Synthesis of a reactive oxygen species-responsive doxorubicin derivative

Article abstract of DOI:10.3390/molecules23071809

A heterobifunctional reactive oxygen species (ROS)-responsive linker for directed drug assembly onto and delivery from a quantum dot (QD) nanoparticle carrier was synthesized and coupled to doxorubicin using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC)/sulfo–NHS coupling. The doxorubicin conjugate was characterized using ¹H NMR and LC-MS and subsequently reacted under conditions of ROS formation (Cu²⁺/H₂O₂) resulting in successful and rapid thioacetal oxidative cleavage, which was monitored using ¹H NMR.

Full text of DOI:10.3390/molecules23071809

molecules
Article
Synthesis of a Reactive Oxygen Species-Responsive
Doxorubicin Derivative
James B. Delehanty 1 ID , Shivani Das 1, Efram Goldberg 2, Ajmeeta Sangtani 1 and
D. Andrew Knight 2,
*
ID
1
Center for Bio/Molecular Science and Engineering, Code 6900, Naval Research Laboratory,
Washington, DC 20375, USA; james.delehanty@nrl.navy.mil (J.B.D.); shivanimdas@gmail.com (S.D.);
Ajmeeta.Sangtani@nrl.navy.mil (A.S.)
Chemistry Department, Florida Institute of Technology, 150 West University Boulevard,
Melbourne, FL 32901, USA; egoldberg2012@my.fit.edu
2
*
Correspondence: aknight@fit.edu; Tel.: +1-321-674-8175
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 28 May 2018; Accepted: 18 July 2018; Published: 21 July 2018
Abstract: A heterobifunctional reactive oxygen species (ROS)-responsive linker for directed drug
assembly onto and delivery from a quantum dot (QD) nanoparticle carrier was synthesized and
coupled to doxorubicin using N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride
1
(EDC)/sulfo–NHS coupling. The doxorubicin conjugate was characterized using H NMR and
LC-MS and subsequently reacted under conditions of ROS formation (Cu2+/H2O2) resulting in
successful and rapid thioacetal oxidative cleavage, which was monitored using 1H NMR.
Keywords: doxorubicin; heterobifunctional; linker
1. Introduction
The use of nanoparticles (NPs) for the controlled delivery of drugs to cells and tissues has
emerged as an attractive alternative to traditional systemic delivery of drugs [1]. This is driven largely
by the advantages afforded by NP-based drug delivery, which include (1) large drug payload per
dose, (2) longer circulation time and clearance, and (3) specific drug targeting to diseased tissues.
One of the more attractive aspects of NP-mediated drug delivery is the potential to control the
spatial and temporal release of the NP-appended drug in response to specific triggering stimuli [2].
Semiconductor quantum dots (QDs) are an excellent prototypical NP platform for the delivery of
therapeutics given their bright, photostable luminescence (for tracking) and amenability to decoration
with drug cargos (for delivery) [3]. Doxorubicin is a potent and widely-used anticancer therapeutic,
yet it elicits significant nonspecific cardiotoxicity when its delivery/release is not properly targeted or
controlled [4]. Thus, there is wide interest in modulating its cellular targeting and release.
Here, we have synthesized a heterobifunctional, cleavable linker for the controlled release
of doxorubicin from the surface of a QD carrier. The conjugate bears a reactive oxygen species
(ROS)-responsive linkage with doxorubicin at one end and a protected amine at the other end.
We demonstrate the ability of the ROS-reactive doxorubicin-linker to actively release the drug
moiety in the presence of copper-catalyzed hydrogen peroxide-generated ROS. Acid cleavage
of the trifluoroacetate protecting group yields a reactive amine that can be conjugated (1) to a
peptide backbone prior to peptide self-assembly to the QD surface, or (2) directly to the QD
surface via attachment to reactive “handles” on the termini of QD-capping ligands (e.g., to the
carboxyl groups on dihydrolipoic acid (DHLA) ligands (Figure 1)). When appended onto the
surface of a QD, the ROS-reactive conjugate described herein can be integrated into a multifunctional
phototriggered ensemble where the light-harvesting central QD excites an appended photosensitizer
Molecules 2018, 23, 1809; doi:10.3390/molecules23071809
