P-Stereodefined phosphorothioate analogs of glycol nucleic acids—synthesis and structural properties

Article abstract of DOI:10.1039/c8ra05568h

Enantiomerically pure, protected acyclic nucleosides of the GNA type (glycol nucleic acids) (^GN′), obtained from (R)-(+)- and (S)-(?)-glycidols and the four canonical DNA nucleobases (Ade, Cyt, Gua and Thy), were transformed into 3′-O-DMT-prote

Full text of DOI:10.1039/c8ra05568h

RSC Advances
View Article Online
View Journal | View Issue
PAPER
P-Stereodefined phosphorothioate analogs of
glycol nucleic acids—synthesis and structural
properties†‡
Cite this: RSC Adv., 2018, 8, 24942
*
Agnieszka Tomaszewska-Antczak,
Katarzyna Jastrzebska, Anna Maciaszek,
˛
Barbara Mikołajczyk and Piotr Guga
Enantiomerically pure, protected acyclic nucleosides of the GNA type (glycol nucleic acids) (GN0),
obtained from (R)-(+)- and (S)-(ꢀ)-glycidols and the four canonical DNA nucleobases (Ade, Cyt, Gua
and Thy), were transformed into 30-O-DMT-protected 2-thio-4,4-pentamethylene-1,3,2-
oxathiaphospholane derivatives (OTP-GN0) containing
a second stereogenic center at the
phosphorus atom. These monomers were chromatographically separated into P-diastereoisomers,
which were further used for the synthesis of P-stereodefined “dinucleoside” phosphorothioates
G
GNPST (GN ¼ GA, GC, GG, GT). The absolute configuration at the phosphorus atom for all eight NPS
T
was established enzymatically and verified chemically, and correlated with chromatographic
G
mobility of the OTP-GN0 monomers on silica gel. The NPS units (derived from (R)-(+)-glycidol) were
introduced into self-complementary PS-(DNA/GNA) octamers of preselected, uniform absolute
configuration at P-atoms. Thermal dissociation experiments showed that the thermodynamic
stability of the duplexes depends on the stereochemistry of the phosphorus centers and relative
arrangement of the GN units in the oligonucleotide strands. These results correlate with the changes
of conformation assessed from circular dichroism spectra.
Received 29th June 2018
Accepted 2nd July 2018
DOI: 10.1039/c8ra05568h
rsc.li/rsc-advances
isomers (usually of comparable abundance), which may have
considerably different properties.§ Over several years P-stereo-
dened oligonucleotides (up to 15–18 nt in length) of PS-DNA11
and PS-LNA12 series, were obtained using an oxathiaphospholane
method.13 In several instances these oligomers showed P-stereo-
dependent biological properties.14–16
Introduction
Nucleic acids play a fundamental role in life. These polyanionic
polymers, as well as numerous synthetic analogs, are also used in
biochemistry, molecular biology, and other elds.1 There is also
growing interest in their application in nanotechnology.2,3
Certain applications require nucleic acids with specic modi-
cations, e.g., at the nucleobases, sugars (ribose or deoxyribose), or
inter-nucleotide phosphate linkages.4–6 In phosphorothioate
analogs of DNA (PS-DNA) one of the non-bridging phosphate
oxygen atoms is replaced with a sulfur atom (Fig. 1A).7–9 PS-DNA
oligomers are remarkably resistant towards nucleolytic enzymes.
Although phosphorothioate and phosphate diesters are formally
isoelectronic, in PS-DNA there is a greater density of negative
charge on the sulfur atom.10 A single O / S replacement creates
a new stereogenic center, thus a PS-DNA decamer synthesized
using a non-stereocontrolled method consists of 29 ¼ 512
Recent advances in nucleic acids chemistry enable synthesis
of analogs of DNA or and RNA with improved chemical and
biological stability. These polymers (oen denoted XNA) usually
carry standard nucleobases but have profoundly altered sugar-
phosphate backbones.17 One of the most fundamental
changes is the replacement of the cyclic ribose (deoxyribose)
moiety with an acyclic structure. Because many of those analogs
are poor substrates of nucleases, they are explored in modula-
tion of the properties of nucleic acid and in synthetic biology.4,5
The inherent exibility of acyclic “nucleosides”, combined with
specic “contact points” offered by a given scaffold, make them
suitable for building of molecular devices.18 Glycol nucleic acid
(GNAs; Fig. 1B) attracted our attention because of interesting
physico-chemical properties, which make them potentially
suitable in biotechnology or nanotechnology.19
Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences,
´ ´
Department of Bioorganic Chemistry, Sienkiewicza 112, 90-363 Łodz, Poland.
E-mail: atom@cbmm.lodz.pl
† Dedicated to the memory of our great friend, Dr Andrzej Okruszek.
‡ Electronic supplementary information (ESI) available: Analytical data for 5a–d,
MS data for cGTMPS, preparation of 11c and RP-HPLC proles recorded aer its
§ Theoretical studies on the PS-DNA energy alteration suggest that
RP-phosphorothioate units destabilize the B-helical structure by 2.7
ꢁ
hydrolysis, RP HPLC proles and MALDI-TOF MS data for (GUPS 11
)
dA treated
3.4 kcal molꢀ1, while SP-counterparts stabilize it by ꢀ1.4 ꢁ 2.4 kcal molꢀ1
,
with DBU and for 17. See DOI: 10.1039/c8ra05568h
compared to the reference B-DNA helix.40
24942 | RSC Adv., 2018, 8, 24942–24952
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
Theoretical studies on the inuence of the phosphorothioate
modication on the electronic properties of model single (ss)
and double-stranded (ds) dinucleotides revealed that the elec-
tron migration process through an ss-PS-DNA molecule may be
slowed down or, in extreme cases, quenched.26 Also, interesting
differences in the electron transfer potentials of P-stereodened
chimeric DNA decamers d(CGPSGCCGCCGA), as well as their
duplexes formed with complementary strands d(TCGGCGPS
-
GCCG), were found by cyclic voltammetry assay.27 The cited
above electronic properties of PS-DNA depend on hybridization
status and/or stereochemistry of the phosphorothioate
linkages.
In this work, we wanted to answer a question whether the
stereochemistry of the phosphorus atoms would affect the
hybridization and/or structural properties of PS-GNA oligomers.
Anticipated differences in hybridization properties between RP-
and SP-PS-(DNA/GNA) oligomers and possible P-stereo-
dependent electronic properties may be advantageous for the
applications of P-stereodened PS-GNA units in nanotech-
nology. Toward this goal, we present synthesis and separation
Fig. 1 Structures of (A) P-stereodefined PS-DNA, (B) C-stereodefined
GNA, and (C) P,C-stereodefined PS-(DNA/GNA) oligomers.
The glycol “Gnucleoside” units (GN) can exist in two enan-
tiomeric forms, i.e. (RC)-GN and (SC)-GN (1a and 1b, respectively,
B ¼ Ade, Cyt, Gua, Thy).
of
P-diastereomers
of
2-thio-4,4-pentamethylene-1,3,2-
oxathiaphospholane derivatives of the 30-O-DMT protected
glycidol-derived “Gnucleosides” (OTP-GN0), and enzymatic and
chemical determination of the absolute conguration of the P-
G
atoms in the assembled N0PST “dinucleotides”. Because of
unexpected destructive side-reactions encountered during
oligonucleotide assembly, we synthesized chimeric self-
complementary PS-(DNA/GNA) oligomers (Fig. 1C), which were
subjected to melting experiments and circular dichroism
analysis.
The GNA oligomers hybridize according to the Watson–
Crick's scheme and form highly stable duplexes with a comple-
mentary GNA strand.20 Hybridization with natural nucleic acids
is only observed for (SC)-GNAs (and not (RC)-GNAs) with
complementary ssRNA templates whereas duplexes with ssDNA
are much less stable. The reasons of this difference were thor-
oughly discussed based on X-ray analysis results.21
Results
Because interesting P-stereodependent effects were observed
in PS-DNA (e.g., different thermodynamic stability of antipar-
allel duplexes (PS-DNA : DNA)22 or different propensity for the B
/ Z conversion23), as well as in conformationally more
restricted “locked” PS-LNA,12 we have turned our efforts toward
synthesis of P-stereodened phosphorothioate analogs of con-
formationally exible, glycol-based GNA (PS-GNA). Taking into
account poor hybridization properties of GNA and the fact that
the phosphorothioate modication usually decreases thermal
stability of the modied duplexes{ we assumed that PS-GNA
oligomers would not be able to act as antisense or antigene
probes specically interacting with mRNA or genomic DNA
strands. Nonetheless, certain acyclic, thermally destabilizing
modications (including GNA) enhance RNAi-mediated gene
silencing, because the Ago2 protein favors loading of the 50-end
of the strand from the less stable end of an siRNA duplex.21,24,25
Thus, one cannot reject PS-GNA from future application in the
RNAi methodology.
