × TBE buffer*5 (1 mL) *3 40% (19 : 1) *6 (40 mL) (33.6 g) 10 × TBE buffer*5 (8 mL)

Milli-Q 80 mL mess up ammonium peroxodisulfate (55 mg) TEMED (40 μL) 1.5 mm

2 1 1 × TBE buffer*5 *4 2 M TEAA buffer (1 mL) 0.1 M EDTA [EDTA-4Na (1.81 g) 40 mLmess up ] (0.2 mL) 20 mL mess up *5 10 × TBE buffer [Tris(hydroxymethyl)aminomethane (108 g) (55 g) EDTA-4Na (7.43 g) 1 L ] 10 *6 (190 g) N,N′- (10 g) Milli-Q 500

mL

HPLC

A : 5% /0.1 M TEAA (pH 7.0) B : 50% /0.1 M TEAA (pH 7.0)

1 mL/min

260 nm

Time ( min. ) B solution

0 0

5 10

20 30

30 100

35 10

40 0

51

UV OD260 260 nm (Abs260)

(0.2-1.0) 100 ( l ) 1 cmAbs260 OD260 V

OD260 (Mε-1mL-1cm-1) = Abs260 (Mε-1) · V -1 (mL) · l -1 (cm-1)

Sequence OD260

5′-r(gua gga gua gug aaa ggc c dRHdRH)-3′ 0.147

5’-r(ggc cuu uca cua cuc cua c dRHdRH)-3′ 0.145

5′-F-r(gua gga gua gug aaa ggc c dRHdRH)-3′ 0.251

ε N1pN2pN3p···Nn-1pNn ε260

ε = 2 { ε (N1pN2) +ε (N2pN3) + ···+ε (Nn-1pNn) } - { ε (N2) +ε (N3) + ···+ε (Nn-1) } ε (Nn) Nn ε260 ε (Nn-1pNn) Nn-1pNn ε260

C ( mol/L )

C = Abs260 ·ε260 -1 · 1-1

(1 mL) 260 nm30 pmol (3 mL)

(3 μL) MALDI-TOF/MS

Sequence Calculated Observed

5′-r(gua gga gua gug aaa ggc c dRHdRH)-3′ 6559.9 6559.8

5′-r(ggc cuu uca cua cuc cua c dRHdRH)-3′ 6250.8 6251.0

5′-F-r(gua gga gua gug aaa ggc c dRHdRH)-3′ 7097.1 7096.6

52

3 3

5′ (Renilla luciferasesiRNA ) 300 pmol 1.5 mL

PBS (-) (1 unit/mL, 100 μL) 37 °C 5 min, 10 min, 15 min, 30 min,

60 min 1.5 mL (, 15 μL) 5 μL 100 °C 10

0 min (control)

20%PAGE (Urea) 500 V 20 mA

3 4

600 pmol (10 mM NaH2PO4/Na2HPO4, 100 mM NaCl (pH 7.0)) (200 μL) 3

μM 5 13 160 μL 50% (Tm) 260 nm

Tm

3 5

96 well plate HeLa 40000 cell/mL 100 μL24 h siRNA TE buffer (pH7.0, 100 mM Nacl)

24 siRNA (1.75 nM, 17.5 nM, 175 nM, 10 μL) psi-CHECK (0.1 μg/μL, 1 μL) transfast (1.0 μg/μL, 1.5 μL) Opti-MEM (115 μL) 1.5 mL

96 well plate well

(35 μL) 1 100 μL 24 -80 °C ( ) 12 96 well plate

well Dual glo substrate (25 μL) 10 96 well plate Firefly luciferase Stop and glo substrate (25 μL)

10 Renilla luciferase Renilla luciferaseFirefly luciferase % of control

53

3 6

D-MEM (50 μL, Phenol red ) 300 pmol (50 μL) 1.5 mLBS (50 μL) 37 °C

(1, 3, 6, 12, 24) 600 μL ( , 15 μL) 5 μL -80 °C ( )

20% Native PAGE 500 V 20 mA GelRedLAS-4000 BS

4 1 1 Scheme 4-2 3,5-O-(1,1,3,3-Tetraisopropyl-1,3-disiloxanediyl)-1-deoxy-D-ribofuranose (17)

1-Deoxy-D-ribofuranose (16) (1.34 g, 11.0 mmol) imidazole (2.74 g, 40.2 mmol) DMF (15 mL) 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (3.47 g, 11.0 mmol)

DMF (15 mL) 18

(n-hexane/EtOAc, 10:1)

3,5-O-(1,1,3,3-tetraisopropyl-1,3-disiloxanediyl)-1-deoxy-D-ribofuranose (17) (3.07 g, 82%) 1H NMR (CDCl3) δ 4.25–4.18 (m, 2H, H-1α, H-2), 4.08 (dd, J = 10.0 Hz, 4.4 Hz, 1H, H-1β), 4.01 (dd, J = 12.4 Hz, 3.6 Hz, 1H, H-4), 3.89 (dd, J = 12.4 Hz, 5.6 Hz, 1H, H-3), 3.82–3.77 (m, 2H, H-5), 2.85 (s, 1H, OH), 1.09–0.95 (m, 28H, CH(CH3)2 of TIPDS); 13C NMR (CDCl3) δ 80.7, 73.5, 73.0, 71.0,62.5, 17.4, 17.3, 17.2, 17.0, 17.0, 13.4, 13.1, 12.8, 12.6; MS (ESI) m/z 399 [M+Na]+,HRMS (ESI) Calcd. for C17H36NaO5Si2 [M+Na]+: 399.1999. Found: 399.1961.

Scheme 4-3 2-O-Benzyl-1-deoxy-D-ribofuranose (15)

3,5-O-(1,1,3,3-Tetraisopropyl-1,3-disiloxanediyl)-1-deoxy-D-ribofuranose (17) (1.49 g, 3.96 mmol) DMF (20 mL) 60% NaH (338 mg, 8.45 mmol) benzyl bromide (0.8 mL, 6.74 mmol) 4.5

THF (10 mL) TBAF (1 M solution in THF, 15 mL) 19

(CH2Cl2/acetone, 30:1~2:1)

54

2-O-benzyl-1-deoxy-D-ribofuranose (15) (237 mg, 27%) 1H NMR (DMSO-d6) δ 7.37–7.25 (m, 5H, H-Ph), 4.84–4.76 (m, 1H,

3-OH), 4.68–4.61 (m, 2H, OCH2αPh, 5-OH), 4.54–4.46 (m, 1H, OCH2βPh), 3.97–3.82 (m, 3H, H-1, H-3), 3.66–3.64 (m, 1H, H-2), 3.57–3.34 (m, 3H, H-4, H-5); 13C NMR (CDCl3) δ 137.2, 128.7, 128.2, 127.9, 83.5, 78.2, 72.4, 71.3, 70.5, 62.5; MS (ESI) m/z 247 [M+Na]+, HRMS (ESI) Calcd. for C12H16NaO4 [M+Na]+: 247.0946. Found: 247.0927. Scheme 4-4 2-O-Benzyl-1-deoxy-D-ribofuranose (15)

3,5-O-(1,1,3,3-Tetraisopropyl-1,3-disiloxanediyl)-1-deoxy-D-ribofuranose (17) (62.1 mg, 165 μmol) CH3NO2 (1 mL) benzaldehyde (14 μL, 137 μmol) Et3SiH (26 μL, 163 μmol) -20 ºC FeCl3 (1.1 mg, 7 μmol) CH3NO2 (70 μL)

41 Et3N·3HF (100 μL, 613 μmol) 4 (CH2Cl2/acetone, 50:1~3:1)

2-O-benzyl-1-deoxy-D-ribofuranose (15) (14.5 mg, 47%)

