Photocleavage of o-nitrobenzyl ether derivatives for rapid biomedical release applications

Article abstract of DOI:10.1016/j.bmcl.2006.05.013

The externally controlled cleavage of covalently linked prodrugs, proteins, or solid-phase formulation vehicles offers potential advantages for controlled drug or gene delivery. A series of o-nitrobenzyl ester compounds (1-8) were synthesized to allow a systematic study of photolability. The o-nitrobenzyl ester was strictly required for photolability, while imido esters were not photolabile. The degradation kinetics of 1-o-phenylethyl ester was an order of magnitude faster than that of o-nitrobenzyl ester. Tosylate, phosphate, and benzoate derivatives of 1-o-nitrophenylethyl displayed similar photolability (>80% decomposition within 10 min at 3.5 mW/cm² at 365 nm). O-o-Nitrobenzyl O′,O″-diethyl phosphate displayed the fastest decomposition at photoirradiation condition (3.5 mW/cm², 365 nm) suitable for biological systems. We report the synthesis and photo-decomposition of 1-o-nitrophenylethyl derivatives amenable for the creation of photolabile prodrugs or formulation particles for drug depots, DNA condensation, or tissue engineering applications.

Full text of DOI:10.1016/j.bmcl.2006.05.013

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4007–4010
Photocleavage of o-nitrobenzyl ether derivatives for
rapid biomedical release applications
Moon Suk Kim and Scott L. Diamond*
Institute for Medicine and Engineering, Department of Chemical and Biomolecular Engineering, 1024 Vagelos Research Laboratory,
University of Pennsylvania, Philadelphia, PA 19104, USA
Received 3 April 2006; revised 3 May 2006; accepted 3 May 2006
Available online 19 May 2006
Abstract—The externally controlled cleavage of covalently linked prodrugs, proteins, or solid-phase formulation vehicles offers
potential advantages for controlled drug or gene delivery. A series of o-nitrobenzyl ester compounds (1–8) were synthesized to allow
a systematic study of photolability. The o-nitrobenzyl ester was strictly required for photolability, while imido esters were not pho-
tolabile. The degradation kinetics of 1-o-phenylethyl ester was an order of magnitude faster than that of o-nitrobenzyl ester.
Tosylate, phosphate, and benzoate derivatives of 1-o-nitrophenylethyl displayed similar photolability (>80% decomposition within
10 min at 3.5 mW/cm2 at 365 nm). O-o-Nitrobenzyl O0,O00-diethyl phosphate displayed the fastest decomposition at photoirradia-
tion condition (3.5 mW/cm2, 365 nm) suitable for biological systems. We report the synthesis and photo-decomposition of 1-o-nitro-
phenylethyl derivatives amenable for the creation of photolabile prodrugs or formulation particles for drug depots, DNA
condensation, or tissue engineering applications.
ꢀ 2006 Elsevier Ltd. All rights reserved.
The timed and accurate dosing of a gene or drug at a
specific location in vivo or in vitro presents a challenging
and diverse set of constraints.1 Controlled delivery and
release can be achieved by the use of specific external
stimulation such as temperature, pH, ultrasound, elec-
tric current, or photoexcitation. Caged compounds or
those with photo-cleavable linkers are able to decom-
pose rapidly upon photoirradiation and several biologi-
cal compounds have been examined.2–5 Among them,
biological compounds containing an o-nitrobenzyl
group appear as an attractive candidate to control the
release of gene and drug from a delivery formulation
triggered by photoirradiation.6–8 The photolytic mecha-
nism of o-nitrobenzyl derivatives is not yet fully estab-
lished, although evidence suggests the nitro group is
reduced to nitroso, while the benzyl carbon is oxidized
via an initiation by the attacking nitro-group oxygen.9
of o-nitrobenzyl derivatives allowed substantial improve-
ments of the rates of photocleavage. In this article, we
report in detail the decomposition kinetics associated with
photolysis of o-nitrobenzyl derivatives (Scheme 1).
