Substrates for investigation of DNA polymerase function: Synthesis and properties of 4′-C-alkylated oligonucleotides

Article abstract of DOI:10.1002/ejoc.200200641

In addition to their potential as diagnostic and therapeutic agents, modified oligonucleotides have also been shown to be highly valuable tools for examination of complex biological processes. Carefully designed nucleotide analogues have therefore found c

Full text of DOI:10.1002/ejoc.200200641

FULL PAPER
Substrates for Investigation of DNA Polymerase Function: Synthesis and
Properties of 4Ј-C-Alkylated Oligonucleotides
Ilka Detmer,[a] Daniel Summerer,[a] and Andreas Marx*[a]
Keywords: DNA / DNA structures / Nucleotides / Oligonucleotides / Wittig reactions
In addition to their potential as diagnostic and therapeutic
agents, modified oligonucleotides have also been shown to
be highly valuable tools for examination of complex biolo-
gical processes. Carefully designed nucleotide analogues
have therefore found considerable application in investi-
these analogues into oligonucleotides by use of automated
solid-phase DNA synthesis. We also studied the pairing prop-
erties of 4Ј-alkylated oligonucleotide duplexes in relation to
those of their natural counterparts under a range of buffer
conditions. In general, our studies indicate that 4Ј-alkylation
gations of DNA polymerase function and mechanism. To ex- of thymidines has little effect on nucleoside and oligonucleo-
amine the contribution of primarily steric constraints on DNA
polymerase selectivity, we have developed a new functional
strategy based on the use of modified nucleotide analogues
that differ primarily in their steric demand. Here we report
the efficient synthesis of modified thymidine analogues bear-
ing 4Ј-alkyl groups with varying steric demand, the effects
of 4Ј-alkylation on sugar puckering, and the incorporation of
tide conformation. These results have relevance to the previ-
ously reported action of 4Ј-alkylated nucleotides and oligon-
ucleotides as probes of DNA polymerase function and mech-
anism.
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2003)
Introduction
Apart from their potential as diagnostic and therapeutic
agents, such as in the antisense and antigene strategies,
modified oligonucleotides have also been shown to be
highly valuable tools for investigation of complex biological
processes.[1Ϫ4] This strategy has recently been finding con-
siderable application in investigations of DNA polymerase
function and mechanism.[5Ϫ7] Thus, nucleotide substrates
were at first carefully modified to target specific substrate
features such as hydrogen-bonding capability as precisely
as possible. Subsequent in-depth functional investigation of
various DNA polymerases gave rise to a plethora of new
functional insights difficult or even impossible to access by
other experimental means.
Recently, in order to investigate the contributions of pri-
marily steric constraints on DNA polymerase selectivity, we
have developed a new functional strategy.[8Ϫ12] This strategy
is based on the use of modified nucleotide analogues dif-
fering primarily in their steric demand. This substrate fea-
ture in the nucleotide probes was achieved through substi-
tution of the 4Ј-hydrogens of thymidines with alkyl groups
of gradually increasing steric demand (Figure 1).
Figure 1. Thymidine (left) and 4Ј-alkylated thymidine (right) build-
ing blocks used as steric probes in enzymatic investigations. T: thy-
mine, R: methyl, ethyl, isopropyl, isobutyl, respectively
Thanks to the employment of these probes in functional
enzyme investigations, a number of new insights into DNA
polymerase selectivity mechanisms have been gained. Fur-
thermore, the 4Ј-alkylated probes turned out to enhance
single mismatch discrimination through the polymerase
chain reaction (PCR) significantly.[13] This feature might be
exploitable for the development of highly accurate and ro-
bust analysis of genome variations such as point mutations
and single nucleotide polymorphisms. Here we report a de-
tailed description of the synthesis of 4Ј-alkylated thymid-
ines, the impact of 4Ј-alkylation on sugar puckering, and
the incorporation of these analogues into oligonucleotides.
We also discuss the pairing properties of 4Ј-alkylated oli-
gonucleotide duplexes in relation to those of their natural
counterparts. The experiments reported here show that site-
specifically 4Ј-alkylated oligonucleotides are capable of
building stable duplexes under varying salt conditions and
virtually independently of the position of the 4Ј-modifi-
[a]
´
Kekule-Institut für Organische Chemie und Biochemie,
Universität Bonn,
Gerhard-Domagk-Str. 1, 53121 Bonn, Germany
Fax: (internat.) ϩ 49-(0)228-73-5388
E-mail: a.marx@uni-bonn.de
Eur. J. Org. Chem. 2003, 1837Ϫ1846
DOI: 10.1002/ejoc.200200641
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1837

I. Detmer, D. Summerer, A. Marx
FULL PAPER
cation in the duplex. These findings have several impli-
4Ј-Ethylated and 4Ј-isobutylated thymidines 1b and 1d
cations for our recent studies concerning the usage of 4Ј- were synthesized in high yields from easily available alde-
modified oligonucleotides as steric probes in studies of hyde 6,[18] by sequential Wittig reaction, desilylation, and
DNA polymerase fidelity mechanisms. Thus, the features of subsequent reduction of the aliphatic double bond
4Ј-alkylated oligonucleotides described here indicate that (Scheme 3).
the described action of 4Ј-alkylated probes on DNA poly-
merases can be assigned primarily to steric effects rather
than to dramatically increased propensities of the DNA
substrates to adopt aberrant conformations or to melting
of the duplex promoted by a single 4Ј-modification.
Scheme 3. Synthesis of 4Ј-ethylthymidine 1b and 4Ј-isobutylthymid-
ine 1d; a) CH3PPh3Br, nBuLi, THF, Ϫ78 to 20 °C, 99% (R ϭ H,
Results and Discussion
7a), or (CH3)2CHPPh3I, nBuLi, Et2O, Ϫ78 to 20 °C, 83% (R ϭ
methyl, 7b); b) TBAF, THF; c) Pd/C, H2, CH3OH, 88% (1b) or
89% (1d) over two steps, respectively
Synthesis of 4Ј-Alkylated Thymidines
As reported, 4Ј-alkylated nucleotides have turned out to
be highly valuable tools for investigation of the complex
mechanisms involved in DNA polymerase selectivity. As a
Finally, the synthesis of thymidine analogue 1c, bearing
fundamental prerequisite for these studies, we have devel- a bulky isopropyl group α to the 4Ј-quaternary carbon
oped a synthetic route that allows access to the desired ana- center, was accomplished from the known ketone 8[18] by
logues in the amounts needed for the subsequent envisaged means of a Wittig reaction and subsequent desilylation and
enzyme investigations. In the past, several strategies to syn- hydrogenation of the aliphatic double bond (Scheme 4).
thesize 4Ј-modified nucleosides have been described.[14Ϫ30]
In most of the known procedures the 4Ј-quaternary carbon
center is constructed by means of a mixed aldol reaction
between aldehyde
(Scheme 1).
2 and formaldehyde to yield 3
Scheme 4. Synthesis of 4Ј-isopropylthymidine 1c; a) CH3PPh3Br,
tBuOK, THF, 91%; b) TBAF, THF; c) Pd/C, H2, CH3OH, 99%
(over two steps)
It is noteworthy that the choice of the base was crucial
for this transformation. Thus, employment of reaction con-
ditions similar to those applied in the synthesis of 1b and
1d with nBuLi resulted in little product formation. Bulky
alkoxides have previously been reported to be the bases of
choice in Wittig reactions involving sterically encumbered
substrates.[31]
In order to gain insights into potential effects of the
modifications on the sugar conformations we performed
conformational analysis based on the coupling constants
(Table 1) deduced from 1H NMR spectroscopic data re-
corded in D2O by described methods.[32] Unmodified thym-
idine 1 was found to adopt about 70% southern confor-
mation [(S)-type], while for 1aϪd approximately 50Ϫ60%
(S)-type conformations were observed, indicating that 4Ј-
alkylation had little impact on sugar puckering in solution.
Scheme 1. General scheme for the synthesis of 4Ј-modified thymid-
ines
After protection group manipulations, alcohol 4[18] is ob-
tained and serves as a basis for further functional group
interconversions. Matsuda et al. have recently followed this
route in the synthesis of several 4Ј-modified nucleosides
starting with related compounds like the alcohol 4.[15,16] En-
couraged by these findings, we envisaged the synthesis of
the desired thymidine probes bearing 4Ј-alkyl groups with
varying steric demand. Our synthesis therefore started with
alcohol 4, which was converted into iodide 5 by treatment
with I2, Ph3P, and imidazole at 50 °C. Hydrogenation with
Pd/C in the presence of Et3N and subsequent cleavage of
the silyl ethers with tetrabutylammonium fluoride (TBAF)
gave 4Ј-C-methylthymidine (1a) in good yield (Scheme 2).
Synthesis of Site-Specifically 4Ј-Alkylated Oligonucleotides
Next, in order to incorporate 4Ј-alkylated thymidines
1aϪd into oligonucleotides, 1aϪd were converted into 2-
(cyanoethyl)phosphoramidite building blocks by conversion
into the respective 4,4Ј-dimethoxytrityl ethers 10aϪd and
subsequent phosphitylation to form 11aϪd (Scheme 5).
For the synthesis of oligonucleotides bearing the 4Ј-
modified thymidines at the 3Ј-end of the strand we coupled
Scheme 2. Synthesis of 4Ј-methylthymidine 1a; a) Ph3P, I2, imidaz-
ole, C6H6, 50 °C, 85%; b) Pd/C, H2, EtOH, EtOAc, NEt3; c) TBAF,
THF, 81% (over two steps)
1838
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Eur. J. Org. Chem. 2003, 1837Ϫ1846

Substrates for Investigation of DNA Polymerase Function
FULL PAPER
1
Table 1. Selected H NMR spectroscopic data (in D2O, 500 MHz) for thymidine 1 and 1aϪd
Coupling constants J [Hz]
1Ј-H-2Ј-H
1Ј-H-2ЈЈ-H
2Ј-H-2ЈЈ-H
2Ј-H-3Ј-H
2ЈЈ-H-3Ј-H
1
6.8
7.1
6.5
7.0
6.3
6.7
5.8
6.0
6.8
6.5
n.a.[a]
14.1
14.3
5.4
6.3
6.0
6.9
6.7
5.4
6.7
7.0
4.4
6.6
1a
1b
1c
1d
14.1
n.a.[a]
[a]
n.a.: not accessible.
have recently investigated 4Ј-alkylated oligonucleotide du-
plexes in term of their abilities to form duplexes and have
found that complementary duplexes are formed, with few
overall helix deviations in relation to their unmodified
counterparts.[42] Nevertheless, the introduction of several
modified residues into oligonucleotide duplexes did de-
crease the duplex stability of the respective oligonucleotides.
Here we report on the effect of varying salt concentrations
on duplex stability.
By use of the procedure described above we first synthe-
sized oligonucleotides ON1aϪd and ON2aϪd, bearing one
or four unmodified or 4Ј-modified thymidine residues,
respectively. We subsequently performed thermal denaturat-
ing studies and measured Tm values (Table 2).
