Selective arylthiolane deprotection by singlet oxygen: A promising tool for sensors and prodrugs

Article abstract of DOI:10.1039/c4cc09040c

A routine thioketal protecting group reacts rapidly and selectively with singlet oxygen to reveal ketone products in good (aryl 1,3-dithiolane) to excellent (aryl 1,3-oxathiolane) yields. Arylthiolanes are stable to biologically relevant reactive oxygen species and can be used as a light-activated gating mechanism for activating fluorescent sensors or small molecule prodrugs.

Full text of DOI:10.1039/c4cc09040c

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Selective arylthiolane deprotection by singlet
oxygen: a promising tool for sensors and
prodrugs†‡
Brian M. Lamb*abc and Carlos F. Barbas III§abc
Cite this: Chem. Commun., 2015,
51, 3196
Received 12th November 2014,
Accepted 9th January 2015
DOI: 10.1039/c4cc09040c
www.rsc.org/chemcomm
A routine thioketal protecting group reacts rapidly and selectively with incorporation into numerous small molecules, (c) inert to
singlet oxygen to reveal ketone products in good (aryl 1,3-dithiolane) common biological sources of ROS and (d) cleanly reveal a
to excellent (aryl 1,3-oxathiolane) yields. Arylthiolanes are stable to useful functional group in high yields. Thioketal protecting
biologically relevant reactive oxygen species and can be used as a groups are well known for their stability to hydrolysis, simple
light-activated gating mechanism for activating fluorescent sensors formation, and unique redox potentials allowing for selective
or small molecule prodrugs.
removal and resulting in their ubiquitous use as protecting
groups in organic syntheses. For their removal a number of
Photoswitches for small molecules are integral components of single electron transfer (SET) reagents, and even common
next-generation pharmaceuticals and biomaterials,1 biomolecular name reactions (e.g. Corey-Seebach) are available. In organic
probes,2 and even nanomachines. Many common photoswitches solvents, photoinduced electron transfer (PET) can provide a
are based on derivatives of nitrosoveratryloxycarbonyl groups,3 catalytic mechanism for the removal of some thioketal contain-
cis–trans isomerizing azobenzenes,4 spiro N–O bond cleavage,5 ing substrates and there have even been some reports that
and two-photon actuation strategies.6 Despite the dominance thioketals are 1O2 reactive in organic solvents.9,10 Owing to
of these mostly ultraviolet-actuated photoswitches in many some of these reports and the ubiquitous use of thioketals as
chemical applications, therapeutically relevant switches require protecting groups in organic synthesis, it was sought to ascertain
visible or infrared light for optimal tissue transparency. This has whether thioketals could be tailored as a modular component
led to the development and delivery of more efficient infrared- for 1O2-mediated activation of fluorescent sensors and prodrugs
absorbing photosensitizers producing singlet oxygen (1O2) for of potential compatibility with methods in PDT.
photodynamic therapy (PDT).7,8 Next-generation photosensitizers
To ascertain the optimal structural parameters for 1O2-mediated
possess an enhanced combination of near-infrared light absorp- transformation of thioketals into ketone products, numerous
1
tion, O2 production, cell targeting/penetration, and minimal dark thioketal containing small molecules possessing modifications
toxicity. If combined with a practical, chemoselective and sensi- to adjacent R, R0, and R00 positions were synthesized (Fig. 1A).
tive 1O2-mediated chemical transformation, these developments These compounds were irradiated by 25 000 lumen white light
could lead to new classes of visible/near-infrared photoswitches in buffered protic solvents with known 1O2 producing photo-
compatible with methods in PDT and enabling localized small sensitizers. Reactivity and product yields were assessed in
molecule therapies.
