Syntheses and oligonucleotide incorporation of nucleoside analogues containing pendant imidazolyl or amino functionalities - The search for sequence-specific artificial ribonucleases

Article abstract of DOI:10.1002/ejoc.200500413

Oligonucleotide derivatives containing imidazolyl and amino functionalities have been proposed as artificial ribonucleases that might mimic the activities of RNase A but with greater sequence specificity. Such oligonucleotide reagents would have important

Full text of DOI:10.1002/ejoc.200500413

FULL PAPER
DOI: 10.1002/ejoc.200500413
Syntheses and Oligonucleotide Incorporation of Nucleoside Analogues
Containing Pendant Imidazolyl or Amino Functionalities – The Search for
Sequence-Specific Artificial Ribonucleases
Stephen C. Holmes[a] and Michael J. Gait*[a]
Dedicated to Prof. Dr. Wojciech Stec on the occasion of his 65th birthday
Keywords: Oligonucleotides / Nucleosides / Modified nucleosides / Artificial ribonuclease
Oligonucleotide derivatives containing imidazolyl and amino
functionalities have been proposed as artificial ribonucleases
that might mimic the activities of RNase A but with greater
sequence specificity. Such oligonucleotide reagents would
have important applications as enhanced antisense reagents
that obviate the need to recruit a cellular nuclease or nucle-
ase complex. We present the syntheses of six nucleoside
phosphoramidite analogues containing pendant protected
imidazolyl or amino groups and their incorporation into oli-
gonucleotides. We show that the six functionalised phos-
phoramidites have similar efficiencies of incorporation and
present mass spectral evidence that the composition of an
oligonucleotide library of 81 components is consistent with
the expected mass mixture distribution. Such phosphoramid-
ites are thus suitable starting materials for construction of
mixed oligonucleotide libraries containing a region of imid-
azolyl and amino-modified nucleotides ready for screening
for sequence-specific ribonuclease activity.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2005)
act in a general acid/base catalytic mechanism. In addition,
a positively charged amine side chain (Lys-41) is thought to
stabilise the pentacoordinate transition state of the scissile
phosphate.[7] With RNase A as a model, Bashkin et al. were
first to propose the imidazolyl modification of oligonucleo-
tides as hydrolytic ribonuclease mimics where the RNA hy-
drolysis activity of imidazole is combined with the ability
of oligonucleotides to bind RNA in a sequence-specific
manner.[8] A 2Ј-deoxyuridine derivative modified at the 5-
position was synthesised that incorporated a long linker
arm and a tert-butoxycarbonyl (BOC) protected imidazole
residue.[8,9] The unit was incorporated successfully into an
11-mer oligonucleotide, but no results were published with
regard to RNA hydrolysis. Ushijima et al. used the same
side arm modification at the 5Ј-end of an antisense phos-
phorothioate oligonucleotide to target a gag mRNA se-
quence from the HIV-1 genome.[10] Use of this modification
resulted in a higher anti HIV-1 activity than that from the
unmodified phosphorothioate oligonucleotide.
More recently, several studies have focussed on the incor-
poration of amino/imidazolyl (lysyl/histidyl) functionalities
into different locations in an oligonucleotide. For example,
Reynolds et al. incorporated a non-nucleotidic internal
linker into a methyl phosphonate oligonucleotide and then
modified it with bis-amino, amino plus imidazolyl, or bis-
imidazolyl units.[11] Low levels of site-specific cleavage of an
RNA target were observed with units containing imidazole
Introduction
Synthetic oligonucleotides and their analogues are used
widely to block gene expression in cells both as therapeutics
and for target validation.[1] The most common mechanism
for the inhibition of gene expression is for the binding of
the oligonucleotide to induce RNA cleavage by the recruit-
ment of an endogenous nuclease, such as RNase H (anti-
sense)[2] or the RISC complex (siRNA).[3] In recent years
there have been attempts to develop oligonucleotide-based
artificial nucleases that contain motifs capable of inducing
sequence-specific RNA cleavage without the requirement
for protein recruitment. Such artificial nucleases have usu-
ally included chemically modified residues in order to en-
hance the catalytic cleavage rates.[4] Some approaches to ar-
tificial nucleases have employed a heavy metal ion,[5] or a
redox reagent bound to an oligonucleotide[6] to achieve
RNA targeted cleavage. However, it is unclear whether
these will be useful in vivo since RNA cleavage may require
the addition of exogenous co-factors.
The protein ribonuclease A (RNase A) achieves highly
efficient RNA cleavage by use of a protonated imidazole
group (His-119) and a free imidazole group (His-12) which
[a] MRC-Laboratory of Molecular Biology,
Hills Rd., Cambridge CB2 2QH, UK
Fax: +44-1223-213556
E-mail: mgait@mrc-lmb.cam.ac.uk
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modifications after a 5 day incubation period. However, zole and amino functionalisation.[27] In addition, we have
some other reported bis-imidazolyl oligonucleotide conju- reported recently the synthesis of oligonucleotides contain-
gates have shown no hydrolytic activity.[12] Beban and ing imidazole and amino side chains at the 2Ј-position of
Miller used post-synthetic modification to conjugate imida- uridine.[28] In this paper we describe the synthesis of a
zole units to a 2Ј-modified 2Ј-deoxyuridine nucleoside,[13] number of nucleoside phosphoramidites functionalised with
whilst Polushin conjugated histidine or imidazoleacetic acid imidazolyl and amino groups on heterocyclic base moieties
to the 3Ј-position of thymidine for incorporation at the 3Ј- and their successful incorporation with similar coupling
terminus of oligonucleotides.[14] Unfortunately, no RNA efficiencies into 2Ј-deoxyribo- and into 2Ј-O-methyl ribo-
cleavage assays were reported using these derivatives.
oligonucleotides. These modified nucleosides are suitable
The most successful oligonucleotide-based imidazolyl ar- for use in the synthesis of mixed libraries of functionalised
tificial nucleases to date have targeted specific RNA se- oligonucleotides for the purpose of screening them for
quences within structured tRNAs. Ushijima et al.[15] and RNA cleavage activity.
Beloglazova et al.[16] showed that imidazole units attached
to the ends of oligonucleotides can cleave at particular
Results and Discussion
“fragile” regions within tRNAs, usually between CpA se-
quences. A number of non-oligonucleotide imidazole-con-
taining small molecules have also been designed.[17] Such
artificial nucleases can cleave RNA sequences site-selec-
tively at susceptible scissile phosphates, but lack the ex-
tended sequence specificity of oligonucleotides.
Site-specific cleavage of RNA has been achieved through
the use of amine modifications alone. Endo et al. have syn-
thesised a DNA oligoamine conjugate[18] and showed it
possessed modest RNA hydrolysis capabilities. Verheijen et
al. tethered ethylenediamine to the 5Ј-end of a PNA oligo-
mer and also showed RNA hydrolysis activity.[19] Most re-
cently an (ArgLeu)4-Gly peptide conjugated to a short
DNA was revealed to have RNA hydrolysis activity when
targeting specific HIV-1 and tRNA sequences.[20] Taken to-
gether with Komiyama and Inokawa’s earlier results,[21] the
idea that there are highly susceptible sequences within
structured nucleic acids is reinforced.
Our concept for design of an artificial ribonuclease is
that such a molecule should contain both RNA substrate
binding region(s) and a catalytic region which would con-
tain the modified nucleosides. To maximise the opportuni-
ties to select “active” molecules (i.e. those capable of cata-
lysing RNA hydrolysis), the catalytic region would consist
of combinatorial mixtures of differently modified nucleo-
sides, whereas binding regions would be exactly matched
to the RNA target. Each library of oligonucleotides must
contain an unbiased population of modified and/or un-
modified nucleotides and thus the first requirement is to
ensure that all phosphoramidites used in library synthesis
have similar coupling efficiencies. For example, it is well es-
tablished that coupling efficiencies differ markedly between
2Ј-deoxynucleoside, ribonucleoside or 2Ј-OMe ribonucleo-
side phosphoramidites. In modifying the heterocyclic base,
rather than the sugar moiety, we aimed to minimise these
differences in coupling efficiencies.
Examples of imidazolyl- and amino-modified nucleo-
sides harnessed for use in RNA catalysis also exist through
their conversion into deoxynucleotide triphosphate (dNTP)
analogues and enzymatic incorporation into modified
DNAzymes selected through in vitro selection experi-
ments.[22] The research groups of Perrin,[23] Williams[24] and
Beigelman[25] have reported the synthesis of dNTPs modi-
Synthesis of 5-Carboxamide-Modified Imidazolyl and
Amino dU Analogues
A facile synthesis for the incorporation of a number of
fied with pendant imidazole and amino functionalities. De- carboxamido modifications at the 5-position of uridine has
oxynucleoside analogues containing imidazole and amino been published previously by Dewey et al.[29] In place of
functions have also been designed for use in oligonucleotide formation of a carboxy ester intermediate and then reaction
synthesis.[26] Although to date the cleavage rates of such with an amine,[30] Dewey et al. used a one-pot carboxamid-
DNAzymes have not proved to be particularly high, the ation procedure. 5-Iodouridine carrying various sugar pro-
principle of utilisation of imidazoles and amino functionali- tecting groups was reacted with a number of different
ties in an oligonucleotide background is now well estab- amines to give the desired 5-carboxamido products. How-
lished.
ever, not all products were isolated and yields were not op-
Our aim is to chemically synthesise small synthetic li- timised. Other 5-carbamoyl-2Ј-deoxyuridine analogues,
braries of oligonucleotides containing amino- and imid- such as the ethylenediamine derivative, may be synthesised
azolyl-modified nucleosides and then to screen these se- post-synthetically after the incorporation of 5-methoxycar-
quences for their ability to cleave a desired RNA substrate. bonyl-2Ј-deoxyuridine phosphoramidite into oligonucleo-
A particular advantage of this approach over enzymatic se- tides.[31]
lection methods is that there is no limitation to those few
By use of completely unprotected 5-iodo-2Ј-deoxyurid-
nucleotide analogues that act as substrates for a polymer- ine, we were able to incorporate successfully a histamine
ase-based reaction. Thus it should be possible in principle group through a one-pot carboxamidation procedure. After
to explore a wider chemical space and functionality.
