Supporting Information

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1 S1 Supporting Information The Five - and Six -Membered Conformationally- Locked 2',4'-Carbocyclic ribo-thymidine: Synthesis, Structure and Biochemical Studies Puneet Srivastava, Jharna Barman, Wimal Pathmasiri, leksandr Plashkevych, Małgorzata Wenska and Jyoti Chattopadhyaya * Department of Bioorganic Chemistry, Box 581, Biomedical Center, Uppsala University, SE Uppsala, Sweden. jyoti@boc.uu.se Table of contents Figure S1. 1 H NMR spectrum of 2 Figure S2. 1 H NMR spectrum of 2 Figure S3. 1 H NMR spectrum of 4 Figure S4. 13 C NMR spectrum of 4 Figure S5. 1 H NMR spectrum of 5 Figure S6. 13 C NMR spectrum of 5 Figure S7. 1 H NMR spectrum of 6 Figure S8. 13 C NMR spectrum of 6 Figure S9. 1 H NMR spectrum of 8 Figure S C NMR spectrum of 8 Figure S11. 1 H NMR spectrum of 9 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 * Corresponding author

2 Figure S C NMR spectrum of 9 Figure S13. 1 H NMR spectrum of 10 Figure S C NMR spectrum of 10 Figure S15. HMQC spectrum of 10 Figure S16. HMBC spectrum of 10 Figure S17. 1 H NMR spectrum of 11a/11b Figure S C NMR spectrum of 11a/11b Figure S19. HMQC spectrum of 11a/11b Figure S20. HMBC spectrum of 11a/11b Figure S21. Expansion of the HMBC spectrum of the benzyl protected isomeric sugar-fused carbocyclic 5-membered bicyclo[2.2.1]heptane 11a/11b showing important connectivities for the ring fusion between C2' and C7' Figure S22. 1 H NMR spectrum of 12a/12b Figure S23. Expansions of characteristic regions of the 1 H spectrum (Figure S22) to show the minor isomer 12b Figure S24. Single and double decoupling experiments resolving coupling constants for 12a Figure S25. Experimental and simulated spectra for 12a Figure S26. Single and double decoupling experiments resolving coupling constants for 12b Figure S27. Experimental and simulated spectra for 12b Figure S C NMR spectrum of 12a/12b Figure S29. DEPT spectra for 12a/12b Figure S30. 2D 1 H CSY spectrum of 12a/12b. Figure S31. Expansion of δ 2.7 to 0.7 ppm region of 2D 1 H CSY spectrum of 12a/12b highlighting the cross peaks owing to the major isomer 12a Figure S32. Expansion of δ 2.7 to 0.7 ppm region of 2D 1 H CSY spectrum of 12a/12b showing the cross peaks owing to the minor isomer 12b Figure S33. 2D 1 H TCSY spectrum of 12a/12b. Figure S34. Expansion of the δ 2.7 to 0.7 region of 2D 1 H TCSY spectrum of 12a/12b Figure S35. 1D NESY spectrum of 12a Figure S36. 1D NESY spectrum of 12b Figure S37. 2D HMQC spectrum of 12a/12b Figure S38. 2D HMBC spectrum of 12a/12b S2 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30 S31-S32 S33 S34 S35 S36 S37 S38 S39 S40 S41 S42 S43 S44

3 Figure S39. Expansion of 1 H- 13 C HMBC spectrum of inseparable diastereomeric mixture of compounds 12a/12b Figure S40. 1 H NMR spectrum of 13 Figure S C NMR spectrum of 13 Figure S P NMR spectrum of 14 Figure S43. 1 H NMR spectrum of 15 Figure S C NMR spectrum of 15 Figure S45. 1 H NMR spectrum of 17 Figure S C NMR spectrum of 17 Figure S47. 1 H NMR spectrum of 20 Figure S C NMR spectrum of 20 Figure S49. 1 H NMR spectrum of 21 Figure S C NMR spectrum of 21 Figure S51. 2D HMQC spectrum of 21 Figure S52. 2D HMBC spectrum of 21 Figure S53. 1 H NMR spectrum of 22 Figure S C NMR spectrum of 22 Figure S55. 2D HMQC spectrum of 22 Figure S56. 2D HMBC spectrum of 22 Figure S57. 1H NMR spectrum of 23 Figure S58. Single and double decoupling experiments resolving coupling constants for 23 Figure S59. Experimental and simulated spectra for 23 Figure S C NMR spectrum of 23 Figure S61. DEPT spectra of 23 Figure S62. 2D 1 H CSY spectrum of 23 Figure S63. Expansion of δ 2.5 to 0.7 region of the CSY spectrum of 23 Figure S64. 2D TCSY spectrum of 23 Figure S65. Expansion of δ 2.7 to 0.7 region of the 2D TCSY spectrum of 23. Dashed box shows the spin system to H8' (including H2') and thereby giving the evidence for the ring closure Figure S66. 1D NESY spectra of compound 23. Figure S67. 2D HMQC spectrum of 23 S3 S45 S46 S47 S48 S49 S50 S51 S52 S53 S54 S55 S56 S57 S58 S59 S60 S61 S62 S63 S64 S65 S66 S67 S68 S69 S70 S71 S72 S73

4 Figure S68. 2D HMBC spectrum of 23 Figure S69. Expansion of the HMBC spectrum of carbocyclic-ena-t (23). Figure S70. 1 H NMR spectrum of 24 Figure S C NMR spectrum of 24 Figure S P NMR spectrum of 25 Figure S73. Correlation of experimental 3 J H1',H2' and 3 J H2',H3' vicinal coupling constants in carbocyclic-ena-t (12a,12b) and carbocyclic LNA-T (23) as well as their 2'-and 2'-N analogs (ENA-T, aza-ena-t, LNA-T and 2'-amino LNA-T) Figure S74. verlay of 2500 molecular structures of the carbocyclic-ena-t nucleoside collected every 0.2 ps of the last 500 ps ( ns) of its MD simulation and their analysis Figure S75. Superposition of 10 randomly selected structures during 1ns of the NMR constrained MD simulations of carbocyclic-lna-t (12a and 12b) and carbocyclic-ena-t (23). Figure S76. Plots of percentage remaining of ANs containing 5 - membered carbocyclic nucleoside (ANs 6-9) in human blood serum versus time (h) Figure S77. Plots of percentage remaining of ANs containing 6 - membered carbocyclic nucleoside (ANs 10-13) in human blood serum versus time (h) Figure S78. Plots of percentage remaining of ANs containing 6 - membered aza-ena nucleoside (ANs 14-17) in human blood serum versus time (h) Figure S79. Autoradiograms of 20% denaturing PAGE showing degradation pattern of 5-32 P- labeled ANs in SVPDE (see Table 3 in text for full AN sequence) Figure S80. Plots of percentage remaining of ANs containing 5 - membered carbocyclic nucleoside (ANs 6-9) in snake venom phosphodiesterase versus time (h) Figure S81. Plots of percentage remaining of ANs containing 6 - membered carbocyclic nucleoside (ANs 10-13) in snake venom phosphodiesterase versus time (h) Figure S82. Plots of percentage remaining of ANs containing 6 - membered aza-ena nucleoside (ANs 14-17) in snake venom phosphodiesterase versus time (h) Table S1. Chemical shifts for the five-membered and six-membered fused carbocyclic analogs of ENA and LNA Table S2. Coupling constants for the s 11a, 12a, 12b, 22, and 23 Table S3. Experimental and theoretical 3 J H,H vicinal proton coupling constants, corresponding S4 S74 S75 S76 S77 S78 S79 S80 S81 S82 S83 S84 S85 S86 S87 S88 S89 S90 S91

