Solid-phase synthesis of (poly)phosphorylated nucleosides and conjugates

Article abstract of DOI:10.1002/chem.201101291

Succinyl-cycloSal-phosphate triesters of ribo-and 2′- deoxyribonucleosides were attached to aminomethyl polystyrene as an insoluble solid support and reacted with phosphate-containing nucleophiles yielding nucleoside di-and triphosphates, nucleoside diphosphate sugars, and dinucleoside polyphosphates in high purity after cleavage from the solid support. Here, reactive cycloSal-phosphate triesters were used as immobilized reagents that led to a generally applicable method for the efficient synthesis of phosphorylated biomolecules and phosphate-bridged bioconjugates.

Full text of DOI:10.1002/chem.201101291

DOI: 10.1002/chem.201101291
Solid-Phase Synthesis of (Poly)phosphorylated Nucleosides and Conjugates
Viktoria Caroline Tonn and Chris Meier*[a]
Dedicated to Professor Dr. Dr. Gerhard Bringmann on the occasion of his 60th birthday
Abstract: Succinyl-cycloSal-phosphate triesters of ribo- and 2’-deoxyribonucleo-
sides were attached to aminomethyl polystyrene as an insoluble solid support and
reacted with phosphate-containing nucleophiles yielding nucleoside di- and tri-
phosphates, nucleoside diphosphate sugars, and dinucleoside polyphosphates in
high purity after cleavage from the solid support. Here, reactive cycloSal-phos-
phate triesters were used as immobilized reagents that led to a generally applica-
ble method for the efficient synthesis of phosphorylated biomolecules and phos-
phate-bridged bioconjugates.
Keywords: bioconjugates
ganic chemistry · nucleotide sugars ·
nucleotides · solid-phase synthesis
· bioor-
Introduction
generated NDPs and NTPs in good yields and purity by
using polymer-bound di- and triphosphitylating re-
agents.[12–14] Peyrottes et al. used polyethylene glycol (PEG)
as a soluble support to generate the 5’-mono-, -di-, and tri-
phosphates of (2’-deoxy)cytidine with the nucleoside an-
ACHTUNGTRENNUNG
AHCTUNGTREGcNNUN hored through the amino function of the heterocyclic
base.[15] However, other nucleobases were not included (ade-
nine or guanine) and the method cannot be applied to thy-
mine or uracil. With regard to NDP sugars, a few chemical
and enzymatic methods are available.[16] The chemical meth-
ods are based on mainly two strategies: the pyrophosphate
bond can be formed by reaction of a glycosyl phosphate
with an activated nucleotide (morpholidate,[17,18] imidazoli-
date[19]) or of a nucleoside diphosphate and a glycopyranosyl
bromide.[20,21] Both methods are characterized by low yields
and in the latter case the stereochemical control at the
anomeric center is almost impossible. The enzymatic path-
way—the reaction of a sugar phosphate with a nucleoside
triphosphate—is done by using specific enzymes, which do
not tolerate nucleoside analogues and lead only to moderate
yields.[22] Consequently, some of these methods are special-
ized for the synthesis of one type of compound and are not
generally applicable for various nucleosides and target mole-
cules. Recently, we have reported on a generally applicable
solution-phase method for the syntheses of (poly)phos-
phorylated nucleosides and dinucleoside polyphosphates[23]
and of NDP sugars,[24–26] which is based on cycloSal-nucleo-
tides as activated species. Originally developed as a delivery
system for antivirally active nucleotides,[27] it was shown that
a variety of phosphorylated biomolecules can be synthesized
by starting from these phosphate triester derivatives. Al-
though the reaction of the cycloSal-phosphate triesters and
the phosphate nucleophiles gave convincing results, like
other solution-phase methods, the approaches are often
hampered by the following difficulties: 1) Crude products
often behaved problematically during chromatography so
ACHTUNGTRENNUNGimidazolidates, or phosphoramidates) with nucleophiles
((pyro)phosphate salts, sugar phosphates) or alternatively as
PIII reagents (phosphite triesters) followed by oxidation.[3–7]
In almost all cases, the solubility of the starting materials in
organic solvents is poor, thus conversions are slow and sepa-
ration of the charged and water-soluble product from polar
impurities needed tedious purification that often led to mod-
erate or even poor yields. Concerning solid-phase methods
for the synthesis of NDPs and NTPs, only a few approaches
have been reported. By starting from immobilized nucleo-
sides the methods are characterized by moderate yields and
low purity after cleavage,[3,8–11] whereas only Parang et al.
[a] Dipl.-Chem. V. C. Tonn, Prof. Dr. C. Meier
Organic Chemistry, Department of Chemistry
Faculty of Sciences, University of Hamburg
Martin-Luther-King-Platz 6, 20146 Hamburg (Germany)
Fax : (+49)40-42838-5592
E-mail: chris.meier@chemie.uni-hamburg.de
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201101291.
+
that counterions were to be replaced by Et3NH+- or NH4
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FULL PAPER
ions. 2) (Pyro)phosphate salts and sugar phosphates were
difficult to remove through chromatography on ion-ex-
change columns or RP-18 silica gel. Therefore, time-con-
suming multiple purification steps of crude products were
required, which resulted in a decrease in the yields due to
the instability of some compounds. 3) The use of nucleo-
philes in excess to increase the yields and to accelerate the
reactions amplified the purification problem described
above. For example, in the case of the synthesis of NDP
sugars starting from cycloSal-phosphate triesters and sugar-
1-phosphates in solution, often prior to the purification the
excess of the used sugar-1-phosphate has to be enzymatically
dephosphorylated to obtain the pure products after chroma-
tography.[26,28] This may be tolerable in the case of easily ac-
cessible sugar-1-phosphates but is not attractive in the case
of rare sugar-1-phosphates.
chloro substituent and enabled fast reactions with reactive
nucleophiles (phosphate, pyrophosphate).[23] Next, the syn-
thetic strategy was transferred to the 2’-deoxyribonucleoside
2’-deoxycytidine, the ribonucleoside uridine, the purine nu-
cleoside 2’-deoxyadenosine, and to the nucleoside analogon
5-[(E)-bromovinyl]-2’-deoxyuridine (BVdU) to demonstrate
general applicability. In addition, methylsulfonyl cycloSal-
phosphate triesters of thymidine and uridine were applied
to prove if increased electrophilicity relative to chloro led to
faster reactions with less reactive nucleophiles (sugar phos-
phates, nucleoside monophosphates).
Results and Discussion
Succinyl-cycloSal-nucleotides: The synthesis of 2’/3’-O-suc-
cinyl-cycloSal-nucleotides started with the attachment of the
linker unit to 5’-O-DMTr-protected nucleosides 6–10 yield-
ing 11–15 followed by deprotection of the 5’-O-DMTr-group
(Scheme 2). Then, 3’-O-succinyl-nucleosides 16–20 were
converted to cycloSal-phos-
To circumvent these limitations, we have developed a
method to use cycloSal-phosphate triesters as active ester
reagents in combination with the solid-phase strategy.
Scheme 1 illustrates the general principle of this novel ap-
phate triesters 21a–e and 22a,b
by using the linker unit as a
protecting group during triester
synthesis to prevent unwanted
phosphorylation of the 3’-OH
group. In addition, there was no
or only one purification process
necessary at triester level. The
ribonucleoside uridine was first
acetylated at the 2’- or 3’-OH
group[29] yielding 23 in 74%
yield as a mixture of 2’- and 3’-
O-acetylated uridine. DMTr
protection
methoxytrityl) of the 5’-OH
group—and in the case of 2’-de-
oxycytidine of the amino group
(DMTr=4,4’-di-
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
as well—was performed accord-
ing to literature protocols[30]
forming 6–10 in 64–71% yield.
The attachment of the linker
was achieved by a fast reaction
of the protected nucleosides 6–
Scheme 1. General principle of immobilization and conversion of cycloSal-nucleotides on the basis of 2’-deoxy-
ribonucleosides.
proach on the basis of 2’-deoxyribonucleosides. The proce-
dure consisted of three steps. First, acceptor-substituted and
10 with succinic anhydride in the presence of 1,8-
diazabicyclo[5.4.0]undec-7-en (DBU) followed by protona-
ACHTUNGTRENNUNG
succinyl-linked cycloSal-nucleotides
1
were attached to
tion of the carboxylic function (11–15, 88–99% yield). De-
protection of the 5’-OH group with trifluoroacetic acid
(TFA) led to 16–20 (71–99% yield). Cyclization of salicyl al-
cohols 24 and 25 with PCl3 gave acceptor-substituted chloro-
phosphites 26 and 27 in 86 and 77% yield, respectively.[26]
Compound 24 was previously generated by reduction of 5-
chloro-salicylic acid[31] and salicyl alcohol 25 by oxidation of
6-methylmercapto-2-phenyl-4H-[1.3.2]benzodioxaborine.[24]
5’-O-Deprotected-3’-O-succinyl-nucleosides 16–20 were
treated with chlorophosphites 26 and 27 followed by subse-
quent oxidation with oxone (2KHSO5·KHSO4·K2SO4)[23]
amino-methyl polystyrene 2 as an insoluble solid support via
an amide bond. Secondly, a nucleophile was reacted with
the immobilized cycloSal-triesters 3, which led to the sup-
port-bound target molecules 4. Finally, the cleavage of the
products 5 from the support was carried out under basic
conditions.
The 5-chloro-substituted 3’-O-succinyl-cycloSal-phosphate
triester of thymidine was used as a model compound for the
following conversions. The phosphorus atom showed a suffi-
cient reactivity due to the electron-withdrawing effect of the
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9833

C. Meier and V. C. Tonn
otides with acceptor substitu-
ents that cause an even stronger
activation at the phosphorus
center to react these im-
AHCTUNGTERGmNNUN obilized phosphate triesters
also with less reactive nucleo-
philes (e.g. sugar phosphates or
nuACTHNUTRGENUGcN leotides) in even shorter re-
action times relative to the 5-
chloro-cycloSal-triester. There-
fore, conversions of methylsul-
fonyl-substituted cycloSal-nu-
cleotides 22a,b to (d)NDPs and
(d)NTPs were carried out to
optimize the coupling condi-
tions (Scheme 3).
As an optimized procedure,
shaking the solid support with a
mixture of the corresponding
cycloSal-nucleotide and an
excess of 1-hydroxybenzotria-
zole (HOBt) and N,N’-diisopro-
pylcarbodiimide (DIC), each
three equivalents, in anhydrous
DMF should be carried out.[33]
If water-free reagents are re-
quired, 2-(1H-benzotriazole-1-
yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TBTU) and
4-ethylmorpholine in anhydrous
DMF proved to be suitable for
Scheme 2. Synthesis of 2’- or 3’-O-succinyl-cycloSal-nucleotides 21a–e and 22a–b: i) PCl3, pyridine, Et2O
(THF), À408C (2 h) to RT (2 h), 26 (86%), 27 (77%); ii) DBU, succinic anhydride, CH2Cl2, RT, 45–120 min,
CH3COOH, 11–15 (88–99%); iii) TFA, CH2Cl2, RT, 8–120 min, 16–20 (71–99%); iv) TMOF, pTsOH, RT, 2 h,
23 (74%); v) DMTrCl, pyridine, Et3N, DMAP, RT, 20 h, 10 (64%); vi) 1) CH3CN (DMF), DIPEA, À358C to
RT, 2–3 h; 2) oxone in H2O, À108C, 15 min, 21a–e+22a,b (56–83%). DMAP=4-dimethylaminopyridine;
TMOF=trimethoxyorthoacetate.
yielding crude materials in good purity after extraction ac-
cording to NMR spectroscopic analysis (21a–e and 22a,b,
56–83% yield). Both, chloro and methylsulfonyl-substituted
cycloSal-phosphate triesters 21a–e and 22a,b could be fur-
ther purified by silica gel chromatography after addition of
1% acetic acid to the eluent.
