Improved synthesis of oligonucleotides with an allylic backbone. Oligonucleotides containing acyclic, achiral nucleoside analogues: N-1 or N-9-[3-hydroxy-2-(hydroxymethyl)prop-1-enyl]nucleobases

Article abstract of DOI:10.1039/b517504f

An improved phosphoramidite method is described to prepare oligonucleotides modified with the acyclic, achiral monomers 1. Examination of dimers, prepared on solid support or in solution, showed that phosphortriester dimers containing the allylic unit 1 were unstable towards bases, whereas phosphordiester dimers were stable. Phosphordiester dimers were obtained by replacing cyanoethyl phosphoramidites 2 with phosphoramidites 3, which gave phosphordiesters directly upon oxidation. The phosphordiester dimers were found to be stable towards capping and oxidation, but were somewhat labile towards acids. By reducing the contact time to acids during detritylation it was possible to prepare oligonucleotides containing 4 or 8 modified A, G or T units. The modified oligonucleotides hybridized to complementary DNA and RNA, although with reduced affinity (ΔT_m per modification -1 to -5°C). The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b517504f

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Improved synthesis of oligonucleotides with an allylic backbone.
Oligonucleotides containing acyclic, achiral nucleoside analogues:
N-1 or N-9-[3-hydroxy-2-(hydroxymethyl)prop-1-enyl]nucleobases
Britta M. Dahl, Ulla Henriksen and Otto Dahl*
Received 9th December 2005, Accepted 11th January 2006
First published as an Advance Article on the web 25th January 2006
DOI: 10.1039/b517504f
An improved phosphoramidite method is described to prepare oligonucleotides modified with the
acyclic, achiral monomers 1. Examination of dimers, prepared on solid support or in solution, showed
that phosphortriester dimers containing the allylic unit 1 were unstable towards bases, whereas
phosphordiester dimers were stable. Phosphordiester dimers were obtained by replacing cyanoethyl
phosphoramidites 2 with phosphoramidites 3, which gave phosphordiesters directly upon oxidation.
The phosphordiester dimers were found to be stable towards capping and oxidation, but were
somewhat labile towards acids. By reducing the contact time to acids during detritylation it was possible
to prepare oligonucleotides containing 4 or 8 modified A, G or T units. The modified oligonucleotides
hybridized to complementary DNA and RNA, although with reduced affinity (DTm per modification
−1 to −5 ◦C).
1a should be able to mimic thymidine in both A- and B-type
Introduction
helices.4 Synthetic problems concerning the preparation of the
monomers 1a–f have been solved,6,7 but incorporation into DNA
via the phosphoramidites 2 was troublesome, and DNA oligomers
containing only one or two thymine monomers have hitherto
been studied.5 The resistance of these oligomers to snake venom
phosphordiesterase was satisfactory, but the binding affinity
towards complementary DNA and RNA strands was found to
Modified oligonucleotides are of current interest as potential
therapeutic agents (antisense or antigene drugs).1,2 The oligonu-
cleotides need modification to be resistant to nucleases, but
the modified units should retain the high selectivity and strong
binding of natural DNA and RNA, based on Watson–Crick
(or Hoogsteen) bonding. For some years we have studied mod-
ified nucleosides where the sugar unit is replaced by a simple
achiral unit, N-1 or N-9-[3-hydroxy-2-(hydroxymethyl)prop-1-
enyl]nucleobases 1a–f (Fig. 1).3–7
◦
be reduced compared to native DNA (DTm − 2 to −6.5 C per
modification).5
In our previous study,5 the phosphoramidite 2a was shown to
couple efficiently when activated with tetrazole. An aqueous wash
was introduced to remove unwanted phosphitylation at the bases.
Oxidation with ButOOH instead of I2–H2O–Py was necessary
since the latter reagent cleaved the allylic C–O bond. However,
after detritylation with CCl3COOH–CH2Cl2, the next coupling
gave a low yield (DMT efficiency 80–85%), independent of the
identity of the next phosphoramidite, and prolonged or repeated
couplings did not improve the yield. Removal of the cyanoethyl
groups on the phosphortriesters flanking the modified unit was
problematic, probably due to competing removal of the allylic
modified unit. Thus, aqueous NH3 mainly cleaved the sequences
at the position of the modification, whereas deprotection with neat,
dry Pri2NH (a poorer nucleophile), followed by aq. NH3, did give
some full length product (10–20% after ion exchange purification
and desalting).
This paper describes our attempts to optimise the coupling and
deprotection conditions. Dimers derived from 2a, or from the a-
isomer 2aꢀ to obtain symmetrical dimers to ease the interpretation
of NMR spectra, have been prepared in solution or on solid
support, and their sensitivity to cleavage under different conditions
Fig. 1
=
The central C C bond restricts the molecules conformationally,
and molecular modelling and geometry calculations indicate that
1
examined by 31P and H NMR. It is shown that oxidation with
ButOOH and capping with NMI–Ac2O–Py does not result in
cleavage, but that detritylation with 3% CCl3COOH in CH2Cl2
and in particular removal of the cyanoethyl group with dry
Department of Chemistry, University of Copenhagen, Universitetsparken
5, DK-2100, Copenhagen, Denmark. E-mail: dahlo@kiku.dk; Fax: (+45)
35320212; Tel: (+45) 35320176
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Pri2NH results in partial cleavage. To avoid the latter step,
the phosphoramidites 3a and 3aꢀ with N-methyl-N-(2-pyridyl)-
2-aminoethyl instead of 2-cyanoethyl have been tested in dimer
experiments. The N-methyl-N-(2-pyridyl)-2-aminoethyl protect-
ing group has been introduced by Beaucage et al. and shown
to eliminate spontaneously upon oxidation of phosphorus by
a cyclodeesterification reaction.8 Pure modified phosphordiester
dimers are formed from phosphite dimers prepared from 3aꢀ and
are shown to be stable to aq. NH3, but acids used for detritylation
results in minor cleavage. The phosphoramidites 3a, 3b, and
3e have been used to prepare some more extensively modified
oligonucleotides, which are shown by UV melting experiments to
pair with both DNA and RNA, although with reduced affinity
compared to unmodified sequences.
by column chromatography. This difference in stability reflects the
huge difference in leaving group ability between a phosphortriester
and a phosphite. Oxidation of 4b (dP 141.1) in CH3CN with
ButOOH gave 4c (dP −0.5) without observable byproducts (31P
NMR). The solvent was removed in vacuo and the residue treated
with dry Pri2NH in dry pyridine. The 31P NMR signal from 4c (dP
−0.1) was over 2 h at rt replaced by two new signals (dP − 1.8 and
−2.0) in a ca. 2 : 3 ratio. After evaporation the residue was dissolved
in DMSO-d6 and analysed by 31P and 1H NMR. The phosphorus
spectrum showed two products (dP −0.5 and −0.9, ratio ca. 2 : 3),
and the proton spectrum was in accordance with a ca. 2 : 3 mixture
of 4d and 5b (Scheme 2), containing some free thymine base and
unidentified products. This shows that the reaction with Pri2NH is
far from clean, since in competition with removal of the cyanoethyl
group to give 4d, the allylic modified unit is also removed from
the phosphortriester to give 5b. The dimer 4c was shown by 31P
NMR to be stable to capping conditions (Ac2O–NMI–Py–THF)
for 24 h at rt. However, 4c partly decomposed under detritylation
conditions. When a solution of 4c in CH2Cl2 was treated with 3%
CCl3COOH in CH2Cl2, a precipitate formed. The precipitate was
dissolved in DMSO-d6 and analysed by NMR. The phosphorus
spectrum showed one signal (dP −0.3), while the proton spectrum
was in accordance with a ca. 4 : 3 mixture of 4e and 5c (Scheme 3),
containing some free thymine base and unidentified products. In
D2O the phosphorus spectrum showed the expected two signals
(dP −1.2 and 0.6). The ratio of these two signals changed with
time, leaving only the signal at dP 0.6 after 3 d. At that time
the proton spectrum showed no signals assigned to 4e, while
the signals assigned to 5c remained. This indicates that 4e (dP
−1.2) slowly decomposed to 5c (dP 0.6) in the weakly acidic
solution.