several attempts at the reaction, it became apparent that the
We have already reported the synthesis of some seco- product could be formed in consistently good yield with
pseudonucleoside analogues potentially suitable for imida- different palladium catalysts, but purification by
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Search for Sequence-Specific Artificial Ribonucleases
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Scheme 1.
chromatography was problematic due to the polar nature Synthesis of 6-Modified Imidazolyl and Amino dA
of the product. By protection of the imidazole ring of the Analogues
histamine with a base-labile tert-butoxycarbonyl group
Cosstick and Douglas have synthesised previously the
(BOC) the molecule became more hydrophobic and this fa-
N6-(ethylamino)-2Ј-deoxyadenosine phosphoramidite for
cilitated ready purification. The BOC group is required to
incorporation into a nucleotide dimer for potential cross-
ensure compatibility with oligonucleotide synthesis and pre-
linking studies.[32] Starting with a 6-chloro-3Ј,5Ј-bis(toluoyl)
vent the bis-dimethoxytritylation of the 5Ј-hydroxy and the
derivative,[33] we tried to synthesise the N6-ethylimidazole
imidazole later in the synthesis. The molecule was then eas-
derivative, but found difficulty with purification following
ily converted into the required phosphoramidite in good
sugar deprotection. To avoid this problem, we synthesised
yield (Scheme 1).
6-iodo-3Ј,5Ј-(tetraisopropyldisiloxane-1,3-diyl)-2Ј-deoxyad-
By contrast, and analogous to previously published
enosine following the photo-induced diazotisation pro-
work,[29] the incorporation of an ethylenediamine group by
cedure developed by Nair and Richardson,[34] which was
5-carboxamidation was found to be less straightforward.
also utilised by Cosstick and Douglas in their synthesis.
Efforts using the unprotected 5-iodo-2Ј-deoxyuridine re-
Substitution with either histamine or ethylenediamine pro-
sulted in large amounts of unknown nucleosidic by-prod-
ceeded well and the amino derivatives were protected with-
ucts from the reaction and attempts to optimise the reaction
out prior purification (Scheme 3). Desilylation, dimeth-
by using different palladium() and palladium(0) catalysts
oxytritylation and conversion into the desired phosphoram-
proved ineffective. To avoid these problems, the synthesis
idites were completed in reasonable yields.
was effected via the 3Ј,5Ј-(tetraisopropyldisiloxane-1,3-
diyl)-protected 5-methyl ester 5, which was formed in quan-
titative yield (Scheme 2). This derivative was then reacted
with ethylenediamine and the resultant amine protected
with a trifluoroacetyl group in a one-pot synthesis. Protec-
Synthesis of 4-Modified 6-Methylimidazolyl and Amino dC
Analogues
tion of the terminal amine at this stage also allowed the
The incorporation of 6-Me-2Ј-deoxyuridine (6-Me-dU)
desilylation product to be purified easily by silica gel into oligonucleotides has been found to destabilize DNA
chromatography. The product was then easily converted duplexes by 2–3 °C per modification.[35] In 1H NMR
into the desired phosphoramidite.
(NOE) and molecular modeling experiments, it was re-
Scheme 2.
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S. C. Holmes, M. J. Gait
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Scheme 3.
vealed that this was because 6-Me-dU has a preference to derivative was formed in good yield through transient silyl
adopt a syn conformation of the base rather than the anti protection of the sugar hydroxy groups.[39] This method was
conformation required for Watson–Crick base-pairing in attempted with the free 6-methyl-dU nucleoside, but poor
both A and B form duplexes.[35,36]
yields resulted after amine substitution and relevant pro-
By prevention of non-Watson–Crick base pairing within tecting group incorporation (BOC or TFA). As an alterna-
our catalytic regions, through incorporation of the 6-methyl tive, the 4-tosyl derivative was formed directly from the si-
modification, we aim to encourage the formation of alter- lylated nucleoside and taken in crude form into the next
native, less rigid bulge structures within otherwise duplex step. Amino substitution and protection followed
regions.
(Scheme 4). The three-step reaction proceeded in good yield
6-Methyl-2Ј-deoxyuridine was first synthesised by Holy and it was found that purification could be deferred until
in 1973.[37] However, since then a more convenient synthesis after the desilylation step. The synthesis was also attempted
has been published that proceeds by a lithiation of 3Ј,5Ј- via the 4-chloro derivative, but no increase in overall yield
(tetraisopropyldisiloxane-1,3-diyl)-2Ј-deoxyuridine.[38] We was attained. Again, conversion into the desired phos-
followed this procedure to give the 6-methyl derivative in phoramidites was carried out easily.
good yield.
Synthesis of Modified Oligonucleotides
There are numerous methods of activating the 4-position
of pyrimidines for nucleophilic displacement by amines.
All six phosphoramidites were incorporated successfully
Miah et al. showed a one-pot strategy where the 4-triazole into a central position within a 15-mer oligodeoxynucleo-
Scheme 4.
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tide sequence using standard coupling times and reagents. All six modified phosphoramidites were shown to have
The analogues in each case coupled with Ͼ95% efficiency. slightly reduced coupling efficiencies when compared to 2Ј-
The oligonucleotides were deprotected by use of saturated deoxyuridine phosphoramidite. For example, with dU
methanolic ammonia overnight at room temperature. Ion- phosphoramidite and the phosphoramidite 29, the inte-
exchange HPLC of the oligonucleotides showed practically grated ratio of peak areas was 1.6:1 (Figure 1). All other
single product peaks and MALDI-TOF mass spectrometry
revealed all oligonucleotides had the desired mass (Table 1).
Table 1. Calculated and observed masses for 15-mer deoxy oligonu-
cleotides containing a single incorporation of each of the modified
nucleoside phosphoramidites.
Sequence 5Ј-ATC TGT AXT CCG TCA
Calcd.
mass
Obsd.
mass
Oligo 1: X = nucleoside derivative 3
Oligo 2: X = nucleoside derivative 9
Oligo 3: X = nucleoside derivative 15
Oligo 4: X = nucleoside derivative 19
Oligo 5: X = nucleoside derivative 25
Oligo 6: X = nucleoside derivative 29
4640
4589
4668
4617
4612
4561
4639
4591
4669
4619
4610
4559
To investigate the coupling efficiencies of the modified
phosphoramidites, an equimolar ratio of modified phos-
phoramidite together with 2Ј-deoxyuridine phosphoramid-
ite was mixed together and used in a single coupling reac-
tion within a 15-mer oligonucleotide. Because the inclusion
of the amino or imidazole functionalities reduces retention
of the oligonucleotide on ion-exchange HPLC, the ratio of
modified oligonucleotide to dU-containing oligonucleotide
Figure 1. Ion-exchange HPLC chromatograph showing the relative
incorporation of phosphoramidite 29 and 2Ј-deoxyuridine (dU)
phosphoramidite into a 15-mer DNA sequence. Peak A contains
the amino-modified nucleoside and the larger peak B contains the
unmodified dU. There is an integrated peak area ratio of about 1:
can be observed by integration of the two separate peaks. 1.6.
Table 2. The 81 different sequences formed from a mixture of three different phosphoramidites (represented here as 1, 2, 3) inserted over
four degenerate positions and their respective masses. The theoretical masses are calculated for the oligonucleotide sequence 2Ј-OMe-
GUC CUN NNN ACU CC where N contains a mixture of dU and the 5-carboxamide-modified amino and imidazolyl dU analogues
(represented by 1, 2 and 3, respectively). Although there are 81 different sequences, there are only 15 different masses.
Degenerate sequence
Theoretical mass
Degenerate sequence
Theoretical mass
Degenerate sequence
Theoretical mass
1111
1112
1113
1121
1122
1123
1131
1132
1133
1211
1212
1213
1221
1222
1223
1231
1232
1233
1311
1312
1313
1321
1322
1323
1331
1332
1333
4363
4449
4500
4449
4535
4586
4500
4586
4637
4449
4535
4586
4535
4621
4672
4586
4672
4723
4500
4586
4637
4586
4672
4723
4637
4723
4774
2111
2112
2113
2121
2122
2123
2131
2132
2133
2211
2212
2213
2221
2222
2223
2231
2232
2233
2311
2312
2313
2321
2322
2323
2331
2332
2333
4449
4535
4586
4535
4621
4672
4586
4672
4723
4535
4621
4672
4621
4707
4758
4672
4758
4809
4586
4672
4723
4672
4758
4809
4723
4809
4860
3111
3112
3113
3121
3122
3123
3131
3132
3133
3211
3212
3213
3221
3222
3223
3231
3232
3233
3311
3312
3313
3321
3322
3323
3331
3332
3333
4500
4586
4637
4586
4672
4723
4637
4723
4774
4586
4672
4723
4672
4758
4809
4723
4809
4860
4637
4723
4774
4723
4809
4860
4774
4860
4911
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coupling ratios were between 1.5–1.7:1 (unmodified: modi- test library was synthesised with the terminal dimethoxytri-
fied).
tyl group left on which was then removed during the course
of purification.
