Studies Towards the Large Scale Chemical Synthesis of the Precursors of Ribonucleosides-3′,4′,5′,5″-2H4 and -2′,3′,4′,5′,5″-2H5

Article abstract of DOI:10.1081/NCN-120026632

A summary delineating the large scale synthetic studies to prepare labeled precursors of ribonucleosides-3′,4′,5′,5″- ²H₄ and -2′,3′,4′,5′,5″ -²H₅ from D-glucose is presented. The recycling of deuterium-labeled

Full text of DOI:10.1081/NCN-120026632

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Studies Towards the Large Scale Chemical Synthesis of
the Precursors of Ribonucleosides-3′,4′,5′,5″- 2H
4
and -2′,3′,4′,5′,5″- 2H
5
András Földesi a b & Jyoti Chattopadhyaya a
a Department of Bioorganic Chemistry , Biomedical Center , University of Uppsala , Uppsala,
Sweden
b Department of Bioorganic Chemistry , Biomedical Center , University of Uppsala , Box 581,
Uppsala, SE-751 23, Sweden
Published online: 19 Dec 2011.
To cite this article: András Földesi & Jyoti Chattopadhyaya (2003) Studies Towards the Large Scale Chemical Synthesis of
the Precursors of Ribonucleosides-3′,4′,5′,5″- 2H and -2′,3′,4′,5′,5″- 2H , Nucleosides, Nucleotides and Nucleic Acids,
4
5
22:12, 2093-2104, DOI: 10.1081/NCN-120026632
To link to this article: http://dx.doi.org/10.1081/NCN-120026632
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, No. 12, pp. 2093–2104, 2003
Studies Towards the Large Scale Chemical Synthesis of
the Precursors of Ribonucleosides-30,40,50,500-2H4
#
and -20,30,40,50,500-2H5
*
Andras Foldesi and Jyoti Chattopadhyaya
´
¨
Department of Bioorganic Chemistry, Biomedical Center,
University of Uppsala, Uppsala, Sweden
ABSTRACT
A summary delineating the large scale synthetic studies to prepare labeled precur-
sors of ribonucleosides-30,40,50,500-2H4 and -20,30,40,50,500-2H5 from D-glucose is
presented. The recycling of deuterium-labeled by-products has been devised to
give a high overall yield of the intermediates and an expedient protocol has
been elaborated for the conversion of 3-O-benzyl-a,b-D-allofuranose-3,4-d2 6 to
1-O-methyl-3-O-benzyl-2-O-t-butyldimethylsilyl-a,b-D-ribofuranose-3,4,5,50-d4 16
(precursor of ribonucleosides-30,40,50,500-2H4) or to 1-O-methyl-3,5-di-O-benzyl-
a,b-D-ribofuranose-3,4,5,50-d4 18 (precursor of ribonucleosides-30,40,50,500-2H4).
Key Words: Isotope labeling; Site specific; Stable isotope; Deuterium;
Ribofuranose; Deuterated nucleosides.
#In honor and celebration of the 70th birthday of Professor Leroy B. Townsend.
´
*Correspondence: Andras Fo¨ldesi, Department of Bioorganic Chemistry, Box 581, Biomedi-
cal Center, University of Uppsala, Uppsala SE-751 23, Sweden; Fax: +46-18-554495; E-mail:
jyoti@bioorchem.uu.se.
2093
DOI: 10.1081/NCN-120026632
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

¨
Foldesi and Chattopadhyaya
2094
INTRODUCTION
Amongst various physicochemical methods used to elucidate the correlation
between structural motifs and biological functions of oligo-DNA or -RNA, NMR
spectroscopy has emerged as particularly important since it gives insight into the
structure and its time dependent change (dynamics) at atomic resolution under a
quasi-physiological condition. In order to overcome molecular size related problems
such as spectral overlap and the deleterious effect of nuclear relaxation, different iso-
tope labeling techniques have been devised[1–3] and exploited.[4] Sequence specific
deuteration[5] has been proven to facilitate the NMR structure determination of large
RNAs[6] and DNAs.[7]
Incorporation of the 3,4,5,50-d4 labeled nucleoside blocks shows some consider-
able advantages in the NMR structure elucidation:[8] It reduces the spectral crowding
in the d 4–5 ppm ribose region. The H10-H20 nOe cross-peaks can unambiguously be
identified and the effective T1 and T2 relaxation times for the d4-RNA were increased
by a factor of 2 compared to unlabelled RNA, which is consistent with our earlier
findings on labeled oligo-DNA.[6d]
It was also expected that this reduction of the relaxation efficiency could signifi-
cantly improve the NOESY spectra of larger RNAs. Later it has been shown that
13
uniform C- and 3,4,5,50-d4 double labelling[9] can be used for the simplification of
the relatively crowded C20=C30 region in the 13C dimension of 13C edited 2D spectra.
