Oligonucleotide analogues with integrated bases and backbone: Part 20 - Hydrazide- and amide-linked analogues. 1. Design and synthesis of monomeric building blocks

Article abstract of DOI:10.1002/hlca.200900047

Hydrazide- and amide-linked oligonucleoside analogues with integrated bases and backbone were designed to allow for a rapid synthesis of long and water-soluble oligomers. The uracil-, cytosine-, and adenine-derived hydrazide building blocks 13-15 were synthesized by nucleophilic substitution with the hydrazine 23 of the halides 19, 28, and 34, derived from the alcohols 18, 27, and 33, respectively, while the uracil-, cytosine-, and adenine-derived amide building blocks 45-47 were synthesized by a Curtius degradation of the carboxylic acids 51, 56, and 61. These acids were obtained by Wittig reaction of the aldehydes 49, 53, and 58. The guanine-derived monomers 44 and 48 were synthesized by reductive cyclisation of the nitroso amides 38 and 63, respectively, resulting from acylation of the known 2,6-diamino-4-(benzyloxy)-5- nitrosopyrimidine (37).

Full text of DOI:10.1002/hlca.200900047

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Helvetica Chimica Acta – Vol. 92 (2009)
Oligonucleotide Analogues with Integrated Bases and Backbone
Part 201)
Hydrazide- and Amide-Linked Analogues. 1. Design and Synthesis of Monomeric
Building Blocks
by Manuel Peifer2), Fabio De Giacomo2), Martin Schandl3), and Andrea Vasella*
Laboratorium fꢀr Organische Chemie, ETH Zꢀrich, Wolfgang-Pauli-Strasse 10, CH-8093 Zꢀrich
(e-mail: vasella@org.chem.ethz.ch)
Hydrazide- and amide-linked oligonucleoside analogues with integrated bases and backbone were
designed to allow for a rapid synthesis of long and water-soluble oligomers. The uracil-, cytosine-, and
adenine-derived hydrazide building blocks 13 – 15 were synthesized by nucleophilic substitution with the
hydrazine 23 of the halides 19, 28, and 34, derived from the alcohols 18, 27, and 33, respectively, while the
uracil-, cytosine-, and adenine-derived amide building blocks 45 – 47 were synthesized by a Curtius
degradation of the carboxylic acids 51, 56, and 61. These acids were obtained by Wittig reaction of the
aldehydes 49, 53, and 58. The guanine-derived monomers 44 and 48 were synthesized by reductive
cyclisation of the nitroso amides 38 and 63, respectively, resulting from acylation of the known 2,6-
diamino-4-(benzyloxy)-5-nitrosopyrimidine (37).
Introduction. – Nucleic acids, oligonucleotides, and their analogues are charac-
terized by a contiguous backbone. The only exception are oligonucleotide analogues
with integrated bases and backbone (ONIBs) that replace the backbone by a linker
between C(5’) of the monoribonucleoside building blocks, and either C(6) of an
adjacent pyrimidine or C(8) of an adjacent purine base [2]. Several partially protected,
self-complementary adenosine- and uridine-derived dinucleosides of this type pair in
CHCl3 solution, i.e., form cyclic duplexes, as shown for dinucleosides 1 – 4 possessing an
oxymethylene- [3], thiomethylene- [4], (Z)-ethenylene [5], or ethynylene linker [6]. A
detailed conformational analysis rationalized the formation of cyclic duplexes by self-
complementary oxymethylene- and thiomethylene-, but not by ethylene-linked
dinucleotides 5 [7], and predicted the structure of the energetically feasible cyclic
duplexes [4]. The result of this analysis has been confirmed by the conformational
analysis of thiomethylene-linked tetramers, evidencing the formation of an incipient A-
type helix, characterised by a syn-conformation of the nucleobases, Watson – Crick or
reverse-Hoogsteen base pairing, and a large twist angle [8].
A priori, it should be possible to vary the structure of the linker between the
nucleobases within broad limits, provided that the resulting di- and oligonucleoside
monoplexes adopt an energetically not disfavoured conformation that allows duplex
1
)
)
)
Part 19 [1].
2
3
Taken in part from the projected Ph.D. thesis of M. P. and F. D. G.
Taken from the diploma thesis of M. S.
ꢁ 2009 Verlag Helvetica Chimica Acta AG, Zꢀrich

Helvetica Chimica Acta – Vol. 92 (2009)
1135
formation by base pairing and base stacking. This implies that ONIBs are not restricted
to ribonucleosides, and that the ribosyl units may be replaced by other structural
elements. We intended to design ONIBs that are devoid of a carbohydrate moiety to
evaluate the scope within which the architecture of ONIBs can be realised. Association
of ONIBs in aqueous solution, and their interaction with DNA and RNA will require
longer oligonucleotide analogues. The linker should thus allow for an efficient synthesis
of such analogues, and ensure that their monoplexes and duplexes are water-soluble.
These requirements suggested an amide bond as central connecting element between
monomeric units. Similarly as for PNAs [9][10], this choice should allow assembling
oligonucleotide analogues by one of the numerous methods for peptide coupling in
solution and on a solid support. To attain a sufficient water-solubility of the analogues,
we planned to introduce a side chain terminating with a carboxylate group. We thought
to attach this side chain to a second N-atom of the linker, thus avoiding elements of
chirality. To test if these precautions are indeed necessary, we planned to also
synthesize oligonucleotide analogues where the substituted second N-atom is replaced
by a CH2 group. We describe the design of the new analogues and the synthesis of the
monomeric building blocks required for their preparation in solution and on a solid
support.
Results and Discussion. – 1. Design of Peptide-Linked ONIBs. For the design of
peptide-linked ONIBs, we considered coupling amino and carboxy fragments derived
from purine bases substituted at C(8) and N(9), and from pyrimidine bases substituted
at C(6) and N(1). The above mentioned aim of introducing a second N-atom
substituted by a carboxyalkyl group suggested to synthesize analogues linked by N-

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acyl-N’-(carboxyalkyl)hydrazines. With the help of Maruzen models, we examined a
range of analogues possessing such linkers differing in length and in the position of the
N-acylhydrazine moiety. Among the analogues examined, those represented by a UA
dimer (Fig. 1,a), in which the hydrazide moiety is separated on each side by a CH2
group from the nucleobase, appeared to readily form cyclic duplexes via Watson –
Crick-type base pairing and base stacking at distances of 3.6 – 4 ꢂ, somewhat larger
than those in A-, B- and Z-DNA (3.3 – 3.4 ꢂ) [11]. These oligonucleotide analogues
were studied in more detail by evaluating the energetically most favourable torsion
angles of the monoplex (Fig. 1,b), and comparing them to the torsion angles required
to form a cyclic duplex, as shown by the conformer in Fig. 1,c4). Whenever possible, we
referred to experimental data, and calculated the energy change resulting from a larger
deviation of a torsion angle from its energetically preferred value.
Fig. 1. a) Definition of the torsion angles and numbering4) of a hydrazide-linked pyrimidine-purine (UA)
dimer. b) Conformation of the UA dimer with energetically preferred torsion angles k – z. c) Conformation
of the UA dimer with torsion angles k – z, required for pairing.
The lone pairs of the tetrahedral and trigonal N-atoms of hydrazides prefer to be
orthogonal to each other [12 – 14], resulting in the four energetically preferred
conformers 6 – 9 (Fig. 2) with an (E)- or (Z)-configuration of the NꢀC(O) moiety, and
a syn- or anti-orientation of the nonbonding, doubly occupied orbital of the tetrahedral
N-atom relative to the acyl moiety. The (Z)-configuration is favoured in polar solvents
4
)
An arbitrary numbering of the linker is used to simplify the discussion within the conformational
analysis.

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1137
and with increasing size of R3 [12][15]5), while the syn-conformation is favoured with
increasing size of R1 and R2. A crystal structure search in the Cambridge Structural
Database (CSD) for disubstituted hydrazides (R1 ¼ any C-substituent possessing at
least one a-H-atom; R2 ¼ H; R3 ¼ any C-substituent) resulted in seven structures with
one (E)-syn , three (Z)-syn , and three (Z)-anti conformers. The search for
trisubstituted hydrazides (R1, R2 ¼ any C-substituent possessing at least one a-H-
atom; R3 ¼ any C-substituent) resulted in 14 structures with 13 (Z)-syn- and one (E)-
syn-conformer. The data show the preference of the trisubstituted hydrazide linker for
the (Z)-syn-conformation, with an average value for the torsion angles l and e1/e1 of
ꢁ 172 ꢁ 58 and ꢁ 117 ꢁ 118, respectively.
Fig. 2. The four energetically preferred conformers of substituted hydrazides
An antiperiplanar orientation of C(6) and either N(3’) or CH2(1’’) of the UA dimer
(Fig. 1) results in two energetically preferred conformations around the CH2(1’)ꢀN(2’)
bond; one with i2 ¼ 608 and the other with i2 ¼ 1808. From the twelve crystal structures
of trisubstituted hydrazides (R1 ¼ any substituted-phenyl substituent; R2 ¼ any C-
substituent possessing at least one a-H-atom; R3 ¼ any C-substituent) in the CSD, three
adopt i2 ¼ꢁ 75 ꢁ 1 and 9 show i2 ¼ꢁ 171 ꢁ 58. The preference for the larger value can be
explained by a less destabilising synclinal interaction between C(6) and the sp2-
hybridised N(3’), as compared to a synclinal interaction between C(6) and the sp3-
hybridised CH2(1’). The average value for k, as defined in Fig. 1,a, is ꢁ 44 ꢁ 138, but
conformational changes by rotation about the C(6)ꢀCH2(1’) bond are expected to be
readily allowed [4].
To find the energetically most favoured torsion angles x and z, we referred to eight
PNA structures possessing an acetamido group on N(1/9)6) as determined by 2D-NMR
[17][18] and by crystal-diffraction [19 – 24] (Protein Data Bank (PDB)). We found
average values of ca. 1808 and ꢁ 908, respectively. An average value of ꢁ 90 ꢁ 258 for z
was found for 36 crystal structures of N(1/9)-(carbonylmethyl)-substituted nucleobases
in the CSD, while twelve of the 36 crystal structures possessing an acetamido
substituent showed average values for x ¼ꢁ 162 ꢁ 188, corresponding to an antiperi-
planar conformation of NH(3’) and N(9).
If R3 ¼ H, there are usually (E)/(Z) mixtures observed [14][16].
5
)
)
6
N(1/9) and C(6/8) denotes N(1) and C(6) of a pyrimidine and N(9) and C(8) of a purine base, resp.

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Stacking of the nucleobases and the right geometry for Watson – Crick base pairing
required adjusting the torsion angles k, x, and z from ꢀ 50, 180, and ꢀ 90 to ꢀ 70,
þ 70, and ꢀ 1008, respectively (Fig. 1,b ! c). We assumed that changing k and z from
their energetically ideal values will result in only little torsional strain, and that the
energy to be paid would be overcompensated by the H-bonds between the nucleobases
[11][25], and by base stacking [26]. However, calculations7) to evaluate the energy to
be paid for the adjustment of the torsion angle x from 1808 to þ 708 gave contradictory
results8). It is thus not possible to specify an overall energy value to be paid for the
conformational transition from conformer b to c in Fig. 1.
Modelling studies of various dimers suggested that stacking interactions depend on
the sequence, and decrease in the order UA ꢂ UU ꢂ AA > AU. To evaluate the
formation of duplexes between longer oligonucleotide analogues, we minimised the
conformational energy of the octamer U4A4 (Fig. 3)9). The model suggested Watson –
Crick-type base pairing and base stacking, with the hydrazide linker adopting a
conformation with torsion angles k – z as deduced for the cyclic duplex (Fig. 1,c). The
Fig. 3. Model of the hydrazide-linked octamer U4A4. a) Side view. b) Top view.
7
)
)
Ab initio calculations using Spartan ꢃ06 and the 6-31G* basis set.
8
The conformational energies of N-methyl-2-(6-methyluracil-1-yl-)acetamide and N-methyl-2-(8-
methyladenine-9-yl-)acetamide were minimised, resulting in an energy minimum of x ¼ ꢀ708 (cf.
experimentally favoured: x ¼ 1808). The value of x ¼ 708, required for duplex formation,
corresponds to a calculated energy maximum.
Amber* force-field calculation using Macromodel 7.0, with the H-bonds between the nucleobases
constrained.
9
)

Helvetica Chimica Acta – Vol. 92 (2009)
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resulting double helix (racemic mixture of right- and left-handed helices) is
characterised by a rise of 3.6 – 4 ꢂ per base pair (cf. A- and B-DNA: 2.8 and 3.3 –
3.4 ꢂ), a pitch of ca. 39 ꢂ (cf. A- and B-DNA: 34 ꢂ), and a twist angle of ca. 288
(cf. A-DNA: 318; B-DNA: 368).
The results of the conformational analysis encouraged us to synthesize hydrazide-
linked base-backbone integrating analogues.
We also considered it instructive to evaluate analogues that are devoid of the
carboxymethyl side chain and replace the N-unsubstituted hydrazide in the linker by an
isosteric peptide moiety. We assumed the energetically preferred torsion angles k, l, x,
and z to be about the same as for a de(carboxymethyl)-hydrazide linker, and restricted
the conformational analysis of the amide linker to evaluating the torsion angles i and e
(Fig. 4).
Fig. 4. a) Definition of the torsion angles and numbering4) of an amide-linked pyrimidine-purine (UA)
dinucleoside. b) Conformation of the UA dinucleoside with the energetically preferred torsion angles e and i.
c) Conformation of the UA dinucleoside with torsion angles e and i, required for pairing.
We expected the fragment C(6)ꢀCH2(1’)ꢀCH2(2’)ꢀNH(3’) to adopt an energeti-
cally preferred antiperiplanar arrangement (i ¼ 1808). To find the energetically most
favoured torsion angle e, we again referred to the eight PNA structures resolved by 2D-
NMR and crystal diffraction [17 – 24], and calculated the preferred conformation of N-
ethylacetamide7). We obtained an average value of e ¼ꢁ 908 from inspecting the PDB

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data and from the calculations. The resulting conformation of the amide-linked UA
dimer monoplex is shown in (Fig. 4,b).
Formation of a cyclic duplex requires a synclinal conformation of C(6) and NH(3’)
(i ¼ ꢀ608), and a conformational adjustment from e ¼ þ908 to e ¼ þ1008. The required
deviation of the other torsion angles from their energetically preferred values appeared
to be very similar to the hydrazide linker (Fig. 1,b ! c). The energy difference between
the antiperiplanar and synclinal conformers appeared to be low, as estimated from a
calculation for N-(2-phenethyl)acetamide 10 (0.25 kcal/mol). Similarly, the calculated
energy difference between the antiperiplanar and synclinal conformations of the
acetamides 11 and 12, derived from uracil and adenine, amounts to 1.1 and 0.23 kcal/
mol, respectively.
We considered the stabilisation by base pairing and base stacking in a cyclic duplex
to well overcompensate the destabilisation of the synclinal conformer, and thus decided
to also synthesize such amide-linked base-backbone integrating analogues, and to
analyse their association10).
2. Synthesis of the Monomeric Building Blocks for the Hydrazide-Linked
Oligonucleotide Analogues. We chose to synthesize the envisaged hydrazide-linked
oligonucleosides by N-acylation of a hydrazine, deriving the required hydrazines and
carboxylates from the fully protected monomeric building blocks 13, 14, 15, and 16. We
temporarily protected the C- and the N-terminus as tert-butyl ester and (9H-fluoren-9-
yl)methyl carbazate (Fmoc), respectively. The guanine-derived monomer was
temporarily protected by O-benzylation. The side chain was permanently protected
as ethyl ester, and the exocyclic amino group of the cytosine and adenine nucleobases
as benzyloxy carbamates (Cbz). The exocyclic amino group of 16 did not require
protection (see below).
The carboxylic acids 21, 29, 36, and 44 were to be obtained by cleaving the tert-butyl
esters, and the amines 20 and 35 by cleaving the N-Fmoc groups. At this stage, we did
not yet remove the Fmoc group from 14 and 16, as we began our investigations by
synthesising di- and tetramers derived from uracil and adenine.
To prepare the fully protected uracil-derived building block 13 (Scheme 1), we
hydroxymethylated the tert-butyl uracil-1-acetate (17) [28] by deprotonation at C(6)
with lithium diisopropylamide (LDA), formylation with DMF, hydrolysis, and
reduction of the resulting aldehyde with NaBH4, to provide alcohol 18 in 45% yield
along with 43% of starting material 17. Attempts to optimise the C(6)-hydroxymethy-
lation by deprotonating 17 with lithium 2,2,6,6-tetramethylpiperidide, lithium hexa-
10
)
To enhance the water-solubility of the amide-linked analogues, we envisaged attaching lysine
residues to the C-terminus, considering the known effect of such a modification on the water-
solubility of PNA oligomers [9][27].

Helvetica Chimica Acta – Vol. 92 (2009)
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methyldisilazide, or triphenylmethyl lithium, and by replacing DMF by N-methyl-
formanilide or N-formylmorpholine did not increase the yield of 18. The alcohol 18 was
converted in 73% yield into the alkyl bromide 19 by mesylation with methanesulfonic
anhydride in THF, followed by addition of a solution of LiBr in DMF. The
bromomethyl derivative 19 was best substituted by treatment with a twofold excess
of the Fmoc-protected ethyl hydrazinoacetate 2311) in DMF, in the absence of base.
This provided the fully protected uracil-derived 13 in a yield of 88%. Substitution of 19
in the presence of Hꢀnigꢄs base, pyridine, 2,6-di(tert-butyl)-4-methylpyridine, or K2CO3
resulted in a less clean reaction, entailing partial loss of the Fmoc group. The desired
monomers 20 and 21 were obtained in high yields by removing the Fmoc group with
piperidine in THF, and by cleaving the tert-butyl ester with TFA in the presence of
Et3SiH.
The fully protected cytosine-derived building block 14 was synthesized in a similar
way as 13 (Scheme 1). To prepare the Cbz-protected cytosine 25, we modified the
published procedure [30][31] by deprotonating a suspension of cytosine in DMF with
NaH instead of using pyridine, and adding a slight excess of ClCOOBn. This raised the
yield of 25 from 43 – 56% to 82% (batch size of 4 to 40 g). The Cbz-protected tert-butyl
cytosine-1-acetate 26 was obtained in 68% yield by alkylation of 25 with ClCH2COO-
(t-Bu) [30]. Hydroxymethylation of 26 at C(6), similarly as described for 17, gave the
alcohol 27 in 51% yield besides 27% of starting material 26. Mesylation of 27, followed
by addition of LiBr in DMF, provided the bromo derivative 28 that was directly treated
with the Fmoc-protected ethyl hydrazinoacetate 23 to yield 57% of the fully protected
cytosine-derived building block 14. Cleavage of the tert-butyl ester with TFA in the
presence of a large excess of Et3SiH to avoid partial loss of the Cbz group [30] afforded
82% of the carboxylic acid 29.
11
)
We obtained 23 in 78% yield by treating ethyl hydrazinoacetate hydrochloride (22) [29] with N-
succinimidoyl (9H-fluoren-9-yl)methylcarbonate (Fmoc-OSu) and N-methylmorpholine (NMM) in
THF.

