A Ni(salen)-biotin conjugate for rapid isolation of accessible DNA [11]

Full text of DOI:10.1021/ja0018670

9046
J. Am. Chem. Soc. 2000, 122, 9046-9047
A Ni(Salen)-Biotin Conjugate for Rapid Isolation of
Accessible DNA
Xiang Zhou, Jason Shearer, and Steven E. Rokita*
Department of Chemistry and Biochemistry
UniVersity of Maryland, College Park, Maryland 20742
ReceiVed May 30, 2000
ReVised Manuscript ReceiVed August 7, 2000
Figure 1. Ni(salen) probes for nucleic acid structure.
The oxidation and coordination chemistry of transition metal
ions offer substantial opportunities for selective recognition and
modification of nucleic acids.1 In particular, certain nickel com-
plexes have demonstrated alternative abilities to oxidize and
couple with highly accessible guanine residues in RNA and DNA
after addition of O2 and sulfite2,3 or peracids such as monoper-
oxysulfate or monoperoxyphthalate.4,5 Adducts formed between
guanine and nickel complexes based on a salen ligand (ethylene-
N-N′-bis(salicyaldimine)) strongly inhibit polynucleotide elonga-
tion catalyzed by reverse transcriptase and DNA polymerase and
consequently allow for sensitive detection through primer exten-
sion assays.5,6
The salen ligand serves two functions by activating the redox
chemistry of the bound Ni(II) and generating a ligand-centered
radical for addition to guanine.5,7 Direct coordination between a
transient Ni(III) intermediate and accessible N7 sites on guanine
appears to enhance the specificity.8 Previous investigations have
focused on the water-soluble salen complex 1, NiTMAPES
(Figure 1), but a wide range of useful derivatives can be
envisioned including a salen-peptide hybrid.3,5,9 These nickel
salens appear unique in their ability to couple with their targets
rather than promote direct strand scission as common to the salen
complexes of Mn, Co, and Cu.5b
Although footprinting well-defined polynucleotides with NiT-
MAPES may be routine with piperidine cleavage or primer
extension, obtaining the actual products of coupling can be an
arduous task. As the heterogeneity of the system increases, even
footprinting becomes difficult. This type of problem is common
in molecular biology, and its solution often relies on a broad range
of techniques based on biotin. Labeling, detecting, and purifying
any biotinylated species regardless of its complexity is made
possible by the extraordinary affinity of biotin for avidin and
streptavidin.10,11 The properties of a combined Ni(salen)-biotin
Figure 2. Synthesis of a Ni(salen)-biotin conjugate with solubility in
water. Reagents and conditions: (a) Et3N, ClCOOEt, NH3, THF, 10 °C
to room temperature, 56%. (b) (CF3CO)2O, Et3N, THF, N2, 0° C, 87%.
(c) H2, Ra-Ni, 6.8 atm, NH3, MeOH, 3 days, 72%. (d) H2, Pd/C, MeOH,
room temperature, overnight, 82%. (e) 2,4-dihydroxybenzaldehyde, Ni-
(OAc)2, EtOH, reflux, N2, 3 h, 78%. (f) TFA, CH2Cl2, room temperature,
0.5 h, 86%. (g) BNHS, Et3N, DMF, 4° C, 3 days, 22%. (h) Br(CH2)3N-
(CH3)3Br, DMF, room temperature, 3 days, 80%.
conjugate should then have the potential to diagnosis and isolate
genomic sequences that contain guanine residues in unusual and
accessible structures. This communication describes the first
synthesis of such a conjugate and its initial characterization with
a model oligonucleotide.
(1) Sigel, H.; Sigel, A., Eds. : Metal Ions in Biological Systems; Marcel
Dekker: New York, 1996; Vols. 32 and 33. (b) Pyle, A. M.; Barton, J. K.
Prog. Inorg. Chem. 1990, 38, 413-475.
Early synthetic targets were designed in direct analogy to
TMAPES and were expected to support a range of strategies for
biotinylation. However, the instability and low reactivity of the
essential intermediates precluded this scheme. These problems
were avoided in a subsequent approach that relied on a triamine
first developed by the laboratory of Bailly for use in construction
of Cu(salen) derivatives.12 NR-Z-Nꢀ-BOC-L-lysine 3 was converted
(2) Muller, J. G.; Hickerson, R. P.; Perez, R. J.; Burrows, C. J. J. Am.