4 1 2 Scheme 4-5 2-O-Benzyl-1-deoxy-5-O-(4,4′-dimethoxytrityl)-D-ribofuranose (19)

2-O-Benzyl-1-deoxy-D-ribofuranose (15) (218 mg, 972 μmol) pyridine (5 mL) 4,4′-dimethoxytrityl cloride (401 mg, 1.18 mmol) pyridine (5 mL)

14

(CH2Cl2/acetone, 40:1~20:1) 2-O-benzyl-1-deoxy-5-O- (4,4′-dimethoxytrityl)-D-ribofuranose (19) (499 mg, 65%) 1H NMR (CDCl3) δ 7.43 (d, J = 7.4 Hz, 2H, CH of DMTr), 7.39–7.17 (m, 7H, CH of DMTr), 6.81 (d, J = 7.4 Hz, 4H, CH of

DMTr), 4.63 (dd, J = 24.8 Hz, 11.6 Hz, 2H, OCH2Ph), 4.18–4.06 (m, 3H, H-1α, H-2, H-3), 3.98–3.95 (m, 1H, H-4), 3.90 (dd, J = 9.6 Hz, 4.6 Hz, 1H, H-1β), 3.78 (s, 6H, OCH3), 3.32 (dd, J = 10.1 Hz, 3.4 Hz, 1H, H-5α), 3.11 (dd, J = 10.1 Hz, 4.4 Hz, 1H, H-5β), 2.65 (d, J = 6.4 Hz, 1H, OH); 13C NMR (CD3OD) δ 158.4, 144.9, 137.3, 136.1, 136.0, 130.1, 128.6, 128.2, 128.1, 127.9, 127.8, 126.7, 113.0, 86.0, 83.3, 78.2, 77.2, 72.3, 72.3, 70.1, 64.3, 55.2; MS (ESI) m/z 549 [M+Na]+, HRMS (ESI) Calcd. for C33H34NaO6 [M+Na]+: 549.2253. Found: 549.2270.

55

2-O-Benzyl-3-O-[(2-Cyanoethoxy)(N,N-diisopropylamino)phosphanyl]-1-dideoxy- 5-O-(4,4′-dimethoxytrityl)-D-ribofuranose (20)

2-O-Benzyl-1-deoxy-5-O-(4,4′-dimethoxytrityl)-D-ribofuranose (19) (196 mg, 372 μmol) THF (2 mL) DIPEA (450 μL, 2.65 mmol) 2-cyanoethyl diisopropylchlorophosphoamidite (200 μL, 897 μmol) 3

(n-hexane/EtOAc, 3:1) 2-O-benzyl-3-O-[(2-cyanoethoxy)(N,N-diisopropylamino)phosphanyl]-1-deoxy-5-O-(4,4′-dimethoxytrityl)-

D-ribofuranose (20) (262 mg, 97%) 31P NMR (160 MHz, CDCl3) δ 149.6, 149.1.

RH

OBn RHOBn

(NTS H-6) 1 μmol 50 mM RHOBn 20 75 mM MeCN

/40% (1:1) (1.2 mL) 60 ºC 30

1.5 mL/ (3:1) DMSO (100 μL)

Et3N·3HF (125 μL) 65 ºC 2.53 M (25 μL) 15 (1 mL) 30 -80 ºC 15000 rpm 10 min 4 ºC

(750 μL) loading solution (200 μL) 20% PAGE*3 (500 V, 20 mA)

(20 mL) Sep-Pak (15 mL) 50% CH3CN in (3 mL)

56

UV OD260 3 2 OD260


Sequence OD260

5′-r(gua gga gua gug aaa ggc c RHOBnRHOBn)-3′ 0.373

5’-r(ggc cuu uca cua cuc cua c RHOBnRHOBn)-3′ 0.238

5′-F-r(gua gga gua gug aaa ggc c RHOBnRHOBn)-3′ 0.322

ε

3 2

3 2 MALDI-TOF/MS


5′-r(gua gga gua gug aaa ggc c RHOBnRHOBn)-3′ 6772.0 6772.8

5’-r(ggc cuu uca cua cuc cua c RHOBnRHOBn)-3′ 6462.9 6463.0

5′-F-r(gua gga gua gug aaa ggc c RHOBnRHOBn)-3′ 7309.1 7309.1

5 1 3

3 3

5 1 4

3 4

5 1 5

3 5

57

4 1 6

3 6

4 1 7 Argonaute2-PAZ

hAgo2-PAZ LB/Amp (20 mL) Hs Argonaute 2 PAZ

BL21(DE3) 37 C 2 LB/Amp (1 L) 10 mL 37˚C 180 rpm OD600 = 0.8

1M IPTG (1 mL) 20˚C 180 rpm 6 4˚C 6000 rpm 10 Buffer A [20 mM Tris-

HCl (pH 8.0), 500 mM NaCl, 5 mM imidazole, 1 mM phenylmethylsulfonyl fluoride] 5 sec×20 4˚C 10000 rpm

15 Buffer ATALON® Metal Affinity Resin Buffer A (5 bet ) Wash Buffer [20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 10 mM imidazole, 1 mM phenylmethylsulfonyl fluoride] (5 bet )

Elution Buffer [20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 200 mM imidazole, 1 mM phenylmethylsulfonyl fluoride] (5 bet )

(30 kDa, 10 kDa) 100 μLSDS-PAGE

30% (3.6 mL) 1.5 M Tris-HCl pH 8.8/0.4% SDS (1.8 mL) Milli-Q (1.8 mL) 10% APS (40 μL) TEMED (6 μL) 15%

30% (252 μL) 0.5 M Tris-HCl (pH 6.8)/0.4% SDS (420 μL) Milli-Q (1 mL) 10% APS (8 μL) TEMED (4 μL)

Loading dye 100˚C10 (500 V, 20 mA) 1

Running Buffer10 CBB 30 MilliQ

LAS-4000

hAgo2–PAZ 1.5 mL dsRNA (10 μM, 1μL) (20 mM

Tris-HCl, 100 mM NaCl, 14 μL) hAgo2-PAZ (10 μM,

58

5 μL) 37 ºC 10 50% (20 μL) 10 10% native

PAGE (500 V, 21 mA) 30 GelRed 15 LAS-4000

4 2 1 Scheme 4-6 2-O-Propargyl-1-deoxy-D-ribofuranose (21)

3,5-O-(1,1,3,3-Tetraisopropyl-1,3-disiloxanediyl)-1-deoxy-D-ribofuranose (17) (1.91 g, 5.07 mmol) DMF (25 mL) 60% NaH (223 mg, 5.58 mmol) propargyl bromide (500 μL, 6.60 mmol) 3

THF (10 mL) TBAF (1 M solution in THF, 15 mL) 1

(CHCl3/MeOH, 50:1~10:1) 2-O-propargyl-1-deoxy-D-ribofuranose (21) (330 mg, 38%)

1H NMR (CDCl3) δ 4.38–3.65 (m, 5H), 3.92–3.65 (m, 3H), 3.74–3.65 (m, 1H), 2.72–2.66 (m, 1H), 2.58 (d, 1H), 2.50 (t, 1H); 13C NMR (CDCl3) δ 83.4, 78.2, 75.4, 71.3,

62.3, 58.8, 57.7.

