Selective inhibition of bacterial and human topoisomerases by N-arylacyl O-sulfonated aminoglycoside derivatives

Article abstract of DOI:10.1021/ml3004507

Numerous therapeutic applications have been proposed for molecules that bind heparin-binding proteins. Development of such compounds has primarily focused on optimizing the degree and orientation of anionic groups on a scaffold, but utility of these polya

Full text of DOI:10.1021/ml3004507

Letter
pubs.acs.org/acsmedchemlett
Selective Inhibition of Bacterial and Human Topoisomerases by
N‑Arylacyl O‑Sulfonated Aminoglycoside Derivatives
Amanda M. Fenner,† Lisa M. Oppegard,‡ Hiroshi Hiasa,‡ and Robert J. Kerns*,†
†Division of Medicinal and Natural Products Chemistry, Department of Pharmaceutical Sciences and Experimental Therapeutics,
University of Iowa, Iowa City, Iowa 52242, United States
‡Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, United States
S
* Supporting Information
ABSTRACT: Numerous therapeutic applications have been
proposed for molecules that bind heparin-binding proteins.
Development of such compounds has primarily focused on
optimizing the degree and orientation of anionic groups on a
scaffold, but utility of these polyanions has been diminished by
their typically large size and nonspecific interactions with many
proteins. In this study, N-arylacyl O-sulfonated aminoglycosides
were synthesized and evaluated for their ability to selectively
inhibit structurally similar bacterial and human topoisomerases. It is demonstrated that the structure of the aminoglycoside and of
the N-arylacyl moiety imparts selective inhibition of different topoisomerases and alters the mechanism. The results here outline
a strategy that will be applicable to identifying small, structurally defined oligosaccharides that bind heparin-binding proteins with
a high degree of selectivity.
KEYWORDS: Topoisomerase inhibitor, glycosaminoglycan mimic, sulfated oligosaccharide, aminoglycoside derivatives,
ciprofloxacin-induced DNA cleavage
opoisomerases are enzymes that wind and unwind DNA
acyl groups to afford reduced-charge structures capable of
Tby breaking and then religating the DNA. Type II competing with unmodified heparin for binding endogenous
HS-binding proteins.14,15 Replacement of N-sulfo groups with
topoisomerases, such as DNA gyrase and topoisomerase IV
select N-aryl groups afforded heparin derivatives with
maintained or increased affinity for HS-binding proteins.14−16
As an intracellular target, topoisomerases are not readily
accessed by sulfated saccharides. However, there is a significant
degree of structural homology among topoisomerases,17 which
makes topoisomerase inhibition a rigorous test for evaluating
new types of GAG mimics for their ability to selectively interact
with structurally similar heparin-binding proteins.
In this study, a panel of bacterial (E. coli gyrase and E. coli
Topo IV) and human (hTopo I and hTopo II) topoisomerases
was used to evaluate N-arylacyl O-sulfonated aminoglycoside
derivatives for their ability to selectively inhibit the different
topoisomerases. The goal of this work was to determine the
level of selectivity that could be achieved for these novel
trisaccharide and tetrasaccharide heparin mimics to inhibit
function of such structurally similar heparin-binding proteins
and to test this strategy for creating novel, small heparin mimics
that selectively bind heparin and modulate function of heparin-
binding proteins. In addition, the identification of derivatives
that inhibit topoisomerases would represent new lead structures
for further elaboration as possible anticancer or antibacterial
agents.
