A library approach to the discovery of small molecules that recognize RNA: Use of a 1,3-hydroxyamine motif as core

Article abstract of DOI:10.1021/ja980826p

A library of compounds based upon an aminoglucopyranoside core has been developed and screened for binding to RNA and specifically to 16S ribosomal RNA. The title molecules simplify the complexity of naturally occurring aminoglycoside antibiotics by embod

Full text of DOI:10.1021/ja980826p

J. Am. Chem. Soc. 1998, 120, 8319-8327
8319
A Library Approach to the Discovery of Small Molecules That
Recognize RNA: Use of a 1,3-Hydroxyamine Motif as Core
Chi-Huey Wong,* Martin Hendrix, David D. Manning, Christoph Rosenbohm, and
William A. Greenberg
Contribution from the Department of Chemistry and The Skaggs Institute for Chemical Biology,
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
ReceiVed March 12, 1998
Abstract: A library of compounds based upon an aminoglucopyranoside core has been developed and screened
for binding to RNA and specifically to 16S ribosomal RNA. The title molecules simplify the complexity of
naturally occurring aminoglycoside antibiotics by embodying a putative recognition motif found within these
structures, namely, a 1,3-hydroxyamine. The core pyranoside bearing the hydroxyamine motif was structurally
varied at two points through a combinatorial approach utilizing acylation and reductive amination protocols.
The aminoglycoside mimetics were screened in an automated assay based upon surface plasmon resonance
(SPR), and some were found effective at binding a 27-nucleotide model (AS-wt) of A-site 16S RNA as well
as a drug-resistant mutant RNA in the micromolar range.
Introduction
development of new, rationally designed RNA binders. Fur-
thermore, no structural information exists for many possible
RNA targets, although RNA structural biology is a rapidly
emerging field. In this situation, combinatorial chemistry may
be an invaluable tool to discover new lead structures for specific
recognition of RNA sequences.8
Among the natural products that recognize RNA specifically,
the class of aminoglycoside antibiotics stands out. These basic
polyamino saccharides have been known for over 50 years as
potent antibacterial drugs.9 Their mechanism of action involves
binding to the ribosomal RNA of bacteria, thereby interfering
with protein biosynthesis. For one subgroup of aminoglycosides
which encompasses the widely used drugs gentamicin, kana-
mycin, and neomycin B, the target site has been localized to
the ribosomal decoding region.10 Recently, the solution structure
of paromomycin bound to a model of this site has been solved
and provided the first structural characterization of a specific
aminoglycoside-RNA recognition event.11
A wealth of information has been discovered in the past 15
years which has fundamentally changed our perspective of the
biological role of RNA. It is now clear that RNA plays a central
part in many processes of life. In addition to its well-known
role as an intermediary in the flow of genetic information, it is
crucial to many catalytic processes.1 It has the ability to cleave
itself during the process of splicing or it can act catalytically to
cleave other RNA sequences.2,3 In concert with proteins, it
forms the catalytic machinery of the ribosome.4
This increased awareness of the central role of RNA has led
to the realization that RNA is a potential drug target that has
thus far remained largely unexplored.5 There is a host of natural
products targeting the ribosome, and RNA binding seems to be
central to their function.6 Other potential RNA targets include
sites of RNA-protein interactions governing transcriptional
activation or repression, splicing, nuclear export, or the packag-
ing of RNA virus particles.7
(7) (a) Zapp, M. L.; Green, M. R. Nature 1989, 342, 714-716. (b) Daly,
T. J.; Cook, K. S.; Gray, G. S.; Maione, T. E.; Rusche, J. R. Nature 1989,
342, 816-819. (c) Malim, M. H.; Hauber, J.; Le, S.-Y.; Maizel, J. V.;
Cullen, B. R. Nature 1989, 338, 254-257. (d) Malim, M. H.; Cullen, B.
R. Mol. Cell. Biol. 1993, 13, 6180-6189. (e) Fischer, U.; Meyer, S.; Teufel,
M.; Heckel, C.; Lu¨hrmann, R.; Rautmann, G. EMBO J. 1994, 13, 4105-
4112. (f) Wen, W.; Meinkoth, J. L.; Tsien, R. Y.; Taylor, S. S. Cell 1995,
82, 463. (g) Fischer, U.; Huber, J.; Boelens, W. C.; Mattaj, I. W.; Lu¨hrmann,
R. Cell 1995, 82, 475. (h) Bogerd, H. P.; Fridell, R. A.; Madore, S.; Cullen,
B. R. Cell 1995, 82, 485. (i) Stutz, F.; Neville, M.; Rosbach, M. Cell 1995,
82, 495.
(8) For some recent combinatorial strategies designed to target RNA,
see: (a) Park, W. K. C.; Auer, M.; Jaksche, H.; Wong, C.-H. J. Am. Chem.
Soc. 1996, 118, 10150-10155. (b) Wuonola, M. A.; Powers, D. G. Use of
combinatorial libraries in the discoVery and deVelopment of noVel anti-
infectiVes; Chaiken, I. M., Janda, K. D., Eds.; Diversity Comb. Chem., Libr.
Drug Discovery, Conf.; American Chemical Society: Washington, DC,
1996; pp 284-297. (c) Cload, S. T.; Schepartz, A. J. Am. Chem. Soc. 1994,
116, 437. (d) Harada, K.; Martin, S. S.; Frankel, A. D. Nature 1996, 380,
175.
Despite the number of RNA targets available for drug
intervention, there remains a poor understanding of the principles
governing small molecule RNA recognition which hinders the
(1) Eckstein, F., Lilley, David M. J., Eds. Catalytic RNA; Vol. 10:
Nucleic Acids and Molecular Biology, Springer-Verlag: Berlin, 1996.
(2) (a) von Ahsen, U.; Davies, J.; Schroeder, R. Nature 1991, 353, 368.
(b) von Ahsen, U.; Davies, J.; Schroeder, R. J. Mol. Biol. 1992, 226, 935.
(c) von Ahsen, U.; Noller, H. F. Science 1993, 260, 1500. (d) Rogers, J.;
Davies, J. Nucleic Acids Res. 1994, 22, 4983.
(3) (a) Stage, T. K.; Hertel, K. J.; Uhlenbeck, O. C. RNA, 1995, 1, 95.
(b) Clouet-d’Orval, B.; Stage, T. K.; Uhlenbeck, O. C. Biochemistry 1995,
34, 11186. (c) Wang, H.; Tor, Y. J. Am. Chem. Soc. 1997, 119, 8734. (d)
Wang, H.; Tor, Y. Bioorg. Med. Chem. Lett. 1997, 7, 1951.
(4) For a review, see: (a) Green, R.; Noller, H. F. Chem. Biol. 1997,
66, 679-716. (b) Noller, H. F. Annu. ReV. Biochem. 1991, 60, 191-227.
(5) Pearson, N. D.; Prescott, C. D. Chem. Biol. 1997, 4, 409-414.
(6) Reviews: (a) Cundliffe, E. In The Ribosome: Structure, Function
and EVolution; Hill, W. E., et al., Eds.; American Society for Microbiol-
ogy: Washington, DC, 1990; pp 479-90. (b) Gale, E. F.; Cundliffe, E. In
The Molecular Basis of Antibiotic Action; Reynolds, P. E., Richmond, M.
H., Waring, M. J., Eds.; John Wiley and Sons: London, 1981. (c) Cundliffe,
E. In Ribosomes: Structure, Function and Genetics; Chambliss, G. R., et
al., Eds.; University Park Press: Baltimore, 1980, pp 555ff.
(9) Umezawa, H.; Hooper, I. R., Eds. Aminoglycoside Antibiotics;
Springer-Verlag: New York, Heidelberg, 1982.
(10) Moazed, D.; Noller, H. F. Nature 1987, 327, 389-394.
(11) Fourmy, D.; Recht, M. I.; Blanchard, S. C.; Puglisi, J. D. Science
1996, 274, 1367-1371.
S0002-7863(98)00826-9 CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/06/1998

8320 J. Am. Chem. Soc., Vol. 120, No. 33, 1998
Wong et al.
paromomycin complexed with a model of the E. coli decoding
region A-site RNA, the core of the library may be aligned with
the 2-deoxy-2-aminoglucopyranoside ring of paromomycin.
