Synthesis of PS/PO-chimeric oligonucleotides using mixed oxathiaphospholane and phosphoramidite chemistry

Article abstract of DOI:10.1039/c4ob01837k

Chimeric oligonucleotides containing phosphodiester and phosphorothioate linkages have been obtained using the solid phase synthesis. The oligonucleotide parts possessing natural internucleotide phosphate bonds were assembled using commercially available

Full text of DOI:10.1039/c4ob01837k

Organic &
Biomolecular Chemistry
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PAPER
Synthesis of PS/PO-chimeric oligonucleotides
using mixed oxathiaphospholane and
phosphoramidite chemistry†
Cite this: Org. Biomol. Chem., 2015,
3, 269
1
a
a,b
Ewa Radzikowska and Janina Baraniak*
Chimeric oligonucleotides containing phosphodiester and phosphorothioate linkages have been obtained
using the solid phase synthesis. The oligonucleotide parts possessing natural internucleotide phosphate
bonds were assembled using commercially available nucleoside 3’-O-(2-cyanoethyl-N,N-diisopropyl-
amino)phosphoramidites 7 whereas the phosphorothioate segment was built using nucleoside 3’-O-
(2-thio-1,3,2-oxathiaphospholanes) 3. The oxidation steps, crucial for the conversion of phosphite lin-
Received 28th August 2014,
Accepted 14th October 2014
kages into the phosphate moieties, were conducted using tert-butylperoxy-trimethylsilane, and
this reagent was not harmful to the diester phosphorothioate linkages. When P-diastereopure nucleoside
DOI: 10.1039/c4ob01837k
3
’-O-(2-thio-1,3,2-oxathiaphospholane) monomers were employed the resulting chimeric backbone
www.rsc.org/obc
retained the P-stereoregularity of the phosphorothioate units.
one of the nonbridging oxygen atoms with a sulfur atom, are
the major representatives of first generation DNA analogs.
PS-ODNs display several attractive features like nuclease
Introduction
4
The oligonucleotide based approach for downregulation of
gene expression in cells is based on delivery of a synthetic resistance, activation of RNase H, and good pharmacokinetic
5
oligonucleotide complementary to a target DNA or mRNA, properties. Their limitations include sequence-nonspecific
with the intention to block either inhibition of transcription/ binding to cellular proteins and concentration dependent
6
translation (steric blockage) or enzymatic cleavage of the target competitive inhibition of many nucleases and polymerases.
1
mRNA (e.g. activation of RNase H). Both methodologies have Because the uniform modification of oligonucleotides with
gained remarkable attention and synthetic oligonucleotides phosphorothioate linkages is not required to confer enhanced
7
have become an important class of potential therapeutic stability, several efforts have been made to synthesize
agents for the treatment of viral infections and genetic dis- “chimeric” oligonucleotides possessing natural phosphate and
2
eases. Unfortunately, both approaches have proven to be chal- phosphorothioate internucleotide linkages in the same mole-
8
lenging in practical applications. A good oligonucleotide agent cule (PS/PO-ODNs). Two main types of such constructs have
should be resistant to exo- and/or endonucleases, and should been reported. The first one consists of oligomers possessing
9
have suitable pharmacological and pharmacokinetic profiles, the PS-linkages in alternate positions, while in the second the
as well as high affinity for the target. However, susceptibility oligomer is composed of oligophosphate and oligophos-
8
a,b,c
of unmodified phosphodiester oligonucleotides (PO-ODNs) to phorothioate clusters.
In the latter type it was assumed
nucleolytic degradation by intracellular nucleases made them that the unmodified segments would preserve good hybridiz-
virtually useless for therapeutic purposes. To alleviate that ation properties of natural oligonucleotides, while the phos-
problem, many chemical modifications have been introduced phorothioate segments would enhance the in vivo stability
to the natural phosphodiester backbone to increase the stabi- towards exo- and endonucleases compared to the unmodified
3
lity of oligonucleotides in body fluids. Among them, phos- PO-Oligos. Thus, the chimeric PS/PO-Oligos were expected
8
phorothioate oligonucleotides (PS-ODNs), created by replacing to be advantageous over PS-Oligos with increased affinity
towards the target oligonucleotides and reduced the non-
specific interactions with proteins.
a
The actual biological activity of the oligonucleotide phos-
phorothioates (e.g., interactions with proteins or nucleic acids)
may depend on stereochemical factors because the phos-
Department of Bioorganic Chemistry, Center of Molecular and Macromolecular
Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Łódź, Poland
b
Institute of Chemistry, Environmental Protection and Biotechnology, Jan Długosz
University, Armii Krajowej 13/15, 42-201 Częstochowa, Poland.
