Synthesis of glycol nucleic acids

Article abstract of DOI:10.1055/s-2006-926313

Starting from glycidol, the synthesis of dimethoxytritylated glycol nucleoside phosphoramidites of adenine (A), thymine (T), uracil (U), guanine (G), and cytosine (C) is reported. These phosphoramidites are the building blocks for the automated solid phas

Full text of DOI:10.1055/s-2006-926313

PAPER
645
Synthesis of Glycol Nucleic Acids
Synthesis of
G
ly
i
col
N
u
l
cleic
A
u
cids Zhang, Adam E. Peritz, Patrick J. Carroll, Eric Meggers*
Department of Chemistry, University of Pennsylvania, 231 S. 34th Street, Philadelphia, PA 19104, USA
Fax +1(215)7460348; E-mail: meggers@sas.upenn.edu
Received 20 August 2005
Abstract: Starting from glycidol, the synthesis of dimethoxytrityl-
O
B
O
ated glycol nucleoside phosphoramidites of adenine (A), thymine
(T), uracil (U), guanine (G), and cytosine (C) is reported. These
phosphoramidites are the building blocks for the automated solid
phase synthesis of glycol nucleic acids (GNA) oligonucleotides and
it is demonstrated that derived GNA duplexes with completely acy-
clic backbones considerably exceed the thermal stabilities of analo-
gous DNA duplexes.
B
H
B
O
O
H
H(OH)
O
O
O
O
–O
P
O
–O
O
–O
P
P
O
B
O
B
O
O
B
H
H
H(OH)
O
O
Key words: GNA, glycol nucleic acid, glycol nucleotides, acyclic
nucleic acid backbone, epoxide ring opening
O
O
–O
O
–O
O
–O
P
P
P
B
O
B
O
O
B
O
H
H
O
O
Since Watson and Crick unraveled the double helix struc-
ture of DNA more than 50 years ago, DNA has become a
target of extensive chemical modification with the aim of
understanding and altering its properties.1–9 Our group is
interested in designing structurally simplified artificial
oligonucleotides that can still form stable duplexes in a
programmable fashion. Along these lines, we recently
succeeded in designing a glycol nucleic acid (GNA) with
a stripped down acyclic backbone.10 GNA may display the
most atom economical solution for a functional nucleic
acid backbone.
H(OH)
DNA (RNA)
O
(R)-GNA
(S)-GNA
Figure 1 Comparison of the constitutions of DNA and RNA, with
both enantiomers of glycol nucleic acid (GNA).
communication by Acevedo and Andrews, in which no
experimental details were provided.21
The glycol nucleosides are accessible starting from the
commercially available enantiomerically pure (R)-(+)-
and (S)-(–)-glycidols. (R)-(+)-Glycidol yields (S)-glycol
nucleotides and (S)-GNA, and (S)-(–)-glycidol yields (R)-
glycol nucleotides and (R)-GNA.10 The synthetic route
starting with (R)-(+)-glycidol (1) is shown in Scheme 1.
Tritylation of 1 with DMTrCl provides the tritylated (S)-
glycidol 2 in quantitative yield.23 We generally prefer to
use the tritylated glycidol as a substrate for the following
epoxide ring opening with nucleobases or their deriva-
tives because it simplifies the purification. Accordingly,
reactions of 2 with thymine (3), uracil (4), N-benzoylcy-
tosine (5), and adenine (6), in the presence of around 0.2
equivalents of sodium hydride, afford in a regioselective
and stereospecific fashion the compounds 712 (49%), 8
(61%), 9 (59%), and 10 (51%), respectively (Schemes 1
and 2). The pyrimidine nucleosides are subsequently di-
rectly converted to the phosphoramidites (S)-T12 (77%),
(S)-U (65%), and (S)-C (66%) by reacting with 2-cyano-
ethyl-N,N-diisopropylchlorophosphoramidite (11) in the
presence of Hünig’s base (Scheme 1). The adenine nucle-
oside 10 is first benzoylated at the exocyclic amino group
to 12 (71%) before being converted to the phosphoramid-
ite (S)-A (73%) (Scheme 2).
We here give a full account of the syntheses of dimeth-
oxytritylated glycol phosphoramidites of adenine (A),
thymine (T), uracil (U), guanine (G), and cytosine (C),
which serve as the building blocks for the automated solid
phase synthesis of GNA oligonucleotides (Figure 1). We
demonstrate that GNA oligonucleotides can be synthe-
sized in high yields and derived duplexes of antiparallel
GNA strands considerably exceed the thermal stabilities
of analogous DNA duplexes.10–12
The synthesis of 2,3-dihydroxypropyl derivatives of nu-
cleobases (glycol nucleosides/nucleotides) has been re-
ported from various starting materials,12–22 most notably,
from isopropylideneglycerol by tosylation of the hydroxyl
group followed by nucleophilic substitution with a nucle-
obase or nucleobase derivative,12–18 or in a different strat-
egy, by direct ring-opening of glycidol with nucleobases
or nucleobase derivatives.19–21
We were attracted by the shorter and more straightforward
approach starting with ‘spring-loaded’ glycidol.19–21 The
presented synthetic schemes are related to a published
For the synthesis of the guanine glycol nucleoside, we
were not able to obtain the desired nucleoside by ring
opening of 2 with guanine or protected guanines. Howev-
er, reaction of 2-amino-6-chloropurine (13) with (R)-(+)-
SYNTHESIS 2006, No. 4, pp 0645–0653
Advanced online publication: 25.01.2006
DOI: 10.1055/s-2006-926313; Art ID: M05505SS
© Georg Thieme Verlag Stuttgart · New York
x
x
.
x
x
.2
0
0
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646
L. Zhang et al.
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HO
O
1
Cl
P
CN
DMTrCl
pyridine
100%
O
O
N
N
DMTrO
O
N
O
H
11
O
N
O
N
O
2
DMTrO
H
O
CN
H
N
O
NaH
(i-Pr)2NEt
77%
DMTrO
N
P
O
(0.21 equiv)
49%
H
HO
N
7
3
(S)-T
O
O
O
N
O
H
N
N
11
2
DMTrO
H
N
O
H
O
N
N
CN
O
DMTrO
(i-Pr)2NEt
65%
NaH
(0.17 equiv)
61%
H
P
O
HO
N
4
8
(S)-U
Ph
Ph
Ph
O
O
N
O
NH
N
NH
N
NH
N
2
11
N
N
DMTrO
O
CN
DMTrO
(i-Pr)2NEt
66%
NaH
O
H
O
O
(0.20 equiv)
59%
HO
5
P
9
O
N
(S)-C
Scheme 1 Synthesis of pyrimidine glycol nucleotides. Shown is the route from (R)-(+)-glycidol leading to (S)-glycol nucleosides and (S)-
phosphoramidites.
glycidol (1) in the presence of substoichiometric amounts derivatized glycol nucleosides verified the desired regio-
of K2CO3 provides the ring-opened product 14 in 39% selectivities of the epoxide ring openings (Figure 2).
