Platinum-Triggered Bond-Cleavage of Pentynoyl Amide and N-Propargyl Handles for Drug-Activation

Article abstract of DOI:10.1021/jacs.0c01622

The ability to create ways to control drug activation at specific tissues while sparing healthy tissues remains a major challenge. The administration of exogenous target-specific triggers offers the potential for traceless release of active drugs on tumor sites from antibody-drug conjugates (ADCs) and caged prodrugs. We have developed a metal-mediated bond-cleavage reaction that uses platinum complexes [K2PtCl4 or Cisplatin (CisPt)] for drug activation. Key to the success of the reaction is a water-promoted activation process that triggers the reactivity of the platinum complexes. Under these conditions, the decaging of pentynoyl tertiary amides and N-propargyls occurs rapidly in aqueous systems. In cells, the protected analogues of cytotoxic drugs 5-fluorouracil (5-FU) and monomethyl auristatin E (MMAE) are partially activated by nontoxic amounts of platinum salts. Additionally, a noninternalizing ADC built with a pentynoyl traceless linker that features a tertiary amide protected MMAE was also decaged in the presence of platinum salts for extracellular drug release in cancer cells. Finally, CisPt-mediated prodrug activation of a propargyl derivative of 5-FU was shown in a colorectal zebrafish xenograft model that led to significant reductions in tumor size. Overall, our results reveal a new metal-based cleavable reaction that expands the application of platinum complexes beyond those in catalysis and cancer therapy.

Full text of DOI:10.1021/jacs.0c01622

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Article
Platinum-triggered Bond-cleavage of Pentynoyl
amide and N-propargyl handles for Drug-Activation
Bruno L. Oliveira, Benjamin J. Stenton, Unnikrishnan V. B., Cátia Rebelo de Almeida, João
Conde, Magda Negrão, Felipe Silveira de Souza Schneider, Carlos Cordeiro, Miguel Godinho
Ferreira, Giovanni F. Caramori, Josiel B Domingos, Rita Fior, and Gonçalo J.L. Bernardes
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c01622 • Publication Date (Web): 26 May 2020
Downloaded from pubs.acs.org on May 26, 2020
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Platinum-triggered Bond-cleavage of Pentynoyl amide and N-propar-
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Bruno L. Oliveira,1,2‡* Benjamin J. Stenton, Unnikrishnan V. B., Cátia Rebelo de Almeida, João Conde, Magda
1‡
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Negrão, Felipe S. S. Schneider, Carlos Cordeiro, Miguel Godinho Ferreira, Giovanni F. Caramori, Josiel B
Domingos, Rita Fior and Gonçalo J. L. Bernardes
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3*
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Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028, Lisboa,
Portugal
Champalimaud Centre for the Unknown, Champalimaud Foundation, Av. Brasilia, 1400-038 Lisboa, Portugal
4 Department of Chemistry, Federal University of Santa Catarina - UFSC, Campus Trindade, Florianopolis - SC, 88040-900, Brazil
Laboratório de FT-ICR e Espectrometria de Massa Estrutural, Faculdade de Ciências da Universidade de Lisboa, Campo-Grande,
3
́
5
1
749-016 Lisboa, Portugal
6
Institute for Research on Cancer and Aging of Nice (IRCAN), INSERM U1081, UMR7284 CNRS, 06107 Nice, France
ABSTRACT: The ability to create ways to control drug activation at specific tissues while sparing healthy tissues remains a major challenge.
The administration of exogenous target-specific triggers offers the potential for traceless release of active drugs on tumor sites from antibody-
drug conjugates (ADCs) and caged prodrugs. We have developed a metal-mediated bond-cleavage reaction that uses platinum com-
plexes[K
2
PtCl or Cisplatin (CisPt)] for drug activation. Key to the success of the reaction is a water-promoted activation process that triggers
4
the reactivity of the platinum complexes. Under these conditions the decaging of pentynoyl tertiary amides and N-propargyls occurs rapidly in
aqueous systems. In cells, the protected analogues of cytotoxic drugs 5-fluorouracil (5-FU) and monomethyl auristatin E (MMAE) are partially
activated by non-toxic amounts of platinum salts. Additionally, a non-internalizing ADC built with a pentynoyl traceless linker that features a
tertiary amide protected MMAE was also decaged in the presence of platinum salts for extracellular drug release in cancer cells. Finally, CisPt-
mediated prodrug activation of a propargyl derivative of 5-FU was shown in a colorectal zebrafish xenograft model that led to significant reduc-
tions in tumor size. Overall, our results reveal a new metal-based cleavable reaction that expands the application of platinum complexes beyond
those in catalysis and cancer therapy.
1. INTRODUCTION
developed with an array of applications that range from in situ acti-
12–15
The targeting of potent drugs with tumor-specific ligands is a eessen-
tial feature of drug delivery and cancer therapy. Notable in this field
vation of prodrugs to the gain-of-function study on proteins.
1
Although the recent results with non-internalizing ADCs for click-
triggered drug release show promise, there are several issues that re-
main to be improved, such as the lack of tumor-selectivity of the
chemical triggers or their short in vivo retention times that may result
in reduced release of the cytotoxic payloads and thus lower efficacy.
Additionally, in such applications the tumor payload concentration
is determined by the cell-surface antigen expression, which in some
are antibody-drug conjugates (ADCs) that use an antibody to
transport a drug to cancerous cells and endogenously release it by
hydrolysis (low pH, reduction of disulfide bonds) or by proteolysis
2,3
(
e.g. cathepsin B protease). Although the cleavage of ADC linkers
with endogenous triggers is the simplest method for drug release, ex-
ternal small-molecule triggers for extracellular drug release may be
more advantageous because they avoid any disparity in cleavage
rates caused by variable biology across subjects, and drug release is
7, 16
cases may be too low to achieve a useful therapeutic response.
This contrasts with the use of internalizing ADCs that have an accu-
mulative effect inside the tumor cells.
