Synthesis and evaluation of hexitol nucleoside congeners as ambiguous nucleosides

Article abstract of DOI:10.1016/j.tetlet.2007.01.111

A series of anhydrohexitol nucleoside congeners was synthesized as ambiguous or so-called universal nucleosides and was evaluated for their hybridization potential and discrimination properties. The 1,5-anhydro-2,3-dideoxy-2-(5-nitroindazol-1-yl)-d-arabin

Full text of DOI:10.1016/j.tetlet.2007.01.111

Tetrahedron Letters 48 (2007) 2143–2145
Synthesis and evaluation of hexitol nucleoside congeners
as ambiguous nucleosides
Catia Lambertucci,a,b Guy Schepers,b Gloria Cristalli,a
Piet Herdewijnb and Arthur Van Aerschotb,*
a
`
Dipartimento di Scienze Chimiche, Universita di Camerino, I-62032 Camerino (MC), Italy
bLaboratory of Medicinal Chemistry, Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Received 23 November 2006; revised 16 January 2007; accepted 19 January 2007
Available online 25 January 2007
Abstract—A series of anhydrohexitol nucleoside congeners was synthesized as ambiguous or so-called universal nucleosides and was
evaluated for their hybridization potential and discrimination properties. The 1,5-anhydro-2,3-dideoxy-2-(5-nitroindazol-1-yl)-D-
arabino-hexitol 4e showed the lower spread in Tm values upon hybridization to the natural bases, with minimal destabilization,
and therefore behaved as a true ambiguous nucleoside.
Ó 2007 Published by Elsevier Ltd.
1. Introduction
2. Results and discussion
In the design of oligonucleotide primers or probes, a
nucleoside pairing equally well with all four natural
bases, a so-called universal or ambiguous nucleobase,
could be used to overcome problems due to degeneracy
of the genetic code or to incomplete peptide sequence
data. Over the last years, different surrogate bases have
been evaluated as universal or degenerate nucleosides,
which either cannot associate through hydrogen bond-
ing but provide a polarized stackable heterocycle, like
5-nitroindazole and 4-nitroimidazole, or allow a flexible
hydrogen bonding pattern mimicking natural bases, like
the azole carboxamide derivatives.1,2 On the other hand,
duplex stability of nucleic acids can be increased by the
modifications of the carbohydrate moiety, like in con-
strained hexitol nucleic acids.3 Therefore, a series of
analogs (4a–c) was prepared bearing a 5-nitroindazole,
4-nitroimidazole or 1,2,4-triazole-3-carboxamide as the
base moiety attached to 1,5-anhydro-3-deoxy-D-glucitol
as the sugar part. Following incorporation into oligo-
deoxynucleotides, their base pairing and discriminatory
properties have been evaluated.
The synthesis of the nucleoside congeners was accom-
plished by reacting tosylated precursor 2 with the differ-
ent heterocyclic bases (1a–c) in DMF using NaH as
basic catalyst (Fig. 1). Heating for 2 h at 110 °C with
nitroindazole afforded a mixture of the N1- and N2-
alkylated congeners (3a in 47% and 3b in 35%, respec-
tively). Prolonged heating (60 h) proved necessary for
alkylation with nitroimidazole (yielding 3c), and a lower
temperature of 90 °C was used with the triazole methyl-
carboxylate affording 3d. In addition, small amounts of,
respectively, the 5-nitroimidazolyl and the triazol-4-yl
congeners were obtained and were isolated by column
chromatography. Benzylidene deprotection with 80%
acetic acid at 70 °C for 1 h afforded 4a,c,e in about
85% yield.4 Treatment of 3d with methanolic ammonia
produced the required amide 3e. Dimethoxytritylation
and phosphitylation afforded the required phosphorami-
dites 6a,c,e.5 However, phosphitylation of tritylated 5e
was complicated by additional phosphitylation of the
carboxamide, reducing the yield to 38%. Phosphityl-
ation of carboxamide moieties of heterocyclic bases
was described before as a possible complication.6 Stan-
dard oligonucleotide assembly, deprotection, and purifi-
cation by anion exchange chromatography, afforded the
desired oligonucleotides, which were characterized by
HPLC–MS as described before.7 While phosphitylation
of 5e was troublesome, assembly using the unprotected
carboxamide 6e, afforded flawless coupling upon
Keywords: Universal nucleosides; Anhydrohexitol nucleosides; Duplex
stability; Ambiguous base; Degenerate nucleosides.
