A phenacrylate scaffold for tunable thiol activation and release

Article abstract of DOI:10.1039/c4cc07343f

A thiol-selective 2-methyl-3-phenacrylate scaffold with spatiotemporal control over delivery of a cargo is reported. The half-lives of decomposition could be tuned from 30 min to 1 day and the scaffold's utility in thiol-inducible fluorophore release in cell-free as well as within cells is demonstrated.

Full text of DOI:10.1039/c4cc07343f

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DOI: 10.1039/C4CC07343F
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A Phenacrylate Scaffold for Tunable Thiol Activation
and Release
Cite this: DOI: 10.1039/x0xx00000x
Rathinam K. Sankar,‡,a Rohan S. Kumbhare,‡,a Allimuthu T. Dharmarajaa and
Harinath Chakrapani*,a
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
www.rsc.org/
A
thiol-selective 2-methyl-3-phenacrylate scaffold with with a biological thiol can be modulated by simple structural
spatiotemporal control over delivery of a cargo is reported. modifications.
The half-lives of decomposition could be tuned from 30 min A 2-methyl-3-phenacrylate-based scaffold was considered for
to day and the scaffold’s utility in thiol-inducible tunable reactivity with thiols (Scheme 1). The core consists of a 2-
fluorophore release in cell-free as well as within cells is methylene-3-arylacrylate functionality connected with a phenol-
1
demonstrated.
based self-immolative linker, which can be attached to a drug or
fluorophore that is to be released. Michael addition23 of a thiol
produces an enol(ate), which can rearrange to produce a self-
immolative phenolate; rearrangement of this intermediate would
Selective and covalent modification of thiols has numerous
applications in biochemistry, imaging and medicine.1-3 Thiols in
proteins or peptides can be conjugated to a small molecule4-5 or a
macromolecule and is useful for interrogating cellular events,
studying enzyme function and in drug discovery.6-10 Cancers, for
example, have elevated thiol levels in comparison with their normal
paired tissue.3, 11-13 Hence, this property can be exploited for directed
delivery of drugs or fluorophores (for imaging) inside cells.3, 11-12, 14
Here, a bioactive molecule or fluorophore is conjugated to a scaffold
which is stable in buffer. Upon selective reaction with a thiol the
active drug molecule or fluorophore is released.15-16 The relative
abundance of thiols in cancers might facilitate site-specific
localization of the biomolecule. Among the methods available for
thiol-mediated activation and release, the disulfide-based scaffold,
which is cleaved by glutathione to initiate a rearrangement leading
result in the release a leaving group, LG.
been frequently used for labeling thiols in biomolecules24-25
supporting direct SN2 attack on the ethereal carbon, which
-Halo acetamides have
a
produces a phenolate and subsequent rearrangement can release
the bioactive molecule LG. The carbonyl group is distant from the
reaction centre but remains in conjugation while the aryl group
communicates with the ether carbon and together these groups
might help tuning of reaction rates. The p
leaving group ability of the linker can also be modulated by changing
substituent to attain the desired reaction rate and hence
Ka of the phenol and hence
X
spatiotemporally controlled release
to drug or fluorophore release, is widely used for drug delivery and
17-19
imaging.2,
However, disulfides are susceptible to cleavage not
just by glutathione but also by reductases20 and this scaffold does not
have any structural handle for controlling rate of release of the