E-mail: baraniak@cbmm.lodz.pl
10
phorus atoms in the PS-linkages are stereogenic. One has to
1
1
keep in mind that the phosphoramidite
and H-phos-
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
1
2
c4ob01837k
phonate methodologies (commonly used to prepare PS-Oligos)
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n
are nonstereospecific and give a mixture of 2 diastereomers,
where n is the number of internucleotide phosphorothioate
functions. Thus, various methods have been elaborated to
synthesize these P-chiral oligonucleotide analogs in a stereo-
1
3
controlled manner, among them the oxathiaphospholane
1
4
method developed by Stec et al., the method utilizing nucleo-
side 3′-O-(3-N-acyl)oxazaphospholidine derivatives as monomer
1
5
units, and the method based on a stereoselective synthesis of
1
6
nucleoside 3′-O-oxazaphospholidine monomers are the most
significant.
All these three methodologies can be used to synthesize
PO/PS chimeric oligonucleotides although only the oxathia-
phospholane method requires an additional set of 2-oxo-1,3,2-
oxathiaphospholane monomers for the incorporation of
1
4b
PO-linkages.
Taking into account the commercial avail-
ability of phosphoramidite nucleotides, it was tempting to
combine the oxathiaphospholane method with the phosp-
horamidite one in order to obtain oligonucleotides having a
stereoregular phosphate/phosphorothioate chimeric backbone.
Unfortunately, the oxathiaphospholane method is incompatible
with the phosphoramidite method of DNA synthesis, because
the internucleotide phosphorothioate linkages (assembled
Scheme 1 Synthesis of the model phosphorothioate dinucleotide 2
and its instability towards TBHP.
In order to decrease the reactivity of TBHP, it was trans-
using the oxathiaphospholane monomers) are diester moieties formed into tert-butylperoxy-trimethylsilane (tert-BuOOSiMe
,
3
and they easily get oxidized during the treatment with an 6). This reagent has been successfully applied by Salamończyk
1
7
iodine–base–water solution, which is a typical way for the for selective oxidation of phosphite groups in dendrimers
III
conversion of unstable phosphite (P ) linkages into the stable already possessing diester P = S and P = Se branching units
V
20
phosphate (P ) moieties. To solve this problem one had to find within the same molecule. The trimethylsilane modification
an effective and strictly chemoselective reagent to oxidize made 6 to be much less reactive than 1, thus 50 equiv. of the
the trivalent phosphorus atoms leaving untouched the labile oxidant and 30 minutes time were needed for the quantitative
phosphorothioate diesters present in the growing oligomer oxidation of the model dithymidyl phosphite. It was demon-
3
1
molecule. Here we present our results on making the oxathia- strated by P NMR spectroscopy that under these conditions
phospholane and phosphoramidite protocols compatible with no desulfurization of 2 occurred and that the phosphorothio-
the selection of tert-butylperoxy-trimethylsilane as an oxidizing ate function in 2 was not temporarily protected (silylated) with
reagent.
the trimethylsilyl group delivered by 6 (see ESI†).
Having defined the conditions suitable for the reaction in
solution, the analogous reaction was executed in solid-phase
experiments on a 1 µmol scale. The reaction pathway leading
to the desired PS/PO-ODNs is shown in Scheme 2. Commer-
cially available nucleoside 3′-O-(2-cyanoethyl-N,N-diisopropyl-
Results and discussion
Searching for mild oxidizing systems we focused our attention amino)phosphoramidites 7 were used for the introduction of
to organic hydroperoxides. For initial studies we selected a natural phosphate nucleotide units. In turn, phosphorothioate
solution of tert-butyl hydroperoxide (TBHP, 1) in decane. Its nucleotide parts were introduced using nucleoside oxathiaphos-
III
ability to oxidize P triesters to the corresponding phosphates pholane monomers 3 (Table 1), which were obtained in the
1
8
19
14b
was reported inter alia by Hayakawa et al. and Engels et al.
modified manner compared to the published procedure.
To assess the chemical stability of the phosphorothioate di- In preliminary experiments the monomers 3 were used as
esters towards TBHP, the model phosphorothioate dinucleotide mixtures of P-diastereomers.
DMT
31
(Scheme 1,
T TOAc, 2; P NMR (CDCl ) two resonance
Using the procedure presented in Scheme 2, a series of pro-
PS
3
signals with chemical shifts at ∼55 ppm) was synthesized on tected chimeric PS/PO-oligonucleotides 13 was synthesized
the reaction of thymidine-3′-O-(2-thio-4,4-dimethyl-1,3,2- (Table 2). Starting from the 5′-end, the oligomers contained
oxathiaphospholane) (3d) with 3′-O-acetyl-thymidine (4), and triester phosphate internucleotide linkages (1–5 units),
its acetonitrile solution was treated with 10 equiv. of 1 at followed by 1–3 diester phosphorothioate internucleotide
3
1
ambient temperature. After 30 minutes the P NMR analysis linkages (Table 2). The starting nucleoside 8 was anchored to
of the reaction mixture showed the presence of the oxidized the controlled pore glass support using a DBU-resistant sarco-
DMT
31
21
T
PO
T
OAc (5, ca. 33%; P NMR (CD
3
CN) δ ∼ 0 ppm), but sinyl-succinoyl linker. To build the phosphorothioate seg-
when 50 equiv. of TBHP was used complete disappearance of 2 ments, the oxathiaphospholane derivatives 3 were condensed
was observed after 30 minutes (see ESI†).