yield (Scheme 2). Acid hydrolysis of the chloride leads to
In order to synthesize oligonucleotides entirely composed
out of glycol nucleotides on solid support, we next deriva-
tized long chain alkylamine controlled pore glass
(LCAA–CPG) with glycol nucleosides. For this, the trityl-
ated nucleosides 7–9, 12, and 17 were first converted to
the succinates in a standard protocol by reacting with suc-
cinic anhydride and triethylamine, followed by amide
coupling with LCAA–CPG under activation with 1-ben-
1515 (82%), followed by protection of the exocyclic amino
group with an isobutyryl group to afford 16 (70%). After
tritylation to 17 with DMTrCl in anhydrous pyridine in
75% yield, reaction with 11 in the presence of Hünig’s
base provides the final phosphoramidite (S)-G. It has to be
noted that we were not able to run (S)-G over a silica gel
column without decomposition. Instead, we developed a
precipitation protocol which provides purified (S)-G in a
yield of 45%.
zotriazole and 1,3-diisopropylcarbodiimide, yielding typ-
ical loadings of 60–70 mmol/g.24,25
Overall, the pyrimidine phosphoramidites (S)-T, (S)-U,
and (S)-C can be synthesized from commercially avail-
able starting materials in three steps (overall yields of
Oligonucleotides 21–28 (Table 1) were synthesized on
these derivatized CPG supports with standard protocols
for 2-cyanoethyl phosphoramidites, except that the cou-
38%, 40%, and 39%, respectively), the adenine glycol nu-
pling time was increased to three minutes. Under these
cleotide (S)-A in four steps (overall yield of 26%), and the
conditions, no differences in coupling efficiencies be-
tween the glycol nucleoside phosphoramidites and com-
mercial 2¢-deoxyribonucleosyl phosphoramidites could
be detected. Typically, glycol oligonucleotides were syn-
guanine glycol nucleotide (S)-G in five steps (overall
yield of 8%). The enantiomeric compounds (R)-T, (R)-U,
(R)-C, (R)-A, and (R)-G are accessible in the same fash-
ion by starting from (S)-(–)-glycidol. Crystal structures of
thesized in trityl-on mode and cleaved from the resin with
concentrated ammonia at 55 °C for 12 hours. Tritylated
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Synthesis of Glycol Nucleic Acids
647
oligonucleotides were first purified by C18 reverse phase melting and weaker hyperchromicities are observed with
HPLC and then detritylated with 80% acetic acid. The the single strands 21 and 22 alone (Figure 3, dashed and
strands were again purified, this time with a Waters dotted curves).
XTerra column at elevated temperature (55–60 °C). This
These conclusions are confirmed by circular dichroism
XTerra column combines reverse-phase with ion-pairing
(CD) measurements. The CD spectra of a 1:1 mixture of
chromatography and gives superior resolution in eliminat-
GNA strands 21:22 and the individual strands are shown
in Figure 4 and demonstrate strongly increased CD sig-
nals at around 205, 220, and 275 nm upon mixing of 21
and 22, supporting the formation of a helical duplex.
ing shorter failure sequences. In our hands, regular reverse
phase HPLC columns were not able to discriminate be-
tween glycol oligonucleotides that differ in the length by
just one nucleotide unit. Typical yields of pure GNA
It is intriguing that the thermal stabilities of GNA duplex-
strands were in the range of 20% to 40% based on the
es exceed the stabilities of analogous DNA duplexes
amount and loading of the solid supports.
(Table 1, entries a and b). For example, the GNA duplex
21:22 (Table 1, entry a) is thermally more stable with
DTM = 25 °C under our experimental conditions (10 mM
sodium phosphate, pH 7.0, 100 mM NaCl, 2 mM each
strand) compared to a DNA duplex of the same sequence.
Similarly, the GNA duplex 23:24 is thermally significant-
GNA strands are stable in buffered solution at room tem-
perature. For example, no decomposition can be detected
by HPLC with an analytical XTerra column of a T6-GNA
strand in 10 mM sodium phosphate buffer (pH 7.0) during
a time period of two days.
We next investigated duplex formation of GNA strands ly more stable compared to the analogous DNA duplex
with temperature-dependent UV spectroscopy at 260 nm. (DTM = 20 °C). This is a surprising result considering the
Mixtures (1:1) of complementary strands 21:22, 23:24, simplicity and acyclic nature of the backbone.
25:26, and 27:28 all yield characteristic sigmoidal melting
curves, thus indicating cooperative melting of GNA du-
plexes. The UV-melting curve of the 15mer duplex 21:22
is displayed in Figure 3. For comparison, no sigmoidal
It has been widely assumed that nucleic acid analogues
containing a phosphodiester backbone need to be cyclic in
order to produce the required conformational preorganiza-
tion for duplex formation.26,27 This conclusion emerged
Ph
Ph
NH2
DMTrO
O
N
NH
11
(TMSCl)
PhCOCl
O
N
NH
NH2
N
N
N
N
O
(i-Pr)2NEt
2
N
N
N
N
N
N
N
DMTrO
71%
73%
N
NaH
(0.2 equiv)
51%
DMTrO
N
N
CN
DMTrO
HO
H
O
10
12
6
HO
P
O
N
(S)-A
O
N
HO
Cl
N
N
Cl
N
N
O
NH
NH2
N
1
1 N HCl
82%
N
N
N
NH2
HO
HO
HO
K2CO3
(0.17 equiv)
39%
N
N
NH2
HO
H
15
14
13
(TMSCl)
i-PrCOCl
pyridine
70%
O
N
N
NH
11
O
O
N
DMTrCl
pyridine
(i-Pr)2NEt
N
NH
N
N
N
NH
NH
NH
NH
DMTrO
O
O
P
N
N
75%
45%
HO
CN
DMTrO
O
O
O
HO
HO
N
16
17
(S)-G
Scheme 2 Synthesis of purine glycol phosphoramidites for the automated solid phase oligonucleotide synthesis. Shown is the route from
(R)-(+)-glycidol leading to (S)-glycol nucleosides and (S)-phosphoramidites.
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648
L. Zhang et al.
PAPER
Figure 2 Crystal structures of derivatives of glycol nucleosides of G, T, A, and C. For the synthesis of 16 see Scheme 2. Crystals of 16 were
grown from ethanol. Compound 18 was derived from 7 (Scheme 1) by detritylation with 5% trichloroacetic acid in CH2Cl2, followed by
acetylation with acetic anhydride in pyridine. Crystals were grown from ethanol. Compound 19 was derived from 12 (Scheme 2) by detritylation
with 80% aq acetic acid. Crystals were grown from methanol. Compound 20 was derived from 9 (Scheme 1) by detritylation with 5% tri-
chloroacetic acid in CH2Cl2. Crystals were grown from hot ethanol, upon which the benzoyl group was cleaved off. X-ray intensity data were
collected on a Rigaku Mercury CCD area detector employing graphite-monochromated Mo-Ka radiation at a temperature of 143 K and the
structures were solved by direct methods.
partly from experimental observations in which single or
multiple acyclic nucleotides incorporated into DNA re-
sulted in a strong destabilization of the duplex structure.
However, since this has been also observed for glycol nu-
cleotides in DNA,12 a reinvestigation of completely artifi-
cial backbones with different acyclic nucleotides may
lead to interesting results.
Table 1 Synthesized GNA Oligonucleotides 21–28 and Thermal
Stabilities of Derived GNA Duplexesa
Entries Duplexes
TM (°C)b
71 (46)
a
b
c
d
3¢-CACATTATTGTTGTA-2¢ (21)
2¢-GTGTAATAACAACAT-3¢ (22)
3¢-AATATTATTATTTTA-2¢ (23)
2¢-TTATAATAATAAAAT-3¢ (24)
51 (31)
32
Figure 3 UV melting curves (260 nm) of a 1:1 mixture of GNA
strands 21:22 (2 mM each), and the individual single strands 21 (2
mM) and 22 (2 mM). A DNA duplex (2 mM each strand) with the same
sequence is shown in red. Experiments were performed in 10 mM so-
dium phosphate (pH 7.0) with 100 mM NaCl.