Results and discussion
Dimer experiments with cyanoethyl phosphoramidites 2a and 2aꢀ
Initial experiments to prepare a dimer from 2a and CPG-bound
dT, using the protocol described earlier,5 gave ca. 25% of the dimer
4a (dP 0.7) and ca. 75% of 5ꢀ-pdT 5a (dP 4.2), according to 31P NMR
−
in D2O (Scheme 1). The products were verified by ES MS (4a
found 515.1, calc. 515.1; 5a found 321.0, calc. 321.0) and CE,
where the major peak coeluted with authentic 5ꢀ-pdT (dP ca. 4.3,
pH-dependent). Clearly the dimer 4a largely decomposed, but at
which step(s) was unknown. In order to clarify this another dimer
4c was prepared by solution chemistry (Scheme 2). Since 4c was
rather labile and attempts to purify it failed, all experiments were
done on 4c, freshly prepared from 4b. The phosphite dimer 4b,
prepared from the a-amidite 2aꢀ, was stable and could be purified
Scheme 1 Reagents: 1. tetrazole, 2. ButOOH, 3. CCl3COOH, 4. Pri2NH, 5. aq. NH3.
Scheme 2
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Scheme 3
Scheme 4
Solid phase experiments with N-methyl-N-(2-pyridyl)-2-
Dimer experiments with N-methyl-N-(2-pyridyl)-2-aminoethyl
phosphoramidites 3a and 3aꢀ
aminoethyl phosphoramidite 3a
The phosphoramidites 3a and 3aꢀ were prepared from crude
[N-methyl-N-(2-pyridyl)-2-aminoethyl] N,N,Nꢀ,Nꢀ-tetraisoprop-
ylphosphorodiamidite8 and DMT-protected 1a in 50–60% yield.
The a-amidite 3aꢀ was used to prepare the stable symmetrical
dimer phosphite 4f (Scheme 4). Oxidation of 4f (dP 140.9) in
CH3CN with ButOOH gave 4g (dP 0.2), which spontaneously
lost the protecting group to give 4d (dP 0.0) with t1/2 ca. 20 min
at 25 ◦C. Both compounds were formed without byproducts
(31P NMR). Extraction of a solution of 4d in CH2Cl2 with aq.
NaHCO3–Na2SO3 followed by removal of the DMT groups with
80% aq. CH3COOH gave reasonably pure 4h (Scheme 5) as the
sodium salt in 95% yield. The dimer 4h was used to evaluate
the stability of phosphordiester-linked modified units towards
aq. NH3. A solution of 4h in 32% aq. NH3 in a sealed NMR
tube was kept at 55 ◦C, and the 31P NMR spectrum recorded
at intervals. A very slow decomposition was observed, 4h (dP
0.5) being transformed to a new compound (dP 5.1, t, J 7 Hz),
probably 5d (Scheme 5), with t1/2 ca. 430 h. Since this corresponds
to only 0.8% decomposition in 5 h at 55 ◦C we conclude that
the phosphordiester linkage flanking the modified unit is stable
enough under normal deblocking conditions to remove the usual
base protecting groups.
A dimer 4i was prepared from 3a (50 lmol) and polystyrene-
bound dT (10 lmol) (Scheme 6). After oxidation the support was
kept overnight in CH3CN to allow for complete cycloelimination
of the phosphorus protecting group. The support was dried and
divided in ca. 2 lmol portions. One portion of 4i was treated
with NH3 to give 4j, which was analysed by 1H and 31P NMR in
D2O. Apart from 4j (dP 1.2) it contained ca. 10% 4a (dP 1.3) and
ca. 3% 5a (dP 4.9, t, J 6 Hz). Spontaneous loss of DMT from a
support-bound oligonucleotide on standing is well known, but the
formation of 5a indicates that ca. 3% decomposition occurs during
coupling. Another portion of 4i was treated with NH3, followed
by 80% aq. CH3COOH (steps 3 and 4 in Scheme 6) to give 4a,
shown by 31P NMR to contain ca. 10% 5a. This indicates that
the diester dimer 4a, like the triester dimer 4e before, is not stable
under acidic conditions. A third portion of 4i was detritylated
on the synthesizer with 3% CCl3COOH in CH2Cl2 for 2 min,
then immediately washed with 1 M Pri2NH in CH3CN for 2 min,
to give 4k (Scheme 7). This compound was coupled with the
Beaucage phosphoramidite 6a to give the trimer 7 after oxidation,
cycloelimination overnight, detritylation followed immediately by
a Pri2NH wash as above, and cleavage from the support by aq.
NH3. NMR analysis in D2O showed 7 (dP 1.1 and 0.1, 1 : 1)
contaminated with ca. 7% 5a (dP 4.9) and ca. 1% of probably
3ꢀ-pdT 5e (dP 4.4).
The experiments described above show that phosphoramidites
like 3a, which spontaneously eliminate the phosphorus protection
group upon oxidation, are in this context wastly superior to
cyanoethyl phosphoramidites like 2a, because a basic elimination
step is avoided. Oligonucleotides which contain allylic phospho-
rtriesters like those described here are cleaved by bases even as
weakly nucleophilic as Pri2NH. However, when the phosphate
Scheme 5
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Scheme 6 Reagents and conditions: 1. tetrazole 10 min, then aq. wash; 2. ButOOH 30 min, then CH3CN wash, then 20 h wait; 3. 32% aq. NH3 2 h rt,
then evap.; 4. 80% aq. CH3COOH 30 min, then evap., extraction of DMTOH with ether, evap.
where N* are the allylic units corresponding to 1. Attempts to
prepare dT*13T gave a very impure product which was not purified,
and attempts to use the 5-methylcytosine amidite 2d to obtain
oligonucleotides containing C* were abandoned because the prod-
ucts even with one modified C were very impure. This is in accord
with the high tendency of the cytosine compounds 1c, 1d, and
their N-protected derivatives to cyclise.7 The A* phosphoramidite
3b and the G* phosphoramidite 3e were prepared in the same
way as the T* phosphoramidite 3a, and used together with the
Beaucage T phosphoramidite 6a and the A analogue 6b10 which are
necessary as the 10th base in the 14-mers. The remaining T and A
phosphoramidites were commercial cyanoethyl phosphoramidites
The coupling cycle used to introduce N* was modified in
several ways from a normal phosphoramidite cycle. A normal
coupling with tetrazole as activator was followed by a 2-min
aqueous wash to remove amidites that had reacted at the bases.
