Synthesis and characterization of an O6-2′-deoxyguanosine- alkyl-O6-2′-deoxyguanosine interstrand cross-link in a 5′-GNC motif and repair by human O6-alkylguanine-DNA alkyltransferase

Article abstract of DOI:10.1039/c0ob00093k

O⁶-2′-Deoxyguanosine-alkyl-O⁶-2′- deoxyguanosine interstrand DNA cross-links (ICLs) with a four and seven methylene linkage in a 5′-GNC- motif have been synthesized and their repair by human O6-alkylguanine-DNA alkyltransferase (hAGT

Full text of DOI:10.1039/c0ob00093k

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and characterization of an O6-2¢-deoxyguanosine-alkyl-O6-2¢-
deoxyguanosine interstrand cross-link in a 5¢-GNC motif and repair by human
O6-alkylguanine-DNA alkyltransferase†
Francis P. McManus,a Qingming Fang,b Jason D. M. Booth,a Anne M. Noronha,a Anthony E. Peggb and
Christopher J. Wilds*a
Received 7th May 2010, Accepted 28th June 2010
DOI: 10.1039/c0ob00093k
O6-2¢-Deoxyguanosine-alkyl-O6-2¢-deoxyguanosine interstrand DNA cross-links (ICLs) with a four
and seven methylene linkage in a 5¢-GNC- motif have been synthesized and their repair by human
O6-alkylguanine-DNA alkyltransferase (hAGT) investigated. Duplexes containing 11 base-pairs with
the ICLs in the center were assembled by automated DNA solid-phase synthesis using a cross-linked
2¢-deoxyguanosine dimer phosphoramidite, prepared via a seven step synthesis which employed the
Mitsunobu reaction to introduce the alkyl lesion at the O6 atom of guanine. Introduction of the four
and seven carbon ICLs resulted in no change in duplex stability based on UV thermal denaturation
experiments compared to a non-cross-linked control. Circular dichroism spectra of these ICL duplexes
exhibited features of a B-form duplex, similar to the control, suggesting that these lesions induce little
overall change in structure. The efficiency of repair by hAGT was examined and it was shown that
hAGT repairs both ICL containing duplexes, with the heptyl ICL repaired more efficiently relative to
the butyl cross-link. These results were reproducible with various hAGT mutants including one that
contains a novel V148L mutation. The ICL duplexes displayed similar binding affinities to a C145S
hAGT mutant compared to the unmodified duplex with the seven carbon containing ICLs displaying
slightly higher binding. Experiments with CHO cells to investigate the sensitivity of these cells to
busulfan and hepsulfam demonstrate that hAGT reduces the cytotoxicity of hepsulfam suggesting that
the O6-2¢-deoxyguanosine-alkyl-O6-2¢-deoxyguanosine interstrand DNA cross-link may account for at
least part of the cytotoxicity of this agent.
implicated in the removal of the damage.3 Understanding the
Introduction
molecular basis of how ICL repair occurs will play an important
Interstrand cross-links (ICLs) that are formed in DNA as a
role towards the development of new chemotherapeutic agents
consequence of the action of bifunctional alkylating agents
that may evade this process, thus increasing the efficacy of these
represent some of the most toxic lesions encountered by cells due
drugs.
to the obstruction of unwinding of the two strands, critical to the
One approach employed in elucidating the roles various DNA
processes of DNA replication, transcription and recombination.1
repair pathways contribute in removing DNA ICLs involves
Interference with these key cellular processes by the presence of
the use of chemically synthesized oligonucleotides that con-
ICLs is the basis of the mechanism of action of bifunctional
tain representative lesions formed by bifunctional alkylating
chemotherapeutics.4,5,6 These oligonucleotides are designed to
contain lesions linking specific atoms in DNA in well defined
orientations and can be incorporated into plasmids for DNA
repair experiments. This approach uses solid-phase synthesis
which generates sufficient amounts of ICL DNA to enable
structural studies and repair assays.7,8,9,10,11
Some of these ICLs are challenging to prepare synthetically.
For example, the bifunctional alkylating agent hepsulfam (1,7-
heptanediol disulfamate) which has been investigated clinically,12
has been shown to form a cross-link between the N7 atoms
of guanines in 5¢-GNC sequences (Fig. 1) as demonstrated
through the use of mass spectrometry and identification of
1,7-bis(guanyl)heptane.13 N7-alkylated guanines are chemically
unstable, for example the presence of N7-methylguanine can result
in an apurinic site in DNA or undergo ring opening to yield the
ring opened formamido pyrimidine (Fapy) derivative.14,15
alkylating agents, such as mechlorethamine, that are used as cancer
therapeutics.2 However, the potency of these agents is reduced by
the ability of cancer cells to repair the lesions resulting in an overall
resistance to therapy.
In eukaryotic cells the repair of ICLs is a complex process with
numerous repair pathways including nucleotide excision repair,
homologous recombination and non-homologous end joining
aDepartment of Chemistry and Biochemistry, Concordia University, 7141
Sherbrooke St. West, Montre´al, QC, Canada, H4B 1R6. E-mail: cwilds@
alcor.concordia.ca; Fax: 514-848-2868; Tel: 514-848-2424 ext. 5798
bDepartments of Cellular and Molecular Physiology and Pharmacology, The
Pennsylvania State University College of Medicine, PO Box 850, Hershey,
PA, USA, 17033
† Electronic supplementary information (ESI) available: 1H NMR and 31
P
NMR of compounds 6a, 6b, 7a and 7b, HPLC chromatographs and MS
spectra for oligonucleotide characterization and repair and binding data
of hAGT to the ICL. See DOI: 10.1039/c0ob00093k
4414 | Org. Biomol. Chem., 2010, 8, 4414–4426
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transfer of the alkyl group from the O6 position of guanine to a
C145 residue occurs.22 The protein can only act once due to the
irreversible alkylation of C145, whereupon the protein is degraded
by the ubiquitin pathway.23 hAGT has been shown not only to
repair small adducts at the O6 position of guanine such as a methyl
group but also larger groups such as benzyl and 4-(3-pyridyl)-4-
oxobutyl.24 The study of these novel ICL substrates to undergo
repair by hAGT will contribute to the range of DNA substrates
that can undergo repair via this pathway.
Results and discussion
Syntheses and characterization of the ICL duplexes
Fig. 1 Structures of the (a) N7-2¢-deoxyguanosine-heptyl-N7-2¢-deoxyg-
uanosine cross-link induced by hepsulfam, (b) the O6-2¢-deoxyguanosine-
alkyl-O6-2¢-deoxyguanosine cross-link (where n = 3 or 6) and oligomers (c)
XL4 and XL7.
Interstrand cross-linked DNA repair studies require access to
substrates of well defined structure. The bifunctional alkylating
agent hepsulfam has been shown to alkylate specifically at the
N7 atom of 2¢-deoxyguanosine in sequences containing a 5¢-
GNC motif placing a heptyl linkage between the two guanines.13
Because they are synthetically challenging to prepare, we have
focused on the synthesis of a cross-link that joins the O6 atoms
in this particular sequence motif. It should be emphasized that
this specific cross-link has not been identified as a product
of alkylation by hepsulfam. The methodology to produce this
ICL was used to prepare duplexes containing alkyl linkages of
various lengths to investigate the affect of linker length on ICL
stability, structure and repair by hAGT. The chemotherapeutic
agent busulfan (1,4-butanediol dimethanesulfonate) is another
example of a bifunctional alkylating agent that has been used
for the treatment of chronic myelogenous leukemia.25 This agent
introduces a butyl linkage between the nucleobases with the
demonstration that this agent reacts with guanosine to form
1,4-di(7-guanosinyl)butane.26 Mass spectrometry of the reaction
products of busulfan with oligonucleotides suggested that this
agent forms intrastrand cross-links with 5¢-GA-3¢ sequences.27
The structure of the O6-2¢-deoxyguanosine-alkyl-O6-2¢-
deoxyguanosine cross-link and the sequences containing the
butyl and heptyl cross-links (XL4 and XL7) are shown in
Fig. 1. The synthesis of the cross-linked amidites 7a and 7b
were performed according to the synthetic pathway in Scheme
1. Starting from commercially available 5¢-O-dimethoxytrityl-
N2-phenoxyacetyl-O6-2¢-deoxyguanosine the free 3¢-alcohol was
protected as an allyloxycarbonyl ester 1 using allyl 1-benzotriazoyl
carbonate.28 The adducts 2a and 2b were prepared by introduction
of 1-tert-butyldiphenylsilyloxy-butan-4-ol or 1-tert-butyldiphenyl
silyloxy-heptan-7-ol at the O6 position of 1 via the Mitsunobu
reaction.18,20 Removal of the tert-butyldiphenylsilyl protecting
group on compounds 2a and 2b was accomplished with TBAF
at room temperature for 30 min to yield the adducts 3a and
3b. The formation of dimers 5a or 5b were achieved via a
second Mitsunobu reaction using 5¢-O-dimethoxytrityl-3¢-O-tert-
butyldimethyl-silyl-2¢-deoxyguanosine (4) in yields of 63 and
71%, respectively. Amidite precursors 6a and 6b required the
removal of the 3¢-O-alloxycarbonyl group from 5a or 5b with
palladium (0) tetrakistriphenylphosphine in THF at room tem-
perature. These were then converted to phosphoramidites 7a and
7b using a slight excess of N,N-diisopropylamino cyanoethyl
phosphonamidic chloride and isolated by hexane precipitation.
The isolated phosphoramidites 7a and 7b were analyzed by mass
In order to prepare oligonucleotides containing a cross-link that
models the orientation of the lesion formed by hepsulfam, our
group has explored methodologies to prepare ICL DNA that links
the O6 atoms of 2¢-deoxyguanosine.16 The O6 atom of guanine is
a known site of alkylation by chemotherapeutic drugs such as the
methylating agent temozolomide and the chloroethylating agent
carmustine which proceeds to form a guanine-cytosine ICL.17
Different chemical groups at the O6 position of guanine have
been incorporated by organic synthesis for numerous purposes
including protection during oligonucleotide synthesis and as sub-
strates for DNA repair studies.18,19 Examples of synthetic methods
that have been employed to introduce these groups include the
post-synthetic modification of oligonucleotides containing the 2¢-
deoxyribonucleoside of 2-amino-6-methylsulfonylpurine and the
Mitsunobu reaction.19,20
Using a combination of solution and solid-phase synthesis,
a 5¢-GNC ICL has been prepared (Fig. 1) which required
a O6-2¢-deoxyguanosine-alkyl-O6-2¢-deoxyguanosine phosphor-
amidite that contains various protecting groups to permit or-
thogonal removal, enabling a unit of asymmetry to be engineered
in the final ICL DNA duplex to allow for a clinically relevant
staggered 1,3 orientation. The synthetic scheme to produce this
amidite involved the Mitsunobu reaction which was selected due
to its mild conditions, compatibility with all protecting groups
employed and direct route to introduce alkyl linkers of various
lengths. This method allows for the production of ICL duplexes
to explore the influence of linker length in substrates of defined
structure and for the systematic investigation of susceptibility to
DNA repair mechanisms.
