Oligonucleotide analogues with integrated bases and Backbone. Part 28 1): Hydrazide- and amide-linked analogues. 2. di-, tetra-, octa-, and decamers: Synthesis and association

Article abstract of DOI:10.1002/hlca.201100124

The protected hydrazide-linked uracil- and adenine-derived tetranucleoside analogues 17, 19, and 21 were synthesized in solution by coupling the dimeric hydrazines 6 and 10 with the carboxylic acids 7, 11, and 16. These hydrazines and acids were obtained

Full text of DOI:10.1002/hlca.201100124

Helvetica Chimica Acta – Vol. 94 (2011)
1153
Oligonucleotide Analogues with Integrated Bases and Backbone
Part 281)
Hydrazide- and Amide-Linked Analogues. 2. Di-, Tetra-, Octa-, and Decamers:
Synthesis and Association
by Fabio De Giacomo2), Manuel Peifer2), Zrinka Rajic3), and Andrea Vasella*
Laboratorium fꢀr Organische Chemie, ETH Zꢀrich, Wolfgang-Pauli Strasse 10, CH-8093 Zꢀrich
(e-mail: vasella@org.chem.ethz.ch)
The protected hydrazide-linked uracil- and adenine-derived tetranucleoside analogues 17, 19, and 21
were synthesized in solution by coupling the dimeric hydrazines 6 and 10 with the carboxylic acids 7, 11,
and 16. These hydrazines and acids were obtained by partially deprotecting the hydrazines 5, 9, and 15,
and these were prepared by coupling the hydrazines 3 and 14 with the carboxylic acids 4 and 8. The crystal
structure analysis of the fully protected UU dimer 5 showed the formation of an antiparallel cyclic duplex
with the uracil units H-bonded via HꢀN(3) and O¼C(2). Stacking interactions were observed between
the uracil units with a buckle twist of 30.98, and between the uracil unit II and the fluoren-9-yl group of
Fmoc (¼ 9H-fluoren-9-yl)methoxycarbonyl). The hydrazide HꢀN(3’) and the C¼O group of Fmoc form
an intramolecular H-bond. The uracil- and adenine-derived, water-soluble hydrazide-linked self-
complementary octamers 23 – 32 and the non-self-complementary uracil derived decamer 33 were
obtained by coupling the carboxylic acids 4 and 8 on a solid support. 1H-NMR Analysis in CDCl3,
mixtures of CDCl3 and (D6)DMSO, and (D8)THF showed that the partially deprotected dimers 5, 6, 12,
and 13 form weakly associated linear duplexes. The partially deprotected tetramers 17 and 18 do not
associate. The hydrazide-linked octamers 23 – 32 do not stack in aqueous solution, and the non-self-
complementary decamer 33 does not stack with the complementary strands of DNA 43 and RNA 42. The
Cbz-protected amide-linked octamers 51 – 56 derived from uracil, adenine, cytosine, and guanine were
obtained as the main products by solid-phase synthesis from the carboxylic acids 46 – 49. The fully
deprotected amide-linked octamers proved insoluble, and could neither be purified nor analysed.
Introduction. – We already detailed the reasons for our interest in novel
oligonucleotide analogues with integrated bases and backbone (ONIBs) [2]. Briefly
put, we wished to modify the structure of previously synthesized ONIBs [3 – 12] in
omitting the ribosyl moiety and choosing an advantageous linker to simplify the
synthesis. We thereby intended to further explore the limits within which the structure
of ONIBs can be varied while maintaining their pairing properties, and to obtain
ONIBs that pair in aqueous solution. To reach these goals, we designed ONIBs
characterized by hydrazide and by amide linkers, as illustrated by the general structures
1 and 2, respectively.
We planned to prepare di- and tetramers in solution and higher oligomers on a solid
support, to combine the advantages of each method [13][14]. Similarly to the synthesis
1
)
)
)
Part 27 [1].
2
3
Taken in part from the Ph.D. thesis of M. P. and F. D. G.
Doctoral exchange student, on leave of absence from the University of Zagreb.
ꢁ 2011 Verlag Helvetica Chimica Acta AG, Zꢀrich

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of peptide nucleic acids (PNAs), the synthesis of the higher oligomers could follow
either a Boc- [15] or an Fmoc-based [16] strategy; we opted for the Fmoc strategy that
allows for deprotection under milder conditions.
The synthesis in solution should lead to sufficient amounts of di- and tetramers to
analyse their association in organic solvents by 1H-NMR spectroscopy, as described for
earlier types of ONIBs [4][8][9][17]. Much smaller amounts of material are obtained
by synthesis on solid support, and the association of the self-complementary octamers
in aqueous solution has to be analysed on the basis of their temperature-dependent UV
spectra [18] to assess p-stacking, the major stabilising force in aqueous solvents [19].
We also intended to test for cross-pairing of a non-self complementary hydrazide-
linked decamer with complementary strands of RNA and DNA.
We already reported the synthesis of the required monomeric building blocks [2],
and now describe the synthesis of uracil- and adenine-derived, hydrazide-linked di-,
tetra-, octa-, and decamers and of amide-linked octamers.
Results and Discussion. – Synthesis of the Hydrazide-Linked Dimers in Solution.
The hydrazide-linked fully protected UU dimer 5, UA dimer 9, and AA dimer 15 were
synthesized by N-acylation of the previously described uracil-derived hydrazine 3 and
the adenine-derived analogue 14 with the carboxylic acids 4 and 8 [2] (Scheme 1)4),
using HBTU5) as coupling agent in combination with Hꢀnigꢂs base in DMF. The Fmoc
groups were removed with piperidine in DMF, and the tert-butyl esters were cleaved by
the action of trifluoroacetic acid (TFA) in the presence of Et3SiH in CH2Cl2, adding a
large excess of Et3SiH when cleaving the tert-butyl ester group of Cbz-protected dimers
to avoid partial loss of the Cbz groups [2][16] that were removed by Pd-catalyzed
hydrogenolysis.
Coupling the hydrazine 3 with the benzotriazol-1-yl ester derived from acid 4
(Scheme 1) yielded 98% of the fully protected UU dimer 5. We similarly coupled the
hydrazines 3 and 14 with the acid 8 to obtain the fully protected UA dimer 9 (77%) and
4
)
The structures of the hydrazide- and amide-linked oligonucleotide analogues are drawn with the
linker connecting C(6) or C(8) of a nucleobase on the left to N(1) or N(9) of the nucleobase on the
right, by analogy to the drawing convention adopted for the other ONIBs [3 – 5][7 – 12]. The base
sequence is, therefore, given from the C- to the N-terminus, in contradistinction to peptides and
PNAs [20], with the base units numbered (roman numerals) from left to right.
O-(Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate. There was no advantage
in using the more expensive HATU (¼O-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluro-
nium hexafluorophosphate).
5
)

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1155
Scheme 1
a) HBTU, HOBt, iPr2NEt, DMF; 98% of 5; 77% of 9. b) Piperidine, DMF; 99% of 6; 80% of 10; 80%
of 13. c) F3CCOOH (TFA), Et3SiH, CH2Cl2; 99% of 7; 98% of 11; 69% of 16. d) 1. Pd(OAc)2, H2,
MeOH/CH2Cl2 1:1; 2. 9, MeOH/CH2Cl2 1:1; 94%.
AA dimer 15, respectively. Removing the Fmoc group of 5 yielded 99% of the dimeric
hydrazine 6. Similarly, 9 yielded 80% of 10.
Cleaving the tert-butyl esters 5 and 9 afforded the dimeric carboxylic acids 7 (98%)
and 11 (84%), respectively. Crude 7 was sufficiently pure for the next step, while acid 11
was purified by MPLC. Cleaving the tert-butyl ester 15 gave acid 16 (78% from 14). Pd-
Catalysed debenzyloxycarbonylation of 9 yielded 94% of 12. The reaction was slow
and required 30 h for completion, even in the presence of a stoichiometric amount of
Pd(OAc)2. Removing the Fmoc group of 12 provided the hydrazine 13 in almost
quantitative yield.

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Slow evaporation of a solution of 5 in EtOH afforded crystals that were suitable for
X-ray analysis (Fig. 1)6).
Each molecule of 5 in the unit cell is H-bonded via HꢀN(3) and O¼C(2) to the
enantiomeric conformer (ꢃinvertomerꢂ, with the tetrahedral N-atom of the hydrazide
group as a centre of chirality), forming an antiparallel cyclic duplex (Fig. 1,a)). The
planes of the uracil rings of units I and II form an angle of 30.98, corresponding to a
buckle twist. The O¼C(4) group of units I in the duplex accept a H-bond from H2O,
whereas the O¼C(4) group of units II accept a H-bond from EtOH.
The fluorenyl and the uracil group of unit II of 5 in adjacent unit cells stack, with a
distance of 3.4 ꢄ between the planes of the aromatic rings (Fig. 1,b)). The hydrazide
N(3’)ꢀH forms an intramolecular H-bond to the Fmoc C¼O group (distance NH ···
O ¼ 2.1 ꢄ). A further H-bond is suggested by the distance of 2.3 ꢄ between HꢀC(5)
and O¼C(4) of units II [21].
A comparison of the conformation of crystalline 5 with the calculated conformation
of a hydrazide-linked dimer lacking the fluorenyl group is far from obvious, considering
the stacking interactions and the intramolecular H-bond in the crystal. Even so, the
torsion angles k, i1, i2, e2, e2, l, and z of 5 in the solid state agree reasonably well with
the calculated torsion angles for the cyclic duplex (ꢁ238) [2] (Table), with a significant
deviation (of 428) for the torsion angle x.
Table. Torsion Angles for the Crystal Structure of 5 and Predicted Torsion Angles for the Hydrazide
Linker
Torsion Angle
Observed in the Crystal Structure of 5 [8]
Predicted [2] [8]
k
i1
i2
e1
e2
l
þ 58
þ 52
þ 70
þ 60
180
ꢀ 120
þ 120
180
þ 174
ꢀ 140
þ 97
180
x
ꢀ 28
ꢀ 70
z
þ 115
þ 100
The torsion angles e1, e2, and l confirm the expected (Z)-syn-conformation of the
hydrazide, and the values found for i1 and i2 confirm the expected antiperiplanar
orientation of C(6/I) and CH2(1’’) [2].
The intermediate synperiplanar/synclinal arrangement of NH(3’) and N(1/II) (x ¼
ꢀ288) may well result from the intramolecular H-bond between NꢀH(3’) and the
carbazate C¼O group. This H-bond can no longer be formed when the torsion angle x
in the crystal structure is changed from the observed ꢀ 288 to the calculated ꢀ 708.
6
)
The crystallographic data have been deposited with the Cambridge Crystallographic Data Centre as
deposition No. CCDC-811879. Copies of the data can be obtained free of charge via http://
www.ccdc.cam.ac.uk/data_request/cif (or from the Cambridge Crystallographic Data Centre, 12
Union Road, Cambridge CB21EZ (fax: þ 44(1223)336033; e-mail: deposit@ccdc.cam.ac.uk).

Helvetica Chimica Acta – Vol. 94 (2011)
1157
Fig. 1. a) Pairing between the enantiomers (invertomers) of 5 in a cyclic duplex involving HꢀN(3) and
O¼C(2) of units I and II (bold dashed lines); H-bonds between enantiomers of 5 involving HꢀC(5) and
O¼C(4) of units II (dashed lines); H-bonds of O¼C(4/I) with H2O, and O¼C(4/II) with EtOH (dashed
lines); values for the torsion angles of the hydrazide linker. The tBu, Et, and Fmoc groups are omitted for
clarity. b) Stacking of the fluorenyl groups and uracil units II of enantiomers of 5; intermolecular H-bonds
between HꢀC(5) and O¼C(4) of units II of the enantiomers of 5; intramolecular H-bonds between
HꢀN(3’) and the carbazate C¼O group.

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To check for stacking interactions in solution, we recorded UV spectra of 5 in
EtOH and CHCl3 while varying the temperature. Specific stacking interactions in
CHCl3 were expected to go along with a strong association via H-bonding. As the
association proved weak (see below), suggesting the formation of mostly linear
duplexes, stacking interactions were not considered specific. The data are discussed
below, in the context of the analyses of the self-association of 5 in solution.
Solution Synthesis of Hydrazide-Linked Tetramers. The hydrazide-linked UUUU
tetramers 17 and 18, the UAUA tetramers 19 and 20, and the UUAA tetramers 21 and
22 were synthesized similarly as the dimers (Scheme 2). We coupled the dimeric
hydrazines 6 and 10 with the dimeric carboxylic acids 7, 11, and 16 to provide the fully
protected U4 tetramer 17 (69%), (UA)2 tetramer 19 (89%), and U2A2 tetramer 21
(72%), respectively. Tetramer 17 proved significantly more polar than dimer 5 and
more difficult to purify by silica-gel chromatography. Removing the Fmoc group of 17
provided hydrazine 18. Isothermal diffusion of Et2O into a saturated solution of 18 in
MeOH precipitated 64% of pure 18. Hydrogenolysis of 19 and 21 in the presence of a
large amount of Pd(OAc)2 provided 65% of 20 after seven days, and 69% of 22 after
six days, respectively, both tetramers requiring purification by MPLC.
Solid-Phase Synthesis of Hydrazide-Linked Octa- and Decamers. 1. Optimisation of
the Reaction Conditions. We synthesised the hydrazide-linked uracil- and adenine-
derived octa- and decamers on the standard Rink amide 4-methylbenzhydrylamine
(MBHA) polystyrene resin [22]. To establish appropriate reaction conditions, we
coupled the uracil-derived monomers 3 and 4 in DMF, NMP (¼ N-methylpyrrolidin-2-
one), or DMSO, activating 4 with either HBTU [23], HATU [24], HCTU7) [25], or
PyBOP8) [26], always in combination with Hꢀnigꢂs base. Performing the coupling in
DMSO and activating the carboxylic acid with HATU resulted in the cleanest and
fastest reaction. The superior properties of DMSO were confirmed by test syntheses on
the solid phase using either DMF, NMP/DMSO 8 :2 [27], 0.8m LiCl in DMF [28], and
DMSO [29]. The N-Fmoc groups were removed using DBU9). The N-termini of the
final oligomers were capped by N-acetylation with Ac2O/Hꢀnigꢂs base before their
cleavage from the resin with TFA/iPr3SiH 97:3.
2. Synthesis of Octamers Derived from Uracil and Adenine. According to our
modelling [2], the strength of association of the hydrazide-linked octamers derived
from uracil and adenine will depend on the sequence, as the extent of base stacking
decreases in the order UA > UU > AA > AU. To test this modelling result, we
synthesized the self-complementary (UA)4 octamer 24, U4A4 octamer 26, (U2A2)2
octamer 28, and (A2U2)2 octamer 30. To evaluate pairing with RNA and DNA, we also
synthesized the non-self-complementary U10 decamer 33.
We synthesized the octamers 24 – 30 on the Rink amide MBHA resin [22], coupling
the acids 4 and 8 with HATU and Hꢀnigꢂs base in DMSO (Scheme 3) and removing the
Fmoc groups by repetitive short treatments with dilute solutions of DBU, each one
followed by washing the resin with DMSO to prevent any addition of the terminal
hydrazine to dibenzofulvene (DBF). A high concentration (0.5m) of the activated
7
)
)
)
O-(6-Chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate.
(Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate.
1,8-Diazabicyclo[5.4.0]undec-7-ene.
8
9

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1159
Scheme 2
a) HBTU, HOBt, EtNiPr2, DMF; 69% of 17; 89% of 19; 72% of 21. b) Piperidine, DMF; 64%. c) 1.
Pd(OAc)2, H2, MeOH/CH2Cl2 1:1; 2. 19 or 21, MeOH/CH2Cl2 1:1; 65% of 20; 69% of 22.

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Scheme 3
a) Solid-Phase Synthesis. Loading and coupling: Rink amide MBHA polystyrene resin, 4 or 8, HATU,
Hꢀnigꢂs base, DMSO. N-Fmoc deprotection: DBU, DMSO. Capping: Ac2O, Hꢀnigꢂs base, N-
methylpyrrolidin-2-one (NMP). Cleavage from the resin: TFA/iPr3SiH 97:3. 2% overall yield of 33.
b) 1. Pd(OAc)2, MeOH, H2. 2. 23, MeOH; 5% overall yield of 24. c) 1. TFA/iPr3SiH 97:3, 808; 2.
Amberlite IRA-68, MeCN/H2O 1:1; 5% overall yield of 26 and 30; 8% overall yield of 28. Yields refer to
the loading of the resin (0.72 mmol/g). d) NH3, H2O; quant. yield. e) 1. LiOH, H2O; 2. Amberlite IRA-
120; quant. yield.

