Synthesis of neomycin-DNA/peptide nucleic acid conjugates

Article abstract of DOI:10.1081/CAR-200059973

Syntheses of neomycin conjugates of DNA and peptide nucleic acid (PNA) is reported. The DNA and PNA conjugates were prepared by coupling neomycin isothiocyanate with the amino group of DNA/PNA in order to form a thiourea linkage. The use of neomycin isoth

Full text of DOI:10.1081/CAR-200059973

This article was downloaded by: [The University of Manchester Library]
On: 04 December 2014, At: 10:33
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Carbohydrate Chemistry
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/lcar20
Synthesis of Neomycin‐DNA/Peptide
Nucleic Acid Conjugates
I. Charles a & Dev P. Arya a
a Laboratory of Medicinal Chemistry, Department of Chemistry ,
Clemson University , Clemson, SC, USA
Published online: 16 Aug 2006.
To cite this article: I. Charles & Dev P. Arya (2005) Synthesis of Neomycin‐DNA/Peptide Nucleic Acid
Conjugates, Journal of Carbohydrate Chemistry, 24:2, 145-160, DOI: 10.1081/CAR-200059973
To link to this article: http://dx.doi.org/10.1081/CAR-200059973
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
howsoever caused arising directly or indirectly in connection with, in relation to or arising
out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions

Journal of Carbohydrate Chemistry, 24:145–160, 2005
Copyright # Taylor & Francis, Inc.
ISSN: 0732-8303 print
DOI: 10.1081/CAR-200059973
Synthesis of Neomycin-DNA/
Peptide Nucleic Acid
Conjugates
I. Charles and Dev P. Arya
Laboratory of Medicinal Chemistry, Department of Chemistry, Clemson University,
Clemson, SC, USA
Syntheses of neomycin conjugates of DNA and peptide nucleic acid (PNA) is reported. The
DNA and PNA conjugates were prepared by coupling neomycin isothiocyanate with
the amino group of DNA/PNA in order to form a thiourea linkage. The use of
neomycin isothiocyanate as an electrophilic reagent for a general route to aminoglyco-
side-coupled nucleic acid biopolymers is described.
Keywords Neomycin, PNA, DNA, Neomycin isothiocyanate
INTRODUCTION
Aminoglycoside antibiotics (Figs. 1 and 2), containing a unique polyamine/
carbohydrate structure, have attracted considerable attention because of
their specific interactions with RNA. Their antibacterial activity is mainly
exerted by their binding to the RNA of the bacterial ribosome,[1] and thus inter-
fering with the mRNA translation process. The miscoding causes membrane
damage, which eventually disrupts cell integrity, leading to bacterial cell
death.[2–5]
Aminoglycosides bind to various RNA molecules such as ribosomal RNA,
50-untranslated region of thymidylate synthase mRNA,6 both the Rev
response element and the transactivating response element RNA motifs[7–9]
of HIV-1, and a variety of catalytic RNA molecules such as group I
introns,[10] ribonuclease
P
RNA,[11] hairpin ribozyme,[12] hammerhead
ribozyme,[13] and hepatitis delta virus ribozyme.[14,15]
Received November 23, 2004; accepted December 19, 2004.
Address correspondence to Dev P. Arya, Laboratory of Medicinal Chemistry, Depart-
ment of Chemistry, Clemson University, Clemson, SC 29634, USA. E-mail: dparya@
clemson.edu
145

I. Charles and D. P. Arya
146
Figure 1: Structures/pKas of aminoglycosides with a central ribose.
Aminoglycosides binding to HIV-1 RNA molecules prevents binding of the
cognate viral proteins Tat and Rev to TAR[16] and RRE[7] respectively.[17,18]
While neomycin stabilizes DNA triplex structures,[19] it (Fig. 1) does not
affect DNA duplex stability (under physiologic ionic conditions).[19] Our
previous work has also suggested that aminoglycoside specificity (neomycin
in high nM-low mM range) may be for nucleic acid forms that show some
features characteristic of an A-type conformation rather than for naturally
occurring RNA.[20] This finding suggested that aminoglycoside-DNA conju-
gates could be effective models for targeting nucleic acids’ sequence specifically
(via a hybrid duplex or triplex formation). Covalent attachment of a ligand to
an oligonucleotide (ODN) increases the stabilization induced by other nucleic
acid-stabilizing agents such as spermine,[21,22] acridine,[23] Hoechst 33258,[24]
and psoralen.[25] Conjugation of an aminoglycoside to an ODN can assist in
the following processes: (1) delivery of aminoglycoside to a specific DNA/
RNA site; (2) increased stabilization induced by this hybrid conjugate; and
(3) increased cellular permeability/site-specific delivery of the ODN.
Additionally, since the polyanionic nature of the DNA/RNA backbone is a
major deterrent in forming duplexes and triplexes, peptide nucleic acid (PNA),
a neutral backbone containing oligonucleotide, has been developed.[26–28] The
hybrid complexes formed with PNA and DNA/RNA exhibit extraordinary

