RNA-selective modification by a platinum(II) complex conjugated to amino- and guanidinoglycosides

Article abstract of DOI:10.1002/anie.200461182

Revving up the RNA response: Binding of the anticancer drug cisplatin (cis-Pt) to nucleic acids is believed to favor DNA over RNA. To "reverse" the nucleic acid selectivity of cis-Pt, amino- and guanidinoglycoside-Pt^II conjugates (1 and 2) have

Full text of DOI:10.1002/anie.200461182

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conjugates have been prepared in which the cis-Pt moiety has
been tethered to high-affinity nucleic acid ligands (for
example, acridine, ethidium bromide, peptides, and short
oligonucleotides).[7] As part of our program aimed at advanc-
ing RNA as a drug target,[8] we have become interested in
devising methods to “reverse” the nucleic acid selectivity of
cis-Pt. Since RNA–protein complexes serve key functions in
the life cycle of many retroviruses, RNA-selective delivery of
reactive PtII complexes may provide a novel mechanism for
the inactivation of pathogen-specific RNA.
HIV-1 replication is critically dependent on the nuclear
export of intron-containing viral RNA by the Rev protein.[9]
The high-affinity Rev binding site within the Rev response
element (RRE) is located at a G-rich bulge that is present in
the minimized construct RRE JW shown in Figure 1.[10] Rev–
RNA-Selective PtII Conjugates
RNA-Selective Modification by a Platinum(ii)
Complex Conjugated to Amino- and
Guanidinoglycosides**
Figure 1. Secondary structures of the RRE constructs used. RRE JW is
a minimized all-ribo Rev-binding RRE construct.[10] All-ribo 3I RRE JW
contains three inosine (I) residues substituting the corresponding G
residues. dRRE JW is an all-deoxyribo DNA analogue of RRE JW. The
numbering used is in accordance with ref. [10].
Jꢀrgen Boer, Kenneth F. Blount, Nathan W. Luedtke,
Lev Elson-Schwab, and Yitzhak Tor*
Binding of the anticancer drug cisplatin (cis-Pt) to nucleic
acids is believed to favor DNA over RNA.[1] Covalent
modification of DNA produces intrastrand cross-links (at
GpG, ApG, and GpNpG; N = A, C, G, or T),[2] and
interstrand cross-links (at GG).[3] Cisplatin reacts with other
cellular components including proteins,[4] phospholipids,[5]
and small molecules.[6] To enhance DNA targeting, numerous
RRE recognition has served as an important paradigm for
exploring the competitive inhibition of RNA–protein inter-
actions.[11] Aminoglycoside antibiotics,[12] guanidinoglyco-
sides,[13] and other small molecules that exhibit RNA over
DNA selectivity have been shown to bind to the RRE and
displace Rev through noncovalent interactions.[14] We
hypothesized that, by using PtII derivatives to covalently
cross-link aminoglycosides and guanidinoglycosides to the
RRE, viral RNA could be permanently damaged. Toward this
end, we have developed RNA-selective PtII conjugates.
Herein we report the synthesis and RRE selectivity of two
conjugates that, to our knowledge, represent the first
examples of RNA-selective cross-linking agents.
