Synthesis, Structural, and Conformational Analysis of 4′-C-Alkyl-2′-O-Ethyl-Uridine Modified Nucleosides

Article abstract of DOI:10.1002/ejoc.202001348

Sugar modifications have attracted much attention due to their potential structural and functional influence on therapeutic nucleic acids. Herein, we report the synthesis of dual modified 4′-C-azidomethyl-2′-O-ethyl-uridine (4′-AzM-2′-OEt?U) and 4′-C-aminomethyl-2′-O-ethyl-uridine (4′-AM-2′-OEt?U) nucleosides using linear multi-step synthesis (16 linear steps). Additionally, we report an alternative route for the synthesis of 2′-O-ethyl-uridine nucleoside which has been achieved in three steps with an overall yield of 40 %. X-ray structure of 4′-AzM-2′-OEt?U illustrates that the nucleoside adopts C2′-endo (South) conformation having a DNA-type glycosidic bond (χ) angle of ?116.01°. Computational studies revealed the C2′-endo and C4′-exo conformations for 4′-AzM-2′-OEt?U and 4′-AM-2′-OEt?U free nucleosides, respectively. The C4′-exo conformation in 4′-AM-2′-OEt?U free nucleoside is collectively stabilized by various non-covalent interactions between positively charged aminomethyl and 2′,3′-hydroxyl groups. Insights into the structural and conformational analysis of dual sugar modified nucleosides and oligonucleotides will be helpful in the rational design of modified nucleosides and therapeutic oligonucleotides.

Full text of DOI:10.1002/ejoc.202001348

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Synthesis, Structural, and Conformational Analysis of 4’-C-
Alkyl-2’-O-Ethyl-Uridine Modified Nucleosides
Rahul R. Nikam+,[a] S. Harikrishna+,[b] and Kiran R. Gore*[a, c]
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Sugar modifications have attracted much attention due to their
potential structural and functional influence on therapeutic
nucleic acids. Herein, we report the synthesis of dual modified
4’-C-azidomethyl-2’-O-ethyl-uridine (4’-AzM-2’-OEtÀ U) and 4’-C-
aminomethyl-2’-O-ethyl-uridine (4’-AM-2’-OEtÀ U) nucleosides
using linear multi-step synthesis (16 linear steps). Additionally,
we report an alternative route for the synthesis of 2’-O-ethyl-
uridine nucleoside which has been achieved in three steps with
an overall yield of 40%. X-ray structure of 4’-AzM-2’-OEtÀ U
illustrates that the nucleoside adopts C2’-endo (South) con-
formation having a DNA-type glycosidic bond (χ) angle of
À 116.01 . Computational studies revealed the C2’-endo and
C4’-exo conformations for 4’-AzM-2’-OEtÀ U and 4’-AM-2’-OEtÀ U
free nucleosides, respectively. The C4’-exo conformation in 4’-
AM-2’-OEtÀ U free nucleoside is collectively stabilized by various
non-covalent interactions between positively charged
aminomethyl and 2’,3’-hydroxyl groups. Insights into the
structural and conformational analysis of dual sugar modified
nucleosides and oligonucleotides will be helpful in the rational
design of modified nucleosides and therapeutic oligonucleo-
tides.
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Introduction
that stabilize the C3’-endo conformation enhance the thermo-
dynamic stability of RNA duplexes.[17,18]
In recent years, nucleic acid drugs have shown enormous
potential for treating a wide range of diseases.[1] Several nucleic
acid-based drug candidates are currently in the final stages of
clinical trials.[2–4] Numerous chemical modifications incorporated
in the ribose has enhanced the therapeutic performance of
various gene silencing agents such as antisense oligonucleo-
tides, ribozymes, and siRNAs.[5–9] Also, chemical modifications in
the sugar ring of therapeutic oligonucleotides have succeeded
in overcoming key challenges such as nuclease instability, off-
target effects, activation of immune responses, and in vivo
delivery.[10,11] Investigation of sugar puckering of chemically
modified nucleosides is essential since sugar conformation can
alter the structural stability and functional role of the
duplex.[11–13] Various 2’-substituted sugar modifications such as
2’-O-methyl (2’-OMe), 2’-Fluoro (2’-F), 2’-methoxyethyl (2’-MOE),
2’-O-allyl, 2’-azido, 2’-O-cyanoethyl, and 2’-O-ethylamine tune
the sugar puckering to C3’-endo resulting in an improved RNA:
RNA, RNA:DNA duplex stability.[14–16] As the C3’-endo conforma-
tion is preferred in an A-type RNA duplex, sugar modifications
2’-modifications such as 2’-F, 2’-OMe, and 2’-MOE have
attracted attention and have been explored in clinically
approved AONs and siRNAs drugs.[19,20] Also, dual modifications
at 2’- and 4’- sugar positions such as 4’-C-aminomethyl-2’-O-
methyl (4’-AM-2’-OMe), 4’-C-aminoethyl-2’-O-methyl (4’-AMEt-
2’-OMe), and 4’-C-aminopropyl-2’-O-methyl (4’-AMPr-2’-OMe)
analogs have adopted C2’-endo (South, DNA-type) sugar
conformation.[21,22] In contrast, other bi-functional sugar mod-
ifications such as 4’-C-aminomethyl-2’-deoxy-2’-fluoro (4’-AM-2’-
F), 4’-C-aminoethyl-2’-deoxy-2’-fluoro (4’-AMEt-2’-F), 2’,4’-diF, 2’-
OMe-4’-F, 2’-F-4’-OMe, 2’,4’-diOMe, 2’-F-4’-OMe-araU, 2’,4’-diF-
araU tuned the sugar conformation predominantly toward C3’-
endo (North, RNA-type), which has been attributed to the small
size, high electronegativity of fluorine, and additional CÀ H···F
interactions.[23–30] From the above observations, it is clear that 2’
and 4’- modifications can alter the sugar conformation,
structural stability, and functional role of the duplex.