Neomycin-DNA/Peptide Nucleic Acid Conjugate Synthesis
147
Figure 2: Structures of aminoglycosides (kanamycin and gentamicin families).
thermal stability and are independent of ionic strength.[26] PNA is not suscep-
tible to hydrolytic (enzymatic) cleavage,[29,30] which displays its potential in
antisense therapeutics. The effective delivery[31,32] of these oligomers to their
respective targets has been achieved with the help of their neutral and lipophi-
lic nature. Because of their significant effects on replication, transcription, and
translation processes as well as their high affinity, high specificity, and high
stability with DNA/RNA observed in in vitro experiments,[33] PNA oligomers
are strong candidates for effective antigene and antisense drug design.
Herein, we describe a general strategy for the solid phase synthesis of cova-
lently attaching aminoglycosides (neomycin) to nucleic acids and analogs
(DNA and PNA).
RESULTS AND DISCUSSION
(1) Covalent Attachment of Neomycin to a DNA Oligomer
The following recognition elements were kept in mind for the design of the
conjugates: The amino groups on rings I, II, and IV (neomycin, Fig. 1, Sch. 1a)

I. Charles and D. P. Arya
148
Scheme1a: Synthesis of neomycin-DNA conjugate.
are necessary in stabilizing and recognizing various nucleic acid forms (amino-
glycosides without any of these amines do not stabilize rRNA, DNA/RNA tri-
plexes as efficiently).[19,34–37] The conjugates based on aminoglycosides must
then retain these amines. The 500-OH on ring III (neomycin) was thus chosen
to provide the linkage to the nucleic acids. The key synthetic step involves
coupling of neomycin isothiocyanate with 50-amino group of the modified
DNA to give the isothiourea linkage (Sch. 1a).
Synthesis of neomycin isothiocyanate[36] 3 (Sch. 1b) was initiated by
conversion of the natural product-neomycin to 500-deoxy-500-amino neomycin 2
(Sch. 1b). Reaction of this amine and 1,10-thiocarbonyldi-2(1H)pyridone (TCDP)
in the presence of 4-N,N-dimethylaminopyridine (DMAP) leads to the formation
of isothiocyanate, which can be easily followed by IR spectroscopy (Fig. 3).
The IR of neomycin isothiocyanate shows the appearance of a new peak at
1/l ¼ 2305.50 cm21, which corresponds to the isothiocyanate group.
The DNA oligomer was modified at the 50-end by introducing 50-amino-50-
deoxythymidine 5 (Sch. 2) at the end of the oligomer synthesis. The synthesis
of 50-amino-50-deoxythymidine 5 from thymidine 4 is described in Scheme. 2.
The amino group was protected with 4-methoxyphenyldiphenylmethyl

Neomycin-DNA/Peptide Nucleic Acid Conjugate Synthesis
149
Scheme1b: Reaction conditions: (i) (a) (Boc)2O, DMF, H2O, Et3N, 608C, 5 hr 60%; (b) 2,4,6-
triisopropylbenzenesulfonyl chloride, pyridine, rt, 40 hr, 50%; (c) H2NCH2CH2SH, NaOEt/EtOH, rt,
18 hr, 50%; (ii) 1,10-thiocarbonyldi-2(1H)pyridone, 4-N,N-dimethylaminopyridine and CH2Cl2
(12 hr, rt 60%).
(MmTr) group, which was followed by phosphitylation using standard phos-
phoramidite chemistry to get 6 (Sch. 2) in good yields.
The synthesis of dT15 7 was carried out on a CPG column using standard
phosphoramidite chemistry. After oligomer synthesis, the di(p-methoxyphenyl)
Figure 3: IR of neomycin isothiocyanate (recorded as a solution in CCl4, and then manually
subtracting the CCl4 peaks to get neomycin isothiocyanate 3 spectrum).

