The 5-methylisocytosine β-D-ribopyranosyl (4′ → 2′)-oligonucleotide

Article abstract of DOI:10.1002/hlca.200690024

In the context of Eschenmoser's work on pyranosyl-RNA ('p-RNA'), we investigated the synthesis and base-pairing properties of the 5-methylisocytidine derivative. The previously determined clear-cut restrictions of base-pairing modes of p-RNA had led to th

Full text of DOI:10.1002/hlca.200690024

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Helvetica Chimica Acta – Vol. 89 (2006)
The 5-Methylisocytosine b-D-Ribopyranosyl (4’ ! 2’)-Oligonucleotide
by Jan W. Batsa) and Christian Miculka*b)1)
a) Institute of Organic Chemistry and Chemical Biology, University of Frankfurt, Marie-Curie-Strasse 11,
D-60439 Frankfurt am Main
b) Institute of Pharmaceutical Chemistry, University of Frankfurt, Marie-Curie-Strasse 9, D-60439
Frankfurt am Main
In the context of Eschenmoser’s work on pyranosyl-RNA (‘p-RNA’), we investigated the synthesis
and base-pairing properties of the 5-methylisocytidine derivative. The previously determined clear-cut
restrictions of base-pairing modes of p-RNA had led to the expectation that a 5-methylisocytosine b-
D-ribopyranosyl (=D-pr(MeisoC)) based (4’ ! 2’)-oligonucleotide would pair inter alia with D-pr(isoG)
and L-pr(G) based oligonucleotides (D-pr and L-pr=pyranose form of D- and L-ribose, resp.). Remarka-
bly, we could not observe pairing with the D-pr(isoG) oligonucleotide but only with the L-pr(G) oligonu-
cleotide. Our interpretation concludes that this – at first hand surprising – observation is caused by a
change in the nucleosidic torsion angle specific for isoC.
1. Introduction. – More than 40 years ago, the noncanonical Watson–Crick pair of
isocytidine with isoguanosine (Fig. 1) was proposed as an additional component of
an early genetic system [1]. In RNA, isocytosine (isoC) pairing with isoguanosine
[2–6], purine-2,6-diamine ribofuranoside [7], and guanosine [8][9] and functional con-
sequences for the isoC-containing RNA have been investigated. In the context of broad
experimental work led by Eschenmoser on the synthesis and determination of base-
pairing properties of b-D-ribopyranosyl (4’ ! 2’)-oligonucleotides (=‘pyranosyl-
RNA’ or ‘p-RNA’) [10], this paper describes the synthesis of the corresponding 5-
methylisocytosine b-D-ribopyranosyl (4’ ! 2’)-oligonucleotide. The switch from isoC
to the derivative MeisoC was done for synthetic reasons since the 5-methyl-substituted
moiety had been shown to be chemically more stable [3]. The preparation of the b-D-
ribopyranosyl nucleoside D-pr(MeisoC)2) containing the protected 5-methylisocytosine
was performed based on extensive experiences from the syntheses of other nucleobases
[10]. The results of the determination of the base-pairing properties of this oligonucleo-
tide have been reported previously in [11] and will be complemented by a broader
interpretation.
2. Results. – Synthesis of the MeisoC building block 9 amenable to automated oligo-
nucleotide synthesis followed the previously established route [10] for the cytosine
building block (Scheme). The commercially not available 5-methylisocytosine had to
1
)
Current address: Intervet Innovation GmbH, Zur Propstei, D-55270 Schwabenheim (phone: +49-
6130-948-333; fax: +49-6130-948-533; e-mail: christian.miculka@intervet.com).
The abbreviations ‘D-pr’ and ‘L-pr’ stand for the pyranose form of D- and L-ribose, respectively.
2
)
© 2006 Verlag Helvetica Chimica Acta AG, Zürich

Helvetica Chimica Acta – Vol. 89 (2006)
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Fig. 1. Complementary Watson–Crick-like donor/acceptor pattern of isoC and isoG
be synthesized by basic condensation from methyl formate, ethyl propanoate, and gua-
nidine with sodium methanolate following [12]. Then 5-methylisocytosine was benzo-
ylated to the previously unknown N2-benzoyl-5-methylisocytosine (1), the solubility
of which – in comparison to N4-benzoylcytosine – in organic solvents was significantly
lower, requiring a change of the usual workup procedure [13]. Nucleosidation following
a modified Vorbrüggen approach [14] by using an a/b-mixture of tetra-O-benzoyl-D-
ribopyranose as starting material furnished mainly b-D-pyranoside 2, containing only
minor amounts of furanoside. Both the silylation of the free nucleobase with
bis(trimethylsilyl)acetamide and the Lewis acid catalyzed nucleosidation with trime-
thylsilyl trifluoromethanesulfonate proceeded very fast in comparison to other nucleo-
sidations in the p-RNA series. Regioselective cleavage of the sugar benzoyl groups left
the N2-benzoyl group intact and led to sugar-unprotected nucleoside 3.
Assignment of the configuration at the anomeric center of 3 is based on the characteristic value of
the 1H-NMR coupling constant of 9.4 Hz between the anomeric proton HÀC(1’) and the H-atom at C(2’)
of 3, both occupying axial positions at the pyranose chairs. Additional NOE experiments established the
constitution of 3 with respect to the anomeric bond: As shown in Fig. 2, the NOE signal between HÀC(6)
and HÀC(2’) confirms the N1-constitution of the nucleoside; the additional NOE signal between HÀ
C(1’) and HÀC(5’) supports the 4C1 conformation of the pyranose chair.
Fig. 2. Interpretation of NOE signals of 3
Taking into account the necessity to cleave the N2-benzoyl group after synthesis of
the oligonucleotide, we tested the anticipated cleavage methods: Both the treatment
with aqueous hydrazine at 08 or conc. aqueous ammonia at 608 afforded de-benzoylated
3. As expected, the ammonia procedure also led to formation of the thymine derivative
as by-product by concomitant deamination of the nucleobase. The subsequent reaction
steps followed the procedures worked out for the other nucleobases [10], i.e., formation
of the 3’,4’-acetal 4 as a diastereoisomer mixture, 2’-benzoylation (! 5), deacetalation
(! 6), and regioselective dimethoxytritylation at OÀC(4’) (! 7). The 2’ ! 3’ benzoyl

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Scheme. Preparation of isoC Building Block 9 and Solid-Phase-Bound Unit 11 for Automated Synthe-
sis
BSA=N,O-bis(trimethylsilyl)acetamide, TMSOTf=CF3SO3SiMe3, DMAP=N,N-dimethylpyridin-4-
amine, Py=pyridine, DMT=4,4’-dimethoxytrityl=(MeO)2Tr, LCAA-CPG=long-chain aminoalkyl
controlled pore glass.