Assuming this possible alignment, molecular modeling sug-
gested the R-anomeric substituent would be poised to make
hydrogen bonding contacts to acceptors on the bases in the major
groove of the RNA, especially G1494 and U1495, bases known
to be critical for natural aminoglycoside recognition (Figure 3).14
The acyl substituent in the 2-position could make contacts with
the phosphate backbone of the conserved internal loop.
Results and Discussion
Figure 1. Hydroxyamines: a motif for RNA recognition. Phosphodi-
esters and the Hoogsteen face of guanosine represent bidentate RNA
hydrogen bond acceptors that may be recognized by hydroxyamines.
To construct the library, we designed a suitably protected
building block (5, Scheme 1) which would incorporate a free
amine for the introduction of an acyl residue and a masked
aldehyde for a subsequent reductive amination. The latter is
provided in the form of an allyl group which can be cleaved by
ozonolysis to reveal the aldehyde.
A convenient starting material for the synthesis of 2-amino
sugars is N-acetylglucosamine (1). This was converted to the
allyl glycoside by Fischer glycosidation.15 An anomeric mixture
(7:1 ) R:â) is obtained, from which pure 2 can be isolated by
peracetylation, chromatographic separation, and subsequent
deacetylation. Selective tosylation of the primary alcohol and
displacement with sodium azide led to the 6-azido glucoside 4.
The N-acetyl group of 4 was cleaved under basic conditions by
refluxing with aqueous barium hydroxide to provide the desired
building block 5. For the large scale synthesis of this building
block, formation of the allyl glycoside, tosylation, and azide
displacement were carried out without the purification of any
intermediates. At the stage of azide 4, chromatography then
afforded a clean, anomerically pure compound.
Several approaches exist for the construction of libraries. A
strategic choice must be made between the synthesis of
individual compounds or compound mixtures. We expected that
specific recognition of RNA would be difficult to achieve and
were aware that even the selectivity of the naturally occurring
aminoglycosides for their decoding region target site is only 5-
to 20-fold.14b,16 This kind of moderate specificity would likely
be obscured by overwhelming nonspecific binding when screen-
ing mixtures of compounds. We therefore decided to synthesize
all library members individually using a parallel solution phase
approach.
Scheme 2 shows the series of four combinatorial steps leading
from the key intermediate 5 to the final deprotected library
compounds. For the initial acylation, four Z-protected amino
acid N-hydroxysuccinimide esters were used to provide amino
acyl derivatives 6-9. These compounds were individually
purified and then subjected to the remaining three combinatorial
steps. Treatment with ozone and subsequent quenching with
dimethyl sulfide led to the clean production of aldehydes 10-
13 which were reacted with any of six different amines (a-f).
These included four amino acid carboxamides (a-d) as well
as benzylamine (e) and mono-Z-protected ethylenediamine (f).
The reductive amination products 14-17a-f were then depro-
tected by hydrogenolysis in aqueous acetic acid using palladium
hydroxide on carbon as a catalyst.
Figure 2. Library design.
We have used the structure of aminoglycosides as inspiration
in our search for general motifs that may be suitable for RNA
recognition.12 In this regard, we have identified 1,2- and 1,3-
hydroxyamines as an interesting binding motif.13 Hydroxyamines
are bidentate hydrogen bond donors that can interact with
bidentate hydrogen bond acceptors found within RNA structure.
These include backbone phosphodiesters and the Hoogsteen face
of guanosine. Using model systems we have shown that, in
particular, 1,3-hydroxyamines with a gluco configuration are
strong binders for phosphodiesters (Figure 1).
Constructing combinatorial libraries incorporating the hy-
droxyamine motif may therefore lead to the discovery of
compounds that bind to RNA. We designed an approach that
would take advantage of the hydroxyamine motif as a central
core and would utilize straightforward coupling chemistry to
attach readily available building blocks. As shown in Figure
2, the gluco-configured 1,3-hydroxyamine is the core structure
of our library. Two points of variation are introduced, at the
anomeric position and at position 2 of the glucose ring through
reductive amination and acylation, respectively.
Although this approach is general, the particular design
chosen here includes a structural bias for recognition of the
decoding region A-site 16S ribosomal RNA (16S rRNA). Based
on the available NMR structure11 of the aminoglycoside
(12) For other approaches to RNA binders, see: (a) Wilson, W. D.;
Ratmeyer, L.; Zhao, M.; Strekowski, L.; Boykin, D. Biochemistry 1993,
32, 4098-4104. (b) McConnaughie, A. W.; Spychala, J.; Zhao, M.; Boykin,
D.; Wilson, W. D. J. Med. Chem. 1994, 37, 1063-1069. (c) Zhao, M.;
Ratmeyer, L.; Peloquin, R. G.; Yao, S.; Kumar, A.; Spychala, J.; Boykin,
D. W.; Wilson, W. D. Bioorg. Med. Chem. 1995, 3, 785-794. (d)
Fernandez-Saiz, M.; Schneider, H.-J.; Sartorius, J.; Wilson, W. D. J. Am.
Chem. Soc. 1996, 118, 4739-4745. (e) Mei, H.-Y.; Cui, M.; Lemrow, S.
M.; Czarnik, A. W. Bioorg. Med. Chem. 1997, 5, 1185. (f) Zapp, M. L.;
Young, D. W.; Kumar, A.; Singh, R. Boytein, D. W.; Wilson, W. D.; Green,
M. R. Bioorg. Med. Chem. 1997, 5, 1149. (g) Perreault, D. M.; Cabell, L.
A.; Anslyn, E. V. Bioorg. Med. Chem. 1997, 5, 1197. (h) Gilbert, B. A.;
Sha, M.; Wathen, S. T.; Rando, R. R. Bioorg. Med. Chem. 1997, 5, 1115.
(i) Lim, A. C.; Barton, J. K. Bioorg. Med. Chem. 1997, 5, 1131.
(13) Hendrix, M.; Alper, P. B.; Priestley, E. S.; Wong, C.-H. Angew.
Chem., Int. Ed. Engl. 1997, 36, 95-98.
The three-step sequence was carried out without purification
of any intermediates. Instead, the final compounds were purified
(14) (a) Recht, M.; Fourmy, D.; Blanchard, S. C.; Dahlquist, K. D.;
Puglisi, J. D. J. Mol. Biol. 1996, 262, 421. (b) Wong, C.-H.; Hendrix, M.;
Priestley, E. S.; Greenberg, W. A. Chem. Biol., in press.
(15) Lee, R. T.; Lee, Y. C. Carbohyd. Res. 1974, 37, 193-201.
(16) Alper, P. B.; Hendrix, M.; Priestly, E. S.; Wong, C.-H. J. Am. Chem.
Soc. 1998, 120, 1965-1978.

Motif for RNA Recognition
J. Am. Chem. Soc., Vol. 120, No. 33, 1998 8321
Figure 3. A model of compound 20a with As-wt.
Scheme 1
by ion-exchange chromatography using Amberlite CG-50 resin
(a carboxylic acid containing resin) with aqueous ammonia as
the eluent. This procedure afforded final compounds of
this case an antibiotic assay.16 We have chosen the former test
as our preferred method of screening at this stage, as it can
provide a straightforward assessment of the desired RNA
binding properties without being obscured by other issues such
as metabolic stability or cellular uptake. Moreover, the SPR
assay has been automated so that up to 12 compounds each at
four concentrations may be screened against three separate RNA
sequences over a 24-h period (Figure 4).
1
excellent purity as judged by H NMR and 13C NMR. All
library members were also characterized by electrospray mass
spectrometry, which provided further evidence of the identity
of each compound.
Library Evaluation by SPR
The assay measures the change in SPR angle that occurs upon
the binding of an analyte to RNA immobilized on a sensor chip.
The RNAs required for immobilization were prepared enzymati-
cally by runoff transcription (Scheme 3). Briefly, RNAs were
fabricated by the action of T7 RNA polymerase on an appropri-
ate DNA template primed with guanosine 5′-monophospho-
rothioate (GMPS).18 Subsequent alkylation of the product 5′-
We envisioned two possible ways in which to evaluate the
library compounds: a direct testing of their RNA binding
affinities to various target RNAs using our surface plasmon
resonance (SPR) assay17 or a screen for biological function, in
(17) Hendrix, M.; Priestley, E. S.; Joyce, G. F.; Wong, C.-H. J. Am.
Chem. Soc. 1997, 119, 3641-3648.

8322 J. Am. Chem. Soc., Vol. 120, No. 33, 1998
Wong et al.