Several criteria have guided the design of the glycoside–
PtII conjugates: a) Established RNA-selective scaffolds (for
example, aminoglycosides or guanidinoglycosides) are chosen
to secure preferential RNA over DNA binding; b) the
polycationic scaffold is modified by substitution of a hydroxy
group, rather than a chargeable group that is likely to be
[*] Dr. J. Boer, Dr. K. F. Blount, N. W. Luedtke, L. Elson-Schwab,
Prof. Dr. Y. Tor
Department of Chemistry and Biochemistry
University of California, San Diego
La Jolla, CA 92093-0358 (USA)
Fax: (+1)858-534-0202
E-mail: ytor@ucsd.edu
[**] K.F.B. and J.B. contributed equally to this work. We thank the
National Institutes of Health (Grant nos.: AI47673 to Y.T. and
GM065652 to K.B.), the Universitywide AIDS Research Program
(Grant no.: ID01-SD-027), and the Deutsche Forschungsgemein-
schaft (postdoctoral fellowship to J.B.) for support.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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DOI: 10.1002/anie.200461182
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essential for RNA binding; c) a short linker between the Pt
readily removed, and the desired Boc-protected perguanidi-
nylated 5’’-azido neomycin 7 is obtained in excellent yield
(99%; see Scheme 2). Reduction to the corresponding amine
under Staudinger conditions provides 8 in 75% yield.[19]
Condensation of the free amine with Na,Nb-di-Fmoc-(S)-
2,3-diaminopropionic acid by using TBTU/HOBT, followed
by deprotection of the Fmoc groups with piperidine, gives 9 in
63% yield for the two steps. The reaction of the free chelating
1,2-diamino group with potassium tetrachloroplatinate in a
two-phase CHCl3/H2O system, followed by TFA-mediated
deprotection of the Boc groups, leads to the desired guani-
dinylated aminoglycoside–PtII conjugate 2 as the TFA salt in
94% yield.[20]
To explore the ability of 1 and 2 to form covalent cross-
links with RRE JW, denaturing polyacrylamide gel shift
electrophoresis (DPAGE) has been utilized. Upon titration
of RRE JW with 1 and 2, DPAGE shows multiple retarded
bands, a result indicating that the PtII conjugates covalently
bind the RRE (Figure 2a).[21] Addition of neomycin B, the
parent aminoglycoside, produces no such effect (Fig-
ure 2b).[21] The reaction of 1 and 2 with the RRE is rapid
and the adducts formed are stable under these denaturing
conditions (Figure 2b).[22] According to DPAGE analysis,
both conjugates covalently modify the RRE with comparable
efficiencies (Figure 2a). Importantly, covalent modification
takes place even in the presence of a vast excess of calf thymus
DNA (CT DNA; Figure 2c). Approximately 5000-fold
nucleotide excess of CT DNA must be added to prevent
covalent-adduct formation between the platinated glycosides
1 and 2 and the RRE (Figure 2c). This competition experi-
center and the RNA ligand is chosen to ensure enough
conformational flexibility while maintaining proximity of the
reactive metal center to the RNA-selective scaffold; and d) a
chelating diamine is selected to secure PtII coordination to the
RNA-selective binder. With these considerations in mind, PtII
conjugates of neomycin B (1, Scheme 1) and guanidinoneo-
mycin B (2, Scheme 2) were selected as our first-generation
RNA-selective modifiers.
The synthesis of both derivatives starts with 5’’-2,4,6-
triisopropylbenzenesulfonate N-Boc-protected neomycin
3.[15] Treatment of 3 with excess sodium azide affords the
desired monoazide 4 in 89% yield (Scheme 1).[16] Catalytic
hydrogenation of 4 quantitatively gives the corresponding
amine 5, which is coupled to Na,Nb-di-Fmoc-(S)-2,3-di-
aminopropionic acid by using TBTU and HOBT. Cleavage
of the Fmoc groups under basic conditions affords the
neomycin derivative 6, which presents a bidentate coordina-
tion site for platinum. Treatment with potassium tetrachloro-
platinate in DMF/H2O leads to the formation of the fully
protected aminoglycoside conjugate. Deprotection with 2m
HCl in dioxane/water affords the desired PtII derivative 1 as
the chloride salt in 95% yield (from 6).[16]
The 5’’-azido neomycin derivative 4 is a common pre-
cursor for the synthesis of the neomycin–PtII and guanidino-
neomycin–PtII conjugates, 1 and 2, respectively. Thus, after
removal of the six Boc protecting groups in 4 with 50% TFA/
CH2Cl2, the crude 5’’-azido neomycin is dissolved in MeOH/
NEt3 and treated with excess N,N’-di-Boc-N’’-triflylguanidine,
a powerful guanidinylating reagent.[17,18] Excess reagent is
Scheme 1. Synthesis of 1: a) NaN3, DMF/H2O, 89%; b) H2, Pd/C, 99%; c) 1. Na,Nb-di-Fmoc-(S)-2,3-diaminopropionic acid, TBTU, HOBT, 2,4,6-
collidine, DMF; 2. piperidine, DMF, 80%; d) 1. K2PtCl4, DMF/H2O; 2. 2m HCl/dioxane, 95%. Boc=tert-butoxycarbonyl, DMF=N,N-dimethylform-
amide, Fmoc=9-fluorenylmethoxycarbonyl, HOBT=1-hydroxy-1H-benzotriazole, TBTU=benzotriazol-1-yl-N-tetramethyluronium tetrafluoroborate.
