Syntheses of inhibitors for the enzyme thymidine 5'-diphosphate-glucose-4,6-dehydratase (EC 4.2.1.46)

Article abstract of DOI:10.1016/S0008-6215(99)00074-9

Inhibitors of the enzyme thymidine 5'-diphosphate-glucose-4,6-dehydratase (EC 4.2.1.46) were synthesised starting from thymidine, 2-deoxy-uridine and -cytidine. The diphosphate moieties have either been replaced by methylene diphosphonate or phosphoryl hy

Full text of DOI:10.1016/S0008-6215(99)00074-9

www.elsevier.nl/locate/carres
Carbohydrate Research 318 (1999) 38–51
Syntheses of inhibitors for the enzyme thymidine
5%-diphosphate-glucose-4,6-dehydratase (EC 4.2.1.46)
Andreas Naundorf a, Werner Klaffke b,*
a Uni6ersita¨t Hamburg, Institut fu¨r Organische Chemie, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany
b Unile6er Research, Oli6ier 6an Noortlaan 120, NL-3133AT Vlaardingen, The Netherlands
Received 21 December 1998; accepted 15 March 1999
Abstract
Inhibitors of the enzyme thymidine 5%-diphosphate-glucose-4,6-dehydratase (EC 4.2.1.46) were synthesised starting
from thymidine, 2-deoxy-uridine and -cytidine. The diphosphate moieties have either been replaced by methylene
diphosphonate or phosphoryl hydroxyacetic acid to yield inhibitors in a similar range to thymidine 5%-diphosphate
itself. Spacers were attached to various positions, of which those at C-5 of the nucleobase were suitable analogues
and showed a mixed type of inhibition characteristics, whereas attachment at other sites led to marginally active or
inactive inhibitors. These investigations will prepare the ground for developing a purification procedure based on
affinity chromatography. © 1999 Elsevier Science Ltd. All rights reserved.
Keywords: dTDP-Glucose-4,6-dehydratase (EC 4.2.1.46); Nucleoside diphosphate; Diphosphate analogues; Spacer-modified
nucleobases
genated glycosyl donors starting from rela-
1. Introduction
tively easily accessible thymidine 5%-diphos-
phate-(TDP) glucose derivatives. Preparative
syntheses of various intermediates along such
enzymatic routes have been previously re-
ported by us and others [2–6].
The enzymatic synthesis of activated deoxy
sugars is still underdeveloped if compared
with the vast number of reports on mam-
malian oligosaccharides. One of the obstacles
is the low availability of glycosyl donors, i.e.,
the nucleoside diphosphosugars and the re-
spective enzymes to produce them. In the past
few years cloning techniques have made those
gene clusters available, which encode for the
biosynthesis of bacterial deoxysaccharide
structures [1,2]. We embarked on a pro-
gramme to develop the synthesis of the nu-
cleoside-diphosphate activated and deoxy-
Early reports on the inhibition of TDP-glu-
cose-4,6-dehydratase by TDP have prompted
us to forward the idea of developing ana-
logues of the latter suitable for affinity chro-
matography. This paper, therefore, will report
on the synthesis of such analogues and their
suitability to interfere with the substrate,
TDP-glucose, in the dehydratase reaction.
2. Discussion
* Corresponding author. Present address: Organisch-
Chemisches Institut der Westfa¨lischen Wilhelms-Universita¨t,
Corrensstraße 40, D-48149 Mu¨nster, Germany.
E-mail address: werner.klaffke@uni-muenster.de (W.
Klaffke)
As outlined in Fig. 1, five distinct positions
can be envisaged for the attachment of a
spacer by which the nucleoside can be linked
0008-6215/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00074-9

A. Naundorf, W. Klaffke / Carbohydrate Research 318 (1999) 38–51
39
triester 4 were found to be prone to hydroly-
sis, all respective chromatographic steps had
to be carried out using anhydrous solvents
and dried silica gel. Controlled hydrolysis to
give the target compound, phosphoglycolate
5, was achieved by automatic addition of
aqueous LiOH at pH 12.5 and the cleavage
was carefully checked for completion by TLC.
After having developed the chemistry for
the diphosphate mimics, the attachment of
spacers having a chain length of 6–8 carbon
atoms to the different positions of the nucle-
otide (Fig. 1) was next on the agenda. Cou-
pling of Z-protected amino hexanoic acid to
5%-O-TBDMS protected thymidine was carried
out in analogy to the procedure described by
Safarti and co-workers [7] for 3%-O-(6-amino-
hexanoyl)-dTTP. Compound 7 was phospho-
rylated after O-desilylation and subjected to
pyrophosphorylation according to the proce-
dure reported by Davisson et al. [8] to give the
target compound, TDP-3%-hexanoate 8 in 53%
yield, which could be unequivocally character-
onto a solid support. The programme towards
the development of suitable affinity materials
therefore consists of three goals: (i) the re-
placement of the diphosphate by a more stable
isosteric group, (ii) the attachment of bifunc-
tional spacers, and (iii) the measurement of
kinetic parameters of the synthesised N
diphosphate analogues in the dehydratase
reaction.
Substitution of the bridging oxygen atom in
the pyrophosphate by a methylene group
yielded a more stable phosphonate (2). This
was obtained from tosylate 1 by direct nucle-
ophilic replacement with methylene diphos-
phonate according literature procedures [8,21].
The diphosphate mimic, compound 5, has
been addressed based on the notion that in
TDP-glucose only two negative charges are
presented to the enzyme’s active site. For TDP
as an inhibitor, it is reasonable to assume that
a third negative charge will only have minor
impact in the binding. For the synthesis of 5,
3%-O-benzoyl-thymidine was converted to give
phosphoamidite 2. By acid catalysis, the amid-
ite was substituted by methyl glycolate and
subsequently oxidised to yield the correspond-
ing triester 4. Since both phoshoamidite 2 and
1
ised by H and 13C NMR spectroscopy, and
FABMS.
It was conceivable that compound 8 or its
immobilised counterpart would be sensitive to
Fig. 1. Nucleotide positions suitable for spacer attachment.

40
A. Naundorf, W. Klaffke / Carbohydrate Research 318 (1999) 38–51
Scheme 1.
saponification due to any residual esterase
activity in the protein extracts. A preliminary
testing with commercially available lipases
(PPL, CCL) indeed confirmed this assump-
tion. Derivative 8 decomposed within 24 h in
a 50 mM phosphate buffer (pH 7.0) at 37 °C
and therefore showed no advantage over the
previously tested TDP-hexanolamine 6 [22]
carrying a pyrophosphate linker sensitive to
hydrolysis (Scheme 1).
For the stable linkage (type ‘A’, see Fig. 1),
5-iodouridine was subjected to a Heck reac-
tion. This was carried out in anhydrous tri-
ethylamine with 1-trifluoroacetamido-oct-7-in
and 11 in the presence of tris(triphenylphos-
phine) palladium(II) chloride/copper(I) iodide
to yield 12 in 85% yield. Upon pyrophospho-
rylation and de-esterification, the 5%-diphos-
phate 18 could be isolated in 51% yield.
Compound 12 also served as a starting
point for the synthesis of diphosphate ana-
logue 16. Phosphoamidation of 12 was fol-
lowed by substitution with methyl glycolate
and oxidation to furnish triester 15. This, in
turn, was treated similarly to the non-spacer
modified compound 4. Simultaneous cleavage
of all three blocking groups, 3-benzoate, cya-
noethyl ester, and methyl ester, delivered the
deblocked 5-octinyl spacered glycolate mimic
16 in 91% yield with a dU content of 72%
(Scheme 2). The structure was fully assigned
by NMR spectroscopy, with a single 31P
NMR signal at +1.23 ppm decisive for
phophodiesters. The intactness of the ester
was underlined by a [M−Li] ESPMS peak at
m/z 488.1.
Trifluoroacetamido-1-iodo-hexane
[9,10]
proved very suitable for the envisaged
derivatisation at N3. N-Alkylation of 3%-O-
benzoylthymidine in N,N-dimethylformamide,
under carefully optimised Purdie conditions to
avoid benzoyl migration, gave rise to the
spacer-bridged derivative 17. The site of alky-
lation was indicated by 13C NMR: whereas a
derivatisation at positions different from N3
would be indicated by a shift at C-5, the

