Recognition of HIV TAR RNA by triazole linked neomycin dimers

Article abstract of DOI:10.1016/j.bmcl.2011.06.058

A series of neomycin dimers have been synthesized using 'click chemistry' with varying linker functionality and length to target the TAR RNA region of HIV virus. TAR (trans activation response) RNA region, a 59 base pair stem loop structure located at 5′-

Full text of DOI:10.1016/j.bmcl.2011.06.058

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4788–4792
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Recognition of HIV TAR RNA by triazole linked neomycin dimers
⇑
Sunil Kumar, Dev P. Arya
Laboratory of Medicinal Chemistry, Department of Chemistry, Clemson University, Clemson, SC 29634, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of neomycin dimers have been synthesized using ‘click chemistry’ with varying linker function-
ality and length to target the TAR RNA region of HIV virus. TAR (trans activation response) RNA region, a
59 base pair stem loop structure located at 50-end of all nascent HIV-1 transcripts interacts with a key
regulatory protein, Tat, and necessitates the replication of HIV-1 virus. Neomycin, an aminosugar, has
been shown to exhibit more than one binding site with HIV TAR RNA. Multiple TAR binding sites of neo-
mycin prompted us to design and synthesize a small library of neomycin dimers using click chemistry.
The binding between neomycin dimers and HIV TAR RNA was characterized using spectroscopic tech-
niques including FID (Fluorescent Intercalator Displacement) titration and UV-thermal denaturation.
UV thermal denaturation studies demonstrate that neomycin dimer binding increase the melting temper-
ature (Tm) of the HIV TAR RNA up to 10 °C. Ethidium bromide displacement titrations revealed nanomolar
IC50 between neomycin dimers and HIV TAR RNA, whereas with neomycin, a much higher IC50 in the
micromolar range is observed.
Received 4 May 2011
Revised 10 June 2011
Accepted 13 June 2011
Available online 21 June 2011
Keywords:
RNA
DNA
Ó 2011 Elsevier Ltd. All rights reserved.
RNA–protein interactions are essential for regulation of many
important biological processes such as translation, RNA splicing,
and transcription.1 An important example involved in the mecha-
nism of regulatory process of human immunodeficiency
virus type 1 (HIV-1). TAR RNA (trans activation responsive region),
a 59 base stem-loop structure located at the 50-end of the nascent
viral transcripts,2 interacts with Tat protein, (a 86 amino acid
protein) and regulates the transcription of HIV virus. TAR RNA–Tat
protein interaction is an essential interaction in the replication cycle
of HIV virus. TAR, a 29-mer RNA, used in this study is the shorter
version of 59-mer. It contains the binding site for Tat protein (the
tri-nucleotide bulge), along with the wild type TAR RNA core. HIV
TAR structure is comprised of two stems (upper and lower), a three
nucleotide bulge region, and a hairpin (for structures, see Scheme 1).
An arginine rich domain of Tat protein interacts with the tri-
nucleotide bulge (U23, C24, and U25) of TAR RNA3 and causes a
substantial enhancement in the transcript level (ꢀ100-fold).1 NMR
studies have shown that complexation takes place specifically
between arginine residue of Tat protein and a guanine base in the
major groove of TAR RNA.4 Disruption of TAR RNA–Tat interaction
represents an attractive strategy to inhibit viral replication. Several
molecules have been investigated to inhibit TAR RNA–Tat interac-
tion. These include intercalators,5 (ethidium bromide6 and profla-
vine), DNA minor groove binders7 (Hoechst 33258, and DAPI),
phenothiazine,8 argininamide,9 peptides,10 peptidomimetics,11 and
aminoglycosides.12
Aminoglycosides are naturally occurring aminosugars linked
through glycosidic linkages, known to bind to a wide variety of
RNA structures.13 In an attempt to achieve higher RNA binding
affinity and to explore multiple binding sites on RNA targets, the
homo and hetero dimeric units of aminoglycosides14,15 (tobramy-
cin, neamine, neomycin B, and kanamycin A) have been synthe-
sized with various linker length and functionalities through
disulfide bond formation. These aminoglycosides exhibit higher
binding affinity towards dimerized A-site 16 S construct and RRE
RNA than their corresponding monomeric aminoglycoside units.
