Inhibition of (cytosine C5)-methyltransferase by oligonucleotides containing flexible (cyclopentane) and conformationally constrained (bicyclo[3.1.0]hexane) abasic sites

Article abstract of DOI:10.1081/NCN-100002319

Pseudorotationally locked sugar analogues based on bicyclo[3.1.0]-hexane templates were placed in DNA duplexes as abasic target sites in the M.HhaI recognition sequence. The binding affinity of the enzyme increases when the abasic site is constrained to t

Full text of DOI:10.1081/NCN-100002319

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INHIBITION OF (CYTOSINE C5)-METHYLTRANSFERASE
BY OLIGONUCLEOTIDES CONTAINING FLEXIBLE
(CYCLOPENTANE) AND CONFORMATIONALLY
CONSTRAINED (BICYCLO[3.1.0]HEXANE) ABASIC SITES
Victor E. Marquez , Peiyuan Wang , Marc C. Nicklaus , Martin Maier a , Muthiah
Manoharan a , Judith K. Christman b , Nilesh K. Banavali c & Alexander D. Mackerell Jr. d
a ISIS Pharmaceuticals, Inc. , 2292 Faraday Ave., Carslbad, California, 92008, U.S.A.
b Department of Biochemistry and Molecular Biology and UNMC/Eppley Cancer Center ,
University of Nebraska Medical Center , 600 South 42nd St., Nebraska, 68198, U.S.A.
c Department of Pharmaceutical Sciences , School of Pharmacy, University of Maryland ,
Baltimore, Maryland, 21201, U.S.A.
d Department of Pharmaceutical Sciences , School of Pharmacy, University of Maryland ,
Baltimore, Maryland, 21201, U.S.A.
Published online: 07 Feb 2007.
To cite this article: Victor E. Marquez , Peiyuan Wang , Marc C. Nicklaus , Martin Maier , Muthiah Manoharan ,
Judith K. Christman , Nilesh K. Banavali & Alexander D. Mackerell Jr. (2001) INHIBITION OF (CYTOSINE C5)-
METHYLTRANSFERASE BY OLIGONUCLEOTIDES CONTAINING FLEXIBLE (CYCLOPENTANE) AND CONFORMATIONALLY
CONSTRAINED (BICYCLO[3.1.0]HEXANE) ABASIC SITES, Nucleosides, Nucleotides and Nucleic Acids, 20:4-7, 451-459, DOI:
10.1081/NCN-100002319
To link to this article: http://dx.doi.org/10.1081/NCN-100002319
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 451–459 (2001)
INHIBITION OF (CYTOSINE
C5)-METHYLTRANSFERASE BY
OLIGONUCLEOTIDES CONTAINING FLEXIBLE
(CYCLOPENTANE) AND CONFORMATIONALLY
CONSTRAINED (BICYCLO[3.1.0]HEXANE)
ABASIC SITES
Victor E. Marquez,1,∗ Peiyuan Wang,1 Marc C. Nicklaus,1
Martin Maier,2 Muthiah Manoharan,2 Judith K. Christman,3,∗
Nilesh K. Banavali,4 and Alexander D. Mackerell, Jr.4,∗
1Laboratory of Medicinal Chemistry, Division of Basic Sciences,
NCI-FCRDC, Boyles St., Bldg. 376, Frederick, Maryland 21702
2ISIS Pharmaceuticals, Inc., 2292 Faraday Ave., Carslbad, California 92008
3Department of Biochemistry and Molecular Biology and
UNMC/Eppley Cancer Center, University of Nebraska Medical Center,
600 South 42nd St., Nebraska 68198
4Department of Pharmaceutical Sciences, School of Pharmacy,
University of Maryland, Baltimore, Maryland 21201
ABSTRACT
Pseudorotationallylockedsugaranaloguesbasedonbicyclo[3.1.0]-hexanetem-
plates were placed in DNA duplexes as abasic target sites in the M.HhaI recogni-
tion sequence. The binding affinity of the enzyme increases when the abasic site
is constrained to the South conformation and decreases when it is constrained
to the North conformation. A structural understanding of these differences is
provided.
Several lines of evidence suggest that the control of gene expression in mam-
malian cells is related to the pattern of DNA methylation found in more than 70%
∗Corresponding authors.
451
ꢀ
C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