The ability of wild-type human O6-alkylguanine-DNA alkyl-
transferase (hAGT) and some mutants to repair these ICLs was
also investigated. O6-Alkylguanine-DNA alkyltransferases (AGT)
are a class of repair proteins that are found in numerous organisms
whose role is to repair alkylation at the O6 position of the
guanine base and thus they play an important role in maintaining
genomic integrity.21 Alkylation at the O6 position of guanine can be
mutagenic, disrupting normal Watson–Crick base pairing leading
to point mutations which may be fatal. The mechanism by which
hAGT repairs alkylated DNA involves flipping the alkylated base
out of the duplex and into the active site of the protein where
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Scheme 1 Synthesis of O6G-alkyl-O6G amidite 7a (n = 2) and 7b (n = 5). (i) Alloc-OBt, CH2Cl2/pyridine (9 : 1), 12 h; (ii) 4-(tert-butyldiphenylsiloxy)
butanol or 7-(tert-butyldiphenylsiloxy) heptanol (1.1 eqv.), PPh3 (1.25 eqv.), DIAD (1.2 eqv.) (iii) TBAF (1 M in THF), 30 min; (iv) 4 (1.01 eqv.),
PPh3 (1.25 eqv.), DIAD (1.2 eqv.), 1 h; (v) Pd(PPh3)4 (5 mol%), PPh3 (0.25 eqv.), n-butylamine/formic acid (1 : 1, 2.5 eqv.), THF, 20 min; (vi)
N,N-diisopropylamino cyanoethyl phosphonamidic chloride, DIPEA, THF, 30 min.
spectrometry and were found to have the expected molecular
masses. 31P NMR analysis of these phosphoramidites revealed
the presence of two signals for 7a (143.92 and 144.14 ppm)
and 7b (143.91 and 144.11 ppm) in the region diagnostic for a
phosphoramidite.
The synthesis of the 1,3-GNC engineered ICL using phospho-
ramidite dimers 7a or 7b required three different protecting groups
around the cross-linked site enabling an orthogonal approach for
their removal. This would allow for the synthesis of three different
branches in terms of sequences around the site of the ICL. The
Mitsunobu reaction was chosen for its specificity at the O6 position
Fig. 2 Methodology to construct the cross-linked duplexes XL4 and XL7
via solid phase synthesis: (i) coupling of amidite 7a or 7b; (ii) extension with
3¢-O-2¢-deoxyphosphoramidites followed by capping with phenoxyacetic
anhydride; (iii) removal of the 3¢-O-tert-butyldimethylsilyl group with
of a protected 2¢-deoxyguanosine18,20 and the versatility of this
method which allows for the synthesis of cross-links of various
lengths, depending on the diol linker used. This reaction was
employed twice in the synthetic pathway outlined in Scheme 1,
TEA·3HF; (iv) extension with 5¢-O-2¢-deoxyphosphoramidites and (v)
cleavage from the solid support.
first for the synthesis of the monomers 2a and 2b containing the
four and seven carbon linkers at the O6 atom and later in the
scheme for dimers 5a and 5b. The dimerization reaction to give
dimer 5b proceeded in a slightly higher yield relative to 5a (71
versus 63%), presumably due to the reduced steric hindrance of
the longer alkyl linker of 3b that reacts with 4.
repetitive coupling of 5¢-O-2¢-deoxyphosphoramidites at the 3¢-
end of intermediate Y-OH gave full length ICL duplexes XL4 and
XL7.
Solid phase synthesis of XL4 and XL7 was performed on
a 1 mmol scale using phosphoramidites 7a or 7b according
to Fig. 2. The first arm of the duplex was synthesized on
a polystyrene support using commercially available 3¢-O-2¢-
deoxyphosphoramidites. Coupling of the cross-linked phospho-
ramidites 7a or 7b introduced the ICL at the 5¢-ends to the linear
oligomers. The dimethoxytrityl groups of the dimer moiety were
removed by brief treatment with 3% TCA in methylene chloride to
expose the 5¢-OH groups from which the second and third arms of
the duplex were then synthesized with a noted increase in the trityl
conductivity reading on the synthesizer. Repetitive coupling with
protected 3¢-O-2¢-deoxyphosphoramidites gave, after detritylation
and capping (phenoxyacetic anhydride), the branched oligomer
Y-TBS (Fig. 2). The 3¢-O-TBS group was then removed from Y-
TBS by treating the support with anhydrous triethylamine for 16
h followed by TEA·3HF twice for 30 min at room temperature.
C-18 Reversed-phase HPLC analysis of the deprotected interme-
diate Y-OH revealed complete removal of the silyl group with the
shift of the major peak from 11.2 to 8.2 min in the case of the
butyl containing cross-link (see ESI†). Continued synthesis with
The solid-phase synthesis of the ICL containing oligonu-
cleotides XL4 and XL7 required some changes from the stan-
dard synthesis cycle and reagents employed during solid-phase
oligonucleotide synthesis. First, synthesis was performed using a
polystyrene rather than controlled-pore glass (CPG) solid-support
due to the incompatibility of TEA·3HF with the latter.5 As an
added precaution, the cyanoethyl protecting groups were removed
using TEA as it has been observed that prolonged fluoride treat-
ment using TBAF could lead to chain cleavage.29 Phenoxyacetic
anhydride rather than acetic anhydride was used as capping
reagent due to a undesirable N-acetylation reaction that has
been observed with ‘fast-deprotecting’ amidites.30 Additionally,
the protection of the exocyclic amine functionalities of the cross-
linked guanosine bases with the phenoxyacetyl group would allow
for milder deprotection conditions and ease of its removal at the
final stage.
Chain assembly in the 3¢ to 5¢ direction proceeded smoothly
using 3¢-O-deoxyphosphoramidites and the cross-linked dimers
7a and 7b with essentially quantitative coupling observed by
conductivity measurements to assess the trityl values. A higher
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Table 1 Amounts, retention times, nucleoside ratios and mass spectral
data for cross-linked duplexes XL4 and XL7
ICL duplexes XL4 and XL7. The ICL oligomers XL4 and
XL7 required additional time for digestion compared to single
stranded oligonucleotides (48 h as opposed to 30 min). ESI mass
spectrometry of the duplexes XL4 and XL7 had masses of 6726.8
and 6769.0 Da, consistent with the theoretical values for the cross-
linked duplexes.
Ratios
Mass
Retention
Duplex A260 Unitsa timeb
Nucleoside exp obs exp
obs
XL4
141.8 (36.9) 23.7
dC
dG
6.0 6.1 6727.5 6726.8
4.0 4.2
dT
dA
5.0 5.0
5.0 5.1
UV thermal denaturation and circular dichroism spectra (CD) of
ICL duplexes
dG-dG
dC
dG
1.0 1.1
6.0 5.8 6769.6 6769.0
4.0 4.1
XL7
108.6 (39.1) 25.5
dT
dA
dG-dG
5.0 5.0
5.0 5.1
1.0 1.1
UV denaturation experiments were carried out to assess the
effect of the alkyl linkers on duplex stability. The UV thermal
denaturation (Tm) curves for the ICL duplexes XL4, XL7, and
the corresponding non-cross-linked control duplex all exhibited a
sigmoidal denaturation profile with similar hyperchromicities for
the transition curves (see ESI†). The melting temperature for XL4,
XL7 and the non-cross-linked control were all similar with a value
of ~48 ◦C.
a Amount of crude ICL duplex purified by SAX HPLC. The numbers
in parentheses indicate the amount of pure duplex obtained. b Retention
times (min) of ICL duplexes on SAX HPLC using a 0.0–0.5 M linear
gradient of sodium chloride.
When this ICL was present as a directly opposed O6-2¢-
deoxyguanosine-heptyl-O6-2¢-deoxyguanosine mismatch in an 11-
bp duplex, an increase in Tm of 23 ◦C over the control duplex
was observed.16 UV thermal denaturation experiments with
oligonucleotides containing an O6-methyl-2¢-deoxyguanosine/2¢-
deoxycytosine base pair have shown that a single O6-methyl
substitution reduced duplex stability by at least 18 ◦C per
substitution in 1 M NaCl buffer and DNA duplexes containing
two of these alkylated lesions were found to have a Tm 40 ◦C lower
than that of the unmodified duplex.32,33,34 It is believed that the
destabilizing nature of the O6-alkyl lesions are counterbalanced
by the preorganization of the covalently linked strands of the
duplex resulting in a stability comparable to the non-cross-linked
control.5,16
The CD spectra of cross-linked duplexes XL4, XL7 and the non-
cross-linked control were recorded at 10 ◦C (spectra are shown
in ESI†). In all cases, the CD spectra of the duplexes exhibited
signatures characteristic of B-form DNA with a positive maximum
peak centered around 275 nm, a negative peak at approximately
250 nm and a cross over around 260 nm.35,36 The CD spectra
of non-cross-linked controls revealed some minor differences,
particularly a reduction of the signal at 275 nm.
concentration (0.15 M) and longer coupling time (10 min) was
used for phosphoramidites 7a and 7b to ensure a high coupling
efficiency due to their larger size compared to the standard 3¢-
O-deoxyphosphoramidites. At this point, capping with phenoxy-
acetyl anhydride gave intermediate Y-TBS (Fig. 2) which was
desilylated to give the free 3¢OH functionality. The efficiency of
this step was readily monitored by C-18 reversed phase HPLC
(see ESI†). A small amount (approximately 1 mg) of the solid
support was deprotected using NH4OH/ethanol (3 : 1), milder
conditions that have been developed for the deprotection of RNA
to ensure that the TBS groups are not removed.31 The removal
of the TBS group decreases the hydrophobicity of the oligomer
and a change in the retention time for the oligomer from 11 to
8 min on the C-18 reverse phase HPLC indicates removal to give
the more hydrophilic Y-OH intermediate (Fig. 2). The final step
in the assembly of XL4 and XL7 is continued chain extension
with 5¢-O-2¢-deoxyphosphoramidites. Cleavage and deprotection
of XL4 and XL7 were performed with concentrated ammonium
hydroxide/ethanol (3 : 1) at 55 ◦C for 4 h to yield 141.8 and 108.6
OD of material, respectively (see Table 1). Analysis by SAX HPLC
analysis revealed that the major product was the desired ICL
whose retention times are shown in Table 1 (see ESI for HPLC
traces of crude and purified material†). The combination of mild
deprotection conditions and bulky nature of the adduct that the
ICL represents at the O6 position may account for the stability
under these deprotection conditions. Purification by SAX HPLC,
followed by desalting gave XL4 and XL7 in approximately 26 and
36% overall isolated yields.