A. Naundorf, W. Klaffke / Carbohydrate Research 318 (1999) 38–51
41
carbon signals for the nucleobase in 17 re-
mained indistinguishable from literature data
[11,12]. However, a value of 40.1 ppm for C-1
of the hexyl spacer confirmed the expected
N-alkylation. Diphosphate 18 was obtained
after treatment with the tributylammonium
pyrophosphate and ester cleavage under con-
trolled conditions in aqueous LiOH at pH 12
in 72% yield (Scheme 3).
The last derivative that has been the focus
of our interest was the attachment of a linker
at position ‘B’ (cf. Fig. 1). As a starting point
for the synthesis, 2%-deoxycytidine was chosen.
Silylation gave the 5%-O-TBDMS ether, which
was impossible to purify, but was instead re-
acted further to yield the bis(silyl) ether ac-
cording to the ‘transient protection method’
[13]. The crude compound was, after
Mukaiyama activation of the acid and follow-
ing aqueous work-up, isolated as amide 19.
Benzoylation proceeded smoothly, but purifi-
cation on silica gel was problematic in that it
resulted in ester cleavage. It was conceived
that the observed unexpected side reactions
for acyl groups at 3%-OH are based on a
conformational change in the furanoid ring,
which is exerted by the base on the sugar [14].
Compared to dU and dT derivatives, the dC-
ribose showed a different coupling pattern for
H-1% and H-3%. The O-desilylated derivative,
however, was again stable, and benzoate 20
could be purified after previous cleavage of
the 5-O-silyl ether. Conversion of the alcohol
to give diphosphate 21 went along the stan-
dard pyrophosphorylation procedure subse-
quently followed by hydrazinolysis of the
3-benzoate [15] (Scheme 4).
Synthesised TDP analogues were evaluated
in a 50% inhibition test at constant enzyme
concentration and under substrate saturation
(approx 3–4×KM). The respective concentra-
tions of TDP analogues were varied between
100 and 2000 mM, and from a plot of DE
versus [I] the 50% inhibition value was esti-
mated. From this first set of experiments it
became evident that compounds 8, 18, and 21
were not accepted at all by the enzyme and
therefore the substitution at neither 3%-OH of
the ribose nor the attachment of any sub-
stituent at a position of the nucleobase other
Scheme 2.

42
A. Naundorf, W. Klaffke / Carbohydrate Research 318 (1999) 38–51
Scheme 3.
than C-5 would yield effective inhibitors (2, 5,
6, 13, 16).
and
app
max
V
=Vmax/(1+[I]/Ki%)
However, all other derivatives were sub-
jected to further evaluation following the stan-
dard assaying protocol described earlier [16].
Apart from pyrophosphate, which was a clear
uncompetitive inhibitor, all other derivatives
showed a mixed type of inhibition. From ob-
served apparent Vmax and KM values, Ki and
Ki% are defined by the following two
equations:
After rearrangement, the values for Ki and Ki%
were defined as
Ki=[I]/{(V /KMapp)/(Vmax/KM)−1}
app
max
and
app
max
Ki%=[I]/(V /Vmax)−1
where Ki%“ꢀ for competitive inhibition,
Ki“ꢀ for uncompetitive inhibition, and Ki =
Ki% for non-competitive inhibition.
app
(V /KMapp)=(Vmax/KM)/(1+[I]/Ki)
max
Fig. 2. Competitive (Ki) and uncompetitive (K%) contributions to the mixed inhibition observed for compounds 2, 5, 6, 13, and 16
i
(assayed according to Ref. [16]).

A. Naundorf, W. Klaffke / Carbohydrate Research 318 (1999) 38–51
43
Scheme 4.
plex compared with the enzyme–substrate
complex.
Fig. 2 depicts all resulting data for Ki and Ki%;
bargraphs in grey represent the uncompetitive
and those in black the competitive contribu-
tions to the mixed inhibition. Values tending to
ꢀ indicate that the dissociation of the enzyme–
inhibitor complex prevails and therefore the
association is not within the measurable data
range.
Conclusively, it turned out that derivatisation
at C-5 of the nucleobase was yielding good
analogues with values in the same order of
magnitude as that of TDP. Furthermore, an
interesting avenue has been pursued with the
2-phosphoacetic acid derivatives, which showed
even better inhibition properties than the parent
compound. On the basis of these results, exper-
iments are underway to develop an affinity
chromatography protocol for the facile purifi-
cation of the title enzyme.
As explained above, the tested derivatives
were not displaying a simple inhibition pattern,
but instead they were showing a complex pat-
tern of mixed inhibition. This can be taken as
evidence for a sequential, ordered mechanism
[17]. Given this case, Ki represents the compe-
tition between substrate and inhibitor for the
enzyme in the same state, whereas Ki% represents
the competition of substrate and inhibitor for
the enzyme in different states. As a consequence
of the postulated ordered mechanism, the en-
zyme is yielded in different activation levels
after recombination of the apo-enzyme with
NAD+ or NADH [18]. A final proof, however,
would be the observation of an uncompetitive
substrate inhibition, not yet described for the
enzyme under investigation. The experiments
did underpin such behaviour, since the reaction
rate was leveling off after the optimum sub-
strate concentration of 600 mM was exceeded.
Some inhibitors showed at low substrate
concentrations between 25 and 100 mM an
overestimated reaction rate, which was in-
versely proportional to the duration of preincu-
bation of enzyme and inhibitor (Table 1). This
was taken as a clear indication for a slow
formation of the enzyme–inhibitor com-
3. Experimental
General procedures
5%-Silylation of nucleosides. tert-Butyldi-
methylchlorosilane (760 mg, 5 mmol, 1.0
equiv) or tert-butyldiphenylchlorosilane (1.30
mL, 5 mmol, 1.0 equiv) and imidazole (680
mg, 10 mmol, 2 equiv) were dissolved in an-
hyd DMF (20 mL), and to the stirred solution
the nucleoside (5 mmol) was added. After 3 h
at room temperature (rt), the solvent was
evaporated in vacuo. Derivatives of thymidine
and uridine were isolated after column chro-
matography, nucleosides furnished with an ex-
ocyclic amino group were further protected
according to the described transient protection
method [13].
Benzoylation. The alcohol (5 mmol) was
dissolved in anhyd pyridine (10 mL), and
stirred with benzoylchloride (0.7 mL, 6 mmol,
1.2 equiv) at rt. Upon completion of the reac-

