Synthesis and stability of a 2′-O-[N-(aminoethyl)carbamoyl] methyladenosine-containing dinucleotide

Article abstract of DOI:10.1002/ejoc.201300699

Working towards the synthesis of 2′-O-[N-(aminoethyl)carbamoyl] methyl-modified di- and oligonucleotides, we have synthesised a protected 2′-O-[N-(aminoethyl)carbamoyl]methyl-modified adenosine where the modification is introduced in a convenient one-pot three-step procedure. The corresponding H-phosphonate building block was also synthesised, and from this intermediate, a 2′-O-[N-(aminoethyl)carbamoyl]methyl-containing dinucleotide could be made. We also performed studies on the chemical and enzymatic stability of this dinucleotide. The dinucleotide was subjected to different ammonolysis and other basic conditions, and HPLC analysis showed that the modification was intact to most conditions, but that there was some minor hydrolysis when NH₃ (concd. aq.) was used at 55 °C. Under several other sets of conditions, including saturated NH₃ in methanol, and ethylenediamine, the amide remained intact. Treatment of the dinucleotide with Phosphodiesterase I from Crotalus adamanteus venom and Phosphodiesterase II from bovine spleen showed that the N-(aminoethyl)carbamoylmethyl moiety gives the phosphodiester linkage substantial protection against enzymatic degradation; the phosphodiester was not degraded by PDE II at all after seven days. A 2′-O-[N-(aminoethyl)carbamoyl]methyl-modified adenosine and a corresponding dinucleotide were synthesised. Hydrolysis (1-2 %) was observed in concentrated aqueous NH₃ at 55°C, but under several other sets of reaction conditions, the amide remained intact. The N-(aminoethyl) carbamoylmethyl moiety gave substantial protection against enzymatic degradation by nucleases from snake venom and bovine spleen. Copyright

Full text of DOI:10.1002/ejoc.201300699

FULL PAPER
DOI: 10.1002/ejoc.201300699
Synthesis and Stability of a 2Ј-O-[N-(Aminoethyl)carbamoyl]methyladenosine-
Containing Dinucleotide
Stefan Milton,[a] Charlotte Ander,[a] Dmytro Honcharenko,[a] Małgorzata Honcharenko,[a]
Esther Yeheskiely,[a] and Roger Strömberg*[a]
Keywords: Nucleosides / Nucleotides / Alkylation / Hydrolysis / Hydrolases
Working towards the synthesis of 2Ј-O-[N-(aminoethyl)-
carbamoyl]methyl-modified di- and oligonucleotides, we
and HPLC analysis showed that the modification was intact
to most conditions, but that there was some minor hydrolysis
when NH3 (concd. aq.) was used at 55 °C. Under several
other sets of conditions, including saturated NH3 in meth-
anol, and ethylenediamine, the amide remained intact. Treat-
ment of the dinucleotide with Phosphodiesterase I from
Crotalus adamanteus venom and Phosphodiesterase II from
bovine spleen showed that the N-(aminoethyl)carbamoyl-
methyl moiety gives the phosphodiester linkage substantial
protection against enzymatic degradation; the phosphodies-
ter was not degraded by PDE II at all after seven days.
have synthesised
a protected 2Ј-O-[N-(aminoethyl)carb-
amoyl]methyl-modified adenosine where the modification is
introduced in a convenient one-pot three-step procedure.
The corresponding H-phosphonate building block was also
synthesised, and from this intermediate, a 2Ј-O-[N-(amino-
ethyl)carbamoyl]methyl-containing dinucleotide could be
made. We also performed studies on the chemical and enzy-
matic stability of this dinucleotide. The dinucleotide was sub-
jected to different ammonolysis and other basic conditions,
of recognition by native enzymes, and therefore more modi-
fications are allowed. A large number of modifications of
oligonucleotides have been explored in the development of
oligonucleotides for biotechnology or therapy. Important
features of such modifications are affinity for the target mo-
lecule, preferably a low-cost manufacturing process, sta-
bility against enzymatic degradation, and uptake into cells.
In particular, 2Ј-O-alkyloligoribonucleotides[6] are a class of
modified oligonucleotides that has attracted great interest.
To modify the 2Ј-position has several advantages, including
the fact that low-cost starting materials can be used. In con-
trast to 2Ј-deoxynucleosides, the electron-withdrawing
groups in the 2Ј-position affect the pseudoaromatic charac-
ter of the nucleobase,[7] the conformational equilibrium in
the ribose moiety is also pushed towards the north (C3Ј-
endo) conformations, as found in the A-form geometry of
RNA duplexes, which typically leads to more stable du-
plexes with the target RNA.[8] A number of 2Ј-O-alkyloligo-
ribonucleotide modifications have, in contrast to results
with DNA, been shown to give increased stability of du-
plexes with RNA.[9] The 2Ј-O-carbamoylmethyl (CM)
modification[10] caught our attention, and we have recently
shown that this is highly resistant to enzymatic degrada-
tion.[11] Another interesting feature of the 2Ј-O-carbamo-
ylmethyl group is that, depending on the synthetic route
used, it can be readily functionalised with different substitu-
ents on the amide nitrogen.[12] An interesting substitution
of the CM that should give even higher hydrogen-bonding
potential (e.g., with the hydration network), as well as open-
Introduction
Synthetic nucleic acids have been and still are crucial for
the development of life science research, and modified oli-
gonucleotides are developed as means to treat patients with
genetic diseases. Most oligonucleotide therapies, including
siRNA[1] (short interfering RNA) and antisense technol-
ogies,[2] including splice-switching,[3] are limited by e.g., the
lability of oligonucleotides in biological fluids, and poor de-
livery to the site of action. Efficiency in the regulation of
gene expression is more readily achieved if turnover of the
target RNA is obtained. This can occur if native enzymes
(e.g., RNAse H for antisense, and RISC complex for
siRNA) can recognise the relevant oligonucleotide complex.
