2′-O-aminoethyl oligoribonucleotides containing novel base analogues: Synthesis and triple-helix formation at pyrimidine/purine inversion sites

Article abstract of DOI:10.1002/ejoc.200600182

The synthesis of a common sugar intermediate for the 2′-aminoethyl- ribonucleoside synthesis in 9 steps and an overall yield of 33 % starting from D-ribose is described. This intermediate was successfully used for the synthesis of the 2′-aminoethyl-ribonucleosides containing the bases thymine (t), 5-methylcytosine (c), 5-methyl-2-pyrimidinone (x), 2-aminopurine (ap) and guanine (g). These were subsequently transformed into the corresponding cyanoethyl phosphoramidite building blocks for oligonucleotide synthesis. 2′-Aminoethyl oligonucleotide 15-mers were prepared with a DNA synthesizer, and an optimized post-synthetic deprotection protocol has been developed which prevents cyanoethylation of the 2′-amino side chains during conventional ammonia deprotection. A series of fully modified, triplex forming 2′-aminoethyl oligoribonucleotides (2′AE-TFOs) were prepared in which x was designed to bind to CG inversion sites and ap as well as g to TA inversion sites on a double-helical DNA target. The affinity of x-CG base-triple formation in different sequence contexts was assessed by UV- and CD melting analysis. It was found that TFO 15-mers containing up to 5 x residues still form stable triplexes even in the case where all x residues are consecutively arranged in the TFO. The nearest neighbor properties of x have been probed and it was found that triplex stability decreases in the local sequence order -txt- > -txc- ? -cxc-. TFOs containing ap and g were found to bind to their DNA targets with TA inversion sites with less affinity and less selectivity compared to TFOs containing the corresponding deoxyribonucleosides, irrespective whether they were incorporated in TFOs with a DNA or a 2′-AE-RNA backbone. The obtained data suggest that guanine-TA or aminopurine-TA base-triple formation is strongly sensitive to TFO conformation and more efficient in TFOs with a DNA than an RNA backbone. Wiley-VCH Verlag GmbH & Co. KGaA, 2006.

Full text of DOI:10.1002/ejoc.200600182

FULL PAPER
DOI: 10.1002/ejoc.200600182
2Ј-O-Aminoethyl Oligoribonucleotides Containing Novel Base Analogues:
Synthesis and Triple-Helix Formation At Pyrimidine/Purine Inversion Sites
Sabrina Buchini[a] and Christian J. Leumann*[a]
Keywords: Antisense agents / Antigene agents / DNA recognition / oligonucleotides/ DNA triple helix
The synthesis of a common sugar intermediate for the 2Ј-
aminoethyl-ribonucleoside synthesis in 9 steps and an overall
yield of 33% starting from D-ribose is described. This inter-
sequence contexts was assessed by UV- and CD melting
analysis. It was found that TFO 15-mers containing up to 5 x
residues still form stable triplexes even in the case where all
x residues are consecutively arranged in the TFO. The near-
est neighbor properties of x have been probed and it was
found that triplex stability decreases in the local sequence
order -txt- Ͼ -txc- ϾϾ -cxc-. TFOs containing ap and g were
found to bind to their DNA targets with TA inversion sites
with less affinity and less selectivity compared to TFOs con-
taining the corresponding deoxyribonucleosides, irrespective
whether they were incorporated in TFOs with a DNA or a 2Ј-
AE-RNA backbone. The obtained data suggest that guanine-
TA or aminopurine-TA base-triple formation is strongly sen-
sitive to TFO conformation and more efficient in TFOs with
a DNA than an RNA backbone.
mediate was successfully used for the synthesis of the 2Ј-ami-
noethyl-ribonucleosides containing the bases thymine (t), 5-
methylcytosine (c), 5-methyl-2-pyrimidinone (x), 2-aminopu-
rine (ap) and guanine (g). These were subsequently trans-
formed into the corresponding cyanoethyl phosphoramidite
building blocks for oligonucleotide synthesis. 2Ј-Aminoethyl
oligonucleotide 15-mers were prepared with a DNA synthe-
sizer, and an optimized post-synthetic deprotection protocol
has been developed which prevents cyanoethylation of the
2Ј-amino side chains during conventional ammonia depro-
tection. A series of fully modified, triplex forming 2Ј-aminoe-
thyl oligoribonucleotides (2ЈAE-TFOs) were prepared in
which x was designed to bind to CG inversion sites and ap
as well as g to TA inversion sites on a double-helical DNA
target. The affinity of x-CG base-triple formation in different
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2006)
Introduction
Triple-helix forming oligonucleotides (TFOs) received constrained TFOs (e.g. locked nucleic acids, LNA) increase
considerable attention in the past for their ability to target the thermal and thermodynamic stability of corresponding
double-stranded DNA sequence-specifically and to modu- parallel triplexes at neutral pH, presumably by entropic sta-
late gene expression.[1–6] Two triple-helical binding motifs bilization.[10] Another successful approach consists in the
are known. In the parallel binding motif a pyrimidine-rich dual recognition of a DNA duplex by specific base–base
third strand runs parallel to the target purine strand and contacts and concomitant non-specific salt-bridge forma-
forms Hoogsteen C+-GC and T-AT base triples. In the anti- tion between a positively charged ammonium function on
parallel motif a purine-rich third strand is oriented antipar- the TFO and a duplex phosphate group, as in the case of
allel to the duplex purine strand and forms reverse Hoogs- the 2Ј-O-(2-aminoethyl)-RNA (2Ј-AE-RNA) developed by
teen A-AT or T-AT and G-GC base triples. Despite of the Cuneoud and co-workers.[11] TFOs carrying this substitu-
potential benefits, a number of drawbacks limite the use of tion showed enhanced kinetics of triplex formation and a
TFOs. Among those are the low thermal stability of tri- strongly increased stability of the resulting complex at phys-
plexes and the restriction of duplex recognition to homopu- iological pH and at low Mg2+ concentration. The dual re-
rine/homopyrimidine sequence tracts.[7–9]
In the last years new concepts have successfully been de- sugar modifications by other groups and by ourselves.[12,13]
cognition concept has meanwhile been applied to different
veloped and applied in order to overcome the stability
problem. It was shown, for example, that conformationally
The sequence restriction problem is by far more difficult
to solve because of the poor recognition of pyrimidine
bases, which display only one H-bond donor- or acceptor
site available for TFO binding in the major groove. The
most stable base triples with natural bases in the TFO that
recognize pyrimidines in the parallel motif are the T-CG
[a] Department of Chemistry & Biochemistry, University of Bern,
Freiestrasse 3, 3012 Bern, Switzerland
Fax: +41-31-631-3422
E-mail: leumann@ioc.unibe.ch
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Figure 1. Chemical structures of the 2Ј-AE nucleotides prepared and used in this study together with the putative x-CG, and g/ap-TA
base triple.
and the G-TA triple.[14–16] Their structures were firmly es-
Results
Sugar Building Block
tablished by NMR analysis.[17,18] A search for novel nucleo-
base analogues identified a few interesting candidates as, for
example, 5-methyl-2-pyrimidinone[19] or 2-pyridone[20] that
The synthesis of 2Ј-aminoethoxy (2Ј-AE) nucleosides was
recognize CG inversion sites with high selectivity. Triplex first reported in 1998 by Cuenoud and co-workers.[11] Their
stability in these cases, however, is beyond what is needed synthesis of the 2Ј-O-aminoethyl-modified thymidine and 5-
for generating a biological response. Selective TA recogni- methylcytidine building blocks started from protected ri-
tion is even more challenging because of the presence of the bothymidine. Initial attempts to prepare the monomer x by
methyl group on thymine which sterically clashes into the either modifying the base of a 2Ј-aminoethyl ribothymidine
backbone of the TFO. Modified bases are available also intermediate or by coupling 5-methyl-2-pyrimidinone to
here,[21–23] but improvement, especially in terms of selectiv- peracetylated ribose before introduction of the aminoethyl
ity, is desperately needed.
side-chain failed because of the instability of the base and/
The combination of such modified bases with the dual or the glycosidic bond during the following transforma-
recognition concept was recently found to be a viable solu- tions. An alternative pathway was therefore developed
tion to the affinity problem in pyrimidine–purine inversion based on the sugar intermediate 10, which allows the intro-
site recognition. In preliminary work, we have shown that duction of various bases into the 2Ј-O-aminoethyl nucleo-
the combination of the CG selective base 5-methyl-2-pyrim- side scaffold at a late stage during synthesis. (Scheme 1).
idinone with the 2Ј-AE-RNA backbone is formidably
The known methylriboside 2 was initially prepared from
suited to overcome the low affinty of TFOs without com- -ribose (1) with 2% HCl or concd. H2SO4 in MeOH ac-
promising with selectivity, thus extending the amenable tri- cording to known protocols[28,29] but was only obtained in
plex recognition range from two to three base pairs.[24,25] poor yields (20–50%). The use of a resin-bound acid
More recently, Fox and co-workers found, that by careful (Dowex-50 H+) in MeOH at 4 °C overnight greatly en-
combination of sugar and base-modified nucleosides, in- hanced the practicality of this reaction and lead to riboside
cluding the 2Ј-AE modification, all four natural bases can 2 (predominantly the β anomer) in almost quantitative
be recognized at nearly neutral pH.[26,27]
yield. Compound 2 was then TIPS-protected in pyridine to
Here we present now a full account on the synthesis of afford the intermediate 3 (61%), which was subsequently
the novel 2Ј-AE nucleosides x, ap and g, together with an alkylated using bromo methylacetate (Ǟ 4). Reduction
alternative, more convergent synthesis of the already known of the methyl ester with LiBH4 in THF/MeOH lead to
2Ј-AE monomers t and c (Figure 1). Furthermore, we re- compound 5 in excellent yield (99%). After activation
port on the preparation of the corresponding 2Ј-AE oligori- of the alcohol by tosylation (Ǟ 6) and subsequent sub-
bonucleotides by standard phosphoramidite chemistry, an stitution with LiN3 (Ǟ 7), the azide group was reduced to
improved deprotection protocol for fully modified 2Ј-AE the amine 8 under Staudinger conditions. Subsequent TFA
oligoribonucleotides as well as on the CG recognition prop- protection afforded compound 9 in very good yield (93%).
erties of x in various sequence contexts and the TA-recogni- Intermediate 9 was then treated with a mixture of
tion properties of ap and g.
Ac2O/AcOH/H2SO4 and converted into the triacetate
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Scheme 1. Synthesis of the sugar building block 9. a) Dowex-50 H+, MeOH, overnight, 4 °C. b) TIPS-Cl, pyridine, 1 h, room temp. c)
BrCH2CO2Me, NaH, DMF, 7 h, –10 °C Ǟ room temp. d) LiBH4, THF/MeOH, 40 min, 0 °C. e) TsCl, NEt3, DMAP, CH2Cl2, overnight,
room temp. f) LiN3, DMF, 1 h, 100 °C. g) i. Ph3P, pyridine, 3 h, room temp. ii. NH3 33%, 4 h, room temp. h) Et3N, CF3CO2Et, 4 h,
room temp. i) Ac2O/AcOH/H2SO4, overnight, room temp.
10 (α/β 3:1), the central intermediate for the subsequent
nucleosidations.[30,31]
Synthesis of 2Ј-AE Nucleosides and Phosphoramidite
Building Blocks
For the preparation of nucleoside x the corresponding
base 13 had to be synthesized in two steps starting from
commercially available propionaldehyde diethyl acetal (11)
(Scheme 2).[32,33]
A modified procedure involving the
Vilsmeier–Haack–Arnold acylation[34] was used to prepare
a mixture of 12a and 12b in a ratio of 1:2, respectively. The
poor yield in this step could be compensated for by per-
forming the reaction at a relatively high scale. This mixture
was then treated with urea in the presence of Na in EtOH,
followed by acidification to afford compound 13 in accept-
able yield. The benzoyl-protected methylcytosine 17 was
available in 3 steps from thymine (14) in an overall yield of
40% using a modified literature procedure.[35]
Scheme 2. Synthesis of bases. a) i. POCl3, DMF, 2 h, 70 °C. ii. H2O,
K2CO3, overnight. b) i. Na, Urea, EtOH, 2d, reflux. ii. HCl 25%,
1 d, reflux. c) POCl3, Et3N, 1H-1,2,4-triazole, CH3CN, 5–7 h, room
temp. Ǟ reflux. d) NH3 33%, 3 h, reflux. e) Bz2O, DMAP, CH3CN,
7 h, reflux.
Coupling of 13, thymine (14) and 5-methyl-N4-benzoyl-
cytosine (17) with the key intermediate 10 under phosphoramidites were obtained as pure β anomers and the
1
Vorbrüggen conditions afforded the corresponding nucleo- configuration at the anomeric center was confirmed by H
sides 18–20 in respectable yields (Scheme 3). After deacety- NMR-NOE experiments on the level of the acetylated nu-
lation, tritylation and phosphitylation, the phosphoramid- cleosides or of the phosphoramidites (see Experimental Sec-
ite building blocks 27–29 were obtained. While these stan- tion).
dard transformations worked out very well in the x- and t
The 2Ј-AE-aminopurine ap nucleoside was prepared fol-
series we noted that the deacetylation of the c intermediate lowing a nucleosidation procedure developed earlier in our
20 proved more tedious than expected because of the insta- laboratory.[36] The N2-acetyl-protected base 2-amino-6-
bility of the benzoic amide function at N4. After consider- chloro-9H-purine[37] was persilylated with BSA and sub-
able optimization, deacetylation in an ethanolic solution of sequently coupled to the sugar 10 with TMSOTf as Lewis
NaOH in pyridine/EtOH for a few minutes worked best and acid to afford the nucleoside 30 in good yield (Scheme 4).
led to the desired compound 23 in acceptable yield. All After dechlorination by catalytic hydrogenation, the pro-
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2Ј-O-Aminoethyl Oligoribonucleotides Containing Novel Base Analogues
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Scheme 3. Synthesis of nucleosides and phosphoramidites 27–29. a) 13, BSA, SnCl4, CH3CN, 2 h, 0 °C. b) 14, BSA, SnCl4, CH3CN, 4 h,
0 °C Ǟ room temp. c) 17, HMDS, TMS-Cl, TMS-Tf, CH3CN, 2 h, room temp. d) Na2CO3, MeOH, 15 min to 2 h, room temp. e) 50%
ethanolic solution of NaOH (2  NaOH/EtOH, 1:1), pyridine/EtOH, 1:2, 5–20 min, room temp. f) DMT-Cl, pyridine, 3 to 6 h, room
temp. g) iPr2NEt, CEP-Cl, THF, 2 to 3 h, room temp.