Immobilization of succinyl-cycloSal-nucleotides and their
conversions: For immobilization, 2’/3’-O-succinyl-cycloSal-
nucleotides 21a–e and 22a,b were anchored to aminomethyl
polystyrene 2 through an amide bond and the loading of the
amino groups present on the support was determined by the
Kaiser test.[32] It was very important for the success of the
following reaction and particularly for the high purity of the
cleaved target molecule that the coupling conditions of the
cycloSal-triester to the support were mild enough to allow
the immobilization of even highly reactive triesters. Conse-
quently, the purity of cleaved target molecules judged by
31P NMR spectroscopic analysis is a result of both, stability
of the triester during the coupling process and conversion of
the triester to the target molecule. For reactions of cycloSal-
nucleotides with reactive nucleophiles like phosphate salts,
the reactivity of the phosphorus center caused by chloro as
an acceptor-substituent (21a–e) is strong enough for short
Scheme 3. Immobilization of cycloSal-nucleotides 21a–e and 22a,b and
their conversion to (d)NDPs 30a–e, (d)NTPs 31a–d, and (d)NTP ana-
logues 32a,b: i) HOBt, DIC, DMF, 16 h–2 d, RT; ii) TBTU, 4-ethylmor-
pholine, DMF, 2 d, RT; iii) [(nBu)4N]H2PO4 (33), DMF, 16 h, RT; iv) for
Z=O: [(nBu)4N]2H2P2O7 (34), for Z=CH2: [(nBu)4N]2H2PO3CH2PO3
(35), DMF, 16 h, RT; v) 25% aqueous NH3, 2 h, 508C; vi) CH3OH/H2O/
Et3N 7:3:1, 20 h, RT; vii) 3% TCA in CH2Cl2, 24 h, RT.
re
ACHTUNGTRENNUNGaction times and high conversion rates. However, the prin-
ciple of the new approach was transferred to cycloSal-nucle-
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Solid-Phase Synthesis of (Poly)phosphorylated Nucleosides and Conjugates
FULL PAPER
the amide bond formation as well.[34] By using these condi-
tions, cycloSal-nucleotides 21a–e and 22a,b were immobi-
lized resulting in 28a–e and 29a,b. The synthesis of TDP
30a by starting from immobilized 5-chloro-cycloSal-thymi-
dine monophosphate (28a) showed purity as good as with
the analogous reaction (both 90%) by using HOBt/DIC for
the immobilization. However, for 5-methylsulfonyl-cycloSal
triester, the highest purity of TDP 30a (81%) was obtained
by using three equivalents HOBt/DIC and a short reaction
time (20 h) for triester immobilization. As a consequence,
the latter reaction conditions were used for further experi-
ments.
Table 1. Purity of (d)NDPs 30a–e and (d)NTPs 31a–d and 32a,b after
cleavage.
Starting
Coupling
Cleavage[a] Purity
[%]
cycloSal-triester conditions[a]
TDP 30a
TDP 30a
2’-dADP 30b
2’-dCDP 30c
BVdUDP 30d
UDP 30e
28a
29a
28b
28cdepr.
28d
29b
28a
28b
28d
28e
28a
i) or ii)
v)
v)
v)
v)
v)
vi)
v)
v)
v)
vi)
v)
vi)
90
81
88
44
91
63
90
78
90
78
78
75
i)
i)
i)
i)
ii)
i)
i)
i)
i)
i)
i)
TTP 31a
2’-dATP 31b
BVdUTP 31c
UTP 31d
TDPCH2P 32a
UDPCH2P 32b 28e
Concerning the immobilized 5-chloro-cycloSal-N4-(4,4’-di-
methoxytrityl)-3’-O-succinyl-2’-deoxycytidine
monophos-
[a] Conditions described in Scheme 3.
phate (28c) the DMTr-protection group was removed on
solid support by addition of trichloroacetic acid (TCA)
yielding 28cdepr.. The synthesis of (d)NDPs 30a–e, (d)NTPs
31a–d, and the triphosphate analogues 32a,b started with
the preparation of reactive phosphate nucleophiles. Here, an
ion-exchange of phosphoric acid, Na2H2P2O7, and
Na2H2PO3CH2PO3 by using ion-exchange resin Dowex
(50WX8) followed by titration with [(nBu)4N]OH into their
tetra-n-butyl ammonium-salts 33–35 was conducted.[34] The
solutions were titrated until a pH value of 5–6 was reached
to ensure that the linker remained stable. In preliminary ex-
periments a cleavage was observed in cases in which the
phosphate salts were titrated to a pH value of 7.
that a 2’-dCDP-conjugate 36 was formed. After separation
of the two diphosphates and isolation of the 2’-dCDP-conju-
gate 36 and 2’-dCDP 30c in a ratio of 1:1 it was assumed
that the 2-quinone methide 37 released during the nucleo-
philic attack of the phosphate 33 reacted with another phos-
phate to a substituted phosphate nucleophile 38 in a ratio of
1:1 to the target molecule 2’-dCDP 30c (Scheme 4). Nucleo-
phile 38 reacted probably with the immobilized triester
28 cdepr. with formation of the 2’-dCDP-conjugate 36. This
result was reproducible and astonishingly only observed in
the synthesis of 2’-dCDP 30c.
Extensive drying of the resins 28a–e and 29a,b in vacuo
and of the phosphate salts 33–35 first in vacuo followed by
storage over activated molecular sieves as a solution in an-
hydrous DMF was essential for the prevention of byprod-
ucts. Generally, the excess of phosphate salt used for the
conversion was six to ten equivalents, only in the case of
BVdU more was used due to the expensive starting materi-
al. After a reaction time of 16 h, simple washing of the poly-
mer-bound target molecules allowed complete removal of
the excess of phosphate salts and of the released salicyl alco-
hols. In contrast, solution-phase synthesis of these molecules
requires time-consuming purification steps. Cleavage from
the support was done with aqueous ammonia at 508C in the
case of 2’-deoxynucleotides. For products based on ribonu-
cleosides with acetyl protecting groups and NDP sugars with
acetyl groups in the sugar moiety the cleavage was achieved
with a mixture of CH3OH, H2O, and Et3N (7:3:1, v/v/v) at
room temperature, which allowed a cleavage of the acetyl
protecting groups in the form of methyl acetate or acetic
acid at the same time as the solid support. The methyl ace-
tate or acetic acid was removed in vacuo. The linker re-
mained attached to the resin thus enabling separation of
final products by simple filtration. Freeze-drying of the
cleavage solutions yielded target molecules directly in high
purity.
Scheme 4. Synthesis of 2’-dCDP 30c and assumed byproduct 2’-dCDP-
conjugate 36.
The purities of the obtained (d)NDPs 30a–e and (d)NTPs
31a–d and 32a,b were analyzed by 31P NMR spectroscopic
analysis in all cases because the triesters were immobilized
as pure materials and consequently all cleavage products
should contain phosphorus (Table 1). Often in solid-phase
strategies, HPLC analysis of the final products is used to de-
termine the purity of the released product. In our case, we
Following this protocol, the diphosphates of thymidine, 2’-
deoxyadenosine, BVdU, and uridine 30a,b,d,e were ob-
tained in 63–91% purity after cleavage and 2’-deoxycytidine
diphosphate (2’-dCDP) 30c in 44% purity, which was excep-
tionally low (Table 1). In this particular case, it was observed
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C. Meier and V. C. Tonn
decided to use 31P NMR spectroscopy because the only
phosphorus-containing compound attached to the support is
the cycloSal-phosphate triester and after the reaction only
the products of the reaction of the cycloSal-triester and the
nucleophile or known phosphorus-containing byproducts are
released from the support. The advantage of using 31P NMR
spectroscopy is that the spectra are fast and easily conduct-
ed, whereas HPLC analysis would necessarily need for each
class of product an individual elution method. Moreover,
the spectroscopic method enabled the integration of the by-
products signals and thus gives immediately the ratio of the
product to the byproducts.
To further increase the purity of the target compounds,
crude products were easily purified by a rapid chromatogra-
phy on RP-18 silica gel. Therefore, the crude products were
converted into their Et3NH+ salts with ion-exchange resin
Dowex (50WX8) because Et3NH+ as the counterion turned
out to be superior for RP-chromatography relative to, for
example, (nBu)4N+. These procedures were found to be
enormously simplified relative to the purification of the
crude products obtained by solution-phase syntheses due to
the absence of phosphate salts. Six of the compounds pre-
sented in Table 1 (30b,c,e, 31b, and 32a,b) haven’t been syn-
thesized with cycloSal techniques in solution-phase chemis-
try yet. The purification of a crude product that showed a
purity of 90% gave a pure product in 80% chemical yield.
Moreover, the reported procedure was applied to the con-
version of immobilized cycloSal-NMPs 28a,b and 29a,b, to
NDP sugars 39–41, and dinucleoside diphosphates 42 and 43
(Scheme 5).
sion of methylsulfonyl-substituted cycloSal-nucleotides
showed higher purity of products if Et3NH+ was used as
counterion of the nucleophile. For the NDP sugar syntheses
tetra-O-acetyl-b-d-glucosyl-1-phosphate (44) and tetra-O-
acetyl-a-d-galactosyl-1-phosphate (45) that were prepared
according to well-established methods were used,[24] whereas
commercially available Na2UMP was used for the ion-ex-
change to generate 46.
The protocols were identical to the protocol described for
the (d)NDPs and (d)NTPs including drying of salts and
resins, the addition of nucleophile solutions to the resins fol-
lowed by washing and cleavage procedures. On this route,
TDP-b-d-glucose 39, UDP-b-d-glucose 40, thymidine-uri-
dine-5’,5’-diphosphate 42, and 2’-deoxyadenosin-uridine-
5’,5’-diphosphate 43 were synthesized in good conversions of
the corresponding immobilized 5-chloro- or 5-methylsulfon-
yl-substituted cycloSal-nucleotide (purities of 61–78% ac-
cording to 31P NMR spectroscopy), whereas only TDP-a-d-
galactose 41 showed a lower purity (43%; Table 2). It
became apparent that these reactions required longer reac-
tion times relative to the di- and triphosphate syntheses al-
Table 2. Purity of NDP-sugars 39–41 and dinucleoside diphosphates 42
and 43 after cleavage.
Starting
cycloSal-
triester
Coupling
Cleavage[a]
Purity
[%]
conditions[a]
TDP-b-d-glucose 39
TDP-b-d-glucose 39
UDP-b-d-glucose 40
TDP-a-d-galactose 41
Tp2U 42
28a
29a
29b
29a
28a
29a
28b
i)
i)
i)
i)
i)
i)
i)
vi)
vi)
vi)
vi)
v)
78
60
61
43
78
70
70
Generally, nucleophiles show higher nucleophilicity in
their (nBu)4N+ form relative to their Et3NH+ or Na+ salts.
Therefore, the majority of reactions have been carried out
with (nBu)4N+ phosphate salts. On the other hand, conver-
Tp2U 42
2’-dAp2U 43
v)
v)
[a] Conditions described in Scheme 3.
though again the (nBu)4N+
salts of the nucleophiles were
A
ACHTUNGTRENNUNG
used. Interestingly, comparing
the two electron-withdrawing
groups in the cycloSal-moiety,
no advantage could be attribut-
ed to the stronger methylsul-
fonyl group. The dinucleoside
diphosphate 43 hasn’t been syn-
thesized with cycloSal tech-
niques in solution-phase before.