4 2 2

Scheme 4-7 2-O-Propargyl-1-deoxy-5-O-(4,4′-dimethoxytrityl)-D-ribofuranose (22)

2-O-Propargyl-1-deoxy-D-ribofuranose (21) (259 mg, 1.50 mmol) pyridine (5 mL) 4,4′-dimethoxytrityl chloride (611 mg, 1.80 mmol) pyridine (5 mL)

14

(n-

hexane/EtOAc, 5:1~2:1) 2-O-propargyl-1-deoxy-5-O-(4,4′-dimethoxytrityl)-D-ribofuranose (22) (316 mg, 44%) 1H NMR (CDCl3) δ 7.46–7.18 (m, 9H), 6.89–6.81 (m, 4H), 4.35–4.23 (m, 2H), 4.19–4.07 (m, 2H), 3.97–3.88 (m, 2H), 3.80–3.79 (m, 7H), 3.35–3.12 (m, 2H), 2.52–2.48 (m, 2H); 13C NMR (CDCl3) δ 158.4, 144.9, 136.1, 136.0, 130.1, 128.2, 127.8, 126.7, 113.5, 113.1, 86.1, 83.1, 79.1, 78.3, 75.2, 72.3, 69.9, 64.3, 57.7, 55.2. MS (ESI) m/z 497 [M+Na]+, HRMS (ESI) Calcd. for C29H30NaO6 [M+Na]+ : 497.1940. Found 497.1918.

59

2-O-Propargyl-3-O-[(2-Cyanoethoxy)(N,N-diisopropylamino)phosphanyl]-1-dideoxy- 5-O-(4,4′-dimethoxytrityl)-D-ribofuranose (23)

2-O-Propargyl-1-deoxy-5-O-(4,4′-dimethoxytrityl)-D-ribofuranose (22) (196 mg, 372 μmol) THF (2 mL) DIPEA (0.4 mL, 2.35 mmol) 2-cyanoethyl

diisopropylchlorophosphoamidite (200 μL, 897 μmol) 1

(n-hexane/EtOAc, 5:1~2:1) 2-O- propargyl-3-O-[(2-cyanoethoxy)(N,N-diisopropylamino)phosphanyl]-1-dideoxy-5-O-

(4,4′dimethoxytrityl)-D-ribofuranose (23) (200 mg, 77%) 31P NMR (160 MHz, CDCl3) δ 150.00, 149.86.

RHOPg

RHOPg (NTS H-6) 1 μmol

50 mM RHOPg 23 75 mM MeCN5 2

UV OD260 260 nm (Abs260)

(0.2-1.0) 100 ( l ) 1 cmAbs260 OD260 V


Sequence OD260

5′-r(gua gga gua gug aaa ggc c RHOPg)-3′ 0.371

ε

3 2

3 2 MALDI-TOF/MS


5′-r(gua gga gua gug aaa ggc c RHOPg)-3′ 6433.9 6433.6

60

4 2 3 Scheme 4-8 4-Fluorobenzyl azide (25)

4-Fluorobenzyl chloride (520 mg, 3.60 mmol) DMF (7 mL) NaI (55.1 mg, 368 μmol) NaN3 (465 mg, 7.15 mmol) 60 ºC 3

(n-hexane/CH2Cl2, 50:1~20:1) 4-fluorobenzyl azide (25) (431 mg, 79%) 1H NMR (CDCl3) δ 7.32–

7.27 (m, 2H), 7.10–7.05 (m, 2H), 7.50–7.44 (m, 2H), 4.32 (s, 2H); 13C NMR (CDCl3) δ 162.6 (d, J = 245 Hz), 131.1, 130.0 (d, J = 7.6 Hz), 115.8 (d, J = 21.9 Hz), 54.0.

1-Azido-1-deoxy-2,3,4,6-tetra-O-acetyl-β-D-glucopyranose (26) Penta-O-acetyl-β-D-glucopyranose (30) (783 mg, 3.01 mmol) CH2Cl2 (10 mL)

FeCl3 (36.8 mg, 227 μmol) 5 TMSN3 (400 μL, 3.04 mmol) 2

(n-hexane/EtOAc, 5:1~2:1) 1-azido-1-deoxy-2,3,4,6-tetra-O-acetyl-β-D-

glucopyranose (26) (700 mg, 94%) 1H NMR (CDCl3) δ 5.22 (t, J = 9.6 Hz, 1H), 5.11 (t, J = 9.6 Hz, 1H), 4.65 (d, J = 8.4 Hz, 1H), 4.28 (dd, J = 12.4, 4.8 Hz, 1H), 4.17 (dd, J = 12.8, 2.4 Hz, 1H), 3.79 (ddd, J = 7.4, 5.0, 2.2 Hz, 1H), 3.82–3.77 (m, 1H), 2.10 (s, 3H), 2.08 (s, 3H), 2.03 (s, 3H), 2.01 (s, 3H); 13C NMR (CDCl3) δ 170.6, 170.1, 169.3, 169.2, 87.9, 74.0, 72.6, 70.6, 67.8, 61.6, 20.7, 20.5.

1-Azido-1-deoxy-β-D-glucopyranose (27) 1-Azido-1-deoxy-2,3,4,6-tetra-O-acetyl-β-D-glucopyranose (26) (61.7 mg, 165 μmol)

9.4 mM (18 mL) 2 1-azido-1-deoxy-β-D-

glucopyranose (27) (29.3 mg, 86%) 1H NMR (DMSO-d6) δ 4.90 (d, J = 4.9 Hz, 1H), 4.87 (d, J = 4.9 Hz, 1H), 4.85 (d, J = 4.9 Hz, 1H), 4.08 (d, J = 4.1 Hz, 1H), 3.77–3.63 (m, 2H), 3.43–3.39 (m, 2H), 3.37–3.33 (m, 2H), 2.94–2.91 (m, 1H).

1-Naphtalenemethylazide (22)

1–Naphthaldehyde (31) (510 mg, 3.27 mmol) MeOH (10 mL) NaBH4 (269 mg, 7.11 mmol) 2

61

DMF (10 mL) CBr4 (2.18 g, 6.57 mmol) PPh3 (1.72 g, 6.56 mmol) 5NaN3 (429 mg, 6.60 mmol) 14

(n-hexane/CH2Cl2, 50:1~20:1) 1-naphtalenemethylazide (28) (465 mg, 78%) 1H NMR (CDCl3) δ 8.04 (d, J = 8.8 Hz, 2H), 7.92–7.85 (m, 2H), 7.61–7.52 (m, 2H), 7.50–7.44 (m, 2H), 4.78 (s,

2H); 13C NMR (CDCl3) δ 133.9, 131.3, 130.9, 129.4, 128.8, 127.3, 126.7, 126.2, 125.2, 123.5, 53.0.

RHOPg CuAAC

CuAAC 1.5 mL 3′ RHOPg (100 μM

solution in Milli-Q , 20 μL) (10 mM solution in DMSO, 1 μL) (1M solution in Milli-Q , pH 7.0) CH3CN (1 μL)

CuSO4•5H2O (100 mM solution in Milli-Q , 1 μL) Na ascorbate (100 mM solution in Milli-Q , 1 μL) 15

Milli-Q (300 μL) (TOSMIC, 0.2 μm PVDF) HPLC

A : 5% /0.1 M TEAA (pH 7.0) B : 50% /0.1 M TEAA (pH 7.0)

1 mL/min

260 nm

Time ( min. ) B solution

0 5

5 5

30 10

35 30

40 100

62

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Shaikh, S.; Zhang, L.; van der Sluis, J. R.; Jung, E. M.; Akinc, A.; Hutabarat, R.; Kuchimanchi, S.; Fitzgerald, K.; Zimmermann, T.; van Berkel, J. C. T.; Maier, A. M.; Rajeev, G. K.; Manoharan, M. J. Am. Chem. Soc. 2014, 136, 16958–16961.

