New targeting system for antimycotic drugs: β-Glucosidase sensitive Amphotericin B-star poly(ethylene glycol) conjugate

Article abstract of DOI:10.1016/j.bmcl.2008.03.065

A new targeting potentially intravenous conjugate Amphotericin B (AMB)-star poly(ethylene glycol) (sPEG) (M = 25,160) has been synthesized and characterized. It contains a β-d-glucopyranoside molecular switch which is sensitive to β-glucosidases (E.C.3.2.1.21). The β-glucosidase-catalyzed release of AMB from the polymeric carrier was proved in vitro by means of spectrophotometry and HPLC.

Full text of DOI:10.1016/j.bmcl.2008.03.065

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2952–2956
New targeting system for antimycotic drugs: b-Glucosidase
sensitive Amphotericin B–star poly(ethylene glycol) conjugate
a,
a
a
a
b
*
ˇ
´
´
´
˚
´
Milosˇ Sedlak, Pavel Drabina, Elisˇka Bılkova, Petr Simunek and Vladimır Buchta
a
ˇ
Department of Organic Chemistry, Faculty of Chemical Technology, University of Pardubice, Cs. legiı´ 565,
532 10 Pardubice, Czech Republic
bDepartment of Clinical Microbiology, Medicinal Faculty of Charles University, Sokolska´ 581, 500 05 Hradec Kra´love´, Czech Republic
Received 18 February 2008; revised 20 March 2008; accepted 22 March 2008
Available online 27 March 2008
Abstract—A new targeting potentially intravenous conjugate Amphotericin B (AMB)–star poly(ethylene glycol) (sPEG)
(M = 25,160) has been synthesized and characterized. It contains a b-D-glucopyranoside molecular switch which is sensitive to b-
glucosidases (E.C.3.2.1.21). The b-glucosidase-catalyzed release of AMB from the polymeric carrier was proved in vitro by means
of spectrophotometry and HPLC.
ꢀ 2008 Elsevier Ltd. All rights reserved.
Amphotericin B (AMB) (Fig. 1) is a polyene macrocyclic
membrane-active antifungal antibiotic drug used in
treatment of system fungal infections.1 It is a potent sal-
vaging medical drug that is applied in the cases of immu-
nosuppressive patients after transplantations of organs,
those with acquired immune deficiency syndrome
(AIDS) or with tumors and hematological malignities.
However, the clinical application of AMB is restricted
by both its poor solubility in water and some side effects,
particularly nephrotoxicity.1 The pharmaceutical dosage
forms used in practice are non-covalent complexes of the
type Amphotericin B lipid complex (ABLC), which are
colloidal systems composed of biodegradable phospho-
lipid matrixes.2 These types of formulations represent
a solution to the solubility problems of AMB, but the re-
lease of AMB takes place already in the patient’s blood
circulation system, which results in a general impact on
his/her organism inclusive of the undesirable side effects.
lowered pH value due to pathological processes.4 The
rate of release of AMB (acid-catalyzed hydrolysis of
imine linkage) is controlled by the pH value of the milieu
and by substitution of benzene ring attached to the poly-
meric carrier.3c,d
The aim of this work was to prepare and characterize a
conjugate that would release AMB selectively—only in
the area of the attacked organ. The proposed structure
of conjugate (AMB4–sPEG) (Fig. 1) was inspired by
the finding that many parasitic fungal pathogens, such
as species of Aspergillus, Candida or Trichosporon fami-
lies possess specific hydrolases b-glucosidases
(E.C.3.2.1.21) in their enzymatic outfit.5
In the case of human organism, b-glucosidases only oc-
cur in some bacteria present in intestinal micro-flora.6
This finding was previously used for achieving con-
trolled release of some drugs from per oral intestinal
pro-drugs.6 However, there exists no natural occurrence
of b-glucosidases in healthy human tissues.7 The basic
linker in AMB4–sPEG is the b-glucosidic bond of a
substituted phenyl-b-D-glucopyranoside molecule. With
regard to its biocompatibility and binding capacity, we
chose star poly(ethylene glycol) [pentaerythritol
poly(ethylene glycol)ether, M = 20,000] (sPEG) as the
polymeric carrier.8 The AMB4–sPEG conjugate involves
two types of carbamate bond: the connecting units is
linked to polymer by stable carbamate bond, while
AMB is linked to the phenyl-b-D-glucopyranoside frag-
ment by unstable carbamate bond. The enzymatic
The efforts to increase the therapeutic index of AMB led
to development of conjugates with polymeric carriers:
polysaccharides,3a
poly(ethylene
glycol)3b,c
and
poly(ethylene glycol)-b-poly(L-lysine).3d The last men-
tioned types of conjugates3c,d were suggested with the
aim to achieve the release of AMB from polymeric car-
rier in a targeted way, that is, in the areas having locally
Keywords: Drug targeting; Amphotericin B; Controlled release in vitro.
