The P-stereocontrolled synthesis of PO/PS-chimeric oligonucleotides by incorporation of dinucleoside phosphorothioates bearing an O-4-nitrophenyl phosphorothioate protecting group

Article abstract of DOI:10.1002/ejoc.200400910

The synthesis of protected model dinucleoside (3′,5′)-O-aryl phosphorothioates, their separation into pure diastereomers, and their successful incorporation into oligonucleotides followed by stereospecific deprotection of the O-aryl phosphorothioate function with oximate ion (inversion) enables the preparation of chimeric PO/PS-oligonucleotides with a predetermined sense of P-chirality at each internucleotide phosphorothioate position. The absolute configuration at the phosphorus of the internucleotide O-aryl phosphorothioate in "dimeric building blocks" has been assigned. The 3′-terminal S_P-phosphorothioate linkages effectively protect such chimeric constructs from degradation by human plasma exonuclease. Wiley-VCH Verlag GmbH & Co. KGaA, 2005.

Full text of DOI:10.1002/ejoc.200400910

FULL PAPER
The P-Stereocontrolled Synthesis of PO/PS-Chimeric Oligonucleotides by
Incorporation of Dinucleoside Phosphorothioates Bearing an O-4-Nitrophenyl
Phosphorothioate Protecting Group
Lucyna A. Wozniak,[a] Marcin Góra,[a] Małgorzata Bukowiecka-Matusiak,[a]
Sophie Mourgues,[b] Geneviève Pratviel,[b] Bernard Meunier,[b] and Wojciech J. Stec*[a]
Keywords: Antisense agents / Nucleosides / Oligonucleotides / Stereochemistry
The synthesis of protected model dinucleoside (3Ј,5Ј)-O-aryl
phosphorothioates, their separation into pure diastereomers,
and their successful incorporation into oligonucleotides fol-
lowed by stereospecific deprotection of the O-aryl phos-
phorothioate function with oximate ion (inversion) enables
the preparation of chimeric PO/PS-oligonucleotides with a
predetermined sense of P-chirality at each internucleotide
phosphorothioate position. The absolute configuration at the
phosphorus of the internucleotide O-aryl phosphorothioate
in “dimeric building blocks” has been assigned. The 3Ј-ter-
minal SP-phosphorothioate linkages effectively protect such
chimeric constructs from degradation by human plasma ex-
onuclease
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2005)
porated into a growing oligonucleotide chain by applying
routine phosphoramidite chemistry.[6]
Introduction
The development of antisense strategies[1] has prompted
The demand for chimeric oligonucleotides increased after
the design of the so-called “chimeric PO/PS-oligonucleo- reports indicating that internucleotide phosphorothioates of
tides” containing phosphorothioates incorporated at the de- SP-configuration located at the 3Ј-end of such chimeric PO/
sired positions of a oligonucleotide chain, with the expecta- PS-Oligos confer complete resistance towards human
tion that their properties as antisense therapeutics will be plasma 3Ј-exonucleases.[7,8] These observations kindled
improved. The advantage of such chimeric PO/PS-Oligos hopes for the evaluation of more-effective, second-genera-
over PS-Oligos,[2] which were successfully established as tion antisense constructs and brought new challenges for
first-generation antisense drugs, involves their increased af- the design and efficient synthesis of precursors of dinucleo-
finity towards target sequences of selected mRNA and low- side phosphorothioates of SP-configuration, which can then
ered affinity towards proteins. PO/PS-Oligos are notably be used for further incorporation into the growing oligonu-
more resistant towards cellular exo- and endonucleases[3] cleotide chain at the desired positions.
compared to the unmodified PO-Oligos.[4] A satisfactory
approach to the synthesis of oligonucleotides modified at
the selected internucleotide linkage with phosphorothioates
of predetermined sense of P-chirality[5] involves HPLC sep-
aration of the chimeric oligonucleotides or the synthesis of
the appropriately protected dinucleotide phosphorothioates
of known absolute configuration at the P-atom. Using so-
called block condensation, after suitable activation at the
3Ј-O-position, these dimeric building blocks may be incor-
[a] Centre of Molecular and Macromolecular Studies, Polish Acad-
In this laboratory, numerous dinucleoside phos-
emy of Sciences, Department of Bioorganic Chemistry,
phorothioate O-alkyl esters of general structure 1 (R =
CH3, C2H5, iPr) have been prepared by nonstereospecific
methods.[9] Since their synthesis and separation into pure
diastereoisomers is difficult, we recently turned our atten-
tion to dinucleoside phosphorothioates 2, where the phos-
phorothioate function carries an O-aryl protecting group.
