Conformationally Constrained 2'-N,4'-C-Ethylene Bridged...

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S1 Supporting Information Conformationally Constrained 2'-N,4'-C-Ethylene Bridged Thymidine (Aza-ENA-T): Synthesis, Structure, Physical and Biochemical Studies of Aza- ENA-T Modified Oligonucleotides Oommen P. Varghese, Jharna Barman, Wimal Pathmasiri, Oleksandr Plashkevych, Dmytro Honcharenko and Jyoti Chattopadhyaya * Department of Bioorganic Chemistry, Box 581, Biomedical Center, Uppsala University, SE-75123 Uppsala, Sweden. [email protected] Table of Content Figure S1. Structures of North-type conformationally-constrained α/β-D/L-pentofuranosyl nucleoside derivatives ………………………………………………………………....S4 Discussion S1. Conformationally constrained nucleosides and their T m properties……………..S4 Discussion S2. The properties of ENA containing oligos. …………….…………………….…..S6 Figure S2. 1 H- 13 C HMBC spectrum of C in Scheme 1 [Inset (i)]; 1 D selective NOSEY spectrum of C [Inset (ii)]. ……………………………………………………………………….….S7 Discussion S3. Assignments of proton resonances in 12a, 12b and 13. …………………...……S8 Discussion S4. The 3 J H,H simulation and nOe studies of 12a and 12b and 13. …………….……S10 Figure S3. The CD spectra of aza-ENA-T modified AON/DNA and AON/RNA duplexes. …..S11 Discussion S5. Circular Dichroism (CD) analysis of the aza-ENA-T modified AON/DNA and AON/RNA duplexes. ………………………………………………………..………...S12 Table S1. Observed cleavage rate and relative cleavage rate (with respect to control native oligonucleotide) for RNase H digestion as well as T m (°C) found for the AON/RNA duplexes compared to the native counterpart. ………………………………………...S12 Discussion S6. Experimental methods. ……………………………………………………...S13 Discussion S7. Theoretical calculations. ………………………………………………….…S16 Corresponding author

Transcript of Conformationally Constrained 2'-N,4'-C-Ethylene Bridged...

S1

Supporting Information

Conformationally Constrained 2'-N,4'-C-Ethylene Bridged Thymidine (Aza-ENA-T): Synthesis,

Structure, Physical and Biochemical Studies of Aza-ENA-T Modified Oligonucleotides

Oommen P. Varghese, Jharna Barman, Wimal Pathmasiri, Oleksandr Plashkevych,

Dmytro Honcharenko and Jyoti Chattopadhyaya*

Department of Bioorganic Chemistry, Box 581, Biomedical Center,

Uppsala University, SE-75123 Uppsala, Sweden.

[email protected]

Table of Content

Figure S1. Structures of North-type conformationally-constrained α/β-D/L-pentofuranosyl

nucleoside derivatives ………………………………………………………………....S4

Discussion S1. Conformationally constrained nucleosides and their Tm properties……………..S4

Discussion S2. The properties of ENA containing oligos. …………….…………………….…..S6

Figure S2. 1H-13C HMBC spectrum of C in Scheme 1 [Inset (i)]; 1 D selective NOSEY spectrum of

C [Inset (ii)]. ……………………………………………………………………….….S7

Discussion S3. Assignments of proton resonances in 12a, 12b and 13. …………………...……S8

Discussion S4. The 3JH,H simulation and nOe studies of 12a and 12b and 13. …………….……S10

Figure S3. The CD spectra of aza-ENA-T modified AON/DNA and AON/RNA duplexes. …..S11

Discussion S5. Circular Dichroism (CD) analysis of the aza-ENA-T modified AON/DNA and

AON/RNA duplexes. ………………………………………………………..………...S12

Table S1. Observed cleavage rate and relative cleavage rate (with respect to control native

oligonucleotide) for RNase H digestion as well as ∆Tm (°C) found for the AON/RNA

duplexes compared to the native counterpart. ………………………………………...S12

Discussion S6. Experimental methods. ……………………………………………………...S13

Discussion S7. Theoretical calculations. ………………………………………………….…S16

• Corresponding author

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Figure S4. 20% PAGE analysis of RNase H digestion of 5'-32P-labeled AONs 1 to 5 at enzyme

concentration of 0.08 U………………………………………………………......…S17

Figure S5. 20% denaturing PAGE, showing the degradation pattern of 5'-32P-labeled AON 1 to 5

in human serum……………………………………………………………….……..S18

Figure S6 to S8. 1H-NMR experimental and simulated spectra of compound 12a, 12b

and 13. …………….………………………………………………………..…….S19 –S21

Table S2 to S5. 1H, 13C chemical shifts and coupling constants of 12a, 12b, 13 and ENA.S22 –S25

Figure S9 to S14. 13C, 1D NOE, HSQC, HMBC, DQF-COSY and 1H Homodecoupling NMR

spectra of compound C in Scheme 1…………………………….………….…......S26 - S34

Figure S15-S21. 13C NMR spectra of compounds 4, 6 to 11.. ……………………......……S35 –S41

Figure S22-S25. 13C, HSQC, HMBC, COSY respectively NMR spectra of 12a. ……....…S42 –S45

Figure S26-S30. 13C, TOCSY, HSQC, HMBC, COSY, respectively NMR spectra of 12b. S46 –S50

Figure S31-S33. 13C NMR spectra of compounds 14, 15 and 16. …………………….…...S51 –S53

Figure S43. 31P NMR spectrum of compound 17……………………………………..…....S54

Figure S35-S38. 13C, 13C DEPT, HSQC, HMBC, and COSY NMR spectra of 13. …...…..S55 –S58

Figure S39-S40. 13C NMR spectra of compounds 18 and 19. …………………………….S59 – S60

Figure S41. 31P NMR spectrum of compounds 20. ……………………………….…..…...S61

Figure S42. Population distributions of the H7'-C7'-N-H and H7''-C7'-N-H torsions in the N-H

axial (12b) and equatorial (12a) diastereomers during the last 0.5 ns of constrains-free

MD simulations. ……………………………………………………………….......S62

Figure S43. Time-dependent equilibration of 12b into 12a in CDCl3 to calculate corresponding

unimolecular rate and equilibrium constants. …………………………...…..….…S63

Discussion S8. Generalized Karplus parameterization…………………………………......S64

Table S6. Experimental 3JH,H vicinal proton coupling constants, corresponding ab initio and MD

(highlighted in blue) φH,H torsions and respective theoretical 3JH,H obtained using Haasnoot-

de Leeuw-Altona generalized Karplus equation taking into account β substituent

correction. ……………………………………………………………………….…S65

Table S7. Sugar torsions (νννν0 - νννν4 ), pseudorotational phase angle (P), sugar puckering amplitude

(φφφφm), backbone (γγγγ, δδδδ) and glycoside bond (χχχχ), as well as selected torsions (C3'-C2'-O2'

(N2')-C7') and C3'-C4'-C6'-C7') characterizing six-member piperidino heterocycle in 3',5'-

bis-OBn protected and de-protected aza-ENA-T (12a,12b,13) and ENA. ……..…S66

Figure S44: Experimental 3JH1',H2' and 3JH2',H3' vicinal coupling constants of the ENA, aza-ENA-T,

LNA and 2'-amino LNA analogs shown together with contours of theoretical 3JH1',H2' vs. 3JH2',H3' dependencies at fixed sugar puckering amplitudes. ………………….…S67

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Figure S45: Elucidation of the piperidino ring conformation based on experimental 3JH6a/e',H7a/e'

coupling constants (horizontal lines) of the equatorial/axial H6e'/H6a' and H7e'/H7a' protons

of 13, and the theoretical Haasnoot-de Leeuw-Altona generalized Karplus dependences

(ideal sp3 hybridization of C is assumed) for all possible H6'(H6'')-H7'(H7'') vicinal

couplings. ……………………………………………………………………….S68

Figure S46. Superposition of 10 randomly selected structures during last 400 ps of the

unconstrained 2 ns MD simulations of 3',5'-bis-OBn protected (12a,12b) and de-protected

(13) aza-ENA-T as well as the total average RMSds (in Å). …………………..S69

Figure S47-S48. NMR titration plots of pH vs chemical shift of specified marker proton to obtain

pKas of 2'-amine protonation and N3 de-protonation in aza-ENA-T and 2'-amino-LNA-T as

well as the respective Hill plot analysis. ………………………………………..S70-S71

Figure S49. Molecular mechanics (Amber force field) optimized stereochemical representations of

diastereomers of stable fused product (C) (Scheme 1). …………………………S72

References of SI. ……………………………………………………………………….…S73

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(S1) Conformationally constrained nucleosides and their Tm properties:

Incorporation of the North-type sugar conformationally constrained nucleotides (-1° < P <

34°) into AON has been found to be an excellent antisense strategy to induce several favorable

properties to the modified AONs compared to the native counterparts. These nucleotides have

allowed highly favored duplex formation with complementary RNA as they had RNA-like (C3'-

endo) sugar conformation1-3 and have shown enhanced stability towards the endo and exonucleases

in the blood serum. Notable examples are the 2',4'-bridged nucleosides such as: the 2'-O,4'-C-

methylene bridged nucleoside (LNA/BNA) (structure A in Figure S1), reported independently by

Wengel et al.1 and Imanishi et al.4 having furanose fused [2.2.1] five-membered ring. LNA

incorporated oligonucleotides showed unprecedented level of affinity towards complementary RNA

(∆Tm ~ +4° to +8 °C per modification) and a moderate increase of +3 to +5 °C per modification

towards complementary DNA.1 Wengel’s group has also reported increased affinity towards

complementary RNA for other LNA analogs having 2'-thio5 (∆Tm ~ +8 °C per modification), 2'-

amino6, 7 (structure B in Figure S1, ∆Tm ~ +6 to +8 °C per modification), xylo-LNA8, 9 (structure C

in Figure S1, ∆Tm ~ -4 °C per modification) and α-L-LNA8, 10 (structure D in Figure S1, ∆Tm ~ +4 to

