A stable DNA duplex containing a non-hydrogen-bonding and non-shape-complementary base couple: Interstrand stacking as the stability determining factor

Article abstract of DOI:10.1002/1521-3773(20010817)40:16<3012::AID-ANIE3012>3.0.CO;2-Y

The stabilizing effect of a dG:dC base-pair can also be imparted to a DNA duplex by a non-hydrogen- bonding, non-shape-complementary nucleoside analogue when interstrand stacking interactions come into play. This is the case, for example, with dBP, which

Full text of DOI:10.1002/1521-3773(20010817)40:16<3012::AID-ANIE3012>3.0.CO;2-Y

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A Stable DNA Duplex Containing
a Non-Hydrogen-Bonding and
Non-Shape-Complementary Base
Couple: Interstrand Stacking as the
Stability Determining Factor
N
N
N
N
N
O
BnO
BnO
Si
O
O
N
e,f
O
c,d
O
a,b
O
+
32%
Christine Brotschi, Adrian Häberli, and
Christian J. Leumann*
46%
51%
Si
O
OH
BnO OBn
I
BnO OBn
2
3
4
5
Hydrogen-bonding and stacking interac-
tions between nucleobases are the major
components of the noncovalent forces that
stabilize the DNA and RNA double helix.[1, 2]
The relative contribution of each factor to the
stability has been a matter of debate since the
discovery of the structure of the double helix.
Recent reports on stable duplex formation
and selective DNA-polymerase-mediated rep-
lication of oligodeoxynucleotides that contain
hydrophobic, non-hydrogen-bonding base
pairs led to a new twist in this discussion,[3±14]
and have triggered a re-evaluation of the im-
portance of aromatic stacking interactions and
shape selectivity in the base-pair formation.[15]
In a research program devoted to the
introduction of specific metal-binding sites
into DNA,[16±18] we became interested in the
synthesis and incorporation into oligonucleo-
tides of the nucleoside analogue 1 (dBP; see
N
N
N
N
DMTO
iPr2N
N
N
O
O
HO
Si
h,i
g
O
O
52%
O
O
95%
P
Si
O
OH
OCH2CH2CN
6
1
7
(dBP)
Scheme 1. Reagents and conditions: a) 3 (1.0 equiv), nBuLi (1.0 equiv), THF, 788C, 2 h, then
(0.9 equiv),
788C !RT, 16 h; b) Et3SiH (5 equiv), BF3 ´ OEt (5 equiv), CH2Cl2,
2
788C !RT, 16 h; c) BBr3 (3.4 equiv), CH2Cl2, 788C, 4 h; d) 1,3-dichloro-1,1,3,3-tetraiso-
propyldisiloxane (1.0 equiv), pyridine, RT, 5 h; e) 1,1'-thiocarbonyl diimidazole (1.2 equiv),
CH3CN, RT, 18 h; f) Bu3SnH (1.6 equiv), AIBN (1.5 equiv), toluene, 808C, 2 h; g) (HF)3 ´ NEt3
(1.0 equiv), THF, RT, 18 h; h) 4,4'-dimethoxytrityl (DMT) chloride (1.2 equiv), pyridine, RT,
16 h; i) iPr2NEt (3 equiv), [(iPr2N)(NCCH2CH2O)P]Cl (1.3 equiv), THF, RT, 3 h. AIBN ˆ a,a'-
azobisisobutyronitrile.
Scheme 1), which contains a 2,2'-bipyridyl (BP) unit as a base-
surrogate.[19] Herein we report the efficient self-recognition of
dBP within a DNA duplex, with high selectivity and stability
in the absence of transition metal ions.
The synthesis of the corresponding deoxy-C-nucleoside 1
started from 2,3,5-tri-O-benzyl-d-ribonolactone 2[20] and 4-io-
do-2,2'-bipyridine 3[21, 22](Scheme 1) and followed established
routes in C-glycoside synthesis (Scheme 1):[23, 24] Lithiation of
3 followed by addition to lactone 2 resulted in the inter-
mediate formation of the corresponding hemiacetals, which
upon reduction with Et3SiH afforded selectively the b-
isomeric C-glycoside 4 (1H NMR NOE). After a series of
typical transformations for the selective removal of the 2'-
hydroxy group, C-glycoside 1 was obtained and subsequently
converted into the corresponding phosphor-
HO
amidite building block 7.
