Solution-phase synthesis of short oligo-2'-deoxyribonucleotides by using clustered nucleosides as a soluble support

Article abstract of DOI:10.1002/ejoc.201300864

5'-O-(4,4'-Dimethoxytrityl)thymidine was attached to a pentaerythrityl- derived core, and the resulting tetravalent nucleoside cluster and the next dendritic generations served as a soluble support for the synthesis of short oligo-2'-deoxyribonucleotides

Full text of DOI:10.1002/ejoc.201300864

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FULL PAPER
DOI: 10.1002/ejoc.201300864
Solution-Phase Synthesis of Short Oligo-2Ј-deoxyribonucleotides by Using
Clustered Nucleosides as a Soluble Support
Vyacheslav Kungurtsev,[a] Jouni Laakkonen,[a] Alejandro Giminez Molina,[a] and
Pasi Virta*[a]
Keywords: Synthetic methods / Template synthesis / Oligonucleotides / Solution-phase synthesis
5Ј-O-(4,4Ј-Dimethoxytrityl)thymidine was attached to
pentaerythrityl-derived core, and the resulting tetravalent
nucleoside cluster and the next dendritic generations served
a
(1.5 equiv. per 5Ј-OH group) of the standard phosphoramidite
building blocks proved efficient, and the products could be
purified by quantitative precipitation from methanol. Am-
as a soluble support for the synthesis of short oligo-2Ј-deoxy- monolysis released nearly homogeneous oligonucleotides
ribonucleotides in solution. Couplings using a small excess
(CCT, GCT, ACT, and AGCCT) in high yields.
short oligonucleotides. The growing oligonucleotides, which
are tetrahedrally branched from a pentaerythritol-derived
core, were found to precipitate virtually quantitatively from
methanol; a feature that could be utilized as a purification
step in the liquid-phase synthesis. Two precipitations are
needed in each coupling cycle when using commercially
available phosphoramidite building blocks: one after the
oxidation step and the second after removal of the 5Ј-O-
(4,4Ј-dimethoxytrityl) protecting group. The atom economy
is good, because the support is small and still bears four
oligonucleotides. The efficiency of coupling is as high as for
previously described liquid-phase methods.[4–14] Advan-
tageously, the small size and radial-symmetric structure of
the support allows easy MS, NMR and HPLC analysis of
the products (and side-products). Structurally related
branched oligonucleotides have recently been described by
the group of Richert,[15,16] but, instead of synthetic applica-
tions, prefabricated oligonucleotide segments (dimers and
trimers) were attached to a tetravalent core structure (5–
10 μmol scale) for nanochemical purposes.
Introduction
Oligonucleotides are currently prepared by phosphor-
amidite chemistry on a solid support both in the labora-
tory[1,2] and on a large scale.[3] Although this will likely re-
main the case for the near future, there seems to be space
for the development of a solution-phase synthesis for spe-
cific purposes. Short oligonucleotides are sometimes needed
in research laboratories in a scale of hundreds of milligrams
for DNA-based material chemistry and for spectroscopic
and physical organic studies on structure, modification and
interaction with protein fragments or small molecular enti-
ties, including metal complexes. Convenient solution-phase
methodology would undoubtedly be useful for the prepara-
tion of such oligonucleotides in-house without any special
equipment. In fact, numerous strategies for the elongation
of oligonucleotides on soluble supports have been devel-
oped. Phosphoramidite,[4] H-phosphonate,[5] and phospho-
triester[6,7] chemistries have been used for the solution-phase
synthesis of oligonucleotides and their monothioate ana-
logues[8–10] on a soluble polyethylene glycol (PEG) sup-
port.[11] Once the coupling is performed, the PEG-sup-
ported oligonucleotides may be isolated by precipitation in
diethyl ether. More recently, oligonucleotides have been at-
tached to an imidazolium tetrafluoroborate,[12] to an ada-
mantyl tag,[13] and to acetylated and methylated β-cyclodex-
trin[14] for the same purpose, facilitating purification of oli-
gonucleotides by precipitation, extractive workup, and flash
chromatography, respectively.