I. Charles and D. P. Arya
150
Scheme 2: Reaction conditions: (i) tetrachlorophthalimide, PPh3, DIAD, and THF (97%); (ii)
ethylenediamine and THF; (iii) 4-methoxyphenyldiphenylmethyl (MmTr) chloride,
triethylamine, 4-N,N-dimethylaminopyridine, and pyridine (60% for two steps); (iv)
CNCH2CH2OP[N(iPr)2]2, bis(diisopropylammonium)tetrazolide, and CH2Cl2 (61%).
phenylmethyl (DMTr) group on the 50-hydroxyl group of dT15 was deprotected
with 4% CCl3CO2H/CH2Cl2. MmTr-protected 50-amino-50-deoxythymidine
phosphoramidite 6 was coupled with the 50-hydroxyl group of dT15 7 in the
presence of 1H-tetrazole as a coupling reagent with extended coupling time
(20 min). After oxidation and capping steps, 50-MmTr group was deprotected
with 4% CCl3CO2H/CH2Cl2 to give 8. The CPG column containing the
oligomer 8 was then dried with argon gas.
The 50-amino group of the oligomer was reacted with a 0.1 M solution of
neomycin isothiocyanate 3 and 10 mol% DMAP. After 12 hr, the column was
washed with 5 ꢀ 1 mL of CH2Cl2 and dried by flushing with argon gas. The con-
jugate 9 was then detached from the solid support using NH4OH and purified
by preparative reverse phase HPLC (RP-HPLC) using a triethylammonium
acetate buffer system. The dried sample was treated with a 1,4-dioxane
solution containing 5% CF3CO2H and 1% m-cresol (v/v/v %). After 30 min,
the deprotected conjugate 10 was precipitated and washed with excess
diethyl ether. The deprotected conjugate 10 was finally purified by
preparative anion exchange HPLC using a Tris.HCl buffer system (Fig. 4).
Conjugate 10 elutes with a retention time of 6.07 min, whereas the noncon-
jugated dT16 elutes at 7.22 min. All major fractions were pooled together and
checked for their identity using MALDI-TOF mass spectrometry (Fig. 5). The
expected mass for neomycin-DNA conjugate is m/z 6202.30 Da and the
MALDI-TOF mass spectrum showed a peak at m/z 6202.90 Da, confirming
the identity of the desired compound.
(2) COVALENT ATTACHMENT OF AMINOGLYCOSIDE TO
PNA OLIGOMER
The 50-amino group of T10 PNA 11 (Sch. 4) was similarly reacted with neomycin
isothiocyanate in the presence of DMAP and pyridine to give PNA-neomycin
conjugate connected through a thiourea linkage. T10 PNA 11 was first syn-
thesized using the standard PNA chemistry protocols and deprotected from
solid support.

Neomycin-DNA/Peptide Nucleic Acid Conjugate Synthesis
151
Figure 4: HPLC profiles for dT16-neomycin conjugate with Boc protected neomycin [9, plot
(a)], dT16-neomycin conjugate [10, plot (b)], and dT16 [plot (c)]; HPLC conditions for plot (a):
buffer A: 100 mM of triethylammonium acetate in 10% of acetonitrile, pH ¼ 7.0; buffer A in 90%
of acetonitrile; 10–50% of buffer B over buffer A during 20 min; flow rate was 1.5 mL/min;
conditions for plot (b) and (c): buffer A: 25 mM of tris-HCl and 1 mM of EDTA, and pH ¼ 8.0;
buffer B: buffer A þ 1M of NaCl, and pH ¼ 8.0; 30% of buffer B in buffer A for 2 min; 30–50%
during 18 min; flow rate was 0.75 mL/min.
After precipitation, the sample was dried and stirred with a pyridine
solution containing neomycin isothiocyanate and DMAP for 12 hr. The solution
was evaporated and washed with diethyl ether. Boc groups on neomycin were
deprotected with trifluoroacetic acid/m-cresol (4 : 1) in methylene chloride

I. Charles and D. P. Arya
152
Figure 5: MALDI-TOF profiles for dT16-neomycin conjugate. MALDI-TOF mass was recorded
with picolinic acid as a matrix.
Scheme 3: Synthesis of PNA T10-neomycin conjucate.

