Effects of aromatic substituents on the photocleavage of 1-acyl-7-nitroindolines

Article abstract of DOI:10.1016/S0040-4020(00)00745-6

Photolysis of 1-acyl-7-nitroindolines in aqueous solution gives a carboxylic acid and a 7-nitrosoindole. These compounds are useful as photolabile precursors of carboxylic acids, particularly neuroactive amino acids. The effect of electron-donating substituents at the 4-position has been explored for its effect on photolysis efficiency. 4-Methoxy substitution improved the photolysis efficiency >2-fold but the 4-dimethylamino analogue was essentially inert. A 5-alkyl substituent, that blocks unwanted nitration at this position, reduced the beneficial effect of the 4-methoxy group. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00745-6

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 8197±8205
Effects of Aromatic Substituents on the Photocleavage
of 1-Acyl-7-nitroindolines
*
George Papageorgiou and John E. T. Corrie
National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
Received 30 June 2000; revised 31 July 2000; accepted 17 August 2000
AbstractÐPhotolysis of 1-acyl-7-nitroindolines in aqueous solution gives a carboxylic acid and a 7-nitrosoindole. These compounds are
useful as photolabile precursors of carboxylic acids, particularly neuroactive amino acids. The effect of electron-donating substituents at the
4-position has been explored for its effect on photolysis ef®ciency. 4-Methoxy substitution improved the photolysis ef®ciency .2-fold but
the 4-dimethylamino analogue was essentially inert. A 5-alkyl substituent, that blocks unwanted nitration at this position, reduced the
bene®cial effect of the 4-methoxy group. q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
l-glutamate from 1a, have been reported in conference
proceedings.4
Many compounds have been developed in the past two
decades that, upon photolysis with a pulse of near-UV
light, release biological effector species at or near their
site of action within living preparations on a ms to ms
time scale. These photolabile precursors are used particu-
larly because they enable study of fast biological processes
without interference by diffusion kinetics. The concept is
covered in a number of reviews.1 Despite these successes,
there has been a lack of reagents capable of rapid and
ef®cient release of amino acids, such as l-glutamate, that
are key neurotransmitters in synapses of the central nervous
system (see Ref. 2 for a survey of previous work). Recently
we described the 7-nitroindolyl derivative 1a as a particu-
larly promising reagent for rapid photolytic generation of
l-glutamate.3 Initial neurophysiological studies in mam-
malian brain slices, triggered by photochemical release of
Our exploration of this photochemistry originated from the
known photohydrolysis of 1-acyl-5-bromo-7-nitroindolines,
which yields 5-bromo-7-nitroindoline and a carboxylic acid
when irradiated in an aprotic organic solvent containing
,1% water (Scheme 1a).5 Under identical conditions, we
observed the same photohydrolysis when the bromine
substituent was replaced by a methoxycarbonylmethyl
group as in 1.3 However, in fully aqueous solution the reac-
tion took an entirely different course to yield a carboxylic
Scheme 1. Photolytic reactions of 1-acyl-7-nitroindolines: (a) in aprotic organic solvent containing a trace of water; and (b) in fully aqueous solution.
Keywords: amino acids and derivatives; indoles; indolines; photochemistry.
* Corresponding author. Tel.: 144-20-8959-3666, ext. 2276; fax: 144-20-8906-4419; e-mail: jcorrie@nimr.mrc.ac.uk
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00745-6

8198
G. Papageorgiou, J. E. T. Corrie / Tetrahedron 56 (2000) 8197±8205
acid and the 5-substituted 7-nitrosoindole 2 (Scheme 1b),
without incorporation of solvent water into the products.3
Studies of this mechanistic dichotomy are in progress, but
the present paper describes efforts to optimise the ef®ciency
of the aqueous photoreaction by exploring different sub-
stituents on the aromatic ring. Good photolysis ef®ciency
is important in biological experiments in order to maximise
conversion by a single light ¯ash. For simplicity, all the
optimisation experiments were carried out with N-acetyl
compounds.
Ef®ciency in single ¯ash experiments is normally deter-
mined by a combination of the absorption coef®cient at
the wavelength of the irradiating light and the product
quantum yield. Our strategy was focused on enhancing the
absorption coef®cient by introducing electron-donating
substituents para to the nitro group. The results encompass
a range of chemistry of 4-substituted indoles and indolines
and in practice the effects on photolysis ef®ciency were
mediated by changes in both product quantum yield and
absorption coef®cient.
Figure 1. X-Ray crystal structure of compound 7, showing 30% thermal
ellipsoids.
Results and Discussion
Our ®rst goal was to study the effect of a 4-methoxy sub-
stituent, i.e. compound 3, and initially we followed an
analogous route to that used for synthesis of 1.3 Thus
1-acetyl-4-methoxyindoline 4 was treated with acetyl
chloride under Friedel±Crafts conditions and the product
ketone was oxidised with thallium(III) nitrate6 to give the
corresponding methyl indolineacetate. Nitration (NaNO3±
TFA) gave a product that was initially assumed to be the
required compound 3, but this material was photostable and
we were concerned that the ®rst electrophilic substitution
could have taken place at the 7-position rather than at C-5 as
expected. Single crystal X-ray diffraction analysis of the
²
nitrated product (Fig. 1) con®rmed this . The true sequence
is therefore as shown in Scheme 2 to yield the 5-nitro
compound 7.
Scheme 2. Reagents: (i) MeCOCl, AlCl3; (ii) Tl(III)NO3, MeOH; (iii)
NaNO3, TFA.
electrophile in the nitration reaction.7 Comparative
photolysis of aqueous solutions of 1b and 9 at equal concen-
trations indicated that the latter photolysed with ,3-fold
greater ef®ciency, as assessed by spectroscopic changes
upon progressive photolysis (Fig. 2). A more quantitative
measurement is described below, but the effect was
suf®ciently marked to warrant further exploration of sub-
stituent effects, especially as the isosbestic point in the
progressive photolysis spectra (Fig. 2b) suggested that the
photochemical reaction took place cleanly, as previously
demonstrated for 1a.3
Since Friedel±Crafts acylation of 4 was regiospeci®c at the
7-position, we examined direct nitration of 4 in the hope of
obtaining only a compound with the nitro group at C-7.
However, nitration of 4 gave the 5- and 7-nitro compounds
8 and 9 in equal proportion. Nevertheless, the two
compounds were readily separated by chromatography
and the regiochemistry of 9 was assigned by observation
of a strong NOE enhancement between the methoxy protons
and the adjacent aromatic proton. The lack of regioselec-
tivity in this reaction compared to the Friedel±Crafts acyl-
ation can be explained by higher reactivity of the
²
Crystallographic details will be deposited at the Cambridge Crystallo-
graphic Data Centre.