5 ab initio and MD (highlighted in blue) φ H,H torsions obtained using Haasnoot-de Leeuw-Altona generalized Karplus equation Table S4. Sugar torsions (ν 0 - ν 4 ), pseudorotational phase angle (P), sugar puckering amplitude (φ m ), backbone (γ,δ) and glycoside bond (χ), as well as selected torsions* (C3'-C2'- X2'-Y' and C3'-C4'-C6'-C7') characterizing five- and six-membered ring in carbocyclic-lna-t (major and minor isomers, 12a and 12b), carbocyclic-ena-t (23), aza-ena-t, ENA-T 1, 2'- amino-lna-t 2 and LNA-T 3 compounds. Table S5. Solvation energy (E solv ) calculated using Baron and Cossi s implementation of the polarizable conductor CPCM model Discussion S1. NMR Characterization of five-membered fused carbocyclic-lna-t (12a & 12b) and the six-membered carbocyclic-ena-t (23). Discussion S2. Molecular structures of carbocyclic-lna-t and carbocyclic-ena-t based on NMR, ab initio and MD calculations References for SI S5 S92 S93 S95 S100 S102

6 Bn H Bn 2 Figure S1. 1 H NMR spectrum of 2. S6

7 Figure S2. 13 C NMR spectrum of 2. Bn H Bn 2 S7

8 Bn Bn 4 Figure S3. 1 H NMR spectrum of 4. S8

9 Bn Bn 4 Figure S4. 13 C NMR spectrum of 4. S9

10 Bn H Bn 5 Figure S5. 1 H NMR spectrum of 5. S10

11 Bn H Bn 5 Figure S6. 13 C NMR spectrum of 5. S11

12 Bn Bn 6 Figure S7. 1 H NMR spectrum of 6. S12

13 Figure S8. 13 C NMR spectrum of 6. Bn Bn 6 S13

14 NH N Bn Bn Ac 8 Figure S9. 1 H NMR spectrum of 8. S14

15 NH N Bn Bn Ac 8 Figure S C NMR spectrum of 8. S15

16 NH N Bn Bn H 9 Figure S11. 1 H NMR spectrum of 9. S16

17 NH N Bn Bn H 9 Figure S C NMR spectrum of 9. S17

18 NH N Bn Bn CSPh 10 Figure S13. 1 H NMR spectrum of 10. S18

19 Figure S C NMR spectrum of 10. S19

20 S20 CH 2 (Ph) Bn CH 2 (Ph) Me(T) H6 PTC H3 H1 H2 H8 H5 H5 H7 H6 H6 Bn N NH Bn CSPh 10 Me(T) C6 CH 2 Ph(C5 ) + C5 CH 2 Ph(C3 C3 C2 C4 C1 C5 C8 PTC PTC + Bn C7 Bn C6 C4 Bn C2 CS Figure 15. HMQC spectrum of 10.

21 NH CH 2 (Ph) Bn CH 2 (Ph) H6 PTC H3 H1 H2 H8 H5 H5 H7 H6 H6 Me(T) Bn S21 N NH Bn CSPh 10 Me(T) C6 CH 2 Ph(C5 ) + C5 CH 2 Ph(C C3 C2 C4 C1 C5 C8 PTC PTC + Bn C7 C6 Bn C4 Bn C2 CS Figure S16. HMBC spectrum of 10.

22 NH N Bn Bn CH 3 Figure S17. 1 H NMR spectrum of 11a/11b. S22

23 NH N Bn Bn CH 3 Figure S CNMR spectrum of 11a/11b. S23

24 S24 NH Bn N Bn CH 3 Figure S19. HMQC spectrum of 11a/11b..

25 S25 NH Bn N Bn CH 3 Figure S20. HMBC spectrum of 11a/11b.

26 Figure S21. Expansion of the HMBC spectrum of the benzyl protected isomeric sugar-fused carbocyclic 5-membered bicyclo[2.2.1]heptane 11a/11b showing important connectivities for the ring fusion between C2' and C7'. Text and boxes in violet represents the major diastereomer 11a whereas those in red shows the minor diastereomer 11b. H2' overlaps with H7'. Therefore the fully de-protected 12a/12b were used for the full characterization (see text). S26

27 NH H N H CH 3 12a/12b Figure S22. 1 H NMR spectrum (in DMS-d6)of 12a/12b. S27

28 S28 NH H N H CH 3 12a/12b Figure S23. Expansions of characteristic regions of the 1 H spectrum of 12a/12b in Figure S22 to show the minor isomer 12b.

29 S29 A B H7' (m) CH3(C-7') (d) H7' (m) H6'(dd) H6'' (dd) H7' (qd) (dt) Decoupling Decoupling ppm 1.15 ppm ppm 1.88 ppm 0.95 ppm C H6'(dd) CH3 (C-7') (d) D H7' (m) CH3(C-7' )(d) H6'' (dd) H7' (m) H7' (dd) H7' (dd) Decoupling Decoupling 2.60 ppm 1.9 ppm 1.15 ppm ppm 1.15 ppm 0.92 ppm Figure S24. Single and double decoupling experiments resolving coupling constants for 12a: (A) Irradiation at CH3(C7') simplified the multiplet of H7' into a dt (J = 10.6, 4.5, 4.4 Hz) i.e. 3 JH7',H6', 3 JH7',H6'', 3 JH7',H2', respectively. (B) Double irradiation at both H6' and H6'' simplified the multiplet of H7' into a qd (J = 7.3, 4.5 Hz) i.e. 3 JH7',CH3(C7'), 3 JH7',H2'', respectively. (C) Double irradiation at both H6' and CH3(C7') simplified the multiplet of H7' into a dd (J = 5.5, 4.5 Hz) i.e. 3 JH7',H6'', 3 JH7',H2'', respectively. (D) Double irradiation at both H6'' and CH3(C-7') simplified the multiplet of H7' into a dd (J = 10.6, 4.5 Hz) i.e. 3 JH7',H6', 3 JH7',H2', respectively.

30 A : H7' B : H6' C : H2' D : H6'' Figure S25. Experimental and simulated spectra for 12a. Upper one represents the experimental spectrum while the lower one represents the simulated spectrum of H7' (A), H6' (B), H2' (C), and H6'' (D). S30

31 S31

32 Figure S26. Single and double decoupling experiments resolving coupling constants for 12b. Inset A: Irradiation upon H3' simplifies H2' into a singlet. Inset B: Irradiation upon H7' does not change the doublet of H2' indicating that H2' is not coupled with H7'. Inset C: Irradiation upon CH3(C7') gives a dd for H7' (i.e. 3 JH7',H6' and 3 JH7',H6'' ). Inset D: Double irradiation upon H6' and H6'' gives a quartet for ( 3 JH7',Me(C7')). Inset E: Double irradiation at CH3(C7') and H6'' gives a doublet for H7' ( 3 JH7',H6'). Inset F: Double irradiation at CH3(C7') and H6' gives a doublet for H7' ( 3 JH7',H6''). Inset G: Double irradiation at H2' and H6' gives a dq for H7' ( 3 JH7',H6' and 3 JH7',Me(C7')). Inset H: Double irradiation at H2' and H6'' gives a q for H7' ( 3 JH7',Me(C7')). Inset I: Double irradiation at CH3(C7') and H6' gives a d for H7' ( 3 JH7',H6'')). Inset J: Double irradiation at CH3(C7') and H6' gives a d for H7' ( 3 JH7',,H6'')). S32

33 A : H2' B : H7' C : H6'' D : H6' Figure S27. Experimental and simulated spectra for 12b. Upper one represents the experimental spectrum while the lower one represents the simulated spectrum of H2' (A), H7' (B), H6'' (C), and H6' (D). S33

34 Figure S C NMR spectrum (DMS-d 6).of 12a/12b H S34 NH N H CH 3 12a/12b

35 S35 DEPT 135 DEPT 90 DEPT 45 C4 C2 C6 C5 C4' C1' C3' C5' C2' C7' CH3(C7') CH3(T) C6' 13C Full Spectrum ppm NH H N Figure S29. DEPT spectra (in DMS-d 6 ) and assignment for 12a/12b. H CH 3 12a/12b

36 S36 NH H N H CH 3 12a/12b Figure S30. 2D 1 H CSY spectrum (in DMS-d 6 )of 12a/12b.. Highlighted region is given below in Figure S31.