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452
MARQUEZ ET AL.
of the CpG residues present in the genome (1). The methylation of DNA, which
involves the covalent addition of a methyl group to the 5-position of the cytosine
ring, is a post-replicative modification since all mammalian DNA is synthesized
with deoxycytidine triphosphate as a precursor.
The replication of a DNA that is methylated on both chains at CpG doublets
produces two new hemimethylated helices. Subsequently, an enzyme with prefer-
ential affinity for hemimethylated sites (maintenance DNA methylase or DNMT1)
preserves the methylation pattern with high fidelity from one cell generation to the
next (2). Different protooncogenic pathways can up-regulate DNMT1 expression
(3) and high levels of DNMT1 RNA have been observed in many cancer cells
(4). The resulting hypermethylation at CpG islands in the promoter region of a
growth-regulatory gene can silence its expression and provide a growth advantage
to transformed cells. However, since methylation changes are reversible, inhibi-
tion of aberrant DNMT1 activity could reactivate the expression of the growth-
regulatory gene, highlighting the importance of DNMT1 as a therapeutic target
(1).
The mechanism of the methylation reaction proceeds via a 5,6-dihydropyrimi
dine intermediate formed after nucleophilic attack of an enzyme’s cysteinyl thio-
late group on carbon 6 (C6) of the target cytosine (5,6). After methyl transfer from
S-adenosyl-L-methionine (AdoMet) to C5, sp3 to sp2 rehybridization occurs by re-
moval of the C5 hydrogen (5,6). This transient 5,6-dihydropyrimidine intermediate
has been trapped with oligodeoxynucleotides (ODNs) containing 5-fluorocytosine
as a suicide substrate, and crystal structures of the chemically trapped complex with
prokaryotic methylases (M.HhaI and M.HaeIII) reveal that the target cytosine ex-
hibits a flipped-out conformation (7,8). ODNs containing 5-fluorocytosine can thus
function as stoichiometric inhibitors of the enzyme (9). An ideal ODN inhibitor,
however, should not be a substrate and should exhibit a high affinity for the enzyme
with a slow off rate. Since the tightest binding of M.HhaI occurs when either the
target base or the target nucleotide is missing (abasic site or gap) ODNs containing
abasic tetrahydrofuran (THF) sites replacing the target cytosine (C) are considered
ideal candidates (10). Furthermore, crystallographic analysis of the ternary com-
plex containing M.HhaI, the cofactor S-adenosylhomocysteine (AdoHcy) and an
ODN with a THF abasic target site reveal that the flexible sugar surrogate (THF)
is also in a flipped-out conformation (11). In this crystal structure with a resolution
❛
of 2.3 A(11), the conformation of the THF appears to adopt a North conformation
as defined in the pseudorotational cycle (12). Since in the most common form of
DNA (B-DNA), the sugar moieties favors the antipodal South conformation, we
decided to investigate the effect of locking the conformation of the target abasic site
(North or South) on the interaction with M.HhaI. Our results demonstrate that the
binding affinity of M.HhaI for abasic sites increases when the abasic target sugar
is constrained to the South and decreases when it is constrained to the North. The
use of the bicyclo[3.1.0]hexane template for the construction of conformationally
locked abasic sites, as well as the apparent discrepancy with the crystal structure of
M.HhaI complexed with an ODN containing a North THF conformation site will
be discussed.

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INHIBITION OF (CYTOSINE C5)-METHYLTRANSFERASE
453
THE DESIGN OF TARGET ODN SEQUENCES
ODN 13-mers containing abasic pseudosugars (X) in place of the target
C residue in the M.HhaI recognition sequence (5ꢁ-GXGC-3ꢁ) were synthesized
(Fig. 1). Except for the nature of X, these 13-mer ODNs are identical to the ones
used in the elucidation of the ternary structure of M.HhaI with the cofactor AdoHcy
and DNA (ODNs, X = 5-fluorocytosine (7) or X = THF (11)). When the com-
plementary strand contains a 5-methylcytosine (M in the sequence 5ꢁ-GMGC-3ꢁ)
the residue X indicated by the arrow becomes the only target for flipping. ODNs
incorporating pseudosugars locked into one the two antipodal North and South
hemispheres of the pseudorotational cycle (Fig. 2) were compared with an ODN
containing the flexible cyclopentane ring. The stable pseudoboat conformation of
the bicyclo[3.1.0]hexane template ensures that the conformations remain locked
North and South as depicted in Figure 2 (13).
CHEMISTRY
(1S,2R)-2-[(Benzyloxy)methyl] cyclopent-3-enol (1) was selected as the chi-
ral starting material for the synthesis of the conformationally locked South- and
North-type phosphoramidites 5 an 15 (Schemes 1 and 2). Compound 1 was pre-
pared by a modification of the procedure originally reported by Biggadike et al. (14).
Synthesis of the South-type phosphoramidite 5 started with homoallylic hydroxyl-
directed cyclopropanation of 1 under Simmons-Smith conditions with diethylz-
inc and diiodomethane to give carbobicyclic intermediate 2 (Scheme 1). Depro-
tection of the benzyl group with palladium black in methanol provided diol 3,
which after dimethoxytritylation produced the protected alcohol 4. The South-type
phosphoramidite 5 was finally completed from 4 using standard reaction conditions
with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite.
Figure 1. Modified ODN structures with abasic moieties at the M.HhaI site consisting of a flexible
cyclopentane pseudosugar and two conformationally rigid bicyclo[3.1.0]hexane templates locked in
the North and South conformations.

