Development of a new enzyme-responsive self-immolative spacer conjugate applicable to the controlled drug release

Article abstract of DOI:10.1016/j.bmc.2012.04.012

A new self-immolative spacer conjugate based on a chemical adaptor unit aiming at controlled releasing drugs was designed and synthesized. It releases a fluorophore which was used as a model drug via a spontaneous cyclization mechanism after cleavage of an enzyme substrate. This system was proved to be stable under physiological conditions and only decomposed triggered by enzyme. It provides a generic linkage allowing connection of a variety of drugs and targeted devices to the chemical adaptor.

Full text of DOI:10.1016/j.bmc.2012.04.012

Bioorganic & Medicinal Chemistry 20 (2012) 3465–3469
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of a new enzyme-responsive self-immolative spacer conjugate
applicable to the controlled drug release
⇑
Hui-juan Jin , Jing Lu, Xue Wu
Key Laboratory of Natural Resources of Changbai Mountain & Functional Molecules, Yanbian University, Ministry of Education, Yanji 133002, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A new self-immolative spacer conjugate based on a chemical adaptor unit aiming at controlled releasing
drugs was designed and synthesized. It releases a fluorophore which was used as a model drug via a
spontaneous cyclization mechanism after cleavage of an enzyme substrate. This system was proved to
be stable under physiological conditions and only decomposed triggered by enzyme. It provides a generic
linkage allowing connection of a variety of drugs and targeted devices to the chemical adaptor.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 26 January 2012
Revised 4 April 2012
Accepted 5 April 2012
Available online 19 April 2012
Keywords:
Drug release
Enzyme-responsive spacer
Self-immolative spacer
Conjugate
Controlled release
1. Introduction
ously cyclizes to form piperazine-2,5-dione derivative (13) and the
model drug releases from the spacer conjugate at the same time.
Delivering a parent drug via prodrug or reversible carrier-drug
conjugate has been considered as one of the most important and
promising therapeutic tool in nanomedicine, especially in cancer
therapy and typically being associated with severe side effects of
the drugs.1–5 The free parent drug was bounded to the linker or
carrier and could be released at the target site activated by site-
specific enzyme under physiological conditions to exert a con-
trolled drug release.6–17 This way, the use of enzymes to release
the drug could be carried out under very mild neutral and aqueous
conditions and the side effect or drug toxicity could be avoided.
From the chemical concept, one of the component of the pro-
drug or reversible carrier-drug bioconjugate is self-immolative
spacer which should be designed rationally in order to be applied
in the different usages. The spacer can incorporate three parts: tar-
get device, site-specific activation trigger and parent drug.18–22
2. Results and discussion
2.1. Synthesis
For the purpose of constructing three-components spacer con-
jugate with the respective required functions, 2-amino-3-mercap-
topropionic acid (L-cysteine) is chosen as the central core. It has
three different functional groups, SH, CO2H and NH2, suitable for
linkage. In here, this conjugate includes enzyme substrate, reporter
and solubilizer. A phenylacetamide group, known to be a substrate
for the enzyme penicillin-G-amidase from Escherichia coli (PGA), is
used as trigger and is attached to amino group through glycine
moiety. Cleavage of the enzyme substrate triggers the release of
the reporter from the spacer conjugate. 7-Amino-3-trifluorometh-
ylcoumarin is selected as the reporter unit and linked to spacer via
carbamate bond. With the concept of attaching the molecules
bearing different functional groups to the self-immolative spacer
In here, we choose an amino acid L-cysteine bearing three dif-
ferent functional groups as central core and design an enzyme-
responsive self-immolative spacer conjugate (Fig. 1). Solubilizer,
enzyme substrate and model drug are conjugated to the corre-
sponding position of the central core in which tris-TEG as solubiliz-
er, phenylacetyl group as enzyme substrate and fluorescent 7-
amino-4-trifluoromethylcoumarin (AFC) as model drug. Cleaving
the enzyme substrate can generate an amine group that spontane-
conjugate, 4-hydroxymethylphenol is selected between L-cysteine
and reporter. Therefore, diverse molecules containing NH2, CO2H,
or OH moieties can be coupled to 4-hydroxy group through carba-
mate, ester, and carbonate bonds, respectively. To improve the sol-
ubility of the spacer conjugate in aqueous circumstance tris-
triethylene glycol chains are introduced in here.