Scheme 7 Reagents: 1. 6a + tetrazole; 2. ButOOH, then wait; 3.
Capping was performed normally, followed by normal oxidation
with ButOOH. A wait step of 0.5 h was then introduced to allow
the spontaneous elimination of the phosphorus protection group
to occur (experiments with 1 h and 2 h wait did not improve the
yields). Detritylation with 3% CCl3COOH in CH2Cl2 and CH3CN
wash for a total of 1 min was immediately followed by a wash with
0.1 M Pri2NH in CH3CN to remove acid which partially cleaves the
strand at allylic P–O positions. In case of the 14-mers, a standard
cycle was used for the first 4 couplings with normal cyanoethyl
phosphoramidites, but for the last 5 unmodified couplings the
basic wash after detrylation was included, and the 0.5 h wait
was used for the couplings with 6. A stepwise DMT efficiency of
92–94% was obtained for coupling with the N* phosphoramidites,
and 94–96% for the couplings with 6 and the remaining cyanoethyl
phosphoramidites, compared to ca. 99% for the first four cyano-
ethyl phosphoramidites. The support-bound oligonucleotides
were left overnight to complete the elimination of the N-methyl-N-
(2-pyridyl)-2-aminoethyl groups and then treated with conc. aq.
NH3 at 55 ◦C for 8 h (2 h at 25 ◦C for dT5T*4T5). After evaporation
the crude oligonucleotides were purified by reverse phase HPLC,
and the crude and purified products analysed by ion exchange
HPLC. Aproximate yields and MALDITOF MS data are given
in Table 1, and an HPLC profile of a crude product is shown
in Fig. 2.
CCl3COOH, then Pri2NH; 4. aq. NH3.
linkages are converted to phosphordiesters, they are highly inert
towards bases including aq. NH3. The allylic phosphordiesters are
likewise stable towards other conditions during oligonucleotide
synthesis (capping, oxidation) except the acidic treatment during
detritylation. The strongly acidic conditions necessary to remove
DMT probably leads to protonation of the phosphordiester
group,9 thereby converting it into a good leaving group (like a
phosphortriester group). This results in partial cleavage of the
allylic P–O bond during detritylation. The cleavage is minimised by
a short exposure to 3% CCl3COOH in CH2Cl2 (1–2 min) followed
immediately by a Pri2NH wash. By this procedure a trimer 7
could be prepared with concomitant cleavage of only 3–4% per
detritylation. These improved synthesis conditions were used to
prepare some oligonucleotides containing 4 or 8 modified units,
as described below.
Solid phase oligonucleotide synthesis using N-methyl-N-
(2-pyridyl)-2-aminoethyl phosphoramidite 3a, 3b, and 3e
The oligonucleotides prepared in this study are the 14-mers
dT5T*4T5, dA5A*4A5, and the 9-mer dG*T*G*A*T*A*T*G*C,
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Table 1 Yields, purities, and MS data for the modified oligonucleotides
Scale/lM
Crude yield, OD
Purity (%)
Purified yield, OD
Purity (%)
MS (calc.)
dT5T*4T5
dA5A*4A5
dG*T*G*A*T*A*T*G*C
0.25
0.25
1.0
12
21
51
34
40
14
2.5
4
5
85
85
90
4074 (4074)
4202 (4200)
2514 (2511)
1. Previous attempts to incorporate this monomer by standard
phosphoramidite chemistry gave low yields, and we have examined
the reasons for the low yields by dimer experiments analysed
by NMR. It is shown that cyanoethyl phosphoramidites 2 gave
phosphortriesters which largely decomposed during removal of
the cyanoethyl group with base, because the allylic backbone in
the phosphortriester, e.g. 4c, was very sensitive to basic conditions.
A simple allyl group is known to be removable with aq. ammonia
from allyl dinucleoside phosphates,13 and in diallyl 2-cyanoethyl
phosphate, the cyanoethyl group is removed cleanly to give diallyl
phosphate with aq. ammonia.14 It was unexpected that the allylic
backbone was more sensitive to base than a simple allyl group.
Since phosphordiester anions containing two modified units, e.g.
4h, are very resistant to bases, we decided to replace the cyanoethyl
protection group with a group which spontaneously eliminated
after oxidation, without base treatment. The N-methyl-N-(2-
pyridyl)-2-aminoethyl group introduced by Beaucage et al.8 was
chosen, and NMR experiments showed that phosphoramidites
3 containing this protection group gave high yields of modified
dimers. The dimers were stable to oxidation and capping condi-
tions, as well as aq. ammonia necessary to remove base protection
groups. However the strong acid conditions necessary to remove
the DMT protection groups resulted in some cleavage, probably
because protonation of the phosphordiester group leads to some
elimination of the allylic modified unit. By introducing a basic
wash immediately after the DMT removal step the cleavage could
be reduced to 3–4% per coupling cycle.
Fig. 2 Ion exchange HPLC profile of crude dG*T*G*A*T*A*T*G*C.
Hybridization studies
The ability of the modified oligonucleotides to bind to com-
plementary DNA and RNA was examined by thermal melting
temperature (Tm) measurements at 260 nm. The results are given
in Table 2, together with previous results.5
From the data in Table 2 it is seen that one modification in the
middle of a 14-mer gives a depression of the melting temperature
of 5–6 ◦C, but that four modifications are better tolerated. In
particular four modified As are well tolerated with a depression
of only 1–2 ◦C per modification. It was of interest to see whether
a fully modified sequence would bind better, as is known in cases
where the modified unit deviates strongly from natural nucleosides,
e.g. phosphonate analogues of PNA11 and xylo-LNA.12 However,
this is not the case here. The mixed sequence 9-mer with eight
modifications binds with a depression of 2–3 ◦C per modification,
only marginally better than those with four modifications.
Using these improved conditions we were able to prepare
oligonucleotides modified with 4 or 8 modifications from
monomers 3a, 3b, and 3e containing T, A and G. The correspond-
ing modified MeC monomer could not be incorporated in useful
amounts, probably because it cyclised after removal of the DMT
group. The oligonucleotides modified with 4 or 8 consecutive
modifications hybridized to both DNA and RNA complements,
but with reduced melting temperatures compared to unmodified
DNA. The reduction in Tm per modification for the mixed 9-mer
with 8 modifications (2–3 ◦C) was less than previous results with
Conclusion
This paper describes our efforts to improve the preparation of
oligonucleotides modified with the acyclic, achiral monomers
Table 2 Hybridization data (Tm, ◦C) for modified and unmodified oligodeoxyribonucleotides with DNA and RNA complementsa
b
b
c
b
b
dA14
DTm
rA14
DTm
dA6A*A7
DTm
dA5A*4A5
DTm
dT14
rU14
dT7T*T6
dT11T*2T
dT5T*4T5
36
—
—
−5
34
—
—
−6
31
12
N.d.
N.d.
N.d.
−5
0
—
—
—
27
9
N.d.
N.d.
11
−2
−1
—
—
—
12
N.d.