3¢-AAUAUUAUUAUUUUA-2¢ (25)
2¢-UUAUAAUAAUAAAAU-3¢ (26)
3¢-AAAAAAAAAAAAAAA-2¢ (27)
2¢-TTTTTTTTTTTTTTT-3¢ (28)
62
a Measured in 10 mM sodium phosphate (pH 7.0) with 100 mM NaCl.
b Melting points of the analogous DNA duplexes are in brackets.
NMR spectra were recorded on a Bruker AM-500 (500 MHz) spec-
trometer. Low-resolution mass spectra were obtained on an LC plat-
form from Micromass using ESI technique. High-resolution mass
spectra were obtained with a Micromass AutoSpec instrument using
either CI or ES ionization. Infrared spectra were recorded on a Per-
kin-Elmer 1600 series FTIR spectrometer. Solvents and reagents
were used as supplied from Aldrich or Acros. All non-aqueous op-
erations were carried out under a dry argon atmosphere.
In summary, we have presented a full account of the syn-
theses of acyclic nucleic acids. Building blocks for auto-
mated solid phase synthesis are accessible in an
economical fashion by nucleophilic ring opening of trityl-
ated glycidol (A, T, C, U) or glycidol (G). GNA forms du-
plexes that are thermally significantly more stable
compared to analogous DNA duplexes. The completely
acyclic nature of the GNA backbone renders it currently
the most economical solution for a phosphodiester bond
containing nucleic acid backbone. Efforts to functionalize
GNA by introducing artificial base pairs into this new du-
plex scaffold are underway.
Compound 221
To a solution of (R)-(+)-glycidol 1 (1.0 mL, 15.1 mmol) and Et3N
(5.4 mL, 40.7 mmol) in CH2Cl2 (34 mL) was added DMTrCl (6.45
g, 19 mmol). After 12 h, the reaction mixture was poured into sat.
aq NaHCO3 (50 mL). The organic layer was evaporated, the residue
was dissolved in EtOAc, washed with aq sat. NaHCO3, and purified
by chromatography over silica gel eluting with hexanes–EtOAc–
Et3N (10:1:0.01), affording compound 2 as an oil (5.98 g, quantita-
tive yield).
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Synthesis of Glycol Nucleic Acids
649
afford compound 8 as a colorless foam (1.04 g, 61%); [a]D20 –33.4
(c = 1, MeOH).
IR (thin film): 1682, 1607, 1509, 1462, 1385, 1301, 1250, 1177,
1089, 1032, 912, 827, 728, 668, 590 cm–1.
1H NMR (500 MHz, CDCl3): d = 7.35 (m, 2 H), 7.22 (m, 6 H), 7.16
(t, J = 7.2 Hz, 1 H), 7.10 (d, J = 7.9 Hz, 1 H), 6.77 (d, J = 8.8 Hz, 4
H), 5.49 (d, J = 7.9 Hz, 1 H), 3.99 (m, 2 H), 3.72 (s, 6 H), 3.57 (dd,
J = 8.0, 14.7 Hz, 1 H), 3.13 (m, 2 H).
13C NMR (125 MHz, CDCl3): d = 163.7, 158.6, 151.5, 145.9, 144.3,
135.5, 129.9, 128.0, 127.9, 127.0, 113.3, 101.5, 86.5, 69.2, 64.2,
55.2, 51.5.
HRMS: m/z [M + Na+] calcd for C28H28N2O6Na: 511.1845; found:
511.1853.
Compound 9
A mixture of 5 (2.1 g, 9.8 mmol) and NaH (60% in mineral oil, 30
mg, 1.98 mmol) in DMF (20 mL) was stirred at r.t. for 1 h. The trit-
ylated glycidol 2 (3.29 g, 8.75 mmol) in DMF (20 mL) was added,
and the resulting mixture was heated at 110 °C for 20 h. The DMF
was evaporated, the residue was extracted with EtOAc, and purified
by chromatography over silica gel eluting with hexanes–acetone–
Et3N (3:2:0.01) to afford compound 9 as a colorless foam (3.11 g,
59%); [a]D20 –27.9 (c = 1, MeOH).
Figure 4 CD spectra of a 1:1 mixture of GNA strands 21:22 (4 mM
each), and the individual single strands 21 (4 mM) and 22 (4 mM). A
DNA duplex (4 mM each strand) with the same sequence is shown in
red. Experiments were performed in 10 mM sodium phosphate (pH
7.0) with 100 mM NaCl.
IR (thin film): 1608, 1508, 1457, 1300, 1249, 1176, 1069, 1035,
830, 702 cm–1.
1H NMR (500 MHz, CDCl3): d = 7.40 (m, 2 H), 7.29 (m, 4 H), 7.23
(m, 2 H), 7.15 (m, 1 H), 6.77 (d, J = 8.9 Hz, 4 H), 3.73 (s, 6 H), 3.26
(m, 1 H), 3.06 (m, 2 H), 2.71 (m, 1 H), 2.56 (dd, J = 2.2, 5.2 Hz, 1
H).
13C NMR (125 MHz, CDCl3): d = 158.4, 144.7, 135.9, 135.8, 129.8,
128.0, 127.6, 126.6, 113.0, 86.0, 64.5, 54.9, 50.9, 44.3.
HRMS: m/z [M + Na]+ calcd for C24H24O4Na: 399.1572; found:
399.1581.
IR (thin film): 1654, 1621, 1607, 1558, 1508, 1484, 1357, 1297,
1250, 1178, 1091, 1027, 826, 796, 682, 668, 648, 594 cm–1.
1H NMR (500 MHz, CDCl3): d = 7.85 (m, 2 H), 7.56 (d, J = 7.3 Hz,
1 H), 7.52 (tt, J = 2.1, 7.4 Hz, 1 H), 7.42 (t, J = 7.7 Hz, 1 H), 7.36
(m, 2 H), 7.32 (d, J = 7.1 Hz, 1 H), 7.20–7.26 (m, 7 H), 7.14 (tt, J =
1.6, 7.3 Hz, 1 H), 6.76 (d, J = 8.6 Hz, 4 H), 4.29 (dd, J = 2.9, 13.6
Hz, 1 H), 4.18 (m, 1 H), 3.75 (dd, J = 7.3, 13.6 Hz, 1 H), 3.71 (s, 6
H), 3.19 (dd, J = 5.1, 9.6 Hz, 1 H), 3.08 (dd, J = 5.8, 9.6 Hz, 1 H).
13C NMR (125 MHz, CDCl3): d = 162.4, 158.6, 150.4, 144.5, 135.6,
135.5, 133.1, 133.0, 129.9, 128.92, 128.0, 127.9, 127.6, 126.9,
113.2, 96.5, 86.3, 68.9, 64.3, 55.16, 54.3.
Compound 712
HRMS: m/z [M + H+] calcd for C35H34N3O6: 592.2448; found:
592.2454.
A mixture of thymine (3; 0.63 g, 5.0 mmol) and NaH (60% in min-
eral oil, 42 mg, 1.06 mmol) in DMF (8 mL) was stirred at r.t. for 2
h. The tritylated glycidol 2 (1.79 g, 4.76 mmol) was added in DMF
(10 mL) and the resulting mixture was heated at 110 °C for 20 h.