37 Figure S31. Expansion of δ 2.7 to 0.7 ppm region of 2D 1 H CSY spectrum of 12a/12b highlighting the cross peaks owing to the major isomer 12a. S37

38 Figure S32. Expansion of δ 2.7 to 0.7 ppm region of 2D 1 H CSY spectrum of 12a/12b showing the cross peaks owing to the minor isomer, 12b. S38

39 Figure S33. 2D 1 H TCSY spectrum (in DMS-d 6 ) of 12a/12b. Highlighted region is shown below in Figure S34. S39

40 Figure S34. Expansion of δ 2.7 to 0.7 region of the 2D 1 H TCSY spectrum of 12a/12b. Dashed box shows the spin systems to H7' (including H2') and thereby giving the evidence for the ring closure. S40

41 Figure S35. 1D NESY spectra (in DMS-d 6 ) of compound 12a. Irradiation at CH 3 (C7') (shown by thick arrow) results in ne enhancement of 6.5 % on H1' indicating the close proximity of H1' and C7'- methyl (less then 3 Å). Taking into account that ab initio calculated H1'-CH 3 (C7') distances are ca. 4.4 Å and 2.8 Å in case of S and R configuration of C7', respectively, the ne enhancement found confirms the R-configuration for C7' in the major diastereomer 12a. Irradiation upon H3' shows the enhancement on H6 indicating that 1-thyminyl moiety is in the β-configuration and anti conformation. S41

42 S42 H6 H1' H3' 1.8% H7' 4.5% H3' 11% Me(T) 8.7% Figure S36. 1D NESY spectra (in DMS-d 6 ) of compound 12b. Irradiation at H1' (shown by thick arrow) results in ne enhancement of 4.5% on H7' indicating the close proximity of H1' and H7' (less then 3 Å). Taking into account that ab initio calculated H1'-CH 3 (C7') distances are ca. 2.2 Å and 3.7 Å in case of S and R configuration of C7', respectively, the obtained ne enhancement confirms S-configuration for C7 in the minor diastereomer 12b. Irradiation upon H6' shows the enhancement on H3' indicating that 1-thyminyl moiety is in the β-configuration and anti conformation.

43 Figure S37. 2D HMQC spectrum (in DMS-d 6 ) of 12a/12b. Corresponding peaks for the minor diastereomer 12b are shown in red. S43

44 Figure S38. 2D HMBC spectrum (in DMS-d 6 ) of 12a/12b. Highlighted region is shown below in Figure S39. Corresponding peaks for the minor diastereomer 12b are shown in red. S44

45 A S45 B C Figure S39. Expansion of 1 H- 13 C HMBC spectrum of inseparable diastereomeric mixture of compounds 12a/12b (in D 2 ) showing the 3 J H,C and 2 J H,C connectivities, which give the unequivocal evidence supporting the formation of the isomeric sugar-fused carbocyclic 5-membered bicyclo[2.2.1]heptane, 12a / 12b, in which the spatial orientation of the methyl group determines configuration of the chiral center C7' in 12a and 12b as R and S, respectively. All NMR experiments for assignments are shown Figures S22-S38. Inset A: Cross peaks a k for the major diastereomer 12a, and the cross peaks a m for the minor diastereomer 12b, show the respective 3 J H,C and 2 J H,C connectivities. Important correlations showing the carbocyclic ring-fusion between C2 and C7 for the major isomer 12a are shown in the violet boxes, whereas those for the minor isomer 12b are indicated in the red. Inset B: Important long range connectivities for diastereomer 12a are in the square boxes marked with a, b, c, and d. Inset C: Important long range connectivities for diastereomer 12b: a, b, c, d, and j.

46 NH N DMTr H CH 3 13a/13b Figure S40. 1 H NMR spectrum of 13a/13b. S46

47 NH N DMTr H CH 3 13a/13b Figure S C NMR spectrum of 13a/13b. S47

48 Figure S P NMR spectrum of 14a/14b. DMTr N P NH N CH 3 CN 14a/14b S48

49 Bn H Bn 15 Figure S43. 1 H NMR spectrum of 15. S49

50 Figure S C NMR spectrum of 15. Bn H Bn 15 S50

51 Figure S45. Bn Bn 17 1 H NMR spectrum of 17. S51

52 Figure S C NMR spectrum of 17. Bn Bn 17 S52

53 NH N Bn Bn 20 H Figure S47. 1 H NMR spectrum of 20. S53

54 Figure S C NMR spectrum of 20. Bn N Bn 20 H NH S54

55 NH N Bn Bn 21 CSPh Figure S49. 1 H NMR spectrum of 21. S55

56 ppm S NH N Bn Bn 21 CSPh Figure S C NMR spectrum of 21.

57 S57 NH Bn N Bn CSPh 21 Figure S51. HMQC spectrum of 21.

58 S58 NH Bn N Bn CSPh 21 Figure S52. HMBC spectrum of 21.

59 NH N Bn Bn CH3 22 Figure S53. 1 H NMR spectrum of 22. S59

60 ppm S Bn Figure S54. 1 H NMR spectrum of 22. NH N Bn CH3 22

61 S61 NH Bn N Bn 22 CH3 Figure S55. 1 H NMR spectrum of 22.

62 S62 NH Bn N Bn 22 CH3 Figure S56. HMBC spectrum of 22.

63 NH N H H CH3 23 Figure S57. 1H NMR spectrum of 23. S63

64 S64 A B C D E F Figure S58. Single and double decoupling experiments resolving coupling constants: (A) Irradiation at CH 3 (C8') simplified the multiplet of H8' into a ddd (J = 11.8, 5.3, 2.0 Hz) i.e. 3 J H8',H7'', 3 J H8',H7', 3 J H8',H2', respectively. (B) Irradiation at H2' simplified the multiplet of H8' into a qdd (J = 7.0, 11.8, 5.3 Hz) i.e. 3 J H8',CH3(C8'), 3 J H8',H7'', 3 J H8',H7' respectively. (C) Irradiation at H8' simplified the multiplet of H2' into a d (J = 5.2 Hz) i.e. 3 J H2',H3'. (D) Irradiation at H7' simplified the multiplet of H2' into a dd (J = 5.2, 2.0 Hz) i.e. 3 J H2',H3', 3 J H2',H7', respectively. (E) Irradiation at both H2' and CH 3 at C-8' simplified the multiplet of H8' into a dd (J = 11.8 and 5.3 Hz) i.e. 3 J H8',H7'' and 3 J H8',H7, respectively(f) Irradiation at both H8' and H7'simplified the multiplet of H2' into a d (J = 5.2 Hz) i.e. 3 J H2',H3'.