Early observations by Reese and Serafinowska that O-aryl
Sienkiewicza 112, 90-363 Łódz´, Poland
Fax: +48-42-681-9744
E-mail: wjstec@bio.cbmm.lodz.pl
[b] Laboratoire de Chimie de Coordination du CNRS,
205 route de Narbonne, 31077 Toulouse cedex 4, France
Fax: +33-5-61553033
E-mail: bmeunier@lcc-toulouse.fr
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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DOI: 10.1002/ejoc.200400910
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The P-Stereocontrolled Synthesis of PO/PS-Chimeric Oligonucleotides
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Scheme 1. Reagents and conditions: (i) 4 (2.1 equiv.), pyridine, room temp, 0.5 h; (ii) 6 in pyridine/dioxane, added dropwise, room temp,
1 h; (iii) nucleoside (N-protected) 7 (1.1 equiv.), Me-Imi (4 equiv.), room temp, 1.5 h; (iv) silica-gel column chromatography and separation
of diastereomers; (v) phosphitylation: (iPr2N)2POCH2CH2CN (2 equiv.), 1H-tetrazole (4 equiv.), MeCN, 1 h; silica-gel column chromatog-
raphy purification.
protecting groups at internucleotide phosphorothioates can 5Ј,5ЈЈ protons and the aromatic protons of the aryl group
be selectively removed stereospecifically by treatment with of the internucleotide bond (ROESY spectra), we con-
oximate ion were encouraging in this respect.[10] We also cluded that the FAST eluting isomer 2a has the SP-configu-
expected that separation of the diastereoisomers of 2 ration, whereas the SLOW eluting counterpart 2a has the
would be more effective. To prove these hypotheses we pre- RP-configuration. It is worth mentioning that for dinucleo-
pared two pairs of the protected dinucleoside O-aryl tides with an aryloxy group like 2-chlorophenoxy (data not
phosphorothioates
2
as model compounds, namely presented here) or 4-nitrophenoxy linked to the internucleo-
DMT
T
T (2a) and DMTTPS(O-Aryl)CBz (2b) using O,O- tide phosphorothioate, the simplified but reliable assign-
PS(O-Aryl)
di(hydroxybenzotriazoyl) O-4-nitrophenyl phosphorothio- ment of absolute configuration is feasible. Magnetic shield-
ate (5) as a coupling reagent (Scheme 1).[11]
ing (ring-current) effects carry useful structural information
about the site-specific proton shifts, and have been observed
for a large number of proteins and nucleic acids.[14] For a
partially protected diastereomeric 2, the shielding effect of
the aromatic protecting group strongly influences the chem-
ical shifts of H1Ј (δ = 6.38 ppm for SLOW-2a and δ =
6.46 ppm for FAST-2a), H3Ј (δ = 5.37 ppm for SLOW-2a
and δ = 5.54 ppm for FAST-2a), and H2Ј of the thymidine.
An analogous analysis was performed for isomeric pro-
tected phosphotriesters 2b, and established the RP-configu-
ration for SLOW-2b, and the SP-configuration for FAST-2b
(see Figures 1 and 2).
Results and Discussion
A one-pot reaction, as depicted in Scheme 1, yielded di-
nucleotides 2a and 2b as a diastereomeric mixture in an
approximate 1:1 ratio, as estimated by 31P NMR spec-
troscopy, with an average yield of 65–70% (calculating from
starting 5Ј-O-DMT thymidine). These dinucleotides were
purified and separated into their diastereomers FAST-2 and
SLOW-2 by silica-gel column chromatography.
The assignment of the absolute configuration of isomeric
FAST- and SLOW-2[12] was performed by employing 2D
Semi-empirical PM3 and molecular mechanics calcula-
NMR techniques, with use of the Pulse Field Gradient tions (using the MM2 force field)[15] revealed that for (RP)-
(PFG) system to reduce the measurement time and to im- 2 the distances between the aryl group and H1Ј and H3Ј
prove the quality of the spectra (e.g. by reduction of T1 exceed 5.0 Å, and beyond this distance the shielding effect
noise).[13] From the analysis of the interactions between the of the ring (if any) is very small,[14] which supports the
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W. J. Stec et al.
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1
Figure 1. H NMR spectra (500 MHz, in CDCl3) of (a) FAST-2b and (b) SLOW-2b.
1
Figure 2. Fragment of H NMR spectra (500 MHz, in CDCl3) showing the H2Ј region of (a) FAST-2b and (b) SLOW-2b.