44
A. Naundorf, W. Klaffke / Carbohydrate Research 318 (1999) 38–51
N-Detrifluoroacetylation and 3%-O-debenz-
oylation were achieved quantitatively by sa-
ponification with aq LiOH at pH 12 in 2 h.
The resulting solutions were directly fraction-
ated over Sephadex G10.
tion, controlled by TLC to prevent aminoacy-
lation of thymidine and uridine derivatives,
the reaction was stopped by evaporation of
the solvent in vacuo, the mixture was diluted
with CH2Cl2 (200 mL), and washed three
times with a satd aq soln of NaHCO3. The
organic layer was dried over MgSO4, filtered
and concentrated to leave a raw product,
which was purified over SiO2 using the given
eluents.
Desilylation. The silylated compound (5
mmol) was dissolved in anhyd THF (10 mL)
to which TBAF·3H2O (1.74 g, 5.5 mmol, 1.1
equiv) was added. Depending on the amount
of water, reaction times ranged from 1 to 5 h.
The solution was evaporated and purified on
SiO2 using the specified eluents.
5%-Tosylation of nucleosides. The 5%-un-
blocked nucleoside (1 mmol) was dissolved in
CH2Cl2 (20 mL) together with DMAP (160
mg, 1.3 mmol, 1.3 equiv), and p-toluenesul-
fonyl chloride (230 mg, 1.3 mmol, 1.2 equiv)
and stirred for 15 h at rt, before passed over a
short SiO2 column (20:1 CH2Cl2–MeOH).
The product fractions were pooled and con-
centrated, the product was precipitated from
CH2Cl2 with n-hexane.
Nucleoside diphosphates. Tetrasodium py-
rophosphate (5.0 g) was dissolved in warm
distilled water (50 mL), and applied to an
ion-exchanger column Dowex 50W (pyri-
dinium-form, 4×10 cm). The eluate was
lyophilised and applied to a second ion-ex-
change step (Dowex 50W, tributylammonium
form, equilibrated with MeOH, 2×8 cm).
The eluate was concentrated in vacuo and
dried over P2O5.
The N4-acylated deoxycytidine derivative 20
was dissolved in anhyd DMF (20 mL) to
which hydrazine hydrate (150 mL, 3 mmol, 3
equiv) was added. After 15 h, the solvent was
evaporated (TB30 °C) and the residue was
subjected to Sephadex G10 chromatography.
Phosphates were purified by chromatogra-
phy over Dowex 2×8 (Cl−-form) and eluted
by a linear gradient 0“0.8 M LiCl. Product-
containing fractions are pooled, lyophilised
and desalted by passing twice over Sephadex
G10. In cases where the counterion has not
been exchanged completely, the TDP deriva-
tives were passed additionally over Dowex
50W (Li+-form).
Estimation of 50% inhibition. According to
Ref. [18], TDP-4-ketoglucose is measured
photometrically at constant enzyme concen-
tration under substrate saturation (3–4×KM).
The respective TDP analogue was added in
concentrations between 100 and 2000 mM.
Buffer: Tris–HCl (50 mM, pH 7.6), EDTA (2
mM). Substrate: TDP-glucose (Sigma, 14.6
mg) dissolved in bidistilled water (1 mL), re-
sulting in an assay concentration of 0.4 mM,
equivalent to 4KM. Enzyme: 4,6-dehydratase
(3–5 mU). Inhibitor: the respective TDP ana-
logue (15 mg) was dissolved in bidistilled wa-
ter (1 mL) and the thymidine content was
estimated photometrically by comparison with
a TMP standard solution.
The pipetting scheme is shown in Table 2.
The concentration at which the enzyme ac-
tivity had dropped to 50% was read directly
from a DE versus [I] plot.
Inhibition constants. At given enzyme and
inhibitor concentrations, the turnover to give
TDP-6-deoxy-4-keto-glucose was estimated
for substrate concentrations between 25 and
The 5%-unblocked nucleoside (1 mmol), pre-
viously prepared and dried pyrophosphate
(3.0 g, approx 3 equiv), and N,N%-dicyclo-
hexylcarbodiimide (2.06 g, 10 mmol, 10 equiv)
were dissolved in anhyd pyridine (20 mL) and
stirred for 5 days at rt. After evaporation of
the solvent, the residue was taken up at 0 °C
in aq LiOH (0.1 N, 10 mL) and the suspen-
sion was quickly adjusted to pH 8, before
filtered over a Bu¨chner funnel. The precipitate
was washed with 1:1 water–MeOH, and the
filtrate was concentrated and finally
lyophilised.
Table 1
Reaction rates of inhibitors as a function of preincubation
time
Preincubation (min)
blank
0
5
15
mmol product/min mL 0.003 0.0018 0.0014 0.0008

A. Naundorf, W. Klaffke / Carbohydrate Research 318 (1999) 38–51
45
Table 2
Pipetting scheme for estimating 50% inhibition
Table 3
Pipetting scheme for determining inhibition constants
Step
Assay soln (mL)
Blind soln
Step
Assay soln (mL)
Sample aliquot
Tris–buffer
Inhibitor
480−X
X
480−X
X
Tris–buffer
Inhibitor
1990–I–S
I
TDP-Glc soln
10
10
TDP-Glc soln
S
Incubate for 1 min at 37 °C
NaOH (0.2 N)
Incubate for 1 min at 37 °C
Enzyme soln 10
Mix, take sample for control and incubate at 37 °C
500
10
Enzyme soln
Incubate for 15 min at 37 °C
NaOH (0.2 N) 500
10
Take aliquot
200
800
NaOH (0.1 N)
Incubate for 15 min at 37 °C; measure extinction against
blind control
Incubate for 15 min at 37 °C; measure extinction against
blind control at 318 nm
1000 mM (approx 0.5–10×KM). Buffer: Tris–
HCl (50 mM, pH 7.6), EDTA (2 mM). Sub-
strate: TDP-glucose (Sigma, 14.6 mg)
dissolved in bidistilled water (1 mL); per assay
(2 mL) were used 100 mL (1000 mM), 40 mL
(400 mM), 20 mL (200 mM), 10 mL (100 mM), 5
mL (50 mM), 2.5 mL (25 mM). Enzyme: 4,6-de-
hydratase (10–15 mU each, similar batches).
Inhibitor: the respective TDP analogue (15
mg) was dissolved in bidistilled water (1 mL)
and the thymidin content was estimated pho-
tometrically at 267 nm (or 270 and 290 nm,
respectively) by comparison with a TMP
standard solution. Inhibitor concentrations
were: pyrophosphate (2000 mM), TDP (250
mM), 3 (1000 mM), 5 (100 mM), 6 (500 mM), 13
(1000 mM), 16 (500 mM).
residue dried over P2O5 in vacuo. 3%-O-Ben-
zoyl-5%-O-p-toluenesulfonyl-thymidine (1, 500
mg, 0.5 mmol) [20] and the previously pre-
pared phosphonate (1.50 g, approx 1.5 equiv)
were suspended in anhyd DMF (5 mL), and
stirred for 36 h at 80 °C under an Ar atmo-
sphere. After concentration in vacuo, the
residue was dissolved in ice-cold aq LiOH (0.5
N, 10 mL), and further LiOH was added at
pH 12 to saponify the benzoate completely.
The resulting solution was directly fraction-
ated on a Sephadex G10 column, the product-
containing fractions were further purified by
anion-exchange chromatography, as described
for nucleoside diphosphates under the general
procedures (vide supra). After final lyophilisa-
tion, 2 (460 mg, 57%, photometrically deter-
mined dT content: 52%) was obtained as its
The pipetting scheme is shown in Table 3.
Control: subsequently upon addition of en-
zyme solution, the control is quenched by
addition of NaOH.
1
dilithium salt. H NMR and Rf were in agree-
ment with the specifications given in Ref. [21].
13C NMR (100 MHz, D2O): l 162.7 (C-4),
154.2 (C-2), 139.9 (C-6), 114.2 (C-5), 87.8 (d,
C-4%), 87.4 (C-1%), 73.4 (C-3%), 66.2 (d, C-5%),
40.8 (C-2%), 31.3 (t, P–CH2–P), 14.1 (CH3);
JP,C,P 119.0, J4%,P 8.6, J5%,P 5.7 Hz. FABMS−:
m/z 399.2 [M3− +2H+]−.
3%-O-Benzoyl-thymidine 5%-[(2-cyanoethyl)-
N,N-diisopropyl] phosphoramidite (3).—Com-
pound 1 (1.18 g, 3.25 mmol) was dissolved in
anhyd MeCN (20 mL); under an Ar atmo-
sphere N,N-diisopropylethylamine (1.08 mL,
6.34 mmol, 1.95 equiv) and 2-cyanoethoxy-
N,N-diisopropylchlorophosphoamidite (1.0 g,
4.22 mmol, 1.3 equiv) were added. After stir-
ring for 30 min, the solvent was removed
under reduced pressure and the resulting
Molar extinction for enolised ulose m318
=
4800 cm2/mmol. Product concentration in as-
say solution (200 mL aliquot) was
c
prod =(DE×1.042) mmol/mL. From a [prod]
versus t plot, initial rates were determined and
the respective constants were estimated by hy-
perbolic regression analysis according to the
Marquart algorithm [19].
Thymidine 5%-dilithiummethylenediphospho-
nate (2).—Methylene diphosphonic acid (1.0
g, 5.6 mmol) was dissolved in 1:1 pyridine–
water (3 mL), and passed over a Dowex 50W
ion-exchange column (tributylammonium
form, 2×8 cm, previously equilibrated over
MeOH). The filtrate was concentrated, the