Modifications at 2’ and 4’ positions in the ribose ring have a
profound effect on conformational preferences and the bio-
logical properties of therapeutic oligonucleotides. Herein, we
report the linear synthesis of dual sugar modified 4’-C-
azidomethyl-2’-O-ethyl-uridine A (4’-AzM-2’-OEtÀ U) and 4’-C-
aminomethyl-2’-O-ethyl-uridine B (4’-AM-2’-OEtÀ U) nucleosides
(Figure 1) (total 16 steps). X-ray crystal structure of dual sugar
modified 4’-AzM-2’-OEtÀ U nucleoside has been investigated to
determine the effect of dual sugar modifications on sugar
puckering. We also report a new route for synthesizing 2’-O-
ethyl-uridine nucleoside C (2’-OEtÀ U) in just 3 steps (Figure 1).
The synthesis of 2’-O-methyl and 2’-O-ethyl uridine ribonucleo-
side phosphoramidites and their incorporation into oligoribonu-
cleotides were reported previously in the literature.[31–33] To the
best of our knowledge, the influence of 2’-O-ethyl modification
on sugar conformation has not been studied so far. In this
[a] R. R. Nikam,+ Prof. K. R. Gore
Department of Chemistry, University of Mumbai,
Mumbai-400098, India
E-mail: kiran@chem.iitkgp.ac.in
kiranrgore@gmail.com
http://www.chemistry.iitkgp.ac.in/professor/kiran
[b] Dr. S. Harikrishna+
Center for Structural Biology, Vanderbilt University,
Nashville, Tennessee 37232, United States
[c] Prof. K. R. Gore
Department of Chemistry, Indian Institute of Technology Kharagpur
Kharagpur, West Bengal-721302, India
[+] R. R. Nikam and S. Harikrishna contributed equally to this work.
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/ejoc.202001348
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Figure 1. Structures of various modified (dual/mono) and unmodified uridine analogs studied in this report.
context, synthesis and sugar conformational analysis of 2’-O-
Results and Discussion
ethyl modified nucleosides may provide useful insights for the
rational design of chemically modified therapeutically appealing
oligonucleotides. Also, the 4’-C-azidomethyl group in 4’-AzM-2’-
OEtÀ U would open up new opportunities for conjugating the
lipophilic or fluorogenic moiety using click chemistry.[34,35] The
sugar conformation and Watson-Crick base pairing in the
oligonucleotide system have been assessed using DFT calcu-
lations, molecular dynamics (MD) simulations, and umbrella
sampling simulations.
Besides, a detailed comparative study of dual modified 4’-
AzM-2’-OEtÀ U, 4’-AM-2’-OEtÀ U, 4’-AM-2’-OMeÀ U, mono modi-
fied 2’-OEtÀ U, 2’-OMeÀ U, 2’-FÀ U, 4’-AzMÀ U, 4’-AMÀ U; and
unmodified 2’-OH uridine nucleoside analogs (A-I) have been
performed to distinguish the effects of 2’- and 4’-substituents
on the sugar conformational preferences (Figure 1).
Synthesis of 4’-C-Azidomethyl-2’-O-ethyl (5),
4’-C-Aminomethyl-2’-O-ethyl (6) and 2’-O-ethyl (10) Uridine
nucleosides
The synthesis of 4’-C-azidomethyl-2’-O-ethyl-U (5) and 4’-C-
aminomethyl-2’-O-ethyl-U (6) was achieved from 4’-C-
azidomethyl-uridine nucleoside precursor 2 (Scheme 1). The
intermediate 4’-C-azidomethyl-uridine nucleoside 2 was synthe-
sized from D-glucose in 12 steps according to reported
procedures.[36–38] Uridine nucleoside 2 was deacetylated using
1 M sodium methoxide in methanol for 3 h at room temper-
ature to yield 2’-hydroxy nucleoside 3. Crude compound 3 was
directly treated with ethyl iodide and sodium hydride (NaH) in
THF at room temperature for 12 h to obtain 2’-O-ethyl nucleo-
side 4 in 81% yield. Compound 4 was further debenzylated
Scheme 1. Synthesis of 4’-AzM-2’-OEt and 4’-AM-2’-OEt uridine nucleosides 5, 6; Reagents and conditions (i) NaOMe, MeOH, rt, 3 h; (ii) ethyl iodide, NaH, THF,
°
°
°
rt, 12 h (81% after two steps); (iii) BCl3, DCM,À 78 C for 3 h and À 30 C for 5 h (72%); (iv) PPh3, THF, H2O, 45 C, 3 h (86%); (v) DMTrÀ Cl, pyridine, rt, 4 h (88%);
°
°
(vi) ethyl iodide, K2CO3, THF, 50 C, 36 h (53%); (vii) 3% TFA in DCM, 0 C, 15 min (85%).
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°
using 1 M BCl3 in DCM using DCM as solvent at À 78 C for 3 h
showed its characteristic peak in a slight deshielding region
around 63 ppm, whereas for compound 9a, the À N3À CH2
carbon peak was observed in the shielding region around
36 ppm. Compound 9 was further confirmed by HETCOR
spectra wherein the 1H peak for À OÀ CH2 protons showed
correlation with corresponding carbon peak at 63 ppm. Finally,
compound 9 was subjected to DMT deprotection using 3%
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5
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and at À 30 C for 5 h to obtain the debenzylated nucleoside 5
in 72% yield. Further, compound 5 was treated with PPh3 and
°
H2O in THF at 45 C for 3 h to furnish the final nucleoside
compound 6 in 86% yields. The synthesis of 2’-O-ethyl-uridine
10 was started from commercially available uridine nucleoside
7 (Scheme 2). Synthesis of 2’-O-ethyl-uridine 10 was achieved in
three steps with an overall yield of around 40%. The synthesis
of DMT protected precursor 8 was achieved using uridine
nucleoside with DMTrÀ Cl in pyridine at room temperature for
4 h.