Although a library containing a mixture of three phos-
phoramidites inserted over four degenerate positions creates
Synthesis of an 81-Component Oligonucleotide Library
To ensure the suitability of our modified phosphoramid- a possible 81 different sequences, the members of that li-
ites in the production of oligonucleotide libraries, a number brary only possess 15 different masses (Table 2). This makes
of simplified test oligonucleotide libraries were synthesized characterization of the libraries a realistic possibility. Al-
by the standard oligonucleotide synthesis procedures used though the theoretical and experimental MALDI-TOF
previously. A selection of the modified phosphoramidites mass spectra of each test library were not identical they
and dU were coupled in a number of degenerate positions revealed qualitative consistencies with the masses and their
within 2Ј-O-methyl oligonucleotides using a 1.6:1 (modi- associated relative intensities (An example is shown: Fig-
fied:unmodified phosphoramidite) molar ratio. To enable ure 2). This suggests the phosphoramidites were all incor-
reversed phase cartridge purification of the mixtures, each porated and with similar coupling efficiencies. It is hoped
Figure 2. Theoretical (a) and experimental (b) mass spectra of an 81 member test library having the sequence 2Ј-OMe-
GUC CUN NNN ACU CC where N contains a mixture of dU and the 5-carboxamide-modified amino and imidazolyl dU analogues.
The theoretical masses are calculated in Table 2.
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(1 mL) was added and the reaction mixture again concentrated to
an oil under vacuum before being re-dissolved in the minimum
amount of a 2% solution of methanol in DCM and applied to a
silica gel column. The product was eluted using a gradient of 3–
8% methanol in DCM. Desired fractions were pooled and concen-
trated under vacuum to give a pale yellow powder (2.41 g;
that they can therefore be used in more complicated library
syntheses.
Conclusions
1
5.18 mmol; 62%). H NMR (300 MHz, DMSO, 25 °C): δ = 11.81
We have synthesised phosphoramidites of six nucleoside
analogues containing protected imidazolyl or amino func-
tionalities on the heterocyclic bases and shown efficient in-
corporation into oligonucleotides with similar efficiencies.
These nucleoside analogues are also shown to be suitable in
principle for use in synthesis of modified oligonucleotide
libraries of mixed sequences. The synthesis of appropriate
oligonucleotide libraries in various formats and testing for
RNA cleavage is currently underway.
(br., 1 H, NH-3), 8.77 (t, 3JH,H = 5.51 Hz, 1 H, NH–CH2), 8.67 (s,
1 H, H-6), 8.09 (s, 1 H, NCHN), 7.29 (s, 1 H, NCHC), 6.10 (t,
3
3JH,H = 6.6 Hz, 1 H, H-1Ј,), 5.25 (d, JH,H = 4.1 Hz, 1 H, 3Ј-OH),
3
4.97 (t, JH,H = 4.7 Hz, 1 H, 5Ј-OH), 4.21 (m, 1 H, H-4Ј), 3.84 (m,
1 H, H-3Ј), 3.53 (m, 2 H, H-5Ј, H5ЈЈ), 3.27 (m, 2 H, NHCH2CH2),
2.67 (t, 3JH,H = 6.8 Hz, 2 H, NHCH2CH2), 2.19–2.11 (m, 2 H, H2Ј,
H2ЈЈ), 1.55 [m, 9 H, C(CH3)3] ppm. 13C NMR (300 MHz, DMSO,
25 °C): δ = 162.4 (C-4), 154.4 (NH–CO), 150.1 (C-6), 146.6
(CH2C), 141 (NCHN), 114.4 (NCHC), 106 (C-5), 88 (C-4Ј), 86 (C-
1Ј), 86.2 [C(CH3)3], 91 (C-3Ј), 62.0 (C-5Ј), 46.6 (C-2Ј), 38.7
(NHCH2CH2), 28.6 (NHCH2CH2), 28 [C(CH3)3]. MS (ES+): m/z
= 466 [MH+], 366, 242, 229, 102. HRMS: m/z calculated for
C20H28N5O8 [MH+]: 466.1938, found 466.1949. TLC (DCM/meth-
anol, 9:1): Rf = 0.7.
Experimental Section
All reagents and solvents used in chemical synthesis were pur-
chased from Sigma–Aldrich or Lancaster. Methanol and ethanol
were distilled from over magnesium and stored with 4-Å molecular
sieves under argon. Dimethylformamide (DMF), pyridine and
dichloromethane (DCM) were purchased in their anhydrous form
and used as received. TLC was carried out on pre-coated F254
silica plates and column chromatography with BDH “Silica gel for
flash chromatography”. 1H and 13C NMR spectra were recorded
with a Bruker DRX 300 spectrometer. Chemical shifts (δ) for 1H
and 13C are referenced to internal solvent resonances and reported
relative to SiMe4. High resolution mass spectra were recorded with
a Bio-Apex II FT-ICR spectrometer.
Synthesis
of
5-{2-[1-(tert-Butoxycarbonyl)imidazol-4-
yl]ethylaminocarbonyl}-2Ј}-5Ј-dimethoxytrityl-2Ј-deoxyuridine (2):
The starting nucleoside 1 (2.0 g; 4.30 mmol) was dissolved in anhy-
drous pyridine (20 mL) and placed in a dry flask under argon. Di-
methoxytrityl chloride (1.60 g; 4.73 mmol; 1.1 equiv.), dissolved in
a 1:1 mixture of anhydrous DCM and pyridine (10 mL), was slowly
added to the stirring nucleoside solution. After 18 hours the reac-
tion was quenched by the addition of anhydrous methanol (1 mL)
and the reaction mixture was concentrated to an oil under vacuum.
The oil was then re-dissolved in DCM (100 mL) and washed with
saturated sodium hydrogen carbonate solution (satd. NaHCO3)
(100 mL) and brine (100 mL). The organics were dried with sodium
sulfate and then re-concentrated under vacuum to an oil before
being purified by silica gel flash column chromatography eluting
with a gradient of 1–4% methanol in DCM. Desired fractions were
concentrated under vacuum to give a white foam (2.0 g; 2.62 mmol;
61%). 1H NMR (300 MHz, DMSO, 25 °C): δ = 11.71 (br., 1 H,
Oligonucleotide Synthesis and Characterisation: Oligonucleotide
synthesis was carried out with an Applied Biosystems 394 DNA/
RNA synthesiser on a 1 µ scale under standard synthetic pro-
cedures based on phosphoramidite chemistry. Purification was car-
ried out by ion-exchange HPLC using a Dionex NucleoPac PA-100
column with a linear gradient of 15–55% buffer B over 20 minutes
[Buffer A: sodium perchlorate (1 m), Tris·HCl pH 6.8 (20 m),
25% formamide. Buffer B: Sodium perchlorate (400 m), Tris·HCl
pH 6.8 (20 m), 25% formamide]. Samples were then desalted by
dialysis and lyophilised. Analytical reversed-phase HPLC was car-
ried out, where necessary, with a Phenomonex Luna C-18(2) col-
umn eluting with a gradient of acetonitrile in 0.1  triethylammo-
nium acetate buffer (pH, 7.0). MALDI-TOF mass spectra were ob-
tained with a Voyage-DE BioSpectrometry Workstation (PerSep-
tive Biosystems) in positive ion mode using a 1:1 mixture of 2,6-
(dihydroxy)acetophenone (40 mg mL–1 in MeOH) and aqueous di-
ammonium citrate (80 mg mL–1) as matrix.
3
NH-3), 8.80 (t, JH,H = 5.5 Hz, 1 H, NHCH2), 8.41 (s, 1 H, H-6),
8.11 (s, 1 H, NCHN), 7.36–7.18/6.91–6.85 (m, 14 H, 13×Ar-H,
3
1×NCHC), 6.05 (t, JH,H = 6.6 Hz, 1 H, H-1Ј), 5.34 (br., 1 H, 3Ј-
OH), 4.24 (m, 1 H, H-4Ј), 3.90 (m, 1 H, H-3Ј), 3.69 (s, 6 H, 2×Ar–
OCH3), 3.52 (m, 2 H, H-5Ј, H5ЈЈ), 3.16 (m, 2 H, NHCH2CH2),
2.68 (t, 3JH,H = 6.8 Hz, 2 H, NHCH2CH2), 2.22–2.12 (m, 2 H, H2Ј,
H2ЈЈ), 1.54 [m, 9 H, C(CH3)3] ppm. MS (ES+): m/z = 768 [MH+],
668, 303, 242, 102. HRMS: m/z calculated for C41H46N5O10 [MH+]
768.3245, found 768.3250. TLC (DCM/methanol, 9:1): Rf = 0.7.
Generic Synthesis of 3Ј-O-ß-Cyanoethyl-N,N-diisopropylphos-
phoramidites 3, 9, 15, 19, 25 and 29 from the Corresponding 5Ј-
Dimethoxytrityl Compounds 2, 8, 14, 18, 24 and 28: The starting
dimethoxytritylated compound (Ϸ 0.5–0.6 mmol) was dried in
Synthesis
of
5-{2-[1-(tert-Butoxycarbonyl)imidazol-4-
yl]ethylaminocarbonyl}-2Ј-deoxyuridine (1): 5-Iodo-2Ј-deoxyuridine
(3.0 g; 8.47 mmol) was suspended in anhydrous DMF (30 mL) and
histamine (2.82 g; 25.4 mmol; 3.0 equiv.), triethylamine (7.1 mL; vacuo overnight before being dissolved in anhydrous DCM (5 mL)
50.8 mmol; 6.0 equiv.) and tetrakis(triphenylphosphane)palladium
(489 mg; 0.42 mmol; 0.05 equiv.) were added. The reaction mixture
was incubated in a Parr High-Pressure reaction vessel at 70 °C un-
der 90 psi carbon monoxide for 18 h. The reaction mixture was
then cooled to room temperature before being filtered through ce-
lite and the filtrate concentrated under vacuum to an oil. The oil
was then taken up in anhydrous DMF (50 mL) and to the stirring
solution under argon was added triethylamine (3.54 mL;
25.4 mmol; 3.0 equiv.) and tert-butyl dicarbonate (2.78 g;
12.7 mmol; 1.5 equiv.). After 15 minutes anhydrous methanol
and diisopropylethylamine (4 equiv.). The solution was then placed
under argon, before the addition of 2-cyanoethyl diisopropyl-
chlorophosphoramidite (1.5 equiv.). After 20 minutes the reaction
was quenched by the addition of distilled methanol (0.2 mL) and
the organics diluted by the addition of ethyl acetate (15 mL). The
solution was then washed with 10% sodium carbonate solution
(15 mL) and brine (15 mL) before being separated, dried with so-
dium sulfate and concentrated in vacuo. The residue was purified
by silica gel chromatography eluting with a mixture of DCM, ethyl
acetate and triethylamine (45:45:10 to 80:10:10). Desired fractions
Eur. J. Org. Chem. 2005, 5171–5183
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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5177

S. C. Holmes, M. J. Gait
FULL PAPER
were combined and concentrated under vacuum yielding a white
foam (75–90%).