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The P-Stereocontrolled Synthesis of PO/PS-Chimeric Oligonucleotides
FULL PAPER
above assignment. Based upon these findings we assigned question arose, however, whether substitution of the phos-
the absolute configuration of the precursors of dimeric phoryl oxygen by sulfur could modify this proposed depro-
building blocks FAST-2 as SP and SLOW-2 as RP.
tection mechanism, and whether a different mechanism may
operate. This different mechanism could involve attack of
the hydroxide at the phosphorus center of the oxime triester
(Scheme 2, path b), analogous to the mechanism demon-
strated by Bunton and Ihara for 4-nitrophenyl diphenyl-
Stereochemistry of Deprotection of the Phosphorothioate
Triesters 2
Deprotection procedures for the aryl protecting groups phosphate.[22] To distinguish between the two possible
of the internucleotide phosphotriester linkages in phos- mechanisms, the deprotection of the triester 2 was per-
phates were developed in the 1970s and 1980s with the dis- formed in a solution containing [18O] water, such that the
covery of the phosphotriester approach for RNA synthe- product of hydrolysis of the intermediary oxime ester 11
sis.[16] The application of the conjugated bases of oximes, in (path b) should be labeled with 18O. It was found that after
particular syn-4-nitrobenzaldoximate and pyridine-carbox- deprotection in dioxane/H218O (99.8% atom H218O, 4:1
aldoximate, as effective deprotecting reagents for model 2- v/v), no incorporation of 18O into phosphorothiate 10 was
chlorophenyl diethylphosphates and 4-nitrophenyl di- detected by FAB-MS. The assignment of the absolute con-
phenylphosphates, was studied by Reese et al.[17] It was also figuration of the completely deprotected dinucleotide
confirmed that the removal of a 2,5-dichlorophenyl protect- (3Ј,5Ј)-phosphorothioates 12 d(TPSC) was performed by en-
ing group from the phosphorothioate internucleotide bond zymatic digestion followed by HPLC analysis of the prod-
is stereospecific.[10,18] However, since the absolute configu- ucts.[23,24]
ration of the starting phosphotriester was not known, it was
not possible to assign the stereochemical pathway of the photriester FAST-2b, was completely degraded by the RP-
deprotection process. specific Crotalus adamanteus snake venom phosphodiester-
Fully deprotected FAST-12b,[25] obtained from the phos-
In our experiments, the deprotection of the dia- ase (Capd svPDE) within 18 hours, and was not degraded
stereomeric mixture enriched in FAST (TPSCBz) isomer by the SP-specific nuclease P1 (nP1). Accordingly, the phos-
(SP)-2b [dr Ͼ 98%; δ = 62.54 (major), 62.12 ppm (minor)] phorothioate SLOW-12b was not degraded by nuclease
was performed in dioxane/water solution in the presence of Capd, but was hydrolyzed by nP1.[26] The above results con-
an excess of syn-4-nitrobenzaldoxime and 1,1,3,3-tet- firm that the SLOW isomer (RP)-2b, as assigned by NMR
ramethylguanidine (TMG). This reaction was followed by analysis, was deprotected with oxime/TMG to give the cor-
31P NMR spectroscopy and was complete within two responding diester phosphorothioate (SP)-12b, while the
hours.[19] Similar stereospecific deprotection was performed FAST isomer (SP)-2b is the precursor of the isomer (RP)-
with the SLOW isomer (RP)-2b (dr = 94%). In the process 12b. The inversion of configuration supports a mechanism
of these deprotections no other signals were observed in involving attack of the oximate anion at the triester phos-
the NMR spectrum: only substrates 2b and the deprotected phorothioate center, followed by β-elimination of benzo-
phosphorothioate diesters (RP)-10 (δ = 57.37 ppm) and nitrile (path a). Since the oximate ion is a classical α-nucleo-
(SP)-10 (δ = 57.5 ppm), respectively, were detected.
phile bearing nonbonding pairs of electrons adjacent to the
It was postulated earlier that in the rate-determining step nucleophilic center,[27] with a reactivity greater than what
the conjugate base of the oxime attacks the phosphorus would be expected from the pKa value of 9.95,[20] it attacks
atom of the corresponding triester phosphate to form the the phosphorus atom with inversion of configuration. 31P
intermediate oxime ester 11.[20] The intermediate 11 rapidly NMR studies of the deprotection reaction did not indicate
fragments to form the substituted benzonitriles and the cor- formation of any intermediate, only formation of the depro-
responding phosphodiester 10 (Scheme 2, path a).[21] The tected phosphodiesters. This observation is consistent with
Scheme 2. Oximate-mediated removal of aryl protecting groups; path a: base-catalyzed β-elimination of 4-nitrobenzonitrile from the
oxime ester 11 (retention of configuration); path b: hydrolysis of the oxime ester 11 (inversion of configuration).
