A short and efficient synthesis of the tRNA nucleosides PreQ0 and archaeosine

Article abstract of DOI:10.1039/b713309j

Modified nucleosides in tRNAs play an important role in the translational process. They fine tune the codon-anticodon interactions and they influence the folding and stabilisation of the tRNA structure. Herein, we present a novel synthetic route to the highly modified nucleosides PreQ₀ and archaeosine. The synthesis involves coupling of a protected 7-cyano-7- deazaguanosine nucleobase with a TBDMS and isopropylidene protected chloro-ribose unit yielding the PreQ₀ nucleoside after deprotection. This PreQ₀ nucleoside is then used as the starting material for the synthesis of archaeosine providing the first total synthetic access to this hypermodified RNA nucleoside. The Royal Society of Chemistry 2007.

Full text of DOI:10.1039/b713309j

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www.rsc.org/obc | Organic & Biomolecular Chemistry
A short and efficient synthesis of the tRNA nucleosides PreQ0 and archaeosine
Tobias Bru¨ckl, Florian Klepper, Katrin Gutsmiedl and Thomas Carell*
Received 29th August 2007, Accepted 27th September 2007
First published as an Advance Article on the web 12th October 2007
DOI: 10.1039/b713309j
Modified nucleosides in tRNAs play an important role in the translational process. They fine tune the
codon–anticodon interactions and they influence the folding and stabilisation of the tRNA structure.
Herein, we present a novel synthetic route to the highly modified nucleosides PreQ0 and archaeosine.
The synthesis involves coupling of a protected 7-cyano-7-deazaguanosine nucleobase with a TBDMS
and isopropylidene protected chloro-ribose unit yielding the PreQ0 nucleoside after deprotection. This
PreQ0 nucleoside is then used as the starting material for the synthesis of archaeosine providing the first
total synthetic access to this hypermodified RNA nucleoside.
elusive until 1993, when Gregson et al. used mass spectrometric
Introduction
methods to elucidate the molecular structure.12 These authors
Transfer ribonucleic acids (tRNAs) contain more than one
compared the mass spectrometric data of the natural product
hundred modified nucleosides, which complement the four major
RNA nucleosides A, C, G and U.1 These modified nucleosides
with data from a small synthetic sample of archaeosine (7 lg)
obtained through transformation of 1 mg of the natural product
seem to be involved in the fine tuning of codon–anticodon
toyocamycin into archaeosine.
interaction at the ribosome and also influence folding and stability
of tRNAs.2–10 However, in most cases the exact role of the modified
Results and discussion
RNA nucleosides is not known. We have recently started a
synthetic program aimed at the preparation of the hypermodified
tRNA nucleosides queuosine11 and archaeosine in order to
investigate in detail their biosyntheses and their function in the
translational process. Herein, we report an efficient synthesis of the
tRNA nucleosides archaeosine 1, a hypermodified RNA nucleo-
side so far found only in archae bacteria,12 and PreQ0 2, which is a
biosynthetic precursor for archaeosine.5,7,10 Archaeosine occupies
position 15 in the majority of archaeal tRNAs (Scheme 1).7 It is
consequently a quite abundant hypermodification, which seems
to stabilize the 3-dimensional fold of these tRNAs. After being
discovered in 198213 the structure of the nucleoside remained
We decided to synthesize the target compound archaeosine 1 from
the nucleoside PreQ0 2 by conversion of the nitrile to an amidine.
The synthesis of 2 was thought to be possible via a glycosylation
of the heterocyclic precursor 3 with the protected sugar 4 as shown
in Scheme 2.
Scheme 2
Synthesis of the heterocyclic building block 3 started from
chloroacetonitrile 5. Reaction of 5 with formic acid methylester
and 2,6-diaminopyrimidine-4-one in a one pot procedure as
established by Migawa et al. gave nitrile nucleobase 6 (Scheme 3).14
Pivaloyl protection of 6 yielded compound 7.15 Subsequently,
the heterocycle 7 was activated for the subsequent glycosylation
reaction by replacement of the hydroxyl group at the aromatic
system for chlorine.16–19 By that route 3 was obtained in 21% overall
yield in only four steps.