G. Papageorgiou, J. E. T. Corrie / Tetrahedron 56 (2000) 8197±8205
8199
We ®rst considered whether a compound similar to our
original target 3 could be easily accessed, since a 5-sub-
stituent would eliminate the problem of regioselectivity
during nitration. For simplicity in this phase of the work,
we used a 5-methyl substituent (Scheme 3). The required
indole 11 was prepared in poor yield from 2,6-dimethyl-3-
nitroanisole 10 by a Leimgruber±Batcho synthesis,8
reduced to the indoline 12 with NaBH3CN±acetic acid9
and acetylated to give 13. Nitration with NaNO3±TFA
then gave the single isomer 14. Photolysis of 14 in aqueous
solution was ,2-fold less ef®cient than for 9, probably
because the 5-methyl group in 14 sterically inhibits inter-
action between the methoxy group and the aromatic ring.
The implication of these combined results is that an
electron-releasing group at C-4 has a bene®cial effect on
photolysis ef®ciency and that the effect is greater without
a C-5 substituent. On these grounds we decided to examine
the effect of a 4-dimethylamino group alone.
Scheme 3. Reagents: (i) (MeO)2CHNMe2, pyrrolidine; (ii) Fe, silica gel,
toluene, HOAc; (iii) NaBH3CN, HOAc; (iv) Ac2O, HOAc; (v) NaNO3,
TFA.
Figure 2. UV±Vis spectra showing the time course for photolysis of: (a)
compound 1b, and (b) compound 9, both in neutral aqueous solution. The
numbers on the traces show the cumulative irradiation time in minutes.
Synthesis of the required 7-nitroindoline 22 (Scheme 4)
required some changes to the route used for the compounds
that had a 4-methoxy substituent. The Leimgruber±Batcho
Scheme 4. Reagents: (i) (Me2N)3CH, DMF; (ii) semicarbazide, aq. HCl; (iii) H2, Pd±C; (iv) BH3´Me2S, TFA; (v) Ac2O, HOAc; (vi) NaNO3, TFA; (vii) MeOH,
aq. HCl; (viii) chromatography; (ix) MeCOCl, HOAc.

8200
G. Papageorgiou, J. E. T. Corrie / Tetrahedron 56 (2000) 8197±8205
procedure with DMF dimethyl acetal was ineffective when
applied to 2-dimethylamino-6-nitrotoluene 15. However, a
modi®ed procedure10 using tris(dimethylamino)methane
and isolation of the semicarbazone 16 formed from the
intermediate enamine was more satisfactory. Hydrogenation
of 16 gave 4-(N,N-dimethylamino)indole 17 in excellent
yield. Reduction of 17 to the corresponding indoline with
NaBH3CN±acetic acid, as used with the methoxy
compounds, was unsatisfactory but was cleanly effected
with BH3´Me2S in TFA.11 The product indoline was imme-
diately acetylated to give 19 in acceptable overall yield.
Photolysis of 25 was monitored by reverse-phase HPLC to
quantify the extent of reaction and the photolysed solution
was also analysed for free glutamate. The measured amount
of glutamate was in the range 88±93% of that expected from
the amount of starting material consumed. Given the 1:1
stoichiometry previously demonstrated for glutamate photo-
release from 1a, isolation of the comparable nitrosoindole
by-product (see below) and the very clean conversion seen
spectroscopically on progressive photolysis of the acetyl
derivative 9 (Fig. 2b), we consider glutamate photo-libera-
tion from 25 to be stoichiometric within the experimental
errors of the analysis. The product quantum yield, measured
using laser ¯ash irradiation at 347 nm was 0.085, almost
twice that of the previous compound 1a under the same
conditions. Comparative photolysis of 1a and 25 using solu-
tions of equal concentration showed that 25 was converted
with 2.2-fold greater ef®ciency under the particular experi-
mental conditions (continuous irradiation, 350 nm lamps,
Rayonet photochemical reactor).
Nitration of 19 (NaNO3±TFA) gave a complex mixture,
from which the unwanted 5-nitro isomer 20 could be
isolated by chromatography. We were unable to isolate
other components in pure form and the remaining mixture
was subjected to acid hydrolysis, after which two com-
ponents could be separated. One was characterised as 5,7-
dinitroindoline 21 while the second, after re-acetylation,
gave the required 7-nitro compound 22. Although the over-
all yield of 22 via this route was very poor, suf®cient
material was obtained to investigate its properties. First,
NOE experiments in which the dimethylamino group of
20 or 22 was irradiated showed strong enhancement of
one aromatic proton signal for 22 but had no effect on either
aromatic proton for 20, thereby con®rming the assigned
regiochemistry. Disappointingly, near-UV irradiation of an
aqueous solution of 22 showed no change in the UV±Vis
spectrum over time periods longer than those in which
comparable solutions of 1b, 9 or 14 were completely trans-
formed. As our aim in the present work was speci®cally to
seek substituents that would enhance photolysis ef®ciency,
we have not further investigated this compound. However,
in retrospect it seems likely that formation of a low-lying
triplet state (as is known to occur with high ef®ciency for
N,N-dialkyl-4-nitroanilines12) is responsible for quenching
the photoreactivity.
To characterise the photo-product of the methoxynitro-
indolyl group, an aqueous solution of the glutarate 26 was
irradiated until UV analysis indicated that the starting
material was consumed. The isolated by-product had
properties fully consistent with 4-methoxy-7-nitrosoindole
27. This result suggests that the methoxy substituent does
not alter the photolysis mechanism in aqueous solution from
that followed by our earlier nitroindolines such as 1.
The experiments described above de®ne that the sub-
stitution pattern of 9 is optimal for ef®ciency of photolysis.
At this point we had assumed the spectroscopic changes
produced upon photolysis of 9 (Fig. 2b) indicated that its
photochemistry in aqueous solution was the same as
previously demonstrated for compounds of the general
structure 1.3 With the substitution pattern optimised, we
needed to establish that photolysis of 9 did indeed proceed
with high stoichiometry for carboxylate release upon
photolysis and that a nitrosoindole by-product analogous to
2 was formed.
Further work with these compounds will aim to optimise the
nitration step to avoid or minimise formation of unwanted
5-nitro isomers such as 8. This would facilitate access to
these more ef®cient methoxy-substituted caged species.
Biological testing of the new caged glutamate 25 will be
reported in a future communication.
To investigate these issues we required the glutamate and
glutarate derivatives 25 and 26, respectively. The glutamate
derivative was needed to enable easy quanti®cation of the
released amino acid, as in our previous work,3 while the
glutarate derivative was a reasonably easily accessed
compound that was water soluble at pH 7 and that enabled
preparative scale photolysis for isolation of the anticipated
nitrosoindole by-product. Each compound was prepared by
appropriate acylations of 4-methoxyindoline to give the
compounds 23 and 24 which were subsequently nitrated as
for the preparation of 9. As expected, nitration yielded both 5-
and 7-nitro compounds in each case but the mixtures were
separable by appropriate chromatography (see Experimental).
Experimental
General procedures
General experimental details were as previously described2,3
except that high ®eld H NMR spectra were recorded on a
1
Varian Unityplus 500 spectrometer. IR spectra were
determined for Nujol mulls unless otherwise speci®ed.