*
Corresponding author. Tel.: +420 466 037 015; fax: +420 466 037
068; e-mail: milos.sedlak@upce.cz
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.065

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M. Sedlak et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2952–2956
2953
Z
Z
O
O
Z
n
O-CO-NH-AMB
O
O
O
O
n
n
OH
O
O
HO
OH
O
O
Z
HO
HN
Z =
O
n
OH
AMB4— sPEG :
sPEG:
Star poly(ethylene glycol): (
)
Z = H:
OH
H3C
HO
O
OH
CO2H
O
OH OH
OH OH
O
O
CH3
H
H3C
CH3
OH
O
Amphotericin B (AMB)
NH2
OH
Figure 1. Structure of b-glucosidase sensitive Amphotericin B–star poly(ethylene glycol) conjugate (AMB4–sPEG).
hydrolysis of b-glucosidic bond should produce glucose,
and the subsequent 1,6-elimination should give the car-
bamic acid of Amphotericin B, which is quickly decom-
posed to release free Amphotericin B. The remaining
molecule of polymeric carrier with covalently bound
fragment of substituted 4-hydroxybenzylalcohol is then
excreted from the organism (Scheme 1).
The b-configuration of glucopyranosides 1 and 2 was
confirmed by 1H–13C HMQC NMR spectra.13 The ano-
meric carbon atom in derivative 1 exhibits a shift at
98.4 ppm, which corresponds with the doublet of ano-
meric hydrogen atom at 5.68 ppm with the coupling
constant J = 7.9 Hz. Derivative 2 exhibits the anomeric
carbon atom at 99.1 ppm, which corresponds with the
doublet of anomeric hydrogen atom at 5.33 ppm
(J = 7.9 Hz). The values of coupling constant of ano-
meric hydrogen atom coupled with the neighboring vic-
inal hydrogen atom indicate that the two atoms are in
axial positions, that is, the glucose fragment of both
derivatives 1 and 2 assumes the b-configuration [b-
4C1(D)].
Similar principle of activation for low-molecular pro-
drugs was described earlier in the case of hydrolysis of
b-D-glucuronides, where b-D-glucuronic acid is split off
by action of b-D-glucuronidases (E.C.3.2.1.31). This
principle is being used in the so-called Antibody Direc-
ted Enzyme Prodrug Therapy (ADEPT).9
The key part of the AMB4–sPEG molecule is the en-
zyme-sensitive linker derived from substituted b-D-
glucopyranoside, which was prepared by the following
reaction sequence.10–12 First, the Koenigs–Knorr reac-
tion10 of 2,3,4,6-tetra-O-acetyl-a-d-glucopyranosyl bro-
mide with 4-hydroxymethyl-2-nitrophenol9c was used
to prepare 1-(4-hydroxymethyl-2-nitrophenyl)-(2,3,4,6-
tetra-O-acetyl)-b-D-glucopyranoside (1).11 The hydroge-
nation of glucopyranoside 1 on Adams catalyst gave 1-
(2-amino-4-hydroxymethylphenyl)-(2,3,4,6-tetra-O-acet-
yl)-b-D-glucopyranoside (2)12 (Scheme 2).