An alternative approach, independent of native enzymes, is
the use of oligonucleotide-based artificial nucleases
(OBANs)[4,5] with the aim of producing artificial enzymes
that can cleave mRNA sequences arising from genetic or
viral diseases. This allows modifications that give stable oli-
gonucleotides that are not degraded by host enzymes. The
approaches of splice-correction or splice-switching,[3] inter-
est in which is currently growing, are typically independent
[a] Department of Biosciences and Nutrition, Karolinska
Institutet, Novum,
Hälsovägen 7–9, 14183 Huddinge, Sweden
E-mail: roger.stromberg@ki.se
http://www.bionut.ki.se/groups/rst/
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201300699.
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Synthesis and Stability of a Dinucleotide
ing the possibility for electrostatic interactions, is a 2Ј-O- our investigation of the AECM modification further, start-
[N-(aminoethyl)carbamoyl]methyl (AECM) modification ing with the modification of adenosine.
(Figure 1).
In this paper, we report the synthesis of 2Ј-O-[N-(amino-
ethyl)carbamoyl]methyladenosine building blocks, and also
the synthesis of a AECM-A-containing dinucleotide that is
used as a model compound for further studies. In a pre-
vious study of a CM-modified dinucleotide, we found that
treatment with concentrated aqueous ammonia resulted in
partial hydrolysis of the carbamoylmethyl group.[10] The
stability of the 2Ј-O-AECM group under several different
sets of basic conditions that are often used for the final
deprotection of oligonucleotides was therefore investigated.
In addition, we also subjected the AECM-containing dinu-
cleotide to spleen phosphodiesterase II and snake-venom
phosphodiesterase I to see whether this modification ren-
dered the dinucleotide even more stable than the CM modi-
fication.
Figure 1. The 2Ј-O-[N-(aminoethyl)carbamoyl]methyl modifica-
tion.
The fact that 2Ј-O-aminopropyl-RNA carrying a posi-
tively charged ammonium group is resistant to nuclease de-
gradation is of additional interest.[13] As the uncharged CM
modification already results in substantial stabilisation, the
combination of CM with a charged ammonium group in
AECM seemed to be worth testing. While we were pursuing
our studies on the AECM modification, it was reported
Results and Discussion
2Ј-O-Alkyladenosines have been obtained by direct alkyl-
that oligonucleotides containing a single 2Ј-O-[N-(amino- ation of unprotected adenosine, e.g., using sodium hydride
ethyl)carbamoyl]uridine moiety gave a substantial decrease and an alkyl halide in DMF.[15,16] We chose to alkylate 5Ј-
in the melting point of duplexes with both DNA and O-(4-monomethoxytrityl)adenosine (1; Scheme 1) due to its
RNA.[14] We found this result unexpected, surprising, and higher solubility in organic solvents and also to the some-
unconvincing so we were not discouraged from pursuing what higher selectivity for the 2Ј-O-alkylated product over
Scheme 1. Synthesis of 2Ј-O-[N-(aminoethyl)carbamoyl]methyl-adenosine H-phosphonate building block 7.
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the 3Ј-O-alkylated compound compared to when unprotec- product with ethylenediamine; 3) Treatment with TFAA af-
ted adenosine is used. Compound 1 was dissolved in THF ter evaporation of the excess ethylenediamine. The product
and treated with dimsyl sodium[17] as a soluble base in THF was then subjected to chromatography on silica to give
to give the corresponding 2Ј-O-alkoxide.[18] Allyl bromo- compound 4 in 77% isolated yield (Scheme 2; the 2Ј-substi-
acetate was then added, and after isolation, methyl ester 2 tution was confirmed by 2D NMR spectroscopy, see Sup-
(transesterification with methanol apparently occurred dur- porting Information). This procedure was more convenient
ing chromatography in CH2Cl2/methanol, possibly via the and gave a somewhat higher overall yield than the sequence
2Ј,3Ј-lactone) was obtained (Scheme 1; substitution of the with isolation after each step. Following a similar one-pot
isolated compound on O-2Ј was confirmed by 1H–13C procedure, a substrate for Cu-catalysed 1,3-dipolar cycload-
correlated NMR spectroscopy, see Supporting Infor- dition (“click chemistry”),[20] the corresponding 2Ј-O-[N-
mation). Aminolysis of 2 was performed with ethylenedi- (azidoethyl)carbamoyl]methyl nucleoside, can be made con-
amine in methanol to give compound 3. Further treatment veniently from 1 and azidoethylamine.[21,22]
of 3 with trifluoroacetic anhydride (TFAA) gave compound
To investigate the stability of the 2Ј-O-AECM function-
4, and after protection of the remaining hydroxy group with ality to different sets of basic reaction conditions with a
trimethylsilyl chloride (TMSCl), protection of N-6 by treat- vicinal phosphodiester group, as well as to find out the ef-
ment with butyric anhydride gave 5 (Scheme 1). Removal of fect of the modification on the degradation by phosphate-
the 3Ј-O-TMS group gave 6, and subsequent phosphonyl- cleaving enzymes, a 2Ј-O-AECM dinucleotide was synthe-
ation using PCl3/imidazole[19] gave H-phosphonate building sised using H-phosphonate chemistry.[23] AECM-modified
block 7 (Scheme 1).