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Scheme 2. Synthesis of 2: a) 1. TFA/CH2Cl2; 2. N,N’-di-Boc-N’’-triflylguanidine, Et3N, MeOH, 99%; b) Ph3P, THF/H2O, 75%; c) 1. Na,Nb-di-Fmoc-
(S)-2,3-diaminopropionic acid, TBTU, HOBT, 2,4,6-collidine, DMF/CHCl3; 2. piperidine, DMF, 63%; d) 1. K2PtCl4, H2O/CH2Cl2, nBu4NCl; 2. TFA/
CH2Cl2, 94%. TFA=trifluoroacetic acid.
ment indicates a significant preference of these PtII–glycoside
conjugates for the RRE over CT DNA.
MALDI-TOF mass spectrometry is a strongly denaturing
technique, these mass spectral data confirm the specific
covalent-adduct formation of platinated glycosides to the all-
ribo RRE JW.[25]
To explore the nature of the modification induced by the
platinated conjugates, nonlabeled RRE JW (20 mm) was
incubated with 1 (1.5 equiv) for 4 h, and the desalted reaction
mixture was analyzed by MALDI-TOF mass spectrometry
(Figure 3a,b). Under these conditions, the primary species
observed (m/z 11914) represents a 1:1 covalent adduct,
1·RRE, with both chloride ions displaced (expected m/z
11911).[23] Smaller amounts of both unmodified RRE JW
(m/z 11023) and doubly conjugated (1)2·RRE (m/z 12806;
expected m/z 12800) are also observed (Figure 3b). As shown
in Figure 3c, a similar reactivity pattern is established for the
guanidinoneomycin–PtII conjugate 2 under the same condi-
tions, where the dominant species observed is the singly
conjugated product 2·RRE (m/z 12164; expected m/z 12164).
As with 1, small amounts of the unmodified RRE JW and
doubly modified product (2)2·RRE (m/z 13304; expected m/z
13303) are detected (Figure 3c). In contrast, treatment of 1
with 3I RRE JW or dRRE JW (see Figure 1 for sequences)
under similar conditions does not yield a significant fraction
of covalent adducts (not shown). Since neomycin displays
lower affinity to these analogous nucleic acids than to
RRE JW,[16] a correlation between specific binding and
conjugation is implied.[24] When it is taken into account that
To identify the modification site of the PtII conjugates 1
and 2 within RRE JW, the mixtures represented by the
spectra in Figures 3b and c were 5’-32P-labeled and fraction-
ated by using preparative 20% DPAGE. A representative
purification gel is shown in Figure 3d. Whereas the 1·RRE
adduct ran as primarily one shifted band, addition of 2 to the
RRE yielded two separable bands (marked 2·RREa and
2·RREb). Once labeled and isolated, these platinated RRE
adducts were characterized by alkaline hydrolysis mapping
(Figure 4). Covalent binding of the platinated glycosides to
the RRE not only increases the mass of the RNA but also
considerably diminishes its overall negative charge. This
reduces the mobility of each hydrolyzed RNA fragment that
contains the PtII conjugate, whereas unmodified RNA frag-
ments migrate identically to the hydrolyzed control. Thus, a
discontinuity in the ladder points to the site of RNA–ligand
conjugation. For 1, this binding site is localized at the 3’-side
of the tetraloop, a fact suggesting coordination to A62, A63,
and G64 (Figure 4a). This putative binding site is consistent
with the coordination to purines that is seen for cis-Pt binding
