An amino acid-based heterofunctional cross-linking reagent

Article abstract of DOI:10.1007/s00726-014-1685-3

We describe the synthesis and characterization of a new lysine-based heterofunctional cross-linking reagent. It carries two readily available aminooxy functionalities and an activated and protected thiol group that is capable of generating reducible disulfides, the former enable bioorthogonal modification of ketones and aldehydes by the formation of an oxime bond. The efficacy of the linker was proven by coupling two doxorubicin molecules to the functionalized amino acid core and the subsequent bioconjugation of this drug conjugate with a thiolated antibody. Graphical Abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00726-014-1685-3

Amino Acids
DOI 10.1007/s00726-014-1685-3
ORIGINAL ARTICLE
An amino acid-based heterofunctional cross-linking reagent
Marco Lelle Kalina Peneva
•
Received: 15 October 2013 / Accepted: 24 January 2014
Ó Springer-Verlag Wien 2014
Abstract We describe the synthesis and characterization
of a new lysine-based heterofunctional cross-linking
reagent. It carries two readily available aminooxy func-
tionalities and an activated and protected thiol group that is
capable of generating reducible disulfides, the former
enable bioorthogonal modification of ketones and alde-
hydes by the formation of an oxime bond. The efficacy of
the linker was proven by coupling two doxorubicin mole-
cules to the functionalized amino acid core and the sub-
sequent bioconjugation of this drug conjugate with a
thiolated antibody.
enzyme (Tong et al. 2013) or antibody-nanoparticle
(Ackerson et al. 2006) conjugates. Their widespread
application in scientific studies not only opened the
opportunity to explore fluorescently labeled enzymes as
diagnostic tools in clinical testing, but also led to numerous
advantages in targeted drug delivery systems (Gudmand
et al. 2010; Zhao et al. 2012).
The conjugation between two or more (bio)molecules is
typically achieved by the incorporation of mutually reac-
tive groups into the individual components and the sub-
sequent coupling in solution thereby leading to the
formation of amide, oxime, hydrazone, thioether or disul-
fide bonds (Clave et al. 2008; Singh et al. 2006). Two
important requirements must be fulfilled for bioconjuga-
tion: (1) the coupling reactions have to be performed in
aqueous media at ambient temperature, due to the intrinsic
sensitivity of many biological systems against organic
solvents and elevated temperatures; (2) the conjugation
needs to be highly selective for equimolar proportions of
the reactants (Aslam and Dent 1998; Mark 2011).
Keywords Doxorubicin ꢀ Cross-linker ꢀ Antibody drug
conjugate ꢀ Bioconjugation ꢀ Anthracycline ꢀ
Chemoselective
Introduction
Bioconjugation has evolved as a key technique in life
sciences for the linkage of two or more molecules, from
either natural or synthetic origin, to create novel hybrid
materials having the combined properties of its individual
components (Hermanson 2008; Hackenberger and Sch-
warzer 2008). These substances encompass diverse com-
plex structures such as peptide-oligonucleotide (Astriab-
Fisher et al. 2002), protein-drug (Kratz 2002), polymer-
In this regard, cross-linkers have played an undisputed
role for the linkage of various biomolecules and have
attracted continuous interest in biochemistry, chemical
biology and organic chemistry. Heterofunctional cross-
linking reagents that contain a carbonyl-reactive and a
sulfhydryl-reactive functionality are relatively new and hold
potential to conjugate carbohydrate-containing molecules to
thiol-containing proteins or macromolecules. Depending on
the reactive group used either stable thioether or cleavable
disulfide bonds can be created (Smith et al. 2010; Chalker
et al. 2009). Cleavable disulfide bonds can be easily
achieved by employing cross-linkers that carry thiol-disul-
fide exchange functional groups like 2-pyridyl disulfides.
The reaction of thiols with 2-pyridyl disulfides is well suited
for bioconjugation as it undergoes an irreversible disulfide
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-014-1685-3) contains supplementary
material, which is available to authorized users.