Scheme 5. Synthesis of 4Ј-alkylated building blocks suitable for
automated solid-phase DNA synthesis; a) DMTrCl, pyridine,
DMAP, 76Ϫ88%; b) (iPr2N)(NCCH2CH2O)PCl, iPr2NEt, CH2Cl2,
79Ϫ96%; c) succinylated long-chain alkylamine-modified con-
trolled pore glass (LCAA-CPG), DMAP, EDC, NEt3, pyridine;
then 4-nitrophenol, then piperidine, then acetic anhydride,
DMAP, pyridine
Table 2. Thermal denaturating experiments (Tm/°C values shown)
of unmodified and 4Ј-alkylated oligonucleotides at varied buffer
conditions
Oligonucleotides
0.1  NaCl 1  NaCl 4  NaCl
4,4Ј-dimethoxytrityl ethers 10aϪd to succinylated long-
chain alkylamine-controlled pore glass (LCAA-CPG) sup-
port by standard procedures.[33] 4Ј-Modified oligonucleot-
ides were synthesized by automated DNA synthesis from
commercially available 2-(cyanoethyl)phosphoramidites
and the modified building blocks. A standard method for 2-
(cyanoethyl)phosphoramidites was used, with the exception
that the coupling times of and from the modified nucleot-
ides were extended to 10 min. The yields for modified oli-
gonucleotides are similar to those obtained for unmodified
oligonucleotides.
5Ј-d(ATA GCTR AAG ACC)
3Ј-d(TAT CGA TTC AGG)
ON1 (R ϭ 4Ј-H)
ON1a (R ϭ 4Ј-methyl)
ON1b (R ϭ 4Ј-ethyl)
ON1c (R ϭ 4Ј-isopropyl)
ON1d (R ϭ 4Ј-isobutyl)
5Ј-d(CGC GA A TRTRC GCG)
3Ј-d(GCG CTRTRA A G CGC)
ON2 (R ϭ 4Ј-H)
ON2a (R ϭ 4Ј-methyl)
ON2b (R ϭ 4Ј-ethyl)
39.6
38.1
38.7
37.2
38.1
47.6
47.1
47.4
46.6
48.3
45.4
44.3
44.4
43.8
45.3
54.7
49.4
47.2
45.1
46.4
60.8
57.8
53.7
53.8
56.2
56.0
51.4
49.7
48.4
52.7
ON2c (R ϭ 4Ј-isopropyl)
ON2d (R ϭ 4Ј-isobutyl)
Biophysical Properties of Duplexes Containing 4Ј-Alkylated
Oligonucleotides
We next investigated the influence of 4Ј-alkyl groups in
oligonucleotides on duplex stability. The presence of inten-
Each measured profile obtained from thermal denatur-
sive hydration or metal-ion binding throughout the grooves ation studies showed a single transition. We found that the
of DNA double helices is well known from structural presence of a single modification in the helix did not signifi-
investigations.[34Ϫ39] Recent functional investigations em- cantly alter the duplex stability from that of the unmodified
ploying chemical probes constructed on the principle of oligonucleotides ON1. The Tm value was most significantly
modified nucleobases with deleted hydrogen bonding sites decreased when 4Ј-isopropylated thymidine residues (ON1c)
have suggested that minor groove hydration has impli- were present. However, four successive modified 4Ј-alkyl
cations for DNA duplex stability.[40,41] However, alkyl residues pointing into the minor groove, as present in
groups covalently attached at the 4Ј-positions of 2Ј-deoxyri- ON2aϪd, have more significant effects on duplex stability
bose residues should point towards the minor groove of (Table 2). 4Ј-Ethyl- and 4Ј-isopropyl substituents appear to
double-stranded DNA and so should interfere with ligand affect thermal stability most significantly in the sequence
binding in the groove through different mechanisms. We context investigated. Interestingly, at the highest salt con-
Eur. J. Org. Chem. 2003, 1837Ϫ1846
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1839

I. Detmer, D. Summerer, A. Marx
FULL PAPER
centration employed, 4Ј-isobutylated oligonucleotide ON2d
showed the highest stability among the modified oligonucle-
otides, indicating that the presence of potential steric con-
straints in the minor groove might be compensated by other
effects such as hydrophobic interactions. In general, the
thermal denaturating studies are a further indication that
oligonucleotides are able to form stable duplexes at high
and low salt concentrations even with disruption of the hy-
dration spine.
We have recently reported on the positional effects of
site-specifically 4Ј-methylated primer template complexes
on DNA polymerase function.[9,10,13] Thus, 4Ј-methylation
is able to decrease DNA polymerase elongation efficiency
by factors of up to several thousand times relative to the
efficiencies of the unmodified primer template complexes.
It was also found that the observed effects are strongly de-
Figure 2. Circular dichroism spectra of native and 4Ј-methylated
oligonucleotides; spectra of native oligonucleotides are displayed as
pendent on the position of the modification within the closed circles, 4Ј-methylated as open triangles; A: ON3, B: ON4,
primer template stem of the DNA substrate.[9,10] Here we
C: ON5, D: ON6
have investigated whether 4Ј-methyl modifications cause
melting or aberrant conformations of the duplex depending
mechanisms. The results indicate that the introduction of a
on differential positioning of the modifications within the
single 4Ј-methyl modification in double-stranded DNA has
DNA duplex. For these investigations we synthesized oli-
little effect on intrinsic duplex properties such as overall
gonucleotides ON3؊6, in the same sequence context as ap-
helix conformation and stability, regardless of the position
plied in our previous functional enzyme studies, and sub-
within the duplex. Thus, the effects of 4Ј-alkylated probes
sequently studied their apparent duplex properties. The
on DNA polymerase function[9,10,13] can probably be attri-
data derived from thermal denaturation studies indicate
buted primarily to steric effects rather than to dramatically
little effect on duplex stability of the position of the 4Ј-
increased propensities of the DNA substrates to adopt ab-
modification relative to that of the unmodified duplex
errant conformations or to promotion of melting of the du-
(Table 3).
plex by a single 4Ј-modification.
Table 3. Thermal denaturating experiments (Tm/°C values shown)
of oligonucleotides bearing unmodified or 4Ј-methylated thymid-
ines at different positions
Conclusion
Oligonucleotides[a]
4Ј-H
4Ј-methyl
We describe an efficient synthesis of modified thymidine
analogues bearing 4Ј-alkyl groups of varying steric demand.
These analogues adopt sugar conformations similar to
those of unmodified thymidine, as deduced from NMR
spectroscopic data analysis. The 4Ј-alkylated thymidines
were converted into building blocks suitable for automated
solid-supported oligonucleotide synthesis. Several site-
specifically 4Ј-modified oligonucleotide duplexes were syn-
thesized in yields comparable to those obtained for unmodi-
fied strands. Subsequently, duplex properties such as sta-
bility and helix conformation were investigated in relation
to those of the unmodified strands. It was found that du-
plexes comprising 4Ј-alkylated thymidine residues were able
to form stable duplexes at various salt concentrations. Ad-
ditionally, little effect of the positioning of 4Ј-methylated
thymidines within DNA duplexes was found. Taken to-
gether, these studies indicate that 4Ј-alkylation of thymid-
ines has little effect on nucleoside and oligonucleotide con-
formation.
ON3
5Ј-d(TGA CAG ACA TR)
3Ј-d(ACT GTC TGT A CT GTC TGC)
ON4
41.7
49.1
50.9
57.6
43.0
47.7
51.9
57.4
5Ј-d(TGA CAG ACA TRG)
3Ј-d(ACT GTC TGT A CT GTC TGC)
ON5
5Ј-d(TGA CAG ACA TRGA)
3Ј-d(ACT GTC TGT A CT GTC TGC)
ON6
5Ј-d(TGA CAG ACA TRGA C)
3Ј-d(ACT GTC TGT A CT GTC TGC)
[a]
Buffer contained 1  NaCl, 20 m KH2PO4 (pH ϭ 7.0).
We next investigated whether 4Ј-methylation has a signifi-
cant impact on overall DNA helix conformation and exam-
ined the circular dichroism (CD) characteristics of all native
and 4Ј-methylated oligonucleotides (Figure 2).
The corresponding duplexes bearing 4Ј-methyl groups
yield nearly superimposable CD spectra, indicating little if
any dependence of overall helix conformation on the pres-
ence of 4Ј-methyl groups at differential positions. The re-
sults reported here have several implications for our recent
studies concerning the usage of 4Ј-alkylated oligonucleot-
Experimental Section
General: All temperatures quoted are uncorrected. All reagents are
ides as steric probes in studies of DNA polymerase fidelity commercially available and were used without further purification.
1840  2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.eurjoc.org
Eur. J. Org. Chem. 2003, 1837Ϫ1846

Substrates for Investigation of DNA Polymerase Function
FULL PAPER
Solvents were purchased over molecular sieves (Fluka) and were
used directly without further purification unless otherwise noted.
All reactions were conducted with rigorous exclusion of air and
moisture. IR spectra: PerkinϪElmer Lambda 2 FTIR spectropho-
tometer. NMR spectra: Bruker with the solvent peak as internal
standard. FAB MS: Concept 1 H, matrix: 3-nitrobenzyl alcohol
(NBA) and NBA ϩ KCl. Microanalysis was performed at the Kek-
(C-2), 166.8 (C-4) ppm. IR (KBr): ν˜ ϭ 3418, 1700, 1475, 1276,
1062, 860, 775 cmϪ1. MS (FAB): m/z (%) ϭ 257 (19) [M ϩ Hϩ].
C11H16N2O5 1/3H2O (262.11): calcd: C 50.38, H 6.41; found C
50.78, H 6.61.
3Ј-O-tert-Butyldimethylsilyl-5Ј-O-tert-butyldiphenylsilyl-4Ј-C-
(vinyl)thymidine (7a): MePPh3Br (1.88 g, 5.25 mmol) was sus-
pended in dry THF (50 mL) under Ar and treated at 0 °C with
nBuLi (1.75 mL of a 2.5  solution in hexanes, 4.38 mmol). After
the mixture had been stirred for 5 min the ice bath was removed
and stirring was continued at 20 °C for 1 h. The yellow reaction
mixture was then cooled to Ϫ78 °C, aldehyde 6 (1.09 g, 1.75 mmol)
in dry THF (15 mL) was added, stirring at Ϫ78 °C was continued
for 30 min, and the mixture was then allowed to warm to 0 °C.
After stirring for a further 3 h, the reaction mixture was quenched
with concentrated NH4Cl solution and extracted with diethyl ether.
´
ule-Institut für Organische Chemie und Biochemie, Bonn Univer-
sity. Flash chromatography: Merck silica gel G60 (230Ϫ400 mesh).
Thin layer chromatography: Merck precoated plates (silica gel 60
F
254). MALDI-ToF MS analysis of oligonucleotides was conducted
by Metabion, Germany.
Abbreviations: TBDPS ϭ tert-butyldiphenylsilyl, TBS ϭ tert-
butyldimethylsilyl.
