6′-Fluoro[4.3.0]bicyclo nucleic acid: Synthesis, biophysical properties and molecular dynamics simulations

Article abstract of DOI:10.3762/bjoc.14.288

Here we report on the synthesis, biophysical properties and molecular modeling of oligonucleotides containing unsaturated 6'-fluoro[4.3.0]bicyclo nucleotides (6'F-bc4,3-DNA). Two 6'F-bc4,3 phosphoramidite building blocks (T and C) were synthesized startin

Full text of DOI:10.3762/bjoc.14.288

6’-Fluoro[4.3.0]bicyclo nucleic acid: synthesis, biophysical
properties and molecular dynamics simulations
Sibylle Frei, Andrei Istrate and Christian J. Leumann*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2018, 14, 3088–3097.
Department of Chemistry and Biochemistry, University of Bern,
Freiestrasse 3, 3012 Bern, Switzerland
doi:10.3762/bjoc.14.288
Received: 26 September 2018
Accepted: 29 November 2018
Published: 20 December 2018
Email:
Christian J. Leumann* - christian.leumann@dcb.unibe.ch
*
Corresponding author
Associate Editor: S. Flitsch
Keywords:
© 2018 Frei et al.; licensee Beilstein-Institut.
License and terms: see end of document.
DNA/RNA affinity; fluorinated cyclopropanes; fluorinated nucleic
acids; molecular dynamics simulations; sugar modified nucleosides
Abstract
Here we report on the synthesis, biophysical properties and molecular modeling of oligonucleotides containing unsaturated
6
’-fluoro[4.3.0]bicyclo nucleotides (6’F-bc4,3-DNA). Two 6’F-bc4,3 phosphoramidite building blocks (T and C) were synthesized
starting from a previously described [3.3.0]bicyclic sugar. The conversion of this sugar to a gem-difluorinated tricyclic intermedi-
ate via difluorocarbene addition followed either by a NIS-mediated or Vorbrüggen nucleosidation yielded in both cases the
β-tricyclic nucleoside as major anomer. Subsequent desilylation and cyclopropane ring opening of these tricyclic intermediates
afforded the unsaturated 6’F-bc4,3 nucleosides. The successful incorporation of the corresponding phosphoramidite building blocks
into oligonucleotides was achieved with tert-butyl hydroperoxide as oxidation agent. Thermal melting experiments of the modified
duplexes disclosed a destabilizing effect versus DNA and RNA complements, but with a lesser degree of destabilization versus
complementary DNA (ΔTm/mod = −1.5 to −3.7 °C). Molecular dynamics simulation on the nucleoside and oligonucleotide level
revealed the preference of the C1’-exo/C2’-endo alignment of the furanose ring. Moreover, the simulation of duplexes with comple-
mentary RNA disclosed a DNA/RNA-type duplex structure suggesting that this modification might be a substrate for RNase H.
Introduction
A powerful strategy for the treatment of various disorders like ment of the endonuclease RNase H1 which selectively cleaves
cancer, viral and inherited diseases is the use of therapeutic the RNA strand of a DNA/RNA hybrid duplex [8]. To activate
antisense oligonucleotides (AONs) [1-4]. These short, synthetic this process, fully modified DNA-like oligonucleotides (ONs)
fragments bind through Watson–Crick base pairing to cellular or gapmer AONs are used [9,10]. However, the widespread use
RNA, thus modulating or silencing the gene expression through of oligonucleotide-based therapeutics is limited by several
various mechanisms [5-7]. One mode of action is the recruit- factors amongst which the cellular delivery, the biostability, the
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affinity and specificity for target RNA sequences are crucial [42]. In the case of tc-DNA, C(6’) fluorination resulted in a
[
11]. A powerful strategy to increase the affinity for comple- similar affinity to RNA as standard tc-DNA suggesting no
mentary RNA and to improve the ON biostability is the confor- contribution of a fluorine pseudohydrogen bond as in bc-DNA
mational restriction of the flexible backbone by additional ring [43]. Also a similar affinity to complementary DNA or RNA
systems and bridges [12]. Prominent members of this class of than the non-fluorinated tc-DNA was observed in fully modi-
conformationally restricted nucleic acids are locked nucleic fied 2’F-tc-ANA sequences [44]. In contrast, a significant
acids (LNAs) [13-15], hexitol nucleic acids (HNAs) [16,17], stabilisation with RNA targets resulted in the case of the 2’F-tc-
cyclohexenyl nucleic acids (CeNAs) [18,19], tricyclo-DNAs RNA modification due to conformational control of the fura-
(
tc-DNAs, Figure 1) [20-22], and [4.3.0]bicyclo-DNAs (bc4,3- nose conformation [45]. To further widen the scope of fluori-
DNAs, Figure 1) [23].