Metal-mediated decaging of prodrugs has been more extensively re-
ported than small-molecule-mediated decaging. Unlike chemical
triggers, transition metals can be catalytic, which allows their use in
sub-stoichiometric amounts. In these cases, only very small amounts
of catalytic metal are required to achieve the desired pharmacologic
effect, thereby reducing toxicity and side reactions.
was recently demonstrated by Weissleder and co-workers by using
palladium nanoparticles that accumulate in tumor cells and serve as
cellular catalysts for the activation of different model prodrugs and
4–7
7, 16–17
not dependent on the concentrations of cellular triggers. In fact,
ADCs built with protease cleavable linkers for drug release have
been shown recently to not depend on the cathepsin B protease
18
4
function for efficient and targeted cancer-cell killing. The promise
of controlled prodrug activation has fueled research into new trig-
gers that enable bond-cleavage reactions to unleash bioorthogonal
protecting groups, which deactivate otherwise potent drugs.
Robillard and co-workers pioneered the development of tetrazine-
triggered drug delivery from ADCs. By using a non-internalizing
ADC consisting of a diabody conjugated to trans-cyclooctene-linked
drug monomethyl auristatin E (MMAE), the allylic carbamate-
containing linker can rapidly react with a tetrazine through an in-
8
19–20
This feature
9
10
21
resulted in tumor growth inhibition.
Palladium-mediated decaging is indeed the most studied method for
prodrug activation, which relies on the cleavage of terminal propar-
10,11
verse-electron-demand Diels–Alder reaction.
The drug is re-
leased within the extracellular tumor environment and showed effi-
gylic and allylic carbamates moieties introduced into small molecule
10
21–26
cacy in delaying tumor growth in xenograft mice models. Other
chemical- or light-mediated decaging reactions have also been
drugs.
Recently, our group developed an internal bifunctional
thioether propargyl carbamate linker with a conjugating unit for
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protein modification and MMAE for palladium-mediated drug re-
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lease from a nanobody-drug conjugate in cellular systems. Other
28–30 31–32
metals, such as ruthenium
and gold,
have been also explored
for cleavage and drug release. One of the latest additions to this field
33
was the recent report by Peng Chen and co-workers, on a copper-
releasable reaction for protein gain-of-function and drug activation.
Together these examples highlight the potential of metal-mediated
cleavage as a means to achieve controlled and chemically defined
drug release.
Whereas the utility of the above-mentioned metals for decaging ap-
plications has been extensively demonstrated, other metals have not
yet been sufficiently explored. For instance, platinum is widely used
in catalysis, but has found few applications in chemical biology pos-
sibly as a result of its intrinsic cytotoxicity. However, in the context
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Scheme 1. Platinum-mediated Bioorthogonal Bond Cleavage.
34
Secondary amines protected in the form of a tertiary pentynoyl am-
ide (top) or N-propargyl (bottom) can be selectively deprotected by
platinum reagents like chemotherapeutic drug CisPt. This strategy
was explored for drug activation of the protected MMAE and 5-FU
drugs and extended for drug release from an ADC in cancer cells. Ul-
timately, CisPt-mediated activation of a “5-FU-propargyl prodrug”
was evaluated in a zebrafish xenograft model for treatment of colo-
rectal cancer.
of cancer therapy we hypothesized that the use of platinum com-
35–36
plexes [e.g. Cisplatin (CisPt) used in the clinic]
as catalysts for
cleavage reactions could be propitious for bioorthogonal activation
of prodrugs in tumor cells.
“
Bioorthogonal” is perhaps unfitting terminology for a compoun37d–
known to react with water, nucleic acids, amino acids and proteins.
38
However, CisPt is one of the most commonly used chemotherapy
39–40
2. RESULTS AND DISCUSSION
drugs, being used to treat up to 20% of cancer patients.
CisPt was
Engineering of a Platinum-mediated Decaging Reaction. From
studies on the reactivity of platinum complexes, it is apparent that
platinum shares many of its reactions with similar complexes of
deemed a suitable reagent for the development of a drug decaging
reaction because it is highly reactive (half-life in humans of »30
4
1–42
min),
accumulates in the tumor and most importantly, is not pre-
48
43–44
gold. We therefore searched the literature for reactions with Au and
Pt that would function at room temperature, in aqueous media and
with likely fast kinetics to adapt for CisPt-mediated bioorthogonal
decaging reactions. The cyclization of 4-pentynoic acid is well
known to proceed quickly in aqueous media with reaction times
sent in human biology.
In this way, the activation of prodrugs at
the tumor site when the chemical trigger has already accumulated
may represent a major achievement. It may be conceivable for metal
4
1, 45–46
concentrations to reach 0.25 to 3.7 µgs per g of tumor.
For a 1
3
cm tumor (approximately 1 g wet weight) the concentration of
4
9–50
ranging from minutes to a few hours
and has even been demon-
47
CisPt is estimated to be 0.83–12.3 µM.
strated with platinum (II and IV) anti-cancer complexes (Figure
a). Given the previous studies, a metal-catalyzed mechanism was
Therefore, we were interested in investigating new biorthogonal
cleavage reactions catalyzed by platinum for applications in prodrug
activation. Here, we demonstrate that pentynoyl tertiary amide and
N-propargyl handles introduced into small-molecule drugs are suc-
cessfully decaged in aqueous solution and cell media by using non-
toxic amounts of platinum salts (Scheme 1). This strategy was suc-
cessfully applied to small molecule prodrug activation (MMAE and
51
1
devised whereby a carbamate carbonyl could be used as an internal
nucleophile to cause carbocyclisation followed by release of a sec-
ondary amine (Figure 1b). Working on this hypothesis, we synthe-
sized the terminal propargyl carbamate 3a (Figure 1c) to verify if
the carbonyl could act as a nucleophile and attack the alkyne to sub-
sequently release morpholine 6a in the presence of K
served conversions of 20 and 61% for reactions carried out in
O/CD OD (3:1) with 0.1 and 2 equiv. of metal salt, respectively
Figure 1d, Entry 1 and 2; Figures S1 and S2). Similar yields were
found for the reaction with NaAuCl (Figure S3). In contrast, if an
2 4
PtCl . We ob-
5-FU), and further extended to drug release from a non-internalizing
ADC, in cancer cells. Finally, we show that CisPt-mediated bond
cleavage can be used to activate a 5-FU prodrug in a zebrafish xeno-
graft model for treatment of colorectal cancer.
