A short path synthesis of [13C/15N] multilabeled pyrimidine nucleosides starting from glucopyranose nucleosides

Article abstract of DOI:10.1021/jo0205098

The synthesis of fully [¹³C/¹⁵N] labeled pyrimidine nucleosides has been achieved from ¹³C-glucose and labeled nucleobases. The reaction scheme leads directly to the protected nucleosides without the need for the inversion

Full text of DOI:10.1021/jo0205098

A Sh or t P a th Syn th esis of [13C/15N] Mu ltila beled P yr im id in e
Nu cleosid es Sta r tin g fr om Glu cop yr a n ose Nu cleosid es
Irene M. Lagoja,† Sylvie Pochet,‡ Valerie Boudou,† Roy Little,† Eveline Lescrinier,†
J ef Rozenski,† and Piet Herdewijn*,†
Laboratory of Medicinal Chemistry, Rega Institute for Medical Research,
Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium,
and Institut Pasteur, 28 rue du Dr Roux, F-75724 Paris Cedex, France
piet.herdewijn@rega.kuleuven.ac.be
Received August 1, 2002
The synthesis of fully [13C/15N] labeled pyrimidine nucleosides has been achieved from 13C-glucose
and labeled nucleobases. The reaction scheme leads directly to the protected nucleosides without
the need for the inversion of configuration of C-3 of 13C-glucose. This was achieved by an oxitative
ring-opening reaction removing the carbon with the wrong configuration.
In tr od u ction
preparing virtually any conceivable isotopomer of a
labeled nucleoside.9 The key step in the chemical prepa-
ration of fully labeled nucleosides is the synthesis of
protected [13C5]-D-ribose, which starts with the easily
available [13C6]-D-glucose. The disadvantage of the pub-
lished methods is the multiple steps needed to convert
[13C6]-D-glucose to [13C5]-D-ribose. The reason for this is
that one of the carbon atoms, (C-3) of D-glucose, has to
be inverted from configuration while another carbon atom
(C-6) has to be removed. An economically much more
profitable approach would favor the removal of the carbon
with the wrong configuration, i.e., C-3 of D-glucose.
Theoretically, this can be achieved by starting with a
glucopyranose nucleoside via an oxidative ring-opening-
reductive ring-closure procedure removing C-3 of
D-glucose.
We have proven the feasibility of this approach by
examples in the pyrimidine series, i.e., the synthesis of
[1′,2′,3′,4′,5′-13C5]-5′-monomethoxytrityl-[6-13C,1,3-15N2]-
uridine [13] and [1′,2′,3′,4′,5′-13C5]-5′-monomethoxytrityl-
[6-13C,1,3,4-15N3]-N4-benzoylcytidine [14] in 6 and 7 steps,
respectively, starting from [13C6]-D-glucose and labeled
nucleic base.
Full determination of the tertiary structure of DNA or
RNA molecules by NMR techniques needs two sorts of
information: the conformation of each nucleotide unit
and the spatial proximity between the nucleotidyl pro-
tons. Such information is available from 2D- and 3D-
NMR experiments1 for smaller oligonucleotides, but it is
difficult to collect this information for larger oligomers
due to spectral overlap and line broadening. To overcome
these difficulties and to allow the conformational analysis
of a large, biologically functional DNA or RNA, the
introduction of H-, 13C, and 15N-labeled oligonucleotide
2
building blocks is paramount for structure elucidation.
Therefore, with the help of specific 15N and 13C labeling,
key information on local interactions such as hydrogen
bonding,2 protonation,3 hydration,4 ligand interactions,5
and stacking6 on large oligomers can be provided. A
bottleneck in NMR research on nucleic acid structures
and dynamics is the availability of these isotopically
labeled materials in larger amounts and at reasonable
costs. 13C/15N-labeled nucleic acids can be obtained by an
enzymatic approach,7 in a chemical manner, or by a
combination of both.8
One of the pronounced advantages of the chemical
synthesis of labeled nucleosides is the capability of
Resu lts a n d Discu ssion
Uracil [1] was synthesized by hydrogenation of
R-cyanoacetylurea in the presence of a nickel catalyst.10
This three-step synthesis has been adapted to introduce
carbon11 as well as nitrogen labels.12 The only restriction
is that both ring nitrogens are simultaneously introduced
so that selective labeling of one of them is not possible.
Total synthesis of cytosine [5] suffers from the disadvan-
* To whom correspondence should be addressed.
† Katholieke Universiteit Leuven.
‡ Institut Pasteur.
(1) (a) Tate, S.-i.; Ono, A.; Kainosho, M. J . Magn. Res. Ser. B 1995,
106, 89-91. (b) Szyperski, T.; Ono, A.; Ferna´ndez, C.; Iwai, H.; Tate,
S.-i.; Wu¨thrich, K.; Kainosho, M. J . Am. Chem. Soc. 1997, 119, 9901-
9902.
(2) Goswami, B.; Gaffney, B. L.; J ones, R. A. J . Am. Chem. Soc. 1993,
115, 3832-3833.
