Homopurine: R P-stereodefined phosphorothioate analogs of DNA with hampered Watson-Crick base pairings form Hoogsteen paired parallel duplexes with (2′-OMe)-RNAs

Article abstract of DOI:10.1039/c8ob03112f

3′-O-(2-Thio-4,4-pentamethylene-1,3,2-oxathiaphospholane) derivatives of 5′-O-DMT-N⁶-methyl-deoxyadenosine and 5′-O-DMT-N²,N²-dimethyl-O⁶-diphenylcarbamoyl-deoxyguanosine (OTP-N^Y, N^Y = DMT-^m6dA or DMT-^m,m2dG^DPC) were synthesized, resolved onto pure P-diastereomers, and used in P-stereocontrolled synthesis of dinucleoside 3′,5,-phosphorothioates N^X_PST (N^X = ^m6dA or ^m,m2dG), in which the absolute configuration of the stereogenic phosphorus atom was established enzymatically. Diastereomerically pure OTP-N^Y and standard OTP-N (N = DMT-dA^Bz or DMT-dG^Bz,DPC) were used in the synthesis of chimeric R_P-stereodefined phosphorothioate oligomers ((R_P-PS)-DN(N^X)A) with hampered Watson-Crick base pairings. It was found that the ^m6dA units slightly reduce the thermodynamic stability of antiparallel duplexes formed with RNA and (2′-OMe)-RNA matrices, whereas ^m,m2dG units prevent their formation. The ^m6dA units stabilize (by up to 4.5 °C per modified unit) the parallel duplexes formed by (R_P-PS)-DN(N^X)A with Hoogsteen-paired (2′-OMe)-RNA templates compared to the analogous reference duplex containing only unmodified nucleobases. In contrast, the ^m,m2dG units destabilize such duplexes by up to 3 °C per modified unit. Both units prevent the formation of the corresponding parallel triplexes.

Full text of DOI:10.1039/c8ob03112f

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Homopurine RP-stereodefined phosphorothioate analogs of DNA
with hampered Watson-Crick base pairings form Hoogsteen
paired parallel duplexes with (2’-OMe)-RNAs.†
Received 00th January 20xx,
Accepted 00th January 20xx
Anna Maciaszek*a, Katarzyna Jastrzębskaa, Piotr Gugaa
DOI: 10.1039/x0xx00000x
3’-O-(2-thio-4,4-pentamethylene-1,3,2-oxathiaphospholane) derivatives of 5’-O-DMT-N6-methyl-deoxyadenosine- and 5’-
www.rsc.org/
O-DMT-N2,N2-dimethyl-O6-diphenylcarbamoyl-deoxyguanosine (OTP-NY, NY=DMT-m6dA or DMT-m,m2dGDPC
)
were
synthesized, resolved onto pure P-diastereomers, and used in P-stereocontrolled synthesis of dinucleoside 3’,5,-
phosphorothioates NXPST (NX= m6dA or m,m2dG), in which the absolute configuration of the stereogenic phosphorus atom
was established enzymatically. Diastereomerically pure OTP-NY and standard OTP-N (N=DMT-dABz or DMT-dGBz,DPC) were
used in synthesis of chimeric RP-stereodefined phosphorothioate oligomers ((RP-PS)-DN(NX)A) with hampered Watson-
Crick base pairings. It was found that the m6dA units slightly reduce thermodynamic stability of antiparallel duplexes
formed with RNA and (2’-OMe)-RNA matrices, whereas m,m2dG units prevent their formation. The m6dA units stabilize (by
up to 4.5°C per modified unit) parallel duplexes formed by (RP-PS)-DN(NX)A with Hoogsteen-paired (2’-OMe)-RNA
templates compared to the analogous reference duplex containing only unmodified nucleobases. On contrary, the m,m2dG
units destabilize such duplexes by up to 3°C per modified unit. Both units prevent the formation of the corresponding
parallel triplexes.
form thermally highly stable parallel triplexes RNA&(RP-PS)-
DNA:RNA19 (the “&” sign indicates parallel strands orientation)
and duplexes RNA&(RP-PS)-DNA.20 Notably, RNA&(RP-PS)-DNA
Introduction
Over more than thirty years two main approaches have been
tested to modulate protein biosynthesis, i.e., an antigene
strategy aimed to prevent the transcription of double-stranded
genome DNA1,2,3 and an antisense strategy intended to
are more stable than the corresponding DNA:RNA antiparallel
Watson-Crick (WC) paired duplexes and the stabilizing effects
originate not only from Hoogsteen interactions21 but also from
water bridges, which are supposed to span (via charge assisted
hydrogen bonds22) the O2 atoms in pyrimidines and the sulfur
atoms of the phosphorothioate moieties23,‡. Such (RP-PS)-DNA
probes upon efficient and sequence-specific hybridization with
complementary RNA molecules, might inhibit biosynthesis of
unwanted proteins or reverse transcription of RNA24. To form
capture and destroy target mRNA4,5,6
. More recently
approaches based on RNA interference (RNAi)7 and
CRISPR/Cas9 system8 have been developed. To make DNA
oligonucleotides
more
stable
towards
nucleases
phosphorothioate analogs (PS-DNA)9 were tested. They have a
non-bridging phosphate oxygen atom substituted with a sulfur
atom and this substitution creates a stereogenic center (see
Scheme 2). Standard phosphoramidite or H-phosphonate
methods furnish oligomers which typically consist of hundreds
of P-diastereomers.10 To date P-stereodefined PS-DNA have
been mainly prepared by the oxathiaphospholane approach
(developed in this laboratory)11, although other methods have
also been published.12 In many instances PS-DNA exhibited P-
stereodependent biological13-15 and/or physico-chemical
properties16,17,18. Homopurine (RP-PS)-DNA were found to
a perfect parallel triplex RNA&(RP-PS)-DNA:RNA (e.g. I), an RNA
molecule has to contain an 8-12 nt homopyrimidine segment
of palindromic sequence, so the same RNA oligomer may serve
as the Hoogsteen paired (Hp) and Watson-Crick paired (WCp)
component. However, such tracts are not frequent in a cellular
or viral RNA pool.
Hp-RNA 5’-r(CUCUUUUUCUC)-3’ WCp-RNA≡Hp-RNA
PS-DNA 5’-d(GAGAAAAAGAG)-3' - a palindromic sequence
WCp-RNA 3’-r(CUCUUUUUCUC)-5’
(I)
Homopyrimidine non-palindromic segments may form parallel
duplexes RNA&(RP-PS)-DNA, which are reasonably stable,
although still less stable than the corresponding RNA&(RP-PS)-
DNA:RNA triplexes.20 Hence, a non-palindromic (RP-PS)-DNA
probe may be less specific, because the intended parallel
duplex RNA&(RP-PS)-DNA may recruit a non-target WCp-RNA
,
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to form a perfect, thermodynamically more stable, yet we decided to verify the aforementioned hypothesis using (2’-
View Article Online
DOI: 10.1039/C8OB03112F
24
unwanted triplex (e.g. II).
