Trimethyl-substituted carbamate as a versatile self-immolative linker for fluorescence detection of enzyme reactions

Article abstract of DOI:10.3390/molecules25092153

Self-immolative linker is a useful building block of molecular probes, with broad applications in the fields of enzyme activity analysis, stimuli-responsive material science, and drug delivery. This manuscript presents N-methyl dimethyl methyl (i.e., trimethyl) carbamate as a new class of self-immolative linker for the fluorescence detection of enzyme reactions. The trimethyl carbamate was shown to spontaneously undergo intramolecular cyclization upon formation of a carboxylate group, to liberate a fluorophore with the second time rapid reaction kinetics. Interestingly, the auto-cleavage reaction of trimethyl carbamate was also induced by the formation of hydroxyl and amino groups. Fluorescent probes with a trimethyl carbamate could be applicable for fluorescence monitoring of the enzyme reactions catalyzed by esterase, ketoreductase, and aminotransferase, and for fluorescence imaging of intracellular esterase activity in living cells, hence demonstrating the utility of this new class of self-immolative linker.

Full text of DOI:10.3390/molecules25092153

molecules
Article
Trimethyl-Substituted Carbamate as a Versatile
Self-Immolative Linker for Fluorescence Detection of
Enzyme Reactions
Noriaki Nakamura, Shohei Uchinomiya, Kazuya Inoue and Akio Ojida *
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan;
jokiuhj@gmail.com (N.N.); uchinomiya@phar.kyushu-u.ac.jp (S.U.); inoue.kazuya.751@s.kyushu-u.ac.jp (K.I.)
* Correspondence: ojida@phar.kyushu-u.ac.jp; Tel.: +81-92-642-6596
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Takuya Terai
Received: 21 April 2020; Accepted: 2 May 2020; Published: 5 May 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Self-immolative linker is a useful building block of molecular probes, with broad applications
in the fields of enzyme activity analysis, stimuli-responsive material science, and drug delivery.
This manuscript presents N-methyl dimethyl methyl (i.e., trimethyl) carbamate as a new class of
self-immolative linker for the fluorescence detection of enzyme reactions. The trimethyl carbamate was
shown to spontaneously undergo intramolecular cyclization upon formation of a carboxylate group,
to liberate a fluorophore with the second time rapid reaction kinetics. Interestingly, the auto-cleavage
reaction of trimethyl carbamate was also induced by the formation of hydroxyl and amino groups.
Fluorescent probes with a trimethyl carbamate could be applicable for fluorescence monitoring of the
enzyme reactions catalyzed by esterase, ketoreductase, and aminotransferase, and for fluorescence
imaging of intracellular esterase activity in living cells, hence demonstrating the utility of this new
class of self-immolative linker.
Keywords: fluorescent probe; self-immolative linker; trimethyl carbamate; enzyme detection
1. Introduction
Fluorescent probes are invaluable chemical tools for the detection of enzyme activities in biological
research. To date, a variety of small-molecule fluorescent probes have been developed to detect various
enzyme reactions, such as esterase/protease-catalyzed hydrolysis and oxidoreductase-catalyzed redox
reactions [1–4]. These probes are, in most cases, designed as fluorogenic substrates that emit bright
fluorescence in response to enzyme-mediated structural change. To achieve precise and sensitive
detection in biological systems, the probe should serve as an enzyme substrate and be efficiently
converted to a fluorescent product. In addition, the probe should be sufficiently stable under biological
conditions to avoid non-enzymatic degradation, such as hydrolysis, which would generate unwanted
background fluorescence signal. To fulfill these requirements, several types of self-immolative linker
have been devised and exploited as a building unit in probe design [5–7]. In this molecular design,
self-immolative linker is inserted between fluorophore and reactive group as a functional adaptor.
Upon enzymatic transformation, structural change on the reactive group triggers spontaneous cleavage
of the linker unit to liberate a fluorophore with bright emission. Representative self-immolative linkers,
such as p-hydroxybenzyl group and trimethyl lock system are shown in Figure 1a,b [8–10]. Although
these linkers have been widely used in fluorescent probe design, there is still need for development
of new type of self-immolative linker, which would allow us to sense various types of enzymatic
reactions with enhanced enzyme selectivity, higher reaction kinetics, and greater chemical stability.