Spacer conjugate 1 was synthesized according to the route
shown in Scheme 1. Compound 2 containing enzyme substrate
⇑
Corresponding author. Tel.: +86 13844371179.
E-mail address: jinhj@ybu.edu.cn (H. Jin).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.012

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H. Jin et al. / Bioorg. Med. Chem. 20 (2012) 3465–3469
Figure 1. Structure of enzyme-responsive self-immolative conjugate 1.
Scheme 1. Synthesis of conjugate 1.
was formed by reacting glycine with phenylacetyl chloride in the
basic acetone solution. The functional group SH and CO2H of -cys-
2.2. Release studies
L
teine were protected with triphenylmethyl and methyl ester suc-
cessively to afford 4, which was further coupled with 2 to
produce compound 5. Hydrolysis of 5 gave carboxylic acid 6, which
was coupled with compound 7 (TBDMS-protected 4-hydroxymeth-
ylphenol) via EDC/DMAP condition to generate compound 8. De-
protecting of TBDMS group selectively using acidic amberlyst-15
afforded hydroxyl derivative 9. The benzylic alcohol was reacted
with 7-amino-4-trifluorocoumarin isocyanate to give 10, in which
AFC was attached to the spacer through stable carbamate forma-
tion. The trityl group was removed from 10 in 50% TFA/DCM solu-
tion catalyzed by triethylsilane (TES) to generate mercapto
derivative 11, which was then reacted with 12 to give the target
compound 1. Compound 12 was prepared from 3,4,5-tris(2-(2-(2-
methoxyethoxy)ethoxy)ethoxy)benzoic acid with S-(2-pyridyl-
thio)cysteamine via amide formation reaction using EDC/HOBt.
Conjugate 1 was incubated in phosphate buffer solution (PBS,
pH 7.4) at 37 °C (with or without PGA) and tested whether the re-
porter (AFC) can be released after activated by PGA which process
was monitored by fluorescence and UV-vis spectroscopy. Accord-
ing to our design (Fig. 2), the phenylacetamide group can be recog-
nized by enzyme PGA and the amide bond will be broken firstly.
The generated amine will attack ester bond and occurs tandem
cyclization reaction to generate piperazine-2,5-dione derivative
(13) and the ester bond breaks. Then 1,6-elimination will occur
within phenyl ring, and do expulsion of CO2 concomitantly. Fol-
lowing this, the reporter AFC can be released from the spacer con-
jugate immediately.
The fluorescence spectrum of conjugate 1 exhibited as strong
emission band at 391 nm and no significant fluorescence emission
at 494 nm (AFC fluorescence band, shown in Figure 3A, black line).

H. Jin et al. / Bioorg. Med. Chem. 20 (2012) 3465–3469
3467
Figure 2. Enzyme-responsive self-immolative mechanism of conjugate 1.
In order to test whether the AFC can be released from conjugate 1
by the catalytic activity of PGA, we incubated conjugate 1 in PBS
(pH 7.4) at 37 °C in the presence of PGA and followed the process.
As shown in Figure 3A, along with the time consuming the emis-
sion band of 391 nm decreased and a new strong band generated
at 494 nm which indicated that the catalytic cleavage of the phen-
ylacetamide bond and the release of free AFC do occurred. For fur-
ther evaluating the kinetic behavior of conjugate 1 releasing
reporter, the maximum intensities of the band at 494 nm were
plotted as a function of the time (Fig. 3B). The maximum fluores-
cence intensity was reached after about 4 h which showed that
the most reporters can be released during 4 h. Importantly, no
spontaneous AFC release was observed in the absence of the
enzyme.
In addition, GC–MS analysis of the enzyme catalytic hydrolysis
mixture of 1 was performed to confirm the enzyme-responsive re-
leased mechanism. The results proved the existence of the decom-
position fragments, phenylacetic acid (tR = 7.46 min, [M] = 136.05),
4-hydroxymethylphenol (tR = 10.21 min, [M] = 124.05), and AFC
(tR = 13.57 min, [M] = 229.04) and the efficacy of this spontaneous
release process.
