Efficient preparation of photolabile agent mni-glu by regioselective nitration of 4-methoxyindoline derivative

Article abstract of DOI:10.1080/00397910902883645

The present work investigates the nitration of 4-methoxy indoline derivative with different reagents and optimized conditions. We now report a simple methodology for nitration employing claycop in the presence of amine, which offers significant improvement with regard to the yield and regioselectivity, and a much more favorable isomeric ratio of products was obtained under mild conditions. Therefore, a useful method for preparation of a photolabile L-glutamate derivative was concurrently established.

Full text of DOI:10.1080/00397910902883645

This article was downloaded by: [University of Wisconsin-Milwaukee]
On: 10 October 2014, At: 10:53
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954
Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH,
UK
Synthetic Communications: An
International Journal for Rapid
Communication of Synthetic
Organic Chemistry
Publication details, including instructions for
authors and subscription information:
http://www.tandfonline.com/loi/lsyc20
Efficient Preparation of
Photolabile Agent MNI-glu by
Regioselective Nitration of 4-
Methoxyindoline Derivative
Xiaowei Wang a , Yu Gao a , Yang Xu a , Li Li a , Zhili
Zhang a & Junyi Liu a b
a Department of Chemical Biology, School of
Pharmaceutical Sciences , Peking University ,
Beijing, China
b State Key Laboratory of Nature and Biomimetic
Drugs , Peking University , Beijing, China
Published online: 16 Oct 2009.
To cite this article: Xiaowei Wang , Yu Gao , Yang Xu , Li Li , Zhili Zhang & Junyi Liu
(2009) Efficient Preparation of Photolabile Agent MNI-glu by Regioselective Nitration
of 4-Methoxyindoline Derivative, Synthetic Communications: An International Journal
for Rapid Communication of Synthetic Organic Chemistry, 39:22, 4030-4038, DOI:
10.1080/00397910902883645
To link to this article: http://dx.doi.org/10.1080/00397910902883645
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the
information (the “Content”) contained in the publications on our platform.
However, Taylor & Francis, our agents, and our licensors make no

representations or warranties whatsoever as to the accuracy, completeness,
or suitability for any purpose of the Content. Any opinions and views
expressed in this publication are the opinions and views of the authors, and
are not the views of or endorsed by Taylor & Francis. The accuracy of the
Content should not be relied upon and should be independently verified with
primary sources of information. Taylor and Francis shall not be liable for any
losses, actions, claims, proceedings, demands, costs, expenses, damages,
and other liabilities whatsoever or howsoever caused arising directly or
indirectly in connection with, in relation to or arising out of the use of the
Content.
This article may be used for research, teaching, and private study purposes.
Any substantial or systematic reproduction, redistribution, reselling, loan,
sub-licensing, systematic supply, or distribution in any form to anyone is
expressly forbidden. Terms & Conditions of access and use can be found at
http://www.tandfonline.com/page/terms-and-conditions

Synthetic Communications1, 39: 4030–4038, 2009
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397910902883645
Efficient Preparation of Photolabile Agent
MNI-glu by Regioselective Nitration
of 4-Methoxyindoline Derivative
Xiaowei Wang,1 Yu Gao,1 Yang Xu,1 Li Li,1 Zhili Zhang,1
and Junyi Liu1,2
1Department of Chemical Biology, School of Pharmaceutical Sciences,
Peking University, Beijing, China
2State Key Laboratory of Nature and Biomimetic Drugs, Peking
University, Beijing, China
Abstract: The present work investigates the nitration of 4-methoxy indoline
derivative with different reagents and optimized conditions. We now report a
simple methodology for nitration employing claycop in the presence of amine,
which offers significant improvement with regard to the yield and regioselectivity,
and a much more favorable isomeric ratio of products was obtained under mild
conditions. Therefore, a useful method for preparation of a photolabile
L-glutamate derivative was concurrently established.
Keywords: Aromatic nitration, caged L-glutamate, claypop, nitroindolines,
photorelease agent
INTRODUCTION
Photorelease of biologically signaling molecules from photocleavable
(caged) carriers is now a standard technique for physiologists, neuro-
scientists, and biochemists, but rapid and efficient release of these
molecules faces lots of problems including lack of suitable carriers. In
Received November 12, 2008.
Address correspondence to Xiaowei Wang, Department of Chemical Biology,
School of Pharmaceutical Sciences, Peking University, Beijing 100191, China.
E-mail: xiaoweiwang@bjmu.edu.cn
4030

