Targeted Shiga toxin-drug conjugates prepared via Cu-free click chemistry

Article abstract of DOI:10.1016/j.bmc.2015.10.010

The main drawback of the anticancer chemotherapy consists in the lack of drug selectivity causing severe side effects. The targeted drug delivery appears to be a very promising strategy for controlling the biodistribution of the cytotoxic agent only on malignant tissues by linking it to tumor-targeting moiety. Here we exploit the natural characteristics of Shiga toxin B sub-unit (STxB) as targeting carrier on Gb3-positive cancer cells. Two cytotoxic conjugates STxB-doxorubicin (STxB-Doxo) and STxB-monomethyl auristatin F (STxB-MMAF) were synthesised using copper-free 'click' chemistry. Both conjugates were obtained in very high yield and demonstrated strong tumor inhibition activity in a nanomolar range on Gb3-positive cells.

Full text of DOI:10.1016/j.bmc.2015.10.010

Bioorganic & Medicinal Chemistry 23 (2015) 7150–7157
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Targeted Shiga toxin–drug conjugates prepared via Cu-free click
chemistry
Vesela Kostova, Estelle Dransart, Michel Azoulay, Laura Brulle, Siau-Kun Bai, Jean-Claude Florent,
⇑
Ludger Johannes, Frédéric Schmidt
Institut Curie, CNRS, UMR 3666/INSERM U1143, 26 rue d’Ulm, 75248 Cedex 05 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The main drawback of the anticancer chemotherapy consists in the lack of drug selectivity causing severe
side effects. The targeted drug delivery appears to be a very promising strategy for controlling the biodis-
tribution of the cytotoxic agent only on malignant tissues by linking it to tumor-targeting moiety. Here
we exploit the natural characteristics of Shiga toxin B sub-unit (STxB) as targeting carrier on Gb3-positive
cancer cells. Two cytotoxic conjugates STxB–doxorubicin (STxB–Doxo) and STxB–monomethyl auristatin
F (STxB–MMAF) were synthesised using copper-free ‘click’ chemistry. Both conjugates were obtained in
very high yield and demonstrated strong tumor inhibition activity in a nanomolar range on Gb3-positive
cells.
Received 21 July 2015
Revised 5 October 2015
Accepted 6 October 2015
Available online 8 October 2015
Keywords:
Targeted drug delivery
Shiga toxin
Copper-free click
Doxorubicin
Ó 2015 Elsevier Ltd. All rights reserved.
Auristatin
1. Introduction
agents for antitumoral treatment of Hodkins lymphoma and breast
cancer.5,6
The development of new potent anticancer treatments has
always presented a challenge, especially in cancer chemotherapy,
where the lack of drug selectivity decreases the efficiency of the
therapy leading to systematic toxicity and side effects. However
targeted drug delivery was designed to circumvent this draw-
back.1,2 The current strategies rely on the difference of the
expressed surface receptors, metabolism profile and site location
of the normal or cancerous cells. Indeed, the overexpression of cell
surface molecules by tumor cells allows the use of specific ligands.
To target only malignant tissues, the drug delivery systems are
based on the coupling of cytotoxic drugs to tumor targeting carri-
ers such as proteins, peptides, nucleic acid, carbohydrate and small
molecules.3,4 The most widely used targeting moieties are mono-
clonal antibodies (mAb). They are linked to the anticancer agent
by lysine- or cysteine-based conjugation using different linker
types: protease-, pH-, reductive cleavable linkers allowing the drug
release inside the cell or in some case non-cleavable linker as
thioether. In the last years two antibody–drug conjugates (ADCs)
have been approved by FDA: brentuximab vedotin (AdcetrisÒ) in
2011 and ado-trastuzumab emtansyne (KadcylaÒ) in 2013.5 They
are considered as an essential improvement of chemotherapeutic
The mAb antibody has to fulfill a list of requirements such as
being selective for tumor-associated antigens and humanized to
minimize immunogenicity.7–9 The antigen to which it is targeted
should be highly expressed on the cell surface to enable efficient
internalization and delivery of the cytotoxic payload. The anti-
cancer agents typically used in ADCs are highly potent because
the drug concentration is limited by antigen expression on the cel-
lular membrane (typically less than 105 per cell).10 After cellular
binding, ADCs are taken up by endocytosis and follow the lysoso-
mal pathway where they undergo degradation, leading to the
release of the cytotoxic drug. The linker between the antibody
and the anticancer agent should be stable in circulation and
efficiently cleaved inside tumor cells.11–13 Another challenge for
ADC preparation is to control the drug-to-antibody ratio (DAR)
avoiding heterogeneous mixtures of conjugate species.
Natural ligands for cancer cell markers represent alternatives to
antibodies. One of these is the B-subunit of Shiga toxin. This toxin
is produced by Shigella dysenteriae, while enterohemorrhagic
strains of Escherichia coli produce a family of Shiga-like toxins (or
verotoxins). All these toxins are composed of toxic A-subunits
and non-toxic receptor-binding B-subunits (STxB). STxB is
a
homopentameric protein composed of five identical B-fragments
of 7.7 kDa. STxB binds to a glycosphingolipid, globotriaosylce-
ramide (Gb3), that is, overexpressed by human tumor cells.14–19
STxB possesses 15 Gb3 binding sites, and the apparent affinity for
⇑
Corresponding author. Tel.: +33 156 246 664; fax: +33 156 246 6 31.
