Synthesis of hapten-phosphoramidites from 2′-deoxyuridine

Article abstract of DOI:10.1016/S0040-4020(03)00875-5

A total of 11 novel phosphoramidites, 3a-d, 4a-d and 14a-c were prepared from 2′-deoxyuridine functionalized at 5 and 6-position of the pyrimidine ring with hapten reporter groups, e.g. adamantane, carbazole, dansyl and dabsyl, suitable for use in immunod

Full text of DOI:10.1016/S0040-4020(03)00875-5

Tetrahedron 59 (2003) 5749–5761
Synthesis of hapten–phosphoramidites from 20-deoxyuridine
*
Maciej Adamczyk, Srinivasa Rao Akireddy, Phillip G. Mattingly and Rajarathnam E. Reddy
Department of Chemistry (09MD, Bldg AP20), Diagnostics Division, Abbott Laboratories 100 Abbott Park Road,
Abbott Park, IL 60064-6016, USA
Received 8 April 2003; revised 14 May 2003; accepted 27 May 2003
Abstract—A total of 11 novel phosphoramidites, 3a–d, 4a–d and 14a–c were prepared from 20-deoxyuridine functionalized at 5 and
6-position of the pyrimidine ring with hapten reporter groups, e.g. adamantane, carbazole, dansyl and dabsyl, suitable for use in
immunodetection nucleic acid testing assays. q 2003 Elsevier Science Ltd. All rights reserved.
1. Introduction
reporter group after isolation and purification. Most often
nucleoside phosphoramidites bearing the linkers are used.18
Examples include phosphoramidites of 5-(trifluoroacetyl-
amidoalkyl)-,19–25 5-(alkylthio)-,26 5-(alkyldiol)-,27 5-(car-
Molecular diagnostics is the fastest growing area in the field
of clinical analysis.1 The completion of the Human Genome
Mapping Project (HGMP)2,3 has accelerated identification
of new nucleic acid markers useful in the diagnosis of
disease states and monitoring the therapy for those ailments.
Concurrently, nucleic acid testing (NAT) has progressed
from a laborious manual process using radiolabeled
reagents to highly automated high throughput screening
(HTS). At the heart of the current technology for detection
of specific nucleic acid sequences, is the preparation of
probes bearing reporter groups.4–6 The reporter groups
employed are as varied as the assay formats.7 Beacon8–11
and Taqman12 formats require dual-labeled fluorophore/
quencher probes, while hybridization protection assays
make use of chemiluminescent probes.13 In the Abbott LCx
system (Fig. 1) the reporter groups are haptens,14 i.e. low
molecular weight compounds that bind to specific anti-
bodies. In that system oligonucleotide probes complemen-
tary to a target sequence are differentially labeled with
haptens Ha and Hb. The ligase chain reaction (LCR)15–17
joins the probes, which then become viable targets in the
next round of amplification. At the completion of target
amplification, the ligated probes are captured using anti-
hapten Ha antibody-coated microparticles and detected with
an anti-hapten Hb antibody-alkaline phosphatase conjugate
using 4-methylumbilliferone phosphate (MUP) substrate.
boxymethyl)-,28,29
deoxythymidine,
and
N 4-
(trifluoroacetylamidoalkyl)deoxycytidine.21,30,31 Alterna-
tively, the linker can be introduced on the phosphate
backbone of the probe via H-phosphonate chemistry,4,5,32 or
non-selectively via transamination of cytosine residues.33,34
The integrated methodology introduces the reporter group
during the synthesis of the probe. This can be done
enzymatically using 5-substituted dUTP35–38 or N 4-sub-
stituted 5-methyl dCTP,39 but more control is achieved with
automated synthesis using phosphoramidites. Recent
examples of modified-nucleoside phosphoramidites in this
category include 5-substituted deoxythymidine bearing
photolabels,40 spin-labels,41 thymine,42 and metal
chelators.43
We have detailed the synthesis of hapten-bearing, non-
nucleoside pho0sphoramidites useful for the preparation of
50-44–48 and 3 -haptenated probes.44,46–48 A preliminary
communication on hapten phosphoramidites based on
6-substituted-20-deoxyuridine has recently appeared.49
Here we present the full report 0for the preparation and
characterization of 6-haptenated-2 -deoxyuridine phosphor-
amidites. Additionally, novel 5-haptenated-20-deoxyuridine
phosphoramidites are also described.
Many strategies have been reported for labeling oligo-
nucleotides probes, but they can be broadly grouped into
either post-synthetic or integrated approaches. In the
former, specific linker functional groups are incorporated
into the probes that can be further elaborated with the
2. Results and discussion
2.1. 5-[(2E)-N-(Alkyl)prop-2-enamidyl]-20-deoxyuridine
hapten phosphoramidites
Introduction of reporter groups at the C-5 position of 20-
deoxyuridine has been often cited as the preferred
regiochemistry to minimize unfavorable steric interactions
Keywords: 20-deoxyuridine; haptens; phosphoramidites.
*
Corresponding author. Tel.: þ1-847-937-0225; fax: þ1-847-938-8927;
e-mail: maciej.adamczyk@abbott.com
0040–4020/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)00875-5

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M. Adamczyk et al. / Tetrahedron 59 (2003) 5749–5761
Figure 1. NAT Signal amplification and antibody capture detection.

M. Adamczyk et al. / Tetrahedron 59 (2003) 5749–5761
5751
and perturbation of Watson–Crick base-pairing18 during
hybridization of probes containing modified bases. How-
ever, the exact nature of the substituent can have a positive
or negative effect on the stability of the duplex formed.25
The commercial availability of 5-substituted-20-deoxy-
uridine derivatives 1 and 2 (Scheme 1) has added to their
popularity. To introduce the required modifications to
enable various immunodetection formats, 1 and 2 were
coupled (HOBt, EDAC) with our desired haptens, 2-[3-(4-
nitrophenyl)-1-adamantyl]acetic acid (5a)48 and 4-(9H-
carbazol-2-yloxy)butanoic acid (5b),48 as shown to produce
intermediates 1a,b and 2a,b, respectively in good to
excellent yield and purity. The dansyl (5c) and dabsyl
(5b) moieties are dual reporter groups. Both can function as
haptens, while dansyl is also a fluorescent label and dabsyl
is an efficient fluorescence quencher. The dansyl 1c, 2c and
dabsyl 1d, 2d derivatives were prepared by sulfonylation of
amines 1 and 2 with the corresponding sulfonyl chlorides in
THF/aqueous sodium carbonate in good yield. Conversion
to 30-position phosphoramidites 3a–d, 4a–d was accom-
plished by treatment with 2-cyanoethyl N,N-diisopropyl-
chlorophosphoramidite in THF in the presence of
diisopropylethylamine in 41–77% yield. In most cases
flash silica chromatography was sufficient to provide
phosphoramidites in greater than 95% purity. Dansyl (3c
and 4c) and dabsyl phosphoramidites (4d) required
additional purification by preparative reverse phase HPLC
to reach that level of purity.
2.2. 6-[(2E)-N-(Alkyl)prop-2-enamidyl]-20-deoxyuridine
hapten phosphoramidites
20-Deoxyuridine phosphoramidites labeled at position C-6
have only recently been described.49 Unlike the C-5
analogues, the stability of hybridized probes containing
the modified bases might be even more sensitive to the
Scheme 1.

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M. Adamczyk et al. / Tetrahedron 59 (2003) 5749–5761
substituent. The work of Sanghvi, et al.50 indicated that even
introduction of a methyl group at that position destabilized
duplexed oligos. However, it appears that any destabiliza-
tion due to base modification can be accommodated in the
overall probe design, for example, by limiting the placement
of the modified base to the 30 or 50 termini or adjusting the
sequence length to achieve the desired hybridization
stringency. Our strategy for synthesis of phosphoramidites
14a–c from 20-deoxyuridine first involved the introduction
of a handle [e.g. ester group in compound 8] at position-6,
which could be conjugated to the haptenic group via suitable
linking arm. Thus, the 30- and 50-hydroxyl groups in 20-
deoxyuridine (Scheme 2) were protected with the 1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl (TIPDS)7 group to give 6,
which was then treated with LDA and DMF in the presence
of HMPA.51,52 The crude reaction mixture was treated with
acetic acid at 2788C to give the aldehyde (7) in 52% yield
after purification by silica gel column chromatography. The
aldehyde 7 was then subjected to the Wittig reaction with
methyl(triphenylphosphoranylidene)acetate in benzene to
afford the unsaturated ester 8 in almost quantitative yield as
the E-isomer (.98%). The next step was to exchange the
silyl protective group at the 50-position of 8 to the 4,40-
dimethoxytrityl group (DMT), which is compatible with
automated oligonucleotide synthesis. Thus, the TIPDS
protective group in 8 was hydrolyzed by treatment with
tetra-n-butylammonium fluoride in THF and the crude
compound was purified by silica gel column chromato-
graphy to afford diol (þ)-9 in excellent yield (98%). The
50-hydroxyl group in 9 0 was protected as the DMT ether
by treatment with 4,4 -dimethoxytrityl chloride in the
presence of silver nitrate and pyridine. The crude
compound was purified by silica gel column
chromatography (ethyl acetate/triethylamine/methanol,
97:2:1) to afford ester 10.
The next step in the synthesis of probes 14a–c was to
conjugate ester 10 to the hapten reporter groups via a linking
arm (Scheme 2). Thus, 10 was subjected to hydrolysis by
lithium hydroxide in aqueous THF and the resulting crude
acid (11) was conjugated with amines 12a–c using HOBt
and EDAC in anhydrous DMF. The conjugates 13a–c were
isolated in moderate yield and high purity after purification
by silica gel column chromatography. Finally, 13a–c were
Scheme 2.