These results and the idea that the synthesis of 30,40,50,500-2H4-nucleosides could be
extended to the stepwise synthesis of 20,30,40,50,500-2H5 derivatives for the creation
of NMR invisible stretches in our Uppsala ‘‘NMR-window’’ approach[5a-b,6c–e]
prompted us to develop their D-glucose based preparation.[10] We herein report on
the large scale preparation of the appropriately protected 3,4,5,50-2H4-ribofuranoside
precursors.
RESULTS AND DISCUSSION
The synthesis of 3-O-benzyl-1,2-O-isopropylidene-ribofuranose-2H4 (10) follows
our published procedure[10] with two important modifications (Sch. 1, the scales indi-
cated for the particular reactions are the sums of 4–5 simultaneous reactions). Due to
the long equilibration (22 days) time required to convert the C4-protio counterpart
of 8 to 8,[10] the deuterium incorporation at C4 was achieved upon a treatment of
3-ulose 2 in pyridine-2H2O at 95ꢀC (see experimental section).[11] In order to achieve
the required >97 atom% isotope substitution, a cycle consisting of 20 min heating at
95ꢀC and 1-day standing at room temperature was repeated 6 times. After the third
cycle of exchange, the mixture of solvents was removed in vacuo and a fresh mixture
of pyridine-2H2O was added. This procedure is somewhat lengthier but gives signifi-
cantly higher deuterium isotope incorporation (>97 atom% isotope substitution)
than the published procedure which involved a cycle of 5 min heating at 95ꢀC and
18 h standing at room temperature and change the exchange media after each
cycle.[11] The latter procedure gives however only 95 atom% isotope substitution
2
and consumes more H2O than the our present procedure. It should be mentioned
that during the deuterium equilibration, the C4 center of ketone 2 is epimerized to

Synthesis of Labeled Ribonucleoside Precursors
2095
Scheme 1. Abbreviations: Bn ¼ benzyl. Conditions: (i) PDC, acetic anhydride, dry dichloro-
methane (DCM), reflux, 3 h; (ii) Pyridine=2H2O (3.2 mL=0.8 mL=mmol), 95 ꢀC for 20 min,
then 1 day, r.t., repeated 6 times; (iii) LiAlD4 in dry diethyl ether or NaBD4 in ethanol, r.t.;
(iv) BnCl, NaH in dry acetonitrile, r.t., overnight; (v) 80% aqueous acetic acid, r.t., 16–18 h;
(vi) NaIO4, ethanol=H2O (1:1, v=v), r.t., 3.5 h; (vii) bromine in methanol=H2O=NaHCO3,
r.t, 4 h; (viii) LiAlD4, dry diethyl ether, r.t, 4 h.
give gulo counterpart of 3 along with the major allo=gluco product 3. After the reduc-
tion, the mixture of 1,2:5,6-di-O-isopropylidene-a-D-gulofuranose-3,4-2H2 diastereo-
mer (ca 10%) and 1,2:5,6-di-O-isopropylidene-a-D-allofuranose-3,4,2H2 (4) (allo is
formed over gluco owing to the hydride delivery from the b-face) was separated
by column chromatography to give the desired allofuranose 4 (85% in 2-steps) and
1,2:5,6-di-O-isopropylidene-a-D-gulofuranose-3,4-2H2.
Another important modification was to separate the low Rf compound 6 formed
in 8% yield from the desired product 7 in the acid deprotection of 5 [step (v)]. This
was achieved in the following manner: after the removal of the volatile matter, the
residual deprotected mixture still contained acetic acid which was first carefully
removed by coevaporation with toluene. The acid-free residue was then partitioned
between water and dichloromethane. From the aqueous phase the 3-O-benzyl deri-
vative 6 could be recovered in substantially pure form, which is an important

¨
Foldesi and Chattopadhyaya
2096
Scheme 2. Abbreviations: Bn ¼ benzyl, TBDMS ¼ tert-butyldimethylsilyl. Conditions: (i)
conc. H2SO4 in dry methanol, 4ꢀC, 4 days; (ii) NaIO4, ethanol=H2O (1:1, v=v), r.t, 3.5 h;
(iii) bromine in methanol=H2O=NaHCO3, r.t., 4 h; (iv) TBDMS-Cl, pyridine, AgNO3 in dry
THF; (v) LiAlD4, dry diethyl ether, r.t, 4 h; (vi) BnBr, NaH in dry acetonitrile, r.t., overnight;
(vii) 1.0 M TBAF in dry THF.
improvement because this by-product bears 2 deuterium labels, and in the large scale
synthesis this 8% by-product accumulates to be a considerable amount (ꢁ28 mmol).