E-mail address: Frederic.Schmidt@curie.fr (F. Schmidt).
http://dx.doi.org/10.1016/j.bmc.2015.10.010
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

V. Kostova et al. / Bioorg. Med. Chem. 23 (2015) 7150–7157
7151
cells is in order of 109 MÀ1. Once STxB recognizes Gb3, the complex
clusters on cell surface and induces membrane bending as a first
step of its clathrin-independent uptake into cells.14 The inter-
nalised protein then traffics through early and recycling endo-
somes and the trans-Golgi network (TGN) to the ER. This
unconventional itinerary, termed the retrograde transport route,
bypasses the late degrading environment of the late endocytic
pathway.14 STxB might therefore be used as a delivery tool to
selectively transport cytotoxic compounds inside Gb3-positive
cells. For chemical cross-linking, a STxB variant is used to which
a cysteine residue was added onto the C-terminus of each B-frag-
ment, producing free thiol groups. In order to create STxB–drug
conjugates, the conjugation reaction between STxB and prodrug
must be accomplished in mild conditions in aqueous medium to
preserve the integrity, the native conformation and functionality
of the delivery tool. Unpublished studies performed in our team
have demonstrated the loss of the STxB functionality when it
was coupled to anticancer agent by copper catalyzed click reaction.
Hence, bioorthogonal copper-free ‘click’ chemistry appears to be
appropriate for the coupling of STxB to an anticancer agent.20,21
The copper-free Huisgen [3+2] cycloaddition was reported as a
very efficient reaction between cyclooctyne and azide where the
ring strain of the alkyne drives the reaction forward without the
use of catalyst.24 Cyclooctyne and azide are inert to other function-
alities present in biomolecules. Many biological applications have
been reported where this coupling method did not interfere with
protein structure and functions.22,23 This chemistry thus proved
to be adequate in satisfying many criteria such as biocompatibility,
selectivity and especially high yield of coupling, which could avoid
further protein purification problems.25 Consequently, the copper-
free Huisgen [3+2] cycloaddition appears to be a suitable choice for
our carrier.
Here we explore the effectiveness of Gb3-targeting delivery
system using STxB conjugates prepared by versatile and modular
copper-free click-based method. The targeting moiety is first
functionalized with heterobifunctional linker 1 defined by malei-
mide functionalised monofluorocyclooctyne (MFCO) (Scheme 1).
In this way the genetically modified STxB/Cys with 5 thiol func-
tionalities is linked to the maleimide by thioether bond. In a second
step, the click coupling between STxB–MFCO and azido-biotin 2 or
the azido-prodrugs 3, 4 are performed. The active principles of
azido-prodrugs 3 and 4 are linked to STxB via their amine function-
alities. Their scaffold was designed with disulfide bonds necessary
to trigger, after reduction, the self-immolative mechanism and the
drug release. The STxB–biotin conjugate is used for proof of con-
cept demonstration. The two other STxB–drug conjugates aim to
validate the efficiency of the targeting strategy, the drug releasing
mechanism and optimize the anticancer treatment effect by intro-
ducing a highly potent anti-tumoral agent.
The anticancer agents chosen for the study are doxorubicin
(Doxo) and monomethylauristatin F (MMAF) both containing free
amine function in their respective structures. Doxo is a topoiso-
merase II inhibitor having DNA intercalating properties. The com-
pound possesses high water solubility, but its use in the clinics is
limited by detrimental side effects such as cardiotoxicity. MMAF
is a derivative of the natural product dolostatin 10 with potent
microtubulin inhibition activity. Its C-terminal domain is nega-
tively charged at physiological pH, which makes MMAF little prone
to cross the cell membrane spontaneously. Previous studies have
shown the potentiated inhibition activity of its analog monomethyl
auristatin E (MMAE) via antibody–drug conjugate (ADC), suggest-
ing that impaired translocation capabilities of MMAF are the rea-
son for its mild activity.11 It has been demonstrated as well that
the amine functions of Doxo and MMAF can be manipulated in
order to create prodrugs for which the cytotoxic principle is
released only once they target cells are reached.11,26
2. Results and discussion
2.1. Proof of concept: Synthesis of STxB–biotin conjugate
STxB–biotin conjugate was synthesized to test protein function-
ality after using copper-free cycloaddition on STxB. MFCO was cho-
sen because of the balance between reactivity and synthesis
accessibility of the molecule. The synthesis of bifunctional linker
1 started by preparation of MFCO 5 following a four step synthesis
scheme described by Pigge from commercially available
cyclooctanone.27 The acid function of MFCO was coupled to the
amine of a previously synthesised N-(2-aminoethyl)maleimide 6.
Compound 6 was obtained in two steps from Mitsunobu reaction
between maleimide and Boc-ethanolamine followed by deprotec-
tion28 (Scheme 2).
Several studies have reported side reactions of a thiol function
on an alkyne.29 To confirm the selectivity of MFCO toward the
cycloaddition, we tested the reactivity of monofluorocyclooctyne
5 in the presence of cysteine methyl ester in organic medium.
The reaction did not show any progress after 48 h at room temper-
ature. Based on these results, compound 1 went through Michael
addition with the free sulfhydryl residues of each STxB/Cys mono-
mer. In parallel an azide-functionalised biotin 2 was obtained from
peptide coupling between biotin and 1-amino-11-azido-3,6,9-tri-
oxaundecane chosen for its hydrosolubility and commercial
availability.30 Then the intermediate STxB–MFCO 7 was coupled
by copper free Huisgen [3+2] cycloaddition to linker 2 to afford
conjugate 8 (Scheme 2). The yield and purity of each step were
monitored by LC–MS analysis. The yields of the two steps were
respectively of 85% and 95% for a 1:1 stoichiometry between biotin
and STxB monomer.