OMe)-RNA oligomers. As was reported before, dodecameric
parallel duplexes and triplexes consisting of (RP-PS)-DNA and
(2’-OMe)-RNA strands were thermally so stable that the Tm’s
often exceeded 80°C. Thus here we dealt mostly with the
oligomers 9-nt long.
Hp-RNA 5’-r(CUCCUUUUUCUC)-3’ -target RNA
PS-DNA 5’-d(GAGGAAAAAGAG)-3' - non-palindromic
WCp-RNA 3’-r(CUCCUUUUUCUC)-5’ - non-target RNA
(II)
To eliminate this drawback we synthesized non-palindromic
(RP-PS)-DN(NX)A oligomers, where “NX” indicates the presence
of nucleotide units with N6-methyl-deoxyadenosine (m6dA) or
R
N
O
Hp-RNA
N
WCp-RNA
O
O
H
X
CH3
N
H
N2,N2-dimethyl-deoxyguanosine (mm2dG) (
1 or 2, respectively,
N
N
N
N
H
N
Chart 1). We hypothesized that these oligomers should have
reduced ability to form the classical WC hydrogen bonds, and
retained ability to interact according to the normal Hoogsteen
hydrogen bonding scheme. Our assumption was based on
reports showing that m6A–U pairing in the antiparallel duplex is
accompanied by the rotation (with 0.5–1.7 kcal/mol energetic
toll) of the methylamino group from its energetically preferred
syn geometry on the Watson–Crick face to the higher-energy
anti conformation, thus the methyl group is directed towards
the major groove.25,26 Also it was reported that m6A in unpaired
positions, where the rotation of the methylamino group is
unnecessary, exerts base stacking considerably more strongly
than the unmodified base.26 We appreciated the geometric
suitability of the energetically preferred syn-rotamer for the
intended formation of a parallel duplex (Chart 1, an upper
panel).
R
O
N
dR
N6-methyl-deoxyadenosine (m6dA,
1
)
H
R
H
N
WCp-RNA
H
N
N
N+
H
Hp-RNA
H
O
N
N
O
N
R
H
N
N
O
N
X
CH3
N
dR
CH3
N2,N2-dimethyl-deoxyguanosine (mm2dG, 2)
Chart 1. Visualization of possible Hoogsteen and Watson-Crick hydrogen bonds for the
nucleosides and methylated within the nucleobases. ribose, dR 2’-
1
2
R
=
=
deoxyribose. The red X marks indicate places where typical WC hydrogen bonds cannot
be formed.
Using N2,N2-dimethyl-deoxyguanosine one has to take into
account the steric hindrance caused by the exocyclic –NMe2
group. Wang and Schaefer summarized literature data and
provided theoretical calculations demonstrating the intrinsic
nonplanarity of the exocyclic –NH2 group (a significant part of
sp3 hybridization of the N2 atom) for the unmodified guanine.
The authors analyzed all three natural nucleosides possessing
the exocyclic –NH2 groups and found a barrier to planarization
(sp3→sp2) of 0.74 kcal mol−1 for guanine, compared to 0.02
kcal mol−1 for adenine and 0.03 kcal mol−1 for cytosine.27 Thus,
the electron pair of N2 atom does not participate in the
aromatic system and only one of the attached hydrogens can
reside close to the plane of the ring, as the N1-C2-N2-H21 and
N1-C2-N2-H22 torsion angles of -31.7° and -169.4°, respectively,
were found.28 Earlier an energy barrier of 1.6 kcal mol−1 for
planarization of guanine was predicted and rationalized by the
repulsion between the N1-H hydrogen and the amino group
hydrogen atoms.29 However, we did not find in the literature
any data on possible influence of this modification on
interactions at the Hoogsteen face of parallel duplexes.
Results and Discussion
Synthesis of P-diastereomerically pure 3’-O-(2-thio-4,4-
pentamethylene-1,3,2-oxathiaphospholane) derivatives of 5’-O-
DMT-N6-methyl-deoxyadenosine and 5’-O-DMT- N2,N2-dimethyl-
O6-diphenylcarbamoyl-deoxyguanosine.
N6-methyl-deoxyadenosine (
1
,
m6dA) was bought from a
commercial source. N2,N2-dimethyl-deoxyguanosine (
2,
mm2dG)
was synthesized according to the literature method.30 In brief,
(Scheme 1S, ESI) commercially available 3’,5’-O,O-diacetyl-
deoxyguanosine was transiently protected at O6 with TMS-Cl,
followed
by
reaction
with
1,3-benzodithiolylium
tetrafluoroborate (BDTF, Nakayama’s reagent) and reduction
of the N2-(1,3-benzodithiol-2-yl) intermediate with tributyltin
hydride. The resultant N2-methyl derivative was subjected to
the same procedure to yield 3’,5’-O,O-diacetyl-N2,N2-dimethyl-
deoxyguanosine, which was further deacetylated with 30% aq.
NH4OH to yield N2,N2-dimethyl-deoxyguanosine. Compounds
1
and
2 were routinely converted into the corresponding 5’-O-
DMT derivatives
3
and
and with 2-chloro-4,4-pentamethylene-
) in pyridine in the presence of
elemental sulfur yielded the OTP monomers and 7*, which
4
(Scheme 1), respectively.
Phosphitylation of
3
4
Here we present preparation and indirect determination of
absolute configuration of the P atoms in newly obtained 5’-O-
DMT-3’-O-(2-thio-4,4-pentamethylene-1,3,2-
1,3,2-oxathiaphospholane11
(5
6
oxathiaphospholane) derivatives of m6dA and mm2dG
were isolated on a silica gel column. Because numerous
attempts at chromatographic separation of the P-
diastereomers of 7* were unsuccessful, a reaction at the O6
atom with diphenylcarbamoyl chloride31 was performed to
(monomers
6 and 7
, OTP-NY, Scheme 1), as well as synthesis of
model (RP-PS)-DN(NX)A and (SP-PS)-DN(NX)A oligomers, and
assessment of composition, thermal stability and
conformation of their complexes formed with Hp and WCp
RNA and (2’-OMe)-RNA strands. Because of hydrolytic
instability of RNA oligomers at elevated temperatures and
relatively poor cooperativity of melting of RNA&(RP-PS)-DNA20
yield
and 7) was done using a silica gel Phenomenex Luna column
7
. Efficient separation of the P-diastereomers of OTP-NY
(6
(5µm) and ethyl acetate/hexane mixtures, and their 31P NMR
spectra recorded in CDCl3 contained signals at δ 104.5 - 105.3
2 | J. Name., 2012, 00, 1-3
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ppm. The optimized conditions were successfully transferred Solid phase synthesis of P-stereodefined PVSie-wDANrtiAcle Oannlinde
X
DOI: 10.1039/C8OB03112F
on a preparative HPLC silica gel column.
chimeric PS-DN(N )A oligomers and complementary DNA and
RNA strands.