Scheme 4. Synthesis of phosphoramidites 34 and 37. a) i) 2-aminoacetyl-6-chloropurine, BSA, ClCH2CH2Cl, 80 °C, 20 min. ii) 1,
TMSOTf, toluene, 80 °C, 3 h. b) Et3N, Pd/C, H2, EtOAc, room temp., 5 h. c) Na2CO3, MeOH, 1 h, room temp. d) DMTr-Cl, mol. sieves,
pyridine, 4 h, room temp. e) iPr2NEt, CEP-Cl, mol. sieves, THF, 1.5 to 3 h, room temp., 83%. f) NC(CH2)2OH, Cs2CO3, THF, 0 °C, 2 h.
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tected nucleoside 31 was obtained. Assignment of the β-N9- oligonucleotides. One problem is the known lability of the
1
configuration was achieved by 13C NMR, H NMR-NOE 5-methyl-2-pyrimidinone base in x when treated with
experiments and by comparison with literature data.[36] Af- concd. ammonia.[41,42] The lability is most likely due to nu-
ter removal of the acetyl groups, the ap nucleoside 32 was cleophilic addition of ammonia at C6 followed by depyrimi-
converted into the phosphoramidite 34 under standard con- dination, leading to an abasic site prone to chain cleavage.
ditions.
This depyrimidination and strand cleavage could mostly be
The regioselectivity for the glycosylation of guanine un- circumvented by using concd. NH3 at room temperature for
der Vorbrüggen conditions is a known problem in nucleo- 2 hours or by using a 40% aqueous solution of CH3NH2 at
side synthesis. Aware of the fact that the N9 isomer is fa- room temperature for 3 hours.
vored under thermodynamic control, we initially performed
Another complication was encountered during deprotec-
the nucleosidation reaction using similar conditions as for tion of fully modified 2Ј-AE oligonucleotides. The RP-
the synthesis of 31 with TMSOTf as Lewis acid in toluene HPLC traces of crude oligonucleotides indicated a compli-
at 80 °C for 2–6 hours and three different persilyated nuc- cated mixture of products, which could not be explained on
leobases: N2-(acetyl)guanine, N2-acetyl-O6-(diphenylcar- the basis of the trityl assays of the corresponding syntheses
bamoyl)guanine and N2-isobutyryl-O6-[2-(p-nitrophenyl)- (Figure 2, a).
ethyl]guanine. The latter two nucleobase derivatives are
MALDI-TOF MS analysis of the major peaks revealed
known for their ability to direct the nucleosidation towards a mixture of oligonucleotides with masses that were higher
the N9 position.[38,39] Unfortunately, in all cases only anom- by m/z 53 or multiples of it compared to the expected mass.
eric mixtures of N7 nucleosides were observed. Increasing We hypothesized that the increase in molecular mass was
the reaction time from 6 to 15 hours in the case of N2-(ace- due to one or multiple additions of acrylonitrile, arising
tyl)guanine as base lead to the corresponding β-N9 nucleo- from the deprotection of the cyanoethyl phosphate triester,
side, but only in 15% yield. Clearly an alternative had to be to the already deprotected amine function of the aminoe-
found.
Rosenbohm et al. reported the preparation of guanine
thyl side chain by Michael addition (Scheme 5).
nucleosides starting from 2-amino-6-chloro-9H-purine de-
rivatives by nucleophilic substitution of chlorine with cyan-
oethanol under basic conditions followed by in situ β elimi-
nation.[40] We applied these conditions to the intermediate
30 and isolated the O-deacetylated guanine nucleoside 35 in
a remarkable 58% yield. Subsequent transformation into
the phosphoramidite 37 proceeded smoothly on standard
protocols.
Scheme 5. Proposed mechanism for the reaction of acrylonitrile
with the amino side chain.
Two different strategies were tested in order to prevent
this alkylation of the side chains by acrylonitrile during the
ammonia treatment. The use of acrylonitrile scavengers
Synthesis, Deprotection and Purification of 2Ј-AE
Oligonucleotides
2Ј-AE-RNA oligonucleotides were synthesized on a (like for instance thiophenol and tBuNH2) together with
1.0 µmol scale on an automated DNA synthesizer using NH3 was not satisfactory. Thus a two-step deprotection
standard phosphoramidite chemistry. Higher concentra- procedure was developed consisting of removal of the cyan-
tions (0.12 ) of the 2Ј-AE phosphoramidites were used for oethyl phosphate protecting groups via β elimination with
obtaining coupling yields Ͼ95%. Various difficulties were a non-nucleophilic, sterically hindered base, like TBD or
encountered during the deprotection of the 2Ј-AE-RNA DBU, prior to standard deprotection of the remaining pro-
Figure 2. RP-HPLC traces of TFO T2: comparison between two different deprotection protocols. a) standard: NH3 33%, room temp.,
2 h. b) modified: DBU 1  in CH3CN, room temp., 2 h followed by NH3 33%, room temp., 2 h.
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tecting groups and cleavage from the solid support with limitations of the generally very strong CG recognition by
NH3. Indeed these new, fast and mild conditions (DBU 1  x in fully modified AE-TFOs it was necessary to address
or TBD 1  in CH3CN, room temp., 2 h followed by NH3 possible sequence effects. Therefore, a further series of 2Ј-
33%, room temp., 2 h) allowed the exclusive formation of AE-RNA TFOs were investigated with the aim of studying
the expected deprotected oligonucleotides (Figure 2, b).
nearest neighbor effects of the bases 3Ј and 5Ј to x on tri-
A further property of fully modified 2Ј-AE oligonucleo- plex stability. Sequence information and Tm data of the in-
tides, worth to mention, is their low solubility at neutral vestigated TFOs are summarized in Table 1.
pH, due to their zwitterionic nature. The handling of fully
With a UV melting-curve analysis we found that only
modified 2Ј-AE-RNA oligonucleotides proved therefore to TFO T4 bound to its DNA target with relatively low affin-
be quite difficult and often resulted in poor yields for that ity compared to all other TFOs. We reasoned that local
reason. We found it advantageous to heat stock solutions repulsion of the accumulated positive charges at consecu-
up to 90° for a few minutes before handling.
tive c residues in T4 might be responsible for this. It is
known that contiguous cytosine units in a TFO decrease
the affinity of the third strand both for RNA and for DNA
TFOs.[43] However, sequence effects on 2Ј-AE-RNA/DNA
Recognition of CG Inversion Sites
Tm data and selectivity studies with the TFOs T1–T3 interactions even within the context of canonical base re-
were described before[25] and are reproduced in Table 1 for cognition (t-AT, c+-G-C) have not been addressed so far in
comparison only. In order to further explore scope and the literature. In order to determine whether this Tm de-
Table 1. Tm data for third-strand melting from UV melting experiments (λ = 260 nm).[a]
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pression is base- or backbone-dependent we prepared the curve of T5 at 220 nm (Figure 3, top left) led to a sigmoidal
TFO T7 and, as a DNA control, TFO T6. The melting curve with an approximated Tm value of 66 °C, which is in
profile of the natural sequence T6 showed a relatively low good agreement with the value determined by UV melting
Tm around 34 °C, which was still higher than the value de- experiments. Temperature-dependent CD measurements
termined for T4 (∆Tm = ca. –14 °C). Interestingly the fully were performed as well on triplex T8, containing three con-
modified AE-TFO T7 formed a triplex (confirmed by gel tiguous x units (Figure 3, bottom left). Also here a Tm of
electrophoresis and CD experiments, data not shown) hav- 66 °C, comparable to that measured by UV experiments
ing a much higher stability than the corresponding duplex (Table 1) was observed. The same experiment was repeated
(∆Tm = +11 °C). On the basis of these observations we con- for triplex T9, containing five x units in a row. Monitoring
clude that not the backbone but the base modification ac- of the temperature dependence of the CD intensity at
counts for the sequence effect observed with TFO T4. Thus 215 nm (Figure 3, top right) resulted again in a sigmoidal
as a rule of thumb it emerges that x binds very strongly to curve with a Tm comparable to that of the UV melting
CG interruptions when placed between two thymines, still curve. In all cases the CD spectra of the triplexes differed
strongly when placed between one thymine and one methyl- markedly from the calculated sum of the spectra of the sin-
cytosine and relatively weakly when inserted between two gle strands and the duplexes alone at room temperature and
5-methylcytosines as nearest neighbor bases. Interstingly they matched only at 85 °C, thus validating the triplex spec-
enough x-residues at the 3Ј- or 5Ј-side of x, as in the case ificity for the observed transitions.
of the consecutively modified TFOs T8 and T9 are also well
tolerated.
In the UV melting curves, the determination of third-
strand dissociation was not completely unambiguous in
TA Recognition
many cases because of considerable overlap with target du-
We tested the TA recognition properties of the 2Ј-AE
plex melting. While we have already proven triplex forma- nucleosides ap and g and compared them to the corre-
tion by gel shift assays for the cases T1–T3,[17] we were in sponding deoxy-TFOs carrying a deoxynucleoside with the
need for additional proof in the cases of T5, T8, and T9. same base. Table 2 contains the sequence information and
CD spectroscopy is perfectly suited for monitoring triplex affinity data for the triplexes investigated. T10 is a control
formation. Especially the range around 220 nm is very sen- TFO with the DNA backbone, containing a deoxy G resi-
sitive for triplex structures, which typically show strong due instead of one of the modifications. As expected this
negative cotton effects while duplexes do not. A CD melting TFO showed the highest affinity towards a target where G
Figure 3. CD melting curves of triplex T5 (top, left) T8 (bottom left) and T9 (top, right) at the indicated wavelength. Buffer: c (single
strand) = 1.6 µ (T5), 3.8 µ (T8) and 1.6 µ (T9) in 10 m sodium cacodylate, 100 m NaCl, 0.25 m spermine, pH 7.0.
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is opposite the TA base pair with a third strand Tm of be observed while no such transition occurred in the case
41 °C. All other possible base pairs are discriminated by of T15 (g).
5–11 °C in Tm. TFO T11 is again a deoxyoligonucleotide
containing a 2Ј-deoxy-2-aminopurine (AP) nucleoside. Rel-
ative to guanine it preserves the 2-amino function necessary
for T recognition (Figure 1) but lacks the O6 carbonyl
Discussion and Conclusions
group. Also in this case the priority binding site of AP is a
We demonstrated before that the base 5-methyl-2-pyrimi-
TA base pair with a Tm of 38 °C. All other base pairs are dinone in the context of parallel binding deoxyribo-TFOs
again discriminated by 4–6 °C. From these experiments we recognizes a CG base pair selectively albeit with low affin-
confirm the high selectivity of G for a corresponding TA ity.[14] We assume that this base recognizes cytosine by one
base pair in a parallel TFO with the DNA backbone also conventional H-bond probably with the assistance of a non-
in this particular sequence context. Furthermore we find conventional C–H···O hydrogen bond (Figure 1). We rea-
that AP can act as a substitute for G showing essentially soned that the combination of this base with a 2Ј-AE ribose
the same recognition pattern albeit with slightly lower affin- unit, as in x, might enhance triplex stability due to binding
ity and lower selectivity.
assistance from the aminoethyl side chain. Fully aminoethy-
In a next step, we investigated DNA-TFOs containing lated TFOs containing x confirmed the high selectivity for
one insertion of either a g or an ap unit (T12 and T13). We CG recognition and, remarkably enough, bound very
observed a drop in Tm by 9 °C and 4 °C, respectively, when strongly to their duplex target even in the presence of five
positioning these residues opposite to a TA base pair. With x modifications corresponding to 33% pyrimidine content
this drop, the selectivity relative to any of the Z/W base in the target site.[25] In this study, we further confirmed the
pairs in the duplex target is abolished, indicating no specific high affinity of x for CG inversion sites in different se-
g-TA or ap-TA interaction. In order to test the effect quence contexts with up to 5 x residues in a 15mer TFO.
of multiple substitutions in a fully modified 2Ј-AE-TFO, Interestingly, we found that the distribution of x within the
T14 and T15 were hybridized to the corresponding DNA sequence (scattered or consecutive) did not much alter the
target showing TA base pairs opposite the g or ap residues. affinity pattern of a corresponding TFO. On the other
Only in the case of T14 (ap) could third strand melting hand, it appeared that nearest neighbor bases play an im-
Table 2. Sequence information and Tm data for third-strand melting from UV melting experiments (λ = 260 nm).[a]
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tilled before use. Reagents were purchased from Fluka AG, Aldrich
and Merck (highest quality available). NMR spectra were mea-
sured on a Bruker AC-300 or a Bruker DRX-400 spectrometer. 1H
NMR spectra were recorded at 300 MHz or 400 MHz: δ in ppm
relative to residual non- or partially deuterated solvent (CHCl3 =
7.27, [D5]DMSO = 2.50, CD2HOH = 3.35, HDO = 4.65), J in Hz.
13C NMR spectra were recorded at 75 MHz or 100 MHz: δ in ppm
relative to non- or partially deuterated solvent (CHCl3 δ = 77.00,
[D5]DMSO δ = 39.70, CD2HOH δ = 49.30). 19F NMR spectra
were recorded at 376 MHz: δ in ppm relative to 0.1% TFA/acetone
portant role, and triplex stability decreases in the following
local sequence context: -txt- Ͼ -txc- ϾϾ -cxc-. From our
investigations we can exclude this sequence dependence to
be due to the 2Ј-AE-RNA backbone, as it is not observed
in AE-TFOs with only regular bases c and t. Thus it is a
direct consequence of the base modification. With the ex-
periments reported here we have been able to define the
basic rules for designing high-affinity TFOs for recognition
of CG inversion sites by x. Further work is now concerned
with its use in triplex-mediated regulation of cellular pro- as external standard. 31P NMR spectra were recorded at 162 MHz:
δ in ppm relative to 85% H3PO4 as external standard. Difference
NOE experiments were recorded at 400 MHz. ESI-MS: VG plat-
form Fisons instruments. HR-MS: Applied Biosystem, Sciex
QSTAR Pulsar. TLC was performed using pre-coated silica gel
plates SIL-G-25 UV254 from Macherey–Nagel. Visualization by
UV (254 nm and/or 366 nm) and/or by Ce(SO4)2/phosphomolybdic
acid staining.
cesses.