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molecule to generate ROS, leading to the combined, multistage generation of ROS coupled with
doxorubicin release.
Figure 1. The linker-doxorubicin (DOX) conjugate can be conjugated (a) to a peptide prior
to the assembly of the peptide onto the quantum dot (QD) surface or (
b) directly to a
QD-capping ligand, such as dihydrolipoic acid (DHLA). EDC—N-(3-dimethylaminopropyl)-
0
N -ethylcarbodiimide hydrochloride.
2. Results and Discussion
The method we chose for the preparation of an ROS-responsive doxorubicin conjugate is shown
in Figure 2a, and required the synthesis of a doubly protected heterobifunctional linker 3 containing
a dithioacetal group, which would undergo cleavage when exposed to reactive oxygen species.
Protection of the carboxylic acid group of the linker was achieved using a methyl ester and the amine
functionality was protected using a trifluoroacetate group. This strategy has been described previously
by Ling et al. [
conditions gave 2,2,2-trifluoro-N-(2-mercaptoethyl)acetamide
the reaction conditions gave a modest improvement of yield described previously in the literature
(65%) [ ]. The doubly protected S, S-thioacetal was then synthesized via the reaction of with
acetone and methyl 3-mercaptopropionate under Lewis acidic conditions using boron trifluoride
etherate. Efficient deprotection of using pig liver esterase in PBS buffer gave the amine protected
thioketal with a free COOH group, which would allow conjugation to amines via amide coupling.
5]. The reaction of cysteamine hydrochloride and ethyl trifluoroacetamine under basic
1
in 86% yield. Slight modification of
6
2
1
2
3
A number of amide coupling methods have previously been reported for attaching doxorubicin to
carboxylic acids. For example, dicyclohexylcarbodiimide (DCC) was used to attach doxorubicin
to poly(amidoamine) dendrimer encapsulated gold nanoparticles [
doxorubicin conjugates were prepared via reaction of doxorubicin with fatty acids in the presence
of the coupling agent HBTU [
]. We chose to use N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide
hydrochloride (EDC)/sulfo–NHS coupling to attach doxorubicin to the ROS-responsive linker
7]. Monosubstituted fatty acyl
8
3,
as shown in Figure 2b, because of the ease of work-up and high coupling efficiency. A ten-fold excess
of the linker was used for the reaction with doxorubicin performed in DMF at room temperature.
After work-up involving extraction of the conjugate into CH2Cl2, and purification over silica gel,
compound
4 was isolated in 50% yield. Characterization of 4
was achieved using 1H NMR and mass
spectrometry (see Supplementary Materials, Figures S1 and S2), however, our attempts to obtain a 13
NMR spectrum with suitable signal/noise were unsuccessful.
C
To demonstrate the response of the conjugate to reactive oxygen species, which would result
1
in cleavage and release of doxorubicin, an in situ H NMR experiment was performed in which
reactive ROS were catalytically generated using copper(II) ion and hydrogen peroxide in CD3OD
as described by Ling et al. [5]. The cupric ion–hydrogen peroxide reaction follows typical Fenton
behavior, generating superoxide radicals, molecular oxygen, and hydroxyl radicals according to
Equations (1)–(3) [9].
Briefly, a solution of doxorubicin conjugate
4 in CD3OD was placed in an NMR tube, and a
spectrum was recorded that showed the presence of methyl protons at 1.5 ppm due to the dimethyl

Molecules 2018, 23, 1809
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thioacetal group. On addition of hydrogen peroxide and a catalytic amount of copper chloride,
1
a second H NMR spectrum was recorded after 30 min. Reduction in intensity of methyl protons
indicated successful cleavage of the thioacetyl group (from peak integration, 80% reduction in total
amount of CH3 protons). After 8 h, a final NMR spectrum showed complete disappearance of the
dimethyl thioacetal group (Figure 3).
Cu2+ + H2O2
2O2 + 2H+
Cu+ + H2O2
Cu+
H 2O2 + O2
Cu2+
HO + HO
+
2H+
+
O2
(1)
(2)
(3)
+
(a)
(b)
Figure 2.
(
a
) Synthesis of heterobifunctional linker
3
; (
i
) Et3N, MeOH; (ii) acetone, BF3 Et2O, CH3CN,
·
◦
0 C; (iii) porcine liver esterase, acetone, PBS. (
doxorubicin conjugate 4.
b) Synthesis of reactive oxygen species (ROS)-responsive