3Ј-O-tert-Butyldimethylsilyl-5Ј-O-tert-butyldiphenylsilyl-4Ј-(C-iodo-
methyl)thymidine (5): Ph3P (50.4 mg, 0.19 mmol), imidazole The combined extracts were dried over MgSO4, concentrated, and
(13.0 mg, 0.19 mmol), and 4 (100 mg, 0.16 mmol) were dissolved in purified by column chromatography on silica gel (tert-butyl methyl
dry benzene (2 mL), and iodine (45.7 mg, 0.18 mmol) was added ether/cyclohexane, 3:2) to give 7a (1.08 g, 99%) as a colorless foam.
in a stream of argon. The flask was sealed and heated to 50 °C,
and stirring was continued for 18 h. After cooling to 20 °C, the
mixture was poured into a solution of concentrated NaHCO3/
Rf ϭ 0.62 (ethyl acetate/cyclohexane, 1:1). 1H NMR (400 MHz,
CDCl3): δ ϭ 0.06 (s, 6 H, Me-Si), 0.87 (s, 9 H, tBu-Si), 1.09 (s,
9 H, tBu-Si), 1.51 (d, J ϭ 1.3 Hz, 3 H, 5-Me), 2.16 (ddd, J ϭ 13.2,
Na2S2O3 and extracted three times with CH2Cl2. The combined 7.2, 5.0 Hz, 1 H, 2ЈЈ-H), 2.28 (ddd, J ϭ 13.3, 6.7, 6.7 Hz, 1 H, 2Ј-
extracts were dried over MgSO4, concentrated, and purified by
flash column chromatography (tert-butyl methyl ether/cyclohexane,
H), 3.70 (s, 2 H, 5Ј-H), 4.74 (dd, J ϭ 6.9, 6.9 Hz, 1 H, 3Ј-H), 5.22
(dd, J ϭ 10.9, 1.8 Hz, 1 H, 4Ј-CHϪCH2), 5.44 (dd, J ϭ 17.4,
1.7 Hz, 1 H, 4Ј-CHϪCH2), 5.83 (dd, J ϭ 17.3, 10.9 Hz, 1 H, 4Ј-
1:3 Ǟ 1:1) to yield 5 (100 mg, 85%) as a colorless foam. Rf ϭ 0.46
1
(ethyl acetate/cyclohexane, 1:2). H NMR (400 MHz, CDCl3): δ ϭ CHϪCH2), 6.26 (dd, J ϭ 7.1, 5.1 Hz, 1 H, 1Ј-H), 7.33Ϫ7.44 (m, 6
0.07 (s, 3 H, SiMe), 0.08 (s, 3 H, SiMe), 0.90 (s, 9 H, SiCMe3), 1.09
(s, 9 H, SiCMe3), 1.64 (d, J ϭ 1.3 Hz, 3 H, 5-Me), 2.14 (ddd, J ϭ
H, Harom), 7.51 (q, J ϭ 1.2 Hz, 1 H, 6-H), 7.62Ϫ7.69 (m, 4 H,
H
arom), 8.72 (s, 1 H, NH) ppm. 13C NMR (101 MHz, CDCl3): δ ϭ
13.4, 8.4, 5.8 Hz, 1 H, 2ЈЈ-H), 2.36 (ddd, J ϭ 13.4, 5.7, 2.4 Hz, 1 H, Ϫ5.0 (Me-Si), Ϫ4.5 (Me-Si), 11.9 (5-Me), 17.9 (CMe3), 19.5
2Ј-H), 3.31 (d, J ϭ 11.0 Hz, 1 H, 5ЈЈ-H), 3.48 (d, J ϭ 10.9 Hz, 1 (CMe3), 25.7 (CMe3), 27.1 (CMe3), 40.6 (C-2Ј), 65.6 (C-5Ј), 71.0
H, 5Ј-H), 3.83 (d, J ϭ 1.5 Hz, 2 H, 4Ј-CH2), 4.56 (dd, J ϭ 5.7, (C-3Ј), 82.8 (C-1Ј), 89.3 (C-4Ј), 111.1 (C-5), 116.5 (4Ј-CHϪCH2),
2.4 Hz, 1 H, 3Ј-H), 6.29 (dd, J ϭ 8.5, 5.6 Hz, 1 H, 1Ј-H), 7.21 (q, 127.9, 127.9, 130.0, 130.1, 132.6, 133.0 (Carom), 134.2 (4Ј-
J ϭ 1.3 Hz, 1 H, 6-H), 7.33Ϫ7.49 (m, 6 H, Harom), 7.61Ϫ7.69 (m, CHϪCH2), 135.3 (Carom), 135.4 (C-6), 135.5 (Carom), 150.3 (C-2),
4 H, Harom), 8.26 (s, 1 H, NH) ppm. 13C NMR (101 MHz, CDCl3): 163.7 (C-4) ppm. IR (KBr): ν˜ ϭ 2930, 2857, 1699, 1469, 1428,
δ ϭ Ϫ4.9 (Me-Si), Ϫ4.6 (Me-Si), 7.6 (4Ј-CH2), 12.2 (5-Me), 18.0 1279, 1116, 1068, 939, 837, 778, 740, 703, 609 cmϪ1. MS (FAB):
(CMe3), 19.4 (CMe3), 25.7 (CMe3), 27.0 (CMe3), 41.6 (C-2Ј), 68.2 m/z (%) ϭ 621 (28) [M ϩ Hϩ]. C34H48N2O5Si2 (620.9): calcd. C
(C-5Ј), 72.6 (C-3Ј), 84.1 (C-1Ј), 86.9 (C-4Ј), 111.1 (C-5), 128.0, 65.77, H 7.79, N 4.51; found C 65.84, H 7.73; N 4.17.
128.0, 130.2, 130.2, 132.2, 132.7 (Carom), 135.0 (C-6), 135.4, 135.7
4Ј-C-(Ethyl)thymidine (1b): Nucleoside 7a (982 mg, 1.58 mmol) was
dissolved in THF (10 mL), and a nBu4NF solution in THF (1 ,
3.50 mL, 3.50 mmol) was added at 20 °C. After stirring for 3 h the
reaction mixture was poured on a silica column without further
workup. Short column chromatography (ethyl acetate Ǟ ethyl acet-
ate/methanol, 10:1) yielded the crude product as a gum that could
be used directly in the subsequent reaction. The residue was dis-
solved in methanol (15 mL) under an Ar atmosphere, and 10% Pd/
C (150 mg) was added. The reaction mixture was subsequently
floated with hydrogen (balloon). After stirring under a hydrogen
(Carom), 149.9 (C-2), 163.3 (C-4) ppm. IR (KBr): ν˜ ϭ 3049, 2928,
2855, 1692, 1471, 1113, 834, 701 cmϪ1. MS (FAB): m/z (%) ϭ 735
(81) [M ϩ Hϩ].
4Ј-(C-Methyl)thymidine (1a): Compound 5 (2.85 g, 3.87 mmol) and
10% Pd/C (1.15 g) were placed under argon in a flask, and ethyl
acetate (8.40 mL), ethanol (8.40 mL), and Et3N (0.86 mL,
6.19 mmol) were added. The reaction mixture was subsequently
floated with hydrogen (balloon). After 7 days stirring under hydro-
gen atmosphere at 20 °C, the mixture was diluted with CH2Cl2 and
filtered through a short silica column. After removal of the solvent atmosphere for 2 h, the reaction mixture was poured on a silica
the residue was employed directly in the subsequent transform- column without further workup. Short-column flash chromatogra-
ation, being dissolved in THF (26 mL) and a nBu4NF solution in phy (ethyl acetate Ǟ ethyl acetate/methanol, 10:1) yielded 1b
THF (1 , 8 mL, 8 mmol) being added at 25 °C. After stirring for (375 mg, 88%) as a colorless foam. Rf ϭ 0.16 (ethyl acetate). 1H
3 h the reaction mixture was poured on a silica column without
further workup. Flash chromatography (ethyl acetate Ǟ ethyl acet-
ate/methanol, 10:1) yielded 1a (799 mg, 81%) as a colorless foam.