Also, aminoglycoside dimers exhibit 20- to 12000-fold higher
inhibitory effects towards the catalytic function of Tetrahymena
ribozyme than the monomeric units.15 A neomycin neomycin
dimer synthesized with an intercalator, naphthalenediimide, as
linker, showed 35-fold higher affinity than neomycin towards A-
site 16S RNA.16 We have recently reported that a neomycin dimer
with a flexible thiourea linker showed nanomolar binding affinity
towards AT rich DNA duplexes.17,18 These reports have consider-
ably expanded the potential of aminoglycosides in shape recogni-
tion of nucleic acids.19–28
Neomycin (Scheme 1), an aminoglycoside, is known to bind var-
ious RNA structures, such as the 50-untranslated region of thymi-
dylate synthase mRNA,29 rev response element (RRE) and the
trans activation responsive RNA (TAR RNA) region of HIV-1,30,31
kissing loop formed by DIS (dimerization initiation site) of genomic
HIV RNA,32 group I introns,33 ribonuclease P RNA,34 hairpin ribo-
zyme35 and hammerhead ribozyme.36
Among all the aminoglycosides, neomycin has shown the high-
⇑
Corresponding author.
E-mail address: dparya@clemson.edu (D.P. Arya).
est inhibitory effect towards TAR RNA (Kd < 1 l
M).12 ESI-MS37 and
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.058

S. Kumar, D. P. Arya / Bioorg. Med. Chem. Lett. 21 (2011) 4788–4792
4789
Hairpin Region
G G
U
G
A
C
NH2
C
G
O
G C
Stem
HO
HO
A U
H2N
region
U G C
NH2
H2N
O
O
Upper
C
O
HO
Bulge
U
OH
A U
G C
A U
C G
G C
G C
5' 3'
OH
NH2
OH
O
Lower Stem
O
OH
H2N
Neomycin
TAR RNA
HO
NH2
OH
O
NH2
O
O
HO
H2N
N
N
HO
HO
H2N
N
O
NH2
H2N
O
HO
O
X
N
O
O
O
NH2
OH
N
OH
N
H2N
OH
OH
NH2
NH2
OH
O
H2N
O
12 HCl
O
OH
H2N
X represents variation in linker length
and/or functionality
Neomycin dimer
Scheme 1. Structures of small molecule ligands (neomycin and neomycin dimer) and TAR RNA used in the study.
ribonuclease protection experiments38 have suggested that the
probable binding site of neomycin lies in the stem region just be-
low the tri-nucleotide bulge in TAR RNA. Further ESI-MS experi-
ments and gel shift assays have revealed the existence of three
neomycin binding sites in HIV TAR RNA.37 To achieve higher bind-
ing affinity and explore the multiple binding sites on HIV TAR RNA,
we have designed and synthesized a series of neomycin dimers
using click chemistry. Herein, we report synthesis and TAR RNA
binding studies of a series of triazole linked neomycin dimers
(for structure see Scheme 1) with various linker lengths and flexi-
bility to optimize the binding affinity.
Synthesis of neomycin dimers using click chemistry: We have used
click chemistry39 to synthesize a number of neomycin dimers. A
number of commercially available dialkynes were reacted with 2
mole equivalent of boc-protected neomycin-500-azide, in the pres-
ence of CuI, toluene, and DIPEA, to form the neomycin dimers
(see Scheme 2). Two additional dialkynes were synthesized to
achieve the longer linker length between two neomycin units
(DPA58 and DPA60, Table 1).
Neomycin dimers significantly enhance thermal stability of HIV
TAR RNA: UV thermal denaturation studies were carried out to
monitor the effect of neomycin dimers on HIV-1 TAR RNA. Figure
1 shows the representative plot of UV denaturation profile of HIV
TAR RNA in the absence and presence of neomycin dimer (left)
and neomycin (right) at a HIV-1 TAR RNA/ligand ratio of 1. The
UV thermal denaturation temperature of HIV-1 TAR RNA and
HIV-1 TAR RNA–ligand complexes were determined using first
derivative plots and the results are summarized in Table 1. The
UV melting temperature (Tm) of HIV 1 TAR RNA in the absence of
ligand is 68.9 °C (100 mM KCl, pH 6.8).