Scheme 1
Ludwig-Maximilians University Munich, Butenandtstr. 5-13, Haus F, 81377
Munich, Germany. E-mail: Thomas.carell@cup.uni-muenchen.de; Fax: +49
(0)89 2180 77756; Tel: +49 (0)89 2180 77755
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spectrum. Furthermore, interactions between C(6)H and C1ꢀ
(Scheme 4a) were clearly detected. This unequivocally determines
the constitution of the nucleoside 11. The sugar and the nucleobase
moiety are connected via a bond between C1ꢀ and N7.
Scheme 4 (a) Nucleoside 11 highlighting the systematic numbering of the
nucleobase and of C(1ꢀ)H; (b) nucleoside 11 displaying the sugar and the
nucleobase protons together with observed NOESY interactions.
The corresponding NOESY spectrum proved the b-confi-
guration of the obtained nucleoside 11. A strong interaction
between C(6)H and C(2ꢀ)H was observed. Additional interac-
tions (C(6)H–C(3ꢀ)H and C(6)H–C(5ꢀ)H) usually observed for
b-configured nucleosides and the absence of typical interactions
(C(6)H–C(4ꢀ)H) in a-nucleosides further supported the stereo-
chemical assignment (Scheme 4b). Another indication for the b-
configuration of 11 is the small C(1ꢀ)H–C(2ꢀ)H coupling constant
(1 Hz), which is in the typical range for b-configured nucleosides.
a-Configured nucleosides usually show larger coupling constants.
These spectroscopic data, in our eyes, prove the formation of the
glycosidic bond between C1ꢀ and N7 and the b-configuration of
the obtained nucleoside 11.
Scheme
3 Synthesis of PreQ0 2: (a) HCOOMe, NaOMe, THF;
(b) NaOAc, 2,6-diaminopyrimidine-4-one, water, THF, 38%; (c) trimethyl-
acetyl chloride, pyridine, 75%; (d) POCl3, TEBACl, dimethylaniline,
acetonitrile, 75%; (e) acetone, conc. HCl, 87%; (f) imidazole, TBDMSCl,
DMF, 83%; (g) tetrachloromethane, HMPA; (h) NaH, acetonitrile, 20%;
(j) Et3N, DABCO, NaOAc, DMF, 81%; (k) trifluoroacetic acid, water,
77%; (l) 28% ammonia in water, 99%.
Conversion of the chloro to the needed oxo substituent in
position 4 of the nucleoside 11 was achieved with DABCO, Et3N
and NaOAc yielding 12 (Scheme 3).25 The protecting groups at the
sugar moiety of the nucleoside 12 were removed using a water–
trifluoroacetic acid mixture to give 13. The best method for the
deprotection of the amine in position 2 of the nucleoside 13 proved
to be 28% ammonia in water. By this method the conversion of 13
to PreQ0 2 was quantitative. The transformation of 11 to PreQ0
2 was therefore possible with 62% yield in only three steps. The
analytical data obtained from compound 2 compared very well
with those published by Kondo et al.26 and Cheng et al.27,28
The final transformation of PreQ0 to archaeosine turned out
to be the second critical step. A synthesis of the archaeosine
base only was reported by Hashizume and McCloskey.29 They
converted the nitrile nucleobase 6 into the corresponding amidine
with trimethylaluminium and ammonium chloride. This method,
however, failed in our hands with compound 2 as the starting
material. Also, the conversion of the nitrile to the amidine with
hydroxylamine followed by reduction with palladium on charcoal
or Raney nickel24,30,31 gave no conversion to compound 1. Wefinally
used the classical Pinner reaction32–36 for the desired transforma-
tion. Treatment of a solution of 2 in methanol with HCl (g) for 3 h,
followed by the removal of the solvent and stirring of the resulting
white solid in 7 N ammonia in methanol indeed gave the title
compoundarchaeosine 1 as a white solid (Scheme 5). 1 was purified
by reversed phase HPLC with a 0.1 M triethylammonium acetate
buffer. The resulting white solid turned out to be archaeosine
obtained as its acetate salt plus a small amount of additional
The second building block 4 was synthesized starting from
ribose 8. Protection of the 2ꢀ,3ꢀ-OH groups with acetone gave
compound 9.20,21 Treatment of the crude product with TBDMSCl
provided the 5ꢀ-O-protected sugar 10 in 72% yield. Conversion of
10 into the glycosyl donor 4 was achieved with HMPA and CCl4.22
Due to the lability of compound 4, it was used for the glycosylation
reaction without further purification.