O
O
HN
HO
O
O
O
O
NH
N3
O
N
N
N
H
F
H
1
S
O
mic
plas
um
icul
Ret
ndo
E
2
Plasma membrane
O
O
STxB
lgi
Go
HS
A
OH
OH
O
O
O
O
O
O
N
O
H
O
S
S
STxB
STxB
S
T
S
N
O
O
STxB
O
x
N3
N3
B
O
F
S
HO
T
x
s
eu
cl
u
N
B
N
Gb3
N
H
3
O
O
Early
endosome
HO
C
B
N3
O
O
H
H
S
N
N
N
O
S
N
N
N
O
F
N
Me OMeO
S
N
N3
N
OMeO
CO2H
N
H
Me O
O
4
Scheme 1. Click synthesis and retrograde delivery strategy. (A) Bifunctional linker 1 coupling with STxB/Cys, (B) Cu-free click ligation with azido-probiotin 2 or
azido-prodrug 3 and 4, (C) tumor-specific drug delivery induced by STxB on positive Gb3 cells.

7152
V. Kostova et al. / Bioorg. Med. Chem. 23 (2015) 7150–7157
O
O
F
O
(i)
OH
O
O
O
F
5
N
(iii)
N
H
O
O
O
NH3 CF3CO2
(ii)
1
NHBoc
N
NH
HO
O
6
O
O
NH
HN
O
4
S
S
O
N
O
O
O
N
N
N
S
O
F
O
F
O
H
3
N
N
N
(iv)
(v)
N
N
H
N
H
F
H
O
O
O
1
7
8
Scheme 2. Synthesis of bifunctional linker 1 and STxB–biotin conjugate 8. Reagents and conditions: (i) (a) Selectfluor,CH3CN, reflux, (93%); (b) KHMDS, Tf2NPh, THF, À80 °C,
(60%); (c) LiOH, THF, (92%); (ii) (a) DEAD, PPh3, THF, À78 °C, (80%); (b) TFA, CH2Cl2, (87%); (iii) (a) PFP–TFA, DIPEA, CH2Cl2; (b) DIPEA, DMF, (78%); (iv) STxB/Cys, PBS buffer,
21 °C, 18 h, (85%); (v) 2, PBS buffer, 21 °C, 18 h, (95%).
2.2. Biological assessment of STxB–biotin conjugate
the drug delivery. Previous work of the laboratory31 demonstrated
that a 2-amino-ethanethiol can release a phenol through disulfide
reduction. If the phenol is a 4-hydroxybenzyl alcohol, it can be
used to release the drug amino group. Based on the literature, we
suggest the following mechanism of drug release32 (Scheme 4).
Firstly we prepared linker 3 starting by deacetylation dimeriza-
tion of compound 9 previously synthesized in two steps from 2-
chlorethoxyethanol, affording derivative 1033 (Scheme 5). The next
step was a dismutation between 10 and 1131 prepared from
commercially available methylaminoethanol. The corresponding
tert-butoxycarbonyl derivative 12 went through a classical depro-
tection in acidic conditions. The obtained salt 13 was condensed
with p-nitrophenyl carbonate derivative 22 previously obtained
in two steps from commercial 4- hydroxybenzyl alcohol.32 The
instability of the TBDMS ether in mild acid conditions was used
to deprotect the primary alcohol of 14 so it could be reactivated
by 4-nitrophenyl chloroformate to afford final activated linker
15. Doxorubicin was then coupled to spacer 15 in basic conditions
giving derivative 3 (Scheme 6). Compound 3 was finally coupled to
STxB–biotin conjugate was tested for its ability to be internal-
ized into Gb3-positive cells and to follow the retrograde trafficking
route. To validate the concept, the unmodified STxB/Cys and conju-
gate 8 were followed by immunofluorescence analysis on HeLa
cells after 45 min incubation at 37 °C. Co-localization of conjugate
8 with the Golgi marker giantin, was similar to this observed for
the non-modified STxB/Cys (Scheme 3).
The cellular uptake characteristics of STxB–biotin were obvi-
ously preserved and the carrier was functional. We concluded that
the copper free cycloaddition was compatible with STxB targeting
strategy and it can be used for the design of STxB–prodrug
conjugates.
2.3. Design and synthesis of STxB–Doxo conjugate
To exploit some of the previous mentioned properties of STxB
and release a drug inside Gb3 positive tumors cells after carrier-
mediated targeting, we designed and synthesized conjugate 16
(Scheme 6). We choose to rely on disulfide bonds able to be reduc-
tively cleaved and to trigger drug release. Doxorubicin (Doxo)
appeared to be a good candidate for testing the targeting strategy
showing good hydrosolubility and cytotoxic activity toward HT 29
colon carcinoma cell lines. However the drug itself does not pos-
sess a thiol so we built a spacer able to liberate the amino-bearing
anticancer agent after cleavage of the disulfide. The scaffold of the
linker is defined by an azide function that is linked by a small PEG
to the disulfide bond, followed by a biocleavable spacer enabling
STxB–MFCO
7 in PBS buffer and MALDI-TOF spectrometry
validated the formation of the conjugate.
The mass spectrum displayed a major peak for the final conju-
gate 16 and a minor peak at 8646 Da. This minor peak could be
attributed to a byproduct of the conjugate without the aromatic
planar chromophore of doxorubicin, probably due to the photosen-
sitivity of the drug. The conjugate 16 was produced with the
maximum substitution (5 molecules of doxorubicin per pentamer
of STxB). The yield for the final click reaction was 89 % as
determined by LC–MS analysis.
HO
O
O
O
O
O
O
O
N
O
OH
OH
H
O
S
S
N
O
N3
HO
3
O
HO
Disulfide reduction
O
OH
N
S
CO2
H2N
Doxorubicin
Scheme 3. Retrograde trafficking of STxB and STxB–biotin conjugate. STxB or
STxB–biotin were bound to HeLa cells at 4 °C, followed by incubation for 45 min at
37 °C. Cells were fixed and labeled for the indicated markers. Top: STxB; bottom:
STxB–biotin conjugate 8. STxB: green; giantin: red; Biotin: blue; MRG: merge.