Contrary to the beneficial effect of the 2ЈAE modifica-
tion for CG recognition were the findings with TFOs con-
taining ap and g residues targeted to TA inversion sites. The
pairing properties of ap and g to all four Watson–Crick
base pairs on a DNA target duplex clearly showed that
these bases in the context of the 2ЈAE-backbone have lost
their selectivity and affinity towards the TA inversion site.
From the described experiments it appears that local con-
formational effects in the TFO may play an important role
in guanine-TA base-triple formation. For example, the
change of sugar pucker from 2Ј-endo to 3Ј-endo, as in the
case of the 2ЈAE ribonucleotides can lead to a local distor-
tion of the TFO backbone, which is incompatible with the
formation of the g-TA or ap-TA base triple and the salt
bridge of the side chain with a duplex phosphate unit. Bind-
ing data on TFOs with an unmodified RNA backbone con-
taining a rG-TA base triple are unfortunately not available
as they would help identifying the effect of the aminoethyl
side chain. It seems unlikely, however, that the side chain
alone is responsible for this large drop in binding efficiency
for steric reasons.
The O6 carbonyl group of guanine has some influence
on the energetics of base-triple formation although it is not
directly involved in specific H-bond formation. With TFOs
in the DNA backbone, deletion of this functional group
leads to reduced affinity and reduced selectivity. In the con-
text of 2Ј-AE-TFOs the situation is opposite in the sense
that the removal of O6 is beneficial for triplex formation. A
clear explanation for this fact is elusive. However, it seems
plausible that hydration effects in the major major groove,
or direct or indirect contacts between third strand and pyr-
imidine-rich strand of the target duplex can account for
this, given that the width and dimensions of this groove
changes considerably by changing the third strand confor-
mation from B-type to A-type. In order to interpret this
fact in more detail, a high resolution structure of a G-TA
triplex in which the Hoogsteen strand carries the RNA
backbone is needed. Unfortunately such a structure has not
yet been solved. In any case, to fully exploit the dual re-
cognition features of 2Ј-AE-RNA with respect to recogni-
tion of TA inversion sites, the design of novel bases is
clearly indicated.
Methyl
D
-Ribofuranoside (2):[28,29] To a solution of -ribose (1)
(20 g, 133 mmol) in MeOH (400 mL) was added Dowex-50 (H+
form, 10–20% w/w) at 0 °C. The mixture was stirred at 4 °C over-
night. The solution was then filtered through celite, concentrated
to dryness and crude 2 (predominantly β anomer) was obtained as
a colorless oil in quantitative yield. Rf (β) = 0.3 (EtOAc/EtOH,
1
9:1). H NMR (300 MHz, CDCl3): δ = 3.25 (s, 3 H, OCH3), 3.46
(dd, JH,H = 6.4, 12.5 Hz, 1 H, 5-H), 3.65 (dd, JH,H = 3.1, 12.5 Hz,
1 H, 5-H), 3.83–3.89 (m, 2 H, 3-H, 4-H), 4.01 (dd, JH,H = 4.8,
7.0 Hz, 1 H, 2-H), 4.75 (s, 1 H, 1-H) ppm. 13C NMR (75 MHz,
CDCl3): δ = 55.5 (s, OCH3), 63.1 (C-5), 71.1 (C-3), 74.6 (C-2), 83.2
(C-4), 108.3 (C-1) ppm. HR-MS (ESI+): 187.0586 [M+ +Na] (calcd.
for C6H12NaO5: 187.0582).
Methyl 3,5-O-(1,1,3,3-Tetraisopropyldisiloxane-1,3-diyl)-β-D-ribofu-
ranoside (3): Riboside 2 (3.65 g, 22.2 mmol) was dissolved in dry
pyridine (60 mL). The solution was cooled to 0 °C and TIPS-Cl
(8.42 g, 26.7 mmol) was added dropwise. After stirring for 1 h at
room temp., the solution was diluted with EtOAc (300 mL) and
washed with H2O (2×180 mL) and a satd. NaHCO3 (2×180 mL).
The combined organic layers were dried with MgSO4, filtered and
concentrated to dryness and purified by column chromatography
(EtOAc/hexane, 1:9) to give 3 (5.54 g, 61%) as colorless oil. Rf =
Experimental Section
General: Reactions were carried out under Ar in anhydrous sol-
vents. Solvents for extractions were technical grade and were dis-
1
0.5 (EtOAc/hexane, 1:9). H NMR (300 MHz, CDCl3): δ = 1.03–
3160
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 3152–3168

2Ј-O-Aminoethyl Oligoribonucleotides Containing Novel Base Analogues
1.11 (m, 28 H, TIPS), 3.33 (s, 3 H, OCH3), 3.71–3.79 (m, 1 H, 5- Methyl 2-O-(2-Azidoethyl)-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-
FULL PAPER
H), 3.99–4.12 (m, 3 H, 5-H, 2-H, 4-H), 4.49–4.52 (m, 1 H, 3-H),
1,3-diyl)-β-D-ribofuranoside (7): Compound 6 (29.76 g, 49.2 mmol)
4.83 (s, 1 H, 1-H) ppm. 13C NMR (75 MHz, CDCl3): δ = 13.2– was dissolved in dry DMF (350 mL). LiN3 (4.91 g, 98.3 mmol) was
18.2 (TIPS), 55.6 (OCH3), 66.9 (C-5), 75.7 (C-3), 76.4 (C-2), 83.4 added and the solution was heated to 90–100 °C for 1 h. The reac-
(C-4), 107.9 (C-1) ppm. HR-MS (ESI+): 429.1781 [M+ +Na] (calcd. tion mixture was cooled to room temp., diluted with MeOH
for C18H38NaO6Si2: 429.2104).
(440 mL) and then evaporated to dryness. The residue was taken
up with tert-butyl methyl ether (600 mL) and washed with H2O.
The organic layer was dried with MgSO4 and evaporated to give
compound 7 (23.8 g, quantitative yield) as a white solid. Rf = 0.5
Methyl 2-O-(Methoxycarbonylmethyl)-3,5-O-(1,1,3,3-tetraisoprop-
yldisiloxane-1,3-diyl)-β-D-ribofuranoside (4): To a solution of 3
(22.97 g, 56.5 mmol) in dry DMF (134 mL) was added at –10 °C
BrCH2CO2CH3 (26.2 mL, 282 mmol), followed by portionwise ad-
dition of NaH (4.97 g, 124 mmol) over 1.5 h. After stirring for 7 h,
the reaction mixture was carefully quenched with satd. NH4Cl
(380 mL), extracted with EtOAc (350 mL) and the organic phase
was washed five times with H2O. After drying with MgSO4, the
organic layer was evaporated and the residue purified by
chromatography (EtOAc/hexane, 1:6) to give compound 4 (19.18 g,
71%) as a white solid. Rf = 0.4 (EtOAc/hexane, 1:6). m.p. 61–64 °C.
1H NMR (300 MHz, CDCl3): δ = 1.04–1.08 (m, 28 H, TIPS), 3.34
(s, 3 H, OCH3), 3.75 (s, 3 H, OCH3 chain), 3.79–4.07 (m, 4 H, 2-
H, 4-H, 5-H2), 4.41 (dd, JH,H = 16.9, 37.1 Hz, 2 H, 1Ј-H), 4.50 (dd,
JH,H = 4.2, 7.5 Hz, 1 H, 3-H), 4.88 (s, 1 H, 1-H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 13.4–18.1 (TIPS), 52.5 (OCH3 chain), 55.4
(OCH3), 64.4 (C-5), 70.0 (C-1Ј), 74.8 (C-3), 81.5 (C-2), 83.9 (C-4),
106.6 (C-1), 171.6 (CO) ppm. HR-MS (ESI+): 501.2168 [M+ +Na]
(calcd. for C21H42NaO8Si2: 501.2315).
1
(EtOAc/hexane, 1:8). H NMR (400 MHz, CDCl3): δ = 1.04–1.10
(m, 28 H, TIPS), 3.28–3.46 (m, 2 H, 2Ј-H), 3.34 (s, 3 H, OCH3),
3.71–3.75 (m, 1 H, 1Ј-H), 3.77 (d, JH,H = 4.0 Hz, 1 H, 2-H), 3.88
(dd, JH,H = 5.7, 11.9 H, 1 Hz, 5-H), 4.01 (dd, JH,H = 4.4, 15.4 Hz,
1 H, 5-H), 4.06 (dd, JH,H = 2.9, 5.9 Hz, 1 H, 4-H), 4.12–4.19 (m,
1 H, 1Ј-H), 4.49 (dd, JH,H = 4.4, 8.1 Hz, 1 H, 3-H), 4.79 (s, 1 H,
1-H) ppm. 13C NMR (100 MHz, CDCl3): δ = 13.4–18.1 (TIPS),
51.7 (C-2Ј), 55.4 (OCH3), 64.3 (C-5), 71.3 (C-1Ј), 74.6 (C-3), 81.5
(C-4), 84.4 (C-2), 106.6 (C-1) ppm. HR-MS (ESI+): 498.2419
[M+ +Na] (calcd. for C20H41N3O6Si2: 498.2431).
Methyl 2-O-(2-Aminoethyl)-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-
1,3-diyl)-β-D-ribofuranoside (8): To a solution of compound 7
(1.1 g, 2.3 mmol) in dry pyridine (10 mL) was added PPh3 (1.5 g,
5.7 mmol). The evolution of a gas was observed indicating the
forthcoming of the reaction. The resulting yellow solution was
stirred at room temp. for 3 h, when concentrated NH3 33%
(3.5 mL) was added and stirring continued for another 4 h. The
formed precipitate (Ph3P=O) was filtered off, the solvent evapo-
rated, the residue taken up with EtOAc (30 mL) and washed with
H2O (10 mL). The organic layer was dried with MgSO4 and evapo-
rated to give compound 8 as a white solid (quant.) that was used
in the next transformation without further purification. Rf = 0.6
(CH2Cl2/MeOH, 5:1). 1H NMR (300 MHz, CDCl3): δ = 1.03–1.09
(m, 28 H, TIPS), 2.79–2.92 (m, 2 H, 2Ј-H), 3.34 (s, 3 H, OCH3),
3.60–3.66 (m, 1 H, 1Ј-H), 3.73 (d, JH,H = 4.4 Hz, 1 H, 2-H), 3.82–
Methyl 2-O-(2-Hydroxyethyl)-3,5-O-(1,1,3,3-tetraisopropyldisilox-
ane-1,3-diyl)-β-
D-ribofuranoside (5): Compound
4
(15.08 g,
31.5 mmol) was dissolved in dry MeOH/THF (120:30 mL). The
solution was cooled to 0 °C and LiBH4 (2.74 g, 125.8 mmol) was
added portionwise over 40 min. After stirring for another hour, the
reaction was carefully quenched with satd. NH4Cl (400 mL) and
extracted with EtOAc (240 mL). The organic layer was washed with
a satd. NaCl (100 mL), dried with MgSO4 and filtered. Evapora-
tion of the solvent gave compound 5 (14.0 g, 99%) as a white solid,
which could be used in the next step without further purification.
Rf = 0.1 (EtOAc/hexane, 1:9). 1H NMR (500 MHz, CDCl3): δ =
1.04–1.10 (m, 28 H, TIPS), 3.33 (s, 3 H, OCH3), 3.71–3.76 (m, 3
H, 2-H, 2Ј-H), 3.80–3.91 (m, 3 H, 5-H, 1Ј-H), 3.97–4.05 (m, 2 H,
4-H, 5-H), 4.51 (dd, JH,H = 4.2, 7.8 Hz, 1 H, 3-H), 4.78 (s, 1 H, 1-
H) ppm. 13C NMR (125 MHz, CDCl3): δ = 13.4–18.2 (TIPS), 55.3
(OCH3), 62.4 (C-2Ј), 64.3 (C-5), 73.8 (C-1Ј), 74.2 (C-3), 81.7 (C-4),
84.3 (C-2), 106.6 (C-1) ppm. HR-MS (ESI+): 451.2536 [M+ +H]
(calcd. for C20H42O7Si2: 451.2547).
3.90 (m, 2 H, 5-H), 3.98–4.04 (m, 2 H, 4-H, 1Ј-H), 4.48 (dd, JH,H
=
4.4, 7.7 Hz, 1 H, 3-H), 4.77 (s, 1 H, 1-H) ppm. 13C NMR (75 MHz,
CDCl3): δ = 13.4–18.2 (TIPS), 42.8 (C-2Ј), 55.3 (OCH3), 64.6 (C-
5), 74.4 (C-1Ј), 74.5 (C-3), 81.8 (C-4), 84.0 (C-2), 106.7 (C-1) ppm.
MS (ES+): m/z (%) 450 (5) [M+ +H], 406 (94), 260 (46), 205 (53),
135 (57), 44 (100).
Methyl 3,5-O-(1,1,3,3-Tetraisopropyldisiloxane-1,3-diyl-2-O-(2-tri-
fluoroacetamido)ethyl-β-D-ribofuranoside (9): Compound 8 (19.0 g,
42.2 mmol) was dissolved in CF3CO2Et (350 mL). Freshly distilled
Et3N (7 mL, 50.2 mmol) was added and the solution was stirred at
room temp. for 4 h. After evaporation to dryness the residue was
taken up with CH2Cl2 (300 mL), washed with H2O (2×200 mL)
and dried with MgSO4. The crude product was purified by column
chromatography (EtOAc/hexane, 1:5) to give compound 9 (21.45 g,
93%) as a white solid. Rf = 0.6 (EtOAc/hexane, 1:4). m.p. 50–52 °C.
1H NMR (400 MHz, CDCl3): δ = 1.01–1.09 (m, 28 H, TIPS), 3.33
(s, 3 H, OCH3), 3.58 (dd, JH,H = 5.0, 10.1 Hz, 2 H, 2Ј-H), 3.73 (d,
JH,H = 4.4 Hz, 1 H, 2-H), 3.75–3.81 (m, 1 H, 1Ј-H), 3.86–4.03 (m,
4 H, 4-H, 5-H, 1Ј-H), 4.51 (dd, JH,H = 4.4, 7.7 Hz, 1 H, 3-H), 4.75
(s, 1 H, 1-H), 6.92 (s, 1 H, NH) ppm. 13C NMR (100 MHz,
CDCl3): δ = 13.3–18.1 (TIPS), 40.6 (C-2Ј), 55.4 (OCH3), 64.2 (C-
5), 69.4 (C-1Ј), 74.3 (C-3), 81.8 (C-4), 83.8 (C-2), 106.2 (C-1), 118.4
(CF3), 157.6 (CO) ppm. 19F NMR (376 MHz, CDCl3): δ = –76.40
ppm. HR-MS (ESI+): 568.2330 [M+ +Na] (calcd. for
C22H42F3NO7Si2: 568.2349).
