Towards a DNA-like duplex without hydrogen-bonded base pairs

Article abstract of DOI:10.1002/1521-3773(20020902)41:17<3203::AID-ANIE3203>3.0.CO;2-K

The inverse quadrupolar moments of the phenyl and pentafluorophenyl residues in the non-hydrogen-bonded, artificial base pair shown here promotes strong intramolecular stacking interactions in oligonucleotide duplexes. The greater the number of natural ba

Full text of DOI:10.1002/1521-3773(20020902)41:17<3203::AID-ANIE3203>3.0.CO;2-K

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Towards A DNA-Like Duplex without
Hydrogen-Bonded Base Pairs**
Gÿrald Mathis and J¸rg Hunziker*
The creation of alternative base pairs to expand the genetic
13]
alphabet has recently been the focus of intensive research.[1
Initial work was based on the systematic permutation of
hydrogen bond donor and acceptor arrangements within the
3]
pyrimidine and purine nuclei.[1 In subsequent studies the
Watson Crick complementarity paradigm was abandoned
9]
altogether.[4 It was shown that aromatic entities devoid of
hydrogen bonding functional groups can satisfy at least part of
the criteria for replicable base pairs, that is, thermodynamic
stability of the resulting duplex, orthogonality to natural
nucleobases, and enzymatic processability. Such nonpolar
base pair isosteres are held together by hydrophobic dis-
persion forces which act predominantly along the base stack
but less so at the interface between the bases. In an attempt to
Figure 1. Details of the X-ray crystal structure of the solid 1:1 complex
formed from benzene and hexafluorobenzene (grey: carbon, white:
hydrogen, green: fluorine). The side view (top) shows the stacks of
alternating composition. The aromatic molecules are tilted with respect to
the stack axis, which leads to lateral alternation within the plane of
individual benzene or hexafluorobenzene molecules (bottom).
orient the base base attraction perpendicular to the helix axis,
13]
chelating nucleosides have been developed.[10
However,
such metal-mediated base pairs can hardly be amenable to
enzymatic replication.
In the search for alternative base pair design principles we
were particularly intrigued by the interaction between ben-
18]
zene and hexafluorobenzene.[14 The two compounds, both
F
liquids at room temperature, form a solid aggregate[19,20] which
is characterized not only by p stacks of alternating compo-
nents but by the lateral alternation between hexafluoroben-
zene and benzene rings as well (Figure 1). The inverse
quadrupolar moments of benzene and hexafluorobenzene
lead to edge-to-edge attractive intermolecular forces which
could be utilized in designing novel base pairs, such as the one
formed between C-nucleosides 1 and 2 (Scheme 1). Here we
report on the synthesis and the promising pairing properties of
oligonucleotides containing 1 and 2.
F
F
F
O
O
HO
HO
F
HO
HO
1
2
DNA
F
O
F
DNA
O
F
F
O
The synthesis of phenyl-2-deoxyriboside 2 and its incorpo-
ration into oligonucleotides has been described.[21] The novel
pentafluorophenyl congener 1 was prepared from 2-deoxy-
ribonolactone following a literature strategy (Scheme 2).[22]
Commercially available bromopentafluorobenzene (4) was
lithiated and then the silyl-protected 2-deoxyribonolactone
3[22] was added. The resulting hemiacetal was reduced in situ
with Et3SiH to give an inseparable 9:1 mixture of the two
anomers 5 (b) and 6 (a) in low yield. These compounds could
be separated by chromatography after deprotection of the
deoxyribose moiety and reprotection as 5’-dimethoxytrityl
ethers. At this stage the configuration of the two isomers
could be assigned by 1H NMR NOE experiments.[23] The
pentafluorophenyl-b-d-riboside phosphoramidite 8 was then
obtained by standard phosphitylation.[24] Oligonucleotides
O
DNA
O
F
DNA
O
Scheme 1. Structures of the pentafluorophenyl-b-d-deoxyriboside (F5, 1)
and phenyl-b-d-deoxyriboside (P, 2) used to investigate the stability of F5
P
base pairs in oligonucleotide duplexes.
containing 1 and 2 (Tables 1 and 2) were prepared using a
regular solid-phase DNA synthesis protocol.[24,25]
When incorporated into a 10-mer duplex, a single penta-
fluorophenyl phenyl pair (F5 P) leads to a strong decrease in
duplex stability, as judged by the melting temperature Tm
(Table 1). The stability of the novel base pair is intermediate
to that of the relatively well-tolerated mismatched pairs A G
and G G as well as the wobble pair G T and all other more
deleterious mismatches between natural bases. The interac-
tion between the phenyl and the pentafluorophenyl units is
stronger than those with any of the natural bases. Unfortu-
[*] Priv.-Doz. Dr. J. Hunziker, Dipl.-Chem. G. Mathis
Department of Chemistry and Biochemistry
University of Bern
nately, there is no or only a slight discrimination between F5
P
Freiestrasse 3, 3012 Bern (Switzerland)
Fax : (þ 41)31-631-8057
and the corresponding self-pairs P P and F5 F5 in this
sequence context.