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Scheme 1
a) 1. LDA (¼ lithium diisopropylamide), THF, ꢀ 708, 2 h; 2. DMF, ꢀ 708; 3. AcOH, EtOH; 4. NaBH4;
45% of 18; 43% of 17. b) 1. Ms2O, EtN(i-Pr)2, THF; 2. LiBr, DMF; 73%. c) 23, DMF; 88%. d)
Piperidine, THF; 96%. e) TFA (¼CF3COOH), Et3SiH, CH2Cl2; 99%. f) Fmoc-OSu (Fmoc ¼ (9H-
fluoren-9-yl)methoxycarbonyl), NMM (¼ N-methylmorpholine), THF; 78%. g) 1. NaH, DMF; 2.
ClCO2Bn; 82%. h) ClCH2CO2(t-Bu), K2CO3, Cs2CO3, DMF; 68%. i) 1. LDA, THF, ꢀ 708; 2. DMF,
ꢀ 708; 3. AcOH, EtOH; 4. NaBH4; 51% of 27; 27% of 26. j) 1. Ms2O (Ms ¼ mesyl ¼ methanesulfonyl),
THF; 2. LiBr, DMF. k) 23, DMF; 57% from 27. l) TFA, Et3SiH, CH2Cl2; 82%. Cbz ¼ (Benzyloxy)car-
bonyl.
Crystallisation of the fully protected building block 13 from MeOH afforded
crystals suitable for X-ray analysis. Fig. 5 shows capped sticks representations of the
crystal structure.
A comparison of the torsion angles of crystalline 13 with those predicted is not
straightforward, as two molecules of crystalline 13 are linked by four intermolecular H-
bonds (two H-bonds each between the carbazate NH and C¼O groups of one molecule
with the C(4)¼O and N(3)ꢀH groups of the second one) with a stacking distance of
3.3 ꢂ between the uracil planes. Even so, the torsion angles k, i1, i2, x, and z of
crystalline 13 deviate by less than ꢁ 68 from those derived by conformational analysis
of the hydrazide linker in the monoplex (Table).
The largest deviations from the values predicted for the monoplex were found for
the torsion angles l, e1, and e2 (Dl ¼ 1668, De1 ¼ 1868, and De2 ¼ 1838), corresponding to
an (E)-anti-conformation of 13. This conformation appears to be favoured by the

Helvetica Chimica Acta – Vol. 92 (2009)
1143
Fig. 5. Crystal structure of 13. a) Capped sticks representation indicating the torsion angles k – z. b)
Intermolecular H-bonding between two molecules (the 9H-fluorene-9-yl groups are deleted for the sake
of clarity).
intermolecular H-bonds and by a stabilising face-to-edge-type interaction between
HꢀC(5) of the uracil moiety and one aromatic ring of the fluorenyl group. The distance
of 2.65 ꢂ between HꢀC(5) and the s-plane of a fluorenyl benzene group, and their
almost orthogonal arrangement are in agreement with such an interaction that may
stabilise the conformation by ca. 2.5 kcal/mol (cf. [32]).
To prepare the Cbz-protected adenine 31, we modified the published procedure
[30] (Scheme 2), deprotonating adenine with NaH and adding 1.1 instead of 2.2 equiv.
of ClCOOBn. This raised the yield from 44 to 76% (batch size of 5 – 80 g). Alkylation
of 31 with ClCH2COO(t-Bu) [30] yielded 78% of 32. Hydroxymethylation of 32 at
C(8) yielded 76% of the 8-hydroxymethyl derivative 33, besides small amounts of
starting material. Mesylation of 33, followed by addition of LiBr in DMF provided
85% of the bromo derivative 34 that was treated with hydrazinoacetate 23 in DMF to

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Table. Torsion Angles of the Crystal Structure of 13 and Comparison with the Torsion Angles Derived for
the Hydrazide Linker
Torsion Observed in the
angles
Evaluated by conformational analysis of the hydrazide linker
crystal Structure of 13a) [8]
Monoplexb) [8]
Duplexb) [8]
k
i1
i2
e1
e2
l
ꢀ 65
ꢀ 59
þ 173
ꢀ 66
þ 63
ꢀ 14
ꢀ 178
ꢀ 95
ꢀ 50
ꢀ 60
180
þ 120
ꢀ 120
180
180
ꢀ 90
ꢀ 70
ꢀ 60
180
þ 120
ꢀ 120
180
x
þ 70
z
ꢀ 100
a) Torsion angles according to definition in Fig. 5. b) Torsion angles according to definition in Fig. 1.
yield 85% of the fully protected adenine-derived building block 15. The hydrazine 35
was prepared in 85% yield by removing the Fmoc group of 15 with piperidine, and the
carboxylic acid 36 was obtained in a yield of 98% by cleaving the tert-butyl ester with
TFA, similarly as described above for 29.
For the synthesis of the guanine-derived carboxylic acid 44 (Scheme 2), we acylated
the NH2 group at C(6) of 2,6-diamino-4-(benzyloxy)-5-nitrosopyrimidine (37) [33],
with acetoxyacetyl chloride in THF to obtain the 6-(acylamino)-5-nitrosopyrimidine 38
(84%). Reductive cyclisation of 38 with Ph3P in o-xylene [34] yielded almost
quantitatively the 8-(acetoxymethyl)purine 39. Alkylation of 39 with ClCH2COO(t-
Bu) gave 40 and 41 in a ratio of 3 :1, the regioselectivity being very similar to the one of
the alkylation of 2-amino-6-chloropurine [30][31]. The desired regioisomer 40 was
isolated in 69% yield by silica-gel chromatography, and its structure was assigned on the
basis of the cross-peak between C(8) and CH2ꢀN(9) in the HMBC spectrum.
Deacetylation of 40 led in 94% to the alcohol 42 that was treated with MsCl/LiCl to
provide the chloromethyl derivative 43. The yield was 76% on a 0.6 g scale, and 47% on
a 16 g scale. On the large scale, the reaction was somewhat less clean, and, more
significantly, purification of 43 by silica-gel chromatography proved difficult due to the
low solubility of 43 in organic solvents and its progressive decomposition on silica gel
when MeOH was used as an eluent. Attempts to prepare 43 or the analogous bromo
derivative by mesylating 42 with Ms2O, followed by treatment with LiCl or LiBr, as for
the synthesis of 19, 28, and 34, did not meet with success. Alkylation of the protected
ethyl hydrazinoacetate 23 with 43 under the conditions used for the preparation of 13,
14, and 15 failed, as 43 did not react with 23 in the absence of a base. Addition of
Hꢀnigꢄs base induced partial cleavage of the Fmoc group over longer reaction times.
However, coupling the chloro compound 43 with 23 in the presence of Bu4NBr and 2,6-
lutidine in DMSO was successful if slow, requiring 3 d for complete consumption of
starting materials. The resulting fully protected guanine-derived building block was
directly treated with TFA, similarly as described above for the other monomers, to
cleave both the tert-butyl ester and the benzyl ether groups [31]. The carboxylic acid 44
was obtained in a yield of 91% from 43.

Helvetica Chimica Acta – Vol. 92 (2009)
1145
Scheme 2
a) 1. NaH, DMF; 2. ClCO2Bn; 76%. b) ClCH2CO2(t-Bu), K2CO3, Cs2CO3, DMF; 78%. c) 1. LDA, THF,
ꢀ 768; 2. DMF, ꢀ 768; 3. AcOH, EtOH; 4. NaBH4; 76%. d) 1. Ms2O, EtN(i-Pr)2, CH2Cl2; 2. LiBr, DMF;
85%. e) 23, DMF; 85%. f) Piperidine, THF; 86%. g) TFA, Et3SiH, CH2Cl2; 98%. h) ClCOCH2OAc,
THF; 84%. i) Ph3P, o-Xylene, 1208; 99%. j) ClCH2CO2(t-Bu), K2CO3, DMF; 69% of 40. k) K2CO3,
MeOH/H2O; 94%. l) 1. MsCl, EtN(i-Pr)2, DMF; 2. LiCl; 47%. m) 23, Bu4NBr, 2,6-lutidine, DMSO. n)
TFA, Et3SiH, CH2Cl2; 91% from 43.
We originally intended to protect the NH2 group at C(2) of the guanine-derived
monomer by benzyloxycarbonylation to avoid acylating this amino group during
formation of the hydrazide bond. However, attempts at introducing the Cbz group in
38, 40, and 44 failed under a variety of conditions, such as those applied for the
protection of adenine and cytosine, the use of Rapoportꢄs reagent (1-[(benzyloxy)-
carbonyl]-2-ethylimidazolium tetrafluoroborate) [35], or of ClCOOBn in the presence
of KH and 18-crown-6 [30][36]. Fortunately, it turned out that this amino group did not
require protection, as 40 did neither react with Ac2O, nor with 21 and 36 using HATU in
the presence of Hꢀnigꢄs base, similarly as it was reported for some guanine-derived
monomers in the synthesis of PNA [31][37].
3. Synthesis of the Building Blocks Required for the Preparation of Amide-Linked
Oligonucleotide Analogues. We chose the same protecting groups for the fully
protected building blocks 45, 46, 47, and 48 as for the hydrazide building blocks.

1146
Helvetica Chimica Acta – Vol. 92 (2009)
Formylation of the tert-butyl acetate 17 provided 37% of 49 (Scheme 3). Wittig
reaction of 49 with benzyl (triphenylphosphoranylidene)acetate in THF yielded 76%
of the acrylate 50. Hydrogenation in the presence of Pd/C in THF/MeOH reduced the
olefinic C¼C bond and cleaved the benzyl ester of 50 to give the carboxylic acid 51 in
practically quantitative yield. The acyl azide was generated from 51 via the mixed
anhydride with ClCOOEt in THF. The crude acyl azide obtained upon aqueous workup
was heated in dry dioxane in the presence of 9H-fluorene-9-methanol to give the fully
protected uracil-derived building block 45 (90% from 51). Treatment of 45 with TFA
cleaved the tert-butyl ester, and yielded 72% of the carboxylic acid 52.
The fully protected cytosine-derived building block 46 was similarly synthesized
(Scheme 3). Aldehyde 53 was obtained in 64% yield by formylating the Cbz-protected
tert-butyl cytosine-1-acetate 26. Wittig reaction gave the methyl acrylate 5412) that
could not be completely separated from Ph3PO when the reaction was performed on a
scale of 7 g (97% of 54/Ph3PO). Reduction of the olefinic C¼C bond of 54 · Ph3PO
with NaBH4 in MeOH gave 98% of 55/Ph3PO, and saponification with LiOH in H2O/
THF afforded 83% of the carboxylic acid 56 that was separated from Ph3PO by
chromatography. The fully protected cytosine-derived building block 46 was obtained
in 57% yield by Curtius degradation and interception of the isocyanate with 9H-
fluorene-9-methanol, besides 29% of starting material 56. The tert-butyl ester was again
cleaved by the action of TFA in the presence of a large excess of Et3SiH to provide the
carboxylic acid 57 in almost quantitative yield.
Preparation of the fully protected adenine-derived building block 47 (Scheme 4)
began by formylating the Cbz-protected tert-butyl adenine-9-acetate 32, similarly as
described above, to give aldehyde 58 (58%). The Wittig reaction to provide methyl
acrylate 59 (90%) was performed similarly as described for 54. The olefinic C¼C bond
of 59 was reduced with NaBH4 in MeOH, and the methyl ester was saponified with
12
)
The methyl ester was chosen, as hydrogenolytic cleavage of the benzyl ester was expected to cleave
also the NCbz group.

Helvetica Chimica Acta – Vol. 92 (2009)
1147
Scheme 3
a) 1. LDA, THF, ꢀ 768; 2. DMF, ꢀ 768; 3. AcOH; 37%. b) Ph3PCHCO2Bn, THF; 76%. c) H2,
Pd(OAc)2, MeOH/THF; 100%. d) 1. ClCO2Et, EtN(i-Pr)2, THF; 2. NaN3, H2O; 3. 9H-fluorene-9-
methanol, dioxane, 1008; 90%. e) TFA, Et3SiH, CH2Cl2; 72%. f) 1. LDA, THF, ꢀ 768; 2. DMF, ꢀ 768; 3.
AcOH, EtOH; 64%. g) Ph3PCHCO2Me, THF; 97%. h) NaBH4, MeOH; 98%. i) LiOH, THF/H2O;
83%. j) 1. ClCO2Et, EtN(i-Pr)2, THF/acetone; 2. NaN3, H2O; 3. 9H-fluorene-9-methanol, dioxane, 808;
57%. k) TFA, Et3SiH, CH2Cl2; 98%.
LiOH in H2O/THF 4 :1 to yield 73% of 60 and 81% of 61. The resulting carboxylic
acid 61 was converted to the acyl azide that was subjected to a Curtius degradation,
intercepting the resulting isocyanate with 9H-fluorene-9-methanol. This provided 52%
of the fully protected adenine-derived building block 47, besides 13% of starting
material 61 resulting from hydrolysis of the acyl azide during aqueous workup. Cleaving
the tert-butyl ester with TFA in the presence of a large excess of Et3SiH afforded the
carboxylic acid 62 in 98% yield.
To prepare the fully protected guanine-derived building block 48, we acylated 2,6-
diamino-4-(benzyloxy)-5-nitrosopyrimidine (37) [33] with N-phthaloyl-b-alanyl chlor-
ide (69) [38] to obtain the nitrosoamide 63 in 94% yield (Scheme 4). Its reductive
cyclisation to the 8-(phthalimidoethyl)purine 64 using Me3P proceeded at a lower
temperature (608 instead of 1208) and more cleanly than the cyclisation of 63 using
Ph3P. The crude C(8)-substituted guanine 64 was alkylated with ClCH2COO(t-Bu), to
provide a ca. 3 :1 mixture of the N(9)- and N(7)-substituted regioisomers 65 and 66,
respectively. Their structure was assigned on the basis of the cross-peak between C(8)
and CH2ꢀN(9) in the HMBC spectrum. The desired 65 was obtained pure by
chromatography (51% from 63). Hydrazinolytic dephthaloylation in EtOH yielded
86% of 67, and Fmoc protection gave the fully protected guanine-derived building
block 48 in 86% yield. The tert-butyl ester and the benzyl ether groups of 48 were
cleaved by treatment with TFA/Et3SiH to yield 98% of the carboxylic acid 68.

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Helvetica Chimica Acta – Vol. 92 (2009)
Scheme 4
a) 1. LDA, THF, ꢀ 768; 2. DMF, ꢀ 768; 3. AcOH, EtOH; 58%. b) Ph3PCHCO2Me, THF; 90%. c)
NaBH4, MeOH; 73%. d) LiOH, THF/H2O; 81%. e) 1. ClCO2Et, EtN(i-Pr)2, THF/acetone; 2. NaN3,
H2O; 3. 9H-fluorene-9-methanol, dioxane, 808; 52%. f) TFA, Et3SiH, CH2Cl2; 98%. g) Phth-b-Ala-Cl
(Phth ¼ phthaloyl; 69), THF; 94%. h) Me3P, toluene, 608. i) ClCH2CO2(t-Bu), K2CO3, Cs2CO3, DMF;
51% of 65. j) N2H4, EtOH; 86%. k) Fmoc-OSu, THF; 86%. l) TFA, Et3SiH, CH2Cl2; 98%.
We thank Dr. B. Bernet for his contribution to the conformational analyses and for checking the
manuscript, and the ETH-Zꢀrich and Syngenta, Basel, for generous support.
Experimental Part
General. THF was distilled from Na/benzophenone, CH2Cl2, MeOH, DMF, pyridine, EtN(i-Pr)2,
and (i-Pr)2NH from CaH2. Reactions were run under N2. Qual. TLC: precoated silica-gel plates (Merck
silica gel 60 F 254); detection by UV light at 254 nm wavelength and by spraying with ꢃmostainꢄ 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 indol-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.

Helvetica Chimica Acta – Vol. 92 (2009)
1149
tert-Butyl 6-[(1-(2-Ethoxy-2-oxoethyl)-2-{[(9H-fluoren-9-yl)methoxy]carbonyl}hydrazino)methyl]-
uracil-1-acetate (13). A soln. of 23 (19.6 g, 58 mmol) in DMF (150 ml) was treated dropwise with a soln.
of 19 (11.7 g, 37 mmol) in DMF (100 ml), stirred at r.t. for 2 d, treated with sat. NaHCO3 soln. (300 ml),
and extracted with AcOEt (5 ꢃ 200 ml). Drying of the combined org. layers (MgSO4), filtration,
evaporation, and FC (AcOEt/cyclohexane 1:1 ! 4 :1) gave 13 (18.6 g, 88%). White powder. Rf (AcOEt/
cyclohexane 1:1) 0.21. M.p. 196 – 1998. UV (CHCl3): 267 (28700), 302 (5700). IR (ATR): 3270w, 3151w,
3094w, 2980w, 1740s, 1702s, 1677s, 1466m, 1448m, 1409m, 1391m, 1365m, 1326m, 1232s, 1207s, 1159s,
1
1126m, 1102w, 1065m, 1028w, 1001w, 979w, 933w, 870w, 846w, 830m. H-NMR (300 MHz, (D6)DMSO;
assignments based on a HSQC and a HMBC spectrum): 11.44 (s, HꢀN(3)); 8.91 (br. s, NHCO2); 7.89 –
7.29 (m, 8 arom. H); 5.60 (br. s, HꢀC(5)); 4.91 (br. s, CH2ꢀN(1)); 4.27 (br. s, CH2ꢀC(9’)); 4.18 (br. s,
HꢀC(9’)); 4.09 (q, J ¼ 7.0, MeCH2O); 3.72 (br. s, CH2ꢀC(6), NCH2CO2Et); 1.40 (s, tBu); 1.19 (t, J ¼ 7.2,
MeCH2O). 1H-NMR (300 MHz, (D6)DMSO, 1008): 11.00 (s, HꢀN(3)); 8.38 (s, NHCO2); 7.85 – 7.29 (m,
8 arom. H); 5.55 (s, HꢀC(5)); 4.81 (s, CH2ꢀN(1)); 4.37 (d, J ¼ 6.3, CH2ꢀC(9’)); 4.21 (t, J ¼ 6.5,
HꢀC(9’)); 4.13 (q, J ¼ 6.9, MeCH2O); 3.72, 3.60 (2s, CH2ꢀC(6), NCH2CO2Et); 1.43 (s, tBu); 1.23 (t, J ¼
6.9, MeCH2O). 13C-NMR (75 MHz, (D6)DMSO; assignments based on a HSQC and a HMBC
t
spectrum): 168.88 (s, CO2Et); 167.87 (s, CO2 Bu); 162.23 (s, C(4)); 154.98, 150.71 (2s, C(2), NHCO2);
151.46 (s, C(6)); 143.43 (2s); 140.56 (2s); 127.54 (2d); 126.99 (2d); 124.90 (2d); 119.99 (2d); 103.79 (d,
C(5)); 81.54 (s, Me3C); 65.61 (t, CH2ꢀC(9’)); 60.38 (t, MeCH2O); 57.57 (t, CH2ꢀC(6)); 56.61 (t,
NCH2CO2Et); 46.59 (d, C(9’)); 45.26 (t, CH2ꢀN(1)); 27.65 (q, Me3C); 14.07 (q, MeCH2O). ESI-MS: 601
t
t
(100, [M þ Na]þ), 545 (23, [M ꢀ Bu þ H þ Na]þ), 523 (11, [M ꢀ Bu þ 2 H]þ). Anal. calc. for C30H34N4O8
(578.61): C 62.27, H 5.92, N 9.68; found: C 62.32, H 6.15, N 9.91.
X-Ray Analysis of 13. Crystals of 13 were obtained by slow evaporation of a soln. of 13 in MeOH
(dimensions of the analysed crystal: cube 0.4 ꢃ 0.22 ꢃ 0.04 mm). C30H34N4O8, Mr ¼ 578.62, triclinic P1,
a ¼ 10.7336(7), b ¼ 11.0417(9), c ¼ 13.1991(9) ꢂ, a ¼ 75.096(4), b ¼ 74.695(4), g ¼ 84.237(4)8, V ¼
1457.2(2) ꢂ3, Z ¼ 2, Dx ¼ 1.319 Mg · mꢀ3. The reflections were measured on a KappaCCD diffractometer,
with MoKa radiation l ¼ 0.71073 ꢂ. Cell parameters from 9199 refl., q ¼ 0.998 – 24.1088, m ¼ 0.097 mmꢀ1
,
T¼ 223 K. 6105 Measured reflections, 4217 independent reflections, 2837 observed reflections (>2s(I)).
Refinement on F2: full-matrix least-squares refinement, R(all) ¼ 0.1095, R(gt) ¼ 0.0700. All diagrams
and calculations were performed using maXus (Bruker Nonius, Delft and MacScience, Japan). The
program SIR97 was used to solve the structure and the program SHELXL-97 to refine it.
tert-Butyl N4-[(Benzyloxy)carbonyl]-6-[(1-(2-ethoxy-2-oxoethyl)-2-{[(9H-fluoren-9-yl)methoxy]-
carbonyl}hydrazino)methyl]cytosine-1-acetate (14). A soln. of 27 (8.5 g, 22 mmol) in THF (85 ml) was
cooled to 08, treated with EtNiPr2 (11.4 ml, 65 mmol), stirred for 30 min, treated dropwise with a soln. of
Ms2O (11.4 g, 65 mmol) in THF (50 ml), and stirred for 2.5 h while being allowed to reach r.t. The soln.
was cooled to 08, treated dropwise with a soln. of LiBr (9.5 g, 109 mmol) in DMF (85 ml), stirred for 3 h
while being allowed to reach r.t., cooled to 08, treated with sat. NaHCO3 soln. (100 ml), and extracted
with AcOEt (3 ꢃ 300 ml). The combined org. layers were dried (MgSO4), filtered, and evaporated. A
soln. of the residue in DMF (60 ml) was treated portionwise with 23 (18.6 g, 55 mmol), stirred for 12 h at
r.t., and diluted with AcOEt (100 ml) and sat. NaHCO3 soln. (100 ml). After separation of the layers, the
aq. layer was extracted with AcOEt (3 ꢃ 200 ml). Drying of the combined org. layers (MgSO4),
filtration, evaporation, and FC (AcOEt/cyclohexane 1:2 ! 4 :1) gave 14 (8.8 g, 57%). White powder. Rf
(AcOEt/cyclohexane 2 :1) 0.48. UV (MeOH): 254 (24400), 300 (13600). IR (ATR): 3217w (br.), 2979w,
1736s, 1704m, 1668m, 1616m, 1567m, 1498m, 1450m, 1415m, 1389s, 1369m, 1324w, 1192s, 1152s, 1077m,
1052m, 1028m, 971w, 862w, 817w. 1H-NMR (300 MHz, (D6)DMSO): 10.71 (s, NHꢀC(4)); 8.94 (s,
NHꢀN); 7.86 – 7.24 (m, 13 arom. H); 7.04 (s, HꢀC(5)); 5.09 (s, CH2ꢀN(1)); 4.97 (s, PhCH2); 4.21 (br. s,
CH2ꢀC(9’)); 4.14 – 4.07 (m, MeCH2O, HꢀC(9’)); 3.90 (s, CH2ꢀC(6)); 3.79 (s, NCH2CO2Et); 1.41 (s,
Me3C); 1.20 (t, J ¼ 7.2, MeCH2O). 13C-NMR (100 MHz, (D6)DMSO; assignments based on a HSQC and
t
a HMBC spectrum): 169.08 (s, CO2Et); 167.81 (s, CO2 Bu); 162.35 (s, C(4)); 155.93 (s, C(2)); 155.14 (s,
NꢀNCO2, C(6)); 152.87 (s, C(4)NCO2); 143.57 (2s); 140.64 (2s); 135.76 (s); 128.37 (2d); 128.08 (2d);
127.89 (2d); 127.57 (2d); 126.98 (d); 124.90 (2d); 119.99 (2d); 96.97 (d, C(5)); 81.39 (s, Me3C); 66.39 (t,
PhCH2); 65.69 (t, CH2ꢀC(9)); 60.38 (t, MeCH2O); 57.78, 56.63 (2t, CH2ꢀC(6), NCH2CO2Et); 46.70 (t,
CH2ꢀN(1)); 46.48 (d, C(9’)); 27.60 (q, Me3C); 13.94 (q, MeCH2O). HR-MALDI-MS: 750.2524 (12,
[M þ K]þ, C38H41KN5O9þ ; calc. 750.2536), 734.2770 (10, [M þ Na]þ, C38H41N5NaOþ9 ; calc. 734.2796),