to DNA.[2a] Interestingly, a distinct gap is observed in the
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Figure 2. DPAGE radiogram with 25 nm 5’-32P-labeled RRE JW.[16]
a) Lane 1: RNA only; lanes 2–9: increasing concentrations of 1 (0.050,
0.10, 0.50, 1.0, 2.5, 5.0, 7.5, and 10 mm, respectively); lane 10: RNA
only; lanes 11–16: increasing concentrations of 2 (0.050, 0.10, 0.50,
1.0, 2.5, and 5.0 mm, respectively). b) Lanes 1–4: RNA with increasing
concentrations of neomycin B (0.625, 1.25, 12.5, and 125 mm, respec-
tively); lanes 5–9: RNA with 625 nm of 1 after incubation (after 0.5, 1,
2, 3, 4 and 5 h, respectively). c) Lane 1: RNA only; lanes 2–8: RRE JW
with 1 mm 1 and increasing concentrations of calf thymus DNA (0, 8.5,
85, 425, 850 mm, 4.25 and 8.5 mm base pairs, respectively); lane 9:
RNA only; lanes 10–17: RRE JW with 1 mm 2 and increasing calf
thymus DNA (0, 8.5, 85, 425, 850 mm, 4.25 and 8.5 mm base pairs,
respectively).
hydrolytic map of 2·RREb (Figure 4a), a result that suggests
an intramolecular cross-link between nucleosides at the
boundaries of the gap, as has been previously observed for
other RNA–RNA cross-links.[26] These results suggest the
formation of an intramolecular cross-link between approx-
imately G48 and G67. This is consistent with the known
propensity of cis-Pt to form cross-strand purine–purine cross-
links.[2a] Examination of the NMR spectrum of the RRE
reveals that the N7 positions of these two nucleotides are
favorably placed for forming this interstrand cross-link
(Figure 4c).[27]
Figure 3. MALDI-TOF MS analysis of 20 mm RRE JW a) alone, b) with
30 mm of 1, and c) with 20 mm of 2. See the Experimental Section for
procedures. d) Denaturing gel purification of 5’-32P-labeled RRE JW and
RRE JW adducts. The two adducts isolated are indicated (2·RREa and
2·RREb).
coefficient of 6550 cmÀ1mÀ1 per base. The extinction coefficient for
all RRE JW constructs was calculated as 371000 cmÀ1mÀ1
.
In summary, new PtII conjugates of neomycin B and
guanidinoneomycin B are found to generate site-specific,
RNA-selective, covalent cross-links to the RRE. The use of
RNA-selective scaffolds, such as aminoglycosides or guanidi-
noglycosides, is demonstrated to impart the necessary RNA
selectivity upon the cis-Pt moiety. This observation serves as
an important precedent for the selective covalent modifica-
tion of therapeutically relevant RNA targets.
DPAGE analysis: For evaluating the cross-linking ability of 1 and
2, RNAwas prefolded by being heated to 658C for 5 minutes and then
cooled to room temperature for 20 min. A final concentration of
25 nm prefolded RRE JW containing
a
trace of 5’-32P-labeled
RRE JW (ꢀ 5000 cpm) was then incubated with the indicated
amount of 1 or 2 in 10 mm sodium phosphate buffer (pH 7.0)
containing 100 mm sodium perchlorate. Where appropriate, calf
thymus DNA was added to the folded RRE JW prior to the addition
of 1 or 2. After 2 h at room temperature, cross-linking reactions were
diluted with an equal volume of formamide loading dye, heated to
908C for 3 min, and loaded immediately onto a 20% denaturing
polyacrylamide gel.