Results and Discussion
Synthesis of Tetravalent Nucleoside Cluster 9
Pentaerythrityl-derived tetraazide 5 meets the require-
ments for a good branching unit (Scheme 1); it is a compact
and radial-symmetric structure, its synthesis is straightfor-
ward, and clustering of the first nucleoside on this
branching unit may be efficiently carried out. Additionally,
the residue of the core structure (17 see Scheme 2 below)
may be isolated from the deprotected oligonucleotides by
simple filtration, because it precipitates in aqueous ammo-
We now report a novel soluble-support-based strategy
with properties that are highly useful for the synthesis of
[a] Department of Chemistry, University of Turku,
20014 Turku, Finland
E-mail: pamavi@utu.fi
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201300864.
nia.
Tetrakis{[4-(chloromethyl)phenoxy]methyl}methane
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Scheme 1. Reagents and conditions: (i) 4-hydroxybenzaldehyde, KOH, TBAI, DMF, 120 °C; (ii) NaBH4, MeOH at room temperature;
(iii) SOCl2, dioxane, 70 °C; (iv) NaN3, DMF; (v) pent-4-ynoic anhydride, DMAP, pyridine; (vi) 5Ј-O-DMTr-3Ј-O-(pent-4-ynoyl)thymidine,
CuSO4, sodium ascorbate, H2O, dioxane, 40 °C; (vii) HCl (13 mmolL–1) in MeOH/CH2Cl2 (1:1 v/v) at room temperature.
(4) was prepared from pentaerytrityl tetrabromide (1) ac- in the crude product mixtures. The desired products were
cording to a reported procedure[17] (with some modifica- obtained quantitatively.
tions), and the benzylic chlorides were then replaced by
azides to give 5 in 55% overall yield. 5Ј-O-DMTr-thymidine
The Oxidation
(6) was acylated to give pent-4-ynoic anhydride (7)[18] and
Phosphoramidite coupling results in the formation of PIII
attached to 5 by using the click reaction.[19] It is worth not-
ing that a relatively small excess of the alkyne-derived
phosphite triesters, which should be subsequently oxidized
to stable PV phosphotriesters. When conducted on a solid
nucleoside 7 (1.25 equiv. per azide group) could be used,
phase, the oxidation may be carried out by simply using an
and the remaining excess could be recovered. Thus, wastage
excess of oxidant (usually aqueous iodine[1,2]), which can
then be removed by extensive washings; however, this extra
step complicates the synthesis in solution. The oxidant rea-
of the first nucleoside was modest, with the overall yield of
8 from the commercially available nucleoside 6 being 98%.
The DMTr groups of cluster 8 were removed by a carefully
gents should be quenched and/or removed at the latest prior
to the next coupling (cf. 20 in Figure 1). In spite of the
promising results obtained by using alternative oxidants
(e.g., iodobenzene diacetate,[20] tetrabutylammonium per-
iodate,[21] N-oxides, peroxides,[21,22] and a mixture of carbon
tetrachloride, N-methylmorpholine and water[23,24]) conven-
tional aqueous iodine was applied. Once the phosphoram-
idite coupling was completed, an aqueous solution of iodine
(0.2 molL–1 I2 in pyridine/H2O/THF, 2:4:8 v/v/v, added un-
til the dark color remained; see conversion of 10 into 11 in
Scheme 2 and Figure 1, plots i–ii) was added to the crude
mixture, and the oxidation was quenched by the addition
of trimethyl phosphite (1.0 molL–1 in DMF, added until the
dark color disappeared). Precipitation in methanol removed
all traces of reagents and nucleoside derivatives (cf. 18–22
in Figure 1) and gave the desired phosphotriesters, which
were then subjected to the detritylation step.
treatment with HCl (13 mmolL–1) in a mixture of MeOH/
CH2Cl2 (1:1 v/v; see details for the detritylation below),
which gave 9 in 78% yield.