3. Conclusion
0
A
0.5h
1000
800
600
400
200
0
1h
In conclusion, a new three-component enzyme-responsive self-
immolative cyclization spacer conjugate based on L-cysteine as the
1.5h
2h
391 nm
2.5h
3h
central core that allowed the release of fluorophore AFC triggered
by enzyme has been designed and synthesized. This spacer system
was proved to be stable under physiological conditions and only
decomposed trigger by enzyme. It provides a generic linkage allow-
ing connection of a variety of drugs and target devices to the chem-
ical adaptor. Now the solubilizer was displaced by mPEG and
further to form amphiphilic molecular and some continuous works
about amphiphilic drug carriers have been carried out in our lab.
3.5h
4h
4.5h
5h
5.5h
6h
494 nm
6.5h
7h
7.5h
8h
8.5h
9h
9.5h
10h
4. Experimental
350
400
450
500
550
600
650
All commercial available chemical reagents were purchased
from Aladdin, Alfa, GL (Shanghai, China) companies. Enzyme PGA
was purchased from Zhejiang Shunfeng Haider Company. 1H
Wavelength (nm)
NMR and 13C NMR spectra were recorded using
a AV300
600
500
400
300
200
100
0
B
(300 MHz, Bruker) spectrometer. The chemical shifts (ppm) were
expressed in d relative to TMS and the coupling constants J in Hz.
The MALDI-TOF MASS data were recorded using a MALDI AXI-
MA-CFR (PLUS, Japan; Bruker, Germany). Anhydrous solvent were
distilled as follows: tetrahydrofuran (THF) from sodium/benzophe-
none; dichloromethane (DCM) from calcium hydride; methanol
from magnesium.
with enzyme PGA
4.1. Chemistry
4.1.1. 2-(2-Phenylacetamido)acetic acid (2)
PBS buffer only
10
L-Glycin (100 mg, 1.33 mmol) was dissolved in 2 M sodium
hydroxide solution (2 mL), then acetone (2 mL) was added and
the mixture was cooled under ice-bath. Phenyl acetyl chloride
(161 lL, 1.21 mmol) was added dropwise at the same temperature,
0
1
2
3
4
5
6
7
8
9
Time (h)
followed by warming to room temperature. After 6 h, the solvent
was evaporated off, and the residue was adjust to pH = 2–3 using
1 M HCl and extracted with ethyl acetate. The combined organic
Figure 3. (A) Fluorescence emission spectrum of conjugate 1 with enzyme PGA
incubated at 37 °C in 10% DMSO–PBS buffer. (B) Fluorescence emission time-course
of conjugate 1 with or without enzyme.

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H. Jin et al. / Bioorg. Med. Chem. 20 (2012) 3465–3469
layer was dried over anhydrous magnesium sulfate, filtered, and
concentrated to give 2 (190 mg, 81%) as a white solid. 1H NMR
(300 MHz, acetone-d6): d = 7.41–7.20 (m, 5H), 3.95 (d, J = 5.74 Hz,
1H), 3.59 (s, 2H). 13C NMR (75 MHz, DMSO-d6): d = 717.53,
170.81, 136.32, 129.25, 128.36, 126.55, 42.15, 40.95. MALDI-TOF-
MS: Calcd for C10H12NO3 194.1. Found 194.1 [M+H]+.
91%) as a yellow solid. 1H NMR (300 MHz, CDCl3): d = 7.37–7.14 (m,
20H), 6.74 (d, J = 7.08 Hz, 1H), 6.53 (s, 1H), 4.29 (t, J = 12.6 Hz,
6.30 Hz, 1H), 3.82 (d, J = 5.34 Hz, 1H), 3.77 (d, J = 5.25 Hz, 1H),
3.54 (s, 2H), 2.68 (dd, J = 12.8 Hz, 5.94 Hz, 1H), 2.59 (dd,
J = 12.8 Hz, 8.04 Hz, 1H). 13C NMR (75 MHz, CDCl3): d = 172.76,
172.55, 169.34, 144.20, 134.30, 129.45, 129.35, 128.80, 128.00,
127.21, 126.85, 67.01, 60.52, 51.58, 42.80, 33.26. MALDI-TOF-MS:
Calcd for C32H30N2NaO4S 561.2. Found 561.2 [M+Na]+.