MALDI-TOF MS experiments: A solution of RRE JW (75 mL of
a 40 mm solution) in 10 mm tris(hydroxymethyl)aminomethane (Tris;
pH 7.5) with 50 mm NaCl was heated to 658C for 5 min and cooled at
room temperature for 20 min. This solution was diluted with
Experimental Section
RNA: Oligonucleotides were purchased from Dharmacon Research,
deprotected, and purified by using standard protocols. A solution of
calf thymus DNA was purchased from Gibco BRL (catalogue
no.: 15633-019) and quantified by using an average extinction
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in water (50 mL). Alkaline hydrolysis reactions were
conducted in a 50 mm sodium bicarbonate buffer
(pH 9.0; total volume 8 mL) and contained 5’-32P-
labeled RRE JW (ꢀ 60000 cpm). These reactions
were heated to 908C for 6.5 min and immediately
quenched on ice for 10 min. Following a brief spin,
samples were 1:1 diluted with formamide loading
dye and 1.5 mL were loaded onto a 20% polyacryl-
amide gel. Ribonuclease T1 reactions contained
38000 cpm of each RRE JW adduct and 1 unit of
ribonuclease T1 (Boehringer Mannheim) in a so-
lution of 20 mm sodium citrate (pH 5.0), 1 mm
ethylenediaminetetraacetate (EDTA), 3m urea,
and 0.04 mgmLÀ1 yeast torula RNA (Ambion).
These reactions were incubated at 658C for
30 minutes, then diluted into an equal volume of
formamide loading dye and separated with 20%
denaturing polyacrylamide gels. All gels were run
for approximately 3 h at 15 W and analyzed by using
a Molecular Dynamics Storm Phosphorimager.
Received: July 3, 2004
Published online: January 3, 2005
Keywords: aminoglycosides · antiviral agents ·
.
drug design · platinum · RNA
[1] E. R. Jamieson, S. J. Lippard, Chem. Rev. 1999,
99, 2467 – 2498.
[2] a) A. M. J. Fichtinger-Schepman, J. L. van
der Veer,
J. H. J.
den Hartog,
P. H. M.
Lohman, J. Reedijk, Biochemistry 1985, 24,
707 – 713; b) A. Eastman, Biochemistry 1986,
25, 3912 – 3915.
Figure 4. Alkaline hydrolytic footprint of RRE JW and its platinated adducts.[16] a) “std”
lanes are untreated RNA, “OHÀ” lanes show RNA after alkaline hydrolysis, and “T1”
lanes show RNA after cleavage by ribonuclease T1 under denaturing conditions. The con-
jugation site of 1 is shaded. An interstrand cross-link (dotted line) is observed between
approximately G48 and G67 (shown in red). b) Conjugation sites are shown in the context
of the secondary structure of RRE JW. c) The three-dimensional structure of the RRE
reveals that the N7 positions of G48 and G67 (red space-filling representation) are near
each other in space and well-positioned for forming an interstrand cross-link.
[3] H. Huang, J. Woo, S. C. Alley, P. B. Hopkins,
Bioorg. Med. Chem. 1995, 3, 659 – 669.
[4] R. N. Bose, Mini-Rev. Med. Chem. 2002, 2,
103 – 111.
[5] G. Speelmans, R. W. Staffhorst, K. Versluis, J.
Reedijk, B. de Kruijff, Biochemistry 1997, 36,
10545 – 10550.
[6] T. Ishikawa, F. Ali-Osman, J. Biol. Chem. 1993,
268, 20116 – 20125.