HO
Scheme 4. Release mechanism of doxorubicin by reduction of disulfide bond
followed by 1,6-elimination.

V. Kostova et al. / Bioorg. Med. Chem. 23 (2015) 7150–7157
7153
O
O2N
O
O
OTBDMS
N
S
Boc
O
11
22
O
(i)
(ii)
(iii)
O
O
S
N3
O
S
Boc
O
N3
S
O
N3
S
N
N3
S
9
10
12
O
NO2
O
O
OTBDMS
O
(iv)
(v)
O
S
O
S
S
13
N3
S
N
O
N3
N
O
14
15
Scheme 5. Azido-linker 15 synthesis. Reagents and conditions: (i) MeONa (0.5 M), MeOH, rt, overnight under air (70%); (ii) 11, MeONa, MeOH, rt, (72%); (iii) (a) TFA, DCM, rt,
(quant.); (iv) 22, Et3N, DMF, rt, (87%); (v) (a) HCl (12 N) at 10% in EtOH, rt, (95%); (b) pNO2PhOCOCl, Et3N, DCM, rt, (66%).
HO
NO2
O
O
O
OH
OH
O
O
O
O
O
N
O
(i)
O
O
O
H
O
S
O
S
S
N
O
N3
N3
S
N
O
HO
15
3
O
(ii)
HO
HO
O
O
OH
O
O
O
O
N
O
H
S
O
O
S
N
S
N
O
N
N
O
HO
N
N
H
OH
O
F
O
O
HO
16
Scheme 6. Synthesis of STxB–doxorubicin conjugate 16. Reagents and conditions: (i) doxorubicin, Et3N, DMF, (50%); (ii) compound 7, PBS buffer, 21 °C, 18 h, (89%).
Table 1
2.4. Cytotoxicity of STxB–Doxo conjugate
IC50 values of STxB–Doxo conjugate 16 on Gb3+ versus Gb3À (PPMP treated) HT29
cells in comparison to untargeted doxorubicin and pro-Doxo 3
The cytotoxic activity of STxB–Doxo conjugate was tested using
a MTT colorimetric assay on Gb3 positive colorectal carcinoma cells
HT29. To generate a Gb3-negative control condition, HT29 cells
were treated with the glycosylceramide synthetase inhibitor
1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) to
inhibit glycosphingolipid synthesis. Cytotoxicity assay revealed
the specificity of the STxB targeting strategy, showing cytotoxic
activity of STxB–Doxo conjugate 16 only on Gb3 positive HT29
cells. As expected the toxicity of free Doxo and the linker 3 was
Gb3 independent (Scheme 7). The IC50 values of free doxorubicin
and conjugate 16 were in the same range of 84.8 and 22.5 nM,
respectively, (Table 1). This result demonstrates that the
self-immolative drug release mechanism is functional.
Cytotoxic activity on HT 29
IC50 (nM) Gb3+
IC50 (nM) Gb3À
25.9
10,000
—
Doxorubicin
Prodrug 3
STxB–Doxo 16
84.8
6000
22.5
The experiment confirmed the functionality and the efficiency of
the STxB drug delivery strategy with an increased activity of the
anticancer agent on tumor cells. The intracellular uptake of conju-
gate was tested in vitro and confirmed the protein integrity of 16.
2.5. Design and synthesis of STxB–MMAF conjugate
To optimize the established targeting strategy we replaced dox-
orubicin with a more potent anticancer agent, monomethyl auris-
tatin
F (MMAF), using the same self-immolative spacer 15
(Scheme 8). We proceed with coupling between derivative 15
and MMAF in basic conditions affording compound 23. The cop-
per-free reaction between STxB–MFCO 7 and 23 gave less than
10% yield. It seemed likely that a solubility problem could explain
the low yields. Assays were therefore performed at different
temperatures, buffer conditions, linker arms, and STxB–MFCO
concentrations, but reactivity remained similarly low. A second
hypothesis was that steric hindrance at the azide group position
prevented the cycloaddition with MFCO, due to a conformational
restriction resulting for example from
p–p stacking between the
two aromatic rings in the structure of compound 23 (Scheme 8).
In order to test this hypothesis, we replaced the phenyl carba-
mate by a simple carbamate linked to the amine extremity of
MMAF.34 The synthesis of the linker 4 started with hydrolysis of
Scheme 7. Comparison of cytotoxicity of the conjugate STxB–Doxo 16 on Gb3+ and
Gb3À (PPMP treated) HT29 cells.

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V. Kostova et al. / Bioorg. Med. Chem. 23 (2015) 7150–7157
NO2
O
O
O
H
H
N
N
N
O
O
O
O
O
N
N
O
S
(i)
O
S
Me
O
Me OMe O
OMe
O
CO2H
S
N
O
N3
S
N
O
N3
23
15
(ii)
O
O
H
H
N
N
N
N
O
O
N
S
O
N
O
OMe
O
CO2H
Me OMe
O
S
Me
O
N
N
S
N
O
O
F
N
N
H
O
17
Scheme 8. Synthesis of STxB–MMAF conjugate 17. Reagents and conditions: (i) MMAF, Et3N, DMF, rt, overnight, (60%); (ii) compound 7, PBS buffer, 21 °C, 18 h, (10%).