Chem. Soc. 1997, 119, 1501-1506.
(3) Stemmler, A. J.; Burrows, C. J. J. Am. Chem. Soc. 1999, 121, 6956-
6957.
(4) Burrows, C. J.; Rokita, S. E. Acc. Chem. Res. 1994, 27, 295-301. (b)
Burrows, C. J.; Rokita, S. E. In Metal Ions in Biological Systems; Sigel, H.,
Sigal, A., Eds.; Dekker: New York, 1996; Vol. 32, Chapter 18, pp 537-560.
(c) Rokita, S. E.; Burrows, C. J. In Current Protocols in Nucleic Acid
Chemistry; Beaucage, S. I., Bergstrom, D. E., Glick, G., Jones, R. A., Eds.;
Wiley: New York, 2000; Chapter 6.4.
to its nitrile derivative 4 as described previously (Figure 2).12
A
subsequent procedure requiring hydrogenation under high pressure
was avoided by use of two sequential hydrogenations in the
presence of Raney nickel and then Pd/C to yield the diamine 5
(see Supporting Information for experimental details). The BOC-
protected salen derivative 6 was formed by condensation of this
diamine under N2 with 2 equiv of 2,4-dihydroxybenzaldehyde in
the presence of Ni(OAc)2. Deprotection by standard conditions
(dry TFA/CH2Cl2) and coupling with the commercially available
(5) Muller, J. G.; Paikoff, S. J.; Rokita, S. E.; Burrows, C. J. J. Inorg.
Biochem. 1994, 54, 199-206. (b) Muller, J. G.; Kayser, L. A.; Paikoff, S. J.;
Duarte, V.; Tang, N.; Perez, R. J.; Rokita, S. E.; Burrows, C. J. Coord. Chem.
ReV. 1999, 185-186, 761-774.
(6) Woodson, S. A.; Muller, J. G.; Burrows, C. J.; Rokita, S. E. Nucleic
Acids Res. 1993, 21, 5524-5526. (b) Pan, J.; Woodson, S. A. J. Mol. Biol.
1998, 280, 597-609.
(7) Goldsby, K. A. J. Coord. Chem. 1988, 19, 83-90. (b) Goldsby, K. A.;
Blaho, J. K.; Hoferkamp, L. A. Polyhedron 1989, 8, 113-115.
(8) Shih, H.-C.; Kassahun, H.; Burrows, C. J.; Rokita, S. E. Biochemistry
1999, 38, 15034-15042.
(11) Diamonds, E. P.; Christopoulos, T. K. Clin. Chem. 1991, 37, 625-
636. (b) Wilchek, M.; Edward, E. A., Eds. Methods Enzymol. 1990, 184, entire
volume.
(12) Routier, S.; Bernier, J.-L.; Waring, M. J.; Colson, P.; Houssier, C.;
Bailly, C. J. Org. Chem. 1996, 61, 2326-2331. (b) Routier, S.; Cotelle, N.;
Catteau, J.-P.; Bernier, J.-L.; Waring, M. J.; Riou, J.-F.; Bailly, C. Bioorg.
Med. Chem. 1996, 4, 1185-1196.
(9) Shearer, J. M.; Rokita, S. E. Bioorg. Med. Chem. Lett. 1999, 9, 501-
504.
(10) Launer, H. F.; Fraenkel-Conrat, H. J. Biol. Chem. 1951, 193, 125-
132. (b) Green, N. M. Biochem. J. 1963, 89, 585-620. (c) Green, N. M. AdV.
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10.1021/ja0018670 CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/31/2000

Communications to the Editor
J. Am. Chem. Soc., Vol. 122, No. 37, 2000 9047
(Figure 3, lane 4). This yield decreased to 5% when the ionic
strength of the mixture was increased by addition of 100 mM
NaCl. In either case, reaction remained specific for the sole
guanine residue as indicated below. Biotinylated DNA generated
on a preparative scale sufficient for mass spectrometry was
conveniently separated from its parent oligonucleotide with a
monomeric avidin matrix (Pierce) and desalted with reverse-phase
(C-18) chromatography (Figure 3, lane 5). These same procedures
can also be expected to function equivalently on an analytical
scale. Treatment of the isolated adduct with hot piperidine was
subsequently investigated since this condition had previously
induced a diagnostic strand scission of adducts formed by
NiTMAPES 1.5 The product formed by conjugate 2 demonstrated
similar lability in the presence of hot piperidine. The resulting
oligonucleotide fragment comigrated with the guanine standard
produced by Maxam-Gilbert sequencing (Figure 3, lanes 6 and
7, respectively).14 As expected, the parent oligonucleotide exhib-
ited no sensitivity to piperidine (Figure 3, lane 8).