pH 7.4 buffered aqueous–ethanolic solutions to maximize the
1
Ideally a O2-mediated photoswitch should be (a) stable in probability that the observed reactions and mechanisms would
aqueous or serum-containing solutions, (b) modular for simple proceed similarly in an in vivo like environment. Good to
excellent yields and rapid formation of ketone product were
observed with arylthiolanes (Fig. 1, 1a–5a, 8a, 14), especially in
the presence of a strong electron donor (4a). Aryldithiolanes
a The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla,
CA, 92037, USA. E-mail: bmlamb@scripps.edu
b Department of Chemistry, The Scripps Research Institute, La Jolla, CA, 92037, USA
c The Scripps Research Institute, 10550 N. Torrey Pines Rd, La Jolla, CA, 92037,
USA
(1b–5b) and aryldithianes (6a, 6b, and 7) exhibited reduced
reaction rates and yields of ketone product. Thioketal reactivity
was only observed in the presence of a-thioether protons and
yields of ketone product are minimal in the absence of an a-aryl
substituent to the thioketal or when a competing a-thioether
proton is present (9–13). These results differ from SET mecha-
† B. M. L. dedicates this work to the memory of Carlos F. Barbas III, an
outstanding mentor and friend.
‡ Electronic supplementary information (ESI) available: Synthetic schemes, spec-
tral characterization. See DOI: 10.1039/c4cc09040c
§ C. F. B. passed away June 24, 2014, during the preparation of the manuscript. nisms for removal of thioketals in polar aprotic solvents and
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Fig. 1 Optimization of thioketal removal with 1O2. (A) General synthetic scheme for producing thioketals and 1O2-mediated revealing of ketone
substrates. (B) Degradation rates and yields of ketone product derived from photo-oxidation of selected thioketal substrates. Standard conditions: 2 mM
thioketal, 7 : 3 EtOH/H2O + 50 mM TrisHCl pH 7.4 and 5 mol% photosensitizer (MB = methylene blue, RB = rose bengal, mPPa = methylpyropheo-
phorbide a), exposed to 25 000 lumen compact fluorescent light. Reported yields from HPLC. * NMR yields. a Substrate was oxidized under an oxygen
atmosphere.
are more consistent with mechanistic observations associated
with the reactivity of ethyl disulfide, 1,3-dithianes, and various
thioethers with O2.9 Specifically, O2 is expected to produce a
peroxysulfonium ylide intermediate with reactivity strongly
dependent on an aprotic or protic environment. Generally,
1O2 oxidation yields sulfone or sulfoxide products in aprotic
solvents, whereas protic conditions tend to quench the ylide,
leading to alternative reactivity. In agreement with these reactive
proclivities, photo-oxidation with rose bengal (RB), methylene blue
(MB), or methyl pyropheophorbide a (mPPa) in polar aprotic
solvents exclusively provided sulfoxide products, whereas the same
reaction in protic solvent led exclusively to ketone product. Under
protic conditions an oxidized sulfide byproduct was characterized
by NMR and MS analysis suggestive of an elimination mechanism
(ESI,‡ Fig. S1). From the data in Fig. 1, it was determined that an
optimal thioketal structure for 1O2-mediated transformation of
thioketals into ketone products in buffered aqueous solvents pos-
sessed (1) activated a-aryl groups, (2) a single pair of a-thioether
protons, and an 1,3-oxathiolane thioketal (exemplified by 4a).
Singlet oxygen is one of several classes of ROS, which includes
superoxide, peroxides, nitric oxide, and various oxygen radical
substituents. To assess the specificity of the 1,3-dithiolane and
1
1
Fig. 2 Assessment of arylthiolane reactivity with ROS. (A) General reaction
for the production of the fluorescent ketone product. (B) Arylthiolane
degradation by ROS. Standard conditions: 0.5 mM arylthiolane 8a/b, 7 : 3
EtOH/H2O + 50 mM TrisHCl pH 7.4. ROS conditions: 5 mol% RB or MB
photosensitizer + 25 000 lumen compact fluorescent light, 100 mM H2O2,
or 10 mM FeCl3 and 100 mM H2O2 (Fenton).
