Tuning single nucleotide discrimination in polymerase chain reactions (PCRs): Synthesis of primer probes bearing polar 4′-C-modifications and their application in allele-specific PCR

Article abstract of DOI:10.1002/chem.200401114

There are many methods available for the detection of nucleotide variations in genetic material. Most of these methods are applied after amplification of the target genome sequence by the polymerase chain reaction (PCR). Many efforts are currently underwa

Full text of DOI:10.1002/chem.200401114

FULL PAPER
Tuning Single Nucleotide Discrimination in Polymerase Chain Reactions
(PCRs): Synthesis of Primer Probes Bearing Polar 4’-C-Modifications and
Their Application in Allele-Specific PCR
Jens Gaster and Andreas Marx*[a]
Abstract: There are many methods
available for the detection of nucleo-
tide variations in genetic material.
Most of these methods are applied
after amplification of the target
genome sequence by the polymerase
chain reaction (PCR). Many efforts are
currently underway to develop tech-
niques that can detect single nucleotide
variations in genes either by means of,
or without the need for, PCR. Allele-
specific PCR (asPCR), which reports
nucleotide variations based on either
the presence or absence of a PCR-am-
plified DNA product, has the potential
to combine target amplification and
analysis in one single step. The princi-
ple of asPCR is based on the formation
of matched or mismatched primer–
target complexes by using allele-specif-
ic primer probes. PCR amplification by
a DNA polymerase from matched 3’-
primer termini proceeds, whereas a
mismatch should obviate amplification.
Given the recent advancements in real-
time PCR, this technique should, in
principle, allow single nucleotide varia-
tions to be detected online. However,
this method is hampered by low selec-
tivity, which necessitates tedious and
costly manipulations. Recently, we re-
ported that the selectivity of asPCR
can be significantly increased through
the employment of chemically modi-
fied primer probes. Here we report fur-
ther significant advances in this area.
We describe the synthesis of various
primer probes that bear polar 4’-C-
modified nucleotide residues at their 3’
termini, and their evaluation in real-
time asPCR. We found that primer
probes bearing a 4’-C-methoxymethy-
lene modification have superior prop-
erties in the discrimination of single
nucleotide variations by PCR.
Keywords: DNA
replication
·
·
·
genomics oligonucleotides
·
polymerase chain reactions
polymorphism
Introduction
vance this field. Many methods for the detection of nucleo-
tide variations have been described; however, because these
exhibit a range of advantages and disadvantages, no general
methodology has prevailed.[10–15] Most of these methods are
applied after amplification of the target genome sequence
by the polymerase chain reaction (PCR).[10–15]
Allele-specific PCR (asPCR), which reports nucleotide
variations based on either the presence or absence of a
DNA amplification product, has the potential to combine
target amplification and analysis in one single step.[16–23] The
principle of asPCR is based on the formation of matched or
mismatched primer–target complexes through the use of
allele-specific primer probes. PCR amplification catalyzed
by a DNA polymerase proceeds from matched 3’-primer ter-
mini, whereas a primer–template mismatch should obviate
amplification. However, there have been numerous reports
indicating low selectivity of this approach, which necessi-
tates the laborious and costly optimization of buffer condi-
tions, as well as sequence and assay design.[20–23]
The deciphering of the human genome sequence has facili-
tated studies of genome variation between individuals and
the influence of these differences, such as single nucleotide
polymorphisms (SNPs), on the predisposition to disease, and
on the effects of drugs on different patients. So far, nearly
1.8 million SNPs have been discovered and characterized.[1]
A specific knowledge of clinically relevant nucleotide varia-
tions may enable a particular therapy to be adapted to the
respective genetic make-up of the patient, and should help
to predict individual drug efficacies and/or toxicities.[2–15]
Methods that permit the efficient and cost-effective diagno-
sis of relevant single nucleotide variations will further ad-
[a] Dipl.-Chem. J. Gaster, Prof. Dr. A. Marx
Fachbereich Chemie, Universitꢀt Konstanz
Universitꢀtsstrasse 10, 78457 Konstanz (Germany)
Fax : (+49)7531-88-5140
E-mail: andreas.marx@uni-konstanz.de
Chem. Eur. J. 2005, 11, 1861 – 1870
DOI: 10.1002/chem.200401114
ꢁ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1861

Recently, we and others have reported that the selectivity
of asPCR can be significantly increased by employing chem-
ically modified primer probes.[24–28] By using primer probes
that bear small, nonpolar 4’-C-modifications at their 3’ ter-
mini, together with a commercially available 3’-5’-exonu-
clease-deficient variant of a DNA polymerase from Thermo-
coccus litoralis (Vent (exoÀ) DNA polymerase),[24–26] we ob-
served a significantly higher amplification selectivity than
was obtained with unmodified primer probes. We were able
to show that detection of single nucleotide variations could
be diagnosed by using real-time PCR,[29] in which fluores-
cent SybrGreen I detection provided a rapid and convenient
tool to detect and analyze the degree of allele discrimina-
tion.
Here we describe the synthesis of various primer probes
that bear polar 4’-C-modified nucleotide residues at their 3’
termini, and the evaluation of these probes by using real-
time asPCR. We discovered that primer probes bearing a 4’-
C-methoxymethylene modification have superior properties
in the discrimination of single nucleotide variations by PCR.
Scheme 1. a) NaH, MeI, THF (2a: 75%); b) NaH, BnBr/NaI, THF (2b:
40%); c) i. Tf2O, pyridine, CH2Cl2, ii. PhOH, NaH, DMF (2c: 50%);
d) 1m TBAF, THF; e) DMTCl, DMAP, pyridine (3a: 54%, 3b: 27%, 3c:
55%); f) EDC, DMAP, succinylated LCAA-CPG, pyridine; then 4-nitro-
phenol; then piperidine; then acetic anhydride/pyridine/THF (Cap A)
and 1-methylimidazole/THF (Cap B); g) i. oligonucleotide synthesis,
ii. 33% NH4OH. TBS=tert-butyldimethylsilyl; TBDPS=tert-butyldiphe-
nylsilyl; DMT=4,4’-dimethoxytrityl; DMAP=4-(N,N-dimethylamino)-
pyridine; LCAA-CPG=long-chain alkyl amine-modified controlled pore
glass; EDC=1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochlo-
ride; TBAF=tetra-n-butylammonium fluoride.
Results and Discussion
Synthesis of 4’-C-modified primer probes: To explore the ef-
fects of polar 4’-C-substituents on the selectivity of single
nucleotide discrimination in PCR we synthesized a variety
of 4’-C-modified oligonucleotides. Our synthetic approach
uses the known 4’-C-modified thymidine (1) as a versatile
starting point for further diversification (Scheme 1). Com-
pound 1 was chosen as it is readily available in gram quanti-
ties from a procedure published by Giese and co-workers.[30]
Compound 1 was converted into its methyl and benzyl
ether (2a–b) by using the respective methyl or benzyl halo-
genides. The phenyl ether 2c was synthesized by conversion
of the alcohol 1 into the triflate[31,32] and subsequent treat-
ment with sodium phenolate. Desilylation and 5’-O-trityl-
ation of 2a–c yielded 3a–c, which were subsequently cou-
pled to a succinylated long-chain alkyl amine-modified con-
trolled pore glass (LCAA-CPG) support.[33] The solid sup-
ports 4a–c were used in standard automated DNA synthesis
to yield oligonucleotides 5–7 that carry the respective, modi-
fied thymidine moiety at their 3’ termini.[33] We also synthe-
sized oligonucleotides bearing 4’-C-hydroxymethylene moi-
eties at their 3’ termini by following a protocol published by
Wengel and co-workers.[34] Attempts to synthesize the 4’-C-
ethoxymethylene analogue from 1 failed; therefore, we de-
veloped the synthesis by the de novo construction of the nu-
cleoside. This synthesis starts with the known ribose deriva-
tive 8 that was readily alkylated to form ethyl ether 9
(Scheme 2).[35,36]
transformed into 13 by 5’-O-tritylation. Following coupling
to a solid support, oligonucleotide synthesis was readily ach-
ieved by using 14 to yield 15.
Next, we investigated the synthesis of various caboxylate-
modified oligonucleotides from 1 (Scheme 3). Oxidation and
ester formation yielded the methyl ester 16.[39] Protection
group manipulations produced the 5’-O-tritylated building
block 17 that was subsequently coupled to a solid support to
yield 18. This was followed by automated DNA synthesis.
To vary the carboxylate functionality, the resin-bound oligo-
nucleotides were cleaved from the support and then depro-
tected under different conditions. For example, to obtain the
carboxylate functionality in 20 the resin was treated with
0.5m NaOH; for the synthesis of the amide 21, standard de-
protection with concentrated ammonia was employed; and
to maintain the methyl ester functionality in 22, 2m NaOMe
was used to cleave the oligonucleotides from the solid sup-
port, and also the protection groups. The integrities of the
oligonucleotides were confirmed by high resolution mass
spectrometric analysis (HRMS).
Protection group manipulations involved cleavage of the
acetyl group and conversion to the respective acetates.[35]
The nucleobase was introduced by using a standard protocol
for Vorbrꢂggen glycosylation to yield 10.[37,38] Next, saponifi-
cation and deoxygenation of the 2’-hydroxyl function was
conducted to yield 11. Deprotection gave 12, which was
Single nucleotide discrimination by real-time PCR using un-
modified versus 4’-C-modified primer probes: We conducted
two reactions simultaneously and in parallel: One PCR tem-
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Chem. Eur. J. 2005, 11, 1861 – 1870

Single Nucleotide Discrimination in PCR
FULL PAPER
Scheme 3. a) i. PDC, powdered molecular sieve (4 ꢃ), DMF, ii. EDC,
DMAP, MeOH, CH2Cl2, 44% over both steps; b) 1m TABF, THF;
c) DMTCl, DMAP, pyridine, 56% over both steps; d) EDC, DMAP, suc-
cinylated LCAA-CPG, pyridine; then 4-nitrophenol; then piperidine;
then acetic anhydride/pyridine/THF (Cap A) and 1-methylimidazole/
THF (Cap B); e) i. oligonucleotide synthesis, ii. 0.5m NaOH, then 2m
TEAA (pH 7) to yield 20; f) i. oligonucleotide synthesis, ii. 33% NH4OH
to yield 21; g) i. oligonucleotide synthesis, ii. 2m NaOMe, MeOH, then
2m TEAA (pH 7) to yield 22. PDC=pyridinium dichromate; TEAA=
triethylammonium acetate buffer.
Scheme 2. a) NaH, EtI, DMF, 63%; b) i. 80% AcOH, TFA, ii. Ac2O,
DMAP, pyridine; c) thymine, BSA, TMSOTf, ACN, 32% over all steps;
d) NaOMe, MeOH; e) PhOCSCl, DMAP, ACN; f) AIBN, nBu3SnH, tol-
uene, 65% after both steps; g) i. 1m TBAF, THF, ii. Pd(OH)2/C, H2, etha-
nol, 69% over both steps; h) DMTCl, DMAP, pyridine, 25%; i) EDC,
DMAP, succinylated LCAA-CPG, pyridine; then 4-nitrophenol; then pi-
peridine; then acetic anhydride/pyridine/THF (Cap A) and 1-methylimi-
dazole/THF (Cap B); j) i. oligonucleotide synthesis, ii. 33% NH4OH.