285
295
15
315
315
−2.5
−3.5
−2.5
−5
22
b
b
dGCATATCAC
33
8
DTm
—
−3
rGCAUAUCAC DTm
31
12
dGTGATATGC
dG*T*G*A*T*A*T*G*C
—
−2
a Tm was determined by measuring absorbance at 260 nm against increasing temperature (1 ◦C steps) on equimolar mixtures (3 lM in each strand) of
the modified oligomer and its complementary DNA or RNA strand in medium salt buffer (10 mM Na2HPO4, 100 mM NaCl, 0.1 mM EDTA, pH 7.0).
b Change in Tm per modification. c Prepared from 2b as described earlier.5
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◦
one modification (5–6 C), and the purines A and G seem to be
26%, Rf = 0.29), all colourless solids. (E): NMR (DMSO-d6): dH
11.39 (1H, s, NH), 7.43–6.89 (14H, m, Ar + H-6), 6.65 (1H, s,
better tolerated than T.
=
Our original goal—to prepare a simple acyclic, achiral sub-
stitute for the natural nucleosides—has been achieved, but with
limited success. The monomers 1 are not simple to prepare,
and they are difficult to incorporate as phosphoramidites in
oligonucleotides because of their allylic structure. Since their
affinities towards DNA and RNA are lower than unmodified
DNA, we have decided to stop further work in this area.
NCH C), 4.90 (1H, t, J 5.3, OH), 3.92 (2H, d, J 5.3, CH2OH),
3.74 (6H, s, OCH3), 3.70 (2H, s, CH2ODMT), 1.78 (3H, d, J 1.2,
T–CH3). FAB+ MS: 515.2 (M + H+ calc. 515.2). (Z): NMR
(DMSO-d6): dH 11.30 (1H, s, NH), 7.31–6.86 (14H, m, Ar + H-6),
=
6.49 (1H, s, NCH C), 5.15 (1H, t, J 5.0, OH), 4.16 (2H, d, J 5.0,
CH2OH), 3.73 (6H, s, OCH3), 3.47 (2H, s, CH2ODMT), 1.60 (3H,
d, J 1.2, T–CH3). dC 164.1, 158.9, 150.4, 144.5, 140.5, 136.1, 135.5,
130.1, 128.2, 127.3, 124.8, 113.5, 110.6, 87.3, 63.6, 58.9, 55.5, 12.6.
FAB+ MS: 515.2 (M + H+ calc. 515.2). The Z configuration of
the latter product was determined by 1H NMR NOE effects from
Experimental
The compounds 1a,6 6-N-(4-tert-butylbenzoyl)-9-[3-hydroxy-
2(hydroxymethyl)prop-1-enyl]adenine,6 1-[3-benzyloxy-2-(benzyl-
oxymethyl)prop-1-enyl]-N-isobytyrylguanine,6 [N-methyl-N-(2-
pyridyl)-2-aminoethyl] N,N,Nꢀ,Nꢀ-tetraisopropylphosphorodi-
amidite,8 6a,8 and 6b10 were prepared according to literature
procedures. Other chemicals were 97–99% pure from Aldrich,
Fluka, Sigma, or Merck. Solvents were HPLC grade from
LABSCAN. CH2Cl2, DMF, pyridine, Et3N and CH3CN were
=
NCH C to CH2OH, and 1-alkylation of T by NOE effects from
=
NCH C to H-6.
(E)- and (Z)-6-N-(4-tert-Butylbenzoyl)-9-[2-
(dimethoxytrityloxymethyl)-3-hydroxyprop-1-enyl]adenine
To a solution of dry 6-N-(4-tert-butylbenzoyl)-9-[3-hydroxy-
2(hydroxymethyl)prop-1-enyl]adenine (0.271 g, 0.710 mmol) in
dry pyridine (20 ml) was added dropwise a solution of DMTCl
(0.287 g, 0.85 mmol) in dry pyridine (4 ml), and the mixture
was stirred in the dark under N2 for 3 h at rt. Sat. aqueous
NaHCO3 (1 ml) was added and the solvents removed in vacuo.
The residue was dissolved in acetone (15 ml), filtered, and acetone
removed in vacuo to give the crude product (0.58 g), a mixture
of hydrolysed DMTCl, the bis-, the two mono-dimethoxytrityl
derivatives, and the starting material. This mixture was resolved
into the components by normal gravity column chromatography,
eluted with first EtOAc–hexane–MeOH–Et3N 49 : 49 : 1 : 1
v/v/v/v, then EtOAc–MeOH–Et3N 98 : 1 : 1 v/v/v, and last
EtOAc–MeOH–Et3N 79 : 20 : 1 v/v/v, to give the di-DMT
derivative (0.164 g, 23%, Rf = 0.35 in EtOAc–hexane–MeOH–
Et3N 49 : 49 : 1 : 1 v/v/v/v), the (E)-product (0.150 g, 31%,
Rf = 0.55 in EtOAc–MeOH–Et3N 98 : 1 : 1 v/v/v), the (Z)-
product (0.119 g, 24%, Rf = 0.16 in EtOAc–MeOH–Et3N 98 :
1 : 1 v/v/v), and the starting material (0.028 g, 10%, Rf = 0.36
in EtOAc–MeOH–Et3N 79 : 20 : 1 v/v/v), all colourless solids.
(E): NMR (DMSO-d6): dH 11.13 (1H, s, NH), 8.79 (1H, s, H-2),
8.53 (1H, s, H-8), 8.00 (2H, d, J 8.2, tert-butylbenzoyl), 7.59–7.25
˚
dried over molecular sieves (4 A from Grace Davison) to a
water content below 20 ppm, measured on a Metrohm 652 KF-
Coulometer. TLC was run on Merck 5554 silica 60 aluminium
sheets, LC on either Merck 9385 silica 60 (0.040–0.063 mm) for
normal gravity chromatography, or Merck 15111 silica 60 (0.015–
0.040 mm) for dry column vacuum chromatography.15 RP HPLC
purifications were done on a Waters Prep LC 4000 System using
a Xterra MS C18-column (10 lm, 7.8 × 150 mm) with a gradient
of 1.38% CH3CN per. min and UV detection at 254 nm. Ion
exchange HPLC analysis were performed on a LaChrom D-7000
System using a Gen-Pak Fax column (4.6 × 100 mm) with a
gradient of 1.07% 2 M NaCl per. min and UV detection at 254 nm.
NMR spectra (reference tetramethylsilane for dH and dC, external
85% H3PO4 for dP, J values are given in Hz) were run on a Varian
Mercury 300 MHz spectrometer. FAB MS data were obtained on a
JEOL HX 110/110 Mass Spectrometer with m-NBA as the matrix,
ES MS data on a Micromass Q-Tof Mass Spectrometer, and
MALDITOF MS data on a Bruker Ultraflex II TOF/TOF System
using a HPA matrix. Thermal melting temperature measurements
were performed on a Cary 300 Version 9.00 UV spectrometer.
=
(12H, m, Ar + C CH), 6.94 (4H, d, J 8.8, Ar), 5.07 (1H, t, J 5.3,
OH), 4.01 (2H, d, J 5.3, CH2OH), 3.87 (2H, s, CH2ODMT), 3.75
(6H, s, OCH3), 1.34 (9H, s, But). The (E) konfiguration was proven
by NOE (irradiation at CH2OH gave 5% enhancement of H-8).
