Biopolymers Peptide Science
Supporting Information
Design, synthesis, in vitro stability and cytostatic effect of multifunctional
anticancer drug-bioconjugates containing GnRH-III as a targeting moiety
Ulrike Leurs1, Gábor Mező2, Erika Orbán2, Peter Öhlschläger3, Andreas Marquardt4 and
Marilena Manea 1,5*
1
2
3
Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of
Chemistry, University of Konstanz, 78457 Konstanz, Germany
Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös Loránd
University, 1117 Budapest, Hungary
Laboratory of Immunology, Department of Biology, University of Konstanz, 78457 Konstanz,
Germany
4
Proteomics Facility, University of Konstanz, 78457 Konstanz, Germany
5
Zukunftskolleg, University of Konstanz, 78457 Konstanz, Germany
*Corresponding author:
Dr. Marilena Manea, University of Konstanz, Zukunftskolleg and Department of Chemistry,
Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Universitätsstrasse 10,
78457 Konstanz, Germany; Phone: (+49)-7531-88-2285; Fax: (+49)-7531-88-3097
E-mail: marilena.manea@uni-konstanz.de
Table of Contents
RP-HPLC profile and ESI-ion trap mass spectrum of GnRH-III[4,8Lys(Dau=Aoa)] .................................. S3
RP-HPLC profile and ESI-ion trap mass spectrum of GnRH-III[8Lys(Dau=Aoa- Lys(Dau=Aoa))] ......... S3
RP-HPLC profile and ESI-ion trap mass spectrum of GnRH-III[8Lys(Dau=Aoa)] .................................... S4
Stability of GnRH-III[4,8Lys(Dau=Aoa)] in human serum. ......................................................................... S5
Stability of GnRH-III[8Lys(Dau=Aoa- Lys(Dau=Aoa))] in human serum. ................................................ S6
Degradation of daunorubicin-GnRH-III derivative bioconjugates in the presence of rat liver lysosomal
homogenate. ................................................................................................................................................ S7
S2
30.3
3+
822.7
*
3+
Intens.
x 107
779.7
3
2+
1233.5
2
1
MW (calc.) 2465.57
MW (exp.) 2465.07
400
600
800
1000
1200
1400 1600
m/z
time [min]
Figure 1 RP-HPLC profile and ESI-ion trap mass spectrum of GnRH-III[4,8Lys(Dau=Aoa)]. The
asterisk indicates the loss of daunosamine.
29.00
Intens.
x 107
3+
852.0
2
2+
1277.2
1
4+
639.8
500
MW (calc.) 2553.66
MW (exp.) 2553.53
1000
1500
2000
m/z
time [min]
Figure 2 RP-HPLC
profile
and ESI-ion
trap
mass
spectrum
of
GnRH-III[8Lys(Dau=Aoa-
Lys(Dau=Aoa))].
S3
100
90
26.5
6
80
mAU [280 nm]
3+
614.7
Intens.
x106
2+
921.5
70
60
4
50
40
30
2
*
20
3+
565.7
10
*
2+
856.6
MW(calc): 1840.75
MW(exp): 1841.05
0
0
10
15
20
25
30
35
40
45
50
55
200
400
600
800
1000
1200
1400
1600
1800 m/z
Time(min)
Figure 3
RP-HPLC profile and ESI-ion trap mass spectrum of GnRH-III[8Lys(Dau=Aoa)]. The asterisk
indicates the loss of daunosamine.
S4
A
B
□
764.8
Intens.
x104
□
Intens.
x104
764.7
2.0
*
4
3+
779.9
3
1.5
3+
823.0
□
1.0
2
□
887.0
1
500
600
700
800
900
**
2+
*
1104.5 2+
1169.0
1000
1100
886.9
561.6
1200
786.7
0.5
473.7
1300 m/z
625.6
822.6
0.0
300
400
500
600
700
800
900
1000 1100 1200 m/z
Figure 4 Stability of GnRH-III[4,8Lys(Dau=Aoa)] in human serum.
(A) ESI-ion trap mass spectrum was recorded after 24 hours of incubation of the bioconjugate
in human serum at 37°C. Mass spectrum was averaged over the chromatographic window
where the compounds eluted. The loss of daunosamine is indicated by an asterisk (-129) or
double asterisk (-147). The squares indicate ions originating from human serum.
GnRH-III[4,8Lys(Dau=Aoa)]
MWcalc=2465.57
MWexp=2465.40
(B) ESI-ion trap mass spectrum of human serum recorded under the conditions described
above.
S5
A
Intens.
x104
B
□
Intens.
600
4+
639.4
735.5
500
2.0
3+
851.8
1.5
400
689.4
300
□
1.0
588.7
200
□
735.6
0.5
1032.9
843.0
□
100
1162.0 1238.6
1032.9
0
500
600
700
800
900
1000
1100
m/z
500
600
700
800
900
1000 1100 1200 1300 1400 m/z
Figure 5 Stability of GnRH-III[8Lys(Dau=Aoa- Lys(Dau=Aoa))] in human serum.
(A) ESI-ion trap mass spectrum was recorded after 24 hours of incubation of the bioconjugate
in human serum at 37°C. Mass spectrum was averaged over the chromatographic window
where the compounds eluted. The squares indicate ions originating from human serum.
GnRH-III[8Lys(Dau=Aoa-Lys(Dau=Aoa))]
MWcalc=2553.66
MWexp=2553.00
(B) ESI-ion trap mass spectrum of human serum recorded under the conditions described
above.
S6
A
B
*
**
1+
584.7
Intens.
x 104
Intens.
x 104
3+
771.8
0.6
*
1+
729.2
0.4
1.0
2+
1016.5
*
1.5
1+
600.4
*
2+
2+
878.5 952.0
2+
732.8
3+
851.9
*
**
3+
803.0
2+
659.1
*
0.2
*
*
1+
1034.5
2+
644.1
500
Figure 5
700
900
2+
704.9
0.5
**
**
1+
**
1+
1+
1334.7
1181.5
1278.6
1+
1163.6
1100
1300
m/z
400
600
800
1000
1200
1400
1600 m/z
Degradation of daunorubicin-GnRH-III derivative bioconjugates in the presence of rat liver
lysosomal homogenate.
ESI-ion trap mass spectra were recorded after 24 hours of incubation of the bioconjugates in
the presence of rat liver lysosomal homogenate (1:1 ratio, w/w) at 37°C. Mass spectra were
averaged over the chromatographic window where the compounds eluted. The asterisk
indicates the loss of daunosamine; the double asterisk indicates the loss of two daunosamine
moieties.
(A)
GnRH-III[4,8Lys(Dau=Aoa)]
(B)
GnRH-III[8Lys(Dau=Aoa-Lys(Dau=Aoa))]
S7