These results suggests that the cross-links had minimal effect
on the global B-form structure. NMR studies performed with
dodecanucleotides containing a O6meG·C base pair implicate the
formation of a wobble base pair, with the methylated guanine
sliding towards the major groove and the cytosine oriented towards
the minor groove. This base pair is stacked in the duplex between
the flanking base pairs inducing only a small conformational
change from the wild-type duplex.37 In a standard B-form duplex,
the distance between the O6–O6 atoms in a 5¢-GNC sequence
These ICL duplexes were digested to the constituent nucleosides
with a combination of snake venom phosphodiesterase and calf
intestinal phosphatase and analyzed by C-18 reversed phase
HPLC (see ESI for HPLC traces†). In addition to the four
standard 2¢-deoxynucleosides, one additional peak was observed
with a retention time of 18.1 min for XL4 and 25.5 min for
XL7. These additional peaks had retention times identical to
completely deprotected dimers of 6a and 6b. As shown in Table 1,
the ratios of the component 2¢-deoxynucleosides and cross-linked
nucleosides were consistent with the theoretical composition of
˚
is approximately 6.4 A. The O–O distance in the fully extended
˚
1,4-butanediol and 1,7-heptanediol is 6.2 and 9.9 A, respectively,
which is sufficient to span the distance linking the two O6 atoms in a
5¢-GNC sequence motif without inducing a significant structural
change. Molecular models for the oligomers XL4, XL7 and the
non-cross-linked control duplex that were geometry optimized
using the AMBER forcefield suggest that the alkyl linkers are
oriented towards the major groove and do not greatly distort the
duplex (see ESI†).
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ICL repair assay with hAGT
virtually identical results obtained, indicating that the reaction
was complete as monitored by denaturing PAGE (Fig. 3). Lanes
1 and 2 contain 2 pmol of ssDNA corresponding to the sequence
of one of the repaired strands with the only difference being the
presence of hAGT in lane 2. Lanes 3 and 8 contain 2 pmol of XL4
and XL7, respectively, representing the unrepaired ICL duplexes
with a reduced mobility relative to that of completely repaired
substrate. Lanes 4 and 9 contain 60 pmol of hAGT with 2 pmol of
either XL4 (lane 4) or XL7 (lane 9). In both cases two additional
bands are seen, one that migrates more quickly corresponding
to the completely repaired single stranded product and the other
at the top of the gel with significantly reduced mobility which is
likely a single hAGT-bound covalently to a DNA species, a median
repair product. For both XL4 and XL7, no repair products are
observed with 60 pmol of the mutants C145S (lanes 5 and 10),
while P140A (lanes 6 and 11) or V148L (lanes 7 and 12) show
very minimal repair of XL7 and no repair of XL4. The repair
assays demonstrate that both XL4 and XL7 are repaired by hAGT
with XL7 undergoing a greater amount of repair (with 57.0%
of ICLs repaired) over XL4 (31.4% of ICLs repaired) based on
quantitation of ICLs remaining as measured by the relative counts
of the labelled products present on the gel by ImageQuantTM (see
ESI†).
Repair assays were performed on the ICLs containing oligomers
XL4 and XL7 with wild-type hAGT and the mutants C145S,
P140A and V148L. The C145S mutant was selected as a negative
control due to its lack of alkyltransferase activity and for the
investigation of protein binding to the O6-dG-alkyl-O6-dG ICLs.38
The P140A mutation has been investigated for the influence of
reduced size of the active site pocket on hAGT activity.39,40 In
addition, some AGTs such as the E.coli Ada-C, have alanine in
place of proline in the equivalent position of hAGT. The V148L
mutant was engineered to probe the effect of the side chain of V148
on the repairing ability of the protein. This mutant was selected
as little is known about this amino acid other than the carbonyl
group of V148 has been shown in wild-type hAGT to accept a H-
bond from the N2 atom of a bound guanine base.22,41 We suspected
that substituting Val to a larger amino acid would reduce the size
of the active site pocket yielding a protein with the same properties
as P140A.
All protein (wild-type hAGT, C145S, P140A and V148L) masses
were verified by ESI-MS and were in agreement with the calculated
values (see ESI†). The secondary structure of the proteins by
far UV CD was examined to ensure that the mutations did not
compromise the structure. CD scans of the P140A and V148L
hAGT mutants revealed similar secondary structures to the wild-
type hAGT due the highly conservative nature of these mutations
(see ESI†). The C145S mutation had a greater impact on the
global secondary structure of the protein. Although the size of
the cysteine and serine side groups are relatively similar, the H-
bonding capabilities of these two amino acids differ.
The stability of the proteins (wild-type hAGT, C145S, P140A
and V148L) were assessed via thermal denaturation studies
to ensure that the proteins were properly folded at the assay
temperature (37 ◦C) by monitoring the a-helical content of the
protein at 222 nm as a function of increasing temperature. The
thermal denaturation studies demonstrated that all hAGT proteins
were still properly folded at 37 ◦C due to their Tm’s being well
above 37 ◦C. Thermal denaturation studies of the wild-type hAGT
correlated with values reported for complete inactivation of the
◦
protein at 60 C.42 All mutations destabilized the protein based
Fig. 3 Repair of XL4 and XL7 by wild-type hAGT, C145S, P140A
and V148L. Denaturing PAGE of repair reactions as described in the
experimental section: Lane 1, 2 pmol Control; lane 2, 2 pmol Control + 60
pmol hAGT; lane 3, 2 pmol XL4; lane 4, 2 pmol XL4 + 60 pmol hAGT;
lane 5, 2 pmol XL4 + 60 pmol C145S; lane 6, 2 pmol XL4 + 60 pmol
P140A; lane 7, 2 pmol XL4 + 60 pmol V148L; lane 8, 2 pmol XL7; lane
9, 2 pmol XL7 + 60 pmol hAGT; lane 10, 2 pmol XL7 + 60 pmol C145S;
lane 11, 2 pmol XL7 + 60 pmol P140A; lane 12, 2 pmol XL7 + 60 pmol
V148L.
on CD spectra where the stability of the secondary structure of
the protein was compromised (see ESI†). Presumably, the presence
of an additional methylene group in the V148L mutant affected
protein stability negatively while the P140A mutant exhibited a
smaller entropy increase upon folding for the alanine versus proline
substitution leading to a reduced protein stability.
The intrinsic fluorescence of the hAGT proteins were inves-
tigated by monitoring the local environments containing both
tryptophan and tyrosine residues using an excitation wavelength
of 280 nm while in the case of tryptophan alone the excitation
wavelength of 295 nm was used. It is clear from the emission
wavelengths and intensities that the mutations had no dramatic
affect on the tertiary structure of the protein with V148L having
a slight impact on tertiary structure of the protein relative to the
other mutants (see ESI†).
For the repair assays involving XL4 and XL7, the samples were
denatured in stop reaction buffer before being loaded on a 7 M
urea denaturing gel to prevent self-complexation of the repaired
DNA strands which could complicate determination of ratios of
the repaired intermediate and fully repaired product versus intact
(unrepaired) ICLs (Scheme 2).
The inactive C145S mutant was unable to repair both ICL
duplexes as expected due to the lack of the active site Cys residue
which is known to be the alkyl group acceptor. The other P140A
and V148L mutants showed no repair of XL4 and very poor
The 5¢-32P labeled ICL DNA duplexes XL4 and XL7 (2 pmol)
were incubated with 60 pmol of either the wild-type hAGT or
the mutants (C145S, P140A and V148L) overnight at 37 ◦C to
determine repair. Trials for either 14 or 16 h were performed with
4418 | Org. Biomol. Chem., 2010, 8, 4414–4426
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Table 2 Hill factor and monomeric Kd values of C145S hAGT-DNA
complexes
DNA
Cooperativity Factor
Kd /mM
Control
XL4
XL7
1.41 0.06
1.75 0.13
1.32 0.06
8.36 1.11
9.87 1.22
9.67 1.12
hAGT preferentially repaired the median product over the initial
substrate the median product should not increase with time as the
median product would be consumed as it would be formed.
One possible explanation as to why this reaction does not go
to completion is that the reacted hAGT binds to another lesion
containing ICLs or oligonucleotide in our assay. It is known that
alkylated hAGT can also bind to DNA.45 In vivo, dissociation is
most likely reinforced by ubiquitination resulting in degradation
of the alkylated hAGT.46 In addition, it is possible that the cross-
link may be oriented in an alternate, less reactive conformation,
as has been postulated for O6-(2-hydroxyethyl)guanine and O6-[4-
oxo-4-(3-pyridyl)but-1-yl]guanine containing oligonucleotides.47
Scheme 2 Proposed repair pathway of cross-link species by wild-type
hAGT, P140A and V148L (MP - median product; FP- final product).
repair for XL7 (4.5% and 3.0%, respectively). The diminished
capacity for repair by the P140A mutant has been observed as
it is much less active than wild-type hAGT in dealkylating O6-
benzylguanine, which has been explained by the reduced size of
the binding pocket.43
Time course assays were performed using 60 pmol wild-type
hAGT and 2 pmol of the XL4 and XL7 substrates by quantitating
the amount of unrepaired, partially and fully repaired species (see
ESI†). XL4 was repaired at a much slower rate with 25% repair
of the ICLs occurring in 8.5 h whereas the same level of repair for
XL7 required approximately 1 h.
Binding studies of C145S hAGT mutant using protein titration
Binding cooperativity (Hill factor) and monomeric Kd values
were determined for the non-cross-linked control duplex, XL4
and XL7 with the C145S hAGT mutant (see Table 2 and ESI†).
Similar cooperativity values were obtained for the control duplex
(1.41 0.06) and XL7 (1.32 0.06) whereas this value was found
to be slightly higher for XL4 (1.75 0.13). All three DNA duplexes
showed a similar monomeric dissociation constant ranging from
8.36 for the control duplex to 9.87 mM for XL4 (see ESI).
Experiments to measure cooperativity and dissociation con-
stants for XL4 and XL7 showed only two bands by EMSA
corresponding to the free and bound ICL DNA. The absence
of an intermediate band on the native gel indicates that there is
cooperativity in the binding of hAGT for the ICL DNA. XL7 and
the control duplex showed similar cooperativity and dissociation
constants indicating that the presence of the 7-methylene cross-
link had little influence on hAGT interaction with the DNA.