Thus, it is clear from the above discussion (Sch. 1) that for the improvement of
the overall yield of either 11 or 18, we have two possibilities: (1) recycling the C4 epi-
meric gulo counterpart of 4 or (2) finding an appropriate recycling of 6. Since the
former might be carried out through the equilibration of the gulofuranose-3-ulose,
one practically looses the two deuterium labels already present in the molecule.
Hence it was more attractive to find a salvage pathway for compound 6 (Sch. 2).
The recycling commences (Sch. 2) with the preparation of methyl glucofurano-
side 12 (92%) (from 6) in dry methanol with concentrated H2SO4 at ꢁ4ꢀC. The 13C
chemical shifts for C1 (108.9 ppm for b, 102.4 ppm for a) indicate the presence of the
furanose ring. The free C5-C6 diol system of 12 was cleaved using NaIO4 in ethanol-
water to give the corresponding C5 aldehyde 13 (99%), which was taken to the next
step without further purification. The oxidation of 13 directly to the uronic acid
methyl ester 14 with bromine in methanol-water and in presence of sodium bicarbo-
nate as buffer proceeded with a good yield (81%). According to our earlier find-
ings[10], the protection of the C2-hydroxyl function is mandatory in order to
successfully reduce the methyl ester functionality of 14. The introduction of a 2-O-
TBDMS group made the separation of the anomers also possible by short column
chromatography giving the b-anomer 15a as predominant product (70%) over the
a-anomer 15b (19%). From this point, subsequent reactions were carried out sepa-
rately with the anomerically pure compounds. The ester group of 15a-b was reduced
with lithium aluminium deuteride in dry diethyl ether to give the partially protected
tetradeuterio methyl ribofuranosides 16a (87%) and 16b (72%). The inspection of the
1H-NMR spectrum showed the absence of H5=50 signals revealing >97 atom%

Synthesis of Labeled Ribonucleoside Precursors
2097
incorporation of deuterium at C5. This was further corroborated by the absence of
the C5 signal in the 13C spectra of 16a-b. It is necessary to emphasize here that in the
IR spectra of 16a and 16b we observed C-D stretching bands in the region[12] of
2300–2000 cmꢂ1, which were however not observable for any other deuterated pre-
cursors of 16a=16b. Compounds 16a-b can directly be deprotected to 11 in two steps
giving an additional crop of the methyl ribofuranoside-3,4,5,50-2H4, (ꢁ12 mmol).
For the synthesis of methyl ribofuranoside-2,3,4,5,50-2H5,[5b] which includes an
oxidation-reduction-inversion sequence at C2, the C5-OH was protected as benzyl
ether upon a treatment of 16a-b with NaH and benzyl bromide in dry acetonitrile.
The relatively lower yield for the a-anomer 17b (74%) compared to that of 17a
(96%) might indicate a higher level of steric hindrance at the a-face for this series
of compounds. Removal of the TBDMS protecting group with the usual treatment
with 1M TBAF in dry tetrahydrofurane afforded the deuterated compounds 18a
(95%) and 18b (88%) which are the key intermediates in the synthesis of ribonucleo-
sides-20,30,40,50,500-2H5.[5b] Compounds 18a-b were obtained in an amount (10.5 mmol)
which represents ꢁ3% overall improvement on the described ꢁ270 mmol scale
synthesis of this precursor 18.
EXPERIMENTAL SECTION
Pyridine was distilled after refluxing with calcium hydride and it was kept over
˚
molecular sieves (4A). Dichloromethane, 1,2-dichloroethane and acetonitrile were
stirred with phosphorus pentoxide overnight and distilled in nitrogen atmosphere.
Toluene was refluxed with calcium hydride followed by distillation. The chromato-
graphic separations were performed on Merck G60 silica gel. Thin layer chromato-
graphy (TLC) was performed on Merck pre-coated silica gel 60 F254 glass backed
plates developed in following systems: (A) methanol-DCM (10:90, v=v), (B) ethyl
acetate-cyclohexane (1:3, v=v) (C) methanol-DCM (5:95), (D) ethyl acetate-cyclo-
1
hexane (1:1, v=v). H-NMR spectra were recorded with Jeol GX 270 spectrometer
at 270.17 MHz, using TMS (0.0 ppm) peak as internal standards. 13C-NMR spectra
were recorded with Jeol GX 270 spectrometer at 67.9 MHz using the central peak of
CDCl3 (76.9 ppm) as internal standard. Chemical shifts are reported in ppm (d scale).