Interestingly, we isolated the
1,2-cyclophosphate of a-ga-
AHCTUNGTRENGNlUN actose and thymidine-5’-mono-
phosphate after the cleavage of
TDP-a-d-galactose 41 with
CH3OH, H2O, and Et3N (7:3:1,
v/v/v) at room temperature
from the solid support. Thus,
the cleavage conditions seems
to lead to the cleavage of the
Scheme 5. Conversion of acceptor-substituted immobilized cycloSal-NMPs 28a,b and 29a,b into NDP-sugars
39–41 and dinucleoside diphosphates 42 and 43: i) (Et3NH)-tetra-O-acetyl-b-d-glucosyl-1-phosphate (44a),
DMF, 4 d, RT; ii) [(nBu)4N]-tetra-O-acetyl-b-d-glucosyl-1-phosphate (44b), DMF, 5 d, RT; iii) (Et3NH)-tetra-
O-acetyl-a-d-galactosyl-1-phosphate (45), DMF, 4 d, RT; iv) [(nBu)4N]UMP (46), DMF, 4–5 d, RT; v) 25%
aqueous NH3, 2 h, 508C; vi) CH3OH/H2O/Et3N 7:3:1, 20 h, RT.
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Solid-Phase Synthesis of (Poly)phosphorylated Nucleosides and Conjugates
FULL PAPER
to use. CH2Cl2, ethyl acetate, and methanol (CH3OH) for chromatogra-
phy and extractions were distilled before used. Solid-phase aminomethyl
polystyrene was stored at À268C under a nitrogen atmosphere. Column
chromatography was performed by using Merck silica gel 60, 230–400
mesh. Reversed-phase column chromatography was performed on re-
verse-phase silica gel with distilled water as the eluent at room tempera-
ture in a glass column. Ion exchange was executed by Dowex (50WX8,
100–200 mesh, ion-exchange resin) in a glass column. Some separations
were performed on a chromatotron by using glass plates coated with 1, 2,
or 4 mm layers silica gel (60 PF254) containing a fluorescent indicator.
Analytical TLC was performed on Merck precoated aluminum plates 60
F254 with a 0.2 mm layer of silica gel containing a fluorescent indicator.
Sugar-containing compounds were visualized with sugar spray reagent (4-
methoxybenzaldehyde (0.5 mL), ethanol (9 mL), concd sulfuric acid
(0.5 mL), and glacial acetic acid (0.1 mL)).
pyrophosphate bond in the previously formed NDP sugars
as reported by Kosma et al. for 2,3,4,6,7-penta-O-acetyl-
ADP-b-d- and -l-heptopyranoses.[35] Consequently, new im-
proved cleavage conditions for NDP sugars should avoid
this decomposition and yield the target molecules in higher
purity.
Comparing the synthesis in solution and the solid-phase
synthesis of the compounds described above,[23–26] the
number of synthesis steps to get to the reactive cycloSal-nu-
cleotides is identical. In solution, the 2’- and/or 3’-OH group
has to be protected, whereas for solid-phase synthesis this
position is used for the attachment of the linker unit. Al-
though there is the additional step for the immobilization of
the cycloSal-nucleotides, this method yielded the target mol-
ecules in very high purities directly after cleavage in the
cleavage solution without impurities of the phosphate nucle-
ophiles. Generally, (d)NDPs and (d)NTPs are obtained in a
purity of ~90%. In contrast, the solution-phase method re-
quired the additional step of deprotection and (sometimes)
multiple time-consuming purification procedures on RP-18
silica gel to remove the phosphate nucleophiles and the by-
products. Particularly interesting might be to use the devel-
oped method in the context of immobilized biooligomers.
DNA or RNA strands might be modified at the 5’-end, for
example, as their (poly)phosphates. This type of compounds
cannot be prepared by solution-phase strategies. Work along
this line is currently undergoing in our laboratories.
Instrumentation: 1H NMR spectroscopy was carried out by using
a
Bruker AMX 400 at 400 MHz or Bruker AV 400 at 400 MHz. 13C NMR
spectra were recorded on a Bruker AMX 400 at 101 MHz or Bruker AV
400 at 101 MHz. 31P NMR spectra were recorded on a Bruker AMX 400
at 162 MHz (H3PO4 as external standard). The spectra were recorded at
1
room temperature. All H-, 31P-, and 13C NMR chemical shifts are quoted
in parts per million (ppm) downfield from tetramethylsilane and calibrat-
ed on solvent signals. All 13C- and 31P NMR spectra were recorded in the
proton-decoupled mode. HRMS were obtained with a VG Analytical
VG/70–250F spectrometer (FAB, matrix was m-nitrobenzyl alcohol). For
lyophilization of aqueous solutions, a Christ-Alpha 2–4 lyophilizer was
used. For shaking of solid-phase reactions, a Heidolph Polymax 1040
shaker with Incubator 1000 was used.
General procedure 1 for the synthesis of 5’-O-(4,4’-dimethoxytrityl)-pro-
tected and 2’/3’-O-succinyl-linked nucleosides 11–15: The reaction was
carried out under a nitrogen atmosphere. DBU (1 equiv) was added to a
stirred solution of DMTr-protected nucleoside (1 equiv) and succinic an-
hydride (1.5 equiv) in CH2Cl2 at room temperature. After complete con-
version of starting material (15–120 min) acetic acid (2 equiv) was added
and stirred for 10 min. The reaction mixture was washed with water (3ꢂ)
and the combined aqueous layers were extracted with CH2Cl2 (2ꢂ). The
combined organic layers were dried over sodium sulphate and the solvent
was removed in vacuo yielding the product as a solid.
Conclusion
This novel method combines the efficient conversion of cy-
cloSal-nucleotides into (d)NDPs, (d)NTPs, and (d)NDP
sugars and dinucleoside diphosphates with the easy-to-per-
form and time-saving purification resulting from the use of
solid-phase strategies.
5-Acceptor-substituted (chloro or methylsulfonyl) cyclo-
Sal-nucleotides of ribo-, 2’-deoxyribonucleosides and of the
nucleoside analogon BVdU that contained a succinyl-linker
unit at the 2’- or 3’-OH group were synthesized and easily
attached to aminomethyl polystyrene followed by a fast and
reliable conversion into several different types of phos-
phorylated nucleosides and bioconjugates. Importantly, the
disclosed method offered an access for the synthesis of a va-
riety of biologically important compounds by the same
chemical approach.
General procedure 2 for the synthesis of 2’/3’-O-succinyl-linked nucleo-
sides 16–20
Method A: The corresponding compound was dissolved in CH2Cl2/
CH3OH 7:3 and treated with TFA (3–3.5 equiv). After stirring for 1–2 h
at room temperature, toluene was added and the solvents were removed
in vacuo. The crude product was purified on a chromatotron with a gradi-
ent of methanol in CH2Cl2 (0–10%).
Method B: The corresponding compound was dissolved in CH2Cl2 and
treated with TFA (3–3.5 equiv). After stirring for 30 min at room temper-
ature, methanol was added and stirred for 10 min. Then, toluene was
added and the solvents were removed in vacuo. The crude product was
purified on a chromatotron with a gradient of methanol in dichlorome-
thane (0–10%).
Method C: The corresponding compound was dissolved in dichlorome-
thane/methanol 7:3 and 6% TFA. After stirring for 8 min at room tem-
perature, 7m methanolic NH3 solution was added until the solution was
neutralized. Subsequently, the solvents were removed in vacuo and the
residue was dissolved in ethyl acetate and washed with water. The aque-
ous layer was extracted with ethyl acetate and the combined organic
layers were dried over sodium sulphate, filtrated, and the solvent re-
moved in vacuo. The crude product was purified on a chromatotron with
a gradient of methanol in dichloromethane (0–10%).
Experimental Section
General: In the case of water-sensitive compounds, the reactions were
performed in flame-dried glassware under a nitrogen atmosphere. There-
fore, solvents were dried as follows: acetonitrile (CH3CN) and dichloro-
methane (CH2Cl2) were distilled from calcium hydride under nitrogen
and stored over activated molecular sieves (CH3CN 3 ꢁ and CH2Cl2
4 ꢁ), N,N-diisopropylethylamine (DIPEA) was distilled from sodium,
DBU was distilled and stored over activated 4 ꢁ molecular sieves, and
DMF was stored over activated 4 ꢁ molecular sieves, respectively, prior
General procedure 3 for the synthesis of 2’/3’-O-succinyl-linked 5-chloro-
or 5-methylsulfonyl-substituted cycloSal-nucleotides 21a–e and 22a,b: 3’-
O-Succinyl-linked nucleoside 16–19 or 2’- or 3’-O-acetylated succinyl-
linked nucleoside 20 (1 equiv) was dissolved in acetonitrile (and dime-
thylformamide if required) and the reaction mixture was cooled to
À308C. DIPEA (2 equiv) was added and after 10 min a solution of 5-ac-
ceptor-substituted saligenyl chlorophosphite 26 or 27 (1.3–1.6 equiv) in
acetonitrile was added dropwise over 20 min. After removing cooling,
Chem. Eur. J. 2011, 17, 9832 – 9842
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9837

C. Meier and V. C. Tonn
the reaction mixture was stirred for 2–3 h and then cooled to À108C.
Oxone (2 equiv/phosphite) dissolved in cold water was added and the re-
action mixture was stirred for 15 min without cooling. The organic layer
was directly washed with cold water (2ꢂ) and the combined aqueous
layers were extracted with ethyl acetate (1ꢂ). The combined organic
layers were dried over sodium sulphate and the solvent was removed in
vacuo. The residue was lyophilized from H2O/CH3CN 2:1) yielding prod-
ucts as colorless foams that were determined to be pure by 31P NMR
spectroscopy. For higher purity, the crude products were purified on a
chromatotron with a gradient of methanol in dichloromethane (0–10%)
and 1% acetic acid.
5 mL). Washing and cleavage solution were combined and lyophilized to
afford target molecules as crude products.
General procedure 7 for the purification of target molecules: If desired,
a crude product was converted into its corresponding Et3NH+ salt by ion
exchange with Dowex (50WX8, 100–200 mesh, ion-exchange resin).
Dowex was first adjusted to the protonated form with hydrochloric acid,
then crude product was eluted with water, THF (2 mL) was added, and
the solution neutralized with Et3N. Freeze-drying yielded the Et3NH+
salt of the target molecule that was purified by RP-18-chromatography
with water as the eluent.
5’-O-(4,4’-Dimethoxytrityl)-3’-O-succinyl-thymidine (11): The reaction
was performed according to general procedure 1 by using 6 (4.00 g,
7.34 mmol), succinic anhydride (1.10 g, 11.0 mmol), DBU (1.10 mL,
7.36 mmol), acetic acid (841 mL, 14.7 mmol), and CH2Cl2 (85 mL) within
a reaction time of 45 min yielding a rose solid (4.75 g, 7.11 mmol, 97%).
Rf (CH2Cl2/CH3OH 9.5:0.5, 0.1% Et3N)=0.22; 1H NMR (400 MHz,
[D6]DMSO): d=12.25 (s, 1H; COOH), 11.39 (s, 1H; NH), 7.52 (s, 1H;
General procedure 4 for immobilization of 5-acceptor-substituted 2’/3’-O-
succinyl-linked cycloSal-nucleotides 21a–e and 22a,b
Method A: 3’-O-Succinyl-linked 5-acceptor-substituted cycloSal-nucleo-
tide (1.1 equiv) and solid-support aminomethyl polystyrene 2 (1 equiv)
were dried in vacuo for at least 2 h. The resin was swollen in DMF
(1 mL/70 mg) for 30 min and the phosphate triester, 1-hydroxybenzotria-
zole (HOBt, 3.3 equiv) and N,N’-diisopropylcarbodiimide (DIC,
3.3 equiv) were added and the mixture was shaken for at least for at 20 h
at room temperature (complete loading checked by the Kaiser test[31]).