Composition of the isolated adduct was confirmed by mass
spectral analysis. Electrospray mass spectrometry identified a
major species of mass 6377 that is consistent with the oligo-
nucleotide + Ni(salen) + oxygen. The additional oxygen was
anticipated from MMPP-dependent oxidation of the biotin sulfide.
Incubation of the unconjugated biotin under conditions analogous
to the DNA experiments above led to formation of biotin
R-sulfoxide as indicated by comparison to a standard produced
by known reaction with H2O2.15 This oxidation did not signifi-
cantly interfere with avidin affinity chromatography since the
R-sulfoxide product retains high affinity for avidin in contrast to
the â-sulfoxide derivative.16
Figure 3. Phosphorimage of a denaturing polyacrylamide gel (20%, 7
M urea) used to identify the high molecular weight conjugate formed
between DNA and Ni(salen)-biotin. The 5′-[32P] labeled oligodeoxy-
nucleotide (18′mer), 5′-d(AAAATATCAGATCTAAAA) (12 µM, 6 nCi),
in 10 mM sodium phosphate pH 7 (lane 1) was alternatively incubated
with Ni(salen)-biotin 2 (50 µM, lane 2), MMPP (120 µM, lane 3), and
their combination (lane 4). Adduct formation was repeated on a
preparative scale, and the product was isolated from a monomeric avidin
affinity column for mass spectrometry. This material was also 5′-[32P]
labeled and analyzed before (lane 5) and after piperidine treatment (0.2
M, 90° C, 30 min, lane 6). A standard G-lane was generated by dimethyl
sulfate as described by Maxam and Gilbert (lane 7).14 As a control, the
parent oligonucleotide was also treated with piperidine (0.2 M, 90° C,
30 min, lane 8).
The ability of the nickel complex to deliver a biotin tag to
accessible guanine residues should allow for future isolation and
characterization of noncanonical structures in complex systems
such as chromatin. Few alternatives are currently available that
share both a dependable conformational specificity and an easy
assimilation into the wide ranging and powerful techniques based
on biotin recognition.
N-hydroxysuccinimide ester of biotin (BNHS) produced the
neutral and water-insoluble derivative 7. The obligatory solubility
in water was achieved through alkylation of the two free phenolic
oxygens with 3-bromopropyltrimethylammonium bromide.13 The
desired perchlorate salt of 2 was precipitated by addition of KClO4
and recrystallized from MeOH/Et2O. Data from electrospray mass
1
spectroscopy as well as H and 13C NMR were consistent with
Acknowledgment. This research was supported in part by the National
Institute of Health (GM-47531). We thank Ms. R. P. Hickerson, Ms. O.
Kornyushynay, Dr. J. G. Muller, and Professor C. J. Burrows for helpful
discussion and technical assistance.
the desired product.
Incubation of a model oligonucleotide (18′mer, one central G)
with the Ni(salen)-biotin conjugate 2 (50 µM) or the magnesium
salt of monoperoxyphthalic acid (MMPP) (120 µM) in 10 mM
sodium phosphate pH 7 generated no apparent products as
detected by denaturing polyacrylamide gel electrophoresis (Figure
3, lanes 2 and 3). However, their combined presence produced a
high molecular weight DNA adduct in approximately 60% yield
Supporting Information Available: Synthesis and characterization
of 4-7 and ESI-MS data for oligonucleotide adduct (PDF). This material
is available free of charge via the Internet at http://pubs.acs.org.
JA0018670
(14) Maxam, A. M.; Gilbert, W. Methods Enzymol. 1980, 65, 499-560.
(15) Melville, D. B. J. Biol. Chem. 1954, 208, 495-500.
(16) Garlick, R. K.; Giese, R. W. Biochem J. 1990, 268, 611-613.
(13) Kayser, L. A., M.S. Thesis, University of Utah, 1997.