Kanamycin A 6′-pyrenylamide: A selective probe for heparin detection

Article abstract of DOI:10.1016/j.tetlet.2012.03.123

We have synthesized a novel derivative of kanamycin A bearing a pyrenyl fluorophore which can be used for the specific recognition of heparin in EtOH:H₂O (1:3) through formation of a 2:1 complex. The receptor shows specific optical signalling for heparin, but exhibits no significant changes on addition of various other biological molecules.

Full text of DOI:10.1016/j.tetlet.2012.03.123

Tetrahedron Letters 53 (2012) 2864–2867
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Kanamycin A 60-pyrenylamide: a selective probe for heparin detection
Santosh V. Nalage a, Sidhanath V. Bhosale a,c, , Suresh K. Bhargava b, Sheshanath V. Bhosale b,
⇑
⇑
a Department of Organic Chemistry, North Maharashtra University, Jalgaon, India
b School of Applied Sciences, RMIT University, GPO Box 2476V, Melbourne, Vic. 3001, Australia
c Polymer and Functional Materials, Indian Institute of Chemical Technology, Hyderabad, AP 500 607, India
a r t i c l e i n f o
a b s t r a c t
Article history:
We have synthesized a novel derivative of kanamycin A bearing a pyrenyl fluorophore which can be used
for the specific recognition of heparin in EtOH:H2O (1:3) through formation of a 2:1 complex. The recep-
tor shows specific optical signalling for heparin, but exhibits no significant changes on addition of various
other biological molecules.
Received 23 November 2011
Revised 18 March 2012
Accepted 29 March 2012
Available online 4 April 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Biosensor
Heparin
Aminoglycoside
Pyrene
Fluorescence
UV–vis spectroscopy
Fluorescent molecules have been central to the development of
highly sensitive and selective sensors for the detection of cations,
anions and neutral molecules, particularly in bioimaging and bio-
molecule detection.1,2 Heparin is a widely used pharmacological
agent for anticoagulation during surgery.3 Heparin works as a
modulator and effector, disrupting important signalling through
binding to molecules such as antithrombin III leading ultimately
to the inactivation of factor Xa.4 Heparin, a highly sulfated amino-
glycan, is also a highly negatively-charged biopolymer at physio-
logical pH. Several methods for heparin detection have been
described including the use of surface plasmon resonance, colori-
metric changes and indicator displacement assay approaches.5,6
In addition, there have been several reports on heparin binding
to various synthetic receptors7 and polymeric conjugates.8–10 The
drawbacks associated with many of these approaches, such as poor
selectivity and sensitivity and the use of short emission wave-
lengths, mean that simple, practical sensors for the detection of
heparin are still needed.
compromised by oto- and nephrotoxicity and the rapid emergence
of bacterial resistance. However, their propensity to bind strongly
to negatively-charged species has led to studies in gene transfec-
tion, micelle formation and in forming fibres on DNA, amongst oth-
ers.12–14
Herein, we report that a new kanamycin A derivative 1 bearing
a pyrenyl fluorophore chromophore is highly selective and sensi-
tive for heparin detection by both colorimetric and fluorometric
analyses. Derivative 1 contains two kanamycin A moieties that
could function as binding sites and a built-in pyrene component
which should act as a colorimetric reporter of any binding events
(Fig. 1). The conjugate kanapy-60 (kanamycin A-pyrene) 1 bears
three important features: (1) polar hydroxy head groups at the
periphery for hydrogen bonding, (2) four amino functional groups
for interaction with anionic species and (3) a pyrenyl moiety which
plays the role of both colorimetric and fluorometric reporter upon
binding. It is the polyamine unit (kanamycin, with its pyrene fluo-
rophore, i.e. kanapy-60) that aggregates around the polyanionic
heparin, bringing them into close proximity.
On the other hand, aminoglycoside antibiotics and their deriva-
tives act as key components for new applications. Examples includ-
ing kanamycin A are a group of clinically important antibacterial
drugs used for the treatment of Gram-negative infections.11 Their
widespread use over the past decade has been significantly
Kanamycin A 60-pyrenylamide was prepared in 65% yield by
condensation of kanamycin A disulfate (2) with pyrene butyric acid
3 in the presence of O-(benzotriazol-1-yl)-N,N,N0,N0-bis(tetrameth-
ylene)uranium hexafluorophosphate (HBPyU) and sodium bicar-
bonate in a 1:1 mixture of distilled deionized water and THF at
room temperature (Scheme 1). The major product shows coupling
at the 60-position only, in line with the earlier results.15,16
The ability of 1 to complex with biological molecules was
explored with UV–vis absorption and fluorescence spectrometry.
⇑
Corresponding authors. Fax: +91 257 2258403 (Sidhanath V.B.); fax: +61 3
990252680 (Sheshanath V.B.).
E-mail addresses: sidhanath.bhosale@gmail.com (S.V. Bhosale), bsheshanath@
gmail.com (S.V. Bhosale).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.03.123