Regioselective Nitration of 4-Methoxyindoline
4031
1999, Papageorgiou et al. developed a series of 7-nitroindolyl derivatives
that rapidly and efficiently released carboxylates, such as L-glutamate, a
key neurotransmitter, in synapses of the central nervous system.[1] To
enhance the photorelease efficiency, many research groups focused on this
kind of compound, especially 1-glutamine-4-methyloxy-7-nitroindoline
(MNI-glu), a practical reagent that could improve the photolytic
efficiency by two-folds.[2] At the same time, different methods have been
developed for the nitration on the aromatic ring. Fedoryak[3] and
coworkers used the traditional AgNO3 method to perform the 7-nitration
on the indole ring. The yield was blocked by side reactions including
5-nitro isomer (3b) and partial nitration of the Boc-protected amino
function in the side chain. Hereafter, claycop-acetic anhydride, as a mild
selective nitration reagent, was used to successfully decrease the amount
of 5-nitro isomer,[4] but the N-nitroamide by-products still existed in
a significant quantity. Herein, our strategy focused on giving more
favorable regioselectivity on the 7-nitro isomer (3a) and avoiding the side
reaction by optimizing experimental conditions and introducing amines as
substrate of transition of the nitro group.
RESULTS AND DISCUSSION
Recently, a study found that 4-methoxy-7-nitroindoline glutamate has a
remarkable photolytic efficiency.[2] Herein, we report a regioselective
synthesis of the title compound from commercially available 4-methoxyin-
dole. The synthetic route was outlined in Scheme 1. 4-methoxyindole was
reduced in quantitative yield using sodium cyanoborohydride (NaBH3CN)
in acetic acid to 4-methoxyindoline, which was coupled to N-tert-BOC-L-
glutamic acid a-tert-butyl ester under standard 1,3-dicyclohexylcarbodii-
mide (DCC) coupling conditions to give methoxyindoline 2. Nitration of
compound 2 was accomplished to yield nitro methoxyindoline 3, which
was deprotected by trifluoroacetic acit (TFA) treatment to obtain
MNI-glu. However, the advantage could not readily be exploited because
of the very poor yield for introduction of the nitro group. Although the
detailed photolytic mechanism is not known, oxygen transfer takes place
from the 7-nitro group to the adjacent 1-acyl group, so the 5-nitro isomer
is not useful. Therefore, nitration of aromatic rings has received consider-
able attention for the regioselectivity. Some research groups tried to block
the 5-position with a suitable substituent, such as 5-methyl group, but this
caused a significant reduction in photoefficiency, which could be ascribed
to steric inhibition of the resonance interaction between the methoxy group
and the aromatic ring.[5] Thus, it is very important and urgent to explore a
practical nitration agent and optimize the conditions.

4032
X. Wang et al.
Scheme 1. Synthetic route of MNI-glu.
We screened a wide range of nitration reagents and observed that
with excess AgNO3–acetic chloride, a complex mixture of products, which
included the 7- and 5-nitro isomers in a ratio of nearly 1:1 together with a
large part of additional products, was obtained under traditional conditions.
In a previous report, the by-products were a mixture of N-nitroso and
N-nitro species on the nitrogen of the protected amino acid residue.[6]
According to our experience, far more excess AgNO3 may cause the
problem. Therefore, we decreased the amount of AgNO3 involved in this
reaction from 2.5 to 1.0 equiv (Table 1), and performed the reaction at
Table 1. Nitration of 2 with AgNO3=CH3COCl method
Yield of products 3 (%)
Time
Ratio Temp. Solvent (h) (7-isomer) (5-isomer) (N-nitro)
3a
3b
3c
Reactant
2 þ AgNO3 1:2–2.5
2 þ AgNO3 1:1.02
2 þ AgNO3 1:1.02
2 þ AgNO3 1:1.02
rt
rt
CH3CN
CH3CN
1
1
1
48
14
37
64
—
10
20
10
—
63
25
6
0ꢀC CH3CN
0ꢀC
CCl4
—