Optical rotation data were measured on Perkin-Elmer 241 polarimeter. Infrared
spectra were recorded with a Perkin-Elmer 298 spectrometer.
1,2:5,6-Di-O-isopropylidene-a-D-allofuranose-3,4-2H2 (4). Pyridinium dichro-
mate (46.0 g, 72.2 mmol) was added to dry dichloromethane (420.0 mL) followed
by acetic anhydride (37.0 mL, 227.3 mmol). To the stirred suspension 1,2:5,6-di-O-
isopropylidene-a-D-glucose (1) (31.7 g, 121.8 mmol) was added and the mixture
was boiled at ꢁ75ꢀC for 3.5 h. The mixture was cooled to room temperature, diluted
with ethyl acetate and the precipitate was filtered. The filtered solid was washed with
ethyl acetate then the solvent was evaporated and the oil was repeatedly co-evapo-
rated with toluene. Diethyl ether was added, and the solution was filtered again.
After evaporation of the solvent, this procedure was repeated once more to give
1
an oily product (25.7 g, 82%). H-NMR (CDCl3): 6.14 (d, JH1,H2 ¼ 4.4 Hz, 1H) H-1;
4.4–3.9 (m, 5H) H-2,4,5,6,60; 1.46, 1.44, 1.34 (3xs, 12H) 4xCH3. The procedure was

¨
Foldesi and Chattopadhyaya
2098
repeated on scales (18.6 g, 31.7 g and 2 ꢃ 25.6 g, altogether 512.0 mmol) of 1 giving
altogether 116.6 g (451.6 mmol) ketone 2. 3-Ulose 2 (33.9 g, 131.2 mmol) was dis-
2
solved in a mixture of dry pyridine (420 mL and H2O (105 mL). The solution was
heated at ꢁ95 ꢀC for 20 min, then kept at room temperature till next day. This
was repeated twice more, then the solvent was removed and the same mixture of
fresh solvent was added. After three heating an wait periods, reduction of an aliquot
with LiAlD4 showed appropriate level of deuteration (>97 atom%). Solvents were
removed, the residual oil was co-evaporated with dry toluene (3x), then it was dis-
solved in dry ether (300 mL) and reduced with LiAlD4 (2.05 g, 48.8 mmol) to give
1
compound 4 (30.9 g, 90%). H-NMR (CDCl3): 5.82 (d, JH1,H2 ¼ 3.8 Hz, 1H) H-1;
0
4.61 (d, 1H) H-2; 4.31 (t, 1H) H-5; 4.11–3.99 (m, JH6,H5 ¼ 6.6 Hz, JH6 ,H5 ¼ 6.6 Hz,
0
0
JH6,H6 ¼ 8.5 Hz, 2H) H-6=6 ; 2.54 (s, 1H) OH; 1.58, 1.47, 1.39, 1.38 (4xs, 12H)
4xCH3. 13C-NMR (CDCl3): 112.7 (1,2-O-C[CH3]2); 109.7 (5,6-O-C[CH3]2); 103.8
(C-1); 78.8 (C-2); 75.4 (C-5); 65.7 (C-6); 26.5, 26.4, 26.2, 25.2, (4xCH3).
1-O-Methyl-3-O-benzyl-a,b-D-allofuranose-3,4-d2 (12). Sugar derivative
6
(7.73 g, 28.6 mmol) was dissolved in dry methanol (100 mL) and conc. sulfuric acid
(500 mL) was added at 0ꢀC. The reaction mixture was kept in a refrigerator at 4ꢀC
for 4 days. Solid NaHCO3 was added for neutralization of the acid. After filtering
away the excess solid salt, the methanol was evaporated, the residue was taken up
in dichloromethane and extracted with aqueous sat. sodium bicarbonate. The
organic phase was dried over MgSO4, filtered and evaporated to obtain 12 (7.55 g,
1
92%) as a white solid. Rf: 0.55 (System A). H-NMR (CDCl3): 7.42–7.30 (m, 5H)
benzyl; 4.86 (d, JH1,H2 ¼ 4.6 Hz,) H-1 a; 4.85 (d, JH1,H2 ¼ 0.6 Hz), H-1 b; 4.77–4.55
(m, 2H) CH2-benzyl; 4.09 (m) H-2 a; 4.02 (s) H-2 b; 3.83–3.55 (m, 3H) H-5, H-
6,60; 3.46 (s) OCH3 a; 3.38 (s) OCH3 b. 13C-NMR (CDCl3): 137.5, 136.7, 128.6,
128.4, 128.3, 128.1, 128.0, 127.9 (benzyl), 108.9 (C-1 b), 102.4 (C-1 a), 73.2 (C-2),
72.9 (Ph-CH2, b), 72.8 (Ph-CH2, a), 72.2 (C-5 b), 71.6 (C-5 a), 63.2 (C-6), 55.6
(OCH3).