hance the thermal stabilization of HIV TAR RNA from ꢀ3 to
10 °C; depending on the linker length and structure (Table 1), (3)
The UV thermal denaturation profiles of HIV-1 TAR RNA in the
presence of neomycin dimers show only one transition, suggestive
of specific binding at a single site. (4) A clear trend is observed
between the linker length of neomycin dimers and the thermal sta-
bility of HIV 1 TAR RNA, such that the dimers with shorter linker
length show higher thermal stabilization of HIV-1 TAR RNA, as
opposed to neomycin dimers with longer linkers (Table 1). (5)
There is very little further increase (1–2 °C) in the thermal stabil-
ization of HIV-1 TAR RNA at higher stoichiometric ratio of all neo-
mycin dimers (dimer/RNA ratio = 2:1), indicative of a non-specific
electrostatic interaction at higher concentrations of neomycin
dimers (data not shown).
CD spectroscopy studies to characterize the binding between neo-
mycin dimer and HIV-1 TAR RNA: Previous studies have revealed
that there is a substantial change in the structure of HIV TAR
RNA induced by Tat protein and other ligands which can be mon-
itored by CD spectroscopy.40,41 Therefore, CD studies were carried
out to monitor the binding interaction between neomycin dimer
and HIV-1 TAR RNA. As depicted in Figure 2, the CD spectrum of
HIV 1 TAR RNA alone is representative of an A-form RNA. The CD
spectrum of HIV TAR RNA contains a strong positive band near
265 nm, a fairly intense negative band around 215 nm, and a weak
negative band around 240 nm.42 A CD titration was performed be-
tween neomycin dimer and HIV TAR RNA by continuous addition
of neomycin dimer in HIV TAR RNA solution (Fig. 2A). The shape
of both the CD spectra, in the absence and presence of neomycin
dimer are similar suggesting that the overall conformation of HIV
TAR RNA is conserved.
UV thermal denaturation of HIV-1 TAR RNA in the absence and
presence of neomycin monomer and dimers was performed. A few
trends can be identified: (1) There is very little change observed in
the Tm of HIV-1 TAR RNA in the presence of neomycin (ꢀ0.2 °C),
which lies within experimental error. (2) Neomycin dimers en-
There are, however, some changes in the spectrum of HIV TAR
RNA upon addition of neomycin dimer. The alteration of the CD
spectrum of HIV TAR RNA is indicative of the interaction between
HIV TAR RNA and neomycin dimer. The binding of neomycin dimer
causes a gradual decrease in the peak at 210 and 225 nm upon

4790
S. Kumar, D. P. Arya / Bioorg. Med. Chem. Lett. 21 (2011) 4788–4792
NH2
O
HO
H2N
HO
HO
NH2
H2N
O
O
O
OH
OH
NH2
O
OH
O
H2N
OH
a-c
NHR
O
HO
HO
RHN
NHR
RHN
O
R = Boc
OH
O
N3
NHR
O
OH
OH
O
O
RHN
OH
d-e
HO
O
NH2
OH
NH2
O
O
HO
HO
H2N
H2N
HO
N
N
NH2
O
H2N
HO
O
N
X
N
N
NH2
O
O
O
NH2
O
N
OH
OH
H2N
OH
OH
NH2
OH
O
H2N
O
O
H2N
12 HCl
OH
Scheme 2. Reagents and conditions: (a) (Boc)2O, DMF, H2O, Et3N, 60 °C, 5 h, 60%; (b) TPS–Cl, pyridine, rt, 40 h, 50%; (c) NaN3, DMF/H2O (10:1), 12 h, 90 °C, 90%; (d) toluene,
CuI, DIPEA, 90 °C, 18 h, 85–92%; (e) 4 M HCl/dioxane, rt, 5 min, 76–95%.
Table 1
Table of UV thermal denaturation of ligand/HIV TAR RNA complexes at rdd = 1 and
D
Tm
Neomycin dimer
Structure of the linker (–x–, from Schemes 1 and 2)
Tm
DTm
HIV-1 TAR RNA
DPA 51
DPA 52
NA
–O–
–CH2–
68.9
79.1
78.2
NA
10.2
9.3
DPA 65
78.2
9.3
DPA 53
DPA 54
78.5
77.1
9.6
8.2
–CH2CH2–
H2
C
DPA 55
76.4
7.6
C
H2
H2
C
DPA 56
DPA 58
74.9
74.3
6.1
5.4
C
H2
O
O
4
O
O
DPA 60
72.1
69.1
3.3
0.2
6
Neomycin
NA
Buffer conditions: 100 mM KCl, 10 mM SC, 0.5 mM EDTA, pH 6.8. HIV TAR RNA = 1
Neomycin = 1 M.