+5 °C per modification) function. Wang and co workers have reported 2',4'-C-bridged 2'-

deoxynucleotides2, 11 (structure E in Figure S1), which showed moderate affinity to RNA (∆Tm ~

+1.9 to +3.3 °C per modification). Other bridged nucleosides having 2',4'-C-propylene-bridged 2'-

deoxynucleoside12 (structure F in Figure S1) have also been reported which showed unfavourable

BO

OO

OB

O

NHO

O

BO

O

O

BO

OO

O

O

BO

O

O

BO

O

O

O

BO

O

O

O

BO

OO

O

P OO P OOP OO

P OO

P OO P OOP OO

P OO

E

G

1'2'3'4'

5'

A B C D

F H

BO

OO

O

P OO

BO

HNO

O

P OO

I J

Figure S1: Structures of North-type conformationally-constrained α/β-D/L-pentofuranosyl nucleoside derivatives

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duplex formation with complementary RNA (∆Tm ~ -2.3 °C per modification). We have previously

reported, 1',2'-bridged oxetane13, 14 (structure G in Figure S1), and azetidine15 (structure H in Figure

S1), North–type conformationally constrained nucleotides which have shown sequence-specific

target affinity in the following manner: ∆Tm drop for the AON/RNA duplexes of ~ -5 °C per

oxetane-T, ~ -3 °C per oxetane-C and no Tm drop for oxetane-A and oxetane-G13 modification, and -

4 °C for azetidine-T/U and to -2 °C for azetidine-C15 modification.

Another North-constrained sugar modification recently reported by Koizumi et al.3 is the 2'-

O,4'-C-ethylene bridged nucleoside3, 16 (ENA) (structure I in Figure S1), which showed target

affinity with complementary RNA (∆Tm ~ +3.5 to +5.2 °C per modification) as high as that of the

isosequential LNA16. The same group reported 2'-O,4'-C-propylene bridged nucleoside (PrNA)3

(structure J in Figure S1) which on the other hand did not show appreciable Tm enhancement (∆Tm ~

-0.5 °C per modification).

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(S2) The properties of ENA containing oligos

High target affinity has also been reported17 for the triplex formation by ENA monosubstituted

oligopyrimidine 2'-deoxynucleotides efficiently binding to dsDNA at physiological pH.17

ENA/DNA chimeric AONs have also been used to study the RNase H mediated antisense activity.18

More than 90% inhibition of VEGF mRNA production was observed after RT-PCR analysis18 when

these AONs were used against vascular endothelial growth factor (VEGF) mRNA in A549 lung

cancer cells in the presence of a cationic polymer. Incorporation of the ENA residues at 3'- and 5'-

ends of AONs that specifically target the genes of rat organic anion transporting polypeptide (oatp)

subtype resulted in enhanced inhibitory activity mediated by RNase H with high selectivity19. AONs

having 2'-O-methyl RNA/ENA chimera were found to be 40 times more effective in exon-19-

skipping compared to conventional phosphorothioate AONs associated with human dystrophin

gene.20 Similarly, the RNA/ENA chimera duplexes were also found to induce skipping of the exon-

41 containing the nonsense mutation which suggested that such design of ENA incorporated AONs

could be used to promote dystrophin expression in mycocytes of Duchenne Muscular Dystrophy

(DMD) patients.21 The RNAi effect of chemically synthesized siRNA targeting the mRNA of Jun

dimerization protein-2 (JDP2) with ENA modification at the 3'-end of either sense or antisense

strands completely abolished RNAi activity.22

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ppm

5.75.85.96.0 ppm

60

65

70

Figure S2. Inset (i): 1H-13C HMBC spectrum showing the long range through-bond connectivities

between C2'/H6' (2JCH), C3'/ H6' (3JCH), and C5'/H6' (3JCH) for 2',4'-cyanomethylene bridged

carbocyclic thymine nucleoside (compound C, Scheme 1), Inset (ii): 1D selective NOESY spectrum of

compound C (Scheme 1) showing enhancements at H6 (7.5%), H6' (3.8%), and H2' (4.3%) upon

irradiation at H3'.

(i)

OBnO

OBn

NH

N

O

O

NC

1'2'

3'4'

5'

6'

C2'

C1'

C3'

C5'

H6'

(ii)

C (from Scheme 1)

4.0 ppm5.0 ppm7 ppm 3.4 ppm

H3' H2' H6' H6

7.5% 3.8% 10%

Bn

4.3% Bn

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(S3) Assignments of proton resonances for 12a, 12b and 13

(A) Major isomer 12a

The upfield H6e' proton (δ1.31) of 12a was distinguished from the H6a' proton (δ2.02) by the fact

that the former has only a smaller 3JH,H coupling of 4.8 Hz beside a geminal coupling of 13 Hz,

whereas H6a' proton has a large trans 3JH,H coupling of 11.6 Hz and a cisoid 3JH,H coupling of 6.7 Hz.

This assignment of H6a'/H6e' protons allowed us to identify immediately the H7a'/H7e' protons (at

δ3.13 and δ3.02) by a double-decoupling experiment (decoupled simultaneously at δ2.02 and

δ1.31). Similarly, a stepwise decoupling of either H6e' or H6a' proton led us to determine the vicinal

coupling constant for H7e' or H7a' proton. The four-line multiplet (doublet of doublet, 3JH-7e,H-6a = 6.5

Hz and 2JH-7a',H-7e'= 13.3 Hz) at δ3.02 was thus assigned to H7e' and the eight-line multiplet at δ 3.13

(doublet of doublet of doublet, 3JH7a',H6a' = 11.6 Hz, 3JH7a',H6e'= 4.8 Hz, 2JH7a',H7e'= 13.3 Hz) was

assigned to H7a'. This shows that the dihedral angle between H6e' and H7e', φ[H7e'-C7'-C6'-H6e'], is

close to 90°. These assignments were further confirmed by detailed proton spin-spin simulation

experiments (see Figure S3-S5 in SI) as well by 2D COSY spectra (see Figure S20 in SI). There was

no 3JH7a',H7e' coupling observed with the vicinal N-H proton, suggesting that the N-H proton is

presumably cisoid with both H7a'/H7e' protons (i.e. N-H proton is most probably equatorial).

Furthermore, the HMQC and HMBC spectra allowed us to correlate the 1H chemical shifts with the 13C chemical shifts. The HMBC spectrum showed that only H7e' had correlations with C2' but not

between H7a' and C2'. This suggested that the dihedral angle between H7e' and C2', φ[H7e'-C7'-N-

C2'], is close to180°, whereas H7a' and C2', φ[H7a'-C7-N'-C2'], is about 90°. The presence of long-

range HMBC correlation of H7e' with C2' also unequivocally showed that the six-membered

piperidino [3.2.1] ring fused with the pentofuranose ring has indeed been formed in the ring-closure

reaction (11 → 12a/12b, Scheme 2).

(B) Minor isomer 12b

Analogous to the assignment of the axial and equatorial H6' protons of the piperidino ring, the H6e'

proton (δ1.53) of 12b was distinguished from the H6a' proton (δ2.04) by the fact that the former has

only a smaller 3JH,H coupling of 5.3 Hz beside a geminal coupling of 13.4 Hz, whereas H6a' proton

has a large trans 3JH,H coupling of 11.9 Hz and cisoid 3JH,H coupling of 6.7 Hz. A set of double and

single decoupling experiments at the centre of multiplets of H6a' and/or H6e' protons gave the

assignment of both the H7a' and H7e' protons. From the geometrical point of view the H7a' is

expected to have different couplings with H6a' (φ[H7a'-C7'-C6'-H6a'] is close to 180°, hence 3JH,H

coupling should be large) and H6e' (φ[H7a'-C7'-C6'-H6e'] is close to 55° hence 3JH,H coupling should

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be small). The 3JH,H coupling constant between equatorial H7e' and H6a' is expected to be medium

(φ[H7e'-C7'-C6'-H6a'] is about 40°), while that between H7e' and H6e' is expected to be zero (φ[H7e'-

C7'-C6'-H6a'] is close to 90°). Thus, the H7a' has been assigned to the 12-line multiplet (doublet of

triplet of doublet) at δ 2.98 because it has three distinguished vicinal couplings (3JH7a', H6a' = 11.9 Hz

and 3JH7a', H6e' = 5.2 Hz and 3JH7a', NHa= 11.9 Hz) besides the geminal coupling (2JH7a', H7e'= 14.2 Hz).

The H7e' has been unambiguously assigned to the 8-line multiplet (doublet of doublet of doublet) at

δ3.26 because it had only two vicinal couplings (3JH7e', H6a' = 6.4 Hz and 3JH7e', NHa= 3.9 Hz) besides

the geminal coupling (2JH7a', H7e' = 14.2 Hz). The 3JH7a' coupling observed with the vicinal NHa proton

of 11.9 Hz and 3JH7e' coupling with the vicinal NHa proton of 3.9 Hz suggested that the NH proton is

presumably transoid with respect to H7a' and cisoid with H7e' protons (i.e. NHa proton is most

probably axial). 2D COSY spectrum (see Figure S25 in SI) for the minor isomer 12b again was used

to confirm that the H6e' is only coupled with H7a'. 1D selective TOCSY experiment confirmed the

spin system for H7a', H7e', H6a', H6e', as well as that of the NHa proton at δ4.55 ppm (Figure S22 in

SI). Furthermore, HMBC spectrum confirmed the assignment of H7e' by having its correlation with

C2' but not between H7a' and C2' (Figure 3). This furthermore proved that the dihedral angle

between H7e' and C2', φ[H7e'-C7'-N-C2'], is ≈180°, whereas H7a' and C2', φ[H7a'-C7'-N-C2'], is in

the region of ≈90°.