Neomycin-DNA/Peptide Nucleic Acid Conjugate Synthesis
153
(v/v%). Conjugate 12 (Sch. 3) was then purified with RP-HPLC using a trifluoro-
acetic acid buffer system (Fig. 6).
HPLC purification and characterization of T10 PNA 11 (Sch. 3) has been
found to be difficult because of poor solubility in water and self-aggregation
even at lower PNA concentrations (50 mM). After introducing one lysine
residue at the 30-end, T10 PNA solubility increases and self-aggregation also
decreases.[26,38–42] Conjugation of T10 PNA with neomycin remarkably
increased the solubility, and conjugate 12 (Sch. 3) did not self-aggregate even
at high PNA concentrations.
This is most likely due to the introduction of six amino groups in the con-
jugate. The identity of the molecule was confirmed with MALDI-TOF
(Fig. 7). The mass spectrum of the conjugate 12 (Sch. 3) showed a molecular
ion peak at m/z 3398.37 Da (calculated m/z ¼ 3398.22 Da).
A few other PNA sequences that are complementary to biologically import-
ant DNA/RNA sequences were prepared, and their neomycin conjugates were
also successfully prepared.[43–48] The synthesis and purification were carried
out in a manner similar to that of PNA T10-neomycin conjugate 12. The
HPLC retention times of these PNAs with and without neomycin conjugation
are listed in Table 1.
EXPERIMENTAL
All commercial reagents were used without further purification. Pyridine,
methylene chloride, and N,N-dimethylformamide (DMF) were refluxed over
Figure 6: HPLC profile for T10-Neo (12) and T10-LysNH2; buffer conditions for HPLC: buffer A,
0.1% of trifluoroacetic acid in water; buffer B, 0.08% of trifluoroacetic acid in acetonitrile: for
PNA, 0–100% buffer B during 7 min; for PNA conjugate, 0–30% of buffer B over buffer A during
13 min; 30–100% buffer B during 2 min.

I. Charles and D. P. Arya
154
Figure 7: MALDI-TOF profile for PNAT10-neomycin conjugate 12. a-Cyano-4-hydroxycinnamic
acid was used as a matrix.
calcium hydride and distilled. All reactions were carried out in oven-dried
glassware under N2/argon atmosphere. All reactions were monitored by
thin-layer chromatography (TLC) with precoated silica gel on glass plates.
Visualization of TLC plates was achieved with either UV light or staining sol-
utions such as p-anisaldehyde or phosphomolybdic acid followed by heating the
˚
plate with a heating gun. ICN silica gel 32–63 (60 A) was used for column
1
chromatography. H NMR spectra were recorded either using a 300 MHz or a
500 MHz NMR. IR was recorded on a Nicolet Magna-IRTM spectrometer-550
either as a solution of 1,2-dichloroethane or CCl4, and the peaks corresponding
to the solvent were subtracted manually. MALDI-TOF mass was recorded on a
Bruker Daltonics OmniFLEXTM Bench-Top MALDI-TOF mass spectrometer.
Table 1: HPLC retention times of PNA and PNA-neomycin conjugates.
Retention Time (min)
Serial
No.
PNA Sequence
PNA
Conjugate
1
2
3
4
5
6
HTCTCCCTCTCLysNH2[43]
HAGCGTGCGCCATCCCLysNH2[44]
HAGATCTTGGAGTGCGLysNH2[45]
HTAAACLysNH[246]
4.6
8.83
8.98
8.97
8.65
8.98
8.82
4.62
4.64
4.52
4.51
4.50
HCCTAGGAGGAATLysNH2[46]
HCCGGCNH[247]
Buffer conditions: buffer A: 0.1% of TFA in water; buffer B: 0.1% of TFA in acetonitrile; for PNA,
0–100% buffer B during 7 min; 508C; for PNA conjugate, 0–30% of buffer B over buffer A during
13 min; 30–100% buffer B during 2 min, 658C.