with the 5′-hydroxyl group of the growing oligomers. Then, the
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PO-segments were assembled via the phosphoramidite
method using a 20-fold molar excess of the phosphoramidites
7
for each coupling, and at each of those cycles the phosphite
moieties in oligonucleotide derivatives 11 were oxidized
−
1
using a 50-fold molar excess (concentration: 0.33 μmol ml
of tert-BuOOSiMe (6) over 30 minutes to form the corres-
ponding phosphates 12. Similarly, a 15-mer
) T T T T was obtained containing an octameric phos-
)
3
DMT
PS PS PS
T T T -
(
T
PO 8 PS PS PS
phate segment at the middle of the chain and trimeric phos-
phorothioate segments at, both, 3′- and 5′-ends. Finally, the
oligomers 12 were deprotected with concentrated NH OH to
4
form DMT-tagged chimeric PS/PO oligonucleotides 13, which
without any further purification were subjected to MALDI-TOF
MS analysis to assess the content of the oxidized products. For
these compounds the molecular ions were accompanied by
ions at m/z = M-304, attributed to the oligomers being partially
detritylated due to the acidity of the matrices used.
Starting from pure P-diastereomers of 3, chimeric PS/
PO-Oligos possessing stereodefined phosphorothioate linkages
1
4b
(
of either R or S configuration)
were synthesized (entries
P
P
15–19) and the MS data (Table 2) are given for the detritylated
oligomers. During each synthesis of PS/PO-Oligo, the
dimethoxytrityl cation absorbance was monitored and the
crude product was characterized by means of RP HPLC
+
(
see ESI†). The repetitive yields calculated on the DMT cation
absorbance decay were in the range of 94–96%.
One might consider that the yield of the synthesis of 13n
should be lower than those for the other assembled oligomers.
This is because, in this case, the attachment of the last PS
segment by the oxathiaphospholane method inevitably leads
to the elimination of the 2-cyanoethyl groups from the PO
units and such generated phosphodiester linkages may be sus-
ceptible to side reactions. However, this can be avoided,
because exactly the same situation had taken place during the
synthesis of chimeric PS/PO-Oligos based only on the oxathia-
1
4b
phospholane method
where the by-products related to
transformations of the phosphodiester linkages were not
observed.
3
1
Scheme 2 Synthesis of PS/PO-Oligos. Reagents: (i) 3, DBU, CH
3
CN; (ii)
An exemplary P NMR spectrum (recorded for crude 13r)
CHCl
SiMe
2
COOH in CH
2
Cl
2
; (iii) 7, 1H-tetrazole, CH
3
CN; (iv) tert-BuOO- indicates the presence of two groups of signals at the expected
3
, (6); (v) m-fold repetition of steps ii–iv; (vi) piperidine, NH OH.
4
intensity ratio 3 : 5, corresponding to the phosphorothioate
and phosphate moieties (δ 55.50 ppm and −0.97 ppm,
respectively, Fig. 1).
Analysis of the MALDI-TOF MS data revealed that in the
majority of spectra the signal corresponding to the molecular
ion (taken as 100%) was accompanied by a signal at m/z (M-16)
of low intensity (typically <10%), which we attributed to the
molecules with one of the sulfur atoms being randomly
replaced with an oxygen atom. However, if the PS→PO
Table 1 Physicochemical characteristic of compounds 3a–d
3
1
Chromatographic mobility vs.
P NMR
Yield
Compound configuration at P atom
3
(CDCl ) δ (ppm) (%)
exchange was caused by tert-BuOOSiMe , the phosphorothio-
3
3a (Ade)
3b (Cyt)
3c (Gua)
3d (Thy)
“Fast” (S
Slow” (R
“Fast” (S
Slow” (R
“Fast” (S
Slow” (R
“Fast” (R
Slow” (S
P
)
103.57
103.65
104.09
104.34
103.78
103.88
106.70
106.73
42
33
45
30
20
26
49
44
ate diester linkages should be exposed to a cumulative treat-
ment over up to eight synthetic cycles. Thus, one should
observe a correlation between the number of phosphoramidite
condensation/oxidation steps and the extent of the PS→PO
exchange. But none of the recorded spectra contained signals
attributable to the oxidation of two (m/z = M-32) or three (m/z =
“
P
)
P
)
)
)
)
P
“
P
“
P
P
)
“
P
)
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Table 2 The sequences and MS analysis of the synthesized PS/PO-Oligos 13
Theoretical
mass (Da)
Observed mass
MALDI-MS m/z (M − 1)
Peak (M − 16)
a
Entry
Comp
Sequence
content (%)
DMT
b,d, f
1
2
3
4
5
6
7
8