bioactive small molecule. Hence, this method might not be suitable
for slow release of drugs, for example. Finn and coworkers reported
an oxanorbornadiene-based methodology for thiol activation and
release21-22 where the rate of drug release could be tuned by
structural modifications to the scaffold and is well suited for release
of a fluorophore, possibly for imaging. For applications in drug
release, however, this scaffold may have limited utility as the drug is
still conjugated to a furan. Here, we report results of our design,
synthesis and evaluation of a novel scaffold whose reaction rates
Scheme 1. Proposed 2‐methyl‐3‐phenacrylate scaffold for tunable thiol
activation. LG = leaving group
In order to synthesize this scaffold, bromide 2a was prepared
from the corresponding alcohol 1a by a PBr3-mediated bromination
reaction (Table 1, entry 1).26-27 The alcohol 1a was in turn prepared by
a Baylis-Hillman reaction of benzaldehyde and methyl acrylate.28-29
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Next, 2a was reacted with various phenols to produce the
corresponding phenol ethers 4a
(Table 2, entries 1-6).30
-9a in yields ranging from 75-95%
View Article Online
DOI: 10.1039/C4CC07343F
Table 1. Synthesis of bromides 2a-2f
O
OH
O
PBr3
OMe
OMe
DCM
0 oC to RT
E
E
Br
1a-1f
2a-2f
Entry
E
H
NO2
CN
Br
Reactant
Product
2a
2b
2c
2d
% Yield
79
89
71
71
1
2
3
4
5
6
1a
1b
1c
1d
1e
1f
Figure 1. (a) A comparison of GSH‐mediated decomposition of 4a
by HPLC analysis of reaction mixtures after 4 h and the reported pKas of the
corresponding leaving groups, 3a 3f (b) Time courses of GSH‐mediated
decomposition of 8a and formation of 3e were monitored by HPLC analysis.
Curve fitting gave rate constants for decomposition of 8a and formation of 3e
(c) Linear Free Energy Relationship Analysis of log(kx kH) obtained from rate
8f (Table 3) and Hammett
‐9a monitored
F
OMe
99
66
2e
2f
‐
;
;
Table 2. Synthesis of 4a-9a, 8b-8f and 10.
/
constants for thiol‐mediated decomposition of 8a
‐
X
Y
constants.
OMe
O
X
Y
O
O
Z
OMe
+
HO
Z
When 8c was reacted with thiophenol in pH 7.4 buffer, we find
3h (Figure 2) as the major product supporting direct displacement as
the dominant pathway (ESI, Figure S2). The literature contains
numerous examples of Michael attack by thiols suggesting the
Cs2CO3
CH3CN
E
Br
2a-2f
3a-3g
E
likelihood of this pathway.1,
If the rate determining step is
3,
5
Entry
1
E
H
H
H
H
H
H
RBr
2a
2a
2a
2a
2a
2a
2b
2c
2d
2e
2f
X
H
Br
H
H
Y
H
H
OMe
H
H
H
H
H
H
H
H
H
Z
Me
Me
H
CN
Me
NO2
Me
Me
Me
Me
ArOH
Prod
4a
5a
6a
7a
8a
9a
8b
8c
conjugate addition of the thiol, the rate of decomposition of the
compound must: (a) be significantly different from the rates of
appearance of phenolate; and (b) not depend on the leaving group
3a
3b
3c
3d
3e
3f
3e
3e
3e
3e
3e
3g
2
3
4
5
6
7
8
9
ability. However, due to nearly identical rates of decomposition (kx
Table 3) as well as release ( f, Table 3) and the dependence on the
leaving group (Figure 1a), it appears that N2 (or possibly N2’
,
NO2
H
k
NO2
CN
Br
NO2
NO2
NO2
NO2
NO2
NO2
S
S
followed by rapid thiol exchange) for displacement of the leaving
group by GSH is more likely (ESI, Figure S3). Furthermore, our results
are consistent with a previous report conducted with 2a and sodium
azide as the nucleophile, where the authors did not detect the
Michael-attack intermediate.27
8d
8e
8f
10
11
12
F
OMe
CN
Me
CH2OH
2c
8g
In order to study if these compounds were capable of reacting
with glutathione, 4a 9a were reacted with 10 eq. GSH in pH 7.4
buffer at 37 C and the amount of compound remaining was
-
Table 3. Glutathione-mediated decomposition of 8a-8f and release of 3e.
