Synthesis of short oligodeoxyribonucleotides by phosphotriester chemistry on a precipitative tetrapodal support

Article abstract of DOI:10.1002/ejoc.201301352

Short oligodeoxyribonucleotides have been assembled from appropriately protected nucleoside 3′-(benzotriazol-1-yl 2-chlorophenyl phosphate) derivatives on hundred-milligram scales on a soluble tetrakis-O-(4- azidomethylphenyl)pentaerythritol support beari

Full text of DOI:10.1002/ejoc.201301352

SHORT COMMUNICATION
DOI: 10.1002/ejoc.201301352
Synthesis of Short Oligodeoxyribonucleotides by Phosphotriester Chemistry on
a Precipitative Tetrapodal Support
Vyacheslav Kungurtsev,[a] Pasi Virta,[a] and Harri Lönnberg*[a]
Keywords: Oligonucleotides / Synthetic methods / Protecting groups / Soluble supports
Short oligodeoxyribonucleotides have been assembled from
appropriately protected nucleoside 3Ј-(benzotriazol-1-yl 2-
chlorophenyl phosphate) derivatives on hundred-milligram
scales on a soluble tetrakis-O-(4-azidomethylphenyl)pentae-
rythritol support bearing four 3Ј-O-(4-pentynoyl)thymidines.
Small-molecule reagents and waste were removed by two
precipitations of the support-bound growing oligonucleo-
ing group and the second after coupling. The building blocks
were obtained by one-step phosphorylation of commercially
available protected nucleosides with bis(benzotriazol-1-yl) 2-
chlorophenyl phosphate. Removal of the phosphate protect-
ing groups from the completed chain with (E)-2-nitrobenzal-
doxime followed by conventional ammonolysis allowed pre-
cipitation of the oligonucleotide as the sodium salt with eth-
tides with methanol, the first after removal of the 5Ј-protect- anol.
phosphate) building blocks appears particularly interesting.
Introduction
We have now applied this method to the preparation of
short oligodeoxyribonucleotides on our recently reported
soluble support,[12] tetrakis-O-(4-{4-[3-(thymidin-3Ј-yl)-3-
oxopropyl]-1,2,3-triazol-1-ylmethyl}phenyl)pentaerythritol
(1; Figure 1). The usefulness of this support stems from its
quantitative precipitation with MeOH. Accordingly, each
coupling cycle contains only two precipitations, one after
removal of the 5Ј-protecting group and the second after the
coupling. All the small-molecule compounds remain in
solution, while the support precipitates quantitatively. By
ammonolytic release, nearly homogeneous heterotrimers
were obtained in 70% isolated yield and a pentamer in 55%
yield. In spite of the simplicity of the synthesis, the yields
favorably compete with the previously described ap-
proaches. Interestingly, a related branched core structure
has previously been used for immobilization of prefabri-
cated dimers and trimers as building blocks of nanoscale
objects.[23,24]
Oligonucleotides are usually prepared on a solid support
by the so-called phosphoramidite strategy, which is based
on formation of internucleosidic P–O bonds at the PIII level,
followed by oxidation of the resulting phosphite triester to
the phosphate triester immediately after each coupling
step.[1–3] Virtually the same chemistry is used in lab-scale
synthesis[4] and in large-scale production.[5] For in-house
preparation of short oligonucleotides on hundred-milligram
scales, which on a lab-scale synthesizer requires dozens of
repetitions, the application of a similar strategy in solution
on a soluble support has been attempted.[6–15] The need for
oxidation after each coupling step unfortunately increases
the number of steps in the coupling cycle. Whereas this is
not a problem with solid-supported synthesis, for which
waste and excess monomeric blocks and activators can be
removed by simple washing, it considerably complicates the
synthesis in solution. In fact, the “outdated” phosphotri-
ester strategy,[16,17] based on coupling of PV blocks and
hence avoiding the oxidation step, might offer a more
straightforward procedure for synthesis in solution. This
chemistry has previously been applied to the synthesis of
oligonucleotides on a soluble poly(ethylene glycol) sup-
port[18,19] by using either 1-hydroxybenzotriazole[20–22] or 1-
(mesitylsulfonyl)-3-nitro-1H-1,2,4-triazole (MSNT)[18] acti-
vation. Among these methods, the approach based on cou-
pling of prefabricated 3Ј-(benzotriazol-1-yl 2-chlorophenyl
Figure 1. Structure of the soluble support and the building-blocks
employed.