Helvetica Chimica Acta – Vol. 94 (2011)
1161
carboxylic acid was crucial to drive the couplings to completion. Coupling of the uracil-
derived carboxylic acid 4 proceeded cleanly and completely within 4 h. In contra-
distinction, coupling of the adenine-derived carboxylic acid 8 was slow, and we detected
significant amounts of truncated sequences. The coupling efficiency was neither
improved by changing the loading of the resin (as evidenced by similar results obtained
from loadings at 0.72 and 0.1 mmol/g of free amino groups on the resin), nor by
coupling at 358 rather than at room temperature. When the final capping by acetylation
of the N-terminus was omitted, and the polymer-bound oligomer directly exposed to
the cleavage conditions (TFA/iPr3SiH (TIPS) 97:3), we obtained a complex mixture of
shorter and longer oligomers, as judged by LC/MS analysis. The mass corresponding to
the individual peaks in the chromatogram agreed well with the formation of trans-
hydrazidation products resulting from the consecutive hydrazinolysis of (protonated)
hydrazide groups by the uncapped terminal hydrazino group resulting in the observed
mixture of products10).
Not unexpectedly, the Cbz groups were partially removed during the acidic
cleavage of the octamers from the support. The crude octamers 23, 25, 27, and 29 were,
therefore, deprotected either by Pd-catalysed hydrogenolysis, or by treatment with
TFA/TIPS 97:3 at reflux temperature (Scheme 3).
The crude octamers were purified by preparative HPLC. For 24, 26, 28, and 30, we
used an amino phase and for 33 a reversed phase. The low yields of 5 – 8% for 24 – 30
may in part be due to incomplete couplings of the adenine-derived monomer 8, while
incomplete detachment from the support is evidenced by the low mass balance of 40 –
50%. The even lower yield (2%) of 33 results from its difficult chromatographic
purification, 33 running close to the truncated sequences.
The self-complementary octameric analogues 24, 26, 28, and 30, and the non-self-
complementary decameric 33 were sufficiently well water-soluble to prepare 10 mm
solutions, required to test for stacking in aqueous solution by temperature-dependent
UV spectroscopy [18]. To test the influence of the side-chain polarity on the
association, we transformed the EtOCO groups of 30 to NH2CO and to COOH groups
(Scheme 3). Aminolysis of 30 to 31, and hydrolysis of 30 to 32 proceeded cleanly, and
the products did not require purifying.
3. Attempted Synthesis of Octamers Derived from Cytosine and Guanine. We also
applied the optimised conditions for coupling, deprotection, and capping (see above) to
synthesize the cytosine- and guanine-derived octamers. Besides the Rink amide MBHA
resin, we tested the syntheses on a Sieber amide polystyrene resin [30] that allows for
cleavage of the oligomers under milder conditions, using CH2Cl2/TFA 99 :1.
Prior to the synthesis of the (CCbzG)4 octamer 40, we tested whether the H2NꢀC(2)
group of guanine was likely to cause an aminolysis of the hydrazide groups during the
cleavage of 40 from the solid support. For this purpose, we synthesized the ACbzGU
trimer 35 (Scheme 4), sequentially coupling the hydrazino acids 4, 8, and 34 by
activation with HATU in the presence of Hꢀnigꢂs base in DMSO, followed by removing
the Fmoc groups with dilute solutions of DBU in DMSO, similarly as described for the
10
)
An analogous trans-amidation, however, was never observed during the synthesis of the amide-
linked analogues (see below). This difference is rationalized by the higher electrophilicity of the
monoprotonated hydrazides and the nucleophilic properties of the monoprotonated hydrazines.

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Scheme 4
a) Solid-Phase Synthesis. Loading and coupling: Rink amide 4-methylbenzhydrylamine (MBHA)
polystyrene or the Sieber amide polystyrene resin, 8, 34, or 4, HATU, Hꢀnigꢂs Base, DMSO. N-Fmoc
deprotection: DBU, DMSO. Capping: Ac2O, Hꢀnigꢂs base, NMP. Cleavage from the resin: Rink amide:
TFA/iPr3SiH 97:3; Sieber amide: CH2Cl2/TFA 99 :1.
synthesis of the octamers derived from uracil and adenine (see above). The synthesis
on a Rink amide MBHA resin led to small amounts of shorter and longer oligomers
besides the desired 35, even when the N-terminal hydrazino group was capped, while
the synthesis on the Sieber amide resin gave exclusively the desired 35. This observation
can be explained by an aminolysis/trans-hydrazidation sequence, analogous to the
strand-breaking mechanism described above, but involving the H2NꢀC(2) group of
guanine.
These side-products were not formed under the mild conditions required for the
cleavage of the trimer from the Sieber amide resin (CH2Cl2/TFA 99 :1). We did not
observe the acetylation of C(2)ꢀNH2 during capping, as it was reported for the
synthesis of PNA under similar conditions [31].
We then set off to synthesize the (CCbzG)4 octamer 40 on the Rink amide MBHA
[22] and also on the Sieber amide resin [30] (Scheme 5), coupling the acids 36 and 34,
similarly as described for the synthesis of the octamers derived from uracil and adenine.

Helvetica Chimica Acta – Vol. 94 (2011)
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Scheme 5
a) Solid-Phase Synthesis. Loading and coupling: Rink amide MBHA polystyrene or the Sieber amide
polystyrene resin, 36 or 34, HATU, Hꢀnigꢂs base, DMSO. N-Fmoc deprotection: DBU, DMSO. Capping:
Ac2O, Hꢀnigꢂs base, NMP. Cleavage from the resin: Rink amide: TFA/iPr3SiH 97:3; Sieber amide:
CH2Cl2/TFA 99 :1.
The expected octamer 40 was removed from the Rink amide MBHA resin by
treatment with TFA/TIPS 97:3, but LC/MS analysis revealed not even a trace of the
expected product. The analogous synthesis on the Sieber amide resin, monitoring the
progress of coupling and deprotection, and the purity of the oligomers resulting after
every second cycle by LC/MS analysis, led to the dimer 37 and the tetramer 38, besides
only small amounts of impurities. The hexamer 39, however, could hardly be identified
by LC/MS analysis, and the octamer 40 could not be detected, even after washing the
resin with H2O, MeOH, EtOH, and DMSO. As the LC/MS analysis requires a sufficient
solubility of the octamers in MeCN/H2O, while their cleavage from the support requires
that they are soluble in CH2Cl2/TFA 99 :1, octamer 40 may well have been formed, but
was either not cleaved from the support, or did not dissolve in at least one of the solvent
mixtures.
Self-Association of the Hydrazide-Linked UU Dimers. The UU dimer 5 was
sufficiently soluble in CDCl3 to follow the dependence of the chemical shift of the

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Fig. 2. SCCs for HꢀN(3) of the UU dimers 5 and 6 in CDCl3, CDCl3/(D6)DMSO 95 :5, and in (D8)THF
(solid lines: fitted curve); values for the association constant and thermodynamic parameters of 5 in
CDCl3; structure of the U monomer 41
HꢀN(3) signals of solutions between the concentrations of 50 and 0.5 mm (Fig. 2). The
1H-NMR signals of 5 in CDCl3 were broad, and HMBC spectra did not allow assigning
the two HꢀN(3) signals to the individual uracil units. Their concentration and
temperature dependence was, however, very similar, resulting in almost identical
association constants and thermodynamic parameters of association, so that only the
data derived from one of the HꢀN(3) are shown in Fig. 2. The association of 5 is weak,
with Kass ¼ 74(ꢂ4) mꢀ1 11) (DG295 ¼ ꢀ2.5 kcal/mol). The enthalpy of association
(DHass ¼ ꢀ7.6 kcal/mol)12) and the shape of the ꢃshift-concentration curveꢂ (SCC)
evidence the formation of (one or several) linear duplexes and higher associates.
According to the vapour-pressure osmometric (VPO) determination of the apparent
molecular mass of 5 at five concentrations between 10 and 50 mm in CHCl3, the ratio
between the apparent molecular mass and the formula weight increased from 1.23
(10 mm) to 1.74 (50 mm), confirming the (partial) formation of linear duplexes and
11
)
)
Determined from the monoplex shift by extrapolation to c ¼ 0 and from the duplex shift by
extrapolation to c ¼1 .
12
Determined by vanꢁt Hoff analysis, based upon the temperature dependence of the association at a
concentration of 10 mm (7 – 508).

Helvetica Chimica Acta – Vol. 94 (2011)
1165
higher associates. These results do not reflect the H-bonding pattern found in the
crystal structure of 5, where two base pairs of 5 are H-bonded in a cyclic duplex
involving the HꢀN(3) and the O¼C(2) groups of the two uracil units.
A ROESY spectrum of 5 was recorded at a 100 mm concentration in CDCl3 to
obtain information about the structure of the associates of 5 in CDCl3. However, no
cross-peaks were detected for the hydrazide NH, HꢀN(3), HꢀC(5), or CH2ꢀN(1),
possibly on account of the broad signals. Only weak and insignificant cross-peaks were
detected for the (CH2)2ꢀN groups and the aromatic H-atoms of the fluorenyl group.
The chemical shift of the hydrazide NH of 5 changed only from 8.66 to 8.16 ppm by
lowering the concentration from 50 to 0.5 mm in CDCl3. These values are typical for 1-
acyl-2-alkylhydrazines [32], and evidence that the hydrazide NH is neither involved in
a significant intermolecular nor in an intramolecular H-bond, as in the crystal structure
of 5.
We interpret the broad signals in the 1H-NMR spectra of 5 in CDCl3 as reflecting
rotational equilibria about the hydrazide and the carbazate CꢀN bonds, and the
presence of several weak associates. The assumption of rotational equilibria is
confirmed by the 1H-NMR spectra in (D6)DMSO, where there should be no
association. Although the spectra show sharper peaks, they display two sets of signals,
probably due to hydrazide (E)- and (Z)-isomers, as evidenced by the splitting of the
hydrazide NH signal into two peaks in a 6 :4 ratio. A similar ratio was found for the
other split signals. As the (Z) conformation of hydrazides is favoured in polar solvents
and with increasing size of the substituents [33], we assume that the (Z)-isomers also
dominate in (D6)DMSO. Heating a solution of 5 in (D6)DMSO to 1008 led to
coalescence.
1
The H-NMR spectra of the partially deprotected UU dimer 6 in CDCl3 showed
strong signal broadening. The two HꢀN(3) resonate as one broad signal that could only
be followed in the concentration range of 9.5 to 100 mm. At lower concentrations, it
could no longer be detected. The chemical shift of HꢀN(3) of 6 hardly changed in the
mentioned concentration range (Fig. 2). This evidences either a very strong or no
association. The downfield shift (Dd ¼ 2.7 ppm) for HꢀN(3) of 6 relative to the
monomer 41 at a concentration of 0.5 mm (Fig. 2) suggests a very strong association of
6. 1H-NMR Spectra of 6 were, therefore, recorded in CDCl3/(D6)DMSO 95 :5, DMSO
weakening the association. As expected, sharper signals were observed in this solvent
mixture, and the two HꢀN(3) gave rise to a single broad signal. The HꢀN(3) signal was
followed between 50 and 0.9 mm 6 (Fig. 2), but its chemical shift changed only from
10.47 to 10.16 ppm, and is similar to that of 41 at 0.5 mm in CDCl3/(D6)DMSO 95 :5
(9.94 ppm), evidencing that 6 hardly associates in this solvent mixture. Considering this
result and the observation of a single signal for the two HꢀN(3), it appears highly
improbable that 6 associates strongly in pure CDCl3. The large value for d(HꢀN(3)) in
CDCl3 (10.6 ppm) and its concentration independence within the mentioned concen-
tration range suggest that HꢀN(3) is involved in an intramolecular H-bond, although
the H-bond acceptor is not clear.
The concentration dependence of d(HꢀN(3)) of 6 in (D8)THF (Fig. 2) is
characterized by an almost constant chemical shift (10.4 ppm), and the comparison
with d(HꢀN(3)) of 41 at a concentration of 0.5 mm (10.28 ppm) evidences that 6 does
not associate in THF solution.

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1
The H-NMR spectra of 6 in pure (D6)DMSO exhibited two sets of signals,
similarly as observed for 5, suggesting (E)- and (Z)-hydrazide isomers. Heating to 1008
led to coalescence.
To further test if stacking interactions are present in solutions of either 5 and/or 6, as
compared to those found in the crystal structure of 5, we recorded the UV spectra of
these dimers in EtOH at 0 and 708, and in CHCl3 at 0 and 558 (Fig. 3). Stacking
interactions were expected to be favoured over H-bonding in EtOH, whereas weaker
stacking was expected in CHCl3. The UV absorption of both 5 and 6 in both solvents
depends on the temperature. The difference of the extinction coefficient of 5 in EtOH
at 0 and 708 (Fig. 3,a)) at lmax 300 nm (De ¼ 1340 l · molꢀ1 · cmꢀ1) indicates inter- or
intramolecular stacking involving the fluorenyl group, and De ¼ 3580 l · molꢀ1 · cmꢀ1 at
lmax 266 nm indicates inter- or intramolecular stacking, either involving the fluorenyl
and the uracil groups, or the uracil groups only. A similar temperature dependence of
the extinction coefficient of 5 was observed in CHCl3 solution (Fig. 3,b) at lmax 266 nm
(De ¼ 2700 l · molꢀ1 · cmꢀ1), but a smaller dependence was found at lmax 300 nm (De ¼
700 l · molꢀ1 · cmꢀ1), suggesting weaker stacking interactions of the fluorenyl group as
compared to stacking involving the uracil units. The UV spectra of 6 in EtOH showed
also a temperature dependence of the absorption at lmax 266 nm (De ¼ 2060 l · molꢀ1
·
cmꢀ1) that suggests intra- or intermolecular stacking of the uracil groups. A somewhat
smaller temperature dependence was found for the absorption of 6 in CHCl3 at lmax
266 nm (De ¼ 950 l · molꢀ1 · cmꢀ1).
Fig. 3. Extinction coefficient of the UU dimers 5 and 6 in a) 10ꢀ5 m EtOH solution at 0 and 708, and in
b) 10ꢀ5 m CHCl3 solution at 0 and 558
We conclude that the temperature dependence of the extinction coefficients of 5
and 6 denotes non-specific stacking interactions, as there is no evidence for specific
stacking in these cyclic duplexes in view of the evidence for the formation of only linear
duplexes and possibly higher associates obtained from the 1H-NMR analyses discussed
above. The similar temperature dependence of the extinction coefficients in EtOH and
in CHCl3 also suggests non-specific stacking interactions.
Self-Association of Hydrazide-Linked UA Dimers. A solution of the UA dimer 12 in
CDCl3 displayed very broad 1H-NMR signals. The HꢀN(3) signal of 12 at a

Helvetica Chimica Acta – Vol. 94 (2011)
1167
concentration of 50 mm was only just discernible from the base line, resonating
between ca. 12.4 and ca. 11.6 ppm. The shift to ca. 12 ppm for HꢀN(3) of 12 (50 mm), as
compared to d(HꢀN(3)) 7.91 ppm of the U monomer 41 (0.5 mm), suggests the
involvement of HꢀN(3) in a H-bond and, therefore, association. However, dilution
resulted in the disappearance of the peak, and it was not possible to follow its
concentration dependence.
1
As expected, sharper signals characterize the H-NMR spectra of 12 in CDCl3/
(D6)DMSO 95 :5, and the concentration dependence of d(HꢀN(3)) was determined
between 50 and 1.7 mm (Fig. 4). A small association constant Kass ¼ 191(ꢂ 83) mꢀ1
(DG295 ¼ ꢀ3.1 kcal/mol) resulted from the shift-concentration data, suggesting the
formation of mainly linear aggregates, in agreement with the shape of the SCC and the
thermodynamic parameters of association (DHass ¼ ꢀ10.1 kcal/mol, DSass ¼ ꢀ24.2 e.u.,
DGass ¼ ꢀ3.0 kcal/mol), as determined by a vanꢁt Hoff analysis of a 10 mm solution
(T¼ 7 – 508).
This result leads to the conclusion that the UA dimer 12 associates in CDCl3. The
shift to lower fields of the HꢀN(3) signal of 12 in CDCl3 at a concentration of 50 mm
(ca. 12.0 ppm) relative to the corresponding N-Fmoc-protected UU dimer 5 at the same
concentration (9.8 ppm) evidences a stronger association of 12.
Fig. 4. SCCs for HꢀN(3) of the UA dimers 12 and 13 in CDCl3/(D6)DMSO 95 :5 and 90 :10, and in
(D8)THF (solid lines: fitted curve); values for the association constants and thermodynamic parameters
of 12 in CDCl3/(D6)DMSO 95 :5

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The sharpening of the 1H-NMR signals of 12 upon addition of 5% DMSO suggests
that the broad signals in CDCl3 solution result from the formation of several types of
associates (partially dissociating upon the addition of DMSO) and to equilibria of
diastereoisomers resulting form the restricted rotation about the hydrazide and
1
carbazate CꢀN bonds. H-NMR Spectra in pure (D6)DMSO could not be used to
detect if the hydrazides from (E)- and (Z)-isomers in the absence of pairing, as 12
degraded slowly, partial loss of the NFmoc group being observed by TLC.
Two sets of signals for 12 were observed in (D8)THF, in agreement with a mixture of
(E)- and (Z)-isomers. The weak concentration dependence of d(HꢀN(3)) of 12 in
(D8)THF (Fig. 4) resulted in an insignificant association constant of 4.2(ꢂ1) mꢀ1.
The partially deprotected UA dimer 13 was insoluble in CDCl3 and poorly soluble
in CDCl3/(D6)DMSO 95 :5. A 1H-NMR spectrum of a saturated solution of 13 in this
solvent mixture (ca. 10 mm) showed broad signals, with the one of HꢀN(3) barely
discernible between ca. 11.8 and ca. 11.0 ppm. In CDCl3/(D6)DMSO 90 :10, the
1H-NMR spectrum of 13 displayed sufficiently sharp signals to determine the
concentration dependence of d(HꢀN(3)) between 50 and 1 mm (Fig. 4), resulting in
a very small association constant of 11(ꢂ1) mꢀ1.
1
Self-Association of Hydrazide-Linked Tetranucleotide Analogues. The H-NMR
spectra of the N-Fmoc-protected U4 tetramer 17 in CDCl3 is characterized by strong
signal broadening. We could only tentatively assign the broad signals, with the fluorenyl
signals appearing as three broad peaks between 7.8 and 7.2 ppm, those of all CH2 groups
t
as a single, very broad peak at 4.6 – 3.2 ppm, and those of Me and Bu groups as two
broad peaks between 1.6 and 1.0 ppm. No other signals were detected. Similarly broad
1
signals characterized the H-NMR spectra of 17 in CDCl3/(D6)DMSO 95 :5, while
sharper signals for solutions in CDCl3/(D6)DMSO 90 :10 allowed us to follow the
concentration dependence of d(HꢀN(3)). The four HꢀN(3) of 17 (50 and 0.6 mm)
resonate as a single, broad, concentration independent signal (Fig. 5). The chemical
shift of 10.6 – 10.5 ppm is about the same as that for HꢀN(3) of the U monomer 41
(0.5 mm), evidencing that 17 does not associate in CDCl3/(D6)DMSO 90 :10.
The similar, concentration-independent chemical shift (10.5 – 10.4 ppm) of HꢀN(3)
of 17 and of 41 (10.28 ppm) in (D8)THFat a concentration of 0.5 mm evidences that 17
does not associate in THF (Fig. 5).
Very similar results were obtained for the partially deprotected U4 tetramer 18 in
CDCl3/(D6)DMSO 90 :10, and in (D8)THF at concentrations between 25 and 1 mm.
The insensitivity of d(HꢀN(3)) to the change of concentration evidences that 18 does
not associate in these solvents. The tetramer 18 proved too poorly soluble in CDCl3 to
establish a SCC, and the spectra of saturated solutions in CDCl3/(D6)DMSO 95 :5 were
too broad to be analysed.
Similarly, we could not derive a SCC for the self-complementary U- and A-derived
tetramers 20 and 22 (Scheme 2). They proved too poorly soluble in CDCl3, CDCl3/
1
(D6)DMSO 95 :5, and (D8)THF, and the H-NMR signals of solutions in CDCl3/
(D6)DMSO 90 :10 were too broad. The spectra of 20 and 22 in (D6)DMSO (where no
association is expected) are characterized by sharper, but complex signals.
In summary, the hydrazide-linked di- and tetramers do not pair in any of the
solvents studied, in contradistinction to the formation of a cyclic duplex of the UU
dimer 5 in the solid state. The signal splitting in the 1H-NMR spectra in (D6)DMSO or