Helvetica Chimica Acta – Vol. 89 (2006)
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migration was slower than in the cytosine series: The reaction mixture was worked up
after 72 h and delivered almost equal amounts of the desired 3’-benzoate 8 and starting
material 7. Separation could be achieved easily by chromatography as 7 and 8 showed
distinct differences in retention in comparison to the cytosine derivative. Constitutional
uniformity was confirmed by the shift of the characteristic dd-signal of HÀC(5’) from d
3.39 (2’-benzoate 7) to 2.82 (3’-benzoate 8). The 3’-benzoate 8 served as educt for the
building blocks for the automated synthesis: phosphitylation with allyl diisopropyl-
phosphoramidochloridite led to 9 as a diastereoisomer mixture, and conversion with
bi-activated heptanedioic acid (! 10) and long chain alkylamino controlled pore
glass led to 11, the protected isoC unit coupled to the solid support.
The main diastereoisomer of the mixture 4 happened to crystallize and was sub-
jected to X-ray structure analysis; it established the ‘endo’-configuration of the 3’,4’-
acetal moiety (see below).
Automated synthesis of the b-D-ribopyranosyl (4’ ! 2’)-oligonucleotide followed
previous experiences (Scheme 4 in [10]) and was performed on a 1-mmol scale in the
‘trityl on’ mode with a DNA/RNA synthesizer applying modifications to the manufac-
turer’s protocol for DNA synthesis, mainly with respect to extending the coupling step,
the capping step, and the detritylation step because of the reactions being performed at
the secondary 4’-OH rather than at a primary 5’-OH in DNA. Not optimized coupling
efficiencies in the synthesis of protected D-pr(MeisoC8) were 85–87%, these values
being lower than in the other p-RNA-syntheses. The allyl protecting groups were
removed by treatment with tetrakis(triphenylphosphine)palladium. Cleavage of the
dimethoxytritylated oligonucleotide from the glass support with concomitant deben-
zoylation was achieved by a comparably long treatment with aqueous hydrazine solu-
tion at 48, which suggests low reactivity of the benzoyl group at the isoC nucleobase.
This reaction had to be monitored carefully by HPLC: After 15 h, no free oligonucleo-
tide could be detected, after 65 h, the oligonucleotide was fully deprotected, after 115 h,
the HPLC showed substantial strand cleavage. The still dimethoxytritylated oligonu-
cleotide was purified by prep. reversed-phase HPLC, detritylated, resubjected to
prep. HPLC, and desalted. The calculated molecular mass of D-pr(MeisoC8) was con-
firmed by MALDI-TOF-MS.
Base-pairing properties of D-pr(MeisoC8) alone (10 mM) and with D-pr(isoG8),
L-pr(G8), D-pr(C8), and D-pr(G8) (5 mM each) were determined under previously estab-
lished standard conditions (0.15M NaCl, 0.01M Tris·HCl, pH 7.0 in H2O) by observing
UVabsorption as a function of temperature3). D-pr(MeisoC8) alone and in an equimolar
mixture with D-pr(G8) did not show any cooperative behavior. Also, we could not
observe any pairing between D-pr(MeisoC8) and D-pr(C8) both under neutral and weakly
acid conditions. D-pr(MeisoC8) did not pair with D-pr(isoG8), as was confirmed by meas-
uring the absorption at three different wavelengths (290, 280, and 245 nm), at three dif-
ferent concentrations, i.e., 5 mM, 0.5 mM, and 0.25 mM each, and at pH 5.5 additionally. As
expected, D-pr(MeisoC8) paired with L-pr(G8), Tm 248 [11].
The oligonucleotides for base-pairing experiments with D-pr(MeisoC8) were kindly provided by col-
leagues from Eschenmoser’s ETH group. Originally planned base pairing with partner L-pr(isoG8)
was not performed since this partner was not available anymore when this work was carried out.
3
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3. Discussion. – In our hands, the nucleobase MeisoC behaved very much like C in
the synthetic workflow. Main differences were the significantly lower solubility of
N2-benzoyl-5-methylisocytosine (1) in comparison to N4-benzoylcytosine in organic
solvents, the faster nucleosidation (1 ! 2), the slower 2’ ! 3’-benzoyl migration (7 !
8), and the slower N2-debenzoylation of the octameric oligonucleotide in comparison
to other nucleobases in the p-RNA series. The chemical shift of the anomeric proton
HÀC(1’) of 2 to lower fields (d 6.99) in comparison to the other nucleosides in the p-
RNA series (from [10], d 6.38 to 6.67) seems noticeable but can be explained by the
influence of the benzoyl group at the exocyclic N-atom.
The molecular conformation of the ‘endo’-isomer of 4 observed in the crystal struc-
ture is shown in Fig. 3. The six-membered ribopyranose ring approximately has a boat
conformation with atoms C(2) and C(5) about 0.61 Å outside the best plane C(3)À
C(4)ÀO(2)ÀC(1) (arbitrary atom numbering, see Fig. 3). The boat conformation is sta-
bilized by the acetal ring, which forces the C(3)ÀC(4) bond to be almost eclipsed (tor-
sion angle C(2)ÀC(3)ÀC(4)ÀC(5) 6.9(2)8). The five-membered acetal ring has a con-
formation intermediate between a C6 envelope and a C6,O3 twist. The proton expected
at the N-benzoyl group (N(3)) has migrated to the isocytosine group (N(2)). It is
involved in an intramolecular H-bond (N(2)ÀH(02)···O(7) with N(2)ÀH(02) 0.87(2)
Å, H(02)···O7 1.84(2) Å, N(2)···O(7) 2.558(2) Å, and N(2)ÀH(02)ÀO(7) 139(2)8).
The C(17)ÀN(3) bond length of 1.315(2) Å corresponds to a double bond. The N-ben-
zoyl-5-methylisocytosine group is almost planar (mean deviation from plane: 0.032 Å).
It has a syn-periplanar conformation with respect to the C(1)ÀH(1) bond, resulting in a
short intramolecular contact distance of only 2.27(2) Å between H(1) and N(3) (torsion
angle H(1)ÀC(1)ÀN(1)ÀC(17) 128). The OH group at C(2) donates an intermolecular
H-bond to the MeO group of a neighboring molecule.
The pairing properties of p-RNA and the restrictions on pairing modes have been
discussed in extenso [10] and will not be repeated here. Of all base-pairing experiments
with D-pr(MeisoC8), only pairing between D-pr(MeisoC8) and L-pr(G8) was observed,
albeit this pairing was significantly weaker than in the corresponding D-pr(C8)·
L-pr(isoG8) duplex. The absence of pairing of D-pr(MeisoC8) alone and of D-pr(MeisoC8)
with D-pr(G8) was in line with the expected inability of the nucleobases to meet the con-
stitutional requirements of a Watson–Crick pairing. The absence of pairing between
D-pr(MeisoC8) and D-pr(C8) also under weakly acidic conditions can be explained by
the lack of interstrand stacking between pyrimidine bases, which would contribute to
the stability of the duplex.
The surprise that D-pr(MeisoC8) did not pair with D-pr(isoG8) was based on expec-
tations arising from knowledge of one of the strongest pairings in the p-RNA series,
namely D-pr(C8) with D-pr(G8) (Tm 828 at 2.5 mM each). Here we would like to elaborate
on the original assumption (see comment [32] in [11]) that a sterically induced propeller
twist could hinder the duplex formation of D-pr(MeisoC8) and D-pr(isoG8): Fig. 4 illus-
trates the interdependency of the nucleosidic torsion angle c and pairing of isochiral
(left) and heterochiral (right) strands. In the idealized case of c=À1208 for the D-con-
figured strand (+1208 for the L-configured strand), base planes of opposite strands are
perfectly aligned for Watson–Crick pairing (isochiral case, D/D, left column) or reverse
Watson–Crick pairing (heterochiral case, D/L, right column). Inclination of the nucleo-
bases’ upper rim towards the pyranose O-atom in one strand by changing the nucleo-