NMR (400 MHz, CD3OD): δ ϭ 0.95 (dd, J ϭ 7.5, 7.5 Hz, 3 H, 4Ј-
CH2-Me), 1.58 (dq, J ϭ 14.5, 7.4 Hz, 1 H, 4Ј-CH2-Me), 1.71 (dq,
J ϭ 14.7, 7.4 Hz, 1 H, 4Ј-CH2-Me), 1.87 (d, J ϭ 1.1 Hz, 3 H, 5-
R
f ϭ 0.15 (ethyl acetate). 1H NMR (400 MHz, CD3OD): δ ϭ 1.15 Me), 2.29 (ddd, J ϭ 13.6, 6.6, 5.0 Hz, 1 H, 2ЈЈ-H), 2.33 (ddd, J ϭ
(s, 3 H, Me), 1.86 (q, J ϭ 1.1 Hz, 3 H, 5-Me), 2.32 (dd, J ϭ 6.2, 13.6, 6.5, 6.5 Hz, 1 H, 2Ј-H), 3.57 (d, J ϭ 11.8 Hz, 1 H, 5ЈЈ-H),
6.2 Hz, 1 H, 2ЈЈ-H), 2.32 (dd, J ϭ 6.2, 6.2 Hz, 1 H, 2Ј-H), 3.55 (d, 3.67 (d, J ϭ 11.8 Hz, 1 H, 5Ј-H), 4.45 (dd, J ϭ 6.4, 5.0 Hz, 1 H,
J ϭ 11.8 Hz, 1 H, 5ЈЈ-H), 3.59 (d, J ϭ 11.8 Hz, 1 H, 5Ј-H), 4.39
(dd, J ϭ 5.9, 5.9 Hz, 1 H, 3Ј-H), 6.19 (dd, J ϭ 6.3, 6.3 Hz, 1 H,
3Ј-H), 6.19 (dd, J ϭ 6.5, 6.5 Hz, 1 H, 1Ј-H), 7.87 (q, J ϭ 1.2 Hz,
1 H, 6-H) ppm. 13C NMR (101 MHz, CD3OD): δ ϭ 8.8 (4Ј-CH2-
1Ј-H), 7.88 (q, J ϭ 1.2 Hz, 1 H, 6-H) ppm. 13C NMR (101 MHz, Me), 12.7 (5-Me), 25.3 (4Ј-CH2-Me), 41.8 (C-2Ј), 65.3 (C-5Ј), 73.0
CD3OD): δ ϭ 12.7 (5-Me), 18.2 (4Ј-Me), 41.4 (C-2Ј), 67.8 (C-5Ј),
(C-3Ј), 85.5 (C-1Ј), 91.1 (C-4Ј), 111.6 (C-5), 138.7 (C-6), 152.7 (C-
72.6 (C-3Ј), 85.3 (C-1Ј), 89.3 (C-4Ј), 111.6 (C-5), 138.7 (C-6), 152.7 2), 166.8 (C-4) ppm. IR (KBr): ν˜ ϭ 3443, 2927, 1687, 1473, 1272,
Eur. J. Org. Chem. 2003, 1837Ϫ1846
www.eurjoc.org
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1841

I. Detmer, D. Summerer, A. Marx
FULL PAPER
1070, 776 cmϪ1. MS (FAB): m/z (%) ϭ 271 (18) [M ϩ Hϩ]. °C, compound 6 (600 mg, 0.96 mmol) in dry diethyl ether (5 mL)
C12H18N2O5 (270.3): calcd. C 53.33, H 6.71; found C 53.01, H 6.73. was added, stirring was continued at Ϫ78 °C for 15 min, and the
reaction mixture was subsequently allowed to warm to 20 °C. After
3Ј-O-tert-Butyldimethylsilyl-5Ј-O-tert-butyldiphenylsilyl-4Ј-(C-iso-
propenyl)thymidine (9): MePPh3Br (2.00 g, 5.60 mmol) and tBuOK
(550 mg, 4.90 mmol) were suspended in dry THF (20 mL) under
Ar and stirred at 20 °C for 1 h. Compound 8 (890 mg, 1.40 mmol)
stirring for a further 30 min, the reaction mixture was cooled to
Ϫ78 °C and afterwards quenched with concentrated NH4Cl solu-
tion. After warming to 20 °C, the mixture was extracted with
CH2Cl2. The combined extracts were dried over MgSO4, concen-
in dry THF (5 mL) was then added to the reaction mixture. After
trated, and purified by flash column chromatography (tert-butyl
stirring for 14 h at 20 °C, the reaction mixture was quenched with
methyl ether/cyclohexane, 1:4 Ǟ 1:3) to yield 7b (522 mg, 83%) as
concentrated NH4Cl solution and extracted with CH2Cl2. The
a colorless foam. Rf ϭ 0.14 (tert-butyl methyl ether/cyclohexane,
combined extracts were dried over MgSO4, concentrated, and puri-
fied by column chromatography on silica gel (ethyl acetate/cyclo-
1
1:4). H NMR (400 MHz, CDCl3): δ ϭ 0.07 (s, 3 H, Me-Si), 0.08
(s, 3 H, Me-Si), 0.88 (s, 9 H, tBu-Si), 1.08 (s, 9 H, tBu-Si), 1.53 (d,
J ϭ 1.3 Hz, 3 H, 5-Me), 1.67 (d, J ϭ 1.4 Hz, 3 H, 4Ј-CHCMe2),
1.72 (d, J ϭ 1.3 Hz, 3 H, 4Ј-CHCMe2), 2.10 (ddd, J ϭ 13.2, 7.8,
hexane, 1:3) to give 9 (810 mg, 91%) as a colorless foam. Rf ϭ 0.31
1
(ethyl acetate/cyclohexane, 1:3). H NMR (400 MHz, CDCl3): δ ϭ
Ϫ0.02 (s, 3 H, Me-Si), 0.04 (s, 3 H, Me-Si), 0.85 (s, 9 H, tBuϪSi),
4.5 Hz, 1 H, 2ЈЈ-H), 2.27 (ddd, J ϭ 13.3, 7.6, 7.6 Hz, 1 H, 2Ј-H),
1.09 (s, 9 H, tBuϪSi), 1.60 (q, J ϭ 1.6 Hz, 3 H, 5-Me), 1.66 (m, 3
3.59 (d, J ϭ 11.8 Hz, 1 H, 5ЈЈ-H), 3.85 (d, J ϭ 11.6 Hz, 1 H, 5Ј-
H, 4Ј-CCH2Me), 2.26 (ddd, J ϭ 12.8, 6.1, 3.3 Hz, 1 H, 2ЈЈ-H), 2.30
H), 4.67 (dd, J ϭ 7.7, 7.7 Hz, 1 H, 3Ј-H), 5.30 (dq, J ϭ 1.4, 1.4 Hz,
(ddd, J ϭ 12.8, 7.7, 5.0 Hz, 1 H, 2Ј-H), 3.81 (d, J ϭ 11.2 Hz, 1 H,
1 H, 4Ј-CHCMe2), 6.09 (dd, J ϭ 7.6, 4.4 Hz, 1 H, 1Ј-H), 7.32Ϫ7.45
5ЈЈ-H), 3.94 (d, J ϭ 11.4 Hz, 1 H, 5Ј-H), 4.44 (dd, J ϭ 4.8, 3.4 Hz,
(m, 6 H, Harom), 7.39 (q, J ϭ 1.3 Hz, 1 H, 6-H), 7.64Ϫ7.70 (m, 4
1 H, 3Ј-H), 4.87 (m, 2 H, 4Ј-CCH2Me), 6.34 (dd, J ϭ 7.6, 6.1 Hz,
H, Harom), 8.18 (s, 1 H, NH) ppm. 13C NMR (101 MHz, CDCl3):
1 H, 1Ј-H), 7.35Ϫ7.47 (m, 6 H, Harom), 7.61Ϫ7.66 (m, 4 H, Harom),
δ ϭ Ϫ4.9 (Me-Si), Ϫ4.5 (Me-Si), 11.9 (5-Me), 18.0 (CMe3), 18.6
7.67 (q, J ϭ 1.5 Hz, 1 H, 6-H), 8.14 (s, 1 H, NH) ppm. 13C NMR
(4Ј-CHCMe2), 19.5 (CMe3), 25.7 (CMe3), 27.1 (CMe3), 27.6 (4Ј-
(101 MHz, CDCl3): δ ϭ Ϫ5.1 (Me-Si), Ϫ4.4 (Me-Si), 12.0 (5-Me),
CHCMe2), 40.1 (C-2Ј), 64.1 (C-5Ј), 70.1 (C-3Ј), 82.2 (C-1Ј), 88.0
17.9 (CMe3), 19.4 (CMe3), 20.9 (4Ј-CCH2Me), 25.6 (CMe3), 27.1
(C-4Ј), 110.9 (C-5), 118.4 (4Ј-CHCMe2), 127.8, 127.9, 129.9, 130.0,
(CMe3), 41.7 (C-2Ј), 67.6 (C-5Ј), 74.3 (C-3Ј), 84.5 (C-1Ј), 92.9 (C-
132.9, 133.2, 135.4, 135.5 (Carom), 135.7 (C-6), 139.6 (4Ј-CHCMe2),
4Ј), 110.8 (C-5), 112.5 (4Ј-CCH2Me), 128.0, 128.0, 130.1, 130.3,
150.1 (C-2), 163.4 (C-4) ppm. IR (KBr): ν˜ ϭ 3050, 2929, 2857,
132.4, 132.8, 135.4, 135.6 (Carom), 135.7 (C-6), 143.0 (4Ј-CCH2Me),
1701, 1470, 1428, 1279, 1113, 1062, 1008, 949, 838, 779, 741, 702,
150.1 (C-2), 163.5 (C-4) ppm. MS (FAB): m/z (%) ϭ 635 (26) [M
611 cmϪ1
. MS (FAB): m/z (%) ϭ 649 (18) [M ϩ
Hϩ].
ϩ Hϩ]. C35H50N2O5Si2 (635.0): calcd. C 66.21, H 7.94, N 4.41;
C36H52N2O5Si2 (649.0): calcd. C 66.63, H 8.08, N 4.32; found C
66.78, H 8.08, N 3.98.
found C 66.24, H 7.94, N 4.21.
4Ј-C-(Isopropyl)thymidine (1c): Compound 9 (768 mg, 1.21 mmol)
was dissolved in THF (10 mL) and a nBu4NF solution in THF (1
, 2.70 mL, 2.70 mmol) was added at 20 °C. After stirring for 3 h,
the reaction mixture was poured on a silica column without further
workup. Short-column chromatography (ethyl acetate Ǟ ethyl acet-
ate/methanol, 10:1) yielded the crude product as a gum that could
be used directly in the subsequent reaction. The residue was dis-
solved in methanol (15 mL) under an Ar atmosphere, and 10% Pd/
C (150 mg) was added. The reaction mixture was subsequently
floated with hydrogen (balloon). After stirring under a hydrogen
atmosphere for 2 h, the reaction mixture was poured on a silica
column without further workup. Short-column chromatography
(ethyl acetate/cyclohexane, 10:1 Ǟ ethyl acetate/methanol, 10:1)
yielded 1c (342 mg, 99%) as a colorless foam. Rf ϭ 0.23 (ethyl acet-
ate). 1H NMR (300 MHz, CD3OD): δ ϭ 1.00 (d, J ϭ 7.0 Hz, 3 H,
4Ј-CHMe2), 1.03 (d, J ϭ 7.2 Hz, 3 H, 4Ј-CHMe2), 1.83 (d, J ϭ
1.2 Hz, 3 H, 5-Me), 2.17Ϫ2.29 (m, 1 H, 4Ј-CHMe2), 2.23 (ddd, J ϭ
13.2, 6.0, 2.7 Hz, 1 H, 2ЈЈ-H), 2.42 (ddd, J ϭ 13.3, 8.2, 6.0 Hz, 1
H, 2Ј-H), 3.72 (s, 2 H, 5Ј-H), 4.54 (dd, J ϭ 6.1, 2.7 Hz, 1 H, 3Ј-
H), 6.19 (dd, J ϭ 8.3, 5.9 Hz, 1 H, 1Ј-H), 7.90 (q, J ϭ 1.2 Hz, 1
H, 6-H) ppm. 13C NMR (75 MHz, CD3OD): δ ϭ 12.8 (5-Me), 18.7
(4Ј-CHMe2), 19.3 (4Ј-CHMe2), 32.3 (4Ј-CHMe2), 43.1 (C-2Ј), 64.1
(C-5Ј), 74.4 (C-3Ј), 86.1 (C-1Ј), 93.2 (C-4Ј), 111.6 (C-5), 139.1 (C-
6), 152.9 (C-2), 166.8 (C-4) ppm. IR (KBr): ν˜ ϭ 3444, 2964, 1686,
1474, 1275, 1206, 1079, 973, 869, 775 cmϪ1. MS (FAB): m/z (%) ϭ
285 (30) [M ϩ Hϩ]. C13H20N2O5·0.5H2O (293.3): calcd. C 53.23,
H 7.22, N 9.55; found C 53.51, H 7.36, N 9.29.