Helvetica Chimica Acta – Vol. 94 (2011)
1169
Fig. 5. SCCs for HꢀN(3) of the U4 tetramers 17 and 18 in CDCl3/(D6)DMSO 90 :10 and in (D8)THF
(solid lines: fitted curve)
in (D8)THF evidences (E)- and (Z)-hydrazide isomers, and the broad 1H-NMR signals
in the less polar solvents or solvent mixtures suggest the formation of equilibrating
linear associates.
Analysis of Association of the Hydrazide-Linked Octamers. We tested stacking of
the self-complementary octamers 24 – 32 by monitoring the UV absorption of a 10 mm
solution in H2O or in 10 mm Na-phosphate buffer at pH 7 at 260 nm, increasing the
temperature from 4 to 808 in increments of 0.18/min, and then cooling the solution to 48
[18]. We also recorded a UV melting curve of a mixture of 28 (U2A2)2 and 30 (A2U2)2
(c ¼ 5 þ 5 mm), allowing for hetero-association with parallel strand orientation. The
results from the heating and cooling experiments were identical. The absence of any
hyper- or hypochromic effect for 24 – 32, at 260, 270, 280, and 290 nm, and at
concentrations of 10 – 100 mm shows the absence of any intra- or intermolecular
stacking. This conclusion is confirmed by the ESI-MS of 30, 31, and 32 at a
concentration of 1 mm, where only peaks of the singly or doubly charged monoplexes
were detected.
The absence of stacking interactions suggests the absence of pairing. In addition to
the unfavourable conformation for the formation of H-bonds between the nucleobases
that may disfavour pairing of the hydrazide-linked di- and tetramers in organic solvents,

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stacking in H2O may be disfavoured by the calculated [2] large distance between the
planes of the nucleobases (cf. hydrazide analogues: 3.6 – 4 ꢄ; A-DNA and B-DNA:
3.3 – 3.4 ꢄ) and the energetic penalty required for reducing it, considering the rather
rigid linker. This penalty is expressed by the buckle twists in the crystal structure of 5
(Fig. 1), and visible in the energetically minimized octamer U4A4 in [2].
Association of 33 with DNA and RNA. To test for hetero-association of the
hydrazide-linked U10 decamer 33 with the complementary strands 43 of DNA and 42 of
RNA (A10), we monitored the UVabsorption, while heating and cooling the solution of
the mixtures 33/43 and 33/42 (c ¼ 5 mm each; 10 mm Na-phosphate buffer: 100 mm NaCl
and 0.1 mm EDTA, pH 7) between 48 and 808. As a negative control, we recorded UV
melting curves of the individual, non-self-complementary strands (c ¼ 5 mm) in the
same temperature range (Fig. 6).
Fig. 6. Temperature dependence of the UV absorption of the decamers 33, 43, and 42. Recorded in 10 mm
Na-phosphate buffer (100 mm NaCl, 0.1 mm EDTA), c ¼ 5 þ 5 mm, l ¼ 260 nm, 1-cm cell.
The smooth, not completely straight curves of the individual decamers 43, 42, and
33 indicate unspecific stacking. The curves for the mixtures 33/43 and 33/42 represent
an overlap of the individual curves.
Considering that a sufficient degree of flexibility of the linker is crucial for the
association into helical structures, we conclude that peptide-linked ONIBs require a
longer linking element, comprising functional groups that allow for conformational
changes and induce a conformation of the monoplex that favours pairing. Such peptide-
linked ONIBs may lead to a large twist angle, and we intend to explore if such a large
twist angle will allow for a sufficient degree of stacking to favour pairing.
Attempted Synthesis of Amide-Linked Octamers. We attempted to synthesize the
self-complementary octamer 50 (U4ACbz4) on the Rink amide MBHA resin [22] under
similar conditions as described for the synthesis of the hydrazide-linked octamers, but
with the necessity of coupling the acids 46 and 48 twice at 358 for 8 – 10 h each time to

Helvetica Chimica Acta – Vol. 94 (2011)
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realize satisfactory coupling yields. In spite of this, we did not detect 50 by LC/MS
analysis. This may well reflect a poor solubility of the product in H2O, resulting from
replacing an N-alkyl group of the hydrazides by a CH2 group, and omitting the side
chain. We, therefore, synthesized the octamers 51 – 56 (Scheme 6), modified at the N-
and C-terminus by incorporation of lysine and aspartic acid residues, as lysine is well-
known to increase the solubility of PNA oligomers in H2O [34]. The amino acids were
used in a fivefold excess and doubly coupled for 30 min.
The U-, A-, C-, and G-derived self-complementary benzyloxycarbonylated
octamers 51 – 56 were isolated as the main products of the synthesis on a Rink amide
MBHA resin, besides truncated sequences resulting from incomplete coupling of the
purine-derived monomers (Scheme 6). However, debenzyloxycarbonylation of these
Scheme 6
a) Solid-Phase Synthesis. Loading and coupling: Rink amide MBHA polystyrene resin, 46, 47, 48, 49, 44,
or 45, HATU, Hꢀnigꢂs base, DMSO. N-Fmoc deprotection: DBU, DMSO. Capping: Ac2O, Hꢀnigꢂs base,
NMP. Cleavage from the resin: TFA/iPr3SiH 97:3.

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octamers under hydrogenolytic or acidic conditions did not lead to the desired
deprotected octamers, probably owing to their insolubility in aqueous solution.
We thank Dr. Bruno Bernet for checking the experimental data, Dr. Bernd Schweizer for the crystal
structure analysis, and Syngenta AG, Basel, for generous support.
Experimental Part
General. THF was distilled from Na/benzophenone, and CH2Cl2, MeOH, DMF, pyridine, EtNiPr2,
i
and Pr2NH from CaH2. Reactions were run under N2. Qual. TLC: precoated silica-gel plates (Merck
silica gel 60 F254); detection by UV light at 254 nm wavelength and by spraying with ꢃmostainꢂ, and
heating. Flash chromatography (FC): silica gel Fluka 60 or Merck 60 (0.04 – 0.063 mm). UV Spectra:
10ꢀ5 m solns. in CHCl3 or MeOH at 208 in a 1-cm Suprasil cell. FT-IR: solid state (ATR). 1H- and
13C-NMR: at 300 or 400 MHz and 75 or 100 MHz, resp. MS: Matrix-assisted laser desorption ionization
time-of-flight mass spectrometry (MALDI-TOF) with 0.05m indole-3-acrylic acid (IAA) in THF, or with
0.05m a-cyano-4-hydroxycinnamic acid (CCA) in MeCN/EtOH/H2O, and high-resolution (HR)
MALDI-TOF with 0.05m 2,5-dihydrobenzoic acid (DHB) in THF.
General Procedure for Concentration- and Temperature-Dependent NMR Studies. NMR Experi-
ments were performed at 295 K and at 300 MHz in CDCl3, 5% (D6)DMSO/CDCl3, 10% (D6)DMSO/
CDCl3, or (D8)THF (passed through basic aluminium oxide and dried over mol. sieves (4 ꢄ) prior to
use). Experiments started at the highest concentration, with stepwise replacement of 0.2 ml of the 0.7-ml
soln. with 0.2 ml of solvent. The data were analysed by non-linear least-squares fitting using MATLAB
(trust-region algorithm); the parameters were Kass, d(HꢀN(3)), c ¼ 0 mm, and d(HꢀN(3)), c ¼1 ). The
thermodynamic parameters were determined by vanꢁt Hoff analysis. The uracil d(HꢀN(3) was monitored
at 7, 15, 22, 30, 40, and 508, and at a fixed concentration of 10 mm.
General Procedure for Temperature-Dependent UV Spectroscopy. UV Experiments were performed
by equilibrating 10 mm soln. of the oligomers in H2O or in 10 mm Na-phosphate buffer (100 mm NaCl and
0.1 mm EDTA, pH 7) for 30 min at 48, heating to 808, and cooling the soln. back to 48 (rate of 0.18/min).
The required UVabsorption at l 260 nm to obtain 5 or 10 mm soln. was calculated by LambertꢀBeerꢂs law
via the extinction coefficients of the oligomers, which were in turn calculated by summing up the
extinction coefficients of the monomers (U(T): 8.7, A: 15.4 ml/mmolꢀ1 cmꢀ1).
tert-Butyl 6-{{1-(2-Ethoxy-2-oxoethyl)-2-[(9H-fluoren-9-yl)methoxycarbonyl]hydrazino}methyl}ur-
acil-1-acetyl-(12 ! 63-N)-6-{[1-(2-ethoxy-2-oxoethyl)hydrazino]methyl}uracil-1-acetate (¼(9H-Fluoren-
9-yl)methyl 2-[(3-{2-[2-({3-[2-(tert-Butoxy)-2-oxoethyl]-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl}-
methyl)-2-(2-ethoxy-2-oxoethyl)hydrazinyl]-2-oxoethyl}-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)-
methyl]-2-(2-ethoxy-2-oxoethyl)hydrazinecarboxylate; 5). A soln. of 4 (3.9 g, 7.5 mmol) in DMF (120 ml)
was cooled to 08, treated with HOBt (1.1 g, 8.1 mmol) and HBTU (3.2 g, 8.4 mmol), stirred for 1.5 h at
r.t., treated with a soln. of 3 (2.5 g, 7.0 mmol) in DMF (25 ml) and with EtNiPr2 (1.5 ml, 8.6 mmol), stirred
for 20 h, treated with sat. NaHCO3 soln. (150 ml), and extracted with AcOEt (5 ꢃ 150 ml). Drying of the
combined org. layers (MgSO4), filtration, evaporation, and FC (CH2Cl2/MeOH 97:3 ! 90 :10) gave 5
(5.95 g, 98%). White powder. Rf (CH2Cl2/MeOH 9 :1) 0.54. M.p. (of a small batch crystallized from
EtOH) 130 – 1328. UV (CHCl3): 267 (35600), 301 (5300). IR (ATR): 3224w (br.), 2980w, 1675s (br.),
1627m, 1451m, 1421m, 1389m, 1228m, 1204m, 1153s, 1072w, 1024w, 980w, 930w, 822w. 1H-NMR
(400 MHz, (D6)DMSO; ca. 2 :1 mixture of rotamers; assignments based on a DQFCOSY, a HSQC, and a
HMBC spectrum): 11.39 (0.6 H), 11.34 (0.6 H), 11.29 (0.8 H) (3s, 2 HꢀN(3)); 9.66 (0.4 H), 8.90 (0.6 H)
(2s, HNꢀNCH2C(6/I)); 8.85 (s, HNꢀNCH2C(6/II)); 7.88 – 7.30 (m, 8 arom. H); 5.63 (s, 0.7 HꢀC(5/I)); 5.57
(s, 0.9 HꢀC(5/II)); 5.52 (s, 0.3 HꢀC(5/I), 0.1 HꢀC(5/II)); 5.31 (br. d, J ¼ 17.2, 0.2 CH2ꢀN(1/II)); 5.22 (br.
d, J ¼ 17.2, 0.2 CH2ꢀN(1/I)); 4.94 (s, 0.6 CH2ꢀN(1/I)); 4.81 (br. s, 0.5 CH2ꢀN(1/II)); 4.71 (br. d, J ¼ 17.2,
0.3 CH2ꢀN(1/II)); 4.60 (br. d, J ¼ 17.2, 0.2 CH2ꢀN(1/I)); 4.27 (br. s, CH2ꢀC(9’)); 4.18 (br. s, HꢀC(9’));
4.08 – 4.05 (m, 2 MeCH2O); 3.98 (br. s, 0.3 H); 3.80 (br. s, CH2ꢀC(6/I)); 3.76 (br. s, CH2NCH2C(6/I));
3.73, 3.69 (2 br. s, CH2ꢀC(6/II), CH2NCH2C(6/II)); 3.56 (br. s, 0.3 H); 3.52 (br. s, 0.3 H); 1.43 (3 H), 1.39
(6 H) (2s, t-Bu); 1.20 – 1.11 (m, 2 MeCH2O). 13C-NMR (100 MHz, (D6)DMSO; ca. 2 :1 mixture of

Helvetica Chimica Acta – Vol. 94 (2011)
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rotamers; assignments based on a HSQC and a HMBC spectrum): signals of the major rotamer: 169.22,
t
169.05 (2s, 2 CO2Et); 168.10 (s, CO2 Bu); 166.95 (s, C(O)CH2N(1/II)); 81.52 (s, Me3C); signals of the
t
minor rotamer: 170.23 (s, C(O)CH2N(1/II)); 169.36, 169.31 (2s, 2 CO2Et); 167.94 (s, CO2 Bu); 81.67 (s,
Me3C); signals of both rotamers: 162.51, 162.47 (2s, 2 C(4)); 155.22 (br. s, NCO2); 151.70, 151.63, 151.60,
151.22, 149.75 (5s, 2 C(2), C(6/II)); 150.69 (s, C(6/I)); 143.62 (2 br. s); 140.71 (2 br. s); 127.66 (2 br. d);
127.10 (2 br. d); 125.08 (2 br. d); 120.08 (2 br. d); 105.33, 103.86, 103.77, 103.39 (4d, 2 C(5)); 65.68 (t,
CH2ꢀC(9’)); 60.49, 60.36, 60.32, 60.26, 59.25 (5t, 2 MeCH2O, CH2NCH2C(6/II)); 57.51 (br. t, CH2ꢀC(6/
II)); 56.54 (t, CH2NCH2C(6/I)); 56.41 (t, CH2ꢀC(6/I)); 46.58 (d, C(9’)); 45.21 (t, CH2ꢀN(1/I)); 44.03 (t,
CH2ꢀN(1/II)); 27.59 (q, Me3C); 13.99, 13.94, 13.85 (3q, 2 MeCH2O). HR-MALDI-MS: 883.3248 (100,
t
[M þ Na]þ, C41H48N8NaO1þ3 ; calc. 883.3233), 827.2614 (41, [M ꢀ Bu þ H þ Na]þ, C37H40N8NaO1þ3 ; calc.
t
t
827.2602), 805.2819 (75, [M ꢀ Bu þ 2 H]þ, C37H41N8O1þ3 ; calc. 805.2782), 583.2117 (30, [M ꢀ Bu ꢀ
Fmoc þ 3 H]þ, C22H31N8O1þ1 ; calc. 583.2101). Anal. calc. for C41H48N8O13 · CH4O (892.91): C 56.50, H
5.87, N 12.55; found: C 56.70, H 5.76, N 12.69.
Crystal Structure Analysis of 5. Crystals of 5 were obtained by slow evaporation of a soln. of 5 in
EtOH (dimensions of the analysed crystal: cube 0.3 ꢃ 0.2 ꢃ 0.1 mm). C41H48N8O13 · C2H6O · H2O, Mr
ꢀ
924.96, triclinic P1, a ¼ 14.0050(3), b ¼ 14.0670(3), c ¼ 15.0703(4) ꢄ, a ¼ 63.6474(11), b ¼ 72.1477(10),
g ¼ 61.3441(12)8, V¼ 2317.94(9) ꢄ3, Z ¼ 2, Dx ¼ 1.325 Mg mꢀ3. The reflections were measured on a
KappaCCD diffractometer, with MoKa radiation, l ¼ 0.71073 ꢄ. Cell parameters from 32355 refl., q ¼
2.425 – 27.4858, m ¼ 0.101 mmꢀ1, T¼ 203 K. 18629 measured reflections; 10576 independent reflections;
7455 observed reflections (>2s(I)). Refinement on F2: full-matrix least-squares refinement, R(all) ¼
0.1096, R(gt) ¼ 0.0804. All diagrams and calculations were performed using maXus (Bruker Nonius,
Delft & MacScience, Japan). The program SIR97 was used to solve the structure and the program
SHELXL-97 to refine it.
tert-Butyl 6-{[1-(2-Ethoxy-2-oxoethyl)hydrazino]methyl}uracil-1-acetyl-(12 ! 63-N)-6-{[1-(2-ethoxy-
2-oxoethyl)hydrazino]methyl}uracil-1-acetate (¼ Ethyl (1-({3-[2-(tert-Butoxy)-2-oxoethyl]-2,6-dioxo-
1,2,3,6-tetrahydropyrimidin-4-yl}methyl)-2-{[6-{[1-(2-ethoxy-2-oxoethyl)hydrazinyl]methyl}-2,4-dioxo-
3,4-dihydropyrimidin-1(2H)-yl]acetyl}hydrazinyl)acetate; 6). A soln. of 5 (1 g, 1.2 mmol) in DMF
(10 ml) was treated dropwise with piperidine (0.6 ml, 6.1 mmol), stirred for 2 h at r.t., and evaporated.
The solid residue was washed with Et2O (8 ꢃ 10 ml). The solid was suspended in CH2Cl2 (100 ml) and
filtered. Evaporation of the filtrate gave 6 (0.74 g, 99%). White powder. Rf (CH2Cl2/MeOH 9 :1) 0.22.
M.p. 153 – 1578. UV (CHCl3): 266 (19600). IR (ATR): 3200w (br.), 2981w, 2808w, 1673s (br.), 1461m,
1419m, 1389m, 1370m, 1294w, 1235m, 1200m, 1154s, 1074w, 1024w, 980w, 927w, 819m. 1H-NMR
(400 MHz, (D6)DMSO; ca. 2 :1 mixture of rotamers; assignments based on a DQFCOSY, a HSQC, and a
HMBC spectrum): 11.29 (br. s, 2 HꢀN(3)); 9.55 (0.6 H), 8.89 (0.4 H) (2s, HNꢀNCH2C(6/I)); 5.61 (s,
0.7 HꢀC(5/I)); 5.58, 5.57 (2s, 0.7 HꢀC(5/II), 0.3 HꢀC(5/I)); 5.54 (s, 0.3 HꢀC(5/II)); 5.07 (br. d, J ¼ 17.2,
0.2 CH2ꢀN(1/I)); 5.03 (br. d, J ¼ 17.2, 0.2 CH2ꢀN(1/II)); 4.90 (s, 0.6 CH2ꢀN(1/I)); 4.60 – 4.53 (m,
0.8 CH2ꢀN(1/II), 0.2 CH2ꢀN(1/I)); 4.14, 4.10, 4.08 (3q, J ¼ 7.1, 2 MeCH2O); 3.92 (s, 0.1 H); 3.88 (s,
0.3 H); 3.83 (s, 0.4 H); 3.80 (s, CH2ꢀC(6/I)); 3.77 (s, CH2NCH2C(6/I)); 3.73 (br. s, NH2); 3.59 (s, 0.3 H);
3.56 (s, 0.4 H); 3.51 (s, CH2ꢀC(6/II)); 3.48, 3.47, 3.46 (3s, 0.9 H); 3.44 (s, CH2NCH2C(6/II)); 3.43 (s,
0.2 H); 1.43 (3 H), 1.39 (6 H) (2s, t-Bu); 1.23 – 1.18 (m, 2 MeCH2O). 13C-NMR (100 MHz, (D6)DMSO;
ca. 2 :1 mixture of rotamers; assignments based on a HSQC and a HMBC spectrum): signals of the major
t
rotamer: 169.93 (s, C(O)CH2NCH2C(6/II)); 169.27 (s, C(O)CH2NCH2C(6/I)); 168.03 (s, CO2 Bu); 166.70
(s, C(O)CH2N(1/II)); 162.69 (s, C(4/II)); 162.48 (s, C(4/I)); 152.10 (s, C(6/II)); 151.81, 151.67 (2s, 2 C(2));
150.76 (s, C(6/I)); 81.55 (s, Me3C); 60.38 (t, CH2ꢀC(6/II)); 56.54 (t, CH2NCH2C(6/I)); 56.27 (t, CH2ꢀC(6/
I)); 27.60 (q, Me3C); signals of the minor rotamer: 170.11 (s, CO2Et); 169.98 (s, C(O)CH2N(1/II)); 167.72
t
(s, CO2 Bu); 162.74 (s, C(4/II)); 162.44 (s, C(4/I)); 152.60 (s, C(6/II)); 151.77, 151.61 (2s, 2 C(2)); 149.74 (s,
C(6/I)); 81.81 (s, Me3C); 57.59 (br. t, CH2ꢀC(6/I)); 27.63 (q, Me3C); signals of both rotamers: 103.72 (d,
2 C(5)); 60.62, 60.34, 60.23, 60.17, 60.02, 59.97, 59.93, 59.20 (8t, 2 MeCH2O, 2 CH2NCH2C(6), CH2ꢀC(6/
II)); 45.22 (t, CH2ꢀN(1/I)); 43.94 (t, CH2ꢀN(1/II)); 14.05, 13.96 (2q, 2 MeCH2O). HR-MALDI-MS:
661.2541 (44, [M þ Na]þ, C26H38N8NaO1þ1 ; calc. 661.2552), 639.2733 (33, [M þ H]þ, C26H39N8O1þ1 ; calc.
t
639.2733), 605.1911 (16, [M ꢀ Bu þ H þ Na]þ, C22H30N8NaO1þ1 ; calc. 605.1921), 583.2101 (100, [M ꢀ
tBu þ 2 H]þ, C22H31N8O1þ1 ; calc. 583.2101).