The Phosphoramidite Coupling
For each coupling, 6.0 equiv. of a nucleoside phos-
phoramidite [2-cyanoethyl DMTrT, DMTrdCBz, DMTrdGiBu or
DMTrdABz N,N-diisopropylphosphoramidite, 1.5 equiv. per
5Ј-OH group, 0.13 molL–1 solution in N,N-dimethylform-
amide/acetonitrile (1:1 v/v)] activated by 6.0 equiv. of 4,5-
dicyanoimidazole (DCI) was used to give the desired phos-
phite triester (2 h at room temperature under N2). Comple-
tion of the reactions were verified by RP HPLC analysis
(Figure 1, plots i–iii). Incomplete couplings, if present, were
observed as sets of un-, mono-, di- and trisubstituted start-
ing materials. The major byproducts were, as expected,
hydrolyzed phosphoramidites (i.e., H-phosphonates such as
19), but traces of oxidized phosphoramidites (i.e., phos-
phoramidates such as 20) and moderate amounts of unre-
The Detritylation
In the standard solid-phase synthesis of oligonucleotides,
acted phosphoramidites (such as 18) could also be detected deprotection of the DMTr group is carried out by an expo-
2
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Solution-Phase Synthesis of Oligo 2Ј-Deoxyribonucleotides
Figure 1. RP HPLC chromatograms of the crude product mixtures (for the reaction conditions and structures of 10–16, see Scheme 2).
Details of RP HPLC conditions A (plots i–iii), B (plots iv–vii), and C (plots vii–xi) are provided in the Exp. Sect. The identities of all
species in the RP HPLC profiles were verified by MS (ESI) analysis.
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Scheme 2. Reagents and conditions: (i) Phosphoramidite building block (6.0 equiv.), DCI (6 equiv.), DMF, acetonitrile, under N2; (ii) I2
(1.3 equiv.), pyridine, H2O, THF [the reaction was quenched by the addition of (MeO)3P and the product was precipitated in MeOH].
(iii) HCl (13 mmolL–1) in MeOH/CH2Cl2 (1:1 v/v) (the reaction mixture was neutralized with pyridine and the product precipitated in
MeOH). (iv) aq. NH3 (33%).
4
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Solution-Phase Synthesis of Oligo 2Ј-Deoxyribonucleotides
sure to a short treatment of tri- or dichloroacetic acid in plot x) of the chromatograms, but nearly homogeneous
dichloromethane.[1,2] The acidic medium is then rapidly re- products (GCT, AGT, CCT, AGCCT) could be obtained
moved by extensive washings, which minimizes the time for (still containing released benzamide, d in Figure 1, plot xi).
undesired N-glycosyl cleavage.[25] The reversibility of the re- According to UV absorbance, overall yields (from 9) for the
action makes the situation more challenging in solution. trimers were 67–73% and 43% for the pentamer. The white
The reactive DMTr cation needs a scavenger, and the acid precipitate obtained upon ammonolysis was also charac-
treatment should additionally be neutralized either by ad- terized, and was found to be the expected tetrakis[(4-{[4-(3-
dition of a base or by a basic extractive workup. The appli- amino-3-oxopropyl)-1H-1,2,3-triazol-1-yl]methyl}phenoxy)
cability of three volatile acids, 1,1,1,3,3,3-hexafluoro-2- methyl]methane (17 in Scheme 2).
propanol (HFIP),[26] aqueous acetic acid, and dilute HCl
(in MeOH/CH2Cl2, 1:1 v/v) were initially evaluated for the
detritylation. The progress of the reactions were roughly
monitored by TLC and, after precipitation, the product dis-
Conclusions
The application of clustered nucleosides as a soluble sup-
port for the synthesis of short oligo-2Ј-deoxynucleotides in
solution has been described. Couplings using a small excess
(1.5 equiv. of a nucleoside phosphoramidite per 5Ј-OH
group) of the standard phosphoramidite building blocks
proved efficient, and purification of the products could be
carried out by simple precipitation (over 90% of the prod-
ucts usually gained) in methanol. Ammonolysis gave nearly
homogeneous nucleotide trimers in ca. 70% yield and a
pentamer in 43% yield. It is noteworthy that the couplings
were nearly quantitative (according to RP HPLC profiles
in Figure 1, plots viii–xi), and the yields indicate that the
overall isolation of the products was comparable to those
obtained by other liquid-phase methods.[4–14] Problems re-
lated to standard protecting groups (premature cleavage of
cyanoethyl and Bz protections and susceptibility of dA for
depurination upon DMTr removal) arose, but the principle
of the strategy with more compatible protecting groups[14]
and/or with an alternative coupling chemistry may be very
useful for the production of short oligonucleotides on a
scale of hundreds of milligrams without the need for any
special equipment.