°
determined. A plot of % remaining versus pKa31of the corresponding
phenol leaving group shows a nearly linear relationship (Figure 1a)
suggesting that reactivity with thiols could be tuned.
b
a
0
0.77
0.67
0.23
0.05
0.29
Compd
8a
8b
8c
8d
E
H
NO2
CN
Br
kX
t1/2, hc
9.6
0.5
0.8
3.15
4.9
kfd
0.077
1.44
1.20
0.20
% Yielde
0.072
1.32
0.83
0.22
0.128
0.0274
78
88
79
73
84
79
Due to ease of monitoring of the product 3e formed during thiol-
mediated activation, we chose 8a for further studies. A time course
for decomposition of 8a (Figure 1b) in the presence of glutathione
F
OMe
0.124
0.030
8e
8f
25
aHammett constant. bPseudo first-order rate constant for decomposition
expressed in h1 was obtained by curve fitting (R2 ≥ 0.995, P-value <0.001,
see ESI Table S1); cHalf-life of decomposition of the compound; dFirst-order
rate constant for formation of 3e in h1 was determined by curve fitting (R2 ≥
0.995, P-value <0.001, see ESI Table S1); eMaximum yield of the phenol
obtained during the decomposition.
was conducted and a pseudo first order rate constant kx for loss of 8a
was found as 0.072 h-1, with a half-life of 9.6 h (Table 3). The formation
of 3e was monitored during the aforementioned reaction and a rate
constant of 0.072 h-1 and with an excellent yield of 3e (78%, Table 3).
We next tested if changing electronics on the aryl ring of this
scaffold influenced rates of thiol activation and release. Bromides
with electron withdrawing and electron donating groups attached to
the aryl group were prepared (Table 1); these bromides were reacted
The half-lives of decomposition of 8a-8f were found to be 30
min–1 day (Table 3) suggesting a high degree of tunability of release
of the free phenolate. The yields of 3e during decomposition was
>75% in a majority of the cases supporting the possibility of efficient
release upon activation by a thiol (Table 3). Linear free energy
with 3e to give 8b
Glutathione-mediated decomposition of these compounds (see ESI)
was conducted and rate constants ( x) for decomposition as well as
formation of 3e f) were determined (Table 3).
-8f in excellent yields (Table 2, entries 7-11).
k
relationship analysis (Figure 1c) of rates of decomposition of 8a-8f
(
k
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showed a nearly linear relationship between Hammett constant
and log(kx H). Linear regression analysis yielded a -value of +1.54 (
= 0.9944) supporting a moderate but predictable electronic effect on dimethylaminocoumaric acid (11), a fluorophore was carried out with

Next, in order to test the suitability of this scaffold for activation
/k

R
and release of a fluorophore, a coupling reaction of N,N-
View Article Online
DOI: 10.1039/C4CC07343F
thiol-mediated decomposition of phenacrylates (Figure 1c).
3g to produce 12 34-35
buffer while 11 is highly fluorescent under these conditions.36 When
reacted with thiol, decomposition of 12 should generate
.
The ester 12 is weakly fluorescent in aqueous
O
O
NH2
a
a
OMe
Ph
OMe
phenolate, which would self-immolate to generate 11 leading to a
significant increase in fluorescence intensity (Scheme 1). We studied
the reaction of 12 with 2-mercaptoethanol in methanolic pH 7.4
buffer and we found a gradual increase in fluorescence during 5 h
(Figure 4a) attributable to the formation of 11 (Figure 4a, inset). The
yield of 11 under these conditions was estimated as 76% (calculated
from a calibration curve for 11, see ESI, Figure S5) suggesting the
potential for temporally controlled and efficient release.
NC
S
NC
O
O2N
3h
OMe
MeO
10
CN
O
O
O
O
O
NH
O
O2N
O
OH
O
O
N
O
O
N
OMe
9
11
12
`
Figure 2. Compounds 3h, 9, 10, 11 and 12.