4Ј-C-(Isobutyl)thymidine (1d): Compound 7b (1.07 g, 1.65 mmol)
was dissolved in THF (15 mL), and a nBu4NF solution in THF (1
, 3.63 mL, 3.63 mmol) was added at 20 °C. After stirring for 4 h,
the reaction mixture was poured on a silica column without further
workup. Short-column chromatography (ethyl acetate Ǟ ethyl acet-
ate/methanol, 10:1) yielded the crude product as a gum that could
be used directly in the subsequent reaction. The gum was dissolved
in methanol (15 mL) under an argon atmosphere, and 10% Pd/C
(150 mg) was added. The reaction mixture was subsequently
floated with hydrogen (balloon). After stirring under a hydrogen
atmosphere for 48 h, the reaction mixture was poured on a silica
column without further workup. Short column chromatography
(ethyl acetate/cyclohexane, 10:1 Ǟ ethyl acetate/methanol, 10:1)
yielded 1d (438.7 mg, 89%) as a colorless foam. Rf ϭ 0.19 (ethyl
1
acetate). H NMR (300 MHz, CD3OD): δ ϭ 0.96 [d, J ϭ 6.6 Hz,
3
H, 4Ј-CH2ϪCH(Me)2], 0.99 [d,
J
ϭ
6.6 Hz,
3
1
1
H, 4Ј-
H, 4Ј-
H, 4Ј-
CH2ϪCH(Me)2], 1.46 [dd,
CH2ϪCH(Me)2], 1.55 [dd,
J
J
ϭ
14.8, 5.2 Hz,
14.9, 7.2 Hz,
ϭ
CH2ϪCH(Me)2], 1.78 [dddq, J ϭ 6.7, 6.8, 6.8, 5.0 Hz, 1 H, 4Ј-
CH2ϪCH(Me)2], 1.83 (d, J ϭ 1.3 Hz, 3 H, 5-Me), 2.30 (dd, J ϭ
6.2, 6.2 Hz, 1 H, 2ЈЈ-H), 2.30 (dd, J ϭ 6.2, 6.2 Hz, 1 H, 2Ј-H), 3.58
(d, J ϭ 11.5 Hz, 1 H, 5ЈЈ-H), 3.75 (d, J ϭ 11.7 Hz, 1 H, 5Ј-H), 4.47
(dd, J ϭ 6.0, 6.0 Hz, 1 H, 3Ј-H), 6.18 (dd, J ϭ 6.4, 6.4 Hz, 1 H,
1Ј-H), 7.90 (q, J ϭ 1.2 Hz, 1 H, 6-H) ppm. 13C NMR (75 MHz,
CD3OD): δ ϭ 12.8 (5-Me), 24.9 [4Ј-CH2ϪCH(Me)2], 25.5 [4Ј-
CH2ϪCH(Me)2],
25.8
[4Ј-CH2ϪCH(Me)2],
40.7
[4Ј-
CH2ϪCH(Me)2], 41.3 (C-2Ј), 65.6 (C-5Ј), 73.1 (C-3Ј), 85.3 (C-1Ј),
90.9 (C-4Ј), 111.5 (C-5), 138.7 (C-6), 152.7 (C-2), 166.8 (C-4) ppm.
3Ј-O-tert-Butyldimethylsilyl-5Ј-O-tert-butyldiphenylsilyl-4Ј-C-(2-
methylpropenyl)thymidine (7b): Isopropylphosphonium iodide
(1.78 g, 4.12 mmol) was suspended in dry diethyl ether (20 mL) un-
der an Ar atmosphere, and the mixture was treated at 20 °C with
IR (KBr): ν˜ ϭ 3423, 2955, 1 687, 1473, 1273, 1066, 963, 778 cmϪ1
.
MS (FAB): m/z (%) ϭ 299 (6) [M ϩ Hϩ]. C14H22N2O5 (298.3):
calcd. C 56.36, H 7.43, N 9.39; found C 56.08, H 7.61, N 9.13.
nBuLi (1.34 mL of a 2.5  solution in hexanes, 3.36 mmol). After General Procedure for 5Ј-O-Dimethoxytritylation of 4Ј-Modified
stirring for 45 min the dark red reaction mixture was cooled to Ϫ78 Thymidines 1aϪd: The respective nucleoside 1aϪd was coevapo-
1842
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2003, 1837Ϫ1846

Substrates for Investigation of DNA Polymerase Function
FULL PAPER
rated twice with pyridine and dissolved in pyridine (5 mL/mmol),
and 4,4Ј-dimethoxytrityl chloride (1.5Ϫ2.0 equiv.) and a catalytic
amount of 4-(dimethylamino)pyridine were then added at 20 °C.
After TLC analysis indicated complete consumption of the starting
material (3Ϫ5 h), the reaction was quenched by addition of access
methanol and stirring was continued for 30 min. The reaction mix-
ture was then poured into saturated NaHCO3 solution and ex-
tracted with CH2Cl2. The combined extracts were dried over
MgSO4, concentrated, and purified by column chromatography on
silica gel (ethyl acetate/cyclohexane, 2:1 Ǟ 3:1 containing 1% Et3N)
to yield the product as a colorless foam.
(C-5), 113.4 (Carom), 127.9, 128.1, 128.3, 129.0, 135.3, 135.5 (Carom),
135.8 (C-6), 144.7 (Carom), 150.5 (C-2), 158.3 (Carom), 163.8 (C-4)
ppm. IR (KBr): ν˜ ϭ 3442, 2956, 2834, 1690, 1607, 1509, 1465,
1251, 1176, 1033, 828, 755, 729, 697 cmϪ1. MS (FAB): m/z (%) ϭ
586 (14) [M ϩ H]ϩ. C34H38N2O7·0.75 H20 (600.2): calcd. C 68.04,
H 6.59, N 4.67; found C 68.02, H 6.84, N 4.48.
5Ј-O-Dimethoxytrityl-4Ј-C-(isobutyl)thymidine (10d): Compound
1d (100 mg, 0.36 mmol) was converted into 10d (165 mg, 75%).
Rf ϭ 0.42 (ethyl acetate/cyclohexane, 3:1). 1H NMR (400 MHz,
[D6]DMSO): δ ϭ 0.75 (d, J ϭ 6.2 Hz, 3 H, 4Ј-CH2ϪCH(Me)2],
0.77 [d, J ϭ 6.2 Hz, 3 H, 4Ј-CH2ϪCH(Me)2], 1.37 (d, J ϭ 1.1 Hz,
5Ј-O-Dimethoxytrityl-4Ј-C-(methyl)thymidine (10a): Compound 1a 3 H, 5-Me), 1.40Ϫ1.53 [m, 3 H, 4Ј-CH2ϪCH(Me)2], 2.19 (ddd, J ϭ
(200 mg, 0.78 mmol) was converted into 10a (383 mg, 88%). Rf ϭ
0.23 (ethyl acetate/cyclohexane, 2:1). 1H NMR (400 MHz,
[D6]DMSO): δ ϭ 1.08 (s, 3 H, 4Ј-Me), 1.42 (d, J ϭ 1.1 Hz, 3 H, 5-
13.5, 6.7, 5.1 Hz, 1 H, 2ЈЈ-H), 2.23Ϫ2.31 (m, 1 H, 2Ј-H), 3.03 (d,
J ϭ 9.5 Hz, 1 H, 5ЈЈ-H), 3.16 (d, J ϭ 9.5 Hz, 1 H, 5Ј-H), 3.73 (s, 6
H, Me-O), 4.52 (dd, J ϭ 6.8, 5.1 Hz, 1 H, 3Ј-H), 5.25 (s, 1 H, OH),
Me), 2.22Ϫ2.29 (m, 2 H, 2Ј-H), 3.02 (d, J ϭ 9.9 Hz, 1 H, 5Ј-H), 6.07 (dd, J ϭ 6.6, 6.6 Hz, 1 H, 1Ј-H), 6.86Ϫ6.91 (m, 4 H, Carom),
3.07 (d, J ϭ 9.9 Hz, 1 H, 5ЈЈ-H), 3.74 (s, 6 H, Me-O), 4.37 (dd, J ϭ
7.21Ϫ7.41 (m, 9 H, Carom), 7.48 (q, J ϭ 1.3 Hz, 1 H, 6-H), 11.25
11.6, 6.3 Hz, 1 H, 3Ј-H), 5.24 (d, J ϭ 5.2 Hz, 1 H, OH), 6.10 (dd, (s, 1 H, NH) ppm. 13C NMR (101 MHz, [D6]DMSO): δ ϭ 11.8 (5-
J ϭ 6.4, 6.4 Hz, 1 H, 1Ј-H), 6.85Ϫ6.91 (m, 4 H, Carom), 7.23Ϫ7.42 Me), 23.6 [4Ј-CH2ϪCH(Me)2], 24.0 [4Ј-CH2ϪCH(Me)2], 25.0 [4Ј-
(m, 9 H, Carom), 7.45 (q, J ϭ 1.3 Hz, 1 H, 6-H), 11.27 (s, 1 H, NH)
CH2-CH(Me)2], 55.2 (OMe), 65.9 (C-5Ј), 71.7 (C-3Ј), 82.7 (C-1Ј),
86.2 (C-4Ј), 87.9 [CPh(C6H4OMe)2], 109.5 (C-5), 113.4 (Carom),
ppm. 13C NMR (101 MHz, [D6]DMSO): δ ϭ 12.1 (5-Me), 18.5 (4Ј-
Me), 55.5 (OMe), 60.2 (C-5Ј), 71.2 (C-3Ј), 82.8 (C-1Ј), 86.2 (C-4Ј), 127.0, 127.9, 128.0, 129.9, 135.5, 135.5, (Carom), 135.7 (C-6), 144.8
86.6 [CPh(C6H4OMe)2], 109.7 (C-5), 113.6 (Carom), 127.2, 128.2, (Carom), 150.5 (C-2), 158.3 (Carom), 163.8 (C-4) ppm. IR (KBr): ν˜ ϭ
128.3, 130.2, 135.7, 135.9 (Carom), 136.1 (C-6), 145.2 (Carom), 150.8 3480, 2951, 1698, 1607, 1509, 1471, 1298, 1251, 1176, 1131, 1033,
(C-2), 158.6 (Carom), 164.1 (C-4) ppm. IR (KBr): ν˜ ϭ 3554, 3037,
966, 901, 830, 755, 699 cmϪ1. MS (FAB): m/z (%) ϭ 600 (17) [Mϩ].
2835, 1694, 1606, 1509, 1471, 1371, 1299, 1250, 1176, 1117, 1081, C34H38N2O7. 0.25 H2O (605.2): calcd. C 69.45, H 6.75, N 4.63;
1034, 831, 754, 700 cmϪ1. MS (FAB): m/z (%) ϭ 558 (25) [M ϩ found C 69.55, H 6.75, N 4.30.
H]ϩ. C32H34N2O7 (558.6): calcd. C 68.80, H 6.13, N 5.01; found C
General Procedure for the Synthesis of Phosphoramidites 11aϪd:
The respective nucleoside 10aϪd was coevaporated twice with tolu-
68.49, H 6.10, N 4.86.