E-mail: juerg.hunziker@ioc.unibe.ch
Moving to a self-complementary 10-mer duplex with
several F5 P base pairs dramatically changes the picture.
[**] This work was supported by the Swiss National Science Foundation
(grant no. 20-61730.00).
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Table 1. The Tm values [8C] determined from UV melting experiments for the 10-mer DNA duplex 9 with varying bases in positions X and Y.[a]
A
T
G
C
F5
P
A
T
G
C
F5
P
29.0
45.0
35.6
29.8
25.0
25.1
44.3
27.1
36.1
28.0
22.0
21.0
36.2
35.5
38.9
50.0
28.7
27.2
28.1
27.1
49.0
25.1
21.1
17.6
25.0
22.0
25.0
24.1
29.9
30.0
25.1
21.1
23.0
24.1
30.6
30.0
[a] Duplex concentration: 4 mm; buffer: 10 mm NaH2PO4, 150 mm NaCl, pH 7.0; detection at 260 nm, Tm ¼ mean value of three melting curves, heating rate ¼
0.58CminÀ1
.
F
F
a duplex which is more stable than its natural
counterpart with four A T pairs (DTm/mod.
þ 1.5K). The beneficial mutual interaction is
unique to the phenyl and pentafluorophenyl resi-
dues in this system. The duplexes 152 and 162 with
four F5 G and F5 A pairs, respectively, are desta-
bilized in comparison.
F
F
F
F
O
O
O
O
O
F
F
F
F
a,b
(iPr)2Si
(iPr)2Si
+
20%
O
O
O
(iPr)2
O
Si
Si
(iPr)2
Br
3
4
5 / 6
The differences in duplex stability of sequences
with one, two, or four consecutive F5 P base pairs
may reflect the altered geometry of this artificial
base pair. The contact between phenyl and penta-
fluorophenyl in an optimal orientation likely
requires a backbone distance shorter than the
11 ä of a standard pyrimidine purine base pair.
The backbone may only adapt to this if several
F5 P pairs are arranged in a row.[26]
Measuring the thermal stability of 10-mer du-
plexes containing more than four F5 P pairs meets
technical hurdles. The UV absorption of C-nucleo-
sides 1 and 2 is weak over the wavelength range of
the melting experiments. In essence, only the
destacking of natural bases is detected, as can be
F
F
F
F
F
F
F
F
F
O
O
HO
DMTO
d,e
c
F
(82%)
(82%)
HO
O
P
CN
(iPr)2N
O
8
1 / 7
Scheme 2. a) Compound 4 (2.0 equiv), BuLi (1.8 equiv), Et2O, À788C, 1 h, then 3
(1.0 equiv) in Et2O, À788C, 2.5 h; b) Et3SiH, BF3¥Et2O, CH2Cl2, À788C, 12 h; c) Bu4NF,
THF, RT, 2 h; d) DMT-Cl, N,N-dimethylaminopyridine, pyridine, RT, 3 h;
e) [(iPr2N)(NCCH2CH2O)P]Cl, iPr2NEt, THF, RT, 1 h. DMT¼ 4,4’-dimethoxytrityl.
With two such pairs in a central position (duplex 122) there is
still a decrease in thermal stability (DTm/mod. À4.0K
compared to A T, Table 2), however, to a considerably lower
degree than in the case of a single substitution (DTm/mod.
À15K). Introducing four consecutive F5 P pairs (132) leads to
seen from the drop in hyperchromicity in duplexes 122
(14.5%, Table 2), 132 (8%), 182 (7.5%), and 212 (À1%). In
addition, duplex 182 with six alternating F5 P base pairs does
not show a sigmoidal curve indicative of a cooperative
transition.
The strength and the directionality of the interaction
between the phenyl and pentafluorophenyl residues is re-
vealed in concentration variation experiments, which also
yield the thermodynamic parameters of duplex formation
(Table 3). A single F5 P base pair in the duplex d(CTGAPTC-
GAC)¥d(GTCGAF5TCAG) (24¥25) leads to a decrease in the
enthalpy of duplex formation compared to the unmodified
reference, which cannot be compensated by a more favorable
pairing entropy. The situation is different in the self-comple-
mentary duplex d(CTGF5PF5PCAG)2 (13)2. The pairing
enthalpy in this system is almost 40% higher than for the
reference d(CTGATATCAG)2 (10)2, which can most likely be
attributed to the extraordinarily strong stacking interactions
between alternating phenyl and pentafluorophenyl bases.
Support for this notion stems from the following observations.
