Hapten-phosphoramidites based on 6-[(2E)-N-(hexyl)prop-2-enamidyl]-2′-deoxyuridine

Article abstract of DOI:10.1016/S0040-4039(01)01091-7

Novel hapten-phosphoramidites 11a-c were prepared from 2′-deoxyuridine (2) by functionalization at the 6-position and subsequent conjugation with adamantane, carbazole and dansyl reporter groups in good overall yield.

Full text of DOI:10.1016/S0040-4039(01)01091-7

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 5621–5623
Hapten-phosphoramidites based on
6-[(2E)-N-(hexyl)prop-2-enamidyl]-2%-deoxyuridine
Maciej Adamczyk,* Srinivasa Rao Akireddy, Phillip G. Mattingly and Rajarathnam E. Reddy
Dept. 9NM, Bldg AP20, Diagnostics Division, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064-6016, USA
Received 30 April 2001; accepted 8 June 2001
Abstract—Novel hapten-phosphoramidites 11a–c were prepared from 2%-deoxyuridine (2) by functionalization at the 6-position
and subsequent conjugation with adamantane, carbazole and dansyl reporter groups in good overall yield. © 2001 Elsevier
Science Ltd. All rights reserved.
The inclusion of reporter groups in synthetic oligonu-
O
cleotides is essential for the detection, quantification
H
and identification of nucleic acid target sequences as a
part of medical diagnoses or characterization of bio-
technological processes.1–3 Such reporter molecules are
a diverse group that include fluorescent4 and chemilu-
minescent species5 that can be directly measured, and
other low molecular weight compounds that facilitate
indirect detection. The latter category includes species
with high affinity receptors (biotin/avidin)6–9 and an
assortment of haptens (molecules that can be measured
by immunoassays).10 The reporter groups are con-
veniently introduced by solid-phase phosphoramidite
chemistry using both nucleoside6,9,11–14 and non-
nucleoside scaffolds.2,15 The nucleoside scaffold
approach first described by Ruth16,17 utilized an amino-
containing linker at the 5-position of deoxyuridine to
which the reporter group is attached. However, func-
tionalization of deoxyuridine at the 6-position for this
purpose has not been exploited. An explanation may lie
in the report that 6-methyl-2%-deoxyuridine lowers the
melt temperature (Tm) of some antisense oligonucle-
otides by varying degrees depending on the overall
sequence, placement within the sequence and frequency
of substitution.18,19 The authors believed that 6-Me-dU
adopted a syn conformation which was unfavorable for
Watson–Crick hydrogen bonding. In light of our suc-
cess incorporating reporter groups into oligonucleotide
probes using non-nucleoside scaffolds4,10 which also lack
Watson–Crick hydrogen bonding, and using them in
hybridization assays, we proceeded to prepare a series
of hapten-phosphoramidites (11a–c) with a common
scaffold, 6-[(2E)-N-(hexyl)prop-2-enamidyl)]-2%-deoxy-
uridine (1).
N
O
NH(CH2)6
O
N
O
HO
OH
1
Thus, 3%- and 5%-hydroxyl groups of 2%-deoxyuridine (2)
(Scheme 1) were protected with 1,1,3,3-tetraisopropyl
disioxane-1,3-diyl (TIPDS)20,21 group to give 3, which
was then deprotonated at the 6-position using lithium
diisopropylamide and reacted with DMF in the pres-
ence of HMPA.13 The crude reaction mixture was
treated with acetic acid at −78°C to give the aldehyde
(4) in 52% yield after purification by silica-gel column
chromatography (unreacted starting material 3, could
be recovered and recycled). The aldehyde 4 was then
subjected to a Wittig reaction with methyl (tri-
phenylphosphoranylidene)acetate in benzene to afford
unsaturated ester 5 in almost quantitative yield as a
E-isomer (>98%). The next step was to exchange the
silyl protective group at the 5%-position of 5 to a 4,4%-
dimethoxytrityl group (DMT), which is compatible for
automated oligonucleotide synthesis. Thus, TIPDS pro-
tective group in 5 was hydrolyzed by treatment with
tetra-n-butylammonium fluoride in THF and the crude
product was purified by silica-gel column chromatogra-
phy to afford diol (+)-6 in excellent yield (98%). The
5%-hydroxyl group in 6 was protected as DMT ether by
treatment with 4,4%-dimethoxytrityl chloride in the pres-
ence of silver nitrate and pyridine. The crude com-
* Corresponding author. E-mail: maciej.adamczyk@abbott.com
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01091-7