1-O-Methyl-3-O-benzyl-a,b-D-ribo-pentodialdo-1,4-furanose-3,4-d2 (13). NaIO4
(5.9 g, 27.7 mmol) was dissolved in a mixture of ethanol (75.0 mL) and water
(75.0 mL). This solution was added to sugar 12 (7.55 g, 26.4 mmol) and the reaction
mixture was stirred for 3.5 h at ambient temperature. The white precipitate was fil-
tered and a few drops of ethylene glycol were added to the filtrate. The precipitate
was filtered again and the solution phase was evaporated. Ethanol was added to
the residue and the precipitate was filtered and this procedure was repeated until
no further precipitation occurred. The oily residue was kept on an oil pump to give
aldehyde 13 (6.68 g, 99%).
Methyl 1-O-Methyl-3-O-benzyl-a-b-D-ribofuranuronate-3,4-d2 (14). The crude
deuterated aldehyde 13 (6.68 g, 26.3 mmol) was dissolved in mixture of methanol
(51.5 mL) and H2O (5.7 mL) to obtain a 0.5 M solution. NaHCO3 (91.6 g,
114.4 mmol) was added followed by a solution of bromine (14.7 mL) in a mixture
of methanol (129 mL) and H2O (14 mL). The reaction mixture was stirred for 4 h
at ambient temperature, then a small amount of Na2SO3 was added and the reaction
mixture was partitioned between water and DCM. The organic phase was dried over

Synthesis of Labeled Ribonucleoside Precursors
2099
MgSO4, filtered and evaporated. The residue was separated on silica gel column
yielding compound 14 as yellow oil (6.0 g, 81%). Rf: 0.51 (a), 0.60 (b) (System D).
IR nmax (neat): 3490, 3082, 3059, 3025, 2994, 2950, 2926, 2835, 1745, 1493, 1450,
1
1434, 1279, 1192, 1140, 1110, 1080, 1050, 989, 960 cmꢂ1. H-NMR (CDCl3): 7.4–
7.3 (m, 5H) Ph-CH2; 5.02 (d, JH1,H2 ¼ 4.8 Hz, 1H) H-1 a; 4.94 (d, JH1,H2 ¼ 0.6 Hz,
1H) H-1 b; 4.75–4.63 (m, 2H) Ph-CH2; 4.14 (dd, 1H) H-2 a; 4.02 (d, 1H) H-2 b; 3.77
(s, 3H) C(O)OCH3 b; 3.74 (s, 3H) C(O)OCH3 a; 3.50 (s, 3H) OCH3 a; 3.40 (s, 3H)
OCH3 b; 2.91 (d, JOH,H2 ¼ 11.4 Hz, 1H) OH a; 2.74 (d, JOH,H2 ¼ 2.6 Hz, 1H) OH b.
13C-NMR (CDCl3): 171.7 (C ¼ O b), 170.6 (C ¼ O a), 137.0, 136.7, 128.5, 128.2,
127.9 (Ph-CH2); 109.0 (C-1 b); 103.1 (C-1 a); 73.2 (C-2), 73.0, 72.9, 71.2 (Ph-CH2);
55.9 (OCH3 a), 55.2 (OCH3 b), 52.4 (C(O)OCH3 a), 52.2 (C(O)OCH3 b).
Methyl 1-O-Methyl-3-O-benzyl-2-O-t-butyldimethylsilyl-b-D-ribofuranuronate-
3,4-d2 (15a) and Methyl 1-O-Methyl-3-O-benzyl-2-O-t-butyldimethylsilyl-a-D-ribo-
furanuronate-3,4-d2 (15b). The ester 14 (4.61 g, 16.3 mmol) was dissolved in dry
THF (130 mL). Dry pyridine (7.3 mL) was added, followed by silver nitrate
(4.23 g, 27.5 mmol). This mixture was stirred for 15 min, then t-butyldimethylsilyl
chloride (3.9 g, 25.9 mmol) was added. The reaction mixture was stirred in darkness
for 4 h, when more silver nitrate (1.27 g, 8.3 mmol) and t-butylmethylsilyl chloride
(0.74 g, 4.9 mmol) were added. After overnight stirring the solution was filtered
through a Celite bed, which was washed with DCM and the filtrates were evapo-
rated. The residue was dissolved in DCM, and washed with sat. NaHCO3, water
and dried over MgSO4. The solvent was evaporated and the residue was subjected
to short column chromatography to give compounds 15a (4.52 g, 69.6%) and 15b
(1.22 g, 18.8%). Compound 15a: Rf: 0.60 (System B). [a]D25 þ 13 (c, 0.39, CHCl3).