With these results in hand, we tested if the scaffold was selective
towards thiols. When 8b was reacted with amino acids that do not
contain a free thiol, we found no evidence for decomposition after 4
h (see ESI, Figure S1); whereas this compound was nearly completely
decomposed when this reaction was conducted with glutathione
(Table 2) suggesting a high degree of selectivity towards activation
by biological thiols (see ESI, Figure S1).
Figure 4. (a) Decomposition of 12 in the presence of 2‐mercaptoethanol (MSH,
25 eq.) to release of 11 was monitored by fluorescence measurements over
several hours. Inset: time course of 11 generated during this experiment was
estimated based on a calibration curve for authentic 11 (see ESI, Figure S5). (b)
Solutions of 12 incubated in pH 7.4 alone and in the presence of MSH (25 eq.)
after 8 h. (c) Human cervical cancer HeLa cells were incubated with DMSO (Ctrl)
and 12 (10
(NEM, 100

M); + NEM indicates cells were treated with N‐ethylmaleimide

M) for 30 min followed by addition of 12 (10 M). Data presented is

for fluorescence measured after 2 h. (d) Live cell images of HeLa cells incubated
with 12 (10 μM) for 2 h at 37 °C: (i) brightfield, (ii) fluorescence and (iii) overlay.
Figure 3. Representative HPLC traces for decomposition of
glutathione (GSH, 10 eq.) to release of 10. The formation of an intermediate was
9 in the presence of
observed by HPLC analysis and MS analysis of this fraction suggested that this
intermediate was 3i. Ctrl is
aforementioned decomposition of
9
incubated in buffer for 4 h. (b) Time course of the
in the presence of GSH.
Under similar conditions, no significant increase in fluorescence
was observed in the absence of a thiol (Figure 4b) or in the presence
of esterase (see ESI) supporting the thiol-selectivity of the trigger.
A cell viability assay using human cervical cancer HeLa cells
9
In order to test the suitability of this scaffold for release of a
bioactive molecule, 4-(hydroxymethyl)-2-nitrophenol32-33
3g, Table 2,
entry 12) was prepared using a reported procedure. Reaction of 3g
with 2c gave 8g (Table 2, entry 12). This alcohol was further reacted
with 4-nitrophenyl (4-methoxyphenyl)carbamate to give
This compound upon exposure to thiol should undergo
decomposition to release the 2-nitrophenolate intermediate 3i
which in turn would undergo decomposition to produce CO2 and 4-
anisidine 10 (Figure 2). HPLC analysis of the reaction mixture of and
GSH showed gradual decomposition of during 4 h and concomitant
(
showed that the IC50 of this compound was >25 M suggesting that
this scaffold is itself not significantly toxic (see ESI, Figure S6).
Treatment of Hela cells with 12 and subsequent fluorescence
measurement showed enhanced fluorescence attributable to the
9 (Figure 2).
a
formation of 11 (Figure 4c). HeLa cells were incubated with
N-
,
ethylmaleimide (NEM), a quencher of free thiols for 30 min followed
by addition of 12. Here, we find significant decrease in fluorescence
indicating thiol activation as a mechanism for fluorophore release in
cells (Figure 4c). A similar experiment conducted using 12 in serum
9
9
formation of 10 (Figure 3a). In addition, the formation of an
intermediate was observed (Figure 3b); mass spectrometry of this
intermediate supported 3i (ESI, Figure S4) thus providing further
evidence for the proposed mechanism (ESI, Figure S3). In the absence
starved HeLa cells showed
a dose-dependent increase in
fluorescence supporting activation by thiols within cells to release
the fluorophore (see ESI, Figure S7).
Finally, HeLa cells were treated with 12 and fluorescence
microscopy revealed increased fluorescence corresponding to 11
of glutathione, we found
period (Ctrl, Figure 3).