The resin was collected by filtration and washed with DMF and CH2Cl2
followed by drying in vacuo for several hours.
H6), 7.38–7.23 (m, 9H; DMTr-H), 6.89 (d, 4H; DMTr-H, 3JHH =7.7 Hz),
3
6.22 (dd, 1H; H1’, 3JHH =8.5, 3JHH =6.0 Hz), 5.30 (d, 1H; H3’, JHH
=
6.4 Hz), 4.07–4.04 (m, 1H; H4’), 3.74 (s, 6H; 2ꢂOCH3,DMTr), 3.34–3.30
(m, 1H; H5’a), 3.22 (dd, 1H; H5’b, 2JHH =10.3, 3JHH =2.9 Hz), 2.55–2.50
(m, 4H; H2’’, H3’’), 2.50–2.44 (m, 1H; H2’a), 2.33–2.29 (dd, 1H; H2’b,
3
2JHH =13.3, JHH =5.8 Hz), 1.42 ppm (s, 3H; H7).
Method B: Succinyl-linked 5-acceptor-substituted cycloSal-nucleotide
(1.2 equiv) and solid-support aminomethyl polystyrene 2 (1 equiv) were
dried in vacuo for at least 2 h. 4-Ethylmorpholine (0.98 equiv) was dis-
solved in DMF (2 mL/10 mL base) and stored over activated molecular
sieves 4 ꢁ for 1 h. The resin was swollen in DMF (1 mL/70 mg) for
30 min and TBTU (1.2 equiv) was added to the 4-ethylmorpholine solu-
tion and stored for 15 min. This solution was added to the resin and the
mixture was shaken for 1–2 d at room temperature (complete loading
checked by the Kaiser test[31]). The resin was collected by filtration and
washed with DMF and CH2Cl2 followed by drying in vacuo for several
hours.
5’-O-(4,4’-Dimethoxytrityl)-3’-O-succinyl-2’-deoxyadenosine (12): The re-
action was performed according to general procedure 1 by using
7
(685 mg, 1.24 mmol), succinic anhydride (187 mg, 1.87 mmol), DBU
(185 mL, 1.24 mmol), acetic acid (142 mL, 2.47 mmol), and CH2Cl2
(10 mL) within a reaction time of 75 min yielding a rose solid (763 mg,
1.17 mmol, 94%). Rf (CH2Cl2/CH3OH 9:1, 0.1% Et3N)=0.38; 1H NMR
(400 MHz, [D6]DMSO): d=12.30 (s, 1H; COOH), 8.25 (s, 1H; H8), 8.03
3
(s, 1H; H2), 7.34–7.19 (m, 9H; DMTr-H), 6.82 (d, 2H; DMTr-H, JHH
=
3
8.6 Hz), 6.80 (d, 2H; DMTr-H, 3JHH =8.6 Hz), 6.37 (dd, 1H; H1’, JHH
=
7.3, 3JHH =6.6 Hz), 5.41–5.39 (m, 1H; H3’), 4.18–4.15 (m, 1H; H4’), 3.72
(s, 6H; 2ꢂOCH3,DMTr), 3.31–3.17 (m, 2H; H5’), 2.58–2.53 (m, 4H; H2’’,
H3’’), 2.47–2.45 ppm (m, 2H; H2’).
General procedure 5 for the synthesis of nucleoside di- and triphosphates
30a–e, 31a–d, 32a,b, nucleoside diphosphate sugars 39—41 and dinucleo-
side polyphosphates 42 and 43: Tetra-n-butylammonium salts of phos-
phate 33, pyrophosphate 34, and methylenpyrophosphate 35 were pre-
pared by ion-exchange chromatography with Dowex (50WX8, 100–200
mesh, ion-exchange resin) of their protonated and sodium forms, respec-
tively, and titration with tetra-n-butylammonium hydroxide to a pH value
of 5–6. Tetra-O-acetyl-b-d-glucose (44a) and tetra-O-acetyl-a-d-galactose
(45) were used in their triethylammonium forms, but for conversion of
chloro-substituted cycloSal-nucleotide, tetra-n-butylammonium tetra-O-
acetyl-b-d-glucose (44b) was used (also prepared by ion exchange).
Na2UMP was converted into the tetra-n-butylammonium form 46. The
lyophilized salts were dried for 2 d in vacuo and afterwards dissolved in
DMF stored over activated molecular sieves 4 ꢁ 2 h before the reaction.
The immobilized cycloSal-nucleotides 28a,b and 29a,b were coevaporat-
ed with CH3CN (3ꢂ) and dried in vacuo for several hours. The resin was
swollen in DMF (analogue the general procedure 4 for immobilization of
cycloSal-triesters, see above) and the solution of phosphate- or pyrophos-
phate salt 33–35 (5.5–9.7 equiv, for BVdU-triester 28d 20 or 23 equiv as
an exception) or sugar- or nucleoside monophosphate 44–46 (3.4–
8.2 equiv) was added. The mixture was shaken for 16 h in the case of the
di- and triphosphates and for the others as described in the respective
protocol (4—5 d). Afterwards, the resin was washed with DMF, CH2Cl2,
and water.
N4--5’-O-Bis(4,4’-dimethoxytrityl)-3’-O-succinyl-2’-deoxycytidine
(13):
The reaction was performed according to general procedure 1 by using 8
(1.82 g, 2.19 mmol), succinic anhydride (328 mg, 3.28 mmol), DBU
(328 mL, 2.19 mmol), acetic acid (250 mL, 4.37 mmol), and CH2Cl2
(12 mL) within a reaction time of 120 min yielding a rose solid (2.01 g,
2.16 mmol, 99%). Rf (CH2Cl2/CH3OH 8.5:1.5, 0.1% Et3N)=0.55;
1H NMR (400 MHz, [D6]DMSO): d=12.21 (s, 1H; COOH), 8.38 (s, 1H;
NH), 7.51 (d, 1H; H6, 3JHH =7.1 Hz), 7.37–7.12 (m, 18H; DMTr-H), 6.90
(d, 4H; DMTr-H, 3JHH =8.4 Hz), 6.89 (d, 4H; DMTr-H, 3JHH =8.6 Hz),
3
6.17 (d, 1H; H5, 3JHH =7.6 Hz), 6.08 (dd, 1H; H1’, 3JHH =7.2, JHH
=
6.8 Hz), 5.25–5.15 (m, 1H; H3’), 4.05–3.97 (m, 1H; H4’), 3.75 (s, 6H; 2ꢂ
OCH3,DMTr), 3.72 (s, 6H; 2ꢂOCH3,DMTr), 3.30–3.18 (m, 2H; H5’), 2.28–
2.16 ppm (m, 2H; H2’).
5’-O-(4,4’-Dimethoxytrityl)-3’-O-succinyl-BVdU (14): The reaction was
performed according to general procedure 1 by using
9 (271 mg,
0.426 mmol), succinic anhydride (77 mg, 0.77 mmol), DBU (64 mL,
0.43 mmol), acetic acid (49 mL, 0.85 mmol), and CH2Cl2 (5 mL) within a
reaction time of 15 min yielding a colorless solid (277 mg, 0.377 mmol,
88%). Rf (CH2Cl2/CH3OH 9:1, 0.1% Et3N)=0.37; 1H NMR (400 MHz,
[D6]DMSO): d=12.26 (s, 1H; COOH), 11.68 (s, 1H; NH), 7.81 (s, 1H;
3
H6), 7.37–7.18 (m, 10H; DMTr-H, H8), 6.88 (dd, 4H; DMTr-H, JHH
=
8.9, 4JHH =2.2 Hz), 6.37 (d, 1H; H7, 3JHH =13.5 Hz), 6.19 (dd, 1H; H1’,
3JHH =6.9, 3JH,H =7.3 Hz), 5.26–5.22 (m, 1H; H3’), 4.10–4.08 (m, 1H;
H4’), 3.74 (s, 6H; 2ꢂOCH3,DMTr), 3.35–3.32 (m, 1H; H5’a), 3.23 (dd, 1H;
H5’b, 2JH,H =10.6, 3JHH =3.2 Hz), 2.55–2.47 (m, 5H; C2’’, C3’’, H2’a),
General procedure 6 for the cleavage of target molecules
Method A: 25% aqueous NH3 solution (5 mL) was added to the washed
immobilized target molecule. After 2 h shaking at 508C, the resin was
collected by filtration and washed with water (10ꢂ5 mL). Washing and
cleavage solution were combined and lyophilized to afford target mole-
cules as crude products.
2
3
4
2.35 ppm (ddd, 1H; H2’b, JHH =14.1, JHH =6.0, JHH =2.0 Hz).
5’-O-(4,4’-Dimethoxytrityl)- 2’- or 3’-O-acetyl- 2’- or 3’-O-succinyl-uridine
(15): The reaction was performed according to general procedure 1 by
using 10 (1.69 g, 2.87 mmol), succinic anhydride (430 mg, 4.30 mmol),
DBU (437 mL, 2.88 mmol), acetic acid (330 mL, 5.76 mmol), and CH2Cl2
(30 mL) within a reaction time of 120 min yielding a rose solid (1.94 g,
2.82 mmol, 98%) as a mixture of 5’-O-DMTr-protected 2’-O-acetyl-3’-O-
succinyl-uridine and 3’-O-acetyl-2’-O-succinyl-uridine. Rf (CH2Cl2/
Method B: CH3OH (5 mL), H2O (2 mL), and Et3N (0.7 mL) were added
to the washed immobilized target molecule. After 16–48 h shaking at
room temperature, the resin was collected by filtration. CH3OH and
Et3N were removed in vacuo and the resin was washed with water (10ꢂ
9838
www.chemeurj.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 9832 – 9842

Solid-Phase Synthesis of (Poly)phosphorylated Nucleosides and Conjugates
FULL PAPER
CH3OH 9:1, 0.1% Et3N)=0.38; 1H NMR (400 MHz, [D6]DMSO): d=
5-Chloro-cycloSal-3’-O-succinyl-thymidine monophosphate (21a): The
reaction was performed according to general procedure 3 by using 16
(1.59 g, 4.65 mmol), DIPEA (1.62 mL, 9.29 mmol), 5-chloro-saligenyl
chlorophosphite (26) (1.35 g, 6.04 mmol), oxone (7.42 g, 12.1 mmol),
CH3CN (40 mL), and DMF (2 mL) within a reaction time of 2.5 h yield-
ing a colorless foam (1.65 g, 3.03 mmol, 65%). Rf (CH2Cl2/CH3OH 9:1,
0.1% acetic acid)=0.48; 1H NMR (400 MHz, [D6]DMSO): d=12.21 (s,
2H; 2ꢂCOOH), 11.34 (s, 2H; 2ꢂNH), 7.49–7.41 (m, 6H; 2ꢂH6, 2ꢂ
H4ar, 2ꢂH6ar), 7.18–7.15 (m, 2H; 2ꢂH3ar), 6.18–6.13 (m, 2H; 2ꢂH1’),
5.55–5.40 (m, 4H, 2ꢂCH2-benzyl), 5.20–5.16 (m, 2H; 2ꢂH3’), 4.44–4.31
(m, 4H; 2ꢂH5’), 4.16–4.11 (m, 2H; 2ꢂH4’), 2.58–2.48 (m, 8H; 2ꢂH2’’,
2ꢂH3’’), 2.39–2.33 (m, 2H; 2ꢂH2’a), 2.26 (dd, 2H; 2ꢂH2’b, 2JHH =13.0,
3JHH =5.0 Hz), 1.76 (s, 3H; CH3), 1.73 ppm (s, 3H; CH3); 31P NMR
(162 MHz, [D6]DMSO): d=À9.47 (s), À9.65 ppm (s) (2 diastereomers in
a ratio of 1.0:1.0).