BSA=N,O-bis(trimethylsilyl)acetamide; AIBN=2,2’-azobisisobutyroni-
trile.
Table 1. DCt values obtained by using unmodified or 4’-C-modified
primer probes and DNA template FarA versus FarG.
5’–AGGTR (Primer probe)
3’–TCCNACTAA– (Template)[a]
R
Ct(N=A)
DCt
Far 1
Far 2
Far 3
Far 4
Far 5
Far 6
Far 7
Far8
Far9
H
17
17
19
0
2.5
9
–
–
–
–
7
–
CH2OH
plate contained a dA residue in the position opposite to the
3’-terminal thymidine in the primer probe.[40] The other ex-
periment employed the same primer probe and template
strand; however, the sequence of the template included a
dA-to-dG mutation opposite to the 3’-terminal thymidine
moiety. Both set-ups contained the same reverse primer.
The progress of the PCR was analyzed in real-time by using
appropriate thermocycler equipment to measure the fluores-
cence of SybrGreen I in response to double-stranded DNA
binding. We compared the efficacy of amplification of the
respective primer probe by using Vent (exoÀ) DNA poly-
merase. Two crucial parameters were identified: the thresh-
old crossing point (Ct) as a measure of amplification effi-
ciency, and the difference in threshold crossing points (DCt)
of canonical versus noncanonical primer–template amplifica-
tion as an indication of single nucleotide discrimination. The
results are shown in Table 1 and, in part, in Figure 1.
CH2OCH3
CH2OCH2CH3
CH2OBn
CH2OPh
CO2H
n.a.[b]
n.a.[b]
n.a.[b]
n.a.[b]
22
C(O)NH2
C(O)OCH3
n.a.[b]
[a] N=A or G for FarA or FarG, respectively. [b] n.a.: no amplification
after 40 cycles of PCR.
that bear bulky ethers, methyl ester, or carboxylate 4’-C-
modifications resulted in no significant amplification after
40 PCR cycles. The smaller 4’-C-hydroxymethylene func-
tional group failed to induce significant discrimination be-
tween match and mismatch; however, the 4’-C-methoxy-
methylene modification gave superior results in the discrimi-
nation of single nucleotide variations by PCR.
Clearly, amplification efficiency and the ability to discrim-
inate between single nucleotide mismatches varies according
to the modified residue employed. The use of thymidines
Next, we studied the properties of 4’-C-methoxymethy-
lene-modified primer probes in several clinically relevant se-
Chem. Eur. J. 2005, 11, 1861 – 1870
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J. Gaster and A. Marx
Figure 1. Results of real-time PCR experiments obtained by using primer
probes bearing unmodified or 4’-C-modified thymidine residues at the 3’
terminus of the primer (R: 4’-C-modification as indicated in Table 1).
PCR amplification in the presence of Far primer (as indicated), and
target template FarA (solid line) or FarG (dashed line). All experiments
were conducted under identical reaction conditions containing equal
amounts of dNTPs, DNA substrates, and Vent (exoÀ) DNA polymerase.
Scheme 4. a) TPSCl, DMAP, Et3N, ACN; b) 33% NH4OH/ACN;
c) Bz2O, DMAP, pyridine, 63% over all steps; d) 1m TBAF, THF;
e) DMTCl, DMAP, pyridine, 71% over both steps; f) EDC, DMAP, suc-
cinylated LCAA-CPG, pyridine; then 4-nitrophenol; then piperidine;
then acetic anhydride/pyridine/THF (Cap A) and 1-methylimidazole/
THF (Cap B); g) i. oligonucleotide synthesis, ii. 33% NH4OH. TPSCl=
2,4,6-triisopropylbenzenesulfonyl chloride.
quences,[41,42] and compared their properties with those of
the recently identified, respective 4’-C-vinylated probes.[25]
The results are depicted in Table 2.
Table 2. DCt values obtained by using unmodified or 4’-C-modified
primer probes in various sequence contexts.
We synthesized primer probes for the same sequence as that
described above, and used real-time PCR to compare their
efficiency in the discrimination of single nucleotide varia-
tions with those of an unmodified probe. We found that, as
above, the use of modified probes led to a significant in-
crease in selectivity in the detection of single nucleotide var-
iants within PCR targets (Table 3).
R
H[a]
CH=CH2
CH2OCH3
FarA vs FarG
LeiA vs LeiG
DPyDA vs DPyDG
0
8
9
1
10
12
1.5
4.5
14
[a]
[a] Results from ref. [25].
Interestingly, for all of the sequences investigated, the 4’-
C-methoxymethylene-modified probes gave results superior
to those of the known 4’-C-vinyl-modified probes.
Table 3. DCt values obtained by using unmodified or 4’-C-modified
primer probes containing 26 in various sequence contexts.
FarG vs. FarA
LeiG vs. LeiA
DPyDG vs. DPyDA
Synthesis of 4’-C-methoxymethylene-5-methyl cytidine and
its properties as primer probes: To perform genotyping ex-
periments we synthesized the respective cytidine derivatives.
Because 4’-C-methoxymethylene thymidine derivatives dis-
played the most discriminative properties (see above), we
focused on the synthesis of the respective 2’-deoxycytidine
derivatives. Our synthetic strategy was based on the conver-
sion of uridine or thymidine derivatives into the respective
cytidine analogues.[43–45] Thus, 2a was converted into 23 by
treatment with the 2,4,6-triisopropylbenzenesulfonyl chlo-
ride (TPSCl)-Et3N-DMAP system, and subsequent aminoly-
sis and benzoylation of the exocyclic amino function. Stan-
dard protection group manipulations yielded 24, which was
coupled to a solid support (25), from which oligonucleotides
bearing 4’-C-methoxymethylene-5-methyl cytidine residues
at the 3’ terminus (26) were readily available (Scheme 4).
dC
26
0
10
1
13
1
8
These results demonstrate that chemically modified
primer probes are amplified in matched primer–template
complexes significantly more efficiently than do their un-
modified counterparts.
Conclusion
We have shown that primer probes bearing certain polar 4’-
C-modifications can significantly increase single nucleotide
discrimination by PCR. The 4’-C-methoxymethylene-modi-
fied nucleotides were shown to be ideally suited for this pur-
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Single Nucleotide Discrimination in PCR
FULL PAPER
(dd, 3J=3J=6.7 Hz, 1H; H-1’), 7.81 ppm (d, 4J=1.1 Hz, 1H; H-6);
13C NMR (100.6 MHz, CD3OD): d=12.6 (CH3), 41.6 (C-2’), 59.9
(OCH3), 64.6 (C-5’), 73.1 (4’-C-CH2), 73.8 (C-3’), 86.1 (C-1’), 90.1 (C-4’),
111.7 (C-5), 138.4 (C-6), 152.6 (C-2), 166.4 ppm (C-4); FAB MS (3-NBA
matrix): m/z: 287.1 [M+H]+. The alcohol (53.7 mg, 0.19 mmol) was dis-
solved in pyridine (1 mL), 4,4’-dimethoxytrityl chloride (DMTCl) and a
catalytic amount of 4-(dimethylamino)pyridine (DMAP) were added at
08C, and the cooled solution was stirred for 30 min. The reaction mixture
was then allowed to warm up to room temperature and stirring was con-
tinued for 4 h. The reaction was quenched by the addition of methanol
(1.5 mL) and stirring was continued for 30 min. The solvent was removed
in a vacuum and the residue was purified by flash column chromatogra-
phy (SiO2, cyclohexane/ethyl acetate 3:7+1% Et3N). Compound 3a was
isolated as a white foam (60 mg, 0.10 mmol, 54%); Rf =0.28 (cyclohex-
pose. These compounds are readily available and can be in-
corporated into DNA strands using standard oligonucleotide
chemistry. The systems described supersede recently report-
ed approaches and should be useful for the direct diagnosis
by PCR of single nucleotide variations, such as single nu-
cleotide polymorphisms or point mutations, without the
need for further time-consuming and costly post-PCR analy-
ses.
Experimental Section
1
ane/ethyl acetate 3:7); H NMR (400 MHz, CDCl3): d=1.49 (s, 3H; CH3-
5), 2.37–2.51 (m, 2H; H-2’a, H-2’b), 3.34 (d, 2J=9.9 Hz, 1H; H-5’a), 3.39
General: All temperatures quoted are uncorrected. All reagents were ob-
tained commercially and used without further purification. Solvents were
purchased over molecular sieves (Fluka) and were used directly without
further purification unless otherwise stated. All reactions were conducted
under the rigorous exclusion of air and moisture. NMR spectroscopy:
Bruker DPX300, DPX400, DRX500 with the solvent peak as internal
standard. Fast atom bombardment mass spectrometry (FAB MS): Con-
cept 1H (Kratos), matrix: 3-nitrobenzyl alcohol (3-NBA). Flash chroma-
tography: Merck silica gel G60 (230–400 mesh). Thin-layer chromatogra-
phy: Merck precoated plates (silica gel 60 F254). MALDI-ToF MS analysis
of oligonucleotides was conducted by Metabion, Germany. Electrospray
ionization Fourier transform ion cyclotron resonance mass spectrometry
(ESI-FTICR MS) (Bruker APEX) was performed at the Kekulꢄ-Institut
fꢂr Organische Chemie und Biochemie, Bonn University.
2
2
(s, 3H; OCH3), 3.42 (d, J=9.9 Hz, 1H; H-5’b), 3.60 (d, J=10.0 Hz, 1H;
4’-C-CH2a), 3.65 (d, 2J=10.0 Hz, 1H; 4’-C-CH2b), 3.84 (s, 6H; OCH3),
4.66 (dd, J=6.6 Hz, J=4.2 Hz, 1H; H-3’), 6.39 (dd, J=3J=6.6 Hz, 1H;
H-1’), 6.91–7.50 (m, 13H; Ar), 7.63 ppm (s, 1H; H-6); 13C NMR
(100.6 MHz, CDCl3): d=12.4 (CH3), 41.9 (C-2’), 55.9 (OCH3), 59.9
(OCH3), 66.5 (C-5’), 73.6 (4’-C-CH2), 74.3 (C-3’), 86.1 (C-1’), 88.4 (C-4’),
89.7 (CAr3), 114.3 (C-5), 128.2, 129.0, 129.6, 131.59, 131.6 (Ar), 137.0 (C-
6), 137.1, 137.7 (Ar), 146.2 (C-2), 160.5 ppm (C-4); FAB MS (3-NBA
matrix): m/z: 588.1 [M+H]+, 303.1 [DMT+].