M. Lelle ꢀ K. Peneva (&)
Max Planck Institute for Polymer Research,
Ackermannweg 10, 55128 Mainz, Germany
e-mail: peneva@mpip-mainz.mpg.de
123

M. Lelle, K. Peneva
O
O
O
O
O
O
H
H
a
b
O
N
N
S
O
O
N
O
N
H
S
N
O
N
OH
O
N
O
H
H
3
5
O
O
2
4
O
N
1
HN
O
SH
S
ClH N
ClH N
S
3
3
O
Scheme 1 Preparation of the reactive precursors for the modification
of lysine. a NHS (1.05 eq), DIC (1.15 eq), dichloromethane (DCM),
argon, 4 h, r.t., 92 % b 2,2 -dipyridyl disulfide (6 eq), methanol,
argon, overnight, r.t., quantitative yield
HN
O
0
O
Fig. 1 Heterofunctional cross-linker 1
Results and discussion
exchange with a free sulfhydryl group and also enables
monitoring of the coupling by measuring the characteristic
absorbance of the released pyridine-2-thione at 343 nm
Synthesis of lysine-based heterofunctional cross-linking
reagent 1
(
Carlsson et al. 1978). Moreover, cleavable disulfides can
To make the amino acid lysine accessible for bioorthogonal
reactions the corresponding carbonyl-reactive functional-
ities had to be introduced. The preparation of the N-
hydroxysuccinimide (NHS) active ester 3 from the com-
mercially available (Boc-aminooxy)acetic acid was
accomplished as described before (Deroo et al. 2003). The
activated thiol of cysteamine hydrochloride 4 was obtained
by an irreversible disulfide exchange (Scheme 1) (van der
Vlies et al. 2010). The reaction was carried out overnight in
serve as intracellular delivery systems due to the reductive
environment present in the cytosol (Ducry and Stump 2010;
Meister and Anderson 1983).
Chemoselective oxime ligation between aminooxy
components and aldehydes or ketones is a valuable conju-
gation reaction that also belongs to the repertoire of bio-
orthogonal chemistry. The a-effect in aminooxy
compounds enables condensation with carbonyls in a highly
efficient manner, even in aqueous media (Sander and Jencks
0
methanol with an excess of 2,2 -dipyridyl disulfide to avoid
1
968). In addition, nucleophilic catalysis of the oxime for-
side reactions by a stepwise addition of cysteamine
hydrochloride. After twofold precipitation in cold diethyl
ether, 2-(2-pyridyldithio)ethylamine hydrochloride 5 was
obtained in quantitative yield and used without further
purification. Compounds 3 and 5 were subsequently cou-
pled to lysine via amide bond formation to create the
reactive side chains on the amino acid.
mation by aniline can further accelerate the condensation,
so that complex biological entities can be coupled to each
other within a few minutes (Dirksen et al. 2006).
Heterofunctional cross-linkers, which contain three
functional groups, are a relatively small but important cat-
egory of bioconjugation reagents that are often based on the
chemical functionalization of an amino acid with at least
two bioorthogonal functionalities (Smeenk et al. 2012;
Clave et al. 2010). Natural amino acids such as glutamic
acid, cysteine or lysine exhibit already different functional
groups, however, the preparation of heterofunctional cross-
linking reagents often requires enormous synthetic efforts
and frequently involves several synthetic steps.
Lysine bears three functional groups, which can be
easily and selectively modified if appropriate protective
groups are utilized. To prevent side reactions during the
amide bond formation either the two amino groups or the
0
carboxylic acid has to be protected. Hence the N,N -di-Boc-
L-lysine dicyclohexylammonium salt was reacted initially
with 5 to introduce the activated thiol on the cross-linker
precursor 7. The reaction was carried out in N,N-dimeth-
ylformamide (DMF) with an excess of N,N-diisopropyl-
ethylamine (DIPEA) as base to maintain the free amine of
2-(2-pyridyldithio)ethylamine hydrochloride. The amide
bond formation between the protected amino acid deriva-
Herein we report the synthesis and characterization of a
new heterofunctional cross-linker 1 which employs the
scaffold of the amino acid lysine. The reagent carries two
aminooxy groups for bioorthogonal modification of car-
bonyls and a 2-pyridyl disulfide for the chemoselective
ligation to thiol-containing biomolecules (Fig. 1). The
potency of this cross-linking reagent is demonstrated by the
efficient coupling to the anthracycline doxorubicin and
immunoglobulin G (IgG).