NMR (CDCl3): dH 9.08 (1H, s, NH), 8.79 (1H, s, H-2), 8.13 (1H, s,
H-8), 7.97 (2H, d, J 8.2, tert-butylbenzoyl), 7.56–7.24 (11H, m, Ar),
(E)- and (Z)-1-[2-(Dimethoxytrityloxymethyl)-3-hydroxyprop-1-
enyl]thymine
To a solution of dry 1a (0.212 g, 1.00 mmol) in dry pyridine (4 ml)
was added dropwise a solution of DMTCl (0.34 g, 1.00 mmol)
in dry pyridine (4 ml), and the mixture was stirred in the dark
under N2 for 2 h at rt. Sat. aqueous NaHCO3 (1 ml) was added
and the solvents removed in vacuo. The residue was dissolved in
a mixture of CH2Cl2 (25 ml) and water (10 ml), the phases were
separated, the CH2Cl2 phase extracted with water (2 × 3 ml),
and the combined aq. phases extracted with CH2Cl2 (5 ml). The
CH2Cl2 phase was dried (MgSO4) and the solvent removed in
vacuo to give the crude product (0.54 g), a mixture of hydrolysed
DMTCl, the bis-, and the two mono-dimethoxytrityl derivatives,
which was resolved into the components by normal gravity column
chromatography, eluted with EtOAc–MeOH–Py 98 : 1 : 1 v/v/v,
to give the di-DMT derivative (0.176 g, 22%, Rf = 0.61), the (E)-
product (0.178 g, 35%, Rf = 0.45), and the (Z)-product (0.135 g,
=
6.94 (1H, s, C CH), 6.86 (4H, d, J 8.5, Ar), 4.31 (1H, t, J 6.7,
OH), 4.08 (2H, s, CH2ODMT), 3.96 (2H, d, J 6.7, CH2OH), 3.80
(6H, s, OCH3), 1.37 (9H, s, But). dC 164.5, 158.8, 156.8, 153.0,
151.8, 150.3, 144.7, 143.4, 139.0, 135.8, 130.7, 130.1, 128.1, 127.9,
127.2, 126.0, 122.7, 116.9, 113.4, 87.3, 64.5, 57.6, 55.4, 35.3, 31.2.
FAB+ MS: 684. 5 (M + H+ calc. 684.3). (Z): NMR (DMSO-
d6): dH 11.08 (1H, s, NH), 8.67 (1H, s, H-2), 8.25 (1H, s, H-8),
7.99 (2H, d, J 8.5, tert-butylbenzoyl), 7.59–7.07 (11H, m, Ar),
=
7.03 (1H, s, C CH), 6.77 (4H, d, J 8.4, Ar), 5.35 (1H, t, J 5.3,
OH), 4.30 (2H, d, J 5.3, CH2OH), 3.70 (6H, s, OCH3), 3.63 (2H, s,
CH2ODMT), 1.34 (9H, s, But). The (Z) konfiguration was proven
by NOE (irradiation at CH2ODMT gave 2% enhancement of H-
8). NMR (CDCl3): dH 9.0 (1H, br s, NH), 8.75 (1H, s, H-2), 7.97
1120 | Org. Biomol. Chem., 2006, 4, 1115–1123
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=
(2H, d, J 8.2, tert-butylbenzoyl), 7.96 (1H, s, H-8), 7.55–7.18 (11H,
br s, NCH C), 5.31 (1H, t, J 5.2, OH), 4.27 (2H, dd, J 5.2 and
1.5, CH2OH), 3.70 (6H, s, OCH3), 3.56 (2H, s, CH2ODMT), 2.74
(1H, septet, J 6.7, CH(CH3)2), 1.11 (6H, d, J 6.7, CH(CH3)2). The
(Z) configuration was proven by NOE (irradiation at CH2OH gave
=
m, Ar), 7.07 (1H, s, C CH), 6.77 (4H, d, J 9.1, Ar), 4.49 (2H, s,
CH2OH), 3.83 (2H, s, CH2ODMT), 3.75 (6H, s, OCH3), 1.37
(9H, s, But). dC 164.5, 158.9, 156.8, 153.2, 151.9, 149.8, 144.4,
142.6, 136.8, 135.4, 131.0, 130.1, 128.2, 128.1, 127.9, 127.3, 126.1,
122.4, 118.3, 113.5, 87.4, 63.8, 59.7, 55.5, 35.4, 31.4. FAB+ MS:
684.6 (M + H+ calc. 684.3).
=
5% enhancement of NCH C). dC 180.2, 158.1, 154.8, 148.1, 144.6,
140.2, 138.8, 135.1, 129.5, 128.9, 127.8, 127.4, 126.7, 117.4, 113.1,
85.9, 61.0, 58.6, 55.0, 34.8, 18.8. FAB+ MS: 610.2 (M + H+ calc.
610.3). (E): NMR (CDCl3): dH 12.24 and 9.90 (2 × 1H, 2 × br s,
NH), 7.78 (1H, s, H-8), 7.43–7.14 (9H, m, Ar), 6.80 (4H, d, J 8.8,
1-[3-Hydroxy-2-(hydroxymethyl)prop-1-enyl]-N-2-
isobutyrylguanine
=
Ar), 6.71 (1H, s, NCH C), 4.04 and 3.99 (2 × 2H, 2 × s, CH2O),
3.76 (6H, s, OCH3), 2.73 (1H, septet, J 6.8, CH(CH3)2), 1.20 (6H,
d, J 6.8, CH(CH3)2). FAB+ MS: 610.1 (M + H+ calc. 610.3).