9
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
13m
13n
13o
13p
13r
13s
13t
T
T
T
T
T
PO
PO
PO
PO
PO
T
T
T
T
T
PS
POdCPS
POdGPS
T
PS
T
1488
1457
1497
1792
2114
1812
2066
1764
2167
2183
2454
3062
2964
4896
2749
2677
2677
2797
2722
1488.4
1456.8
1496.8
1791.7
2113.1
1811.3
2064.9
1762.7
2167.0
2183.0
2454.1
3061.5
2963.5
4895.1
2751.1
2674.9
2674.3
2797.4
2721.5
0
DMT
DMT
DMT
DMT
c,d
T
T
3.7
5.6
3.9
4.8
c,d
b,d
PO
T
T
PS
T
PS
PS
PS
POd(CPS
POd(CPS
T
A
c,d
PO
PO
A
PS
T
OH
c,d
T
PO
T
PO
PO
PO
T
PO
A
A
PS
T
C
7.0
DMT
b,d
c,d
T
PO
T
T
T
PS)T
4.3 ; 6.0
c,d
OH
b,d
T
PO
T
C
PO
PS)T
6.3 b,;d11.0
10.6
DMT
DMT
DMT
DMT
DMT
DMT
d(GPO
G
G
PO
A
A
PO
A
A
A
PSA)
c,d
1
1
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
8
9
d(GPO
PO
PO
PS
A
PSA)
8.0
1.3
0
b,d
T
T
T
T
PO
T
PO
T
PO
T
PO
T
PO
T
PO
T
POd(APS
A
PS
POd(APS
PO
POd(CPS
PS PS
PO
POd(APS
PO
POd(CPS
PO
PO
PO
A
A
PSA)
PSA)
PSC)
b,d, f
PO
T
T
PS
A
C
b,d
PO
T
PS
C
25.9
b,d, f
PS
T
PS
T
PS(TPO
)
8
T
T
T
PS
T
0
b,d
b,e
[all-Sp]-TPO
[all-Sp]-TPO
[all-Rp]-TPO
[all-Sp]-TPO
[all-Rp]-TPO
T
T
T
T
T
PO
PO
PO
PO
PO
T
PO
PO
PO
PO
PO
T
T
T
T
T
T
T
T
A
C
C
G
PS
A
PS)T
PS)T
PS)T
PS)T
20.0 ; 16.0
b,d
T
T
T
T
PS
C
15.0
b,d
b,e
POd(CPS
POd(GPS
PS
C
5.6 ; 3.0
b,d
PS
G
18.0
b,d
T
T
PO
T
PS
T
PS
T
PS
T
6.0
a
d
b
c
The intensities of the (M − 1) peaks were taken as 100%. HPA (3-hydroxypicolinic acid) matrix. THA (2′,4′,6′-trihydroxyacetophenone) matrix.
e
f
Voyager-Elite MALDI-TOF mass spectrometer. SHIMADZU Axima Performance MALDI-TOF mass spectrometer. Below the detection limit.
3
1
PO PO PO PS
Fig. 1 202 MHz P NMR spectrum of crude [all-Rp]-TPOT T T TPOd(CPSC CPS)T (13r).
M-48) P-S linkages in a single molecule. Moreover, the MS ana- signal (M-16) was observed compared to that detected for the
lysis of 13k (entry 11) showed the presence of the M-16 signal, HPA matrix (entry 7 or 8).
whereas in the MS spectra recorded for 13l (which contains
It is worth noting that when oligo 13o-mix was synthesized
one more thymidine phosphate residue) and for 13n (eight using only commercially available deoxyribonucleoside
1
Bz
consecutive oxidation steps) the relevant M-16 signals were not phosphoramidites 7 (B = Thy, Ade ) and either 3H-1,2-benzo-
detected at all. On the other hand, it has been noticed that the dithiol-3-one1,1-dioxide or iodine–water to oxidize the trivalent
extent of desulfurization for a given oligomer varied depending phosphorus atoms, MALDI-TOF MS analysis of deprotected
on the spectrometer and on the matrix used for MS analysis oligo revealed desulfurization of phosphorothioate diesters to
(entry 15 or 17). When THA was employed, a more intense the extent of 22%.
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Therefore, these studies have shown that to a certain extent <10%) attributed to the molecules with one of sulfur atoms
the desulfurization occurred under the MALDI-TOF MS operat- along the chain randomly replaced with oxygen were present
ing conditions. To the best of our knowledge it is the first in the majority of MALDI-TOF MS spectra. But, in our opinion,
example demonstrating that unexpected desulfurization the occurrence of desulfurization during the recording of the
can take place even under common negative MALDI-TOF MS spectrum seems to be the most likely explanation of this obser-
operating conditions. On the other hand desulfurization of vation because the extent of desulfurization varied depending
phosphorothioate oligonucleotides occurring during negative on the spectrometer and on the matrix used. No data on this
electrospray ionization (ESI-MS) has been described in the subject have been found in the literature. Additionally, phos-
2
2
2 2
literature. Thus, a comparative analysis of the desulfurization gene present in the reagent (3% TCA in CH Cl ) used for the
of oligo 13k (entry 11) based on spectra recorded by means of 5′-deblocking step in solid phase oligonucleotide synthesis
ESI and MALDI techniques (see ESI†) revealed the greater seems to be in part responsible for the desulfurizing of the
amount of its desulfurization product in the ESI-MS spectrum PS-linkages. Thus the use of toluene instead of CH Cl may be
2 2
(
ca. 8% vs. 1.3%).
beneficial in the preparation of PS/PO-Oligos.