1150
Helvetica Chimica Acta – Vol. 92 (2009)
t
712.2976 (100, [M þ H]þ, C38H42N5O9þ ; calc. 712.2977), 656.2324 (21, [M ꢀ Bu þ 2 H]þ, C34H34N5Oþ9 ;
calc. 656.2351).
tert-Butyl N6-[(Benzyloxy)carbonyl]-8-[(1-(2-ethoxy-2-oxoethyl)-2-{[(9H-fluoren-9-yl)methoxy]-
carbonyl}hydrazino)methyl]adenine-9-acetate (15). A soln. of 23 (7.15 g, 21.0 mmol) in DMF (200 ml)
was treated dropwise with a soln. of 34 (9.1 g, 21.0 mmol) in DMF (70 ml) and stirred for 16 h at r.t. The
soln. was poured on sat. NaHCO3 soln. and extracted with AcOEt (5ꢃ). The combined org. fractions
were washed with brine (5ꢃ), dried (MgSO4), and evaporated. FC (AcOEt/cyclohexane 1:1) gave 15
(12.0 g, 85%). White foam. Rf (CH2Cl2/MeOH 19 :1) 0.38. UV (MeOH): 230 (5740), 266 (37620), 212
(54720). IR (ATR): 3191w, 3063w, 3033w, 2979w, 2942w, 2903w, 1733m, 1613m, 1590m, 1538w, 1497w,
1477w, 1450m, 1392w, 1368m, 1321w, 1299w, 1237m, 1201s, 1151s, 1102m, 1041m, 1029m, 1006w, 972w,
847w, 758m, 739s, 697m. 1H-NMR (400 MHz, (D6)DMSO): 10.65 (br. s, NHꢀC(6)); 8.83 (br. s,
NHFmoc); 8.59 (s, HꢀC(2)); 7.85 – 7.15 (m, 13 arom. H); 5.48 (br. s, CH2ꢀN(9)); 5.20 (s, PhCH2); 4.31
(br. s, CH2ꢀC(8)); 4.22 (d, J ¼ 6.5, CH2ꢀC(9’)); 4.15 – 4.05 (m, MeCH2O, HꢀC(9’)); 3.73 (br. s,
NCH2CO2Et); 1.41 (s, Me3C); 1.19 (t, J ¼ 7.1, MeCH2O). 13C-NMR (100 MHz, (D6)DMSO): 168.97 (s,
t
CO2Et); 167.05 (s, CO2 Bu); 154.97 (s, NNHCO2); 153.67 (s, NHCO2Bn); 151.96 (s, C(4)); 151.48 (s,
C(8)); 150.50 (d, C(2)); 148.81 (s, C(6)); 143.48 (2s); 140.60 (2s); 136.30 (s); 128.28 – 124.90 (11d); 121.92
(s, C(5)); 119.96 (2d); 82.10 (s, Me3C); 66.17 (t, PhCH2); 65.49 (t, CH2ꢀC(9’)); 60.27 (t, MeCH2O); 57.52,
53.79 (2t, CH2ꢀC(8), NCH2CO2Et); 46.49 (d, C(9’)); 44.42 (t, CH2ꢀN(9)); 27.55 (q, Me3C); 13.91 (q,
MeCH2O). HR-MALDI-MS: 774.2650 (8, [M þ K]þ, C39H41KN7O8þ ; calc. 774.2654), 758.2927 (61, [M þ
Na]þ, C39H41N7NaOþ8 ; calc. 758.2914), 736.3075 (100, [M þ H]þ, C39H42N7O8þ ; calc. 736.3095), 702.2270
t
t
(18, [M ꢀ Bu þ H þ Na]þ, C35H33N7NaO8þ ; calc. 702.2288), 680.2442 (22, [M ꢀ Bu þ 2 H]þ, C35H34N7Oþ8 ;
calc. 680.2469), 628.2511 (14, [M ꢀ BnOH þ H]þ, C32H34N7O7þ ; calc. 628.2520), 594.1703 (2, [M ꢀ
BnOH ꢀ Bu þ H þ Na]þ, C28H25N7NaO7þ ; calc. 594.1713), 572.1875 (9, [M ꢀ BnOH ꢀ Bu þ 2 H]þ,
C28H26N7Oþ7 ; calc. 572.1894). Anal. calc. for C39H41N7O8 (735.78): C 63.66, H 5.62, N 13.33; found: C
63.43, H 5.67, N 13.16.
t
t
i
tert-Butyl 6-(Hydroxymethyl)uracil-1-acetate (18). A soln. of Pr2NH (29.7 ml, 210 mmol) in THF
(200 ml) was cooled to ꢀ 678, treated dropwise with 1.6m BuLi in hexane (131 ml, 210 mmol), stirred for
20 min, warmed to 08, stirred for 20 min, cooled to ꢀ 688, and treated dropwise with a soln. of 17 (9.5 g,
42 mmol) in THF (225 ml). After stirring for 2 h at ꢀ 708, the soln. was treated dropwise with DMF
(65 ml, 840 mmol), stirred for another 1.5 h at ꢀ 708, treated dropwise with AcOH (24 ml, 420 mmol),
diluted with EtOH (200 ml), allowed to reach 08, treated portionwise with NaBH4 (4.8 g, 126 mmol),
stirred for 30 min, treated with sat. NH4Cl soln. (500 ml), allowed to reach r.t., and extracted with CH2Cl2
(3 ꢃ 300 ml). Drying of the combined org. layers (MgSO4), filtration, evaporation, and FC (CH2Cl2/
MeOH 97:3 ! 92 :8) gave 17 (4.1 g, 43%) and 18 (4.8 g, 45%).
Data of 18. White powder. Rf (CH2Cl2/MeOH 9 :1) 0.40. M.p. 174 – 1758. UV (CHCl3): 264 (9500).
IR (ATR): 3461w, 3142w, 3090w, 3006w, 2978w, 2927w, 2874w, 2808w, 1745m, 1689s, 1656m, 1617w,
1476m, 1427m, 1397m, 1378m, 1367m, 1232m, 1206m, 1158s, 1078w, 1044m, 994m, 980w, 930w, 871m,
1
851m, 823w. H-NMR (300 MHz, (D6)DMSO): 11.36 (s, NH); 5.77 (t, J ¼ 5.6, OH); 5.65 (s, HꢀC(5));
t
4.44 (s, NCH2); 4.21 (d, J ¼ 5.1, CH2ꢀC(6)); 1.41 (s, Bu). 13C-NMR (75 MHz, (D6)DMSO): 167.29 (s,
CO2); 162.84 (s, C(4)); 156.39 (s, C(2)); 151.65 (s, C(6)); 98.89 (d, C(5)); 81.86 (s, Me3C); 59.00 (t,
CH2OH); 44.54 (t, NCH2); 27.63 (q, Me3C). HR-EI-MS: 256.1055 (3, Mþ, C11H16N2O5þ ; calc. 256.1059),
t
t
200.0430 (2, [M ꢀ Bu þ H]þ, C7H8N2O5þ ; calc. 200.0433), 183.0399 (15, [M ꢀ BuO]þ, C7H7N2Oþ4 ; calc.
t
183.0400), 156.0530 (34, [M ꢀ CO2 Bu þ H]þ, C6H8N2O3þ ; calc. 156.0535), 57.0719 (100, tBuþ, C4Hþ9 ; calc.
57.0699). Anal. calc. for C11H16N2O5 (256.26): C 51.56, H 6.29, N 10.93; found: C 51.66, H 6.30, N 10.80.
tert-Butyl 6-(Bromomethyl)uracil-1-acetate (19). A soln. of 18 (12.6 g, 49 mmol) and EtNiPr2
(17.1 ml, 98 mmol) in THF (320 ml) was cooled to 08, treated dropwise with a soln. of Ms2O (12.9 g,
74 mmol) in THF (30 ml), and stirred for 2 h. The soln. was allowed to reach r.t., treated dropwise with a
soln. of LiBr (8.5 g, 98 mmol) in DMF (120 ml), stirred for 4 h, treated with brine (300 ml), and extracted
with AcOEt (3 ꢃ 300 ml). Drying of the combined org. layers (MgSO4), filtration, evaporation, and
crystallisation from MeOH gave 19 (11.5 g, 73%). White crystals. Rf (CH2Cl2/MeOH 95 :5) 0.40. M.p.
232 – 2408. UV (CHCl3): 277 (12270). IR (ATR): 3003w, 2815w, 1737s, 1720s, 1697s, 1607w, 1471m, 1459w,
1414s, 1385m, 1372m, 1234m, 1198w, 1167m, 1140m, 992w, 923w, 883m, 866m, 821w. 1H-NMR (300 MHz,
t
CDCl3): 8.59 (br. s, NH); 5.81 (d, J ¼ 2.1, HꢀC(5)); 4.65 (s, NCH2); 4.02 (s, CH2ꢀC(6)); 1.49 (s, Bu).

Helvetica Chimica Acta – Vol. 92 (2009)
1151
13C-NMR (75 MHz, (D6)DMSO): 166.28 (s, CO2); 162.10 (s, C(4)); 151.34, 151.24 (2s, C(2), C(6)); 103.13
(d, C(5)); 81.72 (s, Me3C); 45.63 (t, NCH2); 28.10 (t, CH2Br); 27.67 (q, Me3C). HR-EI-MS: 320.0186 (2,
Mþ, C11H1581BrN2O4þ ; calc. 320.0195), 318.0210 (2, Mþ, C11H1579BrN2O4þ ; calc. 318.0215), 219.9657 (14,
t
t
[M ꢀ CO2 Bu þ H]þ, C6H781BrN2O2þ ; calc. 219.9670), 217.9675 (14, [M ꢀ CO2 Bu þ H]þ, C6H779BrN2Oþ2 ;
calc. 217.9691), 57.0711 (100, tBuþ, C4H9þ ; calc. 57.0699). Anal. calc. for C11H15BrN2O4 (319.15): C 41.40,
H 4.74, N 8.78; found: C 41.69, H 4.82, N 8.72.
tert-Butyl 6-{[1-(2-Ethoxy-2-oxoethyl)hydrazino]methyl}uracil-1-acetate (20). A soln. of 13 (5 g,
8.6 mmol) in THF (150 ml) was treated dropwise with piperidine (1.3 ml, 13.1 mmol), stirred for 8 h at
r.t., and evaporated. The solid residue was washed with Et2O (5 ꢃ 50 ml). The solid was suspended in
CH2Cl2 (200 ml) and filtered. Evaporation of the filtrate gave 20 (3.0 g, 96%). White powder. Rf (CH2Cl2/
MeOH 95 :5) 0.23. M.p. 133 – 1348. UV (CHCl3): 267 (8300). IR (ATR): 3178w (br.), 2980w, 1730s, 1687s,
1
1624w, 1458m, 1419m, 1390m, 1370m, 1235m, 1199m, 1156s, 1103w, 1026w, 926w, 865w, 819w. H-NMR
(300 MHz, (D6)DMSO; assignments based on a HSQC and a HMBC spectrum): 11.37 (s, HꢀN(3)); 5.60
(s, HꢀC(5)); 4.65 (s, CH2ꢀN(1)); 4.10 (q, J ¼ 7.2, MeCH2O); 3.68 (s, NH2); 3.63 (s, CH2ꢀC(6)); 3.49 (s,
t
NCH2CO2Et); 1.41 (s, Bu); 1.21 (t, J ¼ 6.9, MeCH2O). 13C-NMR (75 MHz, (D6)DMSO; assignments
t
based on a HSQC and a HMBC spectrum): 169.76 (s, CO2Et); 167.31 (s, CO2 Bu); 162.39 (s, C(4)); 151.65
(s, C(6)); 151.59 (s, C(2)); 103.70 (d, C(5)); 81.31 (s, Me3C); 60.10 (t, CH2ꢀC(6)); 60.00 (t, MeCH2O);
59.84 (t, NCH2CO2Et); 45.32 (t, CH2ꢀN(1)); 27.68 (q, Me3C); 14.12 (q, MeCH2O). HR-ESI-MS:
t
379.1589 (82, [M þ Na]þ, C15H24N4NaO6þ ; calc. 379.1588), 323.0962 (100, [M ꢀ Bu þ H þ Na]þ,
t
C11H16N4NaOþ6 ; calc. 323.0962), 301.1151 (18, [M ꢀ Bu þ 2 H]þ, C11H17N4Oþ6 ; calc. 301.1143). Anal.
calc. for C15H24N4O6 (356.37): C 50.55, H 6.79, N 15.72; found: C 50.71, H 6.71, N 15.70.
tert-Butyl 6-[(1-(2-Ethoxy-2-oxoethyl)-2-{[(9H-fluoren-9-yl)methoxy]carbonyl}hydrazino)methyl]-
uracil-1-acetic Acid (21). A suspension of 13 (3.2 g, 5.5 mmol) in CH2Cl2 (50 ml) was treated dropwise
with Et3SiH (1.1 ml, 6.9 mmol) and TFA (12.3 ml, 166 mmol) and stirred for 4.5 h at r.t. After
evaporation, the solid residue was washed with Et2O (10 ꢃ 50 ml). Filtration gave 21 (2.9 g, 99%). White
powder. Rf (AcOEt/MeOH/H2O 80 :15 :5) 0.27. M.p. 234 – 2368. UV (MeOH): 264 (32400), 299 (9400).
IR (ATR): 3383w, 3270 – 2410w (br.), 3021w, 1734m, 1710s, 1665s, 1469m, 1448w, 1417s, 1390m, 1333m,
1300w, 1270w, 1211s, 1176m, 1141m, 1064m, 1016m, 984w, 957w, 933w, 886m, 858m, 832m. 1H-NMR
(300 MHz, (D6)DMSO; assignments based on a HSQC and a HMBC spectrum): 12.97 (br. s, CO2H);
11.42 (d, J ¼ 2.1, HꢀN(3)); 8.89 (br. s, NHCO2); 7.89 – 7.29 (m, 8 arom. H); 5.59 (br. s, HꢀC(5)); 4.89 (br.
s, CH2ꢀN(1)); 4.27 (br. s, CH2ꢀC(9’)); 4.17 (br. s, HꢀC(9’)); 4.09 (q, J ¼ 7.1, MeCH2O); 3.78, 3.73 (2 br.
s, CH2ꢀC(6), NCH2CO2Et); 1.18 (t, J ¼ 7.1, MeCH2O). 1H-NMR (300 MHz, (D6)DMSO, 808): 11.10 (s,
HꢀN(3)); 8.48 (br. s, NHCO2); 7.86 – 7.29 (m, 8 arom. H); 5.53 (s, HꢀC(5)); 4.82 (s, CH2ꢀN(1)); 4.34 (d,
J ¼ 6.6, CH2ꢀC(9’)); 4.20 (t, J ¼ 6.5, HꢀC(9’)); 4.12 (q, J ¼ 7.0, MeCH2O); 3.72, 3.61 (2s, CH2ꢀC(6),
NCH2CO2Et); 1.21 (t, J ¼ 7.2, MeCH2O). 13C-NMR (75 MHz, (D6)DMSO; assignments based on a
HSQC and a HMBC spectrum): 170.08 (s, CO2H); 168.87 (s, CO2Et); 162.23 (s, C(4)); 154.96 (s,
NHCO2); 151.50, 150.82 (2s, C(2), C(6)); 143.42 (2s); 140.54 (2s); 127.51 (2d); 126.96 (2d); 124.93 (2d);
119.97 (2d); 103.66 (d, C(5)); 65.57 (t, CH2ꢀC(9’)); 60.37 (t, MeCH2O); 57.50, 56.55 (2t, CH2ꢀC(6),
NCH2CO2Et); 46.56 (d, C(9’)); 44.70 (t, CH2ꢀN(1)); 14.06 (q, MeCH2O). HR-MALDI-MS: 545.1646
(100, [M þ Na]þ, C26H26N4NaO8þ ; calc. 545.1643). Anal. calc. for C26H26N4O8 (522.51): C 59.77, H 5.02, N
10.72; found: C 59.49, H 5.03, N 10.65.
Ethyl 2-(2-{[(9H-Fluoren-9-yl)methoxy]carbonyl}hydrazine)acetate (23). A suspension of 22
(30.0 g, 194 mmol) in THF (1 l) was cooled to 08, treated with 4-methylmorpholine (NMM; 21.3 ml,
194 mmol), then dropwise within 2.5 h with a soln. of Fmoc-OSu (59.5 g, 176 mmol) in THF (500 ml) at
08, and stirred for 3 h. The precipitate was filtered off and washed with THF. After evaporation of the
filtrate, the residue was poured on H2O. Extraction with AcOEt (3ꢃ), washing of the combined org.
layers with brine (3ꢃ), drying (MgSO4), evaporation, and crystallisation from AcOEt/cyclohexane gave
23 (46.7 g, 78%). Colourless needles. Rf (CH2Cl2/MeOH 98 :2) 0.40. M.p. 112 – 1138. UV (CHCl3): 268
(19231), 290 (5154), 301 (5923). IR (ATR): 3392m, 3229w, 3037w, 2981w, 2893w, 1733s, 1709s, 1552w,
1487m, 1463m, 1449m, 1398w, 1371m, 1350w, 1279m, 1210s, 1178m, 1127m, 1104m, 1079m, 1048m, 1031m,
995w, 954w, 938w, 892w, 861m. 1H-NMR (300 MHz, CDCl3): 7.78 – 7.29 (m, 8 arom. H); 6.66 (br. s,
NHC¼O); 4.45 (d, J ¼ 6.9, CH2ꢀC(9)); 4.26 – 4.19 (m, MeCH2O, NHCH2, HꢀC(9)); 3.65 (br. s,
NHCH2); 1.30 (t, J ¼ 7.1, MeCH2O). 1H-NMR (400 MHz, (D6)DMSO): 8.75 (br. s, NHC¼O); 7.90 – 7.30