To a stirred solution of 1-[3-benzyloxy-2-(benzyloxymethyl)prop-
1-enyl]-N-isobytyrylguanine (0.703 g, 1.44 mmol) in dry CH2Cl2
(25 ml) under N2 at 0 ◦C was added dropwise BCl3 (1 M in
CH2Cl2, 15 ml, 15 mmol). After stirring for 1 h at 0 ◦C, MeOH–
CH2Cl2 (1 : 1, v/v, 5 ml) was added, and the solvents removed in
vacuo. The residue was dissolved in MeOH–CH2Cl2 (1 : 1, v/v,
20 ml) and solid NaHCO3 was added to pH 7. The solids were
removed by filtration and washed with MeOH–CH2Cl2 (1 : 1,
v/v, 25 ml). The combined filtrates were concentrated in vacuo,
and the residue partitioned between H2O (75 ml) and CHCl3
(50 ml). The aqueous phase was extracted with CHCl3 (2 × 50 ml)
and evaporated in vacuo. Purification of the residue by normal
gravity column chromatography (elution with CH2Cl2–MeOH
9 : 1 and 4 : 1, v/v) yielded the title compound as colourless
crystals (0.390 g, 88%, Rf = 0.13 in CH2Cl2–MeOH (9 : 1)),
mp 205–206 ◦C (Found: C, 50.5; H, 5.5; N, 22.5. C13H17N5O4
requires C, 50.8; H, 5.6; N, 22.8%). NMR (DMSO-d6): dH 12.07
and 11.76 (2 × 1H, 2 × s, NH), 8.05 (1H, s, H-8), 6.80 (1H,
(E)-1-[3-(Dimethoxytrityloxy)-2-[2-cyanoethoxy(diisopropylamino)-
phosphinoxymethyl]prop-1-enyl]thymine (2a)
To a stirred solution of dry (Z)-1-[2-(dimethoxytrityloxymethyl)-
3-hydroxyprop-1-enyl]thymine (207 mg, 0.40 mmol) in dry CH2Cl2
(10 ml) under N2 was added dry Et3N (0.19 ml, 0.80 mmol), fol-
lowed by 2-cyanoethyl N,N-diisopropylphosphoramidochloridite
(114 mg, 0.48 mmol). After 2 h at rt the solution was diluted with
CH2Cl2 (10 ml), extracted with sat. aq. NaHCO3 (3 × 10 ml), dried
(MgSO4) and evaporated in vacuo to an oil. Purification by normal
gravity column chromatography (elution with EtOAc–Et3N 99 :
1 v/v) gave the product which was dissolved in EtOAc (2 ml),
◦
precipitated from hexane (100 ml) at 0 C, and lyophilised from
dry CH3CN (10 ml). Yield 194 mg (68%) of a colourless foam,
Rf 0.53 (EtOAc–Et3N 99 : 1 v/v). NMR (CDCl3): dH 7.40–7.18
(10H, m, Ar + NH), 7.14 (1H, s, H-6), 6.81 (4H, d, J 8.8, Ar), 6.70
=
=
(1H, s, NCH C), 4.38 (2H, AB of ABX system, D = 27.5 Hz, JAB
br s, NCH C), 5.19 and 5.06 (2 × 1H, 2 × t, 2 × J 5, OH),
=
13.4, JAX = JBX = 8.2, CH2OP), 3.90–3.53 (6H, m, CH2ODMT,
CH2CH2CN, CHMe2), 3.78 (6H, s, CH3O), 2.61 (2H, t, J 6.3,
CH2CH2CN), 1.71 (3H, s, T-CH3), 1.19 (12H, “t”, J 7.0). dC 164.1,
158.9, 150.2, 144.6, 140.7, 135.6, 133.3, 133.2, 130.2, 128.2, 128.1,
127.3, 125.3, 113.4, 110.4, 87.1, 64.0, 63.7, 58.9, 58.8, 55.5, 43.5,
43.4, 24.9, 24.8, 20.7, 20.6, 12.6. dP 149.8. FAB+ MS: 715.3 (M +
H+ calc. 715.3).
4.21 (2H, dd, J 5 and 1.8, CH2C CH), 3.99 (2H, d, J 5,
=
CH2C CH), 2.74 (1H, septet, J 6.7, CH(CH3)2), 1.11 (6H, d,
J 6.7, CH(CH3)2). dC 180.1, 154.9, 148.7, 148.2, 139.5, 139.2, 119.7,
115.6, 60.8, 56.2, 34.7, 18.8. FAB+ MS: 308.2 (M + H+ calc. 308.1).
(E)- and (Z)-2-N-Isobutyryl-9-[2-(dimethoxytrityloxymethyl)-3-
hydroxyprop-1-enyl]guanine
To a solution of 2-N-isobutyryl-9-[3-hydroxy-2-(hydroxymethyl)-
prop-1-enyl]guanine (0.383 g, 1.2 mmol) in dry pyridine (15 ml)
was added dropwise a solution of DMTCl (0.380 g, 1.1 mmol) in
dry pyridine (5 ml), and the mixture was stirred in the dark under
N2 for 48 h at rt. Sat. aqueous NaHCO3 (1 ml) was added and
the solvents removed in vacuo to give the crude product, a mixture
of hydrolysed DMTCl, the bis-, the two mono-dimethoxytrityl
derivatives, and the starting material. This mixture was subjected
to normal gravity column chromatography, eluted with EtOAc–
MeOH–Et3N (94 : 5 : 1, 89 : 10 : 1 and 79 : 20 : 1, v/v/v) to
separate the starting material (0.132 g, 35%, Rf = 0.04, the Rf
values given are in EtOAc–MeOH–Et3N (94 : 5 : 1, v/v/v)) from
the compounds containing DMT. The fractions containing the
bis- and the two mono-dimethoxytrityl derivatives were resolved
into the components by dry column vacuum chromatgraphy (0–
99% EtOAc in toluene, 10% increments, followed by 1–5% MeOH
in EtOAc, 1% increments, all containing 1% Et3N) to give the
di-DMT derivative (0.145 g, 13%, Rf = 0.58), the (Z)-product
(0.196 g, 26%, Rf = 0.26) and the (E)-product (0.125 g, 16%,
Rf = 0.32), all as slightly yellow foams. (Z): NMR (DMSO-d6): dH
12.06 and 11.75 (2 × 1H, 2 × s, NH), 7.73 (1H, s, H-8), 7.27–7.14
(5H, m, Ar), 6.94 (8H, AB system, D 96.0 Hz, J 8.8, Ar), 6.83 (1H,
(Z)-1-[3-(Dimethoxytrityloxy)-2-[2-cyanoethoxy(diisopropylamino)-
phosphinoxymethyl]prop-1-enyl]thymine (2aꢀ)
This compound was prepared in the same way as 2a from (E)-1-
[2-(dimethoxytrityloxymethyl)-3-hydroxyprop-1-enyl]thymine in
70% yield, Rf 0.13 (EtOAc–hexane–Et3N 49 : 49 : 2 v/v/v). NMR
(CDCl3): dH 7.39–7.14 (10H, m, Ar + NH), 7.12 (1H, q, J 1.2,
=
H-6), 6.76 (4H, d, J 8.8, Ar), 6.70 (1H, s, NCH C), 4.11–3.96
(2H, m, CH2OP), 3.80–3.36 (6H, m, CH2ODMT, CH2CH2CN,
CH(CH3)2), 3.72 (6H, s, CH3O), 2.41 (2H, t, J 6.3, CH2CH2CN),
1.87 (3H, d, J 1.2, T–CH3), 1.08–0.95 (12H, m, CH(CH3)2). dP
150.7.
(E)-9-[3-(Dimethoxytrityloxy)-2-[2-cyanoethoxy(diisopropylamino)-
phosphinoxymethyl]prop-1-enyl]-N-6-(4-tert-butylbenzoyl)adenine
(2b)
This compound was prepared in the same way as 2a from
(Z)-1-[2-(dimethoxytrityloxymethyl)-3-hydroxyprop-1-enyl]-N-6-
(4-tert-butylbenzoyl)adenine in 72% yield, Rf 0.22 (EtOAc–
hexane–Et3N 49 : 49 : 2 v/v/v). NMR (CDCl3): dH 9.16 (1H, br s,
NH), 8.77 (1H, s, H-2), 8.02 (1H, s, H-8), 7.99 (2H, d, J 8.5,
tert-butylbenzoyl), 7.54 (2H, d, J 8.5, tert-butylbenzoyl), 7.42–7.19
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=
(9H, m, Ar), 7.14 (1H, br s, C CH), 6.79 (4H, d, J 8.2, DMT),
4.53 (2H, AB of ABX system, D = 30.4 Hz, JAB 14.1, JAX = JBX
NH), 8.76 (1H, s, H-2), 8.10–8.08 (1H, m, Ar), 7.97 (1H, s, H-8),
7.96 (2H, d, J 8.5, Ar), 7.55 (2H, d, J 8.5, Ar), 7.40–7.14 (10H,
=
=
7.3, CH2OP), 4.00–3.62 (6H, m, CH2ODMT, CH2CH2CN,
CHMe2), 3.76 (6H, s, CH3O), 2.65 (2H, t, J 6.2, CH2CH2CN),
1.38 (9H, s, tert-butyl), 1.24 + 1.23 (12H, 2 × d, J 7.0, CHMe2).
dC 164.5, 158.7, 156.5, 153.0, 151.7, 149.7, 144.4, 142.5, 135.3,
135.3, 134.2, 134.1, 130.9, 130.0, 128.0, 127.9, 127.8 127.0, 125.8,
122.2, 118.6, 117.6 113.3, 87.0, 63.8, 63.6, 59.0, 58.7, 58.5, 55.3,
43.4, 43.2, 35.1, 31.2, 24.8, 24.7,24.7, 24.6 20.5, 20.4. dP 150.0.