(3) Wang, C.; Gao, H.; Gaffney, B. L.; J ones, R. A. J . Am. Chem.
Soc. 1991, 113, 5486-5488.
(7) Scott, L. G.; Tolbert, T. J .; Williamson, J . R. Methods Enzymol.
2000, 317, 18-38.
(8) Miyazaki, T.; Sato, H.; Sakakibara, T.; Kajihara, Y. J . Am. Chem.
Soc. 2000, 122, 5678-5694.
(9) (a) Tate, S.; Kubo, Y.; Ono, A.; Kainosho, M. J . Am. Chem. Soc.
1995, 117, 7277-7278. (b) Oogo, Y.; Ono, (Mei) A.; Ono, (Sho) A.;
Kainosho, M. Tetrahedron Lett. 1998, 39, 2873-2876.
(10) Rupe, H.; Metzger, A.; Vogler, V. Helv. Chim. Acta 1925, 8, 848.
(4) Gaffney, B. L.; Wang, C.; J ones, R. A. J . Am. Chem. Soc. 1992,
114, 4047-4050.
(5) Rhee, Y. S.; Wang, C.; Gaffney, B. L.; J ones, R. A. J . Am. Chem.
Soc. 1993, 115, 12607-12608.
(6) (a) Zhang, X.; Gaffney, B. L.; J ones, R. A. J . Am. Chem. Soc.
1997, 119, 6432-6433. (b) Zhang, X.; Gaffney, B. L.; J ones, R. A. J .
Am. Chem. Soc. 1998, 120, 615-618.
10.1021/jo0205098 CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/29/2003
J . Org. Chem. 2003, 68, 1867-1871
1867

Lagoja et al.
SCHEME 1. Syn th esis of La beled Cytosin e [4]a ,b
a
The labeled positions are indicated with an asterisk. bReagents and conditions: (i) POM-Cl, K2CO3, DMF, 54%; (ii) 3NT, TsCl, diphenyl
*
phosphate, pyridine, 83%; (iii) NH4Cl, KOH aq, MeCN, THF, 90 %; (iv) 0.5 N KOH, DMF, 82 %; (v) BzCl, pyridine, 76%.
SCHEME 2. Syn th esis of [1′,2′,3′,4′,5′-13C5]-5′-O-Mon om eth oxytr ityl-[6-13C,1,3-15N2]-u r id in e [13]a a n d th e
str u ctu r e of [1′,2′,3′,4′,5′-13C5]-5′-O-Mon om eth oxytr ityl-[6-13C,1,3,4-15N3]-N4-ben zoylcytosin e [14]
a
Reagents and conditions: (i) Ac2O, pyridine 98 %; (ii) [6-13C,1,3-15N2]-uracil, BSA, TMSOTf, ClCH2CH2Cl, 87 %; (iii) NH3/MeOH,
97%; (iv) MMTrCl, pyridine, Et3N, 90%; (v) Pb(OAc)4, CH2Cl2, 90 %; (vi) Bu3SnH, dioxane, AIBN, 68%.
tage of the formation of isocytosine13 as byproduct and
inferior yields of the desired compound.14 By condensation
of urea with cyanoacetal in the presence of sodium
butoxide, cytosine can be obtained in an overall yield of
56%.15 It is much more efficient, however, to start from
the more readily available uracil base [1] and convert it
into the cytosine base [5] (Scheme 1). Starting from
[6-13C,1,3-15N2]-uracil [1], first N-1 was protected to yield
[6-13C,1,3-15N2]-1-pivaloyloxymethyluracil [2]. For the
introduction of the exocyclic amino group 3-nitro-1,2,4-
triazole (NT)16 is introduced into the C-4 position of the
protected uracil [2], which is subsequently displaced by
15NH3 to obtain 1-[6-13C,1,3,4-15N3]-pivaloyloxymethylcy-
tosine [4]. Deprotection, followed by benzoylation of the
exocyclic amino group yields [6-13C,1,3,4-15N3]-N4-ben-
zoylcytosine [6], which is suitable for Vorbruggen con-
densation.17
The published methods to synthesize [13C5]-D-ribose all
start from labeled glucose.18 They differ from each other
by (a) first shortening of the side chain of protected
glucofuranose by a NaIO4-NaBH4 reaction followed by
inversion of configuration at C-3, using a nucleophilic
substitution reaction,19 (b) first inversion of configuration
at C-3 by a PDC-NaBH4 reaction and then oxidative
cleavage of the side chain20 of the protected glucofura-
nose, and (c) a one-pot procedure of oxidatively cleaving
the diol and reducing simultaneously both the 3-keto
function and the 5-aldehyde group.21 A shorter route,
which we present here, starts from glucopyranose nu-
cleosides. This method is outlined for the synthesis of
[1′,2′,3′,4′,5′-13C5]-5′-monomethoxytrityl-[6-13C,1,3-15N2]-
uridine [13] and [1′,2′,3′,4′,5′-13C5]-5′-monomethoxytrityl-
[6-13C,1,3,4-15N3]-N4-benzoylcytidine [14]. In this case the
C-3 of glucose (i.e., the carbon with the wrong configu-
ration) is removed, which means that the inversion
reaction can be omitted. The reaction sequence (Scheme
2) leads directly to the 5’-O-monomethoxytrityl protected
ribonucleosides (Scheme 2).