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Helvetica Chimica Acta – Vol. 94 (2011)
6-[(1-(2-Ethoxy-2-oxoethyl)-2-{[(9H-fluoren-9-yl)methoxy]carbonyl}hydrazino)methyl]uracil-1-
acetyl-(12 ! 63-N)-6-{[1-(2-ethoxy-2-oxoethyl)hydrazino]methyl}uracil-1-acetic Acid (¼ [6-{[1-(2-
Ethoxy-2-oxoethyl)-2-{[6-({1-(2-ethoxy-2-oxoethyl)-2-[(9H-fluoren-9-ylmethoxy)carbonyl]hydrazinyl}-
methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl]acetyl}hydrazinyl]methyl}-2,4-dioxo-3,4-dihydropyri-
midin-1(2H)-yl]acetic acid; 7). A soln. of 5 (1 g, 1.2 mmol) and Et3SiH (0.2 ml, 1.3 mmol) in CH2Cl2
(10 ml) was treated dropwise with TFA (2.6 ml, 35 mmol) and stirred for 2.5 h at r.t. After evaporation,
the solid residue was washed with Et2O (8 ꢃ 10 ml). Filtration gave 7 (0.92 g, 98%). White powder. Rf
(AcOEt/MeOH/H2O 7:2 :1) 0.33. M.p. 148 – 1528. UV (MeOH): 266 (34600), 300 (5800). IR (ATR):
3680 – 2140w (br.), 3213w (br.), 2984w, 2808w, 1673s (br.), 1463m, 1450m, 1391m, 1292w, 1204s, 1158m,
1105w, 1072w, 1023w, 983w, 958w, 929w, 893w, 827w. 1H-NMR (400 MHz, (D6)DMSO; ca. 2 :1 mixture of
rotamers; assignments based on a DQFCOSY, a HSQC, and a HMBC spectrum): 11.36 (0.7 H), 11.34
(0.6 H), 11.28 (0.7 H) (3s, 2 HꢀN(3)); 9.66 (0.4 H), 8.88 (0.6 H) (2s, HNꢀNCH2C(6/I)); 8.85 (s,
HNꢀNCH2C(6/II)); 7.88 – 7.30 (m, 8 arom. H); 5.62 (s, 0.7 HꢀC(5/I)); 5.58 (s, 0.9 HꢀC(5/II)); 5.54 (s,
0.3 HꢀC(5/I), 0.1 HꢀC(5/II)); 5.28 (0.2 H), 5.06 (0.2 H), 4.73 (0.3 H), 4.63 (0.3 H) (4 br. d, J ¼ 17.4,
0.5 CH2ꢀN(1/I), 0.5 CH2ꢀN(1/II)); 4.86 (s, 0.5 CH2ꢀN(1/I)); 4.80 (s, 0.5 CH2ꢀN(1/II)); 4.27 (br. s,
CH2ꢀC(9’)); 4.18 (br. s, HꢀC(9’)); 4.07 (q, J ¼ 6.9, 2 MeCH2O); 3.97 (br. s, 0.3 H); 3.93 (br. s, 0.4 H); 3.85
(s, CH2ꢀC(6/I)); 3.79 (s, CH2NCH2C(6/I)); 3.73, 3.71, 3.69 (3 br. s, CH2ꢀC(6/II), CH2NCH2C(6/II));
1.21 – 1.11 (m, 2 MeCH2O). 13C-NMR (100 MHz, (D6)DMSO; ca. 2 :1 mixture of rotamers; assignments
based on a HSQC and a HMBC spectrum): signals of the major rotamer: 170.23 (s, CO2H); 167.05 (s,
C(O)CH2N(1/II)); signals of the minor rotamer: 170.31, 170.13 (2s, CO2H, C(O)CH2N(1/II)); signals of
both rotamers: 169.33 (s, C(O)CH2NCH2C(6/II)); 169.10 (s, C(O)CH2NCH2C(6/I)); 162.59, 162.56,
162.51 (3s, 2 C(4)); 155.25 (br. s, NCO2); 151.79, 151.74, 151.63, 151.29, 149.95 (5s, 2 C(2), C(6/II)); 151.08
(br. s, C(6/I)); 143.65 (2 br. s); 140.74 (2s); 127.70 (2s); 127.15 (2s); 125.15 (2 br. s); 120.12 (2s); 103.89,
103.47, 103.26 (3d, 2 C(5)); 65.69 (br. t, CH2ꢀC(9’)); 60.56, 60.42, 60.37, 60.30, 59.14, 57.59 (6t, 2 MeCH2O,
CH2ꢀC(6/II), CH2NCH2C(6/II)); 56.80 (t, CH2NCH2C(6/I)); 56.32 (t, CH2ꢀC(6/I)); 46.62 (d, C(9’));
44.77, 44.08, 43.94 (3 br. t, 2 CH2ꢀN(1)); 14.03, 14.02, 13.99, 13.92 (4q, 2 MeCH2O). HR-MALDI-MS:
827.2609 (52, [M þ Na]þ, C37H40N8NaO1þ3 ; calc. 827.2607), 805.2791 (40, [M þ H]þ, C37H41N8O1þ3 ; calc.
805.2788).
tert-Butyl 8-[(1-(2-Ethoxy-2-oxoethyl)-2-{[(9H-fluoren-9-yl)methoxy]carbonyl}hydrazino)methyl]-
N6-(benzyloxycarbonyl)adenine-9-acetyl-(92 ! 63-N)-6-{[1-(2-ethoxy-2-oxoethyl)hydrazino]methyl}ura-
cil-1-acetate (¼(9H-Fluoren-9-yl)methyl 2-[(6-{[(Benzyloxy)carbonyl]amino}-9-{2-[2-({3-[2-(tert-bu-
toxy)-2-oxoethyl]-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl}methyl)-2-(2-ethoxy-2-oxoethyl)hydrazin-
yl]-2-oxoethyl}-9H-purin-8-yl)methyl]-2-(2-ethoxy-2-oxoethyl)hydrazinecarboxylate; 9). A soln. of
3
(1 g, 2.8 mmol), 8 (2.1 g, 3.1 mmol), and EtNiPr2 (0.53 ml, 3.1 mmol) in DMF (20 ml) was cooled to 08,
treated with a soln. of HBTU (1.17 g, 3.1 mmol) in DMF (5 ml), stirred for 2.5 h at r.t., treated with sat.
NaHCO3 soln. (100 ml), and extracted with AcOEt (3 ꢃ 100 ml). Drying of the combined org. layers
(MgSO4), filtration, evaporation, FC (CH2Cl2/MeOH 95 :5 ! 93 :7), and MPLC (CH2Cl2/MeOH
96 :4 ! 94 :6, flow: 30 ml/min) gave 9 (2.2 g, 77%). White powder. Rf (CHCl3/MeOH 95 :5) 0.23. M.p. >
1128 (dec.). UV (CHCl3): 269 (49200), 301 (6000). IR (ATR): 3230w (br.), 2980w, 1728s (br.), 1687s
(br.), 1612m, 1592m, 1533w, 1495w, 1451m, 1423m, 1392m, 1368m, 1321w, 1202s, 1154s, 1103m, 1075w,
1028m, 978w, 844m. 1H-NMR (400 MHz, (D8)THF; ca. 2 :1 mixture of rotamers; assignments based on a
HSQC and a HMBC spectrum): 10.53 (0.3 H), 10.41 (0.7 H) (2s, HꢀN(3/I)); 9.57 (s, HNꢀC(6/II)); 9.52
(0.3 H), 9.09 (0.7 H) (2s, HNꢀNCH2C(6/I)); 8.50 (0.7 H), 8.46 (0.3 H) (2s, HꢀC(2/II)); 8.23 (0.3 H), 8.18
(0.7 H) (2s, HNꢀNCH2C(8/II)); 7.74 – 7.16 (m, 13 arom. H); 5.90, 5.43 (2 br. d, J ¼ 17.6, 0.25 CH2ꢀN(1/I),
0.25 CH2ꢀN(9/II)); 5.32 (br. s, 0.5 CH2ꢀN(9/II)); 5.18 – 5.12 (m, PhCH2 , 0.25 CH2ꢀN(1/I) or
0.25 CH2ꢀN(9/II)); 4.96 – 4.89 (m, 0.5 CH2ꢀN(1/I), 0.25 CH2ꢀN(1/I) or 0.25 CH2ꢀN(9/II)); 5.60
(0.3 H), 5.55 (0.7 H) (2s, HꢀC(5/I)); 4.44 (br. s, CH2ꢀC(8/II)); 4.28 (0.6 H), 4.24 (1.4 H) (2d, J ¼ 7.2,
CH2ꢀC(9’)); 4.20 – 4.09 (m, 2 MeCH2O, HꢀC(9’)); 4.06 (br. s, 0.2 H); 4.00 (s, 0.4 H); 3.97 (s, CH2ꢀC(6/
I)); 3.88 (s, CH2NCH2C(6/I)); 3.79 (br. s, CH2NCH2C(8/II)); 3.69 (br. s, 0.6 H); 1.47 (3 H), 1.37 (6 H) (2s,
t-Bu); 1.29 – 1.16 (m, 2 MeCH2O). 13C-NMR (100 MHz, (D8)THF; ca. 2 :1 mixture of rotamers;
assignments based on a HSQC and a HMBC spectrum): signals of the major rotamer: 170.75, 170.49 (2s,
t
2 CO2Et); 169.35 (s, CO2 Bu); 167.67 (s, C(O)CH2N(9/II)); 163.04 (s, C(4/I)); 156.60 (s, NꢀNCO2);
154.68 (s, C(4/II)); 152.98 (d, C(2/II)); 152.67 (s, C(2/I)); 152.32 (s, NCO2ꢀC(6/II)); 151.30 (s, C(6/I));