Scheme 2
Scheme 3
Figure 4. Automated analysis of aminoglycoside mimetics by surface
plasmon resonance. Zones A-D represent four distinct regions of the
SPR sensorchip that may be individually modified with RNA and
simultaneously analyzed for aminoglycoside binding. Twelve ami-
noglycoside mimetics each at four concentrations may be screened in
a 24-h period. For our studies, zones A-C contain immobilized
biotinylated RNAs (AS-wt, U1406A, U1495A), while zone D was left
unmodified to measure background binding.
phosphorothioate modified transcripts with a biotinylated
iodosuccinimide linker affords biotinylated RNAs which when
purified by gel electrophoresis were suitable for immobilization
on streptavidin-coated sensor chips.
The library compounds were tested for RNA binding with a
set of A-site related RNAs (Figure 5).14,19 Briefly, AS-wt and
AS-U1406A are model systems of the aminoglycoside binding
site found in the E. coli ribosomal decoding region A-site. They
form specific complexes with a number of aminoglycosides,
particularly the 4,5-linked 2-deoxystreptamine derivatives of the
neomycin class. The mutant AS-U1495A no longer possesses
this specificity and serves as a negative control for aminogly-
coside binding.
tested at a range of concentrations between 3 and 100 µM. Kd’s
were derived from a model that includes terms for specific and
uniform multisite binding modes (see eq 3, Experimental
Section). Representative data from the SPR screen are shown
in Figure 6.
There is considerable variation in the affinity of individual
library members for the A-site related RNAs. Within a series
having the same acyl residue, all compounds have the same
number of amines, except for the last entry in each series (f,
the condensation product with ethylenediamine), thus enabling
comparison between compounds of the same charge. In general,
compounds 19-21a,e with NHCH2CONH2 and NH2 linked to
the anomeric center have the highest RNA binding affinity.
Compounds with aliphatic hydrophobic residues (alaninamide,
leucinamide) show much weaker binding. Interestingly, phe-
nylalaninamide is noticeably better in affinity than leucine in
the first two acyl series (i.e., those with glycine or alanine as
the acyl residue), but noticeably worse in the third series.
Table 1 shows the SPR assay results obtained with AS-wt
for library compounds 18-21a-f. The library compounds were
(18) (a) Milligan, J. F.; Groebe, D. R.; Witherell, G. W.; Uhlenbeck, O.
C. Nucleic Acids Res. 1987, 15, 8783. (b) Milligan, J. F.; Uhlenbeck, O. C.
Methods Enzymol. 1989, 180, 51.
(19) Miyaguchi, H.; Narita, H.; Sakamoto, K.; Yokoyama, S. Nucleic
Acids Res. 1996, 24, 3700.

Motif for RNA Recognition
J. Am. Chem. Soc., Vol. 120, No. 33, 1998 8323
Figure 5. Model RNA sequences and their relationship to wild-type
A-site 16s rRNA. AS-wt and AS-U1406A retain the internal loop
necessary for specific binding profiles observed in natural aminogly-
cosides. Mutant AS-U1495A binds aminoglycosides nonspecifically
and serves as a negative control for aminoglycoside binding.
Figure 6. Representative SPR binding data for AS-wt, U1406A, and
U1495A.
Table 1. Screening of the Library against A-Site Related RNAs
than the alanine side chain. For example, glycine derivative
18d is 5-fold more potent than alanine derivative 19d. As
expected, compounds possessing either a lysine or arginine as
the N-acyl side chain are generally more potent than those
displaying alanine or glycine, albeit their affinities for the RNA
models is similar. These results are presented in a chart shown
in Figure 7 to better understand the effects of substituents on
binding.
Specificity
R1
R2
As-wta U1406Aa U1495Aa
factorb
18a Gly GlyNH2
130
170
>250
70
100
50
90
140
>250
35
100
200
>250
70
110
50
0.8
1.2
18b
18c
18d
18e
18f
AlaNH2
LeuNH2
PheNH2
NH2
1
1.1
1
90
50
NH(CH2)2NH2
19a Ala GlyNH2
40
30
40
1
The A-site related RNA sequences can discriminate between
individual library members. Additionally, several of the library
compounds (20a, 20e, 20f, 21f) have affinities similar to that
of the antibiotic neamine which has a dissociation constant of
5 µM16 as measured by the SPR assay. This can be taken as
an indication that the hydroxyamine structure chosen in the
scaffold design is an effective core structure to achieve high
affinity RNA binding. Whereas the library compounds show
generally the same affinity for all three A-site variants, natural
aminoglycosides such as neomycin B and synthetic analogues
show noticeable differences in affinity for these RNAs, with
specificities of 5-20-fold.14b,16 Molecular modeling suggests
these molecules could have specific interactions with conserved
bases within the loop region of AS-wt (Figure 3). Perhaps the
low specificity observed for the library compounds is related
to the flexible nature of side chains R1 and R2.
Yeast ribosomal RNA in which the 1409-1491 base pair is
disrupted imparts resistance to a wide spectrum of aminogly-
coside antibiotics.21 To address the effectiveness of our
aminoglycoside mimetics against drug-resistant strains we
examined one mutant (AS-res)14a RNA containing a mismatched
base pair across the 1409-1451 junction (Figure 8). The
affinity for this sequence remained in the micromolar range and
was only about 2-fold lower with regard to specificity for AS-
wt. Although the mimetics discovered in this study have low
specificity for the A-site, they are shown to be effective against
the drug-resistant mutant AS-res. It would be of interest to see
if the mimetics would bind other RNA sequences, and work is
in progress to investigate this specificity issue.
19b
19c
19d
19e
19f
AlaNH2
LeuNH2
PheNH2
NH2
>250
>500
210
130
70
30
90
>250
>250
15
15
60
100
>250
130
50
>250
>500
170
130
70
25
70
190
>250
10
15
50
70
220
90
40
15
>250
>500
170
110
70
40
70
150
>250
10
15
40
60
200
90
30
6.0
0.8
0.8
1
1.3
0.8
0.6
NH(CH2)2NH2
20a Lys GlyNH2
20b
20c
20d
20e
20f
AlaNH2
LeuNH2
PheNH2
NH2
0.7
1
NH(CH2)2NH2
21a Arg GlyNH2
0.7
0.6
0.8
0.7
0.6
0.6
21b
21c
21d
21e
21f
AlaNH2
LeuNH2
PheNH2
NH2
NH(CH2)2NH2
10
a Kd values are reported in µM. b The specificity factor is the ratio
of the Kd for the negative control and the Kd for the wild-type RNA
model, Kd(U1495A)/Kd(AS-wt).
The variation observed for the affinities of the RNAs for
closely related library members is quite stunning in some cases.
Thus, introduction of a single methyl group into compound 19a
to form 19b led to a greater than 8-fold drop in affinity with
the difference to leucine even larger. Thus, small R2 substituents
seem important for high RNA binding affinity in this class of
compounds. Introduction of an additional amine into the R2
substituent was anticipated to increase binding by at least a factor
of 10 as is commonly observed for the natural aminoglycosides.14b
However, only a factor of 2 increase in binding was observed
in the 18 and 19 acyl series (mimetics e and f). For compounds
possessing an extra charge (compounds 20 and 21), the effect
was similar, with less than a 10-fold enhancement observed.
Possibly the proximity of amines in the ethylenediamine chain
perturbs the system so that only one can be significantly
protonated at pH 7.4, at which the assay is conducted.20
Comparison of the N-acyl side chains among similarly
charged derivatives indicates that glycine is slightly more potent
Conclusion
We have demonstrated that a library of small molecule RNA
binders can be assembled rapidly in a parallel solution phase
approach. An interesting range of RNA binding affinities were
obtained with the library compounds, spanning about 2 orders
of magnitude. The hydroxyamine core structure is effective
(20) Ethylenediamine: pKa1 ) 9.93, pKa2 ) 6.85. Martell, A. E.; Smith,
R. M. Critical Stability Constants; Plenum Press: New York, 1974.
(21) Li, M.; Tzagoloff, A.; Underbrink-Lyon, K.; Martin, N. J. Biol.
Chem. 1982, 257, 5921-5928.

8324 J. Am. Chem. Soc., Vol. 120, No. 33, 1998
Wong et al.