DMTO
B'
Cl
P
The
reference
“canonical”
(a 9-mer,
PS-oligomers
12R and
(RP-PS)-
(RP-PS)-
O
DMTO
B'
O
S8
Py
separation
d(GAAAGAGAG)
)
6f and 6s
+
S
O
O
B' = m6Ade
S
S
of P-epimers
of 6
d(GAGGAAAGAGAG) (a 12-mer, 16R20) were synthesized24
manually at a 1 µmol scale using the P-diastereomerically pure
OH
R
R'
P
5
O
3 B' = m6Ade,
or
4 B' = m,m2Gua
R
R'
OTP-dABz and OTP-dGiBu,DPC (OTP-N, analogs of
6 and 7,
respectively, obtained according to Ref. 11). To prevent the
DBU-promoted cleavage of the standard lcaa linker, the first
nucleoside was attached to the solid support via a DBU-
6 or 7* (RP + SP)
DPC-Cl/Py
R,R'= -(CH2)5-
separation
resistant sarcosine linker.34 Using OTP-N and OTP-NY 6f
,
6s
P-stereodefined “chimeric” PS-DN(NX)A 9-mer
oligonucleotides, i.e. (RP-PS)- and (SP-PS)-d(GAXAXAGAXGAXG)
, 7f,
7f and 7s
B' = m,m2GuaDPC
7
of P-epimers
or 7s
,
of 7
Scheme 1. Synthesis of the oxathiaphospholane monomers 6 and 7.
(
13R and 13S
,
dAX=m6dA), (RP-PS)- and (SP-PS)-
d(GXAAAGXAGXAG) (14R and 14S, dGX=m,m2dG), as well as a 12-
mer (RP-PS)-d(GAXGGAXAXAXGAXGAXG) (15R, dAX=m6dA) were
obtained according to the published protocol.11 Routine
coupling steps were done using 20 mg of OTP-N and OTP-NY
monomers and 0.15M DBU in total 150 µl of anhydrous
Selected physico-chemical data for the fast- and slow-eluting
P-diastereomers (6f, 6s, and 7f, 7s) are given in Table S1 (ESI).
1
The relevant HR MS, 31P NMR, H NMR, and 13C NMR spectra
are shown in Figures 1S-4S (ESI).
Assignment of the absolute configuration of the P-atom in
NXPST obtained using OTP-NY.
acetonitrile. In the syntheses of 13-15 double coupling (20 mg
+ 20 mg) of OTP-dGiBu,DPC or OTP-dGY was applied. Decay of the
trityl cation absorption was measured photometrically at 503
nm and overall 88-90% repetitive yield was noted. This was
slightly less than 92% observed for OTP-N, but the protocol
was not optimized.
P-diastereomerically pure 6f
3’-O-acetyl-thymidine in the presence of DBU to yield the
corresponding protected NYPSTAc
8f 8s 9f 9s, respectively,
, 6s, 7f, and 7s were reacted with
(
,
,
,
Scheme 2), which after routine two-step deprotection
furnished the phosphorothioates NXPST (10f,s and 11f,s). The
products were isolated by RP-HPLC and identified by MALDI-
TOF MS (Figure 5S, ESI). The diastereomeric NXPST were treated
with snake venom phosphodiesterase (svPDE, an RP-specific
nuclease) and nuclease P1 (nP1, an SP-specific nuclease).32,33 It
was found that 10f and 11f (obtained from fast-eluting 6f and
7f, respectively) were hydrolyzed by svPDE (and not by nP1),
so were assigned an RP configuration. Compounds 10s and 11s
(obtained from slow-eluting 6s and 7s) were hydrolyzed by nP1
(and not by svPDE), thus were assigned an SP configuration.
Because the condensation step is stereoretentive these
absolute configurations can be assigned to the phosphorus
atoms in the parent OTP-NY monomers.
After each synthesis was complete, the DMT-tagged oligomer
was cleaved from the support and the N-protecting groups (Bz,
iBu and DPC) were removed using routine treatment with
conc. NH4OH. The oligonucleotides were isolated by RP-HPLC,
then detritylated with 50% acetic acid, purified by RP-HPLC
and analyzed by MALDI-TOF MS (Figure 6S, ESI). The syntheses
furnished 6-13 OD260 of each oligomer.
An unmodified oligomer d(GAGGAAAGAGAG) (a 12-mer, PO-
1620) and oligomers complementary to 12
-
16, i.e. Hp-RNA (hR
and hR12; a 9-mer and a 12-mer complementary to 12R 14R
and 15R 16R, respectively) and WCp-RNA (wR and wR12),
-
-
their 2’-OMe congeners Hp-(2’-OMe)-RNA (hM and hM12) and
WCp-(2’-OMe)-RNA (wM and wM12), and WCp-DNA wD12
were prepared using standard phosphoramidite monomers
and the protocols recommended by their manufacturers.
Thermal stability of complexes formed by (SP-PS)-DN(NX)A.
Protonation of the N3 atom of cytidine is crucial for the
Hoogsteen interactions (Chart 1), which are effectively
developed at pH<5.5. Although one can assume that at pH
close to neutral the protonation may be by far incomplete,
NMR studies revealed that in a Py&Pu:Py DNA parallel triplex
the cytosines involved in Hoogsteen interactions are
protonated at pH 7.2.35 This “enhanced basicity” was observed
earlier for RNA&(RP-PS)-DNA:RNA and RNA&(RP-PS)-DNA
complexes.20,23 Following this scheme, in the present research
the oligonucleotide samples were dissolved in pH 7.2 or pH 5.4
buffer containing 10 mM Tris-HCl, 100 mM NaCl, and 10 mM
MgCl2. The annealing was done from 85°C to 15°C with a
temperature gradient of 1°C/min. Then the melting profiles
were recorded over a 15→85°C range (0.5°C/min) and melting
DMTO
B'
Thy
B'
HO
O
O
DMTO
O
R
OAc
O
O
O
S
S
S
R'
-
S
P
P
-S
R
DBU, CH3CN
Thy
O
O
O
R'
R
R'
OAc
6f, 6s, 7f, or 7s
BX
B'
O
DMTO
HO
O
deprotection
S-
O
S-
O
O
P
P
Thy
Thy
O
O
O
O
O
10f, 10s: BX
11f, 11s: BX
=
=
m6Ade
mm2Gua
8f, 8s, 9f, or 9s
OAc
OH
Scheme 2. Synthesis of NXPST (10 and 11) and a simplified mechanism of the DBU-
promoted condensation step.