Further, compound 8 was subjected to 2’-ethylation using
ethyl iodide, and K2CO3 in THF resulted in the formation of
desired 2’-O-ethyl uridine nucleoside 9 along with undesired N3-
ethyl uridine compound 9a (Scheme 2). The desired compound
9 was obtained in 53% yield and undesired compound 9a was
obtained in 38% yield. The desired product 9 was confirmed by
1H, 13C, HETCOR, and HRMS data (Compound 9 and 9a,
Supporting Information). In 13C spectra, the À OÀ CH2 carbon
°
trifluoroacetic acid in DCM at 0 C for 15 min to afford final
nucleoside compound 10 in 85% yield.
X-ray crystal studies of 4’-AzM-2’-OEt-U. The molecular
structure of 4’-AzM-2’-OEtÀ U nucleoside was determined using
single-crystal X-ray diffraction in order to investigate the effect
of 4’-C-azidomethyl and 2’-OEt group on sugar puckering
(Figure 2).
The single crystal of 4’-AzM-2’-OEtÀ U nucleoside was
obtained by slow evaporation in a solution of MeOH and
hexane in a ratio of 8:2 at room temperature. The selected
crystal data, data collection, and refinement parameters are
listed in Table S2, Supporting Information. Various torsional
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Scheme 2. Synthesis of 2’-OEt uridine nucleoside 10; Reagents and conditions (v) DMTrÀ Cl, pyridine, rt, 4 h (88%); (vi) ethyl iodide, K2CO3, THF, 50 C, 36 h
°
(53%); (vii) 3% TFA in DCM, 0 C, 15 min (85%).
Figure 2. Crystal structure (ORTEP) of 4’-AzM-2’-OEt-uridine 5. (CCDC-1982412). Colour pattern in crystal structure: grey denotes carbon atom; red is oxygen
atom, and blue is nitrogen atom.
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angles (ν0- ν4), were measured and νmax (puckering amplitude),
conformation, JH1’À H2’ value is estimated to be closer to
10 Hz.[43,44] Based on NMR studies, it was observed that 4’-AzM-
2’-OEt and 4’-AM-2’-OEt-uridine nucleosides predominantly
adopt South conformation, and 2’-O-ethyl-uridine nucleoside
adopts exclusively North conformation, in solution. Moreover,
Ueno Y. and coworkers have also obtained similar results for
analogous compounds using NMR measurements.[22,24] The
calculated percentages of sugar conformations for 4’-C-azi-
doethyl-2’-O-methyluridine and 4’-C-azidopropyl-2’-O-meth-
yluridine compounds, observed ~69% South conformations for
both the compounds.[22,24] These results confirm that the 4’-
modification helps to alter the sugar conformation exclusively
towards the South conformation.
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and pseudorotational angle P were calculated. The dihedral
parameters which define the internal rotation of the sugar ring
is mentioned in Table S3, Supporting Information. The pseudor-
otation angle (P) calculated using the PROSIT tool[39,40] by NCI/
CADD group (Table S4, Supporting Information). The χ angle
(O4’À C1’À N1À C2) adopts a value at À 116.03, which indicated
C2’-endo sugar conformation. The predicted χ angle for typical
°
DNA type conformation is À 120 , and for typical RNA type
°
conformation is À 160 .
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The calculated pseudorotational phase angle P value at
169.4 indicated that 4’-AzM-2’-OEtÀ U nucleoside favored C2’-
endo conformation. Based on the parameters mentioned in
Table S3, Supporting Information, it is clear that the sugar
conformation of 4’-AzM-2’-OEtÀ U nucleoside favors C2’-endo
(South). In our earlier reports, we have studied the sugar
puckering for the 4’-AM-2’-OMeÀ U and 4’-AM-2’-FÀ U
modifications.[23] The torsional angles and P values for all four
dual modifications are mentioned in Table 1. The data confirms
that the value of pseudorotation phase angle (P) of 4’-AM-2’-
Sugar conformation and electronic effects. Here, we have
calculated the stereoelectronic preference of sugar conforma-
tion of our modified free nucleosides based upon the reported
protocol (Figure S1, Supporting Information).[44] The strength of
°
the anomeric effect is as follows 4’-F (P=55.5 ) >4’-AM-2’-OEt
°
°
(P=171.5 ) >4’-AzM-2’-OEt (P=169.4 ). The anomeric effect is
weaker due to the presence of 4’-C-aminomethyl and 4’-C-
azidomethyl group since the lone pair orbital interaction is
weak between O4’ and C4’.[45–47] The 4’-modifications used in
our study show weak hyperconjugation and anomeric effect,
responsible for the South conformation (Table S1, Supporting
Information). The superposition of the 4’-AzM-2’-OEt crystal
structure and the lower energy model shows the overall
orientation and sugar puckering is similar. The potential energy
surface of the nucleosides along the pseudorotational phase
angle (Figures S2-S10, Supporting Information) calculated utiliz-
ing the second-order Møller–Plesset perturbation theory (MP2)
and the 6–311G* basis set (vacuum phase) in Gaussian09
package. In the case of 4’-AzM-2’-OEt nucleoside, a single low
energy profile was observed at C2’-endo orientation. The two
low energy regions were observed in the energy profile of 4’-
AM-2’-OEt nucleoside in C2’-endo and C4’-exo orientation, with
~ <1 kJ/mol lower energy in the C2’-endo orientation (Figure 3).
The C4’-exo is collectively stabilized by the formation of the
following non-covalent interactions (i) 4’-C-aminomethyl and 3’-
hydroxyl form H-bond at a distance of ~2.0 Å, (ii) 2’-hydroxyl
and 3’-hydroxyl form H-bond at a distance of ~2.0 Å, and (iii) C-
H…O interaction between carbonyl in the base and the 2’-O-
ethyl modification (iv) 4’-C-aminomethyl and 5’-hydroxyl ~2.0 Å.