9 to be stable in buffer during this time
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supporting localization of this fluorophore within cells (Figure 4d). 17. Z. B. Zheng, G. Zhu, H. Tak, E. Joseph, J. l. Eiseman and D. J.
Together our data suggests that this scaffold is capable of
permeating cells to deliver a bioactive cargo such as a fluorophore.
In summary, we report a novel scaffold that is suitable for thiol-
Creighton, Bioconjug. Chem., 2005, 16, 598-607.
18. L. R. Jones, E. A. Goun, R. Shinde, J. B. Rothbard, C.VHiew. CArotinclteaOgnalinnde
DOI: 10.1039/C4CC07343F
P. A. Wender, J. Am. Chem. Soc., 2006, 128, 6526-6527.
mediated activation and release of a drug or fluorophore with a high 19. S. Chen, X. Zhao, J. Chen, J. Chen, L. Kuznetsova, S. S. Wong and I.
degree of thiol-selectivity and half-lives ranging from 30 min to 1 Ojima, Bioconjug. Chem., 2010, 21, 979-987.
day.37-38 The most common methodology available i.e. the disulfide- 20. A. Meister, J. Biol. Chem., 1988, 263, 17205-17208.
based method has no structural handle for rate control. It is 21. V. Hong, A. A. Kislukhin and M. G. Finn, J. Am. Chem. Soc., 2009,
anticipated that the method presented here will be useful for
131, 9986-9994.
selective thiol modification, thiol-mediated drug release as well as 22. A. A. Kislukhin, C. J. Higginson, V. P. Hong and M. G. Finn, J. Am.
cellular imaging.25
Chem. Soc., 2012, 134, 6491-6497.
Notes and references
23. C. Yin, F. Huo, Z. J, R. Martinez-Manez, Y. Yang, H. Lv and S. Li,
Chem. Soc. Rev., 2013, 42, 6032-6059.
aIndian Institute of Science Education and Research Pune, Dr. Homi Bhabha
Road, Pashan Pune 411 008, Maharashtra, India. Fax: 91 20 2589 9790;
Tel: 91 20 2590 8090; E-mail: harinath@iiserpune.ac.in.
24. L. A. Marcaurelle, M. R. Pratt and C. R. Bertozzi, ChemBioChem
2003, , 224-228.
,
4
† These authors contributed equally
25. R. E. Hansen and J. R. Winther, Anal. Biochem., 2009, 394, 147-158.
26. Y. Zulykama and P. T. Perumal, Aus. J. Chem., 2007, 60, 205-210.
27. M. M. Sa, M. D. Ramos and L. Fernandes, Tetrahedron, 2006, 62
11652-11656.
‡
Electronic Supplementary Information (ESI) available: [Compound
characterization data, assay protocols and spectra]. See
DOI: 10.1039/c000000x/
,
28. D. Basavaiah, A. J. Rao and T. Satyanarayana, Chem. Rev., 2003,
103, 811-892.
1. C. Yin, F. Huo, J. Zhang, R. Martinez-Manez, Y. Yang, H. Lv and S.
Li, Chem. Soc. Rev., 2013, 42, 6032-6059.
29. C. G. L. Junior, F. P. L. Silva, R. G. d. Oliveira, F. L. Subrinho, N. G.
2. M. H. Lee, Z. Yang, C. W. Lim, Y. H. Lee, S. Dongbang, C. Kang
and J. S. Kim, Chem. Rev., 2013, 113, 5071-5109.
d. Andrade and M. L. A. A. Vasconcellos, J. Brazil. Chem. Soc.
2011, 22, 2220-2224.
,
,
3. X. Chen, Y. Zhou, X. Peng and J. Yoon, Chem. Soc. Rev., 2010, 39
,
30. A. B. Charette, B. Cote, S. Monroc and S. Prescott, J. Org. Chem.
1995, 60, 6888-6894.
2120-2135.