M. Adamczyk et al. / Tetrahedron 59 (2003) 5749–5761
5753
treated with 2-cyanoethyl N,N-diisopropylchlorophosphor-
amidite in THF in the presence of diisopropylethylamine to
afford the probes 14a–c in 37–52% yield as mixtures of
diastereomers following reverse phase HPLC purification.
furanyl)-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinyl]-2-
propenamide (1) and (E)-N-(6-amino-hexyl)-3-[1-((4S,5R)-
5-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4-
hydroxytetrahydro-2-furanyl)-2,4-dioxo-1,2,3,4-tetrahydro-
5-pyrimidinyl]-2-propenamide (2) were purchased from
Berry & Associates, Inc. (Dexter, MI). 2-[3-(4-Nitro-
phenyl)-1-adamantyl]acetic acid (5a), 4-(9H-carbazol-2-
yloxy)butanoic acid (5b) were prepared according to our
published procedure.48 2-Cyanoethyl N,N-diisopropyl-
chlorophosphoramiditewas purchased from ChemGenes,
Inc. (Ashland, MA).
In summary, 11 new oligonucleotide building blocks,
phosphoramidites 3a–d, 4a–d and 14a–c, were prepared
from 20-deoxyuridine modified with an amino-terminated
linker from either the 5 or 6-position. When paired with a
complementary antibody, probes bearing adamantyl, carba-
zole, dansyl, and dabsyl haptens serve as powerful
immunodetection reagents employed in the Abbott LCx.
Moreover, other haptens can be likewise incorporated t0o
further expand the utility of 5- and 6-substituted 2 -
deoxyuridine phosphoramidites, i.e, multianalyte detection,
incorporation of internal controls, etc. The fluorescent
properties of probes built from dansyl phosphoramidites 3c,
4c, 14c and the quenching ability of dabsyl phosphoramidite
3d, 4d-derived probes are attractive for construction of a
variety of donor-quencher based homogeneous NAT
formats such as Beacon8–11 and Taqman.12
3.2. Preparation of 5-haptenated-50-O-dimethoxytrityl-
20-deoxyuridine 1a,b and 2a,b
Hapten carboxylic acid (5a or b, 3 mmol), N-hydroxy-
benzotriazole (HOBt, 0.61 g, 4.5 mmol, 150 mol%), and
triethylamine (1.7 mL, 12 mmol, 400 mol%) were sequen-
tially added to a suspension of the 5-aminoalkyl substituted
20-deoxyuridine derivative (1 or 2, 3 mmol) in dichloro-
methane or dimethylformamide (30 mL). 1-Ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDAC,
0.86 g, 4.5 mmol, 150 mol%) was added and the resulting
solution was stirred at ambient temperature for 18 h. The
solvent was then removed on a rotary evaporator under
reduced pressure. The residue was purified by silica
gel chromatography (300 g), eluting with ethyl acetate/
methanol/triethylamine (v:v:v shown). The material was
concentrated on a rotary evaporator under reduced pressure
and then the residue was repeatedly co-evaporated with
toluene (5£50 mL) followed by dichloromethane
(5£50 mL). Analytical reversed-phase HPLC [acetonitrile/
0.1 M aq triethylammonium acetate, v:v (shown). 2.0 mL/
min, 225 nm].
3. Experimental
3.1. General methods and materials
The 1H, 13C and 31P NMR were recorded on a Varian
Gemini spectrometer (300 MHz) and the chemical shifts for
1H and 13C (d) were reported in ppm relative to TMS and
coupling constants (J) were reported in Hz; 31P chemical
shifts are relative to phosphoric acid. Electrospray ioniz-
ation mass spectrometry (ESI-MS) was carried out on a
Perkin–Elmer (Norwalk, CT) Sciex API 100 Benchtop
system employing the Turbo Ionspray ion source. High-
resolution mass spectra (HRMS) were obtained on a
Nermang 3010 MS-50 or JEOL SX102-A mass spec-
trometers. Column chromatography was performed on silica
gel, Merck grade 60 (230–400 mesh). THF was freshly
distilled from a solution of sodium benzophenone ketyl.
Dichloromethane was freshly distilled from CaH2 under
nitrogen. All reagents were purchased from Aldrich
Chemical Co. (Milwaukee, WI) or Sigma Chemical Co.
(St. Louis, MO) and used without purification, except where
noted. All the solvents employed were of HPLC grade
purchased from EM Science (Gibbstown, NJ) and used as
received. Analytical reverse phase (RP) HPLC was
performed using a Waters (Milford, MA) mBondapak
RCM C18 10m (8£100 mm) column (solvent ratio v/v
reported) unless otherwise stated. Preparative reverse phase
(RP) HPLC was performed using a Waters mBondapak
RCM C18 10m (40£100 mm) column (solvent ratio v/v
reported) unless otherwise stated. Optical rotations were
measured on Autopol III polarimeter from Rudolph
Research (Flanders, NJ). Melting points were recorded in
open capillary tubes on an Electrothermal Melting Point
Apparatus (Barnstead International, Dubuque, IA) and were
uncorrected. IUPAC names were obtained using the
ACD/ILab Web service version 3.5 at http://www.acdlabs.
com/ilab/.
3.2.1. (1)-(E)-3-[1-((4S,5R)-5-{[Bis(4-methoxyphenyl)-
(phenyl)methoxy]methyl}-4-hydroxytetrahydro-2-fura-
nyl)-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinyl]-N-[2-
({2-[3-(4-nitrophenyl)-1-adamantyl]acetyl} amino)-
ethyl]-2-propenamide (1a). Chromatography (3–6%
MeOH in ethyl acetate containing 2% Et3N). Yield (84%
as a colorless glassy material). Mp 145–558C. Analytical
RP HPLC [75:25] 2.90 min, 95.2%. [a]2D3¼þ5.6 (c 1.03,
1
MeOH). H NMR (CDCl3) d 8.10–8.04 (m, 2H), 7.87 (s,
1H), 7.45–7.02 (m, 11H), 6.83 (d, 4H, J¼6.0 Hz), 6.78–
6.70 (m, 1H), 6.55 (dist t, 1H), 6.30 (t, 1H, J¼4.0 Hz),
4.53–4.47 (m, 1H), 4.22–4.16 (m, 1H) 3.75 (s, 3H), 3.74 (s,
3H), 3.52–3.34 (m, 6H), 2.66–2.56 (m, 1H), 2.28–2.16 (m,
1H), 2.12 (br s, 2H), 2.01 (s, 2H), 1.88–1.67 (m, 8H), 1.66–
1.45 (m, 6H). 13C NMR (CDCl3) d 171.7, 167.5, 161.8, 158.7,
158.6, 158.0, 149.4, 145.8, 144.4, 141.2, 135.5, 135.4, 132.1,
130.0, 129.9, 128.0, 127.9, 127.1, 125.9, 123.4, 122.4, 113.4,
110.2, 86.8, 86.7, 86.2, 72.4, 64.4, 63.7, 60.4, 55.2, 51.1, 47.3,
41.9, 41.6, 41.4, 40.3, 38.7, 37.7, 35.5, 33.6, 30.6, 28.9, 21.0,
19.1, 14.2, 13.7. ESI-MS(m/z)941(MþH)þ, 958(MþNH4)þ,
1898 (2MþNH4)þ. HRMS (FAB, m/z) calcd for
C53H57N5O11Na, 962.3949 (MþNa)þ; observed, 962.3947.
3.2.2. (2)-(E)-3-[1-((4S,5R)-5-{[Bis(4-methoxyphenyl)-
(phenyl)methoxy]methyl}-4-hydroxytetrahydro-2-fura-
nyl)-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinyl]-N-(2-
{[4-(9H-carbazol-2-yloxy)butanoyl]amino} ethyl)-2-pro-
penamide (1b). Chromatography (88:10:2). Yield (63%
(E)-N-(2-Aminoethyl)-3-[1-((4S,5R)-5-{[bis(4-methoxy-
phenyl) (phenyl)methoxy]methyl}-4-hydroxy-tetrahydro-2-

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M. Adamczyk et al. / Tetrahedron 59 (2003) 5749–5761
colorless glassy material. Mp 164–668C. Analytical RP
HPLC [50:50] 7.96 min, 97%. [a]2D3¼23.39 (c 0.765,
MeOH). 1H NMR (CDCl3) d 8.59 (br s, 1H), 7.86–7.73 (m,
3H), 7.36–7.05 (m, 11H), 6.80–6.62 (m, 9H), 6.15–6.06 (m,
1H), 4.42–4.37 (m, 1H), 4.15–4.08 (m, 1H), 3.86–3.79 (m,
2H), 3.65 (s, 6H), 3.41–3.22 (m, 6H), 2.58–2.42 (m, 1H),
2.36–2.24 (m, 2H), 2.18–1.97 (m, 2H). 13C NMR (CDCl3) d
173.8, 167.6, 162.2, 158.5, 158.0, 149.4, 144.4, 140.9, 139.6,
135.5, 135.4, 129.9, 129.0, 128.2, 127.9, 127.0, 125.2, 124.4,
123.1, 120.8, 119.2, 116.8, 113.3, 110.6, 109.8, 108.4, 95.4,
86.6, 86.3, 77.2, 67.2, 63.3, 55.1, 52.3, 45.8, 32.8, 25.2, 10.2,
7.1. ESI-MS (m/z) 894 (MþH)þ, 995 (MþEt3NH)þ.
added to a suspension of the 5-aminoalkyl substituted 20-
deoxyuridine derivative (1 or 2, 3 mmol) in aqueous
tetrahydrofuran (40%, 32 mL). An additional amount of
tetrahydrofuran (18 mL) was added and the reaction mixture
was stirred for 30 min. Saturated aq sodium chloride
solution (36 mL) was added to the reaction mixture then
extracted with ethyl acetate (300 mL). The organic layer
was separated and washed with brine (3£50 mL), dried over
anhydrous sodium sulfate and concentrated on a rotary
evaporator.
The residue was purified by silica gel chromatography
(300 g), eluting with ethyl acetate/methanol/triethylamine
(v:v:v shown). The eluted material was evaporated on a
rotary evaporator under reduced pressure then the residue
was repeatedly co-evaporated with toluene (5£50 mL) and
dichloromethane (5£50 mL). Analytical reversed-phase
HPLC [acetonitrile/0.1 M aq triethylammonium acetate,
v:v (shown), 2.0 mL/min].
3.2.3. (1)-(E)-3-[1-((4S,5R)-5-{[Bis(4-methoxyphenyl)-
(phenyl)methoxy]methyl}-4-hydroxytetrahydro-2-fura-
nyl)-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinyl]-N-[6-
({2-[3-(4-nitrophenyl)-1-adamantyl]acetyl} amino)-
hexyl]-2-propenamide (2a). Chromatography (3–6%
MeOH in ethyl acetate containing 2% Et3N). Yield (86%,
colorless glassy material). Mp 125–358C. Analytical RP
HPLC [75:25] 3.82 min, .99%. [a]2D3¼þ7.9 (c 0.9,
3.3.1. (1)-(E)-3-[1-((4S,5R)-5-{[Bis(4-methoxyphenyl)-
(phenyl)methoxy]methyl}-4-hydroxytetrahydro-2-fura-
nyl)-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinyl]-N-[2-
({[5-(dimethylamino)-1-naphthyl]sulfonyl} amino)-
ethyl]-2-propenamide (1c). Chromatography (2–4%
MeOH in ethyl acetate containing 2% of Et3N). Yield
(58%, pale yellow powder). Mp 145–498C. Analytical RP
HPLC [75:25] 2.48 min, .99%. [a]2D3¼þ11.8 (c 0.87,
acetonitrile/MeOH 9:1). 1H NMR (CDCl3þ5 drops of
CD3OD) d 8.53 (d, 1H, J¼8.7 Hz), 8.27 (d, 1H, 8.4 Hz),
8.20 (dd, 1H, J¼7.2, 1.2 Hz), 7.88 (s, 1H), 7.57–7.17 (m,
14H), 6.91 (d, 1H, J¼15.3 Hz), 6.81 (d, 4H, J¼8.7 Hz), 6.61
(dd, 1H, J¼15.3, 2.1 Hz), 6.36–6.32 (m, 1H), 6.27 (t, 1H,
J¼7.2 Hz), 4.44–4.39 (m, 1H), 4.14–4.09 (m, 1H), 3.75 (s,
3H), 3.74 (s, 3H), 3.44–3.16 (m, 6H), 2.87 (s, 6H), 2.52–
2.44 (m, 1H), 2.28–2.18 (m, 1H). 13C NMR (CDCl3þ5
drops of CD3OD) d 166.8, 162.0, 158.3, 158.2, 151.5, 149.3,
144.1, 140.7, 135.3, 135.2, 134.3, 132.1, 130.0, 129.7,
129.6, 129.2, 128.9, 128.0, 127.7, 127.6, 126.8, 122.8,
121.1, 118.6, 115.0, 113.0, 109.7, 86.4, 86.3, 85.6, 71.1,
63.3, 54.8, 44.9, 42.1, 40.8, 39.0. ESI-MS (m/z) 876
(MþH)þ, 898 (MþNa)þ, 1752 (2MþH)þ. HRMS (FAB,
m/z) calcd for C47H49N5O10SNa, 898.3092 (MþNa)þ;
observed, 898.3090.
1
MeOH). H NMR (CDCl3) d 8.14–8.11 (m, 2H), 7.90 (s,
1H), 7.50–7.10 (m, 11H), 6.82 (d, 4H, J¼8.4 Hz), 6.63 (d,
1H, J¼15.3 Hz), 6.33 (t, 1H, J¼6.6 Hz), 5.74 (t, 1H,
J¼5.7 Hz), 5.24 (t, 1H, J¼5.44 Hz), 4.53–4.47 (m, 1H),
4.10–4.08 (m, 1H), 3.76 (s, 6H), 3.52–3.04 (m, 6H), 2.66–
2.56 (m, 1H), 2.56–2.46 (m, 1H), 2.32–1.56 (m, 12), 1.52–
1.18 (m, 14H). 13C NMR (CDCl3) d 171.2, 166.3, 161.9,
159.1, 158.5, 149.5, 146.3, 144.9, 140.5, 135.9, 135.8,
132.1, 130.4, 130.3, 128.5, 128.4, 127.6, 126.4, 123.8,
122.5, 113.8, 110.8, 87.1, 86.6, 85.9, 72.4, 64.0, 60.8, 55.7,
51.7, 48.0, 46.5, 42.4, 42.0, 41.6, 39.6, 39.4, 38.2, 36.1,
34.1, 29.8, 29.7, 29.4, 26.6, 26.4, 21.5, 19.5, 14.6, 14.1. ESI-
MS (m/z) 997 (MþH)þ, 1014 (MþNH4)þ, 2010
(2MþNH4)þ. HRMS (FAB, m/z) calcd for C57H65N5O11Na,
1018.4574 (MþNa)þ; observed, 1018.4573.
3.2.4. (1)-(E)-3-[1-((4S,5R)-5-{[Bis(4-methoxyphenyl)-
(phenyl)methoxy]methyl}-4-hydroxytetrahydro-2-fura-
nyl)-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinyl]-N-(6-
{[4-(9H-carbazol-2-yloxy)butanoyl]amino} hexyl)-2-pro-
penamide (2b). Chromatography (88:10:2). Yield (72%
colorless glassy material). Mp 144–468C. Analytical RP
HPLC [50:50] 10.7 min, 99%. [a]2D3¼þ6.0 (c 1.0, MeOH).
1H NMR (CDCl3) d 9.06 (s, 1H), 7.91–7.82 (m, 3H), 7.38–
7.19 (m, 11H), 6.83–6.72 (m, 7H), 6.27 (t, 1H, J¼6.3 Hz),
6.05–5.99 (m, 1H), 5.60–5.55 (m, 1H), 4.48–4.42 (m, 1H),
4.13–4.07 (m, 1H), 3.98–3.92 (m, 1H), 3.67 (s, 6H), 3.37–
3.28 (m, 2H), 3.18–2.94 (m, 3H), 2.51–2.32 (m, 3H), 2.22–
2.01 (m, 3H), 1.31–0.96 (m, 10H). 13C NMR (CDCl3) d
172.8, 166.3, 161.9, 158.6, 158.1, 149.3, 144.4, 141.0, 140.5,
139.7, 135.5, 131.9, 129.9, 128.8, 129.0, 128.0, 127.9, 127.1,
124.4, 123.3, 122.1, 120.8, 119.2, 119.1, 117.0, 113.3, 110.6,
110.3,108.4,95.3, 86.6, 86.2, 85.6, 77.2, 71.8, 66.9, 63.6, 55.2,
41.0, 39.2, 33.0, 29.2, 26.0, 25.3. ESI-MS (m/z) 950 (MþH)þ,
972 (MþNa)þ. HRMS (FAB, m/z) calcd for C55H59N5O10Na,
972.4154 (MþNa)þ; observed, 972.4149.
3.3.2. (1)-(E)-3-[1-((4S,5R)-5-{[Bis(4-methoxyphenyl)-
(phenyl)methoxy]methyl}-4-hydroxytetrahydro-2-fura-
nyl)-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinyl]-N-(2-
{[(4-{(E)-2-[4-(dimethylamino)phenyl] diazenyl}phe-
nyl)sulfonyl]amino}ethyl)-2-propenamide (1d). Chroma-
tography (94:4:2). Yield (90%). Mp 182–848C. Analytical
RP HPLC [55:45, 254 nm] 7.16 min, 98%. [a]2D3¼þ107.0 (c
0.86, MeOH). 1H NMR (CDCl3) d 8.39–8.20 (m, 2H),
7.98–7.76 (m, 6H), 7.41–7.05 (m, 12H), 6.82–6.63 (m,
5H), 6.18–6.06 (m, 1H), 4.43–4.05 (m, 3H), 3.66 (s, 6H),
3.42–3.20 (m, 3H), 3.08 (s, 6H), 3.04–2.86 (m, 3H), 2.64–
2.51(m, 2H), 2.25–2.12(m, 2H). 13C NMR (CD3CN) d 167.8,
162.4, 159.6, 156.2, 154.4, 150.1, 145.9, 144.1, 142.9, 140.8,
136.8, 136.7, 133.8, 130.9, 130.9, 129.0, 128.9, 128.9, 127.9,
126.3, 123.3, 122.2, 114.1, 112.6, 110.5, 87.2, 87.1, 86.2, 71.8,
64.5, 55.9, 44.3, 41.0, 40.5, 39.8. ESI-MS (m/z) 930 (MþH)þ,
1860 (2MþH)þ. HRMS (FAB, m/z) calcd for C49H52N7O10S,
930.3491 (MþH)þ; observed, 930.3499.
3.3. Preparation of 5-haptenated-50-dimethoxytrityl-20-
deoxyuridine 1c,d and 2c,d
Hapten sulfonyl chloride (5c or d, 3 mmol) and sodium
carbonate (1.27 g, 12 mmol, 400 mol%) were sequentially