The DMF was evaporated, the residue was extracted with EtOAc,
and purified by silica gel chromatography eluting with hexanes–
EtOAc–Et3N (1:2:0.01), affording compound 7 as a colorless foam
(1.22 g, 49%); [a]D20 = –27.1 (c = 1, MeOH).
Compound 10
A mixture of 6 (1.3 g, 9.6 mmol) and NaH (60% in mineral oil, 78
mg, 1.95 mmol) in DMF (15 mL) was stirred at r.t. for 2 h. The trit-
ylated glycidol 2 (3.4 g, 9.04 mmol) in DMF (25 mL) was added,
and the resulting solution was heated at 105 °C for 15 h. The DMF
was evaporated, the residue was extracted with EtOAc, and purified
by chromatography over silica gel, first eluting with hexanes–
EtOAc–Et3N (1:2:0.01), then with EtOAc–Et3N (100:1) affording
compound 10 as a colorless foam (2.5 g, 51%); [a]D20 = –7.8 (c = 1,
MeOH).
IR (thin film): 3444, 3199, 3061, 2933, 1681, 1607, 1510, 1464,
1302, 1249, 1177, 1034, 831, 582 cm–1.
1H NMR (500 MHz, CDCl3): d = 7.35 (d, J = 8.0 Hz, 2 H), 7.23 (d,
J = 8.5 Hz, 4 H), 7.19 (t, J = 7.8 Hz, 2 H), 7.12 (t, J = 7.1 Hz, 1 H),
6.99 (s, 1 H), 6.74 (d, J = 8.6 Hz, 4 H), 3.96–4.02 (m, 2 H), 3.69 (s,
6 H), 3.55 (dd, J = 7.3, 13.8 Hz, 1 H), 3.09 (m, 2 H), 1.72 (s, 3 H).
13C NMR (125 MHz, CDCl3): d = 164.8, 158.8, 152.1, 144.9, 142.4,
136.0, 130.2, 128.3, 128.1, 127.1, 113.5, 110.1, 86.6, 69.4, 64.9,
55.4, 52.0, 12.4.
IR (thin film): 3120, 1641, 1602, 1509, 1444, 1301, 1248, 1176,
1069, 1032, 829, 729 cm–1.
1H NMR (500 MHz, CDCl3): d = 8.17 (s, 1 H), 7.64 (s, 1 H), 7.32
(d, J = 8.3 Hz, 2 H), 7.19 (m, 6 H), 7.13 (t, J = 7.7 Hz, 1 H), 6.73 (d,
J = 8.9 Hz, 4 H), 5.94 (s, 2 H), 4.33 (dd, J = 2.4, 14.2 Hz, 1 H), 4.20
(dd, J = 7.0, 14.2 Hz, 1 H), 4.12 (m, 1 H), 3.71 (s, 6 H), 3.19 (dd,
J = 5.4, 9.6 Hz, 1 H), 3.01 (dd, J = 6.2, 9.6 Hz, 1 H).
HRMS: m/z [M + Na]+ calcd for C29H30N2O6Na: 525.2002; found:
525.2019.
13C NMR (125 MHz, CDCl3): d = 158.5, 155.5, 152.5, 150.0, 144.5,
141.6, 135.7, 135.6, 129.9, 128.0, 127.9, 126.9, 119.3, 113.2, 86.4,
69.4, 64.5, 55.2, 48.2.
HRMS: m/z [M + Na+] calcd for C29H29N5O4Na: 534.2117; found:
534.2137.
Compound 8
A mixture of 4 (0.5 g, 4.5 mmol) and NaH (60% in mineral oil, 30
mg, 0.76 mmol) in DMF (10 mL) was stirred at r.t. for 1 h. The trit-
ylated glycidol 2 (1.42 g, 3.77 mmol) in DMF (10 mL) was added,
and the resulting mixture was heated at 110 °C for 20 h. The DMF
was evaporated, the residue was extracted with EtOAc, and purified
by chromatography over silica gel eluting first with hexane–
EtOAc–Et3N (1:2:0.01), and thereafter with EtOAc–Et3N (100:1) to
Compound (S)-T12
To a solution of 7 (1.1 g, 2.2 mmol) and N,N-diisopropylethylamine
(2.2 mL, 11.7 mmol) in CH2Cl2 (36 mL) was added 2-cyanoethyl
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650
L. Zhang et al.
PAPER
N,N-diisopropylchlorophosphoramidite (1 mL, 4.5 mmol). After 2
h, the reaction mixture was poured into sat. aq NaHCO3 (35 mL)
and extracted with CH2Cl2 (2 × 25 mL). The organic layer was evap-
orated and the residue was purified by chromatography over silica
gel eluting with hexanes–EtOAc–Et3N (1:2:0.01), affording com-
pound (S)-T as a colorless foam (1.18 g, 77%).
(2:1:0.01), affording compound (S)-C as a colorless foam (0.83 g,
66%).
IR (thin film): 2960, 1693, 1667, 1623, 1606, 1556, 1509, 1486,
1420, 1360, 1300, 1250, 1179, 1076, 1032, 979, 828, 705 cm–1.
1H NMR (500 MHz, CDCl3): d = 8.64 (br, 2 H), 7.85 (d, J = 7.1 Hz,
4 H), 7.56 (m, 4 H), 7.40–7.46 (m, 8 H), 7.29 (m, 9 H), 7.20–7.25
(m, 5 H), 7.15 (m, 2 H), 6.77 (m, 8 H), 4. 25–4.37 (m, 4 H), 3.45–
3.85 (m, 10 H), 3.73 (s, 6 H), 3.72 (s, 6 H), 3.35 (dd, J = 4.6, 10.0
Hz, 1 H), 3.21 (dd, J = 4.5, 9.9 Hz, 1 H), 3.16 (dd, J = 2.8, 10.0 Hz,
1 H), 3.09 (dd, J = 4.4, 10.0 Hz, 1 H), 2.62 (dt, J = 6.8, 16.8 Hz, 1
H), 2.51 (dt, J = 5.9, 16.8 Hz, 1 H), 2.37 (t, J = 6.5 Hz, 2 H), 1.08
(m, 24 H).
13C NMR (125 MHz, CDCl3): d = 162.3, 162.2, 158.5, 150.5, 150.4,
144.6, 135.8, 135.7, 135.63, 135.56, 133.1, 133.0, 130.1, 130.03,
129.99, 128.9, 128.1, 128.0, 127.8, 127.5, 126.81, 126.77, 117.8,
117.4, 113.1, 96.0, 95.8, 86.1, 86.0, 70.4, 70.3, 69.7, 69.6, 64.1,
63.8, 58.5, 58.3, 58.2, 58.1, 55.2, 53.6, 53.3, 43.3, 43.2, 43.1, 24.61,
24.55, 24.51, 20.13, 20.07, 20.01.
IR (thin film): 2965, 1685, 1607, 1508, 1463, 1364, 1300, 1250,
1178, 1033, 978, 885, 830, 704, 582 cm–1.
1H NMR (500 MHz, CDCl3): d = 7.38–7.40 (m, 4 H), 7.27 (m, 8
H), 7.23 (m, 4 H), 7.16 (m, 2 H), 6.99 (d, J = 1.1 Hz, 1 H), 6.98 (d,
J = 1.1 Hz, 1 H), 6.77 (m, 8 H), 4.16 (m, 3 H), 4.00 (dd, J = 4.7, 13.9
Hz, 1 H), 3.47–3.78 (m, 10 H), 3.74 (s, 6 H), 3.73 (s, 6 H), 3.24 (dd,
J = 5.1, 9.9 Hz, 1 H), 3.19 (dd, J = 3.3, 9.9 Hz, 1 H), 3.14 (dd, J =
4.9, 10.0 Hz, 1 H), 3.09 (dd, J = 4.8, 10.0 Hz, 1 H), 2.53 (m, 2 H),
2.34 (t, J = 6.4 Hz, 2 H), 1.78 (d, J = 1.0 Hz, 3 H), 1.77 (d, J = 1.0
Hz, 3 H), 1.04–1.11 (m, 24 H).