D
2
3
(
4
aliphatic carbamate with no propargyl handle is used (compound
S1, Supporting Information) under the same decaging conditions
the free amine is not released (Figures S4 and S5, respectively).
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Figure 1. Platinum-mediated Decaging Reaction Engineering. a. The cyclization of 4-pentynoic acid is known to proceed rapidly in aqueous
media with gold and platinum complexes. b. The proposed reaction uses a carboxamide as an internal nucleophile that cyclizes and displaces
the secondary amine leaving group, which could be a drug or a fluorophore. c. Model compounds with alkyne amide or carbamate were used
1
1
to survey the decaging reaction. d. Efficiency of the cleavage reaction under different conditions was assessed by H NMR spectroscopy. e. H
NMR spectroscopy of the decaging of the tertiary amide 4a in the presence of a catalytic amount of K
2
PtCl . The reaction generates a cyclized
4
1
intermediate that undergoes hydrolysis to release morpholine 6a. General procedure for determining decaging conversion by H NMR spec-
troscopy: carbamate and amide compounds (10 mgs) were dissolved in MeOD (0.2 mL) and metal complexes (0.1 or 2 equiv.) were added in
D O (0.6 mL) at room temperature in an open vessel for 14 h. The reactions were transferred to an NMR spectroscopic tube and sealed. Con-
2
version was calculated based on the relative ratios of methylene peaks resulting from the starting material and the released amine product.
Numerical data are the mean of 2 or 3 replicates.
According to these observations, it should also be possible to decage
tertiary amides to release secondary amines (Figure 1c). This was
an attractive prospect because amides are often much more stable
than their corresponding carbamates. Decaging of pentynoyl ter-
tiary amide 4a was monitored by NMR spectroscopy over time and
was proved to have comparable rates and yields to the corresponding
carbamate 3a (Figure 1d, Entry 4; Figure 1e). Importantly, the re-
action proceeds with sub-stoichiometric amounts of the metal com-
plex (Figure 1d, Entry 3; Figure S6). The reactions were also suc-
S2) was reacted with K
2
PtCl
4
and NaAuCl under similar conditions
4
(Figures S9,10). We found that the reaction proceeds with much
lower extends of decaging, likely due to the amide nitrogen compet-
ing as a nucleophile to yield a stable cyclized product, and thus a
smaller yield of released amine.
52
Mechanistic and Kinetic Studies of the Platinum-mediated
Decaging Reaction. To further study the platinum decaging reac-
tion, the pentynoyl tertiary amide was conjugated to a naph-
thalimide-based fluorophore to generate fluorescent quenched
cessfully trialed with K
2
PtCl as a representative Pt(IV) species (Fig-
6
ure 1d, Entries 5 and 6; Figure S7) and NaAuCl
4
(Figure S8) with
probe 7 (Figure 2a, see Supporting Information for synthetic de-
53–54
good yields.
tails).
The reaction was then monitored by the increase in fluo-
Overall, these results are important because they demonstrate a
decaging reaction of stable protected tertiary amides by using sub-
stoichiometric amounts of platinum complexes that could function
in water, open air, and without need of extreme temperatures or
complex ligands. It is important to note that even after all starting
material has been consumed (as evidenced by loss of terminal alkyne
proton), not all of it has decomposed to release the amine (Figure
rescence upon removal of the protecting group to form fluorescent
probe 8. With 50 equiv. of K PtCl or CisPt, we found that the fluo-
rescence was restored over a period of 200 min for K PtCl and 300
min for CisPt (Figure 2b), with complete consumption of 7 and for-
mation of corresponding “turned ON fluorophore”, as indicated by
LC-MS analysis (Figure S11). For both metals the conversion was
accompanied by an initial steady state followed by a marked increase
of the fluorescence, which suggested the formation of an activated
intermediate. Indeed, it is known that platinum complexes form a se-
ries of reactive intermediates by successive replacement of the
2
4
2
4
1
e). This lack of conversion is likely due to side reactions, and nucle-
ophilic attack on the alkyne, which seems the most plausible mecha-
nism. To elucidate this a pentynoyl secondary amide (compound
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PtCl
5
5–57
chloro ligands by water or hydroxyl groups.
We hypothesized
the turn-on half-time from t1/2 = 171 min to t1/2 = 30 min for K
2
4
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that formation of such an aqua intermediate early on could be re-
sponsible for the activation of the platinum complexes. This hypoth-
esis was verified by using LC-MS studies to follow formation of
and from t1/2 = 276 min to t1/2 = 60 min for “CisPt” (Figure 2c and
Table S1). Accordingly, if the reaction was performed in pure DMF,
formation of the decaged probe was not observed (50 equiv. of
2 4
K PtCl - and CisPt-aqua complexes over time, which occurred
K
2
PtCl or CisPt for 14 h at 37 °C). This result is in agreement with
4
within 6 h (Figures S12,13). Consistent with this hypothesis, plati-
num salts failed to form the aqua complexes when incubated in the
presence of N,N-dimethylformamide (DMF). Based on these obser-
vations we further studied the kinetics of the releasing reaction after
formation of the aqua complexes (6 h in water/DMF at 37 °C). As
expected, activation of the platinum salts significantly accelerated
previous LC-MS studies that suggested the requirement of water to
generate the active catalyst. The activation of metal chloride in aque-
ous solvents has few precedents but has been reported for gold com-
58
plexes. This effect is explained by facilitated ionization of the M–
Cl bonds in water.