0
0
tive and 5 was achieved with a slight excess of N,N,N ,N -
tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate
(TSTU) as coupling reagent. In this initial step the in situ
formation of the NHS active ester was mediated by TSTU
1
23

An amino acid-based heterofunctional cross-linking reagent
O
H2
N
H
N
H
N
O
S
O
N
H
S
N
O
H
O
O
a
O
N
S
b,c
O
N
H
S
N
H
N
5
3
O
O
O
1
HN
O
O
O
6
7
HN
O
HN
O
HN
O
O
O
O
0
Scheme 2 Synthesis of heterofunctional cross-linker 1. a N,N -di-Boc-L-lysine dicyclohexylammonium salt (1 eq), 5 (1.1 eq), TSTU (1.1 eq),
DIPEA (4 eq), DMF, argon, 4 h, r.t., 78 % b DCM/TFA (1:1), 1 h, r.t., quantitative yield (c) DIPEA (10 eq), 3 (2 eq), DMF, argon, 3 h, r.t., 81 %
and the desired product was formed within 4 h. To obtain
the final cross-linking reagent from 7, two straightforward
consecutive steps were applied as depicted on Scheme 2.
At first the a and e amino groups of the lysine derivative
were deprotected. Among the numerous deprotection
conditions for the Boc group, a solution of equal amounts
of dichloromethane (DCM) and trifluoroacetic acid (TFA)
was utilized and the deprotection occurred quantitatively
within 1 h (Wuts and Greene 2006). After removal of the
solvent, the residue was dried for 24 h in high vacuum to
remove the remaining TFA. The latter can easily adhere to
the oily material causing side reactions, such as deprotec-
tion and polymerization of 3 during the subsequent reaction
step. The residual oil was dissolved in dry DMF and the pH
of the solution was adjusted to 8 with DIPEA. To this
solution, two equivalents of 3 were added and the reaction
mixture was stirred for 3 h under argon atmosphere. The
heterofunctional cross-linker 1 was obtained in high yield
as colorless solid, which was characterized, by NMR
spectroscopy and MALDI-TOF mass spectrometry. The
new linker can thus be synthesized on a gram scale with
O
O
OH
O
OH
OH
H
O
OH
O
O
8
OH
NH
2
Fig. 2 Reactive sites for chemical modification of doxorubicin
breast cancer, childhood solid tumors, soft tissue sarcomas
and aggressive lymphomas (Minotti et al. 2004). However,
the therapeutic efficacy of many anthracyclines is restricted
by the onset of drug resistance and poor tumor targeting.
Different modifications of doxorubicin, like attachment to
polymers or targeting molecules, have been carried out to
overcome the poor selectivity that results in low thera-
peutic indexes and substantial toxicities to healthy tissues
5
8 % yield within four steps from readily available com-
mercial materials. Due to the high reactivity of free am-
inooxy groups towards omnipresent electrophiles (e.g.,
laboratory atmosphere) the Boc protective group of 1 was
cleaved immediately before the corresponding bioconju-
gation (Viault et al. 2013). Otherwise, the obtained cross-
linker can be stored for several months without any
decomposition.
(
Casi and Neri 2012). Doxorubicin bears three different
functional groups, which are accessible to chemical mod-
ifications: a primary alcohol, an amine and an aliphatic
keto group (Fig. 2). The functionalization of the alcohol as
well as the amine is often not suitable for biological and
therapeutic applications due to the rapid hydrolysis of
esters in the blood stream and the reduced DNA interca-
lation profile of the amine-modified doxorubicin (Mezo
et al. 2008). The herein described heterofunctional cross-
linker is well suited for the functionalization of doxorubi-
cin utilizing the aliphatic ketone via bioorthogonal oxime
bond formation and thus leaving the amine or other func-
tional groups intact.