For the crucial step of the synthesis, the glycosylation, initially
1-acetoxyribose-2,3,5-tribenzoate as glycosyl donor was reacted
with compound 6 under Lewis acid catalysis.23 This route, however,
turned out to give an inseparable mixture of two products.
NMR measurements indicated formation of a nucleoside with
the sugar attached to the exocyclic amino group. Additionally,
when 3 was reacted under these conditions no conversion was
observed. Ramasamy et al.24 proposed a different glycosylation
route using 4 and a twofold excess of NaH and 4-chloro-2-amino-
7H-pyrrolo[2,3-d]pyrimidine. Deprotonation of two equivalents
of 3 with NaH and subsequent addition of 4 to the reaction
mixture provided the nucleoside 11 in 16% yield. In our hands,
the best yield (20%) was obtained when a 1.4 fold excess of NaH
and of th◦e glycosyl donor were used (Scheme 3). Slow addition
of 4 at 0 C and rigorously dried solvents and reagents are very
important for the success of the reaction.
The configuration of the obtained product 11 was verified by
HMBC and NOESY spectroscopy. Clear interactions could be
observed between C(1ꢀ)H and C6, C5 and C7a in the HMBC
3822 | Org. Biomol. Chem., 2007, 5, 3821–3825
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dH (600 MHz, [D6]DMSO) 6.40 (2 H, s, NH2), 7.63 (1 H, s, C(6)H),
10.72 (1 H, s, N(7)H) and 12.00 (1H, s, N(3)H); dC (150 MHz,
[D6]DMSO) 90.7 (C5), 105.0 (C4a), 121.7 (CN), 133.7 (C6), 157.7
(C7a), 159.7 (C2) and 163.8 (C4); m/z (ESI−) 174.0426 (M−,
C7H4N5O− requires 174.0421).
5-Cyano-3,4-dihydro-2-pivaloylamino-7H-
pyrrolo[2,3-d]pyrimidine-4-one 7
6 (1.5 g, 8.6 mmol) was dissolved in pyridine (10 cm3) and
slowly treated with trimethylacetyl chloride (3.1 g, 26 mmol).
The resulting suspension was stirred for 2 h at 85 ◦C. After
cooling to room temperature a white solid precipitated, which
was removed by filtration. The resulting solution was neutralised
with 7 N ammonia in methanol and left to stand at 4 ◦C for
16 h. The resulting precipitate was collected and washed with
ethanol (20 cm3) and diethyl ether (10 cm3) to give the pivaloyl
protected nucleobase 7 (1.7 g, 75%). Rf = 0.2 (DCM–MeOH, 20 :
1); mmax/cm−1 3125 vs (NH), 2228 s (CN), 1684 s (CO), 1646 vs
(CC), 1608 vs, 1579 vs (NH), 1409 vs (CH3), 1239 s (CN), 1176 s
(CN), 938 w (CH) and 782 s (CH); dH (600 MHz, [D6]DMSO)
1.25 (9 H, s, CH3), 7.93 (1 H, s, C(6)H), 11.00 (1 H s, N(7)H),
12.11 (1 H, s, N(3)H) and 12.65 (1 H, s, NHPiv); dC (150 MHz,
[D6]DMSO) 27.0 (C(CH3)3), 40.7 (C(CH3)3), 86.9 (C5), 103.7
(C4a), 115.9 (CN), 130.9 (C6), 149.1 and 149.1 (C2 and C7a),
156.4 (C4) and 181.7 (COC(CH3)3); m/z (ESI+) 259.1088 (M+,
Scheme 5 Synthesis of archaeosine 1: (i) HCl (g), methanol (ii) 7 N
ammonia in methanol, 30%.
triethylammonium acetate. Attempts to completely remove the
excess triethylammonium acetate under high vacuum caused
degradation of archaeosine 1 partly back to PreQ0 2. However,
the analytical data obtained for the product were in excellent
agreement with those presented by Gregson et al.12
In summary, we were able to establish a short and efficient
synthesis of PreQ0 (2% overall yield, 11 steps) and archaeosine
(0.6% overall yield, 12 steps).
Together with our recently presented work on the synthesis
of queuosine,11 these syntheses allow preparation of most of
the deazaguanosine modified tRNA nucleosides in reasonable
quantities. Our future aim is to incorporate these deazaguanosine
derived nucleosides into RNA strands via RNA solid phase
synthesis in order to unravel their functions in tRNA.
+
C12H13N5O2 requires 259.1069).