(C) De-protected piperidino [3.2.1] constrained nucleoside 13:

The H6e' proton (δ1.17) of 13 was distinguished from the H6a' (δ1.78) by the fact that the former has

only a smaller 3JH,H coupling of 4.6 Hz besides a geminal coupling of 12.9 Hz, whereas H6a' proton

has a large trans 3JH,H coupling of 13.0 Hz and cisoid 3JH,H coupling of 6.8 Hz. A set of double and

single decouplings at the centre of multiplets of H6a' and/or H6e' protons gave both the assignment of

H7a' and H7e' protons as well as their vicinal couplings. The H7a' was assigned to the six-line

multiplet (doublet of triplet) at δ2.95 because of two vicinal couplings (3JH7a', H6a' = 13 Hz and 3JH7a',H6e' = 4.8 Hz) besides the geminal coupling (2JH7a', H7e' = 12.8 Hz) and H7e' was assigned to the

four-line multiplet (doublet of doublet) at δ2.87. The four lines of H7e' were due to one vicinal

coupling (3JH7e', H6a' = 6.6 Hz) besides geminal coupling (2JH7a', H7e' (gem) = 12.8 Hz). No couplings

were observed between H7e' and H6e' confirmed by 2D COSY experiments. This shows that the

dihedral angle between H6e' and H7e', φ[H7e'-C7'-C6'-H6e'], is about 90°. HMBC spectrum

confirmed the assignment of H7e' by having its correlation with C2' but not between H7a' and C2'

(Figure S32 in SI). This furthermore proved that the dihedral angle between H7e' and C2', φ[H7e'-

C7'-N-C2'], is ≈180°, whereas H7a' and C2', φ[H7a'-C7'-N-C2'], is in the region of ≈90°.

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(S4) The 3JHH simulation and nOe Studies of 12a and 12b and 13 All apparent coupling constants observed and simulated using MestRec software23 (Figures S3-S5

and Table S3 in SI) suggested that the H7a' and the NH are in trans-diaxial position in the minor

isomer 12b which is only possible when the NH is axial (NHa). On the other hand, absence of NH

coupling with H7a' or H7e' showed that the dihedral angle, φ[H7a'(H7e')-C7'-NHe] is in the region of

80-90°, which means that N-H is in equatorial position (NHe) in the major isomer 12a. This is

further evidenced by the fact that the chemical shift of H7a' is more upfield compared to that of H7e' in the minor isomer 12b because H7a' is in the cisoid orientation with the nitrogen lone-pair.

Similarly, the situation is reversed in the major isomer 12a by the fact that the nitrogen lone-pair is

now cisoid to H7e', hence it is more upfield shifted than that of H7a'. The absence of H1' and H2'

coupling constant in 12a and 12b indicate that this locked nucleoside has a unique C3'-endo type

conformation as observed in LNA and ENA analogs. Strong nOe contacts (10% to 11%) between

H6 and H3' for both 12a and 12b further confirms that the sugar is indeed fixed in North

conformation as observed for other North-locked nucleoside such as ENA3, 16 and LNA1 (8 to 9%

nOe between H-6 and H3').

In the fully de-protected nucleoside 13 in DMSO-d6, the H7e' at δ2.87 and H7a' at δ2.95

showed a difference in their multiplicities: The multiplet at δ2.95 for H7a' appeared as a well-

resolved 6-line spectrum with 2JH7a', H7e' (gem) = 12.8 Hz, 3JH7a', H6a' = 13.0 Hz, and 3JH7a', H6e' = 4.8 Hz,

while the multiplet at δ2.87 for H7e' was a 4-line spectrum with 2JH7a', H7e' (gem) = 12.8 Hz, and 3JH7e',H6a' = 6.8 Hz. On the other hand, absence of N-H coupling with H7a' or H7e' suggests that the

dihedral angle, φ[H7a' (H7e')-C7'-NHe] is in the region of 80-90°, which means that N-H is in the

equatorial position (NHe) in the fully de-protected major isomer 13. This is further evidenced by the

fact that the chemical shift of H7e' is more upfield compared to that of H7a' because H7e' is in the

cisoid orientation with the nitrogen lone-pair. The vicinal and geminal coupling constants and the

multiplicities in the 1H spectra as well as the resemblance of the HMBC spectra of 13 with those of

12a shows that the conformation of fully de-protected major isomer is similar to that of 12a.

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Figure S3: The CD spectra of aza-ENA-T modified 15mer AONs (AON 2-5) as duplex with (A) complementary DNA, and (B) with complementary RNA in comparison with the native counterpart (AON 1).

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(S5) Circular Dichroism (CD) analysis of the aza-ENA-T modified AON/DNA

and AON/RNA duplexes. Since CD spectroscopy is an effective tool to measure the global helical conformation of nucleic

acids, we have recorded the CD spectra of the aza-ENA-T modified 15mer AON/DNA and

AON/RNA hybrids (Table 1 and Figure 7). No significant change in ellipticity of the homo or the

hetero duplexes was observed in comparison with the native counterpart (duplex with AON 1 +

DNA or RNA) (Figure 7). This result is similar to what was observed for doubly modified ENA

duplexes.3 This means that single modifications with North locked aza-ENA-T nucleoside do not

alter the overall global structure compared to that of the native counterpart, whereas the differences

in the local conformational changes could easily be identified by RNase H digestion experiment (see

next section).

Table S1: Observed cleavage rate and relative cleavage rate (with respect to control native oligonucleotide) for RNase H digestion as well as ∆Tm (°C) found for the AON/RNA duplexes compared to the native counterpart

AON kobs (min-1) ∆∆∆∆Tm (°C) krel

1 (Native) 0.050 (±0.004) 0 1

2 0.055 (±0.007) 4 1.10

3 0.094 (±0.029) 2.5 1.88

4 0.210 (±0.029) 3.5 4.20

5 0.047 (±0.008) 4.0 0.94

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(S6) Experimental Methods:

(a) General Experimental Methods

Chromatographic separations were performed on Merck G60 silica gel. Thin layer

chromatography (TLC) was performed on Merck pre-coated silica gel 60 F254 glass-backed plates. 1H NMR spectra were recorded at 270.1 MHz, 500 MHz and 600 MHz respectively, using TMS (0.0

ppm) or methanol (3.4 ppm) as internal standards. 13C NMR spectra were recorded at 67.9 MHz,

125.7 MHz and 150.9 MHz respectively, using the central peak of CDCl3 (76.9 ppm) as an internal

standard. 31P NMR spectra were recorded at 109.4 MHz using 85% phosphoric acid as external

standard. Chemical shifts are reported in ppm (δ scale). Compound names for the bicyclic structures

are given according to the von Baeyer nomenclature. MALDI-TOF mass spectra were recorded in

positive ion mode for oligonucleotides and for other compounds as indicated. The mass

spectrometer was externally calibrated with a peptide mixture using alpha-cyano-4-

hydroxycinnamic acids as matrix. Thermal denaturation experiments were performed on a PC-

computer interfaced UV/VIS spectrophotometer with Peltier temperature controller.

(b) Synthesis, Deprotection and Purification of Oligonucleotides.

All oligonucleotides were synthesized using an automated DNA/RNA synthesizer by Applied

Biosystems, model 392. For modified AONs containing aza-ENA-T units, fast deprotecting

phosphoramidites (nucleobases were protected using the following groups: Ac for C, iPr-PAC for G,

and PAC for A) were used. The AONs were de-protected at room temperature by aqueous NH3

treatment for 24 h. All AONs and the target RNA were purified by 20% polyacrylamide/7M urea)

PAGE, extracted with 0.3 M NaOAc, desalted with C18-reverse phase cartridges and their purity

(greater than 95%) was confirmed by PAGE.

(c) UV Melting Experiments.

Determination of the Tm of the AON/RNA hybrids was carried out in the following buffer: 57 mM

Tris-HCl (pH 7.5), 57 mM KCl, 1 mM MgCl2. Absorbance was monitored at 260 nm in the

temperature range from 20 °C to 70 °C using UV spectrophotometer equipped with Peltier

temperature programmer with the heating rate of 1 °C per minute. Prior to measurements, samples

(1 µM of AON and 1 µM RNA mixture) were pre-annealed by heating to 80 °C for 5 min followed

by slow cooling to 4 °C and 30 min equilibration at this temperature.

(d) CD Experiments.

S14

CD spectra were recorded from 300 to 200 nm in 0.2 cm path length cuvettes. Spectra were

obtained with a AON/RNA duplex concentration of 5 µM in 57 mM Tris-HCl (pH 7.5), 57 mM

KCl, 1 mM MgCl2. All the spectra were measured at 25 °C and each spectrum is an average of 5

experiments from which CD spectrum of the buffer was subtracted.

(e) 32P Labeling of Oligonucleotides.

The oligoribonucleotide, oligodeoxyribonucleotides were 5'-end labeled with 32P using T4

polynucleotide kinase and [γ-32P]ATP by standard procedure. Labeled AONs and RNA were

purified by 20% denaturing PAGE and specific activities were measured using Beckman LS 3801

counter.

(f) RNase H Hydrolysis Experiment.

The source of RNase H1 (obtained from Amersham Bioscience) was Escherichia coli containing

clone of RNase H gene. The solutions of 15mer AON (1→5)/RNA duplex: [AON] = 10-6 M, [RNA]

= 10-7 M in a buffer, containing 20 mM Tris-HCl (pH 8.0), 20 mM KCl, 10 mM MgCl2, 0.1 mM

EDTA and 0.1 mM dithiothreitol (DTT) at 21 °C in 30 µL of the total reaction volume have been

used. The percentage of RNA cleavage was monitored by gel electrophoreses as a function of time

(0-60 min), using 0.08 U and 0.12 U of RNase H.

(g) Exonuclease Degradation Studies.