Conjugation of 1 to two doxorubicin molecules
To test the efficacy of 1 for bioconjugation, a model drug,
namely doxorubicin was attached to the cross-linker. The
anthracycline doxorubicin is a widely applied anticancer
drug and is an essential component for the treatment of
123

M. Lelle, K. Peneva
O
O
O
OH
HO
O
H
O
N
O
N
S
O
N
H
N
H
O
S
H
H
HO
O
N
N
S
O
O
O
N
H
S
N
H
H N
2
O
O
a,b
HO
HN
O
8
9
1
HN
O
O
N
OH
OH
O
HN
O
HO
O
H
O
O
OH
O
OH
NH2
O
O
Scheme 3 Bioorthogonal modification of doxorubicin 8 with cross-linking reagent 1. a DCM/TFA (1:1), 1 h, r.t., quantitative yield
b doxorubicin hydrochloride (2.2 eq), DMF/0.4 M sodium acetate buffer (1:1)—pH 4.8, 24 h, r.t., 71 %
Doxorubicin hydrochloride was dissolved in a mixture
of DMF and 0.4 M sodium acetate buffer (pH 4.8) and was
added to the deprotected cross-linker. The slightly acidic
reaction conditions and the excess of doxorubicin enabled
full conversion of the reactants, as seen by reversed phase
high performance liquid chromatography (RP-HPLC). The
reaction mixture was first purified by gel permeation
chromatography (GPC) with Bio-Gel P-2 as stationary
phase and triethylammonium acetate (TEAA) buffer (pH 7)
as eluent, to remove the excess of doxorubicin. The iso-
lated material was further purified by preparative RP-
HPLC to obtain 9 as a red solid in 71 % yield. The product
of the bioorthogonal modification 9 was characterized by
conjugates play a crucial role in the targeted delivery of
drugs to the tumor environment. This emerging class of
bioconjugates combines the targeting properties from the
antibody with the cytotoxic properties of the utilized drug,
mediated by a cross-linking reagent. Usually the cross-lin-
ker enables hydrolytic or reductive cleavage of the drug
within the tumor site (Ducry and Stump 2010; Alley et al.
2010). The synthesized activated thiol-containing doxoru-
bicin bioconjugate 9 gives the opportunity to access such
antibody drug conjugates. Nevertheless, most of the anti-
bodies do not exhibit free sulfhydryl groups for bioconju-
gation. Typically the cysteine residues of proteins are
buried as disulfides, which are very important for the pro-
tein structure and function (Brocchini et al. 2008). To make
the disulfides accessible for bioconjugation chemistry,
reducing agents such as dithiothreitol or tris(2-carboxy-
ethyl)phosphine are applied, that can alter the native protein
or antibody, respectively. In contrast to disulfides, numer-
1
H NMR spectroscopy and mass spectrometry. Additional
2
D-NMR experiments (COSY, NOESY and TOCSY)
allowed the assignment of all proton signals from the
obtained doxorubicin bioconjugate. Moreover, 1 mM
solution of 9 in phosphate buffered saline (pH 7.4) was
analyzed by RP-HPLC at the characteristic absorption of
the drug at 480 nm (supplementary data). The isolated
doxorubicin conjugate exhibits a retention time of 23.2 min
and a purity of 95.1 % according to the peak integral
0
ous amino groups from lysine are exposed on the antibody s
surface and they can be selectively modified with 2-imi-
0
nothiolan 11, also known as Traut s reagent, to introduce a
precise number of sulfhydryl groups on the proteins surface
(Hermanson 2008). These sulfhydryls can be coupled to
thiol reactive groups like the 2-pyridyl disulfide of 9.
Therefore, IgG 10 was reacted with 10 equivalents of 11 at
(
Scheme 3).
Site-selective modification of immunoglobulin G
37 °C for one and a half hours in an aqueous buffer solution.
To prove the suitability of the activated thiol-containing
doxorubicin bioconjugate for site-selective attachment to
proteins, 9 was reacted with thiolated IgG. Antibody drug
This was done according to a literature procedure and led to
3–4 free thiol groups on the antibody’s surface (Ji et al.
2010). 12 was obtained in quantitative yield and was always
1
23

An amino acid-based heterofunctional cross-linking reagent
S
NH2
Cl
11
a
1
0
12
H
N
HS
NH2
Cl
3
-4
O
O
O
OH
HO
O
1
3
H
H
O
b
N
N
S
N
H
O
N
H
S
HO
9
O
O
NH2
Cl
H N
OH
2
HO
HN
O
3-4
O
N
OH
OH
HO
H
O
O
OH
O
OH
NH2
O
O
Scheme 4 Bioconjugation of IgG with 9. a 11 (10 eq), 0.1 M PBS buffer (pH 7.2—0.15 M NaCl, 10 mM EDTA), 90 min, 37 °C, quantitative
yield b 9 (20 eq), 0.1 M PBS buffer (pH 7.4), 3 h, 37 °C, quantitative yield
kept in an aqueous buffer solution to prevent degradation.
Nevertheless, this solution was not stored, but directly used
for bioconjugation with 9 to avoid side reactions involving
the free sulfhydryl groups on the antibody’s surface. Hence,
a solution of 9 in 500 ll of phosphate buffered saline (PBS)
was prepared immediately and added to the thiolated anti-
body, the reaction mixture was incubated for 3 h at 37 °C.