Methyl 3,5-O-(1,1,3,3-Tetraisopropyldisiloxane-1,3-diyl)-[2-O-(2-p-
tolylsulfonyloxyethyl]-β-D-ribofuranoside (6): Compound 5 (24.03 g,
53.3 mmol) was dissolved in dry CH2Cl2 (420 mL). TsCl (22.3 g,
116.9 mmol), Et3N (17.8 mL, 127.7 mmol) and DMAP (0.65 g,
5.3 mmol) were added to the yellow solution. After stirring over-
night, the solution was diluted with H2O (200 mL), extracted with
CH2Cl2 (3×300 mL) and the organic layer was dried with MgSO4.
Evaporation of the solvent and purification by column chromatog-
raphy (EtOAc/hexane, 1:8) afforded 6 (29.76 g, 92%) as a white
1
solid. Rf = 0.4 (EtOAc/hexane, 1:6). H NMR (400 MHz, CDCl3):
δ = 0.98–1.07 (m, 28 H, TIPS), 2.45 (s, 3 H, CH3-Ts), 3.33 (s, 3 H,
OCH3), 3.69 (d, JH,H = 4.0 Hz, 1 H, 2-H), 3.73–3.90 (2m, 3 H, 4-
H, 5-H, 2Ј-H), 3.93–3.97 (m, 1 H, 5-H), 4.08–4.18 (m, 3 H, 1Ј-H,
2Ј-H), 4.42 (dd, JH,H = 4.2, 7.5 Hz, 1 H, 3-H), 4.61 (s, 1 H, 1-H),
7.33 (d, JH,H = 7.7 Hz, 2 H, H-Ts), 7.80 (d, JH,H = 8.4 H, 2 Hz,
H-Ts) ppm. 13C NMR (100 MHz, CDCl3): δ = 13.3–18.1 (TIPS),
22.3 (CH3 Ts), 55.3 (OCH3), 64.4 (C-5), 69.6 (C-2Ј), 70.8 (C-1Ј),
74.8 (C-3), 81.5 (C-4), 84.3 (C-2), 106.6 (C-1), 128.7–130.5 (Ts), 1,3,5-Tri-O-acetyl-2-O-(2-trifluoroacetamido)ethyl-β-
D
-ribofurano-
side (10): A solution of 9 (3.87 g, 7.1 mmol) in Ac2O (80 mL) and
AcOH (80 mL) was stirred for 1 h, before concd. H2SO4 (0.8 mL)
133.7 (C-SO2), 145.4 (C-CH3) ppm. HR-MS (ESI+): 605.2610
[M+ +H] (calcd. for C27H48O9SSi2: 605.2635).
Eur. J. Org. Chem. 2006, 3152–3168
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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3161

S. Buchini, C. J. Leumann
FULL PAPER
was added. The clear yellowish solution was then stirred at room
temp. overnight. The reaction mixture was then added dropwise to
400 mL of satd. NaHCO3 and extracted with CHCl3 (3·1000 mL).
The combined organic layers were dried with MgSO4, the solvent
was evaporated and the crude product was purified by column
chromatography (EtOAc/CH2Cl2, 1:1) to yield compound 10
(2.68 g, 91%) in an anomeric ratio of α/β, 3:1. Rf = 0.6 (α anomer),
0.4 (β anomer) (EtOAc/ CH2Cl2 1:4). α Anomer: 1H NMR
(400 MHz, CDCl3), δ = 2.09–2.14 (m, 9 H, CH3 Ac), 3.53–3.82 (m,
4 H, 1Ј-H, 2Ј-H), 4.10–4.16(m, 2 H, 2-H, 5-H), 4.32–4.52 (m, 2 H,
4-H, 5-H), 5.07 (dd, JH,H = 5.2, 6.3 Hz, 1 H, 3-H), 6.11 (s, 1 H, 1-
H), 6.78 (s, 1 H, NH) ppm. 13C NMR (100 MHz, CDCl3): δ =
21.4, 21.6, 21.8 (CH3 Ac), 40.2 (C-2Ј), 64.4 (C-5), 69.5 (C-1Ј), 72.9
(C-3), 80.4 (C-2), 81.6(C-4), 99.8 (C-1), 118.4 (CF3), 169.2, 170.3,
6) ppm. HR-MS (ESI+): 110.048 (M+ +H) (calcd. for C5H6N2O:
110.048).
4-Triazolylthymine (15):[35] 1H-1,2,4-Triazole (6.9 g, 99.9 mmol)
was suspended in dry CH3CN (80 mL). The suspension was cooled
to 0 °C and POCl3 (3 mL, 32.8 mmol) was added followed by drop-
wise addition of dry Et3N (14 mL, 100 mmol). The suspension was
stirred at 0 °C for about 40 min and then at room temp. for another
30 min. Thymine (14) (1.8 g, 14.3 mmol) was then added and the
yellowish suspension was stirred for another 4 h at room temp. and
5 h under reflux. The mixture was cooled to room temp., H2O
(2.5 mL) was added and stirring was continued for 10 min. The
mixture was then filtered and the yellow residue was resuspended
in H2O (40 mL) and stirred for 40 min. The filtrate was evaporated
and the residue resuspended in H2O (40 mL) and stirred for 20 min.
The combined aqueous suspension were filtered and rinsed with
H2O. Further product could be isolated from the filtrate after
standing overnight. Compound 15 was obtained in 59 % yield
1
170.9 (CO of Ac) ppm. β Anomer: H NMR (400 MHz, CDCl3): δ
= 2.10, 2.12, 2.14 (3s, 9 H, CH3 Ac), 3.49 (dd, JH,H = 5.2, 9.6 Hz,
2 H, 2Ј-H), 3.73 (t, JH,H = 5.0 Hz, 2 H, 1Ј-H), 3.98 (dd, JH,H = 4.4,
6.6 Hz, 1 H, 2-H), 4.12–4.18 (m, 1 H, 5-H), 4.31 (dd, JH,H = 3.3,
12.1 Hz, 1 H, 5-H), 4.50 (dd, JH,H = 3.7, 7.0 Hz, 1 H, 4-H), 5.20
(dd, JH,H = 2.9, 6.6 Hz, 1 H, 3-H), 6.35 (d, JH,H = 6.4 Hz, 1 H, 1-
H) ppm. 13C NMR (100 MHz, CDCl3): δ = 21.3, 21.4, 21.7 (CH3
Ac), 40.5 (C-2Ј), 64.1 (C-5), 70.6 (C-3), 71.1 (C-1Ј), 79.5 (C-2),
81.8(C-4), 95.9 (C-1), 171.0, 171.2, 171.7 (CO of Ac) ppm. 19F
NMR (376 MHz, CDCl3): δ = –76.20 ppm. MS (EI+): m/z (%) =
416 (10) [M+ +H], 356 (100), 279 (38), 236 (87).
1
(1.5 g) as a yellow solid. Rf = 0.4 (EtOAc/MeOH, 5:1). H NMR
(300 MHz, DMSO): δ = 2.25 (s, 3 H, CH3), 8.10 (s, 1 H, 6-H), 8.34
(s, 1 H, triazole), 9.27 (s, 1 H, triazole) ppm. 13C NMR (75 MHz,
DMSO): δ = 15.8 (CH3), 145.3 (C-6), 151.1 (C-4), 153.4 (triazole),
158.8 (C-2) ppm. MS (EI+): m/z (%) = 177 (30) [M+ +H], 86 (100),
69 (59).
5-Methylcytosine (16):[35] Compound 15 (1.6 g, 9.0 mmol) was sus-
pended in concentrated NH3 (33%, 51 mL) and refluxed for 3 h.
3-Ethoxy-2-methylprop-2-enal (12a) and 3-Dimethylamino-2-meth- The reaction mixture was cooled to room temp., evaporated and
ylprop-2-enal (12b):[34] DMF (62 mL) was added dropwise at 0 °C the residue dissolved in hot EtOH (the insoluble solid was filtered
to POCl3 (64 mL) under vigorous stirring such that the temperature
off). Cold acetone (40 mL) was added at room temp. and the
never exceeded 30 °C. The ice bath was then replaced by a water
bath of 40 °C and propionaldehyde diethylacetal (53 mL) was as white solid. Rf = 0.1 (EtOAc/MeOH, 1:1). H NMR (300 MHz,
formed precipitate collected by filtration to obtain 16 (1.0 g, 88%)
1
added slowly accompanied with an exothermic reaction (30 °C Ǟ
85 °C). The reaction temperature was maintained at 60–70 °C by
dropwise addition of propionaldehyde diethylacetal and under ice-
cooling. The now dark brown solution was further heated to 70 °C
for 2 h. After cooling to room temp. the mixture was poured onto
ice (500 g), and left overnight. Anhydrous K2CO3 was added until
pH ca. 9, followed by H2O (350 mL) to dissolve the precipitated
salts. The aqueous solution was then extracted with CH2Cl2
(5×100 mL). The combined organic layers were dried with K2CO3
and the solvents evaporated. A mixture of 12a and 12b in a ratio
of 1:2 ester/amine (16.5 g, 22%) was obtained as a dark red liquid.
1H NMR (300 MHz, CDCl3): δ = 1.34 (t, JH,H = 7.2 Hz, 3 H, 5-
H), 1.62 (s, 3 H, 6-H), 1.88 (s, 3 H, 12-H), 3.10 (s, 6 H, 10-H, 11-
H), 4.12 (q, JH,H = 7.2 Hz, 2 H, 14.2 Hz, 4-H), 6.48 (s, 1 H, 9-H),
6.94 (s, 1 H, 3-H), 8.80 (s, 1 H, 7-H), 9.17 (s, 1 H, 1-H) ppm. 13C
NMR (75 MHz, CDCl3): δ = 6.2 (C-5), 8.5 (C-6), 15.1 (C-12), 42.9
(C-10, C-11), 70.8 (C-4), 108.7 (C-9), 119.8 (C-3), 159.9 (C-8), 167.7
(C-2), 190.9 (C-7), 191.6 (C-1) ppm.
DMSO, 25 °C): δ = 1.82 (s, 3 H, CH3), 3.55 (s, 2 H, NH2), 7.35 (s,
1 H, C6-H) ppm. 13C NMR (75 MHz, DMSO, 25 °C): δ = 12.7
(CH3), 100.6 (C5), 141.5 (C6), 154.8 (C2), 164.8 (C4) ppm. MS
(EI+): m/z (%) = 125 (100) [M+ +H], 81 (23), 70 (38), 54 (33).
N-Benzoyl-5-methylcytosine (17):[35] To a suspension of 16 (1.0 g,
8.0 mmol) in dry CH3CN (32 mL) was added Bz2O (2.2 g,
9.7 mmol), followed by DMAP (0.2 g, 1.6 mmol). The reaction
mixture was refluxed for 7 h, when EtOH (21 mL) was added to
the hot solution. The mixture was cooled to room temp., the solid
was filtered off and washed with EtOH and Et2O. Compound 17
(1.4 g, 76%) was obtained as white solid. Rf = 0.1 (EtOAc/hexane,
1
1:1). H NMR (300 MHz, DMSO): δ = 1.98 (d, JH,H = 0.7 Hz, 3
H, CH3), 7.45–7.50 (m, 2 H, Bz-H meta), 7.56 (d, JH,H = 7.3 Hz,
1 H, Bz-H para), 7.65 (d, JH,H = 0.8 Hz, 1 H, 6-H), 8.18 (d, JH,H
= 7.2 Hz, 2 H, Bz-H ortho), 11.53 (s, 1 H, NH), 12.98 (s, 1 H, NH)
ppm. 13C NMR (75 MHz, DMSO): δ = 12.8 (CH3), 128.5 (Bz),
129.5 (Bz), 132.5 (C-6) ppm. MS (EI+): m/z (%) = 229 (43)
[M+ +H], 152 (18), 105 (100), 77 (60).