[a] Department of Chemistry, University of Turku,
20014 Turku, Finland
E-mail: harlon@utu.fi
Results and Discussion
The tetravalent nucleoside cluster 1 has been shown to
be a promising soluble support for the synthesis of oligonu-
http://www.utu.fi/en/units/sci/units/chemistry/research/
orgchem/bioorganic/Pages/home.aspx
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201301352.
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Synthesis of Short Oligodeoxyribonucleotides
cleotides in solution.[12] It has radial symmetric structure an oil. The residue was dissolved in a minimal amount of
that allows the homogeneity of the products to be verified the mixture of MeOH and dichloromethane and precipi-
after each coupling by NMR spectroscopic analysis, in ad- tated by diluting with MeOH. As seen from Figure 3, the
dition to HPLC and mass spectrometry. The structure itself detritylated support precipitated virtually quantitatively.
is very stable, allowing storage at ambient temperature for The coupling and detritylation steps described above were
a long time (no sign of degradation was observed after then repeated to obtain support-bound trimers 18–21, still
8 months). Quantitative precipitation of the support-bound bearing the phosphate and base moiety protecting groups
oligonucleotide cluster with MeOH allows facile removal of (see Table 1 for ESI-MS data). The 2-chlorophenyl protect-
monomeric reagents and waste. Although the application ing groups were then removed with the tetramethylguanid-
of phosphoramidite chemistry on this support allowed the ium salt of (E)-2-nitrobenzaldoxime in aqueous dioxane,[25]
preparation of a heteropentamer in 45% isolated yield, the and the base-moiety protecting groups were removed with
protocol employed still suffered from some minor side reac- 25% aqueous ammonia at 55 °C. The precipitated support
tions, including premature cleavage of the 2-cyanoethyl and was removed by filtration, and the filtrate was concentrated
benzoyl protecting groups and depurination of 2Ј-deoxyad- to dryness under reduced pressure. The residue was dis-
enosine upon acidolytic removal of the 5Ј-O-(4,4Ј-dimeth- solved in aq. MeCN, aq. NaOAc was added, and the oligo-
oxytrityl) group. For these reasons, and to avoid the oxi- nucleotide was precipitated by diluting with EtOH. Trimers
dation step, the feasibility of applying phosphotriester
chemistry on this support by using 3Ј-(benzotriazol-1-yl 2-
chlorophenyl phosphotriesters) as building blocks has been
studied. The results are described herein.
Commercially available appropriately protected nucleo-
sides, 5Ј-O-(4,4Ј-dimethoxytrityl)-N6-benzoyl-2Ј-deoxyad-
enosine, N4-benzoyl-2Ј-deoxycytidine, N2-isobutyryl-2Ј-de-
oxyguanosine and thymidine, were converted into their 3Ј-
(benzotriazol-1-yl 2-chlorophenyl phosphates) (2–5; Fig-
ure 1) by treatment with bis(benzotriazol-1-yl)(2-chloro-
phenyl)phosphate (1.1 equiv.) in 1,4-dioxane.[22] The
stock solution of the latter reagent (0.2 molL–1) was ob-
tained by allowing commercial 2-chlorophenyl phosphoro-
dichloridate to react with 1-hydroxybenzotriazole (2 equiv.)
in dioxane in the presence of pyridine (2 equiv.). The re-
sulting pyridinium chloride precipitate was removed by fil-
tration, and the filtrate was divided into small portions and
stored at –20 °C. As indicated earlier,[22] the stoichiometry
upon preparation of 2–5 is of crucial importance: excess
nucleoside results in formation of a 3Ј,3Ј-dinucleosidephos-
phate, whereas excess phosphorylating agent may lead to
phosphorylation of the 5Ј-OH of the supported nucleotide.
Of these potential side reactions, only dimer formation was
sometimes detected. Fortunately, the presence of a 3Ј,3Ј-di-
mer did not markedly affect subsequent coupling. To
achieve sufficient accuracy, a gravimetric method was ap-
plied to the preparation and dosage of the stock solution.
Four trimers and one pentamer containing all four 2Ј-
deoxynucleosides were prepared (Scheme 1). Support 1,
bearing four thymidine residues, was prepared as described
previously.[12] To achieve the first coupling, benzotriazole-
activated building block (2–5; 8 equiv.; i.e., 2 equiv. per sup-
port-bound thymidine), and 1-methylimidazole (10 equiv.)
were added in dioxane onto the dried support under nitro-
gen. After 2 h of stirring, the support was precipitated by
diluting the reaction mixture with 10 volumes of MeOH.