3
12.27 (s, 2H; COOH), 11.45 (s, 2H; NH), 7.67 (d, 2H; H6, JHH
=
8.1 Hz), 7.39–7.22 (m, 18H; DMTr-H), 6.91–6.88 (m, 8H; DMTr-H), 5.91
(d, 1H; H1’a, 3JHH =5.5 Hz), 5.88 (d, 1H; H1’b, 3JHH =4.8 Hz), 5.54–5.48
(m, 4H; H2’, H5), 5.46–5.39 (m, 2H; H3’), 4.19–4.14 (m, 2H; H4’), 3.74
(s, 12H; 4ꢂOCH3,DMTr), 3.36 (dd, 2H; H5’a, 2JHH =10.6, 3JHH =5.0 Hz),
3.25 (dd, 2H; H5’b, 2JHH =10.8, 3JHH =3.3 Hz), 2.59–2.45 (m, 8H; C2’’,
C3’’), 2.06 (s, 3H; CH3,acetyl), 2.04 ppm (s, 3H; CH3,acetyl).
3’-O-Succinyl-thymidine (16): The reaction was performed according to
general procedure 2A by using 11 (4.93 g, 7.64 mmol), TFA (2.07 mL,
26.8 mmol), CH2Cl2/CH3OH (100 mL), and toluene (5 mL) within a re-
ACHTUNGTRENNUNGaction time of 2 h yielding a pale-orange solid (1.79 g, 5.22 mmol, 71%).
Rf (CH2Cl2/CH3OH 9:1)=0.24; 1H NMR (400 MHz, [D6]DMSO): d=
4
12.28 (s, 1H; COOH), 11.32 (s, 1H; NH), 7.74 (d, 1H; H6, JHH
=
1.2 Hz), 6.18 (dd, 1H; H1’, 3JHH =8.7, 3JHH =5.9 Hz), 5.24–5.20 (m, 2H;
H3’, H5’-OH), 3.97–3.95 (m, 1H; H4’), 3.63–3.61 (m, 2H; H5’), 2.57–2.47
5-Chloro-cycloSal-3’-O-succinyl-2’-deoxyadenosine
monophosphate
(21b): The reaction was performed according to general procedure 3 by
using 17 (359 mg, 1.02 mmol), DIPEA (355 mL, 2.04 mmol), 5-chloro-sali-
genyl chlorophosphite (26) (296 mg, 1.33 mmol), oxone (1.63 g,
2.65 mmol), CH3CN (18 mL), and DMF (2 mL) within a reaction time of
2.5 h yielding a colorless foam (417 mg, 0.753 mmol, 74%). Rf (CH2Cl2/
CH3OH 8:1, 0.1% acetic acid)=0.20; 1H NMR (400 MHz, [D6]DMSO):
d=12.26 (s, 1H; COOH), 12.25 (s, 1H; COOH), 8.30 (s, 1H; H2), 8.29
(s, 1H; H2), 8.12 (s, 1H; H8), 8.11 (s, 1H; H8), 7.41–7.32 (m, 8H; 2ꢂ
H4ar, 2ꢂH6ar, 2ꢂNH2), 7.13 (d, 1H; H3ar, 3JHH =8.7 Hz), 6.97 (d, 1H;
H3ar, 3JHH =8.8 Hz), 6.38–6.33 (m, 2H; 2ꢂH1’), 5.49–5.31 (m, 6H; 2ꢂ
CH2-benzyl, 2ꢂH3’), 4.46–4.35 (m, 4H; 2ꢂH5’), 4.23–4.22 (m, 2H; 2ꢂ
H4’), 3.12–3.06 (m, 2H; 2ꢂH2’a), 2.59–2.48 ppm (m, 10H; 2ꢂH2’’, 2ꢂ
H3’’, 2ꢂH2’b); 31P NMR (162 MHz, [D6]DMSO): d=À10.48 (s),
À10.54 ppm (s) (2 diastereomers in a ratio of 1.0:1.0).
(m, 4H; H2’’, H3’’), 2.34–2.25 (m, 1H; H2’a), 2.24–2.18 (ddd, 1H; H2’b,
4
2JHH =14.0, 3JHH =5.9, 4JHH =1.7 Hz), 1.78 ppm (d, 3H; CH3, JHH
=
1.0 Hz).
3’-O-Succinyl-2’-deoxyadenosine (17): The reaction was performed ac-
cording to general procedure 2A by using 12 (733 mg, 1.12 mmol), TFA
(260 mL, 3.37 mmol), CH2Cl2/CH3OH (15 mL), and toluene (3 mL) within
a reaction time of 1 h yielding a colorless solid (389 mg, 1.11 mmol,
99%). Rf (CH2Cl2/CH3OH 9:1)=0.18; 1H NMR (400 MHz, [D6]DMSO):
d=12.28 (s, 1H; COOH), 8.43 (s, 1H; H8), 8.22 (s, 1H; H2), 7.74 (s, 2H;
NH2), 6.37 (dd, 1H; H1’, 3JHH =8.5, 3JHH =6.0 Hz), 5.38 (d, 1H; H3’,
3JHH =5.6 Hz), 4.10–4.08 (m, 1H; H4’), 3.66 (dd, 1H; H5’a, 2JHH =12.0,
3JHH =3.9 Hz), 3.60 (dd, 1H; H5’b, 2JHH =12.0, 3JHH =4.1 Hz), 2.99–2.96
(ddd, 1H; H2’a, 2JHH =14.4, 3JHH =8.5, 3JHH =6.2 Hz), 2.60–2.44 ppm (m,
5H; H2’b, H2’’, H3’’).
5-Chloro-cycloSal-N4-(4,4’-dimethoxytrityl)-3’-O-succinyl-2’-deoxycyti-
dine monophosphate (21c): The reaction was performed according to
general procedure 3 by using 18 (374 mg, 0.594 mmol), DIPEA (207 mL,
N4-(4,4’-Dimethoxytrityl)-3’-O-succinyl-2’-deoxycytidine (18): The re-
ACHTUNGTRENNUNG
1.19 mmol),
5-chloro-saligenyl
chlorophosphite
(26)
(172 mg,
a
0.771 mmol), oxone (0.95 g, 1.55 mmol), CH3CN (23 mL), and DMF
(4 mL) within a reaction time of 3 h yielding a colorless foam (409 mg,
0.491 mmol, 83%). Rf (CH2Cl2/CH3OH 8:1, 0.1% acetic acid)=0.41;
0.524 mmol, 89%). Rf (CH2Cl2/CH3OH 8:2, 0.1% Et3N)=0.50; 1H NMR
(400 MHz, [D6]DMSO): d=12.23 (s, 1H; COOH), 8.40 (s, 1H; NH), 7.74
(d, 1H; H6, 3JHH =7.4 Hz), 7.29–7.13 (m, 9H; DMTr-H), 6.84 (d, 4H;
DMTr-H, 3JHH =8.3 Hz), 6.27 (d, 1H; H5, 3JHH =7.3 Hz), 6.08 (dd, 1H;
H1’, 3JHH =7.1, 3JHH =6.8 Hz), 5.18 (d, 1H; H3’, 3JHH =4.1 Hz), 5.12 (s,
1H; 5’-OH), 3.95–3.90 (m, 1H; H4’), 3.72 (s, 6H; 2ꢂOCH3,DMTr), 3.61–
3.51 (m, 2H; H5’), 2.55–2.47 (m, 4H; H2’’, H3’’), 2.19–2.07 ppm (m, 2H;
H2’).
1H NMR (400 MHz, [D6]DMSO): d=12.22 (s, 2H; 2ꢂCOOH), 8.43 (s,
2H; 2ꢂNH), 7.49 (d, 1H; H6, 3JHH =7.2 Hz), 7.49 (d, 1H; H6, JHH
=
3
7.5 Hz), 7.45–7.06 (m, 24H; 2ꢂH4ar, 2ꢂH6ar, 2ꢂH3ar, 18ꢂDMTr-H),
6.91–6.82 (m, 8H, 8ꢂDMTr-H), 6.25 (d, 1H; H5, 3JHH =7.6 Hz), 6.23 (d,
3
1H; H5, JHH =7.4 Hz), 6.06–6.02 (m, 2H; 2ꢂH1’), 5.54–5.38 (m, 4H; 2ꢂ
CH2-benzyl), 5.12–5.08 (m, 2H; 2ꢂH3’), 4.38–4.26 (m, 4H; 2ꢂH5’), 4.09–
4.04 (m, 2H; 2ꢂH4’), 3.72 (s, 12H; 4ꢂOCH3,DMTr), 2.56–2.45 (m, 8H; 2ꢂ
H2’’, 2ꢂH3’’), 2.17–2.14 ppm (m, 4H; 2ꢂH2’); 31P NMR (162 MHz,
[D6]DMSO): d=À10.31 (s), À10.46 ppm (s) (2 diastereomers in a ratio
of 0.9:1.0).
3’-O-Succinyl-BVdU (19): The reaction was performed according to gen-
eral procedure 2B by using 14 (270 mg, 0.367 mmol), TFA (95 mL,
1.2 mmol), CH2Cl2 (5 mL), and then CH3OH (5 mL) and toluene (5 mL)
within a reaction time of 30 min yielding a pale-orange solid (157 mg,
0.362 mmol, 99%). Rf (CH2Cl2/CH3OH 9:1)=0.10; 1H NMR (400 MHz,
[D6]DMSO): d=12.26 (s, 1H; COOH), 11.64 (s, 1H; NH), 8.08 (s, 1H;
H6), 7.26 (d, 1H; H8, 3JHH =13.6 Hz), 6.85 (d, 1H; H7, 3JHH =13.6 Hz),
5-Chloro-cycloSal-3’-O-succinyl-BVdU monophosphate (21d): The re-
AHCTUNGTREGaNNUN ction was performed according to general procedure 3 by using 19
(130 mg, 0.300 mmol), DIPEA (105 mL, 0.600 mmol), 5-chloro-saligenyl
chlorophosphite (26) (100 mg, 0.450 mmol), oxone (553 mg, 0.900 mmol),
CH3CN (5 mL), and DMF (0.6 mL) within a reaction time of 3 h yielding
a colorless foam (125 mg, 0.197 mmol, 66%). Rf (CH2Cl2/CH3OH 9:1,
0.1% acetic acid)=0.54; 1H NMR (400 MHz, [D6]DMSO): d=12.27 (s,
2H; COOH), 11.65 (s, 2H; 2ꢂNH), 7.86 (s, 1H; H6), 7.84 (s, 1H; H6),
7.44–7.37 (m, 4H; 2ꢂH4ar, 2ꢂH6ar), 7.30 (d, 1H; H8, 3JHH =13.7 Hz),
7.29 (d, 1H; H8, 3JHH =13.6 Hz), 7.16 (d, 1H; H3ar, 3JHH =8.6 Hz), 7.14
(d, 1H; H3ar, 3JHH =9.5 Hz), 6.85 (d, 1H; H7, 3JHH =13.6 Hz), 6.83 (d,
1H; H7, 3JHH =13.6 Hz), 6.17–6.11 (m, 2H; 2ꢂH1’), 5.55–5.39 (m, 4H;
2ꢂCH2-benzyl), 5.23–5.18 (m, 2H; 2ꢂH3’), 4.46–4.33 (m, 4H; 2ꢂH5’),
4.22–4.17 (m, 2H; 2ꢂH4’), 2.58–2.48 (m, 8H; 2ꢂH2’’, 2ꢂH3’’), 2.44–
2.30 ppm (m, 4H; 2ꢂH2’); 31P NMR (162 MHz, [D6]DMSO): d=À10.05
(s), À10.14 ppm (s) (2 diastereomers in a ratio of 1.0:0.7).