1,7-Diacetyl-4-methoxyindoline 5. 1-Acetyl-4-methoxy-
indoline 4 was prepared from 4-methoxyindole8 by

G. Papageorgiou, J. E. T. Corrie / Tetrahedron 56 (2000) 8197±8205
8201
reduction to the indoline9 and acetylation, mp 115±1168C
(lit. mp13 1128C). A stirred solution of 4 (4.02 g, 21 mmol)
in dry 1,2-dichloroethane (30 mL) was cooled in ice and
acetyl chloride (2.1 mL, 30 mmol) was added, followed
by portionwise addition of powdered anhydrous AlCl3
(8.4 g, 63 mmol). The mixture was stirred at room tempera-
ture overnight, then heated at 508C for 2 h. The red/brown
slurry was poured into acidi®ed cold water (200 mL) and
extracted with CH2Cl2. The combined organic phases were
washed with 0.5 M aq. NaOH and brine, dried and evapo-
rated to give a brown oil. Flash chromatography (EtOAc)
followed by trituration with Et2O afforded 5 as pale yellow
needles (2.22 g, 54%), mp 88±898C: IR: nmax/cm21 1670,
the mixture was stirred for 2 h, allowing the temperature
to rise to 2108C. The solution was poured into ice-cold
water and extracted with EtOAc. The combined organic
phases were washed with saturated aq. NaHCO3 and
brine, dried and evaporated to give a brown viscous oil.
Flash chromatography (EtOAc±light petroleum (1:1))
afforded two products. The less polar product was the
7-nitro isomer 9 as yellow crystals (43 mg, 36%) mp
180±1828C (EtOAc±light petroleum) and the more polar
was the 5-nitro isomer 8 as yellow crystals (44 mg, 37%)
mp 101±1028C (EtOAc±light petroleum).
The compound
8 had UV: lmax (EtOH)/nm 238
1
1650, 1600, 1355, 1270, 1095; H NMR (90 MHz): d 7.33
(e/M21 cm21 9100), 327 (8600); lmax (EtOH±25 mM Na
phosphate, pH 7.0 (5:95))/nm 238 (e/M21 cm21 8550),
341 (9200); IR: nmax/cm21 1665, 1600, 1520, 1385, 1335;
1H NMR (500 MHz): d 8.01 (1H, d, Jˆ8.5 Hz, H7), 7.86
(1H, d, Jˆ8.5 Hz, H6), 4.18 (2H, t, Jˆ8.5 Hz, H2), 3.93
(3H, s, OMe), 3.28 (2H, t, Jˆ8.5 Hz, H3), 2.26 (3H, s,
Ac). Calcd for C11H12N2O4: C, 55.93; H, 5.12; N, 11.85.
Found: C, 55.89; H, 5.10; N, 11.82.
(1H, d, Jˆ8.3 Hz, H6), 6.61 (1H, d, Jˆ8.3 Hz, H5), 4.17
(2H, t, Jˆ8.1 Hz, H2), 3.85 (3H, s, OMe), 3.05 (2H, t,
Jˆ8.1 Hz, H3), 2.44 (3H, s, Ac), 2.21 (3H, s, Ac). Calcd
for C13H15NO3: C, 66.94; H, 6.48; N, 6.00. Found: C, 66.89;
H, 6.50; N, 5.95.
Methyl 1-acetyl-4-methoxyindoline-7-acetate 6. Thallium-
(III) nitrate trihydrate (3.21 g, 7.1 mmol) was added to a
solution of 5 (1.65 g, 7.1 mmol) in MeOH (70 mL) that
contained perchloric acid (60% w/w, 1.5 mL) and the
mixture was stirred at room temperature for 4 h. The pre-
cipitated solid was ®ltered off and the ®ltrate concentrated to
,10 mL, diluted with EtOAc (50 mL) and washed with
water. The aqueous phase was extracted with EtOAc and
the combined organic phases were washed with saturated
aq. NaHCO3 and brine, dried and evaporated to give a
viscous oil. Flash chromatography (EtOAc±light petroleum
(4:1)) gave 6 as white crystals (0.88 g, 47%), mp 80±818C
(EtOAc±light petroleum): IR: nmax/cm21 1730, 1660, 1615,
The compound
9 had UV: lmax (EtOH)/nm 248
(e/M21 cm21 18300), 298 (4700), 335sh (3600); lmax
(EtOH±25 mM Na phosphate, pH 7.0 (5:95))/nm 246
(e/M21 cm21 17500), 322 (4800); IR: nmax/cm21 1670,
1605, 1520, 1340, 1275; 1H NMR (500 MHz): d 7.75
(1H, d, Jˆ9.0 Hz, H6), 6.63 (1H, d, Jˆ9.0 Hz, H5), 4.23
(2H, t, Jˆ8.1 Hz, H2), 3.91 (3H, s, OMe), 3.08 (2H, t,
Jˆ8.1 Hz, H3), 2.24 (3H, s, Ac). Calcd for C11H12N2O4:
C: 55.93; H, 5.12; N, 11.85. Found: C, 56.05; H, 5.12; N,
11.80.
1
1500, 1390, 1345, 1270, 1150; H NMR (90 MHz): d 7.04
2,6-Dimethyl-3-nitroanisole 10. A mixture of 2,6-methyl-
3-nitrophenol (4.18 g, 25 mmol; prepared from 2,6-
dimethyl-3-nitroaniline according to the general method
described14) and anhydrous K2CO3 (4.15 g, 30 mmol) in
acetone (100 mL) was treated with dimethyl sulfate
(2.84 mL, 30 mmol) and the mixture was heated under
re¯ux. The progress of the reaction was followed by TLC
(EtOAc±light petroleum (1:3)). Further aliquots of dimethyl
sulfate (2£2 mL) were added after 4 and 6 h and the mixture
was re¯uxed overnight. After cooling to room temperature
the solid was ®ltered and washed with acetone and the
combined ®ltrate was evaporated. The residue was
dissolved in conc. aq. ammonia (100 mL) to destroy excess
dimethyl sulfate, stirred at room temperature for 30 min and
extracted with ether. The combined organic phases were
washed with 2 M aq. HCl, 1 M aq. NaOH and brine, dried
and evaporated to give a brown oil (4.30 g). TLC indicated
the presence of starting phenol. The product was dissolved
in light petroleum (100 mL), washed with Claisen's alkali15
(2£100 mL) and the petroleum phase was dried and evapo-
rated to give a brown oil (4.08 g). Flash chromatography
(EtOAc±light petroleum (1:9)) afforded 10 as an oil
(3.43 g, 76%) that was used without further puri®cation;
1H NMR (90 MHz): d 7.60 (1H, d, Jˆ8.3 Hz, H4), 7.11
(1H, d, Jˆ8.3 Hz, H5), 3.74 (3H, s, OMe), 2.48 (3H, s,
Me), 2.35 (3H, s, Me).
(1H, d, Jˆ8.3 Hz, H6), 6.63 (1H, d, Jˆ8.3 Hz, H5), 4.06
(2H, t, Jˆ8.1 Hz, H2), 3.81 (3H, s, OMe), 3.76 (2H, s,
ArCH2), 3.71 (3H, s, CO2Me), 2.96 (2H, t, Jˆ8.1 Hz, H3),
2.24 (3H, s, Ac). Calcd for C14H17NO4: C, 63.86; H, 6.51; N,
5.32. Found: C, 63.60; H, 6.50; N, 5.28.
Methyl 1-acetyl-4-methoxy-5-nitroindoline-7-acetate 7.
