Polycyclic aromatic DNA-base surrogates: High-affinity binding to an adenine-specific base-flipping DNA methyltransferase

Article abstract of DOI:10.1002/anie.200219972

Filling the hole is a strategy that confers high-affinity DNA binding to the M·TaqI DNA methyltransferase. Aromatic base surrogates (e.g. pyrene, red in picture) were introduced into DNA by means of organocuprate-mediated C-glycosylations. A new competitive binding assay revealed that DNA with aromatic base surrogates placed opposite to the target base binds to M·TaqI with up to 400-fold-enhanced affinity.

Full text of DOI:10.1002/anie.200219972

Communications
DNA–Protein Interactions
DNA. The cytosine-specific DNA MTases M·HhaI[4] (Fig-
ure 1A) and M·HaeIII[5] (Figure 1B) either only insert an
amino acid side chain into the opened space or insert an
amino acid side chain in addition to rearranging the base
pairing. This is in contrast to the adenine-specific DNA
MTase M·TaqI[6] (Figure 1C). In this case, the formed
unpaired partner base is inserted into the opened space,
resulting in interstrand stacking.
Polycyclic Aromatic DNA-Base Surrogates:
High-Affinity Binding to an Adenine-Specific
Base-Flipping DNA Methyltransferase**
Christine Beuck, Ishwar Singh, Anupam Bhattacharya,
Walburga Hecker, Virinder S. Parmar, Oliver Seitz,* and
Elmar Weinhold*
DNA methylation is an important biological
event that serves diverse cellular functions
such as protection against endogenous
restriction endonucleases, direction of
DNA-mismatch repair, as well as regulation
of gene expression and DNA replication.[1]
DNA methylation is catalyzed by DNA
methyltransferases (MTases), which bind to
specific DNA sequences and transfer a
methyl group from S-adenosyl-l-methionine
to the exocyclic amino groups of adenine or
cytosine or to C5 of cytosine.[2] Interestingly,
methylation is commenced by flipping the
target nucleotide completely out of the
helix.[3] The energy cost for disrupting Wat-
son–Crick hydrogen bonds and base-stack-
ing interactions is mainly compensated by
specific binding of the target bases within the
active sites of the enzymes and by stabilizing
the formed apparent abasic site. Three
different mechanisms of abasic-site stabili-
zation have been observed in crystal struc-
tures of DNA MTases complexed with
Figure 1. DNA structures found in MTase–DNA complexes with the target extrahelical
nucleotide in blue and the orphaned nucleobase in red. A) In the M·HhaI structure,
Gln237 (represented as a ball-and-stick model) partially occupies the opened space after
cytosine flipping and forms hydrogen bonds with the orphaned guanine. B) M·HaeIII
inserts Ile221 (represented as a ball-and-stick model) into the DNA, and the orphaned
guanine pairs with the 3’-cytosine that flanks the flipped target cytosine. C) In M·TaqI, the
orphaned thymine is displaced towards the center of the DNA helix. As a result, inter-
strand stacking with the 5’ neighboring base of the target adenine occurs. D) A modeled
replacement of the orphaned thymine in the M·TaqI-DNA structure suggests that aro-
matic base surrogates such as pyrene (red) fit well into the cavity left by the flipped
target adenine and potentially improve interstrand stacking. This filling of the apparent
abasic site is likely to contribute to a tightening of the enzyme–DNA complex.
[*] Prof. Dr. O. Seitz
Institut für Chemie
Inspection of the crystal structure of M·TaqI suggested
that interstrand stacking could be improved by replacing the
partner thymine with larger aromatic DNA-base surrogates.