phosphate/perchlorate buffer (75 mL) to yield final concentrations of
10 mm sodium phosphate (pH 7.4), 100 mm sodium perchlorate, and
either 30 mm 1 or 20 mm 2. These reaction mixtures were incubated at
room temperature for 4 h, at which point they were desalted by
passing through a G-25 spin column (Amersham–Pharmacia) that
had been equilibrated with water. After desalting, the reactions were
lyophilized overnight and resuspended in water (20 mL). To confirm
the addition of platinated conjugates, an aliquot was fractionated on a
20% DPAGE gel and visualized with Stains-all (Acros). For MALDI-
TOF MS analysis, 2 mL of each sample was combined with 1 mm
ammonium citrate buffer (PE Biosystems), 1 mL of 75 mm DNA
standard (5’-AGGCATGCAAGCTTGAGTATTCTAT-3’), and 6 mL
of saturated 3-hydroxypiccolinic acid. After desalting with an ion-
exchange resin (PE Biosystems), the sample was spotted onto a gold-
coated MALDI plate and dried on a 558C heat block. MALDI-TOF
[7] Selected examples: a) B. E. Bowler, L. S. Hollis, S. J. Lippard, J.
Am. Chem. Soc. 1984, 106, 6102 – 6104; b) B. D. Palmer, H. H.
Lee, P. Johnson, B. C. Baguley, G. Wickham, L. P. G. Wakelin,
W. D. McFadyen, W. A. Denny, J. Med. Chem. 1990, 33, 3008 –
3014; c) D. Gibson, K. F. Gean, J. Katzhendle, R. Ben-Shoshan,
A. Ramu, I. Ringel, J. Med. Chem. 1991, 34, 414 – 420; d) J.
Whittaker, W. D. McFadyen, G. Wickham, L. P. Wakelin, V.
Murray, Nucleic Acids Res. 1998, 26, 3933 – 3939; e) L. C. Perrin,
C. Culliname, W. D. McFayden, D. R. Phillips, Anti-Cancer Drug
Des. 1999, 14, 243 – 252; f) Y. S. Chen, M. J. Heeg, P. G.
Brauschweiger, W. H. Xie, P. G. Wang, Angew. Chem. 1999,
111, 1882 – 1884; Angew. Chem. Int. Ed. 1999, 38, 1768 – 1769;
g) M. S. Robillard, A. R. P. M. Valentijn, N. J. Meeuwenoord,
G. A. van der Marel, J. H. van Boom, J. Reedijk, Angew. Chem.
2000, 112, 3226 – 3229; Angew. Chem. Int. Ed. 2000, 39, 3096 –
3099; h) Y. Mikata, Y. Shinohara, K. Yoneda, Y. Nakamura, I.
Brudzinska, T. Tanase, T. Kitayama, R. Takagi, T. Okamoto, I.
Kinoshita, M. Doe, C. Orvig, S. Yano, Bioorg. Med. Chem. Lett.
2001, 11, 3045 – 3047; i) S. K. Sharma, L. W. McLaughlin, J. Am.
Chem. Soc. 2002, 124, 9658 – 9659; j) M. S. Robillard, N. P.
Davies, G. A. van der Marel, J. H. van Boom, J. Reedijk, V.
Murray, J. Inorg. Biochem. 2003, 96, 331 – 338; k) R. J. Heetebrij,
M. de Kort, N. J. Meeuwenoord, H. den Dulk, G. A. van der
spectra were collected on
a PE Biosystems Voyager-DE STR
MALDI-TOF spectrometer in positive-ion, delayed-extraction
mode. By calibrating all spectra relative to the +1 and +2 ions of
the internal DNA standard, the observed mass of RRE JW and its
adducts should have a resolution of Æ 4 mass units.
Footprinting analysis: 1·RRE and 2·RRE (3 mL) from the
MALDI-TOF MS analysis described above were labeled with
T4 polynucleotide kinase (New England Biolabs) and purified by
using standard protocols.[16] Purified RNA adducts were resuspended
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Communications
Marel, J. H. van Boom, J. Reedijk, Chem. Eur. J. 2003, 9, 1823 –
1827.
[8] a) Y. Tor, ChemBioChem 2003, 4, 998 – 1007; b) N. W. Luedtke,
Y. Tor in Small Molecule DNA and RNA Binders: From
Synthesis to Nucleic Acid Complexes (Eds.: M. Demeunynck,
C. Bailly, D. Wilson), Wiley-VCH, Weinheim, 2003, pp. 18 – 40.
[9] a) V. W. Pollard, M. H. Malim, Annu. Rev. Microbiol. 1998, 52,
491 – 532; b) A. D. Frankel, J. A. T. Young, Annu. Rev. Biochem.