To a stirred solution of NaNO3 (75 mg, 0.9 mmol) in TFA
(4 mL) was added 6 (211 mg, 0.8 mmol). The mixture was
stirred at room temperature for 4 h and the red/brown solu-
tion was poured into ice-cold water and extracted with
EtOAc. The combined organic phases were washed with
saturated aq. NaHCO3 and brine, dried and evaporated.
The residue was ¯ash chromatographed (EtOAc) and
crystallised (EtOAc±light petroleum) to give 7 as pale
yellow needles (90 mg, 36%), mp 181±1828C: UV: lmax
(EtOH)/nm 314 (e/M21 cm21 7250); lmax (EtOH±25 mM
Na phosphate, pH 7.0 (5:95))/nm 325 (e/M21 cm21 7370);
IR: nmax/cm21 1735, 1680, 1615, 1575, 1515, 1380, 1320,
1
1200, 1165; H NMR (90 MHz): d 7.74 (1H, s, H6), 4.16
(2H, t, Jˆ8.1 Hz, H2), 3.90 (3H, s, OMe), 3.78 (2H, s,
ArCH2), 3.69 (3H, s, CO2Me), 3.15 (2H, t, Jˆ8.1 Hz, H3),
2.27 (3H, s, Ac). Calcd for C14H16N2O6: C, 54.54; H, 5.23;
N, 9.09. Found: C, 54.59; H, 5.24; N, 9.03.
1-Acetyl-4-methoxy-5-nitroindoline 8 and 1-acetyl-4-
methoxy-7-nitroindoline 9. 1-Acetyl-4-methoxyindoline
4 (96 mg, 0.5 mmol) was added to a stirred solution of
NaNO3 (43 mg, 0.5 mmol) in TFA (2 mL) at 2158C and
4-Methoxy-5-methylindole 11. To a stirred solution of 10
(3.26 g, 18 mmol) in dry DMF (36 mL) were added DMF
dimethyl acetal (94%; 2.51 g, 19.8 mmol) and pyrrolidine

8202
G. Papageorgiou, J. E. T. Corrie / Tetrahedron 56 (2000) 8197±8205
(1.8 mL). The mixture was stirred under nitrogen at 1258C
for 3 h, then cooled to room temperature and concentrated in
vacuo. The oily residue was dissolved in toluene±acetic
acid (5:3, 36 mL) and added to a stirred mixture of iron
powder (18 g) and silica gel (70±230 mesh, 45 g) in
toluene±acetic acid (5:3, 235 mL) under nitrogen. The
mixture was heated under re¯ux for 1 h, cooled to rt, diluted
with CH2Cl2 and ®ltered. The ®lter cake was washed
thoroughly with CH2Cl2 and the combined ®ltrates were
washed successively with aq. Na2S2O5, saturated aq.
NaHCO3 and brine, dried and evaporated. Flash chroma-
tography (CH2Cl2±light petroleum (3:2)) gave 11 as a
colourless viscous oil (0.48 g, 16%) that was used without
Calcd for C12H14N2O4: C, 57.59; H, 5.64; N, 11.19. Found:
C, 57.68; H, 5.62; N, 11.20.
Comparative photolysis of 1-acetyl-4-methoxy-7-
nitroindoline 9 and methyl 1-acetyl-7-nitroindoline-5-
acetate 1b
Separate solutions (1 mL) of 9 and 1b (each 89.6 mM in
EtOH±25 mM Na phosphate, pH 7 (5:95)) in 1 cm path
length cells were irradiated simultaneously for intervals of
up to 8 min in
a Rayonet Photochemical Reactor
(16£350 nm lamps) and UV±Vis spectra were recorded
after each time interval (see Fig. 2).
1
further puri®cation; H NMR (90 MHz): d 8.10 (1H, br s,
NH), 7.08±6.88 (3H, m, ArH), 6.64±6.50 (1H, m, H3), 3.98
(3H, s, OMe), 2.32 (3H, s, Me).
Comparative photolysis of 1-acetyl-4-methoxy-7-
nitroindoline 9 and 1-acetyl-4-methoxy-5-methyl-7-
nitroindoline 14
4-Methoxy-5-methylindoline 12. NaBH3CN (0.75 g,
9 mmol) was added portionwise over 10 min to a solution
of 11 (0.48 g, 2.98 mmol) in acetic acid (20 mL) (exo-
thermic reaction) and the mixture was stirred at room
temperature for 0.5 h. Water (3 mL) was added and the
solvent was removed under reduced pressure. The residue
was dissolved in EtOAc and washed with saturated aq.
NaHCO3 and brine, dried and evaporated to give 12 as a
viscous oil (449 mg, 92%) that was used immediately with-
out puri®cation; 1H NMR (90 MHz): d 6.80 (1H, d, Jˆ9 Hz,
H6), 6.31 (1H, d, Jˆ9 Hz, H7), 3.76 (3H, s, OMe), 3.56
(2H, t, Jˆ8 Hz, H2), 3.12 (2H, t, Jˆ8 Hz, H3), 2.14 (3H,
s, Me).
Separate solutions (1 mL) of 9 and 14 (each 74.8 mM in
EtOH±25 mM Na phosphate, pH 7 (5:95)) in 1 cm path
length cells were irradiated simultaneously for intervals of
up to 6 min as described above and UV±Vis spectra were
recorded after each time interval (data not shown).
N,N-Dimethyl-2-methyl-3-nitroaniline 15. This com-
pound has been prepared previously16 but the amount of
dimethyl sulfate speci®ed was insuf®cient to achieve full
alkylation. The procedure given here is satisfactory.
2-Methyl-3-nitroaniline (20.9 g, 137 mmol) was added to
a
solution of K2CO3 (47.5 g, 343 mmol) in water
(130 mL) and the mixture was heated to 1008C. Dimethyl
sulfate (36.4 g, 27.3 mL, 288 mmol) was added slowly over
1 h the mixture was heated overnight. The progress of the
reaction was monitored by TLC (EtOAc±light petroleum
(1:3)). More K2CO3 (47.5 g) and dimethyl sulfate (27 mL)
were added and heating was continued until all the starting
material was consumed. The solution was then cooled to rt,
diluted with water and extracted with Et2O. The combined
organic phases were evaporated, treated with conc. aq.
ammonia (100 mL) and stirred at room temperature for
20 min. The solution was concentrated, diluted with water
and extracted with Et2O. The combined organic phases were
dried evaporated and the residual oil was dissolved in Ac2O
(50 mL) and stirred at room temperature overnight (to
acetylate the monomethyl product). The solution was
concentrated under reduced pressure and the residue was
dissolved in Et2O and washed with 1 M aq. HCl. The
aqueous phase was basi®ed to pH 13 with 2 M aq. NaOH
and extracted with Et2O. The organic extract was washed
with brine, dried and evaporated and the residue was
distilled under reduced pressure to give 15 as a yellow oil
(17.04 g, 69%), bp 1108C/0.7 mmHg (lit.17 bp: 191±1928C/
100 mmHg); IR: nmax/cm21 (®lm) 2940, 2830, 2780, 1600,
1520, 1350, 1285, 1145, 1045, 955, 815; 1H NMR
(90 MHz): d 7.19±7.49 (3H, m, ArH), 2.72 (6H, s,
NMe2), 2.42 (3H, s, Me).