11. Deleavay, F. G.; Damha, J. M. Chem. Biol. 2012, 19, 937–954. 12. Ueno, Y.; Inoue, T.; Yoshida, M.; Yoshikawa, K.; Shibata, A.; Kitamura, Y.; Kitade, Y.

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1. Practical and Reliable Synthesis of 1,2-Dideoxy-D-ribofuranose and its Application in RNAi

Studies

Yuki Nagaya, Yoshiaki Kitamura, Remi Nakashima, Aya Shibata, Masato Ikeda,

Yukio Kitade.

Nucleosides Nucleotides Nucleic Acids. 2016, 35, 64−75.

2. Introduction of 2-O-benzyl abasic nucleosides to the 3′-overhang regions of siRNAs greatly improves nuclease resistance

Yuki Nagaya, Yoshiaki Kitamura, Aya Shibata, Masato Ikeda, Yukihiro Akao, Yukio Kitade.

Bioorg. Med. Chem. Lett. 2017, 27, 5454−5456.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS, VOL. , NO. , –http://dx.doi.org/./..

Practical and Reliable Synthesis of,-Dideoxy-D-ribofuranose and its Application in RNAiStudies

Yuki Nagayaa, Yoshiaki Kitamurab,c, Remi Nakashimab, Aya Shibatab,c,Masato Ikedaa,b,c, and Yukio Kitadea,b,c

aUnited Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, -Yanagido, Gifu, Japan; bDepartment of Biomolecular Science, Graduate School of Engineering, GifuUniversity, - Yanagido, Gifu, Japan; cDepartment of Chemistry and Biomolecular Science, Faculty ofEngineering, Gifu University, - Yanagido, Gifu, Japan

ARTICLE HISTORYReceived May Accepted October

KEYWORDSAbasic nucleoside; glycal;oligonucleotide; siRNA; RNAi

ABSTRACTWedeveloped a practical and reliablemethod for synthesizing anabasic deoxyribonucleoside, 1,2-dideoxy-D-ribofuranose (dRH) viaelimination of nucleobase from thymidine. To synthesize oligonu-cleotides bearing dRH by the standard phosphoramidite solid-phase method, dRH was converted to the corresponding phos-phoramidite derivative and linked to a solid support (controlledpore glass resin). Chemically modified small interfering RNAs (siR-NAs) possessing dRH at their 3′-overhang regions were synthe-sized. Introducing dRH to the 3′-end of the antisense strand ofsiRNA reduced its knockdown effect.

Introduction

The abasic deoxyribonucleoside 1,2-dideoxy-d-ribofuranose (dRH; 1) is a struc-tural analogue of sugar residue at natural apurinic/apyrimidinic (AP) sites, whichresults from the hydrolysis of the N-glycosidic bond of nucleotides in DNA. Sev-eral oligonucleotides (ONs) containing dRH have been synthesized to study DNArepair mechanisms[1–5], catalytic activity of ribozymes[6–9], and RNA interference-based therapy.[10,11] An AP site containing dRH provides a unique hydrophobicbinding pocket in DNA duplex.[12] Several researchers have developed functionalprobes bearing dRH by exploiting its chemical properties.[12–14] In addition, Koolet al. reported various oligodeoxy-fluoroside sensors incorporating dRH as a non-fluorescent spacer,[15,16] and dRH is a well-known building block for synthesizingcarbapenem antibiotics.[17,18]

Several synthetic approaches to dRH have been developed (see Figure 1), includ-ing the reduction of 2-deoxy-d-ribose (a)[2,19] or its furanosyl derivatives, such asfuranosyl chloride (b),[1,20,21] methyl furanoside (b),[1,22,23] thiofuranoside (c),[24]

CONTACT Yukio Kitade [email protected] . Department of Chemistry and Biomolecular Science, Faculty ofEngineering, Gifu University, - Yanagido, Gifu -, Japan.© Taylor & Francis Group, LLC

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NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS 65

Figure . Approaches for the synthesis of dRH.

and furanose erythro-glycals (furanoid glycals) (d).[25,26] However, most of theexisting protocols are not practical. Chenault et al. synthesized dRH from 2-deoxy-d-ribose (a) in high yield, although an excess of ion exchange resin is required toobtain dRH.[23] Beigelman and co-workers reported a simple synthetic method fordRH precursors via a furanoid glycal, but preparation of the furanoid glycal waschallenging and derivation into dRH was not reported.[27] Ferrero et al. describeda biocatalytic procedure for preparing dRH precursors from readily available mate-rials that is useful in ON synthesis, but the protocol requires multiple steps.[28] Tostudy several ONs containing dRH for ON-based therapeutics, a practical and reli-able synthesis of dRH is needed.

Here we describe a practical and reliable procedure for synthesizing dRH (1)from 5′-O-silylated thymidine via the corresponding furanoid glycal by elimina-tion of nucleobase (Scheme 1). We also studied the biological properties of smallinterfering RNAs (siRNAs) bearing dRH dimer instead of the natural nucleotides atthe 3′-overhang region of siRNAs.

Results and discussion

Synthesis of 1,2-dideoxy-D-ribofuranose and its derivatives

First, 5′-O-(tert-butyldiphenylsilyl)thymidine (2),[25] which can be easily preparedfrom thymidine, was reacted with ammonium sulfate ((NH4)2SO4) in 1,1,1,3,3,3-hexamethyldisilazane (HMDS) under reflux to give the corresponding 3,5-O-disilylglycal 3. Although Pedersen’s protocol[25] gave the corresponding furanderivative 3′[29] as a major product rather than the desired glycal 3, optimizationof concentration, amount of (NH4)2SO4, and reaction time provided the desiredglycal 3 almost quantitatively. Subsequently, the trimethylsilyl (TMS) group at the3-positionwas removedwith potassium carbonate to produce 5-mono-silylated gly-cal 4. Han and Lowary reported obtaining 5 in 87% yield from 4with Pd/C-catalyzedhydrogenation in MeOH;[30] in contrast, we found that hydrogenation of olefin in4 under the above conditions gave the desired 5-O-TBDPS-dRH 5 in 36% yield,along with a tetrahydrofuran derivative 5′[29] (31%) produced from the Ferrierrearrangement[31] followed by hydrogenation of resulting olefin. We could

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66 Y. NAGAYA ET AL.

completely suppress the side reaction by using i-PrOH, a bulkier alcohol as sol-vent, and could isolate 5 (95% yield from 2) as a sole product. Finally, desi-lylation by treatment with tetra-n-butylammonium fluoride (TBAF) provided 1quantitatively.

For incorporation of 1 into ONs by the standard phosphoramidite solid-phase method, 1 was converted to the corresponding phosphoramidite deriva-tive 7 and the solid support 8 on controlled pore glass (CPG). Treatment of1 with 4,4′-dimethoxytrityl (DMTr) chloride in DMF/pyridine gave the corre-sponding 5-DMTr derivative 6, which was phosphorylated with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite to produce 7 quantitatively. Compound 6 wassuccinated, and the resulting succinate was linked with CPG to generate solid sup-port 8 (54.5 μmol/g).

RNA interfering (RNAi) study

Small interfering RNAs inhibit gene suppression by RNAi and thus hold greatpromise as nucleic acid drugs. We have reported the synthesis and biological prop-erties of siRNAs chemically modified at their 3′-overhang regions.[32–37] In an RNAistudy, an overhang of two thymidine residues (dTdT) at the 3′-end of siRNA is gener-ally used for nuclease protection. Here we synthesized siRNA incorporating an aba-sic deoxyribonucleoside (dRH) dimer at this position to investigate the effects of lackof nucleobase moiety at the two nucleotides of the 3′-overhang region (Figure 2).