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W. J. Stec et al.
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a rate-determining stereoinversion step of the transesterifi- ments with snake venom phosphodiesterase (svPD) and
cation reaction, followed by fast β-elimination of the corre- 50% human serum.[8] We found that oligonucleotides 14
sponding benzonitrile and formation of the internucleotide and 15 protected by the 3Ј-terminal SP-phosphorothioate
phosphorothioate.
function remained almost intact (96%) after two hours in-
cubation with svPD, and were stable in more than 60% af-
ter eight hours of incubation in 50% human serum at 37 °C.
In control experiments under the same conditions unmodi-
fied octanucleotide d(TCTTTTTC) was completely de-
graded within one hour of incubation. Comprehensive
analysis of such protection is given elsewhere.[28]
Synthesis of Chimeric Oligonucleotides with Incorporated
Phosphorothioate Funcionalities of Predetermined Absolute
P-Configuration
To check the applicability of the diastereomerically pure
dimeric building blocks with O-aryl protection of the in-
ternucleotide phosphorothioates for synthesis of chimeric
PO/PS oligonucleotides we prepared representative chim-
eric constructs (Table 1). The partially protected phos-
photriesters 2b were synthesized, separated into FAST- and
SLOW-eluting diastereoisomers (vide supra, Scheme 1),
phosphitylated at the 3Ј-OH position, and purified by sil-
ica-gel column chromatography. The corresponding 3Ј-O-
(β-cyanoethyl-N,N-diisopropyl phosphoramidites) (9b) were
used for the coupling reactions with the CPG-bound oligo-
nucleotides prepared by phosphoramidite chemistry. All
oligomers were prepared on a 1-μmol scale with an ABI 394
Synthesizer (Applied Biosystems Inc., Foster City, USA).
Incorporation of 9b was achieved by prolonging the coup-
ling time to 10 min, without further modifications of the
standard protocol.
Conclusions
The data presented here reveal that the partially pro-
tected diastereomerically pure dinucleoside (3Ј,5Ј)-phos-
phorothioates 2 with 4-nitrophenyl groups protecting the
internucleotide phosphorothioate centers are convenient
substrates for the “dimeric building blocks” strategy. They
can be efficiently used for incorporation of dia-
stereomerically pure phosphorothioate linkages into the
chimeric oligonucleotides using standard phosphoramidite
chemistry. The use of the aryl protecting groups, and the 4-
nitrophenyl group in particular, allows for separation of the
dinucleoside (3Ј,5Ј)-O-aryl-phosphorothioates into dia-
stereomerically pure isomers by silica-gel column
chromatography. These protecting groups can be removed
from dinucleotides 2 and from chimeric oligonucleotides
13–20 in a stereospecific manner, with inversion of configu-
ration at the internucleotide phosphorothioate center, by
treatment of the LCAA CPG-bound oligonucleotides with
4-nitrobenzyloximate ions, before standard deprotection
procedures. The partially protected phosphotriesters
SLOW-2 are precursors for the SP-phosphorothioate dies-
ters, whereas FAST-2 serve as precursors of RP internucleo-
tide phosphorothioate diesters.
Table 1. Chimeric oligonucleotides obtained by the phosphoramid-
ite method with incorporated dimeric building blocks.
Chimeric oligonucleotides with
the specified modifications
Absolute MALDI TOF[b]
conf.
13 5Ј-TPSCTTTT-3Ј[a]
13 5Ј-TPSCTT TT-3Ј
14 5Ј-TCTTTTTPSC-3Ј
15 5Ј-TPSCTTTTTPSC-3Ј
16 5Ј-TPSCTCCCAGCGTGCGCCAT-3Ј
17 5Ј-TPSCTCCCAGCGTGCGCCAT-3Ј
18 5Ј- TGACGTPSCATTTTTGACGTCA-3Ј SP
19 5Ј- TGACGTPSCATTTTTGACGTCA-3Ј RP,SP
20 5Ј-TPSCTCCCAGCGTGCGCCATPSC-3Ј SP
RP
SP
SP
SP
RP,SP
SP
(1763) 1762
(1763) 1762
(2373) 2373
(2373) 2373
(5424) 5425
(5424) 5424
(6414) 6413[d]
(6111) 6111
(5729) 5725
[c]
Experimental Section
[a] TPSC-dinucleoside (3Ј,5Ј)-phosphorothioate. [b] Theoretical
molecular masses are given in parentheses. [c] Obtained according
to the standard phosphoramidite procedure with a single sulfuri-
zation step. [d] Contains a DMT group.