1-Acetyl-4-methoxy-5-methylindoline 13. Crude 4-meth-
oxy-5-methylindoline 12 (449 mg, 2.75 mmol) was
dissolved in a mixture of acetic anhydride (10 mL) and
glacial acetic acid (10 mL) and the mixture was heated
under re¯ux for 3 h, then cooled to room temperature and
concentrated. The residue was dissolved in EtOAc and
washed with water, saturated aq. NaHCO3 and brine, dried
and evaporated to give 13 as white crystals (388 mg, 69%),
mp 139±1408C (EtOAc±light petroleum); IR: nmax/cm21
1
1650, 1610, 1335, 1220, 1080; H NMR (90 MHz): d 7.84
(1H, d, Jˆ8.3 Hz, H7), 6.99 (1H, d, Jˆ8.3 Hz, H6), 4.05
(2H, t, Jˆ8.3 Hz, H2), 3.77 (3H, s, OMe), 3.19 (2H, t,
Jˆ8.3 Hz, H3), 2.23 (3H, s, Me), 2.19 (3H, s, Ac). Calcd
for C12H15NO2: C, 70.22; H, 7.37; N, 6.82. Found: C, 70.17;
H, 7.37; N, 6.85.
1-Acetyl-4-methoxy-5-methyl-7-nitroindoline 14. To a
solution of NaNO3 (140 mg, 1.65 mmol) in TFA (5 mL)
was added 13 (308 mg, 1.5 mmol) and the mixture was
stirred at room temperature for 3 h. The solution was poured
into ice-cold water and extracted with EtOAc. The
combined organic phases were washed with saturated aq.
NaHCO3 and brine, dried and evaporated to give a red
viscous oil which after trituration with Et2O±light petro-
leum gave 14 as red microcrystals (155 mg, 41%), mp
153±1548C (EtOAc±light petroleum); UV (EtOH):
l
(EtOH±25 mM Na phosphate, pH 7.0 (1:9)): lmax/nm 246
max/nm 247 (e/M21 cm21 22600), 329 (3100); UV
2-(2-Dimethylamino-6-nitrophenyl)acetaldehyde semi-
carbazone 16. To a stirred solution of 15 (5.40 g,
30 mmol) in dry DMF (30 mL) was added tris(dimethyl-
amino)methane (6.54 g, 45 mmol) and the mixture was
heated under nitrogen to 1158C (internal temp.) for 4 h.
The progress of the reaction was followed by the
(e/M21 cm21 18900), 338 (3300); IR: nmax/cm21 1670,
1600, 1515, 1375, 1335; H NMR (90 MHz): d 7.52 (1H,
1
s, H6), 4.22 (2H, t, Jˆ8.1 Hz, H2), 3.86 (3H, s, OMe), 3.23
(2H, t, Jˆ8.1 Hz, H3), 2.25 (3H, s, Ac), 2.22 (3H, s, Me).

G. Papageorgiou, J. E. T. Corrie / Tetrahedron 56 (2000) 8197±8205
8203
consumption of the starting nitroaniline, monitored by TLC
(EtOAc±light petroleum (1:3)). The dark red solution was
cooled in an ice bath, diluted with DMF (20 mL) and a
solution of semicarbazide hydrochloride (3.51 g,
31.5 mmol) and conc. HCl (5.4 mL) in water (50 mL) was
added. Stirring was continued for 30 min and the pH was
raised to 5.0. After a further 30 min at 08C the pH was raised
to 7.5. The precipitated yellow solid was ®ltered, washed
with ice-cold water and ether and dried in vacuo. Recrys-
tallisation from aq. EtOH afforded 16 as yellow microcrys-
tals (3.93 g, 49%), mp 156±1578C; IR: nmax/cm21 3450,
C12H16N2O: C, 70.56; H, 7.89; N, 13.71. Found: C, 70.39;
H, 7.91; N, 13.64.
Nitration of 1-acetyl-4-(N,N-dimethylamino)indoline. To
a stirred solution of NaNO3 (204 mg, 2.4 mmol) in TFA
(20 mL), cooled to 2108C, was added 1-acetyl-4-N,N-
dimethylaminoindoline 19 (408 mg, 2 mmol) and the
mixture was stirred for 5 h, keeping the temperature
below 08C. The solution was poured into ice-cold water
and extracted with EtOAc. The combined organic phases
were washed with saturated aq. NaHCO3 and brine, dried
and evaporated to give a red viscous oil (640 mg). Flash
chromatography (EtOAc±light petroleum (9:1)) gave two
fractions. The ®rst was 1-acetyl-4-N,N-dimethylamino-5-
nitroindoline 20 as ®ne orange needles (74 mg, 15%), mp
1
3160, 1720, 1600, 1525, 1350; H NMR (500 MHz): d
(,1:3 mixture of syn and anti stereoisomers) 9.91 and
9.69 (1H, 2£s, NH), 7.71 and 7.53 (1H, 2£dd, Jˆ1.2,
7.9 Hz, H5) 7.45 and 7.37 (1H, 2£t, Jˆ7.9 Hz, H4), 7.42
(1H, dd, Jˆ1.2, 7.9 Hz, H3), 7.32 and 6.64 (1H, 2£t,
Jˆ4.9 Hz, CHN), 5.91 and 5.53 (2H, 2£br s, NH2),
4.02 and 3.84 (2H, 2£d, Jˆ4.9 Hz, ArCH2), 2.79
and 2.69 (6H, 2£s, NMe2). Calcd for C11H15N5O3:
C, 49.80; H, 5.70; N, 26.40. Found: C, 49.65; H, 5.70; N,
26.14.
163±1648C (EtOAc±light petroleum); UV (EtOH): lmax
/
nm 245 (e/M21 cm21 25300), 266 (24250), 332 (11550);
(EtOH±25 mM Na phosphate, pH 7.0 (2.5:97.5)): lmax/nm
248 (e/M21 cm21 24800), 356 (11300); IR: nmax/cm21
1665, 1595, 1385, 1305; 1H NMR (500 MHz): d 7.93
(1H, d, Jˆ8.5 Hz, H7), 7.63 (1H, d, Jˆ8.5 Hz, H6), 4.14
(2H, t, Jˆ8.5 Hz, H2), 3.19 (2H, t, Jˆ8.5 Hz, H3), 2.80 (6H,
s, NMe2), 2.24 (3H, s, Ac). Calcd for C12H15N3O3: C, 57.82;
H, 6.07; N, 16.85. Found: C, 57.76; H, 6.13; N, 16.64.
