Oligonucleotide analogues with integrated bases and backbone. Part 30: Synthesis and association of a self-complementary thiomethylene-linked octanucleoside

Article abstract of DOI:10.1002/hlca.201300043

The protected G*[s]C*[s]U*[s]A*[s]U*[s] A*[s]G*[s]C* octanucleoside 24 was prepared by S-alkylation of the thiolate derived from tetranucleoside 23 with the methanesulfonate 22, and transformed to the silylated and isopropylidenated 25, and further into t

Full text of DOI:10.1002/hlca.201300043

Helvetica Chimica Acta – Vol. 96 (2013)
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Oligonucleotide Analogues with Integrated Bases and Backbone
Part 301)
Synthesis and Association of a Self-Complementary Thiomethylene-Linked
Octanucleoside
Dieter Seebach mit den besten Glꢀckwꢀnschen zugeeignet
by Lorenz Herdeis, Siji Thomas, Bruno Bernet*, and Andrea Vasella*
Laboratorium fꢀr Organische Chemie, ETH Zꢀrich, Wolfgang-Pauli Strasse 10, CH-8093 Zꢀrich
The protected G*[s]C*[s]U*[s]A*[s]U*[s]A*[s]G*[s]C* octanucleoside 24 was prepared by S-
alkylation of the thiolate derived from tetranucleoside 23 with the methanesulfonate 22, and transformed
to the silylated and isopropylidenated 25, and further into the fully deprotected octanucleoside 26.
Compound 22 was derived from the methoxytrityl-protected tetranucleoside 21, and 21 was obtained by
S-alkylation of the thiolate derived from the dinucleoside 19 with methanesulfonate 17 derived from 16
by detritylation and mesylation. Similarly, tetranucleoside 23 resulted from S-alkylation of the thiolate
derived from 18 with the methanesulfonate 20 derived from 19. Dinucleosides 16 and 18 resulted from S-
alkylation of the thiolate derived from the known cytidine-derived thioacetate 15 with the C(8)-
substituted guanosine-derived methanesulfonates 12 and 14, respectively, that were synthesized from the
protected precursors 4 and 7 by formylation, reduction, protection, and mesylation.
The structures of the duplexes of 25 and 26 were calculated using AMBER* modelling and based on
the known structure of the core tetranucleoside U*[s]A*[s]U*[s]A*. The former shows a helix with a
bent helix axis and strong buckle and propeller twists, whereas the latter is a regular, right-handed, and
apparently strain-free helix. In agreement with modelling, the silylated and isopropylidenated
octanucleoside 25 in (CDCl2)2 solution led to a mixture of associated species possessing at most four
WatsonꢀCrick base pairs, while the fully deprotected octanucleoside 26 in aqueous medium forms a
duplex, as evidenced by a decreasing CD absorption upon increasing the temperature and by a UV-
melting curve with a melting temperature of ca. 108 below the one of the corresponding RNA octamer,
indicating cooperativity between base pairing and base-pair stacking.
Introduction. – Our investigation of analogues that replace the contiguous
backbone of oligonucleotides by linking elements between pairs of nucleobases
(ONIBs)2) has shown that self-complementary and non self-complementary, partially
protected di- and tetranucleosides, possessing a variety of linking elements, undergo
pairing in CDCl3 solution [2 – 9]. Pairing (i.e., the formation of cyclic duplexes)
depends on the sequence of the nucleobases, and on the constitution and conformation
of the linking elements. Pairing requires a syn conformation, and involves Watsonꢀ
Crick and/or Hoogsteen-type H-bonds. In some ONIBs, pairing in CDCl3 is
accompanied by stacking of the nucleobases, as evidenced by CD spectroscopy.
1
)
)
Part 29: [1].
2
Abbreviation of the originally suggested term ꢂOligoNucleotides Integrating Backbone and basesꢃ.
ꢁ 2013 Verlag Helvetica Chimica Acta AG, Zꢀrich

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Two types of linkers have proven of special interest for our goal of making ONIBs
that pair in aqueous solutions. In the corresponding ONIBs, two adjacent nucleobases
are either joined by S-methylene-5-thioribosyl units [4 – 8], or by S-methylene-l-
cysteine-containing dipeptidyl units [1]. We have already shown that octameric ONIBs
of the latter type associate in aqueous solution in a sequence-specific, reversible way to
form a left-handed double helix with antiparallel strand orientation, characterized by
melting temperatures and free enthalpies that are higher than those of natural RNA
and DNA of the same sequence [1]. In parallel experiments, we pursued the goal of
making H2O-soluble octameric ONIBs of the former type. Pairing in aqueous solution
of two types of ONIBs should conclusively demonstrate that the structural differ-
entiation of oligonucleotides in a contiguous backbone and appended nucleobases is
neither a prerequisite for pairing, nor for the formation of defined conformers.
The partially protected thiomethylene-linked U*[s]A*[s]U*[s]A*3) tetranucleo-
side 1 pairs in CDCl3 according to a WatsonꢀCrick type to form a right-handed
antiparallel helix, as determined by NMR spectroscopy and force-field calculations [6].
The fully deprotected tetranucleoside, however, proved too poorly soluble in H2O to
investigate its pairing properties.
The structure of the isopropylidene-protected U*[s]A*[s]U*[s]A* tetranucleoside
1 is characterized by a strong bending of the helix axis that leads to a pronounced
inclination of the central base pairs (roll angle of ca. 158) and results in poor base
stacking [6]. This bending appears incompatible with helix formation of longer
oligonucleosides of this type. However, a self-complementary octanucleoside, obtained
by extending the tetranucleoside with cytidine and guanosine derived units, may well be
sufficiently soluble in H2O, considering the H2O solubility of cytidine-derived di- and
tetranucleosides [8], and partial or complete deprotection may confer them a sufficient
flexibility to form a full helix turn, in spite of the strong bending of the helix axis of the
core U*[s]A*[s]U*[s]A* tetranucleoside.
Results and Discussion. – 1. Calculation of Fully Paired Duplex Structures of the
Target Octanucleosides 25 and 26. To evaluate the formation of cyclic duplexes with all
nucleobases involved in base pairing, we calculated the cyclic duplexes of the target
3
)
Conventions for abbreviated notation: The substitution at C(6) of pyrimidines and C(8) of purines is
denoted by an asterisk (*); for example, U* and A* for hydroxymethylated uridine and adenosine
derivatives, respectively. U(*) and A(*) represent both unsubstituted and hydroxymethylated
nucleobases. The moiety linking C(6)CH2 or C(8)CH2 of unit II, and C(5’) of unit I is indicated in
square brackets, i.e., [s] for a S-atom.

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G*[s]C*[s]U*[s]A*[s]U*[s]A*[s]G*[s]C* octanucleosides 25 and 26 (see Scheme 3
below) with the force-field programme AMBER* implemented in Macromodel v.6.0
[10]. The duplex structure of the U*[s]A*[s]U*[s]A* tetranucleoside 1 (1 · 1 ¼ 26C in
[6]) corresponds to the central units III – VI of 25 and 26. It was extented on both sides
by G*[s]C* moieties possessing also WatsonꢀCrick base pairs. This calculation of 25 ·
25 resulted, after releasing all constraints, in a left-handed double helix with eight
WatsonꢀCrick base pairs (H-bond distances of 1.68 – 1.82 ꢄ; Fig. 1,a) in spite of the
strong curvature of 1 · 1. The isopropylidene and silyl protecting groups of 25 · 25 were
Fig. 1. AMBER*-calculated structures of the WatsonꢀCrick base-paired duplexes a) 25 · 25 and b) 26 · 26:
front and top views (for 25 · 25: H-atoms of the protecting groups are omitted for enhanced clarity), and
space-filling (CPK) front view

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removed, and minimisation gave the structure of the duplex 26 · 26 (with shorter H-
bond distances of 1.64 – 1.79 ꢄ) of the fully deprotected octanucleoside 26 (Fig. 1,b).
Both structures 25 · 25 and 26 · 26 contain six units per turn as indicated in the top
view (Fig. 1). Despite the isopropylidene groups, the former duplex possesses a deeper
major groove than the latter one (see CPK structures). The duplex 26 · 26 shows a
regular double helix with a linear helix axis (type-B helix with a small central hole),
whereas the duplex 25 · 25 is less regular and possesses a bent helix axis (inclined to the
right in the top view) that is, however, less pronounced than for the duplex 1 · 1 of the
U*[s]A*[s]U*[s]A* tetranucleoside. In addition, the front view shows a better p-
stacking of the nucleobases (distance between base pairs of 3.1 – 3.5 ꢄ), smaller
propeller twists, and smaller roll angles for 26 · 26 than for 25 · 25. This indicates a severe
backbone strain in the cyclic duplex 25 · 25 which may prevent the formation of a
completely base-paired cyclic duplex. However, the far more regular structure of 26 ·
26, with little or no backbone strain, suggests an easy formation of this duplex. It
appeared worthwhile to synthesize 25 and 26, and to investigate their pairing
properties.
2. Synthesis of G*[s]C*[s]U*[s]A*[s]U*[s]A*[s]G*[s]C* Octanucleosides. We
aimed at a convergent synthesis of the title octanucleoside rather than following the
longer linear strategy underlying the synthesis of the core tetranucleoside 1 [6]. As
conceived, the synthesis required the four dinucleosides 17 – 20 (cf. Scheme 3 below), to
be coupled to form two tetranucleosides, 21 and 23. Coupling of tetranucleoside 22
derived from 21 with 23 should provide the fully protected octanucleoside 24. Thus,
each coupling step requires the directed formation of a thioether by alkylating a
thiolate anion, generated by cleaving the C(5’)ꢀSR group, with a methanesulfonate (or
a similar electrophile) derived from a nucleoside possessing a hydroxymethyl
substituent at C(6) or C(8). Finally, deprotection of 24 should first provide the
isopropylidenated and silylated derivative 25, and then the desired fully deprotected
octanucleoside 26.
The synthesis started with the preparation of the guanosine-derived methanesul-
fonate 12 and 14, corresponding to units VIII and II of the octanucleosides (Scheme 1).
For the synthesis of 12, the known 2’,3’-O-isopropylideneguanosine (2) [11] was
silylated under standard conditions to the thexyl(dimethyl)silyl (¼ TDS) ether 3, and
then transformed according to [12] to the isobutyramide 4. For the synthesis of 14, 2
was directly protected as isobutyramide 5 [13] and further treated with (PhO)3PMeI
[14] to provide iodo derivative 6 that was substituted with the sodium salt of
monomethoxytritylthiol4) to yield 90% of 7.
The protected nucleosides 4 and 7 differ only by the substituent at C(5’), and were
both hydroxymethylated by deprotonation at C(8) with excess LiHMDS, formylation,
and reduction. Formyl derivative 8 obtained from 4 was isolated and reduced with
NaBH4 to hydroxymethyl derivative 95), while the analogous formyl derivative
resulting from 7 was reduced in situ to hydroxymethyl derivative 10. The purine moiety
of 9 and 10 was further protected by alkylation with 2-(4-nitrophenyl)ethanol under
4
)
Monomethoxytritylthiol was synthesized from the corresponding alcohol according to a procedure
developed by Jahn-Hofmann and Engels [15].
Compound 9 was synthesized by Daniel Egli [16].
5
)

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Scheme 1
i
a) TDSCl, 1H-imidazole, DMF; 91%. b) Me3SiCl, pyridine, CH2Cl2, then PrCOCl, then MeOH and
H2O; 84%. c) (iPrCO)2O, 4-(dimethylamino)pyridine (DMAP), pyridine; 95%. d) (PhO)3PMeI,
MeCN; 93%. e) MMTrSH, NaH, THF; 90%. f) Lithium bis(trimethylsilyl)amide (LiHMDS), DMF,
THF, ꢀ 788 ! r.t; 80% of 8. g) NaBH4, EtOH; < 98% of 9, 74% of 10 from 7. h) 1-(Trimethylsilyl)-1H-
imidazole, dioxane, then PPh3, diisopropyl azodicarboxylate (DIAD), 2-(4-nitrophenyl)ethanol. i)
AcOH/H2O 1:1; 88% of 11. j) NH4F, MeOH; 78% of 13. k) MsCl, EtNiPr2, CH2Cl2; 87% of 12, 80% of
14. TDS ¼ thexyl(dimethyl)silyl (¼dimethyl(1,1,2-trimethylpropyl)silyl), MMTr¼ (monomethoxy)trityl
(¼(4-methoxyphenyl)diphenylmethyl).
Mitsunobu conditions [17]. This alkylation required the introduction of a temporary O-
trimethylsilyl group [18]. It was removed immediately upon alkylation using aqueous
AcOH to yield the 2-phenylethyl derivative 11, while 13 was prepared by desilylation
with NH4F in MeOH to avoid any acid-promoted detritylation. The hydroxymethyl

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derivatives 11 and 13 were transformed under standard conditions to the methanesul-
fonates 12 and 14, respectively.
For the synthesis of the G*[s]C* dinucleosides, we deacetylated the known cytidine-
derived thioacetate 15 [8] to generate the corresponding thiolate (Scheme 2). Best
results were obtained by treating 15 with MeSNa in MeOH/THF. The crude thiolate
was S-alkylated with methanesulfonates 12 and 14 to obtain the dinucleosides 16 (71%)
and 18 (65%), respectively. Reductive detritylation of 16 with Cl2CHCOOH in the
i
presence of Pr3SiH, and reaction of the crude alcohol with MsCl/EtNiPr2 gave
methanesulfonate 17.
Scheme 2
a) 1. MeSNa, THF/MeOH 1:1, ꢀ 108; 2. 12 or 14, EtNiPr2, DMSO; 71% of 16, 65% of 18. b) 1.
Cl2CHCO2H, iPr3SiH, CH2Cl2; 2. MsCl, EtNiPr2, CH2Cl2; 84%. Bz ¼ Benzoyl.
The known U*[s]A* dinucleoside 19 [6] played a key role in the synthesis of the
tetranucleosides 21 (! units V– VIII of 24) and 23 (! units I – IV; Scheme 3). It was
transformed to tetranucleoside 21 (65%) by S-deacetylation (best with K2CO3 in
MeOH/CH2Cl2) and treatment of the resulting thiolate with methanesulfonate 17. For
the synthesis of tetranucleoside 23, dinucleoside 19 was transformed under standard
conditions to the methanesulfonate 20. The thiolate required for the formation of
tetranucleoside 23 was generated by reductive O- and S-detritylation of 18, followed by
deprotonation with Hꢀnigꢃs base. Selective S-alkylation of the resulting thiolate with 20
provided tetranucleoside 23 (67%).

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For the synthesis of octanucleoside 24, we removed the O-MMTr group of 21
similarly as described for 19, and transformed the resulting alcohol to methanesulfo-
nate 22 (74%). S-Deacetylation of 23 was first attempted by treatment with MeSNa in
THF/MeOH, but this resulted in low coupling yields, while S-deacetylation with NaOH
in pyridine/EtOH [19], followed by coupling of the resulting thiolate with 22, yielded
61% of the desired octanucleoside 24.
We aimed at a partial deprotection of 24 to obtain the isopropylidenated and
silylated 25, as this octanucleoside might well pair in CHCl3 and conceivably also in
aqueous solution. We first attempted at cleaving the 2-(4-nitrophenyl)ethyl groups of
24 with 0.5m DBU (¼1,8-diazabicycloundec-7-ene) in pyridine [18], but the protracted
dealkylation was accompanied by the formation of substantial amounts of unidentified
side products. However, replacing DBU by 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD)
[20] led to the desired cleavage of the 2-(4-nitrophenyl)ethyl groups. The resulting
crude material was fully debenzoylated by the action of saturated NH3 in MeOH to
yield 50% of 25. Finally, aqueous HCO2H led to desilylation and complete
deisopropylidenation of 25, providing octanucleoside 26 (60%) that was purified by
filtration through a C18 functionalized silica column (Bakerbond C18). The purity of
26 was determined by HPLC/MS (Fig. 2). The high-resolution ESI mass spectrum
shows peaks at m/z 1151.2740, 1162.2639, and 1085.2514 for [M þ 2 H]2þ, [M þ H þ
Na]2þ, and the fragment ion [M ꢀ C5H9O4 þ 3 H]2þ resulting from loss of the ribosyl
moiety of unit VIII, respectively. These ions show the parent peaks of the isotopomers
in the expected ratio (see the Exper. Part). Investigations of the duplex formation of 26
were limited to UVand CD spectroscopy due to the small amount (2 mg), and the fact
that 1D- and 2D-NMR spectra had to be recorded in H2O containing 10 – 20% of an
aprotic cosolvent and at 0 – 58 [21] to detect exchangeable H-atoms.
Fig. 2. HPLC Trace of the completely deprotected octamer 26 after purification (UV detection at 260 nm)
3. Conformation of the 2’,3’-O-Isopropylidenated Guanosine Mononucleosides 2 –
14. The orientation of the guaninyl moiety in 2’,3’-O-isopropylideneguanosines should
show a similar dependence upon the substituents at C(8) and at C(5’) as observed