4.2.2. 2-Amino-3-(tritylthio)propanoic acid (3)
To a solution of
(15 mL) was added triphenyl methanol (1.64 g, 6.30 mmol), fol-
lowed by adding trifluoroboron ethylether (720 L, 5.69 mmol)
L-cysteine (1.0 g, 5.69 mmol) in acetic acid
4.2.6. 4-Tributyldimethylsilylhydroxymethylphenol (7)
4-Hydroxymethylphenol (600 mg, 4.83 mmol) was dissolved in
well-dried THF (5 mL) and cooled to 0 °C in ice-bath. Then imidaz-
ole (658 mg, 9.67 mmol) was added. After stirring for 10 min, tert-
butylchlorodimethylsilane (TBDMSCl, 729 mg, 4.83 mmol) was
added. The reaction was stirred at room temperature for 3 h. The
mixture was quenched with saturated NH4Cl and extracted with
ethyl acetate. The combined organic layer was dried over anhy-
drous magnesium sulfate, filtered and concentrated. The residue
was purified by column chromatography on silica gel (V(EA)/
V(Hexane) = 1:15) to give the desired compound 7 (860 mg, 75%).
1H NMR (300 MHz, CDCl3): d = 7.08 (d, J = 8.08 Hz, 2H), 6.66 (d,
J = 8.10 Hz, 2H), 5.18 (s, 1H), 4.57 (s, 1H), 0.84 (s, 9H), 0.7 (s, 6H).
13C NMR (75 MHz, CDCl3): d = 155.01, 132.89, 128.13, 115.37,
65.16, 26.11, 18.58, À5.03. ESI-MS: Calcd for C13H21O2Si 237.1.
Found 237.1 [MÀH]À.
l
dropwise and the reaction was stirred at room temperature. After
5 h, the reaction mixture was neutralized with saturated sodium
acetate. The resulting precipitate was washed with ethylether
and collected to give the desired compound 3 (1.87 g, 90%) as a
white solid. 1H NMR (300 MHz, acetone-d6): d = 7.46–7.23 (m,
15H), 3.64 (dd, J = 8.10 Hz, 4.20 Hz, 1H), 2.67–2.52 (m, 2H). 13C
NMR (75 MHz, DMSO-d6): d = 169.89, 144.42, 129.25, 128.11,
126.78, 65.95, 53.64, 34.39. MALDI-TOF-MS: Calcd for C22H20NO2S
362.1. Found 362.1 [MÀH]À.
4.2.3. Methyl 2-amino-3-(tritylthio)propanoate (4)
Compound 3 (500 mg, 1.38 mmol) was dissolved in well-dried
methanol (8 mL) and was stirred under reflux catalyzed by concen-
trated sulfuric acid (0.5 mL). After 4 h, the solvent was removed
under reduced pressure and the crude product was extracted with
ethyl acetate and washed with saturated sodium bicarbonate for
several times. The organic layer was dried over anhydrous magne-
sium sulfate, filtered and concentrated to give ester 4 (420 mg,
81%) as a yellow viscous oil. 1H NMR (300 MHz, CDCl3): d = 7.45–
7.18 (m, 15H), 3.66 (s, 3H), 3.20 (q, J = 7.61 Hz, 4.94 Hz, 1H), 2.59
(dd, J = 12.5 Hz, 4.76 Hz, 1H), 2.47 (dd, J = 12.4 Hz, 7.76 Hz, 1H),
1.65 (s, 2H). 13C NMR (75 MHz, CDCl3): d = 173.61, 144.15,
129.11, 127.53, 126.34, 66.31, 53.28, 51.58, 36.46. MALDI-TOF-
MS: Calcd for C23H23KNO2S 416.1. Found 416.1 [M+K]+.
4.2.7. 4-((Tert-butyldimethylsilyloxy)methyl)phenyl 2-(2-(2-
phenylacetamido)acetamido)-3-(tritylthio)propanoate (8)
A mixture of compound 6 (500 mg, 0.93 mmol), 7 (222 mg,
0.93 mmol), EDC (268 mg, 1.40 mmol), HOBt (190 mg, 1.40 mmol)
and TEA (260 lL, 1.86 mmol) in DCM (6 mL) was stirred at room
temperature for 5 h. After completion, the organic layer was
washed with saturated sodium bicarbonate, then dried over anhy-
drous magnesium sulfate, filtered and concentrated. The residue
was purified by column chromatography on silica gel (V(EA)/V(Hex-
ane) = 1:2) to give compound 8 (530 mg, 75%) as a light pink solid.