H-phosphonate 7 was coupled to 3Ј-O-MMT-thymidine 8
An improved three-step one-pot synthesis of compound (MMT = monomethoxytrityl) using bis(2-oxo-3-oxazolid-
4 was also developed: 1) Treatment with potassium tert-but- inyl) phosphinic chloride (OXP),[24] and this was followed
oxide, a soluble and commercially available base, in THF, by oxidation with iodine/pyridine/H2O. Fully protected di-
followed by allyl bromoacetate (or the commercially avail- mer 9 was then subjected to treatment with acetic acid (80%
able methyl bromoacetate); 2) Subsequent treatment of the aq.) to remove the MMT-groups. Final removal of the acyl
Scheme 2. Improved three-step one-pot synthesis of trifluoroacetyl-protected 2Ј-O-(N-aminoethylcarbamoyl)methyl-modified adenosine 4.
Scheme 3. Synthesis of 2Ј-O-[N-(aminoethyl)carbamoyl]methyl-adenosine-containing dinucleotide 10.
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Synthesis and Stability of a Dinucleotide
protection was performed with ethylenediamine (20% in degree of hydrolysis of the 2Ј-O-carboxymethyladenosine
methanol). The crude material was purified using reverse- 3Ј-(thymidine 5Ј-phosphate) moiety was less pronounced
phase (RP) HPLC chromatography to give 2Ј-O-AECM- than for CMAT,[10] and amounted to 1% degradation after
modified dinucleotide 10 (Scheme 3).
24 h (Figure 2, d) and 2% after 48 h (Figure 2, e) upon
The isolated dinucleotide (i.e., 10; AECM-AT) gave a treatment with ammonia (30% aq.) at 55 °C.
clean HPLC-profile (Figure 2, a), with a retention time dif-
Treatment of 10 at room temperature with ethylenedi-
ferent from the previously studied 2Ј-O-carbamoylmeth- amine (20% in methanol) or with saturated methanolic am-
yladenosine 3Ј-(thymidine 5Ј-phosphate)[10] (CMAT, Fig- monia, on the other hand, resulted in a clean product for
ure 2, c) and the hydrolysis product, i.e., 2Ј-O-carboxymeth- which the HPLC chromatogram did not reveal any traces
yladenosine 3Ј-(thymidine 5Ј-phosphate)[10] (COMAT, Fig- of degradation, even after treatment for 48 h (Figure 2f and
ure 2, b).
g). Although the retention times are different for CMAT
2Ј-O-AECM dinucleotide 10 was then subjected to dif- and AECM-AT (Figure 2a and c), we considered that peak
ferent conditions for ammonolysis and aminolysis with eth- overlap could mask the presence of CMAT in the AECM-
ylenediamine. As was seen in the previously reported study AT after treatment with methanolic ammonia. To see
of the CMAT, AECM-dinucleotide 10 was not completely whether we could detect any contamination by CMAT, we
stable under standard deprotection conditions for oligonu- also analysed the samples by MS, and we could not detect
cleotides, i.e., concentrated aqueous ammonia at 55 °C. The any mass peaks corresponding to CMAT but only from the
Figure 2. RP-HPLC analysis of a) 2Ј-O-(N-aminoethylcarbamoyl)-methyladenosine 3Ј-(thymidine 5Ј-phosphate) 10; b) 2Ј-O-carboxymeth-
yladenosine 3Ј-(thymidine 5Ј-phosphate); c) 2Ј-O-carbamoylmethyladenosine 3Ј-(thymidine 5Ј-phosphate); crude mixtures after treatment
of 10 (enlarged chromatograms) with different aqueous or alcoholic ammonia or ethylenediamine solutions: d) NH3 (concd. aq.) at 55 °C,
24 h; e) NH3 (concd. aq.) at 55 °C, 48 h; f) ethylenediamine (20% in methanol), room temp., 48 h; g) methanol saturated with NH3, room
temp., 48 h; h) NH3 (aq.)/ethanol (3:1), room temp., 24 h.
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AECM-AT. Thus, there is no detectable conversion from For comparison, an unmodified dApT dimer was also sub-
AECM-AT into CMAT in methanolic ammonia after 48 h. jected to similar treatment.
Another set of reaction conditions that are sometimes used
Aminoethylcarbamoylmethyl-modified dinucleotide 10 is
for the deprotection of oligonucleotides,[25] concentrated highly resistant to enzyme-catalysed cleavage by snake-
ammonia (30% aq.)/ethanol (3:1) at room temperature, also venom PDE I. Under conditions where the unmodified di-
caused no degradation (Figure 2, h).
mer was completely cleaved in about 2 h, there was still ca.