To maintain full conversion of the free thiols from 12, 20
equivalents of 9 were used for the ligation step. Subse-
quently the excess of 9 was easily removed by GPC on Bio-
Gel P-2, due to the intensive red color of the anthracycline
doxorubicin. The obtained product fraction containing the
doxorubicin carrying IgG 13 was concentrated with ultra-
filtration devices (MWCO 10 kDa) and analyzed by sodium
123

M. Lelle, K. Peneva
observed for the reduction of 13 (lane 5). Moreover, the
right panel has shown no fluorescence related to the two
different chains, but was clearly visible at the low molec-
ular weight part (bottom) of the gel. Furthermore, no
unspecific binding between 9, as well as doxorubicin, and
IgG was observed in a control experiment where the con-
jugation was carried out in the absence of Traut’s reagent
(supplementary data). These results from SDS-PAGE dis-
play the highly efficient cleavage of the disulfide bond
between 9 and 12, which has been generated by the acti-
vated thiol from the cross-linker.
Conclusion
Fig. 3 Comparative study of the antibody drug conjugate 13 and
native IgG by SDS-PAGE. Protein bands were visualized by
Coomassie staining (left panel) and fluorescence upon excitation by
UV-light (right panel)
The synthesis and application of a novel amino acid-based
heterofunctional cross-linker readily available from com-
mercial sources in excellent yield were described. Bioor-
thogonal dimerization of appropriate biomolecules with the
potential for further modification has been demonstrated.
Thereby, stable dimeric drug molecules carrying a pro-
tected and activated thiol could be easily prepared and
employed in the synthesis of important antibody drug
conjugates. The capability of the antibody doxorubicin
conjugate as a potential targeted drug delivery system was
highlighted by the efficient reductive cleavage of the
disulfide bond between the antibody and the drug conju-
gate, respectively. We believe that this new cross-linker
can serve as a powerful reagent in bioconjugation chem-
istry and will contribute to the rapidly expanding field of
chemical biology. This cross-linking reagent can be further
employed in additional ligation reactions, involving
monoclonal antibodies, oligonucleotides, peptides, carbo-
hydrates, polymers or fluorescent probes.
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) (Scheme 4).
SDS-PAGE of the antibody drug conjugate 13 is
depicted on Fig. 3. The left panel displays the obtained
protein bands after staining with Coomassie Brilliant Blue.
The intrinsic fluorescence of doxorubicin was employed to
visualize drug-related bands after gel electrophoresis (right
panel). An appropriate protein ladder for the accomplished
SDS-PAGE was applied in lane 2 and the corresponding
molecular weights are shown next to the gel. In lane 1 the
doxorubicin carrying IgG 13 was analyzed and the
achieved intensive band is in good accordance with the
obtained band from the utilized native antibody 10 depicted
in lane 3 (left panel). Unlike the unmodified antibody the
obtained band from 13 was also visible upon UV radiation
(
bright spot), which distinctly indicates the selective
labeling of IgG with the doxorubicin derivative 9. Fur-
thermore, no low molecular weight doxorubicin-associated
substances were detected in the first lane of the right panel,
which serves as an efficient proof for the materials purity.
The second band from the standard was in good agreement
with the molecular weight of IgG (approximately 150 kDa)
and 13. The modification of the thiolated antibody with 3–4
units of 9 led to minor changes in the molecular weight.
Nevertheless, 13 exhibited a small band above the product
presumably attributed to disulfides of the antibody, which
were observed due to the high gel loading to visualize the
fluorescence of doxorubicin. Compared to the native anti-
body, the reduced IgG (lane 4) possessed two explicit
bands upon treatment with dithiothreitol. This result was as
expected and the obtained bands are corresponding to the
molecular weight of the two light (25 kDa) and heavy
Experimental section
General
All chemicals, solvents and reagents were purchased from
commercial sources (Acros Organics, Alfa Aesar, Appli-
Chem, Deutero, Fisher Scientific, Fluka, Merck, Sigma-
Aldrich) and used without further purification. Doxorubicin
hydrochloride was purchased from Ontario Chemicals, Inc.