5622
M. Adamczyk et al. / Tetrahedron Letters 42 (2001) 5621–5623
O
O
N
H
N
H
O
N
O
[(i-Pr)2ClSi]2O, DMF
Ph3P=CHCO2Me
R
then LDA, THF, DMF
and AcOH
(52% for two steps)
O
N
Benzene, rt, 1 h
(96%)
O
O
HO
Si
O
O
OH
Si
3: R = H
2
4: R = CHO
O
N
O
H
O
N
O
H
N
O
OMe
O
1. TBAF, THF, rt
30 min (98%)
OMe
O
N
O
O
2. DMT-Cl, AgNO3
RO
Si
THF, Py, rt, 4 h
(71%)
O
O
Si
5
6: R = H
OH
7: R = DMT
Scheme 1.
pound was purified by silica-gel column chromatogra-
phy using EtOAc:Et3N:MeOH in 97:2:1 ratio to afford
ester 7 in 76% yield as a colorless solid (mp: 100–
101°C). The ester 7 has the required protective group
(DMT) at 5%-hydroxyl group and free 3%-hydroxy func-
tionality for introduction of 2-(cyanoethyl)-N,N-diiso-
propylphosphoramidite, which are needed for
incorporation of modified nucleotide building block
into oligonucleotide via solid-phase synthesis.
C-6 linking arm containing a terminal amino function-
ality to provide 8a–c.22 The ester 7 was then subjected
to hydrolysis (Scheme 2) with lithium hydroxide in
THF–water and the resulting crude acid was conju-
gated with amines 8a–c using HOBt and EDAC in
anhydrous DMF. The conjugates 9a–c were purified by
silica-gel column chromatography in 25–53% yield.
Finally, 9a–c were treated with (2-cyanoethyl)-N,N-
diisopropylchlorophosphoramidite (10) in THF in
the presence of 4.0 equiv. of diisopropylethylamine
and the crude product was purified by preparative
reversed-phase HPLC23 to afford the phosphoramid-
ites 11a–c in 37–52% yield24 as a mixture of diastereo-
mers.
The next step in synthesis of modified nucleotide
building blocks 11a–c was to conjugate ester 7 to the
hapten reporter groups. Accordingly, adamantane, car-
bazole and dansyl reporter groups were coupled to a
O
H
N
8a–c: H2N(CH2)6-R
NO2
NH(CH2)6-R
LiOH, THF-water
O
N
Ester 7
O
O
then 8a–c, HOBT
EDAC, DMF, rt
DMTO
18–20 h (25–53%)
9a: R = Adamantane
9b: R = Carbazole
9c: R = Dansyl
OH
8a R = NHCOCH2
adamantane
O
N
H
O
N
O
O
N
H
8b R =
8c R =
NH(CH2)6-R
O
NC(CH2)2O
carbazole
P
Cl
DMTO
NHO2S
(i-Pr)2N
10
DIPA, CH2Cl2,
O
O
P
CN
rt, 1-4 h (37-52%)
NMe2
11a: R = Adamantane
11b: R = Carbazole
11c: R = Dansyl
N
dansyl
Scheme 2.