A series of photo-labile compounds (1–8) were synthe-
sized by the reaction of p-toluene sulfonic chloride (tosyl
O
O
NO2
CH2
N
N
O
S
X=
X
O
CH3
O
O
O
1
2
3
NO2
CH2
O2N
CH2
O
P
NO2
CH2
O
P
O
O
O
OCH2CH3
OCH2CH3
We investigated photo-cleavable delivery chemistry suit-
able for photoirradiation in vivo and in vitro.10 The study
5
4
O
P
O
S
O
NO2
Keywords: Photocleavage; o-Nitrobenzyl; Drug delivery.
*
O
OCH2CH3
CH3
O
O
C
Y=
Corresponding author. Tel.: +1 215 573 5702; fax: +1 215 573
7227; e-mail: sld@seas.upenn.edu
OCH2CH3
CH
CH3
O Y
O
6
7
8
Present address: Medicinal Science Division, Korea Research
Institute of Chemical Technology, PO Box 107, Yuseong, Daejeon
305-600, Republic of Korea.
Scheme 1.
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.05.013

4008
M. S. Kim, S. L. Diamond / Bioorg. Med. Chem. Lett. 16 (2006) 4007–4010
NO2
chloride), benzoyl chloride, diethyl phosphorochlori-
date, and phenyl phosphonic dichloride with various
alcohols in the presence of an amine or sodium hydride.
The structures of 1–8 were confirmed by 1H, 13C NMR,
and elemental analysis.11–19
a
O
S
hv
CH2
O
CH3
O
R
O-
O-
N
N
OH
CH
O
CH
O R
Photo-decomposition reactions have employed various
photo-active compounds and various irradiation
protocols for biochemical or chemical research.5 The
photoreactions carried out in the body tend to use
photosource with comparable long wavelength
(k > 300 nm) to minimize damage to tissue.20–22 There-
fore, we have chosen o-nitrobenzyl group which can be
activated by photoirradiation of 365 nm. First, the
photoirradiation of 1–3 was carried out using 365 nm
(1.6 mW/cm2) for 60 min to compare photo-decomposi-
tion of o-nitrobenzyl and imide groups which have
photoactivity. 1H NMR monitored the structural changes
of 1–3 before and after irradiation. N-Hydroxylphthali-
mide tosylate 2 and N-hydroxylnaphthalimide tosylate 3
exhibited no change in 1H NMR after irradiation
(365 nm) for 60 min, while 1 showed the integration
decrease of benzyl proton and a new signal assignable to
methyl proton of p-toluene sulfonic acid formed by
decomposition of 1. Therefore, the integration changes
of 1H NMR were calculated as decomposition degree of 1.
O
R
NO
O
S
b
CH3
O
C
+
HO
H
O
50
40
30
20
10
0
1H NMR
300 nm
a
b
(ppm)
2.5
2.0
365 nm
400 nm
0
10
20
30
40
50
60
Irradiation Time (min)
For photolability, a strict requirement for the –NO2
group was consistent with the need for the formation
of nitroso intermediate to initiate the decomposition
reaction. Compound 1 displayed 6%, 27%, and 43%
decomposition after 60 min irradiation at 400, 365,
and 300 nm (Fig. 1). On irradiation of 1 at 365 nm
(1.6 mW/cm2) in various solvents, the ln[C]/[C]o versus
time plots exhibited the expected linear decrease
(Fig. 2). From the slope of the plots the values of appar-
Figure 1. Decomposition–irradiation time curves of 1 in CD3OD at
300, 365, and 400 nm.
0.0
-0.2
-0.4
-0.6
ent first order dissociation kinetic rate constants (kapp
)
and
were 8.83 · 10ꢀ4
,
1.31 · 10ꢀ4
,
1.63 · 10ꢀ4
,
1.12 · 10ꢀ4 sꢀ1, in CD3OD, DMSO-d6, dioxane-d8,
and CDCl3, respectively (Fig. 2).