(Topo IV), make a double stranded break in DNA and pass
unbroken DNA through the break, creating a net change of two
in the linking number.1 Type I topoisomerases, conversely,
make single strand breaks that allow the DNA to swivel around
the unbroken strand.2,3 Topoisomerases are targets for
antibacterial and anticancer agents.4,5
Heparin, a highly sulfated human glycosaminoglycan (GAG)
has been shown to inhibit topoisomerase I (Topo I), a type I
topoisomerase.6 The polyanionic polysaccharide is thought to
mimic the phosphate backbone of DNA and bind to the DNA
binding site.6 Heparin and heparan sulfate (HS) have been
shown to inhibit type I topoisomerase, while HS has an as-yet
unexplained inductive effect on type II topoisomerases.7−9
Inhibition of topoisomerase with fractionated heparin mixtures
demonstrated that highly sulfated heparin chains are better at
inhibiting Topo I.8 Heparin was also a much more potent
inhibitor of Topo I than HS, a less sulfated, though structurally
similar GAG.9 Other oligosaccharide and polysaccharide GAG
mimics have also been shown to inhibit topoisomerases,
including suramin, a hexa-sulfated poly aromatic com-
pound,10,11 a sulfated polysaccharide isolated from microalgae
in red tide,12 and an unsulfonated dextran derivative that was
modified to various degrees by the addition of aromatic
carboxymethyl-benzylamides.13
Received: January 10, 2013
Accepted: March 25, 2013
Published: March 25, 2013
Previous work in our laboratory evaluated replacement of N-
sulfo groups on heparin and other GAGs with nonanionic-N-
© 2013 American Chemical Society
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Synthesis of the O-sulfonated N-arylacyl aminoglycosides was
accomplished in two steps (Scheme 1, panel A). The
aminoglycosides apramycin, kanamycin, and neomycin were
first converted to their N-acetyl, N-benzoyl (N-bz), N-
benzyloxycarbonyl (N-cbz), and N-phenylacetyl (N-pha)
derivatives by coupling with the corresponding N-(acyl)
succinimide (NHS ester) or acyl chloride under alkaline
conditions. Synthesis and characterization of per N-cbz
derivatives of neomycin,18 kanamycin,19 and apramycin20 have
been reported. The per-N-acyl aminoglycosides were sulfonated
with pyridine sulfur trioxide complex (Pyr•SO3) or chlor-
osulfonic acid (ClSO3H). The degree of sulfonation for each
derivative was determined as previously described (see also
Supporting Information for compound synthesis, purification,
and characterization).21 The acetyl derivatives were not
assigned an average degree of sulfation because they lack a
chromophore for calculating peak areas with HPLC and are
instead characterized by a range of sulfated states (e.g., “3−5”
indicates the sample contains a mixture of tri-, tetra-, and penta-
sulfated states).
Scheme 1. Synthesis of N-Arylacylated O-Sulfonated
a
Aminoglycosides
The N-arylacyl O-sulfonated aminoglycosides were initially
screened to determine inhibition of E. coli gyrase-mediated
supercoiling of relaxed DNA and E. coli Topo IV-mediated
relaxation of supercoiled DNA and decatenation of kinetoplast
DNA (kDNA) (see Figure 1; methods in Supporting
Information). For inhibition of gyrase-mediated supercoiling,
gyrase was incubated with relaxed DNA and either no test
compound or 200 μM of one of the following compounds: ab0,
nz7, nb4.6, aa3−5, or ab4.1 (Figure 1A). In the absence of a
test compound, E. coli gyrase converted relaxed DNA into
supercoiled DNA (lane 2). Of the compounds screened, only
nz7 (lane 4) blocked the supercoiling activity of gyrase. For
Topo IV-mediated relaxation of supercoiled DNA, Topo IV
was incubated with negatively supercoiled DNA in the absence
(lane 2) or presence of 200 μM of one of the test compounds:
ab0, nz7, nb4.6, aa3−5, or ab4.1. Topo IV-mediated relaxation
of supercoiled DNA was inhibited by both nz7 (lane 4) and
nb4.6 (lane 5) (Figure 1B). While nz7 inhibited both
a
(Panel A) Methods used for the per N-acylation and O-sulfonation of
aminoglycosides (kanamycin shown as example). (a) NHS ester,
NaHCO3, H2O, 23 °C, 12-16 h, 30-85%; (b) acyl chloride, NaHCO3,
H2O; (c) i: Pyr·SO3, DMF, anhydrous pyr, 66 °C, 5-7 h; ii: H2O, 10
mM NaOH, 4 °C, 45-65%; (d) i: ClSO3H, pyr, 57 °C, 4-6 h; ii: H2O,
NaHCO3, 35-95%. (Panel B) Structures of apramycin and neomycin.