nated nucleic acid analogs as building blocks for therapeutic
oligonucleotides, we investigated the bc4,3-DNA as scaffold for
the modification. The idea was to place the fluorine atom next
to the internucleosidic linkage. Furthermore, an additional
double bond in the cyclohexane ring was expected to rigidify
the carbocyclic unit and possibly positively impact the duplex
stability. Here we report on the synthesis of the two 6’F-bc4,3
pyrimidine analogs with the base T and C, their incorporation
into DNA, their biophysical properties, as well as a structural
analysis by molecular dynamics simulations of hybrid DNA and
RNA duplexes.
Results and Discussion
Synthesis of the phosphoramidite building
blocks
Our strategy for the construction of the two phosphoramidite
building blocks 10 and 16 envisaged as a key step the forma-
tion of a [4.3.0]bicyclic fluoroenone from a tricyclic siloxydi-
fluorocyclopropane through a ring enlargement via selective
cyclopropane ring opening [46-49]. Consequently, the synthe-
sis started from the previously described bicyclic silyl enol
ethers 1α/β (Scheme 1) [50,51]. The two anomers of 1 were
Figure 1: Chemical structure of selected nucleic acid analogs.
Another strategy to overcome some of the limitations deals with individually transformed into the trimethylsilyl (TMS)-pro-
the insertion of one or several fluorine atom(s) in the sugar tected sugars 2α/β by adapting and improving the already
moiety of the nucleic acid analog. The polar hydrophobic nature existing protocol [50]. The sugars 2α/β were then individually
[
24] of the fluorine atom can positively alter the furanose con- treated with the Ruppert–Prakash reagent (TMSCF3) as difluo-
formation, basicity and/or polarizability of the modified rocarbene precursor and sodium iodide as initiator [52],
nucleotide, and therefore influences the metabolic stability, the furnishing the exo-tricyclic sugars 3α/β as major isomers. The
membrane permeability, the RNA- and protein-binding affinity closer evaluation of this reaction revealed that the type of silyl
of the AON [25-29]. Over the last almost two decades, fluori- enol ether drastically influenced the yield of the corresponding
nated oligonucleotide analogs like 2’-deoxy-2’-fluoro-RNA siloxydifluorocyclopropane. Whereas the TMS enol ethers were
(
(
F-RNA) [26,30,31], 2’-deoxy-2’-fluoroarabino nucleic acid not suitable for the reaction due to instability of the silyl group,
F-ANA) [32-34], 3’-hexitol nucleic acids (FHNA and Ara- the tert-butyldimethylsilyl (TBDMS) enol ethers were poorly
FHNA) [35], 2’-fluorocyclohexenyl nucleic acid (F-CeNA) reactive most likely due to the hindrance of the difluorocarbene
36], and other modifications [37-41] were evaluated for their attack on the double bond. The stereochemistry around the
[
antisense properties. In this context, our research group has cyclopropane ring (endo vs exo) could be assessed by the char-
systematically analyzed the effect of the fluorine atom on the acteristic coupling pattern between the fluorine atom and the
bc-DNA and the tc-DNA scaffold. 6’-β-Fluorination of H-C(1) or C(7) in the endo-tricyclic sugars in the correspond-
[
3.3.0]bicyclo-DNA led to a pseudohydrogen bond interaction ing 1H and 13C NMR spectra (Supporting Information File 1).