Scheme . Reagents and conditions: (a) (NH)SO, HMDS, reflux; (b) KCO, MeOH, rt; (c) H, Pd/C, i-PrOH, rt, %; (d) TBAF, THF, rt, %; (e) DMTrCl, pyridine, rt, quant; (f ) (i-PrN)P(Cl)O(CH)CN, i-PrNEt,THF, rt, quant; (g) (i) succinic anhydride, DMAP, pyridine, rt; (ii) CPG, EDC·HCl, DMF, rt, .μmol/g.

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Figure . Structures of modified siRNAs.

Oligonucleotide synthesis

Using 7 and 8, ONs containing dRH were synthesized using a DNA/RNAsynthesizer (Table 1). The fully protected ONs linked to a solid support were treatedwith concentrated NH4OH/EtOH (3:1, v/v) at room temperature for 12 h and withTBAF (1.0 M solution in THF) at room temperature for 12 h. The ONs were puri-fied by denaturing 20% polyacrylamide gel electrophoresis (PAGE) to isolate thedeprotected ONs bearing abasic deoxyribonucleoside dRH. These ONs were ana-lyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrom-etry (MALDI-TOF/MS), and the observed molecular weights were in good agree-ment with their structures.[38]

Table . Sequences of ONs and siRNAs used in this study.

No. of siRNA No. of ON Sequencea,b

siRNA 9 ON 13 ′-GGCCUUUCACUACUCCUACdTdT-′ON 14 ′-dTdTCCGGAAAGUGAUGAGGAUG-′

siRNA 10 ON 15 ′-GGCCUUUCACUACUCCUACdRHdRH-′ON 16 ′-dRHdRHCCGGAAAGUGAUGAGGAUG-′

siRNA 11 ON 13 ′-GGCCUUUCACUACUCCUACdTdT-′ON 16 ′-dRHdRHCCGGAAAGUGAUGAGGAUG-′

siRNA 12 ON 15 ′-GGCCUUUCACUACUCCUACdRHdRH-′ON 14 ′-dTdTCCGGAAAGUGAUGAGGAUG-′

ON 17 F-′-GUAGGAGUAGUGAAAGGCCdTdT-′ON 18 F-′-GUAGGAGUAGUGAAAGGCCdRHdRH-′

aCapital letters indicate ribonucleosides.bF denotes fluorescein.

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68 Y. NAGAYA ET AL.

Figure . Dual-luciferase assay.

Dual-luciferase assay

The silencing activity of siRNAs 9–12 was examined by a dual-reporter assayusing a psiCHECK-2 vector in HeLa cells. This vector contains the Renilla andfirefly luciferase genes, and the siRNA sequences were designed to target the Renillaluciferase gene. HeLa cells were co-transfected with the vector and the indicatedamount of siRNAs. The signal levels of Renilla luciferase were normalized by thesignal levels of firefly luciferase. All siRNAs effectively reduced luciferase activity ina dose-dependent manner (Figure 3). The silencing activity of siRNA 10 with dRH

dimers was slightly less than that of siRNA 9, which possesses natural thymidines,at 0.1 and 10 nM. Of siRNAs 9–12, the lowest knockdown activity was exhibited byscrambled siRNA 11, which possesses dRHat the 3′-end of the antisense strand, andthe highest was exhibited by scrambled siRNA 12, which bears dRHat the 3′-end ofthe sense strand.

Nuclease resistance

We next investigated the enzymatic stability of ONs against 3′-exonucleases, whichare dominant in human serum. The susceptibility of ONs to snake venom phospho-diesterase (SVPD), which degrades ONs from their 3′-end, was examined. Unmod-ified ON 17 and modified ON 18, labeled with fluorescein at their 5′-ends, wereincubated with SVPD, and the reactions were analyzed by PAGE under denatur-ing conditions. The half-life (t1/2) of unmodified ON 17 was <5 min, and that ofmodified ON 18 was 15 min, indicating that ON 18 carrying an dRH dimer atits 3′-end was significantly more resistant to SVPD than was unmodified ON 17(Figure 4).

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Figure . Nuclease resistance of ON 17 and ON 18 against SVPD.

Experimental

General methods

All reactions were carried out under an argon atmosphere, unless otherwise noted.All reagents and solvents were purchased from commercial vendors and usedwithout further purification unless indicated otherwise. Pyridine was distilled overCaH2 and stored over activated molecular sieves 4 A. 1H and 13C NMR spectrawere recorded on a JEOL JNM AL-400 spectrometer or JNM ECS-400 spectrom-eter (400 MHz for 1H NMR, 100 MHz for 13C NMR, and 162 MHz for 31P NMR).Chemical shifts (δ) were expressed in parts per million (ppm) and internally refer-enced (7.26 ppm for CDCl3 or 3.31 for CD3OD for 1HNMR, 77.0 ppm for CDCl3 or49.0 ppm for CD3OD for 13CNMR and 0.00 ppm for H3PO4/CDCl3 for 31P NMR).Direct analysis in real time (DART) coupled with time-of-flight (TOF)mass spectrawere taken on a JMS T100TD instrument. Electron impact (EI) mass spectra weretaken on a Shimadzu GCMS-QP2010A instrument. Matrix-assisted laser desorp-tion/ionization (MALDI) coupledwith TOFmass spectrawere taken on a ShimadzuAXIMA-CFR plus instrument. Flash column chromatography was performed usingsilica gel 60 N (spherical neutral [63–210 µm]) from Kanto Chemical Co. Inc.

5′-O-(tert-Butyldiphenylsilyl)thymidine (2)[25]

TBDPSCl (600 μL, 2.34 mmol) was added to a solution of thymidine (486 mg,2.01 mmol) and DMAP (49.2 mg, 403 μmol) in pyridine (10 mL), and the mixturewas stirred at room temperature for 15 h. Themixture was partitioned as EtOAc andH2O. The organic layer was washed with saturated aqueous NH4Cl solution andbrine, dried over Na2SO4, and concentrated under reduced pressure. The residuewas purified by column chromatography on silica gel (n-hexane/EtOAc, 3:1˜0:1) togive 2 as a white solid (882 mg, 91%); 1H NMR (CDCl3) δ 9.25 (s, 1H, NH), 7.67–7.64 (m, 4H,H-Ph), 7.50 (d, J= 0.9Hz, 1H,H-6), 7.47–7.37 (m, 6H,H-Ph), 6.43 (dd,J= 8.4 Hz, 5.8 Hz, 1H, H-1′), 4.58–4.56 (m, 1H, H-3′), 4.05–4.03 (m, 1H, H-4′), 3.98(dd, J = 11.8 Hz, 2.7 Hz, 1H, H-5′

α), 3.85 (dd, J = 11.8 Hz, 2.7 Hz, 1H, H-5′β), 2.43

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(ddd, J= 13.8 Hz, 5.8 Hz, 2.3 Hz, 1H, H-2′α), 2.20 (ddd, J= 13.8 Hz, 8.4 Hz, 5.9 Hz,

1H, H-2′β), 1.62 (d, J = 0.9 Hz, 3H, CH3 of thymine), 1.08 (s, 9H, C(CH3)3); 13C

NMR (CDCl3) δ 163.8, 150.5, 135.5, 135.3, 132.8, 132.3, 130.1, 130.0, 128.0, 127.9,111.2, 87.1, 84.7, 72.3, 64.1, 41.0, 27.0, 19.3, 12.1; MS (DART) m/z 504 [M+Na]+,HRMS (DART)Calcd. for C26H32N2NaO5Si [M+Na]+: 503.6180. Found: 503.6163.