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MARQUEZ ET AL.
Figure 2. Fixed locations of bicyclo[3.1.0]hexane envelope conformations (E) in the pseudorota-
tional cycle.
The approach to the North-type phosphoramidite 15 started from chiral inter-
mediate 6 (15), which was easily obtained from compound 1. Treatment of 6 with
PhSeCl/AgCF3CO2 followed by hydrolysis of the trifluoroacetate ester gave the
trans-2-(phenylselenyl)-cyclopentan-1-ol (8). The regio- and stereoselectivity of
this addition reaction has been reported (13). Following oxidation of the phenylse-
lenide group, unidirectional elimination of PhSeOH gave the corresponding allylic
alcohol 9 exclusively. Deoxygenation of this allylic alcohol with sulfur trioxide-
pyridine complex, followed by LiAlH4 reduction afforded compound 10. Allylic
alcohol 11 was obtained after deprotection of 10 with triethylamine trihydrofluo-
ride and subjected to hydroxyl-directed cyclopropanation of under Simmons-Smith
Scheme 1.

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INHIBITION OF (CYTOSINE C5)-METHYLTRANSFERASE
455
Scheme 2.
conditions to give the North-type intermediate 12. Deprotection of the benzyl group,
followed by dimethoxytritylation provided compound 14, which was converted to
the North-type analogue phosphoramidite 15 by the standard methodology.
A synthetic method for the preparation of cyclopentane phosphoramidite 18
has been reported (16). However, as outlined in Scheme 3, a simpler approach from
1 provided the same material.
Phosphoramidites 5, 15 and 18 were used in a conventional DNA synthe-
sizer to prepare the corresponding 13-mers containing the South- (ODN-South),
the North- (ODN-North) and the cyclopentane (ODN-cyclopentane) abasic sites.
The sequence of the target oligos synthesized was 5ꢁ-TGTCAGXGCATGG-3ꢁ
where X corresponds to the abasic sites shown in Figure 1. The coupling yields
for the modified abasic pseudosugars were >95% which represents a significant
improvement from the coupling yields obtained in our first approach (17). All 13-
mer ODNs were annealed with equimolar amounts of the complementary strand
containing the hemimethylated HhaI site (GMGC where M = 5-methylcytosine)
after heating at 90◦C for ten minutes and slowly cooling to room temperature. The
three double-stranded ODNs gave similar melting curves (not shown) with almost
identical Tm values: ODN-South (Tm = 38.2◦C), ODN-North (Tm = 39.6◦C)
and ODN-cyclopentane (Tm = 37.1◦C). Irrespective of their chemical nature, the
presence of an abasic site strongly destabilizes the duplex relative to control ODN

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MARQUEZ ET AL.
Scheme 3.
(X = cytosine, Tm = 56.1◦C) due to the loss of base stacking and Watson-Crick hy-
drogen bonding. The Tm for the ODN-cyclopentane was virtually indistinguishable
from that of the THF-containing ODN (17).
INHIBITION OF M.HhaI METHYLASE
Inhibition assays were performed as described (17) with reaction mixtures
containing 0.5 µg of each ODN, ∼0.6 unit of DNA M.HhaI (1 unit transfers 1 pmol
to the substrate per min) and 2.8 µCi (1Ci = 37 GBq) of [methyl-3H] AdoMet (8
µM). Substrate = AMp:A’; A = 5ꢁ-ATTGMGCATTCMGGATCMGMGATC-3ꢁ
and Aꢁ = 3ꢁ-TAACGCGTAAGGCCTAGGCGCTAG-5ꢁ (target C underlined).
The inhibition curves for M.HhaI transferase activity exhibited by the three
abasic ODNs (Fig. 3) shows that ODN-South has the highest inhibitory activity
(IC50 of 14 nM) followed by ODN-cyclopentane (IC50 of 48 nM). ODN-North, on
the other hand, had no detectable activity below 75 nM. The inhibitory activity of
the flexible ODN-cyclopentane falls between that of ODN-South and ODN-North
and is indistinguishable from that measured for ODN-THF (17).
Native gel-shift assays with M.HhaI (not shown) demonstrated that the ODN-
South was able to form a “closed” complex with the enzyme even in the absence
of cofactor (AdoMet or AdoHcy), whereas the ODN-North remained mainly in the
“open” form (17). Although the binding of ODN-South and ODN-THF appeared
essentially irreversible, ODN-South binds tighter. Indeed, it takes more than 110 h
for 50% dissociation of ODN-South versus 55 h for ODN-THF to dissociate from
M.HhaI when incubated in the presence of a 100-fold excess of cold competitor
ODNs (17).