13C NMR (125 MHz, CDCl3): d = 164.4, 164.4, 158.8, 151.1, 151.0,
144.9, 142.6, 142.5, 136.1, 136.00, 135.95, 130.3, 130.2, 128.4,
128.3, 128.1, 127.11, 127.09, 117.8, 117.6, 113.4, 109.9, 109.7,
86.5, 86.4, 71.4, 71.3, 70.8, 70.7, 64.4, 64.2, 58.6, 58.4, 58.4, 58.3,
55.47, 55.46, 51.91, 51.86, 51.1, 43.35, 43.45, 43.36, 24.92, 24.87,
24.82, 24.79, 24.77, 20.51, 20.45, 20.43, 20.37, 12.4.
31P NMR (121 MHz, CDCl3): d = 149.89.
HRMS: m/z [M + Na+] calcd for C44H50N5O7PNa: 814.3346; found:
814.3340.
31P NMR (121 MHz, CDCl3): d = 150.08, 149.91.
HRMS: m/z [M + Na+] calcd for C38H47N4O7PNa: 725.3080; found:
Compound 12
To compound 10 (2.15 g, 4.2 mmol) in anhyd pyridine (32 mL) was
added trimethylsilyl chloride (2.2 mL, 17.3 mmol). After stirring for
2 h at r.t., the mixture was cooled to 0 °C and benzoyl chloride (0.75
mL, 6.5 mmol) was added dropwise. The mixture was allowed to
warm slowly to r.t. and stirred for an additional 2 h. The reaction
was stopped by the addition of H2O (5 mL) at 0 °C with stirring, and
then concd aq NH3 (10 mL) was added after 15 min. After stirring
for another 30 min, the mixture was extracted with CH2Cl2 (2 × 40
mL). The solvent was evaporated and the residue was purified by
chromatography over silica gel eluting with hexanes–EtOAc–Et3N
(1:2:0.01), then with EtOAc–Et3N (100:1), and finally with EtOAc–
MeOH–Et3N (25:1:0.01), affording compound 12 as a colorless
foam (1.84 g, 71%).
725.3054.
Compound (S)-U
To a solution of 8 (0.5 g, 1.0 mmol) and N,N-diisopropylethylamine
(1.2 mL, 6.4 mmol) in CH2Cl2 (18 mL) was added 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite (0.44 mL, 2 mmol). After 2
h, the reaction mixture was poured into sat. aq NaHCO3 (20 mL)
and extracted with CH2Cl2 (2 × 15 mL). The organic layer was evap-
orated and the residue was purified by chromatography over silica
gel eluting fast with hexanes–EtOAc–Et3N (2:3:0.01), affording
compound (S)-U as a colorless foam (0.46 g, 65%).
IR (thin film): 2968, 1682, 1607, 1507, 1462, 1302, 1251, 1179,
1031, 979, 826, 668, 648 cm–1.
IR (thin film): 3286 (br), 2933, 1704, 1609, 1582, 1510, 1454, 1301,
1250, 1176, 1073, 1033, 910, 828, 798, 727, 705 cm–1.
1H NMR (500 MHz, CDCl3): d = 7.39 (m, 4 H), 7.20–7.29 (m, 12
H), 7.16 (tt, J = 1.9, 7.0 Hz, 2 H), 7.12 (d, J = 7.9 Hz, 1 H), 7.09 (d,
J = 7.9 Hz, 1 H), 6.77 (m, 8 H), 5.49 (d, J = 7.3 Hz, 1 H), 5.47 (d,
J = 7.2 Hz, 1 H), 4.06–4.19 (m, 4 H), 3.47–3.78 (m, 10 H), 3.74 (s,
6 H), 3.73 (s, 6 H), 3.26 (dd, J = 5.2, 9.8 Hz, 1 H), 3.19 (dd, J = 3.4,
9.9 Hz, 1 H), 3.15 (dd, J = 5.2, 10.0 Hz, 1 H), 3.09 (dd, J = 4.5, 10.0
Hz, 1 H), 2.54 (m, 2 H), 2.36 (t, J = 7.5 Hz, 2 H), 1.05–1.11 (m, 24
H).
13C NMR (125 MHz, CDCl3): d = 163.6, 163.5, 158.6, 150.8, 150.6,
146.1, 146.0, 144.5, 144.4, 135.69, 135.65, 135.62, 135.60, 130.03,
129.95, 128.12, 128.05, 127.8, 126.9, 117.7, 117.4, 113.18, 113.15,
101.4, 101.1, 86.2, 86.1, 70.9, 70.8, 70.5, 70.4, 63.8, 63.5, 58.3,
58.2, 58.1, 58.0, 55.2, 51.5, 51.5, 50.9, 43.3, 43.20, 43.15, 43.06,
24.64, 24.61, 24.58, 20.28, 20.22, 20.18, 20.12.
1H NMR (500 MHz, CDCl3): d = 9.12 (br, 1 H), 8.68 (s, 1 H), 8.00
(d, J = 7.5 Hz, 2 H), 7.94 (s, 1 H), 7.57 (t, J = 7.3 Hz, 1 H), 7.47 (t,
J = 7.6 Hz, 2 H), 7.37 (d, J = 7.6 Hz, 2 H), 7.25 (m, 6 H), 7.19 (t,
J = 7.2 Hz, 1 H), 6.78 (d, J = 8.8 Hz, 4 H), 4.45 (dd, J = 2.4, 14.3
Hz, 1 H), 4.28 (dd, J = 7.2, 14.3 Hz, 1 H), 4.17 (m, 1 H), 3.75 (s, 6
H), 3.20 (dd, J = 5.6, 9.6 Hz, 1 H), 3.14 (dd, J = 5.8, 9.6 Hz, 1 H).
13C NMR (125 MHz, CDCl3): d = 164.7, 158.6, 152.2, 152.0, 149.4,
144.4, 144.1, 135.5, 135.4, 133.7, 132.7, 129.9, 128.8, 127.91,
127.85, 127.0, 122.5, 113.2, 86.5, 69.3, 64.5, 55.2, 47.9.
HRMS: m/z [M + Na+] calcd for C36H33N5O5Na: 638.2379; found:
638.2409.
Compound (S)-A
31P NMR (121 MHz, CDCl3): d = 150.06, 149.71.
HRMS: m/z [M + Na+] calcd for C37H45N4O7PNa: 711.2924; found:
To a solution of 12 (1.20 g, 1.95 mmol) and N,N-diisopropylethyl-
amine (2.1 mL, 11.2 mmol) in CH2Cl2 (33 mL) was added 2-cyano-
ethyl N,N-diisopropylchlorophosphoramidite (0.88 mL, 3.97
mmol). After 2 h, the reaction mixture was poured into sat. aq
NaHCO3 (40 mL) and extracted with CH2Cl2 (2 × 35 mL). The or-
ganic layer was evaporated and the residue was purified by chroma-
tography over silica gel eluting with hexanes–EtOAc–Et3N
(1:2:0.01) affording compound (S)-A as a colorless foam (1.16 g,
73%).