6.15 (dd, 1H; H1’, 3JHH =6.2, 3JHH =6.4 Hz), 5.24–5.22 (m, 2H; H3’, 5’-
OH), 4.02–4.00 (m, 1H; H4’), 3.65 (dd, 1H; H5’a, 2JHH =12.1, JHH
=
3
2
3
3.7 Hz), 3.62 (dd, 1H; H5’b, JHH =12.0, JHH =3.9 Hz), 2.57–2.41 (m, 4H;
H2’’, H3’’), 2.35–2.27 ppm (m, 2H; H2’).
2’- or 3’-O-Acetyl- 2’- or 3’-O-succinyl-uridine 20: The reaction was per-
formed according to general procedure 2B by using 15 (1.87 g,
2.72 mmol), TFA (630 mL, 8.18 mmol), CH2Cl2 (10 mL), and then
CH3OH (10 mL) and toluene (5 mL) within a reaction time of 30 min
yielding a pale-yellow solid (897 mg, 2.32 mmol, 85%) as a mixture of 2’-
O-acetyl-3’-O-succinyl-uridine and 3’-O-acetyl-2’-O-succinyl-uridine. Rf
(CH2Cl2/CH3OH 9:1)=0.20; 1H NMR (400 MHz, [D6]DMSO): d=12.27
(s, 2H; 2ꢂCOOH), 11.42 (d, 2H; 2ꢂNH, 4JHH =2.1 Hz), 7.90 (d, 1H;
H6, 3JHH =8.1 Hz), 7.89 (d, 1H; H6, 3JHH =8.2 Hz), 6.03 (d, 1H; H1’,
3JHH =6.1 Hz), 6.00 (d, 1H; H1’, 3JHH =5.6 Hz), 5.72 (dd, 1H; H5, JHH
=
3
5-Chloro-cycloSal-2’- or 3’-O-acetyl-2’- or 3’-O-succinyl-uridine mono-
phosphate (21e): The reaction was performed according to general pro-
cedure 3 by using 20 (345 mg, 0.893 mmol), DIPEA (310 mL, 1.78 mmol),
5-chloro-saligenyl chlorophosphite (26) (259 mg, 1.16 mmol), oxone
(1.43 g, 2.33 mmol), and CH3CN (12 mL) within a reaction time of 2 h
5.1, 4JHH =2.3 Hz), 5.72 (dd, 1H; H5, 3JHH =5.7, 4JHH =2.3 Hz), 5.42 (s,
2H; 2ꢂH5’-OH), 5.36–5.31 (m, 4H; 2ꢂH2’, 2ꢂH3’), 4.13–4.12 (m, 2H;
2ꢂH4’), 3.67–3.60 (m, 4H; 2ꢂH5’), 2.67–2.41 (m, 8H; 2ꢂH2’’, 2ꢂH3’’),
2.09, 2.01 ppm (2ꢂs, 6H; 2ꢂCH3,acetyl).
Chem. Eur. J. 2011, 17, 9832 – 9842
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9839

C. Meier and V. C. Tonn
yielding
a
colorless foam (403 mg, 0.685 mmol, 77%). Rf (CH2Cl2/
H3’), 4.18–4.14 (m, 3H; H4’, H5’), 2.42–2.30 (m, 2H; H2’), 1.91 ppm (d,
3H; CH3, 4JHH =0.9 Hz); 31P NMR (162 MHz, D2O): d=À9.54 (d; Pb,
CH3OH 9:1, 0.1% acetic acid)=0.20; 1H NMR (400 MHz, [D6]DMSO):
d=12.25 (s, 4H; 4ꢂCOOH), 11.48–11.45 (m, 4H; 4ꢂNH), 7.64–7.60 (m,
4H; 4ꢂH6), 7.44–7.40 (m, 8H; 4ꢂH4ar, 4<MxH6ar), 7.20–7.15 (m, 4H;
4ꢂH3ar, 3JHH =8.9 Hz), 5.87–5.83 (m, 4H; 4ꢂH1’), 5.65–5.61 (m, 4H; 4ꢂ
H5), 5.56–5.45 (m, 8H; 4ꢂCH2-benzyl), 5.43–5.39 (m, 4H; 4ꢂH2’), 5.35–
5.26 (m, 4H; 4ꢂH3’), 4.48–4.31 (m, 8H; 4ꢂH5’), 4.26–4.24 (m, 4H; 4ꢂ
H4’), 2.59–2.45 (m, 16H; 4ꢂH2’’, 4ꢂH3’’), 2.06, 2.04 ppm (s, 12H; 4ꢂ
CH3,acetyl); 31P NMR (162 MHz, [D6]DMSO): d=À10.58 (s), À10.60 ppm
(s) (2 pairs of diastereomers).
2
2JPP =20.2 Hz), À11.05 (d; Pa, JPP =20.2 Hz).
Tetra-n-butylammonium-2’-deoxyadenosine-5’-diphosphate (30b): Cyclo-
Sal-nucleotide 21b was immobilized according to general procedure 4A
with aminomethyl polystyrene 2 (75 mg, 83 mmol). By following general
procedure 5, conversion to 2’-dADP 30b was carried out with tetra-n-bu-
tylammonium dihydrogenphosphate (33) (250 mg, 746 mmol, 9.0 equiv).
Cleavage was achieved according to general procedure 6A yielding 31 mg
of 2’-dADP 30b as crude product in 88% purity that was purified by RP-
18-chromatography with (nBu)4N ions as counterions and with water as
the eluent. Analytical data were identical to those found in the literature.
1H NMR (400 MHz, D2O): d=8.42 (s, 1H; H8), 8.12 (s, 1H; H2), 6.40
5-Methylsulfonyl-cycloSal-3’-O-succinyl-thymidine
monophosphate
(22a): The reaction was performed according to general procedure 3 by
using 16 (510 mg, 1.49 mmol), DIPEA (519 mL, 2.98 mmol), 5-methylsul-
fonyl-saligenyl chlorophosphite (27) (610 mg, 2.29 mmol), oxone (2.81 g,
4.57 mmol), CH3CN (40 mL), and DMF (1.7 mL) within a reaction time
3
3
(dd, 1H; H1’, JHH =6.5, JHH =6.5 Hz), 4.79–4.72 (m, 1H; H3’), 4.21–4.10
(m, 3H; H4’, H5’), 3.11 (t, 9H; H1NBu4
H2’a), 2.57–2.52 (m, 1H; H2’b), 1.56 (tt, 9H; H2NBu4
,
3JHH =8.4 Hz), 2.78–2.69 (m, 1H;
3
,
3JHH =8.1, JHH
=
of 2.5 h yielding
a colorless foam (494 mg, 0.839 mmol, 56%). Rf
(CH2Cl2/CH3OH 9:1, 0.1% acetic acid)=0.31; 1H NMR (400 MHz,
3
7.8 Hz), 1.28 (m, 9H; H3NBu4), 0.88 ppm (t, 13.5H; H4NBu4, JHH =7.4 Hz);
31P NMR (162 MHz, D2O): d=À6.61 (d, Pb, 2JPP =22.4 Hz), À10.83 ppm
[D6]DMSO): d=12.28 (s, 2H; 2ꢂCOOH), 11.40 (s, 1H; NH), 11.39 (s,
1H; NH), 7.94–7.90 (m, 4H; 2ꢂH4ar, 2ꢂH6ar), 7.52 (d, 1H; H6, JHH
1.2 Hz), 7.49 (d, 1H; H6, JHH =1.1 Hz), 7.40 (d, 1H; H3ar, JHH =9.2 Hz),
7.37 (d, 1H; H3ar, 3JHH =8.5 Hz), 6.19–6.13 (m, 2H; 2ꢂ H1’), 5.69–5.50
(m, 4H; 2ꢂCH2-benzyl), 5.20–5.18 (m, 2H; H3’), 4.48–4.35 (m, 4H; 2ꢂ
4
2
=
(d, Pa, JPP =22.6 Hz).
4
3
Tetra-n-butylammonium-2’-deoxycytidine-5’-diphosphate (30c): CycloSal-
nucleotide 21c was immobilized according to general procedure 4A with
aminomethyl polystyrene 2 (134 mg, 147 mmol). DMTr-deprotection was
done by addition of TCA (5 mL) in CH2Cl2 (3%) to the resin and shak-
ing the mixture at room temperature for 24 h yielding 28cdepr.. After-
wards the resin was washed with CH2Cl2 (10ꢂ5 mL). By following gener-
al procedure 5, conversion to 2’-dCDP 30c was carried out with tetra-n-
butylammonium dihydrogenphosphate 33 (205 mg, 604 mmol, 4.1 equiv).
Cleavage proceeded according to general procedure 6A yielding 65 mg of
2’-dCDP 30c as crude product in 44% purity that was purified according
H5’), 4.17–4.13 (m, 2H; 2ꢂH4’), 3.22 (s, 6H; 2ꢂCH3ACHTNUTRGENNG(U SO2)), 2.56–2.47
(m, 8H; 2ꢂH2’’, 2ꢂH3’’), 2.41–2.32 (m, 2H; 2ꢂH2’a), 2.30–2.23 (ddd,
2H; 2ꢂH2’b, 2JHH =14.2, 3JHH =6.0, 4JHH =2.2 Hz), 1.75 (d, 3H; CH3,
4JHH =1.0 Hz), 1.72 ppm (d, 3H; CH3, 4JHH =1.0 Hz); 31P NMR
(162 MHz, [D6]DMSO): d=À10.39 (s), À10.56 ppm (s), (2 diastereomers
in a ratio of 1.0:0.9).
5-Methylsulfonyl-cycloSal-2’- or 3’-O-acetyl-2’- or 3’-O-succinyl-uridine
monophosphate (22b): The reaction was performed according to general
procedure 3 by using 20 (328 mg, 0.846 mmol), DIPEA (296 mL,
1.69 mmol), 5-methylsulfonyl-saligenyl chlorophosphite (27) (364 mg,
1.37 mmol), oxone (1.68 g, 2.73 mmol), and CH3CN (20 mL) within a re-
action time of 3 h yielding a colorless foam (385 mg, 0.609 mmol, 72%).