To see to what extent the N-(aminoethyl)carbamoyl- 75% of 10 remaining, even after 7 d (Figure 3, upper part).
methyl moiety would cause retardation of enzyme-catalysed When treated with spleen PDE II, modified dimer 10 was
hydrolysis of the vicinal phosphodiester groups was also of not cleaved at all after 7 d, under conditions where dApT
interest. Thus 2Ј-O-[N-(aminoethyl)carbamoyl]methyladen- was completely cleaved in a few hours (Figure 3, lower
osine 3Ј-(thymidine 5Ј-phosphate) 10 was treated with the part). The resistance to enzymatic degradation is
enzymes Phosphodiesterase I (PDE I) from Crotalus adam- remarkable, and is even greater than that of the stable
anteus venom and Phosphodiesterase II (PDE II) from bo- CMAT dinucleotide,[10] which lacks the aminoethyl substit-
vine spleen, and the mixtures were analysed by RP-HPLC. uent.
Figure 3. Graphs over different timescales showing percentage remaining of 2Ј-O-[N-(aminoethyl)carbamoyl]methyladenosine 3Ј-(thymid-
ine 5Ј-phosphate) 10 (filled symbols and solid lines) after treatment with snake-venom PDE I (upper graphs) or spleen PDE II (lower
graphs). Cleavage of the reference compound dApT under similar conditions is represented by ϫ and + symbols (and dashed lines).
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Synthesis and Stability of a Dinucleotide
anol). Reverse-phase HPLC was carried out with a Jasco apparatus.
For the analysis of the chemical stability reactions, the HPLC appa-
ratus was equipped with a Reprosil Pur-C18 column (3ϫ250 mm).
A 30 min linear gradient from 50 mm triethylammonium acetate
(pH 6.5) to 10% of 50 mm triethylammonium acetate in acetonitrile
was applied at room temperature, with a flow rate of 0.43 mL/min.
UV detection was carried out at 254 nm. In the enzymatic degrada-
tion study, the column and buffers used for the HPLC analysis were
the same, but a different gradient was used (0–10% for 20 min, and
then 10–35% for a further 10 min). All NMR spectra were recorded
using a Bruker AVANCE DRX-400 instrument (400.13 MHz for
1H, 162.00 MHz for 31P, and 100.62 MHz for 13C) in the solvents
specified. 13C and 1H chemical shifts are given in ppm. Most 1H
NMR assignments were made by standard 1H–1H-correlation spec-
troscopy (COSY). Mass spectrometric analysis was carried out with
a Micromass LCT ESI-TOF mass spectrometer, using leucine enke-
phaline as an internal mass standard. Dinucleotides were analysed
in negative mode in acetonitrile/water (1:1 v/v) solutions.
Conclusions
We have described the synthesis of 2Ј-O-[N-(aminoethyl)-
carbamoyl]methyladenosine (AECM-A) building blocks as
well as an AECM-A-containing dinucleotide (AECM-AT).
The preferred method for the crucial introduction of the 2Ј-
modification is the convenient and efficient one-pot three-
step procedure shown in Scheme 2. Synthesis of an AECM-
A H-phosphonate then enabled the preparation of AECM-
AT dinucleotide 10.
These results show that the 2Ј-O-[N-(aminoethyl)carb-
amoyl]methyl modification vicinal to a phosphodiester can
be hydrolysed by concentrated aqueous ammonia, condi-
tions that are commonly used for the complete deprotection
of oligonucleotides. The degradation only occurs to a minor
extent, but a degradation of even 1–2% is undesirable, espe-
cially for a fully modified oligonucleotide. However, this de-
gradation can be avoided by the use of ethylenediamine or 5Ј-O-(4-O-Monomethoxytrityl)-2Ј-O-(methoxycarbonyl)-methyl-
adenosine (2): Compound 1 (2.0 g, 3.7 mmol) was dried by evapora-
tion of added dry MeCN (50 mL). The dried material was dissolved
in distilled THF (200 mL), and dimsyl sodium (2 m solution in
DMSO; 4.44 mmol) was slowly added to the mixture. The mixture
was stirred for 30 min, and then allyl bromoacetate (4.8 mmol) was
added. After 1.5 h, ca. 175 mL of the THF was evaporated under
reduced pressure. CH2Cl2 (400 mL) was added, and the organic
phase was washed with a mixture of brine (300 mL) and H2O
(100 mL). The organic phase was dried with MgSO4 and concen-
trated. The crude product was purified by chromatography on a
methanolic ammonia for deprotection, as under these con-
ditions, the aminoethylcarbamoylmethyl group is stable.
We also showed that the 2Ј-O-aminoethylcarbamoyl
(AECM) moiety, to an even greater extent than the carb-
amoylmethyl modification,[10] protected the dinucleotide
against enzyme-catalysed degradation by snake-venom
phosphodiesterase I, and made it completely resistant to
degradation by spleen phosphodiesterase II. This makes the
AECM an interesting modification to use in constructs for
biotechnological and therapeutic applications such as oligo- silica gel column (CH2Cl2/methanol, 20:1 containing 0.05% trieth-
ylamine). During the chromatography and/or subsequent concen-
nucleotide-based artificial nucleases and antisense oligonu-
cleotides. It would be worth investigating fully or partially
AECM-modified oligonucleotides to see how the modifica-
tion affects duplex stability and biological activity.