In this study, we report a new class of self-immolative linker useful for fluorogenic detection of enzyme
reactions. Fluorescence-based study revealed N-methyl dimethyl methyl (i.e., trimethyl) carbamate as
Molecules 2020, 25, 2153; doi:10.3390/molecules25092153
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an auto-cleavable linker (Figure 1c), which is rapidly cleaved from the fluorophore upon generation of
a free carboxylate. Interestingly, this auto-cleavage reaction was also induced upon formation of an
amino and hydroxylate group on the trimethyl carbamate unit (Figure 1d). Utility of this linker was
demonstrated by the fluorescence monitoring of various enzymatic reactions and by the fluorescence
imaging of intracellular esterase activity in living cells.
Figure 1. Activation mechanisms of self-immolative linkers: (
methide, ( ) acyl transfer of trimethyl lock, and ( ) acyl transfer of the trimethyl carbamate developed
in this study.
a) 1,6-elimination to form quinone
b
c,d
2. Results
2.1. Development of N-Methyl Dimethyl Methyl Carbamate Linker
Our study first focused on the development of a carboxylate-appended carbamate linker, which
would be cleaved by the intramolecular acyl transfer reaction (Figure 1c). To this end, we synthesized
a series of coumarin carbamates, bearing different carboxylate units, and evaluated the rate of
spontaneous degradation under neutral aqueous conditions (50 mM HEPES buffer, pH 7.4) by
fluorescence measurement. The reaction kinetics parameters of coumarin probes are summarized in
Table 1. The non-fluorescent carbamate probe
fluorescence over 24 h, suggesting the spontaneous release of 7-hydroxycoumarin
(half-reaction time: t1/2 = 30 h). However, we found that the introduction of methyl group(s) at
-position of the carboxylate group greatly facilitated the release of , and the half-degradation time
3, bearing glycine linker, showed slow increment of
1
to be sluggish
α
1
(t1/2) of
α
-methyl carboxylate
4
was reduced to 0.58 h. This effect was more prominently observed
in -dimethyl carboxylate
α
,
α
5
(Figure 2), which showed over 5000-fold enhancement of reaction
rate (t1/2 = 21 s) than in
Thorpe-Ingold effect [11
3
. The large rate enhancements in
4 and 5 could be reasonably attributed to
–13], which contributed to increase the population of carboxylate group properly
oriented for intramolecular cyclization due to reactive rotamer effect. In contrast, the corresponding
methyl ester stably existed under neutral conditions, strongly suggesting the participation of
6

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carboxylate group of
5
in the auto-cleavage reaction. Compound 7, consisting of
-dimethyl-
with a moderate reaction kinetics (t1/2 = 31 min). A large increase of reaction rate was also
β
-amino acid unit, was
also stable under neutral conditions, whereas 8, bearing
degraded to
β,
β
β-alanine linker, spontaneously
1
observed in probe 9, possessing rigid azabicyclo[2.2.1]heptene linker (t1/2 = 1.2 min) [12]. On the other
hand, introduction of cyclopropyl [11] and proline linker resulted in deceleration of the degradation of
10 (t1/2 = 11.6 h) and 11 (t1/2 > 100 h), respectively.
Table 1. Summary of the reaction kinetics parameters of coumarin carbamate probes bearing different
linker units a.
1R
2
n.d.
3
4
1.2
0.58
5
120
0.0058
(21 s)
6
n.d.
c
b
b
k (h−1
)
0.023
t1/2 (h)
30
> 100 h
n.d.
(35 min)
1R
7
n.d.
8
1.3
0.53
9
36
0.019
(1.2 min)
10
0.060
11
n.d.
c
c
k (h−1
t1/2 (h)
)
> 100 h
11.6
> 100 h
(32 min)
a
Reaction was monitored by the fluorescence spectral change of coumarin probe. Measurement conditions:
[probe] = 5 µM, 50 mM HEPES, pH 7.4, 37 ◦C, λex = 320 nm. Not determined due to instability of the probe under
measurement conditions. Not determined.
b
c
Figure 2. Fluorescence analysis of spontaneous degradation of
intensity of = 450 nm) released from . ( ) Fluorescence spectral change of
were conducted at 0.5, 1, 1.5, 2, 2.5, and 3 min after addition of into the neutral buffer solution.
Measurement conditions: (5) = 5 µM, 50 mM HEPES, pH 7.4, 37 ◦C, λ = 320 nm.