Experimental
4-Chloro-5-cyano-2-pivaloylamino-7H-pyrrolo[2,3-d]pyrimidine 3
General procedures
7 (200 mg, 0.77 mmol) was suspended in acetonitrile (1.2 cm3) and
dimethylaniline (0.41 cm3, 3.28 mmol), triethylbenzylammonium
chloride (TEBACl) (89 mg, 0.39 mmol) and phosphoryl chloride
(0.72 cm3, 7.70 mmol) were added. The suspension was heated to
90 ◦C for 1 h. The cooled down suspension was concentrated
in vacuo and the resulting oil was cautiously treated with ice.
The acidic mixture was set to pH = 4 with conc. ammonia in
methanol. The precipitate was collected by filtration and washed
with water. Purification by column chromatography (isohexane–
ethyl acetate 3 : 2) gave nucleobase 3 (160 mg, 75%) as a white
solid. Rf = 0.65 (CHCl3–MeOH, 9 : 1); mmax/cm−1 3448 w (NH),
3160 vs (NH), 2231 s (CN), 1708 s (CO), 1563 vs (NH), 1521 s
(NH), 1445 vs (CH3), 1419 vs (CH3), 1271 s (CN), 1164 s (CN)
and 785 s; dH (400 MHz, [D6]DMSO) 1.22 (9 H, s, C(CH3)3), 8.50
(1 H, s, C(6)H), 10.27 (1 H, s, N(7)H) and 13.35 (1 H, s, NHPiv);
dC (100 MHz, [D6]DMSO) 27.4 (C(CH3)3), 40.3 (C(CH3)3), 84.1
(C5), 111.8 and 115.1 (C4a and CN), 138.2 (C6), 151.4 (C4), 153.5
(C7a), 153.8 (C2) and 176.5 (COC(CH3)3); m/z (ESI+) 278.0796
(M+, C12H13ClN5O+ requires 278.0806).
All reactions were performed with dry solvents in dried Schlenk
flasks fitted with a septum under a positive pressure of nitrogen,
unless water was used as a solvent. Air- and moisture-sensitive
liquids and solutions were transferred via syringes. Organic
solutions were concentrated by rotary evaporation below 40 ◦C
under appropriately reduced pressure. Thin layer chromatography
(TLC) was carried out with precoated Merck F254 silica gel plates.
Flash chromatography (FC) was carried out with Merck Silica gel
60 (0.040–0.063 mm). Reagents, dry solvents and starting materials
were obtained from commercial suppliers, and used without
further purification unless otherwise indicated. IR spectra were
recorded on a Bruker IFS 25 and a Bruker IFS 88 spectrometer.
IR data are reported as wave numbers with w (weak), s (strong)
and vs (very strong) indicating the intensity of the absorption
bands. 1H NMR and 13C NMR spectra were recorded with
Varian Mercury 200 (200 MHz and 75 MHz), Bruker AMX
400 (400 MHz and 100 MHz) and Bruker AMX 600 (600 MHz
1
and 150 MHz) spectrometers. Chemical shifts for H NMR are
reported in ppm relative to Me4Si as internal standard. Data
1
for H NMR are reported as follows: chemical shift (d, ppm),
2ꢀ,3ꢀ-O-Isopropylidene-a/b-D-ribofuranose 920,21
integration, multiplicity (s = singlet, d = doublet, t = triplet, m =
multiplet), coupling constant (Hz) and assignment. Data for 13C
NMR are reported in terms of chemical shifts. Mass spectrometry
was performed with Finnigan MAT 95Q, Finnigan MAT 90 and
Thermo Finnigan LTQ-FT mass spectrometers.
D-Ribose 8 (25.0 g, 150 mmol) w◦as suspended in acetone. Conc.
HCl (0.025 cm3) was added at 0 C and the mixture was stirred
for 2 h. The resulting suspension was filtered through Celite and
activated charcoal. Concentration of the resulting solution gave
the isopropylidene protected sugar 9 (33.0 g, 87%). Rf = 0.29
(DCM–MeOH, 20 : 1); dH (200 MHz, [D6]DMSO) 1.31 (3 H, s,
CH3), 1.47 (3 H, s, CH3), 3.72 (2 H, m, C(5ꢀ)H2), 4.40 (1 H, t, J 2,
C(4ꢀ)H), 4.57 (1 H, d, J 6, C(3ꢀ)H), 4.83 (1 H, d, J 6, C(2ꢀ)H) and
5.41 (1 H, s, C(1ꢀ)H).