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INHIBITION OF (CYTOSINE C5)-METHYLTRANSFERASE
457
Figure 3. Inhibition of methyltransferase activity of M.HhaI by ODNs containing abasic sites.
THE FLIPPING MECHANISM
Investigation of the influence of a conformationally locked pseudosugar struc-
ture on the torsional properties of the γ dihedral was performed via ab initio calcu-
lations (Gaussian 94 or Gaussian 98 programs with the HF/6-31+G∗ basis set) of
model compounds A, B, and C (Fig. 4). Since the behavior of the ODN-THF and
ODN-cyclopentane was identical, only the THF moiety was studied as a flexible
abasic site (17).
The γ dihedral energy surface (Fig. 5) for the three abasic sugar moieties was
obtained by constraining the C3ꢁ-C4ꢁ-C5ꢁ-O5ꢁ dihedral in 30◦ increments from 0◦ to
360◦. In all cases, the following additional constraints were included: C4ꢁ-C5ꢁ-O5ꢁ-
P = 168◦, C5ꢁ-O5ꢁ-P-O = 298◦, O5ꢁ-P-O-C = 262◦, and C4ꢁ-C3ꢁ-O3ꢁ-H = 187◦,
Figure 4. Model compounds to study the γ dihedral energy surface in the abasic moieties.

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MARQUEZ ET AL.
Figure 5. Potential energy as a function of the γ dihedral (ꢀ = model A (THF); ꢁ = model B
(North); ꢂ = model C (South).
while the remainder of the molecule was allowed to relax. These constraints corre-
spond to modal values of the β, α, ζ and ε crystallographic dihedral distributions in
B-DNA (12), and are introduced to prevent variations in the other dihedral degrees
of freedom from interfering with the γ energy surface, as previously performed
(18). Due to the flexibility of the THF ring and to allow for a systematic analysis
of the effect of sugar conformation on the behavior of γ , additional ring constrains
were applied to C3ꢁ-C4ꢁ-O4ꢁ-C1ꢁ to explicitly sample the C2ꢁ-endo (South) and
C3ꢁ-endo (North) conformations in A.
Comparison of the three surfaces (Fig. 5) shows the structure of the sugar
moiety to significantly influence the energetic properties. Common to all surfaces
are the three minima that occur around 60◦, 180◦ and 300◦, corresponding to the
+sc, ap and −sc states, respectively. The +sc minimum corresponds to the con-
formation observed in experimental DNA duplex structures (12). For the North
model compound B, the +sc to −sc barrier (ca. 6 kcal/mole) is higher than the
+sc to ap barrier (ca. 4 kcal/mole) and, more importantly, the energy of the −sc
minimum is ca. 6 kcal/mol greater than that of both the THF and South model
compounds, A and C, respectively. These energy differences are suggested to dis-
allow the transition to the −sc conformation for γ in the North DNA system and
prevent flipping. What these results suggest is that flipping probably begins with
the torsion angle γ rotating to the −sc state and proceeding to ap state, which
corresponds to the conformation observed in the crystal structure of the ternary
complex (11). Both of these transitions are permissible for the flexible THF and the
rigid South conformer, and are energetically more favorable than a direct +sc to ap
rotation. After the conclusion of the flipping event, the sugar is able to readjust its
pucker to a North conformation only in the case of the flexible THF. This obviously
cannot occur with the rigid South conformer and suggests that changes in pseu-

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INHIBITION OF (CYTOSINE C5)-METHYLTRANSFERASE
459
dorotation after the flipping event is concluded might be irrelevant for the stability
of the final “closed” conformation of the complex.
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