M. Adamczyk et al. / Tetrahedron 59 (2003) 5749–5761
5755
3.3.3. (1)-(E)-3-[1-((4S,5R)-5-{[Bis(4-methoxyphenyl)-
(phenyl)methoxy]methyl}-4-hydroxytetrahydro-2-fura-
nyl)-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinyl]-N-[6-
({[5-(dimethylamino)-1-naphthyl]sulfonyl} amino)-
hexyl]-2-propenamide (2c). Chromatography (96:2:2).
Yield (94%). Mp 115–168C. Analytical RP HPLC [60:40,
evaporated under reduced pressure then the residue was
repeatedly co-evaporated with toluene (5£50 mL) and
dichloromethane (5£50 mL). Analytical reversed-phase
HPLC [acetonitrile/0.1 M aq triethylammonium acetate,
v:v (shown), 2.0 mL/min].
1
254 nm] 6.4 min, 99%. [a]2D3¼þ4.21 (c 0.83, MeOH). H
3.4.1. 2-{[Bis(4-methoxyphenyl)(phenyl)methoxy]-
methyl}-5-[5-((1E)-3-{[2-({[3-(4-nitrophenyl)-1-adaman-
tyl]acetyl}amino)ethyl]amino}-3-oxoprop-1-enyl)-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl]tetrahydrofuran-
3-yl 2-cyanoethyl diisopropylamidophosphite (3a). Chro-
matography (3–4% MeOH containing 2% Et3N). Yield
(74%, mixture of diastereomers). Analytical RP HPLC
[60:40, 254 nm] 11.34 and 13.88 min, 95%. 1H NMR
(CD3CN) d 8.22–8.14 (m, 2H), 7.84–6.60 (m, 14H), 6.78–
6.70 (m, 2H), 6.32–6.24 (m, 1H), 4.66–4.56 (m, 1H), 4.28–
4.18 (m, 1H), 3.92–3.56 (m, 6H), 3.84 (s, 6H), 3.48–3.28
(m, 6H), 2.76–2.42 (m, 8H), 2.30–1.14 (m, 25H). 31P NMR
NMR (CDCl3) d 8.49 (d, 1H, J¼8.5 Hz), 8.30 (d, 1H,
J¼8.8 Hz), 8.19 (d, 1H, J¼7.1 Hz), 7.91 (s, 1H), 7.51–7.44
(m, 2H), 7.41–7.17 (m, 2H), 7.30–7.10 (m, 11H), 6.81–
6.71 (m, 4H), 6.32 (t, 1H, J¼6.6 Hz), 5.69–5.58 (m, 1H),
4.51–4.45 (m, 1H), 4.2–4.08 (m, 1H), 3.69 (s, 6H), 3.45–
3.24 (m, 2H), 3.08–2.90 (m, 3H), 2.84 (s, 6H), 2.58–2.47
(m, 1H), 2.33–2.21 (m, 1H), 1.42–0.98 (m, 8H). 13C NMR
(CDCl3) d 166.1, 162.1, 158.5, 158.5, 151.8, 149.5, 144.5,
140.6, 135.5, 135.5, 135.0, 131.8, 130.2, 129.9, 129.8,
129.7, 129.6, 129.3, 128.2, 127.9, 127.0, 123.1, 122.0,
118.9, 115.1, 113.3, 110.3, 86.5, 86.2, 85.5, 71.7, 63.6, 55.2,
45.3, 43.0, 40.1, 39.0, 29.1, 28.9, 25.8, 25.7. ESI-MS (m/z)
933 (MþH)þ, 1033 (MþEt3NH)þ. HRMS (FAB, m/z) calcd
for C51H57N5O10SNa, 954.3718 (MþNa)þ; observed,
954.3721.
(CD3CN)
d
148.62, 148.58. ESI-MS (m/z) 1058
(MþNH4)þ. HRMS (FAB, m/z) calcd for C62H74N7O12PNa,
1162.5025 (MþNa)þ; observed, 1162.5041. Anal. calcd for
C62H74N7O12P: C, 65.31%. H, 6.54%. N, 8.60%. P, 2.72%.
Found: C, 65.81%. H, 6.65%. N, 8.13%. P, 3.04%.
3.3.4. (1)-(E)-3-[1-((4S,5R)-5-{[Bis(4-methoxyphenyl)-
(phenyl)methoxy]methyl}-4-hydroxytetrahydro-2-fura-
nyl)-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinyl]-N-(6-
{[(4-{(E)-2-[4-(dimethylamino)phenyl] diazenyl}phe-
nyl)sulfonyl]amino}hexyl)-2-propenamide (2d). Chroma-
tography (94:4:2). Yield (91%). Analytical RP HPLC
[55:45, 254 nm] 13.03 min, 99%. [a]2D3¼þ5.6 (c 0.75,
3.4.2. 2-{[Bis(4-methoxyphenyl)(phenyl)methoxy]-
methyl}-5-[5-((1E)-3-{[2-(9H-carbazol-2-yloxy)ethyl]-
amino}-3-oxoprop-1-enyl)-2,4-dioxo-3,4-dihydropyrimi-
din-1(2H)-yl]tetrahydrofuran-3-yl 2-cyanoethyl
diisopropylamidophosphite
(3b).
Chromatography
(93:5:2). Yield (77% as a mixture of diastereomers).
Analytical RP HPLC [60:40, 254 nm] 12.29 and
14.20 min, 99%. 1H NMR (CDCl3) d 8.61 (d, 1H,
J¼2.2 Hz), 7.89 (d, 1H, J¼7.4 Hz), 7.81 (d, 1H,
J¼8.5 Hz), 7.73 (d, 1H, J¼15.6 Hz), 7.46–7.14 (m, 11H),
7.11–7.00 (m, 1H), 6.87–6.61 (m, 8H), 6.17 (t, 1H,
J¼5.7 Hz), 6.08–5.98 (m, 1H), 4.56–4.47 (m, 1H), 4.23–
4.15 (m, 1H), 3.93 (t, 2H, J¼6.0 Hz), 3.73 (s, 3H), 3.73 (s,
3H), 3.68–3.51 (m, 4H), 3.47–3.34 (m, 1H), 3.33–3.24 (m,
5H), 2.57 (t, 1H, J¼6.3 Hz), 2.41 (t, 1H, J¼6.3 Hz), 2.37–
2.32 (m, 2H), 2.24–2.04 (m, 1H), 1.92–1.61 (m, 2H), 1.98–
1.04 (m, 12H). 31P NMR (CDCl3) d 150.17, 149.95. ESI-MS
(m/z) 1094 (MþH)þ, 1111 (MþNH4)þ; HRMS (FAB, m/z)
calcd for C60H68N7O11PNa, 1116.4607 (MþNa)þ;
observed,1116.4604. Anal. calcd for C60H68N7O11P: C,
65.86%. H, 6.26%. N, 8.96%. P, 2.83%. Found: C, 66.43%.
H, 6.31%. N, 8.47%. P, 3.08%.
1
MeOH). H NMR (CD3CN) d 7.92–7.80 (m, 6H), 7.73 (s,
1H), 7.43–7.38 (m, 2H), 7.32–7.17 (m, 8H), 6.89–6.77 (m,
8H), 6.23–6.14 (m, 2H), 5.67 (t, 1H, J¼6.0 Hz), 4.35–4.31
(m, 1H), 3.98–3.94 (m, 1H), 3.71 (s, 6H), 3.27–3.23 (m,
2H), 3.14–3.05 (m, 2H), 3.06 (s, 6H), 2.89–2.83 (m, 2H),
2.34–2.17 (m, 3H), 1.43–1.27 (m, 4H), 1.26–1.12 (m, 4H).
13C NMR (CD3CN) d 166.7, 162.5, 159.6, 156.2, 154.4,
150.2, 145.9, 144.1, 142.4, 141.3, 136.8, 136.7, 133.0,
130.9, 129.0, 128.9, 128.9, 127.9, 126.4, 123.2, 122.9,
114.1, 112.5, 110.7, 87.2, 87.1, 86.3, 71.8, 64.6, 55.8, 43.8,
41.0, 40.5, 39.7, 30.0, 29.9, 26.8, 26.6. ESI-MS (m/z) 986
(MþH)þ. HRMS (FAB, m/z) calcd for C53H60N7O10S,
986.4117 (MþH)þ; observed, 986.4125.
3.4. Preparation of 5-haptenated-50-O-dimethoxytrityl-
20-deoxyuridine phosphoramidites 3a–d and 4a–d
Diisopropylethylamine (1.4 mL, 8 mmol, 400 mol%) and
2-cyanoethyl
3.4.3. 2-{[Bis(4-methoxyphenyl)(phenyl)methoxy]-
methyl}-5-[5-((1E)-3-{[2-({[5-(dimethylamino)-1-
naphthyl]sulfonyl}amino)ethyl]amino}-3-oxoprop-1-
enyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl]tetra-
hydrofuran-3-yl 2-cyanoethyl diisopropylamidophos-
phite (3c). Chromatography (2–3% MeOH in ethyl
acetate containing 2% Et3N). This material was further
purified by preparative RP HPLC [acetonitrile/H2O 76:24,
45 mL/min, 225 nm]. The product was collected, concen-
trated on rotary evaporator (,358C) and azeotroped with
toluene (5£40 mL) followed by dichloromethane
(5£40 mL). Yield (58%, mixture of diastereomers, pale
yellow solid). Analytical RP HPLC [acetonitrile/water
85:15, 225 nm] 4.04 and 4.80 min, 98.3%. 1H NMR
(CD3CN) d 8.22–8.14 (m, 1H), 7.84–6.60 (m, 18H),
6.78–6.70 (m, 3H), 4.60–4.56 (m, 1H), 4.20–4.08 (m, 1H),
N,N-diisopropylchlorophosphoramidite
(0.67 mL, 3 mmol, 150 mol%) 0were added sequentially to
a solution of 5-haptenated-5 -dimethoxytrityl-20-deoxy-
uridine (1a–d, 2a–d, 2 mmol) dissolved in dichloro-
methane (25 mL) at room temperature under nitrogen.
After stirring the mixture for 1 h, dry methanol (0.08 mL,
2 mmol, 100 mol%) was added, and stirred the reaction for
an additional 30 min. The reaction mixture was diluted with
6% triethylamine in ethyl acetate (265 mL) then washed
sequentially with 10% aq sodium carbonate (3£40 mL) and
brine (3£40 mL). The organic layer was dried over
anhydrous sodium sulfate and concentrated on a rotary
evaporator. The residue was purified by silica gel chroma-
tography (300 g), eluting with ethyl acetate/methanol/
triethylamine (v:v:v shown). The eluted material was