(11) SantaLucia, J ., J r.; Shen, L. X.; Cai, Z.; Lewis, H.; Tinoco, I.,
J r. Nucleic Acids Res. 1995, 23 (23), 4913-4921.
(12) Triplett, J . W.; Mack, S. W.; Smith, S. L.; Digenis, G. A. J .
Labeled Compd. Radiopharm. 1976, 14 (1), 35-41.
(13) Hilbert, G. E.; J ohnson, T. B. J . Am. Chem. Soc. 1930, 52, 1152-
1157.
(18) (a) Lagoja, I. M.; Herdewijn, P. Synthesis 2002, 3, 301-314.
(b) Fo¨ldesi, A.; Trifonova, A.; Kundu, M. K.; Chattopadhyaya, J .
Nucleosides, Nucleotides Nucleic Acids 2000, 19, 1615-1656.
(19) Quant, S.; Wechselberger, R. W.; Wolter, M. A.; Wo¨rner, K. H.;
Schell, P.; Engels, J . W.; Griesinger, C.; Schwalbe, H. Tetrahedron Lett.
1994, 35 (36), 6649-6652.
(20) Milecki, J .; Zamaratski, E.; Maltseva, T. V.; Fo¨ldesi, A.;
Adamiak, R. W.; Chattopadhyaya, J . Tetrahedron 1999, 35, 6603-
6622.
(14) Wheeler, H. L.; J ohnson, T. B. J . Am. Chem. Soc. 1903, 29, 492-
505.
(15) Bendich, A.; Getler, H.; Brown, G. B. J . Biol. Chem. 1949, 177,
565-570.
(16) Kamaike, K.; Takahashi, M.; Utsugi, K.; Tomizuka, K.; Okazaki,
Y.; Tamada, Y.; Kinoshita, K.; Masuda, H.; Ishido, Y. Nucleosides &
Nucleotides 1996, 15 (1-3), 749-769.
(17) (a) Vorbruggen, H.; Krolikiewicz, K.; Bennua, B. Chem. Ber.
1981, 114, 1234-1255. (b) Niedballa, U.; Vorbruggen, H. J . Org. Chem.
1974, 39, 3654-3660.
(21) Agrofoglio, L. A.; J acquinet, J .-C.; Lancelot, G. Tetrahedron Lett.
1997, 38 (8), 1411-1412.
1868 J . Org. Chem., Vol. 68, No. 5, 2003

Synthesis of Multilabeled Pyrimidine Nucleosides
F IGURE 1. Reaction mechanism proposed for the formation of the ribofuranose ring starting from the 1,5-dialdehyde precursor.
F IGURE 2. Chiral HPLC (solvent system ETOH, n-hexane): (left) a 1:1 mixture of D- and L-uridine; (right) a typical run of the
uridine obtained by the ring-opening-ring-closure process.
After monomethoxytritylation of the primary alcohol
function the formed [1’,2′,3′,4′,5′,6′-13C6]-5′-O-monomethoxy-
trityl-â-D-glucopyranosyl-[6-13C,1,3,4-15N2]-N4-benzoyl-
cytosine is suitable to the oxidative ring-opening and
reductive ring-closure sequence obtaining [1′,2′,3′,4′,5′-
13C5]-5′-O-monomethoxytrityl-[6-13C,1,3,4-15N3]-N4-ben-
zoylcytidine [14] in an overall yield of 31%. The use of
periodic acid and its salts or of lead tetraacetate for
cleavage of R-glycol groups was described by Malaprade22
and Criegee.23 Kinetic studies showed, that cis glycols
are oxidized more rapidly than trans glycols.24 To obtain
nucleoside dialdehydes25 from the corresponding glucopy-
ranosides, oxidation with 3 equiv of lead tetraacetate
turned out to be most efficient.26
F IGUR E 3. [1′,2′,3′,4′,5′-13C5]-5′-O-Monomethoxytrityl-[6-
13C,1,3-15N2]-uridine [13]: 13C NMR sugar region.
After acetylation of [13C6]-D-glucose [7] to the corre-
sponding [13C6]-pentaacetyl-D-glucose [8], a Vorbruggen
condensation with [6-13C,1,3-15N2]-uracil [1] is carried out
to obtain [1′,2′,3′,4′,5′,6′-13C6]-(2′,3′,4′,6′-tetraacetyl-â-D-
glucopyranosyl)-[6-13C,1,3-15N2]-uracil [9]. After depro-
tection to the corresponding â-D-glucopyranoside [10],
monomethoxytritylation yielded the 1′,6′-substituted â-D-
glucopyranoside [11], which is the starting compound for
the key step of this synthesissan oxidative ring-opening
and reductive ring-closure sequence obtaining [1′,2′,3′,4′,5′-
13C5]-5′-O-monomethoxytrityl-[6-13C,1,3-15N2]-uridine [13]
in an overall yield of 43% from [13C6]-D-glucose [7]. The
synthesis of [1′,2′,3′,4′,5′-13C5]-5′-O-monomethoxytrityl-
[6-13C,1,3,4-15N3]-N4-benzoylcytidine [14] is carried out in
an analogous way, starting from peracetylated [13C6]-D-
glucose [8] and [6-13C,1,3,4-15N3]-N4-benzoylcytosine [6].