Neomycin-DNA/Peptide Nucleic Acid Conjugate Synthesis
155
˚
Preparative anion-exchange HPLC was carried out on a Phenomenex SAX 80 A
column (10 ꢀ 250 mm, 5 m), and for analytical anion-exchange HPLC, a Waters
Gen-Pak FAX (4.6 ꢀ 100 mm) column was used. Preparative and analytical
˚
RP-HPLC was carried out with Alltima C8 100 A (250 ꢀ 4.6 mm, 10 m) column.
Preparation of neomycin isothiocyanate (3, Sch. 1b):[36] To a stirred
solution of neomycin amine 2 (Sch. 1b) (60 mg, 0.05 mmol) in anhydrous
CH2Cl2 was added 1,10-thiocarbonyldi-2(1H)pyridone (12 mg, 0.05 mmol) and
DMAP (1 mg, 0.008 mmol) under an argon atmosphere. After 13 hr, the
solution was concentrated and loaded on a silica gel column. Flash chromato-
graphy with EtOAc as the eluent gave the required compound neomycin iso-
thiocyanate 3 (Sch. 1b) in 48% yield (30 mg); Rf ¼ 0.55 (10% CH3OH in
1
CH2Cl2); H NMR (300 MHz, CDCl3) d ppm: 1.4 (m, 54H), 1.89 (2H), 2.85–2.9
(4H), 3.0–3.3 (9H), 3.40–3.52 (6H), 3.76 (2H), 3.89 (2H), 4.15 (2H), 4.23 (1H),
4.92 (1H), 5.13 (1H), 5.3 (br, 1H); IR (as a solution in CCl4 followed by subtrac-
tion of solvent peaks) n cm21; 1160.71, 1270.95, 1421.9, 1511.7, 1692.44 (.C55O
stretch), 2305.5 (.N55C55S stretch), 2986.74, 3045.28. Maldi MS: 1315.60;
found: 1334.55 (M þ H2O).
Covalent attachment of neomycin to dT16: The oligonucleotide synthesis
was carried out on an Expedite Nucleic Acid Synthesis System (8909) using
standard phosphoramidite chemistry. The 50-amino-50-deoxythymidine 5
(Sch. 2) was synthesized from thymidine using the literature procedure.[49,50]
The 50-amino deoxythymidine was added at the 50-end of the dT15(7) with the
same phosphoramidite chemistry with 20 min coupling time.
After the oligomer synthesis, the CPG column was dried using argon gas. A
syringe containing 0.1 M pyridine solution of neomycin isothiocyanate 3
(Sch. 1b) and 10 mol% of DMAP was attached to one end of the expedite CPG
column containing the oligomer 8, and another end was attached to an
empty syringe. The solution was transferred from one syringe to another and
left undisturbed for 30 min. Again, the solution was pushed back to the other
syringe and this push-and-pull procedure was repeated every 30 min. After
12 hr, the column was detached from both syringes, washed with 5 ꢀ 1 mL of
CH2Cl2, and dried by flushing with argon gas. Then, conjugate 9 was
detached from the solid support using NH4OH, and the resulting solution
was evaporated. RP-HPLC purification using triethylammonium acetate
buffer gave the conjugate 9. The dried sample was treated with 1 mL of 1,4-
dioxane solution containing 5% CF3CO2H and 1% m-cresol (v/v/v %). After
30 min, the conjugate 10 was precipitated with 10 mL of diethyl ether, and
the precipitate was washed with 3 ꢀ 10 mL of diethyl ether.
Conjugate 10 was purified by preparative anion exchange HPLC using a
tris buffer (buffer A: 25 mM of tris.HCl and 1 mM of EDTA, buffer B; buffer A
and 1 M NaCl; 0–60% of buffer B over buffer A during 60 min). The fractions

I. Charles and D. P. Arya
156
were collected and concentrated. The major fractions were checked for their
identity using MALDI-TOF mass spectrometry.
Covalent attachment of neomycin to T10 PNA oligomer (11): The PNA
oligomer synthesis was carried out on Expedite Nucleic Synthesis System
(8909) using standard PNA chemistry on PAL resin (5-(40-Aminomethyl-30,50-
dimethoxyphenoxy)-valeric acid resin). At the end of the synthesis, the
Fmoc-free 50-amino group containing PNA oligomer 11 was dried by flushing
with argon gas. A portion of the sample was removed and deprotected from
the column by the following procedure. A syringe containing a solution of
CF3CO2H (TFA) and m-cresol (4 : 1, v/v%) was attached to one end of the
PAL resin column and another end was attached to an empty syringe. The
solution was transferred from one syringe to another and left undisturbed
for 30 min. Again, the solution was transferred back to the other syringe and
this push-and-pull procedure was repeated every 30 min. After 90 min, the
syringes were detached from the column, and the column was rinsed with
5 ꢀ 0.3 mL of TFA. T10 PNA 11 (Sch. 3) was then precipitated with ethyl
ether and centrifuged. The precipitate was washed with ethyl ether and
dried. Another portion of the T10 PNA 11 (Sch. 3) was stirred with a pyridine
solution containing 0.1 M solution of neomycin isothiocyanate 3 (Sch. 1b) and
10% of DMAP. After 12 hr, the conjugate was precipitated with 10 mL of
diethyl ether and the precipitate was further washed with 3 ꢀ 10 mL of
diethyl ether. The Boc groups on neomycin amines were deprotected with 1M
HCl/dioxane in the presence of 1,2-ethanedithiol to give 12 (Sch. 3).
Conjugate 12 (Sch. 3) was purified by preparative RP-HPLC using a TFA
buffer system (buffer A: 0.1% of TFA in water, buffer B: 0.1% TFA in aceto-
nitrile). The fractions were collected, concentrated, and pooled together after
checking their identity using HPLC and MALDI-TOF mass spectrometry.
CONCLUSION
In conclusion, a novel synthesis of nucleic acid (PNA/DNA)-neomycin
conjugate is reported. In view of the increased hybrid duplex/triplex stability
observed with neomycin, this synthetic methodology opens up new avenues
in sequence-specific drug interaction with nucleic acids. These conjugates
can be used for targeting DNA/RNA of interest via triplex/duplex formation,
and can be used to bring sequence specificity to aminogycoside–rRNA
interactions. The potential of these conjugates in targeting RNA remains an
attractive option because of the highly conserved nature of the A-site
sequence of the 30S ribosomal subunit aminoglycoside binding site. Addition-
ally, such nucleic acid conjugates may assist in overcoming traditional
enzymatic/efflux-mediated methods of antibiotic resistance.