tribution was quantified by RP HPLC. Although HFIP
(neat) can be used for the detritylation of monomeric nu-
cleosides,[26] the treatment proved ineffective for the nucleo-
side clusters (especially 8). Additionally, partial premature
cleavage of the benzoyl group of Ade (15, Bz of Cyt to a
lesser extent) was observed. A similar trend was observed
with 90% aqueous acetic acid. The reaction rate was suf-
ficient at 50 °C, but a remarkable acid-catalyzed cleavage of
the Bz group of Ade was observed. Carefully adjusted
acidic conditions by using HCl (13 mmolL–1 HCl in
MeOH/CH2Cl2, 1:1 v/v, for 15 min) proved most promising,
although traces of benzoyl removal and depurination of
Ade could be observed (see Figure 1, b and c). After com-
pletion, the reaction mixture (ca. 15 min reaction time) was
neutralized by addition of pyridine and concentrated to an
oil, but not to dryness, because pyridinium hydrochloride
in concentrated solutions catalyzed the reverse reaction.
Precipitation of the products finally removed the reactive
DMTr traces.
Synthesis of Oligonucleotides
The synthesis cycle included phosphoramidite coupling,
oxidation, detritylation, and two precipitations described
above. The steps were repeated to elongate three branched
trimers and one pentamer (13–16 in Scheme 2) on a
27 μmol (13–15) and a 35 μmol scale (16) (0.11 and
0.14 mmol of oligonucleotides). RP HPLC chromatograms
of the crude product mixtures (13–16) are shown in Fig-
ure 1, plots iv–vii. In addition to traces of depurination of
Ade and debenzoylation (Figure 1, b and c, discussed above
for the detritylation), a remarkable premature cleavage of
the cyanoethyl groups was observed (Figure 1, a). Although
the exposed exocyclic amino groups may result in undesired
phosphoramidites, the latter side reaction is quite harmless
for potential further chain elongation.[27] The identities of
13–16 were verified by MS (ESI) analysis, and one of the
clustered trimers (15) was additionally characterized by
NMR spectroscopy (1H, 13C, 31P, COSY and HSQC). Ali-
quots (20 mg) of 13–16 were treated with concentrated
aqueous ammonia, the resulting white precipitate was re-
moved by filtration, and the filtrates were analyzed by RP
HPLC. Traces of incomplete couplings and depurination of
Experimental Section
General Remarks: NMR spectra were recorded with a 500 MHz
spectrometer (Bruker Avance). Chemical shifts are given in ppm
and referenced to the solvent signals.[28] RP HPLC conditions: (A)
gradient elution from 50% acetonitrile in 0.1 molL–1 Et3NH+AcO–
to 100% acetonitrile in 25 min, then continued with acetonitrile;
(B) gradient elution from 25% acetonitrile in 0.1 molL–1
Et3NH+AcO– to 100% acetonitrile in 25 min, then continued with
acetonitrile; (C) gradient elution from 2.5% acetonitrile in
0.1 molL–1 Et3NH+AcO– to 50% acetonitrile in 0.1 molL–1
Et3NH+AcO– in 25 min, then continued with 50% acetonitrile in
0.1 molL–1 Et3NH+AcO–. An analytical C-18 RP column
(250ϫ4.6 mm, 5 μm, flow rate 1.0 mL min–1, λ = 260 nm) was
used.