After full deprotection to the corresponding â-D-glucopy-
ranoside, the exocyclic amino function was benzoylated.
Ring closure to obtain the monomethoxytrityl protected
ribonucleosides was achieved via an intramolecular
pinacol-coupling reaction. This reduction with either
SmI227 or tributyltin hydride shows a high cis-selectivity.
For tributyltin hydride, the reaction is proposed to occur
via an addition of a tin ketyl to a carbonyl group followed
by an intramolecular homolytic substitution step (SH2).28
(22) (a) Malaprade, L. Bull. Soc. Chim. Fr. 1928, 43, 683-696. (b)
Malaprade, L. Compt. Rend. 1928, 186, 382-384.
(23) (a) Criegee Ber. 1931 (64), 260. (b) Criegee Ber. 1932, 65, 1770.
(24) (a) Hockett, R. C.; McClenahan, W. S. J . Am. Chem. Soc. 1939,
61, 1667-1671. (b) Hockett, R. C.; Mowery D. F., J r. J . Am. Chem.
Soc. 1943, 65, 403-409.
(25) (a) Howarth, O.; J ones, A. S.; Walker, R. T.; Wyatt, P. G. J .
Chem. Soc., Perkin Trans. 2 1984, 261-265. (b) Ermolinsky, B. S.;
Mikhailov, S. N. Russ. J . Bioorg. Chem. 2000, 26, 429-449.
(26) Perlin, A. S. J . Am. Chem. Soc. 1954, 76, 5505-5508.
(27) (a) Namy, J . L.; Souppe, J .; Kagan, H. B. Tetrahedron Lett.
1983, 24, 765. (b) Riber, D.; Hazell, R.; Skrydstrup, T. J . Org. Chem.
2000, 65, 5382-5390.
J . Org. Chem, Vol. 68, No. 5, 2003 1869

Lagoja et al.
with 3-nitrotriazole (0.94 g, 8.25 mmol) in pyridine (10 mL) to
form a solution which was again evaporated to an oil. This
was treated with diphenyl phosphate (1.2 g, 4.95 mmol) and
pyridine (10 mL), and the solution was again evaporated to
an oil. To this residual oil was added pyridine (10 mL) and
p-toluenesulfonyl chloride (1.57 g, 8.25 mmol). The solution
was stirred at room temperature for 30 h and then quenched
by the addition of H2O (1 mL). The reaction mixture was
extracted with CH2Cl2 (100 mL) and saturated Na2CO3 (100
mL). After drying over Na2SO4 the solvent was removed in
vacuo to obtain a dark-brown solid (1.10 g, 83%), which was
used without further purification in the preparation of
[6-13C,1,3,4-15N3]-1-pivaloylmethylcytosine [4]. Rf (CH2Cl2/
MeOH 95:5) 0.55; mp 195-197 °C (CH2Cl2); 13C NMR (50
MHz, CDCl3) δ 26.7 (C(CH3)3), 38.9 (C(CH3)3), 71.7 (d, J ) 55
Hz, OCH2N), 96.7 (d, J ) 291 Hz, 5-C), 143.1 ((t), J ) 11 Hz,
2-C), 144.8 (3-C, NT), 147.4 (5-CH NT), 155.4 (J ) 48.6, 6-C),
164.7 (d, J ) 11 Hz, 4-C), 177.8 (CdO); 15N NMR (50 MHz,
DMSO-d6) δ 165.6 (N1), 241.0 (N3); exact mass calcd for
This distinguishes this pinacol cyclization from other
reductive cyclizations of tin ketals and causes the high
cis-selectivity in the cyclization of 1,5-dicarbonyl com-
pounds (Figure 1).
In the case of the furanose sugars, the reaction may
also proceed via a di-enol intermediate. This mechanism
would give rise to a mixture of D- and L-nucleosides.
However, this possibility was ruled out by the fact that
L-nucleosides were not detected during the reaction
(Figure 2).
Figure 3 shows the 13C spectrum of the sugar region
of the obtained MMT-uridine [13]. The isotopic purity
was verified by exact mass spectrometry.
Con clu sion
In summary, the synthesis of fully [13C/15N]-labeled
pyrimidine nucleosides has been achieved from 13C-
glucose and labeled nucleobases. The reaction scheme
leads directly to the monomethoxytrityl-protected nucleo-
sides without the need for the inversion of configuration
of C-3 of 13C-glucose. This was achieved by an oxidative
ring-opening reaction removing the carbon with the
wrong configuration. Due to its shortness and high yield,
this reaction scheme can be considered as a considerable
improvement for existing methods, making labeled nu-
cleotide material more easily available.