1,4-Anhydro-5-O-(tert-butyldiphenyl)-2-deoxy-D-erythro-pent-1-enitol (5)[30]

Amixture of 2 (482mg, 1.00mmol) and (NH4)2SO4 (265mg, 2.01mmol) inHMDS(8mL) was refluxed for 1.5 h. The solvent was removed under reduced pressure, andthe residue was diluted with EtOAc and H2O. The organic layer was washed withbrine and dried over Na2SO4, and concentrated under reduced pressure. A suspen-sion of K2CO3 (28.5 mg, 206 μmol) in MeOH (15 mL) was added to the residuecontaining 3 and stirred at room temperature for 2 h. The reaction mixture waspartitioned as EtOAc andH2O. The organic layer was washed with brine, dried overNa2SO4, and concentrated under reduced pressure. The residuewas filtered throughcolumn chromatography on silica gel (n-hexane/EtOAc, 3:1˜2:1) to give 4 as a yel-low oil. A mixture of 4 and 10% Pd/C (15 wt%) in i-PrOH (10 mL) was vigorouslystirred at room temperature under ambient pressure of hydrogen for 24 h. The reac-tion mixture was filtered through a celite pad, and the filtrate was evaporated underreduced pressure. The residue was purified by column chromatography on silica gel(n-hexane/EtOAc, 7:1˜2:1) to give 5 as a yellow oil (338mg, 94%); 1HNMR (CDCl3)δ 7.70–7.66 (m, 4H, H-Ph), 7.46–7.37 (m, 6H, H-Ph), 4.43–4.40 (m, 1H, H-3), 3.96(dd, J = 8.4 Hz, 5.7 Hz, 2H, H-5), 3.85–3.82 (m, 1H, H-4), 3.78 (dd, J = 10.7 Hz,4.3 Hz, 1H, H-1α), 3.60 (dd, J = 10.7 Hz, 6.4 Hz, 1H, H-1β), 2.20–2.10 (m, 1H,H-2α), 1.93–1.86 (m, 1H, H-2β), 1.07 (s, 9H, C(CH3)3); 13C NMR (CDCl3) δ 135.6,135.5, 133.2, 133.1, 129.8, 129.8, 127.7, 86.1, 77.2, 74.3, 67.1, 64.8, 34.8, 26.8, 19.2;MS(DART) m/z 379 [M+Na]+, HRMS (DART) Calcd. for C21H28NaO3Si [M+Na]+:379.1705. Found: 379.1750.

1,2-Dideoxy-D-ribofuranose (1)[28]

Tetra-n-butylammoniumfluoride (1.0M in THF, 660μL) was added to a solution of5 (215mg, 603μmol) in THF (2.7mL), and themixture was stirred at room temper-ature for 12 h. The mixture was concentrated under reduced pressure. The residuewas purified by column chromatography on silica gel (CH2Cl2/acetone, 5:1˜2:1) togive 1 as a yellow oil (68.8 mg, 97%); 1HNMR (CD3OD) δ 4.21–4.18 (m, 1H, H-3),3.97–3.87 (m, 2H, H-1), 3.75–3.72 (m, 1H, H-4), 3.57–3.49 (m, 2H, H-5), 2.18–2.03(m, 1H, H-2α), 1.88–1.81 (m, 1H, H-2β); 13C NMR (CD3OD) δ 88.1, 73.8, 68.0,63.6, 35.9; MS (DART) m/z 141 [M+Na]+, HRMS (DART) Calcd. for C5H10NaO3[M+Na]+: 141.1209. Found: 141.1250.

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1,2-Dideoxy-5-O-(4,4′-dimethoxytrityl)-D-ribofuranose (6)[24]

A mixture of 1 (101 mg, 853 μmol) and DMTrCl (376 mg, 1.11 mmol) in pyridine(3.3 mL) was stirred at room temperature for 12 h. The reaction mixture was par-titioned as CH2Cl2 and H2O. The organic layer was washed with brine, dried overNa2SO4, and concentrated under reduced pressure. The residue was purified by col-umn chromatography on silica gel (CH2Cl2/acetone, 20:1˜10:1) to give 7 as a yellowoil (265 mg, quant); 1H NMR (400 MHz, CDCl3) δ 7.43 (d, J = 7.8 Hz, 2H, H-Ph),7.36–7.16 (m, 7H, H-Ph), 6.82 (d, J = 9.2 Hz, 4H, H-Ph), 4.30–4.27 (m, 1H, H-3),3.99–3.96 (m, 2H, H-1), 3.90–3.86 (m, 1H, H-4), 3.79 (s, 6H, OCH3), 3.23 (dd, J =9.8 Hz, 4.8 Hz, 1H, H-5α), 3.07 (dd, J = 9.8 Hz, 6.2 Hz, 1H, H-5β), 2.19–2.10 (m,1H, H-2α), 1.92–1.84 (m, 1H, H-2β); 13CNMR (CDCl3) δ 158.4, 144.8, 136.0, 136.0,130.0, 128.1, 127.8, 126.8, 113.1, 86.2, 85.0, 74.6, 67.0, 64.5, 55.2, 34.8; MS (EI) m/z420 (M+)

3-O-[(2-Cyanoethoxy)(N,N-diisopropylamino)phosphanyl]-1,2-dideoxy-5-O-(4,4′-dimethoxytrityl)-D-ribofuranose (7)

i-Pr2NEt (730 μL, 4.18 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (250 μL, 1.12 mmol) were added to a solution of 6(292 mg, 694 mmol) in THF, and stirred at room temperature for 2 h. The reactionmixture was partitioned as CHCl3 and saturated NaHCO3 aqueous solution. Theorganic layer was washed with brine, dried over Na2SO4, and concentrated underreduced pressure. The residue was purified by column chromatography on silicagel (n-hexane/EtOAc, 4:1) to give 7 as a yellow oil; 31P NMR (162 MHz, CDCl3) δ

148.3, 148.6

Solid support synthesisA mixture of 7 (102 mg, 243 μmol), succinic anhydride (112 mg, 1.12 mmol), andDMAP (30.4 mg, 249 μmol) in pyridine (2.4 mL) was stirred for 23 h at room tem-perature. The reaction mixture was partitioned as EtOAc and saturated NaHCO3aqueous solution. The organic layer was washed with brine, dried over Na2SO4, andconcentrated under reduced pressure to give the corresponding succinate. Amino-propyl controlled pore glass (546 mg, 53.5 μmol) was added to a solution of suc-cinate and EDC·HCl (41.6 mg, 217 μmol) in DMF (5.3 mL), and the mixture waskept for 72 h at room temperature. After the resin was washed with pyridine, a cap-ping solution (30 mL, 0.1 M DMAP in pyridine/Ac2O, 9:1, v/v) was added and thewhole mixture was kept for 72 h at room temperature. The resin was washed withpyridine, EtOH, MeCN, and dried in vacuo. The amount of loaded 6 to the solidsupport was 54.5 μmol/g from calculation of released dimethoxytrityl cation by asolution of 70% HClO4/EtOH, 3:2 (v/v).

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Oligonucleotide synthesisSynthesis was carried out with a DNA/RNA synthesizer (ABI Expedite 3400) by thephosphoramidite method. Deprotection of bases and phosphates was performed inconcentrated NH4OH/EtOH, 3:1 (v/v) at room temperature for 12 h. 2′-TBDMSgroups were removed by TBAF (1.0 M in THF) at room temperature for 12 h. Thereaction was quenched with 0.1 M triethylammonium acetate buffer (pH 7.0) anddesalted on a Sep-Pak C18 cartridge. Deprotected ONs were purified by 20% PAGEcontaining 7.0 M urea to give a highly purified ON 15 (8), ON 16 (7), and ON 18(12). The yields are indicated in parentheses as OD units at 260 nm starting from1.0-μmol scale. The extinction coefficients of ONs were calculated from those of themononucleotides and dinucleotides according to the nearest neighbor approxima-tion method.[38]

Mass spectrometric analyses of ONsSpectra were obtained by MALDI-TOF/MS (negative mode). ON 15: calculatedmass, 6250.8; observed mass, 6251.0. ON 16: calculated mass, 6559.9; observedmass, 6559.8. ON 18: calculated mass, 7098.0; observed mass, 7099.4.