5Ј-O-Dimethoxytrityl-4Ј-C-(ethyl)thymidine (10b): Compound 1b
(205 mg, 0.76 mmol) was converted into 10b (332 mg, 76%). Rf ϭ
ene and then dissolved in CH2Cl2 (10 mL/mmol), and N-ethyl di-
isopropylamine (5.0 equiv.) and 2-cyanoethyl-N,N-(diisopropylami-
0.26 (ethyl acetate/cyclohexane, 3:1). 1H NMR (400 MHz, no)chlorophosphite (2.0 equiv.) were then added at 20 °C. After
[D6]DMSO): δ ϭ 0.76 (t, J ϭ 7.5 Hz, 3 H, 4Ј-CH2Me), 1.50 (d, TLC analysis indicated complete consumption of the starting mate-
J ϭ 1.1 Hz, 3 H, 5-Me), 1.66 (m, 2 H, 4Ј-CH2Me), 2.25 (ddd, J ϭ
rial (3Ϫ5 h), the reaction mixture was quenched by addition of con-
13.6, 6.6, 4.6 Hz, 1 H, 2ЈЈ-H), 2.35 (ddd, J ϭ 13.6, 6.8, 6.8 Hz, 1 centrated NaHCO3 solution and extracted with CH2Cl2. The com-
H, 2Ј-H), 3.06 (s, 2 H, 5Ј-H), 3.74 (s, 6 H, Me-O), 4.50 (ddd, J ϭ
6.8, 4.9, 4.9 Hz, 1 H, 3Ј-H), 5.30 (d, J ϭ 5.2 Hz, 1 H, OH), 6.16
bined extracts were dried over MgSO4, concentrated, and purified
by column chromatography on silica gel (ethyl acetate/cyclohexane,
(dd, J ϭ 6.7, 6.7 Hz, 1 H, 1Ј-H), 6.85Ϫ6.92 (m, 4 H, Carom), 2:1 Ǟ 3:1 containing 1% Et3N) to yield the product as a colorless
7.22Ϫ7.41 (m, 9 H, Carom), 7.44 (q, J ϭ 1.3 Hz, 1 H, 6-H), 11.16
(s, 1 H, NH) ppm. 13C NMR (101 MHz, [D6]DMSO): δ ϭ 8.5 (4Ј-
CH2Me), 12.2 (5-Me), 24.7 (4Ј-CH2Me), 55.5 (OMe), 66.1 (C-5Ј),
72.0 (C-3Ј), 83.2 (C-1Ј), 86.3 (C-4Ј), 88.4 [CPh(C6H4OMe)2], 109.8
(C-5), 113.6 (Carom), 127.2, 128.2, 128.3, 130.2, 135.7, 135.9, (Ca-
rom), 136.1 (C-6), 145.2 (Carom), 150.8 (C-2), 158.6 (Carom), 164.1
(C-4) ppm. IR (KBr): ν˜ ϭ 2927, 1686, 1607, 1509, 1464, 1251,
1176, 1034, 828, 700 cmϪ1. MS (FAB): m/z (%) ϭ 572 (13) [M ϩ
H]ϩ. C33H36N2O7 (572.7): calcd. C 69.21, H 6.34, N 4.89; found C
69.00, H 6.66, N 4.58.
foam.
Thymidine Derivative (11a): Compound 10a (350 mg, 0.63 mmol)
was converted into 11a (383 mg, 81%).
Diastereomer a: Rf ϭ 0.47 (ethyl acetate/cyclohexane, 2:1). 1H
NMR (400 MHz, [D6]acetone): δ ϭ 1.19 (d, J ϭ 4.6 Hz, 6 H,
NϪCHMe2), 1.20 (s, 3 H, 4Ј-Me), 1.21 (d, J ϭ 4.6 Hz, 6 H,
NϪCHMe2), 1.46 (d, J ϭ 1.3 Hz, 3 H, 5-Me), 2.51 (dd, J ϭ 6.5,
6.5 Hz, 1 H, 2ЈЈ-H), 2.51 (dd, J ϭ 6.5, 6.5 Hz, 1 H, 2Ј-H), 2.62 (dd,
J ϭ 6.1, 6.0 Hz, 2 H, CH2CN), 3.25 (d, J ϭ 10.0 Hz, 1 H, 5ЈЈ-H),
3.30 (d, J ϭ 10.0 Hz, 1 H, 5Ј-H), 3.60Ϫ3.77 [m, 4 H, N(CHMe2)2,
5Ј-O-Dimethoxytrityl-4Ј-C-(isopropyl)thymidine (10c): Compound POCH2], 3.79 (s, 6 H, Me-O), 4.86 (ddd, J ϭ 10.7, 6.6, 6.6 Hz, 1
1c (200 mg, 0.70 mmol) was converted into 10c (329 mg, 80%).
H, 3Ј-H), 6.24 (dd, J ϭ 6.3, 6.3 Hz, 1 H, 1Ј-H), 6.85Ϫ6.91 (m, 4
Rf ϭ 0.40 (ethyl acetate/cyclohexane, 3:1). 1H NMR (400 MHz, H, Carom), 7.23Ϫ7.42 (m, 9 H, Carom), 7.59 (q, J ϭ 1.2 Hz, 1 H, 6-
[D6]DMSO): δ ϭ 0.78 [d, J ϭ 7.0 Hz, 3 H, 4Ј-CH(Me)2], 0.81 [d, H) ppm. 13C NMR (101 MHz, [D6]acetone): δ ϭ 11.2 (5-Me), 18.2
J ϭ 7.0 Hz, 3 H, 4Ј-CH(Me)2], 1.39 (d, J ϭ 1.1 Hz, 3 H, 5-Me),
2.10Ϫ2.24 [m, 2 H, 2ЈЈ-H, 4Ј-CH(Me)2], 2.35 (ddd, J ϭ 13.4, 8.0,
(4Ј-Me), 19.8 (CH2CN), 23.9 [N(CHMe2)2], 38.6 (C-2Ј), 42.9
[N(CHMe2)2], 54.5 (OMe), 58.6 (CH2OP), 67.1 (C-5Ј), 72.8 (C-3Ј),
6.0 Hz, 1 H, 2Ј-H), 3.08 (d, J ϭ 10.1 Hz, 1 H, 5ЈЈ-H), 3.22 (d, J ϭ 82.8 (C-1Ј), 86.1 (C-4Ј), 86.3 [CPh(C6H4OMe)2], 109.8 (C-5), 113.0
10.2 Hz, 1 H, 5Ј-H), 3.72 (s, 6 H, Me-O), 4.22 (m, 1 H, 3Ј-H), 5.24
(Carom), 117.8 (CN), 126.8, 127.7, 128.2, 130.1, (Carom), 135.4 (C-
(d, J ϭ 5.2 Hz, 1 H, OH), 6.15 (dd, J ϭ 8.0, 6.0 Hz, 1 H, 1Ј-H), 6), 144.9 (Carom), 150.2 (C-2), 158.8 (Carom), 163.2 (C-4) ppm. 31P
6.86Ϫ6.92 (m, 4 H, Carom), 7.21Ϫ7.34 (m, 9 H, Carom), 7.47 (q, J ϭ NMR (162 MHz, [D6]acetone): δ ϭ 151.3 ppm.
1.2 Hz, 1 H, 6-H), 11.27 (s, 1 H, NH) ppm. 13C NMR (101 MHz,
Diastereomer b: Rf ϭ 0.34 (ethyl acetate/cyclohexane, 2:1). 1H
[D6]DMSO): δ ϭ 11.7 (5-Me), 17.9 [4Ј-CH(Me)2], 18.5 [4Ј- NMR (400 MHz, [D6]acetone): δ ϭ 1.11 (d, J ϭ 6.8 Hz, 6 H,
CH(Me)2], 30.8 [4Ј-CH(Me)2], 41.2 (C-2Ј), 55.2 (OMe), 65.0 (C-5Ј), NCHMe2), 1.18 (d, J ϭ 6.8 Hz, 6 H, NCHMe2), 1.20 (s, 3 H, 4Ј-
72.4 (C-3Ј), 83.6 (C-1Ј), 86.8 (C-4Ј), 90.1 [CPh(C6H4OMe)2], 109.6
Me), 1.47 (d, J ϭ 1.3 Hz, 3 H, 5-Me), 2.48 (ddd, J ϭ 13.8, 7.5,
Eur. J. Org. Chem. 2003, 1837Ϫ1846
www.eurjoc.org
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1843

I. Detmer, D. Summerer, A. Marx
FULL PAPER
6.1 Hz, 1 H, 2ЈЈ-H), 2.57 (ddd, J ϭ 14.0, 6.9, 5.7 Hz, 1 H, 2Ј-H),
781.4): calcd. C 64.52, H 6.96, N 7.17; found C 64.81, H 7.26,
2.73Ϫ2.82 (m, 2 H, CH2CN), 3.22 (d, J ϭ 10.0 Hz, 1 H, 5ЈЈ-H), N 7.07.
3.28 (d, J ϭ 10.0 Hz, 1 H, 5Ј-H), 3.58Ϫ3.96 [m, 4 H, N(CHMe2)2,
Thymidine Derivative (11c): Compound 10c (300 mg, 0.55 mmol)
was converted into 11c (340 mg, 79%).
POCH2], 3.78 (s, 6 H, Me-O), 4.81 (ddd, J ϭ 9.8, 7.2, 5.7 Hz, 1 H,
3Ј-H), 6.26 (dd, J ϭ 6.8, 5.9 Hz, 1 H, 1Ј-H), 6.86Ϫ6.91 (m, 4 H,
Carom), 7.20Ϫ7.52 (m, 9 H, Carom), 7.56 (q, J ϭ 1.2 Hz, 1 H, 6-H)
ppm. 13C NMR (101 MHz, [D6]acetone): δ ϭ 12.2 (5-Me), 19.2
(4Ј-Me), 20.8 (CH2CN), 24.8 [N(CHMe2)2], 39.8 (C-2Ј), 44.0
[N(CHMe2)2], 55.5 (OMe), 59.2 (CH2OP), 68.2 (C-5Ј), 74.8 (C-3Ј),
83.7 (C-1Ј), 86.8 (C-4Ј), 87.4 [CPh(C6H4OMe)2], 110.9 (C-5), 114.0
(Carom), 119.0 (CN), 127.7, 128.7, 129.1, 131.1 (Carom), 136.5 (C-
6), 145.9 (Carom), 151.2 (C-2), 158.7 (Carom), 164.2 (C-4) ppm. 31P
NMR (162 MHz, [D6]acetone): δ ϭ 151.6 ppm. IR (mixture of dia-
stereomers, KBr): ν˜ ϭ 2967, 1689, 1608, 1509, 1464, 1365, 1252,
1178, 1033, 978, 878, 830, 727 cmϪ1. MS (mixture of diastereomers,
FAB): m/z (%) ϭ 759 (10) [M ϩ H]ϩ. C41H51N4O8P (mixture of
diastereomers, 758.8): calcd. C 64.89, H 6.77, N 7.38; found C
64.49, H 6.62, N 7.14.