5. (
a) Time-trace plot of the fluorescence
1
(λ
em
5
b
5. Measurements
5
ex
The rapid reaction kinetics of
spontaneous degradation triggered by other functional groups. We thus prepared a set of coumarin
derivatives 12 16 and evaluated their auto-cleavage reactions by fluorescence measurement under
neutral aqueous conditions (50 mM HEPES buffer, pH 7.4). The results are summarized in Table 2. We
found that the amine probe 12 rapidly liberated coumarin (t1/2 = 75 s) while its corresponding N-Boc
derivative 13 remained stable. Interestingly, the hydroxyl derivatives 14 and 15 decomposed with a
moderate reaction kinetics to release (t1/2 = 1.1 and 0.92 h, respectively). On the other hand, the ketone
5 prompted us to evaluate the utility of trimethyl carbamate in
–
1
1

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16 stably existed under neutral aqueous conditions. The results suggested nucleophilic amino and
hydroxyl groups of 12 14, and 15 participated in intramolecular cyclization, thereby inducing the
,
spontaneous release of coumarin 1 (Figure 1d).
Table 2. Summary of the reaction kinetics parameters of trimethyl carbamate probes possessing
different functional groups a.
1R
5
120
12
92
13
n.d.
b
k (h−1
)
0.0058
(21 s)
0.021
(75 s)
t1/2 (h)
> 100 h
1R
14
0.64
1.1 h
15
0.75
0.92 h
16
k (h−1
t1/2 (h)
)
n.d.b
> 100 h
a
Reaction was monitored by fluorescence spectral change of coumarin probe. Measurement conditions:
[probe] = 5 µM, 50 mM HEPES, pH 7.4, 37 ◦C, λex = 320 nm. Not determined.
b
2.2. Fluorescence Detection of Enzyme Reactions with the Trimethyl Carbamate Linker
With the versatile trimethyl carbamate as self-immolative linker in hand, we next evaluated its
utility in fluorescence detection of enzyme reactions. We initially employed the methyl ester
hydrolysis reaction catalyzed by porcine liver esterase (PLE). However, the fluorescence increase was
rather small even after 6 h, suggesting as not-so-suitable substrate of PLE. To facilitate the enzyme
6 for the
6
reaction, we designed the phenyl ester 17 and evaluated its hydrolysis efficiency (Figure 3a). Results
showed the rate of fluorescence increased depending on the concentration of PLE (0–2.0 unit/min)
and the emission intensity increased up to >2000-fold at the reaction saturation point (Figure 3b,c).
The data together suggested that 17 served as a good fluorogenic substrate of PLE. We further evaluated
the hydrolysis efficiency of 17 by other esterases. The data indicated that 17 served as a fluorogenic
substrate of esterases E2 and E3, but not of carboxy esterases 1 and 2, and esterase E1 (Figure 3d).
This enzyme specificity might be attributable to the unique phenyl ester structure of 17 connected to
trimethyl carbamate linker.
Utility of the self-immolative linker was further evaluated in fluorescence detection of enzymatic
reactions catalyzed by transaminase and ketoreductase (Figure 4a). We found that the fluorescence of
keto probe 16 increased upon treatment with ketoreductase K-6 (Figure 4b). The rate of fluorescence
increase (
λ
= 450 nm) depended on the amount of ketoreductase. Fluorescence change was not
em
observed in the absence of NADPH, which is an enzyme co-factor. These results suggested the
reduction of 16 by K-6 to the corresponding alcohol 15, which spontaneously liberated coumarin to
1
exhibit enhanced fluorescence. We also found that 16 served as a substrate of transaminase (Figure 4c).
Treatment of 16 with transaminase TA-3 in presence of methylbenzylamine as a co-substrate induced
an increase in fluorescence while removal of methylbenzylamine from the reaction solution completely
suppressed the increase. Other transaminases TA-1 and TA-2 induced slight fluorescence enhancements.
These data suggested that TA-3 efficiently catalyzed the conversion of 16 to the corresponding amine
12, which spontaneously decomposed to fluorescent coumarin 1.

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Figure 3. Fluorescence detection of esterase-catalyzed hydrolysis of the probe 17. (
reaction of 17, consisting of esterase-catalyzed hydrolysis and subsequent spontaneous degradation.