Helvetica Chimica Acta – Vol. 89 (2006)
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Fig. 3. Molecular conformation of the ‘endo’-isomer of 4 observed in the X-ray crystal structure. 50%
Probability displacement ellipsoids. The H atoms are drawn as small spheres of arbitrary radius.
Arbitrary atom numbering.
sidic torsion angle c to 1808 (middle and lower row, left strand in each case) needs an
opposite change of the nucleosidic torsion angles to À608 in the isochiral pairing part-
ner (middle row, left case, right strand), and the same change of the nucleosidic torsion
angles to 1808 in the heterochiral pairing partner (middle row, right case, right strand)
to keep nucleobases in plane for pairing. Vice versa, the same change of the nucleosidic
torsion angles to 1808 in the isochiral pairing partner (lower row, left case, right strand),
and the opposite change of the nucleosidic torsion angles to +608 in the heterochiral
pairing partner (lower row, right case, right strand) would bring the nucleobases out
of their pairing planes and would constitute the propeller twist. In other words, changes
of nucleosidic torsion angles in the same sense favor pairing of heterochiral strands and
disfavour pairing of isochiral strands, under the condition that all other requirements
for pairing in p-RNA [10] be fulfilled. We assume that our experimental observation
that D-pr(MeisoC8) pairs with L-pr(G8) (heterochiral case) but not with D-pr(isoG8) (iso-
chiral case) can be interpreted accordingly: the main structural difference between the
isocytosine and the canonical nucleobases of relevance for the interpretation of this
observation is the 2-amino group replacing the O-atom. When incorporated into a ribo-
pyranosyl nucleotide, the protons of the isocytosine amino group and the anomeric pro-
ton sustain a steric strain, which can be relieved by a change of the nucleosidic torsion
angle c, presumably specific for the isocytosine moiety in direction and/or extent in
relation to the other canonical pyrimidine nucleobases (see [15] for the furanose-
based nucleotide). In Fig. 4, the pairing of D-pr(MeisoC8) with L-pr(G8) would corre-
spond to the heterochiral anti/anti-case (middle row, right), and the respective isochiral

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Fig. 4. Changes of nucleosidic torsion angle c and their consequences for pairing of isochiral (left) and
heterochiral (right) strands. View for Newman projections is from the duplex centre outwards, in
plane with bases (black wedge).
anti/anti-case (lower row, left) would correspond to the at first hand surprising nonpair-
ing of D-pr(MeisoC8) with D-pr(isoG8). With this interpretation, we would like to com-
plement the findings on base pairing between heterochiral p-RNA strands [11].
This work was funded by the Bundesministerium für Bildung und Forschung (BMBF), project
0311030, and the former Hoechst AG. C. M. would like to express his personal thanks to Prof. Albert
Eschenmoser for allowing publication of these results, Prof. Gerhard Quinkert for initiating the BMBF
project and continuous support, and Prof. Christian R. Noe (now at the University of Vienna) for gener-
ously providing the laboratory infrastructure of the Institute of Pharmaceutical Chemistry at the Univer-
sity of Frankfurt for this work.
Experimental Part
1. General. Solvents: distilled, synthesis grade. Reagents: unless otherwise noted, from Merck, Roth,
Fluka, Riedel-de Haën, Aldrich, Sterling, MWG-Biotech, or Sigma, various grades. TLC: silica gel 60 F254
aluminium plates (Merck); visualization by UV absorption and/or spraying with CeSO4 (1.05 g), ammo-
nium molybdate (2.1 g), and H2SO4 (conc., 6 ml) in 90 ml H2O followed by heating. Flash column chro-
matography (CC): silica gel 60 (0.40Æ0.63 mm, 230Æ440 mesh; Merck) at low pressure (max. 2 bar). Oli-
gonucleotides were synthesized on an Applied-Biosystems 392 DNA/RNA synthesizer. HPLC: Beck-