Tetrakis{[4-(hydroxymethyl)phenoxy]methyl}methane (3): Alcohol 3
was prepared according to the literature,[17] with the exception that
pentaerythrityl tetrabromide (1) was used as a starting material in-
stead of the corresponding tetratosylate. Pentaerythrityl tetrabrom-
ide (1; 5.0 g, 13 mmol), 4-hydroxybenzaldehyde (7.9 g, 65 mmol),
KOH (3.6 g, 65 mmol) and tetrabutylammonium iodide (TBAI;
40 mg, 0.11 mmol) were dissolved in N,N-dimethylformamide
Ade may be seen upon more detailed inspection (Figure 1, (40 mL), and the mixture was stirred at 120 °C overnight, cooled
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to room temperature and poured into water. The crude product was
extracted with dichloromethane, washed with saturated NaHCO3,
dried with Na2SO4, and the solvents were evaporated to dryness.
The residue was purified by silica gel chromatography (MeOH/
CH2Cl2, 5:95 v/v) to yield 2 (6.0 g, 87%) as a white foam. Aldehyde
2 (6.0 g, 11 mmol) was dissolved in methanol (140 mL), and
NaBH4 (5.0 g, 130 mmol) was slowly added at 0 °C. The mixture
was warmed and stirred at room temperature overnight. The reac-
tion was quenched by the addition of water, and the mixture was
concentrated to a viscous oil. The oil was dissolved in ethyl acetate
and washed with water and brine. The organic layer was dried with
Na2SO4, filtered, and the solvents were evaporated to dryness to
give 3 (6.0 g, quant.) as white solid flakes (according to NMR
analysis, further purification was not required). 1H NMR
(500 MHz, CDCl3): δ = 7.24 (d, J = 8.5 Hz, 8 H), 6.90 (d, J =
8.6 Hz, 8 H), 4.53 (s, 8 H), 4.35 (s, 8 H) ppm. 13C NMR (125 MHz,
CDCl3): δ = 158.0, 133.4, 128.1, 114.2, 66.3, 63.6, 44.6 ppm. ESI
(MS): m/z = 583.3 [M + Na]+.
was extracted with ethyl acetate. The ethyl acetate layers were com-
bined, washed with saturated NaHCO3 and brine, dried with
Na2SO4, and the solvents were evaporated to dryness. The residue
was purified by silica gel chromatography (MeOH/Et3N/CH2Cl2,
5:1:95 v/v/v) to give 8 (6.7 g, quant.) as a white foam. 1H NMR
(500 MHz, CDCl3): δ = 7.61 (s, 4 H), 7.40 (m, 8 H), 7.32–7.23 (m,
32 H), 7.18 (m, 8 H), 6.88–6.84 (m, 24 H), 6.39 (dd, J = 7.3, 6.7 Hz,
1 H), 5.47 (br., 4 H), 5.39 (s, 8 H), 4.30 (s, 8 H), 4.12 (br., 4 H),
3.79 (s, 24 H), 3.50–3.44 (m, 8 H), 3.00 (m, 8 H), 2.75 (m, 8 H),
2.43 (m, 8 H), 1.39 (s, 12 H) ppm. 13C NMR (125 MHz, CDCl3):
δ = 172.1, 164.0, 158.9, 158.8, 150.8, 146.3, 144.2, 135.4, 135.3,
135.2, 130.12, 130.09, 129.6, 128.14, 128.05, 127.5, 127.2, 120.9,
115.1, 113.3, 111.7, 87.2, 84.3, 83.9, 75.5, 66.0, 63.7, 55.3, 53.5,
53.5, 44.7, 37.9, 33.5, 20.9, 11.7 ppm. MS (ESI): m/z = 1601.7 [M
+ 2 Na]2+/2.