S. V. Nalage et al. / Tetrahedron Letters 53 (2012) 2864–2867
2865
OH
H2N
HO
O
OH
O
O
H
OH
H2
N
O
OH
O
CH2OSO3
H2N
NH2
H
O
O2C
CO2
OH
OH
HO
H
O
O
O
HO
O
OH
O3S
H
OH
HO
NHSO3
O
O
O
O
HN
OH
O
NH2
OSO3
n
O
HO
H
O
OH
Heparin
Buffer
H2N
OH
OSO3
1
OH
O
HO
HO
O
O
H2N
NH2
O
OH
HN
O
H
HO
S
N
OH
O3
H
OH
HN
O
HO
NH
O
O
O
Figure 1. Schematic illustration of the proposed interactions of heparin with kanapy-60 (1).
OH
OH
HO
OH
O
HO
O
HO
H2N
O
+
NH2
HO
O
OH
O
NH2
.
2H2SO4
H2N
3
2
NaHCO3,
HBPyU (1 equiv)
H2O:THF (1:1)
OH
O
HO
O
OH
HO
O
N
H
HO
H2N
HO
O
OH
NH2
O
1
H2N
Scheme 1. Synthetic route for the synthesis of kanamycin A 6-pyrenylamide (1).
The conjugate kanapy-60 (1) displays two absorption bands at 342
and 328 nm and one weak band at 308 nm in the UV spectrum in
EtOH:H2O (1:3),17 as shown in Figure 2. Upon addition of heparin,
concentration-dependent changes were observed occurring in
intensity and bathochromic shifts leading to a clear isosbestic
point at 348 nm. Figure 2b illustrates the gradual enhancement
in absorption at 358 nm upon interaction of heparin with 1 and
the apparent association constant (Ka) was determined to be
2.1 ꢀ 10ꢁ4 M. The band broadening and red shift in the absorption
of 1 upon the accumulation of heparin can be attributed to inter-
molecular
p–p interactions between two or more pyrene residues
in the complex.
The formation of a pyrene excimer was also supported by fluo-
rescence titration of 1 with heparin. The fluorescence emission
spectrum of kanapy-60 (1) (kexc = 342 nm) consists of two strong
bands at 376 and 400 nm, corresponding to the emission of the
Figure 2. Overall changes in the absorption spectra of 1 (10
heparin (0–1.4 mM) in 10 mM HEPES buffer solution (EtOH:H2O = 1:3). (b) Changes
in the conjugate 1 at relative absorption intensity (I358) on addition of 10 ll aliquots
of heparin.
l
M) upon addition of