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Figure 2. Examination of the Platinum-catalyzed Bioorthogonal Cleavage Reaction. a. Naphthalimide -based fluorogenic probes were
used to study the cleavage efficiency of the platinum reaction for decaging alkyne-containing molecules. The caged naphthalimide derivatives
exhibited high stability in solution and cell media and their quenched fluorescence could be reactivated upon removal of the caging group (λex
=
445 nm, λem = 545 nm). b. Changes in fluorescence intensity during the time course of the decaging reaction between fluorogenic probe 7
and platinum salts (K PtCl /CisPt). c. Determined half-time for the reaction of 7 with activated and non-activated platinum salts. d. Decaging
2
4
kinetics for the pentynoyl amide fluorophore. Rate constants were determined under pseudo first order conditions with a 50 μM final concen-
tration of probe 7 and 10–50 equiv. of aqua platinum metals. e. Kinetics profiles of the decaging reaction in the presence of the metal poisons
CS and EDTA. Error bars represent ± s.d. (n = 3). All experiments were repeated 3 independent times. f. Calculated mechanism for the depro-
2
pargylation reaction catalyzed by Pt with model substrate 4a. Calculations were performed with an implicit solvent model for water. Geometries
and frequencies were calculated with the functional revPBE and, to obtain very accurate energetics, single point energy calculations with
DLPNO-CCSD(T) and counterpoise corrections were employed to suppress basis set superposition errors.
In terms of catalytic activity, the reaction of 7 with 0.3 equiv. of acti-
vated K PtCl complex yielded decaged probe 8 in 98% yield after
before. Finally, the compatibility and efficiency of the reactions were
tested under physiological conditions. The activated aqua com-
plexes were first shown to persist in complete DMEM cell media for
at least 16 h at 37 °C as assessed by LC-MS analysis, although a sig-
nificant decrease in their concentration was observed overtime (Fig-
ure S15). Later, the reactions were shown to proceed in cell media
2
4
72 h at 37 °C (catalyst turnover number 3.3). Upon moving to 2
equiv. of the metal complex, the decaged product was obtained in
quantitative yield after 4 h at 37 °C (Figure S14). As a comparison,
the same study was performed with Pd(OAc) , a standard palladium
2
59
complex for N-depropargylation. Interestingly, the Pd-reaction
proceeded with comparable efficiency, although with slightly better
rates of conversion (>98% yield in 1 h, LC-MS analysis). Of rele-
vance, palladium decaging of alkyne amides has never been reported
with conversions of 69% for K
2
PtCl (50 equiv.) and 17% for CisPt
4
(150 equiv.) after 14 h at 37 °C (Figure S16,17). Similarly, the re-
action was also trialed in high salt concentration buffers with high
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efficiency (t1/2 = 36 min for 50 equiv. of K
2
PtCl
4
and t1/2 = 105 min
based assay was also employed to calculate the second-order rate
constant for the reaction. Accordingly, the calculated rate constant
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for 100 equiv. of CisPt, 37 °C in E3 medium, Figure S18,19).
–1
–1
–1 –1
Having found an efficient platinum complex for decaging pentynoyl
tertiary amides, we turned our attention to the determination of the
rate constant of the reaction (Figure 2d). By fitting the appearance
of 8 in the presence of increasing amounts of metal complexes and
by using pseudo-first order conditions, the reactions were found to
was 0.120 ± 0.001 M s for K
2
PtCl
4
and 0.0160 ± 0.0004 M s for
CisPt (Figure S27). These results show that N-propargyls decage
slower than pentynoyl amides. As a reference, the same study was
performed with palladium complex Pd(OAc) , which behaved
2
slightly better than the platinum salts, to promote formation of 10
–1
–1
–1 –1
have second order rate constants of 0.230 ± 0.004 M s for K
2
PtCl
4
with a second-order rate constant of 0.39 ± 0.015 M s (Figure
–1 –1
and 0.080 ± 0.002 M s for CisPt (Figure 2d). These reaction rates
S28). The reaction was also subjected to CS
2
and EDTA poisoning.
are similar to those reported for other metal-assisted decaging reac-
CS had no effect but EDTA completely inhibited the reaction,
2
27
tions.
which indicates the participation of Pt(II) species (Figure S29 Ta-
ble S4). Based on these results and LC-MS analysis after 2 h of reac-
0
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To determine the nature of active species involved in the decaging
reaction, we performed kinetic experiments with carbon disulfide
tion between K
first turnover of the reaction proceeds as recently disclosed for palla-
2
PtCl and probe 9 (Figure S30), we propose that the
4
(
Figure 2e, Table S2). CS acts as a catalyst poison for homogene-
2
59
ous and heterogeneous Pt(0) reactions, although Pt(II) species are
unaffected. As seen in Figure 2e, the reaction rates are similar with
dium depropargylation, i.e. (i) co-ordination of Pt(II) to alkyne
moiety, (ii) attack of a H O molecule at the propargyl terminal car-
2
2
or without CS . This result can be attributed to the non-involvement
bon to form an enol, (iii) tautomerization to a more stable Pt-alde-
hyde complex and (iv) C–N bond cleavage by either hydrolysis or
beta-N elimination followed by hydration of Pt-complex (Figure
S31). Finally, we investigated the ability of platinum salts to remove
the propargyl protecting group in cells (DMEM) and zebrafish (E3)
media. The reaction with the fluorogenic probe was monitored for
of Pt(0) species in the reaction. However, the reaction rates were
significantly affected by the addition of ethylenediamine tetraacetic
acid (EDTA; Figure 2e), possibly due the participation of Pt(II) in
the reaction.