Stability of the AONs toward 3'-exonucleases was tested using snake venom phosphodiesterase

from Crotalus adamanteus. All reactions were performed at 3 µM DNA concentration (5'-end 32P

labeled with specific activity 50 000 cpm) in 56 mM Tris-HCl (pH 7.9) and 4.4 mM MgCl2 at 21 °C.

Exonuclease concentration of 17 ng/µL was used for digestion of oligonucleotides. Total reaction

volume was 14 µL. Aliquots (3 µL) were taken at 1, 2, 4, and 24 h and quenched by addition to 7 µL

volume of 50 mM EDTA in 80% formamide. Reaction progress was monitored by 20% denaturing

(7 M urea) PAGE and autoradiography.

(h) Stability studies in human serum.

AONs (6 µL) at 2 µM concentration (5'-end 32P labeled with specific activity 90 000 cpm) were

incubated in 26 µL of human serum (male AB) at 21 °C (total reaction volume was 36 µL). Aliquots

(3 µL) were taken at 0, 15 and 30 min, 1, 2 and 9 h, and quenched with 7 µL quenching solution

containing 50 mM EDTA in 80% formamide, resolved in 20% polyacrylamide denaturing (7 M

urea) gel electrophoresis and visualized by autoradiography.

S15

(i) pH-dependent 1H NMR measurement

All NMR experiments were performed using Bruker DRX-500 and DRX-600 spectrometers. The

NMR samples of the aza-ENA-T and 2'-amino-LNA-T were prepared in D2O solution (concentration

of 1 mM in order to rule out any chemical shift change owing to self-association) with δDSS = 0.015

ppm as internal standard. All pH-dependent NMR measurements have been performed at 298 K. The

pH values [with the correction of deuterium effect] correspond to the reading on a pH meter

equipped with a calomel microelectrode (in order to measure the pH inside the NMR tube) calibrated

with standard buffer solutions (in H2O) of pH 4, 7 and 10. The pD of the sample has been adjusted

by simple addition of microliter volumes of NaOD solutions (0.5M, 0.1M and 0.01M). The pH

values are obtained by the subtraction of 0.4 from corresponding pD values [pH = pD – 0.4]. All 1H

spectra have been recorded using 128 K data points and 64 scans.

(j) The pH titration of aromatic protons and pKa determination from Hill plot analysis

The pH titration studies were done over the range of pH (1.8 < pH < 12.2), with [0.2 – 0.3] pH

interval. pH titration studies consist of 17 and 22 data points for pKa determination for protonation

of 2'-amino-group of 2'-amino-LNA-T and aza-ENA-T, respectively, and 23 and 21 data points for

pKa determination of N3(T) de-protonation of 2'-amino-LNA-T and aza-ENA-T, respectively. The

pH-dependent [over the range of pH 1.8 < pH < 12.2, with an interval of pH 0.2 – 0.3] 1H chemical

shifts (δ, with error ± 0.001 ppm) for all compounds have shown a sigmoidal behavior. Values of the

pKas have been determined from the inflection points of the NMR titration curves as well as based

also on the Hill plot analysis using equation: pH = log ((1-α)/α) + pKa, where α represents fraction

of the protonated species. The value of α has been calculated from the change of chemical shift

relative to the de-protonated (D) state at a given pH (∆D = δD – δobs. for de-protonation, where δobs is

the experimental chemical shift at a particular pH), divided by the total change in chemical shift

between neutral (N) and de-protonated (D) state (∆T). So the Henderson-Hasselbach type equation

can then be written as pH = log [(∆T - ∆D)/∆D] + pKa and the pKa is calculated from the linear

regression analysis of the Hill plot.

S16

(S7) Theoretical calculations.

2',4' conformationally constrained aza-ENA-T (3',5'-bis-OBn protected compounds 12a and 12b

and de-protected 13) nucleosides have theoretically simulated to build up their molecular structures

using the following protocol:(i) Derive Initial dihedral angles from the observed 3JH,H using

Haasnoot-de Leeuw-Altona generalized Karplus equation24, 25 (ii) Perform NMR constrained

molecular dynamics (MD) simulation ( 0.5 ns,10 steps) simulated annealing (SA) followed by 0.5 ns

NMR constrained simulations at 298 K using the NMR derived torsional constraints from Step (i) to

yield NMR defined molecular structures of 3',5'-bis-OBn protected (12a, 12b) and de-protected aza-

ENA-T (13). (iii) Acquire 6-31G** Hartree-Fock optimized ab initio gas phase geometries in order

to compare the experimentally derived torsions with the ab initio geometry. (iv) Refine the Karplus

parameters with the help of the NMR-derived and ab initio derived torsions. (v) Analyze the full

conformational hyperspace using 2 ns NMR/ab initio constrained MD simulations of compounds

12a, 12b and 13 followed by full relaxation of the constraints.

The geometry optimizations of the modified nucleosides have been carried out by GAUSSIAN

98 program package26 at the Hartree-Fock level using 6-31G** basis set. The atomic charges and

optimized geometries of compounds 12a, 12b, and 13 were then used as AMBER27 force field

parameters employed in the MD simulations. The protocol of the MD simulations is based on

Cheathan-Kollman's28 procedure employing modified version of Amber 1994 force field as it is

implemented in AMBER 7 program package.27 Periodic boxes containing 1394, 1449, 1076, and

715, TIP3P29 water molecules to model explicit solvent around the 3',5'-bis-OBn protected (N-H

axial and N-H equatorial), and the de-protected (N-H axial and N-H equatorial) aza-ENA-Ts,

respectively, were generated using xleap extending 12.0 Å from these molecules in three dimensions

in both the NMR constrained and unconstrained MD simulations. SA protocol included ten repeats

of 25 ps heating steps from 298 K to 400 K followed by fast 25 ps cooling steps from 400 K to 298

K. During these SA and NMR constrained MD simulations torsional constrains of 50 kcal mol-1 rad-

2 were applied. The constrains were derived from the experimental 3JH1',H2', 3JH2',H3' and available 3JH7a'/H7e',NH coupling constants using Haasnoot-de Leeuw-Altona generalized Karplus equation24, 25

and parameters discussed in the text. Ten SA repeats were followed by 0.5 ns MD run at constant

298 K temperature applying the same NMR constrains.

S1

7

Fi

gure

S4.

Aut

orad

iogr

ams

of 2

0% d

enat

urin

g PA

GE,

sho

win

g th

e cl

eava

ge k

inet

ics o

f 5'-32

P-la

bele

d ta

rget

RN

A b

y E.

coli

RN

ase

H1

in

nativ

e A

ON

1/R

NA

(lan

e 5)

and

the

aza-

ENA

-T m

odifi

ed A

ON

s (2

-4)/R

NA

hyb

rid d

uple

xes

(lane

1 to

4) a

fter 2

, 5, 1

5, 3

5 an

d 60

min

of

incu

batio

n. C

ondi

tions

of c

leav

age

reac

tions

: RN

A (0

.8 µ

M) a

nd A

ON

s (4

µM

) in

buff

er c

onta

inin

g 20

mM

Tris

-HC

l (pH

8.0

), 20

mM

K

Cl,

10 m

M M

gCl2

and

0.1

mM

DTT

at 2

1 °C

; 0.0

8 U

of R

Nas

e H

. Tot

al re

actio

n vo

lum

e 30

µL .

S1

8

Fi

gure

S5.

Aut

orad

iogr

ams o

f 20%

den

atur

ing

PAG

E, sh

owin

g th

e de

grad

atio

n pa

ttern

of 5

'-32P-

labe

led

AO

N 1

to 5

in h

uman

seru

m.

Tim

e po

ints

are

take

n af

ter 0

, 4, 1

0, 2

4, 3

4 an

d 48

h o

f inc

ubat

ion.

S1

9

1.280

1.290

1.300

1.310

1.320

1.330

1.990

2.000

2.010

2.020

2.030

2.040

2.050

2.060

2.990

3.000

3.010

3.020

3.030

3.040

3.050

3.100

3.150

H7 e

' H

7 a'

H6 a

' H

6 e'

1.280

1.290

1.300

1.310

1.320

1.330

1.990

2.000

2.010

2.020

2.030

2.040

2.050

2.060

2.990

3.000

3.010

3.020

3.030

3.040

3.050

3.100

3.110

3.120

3.130

3.140

3.150

3.160

3.170

(A)

1 H-N

MR

spec

trum

(600

MH

z, 2

98K

) of t

he M

ajor

com

poun

d 12

a:

(B)

Sim

ulat

ed 1 H

-NM

R sp

ectr

um o

f the

Maj

or c

ompo

und

12a:

Figu

re S

6. 1 H

-NM

R e

xper

imen

tal a

nd si

mul

ated

spec

tra o

f com

poun

d 12

a

S2

0

Figu

re S

7. 1 H

-NM

R e

xper

imen

tal a

nd si

mul

ated

spec

tra o

f com

poun

d 12

b.

(B)

Sim

ulat

ed 1 H

-NM

R sp

ectr

um

3.210

3.220

3.230

3.240

3.250

3.260

3.270

3.280

2.01

02.

020

2.03

02.

040

2.05

02.

060

2.07

01.5

001.5

101.5

201.5

301.5

404.5

004.5

504.6

002.9

503.0

00

(A) 1 H

-NM

R sp

ectr

um (6

00 M

Hz,

298

K) o

f the

Min

or c

ompo

und

12b:

4.500

4.550

4.600

3.210

3.220

3.230

3.240

3.250

3.260

3.270

3.280

2.010

2.020

2.03

02.0

402.0

502.0

602.0

701.5

001.5

101.5

201.5

301.5

40

Bn-

CH

2 +

NH

a N

Ha

H7 e

' H

7 a'

H6 a

'

H6 e

' CH

3 (T

)

2.950

3.000

S2

1

2.910

2.920

2.930

2.940

2.950

2.960

2.970

2.980

2.850

2.860

2.870

2.880

2.890

1.150

1.160

1.170

1.180

1.190

1.200

1.750

1.760

1.770

1.780

1.790

1.800

1.810

1.150

1.160

1.170

1.180

1.190

1.200

1.750

1.760

1.770

1.780

1.790

1.800

1.810

2.850

2.860

2.870

2.880

2.890

2.910

2.920

2.930

2.940

2.950

2.960

2.970

2.980

H6 a

' H

7 a'

H7 e

' H

6 e'

(A) 1 H

-NM

R sp

ectr

um (6

00 M

Hz,

298

K) o

f the

com

poun

d 13

:

(B) S

imul

ated

1 H-N

MR

spec

trum

:

Figu

re S

8. 1 H

-NM

R e

xper

imen

tal a

nd si

mul

ated

spec

tra o

f com

poun

d 13

.