46
A. Naundorf, W. Klaffke / Carbohydrate Research 318 (1999) 38–51
130.2 (Bz), 130.0 (2 C, o-Bz), 129.0 (2 C,
m-Bz), 117.6 (CN), 111.0 (C-5), 85.1 (C-1%),
82.9 (d, C-4%), 75.3 (C-3%), 68.0 (d, C-5%), 64.0
(d, H2CC(O)OCH3), 63.3 (d, POCH2), 52.1
(C(O)OCH3), 37.0 (C-2%), 19.5 (d, CH2CN),
residue was purified over previously dried
SiO2 (3:1 anhyd toluene–anhyd acetone). The
diastereomeric phosphites 3 were obtained in
1
equal amounts (1.43 g, 81%); H NMR (400
MHz, acetone-d6): l 8.09 (d, 2 H, o-Bz), 7.79
(s, H-6), 7.67 (m, p-Bz), 7.55 (m, 2 H, m-Bz),
6.48 and 6.41 (dd, each 1/2 H, H-1%), 5.65 and
5.62 (m, each 1/2 H, H-3%), 4.43 and 4.41 (m,
each 1/2 H, H-4%), 4.13–3.90 (m, 4 H, H-5a%,
5b%, P(O)CH2), 3.77–3.68 (m, 2 H, 2 iso-
propyl-CH), 2.83–2.79 (m, 2 H, CH2CN),
2.63–2.43 (m, 2 H, H-2a%, 2b%), 1.91 and 1.90
(s, each 3/2 H, CH3), 1.27–1.22 (m, 12 H, 4
isopropyl-CH3); 13C NMR (100 MHz, ace-
tone-d6): l 166.0 (CꢀO), 163.7 (C-4), 150.8
(C-2), 135.8/135.6 (C-6), 13.8 (p-Bz), 130.3
(Bz), 129.9 (2 C, o-Bz), 129.0 (2 C, m-Bz),
118.4 (CN), 110.8 and 110.6 (each C-5), 85.2
and 84.9 (C-1%), 84.4 and 84.3 (each d, each
C-4%), 76.4 and 76.1 (each C-3%), 64.6 and 64.4
(each d, each C-5%), 59.7 and 59.5 (each d,
each POCH2), 43.5 (d, PNCH), 37.8 and 37.5
(each C-2%), 24.5 and 24.4 (2 s, NCH(CH3)2),
20.3 (CH2CN), 12.2 (CH3); J4%,P 9.6, J5%,P 4.3,
2
12.0 (CH3); J4%,P 7.6, J5%,P 5.6, JCH COOMe,P 5.4,
2
2
3
31
3JCOOMe, P 4.8, JPOCH 4.8, JCH CN,P 8.2 Hz; P
2
NMR (81 MHz, acetone-d6): l +0.05, +
0.01. Anal. Calcd for C23H26N3O11P (515.46):
C, 50.10; H, 4.75; N, 7.62; P, 5.62. Found C,
49.72; H, 4.84; N, 7.89; P, 5.51.
Thymidine 5%-monolithium carboxymethyl-
phosphate (5).—Compound 4 (0.95 g, 1.72
mmol) [dissolved in 3:1 EtOH–water (10 mL)
and aq LiOH (0.5 N)] was constantly added at
rt by means of an automatic titrator (pH
12.5). The reaction was controlled by TLC
(5:3:2 2-propanol–EtOH–water, 5% triethy-
lamine, 2% HOAc). The resulting solution was
directly fractionated by Sephadex G10 chro-
matography; product-containing fractions
were additionally desalted over Sephadex G10
or Biogel P2 to yield 5 (870 mg, 93%, photo-
1
metrically determined dT content: 72%); H
3
2
2JPOCH 20.9, JCHCN,P 6.0, JPNCH 12.4 Hz.
3%-O-Benzoyl-thymidine 5%-(2-cyanoethyl)-
methoxycarbonylmethyl phosphate (4).—Com-
pound 3 (1.32 g, 2.41 mmol) and methyl
glycolate (600 mL, 7.8 mmol, 3 equiv) were
dissolved in anhyd MeCN (20 mL) under an
Ar atmosphere. After the addition of 1-H
tetrazole (340 mg, 4.83 mmol, 2 equiv) the
mixture was stirred for 30 min, before tert-
butylyhydroperoxide (70% in water, 850 mL, 3
equiv) was added. After a further 60 min, the
solution was concentrated under reduced pres-
sure (TB30 °C) and the residue was purified
by chromatography over previously dried SiO2
(1:1 anhyd toluene–acetone) to yield 4 (1.06 g,
NMR (400 MHz, D2O): l 7.90 (s, H-6), 6.52
(dd, H-1%), 4.74 (m, H-3%), 4.42 (d, 2 H,
CH2COOH), 4.34 (m, H-4%), 4.28 (m, 2 H,
H-5a%, 5b%), 2.56–2.52 (m, 2 H, H-2a%, 2b%),
2.10 (s, 3 H, CH3); 13C NMR (100 MHz,
D2O): l 176.5 (d, COOH), 167.0 (C-4), 152.2
(C-2), 137.7 (C-6), 112.2 (C-5), 85.8 (d, C-4%),
85.4 (C-1%), 71.4 (C-3%), 65.5 (d, C-5%), 64.2 (d,
OCH2COOH), 39.1 (C-2%), 12.0 (CH3);
2
3JCOOH,P 7.7, JPOCH C(O) 5.7, J4%,P 9.2, J5%,P 5.2
2
Hz. ESIMS− (4.5 kV): m/z 379.2 [M2−
+
H+]−.
Thymidine 5%-dilithium-(6-N-trifluoroaceta-
midohexyl) diphosphate (6).—6-Trifluoroac-
etamido-hexyl-1-phosphate [22] (1.0 g, approx
1.5 mmol, approx 2 equiv) was co-evaporated
with anhyd pyridine to remove traces of wa-
ter, then dissolved in anhyd MeCN (10 mL)
and stirred for 2 days at rt with thymidine-5%-
1
80%); m.p. 60 °C; H NMR (400 MHz, ace-
tone-d6, diastereoisomers are indistinguish-
able): l 8.09 (d, 2 H, o-Bz), 7.70–7.65 (m, 3
H, H-6, p-Bz), 7.54 (m, 2 H, m-Bz), 6.46 (dd,
H-1%), 5.64 (m, H-3%), 4.75 (dd, 2 H, CH2-gly-
colate), 4.54 (m, 2 H, H-5a%, 5b%), 4.49 (m,
H-4%), 4.46–4.39 (m, 2 H, POCH2), 3.75 (d, 3
H, H3COC(O)), 3.01–2.96 (m, 2 H, CH2CN),
2.66–2.53 (m, 2 H, H-2a%, 2b%), 1.88 (s, 3 H,
CH3); 13C NMR (100 MHz, acetone-d6): l
168.8 (d, C(O)OMe), 165.9 (CꢀO), 163.6 (C-
4), 150.8 (C-2), 135.8 (C-6), 133.8 (p-Bz),
monophosphomorpholidate
(commercially
available dicyclohexylurea salt, 500 mg, 0.73
mmol). The solvent was distilled off under
reduced pressure, the residue was then dis-
solved in aq LiOH (0.1 N, 10 mL) at 0 °C and
quickly adjusted with further LiOH to pH 8.0.
The phosphates were separated by anion-ex-
change
chromatography
(Dowex
2×