C
11*CH15N4*N2O5 326.1078, found 326.1103.
P r ep a r a tion of [6-13C,1,3,4-15N3]-1-P iva loylm eth ylcy-
tosin e [4]. A mixture of NH4Cl (0.19 g, 3.3 mmol) and KOH
(0.25 g, 3.4 mmol) in CH3CN (8 mL) was cooled in an ice bath.
Through
a septum was added H2O (5 mL), followed by
triethylamine (0.32 g, 3.12 mmol). To the resulting clear,
colorless solution was added a solution of [6-13C,1,3-15N2]-1-
pivaloyloxymethyl-4-(3’-nitrotriazol-1’-yl)-2-pyrimidone [3] (1.04
g, 3.2 mmol) in CH3CN (8 mL) and H2O (5 mL). The reaction
was allowed to warm to room temperature. After 48 h, the
formation of a precipitate (KCl) was noted and the reaction
mixture was diluted with MeOH to form a solution that was
adsorbed onto 10 g of silica gel and purified by column
chromatography (50 g, silica, 1% MeOH in CH2Cl2). Yield 0.69
g (90%); Rf (CH2Cl2/MeOH 9:1) 0.73; mp 265-266 °C (CH2-
Cl2); 13C NMR (50 MHz, DMSO-d6) δ 26.6 (C(CH3)3), 38.1
(C(CH3)3), 71.8 (d, J ) 54.6 Hz, OCH2N), 94.4 (J ) 267 Hz,
5-C), 145.9 (J ) 48.4, 6-C), 156.8 ((t), J ) 11 Hz, 2-C), 166.4
(d, J ) 55 Hz, 4-C), 177.4 (CdO).
P r epar ation of [6-13C,1,3,4-15N3]-Cytosin e [5]. [6-13C,1,3,4-
15N3]-1-Pivaloylmethylcytosine [4] (0.75 g, 3.30 mmol) was
treated with DMF (25 mL) and 0.5 N KOH (25 mL). The
suspension was stirred at room temperature for 5 days, during
which time all initially insoluble material dissolved. The
reaction mixture was concentrated in vacuo, dissolved in
MeOH, and adsorbed onto 5 g of silica gel and chromato-
graphed from 55 g of silica gel with use of a gradient ranging
from 3% to 12.5% MeOH in CH2Cl2. Yield 0.31 g (82.3%); Rf
(CH2Cl2/MeOH 9:1) 0.80; mp >300 °C; 13C NMR (50 MHz,
DMSO-d6) δ 92.6 (J ) 267 Hz, 5-C), 142.8 (d, J ) 48.4, 6-C),
156.8 ((t), J ) 11 Hz, 2-C), 166.7 (d, J ) 55 Hz, 4-C); 15N NMR
(50 MHz, DMSO-d6) δ 91.3 (d, J ) 5.8 Hz, 4-NH2), 139.7 (d, J
) 11.1 Hz, N1), 209.9 (d, J ) 5.8 Hz, N3); exact mass calcd
for C3*CH6*N3O 116.0455, found 116.0472.
P r ep a r a tion of [6-13C,1,3,4-15N3]-N4-Ben zoylcytosin e [6]-
. [6-13C,1,3,4-15N3]-Cytosine [5] (0.5 g, 4.5 mmol) was suspended
in pyridine (25 mL). Under cooling benzoyl chloride (2.5 g, 17
mmol) was added dropwise. After being stirred at room
temperature for 6 h, the reaction mixture was cooled in an ice
bath and first treated with EtOH (5 mL), then after 20 min
H2O (5 mL) was added. The reaction mixture was allowed to
warm to room temperature with stirring for 18 h. After
filtration and washing with cold H2O (2 × 3 mL) the precipitate
was dried over P2O5. Because of the bad solubility the product
only was verified by MS. Yield 0.65 g (76%); Rf (CH2Cl2/MeOH
9:1) 0.15; mp >300 °C; exact mass calcd for C10*CH13*N3O2
220.0717, found 220.0700.
