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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.
jyoti@boc.uu.se
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
S2
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
S3
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
S4
(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
S5
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).
S6
(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
S7
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
S8
(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
S9
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°.
S10
(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.
S11
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).
S12
(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
S13
(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'
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
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
'
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
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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)
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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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