Tetrakis{[4-({4-[3-(thymidin-3Ј-O-yl)-3-oxoprop-1-yl]-1-H-1,2,3-tri-
azol-1-yl}methyl)phenoxy]methyl}methane (9): Compound 8 (1.2 g,
0.38 mmol) was dissolved in 50 mL of 10 mmolL–1 HCl in MeOH/
CH2Cl2 (1:1 v/v). The mixture was stirred at room temperature for
1 h, neutralized by the addition of pyridine, and the solvents were
evaporated to dryness. Completion of the reaction was verified by
RP HPLC, and the residue was purified by silica gel chromatog-
raphy (MeOH/CH2Cl2, 1:9 v/v) to give 12 (0.58 g, 78%) as a white
foam. 1H NMR (500 MHz, CDCl3): δ = 7.75 (s, 4 H), 7.44 (s, 4
H), 7.14 (d, J = 8.6 Hz, 8 H), 6.84 (d, J = 8.6 Hz, 8 H), 6.19 (dd,
J = 7.3, 7.1 Hz, 4 H), 5.36 (s, 8 H), 5.26 (br., 4 H), 4.25 (s, 8 H),
3.95 (br., 4 H), 3.75 (s, 8 H), 2.93 (dd, J = 7.3, 7.2 Hz, 8 H), 2.68
(dd, J = 7.4, 7.2 Hz, 8 H), 2.23 (m, 8 H), 1.82 (s, 12 H) ppm. 13C
NMR (125 MHz, CDCl3): δ = 176.2, 168.8, 162.9, 154.9, 150.2,
140.4, 133.5, 131.4, 125.6, 119.0, 114.9, 89.1, 88.8, 79.3, 70.3, 65.7,
57.3, 48.7, 41.4, 37.3, 24.5, 15.8 ppm. MS (ESI): m/z = 973.4 [M –
2 H]2–/2.
Tetrakis{[4-(azidomethyl)phenoxy]methyl}methane (5): Alcohol 3
(2.3 g, 4.1 mmol) was dissolved in dioxane (60 mL), and thionyl
chloride (2.4 mL, 31 mmol) was added. The mixture was heated to
reflux with stirring under nitrogen overnight, then the solvents were
evaporated to dryness. The resulting oil (4; 2.9 g) and NaN3 (3.2 g,
49 mmol) were dissolved in N,N-dimethylformamide (40 mL), and
the mixture was stirred at room temperature overnight, poured into
water, and the crude product was extracted with diethyl ether. The
product fractions were combined, dried with Na2SO4, filtered, and
the solvents were evaporated to dryness. The residue was purified
by silica gel chromatography (CH2Cl2/hexane, 1:1 v/v) to give 5
(1.7 g, 63%) as a colorless oil that crystallized spontaneously upon
further storage. 1H NMR (500 MHz, CDCl3): δ = 7.26 (d, J =
8.5 Hz, 8 H), 6.97 (d, J = 8.5 Hz, 8 H), 4.40 (s, 8 H), 4.28 (s, 8 H)
ppm. 13C NMR (125 MHz, CDCl3): δ = 158.8, 129.8, 127.9, 115.0,
66.6, 54.3, 44.8 ppm. HRMS (ESI): calcd. for C33H32ClN12O4 [M
+ Cl]– 695.2358; found 695.2375.
Synthesis of Branched Oligonucleotides 12–16: The dT cluster 9
(0.30 g, 0.15 mmol) and 2-cyanoethyl DMTrdCBz N,N-diisoprop-
ylphosphoramidite (18; 0.77 mg, 0.92 mmol, 6.0 equiv.) were dis-
solved in anhydrous DMF (3.7 mL) and a solution of DCI
(0.25 molL–1 in acetonitrile, 3.7 mL, 6.0 equiv.) was added. The
completed coupling (2 h at room temperature under N2) was veri-
fied by RP HPLC (Figure 1, plot i), and then an aqueous solution
of iodine (0.2 molL–1 I2 in H2O/pyridine/THF, 2:4:8 v/v/v, titrated
until the dark color remained, ca. 1.3 equiv. per mol 18) was added.
After 5 min, a solution of P(OMe)3 (1.0 molL–1 in DMF; titrated
until the dark color disappeared, ca. 0.3 equiv. per mol 18) was
added (complete oxidation was verified by RP HPLC analysis, Fig-
ure 1, plot ii), and the reaction mixture was added to cold methanol
(150 mL). The precipitate was isolated, dissolved in MeOH/CH2Cl2
(1:1 v/v, 100 mL), and HCl in MeOH/CH2Cl2 (0.125 molL–1,
10 mL) was added. Complete detritylation was verified by TLC
analysis (ca. 15 min reaction time), then the mixture was neutral-
ized by addition of pyridine and concentrated to an oil. The oil
was dissolved in a mixture of MeOH/CH2Cl2 (1:1 v/v, 2.0 mL) and
added to cold methanol (150 mL). The precipitate was isolated and
dried under vacuum to give 12 (0.57 g, quant.) as a white powder.