5756
M. Adamczyk et al. / Tetrahedron 59 (2003) 5749–5761
3.80–3.10 (m, 5H), 3.84 (s, 6H), 2.80 (s, 6H), 2.92–2.20
(m, 10H), 1.28–1.05 (m, 12H). 31P NMR (CD3CN) d
148.49. ESI-MS (m/z) 1076 (MþH)þ. Anal. calcd for
C56H66N7O11PS: C, 62.50%. H, 6.18%. N, 9.11%. P, 2.88%.
Found: C, 63.87%. H, 6.23%. N, 8.47%. P, 2.77%.
1168 (MþNH4)þ. HRMS (FAB, m/z) calcd for C64H76N7-
O11PNa (MþNa)þ 1172.5233; observed 1172.5244. Anal.
calcd for C64H76N7O11P: C, 66.83%. H, 6.66%. N, 8.52%.
P, 2.69%. Found: C, 67.35%. H, 6.72%. N, 7.87%. P,
2.47%.
3.4.4. 2-{[Bis(4-methoxyphenyl)(phenyl)methoxy]-
methyl}-5-[5-{(1E)-3-[(2-{[(4-{(E)-[4-(dimethylamino)-
phenyl]diazenyl}phenyl)sulfonyl]amino}ethyl)amino]-3-
oxoprop-1-enyl}-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl]tetrahydrofuran-3-yl 2-cyanoethyl diisopropylamido-
phosphite (3d). Chromatography (96:2:2). Yield (75%).
Analytical RP HPLC [70:30, 254 nm] 6.9 and 7.72 min,
3.4.7. 2-{[Bis(4-methoxyphenyl)(phenyl)methoxy]-
methyl}-5-[5-((1E)-3-{[6-({[5-(dimethylamino)-1-
naphthyl]sulfonyl}amino)hexyl]amino}-3-oxoprop-1-
enyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl]tetra-
hydrofuran-3-yl 2-cyanoethyl diisopropylamido-
phosphite (4c). Chromatography (96:2:2). The material
was further purified by preparative RP HPLC [acetonitrile/
water 78:22, 45 mL/min, 254 nm]. After lyophilization the
yield was 41%. Analytical RP HPLC [67:33, 254 nm] 9.76
1
99%. H NMR (CD3CN) d 9.10 (br s, 1H), 7.88–7.79 (m,
6H), 7.69 (d, 1H, J¼7.9 Hz), 7.44–7.38 (m, 2H), 7.33–7.15
(m, 8H), 6.95–6.74 (m, 8H), 6.36 (q, 1H, J¼5.7 Hz), 6.13
(q, 1H, J¼6.8 Hz), 6.09–6.03 (m, 1H), 4.55–4.44 (m, 1H),
4.15–4.06 (m, 1H), 3.81–3.70 (m, 7H), 3.67–3.48 (m, 3H),
3.33–3.15 (m, 4H), 3.07 (s, 3H), 3.07 (s, 3H), 3.03–2.96
(m, 2H), 2.62 (t, 1H, J¼5.8 Hz), 2.49 (t, 1H, J¼6.0 Hz),
2.45–2.36 (m, 1H), 2.32–2.23 (m, 1H), 1.16–1.12 (m, 9H),
1.02 (d, 3H, J¼6.8 Hz). 31P NMR (CD3CN) d 148.86. ESI-
MS (m/z) 1129 (MþH)þ. HRMS (FAB, m/z) calcd for
C58H69N9O11PS (MþH)þ 1130.4569; observed 1130.4569.
Anal. calcd for C58H69N9O11PS: C, 61.63%. H, 6.06%. N,
11.15%. P, 2.74%. S, 2.84%. Found: C, 62.16%. H, 6.28%.
N, 10.94%. P, 2.48%. S, 2.63%.
1
and 11.24 min, 95%. H NMR (CD3CN) d 9.21 (br s, 1H),
8.52–8.49 (m, 1H), 8.25 (d, 1H, J¼8.5 Hz), 8.16–8.13 (m,
1H), 7.79–7.75 (m, 1H), 7.61–7.52 (m, 2H), 7.44–7.39 (m,
2H), 7.33–7.15 (m, 10H), 7.07–6.95 (m, 1H), 6.90–6.83
(m, 4H), 6.22–6.06 (m, 2H), 5.82 (t, 1H, J¼5.8 Hz), 4.55–
4.46 (m, 1H), 4.17–4.08 (m, 1H), 3.72 (s, 3H), 3.71 (s, 3H),
3.67–3.48 (m, 4H), 3.39–3.23 (m, 2H), 3.03–2.95 (m, 2H),
2.82 (s, 6H), 2.81–2.75 (m, 2H), 2.64–2.46 (m, 1H), 2.44–
2.28 (m, 2H), 1.26–0.93 (m, 20H). 31P NMR (CDCl3) d
149.31. ESI-MS (m/z) 1132 (MþH)þ. HRMS (FAB, m/z)
calcd for C60H74N7O11PSNa (MþNa)þ 1154.4797;
observed 1154.4806. Anal. calcd for C60H74N7O11PS: C,
63.64%. H, 6.59%. N, 8.66%. P, 2.74%. S, 2.83%. Found:
C, 63.68%. H, 6.61%. N, 8.33%. P, 2.46%. S, 2.60%.
3.4.5. 2-{[Bis(4-methoxyphenyl)(phenyl)methoxy]-
methyl}-5-[5-((1E)-3-{[6-({[3-(4-nitrophenyl)-1-adaman-
tyl]acetyl}amino)hexyl]amino}-3-oxoprop-1-enyl)-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl]tetrahydrofuran-
3-yl 2-cyanoethyl diisopropylamidophosphite (4a). Chro-
matography (3–4% MeOH in ethyl acetate containing 2%
Et3N). Yield (64%, mixture of diastereomers). Analytical
3.4.8. 2-{[Bis(4-methoxyphenyl)(phenyl)methoxy]-
methyl}-5-[5-{(1E)-3-[(6-{[(4-{(E)-[4-(dimethylamino)-
phenyl]diazenyl}phenyl)sulfonyl]amino}hexyl)amino]-
3-oxoprop-1-enyl}-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl]tetrahydrofuran-3-yl 2-cyanoethyl diisopropyl-
amidophosphite (4d). Chromatography (97:1:2). The
material was further purified by preparative RP HPLC
(acetonitrile/water 80:20, 45 mL/min at 254 nm). After
lyophilization the yield was 69%. Analytical RP HPLC
[acetonitrile/water 65:35, 254 nm] 17.32 and 20.23 min,
1
RP HPLC [85:15, 225 nm] 5.76 and 6.87 min, 95%. H
NMR (CD3CN) d 8.22–8.14 (m, 2H), 7.84–6.60 (m, 14H),
6.78–6.70 (m, 2H), 6.32–6.24 (m, 1H), 4.66–4.56 (m, 1H),
4.28–4.18 (m, 1H), 3.92–3.56 (m, 6H), 3.84 (s, 6H), 3.48–
3.28 (m, 6H), 2.76–2.42 (m, 8H), 2.30–1.14 (m, 32H). 31P
NMR (CD3CN) d 148.71. ESI-MS (m/z) 1197 (MþH)þ.
HRMS (FAB, m/z) calcd for C66H82N7O12PNa, 1218.5651
(MþNa)þ; observed, 1218.5667. Anal. calcd for
C66H82N7O12P: C, 66.26%. H, 6.91%. N, 8.20%. P,
2.59%. Found: C, 66.95%. H, 7.01%. N, 7.79%. P, 2.97%.
1
95%. H NMR (CD3CN) d 7.92–7.74 (m, 7H), 7.44–7.38
(m, 2H), 7.32–7.16 (m, 8H), 7.50–6.97 (m, 1H), 6.87–6.78
(m, 7H), 6.21–6.09 (m, 2H), 5.72–5.65 (m, 1H), 4.54–4.45
(m, 1H), 4.16–4.06 (m, 1H), 3.72 (s, 3H), 3.71 (s, 3H),
3.68–3.46 (m, 4H), 3.38–3.22 (m, 2H), 3.12–3.06 (m, 1H),
3.06 (s, 6H), 2.89–2.81 (m, 2H), 2.61 (t, 1H, J¼6.0 Hz),
2.48 (t, 1H, J¼5.8 Hz), 2.45–2.27 (m, 2H), 1.42–1.25 (m,
4H), 1.25–1.09 (m, 13H), 1.01 (d, 3H, J¼6.8 Hz). 31P NMR
(CD3CN) d 148.79. ESI-MS (m/z) 1185 (MþH)þ. HRMS
(FAB, m/z) calcd for C62H76N9O11PSNa (MþNa)þ
1208.5015; observed 1208.5030. Anal. calcd for
C62H76N9O11PS: C, 62.77%. H, 6.46%. N, 10.63%. P,
2.61%. S, 2.70%. Found: C, 62.42%. H, 6.56%. N, 10.53%.
P, 2.49%. S, 2.60%.
3.4.6. 2-{[Bis(4-methoxyphenyl)(phenyl)methoxy]-
methyl}-5-[5-((1E)-3-{[6-(9H-carbazol-2-yloxy)hexyl]-
amino}-3-oxoprop-1-enyl)-2,4-dioxo-3,4-dihydropyrimi-
din-1(2H)-yl]tetrahydrofuran-3-yl 2-cyanoethyl
diisopropylamidophosphite
(96:2:2). Yield (67%). Analytical RP HPLC [60:40,
(4b).
Chromatography
1
254 nm] 16.53 and 19.37 min, 99%. H NMR (CDCl3) d
9.45 (s, 1H), 7.94 (d, 1H, J¼7.7 Hz), 7.89 (d, 1H,
J¼8.8 Hz), 7.75 (d, 1H, J¼8.2 Hz), 7.44–7.39 (m, 3H),
7.33–7.10 (m, 9H), 7.40–6.75 (m, 8H), 6.46–6.38 (m, 1H),
6.27–6.14 (m, 2H), 4.54–4.45 (m, 1H), 4.37–4.08 (m, 1H),
4.03 (t, 2H, J¼6.0 Hz), 3.72 (s, 6H), 3.67–3.49 (m, 3H),
3.38–3.22 (m, 2H), 3.16–3.06 (m, 4H), 2.64–2.46 (m, 1H),
2.36–2.27 (m, 2H), 2.18–2.13 (m, 1H), 2.09–2.01 (m, 1H),
1.97–1.92 (m, 2H), 1.43–1.32 (m, 5H), 1.28–1.18 (m, 5H),
1.16–1.11 (m, 9H), 1.02 (d, 3H, J¼6.6 Hz). 31P NMR
(CDCl3) d 148.73, 148.76. ESI-MS (m/z) 1150 (MþH)þ,
3.4.9. 20-Deoxyuridine-30,50-(1,1,3,3-tetraisopropyl)di-
siloxane (6). Imidazole (17.5 g, 256.5 mmol, 450 mol%)
and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (20.0 g,
62.72 mmol, 110 mol%) were added to a solution of 20-
deoxyuridine (13.0 g, 57.01 mmol) in DMF (80 mL) at
room temperature under nitrogen. After stirring for 23 h, the
mixture was poured into water (1.1 L) and extracted with
ethyl acetate (0.8 L). The organic layer was washed with