Helvetica Chimica Acta – Vol. 89 (2006)
225
man-Gold® chromatography system, equipped with a diode array detector; anal. and prep., reversed-
phase column, Spherisorb-S10X RP-C18 (10 mm, 300 Å, packed by Dr. J. Schreiber, ETH Zürich;
220×12 mm); flow 3 ml/min; buffer A: 0.1M Et3N, 0.1M AcOH, H2O, pH 7.0; buffer B: 0.1M Et3N, 0.1M
AcOH, H2O/MeCN 1:4. Concentrations of oligonucleotide solns. were calculated from the UV absor-
bance of the solns. at 260 nm (pH 7) at ca. 808 with the following molar extinction coefficients:
e(pr(C))=7600 l·molÀ1 ·cmÀ1, e (pr(G))=11700 l·molÀ1 ·cmÀ1, e(pr(isoC))=6300 l·molÀ1 ·cmÀ1 (taken
from the furanoid isomer [8]). M.p. uncorrected. NMR: d values in ppm rel. to SiMe4 as internal standard,
J in Hz; 1H assignments, in some cases, based on 1H,1H-COSY. 13C assignments and multiplicities based
on 1H,13C-COSY. FAB-MS: positive-ion mode, VG-ZAB-SEQ double-focusing high-resolution mass
spectrometer, with 3-nitrobenzyl alcohol (3-NBA) as matrix, or alternatively, EI mass spectrometer; in
m/z (intensity in%). Matrix-assisted laser-desorption-ionization time-of-flight mass spectrometry
(MALDI-TOF-MS). Voyager-Elite mass spectrometer (Perseptive Biosystems) with delayed extraction
with 2,4,6-trihydroxyacetophenone (THAP) or 2,5-dihydroxybenzoic acid (DHB) as the matrix with am-
monium citrate added to the sample.
2. Synthesis of 11. N2-Benzoyl-5-methylisocytosine (1). Benzoyl chloride (49.4 g, 0.352 mol, 1.1
equiv.) in abs. pyridine (50 ml) was added within 30 min to a suspension of 5-methylisocytosine (40 g,
0.32 mol, 1.0 equiv.; prepared following the procedure in [12]) under mechanic stirring. Additional ben-
zoyl chloride (4.9 g, 0.035 mol, 0.11 equiv.) was added after 4 h. After 16 h of stirring, 2N HCl (100 ml)
was added, the mixture adjusted to pH 4.5 by dropwise addition of conc. HCl soln., and the precipitate
filtered off after 15 min. The precipitate was washed with hot EtOH (250 ml) and dried in vacuo over-
night: 62 g (90%) of 1. Colorless solid, suitable for the next step. Anal. data were obtained from a sample
recrystallized from CHCl3. Colorless crystals. M.p. 277–2798. TLC (CH2Cl2/MeOH 8 :1): Rf 0.70. UV
(CH2Cl2): 237 (10600), 298 (9200). IR (KBr): 3190m, 3070m, 2970w, 2940w, 2920w, 2880w, 2340w,
1680m, 1650s, 1620s, 1590s, 1560m, 1508m, 1484m, 1448w, 1425w, 1368w, 1290m, 1265m, 1230m, 1161w,
1107w, 1024m, 992w, 959w, 935w, 892w, 827w, 802m, 784m, 745w, 718w, 698m, 676w, 581w. 1H-NMR
(300 MHz, (D6)DMSO): 1.89 (s, Me); 7.49–7.64 (m, arom. H); 7.68 (s, HÀC(6)); 8.06 (d, J=7.2,
13
arom. H); 12.15 (br. s, NH). C-NMR4) (50 MHz, (D6)DMSO): 12.54 (q, Me); 117.15 (s, C(5));
128.37, 128.53, 132.62 (3d, arom. C); 133.94 (s, arom. C); 151.57, 162.32, 171.45 (3s, C(2), C(4), CO).
EI-MS: 229.1 (14, M+), 228.1 (100, [MÀ1]+), 113 (33), 69 (17). Anal. calc. for C12H11N3O2: C 62.87, H
4.84, N 18.33; found: C 62.78, H 4.96, N 18.18.
N2-Benzoyl-5-methyl-1-(2,3,4-tri-O-benzoyl-b-D-ribopyranosyl)isocytosine (2). Bis(trimethylsilyl)-
acetamide (33.5 g, 130 mmol, 2.0 equiv.) was added to a suspension of 1 (14.2 g, 65 mmol, 1.0 equiv.)
and tetra-O-benzoyl-D-ribopyranose (a/b mixture [16]; 37 g, 65 mmol, 1.0 equiv.) in MeCN (350 ml).
After 30 min stirring at 608 (! clear soln.), trimethylsilyl trifluoromethanesulfonate (43.3 g, 195 mmol,
3.0 equiv.) was added in one portion. After 2 h, the mixture was cooled down to r.t., poured on a stirred
mixture of ice (300 g) and solid NaHCO3 (50 g), and extracted with AcOEt (3×400 ml). The combined
org. phase was dried (Na2SO4) and evaporated and the residue filtered over silica gel (250 g, 10×6 cm,
AcOEt/hexanes 1:1): 41.4 g of 2. Slightly yellow oil containing impurities (TLC), suitable for the next
step. Anal. data were obtained from a sample after CC (silica gel, AcOEt/hexanes 2 :3), containing
1
5% of furanoid nucleosides (as determined by H-NMR). Colorless foam. TLC (AcOEt/hexanes 1:1):
1
Rf 0.54. H-NMR (300 MHz, CDCl3): 2.04 (s, Me); 4.38 (t, J=10.9, HaxÀC(5’)); 4.44 (dd, J=5.3, 10.9,
HeqÀC(5’)); 5.50 (dd, J=2.8, 9.5, HÀC(2’)); 5.61 (ddd, J=2.7, 5.7, 8.5, HÀC(4’)); 6.40 (br. t, J=2.4,
HÀC(3’)); 6.99 (m, HÀC(1’), arom. H); 7.2–7.4 (m, arom. H); 7.45–7.6 (m, arom. H); 7.65–7.8 (m,
arom. H); 7.8–7.9 (m, arom. H); 8.05–8.15 (m, arom. H); 8.15–8.25 (m, arom. H); 13.34 (br. s, NH).
13C-NMR (50 MHz, CDCl3): 13.10 (q, Me); 64.43 (t, C(5’)); 66.81, 69.20, 69.30 (3d, C(2’), C(3’), C(4’));
80.62 (d, C(1’)); 115.79 (s, C(5)); 127.89, 128.25, 128.42, 128.50, 129.04, 129.30, 129.60, 129.82, 130.14,
132.06, 133.63, 133.82, 134.26 (13d, arom. C); 136.78 (s, arom. C); 153.73, 160.18 (2s, C(2), C(4));
164.88, 165.06 (2s, CO); 177.23 (s, PhCONH). EI-MS: 673 (11, M+), 672 (24, [MÀ1]+), 281 (10), 255
(29), 253 (12), 228 (11), 227 (11), 121 (16), 97 (14), 77 (30), 69 (100), 61 (15), 60 (18), 59 (11). Anal.
calc. for C38H30N3O9: C 67.85, H 4.50, N 6.25; found: C 67.52, H 4.80, N 6.21.
4
)
C(6) could not be determined.

