Synthesis and molecular modeling of a nitrogen mustard DNA interstrand crosslink

Article abstract of DOI:10.1002/chem.201002041

Nitrogen mustard reloaded: Over 60 years after nitrogen mustards (NMs) were the first agents used to treat tumors by chemotherapy, we provide a method to generate the main DNA adduct formed by NMs and validate them by using molecular dynamics simulations

Full text of DOI:10.1002/chem.201002041

DOI: 10.1002/chem.201002041
Synthesis and Molecular Modeling of a Nitrogen Mustard DNA Interstrand
Crosslink
Angelo Guainazzi,[a] Arthur J. Campbell,[b] Todor Angelov,[c] Carlos Simmerling,*[b] and
Orlando D. Schꢀrer*[a]
Dedicated to Prof. Francis Johnson on the occasion of his 80th birthday
DNA interstrand crosslinks (ICLs) are formed by bifunc-
tional agents with the ability to covalently link two strands
of duplex DNA. ICLs are extremely cytotoxic, since they
block essential processes such as DNA replication and tran-
scription. Based on these properties, agents such as nitrogen
mustards (NM) or cisplatin are widely used in cancer
Here we report the synthesis of a stable NM ICL isostere
(2) and use atomic detail simulations to show that it recap-
tures the structural and dynamic properties of the native
NM ICL 1. To obtain the NM ICL isostere we used a strat-
egy that we recently developed for the synthesis of major
groove ICLs.[4]
chemoACHTUNGTRENNUNG
therapy.[1] ICLs induce a number of biological re-
sponses that counteract the therapeutic effects of crosslink-
ing agents. However, the elucidation of the mechanism by
which these responses cause resistance in tumor cells has
been hampered by the lack of efficient methods to generate
ICLs formed by antitumor agents. Current approaches
toward the synthesis of site-specific ICLs have yielded
mostly models of the clinically relevant lesions or ICLs
formed by endogenous agents.[2] A number of recent studies
have shown that ICLs with different structures are pro-
cessed in distinct ways (see reference [3] for some recent re-
views), emphasizing the need to generate substrates contain-
ing the ICLs formed by the clinically important drugs.
This approach involves a double reductive amination re-
action of two acetaldehyde functionalities (5) linked to the
7-position of G residues on complementary strands of
dsDNA. One dG residue was substituted with 7-deaza-2’-de-
oxyguanosine to counteract the inherent lability of the gly-
cosidic bond in N7-alkylated guanines.[5] This approach al-
lowed the generation of a six-atom ICL by coupling two
acetaldehyde groups with hydrazine. However, we were un-
successful in generating ICL 3 with the five-atom bridge
found in NM ICLs[6] (Scheme 1), possibly because the reduc-
tive amination with ammonia was not powerful enough to
introduce the strain in the DNA caused by the ꢀ7.5 ꢀ
bridge of the NM.[4,7]
[a] A. Guainazzi, Prof. O. D. Schꢁrer
Department of Pharmacological Sciences and Chemistry
Stony Brook University, Stony Brook
NY 11794-3400 (USA)
Fax : (+1)631-632-7546
E-mail: orlando@pharm.stonybrook.edu
[b] A. J. Campbell, Prof. C. Simmerling
Department of Chemistry and Center for Structural Biology
Stony Brook University, Stony Brook
NY 11794-3400 (USA)
Fax : (+1)631-632-1555
E-mail: carlos.simmerling@gmail.com
[c] Dr. T. Angelov
Institute for Molecular Cancer Research
University of Zꢂrich, 8057 Zꢂrich (Switzerland)
We reasoned that an ICL isosteric to those formed by
NM might be formed by reaction of hydrazine with an acet-
aldehyde (5) and a formyl aldehyde derivative (4) of deaza-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201002041.
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COMMUNICATION
Scheme 2. Synthesis of the formyl aldehyde 4: a) vinyl-Sn(Bu)3,
Pd[P(Ph)3]4, toluene, 908C, 43 h, 70%; b) pyridine-2-carboxaldoxime,
N,N,N’,N’-tetramethylguanidine, dioxane, DMF, RT, 42 h, 88%;
c) NaOMe, THF, RT, 5 h, 97%; d) TBDMS-Cl, imidazole, DMF, RT,
16 h, 93%; e) OsO4, NMMO, THF, 08C, 3.5 h, 63%; f) Ac2O, pyridine,
RT, 1 h, 82%; g) TBAF, AcOH, THF, RT, 20 h, 79%; h) DMTr-Cl, pyri-
dine, RT, 1 h, 74%; i) iPr2NP(Cl)OC2H4CN, DIEA, CH2Cl2, RT, 1 h,
72%; j) oligonucleotide synthesis; k) 33% NH4OH, 508C, 12 h; l) an-
nealing; m) NaIO4, RT, 12 h. TBDMS-Cl=tert-butyldimethylsilyl chlo-
ride; NMMO=N-methylmorpholine oxide; TBAF=tetra-n-butylammo-
nium fluoride; DMTr-Cl=4,4’-dimethoxytrityl chloride; DIEA=N,N-
Scheme 1. Formation of NM ICLs by reductive amination was not suc-
cessful using two acetaldehyde precursors 5 and NH4Cl, prompting us to
explore the generation of isostere 2 by linking precursors 4 and 5 with
hydrazine.