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S. V. Nalage et al. / Tetrahedron Letters 53 (2012) 2864–2867
Figure 4. Selectivity of conjugate 1 for heparin upon addition of various biomol-
ecules at concentration of 200 in 10 HEPES buffer solution
(EtOH:H2O = 1:3) to 10 solution of conjugate in 10 HEPES buffer
a
l
M
lM
a
l
M
1
lM
solution (EtOH:H2O = 1:4) at pH 7.4. Excitation wavelength was at kmax = 342 nm,
with a slit width of 2 nm.
Acknowledgements
S.V.N. and Sid. V.B. thank the Department of Science and Tech-
nology, New Delhi, India for financial support under the Fast-Track
Young Scientist Programme (SR/FTP/CS-82/2007). We also
acknowledge Prof. Steven Langford for valuable suggestions.
Supplementary data
Supplementary data associated with this article can be found, in
theonlineversion, at http://dx.doi.org/10.1016/j.tetlet.2012.03.123.
References and notes
Figure 3. (a) Changes in the fluorescence spectra of conjugate 1 (10 lM) upon
titration with heparin (0–200 lM) in 10 mM HEPES buffer solution
1. (a) Buhlmann, P.; Pretsch, E.; Bakker, E. Chem. Rev. 1998, 98, 1593; (b) Prode, L.;
Bolletta, F.; Montalti, M.; Zaccheroni, N. Coord. Chem. Rev. 2000, 205, 59; (c)
Manez, R. M.; Sancenon, F. Chem. Rev. 2003, 103, 4419; (d) Fabbrizzi, L.;
Licchelli, M.; Rabaioli, G.; Taglietti, A. Coord. Chem. Rev. 2000, 205, 85; (e) James,
T. D.; Linnane, P.; Shinkai, S. Chem. Commun. 1996, 281; (f) Takeuchi, M.;
Yamamoto, M.; Shinkai, S. Chem. Commun. 1997, 1731; (g) Yam, V. W.-W.; Kai,
A. S.-F. Chem. Commun. 1998, 109.
2. (a) Zang, L.; Wang, P.; Zhang, H.; Zhuang, X.; Dai, Q.; Liu, W. Org. Lett. 2009, 11,
4294; (b) Zang, L.; Wu, J.; Dai, Q.; Liu, W.; Wang, P.; Lee, C.-S. Org. Lett. 2010, 12,
4014.
3. (a) Friedrich, U.; Blom, A. M.; Dahlback, B.; Villoutreix, B. O. J. Biol. Chem. 2001,
276, 24122; (b) Caldwell, E. E. O.; Nadkarni, V. D.; Fromm, J. R.; Linhardt, R. J.;
Weiler, J. M. Int. J. Biochem. Cell Biol. 1996, 28, 203.
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5. Wiskur, S. L.; Anslyn, E. V. J. Am. Chem. Soc. 2001, 123, 10109.
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(b) Wang, M.; Zhang, D.; Zhang, G.; Zhu, D. Chem. Commun. 2008, 4469; (c)
Wang, S.; Chang, Y.-T. Chem. Commun. 2008, 1173.
(EtOH:H2O = 1:3). (b) Changes in conjugate 1 at relative emission intensity (I472
)
upon addition of 10 ml aliquots of heparin.
pyrene monomer (Fig. 3). The addition of heparin (0–200 lM) led
to quenching of the monomer fluorescence and the appearance
of a new broad band at 472 nm, which is typical of excimer forma-
tion.2 To substantiate the role of kanamycin A, pyrene 3 failed to
show any marked difference in its fluorescence spectrum under
the same conditions. The apparent association constant (Ka) was
determined to be 1.7 ꢀ 10ꢁ4 M using a linear-regression analysis
(see graph displayed in Fig. 3b).
The selectivity of conjugate 1 for heparin in EtOH:H2O (1:3) was
apparent from an investigation of the emission response of other
important biomolecules such as adenosine triphosphate (ATP),
adenosine monophosphate (AMP) and amino acids including tri-
glycine, aspartic acid, ascorbic acid and glutamic acid and also
DNA, pimelic acid, dodecanedioic acid and octadodecanoic acid
(Fig. 4). Such selectivity in a semi-aqueous environment is promis-
ing for the use of kanapy-60 in practical applications.
8. Yi, K.; Liu, B. Adv. Funct. Mater. 2009, 19, 277.
9. Sun, W.; Bandmann, H.; Schrader, T. Chem. Eur. J. 2007, 13, 7701.
10. (a) Forge, A.; Schacht, J. Audio Neuro. Oto. 2000, 5, 3; (b) Magnet, S.; Blanchard, J.
S. Chem. Rev. 2005, 105, 477.
11. Aminoglycoside Antibiotics; Umezawa, H., Hooper, I. R., Eds.; Springer: New
York, Heidelberg, 1982. pp. 1–2.
12. Belmont, P.; Aissaoui, A.; Hauchecorne, M.; Oudrhiri, N.; Petit, L.; Vigneron, J.-
P.; Lehn, J.-M.; Lehn, P. J. Gene. Med. 2002, 4, 517.
In summary, we have reported a novel kanamycin A based fluo-
rescent neutral sensor for optical signalling of heparin in which the
pyrene unit shows optical for both UV/vis-absorption and fluores-
cence. The receptor 1 displayed changes in UV/vis absorption and
fluorescence emission intensities selectively for heparin in buffer
over various biologically active molecules. The sensor should have
potential in various sensing applications such as the study of hep-
arin transport or purification, where the availability of cheap and
easy-to-prepare heparin receptors would be advantageous. We
are currently tailoring these and other kanamycin A based recep-
tors to be specific for other binding events of choice.
13. Gouzy, M.-F.; Lauer, M.; Gonzaga, F.; Schulz, A.; Fuhrhop, J.-H. Langmuir 2002,
18, 10091.
14. Kopaczynska, M.; Lauer, M.; Schulz, A.; Wang, T.; Schaefer, A.; Fuhrhop, J.-H.
Langmuir 2004, 20, 9270.
15. Sainlos, M.; Belmont, P.; Vigneron, J.-P.; Lehn, P.; Lehn, J.-M. Eur. J. Org. Chem.
2003, 10, 2764.
16. Synthesis of kanapy-60 (1): Kanamycin A disulfate (2) (300 mg, 0.44 mmol) and
NaHCO3 (185 mg, 2.20 mmol) were dissolved in 15 ml of distilled H2O with
stirring. Pyrene butyric acid 3 (128 mg, 0.44 mmol) in 12.5 ml of THF was
added at room temperature, followed by addition of HBPyU (190 mg,
0.44 mmol) in small portions. The mixture was stirred for 6 h at room
temperature, the THF evaporated in vacuo, and the aqueous solution
lyophilized. Flash column chromatography on silica gel (CHCl3: MeOH: aq.
NH3) gave the kanapy-60 (1) adduct in 65% yield. 1H NMR (300 MHz; CD3OD;