We also performed computational studies to help understand the re-
action mechanism (Figure 2f). These studies suggest that the most
probable operating reaction pathway of substrate 4a is a stepwise
process involving the coordination of substrate molecule to Pt(II),
followed by an intra-molecular attack of the carbonyl oxygen of the
K
2
PtCl
generally high with the reaction complete in 60 min and 150 min for
PtCl and CisPt, respectively (Figure S32). In DMEM, cleavage
was less efficient with conversion yields of 67% for K PtCl (50
4
and CisPt for 14 h at 37ꢀ°C. Efficiencies in E3 media were
K
2
4
2
4
0
Pt-coordinated substrate (CS ) to the pentynoyl moiety, which
equiv.) and 30% for CisPt (150 equiv.) after 14 h at 37 °C (Figure
S33).
gives five-membered ring intermediate CP. Different pathways to
decomposition of CP were explored (Figure S20); the lowest en-
ergy one was the hydration of CP leading to formation of intermedi-
Platinum-mediated decaging in living cells. To verify whether
platinum-mediated depropargylation would function in cell culture,
a pentynoyl amide derivative of antineoplastic drug MMAE was syn-
thesized. MMAE is the drug present in the ADC brentuximab ve-
dotin that is in clinical use to treat patients with relapsed Hodgkin
ate Q , which readily decomposes to liberate free amine 6a. The
1
metal complex is then recovered in a subsequent step by hydrolysis
of 5 (Figure 2f). The complete calculation for the first reaction turn-
over of the main mechanism is depicted in Figure S21 and Movie.
This mechanism is further supported by the identification of inter-
60
lymphoma and systemic anaplastic large-cell lymphoma, and re-
mains the drug of choice for antibody-targeted therapies. In addi-
tion, a N-propargyl 5-fluorouracil (pFU) derivative was also tested,
which was found to be efficiently decaged and activated with gold
mediate species CS
0
by LC-MS (Figures S22-26). The main differ-
ence observed for the reaction with substrate 3a relative to 4a was
#
–1
the higher free energy of activation (∆∆G = 2.75 kcal mol ) for the
intra-molecular attack of the carbonyl oxygen at the pentynoyl moi-
ety. However, both substrates share the same energy barrier for hy-
dration of CP and release of 6a (Figure S21).
31
25
nanoparticles and palladium complexes. When MMAE-am was
treated in DMF/water (1:1) for 4 h with 10 equiv. of K PtCl , com-
2
4
plete consumption of MMAE-am was seen by LC-MS with 37% re-
lease of MMAE along with the formation of the intermediate Q1s
(Figures S20,34). In a similar fashion, decaging of pFU proceeds
Extending the Decaging Reaction to N-Propargyl Group. Fol-
lowing the discovery of a platinum-cleavable group, we hoped to ex-
tend the scope of handles that could be used for decaging. Metal-
mediated decaging of N-propargyl handles has been widely explored
to modulate the cytotoxic activity of antineoplastic drugs in a con-
with yields of 46% ± 2 and 72% ± 2 for K
2
PtCl and CisPt, after 14 h
4
reaction with 2 equiv., at room temperature and 37 °C, respectively
(Figures S35–37). These prodrugs (MMAE-am 11 and pFU 12,
see the Supporting Information for synthetic details) were reacted
with platinum salts in cell culture in the hope of observing a “turn-
on” of toxicity. Unfortunately, the chemotherapeutic CisPt has a
narrow window of non-toxic concentrations for efficient decaging in
22, 25
trolled manner.
On this basis, we investigated the possibility of
using N-propargyl groups introduced on drugs of interest for pro-
drug activation by using platinum triggers. Firstly, and similarly to
the pentynoyl amide reaction, an N-propargyl group was used to
protect a secondary amine on a naphthalimide derivative to generate
fluorogenic probe 9 (Figure 2a). As described above, we tested the
61
cells. Indeed, CisPt was demonstrated to be toxic in HeLa cells at
concentrations as low as 2.5 µM (Figure S38). On the contrary,
platinum salts K PtCl and K PtCl did not significantly influence
2
4
2
6
reactivity of K
2
PtCl
4
and CisPt before and after formation of the
the viability of HeLa cells at concentrations below 50 μM (Figure
S38). With both prodrugs, an increase of about two-fold in toxicity
could be observed for some of the tested concentrations when re-
acted with K PtCl over 3 days in cell culture (Figure 3a and 3b; e.g.
aqua complexes (6 h incubation at 37 °C in DMF/water). Once
again, dissociation of the chloride anions in water was found to be
crucial for triggering the reactivity of platinum complexes. Indeed,
we found that reactions with aqua complexes are faster according to
2
4
1
nM of MMAE-am and 50 µM of pFU). In contrast, no decrease of
cytotoxicity was observed in cells treated independently with cFU
3, a non decaging control derivative, or in combination with
PtCl (Figure 3b). These control studies indicate that 5-FU was
the calculated half-time for the “fluorescent reactions” (from t1/2
00 ± 3 min to t1/2 = 27 ± 3 min for K PtCl and from t1/2 = 628 ± 51
min to t1/2 = 303 ± 34 min for CisPt; Table S3). The fluorescence-
=
2
2
4
1
K
2
4
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0
not generated because the alkyl handle does not undergo decaging
by K PtCl
purpose because MMAE is a common payload in ADC design. Ide-
ally a CisPt-cleavable ADC would be stable to cleavage by endoge-
nous extra- or intra-cellular conditions. For this reason, we decided
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.
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to use a carbonyl acrylic bioconjugation handle
coupled to
MMAE for antibody modification (Figure 4a; SI for synthesis).
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Figure 3. Platinum-mediated Decaging in Cells. HeLa cells were
incubated with different concentrations of MMAE-am 11 a. or pFU
12 b. for 3 days with or without K
2
PtCl (20 µM, twice a day). Com-
4
pound 13, a non-decaging alkyl-FU derivative, was used as a negative
control. Toxicity was determined by AlamarBlue assay. Error bars
represent ± s.d. (n = 3). Each experiment was repeated three times.