M. Adamczyk et al. / Tetrahedron 59 (2003) 5749–5761
5757
water (0.6 L), brine (0.4 L), dried over anhydrous sodium
sulfate and concentrated on a rotary evaporator. The crude
product was purified by silica gel column chromatography
(40% ethyl acetate in hexane) to afford of 6 (27.0 g,
quantitative). Analytical RP HPLC [acetonitrile/0.05% aq
trifluoroacetic acid 80:20, 2.0 mL/min at 254 nm]
(MþNH4)þ, 1126 (2MþNH4)þ. HRMS (FAB, m/z) calcd
for C25H43N2O8Si2 (MþH)þ 555.2558; observed 555.2537.
3.4.12. (1)-6-[2-(Methoxycarbonyl)ethenyl]-20-deoxy-
uridine (9). Tetrabutylammonium fluoride (1.0 M solution
in THF, 42.6 mL, 42.6 mmol, 200 mol%) was added to a
solution of olefinic ester 8 (11.8 g, 21.3 mmol) in THF
(500 mL) at room temperature under nitrogen. The mixture
was stirred for 40 min then concentrated on a rotary
evaporator. Purification by silica gel column chromatog-
raphy (5% MeOH in ethyl acetate) afforded compound 9
(6.5 g, 98%). Mp 267–88C. Analytical RP HPLC [aceto-
nitrile/0.05% aq trifluoroacetic acid 8:92, 1.0 mL/min,
225 nm] 12.65 min, 98%. [a]2D3¼þ44.3 (c 0.91, MeOH).
1H NMR (CDCl3þ0.1 mL of CD3OD) d 7.59 (d, 1H,
J¼15.6 Hz), 6.41 (d, 1H, J¼15.6 Hz), 6.00 (t, 1H,
J¼7.1 Hz), 5.78 (d, 1H, J¼0.6 Hz), 4.59–4.54 (m, 1H),
3.96–3.87 (m, 2H), 3.84 (s, 3H), 3.75 (dd, 1H, J¼12.1,
3.5 Hz), 2.86–2.76 (m, 1H), 2.18–2.10 (m, 1H). 13C NMR
(CDCl3þ0.1 mL of CD3OD) d 165.5, 162.6, 151.0, 150.6,
135.7, 126.8, 103.3, 87.3, 87.2, 70.6, 62.0, 52.4, 38.1. ESI-
MS (m/z) 311 (M2H)2, 623 (2M2H)2. HRMS (FAB, m/z)
calcd for C13H17N2O7 (MþH)þ 313.1036; observed
313.1029.
1
11.14 min, 99%. [a]D23¼þ34.5 (c 1.5, CHCl3). H NMR
(CDCl3) d 9.85 (br s, 1H), 7.79 (d, 1H, J¼8.2 Hz), 6.06 (dd,
1H, J¼6.8, 1.4 Hz), 5.71 (dd, 1H, J¼8.2, 1.6 Hz), 4.48–
4.39 (m, 1H), 4.14 (dd, 1H, J¼13.2, 1.2 Hz), 4.01 (dd, 1H,
J¼13.2, 2.7 Hz), 3.76 (td, 1H, J¼8.5, 2.7, 2.2 Hz), 2.57–
2.46 (m, 1H), 2.30–2.23 (m, 1H), 1.12–1.07 (m, 6H), 1.06–
0.95 (m, 10H). 13C NMR (CDCl3) d 163.8, 150.2, 139.6,
101.7, 85.0, 84.2, 66.9, 59.9, 39.8, 17.4, 17.3, 17.3, 17.2,
17.0, 16.9, 16.7, 13.3, 12.9, 12.8, 12.3. ESI-MS (m/z) 471
(MþH)þ, 488 (MþNH4)þ, 941 (2MþH)þ.
3.4.10. 6-Formyl-20-deoxyuridine-30,50-(1,1,3,3-tetra0iso-
propyl)disiloxane (7). A mixture of 20-deoxyuridine-3 ,50-
(1,1,3,3-tetraisopropyl)disiloxane (6, 10.0 g, 21.27 mmol)
in THF (100 mL) and HMPA (22.0 mL, 127.6 mmol,
600 mol%) was added to via canula to a solution of LDA
(1 M solution in cyclohexane, 42.5 mL, 63.83 mmol,
300 mol%) in THF (100 mL) at 2788C under nitrogen.
After 30 min, DMF (18.1 mL, 233.9 mmol, 1100 mol%)
was added at 2788C and the stirring was continued for an
additional 2 h. Acetic acid (12.1 mL, 212.7 mmol,
1000 mol%) was added to the reaction at 2788C and the
mixture was poured into water (350 mL). The mixture was
then extracted with ethyl acetate (450 mL). The organic
layer was washed with saturated aq sodium bicarbonate
solution (400 mL), water (500 mL), brine (500 mL), dried
over anhydrous sodium sulfate and concentrated on a rotary
evaporator. The crude product was purified by silica gel
column chromatography (30–40% ethyl acetate in hexanes)
to obtain aldehyde 7 (5.5 g, 52%, colorless glass). 1H NMR
(CDCl3) d 9.72 (s, 1H), 8.89 (brs, 1H), 6.36 (dd, 1H, J¼8.5,
4.9 Hz), 6.09 (s, 1H), 4.82–4.74 (m, 1H), 4.04–3.93 (m,
2H), 3.83–3.78 (d, 1H), 2.72–2.63 (m, 1H), 2.54–2.45 (m,
1H), 1.15–0.92 (m, 16H). 13C NMR (CDCl3) d 184.3,
161.6, 149.3, 148.3, 109.3, 85.8, 85.0, 71.8, 62.2, 40.1, 17.5,
17.4, 17.3, 17.2, 17.1, 16.9, 16.8, 13.3, 13.1, 12.8, 12.5. ESI-
MS (m/z) 516 (MþNH4)þ, 1014 (2MþNH4)þ.
3.4.11. (1)-6-[2-(Methoxycarbonyl)ethenyl]-20-deoxy-
uridine-30,50-(1,1,3,3-tetraisopropyl)disiloxane (8). Methyl
(triphenylphosphoranylidene)acetate (7.37 g, 22.1 mmol,
100 mol%) was added to a solution of aldehyde 7 (11.0 g,
22.1 mmol) in benzene (100 mL) at room temperature under
nitrogen. The mixture was stirred for 2 h then concentrated on
a rotary evaporator. Purification by silica gel column
chromatography (30–40% ethyl acetate in hexanes) to
afforded 8 (11.8 g, 96%, pale yellow solid). Mp 113–1148C.
Analytical RP HPLC [acetonitrile/0.05% aq trifluoroacetic
acid 80:20, 2.0 mL/min, 254 nm] 16.31 min, 98%.
[a]2D3¼þ26.7 (c 0.86, CHCl3). 1H NMR (CDCl3) d 8.15 (br
s, 1H), 7.53 (d, 1H, J¼15.6 Hz), 6.39 (d, 1H, J¼15.6 Hz),
5.93–5.88 (m, 1H), 5.75 (s, 1H), 4.91–4.84 (m, 1H), 4.07–
3.96 (m, 2H), 3.83 (s, 3H), 3.82–3.77 (m, 1H), 2.82–2.75 (m,
1H), 2.38–2.28 (m, 1H), 1.30–1.00 (m, 16H). 13C NMR
(CDCl3) d 165.1, 162.2, 150.9, 149.5, 135.8, 126.7, 102.9,
85.6, 85.2, 73.3, 63.9, 52.3, 39.4, 23.4, 20.1, 17.5, 17, 4, 17.3,
17.2, 17.1, 16.9, 13.2, 12.6, 12.5. ESI-MS (m/z) 572
3.4.13. (1)-50-O-(4,40-Dimethoxytrityl)-6-[2-(methoxy-
carbonyl)ethenyl]-20-deoxyuridine (10). Compound (9
(6.5 g, 20.8 mmol) was dissolved in pyridine (120 mL)
and the mixture was concentrated on a rotary evaporator.
The residue was dissolved in THF (350 mL). Pyridine
(8.4 mL, 104.1 mmol, 500 mol%) and silver nitrate (3.54 g,
20.8 mmol, 100 mol%) were added to the mixture. After
stirring for 5 h at room temperature, the mixture was filtered
through Celite into a saturated aq sodium bicarbonate
solution (300 mL) and the filter cake was washed with 5%
triethylamine in chloroform (50 mL). The filtrate was then
extracted with 5% triethylamine in chloroform (500 mL).
The organic layer was dried over anhydrous sodium sulfate
then concentrated under reduced pressure. Purification by
silica gel column chromatography (ethyl acetate/triethyl-
amine/methanol 97:2:1) to afforded DMT–ether 10 (9.8 g,
76% as a colorless solid). Mp 100–18C. Analytical RP
HPLC [acetonitrile/water 50:50, 2.0 mL/min, 254 nm]
1
7.42 min, 99%. [a]2D3¼þ18.1 (c 0.585, MeOH). H NMR
(CD3CN) d 7.45–7.22 (m, 10H), 6.86–6.81 (m, 4H), 6.41
(d, 1H, J¼15.6 Hz), 6.23–6.18 (m, 1H), 5.81 (d, 1H,
J¼0.6 Hz), 4.24–4.16 (m, 1H), 3.88–3.81 (m, 1H), 3.75 (s,
3H), 3.74 (s, 3H), 3.63 (s, 3H), 3.38–3.32 (m, 1H), 3.23–
3.18 (m, 1H), 2.65–2.50 (m, 1H), 2.14–2.04 (m, 1H). 13C
NMR (CD3CN) d 166.2, 163.0, 159.6, 151.4, 151.3, 146.2,
137.0, 136.9, 136.8, 131.0, 130.9, 129.3, 129.0, 128.7,
127.7, 127.1, 103.9, 86.0, 71.8, 65.4, 55.8, 52.7, 39.1. ESI-
MS (m/z) 637 (MþNa)þ. HRMS (FAB, m/z) calcd for
C34H34N2O9 (M)þ 614.2264; observed 614.2257.
3.4.14. N-(6-Aminohexyl)-2-[3-(4-nitrophenyl)-1-ada-
mantyl]acetamide (12a). tert-Butyl 6-aminohexylcarba-
mate (2.4 g, 11.11 mmol), 3-(4-nitrophenyl)-adamantane-
acetic acid (5a, 3.50 g, 11.11 mmol, 100 mol%), N-hydroxy-
benzotriazole (2.55 g, 16.66 mmol, 150 mol%) and triethyl-
amine (6.20 mL, 44.44 mmol, 400 mol%) were dissolved in
dichloromethane (40 mL) at room temperature under nitro-
gen. To this mixture EDAC (2.13 g, 16.66 mmol, 150 mol%)