Helvetica Chimica Acta – Vol. 94 (2011)
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122.55 (s, C(5/II)); 105.32 (d, C(5/I)); 82.00 (s, Me3C); 61.36, 61.32 (2t, 2 MeCH2O); 57.77 (t,
CH2NCH2C(8/II)); 56.96 (t, CH2NCH2C(6/I)); 56.70 (t, CH2ꢀC(6/I)); 54.30 (t, CH2ꢀC(8/II)); 46.19 (t,
CH2ꢀN(1/I)); 44.70 (t, CH2ꢀN(9/II)); signals of the minor rotamer: 170.99, 170.25 (2s, 2 CO2Et); 168.95
t
(s, CO2 Bu); 162.83 (s, C(4/I)); 156.66 (s, NꢀNCO2); 154.78 (s, C(4/II)); 152.86 (d, C(2/II)); 152.40, 151.73
(2s, C(2/I), C(6/I), NCO2ꢀC(6/II)); 122.47 (s, C(5/II)); 106.54 (d, C(5/I)); 82.33 (s, Me3C); 61.55 (t,
MeCH2O); 59.53, 59.15, 54.70 (3t, CH2NCH2C(8/II), CH2NCH2C(6/I), CH2ꢀC(8/II)); 57.23 (t,
CH2ꢀC(6/I)); 46.27 (t, CH2ꢀN(1/I)); 44.46 (t, CH2ꢀN(9/II)); signals of both rotamers: 150.19, 149.99
(2s, C(6/II), C(8/II)); 145.01 (2s); 142.18 (2s); 137.75 (s); 129.04 (2d); 128.89 (2d); 128.58 (d); 128.29
(2d); 127.73 (2d); 125.99 (2d); 120.51 (2d); 66.95 (t, PhCH2, CH2ꢀC(9’)); 48.06 (d, C(9’)); 28.19 (q,
Me3C); 14.48 (q, 2 MeCH2O). HR-MALDI-MS: 1040.3892 (100, [M þ Na]þ, C50H55N11NaO1þ3 ; calc.
1040.3873), 1018.4082 (59, [M þ H]þ, C50H56N11O1þ3 ; calc. 1018.4054).
6-{[1-(2-Ethoxy-2-oxoethyl)-2-acetylhydrazino]methyl}uracil-1-acetyl-[(12 ! 63-N)-6-{[1-(2-ethoxy-
2-oxoethyl)hydrazino]methyl}uracil-1-acetyl]8-(12 ! 63-N)-6-{[1-(2-ethoxy-2-oxoethyl)hydrazino]meth-
yl}uracil-9-acetamid (¼ Ethyl {1-[(6-{[(Benzyloxy)carbonyl]amino}-9-{2-[2-({3-[2-(tert-butoxy)-2-oxo-
ethyl]-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl}methyl)-2-(2-ethoxy-2-oxoethyl)hydrazinyl]-2-oxoeth-
yl}-9H-purin-8-yl)methyl]hydrazinyl}acetate; 10). A soln. of 9 (800 mg, 0.8 mmol) in DMF (10 ml) was
treated dropwise with piperidine (0.8 ml, 7.9 mmol), stirred for 3.5 h at r.t., and evaporated. MPLC
(CH2Cl2/MeOH 93 :7, flow: 30 ml/min) gave 10 (503 mg, 80%). White powder. Rf (CH2Cl2/MeOH 9 :1)
0.50. M.p. 104 – 1088. UV (CHCl3): 270 (27730). IR (ATR): 3196w (br.), 2979w, 2931w, 1734s, 1687s,
1613m, 1590m, 1535w, 1498w, 1454m, 1424m, 1391m, 1369m, 1321w, 1296w, 1232m, 1201s, 1155s, 1099m,
1027m, 976w, 863w, 820w. 1H-NMR (400 MHz, (D8)THF; ca. 4 :1 mixture of rotamers; assignments
based on a HSQC and a HMBC spectrum): 10.44 (br. s, HꢀN(3/I)); 9.64 (0.8 H), 9.56 (0.2 H) (2 br. s,
HNꢀC(6/II)); 8.97 (0.8 H), 8.27 (0.2 H) (2s, HNꢀNCH2C(6/I)); 8.50 (0.8 H), 8.45 (0.2 H) (2s, HꢀC(2/
II)); 7.46 – 7.23 (m, 5 arom. H); 5.64 (0.2 H), 5.57 (0.8 H) (2s, HꢀC(5/I)); 5.57 (0.2 H), 5.26 (0.3 H), 5.04
(0.3 H), 4.84 (0.2 H) (4 br. d, J ¼ 17.4, 0.5 CH2ꢀN(1/I), 0.5 CH2ꢀN(9/II)); 5.23 (s, PhCH2); 5.12 (s,
0.5 CH2ꢀN(9/II)); 4.90 (s, 0.5 CH2ꢀN(1/I)); 4.19 (s, CH2ꢀC(8/II)); 4.14, 4.13 (2q, J ¼ 7.2, 2 MeCH2O);
3.97 (s, CH2ꢀC(6/I)); 3.93 (0.4 H); 3.88 (s, CH2NCH2C(6/I)); 3.55 (s, CH2NCH2C(8/II)); 3.50 (0.4 H);
1.46 (2 H), 1.37 (7 H) (2s, t-Bu); 1.23 (t, J ¼ 7.2, 2 MeCH2O). 13C-NMR (100 MHz, (D8)THF;
assignments based on a HSQC and a HMBC spectrum): 171.09 (s, C(O)CH2NCH2C(8/II)); 170.57 (s,
t
C(O)CH2NCH2C(6/I)); 169.31 (s, CO2 Bu); 167.44 (s, C(O)CH2N(9/II)); 163.03 (s, C(4/I)); 154.75 (s,
C(4/II)); 152.75 (s, C(8/II)); 152.66 (s, C(2/I)); 152.40 (s, NCO2); 152.26 (d, C(2/II)); 151.32 (s, C(6/I));
150.08 (s, C(6/II)); 137.82 (s); 129.04 (2d); 128.88 (2d); 128.56 (d); 122.74 (s, C(5/II)); 105.21 (d, C(5/I));
82.04 (s, Me3C); 66.94 (t, PhCH2); 61.33, 60.85 (2t, 2 MeCH2O); 60.97 (t, CH2NCH2C(8/II)); 58.48 (t,
CH2ꢀC(8/II)); 56.91 (t, CH2NCH2C(6/I)); 56.49 (t, CH2ꢀC(6/I)); 46.18 (t, CH2ꢀN(1/I)); 44.56 (t,
CH2ꢀN(9/II)); 28.16 (q, Me3C); 14.52, 14.44 (2q, 2 MeCH2O). HR-MALDI-MS: 818.3170 (39, [M þ
Na]þ, C35H45N11NaOþ11 ; calc. 818.3192), 796.3366 (100, [M þ H]þ, C35H46N11O1þ1 ; calc. 796.3373).
8-{{1-(2-Ethoxy-2-oxoethyl)-2-[(9H-fluoren-9-yl)methoxycarbonyl]hydrazino}methyl}-N6-(benzyl-
oxycarbonyl)adenine-9-acetyl-(92 ! 63-N)-6-{[1-(2-ethoxy-2-oxoethyl)hydrazino]methyl}uracil-1-acetic
Acid (¼ [6-{[2-{[6-{[(Benzyloxy)carbonyl]amino}-8-({1-(2-ethoxy-2-oxoethyl)-2-[(9H-fluoren-9-ylme-
thoxy)carbonyl]hydrazinyl}methyl)-9H-purin-9-yl]acetyl}-1-(2-ethoxy-2-oxoethyl)hydrazinyl]methyl}-
2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl]acetic Acid; 11). A soln. of 9 (800 mg, 0.8 mmol) in CH2Cl2
(10 ml) was treated with Et3SiH (0.2 ml, 1.2 mmol) and TFA (1.75 ml, 23.6 mmol), stirred for 3 h at r.t.,
and evaporated. MPLC (CH2Cl2/MeOH 90 :10 ! 80 :20, flow: 30 ml/min) gave 11 (684 mg, 84%). White
powder. Rf (AcOEt/MeOH/H2O 7:2 :1) 0.50. M.p. > 1398 (dec.). UV (CHCl3): 269 (42500), 301 (5200).
IR (ATR): 3510 – 2330w (br.), 3208w (br.), 2982w, 1682s (br.), 1613m, 1530w, 1495w, 1451m, 1416m,
1
1392m, 1322w, 1202s, 1166s, 1103m, 1027m, 826m. H-NMR (500 MHz, (D8)THF; ca. 2 :1 mixture of
rotamers; assignments based on a HSQC and a HMBC spectrum): 10.60 (0.3 H), 10.46 (0.7 H) (2s,
HꢀN(3/I)); 9.61 (br. s, HNꢀC(6/II)); 9.21 (0.7 H), 8.28 (0.3 H) (2s, HNꢀNCH2C(6/I)); 8.51 (0.7 H), 8.48
(0.3 H) (2s, HꢀC(2/II)); 8.21 (br. s, HNꢀNCH2C(8/II)); 7.74 – 7.16 (m, 13 arom. H); 5.94, 5.48 (2 br. d, J ¼
17.0, 0.25 CH2ꢀN(1/I), 0.25 CH2ꢀN(9/II)); 5.64 (0.3 H), 5.58 (0.7 H) (2s, HꢀC(5/I)); 5.34 (br. s,
0.5 CH2ꢀN(9/II)); 5.17 (s, PhCH2); 5.02 (s, 0.25 CH2ꢀN(1/I), 0.25 CH2ꢀN(9/II)); 5.00 (s, 0.5 CH2ꢀN(1/
I)); 4.46 (br. s, CH2ꢀC(8/II)); 4.28 (0.6 H), 4.22 (1.4 H) (2d, J ¼ 7.5, CH2ꢀC(9’)); 4.19 – 4.09 (m,
2 MeCH2O, HꢀC(9’)); 4.01 (s, CH2ꢀC(6/I)); 4.00 (br. s, 0.5 H); 3.88 (s, CH2NCH2C(6/I)); 3.81 (br. s,

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Helvetica Chimica Acta – Vol. 94 (2011)
CH2NCH2C(8/II)); 3.72 (s, 0.7 H); 1.24 – 1.16 (m, 2 MeCH2O). 13C-NMR (125 MHz, (D8)THF; ca. 2 :1
mixture of rotamers; assignments based on a HSQC and a HMBC spectrum): signals of the major
rotamer: 171.01, 170.39 (2s, 2 CO2Et); 163.13 (s, C(4/I)); 156.53 (s, NꢀNCO2); 154.60 (s, C(4/II)); 150.15
(s, C(6/II)); 145.00 (2s); 142.14 (2s); 137.73 (s); 129.03 (2d); 128.90 (2d); 128.56 (d); 128.26 (2d); 127.72
(2d); 126.00 (2d); 122.43 (s, C(5/II)); 120.48 (2d); 61.34, 61.26 (2t, 2 MeCH2O); 56.82 (t, CH2ꢀC(6/I));
54.11 (t, CH2ꢀC(8/II)); 48.02 (d, C(9’)); signals of the minor rotamer: 167.69 (s, C(O)CH2N(9/II)); 162.98
(s, C(4/I)); 156.59 (s, NꢀNCO2); 154.70 (s, C(4/II)); 149.98 (s, C(6/II)); 145.00 (2s); 142.17 (2s); 137.78
(s); 129.01 (2d); 128.86 (2d); 128.51 (d); 128.31 (2d); 127.72 (2d); 125.96 (2d); 122.40 (s, C(5/II)); 120.52
(2d); 61.51, 61.30 (2t, 2 MeCH2O); 58.96 (t, CH2ꢀC(6/I)); 54.46 (t, CH2ꢀC(8/II)); 48.09 (d, C(9’)); signals
of both rotamers: 170.93, 170.73, 170.60, 170.52, 170.43 (5s, 2 CO2Et, CO2H, C(O)CH2N(9/II)); 152.94
(d, C(2/II)); 152.87; 152.70 (s, C(2/I)); 152.28 (s, C(6/II)ꢀNCO2); 151.43, 151.40 (2s, C(6/I), C(8/II));
105.22 (d, C(5/I)); 67.03 (t, PhCH2, CH2ꢀC(9’)); 57.71 (br. t, CH2NCH2C(8/II)); 56.82 (t, CH2NCH2C(6/
I)); 45.25 (t, CH2ꢀN(1/I)); 44.76 (t, CH2ꢀN(9/II)); 14.47, 14.46 (2q, 2 MeCH2O). HR-MALDI-MS:
984.3229 (100, [M þ Na]þ, C46H47N11NaO1þ3 ; calc. 984.3247), 962.3368 (86, [M þ H]þ, C46H48N11Oþ13
;
calc. 962.3428), 876.2634 (29, [M ꢀ BnOH þ Na]þ, C39H39N11NaO1þ2 ; calc. 876.2672), 854.2862 (34, [M ꢀ
BnOH þ H]þ, C39H40N11O1þ2 ; calc. 854.2858).
tert-Butyl 8-{{1-(2-Ethoxy-2-oxoethyl)-2-[(9H-fluoren-9-yl)methoxycarbonyl]hydrazino}methyl}-
adenine-9-acetyl-(92 ! 63-N)-6-{[1-(2-ethoxy-2-oxoethyl)hydrazino]methyl}uracil-1-acetate (¼(9 H-Flu-
oren-9-yl)methyl 2-[(6-Amino-9-{2-[2-({3-[2-(tert-butoxy)-2-oxoethyl]-2,6-dioxo-1,2,3,6-tetrahydropyri-
midin-4-yl}methyl)-2-(2-ethoxy-2-oxoethyl)hydrazinyl]-2-oxoethyl}-9H-purin-8-yl)methyl]-2-(2-ethoxy-
2-oxoethyl)hydrazinecarboxylate; 12). A suspension of Pd(OAc)2 (110 mg, 0.5 mmol) in MeOH/CH2Cl2
1:1 (5 ml) was stirred under H2 for 1 h at r.t., treated with a soln. of 9 (500 mg, 0.5 mmol) in MeOH/
CH2Cl2 1:1 (10 ml), stirred for 30 h, and filtered through Celite. Evaporation of the filtrate and MPLC
(CH2Cl2/MeOH 93 :7, flow: 25 ml/min) gave 12 (410 mg, 94%). White powder. Rf (CH2Cl2/MeOH 9 :1)
0.38. M.p. > 1438 (dec.). UV (CHCl3): 267 (35600), 301 (5000). IR (ATR): 3406w, 3323w, 3270w, 3230w,
3192w (br.), 2977w, 1735s, 1691s, 1666s, 1651s, 1607m, 1582w, 1535w, 1465m, 1436m, 1398m, 1372m,
1325m, 1275m, 1248m, 1232m, 1206s, 1145s, 1079w, 1053w, 1026m, 961w, 944w, 904w, 858w, 836w, 816w.
1H-NMR (400 MHz, (D8)THF; ca. 3 :1 mixture of rotamers; assignments based on a HSQC and a HMBC
spectrum): 10.76 (0.3 H), 10.54 (0.7 H) (2 br. s, HꢀN(3/I)); 9.02 (br. s, HNꢀNCH2C(6/I)); 8.15 (br. s,
HNꢀNCH2C(8/II)); 8.10 (s, HꢀC(2/II)); 7.76 – 7.19 (m, 8 arom. H); 6.45 (br. s, NH2); 5.82 (0.2 H), 5.40
(0.2 H), 5.14 (0.3 H), 4.91 (0.3 H) (4 br. d, J ¼ 17.4, 0.5 CH2ꢀN(1/I), 0.5 CH2ꢀN(9/II)); 5.58 (0.3 H), 5.56
(0.7 H) (2s, HꢀC(5/I)); 5.21 (br. s, 0.5 CH2ꢀN(9/II)); 4.97 (s, 0.5 CH2ꢀN(1/I)); 4.38 (br. s, CH2ꢀC(8/II));
4.29 (0.6 H), 4.23 (1.4 H) (2d, J ¼ 7.0, CH2ꢀC(9’)); 4.21 – 4.08 (m, 2 MeCH2O, HꢀC(9’)); 4.05 (br. s,
0.2 H); 4.00 (br. s, 0.3 H); 3.97 (s, CH2ꢀC(6/I)); 3.86 (s, CH2NCH2C(6/I)); 3.77 (br. s, CH2NCH2C(8/II));
3.67, 3.66, 3.65 (3 br. s, 0.6 H); 1.46 (2 H), 1.38 (7 H) (2s, t-Bu); 1.25 – 1.15 (m, 2 MeCH2O). 13C-NMR
(100 MHz, (D8)THF; ca. 4 :1 mixture of rotamers; assignments based on a HSQC and a HMBC
t
spectrum): signals of the major rotamer: 170.69, 170.51 (2s, 2 CO2Et); 169.36 (s, CO2 Bu); 163.08 (s, C(4/
I)); 156.78 (s, C(6/II)); 156.51 (s, NꢀNCO2); 148.19 (s, C(8/II)); 145.08 (2s); 142.18 (2s); 128.26 (2d);
127.76 (2d); 126.07 (2d); 120.49 (2d); 119.18 (s, C(5/II)); 105.33 (d, C(5/I)); 81.96 (s, Me3C); 61.26 (t,
2 MeCH2O); 56.89 (t, CH2NCH2C(6/I)); 56.69 (t, CH2ꢀC(6/I)); 48.09 (d, C(9’)); 46.20 (t, CH2ꢀN(1/I));
44.63 (t, CH2ꢀN(9/II)); 28.19 (q, Me3C); signals of the minor rotamer: 170.92, 170.58 (2s, 2 CO2Et);
t
169.00 (s, CO2 Bu); 163.03 (s, C(4/I)); 156.67, 156.57 (2s, C(6/II), NꢀNCO2); 150.28 (s); 148.59 (s, C(8/
II)); 145.05 (2s); 142.20 (2s); 128.32 (2d); 127.76 (2d); 125.99 (2d); 120.54 (2d); 119.01 (s, C(5/II)); 106.35
(d, C(5/I)); 82.25 (s, Me3C); 61.49, 61.19 (2t, 2 MeCH2O); 59.31 (t); 59.07 (t); 54.72 (t); 48.16 (d, C(9’));
46.36 (t, CH2ꢀN(1/I)); 44.13 (t, CH2ꢀN(9/II)); 28.23 (q, Me3C); signals of both rotamers: 167.96 (s,
C(O)CH2N(9/II)); 153.87 (d, C(2/II)); 152.75 (s, C(2/I), C(4/II)); 151.33 (s, C(6/I)); 66.94 (t, CH2ꢀC(9’));
57.43 (br. t, CH2NCH2C(8/II)); 54.18 (br. t, CH2ꢀC(8/II)); 14.49, 14.45 (2q, 2 MeCH2O). HR-MALDI-
MS: 906.3488 (100, [M þ Na]þ, C42H49N11NaO1þ1 ; calc. 906.3505), 884.3661 (88, [M þ H]þ, C42H50N11Oþ11
;
t
calc. 884.3686), 850.2872 (12, [M ꢀ Bu þ H þ Na]þ, C38H41N11NaO1þ1 ; calc. 850.2874), 828.3036 (24, [M ꢀ
tBu þ 2 H]þ, C38H42N11O1þ1 ; calc. 828.3054). Anal. calc. for C42H49N11O11 · CH4O (915.96): C 56.39, H 5.83,
N 16.82; found: C 56.30, H 5.76, N 17.01.
tert-Butyl 8-{[1-(2-Ethoxy-2-oxoethyl)hydrazino]methyl}adenine-9-acetyl-(92 ! 63-N)-6-{[1-(2-
ethoxy-2-oxoethyl)hydrazino]methyl}uracil-1-acetate (¼ Ethyl {1-[(6-Amino-9-{2-[2-({3-[2-(tert-bu-