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N2-Benzoyl-5-methyl-1-(b-D-ribopyranosyl)isocytosine (3). Slightly impure 2 (39 g, ca. 58 mmol, 1.0
equiv.) was dissolved in THF/MeOH/H2O 6 :3 :1 (800 ml) and cooled to 08. NaOH (13.9 g, 348 mmol,
6.0 equiv.) was dissolved in H2O/MeOH 3 :6 (90 ml), and added up to 150 ml with THF. The basic
soln. was added to the soln. of 2 within 5 min, and left between À2 and +28 for 10 min. Solid NH4Cl
(37.2 g) was added, the mixture evaporated (bath temp. <408), the residue taken up in toluene (150
ml), and the mixture evaporated. The residue was suspended in CH2Cl2/MeOH 15 :1 (200 ml) and adsor-
bed on silica gel (100 g). CC (silica gel (350 g), 13×5.5 cm, CH2Cl2/MeOH 15 :1 ! 5 :1) within 5.5 l gave
fractions from which 3 precipitated on evaporation as colorless crystals, which were collected and dried in
vacuo: 9.21 g of 3 (used for anal. data). Additional material was obtained from the CC by eluting with
CH2Cl2/MeOH 1:1 (1 l), evaporation, dissolution of the residue in MeOH (80 ml), addition of H2O
(80 ml), separation of the precipitate by filtration, washing with MeOH, and drying in vacuo: 5.17 g of
3. Combined yield: 14.38 g (64%) of 3. Colorless crystals. M.p. 221–2228. TLC (AcOEt/hexanes 1:1):
ꢀ
Rf 0.17. [a]2D0 =+11 (c=0.55, H2O). UV (H2O): 209 (7000), 248 (5100), 298 (9900). IR (KBr): 3375m
(br.), 3075w, 2930w, 1680s, 1655s, 1595s, 1580s, 1555s, 1510w, 1490w, 1475w, 1460w, 1450w, 1400m,
1380s, 1360s, 1350s, 1310w, 1270m, 1235m, 1165w, 1135w, 1095s, 1050s, 1025m, 1000m, 980w, 930w,
1
910w, 885w, 815w, 800w, 790w, 740w, 710s, 685w, 670w. H-NMR (300 MHz, (D6)DMSO): 2.04 (s, Me);
3.6–3.75 (m, HÀC(4’)); 2 HÀC(5’)); 3.86 (m, HÀC(2’)); 4.05 (br. s, HÀC(3’)); 4.88, 5.21 (2m, OH);
6.42 (d, J=9.4, HÀC(1’)); 7.4–7.65 (m, arom. H); 7.94 (s, HÀC(6)); 8.15 (d, J=7.0, arom. H); 13.41
(br. s, NH). 13C-NMR (50 MHz, (D6)DMSO): 12.29 (q, Me); 65.51 (t, C(5’)); 66.45, 67.94, 71.24 (3d,
C(2’), C(3’), C(4’)); 80.75 (d, C(1’)); 113.96 (s, C(5)); 128.18, 128.99, 132.28 (3d, arom. C); 136.83 (s,
arom. C); 137.63 (d, C(6)); 154.35, 159.95 (2s, C(2), C(4)); 176.54 (s, PhCONH). EI-MS: 396 (17), 361
(19, M+), 360 (100, [MÀ1]+), 228 (20), 113 (41), 79 (47), 69 (19). Anal. calc. for C17H19N3O6: C 56.51,
H 5.30, N 11.63; found: C 56.71, H 5.39, N 11.57.
‘endo’/‘exo’-N2-Benzoyl-1-[3,4-O-(4-methoxybenzylidene)-b-D-ribopyranosyl]-5-methylisocytosine
(‘endo’/‘exo’-4). A soln. of 3 (10.2 g, 28.2 mmol, 1.0 equiv.), anisaldehyde dimethyl acetal (12.9 g, 70.6
mmol, 2.5 equiv.), and TsOH·H2O (536 mg, 2.82 mmol, 0.1 equiv.) in DMF (155 ml) was heated at 608
under vacuum (20–30 mbar) until no 3 could be detected. After cooling to 08, the mixture was treated
with solid NaHCO3 (1 g) and then with sat. NaHCO3 soln. (100 ml), and then diluted with CH2Cl2
(500 ml). The org. phase was washed with H2O, dried (Na2SO4), and evaporated: 21.3 g of ‘endo’/
‘exo’-4 as brown oil containing impurities (mainly anisaldehyde dimethyl acetal; TLC), suitable for
the next step. Upon standing, the main diastereoisomer ‘endo’-4 precipitated as crystals. Anal. data
and X-ray analysis (see below) of ‘endo’-4 were obtained from a sample recrystallized from THF. Color-
less crystals. M.p. 233–2348. TLC (CH2Cl2/MeOH 15 :1): Rf 0.48. UV (CH2Cl2): 232 (17000), 248 (14000),
297 (21000). IR (KBr): 3460m (br.), 3070w, 2900w (br.), 1680s, 1570s, 1520m, 1450m, 1400m, 1340s,
1280m, 1250s, 1220m, 1170m, 1110m, 1090s, 1070s, 1030m, 1010m, 920w, 890m, 880m, 860m, 840m,
820m, 770w, 760w, 740m, 710m, 680m, 630w, 580w, 540w. 1H-NMR (300 MHz, CDCl3): 2.01 (s, Me);
1
3.80 (s, MeO); 4.01 (dd, J=5.8, 12.4, HÀC(5’)); 4.08 (dd, J=3.2, 9.2, HÀC(2’)); 4.19 (dd, J=4.7, 12.5,
1HÀC(5’)); 4.58 (dd, J=5.8, 11.2, HÀC(4’)); 4.77 (dd, J=3.5, 6.7, HÀC(3’)); 6.01 (s); 6.75 (d, J=9.6,
HÀC(1’)); 6.75 (d, J=8.7, arom. H); 7.21 (d, J=7.6, arom. H); 7.34 (d, J=1, arom. H); 7.44 (t, J=7.4,
arom. H); 7.56 (d, J=8.7, arom. H); 8.06 (d, J=7.2, arom. H); 13.49 (br. s, NH). 13C-NMR (50 MHz,
CDCl3): 13.24 (q, Me); 55.37 (q, MeO); 67.25 (t, C(5’)); 69.52, 72.36, 76.77 (3d, C(2’), C(3’), C(4’));
81.34 (d, C(1’)); 105.04 (s, C(5)); 114.12 (d); 115.73 (s); 127.97, 128.20, 129.33, 132.25, 134.38 (5d,
arom. C); 136.63 (s, arom. C); 154.63, 160.27, 160.85 (3s, C(2), arom. C, C(4)); 177.15 (s, PhCONH).
EI-MS: 479 (15, M+), 478 (59, [MÀ1]+), 396 (11), 360 (24), 229 (15), 228 (100), 113 (35), 69 (77).
Anal. calc. for C25H25N3O7: C 62.62, H 5.26, N 8.76; found: C 62.13, H 5.44, N 8.63.
Crystal-Structure Determination of ‘endo’-45). C25H25N3O7, Mr 479.49; monoclinic, P21;
a=6.2960(17), b=16.9340(11), c=10.7640(10) Å, b=98.598(15)8, V=1134.7(3) Å3, Z=2, Dx =1.403
g·cmÀ3; m=0.87 mmÀ1, r.t. (293 K). A colorless block with dimensions 0.10×0.32×0.50 mm3 was mea-
5
)
CCDC-282785 contains the supplementary crystallographic data for this paper. These data can be
obtained free of charge from the Cambridge Crystallographic Data Centre via http://www.ccdc.
cam.ac.uk/data_request/cif.