1H NMR (300 MHz, CDCl3): d = 7.24 (m, 24H), 6.50 (d, J = 7.52 Hz,
1H), 6.14 (s, 1H), 4.72 (s, 2H), 4.66 (t, J = 11.5Hz, 6.34 Hz, 1H),
4.12 (d, J = 4.91 Hz, 2H), 3.59 (s, 2H), 2.88 (dd, J = 12.6 Hz, 6.13 Hz,
1H), 2.65 (dd, J = 12.49 Hz, 4.21 Hz, 1H), 0.94 (s, 9H), 0.10 (s, 6H).
13C NMR (75 MHz, CDCl3): d = 171.62, 168.98, 168.79, 149.10,
148.87, 144.09, 139.20, 134.59, 129.39, 129.27, 128.71, 127.97,
127.04, 126.84, 120.89, 67.03, 64.23, 60.29, 51.67, 43.01, 33.49,
25.87, 18.28, -5.32. MALDI-TOF-MS: Calcd for C45H50N2NaO5SSi
781.3. Found 781.3 [M+Na]+.
4.2.4. Methyl 2-(2-(2-phenylacetamido)acetamido)-3-(trityl-
thio)propanoate (5)
Compound 4 was dissolved in DCM (5 mL). EDC (183 mg,
0.95 mmol) and DMAP (97 mg, 0.79 mmol) were added. The reac-
tion was stirred at room temperature for 1 h. Then compound 2
(153 mg, 0.79 mmol) was added and the mixture was stirred for
further 26 h. After completion, the mixture was washed with satu-
rated sodium carbonate. The organic layer was dried over anhy-
drous magnesium sulfate, filtered and concentrated. The residue
was purified by column chromatography on silica gel (V(EA)/
V(Hexane) = 1:1) to give 5 (420.3 mg, 82%) as a white solid. 1H
NMR (300 MHz, CDCl3): d = 7.39–7.18 (m, 20H), 6.22 (d,
J = 7.62 Hz, 1H), 6.06 (s, 1H), 4.46 (dd, J = 12.0 Hz, 5.49 Hz, 1H),
3.84 (d, J = 4.80 Hz, 2H), 3.69 (s, 3H), 3.60 (s, 2H), 2.70 (dd,
J = 12.6 Hz, 6.12 Hz, 1H), 2.58 (dd, J = 12.5 Hz, 4.53 Hz, 1H). 13C
NMR (75 MHz, CDCl3): d = 171.47, 170.57, 168.51, 144.29, 134.54,
129.56, 129.10, 128.12, 127.47, 127.02, 107.84, 67.14, 52.75,
51.36, 43.46, 43.04, 33.59. MALDI-TOF-MS: Calcd for
4.2.8. 4-(Hydroxymethyl)phenyl 2-(2-(2-phenylacetamido)-
acetamido)-3-(tritylthio) propanoate (9)
To a solution of compound 8 (530 mg, 0.70 mmol) in methanol
(10 mL) was added amberlyst-15 (79.5 mg, 15% weight to com-
pound 8). The reaction was stirred at room temperature for 2 h.
The solid was filtered off and the solvent was removed under re-
duced pressure. The residue was purified by column chromatogra-
phy on silica gel (V(EA)/V(Hexane) = 1:2) to give 9 (396 mg, 88%) as
a white foamy solid. 1H NMR (300 MHz, CDCl3): d = 7.20 (m, 24H),
6.41 (d, J = 4.83 Hz, 1H), 4.59 (d, 3H), 3.80 (d, J = 4.98 Hz, 2H), 3.52
(s, 2H). 13C NMR (75 MHz, CDCl3): d = 171.80, 168.97, 168.83,
149.57, 148.26, 144.17, 139.20, 134.43, 129.53, 129.07, 128.18,
128.08, 67.29, 64.45, 51.65, 43.34, 42.98, 33.56. MALDI-TOF-MS:
Calcd for C39H36N2NaO5S 667.2. Found 667.2 [M+Na]+.
C
33H32N2NaO4S 575.2. Found 575.2 [M+Na]+.