S2

2

Tab

le S

2. 1 H

NM

R c

hem

ical

shift

s of 1

2a, 1

2b, 1

3, a

nd E

NA

com

poun

ds

*500

/600

MH

z in

CD

Cl 3

at 2

98K

**

600

MH

z in

DM

SO- d

6 at 2

98K

***

Bio

orga

nic

and

Med

icin

al C

hem

istr

y 11

(200

3) p

2211

-222

6 (8

c:

3,5-

Di-O

-ben

zyl -

2-O

,4-C

-eth

ylen

e-5-

met

hylu

ridi

ne, 9

c: 2

-O,4

-C-E

thyl

ene-

5-m

ethy

luri

dine

)

1 H

12a* (δ δδδ

) 12

b* (δ δδδ

) 13

**(δ δδδ

) EN

A B

n-Pr

otec

ted

(8c)

***

ENA

Dep

rote

cted

(9

c)**

* H

1′ ′′′

5.94

6.

28

5.84

6.

06

6.00

H

2′ ′′′

3.51

3.

57

3.25

3.

94

4.05

H

3′ ′′′

3.98

4.

33

3.99

4.

31

4.09

O

H (3

′ ′′′)

5.

16

H5′ ′′′

3.

71

3.73

3.

57

3.60

? 3.

68

H5′ ′′′

′ ′′′ 3.

56

3.52

3.

50

3.76

? 3.

75

OH

(5′ ′′′)

5.39

H

6′ ′′′

2.03

2.

04

1.78

1.

35

1.33

H

6′ ′′′′ ′′′

1.30

1.

53

1.17

2.

28

1.94

H

7′ ′′′

3.02

3.

25

2.87

4.

10/4

.14

H7′ ′′′

′ ′′′ 3.

14

2.98

2.

95

4.14

/4.1

0 3.

9 –

4.0

NH

4.53

3.

34?

-

CH

2-B

n 4.

68

4.52

4.

64

4.58

-

4.51

4.

54

-

CH

2-B

n 4.

57

4.53

4.

56

4.50

-

4.58

4.

75

- H

6 7.

97

7.77

8.

27

7.91

8.

28

CH

3 (T

) 1.

43

1.50

1.

78

1.41

1.

86

NH

(T)

8.01

7.

96

11.3

0 8.

42

B

n (A

rom

atic

) 7.

38 –

7.2

4 7.

44 –

7.2

2 -

7.3

-

S2

3

Tab

le S

3. 1 H

NM

R C

oupl

ing

cons

tant

s of 1

2a, 1

2b, 1

3, a

nd E

NA

com

poun

ds

*500

/600

MH

z in

CD

Cl 3

at 2

98K

**

600

MH

z in

DM

SO- d

6 at 2

98K

***

Bio

orga

nic

and

Med

icin

al C

hem

istr

y 11

(200

3) p

2211

-222

6 (8

c:

3,5-

Di-O

-ben

zyl -

2-O

,4-C

-eth

ylen

e-5-

met

hylu

ridi

ne, 9

c: 2

-O,4

-C-E

thyl

ene-

5-m

ethy

luri

dine

)

12a*

12

b*

13**

EN

A B

n-Pr

otec

ted

(8c)

***

EN

A D

epro

tect

ed

(9c)

***

1 H

M

ultip

licity

, J

(Hz)

M

ultip

licity

, J

(Hz)

M

ultip

licity

, J

(Hz)

M

ultip

licity

, J

(Hz)

M

ultip

licity

, J

(Hz)

H

1′ ′′′

s s

s s

s H

2′ ′′′

d (3

.9)

d (4

.1)

d ( 3

.9)

d (3

.0)

d (3

.2)

H3′ ′′′

d

(3.9

) d

(4.1

) dd

(3.9

, 5.6

) d

(3.0

) d

(3.2

) O

H (3

′ ′′′)

br

s

H

5′ ′′′

d (1

0.8)

d

(11.

0)

dd (1

2.2,

5.2

) d

(12.

0)

d (1

2.0)

H

5′ ′′′′ ′′′

d (1

0.8)

d

(11.

0)

dd (1

2.2,

4.8

) d

(12.

0)

d (1

2.0)

O

H (5

′ ′′′)

t (

4.96

)

H6′ ′′′

dd

d (1

3.1,

11.

6,

6.7)

dd

d (1

3.4,

11.

9,

6.4)

dt

(12.

9, 1

3.0,

6.

8)

d (1

3.0)

dd

(3.8

, 13)

H6′ ′′′

′ ′′′ dd

(13.

1, 4

.8)

dd (

13.4

, 5.2

) dd

(12.

9, 4

.6)

d (1

3.0)

dd

d (7

.5, 1

1.7,

13)

H7′ ′′′

dd

(13.

3, 6

.5)

ddd

(14.

2, 6

.4,

3.9)

dd

(12.

8, 6

.6)

d (7

.0)

H7′ ′′′

′ ′′′ dd

d (1

3.3,

11.

6,

4.9)

dt

d ( 1

4.2,

11.

9,

5.2)

dt

(12.

8, 1

3.0,

4.

8)

d (7

.0)

m

NH

dd (1

1.9,

3.9

) s

CH

2-B

n d

( 11.

8)

d (1

1.8)

d

(11.

6)

d (1

1.6)

-

d (1

2.0)

d

(12.

0)

- C

H2-

Bn

d (1

1.5)

d

(11.

5)

d (1

1.5)

d

(11.

5)

- d

(12.

0)

d (1

2.0)

-

H6

q (1

.3)

d (1

.2)

s s

d (1

.1)

CH

3 (T

) d

(1.3

) d

(1.2

) s

s d

(0.9

) N

H (T

)

s s

Bn

m

m

S2

4

Tab

le S

4. S

imul

ated

and

exp

erim

enta

l 1 H N

MR

Cou

plin

g co

nsta

nts o

f 12a

, 12b

, and

13

*500

/600

MH

z in

CD

Cl 3

at 2

98K

**

600

MH

z in

DM

SO- d

6 at 2

98K

12a

12b

13

3 JH

,H

Expe

rimen

tal*

Sim

ulat

ed

Expe

rimen

tal*

Sim

ulat

ed

Expe

rimen

tal**

Sim

ulat

ed

3 JH

6a',H

6e'

13.0

5 13

.00

13.4

4 13

.42

12.9

12

.8

3 JH

6e',H

6a'

13.0

6 13

.00

13

.45

12.9

12

.9

3 JH

6a',H

7e'

6.70

6.

65

6.72

6.

35

6.8

6.9

3 JH

6a',H

7a'

11.6

4 11

.63

12.6

8 11

.93

13.0

12

.8

3 JH

6e’,H

7a'

4.76

4.

83

5.

23

4.8

4.6

3 JH

6e',H

7e'

3 JH

7e’,H

6a'

6.50

6.

65

6.35

6.

37

6.6

6.9

3 JH

7e',H

6e’

3 JH

7a',H

6a'

11.5

9 11

.64

11.9

5 11

.92

13.0

12

.7

3 JH

7a',H

6e'

4.87

4.

86

5.25

5.

22

4.6

4.6

3 JH

7a',H

7e'

13.3

1 13

.41

14.1

8 14

.20

12.8

12

.8

3 JH

7e',H

7a'

13.2

8 13

.40

14.2

1 14

.23

12.8

12

.7

3 JH

7a',N

H

11.9

4 11

.92

3 JH

7e',N

H

3.90

3.

94

3 JN

H,H

7a'

11

.91

3 JN

H,H

7e'

3.

91

S2

5

Tab

le S

5. 13

C N

MR

che

mic

al sh

ifts o

f 12a

, 12b

, and

13

*5

00/6

00 M

Hz

in C

DC

l 3 at

298

K *

* 60

0 M

Hz

in D

MSO

- d6 a

t 298

K

13C

12

a*

12b*

13

**

C-1

′ ′′′ 90

.58

82.5

9 87

.15

C-2

′ ′′′ 59

.34

64.2

0 61

.65

C-3

′ ′′′ 71

.90

72.0

8 65

.46

C-4

′ ′′′ 87

.21

82.6

2 87

.09

C-5

′ ′′′ 70

.44

69.5

3 63

.02

C-6

′ ′′′ 27

.66

26.8

0 26

.61

C-7

′ ′′′ 38

.59

45.9

5 38

.74

Bn-

CH

2 73

.56

73.6

9 -

Bn-

CH

2 72

.03

73.6

4 -

Bn

(Aro

mat

ic)

137.

52, 1

37.3

1 12

8.63

– 1

27.3

1 13

6.81

, 136

.02

128.

92 –

127

.66

-

C2

149.

86

149.

18

152.

62

C4

163.

66

163.

48

168.

23

C5

109.

51

110.

29

111.

41

C6

135.

95

135.

65

138.