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Helvetica Chimica Acta – Vol. 92 (2009)
(m, 8 arom. H); 4.88 (br. s, NHCH2); 4.30 (d, J ¼ 7.0, CH2ꢀC(9)); 4.22 (t, J ¼ 6.9, HꢀC(9)); 4.11 (q, J ¼
7.1, MeCH2O); 3.50 (br. s, NHCH2); 1.20 (t, J ¼ 7.1, MeCH2O). 13C-NMR (100 MHz, (D6)DMSO;
assignments based on a HSQC spectrum): 170.27 (s, CO2Et); 156.64 (s, NHC¼O); 143.67 (2s); 140.63
(2s); 127.56 (2d); 126.96 (2d); 125.18 (2d); 120.01 (2d); 65.61 (t, CH2ꢀC(9)); 59.97 (t, MeCH2O); 52.00 (t,
NHCH2); 46.57 (d, C(9)); 13.99 (q, MeCH2O). HR-ESI-MS: 363.1330 (100, [M þ Na]þ, C19H20N2NaO4þ ;
calc. 363.1315). Anal. calc. for C19H20N2O4 (340.37): C 67.05, H 5.92, N 8.23; found: C 66.78, H 5.90, N
8.22.
N4-[(Benzyloxy)carbonyl]cytosine (25). A suspension of NaH (60% in mineral oil, washed with 5 ꢃ
100 ml of pentane, 14.4 g, 360 mmol) in DMF (150 ml) was cooled to 08, treated with cytosine (10 g,
90 mmol), stirred for 1 h, treated with ClCOOBn (13.5 ml, 95 mmol), allowed to reach r.t., stirred for
14 h, and treated carefully with H2O and ice (200 ml). After neutralisation of the resulting soln. with 5m
HCl, a precipitate was formed, which was filtered off, washed with H2O (5 ꢃ 50 ml) and dried to give 25
(18.1 g, 82%). White powder. Spectroscopic data were in agreement with those in [30].
tert-Butyl N4-[(Benzyloxy)carbonyl]cytosine-1-acetate (26) [30]. A suspension of 25 (18 g,
73 mmol), K2CO3 (10.1 g, 73 mmol), and Cs2CO3 (2.4 g, 7.4 mmol) in DMF (250 ml) was cooled to 08,
treated dropwise with ClCH2COOtBu (10.5 ml, 73 mmol), allowed to reach r.t., stirred for 22 h, and
filtered. The combined filtrates were evaporated, and the residue was dissolved in AcOEt (500 ml) and
H2O (100 ml). The layers were separated, and the aq. layer was extracted with AcOEt (2 ꢃ 100 ml).
Drying of the combined org. layers (MgSO4), filtration, evaporation, and crystallisation from toluene
gave 26 (17.9 g, 68%). White crystals. M.p. 169 – 1718 (cf. 168 – 1698 [30]). Spectroscopic data were in
agreement with those in [30].
i
tert-Butyl N4-[(Benzyloxy)carbonyl]-6-(hydroxymethyl)cytosine-1-acetate (27). A soln. of Pr2NH
(32 ml, 244 mmol) in THF (200 ml) was cooled to ꢀ 728, treated dropwise with 1.6m BuLi in hexane
(152 ml, 244 mmol), stirred for 30 min, warmed to 08, stirred for 20 min, cooled to ꢀ 728, and treated
dropwise with a soln. of 26 (17.5 g, 49 mmol) in THF (350 ml). After stirring for 2 h at ꢀ 708, the soln.
was treated dropwise with DMF (75 ml, 969 mmol) and stirred for another 2 h at ꢀ 708. The soln. was
allowed to reach r.t., treated with AcOH (33 ml), diluted with EtOH (300 ml), treated portionwise with
NaBH4 (5.5 g, 145 mmol), stirred for 3.5 h, treated with sat. NH4Cl soln. (500 ml), and extracted with
AcOEt (3 ꢃ 400 ml). The combined org. layers were washed with brine (3 ꢃ 250 ml), dried (MgSO4),
filtered, and evaporated. FC (CHCl3/MeOH 98 :2 ! 95 :5) gave 26 (4.75 g, 27%) and 27 (9.7 g, 51%).
Data of 27. White powder. Rf (CH2Cl2/MeOH 95 :5) 0.33. UV (MeOH): 242 (14200), 295 (7100). IR
(ATR): 3253w (br.), 2978w, 1738m, 1645m, 1610m, 1567m, 1497m, 1455m, 1414m, 1384s, 1368s, 1193s,
1150s, 1105m, 1061m, 1035m, 971w, 912w, 860w, 836w, 818w. 1H-NMR (300 MHz, (D6)DMSO): 10.76 (s,
NHꢀC(4)); 7.41 – 7.33 (m, 5 arom. H); 7.23 (s, HꢀC(5)); 5.89 (t, J ¼ 4.8, OH); 5.19 (s, PhCH2); 4.53 (s,
t
CH2ꢀN(1)); 4.35 (d, J ¼ 5.1, CH2ꢀC(6)); 1.41 (s, Bu). 13C-NMR (75 MHz, (D6)DMSO; assignments
t
based on a HSQC and a HMBC spectrum): 166.72 (s, CO2 Bu); 162.43 (s, C(4)); 161.84 (s, C(6)); 155.59
(s, C(2)); 152.89 (s, NHCO2); 135.82 (s); 128.31 (2d); 127.98 (d); 127.80 (2d); 91.67 (d, C(5)); 81.72 (s,
Me3C); 66.42 (t, PhCH2); 59.18 (t, CH2OH); 45.78 (t, CH2ꢀN(1)); 27.65 (q, Me3C). HR-MALDI-MS:
428.1224 (10, [M þ K]þ, C19H23KN3O6þ ; calc. 428.1218), 412.1478 (18, [M þ Na]þ, C19H23N3NaOþ6 ; calc.
t
412.1479), 390.1660 (100, [M þ H]þ, C19H24N3O6þ ; calc. 390.1660), 334.1035 (21, [M ꢀ Bu þ 2 H]þ,
C15H16N3Oþ6 ; calc. 334.1034).
N4-[(Benzyloxy)carbonyl]-6-[(1-(2-ethoxy-2-oxoethyl)-2-{[(9H-fluoren-9-yl)methoxy]carbonyl}hy-
drazino)methyl]cytosine-1-acetic Acid (29). A soln. of 14 (4.5 g, 6.3 mmol) and Et3SiH (10 ml, 63 mmol)
in CH2Cl2 (60 ml) was cooled to 08, treated dropwise with TFA (18.7 ml, 252 mmol), allowed to reach r.t.,
stirred for 17 h, and evaporated at r.t. The residue was suspended in Et2O (50 ml). Filtration (washing
with 4 ꢃ 50 ml of Et2O) gave 29 (3.4 g, 82%). White powder. Rf (CH2Cl2/MeOH 9 :1) 0.11. UV (MeOH):
255 (21800), 300 (13300). IR (ATR): 3448 – 2170w (br.), 3217w, 2929w, 1726m, 1677m, 1614m, 1568m,
1497m, 1450m, 1415m, 1388m, 1191s, 1082m, 1053m, 1027m, 969w, 930w, 910w, 826w. 1H-NMR
(300 MHz, (D6)DMSO): 10.68 (br. s, NHꢀC(4)); 8.94 (s, NHꢀN); 7.86 – 7.24 (m, 13 arom. H); 7.04 (s,
HꢀC(5)); 5.07 (s, CH2ꢀN(1)); 4.96 (s, PhCH2); 4.20 (br. s, CH2ꢀC(9)); 4.14 – 4.06 (m, MeCH2O,
HꢀC(9’)); 3.94 (s, CH2ꢀC(6)); 3.81 (s, NCH2CO2Et); 1.19 (t, J ¼ 6.8, MeCH2O). 13C-NMR (100 MHz,
(D6)DMSO; assignments based on a HSQC and a HMBC spectrum): 169.95 (s, CO2H); 169.01 (s,
CO2Et); 162.22 (s, C(4)); 155.90 (s, C(2)); 155.18 (s, NꢀNCO2); 155.09 (s, C(6)); 152.84 (s, C(4)NCO2);

Helvetica Chimica Acta – Vol. 92 (2009)
1153
143.51 (2s); 140.57 (2s); 135.71 (s); 128.32 (2d); 128.02 (2d); 127.81 (2d); 127.51 (2d); 126.92 (d); 124.88
(2d); 119.93 (2d); 96.86 (d, C(5)); 66.29 (t, PhCH2); 65.63 (t, CH2ꢀC(9)); 60.34 (t, MeCH2O); 57.67 (t,
NCH2CO2Et); 56.59 (t, CH2ꢀC(6)); 46.40 (d, C(9’)); 46.12 (t, CH2ꢀN(1)); 13.89 (q, MeCH2O). HR-
MALDI-MS: 678.2185 (22, [M þ Na]þ, C34H33N5NaO9þ ; calc. 678.2170), 656.2346 (100, [M þ H]þ,
C34H34N5Oþ9 ; calc. 656.2351).
N6-[(Benzyloxy)carbonyl]adenine (31). NaH (60% dispersion in oil, 94.7 g, 2.37 mol) was washed
with dry Et2O (3ꢃ), dried and cooled to 08. After the addition of DMF (1.5 l), adenine (80 g, 592 mmol)
was added as a solid in several small portions. The suspension was stirred at 08 for 45 min, treated
dropwise with ClCOOBn (88.7 ml, 622 mmol) over 1 h, warmed to r.t., and stirred for 16 h. The yellow
soln. was poured on ice water (2 l), neutralized with conc. HCl (145 ml), and filtered. The white solid was
washed with H2O and Et2O and dried (758, oil pump vacuum) affording 31 (121 g, 76%). White powder.
Spectroscopic data were in agreement with those in [30].
tert-Butyl N6-[(Benzyloxy)carbonyl]adenine-9-acetate (32) [30]. A suspension of 31 (62.3 g,
231 mmol), K2CO3 (40.0 g, 231 mmol) and Cs2CO3 (7.54 g, 23.1 mmol) in DMF (500 ml) was treated
dropwise with ClCH2COOtBu (36.4 ml, 255 mmol) and stirred for 14 h at r.t. The yellow suspension was
poured on H2O and extracted with AcOEt (4ꢃ). The combined org. fractions were washed with brine
(4ꢃ), dried (MgSO4), and filtered. Evaporation of the filtrate and crystallisation from AcOEt/
cyclohexane gave 32 (59.0 g, 78%). Colourless crystals. Spectroscopic data were in agreement with those
in [30].
i
tert-Butyl N4-[(Benzyloxy)carbonyl]-8-(hydroxymethyl)adenine-9-acetate (33). A soln. of Pr2NH
(119 ml, 908 mmol) in THF (750 ml) was cooled to ꢀ 768, treated dropwise with 1.6m BuLi in hexane
(567 ml, 908 mmol), warmed to 08, stirred for 1 h and stored at 48 for 13 h. The soln. was cooled to ꢀ 768
and treated dropwise with a soln. of 32 (58.0 g, 151 mmol) in THF (500 ml) over 1 h. After stirring for 2 h
at ꢀ 768, the soln. was treated dropwise with DMF (567 ml, 3.03 mol) over 30 min and stirred for another
2 h at ꢀ 768. The soln. was treated with AcOH (113 ml, 1.97 mol), allowed to reach r.t., diluted with
EtOH (1.5 l), treated with NaBH4 (22.9 g, 605 mmol) in several small portions, and stirred for 1.5 h. The
suspension was filtered, and the filtrate was evaporated. A suspension of the residue in H2O was
extracted with AcOEt (4ꢃ). The combined org. fractions were washed with brine (5ꢃ), dried (MgSO4),
filtered, and evaporated. FC (AcOEt/cyclohexane 3 :1) gave 33 (49 g, 78%). Yellow foam. Rf (CH2Cl2/
MeOH 19 :1) 0.20. UV (MeOH): 269 (21000), 212 (34080). IR (ATR): 3195w, 2979w, 2937w, 1740s,
1614m, 1590m, 1538w, 1498w, 1453m, 1391w, 1367m, 1322w, 1303w, 1281w, 1208s, 1159s, 1104m, 1039m,
971w, 859w, 844w, 799w, 764w, 743m, 696m, 667m. 1H-NMR (300 MHz, CDCl3; assignments based on a
HSQC and a HMBC spectrum): 9.66 (s, NH); 8.60 (s, HꢀC(2)); 7.29 – 7.24 (m, 5 arom. H); 5.24 (br. s,
t
OH); 5.13 (s, PhCH2); 4.91 (s, CH2ꢀN(9)); 4.74 (s, CH2ꢀC(8)); 1.40 (s, Bu). 13C-NMR (75 MHz,
t
CDCl3; assignments based on a HSQC and a HMBC spectrum): 166.02 (s, CO2 Bu); 153.90 (s, C(8));
152.47 (s, C(4)); 152.30 (d, C(2)); 151.32 (s, NHCO2Bn); 148.59 (s, C(6)); 135.25 (s); 128.38 (2d), 128.27
(3d); 120.11 (s, C(5)); 83.57 (s, Me3C); 67.56 (t, PhCH2); 57.74 (t, CH2ꢀC(8)); 44.31 (t, CH2ꢀN(9)); 27.98
(q, Me3C). HR-MALDI-MS: 452.1329 (4, [M þ K]þ, C20H23KN5O5þ ; calc. 452.1336), 436.1589 (16, [M þ
Na]þ, C20H23N5NaOþ5 ; calc. 436.1597), 414.1766 (100, [M þ H]þ, C20H24N5O5þ ; calc. 414.1777), 396.0702
t
t
(2, [M ꢀ Bu þ H þ K]þ, C16H15KN5Oþ5 ; calc. 396.0710), 380.0963 (5, [M ꢀ Bu þ H þ Na]þ,
C16H15N5NaOþ5 ; calc. 380.0971), 358.1143 (79, [M ꢀ Bu þ 2 H]þ, C16H16N5Oþ5 ; calc. 358.1151). Anal.
t
calc. for C20H23N5O5 (413.43): C 58.10, H 5.61, N 16.94; found: C 58.09, H 5.52, N 16.73.
tert-Butyl N6-[(Benzyloxy)carbonyl]-8-(bromomethyl)adenine-9-acetate (34). A soln. of 33 (13.5 g,
32.7 mmol) in CH2Cl2 (100 ml) was cooled to 08, treated dropwise with a soln. of Ms2O (8.54 g, 49 mmol)
in CH2Cl2 (40 ml), stirred for 1.5 h, treated with a soln. of LiBr (14.2 g, 163 mmol) in DMF (70 ml), and
stirred for another h. The suspension was allowed to reach r.t., stirred for 2 h, and diluted with sat. NH4Cl
soln. The aq. layer was extracted with AcOEt (3ꢃ). The combined org. fractions were washed with brine
(3ꢃ), dried (MgSO4), and evaporated. FC (AcOEt/cyclohexane 2 :1) gave 34 (13.3 g, 85%). White
powder. Rf (CH2Cl2/MeOH 19 :1) 0.33. M.p. 135.6 – 136.58. UV (MeOH): 278 (23540), 212 (28940). IR
(ATR): 3180w, 3117w, 2978w, 2935w, 1772m, 1732s, 1599m, 1581m, 1542m, 1463w, 1455w, 1391w, 1367m,
1335w, 1299w, 1258m, 1240m, 1198s, 1152s, 1102m, 1046m, 1028m, 973w, 940w, 905w, 892w, 860w, 840w,
799m, 743m, 696m, 661w, 613w. 1H-NMR (300 MHz, CDCl3; assignments based on a HSQC and a
HMBC spectrum): 9.28 (br. s, NH); 8.70 (s, HꢀC(2)); 7.33 – 7.31 (m, 5 arom. H); 5.23 (s, PhCH2); 4.97 (s,

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Helvetica Chimica Acta – Vol. 92 (2009)
t
CH2ꢀN(9)); 4.56 (s, CH2ꢀC(8)); 1.43 (s, Bu). 13C-NMR (75 MHz, CDCl3; assignments based on a
t
HSQC and a HMBC spectrum): 165.32 (s, CO2 Bu); 153.05 (d, C(2)); 152.78 (s, C(4)); 151.00 (s,
NHCO2Bn); 149.34 (s, C(8)); 149.20 (s, C(6)); 135.20 (s); 128.42 (3d); 128.30 (2d); 120.98 (s, C(5)); 83.89
(s, Me3C); 67.75 (t, PhCH2); 44.63 (t, CH2ꢀN(9)); 28.01 (q, Me3C); 21.68 (t, CH2ꢀC(8)). HR-MALDI-
MS: 498.0769 (6, [M þ Na]þ, C20H2279BrN5NaO4þ ; calc. 498.0753), 500.0753 (6, [M þ Na]þ,
C20H2281BrN5NaOþ4 ; calc. 500.0732), 476.0931 (86, [M þ H]þ, C20H2379BrN5Oþ4 ; calc. 476.0933),
478.0915 (100, [M þ H]þ, C20H2381BrN5O4þ ; calc. 478.0913), 420.0302 (35, [M ꢀ Bu þ 2 H]þ,
t
C16H1579BrN5Oþ4 ; calc. 420.0307), 422.0287 (33, [M ꢀ Bu þ 2 H]þ, C16H1581BrN5Oþ4 ; calc. 422.0287),
t
t
398.1823 (23, [M ꢀ Br þ H]þ, C20H24N5O4þ ; calc. 398.1828), 342.1210 (6, [M ꢀ Br ꢀ Bu þ 2 H]þ,
C16H16N5Oþ4 ; calc. 342.1202). Anal. calc. for C20H22BrN5O4 (476.32): C 50.43, H 4.66, N 14.70; found:
C 50.52, H 4.75, N 14.53.
tert-Butyl N6-[(Benzyloxy)carbonyl]-8-{[1-(2-ethoxy-2-oxoethyl)hydrazino]methyl}adenine-9-ace-
tate (35). A soln. of 15 (50.0 mg, 68.0 mmol) in DMF (450 ml) was cooled to 08, treated with piperidine
(67.2 ml, 680 mmol), and stirred for 1 h at 08. Evaporation and FC (CH2Cl2/MeOH 98 :2) gave 35 (30.4 mg,
87%). Yellow foam. Rf (CH2Cl2/MeOH 9 :1) 0.47. UV (MeOH): 270 (23760), 211 (37080). IR (ATR):
3317w, 3182w, 2979w, 1737m, 1614m, 1540w, 1500w, 1453m, 1368m, 1302m, 1200s, 1153s, 1101m, 1031m,
1
971m, 850m, 742w, 698w. H-NMR (400 MHz, (D6)DMSO; assignments based on a HMBC spectrum):
10.61 (br. s, NHꢀC(6)); 8.59 (s, HꢀC(2)); 7.50 – 7.30 (m, 5 arom. H); 5.22 (s, PhCH2); 5.17 (s,
CH2ꢀN(9)); 4.20 (s, CH2ꢀC(8)); 4.13 (q, J ¼ 7.1, MeCH2O); 3.73 (br. s, NH2); 3.57 (s, NCH2CO2Et);
1.42 (s, tBu); 1.22 (t, J ¼ 7.1, MeCH2O). 13C-NMR (100 MHz, (D6)DMSO): 169.98 (s, CO2Et); 166.74 (s,
t
CO2 Bu); 153.53 (s, C(4)); 152.09 (s, NHCO2Bn); 151.91 (s, C(8)); 151.36 (d, C(2)); 148.63 (s, C(6));
136.30 (s); 128.31 (2d); 127.87 (d); 127.73 (2d); 122.23 (s, C(5)); 82.00 (s, Me3C); 66.16 (t, PhCH2); 59.96,
59.90 (2t, MeCH2O, NCH2CO2Et); 56.50 (t, CH2ꢀC(8)); 44.47 (t, CH2ꢀN(9)); 27.59 (q, Me3C); 14.04 (q,
MeCH2O). HR-MALDI-MS: 552.1988 (3, [M þ K]þ, C24H31KN7O6þ ; calc. 552.1973), 536.2244 (23, [M þ
Na]þ, C24H31N7NaOþ6 ; calc. 536.2234), 514.2418 (100, [M þ H]þ, C24H32N7O6þ ; calc. 514.2414), 480.1614
t
t
(6, [M ꢀ Bu þ H þ Na]þ, C20H23N7NaO6þ ; calc. 480.1608), 458.1779 (52, [M ꢀ Bu þ 2 H]þ, C20H24N7Oþ6 ;
calc. 458.1788).
N6-[(Benzyloxy)carbonyl]-8-[(1-(2-ethoxy-2-oxoethyl)-2-{[(9H-fluoren-9-yl)methoxy]carbonyl}hy-
drazino)methyl]adenine-9-acetic Acid (36). A soln. of 15 (10.0 g, 13.6 mmol) in CH2Cl2 (140 ml) was
treated with Et3SiH (21.7 ml, 136 mmol) and TFA (41.9 ml, 544 mmol) and stirred for 20 h at r.t. After
evaporation at 308, the residue was treated with iPr2O (250 ml). The precipitate was filtered off, ground,
i
and washed several times with Pr2O. Drying of the solid gave 36 (9.05 g, 98%). White powder. Rf
(CH2Cl2/MeOH 3 :2) 0.54. M.p. 117.1 – 118.38. UV (MeOH): 230 (4740), 266 (31100), 211 (50020). IR
(ATR): 3123w, 3065w, 3036w, 2975w, 2902w, 1733s, 1716s, 1647m, 1619m, 1585m, 1551w, 1479w, 1398w,
1377m, 1288m, 1255m, 1200s, 1162s, 1082m, 1043m, 1030m, 981w, 962w, 797w, 785w, 758m, 742s, 719m,
698m. 1H-NMR (300 MHz, (D6)DMSO; assignments based on a HSQC and a HMBC spectrum): 10.80
(br. s, NHꢀC(6)); 8.85 (br. s, NNH); 8.61 (s, HꢀC(2)); 7.85 – 7.18 (m, 13 arom. H); 5.48 (br. s,
CH2ꢀN(9)); 5.21 (s, PhCH2); 4.35 (br. s, CH2ꢀC(8)); 4.21 (d, J ¼ 6.9, CH2ꢀC(9’)); 4.13 – 4.06 (m,
MeCH2O, HꢀC(9’)); 3.77 (br. s, NCH2CO2Et); 1.18 (t, J ¼ 7.2, MeCH2O). 13C-NMR (75 MHz,
(D6)DMSO; assignments based on a HSQC and a HMBC spectrum): 169.09 (s, CO2H); 168.81 (s,
CO2Et); 155.03 (br. s, NNHCO2); 154.81 (s, C(4)); 153.16 (s, NHCO2Bn); 151.87 (s, C(8)); 150.69 (d,
C(2)); 148.25 (s, C(6)); 143.30 (2s); 140.43 (2s); 136.01 (s); 128.16 – 124.81 (11d); 121.43 (s, C(5)); 119.84
(2d); 66.31 (t, PhCH2); 65.42 (t, CH2ꢀC(9’)); 60.32 (t, MeCH2O); 57.46, 53.50 (2t, CH2ꢀC(8),
NCH2CO2Et); 46.47 (d, C(9’)); 44.01 (t, CH2ꢀN(9)); 14.00 (q, MeCH2O). HR-MALDI-MS: 702.2240
(37, [M þ Na]þ, C35H33N7NaO8þ ; calc. 702.2288), 680.2452 (100, [M þ H]þ, C35H34N7Oþ8 ; calc. 680.2469),
572.1867 (19, [M ꢀ BnOH þ H]þ, C28H26N7O7þ ; calc. 572.1894).
2-{[2-Amino-6-(benzyloxy)-5-nitrosopyrimidin-4-yl]amino}-2-oxoethyl Acetate (38). A suspension
of 37 (21.2 g, 86.6 mmol) in THF (430 ml) was cooled to 08, treated with AcOCH2COCl (11.2 ml,
104 mmol) over 30 min, stirred for 3 h, and evaporated. The residue was treated with H2O. The solid was
filtered off, washed with H2O and Et2O, and dried to give 38 (25.1 g, 84%). Blue powder. Rf (CH2Cl2/
MeOH 19 :1) 0.35. M.p. 177.4 – 178.58. UV (CHCl3): 349 (26420), 255 (19120). IR (ATR): 3437m, 3291w,
3198w, 3143w, 2960w, 1746m, 1718m, 1640m, 1584s, 1546s, 1520m, 1500m, 1469s, 1448s, 1387s, 1336s,
1293s, 1276m, 1243m, 1207s, 1155s, 1083m, 1065s, 1048s, 942m, 912m, 857m, 846m, 827m, 792w, 763m,