Similar conformational preferences were also observed in the
loop structure.[48]
°
°
OMe (171.5 ) is slightly greater than 4’-AzM-2’-OEt (169.4 ) and
°
4’-AM-2’-OEt (166.98 ) compounds reported in this study
(Table 1). The change in P values is attributed to a small
increase in carbon chain length (-ethyl v/s -methyl) at C2’-
position in 4’-AzM-2’-OEt and 4’-AM-2’-OEt nucleosides com-
pared to 4’-AM-2’-OMe. In contrast, P values for 4’-AM-2’-F
indicates C3’-endo (North) conformation predomi-
nantly compared to 4’-AzM-2’-OEt (169.4 ) and 4’-AM-2’-OEt
[23]
°
(33.6 )
°
°
(166.98 ) which preferentially adopt C2’-endo conformation
(Table 1). This dramatic change in sugar conformation is
attributed to the higher electronegativity of fluorine and
intramolecular H- bonding between Fluorine and 4’À CÀ CH2.[23]
NMR conformational analysis. Detailed information regard-
ing the conformation of the ribose ring of 4’-AzM-2’-OEt and 4’-
AM-2’-OEt was obtained using the vicinal spin coupling
constants (JH1’À H2’). In this particular study, the percentage of
North and South conformers in solution was calculated by using
the empirical equation S(%)=10 × JH1’H2’, in which JH1’H2’ is the
1HÀ 1H coupling constant between the H1’ and H2’ protons in
the ribose ring.[41,42] The measured values of JH1’–H2’ coupling
1
constants obtained from basic H NMR experiments for 4’-AzM-
2’-OEt and 4’-AM-2’-OEt found to be ~6.0 Hz and JH1’À H2’
coupling constants for 2’-O-ethyl-uridine nucleoside was found
to be ~3.0 Hz respectively. For nucleosides that are present
predominately in the North conformation, the J value between
H1’ and H2’ is estimated to be less than 4 Hz. For South
Since C4’-exo conformation is a close neighbor of C3’-endo
conformation, the population of C3’-endo conformation may be
Table 1. Sugar endocyclic torsions and P angle of 2’-OMe v/s 2’-OEt v/s 2’-F (dual modifications).
Modification
Torsional angles[a]
ν0
P
[b] [deg]
Sugar puckering
ν1
ν2
ν3
ν4
4’-AM-2’-OMe
4’-AM-2’-F
4’-AzM-2’-OEt
4’-AM-2’-OEt
À 17.6
7.2
34.2
À 36.4
45.8
26.2
À 5.3
33.6
171.5
24.8
169.4
166.98
C2’-endo[23]
C3’-endo[23]
C2’-endo
À 24.3
À 49.6
À 18.2
33.8
À 35.8
26.1
À 5.3
À 20.6
33.9
À 35.2
27.1
À 5.1
C2’-endo
[a] Endocyclic torsional angles are (ν0) (C4’À O4’À C1’À C2’), (ν1) (O4’À C1’À C2’À C3’), (ν2) (C1’À C2’À C3’À C4’), (ν3) (C2’À C3’À C4’À O4’) and (ν4) (C3’À C4’À O4’À C1’); ν0-
ν4 =torsional angles; [b] P=pseudorotation angle.
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Figure 3. Non-covalent interactions in the nucleoside that stabilizes the C4’-exo sugar conformation. (A) The orientation of the nucleoside showed the C4’-exo
sugar conformation of 4’-C-aminomethyl-2’-O-ethyl (4’-AM-2’-OEt), C4’ position is indicated by an arrow. (B) Multiple non-covalent interactions between 4’-C-
aminomethyl and 3’-hydroxyl group, which stabilizes the C4’-exo conformation.
possible at the nucleoside level. To understand the absence of
such conformation in 4’-AM-2’-OMe modification, a similar
analysis was carried out; however, the key C-H…O interaction
becomes weaker, which induces conformational flexibility in
the sugar ring. However, the additional experiments will help to
understand the influence of these non-covalent interactions in
conformational stability.
Molecular modeling studies of oligonucleotides. The effect
of 4’-C-aminomethyl-2’-O-ethyl (4’-AM-2’-OEt) and 4’-C-
azidomethyl-2’-O-ethyl-uridine (4’-AzM-2’-OEt) chemical modifi-
cations on RNA duplex were studied using molecular dynamics
(MD) simulations. Comparisons of MD results for these
modifications executed using results from the MD simulations
of 2’-OH-uridine unmodified and 2’-O-ethyl-uridine (2’-OEt)
modified RNAs[33,49,50] (Figure 4). The C2’-endo conformers of 4’-
AM-2’-OEt and of 4’-AzM-2’-OEt as seen in the stereoelectronic
study was used to calculate the RESP charges using the RED
tools package. The developed structure and dihedral parame-
ters (Supporting Information) were incorporated and MD
simulations (400 ns) were performed using the AMBER16
package.[51] The forcefield used for RNA is ff99OL3.[52,53] The
duplexes investigated in the simulations is the crystal structure
of RNA dodecamer (PDB ID: 1SDR), the modification placed in
strand II (7th position, Table S5).
Single modification in strand II has the least effect on the
overall global A-RNA architecture (Figure 4D and Figure 4E). The
Figure 4. MD simulations result from the 400 ns trajectory. (A) WÀ C base pairing between 4’-AzM-2’-OEt uridine and unmodified adenine. (B) Inter and (C)
intrastrand stacking and phosphate distances around the 4’-AzM-2’-OEt modified region. (D) Superimposed MD simulation snapshots at various times (every
40 ns) retrieved from 400 ns trajectory. Preferred sugar pucker population in the pseudorotation phase wheel from the 400 ns MD trajectory of (E) 4’-AM-2’-
OEt and (F) 4’-AzM-2’-OEt. Atoms are represented in the sticks; modification is highlighted using different colors. Strand I is green and Strand II in cyan color.
Distance between two atoms was represented using dashed lines.