5-Methyl-2-pyrimidinone (13): Dry EtOH (7 mL) was added to Na
(0.3 g, 13 mmol) and the mixture was stirred at room temp. until
the Na was dissolved. After dropwise addition of 12a and 12b
(0.5 g) in EtOH (7 mL), followed by urea (0.3 g, 5 mmol), the mix-
ture was refluxed for two days. HCl 25% was added to adjust the
pH to ca. 2 and refluxing was continued for another day. The mix-
ture was cooled to room temp., the insoluble residues were filtered
off through celite and washed with a small amount of Et2O. The
filtrate was evaporated and purified by column chromatography
(CH2Cl2/MeOH, 4:1). Recrystallization from MeOH afforded com-
pound 13 (0.2 g, 41%) as brown solid. Rf = 0.5 (CH2Cl2/MeOH,
1-[3Ј,5Ј-Di-O-acetyl-2Ј-O-(2-trifluoroacetamido)ethyl-β-D-ribo-
furanosyl]-5-methyl-2-pyrimidinone (18): 5-Methyl-2-pyrimidinone
(13) (0.18 g, 1.6 mmol) was suspended in dry CH3CN (5 mL), BSA
(1 mL, 4.1 mmol) was added and the clear solution was stirred at
room temp. for 1 h. The solution was then cooled to 0 °C and 10
(0.63 g, 1.5 mmol) in dry CH3CN (12 mL) was added, followed by
portionwise addition of SnCl4 (0.21 mL, 1.8 mmol). After stirring
for 2 h at 0 °C, CH2Cl2 (80 mL) was added and the solution washed
with H2O (40 mL) and satd. NaHCO3 (40 mL). The organic layer
was dried with MgSO4, evaporated and the crude product purified
by column chromatography (EtOAc). Compound 18 (0.40 g, 57%)
was obtained as white foam. Rf = 0.1 (EtOAc). 1H NMR
(400 MHz, CDCl3): δ = 2.14–2.17 (m, 9 H, CH3 Ac, CH3 base),
3.53–3.62 (m, 2 H, 2ЈЈ-H), 3.89–3.96 (m, 2 H, 1ЈЈ-H), 4.23 (d, JH,H
1
4:1). M.p. Ͼ 220 °C (n.d.). H NMR (300 MHz, CDCl3): δ = 2.28
(s, 3 H, CH3), 8.72 (s, 2 H, 6-H, 4-H) ppm. 13C NMR (75 MHz,
CDCl3): δ = 13.9 (CH3), 115.6 (C-5), 148.3 (C-2), 160.6 (C-4, C-
3162
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 3152–3168

2Ј-O-Aminoethyl Oligoribonucleotides Containing Novel Base Analogues
= 5.2 Hz, 1 H, 2Ј-H), 4.44 (m, 2 H, 5Ј-H), 4.51–4.54 (m, 1 H, 4Ј- in dry THF (60 mL), iPr2NEt (1.6 mL, 9.3 mmol) was added, fol-
FULL PAPER
H), 4.80 (dd, JH,H = 5.1, 8.8 Hz, 1 H, 3Ј-H), 5.87 (s, 1 H, 1Ј-H),
7.69 (s, 1 H, NH), 7.92 (d, JH,H = 2.6 Hz, 1 H, 6-H), 8.54 (d, JH,H
= 2.9 Hz, 1 H, 4-H) ppm. H NMR-difference NOE (400 MHz): δ
lowed by CEP-Cl (1 mL, 4.5 mmol). After stirring for 2 h at room
temp., CH2Cl2 (200 mL) was added and the organic phase was
washed with satd. NaHCO3 (3×150 mL), dried with MgSO4 and
the solvents evaporated. Column chromatography (EtOAc + 1%
1
= 4.23 (2Ј-H) Ǟ 3.89–3.96 (1ЈЈ-H, 5%), 4.80 (3Ј-H, 12%), 5.87 (1Ј-
H, 5%), 7.92 (6-H, 1%), 4.44 (5Ј-H) Ǟ 4.80 (3Ј-H, 3%), 7.92 (6- Et3N) afforded compound 27 (2.15 g, 78%) as a yellowish foam.
H, 2%), 4.51–4.54 (4Ј-H) Ǟ 5.87 (1Ј-H, 3%), 7.92 (6-H, 1%), 4.80
Rf = 0.4, 0.3 (EtOAc + 1 % TEA). M.p. 70–74 °C. 1H NMR
(3Ј-H) Ǟ 4.23 (2Ј-H, 10%), 4.44 (5Ј-H, 1%), 7.92 (6-H, 4%), 5.87 (400 MHz, CDCl3): δ = 0.98 (d, JH,H = 6.6 Hz, 2 H, CH3 iPr), 1.10,
(1Ј-H) Ǟ 3.89–3.96 (1ЈЈ-H, 2%), 4.23 (2Ј-H, 3%), 4.51–4.54 (4Ј-H,
2%), 7.69 (NH chain, 2%), 7.92 (H6, 2%), 7.92 (H6) Ǟ 2.17 (5-
1.17 (2d, JH,H = 7.0 Hz, 10 H, CH3 iPr), 1.45, 1.50 (2s, 3 H, CH3
base), 2.39, 2.58 (2t, JH,H = 6.1 and 5.9 Hz, 2 H, CH2CN), 3.35–
CH3, 5%), 4.80 (3Ј-H, 4%), 5.87 (1Ј-H, 3%), 8.54 (4-H) Ǟ 2.17 (5- 3.42 (m, 1 H, 5Ј-H), 3.45–3.68 (m, 7 H, 5Ј-H, CH iPr, 2ЈЈ-H, OCH2
CH3, 4%). 13C NMR (100 MHz, CDCl3): δ = 15.2 (CH3 base),
CEP), 3.79, 3.80 (2s, 6 H, OCH3 DMT), 4.06–4.16 (m, 3 H, 2Ј-H,
21.1, 21.5 (CH3 Ac), 40.2 (C-2ЈЈ), 62.4 (C-5Ј), 69.8 (C-1ЈЈ), 70.1 (C- 1ЈЈ-H), 4.25–4.34 (m, 1 H, 4Ј-H), 4.56–4.63 (m, 1 H, 3Ј-H), 5.88,
3Ј), 79.6 (C-4Ј), 81.2 (C-2Ј), 91.8 (C-1Ј), 114.2 (C-5), 118.5 (CF3),
155.7 (C-2), 158.0 (CO chain), 164.4 (C-6), 169.0 (C-4), 170.7, 171.0
(CO of Ac) ppm. 19F NMR (376 MHz, CDCl3): δ = –76.15 ppm.
5.90 (2s, 1 H, 1Ј-H), 6.82–6.87 (m, 4 H, DMT), 7.28–7.36 (m, 7 H,
DMT), 7.42–7.46 (m, 2 H, DMT), 7.91 (m,1 H, NH chain), 8.33
(m, 1 H, 6-H), 8.41–8.44 (m, 1 H, 4-H) ppm. 13C NMR (100 MHz,
HR-MS (ESI+): 488.1257 [M+ +Na] (calcd. for C18H22F3N3O7: CDCl3): δ = 14.3–14.4 (CH3 base), 20.8–20.9 (CH2CN), 25.1–25.4
488.1256).
(CH3 iPr), 40.5–40.6 (C-2ЈЈ), 43.8–43.9 (CH iPr), 55.9–56.0 (OCH3
DMT), 58.1–58.8 (OCH2 CEP), 60.9 (C-5Ј), 69.5 (C-1ЈЈ), 69.6–69.8
(C-3Ј), 81.8 (C-2Ј), 82.6 (C-4Ј), 87.4 (DMT), 91.8–92.3 (C-1Ј),
113.7–113.9 (DMT), 114.5–114.6 (C-5), 118.1 (CF3), 127.8–127.9
(DMT), 128.6–129.0 (DMT), 130.9–131.0 (DMT), 136.0 (DMT),
141.4–141.5 (C-6), 145.0 (DMT), 156.0 (CO chain), 159.4–159.5
(DMT), 168.6–168.7 (C-4) ppm. 19F NMR (376 MHz, CDCl3): δ
= –76.06, –76.02 ppm. 31P NMR (162 MHz, CDCl3): δ = 150.12,
152.54 ppm. HR-MS (ESI+): 884.3609 (M+ + H) (calcd. for
C44H53F3N5O9P: 884.3611).
5-Methyl-1-[2Ј-O-(2-trifluoroacetamido)ethyl-β-D-ribofuranosyl]-2-
pyrimidinone (21): To a solution of 18 (1.84 g, 3.9 mmol) in dry
MeOH (47 mL) was added anhydrous Na2CO3 (180 mg, 10% w/w)
and the mixture was stirred at room temp. for 15 min. The reaction
was then neutralized with DOWEX 50 (H+ form, pH ca. 7), filtered
through celite and the solvents evaporated. Compound 21 (1.5 g,
quantitative yield) was obtained as a white solid and was used for
the next step without further purification. Rf = 0.2 (EtOAc/MeOH,
1
10:1). H NMR (400 MHz, MeOD): δ = 2.17 (s, 3 H, CH3 base),
[3Ј,5Ј-Di-O-diacetyl-2Ј-O-(2-trifluoroacetamido)ethyl-β-D-ribo-
3.49–3.72 (2m, 2 H, 2ЈЈ-H), 3.83–3.92 (m, 2 H, 5Ј-H, 1ЈЈ-H), 3.97
(d, JH,H = 5.1 Hz, 1 H, 2Ј-H), 4.05–4.12 (m, 3 H, 5Ј-H, 4Ј-H, 1ЈЈ-
H), 4.25 (dd, JH,H = 5.0, 9.5 Hz, 1 H, 3Ј-H), 5.92 (s, 1 H, 1Ј-H),
8.52 (d, JH,H = 3.3 Hz, 1 H, 6-H), 8.71 (d, JH,H = 2.2 Hz, 1 H, 4-
H) ppm. 13C NMR (100 MHz, MeOD): δ = 14.50 (CH3 base), 41.0
(C-2ЈЈ), 60.5 (C-5Ј), 68.7 (C-3Ј), 70.2 (C-1ЈЈ), 84.4 (C-2Ј), 85.7 (C-
4Ј), 91.4 (C-1Ј), 115.9 (C-5), 119.7 (CF3), 144.5 (C-4), 157.2 (C-2),
162.2 (C-6) ppm. HR-MS (ESI+): 382.0978 [M+ +Na] (calcd. for
C14H18F3N3O6: 382.1225).
furanosyl]thymine (19): Thymine (14) (1.3 g, 10.3 mmol) was sus-
pended in dry CH3CN (50 mL) and BSA (6.5 mL, 26.6 mmol) was
added. The resulting clear solution was stirred for 1 h at room
temp. Compound 10 (3.55 g, 8.5 mmol) in dry CH3CN (100 mL)
was then added at 0 °C, followed by portionwise addition of SnCl4
(1.32 mL, 11.2 mmol). After stirring for 3 h at room temp., CH2Cl2
(150 mL) was added and the solution was washed with satd.
NaHCO3 (80 mL). The organic layer was dried with MgSO4, evap-
orated to dryness and the crude product purified by column
chromatography (EtOAc/hexane, 3:1) to give compound 19 (2.77 g,
67 %) as white foam. Rf = 0.3 (EtOAc/hexane, 4:1). 1H NMR
(400 MHz, CDCl3): δ = 1.94 (d, JH,H = 1.1 Hz, 3 H, CH3 base),
2.16 (s, 6 H, CH3 Ac), 3.53–3.57 (m, 2 H, 2ЈЈ-H), 3.78–3.82 (m, 2
H, 1ЈЈ-H), 4.12–4.17 (m, 1 H, 2Ј-H), 4.39–4.45 (m, 3 H, 5Ј-H, 4Ј-
H), 4.95 (t, JH,H = 6.0 Hz, 1 H, 3Ј-H), 5.89 (d, JH,H = 3.2 Hz, 1 H,
1Ј-H), 7.33 (s, 1 H, NH chain), 7.36 (d, JH,H = 1.3 Hz, 1 H, 6-H),
9.36 (s, 1 H, NH base).1H NMR-difference NOE (400 MHz): δ =
3.78–3.82 (1ЈЈ-H) Ǟ 3.53–3.57 (2ЈЈ-H, 6%), 4.12–4.17 (2Ј-H, 4%),
5.89 (1Ј-H, 3%), 4.12–4.17 (2Ј-H) Ǟ 3.78–3.82 (1ЈЈ-H, 5%), 4.95
(3Ј-H, 7%), 5.89 (1Ј-H, 3%), 7.36 (6-H, 3%), 4.39–4.45 (5Ј-H) Ǟ
δ = 4.95 (3Ј-H, 2%), 5.89 (1Ј-H, 1%), 7.36 (6-H, 1%), 4.39–4.45
(4Ј-H) Ǟ δ = 4.95 (3Ј-H, 3%), 5.89 (1Ј-H, 2%), 4.95 (3Ј-H) Ǟ 1.94
(Me base, 1%), 4.12–4.17 (2Ј-H, 7%), 4.39–4.45 (5Ј-H, 6%), 7.36
(6-H, 2%), 5.89 (1Ј-H) Ǟ 3.78–3.82 (1ЈЈ-H, 2%), 4.12–4.17 (2Ј-H,
2%), 4.39–4.45 (4Ј-H, 2%), 7.36 (6-H, 3%), 7.36 (6-H) Ǟ 1.94 (Me
base, 3%), 4.12–4.17 (2Ј-H, 2%), 4.95 (3Ј-H, 1%), 5.89 (1Ј-H, 2%).
13C NMR (100 MHz, CDCl3): δ = 13.4 (CH3 base), 21.2, 21.5 (CH3
Ac), 40.2 (C-2ЈЈ), 62.9 (C-5Ј), 69.9 (C-1ЈЈ), 70.7 (C-3Ј), 79.8 (C-4Ј),
81.7 (C-2Ј), 89.6 (C-1Ј), 112.4 (C-5), 134.9 (C-6), 151.4 (C-2), 157.9
(CO chain), 163.9 (C-4), 170.7, 171.2 (CO Ac). 19F NMR
(376 MHz, CDCl3): δ = –76.18. HR-MS (ESI+): 482.1373 (M+ +H)
(calcd. for C18H22F3N3O9: 482.1386).
1-[-5Ј-O-(4,4Ј-Dimethoxytrityl)-2Ј-O-(2-trifluoroacetamido)ethyl-β-
D
-ribofuranosyl]-5-methyl-2-pyrimidinone (24): Compound 21
(1.5 g, 3.9 mmol), co-evaporated from pyridine and dried overnight
at high vacuum, was dissolved in dry pyridine (21 mL) and DMTr-
Cl (2.4 g, 7.1 mmol) was added portionwise over 1.5 h. Stirring was
continued for 5 h in the presence of molecular sieves. CH2Cl2 was
added and the organic layer was washed with satd. NaHCO3, dried
with MgSO4 and the solvents evaporated. Column chromatography
(EtOAc+1% of Et3N) of the crude product afforded compound 24
(2.25 g, 84%) as a white foam. Rf = 0.2 (EtOAc+1% Et3N). 1H
NMR (400 MHz, CDCl3): δ = 1.56 (s, 3 H, CH3 base), 1.77 (s, 1
H, OH), 3.53–3.79 (m, 3 H, 5Ј-H, 2ЈЈ-H), 3.80 (s, 6 H, OCH3
DMT), 4.03 (d, JH,H = 5.1 Hz, 1 H, 2Ј-H), 4.11–4.22 (m, 4 H, 4Ј-
H, 5Ј-H, 1ЈЈ-H), 4.49 (dd, JH,H = 5.5, 8.8 Hz, 1 H, 3Ј-H), 5.85 (s,
1 H, 1Ј-H), 6.83–6.86 (m, 4 H, DMT), 7.24–7.43 (m, 9 H, DMT),
7.99 (s, 1 H, NH), 8.27 (d, JH,H = 2.9 Hz, 1 H, 6-H), 8.42 (d, JH,H
= 3.3 Hz, 1 H, 4-H) ppm. 13C NMR (100 MHz, CDCl3): δ = 14.4
(CH3 base), 40.5 (C-2ЈЈ), 56.0 (OCH3 DMT), 61.6 (C-5Ј), 68.8 (C-
3Ј), 70.3 (C-1ЈЈ), 83.5 (C-2Ј), 83.9 (C-4Ј), 87.5 (DMT), 90.8 (C-
1Ј), 114.0 (DMT), 114.5 (C-5), 127.8 (DMT), 128.8 (DMT), 130.8
(DMT), 136.0 (DMT), 141.6 (C-6), 145.1 (DMT), 155.9 (C-2),
159.4 (DMT), 168.7 (C-4) ppm. MS (ESI+): m/z (%) = 684.22 (100)
[M+ +H], 303.03 (22).