IR nmax (neat): 3082, 3060, 3025, 2992, 2950, 2922, 2854, 1752, 1732, 1491, 1460,
1451, 1432, 1385, 1359, 1286, 1252, 1185, 1139, 1110, 1084, 1052, 1002, 962, 890,
835, 776 cmꢂ1
.
1H-NMR (CDCl3): 7.4–7.3 (m, 5H) Ph-CH2; 4.79 (d, JH1,H2
¼
0.99 Hz, 1H) H-1; 4.69–4.52 (m, 2H) Ph-CH2; 4.12 (d, 1H) H-2; 3.74 (s, 3H)
C(O)OCH3; 3.41 (s, 3H) OCH3; 0.90 (s, 9H) Si-C(CH3)3; 0.09 (2xs, 6H) Si-(CH3)2.
13C-NMR (CDCl3): 172.2 (C¼O), 137.5, 128.2, 127.7 (Ph-CH2); 109.1 (C-1); 74.4
(C-2), 72.4 (Ph-CH2); 55.1 (OCH3), 52.0 (C(O)OCH3), 25.6 (Si-C(CH3)3), 18.1 (Si-
C(CH3)3), ꢂ4.8 & ꢂ4.9 (Si-(CH3)2). Compound 15b: Rf: 0.43 (System B).
[a]D25 þ 103 (c 0.28, CHCl3). IR nmax (neat): 3082, 3060, 3025, 2946, 2922, 2852,
1747, 1491, 1460, 1451, 1432, 1387, 1359, 1280, 1252, 1192, 1139, 1113, 1080,
1064, 1032, 958, 872, 835, 776 cmꢂ1. H-NMR (CDCl3) 7.4–7.3 (m, 5H) Ph-CH2;
1
4.92 (d, JH1,H2 ¼ 4.5 Hz, 1H) H-1; 4.82–4.70 (m, 2H) Ph-CH2; 4.07 (d, 1H) H-2;
3.71 (s, 3H) C(O)OCH3; 3.48 (s, 3H) OCH3; 0.93 (s, 9H) Si-C(CH3)3; 0.08 & 0.11
(2xs, 6H) Si-(CH3)2. 13C-NMR (CDCl3): 171.0 (C¼O), 137.6, 128.1, 128.0, 127.5
(Ph-CH2); 103.8 (C-1); 72.7 (C-2), 72.1 (Ph-CH2); 55.9 (OCH3), 52.2 (C(O)OCH3),
25.7 (Si-C(CH3)3), 18.2 (Si-C(CH3)3); ꢂ4.9 & ꢂ5.0 (Si-(CH3)2).
1-O-Methyl-3-O-benzyl-2-O-t-butyldimethylsilyl-b-D-ribufuranose-3,4,5,50-d4
(16a). Compound 15a (4.6 g, 11.4 mmol) was dissolved in dry diethyl ether
(ꢁ100 mL) and LiAlD4 (239 mg, 5.7 mmol) was added at 0ꢀC. The mixture was stir-
red at r.t. for 2 h, then water was added and the mixture was extracted with DCM.
The organic phase was dried over MgSO4. After evaporation of the volatile matters,

¨
Foldesi and Chattopadhyaya
2100
the residue was purified by column chromatography to obtain compound 16a as col-
ourless oil (3.68 g, 87%). Rf: 0.70 (System C). [a]D25 þ 34 (c 0.36, CHCl3). IR nmax
(neat): 3462, 3085, 3060, 3026, 2950, 2924, 2855, 2198, 2188, 2094, 1494, 1470,
1461, 1452, 1387, 1359, 1301, 1251, 1172, 1135, 1102, 1080, 1062, 980, 935, 889,
838, 775 cmꢂ1
.
1H-NMR (CDCl3): 7.4–7.3 (m, 5H) Ph-CH2; 4.73 (d,
JH1,H2 ¼ 0.99 Hz, 1H) H-1; 4.69–4.43 (m, 2H) Ph-CH2; 4.15 (d, 1H) H-2; 3.39 (s,
3H) OCH3; 0.91 (s, 9H) Si-C(CH3)3; 0.12 & 0.11 (2xs, 6H) Si-(CH3)2. 13C-NMR
(CDCl3): 137.8, 128.3, 127.7, 127.6 (Ph-CH2); 109.1 (C-1); 74.5 (C-2), 72.3 (Ph-
CH2); 55.5 (OCH3), 25.6 (Si-C(CH3)3), 18.1 (Si-C(CH3)3), ꢂ4.8 (Si-(CH3)2).