4.2.5. 2-(2-(2-Phenylacetamido)acetamido)-3-(tritylthio)pro-
panoic acid (6)
Compound 5 (420 mg, 0.76 mmol) was dissolved in methanol
(5 mL) and 2 M sodium hydroxide (1 mL) was added. The reaction
was stirred at room temperature for 1.5 h. After completion, meth-
anol was removed under reduced pressure, and the basic aqueous
layer was washed with ethyl acetate followed by acidified with
1 M HCl. The acidic aqueous layer was extracted with ethyl acetate
and the organic layer was dried over anhydrous magnesium sulfate,
filtered and concentrated to give the desired compound 6 (371 mg,
4.2.9. 4-((2-Oxo-4-(trifluoromethyl)-2H-chromen-7-ylcarba-
moyloxy)methyl)phenyl 2-(2-(2-phenylacetamido)acetamido)-
3-(tritylthio)propanoate (10)
7-Amino-4-trifluoromethylcoumarin (36 mg, 0.16 mmol) was
dissolved in well-dried DCM (2 mL) under nitrogen, then cooled

H. Jin et al. / Bioorg. Med. Chem. 20 (2012) 3465–3469
3469
in ice-bath. TEA (43
l
L, 0.31 mmol) was added followed by tri-
V(MeOH) = 5:1) to give the conjugate 1 (12 mg, 52%). 1H NMR
(300 MHz, CDCl3): d = 7.59 (d, J = 7.76 Hz, 2H), 7.50 (d, J = 7.87 Hz,
2H), 7.42 (d, J = 8.21 Hz, 2H), 7.34 (s, 2H), 7.15 (d, J = 9.56 Hz,
2H), 7.03 (s, 1H), 6.66 (s, 1H), 5.17 (s, 2H), 5.09 (d, J = 6.03 Hz,
1H), 4.19 (m, 6H), 4.17 (d, J = 5.13 Hz, 2H), 3.50-3.85 (m, 29H),
3.37 (s, 10H), 3.33 (dd, J = 6.31 Hz, 1.63 Hz, 1H), 3.00 (q, 2H). 13C
NMR (75 MHz, DMSO-d6): d = 170.58, 169.56, 169.35, 165.71,
159.13, 158.68, 154.93, 153.11, 151.83, 149.67, 143.76, 140.01,
137.86, 136.26, 129.73, 129.58, 129.13, 128.19, 126.36, 121.75,
121.25, 119.26, 115.16, 107.68, 106.29, 105.08, 71.88, 71.31,
70.00, 69.88, 69.79, 69.64, 68.96, 68.40, 58.07, 54.83, 51.72,
42.08, 41.73, 37.29, 36.93. MALDI-TOF-MS: Calcd for
phosgene (55 mg, 0.19 mmol). The yellow solution was stirred at
the same temperature for 1.5 h. Then a solution of 9 (100 mg,
0.16 mmol) in DCM (1 mL) was added and the resulting mixture
was stirred for further 2 h at room temperature. The reaction mix-
ture was washed with saturated sodium bicarbonate. The organic
layer was dried over anhydrous magnesium sulfate, filtered and
concentrated. The residue was purified by column chromatography
on silica gel (V(EA)/V(Hexane) = 1:2) to give 10 (85 mg, 61%) as a
blue solid. 1H NMR (300 MHz, CDCl3): d = 7.40 (m, 24H), 7.05 (d,
J = 8.31 Hz, 2H), 6.67 (s, 1H), 6.40 (d, J = 7.53 Hz, 1H), 6.08 (s, 1H),
5.20 (s, 2H), 4.61 (d, J = 6.03 Hz, 2H), 3.84 (d, J = 5.13 Hz, 2H),
3.59 (s, 2H), 2.88 (dd, J = 12.8 Hz, 6.24 Hz, 1H), 2.65 (dd,
J = 12.6 Hz, 4.47 Hz, 1H). 13C NMR (75 MHz, CDCl3): d = 171.95,
171.39, 168.98, 168.93, 159.54, 155.45, 150.40, 144.13, 141.66,
141.23, 134.40, 133.68, 129.80, 129.52, 129.47, 129.07, 128.21,
127.49, 127.13, 126.00, 119.78, 113.36, 108.68, 106.25, 67.34,
66.75, 60.54, 51.73, 43.32, 43.04, 33.48. MALDI-TOF-MASS Calcd
for C50H40F3N3NaO8S 922.3. Found 922.5 [M+Na]+.
C
61H77F3N4O21S2 1322.5. Found 1323.0 [M]+.