1-{3Ј-O-[2-Cyanoethyl(diisopropylamino)phosphanyl]-5Ј-O-(4,4Ј-di-
[2Ј-O-(2-Trifluoroacetamido)ethyl-β-D-ribofuranosyl]thymine (22):
methoxytrityl)-2Ј-O-(2-trifluoroacetamido)ethyl-β-
5-methyl-2-pyrimidinone (27): To a solution of 24 (2.13 g, 3.1 mmol)
D
-ribofuranosyl}- Compound 19 (2.77 g, 5.7 mmol) was dissolved in dry MeOH
(60 mL), Na2CO3 anhydrous (277 mg, 10% w/w) was added and
Eur. J. Org. Chem. 2006, 3152–3168
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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3163

S. Buchini, C. J. Leumann
FULL PAPER
the mixture was stirred at room temp. for 2 h. The reaction mixture
was neutralized with DOWEX-50 (H+-form, pH ca. 7), filtered
through celite, washed with warm MeOH and the solvents evapo-
rated. Compound 22 (2.52 g, quantitative yield) was obtained as a
white solid, which could be used for the next step without further
iPr), 55.9 (OCH3 DMT), 58.3–58.7 (OCH2 CEP), 61.8–62.0 (C-5Ј),
69.4 (C-1ЈЈ), 70.6–71.3 (C-3Ј), 82.6 (C-2Ј), 83.2 (C-4Ј), 87.5–87.6
(DMT), 89.4–89.7 (C-1Ј), 112.3(C-5), 113.9 (DMT), 118.2–118.4
(CF3), 127.9 (DMT), 128.7–129.0 (DMT), 130.9–131.0 (DMT),
135.6 (C-6), 135.9–136.0 (DMT), 144.9 (DMT), 151.4 (CO chain),
159.4, 164.3 (DMT) ppm. 19F NMR (376 MHz, CDCl3): δ =
–76.06, –76.11 ppm. 31P NMR (162 MHz, CDCl3): δ = 149.18,
1
purification. Rf = 0.1 (EtOAc/hexane, 20:1). H NMR (400 MHz,
MeOD): δ = 1.90 (d, JH,H = 1.1 Hz, 3 H, CH3 base), 3.44–3.63 (m,
2 H, 2ЈЈ-H), 3.74–3.81 (m, 2 H, 5Ј-H, 1ЈЈ-H), 3.84–3.96 (m, 2 H, 150.58 ppm. HR-MS (ESI+): 900.3265 (M+ + H) (calcd. for
5Ј-H, 1ЈЈ-H), 3.99–4.04 (m, 2 H, 2Ј-H, 4Ј-H), 4.30 (t, JH,H = 5.5 H, C44H53F3N5O10P: 900.3560).
1 Hz, 3Ј-H), 5.95 (d, JH,H = 3.6 Hz, 1 H, 1Ј-H), 7.96 (d, JH,H
=
N-Benzoyl-5-methyl -[3Ј,5Ј-Di-O-acetyl-2Ј-O-(2-trifluoroacet-
amido)ethyl-β- -ribofuranosyl]-cytosine (20): To a solution of 10
1.1 Hz, 1 H, 6-H) ppm. 13C NMR (75 MHz, MeOD): δ = 12.8(CH3
base), 41.1 (C-2ЈЈ), 61.8 (C-5Ј), 69.8 (C-1ЈЈ), 70.1 (C-3Ј), 83.9 (C-
4Ј), 86.3 (C-2Ј), 89.3 (C-1Ј), 111.6 (C-5), 138.3 (C-6), 152.7 (C-2),
166.7 (CO) ppm. 19F NMR (376 MHz, MeOD): δ = –73.86 ppm.
HR-MS (ESI+): 398.1185 (M+ + H) (calcd. for C14H18F3N3O7:
398.1175).
D
(4.04 g, 9.7 mmol) in dry CH3CN (80 mL) was added N-benzoyl-
5-methylcytosine 17 (2.68 g, 11.7 mmol), followed by HMDS
(2.4 mL, 11.5 mmol) and TMS-Cl (5.9 mL, 46.6 mmol). The mix-
ture was cooled to –10 °C and SnCl4 (1.2 mL, 10.2 mmol) was
added. After stirring for 1 h at –10 °C and another 2 h at room
temp., the mixture was evaporated, CH2Cl2 (200 mL) was added
and the solution was washed with H2O (90 mL) and satd. NaHCO3
(2×90 mL). The organic layer was dried with MgSO4, evaporated
to dryness and the crude product purified by column chromatog-
raphy (EtOAc/hexane, 1:1) to give compound 20 (4.33 g, 76%) as
[5Ј-O-(4,4Ј-Dimethoxytrityl)-2Ј-O-(2-trifluoroacetamido)ethyl-β-D-
ribofuranosyl]thymine (25): Compound 22 (2.29 g, 5.8 mmol), co-
evaporated from pyridine and dried overnight at the high vacuum,
was dissolved in dry pyridine (30 mL) and DMT-Cl (2.9 g,
8.6 mmol) was added portionwise over 1.5 h. After stirring for an-
other 2 h, CH2Cl2 (200 mL, filtered trough basic Alox) was added
and the mixture was washed with satd. NaHCO3 (2×100 mL) and
dried with MgSO4. Evaporation of the solvent and purification by
column chromatography (EtOAc/hexane, 5:1 + 1% of Et3N) gave
compound 25 (3.56 g, 88%) as a white foam. Rf = 0.1 (EtOAc/
hexane, 5:1 + 1% Et3N). 1H NMR (400 MHz, CDCl3): δ  1.37
(s, 3 H, CH3 base), 2.94 (s, 1 H, OH), 3.45 (dd, JH,H = 2.6, 11.3 Hz,
1 H, 5Ј-H), 3.56-.372 (m, 3 H, 5Ј-H, 2ЈЈ-H), 3.80 (s, 6 H, OCH3
DMT), 3.99–4.04 (m, 3 H, 2Ј-H, 1ЈЈ-H), 4.09 (d, JH,H = 7.0 Hz, 1
H, 4Ј-H), 4.44–4.46 (m, 1 H, 3Ј-H), 5.90 (d, JH,H = 1.9 Hz, 1 H,
1Ј-H), 6.85 (d, JH,H = 8.1 Hz, 4 H, DMT), 7.30–7.32 (m, 7 H,
DMT), 7.40–7.42 (m, 2 H, DMT), 7.74 (d, JH,H = 1.0 Hz, 1 H, 6-
H), 7.89 (t, JH,H = 5.5 Hz, 1 H, NH chain), 10.12 (s, 1 H, NH base)
ppm. 13C NMR (100 MHz, CDCl3): δ = 12.4 (CH3 base), 40.3 (C-
2ЈЈ), 55.9 (OCH3 DMT), 62.2 (C-5Ј), 69.4 (C-3Ј), 69.8 (C-1ЈЈ), 83.9
(C-2Ј), 84.1 (C-4Ј), 87.6 (DMT), 88.9 (C-1Ј), 112.3(C-5), 113.9
(DMT), 118.5 (CF3), 127.9 (DMT), 128.7–128–8 (DMT), 130.7–
130.8 (DMT), 135.7 (C-6), 135.9–136.0 (DMT), 144.9 (DMT),
151.9 (CO chain), 158.4, 158.9, 159.4, 164.6 (DMT) ppm. 19F
NMR (376 MHz, CDCl3): δ = –76.15 ppm. HR-MS (ESI+):
722.2276 [M+ +Na] (calcd. for C35H36F3N3NaO9: 722.2301).
1
a white foam. Rf = 0.5 (EtOAc/hexane, 1:1). H NMR (400 MHz,
CDCl3): δ = 2.15 (d, JH,H = 0.9 Hz, 3 H, CH3 base), 2.16, 2.18 (2s,
6 H, CH3 Ac), 3.46–3.64 (m, 2 H, 2ЈЈ-H), 3.80 (t, JH,H = 5.0 Hz, 2
H, 1ЈЈ-H), 4.19 (dd, JH,H = 3.4, 5.5 Hz, 1 H, 2Ј-H), 4.41–4.46 (m,
3 H, 4Ј-H, 5Ј-H), 5.00 (t, JH,H = 5.9 Hz, 1 H, 3Ј-H), 5.93 (d, JH,H
= 3.6 Hz, 1 H, 1Ј-H), 7.07 (s, 1 H, NH chain), 7.43–7.54 (m, 4 H,
Bz, 6-H), 8.33 (d, JH,H = 7.2 Hz, 2 H, Bz), 13.38 (s, 1 H, NH base)
ppm. 13C NMR (100 MHz, CDCl3): δ = 14.5 (CH3 base), 21.2,
21.5 (CH3 Ac), 40.2 (C-2ЈЈ), 63.0 (C-5Ј), 70.0 (C-1ЈЈ), 70.9 (C-3Ј),
79.9 (C-4Ј), 81.5 (C-2Ј), 89.8 (C-1Ј), 113.2 (C-5), 118.4 (CF3), 128.9,
130.7 (Bz), 133.4 (C-6), 148.7 (CO Bz), 157.8 (C-2), 159.8 (CO
chain), 170.7, 171.1 (CO Ac) ppm. 19F NMR (376 MHz, CDCl3):
δ = –76.22 ppm. HR-MS (ESI+): 585.1818 (M+ +H) (calcd. for
C25H27F3N4O9: 585.1808).
N-Benzoyl-5-methyl-[2Ј-O-(2-trifluoroacetamido)ethyl-β-D-ribo-
furanosyl]cytosine (23): Compound 20 (0.66 g, 1.1 mmol) was sus-
pended in absolute EtOH (3 mL) and pyridine (1.5 mL) was added.
To the clear solution a 50 % ethanolic NaOH solution (3 mL,
NaOH 2 / EtOH abs. 1:1) was added and stirring was continued
for 10 min at room temp. The solution was then neutralized with
DOWEX-50 (H+-form, pHca. 7), filtered through celite, washed
with warm EtOH and the solvents evaporated. The crude product
was purified by column chromatography (EtOAc) to give com-
{3Ј-O-[2-Cyanoethyl(diisopropylamino)phosphanyl]-5Ј-O-(4,4Ј-di-
methoxytrityl)-2Ј-O-(2-trifluoroacetamido)ethyl-β-D-ribofuranosyl}-
thymine (28): To a solution of 25 (3.6 g, 5.1 mmol) in dry THF
(100 mL) was added iPr2NEt (2.9 mL, 16.9 mmol), followed by
CEP-Cl (1.9 mL, 8.5 mmol). After stirring for 3 h, CH2Cl2 (50 mL)
was added and the mixture was washed with satd. NaHCO3
(2×30 mL), dried with MgSO4 and the solvents evaporated. Col-
umn chromatography (EtOAc/hexane, 2:1 + 1 % Et3N) afforded
compound 28 (3.8 g, 82%) as a white foam. Rf = 0.4, 0.3 (EtOAc/
hexane, 3:1 + 1% Et3N). 1H NMR (400 MHz, CDCl3): δ  1.00
(d, JH,H = 7.0 Hz, 2 H, CH3 iPr), 1.16 (dd, JH,H = 6.8, 9.0 Hz, 10
1
pound 23 (0.26 g, 46%) as a yellowish foam. Rf = 0.5 (EtOAc). H
NMR (300 MHz, MeOD): δ = 2.14 (s, 3 H, CH3 base), 3.51–3.67
(m, 2 H, 2ЈЈ-H), 3.83–3.87 (m, 2 H, 1ЈЈ-H), 3.94–4.13 (m, 4 H, 2Ј-
H, 4Ј-H, 5Ј-H), 4.33 (dd, JH,H = 5.5, 7.0 Hz, 1 H, 3Ј-H), 5.99 (d,
JH,H = 2.6 Hz, 1 H, 1Ј-H), 7.50–7.60 (m, 4 H, Bz, 6-H), 8.29 (m, 2
H, Bz) ppm. HR-MS (ESI+): 501.1581 (M+ + H) (calcd. for
C21H23F3N4O7: 501.1597).
N-Benzoyl-5-methyl-[5Ј-O-(4,4Ј-dimethoxytrityl)-2Ј-O-(2-trifluoro-
H, CH3 iPr), 1.33, 1.36 (2s, 3 H, CH3 base), 2.42, 2.64 (2t, JH,H
=
acetamido)ethyl-β-
D-ribofuranosyl]-cytosine (26): Compound 23
6.1 Hz and 5.9 Hz, 2 H, CH2CN), 3.32–3.39 (m, 1 H, 5Ј-H), 3.52-
(0.8 g, 1.6 mmol), co-evaporated from dry pyridine and dried over-
.373 (m, 7 H, 5Ј-H, CH iPr, 2ЈЈ-H, OCH2 CEP), 3.80, 3.81 (2s, 6 night at high vacuum, was dissolved in dry pyridine (16 mL).