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temperatures (Tm) were calculated using the first order
derivative method.
The melting curves recorded for 13S or 14S mixed with each of
the templates (h(R,M), w(R,M)) contained no inflection point
(data not shown) and the observed hyperchromicities were in
View Article Online
DOI: 10.1039/C8OB03112F
1.15
1.05
0.95
0.85
0.75
0.65
0.55
0.45
0.35
hM&12R:wM
hM&12R
12R:wM
hM&14R
hM&13R
a range 0.020.05 A260 units, i.e. <10%, compared to the
average initial absorption 0.53 (±0.05) A260 for the two-strand
systems, and 0.81 (±0.05) A260 for the three-strand systems.
These two facts indicate (as expected) the absence of any
antiparallel and parallel structures.36 The latter were not
formed, because the stabilizing water bridges cannot operate
and only relatively weak Hoogsteen interactions are possible.
Thermal stability and circular dichroism analysis of complexes
formed by (RP-PS)-DN(NX)A.
15
25
35
45
55
65
75
85
Temperature [°C]
The melting curves recorded at pH 7.2 for unmodified PO-16
mixed with wM12 or wR12 showed Tm=43°C or 40°C,
respectively, whereas no transition was observed upon mixing
Figure 1. Melting curves recorded for complexes of 12R, 13R and 14R, with hM and/or
wM in pH 5.4 buffer.
with hM12 or hR12 20
12R
.
The curves obtained for PS-oligomers
Table 1. Melting temperatures and ΔA260 measured in pH 7.2 and 5.4 buffers for
complexes formed by 12R, 13R, and 14R, with the templates hM and/or wM.
-
14R mixed with hR or wR, or for 15R 16R mixed with
-
hR12 or wR12, contained no inflection point (the plots for 15R
are shown in Figure 7S, ESI) and, like for the analogous
mixtures of 13S or 14S, the observed hyperchromicities were
low. Similar results were obtained for those PS-oligomers
mixed in pH 5.4 buffer and the plots for 15R with the reference
2nd or
Tm (°C) /ΔA260 (AU)
pH
7.2
5.4
2nd+3rd
strand
13R
15R
14R
12Ra)
(
m6dA)
(
m6dA)
(
m,m2dG)
nt/nd
ntc/nd
55/0.26
55/0.30
34/0.19
65/0.27
65/0.30
wM
hM
27/ndb
39/0.24
57/0.26
48/0.16
54/0.24
73/0.24
21/nd
29/0.06
curves recorded for hR12&16R20 and 15R
:wD12 are shown in
52/0.20
54/0.27
44/0.14
72/0.20
71/0.22
hM+wM
wM
29/0.07
nt/0.04
48/0.08
48/0.09
Figure 8S (ESI). The base-unmodified 12R formed the most
stable WCp-duplex with wM and Tm=27°C and 48°C were
noted in pH 7.2 and pH 5.4 buffer, respectively24 (Figure 1,
hM
Table 1).‖ For triplexes hM
&
12R
were found, whereas the parallel duplexes hM
the top two strands in III) showed Tm=39°C and 54°C.
:
wM
(
III) Tm=57°C and 73°C
hM+wM
&
12R (built of
a) From Ref. 24, b) nd – not determined, c) nt – no transition observed
As expected, due to N2-CH3↔O=C2 repulsive interactions (see
Hp-RNA 5’-(2’-OMe)-(CUUUUUCUC)-3’
PS-DNA 5’-d(GAAAGAGAG)-3'
WCp-RNA 3’-(2’-OMe)-(CUUUUUCUC)-5’
III
-
-
-
hM
12R
wM
Chart 1, a bottom panel) and the lack of the N2-H---O=C2
hydrogen bonds, the WCp duplexes 14R:wM and 14R:wR were
not formed and the melting curves recorded in pH 7.2 and pH
5.4 buffers showed no inflection point (data not shown).
Oligomers 13R and hM at pH 7.2 and 5.4 formed remarkably
stable complexes with Tm=52°C and 72°C, respectively, and
these values remained virtually unchanged upon addition of
wM. It should be emphasized that these parallel duplexes had
the corresponding Tm’s by 13°C and 18°C higher than the
(
)
The triplexes containing wR instead of wM were by 3-4°C less
stable, indicating that the WCp-(2’-OMe)-RNA strand provides
a more effective stabilizing force.
In neutral and acidic conditions a duplex 13R:wM (in which the
rotamer of 13R shown in Chart 2 is able to provide the
stabilizing WC forces) was less stable than 12R:wM (Tm=21°C
and 44°C‖ vs. 27°C and 48°C, respectively), and in the
experiment at pH 7.2 the ΔA260 values could not be
determined because the low-temperature plateau was not
sufficiently developed. This destabilizing effect was even more
profound for 15R (i.e. 13R elongated with GPSAXPSGPS at the 5’-
reference complexes formed by 12R and hM
.
H3C
N
O
H
N
N
H
N
N
R
end) as for 15R:wM12 the transition was observed only at
acidic pH (Tm=34°C, Figure 9S).
O
N
N
dR
13
wR or wM
Chart 2. A rotamer of N6-methyl-2’-deoxyadenosine that enables the formation of
Watson-Crick hydrogen bonds in the 13:wM duplex.
Because for the formation of the parallel duplexes the rotation
of the N6-Me is unnecessary and the preferred conformation26
is accessible for the Hoogsteen interactions (which in
combination with the water bridges are energetically
considerably favored), the intended preferential complexation
4 | J. Name., 2012, 00, 1-3
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Organic & Biomolecular Chemistry
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ARTICLE
with Hp vs. WCp RNA strands should be achieved with predominantly comes from the water bridges with limited
View Article Online
relatively short (RP-PS)-DN(NX)A probes carrying few m6dA contribution from the Hoogsteen base-pairing. In terms of the
DOI: 10.1039/C8OB03112F
units. However, this idea must be implemented with some intended preferential complexation with Hp vs. WCp RNA
caution, because, rather unexpectedly, compound 15R formed strands the probes of the 14R type will be less practical.
the hM&15R complexes only slightly more stable than
15
hM&13R (ΔTm=3°C) at pH 7.2 and less stable (ΔTm=-7°C) at pH
5.4, but still these complexes were more stable (ΔTm=16°C and
11°C) than those formed by 12R (Figure 2). Upon addition of
10
wM12 the Tm values remained unchanged (Figure 3).