General Remarks: THF was distilled from LiAlH4 directly into the
reaction vessel. NEt3 was distilled from CaH2, and stored under
argon over CaH2 pellets. Dioxane was refluxed with sodium wire
for 3–5 h. 1-Hydroxybenzotriazole was purchased from Fluka
(98%, 13% water content). The protected nucleosides were pur-
chased from JBL, Scientific Inc., or Promega, or were prepared
using standard procedures. HPTLC plates (Silica gel 60F254, pre-
coated for nano-TLC) were purchased from Merck. Glass, gas-tight
syringes (Hamilton), reaction vessels, and magnetic stirrers were
dried overnight under high vacuum.
Since incorporation of the diastereomerically pure phos-
phorothioate at the terminal 3Ј-position of the chimeric oli-
gonucleotide is of special interest due to effective protection
against exonuclease degradation,[7,8] we prepared a CPG
support functionalized with diastereomerically pure
(Ͼ96%) dinucleoside (3Ј,5Ј)-O-arylphosphorothioate 2b
linked to the support through a standard succinyl linker.[4]
The use of (RP)-2b and (SP)-2b allowed the preparation of
supports with a loading capacity of 28.6 and 20.1 μmolg–1,
respectively. These supports were used for synthesis of the
corresponding chimeric oligonucleotides (14, 15, and 20;
Table 1).
Synthesis of 4-Nitrophenyl Phosphorothiodichloridate (3): This com-
pound was prepared as described previously.[26] Yield: 62%. 1H
NMR (CDCl3): δ = 7.51 (dd, JH,H = 7.3, JH,H = 2.3 Hz) 8.32 (dd,
JH,H = 9.3, JH,H = 1.0 Hz) ppm. 31P NMR (CDCl3): δ = 52.25 ppm.
EI-MS: m/z = 271.3, 273.1. M.p. 51–52 °C (ref.[25] 52 °C).
Synthesis of 4-Nitrophenyl O,O-Bis(1-hydroxybenzotriazolyl)phos-
phorothioate (5): 1-Hydroxybenzotriazole hydrate (98% purity,
13% water content, Fluka) was dried by co-evaporation with pyri-
dine (three times), followed by high vacuum drying over P2O5 (16
The enhanced stability of the chimeric oligonucleotides
14 and 15 towards exonucleases was confirmed in experi- hours). Anhydrous 1-hydroxybenzotriazole (1.2 mmol) was dis-
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The P-Stereocontrolled Synthesis of PO/PS-Chimeric Oligonucleotides
FULL PAPER
solved in pyridine (8 mL) and kept overnight with activated 4 Å
molecular sieves (beads 8–12 mesh, Aldrich 4 Å, activated at 220 °C
for 6 h). A solution of 4-nitrophenyl phosphorothiodichloridate
(0.6 mmol) in dioxane (2 mL) was then added dropwise to this solu-
tion and the reaction mixture was stirred for 1 h at room tempera-
ture. The phosphorylating agent 5 (31P NMR: δ = 66.13 ppm; 95%
purity) was used directly for condensation reactions.
stirred at room temperature for 12–22 h, and then evaporated under
reduced pressure. The reaction progress was monitored by HP TLC
(20% MeOH in CHCl3) and 31P NMR spectroscopy. The reaction
was quenched by dilution of the reaction mixture with 500 μL of a
1:1 (v/v) solution of CH3CN/0.1 m TEAB buffer (pH 7). 31P NMR
yield Ͼ 98%. Crude products 10a were analyzed by MALDI MS:
m/z = 863 [M + H+] (calcd: 863). RP HPLC (LC Talk, LDC Ana-
lytical) purification of 10a was carried out: column ECONOSIL
C18 5μ, Buffer A: 0.1 m TEAB, pH 7; Buffer B: 40% MeCN in
buffer A; flow: 1 mLmin–1, linear gradient: 85% A (0.1 m TEAB)
to 100% B over 20 min. (FAST-10a: Rt = 19.13 min; SLOW-10a:
Rt = 19.33 min). Pure fractions of phosphodiesters 10a (DMT-on)
were analyzed by MALDI TOF m/z = 863 [M + H+] (for experi-
ments carried out in H2O and [18O]H2O) ppm. 31P NMR (of reac-
tion mixture): FAST-10a: δ = 57.5 ppm; SLOW-10a: δ = 57.21 ppm.