4-N,N-Dimethylaminoindole 17. A suspension of 16
(6.37 g, 24 mmol) in EtOH (200 mL) was hydrogenated
for 6 h at 60 psi over 10% Pd±C (1.35 g). The reaction
mixture was ®ltered and the ®ltrate evaporated. The residue
was dissolved in EtOAc and washed with saturated aq.
NaHCO3 and brine, dried and evaporated to give a pink
solid. Recrystallisation (EtOAc±light petroleum) followed
by ¯ash chromatography of the residue from the mother
liquor (EtOAc±light petroleum (1:3)) gave 17 as white
crystals (3.30 g, 86%), mp 104±1068C; UV: lmax (EtOH)/
nm 222 (e/M21 cm21 35900), 277 (10700); IR: nmax/cm21
3070, 1605, 1575, 1365; 1H NMR (90 MHz): d 8.12 (1H, br
s, NH), 7.24±6.84 (3H, m, ArH), 6.70±6.46 (2H, m, ArH),
2.99 (6H, s, NMe2). Calcd for C10H12N2: C, 74.97; H, 7.55;
N, 17.48. Found: C, 74.74; H, 7.61; N, 17.48.
The second material eluted (413 mg) was dissolved in a
mixture of MeOH (75 mL), water (15 mL) and conc. HCl
(7.5 mL) and heated under re¯ux for 3 h. The solution was
diluted with water, concentrated in vacuo, basi®ed to pH 12
and extracted with EtOAc. The combined organic phases
were washed with brine, dried and evaporated to give a
yellow solid (399 mg). Flash chromatography (EtOAc±
light petroleum (2:3)) gave two fractions. The ®rst was
processed as described below, while the second was
4-N,N-dimethylamino-5,7-dinitroindoline 21 as bright red
microcrystals (145 mg, 29%), mp 190±1918C (EtOAc±
light petroleum); UV (EtOH): lmax/nm 222 (e/M21 cm21
34100), 355 (46000); (EtOH±25 mM Na phosphate, pH
7.0 (5:95)): lmax/nm 225 (e/M21 cm21 31700), 374
(40600); IR: nmax/cm21 3380, 1590, 1400, 1370, 1320,
1-Acetyl-4-(N,N-dimethylamino)indoline 19. To a solu-
tion of 17 (0.80 g, 5 mmol) in dry THF (15 mL) cooled to
08C under nitrogen was slowly added borane-dimethyl sul-
®de (10 M in THF; 1.90 g, 2.37 mL, 25 mmol). The solution
was then treated with TFA (10 mL, 130 mmol) and the
mixture was stirred at 08C for 1 h, then at room temperature
for 4 h. The progress of the reaction was followed by TLC
(EtOAc±light petroleum (4:1)). After 5 h the solution was
cooled to 08C and more BH3´Me2S (1 mL) was added. After
a further 1 h at room temperature the solution was quenched
with water (5 mL), basi®ed to pH 11 with 2 M aq. NaOH
and extracted with CH2Cl2. The combined organic phases
were washed with brine, dried and evaporated. The residue
was dissolved in a mixture of acetic anhydride (15 mL) and
glacial acetic acid (15 mL), stirred at room temperature
overnight and concentrated in vacuo. The residue was
dissolved in EtOAc and washed with 0.5 M aq. NaOH and
brine, dried and evaporated to give a white solid. Flash
chromatography (EtOAc±light petroleum (4:1)) afforded
19 as white needles (0.77 g, 80%), mp 101±1028C
(EtOAc±light petroleum); IR: nmax/cm21 1655, 1595,
1
1295; H NMR (90 MHz): d 8.48 (1H, s, H6), 6.96 (1H,
br s, NH), 3.92 (2H, t, Jˆ9 Hz, H2), 3.20 (2H, t, Jˆ9 Hz,
H3), 2.91 (6H, s, NMe2). Calcd for C10H12N4O4: C,
47.62; H, 4.80; N, 22.20. Found: C, 47.79; H, 4.81;
N, 22.24.
The ®rst fraction (48 mg) from the above column was
dissolved in a mixture of acetyl chloride (5 mL) and acetic
acid (5 mL) and heated under re¯ux for 6 h. The solution
was poured into ice-water, basi®ed to pH 12 with 2 M aq.
NaOH and washed with CH2Cl2. The combined organic
phases were washed with brine, dried and evaporated to
give a brown viscous oil (66 mg). Flash chromatography
(EtOAc±light petroleum (9:1)) gave 1-acetyl-4-N,N-
dimethylamino-7-nitroindoline 22 as yellow microcrystals
(23 mg, 5%), mp 177±1788C (EtOAc±light petroleum); UV
(EtOH): lmax/nm 227 (e/M21 cm21 35200), 244 (34300)
280 (12600) 373 (16700); UV: (EtOH±25 mM Na
phosphate, pH 7.0 (5:95)) lmax/nm 235 (e/M21 cm21
1
1
1575, 1340, 1310, 790; H NMR (90 MHz): d 7.87 (1H,
30800), 401 (12000); IR: nmax/cm21 1670, 1590, 1375; H
d, Jˆ8.3 Hz, H7), 7.13 (1H, t, Jˆ8.3 Hz, H6), 6.61 (1H,
d, Jˆ8.3 Hz, H5) 4.03 (2H, t, Jˆ8 Hz, H2), 3.10 (2H, t,
H3), 2.76 (6H, s, NMe2), 2.20 (3H, s, Ac). Calcd for
NMR (500 MHz): d 7.72 (1H, d, Jˆ9 Hz, H6), 6.52 (1H, d,
Jˆ9 Hz, H5), 4.20 (2H, t, Jˆ8 Hz, H2), 3.15 (2H, t, Jˆ8 Hz,
H3), 2.96 (6H, s, NMe2), 2.22 (3H, s, Ac). Calcd for

8204
G. Papageorgiou, J. E. T. Corrie / Tetrahedron 56 (2000) 8197±8205
C12H15N3O3: C, 57.82; H, 6.07; N, 16.85. Found: C, 57.66;
H, 6.04; N, 16.80.
1 M aq. NaOH. The solution was washed with ether and
analysed by reverse-phase HPLC (mobile phase 25 mM
Na phosphate, pH 6.0130% MeCN v/v at 1.5 mL/min) to
show two peaks with tR 3.8 and 5.0 min. The solution was
lyophilised and the components were separated by prepara-
tive HPLC. The column was eluted ®rst with 25 mM Na
phosphate, pH 6.0 for 1 h (all ¯ow rates 2.5 mL/min),
then with 25 mM Na phosphate, pH 6.0112.5% MeCN.
Fractions containing the ®rst peak were combined, analysed
and quanti®ed (UV spectroscopy) to give 25 (38 mmol). The
solution was lyophilised and desalted by re-application to
the preparative HPLC column. The column was ®rst eluted
with water for 1 h, then with water120% MeCN. Fractions
containing the product were combined, analysed and quan-
ti®ed (UV spectroscopy) to give a solution of 25 (22 mmol)
that was evaporated and redissolved in a small volume of
Irradiation of 1-acetyl-4-N,N-dimethylamino-7-nitro-
indoline 22
A solution (1 mL) of 22 (33.6 mM in EtOH±25 mM Na
phosphate, pH 7) in a 1-cm path length cell was irradiated
for up to 6 min in a Rayonet Photochemical Reactor
(16£350 nm lamps) and monitored by uv spectroscopy,
which indicated that no photolysis took place.