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Helvetica Chimica Acta – Vol. 96 (2013)
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for adenosines (see [4] and refs. cit. therein), and thus lead to a similar influence
on the chemical shift of HꢀC(2’). 8-Unsubstituted and O(5’)-silylated adenosines
(d(HꢀC(2’)) ¼ 5.20 – 5.30 ppm) in CDCl3 adopt an anti conformation, whereas 8-
unsubstituted and O(5’)-acetylated as well as C(5’)-sulfanylated derivatives
(d(HꢀC(2’)) ꢁ 5.50 ppm) adopt a syn/anti equilibrium together with a gt/tg orientation
of the substituent at C(5’) [4]. Both 8-substituted and O(5’)-unprotected adenosines
(d(HꢀC(2’)) ¼ 5.65 – 5.80 and 5.25 – 5.30 ppm, resp.) adopt a syn conformation, the
former for steric reasons and the latter as the consequence of a strong intramolecular
O(5’)ꢀH ··· N(3) H-bond [4].
In the guanosine series, the chemical shift of HꢀC(2’) is not only influenced by the
orientation of the guanosyl moiety, but also by the N2- and O6-protecting groups (see
Table 3 in the Exper. Part). In CDCl3, the 5’-O-silylated amine 3 and the corresponding
isobutyramide 4 adopt an anti conformation, as evidenced by the upfield shift for
HꢀC(2’) (d(HꢀC(2’)) ¼ 5.15 and 4.99 ppm, resp.) and by a prominent population of the
gg conformation (gg/gt/tg 50 :25 :25 and 60 :25 :156), resp.). As expected, the trityl
thioether 7 adopts a ca. 1:1 gg/gt equilibrium (J(4’,5’a) ¼ 7.1, J(4’,5’b) ¼ 6.6 Hz). The
bulky trityl group entails completely an anti conformation (d(HꢀC(2’) ¼ 5.01 ppm).
The iodo derivative 6 also adopts a ca. 1:1 gg/gt equilibrium, while the downfield shift
for HꢀC(2’) (5.24 ppm) reveals a syn/anti equilibrium, similarly to that observed for the
corresponding adenosine analogue [14]. The intramolecular O(5’)ꢀH ··· N(3’) H-bond
of the alcohol 5 [13] in CDCl3 is evidenced by the gg orientation of HOꢀC(5’)
(J(5’a,OH) ¼ 12.2 Hz) and by the upfield shift for HꢀC(2’), resonating at 5.12 ppm,
resulting from a combination of the syn conformation and a substantial population of
the furanose (S) conformation, similarly to what we had observed for adenosines [4].
The intramolecular H-bond of 2 in (D6)DMSO [22] is completely cleaved, as
evidenced by J(5’a,OH) ¼ J(5’b,OH) ¼ 5.1 Hz. The chemical shift d(HꢀC(2’)) ¼
5.17 ppm suggests an exclusive anti conformation (as compared with 3 in CDCl3),
assuming the absence of a solvent effect.
The 8-substituted guanosines 8 – 14 adopt a syn conformation, evidenced by the
downfield shift for HꢀC(2’). For O6-unsubstituted guanosines, d(HꢀC(2’) ¼ 5.34 ppm
of 10 may be typical; the weak downfield shift for HꢀC(2’) of 9 (5.42 ppm) is probably
due to the change of the solvent from CDCl3 to (D6)DMSO, and the weak upfield shift
for HꢀC(2’) of 8 (5.18 ppm) to the anisotropy of the CHO group. Protection of O6
leads to a stronger downfield shift for HꢀC(2’) of 11 – 14 (5.52 – 5.69 ppm). Here,
d(HꢀC(2’)) is influenced both by the protection of HOCH2ꢀC(8) (downfield shift for
the methanesulfonates) and the substituent at C(5’) (downfield shift for MMTr
thioethers). The thioethers 10, 13, and 14 adopt a ca. 1:1 gt/tg equilibrium, whereas a
significant population of the gg conformation is observed for the silyl ethers 8, 9, 11, and
12 (gg/gt/tg 10 :50 :40 for 8, 15 :40 :45 for 9, 41:32 :27 for 11, and 17:45 :38 for 12)
hinting at a C(2)ꢀNꢀH ··· O(5’) H-bond in the gg conformer. Force-field calculations
(Spartan 08 for Macintosh [23]) confirm the feasibility of such a H-bond.
The furanose ring of 4 and 6 – 14 prefers a (N) conformation as indicated by J(1’,2’)/
J(3’,4’) ¼ 0.3 – 0.8, whereas 3 adopts a 1:1 equilibrium between the (N) and the (S)
conformer.
6
)
Calculated from J(4’,5’a) and J(4’,5’b) with the formulae given in [4].

Helvetica Chimica Acta – Vol. 96 (2013)
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4. Conformation of the Di- and Tetranucleosides 16 – 18 and 20 – 23. The expected
syn conformation of the C(6)- and C(8)-substituted units of the di- and tetranucleosides
16 – 18 and 20 – 23 is evidenced by the downfield shift for HꢀC(2’) (uridine units: 5.19 –
5.24, cytidine units: 5.18 – 5.30, adenosine units: 5.50 – 5.74, guanosine units: 5.58 –
5.89 ppm; see Table 5 in the Exper. Part). The chemical shift d(HꢀC(2’)) of the
guanosine units shows a dependence on the substituents at C(5’): 5.87 – 5.89 ppm for the
TDS silyl ethers 16, 17, 21, and 22 (probably due to the intramolecular C(2)ꢀNꢀH ···
O(5’) H-bond in the gg conformer), 5.67 ppm for MMTr thioether 18, and 5.58 ppm for
the internal thioether 23. The CH2S linker prefers a gt/tg ca. 1:1 conformation, but
sometimes also a substantial preference for the gt conformation is evidenced (e.g., gt/tg
65 :35 for U and C of 22). All furanose rings of 16 – 18 and 20 – 22 prefer a (N)
conformation.
5. Conformation of the Isopropylidenated Octanucleosides 24 and 25. The
isopropylidenated octanucleosides 24 and 25 are well soluble in CDCl3. Whereas the
1H-NMR signals of 24 with protected C, G, and A nucleobases are well-resolved, those
of 25 in CDCl3, CD2Cl2, and (CDCl2)2 are broad, preventing the assignment of
coupling constants. Neither heating (508 in CDCl3) nor cooling (ꢀ 508 in CD2Cl2) led to
a sharpening of the signals. However, well-resolved NMR signals are observed in
(D6)DMSO where 25 is present as a solvated monoplex, as evidenced by d(HꢀN(3/
IV,VI)) ¼ 11.4 and d(HꢀN(1/II,VIII)) ¼ 10.8 ppm (compare with 11.3 and 10.7 ppm,
resp., for the corresponding HꢀN of monouridines and monoguanosines in (D6)DMSO
[6][22]). The octanucleosides 24 in CDCl3 and 25 in (D6)DMSO adopt similar
conformations (see Table 7 in the Exper. Part) as their di- and tetrameric precursors.
6. Pairing of the Isopropylidenated and Silylated Octanucleoside 25 in Chlorinated
Solvents. The 1H-NMR spectrum of 25 in (CDCl2)2 at room temperature shows broad
overlapping signals, and the 13C-NMR spectrum exhibits lines for several sets of slowly
1
equilibrating associates7). Even the H-NMR signals for the TDS group at ca. 0 ppm
(SiMe2) and at 0.7 – 0.9 ppm (SiCMe2CHMe2) evidence a mixture of associates. The
Table 1. NH Signals above 8 ppm of the Silylated and Isopropylidenated Octanucleoside 25 in (CDCl2)2
d [ppm] (H equiv.)
Assignment
13.88 (0.4 H)
13.55 (0.4 H)
(0.2 H)
13.35 (0.4 H)
13.32 (0.2 H)
11.72 (0.4 H)
HꢀN(1) of G or HꢀN(3) of U
HꢀN(1) of G or HꢀN(3) of U
HꢀN(1) of G or HꢀN(3) of U
HꢀN(1) of G or HꢀN(3) of U
HꢀN(1) of G or HꢀN(3) of U
HꢀN(1) of G
(0.2 H)
HꢀN(1) of G
13.64, 13.51, 13.42, 13.06 (each < 0.05 H)
9.92 (0.2 H)
8.93, 8.57 (both 0.1 H)
8.34 – 8.24 (2.4 H)
HꢀN(1) of G or HꢀN(3) of U
HꢀN(3) of U
NH of NH2
NH of NH2, HꢀC(2) of A
7
)
We investigated the pairing of the easily accessible silyl ether 25, although the results of parent di-
and tetranucleosides [4][6] suggest that the corresponding desilylated alcohol should show a far
higher tendency to form cyclic duplexes.

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integral for the SiCMe2CHMe2 moiety was used to gauge the H equivalents for the NH
signals above 8.0 ppm (Table 1). There are three sets of NH signals with an intensity
ratio of 8 :4: < 1. However, only ca. 2.4 of the 4 H-equivalents for HꢀN(1/II,VIII) of
the guanosine and for HꢀN(3/IV,VI) of the uridine moieties were detected between 9.9
and 13.9 ppm; apparently, 1.6 H equivalents are hidden by coalescence.
Despite the broad 1H-NMR signals, we could obtain satisfactory sensitivity-
enhanced 2D-NMR spectra. The CH assignments are taken from the HSQC spectrum
where only cross-peaks for a single associate were observed (Table 9 in the Exper.
Part). Based on characteristic 1H and 13C chemical shifts, the HSQC spectrum allowed
a rough assignment of the CH (i.e., CH(5) of pyrimidines, and CH(1’) to CH(4’)) and
CH2 signals (CH2(5’) and CH2ꢀC(6/8)). In the DQF-COSY spectrum, there are cross-
peaks between the signals for CH(2’) and CH(3’) and for CH(4’) and CH2(5’).
Additionally, there are cross-peaks between the purine CH(1’) and CH(2’), and
between the pyrimidine CH(3’) and CH(4’) signals. This allows to assign sequences
CH(1’)/CH(2’)/CH(3’) and CH(4’)/CH2(5’) for purine units, and sequences
CH(3’)ꢀCH2(5’) for pyrimidine units. The NOESY spectrum allows assignment of
the CH and CH2 signals to individual units, especially by cross-peaks between
CH2ꢀC(6/8) and CH2(5’), between HꢀC(4’) and HꢀC(1’), and between CH2ꢀC(6) and
HꢀC(5) of the pyrimidine units. Thus, the sequence of the nucleobases in 25 is
corroborated by the 2D-NMR spectra. The chemical shifts for HꢀC(2’), resonating at
5.85 – 6.36 ppm (purine bases) and 4.98 – 5.41 ppm (pyrimidine bases) evidence a syn
conformation of all nucleoside units. The signals for HꢀC(5/IV) (7.32 ppm) and
HꢀC(1’/II) (6.66 ppm) are well separated from other signals, and both integrate only
for 0.2 H. This evidences that only 20% of 25 is ꢂvisibleꢃ in the 2D-NMR spectra.
Therefore, and due to the presence of exchange ROE cross-peaks, we renounced an
analysis for the cross-peaks of the NH signals in the ROESY spectrum.
The chemical shifts for HꢀN(1/II,VIII) and HꢀN(3/IV,VI) (signals between 9.9 and
13.9 ppm in Table 1) suggest that three duplexes in a ratio of 8 :4 : < 1 are formed with
2 – 4 units involved in a quite stable base pairing (!visible signals) and four units with a
loose, rapidly exchanging base pairing (!signals hidden by coalescence). According to
the chemical shifts8), the most abundant duplex (d(HN) ¼ 13.88, 13.55, 13.35, and
11.72 ppm) possesses three stable WatsonꢀCrick base pairs and a free guanosine unit9),
and the least abundant duplex (d(HN) ¼ 13.64, 13.51, 13.42, and 13.06 ppm) even four
stable WatsonꢀCrick base pairs. Although a cyclic duplex with eight base pairs cannot
be excluded, it appears rather improbable. The second abundant duplex visible in the
2D-NMR spectra (d(HN) ¼ 13.55, 13.32, 11.72, and 9.92 ppm) possesses only two
stable WatsonꢀCrick base pairs, and both a free guanosine and uridine unit. Tentatively,
one may postulate that units II and III in all three associates are engaged in
WatsonꢀCrick base pairing, and that the terminal guanosine unit I of the two major
8
)
HꢀN(3) of free uridines in CDCl3 resonates at 10.4 – 10.8 ppm [5] and of WatsonꢀCrick U · A base
pairs at 12.5 – 13.5 ppm [4]. HꢀN(1) of guanosine in DMSO resonates at 10.6 – 10.7 ppm [24], of G ·
G base pairs in CDCl3 at 12.0 – 12.1 ppm [25] and in H2O/D2O at 11.0 ppm [24], and of
WatsonꢀCrick G · C base pairs in H2O at 12.4 – 13.7 ppm [26].
9
)
Duplexes possessing both WatsonꢀCrick- and Hoogsteen-type base pairs cannot be formed due to
strongly different radii of the helix [4].

Helvetica Chimica Acta – Vol. 96 (2013)
1247
associates is not involved in base pairing, as it is also the case for uridine unit IV of the
second associate. This result is consistent with the conclusion from the analysis of the
U*[s]A*[s]U*[s]A* tetramer that helical strain prevents the formation of cyclic
duplexes of higher analogues with complete base pairing [6]. A more detailed
characterization of these three associates requires further analytical and spectroscopic
investigations.
WatsonꢀCrick-H-bonded duplexes of U*[s]A* and A*[s]U* dinucleosides show
poorly temperature-dependent CD spectra, as due to large distances between the base
pairs [4]. Similarly, the cyclic duplexes of C*[s]C* and C*[s]C*[s]C*[s]C* di- and
tetranucleosides show no temperature dependence [8]. Hence, a strong CD absorption
of 25 is hardly a strong evidence for a cyclic duplex. The CD spectrum of a 0.1 mm
solution of 25 in CHCl3 at 08 shows a negative Cotton effect (negative maximum at 292
and stronger positive maximum at 270 nm; Fig. 3). Upon increasing the temperature to
508, the negative maximum disappears in favour of a positive shoulder, and the positive
maximum at 270 nm is increased. This evidences the disappearance of one stacked
associate and a stronger persistence of another one, possibly with stacked hydroxy-
methylated cytidine units I, similarly as observed in the C*[s]C*[s]C*[s]C* series [8].
Fig. 3. Temperature-dependent CD spectra in 108 steps from 0 to 508 of the isopropylidenated and silylated
octanucleoside 25 for a 0.1-mm solution in CHCl3
7. Qualitative Pairing Studies of the Fully Deprotected 26 in Aqueous Solutions. The
fully deprotected octanucleoside 26 is sufficiently soluble in H2O to allow investigating
its pairing properties in aqueous solution by CD and UV spectroscopy. The CD
absorbance of 26 in an aqueous solution is strong. Hence, a highly diluted solution (c ¼
4 mm, 10 mm sodium phosphate buffer, 100 mm NaCl, pH 7.0) was analysed, and as a
consequence of this high dilution there was disturbing noise (Fig. 4,a). Heating to 908
led to a strong decrease of the negative Cotton effect. This agrees well with the
dissociation of the cyclic duplex with increasing temperature.
This dissociation of the cyclic duplex is ascertained by a UV melting curve
(Fig. 4,b). The melting temperature and the thermodynamic parameters of the duplex
of 26 were calculated according to the Nearest-Neighbor-Method and compared to

1248
Helvetica Chimica Acta – Vol. 96 (2013)
Fig. 4. a) Temperature-dependent CD spectra in 108 steps from 0 to 908 and b) temperature-dependent UV
spectrum at 260 nm of the deprotected octanucleoside 26 (4 mm in 10 mm sodium phosphate buffer,
100 mm NaCl, pH 7.0)
those of the parent RNA octanucleotide possessing the same sequence [27]; both
duplexes show a similar duplex stability (Table 2). The melting temperature of 26 is
lower by 108 than the one of the analogous RNA octanucleotide, and corresponds to a
lower ꢀ DH298 value (60.1 vs. 71.9 kcal/mol), indicating weaker non-bonding inter-
actions, in keeping with a less efficient base stacking of duplexed 26. The lower ꢀ DH
correlates with a lower ꢀ DS (166.5 vs. 198.6 cal/(mol K)), suggesting a more
favourable conformation of the monoplex (ꢂpreorganisationꢃ). However, the UV
melting was very slowly reversible, showing no hypochromic effect upon cooling from
80 to 48 at a rate of 0.1 8/min. Only prolonged cooling of the sample (at 48 over 12 h)
allowed us to repeat the melting experiment. The reason for the slow re-association
remains speculative. It has been reported that nucleoside analogues with non-ionic
backbones suffer from low solubility in aqueous media, presumably due to poor
solvation, resulting in unspecific self-aggregation [28] as it was observed for peptide-
derived analogues (PNA) [29]. For cysteine-derived, peptide-linked ONIBs [1], an
ionic linking element was essential for pairing in aqueous solution, favouring the
solubility and possibly also an extended conformation [30]. The slow re-association of
26 in aqueous solution may imply that the thermodynamic parameters DG, DH, and DS
(calculated from the melting curve) might not reflect the actual stability of the duplex,
since the ratio between monoplex and duplex does not express an equilibrium situation.
Table 2. UV Melting Temperature and Thermodynamic Data for the Duplex of 26 and of the
Corresponding RNA Analogue rGCUAUAGC
Tm [8]
DG298 [kcal/mol]
DH [kcal/mol]
DS [cal/(mol K)]
26
38.5
48.9
ꢀ 10.5
ꢀ 10.2
ꢀ 60.1
ꢀ 71.9
ꢀ 166.5
ꢀ 198.6
rGCUAUAGC [27]
Conclusions. – CD Spectroscopy and a UV melting curve of the duplex of 26
confirm that the structural differentiation of oligonucleotide analogues into a
contiguous backbone and attached bases is not required for pairing, and this in spite