The next reaction step involves reaction of pentaerythri-
tol tetra(4-nitrophenyl carbonyl) poly(ethylene glycol)
(sPEG–O–CO–O–C6H4–NO2) with glucopyranoside 2
to form a carbamate bond.14 The hydroxymethyl group
of polymer 3 was then activated with 4-nitrophenyl chlo-
roformate and resulting intermediate reacted with
Amphotericin B. The polymer 4 obtained in this way
contains four AMB molecules linked by labile carba-
mate bond.15 Due to the instability of AMB it was
impossible to deprotect the hydroxyl groups of glucose
by means of carboxyesterase (pH 5; 40–50 ꢁC).16 There-
O-CO-NH-AMB
AMB-HN-OC
O
OH
β−glucosidase
OH
O
OH
O
O
HO
HO
HN
HO
HO
HO
H2O
OH
HN
O
OH
O
O
sPEG
O
sPEG
1,6- elimination
AMB4—sPEG
OH
CH2
— CO2
H2O
AMB-NH2
AMB-NH-CO-OH
O
HO
HN
O
HN
O
O
sPEG
O
sPEG
Scheme 1. Principle of release of Amphotericin B form AMB4–sPEG.

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M. Sedlak et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2952–2956
2954
OH
OAc
O
OAc
O
O
i
AcO
AcO
AcO
AcO
NO2
OAc
OAcBr
1
ii
OH
OH
OAc
O
OAc
O
iii
O
O
AcO
AcO
AcO
AcO
HN
O
NH2
OAc
OAc
O
sPEG
3
2
iv
O-CO-NH-AMB
O-CO-NH-AMB
v
OH
OAc
O
O
O
O
HO
HO
AcO
AcO
HN
O
OH
AMB4—sPEG
HN
O
OAc
O
sPEG
4
sPEG
O
Scheme 2. Synthesis of AMB4–sPEG. Reagents and conditions: (i) 2-nitro-4-hydroxymethylphenol/Ag2O/acetonitrile/ultrasound 5 h/25 ꢁC/77%; (ii)
PtO2/H2/ethyl acetate/3 h/25 ꢁC/97%; (iii) sPEG–O–CO–O–C6H4–NO2/DMAP/DMF/48 h/65 ꢁC/84%; (iv) a—4-nitrophenyl chloroformate/toluene/
72 h/25 ꢁC; b—AMB/DMAP/DMF/144 h/25 ꢁC/66%; (v) a—KCN/methanol/5 h/25 ꢁC, b—Amberlite 120-H/methanol/10 min/25 ꢁC/83%.
fore, the deprotection of acetylated hydroxyl groups of
polymer 4 was carried out by catalytic amount of potas-
sium cyanide. Potassium cyanide was removed using of
an ion exchanger after the reaction17 (Scheme 2). The
course of deprotection was monitored by 1H NMR spec-
troscopy. The multiplet of CH3– in acetyl groups at
2.02 ppm gradually disappeared. The final conjugate
AMB4–sPEG was characterized18 by microanalysis
and GPC19 was adopted for determination of Mw. The
HPLC19 was used for estimation of conjugate purity.
The content of free AMB was below 1 mol %. Further-
more, by means of UV–vis spectroscopy (typical max-
ima corresponding to polyene system of AMB18) it
was proved that the content of AMB in the conjugate
AMB4–sPEG corresponds to the molar ratio of 1:4
(sPEG/AMB). The prepared conjugate is very well solu-
ble in water and forms finely opalescent solutions.
Figure 2. Record of time changes of spectrum for enzymatic hydrolysis
of AMB4–sPEG conjugate (3 · 10ꢀ6 M) catalysed by b-glucosidase
The next part of our work was focused on preliminary
in vitro testing of the conjugate synthesized in various
media. First, the stability of conjugate was studied in
phosphate buffer.19 The monitoring of UV–vis spectra
(2 mg/1 mL; 66.6 U/g) in phosphate buffer (pH 7.4, 2 · 10ꢀ2 M) at
37 ꢁC. The inset represents time dependence of absorbance at 409 nm
[s1/2 = (103 4) s].
showed no observable spectral changes in the phosphate
buffer of pH 7.4 during a period of 18–20 h. For a model
medium imitating the tissue attacked by fungal patho-
gen we chose b-glucosidase (E.C.3.2.1.21) (Aspergillus
niger) in phosphate buffer with pH 7.4. Spectral record-
ing of the conjugate showed an increase in the absor-
bance at the wavelengths of 367, 386 and 409 nm
(Fig. 2). Formation of substituted 1,4-quinonemethide
(Scheme 1) should be visible in the wavelength range
of 280–320 nm20; however, this region is overlapped by
the absorption band of b-glucosidase (245–330 nm).