Regioselective Nitration of 4-Methoxyindoline
4033
room temperature. The N-nitro and nitroso by-products did not
dominate the mixture, but the nitration on the 7- and 5- positions showed
less selectivity. Because of the greater thermodynamic stability of 7-nitro
isomer, the regioselectivity could be increased by lowering the reaction
temperature. Meanwhile, it is appeared that formation of by-products
would also be marred. Just as we expected, the best result was obtained
when the reaction was performed at 0ꢀC in acetonitrile. The N-nitro and
nitroso by-products decreased significantly, and the desired 7-nitro
isomer (3a) increased to some extent as indicated by the data presented
in Table 1 (from 37% to 64%).
Furthermore, we found that the nitration process depended not only
on the reaction temperature but also on the polarity of the solvent. The
change of solvent from acetonitrile to tetrachloride decreased the reaction
activity. Therefore, under this circumstance, a polar solvent favored the
reaction more than a nonpolar one (Table 1); the polar solvent may be
better at solvating the intermediate CH3COONO2, stabilizing the
charged NOþ2 electrophile and then reducing the activation energy.
To enhance the ratio of 3a=3b, a mild nitration reagent, ‘‘claycop,’’ was
introduced by Papageorgiou et al.[4] The major advantage is the increasing
regioselectivity, which stems from microporous solids, such as montmoril-
lonite K10 clay, with high diffusivity and shape selectivity. In addition, such
procedures were often found to provide good yields under mild conditions,
reduce the activation energy, and simplify the workup. We used the Laszlo’s
claycop (copper nitrate impregnated on montmorillonite K10) as the
nitration agent and aimed to achieve higher regioselectivity and then
increase the yield of 7-nitro isomer (3a). The experiment results showed that
with the claycop method, the high-selective nitration reagent significantly
increased the 3a=3b isomer ratio, but the 5-nitro isomer 3b still existed in
a small quantity. Moreover, the N-nitro by-product 3c, which was far more
than N-nitroso of amide, formed in considerable amounts.
As discussed previously, lowering the temperature would be
favorable for the reaction, so we carried out the reaction at 0ꢀC and
found that the ratio of 3a=3b increased from 6:1 to 15:1 (Table 2), the
N-nitro by-product decreased a little, but it was far from pure 7-nitro
isomer. Additionally, the reaction time was also profoundly affected by
solvent, and the result was very interesting. When compound 2 reacted
with claycop in tetrachloride, the reaction was almost completed in
12 h and yielded a mixture of 3a and 3b in a 6:1 ratio, strongly favoring
the 7-nitro isomer. In acetonitrile, the reaction was much more sluggish,
and there was no benefit to the product ratio. These observations are the
opposite of the former experiment; a nonpolar solvent would be favor-
able for the reaction, which could probably mean that a different
mechanism dominated in the claycop method.