65 S65 A : H8' B H6' H7' C : H2' D H7'' H6'' Figure S59. Experimental and simulated spectra for 23. Upper one represents the experimental spectrum while the lower one represents the simulated spectrum of H8' (A), H6' and H7' (B), H2' (C), H7'' and H6'' (D).

66 ppm S Figure S C NMR spectrum (in DMS-d6) of 23. H NH N H CH3 23

67 H NH N H CH3 23 Figure S61. DEPT spectra (in DMS-d6) of 23. S67

68 S68 NH H N H 23 CH3 Figure S62. 2D 1 H CSY spectrum (in DMS-d 6 ) of 23. Highlighted region is shown below in Figure S61.

69 S69 NH H N H 23 CH3 Figure S63. Expansion the δ 2.5 to 0.7 region of the CSY spectrum of 23.

70 S70 NH H N H 23 CH3 S63. Figure S64. 2D TCSY spectrum (in DMS-d 6 ) of 23. Highlighted region is shown below in Figure

71 CH3(C8') S71 H8 H2 ' H6' H7' H7'' H6'' CH3(C8') H6'' H7'' H7' H6' H2' H N NH H8' H 23 CH3 Figure S65.Expansion of δ 2.5 to 0.7 region of the 2D TCSY spectrum of 23. Dashed box shows the spin system to H8' (including H2') and thereby giving the evidence for the ring closure.

72 Figure S66. 1D NESY spectra (in DMS-d 6 ) of compound 23. Irradiation at CH 3 (C8') (shown by thick arrow) results in ne enhancement of 3 % on H1' indicating the close proximity of H1' and CH 3 (C8') (less then 3.5 Å). Taking into account that ab initio calculated H1'-CH 3 (C8') distances are ca. 4.6 Å and 3.0 Å in case of S- and R-configuration of C8', respectively, the ne enhancement found confirms the R- configuration for C8' in 23. Irradiation upon H3' shows the enhancement on H6 indicating that 1-thyminyl moiety is in the β-configuration and anti conformation. S72

73 S73 NH H N H 23 CH3 Figure S67. 2D HMQC spectrum (in DMS-d 6 ) of 23.

74 S74 NH H N H 23 CH3 Figure S68. 2D HMBC spectrum (in DMS-d 6 ) of 23. Highlighted region is shown below in Figure S68.

75 Figure 69. Expansion of the HMBC spectrum of carbocyclic-ena-t (23) proving the formation of the oxabicyclo[3.2.1]octane ring system in the ring closure reaction. Inset (A): Important through-bond long-range ( 3 J H,C and 2 J H,C ) correlations confirming the bond formation between C2' and C8' as the result of ring closure forming carbocyclic-ena-t 23 are shown in the violet boxes. Inset (B) Important HMBC long range connectivities for 23 shown in the square boxes in Inset A. S75

76 DMTr Figure S70. NH N H CH H NMR spectrum of 24. S76

77 NH N DMTr H CH3 24 Figure S C NMR spectrum of 24. S77

78 Figure S P NMR spectrum of 25. NC DMTr P N 25 NH N CH 3 S78

79 Figure S73: Correlation of experimental 3 J H1',H2' and 3 J H2',H3' vicinal coupling constants in carbocyclic- ENA-T (12a,12b) and carbocyclic LNA-T (23) as well as their 2'-- and 2'-N analogs (ENA-T, aza- ENA-T, LNA-T and 2'-amino-LNA-T) shown together with contours of theoretical 3 J H1',H2' vs. 3 J H2',H3' dependencies at fixed sugar puckering amplitudes (from 35 to 65 ) calculated using algorithm and Haasnoot-de Leeuw-Altona generalized Karplus equation reported in Ref. 4, 5. S79

80 Figure S74. verlay of 2500 molecular structures of the carbocyclic-ena-t nucleoside collected every 0.2 ps of the last 500 ps ( ns) of its MD simulation. Analysis of these structures shows essentially equimolar dynamics equilibrium (K = ) between two groups of conformers which mainly differ by the relative positions of aglycon (torsion χ) being in antiperiplanar χ ( ap, Conformation A) and anticlinal χ ( ac, Conformation B) conformations. About one third of material in Conformation A group is having γ torsion in +gauche conformation while γ is antiperiplanar in the majority of Conformation A group (74%) and in all s with anticlinal χ (Conformation B) group. S80

81 S81 carbocyclic-lna-t, major (12a) carbocyclic-lna-t, minor (12b) carbocyclic-ena-t (23) RMSd = Å RMSd = Å RMSd = Å (0.291, 0.500) (0.340, 0.593) (0.171, 0.540) Figure S75. Superposition of 10 randomly selected structures during 1ns of the NMR constrained MD simulations of carbocyclic-lna-t (12a and 12b) and carbocyclic-ena-t (23). Total average RMSd (in Å) are shown for all heavy atoms (marked in black) as well as for the all atoms in sugar and carbocyclic moieties (in parentheses marked in red), the base atoms (in parentheses marked in blue).

82 S82 A: AN 6 [3'-d(CTT (5-carbo) CTTTTTTACTTC)] B: AN 7 [3'-d(CTTCTT (5-carbo) TTTTACTTC)] C: AN 8 [3'-d(CTTCTTTT (5-carbo) TTACTTC)] D: AN 9 [3'-d(CTTCTTTTTT (5-carbo) ACTTC)] Figure S76. Percentage of the ANs containing 5-membered carbocyclic-lna-t nucleotide (ANs 6-9) which remain in the human blood serum at 21 C (for PAGE picture see Figure 8, Inset B). Inset A: The AN 6 with the 5-membered carbocyclic-lna modified at position 3 from the 3'-end was degraded to n-1 fragment from the 3'-end in 5 h, which was found to be fully stable for up to 48 h. Inset B: The AN 7 with 5-membered carbocyclic-lna modified at position 6 from 3'-end showed digestion until n 4 fragment from the 3'-end, which was one nucleotide before the modification site, the n-4 fragment was then found to be stable in human serum for 48 h. Inset C: The AN 8 with 5- membered carbocyclic-lna modified at position 8 from 3'-end shows the cleavage up to n-6 fragment from the 3'-end, which was then stable for 48 h. Inset D: The AN 9 with the 5-membered carbocyclic LNA-T modified at position 10 from 3'-end also resisted digestion one nucleotide before the modification to give the n-8 fragment, which was resistant to further digestion by 3'-exonucleases present in the blood serum for 48 h. Since no further cleavage product was found on the PAGE we considered cleavage product n-1 for AN 6, n-4 for AN 7, n-6 for AN 8 and n-8 for AN 9, as 100 %. Note: Cleavage of one nucleotide from the 3'-end is n-1, and the cleavage of two nucleotides from the 3'-end is n-2, and so on where n is the full length AN. (see Experimental Section for details).