1-[S-(4-t-Butoxycarbonyl)-4-(t-butoxycarbonylamino)]-
butanoyl-4-methoxyindoline 23. Crude 4-methoxy-
indoline (149 mg, 1 mmol) was prepared as described
above, dissolved in dry MeCN (8 mL) and treated with
DMAP (366 mg, 3 mmol) and N-BOC-l-glutamic acid
a-t-butyl ester (364 mg, 1.2 mmol), followed by 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide (268 mg, 1.4
mmol). The mixture was stirred at room temperature for
16 h, then evaporated and the residue, dissolved in EtOAc,
was washed with 0.5 M aq. HCl, saturated aq. NaHCO3 and
brine, dried and evaporated to give a viscous oil. Flash
chromatography (EtOAc±light petroleum (3:7)) and tritura-
tion with ether gave 23 as white crystals (315 mg, 72%), mp
131±1328C (EtOAc±light petroleum); IR: nmax/cm21 3350,
1725, 1680, 1660, 1605, 1250, 1180; 1H NMR (500 MHz):
d 7.84 (1H, d, Jˆ8.1 Hz, H7), 7.17 (1H, t, Jˆ8.1 Hz, H6),
6.58 (1H, d, Jˆ8.1 Hz, H5), 5.22 (1H, d, Jˆ7.7 Hz, NH),
4.18±4.26 and 4.10±4.18 (1H, 2£m, CH, rotamers), 4.043
and 4.039 (2H, 2£t, Jˆ8.5 Hz, H2), 3.83 (3H, s, OMe), 3.10
and 2.96 (2H, 2£t, Jˆ8.5 Hz, H3), 2.57±2.43 (2H, m,
COCH2), 2.30±2.23 (1H, m, CH), 2.07±1.99 (1H, m, CH),
1.47 (9H, s, CMe3), 1.42 (9H, s, CMe3). Calcd for
C23H34N2O6: C, 63.57; H, 7.89; N, 6.45. Found: C, 63.42;
H, 7.90; N, 6.41.
1
water for storage at 2208C; H NMR (500 MHz, D2O,
acetone ref.): d 7.82 (1H, d, Jˆ9.1 Hz, H6), 6.93 (1H, d,
Jˆ9.1 Hz, H5), 4.32 (2H, t, Jˆ7.9 Hz, H2), 3.96 (3H, s,
OMe), 3.75 (1H, t, Jˆ5.8 Hz, CH), 3.11 (2H, t, Jˆ7.9 Hz,
H3), 2.78 (2H, t, Jˆ7.5 Hz, COCH2), 2.15±2.19 (2H, m,
CH2CH); HRMS (FAB) m/z 346.1020 (M1Na)1.
C14H17N3O61Na requires 346.1015.
Fractions of the second peak from the original preparative
HPLC, that contained the 5-nitro isomer, were processed
similarly and had H NMR (500 MHz, D2O, acetone ref.):
1
d 7.94 (1H, d, Jˆ9.0 Hz, H7), 7.87 (1H, d, Jˆ9.0 Hz, H6),
4.24 (2H, t, Jˆ8.4 Hz, H2), 3.94 (3H, s, OMe), 3.84 (1H, t,
Jˆ6.2 Hz, CH), 3.31 (2H, t, Jˆ8.4 Hz, H3), 2.72±2.80 (2H,
m, COCH2), 2.14±2.22 (2H, m, CH2CH).
1-(4-Carboxybutanoyl)-4-methoxy-7-nitroindoline 26.
The acid 24 (210 mg, 0.8 mmol) was added to a stirred
solution of NaNO3 (68 mg, 0.8 mmol) in TFA (4 mL),
cooled to 2158C, and the mixture was stirred for 1 h
while allowing the temperature to rise to 2108C. The dark
solution was poured into ice-cold water and extracted with
EtOAc. The combined organic phases were washed with
brine, dried and evaporated to give a dark viscous oil.
Flash chromatography (EtOAc±light petroleum±AcOH
(2.5:7:0.5)) gave two products. The ®rst compound eluted
was mostly the required 7-nitro isomer 26 as a brown
viscous oil (76 mg, 31%) which was photolysed without
further puri®cation (see below); 1H NMR (90 MHz): d
9.98 (1H, br s, CO2H), 7.64 (1H, d, Jˆ8 Hz, H6), 6.58
(1H, d, Jˆ8 Hz, H5), 4.20 (2H, t, Jˆ8.5 Hz, H2), 3.92
(3H, s, OMe), 3.04 (2H, t, Jˆ8.5 Hz, H3), 2.64±2.32 (4H,
m, 2£CH2), 2.00 (2H, quintet, CH2).
1-(4-Carboxybutanoyl)-4-methoxyindoline 24. A solution
of crude 4-methoxyindoline (447 mg, 3 mmol), prepared as
described above, in chloroform (30 mL) was treated with
glutaric anhydride (376 mg, 3.3 mmol) and the solution was
stirred at room temperature for 18 h. The solvent was
removed in vacuo and the residue, dissolved in EtOAc,
was washed with 1 M aq. HCl. The organic phase was
washed with brine, dried and evaporated and the residue was
triturated with ether to give 24 as white crystals (338 mg,
43%), mp 146±1488C (EtOAc±light petroleum); IR: nmax
/
cm21 1700, 1660, 1605, 1250, 1205; H NMR (500 MHz):
1
d 7.82 (1H, d, Jˆ8.1 Hz, H7), 7.15 (1H, t, Jˆ8.1 Hz, H6),
6.58 (2H, d, Jˆ8.1 Hz, H5), 4.07 (2H, t, Jˆ8.5 Hz, H2), 3.83
(3H, s, OMe), 3.10 (2H, t, Jˆ8.5 Hz, H3), 2.51 (2H, t,
Jˆ7.2 Hz, NCOCH2), 2.44 (2H, t, Jˆ7.2 Hz, CH2CO2H)
and 2.02 (2H, quintet, CH2). Calcd for C14H17NO4:
C, 63.87; H, 6.51; N, 5.32. Found: C, 64.01; H, 6.57; N,
5.34.
The material that eluted second was principally the 5-nitro
isomer but was not fully characterised.