4.2. Enzyme activity release
PGA (penicillin-G-amidase, 1 U/lL) was purchased from Zhe-
jiang Shunfeng Haider Co. Ltd. Compound 1 was dissolved in DMSO
(not more than 10%, V/V) and further diluted with 0.1 M PBS (pH
7.4) to give the final stock solution, followed by treating with
PGA. The incubation was kept at 37 °C and the fluorescence spectra
were measured every 30 min by RF-5301PC spectroscopy.
4.2.10. 4-((2-Oxo-4-(trifluoromethyl)-2H-chromen-7-ylcar-
bamoyloxy)methyl)phenyl- 3-mercapto-2-(2-(2-phenylacet-
amido)acetamido)propanoate (11)
To a solution of 10 (20 mg, 0.018 mmol) in DCM (1 mL) was
added trifluoroacetic acid (1 mL), followed by catalytic amount of
triethylsilane. The reaction was stirred at room temperature for
30 min. After completion, the reaction mixture was concentrated
and the residue was purified rapidly by column chromatography
on silica gel (V(EA)/V(Hexane) = 1:2) to give 11 (12 mg, 80%) as a
light blue solid. 1H NMR (300 MHz, DMSO): d = 8.60 (d,
J = 6.99 Hz, 1H), 8.37 (t, J = 5.35 Hz, 1H), 7.71–7.61 (m,overlap,
2H), 7.53–7.47 (m, overlap, 3H), 7.31–7.24 (m, 5H), 7.16 (d,
J = 6.87 Hz, 2H), 6.88 (s, 1H), 5.21 (s, 2H), 4.70 (dt, J = 6.45 Hz,
5.37 Hz, 1H), 3.82 (s, br, 2H), 3.49 (s, 2H), 2.97 (dd, J = 8.34 Hz,
5.10 Hz, 2H), 2.70 (t, J = 8.10 Hz, 1H), MALDI-TOF-MASS: Calcd for
Acknowledgment
The authors appreciate the financial support from the National
Natural Science Foundation of China (No. 20762011).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.04.012.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
C
31H26F3N3NaO8S 680.1. Found 680.1 [M+Na]+.
References and notes
4.2.11. 3,4,5-Tris(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-N-(2-
(pyridin-2-yldisulfanyl)ethyl)benzamide (12)
To a solution of 3,4,5-tris(2-(2-(2-methoxyethoxy)ethoxy)-eth-
oxy)benzoic acid in DCM (2 mL) were added S-(2-pyridylthio)-cys-
teamine23 (100 mg, 0.16 mmol), EDC (38 mg, 0.20 mmol), HOBt
1. Yoo, J. W.; Doshi, N.; Mitragotri, S. Adv. Drug Delivery Rev. 2011, 63, 1247.
2. Bayo-Puxan, N.; Dufresne, M.-H.; Felber, A. E.; Castagner, B.; Leroux, J.-C. J.
Control. Release 2011, 156, 118.
3. Wang, Y. C.; Wang, F.; Sun, T. M.; Wang, J. Bioconjug. Chem. 2011, 22, 1939.
4. Henne, W. A.; Doorneweerd, D. D.; Hilgenbrink, A. R.; Kularatne, S. A.; Low, P. S.
Bioorg. Med. Chem. Lett. 2006, 16, 5350.
5. Ruiz, J. F. M.; Radics, G.; Windle, H.; Serra, H. O.; Simplício, A. L.; Kedziora, K.;
Fallon, P. G.; Kelleher, D. P.; Gilmer, J. F. J. Med. Chem. 2009, 52, 3205.
6. Lee, M. R.; Jung, D. W.; Williams, D.; Shin, I. J. Org. Lett. 2005, 7, 5477.
7. Lee, M. R.; Baek, K. H.; Jin, H. J.; Jung, Y. G.; Shin, I. J. Angew. Chem., Int. Ed. 2004,
43, 1675.