tration, transesterification occurred so that methyl ester 2 (1.45 g,
1
68%) was obtained. H NMR (400 MHz, CDCl3): δ = 3.47 (dd, J
= 10.3 and 4.4 Hz, 1 H, 5Ј-Ha), 3.53 (dd, J = 10.3 and 3.2 Hz, 1
H, 5Ј-Hb), 3.75 (s, 3 H, CH3-trityl), 3.80 (s, 3 H, OMe-carbamoyl),
4.25–4.35 (m, 2 H, CH2-carbamoyl, 4Ј-H), 4.42–4.49 (m, 2 H, CH2-
carbamoyl, 3Ј-H), 4.75 (t, J = 4.7 Hz, 1 H, 2Ј-H), 6.16 (d, J =
6.7 Hz, 1 H, 1Ј-H), 6.30 (br. s, 1 H, 3Ј-OH), 6.82 (d, J = 8.9 Hz, 2
H, trityl), 7.20–7.50 (m, 12 H, trityl), 8.07 (s, 1 H, H2-base), 8.25
(s, 1 H, H8-base) ppm. 13C NMR (100.6 MHz, CDCl3): δ = 51.9,
54.7, 63.0, 67.9, 69.6, 83.5, 83.6, 86.5, 86.7, 112.8, 119.7, 126.7,
127.8, 128.0, 130.4, 134.9, 143.7, 149.4, 155.4, 159.2, 171.2 ppm.
HMQC and HMBC NMR spectra are included in Supporting In-
formation.
Experimental Section
General Materials and Methods: Allyl bromoacetate was synthe-
sised accordingly to a reported procedure.[26] Compound 1 (5Ј-
MMT-A) was prepared following a standard procedure[27] (see also
Supporting Information), and compound 8 (3Ј-MMT-T) was pre-
pared as reported.[28] Other reagents were of commercial grade, and
solvents and concentrated ammonia were of commercial analytical
grade. Solvents were dried over 4 Å (pyridine and CH2Cl2) or 3 Å
(methanol, ethanol, and acetonitrile) molecular sieves. THF and
dioxane were distilled at atmospheric pressure from LiAlH4, prior
to use. Toluene and diethyl ether were dried with sodium wire. Eth-
ylenediamine and triethylamine were distilled from calcium hy-
dride. Silica gel column chromatography was generally carried out
using Matrex silica, 60 Å (35–70 μm, Amicron). Solutions were con-
centrated under reduced pressure at temperatures not exceeding
40 °C. Ammonia (g) for the preparation of an ammonia–methanol
solution was from Aga Gas, Sweden. The 2Ј-O-carbamoylmeth-
yladenosine 3Ј-(thymidine 5Ј-phosphate) (CMAT) and 2Ј-O-carb-
oxymethyladenosine 3Ј-(thymidine 5Ј-phosphate) (COMAT) used
as HPLC references were prepared as reported.[10] The dApT dimer
used for comparison in the enzymatic study was purchased from
Cybergene AB and then further purified by HPLC using the same
gradient as was for the analysis (see below). Both Phosphodiester-
ase enzymes were purchased from Sigma. TLC analysis was carried
out on pre-coated plates, Silica Gel 60 F254 (Merck), with detection
by UV light and/or by charring with sulfuric acid (8% in meth-
5Ј-O-(4-O-Monomethoxytrityl)-2Ј-O-[N-(N-trifluoroacetamido)-
ethyl]carbamoylmethyladenosine (4)
Method A: Ethylenediamine (25 mL) was added to a solution of
compound 2 (15 g, 24.5 mmol) in EtOH (99.5% dried with 3 Å
molecular sieves; 150 mL) at room temperature. The reaction mix-
ture was left overnight at room tempeature. The solvent was evapo-
rated under reduced pressure. Pyridine (50 mL) was added and then
evaporated three times, and then toluene (50 mL) was added and
then evaporated three times. During this process, nitrogen (gas) was
used to equalise the pressure in between each round of evaporation.
The crude material (i.e., 3) was used without further purification.
Crude 3 was then dissolved in CH2Cl2 (500 mL), and triethylamine
(17.49 mL, 125.5 mmol) was added. TFAA (5 mL, 35.97 mmol)
was added over 3 h, and the reaction mixture was left for a further
1 h, after which TLC analysis showed that the reaction was com-
plete. The crude mixture was concentrated under reduced pressure
until 100 mL of the CH2Cl2 remained. The mixture was extracted
with ethyl acetate (350 mL) and washed twice with a mixture of
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R. Strömberg et al.
H2O (100 mL), brine (100 mL), and NaHCO3 (saturated aq.; 3.4 Hz, 1 H, 5Ј-Hb), 3.75 (s, 3 H, CH3-trityl), 4.10–4.27 (m, 3 H,
FULL PAPER
100 mL). Then the organic phase was dried with Na2SO4, filtered,
and concentrated under reduced pressure. The crude product was
purified by silica gel column chromatography (CH2Cl2/MeOH, 10:1
with 0.05% triethylamine) to give 4 (16.24 g, 90% from 2) as a
white foam.
CH2-carbamoyl, 4Ј-H), 4.48–4.60 (m, 2 H, 2Ј-H, 3Ј-H), 6.15 (d, J
= 3.3 Hz, 1 H, 1Ј-H), 6.77 (d, J = 8.8 Hz, 2 H, trityl), 7.15–7.50
(m, 12 H, trityl), 8.25 (s, 1 H, H2-base), 8.65 (s, 1 H, H8-base)
ppm.