Molecules 2018, 23, 1809
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Figure 3. Reactive oxygen species assay: 1H NMR spectra showing disappearance of SC(CH3)2S
methyl protons (pink bars).
3. Materials and Methods
1
All reactions were conducted in oven-dried glassware, under air unless otherwise noted. H NMR
spectra were recorded on Avance Bruker 400-MHz spectrometers. Mass spectra were recorded on an
Agilent LC-mass spectrometer in ESI or APCI modes. Chemical shifts for 1H NMR spectra are reported
(in parts per million) relative the residual solvent. Silica gel (200–300 mesh) was used for column
chromatography. TLC plates were visualized by exposure to iodine vapor. Weighing was performed
on a Mettler Toledo XS105 four-place analytical balance.
Solvents were obtained as follows: dimethylformamide (DMF), dichloromethane, methanol,
acetone, and acetonitrile (Aldrich) were used as received. CDCl3, CD2Cl2, D2O, and CD3OD
(Cambridge Isotopes Laboratory or Aldrich) were used as received.
Reagents and chemicals were obtained as follows: Doxorubicin hydrochloride (Aldrich),
0
N-(3-dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride (Pierce), N-hydroxysulfosuccinimide
sodium salt (Pierce), copper(II) chloride (Aldrich), trimethylsilylpropyl sulfonate (Aldrich), 30%
hydrogen peroxide (Aldrich), silica gel (230–400 mesh, Fisher), and anhydrous sodium sulfate (Aldrich)
were used as received. Heterobifunctional dithioacetal
procedure [1].
3 was prepared according to the literature
Conjugation
of
heterobifunctional
dithioacetal
3
with
doxorubicin:
Compound
3
(5.5 mg, 17 mmol), doxorubicin hydrochloride (1.0 mg, 1.7 mmol),
N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride (EDC) (0.3 mg, 1.7 mmol),
and N-hydroxysulfosuccinimide sodium salt (sulfo-NHS) (0.4 mg, 1.7 mmol) were combined in DMF
(2.0 mL) and stirred at room temperature for 8 h. Then, DI H2O (1 mL) was added and the aqueous
phase was extracted with CH2Cl2 (5
×
1 mL). The CH2Cl2 extracts were combined and dried over
anhydrous Na2SO4. The solvent was removed in vacuo and the resulting red residue was dissolved in
CH2Cl2/MeOH (2 mL, 50/50 v/v) and purified by passing over a silica gel plug (ca. 1 g) and washing
the plug with a further portion of CH2Cl2/MeOH to give a red-orange solution. The solvent was
removed to give
4
as a red solid (0.7 mg, 50% yield). An 1H NMR spectrum recorded in CDCl3 is
1
included in the Supplementary Materials. H NMR
δ 0.80–1.21 (m, 6H), 1.61 (s, 6H), 2.01–2.97 (m, 8H),
3.54–3.82 (m, 11H), 5.23 (m, 2H), 6.81 (s, br., 1H, NH).
Oxidation of 4 using copper(II)-hydrogen peroxide: A 5-mm NMR tube was charged with
conjugate
4 (9.5 mg) and CD3OD (ca. 0.7 mL). A trace of trimethylsilylpropyl sulfonate was added as
an internal reference and an 1H NMR spectrum was immediately recorded. Then, a solution of CuCl2
in 30% H2O2 (15 mL, 200 mM) was added and, after 30 min, a second 1H NMR spectrum was recorded.
Attempts to isolated oxidative products using TLC were unsuccessful.