Figure 7. Stability constants for aminoglycoside mimetics versus three RNA sequences: AS-wt, U1406A, U1495A.
of tosyl chloride (175 mg, 0.918 mmol) was added via syringe. After
1 h, the ice bath was removed and stirring was continued for 12 h. The
reaction was quenched with water and concentrated; the residue was
purified by column chromatography in ethyl acetate/MeOH (0 to 20%)
to afford 200 mg (63%) of 3 as a colorless foam. 1H NMR (400 MHz,
CDCl3) δ ) 7.78 (d, 2H, J ) 8 Hz), 7.32 (d, 2H, J ) 8 Hz), 6.14 (d,
1H, J ) 8.5 Hz), 5.84 (m, 1H), 5.22 (m, 2H), 4.74 (d, 1H, J ) 3.5
Hz), 4.27 (m, 2H), 4.10 (dd, 1H, J ) 13, 5 Hz), 4.0 (m, 1H), 3.90 (dd,
1H, J ) 13, 6.5 Hz), 3.76 (m, 1H), 3.65 (app dd, 1H, J1 ) J2 ) 10
Hz), 3.46 (dd, 1H, J1 ) J2 ) 10 Hz), 2.42 (s, 3H), 2.03 (s, 3H); 13C
NMR (100 MHz, CDCl3) δ ) 172.0, 144.9, 133.2, 132.8, 129.8, 128.0,
118.1, 96.4, 73.5, 70.6, 69.6, 69.1, 68.3, 53.3, 23.2, 21.6; HRMS m/z
found 548.0355 (M + Cs+), calculated 548.0367.
Allyl 6-Azido-6-deoxy-r-D-N-acetylgludopyranoside (4). A solu-
tion of allyl 6-O-tosyl-R-D-N-acetylglucosaminide (3, 190 mg, 0.45
mmol) in DMF containing NaN3 (300 mg, 4.6 mmol) was heated in an
oil bath at 80 °C for 3 h. The solvent was then evaporated and the
residue was purified by column chromatography in ethyl acetate/MeOH
(0 to 20%) to afford 120 mg (91%) of 4 as a white solid. 1H NMR
(400 MHz, CDCl3-10% MeOD) δ ) 6.66 (d, 1H, J ) 9 Hz), 5.81 (m,
1H), 5.25-5.21 (m, 2H), 4.76 (d, 1H, J ) 3.5 Hz), 4.13 (m, 1H), 3.89-
3.96 (m, 2H), 3.67 (ddd, 1H, J ) 10, 6.5, 1.5 Hz), 3.52 (dd, 1H, J )
10, 9 Hz), 3.46 (dd, 1H, J ) 13, 2.5 Hz), 3.40-3.28 (m, 6H), 1.94 (s,
3H); 13C NMR (100 MHz, CDCl3-10% MeOD) δ ) 171.9, 133.2,
117.8, 96.3, 72.2, 71.5, 71.2, 68.2, 53.3, 53.2, 51.4, 22.7; HRMS m/z
found 309.1168 (M + Na+), calculated 309.1175.
Allyl 2-Amino-2-deoxy-6-azido-6-deoxy-r-D-glucopyranoside (5).
A flask fitted with a reflux condenser was charged with allyl 6-deoxy-
6-azido-R-D-N-acetylglucosaminide (4) (960 mg, 3.35 mmol), barium
hydroxide octahydrate (14 g), and water (72 mL). The flask was placed
into a 120 °C oil bath and the mixture stirred for 3 h. After cooling,
the solution was continuously extracted with CH2Cl2 for 20 h to afford
639 mg (78%) of 5 as a colorless oil. 1H NMR (CD3OD) δ ) 5.96
Figure 8. Mimetic binding to a drug-resistant model. Disruption of
the 1409-1491 GC base pair induces broad spectrum aminoglycoside
antibiotic resistance in yeast ribosomal RNA.
for the design of high affinity RNA binders, providing hope
that with the right choice of substituents specificity may be
attained as well.
Experimental Section
General Methods. Reactions requiring anhydrous conditions were
carried out in flame dried glassware under argon. NMR spectra were
recorded on a Bruker AMX-500 at 500 MHz for 1H and 125 MHz for
13C spectra using TMS or residual solvent as reference standards unless
stated otherwise. Mass spectra were recorded on a VG ZAB-ZSE mass
spectrometer.
Allyl 6-O-Tosyl-r-D-N-acetylglucopyranoside (3). Allyl R-D-N-
acetylglucosaminide (2)15 (200 mg, 0.766 mmol) was dissolved in 3
mL of anhydrous pyridine and cooled to 0 °C in an ice bath. A solution

Motif for RNA Recognition
J. Am. Chem. Soc., Vol. 120, No. 33, 1998 8325
(m, 1H), 5.32 (m, 1H), 5.20 (m, 1H), 4.85 (d, 1H, J ) 4 Hz), 4.24 (m,
1H), 4.04 (m, 1H), 3.72 (ddd, 1H, J ) 9.5, 6.5, 2.5 Hz), 3.50-3.37
(m, 3H), 3.23 (dd, 1H, J ) 9.5, 9 Hz), 2.62 (dd, J ) 10, 4 Hz); 13C
NMR (CD3OD) δ ) 135.3, 117.7, 99.4, 75.9, 73.3, 72.8, 69.5, 57.1,
52.8; ESI HRMS m/z found 245.1247 (MH+), calculated 245.1250.
General Procedure for Hydrogenation of Compounds 14-17a-
f. A solution of compounds 14-17 (0.15 mmol) in AcOH (3 mL) and
water (2 mL) was degassed by evacuating and purging with argon
several times. Catalyst (20% Pd(OH)2 on carbon, wet Degussa type,
ca. 20 mg) was added; the flask was carefully evacuated and then
refilled with hydrogen from a balloon. Hydrogen was bubbled though
the solution for ca. 3 min. Then the flask was kept under positive
hydrogen pressure with a hydrogen balloon and stirred for 3-12 h until
reduction was complete (TLC of the products in MeOH/concentrated
NH3 ) 9:1 to 3:1, staining with ninhydrin). The balloon was removed
and the solution purged with argon. Water was added (5 mL) and the
reaction mixture filtered through a Celite pad, which was washed with
water (5 mL). The combined filtrates were concentrated, dissolved in
General Procedure for Acylation of Compound 5 with N-
Hydroxysuccinimide Esters to Afford 2-Acyl Derivatives 6-9. A
solution of allyl 2,6-dideoxy-2-amino-6-azido-glucoside 5 (366 mg, 1.50
mmol) in DMF (6 mL) was cooled to 0 °C and treated with
triethylamine (0.23 mL, 1.7 mmol) followed by a solution of the
succinimide ester (1.5 mmol) in DMF (4 mL). After the reaction was
complete (TLC in CHCl3/MeOH ) 9:1), the solvent was evaporated,
and the residue was taken up in ethyl acetate and washed with saturated
NaHCO3 solution. After evaporation, column chromatography afforded
the 2-acyl derivatives 6-9 in 75-95% yield.
+
water (2 mL), and applied onto an Amberlite CG-50 column (NH4
form, 16 × 1.5 cm) and eluted with a linear gradient of aqueous
ammonia (500 mL total). Gradients of 0 to 1% NH3(aq) (3 charges, 1
secondary amine), 0 to 5% NH3(aq) (3 charges, no secondary amine),
0 to 8% NH3(aq) (4 charges, 1 secondary amine), and 0 to 35% NH3-
(aq) (for arginine-containing derivatives) were employed. Fractions
(5 mL each) were collected with an automatic fraction collector and
the product-containing fractions were pooled and lyophilized. Hydro-
chloride salts of the final products were prepared by adding excess 1
M HCl and lyophilizing again. All compounds were characterized by
1H NMR, 13C NMR, and electrospray MS.
1
Compound 6: H NMR (400 MHz, CD3OD) δ ) 7.36-7.28 (m,
5H), 5.93 (m, 1H), 5.32-5.09 (m, 4H), 4.83 (d, 1H, 3.5 Hz), 4.2 (dd,
1H, J ) 13, 5 Hz), 4.02-3.94 (m, 2H), 3.82 (m, 2H), 3.76-3.72 (m,
1H), 3.65 (dd, 1H, J ) 10.5, 9 Hz), 3.50 (dd, 1H, J ) 13, 2.5 Hz),
3.40 (dd, 1H, J ) 13, 6.5 Hz), 3.35-3.29 (m, 1H); 13C NMR (100
MHz, CD3OD) δ ) 172.4, 159.0, 138.1, 135.2, 129.5, 129.0, 128.9,
118.0, 73.1, 73.0, 72.7, 69.5, 67.8, 55.2, 52.7, 44.8; HRMS m/z found
568.0821, calculated 568.0808.