(Panel C) The O-sulfonation reactions give individual products having
varied degrees of sulfation. For each aminoglycoside, final products
obtained are noted by the core aminoglycoside (n, neomycin; k,
kanamycin; a, apramycin), the N-arylacyl group (a, acetyl; b, benzoyl;
z, benzyloxycarbonyl; p, phenylacetyl), and the calculated average
number of sulfate groups per product obtained (e.g., nz5.6) or range of
sulfate groups for the acetyl derivatives (e.g., aa3-5).
Figure 1. Inhibition of E. coli type II topoisomerases. In all panels, enzyme and test compound (drug) are absent (−) in lane 1, enzyme present (+)
and drug absent in lane 2, and enzyme (arrow) and drug present in lanes 3−7 (in D, 3−13). (Panel A) Supercoiling activity of E. coli gyrase was
blocked by nz7 but not affected by the other modified aminoglycosides. (Panel B) Relaxation activity of E. coli Topo IV was inhibited by either nz7
(lane 4) or nb4.6 (lane 5). (Panel C) Decatenation activity of Topo IV was completely blocked by nz7 (lane 4) and partially blocked by nb4.6 (lane
5). (Panel D) Concentration-dependent inhibition of gyrase supercoiling by nz5.6 and nz7.
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supercoiling by gyrase and relaxation by Topo IV, nb4.6 only
inhibited topo-IV-mediated relaxation. For Topo IV-mediated
decatenation of kDNA, E. coli Topo IV was incubated with
kDNA and 200 μM of one of the test compounds (Figure 1C).
Only nz7 completely blocked the decatenation reaction (lane
4), while nb4.6 partially inhibited decatenation (lane 5). Thus,
Both nz7 and nb4.6 inhibited Topo IV activity in both
relaxation and decatenation assays, though the inhibitory effect
of nb4.6 was more pronounced in the relaxation assay.
The observed differential inhibition of bacterial type-II
topoisomerases by select N-arylacyl O-sulfonated aminoglyco-
sides may be due to the degree of sulfation, the N-arylacyl
moiety, the core aminoglycoside, or a combination these
structural differences. As shown in Figure 1A, nz7 inhibited the
gyrase-mediated supercoiling reaction. Since more highly
sulfated heparin derivatives are more potent inhibitors of
topo I,8 N-cbz O-sulfonated neomycin with an average degree
of sulfation of 5.6 (nz5.6) was screened along with persulfated
nz7 to determine concentration-dependent inhibition of gyrase-
mediated supercoiling of relaxed DNA (Figure 1D). Although
100 μM of nz5.6 only partially inhibited gyrase activity (lane
6), 200 μM of nz5.6 completely inhibited the supercoiling
activity of gyrase (lane 7). Comparatively, 50 μM of nz7
partially inhibited gyrase (lane 11), while 100 μM of nz7
completely inhibited gyrase supercoiling activity (lane 12).
Thus, nz7 is about twice as potent as nz5.6. While this result
demonstrates the importance of sulfate groups for these
particular neomycin derivatives to inhibit gyrase, the overall
data in Figure 1 clearly shows the influence of N-arylacyl
moieties and the core aminoglycoside for selective inhibition of
the bacterial type-II topoisomerases.
heparin-binding proteins have poor affinity for sulfated
polysaccharides less than octasaccharide in length.