P r ep a r a t ion of [1,2,3,4,5,6-13C6]-Glu cosep en t a -O-a c-
eta te [8]. After pyridine (20 mL) was cooled to 0 °C, acetic
anhydride (15 g, 14 mL, 0.15 mmol) was added. The fully
labeled glucose [7] (3.0 g, 0.016 mmol) was added in small
portions at 0 °C. The reaction temperature was slowly allowed
to come to room temperature and stirred overnight. After
Exp er im en ta l Section
1
Ma ter ia ls a n d Gen er a l Meth od s. 13C and H is referred
to TMS, 15N to liquid NH3. Exact mass measurements were
performed on a quadrupole time-of-flight mass spectrometer
equipped with a standard electrospray ionization (ESI) inter-
face. TLC was performed with TLC aluminum sheets and silica
(200-425 mesh) was used for column chromatography. For all
reactions dry (molecular sieve) analytical grade solvents were
used. Solvents for column chromatography were distilled
before use. Isotopically labeled materials were obtained from
Campro Scientific, [6-13C,1,3-15N2]-Uracil [4] was synthesized
according to literature procedures.11,12
P r ep a r a tion of [6-13C,1,3-15N2]-1-P iva loyloxym eth ylu -
r a cil [2]. To a suspension of [6-13C,1,3-15N2]-uracil [1] (0.66 g,
5.86 mmol) and anhydrous K2CO3 (0.68 g, 6.45 mmol) in
anhydrous DMF (80 mL) was added pivaloyloxymethyl chlo-
ride (972 mg, 6.45 mmol). The reaction was stirred at room
temperature for 3 days, protected from atmospheric moisture
by a CaCl2 drying tube. The reaction mixture was concentrated
in vacuo and the resulting residue was extracted continuously
in a Soxhlet extractor with CH2Cl2 (175 mL) for 18 h. The
extract was concentrated and purified by column chromatog-
raphy (silica, CH2Cl2:MeOH 95:5). Besides the desired product
(0.71 g, 56%), 1,3-bispivaloyl derivative (0.06 g, 3%) and
unreacted starting material (0.25 g, 36%) could be isolated.
Rf (CH2Cl2/MeOH 95:5) 0.33; mp 127-128 °C (CH2Cl2); 13C
NMR (50 MHz, DMSO-d6) δ 26.5 (C(CH3)3), 38.2 (C(CH3)3),
70.6 (d, J ) 54.6 Hz, OCH2N), 101.7 (d, J ) 291.4, 5-C), 145.3
(J ) 242.4, 6-C), 155.4 ((t), J ) 11 Hz, 2-C), 163.5 (d, J ) 11
Hz, 4-C), 177.2 (CdO); 15N NMR (50 MHz, DMSO-d6) δ 137.5
(d, J ) 11.8 Hz, N1), 156.1 (s, N3); exact mass calcd for
C9*CH14*N2O4Na 252.0825, found 252.0823.
P r ep a r a tion of [6-13C,1,3-15N2]-1-P iva loyloxym eth yl-4-
(3-n itr otr iazol-1-yl)-2-pyr im idon e [3]. A solution of [6-13C,1,3-
15N2]-1-pivaloyloxymethyluracil [2] (0.93 g, 4.12 mmol) in
pyridine (10 mL) was evaporated to an oil that was treated
(28) (a) Hays, D. S.; Fu, G. C. J . Org. Chem. 1998, 63, 6375-6381.
(b) Bebbington, D.; Bentley, J .; Nilsson, P. A.; Parsons, A. F. Tetra-
hedron Lett. 2000, 41, 8941-8945.
1870 J . Org. Chem., Vol. 68, No. 5, 2003

Synthesis of Multilabeled Pyrimidine Nucleosides
addition of ice-water (60 mL) an oil was formed, which turned
solid with scrapping. The resulting precipitate was filtered off
and recrystallized from water. The R:â enantiomer ratio was
∼1:1 (13C NMR). Yield 6.20 g (97%); Rf (CH2Cl2/MeOH 95:5)
0.63; mp 110-112 °C (H2O); 13C NMR (50 MHz, CDCl3) δ 20.2-
20.5 (5 × CH3 Ac), 169.5-170.7 (5 × CdO Ac), 60.9, 61.8 (d, J
) 44 Hz, CH2-6′), 67.2-71.0 (m, 3 CH, CH-4′,2′,3′), 72.7 (t, J
) 44 Hz, CH-5′), 88.0, 88,6 (d, J ) 44 Hz, CH-1′R), 91.4, 92.0
(d, J ) 44 Hz, CH-1′â); MS (LSIMS; Thgly; m/z (%)) 337 (73.3)
[M + H]+.
mmol) in dry CH2Cl2 (10 mL) was added Pb(OAc)4 (1.33 g, 3
mmol) in dry CH2Cl2 (10 mL) under nitrogen at ambient
temperature. The solution became warm and turned yellow.
After 2 h a colorless precipitate of Pb(OAc)2 was formed.