To identify the other factors which might be responsible
for desulfurization we focused our attention on the paper
published by Beaucage et al. concerning the desulfurization
Experimental
General experimental
2
3
of PS-Oligos synthesized by the phosphoramidite method.
3
1
Their studies based on P NMR revealed that under solid-
phase synthesis conditions the loss of the 2-cyanoethyl protect- The nuclear magnetic resonance spectra were recorded on a
ing group from the triester thiophosphate linkage (occurring Brüker AC-200 instrument (200 MHz, TMS internal standard
3
1
3 4
to the extent of ∼5–13%) depended on the capping reagents for 1H and 85% H PO as the external standard for P)
2
4
being used. Generated in this way phosphorothioate diesters unless stated otherwise. The FAB-MS spectra (13 keV, Cs+)
underwent desulfurization by phosgene detected in, both, were recorded on a Finnigan MAT 95 spectrometer, negative
fresh and aged solutions of the detritylating reagent (3% TCA ion MALDI mass spectra were recorded on a Voyager-Elite
2 2
in CH Cl ). Since the internucleotide phosphorothioate lin- instrument (PerSeptive Biosystems Inc., Framingham, USA)
kages assembled by the oxathiaphospholane method were and on a Shimadzu Axima Performance MALDI-TOF mass
unprotected we have taken into consideration the influence spectrometer, while electrospray mass spectrometry analyses
of the detritylating reagent on the desulfurization of the were done using a Brüker amaZon speed ETD instrument.
PS/PO-Oligos. Hence, oligos 13-e and 13-g were synthesized on A mixture of 0.05 M solution of 2,4,6-trihydroxyacetophenone
the solid support (entries 5 and 7) and MS analysis showed (THA) or 3-hydroxypicolinic acid (HPA) in 50% acetonitrile and
that the observed desulfurization increased for oligos which 0.2 M solution of diammonium hydrogen citrate in water (8 : 1,
were released from the support after removal of the DMT v/v) was used as a matrix. The mass spectrum was accumulated
group (entries 6 and 8).
These examples have shown that the process of detritylation Explorer ver. 4 program (Applied Biosystems, Foster City, CA).
plays a role in the desulfurization process. This phenomenon Deoxyribonucleosides were purchased from Pharma
from at least 50 laser shots and processed using the Data
should not be neglected, because the DMT group is repeatedly Waldhof (Germany). Tetrazole solution, 1,4-diazabicyclo[5.4.0]-
removed during PS/PO-oligonucleotide chain assembly. As a undec-7-ene (DBU), 2-cyanoethanol, succinic anhydride, bis-
remedy to avoid the negative effect of phosgene on the quality (trimethylsilyl)acetamide, acetic anhydride, dimethyl-amino-
of the PS/PO-Oligos a freshly prepared 3% TCA in toluene may pyridine and piperidine were purchased from Aldrich (USA).
be used.
Fmoc-sarcosine was purchased from Bachem (Switzerland).
Long chain alkylamine controlled pore glass (500 Å, mesh
80–120) was supplied by Sigma (USA). Phosphorus trichloride,
ethyl acetate, butyl acetate, chloroform, dichloromethane,
benzene and triethylamine were purchased from POCH
Conclusions
PS/PO-Oligos having a phosphate/stereoregular phosphorothio- (Poland). Elemental sulfur was dried under high vacuum for
ate chimeric backbone have been synthesized using mixed 12 h. Acetonitrile was used as a solvent for DBU and oxathia-
−
1
oxathiaphospholane and phosphoramidite chemistry. The phospholane monomers was dried over P O (5 g L ) and dis-
2
5
oligonucleotide segments possessing natural internucleotide tilled through a 20 cm Vigreux column under an atmosphere
phosphate bonds were assembled using commercially avail- of dry argon and stored over 4 Å molecular sieves. At least one-
able nucleoside 3′-O-(2-cyanoethyl-N,N-diisopropylamino)phos- third of the initial volume must be left in the flask. Acetonitrile
phoramidites 7 whereas phosphorothioate parts were built dried in this way must be transferred using gastight syringes
based on nucleoside-2-thio-1,3,2-oxathiaphospholanes 3. The under an atmosphere of dry argon, or by vacuum line. Phos-
oxidation step, crucial for the formation of PO-linkages, was phoramidites of 5′-O-DMT-nucleosides were purchased from
conducted by means of tert-butylperoxy-trimethylsilane and Glen Research and tert-butyl hydroperoxide [a solution in
phosphorothioate linkages were stable towards this reagent. decane (ca. 5.5 M over molecular sieves 4 Å)] was obtained
2
6
Nonetheless, low intensity signals at m/z (M-16) (typically from Fluka. 2-Chloro-1,3,2-oxathiaphospholane (14)
and
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1
4b
2
-chloro-4,4-dimethyl-1,3,2-oxathiaphospholane (15)
were (s, 3H), 1.39 (s, 3H), FAB-MS m/z: (M − 1) 709. HR-MS
(FAB-MS) m/z for C H N O PS calculated m/z 709.1820,
prepared according to the procedures described previously.