Helvetica Chimica Acta – Vol. 94 (2011)
1177
toxy)-2-oxoethyl]-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl}methyl)-2-(2-ethoxy-2-oxoethyl)hydrazin-
yl]-2-oxoethyl}-9H-purin-8-yl)methyl]hydrazinyl}acetate; 13). A soln. of 12 (290 mg, 0.33 mmol) in DMF
(10 ml) was treated dropwise with piperidine (0.32 ml, 3.3 mmol), stirred for 30 min at r.t., and
evaporated. The solid residue was suspended in Et2O (10 ml) and washed with Et2O (5 ꢃ 10 ml).
Filtration gave 13 (215 mg, 99%). White powder. Rf (CH2Cl2/MeOH 9 :1) 0.28. M.p. > 1858 (dec.). UV
(MeOH): 264 (24800). IR (ATR): 3408w, 3318w, 3198w (br.), 2982w, 2773w (br.), 1733m, 1703s, 1678s,
1647s, 1610m, 1534w, 1456m, 1441m, 1421m, 1394m, 1372m, 1322w, 1292w, 1273w, 1235m, 1196s, 1154s,
1113m, 1075w, 1024m, 1002w, 983w, 960m, 932w, 890w, 844m, 818m. 1H-NMR (300 MHz, (D6)DMSO; ca.
3 :2 mixture of rotamers; assignments based on a HSQC and a HMBC spectrum): 11.40 (br. s, HꢀN(3/I));
9.65 (0.6 H), 8.96 (0.4 H) (2s, HNꢀNCH2C(6/I)); 8.05 (0.6 H), 7.99 (0.4 H) (2s, HꢀC(2/II)); 7.17 (1.2 H),
7.10 (0.8 H) (2 br. s, H2NꢀC(6/II)); 5.62 (0.6 H), 5.59 (0.4 H) (2s, HꢀC(5/I)); 5.39 (br. d, J ¼ 17.7,
0.2 CH2ꢀN(9/II)); 4.91 – 4.87 (br. m, 0.8 CH2ꢀN(9/II)); 5.07 (0.4 H), 4.65 (0.4 H) (2 br. d, J ¼ 17.4,
0.4 CH2ꢀN(1/I)); 4.86 (s, 0.6 CH2ꢀN(1/I)); 4.22 – 4.07 (m, 2 MeCH2O); 4.04 (1.2 H), 3.99 (0.8 H) (2 br. s,
CH2ꢀC(8/II)); 3.92 (0.9 H), 3.78 (1.1 H) (2s, CH2NCH2C(6/I)); 3.86 (0.6 H), 3.64 (0.6 H) (2 br. d, J ¼
14.0, 0.6 CH2ꢀC(6/I)); 3.80 (s, 0.4 CH2ꢀC(6/I)); 3.73 (br. s, H2NꢀN); 3.50 (0.6 H), 3.48 (1.4 H) (2s,
CH2NCH2C(8/II)); 1.43 (3.8 H), 1.35 (5.2 H) (2s, t-Bu); 1.27 – 1.17 (m, 2 MeCH2O). 13C-NMR
(100 MHz, (D6)DMSO; ca. 3 :2 mixture of rotamers; assignments based on a HSQC and a HMBC
t
spectrum): signals of the major rotamer: 167.98 (s, CO2 Bu); 165.92 (s, C(O)CH2N(9/II)); 162.49 (s, C(4/
I)); 155.34 (s, C(6/II)); 151.65 (s, C(2/I)); 151.20 (s, C(4/II)); 150.62 (s, C(6/I)); 148.28 (s, C(8/II)); 117.39
(s, C(5/II)); 103.86 (d, C(5/I)); 81.50 (s, Me3C); 56.20 (t, CH2ꢀC(6/I)); 56.60 (t, CH2ꢀC(8/II)); 45.19 (t,
CH2ꢀN(1/I)); 43.24 (t, CH2ꢀN(9/II)); 27.54 (q, Me3C); signals of the minor rotamer: 169.35 (s,
t
C(O)CH2N(9/II)); 167.66 (s, CO2 Bu); 162.59 (s, C(4/I)); 155.23 (s, C(6/II)); 151.82 (s, C(2/I)); 151.14 (s,
C(4/II)); 149.57 (s, C(6/I)); 148.68 (s, C(8/II)); 117.33 (s, C(5/II)); 105.39 (d, C(5/I)); 81.77 (s, Me3C);
57.64 (t, CH2ꢀC(6/I)); 56.45 (t, CH2ꢀC(8/II)); 45.34 (t, CH2ꢀN(1/I)); 42.67 (t, CH2ꢀN(9/II)); 27.63 (q,
Me3C); signals of both rotamers: 170.20, 170.04, 169.24, 169.23 (4s, 2 CO2Et); 152.27 (d, C(2/II)); 60.66,
60.38, 59.87, 59.84 (4t, 2 MeCH2O); 59.66 (t, CH2NCH2C(8/II)); 59.22 (t, CH2NCH2C(6/I)); 14.06, 14.02,
13.98, 13.95 (4q, 2 MeCH2O). HR-MALDI-MS: 684.2849 (19, [M þ Na]þ, C27H39N11NaO9þ ; calc.
t
684.2824), 662.3000 (100, [M þ H]þ, C27H40N11O9þ ; calc. 662.3005), 606.2385 (41, [M ꢀ Bu þ 2 H]þ,
C23H32N11Oþ9 ; calc. 606.2379).
8-[(1-(2-Ethoxy-2-oxoethyl)-2-{[(9H-fluoren-9-yl)methoxy]carbonyl}hydrazino)methyl]-N6-(ben-
zyloxycarbonyl)adenine-9-acetyl-(92 ! 83-N)-8-{[1-(2-ethoxy-2-oxoethyl)hydrazino]methyl}-N6-[(benzyl-
oxy)carbonyl]adenine-9-acetic Acid (¼(6-{[(Benzyloxy)carbonyl]amino}-8-{[2-{[6-{[(benzyloxy)carbo-
nyl]amino}-8-({1-(2-ethoxy-2-oxoethyl)-2-[(9H-fluoren-9-ylmethoxy)carbonyl]hydrazinyl}methyl)-9H-
purin-9-yl]acetyl}-1-(2-ethoxy-2-oxoethyl)hydrazinyl]methyl}-9H-purin-9-yl)acetic Acid; 16). A soln. of
1513) (1.32 g, 1.12 mmol) in CH2Cl2 (20 ml) was treated with Et3SiH (1.8 ml, 11.2 mmol) and TFA (2.5 ml,
34 mmol), stirred for 2 d at r.t., and evaporated. The solid residue was washed with Et2O (5 ꢃ 20 ml).
MPLC (CH2Cl2/MeOH 85 :15 ! 80 :20, flow: 30 ml/min) gave 16 (863 mg, 69%). Yellow foam. Rf
(CH2Cl2/MeOH 8 :2) 0.57. UV (CHCl3): 270 (54200), 301 (5300). IR (ATR): 3674 – 2351w (br.), 3230w
(br.), 2981w, 1727s (br.), 1612m, 1592m, 1533w, 1497m, 1450m, 1391m, 1320m, 1296m, 1199s, 1163s,
1101m, 1028m, 969w, 897w, 855w. 1H-NMR (400 MHz, (D6)DMSO; ca. 1:1 mixture of rotamers;
assignments based on a HSQC and a HMBC spectrum): 14.22 – 12.45 (br. s, CO2H); 10.67 (0.6 H), 10.61
(0.5 H), 10.55 (0.5 H), 10.48 (0.4) (4s, 2 HNꢀC(6)); 9.81 (0.5 H), 9.13 (0.5 H) (2s, HNꢀNCH2C(8/I));
8.79 (0.5 H), 8.75 (0.5 H) (2s, HNꢀNCH2C(8/II)); 8.63 (0.5 H), 8.56 (0.5 H), 8.39 (0.5 H), 8.08 (0.5 H)
(4s, 2 HꢀC(2)); 7.84 – 7.18 (m, 18 arom. H); 5.86 (0.5 H) (br. d, J ¼ 17.2, CH2ꢀN(9)); 5.46 – 5.35 (1.5 H)
(m, CH2ꢀN(9)); 5.23 (2.1 H) (s, CH2ꢀN(9), PhCH2); 5.18 – 5.16 (3.9 H) (m, CH2ꢀN(9), PhCH2); 4.54
(0.2 H); 4.50 (0.3 H); 4.42 (1.5 H) (s, CH2ꢀC(8)); 4.23 – 4.00 (9.5 H) (m, CH2ꢀC(8), 2 MeCH2O,
CH2ꢀC(9’), HꢀC(9’)); 3.78 (1.0 H), 3.68 (2.0 H) (2s, 2 CH2NCH2C(8)); 3.61 (s, 0.2 H); 3.58 (s, 0.3 H);
1.19 – 1.12 (m, 2 MeCH2O). 13C-NMR (100 MHz, (D6)DMSO; ca. 1:1 mixture of rotamers; assignments
based on a HSQC and a HMBC spectrum): 169.40, 169.22, 169.17, 169.11, 169.00, 168.91, 165.66 (7s,
CO2H, 2 CO2Et, C(O)CH2N(9/II)); 155.07 (br. s, NꢀNCO2); 153.39, 153.29 (2s, 2 C(4)); 152.00, 151.93
(2s, 2 C(6)ꢀNCO2); 151.59 – 151.52, 151.29 – 151.13 (2 br. s, 2 C(8)); 151.00, 150.89, 150.37, 150.21 (4d,
13
)
Dimer 15 was synthesized analogously to dimers 5 and 9, without purification and analysis.

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Helvetica Chimica Acta – Vol. 94 (2011)
2 C(2)); 148.99, 148.86, 148.70, 148.49 (4s, 2 C(6)); 143.52, 143.49 (2s); 140.62, 140.60 (2s); 136.35, 136.26
(2s); 128.32 – 126.91 (several d); 124.99 (br. d); 122.06, 122.00 (2s, 2 C(5)); 119.98 (d); 66.24, 66.11 (2t,
2 PhCH2); 65.55 (br. t, CH2ꢀC(9’)); 60.52, 60.31, 60.24, 60.17 (4t, 2 MeCH2O); 58.45; 56.99, 56.47 (2t,
2 CH2NCH2C(8)); 53.36 (br. t, 2 CH2ꢀC(8)); 46.50, 46.44 (2d, C(9’)); 43.91, 43.70, 43.55 (3t,
2 CH2ꢀN(9)); 13.91 (q, 2 MeCH2O). HR-MALDI-MS: 1141.3921 (17, [M þ Na]þ, C55H54N14NaO1þ3
;
calc. 1141.3887), 1119.4059 (36, [M þ H]þ, C55H55N14O1þ3 ; calc. 1119.4068), 1033.3349 (35, [M ꢀ
BnOH þ Na]þ, C48H46N14NaOþ12 ; calc. 1033.3312), 1011.3539 (100, [M ꢀ BnOH þ H]þ, C48H47N14Oþ12
calc. 1011.3492).
;
tert-Butyl 6-[(1-(2-Ethoxy-2-oxoethyl)-2-{[(9H-fluoren-9-yl)methoxy]carbonyl}hydrazino)methyl]-
uracil-1-acetyl-(12 ! 63-N)-6-{[1-(2-ethoxy-2-oxoethyl)hydrazino]methyl}uracil-1-acetyl-(12 ! 63-N)-6-
{[1-(2-ethoxy-2-oxoethyl)hydrazino]methyl}uracil-1-acetyl-(12 ! 63-N)-6-{[1-(2-ethoxy-2-oxoethyl)hy-
drazino]methyl}uracil-1-acetate (¼(9H-Fluoren-9-yl)methyl 2-[(3-{2-[2-{[3-(2-{2-[(3-{2-[2-({3-[2-(tert-
Butoxy)-2-oxoethyl]-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl}methyl)-2-(2-ethoxy-2-oxoethyl)hydra-
zinyl]-2-oxoethyl}-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)methyl]-2-(2-ethoxy-2-oxoethyl)hydrazin-
yl}-2-oxoethyl)-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]methyl}-2-(2-ethoxy-2-oxoethyl)hydrazinyl]-
2-oxoethyl}-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)methyl]-2-(2-ethoxy-2-oxoethyl)hydrazinecarbox-
ylate; 17). A soln. of 7 (580 mg, 0.72 mmol), HBTU (285 mg, 0.75 mmol), and HOBt (102 mg,
0.75 mmol) in DMF (5 ml) was treated with a soln. of 6 (400 mg, 0.63 mmol) in DMF (5 ml) and EtNiPr2
(130 ml, 0.75 mmol), stirred for 20 h at r.t., treated with sat. NaHCO3 soln. (10 ml), and extracted with
AcOEt (3 ꢃ 20 ml). Drying of the combined org. layers (MgSO4), filtration, evaporation, and FC
(CH2Cl2/MeOH 95 :5 ! 80 :20) gave 17 (619 mg, 69%). White powder. Rf (CH2Cl2/MeOH 85 :15) 0.51.
M.p. > 1528 (dec.). UV (MeOH): 266 (47800), 300 (4800). IR (ATR): 3199w (br.), 2982w, 1673s (br.),
1464m, 1421m, 1393m, 1296w, 1207s, 1153m, 1072w, 1025w, 985w, 961w, 930w, 839s. 1H-NMR (400 MHz,
(D6)DMSO; mixture of rotamers): 11.37 (0.8 H), 11.31 (0.8 H), 11.27 (1.9 H), 11.16 (0.5 H) (4 br. s,
HꢀN(3/I – IV)); 9.73 (0.3 H), 9.65 (0.2 H), 9.62 (0.8 H), 9.57 (0.9 H), 8.90 (0.4 H), 8.86 (0.9 H), 8.83
(0.4 H) (7 br. s, HNꢀNCH2C(6/I – IV)); 7.88 – 7.29 (m, 8 arom. H); 5.63 (0.3 H), 5.61 (0.6 H), 5.58
(0.2 H), 5.55 (2.9 H) (4 br. s, HꢀC(5/I – IV)); 5.23 – 4.56 (m, 3.2 H, CH2ꢀN(1/I – IV)); 4.92 (1.6 H), 4.77
(1 H), 4.76 (0.8 H), 4.74 (1.4 H) (4 br. s, CH2ꢀN(1/I – IV)); 4.25 (br. s, CH2ꢀC(9’)); 4.16 – 4.07 (m,
4 MeCH2O, HꢀC(9’)); 3.79, 3.75, 3.72, 3.69 (4 br. s, CH2ꢀC(6/I – IV), CH2NCH2C(6/I – IV)); 1.43
(1.6 H), 1.42 (1.1 H), 1.38 (5.7 H), 1.37 (0.6 H) (4s, t-Bu); 1.23 – 1.14 (m, 4 MeCH2O). 13C-NMR
(100 MHz, (D6)DMSO; mixture of rotamers): 169.34, 169.32, 169.28, 169.24, 169.20, 169.05, 168.11,
t
167.00 (8s, 4 CO2Et, CO2 Bu, C(O)CH2N(1/II – IV)); 162.49 (br. s, C(4/I – IV)); 155.20 (br. s, NCO2);
151.67, 151.60, 151.14, 150.66 (4s, C(2/I – IV), C(6/I – IV)); 143.63 (2 br. s); 140.70 (2s); 127.66 (2d); 127.11
(2d); 125.10 (2 br. d); 120.09 (2d); 103.87, 103.82, 103.69, 103.66 (4d, C(5/I – IV)); 81.73, 81.52 (2s, Me3C);
60.57, 60.53, 60.45, 60.40, 60.34, 60.29 (6t, 4 MeCH2O); 57.82 – 57.27, 57.10 – 56.14 (2 br. t, CH2ꢀC(6/I – IV),
CH2NCH2C(6/I – IV)); 46.57 (d, C(9’)); 45.22, 43.99, 43.95 (3t, CH2ꢀN(1/I – IV)); 27.63, 27.60 (2q, Me3C);
14.00, 13.96, 13.85 (3q, 4 MeCH2O). HR-MALDI-MS: 1447.5189 (100, [M þ Na]þ, C63H76N16NaO2þ3
;
calc. 1447.5161), 1369.4770 (58, [M ꢀ Bu þ 2 H]þ, C59H69N16O2þ3 ; calc. 1369.4711).
t
tert-Butyl 6-{[1-(2-Ethoxy-2-oxoethyl)hydrazino]methyl}uracil-1-acetyl-(12 ! 63-N)-6-{[1-(2-ethoxy-
2-oxoethyl)hydrazino]methyl}uracil-1-acetyl-(12 ! 63-N)-6-{[1-(2-ethoxy-2-oxoethyl)hydrazino]methyl}-
uracil-1-acetyl-(12 ! 63-N)-6-{[1-(2-ethoxy-2-oxoethyl)hydrazino]methyl}uracil-1-acetate (¼ Ethyl (1-({3-
[2-(tert-Butoxy)-2-oxoethyl]-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl}methyl)-2-{[6-{[1-(2-ethoxy-2-
oxoethyl)-2-{[6-{[1-(2-ethoxy-2-oxoethyl)-2-{[6-{[1-(2-ethoxy-2-oxoethyl)hydrazinyl]methyl}-2,4-dioxo-
3,4-dihydropyrimidin-1(2H)-yl]acetyl}hydrazinyl]methyl}-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl]a-
cetyl}hydrazinyl]methyl}-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl]acetyl}hydrazinyl)acetate; 18). A
soln. of 17 (400 mg, 0.28 mmol) in DMF (5 ml) was treated dropwise with piperidine (0.42 ml,
4.25 mmol), stirred for 4.5 h at r.t., and evaporated. The solid residue was suspended in Et2O (10 ml),
filtered, and washed with Et2O (8 ꢃ 10 ml). Precipitation from MeOH/Et2O gave 18 (217 mg, 64%).
White powder. Rf (CH2Cl2/MeOH 8 :2) 0.61. M.p. > 1778 (dec.). UV (MeOH): 268 (41800). IR (ATR):
3214w (br.), 2982w, 2808w, 1669s (br.), 1528w, 1462m, 1419m, 1390m, 1292w, 1202m, 1154m, 1073w,
1
1024w, 982w, 928w, 838m, 820m. H-NMR (400 MHz, (D6)DMSO; mixture of rotamers): 11.26 (br. s,
HꢀN(3/I – IV)); 9.73 (0.4 H), 9.62 (1 H), 9.57 (0.3 H), 9.49 (0.5 H), 8.90 (0.4 H), 8.86 (0.4 H) (6s,
HNꢀNCH2C(6/I – III)); 5.63 (0.3 H), 5.61 (0.5 H), 5.56 (1 H), 5.55 (0.5 H), 5.54 (0.5 H), 5.53 (0.8 H),