Neomycin-DNA/Peptide Nucleic Acid Conjugate Synthesis
157
ACKNOWLEDGEMENTS
Financial support for this work was provided by NSF-CAREER (CHE/MCB-
0134972) awarded to DPA.
REFERENCES
[1] Noller, H.F. Ribosomal RNA and translation. Annu. Rev. Biochem. 1991, 60,
191–227.
[2] Moazed, D.; Noller, H.F. Interaction of antibiotics with functional sites in 16S ribo-
somal RNA. Nature 1987, 327, 389–394.
[3] Purohit, P.; Stern, S. Interactions of a small RNA with antibiotic and RNA ligands
of the 30S subunit. Nature 1994, 370, 659–662.
[4] Recht, M.I.; Fourmy, D.; Blanchard, S.C.; Dahlquist, K.D.; Puglisi, J.D. RNA
sequence determinants for aminoglycoside binding to an A-site rRNA model oligo-
nucleotide. J. Mol. Biol. 1996, 262, 421–436.
[5] Miyaguchi, H.; Narita, H.; Sakamoto, K.; Yokoyama, S. An antibiotic-binding motif
of an RNA fragment derived from the A-site related region of Escherichia coli 16S
rRNA. Nucleic Acids Res. 1996, 24, 3700–3706.
[6] Tok, J.B.H.; Cho, J.; Rando, R.R. Aminoglycoside antibiotics are able to specifi-
cally bind the 50-untranslated region of thymidylate synthase messenger RNA.
Biochemistry 1999, 38, 199–206.
[7] Zapp, M.L.; Stern, S.; Green, M.R. Small molecules that selectively block RNA
binding of HIV-1 Rev protein inhibit Rev function and viral production. Cell
1993, 74, 969–978.
[8] Mei, H.Y.; Galan, A.A.; Halim, N.S.; Mack, D.P.; Moreland, D.W.; Sanders, K.B.;
Truong, H.N.; Czarnik, A.W. Inhibition of an HIV-1 Tat-derived peptide binding
to TAR RNA by aminoglycloside antibiotics. Bioorg. Med. Chem. Letters 1995, 5,
2755–2760.
[9] Hermann, T.; Westhof, E. Saccharide-RNA recognition. Biopolymers 1998, 48,
155–165.
[10] Chow, C.S.; Bogdan, F.M. A structural basis for RNA-ligand interactions. Chemical
Reviews 1997, 97, 1489–1514.
[11] Mikkelsen, N.E.; Brannvall, M.; Virtanen, A.; Kirsebom, L.A. Inhibition of RNase P
RNA cleavage by aminoglycosides. Proc. Natl. Acad. Sci. USA 1999, 96, 6155–6160.
[12] Earnshaw, D.J.; Gait, M.J. Hairpin ribozyme cleavage catalyzed by aminoglycoside
antibiotics and the polyamine spermine in the absence of metal ions. Nucleic Acids
Res. 1998, 26, 5551–5561.
[13] Tor, Y.; Hermann, T.; Westhof, E. Deciphering RNA recognition: aminoglycoside
binding to the hammerhead ribozyme. Chem. Biol. 1998, 5, R277–R283.
[14] Chia, J.S.; Wu, H.L.; Wang, H.W.; Chen, D.S.; Chen, P.J. Inhibition of hepatitis
delta virus genomic ribozyme self-cleavage by aminoglycosides. J. Biomed. Sci.
1997, 4, 208–216.
[15] Rogers, J.; Chang, A.H.; von Ahsen, U.; Schroeder, R.; Davies, J. Inhibition of the
self-cleavage reaction of the human hepatitis delta virus ribozyme by antibiotics.
J. Mol. Biol. 1996, 259, 916–925.