Synthesis of Dinucleoside (3Ј,5Ј)-Phosphorothioates (2): Nucleosides
were dried by co-evaporation with pyridine (twice) and were kept
under vacuum overnight. 5Ј-O-DMT-thymidine (6; 0.272 g,
0.5 mmol) was dissolved in 2 mL of dioxane and added to the phos-
phorylating agent (prepared as described above). The reaction mix-
ture was stirred at room temperature for 1 h, and reaction progress
was monitored by HP TLC [CHCl3/MeOH, 9:1 (v/v)]. The reaction
mixture containing ester 8 was added to thymidine (2a) or N-ben-
zoyl deoxycytidine (2b) dissolved in 2 mL of pyridine (7,
0.55 mmol) with N-methylimidazole (0.4 g, 180 μL, 5 equiv.). The
resulting mixture was stirred at room temp. for 1.5 hours, then
quenched with NEt3 (0.2 mL), concentrated to 1/4 volume, diluted
with CH2Cl2, and washed with brine (twice) and water. The aque-
ous layers were additionally extracted with chloroform. Combined
organic fractions were dried with MgSO4 and concentrated. Prelim-
inary purification involved a silica-gel chromatography (Kieselgel
60, 230–400 mesh, eluent: chloroform/methanol). Subsequent col-
umn chromatography resulted in diastereomerically pure products.
First: silica gel 60-230-400 mesh; eluent: chloroform/ethanol
0Ǟ5%. Second: silica gel 60 H; eluent: ethyl acetate/methanol
(0Ǟ10% MeOH).
Enzymatic Digestion: Each diastereomer 10 (a and b) (1 mg) was
detritylated, and the fully deprotected dinucleotide 12 (a and b) was
purified by HPLC (LC Talk, LDC Analytical); column ECONO-
SIL C18 5μ, Buffer A: 0.1 m TEAB, pH 7; Buffer B: 40% MeCN
in buffer A; flow: 1 mLmin–1, linear gradient: 100% A (0.1 m
TEAB) to 50% B over 20 min. FAST-12a: Rt = 15.33 min; SLOW-
12a: Rt = 15.21 min (yield 82%). FAST-12b: Rt = 17.03 min;
SLOW-12b: Rt = 17.69 min (yield 84%).
a) 0.1 OD of each diastereomer 12 (a and b) was treated with 1 μg
of nuclease P1 (from Penicillium citricum, Boehringer GmbH, no.
10432620-12) in 1 μL of buffer 10X (100 mm tris-HCl, pH 7.2;
1.0 mm ZnCl2) and 8 μL of H2O at room temperature. Aliquots
(2 μL) were taken after 1 h, 2 h, and 4 h and analyzed by HPLC
FAST-2a: HP-TLC [chloroform/ethanol, 9:1(v/v)] Rf = 0.36. 1H with the same gradient as described below.
NMR (CDCl3): δ = 1.82 (C5-CH3b), 2.18 (H2Јb), 2.37 (H2ЈЈb),
2.46 (H2Јa), 2.65 (H2ЈЈa), 2.93 (3Ј-OH), 3.47 (H5Јa), 4.13 (H4Јb),
4.38 (H4Јa), 4.40, 4.42 (H5Ј,H5ЈЈb), 4.52 (H3Јb), 5.53 (H3Јa), 6.33
(H1Јb), 6.45 (H1Јa), 6.82 (arom. C2-H), 6.84 (arom., C3-H), 7.31
(C6-Hb), 7.56 (C6-Ha) ppm. 31P NMR (CDCl3): δ = 63.43 ppm.
Yield: 23%.
b) 0.1 OD of each diastereomer 12 (a and b) was treated with 0.5 μg
of phosphodiesterase Capd (Crotalus adamanteus venom, Amer-
sham Life Science, no. 108044) in 1 μL of buffer 10X (25 mm Tris-
HCl pH 8.5; 5 mm MgCl2) and 8.5 μL of H2O at 37 °C. The reac-
tion times were 1 h, 2 h, 4 h, and 12 h. Products were analyzed by
HPLC.
1
SLOW-2a: HP-TLC [chloroform/ethanol, 9:1 (v/v)] Rf = 0.30. H
Derivatization of Long-Chain Alkylamine CPG Support with Dinu-
cleoside Phosphorothioate: Succinylated LCAA-CPG (200 mg,
CPG Product no. CPG 00500A), partially protected dinucleoside
(3Ј,5Ј)-phosphorothioate (55 mg, SLOW-2), and DMAP (12 mg)
were dried overnight at high vacuum, followed by addition of 1-(3-
dimethylaminopropyl) ethylcarbodiimide (DEC, 92 mg, 0.05 mm),
pyridine (2 mL), and triethylamine (60 μL). This reaction mixture
NMR (CDCl3): δ = 1.49 (d, C5-CH3a), 1.80 (d, C5-CH3b), 2.25
(m, H2Јb), 2.39 (m, H2ЈЈa), 2.45 (m, H2ЈЈb), 2.72 (dd, H2ЈЈa), 3.35
(dd, H5Јb), 3.46 (dd, H5Јa), 3.53 (s, 3Ј-OH) 4.28 (m, H4Јb), 4.33
(m, H4Јa), 4.45 (dd, H5Ј,H5ЈЈb,a), 4.60 (m, H3Јb), 5.38 (dd, H3Јa),
6.30 (t, H1Јb), 6.38 (dd, H1Јa), 6.82 (m, arom. C2-H), 6.85 (m,
arom., C3-H) ppm. 31P NMR (CDCl3): δ = 63.65 ppm. Yield: 21%.