Compound 7: 1H NMR (400 MHz, DMSO-d6) δ ) 7.75 (d, 1H, J
) 8 Hz), 7.43-7.33 (m, 6H), 5.92 (m, 1H), 5.33-5.29 (m, 2H), 5.15
(d, 1H, J ) 10.5 Hz), 5.03 (m, 2H), 4.90 (d, 1H, J ) 6 Hz), 4.75 (d,
1H, J ) 3.5 Hz), 4.19-4.11 (m, 2H), 4.97 (dd, 1H, J ) 9.5, 5.5 Hz),
3.69-3.61 (m, 2H), 3.52-3.42 (m, 3H), 3.16 (m, 1H), 1.22 (d, 3H, J
) 7 Hz); 13C NMR (100 MHz, DMSO-d6) δ ) 172.9, 155.6, 137.1,
134.3, 128.4, 127.8, 127.7, 117.0, 95.9, 71.6, 70.3, 67.5, 65.3, 53.8,
51.2, 49.9, 18.5.
Compound 8: 1H NMR (DMSO-d6) δ ) 7.76 (d, 1H, 8 Hz), 7.36-
7.25 (m, 6H), 5.86 (m, 1H), 5.30-5.26 (m, 2H), 5.11 (d, 1H, J ) 10.5
Hz), 5.0 (app s, 2H), 4.98 (app s, 2H), 4.85 (broad s), 4.72 (d, 1H, J
) 3.5 Hz), 4.12-3.95 (m, 3H), 3.65 (m, 1H), 3.58 (m, 1H), 3.50-
3.42 (m, 2H), 3.39 (dd, 1H, J ) 13.5, 6.5 Hz), 3.12 (m, 2H), 2.94 (m,
2H), 1.60 (m, 1H), 1.58 (m, 1H), 1.45-1.20 (m, 4H); 13C NMR
(DMSO-d6) δ ) 172.4 156.1, 137.1, 134.3, 128.4, 127.8, 127.7, 117.1,
95.9, 71.6, 70.3, 67.5, 65.3, 65.1, 54.6, 53.7, 51.2, 31.9, 29.2, 22.8.
Compound 9: 1H NMR (CDCl3) δ ) 7.37-7.25 (m, 15H), 6.32 (d,
1H, J ) 5.0 Hz), 5.91 (d, 1H, J ) 3.0 Hz), 5.85-5.75 (m, 1H), 5.28-
5.20 (m, 3H), 5.15-5.02 (m, 4H), 4.72 (broad s, 1H), 4.22-4.16 (m,
1H), 4.08 (dd, 1H, J ) 5.0, 3.1 Hz), 4.00 (t, 1H, J ) 3.1 Hz), 3.94-
3.89 (m, 3H), 3.71-3.67 (m, 1H), 3.53-3.49 (m, 3H), 3.42-3.37 (m,
2H), 1.73-1.62 (m, 8H); 13C NMR (CDCl3) δ ) 173.0, 162.3, 161.4,
137.8, 134.1, 128.5, 128.3, 128.0, 118.2, 96.8, 73.5, 72.2, 71.4, 68.9,
67.8, 66.5, 55.1, 54.2, 52.8, 44.2, 37.5, 31.2, 25.0; ESI HRMS m/z
found 803.3331 (MH+), calculated 803.3364.
1
Compound 18a: H NMR (D2O) δ ) 4.96 (d, 1H, J ) 3.5 Hz),
4.06 (dd, 1H, J ) 10.5, 3.5 Hz), 4.02-3.99 (m, 1H), 3.95 (app d, 2H,
1 Hz), 3.88-3.85 (m, 3H), 3.76-3.72 (m, 2H), 3.43-3.37 (m, 4H),
3.17 (dd, 1H, J ) 13.5, 8.5 Hz); 13C NMR (D2O) δ ) 169.0, 168.1,
98.1, 72.2, 71.4, 68.9, 63.8, 53.9, 48.4, 47.8, 41.3, 41.1; ESI LRMS
m/z found 336 (MH+), calculated 336.
1
Compound 18b: H NMR (400 MHz, D2O) δ ) 4.98 (d, 1H, J )
3.5 Hz), 4.11-4.07 (m, 1H), 3.90-3.87 (m, 3H), 3.79-3.72 (m, 3H),
3.45-3.39 (m, 4H), 3.32-3.28 (m, 1H), 3.19 (dd, 1H, J ) 13, 8.5
Hz), 1.59 (d, 3H, J ) 7 Hz); 13C NMR (100 MHz, D2O) δ ) 169.8,
99.8, 73.9, 73.1, 70.6, 65.6, 58.5, 55.6, 51.3, 48.2, 43.0, 42.8, 17.9;
ESI, LRMS m/z found 350.0 (MH+), calculated 350.0.
1
Compound 18c: H NMR (D2O) δ ) 4.96 (d, 1H, J ) 3.5 Hz),
4.08 (d, 1H, J ) 10.5, 3.5 Hz), 3.99-3.96 (m, 2H), 3.88-3.84 (m,
3H), 3.77-3.71 (m, 2H), 3.44-3.38 (m, 4H), 3.28-3.26 (m, 1H), 3.17
(dd, 1H, J ) 13.5, 8.5 Hz), 1.86-1.81 (m, 1H), 1.76-1.66 (m, 2H),
0.94 (app t, 6H, J ) 7 Hz); 13C NMR (D2O) δ ) 172.0, 168.0, 98.1,
72.2, 71.5, 69.0, 63.7, 60.3, 53.8, 47.1, 41.3, 41.1, 39.6, 25.0, 22.9,
21.8; ESI LRMS m/z found 392.0 (MH+), calculated 392.0.
Compound 18d: 1H NMR (D2O) δ ) 7.45-7.38 (m, 5H), 4.96 (d,
1H, J ) 3.5 Hz), 4.22 (dd, 1H, J ) 9, 6 Hz), 4.08 (dd, 1H, J ) 10.5,
3.5 Hz), 4.01-3.98 (m, 1H), 3.88-3.83 (m, 3H), 3.78-3.71 (m, 2H),
3.45-3.41 (m, 3H), 3.36 (dd, 1H, J ) 13.5, 5.5 Hz), 3.32 (ddd, 1H, J
) 12.5, 8.5, 3 Hz), 3.21-3.16 (m, 2H); 13C NMR (D2O) δ ) 170.9,
168.0, 134.4, 130.3, 129.9, 128.8, 98.1, 72.2, 71.5, 69.0, 63.8, 62.6,
53.8, 47.3, 41.3, 41.1, 36.7; ESI LRMS m/z found 426.0 (MH+),
calculated 426.0.
General Procedure for Ozonolysis of Compounds 6-9.
A
solution of compounds 6-9 (0.15 mmol per reaction) in a total of 7
mL of a MeOH/CH2Cl2 mixture (containing only as much CH2Cl2 as
needed for solubility) was cooled to -78 °C and treated successively
with oxygen, then ozone until the solution was blue. After excess ozone
had been purged with oxygen, dimethyl sulfide was added (200 µL, 3
mmol) and the solution allowed to warm to ambient temperature (ca.
1 h). The solvent was evaporated and the product dried for 1 h under
high vacuum. The crude aldehydes 10-13 were then used in the
reductive amination.
1
Compound 18e: H NMR (D2O) δ ) 4.96 (d, 1H, J ) 3.5 Hz),
4.07 (dd, 1H, J ) 10.5, 3.5 Hz), 3.96 (dt, 1H, J ) 12, 4 Hz), 3.88-
3.83 (m, 3H), 3.75 (dd, 1H, J ) 10.5, 9 Hz), 3.68-3.64 (m, 1H), 3.44-
3.41 (m, 2H), 3.27 (m, 2H), 3.17 (dd, 1 H, J ) 13.5, 8.5 Hz); 13C
NMR (D2O) δ ) 168.1, 98.0, 72.2, 71.4, 68.9, 64.7, 53.9, 41.3, 41.1,
39.9; ESI LRMS m/z found 279.0 (MH+), calculated 279.0.