3
5
38
2
8
2
found 709.1807.
Phosphitylation of the protected nucleosides with 2-chloro-
1
,3,2-oxathiaphospholane (14) and 2-chloro-4,4-dimethyl-1,3,2- Separation of the diastereomers of 3a–d
oxathiaphospholane (15) – a general procedure
A solution of 100–200 mg of monomer 3 in 1.0 mL of an appro-
To a mixture of appropriately protected 2′-deoxyribonucleoside priate eluent was applied onto a column (75 × 2 cm) contain-
Bz
iBu
Bz
(
1 mmol) (A , G , T, or C ), elemental sulfur (2 mmol) and ing 200 g of silica gel (Merck, particle size 200–300 mesh). The
Bz
Bz
iBu
dry pyridine (4 ml) 14 (1.2 mmol; in the case of C , A , G
)
column was eluted with 300 mL of ethyl acetate–butyl acetate
or 15 (1.2 mmol, for T) were added dropwise. The reaction (1 : 1 v/v for the dA and dC derivatives – 3a and 3b), ethyl
mixture was stirred at room temperature for 3 h (monitored acetate (for the dG monomer – 3c) or butyl acetate (for the
by TLC). Then the solvent was removed in vacuo and into it T monomer – 3d) and fractions of 10–12 mL were collected.
the residual acetonitrile (5 mL) was added. Excess sulfur was The control of the eluate was performed on TLC plates.
filtered off and the filtrate was condensed in vacuo. The Typically for the T monomer one passage gave 93% of separ-
product 3 was isolated by silica gel column chromatography ated P-epimers of 100% diastereomeric purity while for dA and
(230–400 mesh, 0→3% methanol in chloroform with 0.1% dC separation was obtained in 75% yield. For the dG derivative
addition of pyridine) as a mixture of diastereomers.
isomers “fast” and “slow” were obtained in 46% summary
yield (both of 100% diastereomeric purity). Yields and
P NMR chemical shifts of the isolated P-epimers of the
compounds 3a–d are given in Table 1.
6
5
3
′-O-(4,4′-Dimethoxytrityl)-N -benzoyl-2′-deoxyadenosine-
′-O-(2-thio-1,3,2-oxathiaphospholane) (3a)
31
3
1
1
Yield 75%; P NMR (CDCl
200 MHz, CD CN, for diastereomeric mixture) δ: 8.56 (s, 1H),
3
) δ: 103.62, 103.71; H NMR
“In Solution” synthesis of phosphorothioate dinucleotide 2
(
3
8
6
.24 (s, 1H), 7.98 (br.d, 2H), 7.63–7.16 (m, 12H), 6.80 (dd, 4H), 5′-O-DMT-thymidine-3′-O-(2-thio-4,4-dimethyl-1,3,2-oxathiaphos-
.44 (t, 1H), 5.61–5.44 (m, 1H), 4.62–4.28 (m, 3H), 4.13–3.98 pholane) (3d, 1 mmol) was dissolved in dry acetonitrile (5 mL),
(m, 1H), 3.73 (s, 6H), 3.39–3.14 (m, 3H), 2.80–2.65 (m, 1H), and then a solution of 3′-O-acetylthymidine (4, 1.1 mmol) and
2
.29–2.21 (m, 1H) FAB-MS m/z: (M − 1) 794.3; HR-MS (FAB-MS) DBU (1.25 mmol) in dry acetonitrile (3 mL) was added. After
m/z for C40
H
37
N
5
O
7
PS
2
calculated m/z: 794.1882, found m/z: 3 hours, the reaction mixture was concentrated under reduced
pressure. The product 2 was isolated by column chromato-
7
94.1872.
graphy (0→12% methanol in chloroform with 0.1% addition
4
5
3
′-O-(4,4′-Dimethoxytrityl)-N -benzoyl-2′-deoxycytidine-
′-O-(2-thio-1,3,2-oxathiaphospholane) (3b)
of pyridine) as a mixture of diastereomers. Yield 85%
31
(
3
769 mg); P NMR (CD CN) δ: 55.26, 55.16 ppm; MALDI-MS
3
1
1
Yield 74%; P NMR (CDCl ) δ: 104.20; 104.31; H NMR m/z: (M − 1) 905.
3
(
7
5
200 MHz CD
3
CN, for diastereomeric mixture) δ: 8.11 (d, 1H),
.93 (d, 2H), 7.64–7.23 (m, 13H), 6.89 (d, 4H), 6.15 (t, 1H),
.29–5.17 (m, 1H), 4.58–4.28 (m, 3H), 3.76 (s, 6H), 3.56–3.44 To bis-(trimethylsilyl)acetamide (1.5 mmol) tert-butyl hydroxy-
Trimethyl(tert-butylperoxy)silane (6)
(
m, 3H), 3.39 (dd, 1H), 2.80–2.64 (m, 1H), 2.50–2.34 (m, 1H) peroxide (1 mmol) was added dropwise at 4 °C. The reaction
FAB-MS m/z: (M 1) 770. HR-MS (FAB-MS) m/z for mixture was stirred at room temperature for 24 hours. The
C H N O PS calculated m/z: 770.1773, found m/z: 770.1760.