Preparation of DMT-protected glycidol “Gnucleosides” (DMT-GN0)
The 30-O-DMT-protected glycidol “Gnucleosides” (DMT-GN0, 4a–c,
Scheme 1) were obtained by a literature method.28 In brief, the
commercially available enantiomerically pure (R)-(+)- and (S)-
(ꢀ)-glycidols 2 were protected with DMT-Cl to yield almost
{ There is only one known exception, where homopurine (RP-PS)-DNA oligomers
form thermally highly stable parallel triplexes (RNA : PS-DNA : RNA)41 and parallel
duplexes (PS-DNA : RNA)42. They are supposed to be stabilized not only by the
Hoogsteen interactions but also by water bridges, which span the O-2 atom in
pyrimidines and the sulfur atom of the phosphorothioate moiety.43
Scheme 1 Synthesis of DMT-GN0 (4a–c) and their conversion into the
oxathiaphospholane monomers 5a–d. In the inset, the numbers in
parentheses indicate the priority of substituents (in terms of the Cahn–
Ingold–Prelog's rules) around the stereogenic centers. The synthesis
of DMT-GGiBu (4d) is shown in Scheme 2.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 24942–24952 | 24943

View Article Online
RSC Advances
Paper
quantitatively the corresponding dimethoxytritylated glycidols carbon center is preselected by the use of a particular enan-
3. The regioselective and stereospecic epoxide ring opening tiomer of glycidol, the number of possible forms is reduced to
reaction with a nucleobase (thymine, adenine or N-benzoylcy- two P-diastereomers. For each compound of 5a–d we were able
tosine) in the presence of NaH afforded DMT-GN0s 4a (B ¼ Thy), to separate these P-diastereomers by semi-preparative HPLC on
4b* (B ¼ Ade) or 4c (B ¼ CytBz).28 DMT-GA (4b*) was next protected a silica gel column. MALDI-TOF MS spectra for fast- and slow-
at the exocyclic amino group with benzoyl chloride to yield eluting 5a are shown in Fig. 5S (ESI),‡ and further character-
DMT-GABz (4b). The guanine derivative 4d was obtained via ization of fast and slow-eluting isomers is given in Table 1S
a different method (Scheme 2), since the literature protocol28 (ESI).‡
didn't work in our hands. In this case we decided to work only
with (R)-(+)-glycidol because the related (SC)-GNA has more
Attempts to prepare crystals of OTP-GN0 for X-ray analysis
useful properties than (RC)-GNA.20 The epoxide 2 was opened
with 2-amino-6-chloropurine to give compound 6, which was
further treated with sodium thiosulfate. The resultant 6-thio-
guanosine derivative 7 was oxidized to GG (8) using hydrogen
peroxide.29 The amino group in 8 was acylated with iso-butyryl
chloride to yield diol 9, which was further protected with
DMT-Cl to furnish DMT-GGiBu (4d).
Our efforts to obtain crystals of 5a–d for X-ray analysis were
unsuccessful. Following the previous studies on crystallization
of the oxathiaphospholane derivatives in DNA11 and LNA12
series, we tried to remove the DMT group in 5a–d to obtain 30-
OH OTP-GN0 derivatives 5*a–d (shown in square brackets in
Scheme 1) using earlier successful p-toluenesulfonic acid
monohydrate, yet with no success. Another protic acid
(dichloroacetic acid), Lewis' acids (ZnCl2, SnCl4), tetra-n-buty-
lammonium peroxydisulfate30 and NaHSO4/SiO2 (ref. 31) did
not work either. In each case we observed the formation of
a very polar compound, and in the case of 5a that polar
compound was isolated and identied by 31P NMR and MS
(Fig. 6S, ESI‡) as the corresponding Gthymidine cyclic 20,30-O,O-
Starting from (R)-(+)- and (S)-(ꢀ)-glycidol (2), the corre-
sponding DMT-GN0s (4) of SC and RC absolute conguration,
respectively, were obtained (see inset in Scheme 1; note that the
methylene group in the oxirane ring (marked as CH2) has
priority over the hydroxymethyl group, as shown by the
numbers in parentheses).
phosphorothioate (cGTMPS) bearing
a 5-membered ring
Preparation of P-diastereomerically pure oxathiaphospholane
derivatives of glycol “Gnucleosides” (OTP-GN0)
(Scheme 3, B ¼ Thy). The reasons of this unexpected elimina-
tion are unclear. It should be noted that the conversion of the
detritylated OTP derivative of dCBz into the corresponding cyclic
30,50-O,O-phosphorothioate, which has an entropically favored
6-membered ring, occurred only aer DBU (1,4-diazabicyclo
[5.4.0]undec-7-ene, an activator of high basicity) was added.32
The possible mechanism of forming this ve membered by-
product is shown in Scheme 3.
The enantiomerically pure “Gnucleoside” substrates (DMT-GN0,
4a–d) were phosphitylated with 2-chloro-4,4-pentamethylene-
1,3,2-oxathiaphospholane (prepared according to ref. 11) in
the presence of DIPEA and elemental sulfur (see Scheme 1).
Aer purication on a silica gel column the derivatives 5a–
d (OTP-GN0) were obtained in good yield (78%, 74%, 71%, 79%,
respectively) and characterized by 31P NMR, 1H NMR, 13C NMR
(for representative spectra see Fig. 1S–3S, ESI‡) and HRMS (for
the spectra of 5a–d see Fig. 4S, ESI‡).
Assignment of the absolute conguration of the P-atom in
G
“dinucleotides” NPST obtained using OTP-GN0
A molecule of OTP-GN0 (5) has two stereogenic centers (at the
carbon atom C-20 and the phosphorus atom, both marked with
asterisks in Scheme 1), therefore, each of 5a–d may exist in four
diastereomeric forms. If the absolute conguration at the
Enzymatic approach. Pure P-diastereomers of 5a–d were
reacted with 30-O-acetyl-thymidine in the presence of DBU to
yield the corresponding protected DMT-GN0PSTAc (10a–d, Scheme
4, Fig. 7S, ESI‡), which aer routine two-step deprotection fur-
G
nished the phosphorothioates NPST (11a–d). The products
were isolated by RP-HPLC and identied by MALDI-TOF MS
(Fig. 8S, ESI‡). In order to assign the absolute conguration at
G
phosphorus atom in diastereomeric NPST (11a–d), the reac-
tions with snake venom phosphodiesterase (svPDE, an RP-specic
nuclease) and nuclease P1 (nP1, an SP-specic nuclease) were
Scheme 3 A possible mechanism of spontaneous oxathiaphospho-
lane ring-opening/cyclization during detritylation of OTP-GT (5a).
Scheme 2 Synthesis of DMT-GGiBu (4d).
24944 | RSC Adv., 2018, 8, 24942–24952
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
Scheme 5 The “inverted” synthesis of GAPST (11*b) using DMT-GABz and
50-OTP-TCamph (12).
Scheme 4 Synthesis of “dinucleotides” GNPST (11a–d) using OTP-GN0
(5a–d) and products of their hydrolysis (if successful) with svPDE.
In next HPLC analyses this mixture of standards was spiked
with a given 11b and it was found that 11b derived from fast-RC-
5b (Fig. 2, panel A) and slow-RC-5b (panel B) (both obtained
from S-(ꢀ)-glycidol) were precursors of the internucleotide
phosphorothioate linkages of SP and RP conguration,
respectively.
The analogous analysis indicated (data not shown) that the
fast- and slow-eluting SC-5b (obtained from R-(+)-glycidol) were
precursors of the internucleotide phosphorothioate linkages of
RP and SP conguration, respectively. In conclusion, the
attempted.33,34 It was found that neither diastereomer of 11 was
hydrolyzed by nP1 to a measurable extent.
The experiments with svPDE gave more useful results. The
HPLC proles recorded aer hydrolysis (Fig. 9S and 10S, ESI‡)
showed that compounds 11a–d obtained from slow-eluting (RC)-
OTP-GN0 (synthesized from S-(ꢀ)-glycidol) or fast-eluting (SC)-
OTP-GN0 (obtained from R-(+)-glycidol) were hydrolyzed to yield
GN (1, Scheme 4) and thymidine 50-O-phosphorothiate (TMPS),
identied by means of RP HPLC (Fig. 11S, ESI‡). Thus the
svPDE-digestable diastereomers were tentatively assigned an RP
conguration. Those obtained from fast-eluting (Rc)-OTP-GN0 or
slow-eluting (SC)-OTP-GN0 remained intact aer 24 h incuba-
tion, so were assigned an SP conguration.
Chemical verication of the enzymatic assignment
Because phosphorothioates 11 are much different from svPDE
natural substrates, one cannot exclude the possibility that these
compounds do not conform the general observation of known
stereoselectivity of the enzyme towards PS-DNA. To strengthen
our stereochemical assignment, a chemical verication was
carried out, based on the “inverted” synthesis of GNPST (11*a–d;
the synthesis of 11*b is shown in Scheme 5). We used fast- and
slow-eluting P-diastereoisomers of the 50-O-oxathiaphospholane
derivative of 30-O-camphanoylated thymidine (50-OTP-TCamph
12), which are known to be the precursors of (SP)-NPST and (RP)-
PST, respectively.35 In the presented example fast-12 was
,
N
reacted with (SC + RC)-DMT-GABz (4b, prepared from racemic
glycidol) to yield a pair of protected DMT-GABzPSTCamph (13b). By
a routine two-step deprotection both isomers of 13b were con-
verted into a pair of SCSP- and RCSP-11*b, but this time the
absolute conguration at P-atoms (i.e. SP) was known.k The
second pair of 11*b consisting of SCRP- and RCRP-isomers was
obtained using slow-eluting 12. The RP and SP pairs of 11*b were
mixed at ca. 1 : 2 molar ratio and we found RP-HPLC conditions
allowing for baseline separation of all four components (Fig. 2,
an inset in the panel A).