The oligonucleotide d(CTGF5PF5PCAG) (13) forms a duplex
at concentrations above 3 mm; below this threshold a mono-
molecular structure–presumably a hairpin–is predominant
Table 2. The Tm values [8C] determined from UV melting experiments for the 10-
mer oligonucleotides 10 21 with several F5 P pairs.[a]
Oligonucleotide
Tm [8C]
DTm/mod. [K]
Hyperchromicity
d(CTGATATCAG) (10)
d(CTGAGCTCAG) (11)
d(CTGAF5PTCAG) (12)
d(CTGF5PF5PCAG) (13)
d(CTGF5F5PPCAG) (14)
d(CTGF5GF5GCAG) (15)
d(CTGF5AF5ACAG) (16)
d(CF5GPATF5CPG) (17)
d(CTPF5PF5PF5AG) (18)
d(CTF5F5F5PPPAG) (19)
d(CF5PF5GCPF5PG) (20)
d(CF5PF5PF5PF5PG) (21)
38.0
27%
23%
14.5%
8 %
8 %
6 %
6.5%
2.5%
7.5%
5%
9%
À1%
48.5
þ 5.3
À4.0
þ 1.5
þ 1.7
À1.1
À0.2
30.0[b]
44.0
44.7[b]
35.8
37.7
[c]
[d]
[d]
23.1[b]
À2.5
[c]
[a] Duplex concentration: 4 mm; buffer: 10 mm NaH2PO4, 150 mm NaCl, pH 7.0;
detection at 260 nm, Tm ¼ mean value of three melting curves, heating rate ¼
0.5KminÀ1. [b] Monomolecular hairpin at 4 mm. [c] No melting transition observed.
[d] No sigmoidal (cooperative) melting transition.
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Table 3. Thermodynamic data for 10-mer DNA duplexes containing F5 P base pairs [1] J. A. Piccirilli, T. Krauch, S. E. Moroney, S. A. Benner, Nature 1990,
and the corresponding references.[a]
343, 33 37.
[2] J. D. Bain, C. Switzer, A. R. Chamberlin, S. A. Benner, Nature 1992,
356, 537 539.
Oligonucleotide
DH8 [kcalmolÀ1]DS8 [calmolÀ1 K]DG8
(258C) [kcalmolÀ1
]
[3] J. J. Voegel, S. A. Benner, Helv. Chim. Acta 1996, 79, 1863 1880; J. J.
Voegel, S. A. Benner, Helv. Chim. Acta 1996, 79, 1881 1898.
[4] T. J. Matray, E. T. Kool, J. Am. Chem. Soc. 1998, 120, 6191 6192.
[5] R. S. Coleman, M. L. Madaras, J. Org. Chem. 1998, 63, 5700 5703.
[6] K. M. Guckian, B. A. Schweitzer, R. X.-F. Ren, C. J. Sheils, D. C.
Tahmassebi, E. T. Kool, J. Am. Chem. Soc. 2000, 122, 2213 2222.
[7] M. Berger, A. K. Ogawa, D. L. McMinn, Y. Wu, P. G. Schultz, F. E.
Romesberg, Angew. Chem. 2000, 112, 3069 3071; Angew. Chem. Int.
Ed. 2000, 39, 2940 2942.
[8] E. Lee Tae, Y. Wu, G. Xia, P. G. Schultz, F. E. Romesberg, J. Am.
Chem. Soc. 2001, 123, 7439 7440.
[9] I. Singh, W. Hecker, A. K. Prasad, V. S. Parmar, O. Seitz, Chem.
Commun. 2002, 500 501.
d(CTGAATCGAC)¥
d(GTCGATTCAG)
(22¥23)
d(CTGAPTCGAC)¥
d(GTCGAF5TCAG)
(24¥25)
d(CTGATATCAG)2 (10)2 À67.5
d(CTGAGCTCAG)2 (11)2À82.3
d(CTGF5PF5PCAG)2 (13)2À94.3
À77.6
À61.5
À213
À175
À13.8
À9.1
À192
À231
À273
À10.1
À13.3
À13.0
[a] Duplex concentration: 1 32 mm; buffer: 10 mm NaH2PO4, 150 mm NaCl, pH 7.0.
(Figure 2). In contrast, the sequences d(CTGF5F5PPCAG)
(14) as well as d(CTGAF5PTCAG) (12) prefer a monomo-
lecular structure with constant Tm up to much higher
concentrations (> 10 mm). In the case of 13 the central portion
of the oligonucleotide seems to be rigidified by the alternating
F5-P-F5-P stack, which is absent or smaller in 12 and 14.[27] The
intramolecular stacking interaction between the phenyl and
pentafluorophenyl residues is therefore more stabilizing than
the lateral, intermolecular one. This is not surprising given the
different sizes of the contacting surfaces in the two dimen-
sions.