Helvetica Chimica Acta – Vol. 92 (2009)
1155
748w, 728s, 695s. 1H-NMR (300 MHz, (D6)DMSO; assignments based on a HSQC and a HMBC
spectrum): 12.33 (s, NH); 8.82 – 8.80 (m, NH2); 7.60 – 7.37 (m, 5 arom. H); 5.65 (s, PhCH2); 5.09 (s,
AcOCH2); 2.18 (s, AcO). 13C-NMR (125 MHz, (D6)DMSO; assignments based on a HSQC and a
HMBC spectrum): 169.88, 169.82 (2s, MeC¼O, NHC¼O); 163.30 (s, C(6)); 138.70 (s, C(4)); 135.60 (s);
128.51 (2d); 128.49 (2d); 128.33 (d); 68.64 (t, PhCH2); 65.08 (t, AcOCH2); 20.34 (q, MeC¼O). HR-
MALDI-MS: 384.0697 (12, [M þ K]þ, C15H15KN5O5þ ; calc. 384.0710), 368.0959 (100, [M þ Na]þ,
C15H15N5NaOþ5 ; calc. 368.0971). Anal. calc. for C15H15N5O5 (345.31): C 52.17, H 4.38, N 20.28; found: C
52.08, H 4.35, N 20.09.
8-(Acetoxymethyl)-6-O-benzylguanine (39). A suspension of 38 (30.2 g, 87.5 mmol) and Ph3P
(55.0 g, 210 mmol) in o-xylene (580 ml) was heated to reflux for 4 h, while the colour changed from blue
to yellow. After cooling the soln. to 48, the resulting suspension was kept at 48 for 36 h and filtered. The
solid was ground and washed with toluene. Crystallisation from MeOH gave 39 (27.1 g, 99%). Colourless
needles. Rf (CH2Cl2/MeOH 19 :1) 0.15. M.p. 193.1 – 194.28. UV (MeOH): 286 (10280), 246 (7700), 209
(31780). IR (ATR): 3485w, 3387m, 3277w, 3089w, 3009w, 3000w, 2932w, 2809w, 2690w, 1739s, 1640m,
1624m, 1592s, 1537w, 1486m, 1450m, 1417s, 1373m, 1352s, 1336m, 1264s, 1224s, 1203m, 1167m, 1100m,
1036s, 1022m, 995s, 973m, 836m, 789m, 762w, 731s, 693m, 670w. 1H-NMR (300 MHz, (D6)DMSO): 12.60
(s, NH); 7.51 – 7.31 (m, 5 arom. H); 6.37 (s, NH2); 5.47 (s, PhCH2); 5.08 (s, CH2ꢀC(8)); 2.08 (s, AcO).
13C-NMR (100 MHz, (D6)DMSO): 169.67 (s, MeC¼O); 159.52 (s, C(6), C(2)); 155.77 (s, C(4)); 144.49 (s,
C(8)); 136.48 (s); 128.21 (2d); 127.83 (3d); 113.33 (s, C((5)); 66.68 (t, PhCH2); 59.22 (t, CH2ꢀC(8)); 20.59
(q, MeC¼O). HR-MALDI-MS: 352.0809 (12, [M þ K]þ, C15H15KN5O3þ ; calc. 352.0812), 336.1062 (32,
[M þ Na]þ, C15H15N5NaO3þ ; calc. 336.1073), 314.1244 (100, [M þ H]þ, C15H16N5Oþ3 ; calc. 314.1253),
254.1029 (38, [M ꢀ AcO]þ, C13H12N5Oþ; calc. 254.1036). Anal. calc. for C15H15N5O3 (313.31): C 57.50, H
4.83, N 22.35; found: C 57.23, H 4.88, N 22.20.
Alkylation of 39. A suspension of 39 (27.2 g, 86.9 mmol) and K2CO3 (36.0 g, 261 mmol) in DMF
(175 ml) was treated dropwise with ClCH2COOtBu (18.6 ml, 130 mmol) over 5 min. The suspension was
stirred for 8 h, diluted with AcOEt (300 ml), and filtered. The filtrate was diluted with sat. NH4Cl soln.
and H2O, and extracted with AcOEt (4ꢃ). The combined org. fractions were washed with brine (5ꢃ),
dried (MgSO4), and filtered. Evaporation and FC (CH2Cl2/MeOH 98 :2 ! 9 :1) gave 40 (25.8 g, 69%)
along with 41.
tert-Butyl 8-(Acetoxymethyl)-6-O-benzylguanine-9-acetate (40). White powder. Rf (CH2Cl2/MeOH
19 :1) 0.35. M.p. 148.0 – 148.58. UV (MeOH): 261 (29760), 209 (48400). IR (ATR): 3486m, 3292w, 3178w,
3119w, 3011w, 2981w, 2937w, 1740s, 1627m, 1615s, 1583s, 1530m, 1494m, 1467m, 1454m, 1438m, 1413m,
1384m, 1368m, 1350s, 1317m, 1260s, 1235s, 1222s, 1212s, 1162s, 1154s, 1112m, 1069m, 1021s, 1005w, 967m,
947m, 916m, 861m, 854m, 842w, 790m, 771w, 752s, 708m, 700m, 676w. 1H-NMR (400 MHz, CDCl3;
assignments based on a HSQC and a HMBC spectrum): 7.81 – 7.27 (m, 5 arom. H); 5.55 (s, PhCH2); 5.22
t
(s, CH2ꢀC(8)); 4.89 (s, NH2); 4.80 (CH2ꢀN(9)); 2.09 (s, AcO); 1.46 (s, Bu). 13C-NMR (100 MHz,
t
CDCl3; assignments based on a HSQC and a HMBC spectrum): 170.24 (s, MeC¼O); 166.38 (s, CO2 Bu);
161.04 (s, C(6)); 159.47 (s, C(2)); 155.34 (s, C(4)); 145.12 (s, C(8)); 136.35 (s); 128.46 (2d); 128.38 (2d);
128.03 (d); 114.60 (s, C(5)); 83.23 (s, Me3C); 68.19 (t, PhCH2); 58.66 (t, CH2ꢀC(8)); 44.29 (t,
CH2ꢀN(9)); 28.00 (q, Me3C); 20.65 (q, MeC¼O). HR-MALDI-MS: 466.1492 (2, [M þ K]þ,
C21H25KN5Oþ5 ; calc. 466.1493), 450.1754 (12, [M þ Na]þ, C21H25N5NaOþ5 ; calc. 450.1753), 428.1931
t
(100, [M þ H]þ, C21H26N5O5þ ; calc. 428.1934), 394.1119 (5, [M ꢀ Bu þ H þ Na]þ, C17H17N5NaOþ5 ; calc.
t
t
394.1127), 372.1293 (93, [M ꢀ Bu þ 2 H]þ, C17H18N5Oþ5 ; calc. 372.1308), 312.1083 (52, [M ꢀ Bu ꢀ
AcO þ H]þ, C15H14N5Oþ3 ; calc. 312.1091). Anal. calc. for C21H25N5O5 (427.45): C, 59.01, H 5.90, N
16.38; found: C 58.81, H 5.93, N 16.09.
tert-Butyl 8-(Acetoxymethyl)-6-O-benzylguanine-7-acetate (41). Yellow powder. Rf (CH2Cl2/MeOH
19 :1) 0.05. M.p. 149.4 – 152.68. UV (MeOH): 301 (8640), 213 (38220). IR (ATR): 3482w, 3296w, 3172w,
2979w, 1740s, 1632s, 1572s, 1511w, 1482m, 1454m, 1432m, 1390m, 1369m, 1326s, 1305w, 1226s, 1175s,
1150s, 1084w, 1065m, 1032m, 970w, 955w, 919w, 895w, 849m, 790m, 743m, 698m, 680w, 655w, 629w.
1H-NMR (400 MHz, (D6)DMSO; assignments based on a HSQC and a HMBC spectrum): 7.52 – 7.32 (m,
5 arom. H); 6.27 (s, NH2); 5.43 (s, PhCH2); 5.23 (s, CH2ꢀC(8)); 5.00 (CH2ꢀN(9)); 2.01 (s, AcO); 1.24 (s,
tBu). 13C-NMR (100 MHz, (D6)DMSO; assignments based on a HSQC and a HMBC spectrum): 169.53
t
(s, MeC¼O); 166.70 (s, CO2 Bu); 162.08 (s, C(2)); 159.74 (s, C(4)); 156.37 (s, C(6)); 150.56 (s, C(8));

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Helvetica Chimica Acta – Vol. 92 (2009)
136.20 (s); 128.34 (2d); 127.96 (d); 127.83 (2d); 107.40 (s, C(5)); 81.80 (s, Me3C); 67.17 (t, PhCH2); 57.31 (t,
CH2ꢀC(8)); 47.38 (t, CH2ꢀN(7)); 27.35 (q, Me3C); 20.34 (q, MeC¼O). HR-MALDI-MS: 466.1494 (12,
[M þ K]þ, C21H25KN5O5þ ; calc. 466.1493), 450.1752 (11, [M þ Na]þ, C21H25N5NaOþ5 ; calc. 450.1753),
428.1933 (100, [M þ H]þ, C21H26N5O5þ ; calc. 428.1934), 394.1127 (2, [M ꢀ Bu þ H þ Na]þ,
t
t
C17H17N5NaOþ5 ; calc. 394.1127), 372.1305 (31, [M ꢀ Bu þ 2 H]þ, C17H18N5Oþ5 ; calc. 372.1308).
tert-Butyl 6-O-Benzyl-8-(hydroxymethyl)guanine-9-acetate (42). A suspension of 40 (14.1 g,
33 mmol) and K2CO3 (4.79 g, 34.7 mmol) in MeOH/H2O 3 :1 (330 ml) was stirred for 5 h at r.t. After
evaporation of MeOH and dilution with sat. NH4Cl soln., the solid was filtered off, washed with H2O, and
dried to give 42 (12.0 g, 94%). White powder. Rf (CH2Cl2/MeOH 19 :1) 0.26. M.p. 216.28 (dec.). UV
(MeOH): 285 (13380), 252 (10780), 211 (32360). IR (ATR): 3482w, 3287w, 3160w, 3110w, 3060w, 2981w,
2931w, 1746s, 1624s, 1587s, 1490m, 1471m, 1445s, 1406m, 1390m, 1363m, 1346m, 1315m, 1253s, 1223s,
1152s, 1115m, 1070s, 1030m, 982m, 937m, 915w, 874m, 841m, 785m, 757m, 749m, 739w, 705s, 666w.
1H-NMR (300 MHz, (D6)DMSO): 7.51 – 7.34 (m, 5 arom. H); 6.52 (s, NH2); 5.60 (t, J ¼ 5.3, OH); 5.50 (s,
PhCH2); 4.82 (s, CH2ꢀN(9)); 4.54 (d, J ¼ 4.7, CH2ꢀC(8)); 1.41 (s, tBu). 13C-NMR (75 MHz,
t
(D6)DMSO): 166.61 (s, CO2 Bu); 159.52, 159.45 (2s, C(2), C(6)); 155.67 (s, C(4)); 149.35 (s, C(8));
136.52 (s); 128.21 (2d); 128.10 (2d); 127.81 (d); 111.85 (s, C((5)); 81.78 (s, Me3C); 66.77 (t, PhCH2); 56.92
(t, CH2ꢀC(8)); 43.83 (t, CH2ꢀN(9)); 27.64 (q, Me3C). HR-MALDI-MS: 424.1386 (3, [M þ K]þ,
C19H23KN5Oþ4 ; calc. 424.1387), 408.1654 (5, [M þ Na]þ, C19H23N5NaO4þ ; calc. 408.1648), 386.1827 (100,
[M þ H]þ, C19H24N5O4þ ; calc. 386.1828), 352.1022 (1, [M ꢀ Bu þ H þ Na]þ, C15H15N5NaOþ4 ; calc.
t
t
352.1022), 330.1192 (54, [M ꢀ Bu þ 2 H]þ, C15H16N5O4þ ; calc. 330.1202). Anal. calc. for C19H23N5O4
(385.42): C, 59.21, H 6.01, N 18.17; found: C 59.28, H 6.07, N 18.18.
tert-Butyl 6-O-Benzyl-8-(chloromethyl)guanine-9-acetate (43). A soln. of 42 (16 g, 41.5 mmol) in
DMF (400 ml) was cooled to 08, treated with EtNiPr2 (10.9 ml, 62.3 mmol), followed by MsCl (4.82 ml,
62.3 mmol) within 10 min, stirred for 1 h at 08, treated with LiCl (8.80 g, 208 mmol), stirred for 30 min,
and poured on H2O. The suspension was extracted with CHCl3/MeOH 95 :5 (1ꢃ), and the aq. suspension
was filtered. The filtrate was extracted with CHCl3/MeOH 95 :5 (4ꢃ). The combined org. fractions were
washed with brine (3ꢃ), dried (MgSO4), and evaporated. The residue was combined with the solid
obtained by the filtration of the aq. suspension, washed with H2O, and dried. FC (CH2Cl2/MeOH
99.75 :0.25 ! 99.5 :0.5) gave 43 (7.90 g, 47%). White powder. Rf (CH2Cl2/MeOH 19 :1) 0.41. M.p. 2108
(dec.). UV (MeOH): 291 (15600), 255 (10020), 211 (32640). IR (ATR): 3479w, 3279w, 3161w, 3109w,
3058w, 3033w, 2975w, 2938w, 1753s, 1621s, 1586s, 1518m, 1493m, 1471m, 1452m, 1439m, 1407w, 1379m,
1367m, 1357m, 1344m, 1318m, 1258s, 1225s, 1151s, 1108m, 1066m, 1030w, 987w, 965w, 937m, 913w, 866m,
858m, 839m, 788m, 771m, 758m, 747m, 732m, 718m, 699s, 684m, 629m. 1H-NMR (300 MHz, CHCl3/
CD3OD): 7.45 – 7.25 (m, 5 arom. H); 5.48 (s, PhCH2); 5.17 (s, NH2); 4.80 (s, CH2ꢀN(9)); 4.67 (s,
t
t
CH2ꢀC(8)); 1.43 (s, Bu). 13C-NMR (75 MHz, CDCl3/CD3OD): 166.04 (s, CO2 Bu); 160.87 (s, C(6));
159.54 (s, C(2)); 155.06 (s, C(4)); 145.27 (s, C(8)); 135.94 (s); 128.30 (2d); 128.19 (2d); 128.00 (d); 113.85
(s, C(5)); 83.58 (s, Me3C); 68.31 (t, PhCH2); 44.35 (t, CH2ꢀN(9)); 37.25 (t, CH2ꢀC(8)); 27.97 (q, Me3C).
HR-MALDI-MS: 426.1332 (3, [M þ Na]þ, C19H22ClN5NaOþ3 ; calc. 426.1309), 404.1491 (100, [M þ H]þ,
C19H23ClN5Oþ3 ; calc. 404.1489), 370.1884 (21, [M ꢀ Bu þ H þ Na]þ, C15H14ClN5NaOþ3 ; calc. 370.0683),
t
348.0860 (76, [M ꢀ Bu þ 2 H]þ, C15H15ClN5O3þ ; calc. 348.0863). Anal. calc. for C19H22ClN5O3 (403.86): C,
t
56.51, H 5.49, N 17.34; found: C 56.75, H 5.62, N 17.26.
8-[(1-(2-Ethoxy-2-oxoethyl)-2-{[(9H-fluoren-9-yl)methoxy]carbonyl}hydrazino)methyl]guanine-9-
acetic Acid (44). A soln. of 43 (7.50 g, 18.5 mmol), 23 (12.6 g, 37.0 mmol), and Bu4NBr (17.9 g,
55.4 mmol) in DMSO (92 ml) was treated with lutidine (6.46 ml, 55.4 mmol), stirred for 72 h at r.t., and
poured on H2O. After extraction with AcOEt (5ꢃ), the combined org. fractions were washed with brine
(5ꢃ), dried (MgSO4), filtered, and evaporated. FC (AcOEt) gave a crude product (tert-butyl acetate)
contaminated with 23, that was used for the next reaction. A soln. of the crude tert-butyl acetate in
CH2Cl2 (180 ml) was treated with Et3SiH (14.8 ml, 92.4 mmol) and TFA (137 ml, 1.85 mmol), and stirred
for 16 h at r.t. After evaporation at 308, the residue was treated with Et2O (200 ml). The precipitate was
filtered off, ground, and washed several times with Et2O. Drying of the solid gave 44 (9.45 g, 91% from
43). White solid. Rf (CH2Cl2/MeOH 3 :2) 0.54. M.p. 186.3 – 192.58. UV (MeOH): 299 (6120), 262
(35920), 210 (51200). IR (ATR): 3318w, 3215w, 3165w, 3066w, 2983w, 2942w, 1724m, 1688s, 1641s, 1582s,
1511w, 1494w, 1478w, 1450m, 1394w, 1373m, 1357m, 1288w, 1203s, 1159m, 1054w, 1025m, 976w, 961w,