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modification makes multiple local conformational changes in
the duplex (Figures S11-S19, Supporting Information). Unlike,
4’-AM-2’-OMe modification, which predominantly prefers C2’-
endo (>80%) sugar puckering, the 4’-AM-2’-OEt found to be
C2’-endo (66%) and C3’-endo (27%) conformers. On the other
hand, 4’-AzM-2’-OEt, the sugar puckering predominately favors
the C2’-endo orientation (92%), mimicking the DNA-like sugar
conformation. The preferred sugar puckering was found in the
northern region (C3’-endo) for 2’-OEt modification and unmodi-
fied 2’-OH RNA, as expected. The introduction of 4’-C-amino-
alkyl modifications favors the sugar puckering toward C2’-endo
has also been observed in the previous studies.[21,22,24,54,55]
Moreover, it is interesting to note that 4’-AM-2’-F adopt C3’-
endo sugar puckering by the formation of C-H…F H-bonding.[23]
These results suggest that sugar conformational transition in
the modified nucleotides also depends on the stereoelectronic
effects of the chemical modification introduced in the ribose.[46]
Therefore, we propose that only bulkiness at C4’-position may
not orient the sugar conformation toward C2’-endo and
bulkiness at C2’-position orient the sugar puckering toward C3’-
endo conformation. Moreover, stereoelectronic effects of the
chemical groups were introduced to play a key role in the
conformational preference of the nucleotide.
angles. Also, the χ angle is similar to that of the DNA-like
1
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4
5
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9
°
conformation (~À 120 ). This deviation was measured in
comparison to 2’-OH/unmodified duplex simulations. The WÀ C
base-pairing energy estimated using umbrella sampling simu-
lations. The results indicate that both 4’-AzM-2’-OEt and 4’-AM-
2’-OEt retains two WÀ C base pairing and the energy (~À 4 kcal/
mol) is comparable to those of unmodified and 2’-OEt modified
base pairs (Figure 4D). The C1’-C1’ distance between 4’-AzM-2’-
OEt-uridine and unmodified adenine is higher (~11.3�1.1 Å)
and flexible in comparison to the unmodified uridine and
adenine system (~10.5�0.7 Å).[56]
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In the case of 4’-AM-2’-OEt uridine and unmodified
adenine, the C1’-C1’ distance is found to be ~11�0.8 Å
(Figure 5A). The distance between the phosphate backbone
and the 4’-azido group is ~4.2�0.3 Å. Stacking interactions
play a significant role in stabilizing nucleic acid structures; we
have analyzed the base-pair parameters to understand the base
stacking interactions. There is a slight deviation in the intra-
strand phosphate distances (~6.5 Å), and also the interstrand
phosphate (~18 Å) distances in both 4’-AzM-2’-OEt and 4’-AM-
2’-OEt modifications in comparison to the unmodified intra and
interstrand phosphate distances 5.5 Å and 17 Å, respectively
(Figure 5B and Figure 5C).
A small deviation in the slide and roll parameters was
observed in 4’-AM-2’-OEt modification, similar to B-DNA
orientation (Figures S21-S23, Supporting Information). As a
consequence, x-displacement showed a deviation from the
unmodified A-RNA duplex. Similarly, 4’-AzM-2’-OEt modification
The analysis of backbone torsional angles reveals that 4’-
AM-2’-OEt adopt RNA-like conformation and 4’-AzM-2’-OEt
adopt DNA-like conformation. A significant deviation in the
backbone is seen in γ and δ angles for 4’-AzM-2’-OEt. In the
case of 4’-AM-2’-OEt, the larger deviation is seen in β and γ
Figure 5. MD simulations result from the 400 ns trajectory. (A) WÀ C base pairing between 4’-AM-2’-OEt uridine and unmodified adenine. (B) Inter and (C)
intrastrand stacking and phosphate distances around the 4’-AM-2’-OEt modified region. (D) Superimposed MD simulation snapshots at various times (every
40 ns) retrieved from 400 ns trajectory. (E) Root-mean-square deviation graph obtained from the MD trajectories of unmodified, 2’-OEt, 4’-AM-2’-OEt, and 4’-
AzM-2’-OEt modified duplex RNAs. (F) The potential mean of force/free energy values as a function of H-bond distance between bases from umbrella
sampling simulations for unmodified and modified base pairs.
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1
following are the signals of the deuterated solvents: CD3OD in H
showed minor deviation in helical base-pair step parameters
and minor groove distances (Figures S21–S23, Supporting
Information). Collectively, these minor conformational changes
may not affect the thermodynamic stability of the siRNA.
However, the introduction of this modification throughout the
siRNA may induce conformational changes in A-RNA architec-
ture.
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5
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NMR spectra at 3.31 ppm, CDCl3 in 13C spectra at 77.2 ppm, and
CD3OD at 49.1 ppm respectively. Multiplicity values of H NMR spin
couplings are denoted as singlet (s), broad singlet (bs), doublet (d),
quartet (q), septet (sep), quintet (quint), doublet of doublets (dd),
and multiplet (m). Coupling constants (J) values are reported in Hz.
High-resolution mass spectra (HRMS) were recorded in positive-ion
electrospray ionization (ESI) mode.
1
Computational methods
9
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All the minimization and MD simulations were carried out in the
AMBER16 package,[51] and analysis was carried out using CPPTRAJ in
AMBER Tools. Figures were rendered using PyMOL 1.2. All quantum
mechanics calculations were carried out using Gaussian D01.