5758
M. Adamczyk et al. / Tetrahedron 59 (2003) 5749–5761
was added. The reaction mixture was stirred for 5 h at room
temperature then was diluted with chloroform (50 mL);
washed with water (50 mL), saturated aq sodium bicarbonate
(50 mL). The organic layer was dried over anhydrous sodium
sulfate and concentrated on a rotary evaporator. The crude
product was purified by silica gel column chromatography
(60% ethyl acetate in hexanes) to afford N-Boc-12a (4.88 g,
85%). Mp 85–68C. Analytical RP HPLC [acetonitrile/0.05%
aq trifluroacetic acid 70:30, 2.0 mL/min, 254 nm] 6.53 min,
99%. 1H NMR (CDCl3) d 8.17–8.12 (m, 2H), 7.53–7.47 (m,
2H), 5.79 (br s, 1H), 4.60 (br s, 1H), 3.24 (d, 1H, J¼6.8 Hz),
3.20 (d, 1H, J¼6.8 Hz), 3.11 (d, 1H, J¼6.3 Hz), 3.06 (d, 1H,
J¼6.3 Hz), 2.25–2.19 (m, 2H), 2.03 (br s, 1H), 1.90–1.85 (m,
4H), 1.82 (br s, 1H), 1.73–1.66 (m, 8H), 1.53–1.42 (m, 13H),
1.37–1.29 (m, 4H). 13C NMR (CDCl3) d 170.5, 158.0, 156.0,
145.8, 129.9, 123.3, 79.0, 51.0, 47.5, 41.9, 41.4, 40.0, 38.9,
37.7, 35.5, 33.5, 29.9, 29.4, 28.9, 28.3, 26.1, 25.8. ESI-MS
(m/z) 514 (MþH)þ, 531 (MþNH4)þ, 536 (MþNa)þ. HRMS
(FAB, m/z) calcd for C29H44N3O5 (MþH)þ 514.3281;
observed 514.3279.
was concentrated and the residue was partitioned between
ethyl acetate (150 mL) and water (100 mL). The organic
layer was separated, washed with brine (75 mL), dried over
anhydrous sodium sulfate and concentrated on a rotary
evaporator. The crude product was purified by silica gel
column chromatography (30% ethyl acetate in hexane) to
afford 2-O-[[6-(tert-butoxycarbonylamino)hexyl]oxy]car-
bazole (3.0 g 61%, pale yellow solid). Mp 129–308C.
Analytical RP HPLC [acetonitrile/0.05% aq trifluoroacetic
acid 65:35, 2.0 mL/min at 225 nm] 11.49 min, 99%. 1H
NMR (CDCl3) d 8.11 (br s, 1H), 7.95 (d, 1H, J¼7.4 Hz),
7.91 (d, 1H, J¼8.5 Hz), 7.38–7.30 (m, 2H), 7.23–7.16 (m,
1H), 6.86–6.81 (m, 2H), 4.55 (br s, 1H), 4.00 (t, 2H,
J¼6.5 Hz), 3.12 (q, 2H, J¼6.3 Hz), 1.85–1.76 (m, 2H),
1.56–1.33 (m, 15H). 13C NMR (CDCl3) d 158.4, 156.0,
140.8, 139.5, 124.4, 123.5, 120.9, 119.4, 117.0, 110.3,
108.7, 95.3, 79.1, 68.1, 40.5, 30.0, 29.1, 28.4, 26.5, 25.7.
ESI-MS (m/z) 383 (MþH)þ, 405 (MþNa)þ, 792
(2MþNH4)þ. HRMS (FAB, m/z) calcd for C23H30N2O3
(M)þ 382.2256; observed 382.2250.
N-Boc-12a (0.163 g, 0.317 mmol) was dissolved in 1,4-
dioxane (10 mL) then treated with hydrogen chloride in
ether (1 M, 5.0 mL) for 45 h at ambient temperature. The
reaction mixture was concentrated to dryness on a rotary
evaporator to give 12a hydrochloride (0.140 g). Analytical
RP HPLC [acetonitrile/0.05% aq trifluoroacetic acid 40:60,
2.0 mL/min at 254 nm] 3.21 min, 98%. 1H NMR (CD3OD)
d 8.19–8.14 (m, 2H), 7.65–7.58 (m, 2H), 3.81 (t, 2H,
J¼7.1 Hz), 2.89 (t, 2H), J¼7.7 Hz), 2.19 (brs, 1H), 2.06
(brs, 1H), 1.96–1.50 (m, 18H), 1.44–1.35 (m, 4H). ESI-MS
(m/z) 414 (MþH)þ; HRMS (FAB, m/z) calcd for
C24H36N3O3 (MþH)þ 414.2757; observed 414.2751.
2-O-[[6-(tert-Butoxycarbonylamino)hexyl]oxy]carbazole
(1.21 g, 3.17 mmol) in 1,4-dioxane (10 mL) was treated
with hydrogen chloride in 1,4-dioxane (4.0 M, 10 mL) for
1 h at ambient temperature. The reaction mixture was
concentrated to dryness on a rotary evaporator to give of the
12b hydrochloride (1.02 g) Analytical RP HPLC [aceto-
nitrile/0.05% aq trifluoroacetic acid 30:70, 2.0 mL/min at
1
225 nm] 11.98 min, 99%. H NMR (CD3OD) d 7.93–7.88
(m, 1H), 7.86 (d, 1H, J¼8.8 Hz), 7.42–7.38 (m, 1H), 7.31–
7.25 (m, 1H), 7.14–7.08 (m, 1H), 6.97 (d, 1H, J¼2.2 Hz),
6.75 (dd, 1H, J¼8.5, 2.2 Hz), 4.04 (t, 2H, J¼6.3 Hz), 2.93
(t, 2H, J¼7.7 Hz), 1.86–1.77 (m, 2H), 1.75–1.65 (m, 2H),
1.60–1.42 (m, 4H). 13C NMR (CD3OD). d 159.4, 142.6,
141.3, 125.3, 124.3, 121.6, 120.0, 119.8, 118.2, 111.4,
109.2, 96.4, 69.3, 40.6, 30.0, 28.3, 27.1, 26.6. ESI-MS (m/z)
283 (MþH)þ; HRMS (FAB, m/z) calcd for C18H23N2O
(MþH)þ 283.1810; observed 283.1804.
3.4.15.
p-Toluenesulfonyl
6-(9H-Carbazol-2-yloxy)hexylamine
(12b).
chloride (3.75 g, 19.77 mmol,
110 mol%) was added to a solution of 6-(tert-butoxycar-
bonylamino)hexanol (3.90 g, 17.97 mmol) in dichloro-
methane (20 mL) and pyridine (4.30 mL, 53.91 mmol,
300 mol%). The mixture was stirred for 20 h at ambient
temperature. The reaction mixture was diluted with
dichloromethane (50 mL), washed with water (100 mL),
0.5N aq HCl (75 mL), saturated aq sodium bicarbonate
(75 mL), dried over anhydrous sodium sulfate and concen-
trated on a rotary evaporator. The crude product was
purified by silica gel column chromatography (30% ethyl
acetate in hexane) to obtain 6-(p-toluenesulfonyloxy)-N-
(tert-butoxycarbonyl)hexylamine (4.8 g, 72%). Analytical
RP HPLC [acetonitrile/0.05% aq trifluoroacetic acid 50:50,
2.0 mL/min at 225 nm] 6.71 min, 99%. 1H NMR (CDCl3) d
7.82–7.75 (m, 2H), 7.37–7.33 (m, 2H), 4.51 (br s, 1H), 4.01
(t, 2H, J¼6.5 Hz), 3.06 (q, 2H, J¼6.6 Hz), 2.45 (s, 3H),
1.68–1.60 (m, 2H), 1.48–1.38 (m, 2H), 1.43 (s, 9H), 1.36–
1.22 (m, 4H). 13C NMR (CDCl3) d 155.9, 144.6, 133.1,
129.8, 127.8, 70.4, 40.3, 29.8, 28.7, 28.4, 26.0, 25.0, 21.6.
ESI-MS (m/z) 372 (MþH)þ, 760 (2MþNH4)þ.
3.4.16. N-(6-Aminohexyl)-5-(dimethylamino)naphthal-
ene-1-sulfonamide (12c). tert-Butyl 6-aminohexylcarba-
mate (2.0 g, 9.26 mmol) and sodium carbonate (3.92 g,
37.03 mmol, 400 mol%) were dissolved in a mixture of
THF/water (4:3, 30 mL). Dansyl chloride (2.49 g,
9.26 mmol, 100 mol%) in THF (17 mL) was added. After
stirring the mixture for 30 min at ambient temperature, 25%
aq sodium chloride (50 mL) was added and the mixture was
extracted with ethyl acetate (50 mL). The organic layer was
washed with 25% aq sodium chloride (50 mL), dried over
anhydrous sodium sulfate and concentrated on a rotary
evaporator. The crude product was purified by silica gel
column chromatography (30–40% ethyl acetate in
hexane) to afford N-Boc-12c (3.9 g, 94%, viscous oil).
Analytical RP HPLC [acetonitrile/0.05% aq trifluoroacetic
acid 50:50, 2.0 mL/min at 254 nm] 10.38 min, 99%. 1H
NMR (CDCl3) d 8.53 (d, 1H, J¼8.5 Hz), 8.31 (d, 1H,
J¼8.5 Hz), 8.26–8.23 (m, 1H), 7.58–7.49 (m, 2H), 7.18 (d,
1H, J¼7.8 Hz), 4.99 (t, 1H, J¼6.0 Hz), 4.52 (br s, 1H),
3.01–2.92 (m, 2H), 2.91–2.84 (m, 8H), 1.43 (s, 9H), 1.37–
1.29 (m, 2H), 1.28–1.21 (m, 2H), 1.17–1.04 (m, 4H). 13C
NMR (CDCl3) d 156.1, 151.9, 134.8, 130.2, 129.8, 129.6,
129.5, 128.3, 123.1, 118.7, 115.1, 79.2, 45.3, 42.9, 40.1,
29.7, 29.3, 28.3, 25.8, 25.8. ESI-MS (m/z) 450 (MþH)þ.
A mixture of 2-hydroxycarbazole (2.20 g, 12.02 mmol),
6-(p-toluenesulfonyloxy)-N-(tert-butoxycarbonyl)hexyl-
amine (4.80 g, 12.93 mmol, 107 mol%), sodium iodide
(1.80 g, 12.02 mmol, 100 mol%) and anhydrous potassium
carbonate (2.50 g, 18.03 mmol, 150 mol%) in 2-butanone
(50 mL) was heated at reflux for 30 h. The reaction mixture