between the fluorine atom and the C(6) hydrogen atom of the
thymine base, fixing the torsion angle χ. As a consequence, an The plan for the pyrimidine nucleoside synthesis comprised the
increased affinity to complementary RNA targets was observed use of the meanwhile well-established β-selective N-iodosuccin-
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Scheme 1: Synthesis of the gem-difluorinated glycal 4 from the silyl enol ethers 1α/β. Reagents and conditions: a) BSA, DCM, rt, 17 h, 86%; b) BSA,
DCM, rt, 18 h, 88%; c) TMSCF3, NaI, THF, 70 °C, 2 h, 71%; d) TMSCF3, NaI, THF, 70 °C, 4 h, 75%; e) TMSOTf, 2,6-lutidine, DCM, 0 °C to rt, 2 h,
41% (4), 39% (5α/β); f) TMSOTf, 2,6-lutidine, DCM, 0 °C to rt, 7 h, 58% (4), 29% (5α/β).
imide (NIS)-mediated addition of a persilylated nucleobase to a (Bu3SnH) generated an anomeric mixture of nucleoside 6α/β
tricyclic glycal [43,45,53,54]. Therefore, the gem-difluorinated with the β-anomer as major component (α/β ratio = 1:4.5 ac-
tricyclic sugars 3α/β were individually reacted with trimethyl- cording to 1H NMR). The inseparable anomers of nucleoside
silyl trifluoromethanesulfonate (TMSOTf) in order to produce 6α/β were subjected to the next reaction step, where the simul-
the corresponding glycal 4. Surprisingly, apart from the desired taneous desilylation and cyclopropane ring opening to the
glycal 4 its hydrolysis products 5α/β were produced as main bicyclic fluoroenone 7 occurred. HF-pyridine smoothly facili-
side products. In the case of the α-tricyclic sugar 3α the ratio of tated this conversion. At this stage the two anomers of fluo-
products 4 to 5α/β could be influenced by the reaction time. A roenone 7 were separable. The configurational assignment of
shorter reaction time furnished the tricyclic alcohols 5α/β as the nucleobase was conducted by 1H,1H-ROESY experiments
major product, while prolongation of the reaction time pro- (Supporting Information File 1). The β-anomer 7β then was
duced the glycal 4 as main component (Table S1, Supporting subjected to Luche reduction [55,56] producing selectively the
Information File 1). Treatment of the glycal 4 with persilylated desired S-configuration at the C(5’) position due to hydride
thymine in the presence of NIS (Scheme 2), followed by radical delivery from the less hindered exo-side of the carbonyl group.
reduction of the iodide intermediate with tributyltin hydride The relative configuration at C(5’) could be assigned by 1H,1H-
Scheme 2: Synthesis of the thymidine phosphoramidite building block 10. Reagents and conditions: a) i) thymine, BSA, NIS, DCM, 0 °C to rt, 4.5 h;
ii) Bu3SnH, AIBN, toluene, 90 °C, 30 min, 70%; b) HF-pyridine, DCM/pyridine 5:1, 0 °C to rt, 1.5 h, 71%; c) CeCl3·7H2O, NaBH4, MeOH, 0 °C, 1 h,
92%; d) DMTr-Cl, pyridine, rt, 3 d, 76%; d) CEP-Cl, DIPEA, THF, rt, 4 h, 62%.
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Beilstein J. Org. Chem. 2018, 14, 3088–3097.
ROESY experiments (Supporting Information File 1). Trityla- ing Information File 1). Tritylation of the nucleoside 14 with in
tion of allylic alcohol 8 with 4,4-dimethoxytrityl chloride situ-prepared 4,4-dimethoxytrityl methanesulfonate (DMTr-
(
DMTr-Cl) afforded intermediate 9 which was subsequently OTf) [58,59] provided the protected derivative 15 which was
phosphitylated with 2-cyanoethyl N,N-diisopropylchlorophos- phosphitylated yielding the cytidine phosphoramidite 16.
phoramidite (CEP-Cl) furnishing thymidine phosphoramidite
1
0.