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5.52 (0.4 H) (7s, HꢀC(5/I – IV)); 5.26 – 4.51 (m, 3.2 H, CH2ꢀN(1/I – IV)); 4.92 (1.4 H), 4.73 (2.1 H), 4.56
(1.3 H) (3 br. s, CH2ꢀN(1/I – IV)); 4.14 – 4.04 (m, 4 MeCH2O); 3.80 (2.4 H), 3.76 (5.1 H), 3.73 (3.7 H),
3.63 (1.6 H), 3.49 (2.8 H), 3.44 (2.4 H) (6 br. s, CH2ꢀC(6/I – IV), CH2NCH2C(6/I – IV), NH2); 1.43
(2.6 H), 1.38 (6.4 H) (2s, t-Bu); 1.23 – 1.14 (m, 4 MeCH2O). 13C-NMR (100 MHz, (D6)DMSO; mixture of
rotamers): 170.18, 170.09, 169.98, 169.35, 169.29, 169.24, 168.14, 167.91, 166.88, 166.82, 166.58 (11s,
t
4 CO2Et, CO2 Bu, C(O)CH2N(1/II – IV)); 162.76, 162.56, 162.54 (3s, C(4/I – IV)); 152.65, 152.12, 151.84,
151.82, 151.71, 151.64, 151.21, 150.71, 149.78 (9s, C(2/I – IV), C(6/I – IV)); 103.84, 103.70 (2d, C(5/I – IV));
81.78, 81.57 (2s, Me3C); 60.62, 60.45, 60.40, 60.34, 60.14, 60.01, 59.23, 56.83 – 56.38 (8 br. t, 4 MeCH2O,
CH2ꢀC(6/I – IV), CH2NCH2C(6/I – IV)); 45.26, 44.06 – 43.66 (2 br. t, CH2ꢀN(1/I – IV)); 27.67, 27.64 (2q,
Me3C); 14.10, 14.00 (2q, 4 MeCH2O). HR-MALDI-MS: 1225.4459 (95, [M þ Na]þ, C48H66N16NaO2þ1
;
t
calc. 1225.4481), 1203.4632 (30, [M þ H]þ, C48H67N16O2þ1 ; calc. 1203.4661), 1169.3814 (100, [M ꢀ Bu þ
t
H þ Na]þ, C44H58N16NaO2þ1 ; calc. 1169.3849), 1147.3969 (95, [M ꢀ Bu þ 2 H]þ, C44H59N16O2þ1 ; calc.
1147.4030).
tert-Butyl 8-{{1-(2-Ethoxy-2-oxoethyl)-2-{[(9H-fluoren-9-yl)methoxy]carbonyl}hydrazino}methyl}-
N6-(benzyloxycarbonyl)adenine-9-acetyl-(92 ! 63-N)-6-{[1-(2-ethoxy-2-oxoethyl)hydrazino]methyl}ura-
cil-1-acetyl-(12 ! 83-N)-8-{[1-(2-ethoxy-2-oxoethyl)hydrazino]methyl}-N6-(benzyloxycarbonyl)adenine-
9-acetyl-(92 ! 63-N)-6-{[1-(2-ethoxy-2-oxoethyl)hydrazino]methyl}uracil-1-acetate (¼(9H-Fluoren-9-yl)-
methyl 2-[(6-{[(Benzyloxy)carbonyl]amino}-9-{2-[2-{[3-(2-{2-[(6-{[(benzyloxy)carbonyl]amino}-9-{2-
[2-({3-[2-(tert-butoxy)-2-oxoethyl]-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl}methyl)-2-(2-ethoxy-2-oxo-
ethyl)hydrazinyl]-2-oxoethyl}-9H-purin-8-yl)methyl]-2-(2-ethoxy-2-oxoethyl)hydrazinyl}-2-oxoethyl)-
2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]methyl}-2-(2-ethoxy-2-oxoethyl)hydrazinyl]-2-oxoethyl}-9H-
purin-8-yl)methyl]-2-(2-ethoxy-2-oxoethyl)hydrazinecarboxylate; 19). A soln. of 10 (400 mg, 0.5 mmol),
11 (580 mg, 0.6 mmol) and EtNiPr2 (0.1 ml, 0.6 mmol) in DMF (3 ml) was cooled to 08, treated dropwise
with a soln. of HBTU (229 mg, 0.6 mmol) in DMF (2 ml), stirred for 2 h, allowed to reach r.t., stirred for
4 h, and treated with sat. NaHCO3 soln. (15 ml) and AcOEt (20 ml). The phases were separated, and the
aq. layer was extracted with AcOEt (2 ꢃ 20 ml). Drying of the combined org. layers (MgSO4), filtration,
evaporation, and MPLC (CH2Cl2/MeOH 95 :5 ! 90 :10, flow: 25 ml/min) gave 19 (777 mg, 89%). White
powder. Rf (CH2Cl2/MeOH 9 :1) 0.43. M.p. > 1608 (partial melting), > 1858 (dec.). UV (CHCl3): 269
(69200), 301 (5900). IR (ATR): 3226w (br.), 2981w, 1682s (br.), 1613m, 1530w, 1498w, 1452m, 1421m,
1392m, 1322w, 1296w, 1204s, 1155s, 1101m, 1027m, 980w, 965w, 897w, 840s. 1H-NMR (400 MHz,
(D6)DMSO; mixture of rotamers; assignments based on a HSQC and a HMBC spectrum): 11.58 (0.5 H),
11.50 (0.2 H), 11.48 (0.2 H), 11.42 (0.1 H), 11.39 (0.4 H), 11.36 (0.1 H), 11.28 (0.2 H), 11.25 (0.1 H),
11.20 (0.1 H), 11.15 (0.1 H) (10 br. s, HꢀN(3/I,III)); 10.64 (0.2 H), 10.61 (0.3 H), 10.55 (0.8 H), 10.54
(0.7 H) (4 br. s, HNꢀC(6/II,IV)); 9.83 (0.2 H), 9.78 (0.4 H), 9.76 (0.5 H), 9.72 (0.3 H), 9.62 (0.2 H), 9.57
(0.2 H), 9.08 (0.3 H), 9.02 (0.4 H), 8.97 (0.6 H), 8.90 (0.1 H), 8.80 (0.8 H) (11 br. s, HNꢀNCH2C(6/I,III),
HNꢀNCH2C(8/II,IV)); 8.51 (0.9 H), 8.47 (0.4 H), 8.45 (0.7 H) (3s, HꢀC(2/II,IV)); 7.86 – 7.19 (m, 18
arom. H); 6.11 – 5.81 (br. m, 0.8 H), 5.38 – 5.31 (br. m, 0.6 H), 5.24 (br. s, 1.3 H), 5.20 (br. s, 2.3 H), 5.18
(br. s, 2.1 H), 5.09 – 5.00 (br. m, 1.5 H), 4.94 – 4.92 (br. m, 0.3 H), 4.86 (br. s, 1.2 H), 4.81 (br. s, 0.6 H),
4.76 – 4.68 (br. m, 1.3 H) (CH2ꢀN(1/I,III), CH2ꢀN(9/II,IV), 2 PhCH2); 5.64 (0.2 H), 5.59 (0.7 H), 5.58
(0.6 H), 5.53 (0.2 H), 5.52 (0.2 H), 5.48 (0.1 H) (6 br. s, HꢀC(5/I,III)); 4.41 – 3.98 (br. m, CH2ꢀC(9’),
HꢀC(9’), 4 MeCH2O, CH2ꢀC(8/II,IV)); 3.89 (0.8 H), 3.86 (0.5 H), 3.84 (0.6 H), 3.80 (1.0 H), 3.78
(0.7 H), 3.76 (1.5 H), 3.74 (1.4 H), 3.71 (2.1 H), 3.67 (1.5 H), 3.61 (1.9 H) (10 br. s, CH2ꢀC(6/I,III),
CH2NCH2C(6/I,III), CH2NCH2C(8/II,IV)); 1.43 (4.8 H), 1.33 (4.2 H) (2s, t-Bu); 1.25 – 1.03 (m,
4 MeCH2O). 13C-NMR (100 MHz, (D6)DMSO; mixture of rotamers; assignments based on a HSQC
and a HMBC spectrum): 169.39, 169.26, 169.17, 169.09, 169.08, 169.05, 168.04, 167.81, 166.75, 165.68 (10s,
t
4 CO2Et, CO2 Bu, C(O)CH2N(1/III), C(O)CH2N(9/II,IV)); 162.59, 162.47 (2s, C(4/I,III)); 155.30 –
154.95 (br. s, NꢀNCO2); 153.51, 153.46, 148.66, 148.59 (4s, C(4/II,IV), C(6/II,IV)); 152.04, 151.60 (2s,
NCO2ꢀC(6/II,IV)); 151.93, 151.88, 151.56, 151.54, 151.52, 151.50, 151.46, 150.42, 149.65 (9s, C(2/I,III),
C(6/I,III), C(8/II,IV)); 150.79, 150.70 (2d, C(2/II,IV)); 143.55, 143.50 (2s); 140.66, 140.60 (2s); 136.35 (s);
128.36 – 126.92 (several d); 125.03, 124.98 (2d); 122.06, 122.02, 121.98, 121.94 (4s, C(5/II,IV)); 120.03,
119.98 (2d); 105.64, 104.08 (2d, C(5/I,III)); 81.69, 81.41 (2s, Me3C); 66.17, 66.13 (2t, 2 PhCH2); 65.58 (t,
CH2ꢀC(9’)); 60.58, 60.40, 60.24, 60.20 (4t, 4 MeCH2O); 58.94 – 56.28 (br. t, CH2ꢀC(6/I,III),
CH2NCH2C(6/I,III), CH2NCH2C(8/II,IV)); 53.79 – 53.23 (br. t, CH2ꢀC(8/II,IV)); 46.52, 46.45 (2d,

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C(9’)); 45.26 (br. t, CH2ꢀN(1/I,III)); 43.55 (t, CH2ꢀN(9/II,IV)); 27.60, 27.50 (2q, Me3C); 13.95, 13.91,
13.86, 13.77 (4q, 4 MeCH2O). HR-MALDI-MS: 1763.6510 (18), 1762.6448 (35), 1761.6415 (31, [M þ
Na]þ, C81H90N22NaOþ23 ; calc. 1761.6441), 1656.5837 (18), 1655.5800 (55), 1654.5768 (100), 1653.5740
(95, [M ꢀ BnOH þ Na]þ, C74H82N22NaO2þ2 ; calc. 1653.5866), 1633.5976 (24), 1632.5952 (44), 1631.5921
(41, [M ꢀ BnOH þ H]þ, C74H83N22O2þ2 ; calc. 1631.6047), 1547.5278 (18), 1546.5244 (44), 1545.5218 (45,
[M ꢀ 2 BnOH þ Na]þ, C67H74N22NaO2þ1 ; calc. 1545.5291).
tert-Butyl 8-{{1-(2-Ethoxy-2-oxoethyl)-2-{[(9H-fluoren-9-yl)methoxy]carbonyl}hydrazino}methyl}-
adenine-9-acetyl-(92 ! 63-N)-6-{[1-(2-ethoxy-2-oxoethyl)hydrazino]methyl}uracil-1-acetyl-(12 ! 83-N)-8-
{[1-(2-ethoxy-2-oxoethyl)hydrazino]methyl}adenine-9-acetyl-(92 ! 63-N)-6-{[1-(2-ethoxy-2-oxoethyl)hy-
drazino]methyl}uracil-1-acetate (¼(9H-Fluoren-9-yl)methyl 2-[(6-Amino-9-{2-[2-{[3-(2-{2-[(6-amino-9-
(2-{2-[(3-[2-(tert-butoxy)-2-oxoethyl]-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)methyl]-2-(2-ethoxy-2-
oxoethyl)hydrazinyl}-2-oxoethyl)-9H-purin-8-yl)methyl]-2-(2-ethoxy-2-oxoethyl)hydrazinyl}-2-oxoeth-
yl)-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]methyl}-2-(2-ethoxy-2-oxoethyl)hydrazinyl]-2-oxoethyl}-
9H-purin-8-yl)methyl]-2-(2-ethoxy-2-oxoethyl)hydrazinecarboxylate; 20). A suspension of Pd(OAc)2
(300 mg, 1.3 mmol) in MeOH/CH2Cl2 1:1 (1 ml) was stirred under H2 for 1 h at r.t., treated with a
soln. of 19 (650 mg, 0.37 mmol) in MeOH/CH2Cl2 1:1 (10 ml), stirred for 7 d, and filtered through Celite.
Evaporation of the filtrate and MPLC (CH2Cl2/MeOH 95 :5 ! 80 :20, flow: 25 ml/min) gave 20 (356 mg,
65%). White powder. Rf (CH2Cl2/MeOH 9 :1) 0.22. M.p. > 1708 (partial melting), > 1958 (dec.). UV
(MeOH): 265 (50700), 300 (4700). IR (ATR): 3323w (br.), 3199w (br.), 2981w, 2804w, 1684s (br.), 1636s,
1605m, 1581m, 1463m, 1449m, 1423m, 1392m, 1375m, 1326m, 1294m, 1203s, 1152s, 1072w, 1024m, 985w,
956w, 856w, 825w. 1H-NMR (300 MHz, (D6)DMSO; mixture of rotamers): 11.53 (0.6 H), 11.44 (0.5 H),
11.40 (0.4 H), 11.29 (0.5 H) (4 br. s, HꢀN(3/I,III)); 9.79 (0.3 H), 9.72 (1.2 H), 9.59 (0.2 H), 9.56 (0.2 H),
9.05 (0.1 H), 8.98 (0.4 H), 8.93 (0.3 H), 8.91 (0.3 H), 8.88 (0.2 H), 8.83 (0.5 H), 8.77 (0.3 H) (11 br. s,
HNꢀNCH2C(6/I,III), HNꢀNCH2C(8/II,IV)); 8.04 (1.3 H), 8.00 (0.7 H) (2s, HꢀC(2/II,IV)); 7.89 – 7.22
(m, 8 arom. H); 7.14 (br. s, 2 NH2); 5.99 – 5.82 (br. m, 0.9 H), 5.38 – 5.19 (br. m, 1.1 H), 5.12 (br. s, 1.4 H),
5.05 – 4.96 (br. m, 1.5 H), 4.87 (br. s, 1.1 H), 4.84 – 4.77 (br. m, 1.1 H), 4.69 (br. s, 0.6 H), 4.63 (br. s, 0.3 H)
(CH2ꢀN(1/I,III), CH2ꢀN(9/II,IV)); 5.62 (0.2 H), 5.59 (0.6 H), 5.55 (0.8 H), 5.51 (0.4 H) (4 br. s, HꢀC(5/
I,III)); 4.33 – 4.00 (br. m, CH2ꢀC(9’), HꢀC(9’), 4 MeCH2O, CH2ꢀC(8/II,IV)); 3.85 (1.2 H), 3.73 (4.9 H),
3.68 (4.0 H), 3.59 (1.9 H) (4 br. s, CH2ꢀC(6/I,III), CH2NCH2C(6/I,III), CH2NCH2C(8/II,IV)); 1.43
(4.8 H), 1.33 (4.2 H) (2s, t-Bu); 1.24 – 1.01 (m, 4 MeCH2O). 13C-NMR (75 MHz, (D6)DMSO; mixture of
rotamers): 169.65, 169.43, 169.32, 169.18, 169.11, 168.10, 167.92, 166.93, 166.57, 166.30, 165.99 (11s,
t
4 CO2Et, CO2 Bu, C(O)CH2N(1/III), C(O)CH2N(9/II,IV)); 162.65, 162.50 (2s, C(4/I,III)); 155.35 (br. s,
NꢀNCO2); 152.53, 152.40, 151.89, 151.84, 151.64, 151.19, 150.90, 150.48, 149.67, 147.47, 147.24 (10s, 1d, C(2/
I,III), C(6/I,III), C(2/II,IV), C(4/II,IV), C(6/II,IV), C(8/II,IV)); 143.58 (br. s); 140.63 (br. s); 127.62 (br.
d); 127.00 (br. d); 125.16, 125.01 (2d); 120.09, 119.96 (2d); 117.33, 117.23 (2s, C(5/II,IV)); 105.60, 104.08
(2 br. d, C(5/I,III)); 81.66, 81.42 (2s, Me3C); 65.61 (t, CH2ꢀC(9’)); 60.56, 60.40, 60.21, 59.88 (4t,
4 MeCH2O); 57.62 – 55.74, 54.47 – 52.89 (2 br. t, CH2ꢀC(6/I,III), CH2NCH2C(6/I,III), CH2ꢀC(8/II,IV),
CH2NCH2C(8/II,IV)); 46.91 – 46.11 (br. d, C(9’)); 45.58 – 44.83 (br. t, CH2ꢀN(1/I,III)); 43.97 – 42.29 (br.
t, CH2ꢀN(9/II,IV)); 27.69, 27.58, 27.44 (3q, Me3C); 13.90 (br. q, 4 MeCH2O). HR-MALDI-MS: 1495.5823
(40), 1494.5767 (89), 1493.5733 (100, [M þ Na]þ, C65H78N22NaO1þ9 ; calc. 1493.5706), 1473.6016 (38),
1472.5949 (86), 1471.5913 (90, [M þ H]þ, C65H79N22O1þ9 ; calc. 1471.5886).
tert-Butyl 8-{{1-(2-Ethoxy-2-oxoethyl)-2-{[(9H-fluoren-9-yl)methoxy]carbonyl}hydrazino}methyl}-
N6-(benzyloxycarbonyl)adenine-9-acetyl-(92 ! 83-N)-8-{[1-(2-ethoxy-2-oxoethyl)hydrazino]methyl}-N6-
(benzyloxycarbonyl)adenine-9-acetyl-(92 ! 63-N)-6-{[1-(2-ethoxy-2-oxoethyl)hydrazino]methyl}uracil-
1-acetyl-(12 ! 63-N)-6-{[1-(2-ethoxy-2-oxoethyl)hydrazino]methyl}uracil-1-acetate (¼(9H-Fluoren-9-yl)-
methyl 2-[(6-{[(Benzyloxy)carbonyl]amino}-9-{2-[2-{[6-{[(benzyloxy)carbonyl]amino}-9-(2-{2-[(3-{2-
[2-({3-[2-(tert-butoxy)-2-oxoethyl]-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl}methyl)-2-(2-ethoxy-2-oxo-
ethyl)hydrazinyl]-2-oxoethyl}-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)methyl]-2-(2-ethoxy-2-oxo-
ethyl)hydrazinyl}-2-oxoethyl)-9H-purin-8-yl]methyl}-2-(2-ethoxy-2-oxoethyl)hydrazinyl]-2-oxoethyl}-
9H-purin-8-yl)methyl]-2-(2-ethoxy-2-oxoethyl)hydrazinecarboxylate; 21). A soln. of 6 (150 mg,
0.23 mmol), 16 (315 mg, 0.28 mmol), and EtNiPr2 (48 ml, 0.28 mmol) in DMF (3 ml) was cooled to 08,
treated dropwise with a soln. of HBTU (107 mg, 0.28 mmol) in DMF (2 ml), stirred for 1 h, allowed to
reach r.t., stirred for 4 h, and treated with sat. NaHCO3 soln. (10 ml) and AcOEt (20 ml). The phases