Helvetica Chimica Acta – Vol. 96 (2013)
1249
of a missing NMR analysis. The release of helical strain upon deisopropylidenation is
favorable for the formation of a cyclic duplex with complete base pairing. The
association of higher isopropylidenated ONIBs linked by thiomethylene groups in
organic solvents is dominated by base pairing, whereas both base pairing and stacking
contribute to the stabilisation of the cyclic duplexes of the corresponding deprotected
ONIBs in aqueous solution, as suggested by force-field calculations.
We thank Prof. Dr. Bernhard Jaun and Dr. Marc-Olivier Ebert for helpful discussions and the Swiss
National Science Foundation and Syngenta AG, Basel, for financial support.
Experimental Part
General. Solvents were distilled, i.e., THF from Na/benzophenone, CH2Cl2, MeOH, DMF, pyridine,
iPr2NH, and EtNiPr2 from CaH2. Reactions were run under Ar or N2. Qual. TLC: precoated silica-gel
plates (Fluka silica gel 60 F 254). Flash chromatography (FC): silica gel Merck 60 (0.04 – 0.063 mm).
HPLC-MS (Waters Atlantis dC18-3, 100 ꢂ 3 mm; MeCN/H2O/HCO2H 10 :90 :0.1 – 95 :5 :0.1; flow rate,
0.2 ml/min; Finnigan LCQ Deca Ion Trap ESI-MS). Optical rotations: 1-dm cell at 258 and 589 nm. FT-
IR Spectra: solid state (ATR). UV Melting-curve experiments: performed by equilibrating a soln. of the
oligonucleotide in the given buffer for 12 h at 48, and heating to 808 at a rate of 0.18/min; melting curves
and thermodynamic parameters were calculated by the ꢂThermal Software Version 02.00ꢃ on a Cary 100
UV spectrometer. 1H- and 13C-NMR spectra: at 300, 400, 500 or 600 MHz and at 75, 100, 125 or 150 MHz,
resp. MS: matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF)
with 0.05m indol-3-acrylic acid (IAA) in THF, or with 0.05m a-cyano-4-hydroxycinnamic acid (CCA) in
MeCN/EtOH/H2O, and HR-MALDI-MS with 0.05m 2,5-dihydroxybenzoic acid (DHB) in THF.
5’-O-[Dimethyl(1,1,2-trimethylpropyl)silyl]-2’,3’-O-isopropylideneguanosine (3). DMF (50 ml,
freshly distilled from CaH2) was transferred via cannula into a 250-ml flat-bottom flask equipped with
mechanical stirring and a drying tube (CaCl2), followed by addition at 268 with 2 [11] (recrystallized from
H2O, finely ground and dried (P2O5); 11.91 g, 36.8 mmol), 1H-imidazole (7.5 g, 110 mmol), and
thexyl(dimethyl)silyl chloride (TDSCl; 7.5 ml, 37.8 mmol, dropwise addn. over 15 min). The mixture was
stirred for 4 h, diluted with CH2Cl2 (300 ml), and washed with H2O (100 ml). The aq. layer was extracted
with CH2Cl2 (2 ꢂ 150 ml). The combined org. layers were washed with brine (3 ꢂ 150 ml) and
evaporated. The residual yellow oil was treated with H2O (100 ml). The precipitate was filtered off by
suction and azeotropically dried with toluene to afford 3 (15.3 g, 91%). White solid. Rf (AcOEt/MeOH/
H2O 7:2 :1) 0.81. M.p. 2548. FT-IR (ATR): 3313w, 3166w, 2956w, 2867w, 1686m, 1630m, 1595m, 1538m,
1487m, 1373m, 1316w, 1251m, 1211m, 1156m, 1073s, 842s, 778s. 1H-NMR (300 MHz, CDCl3): see Table 3;
additionally, 12.06 (br. s, exchanged with D2O, HꢀN(1)); 6.14 (br. s, exchanged with D2O, H2NꢀC(2));
1.61, 1.39 (2s, Me2CO2); 1.59 (sept., J ꢁ 6.9, Me2CH); 0.86 (d, J ¼ 6.8, Me2CHSi); 0.83 (s, Me2CSi); 0.09,
0.08 (2s, Me2Si). 13C-NMR (75 MHz, CDCl3): see Table 4; additionally, 113.81 (s, Me2CO2); 34.05 (d,
Me2CH); 27.22, 25.51 (2q, Me2CO2); 25.29 (s, Me2CSi); 18.51 (q, Me2CSi); 20.31 (q, Me2CH); ꢀ 3.30,
ꢀ 3.43 (2q, SiMe2). HR-ESI-MS: 953.4014 (79, [2M þ Na]þ, C42H70N10NaO10Siþ; calc. 953.4707),
488.2308 (100, [M þ Na]þ, C21H35N5NaO5Siþ; calc. 488.2300).
5’-O-[Dimethyl(1,1,2-trimethylpropyl)silyl]-N2-isobutyryl-2’,3’-O-isopropylideneguanosine (4). Iso-
butyric anhydride (12 ml, 72.3 mmol) and DMAP (120 mg, 1 mmol) were added at 238 to a stirred soln. of
3 (15.3 g, 32.9 mmol) in pyridine (150 ml) in a 250-ml flat-bottom flask, and the mixture was heated to 808
for 13 h. The pale orange soln. was allowed to reach 238, diluted with Et2O (500 ml), and washed with sat.
NaHCO3 soln. (3 ꢂ 100 ml). The combined aq. layers were extracted with Et2O (200 ml). The combined
org. layers were washed with 0.001m HCl (5 ꢂ 150 ml) and H2O (2 ꢂ 200 ml), dried (MgSO4), filtered,
and evaporated. Remaining traces of pyridine were azeotropically removed with toluene. FC (17.6 g,
150 g of silica; AcOEt/cyclohexane 1:1 ! 4 :1) of the orange residue afforded 4 (16.8 g, 95%) as a
colourless glass, which crystallized from Et2O/cyclohexane by slow evaporation of Et2O. White needles.
Rf (MeOH/CH2Cl2 1:9) 0.89. M.p. 134 – 1368. [a]2D5 ¼ ꢀ100.3 (c ¼ 1.0, CHCl3). UV (CHCl3): 286 (12630),
264 (14800). FT-IR (ATR): 3512w, 3416w, 3221w, 3016m, 2963m, 2865w, 1697s, 1605s, 1559m, 1471w,

1250
Helvetica Chimica Acta – Vol. 96 (2013)
Table 3. Selected 1H-NMR Chemical Shifts [ppm] and Coupling Constants [Hz] of the Guanosine
Mononucleosides 3, 4, and 6 – 14 in CDCl3
H-Atom
3
4
6
7a)
8a)
9b)
10a)
11a)
12
13a)
14
HꢀC(8)
7.73
–
–
5.99
5.15
4.90
4.32
3.81
3.75
7.90
–
–
5.92
4.99
4.85
4.32
3.77
3.72
7.84
–
–
5.99
5.24
4.93
4.31
3.37
3.31
7.67
–
–
5.83
5.01
4.56
4.10
2.48
2.41
–
–
4.61
–
–
–
5.52
–
–
5.49
CHaꢀC(8)
CHbꢀC(8)
HꢀC(1’)
HꢀC(2’)
HꢀC(3’)
HꢀC(4’)
HaꢀC(5’)
HbꢀC(5’)
9.83c)
4.79
4.73
6.05
5.34
4.81
3.94
2.52
2.43
4.93
4.87
6.24
5.52
5.11
4.22
3.81
3.75
4.90
4.90
6.11
5.55
5.07
4.01
2.48
2.43
–
4.56
6.27
5.42
5.25
4.05
3.63
3.63
5.50
6.15
5.69
5.15
4.26
3.75
3.70
5.47
6.05
5.59
5.05
3.99
2.51
2.46
6.52
5.18
5.04
4.11
3.82
3.77
d
J(Ha,Hb)
J(1’,2’)
J(2’,3’)
J(3’,4’)
J(4’,5’a)
J(4’,5’b)
J(5’a,5’b)
–
–
–
–
–
14.1
14.0
2.0
6.5
4.1
6.6
14.2
2.9
6.7
4.0
4.4
13.0
2.8
6.6
3.6
5.3
)
12.9
2.1
6.5
3.3
6.4
2.8
6.2
2.8
4.1
4.3
2.7
6.0
3.4
3.5
4.1
2.7
6.6
3.6
7.2
5.1
2.2
6.3
3.1
7.1
6.6
3.1
6.9
4.7
5.5
6.7
1.3
6.3
4.1
6.0
6.0
1.9
6.4
3.4
6.6
8.0
7.9
12.6
5.0
11.2
6.3
11.0
8.2
12.8
d
11.2
11.5
10.6
12.6
10.9
)
12.8
a) Assignments based on a HSQC spectrum. b) In (D6)DMSO. c) CH¼O. d) Not assigned.
Table 4. Selected 13C-NMR Chemical Shifts [ppm] of the Guanosine Mononucleosides 3, 4, and 6 – 14 in
CDCl3
C-Atom
3
4
6
7a)
8a)
9b)
C(2)
C(4)
C(5)
C(6)
153.63
150.85
117.12
158.73
135.81
–
90.20
84.36
81.24
86.92
63.13
148.11c)
148.05c)
121.16
155.78
137.25
–
90.18
84.85
80.23
86.90
63.24
148.46c)
148.26c)
121.82
156.07
138.46
–
90.47
84.48
84.09
86.46
5.70
147.72
147.69
122.01
155.55
137.53
–
90.18
84.51
83.40
85.44
34.94
149.60
150.34
122.64
155.52
143.47
182.39d)
89.08
83.94
81.60
86.76
63.34
147.65
149.11
119.03
155.52
149.80
57.68
89.06
83.86
80.25
86.49
C(8)
CH2ꢀC(8)
C(1’)
C(2’)
C(3’)
C(4’)
C(5’)
62.09
C-Atom
10a)
11a)
12
13a)
14
C(2)
C(4)
C(5)
C(6)
147.46
148.88
119.55
155.63
150.02
57.69
89.37
84.06
83.71
85.73
151.67
153.77
114.77
160.27
152.56
58.38
89.73
83.86
80.45
86.68
62.85
152.40
153.57
114.61
161.08
152.40
63.22
90.31
83.06
81.44
151.37
153.14
114.03
160.11
152.46
57.91
89.56
83.91
83.83
86.51
34.87
152.23
153.25
117.35
161.00
152.23
62.77
90.11
83.82
83.62
86.44
34.80
C(8)
CH2ꢀC(8)
C(1’)
C(2’)
C(3’)
C(4’)
C(5’)
87.55
62.99
34.71
a) Assignments based on a HSQC and a HMBC spectrum. b) In (D6)DMSO. c) Assignments may be
interchanged. d) CHOꢀC(8).

Helvetica Chimica Acta – Vol. 96 (2013)
1251
1403w, 1377w, 1313w, 1221m, 1135m, 1090m, 968w, 837w. 1H-NMR (300 MHz, CDCl3): see Table 3;
additionally, 12.23 (br. s, exchanged with D2O, HꢀN(1)); 10.07 (br. s, exchanged with D2O, HNꢀC(2));
2.77 (sept., J ¼ 6.9, Me2CHC¼O); 1.53 (sept., J ꢁ 6.9, Me2CHCMe2Si); 1.50, 1.28 (2s, Me2CO2); 1.21, 1.19
(2d, J ¼ 6.6, Me2CHC¼O); 0.80 (d, J ¼ 6.9, Me2CHCMe2Si); 0.77, 0.76 (2s, Me2CSi); 0.04, 0.02 (2s, Me2Si).
13C-NMR (75 MHz, CDCl3): see Table 4; additionally, 179.63 (s, NC¼O); 113.92 (s, Me2CO2); 35.97 (d,
Me2CHC¼O); 33.85 (d, Me2CHCMe2Si); 26.99, 25.15 (2q, Me2CO2); 25.15 (s, Me2CSi); 20.17, 20.07 (2q,
Me2CHCMe2Si); 18.95, 18.89 (2q, Me2CHC¼O); 18.31, 18.27 (2q, Me2CSi); ꢀ 3.57, ꢀ 3.71 (2q, SiMe2).
HR-ESI-MS: 537.2930 (29), 536.2893 (100, Mþ, C25H42N5O6Siþ; calc. 536.2899).
N2-Isobutyryl-2’,3’-O-isopropylideneguanosine (5) [13]. A suspension of 2 (24.6 g, 76.1 mmol) in
CH2Cl2 (300 ml) and pyridine (75 ml) was cooled to 08, treated with Me3SiCl (58.4 ml, 457 mmol), stirred
for 6 h at 08, treated dropwise with isobutyryl chloride (8.8 ml, 83.7 mmol), stirred for 10 min at 08 and for
16 h at r.t., treated with MeOH (100 ml), and stirred for 12 h. After evaporation, a soln. of the residue in
CH2Cl2 was washed with H2O (2ꢂ), dried (MgSO4), and evaporated. Filtration through a pad of silica gel
(CH2Cl2/MeOH 95 :5) gave 5 (25.2 g, 84%). Colourless solid.
5’-Deoxy-5’-iodo-N2-isobutyryl-2’,3’-O-isopropylideneguanosine (6). A soln. of 5 (11.8 g, 30 mmol)
in MeCN (7 ml) was treated in portions with (PhO)3PMeI (20.4 g, 45.1 mmol). The mixture was stirred
for 60 min, diluted with MeOH (50 ml), stirred for 30 min, and evaporated. A soln. of the residue in
AcOEt (200 ml) was washed with 10% Na2S2O3 soln., H2O, and brine, dried (MgSO4), and evaporated.
FC (CH2Cl2/MeOH 99 :1 ! 39 :1 ! 9 :1) gave 6 (14.0 g, 93%). Pale-yellow foam. Rf (CH2Cl2/MeOH
19 :1) 0.39. [a]D25 ¼ ꢀ24.2 (c ¼ 1.0, CHCl3). UV (CHCl3): 256 (13940), 284 (10530). IR (ATR): 3143w,
2976w, 2935w, 2874w, 1669s, 1603s, 1556s, 1477m, 1401m, 1374m, 1315w, 1252m, 1208m, 1154s, 1076s,
1
1019m, 969w, 947w, 872m, 856m, 816m, 782m, 750m, 721m. H-NMR (300 MHz, CDCl3): see Table 3;
additionally, 12.17 (br. s, HꢀN(1)); 9.31 (br. s, HNꢀC(2)); 2.71 (sept., J ¼ 6.8, Me2CH); 1.58, 1.36 (2s,
Me2CO2); 1.27, 1.26 (2d, J ¼ 6.8, Me2CH). 13C-NMR (75 MHz, CDCl3): see Table 4; additionally, 179.91
(s, NHC¼O); 114.73 (s, Me2CO2); 36.15 (d, Me2CH); 27.10, 25.28 (2q, Me2CO2); 19.10, 19.05 (2q,
Me2CH). HR-MALDI-MS: 504.0744 (100, [M þ H]þ, C17H23IN5Oþ5 ; calc. 504.0738). Anal. calc. for
C17H22IN5O5 (503.30): C 40.57, H 4.41, N 13.91; found: C 40.86, H 4.52, N 13.94.
N2-Isobutyryl-2’,3’-O-isopropylidene-5’-S-[(4-methoxyphenyl)diphenylmethyl]-5’-thioguanosine (7).
A suspension of 60% NaH in mineral oil (96 mg, 2.4 mmol) in THF (4 ml) was cooled to 08, treated with
a soln. of MMTrSH (735 mg, 2.4 mmol) in THF (4 ml), stirred for 5 min, and treated with 6 (500 mg,
1.0 mmol). The mixture was stirred for 5 min at 08 and for 90 min at r.t., and diluted with sat. NH4Cl soln.
The org. solvents were evaporated, and the aq. phase was extracted with AcOEt (3ꢂ). The combined
org. phases were washed with H2O and brine, dried (MgSO4), and evaporated. FC (CH2Cl2/MeOH 39 :1)
gave 7 (613 mg, 90%). Colourless foam. Rf (CH2Cl2/MeOH 19 :1) 0.48. [a]2D5 ¼ þ9.9 (c ¼ 1.0, CHCl3).
UV (CHCl3): 254 (18690), 282 (12890). IR (ATR): 3152w, 3058w, 2977w, 2934w, 2835w, 1672s, 1604s,
1557s, 1507m, 1484m, 1467m, 1443m, 1413m, 1402m, 1374m, 1304w, 1250m, 1210m, 1181m, 1155m, 1078s,
1033m, 947w, 909w, 874w, 821w, 783w, 758w, 739w. 1H-NMR (400 MHz, CDCl3): see Table 3; additionally,
12.08 (br. s, HꢀN(1)); 8.84 (br. s, HNꢀC(2)); 7.36 – 7.34 (m, 4 arom. H); 7.28 – 7.17 (m, 8 arom. H); 6.79 –
6.75 (m, 2 arom. H); 3.77 (s, MeO); 2.62 (sept., J ¼ 6.8, Me2CH); 1.50, 1.26 (2s, Me2CO2); 1.23, 1.21 (2d,
J ¼ 6.8, Me2CH). 13C-NMR (100 MHz, CDCl3; assignments based on a HSQC and a HMBC spectrum):
see Table 4; additionally, 178.65 (s, NHC¼O); 158.39 (s); 144.69 (2s); 136.39 (s); 130.81, 129.49 – 126.98
(several d); 114.57 (s, Me2CO2); 113.39 (2d); 66.88 (s, Ph2C); 55.40 (q, MeO); 36.60 (d, Me2CH); 27.19,
25.48 (2q, Me2CO2); 19.20, 19.07 (2q, Me2CH). HR-MALDI-MS: 720.2234 (16, [M þ K]þ,
C37H39KN5O6Sþ; calc. 720.2253), 704.2499 (81, [M þ Na]þ, C37H39N5NaO6Sþ; calc. 704.2513), 273.1274
(100, MMTrþ, C20H17Oþ; calc. 273.1274).
5’-O-[Dimethyl(1,1,2-trimethylpropyl)silyl]-8-formyl-N2-isobutyryl-2’,3’-O-isopropylideneguanosine
(8). A cold (08) soln. of hexamethyldisilazane (13.4 ml, 63.5 mmol) in THF (150 ml) was treated with a
soln. of 1.55m BuLi in hexane (246 ml, 381 mmol) during 30 min, cooled to ꢀ 788, and treated dropwise
over 1 h with a soln. of 4 (8.5 g, 15.9 mmol) in THF (80 ml). The slightly yellow soln. was treated with
DMF (31 ml, 400 mmol), allowed to warm to 248, and stirred for 2 h. The mixture was diluted with
AcOEt (750 ml), extracted with 5% aq. NaH2PO4 soln. (2 ꢂ 300 ml) and brine (2 ꢂ 100 ml), and
evaporated. FC (acetone/toluene 1:4) of the residual orange resin afforded 8 (7.15 g, 80%). Colourless
glass. Rf (AcOEt) 0.78. [a]2D5 ¼ þ6.2 (c ¼ 1.0, CHCl3). UV (CHCl3): 335 (18420), 322 (19300), 290 (6040,