lM/strand. Neomycin dimer = 1 lM.
l

S. Kumar, D. P. Arya / Bioorg. Med. Chem. Lett. 21 (2011) 4788–4792
4791
0.45
HIV TAR RNA
TAR RNA + Neomycin (1:1)
HIV TAR RNA
TAR RNA + DPA 52 (1:1)
0.6
0.58
0.56
0.54
0.52
0.5
0.4
0.35
0.3
40
50
60
T (oC)
70
80
90
40
50
60
T (oC)
70
80
90
Figure 1. UV thermal denaturation data of HIV-1 TAR RNA with neomycin dimer (left) and neomycin (right). UV thermal denaturation data of HIV-1 TAR RNA in the absence
(open circle) and presence (solid circle) of ligands (at rdd = 1). Buffer conditions: 100 mM KCl, 10 mM SC, 0.5 mM EDTA, pH 6.8. HIV TAR RNA = 1 M/strand. Neomycin
dimer = 1 M, neomycin = 1
l
l
lM
-18
-20
-22
-24
-26
-28
-30
-32
60
40
20
0
(B)
(A)
increasing
-20
-40
neoneo dimer
0
0.5
1
1.5
2
2.5
3
3.5
200
220
240
260
280
300
molar eq. of neomycin dimer
Wavelength (nm)
Figure 2. CD titration between neomycin dimer and HIV TAR RNA. (A) CD titration of HIV TAR RNA with increasing concentration of neomycin dimer DPA 51. The figure
represents molar ellipticity for CD titration of HIV TAR RNA with neomycin dimer. The continuous changes in the CD spectra correspond to the incremental amount of
neomycin dimer ranging from an rdd of 0–3. (B) A plot of normalized molar ellipticity versus rdd for CD titration of HIV TAR RNA with neomycin dimer. The continuous lines in
the plot reflect the linear least squares fit of each apparent linear domain of the experimental data (filled circles) before and after the apparent inflection point. Molar
ellipticity is per molecule of HIV TAR RNA, and rdd = ratio of the HIV TAR RNA/drug.
1
1
(A)
(B)
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
-0.2
0
0.1
0.2
0.3
0.4
0.5
1
1.5
2
2.5
3
log[DPA56 in µM]
DPA56 [µM]
Figure 3. Representative example of ethidium bromide displacement assays. (A) Decrease in the fluorescence intensity of ethidium bromide–RNA complex with increase in
the concentration of neomycin dimer. (B) The plot for fraction of ethidium bromide displaced from HIV TAR RNA versus the log of concentration of neomycin dimer. The data,
shown with a sigmoidal fit, was used to determine the IC50 value.
incremental addition of neomycin dimer in the HIV TAR RNA solu-
tion. Contrary to Tat protein,42 there is a slight increase in the
intensity at 265 nm along with a shift to longer wavelengths in
the presence of neomycin dimer. The change in the molar
ellipticity of HIV TAR RNA at 265 nm is typical of aminoglyco-
sides–TAR binding. The peak at 265 nm has been attributed to
the sensitivity of base stacking into the bulge region.43 This obser-
vation gives us an insight into the probable binding site of neomy-
cin dimer to HIV TAR RNA. Tat protein binding to the bulge region
of the HIV TAR RNA results in the modification of RNA base stack-
ing and hence causes a significant decrease in the peak intensity
around 265 nm. Neomycin dimer does not induce a significant
change in the peak at 265 nm, suggesting that it does not bind in
the same region as Tat. The CD data is consistent with the model
of approximately one neomycin binding to the lower stem region
of HIV TAR RNA (for structure see Scheme 1), and the second neo-
mycin unit binding to upper stem or the hairpin region, though fur-
ther studies need to be performed to confirm this hypothesis.