FAB+ MS: 884.5 (M + H+ calc. 884.4).
m, Ar), 7.08 (1H, s, NCH C), 6.76 (4H, d, J 9.1, Ar), 6.50–6.44
(2H, m, Ar), 4.45 (2H, AB of ABX system, D = 30.7 Hz, JAB
14.1, JAX = JBX = 7.0, CH2OP), 3.94–3.59 (8H, m, CH2ODMT,
NCH2CH2O, CHMe2), 3.75 (6H, s, CH3O), 3.10 (3H, s, NCH3),
1.37 (9H, s, tert-Bu), 1.20 (6H, d, J 6.7, CHMe2), 1.17 (6H, d,
J 6.7, CHMe2). dP 148.3. FAB+ MS: 965.5 (M + H+ calc. 965.5).
(E)-1-[3-(Dimethoxytrityloxy){N-methyl-N-(2-pyridyl)-2-
aminoethoxy}(diisopropylamino)phosphinoxymethyl]prop-1-enyl]-
N-2-isobutyrylguanine (3e)
(E)-1-[3-(Dimethoxytrityloxy){N-methyl-N-(2-pyridyl)-2-
aminoethoxy}(diisopropylamino)phosphinoxymethyl]prop-1-
enyl]thymine (3a)
This compound was prepared in the same way as 3a from
(Z)-1-[2-(dimethoxytrityloxymethyl)-3-hydroxyprop-1-enyl]-N-2-
isobutyrylguanine in 35% yield after purification by normal
gravity column chromatography (first eluted with EtOAc–Et3N
98 : 2 v/v, then with EtOAc–MeOH–Et3N 93 : 5 : 2 v/v/v).
Colourless foam, Rf 0.32 (EtOAc–Et3N 98 : 2 v/v). NMR (CDCl3):
dH 8.05–8.03 (1H, m, Ar), 7.59 (1H, s, H-8), 7.45–7.16 (10H, m,
To a stirred solution of dry (Z)-1-[2-(dimethoxytrityloxymethyl)-
3-hydroxyprop-1-enyl]thymine (103 mg, 0.20 mmol) in dry
CH3CN (5 ml) under N2 was added [N-methyl-N-(2-pyridyl)-2-
aminoethyl] N,N,Nꢀ,Nꢀ-tetraisopropylphosphorodiamidite (90 mg,
ca. 88% pure, 0.27 mmol), followed by tetrazole (9 mg, 0.13 mmol).
After 2 h at rt the solution was concentrated in vacuo, and
the residue dissolved in EtOAc (10 ml), extracted with sat. aq.
NaHCO3 (2 × 5 ml), the NaHCO3 phase extracted with EtOAc
(5 ml), the EtOAc phase dried (MgSO4) and evaporated in vacuo
to an oil. The crude oil was purified by normal gravity column
chromatography, eluted with EtOAc–heptane–Et3N 58 : 40 : 2
v/v/v, to give the product (80 mg, 50%) as a colourless oil, Rf 0.50
(EtOAc–Et3N 98 : 2 v/v). NMR (CDCl3): dH 8.6 (1H, br, NH),
8.12–8.10 (1H, m, Ar), 7.42–7.18 (10H, m, Ar), 7.10 (1H, d, J 1.2,
=
Ar), 6.86 (1H, s, NCH C), 6.78 (4H, d, J 9.1, Ar), 6.58–6.49
(2H, m, Ar), 4.38 (2H, AB of ABX system, D = 35.5 Hz, JAB
14.7, JAX 8.5, JBX 7.9, CH2OP), 3.92–3.52 (8H, m, CH2ODMT,
NCH2CH2O, CHMe2), 3.77 (6H, s, CH3O), 3.15 (3H, s, NCH3),
2.57 (1H, septet, J 6.7, COCHMe2), 1.21–1.16 (18H, m, CHMe2).
dP 148.2. FAB+ MS: 891.1 (M + H+ calc. 891.4).
2-Cyanoethyl bis-[(E)-2-(dimethoxytrityloxymethyl)-3-
(1-thyminyl)prop-2-enyl] phosphite (4b)
=
H-6), 6.81 (4H, d, J 8.8, Ar), 6.65 (1H, s, NCH C), 6.51–6.47
To a solution of (E)-1-[2-(dimethoxytrityloxymethyl)-3-hydroxy-
prop-1-enyl]thymine (0.206 g, 0.40 mmol) and 2-cyanoethyl
N,N,Nꢀ,Nꢀ-tetraisopropylphosphorodiamidite (0.075 g, 80% pure,
0.20 mmol) in dry CH3CN (5 ml) was added tetrazole (0.035 g,
0.50 mmol). The mixture was stirred under N2 for 2.5 h at rt, and
concentrated in vacuo. The residue was dissolved in a mixture of
EtOAc (20 ml) and sat. aq. NaHCO3 (5 ml), the EtOAc phase
extracted with sat. aq. NaHCO3 (5 ml), the combined aq. phases
extracted with EtOAc (5 ml), and the combined EtOAc phases
dried (MgSO4) and evaporated in vacuo to an oil. The crude oil
was purified by normal gravity column chromatography, eluted
with heptane–EtOAc–MeOH–Py 50 : 43 : 5 : 2 v/v/v/v, to give,
after lyophilisation from CH3CN (10 ml) the product (0.183 g,
81%) as a colourless solid, Rf 0.10 (heptane–EtOAc–MeOH–Py
50 : 43 : 5 : 2 v/v/v/v). NMR (CDCl3): dH 9.0–8.9 (2H, br m,
NH), 7.43–7.18 (18H, m, Ar), 6.91 (2H, d, J 1.2, H-6), 6.82 (8H,
(2H, m, Ar), 4.33 (2H, AB of ABX system, D = 22.3 Hz, JAB
13.8, JAX = JBX = 7.0, CH2OP), 3.86–3.51 (8H, m, CH2ODMT,
NCH2CH2O, CHMe2), 3.77 (6H, s, CH3O), 3.09 (3H, s, NCH3),
1.71 (3H, d, J 1.2, T–CH3), 1.16 (6H, d, J 6.7, CHMe2), 1.15 (6H,
d, J 6.7, CHMe2). dP 148.1. FAB+ MS: 795.3 (M + H+ calc. 796.4).