H, OCH3 DMT), 3.85–4.02 (m, 2 H, 1ЈЈ-H), 4.11–4.14 (m, 1 H, 2Ј- DMTr-Cl (1.08 g, 3.2 mmol) was added portionwise during 1.5 h
H), 4.21–4.28 (m, 1 H, 4Ј-H), 4.48–4.62 (m, 1 H, 3Ј-H), 5.92–5.94 and stirring was continued for another 2 h. CH2Cl2 (100 mL fil-
(m, 1 H, 1Ј-H), 6.82–6.86 (m, 4 H, DMT), 7.27–7.34 (m, 7 H,
tered through basic Alox) was added and the organic layer was
DMT), 7.40–7.44 (m, 2 H, DMT), 7.61 (m, 1 H, NH chain), 7.76 washed with satd. NaHCO3 (2×50 mL), dried with MgSO4 and
(2d, JH,H = 1.1 Hz, 1 H, 6-H), 9.04 (s, 1 H, NH base) ppm. 13C the solvents evaporated. The crude product was purified by column
NMR (100 MHz, CDCl3): δ = 12.3–12.4 (CH3 base), 20.9–21.2 chromatography (EtOAc/hexane, 1:1 + 1% of Et3N) to give com-
(CH2CN), 25.1–25.4 (CH3 iPr), 40.4–40.5 (C-2ЈЈ), 43.8–44.0 (CH pound 26 (0.81 g, 63%) as a yellowish foam. Rf = 0.3 (EtOAc/
3164
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Eur. J. Org. Chem. 2006, 3152–3168

2Ј-O-Aminoethyl Oligoribonucleotides Containing Novel Base Analogues
FULL PAPER
hexane, 1:1 + 1% Et3N). 1H NMR (400 MHz, CDCl3): δ = 1.60 (s,
3 H, CH3 base), 3.45–3.79 (m, 4 H, 2ЈЈ-H, 5Ј-H), 3.81 (s, 6 H,
purified by column chromatography (EtOAc/hexane, 10:1) to give
compound 30 (0.424 g, 68 % yield) as a white foam. Rf = 0.4
OCH3 DMT), 3.95–4.26 (m, 4 H, 2Ј-H, 4Ј-H, 1ЈЈ-H), 4.47 (dd, JH,H (EtOAc/hexane, 10:1). 1H NMR (400 MHz, CDCl3): δ = 2.14, 2.17
= 5.5, 7.4 Hz, 1 H, 3Ј-H), 5.94 (d, JH,H = 2.2 Hz, 1 H, 1Ј-H), 6.87 (2s, 6 H, CH3 Ac), 2.30 (s, 3 H, CH3 base), 3.54–3.64 (m, 2 H, 2ЈЈ-
(dd, JH,H = 1.5, 8.8 H, 4 Hz, DMT), 7.30–7.34 (m, 7 H, DMT),
7.42–7.47 (m, 4 H, Bz, DMT), 7.52–7.56 (m, 1 H, Bz), 7.88 (s, 1 (m, 1 H, 4Ј), 4.89 (dd, JH,H = 2.8, 5.0 Hz, 1 H, 2Ј-H), 5.07 (dd,
H, 6-H), 8.30 (d, JH,H = 7.4 Hz, 2 H, Bz) ppm. 13C NMR
JH,H = 5.3, 6.8 Hz, 1 H, 3Ј-H), 6.08 (d, JH,H = 2.6 Hz, 1 H, 1Ј-H),
H), 3.96–4.02 (m, 2 H, 1ЈЈ-H), 4.39–4.51 (m, 2 H, 5Ј-H), 4.53–4.58
(100 MHz, CDCl3): δ = 13.7 (CH3 base), 40.5 (C-2ЈЈ), 55.9 (OCH3 7.81 (s, 1 H, NH chain), 8.28 (s, 1 H, 8-H), 8.35 (s, 1 H, NH Ac)
DMT), 62.2 (C-5Ј), 69.7 (C-3Ј), 70.1 (C-1ЈЈ), 83.7 (C-2Ј), 84.1 (C- ppm. 13C NMR (100 MHz, CDCl3): δ = 21.2, 21.5 (CH3 Ac), 25.7
4Ј), 87.7 (DMT), 89.3 (C-1Ј), 113.0 (C-5), 114.0 (DMT), 127.9,
(CH3 base), 40.1 (C-2ЈЈ), 62.7 (C-5Ј), 69.5 (C-1ЈЈ), 70.9 (C-3Ј), 80.4
128.8–128.9, 130.6–130.8 (DMT, Bz), 133.3 (C-6), 135.9, 136.0, (C-4Ј), 81.0 (C-2Ј), 89.6 (C-1Ј), 130.0, 143.2 (C-8), 152.0, 152.1,
144.9, 159.5, 159.5 (DMT, Bz) ppm. HR-MS (ESI+): 804.2878
(M+ +H) (calcd. for C41H42F3N4O9: 803.2903).
152.3, 169.1, 171.0, 171.1 (CO Ac, chain) ppm. 19F NMR
(376 MHz, CDCl3): δ = –76.32 ppm.
N2-Acetyl-9-[3Ј,5Ј-di-O-acetyl-2Ј-O-(2-trifluoroacetamido)ethyl-β-
N-Benzoyl-5-methyl-{3Ј-O-[2-cyanoethyl(diisopropylamino)phos-
phanyl]-5Ј-O-(4,4Ј-dimethoxytrityl)-2Ј-O-(2-trifluoroacetamido)-
ethyl-β-D-ribofuranosyl}cytosine (29): To a solution of 26 (0.81 g,
D-ribofuranosyl]purine (31): Compound 30 (0.33 g, 0.6 mmol) was
dissolved in dry MeOH (10 mL). Et3N (0.1 mL, 0.7 mmol) and Pd/
C 10% (0.16 g, 50% w/w) was added and the mixture stirred for 5 h
under H2. The catalyst was filtered off through celite, the solvent
evaporated and the crude product purified by column chromatog-
raphy (EtOAc) to give compound 31 (0.18 g, 58%) as a white solid.
Rf = 0.2 (EtOAc). 1H NMR (300 MHz, CDCl3): δ = 2.14, 2.17 (2s,
6 H, CH3 Ac), 2.34 (s, 3 H, CH3 base), 3.54–3.62 (m, 2 H, 2ЈЈ-H),
3.90–4.03 (m, 2 H, 1ЈЈ-H), 4.38–4.49 (m, 2 H, 5Ј-H), 4.51–4.56 (m,
1 mmol) in dry THF (25 mL) was added iPr2NEt (0.52 mL,
3.0 mmol) followed by CEP-Cl (0.16 mL, 1.5 mmol). After stirring
for 3 h at room temp., CH2Cl2 (90 mL) was added and the organic
phase was washed with satd. NaHCO3 (2 × 30 mL), dried with
MgSO4 and the solvents evaporated. Column chromatography
(EtOAc/hexane, 1:1 + 1% Et3N) of the crude compound yielded
29 (0.86 g, 85%) as a yellowish foam. Rf = 0.5 (EtOAc/hexane, 1:1
+ 1% Et3N). 1H NMR (400 MHz, CDCl3): δ = 1.00 (d, JH,H
=
1 H, 4Ј-H), 4.93 (t, JH,H = 4.2 Hz, 1 H, 2Ј-H), 5.13 (t, JH,H
=
6.8 Hz, 2 H, CH3 iPr), 1.15 (dd, JH,H = 6.8, 9.6 Hz, 10 H, CH3
iPr), 1.50, 1.54 (2d, JH,H = 0.8 Hz, 3 H, CH3 base), 2.41, 2.62 (2t,
JH,H = 5.9 and 6.2 Hz, 2 H, CH2CN), 3.33–3.41 (m, 1 H, 5Ј-H),
3.52-.374 (m, 7 H, 5Ј-H, CH iPr, 2ЈЈ-H, OCH2 CEP), 3.81–3.82 (m,
6 H, OCH3 DMT), 3.92–3.95 (m, 2 H, 1ЈЈ-H), 4.13–4.17 (m, 1 H,
2Ј-H), 4.23–4.31 (m, 1 H, 4Ј-H), 4.52–4.65 (m, 1 H, 3Ј-H), 5.98 (d,
JH,H = 2.3 Hz, 1 H, 1Ј-H), 6.83–6.89 (m, 4 H, DMT), 7.27–7.37
(m, 7 H, DMT), 7.41–7.47 (m, 2 H, DMT), 7.50–7.55 (m, 1 H, Bz),
5.7 Hz, 1 H, 3Ј-H), 6.11 (d, JH,H = 3.3 Hz, 1 H, 1Ј-H), 7.93 (s, 1
H, NH chain), 8.24 (s, 1 H, 8-H), 8.98 (s, 1 H, 6-H), 9.02 (s, 1 H,
NH Ac) ppm. 1H NMR-difference NOE (400 MHz): δ = 3.90–4.03
(1ЈЈ-H) Ǟ 3.54–3.62 (2ЈЈ-H, 4%), 4.91–4.94 (2Ј-H, 5%), 6.10–6.11
(1Ј-H, 3%), 7.93 (NH chain, 1%), 4.38–4.49 (5Ј-H) Ǟ 4.51–4.56
(4Ј-H, 4%), 5.13 (H3Ј, 3%), 4.51–4.56 (4Ј-H) Ǟ 5.13 (3Ј-H, 2%),
6.11 (1Ј-H, 2%), 4.91–4.94 (2Ј-H) Ǟ 3.90–4.03 (1ЈЈ-H, 5%), 5.13
(3Ј-H, 4%), 6.11 (1Ј-H, 4%), 8.24 (8-H, 1%), 5.13 (3Ј-H) Ǟ 4.38–
4.49 (5Ј-H, 2%), 4.51–4.56 (4Ј-H, 2%), 4.91–4.94 (2Ј-H, 1%), 8.24
(8-H, 1%), 6.11 (1Ј-H) Ǟ 3.90–4.03 (1ЈЈ-H, 2%), 4.91–4.94 (2Ј-H,
2%), 8.24 (8-H, 2%), 8.24 (8-H) Ǟ 6.11 (1Ј-H, 3%), 5.13 (3Ј-H,
1%). 13C NMR (75 MHz, CDCl3): δ = 21.2, 21.4 (CH3 Ac), 25.7
(CH3 base), 40.2 (C-2ЈЈ), 63.0 (C-5Ј), 69.5 (C-1ЈЈ), 71.1 (C-3Ј), 80.4
(C-4Ј), 80.9 (C-2Ј), 89.1 (C-1Ј), 118.5 (CF3), 132.4, 143.2 (C-8),
150.5 (C-6), 151.9, 153.1, 158.0, 158.4, 171.0, 171.1 (CO Ac, chain)
ppm. 19F NMR (376 MHz, CDCl3): δ = –76.31 ppm.
7.59 (s, 1 H, NH chain), 7.93–7.95 (m, 1 H, 6-H), 8.30 (d, JH,H
=
1
7.1 Hz, 2 H, Bz), 13.43 (s, 1 H, NH Bz) ppm. H NMR-difference
NOE (400 MHz): δ = 3.33–3.41 (5Ј-H) Ǟ 3.52-.374 (5Ј-H, 17%),
4.23–4.31 (4Ј-H, 6%), 4.52–4.65 (3Ј-H, 2%), 7.41–7.47 and 7.55
7.93 (DMT, Bz, 6%), 7.93–7.95 (6-H, 1%), 8.30 (Bz, 1%), 4.52–4.65
(3Ј-H) Ǟ 4.13–4.17 (2Ј-H, 7%), 7.41–7.47 and 7.55 7.93 (DMT, Bz,
3% and 4%), 7.93–7.95 (6-H, 3%), 8.30 (Bz, 1%), 5.98 (1Ј-H) Ǟ
4.13–4.17 (2Ј-H, 2 %), 7.93–7.95 (6-H, 1 %), 7.93–7.95 (6-H) Ǟ
1.50, 1.54 (Me base, 5%), 4.13–4.17 (2Ј-H, 2%), 4.52–4.65 (H3Ј,
3%), 5.97–5.98 (1Ј-H, 2%), 7.41–7.47 and 7.55 7.93 (DMT, Bz, 4%
and 7%). 13C NMR (100 MHz, CDCl3): δ = 13.53 (CH3 base),
20.9–21.2 (CH2CN), 25.2–25.3 (CH3 iPr), 40.5–40.6 (C-2ЈЈ), 43.8–
44.0 (CH iPr), 56.0 (OCH3 DMT), 58.4–58.7 (OCH2 CEP), 61.8–
61.9 (C-5Ј), 69.4–69.5 (C-1ЈЈ), 70.6–70.7 (C-3Ј), 82.5 (C-2Ј), 83.2
(C-4Ј), 87.6 (DMT), 89.8 (C-1Ј), 113.0 (C-5), 113.9 (DMT), 118.1
(CF3), 127.9 (DMT), 128.7–133.1 (DMT, Bz), 135.9 (C-6), 136.9
(DMT), 144.9 (DMT), 159.4 (DMT), 160.4 (CO) ppm. 19F NMR
(376 MHz, CDCl3): δ = –76.14, –76.10 ppm. 31P NMR (162 MHz,
CDCl3): δ = 149.34, 150.88 ppm. HR-MS (ESI+): 1003.3960
(M+ +H) (calcd. for C51H58F3N6O10P: 1003.3982).
N2-Acetyl-6-chloro-9-[2Ј-O-(2-trifluoroacetamido)ethyl-β-
D-ribo-
furanosyl]purine (32): To a solution of 31 (0.18 g, 0.3 mmol) in dry
MeOH (4 mL) was added anhydrous Na2CO3 (22 mg, 0.2 mmol,
12% w/w) and the suspension was stirred at room temp. for 1 h.
The mixture was then neutralized by addition of DOWEX-50 (H+-
form), filtered through celite and washed with warm MeOH. Evap-
oration of the solvent afforded compound 32 (0.13 g, 86%) as a
white solid that was used without further purification. Rf = 0.1
(EtOAc/MeOH, 10:1). 1H NMR (300 MHz, CDCl3): δ = 2.32 (s, 3
H, CH3 base), 3.50–3.65 (m, 3 H, 1×1ЈЈ-H, 2ЈЈ-H), 3.81–3.99 (m,
5 H, 2Ј-H, 4Ј-H, 5Ј-H, 1×1ЈЈ-H), 4.15 (dd, JH,H = 4.2, 7.2 Hz, 1
H, 3Ј-H), 6.27 (d, J = 2.9 Hz, 1 H, 1Ј-H), 8.72 (s, 1 H, 8-H), 8.94
(s, 1 H, 6-H) ppm. 13C NMR (75 MHz, CDCl3): δ = 24.7 (CH3
base), 41.1 (C-2ЈЈ), 62.3 (C-5Ј), 70.0 (C-1ЈЈ), 70.5 (C-3Ј), 83.9 (C-
4Ј), 87.1 (C-2Ј), 89.0 (C-1Ј), 119.7 (CF3), 132.5, 146.2 (C-8), 150.2
(C-6), 153.1, 154.5, 159.1, 172.3 ppm.