1
Compound 18f: H NMR (400 MHz, D2O) δ ) 4.85 (d, 1H, J )
3.7 Hz), 3.99-3.88 (m, 2H), 3.81-3.71 (m, 3H), 3.69-3.59 (m, 2H),
3.39-3.29 (m, 8H), 3.07 (dd, 1H, J ) 13, 8 Hz); 13C NMR (100 MHz,
D2O) δ ) 169.8, 99.8, 73.8, 73.1, 70.6, 65.4, 55.5, 50.1, 46.9, 43.1,
42.8, 37.8; ESI LRMS m/z found 322 (MH+), calculated 322.
General Procedure for Reductive Amination of Compounds 10-
13. The aldehydes 10-13 (0.15 mmol) were dissolved in MeOH (1
mL) and treated first with a 1 M solution of the amine in MeOH (0.45
mL), then with a 1 M solution of acetic acid in MeOH (0.5 mL), and
finally with a freshly prepared 0.3 M solution of NaCNBH3 in MeOH
(0.22 mL). (If an amine hydrochloride salt was used instead of a free
amine, water was added to the amine solution as needed for solubility,
the amount of AcOH solution was reduced to 0.05 mL, and the amount
of NaCNBH3 solution was increased to 0.25 mL.) After 2 h, water
(0.5 mL) was added and stirring was continued for 20 min, after which
time the mixture was concentrated. The crude products 14-17 were
carried on without further purification.
1
Compound 19a: H NMR (D2O) δ ) 4.95 (d, 1H, J ) 3.5 Hz),
4.08 (q, 1H, J ) 7 Hz) 4.02-3.99 (m, 2H), 3.94 (app d, 2H, J ) 2.5
Hz), 3.87-3.83 (m, 1H), 3.77-3.69 (m, 2H), 3.42-3.36 (m, 2H), 3.15
(dd, 1H, J ) 13.5, 8.5 Hz), 1.52 (d, 3H, J ) 7 Hz); 13C NMR (D2O)
δ ) 172.0, 168.9, 98.0, 72.3, 71.2, 69.0, 63.7, 53.9, 50.0, 48.4, 47.7,
41.1, 17.6; ESI LRMS m/z found 250 (MH+), calculated 250.
1
Compound 19b: H NMR (D2O) δ ) 4.93 (d, 1H, J ) 3.5 Hz),
4.07-3.95 (m, 4H), 3.85-3.81 (m, 1H), 3.73-3.66 (m, 2H), 3.39-
3.33 (m, 3H), 3.25 (ddd, 1H, J ) 12.5, 8.5, 3 Hz), 3.13 (dd, 1H, J )

8326 J. Am. Chem. Soc., Vol. 120, No. 33, 1998
Wong et al.
13.5, 8.5 Hz), 1.53 (d, 3H, J ) 7 Hz), 1.47 (d, 3H, J ) 7 Hz); 13C
NMR (D2O) δ ) 172.8, 172.0, 98.1, 72.4, 71.2, 69.0, 63.9, 56.7, 53.8,
50.0, 46.4, 41.1, 17.6, 16.2; ESI LRMS m/z found 364 (MH+),
calculated 364.
1.41 (m, 2H); 13C NMR (D2O) δ ) 170.8, 98.0, 72.6, 71.1, 70.0, 64.7,
53.9, 41.2, 39.9, 31.2, 27.2, 21.8; ESI LRMS m/z found 350 (MH+),
calculated 350.
1
Compound 20f: H NMR (D2O) δ ) 4.99 (d, 1H, J ) 3.5 Hz),
1
4.15 (app t, 1H, J ) 6.5 Hz), 4.07-4.02 (m, 2H), 3.90 (ddd, 1H, J )
9.5, 6.5, 3 Hz), 3.80 (dd, 1H, J ) 11, 9 Hz), 3.77-3.73 (m, 1H), 3.51-
3.39 (m, 8H), 3.19 (dd, 1H, J ) 13.5, 8.5 Hz), 2.98 (app t, 2H, J ) 7.5
Compound 19c: H NMR (D2O) δ ) 4.95 (d, 1H, J ) 3.5 Hz),
4.07 (q, 1H, J ) 7 Hz), 4.02 (dd, 1H, J ) 11, 3.5 Hz), 3.98-3.94 (m,
2H), 3.86-3.82 (m, 1H), 3.74-3.69 (m, 2H), 3.42-3.36 (m, 3H), 3.25
(ddd, 1H, J ) 13.5, 9, 3.5 Hz), 3.15 (dd, 1H, J ) 13.5, 8.5 Hz), 1.84-
1.78 (m, 1H), 1.74-1.65 (m, 2H), 1.51 (d, 3H, J ) 7 Hz), 0.92 (app
t, 6H, J ) 7 Hz); 13C NMR (D2O) δ ) 172.0, 171.9, 98.1, 72.4, 71.3,
69.0, 63.6, 60.2, 53.8, 50.0, 47.0, 41.1, 39.5, 24.9, 22.8, 21.8, 17.6;
ESI LRMS m/z found 406 (MH+), calculated 406.
Hz), 1.96-1.92 (m, 2H), 1.71-1.66 (m, 2H), 1.49-1.45 (m, 2H); 13
C
NMR (D2O) δ ) 170.8, 98.1, 72.6, 71.1, 69.0, 63.8, 54.0, 53.8, 48.7,
45.3, 41.2, 39.9, 36.2, 31.3, 27.2, 21.9; ESI LRMS m/z found 393
(MH+), calculated 393.
1
Compound 21a: H NMR (D2O) δ ) 4.95 (d, 1H, J ) 3.6 Hz),
Compound 19d: 1H NMR (D2O) δ ) 7.40-7.23 (m, 5H), 4.92 (d,
1H, J ) 3.5 Hz), 4.18 (dd, 1H, J ) 9, 5.5 Hz), 4.05 (q, 1H, J ) 7 Hz),
3.99 (dd, 1H, J ) 11, 4 Hz), 3.97-3.93 (m, 1H), 3.82 (m, 1H), 3.73-
3.67 (m, 2H), 3.39-3.31 (m, 4H), 3.26 (ddd, 1H, J ) 13, 8.5, 3.5),
3.13 (m, 2H), 1.48 (d, 3H, J ) 7 Hz); 13C NMR (D2O) δ ) 171.9,
170.8, 134.4, 130.3, 129.9, 128.8, 98.1, 72.4, 71.3, 69.0, 63.7, 62.5,
53.8, 50.0, 47.2, 41.1, 36.6, 17.6; ESI LRMS m/z found 440 (MH+),
calculated 440.
4.14 (broad s, 1H), 4.04-3.96 (m, 2H), 3.93 (d, 1H, J ) 5.9 Hz), 3.86-
3.79 (m, 1H), 3.75-3.69 (m, 3H), 3.45-3.35 (m, 3H), 3.20-3.12 (m,
4H), 3.02-2.95 (m, 2H), 1.75-1.65 (m, 2H); 13C NMR (D2O) δ )
182.0, 171.4, 158.2, 98.7, 73.2, 71.8, 69.7, 65.8, 64.5, 54.5, 49.0, 48.4,
43.6, 41.8, 29.1, 24.1; ESI HRMS m/z found 435.2671 (MH+),
calculated 435.2682.
1
Compound 21b: H NMR (D2O) δ ) 4.96 (d, 1H, J ) 3.5 Hz),
4.06-4.00 (m, 4H), 3.90 (app dt, J ) 7.0, 2.1 Hz, 1H), 3.76-3.70 (m,
2H), 3.43-3.38 (m, 4H), 3.25-3.15 (m, 5H), 1.74-1.67 (m, 4H), 1.50
(d, J ) 6.8 Hz); 13C NMR (D2O) δ ) 181.9, 173.0, 158.2, 98.8, 73.2,
71.8, 69.7, 64.8, 57.4, 54.6, 54.4, 47.0, 41.9, 29.6, 24.9, 24.1, 16.9;
ESI HRMS m/z found 449.2827 (MH+), calculated 449.2836.
1
Compound 19e: H NMR (400 MHz, D2O) δ ) 4.95 (d, 1H, J )
3.5 Hz), 4.06 (q, 1H, J ) 7 Hz), 4.00 (dd, 1H, J ) 11, 7 Hz), 3.98-
3.93 (m, 1H), 3.85 (ddd, 1H, J ) 10, 8.5, 3 Hz), 3.72 (dd, 1H, J ) 11,
9 Hz), 3.65 (ddd, J ) 11, 7, 4.5 Hz), 3.43-3.37 (m, 2H), 3.28-3.25
(m, 2H), 3.16 (dd, J ) 13, 8.5 Hz), 1.50 (d, 3H, J ) 7 Hz); 13C NMR
(100 MHz, D2O) δ ) 173.7, 99.7, 74.0, 72.9, 70.5, 66.3, 55.5, 51.6,
42.8, 41.5, 19.2; ESI LRMS m/z found 293 (MH+), calculated 293.