Fig. 2 HPLC profiles for the mixture of standards 11*b (an inset in
panel A) and co-injections of 11*b and GAPST (11b) obtained from fast-
and slow-eluting (RC)-OTP-GABz 5b (panels A and B, respectively).
HPLC conditions: a Kinetex column, 250 ꢂ 4.6 mm; flow rate 1
mL minꢀ1, buffer A: 0.1 M TEAB, buffer B: 40% CH3CN in 0.1 TEAB,
gradient: 0–38% of buffer B over 20 min.
k Note, that priority of substituents around the stereogenic phosphorus atom in
GAPST and APST (generally in GNPSN and NPSN) is the same.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 24942–24952 | 24945

View Article Online
RSC Advances
Paper
Table 1 Melting temperatures, hyperchromicity (DA260) and ellipticity values at 254 nm (Q254) for duplexes formed by oligomers 14–19 in pH 7.2
buffer containing 10 mM Tris–HCl, 100 mM NaCl, and 10 mM MgCl2
a
Duplex
P-Chirality
Tm (ꢃC)
DA260 (%)
2.4
Q254b [mdeg]
ꢀ1.6
14 50-(GA TGG CGG CGA T)-30
RP
RP
RP
SP
SP
SP
RP
SP
24
14 30-(T GA C GG C GG T A)-50
G
16 50-(A GT G GC G GC A T)-30
16 30-(T A GC G GC G GT A)-50
14 50-(GA T GG C GG C GA T)-30
16 30-(T A GC G GC G GT A)-50
15 50-(GA TGG CGG CGA T)-30
27
5.9
ꢀ2.9
<12
22
2.0
ꢀ1.1
7.9
ꢀ2.4
15 30-(T GA C GG C GG T A)-50
G
17 50-(A GT G GC G GC A T)-30
17 30-(T A GC G GC G GT A)-50
15 50-(GA T GG C GG C GA T)-30
17 30-(T A GC G GC G GT A)-50
18 50-d(ATGCGCAT)-30
33
>7
ꢀ3.7
20
2.0
ꢀ0.8
29
7.6
ꢀ3.1
18 30-d(TACGCGTA)-50
19 50-d(ATGCGCAT)-30
25
10.7
ꢀ1.7
19 30-d(TACGCGTA)-50
a
b
Temperature gradients of 1ꢃC minꢀ1 for annealing and 0.5ꢃC minꢀ1 for melting were applied. CD spectra were recorded aer 15 minutes
incubation at 15 ꢃC.
experiment shown in Scheme 5, as well as those carried out with analysis showed three ladders of ions corresponding to the
4a, c, d (data not shown) proved that the absolute congura- molecular ion (m/z 3154), the depurinated (due to the acidic
tions at the phosphorus atom in 11a–d assigned using svPDE matrix) derivative (m/z 3020), and the depurinated/hydroxylated
were correct.
oligomer (m/z 3037), all consecutively truncated by up to 5
residues, each of 264 amu (Fig. 14S, ESI‡). The relative inten-
sities of the consecutive bands related to the most intense band
at m/z 3037 are 1 : 0.69 : 0.30 : 0.13 : 0.09 : 0.06, and the other
two sets basically follow this pattern. Thus one can assume that
the cleavage took place at the end of the consecutively truncated
oligomer and not randomly inside the chain. The 264 Da
Attempts at solid phase synthesis of P-stereodened PS-GNA
oligomers
The diastereomerically pure OTP-GN0 monomers 5a–d were
tested in a manually performed, solid-phase synthesis of PS-
GNA oligonucleotides. To prevent the DBU-promoted cleavage
of the standard lcaa linker, the rst nucleoside was attached to
the solid support via a DBU-resistant sarcosine linker.36 Even
with double coupling used at each condensation step (earlier
effectively applied in synthesis of PS-LNA12), only ca. 75%
repetitive yield was achieved (based on a DMT cation assay), so
synthesis of octamers and decamers would provide only ca. 13%
and 7%, respectively, of the theoretical amounts (0.757 ¼ 0.133,
0.759 ¼ 0.07), whereas the amount of actually isolated products
would be further reduced at least by half.37 This is unacceptably
low efficiency, compared to 92–94% repetitive yield noted for
OTP monomers of DNA11 and LNA12 series. To acquire an
insight into a possible mechanism contributing to the problem,
a PS-GNA oligonucleotide (GUPS)11dA has been synthesized
using a modied28 phosphoramidite/sulfurization methodology
G
residue seems to be uridine cyclic 20,30-O,O-phosphorothioate
(cGUMPS, Scheme 3, B ¼ Ura), which is an analog of cGTMPS,
although this time it was formed upon intramolecular attack of
the sequentially released terminal hydroxyl groups at the
phosphorus atom of the nearest internucleotide linkage, with
the concomitant release of next primary hydroxyl group, so the
cleavage process could go further. Undoubtedly, to avoid that
“walking” truncation some mechanistic studies and optimiza-
tion of the synthetic protocol are necessary.
Solid phase synthesis of P-stereodened chimeric PS-(DNA/
GNA) and PS-DNA oligomers
Because of the unsatisfactory coupling efficiency we decided to
synthesize “chimeric” PS-(DNA/GNA) oligonucleotide octamers
containing 3 or 4 GN units, using appropriate OTP-GN0 and OTP-
N0 monomers.** The syntheses were performed manually at a 1
and the sarcosine linker.
A 2-cyanoethyl-N,N-diisopropyl-
phosphoramidite derivative of DMT-GU was used (prepared
according to ref. 28) and >99.2% repetitive yield was noted
(based on a DMT cation assay, Fig. 12S, ESI‡). Aer the
synthesis was complete, the oligomer (still being attached to the
** Because the coupling step performed with an oxathiaphospholane monomer
leads to the phosphodiester product (see Scheme 4), this methodology cannot
be combined with the most frequently used phosphoramidite method of
support) was treated with piperidine/acetonitrile solution (1 M) synthesis of DNA, because the mandatory oxidation step (typically with
I2/H2O/pyridine) would destroy the phosphorothioate linkages present in
to remove the 2-cyanoethyl protecting groups, and the resultant
phosphorothioate diester compound was subjected to a DBU/
acetonitrile solution (1.5 M) for 1 h. The remaining material
a growing oligomer. Also the phosphoramidite based elongation of oligomer
with the intended safe use of sulfurizing reagents (to convert the phosphite
intermediate into the phosphorothioate triester) is difficult because the already
was cleaved from the support and isolated by RP-HPLC (a broad
assembled anionic phosphorothioate backbone interferes with the very sensitive
peak eluting at 15.65 min, Fig. 13S, ESI‡). A MALDI-TOF MS acid/base equilibrium operating during the 1H-tetrazole activation step.44
24946 | RSC Adv., 2018, 8, 24942–24952
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
mmol scale. Selfcomplementary oligomers of a general sequence
PS-d(ATGCGCAT) were synthesized in two DNA/GNA variants
(each chimeric oligomer in two P-stereoregular forms, Table 1),
bearing either purine GNs (PS-GATGGCGGCGAT; RP, 14; SP, 15) or
pyrimidine GNs (PS-AGTGGCGGCAT; RP, 16; SP, 17).†† Only SC-
OTP-GN0 were used in the syntheses. Decay of the trityl cation
absorption aer consecutive coupling steps was measured
photometrically (Fig. 15S, ESI‡). Aer each synthesis was
complete, the DMT tagged oligomer was cleaved from the
support and the N-protecting groups were removed using
routine treatment with conc. NH4OH. The oligonucleotides
were isolated by RP-HPLC, then detritylated with 50% acetic
acid, puried by RP-HPLC (Fig. 16S, ESI‡), and nally charac-
terized by MALDI-TOF MS (Fig. 17S, ESI‡). The syntheses fur-
nished ca. 3 OD260 of each oligomer.
Fig. 3 CD spectra for selfcomplementary oligomers 15–19 (dissolved
in pH 7.2 buffer containing 10 mM Tris–HCl, 100 mM NaCl, and 10 mM
MgCl2) recorded after 15 minutes incubation at 15 ꢃC.
The reference oligomers (RP)-PS-50-d(ATGCGCAT)-30 (18) and
(SP)-PS-50-d(ATGCGCAT)-30 (19) were obtained in an analogous
way (a single coupling was used).
SP-17 was the highest among all the oligomers studied,
including the reference homoduplexes formed by PS-DNA
oligomers 18 and 19. These observations indicate that the
stereochemistry of the phosphorothioate linkages is important
for the mode of hybridization of the oligomers and the resultant
conformation of the duplexes.