Stirring was continued for another 5 h. After the solvent was
reduced on a rotavapor the residue was purified by column
chromatography (silica, 10 × 2 cm, CH2Cl2/MeOH 95:5). The
formation of the dialdehyde has been verified with HR-MS,
but because of the complicated equilibrium observed in the
NMR it is not possible to interpret the NMR spectra in a
straightforward way.25a [1′,2′,3′,4′,5′-13C5]-5′-O-Monomethox-
ytrityl-[6-13C,1,3-15N2]-uridine dialdehyde [12]: Yield 0.49 g
(90%); Rf (CH2Cl2/MeOH 9:1) 0.44; exact mass calcd for
Syn th esis of [1′,2′,3′,4′,5′,6′-13C6]-(2′,3′,4′,6′-Tetr a a cetyl-
â-D-glu cop yr a n osyl)-[6-13C,1,3-15N2]-u r a cil [9]. To the sus-
pension of [6-13C,1,3-15N2]-uracil [1] (0.96 g, 8.32 mmol) and
[13C6]-glucosepenta-O-acetate [8] (3.3 g, 8.32 mmol) in dichlo-
roethane (40 mL) was added BSA (4.8 mL, 20 mmol). The
mixture was stirred under nitrogen at ambient temperature
for 20 min. A clear, colorless solution was obtained. After
addition of TMSOTf (3.60 mL, 20 mmol) under nitrogen the
reaction mixture was heated under reflux for 2 h. After cooling
to ambient temperature the resulting brown mixture was
evaporated in vacuo. The resulting oil was diluted in ethyl
acetate (200 mL) and washed with NaHCO3 (150 mL) and
brine (2 × 100 mL). After the solution was dried over Na2SO4
and the solvent evaporated, the resulted oil was purified by
column chromatography (silica, 24 × 3 cm, ethyl acetate/n-
hexane 2:1). Yield 3.29 g (87.5%); Rf (ethyl acetate/n-hexane
2:1) 0.34; mp 153-155 °C (EtOH); 13C NMR (50 MHz, CDCl3)
δ 20.2, 20.3, 20.4, 20.6 (4 × CH3 Ac), 61.5 (d, J ) 43 Hz, CH6′),
67.8 (t, J ) 43 Hz, CH4′), 69.3 (t, J ) 43 Hz, CH2′), 72.7 (t, J
C
23*C6H26*N2O8Na 545.1774, found 545.1766.
Red u ctive Rin g Closu r e: Typ ica l P r oced u r e. Dialde-
hyde [12] (1 mmol) was dissolved in absolute dioxane (8 mL)
under nitrogen atmosphere. Tri-n-butyltinhydride (0.43 g, 0.4
mL, 1.25 mmol) was added via a septum. The reaction mixture
was heated to 90 °C. A solution of AIBN (0.02 g, 0.12 mmol)
in dioxane (2 mL) was added via the septum. After being
stirred for 30 min at 90 °C the reaction mixture was checked
with TLC (CH2Cl2/MeOH 95:5). If unreacted aldehyde was
monitored on TLC, more AIBN (0.01 g, 0.06 mmol) and tri-n-
butyltinhydride (0.11 g, 0.1 mL, 0.31 mmol) were added and
stirring at 90 °C was continued for another 30 min. After the
solvent was evaporated under reduced pressure the resulting
foam was dissolved in CH2Cl2 (15 mL) and water (5 mL), and
after vigorous stirring for another 3 h KF‚xH2O (0.2 g) was
added to remove all organo-tin compounds, stirring was
continued for another 60 min, a white voluminous precipitate
was formed, after filtration the organic layer was separated,
the filter was washed with CH2Cl2, and the collected organic
layers were dried and purified by column chromatography
(silica, 20 × 3 cm, CH2Cl2 (100 mL) f CH2Cl2/MeOH 95:5).
Final purification to obtain enantiomerically pure nucleosides
[13] and [14], respectively, was carried out with reverse phase
HPLC.
[1′,2′,3′,4′,5′-13C5]-5′-Monomethoxytrityl-[6-13C, 1,3-15N2]-uri-
dine [13]: Yield 0.32 g, (60%); Rf (CH2CL2:MeOH 95:5) 0.41;
mp 100-105 °C (CH2Cl2); 13C NMR (50 MHz, DMSO-d6) δ 55.2
(s, CH3O), 63.0 (d, J ) 43 Hz, CH5′), 69.6 (t, J ) 43 Hz, CH3′),
73.6 (d × d, J ≈ 43 Hz, CH2′), 82.5 (d × d, J ≈ 43 Hz, CH4′),
89.0 (d × d, J 1 ) 12 Hz, J 2 ) 45 Hz, CH1′), 86.3 (C, MMT),
101.6 (d × d, J 1) 14.6 Hz, J 2) 66 Hz, 5-CH), 113.5 (s, 2CH,
AA′BB′, MMT), 124.1-130.3 (CAr, MMT), 134.8 (s, 1C, AA′BB′,
MMT), 140.8 (d, J ) 14.6 Hz, 6-CH), 144.1, 144.4 (s, 2 × 1C,
MMT), 150.7 ((t), J ) 11 Hz, 2-C), 158.5 (s, 4C, AA′BB′, MMT),
163.3 (d, J ) 11 Hz, 4-C); 15N NMR (50 MHz, DMSO-d6) δ
143.5 (N1), 155.2 (N3); exact mass calcd for C23*C6H28*N2O7-
Na 547.1930, found 547.1957.