I. Charles and D. P. Arya
158
[16] Mei, H.Y.; Mack, D.P.; Galan, A.A.; Halim, N.S.; Heldsinger, A.; Loo, J.A.;
Moreland, D.W.; Sannes-Lowery, K.A.; Sharmeen, L.; Truong, H.N.;
Czarnik, A.W. Discovery of selective, small-molecule inhibitors of RNA com-
plexes–I. The Tat protein/TAR RNA complexes required for HIV-1 transcription.
Bioorganic Medicinal Chem. 1997, 5, 1173–1184.
[17] Gao, Y.; Robinson, H.; Wijsman, E.R.; van der Marel, G.A.; van Boom, J.H.;
Wang, A.H.J. Binding of Daunorubicin to b-D-glucosylated DNA found in
protozoa Tryponosoma brucei studied by X-ray crystallography. J. Amer. Chem.
Soc. 1997, 119, 1496–1497.
[18] van Leeuwen, F.; de Kort, M.; van der Marel, G.A.; van Boom, J.H.; Borst, P. The
modified DNA base beta-D-glucosylhydroxymethyluracil confers resistance to
micrococcal nuclease and is incompletely recovered by 32P-postlabeling. Anal.
Biochem. 1998, 258, 223–229.
[19] Arya, D.P.; Coffee, R.L., Jr.; Willis, B.; Abramovitch, A.I. Aminoglycoside-nucleic
acid interactions: Remarkable stabilization of DNA and RNA triple helices by
neomycin. J. Amer. Chem. Soc. 2001, 123, 5385–5395.
[20] Arya, D.P.; Xue, L.; Willis, B. Aminoglycoside (neomycin) preference is for A-form
nucleic acids, not just RNA: results from a competition dialysis study. J. Am.
Chem. Soc. 2003, 125, 10148–10149.
[21] Rajeev, K.G.; Jadhav, V.R.; Ganesh, K.N. Triplex formation at physiological pH:
Comparative studies on DNA triplexes containing 5-Me-dC tethered at N-4 with
spermine and tetraethyleneoxyamine. Nucleic Acids Res. 1997, 25, 4187–4193.
[22] Tung, C.H.; Breslauer, K.J.; Stein, S. Polyamine-linked oligonucleotides for DNA
triple-helix formation. Nucleic Acids Res. 1993, 21, 5489–5494.
[23] Stonehouse, T.J.; Fox, K.R. DNase I footprinting of triple helix formation at poly-
purine tracts by acridine-linked oligopyrimidines: stringency, structural changes
and interaction with minor groove binding ligands. Biochim. Biophys. Acta 1994,
1218, 322–330.
[24] Robles, J.; McLaughlin, L.W. DNA triplex stabilization using a tethered minor
groove binding Hoechst 33258 analogue. J. Amer. Chem. Soc. 1997, 119,
6014–6021.
[25] Perkins, B.D.; Wensel, T.G.; Vasquez, K.M.; Wilson, J.H. Psoralen photo-cross-
linking by triplex-forming oligonucleotides at multiple sites in the human rhodop-
sin gene. Biochemistry 1999, 38, 12850–12859.
[26] Egholm, M.; Buchardt, O.; Nielsen, P.E.; Berg, R.H. Peptide nucleic-acids (Pna) –
oligonucleotide analogs with an achiral peptide backbone. J. Amer. Chem. Soc.
1992, 114, 1895–1897.
[27] Egholm, M.; Nielsen, P.E.; Buchardt, O.; Berg, R.H. Recognition of guanine and
adenine in DNA by cytosine and thymine containing peptide nucleic acids (PNA).
J. Amer. Chem. Soc. 1992, 114, 9677–9678.
[28] Nielsen, P.E.; Egholm, M.; Berg, R.H.; Buchardt, O. Sequence-selective recognition
of DNA by strand displacement with a thymine-substituted polyamide. Science
1991, 254, 1497–1500.
[29] Knudsen, H.; Nielsen, P.E. Antisense properties of duplex- and triplex-forming
PNAs. Nucleic Acids Res. 1996, 24, 494–500.
[30] Hanvey, J.C.; Peffer, N.J.; Bisi, J.E.; Thomson, S.A.; Cadilla, R.; Josey, J.A.;
Ricca, D.J.; Hassman, C.F.; Bonham, M.A.; Au, K.G.; et al. Antisense and
antigene properties of peptide nucleic acids. Science 1992, 258, 1481–1485.