The spectral records clearly show an increase in the
absorption band at 409 nm, which is specific21 for
monomeric AMB. Such increase with time corresponds
with the rate of release of AMB from the carrier. The
rate of release of AMB under the experimental condi-
tions adopted19 obeys the kinetic equation of the pseu-
do-first order, and the half-life of release of AMB is
s
1/2 = (103 4) s. The release of AMB from the conju-
gate was also confirmed by means of HPLC.21 The re-
sults described indicate that the behavior of the
AMB4–sPEG conjugate in the presence of buffered solu-
tion of b-glucosidase corresponds to Scheme 1.

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M. Sedlak et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2952–2956
2955
Weiss, S. I.; Sieverling, N.; Niclasen, M.; Maucksch, C.;
Thunemann, A. F.; Mo¨hwald, H.; Reinhardt, D.; Rose-
¨
In contrast to the pharmaceutical dosage forms of AMB
used for intravenous application nowadays, we have de-
signed a fundamentally new targeting AMB conjugate
that selectively releases AMB by enzymatic action of
b-glucosidase. The results of preliminary study in vitro
allow the presumption that targeted release of AMB will
take place in the area of expected activity of fungal path-
ogen. This new system which we have suggested can be
used more generally, that is, also for other antifungal
medicaments. However, its practical applicability will
need further tests on animal models and their critical
evaluation.
necker, J.; Rudolph, C. Biomaterials 2006, 27, 2302.
10. (a) Koenigs, W.; Knorr, E. Chem. Ber. 1901, 34, 957; (b)
Flowers, H. M. Methods Carbohydr. Chem. 1972, 6, 474.
11. 1-(4-Hydroxymethyl-2-nitrophenyl)-(2,3,4,6-tetra-O-acet-
yl)-b-D-glucopyranoside (1): A mixture of 2,3,4,6-tetra-O-
acetyl-a-d-glucopyranosyl bromide (3.08 g; 7.5 mmol), 4-
hydroxymethyl-2-nitrophenol9c (1.27 g; 7.5 mmol), Ag2O
(7.3 g; 31.5 mmol) and dry acetonitrile (65 mL) was stirred
by means of ultrasound at the temperature of 25 ꢁC for a
period of 5 h. The crude product was purified chromato-
graphically (silica gel/ethyl acetate). Yield: 2.7 g (77%), mp
198–200 ꢁC, TLC: (Silicagel plates Merck), ethyl acetate,
20
RF = 0.55; ½aꢁD +19.4ꢁ (0.5 g/100 mL, CH3OH). 1H NMR
(500.13 MHz, DMSO-d6): 2.03 (s, 3H), 2.06 (s, 3H), 2.07
(s, 3H), 2.09 (s, 3H), 4.17–4.20 (m, 1H), 4.26–4.30 (m, 1H),
4.32–4.35 (m, 1H), 4.57 (d, 2H, J = 5.72 Hz), 5.09 (t, 1H,
J = 9.7 Hz), 5.12–5.16 (m, 1H), 5.44–5.49 (m, 2H), 5.68 (d,
1H, J = 7.9 Hz), 7.44 (d, 1H, J = 8.7 Hz), 7.68 (dd, 1H,
J = 1.9 Hz, 8.7 Hz), 7.85 (d, 1H, J = 1.9 Hz). 13C NMR,
125.76 MHz, DMSO-d6: 20.31, 20.37, 20.48, 20.58, 61.46,
61.68, 67.98, 70.25, 71.24, 71.81, 98.44, 117.94, 122.37,
132.01, 138.54, 140.35, 147.24, 168.94, 169.43, 169.72,
170.13. Calcd for C21H25NO13 (499) (%): C, 50.50; H,
5.05; N, 2.80. Found: C, 50.50; H, 4.97; N, 2.98.