Thermal stability and circular dichroism analysis of
complexes formed by P-stereodened chimeric PS-(DNA/GNA)
octamers 14–17
For the heteroduplexes 14/16 and 15/17, where the GN units
occupy the “face-to-face” positions (see Table 1), rather unex-
pected effects were noted. In both cases very low hyper-
chromicities (2%) and low Tm values were observed. For 14/16 the
Tm value was only roughly assessed at <12 ꢃC, and Tm ¼ 20 ꢃC was
noted for 15/17. For those systems one might expect the presence
of certain amounts of the duplexes formed by 16 (Tm ¼ 24 ꢃC) or
17 (Tm ¼ 33 ꢃC), with the latter duplex expected to be quite
abundant. However, the melting curves did not show the relevant
inection points, as both proles reached a plateau at much
lower temperatures (Fig. 19S,‡ the melting prole for 16 is given
as a reference). Also the corresponding CD spectra (Fig. 4) indi-
cated a transition of both heteroduplexes to the conformation
already observed for 14, with the absence of bands characteristic
for homoduplexes formed by 16 and 17. At that moment we
cannot offer any possible reasons for that behavior.
The P-stereoregular, selfcomplementary chimeric PS-(DNA/
GNA) oligomers 14–17, where the GN units in the related
duplexes occupy “alternate” positions (see the duplex structures
in Table 1), and the reference PS-DNA oligomers 18 and 19 were
dissolved in pH 7.2 buffer containing 10 mM Tris–HCl, 100 mM
NaCl, and 10 mM MgCl2. The annealing was done from 40 ꢃC to
5
ꢃC with a temperature gradient of 1ꢃC minꢀ1. Then the
melting proles were recorded over a 5 / 40 ꢃC range
(0.5ꢃC minꢀ1) and melting temperatures (Tm) were calculated
using the rst order derivative method. For the reference
compounds 18 and 19 we found Tm ¼ 29 ꢃC and 25 ꢃC (Table 1),
hyperchromicity of 7.6% and 10.7% (Fig. 18S, ESI‡) and Q254
ꢀ3.1 and ꢀ1.7 mdeg (Fig. 3), respectively.
¼
The melting curves for homoduplexes formed by 15–17,
showed hyperchromicity (in a range 5.9–7.9%) and cooperativity
similar to 18 and 19 (Fig. 18S, ESI‡). However, for 14 only 2.4%
hyperchromicity was noted and the cooperativity was very poor
(Fig. 19S, ESI‡). This dichotomy was also observed in the cor-
responding CD spectra, which were recorded aer 15 minutes
incubation of the samples at 15 ꢃC. The spectra for homo-
duplexes 15 and 16 were similar to those for 18 and 19, as they
have strong negative bands around 254 nm and weak negative
signals around 280 nm (Fig. 3). In contrast, for compound 17
a positive band around 294 nm (Q294 ¼ 0.52 mdeg) had a wide
shoulder around 280 nm. Interestingly, the CD spectrum for RP-
14 (on contrary to that recorded for the SP-15 counterpart) has
a weak signal at 254 nm and a positive signal around 280 nm, so
one can assume that the conformation got substantially
changed compared to RP-18. Furthermore, the Tm value for SP-
17 was by 6 ꢃC higher than for RP-16 and by 8 ꢃC higher than for
the reference SP-19. Also, ellipticity Q254 ¼ ꢀ3.7 mdeg found for
Thermal stability of complexes formed by PS-(DNA/GNA)
octamers 14–17 with DNA and RNA templates
The unexpected hybridizing properties of chimeric PS-(GNA/
DNA) oligomers prompted us (despite of the most oen
observed destabilizing effect of the phosphorothioate modica-
tion) to investigate the interactions of 14–17 with natural DNA
(DNA, (d(ATGCGCAT), isosequential to 18) and (20-OMe)-RNA
((m)RNA, (20-OMe)-AUGCGCAU)) templates. However, the
results of melting experiments (Table 2S‡) indicate that the Tm
value recorded for the reference homoduplex formed by DNA (43
ꢃ
ꢃC) has been only very little reduced (<2 C) by the presence of
equimolar amount of any of the 14–17 oligomer. In the case of
(m)RNA (Tm ¼ 62 ꢃC) the reduction was only slightly bigger (up to
4 ꢃC for SP-17). Because the melting curves also showed very good
cooperativity (Fig. 20S, ESI‡), one can conclude that the oligo-
mers 14–17 only weakly interact with DNA or (m)RNA, which
preferentially form thermodynamically much more stable
†† For simplicity, in the sequences of chimeric DNA/GNA oligonucleotides 14–17
the letters A, C and G are used instead of dA, dC and dG, respectively.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 24942–24952 | 24947

View Article Online
RSC Advances
Paper
Framingham, MA) operating in the reector mode with the
detection of negative ions, unless otherwise stated. HRMS
measurements were performed on a qTOF Waters ESI SYNAPT
G2-Si high denition mass spectrometer tted with an atmo-
spheric pressure ionization electrospray source (Waters
Corporation, Milford, MA). The instrument was operated in
negative-ion mode, with capillary voltage 2.5 kV, cone 40.0 V,
and source offset 50 V. Desolvation temperature 150 ꢃC was
applied.
Routine UV spectra were recorded on a CINTRA 10e spec-
trophotometer (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 spectropho-
Fig. 4 CD spectra for selfcomplementary oligomers 14, 16, 14/16 and
15/17 (dissolved in pH 7.2 buffer containing 10 mM Tris–HCl, 100 mM
NaCl, and 10 mM MgCl2) recorded after 15 minutes incubation at 15 ꢃC.
tometer CINTRA 4040 (GBC), equipped with
thermocell.
a Peltier
CD measurements were done on a Jasco J-815 dichrograph
using a cuvette with a 5 mm path-length, thermostated (with
a Peltier effect accessory) at 15 C. The spectra were recorded
over a 200–400 nm range with a 1.00 nm bandwidth, at a scan-
ning speed 100 nm minꢀ1, and a data pitch of 0.2 nm. Aer 5
spectra were accumulated, the baseline was subtracted and the
resultant spectrum was smoothed with a means-movement
algorithm using a 5–25 point lter.
homoduplexes. This was conrmed by CD spectra (recorded at
room temperature, Fig. 21S, ESI‡) which showed that the bands
characteristic for the (m)RNA/(m)RNA homoduplex remained
virtually unchanged, except for their lowered intensity resulting
from the reduced by half concentration of (m)RNA.
ꢃ
Preparative HPLC separation of the oxathiaphospholane
monomers was performed using a Shimadzu Prominence HPLC
system consisting of an LC-20AP preparative pump (a 2 mL
sample loop in the Rheodyne valve was used) and an SPD-M20A
UV detector (set at 260 nm) equipped with a 0.2 mm path length
preparative cell. A silica gel column Pursuit XRs (10 mm, 250 ꢂ
Conclusions
P-Diastereomers of OTP-GN0s (obtained from enantiomerically
pure (R)-(+)- and (S)-(ꢀ)-glycidols 2) were chromatographically
separated and used in synthesis of “dinucleotide” phosphor-
G
othioates NPST. The absolute conguration of the P-atom in
GNPST was established by enzymatic and chemical methods.
Then, four self-complementary P-stereodened chimeric oligo-
mers PS-(DNA/GNA) were synthesized, although even double
coupling at the condensation steps utilizing OTP-GN0 led to
a signicant drop in reaction efficiency. Thermal dissociation
experiments showed that the thermodynamic stability of the
duplexes depends on the stereochemistry at phosphorus and
relative arrangement of the GN units in the oligonucleotide
strands. Most interestingly, a duplex formed by 17 (SP)-PS-
(AGTGGCGGCAT) was thermally more stable than that formed by
the corresponding PS-DNA congener 19 (DTm ¼ 8 ꢃC). The
results on thermal stability correlate with the changes of overall
conformation assessed from circular dichroism spectra.
Further studies on the adaptation of the oxathiaphospholane
approach to synthesis of P-stereodened phosphorothioate
glycol nucleic acid are underway.
21.2 mm) was eluted at a ow rate of 25 mL minꢀ1
.
In search for the condition suitable for HPLC separation of
the P-diastereomers of the oxathiaphospholane monomers
˚
a Phenomenex Luna 5u Silica column (100 A; 250 ꢂ 10 mm;
ow rate 5 mL minꢀ1) and a binary Varian HPLC system (two
PrepStar 210 pumps, 25 mL pump heads, a ProStar 320 UV/VIS
detector set at 275 nm) were used.
HPLC solvents and reagents of gradient grade (from Sigma-
Aldrich, Baker or ChemPur) were used.
All moisture-sensitive operations were carried out under
anhydrous argon.
Note: compounds 3, 4a, 4b*, 4c and 4b were obtained
according to ref. 28.
Unmodied DNA d(ATGCGCAT, DNA), and (20-OMe)-RNA
((20-OMe)-AUGCGCAU, (m)RNA) oligonucleotides were synthe-
sized at a 0.2 mmol scale, using commercially available LCA-CPG
supports (Biosearch Technologies, Inc., Petaluma, CA) and
standard DNA and 20-OMe-RNA phosphoramidite monomers
(Glen Research, Sterling, VA).
Experimental section
1H NMR and 31P NMR spectra were recorded using a Bruker
instrument (AV-200, 200 MHz for 1H). Chemical shi values (d)
are given in ppm, relative to internal tetramethylsilane (TMS) or
Dimethoxytritylation of glycidol – synthesis of enantiomeric
compounds 3
1
residual solvent protons for H NMR, and external 85% H3PO4
for 31P NMR.