) 43 Hz, CH3′), 75.0 (t, J ) 43 Hz, CH5′), 80.3 (d × d, J 1
)
14.6 Hz, J 2 ) 45 Hz, CH1′), 103.7 (d × d, J 1) 14.6 Hz, J 2) 66
Hz, 5-CH), 139.2 (d, J ) 14.6 Hz, 6-CH), 150.5 ((t), J ) 11 Hz,
2-C), 162.9 (d, J ) 11 Hz, 4-C), 169.5, 169.6, 169.8, 170.5 (4 ×
CdO Ac); 15N NMR (50 MHz, CDCl3) δ 130.6 (N1), 152.5 (N3);
exact mass calcd for C11*C7H23*N2O11 452.1473, found 452.1510.
Dep r otection : Typ ica l P r oced u r e. A solution of the
tetra-O-acetyl-protected nucleoside [9] (10 mmol) in NH3/
MeOH (20 mL) was stirred overnight at ambient temperature.
After removal of the solvent in vacuo the precipitate was
recrystalized from dichloromethane. [1′,2′,3′,4′,5′,6′-13C6]-(â-D-
Glucopyranosyl)-[6-13C,1,3-15N2]-uracil [10]: Yield 2.77 (98%);
Rf (EE) 0.1; mp 202 °C (MeOH); 13C NMR (50 MHz, DMSO-
d6) δ 60.9 (d, J ) 43 Hz, CH6′), 69.6 (t, J ) 43 Hz, CH4′), 70.8
(t, J ) 43 Hz, CH2′), 76.9 (t, J ) 43 Hz, CH3′), 79.9 (t, J ) 43
Hz, CH5′), 82.5 (d × d, J 1 ) 14.6 Hz, J 2 ) 45 Hz, CH1′), 101.3
(d × d, J 1 ) 14.6 Hz, J 2 ) 66 Hz, 5-CH), 141.0 (d, J ) 14.6 Hz,
6-CH), 151.0 ((t), J ) 11 Hz, 2-C), 163.3 (d, J ) 11 Hz, 4-C);
15N NMR (50 MHz, DMSO-d6) δ 141.2 (N1), 155.4 (N3); exact
mass calcd for C3*C7H15*N2O7 284.1055, found 284.1061.
Mon om eth oxytr ityla tion in th e 6′ P osition : Typ ica l
P r oced u r e. A mixture of glucopyranosylnucleoside (i.e.[10])
(3.5 mmol) and MMTCl (2.16 g, 7.0 mmol) in pyridine (20 mL)
and triethylamine (5 mL) was stirred in an inert atmosphere
at ambient temperature for 18 h. After the solvent was
removed in vacuo the resulted oil was coevaporated with
toluene (4 × 2 mL). The resulting foam was purified by column
chromatography (silica, 15 × 3 cm, CH2Cl2/MeOH 9:1).
[1′,2′,3′,4′,5′,6′-13C6]-6′-O-Monomethoxytrityl-(â-D-glucopyrano-
syl)-[6-13C,1,3-15N2]-uracil [11]: Yield 1.8 g (92%); Rf (CH2Cl2/
MeOH 9:1) 0.31; mp 168-170 °C; 13C NMR (50 MHz, DMSO-
d6) δ 55.1 (s, CH3O), 63.7 (d, J ) 43 Hz, CH6′), 69.6 (t, J ) 43
Hz, CH4′), 70.6 (t, J ) 43 Hz, CH2′), 76.6 (t, J ) 43 Hz, CH3′),
77.9 (t, J ) 43 Hz, CH5′), 82.4 (d × d, J 1 ) 14.6 Hz, J 2 ) 45
Hz, CH1′), 85.8 (C, MMT), 101.3 (d × d, J 1 ) 14.6 Hz, J 2 ) 66
Hz, 5-CH), 113.3 (s, 2CH, AA′BB′, MMT), 126.9-130.3 (CAr,
MMT), 135.4 (s, 1C, AA′BB′, MMT), 141.4 (d, J ) 14.6 Hz,
6-CH), 144.7 (s, 2 × 1C, MMT), 151.0 ((t), J ) 11 Hz, 2-C),
158.3 (s, 4C, AA′BB′, MMT), 163.3 (d, J ) 11 Hz, 4-C); 15N
NMR (50 MHz, DMSO-d6) δ 145.7 (N1), 159.9 (N3); exact mass
calcd for C23*C7H29*N2O8Na2 600.1895, found 600.1864.
Ack n ow led gm en t. We thank the K.U. Leuven for
financial support (GOA 02 /13). I.L. is a research
associate of the Rega foundation. E.L. is a research
associate of the Belgian FWO.
Su p p or tin g In for m a tion Ava ila ble: Spectroscopic data
concerning the synthesis of 4-benzoyl-5′-monomethoxytrityl-
cytosine [14]. This material is available free of charge via the
Internet at http://pubs.acs.org.
Note Ad d ed a fter ASAP P ostin g. The C′2 amd C′3
NMR assignment were incorrect in Figure 3 and the
NMR data fo 13 in the version posted J anuary 29, 2003;
the correct version was posted February 14, 2003.
Oxid a tive Rin g Op en in g: Typ ica l P r oced u r e. To a
solution of the 1’,6’-disubstituated â-D-glucopyranoside [11] (1
J O0205098
J . Org. Chem, Vol. 68, No. 5, 2003 1871