HPLC analysis of the precipitate and the liquid phase veri-
fied completeness of the precipitation. An illustrative exam-
ple is given in Figure 2. The 5Ј-O-DMTr protection was
then removed with HCl in a 1:1 (v/v) mixture of MeOH/
dichloromethane. Upon completion of the removal, the
2-nitrobenzaldoxime tetramethylguanidinium salt in aq. dioxane;
mixture was neutralized with pyridine and concentrated to 2. aq. NH3; (v) aq. MeCN, aq. NaOAc, EtOH, –20 °C.
Scheme 1. Synthesis of oligonucleotides. Reagents and conditions:
(i) 2–5, N-MeIm, dioxane, under N2; (ii) precipitation with MeOH;
(iii) 1. HCl in MeOH/CH2Cl2 (1:1); 2. Py, concentration; (iv) 1. (E)-
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V. Kungurtsev, P. Virta, H. Lönnberg
SHORT COMMUNICATION
22–25 were obtained in 70% isolated yield (determined by Table 1. Negative-ion ESI-MS analysis of the support-bound pro-
tected (18–21 and 26) and released deprotected (22–25 and 27) oli-
gonucleotides.
UV absorption in comparison to support 1). The penta-
meric oligonucleotide 27 was obtained similarly in 55%
Compound Calculated mass
Observed mass
yield. The coupling efficiency and precipitation properties
of the support remained unchanged upon increasing the
chain length. However, we have not attempted to assemble 19
oligonucleotides longer than a pentamer on this support.
The identities of the products were verified by ESI-MS
analysis (Table 1), and their homogeneity was established
by HPLC analysis (Figure 4).
18
1754.9 [(M – 3 H)/3]3–
1754.3 [(M – 3 H)/3]3–
2056.1 [(M – 3 H)/3]3–
1992.1 [(M – 3 H)/3]3–
2008.1 [(M – 3 H)/3]3–
3375.1 [(M – 3 H)/3]3–
849.16 [M – H]–
2056.6 [(M – 3 H)/3]3–
1992.5 [(M – 3 H)/3]3–
2008.6 [(M – 3 H)/3]3–
3376.0 [(M – 3 H)/3]3–
849.18 [M – H]–
20
21
26
22
23
24
25
27
867.21 [M – H]–
867.19 [M – H]–
819.19 [M – H]–
819.20 [M – H]–
899.20 [M – H]–
899.17 [M – H]–
730.14 [(M – 2 H)/2]2–
730.12 [(M – 2 H)/2]2–
Figure 2. (A) HPLC traces for the precipitated support bearing
phosphate-protected 3Ј-TpdCBz-5Ј dimer as a 5Ј-O-DMTr ether (8),
and (B) HPLC traces of the filtrate after the precipitation. For the
chromatographic conditions, see gradient C in the Exp Sect.
Figure 4. HPLC traces of the trimeric (A) and pentameric (B) oli-
gonucleotides prepared. For the chromatographic conditions, see
gradient B in the Exp. Sect.
With the corresponding 1-hydroxybenzotriazole-activated
block 4, this was not necessary.
Conclusions
A convenient phosphotriester approach that allows the
preparation of short oligodeoxyribonucleotides on hun-
Figure 3. (A) HPLC traces for the precipitated support bearing dred-milligram scales in solution has been described. The
phosphate-protected 3Ј-TpdCBz-5Ј dimer having the 5Ј-O-detrityl-
ated (12), and (B) HPLC traces of the filtrate after precipitation.
of appropriately protected nucleoside 3Ј-(benzotriazol-1-yl
For the chromatographic conditions, see gradient A in the Exp.
method is based on 1-methylimidazole-promoted assembly
2-chlorophenyl phosphates)[22] on a soluble tetrakis-O-(4-
Sect.