To a solution of (R)-(+)- or (S)-(ꢀ)-glycidol 2 (1.0 mL, 15.1 mmol)
Mass spectra (FAB-MS) were recorded on a Finnigan MAT 95 and Et3N (5.4 mL, 40.7 mmol) in CH2Cl2 (50 mL) solid DMT-Cl
spectrometer, in the positive and negative ion modes. MALDI- (6.45 g, 19 mmol) was added. Aer 12 h, the reaction mixture
TOF MS analyses of oligonucleotides were performed using was treated with saturated aqueous NaHCO3 (50 mL). The
a
Voyager-Elite instrument (PerSeptive Biosystems Inc., organic layer was separated, dried with MgSO4 and
24948 | RSC Adv., 2018, 8, 24942–24952
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
concentrated. The residue was dissolved in EtOAc and the using a gas-tight Hamilton syringe. The reaction was complete
solution was washed with saturated aq. NaHCO3. The solvent in 5 h and elemental sulfur (64 mg, 2-fold molar excess) was
was evaporated and the product was isolated chromatographi- added. Stirring was continued for 24 h and excess sulfur was
cally on a silica gel column eluted with hexane–EtOAc–Et3N ltered off. Aer evaporation of the solvent, the residue was
(from 95 : 5 : 0.1 to 85 : 15 : 0.1, v/v/v) to give compounds 3 dissolved in chloroform (3 mL) and loaded on a silica gel
(typically 5.4 g, 95% yield).
column. The column was eluted with a mixture of CHCl3 and
iPrOH (a gradient from 100 : 0 to 95 : 5, v/v) containing 0.1% of
pyridine. Appropriate fractions (vide infra) were collected and
the solvent was evaporated under reduced pressure.
5a OTP-GT. (553 mg, 78%); Rf ¼ 0.71 (CHCl3–MeOH 9 : 1, v/v);
HRMS for C36H40N2O7PS2: calc. 707.2015, found 707.2015
(Fig. 4SA, ESI‡); 31P NMR: d 105.8 ppm, d 105.5 ppm (no
deuterated solvent).
Opening of the oxirane ring in 3 with nucleobases – synthesis
of compounds 4a, 4b*, and 4c
A mixture of nucleobase (1 mmol, 126 mg of thymine, 135 mg of
adenine, or 215 mg of N-benzoylcytosine) and NaH (60%
suspension in mineral oil, ꢄ10 mg, 0.2 mmol) in DMF (2 mL)
was stirred at room temperature for 2 h. A solution of dime-
thoxytritylated glycidol 3 (340 mg, 0.9 mmol) in DMF (2–3 mL)
5b OTP-GABz. 607 mg (74%); Rf ¼ 0.75 (CHCl3–MeOH 9 : 1, v/
v); HRMS for C43H43N5O6PS2: calc. 820.2392, found 820.2400
(Fig. 4SB, ESI‡); 31P NMR: d 105.9 ppm, d 105.2 ppm (no
deuterated solvent).
ꢃ
was added and the resulting mixture was heated at 110 C for
20 h. The solvent was evaporated under reduced pressure (an oil
pump), and the residue was dissolved in EtOAc and loaded on
a silica gel column, initially eluted with hexane–EtOAc–Et3N
(1 : 2 : 0.01, v/v/v), then with EtOAc–Et3N (100 : 1, v/v).
4a DMT-GT. 212 mg (47%); Rf ¼ 0.45 (CHCl3–MeOH 9 : 1, v/v);
FAB MS calc. for C29H30N2O6: 502.57, found: 502.4 (+VE), 501.3
(ꢀVE).
5c OTP-GCBz. 566 mg (71%); Rf ¼ 0.69 (CHCl3–MeOH 9 : 1, v/
v); HRMS for C42H44N3O7PS2: calc. 796.2280, found 796.2287
(Fig. 4SC, ESI‡); 31P NMR: d 105.6 ppm, d 105.1 ppm (no
deuterated solvent).
5d OTP-GGiBu. 634 mg (79%); Rf ¼ 0.70 (CHCl3–MeOH 9 : 1,
v/v); HRMS for C40H45N5O7PS2: calc. 802.2498, found 802.2514
Fig. 4SD, ESI;‡ 31P NMR: d 106.1 ppm, d 106.5 ppm (no
deuterated solvent).
4b* DMT-GA. 225 mg (49%); Rf ¼ 0.44 (CHCl3–MeOH 9 : 1, v/
v); FAB MS calc. for C29H29N5O4: 511.59, found: 512.3 (+VE),
510.3 (ꢀVE).
4c DMT-GCBz. 266 mg (50%); Rf ¼ 0.47 (CHCl3–MeOH 9 : 1, v/
v); FAB MS calc. for C35H33N3O6: 591.67, found: 592.1 (+VE),
590.3 (ꢀVE).
Synthesis of (6-chloropurine) Gnucleoside (6)
A mixture of 2-amino-6-chloropurine (169 mg, 1 mmol), glycidol
(73 mL, 1.1 mmol) and K2CO3 (23.5 mg, 0.17 mmol) in DMF (5
mL) was stirred at 100 ꢃC for 18 h. The reaction mixture was
concentrated in vacuo, the residue was dissolved (not easily) in
pyridine and loaded on a silica gel column. Initially the column
was eluted with CHCl3 and later with CHCl3–MeOH (1 : 1, v/v).
N-Benzoylation of DMT-GA – synthesis of compound 4b
To a solution of 4b* (511 mg, 1 mmol) in anhydrous pyridine (10
mL) trimethylsilyl chloride (507 mL, 4 mmol) was added. Aer 2 h
stirring at room temperature the mixture was cooled to 0 ꢃC and
benzoyl chloride (174 mL, 1.5 mmol) was added dropwise. The
mixture was allowed to warm up slowly and was then stirred for
2 h at room temperature. The mixture was cooled to 0 ꢃC and the
reaction was quenched with H2O (5 mL, at 0 ꢃC with stirring), and
aer 15 min conc. aq. NH4OH (10 mL) was added. Aer 40 min
stirring, the mixture was extracted with CH2Cl2 (2 ꢂ 50 mL). The
organic layer was collected, the solvent was evaporated and the
Fractions containing (6-chloropurine) Gnucleoside (6, Rf
0.41 CHCl3–MeOH 9 : 1, v/v) were collected and evaporated to
yield 90 mg (37%). FAB MS (CI): calc. for C8H10ClN5O2 243.6,
found: 244,1 (+VE).
¼
Conversion of (6-chloropurine) Gnucleoside into the 6-thio
derivative 7
residue was puried chromatographically on a silica gel column To a solution of (6-chloropurine) Gnucleoside 6 (243 mg, 1 mmol)
eluted with a CHCl3–iPrOH mixture (gradient from 100 : 0 to in water (25 mL) Na2S2O3 ꢂ 5H2O (1.24 g, 5 mmol) was added
95 : 5, v/v) containing 0.1% of pyridine. Appropriate fraction (vide and the mixture was reuxed with stirring (ca. 16 h) until no
infra) was collected and the solvent was evaporated under starting Gnucleoside was detected by TLC. The mixture was
reduced pressure to yield DMT-GABz: 387 mg (63%, colorless foam); cooled down to room temperature, and extracted with ethyl
Rf ¼ 0.47 (CHCl3–MeOH 9 : 1, v/v); FAB MS calc. for C36H33N5O5: acetate (2 ꢂ 50 mL). The aqueous phase was concentrated under
615.7, found: 616.3 (+VE), 614.2 (ꢀVE).
reduced pressure and the residue was chromatographed on
a reverse phase silica gel column using water as eluent to yield
180 mg of 7 (75%); Rf ¼ 0.55 (i-PrOH/conc. NH4OH/H2O 7 : 1 : 1,
v/v/v); FAB MS (CI): calc. for C8H11N5O2S 241.3 found: 242.0 (+VE).
General procedure for synthesis of compounds 5a–d
To a magnetically stirred solution of 4 (1 mmol; 502 mg for 4a,
615 mg for 4b, 591 mg for 4c or 597 mg for 4d) dried overnight
under high vacuum and anhydrous N,N-diisopropylethylamine
Conversion of (6-thiopurine) Gnucleoside into compound 8
(20% molar excess, 208 mL, 1.2 mmol) in anhydrous methylene A solution of 7 (241 mg, 1 mmol) in NH4OH – 30% H2O2 (5 : 1, v/
chloride (20 mL), 2-chloro-4,4-pentamethylene-1,3,2- v, 8 mL) was allowed to stand at room temperature for 5 h. The
oxathiaphospholane (20% molar excess, 178 mL, 1.2 mmol) solvents were removed under reduced pressure. The obtained
was added dropwise (under dry argon) at room temperature crude residue was puried by crystallization from H2O to give
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 24942–24952 | 24949

View Article Online
RSC Advances
Paper
the corresponding Gnucleoside 8 as white solid material 7.43 (1H, C6–H), 7.40–6.79 (13H, DMT), 5.13–5.06 (1H, C20–H),
(155 mg, 69%). Rf ¼ 0.42 (CHCl3–MeOH 9 : 1, v/v); FAB MS: calc. 4.16–4.15 (2H, C10–H), 4.08–4.05 (2H, P–O–CH2C–S), 3.75 (6H, 2
for C8H11N5O3 225.2, found: 226.2 (+VE), 224.1 (ꢀVE).