It was expected that more complete filling of the apparent
abasic site would lead to tightening of the M·TaqI–DNA
complex (Figure 1D). In addition, we imagined that a
polycyclic base surrogate could destabilize the innerhelical
conformation of the opposing target adenine. The DNA
MTase could thus bind to double-stranded DNA containing
an unstacked target base without having to compensate for
Humboldt-Universität zu Berlin
Brook-Taylor-Strasse 2, 12489 Berlin (Germany)
Fax: (+49)30-2093-7266
E-mail: oliver.seitz@chemie.hu-berlin.de
Prof. Dr. E. Weinhold, C. Beuck
Institut für Organische Chemie, RWTH Aachen
Prof.-Pirlet-Strasse 1, 52056 Aachen (Germany)
Fax: (+49)241-80-92528
E-mail: elmar.weinhold@oc.rwth-aachen.de
W. Hecker
Max-Planck-Institut für molekulare Physiologie
Otto-Hahn-Strasse 11, 44227 Dortmund (Deutschland)
the energy required for disrupting Watson–Crick hydrogen
bonds and base-stacking interactions. These hypotheses were
investigated with DNA substrates that contained pyrenyl, 1-
naphthyl, 2-naphthyl, acenaphthyl, and biphenyl residues
(a–e, Scheme 1) opposite to the target position within the
recognition sequence of M·TaqI.
The synthesis of the required known building blocks 6a–c
and of the new phosphoramidites 6d and 6e relies upon the
formation of a C-glycosidic bond, which we[7] and others[8]
performed by employing the 2-deoxyribose donor 1[9] and
cadmium–organic reagents such as 3a–e as glycosyl acceptors
(Scheme 1).[10] We sought an alternative C-glycosylation
I. Singh, A. Bhattacharya, Prof. Dr. V. S. Parmar
Bioorganic Laboratory, Department of Chemistry
University of Delhi
Delhi 110007 (India)
[**] This work was supported by the DAAD, the DFG, the Indian
Department of Science and Technology, and the Fonds der
Chemischen Industrie. O.S. is grateful for a DFGand Heisenberg
fellowship and C.B. for a doctoral stipend from the Studienstiftung
des Deutschen Volkes. We thank R. S. Goody for helpful discussions
regarding the competitive DNA MTase binding assays.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
3958
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DOI: 10.1002/anie.200219972
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Angewandte
Chemie
Table 2: Thermal stability of duplexes and binding affinities to the
adenine-specific DNA MTase M.TaqI.[a]
Y
X=adenine, 8A
M [8C][b] KA [109 mÀ1 [c]
X=H, 8b
T
]
TM [8C][b]
DTM [K][d]
thymine, 7T
65.9Æ0.2
55.7Æ0.1
61.0Æ0.2
56.1Æ0.2
61.0Æ0.3
57.5Æ0.3
59.0Æ0.4
57.2Æ0.1
58.0Æ0.1
0.05Æ0.02
0.03Æ0.02
0.03Æ0.02
0.04Æ0.02
20Æ10
49.8Æ0.1
50.6Æ0.1
48.7Æ0.2
52.5Æ0.2
63.6Æ0.4
59.0Æ0.3
57.9Æ0.3
56.0Æ0.2
57.8Æ0.2
0.0
+0.8
À1.1
cytosine, 7C
guanine, 7G
adenine, 7A
1-pyrenyl, 7a
4-biphenyl, 7e
acenaphthyl, 7d
1-naphthyl, 7b
2-naphthyl, 7c
+2.7
+13.8
+9.2
+8.1
+6.2
+8.0
3.0Æ1.0
2.0Æ1.0
2.0Æ1.0
1.0Æ0.5
[a] The double-stranded recognition sequence of M·TaqI is boxed.
[b] Measured as denaturation curves at a DNA concentration of 1 mm
in a buffered solution (100 mm NaCl, 10 mm NaH2PO4, pH 7.0). [c] For
experimental details, see Supporting Information. [d] DTM values are
based on the TM value of 8b·7T.