Synthesis of oligonucleotides
A series of oligonucleotides containing single or multiple incor-
Since significant amounts of the alcohols 5α/β were obtained, it porations of the thymidine or cytidine building blocks 10 and 16
was decided to redirect our initial synthetic plan for the cyti- were synthesized to study the pairing properties of the new
dine phosphoramidite. Hence, the sugars 5α/β were first acety- modification with complementary DNA and RNA. At the
lated yielding the intermediate 11α/β (Scheme 3), which is a beginning, the synthesis of the oligonucleotides was conducted
standard glycosyl donor for nucleoside synthesis. The nucleosi- using standard automated phosphoramidite chemistry (for
dation was carried out by applying classical Vorbrüggen condi- details see the experimental part in Supporting Information
tions [57] on the sugars 11α/β, yielding the β-nucleoside 12β as File 1). However, in the synthesis of ON1 and ON2 the yield
major anomer. The α/β-ratio of 1:1.5 was acceptable and the dropped to approximately 40% after the incorporation of the
configuration at the C(1’) was assigned by 1H,1H-ROESY ex- modified unit. The analysis of the crude product by LC–MS
periments (Supporting Information File 1). The gem-difluori- after cleavage from the solid support and deprotection, revealed
nated tricyclic nucleoside 12β was then converted into the the presence of 5’-phosphorylated fragments originating from a
bicyclic fluoroenone 13 via desilylation and ring-enlargement 3’-cleavage of the modification. We propose, that these frag-
by short exposure to HF-pyridine. During the following Luche ments were formed through an E2 elimination during the oxida-
reduction of derivative 13 the benzoyl protecting group of the tion step, most likely on the iodophosphonium ion level
nucleobase was partially removed. As a consequence an addi- (Figure 2) [60]. Based on this hypothesis, we changed the oxi-
tional benzoylation step was needed to obtain the allylic alcohol dation agent from iodine to tert-butyl hydroperoxide (TBHP),
1
4 in high yields. Verification of the configuration at C(5’) was which previously has been successfully applied for the synthe-
again accomplished by 1H,1H-ROESY experiments (Support- sis of iso-tricyclo-T (iso-tc-T) or bcen-T containing oligonucleo-
Scheme 3: Synthesis of the cytidine phosphoramidite building block 16. Reagents and conditions: a) Ac2O, pyridine, 0 °C to rt, 17 h, 87%;
b) N-benzoylcytosine, BSA, TMSOTf, ACN, 0 °C to rt, 3.5 h, 41%; c) HF-pyridine, DCM/pyridine 5:1, 0 °C, 15 min, 91%; d) i) CeCl3·7H2O, NaBH4,
MeOH, −78 °C, 20 min; ii) Bz2O, DMF, rt, 7 h, 94%; e) DMTr-OTf, DCM/pyridine 1:2, rt, 19.5 h, 44%; f) CEP-Cl, DIPEA, THF, rt, 75 min, 43%.
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Figure 2: Proposed mechanism for the formation of the 5’-phosphorylated fragments during the oxidation step in the synthesis of ON1 and ON2.
tides [61]. Indeed, under these conditions high coupling yields mimic. The better tolerance of the cytidine modification in both
(
>98%) of ON3–7 were obtained and the absence of the duplex types could be assigned to the nature of the nucleobase
5
’-phosphorylated fragment was noticed.
and was also observed in the case of the 7’,5’-bc-DNA [62].
Pairing properties with complementary DNA
and RNA
The determination of the base pair selectivity of the 6’F-bc4,3
modification was carried out by UV-melting experiments of
To evaluate the effect of the unsaturated 6’F-bc4,3 modification ON1 with complementary DNA where the mismatched base
on the thermal duplex stability we conducted UV-melting ex- was inserted at the opposing site of the modification (Table S2,
periments of the modified oligonucleotides with DNA and RNA Supporting Information File 1). All three possible mismatches
(
Table 1). These studies showed that ON1–4 bearing the modi- were evaluated. As expected, in all cases the Tm value was sig-
fied thymidine unit exhibited a duplex destabilization when nificantly lowered, with the GT-Wobble pair having the least
paired to DNA (ΔTm/mod = −1.5 to −3.7 °C). The Tm depres- destabilizing effect (−8.5 °C). The Tm depression of the
sion was more pronounced for multiple (ΔTm/mod ≈ −3.5 °C) GT-Wobble pair and the CT-mismatch (−11.0 °C) was in the
than for single (ΔTm/mod ≈ −2.0 °C) inclusions. When the same same range than for the natural duplex. However, a lager Tm
four oligonucleotides were hybridized to RNA the duplex discrimination was found in the TT-mismatch (−14.1 °C) as in
stability further decreased (ΔTm/mod ≈ −4.0 °C). However, the the natural system (−9.7 °C). Taking together, these data
number of modified units seemed not to have an influence on suggest that classical Watson–Crick base pairing also occurs
the Tm value. The three oligonucleotides ON5–7 bearing the with this modification.