Iso-butyrylation of GG – synthesis of compound 9
ꢂ OCH3), 3.33–3.25 (2H, C30–H), 1.89–1.20 (3H, C5–CH3; 10H,
–(CH2)5–“spiro”); 13C NMR d (CDCl3, ppm) 162.6, 157.3, 143.0,
140.0, 134.2, 128.7, 126.7, 126.6, 125.7, 111.9, 78.0, 76.4, 75.7,
75.1, 62.2, 53.9, 36.0, 35.3, 23.9, 22.7, 22.2, 11.0. 5a OTP-GT slow:
Rf ¼ 0.60 (CHCl3–MeOH 9 : 1, v/v); Rt ¼ 21.5 min (EtOAc–hexane
50 : 50, v/v); MALDI TOF MS: calc. for C36H41N2O7PS2 708.8
found: 747.6 ((+); 708.8 + K+); 31P NMR d (CDCl3, ppm) 106.1; 1H
NMR d (CDCl3, ppm) 8.37 (1H, N3–H), 7.40 (1H, C6–H), 7.32–
6.79 (13H, DMT), 5.09–5.02 (1H, C20–H), 4.16–4.11 (2H, C10–H),
4.08–4.05 (2H, P–O–CH2C–S), 3.77 (6H, 2 ꢂ OCH3), 3.38–3.19
(2H, C30–H), 1.86–1.20 (3H, C5–CH3; 10H, –(CH2)5–“spiro”); 13C
NMR d (CDCl3, ppm) 162.6, 157.3, 149.5, 139.8, 128.7, 126.8,
126.7, 111.9, 76.4, 75.7, 75.1, 53.9, 35.7, 23.9, 22.4, 1.0. 5b
OTP-GABz fast: Rf ¼ 0.72 (CHCl3–MeOH 9 : 1, v/v); Rt ¼ 18 min
(EtOAc–hexane 45 : 55, v/v); MALDI TOF MS calc. 821.9 found
822.6 (+); 31P NMR d (CDCl3, ppm) 105.8; 1H NMR d (CDCl3,
ppm) 8.95 (1H, NHCO), 8.75 (1H, C8–H), 8.02 (1H, C2–H), 7.54–
6.78 (18H, DMT, Bz), 5.22–5.15 (1H, C20–H), 4.57–4.54 (2H, C10–
H), 4.08–3.98 (2H, P–O–CH2C–S), 3.77 (6H, 2 ꢂ OCH3), 3.32–
3.30 (2H, C30–H), 1.61–1.23 (10H, –(CH2)5–“spiro”); 13C NMR
d (CDCl3, ppm) 155.0, 140.5, 128.8, 126.3, 124.2, 113.7, 109.4,
75.8, 51.2, 21.2, 20.0, 19.6. 5b OTP-GABz slow: Rf ¼ 0.58 (CHCl3–
MeOH 9 : 1, v/v); Rt ¼ 23 min (EtOAc–hexane 45 : 55, v/v);
MALDI TOF MS calc. 821.9 found 822.6 (+); 31P NMR d (CDCl3,
ppm) 106.6; 1H NMR d (CDCl3, ppm) 8.95 (1H, NHCO), 8.75 (1H,
C8–H), 8.03 (1H, C2–H), 7.54–6.79 (18H, DMT, Bz), 5.23–5.19
(1H, C20–H), 4.56–4.55 (2H, C10–H), 4.09–4.03 (2H, P–O–CH2C–
S), 3.77 (6H, 2 ꢂ OCH3), 3.31–3.30 (2H, C30–H), 1.56–1.21 (10H,
–(CH2)5–“spiro”); 13C NMR d (CD3CN, ppm) 157.5, 143.4, 142.9,
134.1, 128.8, 126.7, 116.0, 111.8, 78.1, 62.1, 53.6, 34.7, 23.5, 22.3.
5c OTP-GCBz fast: Rf ¼ 0.70 (CHCl3–MeOH 9 : 1, v/v); Rt ¼ 27 min
(EtOAc–hexane 55 : 45, v/v); MALDI TOF MS calc. 797.9 found
799.0 (+); 31P NMR d (CDCl3, ppm) 105.1; 1H NMR d (CDCl3,
ppm) 8.70 (1H, NHCO), 7.88–7.85 (2H, C6–H, C5–H), 7.60–6.79
(18H, DMT, Bz), 5.26–5.22 (1H, C20–H), 4.20–4.15 (2H, C10–H),
To a solution of 8 (225 mg, 1 mmol) in anhydrous pyridine (30
mL) trimethylsilyl chloride (1.01 mL, 8 mmol) was added at 0 ꢃC.
The reaction mixture was stirred at 0 ꢃC for 15 min, followed by
ꢃ
40 min at room temperature. The mixture was cooled to 0 C
and iso-butyryl anhydride (415 mL, 2.5 mmol) was added drop-
wise. The mixture was allowed to warm slowly to room
temperature and was stirred for 2 h. The reaction was stopped
by addition of H2O (2 mL) at 0 ꢃC. Aer 10 min, conc. NH4OH (2
mL) was added and the mixture was le for 15 min. The volatile
components were removed in vacuo. The residue was dissolved
in MeOH, loaded onto a silica gel column and the column was
eluted with EtOAc, EtOAc–MeOH (9 : 1, v/v), and nally with
EtOAc–MeOH (9 : 2, v/v) to yield 239 mg (81%) of GGiBu. Rf ¼
0.10 (CHCl3–MeOH 9 : 1, v/v); FAB MS: calc. for C12H17N5O4
295.3, found: 296.1 (+VE), 294.2 (ꢀVE).
Dimethoxytritylation of 9 – synthesis of DMT-GG (compound
4d)
To a solution of 9 (295 mg, 1 mmol) in anhydrous pyridine (4
mL) solid DMT-Cl (406 mg, 1.2 mmol) was added at room
temperature. Aer 2 h, the reaction mixture was concentrated in
vacuo. The residue was puried by chromatography on a silica
gel column, rst eluted with hexane–EtOAc–Et3N (1 : 2 : 0.01, v/
v/v), then with EtOAc–Et3N (100 : 1, v/v), and nally with EtOAc–
DMT-
MeOH–Et3N (25 : 1 : 0.01, v/v/v). The fraction containing
GGiBu (Rf ¼ 0.46 (CHCl3–MeOH 9 : 1, v/v)) was concentrated in
vacuo to yield 430 mg (colorless foam, 72%); FAB MS: calc. for
C
33H35N5O6 597.7 found: 296,1 ([M-DMT]+; +VE).
HPLC separation of P-diastereomers of GNA
oxathiaphospholane monomers 5a–d
A preparative HPLC silica gel column (Pursuit XRs 10 mm, 250 ꢂ 4.10–4.04 (2H, P–O–CH2C–S), 3.76 (6H, 2 ꢂ OCH3), 3.43–3.28
21.2 mm) was equilibrated with a given eluent mixture (see (2H, C30–H), 1.61–1.20 (10H, –(CH2)5–“spiro”); 13C NMR
Table 1) at a ow rate 25 mL minꢀ1 for 20 minutes. A solution of d (CDCl3, ppm) 161.0, 157.3, 148.6, 143.0, 134.2, 134.0, 131.9,
5 (100 mg; 300 mg for 5a) in ethyl acetate (1.5 mL, with 1% TEA) 128.7, 127.8, 126.8, 126.6, 112.0, 85.1, 78.1, 67.7, 62.2, 59.1, 53.9,
was ltered through a membrane lter (0.2 mm pores, organic 50.9, 36.2, 35.2, 23.9, 22.7, 22.2, 12.9. 5c OTP-GCBz slow: Rf ¼
solvent resistant) and the ltrate was applied onto the column. 0.58 (CHCl3–MeOH 9 : 1, v/v); Rt ¼ 31.2 min (EtOAc–hexane
The column was eluted at the ow rate of 25 mL minꢀ1 and 55 : 45, v/v); MALDI TOF MS calc. 797.9 found 799.1 (+); 31P
1
baseline separation of P-diastereomers was achieved. The NMR d (CDCl3, ppm) 105.7; H NMR d (CDCl3, ppm) 8.70 (1H,
collected fractions were concentrated using a rotary evaporator NHCO), 7.84–7.82 (2H, C6–H, C5–H), 7.60–6.80 (18H, DMT, Bz),
with a “cold nger” condenser lled with isopropyl alcohol/dry 5.30–5.21 (1H, C20-H), 4.33–4.26 (2H, C10–H), 4.18–4.08 (2H, P–
ice coolant, with the temperature of a water bath not exceeding O–CH2C–S), 3.77 (6H, 2 ꢂ OCH3), 3.43–3.21 (2H, C30–H), 1.61–
30 ꢃC. Pure P-diastereomers were recovered in ca. 90% and their 1.20 (10H, –(CH2)5–“spiro”); 13C NMR d (CDCl3, ppm) 172.2,
diastereomeric purity was conrmed by 31P NMR analysis 157.3, 148.4, 131.9, 128.8, 127.7, 126.8, 126.6, 126.2, 111.9, 77.9,
(CDCl3, an estimated limit of detection of a minor component 76.4, 75.7, 75.1, 53.9, 50.8, 35.5, 23.9, 22.5, 22.4. 5d OTP-GGiBu
1
0.2%). They gave satisfactory H NMR, 31P NMR, 13C NMR and fast: Rf ¼ 0.65 (CHCl3–MeOH 9 : 1, v/v); Rt ¼ 13.5 min (EtOAc–
MALDI-TOF mass spectra.