The statistical significance of the differences between groups was
evaluated with the unpaired t-test. Statistical results: ns>0.05,
**P≤0.01, ***P≤0.001 and ****P≤0.0001.
It is important to note that for both prodrugs the addition of K
2
PtCl
4
did not restored their toxicity to the level observed for unmodified
MMAE and 5-FU drugs (Figures S39,40). Although a two-fold in-
crease in toxicity for the prodrug activation may look modest, it is
important to mention that this is considered relevant given the slow
reaction rates possible at the low concentration of K
2
PtCl complex
4
tolerated by cells. Indeed, this low reagent concentration was neces-
sary to ensure the platinum complex remained non-toxic. On top of
this, in vitro studies with probes 7 and 9 revealed that the presence
of nucleophiles (e.g. glutathione) ends in lower conversions into the
corresponding decaged products. It should be noted, however, that
even in the presence of high concentrations of glutathione (e.g. 1.5
mM) the reaction still proceeds with moderate rates (t1/2 = 197 min
Figure 4. Platinum-mediated Drug Decaging from a Non-inter-
nalizing ADC. a. Cysteine-selective and irreversible modification of
the non-internalizing antibody F16 (anti tenascin-C) in IgG format
with MMAE conjugating linker 14. IgG(F16) contains a single reac-
tive cysteine at the C-terminal extremity of the light chain ideal for
cysteine-specific modification. Briefly, a solution of F16 (7.1 µM) in
sodium phosphate buffer (NaP ) pH 7.4 was treated with 14 (40
i
for 7 + K
2
PtCl
4
; t1/2 = 246 min for 9 + K
2
PtCl ; Table S5). Regarding
4
equiv.) in MeCN to a final concentration of 10% v/v. The reaction
was heated to 37 ºC for 1 h and reaction progress was monitored by
CisPt, we found that the reaction is more susceptible to the presence
of nucleophiles (e.g. t1/2 = 921 min for 7 + CisPt in the presence of
0.5 mM of glutathione; Table S6). This deactivation of the metals in
the presence of nucleophiles is in line with the modest decaging
yields observed in the cell studies. This is an issue that could be fur-
ther improved, for example, by using platinum-based nanoparticles
known to have reduced toxicity and higher payload concentrations
or by using platinum complexes stabilized with different organic lig-
LC-MS. The ADC was purified by dialysis into fresh NaP
i
buffer pH
7.4 with a 10 kDa MWCO overnight. b. Deconvoluted ESI–MS
mass spectrum of the light-chain of F16. c. Deconvoluted ESI–MS
mass spectrum of the light-chain of F16-14 that shows an exact drug-
to-light-chain ratio of 1. d. Schematic of the platinum-mediated
decaging of MMAE from a non-internalizing ADC. e. Cell viability
of HeLa cells after treatment with F16-14 and subsequent decaging
62
ands in a way to optimize the metal reactivity. Our data, however,
demonstrate that decaging reactions with platinum complexes are
possible in cell culture and could achieve release of sufficient
amounts of the active drug in cells to induce cell death.
efficiency upon treatment with 20 µM K
2
PtCl , twice daily. Cell via-
4
bility was measured at day 3 by using AlamarBlue reagent. The sta-
tistical significance of the differences between groups was evaluated
by using the unpaired t-test. A p value < 0.05 (**) was considered
statistically significant. Error bars represent ± s.d. (n = 3). Experi-
ments were performed three times.
Platinum decaging of ADC. Next, we decided to extend the tertiary
amide caging group for chemically controlled drug-release from an
ADC. The caging group of MMAE-am 11 was adapted for this
6
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To test the susceptibility of the conjugating linker to platinum
decaging, compound 14 and K PtCl (10 equiv.) were incubated in
but the CisPt-treated group showed an increased fluorescence (Fig-
ure 5c). This implies that probe 9 and CisPt are tissue‐permeable
and capable of reacting in vivo.
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DMF/water (1:1) at 37 °C for 18 h and analyzed by LC–MS (Fig-
ure S41). Release of MMAE was observed with complete consump-
tion of 14 along with two potential intermediates (Figure S41). We
then went on and selected the non-internalizing F16 antibody for
modification, which is specific to the alternatively spliced A1 domain
65
of tenascin-C, found overexpressed in most solid tumors. A non-
internalizing ADC ensures that as little ADC as possible will be me-
tabolized by the cells and that the maximum possible drug release is
due to extracellular decaging with platinum complexes. Site-selec-
tive conjugation is expected to occur at the engineered cysteine res-
idues in each light-chain of F16 enabling the construction of a chem-
ically defined ADC. Furthermore, the newly formed C–S bond be-
tween the linker and the antibody is stable and does not undergo
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Figure 5. CisPt Decages the Fluorogenic Probe 9 in vivo.
Zebrafish larvae were exposed to 9 diluted in E3 medium for 24 h,
followed by a 1 h wash in E3 medium. Larvae were randomly distrib-
uted into two conditions: DMSO or CisPt for 24 h a. Confocal image
of zebrafish larvae exposed to 9 + DMSO b. and 9 + CisPt (c).
thiol-exchange reactions as in the case of frequently used malei-
63–64
mides.
Complete conversion to a homogenous ADC was
achieved after reaction of F16 for 1 h at 37 °C with the carbonyl
acrylic MMAE drug linker 14 in sodium phosphate buffer pH 7.4 as
assessed by LC-MS (Figure 4b,c). Importantly, the heavy chain re-
mained unmodified as expected considering the absence of reactive
cysteines in the structure (Figures S42,43). Next, we performed the
decaging in cells to release MMAE from the ADC (Figure 4d). With
a cancer cell line (HeLa cells) as a model, we found F16-14 to be
more toxic to cells at sub-micromolar concentrations in the presence
Before measuring efficacy of CisPt depropargylation, we assessed
the maximum tolerated concentration for each compound: pFU 12,
cFU 13, CisPt, pFU + CisPt and cFU + CisPt in non-tumor zebrafish
larvae (Figure S49). Next, colorectal cancer (CRC) HCT116
67
zebrafish xenografts were generated as previously described.