711.2943.
Compound (S)-C
To a solution of 9 (0.94 g, 1.6 mmol) and N,N-diisopropylethyl-
amine (1.8 mL, 9.6 mmol) in CH2Cl2 (28 mL) was added 2-cyano-
ethyl N,N-diisopropylchlorophosphoramidite (0.66 mL, 3 mmol) at
r.t. After 2.5 h, the reaction mixture was poured into sat. aq
NaHCO3 (20 mL) and extracted with CH2Cl2 (2 × 20 mL). The or-
ganic layer was evaporated and the residue was purified by chroma-
tography over silica gel eluting fast with hexanes–acetone–Et3N
IR (thin film): 2966, 1700, 1608, 1509, 1456, 1302, 1250, 1179,
1034, 978, 830, 728, 582 cm–1.
Synthesis 2006, No. 4, 645–653 © Thieme Stuttgart · New York

PAPER
Synthesis of Glycol Nucleic Acids
651
1H NMR (500 MHz, CDCl3): d = 9.04 (br, 2 H), 8.73 (s, 1 H), 8.71
(s, 1 H), 8.02 (s, 1 H), 7.98 (m, 4 H), 7.97 (s, 1 H), 7.54 (m, 2 H),
7.46 (m, 4 H), 7.39 (m, 4 H), 7.20–7.27 (m, 12 H), 7.16 (m, 2 H),
6.76 (m, 8 H), 4.53 (m, 2 H), 4.44 (m, 2 H), 4.31 (m, 2 H), 3.73 (s,
6 H), 3.72 (s, 6 H), 3.39–3.63 (m, 8 H), 3.24 (dd, J = 4.1, 9.8 Hz, 1
H), 3.15–3.20 (m, 2 H), 3.08 (dd, J = 5.9, 9.9 Hz, 1 H), 2.42 (q, J =
6.4 Hz, 2 H), 2.33 (t, J = 6.4 Hz, 2 H), 1.04 (d, J = 6.8 Hz, 12 H),
1.00 (d, J = 6.8 Hz, 6 H), 0.94 (d, J = 6.8 Hz, 6 H).
13C NMR (125 MHz, CDCl3): d = 164.6, 164.5, 158.5, 152.5, 152.4,
149.2, 144.5, 144.2, 143.9, 135.63, 135.56, 133.8, 132.6, 130.0,
123.0, 128.81, 128.79, 128.1, 128.0, 127.8, 126.9, 126.8, 122.6,
122.5, 117.6, 117.3, 113.4, 86.7, 86.6, 71.5, 71.4, 71.3, 71.1, 64.2,
63.7, 58.1, 58.0, 57.9, 57.8, 55.2, 46.7, 46.6, 46.4, 43.2, 43.1, 43.0,
24.54, 24.51, 24.47, 24.4, 20.18, 20.12, 20.08.
(20:3), and then with EtOAc–MeOH (40:9), affording compound
16 (3.2 g, 70%).
IR (KBr pellet): 3462, 3198, 2933, 1696, 1617, 1562, 1480, 1410,
1323, 1253, 1199, 1164, 1113, 1056, 871, 843, 795, 746 cm–1.
1H NMR (500 MHz, DMSO-d6): d = 12.04 (br, 1 H), 11.64 (br, 1
H), 7.88 (s, 1 H), 5.07 (d, J = 5.4 Hz, 1 H), 4.79 (t, J = 5.4 Hz, 1 H),
4.20 (dd, J = 3.5, 13.9 Hz, 1 H), 3.92 (dd, J = 8.7, 13.9 Hz, 1 H),
3.80 (m, 1 H), 3.40 (m, 1 H), 3.32 (m, 1 H), 2.77 (sept, J = 6.8 Hz,
1 H), 1.11 (d, J = 6.8 Hz, 6 H).
13C NMR (125 MHz, CDCl3): d = 180.1, 154.9, 148.8, 147.7, 140.6,
119.9, 69.6, 63.6, 46.6, 34.7, 18.9.
HRMS: m/z [M + Na+] calcd for C12H17N5O4: 296.1359; found:
296.1358.
31P NMR (121 MHz, CDCl3): d = 150.37, 149.71.
HRMS: m/z [M + Na+] calcd for C45H50N7O6P: 816.3638; found:
Compound 17
To a solution of compound 16 (2.5 g, 8.5 mmol) in anhyd pyridine
(37 mL) was added DMTrCl (3.5 g, 10.3 mmol) at r.t. After 3 h, the
reaction mixture was concentrated in vacuo. The residue was puri-
fied by chromatography over silica gel first eluting with hexanes–
EtOAc–Et3N (1:2:0.01), then with EtOAc–Et3N (100:1), and finally
with EtOAc–MeOH–Et3N (25:1:0.01) to afford compound 17 (3.8
g, 75%) as a colorless foam; [a]D20 = –4.1 (c = 1, MeOH).
816.3648.
Compound 14
A mixture of 13 (2.4 g, 14.2 mmol), 1 (0.96 mL, 14.5 mmol), and
K2CO3 (0.33 g, 2.4 mmol) in DMF (45 mL) was stirred at 90 °C for
14 h. The reaction mixture was concentrated in vacuo, dissolved in
large amounts of MeOH, adsorbed onto silica gel, and then purified
by chromatography over silica gel eluting with EtOAc–MeOH
(10:1), and later with EtOAc–MeOH (20:3), affording compound
14 (1.4 g, 39%).
IR (thin film): 3153, 1675, 1606, 1563, 1510, 1406, 1250, 1176,
1034, 830 cm–1.
1H NMR (500 MHz, CDCl3): d = 7.49 (s, 1 H), 7.39 (d, J = 7.6 Hz,
2 H), 7.26 (dd, J = 2.3, 8.9 Hz, 4 H), 7.20 (t, J = 7.6 Hz, 2 H), 7.13
(t, J = 7.2 Hz, 1 H), 6.74 (d, J = 8.8 Hz, 4 H), 4.39 (m, 1 H), 4.22
(dd, J = 2.2, 14 Hz, 1 H), 3.93 (dd, J = 8.6, 14 Hz, 1 H), 3.71 (s, 6
H), 3.24 (dd, J = 4.4, 9.4 Hz, 1 H), 3.12 (dd, J = 6.3, 9.4 Hz, 1 H),
2.62 (sept, J = 6.9 Hz, 1 H), 1.18 (m, 6 H).
13C NMR (125 MHz, CDCl3): d = 179.0, 158.5, 154.9, 148.3, 147.3,
144.7, 140.7, 135.8, 135.8, 130.0, 128.1, 127.8, 126.8, 119.7, 113.2,
86.2, 68.3, 65.1, 55.2, 48.6, 36.3, 19.0, 18.8.
IR (KBr pellet): 3373, 3210, 2855, 1649, 1614, 1566, 1527, 1481,
1412, 1376, 1302, 1225, 1147, 1110, 1062, 1040, 999, 919, 783,
641 cm–1.
1H NMR (500 MHz, DMSO-d6): d = 8.01 (s, 1 H), 6.86 (br, 2 H),
5.07 (d, J = 5.4 Hz, 1 H), 4.79 (t, J = 5.5 Hz, 1 H), 4.18 (dd, J = 3.4,
13.9 Hz, 1 H), 3.90 (dd, J = 8.6, 13.9 Hz, 1 H), 3.80 (m, 1 H), 3.40
(m, 1 H), 3.31 (m, 1 H).