hexane 85 : 15, v/v); MALDI TOF MS calc. 803.9 found 804.6 (+);
1
5a OTP-GT fast: Rf ¼ 0.68 (CHCl3–MeOH 9 : 1, v/v); Rt ¼ 31P NMR d (CDCl3, ppm) 106.1; H NMR d (CDCl3, ppm) 11.89
19.2 min (EtOAc–hexane 50 : 50, v/v); MALDI TOF MS: calc. for (1H, NHCO), 8.34 (1H, N1–H), 7.58 (1H, C8–H), 7.24–6.76 (13H,
C
36H41N2O7PS2 708.8 found: 747.6 (+); 708.8 + K+); 31P NMR DMT), 5.17–5.10 (1H, C20–H), 4.41–4.31 (2H, C10–H), 4.08–3.94
d (CDCl3, ppm) 105.6; 1H NMR d (CDCl3, ppm) 8.36 (1H, N3–H), (2H, P–O–CH2C–S), 3.76 (6H, 2 ꢂ OCH3), 3.17–3.15 (2H, C30–H),
24950 | RSC Adv., 2018, 8, 24942–24952
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
G
2.53–2.50 (1H, COCH), 2.02–1.50 (10H, –(CH2)5–“spiro”), 1.22– found 519.0 and 519.0. 11b APST: Rt1 ¼ 16.5 min, Rt2
¼
1.19 (6H, 2 ꢂ CH3); 13C NMR d (CDCl3, ppm) 177.0, 157.4, 145.8, 17.0 min; MALDI TOF MS: calc. for C18H25N7O8PS 530.48, found
G
143.0, 138.2, 134.1, 128.7, 126.7, 126.6, 125.7, 111.9, 85.3, 76.4, 528.0 and 528.0. 11c CPST: Rt1 ¼ 15.5 min, Rt2 ¼ 19.0 min;
75.7, 75.1, 67.6, 61.3, 54.0, 35.2, 23.8, 22.4, 17.7. 5d OTP-GGiBu MALDI TOF MS: calc. for C17H23N5O9PS 504.44, found 504.2
G
slow: Rf ¼ 0.50 (CHCl3–MeOH 9 : 1, v/v); Rt ¼ 17 min (EtOAc– and 504.1. 11d GPST: Rt1 ¼ 18.7 min, Rt2 ¼ 18.5 min; MALDI
hexane 85 : 15, v/v); MALDI TOF MS calc. 803.9 found 804.6 (+); TOF MS: calc. for C18H23N7O9PS 544.46, found 544.2 and 544.1.
1
G
31P NMR d (CDCl3, ppm) 106.5; H NMR d (CDCl3, ppm) 11.83 From RC-5a–c substrates: 11a TPST: Rt1 ¼ 19.9 min, Rt2
¼
(1H, NHCO), 7.82 (1H, N1–H), 7.58 (1H, C8–H), 7.30–6.80 (13H, 21.0 min; MALDI TOF MS: calc. for C18H24N4O10PS 519.45,
G
DMT), 4.18–5.16 (1H, C20–H), 4.41–4.31 (2H, C10–H), 4.08–3.94 found 519.3 and 519.0. 11b APST: Rt1 ¼ 17.9 min, Rt2
¼
(2H, P–O–CH2C–S), 3.78 (6H, 2 ꢂ OCH3), 3.19–3.16 (2H, C30-H), 17.6 min; MALDI TOF MS: calc. for C18H25N7O8PS 530.48, found
G
2.61–2.48 (1H, COCH), 2.03–1.58 (10H, –(CH2)5–“spiro”), 1.25– 528.0 and 528.0. 11c CPST: Rt1 ¼ 15.6 min, Rt2 ¼ 18.2 min;
1.22 (6H, 2 ꢂ CH3); 13C NMR d (CDCl3, ppm) 177.0, 157.4, 154.2, MALDI TOF MS: calc. for C17H23N5O9PS 504.44, found 504.1
147.2, 145.8, 143.0, 138.2, 134.0, 128.7, 126.7, 125.7, 111.9, 85.3, and 506.2 (in a positive ions mode).
78.1, 76.3, 75.7, 75.1, 54.0, 35.2, 23.8, 22.3, 17.7.
Hydrolysis of 11 with svPDE
Attempt at detritylation of compound 5a
To a solution of 5a (100 mg) in CH3CN (10 mL) a catalytic 5 mM MgCl2 buffer, 10 mL of the svPDE suspension, and GNPS
The reaction mixture (20 mL) containing 25 mM Tris–Cl (pH 8.5),
T
amount of NaHSO4/SiO2 (prepared according to ref. 38) was (11, 0.2 OD260) was incubated for 24 h at 37 ꢃC. Then, the sample
added. The suspension was stirred for 30 minutes and TLC was heat-denatured (for 1 min at 95 ꢃC) and analyzed by HPLC,
analysis showed no substrate remaining. The product did not using the conditions specied above.
migrate on a TLC plate when a mixture CHCl3 : MeOH (9 : 1)
was applied as an eluent. It was isolated using a CHCl3 : MeOH
gradient 6 : 4/3 : 7 (v/v). MS analysis revealed the presence of
a band at m/z 277. (Fig. 6S, ESI‡).
Hydrolysis of 11 with nuclease P1
The reaction mixture (20 mL) containing 100 mM Tris–Cl (pH
7.2), 1 mM ZnCl2, 10 mL of nuclease P1 suspension, and NPS
(11, 0.2 OD) was incubated for 24 h at 37 C. Then, the sample
was heat-denatured (for 1 min at 95 ꢃC) and analyzed by HPLC,
G
T
ꢃ
General procedure for synthesis of compound 11
A given P-diastereoisomer (fast or slow eluting) of OTP-GN0 5 using the conditions specied above.
(0.04 mmol; 28 mg for 5a, 33 mg for 5b, 32 mg for 5c, or 32 mg for
5d) and 30-O-acetyl-thymidine (2-fold molar excess; 23 mg, 0.08
Chimeric oligonucleotides 14, 15, 16 and 17 – synthesis and
mmol), were dried over P2O5 in a vacuum desiccator for 24 h, and
dissolved in anhydrous acetonitrile (ꢄ1 mL) under dry argon. To
the solution 1,8-diazabicyclo-[5.4.0]undec-7-ene (DBU, 1.1 molar
equivalent) was added and aer 2 h the reaction was complete to
purication
The chimeric oligonucleotides 14–17 were synthesized manu-
ally at a 1 mmol scale. A published protocol for synthesis
utilizing OTP monomers was used,37 except that the coupling of
the OTP-GN0 monomers was extended to 20 min. The rst
nucleoside unit was anchored to the support by a sarcosinyl
linker.36 In the cycles where OTP-GN0 monomers 5 were deliv-
ered, two consecutive coupling steps were executed (20 mg + 20
mg), separated by washing with acetonitrile and drying with
a stream of argon. The coupling efficiency was controlled by
measurement of the DMT cation absorption decay at 504 nm.
All oligomers were puried by reverse phase HPLC (conditions
as above) and analyzed by MALDI-TOF MS. 14: (RP)-
PS-GATGGCGGCGAT; MALDI TOF MS calc. 2353 found 2352.8.
15: (SP)-PS-GATGGCGGCGAT; calc. 2353 found 2352.9. 16: (RP)-
PS-AGTGGCGGCAT; calc. 2395 found 2394.5. 17: (SP)-PS-
AGTGGCGGCAT; calc. 2395 found 2394.1.
give GN0PS Ac 10. The formation of 10 was conrmed by 31P NMR
T
(in CH3CN, no deuterated solvent – the recorded chemical shis
DMT-G
are not precisely reproducible); SC-5 substrates:
T TAc,
PS
d 54.6 ppm (from fast 5a), d 54.9 ppm (from slow); DMT-GABzPSTAc,
d 55.6 ppm (from fast 5b), d 55.9 ppm (from slow); DMT-GCBzPSTAc,
DMT-G
d 56.2 ppm (from fast 5c), d 56.8 ppm (from slow);
G TAc,
PS
d 55.6 ppm (from fast 5d), d 56.7 ppm (from slow); RC-5 substrates:
DMT-G
T TAc, d 56.5 ppm (from fast 5a), d 56.3 ppm (from slow);
PS
DMT-GABzPSTAc d 55.8 ppm (from fast 5b), d 56.0 ppm (from slow);
DMT-GCBzPSTAc d 56.7 ppm (from fast 5c), d 56.3 ppm (from slow).