−
crude product was purified via distillation under reduced
3
9
37
3
8
2
pressure, and a fraction boiling at 40–41 °C/25 mmHg was col-
2
5
3
′-O-(4,4′-Dimethoxytrityl)-N -isobutyryl-2′-deoxyguanosine-
′-O-(2-thio-1,3,2-oxathiaphospholane) (3c)
1
lected: H NMR (CDCl
3
); δ: 1.25 (s, 9H), 0.20 (s, 9H); yield 79%;
CI-MS m/z: (M + 1) 163.
3
1
1
Yield 51%; P NMR (CDCl
200. MHz, CDCl , for diastereomeric mixture) δ: 7.82 (s, 1H),
3
) δ: 103.54, 103.71; H NMR
Solid phase synthesis of PS/PO-Oligos 13
(
3
7.41–7.14 (m, 9H), 6.78 (dd, 4H), 6.22 (t, 1H), 5.42–5.27 (m, All oligonucleotides syntheses were carried out manually. For
1H), 4.55–4.20 (m, 2H), 3.72 (s, 6H), 3.58–3.27 (m, 3H), this purpose gastight syringes were used while all reagents and
3.17–2.99 (m, 1H), 2.69–2.46 (m, 4H), 1.15 (dd, 6H), FAB-MS solvents were stored under an atmosphere of dry argon in vials
m/z: (M – 1) 776.4. HR-MS (FAB-MS) m/z for C H N O PS
2
calculated m/z: 776.1973, found m/z: 776.1978.
capped with rubber septa. A column containing a solid
support was equipped with two Teflon adapters: one for the
insertion of a syringe to deliver reagents and the second with a
needle to allow for safe collection of wastes. After each wash
step the excess of solvent was removed from the column by
3
7
39
5
8
5
′-O-Dimethoxytritylthymidine-3′-O-(2-thio-4,4-dimethyl-1,3,2-
oxathiaphospholane) (3d)
3
1
1
Yield 92%; P NMR (CDCl
3
) δ: 106.71, 106.75; H NMR suction while dry argon was delivered from the opposite side.
200 MHz, CDCl , for diastereomeric mixture) δ: 7.61 (s, 1H), In the column 5′-O-DMT-nucleoside 8 (1 μmol) anchored
.45–7.19 (m, 9H), 6.85 (d, 4H), 6.44 (t, 1H), 5.64–5.50 (m, 1H), to the LCA CPG support using a DBU-resistant sarcosinyl-
(
3
7
4
2
1
.30–4.23 (m, 1H), 4.18–4.11 (m, 1H), 4.08–4.03 (m, 1H), 3.78 succinoyl linker was detritylated with 3% solution of TCA
(s, 6H), 3.59–3.33 (m, 2H), 2.68–2.33 (m, 2H), 1.62 (s, 1H), 1.59 (trichloroacetic acid) in methylene chloride and washed with
274 | Org. Biomol. Chem., 2015, 13, 269–276
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4
mL of dry acetonitrile and then with 4 mL of dry methylene
2 (a) S. Agrawal, Tibitech, 1996, 14, 376; (b) D. E. Szymkowski,
Drug Discovery Today, 1996, 1, 415.
chloride and dried under high vacuum.
For the coupling step, in the case of introduction of phos-
phorothioate internucleotide linkage (PS), a solution of an
appropriate oxathiaphospholane monomer 3 (20 μmol) and
DBU (50 μmol) in dry acetonitrile (150 µL) was prepared and
instantly introduced into the column. After 15 minutes of
intensive swirling the column was washed with dry acetonitrile
3 P. D. Cook, Anti-Cancer Drug Des., 1991, 6, 585.
4 (a) F. Eckstein, Annu. Rev. Biochem., 1985, 54, 367;
(b) F. Eckstein, Antisense Nucleic Acid Drug Dev., 2000, 10,
117.
5 (a) J. M. Campbell, T. A. Bacon and E. Wickstrom,
J. Biochem. Biophys. Methods, 1990, 20, 259;
(b) M. I. Phillips and Y. C. Zhang, Methods Enzymol., 2000,
313, 46; (c) S. T. Crooke, Methods Enzymol., 2000, 313, 3.
6 (a) S. T. Crooke, Therapeutic Application of Oligonucleotides,
R. G. Landes Co., Austin, TX, 1995, pp. 63–79;
(b) M. A. Guvakova, L. A. Yakubov, I. Vlodavsky,
J. L. Tonkinson and C. A. Stein, J. Biol. Chem., 1995, 270,
2620; (c) W. M. Galbraith, W. C. Hobson, D. C. Giclas,
P. J. Schechter and S. Agrawal, Antisense Res. Dev., 1994, 4,
201; (d) D. A. Brown, S.-H. Kang, S. M. Gryaznov,
L. DeDionisio, O. Heidenreich, S. Sullivan, X. Xu and
M. I. Neerenberg, J. Biol. Chem., 1994, 43, 26801.
(4 mL) and dry methylene chloride (4 mL) and dried under
argon. Unreacted 5′-hydroxyl groups were capped using the
standard DMAP–Ac O–lutidine solution in THF.