13C NMR (125 MHz, CDCl3): d = 159.6, 154.3, 149.1, 144.0, 123.2,
69.2, 63.5, 46.5.
HRMS: m/z [M + H+] calcd for C33H36N5O6: 598.2666; found:
598.2687.
Compound 1515
Compound (S)-G
Compound 14 (5.4 g, 22.2 mmol) was stirred in 1 N HCl (195 mL)
at 85 °C for 3 h and then cooled to r.t. The reaction solution was bas-
ified with concd NH4OH to pH 9 and the precipitated product 15
was filtered off as an off-white solid (4.1 g, 82%).
To a solution of 17 (0.5 g, 0.84 mmol) and N,N-diisopropylethyl-
amine (0.22 mL, 1.2 mmol) in THF (7.5 mL) was added 2-cyano-
ethyl N,N-diisopropylchlorophosphoramidite (0.2 mL, 0.9 mmol)
dropwise at 0 °C. The reaction mixture was allowed to warm to r.t.
and was continuously stirred overnight. The reaction mixture was
then poured into sat. aq NaHCO3 (10 mL) and extracted with
CH2Cl2 (2 × 10 mL). The organic layer was concentrated in vacuo,
the residue was dissolved in a small amount of CH2Cl2 (5 mL), and
the solution was added slowly to rapidly stirred pentane (350 mL).
The white precipitate was collected by filtration, re-dissolved in
CH2Cl2 (20 mL), and pentane was added to this solution until it
started to become cloudy. The solution was subsequently stored at
–20 °C for 2 h, during which a yellow oil formed. The remaining
clear solution was decanted and evaporated to afford compound (S)-
G as a light yellow foam (0.3 g, 45%).
IR (KBr pellet): 3130, 1693, 1641, 1542, 1480, 1401, 1234, 1192,
1075, 1042, 812, 779, 764, 696, 634 cm–1.
1H NMR (500 MHz, DMSO-d6): d = 10.55 (s, 1 H), 7.59 (s, 1 H),
6.46 (br, 2 H), 5.04 (br, 1 H), 4.76 (br, 1 H), 4.07 (dd, J = 3.3, 13.6
Hz, 1 H), 3.80 (dd, J = 8.2, 13.5 Hz, 1 H), 3.74 (m, 1 H).
13C NMR (125 MHz, CDCl3): d = 156.7, 153.5, 151.2, 138.2, 116.2,
69.7, 63.4, 46.0.
HRMS: m/z [M + Na+] calcd for C8H12N5O3: 226.0940; found:
226.0949.
Compound 16
IR (thin film): 2968, 1682, 1608, 1558, 1508, 1464, 1407, 1364,
1300, 1251, 1200, 1179, 1156, 1079, 1032, 978, 831, 790, 703 cm–1.
To a mixture of 15 (3.5 g, 15.5 mmol) in anhyd pyridine (100 mL)
was added trimethylsilyl chloride (15 mL, 118 mmol) at 0 °C. The
reaction mixture was stirred at 0 °C for 30 min and thereafter at r.t.
for another 2 h. The mixture was cooled to 0 °C and isobutyryl chlo-
ride (12.9 mL, 77.5 mmol) was added dropwise. The mixture was
allowed to warm slowly to r.t. and was stirred for an additional 4 h.
The reaction was stopped by the addition of H2O (20 mL) at 0 °C.
After 15 min, concd aq NH3 (20 mL) was added and incubated for
30 min. The reaction mixture was concentrated in vacuo. The resi-
due was dissolved in MeOH, adsorbed onto silica gel, and purified
by chromatography over silica gel eluting with EtOAc–MeOH
1H NMR (500 MHz, CDCl3): d = 7.48 (s, 1 H), 7.43 (s, 1 H), 7.36
(m, 4 H), 7.11–7.23 (m, 14 H), 6.72 (m, 8 H), 4.45 (m, 1 H), 4.04–
4.29 (m, 5 H), 3.40–3.80 (m, 10 H), 3.71 (s, 6 H), 3.70 (s, 6 H),
2.97–3.02 (m, 4 H), 2.49–2.57 (m, 4 H), 2.39 (t, J = 5.9 Hz, 1 H),
0.99–1.21 (m, 36 H)
13C NMR (125 MHz, CDCl3): d = 178.7, 178.5, 158.5, 155.61,
155.56, 148.7, 148.5, 147.2, 147.0, 144.64, 144.55, 140.0, 139.7,
135.7, 135.62, 135.56, 129.95, 129.85, 128.0, 127.9, 127.8, 127.7,
126.8, 120.9, 120.7, 118.1, 117.6, 113.1, 113.1, 113.0, 113.0, 86.3,
Synthesis 2006, No. 4, 645–653 © Thieme Stuttgart · New York

652
L. Zhang et al.
PAPER
86.0, 71.2, 71.0, 63.6, 65.4, 57.4, 57.3, 57.2, 57.2, 55.2, 45.6, 43.1,
43.1, 43.03, 42.98, 36.2, 36.0, 24.6, 24.53, 24.49, 24.4, 20.3, 20.2,
18.8.
26
MS (MALDI–TOF): m/z calcd for C114H144N57O70P14: 3866; found:
3868.
31P NMR (121 MHz, CDCl3): d = 149.03, 148.98.
HRMS: m/z [M + Na+] calcd for C42H52N7O6PNa: 820.3564; found:
27
MS (MALDI–TOF): m/z calcd for C120H150N75O58P14: 4005; found:
4006.
820.3589.
Synthesis of the Solid Support
28
MS (MALDI–TOF): m/z calcd for C120H165N30O88P14: 3869; found:
3869.
To compounds 7–9, 12, or 17 (0.5 mmol) were added succinic an-
hydride (0.75 mmol) and Et3N (1.5 mmol) in anhyd CH2Cl2 (5 mL).
The mixture was stirred at r.t. for 4 h. Then, the solution was washed
with 4% citric acid, extracted with CH2Cl2 (2 × 5 mL), dried over
MgSO4, evaporated and used without further purification for the
following coupling to the solid support. For this, a mixture of long
chain alkylamine controlled pore glass (LCAA-CPG, nominal pore
size 500 Å, mesh size 80–120; 500 mg), 1-hydroxybenzotriazole
(0.015 mmol, 2 mg), 1,3-diisopropylcarbodiimide (0.15 mmol, 24
mL), pyridine (0.1 mL), and anhyd MeCN (2 mL) were shaken in a
glass vial at r.t. for 30 min. Next, the succinates of 7–9, 12, or 17
(0.055 mmol) were added and the mixtures were shaken overnight.
The solid support was filtered off, washed with MeOH and CHCl3,
and dried. Unreacted amino groups were acetylated with THF–luti-
dine–Ac2O (8:1:1, 5 mL) and 10% methylimidazole (5 mL) in THF
for 1 h, followed by washing with MeOH and CHCl3. Trityl analysis
yielded a loading of 65 mmol/g (for 7), 69 mmol/g (for 8), 68 mmol/
g (for 9), 71 mmol/g (for 12), and 67 mmol/g (for 17).
DNA strands were purchased from Sigma Genosys (HPLC puri-
fied). Oligonucleotide concentrations were determined by measur-
ing the UV absorbance of aqueous solutions. Extinction coefficients
of oligonucleotides were calculated from increment values of the in-
dividual nucleobases.28
Thermal Denaturation
The melting studies were carried out in 1 cm path length quartz cells
(total volume 325 mL; 200 mL sample solutions were covered by
mineral oil) on a Beckman 800 UV-VIS spectrophotometer
equipped with a thermo-programmer. Melting curves were moni-
tored at 260 nm with a heating rate of 1 °C/min. Melting tempera-
tures were calculated from the first derivatives of the heating
curves. Experiments were performed in duplicate and mean values
were taken.