cytidine modification also expressed a destabilizing pattern with
both complements, but to a lesser amount than in the thymidine The thermodynamic parameters of duplex formation of ON4
series. Again, the Tm depression was higher with complementa- and the corresponding natural sequence versus both comple-
ry RNA, with pronounced sequence effects in the case of single ments were extracted from their melting curves by a known
inclusions. The less destabilizing behaviour of the 6’F-bc4,3- curve fitting methodology (Table S3, Supporting Information
modified oligonucleotides versus complementary DNA sug- File 1) [63]. The comparison of the modified with the natural
gested that this modification more presents a DNA than a RNA duplexes disclosed an entropic stabilization (ΔΔS = +28.9 and
Table 1: Tm and ΔTm/mod data from UV-melting curves (260 nm) of ON1–7 in duplexes with complementary DNA and RNA.
Entry
Sequencea
Tm [°C] vs DNA
ΔTm/mod [°C]
Tm [°C] vs RNA
ΔTm/mod [°C]
ON1
ON2
ON3
ON4
ON5
ON6
ON7
5’-d(GGA TGT TCt CGA)-3’
5’-d(GGA tGT TCT CGA)-3’
5’-d(GGA TGt tCT CGA)-3’
5’-d(GCA ttt ttA CCG)-3’
46.0
47.2
41.3
30.3
47.1
46.8
44.1
−2.7
−1.5
−3.7
−3.4
−1.6
−1.9
−2.3
46.0
47.6
42.0
22.4
46.0
48.3
44.0
−4.0
−2.4
−4.0
−4.4
−4.0
−1.7
−3.0
5’-d(GGA TGT TcT CGA)-3’
5’-d(GGA TGT TCT cGA)-3’
5’-d(GGA TGT TcT cGA)-3’
aLowercase letters: modified nucleotides, capital letters: natural DNA. Total strand conc. 2 μM in 10 mM NaH2PO4, 150 mM NaCl, pH 7.0. Reference
Tm values: DNA1/DNA = 48.7 °C, DNA1/RNA = 50.0 °C, DNA2/DNA = 47.4 °C, DNA2/RNA = 44.4 °C; DNA1 = 5’-d(GGA TGT TCT CGA)-3’, DNA2 =
5’-d(GCA TTT TTA CCG)-3’.
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+
38.0 cal·mol−1·K−1) and an enthalpic destabilization the Southern hemisphere. The lowest energy conformer was as-
(
ΔΔH = +13.8 and +17.0 kcal·mol−1) for both the DNA sociated with the furanose unit in a C2’-endo orientation and the
and RNA complement. This pattern was observed along six-membered ring in a twist-boat conformation (Figure 3b).
the whole bc-DNA series and was attributed to the conforma- Approximately 1 kJ/mol higher in energy was the second
tional restriction of the sugar [62,64]. The Gibbs free energy of conformer where the furanose unit adopted a C3’-exo arrange-
duplex formation corresponded well with the observed Tm ment and the cyclohexene unit a half-chair conformation
values.
(Figure 3c). The C(5’) hydroxy group adopted in both
conformers a pseudoaxial position. Consequently, the torsion
angle γ was aligned in a +sc arrangement (C2’-endo conformer:
CD spectroscopy
Circular dichroism of ON1–7 paired with DNA or RNA was re- 64°; C3’-exo conformer: 83°). The distance between the fluo-
corded to further analyze their helical conformation and to rine atom and the C(5’) oxygen was 3.3 Å in the C2’-endo
compare it with that of the corresponding natural duplexes conformer and 2.9 Å in the C3’-exo conformer.
