Bioorthogonal Removal of 3-Isocyanopropyl Groups Enables the Controlled Release of Fluorophores and Drugs in Vivo

Article abstract of DOI:10.1021/jacs.8b05093

Dissociative bioorthogonal reactions allow for chemically controlling the release of bioactive agents and reporter probes. Here we describe 3-isocyanopropyl substituents as masking groups that can be effectively removed in biological systems. 3-Isocyanopr

Full text of DOI:10.1021/jacs.8b05093

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Communication
Bioorthogonal Removal of 3-Isocyanopropyl Groups Enables
the Controlled Release of Fluorophores and Drugs in Vivo
Julian Tu, MINGHAO XU, Saba Parvez, Randall T Peterson, and Raphael M. Franzini
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b05093 • Publication Date (Web): 21 Jun 2018
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Bioorthogonal Removal of 3-Isocyanopropyl Groups Enables
the Controlled Release of Fluorophores and Drugs In Vivo
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†
,‡
†,‡
§
§
†,*
Julian Tu, Minghao Xu, Saba Parvez, Randall T. Peterson, and Raphael M. Franzini
†
Department of Medicinal Chemistry, College of Pharmacy, University of Utah, 30 S 2000 E, Salt Lake City, UTꢀ
4112, United States
8
§
Department of Pharmacology and Toxicology, College of Pharmacy, University of Utah, 30 S 2000 E, Salt Lake
City, UTꢀ84112, United States
Supporting Information Placeholder
ABSTRACT: Dissociative bioorthogonal reactions allow for
chemically controlling the release of bioactive agents and reporter
probes. Here we describe 3ꢀisocyanopropyl substituents as
masking groups that can be effectively removed in biological
systems. 3ꢀisocyanopropyl derivatives react with tetrazines to
afford 3ꢀoxopropyl groups that eliminate diverse functionalities.
The study shows that the reaction is rapid and can liberate phenols
and amines nearꢀquantitatively under physiological conditions.
The reaction is compatible with living organisms as demonstrated
by the release of a resorufin fluorophore and a mexiletine drug in
zebrafish embryos implanted with tetrazineꢀmodified beads. The
combined benefits of synthetic ease, rapid kinetics, diversity of
leaving groups, high release yields, and structural compactness,
make 3ꢀisocyanopropyl derivatives attractive chemical caging
moieties for uses in chemical biology and drug delivery.
range of leaving groups, lack of toxicity, and extended serum
stability.
Figure 1. Tetrazineꢀmediated release of molecules caged with
3
ꢀisocyanopropyl (ICPr) and 3ꢀisocyanopropylꢀ1ꢀcarbamoyl
(ICPrc) groups.
Here we describe 3ꢀisocyanopropyl (ICPr) and 3ꢀisocyanopropylꢀ
1ꢀcarbamonyl (ICPrc) modifications as masking groups that can
be effectively removed from diverse molecules by bioorthogonal
reactions with 1,2,4,5ꢀtetrazines (Tz; Fig. 1). The design of
ICPr/ICPrc groups is based on the precedence that Tz at room
The ability to chemically control the release of reporter probes,
bioactive
compounds,
and
biomacromolecules
using
bioorthogonal reactions is opening opportunities for the
development of innovative research tools, diagnostics, and
1
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therapeutics. Applications in diverse fields such as biosensing,
temperature converts isonitriles into aldehydes. We rationalized
3
4
cell imaging, gasoemission, and activity control of nucleic
that the acidity of the aldehyde’s αꢀproton would make it possible
to release drugs and reporter molecules via βꢀelimination. Several
reports have previously shown that 3ꢀoxopropyl groups
5
6
acids and proteins exemplify the potential of such
transformations. Moreover, there is an interest in the clinical
translation of dissociative bioorthogonal reactions as chemicallyꢀ
15
spontaneously eliminate diverse functionalities. We anticipated
that ICPr/ICPrc groups would undergo a [4+1] cycloaddition
7
8
responsive antibodyꢀdrug conjugates and prodrugs.
reaction with Tz followed by rapid expulsion of N and formation
The growing demand for clickꢀreactions linked to a payload
release has motivated the invention of several such
transformations. Examples make use of the Staudinger
2
of a pyrazoleꢀimine intermediate. Hydrolysis to the aldehyde will
induce the spontaneous elimination of leaving groups at the Cꢀ1
position of the resulting 3ꢀoxopropyl moiety (Fig. 1).
2b,8a,9
reaction,
inverseꢀelectron
demand
DielsꢀAlder
10
11
cycloaddition, boraneꢀinduced deoxygenation, strainꢀpromoted
3+2] azideꢀalkene cycloaddition,
1
2
[
and metalꢀcatalyzed
Scheme 1. Synthesis of ICPr-tos, ICPr-OH, and ICPr-nc.