A. Naundorf, W. Klaffke / Carbohydrate Research 318 (1999) 38–51
47
8, Cl−-form) and eluted by a linear LiCl-gra-
dient (0“0.8 M). Product-containing frac-
tions were pooled, lyophilised and desalted by
passing twice over Sephadex G10. Product 6
(360 mg, 55%, photometrically determined dT
content: 68%) was obtained after final
H-5a%), 3.97 (dd, H-5b%), 2.65 (ddd, H-2a%),
2.21 (H-2b%), 0.95 (s, 9 H, SiC(CH3)3), 0.20
and 0.19 (each s, 6 H, Si(CH3)2); 13C NMR
(100 MHz, CDCl3): l 166.2 (CꢀO), 159.8 (C-
4), 149.9 (C-2), 144.1 (C-6), 133.6, 129.7, 129.2
and 128.6 (Bz), 86.2 (C-4%), 85.7 (C-1%), 76.2
(C-3%), 68.9 (C-5), 63.7 (C-5%), 38.8 (C-2%), 26.2
((H3C)3)CSi), 18.5 ((H3C)3)CSi), −5.0 and
1
lyophilisation; H NMR (400 MHz, D2O): l
7.94 (s, H-6), 6.52 (dd, H-1%), 4.78 (m, H-3%),
4.37–4.32 (m, 3 H, H-4%, H-5a%, 5b%), 4.08 (dt,
H-1%%), 3.46 (t, H-6%%), 2.56–2.50 (m, 2 H,
H-2a%, 2b%), 2.10 (s, 3 H, CH3), 1.77 (m, 2 H,
H-2%%), 1.71 (m, 2 H, H-5%%), 1.53–1.46 (m, 4 H,
H-3%%, H-4%%); 13C NMR (100 MHz, D2O): l
166.9 (C-4), 151.1 (C-2), 137.9 (C-6), 112.2
(C-5), 85.9 (d, C-4%), 85.3 (C-1%), 71.3 (C-3%),
67.0 (d, C-1%%), 65.7 (d, C-5%), 40.2 (C-6%%), 39.1
(C-2%), 30.1 (d, C-2%%), 28.0 (C-5%%), 26.0 (C-4%%),
24.9 (C-3%%), 12.1 (C-6%%); J1%%,P 6.1, J2%%,P 7.1, J5%,P
6.1, J4%,P 10.2 Hz. ESPMS− (4.5 kV): m/z
596.0 [M2− +H+]−.
−5.2
((H3C)2Si).
Anal.
Calcd
for
C22H29IN2O6Si (572.48): C, 46.16; H, 5.11; I,
22.17; N, 4.89. Found C, 46.03; H, 5.18; I,
21.99; N, 4.88.
3%-O-Benzoyl-2%-deoxy-5-iodouridine (11).—
Compound 10 (720 mg, 1.26 mmol) was desi-
lylated according to the described procedure
(vide supra) and purified by SiO2 chromatog-
raphy (1:1 toluene–EtOAc) to give 11 (540
mg, 94%); m.p. 195 °C, Ref. [24] 183–185 °C;
1H NMR data were in accordance with Ref.
[24]; 13C NMR (100 MHz, acetone-d6): l 165.9
(CO–Bz), 160.1 (C-4), 150.5 (C-2), 145.7 (C-
6), 133.7 (p-Bz), 130.3 (q-Bz), 129.9 (2 C,
o-Bz), 129.0 (2 C, m-Bz), 86.1 (C-4%), 85.9
(C-3%), 68.4 (C-5), 62.4 (C-5%), 38.4 (C-2%).
3%-O-Benzoyl-2%-deoxy-5-(8-N-trifluoroacet-
3%-O-(6%%-N-Benzyloxycarbonylamino)-hexa-
noyl)-thymidine 5%-trilithiumdiphosphate (8).—
Compound 7 [7] (200 mg, 0.40 mmol) was
pyrophosphorylated according to the general
procedures to yield 8 (250 mg, 53%, photo-
1
metrically determined dT content: 56%); H
amido-oct-1-ynyl)uridine
(12).—Previously
NMR (400 MHz, D2O): l 7.66 (s, H-6), 7.38–
7.29 (m, 5 H, Bn), 6.26 (dd, H-1%), 5.40 (m,
H-3%), 5.02 (s, 2 H, Bn–CH2), 4.34 (m, H-4%),
4.18 (m, 2 H, H-5a%, 5b%), 2.93 (m, H-6%%),
2.42–2.31 (m, 4 H, H-2a%, 2b%, H-2%%), 1.87 (s,
3 H, CH3), 1.47 (m, 2 H, H-3%%), 1.27 (m, 2 H,
H-5%%), 1.10 (m, 2 H, H-4%%); 13C NMR (100
MHz, D2O): l 176.4 (C(O)-1%%), 164.8 (C-4),
155.2 (NC(O)O), 152.1 (C-2), 137.5 (C-6),
136.8, 129.1, and 127.8 (Bn), 112.4 (C-5), 85.2
(C-1%), 83.5 (C-4%), 76.2 (C-3%), 67.1 (Bn–CH2),
66.5 (d, C-5%), 40.6 (C-6%%), 36.9 (C-2%), 34.1
(C-2%%), 28.8 (C-5%%), 26.6 (C-4%%), 24.3 (C-3%%),
12.1 (CH3); J4%,P 8.6, J5%,P 4.8 Hz. FABMS−:
m/z 647.3 [M3− +2H+]−.
prepared 5-iodouridine 11 (540 mg, 1.18
mmol), 1-trifluoroacetamido-oct-7-yn (340
mg, 1.53 mmol, 1.3 equiv), tris(triphenylphos-
phine)-palladium(II) chloride (21 mg, 0.03
mmol, 0.025 equiv), and anhyd copper(I)
iodide (23 mg, 0.12 mmol, 0.1 equiv, 4:1 Cu–
Pd) were dissolved in anhyd triethylamine (20
mL). The mixture was stirred under an Ar
atmosphere for 2 h at 50 °C, then concen-
trated under reduced pressure and co-distilled
with toluene. The residue was quenched with
satd aq NaHCO3 (10 mL), and extracted twice
with CH2Cl2 (50 mL each). The organic layer
was dried over MgSO4, concentrated and the
residue was purified by SiO2 chromatography
(1:1 petroleum ether–acetone). Product con-
taining fractions were pooled, concentrated
and crystallised from THF–n-hexane to yield
3%-O-Benzoyl-5%-O-tert-butyldimethylsilyl-2%-
deoxy-5-iodouridine (10).—5-Iodouridine 9
[23] (920 mg, 1.96 mmol) was benzoylated
according to the general procedure and
purified by SiO2 chromatography (2:1
toluene–EtOAc) to give 10 (880 mg, 78%);
1
12 (550 mg, 85%); m.p. 63 °C; H NMR (400
MHz, acetone-d6): l 10.22 (br s, NH), 8.41 (br
s NH), 8.25 (s, H-6), 8.09, 7.67, and 7.55 (d,
m, and m, 5 H, Bz), 6.42 (dd, H-1%), 5.64 (m,
H-3%), 4.55 (br s, HO-5%), 4.33 (m, H-4%), 3.99
(dd, H-5a%), 3.92 (dd, H-5b%), 3.35 (m, 2 H,
H-8%%), 2.65–2.47 (m, 2 H, H-2a%, 2b%), 2.38 (m,
1
m.p. 223 °C; H NMR (400 MHz, CDCl3): l
8.81 (br s, NH), 8.17 (s, H-6), 8.03, 7.58, 7.45
(d, m and m, 2 H, 1 H and 2 H, Bz), 6.39 (dd,
H-1%), 5.49 (m, H-3%), 4.29 (m, H-4%), 4.02 (dd,