3
3
3
4’-C-Benzyloxymethylen-3’-O-tert-butyldimethylsilyl-5’-O-tert-butyldiphe-
nylsilyl thymidine (2b): Sodium hydride (60%, 21 mg, 0.53 mmol) was
added at 08C to a solution of nucleoside 1 (109 mg, 0.17 mmol) dissolved
in THF (2 mL), and the suspension was stirred for 30 min. At À608C
benzyl bromide (104 mL, 0.88 mmol) and a catalytic amount of sodium
iodide were added and stirring was continued for 1 h. The reaction mix-
ture was then allowed to warm up to 08C under continual stirring for 2 h,
after which the reaction was quenched by the addition of methanol
(2 mL). After 30 min the solvent was evaporated, and the acquired resi-
due was dissolved in dichloromethane and then poured onto saturated
aqueous sodium bicarbonate solution. The aqueous layer was extracted
with dichloromethane and the combined organic phase was dried over
MgSO4. Purification of the residue by flash column chromatography
(SiO2, cyclohexane/ethyl acetate 3:7) yielded 2b (49.8 mg, 0.07 mmol,
3’-O-tert-Butyldimethylsilyl-5’-O-tert-butyldiphenylsilyl-4’-C-methoxy-
methylene thymidine (2a): After coevaporation and drying under
vacuum overnight, nucleoside 1 (325 mg, 0.52 mmol) was dissolved in
THF (6 mL). Sodium hydride (44.8 mg, 1.12 mmol) was added at 08C
and the reaction mixture was stirred for 30 min. Iodomethane (162 mL,
2.6 mmol) was added and stirring was continued for 10 h at 08C. The re-
action was quenched by the addition of methanol (2 mL) and then al-
lowed to warm up to room temperature. Saturated aqueous sodium bicar-
bonate solution was added and the aqueous phase was extracted with di-
chloromethane. The combined organic phase was dried (MgSO4) and
concentrated in a vacuum. Purification of the residue by flash column
chromatography (SiO2, ethyl acetate/cyclohexane 1:4–1:1) furnished 2a
as a white foam (250 mg, 0.39 mmol, 75%); Rf =0.64 (ethyl acetate/cyclo-
hexane 1:1); 1H NMR (400 MHz, CDCl3): d=0.03 (s, 3H; SiCH3), 0.07
(s, 3H; SiCH3), 0.90 (s, 9H; SiC(CH3)3), 1.08 (s, 9H; SiC(CH3)3), 1.60 (d,
4J=1.3 Hz, 3H; CH3-5), 2.13–2.20 (m, 1H; H-2’a), 2.26 (ddd, 2J=
13.0 Hz, 3J=5.7 Hz, 3J=2.0 Hz, 1H; H-2’b), 3.29 (s, 3H; OCH3), 3.43 (d,
2J=10.1 Hz, 1H; H-5’a), 3.51 (d, 2J=10.1 Hz, 1H; H-5’b), 3.80 (d, 2J=
10.9 Hz, 1H; 4’-C-CH2a), 3.85 (d, 2J=10.9 Hz, 1H; 4’-C-CH2b), 4.54 (dd,
3J=5.6 Hz, 3J=2.0 Hz, 1H; H-3’), 6.36 (dd, 3J=8.5 Hz, 3J=5.7 Hz, 1H;
H-1’), 7.35–7.67 (m, 11H; Ar, H-6), 8.06 ppm (br s, 1H; NH); 13C NMR
(100.6 MHz, CDCl3): d=À5.0 (SiCH3), À4.5 (SiCH3), 12.2 (CH3), 18.3
(SiC(CH3)3), 19.6 (SiC(CH3)3), 25.9 (SiC(CH3)3), 27.2 (SiC(CH3)3), 41.9
(C-2’), 59.7 (OCH3), 66.0 (C-5’), 73.0 (4’-C-CH2), 73.5 (C-3’), 84.7 (C-1’),
89.3 (C-4’), 111.1 (C-5), 128.17, 128.18, 130.27, 130.34, 132.7, 133.2,
135.64, 135.69, 135.8 (Ar, C-6), 150.3 (C-2), 163.6 ppm (C-4); FAB MS
(3-NBA matrix): m/z: 639.3 [M+H]+.
40%) as
a white foam; Rf =0.33 (cyclohexane/ethyl acetate 7:3);
1H NMR (300 MHz, CDCl3): d=À0.06 (s, 3H; SiCH3), 0.02 (s, 3H;
SiCH3), 0.84 (s, 9H; SiC(CH3)3), 1.07 (s, 3H; SiC(CH3)3), 1.62 (d, 4J=
1.1 Hz, 3H; CH3-5), 2.15 (m, 1H; H-2’a), 2.26 (ddd, 2J=13.0 Hz, 3J=
5.7 Hz, 3J=2.5 Hz, 1H; H-2’b), 3.49 (d, 2J=10.2 Hz, 1H; H-5’a), 3.56 (d,
2J=10.2 Hz, 1H; H-5’b), 3.81 (d, 2J=11.1 Hz, 1H; 4’-C-CH2a), 3.87 (d,
2
2J=11.1 Hz, 1H; 4’-C-CH2b), 4.42 (d, J=12.1 Hz, 1H; CH2Ph), 4.43 (dd,
3
2
3J=8.1 Hz, J=5.7 Hz, 1H; H-3’), 4.53 (d, J=12.1 Hz, 1H; CH2Ph), 6.36
(dd, 3J=8.3 Hz, 3J=5.7 Hz, 1H; H-1’), 7.15–7.66 (m, 16H; Ar, C-6),
8.18 ppm (br s, 1H; NH); 13C NMR (75.5 MHz, CDCl3): d=À5.6
(SiCH3), À4.6 (SiCH3), 12.3 (CH3), 18.2 (SiC(CH3)3), 19.6 (SiC(CH3)3),
25.9 (SiC(CH3)3), 27.2 (SiC(CH3)3), 41.8 (C-2’), 66.0 (C-5’), 70.4 (4’-C-
CH2), 73.4 (C-3’), 73.9 (CH2Ph), 84.7 (C-1’), 89.4 (C-4’), 111.1 (C-5),
127.75. 127.8, 127.9, 128.07, 128.1, 128.2, 128.5, 128.7, 130.28, 130.3, 132.6,
133.1, 135.56, 135.6, 135.75, 135.8, 138.1 (Ar, C-6), 150.3 (C-2), 163.7 ppm
(C-4); FAB MS (3-NBA matrix): m/z: 737.4 [M+Na]+, 715.4 [M+H]+,
657.3 [MÀtBu]+, 607.2 [MÀBnO]+.
5’-O-(4,4’-Dimethoxytrityl)-4’-C-benzyloxymethylene thymidine (3b): A
1m solution of TBAF (140 mL, 0.14 mmol) was added to a solution of nu-
cleoside 2b (44.6 mg, 0.06 mmol) in THF (2 mL) and the mixture was
stirred at room temperature for 7 h. The solvent was removed and the
residue was purified by column chromatography (SiO2, ethyl acetate) to
yield the alcohol as a pale yellow solid (18.6 mg, 0.05 mmol, 83%); Rf =
0.24 (ethyl acetate); 1H NMR (400 MHz, CD3OD): d=1.95 (d, 4J=
1.1 Hz, 3H; CH3-5), 2.36–2.48 (m, 2H; H-2’a, H-2’b), 3.70 (d, 2J=
10.1 Hz, 1H; H-5’a), 3.74 (d, 2J=10.1 Hz, 1H; H-5’b), 3.78 (d, 2J=
5’-O-(4,4’-Dimethoxytrityl)-4’-C-methoxymethylene thymidine (3a):
A
1m solution of TBAF (0.43 mL, 0.43 mmol) was added at 08C to a so-
lution of 2a (123 mg, 0.19 mmol) dissolved in THF (4 mL), and the
cooled solution was stirred for 30 min. The reaction mixture was allowed
to warm up to room temperature and stirring was continued for 3.5 h. A
small amount of silica was added and the mixture was evaporated to dry-
ness. The impregnated silica was coevaporated with toluene and subject-
ed to column chromatography (SiO2, cyclohexane/ethyl acetate 1:9–ethyl
acetate/methanol 9:1) to yield the desired alcohol as a white foam
(54.7 mg, 0.19 mmol, 99%); Rf =0.48 (ethyl acetate/methanol 9:1);
1H NMR (400 MHz, CD3OD): d=1.87 (d, 4J=1.1 Hz, 3H; CH3-5), 2.36
(m, 2H; H-2’a, H-2’b), 3.37 (s, 3H; OCH3), 3.55 (s, 2H; H-5’a, H-5’b),
3.80 (s, 2H; 4’-C-CH2a, 4’-C-CH2b), 4.48 (t, 3J=5.3 Hz, 1H; H-3’), 6.30
2
11.7 Hz, 1H; 4’-C-CH2a), 3.82 (d, J=11.7 Hz, 1H; 4’-C-CH2b), 4.57–4.67
3
(m, 3H; H-3’, CH2Ph), 6.40 (dd, J=3J=6.6 Hz, 1H; H-1’), 7.31–7.45 (m,
5H; Ar), 7.86 ppm (d, 4J=1.1 Hz, 1H; H-6); 13C NMR (100.6 MHz,
CD3OD): d=12.8 (CH3), 41.7 (C-2’), 64.7 (C-5’), 71.5 (4’-C-CH2), 73.0
Chem. Eur. J. 2005, 11, 1861 – 1870
www.chemeurj.org
ꢁ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1865

J. Gaster and A. Marx
(C-3’), 74.9 (CH2Ph), 86.2 (C-1’), 90.1 (C-4’), 111.8 (C-5), 128.8, 128.9,
129.5, 129.6, 138.3, 139.8 (Ar, C-6), 154.0 (C-2), 168.5 ppm (C-4); FAB
MS (3-NBA matrix): m/z: 363.1 [M+H]+. The alcohol (18.5 mg,
0.05 mmol) was dissolved in 0.5 mL pyridine, DMTCl and a catalytic
amount of DMAP were added, and the solution was stirred at room tem-
perature for 19 h. The reaction was then quenched by the addition of
methanol (1 mL) and stirring was continued for 30 min. After evapora-
tion, the residue was subjected to flash column chromatography (SiO2,
cyclohexane/ethyl acetate 1:1+1% triethylamine) to furnish 3b as a yel-
lowish foam (11.4 mg, 0.02 mmol, 33%); Rf =0.64 (ethyl acetate);
1H NMR (300 MHz, CD3OD): d=1.55 (d, 4J=0.9 Hz, 3H; CH3-5), 2.33
(m, 1H; H-2’a), 2.46 (ddd, 2J=13.5 Hz, 3J=6.5 Hz, 3J=3.8 Hz, 1H; H-
2’b), 3.72 (d, J=10.2 Hz, 1H; 4’-C-CH2a), 3.81 (d, J=10.2 Hz, 1H; 4’-C-
CH2b), 4.55 (d, 2J=12.2 Hz, 1H; CH2Ph), 4.62 (d, 2J=12.2 Hz, 1H;
CH2Ph), 4.64 (m, 1H; H-3’), 6.47 (dd, 3J=3J=6.5 Hz, 1H; H-1’), 6.88–
7.53 (m, 18H; Ar), 7.55 ppm (d, 4J=0.9 Hz, 1H; H-6); 13C NMR
(75.5 MHz, CD3OD): d=13.1 (CH3), 42.0 (C-2’), 55.9 (OCH3), 66.7 (C-
5’), 71.8 (4’-C-CH2), 73.8 (C-3’), 74.7 (CH2Ph), 86.2 (C-1’), 88.4 (CAr3),
89.6 (C-4’), 112.0 (C-5), 114.3, 128.2, 128.7, 128.9, 129.0, 129.5, 129.6,
130.6, 131.0, 131.6, 131.62, 137.0, 137.1, 137.2, 139.8 (Ar), 146.3 (C-2),
160.4, 160.5 ppm (Ar, C-4); FAB MS (3-NBA matrix): m/z: 664.3 [M+],
303.1 [DMT+].