Circular Dichroism
Oligonucleotide Synthesis and Purification
CD experiments were performed on an Aviv 62A DS spectrometer.
A 1 cm path length quartz cuvette was used and the solutions were
scanned from 200–320 nm at 25 °C with a time constant of 1 s and
a wavelength step size of 1.0 nm. Each measurement was repeated
5 times and the average was taken.
All oligonucleotides were prepared on an Expedite Nucleic Acid
Synthesizer from ABI. Oligonucleotides were synthesized on 0.2
mmol or 0.5 mmol scales. A standard protocol for 2-cyanoethyl
phosphoramidites (0.05 M) was used, except that the coupling of
the phosphoramidites was extended to 3 min. After the trityl-on syn-
thesis, the resin was incubated with concd aq NH3 at 55 °C for 12 h
and then evaporated. The tritylated oligonucleotides were purified
by C18 reverse phase HPLC with 0.05 M aq TEAA and MeCN as
the eluent (gradient: 5–80% MeCN in 20 min, Varian Dynamax 250
× 10 mm, Microsorb 300-10, C18). The oligonucleotides were then
detritylated with 80% AcOH for 20 min, precipitated with i-PrOH
after addition of NaOAc, and again purified by HPLC. For this pu-
rification step, best purities were obtained with a Waters XTerra
column (MS C18, 4.6 × 50 mm, 2.5 mm) at 55–60 °C with 0.05 M
aq TEAA and MeCN as the eluent (gradient: 3–12% MeCN in 40
min, or 2–11% in 40 min). The identities of all oligonucleotides
were confirmed by MALDI–TOF MS.
Acknowledgment
We thank the University of Pennsylvania, LRSM-MRSEC, and the
ACS Petroleum Research Fund (Type G grant) for supporting this
research. We also thank Sigma-Aldrich for a donation of (R)-(+)-
glycidol and (S)-(–)-glycidol.
References
(1) Watson, J. D.; Crick, F. H. C. Nature (London) 1953, 171,
737.
(2) Freier, S. M.; Altmann, K.-H. Nucleic Acids Res. 1997, 25,
4429.
21
MS (MALDI–TOF): m/z calcd for C118H157N50O76P14: 3926; found:
3925.
(3) Eschenmoser, A. Science 1999, 284, 2118.
(4) Nielsen, P. E. Acc. Chem. Res. 1999, 32, 624.
(5) Kool, E. T. Acc. Chem. Res. 2002, 35, 936.
(6) Leumann, C. J. Bioorg. Med. Chem. 2002, 10, 841.
(7) Henry, A. A.; Romesberg, F. E. Curr. Opin. Chem. Biol.
2003, 7, 727.
22
MS (MALDI–TOF): m/z calcd for C118H154N59O70P14: 3953; found:
3954.
(8) Wagenknecht, H.-A. Angew. Chem. Int. Ed. 2003, 42, 3204.
(9) Benner, S. A. Acc. Chem. Res. 2004, 37, 784.
(10) Zhang, L.; Peritz, A.; Meggers, E. J. Am. Chem. Soc. 2005,
127, 4174.
23
MS (MALDI–TOF): m/z calcd for C120H159N48O76P14: 3924; found:
3927.
(11) The potential stability of duplexes consisting of an acyclic
glycol backbone has been discussed before. See: Wippo, H.;
Reck, F.; Kudick, R.; Ramaseshan, M.; Ceulemans, G.;
Bolli, M.; Krishnamurthy, R.; Eschenmoser, A. Bioorg.
Med. Chem. 2001, 9, 2411.
(12) Glycol nucleotides have been incorporated into DNA
strands and were shown to strongly destabilize duplex
structures. See: Nielsen, P.; Dreiøe, L. H.; Wengel, J.
Bioorg. Med. Chem. 1995, 3, 19.
24
MS (MALDI–TOF): m/z calcd for C120H156N57O70P14: 3951; found:
3951.
25
MS (MALDI–TOF): m/z calcd for C111H141N48O76P14: 3797; found:
3799.
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Synthesis of Glycol Nucleic Acids
653
(13) Holý, A.; Ivanova, G. S. Nucleic Acids Res. 1974, 1, 19.
(14) Holý, A. Collect. Czech. Chem. Commun. 1975, 40, 187.
(15) Holý, A. Collect. Czech. Chem. Commun. 1978, 43, 3103.
(16) Holý, A.; Rosenberg, I.; Dvoꢀáková, H. Collect. Czech.
Chem. Commun. 1989, 54, 2470.
(24) Atkinson, T.; Smith, M. In Oligonucleotide Synthesis: A
Practical Approach; Gait, M. J., Ed.; IRL Press: Oxford,
1984, 35–81.
(25) Song, Q.; Sanghvi, Y. S. Nucleosides, Nucleotides Nucleic
Acids 2001, 20, 1267.
(17) Larsen, E.; Danel, K.; Pedersen, E. B. Nucleosides
Nucleotides 1995, 14, 1905.
(18) Larsen, E.; Danel, K.; Abdel-Aleem, A.-A. H.; Nielsen, P.;
Wengel, J.; Pedersen, E. B. Nucleosides Nucleotides 1995,
14, 1097.
(19) Ueda, N.; Kawabata, T.; Takemoto, K. J. Heterocycl. Chem.
1971, 8, 827.
(20) Seita, T.; Kinoshita, M.; Imoto, M. Bull. Chem. Soc. Jpn.
1973, 46, 1572.
(21) Acevedo, O. L.; Andrews, R. S. Tetrahedron Lett. 1996, 37,
3931.
(22) Seita, T.; Yamauchi, K.; Kinoshita, M.; Imoto, M. Bull.
Chem. Soc. Jpn. 1972, 45, 926.
(23) For assigning the absolute stereochemistry on glycidols, see:
Hanson, R. M. Chem. Rev. 1991, 91, 437.
(26) (a) Schneider, K. C.; Benner, S. A. J. Am. Chem. Soc. 1990,
112, 453. (b) Azymah, M.; Chavis, C.; Lucas, M.; Morvan,
F.; Imbach, J.-L. Nucleosides Nucleotides 1992, 11, 1241.
(c) Vandendriessche, F.; Augustyns, K.; Van Aerschot, A.;
Busson, R.; Hoogmartens, J.; Herdewijn, P. Tetrahedron
1993, 49, 7223. (d) Nielsen, P.; Kirpekar, F.; Wengel, J.
Nucleic Acids Res. 1994, 22, 703. (e) Peng, L.; Roth, H.-J.
Helv. Chim. Acta 1997, 80, 1494.
(27) For the concept of conformational restriction of nucleosides
as a measure for preorganizing oligonucleotide single
strands for duplex formation, see: (a) Tarköy, M.;
Leumann, C. Angew. Chem., Int. Ed. Engl. 1993, 32, 1432.
(b) Steffens, R.; Leumann, C. J. J. Am. Chem. Soc. 1999,
121, 3249. (c) Wengel, J. Acc. Chem. Res. 1999, 32, 301.
(28) Cantor, W. D.; Warshaw, M. M. Biopolymers 1970, 9, 1059.
Synthesis 2006, No. 4, 645–653 © Thieme Stuttgart · New York