diisopropylethylACTHNUTRGNEaNUG mine; ibu=isobtuyryl.
guanine, exploiting the higher reactivity of hydrazine over
ammonia (Scheme 1).[4]
We synthesized formyl aldehyde 4 in an analogous fashion
to the previously reported synthesis of acetaldehyde 5,
masking the aldehyde as a protected diol during solid-phase
DNA synthesis.[4,8] The synthesis started with vinylation of a
protected 7-iodo-7-deazaguanine derivative (6) using a Stille
coupling reaction (Scheme 2) to give compound 7. Oxida-
tion of the allyl group to the diol (8), protection, and func-
tionalization yielded phosphoramidite 9. The aldehyde pre-
cursors were incorporated into complimentary 20-mer oligo-
nucleotides in a 5’-dACTHNUGRTENUNG(GNC) sequence (the preferred se-
quence context for NM ICL formation[6]) by solid-phase syn-
thesis, and the oligonucleotides were deprotected and
annealed.
Figure 1. Analysis of ICL formation by denaturing PAGE with methylene
blue staining. The duplexes, amine used and position of single-stranded
or ICL-containing DNA are indicated. The sequences used were 5’-
d(GTCACTGGTAXACAGCATTG) and 5’-d(CAATGCTXTCTAC-
CAGTGAC) where X represents the modified G. The small amount of
bands running as duplexes in lanes 2 and 5 are likely due to residual
amount of duplex that was not denatured during electrophoresis.
Following oxidation of the diols using periodate, the two
aldehydes were coupled by double reductive amination with
hydrazine and NaBH3CN.[4] The reaction along with appro-
priate controls was analyzed by denaturing PAGE.
As already discussed,[4] ICL formation from two acetalde-
hyde precursors was successful with hydrazine, but not with
ammonium acetate (Figure 1, lanes 2 and 3). However, reac-
tion of a duplex containing 4 and 5 with hydrazine led to
formation of the desired five-atom ICL 2, evidenced by a
band with the same mobility as the previously analyzed
crosslink (Figure 1, lanes 3 and 4). Although the yield of the
five-atom ICL was lower than that of the six-atom ICL
(ꢀ25% vs. ꢀ75%), we were able to isolate the product by
gel purification and electroelution in amounts exceeding
100 nmol. For simplicity of analysis we also synthesized ICL
2 in an 11-mer duplex and unambiguously identified it as
the desired product by ESI-MS (m/z calcd: 6741.23, found:
6741.6, Figure S1 in the Supporting Information). If either
aldehyde precursor was present only on one strand of the
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O. D. Schꢁrer, C. Simmerling et al.
duplex, no significant amounts of the slower migrating spe-
cies were observed, further demonstrating the specificity of
ICL formation (Figure 1, lanes 1 and 5).
twist[11] of the two crosslinked base pairs (Table S1 and Fig-
ure S2 for sequence, in the Supporting Information). Since
molecular mechanics force fields have limited accuracy, it is
more important to note the trends rather than the specific
values. It is well accepted that differences are more reliable
in simulations than absolute values, due to cancellation of
systematic error. The crosslinked guanine 16 that neighbors
the large purine ring of adenine (residue 17) accommodates
the crosslink by buckling of the 7:16 base pair (C: À3.5Æ
0.28, 1: À13.2Æ0.18, and 2: À21.8Æ0.68). The crosslinked
guanine 5 that neighbors the small pyrimidine ring of thy-
mine 6 has more room to move and accommodates the ICL
by an increased propeller twist of the 5:18 base pair (C:
À13.2Æ0.18, 1: À22.3Æ0.28, and 2: À26.9Æ0.88). The addi-
tion of a covalent bridge between both strands of DNA
nearly doubles residue to residue correlation in the central
region of the duplex. Residues 5 and 16 have a correlation
coefficient (r) of 0.29Æ0.04, 0.62Æ0.01, and 0.58Æ0.02 for
C, 1, and 2 respectively (Figure S2 in the Supporting Infor-
mation). ICLs 1 and 2 have a substantial decrease in flexibil-
ity (Figure 2, top) when compared to the uncrosslinked ref-
erence CNT, resulting in the formation of a slight kink in
the duplex of both 1 and 2 to accommodate the ICL
(Figure 2, bottom).