3J=6.5 Hz, 3J=4.6 Hz, 1H; H-3’), 6.44 (dd, 3J=3J=6.7 Hz, 1H; H-1’),
6.95–7.38 (m, 5H; Ar), 7.86 ppm (d, 4J=1.2 Hz, 1H; H-6); 13C NMR
(100.6 MHz, CD3OD): d=12.7 (CH3), 41.7 (C-2’), 64.5 (C-5’), 69.1 (4’-C-
CH2), 73.0 (C-3’), 86.4 (C-1’), 89.7 (C-4’), 111.8 (C-5), 115.8, 122.1, 130.6
(Ar), 138.4 (C-6), 153.4 (C-2), 160.6 (Ar), 167.6 ppm (C-4); FAB MS (3-
NBA matrix): m/z: 349.1 [M+H]+. The alcohol (35 mg, 0.10 mmol) was
dissolved in pyridine (0.5 mL), and DMTCl (86.3 mg, 0.26 mmol) and a
catalytic amount of DMAP were added to the solution. After being stir-
red for 17 h at room temperature, methanol (0.5 mL) was added and stir-
ring was continued for 30 min. The solvent was evaporated in a vacuum
and the resulting residue was subjected to column chromatography (SiO2,
cyclohexane/ethyl acetate 1:1+1% triethylamine–ethyl acetate+1% tri-
ethylamine) to furnish 3c as a slightly yellow foam (38.4 mg, 0.06 mmol,
59%); Rf =0.44 (cyclohexane/ethyl acetate 3:7); 1H NMR (400 MHz,
CDCl3): d=1.59 (d, 4J=1.1 Hz, 3H; CH3-5), 2.35 (m, 1H; H-2a’), 2.55
(ddd, 2J=13.6 Hz, 3J=6.6 Hz, 3J=3.8 Hz, 1H; H-2’b), 3.39 (d, 2J=
9.9 Hz, 1H; H-5a’), 3.55 (d, 2J=9.9 Hz, 1H; H-5’b), 3.82 (s, 6H; OCH3),
4.25 (d, 2J=10.0 Hz, 1H; 4’-C-CH2a), 4.30 (d, 2J=10.0 Hz, 1H; 4’-C-
CH2b), 4.70 (dd, 3J=6.6 Hz, 3J=3.8 Hz, 1H; H-3’), 6.46 (dd, 3J=3J=
6.6 Hz, 1H; H-1’), 6.86–7.50 (m, 18H; Ar), 7.52 ppm (d, 4J=1.1 Hz, 1H;
H-6); 13C NMR (100.6 MHz, CDCl3): d=12.6 (CH3), 41.6 (C-2’) 55.7
(OCH3), 66.0 (C-5’), 68.9 (4’-C-CH2), 73.5 (C-3’), 86.4 (C-1’), 88.2 (C-4’),
89.2 (CAr3), 111.8 (C-5), 114.2, 115.7, 121.9, 128.0, 128.9, 129.4, 130.5,
131.4, 131.5, 136.8, 137.0, 137.2, 146.1 (Ar), 154.2 (C-2), 160.28, 160.3,
160.31 ppm (Ar, C-4); FAB MS (3-NBA matrix): m/z: 673.3 [M+Na]+,
650.4 [M+], 303.1 [DMT+].
2
2
3’-O-tert-Butyldimethylsilyl-5’-O-tert-butyldiphenylsilyl-4’-C-phenoxy-
methylene thymidine (2c): Pyridine (70 mL, 0.89 mmol) was added at 08C
to a solution of nucleoside 1 (212 mg, 0.28 mmol) in dichloromethane
(2 mL). Trifluoromethanesulfonic anhydride (69.5 mL, 0.43 mmol) was
added drop-wise and stirring was continued at 08C for 1.5 h, after which
the reaction was quenched by the addition of saturated NaHCO3 solution
(1 mL). Additional dichloromethane was added and the organic phase
was washed with saturated NaHCO3 and NaCl solution. The organic
phase was dried (MgSO4) and concentrated to give a dark-red residue,
which was used directly in the next reaction step without further purifica-
tion. Sodium hydride (113 mg, 2.84 mmol) was added at À788C to a so-
lution of phenol (267 mg, 2.84 mmol) dissolved in DMF (3 mL), and the
reaction mixture was stirred for 30 min as it warmed up to 08C. The
crude residue was then added and stirred at 08C for 1 h. Next, the mix-
ture was allowed to warm up to room temperature and then heated to
508C for 1.5 h, after which the mixture was poured onto saturated aque-
ous sodium bicarbonate solution. The aqueous phase was extracted with
dichloromethane, and the combined organic phase was dried (MgSO4)
and concentrated. Purification by flash column chromatography (SiO2,
cyclohexane/ethyl acetate 4:1) yielded 2c as a foam (98.4 mg, 0.14 mmol,
50%); Rf =0.61 (ethyl acetate/cyclohexane 3:7); 1H NMR (500 MHz,
CDCl3): d=0.07 (s, 3H; SiCH3), 0.17 (s, 3H; SiCH3), 0.94 (s, 9H;
SiC(CH3)3), 1.16 (s, 9H; SiC(CH3)3), 1.76 (d, 4J=1.0 Hz, 3H; CH3-5),
2.36 (m, 1H; H-2’a), 2.42 (ddd, 2J=13.1 Hz, 3J=5.6 Hz, 3J=1.7 Hz, 1H;
H-2’b), 4.02 (d, 2J=11.2 Hz, 1H; H-5’a), 4.07 (d, 2J=11.2 Hz, 1H; H-
5’b), 4.10 (d, 2J=9.4 Hz, 1H; 4’-C-CH2a), 4.16 (d, 2J=9.4 Hz, 1H; 4’-C-
CH2b), 4.68 (d, 3J=5.2 Hz, 3J=1.7 Hz, 1H; H-3’), 6.52 (dd, 3J=8.7 Hz,
3J=5.6 Hz, 1H; H-1’), 6.86–7.79 (m, 15H; Ar), 7.57 (d, 4J=1.0 Hz, 1H;
H-6), 9.24 ppm (br s, 1H; NH); 13C NMR (125.8 MHz, CDCl3): d=À5.0
(SiCH3), À4.6 (SiCH3), 12.4 (CH3), 18.1 (SiC(CH3)3), 19.6 (SiC(CH3)3),
25.9 (SiC(CH3)3), 27.2 (SiC(CH3)3), 41.9 (C-2’), 66.0 (C-5’), 67.3 (4’-C-
CH2), 73.6 (C-3’), 85.0 (C-1’), 89.1 (C-4’), 111.3 (C-5), 114.4, 121.1, 127.9,
128.0, 128.20, 128.22, 129.0, 130.02, 130.04, 130.4, 131.1, 132.4, 132.9,
135.6 (Ar), 135.8, 135.9 (Ar, C-6), 150.5 (C-2), 158.6 (Ar), 164.3 ppm (C-
4); FAB MS (3-NBA matrix): m/z: 723.2 [M+Na]+, 701.4 [M+H]+, 643.2
[MÀtBu+H]+, 623.1 [MÀPh+H]+.
3-O-Benzyl-5-O-tert-butyldiphenylsilyl-4-C-ethoxymethylene-1,2-isopro-
pylidene-b-d-ribo-pentofuranose (9): Compound 8 (725 mg, 1.32 mmol)
was dissolved in DMF (5 mL) and the solution was cooled to 08C.
Sodium hydride (82.8 mg, 2.07 mmol) was added and the reaction mix-
ture was stirred for 30 min at low temperature. The mixture was then
cooled to À208C, iodoethane (1.07 mL, 13.24 mmol) was added, and stir-
ring was continued for 3 h. The reaction mixture was allowed to warm up
to 08C and stirring was continued for 1 h. The reaction was quenched by
the addition of methanol (5 mL) and then warmed up to room tempera-
ture within 45 min. Saturated NaHCO3 solution was added and the aque-
ous phase was extracted with dichloromethane. The combined organic
phase was dried over MgSO4 and evaporated under reduced pressure.
Column chromatography (SiO2, ethyl acetate/cyclohexane 1:9) furnished
a white solid (9, 479 mg, 63%); Rf =0.51 (ethyl acetate/cyclohexane 1:4);
1H NMR (400 MHz, CDCl3): d=0.98 (s, 9H; SiC(CH3)3), 1.07 (t, 3J=
7.0 Hz, 3H; CH3-ethyl), 1.20 (s, 6H; CH3-acetonide), 3.30–3.48 (m, 2H;
2
2
CH2-ethyl), 3.47 (d, J=10.2 Hz, 1H; H-5a), 3.59 (d, J=10.2 Hz, 1H; H-
5b), 3.96 (d, 2J=11.1 Hz, 1H; 4-C-CH2a), 4.02 (d, 2J=11.1 Hz, 1H; 4-C-
CH2b), 4.08 (d, 3J=5.4 Hz, 1H; H-3), 4.46 (d, 2J=12.3 Hz, 1H; CH2Ph),
4.51 (dd, 3J=5.4 Hz, 3J=4.0 Hz, 1H; H-2), 4.65 (d, 2J=12.3 Hz, 1H;
CH2Ph), 5.68 (d, 3J=4.0 Hz, 1H; H-1), 7.16–7.67 ppm (m, 15H; Ar);
13C NMR (100.6 MHz, CDCl3): d=15.4 (CH3), 19.6 (SiC(CH3)3), 26.6
(CH3), 26.8 (CH3), 27.1 (SiC(CH3)3), 64.9 (C-5), 67.2 (CH2), 72.5 (4-C-
CH2), 72.6 (CH2Ph), 78.4 (C-3), 79.9 (C-2), 87.9 (C-4), 104.5 (C-1), 113.5
(O2C(CH3)2), 127.7, 127.8, 127.9, 128.0, 128.2, 128.5, 128.7, 129.0, 129.7,
131.1, 133.7, 134.0, 135.0, 135.8, 135.9, 136.0, 136.1, 138.3 ppm (Ar); FAB
MS (3-NBA matrix): m/z: 577.3 [M+H]+, 561.2 [MÀMe]+, 519.2
[MÀtBu]+.