83 S83 A: AN 10 [3'-d(CTT (6-carbo) CTTTTTTACTTC)] B: AN 11 [3'-d(CTTCTT (6-carbo) TTTTACTTC)] C: AN 12 [3'-d(CTTCTTTT (6-carbo) TTACTTC)] D: AN 13 [3'-d(CTTCTTTTTT (6-carbo) ACTTC)] Figure S77. Percentage of ANs containing 6-membered carbocyclic nucleotide (ANs 10-13) remaining in the human blood serum at 21 C (for PAGE picture see Inset C in Figure 8). Inset A: The AN 10 with carbocyclic-ena-t modified at position 3 from the 3'-end was degraded to n-1 fragment from the 3'-end in 5 h, which was found to be completely stable for up to 48 h. Inset B: The AN 11 with the carbocyclic-ena-t modified at position 6 from 3'-end showed digestion until n-4 fragment from the 3'-end, which is was one nucleotide before the modification site, the n-4 fragment was then found to be stable in human serum for 48 h. Inset C: The AN 12 with the carbocyclic-ena-t modified at position 8 from 3'-end shows the cleavage up to n-6 fragment from the 3'-end, which was then stable for 48 h. Inset D: The AN 13 with the carbocyclic-ena-t modified at position 10 from 3'-end also resisted digestion one nucleotide before the modification to give the n-8 fragment, which was resistant to further digestion by 3'-exonucleases present in the blood serum for 48 h. Since no further prominent cleavage product was found we considered cleavage product n-1 for AN 10, n-4 for AN 11, n-6 for AN 12, n-8 for AN 13, as 100 %. Note: Cleavage of one nucleotide from the 3'- end is n-1, and the cleavage of two nucleotides from the 3'-end is n-2, and so on where n is the full length AN (see Experimental Section for details).

84 S84 A: AN 14 [3'-d(CTT (aza-ena) CTTTTTTACTTC)] B: AN 15 [3'-d(CTTCTT (aza-ena) TTTTACTTC)] C: AN 16 [3'-d(CTTCTTTT (aza-ena) TTACTTC)] D: AN 17 [3'-d(CTTCTTTTTT (aza-ena) ACTTC)] Figure S78. Percentage of ANs containing 6-membered aza-ena-t nucleotide (ANs 14-17) remaining in the human blood serum at 21 C (for PAGE picture see inset: D in Figure 8). Inset A: The AN 14 with the aza-ena-t modified at position 3 from the 3'-end was degraded from the 3'-end to n- 1 fragment in 5 h, which gradually gave rise to n-2 fragment as well thus showing cleavage just next to the modification site the two fragments however were found to be stable against further hydrolysis for up to 48 h. Inset B: The AN 15 with the aza-ena-t modified at position 6 from 3'-end showed digestion until n-4 fragment from the 3'-end, in addition to slow addition of n-5 cleavage fragment which results from cleavage of phosphodiester bond at 3'-end of the modification as the two fragments were however found to be resistant to further degradation in human serum for 48 h. Inset C: The AN 16 with the aza-ena-t modification at position 8 from 3'-end shows the cleavage up to n-6 fragment from the 3'-end, as well as n-7 fragment which results from cleavage of phosphodiester bond at 3'-end of the modification which were then stable for 48 h. Inset D: The AN 17 with the aza-ena-t modification at position 10 from 3'-end also resisted digestion giving fragment n-8 which is one nucleotide before the site of modification as well as n-9 fragment resulting from cleavage of phosphodiester bond at 3'-end of the modification which were resistant to further digestion by 3'- exonucleases present in the blood serum for 48 h. Since no further prominent cleavage product was found we considered the products (n-1) + (n-2) for AN 14, (n-4) + (n-5) for AN 15, (n-6) + (n-7) for

85 AN 16, (n-8) + (n-9) for AN 17 as 100 %. Note: Cleavage of one nucleotide from the 3'-end is n-1, and the cleavage of two nucleotides from the 3'-end is n-2, and so on where n is the full length AN (see Experimental Section for details). S85 Figure S79. Autoradiograms of 20% denaturing PAGE showing degradation pattern of 5-32 P-labeled ANs in SVPDE (see Table 3 for full AN sequence). Inset A: LNA modified ANs 2-5. Inset B: Carbocyclic LNA modified ANs 6-9. Inset C: Carbocyclic ENA modified ANs Inset D: aza-ena modified ANs Time points are taken after 0 h, 1 h, 2 h, 24 h, 48 h, 72 h of incubation with the enzyme.

86 S86 A: AN 6 [3 -d(ctt (5-carbo) CTTTTTTACTTC)] B: AN 7 [3 -d(cttctt (5-carbo) TTTTACTTC)] C: AN 8 [3 -d(cttcttt T (5-carbo) TTACTTC)] D: AN 9 [3 -d(cttctttttt (5-carbo) ACTTC)] Figure S80. Percentage of ANs containing 5 - membered carbocyclic nucleoside (ANs 6-9) remaining in snake venom phosphodiesterase at 21 C (for PAGE picture see inset B in Figure S69). Inset A: The stability of the AN 6 with the carbocyclic-lna-t modified at position 3 from the 3 - end. The 15mer AN 6 was degraded completely to n-1 fragment from the 3 -end within 24 h, and the n-1 fragment was found to be stable for up to 72 h. Inset B: The stability of the carbocyclic-lna modified AN 7at position 6 from 3 -end showed digestion to fragments n-1, n-2, n-3 until n-4 fragment (one nucleotide before the modification site) from the 3 -end. The n-4 fragment was then found to be stable till 72 h. Inset C: The stability of the 5 - membered carbocyclic-lna modified AN 8 with modification at position 8 from 3 -end shows the cleavage up to n-6 fragment from the 3 -end, which was then stable for 72 h. Inset D: The stability of the AN 9 with the carbocyclic- LNA-T modified with modification at position 10 from 3 -end also resisted digestion one nucleotide before the modification to give the n-8 fragment which was resistant to further digestion even after 72 h.

87 S87 A: AN 10 [3 -d(ctt (6-carbo) CTTTTTTACTTC)] B: AN 11 [3 -d(cttctt (6-carbo) TTTTACTTC)] C: AN 12 [3 -d(cttctttt (6-carbo) TTACTTC)] D: AN 13 [3 -d(cttctttttt (6-carbo) ACTTC)] Figure S81. Percentage remaining of ANs containing 6 - membered carbocyclic nucleoside (ANs 10-13) remaining in snake venom phosphodiesterase at 21 C (for PAGE picture see Inset C in Figure S69). Inset A: The stability of the AN 10 with the carbocyclic-ena-t modified at position 3 from the 3 -end. Note that the 15mer AN 10 was degraded to n-1 fragment from the 3 -end within 24 h, by the 3 -exonucleases activity giving rise to n-1 fragment which was found to be stable for up to 72 h. Inset B: The stability of the 6 - membered carbocyclic-ena (AN 11) modified at position 6 from 3 -end showed digestion to n-1, n-2, n-3 until n-4 (one nucleotide before the modification site) fragments from the 3 -end. The n-4 fragment was then found to be stable till 72 h. Inset C: The stability of the carbocyclic-ena-t modified (AN 12) with modification at position 8 from 3 -end showed the cleavage up to n-6 fragment from the 3 -end, which was then accumulated and resisted further degradation till 72 h. Inset D: The stability of the AN 13 with the carbocyclic-ena-t modified with modification at position 10 from 3 -end also resisted digestion one nucleotide before the modification to give the n-8 fragment which was resistant to further digestion even after 72 h.