(27 mg, 0.20 mmol) and TEA (55 lL, 0.39 mmol). The reaction
was stirred at room temperature for 5 h. After completion, the
reaction mixture was washed with sodium bicarbonate for twice
and the organic layer was dried over anhydrous magnesium sul-
fate, filtered and concentrated. The residue was purified by column
chromatography on silica gel (V(EA)/V(MeOH) = 10:1) to give com-
pound 12 (62 mg, 58%) as a white solid. 1H NMR (300 MHz, CDCl3):
d = 8.40 (d, J = 0.72 Hz, 1H), 8.38 (t, J = 4.02 Hz, 2.40 Hz, 1H), 7.58 (t,
J = 7.38 Hz, 5.58 Hz, 1H), 7.50 (d, J = 8.04 Hz, 1H), 7.17 (s, 2H), 7.13
(t, J = 4.89 Hz, 1.08 Hz, 1H), 4.19 (m, 6H), 3.82 (m, 6H), 3.71 (m, 8H),
3.65 (m, 12H), 3.54 (m, 6H), 3.38 (s, 3H), 3.36 (s, 5H), 3.01 (t,
J = 5.32 Hz, 2.53Hz, 2H). 13C NMR (75 MHz, CDCl3): d = 166.86,
159.03, 152.18, 149.42, 140.98, 136.89, 129.63, 121.10, 120.70,
106.81, 72.11, 71.63, 70.36, 70.22, 69.40, 68.68, 58.71, 53.40,
38.37, 37.71. MALDI-TOF-MASS: Calcd for C35H57N2O13S2 777.3.
Found 777.3 [M+H]+.
8. Biswas, S.; Dodwadkar, N. S.; Sawant, R. R.; Torchilin, V. P. Bioconjug. Chem.
2011, 22, 2005.
9. Thambi, T.; Yoon, H. Y.; Kim, K.; Kwon, I. C.; Yoo, C. K.; Park, J. H. Bioconjug.
Chem. 2011, 22, 1924.
10. Warnecke, A.; Kratz, F. . J. Org. Chem. 2008, 73, 1546.
11. Antczak, C.; Bauvois, B.; Monneret, C. Bioorg. Med. Chem. 2001, 9, 2843.
12. Niculescu-Duvaz, D.; Niculescu-Duvaz, I.; Friedlos, F.; Martin, J.; Spooner, R.;
Davies, L.; Marais, R.; Springer, C. J. Med. Chem. 1998, 41, 5297.
13. Shamis, M.; Lode, H. N.; Shabat, D. J. Am. Chem. Soc. 2004, 126, 1726.
14. Pessah, N.; Reznik, M.; Shamis, M.; Yantiri, F.; Xin, H.; Bowdish, K.; Shomron,
N.; Ast, G.; Shabat, D. Bioorg. Med. Chem. 2004, 12, 1859.
15. Sagi, A.; Segal, E.; Satchi-Fainaro, R.; Shatat, D. Bioorg. Med. Chem. 2007, 15, 3720.
16. Ruiz, J. F. M.; Radic, G.; Windle, H.; Sera, H. O.; Simplicio, A. L.; Kedziora, K.;
Fallon, P. G.; Kelleher, D. P.; gilmer, J. F. J. Med. Chem. 2009, 52, 3205.
17. Paul, G.; Heimink, J.; Koller, H. Chem. Mater. 2008, 20, 5083.
18. Gopin, A.; Rader, C.; Shabat, D. Bioorg. Med. Chem. 2004, 12, 1853.
19. Meyer, Y.; Richard, J. A.; Massonneau, M.; Renard, P. Y.; Romieu, A. Org. Lett.
2008, 10, 1517.
4.2.12. Conjugate 1
20. Jones, G. B.; Crasto, C. F.; Mathews, J. E.; Xie, L. F.; Mitchell, M. O.; El-Shafey, A.;
D’Amoco, A. V.; Bubley, G. J. Bioorg. Med. Chem. 2006, 14, 418.
21. Danieli, E.; Shabat, D. Bioorg. Med. Chem. 2007, 15, 7318.
22. Madec-Lougerstay, R.; Florent, J.-G.; Monneret, C. J. Chem. Soc., Perkin Trans. 1
1999, 1369.
A mixture of compound 11 (12 mg, 0.018 mmol) and compound
12 (12 mg, 0.015 mmol) was stirred in de-oxygen methanol (4 mL)
under nitrogen atmosphere for 24 h. After completion, the solvent
was evaporated off under reduced pressure. The crude product was
purified by column chromatography on silica gel (V(EA)/
23. Ebright, Y. W.; Chen, Y.; Pendergast, P. S.; Ebright, R. H. Biochemistry 1992, 31,
10664.