) Fluorescence spectral change of 17 upon incubation with PLE (1.0 unit) for 0, 0.5, 1, 1.5, 2, 2.5,
3, and 4 h (from bottom to top). ( ) Time-trace plots of change in fluorescence intensity ( = 450
nm) upon incubation of 17 with 0, 0.1, 0.25, 0.5, 1, and 2 units of PLE (from lower to upper curve).
a) Sequential
(
b
c
λ
em
(
d
) Fluorescence intensity change of 17
(
λ
= 450 nm) upon incubation with various esterases for 3 h.
em
Measurement conditions: [17] = 5 µM, 50 mM HEPES, pH 7.4, 37 ◦C, λ = 320 nm.
ex
Figure 4. Fluorescence detection of enzyme-catalyzed conversion of keto probe 16. (a) Sequential
reactions of 16 to release
intensity change ( em = 450 nm) upon incubation of 16 with 0.2, 0.5, 1, 2, and 5 mg/mL of ketoreductase
K-6 (from lower to upper curve) in presence of NADPH. The lowest black line indicates the result with
5 mg/mL of K-6 in absence of NADPH. Measurement conditions: [16] = 10 M, [NADPH] = 0.2 mg/mL,
[glucose] = 10 mg/mL, [glucose dehydrogenase] = 0.2 mg/mL, = 320 nm, 50 mM phosphate buffer,
1, induced by ketoreductase or transaminase. (b) Time-trace plot of fluorescence
λ
µ
λ
ex
pH 8.0, 30 ◦C. (
16 with transaminase TA-3 in the presence or absence of methylbenzylamine (MBA). Measurement
conditions: [16] = 10 M, (transaminase) = 5 mg/mL, (MBA) = 11.5 M, (pyridoxal phosphate) = 0.1 µM,
= 320 nm, 100 mM phosphate buffer, pH 7.0, 30 ◦C.
c) Time-trace plot of fluorescence intensity change (
λ
em
= 450 nm) upon incubation of
µ
µ
λ
ex
Finally, we further evaluated the utility of trimethyl carbamate in the fluorescence imaging of
esterase activity in living cells. We prepared 3-carboxyamide coumarin probe 18, which was able to be

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excited with blue laser (
λex = 405 nm) for confocal fluorescence microscopy analysis (Figure 5). Cell
viability assay revealed that probe 18 did not exert significant cells toxicity at 10
µM (Figure S1). We also
confirmed that 18 was stable in the presence of physiologically relevant concentration of glutathione
(5 mM) under the neutral conditions (Figure S2). When A549 cells were incubated with 18 (10 µM in
HBS) at 37 ◦C, fluorescence due to coumarin gradually increased in a time-dependent manner in the
cytosol of cells. The fluorescence intensity apparently decreased at 4 h when the cells were pre-treated
with phenylmethylsulfonyl fluoride (PMSF) as a pan-esterase inhibitor. The results together suggested
that 18 was hydrolyzed by intracellular esterase(s) to liberate the fluorescent coumarin 1 inside the cells.
O
O
N
H
O
N
O
O
O
O
18
18
Probe
(−)
(−)
(+ )
(+ )
(+ )
(+ )
(+ )
PM SF
(−)
(−)
(−)
0.5 h
2 h
4 h
4 h
Figure 5. Fluorescence detection of intracellular esterase activity in living A549 cells using 18. Cells
were incubated with 18 at the indicated time at 37 ◦C in the presence or absence of PMSF. Conditions:
(18) = 10 µM, (PMSF) = 500 µM, HBS buffer, λ = 405 nm. Scale bar: 20 µm.
ex
3. Materials and Methods
3.1. Fluorescence Measurement and Reaction Kinetics Analysis of the Coumarin Probes
The fluorescence spectrum was measured by a PerkinElmer LS-55 spectrofluorophotometer.
A quartz cell containing the coumarin probe (5
µM) in HEPES buffer (50 mM, pH = 7.4) was placed at
the cell holder and measured the fluorescence (λex = 320 nm) by spectral mode or time-drive mode.
For reaction kinetics analysis, plot of the fluorescence intensity at 450 nm was analyzed by nonlinear
least squares curve-fitting as a first order reaction to obtain rate constant (k, h−1) and t1/2 (h).
3.2. Fluorescence Detection of Esterase Activity Using Probe 17
A HEPES buffer (50 mM, pH = 7.4) containing probe 17 (10
µM) and esterase was incubated at
37 ◦C. The fluorescence spectrum was measured at each reaction time point using PerkinElmer LS-55
spectrofluorophotometer (λ = 320 nm).
ex
3.3. Fluorescence Detection of Ketoreductase Activity Using Probe 16
A phosphate buffer (50 mM, pH = 8.0) containing probe 16 (10 µM), ketoreductase (5 mg/mL,
kindly provided from Amano Enzyme Inc., Aichi, Japan), NADPH (0.2 mg/mL), glucose dehydrogenase
(0.2 mg/mL) and glucose (10 mg/mL) was incubated at 30 ◦C. The fluorescence intensity of the solution

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(λ
em
= 450 nm) was measured at each time point by PerkinElmer EnSpire Multimode Plate Reader
(λex = 320 nm).