Acknowledgments
The authors acknowledge the financial support from the
Science Foundation of the Czech Republic, Grant No.
203/06/0583 and Ministry of Education, Youth and
Sports Project No. MSM 002 162 7501.
Supplementary data
12. 1-(2-Amino-4-hydroxymethylphenyl)-(2,3,4,6-tetra-O-acet-
yl)-b-D-glucopyranoside (2): A solution of glucopyrano-
side 1 (1 g; 2 mmol) in ethyl acetate (150 mL) with an
addition of PtO2 (0.1 g; 0.4 mmol) was hydrogenated
under a mild overpressure of hydrogen (ca 5 kPa) at the
temperature of 25 ꢁC for 3 h. Yield: 910 mg (97%), mp
135–138 ꢁC, TLC: (Silicagel plates Merck), ethyl acetate,
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2008.03.065.
References and notes
20
RF = 0.32; ½aꢁD ꢀ30.4ꢁ (0.5 g/100 mL, CH3OH). 1H NMR
(500.13 MHz, DMSO-d6): 2.03 (s, 3H), 2.06 (s, 3H), 2.08
(s, 3H), 2.09 (s, 3H), 4.10–4.14 (m, 1H), 4.23–4.29 (m, 2H),
4.36 (d, 2H, J = 5.5 Hz), 4.61 (br s, 2H), 5.02–5.06 (m,
2H), 5.08–5.12 (m, 1H), 5.34 (d, 1H, J = 7.9 Hz), 5.5 (t,
1H, J = 9.52 Hz), 6.51 (dd, 1H, J = 1.60 Hz, 8.2 Hz), 6.71
(d, 1H, J = 1.6 Hz), 6.87 (d, 1H, J = 8.2 Hz). 13C NMR
(125.76 MHz, DMSO-d6): 20.43, 20.51, 20.61, 20.66,
61.79, 62.91, 68.28, 70.79, 71.17, 71.79, 99.11, 113.33,
114.51, 115.87, 138.05, 138.19, 142.49, 169.46, 169.70,
170.11. Calcd for C21H27NO11 (469) (%): C, 53.73; H,
5.80; N, 2.98. Found: C, 53.70; H, 5.72; N, 2.88.
1. (a) Trejo, W.; Bennett, R. J. Bacteriol. 1962, 85, 436; (b)
Diamond, R. D. Rev. Infect. Dis. 1991, 13, 480; (c)
Ceregheti, D. M.; Carreira, E. M. Synthesis 2006, 914.
2. (a) Janoff, A. S.; Perkins, W. R.; Saleton, S. L.; Swenson,
C. E. J. Liposome Res. 1993, 3, 451; (b) Ruijgrok, E. J.;
Meis, J. F. G. M. Expert Opin. Emerg. Drugs 2002, 7, 33.
3. (a) Ehrenfreund-Kleinman, T.; Golenser, J.; Domb, A. J.
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Biomaterials 2004, 25, 3049; (b) Sedlak, M.; Buchta, V.;
Kubicova, L.; Simunek, P.; Holcapek, M.; Kasparova, P.
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Bioorg. Med. Chem. Lett. 2001, 11, 2833; (c) Sedlak, M.;
´
Pravda, M.; Staud, F.; Kubicova, L.; Tycova, K.;
Ventura, K. Bioorg. Med. Chem. 2007, 15, 4069; (d)
´ˇ
´
13. Duus, J. Ø.; Gotfredsen, C. H.; Bock, K. Chem. Rev. 2000,
100, 4589.
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´
ˇ´
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Sedlak, M.; Pravda, M.; Kubicova, L.; Mikulcıkova, P.;
Ventura, K. Bioorg. Med. Chem. Lett. 2007, 17, 2554.
4. (a) Mellman, I.; Fuchs, R.; Helenius, A. Ann. Rev.