2
During the introduction of phosphate linkages for the
coupling step a solution of appropriate phosphoramidite
monomer 7 (20 μmol) and 1-H-tetrazole (20 μmol) in dry aceto-
nitrile (150 µL) was prepared and instantly introduced into the
column. After 15 minutes of intensive swirling the column was
washed with dry acetonitrile (4 mL) and dry methylene chlor-
ide (4 mL) and dried under argon. Unreacted 5′-hydroxyl
groups were capped using the standard DMAP–Ac
2
O–lutidine
solution in THF. For oxidation of newly formed phosphite
7 (a) J. P. Shaw, K. Kent, J. Bird, J. Fishback and B. Froehler,
Nucleic Acids Res., 1991, 19, 747; (b) T. M. Woolf,
C. G. B. Jennings, M. Rebagliati and D. A. Melton, Nucleic
Acids Res., 1990, 18, 1763; (c) G. D. Hoke, K. Draper,
S. M. Freier, C. Gonzalez, V. B. Driver, M. C. Zounes and
D. J. Ecker, Nucleic Acids Res., 1991, 19, 5743.
to phosphate groups t-BuOOSiMe (50 μmol) was used as
3
solution in acetonitrile (150 µL).
When the synthesis was complete, the oligomer was cleaved
from the support under standard conditions (25% NH OH,
h) and the protecting groups from nucleobases were
4
2
removed at 55 °C over 12 h. The sample was concentrated
under reduced pressure in a Speed-Vac concentrator.
A single cycle of chain elongation was as follows:
8 (a) C. A. Stein, Ch. Subasinghe, K. Shinozuka and
J. S. Cohen, Nucleic Acids Res., 1988, 16, 3209;
(b) M. K. Ghosh, K. Ghosh and J. S. Cohen, Anti-Cancer
Drug Des., 1993, 8, 15; (c) H. Soreq, D. Patinkin, E. Lev-
Lehman, M. Grifman, D. Ginzberg, F. Eckstein and
H. Zakut, Proc. Natl. Acad. Sci. U. S. A., 1994, 91, 7907;
(d) S. Agrawal, Z. Jiang, Q. Zhao, D. Shaw, Q. Cai, A. Roskey,
L. Channavajjala, C. Saxinger and R. Zhang, Proc. Natl.
Acad. Sci. U. S. A., 1997, 94, 2620; (e) M. C. Uzagare,
K. J. Padiya, M. M. Salunkhe and Y. S. Sanghvi, Bioorg. Med.
Chem. Lett., 2003, 13, 3537; (f) M. A. Maier, A. P. Guzaev
and M. Manoharan, Org. Lett., 2000, 2, 1819;
(g) N. U. Mohe, K. J. Padiya and M. M. Salunkhe, Bioorg.
Med. Chem., 2003, 11, 1419.
1
2
) detritylation, 3% TCA in methylene chloride (2 × 5 ml),
) wash, acetonitrile (4 ml), methylene chloride (4 ml),
drying under high vacuum,
3
4
5
) coupling (monomers 3 or 7),
) wash, acetonitrile (4 ml), methylene chloride (4 ml),
) capping, acetic anhydride/DMAP/2,6-lutidine/THF (0.15 ml;
2
min),
)* oxidation, t-BuOOSiMe
30 min),
6
3
(6) (50 µmol) in acetonitrile (150 µL)
(
7
8
) wash, acetonitrile (4 ml), methylene chloride (4 ml),
) removing of β-cyanoethyl groups, 10% piperidine in aceto-
nitrile (v/v) (1 hour),
) wash, acetonitrile (4 ml), methylene chloride (4 ml),
drying under high vacuum.
Only when phosphoramidite monomers 7 were used.
9 (a) B. T. Monia, J. F. Johnston, H. Sasmor and
L. L. Cummins, J. Biol. Chem., 1996, 271, 14533;
(b) S. V. Patil, R. B. Mane and M. M. Salunkhe, Bioorg. Med.
Chem. Lett., 1994, 4, 2663.
9
*
10 W. J. Stec and A. Wilk, Angew. Chem., 1994, 106, 747,
(
Angew. Chem., Int. Ed. Engl., 1994, 33, 709).
1 (a) L. J. McBride and M. H. Caruthers, Tetrahedron Lett.,
983, 24, 245; (b) S. Beaucage and R. P. Iyer, Tetrahedron,
1992, 48, 2223.
12 (a) B. C. Froehler, Tetrahedron Lett., 1986, 27, 5575;
b) B. C. Froehler, in Methods in Molecular Biology, Vol. 20:
1
Acknowledgements
1
These studies were supported financially by the Statutory
Funds of CMMS PAS.
(
Protocols for Oligonucleotides and Analogues, ed., S. Agrawal,
Humana Press Inc., Totowa, NJ, 1993, pp. 63–80.
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1
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1
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5
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was used
during
the
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1
4
276 | Org. Biomol. Chem., 2015, 13, 269–276
This journal is © The Royal Society of Chemistry 2015