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Helvetica Chimica Acta – Vol. 96 (2013)
shoulder), 277 (4040, shoulder). FT-IR (CHCl3): 3411w, 3194w, 2960w, 2868w, 1726m, 1693s, 1604s, 1555s,
1
1467w, 1429m, 1384w, 1312w, 1091m, 949w, 865w. H-NMR (500 MHz, CDCl3; assignments based on a
HSQC spectrum): see Table 3; additionally, 12.29 (br. s, exchanged with D2O, HꢀN(1)); 9.02 (br. s,
exchanged with D2O, HNꢀC(2)); 2.71 (sept., J ¼ 6.9, Me2CHC¼O); 1.57 (sept., J ¼ 6.8, Me2CHCMe2Si);
1.54, 1.32 (2s, Me2CO2); 1.28, 1.25 (2d, J ¼ 6.9, Me2CHC¼O); 0.85, 0.84 (2d, J ¼ 6.8, Me2CHCMe2Si);
0.830, 0.825 (2s, Me2CSi); 0.08, 0.07 (2s, Me2Si). 13C-NMR (125 MHz, CDCl3; assignments based on a
HSQC and a HMBC spectrum): see Table 4; additionally, 179.01 (s, HNC¼O); 115.05 (s, Me2CO2); 36.60
(d, Me2CHC¼O); 34.07 (d, Me2CHCMe2Si); 27.23, 25.45 (2q, Me2CO2); 25.45 (s, Me2CSi); 20.33 (q,
Me2CHCMe2Si); 19.02 (q, Me2CHC¼O); 18.47, 18.45 (2q, Me2CSi); ꢀ 3.29, ꢀ 3.35 (2q, Me2Si). HR-
MALDI-MS (3-HPA): 564.2850 (100, [M þ H]þ, C26H42N5O7Siþ; calc. 564.2848). Anal. calc. for
C26H41N5O7Si (563.72): C 55.40, H 7.33, N 12.42; found: C 55.68, H 7.33, N 12.27.
5’-O-[Dimethyl(1,1,2-trimethylpropyl)silyl]-8-(hydroxymethyl)-N2-isobutyryl-2’,3’-O-isopropylide-
neguanosine (9) [16]. A suspension of NaBH4 (246 mg, 6.5 mmol) in EtOH (7.7 ml) was treated dropwise
with a soln. of 8 (3.5 g, 6.2 mmol) in EtOH (40 ml), stirred for 30 min, and treated with sat. aq. NH4Cl
soln. (30 ml) and CH2Cl2 (150 ml). The layers were separated, and the aq. layer was extracted with
CH2Cl2 (50 ml). The combined org. layers were dried (MgSO4), filtered, evaporated, and dried in high
vacuum to afford 9 (3.53 g, quant.), which was used without further purification for the next step. White
solid. Rf (AcOEt) 0.31. M.p. 1808 (dec.). [a]2D5 ¼ þ15.9 (c ¼ 1.0, CHCl3). UV (CHCl3): 291 (12150), 262
(14160). FT-IR (CHCl3): 3550w, 3419w, 3206w, 2975s, 1698s, 1606s, 1561s, 1427m, 1377m, 1257m, 1155m,
1080s, 1046s, 974w, 950w, 876m, 837m. 1H-NMR (300 MHz, (D6)DMSO: see Table 3; additionally, 12.09
(br. s, exchanged with D2O, HꢀN(1)); 11.37 (br. s, exchanged with D2O, HNꢀC(2)); 5.70 (br. s, exchanged
with D2O, OH); 2.78 (sept., J ¼ 6.9, Me2CHC¼O); 1.52, 1.33 (2s, Me2CO2); 1.47 (sept., J ¼ 6.9,
Me2CHCMe2Si); 1.14 (d, J ¼ 6.6, Me2CHC¼O); 0.76, 0.75 (2d, J ¼ 6.9, Me2CHCMe2Si); 0.71, 0.70 (2s,
Me2CSi); ꢀ 0.08, ꢀ 0.10 (2s, Me2Si). 13C-NMR (75 MHz, CDCl3): see Table 4; additionally, 179.31 (s,
NC¼O); 114.48 (s, Me2CO2); 36.01 (d, Me2CHC¼O); 33.95 (d, Me2CHCMe2Si); 27.11, 25.36 (2q,
Me2CO2); 25.36 (s, Me2CSi); 20.31, 20.24 (2q, Me2CHCMe2Si); 19.14, 18.94 (2q, Me2CHC¼O); 18.42,
18.39 (2q, Me2CSi); ꢀ 3.42 (q, Me2Si). HR-MALDI-MS (3-HPA): 588.2825 (25, [M þ Na]þ,
C26H43N5NaO7Siþ; calc. 588.2824), 566.3004 (100, [M þ H]þ, C26H44N5O7Siþ; calc. 566.3005), 394.2263
(24).
8-(Hydroxymethyl)-N2-isobutyryl-2’,3’-O-isopropylidene-5’-S-[(4-methoxyphenyl)diphenylmethyl]-
5’-thioguanosine (10). A soln. of hexamethyldisilazane (9.04 g, 56 mmol) in THF (100 ml) was cooled to
08, treated dropwise with 1.6m BuLi in hexane (35 ml, 56 mmol), stirred for 30 min, and cooled to ꢀ 708.
The cold soln. was transferred via a Teflon canula to a cold (ꢀ 788) soln. of 7 (7.64 g, 11.2 mmol) in THF
(100 ml), keeping the temp. of the mixture below ꢀ 688. The mixture was stirred for 1 h at ꢀ 788, treated
with DMF (22 ml, 280 mmol), stirred for 10 min, warmed to 08, stirred for 1 h, and poured into a mixture
of 2m HCl (56 ml), ice, and H2O (100 ml). The mixture was extracted with AcOEt (3ꢂ). The combined
org. phases were washed with 0.1m HCl, H2O (2ꢂ) and brine, dried (MgSO4), and evaporated. A soln. of
the residue in EtOH (120 ml) was added dropwise to a suspension of NaBH4 (424 mg, 11.2 mmol) in
EtOH (100 ml). The mixture was stirred for 30 min and poured into a mixture of 0.1m HCl and ice. The
mixture was extracted with AcOEt (3ꢂ). The combined org. phases were washed with H2O (2ꢂ) and
brine, dried (MgSO4), and evaporated. FC (CH2Cl2/MeOH 24 :1) gave 10 (5.90 g, 74%). Colourless
foam. Rf (toluene/acetone 1:1) 0.50. [a]2D5 ¼ þ74.9 (c ¼ 1.0, CHCl3). IR (ATR): 3550w, 3160w, 2970w,
2931w, 1736m, 1710m, 1676s, 1603s, 1556s, 1507m, 1489m, 1425m, 1373s, 1301w, 1248s, 1216m, 1181m,
1155m, 1075s, 1032s, 949w, 907w, 862m, 820w, 790m, 758w, 725s, 699s. 1H-NMR (400 MHz, CDCl3;
assignments based on a HSQC spectrum): see Table 3; additionally, 12.09 (br. s, HꢀN(1)); 9.44 (br. s,
HNꢀC(2)); 7.33 – 7.15 (m, 12 arom. H); 6.78 – 6.74 (m, 2 arom. H); 4.14 (br. s, OH); 3.77 (s, MeO); 2.65
(sept., J ¼ 6.9, Me2CH); 1.48, 1.25 (2s, Me2CO2); 1.23, 1.21 (2d, J ¼ 6.9, Me2CH). 13C-NMR (100 MHz,
CDCl3; assignments based on a HSQC and a HMBC spectrum): see Table 4; additionally, 179.02 (s,
NHC¼O); 158.27 (s); 144.81, 144.74, 136.55 (3s); 130.73 – 126.81 (several d); 114.54 (s, Me2CO2); 113.39
(2d); 66.56 (s, Ph2C); 55.34 (q, MeO); 36.32 (d, Me2CH); 27.20, 25.55 (2q, Me2CO2); 19.11, 19.05 (2q,
Me2CH). HR-MALDI-MS: 750.2374 (24, [M þ K]þ, C38H41KN5O7Sþ; calc. 750.2358), 734.2606 (52,
[M þ Na]þ, C38H41N5NaO7Sþ; calc. 734.2619), 712.2815 (32, [M þ H]þ, C38H42N5O7Sþ; calc. 712.2799),
273.1270 (100, MMTrþ, C20H17Oþ; calc. 273.1274).

Helvetica Chimica Acta – Vol. 96 (2013)
1253
5’-O-[Dimethyl(1,1,2-trimethylpropyl)silyl]-8-(hydroxymethyl)-N2-isobutyryl-2’,3’-O-isopropyli-
dene-O6-[2-(4-nitrophenyl)ethyl]guanosine (11). A soln. of 9 (3.97 g, 7.01 mmol) in dioxane (40 ml) was
treated with 1-(trimethylsilyl)-1H-imidazole (1.35 ml, 9.11 mmol) and stirred for 90 min. The mixture
was treated with PPh3 (3.67 g, 14 mmol), 2-(4-nitrophenyl)ethanol (2.34 g, 14 mmol), and dropwise with
diisopropyl azodicarboxylate (DIAD, 2.8 ml, 14 mmol), stirred for 2 h, diluted with brine/H2O 1:1, and
extracted with AcOEt (3ꢂ). The combined org. phases were washed with H2O and brine, dried
(MgSO4), and evaporated. A soln. of the residue in AcOH/H2O 1:1 (160 ml) was stirred for 1 h and
neutralized carefully with 5m NaOH. The mixture was extracted with AcOEt (3ꢂ). The combined org.
phases were washed with sat. NaHCO3 soln., H2O, and brine, dried (MgSO4), and evaporated. FC
(pentane/AcOEt 2 :3 ! 1:2) gave 11 (4.42 g, 88%). Yellow foam. Rf (pentane/AcOEt 2 :3) 0.30. [a]D25
¼
þ23.4 (c ¼ 1.0, CHCl3). UV (CHCl3): 274 (27890). IR (ATR): 3296w, 2959w, 2871w, 1721w, 1610m,
1591m, 1517s, 1457m, 1423m, 1381m, 1342s, 1248m, 1216m, 1183m, 1155m, 1071s, 974w, 906w, 828s, 794m,
777m, 745m. 1H-NMR (400 MHz, CDCl3; assignments based on a HSQC spectrum): see Table 3;
additionally, 8.18 – 8.14 (m, 2 arom. H); 7.80 (br. s, HNꢀC(2)); 7.51 – 7.48 (m, 2 arom. H); 4.79 (t, J ¼ 6.9,
CH2CH2O); 3.59 (br. t, J ¼ 5.9, OH); 3.31 (t, J ¼ 6.9, CH2CH2O); 3.15 – 3.01 (br. s, Me2CHC¼O); 1.61,
1.39 (2s, Me2CO2); 1.58 (sept., J ¼ 6.9, Me2CH); 1.28, 1.27 (2d, J ¼ 6.9, Me2CHC¼O); 0.83 (d, J ¼ 6.9,
Me2CH); 0.81, 0.80 (2s, Me2CSi); 0.05, 0.04 (2s, Me2Si). 13C-NMR (100 MHz, CDCl3; assignments based
on a HSQC and a HMBC spectrum): see Table 4; additionally, 175.56 (s, NHC¼O); 146.92, 145.58 (2s);
129.96 (2d); 123.77 (2d); 116.50 (s, Me2CO2); 66.83 (t, CH2CH2O); 35.73 (d, Me2CHC¼O); 35.09 (t,
CH2CH2O); 34.03 (d, Me2CH); 27.20, 25.41 (2q, Me2CO2); 25.46 (s, Me2CSi); 20.33, 20.28 (2q, Me2CSi);
19.39, 19.27 (2q, Me2CHC¼O); 18.41, 18.39 (2q, Me2CH); ꢀ 3.47, ꢀ 3.49 (2q, Me2Si). HR-MALDI-MS:
737.3289 (59, [M þ Na]þ, C34H50N6NaO9Siþ; calc. 737.3301), 715.3482 (100, [M þ H]þ, C34H51N6O9Siþ;
calc. 715.3481). Anal. calc. for C34H50N6O9Si (714.89): C 57.12, H 7.05, N 11.76; found: C 56.85, H 7.19, N
11.48.
5’-O-[Dimethyl(1,1,2-trimethylpropyl)silyl]-N2-isobutyryl-2’,3’-O-isopropylidene-8-{[(methylsulfo-
nyl)oxy]methyl}-O6-[2-(4-nitrophenyl)ethyl]guanosine (12). A soln. of 11 (6.31 g, 8.83 mmol) in CH2Cl2
(150 ml) was cooled to 08, treated with EtNiPr2 (1.70 ml, 9.7 mmol) and dropwise with MsCl (0.75 ml,
9.7 mmol), stirred for 30 min, and poured into H2O. The phases were separated, and the aq. phase was
extracted with CH2Cl2 (2ꢂ). The combined org. phases were washed with H2O (2ꢂ) and brine, dried
(MgSO4), and evaporated. FC (pentane/AcOEt 3 :1 ! 1:1) gave 12 (6.08 g, 87%). Yellow foam. Rf
(pentane/AcOEt 1:2) 0.66. [a]2D5 ¼ þ12.6 (c ¼ 1.0, CHCl3). UV (CHCl3): 275 (27360). IR (ATR): 3300w
(br.), 2960w, 2871w, 1721w, 1610m, 1590m, 1518m, 1459m, 1427m, 1342s, 1246m, 1214m, 1174s, 1155m,
1074s, 1040m, 1000w, 946m, 828w, 796m, 776m, 737m. 1H-NMR (300 MHz, CDCl3): see Table 3;
additionally, 8.20 – 8.15 (m, 2 arom. H); 7.73 (br. s, HNꢀC(2)); 7.52 – 7.47 (m, 2 arom. H); 4.80 (t, J ¼ 6.9,
CH2CH2O); 3.32 (t, J ¼ 6.9, CH2CH2O); 3.08 (s, MsO); 2.99 – 2.88 (br. s, Me2CHC¼O); 1.55, 1.41 (2s,
Me2CO2); 1.53 (sept., J ¼ 6.8, Me2CH); 1.29, 1.27 (2d, J ¼ 6.9, Me2CHC¼O); 0.81 (d, J ¼ 6.8, Me2CH);
0.77, 0.76 (2s, Me2CSi); 0.03 (s, Me2Si). 13C-NMR (75 MHz, CDCl3): see Table 4; additionally, 175.04 (s,
NHC¼O); 145.71, 147.50 (2s); 130.06 (2d); 123.90 (2d); 117.41 (s, Me2CO2); 67.14 (t, CH2CH2O); 38.54 (q,
MsO); 36.19 (d, Me2CHC¼O); 35.15 (t, CH2CH2O); 34.18 (d, Me2CH); 27.33, 25.62 (2q, Me2CO2); 25.37
(s, Me2CSi); 20.39, 20.37 (2q, Me2CSi); 19.51, 19.37 (2q, Me2CHC¼O); 18.54, 18.51 (2q, Me2CH); ꢀ 3.38,
ꢀ 3.41 (2q, Me2Si). HR-MALDIMS: 793.3271 (100, [M þ H]þ, C35H53N6O11SSiþ; calc. 793.3257). Anal.
calc. for C35H52N6O11SSi (792.98): C 53.01, H 6.61, N 10.60; found: C 53.28, H 6.58, N 10.55.
8-(Hydroxymethyl)-N2-isobutyryl-2’,3’-O-isopropylidene-5’-S-[(4-methoxyphenyl)diphenylmethyl]-
O6-[2-(4-nitrophenyl)ethyl]-5’-thioguanosine (13). A soln. of 10 (6.18 g, 8.68 mmol) in dioxane (50 ml)
was treated with 1-(trimethylsilyl)-1H-imidazole (1.34 g, 9.55 mmol) and stirred for 2 h. The mixture was
treated with PPh3 (3.42 g, 13 mmol), 2-(4-nitrophenyl)ethanol (2.18 g, 13 mmol), and dropwise with
DIAD (2.63 ml, 13 mmol), stirred for 4 h, diluted with H2O (100 ml), and extracted with AcOEt (3ꢂ).
The combined org. phases were washed with H2O and brine, dried (MgSO4), and evaporated. A soln. of
the residue in MeOH (50 ml) was treated with NH4F (1.60 g, 43.4 mmol) and stirred for 2 h. The mixture
was poured into H2O and extracted with AcOEt (3ꢂ). The combined org. phases were washed with H2O
and brine, dried (MgSO4), and evaporated. FC (pentane/AcOEt 2 :3) gave 13 (5.82 g, 78%). Yellow
foam. Rf (pentane/AcOEt 2 :3) 0.22. [a]D25 ¼ þ71.6 (c ¼ 1.0, CHCl3). UV (CHCl3): 274 (29340). IR
(ATR): 3300w (br.), 3054w, 2975w, 2931w, 1719m, 1607m, 1592m, 1515s, 1508s, 1458m, 1425m, 1381m,