silica gel flash column chromatography eluting the column in 2%
methanol in DCM. Desired fractions were concentrated to give a
white foam (1.7 g; 2.78 mmol; 73%). 1H NMR (300 MHz, DMSO,
25 °C): δ = 11.88 (br., 1 H, 3-NH), 9.46 (br. t, 1 H, 5-CONH), 8.78
(br. t, 1 H, NHCOCF3), 8.40 (s, 1 H, H-6), 5.98 (d, 1 H, H-1Ј),
4.53 (m, 1 H, H-4Ј), 3.99–3.95 (m, 2 H, H5Ј, H-5ЈЈ), 3.74 (m, 1 H,
H-3Ј), 3.40 (t, 3JH,H = 5.4 Hz, 2 H, CF3CONHCH2), 3.31 (t, 3JH,H
= 5.3 Hz, 2 H, 5-CONHCH2), 2.49–2.37 (m, 2 H, H2Ј, H2ЈЈ), 1.08–
0.96 [m, 28 H, 4×CH(CH3)2]. 13C NMR (300 MHz, DMSO,
25 °C): δ = 163.9 (C-4), 162.7 (NHCO–Ar), 152.2 (NHCOCF3),
150.1 (C-2), 147.1 (C-6), 105.8 (C-5), 86.2 (C-4Ј), 85.4 (C-1Ј), 70.7
(C-3Ј), 62.3 (C-5Ј), 38.1 (CH2–CH2), 17.9/13.5 [CH(CH3)2], 12.8
[CH(CH3)2] ppm. MS (ES+): m/z = 653 [MH+], 242, 180. HRMS:
m/z calculated for C26H44F3N4O8Si2 [MH+] 653.2650, found
653.2646. TLC (DCM/methanol, 49:1): Rf = 0.25.
Synthesis of 5-Iodo-3Ј,5Ј-O-[(tetraisopropyl)disiloxane-1,3-diyl]-2Ј-
deoxyuridine (4): 5-Iodo-2Ј-deoxyuridine (5.0 g; 14.2 mmol) was
dried by repeated co-evaporation from anhydrous pyridine
(3×50 mL) before being dissolved in a further portion of the same
solvent (50 mL) and sealed in a flask under argon. To the stirring
solution was added 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane
(5.0 mL; 15.63 mmol; 1.1 equiv.) and the reaction was left at room
temperature overnight. After concentrating to an oil under vac-
uum, the reaction mixture was re-dissolved in DCM (100 mL) and
washed with satd. NaHCO3 solution (100 mL). The organics were
separated and then washed with brine (100 mL). After a second
separation, the organics were dried with anhydrous sodium sulfate
and then concentrated under vacuum to a foam. Purification was
performed by silica-gel flash column chromatography eluting with
a gradient of 1–4% methanol in DCM. The desired fractions were
Synthesis of 5-[2-(Trifluoroacetylamino)ethylaminocarbonyl]-2Ј-de-
oxyuridine (7): The starting nucleoside 6 (1.7 g; 2.61 mmol) was dis-
solved in anhydrous THF (34 mL) and to the stirring solution was
added TBAF (1  in THF) (6.3 mL; 6.3 mmol; 2.4 equiv.). The re-
action was carefully monitored by TLC and after 2 hours water
(1 mL) was added and the solution partially concentrated under
vacuum before being applied directly to a silica gel column for puri-
fication. The desired product was slowly eluted from the column
using a gradient of 5–10% methanol in DCM (750 mg; 1.83 mmol;
70%). 1H NMR (300 MHz, DMSO, 25 °C): δ = 11.88 (br., 1 H,
concentrated under vacuum to yield
13.8 mmol; 97%). H NMR (300 MHz, DMSO, 25 °C): δ = 11.70
(s, 1 H, NH), 7.94 (s, 1 H, H-6), 5.92 (t, JH,H = 3.0 Hz, 1 H, H-
a white foam (8.2 g;
1
3
1Ј), 4.51 (m, 1 H, H-4Ј), 4.00–3.91 (m, 2 H, H5Ј, H5ЈЈ), 3.69 (m, 1
H, H-3Ј), 2.35–2.20 (m, 2 H, 2×H-2Ј), 1.08 [m, 4 H, 4×CH(CH3)2],
1.05–0.96 [m, 24 H, 4×CH(CH3)2] ppm. 13C NMR (300 MHz,
DMSO, 25 °C): δ = 161.4 (C-4), 150.7 (C-2), 145.6 (C-6), 85.3
(C-5), 84.9 (C-4Ј), 70.2 (C-5Ј), 61.9 (C-3Ј), 40.2 (C-2Ј), 17.9
[CH(CH3)2], 13.2 [CH(CH3)2] ppm. HRMS: m/z calculated for
C21H38IN2O6Si3 [MH+] 597.1313, found 597.1305. TLC (DCM/
methanol, 19:1): Rf = 0.9.
3
3
NH-3), 9.50 (t, JH,H = 4.8 Hz, 1 H, NHCOCF3), 8.82 (t, JH,H
=
3
5.6 Hz, 1 H, 5-CONH), 8.69 (s, 1 H, H-6), 6.11 (t, JH,H = 6.6 Hz,
1 H, H-1Ј), 5.28 (br., 1 H, 5Ј-OH), 5.01 (br., 1 H, 3Ј-OH), 4.22 (m,
1 H, H-4Ј), 3.84 (m, 1 H, H-3Ј), 3.42 (m, 2 H, 2×H-5Ј), 3.40 (m,
2 H, CH2NHCOCF3), 3.32 (m, 2 H, 5-CONHCH2), 2.19–2.09 (m,
2 H, 2×H-2Ј). 13C NMR (300 MHz, DMSO, 25 °C): δ = 163.9 (C-
4), 162.8 (CONH), 150.4 (C-2), 146.8 (C-6), 111.0 (CF3), 106.1 (C-
5), 89.7 (C-4Ј), 87.1 (C-1Ј), 71.3 (C-3Ј), 62.1 (C-5Ј) ppm. MS (ES+):
m/z = 433 [MNa+], 314, 242, 186. HRMS: m/z calculated for
C14H17F3N4O7Na [MNa+] 433.0947, found 433.0957. TLC (DCM/
methanol, 9:1): Rf = 0.25.
Synthesis of 5-Methoxycarbonyl-3Ј,5Ј-O-[(tetraisopropyl)disiloxane-
1,3-diyl]-2Ј-deoxyuridine (5): The starting nucleoside 4 (3.0 g;
5.03 mmol) was dissolved in anhydrous methanol (30 mL) and tri-
ethylamine (1.5 mL) and (bis-benzonitrile)palladium dichloride
(50 mg) was added to the solution. The reaction mixture was heated
at 50 °C under 80 psi carbon monoxide in a Parr pressure reactor
for 18 hours. After this time the reaction mixture was filtered
through celite to remove the palladium catalyst and the filtrate con-
centrated under vacuum to give a white powder. No further purifi-
Synthesis of 5Ј-Dimethoxytrityl-5-[2-(trifluoroacetylamino)ethylami-
nocarbonyl]-2Ј-deoxyuridine (8): The starting nucleoside 7 (1.12 g;
2.73 mmol) was dissolved in anhydrous pyridine (20 mL) and
placed in a dry flask under argon. Dimethoxytrityl chloride (1.02 g;
3.0 mmol; 1.1 equiv.), pre-dissolved in a 1:1 mixture of anhydrous
DCM and pyridine (8 mL), was slowly added to the stirring nucleo-
side solution. After 18 hours the reaction was quenched by the
addition of anhydrous methanol (1 mL) and the reaction mixture
was concentrated to an oil under vacuum. The oil was then re-
dissolved in DCM (100 mL) and washed with satd. NaHCO3 solu-
tion (100 mL) and brine (100 mL). The organics were dried with
sodium sulfate and then re-concentrated under vacuum to an oil
before being purified by silica gel flash column chromatography
eluting with a gradient of 1–4% methanol in DCM. Desired frac-
tions were concentrated under vacuum to give a white foam (1.52 g;
2.14 mmol; 78%). 1H NMR (300 MHz, DMSO, 25 °C): δ = 9.49
1
cation was required (5.0 mmol; 100% yield). H NMR (300 MHz,
CDCl3, 25 °C): δ = 9.02 (br., 1 H, NH), 8.55 (s, 1 H, H-6), 5.99
(m, 1 H, H-1Ј), 4.46 (m, 1 H, H-4Ј), 4.00–3.91 (m, 2 H, H5Ј, H5ЈЈ),
3.82 (s, 3 H, CH3), 3.18 (m, 1 H, H-3Ј), 2.55–2.52 and 2.37–2.34 (m,
2 H, H2Ј, H2ЈЈ), 1.10–1.00 [m, 28 H, 4×CH(CH3)2, 4×CH(CH3)2]
ppm. 13C NMR (300 MHz, CDCl3, 25 °C): δ = 159.6 (C-4), 149.5
(C-2), 147.5 (C-6), 86.0 (C-5), 85.7 (C-4Ј), 68.5 (C-5Ј), 60.9 (C-3Ј),
52.9 (CH3), 40.2 (C-2Ј), 17.5 [CH(CH3)2], 13.4 [CH(CH3)2] ppm.