XL4 had a slightly higher cooperativity of interaction with hAGT,
however, all the measured Hill factors for ICL duplexes XL4 and
XL7 were between 1 and 2 in accordance with previous work
which showed that 1 hAGT protein binds every 4 nucleotides.48
A stoichiometry value of 2 indicates perfect cooperativity, as
previously shown in the case of hAGT for many oligonucleotide
sequences under different assay conditions whereas a value of 1
indicates no cooperativity.48
In the absence of NMR or X-ray structural data for XL4 or
XL7, it can only be speculated that the 4-methylene cross-link
induces a structural change in the DNA that has an influence on
the interaction of the ICL with hAGT contributing to the slight
increase in cooperativity that is observed. It is known that binding
of hAGT to DNA causes structural changes in the DNA, which
may play a role in aiding the rotation of the damage base from the
DNA double helix and subsequently promote hAGT cooperative
binding.22,49 However, the shorter alkyl linker of XL4 versus XL7
may hinder rotation of the alkylated guanine into the active site
contributing to a reduction in repair by hAGT.
The results obtained from the time course experiments for
the 5¢-GNC-3¢ ICLs for XL4 and XL7 follow similar trends to
repair results for the directly opposed O6-dG-alkyl-O6-dG ICL
in a mismatch duplex.44 It was shown that 50% final product
was formed after 2 h for a directly opposed O6-dG-heptyl-O6-dG
mismatch ICL, also observed in the repair of XL7. These results
are surprising, as the ICL spanning a 5¢-GNC-3¢ motif would be
expected to be a less flexible substrate relative to directly opposed
O6-dG-heptyl-O6-dG mismatch ICLs. The mechanism by which
hAGT repairs DNA alkylation involves rotation of the alkylated
base from the DNA duplex from the minor groove into the active
site allowing for transfer of the alkyl group to the residue C145.21
The improved repair of XL7 versus XL4 can be rationalized by
the difference in distances of the cross-link. For the four carbon
linker of XL4, the O–O distance of the fully extended linker is
˚
just sufficient (6.2 A) to span the distance necessary to link the
6
˚
O atoms in a 5¢-GNC sequence (6.4 A). In a geometry optimized
˚
model of XL4, the O–O distance was found to be 4.5 A with some
tilting of the guanine bases towards each other (see ESI†). The
˚
model of XL7 has an O–O distance of 7.1 A with the longer alkyl
chain protruding into the major groove. This would suggest that
rotation of one of the alkylated guanines of XL7 into the active site
of hAGT would be less difficult relative to the more strained XL4
ICLs. These findings demonstrate that there could have been some
distortion in the cross-linked duplexes inherent in the nature of the
substrates prior to the repair process and that XL4 is too short to
fit optimally in the active site relative to the XL7 counterpart.
The formation of the final product occurs at a faster rate
than the formation of the median product for both XL4 and
XL7, as the % final product observed is always greater than the
% median product. Final and median product formation occur
at different rates, suggesting that hAGT preferentially repairs
the median product over the initial substrate as indicated by
the lack of accumulation of median product at any time. If
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Colony forming assay in CHO cells treated with busulfan and
hepsulfam and relevance with O6-dG-alkyl-O6-dG ICLs
structure relative to a non-cross-linked control. Both were also
repaired by hAGT, with efficiency higher for the ICLs containing
the seven methylene linkage. Binding studies suggested similar
affinity of hAGT for both ICLs. Studies with cells treated with
hepsulfam demonstrate that hAGT reduces the cytotoxicity this
bifunctional alkylating agent induces and serves as evidence that
the O6-2¢-deoxyguanosine-alkyl-O6-2¢-deoxyguanosine cross-link
may account for at least part of the cytotoxicity of this agent.
CHO cells that do not express AGT were very sensitive to
killing by hepsulfam (Fig. 4). Expression of hAGT provided a
significant but not complete protection from this agent. This is
consistent with the concept that an O6-2¢-deoxyguanosine-alkyl-
O6-2¢-deoxyguanosine cross-link accounts for at least part of the
cytotoxicity of this agent and that the efficient repair of this
7 carbon adduct by hAGT prevents this killing. There was no
protection from busulfan, which is in accordance with the poor
repair of the 4 carbon adduct produced by this agent.
Experimental
5¢-O-Dimethoxytrityl-N2-phenoxyacetyl-2¢-deoxyguanosine, 3¢-
O-dimethoxytrityl-2¢-deoxyribonucleoside-5¢-O-(b-cyanoethyl-
N,N¢-diisopropyl) phosphoramidites and N,N-diisopropylamino
cyanoethyl phosphonamidic chloride were purchased from
ChemGenes Inc. (Wilmington, MA). 5¢-O-Dimethoxytrityl-
2¢-deoxyribonucleoside-3¢-O-(b-cyanoethyl-N,N¢-diisopropyl)-
phosphoramidites and protected 2¢-deoxyribonucleoside-
polystyrene supports were purchased from Glen Research
(Sterling, Virginia). All other chemicals and solvents were
purchased from the Aldrich Chemical Company (Milwaukee,
WI) or EMD Chemicals Inc. (Gibbstown, NJ). Flash column
chromatography was performed using silica gel 60 (230–400
mesh) obtained from Silicycle (Quebec City, QC). Thin layer
chromatography (TLC) was performed using precoated TLC
plates (Merck, Kieselgel 60 F254, 0.25 mm) purchased from EMD
Chemicals Inc. (Gibbstown, NJ). NMR spectra were recorded
on a Varian INOVA 300 MHz NMR spectrometer at room
temperature. 1H NMR spectra were recorded at a frequency
of 300.0 MHz and chemical shifts were reported in parts per
million downfield from tetramethylsilane. 31P NMR spectra
(1H decoupled) were recorded at a frequency of 121.5 MHz
with H3PO4 used as an external standard. ESI mass spectra
for oligonucleotides were obtained at the Concordia University
Centre for Biological Applications of Mass Spectrometry
(CBAMS) using a Micromass Qtof2 mass spectrometer (Waters)
equipped with a nanospray ion source. The mass spectrometer
was operated in full scan, negative ion detection mode. ESI mass
spectra for small molecules were recorded at the McGill University
Department of Chemistry Mass Spectrometry Facility with a
Finnigan LCQ DUO mass spectrometer in methanol or acetone.
Ampicillin, isopropyl b-D-thiogalactopyranoside (IPTG), and
most other biochemical reagents as well as polyacrylamide gel
materials were purchased from Bioshop Canada Inc (Burlington,
ON). Ni-NTA Superflow Resin was purchased from Qiagen
(Mississauga, ON). Complete, Mini, EDTA-free Protease
Inhibitor Cocktail Tablets were obtained from Roche (Laval, QC)
Nitrocellulose filters (0.20 mm) were obtained from Millipore.
XL-10 Gold E. coli cells were obtained from Stratagene (Cedar
Creek, TX). DpnI, T4 polynucleotide kinase (PNK), Unstained
Protein Molecular Weight Marker and restriction enzymes
EcoRI and KpnI were obtained from Fermentas (Burlington,
ON). [g-32P]ATP was purchased from Amersham Canada Ltd.
(Oakville, ON).
Fig. 4 The effect of the expression of hAGT on sensitivity of CHO cells to
busulfan and hepsulfam. Cell survival was measured with a colony forming
assay as described in the Experimental section. * p<0.05, Compared to
cells expressing hAGT; ** p<0.01, Compared to cells expressing hAGT.
Extensive study of the hAGT reaction has shown that DNA
repair by this protein is specific for adducts on the O6-position
with a minor capability to repair adducts on the O4-position of
thymine. The results shown here using in vitro assays show that
a DNA interstrand cross-link containing 7 carbon atoms linking
two O6-2¢-deoxyguanosine residues is efficiently repaired by hAGT.
Such a cross-link would be expected to be formed in treated cells
by hepsulfam and, although it has not yet been characterized,
the finding that hAGT reduces the cytotoxicity of hepsulfam is
strong evidence that it does occur. Unrepaired DNA interstrand
cross-links are very toxic and only a small number of such cross-
links may be formed. The results also suggest that a high level of
hAGT expression would render tumor cells resistant to therapy
with hepsulfam.
Conclusions
The synthesis of nucleoside dimers containing
a
O6-2¢-
deoxyguanosine-alkyl-O6-2¢-deoxyguanosine cross-link that en-
ables the construction of a 1,3-staggered 5¢-GNC motif by
solid-phase synthesis using phosphoramidite chemistry and an
orthogonal approach to removing protecting groups is described.
These ICLs were obtained in high yield and purity that is required
for DNA repair studies. ICL duplexes containing a four and seven
methylene linkage were found to have similar thermal stability and
3¢-O-alloxycarbonyl-5¢-O-dimethoxytrityl-N2-phenoxyacetyl-2¢-
deoxyguanosine (1)
To
deoxyguanosine (2.15 g, 3.06 mmol) in anhydrous THF/pyridine
a
solution of 5¢-O-dimethoxytrityl-N2-phenoxyacetyl-2¢-
4420 | Org. Biomol. Chem., 2010, 8, 4414–4426
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(9 : 1) was added a catalytic amount of dimethylaminopyridine
(~ 1 mg) followed by allyl 1-benzotriazoyl carbonate (0.88 g,
4.05 mmol) and the mixture was stirred at room temperature for
24 h. The solvent was removed in vacuo, the crude taken up in
CH2Cl2 (50 mL) and washed twice with 5% sodium bicarbonate
(50 mL). The organic layer was then dried over sodium sulfate
and evaporated to near dryness. The crude compound was then
purified by silica gel column chromatography using a gradient of
CH2Cl2–methanol (100 : 0.5 → 100 : 1.4) to yield 1.84 g (76.2%) of
1 as a colorless foam. Rf (SiO2): 0.37 in CH2Cl2–MeOH (20 : 1). 1H
NMR (300 MHz, CDCl3, ppm): 11.79 (s, 1H, NH), 8.99 (s, 1H,
NH), 7.85 (s, 1H, H8), 7.35–7.44 (m, 4H, Ar), 7.18–7.35 (m, 9H,
Ar), 7.12 (t, 1H, Ar), 7.02 (dd, 2H, Ar), 6.82 (dd, 4H, Ar), 6.32
(dd, 1H, H1¢, J = 6.0), 5.90–6.05 (m, 1H, allyl), 5.31–5.40 (m, 2H,
allyl), 4.44–4.50 (m, 1H, H3¢), 4.69 (d, 2H, PhOCH2CO), 4.67 (d,
2H, vinylCH2CO), 4.31–4.36 (m, 1H, H4¢), 3.79 (s, 6H, OCH3),
3.35–3.51 (dd, 2H, H5¢, H5¢), 2.83–2.88 (m, 1H, H2¢), 2.64–2.70
(m, 1H, H2¢). MS (ESI): m/z calcd for C43H42N5O10[M+H]+, 788.3;
found, 788.3.
twice with 5% sodium bicarbonate (50 mL). The organic layer
was then dried over magnesium sulfate, filtered and evaporated to
a solid. The crude compound was purified by silica gel column
chromatography using a gradient of hexanes/ethyl acetate (19 : 1
→ 13 : 7). Triphenylphosphine oxide was precipitated from con-
taminated fractions with diethyl ether and separated by filtration.