4034
X. Wang et al.
Table 2. Nitration of 2 with claycop method
Yield of products 3 (%)
3a 3b 3c
Time
Reactant
Ratioa Temp. Solvent (h) (7-isomer) (5-isomer) (N-nitro)
2 þ claycop
2 þ claycop
2 þ claycop
1:1
1:1
1:1
rt
rt
0ꢀC
CCl4
CH3CN 100
CCl4 48
12
45
36
59
8
6
4
30
35
21
aThe molar ratio was calculated as the amount of nitrate anion contained in
claycop.
Although the claycop nitration provided a satisfactory solution to
the 7- and 5-isomer selectivity, we still encountered the problem of
unwanted partial nitration=nitrosation of the Boc-protected amino func-
tion in the side chain during introduction of the 7-nitro group. In
attempts to suppress these side reactions, other research groups tried to
convert methoxyindoline 2 to its di-Boc derivative,[7] but only a slight
improvement was observed. We decided to introduce the amine as a
nitrotransition agent to the reaction.
We added aromatic and aliphatic amine respectively with 2 and
claycop in one pot at room temperature, with the molar ratio of 1:2:1.
As we know, N-nitrosoamide by-product would be little with claycop
as nitration agent, so major by-products would be 5-nitro isomer 3b
and N-nitroamide 3c. The results showed that the aliphatic amine
decreased the N-nitration by-products more evidently than that of the
aromatic amine, which indicated that t-butylamine favors the nitrotransi-
tion reaction (Table 3). It appears that the formation of the N-nitro and
N-nitrosoamide by-products in small quantities is an inevitable
Table 3. Nitration of 2 with claycop method in the presence of amines at rt
Yield of products 3 (%)
Time
3a
3b
3c
Reactant
Ratioa Solvent (h) (7-isomer) (5-isomer) (N-nitro)
2 þ claycop
1:1
CCl4
12
12
12
45
48
40
8
2
2
30
11
24
2 þ claycop þt-butylamine 1:2:1 CCl4
2 þ claycop þaniline
1:2:1 CCl4
aThe molar ratios were calculated as the amount of nitrate anion contained in
claycop.

Regioselective Nitration of 4-Methoxyindoline
4035
consequence when the substrate contains a secondary amide group.
Further study on the mechanism of nitrotransition will be a future work.
CONCLUSIONS
Our main emphasis was to achieve higher selectivity to meet market
demand. We carried out nitration of compound 2 with different methods
under various conditions. Claycop proved to be a better nitration agent
in terms of regioselectivity. It is noteworthy that under typical claycop
procedure, the 3a and 3b isomers were obtained in 6:1 ratio (i.e., 45%
7-nitro isomer compared to 8% 5-isomer), but the unwanted N-
nitroamide by-product 3c still existed in some quantity. Whereas when
amine was added to the reaction mixture, especially aliphatic amine,
the yield of 3c decreased significantly (from 30% to 11%), while the ratio
of 3a=3b enhanced (to 24). In addition, our results illustrate that tempera-
ture and solvent could affect the reaction profoundly. Decreasing the
reaction temperature would facilitate access to a higher regioselectivity.
Because of different mechanisms of nitration methods, polar and
nonpolar solvents would give opposite outcomes. With claycop as
nitration agents, a nonpolar solvent would be favorable; it is just adverse
to that of the AgNO3=CH3COCl method. In conclusion, a simple
methodology for nitration employing claycop in the presence of aliphatic
amine provides a satisfactory solution to the 7- and 5-nitro isomer
selectivity and offers an interesting chemoselectivity in some instances.
It thus represents a valuable alternative to the existing approaches on
both the laboratory and industrial scales.
EXPERIMENTAL
General
Melting points were determined with an electrothermal capillary
melting-point apparatus and are uncorrected. 1H NMR spectra were
recorded on a Jeol-AL-300 Fourier Transform (FT) spectrometer with
tetramethylsilane (TMS) as the internal standard. The electrospray
ionization=turnover frequency (ESI-TOF) spectra were taken on a Liner
Scieneific LDI-1700 mass spectrometer. Silica gel (0.040–0.064 mm) was
used for column chromatography. Analytical high-performance liquid
chromatography (HPLC) was performed on a Shimazu LC-10AT with
a reverse column of YMC-C8 (4.6 ꢁ 150 mm) at 1 ml=min flow rate.
Some reagents were purchased from Aldrich. Organic solvents were dried