HRMS: m/z calculated for C23H40N2O8 [MH+] 551.2221, found
551.2212. TLC (DCM/methanol, 19:1): Rf = 0.75.
Synthesis of 3Ј,5Ј-O-[(Tetraisopropyl)disiloxane-1,3-diyl]-5-[2-(tri-
fluoroacetylamino)ethylaminocarbonyl]-2Ј-deoxyuridine (6): The
starting nucleoside 5 (2.0 g; 3.8 mmol) was dissolved in anhydrous
DMF (50 mL) and placed under argon. To the stirring solution
was added ethylenediamine (2.5 mL; 38 mmol; 10.0 equiv.) and a
catalytic amount of (dimethylamino)pyridine (DMAP). After 18
hours the reaction mixture was concentrated to an oil under vac-
uum by co-evaporation with toluene. The oil was then re-dissolved
in anhydrous methanol (10 mL) and triethylamine (2.1 mL;
15.2 mmol; 4.0 equiv.) and ethyl trifluoroacetate (0.9 mL;
7.58 mmol; 2.0 equiv.) added. The solution was stirred for 10 min-
utes before being concentrated to an oil under vacuum. The oil was
then re-dissolved in DCM (50 mL) and washed with satd. NaHCO3
solution. The organic layer was separated, dried with anhydrous
sodium sulfate and re-concentrated. Purification was performed by
3
(br., 1 H, NHCOCF3), 8.82 (t, JH,H = 5.5 Hz, 1 H, 5-CONH),
8.42 (s, 1 H, H-6), 7.36–7.18/6.88–6.85 (m, 13 H, Ar-H), 6.08 (t,
3
3JH,H = 6.2 Hz, 1 H, H-1Ј), 5.34 (d, JH,H = 4.4 Hz, 1 H, 3Ј-OH),
4.08 (m, 1 H, H-4Ј), 3.71 (m, 1 H, H-3Ј), 3.39 (m, 2 H,
3
CH2NHCO), 3.31 (m, 2 H, 6-NHCH2), 3.17 (d, JH,H = 3.9 Hz, 2
H, 2×H-5Ј), 2.24–2.17 (m, 2 H, 2×H-2Ј) ppm. MS (ES+): m/z =
735 [MNa+], 529, 490, 380, 303, 284, 143, 102. HRMS: m/z calcu-
lated for C35H35F3N4O9Na [MNa+] 735.2254, found 735.2245.
TLC (DCM/methanol, 9:1): Rf = 0.6.
Synthesis of 3Ј,5Ј-O-[(Tetraisopropyl)disiloxane-1,3-diyl]-2Ј-deoxya-
denosine (10): 2Ј-Deoxyadenosine (10.0 g; 35.2 mmol) was dried by
5178
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© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2005, 5171–5183

Search for Sequence-Specific Artificial Ribonucleases
FULL PAPER
co-evaporation from anhydrous pyridine (3×100 mL) before being
dried overnight in a vacuum oven at 50 °C. The nucleoside was
then suspended in anhydrous pyridine (100 mL) and placed under
argon. 1,3-Dichloro-1,1,3,3-(tetraisopropyl)disiloxane (11.5 mL;
36 mmol; 1.02 equiv.) was carefully added to the suspension and
was left to stir vigorously for two hours. The reaction mixture was
then concentrated to an oil before being re-dissolved in DCM
(200 mL) and washed with satd. NaHCO3 solution. The organic
layer was separated, washed with brine and dried with anhydrous
sodium sulfate. After the organics were re-concentrated under vac-
uum, purification was performed by silica gel flash column
chromatography eluting in a gradient of 0–2% methanol in DCM.
The desired faction were combined and concentrated under vac-
uum to give a white foam (17.7 g; 33.6 mmol; 96%). 1H NMR
(300 MHz, DMSO, 25 °C): δ = 8.20 (s, 1 H, H-2), 8.06 (s, 1 H, H-
8), 7.29 (br., 2 H, NH2), 6.26 (d, 1 H, H-1Ј), 5.18 (m, 1 H, H-4Ј),
3.88 (m, 2 H, H-5Ј, H5ЈЈ), 3.77 (m, 1 H, H-3Ј), 2.64–2.52 (m, 2
H, H-2Ј, H-2ЈЈ), 1.10–1.00 [m, 28 H, 4×CH(CH3)2]. HRMS: m/z
calculated for C22H40N5O4Si2 [MH+] 494.2619, found 494.2621.
TLC (DCM/methanol, 19:1): Rf = 0.4.
2×H-2Ј), 1.63 (s, 9 H, 3×CH3), 1.12–1.05 [m, 28 H, 4×CH(CH3)2]
ppm. 13C NMR (300 MHz, DMSO, 25 °C): δ = 155.3 (C-6), 153.1
(C-2), 148.5 (C=O), 147.5 (C-4), 141.8 (CH2-C), 114.3 (Im-CH),
85.8 (CCH3), 85.2 (C-4Ј), 83.1 (C1Ј), 72.4 (C-2Ј), 63.5 (C-5Ј), 28.2
(CCH3), 27.9 (NHCH2CH2), 18.5/17.9 [CH(CH3)2], 13.5/12.9
[CH(CH3)2] ppm. MS (ES+): m/z = 688 [MH+], 579, 454, 342, 229,
217. HRMS: m/z calculated for C32H54N7O6Si2 [MH+] 688.3674,
found 688.3678. TLC (DCM/methanol, 9:1): Rf = 0.9.
Synthesis of N6-{2-[1-(tert-Butoxycarbonyl)imidazol-4-yl]ethyl}-2Ј-
deoxyadenosine (13): The starting nucleoside 12 (2.1 g; 3.06 mmol)
was dissolved in anhydrous THF (10 mL) and to the stirring solu-
tion was added a 1  solution of tetrabutylammonium fluoride
(TBAF) in THF (7.3 mL; 7.3 mmol; 2.4 equiv.). The reaction was
carefully monitored by TLC and after 20 minutes the solution was
partially concentrated to approximately one quarter of its volume
before being applied directly to a silica gel column for chromato-
graphic purification. After washing the column with DCM, the
product was purified using a gradient of 4–8% methanol in DCM.
Desired fractions were combined and concentrated under vacuum
to give a white foam (1.0 g; 2.21 mmol; 72%). 1H NMR (300 MHz,
DMSO, 25 °C): δ = 8.11 (s, 1 H, H-8), 7.69 (br., 1 H, NH), 7.28 (s,
1 H, H-2), 7.20 (s, 1 H, NCHN), 6.52 (s, 1 H, NCHC), 6.43 (t,
Synthesis of 6-Iodo-3Ј,5Ј-O-[(tetraisopropyl)disiloxane-1,3-diyl]-2Ј-
deoxypurine (11): The starting nucleoside 10 (10.0 g; 19 mmol) was
3
3JH,H = 6.6 Hz, 1 H, H-1Ј), 4.89 (d, JH,H = 5.5 Hz, 1 H, 3Ј-OH),
partially dissolved in
a mixture of diiodomethane (106 mL;
3
1.33 mol; 70 equiv.) and isoamyl nitrite (51.2 mL; 380 mmol;
20 equiv.). The reaction mixture was irradiated at 70 °C for one
hour with “white” light. After this time the reaction mixture was
cooled and was concentrated under vacuum before being re-
disssolved in DCM (250 mL) and washed with satd. sodium metab-
isulfite solution (150 mL) and brine (150 mL) before being dried
with sodium sulfate and concentrated. Purification was performed
by silica gel chromatography eluting with a gradient of 0–2% meth-
anol in DCM. The desired organics were pooled and concentrated
4.89 (t, JH,H = 5.4 Hz, 1 H, 5Ј-OH), 4.31 (m, 1 H, H-4Ј), 3.77 (m,
1 H, H-3Ј), 3.67 (m, 2 H, NHCH2CH2), 3.52–3.30 (m, 2 H, 2×H-
3
5Ј), 2.79 (t, JH,H = 7.0 Hz, 2 H, NHCH2CH2), 2.14–2.10 (m, 2 H,
2×H-2Ј), 1.56 (s, 9 H, 3×CH3) ppm. MS (ES+): m/z = 446 [MH+],
346, 284, 242, 186, 143. HRMS: m/z calculated for C20H28N7O5
[MH+] 446.2152, found 446.2162. TLC (DCM/methanol, 9:1): Rf
= 0.5.
Synthesis of N6-{2-[1-(tert-Butoxycarbonyl)imidazol-4-yl]ethyl}-5Ј-
dimethoxytrityl-2Ј-deoxyadenosine (14): The starting nucleoside 13
(880 mg; 1.77 mmol) was dried by repeated co-evaporation from
anhydrous pyridine (3×20 mL) before being dissolved in a further
portion of the solvent (10 mL). Dimethoxytrityl chloride (687 mg;
2.03 mmol; 1.15 equiv.) was added to the stirring solution under
argon before being left for 48 hours. After this period, a further
portion of dimethoxytrityl chloride (0.1 equiv.) was added and the
reaction monitored by TLC over 4 hours. As no further reaction
seemed to be taking place, the reaction was quenched by the ad-
dition of methanol (1 mL) and the solution was concentrated under
vacuum to give an oil. After dissolving the oil in DCM (50 mL) it
was washed with satd. NaHCO3 solution (50 mL) and re-concen-
trated before being purified by silica gel flash column chromatog-
raphy eluting the desired product in a gradient of 0–4% methanol
to give
a
yellow foam (7.4 g; 11.6 mmol; 61%). 1H NMR
(300 MHz, DMSO, 25 °C): δ = 8.72 (s, 1 H, H-2), 8.54 (s, 1 H, H-
8), 6.37 (d, 1 H, H-1Ј), 5.10 (m, 1 H, H-4Ј), 3.89 (m, 2 H, 2×H-
5Ј), 3.82 (m, 1 H, H-3Ј), 2.94–2.82/2.62–2.57 (m, 2 H, H-2Ј, H-
2ЈЈ), 1.04–1.00 [m, 28 H, 4×CH(CH3)2] ppm. 13C NMR (300 MHz,
DMSO, 25 °C): δ = 147.7 (C-4), 139.6 (C-6), 123.9 (C-5), 85.3 (C-
4Ј), 83.7 (C1Ј), 72.0 (C-2Ј), 63.2 (C-5Ј), 18.0/17.7 [CH(CH3)2], 13.5/
13.0 [CH(CH3)2] ppm. HRMS: m/z calculated for C22H38IN4O4Si2
[MH+] 605.1476, found 605.1486. TLC (DCM/methanol, 19:1): Rf
= 0.95.