The incorporation of 7 into the duplex 8
O
(Table 1) was effected by standard protocols
OH
in automated DNA synthesis.[25] The thermal
φ
stability of the duplexes 8 (X, Yˆ dBP, dA,
dG, dC, dT, and the abasic site analogue f)
was assessed by determination of the melting temperatures
(Tm) by using UV spectroscopy (10 mm NaH2PO4, 150 mm
NaCl, pH 7.0; Figure 1a; Table 1). Inspection of the Tm data
[*] Prof. Dr. C. J. Leumann, Dipl.-Chem. C. Brotschi,
Dipl.-Chem. A. Häberli
~
Figure 1. a) UV melting curves of the duplexes 8 with X ˆ Yˆ dBP ( ),
&
Department of Chemistry and Biochemistry, University of Bern
Freiestrasse 3, 3012 Bern (Switzerland)
Fax : (‡41)31-631-3422
*
X ˆ dBP, Yˆ f ( ), and X ˆ dBP, Yˆ dT ( ); A ˆ standardized absorp-
tion; experimental conditions are described in Table 1; b) CD spectra of
*
*
duplexes 8 with X ˆ Yˆ dBP ( ) and with X ˆ dA, Yˆ dT ( ); c(8) ˆ
E-mail: leumann@ioc.unibe.ch
2.4 mm, buffer described in Table 1).
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Table 1. Sequence of the 19-mer DNA duplex 8 and Tm data for various
combinations of X and Y, as extracted from UV melting curves (260 nm,
1.2 mm, in 10 mm NaH2PO4, 150 mm NaCl, pH 7.0).
5'-GATGACXGCTAGCTAGGAC
3'-CTAC TGYCGATCGATCCTG
8
X
BP
f
A
G
T
C
Y
BP
T
C
A
G
67.4
57.9
59.7
61.4
62.0
58.5
50.1
52.0
52.5
54.8
n.d.[a]
64.0
55.6
57.0
59.2
n.d.[a]
60.0
66.7
59.2
58.6
n.d.[a]
58.5
56.1
64.1
60.1
n.d.[a]
55.7
54.6
55.8
66.0
[a] Not determined.
reveals that the unnatural base pair dBP:dBP in the given
sequence (Tm ˆ 67.48C) is more stable than a dA:dT base pair
by 3.4K, and of similar stability to a dG ± dC base-pair
(‡0.7K). The self-recognition of dBP is selective. When each
of the natural bases is placed opposite a dBP residue in the
duplex, Tm decreases by 5.4 ± 9.5K, which corresponds to the
differences in the free enthalpies of duplex formation (DDG8)
1
of 1.7 ± 3.6 kcalmol (Table 2) relative to the dBP:dBP
duplex. Thus the effect of a dBP:dN base-pair on duplex
stability is similar to that resulting from a mismatched natural
base pair. Importantly, if the dBP residue is located opposite
to an abasic site analogue, a decrease in thermal stability of
8.9K is observed.
Table 2. Two-state model dependent thermodynamic data of duplex
formation obtained from curve fitting of the experimental UV melting
curve.[27] Estimated error: Æ 5%.
Y
DH
[kcalmol
DS
[calK 1 mol
DG258C
[kcalmol ]
1
1
1
(X ˆ BP)
]
]
A
T
G
C
115
106
108
112
113
317
293
296
308
303
21.0
19.1
20.5
20.4
22.7
Figure 2. Structure of a section of duplex 8 (X ˆ Yˆ dBP), which contains
the two dBP residues (bases in yellow) as well as the adjacent natural base
pairs (two on either side). The structure corresponds to the average of the
last 20 ps of an unrestrained molecular dynamics simulation at 300 K on a
200-ps trajectory, using the amber force field incorporated in InsightII/
Discover3 V98.0. Instead of explicit solvent molecules, a distance-depen-
dent permittivity of e ˆ 4r (rˆ distance) was used as a screening function.
Top: view along the helical axis, showing the almost ideal overlap of the
distal pyridine rings of dBP; bottom: view from the major groove side
which shows the stacked dBP base couple within the base stack of the
DNA.
BP
To exclude traces of transition metal ions bound to dBP,
which would thus interfere with the Tm experiments, EDTA
(0.1m) was included in the buffer in control experiments. No
differences in Tm were observed in these cases. Given the low
pKa of the bipyridyl unit (4.3) and the unfavorable geometry
of the ring nitrogen atoms for interresidue hydrogen bonding,
‡
we also exclude the formation of a protonated dBPH :dBP
base pair at pH 7.0.
base-pairs, which is permitted by a rearrangement of the local
backbone conformation. The overall B conformation of the
double helix remains mostly intact, a fact that is fully
supported by the circular dichroism (CD) spectra of the
duplexes 8 with X:Yˆ dA:dT and dBP:dBP (Figure 1b).