Helvetica Chimica Acta – Vol. 92 (2009)
1157
894w, 854w, 782w, 759m, 739s, 695w, 659w, 620w. 1H-NMR (300 MHz, (D6)DMSO; assignments based on
a HSQC and a HMBC spectrum): 13.10 (br. s, CO2H); 10.60 (br. s, HꢀN(1)); 8.76 (s, NNH); 7.88 – 7.18
(m, 8 arom. H); 6.53 (br. s, NH2); 5.14 (br. s, CH2ꢀN(9)); 5.23 – 3.99 (m, CH2ꢀC(8), MeCH2O,
CH2ꢀC(9’), HꢀC(9’)); 3.64 (br. s, NCH2CO2Et); 1.17 (t, J ¼ 7.2, MeCH2O). 13C-NMR (75 MHz,
(D6)DMSO; assignments based on a HSQC and a HMBC spectrum): 169.47, 168.85 (2s, CO2H, CO2Et);
156.16 (s, C(6)); 154.82 (s, NNHCO2); 153.57, 152.46 (2s, C(2), C(4)); 143.42 (2s); 142.94 (s, C(8)); 140.47
(2s); 127.43 – 119.91 (8d); 114.59 (s, C(5)); 65.42 (t, CH2ꢀC(9’)); 60.23 (t, MeCH2O); 57.13 (t,
NCH2CO2Et); 53.60 (t, CH2ꢀC(8)); 46.54 (d, C(9’)); 43.52 (t, CH2ꢀN(9)); 14.03 (q, MeCH2O). HR-
MALDI-MS: 584.1869 (44, [M þ Na]þ, C27H27N7NaO7þ ; calc. 584.1870), 562.2050 (100, [M þ H]þ,
C27H28N7Oþ7 ; calc. 562.2050).
tert-Butyl 6-[2-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)ethyl]uracil-1-acetate (45). A soln. of
51 (2.2 g, 7.48 mmol) in THF (60 ml) was cooled to ꢀ 28, treated dropwise with EtNiPr2 (1.43 ml,
8.23 mmol), stirred for 30 min, treated dropwise with ClCO2Et (1.1 ml, 11.23 mmol), stirred for 1 h,
treated with a soln. of NaN3 (983 mg, 14.97 mmol) in H2O (3 ml), and stirred for another hour at ꢀ 28.
The soln. was treated with ice (50 ml) and extracted with CH2Cl2 (5 ꢃ 50 ml). After drying (MgSO4) of
the combined org. layers and evaporation at r.t., the residue was added to a soln. of 9H-fluorene-9-
methanol (7.35 g, 37.43 mmol) in dioxane (30 ml). Stirring for 1.5 h at 1008, evaporation, and FC (CHCl3/
MeOH 99 :1) gave 45 (3.31 g, 90%). White powder. Rf (CHCl3/MeOH 9 :1) 0.58. M.p. 90 – 938. UV
(CHCl3): 267 (30627), 301 (5577). IR (ATR): 2979w (br.), 1674s (br.), 1619m, 1521w, 1449m, 1414m,
1392m, 1368m, 1235s, 1200w, 1151s, 1085w, 996w, 944w. 1H-NMR (300 MHz, (D6)DMSO, 808): 11.05 (br.
s, HꢀN(3)); 7.84 – 7.30 (m, 8 arom. H); 7.23 (br. s, NHFmoc); 5.47 (s, HꢀC(5)); 4.46 (s, CH2ꢀN(1)); 4.34
(d, J ¼ 6.6, CH2ꢀC(9’’)); 4.19 (t, J ¼ 6.6, HꢀC(9’’)); 3.22 (q, J ¼ 6.9, 2 HꢀC(2’)); 2.53 (t, J ¼ 6.9,
t
2 HꢀC(1’)); 1.41 (s, Bu). 1H-NMR (400 MHz, CDCl3; assignment based on a HSQC and a HMBC
spectrum): 9.69 (br. s, HꢀN(3)); 7.76 – 7.29 (m, 8 arom. H); 5.62 (s, HꢀC(5)); 5.53 (t, J ¼ 5.9, NHFmoc);
4.54 (s, CH2ꢀN(1)); 4.40 (d, J ¼ 6.8, CH2ꢀC(9’’)); 4.19 (t, J ¼ 6.8, HꢀC(9’’)); 3.37 (q, J ¼ 6.3,
2 HꢀC(2’)); 2.60 (t, J ¼ 6.7, 2 HꢀC(1’)); 1.46 (s, Me3C). 13C-NMR (100 MHz, CDCl3; assignment based
t
on a HSQC and a HMBC spectrum): 167.03 (s, CO2 Bu); 162.87 (s, C(4)); 156.46 (s, NHCO2); 154.07 (s,
C(6)); 151.66 (s, C(2)); 143.76 (2s); 141.32 (2s); 127.75 – 120.00 (8d); 101.99 (d, C(5)); 83.44 (s, Me3C);
66.89 (t, CH2ꢀC(9’’)); 47.18 (d, C(9’’)); 45.48 (t, CH2ꢀN(1)); 38.54 (t, C(2’)); 32.62 (t, C(1’)); 27.97
(q, Me3C). HR-MALDI-MS: 530.1695 (51, [M þ K]þ, C27H29N3KO3þ ; calc. 530.1688), 514.1952 (100,
[M þ Na]þ, C27H29N3NaO6þ ; calc. 514.1949), 492.2134 (14, [M þ H]þ, C27H30N3Oþ6 ; calc. 492.2129).
Anal. calc. for C27H29N3O6 (491.54): C 65.97, H 5.95, N 8.55; found: C 65.56, H 5.95, N 8.42.
tert-Butyl N4-[(Benzyloxy)carbonyl]-6-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)ethyl]cy-
tosine-1-acetate (46). A suspension of 56 (1.80 g, 4.17 mmol) in THF/acetone 1:1 (83 ml) was cooled
to ꢀ 108, treated dropwise with EtNiPr2 (800 ml, 4.59 mmol) and ClCOOEt (599 ml, 6.26 mmol), and
stirred for 1 h. The resulting orange soln. was treated with a soln. of NaN3 (553 mg, 8.35 mmol) in H2O
(17 ml) and stirred for 1 h at ꢀ 108. The red soln. was treated with ice/brine and extracted with CH2Cl2
(5ꢃ). The combined org. fractions were dried (MgSO4) and evaporated at r.t. A soln. of the residue in
dioxane (17 ml) was treated with 9H-fluorene-9-methanol (8.19 g, 41.8 mmol) and heated to 808 for 1 h.
Evaporation and FC (CH2Cl2/MeOH 99 :1 ! 98 :2) gave 46 (2.37 g, 57%). Yellow foam. The aq. soln.
from the workup was acidified with 1m HCl and extracted with CHCl3 (5ꢃ). Drying (MgSO4), filtration,
and evaporation gave 56 (528 mg, 29%). Rf (CH2Cl2/MeOH 19 :1) 0.24. UV (MeOH): 299 (15300), 247
(24300), 208 (71100). IR (ATR): 3229w, 2972w, 1739m, 1707m, 1665m, 1607m, 1566m, 1497m, 1450m,
1412m, 1389m, 1368m, 1210s, 1194s, 1151s, 1082m, 1061m, 1003w, 946w, 916w, 858w, 820w, 788w, 758m,
739s, 696m, 620w. 1H-NMR (300 MHz, (D6)DMSO, 808): 10.38 (br. s, NHꢀC(4)); 7.87 – 7.29 (m, 13 arom.
H); 7.18 (br. s, NHFmoc); 6.92 (s, HꢀC(5)); 5.16 (s, PhCH2); 4.61 (s, CH2ꢀN(1)); 4.35 (d, J ¼ 6.5,
CH2ꢀC(9’’)); 4.22 (t, J ¼ 6.5, HꢀC(9’’)); 3.25 (q, J ¼ 6.5, 2 HꢀC(2’)); 2.73 (t, J ¼ 6.2, 2 HꢀC(1’)), 1.42 (s,
t
tBu). 13C-NMR (75 MHz, (D6)DMSO): 166.88 (s, CO2 Bu); 161.94 (s, C(4)); 159.00 (s, C(6)); 155.92 (s,
NHCO2Fm); 155.66 (s, C(2)); 152.82 (s, NHCO2Bn); 143.63 (2s); 140.53 (2s); 135.70 (s); 128.26 – 119.96
(13d); 94.41 (d, C(5)); 81.67 (s, Me3C); 66.36 (t, PhCH2); 65.38 (t, CH2ꢀC(9’’)); 46.68 (d and t, C(9’’) and
C(2’)); 38.49 (t, CH2ꢀN(1)); 32.88 (t, C(1’)); 27.62 (q, Me3C). HR-MALDI-MS: 663.2217 (12, [M þ K]þ,
C35H36KN4Oþ7 ; calc. 663.2221), 647.2487 (21, [M þ Na]þ, C35H36N4NaO7þ ; calc. 647.2482), 625.2661 (100,
t
[M þ H]þ, C35H37N4Oþ7 ; calc. 625.2662), 607.1611 (3, [M ꢀ Bu þ H þ K]þ, C31H28KN4Oþ7 ; calc. 607.1595),

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Helvetica Chimica Acta – Vol. 92 (2009)
t
t
591.1850 (9, [M ꢀ Bu þ H þ Na]þ, C31H28N4NaOþ7 ; calc. 591.1856), 569.2030 (39, [M ꢀ Bu þ 2 H]þ,
C31H29N4Oþ7 ; calc. 569.2036).
tert-Butyl N6-[(Benzyloxy)carbonyl]-8-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)ethyl]ade-
nine-9-acetate (47). A suspension of 61 (2.50 g, 5.49 mmol) in THF/acetone 1:1 (110 ml) was cooled to
ꢀ 108, treated dropwise with EtNiPr2 (1.05 ml, 6.04 mmol) and ClCOOEt (787 ml, 8.23 mmol), and
stirred for 1 h. The resulting yellow soln. was treated with a soln. of NaN3 (714 mg, 10.98 mmol) in H2O
(22 ml) and stirred for another h at ꢀ 108. The greenish soln. was treated with ice/brine and extracted
with CH2Cl2 (5ꢃ). The combined org. fractions were dried (MgSO4) and evaporated at r.t. A soln. of the
residue in dioxane (22 ml) was treated with 9H-fluorene-9-methanol (10.8 g, 45.9 mmol), heated to 808,
stirred for 1 h, and evaporated. FC (CH2Cl2/MeOH 99 :1 ! 98 :2) gave 47 (2.87 g, 52%). White powder.
The aq. layer from the workup was acidified with 1m HCl and extracted with CHCl3 (5ꢃ). The combined
org. fractions were dried (MgSO4), and filtered. Evaporation gave 61 (344 mg, 13%).
Data of 47. Rf (CH2Cl2/MeOH 95 :5) 0.29. M.p. 94.2 – 95.58. UV (CH3OH): 211 (55740), 266 (38540),
300 (6120). IR (ATR): 3338w (br.), 3064w, 3037w, 2980w, 1757m, 1739m, 1713s, 1616s, 1524m, 1491m,
1479m, 1452s, 1426w, 1391m, 1367m, 1322m, 1264m, 1236s, 1206s, 1151s, 1101m, 1070w, 1035m, 987w,
1
969m, 939w, 896w, 858w, 796w, 741s, 699m, 653w, 630w. H-NMR (400 MHz, (D6)DMSO; assignments
based on a HSQC and a HMBC spectrum): 10.57 (s, NHꢀC(6)); 8.57 (s, HꢀC(2)); 7.88 – 7.28 (m, 13
arom. H, NHFmoc); 5.22 (s, PhCH2); 5.02 (s, CH2ꢀN(9)); 4.33 (d, J ¼ 6.8, CH2ꢀC(9’’)); 4.21 (t, J ¼ 6.6,
t
HꢀC(9’’)); 3.47 (q, J ¼ 6.4, 2 HꢀC(2’)); 2.99 (t, J ¼ 7.1, 2 H ꢀC(1’)); 1.40 (s, Bu). 13C-NMR (400 MHz,
t
(D6)DMSO; assignments based on a HSQC and a HMBC spectrum): 166.56 (s, CO2 Bu); 156.05 (s,
NHCO2Fm); 153.67 (s, C(8)); 153.07 (s, C(4)); 152.07 (s, NHCO2Bn); 151.10 (d, C(2)); 148.21 (s, C(6));
143.82 (2s); 140.71 (2s); 136.34 (s); 128.35 – 125.05 (11d); 122.38 (s, C(5)); 120.08 (2d); 82.52 (s, Me3C);
66.22 (t, PhCH2); 65.35 (t, CH2ꢀC(9’’)); 46.69 (d, C(9’’)); 43.82 (t, CH2ꢀN(9)); 37.94 (t, C(2’)); 27.54 (q,
Me3C); 27.29 (t, C(1’)). HR-MALDI-MS: 687.2337 (9, [M þ K]þ, C36H36KN6O6þ ; calc. 687.2333), 671.2588
(58, [M þ Na]þ, C36H36N6NaO6þ ; calc. 671.2594), 649.2774 (100, [M þ H]þ, C36H37N6Oþ6 ; calc. 649.2775),
t
t
615.1965 (9, [M ꢀ Bu þ H þ Na]þ, C32H28N6NaOþ6 ; calc. 615.1968), 593.2142 (16, [M ꢀ Bu þ 2 H]þ,
C32H29N6Oþ6 ; calc. 593.2149), 541.2192 (15, [M ꢀ BnO]þ, C29H29N6O5þ ; calc. 541.2199). Anal. calc. for
C36H36N6O6 (648.71): C 66.65, H 5.59, N 12.95; found C 66.36, H 5.57, N 12.80.
tert-Butyl 6-O-Benzyl-8-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)ethyl}guanine-9-acetate
(48). A soln. of 67 (6.20 g, 15.6 mmol) in THF (100 ml) was cooled to 08, treated dropwise with a cold
(08) soln. of Fmoc-OSu (5.77 g, 17.1 mmol) in THF (60 ml) over 10 min and stirred for 30 min.
Evaporation and FC (AcOEt/cyclohexane 1:1) gave 48 (8.35 g, 86%). White powder. Rf (CH2Cl2/MeOH
19 :1) 0.39. M.p. 139.2 – 141.18. UV (MeOH): 255 (27140), 211 (54660). IR (ATR): 3483w, 3411w, 3312w,
3184w, 2971w, 2943w, 1745m, 1729s, 1635m, 1593s, 1530m, 1489m, 1470m, 1449m, 1436m, 1426m, 1392m,
1377m, 1367m, 1351m, 1331m, 1297m, 1233s, 1195m, 1157m, 1144s, 1098w, 1078m, 1064m, 1042m, 1006m,
980m, 935w, 913w, 878w, 867w, 846m, 832w, 792m, 756s, 738s, 727m, 700m, 669w, 620w. 1H-NMR
(300 MHz, (D6)DMSO, 1008): 7.85 – 7.26 (m, 13 arom. H); 6.97 (s, NHFmoc); 6.02 (s, NH2); 5.53 (s,
PhCH2); 4.74 (s, CH2ꢀN(9)); 4.32 (d, J ¼ 6.5, CH2ꢀC(9’’)); 4.21 (t, J ¼ 6.7, HꢀC(9’’)); 3.43 (q, J ¼ 6.2,
t
2 HꢀC(2’)); 2.84 (t, J ¼ 7.3, 2 HꢀC(1’)); 1.42 (s, Me3C). 13C-NMR (75 MHz, CDCl3): 166.28 (s, CO2 Bu);
159.96 (s, C(6)); 158.69 (s, C(2)); 156.39 (s, NHCO2); 155.32 (s, C(4)); 149.30 (s, C(8)); 143.86 (2s); 141.14
(2s); 136.53 (s); 128.34 – 119.85 (13d); 114.14 (s, C(5)); 83.36 (s, Me3C); 68.10 (t, CH2ꢀC(9’’)); 66.83 (t,
PhCH2); 47.25 (d, C(9’’)); 43.79 (t, CH2ꢀN(9)); 37.76 (t, C(2’)); 28.11 (q, Me3C); 27.69 (t, C(1’)). HR-
MALDI-MS: 659.2344 (7, [M þ K]þ, C35H36KN6O5þ ; calc. 659.2384), 643.2633 (14, [M þ Na]þ,
C35H36N6NaOþ5 ; calc. 643.2645), 621.2817 (100, [M þ H]þ, C35H37N6O5þ ; calc. 621.2825), 603.1751 (1,
t
t
[M ꢀ Bu þ H þ K]þ, C31H28KN6O5þ ; calc. 603.1758), 587.1990 (2, [M ꢀ Bu þ H þ Na]þ, C31H28N6NaOþ5 ;
calc. 587.2019), 565.2167 (30, [M ꢀ Bu þ 2 H]þ, C31H29N6O5þ ; calc. 565.2199). Anal. calc. for C35H36N6O5
t
(620.70): C, 67.73, H 5.85, N 13.54; found: C 67.91, H 5.69, N 13.44.
tert-Butyl 6-Formyluracil-1-acetate (49). A soln. of iPr2NH (35 ml, 265 mmol) in THF (150 ml) was
cooled to ꢀ 768, treated dropwise with 1.6m BuLi in hexane (166 ml, 265 mmol), stirred for 20 min,
warmed to 08, stirred for 15 min, cooled to ꢀ 768, treated dropwise with a soln. of 17 (10 g, 44.2 mmol) in
THF (200 ml), and stirred for 2 h at ꢀ 768. The soln. was treated dropwise with DMF (68 ml, 884 mmol),
stirred for another 2.5 h at ꢀ 768, allowed to reach r.t., treated with AcOH (32 ml) and sat. NH4Cl soln.
(150 ml), and extracted with CHCl3 (4 ꢃ 50 ml). Drying of the combined org. fractions (MgSO4),