*
Corresponding author. Tel.: +32 16 337388; fax: +32 16 337340;
e-mail: arthur.vanaerschot@rega.kuleuven.be
0040-4039/$ - see front matter Ó 2007 Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2007.01.111

2144
C. Lambertucci et al. / Tetrahedron Letters 48 (2007) 2143–2145
Het
Het
O
Het
OTos
i, ii
O
O
O
1a-c +
O
DMTr
O
O
(iii), iv
v, vi
O
O
Ph
O
O
HO
2
3a-d
4a,c,e
6a,c,e
N(iPr)2
HO
Ph
P
CE-O
O
O2N
O2N
N
N
O2N
N
N
N
CH3O
H2N
N
N
Het =
N
N
N
N
N
a
b
c
d
e
Figure 1. Synthetic scheme for preparation of the new anhydrohexitol nucleoside analogs 4a,c,e and their respective amidites 6a,c,e. Reaction
conditions: (i) NaH / DMF, 60 °C, 1 h (1a: 5-nitroindazole; 1b: 4-nitroimidazole; 1c: 1,2,4-triazole-3-methylcarboxylate); (ii) add 2, DMF, T (3a 47%;
3b 35%; 3c 59%; 3d 45%); (iii) NH3/MeOH; (iv) AcOH 80%, 70 °C (4a 87%; 4c 83%; 4e 86% combined); (v) 4,40-dimethoxytrityl chloride (DMTrCl,
1.1 equiv), Py, 3 h (5a 87%; 5c 77%; 5e 80%); (vi) EtN(iPr)2, CH2Cl2, (iPr)2NP(Cl)OCH2CH2CN (6a 89%; 6c 77%; 6e 36%).
monitoring via trityl analysis. Yields for the isolated oli-
gonucleotides (30–45 OD260) proved the same for the
different modifications. Smaller amounts of slower elut-
ing products, however, were noticed, especially with
multiple incorporation of the triazole carboxamide.
d(CACCGXTGCTACC)-30 led to moderately reduced
thermal stability of the resulting duplexes with the
respective natural complements (Table 1). Thus, with
introduction of 5-nitroindazole nucleoside analog (4a),
Tm values in the range of 48.6–50.3 °C were observed,
which proved to be the least destabilizing congener
among the newly tested compounds. In addition, it
showed the least spread in Tm values (DTm of 1.7 °C)
which is a prime requirement for a true universal nucleo-
side. With an average destabilization of 7.8 °C versus
the A/T matched sequence, this analog perfectly
matches the results for the acyclic nitroindazole analog
7 (DTm of 1.7 °C, average destabilization of 7.8 °C,
Fig. 2),6 as well as for the ribosylated 5-nitroindole ana-
log 8 (DTm of 1.0 °C, average destabilization of 8.1 °C).8
The base pairing properties of the analogs were exam-
ined by hybridizing the modified oligomers with natural
DNA strands carrying one of the four natural bases in
juxtaposition to the modification, using temperature-
dependent UV spectroscopy. Hereby, the spread in Tm
is given with Tm for the best hybridizing base minus
Tm for the least hybridizing base.
Compared to the DNA reference,7 introduction of one
of the new nucleosides (4a–c) in the sequence 50-
A shorter acyclic chain carrying the nitroindazole base
under the form of the glycol nucleic acid analogue 9,
was evaluated as well and was prepared9 in analogy with
the report of Zhang et al.10 The higher flexibility of acy-
clic 7 presumably allows optimal positioning of the base
analog, where this seems counterbalanced by the re-
duced entropic penalty upon duplex formation for the
constrained anhydrohexitol 4a, affording the same over-
all outcome. In terms of ambiguity 9 performs even
slightly better with a DTm of only 1.2 °C. However, this
analog gives a larger decrease in hybridization tempera-
ture, probably as of the shorter chain length.
Table 1. Hybridization studiesa for the new anhydrohexitol analogs
(X) within the oligonucleotide duplex 50-d(CACCGXTGCTACC)-30/
30-d(GTGGCYACGATGG)-50
b
c
X
Y =
T
A
C
G
DTm
Average Tm
T
46.8 57.0 44.4 50.9
57.4 46.6 44.7 53.5
50.1 49.4 50.3 48.6
42.5 43.4 42.7 50.1
46.3 47.1 41.8 53.6 11.8
46.2 45.4 45.0 45.0 1.2
A
4a
4c
4e
9
1.7
7.6
49.6
44.7
47.2
45.4
a Tm as determined by taking the first derivative of the melting curve by
slow heating and cooling (0.2 °C/min) in 0.1 M NaCl, potassium
phosphate (0.2 M, pH 7.5) EDTA (0.1 mM).