34

CH

3 11

.84

11.9

8 13

.07

S2

6

160

140

120

100

8060

4020

ppm

11.901

58.84963.68568.30970.77371.69273.22576.71276.96677.22099.290110.330115.106127.833128.072128.239128.478128.543128.571128.601128.630134.483135.764136.648150.237159.953

Figu

re S

9. 13

C sp

ectru

m o

f com

poun

d C

in S

chem

e 1

OBn

O

OBn

NH

NO

O

NC

1'2'

3'4'

5'

6'

C (C

DC

l 3)

S2

7

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

ppm

Figu

re S

10. 1

D n

Oe

spec

trum

of c

ompo

und

C in

Sch

eme

1

OBn

O

OBn

NH

NO

O

NC

1'2'

3'4'

5'

6'

C (C

DC

l 3)

H6

H6'

B

n H

2'

S2

8

pp

m

109

87

65

43

21

0p

pm

160

140

120

100806040200

Figu

re S

11. H

SQC

spec

trum

com

poun

d C

in S

chem

e 1

OBn

O

OB

n

NH

NO

O

NC

1'2'

3'4'

5'

6'

C (C

DC

l 3)

NH

Ph

H-6'

CH2-Ph

H-3' H-5', H-5''

H-2'

CH3 (T)

H6

H-1'

CH

3 (T

)

C-2

' C

-1'

CN

C

-5

C-4

C-2

C-6

Ph

C-5

'

C-6

'

Ph

CH

2-Ph

C-4

'

CH

2-Ph

S2

9

pp

m

98

76

54

32

1p

pm

180

160

140

120

100

80604020

Figu

re S

12. H

MB

C sp

ectru

m o

f com

poun

d C

in S

chem

e 1.

OBn

O

OB

n

NH

NO

O

NC

1'2'

3'4'

5'

6'

C (C

DC

l 3)

NH

Ph

H-6'

CH2-Ph

H-3' H-5', H-5''

H-2'

CH3 (T)

H6

H-1'

CH

3 (T

)

C-2

' C

-1'

CN

C

-5 C

-6Ph

C-5

'

C-6

'

Ph

CH

2-Ph

C

H2-

Ph

S3

0

ppm

12

34

56

78

ppm

1 2 3 4 5 6 7 8

Figu

re S

13a.

DQ

F C

OSY

spec

trum

of c

ompo

und

C in

Sch

eme

1.

OBn

O

OB

n

NH

NO

O

NC

1'2'

3'4'

5'

6'

C (C

DC

l 3)

H6'

H

1'

H3'

H

2'

H6(

T)

H6'

H2'

H3'

H1'

H6(

T)

CH

3(T)

CH

3(T)

H5'

/5'',

2xB

n

H5'

/5'',

2xB

n

S3

1

Figu

re S

13b.

Exp

ansi

on o

f the

DQ

F C

OSY

spec

trum

of c

ompo

und

C in

Sch

eme

1.

ppm

3.5

4.0

4.5

5.0

5.5

6.0

ppm

3.5

4.0

4.5

5.0

5.5

6.0

H6'

H

1'

H3'

H

2'

H2'

H3'

H1'

H6'

H5'

/5'',

2xB

n

OBn

O

OB

n

NH

NO

O

NC

1'2'

3'4'

5'

6'

C (C

DC

l 3)

S3

2

ppm

3.24

3.26

3.28

ppm

5.02

5.04

5.06

5.08

5.10

5.12

5.14

ppm

ppm

5.02

5.04

5.06

5.08

5.10

5.12

5.14

ppm

3.20

3.22

3.24

3.26

3.28

3.30

3.32

ppm

3.85

3.90

3.95

4.00

4.05

4.10

4.15

4.20

ppm

3.20

3.22

3.24

3.26

3.28

3.30

3.32

ppm

3.85

3.90

3.95

4.00

4.05

4.10

4.15

4.20

Figu

re 1

3c. E

xpan

ded

DQ

F-C

OSY

spec

tra sh

owin

g co

uplin

g co

nsta

nts 3 J H

1'H

2' =

3.1

H

z an

d 3 J H

2'H

3' =

8.3

Hz

H

2'

H2'

H1'

H

3'

8.3

Hz

3.1

Hz

8.3

Hz

OBn

O

OB

n

NH

NO

O

NC

1'2'

3'4'

5'

6'

C (C

DC

l 3)

S3

3

5.82

5.83

ppm

5.10

ppm

3.25

ppm

3.95

4.00

ppm

Fi

gure

S14a

. 1 H h

omod

ecou

plin

g sp

ectru

m b

y irr

adia

ting

H3'

of c

ompo

und

C in

Sch

eme

1.

H6'

H1'

H

2'

J 1'-2

' = 2

.2H

z,

J 2'-3

' = 7

.7H

z

H3'

J 1

'-2' =

2.2

Hz,

J 2

'-3' =

7.7

Hz

H1'

H

6'

H2'

O

BnO

OBn

NH

NO

O

NC

1'2'

3'4'

5'

6'

Irra

diat

ion

S3

4

Fi

gure

S14b

. 1 H h

omod

ecou

plin

g sp

ectru

m b

y irr

adia

ting

H6'

of c

ompo

und

C in

Sch

eme

1.

OBn

O

OBn

NH

NO

O

NC

1'2'

3'4'

5'

6'

5.10

ppm

4.00

ppm

3.3

ppm

5.8

5.9

ppm

Irra

diat

ion

H6'

H

1'

H2'

J 1

'-2' =

2.2

Hz,

J 2

'-3' =

7.7

Hz

H3'

J 1

'-2' =

2.2

Hz,

J 2

'-3' =

7.7

Hz

H1'

H

6'

H2'

H

3'

S3

5

Figu

re S

15. 13

C N

MR

spec

trum

of 4

.

O

OO

BnO

OB

nN

C

4

(CD

Cl 3)

S3

6

Fi

gure

S16

. 13C

NM

R sp

ectru

m o

f 6.

O

OAc

BnO

OB

nN

C

NH

NO

O

6 (C

DC

l 3)

S3

7

Fi

gure

S17

. 13C

NM

R sp

ectru

m o

f 7.

O

OH

BnO

OB

nN

C

NH

NO

O

7

(CD

Cl 3)

S3

8

Fi

gure

S18

. 13C

NM

R sp

ectru

m o

f 8.

O

OM

s

BnO

OB

nN

C

NH

NO

O

8 (C

DC

l 3)

S3

9

Fi

gure

S19

. 13C

NM

R sp

ectru

m o

f 9.

OB

nO

OB

nN

C

N

N

O

O

9

(CD

Cl 3)

S4

0

Fi

gure

S20

. 13C

NM

R sp

ectru

m o

f 10.

OB

nO

OB

nN

C

NH

NO

O

HO

10

(CD

Cl 3)

S4

1

Fi

gure

S21

. 13C

NM

R sp

ectru

m o

f 11.

OB

nO

OB

nN

C

NH

NO

O

TfO

11

(CD

Cl 3)

S4

2

170

160

150

140

130

120

110

100

9080

7060

5040

3020

10pp

m

11.866

27.68629.70938.614

59.36270.45971.92972.05373.58276.76677.02077.27484.67587.234

109.534127.638127.800127.911128.082128.170128.524128.658135.969137.334137.542149.883

163.681

Figu

re S

22. 13

C N

MR

spec

trum

of 1

2a.

OBn

O

OBn

NH

NO

O

N

12a

H

S4

3

ppm

98

76

54

32

10

ppm

160

140

120

10080604020

Figu

re S

23. H

SQC

spec

trum

of 1

2a.

OBn

O

OBn

NH

NO

O

N

12a

H

H-6

Ph

H-1'

CH2-Ph

H-3' H-5' H-5'' H-2'

H-7a H-7e'

H-6a

CH3(TH-6a

CH

3 (T

)

C-6

' C

-7'

C-2

' C

-5'

C-4

'

C-5

C-4

C-2

C-6

Ph

Ph

C-3

', C

H2-

Ph

C-1

'

S4

4

ppm

12

34

56

78

9p

pm

160

140

120

10080604020

Figu

re S

24 1 H

-13C

HM

BC

spec

trum

show

ing

the

long

ran

ge th

roug

h-bo

nd c

onne

ctiv

ities

bet

wee

n C

7'/H

2' a

nd C

2'/H

7 e o

f 12a

.

OBn

O

OBn

NH

NO

O

N

12a

H

H-6

Ph

H-1'

CH2-Ph

H-3' H-5' H-2' H-5''

H-7a' H-7e'

H-6a'

H-6e CH3 (T)

CH

3 (T

)

C-6

'

C-7

'

C-2

' C

-5'

C-4

'

C-5

C-4

C-2

C-6Ph

C-3

', C

H2-

Ph

C-1

'

Ph

S4

5

OBn

O

OBn

NH

NO

O

N

12a

H

H-6

Ph

H-1'

CH2-Ph

H-3' H-5' H-2' H-5''

H-7a H-7e'

H-6a'

H-6e' CH3 (T

H-6

e' C

H3

(T)

H-6

a'

H-7

e'

H-7

a'

H-5

'', H

2'

H-5

'

CH

2-Ph

H-1

'

Ph

H-6

Figu

re S

25. C

OSY

spec

trum

of 1

2a.

S4

6

CD

Cl 3

Figu

re S

26. 13

C N

MR

spec

trum

of 1

2b.

160

150

140

130

120

110

100

9080

7060

5040

3020

10ppm

11.979

26.79629.681

45.950

64.20469.53072.08073.64073.68576.74877.00277.25682.58882.623

110.290127.662127.890127.919127.943127.973128.061128.092128.191128.384128.608128.671128.728128.758128.922135.648136.019136.805149.183

163.481

12b

13C

spec

trum

(1H

dec

oupl

ed)

OBn

O

OBn

NH

NO

O

N

12bH

S4

7

2.0

2.5

3.0

3.5

4.0

4.5

ppm

2.0

2.5

3.0

3.5

4.0

4.5

ppm

Figu

re S

27. 1

D T

OC

SY sp

ectru

m o

f 12b

.