Only select N-arylacyl O-sulfonated aminoglycoside that have
both N-arylacyl groups and sulfate groups on a certain
aminoglycoside scaffold inhibited different topoisomerases to
different degrees (Table 1 and Figure 1). This result is
consistent with previous studies for N-desulfonated, N-acyl
derivatives of heparin, where only select N-arylacyl groups
could be substituted in place of N-sulfo groups to maintain or
increase affinity for select heparin-binding proteins.14−16 In
addition to the aryl groups on these tri- and tetra-saccharide-
sized compounds being necessary for topoisomerase inhibition,
these results show that both the structure of the core
aminoglycoside and the structure of the N-arylacyl moiety
imparts selectivity for which the topoisomerase(s) are inhibited.
For example, among the N-cbz O-sulfonated aminoglycosides,
only az4.9 showed greater than 90% inhibition of all four
topoisomerases tested (Table 1). The N-cbz kanamycin
derivative kz6, which is more highly sulfated than az4.9,
showed 75% inhibition of gyrase and greater than 95%
inhibition of hTopo I but did not inhibit either Topo IV or
hTopo II. The cognate N-cbz neomycin derivative, nz5.6,
showed greater than 95% inhibition of gyrase, hTopo II, and
hTopo I, but it did not inhibit Topo IV.
To better understand the mechanism of topoisomerase
inhibition, the N-arylacyl O-sulfonated aminoglycosides were
coadministered with ciprofloxacin to evaluate their ability to
interfere with ciprofloxacin-induced DNA cleavage. Ciproflox-
acin is a known topoisomerase poison that, when incubated
with a bacterial type II topoisomerase and DNA, induces DNA
cleavage by arresting the enzyme and its DNA substrate in a
ternary complex that prevents religation of the double stranded
break in the DNA.23−25
To more fully evaluate the role of sulfate groups, core
structure, and N-arylacyl group on selective topoisomerase
interactions, the complete panel of N-arylacyl and N-acetyl
aminoglycosides, both O-sulfonated and bearing no sulfate,
were screened for inhibitory activity with four topoisomerases:
E. coli gyrase, E. coli Topo IV, hTopo II, and hTopo I (Table 1).
Incubation of E. coli Topo IV with a plasmid DNA affords
mainly supercoiled DNA, and small amounts of nicked and
linear DNA (Figure 2, lane 2). The addition of ciprofloxacin
Table 1. Percent Inhibition of Catalytic Activity for Different
Type I and Type II Topoisomerases
a
drug
gyrase
Topo IV
hTopo II
hTopo I
az4.9
kb6.4
kz6
90%
85%
75%
b
95%
80%
95%
>95%
60%
>95%
>95%
70%
kp5.8
na1−6
nz5.6
np5.9
Figure 2. Select N-arylacyl O-sulfonated aminoglycosides block
ciprofloxacin-induced cleavage of DNA by TopoIV.
>95%
95%
>95%
>95%
>95%
>95%
>95%
a
b
Compounds screened at 200 μM. Blank spaces: <5% inhibition.
stimulates the production of linear, cleaved DNA (Figure 2,
lane 3). Ciprofloxacin-induced DNA cleavage is completely
blocked by nz7 (Figure 2, lane 5), while ab0 and nb4.6
produce a slight decrease (Figure 2, lanes 4 and 6). No effect
on ciprofloxacin-induced DNA cleavage was seen with aa3−5
or ab4.1 (Figure 2, lanes 7 and 8).
The effect of N-arylacyl O-sulfonated aminoglycosides on
ciprofloxacin-induced DNA cleavage by gyrase was similarly
investigated (Table 2). With gyrase, kz6 and nz5.6 completely
blocked the production of ciprofloxacin-induced linear DNA,
while np5.9, kb6.4, and az4.9 partially blocked ciprofloxacin-
induced DNA cleavage. These results indicate that, despite the
structural homologies between gyrase and Topo IV,17
compounds az4.9, kb6.4, and np5.9 block ciprofloxacin-
None of the unsulfated N-arylacyl aminoglycosides (azo, kb0,
kz0, kp0, na0, nz0, and np0) inhibited any topoisomerase
(data not shown). The presence of sulfate groups on the N-acyl
aminoglycosides was not alone sufficient to afford topoisomer-
ase inhibition; neither of the N-acetyl O-sulfonated amino-
glycosides (aa3−5 and na1−6) inhibited any of the top-
oisomerases. Indeed, sulfated dextran oligosaccharides with a
degree of polymerization of 14 are capable of inhibiting hTopo-
I.22 Some DNA-binding and heparin-binding proteins, such as
fibroblast growth factor-I, will bind persulfated disaccharides
like sucrose octasulfate and sulfonated polyaromatics like
suramin with weak affinity. However, most DNA-binding and
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oligosaccharides inhibit the supercoiling activity of gyrase by
blocking the binding of the G-segment.