Scheme 1. a) M=Cd: CdCl2, THF, 40–508C, 2–3 h; M=CuMgX: CuI,
THF, 08C, 5 min; b) M=Cd: 08C–reflux, THF, 1–2 h; M=CuMgX:
THF, 258C, 30 min; c) 1) NaOMe, MeOH, 4 h; 2) DMTr-Cl, DMAP,
EtNiPr2, pyridine, CH2Cl2, 8 h; 3) (iPr2N)2POCH2CH2CN, tetrazole,
CH2Cl2, MeCN, 1–2 h, 59% (6a), 67% (6b), 40% (6c), 80% (6d),
51% (6e). DMTr=dimethoxytrityl, DMAP=dimethylaminopyridine.
affinities of the matched duplex 7T·8A and the duplexes
7C·8A, 7G·8A, and 7A·8A, which contain a mismatched
target adenine. The affinities were determined in a new
competitive fluorescence binding assay by employing a
competitor duplex that contained the fluorescent base
analogue 2-aminopurine at the target position.[12,13] Owing
to a partial overlap of the spectra of the 2-aminopurine and
pyrene fluorophores, this binding assay was not suitable for
the pyrene-containing duplex 7a·8A. Therefore, a pyrene
fluorescence binding assay was performed which employed a
nonfluorescent duplex oligonucleotide as competitor.[13]
M·TaqI binds its unmodified substrate 7T·8A with an
affinity constant of KA = 5 ” 107 mÀ1 and the duplexes 7C·8A,
7G·8A, and 7A·8A containing a mismatched target adenine
with almost the same affinity (Table 2). This similarity
suggests that removal of the Watson–Crick hydrogen bonds
within the target base pair has little effect. However, this
finding differs from results with the cytosine-specific DNA
MTase M·HhaI, in which an up to 20-fold enhanced binding
affinity was observed with duplexes that contained a mis-
matched target cytosine.[14]
In contrast to the duplexes with natural nucleobases,
duplexes with the naphthyl, acenaphthyl, and biphenyl base
surrogates enhanced the binding affinity by a factor of 20–60.
The ꢀ 400-fold enhancement of DNA MTase binding affinity
determined for the duplex 7a·8A containing the pyrenyl
residue was most remarkable. The observed affinity corre-
sponds to a dissociation constant KD = 50 pm.
In principle, the enhancement of binding affinity to
M·TaqI could originate from unstacking the target adenine
in the duplexes 7a–e·8A. This possibility was explored in
previous investigations.[7] Fluorescence studies suggested that
the pyrenyl residue was rather ineffective in disrupting target
base stacking. On the contrary, the biphenyl residue, which
led to a sevenfold lower affinity to M·TaqI, was the most
potent base surrogate in unstacking its partner base. These
results suggest that although binding of DNA to M·TaqI
might profit from an unstacked target base, it is not the
method with the aims of replacing the toxic cadmium and
improving the sometimes low glycosylation yields. Organo-
cuprates are mild and versatile C-nucleophiles in the coupling
with reactive alkyl chlorides.[11] Indeed, the C-glycosylation of
the Normant reagents 4a–e proved superior to the reaction
with the organocadmium reagents 3a–e in terms of both
synthetic practicability and yields (Table 1). The synthesis of
the building blocks 6a–e was completed after removal of the
tolyl protecting groups from 5a–e, regioselective introduction
of the trityl protecting group, and final phosphitylation.
Table 1: Yields of the C-glycosylation reactions.[a]
Organometallic
reagent
5a
5b
5c
5d
5e
Ar2Cd, 3a–e
72%
(4:1)
74%
(3:1)
67%
(4:1)
81%
(4:1)
47%
(2:1)
74%
(3:1)
47%
(2:1)
41%
(2:1)
75%
(2:1)
Ar2CuMgX, 4a–e
[a] The a/b ratio is given in parentheses.