Helvetica Chimica Acta – Vol. 92 (2009)
1159
evaporation, and FC (CH2Cl2/MeOH 99 :1 ! 9 :1) gave 49 (4.15 g, 37%) and 17. White powder. Rf
(CH2Cl2/MeOH 19 :1) 0.18. M.p. 182.5 – 183.58. UV (CHCl3): 306 (68800). IR (ATR): 3196w, 3066w,
2982w, 1736m, 1697s, 1674s, 1615m, 1467m, 1418m, 1384s, 1367s, 1291w, 1232s, 1156s, 1076m, 973m, 920m,
1
867w. H-NMR (300 MHz, CDCl3): 9.48 (s, CHO); 8.69 (br. s, NH); 6.29 (d, J ¼ 2.1, HꢀC(5)); 4.94 (s,
t
CH2ꢀN(1)); 1.47 (s, tBu). 13C-NMR (75 MHz, (D6)DMSO): 188.13 (d, CHO); 167.09 (s, CO2 Bu); 162.43
(s, C(4)); 150.94 (s, C(2)); 146.19 (s, C(6)); 113.39 (d, C(5)); 81.62 (s, Me3C); 44.44 (t, CH2ꢀN(1)); 27.44
t
(q, Me3C). HR-EI-MS: 254.0899 (0.6, Mþ, C11H14N2Oþ5 ; calc. 254.0903), 181.0253 (14, [M ꢀ BuO]þ,
C7H5N2Oþ4 ; calc. 181.0249), 57.0697 (100, tBuþ). Anal. calc. for C11H14N2O5 (254.09): C 51.97, H 5.55, N
11.02; found: C 51.89, H 5.56, N 10.85.
Benzyl (E)-3-(1-{[(tert-Butoxy)carbonyl]methyl}uracil-6-yl)prop-2-enoate (50). A soln. of 49
(3.10 g, 12.19 mmol) and benzyl (triphenylphosphoranylidene)acetate (5.51 g, 13.41 mmol) in THF
(32 ml) was stirred for 5 h at r.t., diluted with H2O (80 ml), and extracted with CHCl3 (4 ꢃ 25 ml). Drying
of the combined organic layers (MgSO4), evaporation, and FC (AcOEt/cyclohexane 1:1) gave 50
(3.58 g, 76%). White powder. Rf (cyclohexane/AcOEt 1:2) 0.58. M.p. 176 – 1778. UV (CHCl3): 241
(8790), 304 (6370). IR (ATR): 3182w, 3058w, 2985w, 2815w, 1740m, 1720m, 1681s, 1611s, 1496w, 1450m,
1412m, 1393m, 1367m, 1311m, 1281m, 1236s, 1202m, 1168s, 1152s, 1041w, 1007m, 995m, 972m, 944w,
1
922w. H-NMR (400 MHz, CDCl3; assignment based on a HSQC and a HMBC spectrum): 9.72 (br. s,
NH); 7.37 (br. s, 5 arom. H); 7.26 (dd, J ¼ 11.6, 0.5, HꢀC(3)); 6.48 (d, J ¼ 11.6, HꢀC(2)); 5.87 (d, J ¼ 0.5,
HꢀC(5’)); 5.24 (s, PhCH2); 4.50 (s, CH2ꢀN(1’)); 1.43 (s, tBu). 13C-NMR (100 MHz, CDCl3; assignment
t
based on a HSQC and a HMBC spectrum): 166.32 (s, CO2 Bu); 164.31 (s, CO2Bn); 162.42 (s, C(4’));
151.12 (d, C(2’)); 150.72 (s, C(6’)); 135.03 (s); 134.34 (d, C(3)); 128.71 (2d); 128.67 (d, C(2)); 128.51 (2d);
128.40 (d); 102.08 (d, C(5’)); 83.66 (s, Me3C); 67.38 (t, PhCH2); 45.98 (t, CH2ꢀN(1)); 27.88 (q, Me3C).
t
HR-EI-MS: 386.1476 (15, Mþ, C20H22N2Oþ6 ; calc. 386.1478), 330.0843 (47, [M ꢀ Bu þ H]þ, C16H14N2Oþ6 ;
t
t
calc. 330.0846), 313.0819 (56, [M ꢀ BuO]þ, C16H13N2O5þ ; calc. 313.0824), 285.0870 (21, [M ꢀ CO2 Bu]þ,
C15H13N2Oþ4 ; calc. 285.0875), 224.0428 (34, [M ꢀ Bu ꢀ OBn þ 2 H]þ, C9H8N2Oþ5 ; calc. 224.0422),
t
195.0404 (40, [M ꢀ Bu ꢀ CO2Bn þ H]þ, C8H7N2O4þ ; calc. 195.0400), 180.0502 (19, [M ꢀ OtBu ꢀ
t
t
CO2Bn þ 2 H]þ, C8H8N2O3þ ; calc. 180.0524), 151.0507 (25, [M ꢀ CO2 Bu ꢀ CO2Bn þ H]þ, C7H7N2Oþ2 ;
calc. 151.0502), 91.0538 (48, C7Hþ7 ), 57.0715 (100, tBuþ), 41.0452 (12, [H2C¼CHꢀCH2]þ). Anal. calc. for
C20H22N2O6 (386.40): C 62.17, H 5.74, N 7.25; found: C 62.04, H 5.75, N 7.13.
3-(1-{[tert-Butoxy)carbonyl]methyl}uracil-6-yl)propanoic Acid (51). A suspension of Pd(OAc)2
(173 mg, 0.77 mmol) in MeOH (30 ml) was stirred for 1 h at r.t. under H2, treated with a soln. of 50
(2.98 g, 7.73 mmol) in THF (50 ml), and stirred for 14 h. Filtration through Celite and evaporation gave
51 (2.30 g, 100%). White powder. Rf (CHCl3/MeOH 3 :1) 0.26. M.p. 169 – 1708. UV (CHCl3): 265 (9587).
IR (ATR): 3116w, 2982w, 2931w, 2870w, 2694w, 2550w, 1745m, 1697s, 1635s, 1472m, 1443m, 1421s, 1393m,
1368s, 1314w, 1288w, 1237s, 1217s, 1191m, 1155s, 1064w, 1037w, 1022w, 1006w, 927m, 862m. 1H-NMR
(300 MHz, (CD3)2CO, spectrum of higher order): 10.11 (br. s, NH); 5.53 (s, HꢀC(5’)); 4.62 (s,
t
CH2ꢀN(1’)); 2.84 – 2.76 (m, 2 HꢀC(2)); 2.72 – 2.65 (m, 2 HꢀC(3)); 1.46 (s, Bu). 13C-NMR (75 MHz,
t
(D6)DMSO): 172.57 (s, CO2H); 167.18 (s, CO2 Bu); 162.21 (s, C(4’)); 155.74 (s, C(6’)); 151.41 (s, C(2’));
99.72 (d, C(5’)); 81.92 (s, Me3C); 45.16 (t, CH2ꢀN(1’)); 30.95 (t, C(2)); 27.62 (q, Me3C); 26.65 (t, C(3)).
t
HR-EI-MS: 298.1160 (3, Mþ, C13H18N2O6þ ; calc. 298.1165), 225.0501 (11, [M ꢀ BuO]þ, C9H9N2Oþ5 ; calc.
t
t
225.0511), 198.0638 (21, [M ꢀ CO2 Bu þ H]þ, C8H10N2O4þ ; calc. 198.0635), 153.0661 (24, [M ꢀ CO2 Bu ꢀ
CO2]þ, C7H9N2Oþ2 ; calc. 153.0659), 57.0715 (100, tBuþ, C4Hþ4 ).
6-[2-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)ethyl]uracil-1-acetic Acid (52). A soln. of 45
i
(3.4 g, 6.91 mmol) in CH2Cl2 (30 ml) was treated with Pr3SiH (7.1 ml, 34.55 mmol) and TFA (15.4 ml,
6.91 mmol), and stirred for 8 h at r.t. After evaporation, the residue was suspended in Et2O (30 ml). The
solid was filtered off, washed with Et2O, and dried to give 52 (2.35 g, 78%). White powder. Rf (CHCl3/
MeOH 3 :2) 0.31. M.p. 147 – 1518. UV (MeOH): 265 (24000), 300 (5000). IR (ATR): 2973w (br.), 1672s
(br.), 1524m, 1449m, 1412m, 1396m, 1335w, 1246m, 1199m, 1148m, 1101w, 1086w, 995w, 931w, 880w.
1H-NMR (300 MHz, (D6)DMSO): 13.19 (br. s, CO2H); 11.35 (s, HꢀN(3)); 7.92 – 7.65 (m, 4 arom. H);
7.48 (t, J ¼ 5.7, NHFmoc); 7.42 – 7.31 (m, 4 arom. H); 5.50 (s, HꢀC(5)); 4.51 (s, CH2ꢀN(1)); 4.32 (d, J ¼
6.9, CH2ꢀC(9’’)); 4.21 (t, J ¼ 6.9, HꢀC(9’’)); 3.18 (q, J ¼ 6.0, 2 HꢀC(2’)); 2.55 (t, J ¼ 6.0, 2 HꢀC(1’)).
13C-NMR (75 MHz, (D6)DMSO): 169.82 (s, CO2H); 162.49 (s, C(4)); 156.19 (s, NHCO2Fm); 154.30 (s,
C(6)); 151.75 (s, C(2)); 143.86 (2s); 140.77 (2s); 127.62 – 120.13 (8d); 100.87 (d, C(5)); 65.54 (t,

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Helvetica Chimica Acta – Vol. 92 (2009)
CH2ꢀC(9’’)); 46.73 (d, C(9’’)); 44.67 (t, CH2ꢀN(1)); 38.03 (t, C(2’)); 31.79 (t, C(1’)). HR-MALDI-MS:
474.1091 (5, [M þ K]þ, C23H21KN3O6þ ; calc. 474.1067), 458.1319 (100, [M þ Na]þ, C23H21N3NaOþ6 ; calc.
458.1323), 436.1509 (17, [M þ H]þ, C23H22N3O6þ ; calc. 436.1509).
i
tert-Butyl N4-[(Benzyloxy)carbonyl]-6-formylcytosine-1-acetate (53). A soln. of Pr2NH (24.1 ml,
184 mmol) in THF (100 ml) was cooled to ꢀ 768, treated dropwise with 1.6m BuLi in hexane (115 ml,
184 mmol), warmed to 08, stirred for 30 min, cooled to ꢀ 768, treated dropwise with a soln. of 26 (11.0 g,
30.6 mmol) in THF (50 ml) over 30 min, and stirred for 2 h. The soln. was treated dropwise with DMF
(47.4 ml, 612 mmol) over 30 min and stirred for another 2 h at ꢀ 768. The soln. was treated with AcOH
(22.8 ml, 398 mmol), allowed to reach r.t., and evaporated. The residue was diluted with H2O and
extracted with AcOEt (4ꢃ). The combined org. layers were washed with brine (5ꢃ), dried (MgSO4),
and evaporated. FC (CH2Cl2/MeOH 97:3 ! 9 :1) gave 53 (7.60 g, 64%). Yellow foam. Rf (CH2Cl2/MeOH
19 :1) 0.21. UV (MeOH): 297 (8560), 244 (16320), 209 (26880). IR (ATR): 3115w, 2997w, 2976w, 2937w,
1740s, 1706m, 1657s, 1612s, 1563m, 1511m, 1455w, 1417m, 1407w, 1379s, 1369m, 1290w, 1226s, 1151s,
1084w, 1064m, 1028w, 994w, 968w, 947w, 927m, 867w, 840m, 781m, 769w, 740s, 715m, 697m, 664w.
1H-NMR (300 MHz, (D6)DMSO): 11.27 (s, NH); 9.68 (s, HꢀC(5)); 7.70 – 7.34 (m, 5 arom. H); 5.24 (s,
PhCH2); 4.85 (s, CH2ꢀN(1)); 1.41 (s, tBu). 13C-NMR (75 MHz, (D6)DMSO): 187.67 (d, CHO); 166.94 (s,
t
CO2 Bu); 163.19 (s, C(4)); 155.01 (s, C(6)); 152.93 (s, NHCO2Bn); 148.91 (s, C(2)); 135.56 (s); 128.33
(2d); 128.09 (2d); 127.90 (d); 102.81 (d, C(5)); 81.69 (s, Me3C); 66.72 (t, PhCH2); 45.55 (t, CH2ꢀN(1));
27.44 (q, Me3C). HR-MALDI-MS: 458.1346 (6, [M þ MeOH þ K]þ, C20H25KN3O7þ ; calc. 458.1330),
444.1184 (7, [M þ H2O þ K]þ, C19H23KN3O7þ ; calc. 444.1173), 442.1599 (14, [M þ MeOH þ Na]þ,
C20H25N3NaOþ7 ; calc. 442.1590), 428.1440 (15, [M þ H2O þ Na]þ, C19H23N3NaOþ7 ; calc. 428.1434),
420.1778 (96, [M þ MeOH þ H]þ, C20H26N3O7þ ; calc. 420.1771), 410.1345 (9, [M þ Na]þ, C19H21N3NaOþ6 ;
calc. 410.1328), 406.1614 (100, [M þ H2O þ H]þ, C19H24N3O7þ ; calc. 406.1614), 388.1508 (34, [M þ H]þ,
t
C19H22N3Oþ6 ; calc. 388.1509), 386.0974 (7, [M þ MeOH ꢀ Bu þ H þ Na]þ, C16H17N3NaOþ7 ; calc.
t
386.0964), 372.0814 (7, [M þ H2O ꢀ Bu þ H þ Na]þ, C15H15N3NaO7þ ; calc. 372.0808), 364.1142 (53,
t
t
[M þ MeOH ꢀ Bu þ 2 H]þ, C16H18N3Oþ7 ; calc. 364.1145), 354.0708 (8, [M ꢀ Bu þ H þ Na]þ,
C15H13N3NaOþ6 ; calc. 354.0702), 350.0982 (74, [M þ H2O ꢀ Bu þ 2 H]þ, C15H16N3Oþ7 ; calc. 350.0988),
t
332.0882 (31, [M ꢀ Bu þ 2 H]þ, C15H14N3O6þ ; calc. 332.0883). Anal. calc. for C19H21N3O6 (387.39): C
t
58.91, H 5.46, N 10.85; found: C 58.63, H 5.53, N 10.55.
Methyl (E)-3-(N4-[(Benzyloxy)carbonyl]-1-{[(tert-butoxy)carbonyl]methyl}cytosin-6-yl)prop-2-
enoate (54). A soln. of 53 (6.80 g, 17.6 mmol) and methyl (triphenylphosphoranylidene)acetate
(6.16 g, 18.4 mmol) in THF (88 ml) was stirred for 3 h at r.t. and evaporated to give a 1:1 mixture 54/
i
Ph3PO (12.5 g, 97%). A small sample was ground and suspended in Pr2O. Filtration and washing with
iPr2O gave pure 54. Yellow powder. Rf (CH2Cl2/MeOH 19 :1) 0.28. M.p. 71.1 – 76.18. UV (MeOH): 314
(6960), 239 (13760), 207 (37740). IR (ATR): 2978w, 2954w, 1736m, 1729m, 1669m, 1602m, 1562m,
1496m, 1454w, 1432w, 1423m, 1387m, 1369m, 1311m, 1276m, 1259m, 1197s, 1175s, 1149s, 1052m, 971w,
947w, 911w, 858w, 819w, 785m, 744m, 696m. 1H-NMR (300 MHz, CDCl3; assignments based on a HSQC
and a HMBC spectrum): 7.93 (br. s, NH); 7.39 – 7.26 (m, 5 arom. H, HꢀC(3), HꢀC(5’)); 6.58 (d, J ¼ 15.7,
HꢀC(2)); 5.20 (s, PhCH2); 4.67 (s, CH2ꢀN(1’)); 3.82 (s, MeO); 1.45 (s, Me3C). 13C-NMR (75 MHz,
t
CDCl3; assignments based on a HSQC and a HMBC spectrum): 166.13 (s, CO2 Bu); 164.91 (s, CO2Me);
161.79 (s, C(4’)); 155.74 (s, C(2’)); 154.48 (s, C(6’)); 152.21 (s, NHCO2Bn); 134.81 (s); 134.09 (d, C(3));
128.83 (d); 128.61 (2d); 128.27 (2d); 93.93 (2d, C(2), C(5’)); 83.39 (s, Me3C); 67.98 (t, PhCH2); 52.40 (q,
MeO); 47.02 (t, CH2ꢀN(1’)); 27.81 (q, Me3C). HR-MALDI-MS: 482.1327 (24, [M þ K]þ, C22H25KN3O7þ ;
calc. 482.1330), 466.1484 (64, [M þ Na]þ, C22H25N3NaOþ7 ; calc. 466.1590), 444.1758 (100, [M þ H]þ,
C22H26N3Oþ7 ; calc. 444.1771), 426.0699 (10, [M ꢀ Bu þ H þ K]þ, C18H17KN3Oþ7 ; calc. 426.0704), 410.0955
t
t
t
(46, [M ꢀ Bu þ H þ Na]þ, C18H17N3NaO7þ ; calc. 410.0964), 388.1127 (82, [M ꢀ Bu þ 2 H]þ, C18H18N3Oþ7 ;
calc. 388.1145).
Methyl 3-(N4-[(Benzyloxy)carbonyl]-1-{[(tert-butoxy)carbonyl]methyl}cytosine-6-yl)propanoate
(55). A soln. of 54/Ph3PO 1:1 (7.60 g, 17.1 mmol of 54) in MeOH (115 ml) was cooled to 08, treated
with NaBH4 (973 mg, 25.7 mmol) in several portions over 20 min and stirred for 1.5 h at 08. The soln. was
poured on H2O and extracted with CH2Cl2 (5ꢃ). The combined org. fractions were washed with brine
(3ꢃ), dried (MgSO4), filtered, and evaporated. Drying of the residue gave a 1:1 mixture 55/Ph3PO
(12.3 g, 98%). A small sample was ground and suspended in iPr2O. Filtration and washing with iPr2O gave

Helvetica Chimica Acta – Vol. 92 (2009)
1161
pure 55. Yellow powder. Rf (CH2Cl2/MeOH 19 :1) 0.25. M.p. 55.3 – 56.18. UV (MeOH): 296 (9460), 241
(16500), 211 (28640). IR (ATR): 2978w, 2953w, 1733s, 1665m, 1607m, 1564m, 1498m, 1453w, 1437w,
1411m, 1389m, 1368s, 1291w, 1207s, 1192s, 1149s, 1061m, 1029w, 984w, 949w, 923w, 896w, 856w, 821w,
1
787m, 770w, 744m, 735m, 695m. H-NMR (300 MHz, CDCl3): 8.05 (s, NH); 7.35 – 7.26 (m, 5 arom. H);
7.09 (s, HꢀC(5’)); 5.17 (s, PhCH2); 4.67 (s, CH2ꢀN(1’)); 3.69 (s, MeO); 2.82 (t, J ¼ 7.7, 2 H ꢀC(3)); 2.68 (t,
t
t
J ¼ 7.7, 2 H ꢀC(2)); 1.43 (s, Bu). 13C-NMR (75 MHz, CDCl3): 171.55 (s, CO2Et); 166.52 (s, CO2 Bu);
161.67 (s, C(4’)); 160.27 (s, C(6’)); 156.31 (s, C(2’)); 152.32 (s, NHCO2Bn); 135.00 (s); 128.53 (2d); 128.47
(d); 128.16 (2d); 93.51 (d, C(5’)); 83.07 (s, Me3C); 67.69 (t, PhCH2); 52.10 (q, MeO); 46.45 (t,
CH2ꢀN(1)); 31.07 (t, C(2’)), 27.82 (q, Me3C); 27.66 (t, C(3’)). HR-MALDI-MS: 484.1498 (8, [M þ K]þ,
C22H27KN3Oþ7 ; calc. 484.1486), 468.1748 (32, [M þ Na]þ, C22H27N3NaO7þ ; calc. 468.1747), 446.1928 (100,
[M þ H]þ, C22H28N3O7þ ; calc. 446.1927), 412.1119 (6, [M ꢀ Bu þ H þ Na]þ, C18H19N3NaOþ7 ; calc.
t
412.1121), 390.1291 (45, [M ꢀ Bu þ 2 H]þ, C18H20N3O7þ ; calc. 390.1301).
t
3-(N4-[(Benzyloxy)carbonyl]-1-{[(tert-butoxy)carbonyl]methyl}cytosin-6-yl)propanoic Acid (56).
a) A soln. of 55 (156 mg, 350 mmol) in THF/H2O 4 :1 (3.5 ml) was cooled to 08, treated with LiOH · H2O
(147 mg, 3.5 mmol) in one portion and stirred for 2 h. Neutralisation with Amberlite IR-120 (prewashed
with MeOH and THF), filtration, evaporation, and FC (CH2Cl2/MeOH 99 :1 ! 9 :1) gave 56 (125 mg,
83%). White powder.
b) A soln. of 55/Ph3PO 1:1 (7.41 g, 16.6 mmol of 55) in THF/H2O 4 :1 (130 ml) was cooled to 08,
treated with LiOH · H2O (6.98 g, 166 mmol) in one portion, and stirred for 3.5 h. Neutralisation with
Amberlite IR-120 (prewashed with MeOH and THF), filtration, evaporation, FC (CH2Cl2/MeOH
99 :1 ! 9 :1), and washing of the residue after evaporation of the fractions containing 56 with AcOEt
gave 56 (2.15 g, 30%). White powder. Rf (CH2Cl2/MeOH 4 :1) 0.56. M.p. 155.6 – 163.18. UV (MeOH):
297 (9200), 240 (14960), 211 (24000). IR (ATR): 2980w, 2933w, 1754m, 1744m, 1698m, 1602m, 1587m,
1511m, 1412m, 1391m, 1373m, 1367m, 1298w, 1213s, 1193s, 1151s, 1067m, 1035w, 982w, 970w, 906w, 862w,
821m, 776m, 768m, 746m, 728m, 697m, 672w. 1H-NMR (300 MHz, (D6)DMSO; assignments based on a
HSQC and a HMBC spectrum): 10.90 (br. s, NH); 7.41 – 7.36 (m, 5 arom. H); 6.99 (s, HꢀC(5’)), 5.18 (s,
t
PhCH2); 4.64 (s, CH2ꢀN(1’)); 2.81 (t, J ¼ 6.9, 2 HꢀC(3)); 2.55 (t, J ¼ 6.9, 2 HꢀC(2)); 1.41 (s, Bu).
13C-NMR (75 MHz, (D6)DMSO; assignments based on a HSQC and a HMBC spectrum): 172.60 (s,
t
CO2H); 166.96 (s, CO2 Bu); 162.077 (s, C(4’)); 161.08 (s, C(6’)); 155.68 (s, C(2’)); 152.92 (s, NHCO2Bn);
135.79 (s); 128.30 (2d); 127.97 (d); 127.75 (2d); 92.95 (d, C(5’)); 81.65 (s, Me3C); 66.29 (t, PhCH2); 46.38
(t, CH2ꢀN(1’)); 30.98 (t, C(2)); 27.41 (q and t, Me3C and C(3)). HR-MALDI-MS: 470.1334 (11, [M þ
K]þ, C21H25KN3O7þ ; calc. 470.1330), 454.1592 (25, [M þ Na]þ, C21H25N3NaOþ7 ; calc. 454.1590),
t
432.1769 (100, [M þ H]þ, C21H26N3O7þ ; calc. 432.1771), 414.0701 (2, [M ꢀ Bu þ H þ K]þ, C17H17KN3Oþ7 ;
calc. 414.0704), 398.0955 (6, [M ꢀ Bu þ H þ Na]þ, C17H17N3NaO7þ ; calc. 398.0964), 376.1131 (46, [M ꢀ
t
tBu þ 2 H]þ, C17H18N3Oþ7 ; calc. 376.1145).
N4-[(Benzyloxy)carbonyl]-6-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)ethyl]cytosine-1-ace-
tic Acid (57). A soln. of 46 (4.38 g, 7.01 mmol) in CH2Cl2 (70 ml) was treated with Et3SiH (11.2 ml,
70.1 mmol) and TFA (21.6 ml, 280 mmol), and stirred for 16 h at r.t. After evaporation at 308, the residue
i
was treated with Pr2O (80 ml). The resulting precipitate was filtered off, ground, and washed several
times with iPr2O. Drying of the solid gave 57 (3.91 g, 98%). White powder. Rf (CH2Cl2/MeOH 3 :2) 0.37.
M.p. 121.7 – 122.38. UV (MeOH): 230 (12700), 265 (17500), 209 (50400). IR (ATR): 2953w, 1719m,
1666m, 1602m, 1569m, 1507m, 1449m, 1414m, 1391m, 1209s, 1191s, 1139m, 1087m, 1066m, 1002m, 908w,
1
826w, 758m, 739s, 695m, 620w. H-NMR (300 MHz, (D6)DMSO; assignments based on a HSQC and a
HMBC spectrum): 11.20 (br. s, NHꢀC(4)); 7.89 – 7.28 (m, 13 arom. H, NHFmoc); 6.97 (s, HꢀC(5)); 5.10
(s, PhCH2); 4.65 (s, CH2ꢀN(1)); 4.31 (d, J ¼ 6.9, CH2ꢀC(9’’)); 4.20 (t, J ¼ 6.5, HꢀC(9’’)); 3.23 (q, J ¼ 6.2,
2 HꢀC(2’)); 2.75 (t, J ¼ 6.5, 2 HꢀC(1’)). 13C-NMR (75 MHz, (D6)DMSO; assignments based on a
HSQC and a HMBC spectrum): 169.19 (s, CO2H); 161.84 (s, C(4)); 159.06 (s, C(6)); 155.92 (s,
NHCO2Fm); 155.63 (s, C(2)); 152.81 (s, NHCO2Bn); 143.61 (2s); 140.51 (2s); 135.68 (s); 128.23 – 119.92
(13d); 94.38 (d, C(5)); 66.33 (t, PhCH2); 65.39 (t, CH2ꢀC(9’’)); 46.66 (d, C(9’’)); 46.06 (t, C(2’)); 38.44 (t,
CH2ꢀN(1)); 32.83 (t, C(1’)). HR-MALDI-MS: 607.1608 (8, [M þ K]þ, C31H28KN4O7þ ; calc. 607.1595),
591.1861 (27, [M þ Na]þ, C31H28N4NaO7þ ; calc. 591.1856), 569.2036 (100, [M þ H]þ, C31H29N4Oþ7 ; calc.
569.2036).