1-(2’-O-Acetyl-3’-O-benzyl-5’-O-tert-butyldiphenylsilyl-4’-C-ethoxymethy-
lene-b-d-ribo-pentofuranosyl)thymine (10): Compound
9
(400 mg,
0.69 mmol) was dissolved in a mixture of 80% acetic acid (10 mL) and
trifluoroacetic acid (500 mL), and the resulting solution was stirred at
room temperature for 4 h. The solvent was then removed under reduced
pressure and the remaining residue was coevaporated with toluene. The
residue was stirred with acetic anhydride (0.7 mL, 7.41 mmol) and a cata-
lytic amount of DMAP in pyridine (5 mL) for 18 h at ambient tempera-
ture, and the reaction mixture was evaporated to dryness. The resulting
residue was diluted by the addition of dichloromethane and then poured
onto aqueous NaHCO3. The aqueous phase was extracted with dichloro-
methane, and the combined organic phase was dried (MgSO4) and con-
centrated. Purification by column chromatography (SiO2, ethyl acetate/
5’-O-(4,4’-Dimethoxytrityl)-4’-C-phenoxymethylene thymidine (3c):
A
1m solution of TBAF (250 mL, 0.25 mmol) was added to a solution of
compound 2c (79.6 mg, 0.11 mmol) in THF (2 mL) and the mixture was
stirred at room temperature for 1 h. The solvent was then evaporated
under reduced pressure and the residue was purified by column chroma-
tography (SiO2, dichloromethane/methanol 9:1). The resulting alcohol
was isolated as a yellowish solid (36.9 mg, 0.11 mmol, 93%); Rf =0.24
(cyclohexane/ethyl acetate 1:9); 1H NMR (400 MHz, CD3OD): d=1.94
4
2
(d, J=1.2 Hz, 3H; CH3-5), 2.43–2.55 (m, 2H; H-2a’, H-2b’), 3.88 (d, J=
11.6 Hz, 1H; H-5’a), 3.91 (d, 2J=11.6 Hz, 1H; H-5’b), 4.17 (d, 2J=
10.0 Hz, 1H; 4’-CH2a), 4.21 (d, 2J=10.0 Hz, 1H; 4’-C-CH2b), 4.66 (dd,
cyclohexane 1:4) yielded the desired bis-acetate as
a white foam
(150.8 mg, 0.24 mmol, 35%); Rf =0.42 (ethyl acetate/cyclohexane 1:4);
1866
ꢁ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2005, 11, 1861 – 1870

Single Nucleotide Discrimination in PCR
FULL PAPER
1H NMR (400 MHz, CDCl3): d=0.97 (s, 9H; SiC(CH3)3), 1.13 (t, 3J=
7.0 Hz, 3H; CH3-ethyl), 1.73 (s, 3H; OCOCH3), 1.98 (s, 3H; OCOCH3),
3.34–3.69 (m, 5H; 4-C-CH2a, 4-C-CH2b, CH2-ethyl, H-5a), 3.73 (d, 2J=
9.9 Hz, 1H; H-5b), 3.76 (d, 2J=11.0 Hz, 1H; CH2Ph), 3.87 (d, 2J=
white foam (119.2 mg, 0.15 mmol, 85%); Rf =0.69 (ethyl acetate/cyclo-
hexane 1:1). A solution of thiocarbonate (113.5 mg, 0.15 mmol) in tolu-
ene (1 mL) was added drop-wise to a solution of 2,2’-azobisisobutyroni-
trile (AIBN, 7 mg, 0.04 mmol) and tri-n-butyltin hydride (120 mL,
0.45 mmol) in anhydrous toluene (0.5 mL) at 858C, and the reaction mix-
ture was heated to reflux for 3 h. The solvent was removed under re-
duced pressure and the residue was purified by chromatography over
silica gel (SiO2, ethyl acetate/cyclohexane 3:7). Compound 11 was ob-
tained as a white foam (63.4 mg, 0.10 mmol, 69%); Rf =0.48 (ethyl ace-
tate/cyclohexane 1:1); 1H NMR (400 MHz, CDCl3): d=1.05 (s, 9H;
SiC(CH3)3), 1.21 (t, J=7.0 Hz, 3H;, CH3-ethyl), 1.91 (d, J=1.0 Hz, 3H;
CH3-5), 2.20 (m, 1H; H-2’a), 2.60 (ddd, 2J=13.4 Hz, 3J=6.5 Hz, 3J=
4.9 Hz, 1H; H-2’b), 3.40–3.84 (m, 6H; 4’-C-CH2a, 4’-C-CH2b, H-5’a, H-
5’b, CH2-ethyl), 4.33 (dd, 3J=6.6 Hz, 3J=4.9 Hz, 1H; H-3’), 4.47 (d, 2J=
11.8 Hz, 1H; CH2Ph), 4.60 (d, 2J=11.8 Hz, 1H; CH2Ph), 6.32 (dd, 3J=
3J=6.5 Hz, 1H; H-1’), 7.22–7.67 (m, 15H; Ar), 7.71 (d, 4J=1.0 Hz, 1H;
H-6), 8.30 ppm (br s, 1H; NH); 13C NMR (100.6 MHz, CDCl3): d=12.8
(CH3), 15.4 (CH3), 19.4 (SiC(CH3)3), 27.1 (SiC(CH3)3), 38.8 (C-2’), 64.3
(C-5’), 67.3 (CH2-ethyl), 72.2 (4’-C-CH2), 72.6 (Bn), 78.8 (C-3’), 84.9 (C-
1’), 88.8 (C-4’), 110.6 (C-5), 127.7, 127.8, 127.9, 127.94, 128.1, 128.6, 128.7,
129.9, 129.95, 133.1, 133.2, 135.6, 135.7, 135.8, 135.9, 136.6 (Ar, C-6),
138.0, 150.4 (C-2), 163.9 ppm (C-4); FAB MS (3-NBA matrix): m/z: 629.3
[M+H]+, 571.2 [MÀtBu]+.
1-(4’-C-Ethoxymethyl-b-d-ribo-pentofuranosyl)thymine (12): A 1m so-
lution of TBAF (220 mL, 0.22 mmol) was added to a solution of 11
(57.6 mg, 0.09 mmol) in 2 mL anhydrous THF and stirring was continued
at ambient temperature for 1 day, after which the solvent was evaporat-
ed. Chromatography over silica (SiO2, ethyl acetate/cyclohexane 8:2) fur-
nished a colorless intermediate, which was used in the next reaction step.
Palladium hydroxide on activated charcoal (20%, 19.5 mg) was added to
the resulting residue, which was then suspended in ethanol (1 mL). The
mixture was degassed, flushed with argon, and placed under a hydrogen
atmosphere. After stirring for 6 h at 608C the catalyst was filtered off by
using celite, then washed with ethanol, and the filtrate was concentrated.
Purification of the residue by flash column chromatography (SiO2, ethyl
3
3
11.0 Hz, 1H; CH2Ph), 4.27 (d, J=5.1 Hz, 1H; H-3), 5.18 (dd, J=5.1 Hz,
3J=1.2 Hz, 1H; H-2), 6.00 (d, 3J=1.2 Hz, 1H; H-1), 7.10–7.66 ppm (m,
15H; Ar); 13C NMR (100.6 MHz, CDCl3): d=15.4 (CH3), 19.5
(SiC(CH3)3), 20.8 (OCOCH3), 21.4 (OCOCH3), 27.0 (SiC(CH3)3), 64.3
(CH2), 67.3 (C-5), 71.7 (4-C-CH2), 73.6 (CH2Ph), 75.2 (C-3), 78.9 (C-2),
87.4 (C-4), 97.9 (C-1), 127.4, 127.7, 127.8, 128.0, 128.5, 129.6, 129.7, 131.0,
133.5, 134.1, 135.78, 135.79, 135.8, 136.2, 138.0 (Ar), 169.4, 169.9 ppm
(OCOCH3); FAB MS (3-NBA matrix): m/z: 619.2 [M+], 561.2
[MÀOAc]+. The bis-acetate (143 mg, 0.23 mmol), thymine (58.9 mg,
0.47 mmol), and N,O-bis(trimethylsilyl)acetamide (BSA, 300 mL,
1.23 mmol) were suspended in acetonitrile (2 mL). The suspension was
stirred at 608C for 30 min until it became completely soluble. The so-
lution was then cooled to 08C, after which trimethylsilyl trifluoromethane
sulfonate (80 mL, 0.44 mmol) was added. The reaction mixture was re-
fluxed for 1 h and saturated aqueous NaHCO3 (10 mL) was added at am-
bient temperature. The organic phase was extracted with dichloro-
methane, then dried (MgSO4) and concentrated. Purification by column
chromatography (SiO2, ethyl acetate/cyclohexane 2:3) afforded a white
solid (10, 144.2 mg, 0.21 mmol, 91%); Rf =0.30 (ethyl acetate/cyclohex-
ane 2:3); 1H NMR (400 MHz, CDCl3): d=0.99 (s, 9H; SiC(CH3)3), 1.17
(t, 3J=7.0 Hz, 3H; CH3-ethyl), 1.84 (d, 4J=1.1 Hz, 3H; CH3-5), 1.86 (s,
3H; OCOCH3), 3.39–3.52 (m, 2H; CH2-ethyl), 3.52 (d, 2J=10.3 Hz, 1H;
4’-C-CH2a), 3.63 (d, 2J=10.3 Hz, 1H; 4’-C-CH2b), 3.65 (d, 2J=11.0 Hz,
3
4
2
3
1H; H-5’a), 3.86 (d, J=11.0 Hz, 1H; H-5’b), 4.29 (d, J=5.7 Hz, 1H; H-
3’), 4.47 (d, 2J=11.5 Hz, 1H; CH2Ph), 4.50 (d, 2J=11.5 Hz, 1H; CH2Ph),
5.26 (dd, 3J=6.2 Hz, 3J=5.7 Hz, 1H; H-2’), 6.08 (d, 3J=6.2 Hz, 1H; H-
1’), 7.12–7.64 (m, 16H; Ar, H-6), 8.63 ppm (br s, 1H; NH); 13C NMR
(100.6 MHz, CDCl3): d=12.8 (CH3), 15.4 (CH3), 19.5 (SiC(CH3)3), 20.8
(COCH3), 27.1 (SiC(CH3)3), 64.2 (C-5’), 67.3 (CH2), 72.6 (4’-C-CH2), 74.8
(CH2Ph), 75.3 (C-3’), 77.9 (C-2’), 85.9 (C-1’), 88.2 (C-4’), 111.3 (C-5),
127.9, 127.92, 127.97, 128.0, 128.2, 128.22, 128.56, 128.6, 130.0, 130.1,
133.0, 133.3, 135.6, 135.8, 135.9, 135.97, 136.0, 137.8 (Ar, C-6), 150.6 (C-
2), 163.8 (C-4), 170.3 ppm (COCH3); FAB MS (3-NBA matrix): m/z:
687.4 [M+H]+, 629.2 [MÀtBu]+, 609.3 [MÀPh]+, 561.2 [MÀthymine]+.
acetate–methanol/ethyl acetate 1:9) yielded 12 as
a colorless solid
(19 mg, 0.06 mmol, 69%); Rf =0.15 (ethyl acetate); 1H NMR (400 MHz,
CD3OD): d=1.32 (t, 3J=7.0 Hz, 3H; CH3-ethyl), 1.95 (d, 4J=1.2 Hz,
3H; CH3-5), 2.39–2.42 (m, 2H; H-2’a, H-2’b), 3.53–3.83 (m, 6H; 4’-C-
CH2a, 4’-C-CH2b, H-5’a, H-5’b, CH2-ethyl), 4.59 (dd, 3J=5.9 Hz, 3J=
4.5 Hz, 1H; H-3’), 6.41 (dd, 3J=3J=6.6 Hz, 1H; H-1’), 7.86 ppm (d, 4J=
1.2 Hz, 1H; H-6); 13C NMR (100.6 MHz, CD3OD): d=12.9 (CH3), 15.7
(CH3), 42.3 (C-2’), 63.8 (C-5’), 68.2 (CH2-ethyl), 73.4 (4’-C-CH2), 73.7 (C-
3’), 86.2 (C-1’), 89.9 (C-4’), 111.5 (C-5), 138.0 (C-6), 153.7 (C-2),
168.0 ppm (C-4); FAB MS (3-NBA matrix): m/z: 340.4 [M+K]+, 286.3
[MÀCH3]+.