5Ј-O-(4,4-Dimethoxytrityl)-3Ј-O-(pent-4-ynoyl)thymidine (7): The
synthesis of 7 has previously been reported by using pent-4-ynoyl
fluoride as a starting material.[18] An anhydride method was, how-
ever, applied in the present study. Pent-4-ynoic acid (3.4 g,
34 mmol) was dissolved in dioxane (40 mL) and dicyclohexylcar-
bodiimide (3.6 g, 17 mmol) was added. The mixture was stirred at
room temperature for 2 h, filtered, and concentrated to a viscous
oil. The resulting pent-4-ynoic anhydride was dissolved in a small
amount of pyridine and added to a mixture of 5Ј-O-(4,4-dimeth-
oxytrityl)thymidine (6; 6.4 g, 12 mmol) in pyridine (70 mL). A cata-
lytic amount of DMAP was added, and the mixture was stirred at
room temperature overnight. The reaction mixture was concen-
trated to a smaller volume, dissolved in ethyl acetate, washed with
saturated NaHCO3, dried with Na2SO4, and the solvents were
evaporated to dryness. The residue was purified by silica gel
chromatography (EtOAc/petroleum ether/Et3N, 70:30:1 v/v/v) to
give 8 (7.3 g, 99%) as a white foam. The authenticity of the product
1
was verified by H, 13C NMR and HRMS (ESI) analysis, and the
data was compared to the reported values.
Tetrakis({4-[(4-{3-[5Ј-O-(4,4-dimethoxytrityl)thymidin-3Ј-O-yl]-3-ox-
oprop-1-yl}-1H-1,2,3-triazol-1-yl)methyl]phenoxy}methyl)methane
(8): Compounds 5 (1.4 g, 2.1 mmol) and 7 (7.4 g, 12 mmol) were
dissolved in dioxane (25 mL). Aqueous solutions of CuSO4
(0.050 molL–1, 2.3 mL, 0.12 mmol) and sodium ascorbate
(0.10 molL–1, 0.60 mL, 0.60 mmol) were added, and the mixture
was stirred at 40 °C for 48 h. The reaction mixture was then con-
centrated to a smaller volume, diluted with water, and the product
Table 1. MS (ESI) data of the branched oligonucleotides.
Compound Calcd. monoisotopic mass
Found mass
12
13
14
15
16
1865.6 [(M – 2 H)/2]2–
1838.2 [(M – 3 H)/3]3–
1846.1 [(M – 3 H)/3]3–
1870.2 [(M – 3 H)/3]3–
2300.6 [(M – 4 H)/4]4–
1866.2 [(M – 2 H)/2]2–
1838.2 [(M – 3 H)/3]3–
1846.2 [(M – 3 H)/3]3–
1870.2 [(M – 3 H)/3]3–
2300.6 [(M – 4 H)/4]4–
6
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Job/Unit: O30864
/KAP1
Date: 26-08-13 17:22:16
Pages: 8
Solution-Phase Synthesis of Oligo 2Ј-Deoxyribonucleotides
The purity of 12 is illustrated in Figure 1, plot iii. Three 0.10 g
batches of 12 (27 μmol) were mixed with 2-cyanoethyl DMTrCBz
,
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DMTrGiBu
and
DMTrABz
N,N-diisopropylphosphoramidite
(160 μmol) in DMF (0.64 mL) and DCI (0.25 molL–1 solution in
acetonitrile, 0.64 mL, 6.0 equiv.) was added to each batch. Once
the phosphoramidite couplings were complete (2 h at room tem-
perature under N2, verified by RP HPLC analysis), the mixtures
were oxidized as described above by using iodine solution
(0.2 molL–1), and the remaining iodine was quenched by the ad-
dition of a solution of trimethyl phosphite (1.0 molL–1 in DMF),
then the mixtures were precipitated in cold methanol (30 mL). The
precipitates were dissolved in MeOH/CH2Cl2 (1:1 v/v, 20 mL), and
HCl in MeOH/CH2Cl2 (0.125 molL–1, 2 mL) was added. After the
detritylation reactions (monitored by TLC, ca. 15 min reaction