Evaporation of the solvent afforded 3.08 g (61.9%) of the product
as a colorless foam. Rf (SiO2): 0.67, hexanes/ethylacetate 2 : 3.
1H NMR (300 MHz, CDCl3, ppm): 8.68 (s, 1H, NH); 8.10 (s, 1H,
H8); 7.66–7.71 (m, 4H, Ar); 7.16–7.46 (m, 18H, Ar); 7.01–7.10 (m,
3H, Ar); 6.79 (dd, 4H, Ar); 6.48 (dd, 1H, H1¢, J = 5.7); 5.89–6.04
(m, 1H, allyl); 5.30–5.46 (m, 2H, allyl); 4.47–4.52 (m, 1H, H3¢);
4.78 (s, 2H, CH2OAr); 4.67 (dt, 2H, vinylCH2CO); 4.59 (d, 2H,
PhOCH2CO); 4.35–4.39 (m, 1H, H4¢); 3.78 (s, 6H, OCH3); 3.67 (t,
2H, CH2OSi); 3.52 (dd, 1H, H5¢); 3.41 (dd, 1H, H5¢); 3.18–3.23 (m,
1H, H2¢); 2.71–2.76 (m, 1H, H2¢); 1.89 (q, 2H, CH2); 1.33–1.62
(m, 6H, (CH2)3); 1.06 (s, 9H, SiC(CH3)3). MS (ESI): m/z calcd
C66H73N5O11SiNa [M+Na]+, 1120.5, found 1120.4.
3¢-O-alloxycarbonyl-5¢-O-dimethoxytrityl-N2-phenoxyacetyl-O6-
(4-hydroxybutyl)-2¢-deoxyguanosine (3a)
3¢-O-alloxycarbonyl-5¢-O-dimethoxytrityl-N2-phenoxyacetyl-O6-
(4-tert-butyldiphenylsiloxybutyl)-2¢-deoxyguanosine (2a)
To a solution of 2a (5.15 g, 5.38 mmol) in anhydrous THF
(10 mL) was added dropwise tetrabutylammonium fluoride (1 M
in THF, 645 mL, 6.45 mmol) and the solution was stirred at
room temperature for 30 min. On completion the reaction was
concentrated, taken up in CH2Cl2 (50 mL), washed twice with 5%
sodium bicarbonate (50 mL), the organic layer dried over sodium
sulfate and evaporated to dryness. The crude was purified by silica
gel column chromatography using a gradient of hexanes/ethyl
acetate (60 : 40 → 20 : 80) to yield 3.25 g (70.3%) of product as a
To a solution of 1 (5.37 g, 6.82 mmol) in anhydrous dioxane
(40 mL) was added 1-tert-butyldiphenylsilylbutanol (1.97 g,
9.63 mmol) and triphenylphosphine (2.55 g, 9.72 mmol) followed
by the dropwise addition of diisopropylazodicarboxylate (1.89 g,
9.34 mmol) introduced in dioxane (4.4 mL). The solvent was
evaporated after 1 h and the crude was taken up in CH2Cl2
(50 mL) and washed twice with 5% sodium bicarbonate (50 mL).
The organic layer was then dried over magnesium sulfate, filtered
and evaporated. The crude compound was purified by silica
gel column chromatography using a gradient of hexanes/ethyl
acetate (19 : 1 → 13 : 7). Triphenylphosphine oxide was pre-
cipitated from contaminated fractions with diethyl ether and
separated by filtration. Evaporation of the solvent afforded 5.16
g (78.8%) of the compound as a colorless foam. Rf (SiO2): 0.67,
hexanes/ethylacetate 2 : 3. 1H NMR (300 MHz, DMSO-d6, ppm):
10.52 (s, 1H, NH), 8.36 (s, 1H, H8); 7.34–7.64 (m, 11H, Ar); 7.11–
7.29 (m, 11H, Ar); 6.89–6.95 (m, 3H, Ar); 6.65–6.74 (dd, 4H, Ar);
6.40 (dd, 1H, H1¢, J = 7.2); 5.84–5.98 (m, 1H, allyl); 5.20–5.35 (m,
3H, allyl, H3¢); 4.97–5.06 (m, 2H, CH2OAr); 4.50–4.64 (m, 4H,
vinylCH2CO, PhOCH2CO); 4.18–4.24 (m, 1H, H4¢); 3.70 (t, 2H,
CH2OSi); 3.67 (s, 3H, OCH3); 3.66 (s, 3H, OCH3); 3.46 (dd, 1H,
H5¢); 3.23–3.27 (m, 1H, H2¢); 3.15 (dd, 1H, H5¢); 2.58–2.63 (m,
1H, H2¢); 1.90 (q, 2H, CH2); 1.67–1.76 (m, 2H, CH2); 0.94 (s, 9H,
SiC(CH3)3). MS (ESI): m/z calcd for C63H67N5O11SiNa [M+Na]+,
1062.5, found 1062.4.
1
colorless foam. Rf (SiO2): 0.50, CH2Cl2–MeOH 10 : 1. H NMR
(300 MHz, DMSO-d6, ppm): 10.58 (s, 1H, NH), 8.41 (s, 1H,
H8); 7.31–7.36 (m, 4H, Ar); 7.18–7.21 (m, 7H, Ar); 6.95–7.02
(m, 3H, Ar); 6.71–6.81 (dd, 4H, Ar); 6.45 (dd, 1H, H1¢, J = 6.9);
5.89–6.04 (m, 1H, allyl); 5.27–5.41 (m, 3H, H3¢, allyl); 4.87–5.15
(m, 2H, CH2OAr); 4.63–4.67 (dt, 2H, vinylCH2CO); 4.59 (d, 2H,
PhOCH2CO); 4.53 (t, 1H, OH); 4.23–4.30 (m, 1H, H4¢); 3.74 (s,
3H, OCH3); 3.73 (s, 3H, OCH3); 3.44–3.55 (m, 3H, CH2OH, H5¢);
3.26–3.35 (m, 1H, H5¢); 3.20 (dd, 1H, H2¢); 2.57–2.69 (m, 1H,
H2¢); 1.90 (q, 2H, CH2); 1.56–1.68 (m, 2H, CH2). MS (ESI): m/z
calcd for C47H49N5O11Na [M+Na]+, 882.3, found 882.2.
3¢-O-alloxycarbonyl-5¢-O-dimethoxytrityl-N2-phenoxyacetyl-O6-
(7-hydroxyheptyl)-2¢-deoxyguanosine (3b)
To a solution of 2b (1.35 g, 1.20 mmol) in anhydrous THF
(10 mL) was added dropwise tetrabutylammonium fluoride (1 M
in THF, 135 mL, 1.35 mmol) and the solution was stirred at
room temperature for 30 min. On completion the reaction was
concentrated, taken up in CH2Cl2 (50 mL) and washed twice
with 5% sodium bicarbonate (50 mL). The organic layer was
then dried over sodium sulfate, evaporated and the crude was
purified by silica gel column chromatography using a gradient of
hexanes/ethyl acetate (60 : 40 → 20 : 80) to yield 0.949 g (89.6%) of
a colorless foam. Rf (SiO2): 0.50, CH2Cl2–MeOH 10 : 1. 1H NMR
(300 MHz, DMSO-d6, ppm): 10.55 (s, 1H, NH), 8.36 (s, 1H, H8);
7.23–7.33 (m, 4H, Ar); 7.10–7.20 (m, 7H, Ar); 6.88–6.98 (m, 3H,
3¢-O-alloxycarbonyl-5¢-O-dimethoxytrityl-N2-phenoxyacetyl-O6-
(7-tert-butyldiphenylsiloxyheptyl)-2¢-deoxyguanosine (2b)
To a solution of 1 (3.48 g, 4.41 mmol) in anhydrous dioxane
(20 mL) was added 1-tert-butyldiphenylsilyloxyheptanol (1.80 g,
4.86 mmol) and triphenylphosphine (1.76 g, 6.71 mmol) followed
by diisopropylazodicarboxylate (1.37 g, 6.46 mmol) introduced
dropwise in dioxane (1.6 mL). The solvent was evaporated after
1 h and the crude was taken up in CH2Cl2 (50 mL) and washed
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Ar); 6.71 (dd, 4H, Ar); 6.40 (dd, 1H, H1¢, J = 5.7); 5.85–6.00
(m, 1H, allyl); 5.31–5.39 (m, 1H, H3¢); 5.22–5.35 (m, 2H, allyl);
5.00 (t, 2H, CH2OAr); 4.60 (d, 2H, vinylCH2CO); 4.52 (d, 2H,
PhOCH2CO); 4.34 (s, 1H, CH2OH); 4.18–4.26 (m, 1H, H4¢); 3.69
(s, 6H, OCH3); 3.42 (dd, 1H, H5¢); 3.30–3.41 (m, 2H, CH2OH);
3.19–3.31 (m, 1H, H2¢); 3.14 (dd, 1H, H5¢); 2.56–2.64 (m, 1H, H2¢);
1.80 (q, 2H, CH2CH2OAr); 1.21–1.47 (m, 6H, (CH2)3). MS (ESI):
m/z calcd for C50H55N5O11Na [M+Na]+, 924.4, found 924.4.
0.00 (s, 3H, SiCH3). MS (ESI): m/z calcd for C92H98N10O18SiNa
[M+Na]+, 1681.7, found 1681.5.