O
O
N3
S
NO2
O
9
(i)
(ii)
O
S
O
S
N3
S
OH
N3
S
O
O
N
S
18
S
S
HO
(iii) 19
21
O
O
H
N
H
N
O
N
N
S
O
N
N3
Me OMe O
OMe O
CO2H
Me
O
4
(iv)
O
N
O
H
N
H
S
N
N
O
O
S
N
N
N
N
S
O
O
F
O
OMe O
CO2H
Me OMe
Me
O
N
N
H
O
20
Scheme 9. Synthesis of STxB–MMAF conjugate 20. Reagents and conditions: (i) MeONa (0.5 M), MeOH, rt, (79%); (ii) pNO2PhOCOCl, Et3N, DCM, rt, (75%); (iii) MMAF, Et3N,
DMF, rt (71%); (iv) compound 7, PBS buffer, 21 °C, 18 h, (94%).
the thioacetate function of 9 generating in situ a free thiol group
that was trapped by nucleophilic attack with disulfide compound
2135 (Scheme 9). The resulting disulfide 18 was activated with
4-nitrophenyl chloroformate to afford the activated linker 19.
The MMAF condensation with compound 19 then afforded the
azide–pro MMAF derivative 4. The synthesis was pursued with
incubation of linker 4 and STxB–MFCO conjugate 7 in PBS buffer
yielding the desired STxB–MMAF conjugate 20. LC–MS analysis
confirmed 94% yield for the final step of copper free Huisgen
cycloaddition [3+2]. This result is consistent with our hypothesis
of steric hindrance lowering the reactivity of the azide group
toward the cycloaddition with MFCO.
In comparison, the untargeted MMAF had an IC50 value of 43 nM.
Conjugate 20 was around 200 folds less toxic on PPMP-treated cells
confirming that STxB conjugation promotes a Gb3-specific cell
internalization of MMAF (Scheme 10). The data also demonstrated
an improved efficiency and highlighted the selectivity of the
STxB-based MMAF delivery strategy, which enhanced the cytotoxic
effect of the anticancer agent on Gb3-positive tumor cells. The
STxB–MMAF conjugate was evaluated in vitro for its intracellular
trafficking characteristics. Accumulation in the Golgi apparatus
2.6. Cytotoxicity of STxB–MMAF conjugate
Conjugate 20 displayed potent cytotoxic activity on
Gb3-expressing HT29 cells with an IC50 value of 8.4 nmol (Table 2).
Table 2
IC50 values of STxB–MMAF conjugate 20on Gb3+ versus Gb3À (PPMP treated) HT29
cells in comparison to untargeted MMAF
Cytotoxic activity on HT 29
IC50 (nM) Gb3+
IC50 (nM) Gb3À
MMAF
STxB–MMAF 20
43.5
8.4
37.5
1500
Scheme 10. Cytotoxicity of the STxB–MMAF conjugate on Gb3+ versus Gb3À
(PPMP treated) HT29 cells.

V. Kostova et al. / Bioorg. Med. Chem. 23 (2015) 7150–7157
7155
Table 3
matrix was composed by
a
solution of sinapinic acid in
IC50 values of STxB–MMAF conjugate 20 on Gb3+ versus Gb3À (PPMP treated) HT29
acetonitrile/TFA. Samples were mixed with the matrix at a ratio
of 1:1. The mixture was spotted onto a MALDI-TOF plate and
allowed to dry.
cells with or without pre-incubation in pure serum (SVF)
Cytotoxic activity on HT 29
IC50 (nM) no pre-
incubation
IC50 (nM) pre-
incubation
Gb3+
Gb3À
Gb3+
Gb3À
4.2. Synthesis and characterization of described compounds
MMAF
64
60
STxB–MMAF 20
6.6
580
5.6
380
4.2.1. 1-Fluoro-cyclooct-2-ynecarboxylic acid [2-(2,5-dioxo-2,5-
dihydro-pyrrol-1-yl)-ethyl]-amide (1)
Pentafluorophenyl trifluoroacetate (0.70 g, 2.52 mmol, 433
was added to a solution of 1-fluorocyclooct-2-ynecarboxylic acid
(0.36 g, 2.12 mmol) and N,N-diisopropylethylamine (0.32 g,
2.52 mmol, 438 L) in dry DCM (21 mL) brought to 0 °C. The
mixture was stirred at room temperature for 3 h and filtered over
silica. The activated acid (0.85 g, 2.55 mmol), N-(2-aminoethyl)-
maleimide 6 (0.34 g, 3.19 mmol) and N,N-diisopropylethylamine
lL)
was as efficient for conjugate 20 as for STxB/Cys, confirming that
the functional integrity of the protein was preserved despite the
presence of the MMAF.
In order to test the stability of the conjugate in the light of
future animal experiments, the cytotoxic activity of conjugate 20
was measured with and without 24 h pre-incubation at 37 °C in
pure SVF serum. We observed similar activity, makes it likely that
the conjugate could be biodistributed in an intact manner before
the liberation of the active principle in cancerous target cells
(Table 3).
5
l
(0.39 g, 3.06 mmol, 533 lL) were agitated in dry DMF (41 mL) for
16 h at room temperature. Distilled water (20 mL) was added.
The organic phase was extracted with DCM (3 Â 30 mL), washed
with brine (80 mL) and dried over MgSO4. After filtration the
mixture was concentrated in vacuum. Purification over silica gel
(EtOAc/hexane, 20:80–30:70) afforded 1 (0.38 g, 61%). Rf = 0.45
(EtOAc/hexane, 1:1). 1H NMR (CDCl3, 300 MHz): d 6.72 (s, 2H),
3.71 (m, 2H), 3.54 (m, 2H), 2.29 (m, 4H), 1.93 (m, 4H), 1.62 (m,
1H), 1.44 (m, 1H); 13C NMR (CDCl3, 75 MHz) d (ppm): 171.4,
169.2 (d), 134.7, 109.7 (d), 96.0, 93.5, 87.2 (d), 46.3 (d), 39.2,
37.5, 34.2, 29.3, 26.0, 21.1; MS (ES+ for C15H17FN2O3): m/z = 293
[M+H+], 315 [M+Na+], calcd 292.