4. S. Krishnan, R. M. Miller, B. Tian, R. D. Mullins, M. P. Jacobson and
J. Taunton, J. Am. Chem. Soc., 2014, 136, 12624-12630.
31. M. D. Liptak, K. C. Gross, P. G. Seybold, S. Feldgus and G. C.
Shields, J. Am. Chem. Soc., 2002, 124, 6421-6427.
5. I. M. Serafimova, M. A. Pufall, S. Krishnan, K. Duda, M. S. Cohen,
R. L. Maglathlin, J. M. McFarland, R. M. Miller, M. Frödin and J.
32. R. Pasceri, D. Siegel, D. Ross and C. J. Moody, J. Med. Chem., 2013,
56, 3310-3317.
Taunton, Nat. Chem. Biol., 2012, 8, 471-476.
33. F. Touzeau, A. Arrault, G. Guillaumet, E. Scalbert, B. Pfeiffer, M.-C.
Rettori, P. Renard and J.-Y. Mérour, J. Med. Chem., 2003, 46, 1962-
1979.
6. V. Chudasama, M. E. Smith, F. F. Schumacher, G. Papaioannou D.
Fau - Waksman, G. Waksman, J. R. Baker and S. Caddick, Chem.
Commun., 2011, 47, 8781-8783.
34. Y. Hori, T. Norinobu, M. Sato, K. Arita, M. Shirakawa and K.
Kikuchi, J. Am. Chem. Soc., 2013, 135, 12360-12365.
35. S. Karthik, B. Saha, S. K. Ghosh and N. D. Pradeep Singh, Chem.
Commun., 2013, 49, 10471-10473.
7. D. Rabuka, J. S. Rush, d. G. W, P. Wu and C. R. Bertozzi, Nat.
Protoc., 2012, 7, 1052-1067.
8. D. A. Shannon, R. Banerjee, E. R. Webster, D. W. Bak, C. Wang and
E. Weerapana, J. Am. Chem. Soc., 2014, 136, 3330-3333.
9. M. H. Potashman and M. E. Duggan, J. Med. Chem., 2009, 52, 1231-
1246.
36. K. Tanabe, N. Hirata, H. Harada, M. Hiraoka and S.-i. Nishimoto,
ChemBioChem, 2008, 9, 426-432.
37. S. R. Malwal, D. Sriram, P. Yogeeswari and H. Chakrapani, Bioorg.
Med. Chem. Lett., 2012, 22, 3603-3606.
10. A. T. Dharmaraja, T. K. Dash, V. B. Konkimalla and H. Chakrapani,
Med. Chem. Commun., 2012, 3, 219-224.
38. S. R. Malwal, D. Sriram, P. Yogeeswari, V. B. Konkimalla and H.
Chakrapani, J. Med. Chem., 2012, 55, 553-557.
11. J. A. Cook, H. I. Pass, S. N. Iype, N. Friedman, W. DeGraff, A.
Russo and J. B. Mitchell, Cancer Res., 1991, 51, 4287-4294.
12. A. K. Godwin, A. Meister, P. J. O'Dwyer, C. S. Huang, T. C.
Hamilton and M. E. Anderson, Proc. Natl. Acad. Sci., 1992, 89
,
3070-3074.
13. R. J. Holland, A. E. Maciag, V. Kumar, L. Shi, J. E. Saavedra, R. K.
Prud'homme, H. Chakrapani and L. K. Keefer, Chem. Res. Toxicol.
2012, 25, 2670-2677.
,
14. S. R. Malwal, A. Labade, A. S. Andhalkar, K. Sengupta and H.
Chakrapani, Chem. Commun., 2014, 50, 11533-11535.
15. H. Chakrapani, A. E. Maciag, M. L. Citro, L. K. Keefer and J. E.
Saavedra, Org. Lett., 2008, 10, 5155-5158.
16. D. Andrei, A. E. Maciag, H. Chakrapani, M. L. Citro, L. K. Keefer
and J. E. Saavedra, J. Med. Chem., 2008, 51, 7944-7952.
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