5
0.6
0
hM&12R
210
230
250
270
290
310
330
0.5
0.4
0.3
0.2
0.1
0
hM&13R
hM&14R, pH 5.4
14R:wM, pH 5.4
hM&13R, pH 5.4
hM&12R, pH 7.2
-5
-10
-15
hM12&15R
hM&12R:wM, pH 7.2
wavelength [nm]
Figure 4. CD spectra recorded for complexes of 12R, 13R and 14R, with hM or wM in
pH 5.4 buffer. The spectrum recorded for the complex hM&12R:wM is given for the
reference.
30 Temper5a0ture [°C] 70
10
90
Figure 2. Melting curves recorded at pH 5.4 for 12R, 13R and 15R mixed with the
corresponding Hp-(2’-OMe)-RNA matrices.
The above explanations are supported by results of analysis of
the investigated complexes by circular dichroism (CD)
spectroscopy (Figure 4). Among the spectra recorded for
hM12&15R:wM12, pH 7.2
dA/dT, pH 7.2
hM12&15R:wM12, pH 5.4
parallel duplexes, those for hM&12R and hM&13R contain
characteristic intense negative signals at 210 nm with Θ
ranging from -5 to -8.5 mdeg. It should be noted, that the
latter value is close to that for a fully developed triplex
2.2
2
dA/dT, pH 5.4
hM&12R:wM at pH 7.2 (Figure 2, the orange dashed line)
containing unmodified nucleobases. These values confirm the
presence of undisturbed Hoogsteen interactions. Also, the
1.8
1.6
1.4
1.2
1
blue-shift of λmax from 280 nm for hM&12R to 263 nm for
hM 13R, together with high intensity and remarkably narrow
&
shape of the band, indicate a higher degree of structuralization
of the latter complex, as expected from the aforementioned
higher Tm value. On the other hand, the spectrum for hM&14R
0.8
10
20
30
40
50
60
70
80
90
(a blue dashed line) contains the negative signal at 210 nm of
much smaller intensity (Θ=-3.2 mdeg) and the band around
270 nm is wide and of the smallest intensity (Θ=4.8 mdeg). The
spectrum is actually similar to that recorded for a mixture 14R
and wM, for which no Tm was found in the melting
experiment. There is also some similarity to the spectrum for a
Temperature [°C]
Figure 3. Melting curves recorded at pH 7.2 and 5.4 for 15R mixed with hM12 and
wM12 matrices and the plots of first derivative dA/dT.
The opposite effect of the N-methylation was observed for the
complexes formed by 14R, as for hM
5.4 Tm’s of 29°C and 48°C were found, respectively, i.e. by 10°C
and 6°C lower than those for the reference complex hM 12R
&14R at pH 7.2 and pH
complex hM&12R, but recorded at pH 7.4, where the overall
stabilization is weaker because of less effective protonation of
the N3 atom in cytosines. These features suggest weak
&
.
(As expected, upon addition of wM the Tm values remained
unchanged.) We assume that the out-of-plane methyl groups
distort the guanine (and perhaps the adenine rings in the
neighboring nucleotides) from the arrangement which allows
for the most effective stacking and Hoogsteen interactions.
structuralization, which can be attributed to
containing the nucleobases poorly stacking and involved in
underdeveloped or marginal hydrogen bonding.
a system
Thus, although the formation of the parallel hM&14R takes
Conclusions
place (an inflection point was found, Figure 1, a blue dashed
line), yet the overall stabilization is low as ΔA260 measured in
pH 7.2 and 5.4 buffers were as low as 0.06 and 0.08,
respectively. We hypothesize that the stabilization
The OTP-NY monomers (NY = DMT-m6dA or DMT-m,m2dGDPC
)
were synthesized and separated into P-diastereomerically pure
species, which were then used in solid phase synthesis of RP-
and SP-stereodefined chimeric PS-DN(NX)A oligomers. The
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Page 6 of 10
ARTICLE
Journal Name
results of melting experiments performed in pH 7.2 and pH 5.4 Phenomenex Luna 5u Silica column (100 Å; 250Vi×ew1A0rticmle Omn;linae
buffers indicate that the m6dA units stabilize the Hoogsteen flow rate 5 ml/min) was utilized. Preparative separation was
DOI: 10.1039/C8OB03112F
paired duplexes with (2’-OMe)-RNA matrices by 3°C and 4.5°C performed using a silica gel column Pursuit XRs (10 μm, 250 ×
per modified unit, respectively, compared to the reference 21.2 mm) eluted at a flow rate of 25 ml/min. HPLC solvents
duplex containing no nucleobase-modified units. On the and reagents of gradient grade (from Sigma-Aldrich, Baker or
contrary, the m,m2dG units destabilize the analogous Hoogsteen ChemPur) were used.
paired duplexes by 3°C and 2°C per modified unit, respectively. Unmodified DNA and RNA oligonucleotides were synthesized
The CD measurements suggest that the destabilization is on
a Gene-World synthesizer (K&A Laborgeraete GbR,
caused by steric hindrance of the N2-methyl groups so the Schaafheim, Germany).
guanine and perhaps the neighboring adenine rings are 5’-O-DMT-N6-methyl-deoxyadenosine (3).
To a solution of N6-methyl-deoxyadenosine (
1, 0.5 g, 1.89
distorted from the arrangement allowing effective stacking
and Hoogsteen interactions. This observations suggests that to
promote selective recruitment of Hp- vs. WCp-RNA into
parallel duplexes the (RP-PS)-DNA probes should possess
rather the m6dA units.
mmol, Barry & Associates Chemicals, Dexter, MI) in anhydrous
pyridine (10 ml) 4,4’-dimethoxytrityl chloride (0.67 g, 1.98
mmol) was added. The mixture was stirred for 6 h at room
temperature (TLC control, Rf=0.51,) and the solvent was
evaporated under reduced pressure. The residue was
dissolved in chloroform (2 ml) containing 0.1% pyridine and
the solution was applied on a silica gel column (230-400 mesh).
The column was eluted with a gradient of methanol (0% to 2%)
in chloroform. The appropriate fractions were collected and
Experimental Section
1H NMR and 31P NMR spectra were recorded using Bruker
instruments (AV-200 or DRX-500, 200 or 500 MHz for 1H).
Chemical shift values (δ) are given in ppm, relative to internal
tetramethylsilane (TMS) or residual solvent protons for 1H
NMR, and external 85% H3PO4 for 31P NMR.
concentrated under reduced pressure to yield
MS (-FAB) m/z 566.2).