3. Conclusion
The work presented herein proposes a novel targeting approach
for amine-containing anticancer agents, using the non-toxic B-sub-
unit of Shiga toxin (STxB) as a delivery tool. We used an efficient
synthetic strategy where protein integrity was preserved by
exploiting copper free Huisgen [3+2] cycloaddition. The catalyst-
free bioorthogonal reaction allowed to obtain very high yields of
coupling between the linkers and STxB. The conjugates STxB–Doxo
and STxB–MMAF demonstrated potent cytotoxic activity and
proved the versatility of the drug releasing mechanism of disulfide
bonds linked to carbamate systems. Our data also highlight that
the nature of carbamate and the structure of the anticancer agent
can influence the coupling yields between STxB and drug linker, for
instance by an imposed conformation. Finally, STxB–Doxo and
STxB–MMAF showed high selectivity and potency on Gb3-positive
HT29 cells, with IC50 values in nanomolar range. These conjugates
are now entering in vivo evaluation in mice.
4.2.2. 1.2-Bis(2-(2-azidoethoxy)ethyl disulfane (10)
To a solution of S-(2-(2-azidoethoxy)ethyl)ethanethiolate 9
(1.89 g, 10 mmol) in dry MeOH (75 mL) purged in argon for
30 min was added NaOMe (2.17 mL, 25% w/v, 10 mmol). The reac-
tion mixture was stirred under air overnight at room temperature.
DOWEX was added until pH 6. After concentration in vacuum, the
crude product was purified on a silica gel column (EtOAc/cyclohex-
ane, 10:90). Disulfide 10 was obtained as yellow oil (2.05 g, 70%).
Rf = 0.4 (EtOAc/cyclohexane, 10:90). 1H NMR (CDCl3, 300 MHz): d
3.76 (t, J = 6.5 Hz, 2H), 3.66 (t, J = 4.9 Hz, 2H), 3.40 (t, J = 6.5 Hz,
2H), 2.90 (t, J = 4.9 Hz, 2H). 13C NMR (CDCl3, 75 MHz): d 69.8,
69.5, 50.7, 38.4. MS (ESI for C8H16N6O2S2): m/z 293 [M+H+], calcd
292.
4. Experimental part
4.1. General chemistry methods
4.2.3. tert-Butyl(2-((2-(2-azidoethoxy)ethyl)disulfanyl)ethyl)
(methyl)carbamate (12)
Reactions were performed under inert atmosphere (nitrogen)
unless otherwise stated. THF was distilled from sodium and ben-
zophenone, CH2Cl2 was distilled from P2O5. DMF and methanol
were stored over molecular sieves. Commercial products were
used without further purification. TLC (0.25 mm silica gel 60-F
plates) was used to follow the reaction. Visualization was accom-
plished with UV light (254 nm), ammonium molybdate or ninhy-
drin. Flash chromatographies were carried out on 320–400 mesh
size silica gel. 1H NMR and 13C NMR spectra were recorded on a
BRUCKER ACP 300 at respectively 300 MHz and 75 MHz. Chemical
shifts are reported in ppm relative to the residual solvent peak as
the internal reference, coupling constants J are given in Hertz. Spin
multiplicities are given with the following abbreviations: s = sin-
glet, d = doublet, dd = doublet of doublet, t = triplet, q = quadruplet,
m = multiplet. Positive and negative electrospray ionization spec-
tra were performed on a WATERS ZQ 2000. High resolution mass
spectra were obtained at the Laboratoire de Spectrométrie de
Masse of Chimie Paris Tech. Mass spectroscopy for compounds
containing a protein moiety was conducted on a Voyager-DE PRO
MALDI-TOF mass spectrometer (Applied Biosystems, Framingham,
USA) operated in the delayed extraction and linear mode. The
NaOMe (0.68 mL of 0.5 M in MeOH, 10 mmol) was added drop-
wise to a stirred solution of S-2-(tert-butoxycarbonyl(methyl)
amino)ethyl ethanethioate 11 (1.2 g, 5.13 mmol) and 10 (1.5 g,
5.13 mmol) in anhydrous MeOH (28 mL). The reaction was stirred
overnight under an argon atmosphere. The residue was purified by
flash chromatography over silica (EtOAc/Hexane, 1:1) to afford 12
as a clear, pale yellow liquid (1.24 g, 72%). Rf = 0.4 (EtOAc/cyclohex-
ane, 1:1). 1H NMR (CDCl3, 300 MHz): d 3.76 (t, J = 6.5 Hz, 2H), 3.66
(t, J = 4.8 Hz, 2H), 3.47 (t, J = 6.6 Hz, 2H), 3.39 (t, J = 6.6 Hz, 2H), 2.90
(t, J = 6.5 Hz, 2H), 2.89 (s, 3H), 2.82 (m, 2H), 1.44 (s, 9H). 13C NMR
(CDCl3, 75 MHz): d 155.5, 80.0, 70.1, 69.9, 69.8, 51.0, 48.75, 43.8,
38.7, 38.6 36.8, 36.6, 35.2, 30.5, 28.8, 27.2. MS (ESI for C12H24N4O3-
S2): m/z 337 [M+H+], calcd 336.