3.4. Fluorescence Detection of Transaminase Activity Using Probe 16
A phosphate buffer (100 mM, pH = 7.0) containing probe 16 (10
kindly provided from Amano Enzyme Inc.), methylbenzylamine (11.5
µ
M), transaminase (5 mg/mL,
M), and pyridoxal phosphate
M) was incubated at 30 ◦C. The fluorescence intensity of the solution (
(100 µ λem = 450 nm) was measured
µ
at each reaction time point using PerkinElmer EnSpire Multimode Plate Reader (λ = 320 nm).
ex
3.5. Cell Culture
A549 cells were cultured in high-glucose Dulbecco’s Modified Eagle medium (DMEM, 4.5 g
of glucose/L, Sigma-Aldrich, St. Louis, United States) supplemented with 10% fetal bovine serum
(FBS), penicillin (100 units/mL) and streptomycin (100 µg/mL) under humidified atmosphere of 5%
CO2 in air. Subculture was performed every 3–4 days from subconfluent (~80%) cultures using
trypsin-EDTA solution.
3.6. Evaluation of Cell Viability after Treatment of A549 Cells with Probe 18
A549 cells seeded on 3.5 cm dish (Falcon) were cultured for 2 days at 37 ◦C in CO2 incubator.
The A549 cells were treated with or without probe 18 (10 µ
M) for 3 h at 37 ◦C in HBS(+) buffer (20 mM
HEPES, 107 mM NaCl, 6 mM KCl, 1.2 mM MgSO4, 2 mM CaCl2, 11.5 mM glucose, adjusted to pH 7.4
with NaOH). The cell viability was evaluated by the standard typan blue assay. Error bars represent
standard deviation from the mean (n = 3).
3.7. Evaluation of Reaction of 18 and Glutathione
A degassed HEPES buffer (50 mM, pH = 7.4) containing probe 18 (10
µM), TCEP (10 mM) and
glutathione (5 mM) was incubated at 37 ◦C for 4 h. The fluorescence spectrum was measured using
PerkinElmer LS-55 spectrofluorophotometer (λ = 405 nm).
ex
3.8. Fluorescence Imaging of Intracellular Esterases in Living A549 Cells
A549 cells seeded on 3.5 cm glass-base dish (Iwaki) were cultured for 2–3 days at 37 ◦C in CO2
incubator. The cells were washed with HBS (+) buffer twice and treated with probe 18 for 4 h at 37 ◦C
in HBS (+) buffer. In control experiment, the cells were pre-treated with PMSF (500
37 ◦C in HBS (+) buffer, and then treated with probe 18 as described above in the presence of PMSF
(500 M). The cells were subjected to fluorescence imaging without wash. Fluorescence imaging was
µM) for 2 h at
µ
performed with confocal microscopy (TCS SP8, Leica microsystems) equipped with HyD detector
(405 nm excitation derived from a semiconductor laser).
4. Conclusions
We have reported the development of trimethyl carbamate as a new class of self-immolative linker.
The rapid-to-moderate reaction kinetics of trimethyl carbamate, in response to the enzyme-catalyzed
structural change, while the high chemical stability of the parent ester and keto probe, allowed us to
detect the activities of different enzymes, including esterase, ketoreductase, and transaminase. We
believe that, as a versatile building block, trimethyl carbamate could widen the choice of available
self-immolative linkers in fluorescent probe design, and thereby contribute to development of
fluorescence-based biological analyses. Not only in enzyme activity analysis, trimethyl carbamate
could also serve as a functional scaffold in stimuli-responsive materials and drug delivery systems.
Supplementary Materials: The following are available online, detail of synthesis of compounds. Figure S1.
Evaluation of cell viability after treatment of A549 cells with probe 18, Figure S2. Evaluation of stability of
coumarin probe 18 in the presence of glutathione.

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Author Contributions: Conceptualization, A.O.; methodology, A.O. and S.U.; software, N.N. and S.U.; validation,
formal analysis, investigation, N.N., S.U. and K.I.; resources, A.O.; data curation, N.N., S.U., K.I. and A.O.;
writing—original draft preparation, A.O.; writing—review and editing, A.O. and S.U.; visualization, A.O. and
S.U.; supervision, A.O.; project administration, A.O.; funding acquisition, A.O. All authors have read and agreed
to the published version of the manuscript.