Neomycin-DNA/Peptide Nucleic Acid Conjugate Synthesis
159
[31] Bonham, M.A.; Brown, S.; Boyd, A.L.; Brown, P.H.; Bruckenstein, D.A.;
Hanvey, J.C.; Thomson, S.A.; Pipe, A.; Hassman, F.; Bisi, J.E.; et al. An assessment
of the antisense properties of RNase H-competent and steric-blocking oligomers.
Nucleic Acids Res. 1995, 23, 1197–1203.
[32] Wittung, P.; Kajanus, J.; Edwards, K.; Haaima, G.; Nielsen, P.E.; Norden, B.;
Malmstrom, B.G. Phospholipid membrane permeability of peptide nucleic acid.
FEBS Lett. 1995, 375, 27–29.
[33] Ray, A.; Norden, B. Peptide nucleic acid (PNA): its medical and biotechnical appli-
cations and promise for the future. FASEB J. 2000, 14, 1041–1160.
[34] Arya, D.P.; Coffee, R.L., Jr. DNA triple helix stabilization by aminoglycoside anti-
biotics. Bioorg. Med. Chem. Lett. 2000, 10, 1897–1899.
[35] Arya, D.P.; Coffee, R.L., Jr.; Charles, I. Neomycin induced hybrid triplex formation.
J. Amer. Chem. Soc. 2001, 123, 11093–11094.
[36] Charles, I.; Xue, L.; Arya, D.P. Synthesis of aminoglycoside-DNA conjugates.
Bioorg. Med. Chem. Lett. 2002, 12, 1259–1262.
[37] Xue, L.; Charles, I.; Arya, D.P. Pyrene-neomycin conjugate: dual recognition of a
DNA triple helix. J. Chem. Soc. Chem. Commun. 2002, 70–71.
[38] Tackett Alan, J.; Corey David, R.; Raney Kevin, D. Non-Watson-Crick interactions
between PNA and DNA inhibit the ATPase activity of bacteriophage T4 Dda
helicase. Nucleic Acids Res. 2002, 30, 950–957.
[39] Corey, D.J. Peptide nucleic acids: expanding the scope of nucleic acid recognition.
TIBTECH 1997, 15, 224–229.
[40] Lesnik, E.; Hassman, F.; Barbeau, J.; Teng, K.; Weiler, K.; Griffey, R.; Freier, S.
Triplex formation between DNA and mixed purine-pyrimidine PNA analog with
lysines in backbone. Nucleosides & Nucleotides 1997, 16, 1775–1779.
[41] Gildea, B.D.; Coull, J.M. Compositions for improving solubility of nucleic acids,
peptide nucleic acids, and polyamides. In Patent Cooperation Treaty Int. Appl.
WO9937670; Boston Probes, Inc.: USA; 1999, , 81.
[42] Gildea, B.D.; Casey, S.; NacNeill, J.; Perry-O’Keefe, H.; Sorensen, D.; Coull, J.M.
PNA solubility enhancers. Tetrahedron Lett. 1998, 39, 7255–7258.
[43] Praseuth, D.; Grigoriev, M.; Guieysse, A.L.; Pritchard, L.L.; HarelBellan, A.;
Nielsen, P.E.; Helene, C. Peptide nucleic acids directed to the promoter of the
alpha-chain of the interleukin-2 receptor. Biochim. Biophys. Acta 1996, 1309,
226–238.
[44] Mologni, L.; Nielsen, P.E.; Gambacorti-Passerini, C. In vitro transcriptional and
translational block of the bcl-2 gene operated by peptide nucleic acid. Biochem.
Biophys. Res. Commun. 1999, 264, 537–543.
[45] Armitage, B.; Koch, T.; Frydenlund, H.; Orum, H.; Schuster, G.B. Peptide nucleic
acid (PNA) DNA hybrid duplexes: intercalation by an internally linked anthraqui-
none. Nucleic Acids Res. 1998, 26, 715–720.
[46] Good, L.; Nielsen, P.E. Inhibition of translation and bacterial growth by peptide
nucleic acid targeted to ribosomal RNA. Proc. Natl. Acad. Sci. USA 1998, 95,
2073–2076.
[47] Kurg, R.; Langel, U.; Ustav, M. Inhibition of the bovine papillomavirus E2 protein
activity by peptide nucleic acid. Virus Res. 2000, 66, 39–50.

I. Charles and D. P. Arya
160
[48] Mologni, L.; leCoutre, P.; Nielsen, P.E.; Gambacorti-Passerini, C. Additive anti-
sense effects of different PNAs on the in vitro translation of the PML/RAR alpha
gene. Nucleic Acids Res. 1998, 26, 1934–1938.
[49] Bannwarth, W. Solid phase synthesis of oligodeoxynucleotides containing phos-
phoramidate internucleotide linkages and their specific chemical cleavage. Helve-
tica Chimica Acta 1988, 71, 1517–1527.
[50] Tetzlaff, C.N.; Schwope, I.; Bleczinski, C.F.; Steinberg, J.A.; Richert, C. A con-
venient synthesis of 50-amino-50-deoxythymidine and preparation of peptide-DNA
hybrids. Tetrahedron Lett. 1998, 39, 4215–4218.