Having verified a strict requirement for 1, phosphate
derivatives were tested for faster dissociation kinetics
in relation to the tosylate of 1–3. The photo-decomposi-
tion was carried out with O-o-nitrobenzyl O0,O00-diethyl
phosphate 4 at 365 nm with different power (0.3, 1.6,
and 3.5 mW/cm2) for 60 min. Decomposition was deter-
mined by the integration decrease of benzyl protons in
1H NMR. Compound 4 showed 84% decomposition at
3.5 mW/cm2 and 78% at 1.6 mW/cm2, while 40% at
0.3 mW/cm2. We compared the photo-decomposition
rate at 365 nm (1.6 mW/cm2) of 4 (one o-nitrobenzyl
group) with that of O,O0-di-o-nitrobenzylphenyl phos-
phonate 5 (two o-nitrobenzyl groups). A similar rate
of decomposition was obtained for 4 and 5 (in 78% at
60 min), even though 5 showed 67% increase of absor-
bance at 365 nm compared with 4.
0
10
20
30
40
50
60
Irradiation Time (min)
Figure 2. The plot for ln[C]/[C]o versus irradiation time of 1 in various
solvents (j) CD3OD, (m) DMSO-d6, (d) dioxane-d8, and (.) CDCl3.
methyl group was introduced at the benzyl position to
increase the decomposition rate. Figure 3 shows the
UV–vis spectra of 1-o-nitrophenylethyl tosylate 6, O-1-
o-nitrophenylethyl O0,O00-diethyl phosphate 7, and
1-o-nitrophenylethyl benzoate 8. The absorbance order
was 7 > 6 > 8 at 365 nm, but the spectra showed almost
identical absorbance from 300 to 450 nm, indicating the
absorbance of 6–8 depended mainly on the 1-o-nitrophe-
nylethyl group not on the other aromatic substitutes.
Long-term exposures at 365 nm can damage cells or
DNA in the body21,23 so maximizing the photocleavage
rate is desirable. Prior studies demonstrated that the
methyl group on the benzyl position enhanced the rela-
tive cleavage kinetics by a factor of 20.23 Therefore, a

M. S. Kim, S. L. Diamond / Bioorg. Med. Chem. Lett. 16 (2006) 4007–4010
4009
3.0
2.5
2.0
1.5
1.0
0.5
0.0
sition than 1–5. The decomposition order was 7 > 6 > 8,
consistent with the absorption strength at 365 nm
(Fig. 3). This result indicated that the decomposition
rate depended most strongly on the nitrobenzyl group
and to a much lesser extent on the substituent groups.
7
Figure 5 shows the HPLC chromatography (acetonitrile
and 1% TFA aqueous [80/20, v/v]) of 6 after irradiation
using 365 nm (3.5 mW/cm2) for 0–10 min. The peak
intensity of 6 decreased with increase of irradiation time,
while the intensity of the new signals ‘a’ and ‘b’, assigned
to o-nitrosoacetophenone and p-toluene sulfonic acid,
respectively, increased (Fig. 5A). The UV spectrum of
signal ‘a’ showed characteristic absorbance of nitroso-
acetophenone at 270–350 nm (Fig. 5B).
6
8
In conclusion, the photo-decomposition of o-nitrobenzyl
and o-nitrophenylethyl derivatives was carried out with
various photosources and power using long wavelength
UV (365 nm). The decomposition rate of o-nitrophenyl-
ethyl derivatives was significantly faster than the rate of
300
350
400
450
Wavelength (nm)
Figure 3. UV–vis spectra of 6–8 (4.4 mM in CDCl3).
The decomposition of 6–8 was monitored using 1H
NMR and HPLC after irradiation. Figure 4 shows the
1H NMR spectra of 6 before and after irradiation. In
the spectrum after irradiation, integration of signals ‘a’
and ‘b’ assignable to methyl and benzyl protons decreas-
es at 1.4 and 4.5 ppm, respectively. Meanwhile, new sig-
nals ‘d’ and ‘e’ appeared and the integration increased
with irradiation time.
NO2
H
C
O
S
O
CH3
NO
CH3
O
O
S
O
6
HO
CH3
+
CH3
C
O
a
b
A 250
b
From the decomposition–irradiation time curves for
6–8, we report the determination of the apparent first
order dissociation kinetic rate constants (kapp) = 4.33 ·
10ꢀ3 sꢀ1 (7), 3.17 · 10ꢀ3 sꢀ1 (6), and 3.05 · 10ꢀ3 sꢀ1 (8).