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Helvetica Chimica Acta – Vol. 96 (2013)
1343s, 1299w, 1246s, 1217s, 1180s, 1155m, 1071s, 1033s, 1000m, 906w, 856m, 822w, 793m, 741m, 724m.
1H-NMR (300 MHz, CDCl3; assignments based on a HSQC spectrum): see Table 3; additionally, 8.19 –
8.15 (m, 2 arom. H); 7.68 (br. s, HNꢀC(2)); 7.52 – 7.49 (m, 2 arom. H); 7.30 – 7.11 (m, 12 arom. H); 6.73 –
6.68 (m, 2 arom. H); 4.86 – 4.74 (m, CH2CH2O); 3.76 (s, MeO); 3.33 (t, J ¼ 6.9, CH2CH2O); 2.92 (t, J ¼
6.2, OH); 2.86 (br. sept., J ¼ 6.5, Me2CH); 1.53, 1.34 (2s, Me2CO2); 1.23 (d, J ¼ 6.9, Me2CH). 13C-NMR
(75 MHz, CDCl3; assignments based on a HSQC and a HMBC spectrum): see Table 4; additionally,
174.66 (s, NHC¼O); 157.91 (s); 146.78, 145.57, 144.70, 144.68, 136.44 (5s); 130.60 – 123.73 (several d);
116.60 (s, Me2CO2); 113.06 (2d); 66.98 (t, CH2CH2O); 66.32 (s, Ph2C); 55.30 (q, MeO); 36.18 (d,
Me2CH); 35.21 (t, CH2CH2O); 27.22, 25.58 (2q, Me2CO2); 19.56, 19.50 (2q, Me2CH). HR-MALDI-MS:
883.3100 (100, [M þ Na]þ, C46H48N6NaO9Sþ; calc. 883.3096). Anal. calc. for C46H48N6O9S (860.99): C
64.17, H 5.62, N 9.76; found: C 63.93, H 5.72, N 9.56.
N2-Isobutyryl-2’,3’-O-isopropylidene-8-{[(methylsulfonyl)oxy]methyl}-5’-S-[(4-methoxyphenyl)di-
phenylmethyl]-O6-[2-(4-nitrophenyl)ethyl]-5’-thioguanosine (14). A soln. of 13 (5.25 g, 6.1 mmol) in
CH2Cl2 (40 ml) was cooled to 08, treated with EtNiPr2 (1.17 ml, 6.7 mmol) and dropwise with MsCl
(0.52 ml, 6.7 mmol), stirred for 45 min, and poured on ice. The phases were separated, and the aq. phase
was extracted with CH2Cl2 (2ꢂ). The combined org. phases were washed with H2O and brine, dried
(MgSO4), and evaporated. FC (pentane/AcOEt 1:1 ! 2 :3) gave 14 (4.58 g, 80%). Yellow foam. Rf
(pentane/AcOEt 2 :3) 0.35. [a]2D5 ¼ þ35.1 (c ¼ 1.0, CHCl3). UV (CHCl3): 276 (27030). IR (ATR): 3400 –
3200w (br.), 2974w, 2934w, 1720w, 1606m, 1594m, 1508s, 1458m, 1427m, 1342s, 1300w, 1247m, 1213s,
1174s, 1154s, 1088m, 1073m, 1033m, 1000m, 969m, 947m, 856m, 822m, 793m, 759w, 742m, 699s. 1H-NMR
(300 MHz, CDCl3): see Table 3; additionally, 8.21 – 8.16 (m, 2 arom. H); 7.68 (br. s, HNꢀC(2)); 7.53 – 7.49
(m, 2 arom. H); 7.29 – 7.13 (m, 12 arom. H); 6.74 – 6.69 (m, 2 arom. H); 4.85 – 4.77 (m, CH2CH2O); 3.76 (s,
MeO); 3.34 (t, J ¼ 6.9, CH2CH2O); 3.04 (s, MsO); 2.85 (br. sept., J ¼ 6.7, Me2CH); 1.55, 1.35 (2s,
Me2CO2); 1.24 (d, J ¼ 6.9, Me2CH). 13C-NMR (75 MHz, CDCl3): see Table 4; additionally, 174.75 (s,
NHC¼O); 158.12 (s); 147.03, 145.52, 145.49, 144.85, 136.56 (5s); 130.71 – 126.60 (several d); 123.87 (2d);
114.39 (s, Me2CO2); 113.17 (2d); 67.15 (t, CH2CH2O); 66.31 (s, Ph2C); 55.28 (q, MeO); 38.40 (q, MsO);
36.16 (d, Me2CH); 35.11 (t, CH2CH2O); 27.10, 25.50 (2q, Me2CO2); 19.43, 19.36 (2q, Me2CH). HR-
MALDI-MS: 961.2873 (100, [M þ Na]þ, C47H50N6NaO11S2; calc. 961.2871). Anal. calc. for C47H50N6O11S2
(939.06): C 60.11, H 5.37, N 8.95; found: C 59.84, H 5.61, N 9.04.
5’-O-[Dimethyl(1,1,2-trimethylpropyl)silyl]-N2-isobutyryl-2’,3’-O-isopropylidene-O6-[2-(4-nitrophe-
nyl)ethyl]guanosine-8-methyl-(81 ! 5’-S)-N4-benzoyl-2’,3’-O-isopropylidene-6-{[(4-methoxyphenyl)di-
phenylmethoxy]methyl}-5’-thiocytidine (16). A soln. of 15 [8] (3.78 g, 5.05 mmol) in THF/MeOH 1:1
(60 ml) was cooled to ꢀ 108, treated dropwise with a 1m MeSNa soln. in MeOH (10.1 ml), stirred for 2 h
at ꢀ 108, and poured into 0.1m HCl (50 ml). After adding brine, the aq. phase was extracted with AcOEt
(3ꢂ). The combined org. phases were washed with H2O and brine, dried (MgSO4), and evaporated. A
soln. of the residue and 12 (4.00 g, 5.05 mmol) in DMSO (15 ml) was degassed, treated with EtNiPr2
(2.64 ml, 15.2 mmol), and stirred for 6 h. Sat. NH4Cl soln. was added, and the aq. phase was extracted
with AcOEt (3ꢂ). The combined org. phases were washed with H2O (3ꢂ) and brine, dried (MgSO4),
and evaporated. FC (toluene/acetone 5 :1 ! 4 :1) gave 16 (5.01 g, 71%). Yellow foam. Rf (toluene/
acetone 4 :1) 0.14. [a]2D5 ¼ ꢀ46.7 (c ¼ 1.0, CHCl3). UV (CHCl3): 267 (47610). IR (ATR): 3500 – 3200w
(br.), 2959w, 2936w, 2870w, 1679m, 1610s, 1587m, 1570m, 1517m, 1511m, 1457m, 1422m, 1372m, 1344s,
1300m, 1249s, 1212s, 1181m, 1154m, 1063s, 1033m, 1000m, 900w, 871w, 829s, 794m, 777m, 745m, 726w.
1H-NMR (500 MHz, CDCl3; assignments based on a HSQC spectrum): see Table 5; additionally, 9.00 (br.
s, BzNH); 8.14 – 8.11 (m, 2 arom. H); 7.93 (br. d, J ¼ 7.2, 2 arom. H); 7.71 (br. s, NHC¼O/II); 7.62 – 7.23
(m, 17 arom. H, HꢀC(5/I)); 6.87 – 6.84 (m, 2 arom. H); 4.74 (t, J ¼ 7.0, CH2CH2O); 3.77 (s, MeO); 3.25 (t,
J ¼ 7.0, CH2CH2O); 3.01 – 2.89 (br. s, Me2CHC¼O); 1.60, 1.42 (6 H), 1.27 (3s, 2 Me2CO2); 1.51 (sept.,
J ¼ 6.9, Me2CHCMe2); 1.26 (d, J ¼ 6.9, Me2CHC¼O); 0.79, 0.78 (2d, J ¼ 6.9, Me2CHCMe2); 0.74, 0.73
(2s, Me2CSi); ꢀ 0.08, ꢀ 0.09 (2s, Me2Si). 13C-NMR (125 MHz, CDCl3; assignments based on a HSQC
and a HMBC spectrum): see Table 6; additionally, 174.96 (s, NHC¼O/II); 166.15 (s, NHC¼O/I); 159.05
(s); 146.89, 145.55, 143.41, 143.31, 134.34 (5s); 133.11 (d and s); 130.56 – 127.36 (several d); 123.74 (2d);
113.83, 113.46 (2s, 2 Me2CO2); 113.46 (2d); 88.52 (s, Ph2C); 66.83 (t, CH2CH2O); 55.23 (q, MeO); 35.91
(d, Me2CHC¼O); 35.10 (t, CH2CH2O); 34.07 (d, Me2CHCMe2); 27.23, 27.11, 25.53, 25.34 (4q, 2 Me2CO2);
25.22 (s, Me2CSi); 20.29, 20.26 (2q, Me2CSi); 19.42, 19.29 (2q, Me2CHC¼O); 18.44, 18.39 (2q,

Helvetica Chimica Acta – Vol. 96 (2013)
1255
Table 5. Selected 1H-NMR Chemical Shifts [ppm] and Coupling Constants [Hz] of the Di- and
a
Tetranucleosides 16 – 18 and 20 – 23 in CDCl3
)
H-Atom
16
17
18 20 21
22
23
Adenosine unit
HꢀC(2)
8.78
5.67
5.64
6.28
5.50
5.18
4.48
3.00
2.86
8.79
4.60
4.55
6.27
5.55
5.12
4.34 – 4.30
2.97 – 2.89
2.97 – 2.89
8.81
5.60
5.60
6.28
5.66
5.12
4.42
2.914
2.87
8.67
4.14
4.05
6.22
5.74
5.17
4.40 – 4.34
2.85 – 2.81
2.85 – 2.81
CHaꢀC(8)
CHbꢀC(8)
HꢀC(1’)
HꢀC(2’)
HꢀC(3’)
HꢀC(4’)
HaꢀC(5’)
HbꢀC(5’)
J(Ha,Hb)
J(1’,2’)
b
13.1
12.1
)
15.3
1.8
6.4
4.3
8.3
4.3
2.0
6.2
4.7
2.3
6.4
4.0
5.8
7.4
1.6
6.3
3.7
J(2’,3’)
J(3’,4’)
J(4’,5’a)
J(4’,5’b)
J(5’a,5’b)
b
b
)
)
)
)
)
)
b
b
b
b
14.8
14.3
Uridine unit
HꢀC(5)
CHaꢀC(6)
CHbꢀC(6)
HꢀC(1’)
HꢀC(2’)
HꢀC(3’)
HꢀC(4’)
HaꢀC(5’)
HbꢀC(5’)
J(Ha,Hb)
J(1’,2’)
J(2’,3’)
J(3’,4’)
J(4’,5’a)
J(4’,5’b)
J(5’a,5’b)
5.03
5.26c)
3.58
3.47
5.77
5.21
4.93
4.20
2.97 – 2.89
2.97 – 2.89
14.6
5.38c)
5.35
3.61
3.45
5.74
5.24
4.78
4.05
3.13
3.13
14.9
< 1.0
6.4
3.9
7.2
3.56c)
3.56c)
5.79
3.53c)
3.53c)
5.78
5.20
4.92
4.26
2.97
5.19
4.82
4.11
3.22 – 3.15
3.22 – 3.15
2.86
b
b
)
)
< 1.0
6.4
< 1.0
6.4
4.2
0.9
6.4
4.3
8.0
5.8
14.4
4.0
b
6.5
)
)
)
b
7.2
6.5
b
b
b
)
)
Cytidine unit
d
d
d
d
HꢀC(5)
CHaꢀC(6)
CHbꢀC(6)
HꢀC(1’)
HꢀC(2’)
HꢀC(3’)
HꢀC(4’)
HaꢀC(5’)
HbꢀC(5’)
J(Ha,Hb)
J(1’,2’)
)
7.65c)
)
)
7.42c)
3.75
3.69
5.92
5.30
5.06 – 5.03
4.30
2.96
2.918
)
4.19c)
4.19c)
5.88
5.24
4.82
4.19
2.93
2.89
5.26
5.19
5.71
5.28
5.01
4.31
2.96
2.94
4.19c)
4.19c)
5.89
5.27
4.98
3.72
3.65
5.90
5.29
5.04
4.29
2.97 – 2.89
2.97 – 2.89
14.6
4.75
4.63
5.68
5.18
4.94
4.40 – 4.34
3.17
2.85 – 2.81
14.5
4.23
2.95
2.92
b
b
)
13.4
)
14.5
< 1.0
6.3
0.7
6.4
4.0
1.0
6.5
3.7
< 1.0
6.1
3.9
< 1.0
6.3
3.7
< 1.0
6.0
4.2
J(2’,3’)
J(3’,4’)
3.7