13
uncaging. The utility of several of these reactions to control the
release of drugs and fluorophores has been demonstrated in vitro
6c,7,8c,13d,13e
and in living vertebrates.
To further advance the scope
of bondꢀcleavage reactions compatible with biological systems,
we were interested to develop structurally compact bioorthogonal
reagents that are facile to synthesize while meeting the key
requirements of rapid reaction kinetics, high release yields, broad
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Figure 2. Tzꢀmediated uncaging of phenols and amines from 3ꢀisocyanopropyl and 3ꢀisocyanopropylꢀ1ꢀcarbamoyl derivatives. (a)
Structures of caged 1,8ꢀnaphthalimide reporter probes and products of their reactions with tetrazine. b) HPLC analysis of tetrazineꢀ
mediated uncaging of ICPrꢀOꢀNA and ICPrꢀNHꢀNA by PEGꢀDPTz (c(probe) = 1 mM, c(Tz) = 2 mM, PBS:DMSO (9:1), T = 37 °C;
DPPA: 3,5ꢀdi(pyridꢀ2ꢀyl)ꢀ1Hꢀpyrazolꢀ4ꢀamine). (c) Timeꢀdependent absorbance changes associated with the reaction of ICPrꢀOꢀNA and
ICPrꢀNHꢀNA with DPTz (c(probe) = 1 mM, c(Tz) = 3 mM, PBS:DMSO (1:1), T = 37 °C). (d) Kinetics of tetrazine consumption and
accumulation of products HOꢀNA and NH ꢀNA (c(probe) = 2 mM, c(Tz) = 0.2 mM, PBS:DMSO (9:1), T = 37 °C).
2
Reagents for the preparation of ICPr/ICPrcꢀderivatives were
straightforwardly accessible (Scheme 1). 3ꢀisocyanoꢀ1ꢀ
tosylpropane (ICPrꢀtos) for the alkylation of phenols and other
nucleophiles could be prepared on a multiꢀgram scale from
inexpensive 3ꢀaminoꢀ1ꢀpropanol in two steps and 62 % yield.
ICPrꢀtos is stable for months at ꢀ20 °C. To mask amines with
ICPrc groups, we prepared 3ꢀisocyanoꢀ4ꢀnitrophenyl carbonate
these reporter molecules, we confirmed that Tz elicits the
traceless removal of ICPr/ICPrc groups from phenols and amines.
Incubation of ICPrꢀOꢀNA and ICPrcꢀNHꢀNA (c = 1 mM) with the
waterꢀsoluble tetrazine PEGꢀDPTz (Fig. 2a; c = 2 mM; T = 37 °C,
PBS:DMSO (9:1, v/v)) led to the complete consumption of the
masked dyes and the formation of the parental fluorophores as
indicated by HPLC analysis (Fig. 2b)
(
ICPrꢀnc) along a similar synthetic route. The ease of preparation
We further monitored ICPr/ICPrc unmasking by UVꢀVis
spectrophotometric analysis (Fig. 2c). The introduced
modifications caused a hypsochromic shift of the absorbance and
emission bands of these fluorophores (Fig. 2a,c). In case of
ICPrꢀOꢀNA (c = 1 mM), the absorbance band with a maximum at
370 nM disappeared rapidly in the presence of excess 3,6ꢀdiꢀ2ꢀ
pyridylꢀ1,2,4,5ꢀtetrazine (DPTz; c = 3 mM, T = 37 °C,
PBS:DMSO (1:1)) concomitant with the appearance of the
absorbance peak characteristic for HOꢀNA (λabs,max = 445 nm) and
an isobestic point at 395 nm. The Tzꢀmediated conversion of
of these molecules favorably contrasts the tedious synthesis of
some bioorthogonallyꢀremovable groups.
To investigate the Tzꢀmediated removal of ICPr/ICPrcꢀgroups,
we synthesized probes that report unmasking by ratiometric
changes in absorbance and fluorescence spectra (Fig. 2).