88 S88 A: AN 14 [3 -d(ctt (aza-ena) CTTTTTTACTTC)] B: AN 15 [3 -d(cttctt (aza-ena) TTTTACTTC)] C: AN 16 [3 -d(cttcttt T (aza-ena) TTACTTC)] D: AN 17 [3 -d(cttctttttt (aza-ena) ACTTC)] Figure S82. Percentage remaining of ANs containing 6 - membered aza-ena nucleoside (ANs 14-17) remaining in snake venom phosphodiesterase at 21 C (see PAGE picture: Inset D in Figure S69). Inset A: The stability of the AN 14 with the aza-ena-t modified at position 3 from the 3 - end. Note that the 15 mer AN 14 was degraded to n-1 as well as n-2 fragment from the 3 -end, within 24 h. Both the fragments then resisted further degradation and were found to be stable for up to 72 h. Inset B: The stability of the aza-ena-t modified (AN 15), modified at position 6 from 3 - end, showed degradation to n-1, n-2, n-3, n-4 until n-5 fragments from the 3 -end. The n-4 fragment, which is one nucleotide before the modification site, along with the n-5 segment were then found to be stable upto 72 h. Inset C: The stability of the aza-ena-t (AN 16) modified with modification at position 8 from 3 -end shows the cleavage up to n-6 fragment from the 3 -end. With time n-7 fragment also appears however both n-6 and n-7 fragments were resistant to further degradation till 72 h. Inset D: The stability of the AN 17 with the aza-ena modified at position 10 from 3 -end also resisted digestion one nucleotide before the modification to give the n-8 fragment as well as some n-9 fragment. Both n-8 and n-9 fragments showed resistance from further till 72 h.

89 S89 Table S1. Chemical shifts for the five-membered and six-membered fused carbocyclic 11a, 12a, 12b, 22, and a (CDCl 3 ) 12a (DMS-d6) 12b (DMS-d6) 22 (CDCl 3 ) 23 (DMS-d6) NH H Bn H1' H3' H(C3') H5' * * H5'' H(C5') Bn(CH 2 ) Bn(CH 2 ) Bn(CH 2 ) Bn(CH 2 ) H7' H8' H2' CH3(T) H6' CH3(C7') CH3(C8') H7'' H6'' *Inseparable

90 Table S2. Coupling constants for 11a, 12a, 12b, 22, and 23. S90 11a (CDCl 3 ) 12a (DMS-d6) 12b (DMS-d6) 22 (CDCl 3 ) 23 (DMS-d6) 4 J H6,CH3(T) J H1',H2' J H6',H6'' J H5',H5'' J H6'',H7' J H6',H7' J H6',H7'' J H6'', H7'' J H7',H7'' J H2',H3' J H2',H7' m J H2',H8' J H7', CH3(C7') J H8', CH3(C8') J H8',H7' J H8',H7'' J Bn J Bn J H2',H6''

91 S91 Table S3. Experimental 3 J H,H vicinal proton coupling constants, corresponding ab initio and MD (highlighted in blue) φ H,H torsions and respective theoretical 3 J H,H obtained using Haasnoot-de Leeuw-Altona generalized Karplus equation 4, 5 taking into account β substituent correction (see text). Compound s 3 J H,H 3 J H,H, calc. Torsion (φ H,H ) Hz 3 J H,H, exp. Hz φ H,H ( ), ab initio φ H,H ( ), MD φ H,H ( ), exp.* Δ 3 J H,H, Hz carbocyclic -LNA-T (major) 12a 3 J H1', H2' H1'-C1'-C2'-H2' ± J H2', H3' H2'-C2'-C3'-H3' ± to J H2', H7' H2'-C2'-C7'-H7' ± to J H6', H7' H6'-C6'-C7'- H7' ± to J H6'', H7' H6''-C6'-C7'-H7' ± to carbocyclic -LNA-T (minor) 12b 3 J H1', H2' H1'-C1'-C2'-H2' ± J H2', H3' H2'-C2'-C3'-H3' ± to J H2', H7' H2'-C2'-C7'-H7' ± J H6', H7' H6'-C6'-C7'- H7' ± to J H6'', H7' H6''-C6'-C7'-H7' ± to J H1', H2' H1'-C1'-C2'-H2' ± J H2', H3' H2'-C2'-C3'-H3' ± to J H2', H8' H2'-C2'-C8'-H8' ± to carbocyclic -ENA-T 3 J H7', H8' H7'-C7'-C8'-H8' ± to J H7'', H8' H7''-C7'-C8'-H8' ± to J H6', H7' H6'-C6'-C7'- H7' ± to J H6', H7'' H6'-C6'-C7'-H7'' ± to J H6'', H7' H6''-C6'-C7'-H7' ± J H6'', H7'' H6''-C6'-C7'-H7'' ± to Non-observable constants are marked in red. * Assuming 0.5 Hz accuracy of experimental 3 J H,H coupling constants

92 S92 Table S4. Sugar torsions (ν0 - ν4 ), pseudorotational phase angle (P), 6 sugar puckering amplitude (φm), 6 backbone (γ,δ) and glycoside bond (χ), as well as selected torsions* (C3'-C2'-X2'-Y' and C3'-C4'-C6'-C7') characterizing five- and six-membered ring in carbocyclic-lna-t (major and minor isomers, 12a and 12b), carbocyclic-ena-t (23), aza-ena-t, ENA-T 1, 2'-amino-LNA-T 2 and LNA-T 3 compounds. The structural parameters are obtained from the ab initio molecular structures as well as from the last 0.5 ns of unconstrained MD simulations (average values and standard deviations are shown in brackets and highlighted in blue) of the respective nucleosides. NH NH NH NH NH NH NH Torsions* H H H 5' N 5' N 5' 4' 6' 3' 2' H 7' 1' Me 4' 1' 3' 2' 4' 3' 6' H Me 7' H H H N 5' N N 6' 7' Me H 8' 1' 2' 4' 3' 1' 2' H 5' N N 4' 1' 3' 2' 6' 7' 6' NH H NH H H H (A) (B) (C) (D) (E) (G) (H) carbocyclic- LNA-T (major) 12a carbocyclic- LNA-T (minor) 12b carbocyclic- ENA-T aza-ena-t ENA-T 2'-amino-LNA-T LNA-T 23 ν0: C4'-4'-C1'-C2' 0.5 (3.4 ± 5.7) 2.8 (5.5 ± 5.6) -0.7 (1.0 ± 7.2) -0.9 (-0.5 ± 6.8) -1.1 (5.8 ± 6.0) -1.6 (-0.7 ± 5.5) -1.6 (0.1 ± 5.6) ν1: 4'-C1'-C2'-C3' (-39.6 ± 4.7) (-41.5 ± 4.5) (-30.4 ± 5.7) (-29.5 ± 5.5) (-34.3 ± 4.9) (-37.7 ± 4.6) (-37.8 ± 4.7) ν2: C1'-C2'-C3'-C4' 53.1 (55.1 ± 3.0) 53.9 (55.9 ± 2.9) 43.5 (45.5 ± 3.5) 43.9 (44.8 ± 3.4) 43.7 (46.1 ± 3.4) 52.6 (56.0 ± 2.8) 53.2 (55.3 ± 3.0) ν3: C2'-C3'-C4'-4' (-55.0 ± 3.6) (-45.5 ± 3.8) (-46.8 ± 4.0) (-47.6 ± 4.0) (-45.3 ± 4.0) (-59.1 ± 3.3) (-57.8 ± 3.4) ν4: C3'-C4'-4'-C1' 26.8 (34.1 ± 5.5) 33.3 (26.6 ± 5.5) 29.2 (29.1 ± 6.4) 29.7 (30.5 ± 6.1) 28.9 (25.2 ± 5.5) 37.0 (39.5 ± 5.2) 37.4 (38.1 ± 5.3) P 14.6 (15.2 ± 5.8) 15.8 (13.1 ± 5.6) 19.4 (17.5 ± 8.4) 19.4 (19.4 ± 8.0) 19.1 (12.1 ± 7.2) 20.0 (19.6 ± 5.3) 19.8 (18.8 ± 5.4)