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4. Conclusions
In summary, we have synthesized a new doxorubicin-conjugated heterobifunctional
ROS-responsive linker molecule using EDC/sulfo–NHS amide bond coupling, in which a carboxylic
acid functional group is coupled to the native primary amine of the anti-cancer drug doxorubicin.
The amine group of the linker is protected by an acid labile trifluoracetate moiety, which can be readily
removed for further conjugation (e.g., to peptides, proteins). The presence of an ROS-responsive
dithioacetal group as part of the linker allows the rapid release of doxorubicin to a target when
exposed to ROS generated in situ. Using 1H NMR, we have demonstrated that the linker is cleaved
readily when exposed to ROS generated from Cu2+/H2O2.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/23/7/1809/
s1, Figure S1 1H NMR spectrum of compound 4, Figure S2. ESI and APCI mass spectra for compound 4.
Author Contributions: Conceptualization, A.S. and D.A.K.; Funding acquisition, J.B.D.; Investigation, S.D., E.G.
and D.A.K.; Methodology, A.S. and D.A.K.; Supervision, J.D.; Writing—original draft, D.A.K.; Writing—review &
editing, D.A.K.
Funding: This work received support from the Base Funding Program at the Naval Research Laboratory. D.A.K.
is grateful to the Office of Naval Research for a Summer Faculty Fellowship. Publication of this article was funded
in part by the Open Access Subvention Fund and Florida Tech Libraries.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to
publish the results.
References
1.
Swain, S.; Sahu, P.K.; Beg, S.; Babu, S.M. Nanoparticles for cancer targeting: Current and future directions.
Curr. Drug Deliv. 2016, 13, 1290–1302. [CrossRef] [PubMed]
2.
Sangtani, A.; Nag, O.K.; Field, L.D.; Breger, J.C.; Delehanty, J.B. Multifunctional nanoparticle composites:
Progress in the use of soft and hard nanoparticles for drug delivery and imaging. Wiley Interdiscip. Rev.
Nanomed. Nanobiotechnol. 2017, 9, 1–23. [CrossRef] [PubMed]
3.
4.
Delehanty, J.B.; Boeneman, K.; Bradburne, C.E.; Robertson, K.; Medintz, I.L. Quantum dots: A powerful tool
for understanding the intricacies of nanoparticle-mediated drug delivery. Expert. Opin. Drug Deliv. 2009, 6,
1091–1112. [CrossRef] [PubMed]
Danz, E.D.B.; Skramsted, J.; Henry, N.; Bennett, J.A.; Keller, R.S. Resveratrol prevents doxorubicin
cardiotoxicity through mitochondrial stabilization and the Sirt1 pathway. Free Radical. Bio. Med. 2009
46, 1589–1597. [CrossRef] [PubMed]
,
5.
6.
7.
8.
9.
Ling, X.; Zhang, S.; Shao, P.; Wang, P.; Ma, X.; Bai, M. Synthesis of a reactive oxygen species responsive
heterobifunctional thioketal linker. Tetrahedron Lett. 2015, 56, 5242–5244. [CrossRef] [PubMed]
Shim, M.S.; Xia, Y. A reactive oxygen species (ROS)-responsive polymer for safe, efficient, and targeted gene
delivery in cancer cells. Angew. Chem. Int. Ed. 2013, 52, 6926–6929. [CrossRef] [PubMed]
Sagita, E.; Djajadisastra, J.; Mutalib, A. Cytotoxic enhancement of doxorubicin in conjugation with PAMAM
G4.5 dendrimer containing gold nanoparticles. Int. J. PharmTech. Res. 2016, 9, 348–356.
Chhikara, B.S.; Jean, N.S.; Mandal, D.; Kumar, A.; Parang, K. Fatty acyl amide derivatives of doxorubicin:
Synthesis and in vitro anticancer activities. Eur. J. Med. Chem. 2011, 46, 2037–2042.
Carvalho do Lago, L.C.; Matias, A.C.; Nomura, C.S.; Cerchiaro, G.J. Radical production by hydrogen
peroxide/bicarbonate and copper uptake in mammalian cells: Modulation by Cu(II) complexes.
Inorg. Biochem. 2011, 105, 189–194. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds are not available from the authors.
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