FAST-2b: HP-TLC [chloroform/ethanol, 9:1 (v/v)] Rf = 0.27 was shaken at 28 °C for 24 h. After the reaction was complete (trityl
[EtOAc/MeOH, 95:5 (v/v)]. 1H NMR (CDCl3): δ = 1.49 (C5-
CH3b), 2.19 (H2Јb), 2.72 (H2ЈЈb), 2.447 (H2Јa), 2.64 (H2ЈЈa), 2.93
assay), the CPG was filtered off, washed with small portions of
MeOH, CH2Cl2 and MeCN, and dried under vacuum. The func-
(3Ј-OH), 3.50 (H5Јa), 4.13 (H4Јb), 4.24 (H4Ј), 4.39 (H5Ј,H5ЈЈb), tionalized support was suspended in acetic anhydride capping rea-
4.48 (H3Јb), 5.35 (H3Јa), 6.28 (H1Јb), 6.40 (H1Јa), 6.82 (arom., C2- gent, stirred for 2 h at room temperature, filtered off, and washed
H), 6.84 (arom., C3-H), 7.31 (C6-Hb), 7.56 (C6-Ha) ppm. 31P
with CH2Cl2. The support was dried at high vacuum and stored
NMR (CDCl3): δ = 60.98 ppm. MS FAB: m/z = 1073.8 [M – H], at –20 °C. The nucleotide loading (28.6 μmolg–1) was determined
1075.9 [M + H] (calcd.: 1074.3). Yield: 27%.
SLOW-2b: HP-TLC [chloroform/ethanol, 9:1 (v/v)] Rf = 0.20. H
by trityl analysis. Yield: 195 mg (95%).
1
Synthesis of Chimeric Oligonucleotides. a) Synthesis of Chimeric
NMR (CDCl3): δ = 1.52 (d, C5-CH3a), 2.28 (m, H2Јb), 2.36 (m, Oligonucleotides with 3Ј-End Modification: Solid support derivat-
H2ЈЈa), 2.69 (dd, H2ЈЈb), 2.82 (m, H2ЈЈb), 3.33 (m, H5Јa), 3.47(dd, ized with 2b as described above was placed in a 1-μmol column
H5Јa), 4.22 (m, H4Јa), 4.31 (m, H4Јb), 4.51 (m, H5Ј, H5ЈЈb), 4.59 and attached to the DNA synthesizer. Subsequent couplings were
(m, H3Јb), 5.28 (m, H3Јa), 6.28 (m, H1Јb), 6.35 (m, H1Јa). 31P routinely performed with commercially available nucleoside 3Ј-
NMR (CDCl3): δ = 61.70 ppm. Yield: 25%.
phosphoramidites according to a standard protocol for the phos-
phoramidite method of oligonucleotide synthesis. Standard RP
HPLC isolation provided pure 14.
Stereochemistry of Removal of the 4-Nitrophenyl Protecting Group:
Each diastereomer of dinucleotide 2a (0.066 m, 0.033 mmol) was
dissolved in dioxane/water (1:2 v/v; 0.5 mL), and a solution of
b) Synthesis of Chimeric Oligonucleotides with 3Ј- and 5Ј-Internucle-
otide Phosphorothioate Modification: Solid support derivatized with
2b as described above was placed in a 1-μmol column and attached
1,1,3,3-tetramethylguanidinium
oximate
(0.6 m,
0.3 mmol,
0.05 mL) was added. The resulting homogeneous solution was
Eur. J. Org. Chem. 2005, 2924–2930
www.eurjoc.org
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2929

W. J. Stec et al.
FULL PAPER
[2]
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DNA Vectors (Ed.: E. Wickstrom), Marcel Dekker, Inc., New
York, 1998.
to the DNA synthesizer. For the synthesis of compounds 15 and
20 the subsequent couplings were routinely performed with com-
mercially available nucleoside 3Ј-phosphoramidites according to a
standard protocol for the phosphoramidite method of oligonucleo-
tide synthesis. 5Ј-O-(4,4Ј-Dimethoxytrityl)-2Ј-thymidyl (3Ј,5Ј)-3Ј-O-
[(4-nitrophenyl)(diisopropylamino)phosphoramidityl]-2Ј-N4-ben-
zoyl-2Ј-deoxycytidine) phosphorothioate (9b, vide infra) was dried
overnight, dissolved in MeCN (0.08 mm), and used as a reagent for
coupling at the synthesizer. The coupling time was 10 min; no other
steps were modified or changed. Average yield of coupling (calcu-
lated from the trityl assay): 98%.