Analysis for a second sequence [50-d(AGTATTGX-
CCTA)-30] afforded the same overall picture with a
spread in Tm of 2.2 °C for congener 4a. Finally, the
self-complementary sequence [50-d(CGCXAATTY-
GCG)-30] reported before11 and comprising two points
b The spread in Tm (DTm) is calculated as the Tm for the highest
melting complementary oligo minus the lowest melting one for each
specific modification.
c Average Tm versus the matched sequence A/T (57.4 °C).
NO2
NO2
NO2
N
N
N
HO
N
N
N
HO
O
HO
HO
OH
HO
7
8
9
Figure 2. Structures for the acyclic nitroindazole congeners 7 and 9 and the ribosylated 5-nitroindole 8.

C. Lambertucci et al. / Tetrahedron Letters 48 (2007) 2143–2145
2145
5.11 (br s, 1H, H-20); 7.91 (d, 1H, J = 9.2 Hz, H-7); 8.22
(dd, 1H, J = 2.2 Hz and 9.2 Hz, H-6); 8.41 (s, 1H, H-3);
8.83 (d, 1H, J = 2.2 Hz, H-4). 13C NMR (DMSO-d6,
200 MHz) d 35.1 (C-30); 54.4 (C-20); 61.4 (C-60); 62.6 (C-
40); 68.0 (C-10); 83.1 (C-50); 111.0 (C-7); 119.2 (C-4); 120.8
(C-6); 123.1 (C-9); 135.8 (C-3); 140.6 (C-8); 141.9 (C-5).
ESMS calcd for C13H16N3O5 ([M+H]+): 294.1090. Found:
294.1086; Compound 4c: 1H NMR (DMSO-d6, 200 MHz)
d 1.88 (m, 1H, H-30a); 2.28 (m, 1H, H-30e); 3.16 (m, 1H,
H-50); 3.55 (m, 3H, H-40 and H-60); 3.77 (m, 1H, H-10a);
4.16 (m, 1H, H-10e); 4.63 (m, 2H, 60-OH and H-20); 4.97
(d, 1H, J = 4.8 Hz, 40-OH); 8.00 (s, 1H, H-2); 8.47 (s, 1H,
of modification per double stranded complex, was stud-
ied with X being one of the different new analogs. How-
ever, even under high salt conditions (1.0 M NaCl) a
mixture of hairpins and duplex structures prevailed, in
contrast with data reported for other modified
analogs.11
3. Conclusions
Three new anhydrohexitol nucleoside analogs were syn-
thesized, characterized, and incorporated into several
oligonucleotide sequences for thermal denaturation
studies. While all modifications destabilized the double
helix upon a single incorporation, 5-nitroindazole con-
gener 4a was the least destabilizing and showed the least
spread in Tm values and therefore is behaving almost
like a true ambiguous nucleoside analog comparable to
the commercially available 5-nitroindazole deoxyribo-
furanoside. The performance of 4a in sequencing and
for PCR primers is under study and is clearly important
in validating its final usefulness as a universal base.
13
H-5). C NMR (DMSO-d6, 300 MHz) d 36.7 (C-30); 54.2
(C-20); 60.2 (C-60 and 40); 67.7 (C-10); 82.7 (C-50); 121.1
(C-5); 136.9 (C-2); 146.7 (C-4). ESMS calcd for
C9H14N3O5 ([M+H]+): 244.0933. Found: 244.0925; Com-
pound 4e: 1H NMR (DMSO-d6, 200 MHz) d 1.89 (m, 1H,
H-30a); 2.50 (m, 1H, H-30e); 3.16 (m, 1H, H-50); 3.41 (m,
1H, H-60); 3.64 (m, 2H, H-40, H-10a); 4.30 (m, 1H, H-10e);
4.60 (t, 1H, J = 6.0 Hz, 60-OH); 4.69 (m, 1H, H-20); 5.00
(d, 1H, J = 5.8 Hz, 40-OH); 7.61 (s, 1H, NH); 7.83 (s, 1H,
NH); 7.85 (s, 1H, H-5). 13C NMR (DMSO-d6, 300 MHz) d
35.5 (C-30); 59.9 (C-20); 61.1 (C-60 and C-40); 67.8 (C-10);
83.5 (C-50); 144.6 (C-5); 156.7 (C-3); 161.0 (CO); ESMS
calcd for C9H15N4O4 ([M+H]+): 243.1093. Found
243.1079.