3 (940 mg, 94%,
5’-O-DMT-N6-methyl-deoxyadenosine-3’-O-(2-thio-4,4-
Mass spectra (FAB-MS) were recorded on a Finnigan MAT 95
spectrometer, in the positive and negative ion modes. MALDI-
TOF MS analyses of oligonucleotides were performed using a
Voyager-Elite instrument (PerSeptive Biosystems Inc.,
Framingham, MA) operating in the reflector mode with the
detection of negative ions, unless otherwise stated. HRMS
measurements were performed on a qTOF Waters ESI SYNAPT
G2-Si high definition mass spectrometer fitted with an
atmospheric pressure ionization electrospray source (Waters
Corporation, Milford, MA). The instrument was operated in a
negative-ion mode, with capillary voltage 2.5 kV, cone 40.0 V,
and source offset 50 V. Desolvation done at 150 °C.
pentamethylene-1,3,2-oxathiaphospholane) (6)
To the magnetically stirred solution of
anhydrous pyridine (12 ml), 2-chloro-4,4-pentamethylene-
1,3,2-oxathiaphospholane11
) (170 μl, 1.16 mmol) was added
3 (0.6 g, 1.06 mmol) in
(5
dropwise (under dry argon) at room temperature using a gas-
tight Hamilton syringe. The reaction was complete in 5 min
and then elemental sulfur (70 mg, 2.19 mmol) was added.
Stirring was continued for 12 h and excess sulfur was filtered
off. After evaporation of the solvent, the residue was dissolved
in chloroform (2 ml) containing 0.1% pyridine and applied on a
230-400 mesh silica gel column eluted with chloroform. The
appropriate fractions (TLC control, Rf=0.81) were collected and
Routine UV spectra were recorded on
a CINTRA 10e
concentrated under reduced pressure to give
MS (+FAB) m/z 774.3).
Preparative separation of P-diastereomers of 6
6 (0.60 g, 73%,
spectrophotometer (GBC, Dandenong, Australia), using a
quartz cuvette of 1 cm path length. UV monitored melting
experiments were carried out in 1 cm path length cells, using a
spectrophotometer CINTRA 4040 (GBC), equipped with a
Peltier thermocell.
CD measurements were done on a Jasco J-815 dichrograph at
room temperature, using a 0.5 cm path-length Hellma quartz
cell. The spectra were recorded over a 200-320 nm range with
a 1.00 nm bandwidth, at a scanning speed 50 nm/min, and a
data pitch of 1 nm. After 3 spectra were accumulated, the
baseline was subtracted and the resultant spectrum was
smoothed with a a Savitzky-Golay algorithm (convolution
width 7).
A chromatogram recorded during search for the conditions
suitable for separation of P-diastereomers is shown in Figure
10S (ESI). For preparative separation a silica gel column Pursuit
XRs (10 μm, 250×21.2 mm) was loaded with 250 mg of
6 and
then eluted with ethyl acetate-hexane (7:3, v/v) at 25 ml min−1.
TLC control of the eluate was performed on HP-TLC plates
using the same eluent (6f Rf=0.39; 6s Rf=0.26). The isomers
were recovered in 80% (ca. 100 mg of each isomer, 100%
diastereomeric purity). For 13C NMR data see ESI. 31P NMR (200
MHz, CDCl3); δ (ppm), 105.096, 105.233. 1H NMR (500MHz,
CDCl3); δ (ppm), 6f: 8.35 (1H, NH), 7.92 (1H, C8-H), 7.41-6.79
(13H, DMT), 6.47 (1H, C1’-H), 5.56 (1H, C3’-H), 4.20-4.16 (2H,
P-O-CH2C-S), 4.12-4.11 (1H, C4’-H), 3.77 (6H, 2xOCH3), 3.44-
3.40 (2H, 5’CH2), 3.18 (3H, NCH3), 2.97-2.95 and 2.78-2.75 (2H,
C2’-H), 1.69-1.23 (10H, -(CH2)5-), 6s: 8.34 (1H, NH), 7.91 (1H,
C8-H), 7.41-6.79 (13H, DMT), 6.46 (1H, C1’-H), 5.56 (1H, C3’-H),
4.21-4.17 (2H, P-O-CH2C-S), 4.11-4.10 (1H, C4’-H), 3.77 (6H,
2xOCH3), 3.42-3.41 (2H, 5’CH2), 3.16 (3H, NCH3), 2.96-2.94 and
For routine analyses TLC silica gel 60 plates F254 developed in
CHCl3/MeOH, 9:1 (v/v) were used. HP TLC silica gel 60 plates
with a UV F254 indicator were used for assessment of
chromatographic separability of P-diastereomers.
For HPLC separation of the P-diastereomers of the monomers
6
and 7, a binary Varian HPLC system (two PrepStar 210
pumps, 25 ml pump heads, a ProStar 320 UV/VIS detector set
at 275 nm) was used. In search for the suitable condition a
6 | J. Name., 2012, 00, 1-3
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ARTICLE
2.77-2.76 (2H, C2’-H), 1.67-1.23 (10H, -(CH2)5-). HRMS: m/z ml, 9,6 mmol) were added. The resulting mixtureVwiewasArrtieclfeluOxnleinde
774.2549 calculated for C39H45N5O6PS2, 6f m/z found 774.2548, under an argon atmosphere for 1 h. TDhOeIn: 10th.1e039s/oClv8OenBt03w11a2Fs
6s m/z found 774.2551,.
removed by evaporation under reduced pressure and the
residue was chromatographed on a silica gel (230-400 mesh)
column with a gradient of methanol (0% to 5%) in CH2Cl2. The
appropriate fractions (TLC control, Rf=0.43) were concentrated
under reduced pressure to give 3’,5’-O,O-diacetyl-N2,N2-
3’,5’-O,O-diacetyl-N2-(1,3-benzodithiol-2-yl)deoxyguanosine
Commercially available (Barry & Associates Chemicals, Dexter,
MI) 3’,5’-O,O-diacetyl-deoxyguanosine (1 g, 2.85 mmol) was
dried by evaporation with anhydrous pyridine (3 times) and
finally suspended in that solvent
trimethylsilyl chloride (0.56 ml, 4.41 mmol) was added under a
dimethyldeoxyguanosine
(0.62 g, 93%, MS (+FAB) m/z 380.3).