time), the mixtures were neutralized by the addition of pyridine,
concentrated to oils, dissolved in MeOH/CH2Cl2 (1:1 v/v, 0.5 mL)
and precipitated in cold methanol (30 mL). The precipitates were
isolated and dried under vacuum to give 13–15 (ca. 0.13 g) as white
powders (88–89%, approximated as homogeneous products). The
branched pentamer 16 was synthesized on a 35 μmol scale (by using
0.070 g of 9) under the conditions described above (the first two
couplings with Cyt repeated) to give 16 (0.22 g, 67%, approximated
as homogeneous product) as a white powder. RP HPLC chromato-
grams of the branched oligonucleotides 13–16 are shown in Fig-
ure 1, plots iv–vii. The authenticity of the products was verified by
ESI (MS) (Table 1). Compound 15 was additionally characterized
by NMR spectroscopic analysis.
[2]
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1986, 27, 4191.
Ammonolysis of Branched Oligonucleotides 13–16: A sample of 13–
16 (20 mg) was treated with concentrated aqueous ammonia
(55 °C, overnight), and the obtained white precipitate was removed
by filtration. The filtrate was concentrated to dryness, dissolved in
water, and analyzed by RP HPLC and UV spectroscopy. The purity
of the desired oligonucleotides is illustrated in Figure 1, plots viii–
xi. According to UV spectroscopic analysis, the trimers (CCT, GCT
and ACT) were obtained in 67–75% yields and the pentamer
(AGCCT) in 43% yield (overall from 9). The filtered white precipi-
tate obtained in the ammonolysis was the released branching unit
1
17. H NMR [500 MHz, [D6]DMSO]: δ = 7.74 (s, 4 H), 7.20 (d, J
[22] R. J. Cvetovich, Org. Process Res. Dev. 2010, 14, 295.
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= 8.2 Hz, 8 H), 6.93 (d, J = 8.2 Hz, 8 H), 5.41 (s, 8 H), 4.23 (s, 8
H), 2.78 (m, 8 H), 2.37 (m, 8 H) ppm. 13C NMR [125 MHz, [D6]
DMSO]: δ = 173.7, 158.7, 146.9, 129.9, 129.0, 122.1, 115.2, 66.5,
52.6, 44.7, 34.9, 21.5 ppm. MS (ESI): m/z = 1049.5 [M + H]+.
Supporting Information (see footnote on the first page of this arti-
cle): 1H and 13C NMR spectra of 5, 8, 9, 15 (supported by HSQC)
and 17 (supported by HMBC) and 31P NMR spectrum of 15.
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Acknowledgments
Received: June 13, 2013
Financial support from the FP7 Marie Curie Actions and from the
Academy of Finland are gratefully acknowledged.
Published Online: ᭿
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
7

Job/Unit: O30864
/KAP1
Date: 26-08-13 17:22:16
Pages: 8
V. Kungurtsev, J. Laakkonen, A. G. Molina, P. Virta
FULL PAPER
Oligonucleotide Synthesis
A liquid-phase method for the synthesis of
short oligonucleotides is described that is
based on the efficient precipitation of tetra-
hedrally branched oligonucleotides in
methanol.
V. Kungurtsev, J. Laakkonen,
A. G. Molina, P. Virta* ..................... 1–8
Solution-Phase Synthesis of Short Oligo-
2Ј-deoxyribonucleotides by Using Clus-
tered Nucleosides as a Soluble Support
Keywords: Synthetic methods / Template
synthesis / Oligonucleotides / Solution-
phase synthesis
8
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Eur. J. Org. Chem. 0000, 0–0