The mixture was concentrated under reduced pressure and treated
(in a tightly closed vessel) with concentrated ammonia solution (2
mL; 10a for 2 h at room temperature, 10b, 10c and 10d for 15 h at
55 ꢃC). Aer evaporation, the DMT moiety was removed with 50%
aqueous acetic acid for 1.5 h at room temperature, followed by
G
Sample preparation and melting UV prole recording
evaporation under reduced pressure. The resultant NPST 11a–
d were isolated using RP-HPLC, a gradient of 0.1 M TEAB, pH 7.3 to The concentration of oligomers was determined by UV absor-
38% CH3CN in 0.1 M TEAB over 20 minutes, elution at 1.0 bance at their lmax in water, using the extinction coefficients
mL minꢀ1: 11a, b, d: an Alltima C18 column, 250 ꢂ 4.6 mm, 5 mm; calculated by the standard method.39 The samples were then
11c: a Clarity C18 column, 250 ꢂ 4.6 mm, 5 mm. Isolated 11 were lyophilized and re-dissolved in pH 7.2 buffer containing 10 mM
characterized by MALDI TOF MS and Rt.
Tris–HCl, 100 mM NaCl, and 10 mM MgCl2. For melting
proles, the oligonucleotides were mixed at 2.0 mM concentra-
G
From SC-5a–d substrates: 11a TPST: Rt1 ¼ 18.5 min, Rt2
¼
19.5 min; MALDI TOF MS: calc. for C18H24N4O10PS 519.45, tion each. (This concentration is also suitable for CD
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 24942–24952 | 24951

View Article Online
RSC Advances
Paper
measurements). First, an annealing step was performed from 20 E. Meggers and L. Zhang, Acc. Chem. Res., 2010, 43, 1092.
40 ꢃC to 5 ꢃC with a temperature gradient of 1ꢃC minꢀ1, followed 21 M. K. Schlegel, D. J. Foster, A. V. Kel'in, I. Zlatev, A. Bisbe,
by a melting step, carried out to 40 ꢃC (or to 85 ꢃC for DNA, (m)
RNA and for mixtures DNA/(m)RNA, 14/(m)RNA and 16/(m)
RNA) with a gradient of 0.5ꢃC minꢀ1. The measurements were
M. Jayaraman, J. G. Lackey, K. G. Rajeev, K. Charisse,
´
J. Harp, P. S. Pallan, M. A. Maier, M. Egli and
M. Manoharan, J. Am. Chem. Soc., 2017, 139, 8537.
done at 260 nm, using a 60 s cycle time and a 2 s integration 22 M. Boczkowska, P. Guga and W. J. Stec, Biochemistry, 2002,
time. The melting temperatures (see Tables 1 and 2S‡) were
calculated using the rst order derivative method.
41, 12483.
23 M. Boczkowska, P. Guga, B. Karwowski and A. Maciaszek,
Biochemistry, 2000, 39, 11057.
24 M. B. Laursen, M. M. Pakula, S. Gao, K. Fluiter, O. R. Mook,
F. Baas, N. Langklær, S. L. Wengel, J. Wengel, J. Kjems and
J. B. Bramsen, Mol. BioSyst., 2010, 6, 862.
25 A. Alagia, M. Terrazas and R. Eritja, Molecules, 2015, 20,
7602.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
26 B. T. Karwowski, Phys. Chem. Chem. Phys., 2015, 17, 21507.
This work was nancially supported by National Centre of 27 W. Lan, Z. Hu, J. Shen, C. Wang, F. Jiang, H. Liu, D. Long,
Science, Poland, grant UMO-2011/03/B/ST5/02670 (to A. T.-A.)
M. Liu and C. Cao, Sci. Rep., 2016, 6, 25737.
28 L. Zhang, A. Peritz, P. J. Carroll and E. Meggers, Synthesis,
2006, 4, 645.
29 Houben-Weyl Methods of Organic Chemistry, ed. E.
Schaumann, 4th edn, Supplement, 1997, vol. E 9b/2, p. 485.
30 S. G. Yang, D. H. Lee and Y. H. Kim, Heteroat. Chem., 1997, 8,
435.
´
´
and by statutory funds of CMMS PAS, Łodz, Poland. The
authors are grateful to Dr Ewelina Wielgus of CMMS PAS for the
recording of mass spectra.
Notes and references
1 O. Khakshoor and E. T. Kool, Chem. Commun., 2011, 47, 31 B. Das, G. Mahender, V. S. Kumar and N. Chowdhury,
7018.
Tetrahedron Lett., 2004, 45, 6709; V. Janardhana Rao,
K. Mukkanti, R. Shankar, N. A. Vekariya and A. Islam,
Rasayan J. Chem., 2013, 6, 15.
2 N. Michelotti, A. Johnson-Buck, A. Manzo and N. Walter,
Wiley Interdiscip. Rev.: Nanomed. Nanobiotechnol., 2012, 4,
139.
3 N. C. Seeman, Nature, 2003, 421, 427.
4 D. H. Appella, Curr. Opin. Chem. Biol., 2009, 13, 687.
32 P. Guga, B. Karwowski, D. Błaziak, M. Janicka, A. Okruszek,
B. R˛ebowska and W. J. Stec, Tetrahedron, 2006, 62, 2698.
33 F. Eckstein, Annu. Rev. Biochem., 1958, 54, 367.
5 V. B. Pinheiro and P. Holliger, Curr. Opin. Chem. Biol., 2012, 34 A. D. Griffiths, B. V. L. Potter and I. C. Eperon, Nucleic Acids
16, 245.
Res., 1987, 15, 4145.
6 A. J. A. Cobb, Org. Biomol. Chem., 2007, 5, 3260.
7 F. Eckstein, Tetrahedron Lett., 1967, 1157.
8 J. Mickleeld, Curr. Med. Chem., 2001, 8, 1157.
9 R. Corradini, S. Sforza, T. Tedeschi and R. Marchelli,
Chirality, 2007, 19, 269.
10 P. A. Frey and R. D. Sammons, Science, 1985, 228, 541.
11 W. J. Stec, B. Karwowski, M. Boczkowska, P. Guga,
M. Koziołkiewicz, M. Sochacki, M. W. Wieczorek and
J. Błaszczyk, J. Am. Chem. Soc., 1998, 120, 7156.
12 K. Jastrz˛ebska, A. Maciaszek, R. Dolot, G. Bujacz and
P. Guga, Org. Biomol. Chem., 2015, 13, 10032.
35 A. Tomaszewska, P. Guga and W. J. Stec, Chirality, 2011, 23,
237.
36 T. Brown, C. E. Pritchard, G. Turner and S. A. Salisbury, J.
Chem. Soc., Chem. Commun., 1989, 891.
37 P. Guga and W. J. Stec, in Current Protocols in Nucleic Acid
Chemistry, ed. S. L. Beaucage, D. E. Bergstrom, G. D. Glick
and R. A. Jones, John Wiley & Sons, Inc., Hoboken, NJ,
USA, 2003, p. 4.17.1.
38 R. K. Kannasani, V. V. Satyanarayana Peruri and
S. R. Battula, Chem. Cent. J., 2012, 6, 136.
39 T. Brown and D. J. S. Brown, in Oligonucleotides and
Analogues: A Practical Approach, ed. F. Eckstein, IRL Press,
Oxford, 1991, p. 20.
´
13 W. J. Stec, A. Grajkowski, M. Koziołkiewicz and B. Uznanski,
Nucleic Acids Res., 1991, 19, 5883.
14 L. Benimetskaya, J. L. Tonkinson, M. Koziołkiewicz, 40 Y.-C. Zhang, J. Liang, P. Lian, Y. Han, Y. Chen, L. Bai,
B. Karwowski, P. Guga, R. Zelser, W. J. Stec and C. A. Stein,
Nucleic Acids Res., 1995, 23, 4239.
Z. Wang, J. Liang, Z. Deng and Y.-L. Zhao, J. Phys. Chem. B,
2012, 116, 10639.
15 A. Krieg, P. Guga and W. J. Stec, Oligonucleotides, 2003, 13, 41 P. Guga, M. Boczkowska, M. Janicka, A. Maciaszek,
491.
B. Nawrot, S. Antoszczyk and W. J. Stec, Pure Appl. Chem.,
2006, 78, 993.
42 P. Guga, M. Janicka, A. Maciaszek, B. Rebowska and
G. Nowak, Biophys. J., 2007, 93, 3567.
43 P. Guga, M. Boczkowska, M. Janicka, A. Maciaszek,
S. Kuberski and W. J. Stec, Biophys. J., 2007, 92, 2507.
16 T. Inagawa, H. Nakashima, B. Karwowski, P. Guga, W. J. Stec,
H. Takeuchi and H. Takaku, FEBS Lett., 2002, 528, 48.
17 V. B. Pinheiro and P. Holliger, Trends Biotechnol., 2014, 32,
321.
18 V. Kumar and V. Kesavan, RSC Adv., 2013, 3, 19330.
19 I. Anosova, E. A. Kowal, M. R. Dunn, J. C. Chaput, W. D. Van 44 A. P. Guzaev and M. Manoharan, J. Org. Chem., 2001, 66,
Horn and M. Egli, Nucleic Acids Res., 2016, 44, 1007.
1798.
24952 | RSC Adv., 2018, 8, 24942–24952
This journal is © The Royal Society of Chemistry 2018