4.2.4. 2-((2-(2-Azidoethoxy)ethyl)disulfanyl)-N-methylethan
aminium 2,2,2 trifluoroacetate (13)
TFA (5 mL) was added to a solution of 12 (1.2 g, 5.06 mmol) dry
DCM (10 mL). The yellow solution was stirred at room temperature
for 1 h. The mixture was evaporated under vacuum. The resulting
product was used without further purification. Rf = 0.0 (acetone/
DCM, 10:90). 1H NMR (CDCl3, 300 MHz): d 3.69 (t, J = 6.6 Hz, 2H),

7156
V. Kostova et al. / Bioorg. Med. Chem. 23 (2015) 7150–7157
3.57 (t, J = 4.8 Hz, 2H), 3.33 (t, J = 6.5 Hz, 2H), 3.26 (t, J = 6.5 Hz, 2H),
2.87 (t, J = 6.6 Hz, 2H), 2.82 (m, 2H), 2.77 (s, 3H). 13C NMR (CDCl3,
75 MHz): d 69.9, 68.0, 50.0, 47.2, 37.1, 33.0, 32.7, 32.1. MS (ESI
for C7H16N4OS2): m/z 237 [M+H+], calcd 236.
MeOH (32 mL) was stirred at room temperature under argon
atmosphere for 1 h. The solution was quenched with AcOH and
evaporated. Then 2-(pyridin-2-yldisulfanyl)ethanol 21 (580 mg,
3.1 mmol) was added to the intermediate diluted in MeOH
(35 mL) and the mixture was stirred overnight at room tempera-
ture. After evaporation, the crude product was purified by column
chromatography over silica (AcOEt/cyclohexane, 10:90–20:80) to
afford product 18 (601 mg, 79%). Rf = 0.51 (Acetone/cyclohexane,
1:1). 1H NMR (CDCl3, 300 MHz): d 3.89 (t, J = 5.7 Hz, 2H), 3.76 (t,
J = 6.6 Hz, 2H), 3.65 (t, J = 4.8 Hz, 2H), 3.39 (t, J = 5.1 Hz, 2H) 2.88
(m, 4H). 13C NMR (CDCl3, 75 MHz): d 69.7, 69.5, 60.2, 50.6, 41.3,
38.2. MS (ESI for C6H13N3O2S2): m/z 224 [M+H+], calcd 223.
4.2.5. 4-(((tert-Butyldimethylsilyl)oxy)methyl)phenyl(2-((2-(2-
azidoethoxy)ethyl)disulfanyl)ethyl)(methyl)carbamate (14)
Triethylamine (1.3 ml, 9.28 mmol) was added dropwise to a
solution of 13 (1.1 mg, 4.64 mmol) in DMF (10 mL) followed by
addition of 4-((tert-butyl dimethyl silyl)oxy)methyl)phenyl(4-
nitrophenyl)carbonate 22 (936 mg, 2.32 mmol). The mixture was
agitated overnight at room temperature. After the evaporation,
the crude product was purified on silica gel (acetone/DCM,
0:100–8:92) to afford colorless oil 14 (1.00 g, 87%). Rf = 0.3 (ace-
4.2.10. 2-((2-(2-Azidoethoxy)ethyl)disulfanyl)ethyl(4-nitrophe-
nyl)carbonate (19)
tone/DCM, 5:95). 1H NMR (CDCl3, 300 MHz):
d 7.32 (d,
J = 15.9 Hz, 2H), 7.08 (d, J = 15.9 Hz, 2H), 4.71 (s, 2H), 3.75 (t,
J = 6.6 Hz, 2H), 3.65 (m, 2H), 3.63 (m, 3H), 3.37 (m, 2H), 3.15–
3.05 (s, 3H), 2.95 (m, 2H), 2.91 (m, 2H), 0.93 (s, 9H), 0.08 (s, 6H).
13C NMR (CDCl3, 75 MHz): d 150.7, 127.9, 126.1, 121.9, 115.6,
69.8, 64.9, 50.6, 38.0, 33.1, 29.16, 0.05. MS (ESI for C21H36N4O4S2-
Si): m/z 501 [M+H+], calcd 500.
Pyridine (421 lL, 5.212 mmol) and 4-nitrophenylchloroformate
(787 mg, 3.9 mmol) were added to solution of 18 (582 mg,
2.61 mmol) in DCM (10 mL). After overnight stirring at room tem-
perature, the residues were purified over silica gel (DCM, 100%) to
give product 19 (765 mg, 75%). Rf = 0.32 (acetone/cyclohexane,
20:80). 1H NMR (CDCl3, 300 MHz): d 8.29 (d, J = 8.9 Hz, 2H), 7.40
(d, J = 9.3 Hz, 2H), 4.55 (t, J = 6.6 Hz, 2H), 3.76 (t, J = 6.6 Hz, 2H),
3.66 (d, J = 4.8 Hz, 2H), 3.39 (d, J = 5.1 Hz, 2H) 3.03(t, J = 6.6 Hz,
2H), 2.94 (t, J = 6.6 Hz, 2H). 13C NMR (CDCl3, 75 MHz): d 156.4,
153.5, 146.8, 125.7, 122.1, 70.9, 69.7, 67.3, 51.0, 38.9, 36.9. MS
(ESI for C13H16N4O6S2): m/z 389 [M+H+], calcd 388.