Table 2. Inhibition of Ciprofloxacin-Induced DNA
Cleavage
a
Studies are currently underway to evaluate selective binding
of these novel small molecule heparin mimics with extracellular
heparin-binding proteins that play a role in a number of disease
states. In addition, while the different topoisomerases were
employed in this study as a class of test proteins to evaluate
selective interaction of test compounds with structurally similar
heparin-binding proteins, select compounds possess unexpect-
edly unique activity. Although these N-arylacyl O-sulfonated
aminoglycosides do not appear to readily enter human or
bacterial cells, as evidenced by a lack of cytotoxicity at 200 μM
(see Supporting Information), understanding how these novel
agents uniquely bind and inhibit the different topoisomerases
will further the design, synthesis, and development of smaller,
lower charge structures that might penetrate cells and have
therapeutic utility as novel topoisomerase inhibitors. The
apparent lack of cellular penetration and cytotoxicity for the
present compounds is a positive characteristic in the context of
pursuing agents to selectively bind and modulate activity of
extracellular heparin-binding proteins.
compound
Ec gyrase
Sa gyrase
Ec Topo IV
az4.9
kb6.4
kz6
-
-
+
+
+
-
-
-
+
+
kp5.8
nz5.6
np5.9
ND
+
ND
+
+
+
-
-
a
ND: not determined; + indicates greater than 95% inhibition.
induced DNA cleavage with Topo IV and not with gyrase and
thus may have different binding sites on Topo IV and gyrase. In
the absence of ciprofloxacin, the N-arylacyl O-sulfonated
aminoglycosides did not induce production of linear DNA,
and thus, they do not act as topoisomerase poisons (see
Supporting Information). This unique ability of some N-
arylacyl O-sulfonated aminoglycosides to block ciprofloxacin-
induced DNA cleavage is unexpected and unusual.
To further test for mechanistic differences, kb6.4 and kz6,
were compared for their ability to inhibit the supercoiling
activity of E. coli gyrase and to block ciprofloxacin-induced
DNA cleavage (Figure 3). At 20 μM, neither kb6.4 nor kz6
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis and purification of N-arylacyl O-sulfonated amino-
glycosides, HPLC and LC−MS chromatograms, and topo-
isomerase assay conditions. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
* (R.J.K.) Tel: +1 319 335 8800. Fax: +1 319 335 8766. E-mail:
robert-kerns@uiowa.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by NIH grant AI087671 to
R.J.K., an NIH Predoctoral Traineeship in the Pharmacological
Sciences to A.M.F. (T32 GM067795), an ACS Division of
Medicinal Chemistry Predoctoral Fellowship sponsored by
Bristol-Myers Squibb to A.M.F., and a Predoctoral Fellowship
from the American Foundation for Pharmaceutical Education
to A.M.F.
Figure 3. Inhibition of gyrase supercoiling does not correlate with the
effect on ciprofloxacin-induced DNA cleavage for different N-arylacyl
O-sulfonated aminoglycosides. (A) Supercoiling reaction with kb6.4
and kz6 at either low (L, 20 μM) or high (H, 200 μM) concentration.
(B) Ciprofloxacin-induced DNA cleavage reaction with kb6.4 and kz6
at 200 μM.