Helvetica Chimica Acta – Vol. 94 (2011)
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were separated, and the aq. layer was extracted with AcOEt (2 ꢃ 20 ml). Drying of the combined org.
layers (MgSO4), filtration, evaporation, and MPLC (CH2Cl2/MeOH 95 :5 ! 80 :20, flow: 30 ml/min)
gave 21 (296 mg, 72%). White powder. Rf (CHCl3/MeOH 9 :1) 0.30. M.p. > 1658 (partial melting),
> 1858 (dec.). UV (CHCl3): 269 (55800), 301 (4900). IR (ATR): 3200w (br.), 2982w, 1730m, 1689s,
1619m, 1534w, 1498w, 1454m, 1421w, 1395m, 1377w, 1324w, 1213s, 1169m, 1156m, 1105w, 1027w, 963w,
839s. 1H-NMR (300 MHz, (D6)DMSO; mixture of rotamers): 11.57 (0.1 H), 11.55 (0.1 H), 11.52 (0.2 H),
11.39 (0.8 H), 11.34 (0.2 H), 11.31 (0.2 H), 11.30 (0.3 H), 11.26 (0.1 H) (8 br. s, HꢀN(3/I,II)); 10.65
(0.5 H), 10.60 (0.7 H), 10.56 (0.3 H), 10.54 (0.2 H), 10.52 (0.3 H) (5 br. s, HNꢀC(6/III,IV)); 9.85 (0.3 H),
9.77 (0.5 H), 9.72 (0.5 H), 9.69 (0.2 H), 9.65 (0.1 H), 9.60 (0.3 H), 9.16 (0.2 H), 9.13 (0.3 H), 9.11
(0.1 H), 9.09 (0.1 H), 9.00 (0.2 H), 8.96 (0.2 H), 8.90 (0.3 H), 8.88 (0.2 H), 8.80 (0.3 H), 8.77 (0.3 H),
8.59 (0.3 H), 8.53 (0.1 H), 8.52 (0.1 H), 8.50 (0.1 H), 8.36 (0.3 H), 8.00 (0.3 H) (22 br. s, HNꢀNCH2C(6/
I,II), HNꢀNCH2C(8/III,IV), HꢀC(2/III,IV)); 8.48 (0.5 H), 8.43 (0.3 H) (2s, HꢀC(2/III,IV)); 7.85 – 7.19
(m, 18 arom. H); 5.89 – 5.81 (br. m, 0.7 H), 5.31 (br. s, 0.3 H), 5.22 (br. s, 1.9 H), 5.17 (br. s, 3.4 H), 5.06 –
4.99 (br. m, 0.9 H), 4.91 (br. s, 1.4 H), 4.70 (br. s, 1.2 H), 4.54 – 4.31 (br. m, 2.2 H) (CH2ꢀN(1/I,II),
CH2ꢀN(9/III,IV), 2 PhCH2); 5.63 (0.1 H), 5.61 (0.6 H), 5.54 (1.0 H), 5.46 (0.3 H) (4 br. s, HꢀC(5/I,II));
4.19 – 4.02 (br. m, CH2ꢀC(9’), HꢀC(9’), 4 MeCH2O, CH2ꢀC(8/III,IV)); 3.73 (6.6 H), 3.70 (3.0 H), 3.67
(2.4 H) (3 br. s, CH2ꢀC(6/I,II), CH2NCH2C(6/I,II), CH2NCH2C(8/III,IV)); 1.40 (2.5 H), 1.37 (6.5 H)
(2s, t-Bu); 1.24 – 1.10 (m, 4 MeCH2O). 13C-NMR (75 MHz, (D6)DMSO; mixture of rotamers): 169.86,
t
169.49, 169.26, 169.08, 168.97, 168.88, 167.90 (7s, 4 CO2Et, CO2 Bu, C(O)CH2N(1/II), C(O)CH2N(9/
III,IV)); 162.34, 162.31 (2s, C(4/I,II)); 154.91 (s, NꢀNCO2); 153.23, 151.76, 151.49, 151.39, 151.08, 150.49,
148.47 (6s, 1d, C(2/I,II), C(6/I,II), NCO2ꢀC(6/III,IV), C(2/III,IV), C(4/III,IV), C(6/III,IV), C(8/
III,IV)); 143.36 (s); 140.46 (s); 136.20 (s); 128.33 (d); 128.13 (d); 127.77 (d); 126.93 (d); 124.99 (d); 121.78
(s, C(5/III,IV)); 119.95 (d); 103.88 (br. d, C(5/I,II)); 81.64, 81.46 (2s, Me3C); 66.13 (br. t, 2 PhCH2); 65.55
(t, CH2ꢀC(9’)); 60.61, 60.49, 60.36, 60.29, 60.04, 59.96 (6t, 4 MeCH2O); 57.91 – 55.33, 54.86 – 52.86 (2 br. t,
CH2ꢀC(6/I,II), CH2NCH2C(6/I,II), CH2ꢀC(8/III,IV), CH2NCH2C(8/III,IV)); 46.90 – 45.82 (br. d,
C(9’)); 45.52 – 44.85 (br. t, CH2ꢀN(1/I,II)); 44.32 – 42.94 (br. t, CH2ꢀN(9/III,IV)); 27.67, 27.57 (2q,
Me3C); 14.09, 13.93 (2q, 4 MeCH2O). HR-MALDI-MS: 1763.6489 (20), 1762.6462 (40), 1761.6439 (42,
[M þ Na]þ, C81H90N22NaO2þ3 ; calc. 1761.6441), 1741.6653 (10), 1740.6619 (21), 1739.6597 (22, [M þ H]þ,
C81H91N22Oþ23 ; calc. 1739.6622), 1656.5888 (14), 1655.5845 (43), 1654.5832 (89), 1653.5835 (100, [M ꢀ
BnOH þ Na]þ, C74H82N22NaO2þ2 ; calc. 1653.5866), 1633.6072 (27), 1632.6042 (56), 1631.6016 (64, [M ꢀ
BnOH þ H]þ, C74H83N22O2þ2 ; calc. 1631.6047), 1547.5434 (10), 1546.5396 (24), 1545.5369 (29, [M ꢀ
2 BnOH þ Na]þ, C67H74N22NaO2þ1 ; calc. 1545.5291).
tert-Butyl 8-{{1-(2-Ethoxy-2-oxoethyl)-2-{[(9H-fluoren-9-yl)methoxy]carbonyl}hydrazino}methyl}-
adenine-9-acetyl-(92 ! 83-N)-8-{[1-(2-ethoxy-2-oxoethyl)hydrazino]methyl}adenine-9-acetyl-(92 ! 63-
N)-6-{[1-(2-ethoxy-2-oxoethyl)hydrazino]methyl}uracil-1-acetyl-(12 ! 63-N)-6-{[1-(2-ethoxy-2-oxoethyl)-
hydrazino]methyl}uracil-1-acetate (¼(9H-Fluoren-9-yl)methyl 2-[(6-Amino-9-{2-[2-{[6-amino-9-(2-{2-
[(3-{2-[2-({3-[2-(tert-butoxy)-2-oxoethyl]-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl}methyl)-2-(2-
ethoxy-2-oxoethyl)hydrazinyl]-2-oxoethyl}-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)methyl]-2-(2-
ethoxy-2-oxoethyl)hydrazinyl}-2-oxoethyl)-9H-purin-8-yl]methyl}-2-(2-ethoxy-2-oxoethyl)hydrazinyl]-
2-oxoethyl}-9H-purin-8-yl)methyl]-2-(2-ethoxy-2-oxoethyl)hydrazinecarboxylate; 22). A suspension of
Pd(OAc)2 (200 mg, 0.89 mmol) in MeOH/CH2Cl2 1:1 (2 ml) was stirred under H2 for 1 h at r.t., treated
with a soln. of 21 (220 mg, 0.13 mmol) in MeOH/CH2Cl2 1:1 (4 ml), stirred for 6 d, and filtered through
Celite. Evaporation of the filtrate and MPLC (CH2Cl2/MeOH 9 :1 ! 8 :2, flow: 25 ml/min) gave 22
(128 mg, 69%). White powder. Rf (CH2Cl2/MeOH 85 :15) 0.44. UV (MeOH): 264 (58100), 300 (5100).
IR (ATR): 3323w (br.), 3195w (br.), 2981w, 2804w, 1682s (br.), 1639s, 1605m, 1581m, 1463m, 1449m,
1421m, 1393m, 1375m, 1326m, 1294m, 1204s, 1152s, 1072w, 1025m, 954w, 855w, 827w. 1H-NMR
(400 MHz, (D6)DMSO; mixture of rotamers): 11.44 (0.2 H), 11.39 (0.3 H), 11.37 (0.6 H), 11.28 (0.8 H),
11.20 (0.1 H) (5 br. s, HꢀN(3/I,II)); 9.78 (0.1 H), 9.75 (0.2 H), 9.73 (0.4 H), 9.68 (0.3 H), 9.66 (0.4 H),
9.64 (0.4 H), 9.04 (0.1 H), 9.01 (0.2 H), 8.92 (0.3 H), 8.88 (0.3 H), 8.85 (0.2 H), 8.81 (0.4 H), 8.77
(0.1 H), 8.75 (0.2 H), 8.71 (0.4 H) (15 br. s, HNꢀNCH2C(6/I,II), HNꢀNCH2C(8/III,IV)); 8.10 (0.4 H),
8.05 (0.7 H), 8.03 (0.3 H), 8.00 (0.3 H), 7.99 (0.3 H) (5s, HꢀC(2/III,IV)); 7.85 – 7.08 (m, 8 arom. H); 7.54
(br. s, 2 NH2); 5.75 (0.1 H), 5.71 (0.2 H), 5.64 (0.3 H), 5.62 (0.2 H), 5.60 (0.4 H), 5.57 (0.2 H), 5.56
(0.3 H), 5.54 (0.1 H), 5.53 (0.1 H), 5.49 (0.1 H) (10 s, HꢀC(5/I,II)); 5.18 (br. s, 0.5 H), 5.14 (br. s, 0.7 H),

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Helvetica Chimica Acta – Vol. 94 (2011)
5.09 (br. s, 0.9 H), 5.06 (br. s, 1.1 H), 4.99 (br. s, 0.5 H), 4.92 (br. s, 0.9 H), 4.91 (br. s, 1.6 H), 4.71 (br. s,
1.4 H), 4.55 – 4.43 (br. m, 0.4 H) (CH2ꢀN(1/I,II), CH2ꢀN(9/III,IV)); 4.27 – 3.84 (br. m, CH2ꢀC(9’),
HꢀC(9’), 4 MeCH2O, CH2ꢀC(8/III,IV)); 3.79 (1.0 H), 3.75 (4.0 H), 3.71 (3.0 H), 3.68 – 3.58 (br. m,
4.0 H) (CH2ꢀC(6/I,II), CH2NCH2C(6/I,II), CH2NCH2C(8/III,IV)); 1.41 (2.0 H), 1.38 (7.0 H) (2s, t-Bu);
1.25 – 1.10 (m, 4 MeCH2O). 13C-NMR (75 MHz, (D6)DMSO; mixture of rotamers): 169.40, 169.36,
t
169.32, 169.23, 169.20, 169.14, 169.11, 169.04, 168.01 (9s, 4 CO2Et, CO2 Bu, C(O)CH2N(1/I,II),
C(O)CH2N(9/III,IV)); 162.64, 162.42, 162.39 (3s, C(4/I,II)); 155.46, 155.38, 155.33, 155.26 (4s,
NꢀNCO2); 152.43, 152.33, 152.22, 152.14, 151.74, 151.61, 151.55, 151.14, 151.08, 150.94 (9s, 1d, C(2/
I,II), C(6/I,II), C(2/III,IV), C(4/III,IV), C(6/III,IV), C(8/III,IV)); 143.53 (br. s); 140.58 (br. s); 127.54
(br. d); 126.95 (br. d); 125.03 (br. d); 119.98 (br. d); 117.34, 117.32 (2s, C(5/III,IV)); 103.74 (br. d, C(5/
I,II)); 81.46 (s, Me3C); 65.63 (t, CH2ꢀC(9’)); 60.44, 60.31, 60.19 (3t, 4 MeCH2O); 56.63 – 56.31 (br. t,
CH2ꢀC(6/I,II), CH2NCH2C(6/I,II), CH2ꢀC(8/III,IV), CH2NCH2C(8/III,IV)); 48.53 (d, C(9’)); 46.51 –
46.45 (br. t, CH2ꢀN(1/I,II)); 45.16 (t, CH2ꢀN(9/III,IV)); 27.55 (q, Me3C); 13.87 (br. q, 4 MeCH2O).
HR-MALDI-MS: 1494.5791 (59), 1493.5733 (100, [M þ Na]þ, C65H78N22NaO1þ9 ; calc. 1493.5706),
1472.6047 (17), 1471.5986 (39, [M þ H]þ, C65H79N22O1þ9 ; calc. 1471.5886).
8-{[1-(2-Ethoxy-2-oxoethyl)-2-acetylhydrazino]methyl}adenine-9-acetyl-[(92 ! 63-N)-6-{[1-(2-
ethoxy-2-oxoethyl)hydrazino]methyl}uracil-1-acetyl-(12 ! 83-N)-8-{[1-(2-ethoxy-2-oxoethyl)hydrazino]-
methyl}adenine-9-acetyl]3-(92 ! 63-N)-6-{[1-(2-ethoxy-2-oxoethyl)hydrazino]methyl}uracil-1-acetamide
(¼ Ethyl {2-Acetyl-1-[(6-amino-9-{2-[2-{[3-(2-{2-[(6-amino-9-{2-[2-{[3-(2-{2-[(6-amino-9-{2-[2-{[3-(2-
{2-[(6-amino-9-{2-[2-{[3-(2-amino-2-oxoethyl)-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]methyl}-2-(2-
ethoxy-2-oxoethyl)hydrazinyl]-2-oxoethyl}-9H-purin-8-yl)methyl]-2-(2-ethoxy-2-oxoethyl)hydrazinyl}-
2-oxoethyl)-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]methyl}-2-(2-ethoxy-2-oxoethyl)hydrazinyl]-2-
oxoethyl}-9H-purin-8-yl)methyl]-2-(2-ethoxy-2-oxoethyl)hydrazinyl}-2-oxoethyl)-2,6-dioxo-1,2,3,6-tetra-
hydropyrimidin-4-yl]methyl}-2-(2-ethoxy-2-oxoethyl)hydrazinyl]-2-oxoethyl}-9H-purin-8-yl)methyl]-2-
(2-ethoxy-2-oxoethyl)hydrazinyl}-2-oxoethyl)-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]methyl}-2-(2-
ethoxy-2-oxoethyl)hydrazinyl]-2-oxoethyl}-9H-purin-8-yl)methyl]hydrazinyl}acetate; 24). a) Solid-
Phase Synthesis. 1. Swelling of the Rink Amide MBHA Resin. The resin (34.7 mg, 0.025 mmol of
reactive sites, loading: 0.72 mmol/g) was treated with CH2Cl2 (5 ml) for 1 h.
2. Fmoc Deprotection of the Rink Amide MBHA Resin. The resin was treated with a soln. of 20%
piperidine in DMSO (0.5 ml) for 10 min, washed with DMSO (5 ꢃ 1 ml), treated with a soln. of 20%
piperidine in DMSO (0.5 ml) for 10 min, and washed with DMSO (10 ꢃ 0.5 ml).
3. Coupling of the 1st Monomer (Double Coupling). The resin was treated with a soln. of 4 (32.7 mg,
0.063 mmol) and HATU (22.8 mg, 0.06 mmol) in DMSO (0.3 ml) and EtNiPr2 (22 ml, 0.13 mmol) for 4 h.
After washing with DMSO (10 ꢃ 0.5 ml), the resin was treated with a soln. of 4 (32.7 mg, 0.063 mmol)
and HATU (22.8 mg, 0.06 mmol) in DMSO (0.3 ml), and EtNiPr2 (22 ml, 0.13 mmol) for 4 h, and washed
with DMSO (10 ꢃ 0.5 ml) and CH2Cl2 (5 ꢃ 1 ml).
4. Acetylation (Capping) of the Unreacted Sites of the Rink Amide MBHA Resin. The resin was
treated with a 0.5m soln. of Ac2O and EtNiPr2 in NMP (0.75 ml) for 15 min, washed with DMSO (5 ꢃ
1 ml), treated with a 0.5m soln. of Ac2O and EtNiPr2 in NMP (0.75 ml) for 15 min, and washed with
DMSO (10 ꢃ 0.5 ml) and CH2Cl2 (5 ꢃ 1 ml).
5. Fmoc Deprotection of the Growing Oligomer. The resin was treated with CH2Cl2 (5 ml) for 1 h
(swelling), then with a soln. of 2% DBU in DMSO (2 ml) for 3 min, washed with DMSO (5 ꢃ 1 ml),
treated with a soln. of 2% DBU in DMSO (2 ml) for 3 min (3ꢃ), and washed with DMSO (10 ꢃ 0.5 ml).
6. Coupling of the 2nd Monomer. The resin was treated with a soln. of 8 (42.5 mg, 0.063 mmol) and
HATU (22.8 mg, 0.06 mmol) in DMSO (0.4 ml), and EtNiPr2 (22 ml, 0.13 mmol) for 4 h, and washed with
DMSO (10 ꢃ 0.5 ml) and CH2Cl2 (5 ꢃ 1 ml).
7. Fmoc Deprotection of the Growing Oligomer. As described under 5.
8. Coupling of the 3rd Monomer. The resin was treated with a soln. of 4 (32.7 mg, 0.063 mmol) and
HATU (22.8 mg, 0.06 mmol) in DMSO (0.3 ml), and EtNiPr2 (22 ml, 0.13 mmol) for 4 h, and washed with
DMSO (10 ꢃ 0.5 ml) and CH2Cl2 (5 ꢃ 1 ml).
9. Fmoc Deprotection of the Growing Oligomer. As described under 5.
10. Coupling of the 4th Monomer. As described under 6.
11. Fmoc Deprotection of the Growing Oligomer. As described under 5.