1
,8ꢀnaphthalimides were modified on 4ꢀOH/4ꢀNH functionalities
2
with ICPr/ICPrc groups (ICPrꢀOꢀNA, ICPrcꢀNHꢀNA; see
Supporting Information for synthesis). A PEG4ꢀgroup at the imine
nitrogen endowed the probes with excellent water solubility. With
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ICPrcꢀNHꢀNA to H NꢀNA, accompanied by an absorbance shift
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from 370 to 430 nm, provided a comparable result with the
exception of a hypsochromic shift at early time points (Fig. 2c),
which indicated the formation of an intermediate with decreased
electronꢀdensity on the amine. The formation of a cyclic 4ꢀ
hydroxyꢀ1,3ꢀoxazinanꢀ2ꢀone at equilibrium with the 3ꢀoxopropyl
carbamate is a possible explanation for this observation as
1
6
reported for related molecules. Measurement of unmasking
yields based on the absorbance intensity of the product indicated
nearꢀquantitative release for the reaction of DPTz with ICPrꢀOꢀ
NA (99.1 ± 4.5%) and ICPrcꢀNHꢀNA (93.5 ± 2.5%). Liberation of
the 1,8ꢀnaphthalimides further led to a strong fluorescence turnꢀon
signal (ICPrꢀOꢀNA: 1210ꢀfold; ICPrcꢀNHꢀNA: 76ꢀfold; (Fig. 2c,
inset). Removal of ICPrꢀgroups also occurred with
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Figure 3. Tetrazineꢀmediated activation of isocyanopropylꢀ
derivatized prodrugs. (a) Structures of drugs caged with
ICPr/ICPrc groups. (b) Release percentages for prodrugs
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ꢀhydroxycoumarin and resorufin (ICPrꢀres) fluorophores (Fig.
(c(prodrug) = 1 mM, c(DPTz) = 2 mM, PBS:DMSO (1:1), T =
S1ꢀ4 in Supporting Information). Based on previous studies of
βꢀeliminations from 3ꢀoxopropyl substituents,
likely that ICPr/ICPrc chemistry can be used to mask diverse
functional groups.
1
5a,17
37 °C). (c) EC50 values in cytotoxicity studies (A549 cells) of
prodrug alone, prodrug + Tz (c = 80 ꢁM) and parent drug (for
doseꢀresponse curves see Fig. S6 in Supporting Information).
it appears
We further studied the kinetics of the Tzꢀinduced release of
phenols and amines from ICPr/ICPrc groups (Fig. 2d). Fitting the
disappearance of PEGꢀDPTz (c = 0.2 mM) absorbance in the
presence of excess ICPrꢀOꢀNA or ICPrcꢀNHꢀNA (c = 2 mM; T =
Considering the appeal of bioorthogonal chemistry for
8c
7
prodrug and therapeutic pretargeting approaches, we evaluated
the reaction in the context of anticancer agents (Fig. 3). We
synthesized ICPr/ICPrcꢀprodrugs of doxorubicin (ICPrcꢀdox),
mitomycin C (ICPrꢀmmc), mercaptopurine (ICPrꢀmp), and SNꢀ38
(ICPrꢀSNꢀ38) by reacting the drugs with ICPrꢀtos or ICPrꢀnc (see
Supporting Information for synthesis). The prodrugs (c = 1 mM)
were incubated with DPTz (c = 2 mM) in PBS:DMSO (1:1; T =
37 °C; 2 mM GSH was added with exception of ICPrꢀSNꢀ38 to
suppress Michael addition of acrolein to 6ꢀmercaptopurine) and
the release was assessed by HPLC (t = 4 h; Fig. S7 in Supporting
Information). Each of the drugs was released in high yields (Fig.
3b; ICPrꢀSNꢀ38, 91 ± 5 %; ICPrcꢀdox, 91 ± 4 %; ICPrꢀmp, 94 ±
3 %; ICPrcꢀmmC = 79 ± 6 %). To confirm that the released
molecules were active, we performed cytotoxicity experiments
with ICPrꢀdox and ICPrcꢀmmc (Fig. 3c). Combinations of the
prodrugs with excess PEGꢀDPTz (c = 80 ꢁM) elicited doseꢀ
dependent cytotoxicity in A549 adenocarcinoma cells. The
potencies of the prodrugs combined with PEGꢀDPTz
(EC50(ICPrcꢀdox) = 0.165 ± 0.035 ꢁM; EC50(ICPrcꢀmmC) =
0.244 ± 0.017 ꢁM) rivaled those of the free drugs (EC50(dox) =
0.144 ± 0.028 ꢁM; EC50(mmC) = 0.191 ± 0.042 ꢁM). In contrast,
the prodrugs alone showed little toxicity in the tested
concentration range (EC50 > 5 ꢁM; Fig. S6) and PEGꢀDPTz was
nonꢀtoxic at concentrations as high as 100 ꢁM. In conclusion,
ICPr/ICPrc modifications can be used to generate tetrazineꢀ
responsive prodrugs for diverse bioactive compounds.
3
7 °C, PBS:DMSO (9:1)) to a pseudoꢀfirst order rate equation
provided the bimolecular rate constants k (ICPrꢀOꢀNA) = 4.0 ±
0.2 M s and k (ICPrcꢀNHꢀNA) = 1.1 ± 0.2 M s , respectively.
Release of the fluorophores in PBS from the postulated
3
the ICPr/ICPrcꢀgroups with PEGꢀDPTz (k1,elim(HOꢀNA) = 1.6 ×
1
with studies of cargo release from such groups.
intermediate was detectable by NMR in DMSOꢀd :D O (9:1) (Fig.
