Synthesis of a reactive oxygen species responsive heterobifunctional thioketal linker

Article abstract of DOI:10.1016/j.tetlet.2015.07.059

A new heterobifunctional reactive oxygen species (ROS) responsive thioketal linker and its synthesis are described. This linker allows for developing new ROS-responsive agents with two distinct functionalities using universal bioconjugation methods. The reaction kinetics of the thioketal cleavage in the presence of ROS is also described.

Full text of DOI:10.1016/j.tetlet.2015.07.059

Tetrahedron Letters xxx (2015) xxx–xxx
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Tetrahedron Letters
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Synthesis of a reactive oxygen species responsive heterobifunctional
thioketal linker
Xiaoxi Ling a, Shaojuan Zhang a, Pin Shao a, Pengcheng Wang b, Xiaochao Ma b, Mingfeng Bai a,
⇑
a Molecular Imaging Laboratory, Department of Radiology, University of Pittsburgh, Pittsburgh, PA 15219, USA
b Center for Pharmacogenetics, Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A new heterobifunctional reactive oxygen species (ROS) responsive thioketal linker and its synthesis are
described. This linker allows for developing new ROS-responsive agents with two distinct functionalities
using universal bioconjugation methods. The reaction kinetics of the thioketal cleavage in the presence of
ROS is also described.
Received 22 June 2015
Accepted 18 July 2015
Available online xxxx
Ó 2015 Published by Elsevier Ltd.
Keywords:
Thioketal
Reactive oxygen species
Stimuli responsive linker
Protecting group
Esterase
Reaction kinetics
Photodynamic therapy (PDT), a non-invasive method for dis-
eases treatment, has gained substantial attention over the last
decade.1 In general, PDT utilizes light and photosensitizers to gen-
erate reactive oxygen species (ROS), which induce oxidative dam-
age toward the target. Recently, Merlin and Murthy reported that
thioketal can be selectively cleaved in the presence of ROS.2 This
structure was later utilized by Liu et al. as the key linker in a
polyprodrug construct, which combines PDT and chemotherapy.3
However, such a homobifunctional thioketal linker is limited in
linking two distinct functionalities because byproducts with two
molecules of the same functionality coupled to one linker are often
collected (Scheme 1). Besides, these homobifunctional linkers
restrict the functional group options of the molecules to be conju-
gated. Here, we report a heterobifunctional thioketal linker that
allows the conjugation of two functionalities (such as a photosen-
sitizer and a targeting ligand) with distinct functional groups. Such
a linker will have great potential in developing ROS-responsive
molecular agents for wide therapeutic and imaging applications.
Initially, we attempted to synthesize the heterobifunctinal
thioketal linker using similar methods as those described for devel-
oping heterobifunctional S,S-thioketal. Early success of such a reac-
tion was reported by Young et al.,4–6 in which aldehydes or ketones
were treated with equal amounts of thioacetic acid and thiols to
yield a mixed acylthio-thioacetal/ketal convertable to asymmetric
dithioacetal/ketals. Later on, McNamara explored a route for an
unsymmetrical dithioacetal7 by generating an O-trimethylsilyl
hemithioacetal intermediate, which was later converted to the
desired product through a low temperature transthioacetalization
reaction catalyzed by boron trifluoride etherate. Additionally,
Morton reported successful interconversion of methoxy acetal
derivatives to thioacetals in good yield.8 Based on these reports,
we opted to prepare an O,S-thioketal as the key precursor of the
heterobifunctional S,S-thioketal, as outlined in Scheme 2. 3-
Mercaptopropionic acid (1) was reacted with excess amount of
2,2-dimethoxypropane in dichloromethane at 40 °C in the pres-
ence of catalytic amount of p-toluenesulfonic acid monohydrate.
Three different ester products,9 O,S-thioketal 2, enol thioether 3,
and S,S-thioketal 4, were separated from the reaction mixture
(structure determined with 1H NMR, data not shown) with the
desired O,S-thioketal 2 as the major product in 90% yield.10
However, the following transthioketalization reaction8 with triflu-
oroacetamide protected b-mercaptoethylamine 5 was unsuccessful
despite several attempts using various acids (p-toluenesulfonic
acid, hydrochloric acid, and aluminum chloride).
In an effort to synthesize the heterobifunctional S,S-thioketal
using an alternative but facile method, we chose to prepare the lin-
ker in a one-pot fashion (Scheme 3).7,11 Stoichiometric amounts of
methyl 3-mercaptopropionate (7), trifluoroacetamide protected b-
mercaptoethylamine 5, and acetone were mixed with boron triflu-
oride etherate7 at 0 °C. The desired product 6 was isolated from
byproducts 8 and 9 in 37% yield. Although this method caps the
⇑
Corresponding author. Tel.: +1 412 624 2565; fax: +1 412 624 2598.
E-mail address: baim@upmc.edu (M. Bai).
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X. Ling et al. / Tetrahedron Letters xxx (2015) xxx–xxx
Scheme 4. Selective deprotection of 6 using PLE.
proceeded. The resulting carboxylic acid 10 can then be coupled
with a desired moiety using classical procedure before the trifluo-
roacetamide protection is removed for the second conjugation.
To validate the linker may be cleaved by ROS, we assessed the