(Z)-1-[3-(Dimethoxytrityloxy){N-methyl-N-(2-pyridyl)-2-
aminoethoxy}(diisopropylamino)phosphinoxymethyl]prop-1-
enyl]thymine (3aꢀ)
This compound was prepared in the same way as 3a from (E)-1-
[2-(dimethoxytrityloxymethyl)-3-hydroxyprop-1-enyl]thymine in
60% yield as a colourless foam, Rf 0.55 (EtOAc–Et3N 98 : 2 v/v).
NMR (CDCl3): dH 9.2 (1H, br, NH), 8.09 (1H, m, Ar), 7.47–
7.19 (11H, m, Ar + H-6), 6.84 (4H, d, J 8.8, Ar), 6.81 (1H, s,
=
NCH C), 6.49 (1H, dd, J 5.0 and 7.0, Ar), 6.42 (1H, d, J 8.8, Ar),
=
4.02 (2H, AB of ABX system, D = 22.4 Hz, JAB 12.0, JAX = JBX
=
d, J 8.8, Ar), 6.62 (2H, s, C CH), 4.23 (4H, d, J 7.0, CH2OP),
6.2, CH2OP), 3.82–3.62 (6H, m, CH2ODMT, NCH2CH2O), 3.78
(6H, s, CH3O), 3.54–3.41 (2H, m, CHMe2), 3.01 (3H, s, NCH3),
1.92 (3H, d, J 0.6, T–CH3), 1.09 (6H, d, J 7.0, CHMe2), 1.03 (6H,
d, J 7.0, CHMe2). dP 149.0. FAB+ MS: 796.4 (M + H+ calc. 796.4).
3.77 (16H, s, CH3O + CH2ODMT), 3.65 (2H, dt, J 7.0 and 6.2,
NCCH2CH2O), 2.35 (2H, t, J 6.2, NCCH2CH2O), 1.86 (6H, br s,
T–CH3). dC 163.9, 158.8, 149.9, 144.6, 140.3, 135.8, 134.0 (d, J 6),
130.1, 128.2, 128.1, 127.1, 124.5, 117.2, 113.4, 113.3, 110.9, 87.0,
63.1, 57.7 (d, J 11), 57.4 (d, J 11), 55.4, 20.2 (d, J 5), 12.4. dP 141.1.
FAB+ MS: 1128.6 (M + H+ calc. 1128.4).
(E)-1-[3-(Dimethoxytrityloxy){N-methyl-N-(2-pyridyl)-2-
aminoethoxy}(diisopropylamino)phosphinoxymethyl]prop-1-enyl]-
N-6-(4-tert-butylbenzoyl)adenine (3b)
Bis-[(E)-2-(dimethoxytrityloxymethyl)-3-(1-thyminyl)prop-2-enyl]
N-methyl-N-(2-pyridyl)-2-aminoethyl phosphite (4f)
This compound was prepared in the same way as 3a from
(Z)-1-[2-(dimethoxytrityloxymethyl)-3-hydroxyprop-1-enyl]-N-6-
(4-tert-butylbenzoyl)adenine in 70% yield as a colourless foam,
Rf 0.60 (EtOAc–Et3N 98 : 2 v/v). NMR (CDCl3): dH 8.9 (1H, br,
To a solution of 3aꢀ (90 mg, 0.11 mmol) in dry CH3CN (2 ml)
was added (E)-1-[2-(dimethoxytrityloxymethyl)-3-hydroxyprop-1-
enyl]thymine (51.5 mg, 0.10 mmol) and tetrazole (7 mg, 1.0 mmol).
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Table 3 Coupling cycle to introduce N*, 0.25 lmol scale
Reagent
Time
3% CCl3COOH in CH2Cl2
0.1 M Pri2NH in CH3CN
Wash CH3CNa
30 s + (5 s wait + 10 s CH3CN) × 2
(15 s + 10 s wait) × 3
3 min
0.2 ml 0.05 M amidite in CH3CN + 0.3 ml
0.50 M tetrazole in CH3CN
Wash CH3CN
6 min manually
1 min
2 min manually
2 min
10% H2O + 2% CAP Ab in THF
Wash CH3CN
CAP A + CAP Bc, 1:1
(30 s + 30 s wait) × 4
Wash CH3CN
1 min
80% ButOOH in But2O, 0.5 M in CH2Cl2–acetone 1 : 1
Wash CH3CN
(6 s + 90 s wait) × 2
1.5 min
Wait (elimination of phosphate protection)
Wash CH3CN
30 min
0.5 min
a Wash CH3CN is a combination of flush, wash, and wait steps. b CAP A = 10% (CH3CO)2O + 10% lutidine in THF. c CAP B = 16% N-methylimidazole
in THF.
The mixture was stirred under N2 for 2.5 h at rt, and concentrated
in vacuo. The residue was dissolved in a mixture of EtOAc (20 ml)
and sat. aq. NaHCO3 (10 ml), the aq. phase extracted with EtOAc
(5 ml), and the combined EtOAc phases dried (MgSO4) and
evaporated in vacuo to an oil. The crude oil was purified normal
gravity column chromatography, eluted with EtOAc–Py 99 : 1 v/v,
to give, after evaporation with CH3CN (2 × 5 ml) the product
(92 mg, 76%) as a colourless foam, Rf 0.42 (EtOAc–Py 99 : 1
v/v). NMR (CDCl3): dH 8.6 (2H, br, NH), 8.04–8.01 (1H, m, Ar),
7.41–7.16 (19H, m, Ar), 6.90 (2H, s, H-6), 6.80 (8H, d, J 8.8, Ar),
Solid phase oligonucleotide synthesis
˚
The syntheses were performed on 500 A CPG supports on
a Biosearch 8750 DNA Synthesizer. The coupling cycle used
for the modified phosphoramidites 3a, 3b, and 3e is given in
Table 3.
Acknowledgements
We thank Ms Suzy W. Lena, Department of Chemistry, University
of Southern Denmark at Odense, and Ms Anette W. Jørgensen,
Department of Biochemistry B, University of Copenhagen, for
expert technical assistance.
=
6.62 (2H, s, C CH), 6.50–6.46 (1H, m, Ar), 6.36–6.33 (1H, m,
Ar), 4.10 (4H, d, J 6.4, CH2OP), 3.78–3.74 (18H, m, CH3O +
CH2ODMT + NCH2CH2O), 3.60 (2H, t, J 6.2, NCH2CH2O),
2.88 (3H, s, CH3N), 1.80 (6H, s, T–CH3). dP 141.1. FAB+ MS:
1209.2 (M + H+ calc. 1209.5).
References
Sodium di-[(E)-2-(hydroxymethyl)-3-(1-thyminyl)prop-2-enyl]
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=
NMR. NMR (D2O): dH 7.42 (2H, s, H-6), 6.61 (2H, s, C CH),
4.40 (4H, d, J 6.2, CH2OP), 4.30 (4H, s, CH2OH), 1.88 (6H, s,
T–CH3). dC 167.2, 152.2, 142.8, 137.3 (J 7.8), 125.1, 111.6, 61.3,
60.4 (J 5.2), 12.0. dP 1.1. FAB− MS: 485.3 (M− calc. 485.1).
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