NH

a H

7 e' H

7 a'

H6 a

' H

6 e'

Sele

ctiv

e Ir

radi

atio

n of

H7 e

'

OBn

O

OBn

NH

NO

O

N

12bH

S4

8

ppm 10

98

76

54

32

10

ppm

140

120

100806040200

OBn

O

OBn

NH

NO

O

N

12bH

CH

3 (T

)

C-6

'

C-7

'

C-2

' C

-5'

C-4

'

C-5

C-2

C-6

Ph C-3

', C

H2-

Ph

C-1

'

Ph

H-6 Ph

H-1

CH2-PhH-3' H-5' H-2' H-5'' H-7e'

H-7a

H-6a' H-6e'

CH3 (T

H-7e'

Figu

re S

28. H

SQC

spec

trum

of 1

2b.

S4

9

ppm

109

87

65

43

21

0pp

m

160

140

120

10080604020

OBn

O

OBn

NH

NO

O

N

12bH

CH

3 (T

)

C-6

'

C-7

'

C-2

' C

-5'

C-4

'

C-5

C-2

C-6Ph

C-3

', C

H2-

Ph

C-1

'

Ph

C-4

H-6 Ph

H-1'

CH2-PhH-3' H-5' H-2' H-5''

H-7a'

H-6a' H-6e'

CH3 (T

H7e'

Figu

re S

29. H

MB

C sp

ectru

m o

f 12b

.

S5

0

OBn

O

OBn

NH

NO

O

N

12bH

H-6 Ph

H-1'

NHa, CH2-Ph H-3'

H-5' H-2' H-5''

H-7a'

H-6a' H-6e'

CH3 (T)

H7e'

H-6

e'

CH

3 (T

)

H-6

a'

H-7

e'

H-7

a'

H-3

' N

Ha.

CH

2-Ph

x 2

H-1

'

Ph

H-6

Figu

re S

30. C

OSY

spec

trum

of 1

2b.

S5

1

Fi

gure

S31

. 13C

NM

R sp

ectru

m o

f 14.

OB

nO

OB

n

NH

NO

O

NP

AC

14

(CD

Cl 3)

S5

2

Fi

gure

S32

. 13C

NM

R sp

ectru

m o

f 15.

OH

O

OH

NH

NO

O

N

PAC

1

5 (C

D3O

D)

S5

3

Fi

gure

S33

. 13C

NM

R sp

ectru

m o

f 16.

OD

MTr

O

OH

NH

NO

O

NP

AC

16

(CD

Cl 3

+ D

AB

CO

)

S5

4

Fi

gure

S34

. 31P

NM

R sp

ectru

m o

f 17.

OD

MTr

O

O

NH

NO

O

N

PO

CN

N

PA

C

17

(CD

Cl 3

+ D

AB

CO

)

S5

5

13.321

26.856

38.989

61.89463.26665.706

87.391

138.595180

170

160

150

140

130

120

110

100

9080

7060

5040

3020

ppm

13.322

26.859

38.990

61.89663.27065.712

87.33687.403

111.658

138.595

152.870

168.476

OH

O

OH

NH

NO

O

NH

13

(DM

SO)

Figu

re S

35. 13

C a

nd 13

C D

EPT

135

NM

R sp

ectra

of 1

3.

S5

6

ppm 10

98

76

54

32

10

ppm

140

120

100806040200

OH

O

OH

NH

NO

O

NH

13 (D

2O)

Figu

re S

36. H

SQC

spec

trum

of 1

3.

CH

3 (T

)

C-6

'

C-7

'

C-2

' C-5

'

C-5

C-2

C-6C-1

', C

-4'

C-3

'H-6

H-1'

H-7e H-2' H-7a

H-6a

H-3' H-5', H-5''

CH3(T) H6e

S5

7

ppm 10

98

76

54

32

10

ppm

160

140

120

100806040200

Fi

gure

S37

. HM

BC

spec

trum

of 1

3.

OH

O

OH

NH

NO

O

NH

13

(D2O

) C

H3

(T)

C-6

' C

-7'

C-2

' C-5

'

C-6

C-4

C-3

'

C-1

', C

-4'

C-5

C-2

H-6

H-1'

H-7a H-2'

H-7e'

H-6a'

H-3' H-5', H-5''

CH3 (T) H6e'

pm

2.80

2.85

2.90

2.95

3.00

ppm

0 0 0 0 0 0 0 0

C6'

C2'

C4'

H7e

H7a

Pa

rt of

the

HM

BC

spec

trum

of

13 in

DM

SO-d

6.

S5

8

ppm

98

76

54

32

1pp

m987654321

Figu

re S

38. C

OSY

spec

trum

of 1

3.

OH

O

OH

NH

NO

O

NH

13 (D

MSO

-d6)

H-6

H-1'

H-7a H-7e'

H-6a'

H-3' H-5', H-5''

CH3 (T)

H6e'

OH-5' OH-3'

H-2'

CH

3 (T

) H

-6a'

H-7

e H

-2'

H-5

',H

-5''

H-6

H-3

'

OH

-3'

OH

-5'

H-6

e'

H-7

a

H-1

'

S5

9

Fi

gure

S39

. 13C

NM

R sp

ectra

of 1

8.

OH

O

OH

NH

NO

O

NC

OC

F 3

1

8 (C

D3O

D)

S6

0

Fi

gure

S40

. 13C

NM

R sp

ectru

m o

f 19.

OD

MTr

O

OH

NH

NO

O

NC

OC

F 3

1

9 (C

DC

l 3 +

DA

BC

O)

S6

1

Fi

gure

S41

. 31P

NM

R sp

ectru

m o

f 20.

OD

MTr

O

O

NH

NO

O

NC

OC

F 3P

OC

NN

2

0 (C

DC

l 3 +

DA

BC

O)

S62

Axial

Torsion angle (o)

-200 -150 -100 -50 0 50 100 150 200

% o

f con

form

atio

ns

0

5

10

15

20

7'-NH7"-NH

Equatorial

Torsion angle (o)

-150 -100 -50 0 50 100 150

% o

f con

form

atio

ns

0

5

10

15

20

7'-NH7"-NH

Figure S42. Population distributions of the H7'-C7'-N-H and H7''-C7'-N-H torsions in the N-H axial (12b) and equatorial (12a) deastereomers during the last 0.5 ns of constrains-free MD simulations. The simulations were started from the axial (upper distribution) as well as from the equatorial (lower distribution) isomers, respectively, but resulted in near equal population of these isomers indicating reversible equilibrium in contradistinction with the experimentally observed preference of the N-H equatorial isomer 12a and observed irreversible transformation of the axial isomer 12b into 12a.

S63

Figure S43 Time-dependent equilibration of 12b to 12a in CDCl3.

R = 1.00k = 1.43e-6 s-1

time (days)0 20 40 60

% 1

2b

50

60

70

80

90

100

110

S64

(S8) Generalized Karplus parameterization

The MD simulations were performed using Amber force field (AMBER 727) and explicit

TIP3P29 aqueous medium (see details in Experimental section). (iii) Acquire 6-31G** Hartree-

Fock optimized ab initio gas phase geometries (by Gaussian 9826) in order to compare the NMR

derived torsions with the ab initio geometry. (iv) Refine the Karplus parameters with the help of

the NMR and ab initio derived torsions. (v) Analyze the full conformational hyperspace using 2

ns NMR/ab initio constrained MD simulations of compounds 12a, 12b and 13 followed by full

relaxation of the constraints. The results of these studies are summarized in Table S6.

(i) Generalized Karplus parameterization

Relevant vicinal proton 3JH1',H2', 3JH2',H3' and 3JH3',H4' coupling constants have been back-calculated

from the corresponding theoretical torsions employing Haasnoot-de Leeuw-Altona generalized

Karplus equation24, 25 taking into account β substituent correction in form: 3J = P1 cos2(φ) + P2 cos(φ) + P3 + Σ ( ∆χi

group (P4 +P5 cos2(ξi φ + P6 |∆χigroup| ))

where P1 = 13.70, P2 = -0.73, P3 = 0.00, P4 = 0.56, P5 = -2.47, P6 = 16.90, P7 = 0.14 (parameters

from Ref.24), and ∆χi group = ∆χi

α- substituent - P7 Σ ∆χi

β- substituent where ∆χi are taken as Huggins

electronegativities.30 Optimized ∆χi group = 1.305 has been used for Naze. Least-square

optimization procedure for the ∆χigroup of nitrogen atom in the piperidino ring will be reported

elsewhere. The other group electronegativities were kept unmodified and their respective

standard values were adopted from Altona et al.24 as follows: C1' (0.738), O4' (1.244), C3'

(0.162), C2' (0.099), O3' (1.3 for OH & 1.244 for OBn), C4' (0.106), C5' (0.218). The correlation

between experimental 3JH1',H2' and 3JH2',H3' vicinal coupling constants of the ENA (compound (I)

in Figure S1), aza-ENA-T (12a, 12b, 13), LNA and 2'-amino LNA analogs (compounds (A) and

(B) in Figure S1) are shown in Inset (A) in Figure S44 together with contours of theoretical 3JH1',H2' vs. 3JH2',H3' dependencies at fixed sugar puckering amplitudes (from 35° to 65°).

S65

Table S6. Experimental 3JH,H vicinal proton coupling constants, corresponding ab initio and MD

(highlighted in blue) φH,H torsions and respective theoretical 3JH,H obtained using Haasnoot-de

Leeuw-Altona generalized Karplus equation24, 25 taking into account β substituent correction (see

text). Non-observable constants are marked in red.