1256
Helvetica Chimica Acta – Vol. 96 (2013)
Table 5 (cont.)
H-Atom
16
17
7.1
7.6
13.5
18
6.9
7.6
13.4
20
21
6.3
6.3
22
8.4
6.5
13.4
23
J(4’,5’a)
J(4’,5’b)
J(5’a,5’b)
6.9
7.4
13.3
9.7
)
b
b
)
13.9
Guanosine unit
CHaꢀC(8)
CHbꢀC(8)
HꢀC(1’)
4.03
3.97
6.27
5.87
5.20
4.22
3.61
3.59
4.08
4.03
4.08
3.97
6.28
5.87
4.08
4.07c)
3.99
6.30
5.89
5.20
4.23
3.63
3.60
14.3
2.1
6.3
3.95
6.20
5.67
5.11
4.00
2.42
2.39
14.3
1.6
6.3
3.98
6.24
5.87
5.18
4.22
3.62
3.60
14.4
2.0
6.3
4.07c)
6.43
HꢀC(2’)
5.58
HꢀC(3’)
5.18
5.36 – 5.34
4.40 – 4.34
2.88
HꢀC(4’)
4.23 – 4.21
3.63 – 3.61
3.60 – 3.57
HaꢀC(5’)
HbꢀC(5’)
2.74
b
J(Ha,Hb)
J(1’,2’)
14.3
14.3
)
2.0
6.2
3.4
5.9
6.4
2.0
6.3
3.4
< 1
J(2’,3’)
J(3’,4’)
5.9
b
3.5
5.8
6.4
10.9
3.1
6.9
8.0
12.8
3.6
5.9
6.4
10.9
)
b
J(4’,5’a)
J(4’,5’b)
J(5’a,5’b)
)
)
)
6.3
6.3
13.9
b
b
10.9
c
a) Assignments based on a HSQC and a HMBC spectrum. b) Not assigned. ) Broad signals. d) Hidden
by aromatic signals.
Me2CHCMe2); ꢀ 3.53 (q, Me2Si). HR-MALDI-MS: 1427.5812 (14), 1426.5776 (47), 1425.5756 (92),
1424.5731 (100, [M þ Na]þ, C74H87N9NaO15SSiþ; calc. 1424.5704), 1404.6024 (10), 1403.5985 (27),
1402.5953 (31, [M þ H]þ, C74H88N9O15SSiþ; calc. 1402.5884), 886.3877 (27), 885.3844 (55, [M ꢀ
C32H26N3O4]þ, C42H61N6O11SSiþ; calc. 885.3883).
5’-O-[Dimethyl(1,1,2-trimethylpropyl)silyl]-N2-isobutyryl-2’,3’-O-isopropylidene-O6-[2-(4-nitrophe-
nyl)ethyl]guanosine-8-methyl-(81 ! 5’-S)-N4-benzoyl-2’,3’-O-isopropylidene-6-(methanesulfonyloxy-
methyl)-5’-thiocytidine (17). A soln. of 16 (625 mg, 446 mmol) in CH2Cl2 (3 ml) was treated with
Cl2CHCO2H (300 ml) and iPr3SiH (274 ml, 1.34 mmol), stirred for 1 h, and poured into sat. NaHCO3 soln.
The phases were separated, and the aq. phase was extracted with CH2Cl2 (2ꢂ). The combined org. phases
were washed with H2O and brine, dried (MgSO4), and evaporated. A soln. of the residue in CH2Cl2
(5 ml) was cooled to 08, treated with EtNiPr2 (81 ml, 491 mmol) and MsCl (38 ml, 491 mmol), stirred for
30 min, and poured into ice/water. After extraction with AcOEt (3ꢂ), the combined org. phases were
washed with H2O and brine, dried (MgSO4), and evaporated. FC (AcOEt/pentane 5 :2 ! 1:0) gave 17
(450 mg, 84%). Yellow foam. Rf (AcOEt/pentane 2 :1) 0.34. [a]2D5 ¼ ꢀ29.7 (c ¼ 1.0, CHCl3). UV
(CHCl3): 270 (52180). IR (ATR): 3400 – 3150w (br.), 2958w, 2936w, 2865w, 1682m, 1612m, 1585m,
1570m, 1519m, 1460m, 1422m, 1370m, 1343s, 1237m, 1212m, 1177m, 1155m, 1067s, 1012m, 1000m, 970m,
948m, 900w, 869w, 829m, 792m, 779m, 735w, 699m. 1H-NMR (500 MHz, CDCl3; assignments based on a
HSQC spectrum): see Table 5; additionally, 8.79 (br. s, BzNH); 8.16 – 8.13 (m, 2 arom. H); 7.90 (br. d, J ¼
7.6, 2 arom. H); 7.73 (br. s, NHC¼O/II); 7.64 – 7.61 (m, 1 arom. H); 7.53 – 7.46 (m, 4 arom. H); 4.77 (t, J ¼
6.9, CH2CH2O); 3.29 (t, J ¼ 6.9, CH2CH2O); 3.20 (s, MsO); 2.98 – 2.90 (br. s, Me2CHC¼O); 1.60, 1.50,
1.42, 1.32 (4s, 2 Me2CO2); 1.51 (sept., J ¼ 6.9, Me2CHCMe2); 1.27, 1.26 (2d, J ¼ 6.9, Me2CHC¼O); 0.78
(d, J ¼ 6.9, Me2CHCMe2); 0.74, 0.73 (2s, Me2CSi); ꢀ 0.07, ꢀ 0.08 (2s, Me2Si). 13C-NMR (125 MHz,
CDCl3; assignments based on a HSQC and a HMBC spectrum): see Table 6; additionally, 174.92 (s,
NHC¼O/II); 166.09 (s, NHC¼O/I); 146.88, 145.59 (2s); 133.52 (d); 132.55 (s); 130.01 – 127.71 (several d);
123.78 (2d); 113.85, 113.84 (2s, 2 Me2CO2); 66.84 (t, CH2CH2O); 38.63 (q, MsO); 35.98 (d, Me2CHC¼O);

Helvetica Chimica Acta – Vol. 96 (2013)
1257
Table 6. Selected 13C-NMR Chemical Shifts [ppm] of the Di- and Tetranucleosides 16 – 18 and 20 – 23 in
a
CDCl3
)
C-Atom
16
17
18
20
21
22
23
Adenosine unit
C(2)
C(4)
C(5)
C(6)
152.68
152.58
124.15
151.37
146.77
62.59
90.07
84.10
84.52
90.29
33.05
152.26
152.26
123.06
149.84
151.45
59.64
89.72
83.71
84.09
88.49b)
33.07
153.38
152.26
122.73
150.47
146.78
62.24
90.05
83.46
83.86
87.73
152.35
152.47
122.31
149.53
152.86
28.23
89.93
83.43
83.86
87.73
C(8)
CH2ꢀC(8)
C(1’)
C(2’)
C(3’)
C(4’)
C(5’)
33.51
32.56
Uridine unit
C(2)
C(4)
C(5)
C(6)
151.16
162.65
104.04
151.64
32.66
91.71
151.06c)
161.64
104.11
150.97
32.80
150.94c)
161.49
104.16
150.91c)
33.11
150.55
162.57
104.17
151.08
32.78
91.68
CH2ꢀC(6)
C(1’)
91.65
91.71
C(2’)
C(3’)
C(4’)
C(5’)
84.98
84.42
88.29
31.53
84.97
83.62
89.68
33.57
84.92
83.62
89.95
33.61
84.97
84.28
88.20
31.33
Cytidine unit
C(2)b)
C(4)
155.43
162.56
97.51
157.38
62.84
93.05
84.54
84.72
88.24
33.81
154.82
162.46
98.31
152.12
64.26
93.01
84.53
84.71
88.92
33.57
155.14
162.39
)
157.47
62.83
93.12
84.70
84.86
88.53
34.83
155.27
161.83
97.85
155.23
161.10
97.98
155.09
162.31
95.21
160.65
60.10
91.68
84.58
84.53
91.51
34.32
d
C(5)b)
C(6)
157.93b)
34.15
158.05b)
34.19
CH2ꢀC(6)
C(1’)
92.56
84.73
84.84
88.92
92.67
84.74
84.89
88.92
C(2’)
C(3’)
C(4’)
C(5’)
33.50
33.29
Guanosine unit
C(2)
C(4)
C(5)
C(6)
151.24
153.73
117.04
160.00
150.67
28.33
89.94
82.86
81.67
151.26
153.75
117.01
159.97
150.48
28.29
89.91
82.87
81.57
151.12
153.52
117.06
159.97
150.36
28.29
89.76
83.69
83.92
86.57
33.75
151.23c)
153.74
117.00
159.95
150.61
28.19
89.89
82.86
81.60
87.71
151.27
153.79
117.03
159.98
150.65
28.19
89.95
82.88
81.66
151.41
153.16
116.90
159.78
150.57
29.04
89.75
83.92
83.86
88.04
34.55
C(8)
CH2ꢀC(8)
C(1’)
C(2’)
C(3’)
C(4’)
C(5’)
87.79
63.17
87.76
63.12
87.73
63.17
63.13
a) Assignments based on a HSQC and a HMBC spectrum. b) Broad signals. c) Assignments may be
interchanged. d) Not assigned.

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Helvetica Chimica Acta – Vol. 96 (2013)
35.10 (t, CH2CH2O); 34.05 (d, Me2CHCMe2); 27.21, 27.02, 25.50, 25.26 (4q, 2 Me2CO2); 25.22 (s, Me2CSi);
20.28, 20.25 (2q, Me2CSi); 19.40, 19.30 (2q, Me2CHC¼O); 18.43, 18.38 (2q, Me2CHCMe2); ꢀ 3.53, ꢀ 3.54
(2q, Me2Si). HR-MALDI-MS: 1247.4035 (28), 1246.4010 (42, [M þ K]þ, C55H73KN9O16S2Siþ; calc.
1246.4018), 1232.4312 (34), 1231.4324 (60), 1230.4301 (84, [M þ Na]þ, C55H73N9NaO16S2Siþ; calc.
1230.4278), 887.3911 (23), 886.3910 (55), 885.3871 (100, [M ꢀ C13H12N3O5S]þ, C42H61N6O11SSiþ; calc.
885.3883). Anal. calc. for C55H73N9O16S2Si (1208.45): C 54.67, H 6.09, N 10.43; found: C 54.94, H 6.05, N
10.30.
N2-Isobutyryl-2’,3’-O-isopropylidene-5’-S-[(4-methoxyphenyl)diphenylmethyl]-O6-[2-(4-nitrophenyl)-
ethyl]-5’-thioguanosine-8-methyl-(81 ! 5’-S)-N4-benzoyl-2’,3’-O-isopropylidene-6-{[(4-methoxyphenyl)-
diphenylmethoxy]methyl}-5’-thiocytidine (18). A soln. of 15 (550 mg, 735 mmol) in THF/MeOH 1:1
(8 ml) was cooled to ꢀ 108 and treated dropwise with a 1m soln. of MeSNa in MeOH (1.47 ml), stirred for
3 h at ꢀ 108, and poured on 0.1m HCl (15 ml). Brine was added, and the aq. phase was extracted with
AcOEt (3ꢂ). The combined org. phases were washed with H2O and brine, dried (MgSO4), and
evaporated. A soln. of the residue and 14 (690 mg, 735 mmol) in DMSO (1.5 ml) was degassed, treated
with EtNiPr2 (385 ml, 2.21 mmol), and stirred for 4 h. After addition of sat. NH4Cl soln., the aq. phase was
extracted with AcOEt (3ꢂ). The combined org. phases were washed with H2O (3ꢂ) and brine, dried
(MgSO4), and evaporated. FC (toluene/acetone 5 :1 ! 4 :1) gave 18 (745 mg, 65%). Yellow foam. Rf
(toluene/acetone 4 :1) 0.18. [a]2D5 ¼ ꢀ18.5 (c ¼ 0.5, CHCl3). UV (CHCl3): 266 (49030). IR (ATR): 3500 –
3200w (br.), 3054w, 2958w, 2930w, 2852w, 1693m, 1676m, 1608s, 1508s, 1484m, 1457m, 1447m, 1422m,
1372m, 1344s, 1300m, 1249s, 1213s, 1180s, 1155m, 1086s, 1063s, 1033s, 1000m, 900w, 869w, 855w, 827m,
1
793m, 743m, 724m, 698s. H-NMR (500 MHz, CDCl3; assignments based on a HSQC spectrum): see
Table 5; additionally, 8.60 (br. s, BzNH); 8.14 – 8.11 (m, 2 arom. H); 7.89 (br. d, J ¼ 6.5, 2 arom. H); 7.62
(br. s, NHC¼O/II); 7.62 – 7.59 (m, 2 arom. H); 7.52 – 7.45 (m, 9 arom. H, HꢀC(5/I)); 7.38 – 7.30 (m, 7 arom.
H); 7.26 – 7.24 (m, 2 arom. H); 7.17 – 7.08 (m, 9 arom. H); 6.87 – 6.83 (m, 2 arom. H); 6.70 – 6.67 (m, 2
arom. H); 4.78 (t, J ¼ 7.0, CH2CH2O); 3.76, 3.74 (2s, 2 MeO); 3.28 (t, J ¼ 7.0, CH2CH2O); 2.89 – 2.80 (br.
s, Me2CH); 1.54, 1.43, 1.35, 1.28 (4s, 2 Me2CO2); 1.22, 1.21 (2d, J ¼ 6.9, Me2CH). 13C-NMR (125 MHz,
CDCl3; assignments based on a HSQC and a HMBC spectrum): see Table 6; additionally, 174.73 (s,
NHC¼O/II); 159.06, 158.01 (2s); 146.92, 145.58, 144.83, 144.81, 143.41, 143.33, 136.59, 134.32 (8s); 133.18
(d); 132.16 (s); 130.63 – 126.52 (several d); 123.78 (2d); 113.86, 113.43 (2s, 2 Me2CO2); 113.46 (2d); 113.07
(2d); 88.56 (s, Ph2C/I); 66.92 (t, CH2CH2O); 66.20 (s, Ph2C/II); 55.23, 55.19 (2q, 2 MeO); 35.92 (d,
Me2CH); 35.15 (t, CH2CH2O); 27.10 (2 C), 25.48, 25.31 (3q, 2 Me2CO2); 19.36, 19.32 (2q, Me2CH);
NHC¼O/I and C(5/I) not assigned due to broadening of the signals. HR-MALDI-MS: 1573.5557 (26),
1572.5548 (61), 1571.5542 (98), 1570.5533 (100, [M þ Na]þ, C86H85N9NaO15S2þ ; calc. 1570.5499).
5’-S-Acetyl-2’,3’-O-isopropylidene-5’-thiouridine-6-methyl-(61 ! 5’-S)-N6-benzoyl-2’,3’-O-isopropyli-
dene-8-{[(methylsulfonyl)oxy]methyl}-5’-thioadenosine (20). A soln. of 19 [6] (1.10 g, 1 mmol) in CH2Cl2
i
(6 ml) was treated with Cl2CHCO2H (0.6 ml) and Pr3SiH (0.62 ml, 3 mmol), stirred for 90 min, and
poured into ice and sat. NaHCO3 soln. The mixture was extracted with CH2Cl2 (3ꢂ). The combined org.
phases were washed with H2O and brine, dried (MgSO4), and evaporated. A soln. of the residue in
CH2Cl2 (6 ml) at 08 was treated with EtNiPr2 (0.19 ml, 1.1 mmol) and dropwise with MsCl (86 ml,
1.1 mmol), stirred for 30 min, and poured into ice/water. The mixture was extracted with CH2Cl2 (3ꢂ).
The combined org. phases were washed with H2O and brine, dried (MgSO4), and evaporated. FC
(AcOEt/pentane 4 :1 ! AcOEt) gave 20 (617 mg, 69%). Colourless foam. Rf (AcOEt/Et2O 2 :3) 0.20.
[a]2D5 ¼ ꢀ41.1 (c ¼ 1.0, CHCl3). UV (CHCl3): 279 (25440). IR (ATR): 3200w (br.), 2987w, 2936w, 1687s,
1610m, 1579w, 1531w, 1502w, 1487m, 1448m, 1430w, 1371m, 1298w, 1243s, 1211m, 1173s, 1156m, 1083s,
1073s, 954m, 856m, 800m, 766m, 705m, 626w. 1H-NMR (400 MHz, CDCl3; assignments based on a HSQC
spectrum): see Table 5; additionally, 10.99 (br. s, HꢀN(3/II)); 10.05 (br. s, BzNH); 8.07 – 8.05 (m, 2 arom.
H); 7.53 – 7.51 (m, 3 arom. H); 3.06 (s, MsO); 2.28 (s, AcS); 1.63, 1.51, 1.38, 1.32 (4s, 2 Me2C). 13C-NMR
(100 MHz, CDCl3; assignments based on a HSQC and a HMBC spectrum): see Table 6; additionally,
194.84 (s, SC¼O); 165.45 (s, NHC¼O); 132.98 (d); 132.85 (s); 128.88 (2d); 128.56 (2d); 115.21, 113.85 (2s,
2 Me2C); 38.63 (q, MsO); 30.66 (q, MeC¼O); 27.31, 27.26, 25.51, 25.37 (4q, 2 Me2C). HR-MALDI-MS:
þ
928.1746 (46, [M þ K]þ, C37H43KN7O13S3
;
calc. 928.1713), 912.1995 (100, [M þ Na]þ,
C37H43N7NaO13S3þ; calc. 912.1973), 890.2163 (98, [M þ H]þ, C37H44N7O13Sþ3 ; calc. 890.2154).