1
1,3-oxathiolanes to degradation by O2 in real time, 8a and 8b
1
were subjected to O2 producing photosensitizers and light or
representative alternative ROS. The ketone product, 6-methoxy-2-
acetonapthhone, is fluorescent UV irradiation, whereas the In the presence of RB, MB, or mPPa, (mPPa not shown in Fig. 2
thioketal and sulfoxide oxidation products lack fluorescence. due to fluorescence overlap), deprotection could be monitored in
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real time via Tecan Fluorimeter (Fig. 2A). Among the fluorescent
sensors, the 1,3-oxathiolane sensor 8a gave the best yields of
ketone product and showed over 20% enhanced reactivity com-
pared to the 1,3-dithiolane sensor 8b (Fig. 2B). Peroxide, super-
oxide, and Fenton conditions (iron3+/peroxide – hydroxyl radical
and superoxide mixtures) were unreactive to the arylthiolanes
over the analysis time. Deprotection of either 8a/b was not
observed even with 4100-fold excess superoxide reagent, either
derived in situ (as part of the Fenton reaction) or in organic
solvent (50-fold excess potassium superoxide and 18-crown-6
ether in refluxing THF, not shown), even over prolonged periods
of time (days). The unreactivity of the thioketal was further
confirmed by HPLC analysis at the end of assay, to ensure the
absence of non-fluorescent oxidation products. Although the
lability of thioketal polymer-based systems to superoxide is
precedented in the literature, we hypothesize that the cyclic
rather than acyclic structure results in differential reactivity of
these thioketals for superoxide or other ROS.11 Finally, since
cellular compartments contain a range of acidic and basic
conditions, the stability of the thioketal protecting group in
pH 1, 7.4, and 13 solutions was investigated. It was observed
in all pH ranges that 8a and 8b were unreactive and stable, with
only slow hydrolysis of 8a observed in strongly acidic solution
(ESI,‡ Fig. S2).
Fig. 3 (A) Schematic for oxidation of 15 to 16 and release of doxorubicin.
(B) Stability of 15 in the presence of photosensitizer/light and fetal bovine
serum. Conditions: 0.5 mM 15 2 : 1 : 1 DMF/PBS/50 mM Tris pH 7.4 or
serum.
It has recently been reported that photosensitizer–prodrug
1
conjugates joined by an aminoacrylate O2-labile linker can be
activated with visible/near-infrared light to site-specifically
eliminate tumors in vitro and in vivo.12,13 It was hypothesized
that an aryl-1,3-oxathiolane linker, based on a serum-induced conditions and shows promise as a modular component in light-
b-elimination mechanism employed by catalytic antibody acti- activated sensors and prodrugs. The thioketal moiety reacts selec-
vated prodrugs and derived from optimal thioketal 4a, could be tively with 1O2 and is stable to other ROS commonly found in
used as a general scaffold for facile creation of 1O2-activated biological settings. When incorporated into a 1O2-labile linker and
prodrugs from small molecules possessing free amines, hydro- conjugated to doxorubicin, photooxidation exclusively led to linker
xyls, or carboxylate functional handles.13 This contrasts with degradation. The simplicity of the synthesis of the thioketals, their
aminoacrylate linkers which are presently limited to the release stability in solution and during synthetic preparation, and the
of phenol products.12 This strategy could significantly expand compatibility with photosensitizers across multiple wavelengths
1
the utility of O2-mediated transformations to cover most small make arylthiolane-based linkers attractive for incorporation into
molecule therapeutics and even enable simple bioconjugations fluorescent sensors for in vivo imaging and localized drug activa-
with oxyamines, hydrazines, or hydrazones.14 A short synthetic effort tion.12,16 We anticipate many applications of arylthiolanes as an
provided prodrug 15 (ESI,‡ Scheme S1), doxorubicin conjugated to an important intramolecular component of light-activated small
optimized 1O2-sensitive arylthiolane linker. Substrate 15 was oxidized molecules, bioconjugated proteins, and key reactive components
to 16 in PBS–Tris–DMF solution and both the arylthiolane degrada- of 1O2-activated liposomal drug delivery vehicles.
tion and appearance of ketone product was observed by HPLC and
We thank The Skaggs Institute for Chemical Biology for funding.
LCMS. Rose bengal and methylene blue photosensitizers and 25000 BML is supported by an American Cancer Society postdoctoral
lumen compact fluorescent white light (Fig. 3B) exposure cleanly fellowship. The authors are indebted to Dr Yoshihiro Ishihara for
removed the 1,3-oxathiolane without modification to doxorubicin. In assistance in the preparation of the manuscript.
the absence of light or photosensitizer, arylthiolane degradation was
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