4036
X. Wang et al.
over anhydrous Na2SO4 and evaporated under reduced pressure.
Claycop was prepared with a 2:3 (w=w) ratio of Cu (NO3)2 and
montmorillonite K10 as described in Ref. 8.
Synthesis of 1-Glutamine-4-methyloxy-7-nitroindoline (4)
4-Methoxyindoline (1)
Sodium cyanoborohydride (NaBH3CN) (1.56 g, 24.8 mmol) was added
portionwise over 20min to a solution of 4-methoxyindole (1.0g,
6.79mmol) in acetic acid (20 mL), and the mixture was stirred at room
temperature for 0.5 h. Water was added, and the solvent was removed
under reduced pressure. The residue was dissolved in ethyl acetate
(25 mL) and washed with saturated NaHCO3 and brine. The organic layer
was dried with anhydrous MgSO4 and concentrated in vacuo to give 1 as
viscous oil (0.98g, 98%), which was used immediately without purification.
1-[S-(4-t-Butoxycarbonyl)-4-(t-butoxycarbonylamino)]-
butanoyl-4-methoxyindoline (2)
1,3-Dicyclohexylcarbodiimide (DCC) (0.29 g, 1.4 mmol), dimethylamino-
pyrimidine (DMAP) (0.37 g, 3.0 mmol), and N-BOC-l-glutamic acid a-t-
butyl ester (0.36 g, 1.2 mmol) were added to a solution of 4-methoxylin-
doline 1 (0.15 g, 1.0 mmol) in acetonitrile (12 mL). The reaction mixture
was stirred at room temperature for 16 h and then filtered. The filtrate
was diluted with CH2Cl2 (25 mL) and washed with saturated NaHCO3
solution, 0.5 N HCl, and brine. The organic layer was dried with anhy-
drous MgSO4 and evaporated to dryness. The crude product was purified
by column chromatography with EtOAc–petroleum ether (60–90ꢀC) to
yield product 2 as white solid (0.34 g, 78%), mp 129–130ꢀC.
Nitration of 2 with Different Agents
AgNO3=CH3COCl Method. Compound 2 (2.17 g, 5.0 mmol) and silver
nitrate (0.86 g, 5.1 mmol) in 20 mL acetonitrile (or CCl4) were stirred at
room temperature (or 0ꢀC) to give a clear solution, and then a solution
of acetyl chloride (0.4 g, 5.1 mmol) was added. A greyish precipitate
formed upon addition of the first few drops. The reaction was complete
when thin-layer chromatography (TLC) showed that compound 2
totally disappeared, which depended on the temperature and solvent
(see Table 1). The mixture was filtered, and the filtrate was washed with

Regioselective Nitration of 4-Methoxyindoline
4037
saturated NaHCO3 solution and brine. The organic layer was dried with
MgSO4 and evaporated to dryness. The crude product was purified by
column chromatography with EtOAc–petroleum ether (60–90ꢀC) to yield
product 3a as yellow solid (for the corresponding yields, see Table 1), mp
142–144ꢀC (lit.[4] 145–147ꢀC).
Claycop Nitration Method. Compound 2 (2.17 g, 5.0 mmol) was added to
a suspension of claycop [1.2 g, 2.55 mmol Cu(NO3)2] in 20 mL CCl4 (or
CH3CN) and acetic anhydride (10 mL) at room temperature (or 0ꢀC).
The mixture was stirred until compound 2 totally disappeared on TLC.
It took 12–100 h typically, as shown in Table 2. The solid was filtered
and washed with EtOAc. The filtrate was washed with saturated
NaHCO3 and brine. The organic layer was dried with MgSO4 and
evaporated to dryness. The crude product was purified by column
chromatography with EtOAc–petroleum ether (60–90ꢀC) to yield pro-
duct 3a as yellow crystals (for the corresponding yields, see Table 2),
mp 143–145ꢀC (lit.[4] 145–147ꢀC).
1H NMR (300 MHz, CDCl3): d ¼ 7.74 (d, J ¼ 9.0 Hz, 1 H, 6-H), 6.63
(d, J ¼ 9.0 Hz, 1 H, 5-H), 5.15 (br s, 1 H, NH), 4.17–4.23 (m, 3 H, CH and
2-H), 3.90 (s, 3 H, OCH3), 3.26 (t, 2 H, 3-H), 2.54–2.60 (t, 2 H, COCH2),
2.26–2.34 and 1.96–2.04 (m, 2 H, CH2), 1.46 (s, 9 H, CH3), 1.42 (s, 9 H,
CH3) ppm; ESI-TOFþ: 480.35 (M þ H)þ.
Hydrolysis of 3a to Prepare 1-[S-(4-Amino-4-
carboxybutanoyl)]-4-methoxy-7-nitroindoline (4)
TFA (1.5 mL in 1 mL CH2Cl2) was added dropwise to a solution of 3a
(0.26 g, 0.5 mmol) in CH2Cl2 (2 mL). The mixture was stirred at room
temperature for 1.5 h and concentrated in vacuo. The residue was dis-
solved in water (20 mL) and adjusted to pH 7.0 with 1 M aq. NaOH.
The solution was washed with ether, lyophilized, and stored at ꢂ20ꢀC
as a pale yellow powder (129 mg, 80%).
1H NMR (300 MHz, D2O): d ¼ 7.57 (d, J ¼ 9.0 Hz, 1 H, H6), 6.67
(d, J ¼ 9.0 Hz, 1 H, H5), 4.11 (t, J ¼ 7.8 Hz, 2 H, H2), 3.76 (s, 3 H,
OMe), 3.51 (t, 1 H, CH), 2.88 (t, J ¼ 7.8 Hz, 2 H, H3), 2.56 (t, 2 H,
COCH2), 1.95 ꢃ 2.08 (m, 2 H, CH2CH).
Nitration of 2 with Claycop Method in the Presence of Amines
Compound 2 (2.17 g, 5.0 mmol) was added to a suspension of claycop
[2.4 g, 5.1 mmol Cu(NO3)2] in CCl4 (25 mL) and stirred at room