48
A. Naundorf, W. Klaffke / Carbohydrate Research 318 (1999) 38–51
2 H, H-3%%), 1.63 (m, 2 H, H-7%%), 1.56 (m, 2 H,
H-4%%), 1.49 (m, 2 H, H-5%%), 1.41 (m, 2 H,
H-6%%); 13C NMR (100 MHz, acetone-d6): l
166.4 (CꢀO), 62.2 (C-4), 150.5 (C-2), 43.2
(C-6), 134.3, 130.9, 130.4, and 129.5 (Bz),
101.2 (C-5), 94.0 (C-2%%), 86.5 (C-4%), 86.3 (C-
1%), 76.6 (C-3%), 73.4 (C-1%%), 62.9 (C-5%), 40.4
(C-8%%), 38.8 (C-2%), 29.4 (C-7%%), 29.2 (C-4%%),
29.0 (C-5%%), 26.9 (C-6%%), 19.8 (C-3%%). Anal.
Calcd for C26H28F3N3O7 (551.52): C, 56.62; H,
5.12; F, 10.33; N, 7.62. Found C, 56.91; H,
5.12; F, 10.74; N, 7.63.
H-1%), 5.66 and 5.61 (m, H-3%), 4.48 and 4.45
(m, H-4%), 4.18-3.92 (m, 4 H, POCH2, H-5a%,
5b%), 3.78-3.69 (m, 2 H, N(CH(CH3)2)2), 3.35
(m, 2 H, H-8%%), 2.85–2.76 (m, 2 H, CH2CN),
2.65 (dd, H-2a%), 2.50 (ddd, H-2b%), 2.41 (m, 2
H, H-3%%), 1.65–1.56 (m, 4 H, H-7%%, H-4%%),
1.51 (m, 2 H, H-5%%), 1.42 (m, 2 H, H-6%%),
1.26–1.24 (m, 12 H, N(CH(CH3)2)2); 13C
NMR (100 MHz, acetone-d6): l 166.4
(C(O)C6H5), 162.6 (C-4), 151.3 (C-2), 142.2
(C-6), 134.2, 130.6, 130.4, and 129.4 (C6H5),
118.7 (CN), 101.2 (C-5), 94.4 (C-2%%), 85.6 and
85.5 (C-1%), 84.9 and 84.8 (each d, C-4%), 76.2
(C-3%), 73.4 (C-1%%), 64.3 and 64.0 (each d,
2%-Deoxy-5-(8-aminooct-1-ynyl)uridine 5%-
trilithiumdiphosphate (13).—According to the
general procedures, compound 12 was py-
rophosphorylated to yield 13 (290 mg, 51%,
photometrically specified dU content: 40%);
UV (200–400 nm): 217 (min), 232 (max), 259
C-5%),
59.7
(d,
POCH2),
43.9
(d,
PN(CH(CH3)2)2), 39.7 (C-8%%), 38.4 and 38.1
(C-2%), 28.9 (C-7%%), 28.8 (C-4%%), 28.6 (C-5%%),
26.4 (C-6%%), 25.0 and 24.9 (N(CH(CH3)2)2),
20.3 (d, CH2CN), 19.4 (C-3%%); J4%,P 8.4, J5%,P
1
(min), 291 (max); H NMR (400 MHz, D2O):
l 8.14 (s, H-6), 6.42 (dd, H-1%), 4.79 (m, H-3%),
4.39–4.28 (m, 3 H, H-4%, H-5a%, 5b%), 3.14 (m,
2 H, H-8%%), 2.60–2.45 (m, 4 H, H-3%%, H-2a%,
2b%), 1.86 (m, 2 H, H-7%%), 1.73 (m, 2 H, H-4%%),
2
3
2
5.0, JPOCH 20.2, JCHCN,P 7.9, JPNCH 12.2 Hz;
31P NMR (81 MHz, acetone-d6): l +149.74,
+149.59. Anal. Calcd for C35H45F3N5O8P
(751.75): C, 55.92; H, 6.03; F, 7.58; N, 9.32; P,
4.12. Found C, 55.78; H, 6.22; N, 9.11.
13
1.66 (m, 2 H, H-5%%), 1.58 (m, 2 H, H-6%%); C
NMR (100 MHz, D2O): l 160.5 (C-4), 156.7
(C-2), 143.8 (C-6), 103.3 (C-5), 97.3 (C-2%%),
85.9 (d, C-4%), 85.8 (C-1%), 74.5 (C-1%%), 70.8
(C-3%), 65.2 (C-5%), 40.2 (C-8%%), 39.8 (C-2%),
27.7 (C-5%%), 27.6 (C-4%%), 26.9 (C-7%%), 25.3 (C-
6%%), 18.5 (C-3%%); J4%,P 8.5, J5%,P 4.8 Hz.
FABMS−: m/z 510.3 [M3− +2H+]−.
3%-O-Benzoyl-2%-deoxy-5 -(8-trifluoroaceta-
midooct-1-ynyl)uridine
5%-(2-cyanoethoxy)-
methoxycarbonylmethyl phosphate (15).—
Compound 14 (420 mg, 0.56 mmol) and
methyl glycolate (150 mL, 1.95 mmol, \3
equiv) were dissolved in anhyd MeCN (20
mL) under an Ar atmosphere. After the addi-
tion of 1-H tetrazole (80 mg, 1.14 mmol, 2
equiv), the mixture was stirred for 30 min,
before tert-butylhydroperoxide (70% in water,
200 mL, \3 equiv) were added. After a fur-
ther 60 min, the solution was concentrated
under reduced pressure (TB30 °C) and the
residue was purified by chromatography over
previously dried SiO2 (2:1 anhyd toluene–ace-
tone) to yield 15 (350 mg, 83%); m.p. 143 °C;
1H NMR (400 MHz, acetone-d6, diastereoiso-
mers were indistinguishable): l 8.45 (br s,
NH), 8.09, 7.68, and 7.55 (C6H5), 7.96 (s,
H-6), 6.41 (dd, H-1%), 5.66 (m, H-3%), 4.78 (dd,
2 H, CH2-glycolate), 4.57 (m, 2 H, H-5a%, 5b%),
4.48–4.40 (m, 4 H, H-4%, POCH2), 3.77 (d, 3
H, H3COC(O)), 3.35 (m, 2 H, H-8%%), 3.03–
2.93 (m, 2 H, CH2CN), 2.71–2.54 (m, 2 H,
H-2a%, 2b%), 2.39 (m, 2 H, H-3%%), 1.66–1.54 (m,
4 H, H-7%%, H-4%%), 1.49 (m, 2 H, H-5%%), 1.38
3%-O-Benzoyl-2%-deoxy-5-(8-trifluoracetam-
idooct-1-ynyl)uridine 5%-[(2-cyanoethyl)-N,N-
diisopropyl] phosphoramidite (14).—Com-
pound 12 (500 mg, 0.91 mmol) was dissolved
in anhyd MeCN (10 mL), and N,N-diiso-
propylethylamine (310 mL, 1.77 mmol, 1.95
equiv) and 2-cyanoethoxy-N,N-diisopropyl-
chlorophosphoamidite (300 mg, 1.18 mmol,
1.3 equiv) were added under an Ar atmo-
sphere. After stirring for 30 min, the solvent
was removed under reduced pressure and the
resulting residue was purified over previously
dried SiO2 (4:1 anhyd toluene–anhyd ace-
tone). The diastereomeric phosphites 14 (590
mg, 86%) were obtained as a clear syrup. Pairs
of diastereomeric signals had an integration
1
1
ratio of 2:3 in H NMR; H NMR (400 MHz,
acetone-d6): l 10.11 (br s, NH), 8.39 (br s,
NH), 8.09, 7.68, and 7.55 (C6H5), 8.06 and
7.99 (each s, H-6), 6.43 and 6.37 (each dd,