Synthesis of N6-{2-[1-(tert-Butoxycarbonyl)imidazol-4-yl]ethyl}-
3Ј,5Ј-O-[(tetraisopropyl)disiloxane-1,3-diyl]-2Ј-deoxyadenosine (12):
The starting nucleoside 11 (3.0 g; 5.06 mmol) was dissolved in an-
hydrous DMF (30 mL) and placed under argon. To the stirring
solution was added histamine (1.68 g, 15.18 mmol; 3.0 equiv.) and
triethylamine (3.06 mL; 30.36 mmol; 6.0 equiv.) and the reaction
mixture left to stir overnight. Di-tert-butyl dicarbonate (5.28 g;
30.36 mmol; 6.0 equiv.) was then added and after 30 minutes anhy-
drous methanol (1 mL) was added to quench the reaction. The
solution was concentrated under vacuum to give an oil before being
taken up in DCM (100 mL) and washed with satd. NaHCO3 solu-
tion. The organics were separated, dried with anhydrous sodium
sulfate and then re-concentrated. Purification was performed by
silica-gel flash column chromatography eluting with a gradient of
0–4% methanol in DCM. Desired fractions were pooled and con-
centrated under vacuum to yield a pale yellow foam (3.41 g;
3.64 mmol; 72%). 1H NMR (300 MHz, CDCl3, 25 °C): δ = 8.35
(br., 1 H, NH), 8.02 (s, 1 H, H-8), 7.93 (s, 1 H, H-2), 7.26 (s, 1 H,
1
(1.12 g; 14.8 mmol; 83%). H NMR (300 MHz, DMSO, 25 °C): δ
= 8.11 (s, 1 H, H-8), 7.69 (br., 1 H, NH), 7.36 (s, 1 H, H-2), 7.33–
7.20/6.84–6.79 (m, 13 H, Ar–H), 7.05 (s, 1 H, NCHC), 6.51 (s, 1
3
H, NCHN), 6.45 (t, 1 H, H-1Ј), 5.32 (d, JH,H = 5.3 Hz, 1 H, 3Ј-
OH), 4.32 (m, 1 H, H-4Ј), 3.90 (m, 1 H, H-3Ј), 3.70 (s, 6 H,
2×CH3), 3.32 (m, 2 H, NHCH2CH2), 3.10 (m, 2 H, 2×H-5Ј), 2.79
3
(t, JH,H = 7.0 Hz, 2 H, NHCH2CH2), 2.22–2.18 (m, 2 H, 2×H-
2Ј), 1.56 (s, 9 H, 3×CH3) ppm. MS (ES+): m/z = 748 [MH+], 648,
546, 380, 303, 189. HRMS: m/z calculated for C41H45N7O7Na
[MNa+] 770.3278, found 770.3270. TLC (DCM/methanol, 19:1): Rf
= 0.5.
Synthesis of 3Ј,5Ј-O-[(Tetraisopropyl)disiloxane-1,3-diyl]-N6-[2-(tri-
fluoroacetylamino)ethylamino]-2Ј-deoxyadenosine (16): The starting
nucleoside 11 (3.0 g; 5.1 mmol) was dissolved in anhydrous DMF
NCHN), 7.17 (s, 1 H, NCHC), 6.27 (d, 1 H, H-1Ј), 4.97 (m, 1 H, (40 mL) and placed under argon. To the stirring solution was
H-4Ј), 4.04 (m, 2 H, NHCH2CH2), 3.89 (m, 3 H, 2×H-5Ј, H-3Ј), added ethylenediamine (6.8 mL; 102 mmol; 2.0 equiv.) and the re-
3
2.93 (t, JH,H = 6.4 Hz, 2 H, NHCH2CH2), 2.69–2.64 (m, 2 H,
action carefully monitored by TLC. After 1 hour the solution was
Eur. J. Org. Chem. 2005, 5171–5183
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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5179

S. C. Holmes, M. J. Gait
FULL PAPER
concentrated to an oil under vacuum and the residue re-dissolved
in DCM (100 mL). The organics were washed with satd. NaHCO3
(100 mL) and brine (100 mL) before being dried with sodium sul-
fate and concentrated to an oil under vacuum. The oil was then
taken up in anhydrous methanol (40 mL) and triethylamine
(14.2 mL; 102 mmol; 20.0 equiv.) and ethyl trifluoroacetate
(3.0 mL; 2.5 mmol; 5.0 equiv.) were added. After 20 minutes the
reaction mixture was once again concentrated under vacuum before
being taken up in DCM (100 mL) and washed with satd. NaHCO3
solution. After the organics were separated, dried with sodium sul-
fate and concentrated to an oil under vacuum, they were purified
by silica gel flash column chromatography eluting with a gradient
of 0–2% methanol in DCM. The desired fractions were concen-
trated to give a pale yellow foam (2.4 g; 3.87 mmol; 76%). 1H
NMR (300 MHz, DMSO, 25 °C): δ = 9.49 (br., 1 H, NHCOCF3),
8.23 (s, 1 H, C-8), 8.12 (s, 1 H, C-2), 7.97 (br., 1 H, 6-NH), 6.27
(d, 1 H, H-1Ј), 5.22 (m, 2 H, 6-NHCH2), 3.90 (m, 2 H, H-5Ј), 3.79
(m, 1 H, H-4Ј), 3.62 (m, 2 H, 6-NHCH2CH2), 3.41 (m, 1 H, H-
3Ј), 2.85–2.81/2.58–2.55 (m, 2 H, H2Ј, H2ЈЈ), 1.10–1.01 [m, 28 H,
4×CH(CH3)2] ppm. 13C NMR (300 MHz, DMSO, 25 °C): δ =
155.5 (C=O / C-6), 152.9 (C-2), 148.6 (C-4), 118.3 (C-5), 85.2 (C-
4Ј), 83.1 (C-1Ј), 72.5 (C-3Ј), 63.6 (C-5Ј), 38.2 (CONHCH2), 17.9–
17.7/13.5–12.9 [CH(CH3)2, CH(CH3)2] ppm. MS (ES+): m/z = 633
[MH+], 522, 377, 342, 229, 185, 143. HRMS: m/z calculated for
C28H42F3N6O5Si2 655.2707, found 655.2710. TLC (DCM/meth-
anol, 19:1): Rf = 0.1.
7.30, 7.23–7.17/6.81–6.76 (m, 13 H, Ar–H), 6.36 (t, 3JH,H = 6.5 Hz,
1 H, H-1Ј), 5.37 (d, JH,H = 4.4 Hz 1 H, 3Ј-OH), 4.46 (m, 1 H, H-
4Ј), 3.96 (m, 1 H, H-3Ј), 3.70 (s, 6 H, 2×OCH3), 3.62, 3.42/3.16
(m, 6 H, 2×H-5Ј, 2×CH2), 2.85/2.34 (m, 2 H, H-2Ј, H-2ЈЈ) ppm.
MS (ES+): m/z = 693 [MH+], 492, 303, 242, 102. HRMS: m/z calcu-
lated for C15H36F3N6O6 [MH+] 693.2648, found 693.2618. TLC
(DCM/methanol, 9:1): Rf = 0.65.
3
Synthesis of 3Ј,5Ј-O-[(Tetraisopropyl)disiloxane-1,3-diyl]-2Ј-deoxyu-
ridine (20): 2Ј-Deoxyuridine (6.89 g; 30.2 mmol) was dried by re-
peated co-evaporation from anhydrous pyridine (3×50 mL) before
being suspended in a further portion of the solvent (100 mL). 1,3-
Dichloro-1,1,3,3-(tetraisopropyl)disiloxane
(10 g;
31.7 mmol;
1.05 equiv.) was slowly added to the stirring suspension under ar-
gon and left for two hours. The pyridine was removed under vac-
uum and the remaining white solid dissolved in DCM (250 mL)
before being washed with satd. NaHCO3 (250 mL) and brine
(250 mL). The organics were separated, dried with sodium sulfate
and concentrated to an oil. Purification was performed by silica
gel flash column chromatography eluting in a gradient of 0–3%
methanol in DCM (13.2 g; 28.3 mmol; 94%). 1H NMR (300 MHz,
3
DMSO, 25 °C): δ = 10.19 (br., 1 H, NH), 7.76 (d, JH,H = 8.0 Hz,
3
3
1 H, H-6), 6.03 (d, JH,H = 6.2 Hz, 1 H, H1Ј), 5.68 (d, JH,H
=
8.0 Hz, 1 H, H-5), 4.41 (m, 1 H, H-4Ј), 4.13–4.09/3.99–3.95 (m, 2
H, H-5Ј, H-5ЈЈ), 3.73 (m, 1 H, H-3Ј), 2.53–2.46/2.27–2.20 (m, 2 H,
H-2Ј, H-2ЈЈ), 1.14–0.98 [m, 28 H, 4×CH(CH3)2] ppm. HRMS:
m/z calculated for C21H39N2O6Si2 [MH+] 471.2347, found
471.2352. TLC (DCM/methanol, 9:1): Rf 0.95
Synthesis of N6-[2-(Trifluoroacetylamino)ethyl]-2Ј-deoxyadenosine
(17): The starting nucleoside 16 (2.25 g; 3.56 mmol) was dissolved
in anhydrous THF (25 mL) and a 1  solution of TBAF in THF
(7.8 mL; 7.8 mmol; 2.2 equiv.) added. The reaction mixture was left
to stir at room temperature for approximately 30 minutes before
being concentrated under vacuum to about one quarter of its origi-
nal volume. This was then applied directly to a silica gel flash col-
umn chromatography column for purification. After washing the
column with DCM, the product was eluted in a gradient of 4–7%
Synthesis of 6-Methyl-3Ј,5Ј-O-[(tetraisopropyl)disiloxane-1,3-diyl]-
2Ј-deoxyuridine (21): The starting nucleoside 20 (14.5 g; 30.8 mmol)
was dissolved in anhydrous THF (100 mL) and sealed in a flask
under argon before being cooled to –78 °C. To the stirring solution
was added lithium diisopropylamide (1.5  solution in hexane)
(100 mL; 154 mmol; 5.0 equiv.) in a manner so as to maintain the
reaction temperature below –70 °C. The solution was then stirred
for one hour before the addition of iodomethane (2.9 mL;
46.2 mmol; 1.5 equiv.) and stirred for a further 2 hours. After this
time, glacial acetic acid (5 mL) was added and the solution warmed
up to room temperature before being concentrated under vacuum.