93 S93 ψm 47.5 (57.3 ± 3.0) 57.8 (57.7 ± 2.9) 47.1 (48.3 ± 3.3) 46.5 (48.0 ± 3.4) 46.3 (47.6 ± 3.3) 55.9 (59.7 ± 2.8) 56.5 (58.7 ± 2.9) 64.5 (53.1 ± 10.7) γ: 5'-C5'-C4'-C3' 64.9 (54.9 ± 9.1) 66.4 (56.3 ± 11.2) 64.1 (53.1 ± 10.7) 63.9 (57.3 ± 9.3) (169.5 ± 51.5) 66.2 (57.2 ± 19.6) 66.6 (58.1 ± 10.3) ( ±10.4) δ: C5'-C4'-C3'-3' 72.7 (60.0 ± 5.3) 63.1 (64.3 ± 5.7) 71.2 (74.5 ± 5.9) 75.6 (74.5 ± 5.9) 76.0 (73.5 ± 5.8) 66.6 (63.9 ± 5.7) 65.8 (63.0 ± 5.8) χ: 4'-C1'-N1-C ( ± 10.1) ( ± 8.8) ( ± 10.8) ( ± 10.8) ( ± 10.9) ( ± 8.7) ( ± 9.9) C3'-C2'-X2'-Y' 64.3 (66.7 ± 5.0) 31.3 (33.7 ± 4.3) 60.9 (67.0 ± 4.8) 68.1 (67.0 ± 4.8) 65.9 (65.1 ± 5.6) 43.1 (39.9 ± 4.4) 38.9 (40.0 ± 5.1) C3'-C4'-C6'-C7' (-57.8 ± 5.1) (-40.2 ± 4.6) (-55.5 ± 5.2) (-55.5 ± 5.2) (-56.1 ± 5.4) (-36.0 ± 4.7) (-35.6 ± 4.6) * X2' represents 2', N2', or C7' where applicable while Y' is C6', C7' or C8' atom which neighbors X2' and belongs to the sugar-fused (hetero)cycle.

94 S94 Table S5. Solvation energy (E solv ) calculated using Baron and Cossi s implementation of the polarizable conductor CPCM model 7 as implemented in Gaussian98. 8 (Polarized solute)-solvent energy, kcal/mol Total non electrostatic energy, kcal/mol Total solvation energy, E solv, kcal/mol ΔE solv, kcal/mol LNA-T '-amino-LNA-T carbocyclic-lna-t ENA-T aza-ena-t carbocyclic-ena-t deoxy-t ribo-t* '-amino-T '-Me-T oxetane-t azetidine-t ribo-t is used as reference for calculation of ΔE solv = E solv () - E solv (reference)

95 Discussion S1. S95 NMR Characterization of five-membered fused carbocyclic-lna-t (12a & 12b) and the sixmembered carbocyclic-ena-t (23). The characterization and conformational analysis of the fused carbocyclic compounds 12a, 12b and 23 have been performed using NMR spectroscopy ( 1 H at 500 and 600 MHz in CDCl 3 /D 2 /DMS-d 6 ) obtained by 1 H homodecoupling experiments, 1D nuclear verhauser effect spectroscopy (1D NESY), 9 2D total correlation spectroscopy (TCSY), 10 2D CSY, and 13 C NMR experiments, including distortionless enhancement by polarization transfer (DEPT), 11 as well as long-range 1 H- 13 C HMBC correlation ( 2 J H,C and 3 J H,C ), 12 and a one-bond heteronuclear multiple-quantum coherence (HMQC) 13 experiments. For the complete 1D and 2D NMR spectral assignments on the basis of the NMR data, as well as the actual NMR spectra for compounds 12a, 12b, and 23 see the Figures S22 S39 and S57-S69). All chemical shifts and coupling constants for compounds 12a, 12b and 23 are shown in Tables S1 and S2. Spin-spin simulations (Figures S27 and S59) to validate experimental coupling constant information were performed by using MestRec 14 software. It should be noted that the first sugar-fused carbocyclic 5-membered bicyclic compound was 3', 5'- Di--benzyl protected nucleoside 11a/11b obtained as a diastereomeric mixture. However, because of the overlap of 1 H NMR peaks for the H7' and H2', which are crucial for the proof of the ring closure between C2' and C7', the de-protected compounds 12a/12b, which have these two crucial peaks unobstructed, has been used for the characterization (for all NMR information see Figures S22-S39). 4.1 Diastereomeric mixture of the 5-membered carbocyclic LNA-T. The 1 H spectrum of 12 shows the presence of two diastereomers, a major (12a) as well as a minor isomer (12b); in proportion ca. 7:3, respectively (Figure S22). Analysis of 1D NE of 12a and 12b allowed us to identify the major and minor diastereoisomers to be in R- and S-configurations, respectively, at C7'.

96 S Major compound 12a Analysis of 1 H and 2D 1 H CSY spectra of the carbocyclic sugar-fused 5-membered compound 12a allowed us to identify H7' (δ2.59) because of its vicinal coupling ( 3 J H,H ) with the CH 3 at C7', H6', H6'', and H2'. Thus the multiplicity of H7' consists of 32 lines centered at δ2.59 [i.e. 3 J H7',H2', 3 J H7',H6', 3 J H7',H6'', and 3 J H7',CH3(C7'), see Tables S 1 and S2 for all 3 J H,H ] resulting from quartet of doublet of doublet of doublet, which were resolved by stepwise 1 H homonuclear single and double decoupling experiments (see Figure S24 for decoupling experiments and Figure S25 for spin-spin simulations). The upfield shift of H6'' (δ0.95, doublet of doublet) of compound 12a could be distinguished from the downfield H6' proton (δ1.91, doublet of doublet) by the fact that the former has a smaller 3 J H,H coupling of 5.0 Hz with H7' ( 3 J H7',H6'' ) beside the geminal coupling of 12.3 Hz, whereas the latter has a large 3 J H,H coupling of 10.6 Hz ( 3 J H7',H6' ) beside the geminal coupling of 12.3 Hz. This shows that the dihedral angle between H7' and H6', φ[h7'-c7'-c6'-h6'], is closer to zero while that between H7' and H6', φ[h7'-c7'-c6'-h6''], is close to ca 180. bservation of the vicinal coupling between H2' and H7', as evidenced by the decoupling experiments (Figure S24 ) and CSY spectra (Figures S30-S32), unequivocally proves that the oxabicyclo[2.2.1]heptane ring system has indeed been formed in the ring-closure reaction to give 12a (Scheme 1). Furthermore 2D TCSY spectrum showed that the H2', H7', H6' and H6'' belong to the same coupled spin system (Figures S33, S34). This evidence was further strengthened by the observation of the long range 1 H- 13 C connectivity of H7' with C2' and C1' ( 2 J H7',C1' ) and that of H2' with CH 3 (C7') ( 3 J H2',CH3(C7') ) in HMBC experiment (Figure S38 and S39). The vicinal coupling constant of 4.5 Hz for 3 J H7',H2' indicates that the dihedral angle between H2' and H7' is close to 45. Strong ne enhancement (12.1 %, corresponds to ca. 2.6 Å) between H6 (thymine) and H3' (Figure S35) of 12a, in addition to 3 J H1',H2' = 0 Hz, further confirms that the sugar is indeed locked in the North conformation as observed for the other North-locked nucleosides such as ENA 1, LNA 3, and aza-