2-Amino-5-cyano-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidine-4-
one 6
14
6 was synthesised according to literature.
m
max
/cm−1 3362 s (NH),
3105 s (NH), 2229 s (conj. CN), 1627 vs (NH2), 1592 vs and 1260 s;
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5ꢀ-O-[(1,1-Dimethylethyl)dimethylsilyl]-2ꢀ,3ꢀ-O-isopropylidene-
a/b-D-ribofuranose 10
J 11 and 6, C(5ꢀ)Hb), 4.12 (1 H, ddd, J 6, 5 and 4, C(4ꢀ)H), 5.21
(1 H, dd, J 6 and 4, C(3ꢀ)H), 5.45 (1 H, dd, J 6 and 1, C(2ꢀ)H),
6.25 (1 H, d, J 1, C(1ꢀ)H), 8.64 (1 H, s, C(6)H) and 10.51 (1 H, s,
NHPiv); dC(100 MHz, [D6]DMSO) −4.9 (SiC(CH3)3(CH3)2), −4.8
(SiC(CH3)3(CH3)2), 18.7 (SiC(CH3)3(CH3)2), 25.9 (exo-C(CH3)2),
26.4 (SiC(CH3)3(CH3)2), 27.3 (COC(CH3)3), 27.6 (endo-C(CH3)2),
40.6 (COC(CH3)3), 64.1 (C5ꢀ), 81.3 (C3ꢀ), 84.4 (C2ꢀ), 84.9(C5),
88.7 (C4ꢀ), 91.3 (C1ꢀ), 113.6 (C(CH3)2), 112.2 and 114.3 (C4a and
CN), 138.9 (C6), 151.7 (C7a), 151.9 and 153.8 (C2 and C4) and
176.6 (COC(CH3)3); m/z (ESI+) 564.2368 (M+, C26H39ClN5O5Si+
requires 564.2404).
9 (33.0 g, 174 mmol) and imidazole (29.0 g, 422 mmol) were
dissolved in DMF (60 cm3). tert-Butyldimethylsilyl chloride
(TBDMSCl) (27.4 g, 182 mmol) was added and the solution
was stirred at room temperature for 16 h. The reaction mixture
was treated with water (100 cm3) and extracted with ethyl acetate
(3 × 250 cm3). The combined organic phases were washed with
water (2 × 120 cm3) and dried with MgSO4. Removal of the solvent
in vacuo yielded the protected sugar 10 (44 g, 83%) as a white
solid, which was used without purification in the next step. An
analytically pure sample was obtained by column chromatography
(isohexane–ethyl acetate, 7 : 1). Rf = 0.6 (CHCl3–MeOH 10 : 1); mp
49.8 ◦C; mmax/cm−1 3345 s (OH), 2939 s (CH), 2861 s (CH), 1382 w
(CH3), 1368 w (CH3), 1258 s (COC), 1084 vs (COC) and 1062 vs
(COC); dH (600 MHz, [D6]DMSO) 0.06 (6 H, s, SiC(CH3)3(CH3)2),
0.88 (9 H, s, SiC(CH3)3(CH3)2), 1.25 (3 H, s, CH3), 1.37 (3 H, s,
CH3), 3.59 (2 H, d, J 7, C(5ꢀ)H2), 3.95 (1 H, t, J 7, C(4ꢀ)H), 4.46
(1 H, d, J 6, C(2ꢀ)H), 4.63 (1 H, d, J 6, C(3ꢀ)H), 5.19 (1 H, d, J
5, C(1ꢀ)H) and 6.43 (1 H, d, J 5, OH); dC (150 MHz, [D6]DMSO)
−5.5 and −0.54 (SiC(CH3)3(CH3)2), 17.9 (SiC(CH3)3(CH3)2), 24.7
(C(CH3)2), 25.8 (SiC(CH3)3(CH3)2), 26.3 (C(CH3)2), 64.2 (C5ꢀ),
81.7 (C3ꢀ), 85.5 (C2ꢀ), 85.8 (C4ꢀ), 101.7 (C1ꢀ) and 111.1 (C(CH3)2);
m/z (ESI−) 303.1626 (M−, C14H27O5Si− requires 303.1633).