Version.[57]
Chemical modifications at 4’ and 2’-positions create a profound
effect on sugar puckering preference as well as the biological
properties of therapeutic oligonucleotides. Here, we have
reported the linear synthesis of 4’-AM-2’-OEtÀ U and 4’-AzM-2’-
OEtÀ U nucleosides. We also reported the synthesis of 2’-OEtÀ U
in only 3 steps with an overall yield of ~40%. X-ray structural
studies of 4’-AzM-2’-OEt uridine revealed that the sugar
conformation drives toward the South-type C2’-endo. Based on
NMR studies, it was observed that 4’-AzM-2’-OEt and 4’-AM-2’-
OEt-uridine nucleosides predominantly adopt a C2’-endo con-
formation. (JH1’H2’ =6.0 Hz, 60% South). Moreover, 2’-OEtÀ U
adopts a C3’-endo sugar conformation. At the double-stranded
RNA level, 4’-AzM-2’-OEt retains the strong South-type pucker-
ing and has a very less disrupting effect on the base pairing
and stacking interactions. Furthermore, positively charged
aminomethyl group in 4’-AM-2’-OEtÀ U modified nucleoside
make multiple non-covalent interactions and stabilize the
unique C4’-exo puckering only in free RNA nucleoside. The C4’-
exo preference of 4’-AM-2’-OEtÀ U needs further investigation
since conformational preferences are unexpected in the
ribonucleoside.[45] However, at the RNA level, such interactions
become weaker, and the C2’-endo sugar conformation is most
preferred. These results suggest that these modifications are
well suited to be incorporated into siRNA guide strands since it
retains the A-form RNA architecture. Comparative studies using
2’-OH, 2’-OEt, and 2’,4’-dual modifications in ribose revealed
that the 4’-modifications have a noteworthy impact on the
sugar puckering. Further investigation of these chemical
modifications in oligonucleotides is under progress in our
laboratory to understand their potential to achieve high gene
silencing efficiency.
Stereoelectronic studies. Nucleosides were initially optimized using
DFT (B3LYP-6-311G+ +, polarized) basis set. The calculations were
performed using the literature-based protocol.[44]
Forcefield development. Initially, along the pseudorotation phase
angle, the modified nucleoside was scanned using DFT (B3LYP-6-
311G+ +) method. The forcefield for the modified nucleosides was
developed using the standard protocol, RED Tool was used to
obtaining the charge. AMBER forcefield with ff99χOL3 correction
was used for RNA.[53]
Classical MD simulations of oligonucleotides. Following oligonu-
cleotide sequence was used for MD simulations of duplex. (Strand I,
5’-UAAGGAGGUGAU-3’; Strand II, 5’-AUCACCUCCUUA-3’). Where,
the modification position is highlighted using bold and underline.
2’-OH, 4’-C-aminomethyl-2’-O-ethyl, 4’-C-azidomethyl-2’-O-ethyl
modified oligonucleotides were utilized for MD simulations.
Modified oligonucleotides were allowed to equilibrate in
a
rectangular box of TIP4PEW explicit solvent neutralized with
monovalent sodium anions. Solute was buffered on all sides with
12 Å solvent. All-atom minimization was carried out in three stages:
First, the solvent was minimized around the restrained RNA with
10000 steps steepest gradient descent followed by 10000 steps of
conjugate gradient descent. Second, the RNA was minimized in the
restrained buffer for 5000 steps steepest gradient descent followed
by 15000 steps of conjugate gradient descent. Finally, the whole
system was unrestrained and underwent 500 steps of steepest
gradient descent followed by 9500 steps of conjugate gradient
descent minimization. Post-minimization, SHAKE was implemented
to constrain covalent bonds to hydrogen atoms. Systems were
slowly heated in the NVT ensemble to 100 K over 50 ps with a 1 fs
time step. Subsequently, systems were heated in NPT ensemble at
1 bar with isotropic position scaling from 100 K to physiologic
temperature (310 K) over 500 ps and 1 fs time step. Production
simulation in the NPT ensemble at 310 K occurred for 300 ns with a
Monte Carlo barostat and 2 fs time step. The temperature was
controlled using Langevin dynamics with a collision frequency of
1 psÀ 1 and a unique random seed for each simulation. Periodic
boundary conditions were imposed on the system throughout
heating and equilibration. Electrostatics were evaluated using the
Particle Mesh Ewald (PME) method and a distance cutoff of 10.0 Å.
Umbrella sampling simulation to study the pseudorotation phase
angle and H-bond free energy was carried out using previous
methods.
Experimental Section
General information: All the chemicals, reagents, and solvents were
purchased from commercial sources. Acetonitrile, THF, DMF, DCM,
DIPEA, pyridine, and Et3N were dried using calcium hydride.
Toluene was dried using powdered calcium chloride; MeOH, and
EtOH over calcium oxide. Acetone was dried using KMNO4 and
CaSO4. Thin-layer chromatography (TLC) was conducted on ready-
made UV active Merck silica gel plates with visualization under
ultraviolet light or by charring the TLC plate into a 5% concentrated
H2SO4 in ethanol (v/v) solution. Silica gel used for column
1-[2’-O-Ethyl-3’,5’-di-O-benzyl-4’-C-azidomethyl-β-D-ribofurano-
syl]-uracil (4): Compound 2 (400 mg, 0.76 mmol) was dissolved in
dry methanol (3 mL) and stirred for 5 min. 1 N NaOMe in MeOH
(1.14 mL) was prepared separately and added to the reaction
mixture. The reaction mixture was stirred at room temperature for
3 h. After completing the reaction confirmed by TLC, MeOH was
1
chromatography comprised of 100–200 mesh size. H NMR (400 or
300 MHz), 13C NMR (100 or 75 MHz) were recorded on a 400 MHz
(Bruker) or a 300 MHz (Bruker) instrument. The chemical shifts in
ppm (δ) are reported downfield from TMS (0 ppm) signal. The
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completely evaporated under reduced pressure, and the reaction
then dissolved in dry THF (10 mL), anhydrous K2CO3 (300 mg,
2.17 mmol) was added under N2 and stirred for 5 min. Then ethyl
iodide (0.24 mL, 2.98 mmol) was added slowly in dropwise under N2
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4
5
6
7
8
9
mixture was extracted with ethyl acetate and water (3×40 mL). All
organic layers were combined, washed with brine (40 mL) and dried
over anhydrous Na2SO4, and concentrated. The crude compound 3
obtained after work-up was co-evaporated with toluene (3×5 mL),
and dried under high vac. Compound 3 was further dried under
°
and reaction was stirred at 50 C for 36 h. After complete
consumption of starting material, the reaction mixture was
extracted with ethyl acetate (3×50 mL) and organic layer washed
with saturated NaHCO3 solution (2×50 mL). The organic layer was
separated, and the aqueous phase was back extracted with ethyl
acetate. All organic layers were combined, washed with brine
(100 mL), dried using anhydrous Na2SO4, and concentrated on
rotavapor to obtain a crude mixture of compound 9 and 9a. Crude
residue was purified by column chromatography (35À 40% ethyl
acetate in pet ether+3% Et3N) to afford desired product 9 as pale
yellow solid (330 mg, 53%) and undesired product 9a (250 mg,
high vacuum for 1 h. Further, crude compound
3 (385 mg,
0.83 mmol) was directly dissolved in dry THF (5 mL), NaH (100 mg,
°
4.18 mmol) was added in two portions at 0 C and stirred for 5 min.