The decomposition rate of 6–8 showed faster decompo-
200
150
100
50
Irradiation (365 nm)
0
30 s
5 m
10 m
a
NO2
b
6
hv (365 nm)
4.4 mM
H
C
O
S
c
O
CH3
CH3
a
O
NO
O
e
O
C
0
d
CH3
+
HO
S
CH3
O
TMS
CHCl3
Retention Time(min)
H2O
c
B
a
(A)
100
b
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
80
60
40
20
0
d
(B)
e
c
a
H2O
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
6
(C)
b
H2O
200
250
300
350
400
Wavelength (nm)
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
Figure 4. 1H NMR spectra of 1-o-nitrophenethyl tosylate 6 before (A)
and after photoirradiation for 6 min (B) and 10 min (C).
Figure 5. HPLC charts for photo-decomposition of 6 (A) and UV
spectra of 6, ‘a’ and ‘b’ (B).

4010
M. S. Kim, S. L. Diamond / Bioorg. Med. Chem. Lett. 16 (2006) 4007–4010
15. Compound 5: yield 63%. 1H NMR (CDCl3): 7.91–7.46 (m,
13H, –(C6H5), –2(NO2C6H4)), 5.55 (m, 4H, 2(–CH2–)).
13C NMR (CDCl3): 153.0, 134.0, 133.1, 131.9, 131.7,
128.8, 128.5, 124.9, 64.4. Anal. Calcd for C20H17N2PO7:
C, 56.08; H, 4.00, N; 6.54. Found: C; 55.92; H; 3.93, N;
6.16.
o-nitrobenzyl derivatives. This detail decomposition
study can serve as the basis for the design of new gene
delivery system (i.e., photolytic cationic polymers) to
control gene release using photoirradiation. Further
research on the synthesis of gene carrier modified with
o-nitrobenzyl derivatives for triggered extracellular or
intracellular DNA release by photoirradiation is now
in progress.
16. 1-o-Nitrophenylethyl alcohol: yield 95%. 1H NMR
(CDCl3): 7.83–7.32 (m, 4H, –(C6H4)), 5.30 (m, 1H,
(–CH–)), 3.16 (br, 1H, OH), 1.46 (d, J = 6.2 Hz, CH3).
17. Compound 6: yield 69%. 1H NMR (CDCl3): 7.91–7.20 (m,
7H, –(C6H4), –(C6H3)), 6.12 (m, 1H, (–CH–)), 2.40 (s, 3H,
CH3), 1.64 (d, J = 6.2 Hz, CH3). 13C NMR (CDCl3):
144.8, 133.8, 133.1, 129.7, 128.7, 128.1, 127.7, 124.2, 75.3,
23.5, 21.5. Anal. Calcd for C15H15NSO5: C; 56.06, H; 4.70,
N; 4.36. Found: C; 56.54; H; 4.65, N; 4.07.
References and notes
1. Fisher, R. S.; Ho, J. CNS Drugs 2002, 16, 579.
2. Epe, B.; Muller, E.; Adam, W.; Saha-Moller, C. R. Chem.
Biol. Interact. 1992, 85, 265.
18. Compound 7: yield 65%. 1H NMR (CDCl3): 7.88–7.34 (m,
4H, –(C6H4)), 5.91 (m, 1H, –CH–), 3.93 (m, 4H, 2CH2),
1.60 (d, J = 6.2 Hz, 3H, CH3), 1.15 (m, 6H, 2CH3). 13C
NMR (CDCl3): 169.4, 146.6, 137.6, 133.5, 128.4, 127.6,
124.1, 71.7, 63.7, 24.1, 15.6. Anal. Calcd for C12H18NPO6:
C; 47.53, H; 5.98, N; 4.62. Found: C; 47.20, H; 6.37, N;
4.58.
3. Givens, R. S.; Kueper, L. W., III Chem. Rev. 1993, 93, 55.
4. Olejnik, J.; Sonar, S.; Krzymanska-Olejnik, E.; Roths-
child, K. J. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 7590.