4.2.6. 4-((((4-Nitrophenoxy)carbonyl)oxy)methyl)phenyl(2-((2-
(2-azidoethoxy)ethyl)disulfanyl)ethyl)(methyl)carbamate (15)
HCl 1% (64 lL) was added to a solution of 14 (1 g, 2.02 mmol) in
EtOH (6.3 mL). The mixture was stirred for 1 h at room tempera-
ture. After evaporation the residue (296 mg, 0.77 mmol) were
diluted in anhydrous DCM (15 mL). To the solution was added
4.2.11. 2-((2-(2-Azidoethoxy)ethyl)disulfanyl)ethyl carbonyle
mono-methylausristatin F (4)
dropwise triethylamine (310 lL, 2.3 mmol) and 4-nitrophenylchlo-
roformate (309 mg, 1.53 mmol). The mixture was stirred for 2 h at
room temperature and purified over silica (EtOAc/cyclohexane,
30:70) to afford compound 15 (280 mg, 66%). Rf = 0.6 (EtOAc/cyclo-
hexane, 10:90). 1H NMR (CDCl3, 300 MHz): d 8.28 (d, J = 9.3 Hz,
2H), 7.46 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 9.3 Hz, 2H), 7.18 (d,
J = 8.4 Hz), 5.27 (s, 2H), 3.75 (t, J = 6.9 Hz, 2H), 3.65 (q, J = 4.2 Hz,
2H), 3.640 (t, J = 5.1 Hz, 2H), 3.38 (q, J = 4.2, 2H), 3.16–3.05 (s,
3H), 2.94 (m, 2H), 2.91 (m, 2H). 13C NMR (CDCl3, 75 MHz): d
155.4, 154.2, 152.1, 151.8, 145.5, 131.0, 130, 125.3, 122.1, 70.3,
69.7, 69.4, 50.3, 48.9, 48.4, 38.1, 36.0. MS (ESI for C22H25N5O8S2):
m/z 551 [M+H+], calcd 552.
Compound 19 (18 mg, 0.046 mmol), MMAF (17 mg,
0.023 mmol), triethylamine (6.8
0.029 mmol) were stirred in THF (600
temperature. After evaporation, the crude product was purified
by chromatography (MeOH/DCM, 0:100–6:94) to afford
(16 mg, 71%). Rf = 0.26 (MeOH/DCM, 5:95). MS (MALDI-TOF+ for
46H76N8O11S2): m/z 1003 [M+Na+], 1019 [M+K+], calcd 980.
lL, 0.050 mmol) and HOBt (4 mg,
lL) overnight at room
4
C
4.3. Coupling with STxB
The coupling reactions were performed with genetically
engineered STxB/Cys containing five C-terminal cysteine residues
purified according to established procedures.36 MALDI-TOF mass
spectrometry was used to follow the formation of STxB-based con-
jugates with error in the range of 5 Da. LC–MS analysis was used
to determine the coupling yield. The cyclooctyne and prodrug com-
pounds were dissolved in DMSO (10% DMSO final concentration in
the reaction volume), and STxB/Cys diluted in a PBS buffer to a final
concentration of 1 mg/mL. To determine the optimal reaction con-
ditions STxB was incubated with tree different molar excess (1, 3
and 9) of cyclooctyne or prodrug per B-fragment monomer (note
that STxB is a homopentamer of 5 B-fragments). Coupling reactions
were carried out for 18 h at 21 °C with stirring, and the conjugates
were dialyzed (10 kDa cut-off) for 3–24 h at room temperature or
4 °C, against water for MALDI analysis, or PBS for in vitro cytotox-
icity testing.
4.2.7. 4-((Carbamoyloxy)methyl)phenyl(2-((2-(2-azidoethoxy)
ethyl)disulfanyl)ethyl)(methyl)carbamate doxorubicin (3)
To a mixture of 15 (20 mg, 0.036 mmol) and doxorubicin
(12 mg, 0.022 mmol) in DMF (1 mL) was added pyridine (3
lL,
0.037 mmol) and triethylamine (3 L, 0.022 mmol). The solution
l
was stirred overnight at room temperature. After evaporation the
residue was purified over silica gel (MeOH/DCM, 0:100–3:97) to
give product 3 (17 mg 81%). Rf = 0.31 (acetone/cyclohexane, 1:1).
MS (MALDI-TOF+ for C43H49N5O16S2): m/z 978 [M+Na+], 994 [M
+K+], calcd 956.
4.2.8. 4-((Carbamoyloxy)methyl)phenyl(2-((2-(2-azidoethoxy)
ethyl)disulfanyl)ethyl)(methyl)monomethylauristatin F (23)
Triethylamine (6.5
0.016 mmol), MMAF (12 mg, 0.016 mmol) in anhydrous DMF
(800 L). The solution was stirred at room temperature overnight.
lL, 0.048 mmol) was added to 15 (8.8 mg,
STxB–MFCO, 7: m/z = 8085 (calcd), 8085 (found); STxB:
m/z = 7793 (calcd), 7792 (found).
l
After evaporation the crude product was purified over silica gel
(MeOH/DCM, 0:100–8:92) to obtain compound 23 (11 mg, 60%).
Rf = 0.23(MeOH/DCM, 10:90). MS (MALDI-TOF+ for C55H85N9O13S2):
m/z 1166 [M+Na+], calcd 1133.
STxB–biotin, 8: m/z = 8529 (calcd), 8531 (found); STxB:
m/z = 7793 (calcd), 7794 (found).
STxB–Doxo, 16: m/z = 9040 (calcd), 9042 (found); STxB:
m/z = 7793 (calcd), 7793 (found).
STxB–MMAF, 17: m/z = 9229 (calcd), 9334 (found); STxB:
m/z = 7793 (calcd), 7796 (found).
STxB–MMAF, 20: m/z = 9065 (calcd), 9069 (found); STxB:
m/z = 7793 (calcd), 7795 (found).
4.2.9. 2-((2-(2-Azidoethoxy)ethyl)disulfanyl)ethanol (18)
A solution of S-2-(2-azidoethoxy)ethyl ethanethioate 9 (648 mg,
3.42 mmol) and sodium methoxide (0.5 M, 3 mL) in anhydrous

V. Kostova et al. / Bioorg. Med. Chem. 23 (2015) 7150–7157
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Supplementary data
36. Mallard, F.; Johannes, L. Shiga Toxin B-Subunit as a Tool to Study Retrograde
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References and notes
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