Diastereomer a: Rf ϭ 0.57 (ethyl acetate/cyclohexane, 2:1). 1H
NMR (300 MHz, [D6]acetone): δ ϭ 0.89 (d, J ϭ 6.8 Hz, 3 H, 4Ј-
CHMe2), 0.96 (d, J ϭ 7.0 Hz, 3 H, 4Ј-CHMe2), 1.23 (d, J ϭ 6.8 Hz,
6 H, NCHMe2), 1.23 (d, J ϭ 6.8 Hz, 6 H, NCHMe2), 1.43 (d, J ϭ
1.3 Hz, 3 H, 5-Me), 2.28 (dq, J ϭ 6.9, 6.9 Hz, 1 H, 4Ј-CHMe2),
2.55 (ddd, J ϭ 13.6, 7.4, 6.1 Hz, 1 H, 2ЈЈ-H), 2.62 (ddd, J ϭ 13.6,
6.5, 3.8 Hz, 1 H, 2Ј-H), 2.70 (dd, J ϭ 5.9, 5.9 Hz, 2 H, CH2CN),
3.34 (d, J ϭ 10.2 Hz, 1 H, 5ЈЈ-H), 3.41 (d, J ϭ 10.2 Hz, 1 H, 5Ј-
H), 3.66 (q, J ϭ 6.8 Hz, 1 H, NCHMe2), 3.69 (q, J ϭ 6.8 Hz, 1 H,
NCHMe2), 3.75Ϫ3.91 (m, 2 H, POCH2), 3.79 (s, 6 H, Me-O), 4.93
(ddd, J ϭ 10.2, 6.1, 4.1 Hz, 1 H, 3Ј-H), 6.29 (dd, J ϭ 7.6, 6.3 Hz,
1 H, 1Ј-H), 6.88Ϫ6.95 (m, 4 H, Carom), 7.25Ϫ7.53 (m, 9 H, Carom),
7.59 (q, J ϭ 1.3 Hz, 1 H, 6-H) ppm. 13C NMR (101 MHz, [D6]ace-
tone): δ ϭ 12.1 (5-Me), 18.5 (4Ј-CHMe2), 18.7 (4Ј-CHMe2), 20.8
(CH2CN), 24.9 [N(CHMe2)2], 40.9 (C-2Ј), 44.0 [N(CHMe2)2], 55.6
(OMe), 59.6 (CH2OP), 65.7 (C-5Ј), 76.2 (C-3Ј), 84.8 (C-1Ј), 88.2
(C-4Ј), 91.4 [CPh(C6H4OMe)2], 110.8 (C-5), 114.0 (Carom), 118.8
(CN), 127.8, 128.7, 129.2, 131.2 (Carom), 136.5 (C-6), 145.7 (Carom),
151.2 (C-2), 159.8 (Carom), 164.2 (C-4) ppm. 31P NMR (162 MHz,
[D6]acetone): δ ϭ 151.0 ppm.
Thymidine Derivative 11b: Compound 10b (250 mg, 0.44 mmol) was
converted into 11b (282 mg, 83%).
Diastereomer a: Rf ϭ 0.51 (ethyl acetate/cyclohexane, 2:1). 1H
NMR (400 MHz, [D6]acetone): δ ϭ 0.83 (dd, J ϭ 7.5, 7.5 Hz, 3 H,
4Ј-CH2Me), 1.20 (d, J ϭ 3.7 Hz, 6 H, NCHMe2], 1.21 (d, J ϭ
3.5 Hz, 6 H, NCHMe2), 1.45 (d, J ϭ 1.3 Hz, 3 H, 5-Me), 1.64 (dq,
J ϭ 14.7, 7.4 Hz, 1 H, 4Ј-CH2Me), 1.79 (dq, J ϭ 14.6, 7.5 Hz, 1
H, 4Ј-CH2Me), 2.51 (dd, J ϭ 6.4, 6.4 Hz, 1 H, 2ЈЈ-H), 2.51 (dd,
J ϭ 6.4, 6.4 Hz, 1 H, 2Ј-H), 2.64 (dd, J ϭ 6.0, 6.0 Hz, 2 H,
CH2CN), 3.23 (d, J ϭ 10.0 Hz, 1 H, 5ЈЈ-H), 3.36 (d, J ϭ 10.0 Hz,
1 H, 5Ј-H), 3.65 (q, J ϭ 3.4 Hz, 1 H, NCHMe2), 3.68 (q, J ϭ
3.4 Hz, 1 H, NCHMe2), 3.72Ϫ3.83 (m, 2 H, POCH2), 3.79 (s, 6 H,
Me-O), 4.96 (ddd, J ϭ 10.8, 6.4, 6.4 Hz, 1 H, 3Ј-H), 6.24 (dd, J ϭ
6.4, 6.4 Hz, 1 H, 1Ј-H), 6.88Ϫ6.93 (m, 4 H, Carom), 7.20Ϫ7.51 (m,
9 H, Carom), 7.57 (q, J ϭ 1.2 Hz, 1 H, 6-H) ppm. 13C NMR
(101 MHz, [D6]acetone): δ ϭ 8.3 (4Ј-CH2Me), 12.1 (5-Me), 20.8
(CH2CN), 24.9 [N(CHMe2)2], 25.8 (4Ј-CH2Me), 39.9 (C-2Ј), 43.9
[N(CHMe2)2], 55.6 (OMe), 59.7 (CH2OP), 66.1 (C-5Ј), 74.5 (C-3Ј),
84.0 (C-1Ј), 87.5 (C-4Ј), 89.0 [CPh(C6H4OMe)2], 110.8 (C-5), 114.0
(Carom), 118.8 (CN), 127.8, 128.7, 129.2, 131.2 (Carom), 136.4 (C-
6), 145.9 (Carom), 151.2 (C-2), 159.8 (Carom), 164.2 (C-4) ppm. 31P
NMR (162 MHz, [D6]acetone): δ ϭ 151.3 ppm.
Diastereomer b: Rf ϭ 0.43 (ethyl acetate/cyclohexane, 2:1). 1H
NMR (300 MHz, [D6]acetone): δ ϭ 0.85 (d, J ϭ 7.0 Hz, 3 H, 4Ј-
CHMe2), 0.94 (d, J ϭ 7.0 Hz, 3 H, 4Ј-CHMe2), 1.17 (d, J ϭ 6.8 Hz,
6 H, NCHMe2), 1.21 (d, J ϭ 6.8 Hz, 6 H, NCHMe2), 1.43 (d, J ϭ
1.3 Hz, 3 H, 5-Me), 2.23 (dq, J ϭ 7.0, 7.0 Hz, 1 H, 4Ј-CHMe2),
2.52Ϫ2.67 (m, 2 H, 2Ј-H), 2.81 (dd, J ϭ 6.3, 6.3 Hz, 2 H, CH2CN),
3.33Ϫ3.41 (m, 2 H, 5Ј-H), 3.65 (q, J ϭ 6.8 Hz, 1 H, NCHMe2),
3.68 (q, J ϭ 6.9 Hz, 1 H, NCHMe2), 3.78 (s, 6 H, Me-O),
3.79Ϫ3.99 (m, 2 H, POCH2), 4.84 (ddd, J ϭ 8.2, 6.0, 3.9 Hz, 1 H,
3Ј-H), 6.31 (dd, J ϭ 7.6, 6.3 Hz, 1 H, 1Ј-H), 6.88Ϫ6.94 (m, 4 H,
Carom), 7.26Ϫ7.53 (m, 9 H, Carom), 7.58 (q, J ϭ 1.3 Hz, 1 H, 6-H)
ppm. 13C NMR (101 MHz, [D6]acetone): δ ϭ 12.0 (5-Me), 18.4
(4Ј-CHMe2), 18.8 (4Ј-CHMe2), 20.9 (CH2CN), 24.9 [N(CHMe2)2],
41.2 (C-2Ј), 44.1 [N(CHMe2)2], 55.6 (OMe), 59.2 (CH2OP), 65.6
(C-5Ј), 76.9 (C-3Ј), 84.6 (C-1Ј), 88.3 (C-4Ј), 90.9 [CPh(C6H4OMe)2],
110.9 (C-5), 114.0 (Carom), 119.0 (CN), 127.8, 128.7, 129.2, 131.2,
(Carom), 136.6 (C-6), 145.7 (Carom), 151.2 (C-2), 159.8 (Carom), 164.2
(C-4) ppm. 31P NMR (162 MHz, [D6]acetone): δ ϭ 152.7 ppm. IR
(mixture of diastereomers, KBr): ν˜ ϭ 2966, 1690, 1608, 1509, 1464,
1365, 1252, 1179, 1037, 979, 893, 829, 727 cmϪ1. MS (mixture of
Diastereomer b: Rf ϭ 0.41 (ethyl acetate/cyclohexane, 2:1). 1H
NMR (400 MHz, [D6]acetone): δ ϭ 0.83 (dd, J ϭ 7.5, 7.5 Hz, 3 H,
4Ј-CH2Me), 1.14 (d, J ϭ 6.8 Hz, 6 H, NCHMe2), 1.19 (d, J ϭ
6.8 Hz, 6 H, NCHMe2), 1.46 (d, J ϭ 1.1 Hz, 3 H, 5-Me), 1.62 (dq,
J ϭ 14.6, 7.3 Hz, 1 H, 4Ј-CH2Me), 1.77 (dq, J ϭ 14.6, 7.3 Hz, 1
H, 4Ј-CH2Me), 2.49 (ddd, J ϭ 13.8, 7.0, 6.9 Hz, 1 H, 2ЈЈ-H), 2.56
(ddd, J ϭ 14.0, 6.8, 5.0 Hz, 1 H, 2Ј-H), 3.21 (d, J ϭ 10.0 Hz, 1 H,
5ЈЈ-H), 3.34 (d, J ϭ 10.0 Hz, 1 H, 5Ј-H), 3.63 [q, J ϭ 6.8 Hz, 1 H,
N(CHMe2)2], 3.67 [q, J ϭ 6.8 Hz, 1 H, N(CHMe2)2], 3.78 (s, 6 H,
diastereomers, FAB): m/z (%)
ϭ
787 (6) [M
ϩ
H]ϩ.
C43H55N4O8P·0.5H2O (mixture of diastereomers, 786.38): calcd. C
64.89, H 7.09, N 7.04; found C 64.87, H 7.17, N 6.96.
Thymidine Derivative 11d: Compound 10d (300 mg, 0.55 mmol) was
Me-O), 3.77Ϫ3.97 [m, 3 H, N(CHMe2)2, POCH2], 4.89 (ddd, J ϭ converted into 11d (537 mg, 96%).