1-{O6-[3¢-O-alloxycarbonyl-5¢-O-dimethoxytrityl-N2-
phenoxyacetyl-2¢-deoxyguanidyl]}-7-{O6-[3¢-O-tert-
butyldimethylsilyl-5¢-O-dimethoxytrityl-N2-phenoxyacetyl-2¢-
deoxyguanidyl]}-heptane (5b)
To a solution of 3b (1.73 g, 1.92 mmol) in anhydrous dioxane
(10.0 mL) was added triphenylphosphine (0.77 g, 2.91 mmol) and
compound 4 (1.54 g, 1.88 mmol). Then diisopropylazodicarboxy-
late (0.59 g, 2.78 mmol) was introduced dropwise in a solution
of dioxane (3 mL). After 30 min the solvent was evaporated, the
crude was taken up in CH2Cl2 (50 mL) and washed twice with 5%
sodium bicarbonate (50 mL). The organic layer was then dried
over sodium sulfate and evaporated to dryness. The crude was
purified by silica gel column chromatography using a gradient of
hexanes/ethyl acetate (1 : 0 → 1 : 4) to yield 2.32 g (71.1%) of a
3¢-O-tert-butyldimethylsilyl-5¢-O-dimethoxytrityl-N2-
phenoxyacetyl-2¢-deoxyguanosine (4)
To
a
solution of 5¢-O-dimethoxytrityl-N2-phenoxyacetyl-2¢-
deoxyguanosine (1.73 g, 2.46 mmol) in anhydrous N,N-
dimethylformamide was added imidazole (1.13 g, 16.64 mmol)
followed by tert-butyldimethylsilyl chloride (1.24 g, 8.23 mmol)
and the mixture was stirred at room temperature for 16 h. The
solvent was removed, and the crude taken up in CH2Cl2 (25 mL)
and washed twice with 5% sodium bicarbonate (25 mL). The
organic layer was then dried over sodium sulfate and evaporated.
The crude was purified by silica gel column chromatography using
a gradient of hexanes/ethyl acetate (5 : 1 → 1 : 3) to yield 1.58 g
(83.2%) of the product which was a colorless foam. Rf (SiO2): 0.55,
1
colorless foam. Rf (SiO2): 0.70, CH2Cl2–MeOH 10 : 1. H NMR
(300 MHz, DMSO-d6, ppm): 10.58 (s, 1H, NH); 10.53 (s, 1H,
NH); 8.39 (s, 1H, H8); 8.35 (s, 1H, H8); 7.10–7.34 (m, 22H, Ar);
6.88–6.97 (m, 6H, Ar); 6.64–6.80 (m, 8H, Ar); 6.40 (dd, 1H, H1¢,
J = 6.0); 6.34 (dd, 1H, H1¢, J = 6.6); 5.85–6.00 (m, 1H, allyl); 5.31–
5.39 (m, 1H, H3¢); 5.21–5.35 (m, 2H, allyl); 4.97–5.03 (m, 4H,
PhOCH2CO); 4.61–4.72 (m, 1H, H3¢); 4.60 (d, 2H, vinylCH2CO);
4.34 (d, 4H, CH2OAr); 4.17–4.26 (m, 1H, H4¢); 3.69–3.77 (m, 1H,
H4¢); 3.68 (s, 12H, OCH3); 3.46 (dd, 1H, H5¢); 3.10–3.32 (m, 4H,
H2¢, H5¢, 2 ¥ H5¢); 2.86–2.98 (m, 1H, H2¢); 2.54–2.65 (m, 1H, H2¢);
2.27–2.38 (m, 1H, H2¢); 1.80 (m, 4H, CH2CH2OAr); 1.41; (m, 6H,
(CH2)3); 0.78 (s, 9H, SiC(CH3)3); 0.00 (s, 3H, SiCH3); -0.05 (s, 3H,
SiCH3). MS (ESI): m/z calcd for C94H104N10O18SiNa [M+Na]+,
1723.7, found 1724.5.
1
CH2Cl2–MeOH 10 : 0.8. H NMR (300 MHz, DMSO-d6, ppm):
11.85 (s, 1H, NH), 11.78 (s, 1H, NH), 8.19 (s, 1H, H8); 7.10–7.36
(m, 12H, Ar); 6.92–7.02 (m, 3H, Ar); 6.74–6.84 (t, 3H, Ar); 6.21
(dd, 1H, H1¢, J = 6.9); 4.85 (d, 2H, PhOCH2CO); 4.49–4.54 (m, 1H,
H3¢); 3.81–3.86 (m, 1H, H4¢); 3.70 (s, 6H, OCH3); 3.05–3.23 (dd,
2H, H5¢, H5¢); 2.72–2.77 (m, 1H, H2¢); 2.32–2.37 (m, 1H, H2¢);
0.79 (s, 9H, SiC(CH3)3); 0.03 (s, 3H, SiCH3); -0.04 (s, 3H, SiCH3).
MS (ESI): m/z calcd for C45H51N5O8SiNa [M+Na]+, 840.3, found
840.2.
1-{O6-[3¢-O-alloxycarbonyl-5¢-O-dimethoxytrityl-N2-
phenoxyacetyl-2¢-deoxyguanidyl]}-4-{O6-[3¢-O-tert-
butyldimethylsilyl-5¢-O-dimethoxytrityl-N2-
phenoxyacetyl-2¢-deoxyguanidyl]}-butane (5a)
1-{O6-[3¢-O-tert-butyldimethylsilyl-5¢-O-dimethoxytrityl-N2-
phenoxyacetyl-2¢-deoxyguanidyl]}-4-{O6-[5¢-O-dimethoxytrityl-
N2-phenoxyacetyl-2¢-deoxyguanidyl]}-butane (6a)
To a solution of 5a (0.97 g, 0.59 mmol) in anhydrous THF (3 mL)
was added triphenylphosphine (23.0 mg, 0.09 mmol), palladium
(0) tetrakistriphenylphosphine (34.0 mg, 0.03 mmol) and a buty-
lamine/formic acid solution (1 : 1, 1.17 mmol) in THF (1 mL).
After stirring the mixture for 30 min the reaction was concentrated
in vacuo and the crude taken up in CH2Cl2 (25 mL) and washed
twice with 5% sodium bicarbonate (25 mL). The organic layer
was dried over sodium sulfate, filtered and evaporated. The crude
was purified by silica gel column chromatography using a gradient
of hexanes/ethyl acetate (3 : 7 → 1 : 9) to give 0.87 g (94.4%) of
colorless product. Rf (SiO2): 0.41 hexanes/ethyl acetate (1 : 9). 1H
NMR (300 MHz, DMSO-d6, ppm): 10.65 (s, 2H, NH); 8.45 (s, 1H,
H8); 8.41 (s, 1H, H8); 7.21–7.38 (m, 22H, Ar); 7.03–6.96 (m, 6H,
Ar); 6.76–6.85 (m, 8H, Ar); 6.40–6.49 (m, 2H, 2 ¥ H1¢); 5.40 (d,
1H, 3¢OH); 5.07 (s, 4H, 2 ¥ PhOCH2CO); 4.64–4.76 (m, 5H, H3¢,
2 ¥CH2CH2OAr); 4.52–4.60 (m, 1H, H3¢); 4.02–4.07 (m, 1H, H4¢);
3.80–3.93 (m, 1H, H4¢); 3.76 (s, 12H, OCH3); 3.26–3.39 (m, 3H, 2
¥ H5¢, H5¢); 3.13–3.17 (m, 1H, H5¢); 2.90–3.03 (m, 3H, 2 ¥ H2¢,
H2¢); 2.40–2.44 (m, 1H, H2¢); 2.03–2.10 (m, 4H, CH2CH2OAr);
0.85 (s, 9H, SiC(CH3)3); 0.07 (s, 3H, SiCH3); 0.00 (s, 3H, SiCH3).
MS (ESI): m/z calcd for C88H95N10O16Si [M+H]+, 1575.7 found
1575.7.
To a solution of compound 3a (1.70 g, 1.98 mmol) in anhy-
drous dioxane (15 mL) was added triphenylphosphine (0.81 g,
3.09 mmol) and compound 4 (1.56 g, 1.93 mmol). Then, diiso-
propylazodicarboxylate (0.64 g, 3.02 mmol) was introduced in a
solution of dioxane (5 mL). The solvent was evaporated after 14
h and the crude was taken up in CH2Cl2 (50 mL) and washed
twice with 5% sodium bicarbonate (50 mL). The organic layer
was then dried over magnesium sulfate and evaporated to dryness,
and then purified by silica gel column chromatography using a
gradient of hexanes/ethyl acetate (1 : 0 → 1 : 5) to yield 2.07 g
(63.1%) of product which was a colorless foam. Rf (SiO2): 0.45,
1
CH2Cl2–MeOH 20 : 1. H NMR (300 MHz, CDCl3, ppm): 8.82
(s, 1H, NH); 8.78 (s, 1H, NH); 8.06 (s, 1H, H8); 8.00 (s, 1H, H8);
6.67–7.45 (m, 22H, Ar); 6.94–6.09 (m, 6H, Ar); 6.64–6.80 (m, 8H,
Ar); 6.42 (m, 2H, 2 ¥ H1¢); 5.86–6.02 (m, 1H, allyl); 5.43–5.51 (m,
1H, H3¢); 5.27–5.44 (m, 2H, allyl); 4.78 (s, 4H, CH2OAr); 4.71 (d,
4H, PhOCH2CO); 4.57–4.69 (m, 2H, vinylCH2CO); 4.52–4.64 (m,
1H, H3¢); 4.31–4.39 (m, 1H, H4¢); 4.03–4.11 (m, 1H, H4¢); 3.75
(s, 12H, OCH3); 3.30–3.56 (m, 4H, 2 ¥ H5¢, 2 ¥ H5¢); 3.00 (m,
1H, H2¢); 2.62–2.76 (m, 2H, H2¢, H2¢); 2.38–2.49 (m, 1H, H2¢);
2.13 (m, 4H, (CH2)2); 0.85 (s, 9H, SiC(CH3)3); 0.04 (s, 3H, SiCH3);
4422 | Org. Biomol. Chem., 2010, 8, 4414–4426
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1-{O6-[3¢-O-tert-butyldimethylsilyl-5¢-O-dimethoxytrityl-N2-
phenoxyacetyl-2¢-deoxyguanidyl]}-7-{O6-[5¢-O-dimethoxytrityl-
N2-phenoxyacetyl-2¢-deoxyguanidyl]}-heptane (6b)
143.91, 144.11. MS (ESI): m/z calcd for C100H117N12O17PSiNa
[M+Na]+, 1839.8, found 1839.5.
Oligonucleotide synthesis, purification and characterization
To a solution of compound 5b (0.52 g, 0.31 mmol) in anhydrous
THF (3 mL) was added triphenylphosphine (24.4 mg, 0.09 mmol),
palladium(0) tetrakistriphenylphosphine (35.2 mg, 0.03 mmol)
and a butylamine/formic acid solution (1 : 1, 0.66 mmol) in THF
(1 mL). After stirring at room temperature for 40 min the reaction
was concentrated, taken up in CH2Cl2 (25 mL) and washed twice
with 5% sodium bicarbonate (25 mL). The organic layer was
then dried over sodium sulfate and evaporated. The crude was
purified by silica gel column chromatography using a gradient of
hexanes/ethyl acetate (1 : 0 → 1 : 4) to yield 0.376 g (76.0%) of
The cross-linked duplexes XL4 and XL7, whose sequences are
shown in Fig. 1, were assembled using an Applied Biosystems
Model 3400 synthesizer on a 1 mmole scale employing standard b-
cyanoethylphosphoramidite cycles supplied by the manufacturer
with slight modifications to coupling times described below.