The combined experimental and modeling results for the
non-hydrogen-bonding dBP-containing oligonucleotide du-
plex 8 led us to conclude that interstrand stacking of two
opposite non-hydrogen-bonded and non-shape-complemen-
tary aromatic residues is an integral part of the duplex
stabilization. A review of the properties of the recently
described duplexes with various hydrophobic base analogues
by Romesberg, Schultz and co-workers,[3, 5, 6] and by Kool and
co-workers[8, 10, 14] is in perfect agreement with the findings
A structural model of a DNA duplex with two opposing
dBP units clearly shows that the two bases can only be
accomodated in the double helix without major backbone
distorsion if the distal pyridyl rings are stacked. Indeed, a
molecular dynamics simulation of the duplex 8 (X ˆ Yˆ dBP)
with the two bipyridyl bases adjacent to each other in the
starting conformation already lead to a partially stacked
geometry (Figure 2, top) during initial energy minimization.
This arrangement remains stable over the whole period of the
200-ps trajectory of the simulation. As can be seen in Figure 2
(bottom), the stacking of the two dBP residues results in an
increase in the distance between the two adjacent dG:dC
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[20] W. Timpe, K. Dax, N. Wolf, H. Weidmann, Carbohydr. Res. 1975, 39,
53 ± 60.
[21] K. M. Guckian, B. A. Schweitzer, R. X.-F. Ren, C. J. Sheils, P. L. Paris,
D. C. Tahmassebi, E. T. Kool, J. Am. Chem. Soc. 1996, 118, 8182 ±
8183.
[22] B. Zhang, R. Breslow, J. Am. Chem. Soc. 1997, 119, 1676 ± 1681.
[23] G. A. Kraus, M. T. Molina, J. Org. Chem. 1988, 53, 752 ± 753.
[24] K. Krohn, H. Heins, K. Wielckens, J. Med. Chem. 1992, 35, 511 ± 517.
[25] The corresponding oligonucleotides of duplex 8 with X ˆ Yˆ dBP
were synthesized and purified as described (I. Pompizi, A. Häberli,
C. J. Leumann, Nucleic Acids Res. 2000, 28, 2702 ± 2708). Building
block 7 was incorporated with coupling yields >98%. Analysis by
reported herein. The shape-complementary difluoroto-
luene:4-methylindole and related base couples,[8, 14] as well
as analogous arrangements of substituted phenyl groups with
geometries that do not allow interstrand stacking destabilize a
DNA duplex.[5] On the other hand, extended aromatic units,
for example, the isocarbostyryls (1-hydroxyisoquinolines)[3, 6]
or pyrene,[10] which have the potential (at least partially) to
stack if they occupy opposite positions in a duplex, show equal
or higher stability than natural base pairs. The importance of
interstrand stacking to duplex stability has also been pointed
out recently in the case of the DNA and RNA analogues
p-RNA and homo-DNA.[26] A high-resolution structural
analysis of a duplex that contains a dBP base pair is currently
in progress.
The reported experimental results provide the basis for the
simple rationale that non-hydrogen-bonding non-shape-com-
plementary base-arrangement in a DNA duplex can attain
similar or even enhanced stabilities relative to a Watson ±
Crick base pair only if interstrand base stacking of such
opposing units is possible. This rationale may prove useful in
further base design for applications in molecular biology or
materials science.
‡
using positive-ion ESI-MS revealed experimental masses ([M‡H]
‡
found 5882.4 and 5784.6) that are in agreement with theory ([M‡H]
calcd 5882.8 and 5784.8, respectively), thus confirming the structural
integrity of the oligonucleotides.
[26] R. Micura, R. Kudick, S. Pitsch, A. Eschenmoser, Angew.Chem. 1999,
111, 715 ± 718; Angew. Chem. Int. Ed. 1999, 38, 680 ± 683, and
references therein.
[27] C. Epple, C. J. Leumann, Chem. Biol. 1998, 5, 209 ± 216.
Efficient Photooxidative Degradation of
Organic Compounds in the Presence of Iron
Tetrasulfophthalocyanine under Visible Light
Irradiation**
Received: March 20, 2001 [Z16817]
Publication delayed at authorsꢁ request
Xia Tao, Wanhong Ma, Tianyong Zhang, and
Jincai Zhao*
[1] C. R. Cantor, P. R. Schimmel in Biophysical Chemistry Part I: The
Conformation of Biological Macromolecules (Ed.: P. C. Vapnek),
Freeman, New York, 1980, pp. 311 ± 341.