Helvetica Chimica Acta – Vol. 96 (2013)
1259
5’-O-[Dimethyl(1,1,2-trimethylpropyl)silyl]-N2-isobutyryl-2’,3’-O-isopropylidene-O6-[2-(4-nitrophe-
nyl)ethyl]guanosine-8-methyl-(81 ! 5’-S)-N4-benzoyl-2’,3’-O-isopropylidene-5’-thiocytidine-6-methyl-
(61 ! 5’-S)-2’,3’-O-isopropylidene-5’-thiouridine-6-methyl-(61 ! 5’-S)-N6-benzoyl-2’,3’-O-isopropylidene-
8-{[(4-methoxyphenyl)diphenylmethoxy]methyl}-5’-thioadenosine (21). A soln. of 19 (1.09 g, 1 mmol) in
MeOH/CH2Cl2 9 :1 (20 ml) was treated with powdered K2CO3 (279 mg, 2 mmol), stirred for 35 min, and
poured into sat. NH4Cl soln. The mixture was extracted with AcOEt (3ꢂ). The combined org. phases
were washed with H2O (2ꢂ) and brine, dried (MgSO4), and evaporated. A soln. of the residue and 17
(1.22 g, 1 mmol) in DMSO (6 ml) was degassed, treated with EtNiPr2 (0.53 ml, 3 mmol), stirred for 6 h,
and poured into sat. NH4Cl soln. The mixture was extracted with AcOEt (3ꢂ). The combined org.
phases were washed with H2O (3ꢂ) and brine, dried (MgSO4), and evaporated. FC (toluene/acetone
3 :1 ! 2 :1) gave 21 (1.41 g, 65%). Yellow foam. Rf (toluene/acetone 5 :2) 0.25. [a]2D5 ¼ ꢀ74.0 (c ¼ 0.5,
CHCl3). UV (CHCl3): 272 (65010). IR (ATR): 3200w (br.), 2934w, 2865w, 1692s, 1607w, 1510m, 1448m,
1421m, 1380m, 1344s, 1248s, 1211s, 1180m, 1155m, 1068s, 1000m, 980m, 902w, 867m, 830m, 794w, 779w,
766w, 746w, 730w, 700m. 1H-NMR (500 MHz, CDCl3; assignments based on a DQF-COSY and a HSQC
spectrum): see Table 5; additionally, 10.10 (br. s, HꢀN(3/II)); 9.35, 8.70 (2 br. s, BzNH); 8.11 – 8.09 (m, 2
arom. H); 8.04, 7.89 (2d, J ¼ 7.3, 4 arom. H); 7.76 (br. s, NHC¼O/IV); 7.58 – 7.38 (m, 15 arom. H, HꢀC(5/
III)); 7.32 – 7.21 (m, 7 arom. H); 4.76 – 4.71 (m, CH2CH2O); 3.76 (s, MeO); 3.27 (t, J ¼ 7.1, CH2CH2O);
3.00 – 2.92 (br. s, Me2CHC¼O); 1.52 (sept., J ¼ 6.9, Me2CHCMe2); 1.59, 1.54, 1.53, 1.48, 1.40, 1.36, 1.33,
1.32 (8s, 4 Me2CO2); 1.25 (d, J ¼ 6.9, Me2CHC¼O); 0.79, 0.78 (2d, J ¼ 6.9, Me2CHCMe2); 0.74, 0.73 (2s,
Me2CSi); ꢀ 0.07, ꢀ 0.09 (2s, Me2Si). 13C-NMR (125 MHz, CDCl3; assignments based on a HSQC and a
HMBC spectrum): see Table 6; additionally, 175.02 (s, NHC¼O/IV); 166.37, 164.69 (2s, NHC¼O/I,III);
158.91 (s); 146.83, 145.57, 143.43, 143.35, 134.25 (5s); 133.42 (d and s); 132.67 (d); 132.45 (s); 130.57 –
127.28 (several d); 123.72 (2d); 114.73, 113.92, 113.83, 113.47 (4s, 4 Me2CO2); 113.40 (2d); 88.21 (s,
Ph2C); 66.82 (t, CH2CH2O); 55.24 (q, MeO); 35.85 (d, Me2CHC¼O); 35.09 (t, CH2CH2O); 34.04 (d,
Me2CHCMe2); 27.24, 27.23, 27.15, 27.07, 25.54, 25.44, 25.21 (2 C) (7q, 4 Me2CO2); 25.20 (s, Me2CSi); 20.28,
20.34 (2q, Me2CSi); 19.41, 19.29 (2q, Me2CHC¼O); 18.43, 18.38 (2q, Me2CHCMe2); ꢀ 3.54 (q, Me2Si).
HR-MALDI-MS: 2179.7910 (24), 2178.7855 (51), 2177.7845 (82), 2176.7841 (100), 2175.7851 (71, [M þ
Na]þ, C108H124N16NaO24S3Siþ; calc. 2175.7798), 886.3902 (74), 885.3870 (74, [M ꢀ C66H63N10O13S2]þ,
C42H61N6O11SSiþ; calc. 885.3883). Anal. calc. for C108H124N16O24S3Si (2154.54): C 60.21, H 5.80, N 10.40;
found: C 59.95, H 5.88, N 10.12.
5’-O-[Dimethyl(1,1,2-trimethylpropyl)silyl]-N2-isobutyryl-2’,3’-O-isopropylidene-O6-[2-(4-nitrophe-
nyl)ethyl]guanosine-8-methyl-(81 ! 5’-S)-N4-benzoyl-2’,3’-O-isopropylidene-5’-thiocytidine-6-methyl-
(61 ! 5’-S)-2’,3’-O-isopropylidene-5’-thiouridine-6-methyl-(61 ! 5’-S)-N6-benzoyl-2’,3’-O-isopropylidene-
8-{[(methylsulfonyl)oxy]methyl}-5’-thioadenosine (22). A soln. of 21 (90 mg, 42 mmol) in CH2Cl2 (2 ml)
was treated with Cl2CHCO2H (0.2 ml) and iPr3SiH (28 ml, 126 mmol), stirred for 1 h, and poured into sat.
NaHCO3 soln. The mixture was extracted with CH2Cl2 (3ꢂ). The combined org. phases were washed
with H2O and brine, dried (MgSO4), and evaporated. A soln. of the residue in CH2Cl2 (1.5 ml) at 08 was
treated with EtNiPr2 (8 ml, 46 mmol) and MsCl (3.6 ml, 46 mmol), stirred for 30 min, and poured into H2O.
The mixture was extracted with CH2Cl2 (3ꢂ). The combined org. phases were washed with H2O and
brine, dried (MgSO4), and evaporated. FC (toluene/acetone 2 :1) gave 22 (61 mg, 74%). Pale-yellow
foam. Rf (toluene/acetone 1:1) 0.54. [a]D25 ¼ ꢀ90.7 (c ¼ 0.5, CHCl3). UV (CHCl3): 272 (72450). IR
(ATR): 3200w (br.), 2936w, 2865w, 1693s, 1606s, 1519m, 1469m, 1457m, 1420m, 1370s, 1344s, 1239s, 1211s,
1175m, 1155m, 1071s, 1000m, 979m, 846m, 855m, 829m, 793m, 779m, 702m. 1H-NMR (600 MHz, CDCl3;
assignments based on a DQF-COSY and a HSQC spectrum): see Table 5; additionally, 9.70 (br. s,
HꢀN(3/II)); 9.18, 8.85 (2 br. s, 2 BzNH); 8.12 – 8.10 (m, 2 arom. H); 8.02, 7.92 (2d, J ¼ 7.4, 4 arom. H);
7.76 (br. s, NHC¼O/IV); 7.61 – 7.42 (m, 8 arom. H); 4.78 – 4.72 (m, CH2CH2O); 3.28 – 3.23 (m,
CH2CH2O); 3.14 (s, MsO); 2.98 – 2.93 (br. s, Me2CHC¼O); 1.50 (sept., J ¼ 6.9, Me2CHCMe2); 1.63, 1.60,
1.55, 1.50, 1.41, 1.40, 1.33 (6 H) (7s, 4 Me2CO2); 1.25 (d, J ¼ 6.9, Me2CHC¼O); 0.79 (d, J ¼ 6.9,
Me2CHCMe2); 0.74, 0.73 (2s, Me2CSi); ꢀ 0.07, ꢀ 0.08 (2s, Me2Si). 13C-NMR (150 MHz, CDCl3;
assignments based on a HSQC and a HMBC spectrum): see Table 6; additionally, 175.05 (s, NHC¼O/
IV); 164.66, 161.10 (2s, C(4/III), NHC¼O); 146.88, 145.62 (2s); 133.42, 132.98 (2d); 133.30, 132.55 (2s);
129.97 – 127.86 (several d); 123.74 (2d); 115.21, 114.02, 113.86, 113.50 (4s, 4 Me2CO2); 66.86 (t,
CH2CH2O); 38.46 (q, MsO); 35.87 (d, Me2CHC¼O); 35.12 (t, CH2CH2O); 34.08 (d, Me2CHCMe2); 27.27,

1260
Helvetica Chimica Acta – Vol. 96 (2013)
27.17, 27.14, 27.11, 25.56, 25.38, 25.24 (2 C) (7q, 4 Me2CO2); 25.24 (s, Me2CSi); 20.31, 20.27 (2q, Me2CSi);
19.41, 19.30 (2q, Me2CHC¼O); 18.45, 18.40 (2q, Me2CHCMe2); ꢀ 3.51 (q, Me2Si); signal of C(4/III)
hidden by the noise. HR-ESI-MS: 1963.6585 (26), 1962.6582 (46), 1961.6549 (82), 1960.6581 (100),
1959.6564 (100, [M þ H]þ, C89H111N16O25S4Siþ; calc. 1959.6553).
5’-S-Acetyl-2’,3’-O-isopropylidene-5’-thiouridine-6-methyl-(61 ! 5’-S)-N6-benzoyl-2’,3’-O-isopropyli-
dene-5’-thioadenosine-8-methyl-(81 ! 5’-S)-N2-isobutyryl-2’,3’-O-isopropylidene-O6-[2-(4-nitrophenyl)-
ethyl]-5’-thioguanosine-8-methyl-(81 ! 5’-S)-N4-benzoyl-6-(hydroxymethyl)-2’,3’-O-isopropylidene-5’-
thiocytidine (23). A soln. of 18 (190 mg, 123 mmol) in CH2Cl2 (2 ml) was treated with CF3CO2H (50 ml)
i
and Pr3SiH (151 ml, 738 mmol), stirred for 45 min, and poured into ice and sat. NaHCO3 soln. The
mixture was extracted with AcOEt (3ꢂ). The combined org. phases were washed with H2O (2ꢂ) and
brine, dried (MgSO4), and evaporated. A soln. of the residue and 20 (109 mg, 123 mmol) in DMSO
(300 ml) was degassed, treated with EtNiPr2 (64 ml, 369 mmol), stirred for 3 h, and poured into sat. NH4Cl
soln. The mixture was extracted with AcOEt (3ꢂ). The combined org. phases were washed with H2O
(3ꢂ) and brine, dried (MgSO4), and evaporated. FC (toluene/acetone 2 :1 ! 1:1) gave 23 (148 mg,
67%). Pale-yellow foam. Rf (toluene/acetone 5 :3) 0.27. [a]2D5 ¼ ꢀ6.3 (c ¼ 0.5, CHCl3). UV (CHCl3): 267
(66670). IR (ATR): 3230w (br.), 2984w, 2931w, 1689s, 1607s, 1519m, 1455m, 1421m, 1380s, 1373s, 1343s,
1
1237s, 1211s, 1155m, 1069s, 998m, 981m, 899w, 865m, 793w, 732w, 702m. H-NMR (500 MHz, CDCl3;
assignments based on a DQF-COSY and a HSQC spectrum): see Table 5; additionally, 10.27 (br. s,
HꢀN(3/IV)); 9.55 (br. s, BzNH); 8.83 (br. s, BzNH, HNꢀC(2/II)); 8.11 – 8.09 (m, 2 arom. H); 8.02, 7.87
(2d, J ¼ 7.5, 4 arom. H); 7.61 – 7.38 (m, 8 arom. H, HꢀC(5/I)); 5.60 (br. s, OH); 4.66 (t, J ¼ 6.8,
CH2CH2O); 3.21 (t, J ¼ 6.8, CH2CH2O); 3.03 – 2.94 (br. s, Me2CHC¼O); 2.33 (s, AcS); 1.61, 1.52, 1.51,
1.47, 1.38, 1.33, 1.31, 1.24 (8s, 4 Me2CO2); 1.22, 1.20 (2d, J ¼ 6.9, Me2CHC¼O). 13C-NMR (125 MHz,
CDCl3; assignments based on a HSQC and a HMBC spectrum): see Table 6; additionally, 194.76 (s,
SC¼O); 166.07, 165.83 (2s, 2 NHC¼O); 146.82, 145.55 (2s); 133.70 (2s); 133.28, 132.52 (2d); 129.85 –
127.72 (several d); 123.74 (2d); 114.74, 113.80, 113.76, 113.40 (4s, 4 Me2CO2); 66.70 (t, CH2CH2O);
35.67 (d, Me2CHC¼O); 35.01 (t, CH2CH2O); 30.60 (q, MeC¼O); 27.15 (3 C), 26.94, 25.32, 25.28, 25.20,
25.02 (6q, 4 Me2CO2); 19.47, 19.33 (2q, Me2CHC¼O). HR-MALDI-MS: 1822.5327 (31), 1821.5295 (72),
1820.5280 (100), 1819.5258 (100, [M þ Na]þ, C82H92N16NaO23Sþ4 ; calc. 1819.5296), 983.2866 (40),
982.2833 (84, [M ꢀ C38H40N9O10S]þ, C44H52N7O13Sþ3 ; calc. 982.2780).
5’-O-[Dimethyl(1,1,2-trimethylpropyl)silyl]-N2-isobutyryl-2’,3’-O-isopropylidene-O6-[2-(4-nitrophe-
nyl)ethyl]guanosine-8-methyl-(8 ! 5’-S)-N4-benzoyl-2’,3’-O-isopropylidene-5’-thiocytidine-6-methyl-
(61 ! 5’-S)-2’,3’-O-isopropylidene-5’-thiouridine-6-methyl-(61 ! 5’-S)-N6-benzoyl-2’,3’-O-isopropylidene-
5’-thioadenosine-8-methyl-(81 ! 5’-S)-2’,3’-O-isopropylidene-5’-thiouridine-6-methyl-(61 ! 5’-S)-N6-ben-
zoyl-2’,3’-O-isopropylidene-5’-thioadenosine-8-methyl-(81 ! 5’-S)-N2-isobutyryl-2’,3’-O-isopropylidene-
O6-[2-(4-nitrophenyl)ethyl]-5’-thioguanosine-8-methyl-(81 ! 5’-S)-N4-benzoyl-6-(hydroxymethyl)-2’,3’-
O-isopropylidene-5’-thiocytidine (24). A soln. of 23 (110 mg, 61 mmol) in degassed pyridine/EtOH 2 :1
(600 ml) was cooled to 08, treated dropwise with a cooled aq. 1m NaOH soln. (300 ml), and stirred for
30 min. The mixture was treated with 2m HCl (150 ml), poured onto ice, and extracted with AcOEt (3ꢂ).
The combined org. phases were washed with H2O (2ꢂ) and brine, dried (MgSO4), and evaporated.
Excess pyridine was removed by co-evaporation with toluene. A soln. of the residue and 22 (120 mg,
61 mmol) in DMSO (600 ml) was degassed, treated with EtNiPr2 (32 ml, 183 mmol), stirred for 6 h, and
poured into ice and sat. NH4Cl soln. The mixture was extracted with AcOEt (3ꢂ). The combined org.
phases were washed with H2O (3ꢂ) and brine, dried (MgSO4), and evaporated. FC (diol phase; AcOEt)
gave 24 (135 mg, 61%). Yellow foam. Rf (diol phase; AcOEt) 0.45. [a]2D5 ¼ ꢀ39.2 (c ¼ 0.5, CHCl3). UV
(CHCl3): 268 (89780). IR (ATR): 3400 – 3200w (br.), 2984w, 2937w, 1690s, 1607s, 1519m, 1458m, 1421m,
1
1380s, 1344s, 1240s, 1212s, 1156m, 1071s, 981m, 866m, 833m, 794w, 704w. H-NMR (600 MHz, CDCl3;
assignments based on a DQF-COSYand a HSQC spectrum): see Table 7; additionally, 12.11, 10.22 (2 br.
s, HꢀN(3/IV,VI)); 9.77, 9.64, 9.52, 8.95 (4 br. s, 4 BzNH); 8.66, 7.80 (2 br. s, NHꢀC(2/II,VIII)); 8.12 – 8.07
(m, 4 arom. H); 8.02, 7.97, 7.91, 7.88 (4d, J ¼ 7.5, 8 arom. H); 7.59 – 7.37 (m, 16 arom. H, HꢀC(5/I,VII));
4.76 – 4.73 (m, CH2CH2O); 4.65 – 4.63 (covered, CH2CH2O); 3.25 (t, J ¼ 7.0, CH2CH2O); 3.21 – 3.18 (m,
CH2CH2O); 2.98 – 2.72 (m, 2 HꢀC(5’/II – VII), 2 Me2CHC¼O); 1.51 (sept., J ¼ 6.9, Me2CHCMe2); 1.60,
1.59 (6 H), 1.54, 1.49 (9 H), 1.39, 1.37, 1.32, 1.31 (6 H), 1.26 (12 H) (9s, 8 Me2CO2); 1.26 – 1.25 (m,
2 Me2CHC¼O); 0.78 (d, J ¼ 6.9, Me2CH); 0.74, 0.73 (2s, Me2CSi); ꢀ 0.07, ꢀ 0.08 (2s, Me2Si). 13C-NMR