1-O-Methyl-3-O-benzyl-2-O-t-butyldimethylsilyl-a-D-ribofuranose-3,4,5,50-d4
(16b). Compound 15b (1.22 g, 3.1 mmol) was reduced with LiAlD4 (65 mg,
1.5 mmol) as described for 15a to get compound 16b as colourless oil (0.83 g,
72%). Rf: 0.65 (System C). [a]D25 þ 149 (c 0.4, CHCl3). IR nmax (neat): 3498, 3082,
3060, 3022, 2945, 2922, 2852, 2198, 2188, 2092, 1494, 1470, 1462, 1453, 1387,
1361, 1251, 1190, 1170, 1111, 1080, 1065, 970, 876, 838, 778 cmꢂ1 1H-NMR
.
(CDCl3): 7.4–7.2 (m, 5H), Ph-CH2; 4.79 (d, JH1,H2 ¼ 4.3 Hz, 1H) H-1; 4.88–4.53
(dd, 2H) Ph-CH2; 4.03 (d, 1H) H-2; 3.46 (s, 3H) OCH3; 0.95 (s, 9H) Si-C(CH3)3;
0.134 & 0.127 (2xs, 6H) Si-(CH3)2. 13C-NMR (CDCl3): 138.5, 128.2, 127.7, 127.5
(Ph-CH2); 103.7 (C-1); 73.5 (C-2), 72.6 (Ph-CH2); 55.5 (OCH3), 25.7 (Si-C(CH3)3),
18.3 (Si-C(CH3)3), ꢂ4.7, ꢂ5.0 (Si-(CH3)2).
1-O-Methyl-3,5-di-O-benzyl-2-O-t-butyldimethylsilyl-b-D-ribofuranose-3,4,5,50-
d4 (17a). Compound 16a (3.68 g, 9.91 mmol) was coevaporated with dry acetoni-
trile then dissolved in the same solvent (33 mL). NaH (286 mg, 11.9 mmol) was added
followed by benzyl bromide (1.42 mL, 11.9 mmol). The mixture was stirred overnight
at r.t., then methanol was added and the stirring was maintained for additional 1 h.
The reaction mixture was poured into sat. NaHCO3 and extracted with DCM. The
pooled organic phase was dried over MgSO4, filtered and evaporated to a syrup.
Purification by column chromatography afforded compound 17a as colourless oil
(4.41 g, 96%). Rf: 0.72 (System B). [a]D25 þ 27 (c 1.23, CHCl3). IR nmax (neat):
3082, 3060, 3022, 2945, 2922, 2848, 2821, 2175, 2150, 2070, 1491, 1468, 1460,
1
1450, 1383, 1358, 1310, 1250, 1200, 1184, 1145, 1050, 959, 890, 832, 772 cmꢂ1. H-
NMR (CDCl3): 7.33–7.3 (m, 10H) 2xPh-CH2; 4.74 (d, JH1,H2 ¼ 1.2 Hz, 1H) H-1;
4.66–4.40 (m, 4H) 2xPh-CH2; 4.12 (d, 1H) H-2; 3.33 (s, 3H) OCH3; 0.91 (s, 9H)
Si-C(CH3)3; 0.10 & 0.096 (2xs, 6H) Si-(CH3)2. 13C-NMR (CDCl3): 138.3, 137.9,
128.2, 127.6, 127.5, 127.46, 127.3 (2xPh-CH2); 108.6 (C-1); 74.1 (C-2), 73.0, 72.1
(2xPh-CH2); 55.0 (OCH3), 25.6 (Si-C(CH3)3), 18.1 (Si-C(CH3)3); ꢂ4.82, ꢂ4.86 (Si-
(CH3)2).
1-O-Methyl-3,5-di-O-benzyl-2-O-t-butyldimethylsilyl-a-D-ribofuranose-3,4,5,50-
d4 (17b). Compound 16b (0.83 g, 2.23 mmol) was coevaporated with dry aceto-
nitrile three times. After dissolving in the same solvent (8 mL), NaH (80 mg,
3.35 mmol) was added followed by benzyl bromide (0.40 mL, 3.35 mmol). The mix-
ture was stirred for 4 h at r.t., then the reaction mixture was poured into sat.