ABBREVIATIONS
■
bz, benzoyl; cbz, benzyloxycarbonyl; cipro, ciprofloxacin;
ClSO3H, chlorosulfonic acid; Ec, E. coli; GAG, glycosamino-
glycan; h, human; HS, heparan sulfate; kDNA, kinetoplast
DNA; N-(acyl)succinimide, R-NHS; pha, phenylacetyl;
Pyr·SO3, pyridine sulfur trioxide complex; topo, topoisomerase
inhibited gyrase supercoiling activity (Figure 3A, lanes 3 and 5),
while at 200 μM, both inhibited the supercoiling activity of
gyrase (Figure 3A, lanes 4 and 6). In contrast, kb6.4 showed no
apparent effect on ciprofloxacin-induced cleavage of DNA
(Figure 3B, lanes 3 and 5), while kz6 blocked the effect of
ciprofloxacin on gyrase-mediated DNA cleavage (Figure 3B,
lanes 3 and 7). This suggests that inhibition of gyrase by kz6
and kb6.4 may occur at different steps during the gyrase-
mediated supercoiling reaction. One possible explanation for
this result would be that kb6.4 does not inhibit the binding of
the G-segment but that it inhibits either the binding of the T-
segment or the binding of ATP. In contrast, kz6 and other
REFERENCES
■
(1) Levine, C.; Hiasa, H.; Marians, K. J. DNA gyrase and
topoisomerase IV: biochemical activities, physiological roles during
chromosome replication, and drug sensitivities. Biochim. Biophys. Acta
1998, 1400, 29−43.
(2) Schvartzman, J. B.; Stasiak, A. A topological view of the replicon.
EMBO Rep. 2004, 5, 256−261.
(3) Wang, J. C. DNA topoisomerases. Annu. Rev. Biochem. 1985, 54,
665−697.
473
dx.doi.org/10.1021/ml3004507 | ACS Med. Chem. Lett. 2013, 4, 470−474

ACS Medicinal Chemistry Letters
Letter
(4) Gellert, M.; O’Dea, M. H.; Itoh, T.; Tomizawa, J. Novobiocin and
coumermycin inhibit DNA supercoiling catalyzed by DNA gyrase.
Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 4474−4478.
(5) Sugino, A.; Peebles, C. L.; Kreuzer, K. N.; Cozzarelli, N. R.
Mechanism of action of nalidixic acid: purification of Escherichia coli
nalA gene product and its relationship to DNA gyrase and a novel
nicking-closing enzyme. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 4767−
4771.
(23) Reece, R. J.; Maxwell, A. DNA gyrase: structure and function.
Crit. Rev. Biochem. Mol. Biol. 1991, 26, 335−375.
(24) Froelich-Ammon, S. J.; Osheroff, N. Topoisomerase poisons:
harnessing the dark side of enzyme mechanism. J. Biol. Chem. 1995,
270, 21429−21432.
(25) Hiasa, H. The Glu-84 of the ParC subunit plays critical roles in
both topoisomerase IV-quinolone and topoisomerase IV-DNA
interactions. Biochemistry 2002, 41, 11779−11785.
(6) Ishii, K.; Katase, A.; Andoh, T.; Seno, N. Inhibition of
topoisomerase I by heparin. Biochem. Biophys. Res. Commun. 1982,
104, 541−547.
(7) Harisi, R.; Dudas, J.; Timar, F.; Pogany, G.; Timar, J.; Kovalszky,
I.; Szendroi, M.; Jeney, A. Invasive growth and topoisomerase-switch
induced by tumorous extracellular matrix in osteosarcoma cell culture.
Cell. Biol. Int. 2005, 29, 959−967.
(8) Ishii, K.; Futaki, S.; Uchiyama, H.; Nagasawa, K.; Andoh, T.
Mechanism of inhibition of mammalian DNA topoisomerase I by
heparin. Biochem. J. 1987, 241, 111−119.
