Guanidinoglycosides: A novel family of RNA ligands [21]

Full text of DOI:10.1021/ja002421m

J. Am. Chem. Soc. 2000, 122, 12035-12036
Scheme 1. Example of Guanidinoglycoside Synthesisa
12035
Guanidinoglycosides: A Novel Family of RNA
Ligands
Nathan W. Luedtke, Tracy J. Baker, Murray Goodman,* and
Yitzhak Tor*
Department of Chemistry and Biochemistry
UniVersity of California, San Diego
La Jolla, California 92093-0358
ReceiVed July 5, 2000
Aminoglycoside antibiotics are a family of structurally diverse
polyamines that have been a central focus of small molecule-
RNA recognition studies over the past decade.1 The antibacterial
activity of these compounds is believed to derive from their
interaction with prokaryotic rRNA.2 More recently, aminoglyco-
sides have been synthetically modified in ongoing efforts to
discover new antiviral and antitumor agents.3 Aminoglycosides
show effective selectivity in their preferential binding of RNA
over DNA,4 but are relatively nonselective in their differentiation
between natural RNAs. Aminoglycosides are reported to bind a
wide range of unrelated RNA structures; including 16S and 18S
rRNAs,2,5 mRNA transcripts,3c tRNA,6 catalytic RNAs,7 and viral
RNAs.8 This general affinity for RNA is related to the ability of
aminoglycosides to bind RNA through electrostatic interactions
mediated by ammonium groups.9
a Reaction conditions: (a) 15 equiv of 6 in 1,4-dioxane/H2O (5:1), 15
equiv of NEt3, 3d, rt. b TFA/CH2Cl2 (1:1).
The guanidinium group plays a key role at many RNA-protein
binding interfaces, including the complexes formed between
transcriptional elongation factors with mRNA, tRNA synthetases
with tRNAs, ribosomal proteins with rRNA, and viral regulatory
proteins with their cognate RNA binding sites.10 In contrast to
ammonium groups, guanidinium groups are highly basic, planar,
and exhibit directionality in their H-bonding interactions. We
hypothesized that the RNA affinity and selectivity of aminogly-
coside-based ligands can be increased by replacing the ammonium
groups with guanidinium groups. In this report, we disclose a
new family of RNA ligands, termed “guanidinoglycosides”, in
which all of the ammonium groups of the natural aminoglycoside
antibiotics have been converted into guanidinium groups (Figure
1).11
butoxycarbonyl-N′′-triflylguanidine 6, a new guanidinylating
reagent.12 This novel reagent facilitates the guanidinylation of
polyfunctional amines in aqueous media and in high yields. For
example, when tobramycin (3a) is treated with an excess of 6 in
a 1,4-dioxane/water mixture, the Boc-protected, fully guanidin-
ylated derivative is obtained (Scheme 1, step a). Subsequent
deprotection of the Boc groups affords guanidino-tobramycin (step
b).13
The HIV-1 Rev-RRE interaction was used to examine the
impact of guanidinylation upon RNA binding, and to probe the
potential antiviral activity of these compounds. The binding of
Rev to the RRE (Rev response element) is responsible for the
export of unspliced and singly spliced HIV genomic RNA out of
the host nucleus.14 This essential protein-RNA interaction re-
mains an important, and nonutilized, therapeutic target. The high-
affinity Rev binding site on the RRE has been localized to the
purine-rich bulge shown in Figure 2.15 The binding of Rev to the
The preparation of guanidinoglycosides has been accomplished
through the treatment of aminoglycosides with N,N′-di-tert-
* Corresponding author. E-mail: ytor@ucsd.edu.
RRE is governed by the arginine-rich fragment, Rev34-50.
16 Key
(1) (a) Michael, K.; Tor, Y. Chem. Eur. J. 1998, 4, 2091-2098. (b) Walter,
F.; Vicens, Q.; Westhof, E. Curr. Opin. Chem. Biol. 1999, 3, 694-704.
(2) Moazed, D.; Noller, H. F. Nature 1987, 327, 389-394.
(3) (a) Kirk, S. R.; Luedtke, N. W.; Tor, Y. J. Am. Chem. Soc. 2000, 122,
980-981. (b) Litovchick, A.; Evdokimov, A. G.; Lapidot, A. Biochemistry
2000, 39, 2838-2852. (c) Sucheck, S. J.; Greenberg, W. A.; Tolbert, T. J.;
Wong, C.-H. Angew. Chem., Int. Ed. 2000, 39, 1080-1084.