The degradation of organic pollutants by photocatalysis[1]
and (photo-)Fenton reactions[2] has been described exten-
sively. Meunier and co-workers reported an efficient oxidative
degradation of trichlorophenol (TCP) in the presence of iron
tetrasulfophthalocyanine ([Fe(PcS)]) and H2O2 in the dark.[3]
In this process the metal ± peroxo species [Fe(OOH)(PcS)] is
involved as an active oxygen intermediate. A merit of this
system is that [Fe(PcS)] is a readily available biomimetic
catalyst that can be fixed onto amberlite and therefore does
not enter into the environment and cause additional pollution.
It should, however, be noted that the solvent employed
contains a large amount of acetonitrile. If water is used as the
sole solvent, the conversion rate of TCP is greatly reduced.[4]
Here we report that when visible light is introduced to an
aqueous system containing test compounds, [Fe(PcS)], and
[2] W. Saenger, Principles of Nucleic Acid Structure, Springer, New York,
1984.
[3] 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.
[4] D. L. McMinn, A. K. Ogawa, Y. Wu, J. Liu, P. G. Schultz, F. E.
Romesberg, J. Am. Chem. Soc. 1999, 121, 11585 ± 11586.
[5] A. K. Ogawa, Y. Q. Wu, D. L. McMinn, J. Liu, P. G. Schultz, F. E.
Romesberg, J. Am. Chem. Soc. 2000, 122, 3274 ± 3287.
[6] Y. Wu, A. K. Ogawa, M. Berger, D. L. McMinn, R. G. Schultz, F. E.
Romesberg, J. Am. Chem. Soc. 2000, 122, 7621 ± 7632.
[7] K. M. Guckian, T. R. Krugh, E. T. Kool, J. Am. Chem. Soc. 2000, 122,
6841 ± 6847.
[8] K. M. Guckian, J. C. Morales, E. T. Kool, J. Org. Chem. 1998, 63,
9652 ± 9656.
[9] T. J. Matray, E. T. Kool, Nature 1999, 399, 704 ± 708.
[10] T. J. Matray, E. T. Kool, J. Am. Chem. Soc. 1998, 120, 6191 ± 6192.
[11] J. C. Morales, E. T. Kool, Nat. Struct. Biol. 1998, 5, 950 ± 954.
[12] J. C. Morales, E. T. Kool, J. Am. Chem. Soc. 2000, 122, 1001 ± 1007.
[13] S. Moran, R. X.-F. Ren, E. T. Kool, Proc. Natl. Acad. Sci. USA 1997,
94, 10506 ± 10511.
[14] B. A. Schweitzer, E. T. Kool, J. Am. Chem. Soc. 1995, 117, 1863 ± 1872.
[15] E. T. Kool, J. C. Morales, K. M. Guckian, Angew. Chem. 2000, 112,
1046 ± 1068; Angew. Chem. Int. Ed. 2000, 39, 990 ± 1009.
[16] E. Meggers, P. L. Holland, W. B. Tolman, F. E. Romesberg, P. G.
Schultz, J. Am. Chem. Soc. 2000, 122, 10714 ± 10715.
[17] K. Tanaka, M. Shionoya, J. Org. Chem. 1999, 64, 5002 ± 5003.
[18] K. Wiederholt, L. W. McLaughlin, Nucleic Acids Res. 1999, 27, 2487 ±
2493.
[19] During the revision of this manuscript, a paper appeared in which two
homo-dBP units that contained a methylene group between the
bipyridyl unit and the C1' of the deoxyribose unit were introduced into
a DNA duplex at opposite positions. A differential increase in duplex
stability in the presence of Cu2‡ ions was observed (H. Weizman, Y.
Tor, J. Am. Chem. Soc. 2001, 123, 3375 ± 3376).
[*] Prof. J. Zhao,[‡] X. Tao, W. Ma, T. Zhang
Laboratory of Photochemistry, Center for Molecular Sciences
Institute of Chemistry, The Chinese Academy of Sciences
Beijing 100080 (China)
Fax : (‡86)10-6487-9375
E-mail: jczhao@ipc.ac.cn
‡
[ ] Current address:
Institute of Photographic Chemistry
Chinese Academy of Sciences
Beijing 100101 (China)
[**] This work was supported financially by NSFC (nos. 29877026,
4001161947, 29725715, and 20077027) and CAS. The authors thank
Dr. Huiyong Zhu, The University of Queensland, Australia, for his
useful discussions.
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