14. Polymer 3: A mixture of pentaerythritol tetra(4-nitro-
phenyl carbonyl) poly(ethylene glycol) M = 20,348 (NOF
Corp.) (1 g; 0.05 mmol), glucopyranoside 2 (1 g; 2 mmol)
and 4-N,N-dimethylaminopyridine (30 mg; 0.25 mmol) in
anhydrous DMF (10 mL) was heated at the temperature
of 65 ꢁC for 100 h. The crude polymer was twice recrys-
ˇ
Biochem. 1986, 55, 773; (b) Ulbrich, K.; Subr, V. Adv.
Drug Delivery Rev. 2004, 56, 1023; (c) Pechar, M.;
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Braunova, A.; Ulbrich, K.; Jelınkova, M.; Rıhova, B.
J. Bioact. Compat. Polym. 2007, 20, 319.
´
´
´
´
1
tallized from propan-2-ol. Yield 920 mg (84%). H NMR
5. Li, Y.-K.; Chir, J.; Chen, F.-Y. Biochem. J. 2001, 355, 835.
6. Hiyarama, F.; Uekama, K.. In Prodrugs: Challenges and
Rewards; Stella, V. J., Borchardt, R. T., Hageman, M. J.,
Oliyai, R., Maag, H., Tilley, J. W., Eds.; Springer: New
York, 2007; Part 1, pp 687–691.
7. Hopfer, U. In Textbook of Biochemistry with Clinical
Correlations.; Devlin, T. M., Ed.; Wiley-Liss: New York,
1992; pp 1079–1080.
8. (a) Greenwald, R. B.; Zhao, H.; Peng, P.; Longley, C. B.;
Dai, Q.-H.; Xia, J.; Martinez, A. Eur. J. Med. Chem. 2005,
40, 798; (b) Fichter, K. M.; Zhang, L.; Kiick, K. L.;
Reineke, T. M. Bioconjugate Chem. 2008, 19, 76.
9. (a) Jung, M. Mini Rev. Chem. 2001, 1, 399; (b) Bouvier, E.;
Thirot, S.; Schmidt, F.; Monneret, C. Org. Biomol. Chem.
2003, 1, 3343; (c) Bouvier, E.; Thirot, S.; Schmidt, F.;
Monneret, C. Bioorg. Med. Chem. 2004, 12, 969; (d)
(500.13 MHz, CDCl3) 2.02 (m, 12H; (CH3)4), 3.42–3.72
(m, 392H; (CH2–CH2–O)98), 3.69 (m, 2H; CH2), 3.75 (m,
2H; CH2), 4.21 (m, 2H; CH2), 4.28 (m, 2H; CH2), 5.01 (m,
2H; CH2), 5.13 (m, 1H; CH), 5.27 (m, 1H; CH), 6.73 (m,
1H; CH) 6.88 (m, 2H; CH2), 7.04 (m, 1H; CH), 7.45 (s,
1H, CONH). Calcd for C997H1920N4O504 (22,030) (%): C,
54.35; H, 8.79; N, 0.25. Found: C, 54.72; H, 8.92; N, 0.36;
Mw/Mn = 1.18.
15. Polymer 4: An anhydrous solution of polymer 3 (1.1 g;
0.05 mmol) and 4-nitrophenyl chloroformate (500 mg;
2.5 mmol) in toluene (40 mL) was kept at the constant
temperature of 50 ꢁC for a period of 48 h, whereupon the
toluene was distilled off, and the residue was mixed with
diethyl ether (250 mL), filtered and washed with diethyl
ether (10 · 50 mL). After drying in desiccator, this inter-

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M. Sedlak et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2952–2956
2956
mediate (ca 1 g) was dissolved in dimethylformamide measured with the CH interaction of 145 Hz.
(8 mL) with addition of Amphotericin
0.20 mmol), 4-N,N-dimethylaminopyridine
B
(185 mg,
(12 mg;
(b) Gel permeation chromatography (GPC) was used for
the estimation of Mw of the polymers. The gel permeation
chromatography measurements of the prepared polymers
3, 4 and conjugate AMB4–sPEG were performed with
HEMA–BIO columns (hydrophilic modification HEMA–
Gel, particle size 10 lL, porosity 40/100/300/1000) at room
temperature using an RI detector and UV–vis detector.
Redistilled water (pH 7.1) was used as the eluent. The
columns were calibrated with a series of standard PEGs
with varying molecular weights (PSS, Polymer Standard
Service GmbH, Mainz, Germany).