9.5, 7.1, 5.1 Hz, 1 H, 3Ј-H), 6.26 (dd, J ϭ 6.7, 6.7 Hz, 1 H, 1Ј-H), Diastereomer a: Rf ϭ 0.54 (ethyl acetate/cyclohexane, 2:1). 1H
6.87Ϫ6.91 (m, 4 H, Carom), 7.19Ϫ7.50 (m, 9 H, Carom), 7.55 (q, J ϭ NMR (300 MHz, [D6]acetone): δ ϭ 0.82 (d, J ϭ 6.2 Hz, 3 H, 4Ј-
1.3 Hz, 1 H, 6-H) ppm. 13C NMR (101 MHz, [D6]acetone): δ ϭ
8.4 (4Ј-CH2Me), 12.1 (5-Me), 20.8 (CH2CN), 24.9 [N(CHMe2)2],
CH2CHMe2), 0.88 (d, J ϭ 6.2 Hz, 3 H, 4Ј-CH2CHMe2), 1.21 (d,
J ϭ 6.8 Hz, 6 H, NCHMe2), 1.22 (d, J ϭ 6.8 Hz, 6 H, NCHMe2),
25.9 (4Ј-CH2Me), 40.1 (C-2Ј), 44.0 [N(CHMe2)2], 55.5 (OMe), 59.2 1.41 (d, J ϭ 1.1 Hz, 3 H, 5-Me), 1.52Ϫ1.67 (m, 3 H, 4Ј-
(CH2OP), 66.2 (C-5Ј), 75.6 (C-3Ј), 83.9 (C-1Ј), 87.5 (C-4Ј), 88.8 CH2CHMe2, 4Ј-CH2CHMe2), 2.50 (dd, J ϭ 6.6, 6.6 Hz, 1 H, 2ЈЈ-
[CPh(C6H4OMe)2], 110.9 (C-5), 114.0 (Carom), 119.0 (CN), 127.7, H), 2.50 (dd, J ϭ 6.6, 6.6 Hz, 1 H, 2Ј-H), 2.64 (dd, J ϭ 6.0, 6.0 Hz,
128.7, 129.2, 131.1 (Carom), 136.5 (C-6), 145.9 (Carom), 151.2 (C-2), 2 H, CH2CN), 3.21 (d, J ϭ 10.0 Hz, 1 H, 5ЈЈ-H), 3.50 (d, J ϭ
159.8 (Carom), 164.2 (C-4) ppm. 31P NMR (162 MHz, [D6]acetone):
10.0 Hz, 1 H, 5Ј-H), 3.67 (q, J ϭ 6.8 Hz, 1 H, NCHMe2), 3.68 (q,
δ ϭ 151.6 ppm. IR (mixture of diastereomers, KBr): ν˜ ϭ 2967, J ϭ 6.9 Hz, 1 H, NCHMe2), 3.72Ϫ3.81 (m, 2 H, POCH2), 3.79 (s,
1689, 1608, 1509, 1464, 1364, 1252, 1179, 1127, 1037, 978, 891, 828, 6 H, Me-O), 5.05 (ddd, J ϭ 10.6, 6.8, 6.8 Hz, 1 H, 3Ј-H), 6.23 (dd,
726, 702 cmϪ1. MS (mixture of diastereomers, FAB): m/z (%) ϭ 773
J ϭ 6.3, 6.3 Hz, 1 H, 1Ј-H), 6.88Ϫ6.94 (m, 4 H, Carom), 7.25Ϫ7.55
(8) [M ϩ H]ϩ. C42H53N4O8P·0.5H2O (mixture of diastereomers, (m, 9 H, Carom), 7.62 (q, J ϭ 1.2 Hz, 1 H, 6-H) ppm. 13C NMR
1844
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2003, 1837Ϫ1846

Substrates for Investigation of DNA Polymerase Function
FULL PAPER
(101 MHz, [D6]acetone): δ ϭ 12.1 (5-Me), 20.8 (CH2CN), 24.2 (4Ј-
were added, and the reaction mixture was stirred under Ar over-
CH2CHMe2), 24.9 [N(CHMe2)2], 25.4 (4Ј-CH2CHMe2), 27.5 (4Ј- night. Afterwards, 4-nitrophenol (0.5 mmol/1.0 g CPG) was added
CH2CHMe2), 40.9 (C-2Ј), 43.9 [N(CHMe2)2], 55.6 (OMe), 59.7 and shaking was continued for an additional 24 h. Piperidine
(CH2OP), 65.9 (C-5Ј), 74.4 (C-3Ј), 83.7 (C-1Ј), 87.6 (C-4Ј), 88.8 (5 mL/1.0 g CPG) was then added, and stirring was continued for
[CPh(C6H4OMe)2], 110.8 (C-5), 114.0 (Carom), 118.8 (CN), 127.8, 5 min. Afterwards, the beads were filtered off and intensively
128.7, 129.2, 131.2 (Carom), 136.4 (C-6), 145.8 (Carom), 151.2 (C-2), washed, first with pyridine, and then with methanol and CH2Cl2.
159.8 (Carom), 164.2 (C-4) ppm. 31P NMR (162 MHz, [D6]acetone):
δ ϭ 151.3 ppm.
After drying, the beads were suspended in pyridine (10 mL/1.0 g
CPG) and acetic anhydride (14 mmol/1.0 g CPG), and a catalytic
amount of DMAP was added. After having been shaken for 5 h,
the beads were filtered off and intensively washed as described
Diastereomer b: Rf ϭ 0.43 (ethyl acetate/cyclohexane, 2:1). 1H
NMR (300 MHz, [D6]acetone): δ ϭ 0.82 (d, J ϭ 6.2 Hz, 3 H, 4Ј-
CH2CHMe2), 0.88 (d, J ϭ 6.2 Hz, 3 H, 4Ј-CH2CHMe2), 1.15 (d, above. After drying, the loading was determined by trityl analysis
J ϭ 6.8 Hz, 6 H, NCHMe2), 1.20 (d, J ϭ 6.8 Hz, 6 H, NCHMe2), of a small portion of the collected beads by known methods.[33]
1.42 (d, J ϭ 1.3 Hz, 3 H, 5-Me), 1.50Ϫ1.68 (m, 3 H, 4Ј-
The integrity of all modified oligonucleotides was confirmed by
CH2CHMe2, 4Ј-CH2CHMe2), 2.46 (ddd, J ϭ 13.7, 6.8, 6.8 Hz, 1 MALDI-ToF MS.
H, 2ЈЈ-H), 2.54 (ddd, J ϭ 14.0, 6.9, 5.6 Hz, 1 H, 2Ј-H), 2.78 (dd,
DNA Thermal Denaturation Studies: Melting curves were recorded
J ϭ 6.0, 6.0 Hz, 2 H, CH2CN), 3.19 (d, J ϭ 10.0 Hz, 1 H, 5ЈЈ-H),
3.47 (d, J ϭ 10.0 Hz, 1 H, 5Ј-H), 3.63 (q, J ϭ 6.8 Hz, 1 H,
NCHMe2), 3.66 (q, J ϭ 6.8 Hz, 1 H, NCHMe2), 3.78 (s, 6 H, Me-
O), 3.80Ϫ3.97 (m, 2 H, POCH2), 4.97 (ddd, J ϭ 9.6, 7.2, 5.6 Hz,
1 H, 3Ј-H), 6.25 (dd, J ϭ 6.5, 6.5 Hz, 1 H, 1Ј-H), 6.88Ϫ6.94 (m, 4
H, Carom), 7.21Ϫ7.54 (m, 9 H, Carom), 7.57 (q, J ϭ 1.3 Hz, 1 H, 6-
H), 9.89 (s, 1 H, NH) ppm. 13C NMR (101 MHz, [D6]acetone):
δ ϭ 12.1 (5-Me), 20.8 (CH2CN), 24.2 (4Ј-CH2CHMe2), 24.9
[N(CHMe2)2], 25.5 (4Ј-CH2CHMe2), 27.5 (4Ј-CH2CHMe2), 41.1
(C-2Ј), 44.0 [N(CHMe2)2], 55.5 (OMe), 59.2 (CH2OP), 66.1 (C-5Ј),
75.6 (C-3Ј), 83.7 (C-1Ј), 87.6 (C-4Ј), 88.5 [CPh(C6H4OMe)2], 110.9
(C-5), 114.0 (Carom), 119.0 (CN), 127.8, 128.7, 129.2, 131.2 (Carom),
136.5 (C-6), 145.8 (Carom), 159.8 (Carom), 164.2 (C-4) ppm. 31P
NMR (162 MHz, [D6]acetone): δ ϭ 151.6 ppm. IR (mixture of dia-
stereomers, KBr): ν˜ ϭ 2964, 1690, 1608, 1509, 1464, 1364, 1252,
1179, 1129, 1033, 979, 899, 831, 702 cmϪ1. MS (mixture of dia-
on a Lambda 2 (PerkinϪElmer) instrument fitted with a PTP-6
temperature-control device. Data were obtained from three individ-
ual cooling/heating cycles. Melting temperatures (Tm/ °C values)
were obtained from the maximum of the first derivative of the melt-
ing curve (A260 vs. temperature). Measurements were conducted in
buffer made up of NaCl (concentrations are indicated in the re-
spective table), 20 m KH2PO4 (pH, 7.0), and containing 600 nM
duplex DNA. The mixtures were heated to 95 °C for 5 min and
allowed to cool slowly to room temperature prior to the melting
curve measurements. A measurement of the buffer was conducted
separately and subtracted from the spectra resulting from the
sample.
CD Spectra: CD spectra were recorded on a Jasco 720 in buffer
made up of 1  NaCl and 20 m KH2PO4 (pH 7.0), which con-
tained 15 µ duplex DNA. The mixtures were heated to 95 °C for
5 min and allowed to cool slowly to room temperature prior to
measurements. A spectrum of the buffer was measured separately
and subtracted from the spectra resulting from the samples. An
average of 12 spectra were recorded in each experiment.
stereomers, FAB): m/z (%)
ϭ
801 (21) [M
ϩ
H]ϩ.
C44H57N4O8P·0.5H2O (mixture of diastereomers, 809.9): calcd. C
65.25, H 7.22, N 6.92; found C 65.16, H 7.21, N 6.92.
Synthesis of 4Ј-Modified Oligonucleotides: The synthesis of oligonu-
cleotides was carried out on a 0.2 µmol scale on an Applied Biosy-
tems Model 392 DNA synthesizer, with commercially available 2-
(cyanoethyl)phosphoramidites. A standard method for 2-(cyanoe-
thyl)phosphoramidites was used, with the exception that the coup-
ling times of and from the modified nucleotides were extended to
10 min. Yields for modified oligonucleotides are similar to those
obtained for unmodified oligonucleotides. After synthesis (trityl-
on) the oligonucleotides were cleaved from the support by treatment
with conc. NH4OH at 55 °C for 12 h. After removal of NH4OH the
residue was purified by RP HPLC (Knauer) with 5Ϫ80% MeCN in
0.1  triethylammonium acetate (TEAA) buffer. Fractions contain-
ing product were freeze-dried, dissolved in 80% acetic acid (20 µL/
OD), and incubated for 20 min at 20 °C. Sodium acetate solution
(3 , 5 µL/OD) and cooled (0 °C) ethanol (60 µL/OD) were added
to the solution, which was stored after intensive mixing at Ϫ80 °C
for 15 min. After centrifugation and decantation, the DNA pellet
was washed with ethanol and the remaining solvent was evapo-
rated. The oligonucleotides were quantified by absorption measure-
ments at 260 nm. Total yields of purified oligonucleotides were in
the 17Ϫ50% range. The integrity of all modified oligonucleotides
was confirmed by MALDI-ToF MS
Acknowledgments
Financial support by the VolkswagenStiftung, the Deutsche For-
schungsgemeinschaft, the Fonds der Chemischen Industrie, and the
Dr. Otto Röhm Gedächtnisstiftung is gratefully acknowledged. We
thank Dr. M. Zelgert for critical reading of the manuscript and
Professor Dr. M. Famulok for his support.
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Received November 15, 2002
1846
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