After re-dissolving the residue in DCM, a sodium hydrogen car-
bonate work-up was performed and the organics re-concentrated
under vacuum. Purification was performed by silica gel flash col-
umn chromatography eluting the column in 30% Ethyl acetate in
hexane and the after the elution of the faster running starting mate-
rial, 60% ethyl acetate in hexane. The desired fractions were con-
centrated to yield a white solid (8.65 g; 17.9 mmol; 58%). 1H NMR
(300 MHz, CDCl3, 25 °C): δ = 9.38 (br., 1 H, NH), 5.95 (m, 1 H,
H-1Ј), 5.51 (s, 1 H, H-5), 4.91 (m, 1 H, H-4Ј), 3.98 (m, 2 H, 2×H-
5Ј), 3.75 (m, 1 H, H-3Ј), 2.87/2.33 (m, 2 H, H-2Ј, H-2ЈЈ), 2.29 (s,
3 H, 6-CH3), 1.54 (s, 9 H, 3×BOC-CH3), 1.11–0.95 [m, 28 H,
4×CH(CH3)2] ppm. 13C NMR (300 MHz, CDCl3, 25 °C): δ =
163.5 (C-4), 153.7 (C-2), 150.4 (C-6), 103.4 (C-5), 86.1 (C-4Ј), 84.7
(C-1Ј), 73.4 (C-3Ј), 64.0 (C-5Ј), 39.6 (C-2Ј), 21.0 (6-CH3), 17.8
[CH(CH3)2], 13.6 [CH(CH3)2] ppm. HRMS: m/z calculated for
C22H40N2O6NaSi2 [MNa+] 507.2323, found 507.2325. TLC (DCM/
methanol, 19:1): Rf = 0.8.
1
methanol in DCM (1.2 g; 3.01 mmol; 85%). H NMR (300 MHz,
DMSO, 25 °C): δ = 9.5 (br., 1 H, NHCOCF3), 8.36 (s, 1 H, H-8),
3
8.20 (s, 1 H, H-2), 7.99 (br., 1 H, 6-NH), 6.34 (t, JH,H = 7.3 Hz,
3
3
1 H, H-1Ј), 5.31 (d, JH,H = 3.9 Hz, 1 H, 3Ј-OH), 5.21 (t, JH,H
=
5.3 Hz, 1 H, 5Ј-OH), 4.39 (m, 1 H, H-4Ј), 3.86 (m, 1 H, H-3Ј),
3.62–3.50 (m, 6 H, 2×H-5Ј, 2×CH2), 2.71 (m, 1 H, H-2Ј), 2.26 (m,
1 H, H-2ЈЈ) ppm. MS (ES+): m/z = 391 [MH+], 280, 275, 229, 164.
HRMS: m/z calculated for C14H18F3N6O4 [MH+] 391.1342, found
391.1331. TLC (DCM/methanol, 9:1): Rf = 0.25.
Synthesis of 5Ј-Dimethoxytrityl-N6-[2-(trifluoroacetylamino)ethyl]-
2Ј-deoxyadenosine (18): The starting nucleoside 17 (1.0 g;
2.51 mmol) was dried by repeated co-evaporation form anhydrous
pyridine (3×20 mL) before being re-dissolved in a further portion
of the solvent (10 mL). Dimethoxytrityl chloride (978 mg;
2.89 mmol; 1.15 equiv.), pre-dissolved in a 1:1 mixture of anhy-
drous pyridine and DCM (5 mL), was slowly added to the stirring
nucleoside solution under argon. After 6 hours the reaction was
quenched by the addition of anhydrous methanol (1 mL) and the
reaction mixture concentrated to an oil under vacuum before being
re-dissolved in DCM (50 mL). This organic solution was then
washed with satd. NaHCO3 solution (50 mL) and brine (50 mL)
before being dried with sodium sulfate and re-concentrated to an
oil. Purification was performed by silica gel flash column
chromatography eluting with a gradient of 0–4% methanol in
DCM. Desired fractions were combined and concentrated under
vacuum to yield a pale yellow foam (1.4 g; 2.0 mmol; 80%). 1H
Synthesis of N4-{2-[1-(tert-Butoxycarbonyl)imidazol-4-yl]ethyl}-6-
methyl-3Ј,5Ј-O-[(tetraisopropyl)disiloxane-1,3-diyl]-2Ј-deoxycytidine
(22): The starting nucleoside 21 (1.1 g; 2.28 mmol) was dissolved in
anhydrous DCM and placed in a flask under argon. To the stirring
solution was added triethylamine (0.35 mL; 5.48 mmol; 2.4 equiv.)
NMR (300 MHz, DMSO, 25 °C): δ = 9.50 (br., 1 H, NHCOCF3), tosyl chloride (433 mg; 2.74 mmol; 1.2 equiv.) and a catalytic
8.13 (s, 1 H, H-8), 8.14 (s, 1 H, H-2), 7.95 (br., 1 H, 6-NH), 7.33– amount of 4-(dimethylamino)pyridine. The solution was left to stir
5180
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© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2005, 5171–5183

Search for Sequence-Specific Artificial Ribonucleases
FULL PAPER
overnight. The reaction mixture was then washed with saturated
NaHCO3 solution, dried with sodium sulfate and concentrated to a
white foam. The tosylated nucleoside was re-dissolved in anhydrous
DMF before histamine (760 mg; 6.84 mmol; 3.0 equiv.) and trieth-
ylamine (1.9 mL; 13.7 mmol; 6.0 equiv.) were added and the solu-
tion stirred at room temperature for 2 hours. Di-tert-butyl dicar-
bonate (3.0 g; 13.7 mmol; 6.0 equiv.) was then added to the reaction
foam on drying (2.28 g; 3.04 mmol; 78%). 1H NMR (300 MHz,
CDCl3, 25 °C): δ = 7.98 (s, 1 H, NCHN), 7.43–7.19/6.80–6.77 (m,
13 H, Ar-H), 7.18 (s, 1 H, NCHC), 6.22 (m, 1 H, H-1Ј), 5.39 (s, 1
H, H-5), 4.68 (m, 1 H, H-4Ј), 3.80 (m, 1 H, H-3Ј), 3.71 (s, 6 H,
2×OCH3), 3.69 (m, 2 H, 4-NHCH2), 3.48–3.40 (m, 2 H, 2×H-5Ј),
2.78 (m, 2 H, 4-NHCH2CH2), 2.61–2.56 (m, 5 H, 6-CH3, 2×H-2Ј),
1.60 (s, 9 H, 3×BOC-CH3) ppm. MS (ES+): m/z = 760 [MNa+],
738 [MH+], 660, 529, 380, 303, 284, 143. HRMS: m/z calculated
for C41H48N5O8 [MH+] 738.3503, found 738.3525. TLC (DCM/
methanol, 19:1 ): Rf = 0.3.
mixture with
a further portion of triethylamine (0.95 mL;
6.84 mmol; 3.0 equiv.). After 30 minutes the reaction was quenched
by the addition of a small amount of anhydrous methanol (1 mL)
and the reaction mixture concentrated to an oil under vacuum. The
oil was taken up in DCM (50 mL) before being washed with satd.
NaHCO3 solution (50 mL) and brine (50 mL). The organics were
separated, dried and concentrated under vacuum. Purification was
performed by silica-gel flash column chromatography eluting with
a gradient of 0–4% methanol in DCM (944 mg; 1.46 mmol; 64%).
1H NMR (300 MHz, CDCl3, 25 °C): δ = 8.11 (s, 1 H, NCHN),
7.75 (br., 1 H, NH), 7.27 (s, 1 H, NCHC), 5.98 (m, 1 H, H-1Ј),
5.47 (s, 1 H, H-5), 5.01 (m, 1 H, H-4Ј), 3.97/3.84 (m, 2 H, H-5Ј,
H-5ЈЈ), 3.85 (m, 1 H, H-3Ј), 3.45 (m, 2 H, NHCH2), 2.71 (t, 2 H,
NHCH2CH2), 2.22 (m, 2 H, 2×H-2Ј), 2.18 (s, 3 H, 6-CH3), 1.52
(s, 9 H, 3×BOC-CH3), 1.14–0.98 [m, 28 H, 4×CH(CH3)2] ppm.
13C NMR (300 MHz, DMSO, 25 °C): δ = 163.9 (C-4), 156.1 (C-6),
153.2 (C-2), 147.6 (NCO), 141.7 (CH2CH2C), 130.3 (Im-NCHN),
128.4 (Im-CCHN), 96.0 (C-5), 86.2 (C-4Ј), 84.7 (C-1Ј), 79.4
[C(CH3)3], 75.4 [C-3Ј], 65.3 (C-5Ј), 28.4 (6-CH3), 17.6 [CH(CH3)2],
13.6 [CH(CH3)2] ppm. MS (ES+): m/z = 678 [MH+], 578, 488, 410,
320, 229, 143. HRMS: m/z calculated for C32H56N5O7Si2 [MH+],
678.3718, found 678.3735. TLC (19:1 DCM: Methanol): Rf 0.25.