S. V. Nalage et al. / Tetrahedron Letters 53 (2012) 2864–2867
2867
25 °C) d: 8.48 (m, 1H, aromatic-H of pyrene), 8.31 (m, 1H, aromatic-H of
pyrene), 7.82–8.16 (m, 1H, aromatic-H of pyrene), 7.60–7.66 (m, 1H, aromatic-
H of pyrene), 7.18–7.25 (m, 3H, aromatic-H of pyrene), 5.54 (anomeric proton,
1H), 5.04–5.30 (4H, from sugar rings), 3.60–3.95 (m, 14H, from sugar rings),
3.11–3.79 (m, 3H, from sugar rings), 2.08–2.36 (m, 8H, from sugar rings and
pyrene chain), 1.71–1.85 (m, 4H, from sugar rings); 13C NMR (75 MHz, DMSO-
d6) d: 27.7, 27.8, 27.9, 28.1, 32.8, 33.0, 35.3, 35.4, 35.8, 36.0, 46.4, 48.3, 50.6,
56.2, 61.2, 70.0, 72.2, 72.7, 73.0, 73.2, 73.3, 73.5, 88.0, 91.2, 112.5, 118.2, 122.4,
123.7, 123.8, 123.9, 124.0, 124.1, 124.6, 124.7, 126.5, 126.6, 126.9, 127.7, 128.6,
cmꢁ1 3271, 3037, 2875, 1641, 1546, 1409, 1350, 1145, 1045, 840; LC–MS (m/z)
calcd for C38H50N4O12 [M+] 754.8, found: 777 (M+Na+), 755 (M+H)+; HRMS (m/
z) calcd for
elemental composition: 60.47% C, 6.68% H, 7.42% N; measured elemental
composition: 60.41% C, 6.64% H, and 7.47% N.
C
38H51N4O12 754.8031, found: 755.8029 (M + H)+. Calculated
17. Preparation of stock solutions: All samples were freshly prepared from stock
solutions. The stock solution of kanapy-60 (1) (10
lM) was prepared in 10 mM
HEPES buffer solution (EtOH:H2O = 1:4) at pH 7.4 for spectral measurements.
The stock solution of heparin (200 mM) was prepared in 10 mM HEPES buffer
solution (EtOH:H2O = 1:3).
129.7, 130.8, 131.3, 131.4, 136.9, 137.2, 137.5, 172.4, 173.0; FT-IR (KBr): mmax
/