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References and notes
120
100
80
60
40
20
0
1. Jones, K. A.; Peterlin, B. M. Annu. Rev. Biochem. 1994, 63, 717.
2. Rana, T. M.; Jeang, K. T. Arch. Biochem. Biophys. 1999, 365, 175.
3. Feng, S.; Holland, E. C. Nature 1988, 334, 165.
4. Puglisi, J.; Tan, R.; Calnan, B.; Frankel, A.; Williamson, J. R. Science 1992, 257, 76.
5. Bailly, C.; Colson, P.; Houssier, C.; Hamy, F. Nucleic Acids Res. 1996, 24, 1460.
6. Ratmeyer, L. S.; Vinayak, R.; Zon, G.; Wilson, W. D. J. Med. Chem. 1992, 35, 966.
7. Dassonneville, L.; Hamy, F.; Colson, P.; Houssier, C.; Bailly, C. Nucleic Acids Res.
1997, 25, 4487.
8. Lind, K. E.; Du, Z.; Fujinaga, K.; Peterlin, B. M.; James, T. L. Chem. Biol. 2002, 9,
185.
9. Tao, J.; Frankel, A. D. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 2723.
10. Hwang, S.; Tamilarasu, N.; Ryan, K.; Huq, I.; Richter, S.; Still, W. C.; Rana, T. M.
Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12997.
11. Athanassiou, Z.; Patora, K.; Dias, R. L. A.; Moehle, K.; Robinson, J. A.; Varani, G.
Biochemistry (N. Y.) 2007, 46, 741.
12. Mei, H.; 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. Bioorg. Med. Chem. 1997, 5, 1173.
13. Thomas, J. R.; Hergenrother, P. J. Chem. Rev. 2008, 108, 1171.
14. Wang, H.; Tor, Y. Bioorg. Med. Chem. Lett. 1997, 7, 1951.
15. Michael, K.; Wang, H.; Tor, Y. Bioorg. Med. Chem. 1999, 7, 1361.
16. Tok, J. B.; Fenker, J. Bioorg. Med. Chem. Lett. 2001, 11, 2987.
17. Arya, D. P.; Coffee, R. L., Jr.; Xue, L. Bioorg. Med. Chem. Lett. 2004, 14, 4643.
18. Kumar, S.; Xue, L.; Arya, D. P. J. Am. Chem. Soc. 2011, 133, 7361.
19. Willis, B.; Arya, D. P. Bioorg. Med. Chem. Lett. 2009, 19, 4974.
20. Xi, H.; Gray, D.; Kumar, S.; Arya, D. P. FEBS Lett. 2009, 583, 2269.
21. Shaw, N. N.; Arya, D. P. Biochimie 2008, 90, 1026.
O
O
-CH2CH2-
O
O
-O-
-CH2-
7
7
7
8
8
10
10
16
20
Linker Length
Figure 4. Bar graph representing the effect of linker length and structure of
neomycin dimers on the binding affinity towards HIV-1 TAR RNA, determined using
ethidium bromide displacement experiments.
22. Shaw, N. N.; Xi, H.; Arya, D. P. Bioorg. Med. Chem. Lett. 2008, 18, 4142.
23. Willis, B.; Arya, D. P. Curr. Org. Chem. 2006, 10, 663.
24. Willis, B.; Arya, D. P. Adv. Carbohydr. Chem. Biochem. 2006, 60, 251.
25. Arya, D. P. Top. Curr. Chem. 2005, 253, 149.
26. Xue, L.; Charles, I.; Arya, D. P. Chem. Commun. 2002, 1, 70.
27. Willis, B.; Arya, D. P. Biochemistry 2010, 49, 452.
Ethidium bromide displacement titrations: To further investigate
the binding between HIV TAR RNA and neomycin dimers, ethidium
bromide displacement experiments44,45 were used (Fig. 3) and the
results are summarized in Figure 4. The binding affinity of neomy-
cin dimers is proportional to the amount of ethidium bromide
displaced from HIV TAR RNA and reflected in the IC50 (the concen-
tration of ligand required to displace 50% ethidium bromide from
HIV TAR RNA) values (Fig. 3).
Ethidium bromide displacement experiments indicate that in
general, neomycin dimers with shorter linker lengths have higher
affinity than neomycin dimers with longer linker towards HIV TAR
RNA. The IC50 values for neomycin dimers fall within a range of
36–99 nM (see Fig. 4), which is in the range of Tat–TAR binding
(IC50 = 56 nm). In comparison to neomycin dimers, neomycin shows
very low affinity towards HIV TAR RNA. The IC50 value of neomycin
towards HIV TAR RNA is 417 115 nM. This trend is similar to what
is observed with UV thermal denaturation experiments.