Dual-luciferase assayHeLa cells were grown at 37°C in a humidified atmosphere of 5% CO2 in air inD-MEM (Wako) supplemented with 10% bovine serum (Sigma). HeLa cells (4 ×104/mL) were transferred to 96-well plates (100 μL per well) 24 h before transfec-tion. They were transfected using TransFast (Promega) according to instructions fortransfection of adherent cell lines. Cells in each well were transfected with a solu-tion (35 μL) of 20 ng of psi-CHECK-2 vector (Promega), the indicated amounts ofsiRNAs, and 0.3 μg of TransFast in Opti-MEM I reduced-serum medium (Invit-rogen), and incubated at 37°C. Transfection without siRNA was used as a control.After 1 h, D-MEM (100 μL) containing 10% bovine serum was added to each well,and the whole was further incubated at 37°C. After 24 h, cell extracts were frozenat −80°C. Activities of firefly and Renilla luciferases in cell lysates were determinedwith a dual-luciferase assay system (Promega). The results were confirmed by at leastthree independent transfection experiments with two cultures each and expressedas an average of mean ± SD from three experiments.

Nuclease resistanceEachON (300 pmol) labeledwith fluorescein at the 5′-endwas incubatedwith SVPD(1 unit/mL) in PBS(–) (100 μL) at 37°C. At appropriate periods, aliquots (5 μL) ofthe reaction mixture were separated and added to a solution of 7.0 M urea (15 μL).The resultingmixtureswere heated at 90°C for 10min and analyzed by electrophore-sis on 20% PAGE containing 7.0 M urea. The labeled ONs in the gel were visualizedwith a lumino image analyzer.

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Conclusions

We demonstrated a practical and reliable method for preparing dRH. To synthe-size ONs bearing dRH by the standard phosphoramidite solid phase method, thecorresponding phosphoramidite derivative and a solid support were prepared. Thesilencing activity of siRNAs containing dRH in their 3′-overhang region was exam-ined using a dual-luciferase assay. Introducing dRH to the 3′-end of the guide strandof siRNAwas found to attenuate its gene silencing effect in an in vitro dual-luciferaseexperiment. It was also found that incorporating dRH into 3′-end enhances thenuclease resistance of ONs. Further study of dRH will contribute to the developmentof ON-based therapeutics.

Acknowledgment

We acknowledge the Division of Instrumental Analysis, Life Science Research Center, Gifu Uni-versity, for the maintenance of instruments.

Funding

This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for thePromotion of Science (JSPS).

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Introduction of 2-O-benzyl abasic nucleosides to the 30-overhang regionsof siRNAs greatly improves nuclease resistance

Yuki Nagaya a, Yoshiaki Kitamura b, Aya Shibata b, Masato Ikeda a,b,c, Yukihiro Akao a, Yukio Kitade b,d,⇑aUnited Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, JapanbDepartment of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, JapancGifu Center for Highly Advanced Integration of Nanosciences and Life Sciences (G-CHAIN), Gifu University, 1-1 Yanagido, Gifu 501-1193, JapandDepartment of Applied Chemistry, Faculty of Engineering, Aichi Institute of Technology, 1247 Yachigusa, Yakusa-cho, Toyota, Aichi 470-0392, Japan

a r t i c l e i n f o

Article history:Received 6 October 2017Revised 26 October 2017Accepted 27 October 2017Available online 28 October 2017

Keywords:RNAsiRNARNAiAbasic nucleoside analoguesNuclease resistance

a b s t r a c t

Chemically modified siRNAs containing 2-O-benzyl-1-deoxy-D-ribofuranose (RHOBn) in their 30-overhang

region were significantly more resistant towards serum nucleases than siRNAs possessing the natural nucle-oside in this region. The knockdown efficacies and binding affinities of these modified siRNAs to the recom-binant human Argonaute protein 2 (hAgo2) PAZ domain were comparable with that of siRNA with athymidine dimer at the 30-end.

� 2017 Elsevier Ltd. All rights reserved.

Small interfering RNAs (siRNAs) inhibit gene expression by RNAinterference (RNAi) and thus have great potential as nucleic aciddrugs.1 RNAi technology is a useful strategy in the fight againstcancers,2 viral infections,3 and other diseases.4 However, naturalRNA strands have many problems that complicate their therapeu-tic application, such as rapid degradation in biological media, non-specific gene silencing (off-target effects), and poor administrationusing existing drug delivery systems. To overcome these problems,artificially modified siRNAs have been developed extensively. AnsiRNA is a short (18–26 nucleotides) double-stranded RNA (dsRNA)containing a 2-nucleotide overhang at the 30-end of each strand.5

Once siRNAs are introduced into a cell by transfection, they areincorporated into a RNA-induced silencing complex (RISC). EachRISC contains a helicase that unwinds the siRNA helix. Uponunwinding, one of the strands, known as an antisense strand (guidestrand), is retained in the RISC. This antisense RNA-RISC, calledmature RISC, binds to and degrades the complementary mRNA tar-get through base-pairing interactions. Eukaryotic translation initi-ation factor 2C2 (EIF2C2, Argonaute protein 2, Ago2), the corecomponent of RISC, is considered to be the major player in RNAi.Ago2 has a conserved structure and includes PAZ, MID, and PIWIdomains. The PAZ domain specifically recognizes the antisense

strand of dsRNA through binding to the 30-overhang region.6,7

The binding site is a hydrophobic pocket composed of aromaticamino acids.7–10

On the basis of these findings, it has been suggested that chem-ical modification of the 30-overhang region is an effective tech-nique for improving the functionality of siRNA for RNAi-basedtherapy.11–25 We previously reported the design and synthesis ofvarious chemically modified functional RNAs bearing nucleic acidmimics at their 30-end.11–15,17,23 As part of our ongoing studies,we found that siRNAs containing 2-O-benzyl-1-deoxy-D-ribofura-nose (RH

OBn; 1) at the 30-ends showed high nuclease resistance anda desirable knockdown effect (Fig. 1).

Synthesis: To synthesize the desired RNAs via the conventionalphosphoramidite method using a DNA/RNA synthesizer, we pre-pared the phosphoramidite derivative of RH

OBn (2) (Scheme 1). First,1-deoxy-D-ribofuranose (RH, 3), which can be prepared from com-mercially available 1-O-acetyl-2,3,5-tri-O-benzoyl-b-D-ribofuranosevia reductive cleavage of the anomeric position,15 was convertedto TIPDS-RH 4 using a disiloxane protection strategy. Subsequently,benzylation with benzaldehyde, Et3SiH, and FeCl3 in CH3NO2,26 fol-lowed by desilylation by treatment with Et3N�3HF, gave 1 in moder-ate yield. Treatment of 1 with 4,40-dimethoxytrityl (DMTr) chloridein pyridine gave the corresponding 5-DMTr derivative 5, whichwas phosphorylated with 2-cyanoethyl N,N-diisopropylchlorophos-phoramidite to produce 2 in 97% yield. Oligonucleotides (ONs) con-taining RH

OBn were synthesized using an automated nucleic acid

https://doi.org/10.1016/j.bmcl.2017.10.0700960-894X/� 2017 Elsevier Ltd. All rights reserved.