Helvetica Chimica Acta – Vol. 96 (2013)
1261
1
Table 7. Selected H-NMR Chemical Shifts [ppm] and Coupling Constants [Hz] of the Octanucleosides
24 and 25a)
H-Atom
24 (CDCl3)
25 ((D6)DMSO)
A(III)
A(V)
A(III)
A(V)
5.67 – 5.64
3.79 – 3.72
3.65 – 3.59
5.67 – 5.64
5.18 – 5.14
4.81
4.15 – 3.98
b
b
HꢀC(5)
CHaꢀC(6)
CHbꢀC(6)
HꢀC(1’)
HꢀC(2’)
HꢀC(3’)
HꢀC(4’)
HaꢀC(5’)
HbꢀC(5’)
J(Ha,Hb)
J(1’,2’)
J(2’,3’)
J(3’,4’)
J(4’,5’a)
J(4’,5’b)
J(5’a,5’b)
)
)
5.80
4.37
4.30 – 4.24
5.80
5.18 – 5.14
4.83
4.15 – 3.98
2.90 – 2.65
2.90 – 2.65
14.1
4.72
4.64
5.73
5.20 – 5.16
4.96 – 4.93
4.42 – 4.34
3.15 – 3.10
3.15 – 3.10
13.2
3.76
3.68
5.92
5.30
5.04
4.33 – 4.28
2.98 – 2.72
2.98 – 2.72
2.90 – 2.65
2.90 – 2.65
)
)
c
c
14.6
< 1
< 1
< 1
6.3
c
)
)
)
)
6.6
3.8
)
)
)
6.4
3.7
)
)
)
c
3.5
)
)
)
c
c
c
c
c
c
c
c
c
c
c
c
)
G(II)
G(VIII)
G(II)
G(VIII)
CHaꢀC(8)
CHbꢀC(8)
HꢀC(1’)
HꢀC(2’)
HꢀC(3’)
HꢀC(4’)
HaꢀC(5’)
HbꢀC(5’)
J(Ha,Hb)
J(1’,2’)
4.11
4.06
6.40
4.09
3.97
6.28
3.91
3.85
6.05
5.39
5.00
4.15 – 3.98
2.90 – 2.65
2.90 – 2.65
14.2
3.91
3.83
6.12
5.49
5.11
4.15 – 3.98
3.67
3.62
14.2
1.1
5.81
5.87
5.35 – 5.33
4.42 – 4.34
2.98 – 2.72
2.98 – 2.72
13.2
5.20 – 5.16
4.26 – 4.17
3.62
3.60
14.5
< 1
1.9
1.1
J(2’,3’)
J(3’,4’)
J(4’,5’a)
J(4’,5’b)
J(5’a,5’b)
5.8
)
)
)
6.3
)
5.9
6.4
10.9
6.5
3.8
)
)
6.2
3.9
7.1
6.0
c
c
c
c
c
c
c
c
)
)
11.3
A(III)
A(V)
A(III)
A(V)
HꢀC(2)
CHaꢀC(8)
CHbꢀC(8)
HꢀC(1’)
HꢀC(2’)
HꢀC(3’)
HꢀC(4’)
HaꢀC(5’)
HbꢀC(5’)
J(Ha,Hb)
J(1’,2’)
8.63
4.09
4.04
6.23
5.71
8.70
4.10
4.02
6.25
5.73
5.20 – 5.16
4.42 – 4.34
2.98 – 2.72
2.98 – 2.72
14.7
8.15
8.15
4.15 – 3.98
4.15 – 3.98
6.21
5.67 – 5.64
5.04 – 5.01
4.30 – 4.24
2.90 – 2.65
2.90 – 2.65
)
< 1
)
)
4.15 – 3.98
4.15 – 3.98
6.21
5.67 – 5.64
5.04 – 5.01
4.30 – 4.24
2.90 – 2.65
2.90 – 2.65
)
< 1
)
)
5.12
4.33 – 4.28
2.98 – 2.72
2.98 – 2.72
13.2
1.5
6.5
c
c
< 1
6.5
)
c
c
J(2’,3’)
J(3’,4’)
c
c
c
3.9

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Helvetica Chimica Acta – Vol. 96 (2013)
Table 7 (cont.)
H-Atom
24 (CDCl3)
25 ((D6)DMSO)
A(III)
A(V)
A(III)
A(V)
c
c
c
c
J(4’,5’a)
J(4’,5’b)
J(5’a,5’b)
)
)
)
)
)
)
)
)
)
)
)
)
c
c
c
c
c
c
c
c
U(IV)
U(VI)
U(IV)
5.72d)
3.79 – 3.72
3.65 – 3.59
5.75
U(VI)
5.63d)
3.79 – 3.72
3.65 – 3.59
5.74
HꢀC(5)
CHaꢀC(6)
CHbꢀC(6)
HꢀC(1’)
HꢀC(2’)
HꢀC(3’)
HꢀC(4’)
HaꢀC(5’)
HbꢀC(5’)
J(Ha,Hb)
J(1’,2’)
J(2’,3’)
J(3’,4’)
J(4’,5’a)
J(4’,5’b)
J(5’a,5’b)
5.33
3.54
3.50
5.77
5.35
3.53
3.48
5.73
5.11
5.20 – 5.16
5.18 – 5.14
5.18 – 5.14
4.91
4.96 – 4.93
4.75 – 4.72
4.15 – 3.98
2.90 – 2.65
2.90 – 2.65
)
)
)
)
)
)
)
4.75 – 4.72
4.15 – 3.98
2.90 – 2.65
2.90 – 2.65
)
)
)
)
)
)
)
4.26 – 4.17
2.98 – 2.72
2.98 – 2.72
14.4
< 1
6.4
4.5
)
4.26 – 4.17
2.98 – 2.72
2.98 – 2.72
14.5
< 1
5.1
)
)
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
)
)
)
)
a) Assignment based on a DQF-COSY and a HSQC spectrum. b) Hidden by aromatic signals. c) Not
assigned. d) Broad signal.
(150 MHz, CDCl3; assignments based on a HMBC and a HSQC spectrum): see Table 8; additionally,
175.72, 175.09 (2s, NHC¼O/II,VIII); 165.76, 162.60 (2s, 4 NHC¼O, C(4/I)); 146.87, 146.85, 145.65, 145.57
133.83 (2 C), 133.75 (2 C) (6s); 133.40, 133.23, 132.64, 132.54 (4d); 129.99 – 127.82 (several d); 123.73
(4d); 114.70, 114.60, 114.00, 113.86, 113.81, 113.70, 113.50, 113.46 (8s, 8 Me2CO2); 66.86, 66.81 (2t,
2 CH2CH2O); 35.88 (br. d, 2 Me2CHC¼O); 35.13, 35.02 (2t, 2 CH2CH2O); 34.08 (d, Me2CHCMe2);
27.27 – 27.13, 25.56 – 25.44 (several q, 8 Me2CO2); 25.09 (s, Me2CSi); 20.30, 20.27 (2q, Me2CSi); 19.47,
19.41, 19.33, 19.29 (4q, 2 Me2CHC¼O); 18.45, 18.41 (2q, Me2CHCMe2); ꢀ 3.51 (q, Me2Si). HR-MALDI-
MS: 3644.1560 (61), 3643.1538 (97), 3642.1424 (100), 3641.1466 (89), 3640.1718 (50, [M þ Na]þ,
C
168H196N32NaO44S7Siþ; calc. 3640.1790).
5’-O-[Dimethyl(1,1,2-trimethylpropyl)silyl]-2’,3’-O-isopropylideneguanosine-8-methyl-(81 ! 5’-S)-
2’,3’-O-isopropylidene-5’-thiocytidine-6-methyl-(61 ! 5’-S)-2’,3’-O-isopropylidene-5’-thiouridine-6-meth-
yl-(61 ! 5’-S)-2’,3’-O-isopropylidene-5’-thioadenosine-8-methyl-(81 ! 5’-S)-2’,3’-O-isopropylidene-5’-thio-
uridine-6-methyl-(61 ! 5’-S)-2’,3’-O-isopropylidene-5’-thioadenosine-8-methyl-(81 ! 5’-S)-2’,3’-O-isopro-
pylidene-5’-thioguanosine-8-methyl-(81 ! 5’-S)-6-(hydroxymethyl)-2’,3’-O-isopropylidene-5’-thiocytidine
(25). Octanucleoside 24 (82 mg, 23 mmol) was dissolved in a 0.5m soln. of 1,5,7-triazabicyclo[4.4.0]dec-5-
ene (TBD) in pyridine (1 ml). The mixture was stirred for 30 min, poured into 0.1m aq. HCl, and
extracted with CH2Cl2 (3ꢂ). The combined org. phases were washed with H2O and brine, dried
(MgSO4), and evaporated. A soln. of the residue in CH2Cl2 (0.4 ml) in a pressure tube was treated with
sat. methanolic NH3 (2 ml). The pressure tube was sealed, and the soln. was stirred for 60 h. After
evaporation, a soln. of the residue in CHCl3 was washed with H2O and brine, dried (MgSO4), and
evaporated. FC (NH2 phase; CHCl3/MeOH 4 :1) gave 25 (31 mg, 50%). Colourless solid. Rf (NH2 phase;
CHCl3/MeOH 4 :1) 0.21. [a]2D5 ¼ ꢀ84.6 (c ¼ 0.25, CHCl3). UV (CHCl3): 268 (73110). IR (ATR): 3332w
(br.), 3173w (br.), 2984w, 2925w, 2854w, 1689s, 1641s, 1532w, 1454w, 1434w, 1373s, 1331w, 1300w, 1259w,

Helvetica Chimica Acta – Vol. 96 (2013)
1263
Table 8. Selected 13C-NMR Chemical Shifts [ppm] of the Octanucleosides 24 and 25a)
C-Atom
24 (CDCl3)
25 ((D6)DMSO)
C(I)
C(VII)
C(I)
C(VII)
C(2)
C(4)
C(5)
C(6)
CH2ꢀC(6)
C(1’)
C(2’)
C(3’)
C(4’)
C(5’)
155.06b)
162.60d)
98.14b)e)
160.52
34.15
155.36b)
c)d)
155.64
165.65
93.13
155.94
59.08
155.64
165.05
95.80
152.07
32.11
95.05b)e)
157.94
34.15
91.79f)
84.89
92.64
84.74
84.89
88.90
91.00
90.74
84.72
84.49d)
84.27d)
87.28
84.64
91.27
34.40
84.39d)
87.52
33.62 – 32.98
33.18e)
33.39e)
G(II)
G(VIII)
G(II)
G(VIII)
C(2)
C(4)
C(5)
C(6)
151.28
151.28
153.78
116.94g)
159.84
150.68
28.06h)
89.94
82.88
81.68
87.73
33.62 – 32.98
153.75f)
151.23
115.40g)
156.53h)
143.23
26.75
153.66f)
151.63
115.37g)
156.51h)
143.17
26.75
88.27
83.23
81.13
87.92
153.18
117.01g)
159.84
150.78
28.20h)
C(8)
CH2ꢀC(8)
C(1’)
C(2’)
C(3’)
C(4’)
C(5’)
89.75
88.44
84.00 – 83.87
84.00 – 83.87
88.53b)
83.57
83.50i)
86.26
33.62 – 32.98
33.11e)
63.35
A(III)
A(V)
A(III)
A(V)
C(2)
C(4)
C(5)
C(6)
152.31
152.31
152.23
121.17i)
149.39
152.47b)
149.63
117.75k)
155.46l)
148.04m)
27.47n)
88.82
82.75o)
83.36i)
85.67p)
32.67e)
152.47b)
149.63
117.64k)
155.41l)
147.86m)
27.25n)
88.76
82.63o)
83.36i)
85.37p)
32.52e)
152.50
122.39i)
149.55
152.67
29.07h)
89.78
83.45
84.00 – 83.87
87.45
C(8)
151.70
h
CH2ꢀC(8)
C(1’)
C(2’)
C(3’)
C(4’)
C(5’)
28.20
89.69
83.60
)
84.00 – 83.87
87.84
33.62 – 32.98
33.62 – 32.98
U(IV)
U(VI)
U(IV)
U(VI)
C(2)
C(4)
C(5)
C(6)
CH2ꢀC(6)
C(1’)
C(2’)
C(3’)
C(4’)
C(5’)
150.92
161.97j)
104.06
150.95
32.80k)
91.66
84.53
83.63
89.86
33.62 – 32.98
150.75
150.83
161.98q)
103.54r)
151.13s)
31.55
150.78
161.94q)
103.43r)
151.09s)
31.55
161.65j)
104.27
150.83
32.68k)
91.37f)
90.81t)
84.27d)
83.71
90.61t)
84.22d)
83.71
84.99
84.00 – 83.87
88.79
88.20
32.52e)
87.41
32.46e)
33.62 – 32.98
a) Assignment based on a HSQC and a HMBC spectrum. b) Broad signals. c) Not assigned. d) –
t) Assignments may be interchanged.

1264
Helvetica Chimica Acta – Vol. 96 (2013)
1209m, 1155m, 1063w, 983m, 930w, 865m, 831m, 796w, 781w, 719w, 682w. 1H-NMR (600 MHz,
(D6)DMSO; assignments based on a DQF-COSYand a HSQC spectrum): see Table 7; additionally, 11.4
(br. s, HꢀN(3/IV,VI); 10.8 (br. s, HꢀN(1/II,VIII)); 7.29, 7.27, 7.22, 7.21 (4 br. s, H2NꢀC(4/I,VII), H2NꢀC(6/
III,V)); 6.64, 6.55 (2 br. s, H2NꢀC(2/II,VIII)); 1.51 (sept., J ¼ 6.9, Me2CHCMe2); 1.52, 1.51 (6 H), 1.46,
1.44, 1.43, 1.42, 1.40, 1.32, 1.30 (6 H), 1.26 (6 H), 1.25, 1.23 (6 H) (12s, 8 Me2CO2); 0.78, 0.77 (2d, J ¼ 6.9,
Me2CHCMe2); 0.73, 0.72 (2s, Me2CSi); ꢀ 0.06, ꢀ 0.09 (2s, Me2Si). See also Table 9. 13C-NMR (150 MHz,
(D6)DMSO; assignments based on a DQF-COSY, HSQC and a HMBC spectrum): see Table 8;
additionally, 113.19 (2 C), 112.77, 112.68, 112.58, 112.41, 111.99, 111.96 (7s, 8 Me2CO2); 33.51 (d,
Me2CHCMe2); 26.91 (2 C), 26.90, 26.86, 26.82 (2 C), 26.81, 26.71, 25.26, 25.03, 25.02, 24.94 (3 C), 24.92,
24.82 (12q, 8 Me2CO2); 24.63 (s, Me2CSi); 20.10, 20.01 (2q, Me2CSi); 18.22, 18.14 (2q, Me2CHCMe2);
ꢀ 3.66, ꢀ 3.72 (2q, Me2Si); signals of C(4/I,VII) and C(6/II,VIII) hidden by the noise. HR-MALDI-MS:
2789.9050 (41), 2788.9020 (71), 2787.9023 (97), 2786.9044 (100), 2785.9017 (59, [M þ Na]þ,
C116H154N30NaO34S7Siþ; calc. 2785.8959).
Table 9. 1H- and 13C-NMR Chemical Shifts [ppm] for the CH and CH2 Groups of the Isopropylidenated
and Silylated Octanucleoside 25 in (CDCl2)2 as Obtained from a HSQC Spectrum (assignments based on
DQF-COSY, TOCSY, and NOESY spectra)
G(VIII) C(VII)
U(VI)
A(V)
U(IV)
A(III)
G(II)
C(I)
HꢀC(5)
C(5)
–
–
6.10
89.6
6.36
80.3
4.94
82.6
4.29
86.0
5.49
99.9
5.59
105.9
5.75
93.0
5.28
–
–
7.32
104.8
5.89
89.5
5.41
84.6
4.98
83.8
4.31
92.9
–
–
–
–
5.71
95.6
5.70
92.1a)
5.32
HꢀC(1’)
5.70
91.6a)
4.98
86.0
4.94
82.6
4.46
90.4
6.41
91.0
5.85
82.3
5.16
83.7
4.42
83.8
6.47
90.0
6.10
80.1
4.70
83.9
3.68
84.2
6.66
89.1
6.36
79.5
4.82
83.6
4.55
84.8
C(1’)
HꢀC(2’)
C(2’)
85.4
85.3
HꢀC(3’)
4.91a)
84.9a)
4.28
4.90a)
85.1a)
4.49
C(3’)
HꢀC(4’)
C(4’)
88.0
93.8
2 HꢀC(5’)
3.80/3.59 3.18/2.85
63.9 35.7
4.00/3.50
30.5
3.02/2.71 2.71/2.65
3.02/2.71 2.56/2.51 2.97
3.15/2.91
33.3
C(5’)
35.8a)
3.79
32.1
4.32/3.93
29.4
30.3a)
34.1 34.6
4.42/3.77 4.13/3.83 4.37/4.15 4.52/4.44
33.2 28.6 30.1 60.0
CH2ꢀC(6/8) 3.86
CH2ꢀC(6/8) 29.5
32.6
a) Assignments in rows may be interchanged.
Guanosine-8-methyl-(81 ! 5’-S)-5’-thiocytidine-6-methyl-(61 ! 5’-S)-5’-thiouridine-6-methyl-(61 !
5’-S)-5’-thioadenosine-8-methyl-(81 ! 5’-S)-5’-thiouridine-6-methyl-(61 ! 5’-S)-5’-thioadenosine-8-meth-
yl-(81 ! 5’-S)-5’-thioguanosine-8-methyl-(81 ! 5’-S)-6-(hydroxymethyl)-5’-thiocytidine (26). A soln. of
25 (4 mg, 1.5 mmol) in HCO2H/H2O 4 :1 (0.3 ml) was stirred for 18 h and evaporated at 238. A soln. of the
residue in 25% aq. NH3 (1 ml) was lyophilized. A soln. of the residue in a mixture of H2O and 25% aq.
NH3 was applied on a Bakerbond C18 column (conditioned with H2O). The column was washed with ca.
5 ml H2O. The product was obtained by elution with H2O/MeCN/NH4OH 4 :1:1. Evaporation of the
solvents gave 26 (2 mg, 60%). Colourless powder. Rf (NH2 phase; MeOH/CHCl3/H2O 3 :1:1) 0.65. tR
(HPLC: Waters Atlantis dC18-3, 100 ꢂ 3 mm; MeCN/H2O/HCO2H 10 :90 :0.1 ! 95 :5 :0.1; flow rate,
0.2 ml/min) 20.37 min. HR-ESI-MS: 1163.2620 (17), 1162.7634 (19), 1162.2639 (16, [M þ H þ Na]2þ
,
C84H105N30NaO34S27þ ; calc. 1162.2671), 1153.7745 (12), 1153.2764 (22), 1152.7762 (62), 1152.2748 (92),
1151.7717 (93), 1151.2740 (100, [M þ 2 H]2þ, C84H106N30O34S27þ ; calc. 1151.2761), 1086.7526 (17),
1086.2519 (23), 1085.7535 (31), 1085.2514 (30, [M ꢀ C5H9O4 þ 3 H]2þ, C79H98N30O30S72þ ; calc.
1085.2550).