(20 ml). To the suspension,
N2,N2-dimethyldeoxyguanosine (2)
dry argon atmosphere. After 15 min stirring at room 3’,5’-O,O-diacetyl-N2,N2-dimethyldeoxyguanosine (0.62 g, 1,63
temperature, the mixture was treated with Nakayama reagent mmol) was treated with concentrated NH4OH (8 ml) at room
(1,3-benzodithiolylium tetrafluoroborate, BDTF, (1.025 g, 4.27 temperature for 2 h The resultant solution was concentrated
mmol). The resultant solution was stirred for 30 min and the under reduced pressure to give N2,N2-dimethyldeoxyguanosine
reaction was quenched with water (4 ml). The mixture was
(2) almost quantitatively (assessed by TLC; MS (+FAB) m/z
extracted with CH2Cl2. The organic layer was washed three 296.2), further used without isolation.
times with water, dried over MgS04, filtered, and concentrated 5’-O-DMT-N2,N2-dimethyldeoxyguanosine (4)
under reduced pressure. The residue was dissolved in CH2Cl2 (2
To
a
suspension of crude (vide supra) N2,N2-
ml) and applied on a 230-400 mesh silica gel column eluted
with a gradient of methanol (0% to 3%) in CH2Cl2. The
appropriate fractions (TLC control, Rf=0.59) were collected and
concentrated under reduced pressure to give the desired
product (1.38 g, 94%, MS (-FAB) m/z 502.1).
dimethyldeoxyguanosine
2
(0.68 g containing, 1.63 mmol of 2)
in dry pyridine (10 ml) dimethoxytrityl chloride (0.61, 1.8
mmol) was added. Stirring was continued at room
temperature for 12 h and the mixture was concentrated under
reduced pressure. The residue was dissolved in chloroform (2
ml) containing 0.1% pyridine and applied on a 230-400 mesh
3’,5’-O,O-diacetyl-N2-methyldeoxyguanosine
To a suspension of 3’,5’-O,O-diacetyl-N2-(1,3-benzodithiol-2- silica gel column, eluted with a gradient of methanol (0% to
yl)deoxyguanosine (1.38 g, 2.68 mmol) in benzene (20 ml) 5%) in chloroform. The appropriate fractions (TLC control,
AIBN (182 mg, 1.1 mmol) and tributyltin hydride (2.95 ml, Rf=0.43) were concentrated under reduced pressure to give
10.97 mmol) were added. The mixture was refluxed under dry (0.47 g, 48% yield, MS (-FAB) m/z 596.3).
argon for 1 h. Then the solvent was evaporated under reduced 5’-O-DMT-N2,N2-dimethyldeoxyguanosine-3’-O-(2-thio-4,4-
pressure and the residue was chromatographed on a silica gel pentamethylene-1,3,2-oxathiaphospholane) (7*)
(230-400 mesh) column with a gradient of methanol (0% to
4
To the magnetically stirred solution of
4 (0.7 g, 1.17 mmol) in
5%) in CH2Cl2. The appropriate fractions (TLC control, Rf=0.47)
were concentrated under reduced pressure to give the desired
compound (0.8 g, 80%, MS (+FAB) m/z 366).
3’,5’-O,O-diacetyl-N2-(1,3-benzodithiol-2-yl)-N2–
methyldeoxyguanosine
14 ml dry pyridine, 1.29 mmol 2-chloro-4,4-penamethylene-
1,3,2-oxathiaphospholane was added dropwise at room
temperature. The reaction was complete in 5 min and then
elemental sulfur (2.34 mmol) was added. Stirring was
continued for 12 h and excess sulfur was filtered off. After
3’,5’-O,O-diacetyl-N2-methyldeoxyguanosine (0.8 g, 2.19 evaporation of the solvent the residue was dissolved in
mmol) was dried by repeated evaporation with dry pyridine (3 chloroform (2 ml) containing 0.1% pyridine and applied on a
times) and dissolved in that solvent (10 ml). Trimethylsilyl 230-400 mesh silica gel column eluted with chloroform. The
chloride (0.450 ml, 3.4 mmol) was added to the solution, and appropriate fractions (TLC control, Rf=0.69) were collected and
the mixture was stirred at room temperature for 30 min. Then concentrated under reduced pressure to give 7* (0.80 g, 85%
BDTF (0.52 g, 2.21 mmol) was added and stirring was yield, MS (+FAB) m/z 804.4).
continued for 1.5 h. Water (3 ml) was added and the mixture Protection of 5’-O-DMT-N2,N2-dimethyldeoxyguanosine-3’-O-(2-
was extracted with CH2Cl2. The organic layer was washed 3 thio-4,4-pentamethylene-1,3,2-oxathiaphospholane) at the O6 site
times with water, dried over MgS04, filtered, and evaporated with diphenylcarbamoyl chloride.
under reduced pressure. The residue was dissolved in CH2Cl2 (2
ml) and applied on a 230-400 mesh silica gel column eluted
To a solution of 7* (0.8 g, 1 mmol) in pyridine (7ml), N,N-
diisopropylethylamine
(0.26
ml,
1.5
mmol)
and
with a gradient of methanol (0% to 2%) in CH2Cl2. The
appropriate fractions (TLC control, Rf=0.57) were concentrated
under reduced pressure to give 3’,5’-O,O-acetyl-N2-(1,3-
benzodithiol-2-yl)-N2-methyl-deoxyguanosine (0.88 g, 78%, MS
(+FAB) m/z 518.2).
diphenylcarbamoyl chloride (0.46 g, 2.0 mmol) were added
with stirring at room temperature. The mixture was stirred for
1h, concentrated, dissolved in chloroform (2 ml) and purified
on a silica gel column with chloroform. The appropriate
fractions (TLC control, Rf=0.8) were concentrated under
3’,5’-O,O-diacetyl-N2,N2-dimethyldeoxyguanosine
reduced pressure to give
7
(0.93 g, 93% yield, MS MALDI TOF
To a suspension of 3’,5’-O,O-diacetyl-N2-(1,3-benzodithiol-2- m/z 997.6).
yl)-N2-methyl-deoxyguanosine (0,88 g, 1.74 mmol) in benzene
(20 ml) AIBN (180 mg, 1.1 mmol) and tributyltin hydride (2.58
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Journal Name
Separation of P-diastereomers of 5’-O-DMT-N2,N2-dimethyl-O6–
DPC-deoxyguanosine-3’-O-(2-thio-4,4-pentamethylene-1,3,2-
oxathiaphospholane)
analyzed using RP-HPLC (a Kinetex C18 column, 5 µm, 250 x 4.6
View Article Online
mm; Phenomenex) with with a gradieDnOt I:o1f0.1b0u3f9f/eCr8OBB0(341012%F
CH3CN in 0.05 M TEAB) in buffer A (0.05 M TEAB pH 7.5) from
0% to 17% B over 15 min, at a flow rate of 1 ml min-1. Peaks
were detected at 260 nm.