azidomethylphenyl)pentaerythritol support bearing four
In addition to 1-hydroxybenzotriazole-activated building thymidines.[12] All small molecules were removed after each
blocks 2–5, more conventional coupling of appropriately coupling and 5Ј-O-detritylation by precipitating the support
protected 2Ј-deoxyribonucleoside 3Ј-(2-chlorophenyl phos- with MeOH. On using 2 equiv. of building blocks per 5Ј-
phates) was attempted by using (mesit-2-yl)sulfonyl chloride OH, a pentamer containing all four nucleosides was ob-
for activation and 1-methylimidazole as a nucleophilic cata- tained in 55% isolated yield. The advantages of this ap-
lyst.[17] 3Ј-TpdC-5Ј and 3Ј-TpdA-5Ј dimers were obtained proach compared to the phosphoramidite method on the
in a still reasonable 85% coupling yield, but upon coupling same support[12] include avoiding the oxidation step and
of the 2Ј-deoxyguanosine block, strong coloration of the preventing premature cleavage of phosphate and base-moi-
reaction mixture was observed (data not shown). In fact, it ety protecting groups. Although depurination is not entirely
has been reported that when this kind of approach is used, avoided during acid-catalyzed removal of the 5Ј-O-DMTr
the O6 atom of the guanine base should be protected.[3] group, the coupling efficiency remains high, enabling iso-
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Synthesis of Short Oligodeoxyribonucleotides
night, collected by centrifugation and dried to give the product
(0.173 g, 90%) as a white solid. The precipitate and supernatant
were analyzed by HPLC and ESI-HRMS.
lated yields that compare favorably with previously de-
scribed approaches.
Assembly of the Support-Bound Oligonucleotides: By using the
methodology described above, the following support-bound oligo-
nucleotides were assembled. AAT cluster 19 was obtained in 82%
yield (0.215 g) from 1 (0.100 g), corresponding to 91% average yield
of a coupling/detritylation cycle. CCT cluster 20 was prepared in
85% yield (0.102 g) from 1 (0.048 g); GGT cluster 21 in 80% yield
(0.352 g) from 1 (0.150 g), and TTT cluster 18 in 82% yield
(0.345 g) from 1 (0.200 g). CGCAT cluster 26 was obtained in 61%
overall yield (0.440 g) starting from support 1 (0.150 g), corre-
sponding to 88% average yield of each coupling/detritylation cycle.
Experimental Section
General: NMR spectra were recorded with a 500 MHz spectrome-
ter. Chemical shifts are given in ppm and referenced relative to
the residual solvent signals.[26] RP HPLC conditions: (A) gradient
elution from 25% MeCN in 0.1 molL–1 Et3NHOAc to 100%
MeCN in 25 min, then continued with MeCN; (B) gradient elution
from 2.5% MeCN in 0.1 molL–1 Et3NHOAc to 50% MeCN in
0.1 molL–1 Et3NHOAc in 25 min, then continued with 50% MeCN
in 0.1 molL–1 Et3NHOAc; (C) gradient elution from 50% MeCN
in 0.1 molL–1 Et3NHOAc to 100% MeCN in 25 min, then contin-
ued with MeCN. An analytical C-18 RP column (250ϫ4.6 mm,
5 μm; flow rate 1.0 mLmin–1; λ = 260 nm) was used. Reactions
were monitored by TLC (Merck, silica gel 60 F254), using short-
wavelength UV or charring with 10% aq. H2SO4 for detection (sys-
tem A: 10% MeOH in CH2Cl2; system B: 8% MeOH in CH2Cl2).
Mass spectra were recorded with a Bruker Daltonics MicrOTOF-
Q spectrometer using ESI ionization. Tetrakis(4-{4-[3-(thymidin-
3Ј-O-yl)-3-oxoprop-1-yl]-1H-1,2,3-triazol-1-yl}methylphenoxy-
methyl)methane (1) was synthesized as reported previously.[12]
General Procedure for Oligonucleotide Isolation: Support-bound tri-
mer 19 (0.035 mmol, 0.215 g) was mixed with the tetramethylguani-
dinium salt of (E)-2-nitrobenzaldoxime (0.3 molL–1, dioxane/water,
9:1, v/v, 8 mL), stirred for 2 h, and then volatiles were removed
under reduced pressure. The residue was diluted with 25% aq. am-
monia (8 mL) and stirred at 55 °C for 4 h. The precipitated support
was removed by filtration, and the filtrate was concentrated and
dried in vacuo to give a yellow oil.
The oily residue was dissolved in a mixture of MeCN and water
(3:7 v/v, 0.5 mL) followed by aqueous sodium acetate (3 molL–1,
1.2 mL). The resulting mixture was diluted with EtOH (16 mL),
vortexed for 20 s, and kept at –20 °C for 1.5 h. The precipitate
formed was collected by centrifugation, washed with EtOH
(10 mL) and dried to give 108 mg of a yellow powder correspond-
ing to 90% isolated yield and 73% overall yield. The precipitate
was dissolved in water (8 mL) and analyzed by UV, HPLC and
ESI-HRMS.