5. Ramamurthy, V.; Schanze, K. S. Organic Molecular
Photochemistry; Marcel Dekker, Inc: New York, 1999.
6. Holmes, C. P.; Jones, D. G. J. Org. Chem. 1995, 60, 2318.
7. Holmes, C. P. J. Org. Chem. 1997, 62, 2370.
8. Smet, M.; Liao, L. X.; Dehaen, W.; McGrath, D. Org.
Lett. 2000, 2, 511.
19. Compound 8: yield 72%. 1H NMR (CDCl3): 8.08–7.25 (m,
9H, –(C6H4), –(C6H5)), 6.57 (m, 1H, (–CH–)), 1.79 (d,
J = 6.2 Hz, CH3). 13C NMR (CDCl3): 165.3, 147.6, 138.0,
133.6, 133.1, 129.5, 128.3, 127.0, 124.4, 68.7, 22.0. Anal.
Calcd for C15H13NO4: C; 66.41, H; 4.83, N; 5.16. Found:
C; 66.76, H; 5.02, N; 4.86.
20. Jean, S.; Bideau, C.; Bellon, L.; Halimi, G.; De Meo, M.;
Orsiere, T.; Dumenil, G.; Berge-Lefranc, J. L.; Botta, A.
Biochim. Biophys. Acta 2001, 1522, 89.
9. McCray, J. A.; Trentham, D. R. Annu. Rev. Biophys.
Biophys. Chem. 1989, 18, 239.
10. Kim, M. S.; Gruneich, J.; Diamond, S. L., in preparation.;
11. Compound 1: yield 51%. 1H NMR (CDCl3): 8.12–7.36 (m,
8H, –2(C6H4)), 5.48 (s, 2H, (–CH2–)), 2.46 (s, 3H, CH3).
Anal. Calcd for C14H13NSO5: C, 54.71; H, 4.26, N; 4.56.
Found: C; 54.68; H; 4.05, N; 4.11.
21. Setlow, R. B.; Grist, E.; Thompson, K.; Woodhead, A. D.
Proc. Natl. Acad. Sci. U.S.A. 1993, 87, 6666.
12. Compound 2: yield 51%. 1H NMR (CDCl3): 7.95–7.43 (m,
8H, –(C6H4), –(C6H4)), 2.50 (s, 3H, CH3). Anal. Calcd for
C15H11NSO5: C; 56.78, H; 3.49, N; 4.41. Found: C; 56.40;
H; 3.28, N; 4.22.
22. Photolysis was performed at a distance of 10 cm using
Rayonet Photochemical Chamber Reactor Model RPR-
100 (Southern New England Ultraviolet Company [0.6–
0.8 mW/cm2]) which was equipped with 12 UV lamps
(each 21 W) used for 300 nm and ENF-240 C (SPEC-
TRONICS Corp., Optical density is 0.3 mW/cm2 at
15 cm), Model XX-15 L (UVP Inc, Optical density is
1.6 mW/cm2 at 15 cm), and Model B-100A Hi Intensity
UV Lamp (UVP Inc, Optical density is 3.5 mW/cm2 at
30 cm) for 365 nm.
13. Compound 3: yield 75%. 1H NMR (CDCl3): 8.65–7.42 (m,
10H, –(C6H4), –(C12H6)), 2.51 (s, 3H, CH3). Anal. Calcd
for C19H13NSO5: C; 62.12, H; 3.57, N; 3.81. Found: C;
61.99; H; 3.30, N; 3.59.
14. Compound 4: yield 31%. 1H NMR (CDCl3): 8.18–7.48 (m,
4H, –C6H4), 5.51 (d, J = 6.9 Hz, 2H, (–CH2–)), 4.18 (m,
4H, 2CH2), 1.35 (m, 6H, 2CH3). Anal. Calcd for
C11H16NPO6: C, 45.68; H, 5.58, N; 4.84. Found: C;
45.50; H; 5.64, N; 4.29.
23. Walker, J. W.; McCray, J. A.; Hess, G. P. Biochemistry
1986, 25, 1799.