M. Adamczyk et al. / Tetrahedron Letters 42 (2001) 5621–5623
5623
Adamantane, carbazole and dansyl haptens (8a–c) form
highly antigenic protein conjugates that elicit selective,
high affinity antibodies.25–27 The distinctive structure of
the haptens ensures minimal cross-reactivity with
potentially interfering substances when they are used in
nucleic acid testing (NAT). The requisite hapten-phos-
phoramidites (11a–c) necessary for NAT were prepared
21. Shimizu, M.; Tanaka, H.; Hayakawa, H.; Miyasaka, T.
Tetrahedron Lett. 1990, 31, 1295–1298.
22. The amine 8a was prepared from 3-(4-nitro-
phenyl)adamantaneacetic10 and 2-amino-6-(tert-butoxy-
carbonyl)aminohexane in two steps (HOBt, Et3N, EDAC
in CH2Cl2 then 6.0 M aq HCl); amine 8b was prepared
from 2-hydroxy-carbazole and 6-(p-toluenesulfonyloxy)-
N-(tert-butoxycarbonyl)aminohexane in two steps (anhy-
drous K2CO3, NaI, methyl ethyl ketone then 4.0 M aq
HCl); amine 8c was prepared from dansyl chloride and
2-amino-6-(tert-butoxycarbonyl)-aminohexane in two
steps (Na2CO3 THF–water then 6.0 M aq HCl).
on
a
6-[(2E)-N-(6-hexyl)prop-2-enamidyl)]-2%-deoxy-
uridine scaffold in good overall yield from 2%-deoxy-
uridine.
23. Preparative reversed-phase (RP) HPLC was carried out
using Waters column (Symmetry, C18, 7.0 mm, 10×100
mm; MeCN:water/75:25, 45.0 mL/min at 225 nm). Ana-
lytical reversed phase (RP) HPLC was carried out using
Waters column (Symmetry, C18, 7.0 mm, 10×100 mm).
24. Selected data for DNA Probes 11a–c: Probe 11a: Analyt-
ical RP HPLC: MeCN:water/76:24, 2.0 mL/min at 225
References
1. Agrawal, S.; Iyer, R. P. Curr. Opin. Biotechnol. 1995, 6,
9–12.
2. Goodchild, J. Bioconjugate Chem. 1990, 1, 165–187.
3. Kricka, L. J. Clin. Chem. 1999, 45, 453–458.
4. Adamczyk, M.; Chan, C. M.; Fino, J. R.; Mattingly, P.
G. J. Org. Chem 2000, 65, 596–601.
1
nm, tR: 11.59 and 14.60 min, 96%; H NMR (CD3CN):
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, 8 H), 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, J=1.4 Hz), 4.56–4.36 (m, 1H), 4.08–3.96 (m,
1H), 3.75–3.62 (m, 8 H), 3.56–3.28 (m, 5H), 3.23–3.08 (m,
5H), 2.75–2.65 (m, 1H), 2.58 (t, 1H, J=6.0 Hz), 2.45 (t,
1H), J=6.0 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, J=6.8 Hz); 31P NMR (CD3CN): 148.63,
148.55; ESI–MS (m/z): 1197 (M+H)+, 1218 (M+Na)+.
Probe 11b: Analytical RP HPLC: MeCN:water/75:25, 2.0
mL/min at 225 nm, tR: 14.47 and 18.14 min, 98%; 1H
NMR (CD3CN): 9.28 (br s, 1H), 7.93 (d, 1H, J=7.7 Hz),
7.89 (d, 1H, J=8.5 Hz), 7.42–7.11 (m, 13H), 6.96 (d, 1H,
J=2.2 Hz), 6.84–6.72 (m, 6H), 6.45–6.39 (m, 1H), 6.17–
6.07 (m, 1H), 5.70 (d, 1H, J=2.5 Hz), 4.55–4.34 (m, 1H),
4.03 (t, 2H, J=6.5 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, J=6.3 Hz), 2.44 (t, 1H, J=6.0 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, J=6.6 Hz); 31P NMR
(CD3CN): 148.66, 148.59; ESI–MS (m/z): 1065 (M+H)+.
Probe 11c: Analytical RP HPLC: MeCN:water/72:28, 2.0
mL/min at 225 nm, tR: 13.98 and 17.76 min, 96%; 1H
NMR (CD3CN): 8.97 (brs, 1H), 8.51 (d, 1H, J=8.5 Hz),
8.27 (d, 1H, J=8.5 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, J=5.5 Hz), 6.44–6.39 (m, 1H),
6.16–6.07 (m, 1H), 5.81 (t, 1H, J=6.0 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,
J=6.0 Hz), 2.45 (t, 1H, J=6.0 Hz), 2.31–2.16 (m, 1H),
1.30–1.20 (m, 4H), 1.12–1.03 (m, 13H), 0.92 (d, 3H,
J=6.6 Hz); 31P NMR (CD3CN): 148.64, 148.55; ESI–MS
(m/z): 1132 (M+H)+.
5. Pringle, M. J. J. Clin. Ligand Assay 1999, 22, 105–122.
6. Cook, F. A.; Vuocolo, E.; Brankel, C. L. Nucleic Acids
Res. 1988, 16, 4077–4095.
7. Coull, J. M.; Weith, H. L; Bischoff, R. Tetrahedron Lett.
1986, 27, 3991–3994.
8. Langer, P. R.; Waldrop, A. A.; Ward, D. C. Proc. Natl.
Acad. Sci. USA 1981, 78, 6633–6637.
9. Telser, J.; Cruickshank, K. A.; Morrison, L. E.; Netzel,
T. L. J. Am. Chem. Soc. 1989, 111, 6966–6976.
10. Fino, J. R.; Mattingly, P. G.; Ray, K. Bioconjugate
Chem. 1996, 7, 274–280.
11. Cruickshank, K. A.; Stockwell, D. L. Tetrahedron Lett.
1988, 29, 5221–5224.
12. Dreyer, G. B.; Dervan, P. B. Proc. Nat. Acad. Sci. USA
1985, 82, 968–972.
13. Kittaka, A.; Asakura, T.; Kuze, T.; Tanaka, H.;
Yamada, N.; Nakamura, K. T.; Miyasaka, T. J. Org.
Chem. 1999, 64, 7081–7093.
14. Muhlegger, K.; Batz, H.-G.; Bohm, S. Nucleosides Nucle-
otides 1989, 8, 1161–1163.
15. Agrawal, S. In Methods in Molecular Biology; Walker, J.
M., Ed. Protocols for oligonucleotide conjugates. Synthe-
sis and analytical techniques; Humana Press: Totowa,
NJ, 1994; Vol. 26.
16. Ruth, J. L. In Oligonucleotides and Analogues. A Practical
Approach; Eckstein, F., Ed. Oligodeoxynucleotides with
reporter groups attached to the base; IRL Press: New
York, 1991.
17. Ruth, J. L.; Morgan, C.; Pasko, A. DNA 1985, 4, 1993.
18. 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.
19. Sanghvi, Y. S.; Hoke, G. D.; Zounes, M. C.; Freier, S.
M.; Martin, J. F.; Chan, H.; Acevedo, O. L.; Ecker, D.
J.; Mirabelli, C. K.; Crooke, S. T.; Cook, P. D.
Nucleosides Nucleotides 1991, 10, 345–346.
25. Mattingly, P. G. US Patent 5,424,414.
20. Tanaka, H.; Hayakawa, H.; Iijima, S.; Haraguchi, K.;
26. Fino, J. R. US Patent 5,464,746.
Miyasaka, T. Tetrahedron 1985, 41, 861–866.
27. Shreder, K. Methods 2000, 20, 372–379.
.