(
Figure S1, Supporting Information File 1). All seven modified
oligonucleotides exhibited a B-type pattern when paired to These two conformers were then used to calculate the atomic
DNA, indicating B-form helices. All modified oligonucleotides charges of the corresponding nucleosides using the R.E.D. III.5
duplexed to RNA disclosed a similar pattern than the natural tools package [66]. The obtained parameters were added to the
hybrid structure, giving evidence of mixed A/B-type helices. Amber94 force field [67] which besides the GROMACS 5.0.6
simulation package [68] was utilized for the molecular dynam-
Molecular modeling
ics simulations. The duplexes investigated in the simulation
To gain more information on the structural features of the 6’F- encompassed: a unmodified DNA strand, ON1, ON4 and a
bc4,3 modification, we performed molecular dynamics simula- fully modified 6’F-bc4,3-DNA strand duplexed to complementa-
tions of the modified duplexes. We first calculated the potential ry DNA and RNA as well as a 6’F-bc4,3-DNA homo-duplex
energy profile versus pseudorotation phase angle of nucleoside (for details on the simulation see the experimental part in Sup-
8
using quantum mechanical methods. The calculations were porting Information File 1).
performed in vacuum with the Gaussian 09 software package
65] utilizing the second order Møller–Plesset perturbation The duplex of the fully modified 6’F-bc4,3-DNA strand with
[
theory (MP2) and the 6-311G* basis set. The energy profile of DNA still featured a B-type helix (Figure 4a) whereas the 6’F-
nucleoside 8 was obtained through a stepwise variation of the bc4,3-DNA/RNA duplex maintained an A-form (Figure 4b).
pseudorotation phase angle P at the range of the maximum Interestingly, the fully modified 6’F-bc4,3-DNA strand exhib-
puckering amplitude νmax and was visualized in the pseudorota- ited almost identical backbone angles and sugar conformation
tion wheel (Figure 3a). The two low energy regions appeared in regardless if paired to DNA or RNA. The preferred sugar
Figure 3: a) Potential energy profile versus pseudorotation phase angle of nucleoside 8 and its two minimal energy conformers: b) C2’-endo and
c) C3’-exo.
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Figure 4: Average structures of the a) 6’F-bc4,3-DNA/DNA, b) 6’F-bc4,3-DNA/RNA, and c) 6’F-bc4,3-DNA/6’F-bc4,3-DNA duplexes obtained from the
last nanosecond of the simulation by firstly extracting a frame each 50 ps and secondly by doing an averaging of them.
arrangement was found in a narrow range in the Southern area Watson–Crick base pairing between the two strands and the
of the pseudorotation wheel (C1’-exo, C2’-endo, Figure 5a and characteristic parameters of a B- or A-type helix, respectively
b), indicating that this modification is a DNA mimic. This (Figures S5–S7, Supporting Information File 1) [69]. Further-
finding is in agreement with the observed Tm values and also more, the examination of the minor groove distances [70]
reflects the entropical stabilization of the duplex structure. The disclosed for the 6’F-bc4,3-DNA/RNA duplex a flexibility
cyclohexene ring of the modified unit adopted either a twist- switching between values of an A- and B-helix (Figure S8, Sup-
boat or a boat alignment in the fully modified strand of both porting Information File 1). This variation of the minor groove
duplex types. Consequently, the fluorine atom was arranged in a distance is thought to play a crucial role for RNase H activation
way that the repulsive electrostatic interactions with the C(5’) [9,71].
oxygen were minimized. The analysis of the backbone torsion
angles revealed that the fused ring system affected all backbone The structure displayed by the fully modified 6’F-bc4,3-DNA
torsion angles (Figure 5c and d). Specifically, the angle α homo-duplex was neither an A- nor B-type helix (Figure 4c).