N2-Acetyl-6-chloro-9-[3Ј,5Ј-di-O-acetyl-2Ј-O-(2-trifluoroacetamido)-
ethyl-β-D-ribofuranosyl]purine (30): BSA (0.74 mL, 3.0 mmol) was
added to a suspension of the nucleobase 2-acetylamino-6-chloropu-
rine (0.256 g, 1.2 mmol) in dry 1,2-dichloroethane (10 mL). The
mixture was heated to 80 °C for 30 min and then evaporated to
dryness. The residue was dissolved in dry toluene (5 mL) and 10
(0.457 g, 2.4 mmol) in dry toluene (5 mL) was added, followed by
TMS-Tf (0.44 mL, 2.4 mmol). The solution was stirred at 80 °C for
2.5 h. The mixture was cooled to room temp., EtOAc (20 mL) was
added and the organic phase was washed with satd. NaHCO3
(2×10 mL) and finally dried with MgSO4. The crude product was
N2-Acetyl-9-[5Ј-O-(4,4Ј-dimethoxytrityl)-2Ј-O-(2-trifluoroacet-
amido)ethyl-β-D-ribofuranosyl]purine (33): Compound 32 (0.13 g,
0.3 mmol) was dissolved in dry pyridine (2 mL) and DMTr-Cl
(0.22 g, 0.6 mmol) was added portionwise over 1.5 h. After stirring
for 4 h at room temp., CH2Cl2 (20 mL, filtered through basic Alox)
was added. The organic layer was washed with satd. NaHCO3
Eur. J. Org. Chem. 2006, 3152–3168
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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3165

S. Buchini, C. J. Leumann
(300 MHz, CDCl3): δ = 2.24 (s, 3 H, CH3 base), 3.36 (dd, JH,H
FULL PAPER
(2×10 mL), dried with MgSO4 and the solvents evaporated. The
=
crude product was purified by column chromatography (EtOAc +
4.5, 10.9 Hz, 1 H, 1×5Ј-H), 3.48–3.54 (m, 1 H, 1×5Ј-H), 3.67–3.68
(m, 2 H, 2ЈЈ-H), 3.77 (s, 6 H, OCH3 DMT), 3.99–4.11 (m, 2 H, 1ЈЈ-
1% of Et3N) to give compound 33 (0.16 g, 74%) as a white foam.
1
Rf = 0.4 (EtOAc + 1% Et3N). H NMR (300 MHz, CDCl3): δ = H), 4.15–4.19 (m, 1 H, 2Ј-H), 4.22 (dd, JH,H = 1.9, 5.3 Hz, 1 H, 4Ј-
2.27 (s, 3 H, CH3 base), 2.85 (s, 1 H, OH), 3.27–3.72, 4.03–4.21,
H), 4.51 (dd, JH,H = 5.5, 7.5 Hz, 1 H, 3Ј-H), 5.91 (d, JH,H = 1.9 Hz,
4.39–4.49 (3m, 9 H, 2Ј-H, 3Ј-H, 4Ј-H, 5Ј-H, 1ЈЈ-H, 2ЈЈ-H), 3.79 (s, 1 H, 1Ј-H), 6.80 (dd, JH,H = 1.7, 8.8 Hz, 4 H, DMT), 7.19–7.33
6 H, OCH3 DMT), 6.11 (d, JH,H = 2.2 Hz, 1 H, H1Ј), 6.75 (d, JH,H (m, 7 H, DMT), 7.43 (d, JH,H = 7.0 Hz, 2 H, DMT), 7.89 (s, 1 H,
= 8.5 Hz, 4 H, DMT), 7.19–7.35 (m, 7 H, DMT), 7.36 (d, JH,H
=
8-H), 8.63 (s, 1 H, NH chain) ppm. 13C NMR (75 MHz, CDCl3):
δ = 24.7 (CH3 base), 40.9 (C-2ЈЈ), 55.9 (OCH3 DMT), 63.5 (C-5Ј),
69.5 (C-1ЈЈ), 70.2 (C-3Ј), 83.5 (C-2Ј), 83.9 (C-4Ј), 87.2 (DMT), 89.1
7.3 Hz, 2 H, DMT), 8.21 (s, 1 H, 8-H), 8.27 (s, 1 H, NH chain),
8.94 (s, 1 H, 6-H), 9.39 (s, 1 H, NH base) ppm. 13C NMR (75 MHz,
CDCl3): δ = 25.6 (CH3 base), 40.3 (C-2ЈЈ), 55.9 (OCH3 DMT), 63.2 (C-1Ј), 113.9 (DMT), 122.2 (C-5), 127.7, 128.6–128.8, 130.7
(C-5Ј), 69.6 (C-1ЈЈ), 70.2 (C-3Ј), 83.7 (C-2Ј), 84.3 (C-4Ј), 87.4 (DMT), 136.4, 137.8 (DMT), 145.2 (C-8), 148.3, 148.5, 156.6,
(DMT), 88.5 (C-1Ј), 114.0 (DMT), 127.7, 128.7–128.8, 130.7
159.3, 173.5 (quaternary C) ppm. HR-MS (ESI+): 767.2672
(DMT), 132.2 (base), 136.1, 136.2 (DMT), 143.6 (C-8), 145.1, 150.2 (M+ +H) (calcd. for C37H37F3N6O9: 767.2652).
(C-6), 152.0, 153.1 (base), 159.3 (CO chain) ppm. MS (ESI+):
N2-Acetyl-9-[3Ј-O-(2-Cyanoethyl(diisopropylamino)phosphanyl)-5Ј-
O-(4,4Ј-dimethoxytrityl)-2Ј-O-(2-trifluoroacetamido)ethyl-β-
m/z (%) = 751.23 (100) [M+ +H].
D
-
N2-Acetyl-9-[3Ј-O-(2-cyanoethyl(diisopropylamino)phosphanyl)-5Ј-
O-(4,4Ј-dimethoxytrityl)-2Ј-O-(2-trifluoroacetamido)ethyl-β-D-ribo-
ribofuranosyl}guanine (37): Compound 36 (0.117 g, 0.15 mmol) was
dissolved in dry THF (4 mL) and iPr2NEt (0.08 mL, 0.5 mmol) was
furanosyl]purine (34): Compound 33 (0.2 g, 0.3 mmol) was dissolved added, followed by CEP-Cl (0.05 mL, 0.2 mmol). After stirring for
in dry THF (8 mL) and iPr2NEt (0.14 mL, 0.8 mmol) was added,
followed by CEP-Cl (0.09 mL, 0.4 mmol). After stirring for 3 h at
room temp., CH2Cl2 (40 mL) was added and the organic phase was
washed with satd. NaHCO3 (2×10 mL), dried with MgSO4 and
the solvents evaporated. Column chromatography (EtOAc/hexane,
1.5 h at room temp., CH2Cl2 (40 mL) was added and the organic
phase was washed with satd. NaHCO3 (2 × 10 mL), dried with
MgSO4 and the solvents evaporated. Column chromatography
(CH2Cl2/MeOH, 30:1 + 1% of Et3N) gave compound 37 (0.21 g,
83%) as a white foam. Rf = 0.3, 0.4 (CH2Cl2/MeOH, 20:1). 1H
1:1 + 1% Et3N) gave compound 34 (0.21 g, 83%) as a white foam. NMR (400 MHz, CDCl3): δ = 1.00 (d, JH,H = 6.8 Hz, 2 H, CH3
Rf = 0.5, 0.6 (EtOAc/hexane, 1:1 + 1% Et3N). 1H NMR (400 MHz,
CDCl3): δ = 1.04 (d, JH,H = 6.8 Hz, 2 H, CH3 iPr), 1.15–1.18 (m,
10 H, CH3 iPr), 2.26 (s, 3 H, CH3 base), 2.37, 2.61 (2t, JH,H = 7.0
iPr), 1.12–1.18 (m, 10 H, CH3 iPr), 2.05, 2.12 (2s, 3 H, CH3 base),
2.36, 2.62 (2t, JH,H = 6.2 and 6.2 Hz, 2 H, CH2CN), 3.52–3.74 (m,
8 H, 5Ј-H, CH iPr, 2ЈЈ-H, OCH2 CEP), 3.79 (m, 6 H, OCH3 DMT),
and 6.0 Hz, 2 H, CH2CN), 3.67–3.74 (m, 8 H, H5Ј, CH iPr, H2ЈЈ, 3.86–3.92 (m, 2 H, 1ЈЈ-H), 4.08–4.25, 4.33–4.42, 4.50–4.54 (3m, 3
OCH2 Phosph.), 3.79 (m, 6 H, OCH3 DMT), 3.84–4.06 (m, 2 H,
1ЈЈ-H), 4.35–4.39, 4.46–4.51, 4.61–4.64 (3m, 3 H, 2Ј-H, 3Ј-H, 4Ј-
H), 6.14–6.16 (m, 1 H, 1Ј-H), 6.80–6.84 (m, 4 H, DMT), 7.27–7.37
H, 2Ј-H, 3Ј-H, 4Ј-H), 5.86, 5.93 (2d, JH,H = 2.6 and 3.6 Hz, 1 H,
1Ј-H), 6.81–6.84 (m, 4 H, DMT), 7.29–7.50 (m, 9 H, DMT), 7.86,
7.88 (2s, 1 H, 8-H) ppm. 31P NMR (162 MHz, CDCl3): δ = 148.88,
(m, 9 H, DMT), 7.73 (s, 1 H, NH chain), 8.23–8.30 (s, 1 H, 8-H), 149.72 ppm. MS (ESI+) m/z 967.37 (M+ +H).
8.59 (s, 1 H, NH base), 8.96–8.97 (s, 1 H, 6-H). 19F NMR
Oligonucleotides: All 2Ј-AE oligonucleotides were prepared from
(376 MHz, CDCl3): δ = –76.20, –76.26 ppm. 31P NMR (162 MHz,
CDCl3): δ = 150.70, 151.10 ppm. HR-MS (ESI+): 973.3609
(M+ +H) (calcd. for C46H54F3N8O9P: 973.3601).
the modified phosphoramidites 27–29 and commercially available
dT solid support (Glen Research) on an Applied Biosystems Expe-
dite 8909 DNA synthesizer by standard solid-phase phosphoramid-
ite chemistry on the 1 µmol scale in the trityl-off mode. The follow-
ing modifications relative to the standard protocols were made: i)
N2-Acetyl-9-[3Ј,5Ј-di-O-acetyl-2Ј-O-(2-trifluoroacetamido)ethyl-β-
D
-ribofuranosyl]guanine (35): To a solution of cyanoethanol
(0.5 mL, 7.3 mmol) in dry THF (4 mL), Cs2CO3 (4 × 0.35 g, the concentrations for 2Ј-AE phosphoramidites were 0.1–0.12 m;
4.3 mmol) was added at 0 °C. The mixture was stirred at room
temp. for about 30 min, then it was cooled to 0 °C and 30 (0.41 g,
0.7 mmol) in dry THF (4 mL) was added dropwise. After stirring
at 0 °C for 1.5 h, the solution was evaporated to dryness. The crude
product was purified by column chromatography (CH2Cl2/MeOH,
ii) 5-ethylthio-1H-tetrazole (0.25  in CH3CN) was used as the
coupling agent; iii) the coupling time for phosphoramidites 27–29,
34, and 37 was extended to 6 min. Deprotection and detachment
from solid support was effected in a two step procedure. First, the
solid support was treated with 1 mL of a 1  solution of DBU in
CH3CN at room temp. for 1.5 h to remove the cyanoethyl protect-
10:1 Ǟ 5:1) to give compound 35 (0.195 g, 58%) as a white solid.
1
Rf = 0.3 (CH2Cl2/MeOH, 5:1). H NMR (300 MHz, CDCl3): δ = ing groups, followed by washing of the solid support with CH3CN
2.27 (s, 3 H, CH3 base), 3.48–3.58 (m, 2 H, 2ЈЈ-H), 3.80–3.92 (m,
and H2O. Then deprotection of the bases and detachment from
4 H, 1ЈЈ-H, 5Ј-H), 4.07–4.11 (m, 1 H, 4Ј-H), 4.38 (t, JH,H = 4.0 Hz, solid support was performed by treatment with 1 mL concd. aque-
1 H, 2Ј-H), 4.46 (t, JH,H = 5.1 Hz, 1 H, 3Ј-H), 6.05 (d, JH,H
=
ous NH3 solution at room temp. for 2 h. The crude oligonucleo-
tides were purified by RP-HPLC (Aquapore RP-300, 7 µm,
220×4.6 mm, Brownlee or NUCLEOSIL 100–5, C18, 220×5 mm,
Macherey–Nagel) and desalted over Sep-Pack C18 cartridges
(Waters). The integrity of all oligonucleotides was confirmed by
ESI-MS. Table 3 shows MS data of the 2Ј-AE oligonucleotides pre-
pared in this way. All oligodeoxynucleotides for the target duplexes
were prepared and HPLC-purified by standard techniques and rou-
tinely analyzed by ESI-MS.
3.9 Hz, 1 H, 1Ј-H), 8.35 (s, 1 H, 8-H) ppm. HR-MS (ESI+):
465.1348 (M+ +H) (calcd. for C16H19F3N6O7: 465.1345).
N2-Acetyl-9-[5Ј-O-(4,4Ј-dimethoxytrityl)-2Ј-O-(2-trifluoroacet-
amido)ethyl-β-D-ribofuranosyl]guanine (36): Compound 35 (0.22 g,
0.5 mmol) was dissolved in dry pyridine (2.4 mL) and DMTr-Cl
(0.19 g, 0.6 mmol) was added portionwise over 1.5 h. After stirring
for 2 h at room temp., CH2Cl2 (20 mL, filtered through basic Alox)
was added. The organic layer was washed with satd. NaHCO3
(2×10 mL), dried with MgSO4 and the solvents evaporated. The
crude product was purified by column chromatography (CH2Cl2/
MeOH, 20:1 + 1% of Et3N) to give compound 36 (0.117 g, 32%)
Thermal Denaturation Experiments: UV melting experiments were
performed with a Varian Cary 100 UV/Vis spectrophotometer
equipped with a temperature controller. Data were collected with
as a yellowish foam. Rf = 0.30 (CH2Cl2/MeOH, 20:1). 1H NMR the Cary WinUV software. All measurements were conducted in a
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Eur. J. Org. Chem. 2006, 3152–3168

2Ј-O-Aminoethyl Oligoribonucleotides Containing Novel Base Analogues
FULL PAPER
Table 3. Experimental and calculated masses of TFOs.
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Acknowledgments
The authors thank the Swiss National Science Foundation (grant-
No. 200020-100178) for financial support of this work.
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Received: March 1, 2006
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Eur. J. Org. Chem. 2006, 3152–3168