S1 in the Supporting Information). To our delight, fluorophore
release in diluted human serum (T = 37 °C, PBS:serum (1:1))
reached completion in few minutes because serum albumin
catalyzes the βꢀelimination reaction.
order rate constants for this step in serum were calculated as
2
ꢀ
1
ꢀ1
ꢀ1 ꢀ1
2
ꢀoxopropyl intermediate was delayed relative to the reaction of
ꢀ4 ꢀ1
ꢀ5 ꢀ1
0 s ; k1 (H NꢀNA) = 5.00 × 10 s ; Fig. 2d) in agreement
,elim 2
15,18
The aldehyde
6
2
1
5b,16,18a
The apparent firstꢀ
ꢀ3 ꢀ1
k1,elim(ICPrꢀOꢀNA) = 4.2 × 10 s and k1,elim(ICPrcꢀNHꢀNA) = 1.6
×
ꢀ
3
ꢀ1
10 s . In the absence of Tz, ICPrꢀOꢀNA and ICPrꢀNHꢀNA
were completely inert to serum for at least three days (Fig. S5 in
Supporting Information). These outcomes demonstrate that stable
ICPr/ICPrcꢀmodifications can be rapidly and nearꢀquantitatively
removed from phenols and amines under physiological
conditions.
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reaction with Tz. In a series of steps, we demonstrated that the
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bimolecular reaction occurred rapidly, that release yields were
nearꢀquantitative, and that the chemistry was compatible with
diverse molecules including reporter fluorophores and cytotoxic
agents. Experiments in zebrafish models exemplified the utility of
the chemistry for in vivo drug and probe release, and efforts to
test the reaction in mammalian cells and other animals are
ongoing. A limitation of the ICPr/ICPrc groups is the delayed
elimination of molecules from the 3ꢀoxopropyl intermediate. In
addition to the possibility of using albumin to accelerate the
release, we will explore simple structural modifications to the
design to afford nearꢀinstantaneous release. The release of
acrolein is a possible limitation of this reaction in drug delivery;
however, acroleinꢀreleasing prodrugs are in clinical use and
addition of Mesna can effectively minimize associated adverse
effects. An intriguing aspect of ICPr/ICPRc groups is their
structural compactness. Such moieties might be engineered into
proteins for chemical control of activity while minimally
disrupting their secondary structure or alternatively be used for
designing prodrugs with little impact on their pharmacokinetics.
The ease of synthesis will further make the outlined chemistry
attractive for diverse applications in chemical biology and smart
therapeutics.
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ASSOCIATED CONTENT
Supporting Information
Figure 4. Release of resorufin and mexiletine inside zebrafish
embryos upon the reaction of ICPr/ICPrc groups with tetrazineꢀ
modified beads (TzꢀPS). (a) Cartoon representation of
experiments involving the implantation of zebrafish with TzꢀPS
followed by incubation with either ICPrꢀrsf for fluorescence
imaging or ICPrcꢀmex leading to a decreased heart rate. (b)
Detection of resorufin fluorescence upon tetrazineꢀmediated
uncaging in zebrafish (scale bar = 200 ꢁm). (c) Fluorescence
increase in zebrafish with either TzꢀPS or unmodified beads in
when incubated with ICPrꢀrsf (c = 10 ꢁM; t = 8 h). (d) Decrease
in heart rate in zebrafish implanted with either TzꢀPS or
unmodified beads treated with ICPrcꢀmex (t = 8 h; normalized to
heart rate at t = 0 h; ** = pꢀvalue < 0.005).
Experimental details, additional data, and characterization of
synthetic intermediates (PDF).
AUTHOR INFORMATION
Corresponding Author
*
raphael.franzini@utah.edu
Author Contributions
‡
These authors contributed equally.
ACKNOWLEDGMENT
R.M.F. gratefully acknowledges financial support from the
University of Utah, the Huntsman Cancer Institute, and the
USTAR initiative. This work was supported by the L. S. Skaggs
Presidential Endowed Chair (R.T.P.).
Having established that it is possible to remove ICPr/ICPrc
groups in aqueous solution at room temperature, we aimed to
demonstrate that this chemistry allows for activation of molecules
in vivo using zebrafish embryos as model organisms (Fig. 4a). A
Tzꢀmodified polystyrene bead (TzꢀPS) was implanted into the
yolk sac of zebrafish embryos followed by incubation with
ICPrꢀrsf (c = 10 µM). Fish implanted with TzꢀPS exhibited a
significantly higher fluorescence staining when exposed to ICPrꢀ
rsf than controls with unmodified beads (5.2ꢀfold, pꢀvalue =
<0.0009, t = 8 h; Fig. 4b,c). To corroborate that ICPr chemistry is
compatible with living systems, we analyzed the
phenomenological effects of releasing an active drug inside
zebrafish (Fig. 4a). We synthesized an ICPrꢀprodrug of mexiletine
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