1-(3’-O-Benzyl-5’-O-tert-butyldiphenylsilyl-4’-C-ethoxymethylene-b-d-
ribo-pentofuranosyl)thymine (11): A solution of nucleoside 10 (132.8 mg,
0.19 mmol) and sodium methoxide (22 mg, 0.41 mmol) in methanol
(2 mL) was stirred at room temperature for 1 h, after which the solvent
was removed in a vacuum. The residue was dissolved in dichloromethane,
poured onto saturated aqueous NH4Cl, and extracted with CH2Cl2. The
organic phase was dried over MgSO4 and concentrated. Chromatography
over silica gel (SiO2, ethyl acetate/cyclohexane 2:3–1:1) yielded the de-
sired alcohol as a white foam (117.1 mg, 0.18 mmol, 94%); Rf =0.39
(ethyl acetate/cyclohexane 1:1); 1H NMR (400 MHz, CDCl3): d=1.00 (s,
1-(5’-O-(4,4’-Dimethoxytrityl)-4’-C-ethoxymethyl-b-d-ribo-pentofurano-
syl)thymine (13): DMTCl (94.7 mg, 0.28 mmol) and a catalytic amount of
DMAP were added to a solution of 12 (16.8 mg, 0.06 mmol) in anhydrous
pyridine (1 mL). After being stirred at 508C for 4 h the reaction was
quenched by the addition of methanol (2 mL), and stirring was continued
for 30 min. The mixture was concentrated and purified by column chro-
matography (SiO2, ethyl acetate/cyclohexane 8:2+1% triethylamine–
3
4
9H; SiC(CH3)3), 1.12 (d, J=7.0 Hz, 3H; CH3-ethyl), 1.83 (d, J=1.1 Hz,
3H; CH3-5), 3.37–3.43 (m, 2H; CH2-ethyl), 3.41 (d, 2J=10.2 Hz, 1H; 4’-
2
2
C-CH2a), 3.46 (d, J=10.2 Hz, 1H; 4’-C-CH2b), 3.68 (d, J=10.9 Hz, 1H;
H-5’a), 3.75 (d, 2J=10.9 Hz, 1H; H-5’b), 4.19 (d, 3J=6.1 Hz, 1H; H-3’),
4.33 (m, 1H; H-2’), 4.60 (d, 2J=11.1 Hz, 1H; CH2Ph), 4.67 (d, 2J=
11.1 Hz, 1H; CH2Ph), 5.88 (d, 3J=4.9 Hz, 1H; H-1’), 7.19–7.64 (m, 15H;
Ar), 7.42 ppm (d, 4J=1.1 Hz, 1H; H-6); 13C NMR (100.6 MHz, CDCl3):
d=12.8 (CH3), 15.3(CH3), 19.3 (SiC(CH3)3), 27.1 (SiC(CH3)3), 64.4 (C-5),
67.3 (CH2), 72.6 (4’-C-CH2), 74.4 (C-2’), 74.9 (CH2Ph), 78.8 (C-3’), 88.3
(C-4’), 91.1 (C-1’), 110.9 (C-5), 127.9, 128.0, 128.06, 128.1, 128.18, 128.2,
128.3, 128.66, 128.7, 130.1, 132.5, 132.6, 135.6, 135.8, 135.86, 135.9, 136.8,
137.6, 137.8 (Ar, C-6), 150.7 (C-2), 163.9 ppm (C-4); FAB MS (3-NBA
matrix): m/z: 645.4 [M+H]+, 587.1 [MÀtBu]+, 567.3 [MÀPh]+. O-Phenyl
chlorothionoformate (30 mL, 0.22 mmol) was added drop-wise to a so-
lution of the alcohol (117 mg, 0.18 mmol) and DMAP (84.2 mg,
0.69 mmol) in acetonitrile (2 mL), and the resulting solution was stirred
at ambient temperature for 1 h. The mixture was then diluted with di-
chloromethane and poured onto saturated aqueous KHSO4. The organic
phase was separated by using dichloromethane, the extracts were dried
over MgSO4 and then concentrated. Purification by column chromatogra-
phy (SiO2, ethyl acetate/cyclohexane 2:8) yielded the thiocarbonate as a
ethyl acetate+1% triethylamine) to furnish 13 as
a foam (8.6 mg,
0.014 mmol, 25%); Rf =0.55 (ethyl acetate); 1H NMR (400 MHz,
CD3OD): d=1.32 (t, 3J=7.6 Hz, 3H; CH3-ethyl), 1.97 (d, 4J=1.1 Hz,
3H; CH3-5), 2.25–2.40 (m, 2H; H-2’a, H-2’b), 3.24 (d, 2J=10.0 Hz, 1H;
2
H-5’a), 3.48 (d, J=10.0 Hz, 1H; H-5’b), 3.56–3.69 (m, 4H; 4’-C-CH2a, 4’-
C-CH2b, CH2-ethyl), 3.85 (s, 6H; OCH3), 4.54 (dd, 3J=5.4 Hz, 3J=
3
4.9 Hz, 1H; H-3’), 6.46 (dd, J=3J=7.0 Hz, 1H; H-1’), 6.91–8.00 ppm (m,
14H; Ar, H-6); 13C NMR (100.6 MHz, CD3OD): d=13.5 (CH3), 15.8
(CH3), 42.1 (C-2’), 55.8 (OCH3), 64.9 (C-5’), 68.2 (CH2), 73.5, 73.7 (C-3’,
4’-C-CH2), 85.9 (C-1’), 87.8 (C-4’), 89.7 (CAr3), 111.8 (C-5), 114.2, 114.3,
127.8, 127.83, 128.8, 128.9, 129.2, 129.6, 130.8, 131.5, 131.6, 137.4, 137.5,
137.6, 137.7 (Ar, C-6), 146.7 (C-2), 160.3 ppm (C-4); FAB MS (3-NBA
matrix): m/z: 603.3 [M+H]+, 303.2 [DMT+].
3’-O-tert-Butyldimethylsilyl-5’-O-tert-butyldiphenylsilyl-4’-C-carboxy-
methyl thymidine (16): Nucleoside 1 (501 mg, 0.80 mmol), powdered mo-
Chem. Eur. J. 2005, 11, 1861 – 1870
www.chemeurj.org
ꢁ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1867

J. Gaster and A. Marx
amine). Nucleoside 17 was isolated as a yellowish foam (26.9 mg,
lecular sieve (4 ꢃ, 626 mg), and pyridinium dichromate (3 g, 7.97 mmol)
were suspended in anhydrous DMF (6 mL), and the mixture was stirred
at room temperature for 3 h. Water (6 mL) and acetic acid (4 mL) were
added and stirring was continued for 30 min. The reaction mixture was
then diluted with ethyl acetate, the precipitate was filtered off and the
aqueous phase was extracted with ethyl acetate. The organic phase was
washed with an aqueous solution of oxalic acid (1.78 g per 50 mL) and
ammonium oxalate (2 g per 50 mL), and the aqueous phase was extracted
with ethyl acetate. The combined organic phase was dried (MgSO4) and
concentrated in a vacuum. Column chromatography (SiO2, cyclohexane/
ethyl acetate 2:3+1% acetic acid) yielded the desired carboxylic acid
(338 mg, 0.52 mmol, 65%) as a yellow foam; Rf =0.81 (cyclohexane/ethyl
acetate 3:7); 1H NMR (400 MHz, CDCl3): d=0.01 (s, 3H; SiCH3), 0.05
(s, 3H; SiCH3), 0.84 (s, 9H; SiC(CH3)3), 1.08 (s, 9H; SiC(CH3)3), 1.52 (d,
4J=1.1 Hz, 3H; CH3-5), 2.23–2.29 (m, 1H; H-2’a), 2.36 (ddd, 2J=
12.9 Hz, J=5.7 Hz, J=2.1 Hz, 1H; H-2’b), 4.07 (d, J=11.4 Hz, 1H; H-
5’a), 4.18 (d, 2J=11.4 Hz, 1H; H-5’b), 4.61 (dd, 3J=5.5 Hz, 3J=2.1 Hz,
1H; H-3’), 6.61 (dd, 3J=8.5 Hz, 3J=5.7 Hz, 1H; H-1’), 7.31–7.67 (m,
11H; Ar, H-6), 8.86 ppm (br s, 1H; NH); 13C NMR (100.6 MHz, CDCl3):
d=À5.1 (SiCH3), À4.7 (SiCH3), 12.1 (CH3), 18.0 (SiC(CH3)3), 19.7
(SiC(CH3)3), 25.7 (SiC(CH3)3), 27.3 (SiC(CH3)3), 41.4 (C-2’), 65.8 (C-5’),
74.3 (C-3’), 86.2 (C-1’), 92.9 (C-4’), 111.6 (C-5), 127.8, 127.9, 128.2, 128.4,
129.9, 130.4, 130.6, 132.3, 132.8, 135.5, 135.7, 135.9, 136.0 (Ar, C-6), 150.3
(C-2), 164.1 (C-4), 172.3 ppm (CO2H); FAB MS (3-NBA matrix): m/z:
661.2 [M+Na]+, 639.3 [M+H]+, 581.2 [MÀtBu]+, 561.2 [MÀPh]+. Anhy-
drous methanol (60 mL, 1.48 mmol) was added at 08C to a solution of the
carboxylic acid (87.8 mg, 0.14 mmol), EDC (166 mg, 0.86 mmol), and
DMAP (50.2 mg, 0.41 mmol) in anhydrous dichloromethane (2 mL). The
reaction mixture was allowed to warm up to room temperature and stir-
ring was continued for 1 day. After being quenched with water (10 mL)
the mixture was extracted with CH2Cl2, the organic phase was dried over
magnesium sulfate, and then evaporated under reduced pressure. Purifi-
cation by flash column chromatography (SiO2, cyclohexane/ethyl acetate
4:1–3:2) furnished 16 as a white solid (60.8 mg, 0.09 mmol, 68%); Rf =
0.45 (ethyl acetate/cyclohexane 2:3); 1H NMR (400 MHz, CDCl3): d=
0.01 (s, 3H; SiCH3), 0.05 (s, 3H; SiCH3), 0.85 (s, 9H; SiC(CH3)), 1.08 (s,
9H; SiC(CH3)3), 1.49 (d, 4J=1.1 Hz, 3H; CH3-5), 2.20 (m, 1H; H-2’a),
2.37 (ddd, 2J=13.1 Hz, 3J=6.0 Hz, 3J=2.8 Hz, 1H; H-2’b), 3.68 (s, 3H;
CO2CH3), 4.06 (d, 2J=11.2 Hz, 1H; H-5’a), 4.18 (d, 2J=11.2 Hz, 1H; H-
5’b), 4.69 (dd, 3J=6.5 Hz, 3J=2.8 Hz, 1H; H-3’), 6.60 (dd, 3J=7.8 Hz,
3J=6.0 Hz, 1H; H-1’), 7.34–7.70 (m, 11H; Ar, H-6), 8.33 ppm (br s, 1H;
NH); 13C NMR (100.6 MHz, CDCl3): d=À5.0 (SiCH3), À4.8 (SiCH3),
12.0 (CH3), 18.1 (SiC(CH3)3), 19.7 (SiC(CH3)3), 25.8 (SiC(CH3)3), 27.3
(SiC(CH3)3), 41.5 (C-2’), 52.3 (CO2CH3), 65.5 (C-5’), 73.9 (C-3’), 86.0 (C-
1’), 92.4 (C-4’), 111.4 (C-5), 128.2, 128.3, 129.0, 130.3, 130.5, 131.1, 132.4,
133.0, 135.5, 135.67, 135.7, 150.2 (C-2), 163.7 (C-4), 170.0 ppm (CO2CH3);
FAB MS (3-NBA matrix): m/z: 653.2 [M+H]+, 595.1 [MÀtBu]+.