1
Compound 21c: H NMR (D2O) δ ) 4.92 (d, 1H, J ) 3.5 Hz),
4.06-3.95 (m, 3H), 3.89-3.82 (m, 2H), 3.73-3.65 (m, 3H), 3.48-
3.40 (m, 3H), 3.31 (app dt, 1H, J ) 10.0, 3.1 Hz), 3.23-3.17 (m, 4H),
1.72-1.62 (m, 5H), 0.95 (t, 6H, J ) 9.9 Hz); 13C NMR (D2O) δ )
182.2, 171.4, 158.6, 98.7, 73.2, 71.9, 69.7, 65.0, 61.0, 54.6, 54.4, 47.7,
41.8, 40.6, 29.7, 25.7, 24.9, 24.4, 23.5, 22.5; ESI HRMS m/z found
491.3289 (MH+), calculated 491.3305.
1
Compound 19f: H NMR (D2O) δ ) 4.92 (d, 1H, J ) 3.5 Hz),
4.10 (q, 1H, J ) 7 Hz), 4.00-3.95 (m, 2H), 3.84-3.81 (m, 1H), 3.74-
3.66 (m, 2H), 3.44-3.34 (m, 8H), 3.13 (dd, 1H, J ) 13, 8.5 Hz), 1.48
(d, 3H, J ) 7.5 Hz); 13C NMR (D2O) δ ) 172.0, 98.1, 72.3, 71.2,
68.9, 63.7, 53.8, 50.0, 48.5, 45.2, 41.1, 36.1, 17.7; ESI LRMS m/z found
336 (MH+), calculated 336.
Compound 21d: 1H NMR (D2O) δ ) 7.28-7.16 (m, 5H), 4.89 (d,
1H, J ) 3.5 Hz), 3.93-3.86 (m, 4H), 3.72-3.68 (m, 2H), 3.62-3.58
(m, 2H), 3.31-3.26 (m, 2H), 3.14-3.02 (m, 7H), 1.50-1.40 (m, 3H);
13C NMR (D2O) δ ) 181.1, 173.3, 171.4, 158.6, 136.2, 130.9, 130.5,
128.1, 98.6, 74.6, 73.1, 69.2, 66.3, 63.9, 55.1, 54.9, 47.5, 41.9, 36.8,
30.2, 25.2, 24.6; ESI HRMS m/z found 525.3133 (MH+), calculated
525.3149.
1
Compound 20a: H NMR (400 MHz, D2O) δ ) 5.00 (d, 1H, J )
3.6 Hz), 4.10-4.06 (m, 3H), 3.99 (app d, 2H, J ) 3 Hz), 3.92-3.87
(m, 1H), 3.87-3.75 (m, 2H), 3.47-3.39 (m, 4H), 3.19 (dd, 1H, J )
13, 8.5 Hz), 2.98 (app t, 2H, J ) 7.5 Hz), 1.96-1.90 (m, 2H), 1.73-
1.65 (m, 2H), 1.52-1.40 (m, 2H); 13C NMR (100 MHz, D2O) δ )
172.4, 170.6, 99.7, 74.2, 72.7, 70.7, 65.4, 55.6, 55.5, 50.0, 49.4, 42.8,
41.5, 32.8, 28.8, 23.5; ESI LRMS m/z found 407 (MH+), calculated
407.
1
Compound 21e: H NMR (D2O) δ ) 4.99 (d, 1H, J ) 3.9 Hz),
4.09-4.05 (m, 2H), 4.02-3.96 (m, 1H), 3.92-3.85 (m, 1H), 3.77 (t,
1H, J ) 12.0 Hz), 3.72-3.66 (m, 1H), 3.47-3.39 (m, 2H), 3.29 (broad
s, 2H), 3.24-3.17 (m, 2H), 1.98-1.91 (m, 2H), 1.72-1.66 (m, 2H);
13C NMR (D2O) δ ) 179.7, 172.2, 99.6, 74.2, 72.7, 70.6, 66.3, 55.5,
42.9, 42.8, 41.6, 30.5, 25.9, 23.2; ESI LRMS m/z found 378 (MH+),
calculated 378.
1
Compound 20b: H NMR (D2O) δ ) 4.99 (d, 1H, J ) 4.0 Hz),
4.11-4.00 (m, 4H), 3.88 (ddd, 1H, J ) 9, 9, 3 Hz), 3.79-3.72 (m,
2H), 3.45-3.39 (m, 3H), 3.29 (ddd, 1H, J ) 13, 9, 3.5 Hz), 3.18 (dd,
1H, J ) 13.5, 9 Hz), 2.97 (app t, 2H, J ) 8 Hz), 1.95-1.91 (m, 2H),
1.70-1.66 (m, 2H), 1.59 (d, 3H, J ) 7 Hz), 1.48-1.43 (m, 2H); 13C
NMR (D2O) δ ) 172.9, 170.7, 98.1, 72.6, 71.1, 69.0, 64.0, 56.8, 54.0,
53.9, 46.4, 41.2, 39.9, 31.2, 27.2, 21.8, 16.2; ESI LRMS m/z found
421 (MH+), calculated 421.
1
Compound 21f: H NMR (D2O) δ ) 4.98 (d, 1H, J ) 3.6 Hz),
4.10-4.00 (m, 3H), 3.91-3.86 (m, 2H), 3.82-3.75 (m, 3H), 3.48-
3.37 (m, 7H), 3.26-3.19 (m, 3H), 3.10 (t, 1H, J ) 4.7 Hz), 1.75-1.66
(m, 2H); 13C NMR (D2O) δ ) 180.9, 176.0, 98.9, 73.2, 71.8, 69.8,
64.5, 59.1, 54.6, 49.2, 42.7, 41.9, 39.2, 29.7, 25.0; ESI HRMS m/z
found 421.2899 (MH+), calculated 421.2887.
1
Compound 20c: H NMR (D2O) δ ) 4.95 (d, 1H, J ) 3.5 Hz),
4.06-4.02 (m, 2H), 3.98-3.95 (m, 2H), 3.84 (ddd, 1H, J ) 9, 9, 5.5
Hz), 3.76-3.70 (m, 2H), 3.42-3.36 (m, 3H), 3.24 (ddd, 1H, J ) 11,
9, 5.5 Hz), 3.15 (dd, 1H, J ) 13, 8.5 Hz), 2.95 (app t, 2H, J ) 7.5
Hz), 1.94-1.88 (m, 2H), 1.85-1.63 (m, 6H), 1.50-1.38 (m, 2H), 0.92
(app t, 6H, J ) 7 Hz); 13C NMR (D2O) δ ) 172.0, 170.7, 98.1, 72.6,
71.2, 69.0, 63.7, 60.3, 53.9, 53.8, 47.1, 41.1, 39.8, 39.5, 31.2, 27.2,
25.0, 22.9, 21.8; ESI LRMS m/z found 463 (MH+), calculated 463.