Bis(benzotriazol-1-yl) 2-Chlorophenyl Phosphate: A solution of 2-
chlorophenyl phosphorodichloridate (7.6 mmol, 1.88 g) in anhy-
drous dioxane (5.75 mL) was added in one portion to a suspension
of 1-hydroxybenzotriazole (15.2 mmol, 2.06 g; dried in vacuo over
P2O5 at 55 °C for 3 d) and pyridine (15 mmol, 1.2 mL) in anhy-
drous dioxane (30 mL). The reaction mixture was stirred for 2 h,
and the precipitate was filtered off under anhydrous conditions to
give a stock solution of bis(benzotriazol-1-yl) 2-chlorophenyl phos-
phate (0.2 molL–1) as a clear colorless liquid. The solution (ρ =
1.057 gmL–1) could be stored for several weeks at –20 °C.
Support-bound trimers 18, 20, 21 and pentamer 26 were isolated
as analytical samples on 1–3 μmol scale. The overall yields starting
from 1 were 69% for 24, 73% for 23, 69% for 25, 70% for 22, and
55% for 27.
General Procedure for the Coupling of 1-Hydroxybenzotriazole-Acti-
vated Phosphotriester Building Blocks: N6-Benzoyl-2Ј-deoxy-5Ј-O-
(4,4Ј-dimethoxytrityl)adenosine (0.41 mmol; 0.27 g) was dried by
coevaporation with anhydrous pyridine (3ϫ 5 mL) and concen-
trated to a small volume followed by addition of the stock solution
of bis(benzotriazol-1-yl) 2-chlorophenyl phosphate in dioxane
(0.41 mmol, 0.2 molL–1, 2.05 mL), giving N6-benzoyl-2Ј-deoxy-5Ј-
O-(4,4Ј-dimethoxytrityl)adenosine 3Ј-(benzotrizol-1-yl 2-chlo-
rophenyl phosphate) (3) in dioxane. The formation of a product
with zero mobility on TLC (system B) indicated that the reaction
was complete. In a separate vessel, support 1 (0.051 mmol, 0.10 g)
was dried by coevaporation with anhydrous pyridine (3ϫ 5 mL),
and then 3 in dioxane and 1-methylimidazole (2 mmol, 0.170 mL)
were added under nitrogen. The reaction mixture was stirred for
2 h to obtain the supported dimer 8, transferred to stoppered
50 mL plastic tubes, and MeOH (46 mL) was added. The precipi-
tate formed was kept at –20 °C overnight, isolated by centrifuga-
tion, and dried to give 8 (0.25 g, 93%) as a white solid. The precipi-
tate and supernatant were analyzed by HPLC to verify the com-
pleteness of precipitation.
General Procedure for the Coupling of Phosphodiester Building
Blocks: Support 1 (0.015 mmol, 30 mg) and the Et3NH+ salt of N4-
benzoyl-5Ј-O-(4,4Ј-dimethoxytrityl-2Ј-deoxycytidine)
3Ј-(2-chlo-
rophenyl phosphate) (0.12 mmol, 114 mg) were dissolved in anhy-
drous pyridine (1 mL), and 2-mesitylsulfonyl chloride (0.22 mmol,
48 mg) and 1-methylimidazole (0.62 mmol, 0.050 mL) were added
under nitrogen. The reaction mixture was stirred overnight, and
then MeOH (10 mL) was added. The precipitate formed was kept
at –20 °C overnight, collected by centrifugation, and dried to give
8 (69 mg, 87%) as a white solid. The precipitate and supernatant
were analyzed by HPLC.
Supporting Information (see footnote on the first page of this arti-
cle): 1H NMR spectra for 18, 19, 21, and 26, 13C NMR spectra for
19 and 26, HSQC spectra for 18, 19, 21, and 26, mass spectra for
18–27.
Acknowledgments
Financial support from the FP7 Marie Curie Actions is gratefully
acknowledged.
General Procedure for Detritylation: Support-bound dimer
7
(0.046 mmol, 0.25 g) was dissolved in a mixture of CH2Cl2 and
MeOH (1:1 v/v, 25 mL), and HCl in MeOH (1.25 molL–1, 0.14 mL)
was added portionwise. The reaction was monitored by TLC (sys-
tem A). Once complete, the reaction mixture was neutralized with
pyridine (1 mL), and the liquid was concentrated. The resulting oil
was dissolved in CH2Cl2/MeOH (1:1, 3 mL), and MeOH was
added (47 mL). The precipitate formed was kept at –20 °C over-
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SHORT COMMUNICATION
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Received: September 6, 2013
Published Online: November 6, 2013
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