Rf (CH2Cl2/CH3OH 9:1, 0.1% acetic acid)=0.26; 1H NMR (400 MHz,
to general procedure 7. Analytical data were identical to those found in
3
the literature. 1H NMR (400 MHz, D2O): d=8.22 (d, 1H; H6, JHH
=
7.9 Hz), 6.32–6.27 (m, 2H; H1’, H5), 4.65–4.60 (m, 1H; H3’), 4.27–4.25
(m, 1H; H4’), 4.22–4.12 (m, 2H; H5’), 3.21 (q, 8H; CH2,Et3N
3JHH
7.3 Hz), 2.53–2.47 (ddd, 1H; H2’a, 2JHH =14.2, 3JHH =6.2, 3JHH =3.8 Hz),
,
=
2.42–2.35 (m, 1H; H2’b), 1.29 ppm (t, 12H; CH3,Et3N,
3JHH =7.3 Hz);
2
31P NMR (162 MHz, D2O): d=À10.83 (d; Pb, JPP =19.3 Hz), À11.38 ppm
[D6]DMSO): d=11.46–11.43 (m, 4H; 4ꢂNH), 7.93–7.91 (m, 8H; 4ꢂH4ar,
4ꢂH6ar), 7.66 (d, 2H; 2ꢂH6, 3JHH =8.1 Hz), 7.64 (d, 2H; 2ꢂH6, JHH
=
3
2
(d; Pa, JPP =19.7 Hz).
8.1 Hz), 7.40 (d, 2H; 2ꢂH3ar, 3JHH =9.2 Hz), 7.37 (d, 2H; 2ꢂH3ar, JHH
=
3
Tetra-n-butylammonium-BVdU-5’-diphosphate (30d): CycloSal-nucleo-
tide 21d was immobilized according to general procedure 4A with amino-
methyl polystyrene 2 (87 mg, 96 mmol). By following general procedure 5,
conversion to BVdUDP 30d was carried out with tetra-n-butylammoni-
um dihydrogenphosphate (33) (760 mg, 2.24 mmol, 23 equiv). Cleavage
was achieved according to general procedure 6A yielding 72 mg of
BVdUDP 30d as crude product in 91% purity. Analytical data were
identical to those found in the literature.[23]
9.2 Hz), 5.89–5.83 (m, 4H; 4ꢂH1’), 5.68–5.62 (m, 12H; 4ꢂCH2-benzyl,
4ꢂH5), 5.45–5.41 (m, 4H; 4ꢂH2’), 5.35–5.26 (m, 4H; 4ꢂH3’), 4.51–4.35
(m, 8H; 4ꢂH5’), 4.30–4.24 (m, 4H; 4ꢂH4’), 3.22 (s, 12H; 4ꢂCH3ACTHNUTRGENNU(G SO2)),
2.58–2.44 (m, 16H; 4ꢂH2’’, 4ꢂH3’’), 2.05, 2.03 ppm (s, 12H; 4ꢂ
CH3,acetyl); 31P NMR (162 MHz, [D6]DMSO): d=À10.70 (s), À10.82 ppm
(s) (2 pairs of diastereomers).
Tetra-n-butylammonium-thymidine-5’-diphosphate (30a)
Method A: 5-Chloro-substituted cycloSal-nucleotide (21a) was im-
ACHTUNGTRENNUNGmobilized according to general procedure 4A with of aminomethyl poly-
Tetra-n-butylammonium-uridine-5’-diphosphate (30e): CycloSal-nucleo-
tide 21e was immobilized according to general procedure 4B with 70 mg
of aminomethyl polystyrene 2 (77 mmol). By following general proce-
dure 5, conversion to UDP 30e was carried out with tetra-n-butylammo-
nium dihydrogenphosphate (33) (250 mg, 746 mmol, 9.7 equiv). Cleavage
was achieved according to general procedure 6B yielding 42 mg of UDP
30e as crude product in 63% purity that was purified according to gener-
al procedure 7. Analytical data were identical to those found in the litera-
ture. 1H NMR (400 MHz, D2O): d=7.93 (d, 1H; H6, 3JHH =8.2 Hz), 5.95
styrene 2 (235 mg, 259 mmol). By following general procedure 5, conver-
sion to TDP 30a was carried out with tetra-n-butylammonium dihydro-
genphosphate (33) (623 mg, 1.84 mmol, 7.1 equiv). Cleavage was ach-
ieved according to general procedure 6A yielding 123 mg of TDP 30a as
crude product in 90% purity.
Method B: 5-Chloro-substituted cycloSal-nucleotide 21a was immobilized
according to general procedure 4B with aminomethyl polystyrene
2
3
3
(d, 1H; H1’, JHH =4.6 Hz), 5.93 (d, 1H; H5, JHH =8.1 Hz), 8.35–8.34 (m,
(70 mg, 77 mmol). By following general procedure 5, conversion to TDP
30a was carried out with tetra-n-butylammonium dihydrogenphosphate
(33) (250 mg, 746 mmol, 9.7 equiv). Cleavage was achieved according to
general procedure 6A yielding 52 mg of TDP 30a as a crude product in
90% purity.
2H; H2’, H3’), 8.25 (ddd, 1H; H4’, 3JHH =5.3, 3JHH =2.8, 3JHH =2.5 Hz),
3
3
4.20 (ddd, 1H; H5’a, 2JHH =11.8, JHH =4.4, JHP =2.4 Hz), 4.15 (ddd, 1H;
H5’b, 2JHH =12.0, 3JHH =5.8, 3JHP =3.1 Hz), 3.16 (q, 10H; CH2,Et3N
7.3 Hz), 1.24 ppm (t, 15H; CH3,Et3NH
D2O): d=À8.56 (d; Pb, JPP =21.8 Hz), À11.27 ppm (d; Pa, JPP =21.9 Hz).
, =
3JHH
,
3JHH =7.3 Hz); 31P NMR (162 MHz,
2
2
Method C: 5-Methylsulfonyl-substituted cycloSal-nucleotide 22a was im-
mobilized according to general procedure 4A with aminomethyl poly-
ACHTUNGTRENNUNGstyrene 2 (87 mg, 95 mmol). By following general procedure 5, conversion
to TDP 30a was carried out with tetra-n-butylammonium dihydrogen-
phosphate (33) (250 mg, 746 mmol, 7.9 equiv). Cleavage was achieved ac-
cording to general procedure 6A yielding 63 mg of TDP 30a as crude
Tetra-n-butylammonium-thymidine-5’-triphosphate (31a): CycloSal-nu-
cleotide 21a was immobilized according to general procedure 4A with
aminomethyl polystyrene 2 (151 mg, 166 mmol). By following general pro-
cedure 5, conversion to TTP 31a was carried out with bis(tetra-n-buty-
lammonium)dihydrogenpyrophosphate
(34)
(751 mg,
1.14 mmol,
product in 81% purity. Analytical data were identical to those found in
6.9 equiv). Cleavage was achieved according to general procedure 6A
yielding 108 mg of TTP 31a as crude product in 90% purity. Analytical
data were identical to those found in the literature. 1H NMR (400 MHz,
4
the literature. 1H NMR (400 MHz, D2O): d=7.73 (d, 1H; H6, JHH
=
1.2 Hz), 6.33 (dd, 1H; H1’, 3JHH =7.2, 3JHH =6.7 Hz), 4.64–4.61 (m, 1H;
9840
www.chemeurj.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 9832 – 9842

Solid-Phase Synthesis of (Poly)phosphorylated Nucleosides and Conjugates
FULL PAPER
D2O): d=7.61 (d, 1H; H6, 4JHH =1.0 Hz), 6.19 (dd, 1H; H1’, 3JHH =6.9,
3JHH =6.8 Hz), 4.55–4.51 (m, 1H; H3’), 4.12–3.96 (m, 3H; H4’, H5’), 3.02
Tetra-n-butylammonium- or triethylammonium-b-d-glucose-thymidine-5’-
diphosphate (39)
(t, 15H; H1NBu4
CH3), 1.48 (tt, 15H; H2NBu4
,
3JHH =8.2 Hz), 2.26–2.16 (m, 2H; H2’), 1.78 (s, 3H;
Method A: 5-Chloro-substituted cycloSal-nucleotide 21a was immobilized
,
3JHH =7.8, 3JHH =7.8 Hz), 1.20 (m, 15H;
according to general procedure 4A with aminomethyl polystyrene
2
H3NBu4), 0.79 ppm (t, 22.5H; H4NBu4,
3JHH =7.4 Hz); 31P NMR (162 MHz,
(130 mg, 143 mmol). By following general procedure 5, conversion to
TDP-b-d-glucose 39 was carried out with tetra-n-butylammonium-tetra-
O-acetyl-b-d-glucose (44b) (789 mg, 1.18 mmol, 8.2 equiv) within a re-
D2O): d=À7.14 (d; Pg, 2JPP =20.4 Hz), À11.47 (d; Pa, 2JPP =20.0 Hz),
2
À22.39 (dd; Pb, 2JPP =20.4, JPP =19.8 Hz).
AHCTUNGTRENNUNG
Tetra-n-butylammonium-2’-deoxyadenosine-5’-triphosphate (31b): Cyclo-
Sal-nucleotide 21b was immobilized according to general procedure 4A
with aminomethyl polystyrene 2 (59 mg, 65 mmol). By following general
procedure 5, conversion to 2’-dATP 31b was carried out with bis(tetra-n-
butylammonium)dihydrogenpyrophosphate (34) (258 mg, 390 mmol,
6.0 equiv). Cleavage was achieved according to general procedure 6A
yielding 20 mg of 2’-dATP 31b as crude product in 78% purity. Analyti-
cal data were identical to those found in the literature.
AHCTUNGTRENNUNG
purity that was purified according to general procedure 7.
Method B: 5-Methylsulfonyl-substituted cycloSal-nucleotide 22a was im-
mobilized according to general procedure 4A with aminomethyl polystyr-
ene 2 (281 mg, 309 mmol). By following general procedure 5, conversion
to TDP-b-d-glucose 39 was carried out with triethylammonium-tetra-O-
acetyl-b-d-glucose (44a) (660 mg, 1.25 mmol, 4.0 equiv) within a reaction
time of 4 d. Cleavage was achieved according to general procedure 6B
yielding 190 mg of TDP-b-d-glucose 39 as crude product in 60% purity
that was purified according to general procedure 7. Analytical data were
identical to those found in the literature.[26] 1H NMR (400 MHz, D2O):
d=7.75 (s, 1H; H6), 6.35 (dd, 1H; H1’, 3JHH =7.2, 3JHH =6.8 Hz), 4.95
(dd, 1H; H1, 3JHH =7.8, 3JHH =7.7 Hz), 4.66–4.61 (m, 1H; H3’), 4.19–4.18
(m, 3H; H4’, H5’), 3.92 (d, 1H; H4, 3JHH =3.2 Hz), 3.82 (dd, 1H; H6a,
Tetra-n-butylammonium-BVdU-5’-triphosphate (31c): CycloSal-nucleo-
tide 21d was immobilized according to general procedure 4A with amino-
methyl polystyrene 2 (87 mg, 96 mmol). By following general procedure 5,
conversion to BVdUTP 31c was carried out with bis(tetra-n-butylammo-
nium)dihydrogenpyrophosphate (34) (1.27 g, 1.92 mmol, 20 equiv). Cleav-
age was achieved according to general procedure 6A yielding 109 mg of
BVdUTP 31c as crude product in 90% purity. Analytical data were iden-
tical to those found in the literature.[23]
3
2JHH =10.3, JHH =6.9 Hz), 3.76–3.67 (m, 3H; H3, H5, H6b), 3.64–3.59 (m,
3
1H; H2), 3.20 (q, 12H; CH2,Et3NH, JHH =7.3 Hz), 2.43–2.31 (m, 2H; H2’),
Tetra-n-butylammonium-uridine-5’-triphosphate (31d): CycloSal-nucleo-
tide 21e was immobilized according to general procedure 4A with amino-
methyl polystyrene 2 (93 mg, 102 mmol). By following general proce-
dure 5, conversion to UTP 31d was carried out with bis(tetra-n-butylam-
monium)dihydrogenpyrophosphate (34) (560 mg, 847 mmol, 8.3 equiv).