Synthesis of 6-Methyl-3Ј,5Ј-O-[(tetraisopropyl)disiloxane-1,3-diyl]-
N4-[2-{trifluoroacetylamino}ethyl]-2Ј-deoxycytidine (26): The start-
ing nucleoside 21 (1.2 g; 2.51 mmol) was dissolved in anhydrous
DCM and placed in a flask under argon. To the stirring solution
was added triethylamine (0.39 mL; 6.02 mmol; 2.4 equiv.) tosyl
chloride (476 mg; 3.01 mmol; 1.2 equiv.) and a catalytic amount of
dimethylaminopyridine. The solution was left to stir overnight at
room temperature. The reaction mixture was then washed with sat-
urated NaHCO3 solution, dried with sodium sulfate and concen-
trated to white foam. The solid was re-dissolved in anhydrous
DMF and ethylenediamine (1.0 mL; 15.06 mmol; 6.0 equiv.) and
triethylamine (2.1 mL; 15.1 mmol; 6.0 equiv.) were added and the
solution stirred at room temperature for 30 minutes. The reaction
mixture was concentrated to an oil and then co-evaporated with
portions of toluene (3×20 mL) to dryness. The solid was re-dis-
solved in anhydrous methanol and ethyl trifluoroacetate (3.0 mL;
25.1 mmol, 10.0 equiv.) and triethylamine added (1.05 mL;
7.52 mmol; 3.0 equiv.). After 20 minutes the reaction mixture was
concentrated to an oil under vacuum. The oil was taken up in
DCM (100 mL) before being washed with satd. NaHCO3 solution
(100 mL) and brine (100 mL). The organics were separated, dried
and concentrated under vacuum. Purification was performed by
silica-gel flash column chromatography eluting with a gradient of
0–4% methanol in DCM (1.24 g; 1.91 mmol; 76%). 1H NMR
(300 MHz, DMSO, 25 °C): δ = 9.49 (br., 1 H, NHCOCF3), 7.77
(br., 1 H, 4-NH), 5.99 (m, 1 H, H-1Ј), 5.48 (s, 1 H, H-5), 5.00 (m,
1 H, H-4Ј), 3.98/3.94 (m, 2 H, 2×H-5Ј), 3.30 (m, 1 H, H-3Ј), 2.71
(m, 2 H, 4-NHCH2), 2.26 (m, 2 H, H-2Ј), 2.20 (s, 3 H, 6-CH3),
Synthesis of N4-{2-[1-(tert-Butoxycarbonyl)imidazol-4-yl]ethyl}-6-
methyl-2Ј-deoxycytidine (23): The starting nucleoside 22 (4.75 g;
7.0 mmol) was dissolved in anhydrous THF (80 mL) and a 1 
solution of TBAF in THF (16.8 mL; 16.8 mmol; 2.4 equiv.) added.
After 30 minutes the reaction mixture was concentrated to an oil
and then loaded directly onto a silica gel chromatography column
for purification. The desired compound was eluted using a gradient
of 4–7% methanol in DCM. Desired fractions were combined and
concentrated under vacuum to yield
a white solid (2.0 g;
4.47 mmol; 64%). 1H NMR (300 MHz, DMSO, 25 °C): δ = 8.11
3
(s, 1 H, NCHN), 7.61 (t, JH,H = 8.0 Hz, 1 H, NH), 7.28 (s, 1 H, 1.11–0.96 [m, 28 H, 4×CH(CH3)2] ppm. MS (ES+): m/z = 645
3
NCHC), 6.11 (m, 1 H, H-1Ј), 5.52 (s, 1 H, H-5), 5.07 (d, JH,H
=
[MNa+], 623 [MH+], 454, 342, 265, 229, 185, 143. HRMS: m/z
4.8 Hz 1 H, 3Ј-OH), 4.80 (m, 1 H, 5Ј-OH), 4.31 (m, 1 H, H-4Ј), calculated for C26H45F3N4O6NaSi2 [MNa+] 645.2727, found
3.65–3.45 (m, 5 H, 2×H-5Ј, H-3Ј, NHCH2), 2.71 (m, 2 H, 645.2719. TLC (DCM/methanol, 9:1): Rf = 0.8.
NHCH2CH2), 2.26 (s, 3 H, CH3), 1.93 (m, 2 H, 2×H-2Ј), 1.54 (s,
9 H, 3×BOC-CH3) ppm. MS (ES+): m/z = 436 [MH+], 343, 320,
Synthesis of 6-Methyl-N4-[2-(trifluoroacetylamino)ethyl]-2Ј-deoxy-
229, 186. HRMS: m/z calculated for C20H29N5O6 [MH+] 458.2016,
cytidine (27): The starting nucleoside 26 (1.90 g; 3.04 mmol) was
found 458.2031. TLC (DCM/methanol, 9:1): Rf = 0.25.
dissolved in anhydrous THF (40 mL) and a 1  solution of TBAF
Synthesis of N4-{2-[1-(tert-Butoxycarbonyl)imidazol-4-yl]ethyl}-5Ј- in THF (7.3 mL; 7.30 mmol; 2.4 equiv.) was added and the solution
dimethoxytrityl-6-methyl-2Ј-deoxycytidine (24): The starting nucleo-
side 23 was dried overnight in a vacuum oven before being dried
further by repeated co-evaporation from anhydrous pyridine
(2×20 mL). It was then dissolved in a further portion of anhydrous
pyridine (10 mL) and sealed in a vessel under argon. Dimethoxytri-
tyl chloride (1.45 g; 4.30 mmol; 1.1 equiv.), pre-dissolved in a 1:1
mixture of anhydrous pyridine and DCM (10 mL), was slowly
added to the stirring solution. After 6 hours the reaction was
quenched by the addition of anhydrous methanol (1 mL) and the
solution concentrated to an oil under vacuum. The oil was taken
up in DCM (50 mL) and washed with satd. NaHCO3 solution
(50 mL) and brine (50 mL) before being separated, dried with so-
dium sulfate and concentrated to a foam under vacuum. Purifica-
tion was performed by silica gel flash column chromatography elut-
ing with a gradient of 0–4% methanol in DCM to yield a yellow
stirred for 10 minutes. The reaction mixture was then partially con-
centrated under vacuum and then applied directly to a silica gel
chromatography column for purification. After eluting the column
with a large volume of DCM (400 mL) the desired product was
eluted using a gradient of 6–8% methanol in DCM. Desired frac-
tions were combined and concentrated under vacuum to yield a
white solid (920 mg; 2.36 mmol; 78%). 1H NMR (300 MHz,
DMSO, 25 °C): δ = 9.49 (br., 1 H, NHCOCF3), 7.71 (br., 1 H, 4-
NH), 6.12 (m, 1 H, H-1Ј), 5.53 (s, 1 H, H-5), 5.08 (d, 1 H, 3Ј-OH,
3
J = 4.9), 4.78 (t, JH,H = 5.4 Hz, 1 H, 5Ј-OH), 4.32 (m, 1 H, H-4Ј),
3.65–3.57/3.51–3.47 (m, 5 H, 2×H-5Ј, H-3Ј, 2×NHCH2CH2), 2.25
(s, 3 H, 6-CH3), 1.94 (m, 2 H, 2×H-2Ј) ppm. MS (ES+): m/z = 403
[MNa+], 288, 265, 186. HRMS: m/z calculated for C14H20F3N4O5
[MH+], 338.1386, found 338.1371. TLC (DCM/methanol, 9:1): Rf
= 0.4.
Eur. J. Org. Chem. 2005, 5171–5183
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5181

S. C. Holmes, M. J. Gait
FULL PAPER
Synthesis of 5Ј-Dimethoxytrityl-6-methyl-N4-[2-(trifluoroacetylam-
ino)ethyl]-2Ј-deoxycytidine (28): The starting nucleoside 27 (850 mg;
2.18 mmol) was dried by repeated co-evaporation form anhydrous
pyridine (3×20 mL) before being dissolved in a further portion of
the solvent (10 mL). Dimethoxytrityl chloride (811 mg; 2.40 mmol;
1.1 equiv.) was added and the reaction mixture stirred under argon
at room temperature overnight. The reaction was quenched by the
addition of anhydrous methanol (1 mL) and concentrated to an oil
under vacuum. After taking the oil up in DCM (50 mL) it was
washed with saturated NaHCO3 solution and brine. The organics
were dried with sodium sulfate and then concentrated to a foam.
Purification was performed by silica gel flash column chromatog-
raphy eluting with a gradient of 0–4% methanol in DCM (1.35 g;
1.95 mmol; 89%). 1H NMR (300 MHz, DMSO, 25 °C): δ = 9.83
(br., 1 H, NHCOCF3), 7.43–7.40, 7.28–7.19, 6.8–6.77 (m, 13 H,
Ar–H), 6.19 (m, 1 H, H-1Ј), 5.52 (s, 1 H, H-5), 4.62 (m, 1 H, H-
4Ј), 3.94 (m, 1 H, H-3Ј), 3.77 (s, 6 H, 2×OCH3), 3.53–3.47 (m, 2
H, 2×H-5Ј), 3.23–3.14, 2.71–2.68 (m, 4 H, NHCH2CH2), 2.19 (s,
3 H, 6-CH3), 1.92 (m, 2 H, 2×H-2Ј) ppm. MS (ES+): m/z = 683
[MH+], 492, 436, 303, 242, 150, 102. HRMS = m/z calculated for
C35H38F3N4O7 [MH+], 683.2720, found 683.2722. TLC (DCM/
methanol, 19:1): Rf = 0.25.
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Received: June 8, 2005
Published Online: September 7, 2005
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www.eurjoc.org
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