M. Adamczyk et al. / Tetrahedron 59 (2003) 5749–5761
5759
HRMS (FAB, m/z) calcd for C23H35N3O4S (M)þ 448.2270;
observed 448.2240.
42.3, 40.0, 39.6, 39.0, 38.8, 36.4, 35.9, 34.4, 30.2, 29.9,
27.1, 27.0. ESI-MS (m/z) 1013 (MþNH4)þ. HRMS (FAB,
m/z) calcd for C57H65N5O11Na (MþNa)þ 1018.4571;
observed 1018.4573.
N-Boc-12c (0.095 g, 0.2117 mmol) was dissolved in
methanol (4.0 mL) then treated with 6N aq HCl (5 mL)
for 30 min at 408C. The mixture was concentrated to
dryness to give of the 12c hydrochloride (0.085 g).
Analytical RP HPLC [acetonitrile/water 20:80, 2.0 mL/
3.5.2. (2E)-3-[3-(5-{[Bis(4-methoxyphenyl)(phenyl)-
methoxy]methyl}-4-hydroxytetrahydrofuran-2-yl)-2,6-
dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]-N-[6-({[5-
(dimethylamino)-1-naphthyl]sulfonyl}amino)hexyl]-
acrylamide (13b). (25%). Mp 113–48C. Analytical RP
HPLC [acetonitrile/water 60:40, 2.0 mL/min at 225 nm]
1
min at 254 nm] 4.99 min, 98%. H NMR (CD3OD) d 8.96
(d, 1H, J¼8.8 Hz), 8.64 (d, 1H, J¼8.8 Hz), 8.40–8.36 (m,
1H), 8.17 (d, 1H, J¼7.7 Hz), 7.93 (d, 1H, J¼8.5 Hz), 7.87
(d, 1H, J¼8.5 Hz), 3.52 (s, 6H), 2.90–2.82 (m, 4H), 1.60–
1.51 (m, 2H), 1.48–1.38 (m, 2H), 1.32–1.23 (m, 4H). 13C
NMR (CD3OD) d 140.2, 131.1, 131.1, 130.7, 128.9, 128.6,
128.1, 127.1, 126.6, 120.7, 43.7, 40.6, 30.5, 28.4, 26.9, 26.8.
ESI-MS (m/z) 350 (MþH)þ, 699 (2MþH)þ. HRMS (FAB,
m/z) calcd for C18H28N3O2S (MþH)þ 350.1902; observed
350.1913.
1
13.68 min, 96%. [a]2D3¼þ3.63 (c 0.55, MeOH). H NMR
(CDCl3þfew drops of CD3OD) d 8.92 (br s, 1H), 7.95–7.88
(m, 2H), 7.44–7.13 (m, 13H), 6.89–6.75 (m, 6H), 6.25 (d,
1H, J¼15.1 Hz), 5.99 (dd, 1H, J¼8.5, 5.2 Hz), 5.62 (s, 1H),
4.49–4.42 (m, 1H), 4.03 (t, 2H, J¼6.3 Hz), 3.91–3.84 (m,
1H), 3.74 (s, 6H), 3.48–3.42 (m, 1H), 3.38–3.24 (m, 3H),
2.82–2.70 (m, 1H), 2.16–2.06 (m, 1H), 1.87–1.78 (m, 2H),
1.62–1.36 (m, 6H). 13C NMR (CDCl3þ0.1 mL of CD3OD)
d 163.7, 163.0, 158.3, 152.2, 149.7, 144.7, 140.9, 140.8,
139.6, 139.5, 136.0, 135.9, 131.4, 130.0, 128.2, 127.6,
126.6, 124.3, 123.2, 120.8, 119.2, 119.1, 117.0, 112.9,
110.3, 108.3, 101.7, 95.4, 86.3, 85.9, 85.1, 71.7, 68.0, 64.4,
55.1, 39.5, 37.7, 29.6, 28.9, 26.2, 25.5. ESI-MS (m/z) 882
(MþNH4)þ, 887 (MþNa)þ; negative ion mode, 863
(M2H)2, 899 (M2Cl)2. HRMS (FAB, m/z) calcd for
C51H52N4O9Na (MþNa)þ 887.3626; observed, 887.3628.
3.5. Preparation of 6-haptenated-[(2E)-N-(alkyl)prop-2-
enamidyl]-20-deoxyuridines 13a–c
A solution of LiOH monohydrate (0.34 g, 8.06 mmol,
330 mol%) in water (75 mL) was added to a solution of
ester (þ)-10 (1.5 g, 2.44 mmol) in THF (90 mL) and the
mixture was stirred for 2 h at ambient temperature. Reaction
was concentrated to dryness and the resulting crude lithium
salt (11) was dissolved in DMF (40 mL) and a solution of
HCl salt of amine 12a–c (120 mol%) and triethylamine
(2.37 mL, 17.08 mmol, 700 mol%) in DMF (40 mL) was
added followed by N-hydroxybenzotriazole (HOBt, 0.49 g,
3.7 mmol, 150 mol%) and 1-ethyl-3-(3-dimethylamino-
propyl)carbodiimide hydrochloride (EDAC, 0.71 g,
0.37 mmol, 150 mol%). The reaction mixture was stirred
for 18–40 h at ambient temperature. Solid sodium
bicarbonate (2.0 g) was added to the reaction and DMF
was removed under reduced pressure. The residue was
partitioned between ethyl acetate (300 mL) and 10%
aqueous sodium bicarbonate solution (250 mL). The
organic layer was dried over anhydrous sodium sulfate,
concentrated on a rotary evaporator and purified by silica
gel column chromatography (ethyl acetate/methanol/
triethylamine 93:5:2) to afford conjugate conjugates 13a–c.
3.5.3. (2E)-3-[3-(5-{[Bis(4-methoxyphenyl)(phenyl)-
methoxy]methyl}-4-hydroxytetrahydrofuran-2-yl)-2,6-
dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]-N-[6-(9H-car-
bazol-2-yloxy)hexyl]acrylamide (13c). (53%, as a pale
yellow solid). Mp 127–88C. Analytical RP HPLC [aceto-
nitrile/0.05% aq trifluoroacetic acid 60:40, 2.0 mL/min,
1
254 nm] 8.88 min, 98%. [a]2D3¼21.1 (c 0.64, MeOH). H
NMR (CD3CN) d 8.51 (d, 1H, J¼8.5 Hz), 8.27 (d, 1H,
J¼8.5 Hz), 8.16 (dd, 1H, J¼7.1, 1.1 Hz), 7.60–7.52 (m,
2H), 7.43–7.17 (m, 11H), 6.82–6.77 (m, 4H), 6.46 (t, 1H,
J¼5.5 Hz), 6.41 (d, 1H, J¼15.4 Hz), 6.11 (dd, 1H, J¼8.5,
4.9 Hz), 5.88 (br s, 1H), 5.70 (s, 1H), 4.32–4.23 (m, 1H),
3.86–3.78 (m, 1H), 3.72 (s, 3H), 3.71 (s, 3H), 3.39–3.32
(m, 1H), 3.21–3.03 (m, 3H), 2.82 (br s, 7H), 2.73–2.56 (m,
2H), 2.13–2.03 (m, 1H), 1.30–1.19 (m, 4H), 1.12–1.02 (m,
4H). 13C NMR (CD3CN) d 164.3, 163.2, 159.5, 153.0,
151.2, 146.2, 137.2, 137.1, 132.0, 131.2, 131.0, 130.9,
130.6, 130.4, 130.0, 129.3, 129.0, 128.7, 127.7, 124.4,
120.0, 116.2, 102.9, 86.8, 86.5, 86.2, 71.9, 65.5, 55.8, 45.7,
43.6, 39.9, 38.9, 29.9, 29.7, 26.7, 26.5. ESI-MS (m/z) 932
(MþH)þ. HRMS (FAB, m/z) calcd for C51H57N5O10SNa
(MþNa)þ 954.3717; observed 954.3718.
3.5.1. (2E)-3-[3-(5-{[Bis(4-methoxyphenyl)(phenyl)-
methoxy]methyl}-4-hydroxytetrahydrofuran-2-yl)-2,6-
dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]-N-[6-({[3-(4-
nitrophenyl)-1-adamantyl]acetyl}amino)hexyl]acryl-
amide (13a). (53%). Mp 109–118C. Analytical RP HPLC
[acetonitrile/water 60:40, 2.0 mL/min at 254 nm]
1
13.44 min, 96%. [a]2D3¼þ1.5 (c 0.975, MeOH). H NMR
(CD3CNþ0.1 mL of CD3OD) d 8.14–8.09 (m, 2H), 7.57–
7.52 (m, 2H), 7.44–7.16 (m, 10H), 6.86–6.78 (m, 4H),
6.73–6.68 (m, 1H), 6.43 (d, 1H, J¼15.1 Hz), 6.12–6.08 (m,
1H), 5.70 (d, 1H, J¼0.6 Hz), 4.30–4.23 (m, 1H), 3.86–3.78
(m, 1H), 3.73 (s, 3H), 3.72 (s, 3H), 3.39–3.32 (m, 1H),
3.20–3.04 (m, 6H), 2.81–2.70 (m, 2H), 2.68–2.56 (m, 1H),
2.16–2.02 (m, 3H), 1.86–1.80 (m, 4H), 1.75–1.72 (m, 2H),
1.69–1.60 (m, 6H), 1.48–1.37 (m, 4H), 1.33–1.25 (m, 4H).
13C NMR (CD3CNþ0.1 mL of CD3OD) d 172.1, 172.0,
164.8, 163.7, 159.6, 153.2, 151.4, 146.4, 137.3, 137.2,
132.2, 131.2, 131.1, 129.2, 128.8, 127.8, 127.2, 124.3,
114.0, 102.9, 87.0, 86.6, 86.4, 71.9, 65.7, 55.9, 51.4, 42.7,
3.6. Preparation of 6-haptenated-50-O-dimethoxytrityl-
20-deoxyuridine phosphoramidites 14a–c
Diisopropylethylamine (0.73 mL, 4.22 mmol, 400 mol%)
and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite
(0.35 mL, 1.58 mmol, 150 mol%) were sequentially added
to a solution of conjugate 13a–c (1.055 mmol) in
dichloromethane (15 mL) at room temperature under
nitrogen. After stirring for 1 h, dry methanol (0.055 mL,
1.37 mmol, 130 mol%) was added and stirring was
continued for an additional 10 min. Triethylamine
(3.7 mL, 26.37 mmol, 2500 mol%) and ethyl acetate