The nucleoside phosphoramidites containing standard protecting
groups were prepared in anhydrous acetonitrile at a concentration
of 0.1 M for the 3¢-O-2¢-deoxyphosphoramidites, 0.15 M for
the cross-linked 3¢-O-2¢-deoxyphosphoramidites (7a and 7b) and
0.2 M for the 5¢-O-2¢-deoxyphosphoramidites. Assembly of se-
quences first involved detritylation (3% trichloroacetic acid [TCA]
in CH2Cl2), followed by nucleoside phosphoramidite coupling
with commercial 3¢-O-2¢-deoxyphosphoramidites (2 min), 5¢-O-2¢-
deoxyphosphoramidites (3 min) or cross-linked phosphoramidites
7a or 7b (10 min); subsequent capping with phenoxyacetic anhy-
dride/pyridine/tetrahydrofuran (1 : 1 : 8, v/v/v; solution A, and
1-methyl-1 H-imidazole/tetrahydrofuran 16 : 84 w/v; solution B)
and oxidation (0.02 M iodine in tetrahydrofuran–water/pyridine
2.5 : 2 : 1) followed every coupling. To complete assembly of the
cross-linked duplexes, the cyanoethyl groups were removed from
the polystyrene-linked oligomers by treating the support with
1 mL of anhydrous triethylamine (TEA) for at least 12 h. The
support was then washed with 30 mL of anhydrous acetonitrile
(ACN) followed by anhydrous THF. The tert-butyldimethylsilyl
(TBS) group was removed from the partial duplex by treating the
support with 2 ¥ 1 mL triethylamine trihydrofluoride (TEA·3HF)
for a total of 1 h. The support was then washed with 30 mL
each of anhydrous THF and ACN followed by drying via high
vacuum (20 min). The final extension of the cross-linked duplex
was then achieved using 5¢-O-2¢-deoxyphosphoramidites with a
total detritylation exposure of 130 s and removal of the 3¢-terminal
trityl group by the synthesizer to yield duplexes XL4 and XL7 on
the solid support.
1
a colorless foam. Rf (SiO2): 0.33 hexanes/ethyl acetate (1 : 4). H
NMR (300 MHz, CDCl3, ppm): 8.76 (s, 1H, NH); 8.67 (s, 1H,
NH); 8.03 (s, 1H, H8); 8.01 (s, 1H, H8); 7.14–7.44 (m, 22H, Ar);
6.97–7.09 (m, 6H, Ar); 6.74–6.84 (m, 8H, Ar); 6.60 (dd, 1H, H1¢,
J = 6.6); 6.43 (dd, 1H, H1¢, J = 6.4); 4.40–4.85 (m, 10H, 2 ¥ H3¢, 2
¥ PhOCH2CO, 2 ¥ CH2OAr); 4.17–4.26 (m, 1H, H4¢); 4.05–4.14
(m, 1H, H4¢); 3.77 (s, 6H, OCH3); 3.75 (s, 6H, OCH3); 3.45 (dd,
1H, H5¢); 3.00–3.38 (m, 3H, H5¢, 2 x H5¢); 3.21 (s, 1H, 3¢OH);
2.56–2.83 (m, 3H, 2 ¥ H2¢, H2¢); 2.40–2.51 (m, 1H, H2¢); 1.90 (m,
4H, CH2CH2OAr); 1.52; (s, 6H, (CH2)3); 0.86 (s, 9H, SiC(CH3)3);
0.00 (s, 3H, SiCH3); -0.05 (s, 3H, SiCH3). MS (ESI): m/z calcd for
C91H100N10O16SiNa [M+Na]+, 1639.7, found 1639.5.
1-{O6-[3¢-O-tert-butyldimethylsilyl-5¢-O-dimethoxytrityl-N2-
phenoxyacetyl-2¢-deoxyguanidyl]}-4-{O6-[5¢-O-dimethoxytrityl-
N2-phenoxyacetyl-2¢-deoxyguanidyl-3¢-O-(b-2-cyanoethyl-N,N¢-
diisopropyl)phosphoramidite]}-butane (7a)
Compound 6a (0.45 g, 0.29 mmol) was dissolved in THF (1.4 mL)
and diisopropylethylamine (0.055 g, 0.43 mmol) followed by N,N-
diisopropylamino cyanoethyl phosphonamidic chloride (0.081 g,
0.343 mmol). The reaction was stirred at room temperature
for 2 h after which it was quenched by the addition of ethyl
acetate (50 mL) and the solution washed twice with 5% sodium
bicarbonate (50 mL). The organic layer was dried over sodium
sulfate, filtered and evaporated. The product was precipitated from
vigorously stirring hexanes to give 0.43 g (86%) of a colorless
powder. Rf (SiO2): 0.64, 0.70 in ethyl acetate. 31P NMR (121.5
MHz, acetone-d6, ppm): 143.92, 144.14. MS (ESI): m/z calcd for
C97H111N12O17PSiNa [M+Na]+, 1797.8, found 1797.6.
The oligomer-derivatized polystyrene beads were transferred
from the reaction column to screw cap microfuge tubes fitted
with teflon lined caps and the oligomer released from the support
and protecting groups removed by treatment with a mixture of
concentrated ammonium hydroxide/ethanol (0.3 mL : 0.1 mL)
◦
for 4 h at 55 C. The cross-linked final products were separated
from pre-terminated products by strong anion exchange (SAX)
HPLC using a Dionex DNAPAC PA-100 column (0.4 cm x
25 cm) purchased from Dionex Corp, (Sunnyvale, CA) with a
linear gradient of 0–50% buffer B over 30 min (buffer A: 100 mM
Tris HCl, pH 7.5, 10% acetonitrile and buffer B: 100 mM Tris HCl,
pH 7.5, 10% acetonitrile, 1 M NaCl) at 40 ◦C. The columns were
monitored at 260 nm for analytical runs or 280 nm for preparative
runs. The purified oligomers were desalted using C-18 SEP PAK
cartridges (Waters Inc.) as previously described.5 The amounts of
purified oligomers obtained are shown in Table 1.
1-{O6-[3¢-O-tert-butyldimethylsilyl-5¢-O-dimethoxytrityl-N2-
phenoxyacetyl-2¢-deoxyguanidyl]}-7-{O6-[5¢-O-dimethoxytrityl-
N2-phenoxyacetyl-2¢-deoxyguanidyl-3¢-O-(b-2-cyanoethyl-N,N¢-
diisopropyl)phosphoramidite]}-heptane (7b)
Compound 6b (0.35 g, 0.22 mmol) was dissolved in THF (1.1 mL)
and diisopropylethylamine (0.042 g, 0.32 mmol) followed by N,N-
diisopropylamino cyanoethyl phosphonamidic chloride (0.061 g,
0.26 mmol). The reaction stirred at room temperature for 2 h after
which it was quenched by the addition of ethyl acetate (40 mL) and
the solution washed twice with 5% sodium bicarbonate (50 mL).
The organic layer was dried over sodium sulfate, filtered and
evaporated. The product was precipitated from vigorously stirring
hexanes to give 0.28 g (71%) of a colorless powder. Rf (SiO2): 0.71,
0.82 in ethyl acetate. 31P NMR (121.5 MHz, acetone-d6, ppm):
The cross-linked oligomers (0.1 A260 units) were characterized
by enzymatic digestion (snake venom phosphodiesterase: 0.28
units and calf intestinal phosphatase: 5 units, in 10 mM Tris,
pH 8.1 and 2 mM magnesium chloride) for a minimum of
◦
36 h at 37 C as previously described.5 The resulting mixture
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using HyperChemTM molecular modeling software. All duplexes
were geometry optimized using the AMBER forcefield.
of nucleosides was analyzed by reversed phase HPLC carried out
R
using a Symmetryꢀ C-18 5 mm column (0.46 ¥ 15 cm) purchased
from Waters Inc, Milford, MA. The C-18 column was eluted with
a linear gradient of 0–60% buffer B over 30 min (buffer A: 50 mM
sodium phosphate, pH 5.8, 2% acetonitrile and buffer B: 50 mM
sodium phosphate, pH 5.8, 50% acetonitrile). The resulting peaks
were identified by co-injection with the corresponding standards
and eluted at the following times: dC (4.6 min), dG (7.9 min), dT
(8.5 min), dA (9.6 min), cross-link dimers (18.1 for the four carbon
and 25.5 min for the seven carbon cross-link), and the ratio of
nucleosides was determined. The results are given in Table 1. The
molecular weights of the cross-linked oligomers were determined
by ESI-MS and these were in agreement with the calculated values
(see ESI for mass spectra†).
Protein expression and purification
Site directed mutagenesis as well as transformation into XL-10 E.
coli cells were performed as directed by the Stratagene manual.
Cells containing either a plasmid coding for wild-type or mutant
hAGT were grown in a 1 L cultures of LB broth + 100 mg mL-1
ampicillin until an OD600 = 0.6 was reached. The cells were induced
◦
with 0.3 mM IPTG, incubated at 37 C for 4 h with shaking at
225 rpm and harvested by centrifugation at 6000 ¥ g at 4 ◦C
for 30 min. Pellets were weighed and resuspended in 5 mL of
buffer [20 mM Tris HCl (pH 8.0), 250 mM NaCl, 20 mM b-
mercaptoethanol supplemented with Complete, Mini, EDTA-free
Protease Inhibitor Cocktail Tablets] for each gram of pellet. The
cells were homogenized by using approximately 20 strokes in a
dounce homogenizer, lysed using two rounds of French press,
centrifuged at 17000 ¥ g for 45 min at 4 ◦C. The lysate was
applied to a pre-equilibrated Ni-NTA Superflow column (100 mL
resuspension buffer) containing 3.5 mL of resin. The lysate was
run twice through the column at 1 mL min-1 to ensure complete
binding. The column was washed with 200 mL resuspension buffer
and 20 mM imidazole at 1 mL min-1, until the OD280 of the
eluant was constant. The protein eluted with 50 mL resuspension
buffer supplemented with 200 mM imidazole at 1 mL min-1.
1 mL fractions were collected and fractions that displayed protein
content (generally fractions 4–11) were pooled followed by dialysis
versus 4 L of dialysis buffer [50 mM Tris HCl (pH 7.6), 250 mM
NaCl, 20 mM b-mercaptoethanol and 0.1 mM EDTA] using 8000
Da cutoff dialysis tubing. Typically a yield of 8–10 mg of purified
protein was obtained per liter of culture inoculated.