Ò
(Guelph, Ontario, Canada). Bio-Gel P-2 was obtained
from Bio-Rad Laboratories and immunoglobulin G
(100 mg/ml; purity C98 %; contains proline and IgA) was a
gift from the University Hospital of Mainz. The ultrafil-
tration devices Amicon Ultra 500 ll (MWCO 10 kDa) were
bought from Millipore and the samples were concentrated
Ò
in an Eppendorf MiniSpin Plus centrifuge. Column chro-
(
50 kDa) chains of the antibody. A similar pattern was
matography purifications were carried out on silica gel from
1
23

An amino acid-based heterofunctional cross-linking reagent
Macherey–Nagel (0.063–0.200 mm) and thin layer chro-
Ò
(4 H, s, CH -CH ), 1.42 (9 H, s, CH ); d (75 MHz;
2
2
3
C
6
DMSO-d ) 170.0, 165.1, 156.6, 80.6, 69.9, 27.9, 25.5; m/z
matography was performed on ALUGRAM SIL G/UV
13
2
54
1
?
(MALDI-TOF) 357.04 [M ? 3Na] .
sheets with appropriate solvents. H and C NMR spectra
were recorded on Bruker AMX 300 and Bruker WS 700
0
2-(2-Pyridyldithio)ethylamine hydrochloride (5). 2,2 -
(
Bruker Avance III) devices. Chemical shifts are expressed
Dipyridyl disulfide (25 g, 113.47 mmol) was gradually
dissolved in 150 ml methanol and degassed in an ultrasonic
bath for half an hour. To this solution cysteamine hydro-
chloride (2.15 g, 18.91 mmol) was slowly added within
30 min. Afterwards the flask was sealed with a septum and
the reaction mixture was stirred overnight under argon. The
yellow solution was precipitated twice in cold diethyl ether
and the product was obtained as a white crystalline solid
in parts per million (ppm) relative to the residual solvent
6
signal: DMSO-d (d = 2.50, d = 39.52) (Gottlieb et al.
H
C
1
997). Coupling constants (J) are given in Hz. MALDI-
TOF mass spectrometry was measured on a Bruker Reflex
II-TOF spectrometer equipped with a 337 nm nitrogen
laser, whereas 2,5-dihydroxybenzoic acid served as a
matrix. RP-HPLC was carried out on a Jasco LC-2000Plus
System, with appropriate diode array detector (MD-2015)
and solvent delivery pumps (PU-2086). Analytical RP-
HPLC was conducted with a ReproSil 100 C18 column
(4.21 g, 18.91 mmol, quantitative yield). d (300 MHz;
H
6
DMSO-d ) 8.55–8.47 (1 H, m, NCH), 8.32 (3 H, br s,
NH ), 7.89–7.72 (2 H, m, SCCH, SCCHCH), 7.42–7.35
3
(
250 9 4.6 mm) with 5 lm particle size as a stationary
(1 H, m, NCHCH), 3.17–3.02 (4 H, m, CH -CH );
2
2
6
phase and a flow rate of 1 ml/min. Purification of the pro-
ducts was performed on a ReproSil 100 C18 column
d (75 MHz; DMSO-d ) 158.1, 149.8, 137.9, 121.6, 120.0,
C
?
37.7, 34.8; m/z (MALDI-TOF) 187.00 [M ? H] .
(
250 9 20 mm) with a flow rate of 15 ml/min and 5 lm
(S)-Di-tert-butyl(6-oxo-6-((2-(2-pyridyldithio)ethyl)
0
silica as stationary phase. The applied eluents were 25 mM
triethylammonium acetate buffer (pH 7) (A) and acetoni-
trile (B). RP-HPLC was accomplished with a linear gradient
from 0 to 65 % B (40 min). All the substances were
detected at 480 nm based on the characteristic absorption of
doxorubicin. Incubation of antibody samples was done in an
amino)hexane-1,5-diyl)dicarbamate (7). N,N -di-Boc-
L-lysine
dicyclohexylammonium
salt
(1.52 g,
2.89 mmol),
5
(707 mg, 3.18 mmol) and TSTU
(955.9 mg, 3.18 mmol) were dissolved in 35 ml anhy-
drous DMF. Afterwards DIPEA (1.49 g, 1.96 ml,
11.55 mmol) was added and the reaction mixture was
stirred for 4 h at room temperature under argon atmo-
sphere. The suspension was diluted with 250 ml of
ethyl acetate and washed three times with 0.2 M
hydrochloric acid. Subsequently the clear solution was
dried with magnesium sulfate and the solvent was
removed under reduced pressure. The obtained residue
was purified by silica gel column chromatography using
ethyl acetate/hexane (3:1) as eluent, whereby the
Ò
Eppendorf Thermomixer compact. SDS-PAGE was car-
Ò
Ò
ried out with NuPAGE Novex 4-12 % Bis–Tris Gels
Ò
(
1.0 mm, 12 well) and NuPAGE MOPS SDS Buffer Kit
from Invitrogen. The reduced samples were obtained with
Ò
NuPAGE Sample Reducing Agent (109). Appropriate
staining of the antibody bands upon SDS-PAGE was
accomplished with Coomassie Brilliant Blue. The obtained
antibody bands were compared to the protein ladder ‘‘No-
Ò
vex Sharp Pre-stained Protein Standard’’ from Invitrogen.