28. Ranjan, N.; Andreasen, K. F.; Kumar, S.; Hyde-Volpe, D.; Arya, D. P. Biochemistry
(N. Y.) 2010, 49, 9891.
29. Tok, J. B.; Cho, J.; Rando, R. R. Biochemistry 1999, 38, 199.
30. Mei, H.; 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.
31. Zapp, M. L.; Stern, S.; Green, M. R. Cell 1993, 74, 969.
32. Bernacchi, S.; Freisz, S.; Maechling, C.; Spiess, B.; Marquet, R.; Dumas, P.;
Ennifar, E. Nucleic Acids Res. 2007, 35, 7128.
33. von Ahsen, U.; Davies, J.; Schroeder, R. J. Mol. Biol. 1992, 226, 935.
34. Mikkelsen, N. E.; Brannvall, M.; Virtanen, A.; Kirsebom, L. A. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 6155.
35. Earnshaw, D. J.; Gait, M. J. Nucleic Acids Res. 1998, 26, 5551.
36. Hermann, T.; Westhof, E. Biopolymers 1998, 48, 155.
37. Sannes-Lowery, K. A.; Mei, H.; Loo, J. A. In. J. Mass Spectrometry 1999, 193, 115.
38. Wang, S.; Huber, P. W.; Cui, M.; Czarnik, A. W.; Mei, H. Biochemistry (N. Y.)
1998, 37, 5549.
39. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angewandte Chemie International Edition
2001, 40, 2004.
40. Long, K. S.; Crothers, D. M. Biochemistry (N. Y.) 1995, 34, 8885.
41. Loret, E. P.; Georgel, P.; Johnson, W. C.; Ho, P. S. Proc. Natl. Acad. Sci. U.S.A. 1992,
89, 9734.
42. Frankel, A. D. Curr. Opin. Genet. Dev. 1992, 2, 293.
43. Wang, S.; Huber, P. W.; Cui, M.; Czarnik, A. W.; Mei, H. Y. Biochemistry (N. Y.)
1998, 37, 5549.
In conclusion, an efficient synthesis of neomycin dimers46 using
click chemistry has been performed. Biophysical results from ethi-
dium bromide displacement and UV thermal denaturation experi-
ments show that the two neomycin binding sites on HIV TAR RNA
are very close to each other as seen by the higher affinity of neomy-
cin dimers with shorter linker length towards HIV TAR RNA. Fur-
ther studies on the effect of these dimers on protein binding and
HIV inhibition are ongoing and will be reported in due course.
44. Luedtke, N. W.; Hwang, J. S.; Glazer, E. C.; Gut, D.; Kol, M.; Tor, Y. ChemBioChem
2002, 3, 766.
45. Boger, D. L.; Fink, B. E.; Brunette, S. R.; Tse, W. C.; Hedrick, M. P. J. Am. Chem. Soc.
2001, 123, 5878.
46. General procedure for synthesis of N-Boc protected neomycin dimers. To a solution
of 500-azide-neomycin
B (62 mg, 0.05 mmol) in dry toluene (5 mL), diyne
(0.025 mmol, 0.50 equiv) was added followed by the addition of CuI (4.76 mg,
0.025 mmol) and DIPEA (6.46 mg, 0.05 mmol). The reaction mixture stirred at
rt for 18 h in the atmosphere of argon. The volatiles were removed under
vacuum. Purification by flash column chromatography (0–10% ethanol in
CH2Cl2) afforded the desired product as a white solid (80–89%).
Acknowledgment
This work was supported by National Institute of Health Grant
(R15CA125724 to D.P.A.)
Deprotection of N-Boc protected neomycin dimers. To a solution of neomycin
dimer (30 mg) in dioxane (3 mL), 4 M HCl/dioxane (1 mL) was added and the
reaction started at room temperature. A white precipitate formed after 15 min.
The reaction mixture was centrifuged and the solid was collected. The solid
was washed with a solution of diethyl ether/hexane (3 ꢁ 5 mL each, v/v). The
solid was then dissolved in water and lyophilized to afford a white solid (90–
95%). For characterization of one of the dimers, please see Supplementary data.
Complete characterization of all the dimers will be reported elsewhere.
Supplementary data
Supplementary data (general synthesis procedures and charac-
terization of one of the ligand) associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2011.06.058.