Helvetica Chimica Acta – Vol. 89 (2006)
227
sured on an Enraf-Nonius-CAD4 diffractometer with Cu-Ka radiation (graphite monochromator). A
hemisphere of reciprocal space was measured up to 2V=1308 with w-scans. Three reference reflections
every 100 min showed no intensity variations. Empirical absorption correction by using y scans of 6
reflections; transmission range: 0.813 to 0.917. Friedel opposites were not merged. Structure determina-
tion by direct methods (SHELXS-97 [17]). H-Atoms were taken from a difference synthesis and were
refined with individual isotropic thermal parameters. Structure refinement on F2 values (SHELXL-97
[17]), number of reflections: 3862, observed reflections with I>2s(I): 3806, number of parameters
394, wR(F2)=0.084, R(F)=0.031, S=1.13, final difference density between À0.10 and +0.12 e·ÅÀ3
.
The absolute configuration was established by anomalous dispersion effects, mainly of the O atoms
(Flack-x=À0.06(13)).
N2-Benzoyl-1-(2-O-benzoyl-b-D-ribopyranosyl)-5-methylisocytosine (6). To a soln. of impure 4 (20.71
g) from the acetalization step (containing max. 11.9 g (24.8 mmol, 1.0 equiv.) of pure 4) and DMAP
(0.303 g, 2.48 mmol, 0.10 equiv.) in pyridine (120 ml) cooled to 08, BzCl (8.72 g, 62 mmol, 2.5 equiv.)
was added within 10 min. After stirring at r.t. for 15 h, the mixture was cooled again, and sat., aq.
NaHCO3 soln. (150 ml) was added. After stirring at r.t. for 30 min, the mixture was extracted with
CH2Cl2 (500 ml), the org. phase extracted with H2O (100 ml), dried (Na2SO4), and evaporated. Residual
H2O was removed by addition of toluene (2×50 ml) and evaporation: 22.41 g of 5 containing impurities
(TLC), which was immediately used for the next step. TLC (CH2Cl2/MeOH 25 :1): Rf 0.57.
CF3COOH (22.6 g, 198 mmol, 8 equiv.) was added to a soln. of impure 5 (22.41 g) in MeOH/THF
10 :1 (275 ml), and the mixture was stirred for 30 min at r.t. After cooling (08), solid NaHCO3 (18.3 g,
218 mmol, 8.8 equiv.) was added. The mixture was left stirring for 30 min at r.t., then evaporated to ca.
50 ml, and adsorbed on silica gel (50 g) by addition of CH2Cl2 (200 ml) and careful evaporation. CC silica
gel (500 g), 26×7 cm, linear gradient CH2Cl2 ! CH2Cl2/MeOH 5 :1 in 5 l) furnished 7.69 g (67% based
on employed 3) of 6. Colorless foam. TLC (CH2Cl2/MeOH 20 :1): Rf 0.20. 1H-NMR (300 MHz, CDCl3):
1.91 (s, Me); 3.45–3.6, 3.95–4.1, 4.1–4.3 (3m, 2 OH, HÀC(4’), 2 HÀC(5’)); 4.57 (br. s, HÀC(3’)); 5.09
(dd, J=2.4, 9.4, HÀC(2’)); 6.9–7.1 (m, arom. H, HÀC(1’)); 7.25–7.5, 7.75–7.85 (2m, arom. H, HÀ
C(6)); 8.25 (d, J=7.1, arom. H); 13.38 (br. s, NH). 13C-NMR (50 MHz, CDCl3): 12.96 (q, Me); 66.51
(t, C(5’)); 66.51, 69.62, 71.55 (3d, C(2’), C(3’), C(4’)); 78.65 (d, C(1’)); 115.18 (s, C(5)); 128.12, 128.29,
129.59, 129.87, 132.32, 133.61, 135.16 (7d, arom. C, C(6)); 136.75 (s, arom. C); 153.41, 160.90 (2s, C(2),
C(4)); 165.15 (s, CO); 177.36 (s, PhCONH). EI-MS: 510 (26), 500 (25), 465 (25, M+), 464 (88,
[MÀ1]+), 228 (50), 121 (100), 113 (17), 69 (21).
N2-Benzoyl-1-{2-O-benzoyl-4-O-[bis(4-methoxyphenyl)phenylmethyl]-b-D-ribopyranosyl}-5-methyl-
isocytosine (7). A mixture of 6 (7.53 g, 16.2 mmol, 1.0 equiv.), (MeO)2TrCl (7.13 g, 21.0 mmol, 1.3 equiv.)
i
and 4-Å molecular sieves (5 g) was suspended in CH2Cl2 (70 ml), and Pr2NEt (5.44 g, 42.1 mmol, 2.6
equiv.) was added with stirring. After 2.5 h, additional (MeO)2TrCl (1.64 g, 4.85 mmol, 0.3 equiv.), 4-Å
i
molecular sieves (1 g), and Pr2NEt (1.05 g, 8.1 mmol, 0.5 equiv.) were added. After a total reaction
time of 3.5 h, CH2Cl2 (200 ml) and sat. NaHCO3 soln. (250 ml) were added. The org. phase was extracted
with 20% aq. citric acid soln. (200 ml) and sat. NaHCO3 soln. (100 ml), dried (Na2SO4), and evaporated:
15.0 g of impure (TLC) 7 as slightly yellow foam, suitable for the next step. A small part of this mixture
was separated by CC (silica gel, AcOEt/hexanes 2 :3) for characterization purposes. Slightly yellow foam.
1
TLC (CH2Cl2/MeOH 20 :1): Rf 0.64. H-NMR (300 MHz, CDCl3): 1.92 (s, Me); 2.60 (s, OH); 3.39 (dd,
J=4.5, 10.3, 1 HÀC(5’)); 3.70 (br. s, HÀC(3’)); 3.82 (s, MeO); 3.9–4.15 (m, HÀC(4’), 1 HÀC(5’));
4.82 (dd, J=2.3, 9.4, HÀC(2’)); 6.91 (dd, J=1.5, 8.8, arom H.); 6.95–7.6 (m, arom. H, therein HÀ
C(1’)); 7.80 (d, J=7.2, arom. H); 8.32 (d, J=7.0, arom. H); 13.28 (br. s, NH). 13C-NMR (50 MHz,
CDCl3): 13.01 (q, Me); 55.29 (q, MeO); 65.21 (t, C(5’)); 68.73, 69.00, 70.78 (3d, C(2’), C(3’), C(4’));
78.31 (d, C(1’)); 87.53 (s); 113.65 (d); 115.18 (s, C(5)); 127.40, 127.81, 128.15, 128.22, 128.34, 128.50,
129.62, 129.82, 129.95, 132.21, 133.53 (11d, arom. C, therein C(6)); 135.61, 135.65, 137.05, 144.75 (4s,
arom. C); 153.56, 159.10, 160.31 (3s, C(2), C(4), arom. C); 165.06 (s, CO); 177.28 (s, PhCONH). EI-
MS: 768 (1.3, M+), 767 (2.5, [MÀ1]+), 465 (13), 464 (45), 372 (33), 272 (13), 229 (15), 228 (100), 215
(17), 121 (28).
N2-Benzoyl-1-{3-O-benzoyl-4-O-[bis(4-methoxyphenyl)phenylmethyl]-b-D-ribopyranosyl}-5-methyl-
isocytosine (8). A mixture of impure 7 (13.76 g) from the last step (max. 11.4 g (14.9 mmol, 1.0 equiv.) of
pure 7), 4-nitrophenol (4.13 g, 29.7 mmol, 2.0 equiv.) and DMAP (2.0 g, 16.4 mmol, 1.1 equiv.) in pyridine