product was obtained as a colorless oil (1.16 g,
6
2
.25 mmol, 78 %). d (300 MHz; DMSO-d ) 8.52–8.43
H
Synthesized compounds
(1 H, m, NCH), 8.05 (1 H, t, J = 5.4 Hz, CHCONH),
7
.88–7.70 (2 H, m, SCCH, SCCHCH), 7.31–7.18 (1 H,
(
(
Boc-Aminooxy)acetic acid N-hydroxysuccinimide ester
3). slurry of N-hydroxysuccinimide (2.79 g,
4.28 mmol) and (Boc-aminooxy)acetic acid (4.42 g,
m, NCHCH), 6.82 (1 H, d, J = 7.8 Hz, CHNH), 6.75
(1 H, t, J = 5.4 Hz, OCONH), 3.89–3.73 (1 H, m,
CHNH), 3.47–3.19 (2 H, m, CH CH S), 2.98–2.77 (4
A
2
2
2
2
3.12 mmol) in 55 ml of dry DCM was prepared initially.
0
H, m, CH CH S, OCONHCH ), 1.58–1.17 (6 H, m,
2 2 2
N,N -diisopropylcarbodiimide
(3.06 g,
3.76 ml,
CHCH CH CH ), 1.36 (18 H, s, CH ); d (75 MHz;
2
2
2
3
C
6
2
4.88 mmol) was added under argon and the clear solution
DMSO-d ) 172.3, 159.0, 155.5, 155.3, 149.6, 137.8,
121.2, 119.3, 77.9, 77.3, 54.4, 39.6, 37.7, 37.5, 31.6,
29.2, 28.3, 28.2, 22.8; m/z (MALDI-TOF) 536.93
0
was stirred for 2 h. Additional N,N -diisopropylcarbodi-
imide (244 mg, 300 ll, 1.99 mmol) was added and the
reaction mixture was stirred for further 2 h. Afterwards the
precipitated urea was filtered off and washed with a small
amount of DCM. The obtained solution was diluted with
?
[M ? Na] .
Lysine-based heterofunctional cross-linker (1).
7
(1.10 g, 2.14 mmol) was dissolved in 15 ml of dry DCM
and the equal amount of TFA was added. Afterwards the
solution was vigorously agitated for 60 min at room tem-
perature and the reagent was removed in vacuo. The
obtained residue was dried for additional 24 h in high
vacuum and subsequently dissolved in 25 ml dry DMF.
2
00 ml of DCM and washed four times with water. The
organic layer was dried with magnesium sulfate and the
solvent was removed in vacuo, to obtain the product as a
colorless solid (6.13 g, 21.27 mmol, 92 %). d (300 MHz;
H
6
DMSO-d ) 10.36 (1 H, s, NH), 4.82 (2 H, s, O-CH ), 2.84
2
123

M. Lelle, K. Peneva
Consecutively DIPEA (2.76 g, 3.63 ml, 21.37 mmol) and 3
1.23 g, 4.27 mmol) were added and the solution was
(2 H, m, CH CHOCHO), 2.24–2.15 (2 H, m, CH CHO-
2
2
(
CHO), 1.89–1.82 (2 H, m, NH CHCH ), 1.68–1.58 (3 H,
2 2
stirred for 3 h under argon at room temperature. The
reaction mixture was diluted with 300 ml ethyl acetate and
washed twice with 0.2 M hydrochloric acid as well as
brine. The organic layer was dried with magnesium sulfate
and the solvent was removed under reduced pressure.
Afterwards the obtained residue was purified by silica gel
column chromatography using ethyl acetate/methanol
m, NH CHCH , CHCH ), 1.48–1.41 (1 H, m, CHCH ),
2 2 2 2
1.36–1.28 (2 H, m, NHCH CH CH ), 1.20–1.12 (8 H, m,
2
2
2
MeCH, NHCH CH CH ); m/z (MALDI-TOF) 1,533.07
2
2
2
?