The phosphoramidites 6a–e were used in the automated
solid-phase synthesis of oligonucleotides 7a–e (Table 2).[7]
First, we investigated whether the presence of the base
surrogates could be tolerated in duplex DNA. The modified
oligonucleotides 7a–e were hybridized with the complemen-
tary strand 8A, which contains a target adenine opposite to
the base surrogates within the 5’-TCGA-3’ recognition
sequence of M·TaqI (Table 2). In all cases the melting
curves exhibited a sigmoid behavior. Substitution of thymine
in 7T by the aromatic base surrogates in 7a–e led to a
decrease in the melting temperature by 4.9–8.7 K.
After having secured the integrity of the duplexes 7a–
e·8A, their binding affinities to the M·TaqI DNA MTase were
investigated in solution and compared with the binding
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ꢀ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3959

Communications
decisive determinant for the observed order of binding
affinities.
Binding of M·TaqI to its target DNA leads to the
aimed at the construction of inhibitors of these interesting
enzymes.
Received: August 14, 2002
formation of an apparent abasic site, which is only partially
filled by the interstrand stacked partner thymine (Figure 1C).
Abasic sites, however, dramatically decrease duplex stability,
as exemplified by the substantial decrease in the melting
temperature that was observed when the target 2’-deoxyade-
nosine in 7T·8A was replaced by the abasic-site analogue 1,2-
dideoxy-d-ribose in 7T·8b (DTM = À16.1 K). This destabili-
zation also occurred with the other natural nucleobases, as
evidenced by similar TM values of duplexes 7C·8b, 7G·8b,
and 7A·8b. We explored whether the base surrogates
opposite to an abasic site are capable of increasing duplex
stability. Indeed, each of the duplexes 7a–e·8b, which contain
an abasic site opposite to any of the five base surrogates,
displayed an enhanced thermal stability relative to the duplex
7T·8b (Table 2). Most importantly, the thermal stabilities of
the abasic site duplexes 7a–e·8b correlate well with the
binding affinities of duplexes 7a–e·8A to M·TaqI (Table 2).
The pyrene residue conferred the highest abasic-site stabili-
zation and led to the highest binding affinity to M·TaqI. Thus,
we propose that the pyrenyl residue enhances the M·TaqI-
binding affinity mainly by an increased compensation of the
energy penalty that arises from the enzyme-induced abasic-
site formation.
Revised: May 14, 2003 [Z19972]
Keywords: abasic sites · DNA methylation · DNA–protein
.
interactions · enzymes · glycosides · nucleic acids
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opposite to an unreactive uracil analogue were reported.[15,16]
Similar to the cytosine-specific DNA MTase M·HhaI, UDG
flips its target base out of the DNA helix and stabilizes the
apparent abasic site by insertion of an amino acid side chain.
In contrast to our binding studies with M·TaqI, only a minor
change in the binding affinity to UDG was conferred by the
pyrenyl residue, which is in agreement with the different
mechanisms of stabilizing the apparent abasic sites. In
M·TaqI, the pyrenyl residue fills the space formed by target
base flipping better than the natural partner thymine, whereas
in UDG a steric interference with the inserting amino acid
side chain is expected. In line with this interpretation, the
weak binding affinity of a UDG variant in which the critical
amino acid side chain is deleted could be restored by
incorporation of a pyrene nucleotide.[15,16] This comparison
between M·TaqI and UDG illustrates that duplexes with
extended aromatic base surrogates opposite to the target base
could be very useful probes to analyze the mechanism of
DNA base-flipping enzymes. In addition, the observed high-
affinity binding the of duplex with a pyrenyl residue is of
interest for the inhibition of adenine-specific DNA MTases
from pathogenic bacteria which are essential for bacterial
virulence.[17]
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In conclusion, we have presented an improved and less
toxic C-glycosylation that is expected to provide a reliable
access to a variety of nonpolar nucleoside analogues. Our
results point out that improved abasic-site stabilization is an
important criterion for high-affinity binding to M·TaqI.
Future work will concern the implementation of this design
approach for the development of high-affinity binders to
other adenine-specific DNA MTases, an endeavor ultimately
3960
ꢀ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
Angew. Chem. Int. Ed. 2003, 42, 3958 –3960