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Helvetica Chimica Acta – Vol. 92 (2009)
i
tert-Butyl N6-[(Benzyloxy)carbonyl]-8-formyladenine-9-acetate (58). A soln. of Pr2NH (14.77 ml,
113 mmol) in THF (80 ml) was cooled to ꢀ 768, treated dropwise with 1.6m BuLi in hexane (70.42 ml,
113 mmol), stirred for 20 min, warmed to 08, stirred for 1 h, and left at 48 for 16 h. The soln. was cooled to
ꢀ 768, treated dropwise with a soln. of 32 (7.20 g, 18.8 mmol) in THF (30 ml), stirred for 2 h, treated
dropwise with DMF (29.08 ml, 376 mmol), and stirred for another 2 h. The soln. was treated with AcOH
(6.45 ml) and allowed to warm to r.t. After evaporation, the residue was diluted with H2O and extracted
with AcOEt (4ꢃ). The combined org. layers were washed with H2O and brine (3ꢃ), dried (MgSO4),
filtered, and evaporated. FC (AcOEt/cyclohexane 3 :1) gave 58 (4.50 g, 58%). Yellow foam. Rf (CH2Cl2/
MeOH 95 :5) 0.28. UV (MeOH): 212 (33160), 269 (21080). IR (ATR): 3239w (br.), 3189w (br.), 2979w,
1742s, 1697m (br.), 1604s, 1583m, 1522w, 1499w, 1454s, 1414w, 1391m, 1367m, 1322w, 1290m, 1282m,
1249m, 1229m, 1198s, 1150s, 1106m, 1029m, 973w, 943w, 897w, 850 m, 801 w, 743m, 696m, 664w. 1H-NMR
(300 MHz, CDCl3): 9.97 (s, CHO); 8.84 (s, HꢀC(2)); 8.78 (s, NH); 7.44 – 7.34 (m, 5 arom. H); 5.30 (s,
t
PhCH2); 5.23 (s, CH2ꢀN(9)); 1.46 (s, Bu). 13C-NMR (75 MHz, CDCl3): 183.68 (d, CHO); 165.58 (s,
t
CO2 Bu); 155.70 (d, C(2)); 152.03 (s, C(4)); 151.63 (s, NHCO2Bn); 150.40 (s, C(6)); 145.43 (s, C(8));
134.95 (s); 128.67 (3d); 128.60 (2d); 121.78 (s, C(5)); 83.68 (s, Me3C); 68.23 (t, PhCH2); 45.51 (t,
CH2ꢀN(9)); 28.07 (q, Me3C). HR-MALDI-MS: 434.1428 (22, [M þ Na]þ, C20H21N5NaO5þ ; calc.
434.1440), 412.1609 (100, [M þ H]þ, C20H22N5O5þ ; calc. 412.1621), 384.1660 (14, [M ꢀ CHO þ 2 H]þ,
t
C19H22N5Oþ4 ; calc. 384.1672), 378.0807 (9, [M ꢀ Bu þ Na þ H]þ, C16H13N5NaOþ5 ; calc. 378.0814),
t
356.0980 (57, [M ꢀ Bu þ 2 H]þ, C16H14N5O5þ ; calc. 356.0995).
Methyl (E)-3-(N6-[(Benzyloxy)carbonyl]-9-{[(tert-butoxy)carbonyl]methyl}adenine-8-yl)prop-2-
enoate (59). A soln. of 58 (7.50 g, 18.2 mmol) and methyl (triphenylphosphoranylidene)acetate
(6.40 g, 19.1 mmol) in THF (90 ml) was stirred for 3 h at r.t. and evaporated. The residue was washed
repetitively with hot cyclohexane to give 59 (7.70 g, 90%). Yellow foam. Rf (CH2Cl2/MeOH 95 :5) 0.38.
UV (MeOH): 205 (26480), 238 (22280), 318 (21760). IR (ATR): 3245w (br.), 3181w (br.), 2980w, 2951w,
1740m, 1719s (br.), 1648w, 1606m, 1586m, 1519w, 1449m, 1437m, 1388w, 1367m, 1295m, 1272m, 1207s,
1
1150s, 1103m, 1037m, 967m, 951w, 897w, 848w, 800w, 746m, 696m, 617w. H-NMR (300 MHz, CDCl3;
assignments based on a HSQC and a HMBC spectrum): 8.72 (s, HꢀC(2’)); 8.62 (s, NH); 7.43 (d, J ¼ 15.4,
HꢀC(3)); 7.45 – 7.31 (m, 5 arom. H); 7.06 (d, J ¼ 15.7, HꢀC(2)); 5.23 (s, PhCH2); 4.93 (s, CH2ꢀN(9’));
t
3.79 (s, MeO); 1.43 (s, Bu). 13C-NMR (75 MHz, CDCl3; assignments based on a HSQC and a HMBC
t
spectrum): 165.80 (s, CO2Me); 165.37 (s, CO2 Bu); 153.32 (d, C(2’)); 152.08 (s, C(4’)); 150.53 (s,
NHCO2Bn); 149.27 (s, C(6’)); 148.00 (s, C(8’)); 135.19 (s); 128.54 (2d); 128.49 (2d); 128.43 (d); 127.84 (d,
C(3)); 126.80 (d, C(2)); 121.79 (s, C(5’)); 84.04 (s, Me3C); 67.69 (t, PhCH2); 52.16 (q, MeO); 43.94 (t,
CH2ꢀN(9’)); 27.78 (q, Me3C). HR-MALDI-MS: 506.1459 (4, [M þ K]þ, C23H25KN5O6þ ; calc. 506.1442),
490.1716 (13, [M þ Na]þ, C23H25N5NaO6þ ; calc. 490.1703), 468.1881 (100, [M þ H]þ, C23H26N5Oþ6 ; calc.
t
t
468.1883), 434.1072 (5, [M ꢀ Bu þ Na þ H]þ, C19H17N5NaOþ6 ; calc. 434.1077), 412.1246 (96, [M ꢀ Bu þ
t
2 H]þ, C19H18N5O6þ ; calc. 412.1257), 368. 1344 (13, [M ꢀ CO2 Bu þ 2 H]þ, C18H18N5Oþ4 ; calc. 368.1359).
Methyl 3-(N6-[(Benzyloxy)carbonyl]-9-{[(tert-butoxy)carbonyl]methyl}adenin-8-yl)propanoate
(60). A suspension of 58 (7.34 g, 15.7 mmol) in MeOH (105 ml) was treated with a minimum of THF
to obtain a soln., cooled to 08, treated with NaBH4 (5.94 g, 157 mmol) in several portions over 20 min,
and stirred for 3 h at 08. The soln. was poured on H2O and extracted with CH2Cl2 (5ꢃ). The combined
org. fractions were washed with brine (3ꢃ), dried (MgSO4), filtered, and evaporated. Drying of the
residue gave 60 (5.41 g, 73%). White foam. Rf (CH2Cl2/MeOH 95 :5) 0.31. UV (MeOH): 211 (35620),
270 (19820). IR (ATR): 3279w (br.), 2980w, 2952w, 1732s, 1609m, 1590m, 1520w, 1497w, 1451m, 1393w,
1366m, 1338w, 1321w, 1301m, 1204s, 1149s, 1102m, 1026m, 971w, 941w, 911w, 894w, 844w, 799w, 737m,
696m, 649w, 619w. 1H-NMR (300 MHz, CDCl3; assignments based on a HSQC and a HMBC spectrum):
8.69 (s, HꢀC(2’)); 8.41 (s, NH); 7.42 – 7.32 (m, 5 arom. H); 5.26 (s, PhCH2); 4.90 (s, CH2ꢀN(9’)); 3.66 (s,
MeO2C); 3.06 – 3.01 (m, 2 HꢀC(3)); 2.98 – 2.93 (m, 2 HꢀC(2)); 1.45 (s, Bu). 13C-NMR (75 MHz,
t
t
CDCl3; assignments based on a HSQC and a HMBC spectrum): 172.53 (s, CO2Me); 165.80 (s, CO2 Bu);
154.09 (d, C(2’)); 152.36 (s, C(4’)); 152.14 (s, C(8’)); 150.70 (s, NHCO2Bn); 147.97 (s, C(6’)); 135.37 (s);
128.57 (2d); 128.52 (2d); 128.41 (d); 120.76 (s, C(5’)); 83.74 (s, Me3C); 67.67 (t, PhCH2); 52.07 (q, MeO);
43.99 (t, CH2ꢀN(9’)); 30.56 (t, C(2)); 28.06 (q, Me3C); 22.54 (t, C(3)). HR-MALDI-MS: 508.1603 (6,
[M þ K]þ, C23H27KN5O6þ ; calc. 508.1598), 492.1855 (37, [M þ Na]þ, C25H27N5NaOþ6 ; calc. 492.1859),
t
470.2031 (100, [M þ H]þ, C23H28N5O6þ ; calc. 470.2040), 436.1225 (12, [M ꢀ Bu þ H þ Na]þ,

Helvetica Chimica Acta – Vol. 92 (2009)
1163
t
C19H19N5NaOþ6 ; calc. 436.1233), 414.1394 (98, [M ꢀ Bu þ 2 H]þ, C19H20N5Oþ6 ; calc. 414.1414), 370.1503
t
(7, [M ꢀ CO2 Bu þ 2 H]þ, C18H20N5O4þ ; calc. 370.1515). Anal. calc. for C23H27N5O6 (469.49): C 58.84, H
5.80, N 14.92; found C 58.55, H 5.71, N 14.75.
3-(N6-[(Benzyloxy)carbonyl]-9-{[(tert-butoxy)carbonyl]methyl}adenin-8-yl)propanoic Acid (61).
A soln. of 60 (5.05 g, 10.8 mmol) in THF/H2O 4 :1 (110 ml) was cooled to 08, treated with LiOH ·
H2O (1.35 g, 32.3 mmol) in one portion, and stirred for 2 h at 08. The resulting white suspension was
neutralized with Amberlite IR-120 (prewashed with MeOH and THF) and filtered. Evaporation of the
filtrate, FC (CH2Cl2/MeOH 97:3 ! 80 :20), and washing the yellow solid product with small amounts of
AcOEt gave 61 (3.98 g, 81%). White foam. Rf (CH2Cl2/MeOH 4 :1) 0.55. M.p. 64.3 – 66.78. UV (MeOH):
212 (35740), 271 (21020). IR (ATR): 3394 – 2778w (br.), 3192w (br.), 2979w, 2938w, 1738s (br.), 1613m,
1595m, 1536w, 1496w, 1453m, 1392w, 1367m, 1323w, 1298w, 1279w, 1207s, 1149s, 1105w, 1022m, 970w,
943w, 848w, 798w, 744m, 696m, 619w. 1H-NMR (300 MHz, (D6)DMSO; assignments based on a HSQC
and a HMBC spectrum): 12.35 (br. s, OH); 10.54 (br. s, NH); 8.57 (s, HꢀC(2’)); 7.47 – 7.33 (m, 5 arom.
H), 5.22 (s, PhCH2); 5.09 (s, CH2ꢀN(9)); 3.07 (t, J ¼ 7.2, 2 HꢀC(3)); 2.82 (t, J ¼ 7.2, 2 HꢀC(2)); 1.42 (s,
tBu). 13C-NMR (75 MHz, (D6)DMSO; assignments based on a HSQC and a HMBC spectrum): 173.03 (s,
t
CO2H); 166.42 (s, CO2 Bu); 154.91 (s, C(8’)); 153.05 (s, C(4’)); 152.06 (s, NHCO2Bn); 150.79 (d, C(2’));
147.90 (s, C(6’)); 136.22 (s); 128.18 (2d); 127.76 (2d); 127.65 (d); 122.22 (s, C(5’)); 82.42 (s, Me3C); 66.15 (t,
PhCH2); 43.78 (t, CH2ꢀN(9)); 38.65 (t, C(2)); 27.57 (q, Me3C); 22.13 (t, C(3)). HR-MALDI-MS:
494.1450 (3, [M þ K]þ, C22H25KN5O6þ ; calc. 494.1442), 478.1700 (24, [M þ Na]þ, C22H25N5NaOþ6 ; calc.
t
478.1703), 456.1880 (91, [M þ H]þ, C22H26N5Oþ6 ; calc. 456.1883), 422.1070 (10, [M ꢀ Bu þ H þ Na]þ,
t
C18H17N5NaOþ6 ; calc. 422.1077), 400.1252 (100, [M ꢀ Bu þ 2 H]þ, C18H18N5O6þ ; calc. 400.1257). 356.1356
(13, [M ꢀ CO2 Bu þ 2 H]þ, C17H18N5O4þ ; calc. 356.1359). Anal. calc. for C22H25N5O6 (455.46): C 58.02, H
t
5.53, N 15.38; found: C 57.99, H 5.45, N 15.15.
N6-[(Benzyloxy)carbonyl]-8-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)ethyl]adenine-9-ace-
tic Acid (62). A soln. of 47 (1.50 g, 2.31 mmol) in CH2Cl2 (23 ml) was treated with HSiEt3 (3.69 ml,
23.1 mmol) and TFA (7.13 ml, 10.6 mmol), and stirred for 16 h at r.t. After evaporation at 308, the residue
was treated with iPr2O (50 ml). The precipitate was filtered off, ground, and washed several times with
iPr2O. Drying of the solid gave 62 (1.36 g, 98%). White solid. Rf (CH2Cl2/MeOH 3 :2) 0.50. M.p. 110.8 –
115.18. UV (MeOH): 210 (49340), 266 (26500), 300 (4240). IR (ATR): 3435 – 2765w (br.), 3036w, 3032w,
2950w, 1713m (br.), 1658m, 1615m, 1529m, 1498w, 1450m, 1414w, 1366w, 1322w, 1207s, 1171s, 1137s,
1106m, 1078w, 1034w, 968w, 900w, 796w, 758m, 739s, 696m, 663w, 620w. 1H-NMR (400 MHz, (D6)DMSO;
assignments based on a HSQC and a HMBC spectrum): 13.48 (br. s, CO2H); 10.64 (br. s, NHꢀC(6));
8.58 (s, HꢀC(2)); 7.88 – 7.28 (m, 13 arom. H, NHFmoc); 5.22 (s, PhCH2); 5.04 (s, CH2ꢀN(9)); 4.32 (d, J ¼
6.8, CH2ꢀC(9’’)); 4.21 (t, J ¼ 6.6, HꢀC(9’’)); 3.48 (q, J ¼ 6.4, 2 HꢀC(2’)); 3.01 (t, J ¼ 7.0, 2 HꢀC(1’)).
13C-NMR (400 MHz, (D6)DMSO; assignments based on a HSQC and a HMBC spectrum): 167.04 (s,
CO2H); 154.23 (s, NHCO2Fm); 152.01 (d, C(2)); 151.22 (s, C(8)); 150.33 (s, C(4)); 149.08 (s, NHCO2Bn);
146.23 (s, C(6)); 142.00 (2s); 138.89 (2s); 134.47 (s); 126.53 – 123.25 (11d); 120.40 (s, C(5)); 118.24 (2d);
64.45 (t, PhCH2); 63.54 (t, CH2ꢀC(9’’)); 44.87 (d, C(9’’)); 41.43 (t, CH2ꢀN(9)); 36.09 (t, C(2’)); 25.45 (t,
C(1’)). HR-MALDI-MS: 615.1972 (44, [M þ Na]þ, C32H28N6NaO6þ ; calc. 615.1968), 593.2143 (100, [M þ
H]þ, C32H29N6Oþ6 ; calc. 593.2149), 549.2227 (14, [M ꢀ CO2 þ H]þ, C31H29N6O4þ ; calc. 549.2250), 485.1569
(30, [M ꢀ BnO]þ, C25H21N6Oþ5 ; calc. 485.1573).
N-[2-Amino-6-(benzyloxy)-5-nitrosopyrimidin-4-yl]-3-(phthalimido)propanamide (63). A suspen-
sion of 37 (17.2 g, 70.2 mmol) in CH2Cl2 (700 ml) was cooled to 08, treated with 69 (19.6 g, 77.2 mmol),
stirred for 5 min, warmed to r.t., stirred for another 2 h, and filtered. The filtrate was washed with 0.1m
NaOH (5ꢃ), dried (MgSO4), filtered, and evaporated. The combined solids of the first filtration and of
the evaporation were dried to afford 63 (29.3 g, 94%). Green powder. Rf (CH2Cl2/MeOH 19 :1) 0.44.
M.p. 170.0 – 175.18. UV (MeOH): 348 (13060), 242 (12760). IR (ATR): 3326w, 3241w, 1771w, 1706s,
1643m, 1593s, 1544s, 1468s, 1449m, 1394s, 1344s, 1309m, 1267m, 1206s, 1190s, 1170s, 1152s, 1111m, 1073m,
1003m, 985w, 972w, 912w, 884w, 870m, 847w, 793m, 774w, 745w, 714s, 699m, 672w, 639w. 1H-NMR
(300 MHz, (D6)DMSO); assignments based on a HSQC and a HMBC spectrum): 12.19 (s, NH); 8.74,
8.65 (2s, NH2); 7.88 – 7.80 (m, 4 arom. H); 7.56 – 7.36 (m, 5 arom. H); 5.61 (s, PhCH2); 3.89 (t, J ¼ 6.9,
2 HꢀC(3)); 3.24 (t, J ¼ 6.9, 2 HꢀC(2)). 13C-NMR (75 MHz, (D6)DMSO); assignments based on a
HSQC and a HMBC spectrum): 172.76 (s, C(1)); 167.38 (2s, 2 C¼O); 163.13 (s, C(6’)); 138.40 (s, C(4’));