⇑ Corresponding author at: Department of Chemistry and Biomolecular Science,Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.

E-mail address: [email protected] (Y. Kitade).

Bioorganic & Medicinal Chemistry Letters 27 (2017) 5454–5456

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/locate /bmcl

synthesizer with phosphoramidite derivative 2 (Table 1). The fullyprotected ONs linked to a solid support were treated with NH4OH/MeNH2 (40% in H2O), 1:1 (v/v) at 65 �C for 30 min and with Et3N�3HFin DMSO at 65 �C for 2.5 h. The crude product can be precipitated byadding 3 M NaOAc, followed by n-BuOH. The mixture was cooled at�80 �C for 12 h and centrifuged at 4 �C, 12,500 rpm, for 30 min. Afterremoval of the supernatant, 70% EtOH was added to the pellet. Theresulting mixture was centrifuged at 4 �C, 12,500 rpm, for 15 min.The supernatant was removed, and the washing step was repeated.Deprotected ONs were purified by denaturing 20% polyacrylamidegel electrophoresis (PAGE) to isolate the desired ONs bearing the RH-OBn modification. These ONs were analyzed by matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS), and the observed molecular weights were in good agree-ment with their expected structures.27

Gene silencing of Renilla luciferase: The silencing activities of thesiRNAs were examined by a dual-reporter assay using the psi-CHECK-2 vector in HeLa cells. This vector contains the Renilla andfirefly luciferase genes, and the siRNA sequences were designed

to target the Renilla luciferase gene. HeLa cells were co-transfectedwith the vector and the indicated amount of each siRNA. The signallevels of Renilla luciferase were normalized to those of firefly luci-ferase. Fig. 2 shows the silencing activities of the siRNAs. Renillaluciferase was suppressed by each siRNA in a dose-dependentmanner. At 1.0 nM and 10 nM, the silencing activity of siRNA 7with RH

OBn dimers was almost equal to that of siRNA 6 with naturalthymidines.

Nuclease resistance: The susceptibilities of the ONs to snakevenom phosphodiesterase (SVPD), a highly active 30-exonuclease,were examined. Thymidine-modified ON 14 and RH

OBn-modifiedON 15, each labeled with fluorescein at their 50-end, were incubatedwith SVPD and the reactions were analyzed by PAGE under denatur-ing conditions. The half-life (t1/2) of ON 14 was <4 min, and that ofON 15 carrying a RH

OBn dimer was 19 min. ON 15 was at least 5 timesmore resistant to the enzyme than thymidine-modified ON 14(Fig. 3). Furthermore, the RH

OBn-modified ON 15 was more resistantto nucleolytic hydrolysis by SVPD than the corresponding modified

Fig. 1. Conceptual diagram of this study.

Scheme 1. Reagents and conditions: (a) TIPDSCl2, imidazole, DMF, rt, 82%; (b) i)PhCHO, Et3SiH, FeCl3, MeNO2, rt; ii) Et3N�3HF, THF, rt, 47%; (c) DMTrCl, pyridine, rt,67%; (d) (i-Pr2N)P(Cl)O(CH2)2CN, i-Pr2NEt, THF, rt, 97%.

Table 1Sequences of ONs and siRNAs used in this study.

No. of siRNA No. of ON Sequence

siRNA 6 ON 10 50-GGCCUUUCACUACUCCUACtt-30

ON 11 30-ttCCGGAAAGUGAUGAGGAUG-50

siRNA 7 ON 12 50-GGCCUUUCACUACUCCUACRHOBnRH

OBn-30

ON 13 30-RHOBnRH

OBnCCGGAAAGUGAUGAGGAUG-50

� ON 14 F-50-GUAGGAGUAGUGAAAGGCCtt-30

� ON 15 F-50-GUAGGAGUAGUGAAAGGCCdRHdRH-30

asiRNA 8 ON 16 50-GGCCUUUCACUACUCCUAC-30

ON 11 30-ttCCGGAAAGUGAUGAGGAUG-50

asiRNA 9 ON 16 50-GGCCUUUCACUACUCCUAC-30

ON 13 30-RHOBnRH

OBnCCGGAAAGUGAUGAGGAUG-50

aCapital letters indicate ribonucleosides and small letters show 20-deoxyribonucleosides.bF denotes fluorescein.

Fig. 2. Dual-luciferase assay.

Fig. 3. Nuclease resistance of ONs against SVPD.

Y. Nagaya et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 5454–5456 5455

ON bearing the parent abasic ribo- and deoxyribonucleoside(Fig. S1).15,23

Serum stability: Next, we tested the stability of chemically mod-ified siRNA with RH

OBn in 50% bovine serum. At various incubationtimes, aliquots of each siRNA were analyzed by 20% native PAGE todetect any degradation products. As shown in Fig. 4, siRNA 7 pos-sessing RH

OBn was more resistant to serum-derived nucleases thanunmodified siRNA 6 with natural thymidines. Unmodified siRNA 6was largely degraded within 1 h, whereas less than 40% of modifiedsiRNA 7 was degraded after 3 h and approximately 15% remainedintact after 24 h incubation (Fig. S2).

Binding affinity with hAgo2 PAZ: We further investigated thebinding affinities of a recombinant hAgo2 PAZ domain protein tothe chemically modified siRNAs (Fig. 5).20 In order to simplify thestudy, we used asymmetric siRNAs (asiRNAs) with an overhangregion at the 30-end of the antisense strand and a blunt end struc-ture at the 30-end of the sense strand (ON 16). The asiRNA 8 con-sists of ON 11 and ON 16, and the asiRNA 9 was duplex of ON 13and ON 16. The addition of hAgo2 PAZ protein resulted in a slightdecrease in the absorbance at 312 nm. The binding affinity ofasiRNA 9 possessing RH

OBn is almost the same as that of asiRNA 8 car-rying a thymidine dimer (Fig. S3). This result suggests that asiRNA 9with RH

OBn can bind the hAgo2 PAZ domain in a very similar mannerto that of asiRNA 8 binding with natural thymidine dimers.

In conclusion, we demonstrated the synthesis of chemicallymodified siRNAs bearing a 2-O-benzylated abasic nucleoside intheir 30-overhang region. It was found that introduction of the aba-sic nucleoside at the 30-end of an siRNA greatly improves resistancetowards various nucleases. Furthermore, the knockdown efficaciesand binding affinities of these modified siRNAs to recombinanthAgo2 PAZ domain were comparable with that of siRNAwith a thy-midine dimer. We believe that this modification method is a usefultechnique for increasing the duration of silencing in vivo.

Acknowledgments

This work was supported by a Grant-in-Aid for Young Scientists(B) (No. 16K18911 (Y.K.)) and a Grant-in-Aid for Scientific Research(B) (No. 24390025 (Y.K.)) from the Japan Society for the Promotionof Science (JSPS). We acknowledge the Division of InstrumentalAnalysis and the Division of Genomics Research, Life ScienceResearch Center, Gifu University, for maintaining the instrumentsused in this study.

A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at https://doi.org/10.1016/j.bmcl.2017.10.070.

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MALDI-TOF/MS (negative mode). ON 12: calculated mass, 6462.9; observedmass, 6461.8. ON 13: calculated mass, 6772.0; observed mass, 6772.8. ON 15:calculated mass, 7310.1; observed mass, 7310.8.

Fig. 4. Serum stability of siRNAs.

Fig. 5. Binding affinities of asiRNAs to the recombinant hAgo2 PAZ domain.

5456 Y. Nagaya et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 5454–5456

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