228
Helvetica Chimica Acta – Vol. 89 (2006)
(160 ml), PrOH (17.9 g, 297 mmol, 20 equiv.), and Et3N (4.5 g, 44.6 mmol, 3.0 equiv.) was stirred at
60–658 for 72 h. The mixture was evaporated and co-evaporated with toluene (50 ml). The residue
was dissolved in CH2Cl2 (400 ml), the soln. washed with 20% aq. citric acid soln. (2×100 ml) and sat.
aq. NaHCO3 soln. (6×200 ml), dried (Na2SO4), and evaporated after addition of silica gel (35 g). The res-
idue was subjected to CC (silica gel (620 g), 25×8 cm, linear gradient AcOEt/hexanes 1:20 ! 1:1 in 4 l,
then isocratic 1:1): 3.67 g (25%) of starting 7 and 3.78 g (32%) 8. Slightly yellowish foam. TLC (AcOEt/
hexanes 1:1): Rf 0.31. 1H-NMR (300 MHz, CDCl3): 1.97 (s, Me); 2.82 (dd, J=5.1, 11.2, 1HÀC(5’));
3.65–3.9, 3.95–4.05 (2m, HÀC(2’), HÀC(4’), 1 HÀC(5’)); 3.79 (s, MeO); 5.95 (br. s, HÀC(3’)); 6.56
(d, J=9.3, HÀC(1’)); 6.75–6.85, 7.1–7.45, 7.5–7.65, 7.7–7.8 (4m, arom. H); 7.97 (d, J=7.2, arom. H);
8.37 (d, J=7.2, arom. H); 13.48 (br. s, NH). 13C-NMR (50 MHz, CDCl3): 13.06 (q, Me); 55.25 (q,
MeO); 66.36 (t, C(5’)); 67.77, 71.28, 73.95 (3d, C(2’), C(3’), C(4’)); 81.63 (d, C(1’)); 87.45 (s); 113.41
(d); 115.59 (s, C(5)); 127.13, 127.98, 128.93, 129.05, 129.95, 130.11, 132.18, 133.64, 134.24 (9d, arom. C,
therein C(6)); 135.76, 135.92, 136.27, 144.83 (4s, arom. C); 154.68, 158.86, 160.09 (3s, C(2), C(4), arom.
C); 166.15 (s, CO); 176.96 (s, PhCONH). EI-MS: 768 (16, M+), 767 (48, [MÀ1]+), 766 (96), 372 (31),
283 (14), 281 (19), 255 (36), 253 (13), 229 (16), 228 (100), 216 (16), 215 (88), 146 (19), 145 (12), 144
(13), 121 (40), 97 (34), 77 (10), 60 (13).
N2-Benzoyl-1-[3-O-benzoyl-4-O-[bis(4-methoxyphenyl)phenylmethyl]-b-D-ribopyranosyl}-5-methyl-
isocytosine 2’-[Prop-2-enyl Diisopropylphosphoramidite] (diastereoisomer mixture; 9). A soln. of 8 (1.92
i
g, 2.5 mmol, 1.0 equiv.) in abs. CH2Cl2 (10 ml) was stirred at r.t. in the presence of Pr2NEt (1.29 g, 10
mmol, 4.0 equiv.) and prop-2-enyl diisopropylphosphoramidochloridite (0.70 g, 3.75 mmol, 1.5 equiv.)
for 2 h. The mixture was then concentrated to 5 ml and directly subjected to CC (silica gel (80 g);
14×4.5 cm, linear gradient hexanes/AcOEt 10 :1 ! 1:1 in 1 l; all eluents contained 2% of Et3N). Product
fractions were evaporated (<408), then co-evaporated with CCl4 (4 ml): 1.90 g (79%) of 9. Colorless
foam. TLC (AcOEt/hexanes/Et3N 39 :59 :2): Rf 0.45. 1H-NMR (300 MHz, CDCl3): 0.68 (d, J=6.7,
Me2CH); 0.87 (d, J=6.7, Me2CH); 0.96 (t, J=6.4); 1.27 (dd, J=3.0, 6.8); 1.95, 1.98 (2s, Me); 2.41, 2.63
(2 dd, J=5.0, 11.0, HÀC(5’)); 3.2–3.45, 3.45–3.85, 3.85–4.2 (3m); 3.78, 3.79 (2s, MeO); 4.4–4.6,
4.95–5.1, 5.15–5.3, 5.3–5.5, 5.55–5.7, 5.85–6.05 (6m); 6.07, 6.29 (br. s, HÀC(3’)); 6.75–6.9, 7.1–7.55,
7.62–7.72 (3m, arom. H, therein HÀC(1’)); 8.13 (d, J=8.0, arom. H); 8.29 (t, J=7.3, arom. H); 13.46
(br. s, NH). 13C-NMR (50 MHz, CDCl3): 13.01, 13.08, 22.90, 22.95, 24.06, 24.14, 24.24, 24.32, 42.78,
43.02, 45.15, 45.27 (12q, Me); 55.21 (q, MeO); 63.11, 63.44, 63.84, 63.98, 64.15, 66.01 (6t, C(5’)); 68.02,
68.11, 70.35, 70.44, 70.62, 73.00, 73.73 (7d); 80.33, 80.49 (2d, C(1’)); 87.38, 87.44, 87.53 (3s); 113.37,
113.45 (2d); 114.69, 114.99 (2s); 115.41, 116.07, 117.32 (3t); 127.02, 127.90, 128.01, 128.71, 128.94,
129.40, 129.74, 130.17, 130.65, 130.79, 131.79, 131.86, 132.99, 133.05, 134.70, 134.86, 135.00, 135.11,
135.17, 135.90 (20d, arom. C); 136.00, 136.06, 136.22, 137.15, 137.24, 145.01, 145.05 (7s, arom. C);
154.14, 154.22, 160.41, 160.54 (4s, C(2), C(4)); 158.81, 158.84 (2s); 165.59 (s, CO); 177.46, 177.56 (s,
PhCONH). FAB-MS: 955 (10, M+), 856 (20), 855 (57), 854 (100), 850 (22), 849 (42), 750 (14), 304
(33), 303 (90), 188 (25), 146 (11), 105 (10), 104 (50), 102 (16).
N2-Benzoyl-1-{3-O-benzoyl-4-O-[bis(4-methoxyphenyl)phenylmethyl]-b-D-ribopyranosyl}-5-methyl-
isocytosine 2’-[(4-Nitrophenyl) Heptanedioate] (10). A soln. of DMAP (52 mg, 0.422 mmol, 1.0 equiv.),
bis(4-nitrophenyl) heptanedioate (1.24 g, 3.08 mmol, 7.3 equiv.), and 9 (324 mg, 0.422 mmol, 1.0 equiv.)
in pyridine (3 ml) was stirred for 17 h. The mixture was evaporated, treated with toluene (6 ml), and
evaporated. The residue was subjected to CC (silica gel (30 g), 12×2.5 cm, linear gradient AcOEt/hex-
1
anes 1:6 ! 1:1 in 0.8 l): 264 mg (61%) of 10. Colorless foam. TLC (AcOEt/hexanes 1:1): Rf 0.49. H-
NMR (300 MHz, CDCl3): 1.05–1.2, 1.3–1.55 (2m); 1.97 (s, Me); 1.95–2.2 (m); 2.41 (t, J=7.4); 2.85
(dd, J=5.0, 11.1, HÀC(5’)); 3.79 (s, MeO); 3.87 (t, J=11.0); 3.95–4.1 (m); 4.95 (dd, J=2.5, 9.5, HÀ
C(2’)); 5.89 (br. s, HÀC(3’)); 6.75–6.85, 6.85–7.05, 7.2–7.3, 7.3–7.45 (4m, arom. H, therein HÀC(1’));
7.59 (t, J=7.7, arom. H); 7.76 (t, J=7.4, arom. H); 8.07 (d, J=7.2, arom. H); 8.15 (d, J=9.1, arom.
H); 8.23 (d, J=9.1, arom. H); 8.33 (d, J=7.2, arom. H); 13.37 (br. s, NH). 13C-NMR (50 MHz,
CDCl3): 13.04 (q, Me); 24.00, 24.02, 28.12, 33.58, 33.79 (5t); 55.26 (q, MeO); 66.31 (t, C(5’)); 67.31,
67.54, 68.70 (3d, C(2’), C(3’), C(4’)); 79.13 (d, C(1’)); 87.48 (s); 113.47 (d); 113.54 (d); 115.28 (s, C(5));
115.63 (d); 122.44, 125.12, 126.17, 127.23, 127.96, 128.07, 128.98, 129.31, 129.90, 130.06, 132.14, 133.64,
134.60 (13d, arom. C, therein C(6)); 135.57, 135.74, 136.75, 144.71, 145.23 (5s, arom. C); 153.58, 160.30