Funding: This research was funded by a Grant-in-Aid for Scientific Research on Innovative Areas “Chemistry for
Multimolecular Crowding Biosystems” (JSPS KAKENHI Grant No. JP17H06349), Grant-in-Aid for Challenging
Exploratory Research (JSPS KAKENHI Grant No. JP17K19203). A.O. acknowledges Naito Science Foundation
and Toray Science Foundation for their financial supports.
Acknowledgments: We would like to thank Amano Enzyme Inc. for kindly providing esterases (E1-E3 and
CSE1-CSE2), ketoreductases K-6 and transaminases (TA1-TA3).
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
Chyan, W.; Raines, R.T. Enzyme-Activated Fluorogenic Probes for Live-Cell and in Vivo Imaging.
ACS Chem. Biol. 2018, 13, 1810–1823. [CrossRef] [PubMed]
Liu, H.W.; Chen, L.; Xu, C.; Li, Z.; Zhang, H.; Zhang, X.B.; Tan, W. Recent progresses in small-molecule
enzymatic fluorescent probes for cancer imaging. Chem. Soc. Rev. 2018, 47, 7140–7180. [CrossRef] [PubMed]
Garland, M.; Yim, J.J.; Bogyo, M.A. Bright Future for Precision Medicine: Advances in Fluorescent Chemical
Probe Design and Their Clinical Application. Cell Chem. Biol. 2016, 23, 122–136. [CrossRef] [PubMed]
Feng, L.; Ning, J.; Tian, X.; Wang, C.; Zhang, L.; Ma, X.; James, T.D. Fluorescent probes for bioactive detection
and imaging of phase II metabolic enzymes. Coord. Chem. Rev. 2019, 399, 213026. [CrossRef]
Gnaim, S.; Shabat, D. Activity-Based Optical Sensing Enabled by Self-Immolative Scaffolds: Monitoring
of Release Events by Fluorescence or Chemiluminescence Output. Acc. Chem. Res. 2019, 52, 2806–2817.
[CrossRef] [PubMed]
6.
Alouane, A.; Labruére, R.; Saux, T.L.; Schmidt, F.; Jullien, L. Self-immolative Spacers: Kinetic Aspects,
Structure-Property Relationships, and Applications. Angew. Chem. Int. Ed. 2015, 54, 7492–7509. [CrossRef]
[PubMed]
7.
8.
9.
Shan, D.; Nicolaou, M.G.; Borchard, R.T.; Wang, B. Prodrug Strategies Based on Intramolecular Cyclization
Reactions. J. Pharm. Sci. 1997, 86, 765–767. [CrossRef] [PubMed]
Haba, K.; Popkov, M.; Shamis, M.; Lerner, R.A.; Barbas, C.F., III; Shabat, D. Single-Triggered Trimeric
Prodrugs. Angew. Chem. Int. Ed. 2005, 44, 716–720. [CrossRef] [PubMed]
Lavine, M.N.; Hoang Trish, T.; Raines, R.T. Fluorogenic Probe for Constitutive Cellular Endocytosis. Chem. Biol.
2013, 20, 614–618. [CrossRef] [PubMed]
10. Chandran, S.S.; Dickson, K.A.; Raines, R.T. Latent Fluorophore Based on the Trimethyl Lock. J. Am. Chem. Soc.
2005, 127, 1652–1653. [CrossRef] [PubMed]
11. Jung, M.E.; Piizzi, G. gem-Disubstituent Effect: Theoretical Basis and Synthetic Applications. Chem. Rev.
2005, 105, 1735–1766. [CrossRef] [PubMed]
12. Lightstone, F.C.; Bruice, T.C. Geminal Dialkyl Substitution, Intramolecular Reactions, and Enzyme Efficiency.
J. Am. Chem. Soc. 1994, 116, 10789–10790. [CrossRef]
13. Bruice, T.C.; Pandit, U.K. The Effect of Geminal Substitution Ring Size and Rotamer Distribution on the
Intramolecular Nucleophilic Catalysis of the Hydrolysis of Monophenyl Esters of Dibasic Acids and the
Solvolysis of the Intermediate Anhydrides. J. Am. Chem. Soc. 1960, 82, 5858–5865. [CrossRef]
Sample Availability: Samples of the compounds are available from the authors.
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