(4) Chen, Q.; Shafer, R. H.; Kuntz, I. D. Biochemistry 1997, 36, 11402-
11407.
guanidinium groups make direct contacts with the RNA platform
and are essential for the specific binding of Rev to the RRE.17
Fluorescence anisotropy has been employed to determine the
affinity of the new derivatives to the RRE in solution.13 The RRE-
bound fluorescent Rev peptide has a slower Brownian tumbling
motion relative to the free peptide. Upon displacement of the
fluorescein-labeled Rev peptide from the RRE by an inhibitor, a
decrease in the anisotropy value is observed. Table 1 (column a)
compares the IC50 values of the guanidinoglycosides to the
aminoglycosides.18 Guanidinylation of kanamycin A, kanamycin
(5) Griffey, R. H.; Hofstadler, S. A.; Sannes-Lowery, K. A.; Ecker, D. J.;
Crooke, S. T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10129-10133.
(6) Kirk, S. R.; Tor, Y. Bioorg. Med. Chem. 1999, 7, 1979-1991.
(7) See ref 1b for a summary of the 11 known autocatalytic RNAs that
bind aminoglycosides.
(8) Zapp M. L.; Stern, S.; Green, M. R. Cell 1993, 74, 969-978. Mei,
H.-Y.; Galan, A. A.; Halim, N. S.; Mack, D, P.; Moreland, D. W.; Sanders,
K. B.; Truong, H. N.; Czarnik, A. W. Bioorg. Med. Chem. Lett. 1995, 5,
2755-2760.
(12) Feichtinger, K.; Sings, H. L.; Baker, T. J.; Matthews, K.; Goodman,
M. J. Org. Chem. 1998, 63, 8432-8439. Baker, T. J.; Goodman, M. Synthesis
1999, 1423-1426.
(9) (a) Hendrix, M.; Priestley, E. S.; Joyce, G. F.; Wong, C.-H. J. Am.
Chem. Soc. 1997, 119, 3641-3648. (b) Wang, H.; Tor, Y. J. Am. Chem. Soc.
1997, 119, 8734-8735. (c) Tor, Y.; Hermann, T.; Westhof, E. Chem. Biol.
1998, 5, R277-R283.
(13) See Supporting Information for experimental details.
(14) Pollard, V. W.; Malim, M. H. Annu. ReV. Microbiol. 1998, 52, 491-
532. Hope, T. J. Arch. Biochem Biophys. 1999, 365, 186-191. Frankel, A.
D.; Young, J. A. T. Annu. ReV. Biochem. 1998, 67, 1-25.
(10) For a review of RNA recognition by arginine-rich peptides, see: Weiss,
M. A.; Narayana, N. Biopolymers 1998, 48, 167-180. For a review of
Protein-RNA Recognition, see: De Guzman, R. N.; Turner, R. B.; Summers,
M. F. Biopolymers 1998, 48, 181-195.
(15) Holland, S. M.; Chavez, M.; Gerstberger, S.; Venkatesan, S. J. Virol.
1992, 66, 3699-3706. Tilley, L. S.; Malim, M. H.; Tewary, H. K.; Stockley,
P. G.; Cullen, B. R. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 758-762.
(16) Kjems, J.; Canlan; B. J.; Frankel, A. D.; Sharp, P. A. EMBO J. 1992,
11, 1119-1129.
(11) For site-specific monoguanidinylation of kanamycin A and gentamicin,
see: Steicher, W.; Loibner, H.; Kildebrandt, J.; Turnowsky, F. Drugs Exp.
Clin. Res. 1983, 9, 591-598. For monoguanidinylation of amikacin, see:
Hoshi, H.; Aburaki, S.; Yamasaki, T.; Naito, T.; Kawaguchi H. J. Antibiot.
1991, 44, 680-682.
(17) Tan, R.; Chen, L.; Buettner, J. A.; Hudson, D.; Frankel A. D. Cell
1993, 73, 1031-1040. Tan, R.; Frankel, A. D. Biochemistry 1994, 33, 14579-
14585. Battiste, J. L.; Mao, H.; Rao, N. S.; Tan, R.; Muhandiram, D. R.;
Kay, L. E.; Frankel, A. D.; Williamson, J. R. Science 1996, 273, 1547-1551.
10.1021/ja002421m CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/17/2000

12036 J. Am. Chem. Soc., Vol. 122, No. 48, 2000
Communications to the Editor
Figure 1. Structures of aminoglycosides (1a-5a) and guanidinoglycosides (1b-5b).
the IC50 values determined by this method. By comparing IC50
values measured in the absence of any competitors to the values
measured in the presence of DNA or RNA, a quantitative measure
for the relative selectivity of each ligand can be made.21
Aminoglycosides have a substantial affinity to poly A‚poly U
duplex RNA (compare column b to c, Table 1).22 Dividing the
IC50 values in column c by column b produces a ratio proportional
to the RRE selectivity of each compound (Table 1, column e).