5760
M. Adamczyk et al. / Tetrahedron 59 (2003) 5749–5761
(100 mL) were then added. The solution was washed with
10% aq sodium carbonate (80 mL), brine (50 mL), dried
over anhydrous sodium sulfate and concentrated on a rotary
evaporator. The crude product was purified by silica gel
column chromatography (ethyl acetate/methanol/triethyl-
amine 96:2:2) to afford 0.86 g of material, which was further
purified by preparative RP HPLC (acetonitrile/water v:v
shown, 45 mL/min, 225 nm). The fractions were concen-
trated on a rotary evaporator to afford 14a–c.
diisopropylamidophosphite (14c). Preparative RP HPLC
(72:28). Yield (37%). Analytical RP HPLC [acetonitrile/
water 72:28, 2.0 mL/min, 225 nm] 13.98 and 17.76 min,
1
96%. H NMR (CD3CN) d 8.97 (br s, 1H), 8.51 (d, 1H,
J¼8.5 Hz), 8.27 (d, 1H, J¼8.5 Hz), 8.18–8.15 (m, 1H),
7.62–7.56 (m, 2H), 7.45–7.40 (m, 2H), 7.34–7.16 (m, 9H),
6.84–6.77 (m, 4H), 6.59 (br q, 1H, J¼5.5 Hz), 6.44–6.39
(m, 1H), 6.16–6.07 (m, 1H), 5.81 (t, 1H, J¼6.0 Hz),
5.70–5.69 (m, 1H), 4.55–4.36 (m, 1H), 4.01–3.93 (m, 1H),
3.75–3.51 (m, 6H), 3.52–3.60 (m, 2H), 3.56–3.24 (m, 4H),
3.31–3.03 (m, 2H), 2.85–2.77 (m, 8H), 2.74–2.64 (m, 1H),
2.57 (t, 1H, J¼6.0 Hz), 2.45 (t, 1H, J¼6.0 Hz), 2.31–2.16
(m, 1H), 1.30–1.20 (m, 4H), 1.12–1.03 (m, 13H), 0.92 (d,
3H, J¼6.6 Hz). 31P NMR (CD3CN) d 148.64, 148.55. ESI-
MS (m/z) 1132 (MþH)þ; negative ion mode, 1130
(M2H)2. HRMS (FAB, m/z) calcd for C60H74N7O11PSNa
(MþNa)þ 1154.4797; observed 1154.4806. Anal. calcd for
C60H74N7O11PS: C, 63.64; H, 6.59; N, 8.66; P, 2.74; S, 2.83.
Found: C, 63.56; H, 6.84; N, 8.56.
3.6.1. 2-{[Bis(4-methoxyphenyl)(phenyl)methoxy]-
methyl}-5-[6-((1E)-3-{[6-({[3-(4-nitrophenyl)-1-adaman-
tyl]acetyl}amino)hexyl]amino}-3-oxoprop-1-enyl)-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl]tetrahydrofuran-
3-yl 2-cyanoethyl diisopropylamidophosphite (14a). Pre-
parative RP-HPLC (76:24). Yield (54%). Analytical RP
HPLC [acetonitrile/water 76:24, 2.0 mL/min, 225 nm]
1
11.59 and 14.60 min, 96%. H NMR (CD3CN) d 9.15 (br
s, 1H), 8.14–8.10 (m, 2H), 7.56–7.52 (m, 2H), 7.45–7.40
(m, 2H), 7.34–7.15 (m, 8H), 6.88–6.78 (m, 5H), 6.50–6.45
(m, 1H), 6.40 (br t, 1H), 6.14–6.06 (m, 1H), 5.69 (d, 1H,
J¼1.4 Hz), 4.56–4.36 (m, 1H), 4.08–3.96 (m, 1H), 3.75–
3.62 (m, 8H), 3.56–3.28 (m, 5H), 3.23–3.08 (m, 5H), 2.75–
2.65 (m, 1H), 2.58 (t, 1H, J¼6.0 Hz), 2.45 (t, 1H),
J¼6.0 Hz), 2.31–2.18 (m, 1H), 2.17–2.11 (m, 2H), 1.89–
1.81 (m, 4H), 1.75 (br s, 1H), 1.69–1.62 (m, 6H), 1.48–1.38
(m, 4H), 1.36–1.27 (m, 4H), 1.14–1.05 (m, 9H), 0.92 (d,
3H, J¼6.8 Hz). 31P NMR (CD3CN) d 148.63, 148.55. ESI-
MS (m/z) 1197 (MþH)þ, 1218 (MþNa)þ; negative ion
mode, 1195 (M2H)2. HRMS (FAB, m/z) calcd for
References
1. Graham, J. Medical Devicelink, 2002. http://www.devicelink.
com/ivdt/archive/02/05/006.html.
2. Venter, J. C.; Adams, M. D.; Myers, E. W.; Li, P. W.; Mural,
R. J.; et al. Science 2001, 291, 1304–1351.
3. McPherson, J. D.; Marra, M.; Hillier, L.; Waterston, R. H.;
Chinwalla, A.; et al. Nature 2001, 409, 934–941.
4. Agrawal, S.; Iyer, R. P. Curr. Opin. Biotechnol. 1995, 6,
12–19.
C66H82N7O12PNa
(MþNa)þ
1218.5651;
observed
1218.5652. Anal. calcd for C66H82N7O12P: C, 66.26; H,
6.91; N, 8.20; P, 2.59. Found: C, 66.41; H, 6.99; N, 8.17; P,
2.46.
5. Agrawal, S. Protocols for Oligonucleotide Conjugates.
Synthesis and Analytical Techniques. In Methods in
Molecular Biology; Walker, J. M., Ed.; Humana: Totowa,
NJ, 1994; Vol. 26.
3.6.2. 2-{[Bis(4-methoxyphenyl)(phenyl)methoxy]-
methyl}-5-[6-((1E)-3-{[6-({[5-(dimethylamino)-1-
naphthyl]sulfonyl}amino)hexyl]amino}-3-oxoprop-1-
enyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl]tetra-
hydrofuran-3-yl 2-cyanoethyl diisopropylamido-
phosphite (14b). Preparative RP HPLC (77:23). Yield
(42%). Analytical RP HPLC [acetonitrile/water 75:25,
6. Goodchild, J. Bioconjugate Chem. 1990, 1, 165–187.
7. Kricka, L. J. Clin. Chem. 1999, 45, 453–458.
8. Zhang, P.; Beck, T.; Tan, W. Angew. Chem. Int. Ed. 2001, 40,
402–405.
9. Fang, X. H.; Li, J. W. J.; Perlette, J.; Tan, W. Hj; Wang, K. M.
Anal. Chem. 2000, 72, 747 A–753 A.
1
2.0 mL/min, 225 nm] 14.47 and 18.14 min, 98%. H NMR
10. Li, J. J; Fang, X.; Schuster, S. M.; Tan, W. Angew. Chem. Int.
Ed. 2000, 39, 1049–1052.
(CD3CN) d 9.28 (br s, 1H), 7.93 (d, 1H, J¼7.7 Hz), 7.89 (d,
1H, J¼8.5 Hz), 7.42–7.11 (m, 13H), 6.96 (d, 1H,
J¼2.2 Hz), 6.84–6.72 (m, 6H), 6.45–6.39 (m, 1H), 6.17–
6.07 (m, 1H), 5.70 (d, 1H, J¼2.5 Hz), 4.55–4.34 (m, 1H),
4.03 (t, 2H, J¼6.5 Hz), 4.0–3.92 (m, 1H), 3.74–3.62 (m,
7H), 3.55–3.31 (m, 5H), 3.27–3.18 (m, 3H), 2.73–2.63 (m,
1H), 2.57 (t, 1H, J¼6.3 Hz), 2.44 (t, 1H, J¼6.0 Hz), 2.30–
2.22 (m, 1H), 1.83–1.74 (m, 2H), 1.56–1.35 (m, 6H), 1.12–
1.03 (m, 9H), 0.92 (d, 3H, J¼6.6 Hz). 31P NMR (CD3CN) d
148.66, 148.59. ESI-MS (m/z) 1065 (MþH)þ; negative ion
mode, 1063 (M2H)2. HRMS (FAB, m/z) calcd for
11. Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303–308.
12. Holland, P. M.; Abramson, R. D.; Watson, R.; Gelfand, D. H.
Proc. Natl. Acad. Sci. USA 1991, 88, 7276–7280.
13. Nelson, N. C.; Cheikh, A. B.; Matsuda, E.; Becker, M. M.
Biochemistry 1996, 35, 8429–8438.
14. Guesdon, J. L. J. Immunol. Methods 1992, 150, 33–49.
15. Marshall, R. L.; Laffler, T. G.; Cerney, M. B.; Sustachek, J. C.;
Kratochvil, J. D.; Morgan, R. L. PCR Methods Appl. 1994, 4,
80–84.
16. Laffler, T. G.; Carrino, J. J.; Marshall, R. L. Ann. Biol. Clin.
(Paris) 1993, 51, 821–826.
C60H69N6O10PNa
(MþNa)þ
1087.4705;
observed
1087.4791. Anal. calcd for C60H69N6O10P: C, 67.65; H,
6.53; N, 7.89; P, 2.91. Found: C, 67.48; H, 6.49; N, 7.60; P,
2.80.
17. Kratochvil, J.; Laffler, T. G. Methods Mol. Biol. 1994, 28,
243–252.
18. Ruth, J. L. Oligodeoxynucleotides with Reporter Groups
Attached to the Base. In Oligonucleotides and Analogues. A
Practical Approach; Eckstein, F., Ed.; IRL: New York, 1991.
19. Ruth, J. L.; Morgan, C.; Pasko, A. DNA 1985, 4, 93.
20. Cook, F. A.; Vuocolo, E.; Brankel, C. L. Nucleic Acids Res.
1988, 16, 4077–4095.
3.6.3. 2-{[Bis(4-methoxyphenyl)(phenyl)methoxy]-
methyl}-5-[6-((1E)-3-{[6-(9H-carbazol-2-yloxy)hexyl]-
amino}-3-oxoprop-1-enyl)-2,4-dioxo-3,4-dihydropyrimi-
din-1(2H)-yl]tetrahydrofuran-3-yl 2-cyanoethyl

M. Adamczyk et al. / Tetrahedron 59 (2003) 5749–5761
5761
21. Telser, J.; Cruickshank, K. A.; Morrison, L. E.; Netzel, T. L.
J. Am. Chem. Soc. 1989, 111, 6966–6976.
Mathis, G. Nucleosides Nucleotides Nucleic Acids 2000, 19,
1463–1474.
22. Bashkin, J. K.; McBeath, R. J.; Modak, A. S.; Sample, K. R.;
Wise, W. B. J. Org. Chem. 1991, 56, 3168–3176.
23. Michel, J.; Moreau, S. Nucleosides Nucleotides 1999, 18,
1633–1637.
38. Sauer, M.; Angerer, B.; Han, K. T.; Zander, C. Phys. Chem.
Chem. Phys. 1999, 1, 2471–2477.
39. Rodriguez-Tanty, C.; Perez, R.; Miranda, J.; Velez-Castro, H.;
Rosado, A.; et al. Nucleosides Nucleotides Nucleic Acids
2001, 20, 1449–1461.
24. Ozaki, H.; Mine, M.; Ogawa, Y.; Sawai, H. Bioorg. Chem.
2001, 29, 187–197.
40. Saintome, C.; Clivio, P.; Fourrey, J.-L.; Woisard, A.; Laugaa,
P.; Favre, A. Tetrahedron 2000, 56, 1197–1206.
41. Giordano, C.; Fratini, F.; Attanasio, D.; Cellai, L. Synthesis
2001, 565–572.
25. Ozaki, H.; Kakamura, A.; Arai, M.; Endo, M.; Sawai, H. Bull.
Chem. Soc. Jpn 1995, 68, 1981–1987.
26. Osborne, S. E.; Cain, R. J.; Glick, G. D. J. Am. Chem. Soc.
1997, 119, 1171–1182.
42. Savy, P.; Benhida, R.; Fourrey, J.-L.; Maurisse, R.; Sun, J.-S.
Bioorg. Med. Chem. Lett. 2002, 12, 981–983.
43. Bashkin, J. K.; Xie, J.; Daniher, A. T.; Sampath, U.; Kao,
J. L.-F. J. Org. Chem. 1996, 61, 2314–2321.
27. Sugiyama, H.; Matsuda, S.; Kino, K.; Qiu-Mei, Z.; Yonei, S.;
Saito, I. Tetrahedron Lett. 1996, 37, 9067–9070.
28. Shinozuka, K.; Umeda, A.; Aoki, T.; Sawai, H. Nucleosides
Nucleotides 1998, 17, 291–300.
44. Adamczyk, M.; Chan, C. M.; Fino, J. R.; Mattingly, P. G.
J. Org. Chem. 2000, 65, 596–601.
29. Kohgo, S.; Shinozuka, K.; Ozaki, H.; Sawai, H. Tetrahedron
Lett. 1998, 39, 4067–4070.
45. Adamczyk, M.; Johnson, D. D.; Mattingly, P. G.; Reddy, R. E.
Org. Prep. Proc. Int. 2001, 33, 505–514.
46. Mattingly, P. G. US Patent 5424414.
30. Urdea, M. S.; Warner, B. D.; Running, J. A.; Stempien, M.;
Clyne, J.; Horn, T. Nucleic Acids Res. 1988, 16, 4937–4956.
31. Sproat, B. S.; Lamond, A. I.; Beijer, B.; Neuner, P.; Ryder, U.
Nucleic Acids Res. 1989, 17, 3373–3386.
47. Fino, J. R. US Patent 5464746.
48. Fino, J. R.; Mattingly, P. G.; Ray, K. Bioconjugate Chem.
1996, 7, 274–280.
32. Tang, J. Y.; Agrawal, S. Nucleic Acids Res. 1990, 18, 6461.
33. Viscidi, R. P.; Connelly, C. J.; Yolken, R. H. J. Clin.
Microbiol. 1986, 23, 311–317.
49. Adamczyk, M.; Akireddy, S. R.; Mattingly, P. G.; Reddy, R. E.
Tetrahedron Lett. 2001, 42, 5621–5623.
34. Adarichev, V. A.; Kalachikov, S. M.; Kiseliova, A. V.;
Dymshits, G. M. Bioconjugate Chem. 1998, 9, 671–675.
35. Muhlegger, K.; Batz, H.-G.; Bohm, S. Nucleosides
Nucleotides 1989, 8, 1161–1163.
50. Sanghvi, Y. S.; Hoke, G. D.; Freier, S. M.; Zounes, M. C.;
Gonzalez, C.; Cummins, L.; Sasmor, H.; Cook, P. D. Nucleic
Acids Res. 1993, 21, 3197–3203.
51. Tanaka, H.; Hayakawa, H.; Iijima, S.; Haraguchi, K.;
Miyasaka, T. Tetrahedron 1985, 41, 861–866.
52. Shimizu, M.; Tanaka, H.; Hayakawa, H.; Miyasaka, T.
Tetrahedron Lett. 1990, 31, 1295–1298.
36. Iverson, B. L.; Dervan, P. B. J. Am. Chem. Soc. 1987, 109,
1241–1243.
37. Alpha-Bazin, B.; Bazin, H.; Guillemer, S.; Sauvaigo, S.;