5. Experimental data for phosphoramidites: Compound 6a:
89% yield; 31P NMR: d 148.11, 148.26; ESMS calcd. for
C43H51N5O8P ([M+H]+): 796.3475. Found 796.3460;
Compound 6c: 77% yield; 31P NMR: d 148.11, 149.15;
ESMS calcd for C39H49N5O8P ([M+H]+): 746.3318.
Found 746.3316; Compound 6e: 38% yield; 31P NMR: d
148.49, 149.03; ESMS calcd for C39H50N6O7P ([M+H]+):
745.3478. Found 745.3478; double phosphitylated 6e
analog: 35% yield; 31P NMR: d 148.55, 148.62, 148.92,
149.09; ESMS calcd for C48H67N8O8P2 ([M+H]+):
945.4557. Found: 945.4555.
6. Van Aerschot, A.; Rozenski, J.; Loakes, D.; Pillet, N.;
Schepers, G.; Herdewijn, P. Nucleic Acid Res. 1995, 23,
4363–4370.
7. Zhou, D.; Lagoja, I. M.; Rozenski, J.; Busson, R.; Van
Aerschot, A.; Herdewijn, P. ChemBioChem. 2005, 6, 2298–
2304.
8. Loakes, D.; Brown, D. M. Nature 1994, 369, 492–493.
9. Alkylation of nitroindazole was accomplished with tolu-
enesulfonylated (S)-isopropylideneglycerol in DMF at
60 °C. 9: 1H NMR (DMSO-d6) d 3.39–3.42 (m, 2H, H-
30), 3.96 (m, 1H, H-20), 4.33–4.44 (dd, 1H, H-10a, J =
7.6 Hz and 14.0 Hz), 4.51–4.60 (dd, 1H, H-10b, J = 3.8 Hz
and 14.0 Hz), 7.79–7.84 (d, 1H, H-6, J = 9.2 Hz); 7.986–
8.04 (dd, 1H, H-7, J = 2.2 Hz and 9.6 Hz), 8.75 (s, 1H,
H-3), 8.89–8.90 (d, 1H, H-9, J = 2.2 Hz); 13C NMR
(DMSO-d6) d 57.2 (C-10), 63.6 (C-30), 70.7 (C-20), 118.1
(C-6); 119.6 (C-7); 119.9 (C-4); 120.7 (C-9), 130.5 (C-3),
142.0 (C-8); 149.2 (C-5). ESMS calcd. for C10H12N3O4
([M+H]+): 238.0828. Found: 238.0821.
Acknowledgments
The authors are indebted to Dr. Rozenski for mass
determinations, and are grateful to Chantal Biernaux
for editorial help. Financial support was obtained from
the K. U. Leuven (GOA) and the Research Foundation-
Flanders (G.0603.06).
Supplementary data
Supplementary data under the form of experimental
procedures and analytical data associated with this arti-
cle are available. Supplementary data associated with
this article can be found, in the online version, at
doi:10.1016/j.tetlet.2007.01.111.
References and notes
1. Loakes, D. Nucleic Acids Res. 2001, 29, 2437–2447.
2. Paul, N.; Nashin, V. C.; Hoops, G.; Zhang, P.; Zhou, J.;
Bergstrom, D. E.; Davisson, V. J. Chem. Biol. 2003, 10,
815–825.
3. Hendrix, C.; Rosemeyer, H.; Verheggen, I.; Seela, F.; Van
Aerschot, A.; Herdewijn, P. Chem. Eur. J. 1997, 3, 110–
120.
4. Analytical data for the deprotected nucleoside analogs 4a:
1H NMR (DMSO-d6, 200 MHz) d 1.99 (m, 1H, H-30a);
2.51 (m, 1H, H-30e); 3.24 (m, 1H, H-50); 3.35 (m, 2H, H-
60); 3.70 (m, 1H, H-40); 3.79 (dd, 1H, J = 2.6 Hz and
12.4 Hz, H-10a); 4.04 (m, 1H, H-10e); 4.55 (t, 1H,
J = 5.9 Hz, 60-OH); 4.97 (d, 1H, J = 5.6 Hz, 40-OH);
10. Zhang, L.; Peritz, A.; Meggers, E. J. Am. Chem. Soc. 2005,
127, 4174–4175.
11. Zhang, P.; Johnson, W. T.; Klewer, D.; Paul, N.; Hoops,
G.; Davisson, V. J.; Bergstrom, D. E. Nucleic Acids Res.
1998, 26, 2208.