[10] M. Shionoya, K. Tanaka, Bull. Chem. Soc. Jpn. 2000, 73, 1945 1954.
[11] E. Meggers, P. L. Holland, W. B. Tolman, F. E. Romesberg, P. G.
Schultz, J. Am. Chem. Soc. 2000, 122, 10714 10715.
[12] H. Weizman, Y. Tor, J. Am. Chem. Soc. 2001, 123, 3375 3376.
[13] C. Brotschi, A. H‰berli, C. J. Leumann, Angew. Chem. 2001, 113,
3101 3103; Angew. Chem. Int. Ed. 2001, 40, 3012 3014.
[14] F. Cozzi, F. Ponzini, R. Annunziata, M. Cinquini, J. S. Siegel, Angew.
Chem. 1995, 107, 1092 1094; Angew. Chem. Int. Ed. Engl. 1995, 34,
1019 1020.
[15] R. E. Gaillard, J. F. Stoddart, A. J. P. White, B. J. Williams, D. J.
Williams, J. Org. Chem. 1996, 61, 4504 4505.
[16] G. W. Coates, A. R. Dunn, L. M. Henling, D. A. Dougherty, R. H.
Grubbs, Angew. Chem. 1997, 109, 290 293; Angew. Chem. Int. Ed.
Engl. 1997, 36, 248 251.
In conclusion, complementary charge distribution as in the
pentafluorophenyl phenyl C-nucleosides 1 and 2 represents a
novel design principle for artificial base pairs. The results from
this study highlight the importance of favorable intrastrand
stacking interactions in the thermodynamic stabilization of
oligonucleotide duplexes. On the other hand, interstrand
stacking has recently been exploited in a base pair formed
between two bipyridine residues.[13] A combination of these
two features could lead to orthogonal, non-hydrogen bonded,
non-shape complementary base pairs. Experiments towards
this end as well as attempts to replicate the pentafluorophe-
nyl phenyl pair by polymerases are currently under way.
[17] F. Ponzini, R. Zagha, K. Hardcastle, J. S. Siegel, Angew. Chem. 2000,
112, 2413 2415; Angew. Chem. Int. Ed. 2000, 39, 2323 2325.
[18] C.-Y. Kim, P. P. Chandra, A. Jain, D. W. Christianson, J. Am. Chem.
Soc. 2001, 123, 9620 9627.
[19] C. R. Patrick, G. S. Prosser, Nature 1960, 187, 1021.
[20] J. H. Williams, J. K. Cockcroft, A. N. Fitch, Angew. Chem. 1992, 104,
1666 1669; Angew. Chem. Int. Ed. Engl. 1992, 31, 1655 1657.
[21] T. A. Millican, G. A. Mock, M. A. Chauncey, T. P. Patel, M. A. W.
Eaton, J. Gunning, S. D. Cutbush, S. Neidle, J. Mann, Nucleic Acids
Res. 1984, 12, 7435 7453.
[22] U. Wichai, S. Wosky, Org. Lett. 1999, 1, 1173 1176.
[23] The tritylated pentafluorophenyl-b-d-deoxyriboside was deprotected
under acidic conditions (CHCl2COOH, pyrrole, CH2Cl2, RT, 20 min)
1
to give anomerically pure 1. H NMR NOE (400 MHz, [D6]DMSO):
irradiation of the signal for H-C(1’) (5.30 ppm) leads to enhancement
of the signal for H-C(4’) (3.75 ppm), and vice versa.
[24] B. A. Conolly in Oligonucleotides and Analogues: A
Practical Approach (Ed.: F. Eckstein), Oxford Uni-
versity Press, Oxford, 1991, pp. 151 183.
Received: March 18, 2002 [Z18923]
[25] The identity of the oligonucleotides synthesized was
confirmed by ESI mass spectrometry.
[26] Substituting the phenyl part of a F5 P base pair by a
naphthalene or indole unit could possibly resolve this
problem. Experiments with the corresponding modi-
fied oligonucleotides are under way.
[27] Experiments with the non-self-complementary 10-
mer duplexes d(CTGF5PF5PGAC)¥d(GTCF5PF5P-
CAG) and d(CTGF5F5F5F5GAC)¥d(GTCPPPPCAG)
were inconclusive since they showed melting temper-
atures below 108C.
Figure 2. Van©t Hoff plot of 1/Tm vs. ln(c) (c in mol LÀ1
) for oligonucleotide
d(CTGF5PF5PCAG) (13). Sequence 13 with four alternating F5 P base pairs in the middle
forms duplexes at concentrations above 3 mm (sloped region, linear regression indicated). In
contrast, sequences d(CTGAF5PTCAG) (12) and d(CTGF5F5PPCAG) (14) form a duplex
only at much higher concentrations.
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