5-Cyano-3,4-dihydro-7-[2ꢀ,3ꢀ-O-isopropylidene-5ꢀ-O-[(1,1-
dimethylethyl)dimethylsilyl]-b-D-ribofuranosyl]-2-pivaloylamino-
7H-pyrrolo[2,3-d]pyrimidine-4-one 12
11 (74.0 mg, 0.13 mmol) was dissolved in DMF (2 cm3). Et3N
(39.7 mg, 0.05 cm3, 0.39 mmol), NaOAc (32.0 mg, 0.39 mmol)
and 1,4-diazabicyclo[2.2.2]octane (DABCO) (15.0 mg, 0.13 mmol)
were added. The mixture was stirred at room temperature for
48 h. Afterwards, water (1 cm3) was added and the solution was
stirred for 1 h. The mixture was extracted with ethyl acetate
(3 × 10 cm3). The combined organic phases were washed with
brine (20 cm3) and dried with MgSO4. Purification via column
chromatography (isohexane–ethyl acetate 3 : 1) gave nucleoside
12 (58 mg, 81%). Rf = 0.56 (isohexane–ethyl acetate 1 : 1); mp
46.5 ◦C; mmax/cm−1 3163 s (NH), 2933 vs (CH), 2232 vs (CN),
1671 vs, 1606 vs, 1542 s (NH), 1250 s, 1077 vs, 833 vs and 780 vs;
dH (400 MHz, [D6]DMSO) 0.02 (6 H, s, SiC(CH3)3(CH3)2), 0.85
(9 H, s, SiC(CH3)3(CH3)2), 1.25 (9 H, s, COC(CH3)3), 1.32 (3 H, s,
exo-C(CH3)2), 1.51 (3 H, s, endo-C(CH3)2), 3.69 (1 H, dd, J 11
and 5, C(5ꢀ)Ha), 3.73 (1 H, dd, J 11 and 4, C(5ꢀ)Hb), 4.14 (1 H,
ddd, J 5, 4 and 4, C(4ꢀ)H), 4.96 (1 H, dd, J 6 and 4, C(3ꢀ)H),
5.13 (1 H, dd, J 6 and 2, C(2ꢀ)H), 6.25 (1 H, d, J 2, C(1ꢀ)H),
8.14 (1 H, s, C(6)H), 11.03 (1 H, s, N(3)H) and 12.24 (1 H, s,
NHPiv); dC(100 MHz, [D6]DMSO) −5.62 (SiC(CH3)3(CH3)2),
−5.59 (SiC(CH3)3(CH3)2), 18.0 (SiC(CH3)3(CH3)2), 25.2 (exo-
C(CH3)2), 25.7 (SiC(CH3)3(CH3)2), 26.2 (COC(CH3)3), 26.9 (endo-
C(CH3)2), 40.6 (COC(CH3)3), 63.0 (C5ꢀ), 80.4 (C3ꢀ), 83.9 (C2ꢀ),
86.3 (C4ꢀ), 87.2 (C5), 88.2 (C1ꢀ), 103.0 (C4a), 113.3 (CN), 114.2
(C(CH3)2), 129.7 (C6), 147.8 (C2), 148.9 (C7a), 155.3 (C4)
and 181.1 (COC(CH3)3); m/z (ESI+) 568.2572 ([M + Na]+,
C26H39N5NaO6Si+ requires 568.2562).
1ꢀ-Chloro-5ꢀ-O-[(1,1-dimethylethyl)dimethylsilyl]-2ꢀ,3ꢀ-
O-isopropylidene-a/b-D-ribofuranose 4
10 (1.12 g, 3.7 mmol) and CCl4 (0.5 cm3, 5.92 mmol) were dissolved
in THF (10 cm3). Hexamethylphosphoramide (HMPA) (0.8 cm3,
4.44 mmol) was added dropwise at −78 ◦C over a period of
5 min. After stirring at −78 ◦C for 2 h, the cooling was removed
upon which a white solid precipitated. The obtained suspension
containing 4 was used in the subsequent reaction in situ.
4-Chloro-5-cyano-7-[2ꢀ,3ꢀ-O-isopropylidene-5ꢀ-O-[(1,1-
dimethylethyl)dimethylsilyl]-b-D-ribofuranosyl]-2-pivaloylamino-
7H-pyrrolo[2,3-d]pyrimidine 11
Acetonitrile (100 cm3) was dried over molecular sieves for 1 h
before addition of 3 (1.10 g, 3.97 mmol) and NaH (95%, 220 mg,
5.56 mmol). The solution was stirred at room temperature for 1 h.