Briefly, 24 h post injection (hpi), xenografts were randomly distrib-
uted into different treatments: DMSO (control), pFU (1.65 mM),
cFU (1.65 mM), CisPt (0.034 mM), pFU + CisPt (1.65 mM + 0.034
mM) and cFU + CisPt (1.65 mM +0.034 mM). Xenografts were an-
alyzed at 4, 6 and 7 days post injection (dpi) i.e. 3, 5 and 6 days post
treatment (dpt), respectively (Figure 6). At 3dpt (4dpi) (Figure 6
I), in the single treatments with pFU or CisPt, we could not observe
any significant reduction of mitotic index (Figure 6 I, n), induction
of apoptosis (activated caspase 3, Figure 6 I, o) or reduction of tu-
mor size (Figure 6 I, p). In contrast, the combinatorial treatment –
pFU + CisPt – induced a significant anti-tumoral synergistic effect
of non-toxic amounts of the platinum complex K
2
PtCl (Figure 4d).
4
This tertiary amide decaging reaction should stimulate platinum-
mediated MMAE delivery from antibodies in the context of targeted
cancer therapeutics. Furthermore, a small model protein (ubiquitin-
66
K63C) engineered with a single cysteine residue was modified with
linker 14 for ease of analysis by LC-MS. When attempting decaging
in vitro with CisPt, loss of MMAE followed by further degradation of
the linker could be observed by LC-MS, which provides further evi-
dence for the efficient release of the secondary amine drug from the
protected tertiary amide protected conjugate (Figures S44-47).
manifested by a »2 fold increase in apoptosis (Figure 6 I, o; DMSO
vs pFU + CisPt **P = 0.0033; pFU versus pFU + CisPt ***P =
0.0006) accompanied by 25% reduction of tumor size (Figure 6 I,
Cisplatin-mediated prodrug decaging in vivo. To test the in vivo
p; DMSO versus pFU + CisPt *P = 0.0279; Figure 6 I, a versus d).
However, if the duration of the treatment is increased 2 (Figure 6
II) or 3 (Figure 6 III) additional days we could detect some toxicity
in single treatment (Figure 6 II, q – s; Figure 6 III, t - v). Neverthe-
less, the combination of pFU with CisPt induced a clear pronounced
anti-tumor synergistic effect. At 5 dpt (6 dpi), the combinatorial
treatment led to a reduction of proliferation (Figure 6 II, q; DMSO
versus pFU + CisPt ****P < 0.0001; pFU vs pFU + CisPt *P =
efficacy of pFU and its combinatorial effect with CisPt, we used the
67
zebrafish larvae xenograft model. This model is a fast in vivo plat-
form with resolution to analyze crucial hallmarks of cancer, such as
metastatic and angiogenic potentials but it is also highly sensitive to
discriminate differential anti-cancer therapy responses with single-
68–71
cell resolution.
We first attempted to visualize the CisPt reaction
by decaging fluorogenic probe 9 in larval zebrafish (Figure 5). This
probe shows an increase in fluorescence of 22-fold upon removal of
the propargyl group (Figure S48). For in vivo imaging, a set of
zebrafish larvae were incubated with probe 9 for 24 h, washed for 1
h in embryonic medium and then further incubated with dimethyl
sulfoxide (DMSO) or CisPt for 24 h (Figure 6a). Probe 9 and CisPt
were used at the highest non-toxic concentration to the zebrafish
0
.0104), a »4 fold increase in cell death by apoptosis (Figure 6 II, r;
DMSO versus pFU + CisPt ****P < 0.0001; pFU versus pFU + CisPt
*
***P < 0.0001) and a »38% reduction of tumor size (Figure 6 II, s;
DMSO versus pFU + CisPt ****P<0.0001; Figure 6 II, e versus h).
Finally, the 6 days of treatment (7 dpi) culminates in a »45% tumor
shrinkage (Figure 6 III, v; DMSO versus pFU + CisPt **P = 0.0010;
Figure 6 III, i versus l).
embryos (9, 1 µM; CisPt, 34 µM; Figure S49). As shown in Figure
5b, the control group displays nearly no background fluorescence,
7
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Figure 6. CisPt-mediated Prodrug Decaging in Zebrafish Xenografts. HCT116 human CRC cells were fluorescently labelled with lipo-
philic CM-DiI (shown in red) and injected into the perivitelline space (PVS) 2 days post-fertilization (dpf) Tg(Fli1:eGFP) zebrafish larvae.
Zebrafish xenografts were randomly distributed into treatment groups, daily treated with DMSO, CisPt, pFU and pFU+CisPt and analyzed at
4, 6 and 7dpi for proliferation, apoptosis and tumor size. At 4dpi, 6dpi and 7dpi zebrafish xenografts were imaged by stereoscope (a–m) and by
confocal microscopy (a’–m’ DAPI plus DiI, a’’–m’’ maximum projection of activated caspase 3). Proliferation (mitotic figures: n; q, *P=0.0104,
*
**P=0.0004, ****P<0.0001; t, **P=0.0023, *** P=0.0002), apoptosis (activated caspase 3: o, **P=0.0033, ***P=0.0006; r, *P=0.0126,
***P<0.0001; u, **P=0.0068) and tumor size (nº of tumor cells: p, *P = 0.0279; s, ****P<0.0001; v, *P=0.0411, **P=0.0010) were analyzed
*
and quantified. Graphs represent fold induction (normalized values to controls) Avg ± SEM. The number of xenografts analyzed is indicated
in the representative images and each dot represents one zebrafish xenograft. Statistical analysis was performed using an unpaired t-test. Statis-
tical results: ns>0.05, *P≤0.05, **P≤0.01, ***P≤0.001 and ****P≤0.0001. All images are anterior to the left, posterior to right, dorsal up, and
ventral down. Scale bar 50 µm.
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Importantly, by comparing the combined treatment of the non-
decaging compound cFU with CisPt to the prodrug pFU with CisPt
it is clear that pFU was able to induce a more significant cytostatic
yield. These results are suggestive of instability of the Pt complexes
probably by formation of bioinorganic complexes. Although the ac-
tive aqua Pt species have a limited lifetime in cell media, they persist
long enough to be partially effective.