N6-Butyryl-5Ј-O-(4-O-monomethoxytrityl)-2Ј-O-[N-(trifluoroacet-
amidoethyl)carbamoyl]methyladenosine (6): Compound 5
(0.085 mmol, 75 mg) was dissolved in CH2Cl2 (dried with 4 Å mo-
lecular sieves; 5 mL), and triethylamine-trihydrofluoride (three
drops) was added to the solution whilst stirring. After 20 min, TLC
showed that the reaction was complete. The solution was washed
with brine (whose pH had been adjusted to 8 with saturated
NaHCO3; 5 mL). The solvent was removed from the organic phase
under reduced pressure to give product 6 (68 mg, 99%) as a white
foam that appeared to be pure by NMR spectroscopic analysis. 1H
NMR (400 MHz, CDCl3): δ = 1.04 (t, J = 7.4 Hz, 3 H, CH3-Bu),
1.75–1.85 (m, 2 H, CH2-Bu), 2.81–2.89 (t, J = 7.4 Hz, 2 H, CH2-
Bu), 3.30–3.55 (m, 6 H, 5Ј-Ha, 5Ј-Hb, CH2-ethylene), 3.75 (s, 3 H,
CH3-trityl), 4.24 (ABq, J = 15.2 Hz, 2 H, CH2-carbamoyl), 4.33–
4.39 (m, 1 H, 4Ј-H), 4.50–4.75 (m, 2 H, 2Ј-H, 3Ј-H), 6.20 (d, J =
3.5 Hz, 1 H, 1Ј-H), 6.75 (d, J = 8.9 Hz, 2 H, trityl), 7.20–7.50 (m,
12 H, trityl), 8.25 (s, 1 H, H2-base), 8.65 (s, 1 H, H8-base) ppm.
Method B – One-Pot Procedure: Compound 1 (2.50 g, 4.77 mmol)
was dried by evaporation of added dry THF (distilled from
LiAlH4), and then the dried material was dissolved in dry THF
(200 mL). Potassium tert-butoxide (0.696 g, 6.20 mmol) was added,
and after 15 min, allyl bromoacetate (1.12 g, 6.20 mmol) was
added. The reaction mixture was stirred for 2 h, after which time
TLC showed that all of compound 1 had been consumed. The THF
was evaporated under reduced pressure, and EtOH (99.8% dried
with 3 Å molecular sieves; 100 mL) and ethylenediamine (2.87 g,
47.7 mmol) were added. The reaction mixture was stirred for 2 h,
and then dry THF (100 mL) was added and the reaction mixture
was left overnight at room temperature. TLC showed complete con-
version of the alkylated products. The solvent was evaporated, and
the excess ethylenediamine was removed by co-evaporation with
added dioxane four times (4ϫ 15 mL). The solid material was dis-
solved in dry CH2Cl2 (200 mL), and triethylamine (1.45 g,
14.31 mmol) and TFAA (1.65 g, 7.85 mmol) were added. TLC
showed that the reaction was complete after 2 h at room tempera-
ture. The reaction mixture was washed with a mixture of water and
brine (1:1; 400 mL). The organic phase was dried with MgSO4 and
concentrated. The crude product was purified by column
chromatography on silica using CH2Cl2/methanol (15:1 with
0.005% triethylamine) as eluent to give 4 (2.7 g, 77%) as a white
foam. (A slightly modified version of this reaction was performed
using methyl bromoacetate instead of the allyl reagent, with the
difference that the ethylenediamine was dried twice by evaporation
of added n-butanol and then three times with dioxane. Compound
N6-Butyryl-5Ј-O-(4-O-monomethoxytrityl)-2Ј-O-[N-(trifluoroacet-
amidoethyl)carbamoyl]methyladenosine 3Ј-H-Phosphonate Trieth-
ylammonium Salt (7): Imidazole (265 mg, 3.95 mmol) was dissolved
in CH2Cl2 (dried with 4 Å molecular sieves; 35 mL). The solution
was chilled to –10 °C, and PCl3 (116 μL, 1.26 mmol) and then tri-
ethylamine (560 μL, 4.04 mmol) were added. The solution was co-
oled down further to –78 °C. Compound 6 (285 mg, 0.359 mmol)
was dissolved in dry CH2Cl2 (3 mL). The solution containing com-
pound 6 was then slowly added by syringe through a septum into
the chilled PCl3/imidazole solution. The mixture was then left for
2 h, during which time it was allowed to reach room temperature.
The mixture was then extracted with CH2Cl2 (15 mL). The organic
phase was washed with triethylammonium hydrogen carbonate (2 m
aq.; 2ϫ 30 mL) and dried with Na2SO4. The solvent was removed
under reduced pressure to give 7 (345 mg, 99%) as a white foam,
and NMR spectroscopic analysis showed that no further purifica-
1
4 was then obtained in a yield of 72%.) H NMR (400 MHz, [D6]-
DMSO): δ = 3.14–3.30 (m, 6 H, CH2-ethylene, 5Ј-Ha, 5Ј-Hb), 3.72
(s, 3 H, CH3-trityl), 4.02–4.18 (m, 3 H, CH2-carbamoyl, 4Ј-H),
4.50–4.63 (m, 2 H, 2Ј-H, 3Ј-H), 5.48 (d, J = 6.4 Hz, 1 H, 3Ј-OH),
6.13 (d, J = 3.1 Hz, 1 H, 1Ј-H), 6.80 (d, J = 9.2 Hz, 2 H, trityl),
7.15–7.38 (m, 12 H, trityl), 8.12 (s, 1 H, H2-base), 8.25 (s, 1 H, H8-
base), 9.48 (br. s, 1 H, NH) ppm. 13C NMR (100.6 MHz, [D6]-
DMSO): δ = 37.2, 39.0, 55.1, 63.5, 69.4, 69.5, 81.8, 82.5, 86.0, 86.3,
113.3, 116.1 (q, J = 285 Hz), 118.3, 127.0, 127.9, 128.0, 130.1,
135.3, 144.5, 149.2, 153.0, 156.5, 156.8 (q, J = 39 Hz), 158.4,
169.5 ppm. Additional NMR spectroscopic data, including HMQC
and HMBC analysis, is given in the Supporting Information.