UV thermal denaturation studies
Molar extinction coefficients for the oligonucleotides and cross-
linked duplexes were calculated from those of the mononucleotides
and dinucleotides according to nearest-neighbour approximations
(M-1 cm-1). Non-cross-linked duplexes were prepared by mixing
equimolar amounts of the interacting strands and lyophilizing the
mixture to dryness. The resulting pellet was then re-dissolved in
90 mM sodium chloride, 10 mM sodium phosphate, 1 mM EDTA
buffer (pH 7.0) to give a final concentration of 2.8 mM duplex.
The cross-linked duplexes were dissolved in the same buffer to
give a final concentration of 2.8 mM. The solutions were then
◦
heated to 90 C for 10 min, cooled slowly to room temperature,
and stored at 4 ◦C overnight before measurements. Prior to the
thermal run, samples were degassed by placing them in a speed-
vac concentrator for 2 min. Denaturation curves were acquired at
260 nm at a rate of heating of 0.5 ◦C min-1, using a Varian CARY
Model 3E spectrophotometer fitted with a 6-sample thermostated
cell block and a temperature controller. The data were analyzed
in accordance with the convention of Puglisi and Tinoco50 and
Protein characterization
transferred to Microsoft ExcelTM
.
All proteins were analyzed by ESI-MS and were prepared by
precipitation using one volume of TCA for every four volumes of
protein. Samples were incubated for 10 min on ice, centrifuged at
4 ◦C for 5 min at 14 krpm on a table-top centrifuge, the supernatant
removed, the pellet washed with 200 mL of cold acetone and
washing repeated. The protein pellet was dried at 95 ◦C for 5 min,
resuspended in a 50% acetonitrile and 1% formic acid solution
at a protein concentration of 10 mM. The samples were analyzed
with a Waters Micromass Q-ToF-2 mass spectrometer operating
in positive-ion mode.
Far-UV circular dichroism (CD) spectra were obtained on a
JASCO 815 spectropolarimeter using a 2 mm path length cell.
The scans were performed at 20 ◦C, by averaging 5 wavelength
scans from 260 to 200 nm (1 nm bandwidth) in 0.2 nm steps at a
rate of 20 nm min-1, and 1 s response at a protein concentration of
5 mM in CD buffer [50 mM potassium phosphate (pH 7.5), 75 mM
NaCl and 2 mM dithiothreitol (DTT)].
Circular dichroism (CD) spectroscopy
Circular dichroism spectra were obtained on a Jasco J-815
spectropolarimeter equipped with a Julaba F25 circulating bath.
Samples were allowed to equilibrate for 5–10 min at 10 ◦C in
90 mM sodium chloride, 10 mM sodium phosphate, 1 mM EDTA
(pH 7.0), at a final concentration of 2.8 mM for the cross-linked
duplexes and ca. 2.8 mM for control duplexes. Each spectrum was
an average of 5 scans. Spectra were collected at a rate of 100 nm
min-1, with a bandwidth of 1 nm and sampling wavelength of
0.2 nm using fused quartz cells (Starna 29-Q-10). The CD spectra
were recorded from 350 to 200 nm at 10 ◦C. The molar ellipticity
was calculated from the equation [f] = e/Cl, where e is the relative
ellipticity (mdeg), C is the molar concentration of oligonucleotides
(moles/L), and l is the path length of the cell (cm). The data were
processed on a PC computer using WindowsTM based software
supplied by the manufacturer (JASCO, Inc.) and transferred into
Microsoft ExcelTM for presentation.
Thermal denaturation curves were obtained by monitoring the
change in mdeg at 222 nm (corresponding to◦the a-helical content
of the protein). The heating rate was set at 15 C h-1 using a Peltier-
type temperature controller, which ranged from 40–70 ◦C. The Tm
was obtained by noting the temperature at which the 1st derivative
of the denaturation plot was the highest.
Molecular modeling of ICL duplexes
The DNA duplex (5¢-dCGATGTCATCG)/(5¢-CGATGA-
CATCG) and cross-linked duplexes XL4 and XL7 were built
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Fluorescence spectra were obtained using a Varian Cary Eclipse
Fluorescence Spectrophotometer in a 10 mm quartz cuvette with
3.5 mM protein in CD buffer at room temperature. Excitation and
emission slits were set at 5 nm with a detector voltage of 650
and the spectra were the average of 10 accumulations. Tyrosine
and tryptophan fluorescence were obtained using an excitation
wavelength of 280 nm and recording the emission spectrum
between 300 and 400 nm. To monitor the tryptophan fluorescence
solely an excitation wavelength of 295 nm was used and the
emission spectrum was monitored between 300 and 400 nm.
in 10 mM Tris acetate (pH 7.6) and 0.25 mM EDTA buffer at 125 V
for 1 h. On completion, the gels were covered and processed as
described above.
Estimates of the monomeric dissociation constant (Kd ) and the
cooperativity factor (n) were obtained from the electrophoretic
mobility shift assays as explained previously.48 The binding of n
moles of hAGT protein [P] to 1 mole of cross-linked DNA [D]
follows the equation: nP + D ↔ PnD. Isolating the variables and
taking the logarithm of the equation yields:
⎛
⎜
⎜
⎞
⎟
⎠
[P D]
n
⎟
⎟
log
= n log [P]free + log Kd
⎟
⎜
⎝
[D]
Denaturing polyacrylamide gel electrophoresis assay of
O6-dG-alkyl-O6-dG interstrand cross-link repair
Plotting log [PnD]/[P] as a function of log [P] gives a slope equal
to n. At half-saturation: log [PnD]/[P] = 0 and observed Kd can be
obtained from the relation observed Kd = -n ln [P]. The monomeric
Kd can be obtained by taking the nth root of the observed Kd .
The cross-linked oligonucleotides XL4 and XL7 as well as the
control DNA (non-cross-linked duplex) were labeled at the 5¢-
end using [g-32P]ATP. 10 mL reaction volumes were used for the
labeling of the DNA. The reaction mixture was comprised of 1 ¥
T4 PNK buffer (Fermentas), 21 mM oligonucleotides, 10 mM cold-
ATP, 1 mL [g-32P]ATP (10 mCi/mL) an◦d 5 units of T4 PNK. The
reaction mixture was incubated at 37 C for 1 h and terminated
by placing the reaction in boiling water for 10 min. 60 pmol of
wild-type and mutant hAGT protein was incubated with 2 pmol
of labeled DNA in a total reaction volume of 15 mL comprised
of hAGT buffer [80 mM Tris HCl (pH 7.6), 0.1 mM EDTA and
5 mM DTT]. The reactions were incubated at 37 ◦C for 14 to
16 h with similar results indicating that the reaction was complete
by 14 h. Denaturing PAGE in 7 M urea was used to analyze the
products. The reaction was terminated by adding 18.2 mL of stop
reaction buffer [81 mM Tris-HCl, 81 mM boric acid, 1.8 mM
EDTA and 1% SDS (pH 8.0) in 80% formamide] to the reaction
tube and heating as above. The samples were loaded onto 14 cm ¥
16 cm, 17% polyacrylamide gels (19 : 1) in the presence of 7 M
urea. The gels were run using 1 ¥ TBE [89 mM Tris-HCl, 89 mM
boric acid, 2 m◦M EDTA (pH 8.0)] for 1.5 h at 700 V, which heated
the gels to 40 C. After electrophoresis, the gels were covered in
Saran wrap and exposed to a storage phosphor screen. The image
was captured using a Typhoon 9400 (GE Healthcare, Piscataway,
NJ) and the counts of the radiolabelled products quantified using
ImageQuantTM (Amersham Biosciences). A control lane was used
as a standard in order to quantitate the other bands on the gel.
For the repair time course assay of the ICLs a 150 mL master mix
composed of 600 pmol of wild-type hAGT and 20 pmol of DNA
substrate in hAGT buffer was prepared. The sample was placed at
37 ◦C. At every time point 7.5 mL (1 pmol of DNA) was removed
from the master mix and placed in a tube with 9.1 mL of stop
reaction buffer and terminated as above.
Colony forming assay in CHO cells treated with busulfan and
hepsulfam
CHO cells transfected with either the empty pCMV vector or
pCMV-hAGT were grown in a-MEM media (Gibco-Invitrogen,
Carlsbad, CA) containing 26 mM NaHCO3, 10% fetal bovine
serum (Atlanta Biologicals, Lawrenceville, CA), 100 U/ml peni-
cillin and 100 mg ml-1 streptomycin. The cells were maintained by
seeding at 1 ¥ 105 cells/25 cm2 flask at weekly intervals. For colony
forming assays, cells were plated at a density of 106 per 25 cm2
flask and 24 h later were treated with different concentrations of
busulfan (Sigma, St Louis, MO) or hepsulfam (NCI, Frederick,
MD) for 4 h. The cells were washed with phosphate buffered saline
(PBS) twice, and then the medium was replaced with fresh medium
and the cells allowed to grow for another 16–18 h. They were then
replated at densities of 250–1000 cells per 25 cm2 flask and grown
for 6–8 days until discrete colonies had formed. The colonies were
washed with 0.9% saline solution, stained with 0.5% crystal violet
and counted.
Acknowledgements
We thank the Natural Sciences and Engineering Research Council
of Canada (NSERC), the Canada Foundation for Innovation
(CFI), the Canada Research Chair (CRC) program (to CJW) and
grant CA-018137 from the NCI, US Public Health Service (to
AEP) for financial support of this project. FPM is the recipient
of graduate fellowships from NSERC, the Fonds Que´be´cois de la
Recherche sur la Nature et les Technologies (FQRNT) and Groupe
de Recherche Axe´ sur la Structure des Prote´ines (GRASP). We
also thank Mr Nadim Saadeh and Dr Bruce Lennox of McGill
University for ESI-MS analysis.
Binding studies of C145S hAGT using protein titration
Reaction tubes consisting of 5 nM DNA and increasing C145S
hAGT concentrations (ranging from 1 to 35.69 mM) were prepared
in a total solution volume of 20 ml of binding buffer [10 mM Tris-
HCl (pH 7.6), 5 mM DTT and 0.1 mM EDTA]. The samples
were allowed to equilibrate on ice for 30 min. Triplicate samples
were incubated for longer (45 min and 2 h) and yielded similar
results indicating that equilibrium had been attained. Each sample
(0.1 pmol) was loaded on 14 cm x 16 cm, 4 ◦C, preequilibrated na-
tive 6% polyacrylamide (75 : 1) gels. The gels were electrophoresed
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