4 (1.80 g, 5.56 mmol) was added dropwise over a period of 20 min
at 0 ◦C. The resulting solution was stirred at room temperature for
16 h. After removal of the solvent in vacuo the product was treated
with water (50 cm3) and ethyl acetate (50 cm3). The layers were
separated and the water layer was extracted with ethyl acetate
(2 × 100 cm3). The combined organic layers were washed with
brine (200 cm3) and dried with MgSO4. The crude mixture was
purified by column chromatography (isohexane–ethyl acetate 7 :
1) to yield nucleoside 11 (441 mg, 20%). An analytically pure
sample was obtained via HPLC (gradient; eluent A: isohexane,
eluent B: ethyl acetate, gradient: 100% A, 0% B → 30% A,
70% B in 45 min, retention time = 24.9 min, Nucleodur 100–
5). Rf = 0.85 (isohexane–ethyl acetate, 1 : 1); dH (400 MHz,
[D6]DMSO) −0.16 (3 H, s, SiC(CH3)3(CH3)2), −0.13 (3 H, s,
SiC(CH3)3(CH3)2), 0.73 (9 H, s, SiC(CH3)3(CH3)2), 1.22 (9 H, s,
COC(CH3)3), 1.32 (3 H, s, exo-C(CH3)2), 1.51 (3 H, s, endo-
C(CH3)2), 3.64 (1 H, dd, J 11 and 5, C(5ꢀ)Ha), 3.67 (1 H, dd,
5-Cyano-3,4-dihydro-2-pivaloylamino-7-b-D-ribofuranosyl-
7H-pyrrolo[2,3-d]pyrimidine-4-one 13
12 (18.0 mg, 0.03 mmol) was dissolved in water (0.15 cm3)
and trifluoroacetic acid (0.35 cm3) was added at 0 ◦C. The
mixture was stirred at 0 ◦C for 4 h before removing the solvent
in vacuo at 0 ◦C. The resulting solid was dissolved in water
(2 cm3) and stirred at room temperature over a period of 10 min.
Concentration yielded a colourless oil, which was purified via
column chromatography (DCM–MeOH 10 : 1). Nucleoside 13
(10 mg, 77%) was obtained as a white solid. Rf = 0.38 (DCM–
◦
MeOH 10 : 1); mp >210 C (decomposition); mmax/cm−1 3482 s,
3417 vs, 3152 s (NH), 2966 s (CH), 2884 s (CH), 2233 s (CN),
1693 vs, 1659 vs, 1598 s (NH), 1557 s (NH), 1538 s (NH), 1411 s
(CH), 1091 s (COC) and 775 s; dH (400 MHz, [D6]DMSO) 1.2
3824 | Org. Biomol. Chem., 2007, 5, 3821–3825
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(9 H, s, COC(CH3)3), 3.5 (1 H, dd, J 12 and 4, C(5ꢀ)Ha), 3.6
(1 H, dd, J 12 and 4, C(5ꢀ)Hb), 3.9 (1 H, ddd, J 4, 4 and 3,
C(4ꢀ)H), 4.1 (1 H, dd, J 5 and 3, C(3ꢀ)H), 4.3 (1 H, dd, J 6
and 5, C(2ꢀ)H), 6.1 (1 H, d, J 6, C(1ꢀ)H), 8.2 (1 H, s, C(6)H),
11.1 (1 H, s, N(3)H) and 12.2 (1 H, s, NHPiv); dC (100 MHz,
[D6]DMSO) 26.9 (COC(CH3)3), 40.7 (COC(CH3)3), 61.9 (C5ꢀ),
71.0 (C3ꢀ), 74.9 (C2ꢀ), 86.1 (C4ꢀ), 87.0 (C1ꢀ), 87.7 (C5), 103.6
(C4a), 115.2 (CN), 130.3 (C6), 149.3 (C7a), 149.7 (C2), 156.1
(C4) and 182.0 (COC(CH3)3); m/z (ESI+) 414.1363 ([M + Na]+,
Acknowledgements
We thank the Deutsche Forschungsgemeinschaft (Leibniz Award
program, SFB 646, Grant CA275/8) for financial support.
Notes and references
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−
C12H15N6O5 requires 323.1109).
This journal is
The Royal Society of Chemistry 2007
Org. Biomol. Chem., 2007, 5, 3821–3825 | 3825
©