C36H36N7O7F3: calcd. C 58.77, H 4.93, N 13.33; found C 58.57, H
5.03, N 13.16.
N6-Butyryl-5Ј-O-(4-O-monomethoxytrityl)-2Ј-O-[N-(trifluoroacet-
amidoethyl)carbamoyl]methyl-3Ј-O-trimethylsilyladenosine (5):
Compound 4 (235 mg, 0.32 mmol) was dried by evaporation of
added pyridine (dried with 4 Å molecular sieves), and the dried
material was dissolved in dry pyridine (3 mL). Trimethylsilyl chlor-
ide (TMSCl; 123 μL, 0.96 mmol) was then added. The reaction
mixture was stirred for 1 h at room temperature. The solution was
then cooled to –10 °C (ice–salt bath), and butyric anhydride (64 μL,
0.39 mmol) was added. The reaction mixture was left overnight.
The solvent was removed under reduced pressure to give the crude
product, which was purified by chromatography on silica gel using
CH2Cl2/methanol (20:1) as eluent to give 5 (169 mg, 60%) as a
white foam. 1H NMR (400 MHz, CDCl3): δ = 0.05 (s, 9 H, Si-Me),
1
tion was needed before the next step. H NMR (400 MHz, CDCl3
plus Et3N): δ = 1.05 (t, J = 7.4 Hz, 3 H, CH3-Bu), 1.20 (CH3 from
Et3N), 1.77–1.86 (m, 2 H, CH2-Bu), 2.82–2.91 (m, 2 H, CH2-Bu
plus CH2 from Et3N), 3.35–3.65 (m, 6 H, 5Ј-Ha, 5Ј-Hb, CH2-ethyl-
ene), 3.77 (s, 3 H, CH3-trityl), 4.03 (ABq, J = 15.8 Hz, 2 H, CH2-
carbamoyl), 4.31–4.40 (m, 1 H, 4Ј-H), 4.87–4.93 (m, 1 H, 2Ј-H),
5.00–5.12 (m, 1 H, 3Ј-H), 6.94 (d, J = 640 Hz, 1 H, P-H), 6.23 (d,
J = 5.5 Hz, 1 H, HЈ-1), 6.82 (d, J = 8.8 Hz, 2 H, trityl), 7.20–7.48
(m, 12 H, trityl), 8.12 (s, 1 H, H2-base), 8.60 (s, 1 H, H8-base)
ppm. 31P NMR: δ = 3.5 ppm. ES-TOF: m/ z calculated for M-,
868.2683, found 868.2664.
2Ј-O-[N-(Aminoethyl)carbamoyl]methyladenosine 3Ј-(Thymidine 5Ј-
Phosphate) (10): H-phosphonate 7 (200 mg, 0.21 mmol) and 3Ј-O-
MMT-thymidine 8 (114 mg, 0.21 mmol) were dissolved in pyridine
(10 mL). Bis(2-oxo-3-oxazolidinyl) phosphinic chloride (OXP;
107 mg, 0.42 mmol) was added, and the solution was then stirred
for 1 h at room temperature. TLC analysis suggested that the for-
mation of 9 was complete. H2O (0.10 mL) and iodine (100 mg) were
added, and the mixture was stirred for a further 35 min. EtOAc
(250 mL) was then added, and the solution was washed with a sul-
fite solution [brine/Na2SO3 (1 m)/H2O, 1:1:1; 2ϫ 50 mL]. The or-
1.03 (t, J = 7.4 Hz, 3 H, CH3-Bu), 1.73–1.82 (m, 2 H, CH2-Bu), ganic phase was dried with Na2SO4 and concentrated under re-
2.89 (t, J = 7.4 Hz, 2 H, CH2-Bu), 3.27 (dd, J = 10.8 and 4.0 Hz,
1 H, 5Ј-Ha), 3.40 (m, 4 H, CH2-ethylene), 3.56 (dd, J = 10.8 and
duced pressure. Traces of pyridine were removed by the evaporation
of added THF (distilled from LiAlH4). The solid material was then
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Synthesis and Stability of a Dinucleotide
suspended in aqueous acetic acid (80%; 10 mL), and the mixture
was kept for 4 h. The solution was partioned between diethyl ether
(30 mL) and H2O (20 mL). The aqueous phase was evaporated un-
der reduced pressure, ethylenediamine (20% in MeOH dried with
3 Å molecular sieves; 8 mL) was added, and the resulting reaction
mixture was left for 21 h at room temperature. The solution was
concentrated under reduced pressure, and then ethanol (99.5 %;
10 mL) was added and then evaporated. This was repeated three
times. Ammonium acetate (10 mg) was added followed by ethanol,
and then the liquid was evaporated. The crude product (i.e., 10;
82 mg, 0.12 mmol, 57%) was of 97% purity (as judged by HPLC),
but to obtain very pure material for the studies of the dimer, a
60 mg portion of the material was further purified by reverse-phase
HPLC to give 10 (40 mg). [Reprosil Pur C-18 column
(20ϫ250 mm) using a 30 min. linear gradient from 50 mm aqueous
triethylammonium acetate (TEAA, pH 6.5) to 50mm TEAA in