97 S97 ENA 15 (8 to 10 % ne between H6 and H3'). This important H6 (thymine) to H3' ne contact shows that the 1-thyminyl moiety is in β-configuration and anti-conformation across the glycoside bond. The fact that the ne enhancement of 6.5 % for H1' upon irradiation on CH 3 at C7' (Figure S35) shows that the methyl group on C7' is in close proximity of H1' (ca. 2.8 Å). This observation is further strengthened by the fact that H7' has a long range 3 J H,C coupling to C1' in HMBC, which suggests that the dihedral angle between H7' and C1', φ[h7'-c7'-c2'-c1'], is about 180, thereby confirming that the H7' is in the pseudoaxial orientation and the CH 3 group is indeed in the pseudo-equatorial position (R configuration at C7') Minor diastereomer 12b Analysis of 1 H spectrum of the minor diastereomer 12b allowed us to identify H7' (δ2.04) because of its vicinal coupling ( 3 J HH ) with the CH 3 at C7', H6' and H6''. Thus the multiplicity of H7' consists of 16 lines centered at δ2.04 [i.e. 3 J H7',H6', 3 J H7',H6'', and 3 J H7',CH3(C7'), see Table S2 for all 3 J H,H ] resulting from quartet of doublet of doublet, which were resolved from stepwise 1 H homonuclear single and double decoupling experiments (Figure S26) as well as by spin-spin simulations (Figure S27). Another interesting observation is that the lack of coupling between H7' and H2', i.e. 3 J H7',H2' = 0. The absence of vicinal coupling between H2' and H7' indicates that the dihedral angle between H2' and H7' is closer to 90. The upfield shift of H6' (δ 1.44, doublet of doublet) of compound 12b could be distinguished from the downfield H6'' proton (δ 1.75, doublet of doublet) by the fact that the former has a smaller 3 J H,H coupling of 5.0 Hz with H7' ( 3 J H7',H6' ) beside the geminal coupling of 12.2 Hz, whereas the latter has a large 3 J H,H coupling of 8.8 Hz ( 3 J H7',H6'' ) beside the geminal coupling of 12.2 Hz. This shows that the dihedral angle between H7' and H6', φ[h7'-c7'-c6'-h6'], is closer to a median value of (135 ) while that between H7' and H6'', meaning that the dihedral angle, φ[h7'-c7'-c6'-h6''], is closer to ca. 0. These assignments were further confirmed by CSY, HMQC, and HMBC experiments (Figures S30-S32, S37, and S38). Furthermore the observation of long range 3 J H,C and 2 J H,C connectivities of H2'-C6', H7'-

98 S98 C2', H7'-C1' unequivocally proves that the oxa-bicyclo [2.2.1] heptane ring system has indeed been formed in the minor diastereomer too as the result of the ring-closure reaction [(Scheme 1), Figure S39]. Strong ne enhancement (11 %, corresponds to ca. 2.2 Å) between H6(thymine) and H3' (Figure S36 ) for the minor diastereomer 12b, in addition to 3 J H1',H2' = 0 Hz, further confirms that the sugar is indeed locked in the North-type conformation as observed for other North-locked nucleosides such as ENA, 1 LNA, 3 and aza-ena. 15 This ne enhancement shows that the 1-thyminyl moiety is in β- configuration and anti conformation across the glycoside bond as seen for the major 12a. The ne enhancement of 4.5 % for H7' (corresponds to ca. 2.4 Å) upon irradiation at H1' (Figure S36) proved that the H7' is in close proximity of H1' thus indicating S-configuration for the C7' chiral center. 4.2 NMR characterization of the carbocyclic-ena-t (23). Examination of 1 H and 2D 1 H CSY spectra for compound 23 (Scheme 2) allowed us to identify H8' (δ2.25) because of its vicinal ( 3 J H,H ) coupling with the CH 3 at C8', H7', H7'', and H2'. Thus the multiplicity of H8' consists of 32 lines centered at δ2.25 [i.e. 3 J H8',H2', 3 J H8',H7', 3 J H8',H7'', and 3 J H8',CH3(C8'), see Table S2 for all 3 J H,H ], which were resolved from the stepwise 1 H homonuclear single and double decoupling experiments (Figure S58). Apparent broad doublet of H2' at δ1.97 indicated that the H2' has some additional coupling(s) to another atom other than H3' and H8'. The decoupling experiment of H7', H7'' at (δ1.57) resolved this broad doublet into a doublet of doublet (Figure S58). This indicated that the peak at δ1.57 has a 4 J H,H coupling to H2' besides the coupling with H3' and H8'. This is only possible when the peak at δ1.57 is in the axial position thus having a W-coupling to H2', and it can be assigned as H7'. The H7'' could be assigned to the multiplet at δ1.23 by comparison of CSY spectra (Figure S62, S63). The multiplicity pattern of H7' (dt, J = 13.4, 5.5, 5.5 Hz) and CSY spectra reveals that only one of the H6'/H6'' has coupling to H7' and it can be assigned to H6' hence 3 J H7',H6'' = 0 Hz ( 3 J H7',H7'' = 13.4 Hz, 3 J H7',H6' = 5.5 Hz. 3 J H7',H8'' = 5.5 Hz). The observation that the 3 J H7',H6'' = 0 Hz indicates that the dihedral

99 S99 angle between H7' and H6'', φ[h7'-c7'-c6'-h6''], is close to 90. Furthermore, the coupling constants of 5.5 Hz for 3 J H7',H6' and 3 J H8',H7' shows that the dihedral angles between H7' and H6', φ[h7'-c7'-c6'-h6'], and H8' and H7', φ[h8'-c8'-c7'-h7'], are close to 45. This is consistent with what we have observed in the coupling patterns for the aza-ena analogue. 15 Vicinal coupling of H2' with H8' as evidenced by decoupling experiments and CSY spectra (Figures S58, S62, S63) unequivocally showed that the bicyclo [3.2.1] octane ring system has indeed been formed in the ring-closure reaction (Scheme 2). 2D TCSY spectrum is also in agreement that the H2', H8', H7', H7'', H6' and H6'' belong to the same spin system (Figures S64, S65), which was confirmed by both homonuclear decoupling experiment as well as by spin-spin simulations (Figures S58, S59). This evidence was further corroborated by the observation of the long range 1 H- 13 C connectivity of H8' with C2' and C1', H7' with C2' and H2' with CH 3 (C8') (Figure S69) in HMBC experiment. Strong ne enhancement (8.6 %, corresponding to ca. 2.6 Å) between H6 (thymine) and H3' (Figure S66) for the compound 23, in addition to 3 J H1',H2' = 0 Hz, further confirms that the sugar is indeed locked in the North-type conformation as observed for 12a and 12b as well as for other North-locked nucleosides such as ENA 1, LNA 3, and aza-ena. 15 This ne contact also shows that the 1-thyminyl moiety is in β-configuration and anti conformation across the glycoside bond. The ne enhancement of 3.0 % for H1' upon irradiation at CH 3 (C8') (Figure S66) proved that the CH 3 (C8') group is in close proximity of H1', thus the C8' chiral center is in R-configuration. The long range 3 J H,C coupling between H7' and C2' in HMBC (Figure S69) suggests that the dihedral angle, φ[h7'-c7'-c8'-c2'], is about 180, whereas that between H7'' and H2' (φ[h7''-c7'-c8'-c2']) is about 90.