A. Naundorf, W. Klaffke / Carbohydrate Research 318 (1999) 38–51
49
(m, 2 H, H-6%%); 13C NMR (100 MHz, acetone-
d6): 168.7 (d, C(O)OCH3), 165.9
mmol, 3 equiv) were dissolved in anhyd DMF
(20 mL). To the solution, kept at 80 °C under
an Ar atmosphere, anhyd silver oxide (330
mg, 1.39 mmol, 0.6 equiv) was added. The
resulting mixture was stirred for 3 h before
being concentrated under reduced pressure
and passed over a SiO2 column (2:1 petroleum
ether–acetone). The product containing frac-
tions were collected, concentrated and recrys-
tallised from CH2Cl2–n-hexane to yield 17
l
(C(O)C5H6), 161.7 (C-4), 149.9 (C-2), 142.2
(C-6), 133.9, 130.1, 130.0, and 129.0 (C6H5),
117.6 (CN), 100.9 (C-5), 94.0 (C-2%%), 85.9
(C-1%), 83.1 (d, C-4%), 75.2 (C-3%), 72.6 (C-1%%),
67.9 (d, C-5%), 64.1 (d, H2CC(O)OCH3), 63.4
(d, POCH2), 52.5 (C(O)OCH3), 39.8 (C-8%%),
37.4 (C-2%), 28.9 (C-7%%), 28.7 (C-4%%), 28.5 (C-
5%%), 26.4 (C-6%%), 19.4 (d, CH2CN), 19.3 (C-3%%);
2
3
1
J4%,P 7.3, J5%,P 5.1, JCH COOMe,P 5.5, JCOOMe,P
(980 mg, 78%); m.p. 109 °C; H NMR (400
2
6.5, 2JPOCH 5.3, 3JCH CN,P 6.9 Hz; 31P NMR (81
MHz, acetone-d6): l 8.09, 7.67, and 7.55 (Bz),
7.89 (s, H-6), 6.48 (dd, H-1%), 5.62 (m, H-3%),
4.46 (br s, HO-5%), 4.20 (m, H-4%), 3.98–3.86
(m, 4 H, H-5a%, 5b%, H-1%%), 3.34 (m, 2 H,
H-6%%), 2.58–2.50 (m, 2 H, H-2a%, 2b%), 1.86 (s,
3 H, CH3), 1.63–1.55 (m, 4 H, H-2%%, H-5%%),
1.43–1.32 (m, 4 H, H-3%%, H-4%%); 13C NMR
(100 MHz, acetone-d6): l 166.3 (CꢀO), 163.6
(C-4), 151.7 (C-2), 151.7 (C-2), 135.2 (C-6),
134.2, 130.8, 130.3, 129.4 (Bz), 110.2 (C-5),
86.4 (C-1%), 86.1 (C-4%), 76.7 (C-3%), 62.9 (C-5%),
41.3 (C-1%%), 40.1 (C-6%%), 38.3 (C-2%), 29.3 (C-
2%%), 28.1 (C-5%%), 27.0 (C-4%), 26.9 (C-3%%), 13.3
(CH3). Anal. Calcd for C25H30F3N3O7
(541.53): C, 55.45; H, 5.58; F, 10.52; N, 7.76.
Found C, 55.31; H, 5.63; F, 10.41; N, 7.61.
N3-(6-Amino-1-hexyl)-thymidine 5%-trilithi-
umdiphosphate (18).—Compound 17 (270 mg,
0.5 mmol) was pyrophosphorylated according
to the general procedures to yield 18 (400 mg,
72%, photometrically determined dT content:
46%); UV (200–400 nm): 207 (max), 238
2
MHz, acetone-d6): l 0.05, 0.16. Anal. Calcd
for C32H36F3N4O12P (756.63): C, 50.80; H,
4.80; F, 7.53; N, 7.40; P, 4.09. Found C,
50.45; H, 4.95; N, 7.76.
2%-Deoxyuridine 5%-monolithium-methoxy-
carbonylmethyl phosphate (16).—Compound
15 (300 mg, 0.40 mmol) was dissolved in 3:1
2-propanol–water (10 mL) and aq LiOH (0.5
N) was constantly added at rt by means of an
automatic titrator (pH 12.5). The reaction was
controlled by TLC (5:3:2 2-propanol–EtOH–
water, 5% triethylamine, 2% HOAc). The re-
sulting solution was directly fractionated by
Sephadex G10 chromatography, product con-
taining fractions were additionally desalted
over Sephadex G10 or Biogel P2 to yield 16
(250 mg, 91%, photometrically determined dU
content: 72%); UV (200–400 nm): 217 (min),
1
233 (max), 259 (min), 291 (max); H NMR
(400 MHz, D2O): l 7.91 (s, H-6), 6.29 (dd,
H-1%), 4.49 (m, H-3%), 4.22 (dd, 2 H,
CH2COOH), 4.12 (m, H-4%), 4.05 (m, 2 H,
H-5a%, 5b%), 2.96 (m, 2 H, H-8%%), 2.38 (m, 2 H,
H-3%%), 2.30–2.21 (m, 2 H, H-2a%, 2b%), 1.64 (m,
2 H, H-7%%), 1.53 (m, 2 H, H-4%%), 1.46–1.34 (m,
1
(min), 267 (max); H NMR (400 MHz, D2O):
l 7.82 (s, H-6), 6.43 (dd, H-1%), 4.76 (m, H-3%),
4.30 (m, 2 H, H-5a%, 5b%), 4.09 (m, H-4%), 4.01
(m, H-1%%), 3.10 (m, H-6%%), 2.50–2.42 (m, 2 H,
H-2a%, 2b%), 2.04 (s, CH3), 1.79–1.66 (m, 4 H,
H-2%%, H-5%%), 1.52 (m, 2 H, H-4%%), 1.47 (m, 2
13
4 H, H-5%%, H-6%%); C NMR (100 MHz, D2O):
l 176.5 (d, COOH), 161.6 (C-4), 156.5 (C-2),
142.7 (C-6), 100.7 (C-5), 95.7 (C-2%%), 85.9 (d,
C-1%), 85.6 (C-4%), 73.2 (C-1%%), 71.2 (C-3%), 65.3
(d, C-5%), 64.3 (d, OCH2COOH), 39.7 (C-8%%),
39.1 (C-2%), 27.7 (C-5%%), 26.8 (C-7%%), 25.3 (C-
13
H, H-3%%); C NMR (100 MHz, D2O): l 166.2
(C-4), 152.2 (C-2), 136.0 (C-6), 111.4 (C-5),
86.4 (C-1%), 85.8 (d, C-4%), 70.9 (C-3%), 65.7
(C-5%), 41.9 (C-1%%), 39.7 (C-6%%), 39.0 (C-2%),
26.9 (C-5%%), 26.8 (C-2%%), 25.8 (C-4%%), 25.6 (C-
3%%), 12.8 (CH3); J4%,P 8.6, J5%,P 4.8 Hz.
FABMS−: m/z 500.2 [M3− +2H+]−.
3
2
6%%), 18.9 (C-3%%); JCOOH,P 9.5, JPOCH C(O) 5.9,
2
J4%,P 8.6, J5%,P 4.8 Hz; 31P NMR (81 MHz,
D2O): l +1.23; ESPMS− (4.5 kV): m/z 488.1
[M−Li]−.
N4-(6-Benzyloxycarbonylaminohexanoyl)-5%-
O-tert-butyldimethylsilyl-2%-deoxycytidine (19).
—A solution of tert-butyldimethylchlorosi-
lane (580 mg, 3.79 mmol, 1.0 equiv) in anhyd
3%-O-Benzoyl-N3-(6-N-trifluoroacetamido-1-
hexyl)thymidine (17).—3%-O-Benzoyl-thymi-
dine (800 mg, 2.31 mmol, Sigma) and 6-N-
trifluoroacetamido-1-iodohexane (2.24 g, 6.93