Helvetica Chimica Acta – Vol. 89 (2006)
229
(2s, C(2), C(4)); 155.51, 158.96 (2s); 161.69, 165.59, 170.84, 171.89 (4s); 177.33 (s, PhCONH). FAB-MS:
1033 (21), 1032 (51), 1031 (78, [M+H]+), 307 (12), 304 (37), 303 (100), 289 (12), 230 (14), 139 (13), 138
(19), 137 (28), 136 (31), 121 (10), 107 (14), 104 (42).
N2-Benzoyl-1-{3-O-[bis(4-methoxyphenyl)phenylmethyl]-b-D-ribopyranosyl}-5-methylisocytosine 2’-
{7-[(LCAA-CPG)amino]-7-oxoheptanoate} (11). A suspension of 10 (80 mg, 0.078 mmol, 1.0 equiv.),
LCAA-CPG (390 mg), iPr2NEt (101 mg, 0.78 mmol, 10 equiv.), and pyridine (31 mg, 0.39 mmol, 5
equiv.) in DMF (1.2 ml) was shaken overnight. The solid support was filtered off, washed consecutively
with DMF (30 ml), MeOH (30 ml), and Et2O (30 ml), dried under high vacuum, suspended in pyridine
(1.5 ml), and treated with Ac2O (0.25 ml) and DMAP (12 mg). After being shaken for 68 h at r.t., the
solid support was filtered off and washed consecutively with DMF (30 ml), MeOH (30 ml), and Et2O
(30 ml). After drying (! 310 mg of 11), the loading density was determined by detritylation of 11 (6.3
mg) with 0.1M TsOH in MeCN (10 ml) and measuring the absorption at 498 nm (e ((MeO)2Tr+
ion)=7·105 l·molÀ1 ·cmÀ1). The loading density was 27 mmol isoC nucleoside/g solid support.
3. Automated Solid-Phase Synthesis and Purification of the Oligonucleotide D-pr(MeisoC8) (see
Scheme 4 in [10]). Oligonucleotide synthesis was carried out on a 1-mmol scale on an Applied Biosys-
tems-392 DNA/RNA synthesizer.
Pre-automation Procedures. The DNA/RNA synthesizer column was filled with 11 (27 mg, 1 mmol).
A soln. of phosphoramidite 9 (112 mg, 0.1175 mmol) in MeCN (1.26 ml; ! 0.1M soln.) was dried (4-Å
molecular sieves, 8–12 mesh, freshly activated) for 3 h at r.t. prior to use.
Activator soln.: A mixture of 0.15M 5-(4-nitrophenyl)-1H-tetrazole and 0.35M 1H-tetrazole in MeCN
was dried over freshly activated 4-Å molecular sieves.
Capping A (THF/Ac2O/lutidine), capping B (THF/1-methyl-1H-imidazol), and oxidizing soln.
(THF/pyridine/H2O/I2) as purchased from MWG-Biotech.
Detritylation reagent: 6% Cl2CHCOOH in CH2Cl2.
The automated ‘trityl on’ synthesis of the oligonucleotide with the DNA/RNA synthesizer was
accomplished with the following modifications to the manufacturer’s protocol for DNA synthesis: 1)
the delivery step of 9 and activator was done within 2.5 s, 2) the coupling step (20 equiv. of 9 each)
was extended to 225 s, 3) the delivery step of activator alone was done within 4 s, 4) the capping soln.
was pumped for 90 s, 5) the detritylation step was conducted within 253 s, and 6) the oxidizer soln.
was pumped within 8 s.
Post-automation Procedure. For allyl deprotection, following Method B in [10], a soln. of Et2NH (50
ml), [Pd0(Ph3P)4] (20 mg), and Ph3P (20 mg) in CH2Cl2 (1.5 ml) was added to the CPG solid support, and
the resulting suspension was vigorously shaken at r.t. for 5 h. The suspension was filtered, the CPG solid
support carefully washed sequentially with CH2Cl2 (10 ml) and acetone (15 ml), treated with 0.1M aq.
NaCS2NEt2 (4.5 ml) at r.t. for 30 min, filtered, and washed again sequentially with H2O (10 ml), acetone
(15 ml), and EtOH (10 ml), and dried under high vacuum. For the detachment from CPG and acyl depro-
tection, the CPG-attached oligonucleotide was suspended in 24% aq. NH2NH2 ·H2O soln. (1.5 ml) and
vigorously shaken at 48 for 70 h. The mixture was diluted with 0.1M aq. Et3NH·HCO3 buffer to a total
volume of 10 ml. A Sep-Pak-C18 cartridge (Waters) was washed three times sequentially with MeCN
(5 ml) and 0.1M aq. Et3NH·HCO3 buffer (5 ml). Alternatingly, the oligonucleotide soln. (1 ml) and
0.1M aq. Et3NH·HCO3 buffer (1 ml) were applied to the cartridge, and subsequently, 0.1M aq.
Et3NH·HCO3 buffer (10 ml) and H2O (10 ml) were used to elute excess hydrazine (UV monitoring).
The oligonucleotide was eluted by applying an H2O/MeCN gradient (5, 10, 25, and 50% MeCN, 10 ml
each). Product fractions were evaporated, and the oligonucleotide was purified by reversed-phase
HPLC (gradient 0–100% eluent B in 100 min; tR of (MeO)2Tr-D-pr(MeisoC8) 47 min). The combined
product fractions from HPLC purification were evaporated and then detritylated by treatment with
80% HCOOH (3 ml; immediate orange color) for 10 min at r.t. The mixture was evaporated, taken
up in H2O (5 ml), evaporated again, taken up in H2O (10 ml) again, extracted with CH2Cl2 (3×2 ml)
to remove the (MeO)2TrOH, evaporated and subjected to prep. reversed-phase HPLC (gradient
0–100% eluent B in 100 min; tR of D-pr(MeisoC8) 21 min). The oligonucleotide was desalted by using
the published method (see [10], Exper. Part, Sect. 7). MALDI-TOF-MS: 2489.3 (C80H113N24O54Pþ7 ;
calc. 2490).

230
Helvetica Chimica Acta – Vol. 89 (2006)
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Received October 17, 2005