A chromatogram recorded during search for the conditions
suitable for separation of P-diastereomers is shown in Figure
11S (ESI). For preparative separation a silica gel column Pursuit
Chemical synthesis of oligonucleotides
XRs (10 μm, 250×21.2 mm) was loaded with 600 mg of
7 and Unmodified RNA and (2’-OMe)-RNA oligomers were
eluted with ethyl acetate-hexane (1:1, v/v). TLC control of the synthesized at a 0.2 μmol scale, using commercially available
eluate was performed on HP-TLC plates using the same eluent LCA-CPG supports (Biosearch Technologies, Inc., Petaluma, CA)
(
7f Rf=0.51; 7s Rf=0.4). The isomers were recovered in 66% (ca. and the standard phosphoramidite monomers (Glen Research,
200 mg of each isomer, 100% diastereomeric purity). For 13C Sterling, VA). Oligonucleotides were routinely deprotected and
NMR data see ESI. 31P NMR (500 MHz, CDCl3); δ (ppm), 7f
isolated using a binary Varian HPLC system, consisting of two
:
104.55, 7s: 105.08. 1H NMR (500MHz, CDCl3); δ (ppm), 7f: 7.78 PrepStar 218 pumps and a ProStar 325 UV/VIS detector set at
(1H, C8-H), 7.39-6.76 (23H, DMT, diphenylcarbamoyl residue), 260 nm. A reverse phase HPLC column (PRP-1, C18, 7 μm,
6.28 (1H, C1’-H), 5.52-5.48 (1H, C3’-H), 4.18-4.14 (2H, P-O- 305×7 mm, Hamilton, Reno, NV) was eluted with a 1% min−1
CH2C-S), 4.12-4.09 (1H, C4’-H), 3.74 and 3.73 (6H, 2xOCH3), gradient of CH3CN in 0.1 M TEAB (pH 7.3) at a flow rate 2.5 ml
3.43-3.30 (2H, 5’CH2), 3.10 (6H, N(CH3)2), 3.01-2.96 and 2.72- min−1. All oligomers were analyzed by MALDI-TOF MS and
2.67 (2H, C2’-H), 1.70-1.25 (10H, -(CH2)5-), 7s: 7.76 (1H, C8-H), polyacrylamide gel electrophoresis (PAGE).
7.39-6.76 (23H, DMT, diphenylcarbamoyl residue), 6.27 (1H,
C1’-H), 5.50-5.48 (1H, C3’-H), 4.20-4.16 (2H, P-O-CH2C-S), 4.11
(1H, C4’-H), 3.73 (6H, 2xOCH3), 3.40-3.33 (2H, 5’CH2), 3.09 (6H,
Conflicts of interest
N(CH3)2), 3.00 and 2.67 (2H, C2’-H), 1.66-1.23 (10H, -(CH2)5-).
HRMS: m/z 999.3339 calculated for C53H56N6O8PS2, 7f m/z
found 999.3337; 7s m/z found 999.3329.
There are no conflicts to declare.
Dinucleoside 3′,5′-phosphorothioates d(NX PST) (NX = m6dA, m,m2dG)
Acknowledgements
Powdered P-diastereomerically pure oxathiaphospholane
monomer 6f,s or 7f,s (10 mg, ca. 0.01 mmol) and 3′-O-Ac-
thymidine (0.02 mmol) mixed in a 2 ml flask were dried at high
vacuum (an oil pump) in a desiccator over 24 h. The flask was
filed with dry argon and 500 μl of dry CH3CN and 2.5 μL of 1,8-
diazabicyclo-[5.4.0]undec-7-ene (DBU, 1.1 molar equivalent
This research was financially supported by The National Science
Centre in Poland, Project Number UMO-2013/11/B/ST5/01581 to
AM. Prof. Dr. Maria Bryszewska and Dr. Katarzyna Miłowska (Lodz
University) are gratefully acknowledged for making available to the
authors an instrument for the CD measurements. We thank Dr.
Ewelina Wielgus of our Centre for the recording of mass spectra.
over
6 and 7) were added. After 2 h the reactions were
complete. Each reaction mixture was concentrated under
reduced pressure and the corresponding 8f,s and 9f,s were
treated (in tightly closed vessels) with concentrated ammonia
solution (1.5 ml) at room temperature for 4 h. After
evaporation of the volatile components, the DMT moiety was
removed with 50% aqueous acetic acid (1.5 h, room
temperature), followed by concentration under reduced
pressure. The corresponding d(NXPST) 10f,s and 11f,s were
isolated in amount of a few OD260 units each using RP-HPLC.
Conditions: a C18 column, 250 × 4.6 mm, 5 μm; elution at 1.0
ml min−1 with a gradient of 0.1 M TEAB, pH 7.3 to 20% CH3CN
in 0.1 M TEAB over 20 minutes.
Notes and references
‡
The mechanism of stabilization (proposed by C. Hélène and
further discussed by others37), based on the hydrogen bonding
between the 2’-OH group of ribose in the Hp strand and the pro-RP
oxygen atom (or the RP sulfur atom in our work) of the
internucleotide bond in a homopurine strand, cannot be taken into
account because parallel triplexes (as well as parallel duplexes)
formed with Hp 2’-OMe-RNA oligomers are thermally more stable.
Also direct interactions pyrimidine-C2=O•••H-S-P(O)(OR)2, analogous
to Cys-S-H•••O=C postulated in proteins38, are less likely because
phosphorothioate diesters at neutral pH exist in practically fully
ionized form.
‖
The observed increase of Tm for 12R:wM and 13R:wM at acidic
Enzymatic reactions
pH 5.4 may be explained by a partial formation (ΔA260=0.16) of a
triplex structure (IV) when the protonated cytosines provided
Hoogsteen interactions additional to those resulting from the
presence of three adenine bases.
To determine the absolute configuration of the phosphorus
atom in dinucleoside 3′,5′-phosphorothioates 10f,s and 11f,s
enzymatic methods based on reactions with RP-specific snake
venom phosphodiesterase (svPDE) and SP-specific nuclease P1
(nP1) were used. For reaction with svPDE (1–3 µg, Sigma-
Aldrich) 50 µM solutions of the substrates in 20 mM Tris–HCl
buffer containing 15 mM MgCl2 (pH 8.0, 25 µl) were prepared.
For reaction with nP1 (0.6 µg, Sigma-Aldrich) 100 mM
CH3COONa buffer containing 1 mM ZnCl2 (pH 7.0, 25 µl) was
used. The mixtures were incubated at 37 °C for 20 h and the
reactions were quenched by cooling on ice. The products were
wM 5’-r(CUCUCUUUC)-3’
12R
wM
5’-d(GAAAGAGAG)-3’
3’-r(CUUUCUCUC)-5’
(IV)
Similar stability enhancement was observed for 16R:wM12
(ΔTm=31°C) and for 16R:wR12 (ΔTm=7°C).24
8 | J. Name., 2012, 00, 1-3
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ARTICLE
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DOI: 10.1039/C8OB03112F
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Organic & Biomolecular Chemistry
Page 10 of 10
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DOI: 10.1039/C8OB03112F
Hp-RNA
R
N
O
H
N
O
CH3
N
O
O
X
N
N
H
H
H
N
O
N
N
H
N
S-
O
R
P
dR
WCp-RNA
O
N6-methyl-deoxyadenosine
At pH 7.2 N6-methyl-dA units present in homopurine RP-stereodefined PS-DNA oligomers stabilize
Hoogsteen paired duplexes with (2’-OMe)-RNA matrices by 3°C per modified unit,