adopted values in the +ap to −sc range which was in contrast to This structure featured a very variable minor groove (≈8 to
the canonical parameters (DNA: ±sc, −ac; RNA +ac, +ap, 18 Å), an increased rise (≈3.4 Å), a positive slide (≈1.6 Å) and a
−sc). The angle β was found in the +ac or anti orientation, most positive roll (≈4.6 Å; Figures S6–S8, Supporting Information
likely due to either the boat or the twist-boat conformation of File 1). As a consequence of the latter the x-displacement
the cyclohexene ring. Furthermore, the angle γ was constrained (≈1.1 Å) was shifted towards a positive value. The sugar con-
to a +sc arrangement as also found in canonical A- or B-type formation in the two strands (Figure S4, Supporting Informa-
helices. The torsion angle ε exhibited values in the ±sc and anti tion File 1) was in the same range (C1’-exo, C2’-endo) as de-
range, whereas the angle ζ adopts all values between 0–360°. scribed above for the hybrid duplexes. The backbone torsion
The reason for the flexibility of the angle ζ might lie in its angles of the homo-duplex exhibited identical conformations in
compensatory nature to balance the constrained backbone both strands (Figure S4, Supporting Information File 1). Some
angles that lay within the carbocyclic system.
variations in the torsion angle ζ (−sc to +ac) and the glycosidic
bond angle χ (200–360°) were observed compared to the fully
The DNA or RNA strand in these hybrid duplexes displayed the modified hybrid duplexes.
same structural preference as in the natural reference structures
(
Figures S2 and S3, Supporting Information File 1). The evalua- The structural data of ON1 and ON4 containing either one or
tion of the base pair body parameters of the 6’F-bc4,3-DNA five consecutive modifications are shown in Figures S2, S3, and
strand hybridized to DNA or RNA revealed the expected S8 in Supporting Information File 1.
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Beilstein J. Org. Chem. 2018, 14, 3088–3097.
Figure 5: Preferred sugar pucker of a) 6’F-bc4,3-DNA/DNA, and b) 6’F-bc4,3-DNA/RNA duplexes and torsion angles of c) 6’F-bc4,3-DNA/DNA, and
d) 6’F-bc4,3-DNA/RNA duplexes extracted from a 100 ns molecular dynamics trajectory.
Conclusion
Mainly this rigidity in combination with the repulsive electro-
In this study, we presented the successful synthesis of the two static interactions of the fluorine atom and the C(5’) oxygen
6
’F-bc4,3 pyrimidine phosphoramidite building blocks 10 and seem to be responsible for the duplex destabilization. Neverthe-
6 starting from a bicyclic silyl enol ether. The key step in the less, the MD simulations pointed to a flexible minor groove
1
synthesis was the transformation of a gem-difluorinated for the modified oligonucleotides hybridized to RNA, indicat-
tricyclic nucleoside into a ring-enlarged bicyclic fluoroenone by ing together with the preferred Southern conformation of the
simultaneous desilylation and cyclopropane ring opening which modified unit, that this modification might be a substrate for
proceeded in high yields. The two phosphoramidite building RNase H.
blocks were successfully incorporated into oligonucleotides by
automated solid-phase DNA synthesis with tert-butyl hydroper-
Supporting Information
oxide as the oxidation agent. The CD spectra of the 6’F-bc4,3-T
or -C-modified oligonucleotides displayed a B-type helix when
Additional tables and figures, the experimental part, as
paired to DNA and an intermediate A/B form when the counter
well as copies of the NMR spectra (1H, 13C, 19F, 31P) of
part was RNA.
the new compounds are given in the Supporting
Information.
The modified oligonucleotides exhibited a significant destabi-
lization versus both complements, but with complementary
DNA being less discriminating (ΔTm/mod = −1.5 to −3.7 °C)
than complementary RNA. This finding indicates that the
Supporting Information File 1
Additional data, experimental part, and NMR spectra.
[https://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-14-288-S1.pdf]
6
’F-bc4,3 modification is more a DNA mimic than an RNA
mimic. In accordance with this were the results obtained
from the molecular dynamics simulation of the duplexes
where the sugar pucker preferably adopted a Southern Acknowledgements
conformation (C1’-exo, C2’-endo). Furthermore the simula- Financial support for this project by the Swiss National Science
tions revealed a very rigid bicyclic sugar system with a dimin- Foundation (grant-no.: 200020_165778) is gratefully acknowl-
ished conformational adaptability of the cyclohexene unit. edged.
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Beilstein J. Org. Chem. 2018, 14, 3088–3097.
ORCID® iDs
22.Goyenvalle, A.; Griffith, G.; Babbs, A.; El Andaloussi, S.; Ezzat, K.;
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