NaHCO3 and extracted with DCM. The pooled organic phase was dried over
MgSO4, filtered and evaporated. Purification by column chromatography afforded

Synthesis of Labeled Ribonucleoside Precursors
2101
compound 17b as colourless oil (0.76 g, 74%). Rf: 0.52 (System B). [a]D25 þ 114 (c 1.13,
CHCl3). IR nmax (neat): 3082, 3059, 3022, 2945, 2920, 2845, 2170, 2155, 2070,
1491, 1468, 1460, 1450, 1383, 1358, 1248, 1194, 1170, 1113, 1086, 1065, 1042, 960,
878, 832, 774 cmꢂ1 1H-NMR (CDCl3): 7.35–7.21 (m, 10H) 2xPh-CH2; 4.79
.
(d, JH1,H2 ¼ 4.4 Hz, 1H) H-1; 4.81–4.40 (m, 4H) 2xPh-CH2; 4.07 (d, 1H) H-2; 3.45
(s, 3H) OCH3; 0.94 (s, 9H) Si-C(CH3)3; 0.12 & 0.10 (2xs, 6H) Si-(CH3)2. 13C-
NMR (CDCl3): 138.6, 137.9, 128.2, 128.1, 127.8, 127.5, 127.3 (2xPh-CH2); 103.5
(C-1); 73.3 (Ph-CH2), 73.2, (C-2); 72.3 (Ph-CH2), 55.5 (OCH3), 25.8 (Si-C(CH3)3),
18.3 (Si-C(CH3)3); ꢂ4.8, ꢂ5.0 (Si-(CH3)2).
1-O-Methyl-3,5-O-dibenzyl-b-D-ribofuranose-3,4,5,50-d4 (18a). Compound 17a
(4.41 g, 9.55 mmol) was dissolved in dry tetrahydrofuran (95 mL). 1 M TBAF in
dry THF (9.55 mL) was added and stirring was maintained for 10 min at ambient
temperature. The mixture was evaporated to an oil. Purification on silica gel afforded
compound 18a (3.15 g, 95%) as colourless oil). Rf: 0.82 (System A). [a]D25 ꢂ27 (c 0.71,
CHCl3). IR nmax (neat): 3450, 3081, 3059, 3022, 2922, 2856, 2824, 2178, 2150, 2072,
1
1491, 1450, 1385, 1366, 1203, 1148, 1050, 1115, 955, 870 cmꢂ1. H-NMR (CDCl3):
7.38–7.23 (m, 10H) 2xPh-CH2; 4.86 (d, JH1,H2 ¼ 0.87 Hz, 1H) H-1; 4.57 & 4.568
(2xs, 4H) 2xPh-CH2; 4.02 (br.s, 1H) H-2; 3.32 (s, 3H) OCH3; 2.70 (br.d,
JOH,H2 ¼ 2.5 Hz, 1H) OH. 13C-NMR (CDCl3): 138.1, 137.0, 128.5, 128.2, 128.1,
127.8, 127.5 (2xPh-CH2); 108.5 (C-1); 73.2, (C-2); 73.1, 72.7 (2xPh-CH2), 54.9
(OCH3).
1-O-Methyl-3,5-O-dibenzyl-a-D-ribofuranose-3,4,5,50-d4 (18b). Compound 17b
(0.75 g, 1.62 mmol) was treated in dry tetrahydrofuran (16 mL) with 1 M TBAF in
dry THF (1.62 mL) as described for 18a. Column chromatography afforded com-
pound 18b (497 mg, 88%) as colourless oil. Rf: 0.83 (System A). [a]D25 þ 121 (c
0.18, CHCl3). IR nmax (neat): 3552, 3080, 3059, 3022, 2922, 2856, 2824, 2170,
1
2140, 2070, 1491, 1450, 1408, 1383, 1366, 1320, 1188, 1148, 1045, 959 cmꢂ1. H-
NMR (CDCl3): 7.37–7.22 (m, 10H) 2xPh-CH2; 4.88 (d, JH1,H2 ¼ 4.7 Hz, 1H) H-1;
4.73 & 4.57 and 4.51 & 4.44 (2xq, JAB ¼ 12.4 & 12.1 Hz, 4H) 2xPh-CH2; 4.11 (dd,
1H) H-2; 3.47 (s, 3H) OCH3; 2.94 (d, JOH,H2 ¼ 11.1 Hz, 1H) OH. 13C-NMR (CDCl3):
137.8, 137.7, 128.3, 128.28, 127.8, 127.7, 127.6, 127.5 (2xPh-CH2); 102.9 (C-1); 73.3,
72.9 (2xPh-CH2), 71.6 (C-2); 55.5 (OCH3)
ACKNOWLEDGMENTS
Authors thank Swedish Natural Science Research Council (NFR contract # K-
KU 12067-300 to AF & K-AA=Ku04626-321 to JC), Philip Morris USA Inc (to JC
and Carl Tryggers Foundation (CTS) (to AF) for generous financial support.
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Received July 29, 2003
Accepted August 20, 2003