0.05 mmol, 60%); Rf =0.45 (cyclohexane/ethyl acetate 1:9); 1H NMR
(400 MHz, CD3OD): d=1.52 (d, 4J=1.2 Hz, 3H; CH3-5), 2.44–2.52 (m,
2
2H; H-2’a, H-2’b), 3.60 (d, 2J=10.0 Hz, 1H; H-5’a), 3.70 (d, J=10.0 Hz,
1H; H-5’b), 3.77 (s, 3H; CO2CH3), 3.84 (s, 6H; OCH3), 4.73 (dd, 3J=
6.4 Hz, 3J=4.8 Hz, 1H; H-3’), 6.62 (dd, 3J=3J=6.8 Hz, 1H; H-1’), 6.90–
7.50 (m, 13H; Ar), 7.61 ppm (d, 4J=1.2 Hz, 1H; H-6); 13C NMR
(100.6 MHz, CDCl3): d=12.4 (CH3), 40.9 (C-2’), 52.7 (CO2CH3), 55.9
(OCH3), 66.6 (C-5’), 74.1 (C-3’), 87.2 (C-1’), 88.3 (CAr3), 93.0 (C-4’),
112.1 (C-5), 114.36, 114.37, 125.3, 128.3, 129.1, 129.6, 131.57, 131.6, 136.8,
136.9, 137.2, 137.9, 146.1, 149.4, 149.6 (Ar, C-6), 152.8 (C-2), 153.0,
160.52, 160.53 (Ar), 167.1 (C-4), 172.3 ppm (CO2CH3); FAB MS (3-NBA
matrix): m/z: 602.3 [M+], 303.1 [DMT+].
4-N-Benzoyl-3’-O-tert-butyldimethylsilyl-5’-O-tert-butyldiphenylsilyl-4’-C-
methoxymethyl-5-methyl cytidine (23): 2,4,6-Triisopropylbenzenesulfonyl
chloride (TPSCl, 95 mg, 0.31 mmol) was added to a solution of 17
(100 mg, 0.16 mmol), DMAP (38.5 mg, 0.32 mmol), and triethylamine
(44 mL, 0.32 mmol) in acetonitrile (1 mL). The mixture was stirred for
1.5 h at 08C, then a solution of 33% NH4OH/CH3CN (1 mL, 1:1) was
added and stirring was continued at 08C for 1.5 h. The reaction mixture
was allowed to warm up to room temperature and stirring was continued
for another 1.5 h. The mixture was then diluted with dichloromethane
and poured onto aqueous KHSO4 solution (50 mL, pH 5). The aqueous
layer was extracted with CH2Cl2, the combined organic phase was dried
over MgSO4, and concentrated. The desired cytidine derivative was ob-
tained following purification by flash column chromatography (SiO2,
ethyl acetate–ethyl acetate/methanol 9:1) as a colorless foam (71 mg,
0.11 mmol, 71%); Rf =0.30 (ethyl acetate/cyclohexane 1:9); 1H NMR
(400 MHz, CD3OD): d=0.16 (s, 3H; SiCH3), 0.19 (s, 3H; SiCH3), 1.00 (s,
9H; SiC(CH3)3), 1.16 (s, 9H; SiC(CH3)3), 1.69 (d, 4J=1.1 Hz, 3H; CH3-
5), 2.25 (ddd, 2J=13.3 Hz, 3J=7.7 Hz, 3J=6.0 Hz, 1H; H-2’a), 2.50 (ddd,
3
3
2
3
3
2J=13.3 Hz, J=5.9 Hz, J=2.9 Hz, 1H; H-2’b), 3.38 (s, 3H; OCH3), 3.61
(d, 2J=9.9 Hz, 1H; H-5’a), 3.65 (d, 2J=9.9 Hz, 1H; H-5’b), 3.92 (d, 2J=
11.0 Hz, 1H; 4’-C-CH2a), 3.95 (d, 2J=11.0 Hz, 1H; 4’-C-CH2b), 4.65 (dd,
3J=6.0 Hz, 3J=2.9 Hz, 1H; H-3’), 6.37 (dd, 3J=7.7 Hz, 3J=5.9 Hz, 1H;
H-1’), 7.45–7.77 (m, 10H; Ar), 7.63 ppm (d, 4J=1.0 Hz, 1H; H-6);
13C NMR (100.6 MHz, CD3OD): d=À4.8 (SiCH3), À4.4 (SiCH3), 13.4
(CH3), 19.1 (SiC(CH3)3), 20.4 (SiC(CH3)3), 26.4 (SiC(CH3)3), 27.7
(SiC(CH3)3), 43.2 (C-2’), 59.8 (OCH3), 66.7 (C-5’), 73.6 (4’-C-CH2), 74.7
(C-3’), 87.2 (C-1’), 90.6 (C-4’), 104.4 (C-5), 129.16, 129.17, 131.35, 131.4,
134.1, 134.5, 136.8, 139.3 (C-6), 158.3 (C-2), 167.4 ppm (C-4); FAB MS
(3-NBA matrix): m/z: 1275.8 [M+H]+, 638.4 [M+H]+; HRMS (ESI):
calcd for C34H50N3O5Si2: 636.3289; found: m/z: 636.3325 [MÀH]À. The
cytidine analogue (61.6 mg, 0.1 mmol), benzoic anhydride (44.9 mg,
0.2 mmol), and a catalytic amount of DMAP were dissolved in anhydrous
pyridine (1 mL). The solution was stirred under argon for 8 h at room
temperature. The solvent was then evaporated and the residue was puri-
fied by column chromatography (SiO2, cyclohexane/ethyl acetate 9:1) to
yield 63.3 mg (0.09 mmol, 88%) of a colorless foam (23); Rf =0.30 (ethyl
acetate/cyclohexane 1:9); 1H NMR (400 MHz, CDCl3): d=0.03 (s, 3H;
SiCH3), 0.07 (s, 3H; SiCH3), 0.90 (s, 9H; SiC(CH3)3), 1.09 (s, 9H;
SiC(CH3)3), 1.79 (d, 4J=1.0 Hz, 3H; CH3-5), 2.19 (ddd, 2J=13.1 Hz, 3J=
8.3 Hz, 3J=5.8 Hz, 1H; H-2’a), 2.36 (ddd, 2J=13.1 Hz, 3J=5.6 Hz, 3J=
5’-O-(4,4’-Dimethoxytrityl)-4’-C-(carboxylic acid methyl ester) thymidine
(17): Compound 16 (55.2 mg, 0.09 mmol) and a 1m solution of TBAF
(200 mL, 0.20 mmol) in anhydrous THF (4 mL) were stirred at room tem-
perature for 1.5 h. The solvent was then evaporated in a vacuum and the
residue was subjected to column chromatography (SiO2, dichlorometh-
ane/methanol 9:1) to yield the desired alcohol as
a colorless solid
2
2.0 Hz, 1H; H-2’b), 3.30 (s, 3H; OCH3), 3.44 (d, J=10.2 Hz, 1H; H-5’a),
(23.8 mg, 0.08 mmol, 93%); Rf =0.24 (dichloromethane/methanol 9:1);
1H NMR (400 MHz, CD3OD): d=1.87 (d, 4J=1.2 Hz, 3H; CH3-5), 2.28–
2.35 (m, 2H; H-2’a, H-2’b), 3.74 (s, 3H; CO2CH3), 3.91 (d, 2J=12.0 Hz,
1H; H-5’a), 3.96 (d, 2J=12.0 Hz, 1H; H-5’b), 4.57 (dd, 3J=6.3 Hz, 3J=
5.2 Hz, 1H; H-3’), 6.48 (dd, 3J=3J=6.6 Hz, 1H; H-1’), 7.73 ppm (d, 4J=
1.2 Hz, 1H; H-6); 13C NMR (100.6 MHz, CD3OD): d=12.6 (CH3), 40.7
(C-2’), 52.7 (CO2CH3), 64.4 (C-5’), 73.5 (C-3’), 87.3 (C-1’), 93.5 (C-4’),
111.9 (C-5), 138.5 (C-6), 152.7 (C-2), 166.9 (C-4), 172.6 ppm (CO2CH3);
FAB MS (3-NBA matrix): m/z: 301.1 [M+H]+, 242.3 [MÀCO2Me+H]+.
DMTCl (51.6 mg, 0.15 mmol) and a catalytic amount of DMAP were
added at room temperature to a stirred solution of the alcohol (22.6 mg,
0.08 mmol) in pyridine (2 mL). After 5 h the reaction was quenched by
the addition of methanol (1 mL) and stirring was continued for 30 min.