Our work was conceived on the hypothesis that platinum complexes
could be used for prodrug activation on tumors during CisPt chem-
otherapy. The instability of the platinum complexes in physiologi-
cal/biological conditions preclude the application envisioned. Fur-
ther studies are needed to obtain Pt complexes compatible for such
in vivo applications, but these results set the stage for future develop-
ments on platinum-mediated decaging reactions.
(
block proliferation) and cytotoxic effect (apoptosis and reduction
of tumor size) than the control cFU at both 5 dpf (6 dpi) and 6 dpt
7 dpi; Figures S50,51). Also, the combined effect of pFU + CisPt
(
was more pronounced than the combination of 5-FU + CisPt, re-
garding proliferation (DMSO versus pFU + CisPt ****P<0.0001;
DMSO versus FU + CisPt *P = 0.0104; Figure S50i) and tumor size
(DMSO versus pFU + CisPt **** P<0.0001; DMSO versus FU +
CisPt *P = 0.0273; Figure S50k). This might be related with the in-
creased permeability of pFU (versus FU), which results in a more
efficient intracellular delivery of FU after Pt decaging. In conclusion,
our results show the efficient activation of the anti-cancer pFU in the
presence of non-therapeutic amounts of the anti-cancer drug CisPt
in an in vivo setting.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Pub-
lications website.
CONCLUSIONS
Detailed methods, characterization data and additional figures (PDF).
Movie with metadynamics calculations (mov).
In summary, we present a new decaging reaction of alkynes with
platinum complexes for the release of secondary amines from other-
wise stable tertiary amides, both in mammalian cell culture and in
living organisms. This reaction was shown to proceed by platinum-
mediated intramolecular cyclization mechanism. Our data suggest
that water, a necessary solvent in chemical biology applications, is
working as a metal-activating agent. Molecular electronic structure
calculations further corroborated the mechanism of the reaction
which was also supported by LC-MS characterization of the inter-
mediates. The reaction can proceed catalytically under certain con-
ditions and was later extended to N-propargyl groups with compara-
ble efficacies to that of palladium-mediated depropargylation. The
caging group was adapted for the synthesis of a non-internalizing
ADC, which results in drug release upon treatment with platinum
complexes in cancer cells. The reaction was also adapted and
demonstrated to function in a colorectal cancer zebrafish xenograft
model with non-toxic amounts of CisPt to activate a prodrug of an-
ticancer agent 5-FU, which led to a significant tumor reduction in
vivo.
AUTHOR INFORMATION
Corresponding Author
*gb453@cam.ac.uk
*rita.fior@research.fchampalimaud.org,
*bruno.oliveira@medicina.ulisboa.pt
Author Contributions
‡
These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This project has received funding from the European Union’s
Horizon 2020 research and innovation programme under the
Marie Skłodowska-Curie grant agreement Nos 675007 and
702574. We also thank the EPSRC (doctoral studentship to
B.S.), FCT Portugal (FCT Investigator to G.J.L.B.,
The work disclosed here represents a significant addition to the
toolbox of decaging strategies for chemical biology applications. In-
deed, the platinum-mediated cleavable reaction can be accom-
plished in aqueous systems having high concentrations of salts with
high yields and reaction rates, similar to those observed for the
standard palladium decaging metal. The reaction is, however, sus-
IF/00624/2015;
FCT
Stimulus
to
B.L.O.,
CEECIND/02335/2017; LISBOA-01-0145-FEDER-022170
and FCT-PTDC/MEC-ONC/31627/2017 to R.F.), CNPq
(PhD scholarship to F. S. S. S., 140485/2017-1; research grant to
G.F.C., 311963/2017-0), the Champalimaud Foundation and
Congento (R.F.) and H2020 (SIMICA, GA 852985, B.L. and
G.J.L.B.; EU_FT-ICR_MS, GA 731077, C.C.) for funding. We
also thank Prof. Dario Neri’s laboratory for the generous gift of
F16 antibody, Dr Ester Jiménez-Moreno for providing ubiquitin,
the Champalimaud Fish and Rodents Facility for their excellent
ceptible to the presence of nucleophiles resulting in slower rates (»
6
–15 times slower). We further demonstrate the compatibility of the
reaction in cellular environments. Although the reaction is suitable
for drug activation on cells inducing cytotoxicity, the presence of a
range of biomolecules/nucleophiles significantly reduces the overall
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animal care, the Portuguese Mass Spectrometry Network
LISBOA-01-0145- FEDER-022125), SeTIC/UFSC, special
16.Zhang, D. A.-O.; Dragovich, P. S.; Yu, S. F.; Ma, Y.; Pillow, T.
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H.; Sadowsky, J. D.; Su, D.; Wang, W.; Polson, A.; Khojasteh, S. C.;
Hop, C., Exposure-Efficacy Analysis of Antibody-Drug Conjugates
Delivering an Excessive Level of Payload to Tissues. Drug Metab.
Dispos. 2019, 47 (10), 1146–1155.
(
staff members (Bruno Amattos, Guilherme Arthur Geronimo
and Stephanie Andreon) for supercomputing resources, infra-
structure and assistance, and Dr Vikki Cantrill for her help with
the editing of this manuscript. G.J.L.B. is a Royal Society Univer-
sity Research Fellow (URF\R\180019) and the recipient of a
European Research Council Starting Grant (TagIt, GA 676832).
1
7.Durbin, K. R.; Phipps, C.; Liao, X., Mechanistic Modeling of
Antibody-Drug Conjugate Internalization at the Cellular Level Reveals
Inefficient Processing Steps. Mol. Cancer Ther. 17 (6), 1341–1351.
18.Yang, M.; Li, J.; Chen, P. R., Transition metal-mediated
bioorthogonal protein chemistry in living cells. Chem. Soc. Rev. 2014,
4
3 (18), 6511–6526.
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