20% acetonitrile, flow rate of 10 mL/min]. 1H NMR (400 MHz,
D2O): δ = 1.77 (s, 3 H, CH3-Thym), 2.28–2.40 (m, 2 H, 2Ј-Ha-
Thym, 2Ј-Hb-Thym), 3.15 (t, J = 5.8 Hz, 2 H, CH2-ethylene), 3.46–
3.55 (m, 2 H, CH2-ethylene), 3.82 (dd, J = 13.0 and 3.0 Hz, 1 H,
5Ј-Ha-Ad), 3.95 (dd, J = 13.0 and 2.2 Hz, 1 H, 5Ј-Hb-Ad), 4.05–
4.20 (m, 3 H, 5Ј-Ha-Thym, 5Ј-Hb-Thym, 4Ј-H-Thym), 4.25 (ABq,
J = 15.3 Hz, 2 H, 2Ј-O-CH2-carbonyl), 4.46–4.51 (m, 2 H, 4Ј-H-
Ad, 3Ј-H-Thym), 4.72 (t, J = 4.8 Hz, 1 H, 2Ј-H-Ad), 4.84–4.90 (m,
1 H, 3Ј-H-Ad), 6.15–6.20 (m, 2 H, 1Ј-H-Ad, 1Ј-H-Thym), 7.56 (s,
1 H, 6-H-Thym), 8.15 (s, 1 H, 2-H-Ad), 8.33 (s, 1 H, 8-H-Ad) ppm.
13C NMR (100.6 MHz, D2O): δ = 11.5, 36.4, 38.7, 39.1, 60.6, 65.0,
69.1, 70.0, 72.9, 81.0, 84.4, 84.8, 84.9, 85.1, 86.8, 111.2, 119.0,
136.7, 148.1, 151.3, 152.5, 155.4, 166.0, 172.4 ppm. (Es–/TOF): m/z
calculated for C24H33N9O12P [M], 670.1986, found 670.1963.
AS-I solutions (0.99 mL) to which enzyme solution ES-I (0.01 mL)
was added. The reactions were incubated at 37 °C, and aliquots of
0.1 mL were taken and quenched at different reaction times. For
the quenching, EDTA solution (75 mm; 0.02 mL) was added to
each aliquot, and the solution was then filtered through a Micron
YM-3-filter. The resulting solutions were then analysed by RP-
HPLC as described above.
For Phosphodiesterase II from Bovine spleen, which cleaves oligo-
nucleotides at the phosphate to give a 3Ј-phosphate and a 5Ј-OH,
the following procedure was used. An assay buffer (pH 6.5) con-
taining succinate buffer (s-2378; 250 mm) was prepared. The 2Ј-O-
CMAT dimer and dApT were each dissolved in the assay buffer to
give assay solutions (AS-II) with concentrations of 0.07 mm. A
stock solution of the enzyme (ES-II) was prepared containing en-
zyme (3 units/mL) in sterile water. For the enzyme assay with PDE
II, Eppendorf tubes were loaded with the respective ApT and
CMAT assay solutions (AS-II; 0.495 mL), and then enzyme solu-
tion ES-II (0.005 mL) was added. The reactions were incubated at
37 °C, and 0.1 mL aliquots were taken and quenched at different
times. To quench the reaction, the enzyme was removed by fil-
tration through a Micron YM-3-filter. The quenched reaction mix-
tures were analysed by RP-HPLC as described above.
Supporting Information (see footnote on the first page of this arti-
cle): Additional NMR spectroscopic data, including 2D 1H–13C
correlation spectra (HMQC and HMBC) of compounds 2 and 4.
Acknowledgments
Financial support from the S. Engkvist Foundation is gratefully
acknowledged.
Procedure for Investigating the Stability of AECM-AT Dimer 10 to
Different Basic Deprotection Solutions: Compound 10 (0.5 mg/
flask) was transferred into different 10 mL conical flasks from an
aqueous stock solution of known concentration. The solutions
were lyophilised, and the dry compound 10 was dissolved in the
different solutions (2 mL of each), which were then left for different
times and at different temperatures (see below). The tests were run
in duplicate. 1) NH3 (aq.) 30%, 24 h, 55 °C; 2) NH3 (aq.) 30%,
48 h, 55 °C; 3) ethylenediamine (20 % in methanol), 24 h, 20 °C;
4) ethylenediamine (20% in methanol), 48 h, 20 °C); 5) NH3 (satu-
rated in methanol), 24 h, 20 °C; 6) NH3 (saturated in MeOH), 48 h,
20 °C; 7) NH4OH (30% aq.)/EtOH (3:1), 24 h, 20 °C. After incu-
bation, each sample was concentrated to dryness under reduced
pressure, then water (2 mL) was added, and the sample was freeze-
dried. The material in each flask was then dissolved in buffer A
and subjected to RP-HPLC analysis as described above.
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Received: May 13, 2013
Published Online: September 13, 2013
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