[3]
[4]
E. Wickstrom, J. Biochem. Biophys. Methods 1986, 13, 97–102.
P. Guga, M. Koziołkiewicz, A. Okruszek, W. J. Stec, in Applied
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722.
M. J. O’Donnel, N. Herbert, L. W. McLaughlin, Oligonucleo-
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[5]
[6]
c) Synthesis of Chimeric Oligonucleotides with “Internal” or 5Ј-Ter-
minal Modification: Diastereomerically enriched isomer SLOW-2b
(RP/SP: 95:5; 20 mg) was co-evaporated with dry MeCN (three
times), dried overnight under high vacuum, and dissolved in dry
MeCN (300 μL). 2-Cyanoethyl tetraisopropylphosphordiamidite
(6.5 μL) was added to this solution, followed by a 0.5 m solution
of 5-ethylthiotetrazol (25 μL). The reaction mixture was stirred for
15 min and used without further purification [31P NMR: δ =
149.26, 148.73, 60.13 ppm for (RP)-9b] for the coupling reaction
on a solid support. A 1-μmol column was used for preparation of
compounds 16–19. Partially synthesized oligonucleotides 18 and 10
were detached from the DNA synthesizer after completion of the
unmodified sequences by the phosphoramidite method, and man-
ual coupling with 9b was performed; reaction time: 10 min. The
consecutive coupling with five nucleosides in the case of com-
pounds 16–19 was performed automatically by phosphoramidite
chemistry after attachment of the column to the DNA synthesizer.
After the synthesis of compounds 16–19 was accomplished, the
DMT group was removed, and the column was detached from the
DNA synthesizer. Average yield of coupling (calculated from the
trityl assay): 98%.
[7]
M. Koziołkiewicz, M. Wojcik, A. Kobylanska, B. Karwowski,
B. Re˛bowska, P. Guga, W. J. Stec, Antisense Nucleic Acid Drug
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M. Gilar, A. Belenky, Y. Budman, D. L. Smisek, A. S. Cohen,
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Chem. Commun. 1983, 591–593.
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rel, W. J. Van Zoest, J. H. Van Boom, Nucleic Acids Res. 1984,
12, 9095–9110.
FAST and SLOW denote diastereomers that elute from a silica
gel column in chloroform/ethanol solvent as first and second
components, respectively.
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d) Deprotection: The solid support-bound oligonucleotides were
treated with a 0.4 m solution of syn-4-nitrobenzaldoxime and
1,1,3,3-tetramethylguanidine in dioxane/water (1 mL) for 1 h, fol-
lowed by treatment with NH4OH at 55 °C for 16 h. Purification of
the oligonucleotides was performed by HPLC (RP-C18 SUPEL-
COSIL, 1 mLmin–1, gradient 2–20% MeCN, 0.1 m TEAB buffer,
pH 7.5). Their purity was additionally confirmed by gel electropho-
resis. Results of MS analysis are included in Table 1.
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[19] Preliminary studies, not described here in detail, with the 2a
isomers allowed the optimization of reaction conditions.
[20] C. B. Reese, Tetrahedron 2002, 58, 8893–8920.
[21] Nucleic Acids in Chemistry and Biology (Eds.: G. M. Blackburn,
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cleic Acids Res. 1991, 19, 5883–5888.
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Acknowledgments
The authors are indebted to The State Committee for Scientific
Research (KBN), the European Commission within the 5th Frame-
work Program (Contract ICA1-CT-2000-70021 Centres of Excel-
lence) and Genta Inc., for financial assistance.
[27] R. M. Tarkka, E. Buncel, J. Am. Chem. Soc. 1995, 117, 1503–
1507.
[28] M. Wójcik, M. Cies´lak, W. J. Stec, J. W. Goding, M. Koziołkie-
wicz, manuscript in preparation.
[1] Applied Antisense Oligonucleotide Technology (Eds.: C. A.
Stein, A. Krieg), Wiley-Liss, New York, 1998.
Received: December 23, 2004
2930
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www.eurjoc.org
Eur. J. Org. Chem. 2005, 2924–2930