reaction kinetics of ROS triggered linker cleavage of thioketal 6
by 1H NMR using simulated ROS condition.17 Thioketal linker 6
was dissolved in a 1:1 mixture of CD3OD and D2O containing a cat-
alytic amount of copper(II) chloride and 3-(trimethylsilyl)propi-
onic-2,2,3,3-d4 acid sodium salt (TMSP) as the internal reference.
Upon addition of hydrogen peroxide solution, ROS was generated
in the presence of copper(II) chloride.18 The decomposition of the
thioketal group and generation of acetone (d = 1.60 and
2.20 ppm, respectively) were observed (see Fig. SI-1) and quanti-
fied using TMSP as the internal reference at 37 °C (see Fig. 1(a)).
During the first 1.9 h, limited amount of the thioketal was con-
verted as the reaction was likely limited by the low concentration
of ROS. This is supported by the slow broadening and disappear-
ance of the hydrogen peroxide signal around 10.8 ppm (see
Fig. SI-2) and poor 1H NMR spectrum resolution which may be
associated with the presence of the oxygen bubble (see Fig. SI-1,
spectra between 0 and 111 min) during this time period. Over
the next hour, the reaction started to accelerate as the concentra-
tion of ROS increased (see Fig. 1(b)), though the reaction kinetics
were not accessible due to the dynamic change of the reaction con-
ditions and limited data. After 2.8 h the concentration of ROS was
presumably high enough that its reaction with the thioketal
became the rate-determining step. Hereafter, this reaction fol-
Scheme 1. Representative examples of ROS responsive linkers.
Scheme 2. Synthesis of thioketal 6 using transthioketalization.
lowed pseudo-first order kinetics with
a rate constant of
1.30( 0.01) Â 10À4 sÀ1 and a half-life of 1.5 h. After 10 h, over
Scheme 3. Synthesis of thioketal 6 using a one-pot method.
yield of the desired heterobifunctional S,S-thioketal 6 at 50%, as
dictated by statistical distribution of the three possible combina-
tions, it is a much more straightforward and easier method with
an acceptable yield.
To prepare the linker for conjugation, it is necessary to selec-
tively remove either the methyl ester or the trifluoroacetamide
protecting group. Both methyl ester and trifluoroacetamide are
popular small molecule protecting groups in bioconjugation that
are relatively stable in many conditions but may be readily hydro-
lyzed in alkaline conditions. Although reports12–14 of selective
deprotection of methyl ester in the presence of trifluoroacetamide
are known, the reaction conditions are not universally transferable
to other structures due to poor efficiency or selectivity.15
Unfortunately, attempts using typical literature conditions
(lithium hydroxide or triethylamine in aqueous methanol) were
unsuccessful. To resolve this chemoselectivity problem, we
decided to use porcine liver esterase (PLE) to remove the methyl
ester (see Scheme 4). PLE earned most of its fame in enzymatic
organic synthesis for its remarkable performance in asymmetric
hydrolysis of methyl esters.16 While enantioselectivity is not a con-
cern in this ester hydrolysis, the PLE worked profoundly well due
to the chemoselectivity associated with enzymatic reactions.
Initial trial of adding PLE to a PBS buffer containing the thioketal
linker precursor 6 in acetone gave the propionic acid product 10
in 32% yield. The reaction yield was later improved to 73% by care-
fully maintaining the pH of the reaction mixture around 7–8 with
the addition of sodium hydroxide solution as the reaction
Figure 1. (a) The natural logarithm of the percentage of residual thioketal signal
from 6 (red; left y-axis) and the percentage of acetone signal (blue; right y-axis) in
simulated ROS environment plotted against time. (b) Zoomed view of the first 3 h of
(a).
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X. Ling et al. / Tetrahedron Letters xxx (2015) xxx–xxx
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96% of thioketal was oxidized. Additional signals which may be
References and notes
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In summary, we have developed a new heterobifunctional ROS-
responsive thioketal linker with a carboxylic acid and a primary
amine group as the conjugation sites. Such a heterobifunctional
linker allows for developing ROS-responsive agents with two dis-
tinct functionalities, therefore, it has great promise in biomedical
applications where on-demand photo activated bond cleavage is
desirable. In addition, we found that PLE could be a useful tool to
selectively remove methyl ester in the presence of trifluoroac-
etamide protecting group. Lastly, kinetic study showed that ROS
effectively cleaved this thioketal linker and the cleavage reaction
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Acknowledgments
This work was supported by the NIH Grant # R21CA174541 (PI:
Bai) and NIH Grant # R01DK090305 (PI: Ma), as well as the startup
fund provided by the Department of Radiology, University of
Pittsburgh. The authors would also like to thank Dr. Damodaran
Krishnan and Dr. Viswanathan Elumalai from the Department of
Chemistry, University of Pittsburgh, for their generous help in the
reaction kinetic study using 1H NMR.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2015.07.
059.
Please cite this article in press as: Ling, X.; et al. Tetrahedron Lett. (2015), http://dx.doi.org/10.1016/j.tetlet.2015.07.059