Molecular modeling was used to validate ICL 2 as a
model for NM 1 and to compare the structural consequen-
ces of the two ICLs with the uncrosslinked control (C) in
identical 11-mer sequences. We used the Amber simulation
package with atomic detail and explicit water (see Support-
ing Information) to validate that 2 gives similar amounts of
distortion when compared to the NM ICL 1 and consistent
differences when both are compared to C.[9] Two indepen-
ACHTUNGTRENNUNGdent simulations of 50 ns were run for each of the 1, 2, and
C systems. The duplex was stable throughout all six simula-
tions.
The N7 to N7 distances of the NM ICL 1 overlapped well
with the C7 to C7 distances of 2; both are restricted as com-
pared to C, which samples a broader range (Figure 2, top).
The decreased distance between the two crosslinked bases
has a direct effect on the local distortion around the ICL,
and is also influenced by the sequence (Figure 2 bottom,
and Table S1 in the Supporting Information). One measure
of distortion is to examine the buckling and propeller
To test for local duplex bending at the crosslink site we
measured an angle that would encompass the tightening of
the 7- to 7-position of residues 5 and 16 (Figure 2, top). The
smaller angles sampled by 1 and 2 suggest that the analogue
slightly bends the duplex outside the crosslink in the same
manner that the NM ICL simulations do (Figure 2, bottom).
The slight bending of the central region of the ICL simula-
tions of 1 and 2 compared to C is qualitatively similar to
past experimental work.[7]
Circular dichroism (CD) spectra were recorded to gain
experimental insight into to what extent the NM ICLs con-
taining oligonucleotide 2 deviates from B-form DNA. The
CD spectra of 2 and an unmodified duplex of the same se-
quence displayed the characteristic features of B-form DNA
(Figure S4 in the Supporting Information), consistent with
our molecular dynamics simulations, indicating the NM ICL
induces only a minor bend in the DNA. It has been shown
that only more dramatic distortions, such as the ones in-
duced by cisplatin ICL result in significant changes in CD
spectra.[2j,12]
In summary, we describe the synthesis of the stable NM
ICL analogue 2 by post-synthetic double reductive amina-
tion. Although this ICL has three atoms substituted with re-
spect to the NM ICL 1 (the two N at the 7-positions of dG
by C and one C by N in the hydrazine-formed bridge), our
molecular dynamics simulations show that the two ICLs
affect DNA structure and motion in equivalent ways. The
availability of stable, site-specific NM ICLs will enable stud-
ies of the structural consequences and biological responses
induced by NM ICLs,[13] more than sixty years after NMs
were the first agents to be used in cancer chemotherapy.[14]
Such studies will provide new insights into how tumors
become resistant to treatment by crosslinking agents.
Figure 2. Data from MD simulations of 2 (black), 1 (blue) and C (red).
Top: Distance measurement marked in pink, C/N7 to C/N7 atoms be-
tween residues 5 and 16. Bottom: Center of mass angle measurement, in
which each point is defined by the heavy atoms of the base pair (high-
lighted in blue in inset picture): point 1 represents 4A and 19T, point 2
represents 6T and 17A and point 3 represents 8A and 15T.[10]
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Oligonucleotide Synthesis
COMMUNICATION
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Experimental Section
Crosslink formation: A solution of the single-strand oligonucleotides
(25 nmol) in NaCl (125 mL, 10 mm) was heated to 958C and allowed to
cool to room temperature over a period of 4 h to allow for duplex forma-
tion. After addition of sodium phosphate buffer (15 mL, 1m, pH 5.4) and
NaIO4 (10 mL, 50 mm) the reaction mixture was kept in the dark over-
night at 48C. Excess NaIO4 was removed by centrifugation through Mi-
crocon columns with a 3 K cutoff (Millipore). Then aqueous hydrazine
(10 mL, 5 mm) and NaCNBH3 (10 mL, 0.5m) were added and the reaction
mixture was left overnight at room temperature in the dark. ICL forma-
tion was assessed by electrophoresis on a denaturing 20% polyacryl-
amide gel. The band containing the crosslinked oligonucleotide was ex-
cised from the gel and the DNA was extracted by electroelution using d-
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Acknowledgements
We are grateful to Robert Rieger and the Proteomic Center of Stony
Brook University for MS spectral analyses supported by grant NIH/
NCRR1 S10 RR023680-1. Support for this project was provided by NSF
0549370 (A.J.C.), NIH GM6167803 (C.S.), the New York State Office of
Science and Technology and Academic Research (NYSTAR), C040069
(O.D.S.), the Swiss Cancer League OCS-01413-080-2003 (O.D.S.) and su-
percomputer resources through the NSF Teragrid MCA02N028 (C.S.),
DOE (DE-AC02–98CH10886), and the State of New York (C.S.).
Keywords: antitumor agents · DNA · molecular dynamics ·
nitrogen mustard · oligonucleotides
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Received: July 19, 2010
Published online: September 14, 2010
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