Cleavage was achieved according to general procedure 6B yielding 50 mg
of UTP 31d as crude product in 78% purity. Analytical data were identi-
cal to those found in the literature. 1H NMR (400 MHz, D2O): d=7.95
(d, 1H; H6, 3JHH =8.1 Hz), 5.99–5.95 (m, 2H; H1’, H5), 4.41–4.35 (m,
2H; H2’, H3’), 4.27–4.23 (m, 3H; H4’, H5’), 3.19–3.12 (m, 28H;
1.93 (s, 3H; CH3), 1.28 ppm (t, 18H; CH3,Et3NH,
3JHH =7.3 Hz); 31P NMR
(162 MHz, D2O): d=À11.48 (d; Pa, 2JPP =18.5 Hz), À12.97 ppm (d; Pb,
2JPP =18.2 Hz).
Triethylammonium-b-d-glucose-uridine-5’-diphosphate (40): CycloSal-nu-
cleotide 22b was immobilized according to general procedure 4A with
aminomethyl polystyrene 2 (27 mg, 30 mmol). By following general proce-
dure 5, conversion to UDP-b-d-glucose 40 was carried out with triethy-
lammonium-tetra-O-acetyl-b-d-glucose
(44a)
(77 mg,
145 mmol,
4.8 equiv) within a reaction time of 4 d. Cleavage was achieved according
to general procedure 6B yielding 15 mg of UDP-b-d-glucose 40 in 61%
purity. Analytical data were identical to those found in the literature.[26]
CH2,Et3NH, H1NBu4), 1.67–1.56 (m, 22H; H2NBu4), 1.37–1.29 (m, 22H;
H3NBu4), 1.25 (t, 9H; CH3,Et3NH
,
3JHH =7.3 Hz), 0.91 ppm (t, 33H; H4NBu4
,
2
3JHH =7.3 Hz); 31P NMR (162 MHz, D2O): d=À10.27 (d; Pg, JPP
=
Triethylammonium-a-d-galactose-thymidine-5’-diphosphate (41): Cyclo-
Sal-nucleotide 22a was immobilized according to general procedure 4A
with aminomethyl polystyrene 2 (359 mg, 395 mmol). By following gener-
al procedure 5, conversion to TDP-a-d-galactose 41 was carried out with
triethylammonium-tetra-O-acetyl-a-d-galactose (45) (720 mg, 1.36 mmol,
3.4 equiv) within a reaction time of 4 d. Cleavage was achieved according
to general procedure 6B yielding 194 mg of TDP-a-d-galactose 41 as
crude product in 43% purity that was purified according to general pro-
cedure 7. Analytical data were identical to those found in the litera-
ture.[26] 1H NMR (400 MHz, D2O): d=7.77 (s, 1H; H6), 6.39–6.35 (m,
1H; H1’), 5.63 (dd, 1H; H1, 3JHP =7.2, 3JHH =3.4 Hz), 4.68–4.63 (m, 1H;
H3’), 4.22–4.21 (m, 3H; H4’, H5’), 3.94–3.89 (m, 1H; H4), 3.89–3.85 (m,
1H; H6a), 3.83–3.78 (m, 2H; H3, H6b), 3.57–3.53 (m, 1H; H2), 3.51–3.46
21.0 Hz), À11.52 (d; Pa, 2JPP =20.3 Hz), À23.15 ppm (dd; Pb, 2JPP =20.4,
2JPP =19.5 Hz).
Tetra-n-butylammonium-thymidine-5’-b,g-methylenetriphosphate (32a):
CycloSal-nucleotide 21a was immobilized according to general procedur-
e 4A with aminomethyl polystyrene 2 (151 mg, 166 mmol). By following
general procedure 5, conversion to thymidine-5’-b,g-methylenetriphos-
phate 32a was carried out with bis(tetra-n-butylammonium)dihydrogen-
methylenpyrophosphate (35) (600 mg, 911 mmol, 5.5 equiv). Cleavage
proceeded according to general procedure 6A yielding 87 mg of thymi-
dine-5’-b,g-methylenetriphosphate 32a as crude product in 78% purity
that was purified by RP-18-chromatography with (nBu)4N ions as coun-
terions and with water as eluent. 1H NMR (400 MHz, D2O): d=7.71 (s,
1H; H6), 6.31 (dd, 1H; H1’, 3JHH =7.1, 3JHH =6.7 Hz), 4.63–4.59 (m, 1H;
H3’), 4.15–4.09 (m, 3H; H4’, H5’), 2.41–2.31 (m, 2H; H2’), 2.27–2.17 (dd,
2H; P-CH2-P, 2JHP =20.0, 2JHP =20.2 Hz), 1.89 ppm (s, 3H; CH3);
31P NMR (162 MHz, D2O): d=13.07 (m; Pg), 11.56 (m; Pb), À11.16 ppm
(m, 1H; H5), 3.23 (q, 8H; CH2,Et3NH,
3JHH =7.3 Hz), 2.43–2.38 (m, 2H;
H2’), 1.96 (s, 3H; CH3), 1.30 ppm (t, 12H; CH3,Et3NH,
3JHH =7.3 Hz);
2
31P NMR (162 MHz, D2O): d=À11.37 (d; Pa, JPP =20.4 Hz), À12.93 ppm
2
(d; Pb, JPP =20.6 Hz).
2
Tetra-n-butylammonium-thymidine-uridine-5’,5’-diphosphate (42)
(d; Pa, JPP =25.9 Hz).
Method A: 5-Chloro-substituted cycloSal-nucleotide 21a was immobilized
Tetra-n-butylammonium-uridine-5’-b,g-methylenetriphosphate (32b): Cy-
cloSal-nucleotide 21e was immobilized according to general procedur-
e 4A with aminomethyl polystyrene 2 (75 mg, 83 mmol). By following
general procedure 5, conversion to uridine-5’-b,g-methylenetriphosphate
32b was carried out with tetra-n-butylammonium dihydrogenmethylen-
pyrophosphate (35) (356 mg, 540 mmol, 6.5 equiv). Cleavage was achieved
according to general procedure 6B yielding 32 mg of 32b as crude prod-
uct in 75% purity that was purified according to general procedure 7.
1H NMR (400 MHz, D2O): d=7.97 (d, 1H; H6, 3JHH =8.2 Hz), 5.99 (d,
1H; H1’, 3JHH =3.8 Hz), 5.97 (d, 1H; H5, 3JHH =8.0 Hz), 4.41–4.37 (m,
according to general procedure 4A with aminomethyl polystyrene
2
(130 mg, 143 mmol). By following general procedure 5, conversion to thy-
midine-uridine-5’,5’-diphosphate 42 was carried out with tetra-n-butylam-
monium-uridine monophosphate (46) (600 mg, 1.06 mmol, 7.4 equiv)
within a reaction time of 4 d. Cleavage was achieved according to general
procedure 6A yielding 100 mg of thymidine-uridine-5’,5’-diphosphate 42
as crude product in 78% purity that was purified by RP-18-chromatogra-
phy with (nBu)4N ions as counterions and with water as the eluent.
Method B: 5-Methylsulfonyl-substituted cycloSal-nucleotide 22a was im-
mobilized according to general procedure 4A with aminomethyl polystyr-
ene 2 (42 mg, 46 mmol). By following general procedure 5, conversion to
thymidine-uridine-5’, 5’-diphosphate 42 was carried out with tetra-n-buty-
lammonium-uridine monophosphate (46) (180 mg, 0.318 mmol, 6.9 equiv)
within a reaction time of 5 d. Cleavage was achieved according to general
2H; H2’, H3’), 4.30–4.26 (m, 1H; H4’), 4.25–4.21 (m, 2H; H5’), 3.20 (q,
2
18H; CH2,Et3NH
20.3 Hz), 1.28 ppm (t, 27H; CH3,Et3NH
,
3JHH =7.3 Hz), 2.35 (dd, 2H; PCH2P, 2JHP =20.4, JHP
=
,
3JHH =7.3 Hz); 31P NMR
(162 MHz, D2O): d=15.36 (m; Pg), 7.98 (m; Pb), À11.20 ppm (d; Pa,
2JPP =26.4 Hz).
Chem. Eur. J. 2011, 17, 9832 – 9842
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9841

C. Meier and V. C. Tonn
procedure 6A yielding 26 mg of thymidine-uridine-5’, 5’-diphosphate 42
as crude product in 70% purity. Analytical data were identical to those
found in the literature.[23] 1H NMR (400 MHz, D2O): d=7.93 (d, 1H;
H6U, 3JHH =8.3 Hz), 7.72 (d, 1H; H6T, 4JHH =1.2 Hz), 6.34–6.30 (m, 1H;
H1’T), 5.95 (d, 1H; H5U, 3JHH =7.9 Hz), 5.94 (d, 1H; H1’U, 3JHH =4.8 Hz),
4.62–4.59 (m, 1H; H3’T), 4.36–4.34 (m, 2H; H2’U, H3’U), 4.27–4.15 (m,
[9] R. K. Gaur, B. S. Sporat, G. Krupp, Tetrahedron Lett. 1992, 33,
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6H; H4’T, H4’U, H5’T, H5’U), 2.37–2.34 (m, 2H; H2’T), 1.92 ppm (d, 3H;
[12] Y. Ahmadibeni, K. Parang, Org. Lett. 2005, 7, 5589–5592.
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2
CH3, 4JHH =1.0 Hz); 31P NMR (162 MHz, D2O): d=À11.41 (d, 1P, JPP
=
2
19.6 Hz), À11.57 ppm (d, 1P, JPP =21.5 Hz).
Tetra-n-butylammonium-2’-deoxyadenosine-uridine-5’,5’-diphosphate
(43): CycloSal-nucleotide 21b was immobilized according to general pro-
cedure 4A with aminomethyl polystyrene 2 (118 mg, 130 mmol). By fol-
lowing general procedure 5, conversion to 2’-deoxyadenosine-uridine-5’,
5’-diphosphate 43 was carried out with tetra-n-butylammonium-uridine
monophosphate (46) (485 mg, 0.858 mmol, 6.6 equiv) within a reaction
time of 5 d. Cleavage was achieved according to general procedure 6A
yielding 33 mg of 2’-deoxyadenosine-uridine-5’,5’-diphosphate 43 as crude
product that was purified according to general procedure 7. 1H NMR
(400 MHz, D2O): d=8.46 (s, 1H; H8A), 8.25 (s, 1H; H2A), 7.68 (d, 1H;
H6U, 3JHH =8.1 Hz), 6.48 (d, 1H; H1A, 3JHH =6.8 Hz), 5.83 (d, 1H; H1’U,
3JHH =4.8 Hz), 5.70 (d, 1H; H5U, 3JHH =8.0 Hz), 4.79–4.69 (m, 1H; H3A),
4.27–4.13 (m, 8H; H4A, H5A, H2’U, H3’U, H4’U, H5’U), 3.20 (q, 12H;
CH2,Et3NH
,
3JHH =7.3 Hz), 2.83 (ddd, 1H; H2’a,A, 2JHH =14.0, 3JHH =6.8,
3
3JHH =6.2 Hz), 2.58 (ddd, 1H; H2’b,A, 2JHH =14.2, 3JHH =6.2, JHH
=
3.5 Hz), 1.28 ppm (t, 18H; CH3,Et3NH,
3JHH =7.3 Hz); 31P NMR (162 MHz,
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Acknowledgements
The authors thank the University of Hamburg for financial support. V.T.
is grateful for a Ph.D. fellowship for the “Fçrderung des wissenschaftli-
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Received: April 28, 2011
Published online: July 15, 2011
9842
www.chemeurj.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 9832 – 9842