Then ethyl iodide (0.33 mL, 2.11 mmol) was added dropwise under
N2, and the reaction was stirred at room temperature for 12 h. After
completing the reaction, a saturated NaHCO3 solution (50 mL) was
added, and the reaction mixture was extracted with ethyl acetate
(3×50 mL). All organic layers were combined, washed with brine
(40 mL), dried over anhydrous Na2SO4, and concentrated under
reduced pressure. Crude residue was purified by column chroma-
tography (15À 20% ethyl acetate in pet ether) to yield compound
4 as white sticky solid (345 mg, 81%, after two steps); Rf= 0.63
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38%); Rf=0.56 (60% ethyl acetate in pet ether); Compound 9: H
NMR (300 MHz, CDCl3): δ 8.11 (d, J=6.0 Hz, 1H), 7.36À 7.21 (m,
10H), 6.83 (d, J=9 Hz, 4H), 5.86 (d, J=3.0 Hz, 1H), 5.67 (d, J=6.0 Hz,
1H), 4.46À 4.32 (m, 5H), 3.78 (bs, 6H), 3.48 (dd, J=3.0 Hz, 12 Hz,
1H), 3.39 (dd, J=3.0 Hz, 12 Hz, 1H), 1.36 (t, J=6.0 Hz, 3H); 13C NMR
(75 MHz, CDCl3): δ 171.9, 158.6, 157.4, 144.2, 142.5, 135.3, 135.2,
130.0, 128.0, 127.9, 127.0, 113.2, 96.0, 92.9, 86.9, 85.4, 71.4, 63.4,
62.6, 55.2, 14.1; HRMS (ESI positive) m/z calcd for C32H34N2O8Na
597.21 [M+Na]+, found [M+Na]+ 597.22.
1
(40% ethyl acetate in pet ether); H NMR (300 MHz, CDCl3): δ 10.09
(s, 1H), 7.94 (d, J=9.0 Hz, 1H), 7.39–7.21 (m, 10H), 6.04 (d, J=3.0 Hz,
1H), 5.17 (d, J=9.0 Hz, 1H), 4.72 (d, J=12.0 Hz, 1H), 4.51À 4.46 (m,
3H), 4.26 (d, J=6.0 Hz, 1H), 4.0–3.86 (m, 4H), 3.61À 3.51 (m, 2H),
3.34 (d, J=12.0 Hz, 1H), 1.25 (t, J=6.0 Hz, 3H); 13C NMR (75 MHz,
CDCl3): δ 163.86, 150.31, 140.36, 137.27, 136.99, 128.63, 128.49,
128.35, 128.15, 128.06, 127.88, 101.73, 89.01, 87.34, 81.52, 75.66,
73.76, 72.90, 70.23, 67.01, 53.18, 15.35; HRMS (ESI positive) m/z
calcd for C26H29N5O6Na 530.2010 [M+Na]+, found [M+Na]+
530.2010.
1
Compound 9a: H NMR (300 MHz, CDCl3): δ 7.82 (d, J=9.0 Hz, 1H),
7.38À 7.21 (m, 10H), 6.84 (d, J=9.0 Hz, 4H), 5.86 (d, J=3.0 Hz, 1H),
5.48 (d, J=6.0 Hz, 1H), 4.41À 4.38 (m, 1H), 4.31-4.29 (m, 1H), 4.22-
4.21 (m, 1H), 3.97 (q, J=6.0 Hz, 2H); 3.78 (s, 6H); 3.50 (dd, J=3.0 Hz,
12 Hz, 1H), 3.42 (dd, J=3.0 Hz, 12 Hz, 1H), 1.22 (t, J=6.0 Hz, 3H); 13
C
1-[2’-O-Ethyl-4’-C-azidomethyl-β-D-ribofuranosyl]-uracil (5): Com-
pound 4 (300 mg, 0.59 mmol) was dissolved in dry DCM (3 mL), to
which 1 M BCl3 in DCM (3.2 mL) was added dropwise under N2 at
NMR (75 MHz, CDCl3): δ 162.5, 158.7, 151.5, 144.2, 137.7, 135.5,
135.1, 130.0, 128.0, 127.9, 127.1, 113.2, 101.7, 91.3, 87.0, 84.3, 76.0,
70.6, 62.3, 55.2, 36.3, 12.7.