Compounds

3JHH Torsion (φφφφH,H)

3JHH, calc. Hz

3JH,H, exp. Hz

φφφφH,H (°°°°), ab initio

φφφφH,H (°°°°), MD φφφφH,H (°°°°), exp. ∆∆∆∆3JH,H,

Hz 3JH1', H2' H1'-C1'-C2'-H2' 1.02 0 87.17 94.8 ± 6.6 88.6 to 92.9 1.023JH2', H3' H2'-C2'-C3'-H3' 3.60 3.90 50.60 46.7 ± 5.8 48.3 to 48.5 -0.303JH7e', H6a' H7e'-C7'-C6'- H6a' 6.81 6.65 43.27 45.1 ± 8.1 44.1 to 44.2 0.163JH7a', H6a' H7a'- C7'-C6'-H6a' 11.54 11.64 159.69 155.7 ± 7.9 160.6 to160.8 -0.103JH7e', H6e' H7e'-C7'-C6'-H6e' 0.30 0 -75.52 -72.3 ± 7.8 -84.6 to -80.3 0.303JH7a', H6e' H7a'-C7'-C6'-H6e' 5.04 4.86 40.90 38.4 ± 7.9 41.8 to 41.9 0.183JH7e', HN H7e'-C7'-N-HN 1.71 0 67.65 3.5 ± 36.3 85.5 to 89.6 1.71

12a (major): [aza-ENA, 3’,5’-bis-OBn, NH-equatorial]

3JH7a', HN H7a'-C7'-N-HN 4.85 0 -50.10 -113.1 ± 36.4 -90.3 to -85.5 4.853JH1', H2' H1'-C1'-C2'-H2' 1.00 0 89.39 95.6 ± 6.8 88.6 to 92.9 1.003JH2', H3' H2'-C2'-C3'-H3' 3.73 3.90 49.41 47.0 ± 5.9 48.4 to 48.5 -0.143JH7e', H6a' H7e'-C7'-C6'- H6a' 6.94 6.35 42.58 43.7 ± 8.5 45.7 to 45.9 0.593JH7a', H6a' H7a'- C7'-C6'-H6a' 11.46 11.92 158.93 153.4 ± 8.4 163.9 to 164.4 -0.463JH7e', H6e' H7e'-C7'-C6'-H6e' 0.26 0 -76.41 -72.0 ± 8.3 -84.6 to -80.3 0.263JH7a', H6e' H7a'-C7'-C6'-H6e' 5.22 5.22 39.94 37.7 ± 8.4 39.9 to 40.0 0.003JH7e', HN H7e'-C7'-N-HN 6.59 3.94 -41.76 11.9 ± 35.4 -55.1 to -55.0 2.65

12b (minor): [aza-ENA-

3’,5’-bis-OBn, NH-axial]

3JH7a', HN H7a'-C7'-N-HN 11.38 11.91 -156.72 -104.2 ± 35.7 -160.4 to -160.3 -0.533JH1', H2' H1'-C1'-C2'-H2' 0.97 0 89.78 96.9 ± 6.8 88.6 to 92.9 0.973JH2', H3' H2'-C2'-C3'-H3' 3.77 3.9 49.69 43.5 ± 5.6 48.7 to 48.9 -0.133JH7e', H6a' H7e'-C7'-C6'- H6a' 7.16 6.7 41.37 42.3 ± 8.2 43.8 to 44.0 0.463JH7a', H6a' H7a'- C7''-C6'-H6a' 11.37 13.0 158.12 152.5 ± 8.0 172.5 to176.4 -1.633JH7e', H6e' H7e'-C7'-C6'-H6e' 0.25 0 -76.75 -74.2 ± 7.9 -84.6 to -80.3 0.253JH7a', H6e' H7a'-C7'-C6'-H6e' 5.04 4.7 40.01 36.1 ± 8.0 42.6 to 42.8 0.343JH7e', HN H7e'-C7'-N-HN 2.13 0 64.74 24.8 ± 30.9 85.5 to 89.6 2.13

13 (major): [aza-ENA,

NH-equatorial]

3JH7a', HN H7a'-C7'-N-HN 4.29 0 -52.96 -91.3 ± 30.8 -90.3 to -85.5 4.29

S66

Table S7. Sugar torsions (νννν0 - νννν4 ), pseudorotational phase angle (P),31 sugar puckering amplitude (φφφφm),31 backbone (γγγγ,δδδδ) and glycoside bond (χχχχ), as well as selected torsions (C3'-C2'-O2' (N2')-C7') and C3'-C4'-C6'-C7') characterizing six-member piperidino heterocycle in 3',5'-bis-OBn protected and de-protected aza-ENA (12a,12b,13) and ENA3 (compound (I) in Figure S1). 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.

Torsions O

OBn

BnON

NH

O

O

NH

1'2'3'4'

5'

6' 7'

O

OH

HON

NH

O

O

NH

1'2'3'4'

5'

6' 7' N-H equatorial (12a) N-H axial (12b) N-H equatorial (13)

νννν0: C4'-O4'-C1'-C2' 3.38 (4.2 ± 6.0) 3.21 (4.2 ± 5.6) -0.91 (-0.5 ± 6.8)

νννν1: O4'-C1'-C2'-C3' -31.88 (-32.9 ± 4.9) -31.28 (-32.9 ± 4.6) -28.21 (-29.5 ± 5.5)νννν2: C1'-C2'-C3'-C4' 46.00 (45.2 ± 3.2) 45.07 (45.1 ± 3.2) 43.87 (44.8 ± 3.4)νννν3: C2'-C3'-C4'-O4' -45.21 (-45.5 ± 3.8) -43.87 (-45.4 ± 3.8) -45.14 (-47.6 ± 4.0)νννν4: C3'-C4'-O4'-C1' 26.83 (26.6 ± 5.5) 26.19 (26.5 ± 5.3) 29.65 (30.5 ± 6.1)

P 14.56 (14.1 ± 7.2) 14.38 (14.0 ± 6.8) 19.38 (19.4 ± 8.0)φφφφm 47.53 (46.9 ± 3.1) 46.53 (46.8 ± 3.2) 46.50 (48.0 ± 3.4)

γγγγ: O5'-C5'-C4'-C3' 45.92 (54.9 ± 9.1) 47.44 (57.3 ± 9.3) 64.53 (53.1 ± 10.7)δδδδ: C5'-C4'-C3'-O3' 72.74 (60.0 ± 5.3) 80.31 (69.4 ± 5.4) 75.61 (74.5 ± 5.9)

χχχχ: O4'-C1'-N1-C2 -164.31 (-158.6 ± 10.1) -163.21 (-154.9 ± 10.5) -156.21 (-157.2 ± 10.8)

C3'-C2'-O2' (N2')-C7' 64.31 (66.7 ± 5.0) 58.56 (66.4 ± 5.2) 68.08 (67.0 ± 4.8)C3'-C4'-C6'-C7' -59.75 (-57.8 ± 5.1) -59.4 (-57.6 ± 5.4) -57.49 (-55.5 ± 5.2)

S67

Figure S44: Experimental 3JH1',H2' and 3JH2',H3' vicinal coupling constants of the ENA (compound (I) in Figure S1), aza-ENA (12a,12b,13), LNA and 2'-amino LNA analogs (compounds (A) and (B) in Figure S1) shown together with contours of theoretical 3JH1',H2' vs. 3JH2',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.24, 25 and using parameters as described in the text..

S68

Torsions H7a'-C7'-C6'-H6a', H7e'-C7'-C6'-H6a', H7a'-C7'-C6'-H6e' and H7e'-C7'-C6'-H6e' in the ideal sp3 environment are related and equal to φ, (φ - 120°), (φ - 120°), and (φ - 240°), respectively.

Figure S45: Elucidation of the piperidino ring conformation based on experimental 3JH6a/e',H7a/e' coupling constants (horizontal lines) of the equatorial/axial H6e'/H6a' and H7e'/H7a' protons of 13, and the theoretical Haasnoot-de Leeuw-Altona generalized Karplus dependences24, 25 (ideal sp3 hybridization of carbon atom is assumed) for all possible H6'(H6'')-H7'(H7'') vicinal couplings. Vertical lines (magenta) point to experimentally derived values of the corresponding torsions only possible for a particular combination of all four experimental 3JH6a/e',H7a/e' coupling constants (see Table S6 for exact φ values).

S69

3',5'-OBn Aza-ENA-T* (12a)

N-H equatorial

3',5'-OBn Aza-ENA-T*

(12b)

N-H axial

Aza-ENA-T

(18)

N-H equatorial

RMSd = 0.417*

(0.078, 0.611,1.306) RMSd = 0.469*

(0.072, 0.704,1.683) RMSd = 0.433 (0.092, 0.656)

* For clarity of the view the 3',5'-OBn protective groups in 12a and 12b are not shown. Figure S46. Superposition of 10 randomly selected structures during last 400 ps of the unconstrained 2 ns MD simulations of 3',5'-bis-OBn protected (12a,12b) and de-protected (13) aza-ENA-T. Total average RMSd (in Å) are shown for all heavy atoms (marked in black) as well as for the heavy atoms in sugar and piperidino moieties (in parentheses marked in red), the base atoms (in parentheses marked in blue), and OBn-groups’ heavy atoms (in parentheses marked in green).

S70

pKa of 2'-amine protonation

Figure S47. NMR titration plots of pH vs chemical shift of specified marker proton to obtain pKa of 2'-amine protonation in aza-ENA-T (panel A) and 2'-amino-LNA-T (panel B) as well as the results of respective Hill plot analysis (panels (a)-(b), pH vs log[(∆Τ-∆)/∆], see Experimental details in SI for details).

(A)

(B)

(a)

(b)

S71

pKa of N3 de-prononation

Figure S48. NMR titration plots of pH vs chemical shift of specified marker proton to obtain pKa of N3 protonation in aza-ENA-T (panel A) and 2'-amino-LNA-T (panel B) as well as the results of respective Hill plot analysis (panels (a)-(b), pH vs log[(∆Τ-∆)/∆], see Experimental details in SI for details).

(A)

(B)

(a)

(b)

S72

Figure S49. Molecular mechanics (Amber force field) optimized stereochemical representations of diastereomers of stable fused product (C) (Scheme 1). Inset A shows major deastereomer having C6' is in R configuration due to observable 4JHH W-coupling with H6' (1.5 Hz). Inset B shows minor diastereomer having C6' is in S configuration (no 4JHH because no W-coupling is possible). R = Bn (not shown for clarity of the picture).

S73

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