50
A. Naundorf, W. Klaffke / Carbohydrate Research 318 (1999) 38–51
matography (2:3 petroleum ether–acetone).
20: 370 mg, 76%; m.p. 157 °C; H NMR (400
DMF (20 mL) was stirred with imidazole (520
mg, 7.58 mmol, 2 equiv) for 20 min at rt, then
2%-deoxycytidine hydrochloride (1.0 g, 3.79
mmol) was added. After 3 h, the reaction
mixture was concentrated under reduced pres-
sure, the residue then suspended in HMDSA
(6.4 mL, 30 mmol, 8 equiv) and stirred for 12
h. After concentration and co-evaporation
with toluene, the residue was dissolved in an-
hyd dioxane (50 mL) with 6-benzyloxycar-
bonylamino hexanoic acid [7] (4.63 g, 17.4
mmol, 4.6 equiv), 2-chloro-N-methylpyri-
dinium iodide (4.85 g, 19.0 mmol, 5 equiv),
and N,N-diisopropylethylamine (6.5 mL, 38
mmol, 10 equiv). The solution was stirred at
50 °C under an Ar atmosphere for 40 h, be-
fore being concentrated under reduced pres-
sure, co-evaporated with toluene and
quenched by the addition of satd aq NaHCO3
(50 mL). The product was extracted with
CH2Cl2 (2×200 mL), the combined organic
layers were dried over MgSO4 and concen-
trated. Purification over SiO2 (1:2 petroleum
ether–acetone) yielded 19 (1.50 g, 67%) as a
1
MHz, CDCl3): l 8.45 (d, H-6), 8.06, 7.54, and
7.42 (C(O)C6H5), 7.48 (d, H-5), 7.34–7.27
(CH2C6H5), 6.37 (dd, H-1%), 5.55 (m, H-3%),
5.05 (s, 2 H, CH2C6H5), 4.35 (m, H-4%), 3.91
(m, 2 H, H-5a%, 5b%), 3.14 (m, 2 H, H-6%%), 2.81
(dd, H-2a%), 2.44–2.32 (m, 3 H, H-2%%, H-2b%),
1.69 (m, 2 H, H-3%%), 1.53 (m, 2 H, H-5%%), 1.37
13
(m, 2 H, H-4%%); C NMR (100 MHz, CDCl3):
l 173.9 (C-1%%), 166.0 (C(O)C6H5), 162.4 (C-4),
161.0 (NC(O)OCH2C6H5), 156.0 (C-2), 144.5
(C-6), 136.4, 132.4, 129.3, 129.2, 128.9, 128.1,
128.0, 127.8, 127.5, and 127.3 (CH2C6H5 and
C(O)C6H5), 96.9 (C-5), 87.1 (C-1%), 85.9 (C-4%),
75.4 (C-3%), 66.0 (CH2C6H5), 61.3 (C-5%), 40.1
(C-6%%), 38.8 (C-2%), 36.7 (C-2%%), 29.0 (C-5%%),
25.7 (C-4%%), 23.9 (C-3%%). Anal. Calcd for
C30H34N4O8 (578.63): C, 62.27; H, 5.92; N,
9.68. Found C, 62.24; H, 5.63, N, 9.53.
(6%% - Benzyloxycarbonylamino - hexanoyl) - 2%-
deoxy-N4-cytidine 5%-trilithiumdiphosphate (21).
—Compound 20 was pyrophosphorylated and
purified as described under the general proce-
dures to give 21 (420 mg, 60%, photometri-
cally estimated dC content: 48%); UV
(200–400 nm): 224 (min), 231 (max), 251
1
clear syrup; H NMR (400 MHz, acetone-d6):
l 9.72 (br s, NH), 8.27 (d, H-6), 7.35–7.26 (m,
6 H, H-5, C6H5–CH2), 6.28 (br s, NH), 6.18
(dd, H-1%), 5.03 (s, 2 H, C6H5–CH2), 4.51 (m,
H-3%), 3.96 (m, H-4%), 3.97 (dd, H-5a%), 3.93
(dd, H-5b%), 3.14 (m, 2 H, H-6%%), 2.53 (m, 2 H,
H-2%%), 2.42 (d, H-2a%), 2.19 (ddd, H-2b%), 1.67
(m, 2 H, H-3%%), 1.53 (m, 2 H, H-5%%), 1.38 (m,
2 H, H-4%%), 0.94 (s, 9 H, SiC(CH3)3), 0.13 and
0.12 (each s, 6 H, Si(CH3)2); 13C NMR (100
MHz, acetone-d6): l 174.2 (C-1%%), 163.4 (C-4),
157.1 (NC(O)OBn), 155.3 (C-2), 145.1 (C-6),
138.5, 129.1, 128.5, and 127.4 (C6H5), 95.9
(C-5), 88.5 (C-4%), 87.3 (C-1%), 71.6 (C-3%), 66.2
(C6H5CH2), 63.0 (C-5%), 42.2 (C-2%), 41.3 (C-
6%%), 37.6 (C-2%%), 30.3 (C-5%%), 26.8 (C-4%%), 26.2
((H3C)3CSi), 25.2 (C-3%%), 18.8 ((H3C)3CSi),
−5.5, −5.6 ((H3C)2Si). Anal. Calcd for
C29H44N4O7Si (588.78): C, 59.16; H, 7.53; N,
9.52. Found C, 59.14; H, 7.66; N, 9.33.
1
(min), 270 (max); H NMR (400 MHz, D2O):
l 7.98 (d, H-6), 7.41–7.31 (m, 5 H, C6H5),
6.37 (dd, H-1%), 6.19 (d, H-5), 5.43 (m, H-3%),
5.03 (s, 2 H, CH2C6H5), 4.41 (m, H-4%), 4.28–
4.19 (m, 2 H, H-5a%, 5b%), 3.37 (m, 2 H, H-6%%),
2.53–2.35 (m, 4 H, H-2%%, H-2a%, 2b%), 1.69 (m,
2 H, H-3%%), 1.61 (m, 2 H, H-5%%), 1.39 (m, 2 H,
H-4%%); 13C NMR (100 MHz, D2O): l 176.4
(C-1%%), 166.5 (C-4), 159.2 (NC(O)OCH2C6H5),
157.8 (C-2), 141.9 (C-6), 137.6, 129.1, and
128.0 (C6H5), 97.3 (C-5), 86.5 (C-1%), 83.8 (d,
C-4%), 76.2 (C-3%), 68.2 (CH2C6H5), 66.2 (d,
C-5%), 40.0 (C-6%%), 37.8 (C-2%), 34.1 (C-2%%), 27.8
(C-5%%), 25.8 (C-4%%), 24.1 (C-3%%); J4%,P 7.0, J5%,P
5.7 Hz. FABMS−: m/z 595.2 [M3− +2H+]−.
N4 - (6 - Benzyloxycarbonylaminohexanoyl)-
Acknowledgements
3%-O-benzoyl-2%-deoxycytidine
(20).—Previ-
ously prepared compound 19 (500 mg, 0.85
mmol) was first benzoylated and thereafter
O-desilylated according to the general proce-
dures (vide supra) to give a raw product,
which was purified by SiO2 column chro-
We wish to express our thanks to Professor
Peter Reeves for the overexpression systems E.
coli K12 (P3378 and P4439). Financial sup-
port by the Deutsche Forschungsgemeinschaft
(grant KL631/4-3) is gratefully acknowledged.

A. Naundorf, W. Klaffke / Carbohydrate Research 318 (1999) 38–51
51
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