The solvent was then removed under reduced pressure and the resulting
residue was separated by flash column chromatography (SiO2, cyclohex-
ane/ethyl acetate 1:9+1% triethylamine–ethyl acetate+1% triethyl-
3.54 (d, 2J=10.2 Hz, 1H; H-5’b), 3.82 (d, 2J=11.0 Hz, 1H; 4’-C-CH2a),
3.90 (d, 2J=11.0 Hz, 1H; 4’-C-CH2b), 4.55 (dd, 3J=5.8 Hz, 3J=2.0 Hz,
1H; H-3’), 6.38 (dd, 3J=8.3 Hz, 3J=5.6 Hz, 1H; H-1’), 7.36–7.68 (m,
16H; Ar, H-6), 8.27 (d, 4J=1.4 Hz, 1H; Ar), 8.29 ppm (d, 4J=1.4 Hz,
1H; Ar); 13C NMR (100.6 MHz, CDCl3): d=À5.0 (SiCH3), À4.5 (SiCH3),
13.4 (CH3), 18.3 (SiC(CH3)3), 19.6 (SiC(CH3)3), 25.9 (SiC(CH3)3), 27.2
(SiC(CH3)3), 42.3 (C-2’), 59.7 (OCH3), 65.9 (C-5’), 72.9 (4’-C-CH2), 73.5
(C-3’), 85.3 (C-1’), 89.7 (C-4’), 111.9 (C-5), 128.18, 128.2, 128.3, 130.0,
130.3, 130.4, 132.6, 132.7, 133.2, 135.6, 135.8, 136.9, 137.5 (Ar, C-6), 148.1
(C-2), 160.0 (C-4), 179.8 ppm (COCH2Ph); FAB MS (3-NBA matrix): m/
z: 742.3 [M+H]+; HRMS (ESI): calcd for C41H55N3O6Si2: 740.3551;
found: m/z: 740.3566 [MÀH]À.
4-N-Benzoyl-5’-O-(4,4’-dimethoxytrityl)-4’-C-methoxymethyl-5-methyl
cytidine (24): A 1m solution of TBAF (0.17 mL, 0.17 mmol) was added to
a solution of 23 (58.8 mg, 0.08 mmol) in 1 mL anhydrous THF and the
1868
ꢁ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2005, 11, 1861 – 1870

Single Nucleotide Discrimination in PCR
FULL PAPER
preparation was stirred at room temperature for 4 h. The solvent was
then removed in a vacuum and the residue was subjected to column chro-
matography (SiO2, ethyl acetate/methanol 9:1) to isolate the desired in-
termediate, which was used in the next reaction step. The residue was co-
evaporated and redissolved in anhydrous pyridine (1 mL). DMTCl
(76.4 mg, 0.23 mmol) and a catalytic amount of DMAP were added to
the resulting solution at 08C. After being stirred for 30 min, the mixture
was allowed to warm up to room temperature and stirring was continued
for 5 h. The reaction was then quenched by the addition of methanol
(4 mL) and after 30 min evaporated to dryness. Purification of the resul-
tant residue by flash column chromatography (SiO2, cyclohexane/ethyl
acetate 2:3+1% triethylamine) furnished 38.5 mg (0.06 mmol, 71%) of a
faint yellowish foam (24); Rf =0.45 (ethyl acetate/cyclohexane 7:3);
1H NMR (400 MHz, CD3OD): d=1.72 (d, 4J=1.0 Hz, 3H; CH3-5), 2.34
(m, 1H; H-2’a), 2.60 (m, 1H; H-2’b), 3.36 (d, 2J=10.0 Hz, 1H; H-5’a),
3.41 (s, 3H; OCH3), 3.47 (d, J=10.0 Hz, 1H; H-5’b), 3.65 (d, J=9.9 Hz,
1H; 4’-C-CH2a,), 3.69 (d, 2J=9.9 Hz, 1H; 4’-C-CH2b), 3.83 (s, 3H;
OCH3), 3.84 (s, 3H; OCH3), 4.67 (dd, 3J=6.8 Hz, 3J=4.4 Hz, 1H; H-3’),
6.20 (dd, 3J=3J=6.5 Hz, 1H; H-1’), 6.90–7.92 (m, 17H; Ar, H-6), 8.08
(dd, 3J=8.5 Hz, 4J=1.3 Hz, 1H; Ar), 8.20 ppm (d, 3J=7.1 Hz, 1H; Ar);
13C NMR (100.6 MHz, CD3OD): d=13.2 (CH3), 42.8 (C-2’), 55.9
(OCH3), 59.9 (OCH3), 66.2 (C-5’), 73.4 (4’-C-CH2), 74.2 (C-3’), 87.1 (C-
1’), 88.3 (C-4’), 89.9 (CAr3), 104.3 (C-5), 114.4, 128.2, 129.0, 129.1, 129.6,
129.63, 129.7, 130.6, 131.5, 131.58, 131.6, 134.3, 136.9, 137.0, 137.1, 137.13,
139.7, 146.2 (Ar, C-6), 158.3 (C-2), 160.5 (C-4), 167.4 ppm (COCH2Ph);
FAB MS (3-NBA matrix): m/z: 588.3 [MÀBz+2H]+, 303.1 [DMT+];
HRMS (ESI): calcd for C40H40N3O8: 690.2815; found: m/z: 690.2835
[MÀH]À.
General procedure for coupling of 5’-O-protected nucleosides to succiny-
lated LCAA-CPG: Compounds 3a–c, 13, 17, and 24 were coupled to suc-
cinylated LCAA-CPG by using standard protocols.[46] Briefly, succinylat-
ed LCAA-CPG, the respective nucleosides 3a–c, 13, 17, or 24, DMAP
(each 0.1 mmol/1.0 g CPG), and EDC (1.0 mmol/1.0 g CPG), were com-
bined, pyridine (10 mL/1.0 g CPG) and NEt3 (80 mL/1.0 g CPG) were
added, and the reaction mixture was shaken under argon overnight.
Next, 4-nitrophenol (0.5 mmol/1.0 g CPG) was added and shaking was
continued for an additional 24 h. Piperidine (5 mL/1.0 g CPG) was
added, and shaking was continued for 5 min. The beads were then fil-
tered off and washed successively with pyridine, methanol, and finally
with CH2Cl2. After drying, the beads were suspended in equal amounts
of acetic anhydride/pyridine/THF (Cap A) and 1-methylimidazole/THF
(Cap B) capping reagents. After shaking for 2 h, the beads were filtered
off and intensively washed as described above. After drying, loading was
determined by trityl analysis of a small portion of the collected beads
(loading range 8.5–31.9 mmolgÀ1).[46]
Real-time PCR experiments: Real-time PCR was performed by using an
iCycler System (Bio-Rad). In brief, the reactions were performed in a
total volume of 50 mL, which contained 4 pmol of the respective tem-
plates in the respective buffers provided by the supplier for Vent (exoÀ)
DNA polymerase (20 mm Tris-HCl (pH 8.8), 10 mm KCl, 10 mm
(NH4)SO4, 2 mm MgSO4, 0.1% Triton X-100). The final mixtures con-
tained dNTPs (200 mm each of dATP, dGTP, dCTP, and TTP), primers
(0.5 mm each of respective primer probe and reverse primer), 1.2 units of
Vent (exoÀ) DNA polymerase (units defined by the supplier, New Eng-
land Biolabs), and a 1:25000 aqueous dilution of a 10000x solution of
SybrGreen I in DMSO (Molecular Probes). All PCR amplifications were
performed by employing the following program: initial denaturation at
958C for 3 min followed by 40 cycles of denaturation at 958C for 30 s,
primer annealing at 558C for 35 s, and extension at 728C for 40 s. The
data presented were obtained from independent measurements of tripli-
cates originating from one master-mix. All experiments were repeated at
least two times. To enable comparison with previous studies, the DNA se-
quences of template target, primer probe, and reverse primer were iden-
tical to those employed previously. Sequences in the Farber disease con-
text: Primer probe 5’-d(CGT TGG TCC TGA AGG AGG ANR), reverse
primer: 5’-d(CGC GCA GCA CGC GCC GCC GT), target template
Far X : 5’-d(CCG TCA GCT GTG CCG TCG CGC AGC ACG CGC
CGC CGT GGA CAG AGG ACT GCA GAA AAT CAA CCT XTC
CTC CTT CAG GAC CAA CGT ACA GAG); X: A, FarA; G, FarG.
Sequences in the Factor V Leiden disease context: Primer probe 5’-
d(CAA GGA CAA AAT ACC TGT ATT CCT NR), reverse primer: 5’-
d(GAC ATC ATG AGA GAC ATC GC), target template LeiX: 5’-
d(GAC ATC ATG AGA GAC ATC GCC TCT GGG CTA ATA GGA
CTA CTT CTA ATC TGT AAG AGC AGA TCC CTG GAC AGG
CXA GGA ATA CAG GTA TTT TGT CCT TG); X: A, LeiA; G,
LeiG. Sequences in the DPyD context: Primer probe DpyDTR: 5’-
d(GTT TTA GAT GTT AAA TCA CAC TTA NR), reverse primer: 5’-
d(AAA GCT CCT TTC TGA ATA TTG AG), target template DPyDX:
5’-d(AAA ATG TGA GAA GGG ACC TCA TAA AAT ATG TCA
TAT GGA AAT GAG CAG ATA ATA AAG ATT ATA GCT TTT
CTT TGT CAA AAG GAG ACT CAA TAT CTT TAC TCT TTC ATC
AGG ACA TTG TGA CAA ATG TTT CCC CCA GAA TCA TCC
GGG GAA CCA CCT CTG GCC CCA TGT ATG GCC CTG GAC
AAA GCT CCT TTC TGA ATA TTG AGC TCA TCA GTG AGA
AAA CGG CTG CAT ATT GGT GTC AAA GTG TCA CTG AAC
TAA AGG CTG ACT TTC CAG ACA ACX TAA GTG TGA TTT
AAC ATC TAA AAC); X: A, DPyDA; G, DPyDG.
2
2
Acknowledgements
Synthesis of 4’-C-modified oligonucleotides: The synthesis of oligonucleo-
tides was performed on a 0.2 mmol scale by using an Applied Biosys-
tems 392 DNA synthesizer and commercially available 2-(cyanoethyl)-
phosphoramidites. A standard method for the synthesis of 2-(cyanoethyl)
phosphoramidites was used, with the exception that the coupling times
for the modified nucleotides were extended to 10 min. Yields for modi-
fied oligonucleotides were similar to those obtained for unmodified oli-
gonucleotides. After synthesis (trityl off) the oligonucleotides were
cleaved from the support by treatment with 33% NH4OH at 558C for
12 h. After removal of NH4OH the residue was purified by preparative
electrophoresis through a 12% polyacrylamide gel containing 8m urea.
For the synthesis of primer probes Far 7 and Far 9, 18 was used for oligo-
nucleotide synthesis and subsequently treated with 0.5m NaOH or 2m
NaOMe (MeOH/H2O 4:1), respectively, for 22 h at room temperature,
then neutralized with 2m triethylammonium acetate (TEAA), and desalt-
ed (NAP-25 column, Amersham Biosciences). After evaporation the resi-
dues were purified by preparative PAGE, as described above. The DNA
oligonucleotides were recovered by standard precipitation with ethanol
in the presence of 0.3m sodium acetate. The oligonucleotides were quan-
tified by measuring absorption at 260 nm. Total yields of purified oligo-
nucleotides were in the range of 25–33%. The integrity of all modified
oligonucleotides was confirmed by performing MALDI-ToF or ESI-
FTICR MS.
We wish to thank M. Strerath for expert support in the acquisition of
real-time PCR data, K.-H. Jung for critical reading of the manuscript,
and M. Engesser, Universitꢀt Bonn, for recording the HRMS data. Fi-
nancial support by the Deutsche Forschungsgemeinschaft, Volkswagen
Foundation, Roche Diagnostics, and Fonds der Chemischen Industrie is
gratefully acknowledged.
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