1998, 67, 1 – 25.
[10] J. L. Battiste, H. Mao, N. S. Rao, R. Tan, D. R. Muhandiram,
L. E. Kay, A. D. Frankel, J. R. Williamson, Science, 1996, 273,
1547 – 1551.
[11] N. W. Luedtke, Y. Tor, Biopolymers 2003, 70, 103 – 119.
[12] M. L. Zapp, S. Stern, M. R. Green, Cell 1993, 74, 969 – 978.
[13] N. W. Luedtke, T. J. Baker, M. Goodman, Y. Tor, J. Am. Chem.
Soc. 2000, 122, 12035 – 12036.
[14] K. Li, T. M. Davis, C. Bailly, A. Kumar, D. W. Boykin, W. D.
Wilson, Biochemistry 2001, 40, 1150 – 1158.
[15] a) K. Michael, H. Wang, Y. Tor, Bioorg. Med. Chem. 1999, 7,
1361 – 1371; b) S. R. Kirk, N. W. Luedtke, Y. Tor, J. Am. Chem.
Soc. 2000, 122, 980 – 981.
[16] See the Supporting Information for detailed experimental
procedures and additional data.
[17] T. J. Baker, N. W. Luedtke, Y. Tor, M. Goodman, J. Org. Chem.
2000, 65, 9054 – 9058.
[18] We have found it is beneficial to install the guanidinium groups
at early stages of the synthetic scheme.
[19] Attempts to reduce the azide by catalytic hydrogenation, a
method that was successfully employed for 4, led to several by-
products.
[20] Both cisplatin conjugates were fully characterized by ESI mass
spectrometry and 1D and 2D 1H and 13C NMR spectroscopy, as
well as by 195Pt NMR spectroscopy.[16] Importantly, the 195Pt
NMR spectra show a single peak at d = À2360 and À2361 ppm,
for 1 and 2, respectively; this indicates a [Cl2PtN2] coordination
sphere.
[21] Noncovalent complexes are denatured under such conditions
and yield no gel-mobility shift. See, for example, Figure 2b
which illustrates the case with neomycin B (a molecule that is
known to associate with the RRE noncovalently).
[22] Note that displacement experiments with BODIPY-labeled
neomycin and CNÀ-treated 1 and 2 indicate that these deriva-
tives have approximately the same affinity to the RRE JW
compared to the nonmodified parent compounds (neomycin B
and guanidinoneomycin B, respectively).[16] This indicates that
the conjugated metal center does not significantly alter the
RNA-binding characteristics of the glycoside skeleton.
[23] The unmodified RNA (m/z 11023) plus
1
(964 for
C26H53N9O13PtCl2) minus two chloride ions (70) yields an
expected mass of m/z 11917. Since the aminoglycoside carries
six positive charges under the acidic conditions of the matrix
used, six fewer protons are required to form a single-charged
positive ion of the Pt-conjugated RRE JW. For an oligonucleo-
tide of this size, MALDI-TOF MS can be expected to show an
accuracy of Æ 4 amu. As such, good agreement between the
expected and experimentally observed mass is established.
[24] Similar mass spectral characterization of 2 with the RRE reveals
its binding and covalent modification are slightly less specific
than those of 1. This agrees with a previous determination of the
RRE-binding specificity of amino- and guanidinoglycosides[13]
and underscores the link between specific binding and platinum
conjugation to the RNA.
[25] Since the mass spectra indicate that both chloride ions are
displaced, a similar mechanism to the coordination of cis-Pt to
DNA is implied.
[26] L. Wang, D. E. Ruffner, Nucleic Acids Res. 1997, 25, 4344 – 4361.
[27] J. A. Ippolito, T. A. Steitz, J. Mol. Biol. 2000, 295, 711 – 717.
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