Upon guanidinylation of kanamycin A, kanamycin B, tobramycin,
and paromomycin, this ratio decreases, suggesting that these
compounds are more selective for the RRE than their amino
precursors.23 For neomycin, this ratio increases upon guanidin-
ylation, indicating a lower RRE selectivity.24 These results
demonstrate that guanidinylation impacts the RNA specificity of
glycoside-based ligands. Both the aminoglycosides and the
guanidinoglycosides show very little affinity for double-stranded
DNA (compare column b to d, Table 1). This suggests that the
core structure of the glycosides, and not the identity of the basic
groups, is responsible for the RNA over DNA selectivity exhibited
by both families of glycosides.25
Figure 2. Minimal binding domains for Rev and RRE. For fluorescence
anisotropy experiments, X ) triphosphate. For solid-phase assay, X )
biotin-streptavidin linkage to solid-support.13,21
Table 1. IC50 Values (µM) as Determined by Fluorescence
Anisotropy and Solid-Phase Assaysa
(c)
solid-phase solid-phase ratio
glycoside anisotropy solid-phase +polyA•polyU +DNA (c)/(b)
(d)
(e)
The transformation of aminoglycosides into guanidinoglyco-
sides has created a new family of compounds for the study of
RNA-small molecule interactions. Our results indicate that
guanidinoglycosides bind RNA preferentially over DNA, and
show selectivity between various RNAs. The higher affinity and
selectivity of guanidinoglycosides to the RRE implicate their
potential use as antiviral agents.
(a)
(b)
1a
1b
2a
2b
3a
3b
4a
4b
5a
5b
750
65
80
3.5
44
3.8
65
18
700
50
80
3.0
45
3.0
55
14
1300
60
160
4.7
140
5.0
110
20
750
55
90
3.5
50
3.5
60
16
1.9
1.2
2.0
1.6
3.1
1.7
2.0
1.4
2.7
5.6
Acknowledgment. We are grateful to the National Institutes of Health
for support (AI 47673 to Y.T., 33452A to T.B.). N.L. is supported by an
NIH Molecular Biophysics Training Grant (GM 08326). We thank NIGU-
Chemie GmbH and the Universitywide AIDS Research Program for
partial support.
6.0
1.3
6.0
0.8
16
4.5
8.0
0.9
Supporting Information Available: General synthetic procedures,
analytical data for all new derivatives, as well as conditions and procedures
for RNA binding assays (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
a The standard devaition of all values is less than (25% of the
reported value.13,22
B, and tobramycin results in a greater than 10-fold increase in
inhibitory activity relative to the parent compounds.19 A 5-fold
increase in activity is observed upon guanidinylation of neomycin
B and paromomycin.20
A novel solid-phase assembly was used to evaluate the RNA
specificity of the new derivatives.21 This assay relies on the
displacement of a fluorescent Rev peptide from a solid-phase
immobilized RRE and can be performed in the presence of
competing nucleic acids.22 Table 1 (columns b-d) summarizes
JA002421M
(20) Both guanidino-tobramycin (3b) and guanidino-paromomycin (4b),
have five guanidinium groups, but guanidino-tobramycin shows a significantly
higher inhibitory activity. This may indicate that the spatial arrangement of
the five guanidinium groups on 3b is better suited for binding the RRE than
the arrangement presented by 4b.
(21) Luedtke, N. W.; Tor, Y. Angew Chem. Intl. Ed. 2000, 39, 1788-
1790.
(22) Experiments with competing nucleic acids contain 15 µg/mL plasmid
DNA (pGEM) or 15 µg/mL of poly A‚poly U duplex RNA (∼50-fold molar
excess of DNA or RNA nucleotides, relative to the RRE).
(23) Similar differences in RRE selectivity were seen when a complex
mixture of mature and pre-tRNAs (at 64 µg/mL) is used instead of poly A‚
poly U. This suggests that the changes in RRE selectivity, observed upon
guanidinylation, is a general phenomenon.
(24) The lower RRE selectivity of guanidinylated neomycin is likely to
reflect a general trend exibited by highly cationic compounds; as the total
charge increases, the specificity for a particular RNA typically decreases.
(25) Our results indicate that both families of glycosides have a higher
affinity to simple duplex RNA (poly A‚poly U) than to simple duplex DNA.
This suggests that the difference between A-form and B-form helical structure
is an important determinant for glycoside recognition.
(18) IC50 values are reported instead of Ki since the number of binding
sites is unknown.
(19) The differences in RRE affinity between kanamycin B and tobramycin
are lost upon guanidinylation. Previous studies suggest that the pKa of the
ammonium at position R1 is lowered by the hydroxyl at position R2 (Figure
1).9b Consequently, kanamycin B has a lower total charge and shows lower
RNA affinity, relative to tobramycin. This postulate is supported by the nearly
identical activities exhibited by guanidino-tobramycin and guanidino-kana-
mycin B, since guanidinium groups are significantly more basic and have
pKa values that are less variable, compared to aliphatic amines.