[M ? Na] .
Thiolated IgG (12). A solution of IgG (5 mg,
0.035 lmol) in 500 ll 0.1 M PBS buffer (pH 7.2) con-
taining 0.15 M NaCl and 10 mM EDTA was prepared. The
thiolation reagent 11 (0.96 mg) was dissolved in 1 ml of
the same buffer mixture and 50 ll (0.348 lmol) was added
to the IgG solution. Afterwards the reaction mixture was
incubated in an Eppendorf Thermomixer (300 rpm) at
37 °C for 90 min. This solution was transferred into 500 ll
ultrafiltration devices Amicon Ultra (MWCO 10 kDa) and
concentrated in an Eppendorf MiniSpin Plus centrifuge
(5 min, 10.000 rpm) to 100 ll. Subsequently 400 ll of
0.1 M PBS buffer (pH 7.4) was added and spun again at
10.000 rpm for 5 min. This procedure was repeated one
more time and the thiolated antibody was obtained in
100 ll of a viscous buffer solution (5 mg, 0.035 lmol,
quantitative yield).
(
(
15:1) and the product was isolated as a colorless solid
6
1.14 g, 1.73 mmol, 81 %). d (300 MHz; DMSO-d )
H
1
0.32 (1 H, s, CONHO), 10.29 (1 H, s, CONHO),
8
.50–8.41 (1 H, m, NCH), 8.24 (1 H, t, J = 5.4 Hz,
CHCONH), 8.10 (1 H, d, J = 8.1 Hz, CHNH), 7.98 (1 H, t,
J = 5.4 Hz, OCH CONH), 7.89–7.70 (2 H, m, SCCH,
2
SCCHCH), 7.29–7.18 (1 H, m, NCHCH), 4.38–4.08 (3 H,
m, CHNH, NHOCH ), 4.12 (2 H, s, NHOCH ), 3.49–3.19
2
2
(
2 H, m, CH CH S), 3.08 (2 H, q, J = 6.6 Hz,
2 2
NHCH CH CH ), 2.89 (2 H, t, J = 6.6 Hz, CH CH S),
2
2
2
2
2
1
.71–1.48 (2 H, m, CHCH ), 1.47–1.34 (2 H, m,
2
NHCH CH CH ), 1.33–1.21 (2 H, m, NHCH CH CH ),
2
2
2
2
2
2
6
1
1
8
2
.40 (18 H, s, CH ); d (75 MHz; DMSO-d ) 171.3, 167.9,
3
C
67.7, 159.1, 157.0, 156.9, 149.6, 137.8, 121.2, 119.3,
IgG doxorubicin conjugate (13).
9
(1.05 mg,
0.6, 80.6, 74.8, 74.6, 52.0, 38.0, 37.8, 37.3, 31.8, 28.7,
?
7.9, 22.7; m/z (MALDI-TOF) 661.11 [M ? H] .
0.695 lmol) was gradually dissolved in 500 ll of 0.1 M
PBS buffer (pH 7.4) and added to 100 ll of the thiolated
IgG (5 mg, 0.035 lmol) solution. The reaction mixture
was incubated in an Eppendorf Thermomixer (300 rpm) for
3 h at 37 °C and subsequently purified by GPC on Bio-Gel
P-2 with 0.1 M PBS buffer (pH 7.4). The obtained red
product fraction was concentrated in an Eppendorf Mini-
Spin Plus centrifuge (10.000 rpm) to 100 ll (5 mg,
0.035 lmol, quantitative yield).
Dimeric doxorubicin bioconjugate (9). The cross-linker
(217.5 mg, 330 lmol) was dissolved in 10 ml of dry
1
DCM and gradually 10 ml of TFA was added. The solu-
tion was stirred for 1 h at room temperature and subse-
quently the solvent mixture was removed under reduced
pressure.
Doxorubicin
hydrochloride
(421.1 mg,
7
26 lmol) was dissolved in 80 ml DMF/sodium acetate
buffer (1:1)—pH 4.8 and was added to the oily residue.
Afterwards the reaction mixture was stirred for 24 h at
room temperature and the crude product was purified by
GPC on Bio-Gel P-2 using 25 mM TEAA buffer (pH 7) as
eluent. The isolated fractions were additionally purified by
RP-HPLC and the product was obtained as a red solid
Conflict of interest The authors declare that they have no conflict
of interest.
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