4-Methoxy-7-nitrosoindole 27. A solution of crude 26
(193 mg, 0.62 mmol) in EtOH (4 mL) was diluted with
50 mM ammonium phosphate, pH 7.0 (76 mL) and irra-
diated for 2 h under nitrogen in a Pyrex ¯ask, using a
100 W mercury arc lamp. The solution was diluted with
water and extracted with EtOAc. The combined organic
phases were washed with saturated aq. NaHCO3 and
brine, dried and evaporated. The residue was ¯ash
chromatographed (EtOAc±light petroleum (1:4)) to give
1-[S-(4-Amino-4-carboxybutanoyl)]-4-methoxy-7-nitro-
indoline 25. The protected glutamate derivative 23
(250 mg, 0.575 mmol) was added to a stirred solution of
sodium nitrate (49 mg, 0.575 mmol) in TFA (5 mL) and
the mixture was stirred at 2158C for 40 min. The dark
solution was concentrated in vacuo and the residue was
dissolved in water (50 mL) and adjusted to pH 6.7 with

G. Papageorgiou, J. E. T. Corrie / Tetrahedron 56 (2000) 8197±8205
8205
27 as small green needles (7 mg, 6%), mp 120±1218C
(Et2O±light petroleum); UV: lmax (EtOH)/nm 232
(e/M21 cm21 4700), 256 (9300), 289 (5500), 392 (13200);
lmax (EtOH±25 mM Na phosphate, pH 7.0 (1:9))/nm
and 6.4 min for the phosphate and 25 respectively and the
corresponding absorption coef®cients at 347 nm were 510
and 4330 M21 cm21. Extents of conversion were 28.4 and
38.1% for the phosphate and 25, respectively.
257 (e/M21 cm21 7500), 300 (6300), 404 (14500); nmax
/
cm21 (CHCl3) 3440, 1600, 1585, 1500, 1490, 1330,
1170, 955, 895; H NMR (500 MHz): d 10.59 (1H, br
1
Acknowledgements
s, H1), 8.97 (1H, d, Jˆ8.5 Hz, H6), 7.13 (1H, t,
J1,2ˆJ2,3ˆ2.7 Hz, H2), 6.91 (1H, d, Jˆ8.5 Hz, H5),
6.66 (1H, t, J1,3ˆ2.7 Hz, H3), 4.16 (3H, s, OMe);
HRMS (FAB) m/z 177.0659 (M1H)1. C9H8N2O21H
requires 177.0664.
We are indebted to Dr Becky Wan for the X-ray structure
determination. We thank Dr K. J. Welham for high reso-
lution mass spectrometric data, Drs V. R. N. Munasinghe
and G. Kelly for recording NMR spectra, Mr P. Sharratt for
amino acid analyses and the MRC Biomedical NMR Centre
for access to facilities.
Photolysis and product analysis for 25
Solutions of 25 (0.45±0.54 mM) in 25 mM ammonium
phosphate, pH 7.0 containing 1 or 5 mM dithiothreitol
were irradiated for varying times (30 or 45 s) in 1 mm
path length cells in a Rayonet Photochemical Reactor
(16£350 nm lamps). Solutions were analysed by reverse-
phase HPLC (Merck Lichrosphere RP8 column; mobile
phase 25 mM Na phosphate, pH 6.0130% MeCN v/v,
¯ow rate 1.5 mL/min) and the extent of photolysis was
determined by comparison of peak areas with those of
unphotolysed controls. Aliquots of the photolysed solutions
were also subjected to quantitative amino acid analysis as
previously described.3 Measured glutamate concentrations
were 88±93% of the values expected from the extent of
photolysis and were not affected by the concentration of
dithiothreitol.
References
1. Adams, S. R.; Tsien, R. Y. Annu. Rev. Physiol. 1993, 55, 755;
Corrie, J. E. T.; Trentham, D. R. In Biological Applications of
Photochemical Switches; Morrison, H., Ed.; Wiley: New York,
1993; pp 243±305; Marriott, G., Ed. Methods in Enzymology;
Academic: New York, 1998; Vol. 291.
2. Corrie, J. E. T.; Papageorgiou, G. Tetrahedron 1999, 55, 237.
3. Papageorgiou, G.; Ogden, D. C.; Barth, A.; Corrie, J. E. T.
J. Am. Chem. Soc. 1999, 121, 6503.
4. Canepari, M.; Papageorgiou, G.; Corrie, J. E. T.; Ogden, D.
Biophys. J. 2000, 78, 355A.
5. Amit, B.; Ben-Efraim, D. A.; Patchornik, A. J. Am. Chem. Soc.
1976, 98, 843.
6. McKillop, A.; Swann, B. P.; Taylor, E. C. J. Am. Chem. Soc.
1971, 93, 4920.
Comparative photolysis of 1a and 25
7. March, J. Advanced Organic Chemistry, 3rd ed.; Wiley: New
York, 1985 (pp 463±466).
Separate solutions of 1a and 25 (each 0.50 mM in 25 mM
ammonium phosphate, pH 7.0 containing 5 mM dithio-
threitol) were irradiated for 30 s (Rayonet, see above) in
1 mm path length cells. The extent of photolysis was deter-
mined by reverse-phase HPLC (column as above, mobile
phase 25 mM Na phosphate, pH 6.0120% MeCN v/v, ¯ow
rate 1.5 mL/min). Retention times were 6.7 and 6.4 min,
respectively and quanti®cation was by measurement of
peak heights. Extents of conversion were 23.3 and 50.5%
for 1a and 25 respectively.
8. Kawase, M.; Sinhababu, A. K.; Borchardt, R. T. J. Heterocycl.
Chem. 1987, 24, 1499; Buchanan, J. G.; Stoddart, J.; Wightman,
R. H. J. Chem. Soc., Perkin Trans. 1 1994, 1417.
9. Gangjee, A.; Vasudevan, A.; Queener, S. F. J. Med. Chem.
1997, 40, 479.
10. Kruse, L. I. Heterocycles 1981, 16, 1119.
11. Maryanoff, B. E.; McComsey, D. F. US Patent 4,210,590;
Chem. Abstr. 1980, 93, 204454.
12. Schuddeboom, W.; Warman, J. M.; Biemans, H. A. M.;
Meijer, E. W. J. Phys. Chem. 1996, 100, 12369.
13. Wieland, T. A.; Unger, O. Chem. Ber. 1963, 96, 253.
14. Beckett, A. H.; Daisley, R. W.; Walker, J. Tetrahedron 1968,
24, 6093.
Quantum yield estimation for 25
A solution of 1-(2-nitrophenyl)ethyl phosphate18 and 25
(each 0.5 mM) and dithiothreitol (5 mM) in 25 mM ammo-
nium phosphate, pH 7.0 was irradiated in aliquots (7£30 ml)
in a 1 mm path length cell, using 347 nm light from a
frequency-doubled pulsed ruby laser (average energy
122 mJ, one pulse per aliquot). The irradiated aliquots
were combined and the extent of photolysis for each
compound was determined by reverse-phase HPLC (column
as above, mobile phase 25 mM Na phosphate, pH 6.0120%
MeCN v/v, ¯ow rate 1.5 mL/min). Retention times were 2.7
15. Feiser, L. F.; Feiser, M. In Reagents for Organic Synthesis,
Wiley: New York, 1967; Vol. 1, p 153.
16. Esser, F.; Stahle, H.; Sullke, S.; Muramatsu, J.; Kitagawa,
H. K.; Uhida, S. German Patent 19,514,579; Chem. Abstr. 1996,
126, 8114.
17. von Tatschaloff, A. J. Prakt. Chem. 1902, 65, 239.
18. Corrie, J. E. T.; Reid, G. P.; Trentham, D. R.; Hursthouse,
M. B.; Mazid, M. A. J. Chem Soc., Perkin Trans. 1 1992, 1015.