0.1 mmol) and trioctylamine (350 mg; 1 mmol). The mix-
ture was stirred in the dark in argon atmosphere at room
temperature for several days. The end of reaction was
estimated by means of GPC monitoring. The crude
polymer was purified by reprecipitation from diethyl
ether. Yield 850 mg (66%); UV–vis: kmax (nm) 346, 367,
386, 409. Calcd for C1198H2204N8O576 (25,830) (%): C,
55.29; H, 8.60; N, 0.43. Found: C, 55.56; H, 8.89; N, 0.59;
Mw/Mn = 1.22.
16. Baba, A.; Yoshioka, T. J. Org. Chem. 2007, 72, 9541.
17. Herzig, J.; Nudelman, A.; Gottlieb, H. E.; Fischer, B.
J. Org. Chem. 1986, 51, 727.
(c) The purity and stability of conjugate AMB4–sPEG was
estimated by HPLC using LiChroCARTꢂ 125 · 4 mm
column packed with LiChrospherꢂ 100 RP–18 e 5 lm
(Merck) and eluted with mobile phase of acetonitrile with
20 mM chelaton II.
18. AMB4–sPEG conjugate: A mixture of polymer 4 (260 mg;
0.01 mmol) and KCN (6.5 mg; 0.1 mmol) in methanol
(10 mL) was stirred at the temperature of 25 ꢁC for a
period of 5 h, whereupon Amberlite IR–120H ion resin
(1 g) was added. After 10 min, the ion exchanger was
removed by decantation, and methanol was distilled off
under reduced pressure at the temperature of 35 ꢁC. Yield
230 mg (83%); UV–vis: kmax (nm) 346, 367, 386, 409.
Calcd for C1198H2204N8O576 (25,160) (%): C, 55.23; H,
8.70; N, 0.45. Found: C, 55.61; H, 9.08; N, 0.52; Mw/
Mn = 1.23.
(d) The stability of AMB4–sPEG conjugate was monitored
spectrophotometrically using an HP UV–vis 8453 Diode
Array apparatus and 1-cm closable quartz cell in the cell
holder kept at the constant temperature of 37 ꢁC. The cell
was charged with 2 mL phosphate buffer added from a
pipete (pH 7.4; 2 · 10ꢀ2 M), and after reaching the said
temperature, 15 lL methanolic solution of AMB4–sPEG
conjugate was injected, so that the final concentration of
the substrate was about 3 · 10ꢀ6 M. The enzymatic
hydrolysis of AMB4–sPEG was monitored by the same
methodology; however, the used solution was b-glucosi-
dase (2 mg/mL) (E.C.3.2.1.21; Fluka; 66.6 U/g; A. niger) in
phosphate buffer (pH 7.4, 2 · 10ꢀ2 M). The measured
absorbance-time (As) dependence was used to calculate the
half-life of enzymatic hydrolysis (s1/2 = ln2/kobs) and the
observed rate constant (kobs (sꢀ1)):kobss = lnDA + const.,
where DA = (A0 ꢀ As).
19. General methods/equipment: (a) The 1H NMR spectra were
calibrated with respect to the middle signal in the multiplet
of solvent (d = 2.55; DMSO-d6) or (d = 7.24; CDCl3). The
1H NMR spectra of polymers were measured with
relaxation delay of 6 s and an acquisition time of 4 s:
Tocco, G.; Begala, M.; Delogu, G.; Meli, G.; Picciau, C.;
Podda, G. Synlett 2005, 1296. The 13C NMR spectra were
measured in standard way using broad-band proton
decoupling and/or pulse sequence APT. The 13C NMR
spectra were calibrated with respect to the middle signal in
the multiplet of solvent (d = 39.6; DMSO-d6) or (d = 77.0;
CDCl3).The pulse sequence gs 1H–13C HMQC was
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20. Pospısek, J.; Pısova, M.; Soucek, M. Collect. Czech. Chem.
Commun. 1975, 40, 142.
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21. Gaboriau, F.; Cheron, M.; Leroy, L.; Bolard, J. J.
Biophys. Chem. 1997, 66, 1.