(9) Kovalszky, I.; Dudas, J.; Olah-Nagy, J.; Pogany, G.; Tovary, J.;
Timar, J.; Kopper, L.; Jeney, A.; Iozzo, R. V. Inhibition of DNA
topoisomerase I activity by heparan sulfate and modulation by basic
fibroblast growth factor. Mol. Cell. Biochem. 1998, 183, 11−23.
(10) Bojanowski, K.; Lelievre, S.; Markovits, J.; Couprie, J.;
Jacquemin-Sablon, A.; Larsen, A. K. Suramin is an inhibitor of DNA
topoisomerase II in vitro and in Chinese hamster fibrosarcoma cells.
Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 3025−3029.
(11) Onda, T.; Toyoda, E.; Miyazaki, O.; Seno, C.; Kagaya, S.;
Okamoto, K.; Nishikawa, K. NK314, a novel topoisomerase II
inhibitor, induces rapid DNA double-strand breaks and exhibits
superior antitumor effects against tumors resistant to other
topoisomerase II inhibitors. Cancer Lett. 2008, 259, 99−110.
(12) Umemura, K.; Yanase, K.; Suzuki, M.; Okutani, K.; Yamori, T.;
Andoh, T. Inhibition of DNA topoisomerases I and II, and growth
inhibition of human cancer cell lines by a marine microalgal
polysaccharide. Biochem. Pharmacol. 2003, 66, 481−487.
(13) Bittoun, P.; Avramoglou, T.; Vassy, J.; Crepin, M.; Chaubet, F.;
Fermandjian, S. Low-molecular-weight dextran derivatives (f-CMDB)
enter the nucleus and are better cell-growth inhibitors compared with
parent CMDB polymers. Carbohydr. Res. 1999, 322, 247−255.
(14) Huang, L.; Kerns, R. J. Diversity-oriented chemical modification
of heparin: Identification of charge-reduced N-acyl heparin derivatives
having increased selectivity for heparin-binding proteins. Bioorg. Med.
Chem. 2006, 14, 2300−2313.
(15) Huang, L.; Fernandez, C.; Kerns, R. J. Different protein-binding
selectivities for N-acyl heparin derivatives having N-phenylacetyl and
heterocycle analogs of N-phenylacetyl substituted in place of N-sulfo
groups. Bioorg. Med. Chem. Lett. 2007, 17, 419−423.
(16) Fernandez, C.; Hattan, C. M.; Kerns, R. J. Semi-synthetic
heparin derivatives: chemical modifications of heparin beyond chain
length, sulfate substitution pattern and N-sulfo/N-acetyl groups.
Carbohydr. Res. 2006, 341, 1253−1265.
(17) Champoux, J. J. DNA topoisomerases: structure, function, and
mechanism. Annu. Rev. Biochem. 2001, 70, 369−413.
(18) Kumar, V.; Remers, W. A. Aminoglycoside antibiotics. 4.
Regiospecific partial synthesis of ribostamycin and 4″-thioribostamy-
cin. J. Org. Chem. 1981, 46, 4298−4300.
(19) Chen, G.; Pan, P.; Yao, Y.; Chen, Y.; Meng, X.; Li, Z.
Regioselective modification of amino groups in aminoglycosides based
on cyclic carbamate formation. Tetrahedron 2008, 64, 9078−9087.
(20) Igarashi, K.; Honma, T. Aprosamine derivatives. Belgian Patent
890.873, Feb 15, 1982.
(21) Fenner, A. M.; Kerns, R. J. Synthesis, separation, and
characterization of amphiphilic sulfated oligosaccharides enabled by
reversed-phase ion pairing LC and LC−MS methods. Carbohydr. Res.
2011, 346, 2792−2800.
(22) Ishii, K.; Futaki, S.; Uchiyama, H.; Nagasawa, K.; Andoh, T.
Mechanism of inhibition of mammalian DNA topoisomerase I by
heparin. Biochem. J. 1987, 241, 111−119.
474
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