°
À 78 C. The reaction mixture was stirred under N2 for 3 h. The
2’-O-ethyl-uridine (10): Compound 9 (100 mg, 0.174 mmol) was
°
reaction mixture was then warmed to À 30 C and stirred further for
°
dissolved in dry DCM (3.0 mL), and stirred at 0 C for 5 min. Then
3% trifluoroacetic acid in DCM (1.0 ml) was added in drop wise
5 h. After completion of reaction, the reaction mixture was
quenched using 1:1 mixture of DCM-MeOH (1 mL). The solvent was
completely evaporated and crude compound obtained was purified
by column chromatography (70% ethyl acetate in pet ether) to
afford compound 5 as white solid (140 mg, 72%); Rf= 0.3 (60%
°
under N2 and reaction was stirred at 0 C for 15 min. After
completion of reaction, without any solvent work-up, the reaction
mixture was directly purified by column chromatography (100%
ethyl acetate+3% Et3N) to afford compound 10 as pale yellow oily
liquid (40 mg, 85%); Rf=0.25 (100% ethyl acetate); 1H NMR
(300 MHz, CD3OD): δ 8.31 (d, J=9.0 Hz, 1H), 5.97 (d, J=6.0 Hz, 1H),
5.81 (d, J=3.0 Hz, 1H), 4.32 (q, J=6.0 Hz, 2H), 4.08À 4.05 (m, 2H),
3.98À 3.95 (m, 1H), 3.82 (dd, J=3.0 Hz, 12 Hz, 1H), 3.68 (dd, J=
3.0 Hz, 12 Hz, 1H), 1.22 (t, J=6 Hz, 3H); 13C NMR (75 MHz, CD3OD): δ
173.3, 158.5, 145.4, 97.3, 92.5, 86.0, 76.5, 70.4, 64.4, 61.6, 14.4; IR
(cmÀ 1): 1645, 1510, 1304, 1248, 1170, 1031, 820, 770, 580; (HRMS
(ESI positive) m/z calcd for C11H16N2O6Na 295.08 [M+Na]+, found
[M+Na]+ 295.09.
ethyl acetate in pet ether); mp 140–142 C; 1H NMR (300 MHz,
°
CDCl3): δ 7.96 (d, J=9.0 Hz, 1H), 5.99 (d, J=6.0 Hz, 1H), 5.64 (d, J=
9.0 Hz, 1H), 4.28 (d, J=6.0 Hz, 1H), 4.11 (dd, J=6.0 Hz, 3.0 Hz, 1H),
3.65À 3.48 (m, 5H), 3.35 (d, J=15.0 Hz, 1H), 1.13 (t, J=6.0 Hz, 3H);
13C NMR (75 MHz, CDCl3): δ 165.99, 152.38, 142.64, 103.15, 89.48,
88.23, 82.99, 71.34, 67.47, 64.53, 53.45, 15.55; IR (cmÀ 1): 2101, 1680,
1454, 1270, 1090, 1064, 731, 697; HRMS (ESI positive) m/z calcd for
C12H17N5O6Na 350.1071 [M+Na]+, found [M+Na]+ 350.1070.
1-[2’-O-Ethyl-4’-C-aminomethyl-β-D-ribofuranosyl]-uracil (6): Diol
compound 5 (100 mg, 0.30 mmol) was dissolved in THF (2.0 mL). To
this, water (0.04 mL) and PPh3 (103 mg, 0.39 mmol) were added
Deposition Number 1982412 (for 5) contains the supplementary
crystallographic data for this paper. These data are provided free of
charge by the joint Cambridge Crystallographic Data Centre and
Fachinformationszentrum Karlsruhe Access Structures service
www.ccdc.cam.ac.uk/structures.
°
simultaneously. The reaction mixture was stirred at 45 C for 3 h.
The solvent was completely evaporated at high vac. and dried
crude compound was purified by column chromatography (100%
ethyl acetate in pet ether+3% Et3N) to give amino nucleoside 6 as
1
white sticky solid (78 mg, 86%). Rf= 0.25 (100% ethyl acetate); H
NMR (300 MHz, CDCl3): δ 8.36 (s, 1H), 7.81 (d, J=9.0 Hz, 1H), 5.94 (d,
J=6.0 Hz, 1H), 5.58 (d, J=9.0 Hz, 1H), 4.30 (d, J=6.0 Hz, 1H), 4.15 (t,
J=6.0 Hz, 1H), 3.55À 3.43 (m, 3H), 3.24 (d, J=15 Hz, 1H), 3.16 (d,
J=12 Hz, 1H), 2.89 (d, J=12 Hz, 1H), 1.03 (t, J=6 Hz, 3H); 13C NMR
(75 MHz, CDCl3): δ 165.85, 156.77, 142.53, 103.46, 89.26, 83.50,
82.67, 78.99, 67.66, 45.60, 15.57; IR (cmÀ 1): 1687, 1510, 1252, 1177,
1027, 832, 702, 584; HRMS (ESI positive) m/z calcd for C12H19N3O6
301.1282, found [M+H]+ 302.1352.
Acknowledgments
This work was financially supported by the Department of Science
and Technology–Innovation in Science Pursuit for Inspired
Research (DST-INSPIRE), Govt. of India (sanction no. DST/INSPIRE/
04/2015/000076). Authors thank the Department of Chemistry,
Indian Institute of Technology Kharagpur, and Department of
Chemistry, University of Mumbai, for providing the infrastructures
and research facilities. Authors thank Dr. Sachin P. Gholap, Tel
2’-O-Ethyl-5’-O-(4,4’-dimethoxytrityl)-uridine (9): Compound
8
(600 mg, 1.05 mmol) was initially co-evaporated with toluene (3
×10 mL) and dried at high vac for 2 h. The dried compound was
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[27] E. Wagner, B. Oberhauser, A. Holzner, H. Brunar, G. Issakides, G.
Aviv University, Israel, for the critical reading of the manuscript.
Authors also thanks Mr. Saurja Dasgupta, Howard Hughes Medical
Institute for critical reading of the manuscript. K.R.G. is the
recipient of the DST-INSPIRE Faculty award. R.R.N. thanks the
Department of Science and Technology–Innovation in Science
Pursuit for Inspired Research (DST-INSPIRE), Govt. of India for
fellowship.
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384.
The authors declare no conflict of interest.
Keywords: Conformational analysis · Nucleoside crystal · 2’-O-
ethyl-uridine · Stereoelectronic effects · Sugar puckering
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Manuscript received: December 26, 2020
Revised manuscript received: January 21, 2021
Eur. J. Org. Chem. 2021, 924–932
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