4038
X. Wang et al.
temperature. Two additional experiments were carried out at the same
time with identical amounts of reagents, but t-butylamine (0.5 ml,
5.0 mmol) and aniline (0.47 ml, 5.0 mmol) were, respectively, added to
such solutions. After 12 h, the mixtures were detected by HPLC with
methanol–water as mobile phase, gradient elution, rate 1 ml=min,
detection wavelength: 314 nm. The results are shown in Table 3.
ACKNOWLEDGMENTS
We thank the National Science Foundation of China and 985 Program of
the Ministry of Education of China for financial support.
REFERENCES
1. Papageorgiou, G.; Ogden, D. C.; Barth, A.; Corrie, J. E. T. Photorelease of
carboxylic acids from 1-acyl-7-nitroindolines in aqueous solution: Rapid and
efficient photorelease of L-glutamate. J. Am. Chem. Soc. 1999, 121, 6503–6504.
2. Papageorgiou, G.; Corrie, J. E. T. Effects of aromatic substituents on the
photocleavage of 1-acyl-7-nitroindolines. Tetrahedron 2000, 56 (41),
8197–8205.
3. Fedoryak, O. D.; Sul, J. Y.; Haydon, P. G.; Ellis-Davies, G. C. R. Synthesis of
a caged glutamate for efficient one- and two-photon photorelease on living
cells. Chem. Commun. 2005, 29, 3664–3666.
4. Papageorgiou, G.; Corrie, J. E. T. Regioselective nitration of 1-acyl-4-
methoxyindolines leads to efficient synthesis of a photolabile L-glutamate
precursor. Synth. Commun. 2002, 32 (10), 1571–1577.
5. Papageorgiou, G.; Corrie, J. E. T. Optimised synthesis and photochemistry of
antenna-sensitised 1-acyl-7-nitroindolines. Tetrahedron 2005, 61, 609–616.
6. Emil, H. W.; Daniel, W. G. The preparation and decomposition of certain
N-nitroamides and N-nitrocarbamates. J. Am. Chem. Soc. 1961, 83, 1191.
7. Papageorgiou, G.; Ogden, D.; Corrie, J. E. T. An antenna-sensitized nitroin-
doline precursor to enable photorelease of L-glutamate in high concentrations.
J. Org. Chem. 2004, 69, 7228–7233.
8. Laszlo, P.; Cornelis, A. ‘‘Claycop,’’ a user-friendly oxidizing and nitrating
reagent. Aldrichim. Acta 1988, 21 (4), 97.