Synthesis of Biotinylated RNA. In vitro transcription reactions
were performed according to the general procedure reported by
Uhlenbeck and co-workers.18 The DNA templates (for AS-wt, 5′-GGC
GUC ACA CCU UCG GGU GAA GUC GCC CCT ATA GTG AGT
CGT ATT A-3′; for AS-U1406A, 5′-GGC GAC ACA CCU UCG
GGU GAA GUC GCC CCT ATA GTG AGT CGT ATT A-3′; for
AS-U1495A, 5′-GGC GUC ACA CCU UCG GGU GAA GAC GCC
CCT ATA GTG AGT CGT ATT A-3′; for AS-res, 5′-GGC GUC AUA
CCU UCG GGU UAA GUC GCC CCT ATA GTG AGT CGT ATT
A-3′) were annealed to a 2-fold excess of 18-mer T7 promoter (5′-
TAA TAC GAC TCA CTA TAG-3′) in H2O by heating to 65 °C and
slow cooling to below 37 °C. The 5′-phosphorothioate transcripts were
generated by incubating the annealed templates (0.2 µM) in 50 mM
Tris (pH 7.5), 15 mM MgCl2, 2 mM spermidine, 5 mM DTT, 2 mM
ATP, 2 mM CTP, 2 mM UTP, 0.2 mM GTP, 4 mM guanosine 5′-
monophosphorothioate (5′-GMPS) with 5 U/µL T7 RNA polymerase
and 0.001 U/µL inorganic pyrophosphatase for 2 h at 37 °C in a total
volume of 100 µL. Additional 1-µL aliquots of 20 mM GTP were
added at 20-min intervals and 500 U of T7 RNA polymerase were
added after 1 h. Reactions were quenched by addition of EDTA,
Compound 20d: 1H NMR (D2O) δ ) 7.44-7.34 (m, 5H), 5.00 (d,
1H, J ) 4.25 Hz), 4.25 (dd, 1H, J ) 11.5, 7 Hz), 4.10-4.01 (m, 3H),
3.92-3.87 (m, 1H), 3.81-3.75 (m, 2H), 3.48-3.39 (m, 4H), 3.36-
3.30 (m, 1H), 3.25-3.18 (m, 2H), 3.00 (app t, 2H, J ) 9.5 Hz), 1.99-
1.94 (m, 2H), 1.76-1.68 (m, 2H), 1.54-1.43 (m, 2H); 13C NMR (D2O)
δ ) 170.8, 170.6, 134.4, 130.3, 129.9, 128.8, 98.1, 72.6, 71.2, 69.0,
63.8, 62.6, 53.9, 53.8, 47.3, 41.1, 39.9, 36.7, 31.2, 27.2, 21.8; ESI LRMS
m/z found 497 (MH+), calculated 497.
1
Compound 20e: H NMR (D2O) δ ) 4.97 (d, 1H, J ) 3.5 Hz),
4.07-4.03 (m, 2H), 3.99-3.95 (m, 1H), 3.86 (ddd, 1H, J ) 9, 5.5, 2.5
Hz), 3.74 (dd, 1H, J ) 10.5, 9 Hz), 3.70-3.65 (m, 1H), 3.45-3.38
(m, 2H), 3.28-3.27 (m, 2H), 3.18 (dd, 1H, J ) 13, 8.5 Hz), 2.96 (app
t, 2H, J ) 7.5 Hz), 1.94-1.90 (m, 2H), 1.71-1.65 (m, 2H), 1.50-

Motif for RNA Recognition
J. Am. Chem. Soc., Vol. 120, No. 33, 1998 8327
extracted with phenol, and precipitated with ethanol. Electrophoresis
was carried out on 20% denaturing polyacrylamide gels. Full-length
transcripts were excised from the gel and eluted into 200 mM NaCl,
10 mM Tris (pH 7.5), 0.5 mM EDTA, and desalted on a Nensorb
column (DuPont/NEN). Typically, a 100-µL transcription reaction
produced 150-500 pmol of RNA 5′-phosphorothioate. The purified
RNA transcripts were resuspended in 18 µL of 100 mM NaH2PO4 (pH
8.0) containing 1 mM EDTA and were treated with 2 µL of 20 mM
biotin-iodoacetamide in DMF (USB). After 2 h at room temperature
(RT), an additional 2 µL of the biotinylation reagent was added and
incubation was continued for 1 h. The RNA was ethanol precipitated
in the presence of glycogen, gel purified, and desalted as described
above.
Immobilization of Biotinylated RNAs. Streptavidin functionalized
BIAcore sensorchips were either obtained directly from Pharmacia
Biosensor AB (sensorchip SA5) or were prepared from carboxymethyl-
ated sensorchips (CM5) by EDC activation followed by injection of
streptavidin (Pierce, immunopure grade) in acetate buffer (10 mM, pH
5) over all four flowcells according to the manufacturers recommenda-
tions. Prior to immobilization, frozen solutions of biotinylated RNA
(1-10 pmol) in 80 µL of buffer (10 mM HEPES, 0.1 mM EDTA, 100
mM NaCl, pH 6.8) were renatured by heating to 80 °C for 2 min
followed by slow cooling to RT. Typically, individual flowcells were
functionalized by injecting 60 µL of RNA buffer using the QUICK-
INJECT command at a flowrate of 2 µL/min, followed by injection of
running buffer. Three flowcells were used to immobilize RNA while
the fourth remained unmodified to serve as a blank control for matrix
affects. Levels of RNA capture and ligand binding were calculated
by subtracting response units of the blank flowcell from response units
in the RNA functionalized flowcells.
General Procedures for SPR Binding Studies. Surface plasmon
resonance measurements were performed using a BIAcore 2000 system
from Pharmacia Biosensor AB. Samples were prepared by serial
dilutions from stock solutions in RNase free microfuge tubes (Ambion)
and were centrifuged at 14 000 rpm for degassing. Unless otherwise
noted, all binding studies were carried out using HBS buffer (Pharmacia
Biosensor AB) which was used as obtained. Magnesium salts were
excluded from the buffer as negligible effects are expected for the
binding of aminoglycoside mimetics to the model sequences.14a All
procedures for binding studies were automated as methods using
repetitive cycles of sample injection and regeneration. Typically, buffer
was injected in the first two cycles to establish a stable baseline value.
Samples were injected at a flowrate of 5-10 µL/min using the
KINJECT command. The aminoglycoside mimetics were injected from
autoclaved 7-mm plastic vials that were capped with pierceable plastic
crimp caps. To minimize carry over, samples were injected in order
of increasing concentration. The running buffer was identical with the
injection buffer. Mimetic binding to three RNA sequences was
monitored at once in three parallel flowcells, with the fourth flowcell
containing no immobilized RNA as a control. Compounds were
assayed at four concentrations (100, 31.6, 10, 3.16 µM) with injection
times 4 min 30 s each. After recording three different series, a control
sample of paromomycin was tested to ensure reproducibility. With
the available autosampler racks, up to 12 compounds can be screened
over a period of 24 h.
Expected values for the equilibrium response of 1 equiv of analyte
were calculated from the formula 0.76 × (current response units of
RNA bound) × (aminoglycoside molecular weight)/(RNA molecular
weight). Binding constants were calculated by fitting the recorded
binding isotherm (equivalents bound versus concentration) to a model
with two modes of binding. The binding constant Kns describes a
general binding mode expected between RNA and aminoglycosides
wherein multiple binding events can occur simultaneously. The
constant Ka represents a tighter binding specific site, if any, for these
ligands. The equation is derived from the third-order general isotherm
(eq 1), modified to group higher order binding events with like binding
constants (K2 ) K3 ) Kns) to give the final eq 3. Grouping Kns binding
terms afforded a more robust computational model that does not report
negative high-order binding constants in response to small measurement
errors. Higher order binding equations (N ) 4, N ) 5) which similarly
grouped higher order binding constants did not produce significantly
more accurate results for conditions in which less than 3 equiv of ligand
was bound to the RNA.
i)1
j)1
i[L]i Kaj
∑ ∏
N
i
equivalents bound )
)
i)1
j)1
1 +
[L]i Kaj
∑ ∏
N
i
Ka1[L] + 2Ka1Ka2[L]2 + 3Ka1Ka2Ka3[L]3
(1)
(2)
1 + Ka1[L] + Ka1Ka2[L]2 + Ka1Ka2Ka3[L]3
K2 ) K3 ) Kns
Ka[L] + 2KaKns[L]2 + 3KaKns2[L]3
1 + Ka[L] + KaKns[L]2 + KaKns2[L]3
equivalents bound ) c +
(3)
where c ) constant, Ka ) equilibrium constant for specific binding,
Kns ) equilibrium constant for multiple site binding, and [L] ) mimetic
concentration. A constant c was added to correct for machine baseline
inconsistencies. Equation 3 was fit to the data using KaleidaGraph
(Abelbeck Software) on a Macintosh computer.
Molecular Modeling. The structure shown is a minimized low
energy conformation selected from 100 000 dynamics iterations at 450
K using Insight/Discover. During the dynamics run and the minimiza-
tion, the coordinates of the 2,6-diaminodideoxy glucopyranoside ring
and the RNA were fixed according to the NMR structure of paromo-
mycin bound to AS-wt published by Puglisi and co-workers.11
Acknowledgment. This work was supported by Novartis.
C.R. thanks Odense Universitet Denmark for financial support.
We thank Ian R. Ollmann for assistance in data analysis.
JA980826P