Synthesis and photolytic evaluation of a nitroindoline-caged glycine with a side chain of high negative charge for use in neuroscience

Article abstract of DOI:10.1016/j.tet.2011.05.045

A photolabile precursor of the neuroinhibitory amino acid glycine has been synthesised with two phosphate groups attached to the indoline nucleus at a 4-alkoxy substituent. In common with the photochemical properties of other 1-acyl-7-nitroindolines, this releases glycine on a sub-μs time scale upon irradiation with near-UV light. The synthetic route previously developed for the preparation of the GABA analogue required some modifications because of the greater hydrolytic sensitivity of the glycine compound. The phosphorylation method used here could be beneficial to the synthesis of other nitroindoline-caged amino acids, especially the related caged GABA derivative. Glycine released by laser photolysis on spinal cord neurons generated fast-rising responses and the pharmacological properties of the reagent are such that it is useful for physiological experiments.

Full text of DOI:10.1016/j.tet.2011.05.045

Tetrahedron 67 (2011) 5228e5234
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Synthesis and photolytic evaluation of a nitroindoline-caged glycine with a side
chain of high negative charge for use in neuroscienceq
,
b
c
George Papageorgiou a , Marco Beato , David Ogden
*
a MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
b Department of Neuroscience, Physiology and Pharmacology, University College London, Gower Street, London WC1E 6BT, UK
c
ꢀ
ꢀ
CNRS Unite 8118, Universite Paris Descartes, 75006 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A photolabile precursor of the neuroinhibitory amino acid glycine has been synthesised with two
phosphate groups attached to the indoline nucleus at a 4-alkoxy substituent. In common with the
photochemical properties of other 1-acyl-7-nitroindolines, this releases glycine on a sub-ms time scale
upon irradiation with near-UV light. The synthetic route previously developed for the preparation of the
GABA analogue required some modifications because of the greater hydrolytic sensitivity of the glycine
compound. The phosphorylation method used here could be beneficial to the synthesis of other
nitroindoline-caged amino acids, especially the related caged GABA derivative. Glycine released by laser
photolysis on spinal cord neurons generated fast-rising responses and the pharmacological properties of
the reagent are such that it is useful for physiological experiments.
Received 21 February 2011
Received in revised form 12 April 2011
Accepted 10 May 2011
Available online 18 May 2011
Keywords:
Photolysis
Caged compounds
Nitroindolines
Phosphorylation
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
a satisfactory extent, they are now commercially available and have
been extensively applied in neuroscience.8 In contrast, caged GABA 3
The technique of liberating biologically active compounds from
photolabile precursors, upon brief irradiation with near-UV light is
a valuable tool for studies of biological processes. The method relies
on the availability of suitable reagents, widely known as ‘caged’
compounds and their syntheses and applications are described in
reviews.1 These reagents must satisfy a number of prerequisites
such as solubility and stability in aqueous media, high purity and
pharmacological inertness prior to photolysis, rapid and efficient
photolysis at neutral pH followed by fast dark reactions after
photolysis, and no release of toxic by-products after photocleavage.
Failure to fulfil any one of the above criteria usually renders a caged
reagent inadequate for application in biological systems.
and caged glycine 4 were found to bind to their respective ionotropic
GABA or glycine receptors on neuronal cells prior to photolysis,
blunting the response to the photolytically released amino acid.4,7 A
separate investigation has indicated that neither caged glycine 4 nor
caged
agent 4 might have useful application in studies of the effects of co-
agonist binding to NMDA receptors.5 Photolytic release of
-alanine
L-aspartate 5 inhibit the NMDA receptor, suggesting that re-
L
from 6 has enabled the observation of alanine-induced pre-steady-
state currents of three different neutral amino acid transporters.6
Photolysis of these reagents in aqueous solution proceeds with
stoichiometric release of the amino acid, accompanied by the corre-
sponding nitrosoindole by-product as shown in Scheme 1. A study of
the photolysis mechanism9 of 1-acyl-7-nitroindolines in aqueous
solution determined the release rate of the amino acid as w5ꢀ106 sꢁ1
at ambient temperature, corresponding to a half-life of w150 ns,
which is sufficiently rapid for mimicking biological processes.
The advantageous photochemical properties and hydrolytic
stability of 1-acyl-7-nitroindolines motivated us to seek means of
eliminating or at least suppressing the adverse pharmacological
properties of 3 and we have previously reported the synthesis and
photochemical evaluation of the modified caged GABA reagent 7.10
The incorporation of a high concentration of negative charge close
to the nitroindoline nucleus did not affect its photochemical
properties. Furthermore, the structure retained the advantages
of hydrolytic stability of the cage and its fast and efficient
During the last decade we have produced
7-nitroindoline-caged neuroactive amino acids, including
mates 12 and2,3 -aminobutyrate(GABA)3andglycine4,4
-aspartate
5,5 and -alanine 6,6 which have been applied in various biological
a
number of
L-gluta-
g
L
L
systems. Detailed pharmacological evaluation has found that caged
glutamates 1 and 2 do not bind to glutamergic receptors prior
to photolysis and are therefore suitable reagents for investigations
of synaptic transmission.4,5,7 They fulfil all the above criteria to
q
The work described here is, in part, the subject of patent applications US Appl.
11/507,961 and EP Appl. 06254350.4.
* Corresponding author. Tel.: þ44 20 8816 2407; fax: þ44 20 8906 4477; e-mail
address: gpapage@nimr.mrc.ac.uk (G. Papageorgiou).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.05.045

G. Papageorgiou et al. / Tetrahedron 67 (2011) 5228e5234
5229
2. Results and discussion
R2
CO2R1
R1
In our previous investigations10 of a synthetic route to 7, we had
found that di-Boc protection of the amino group in a GABA pre-
cursor had been ineffective in avoiding side reactions at this centre,
although the same protection strategy had been effective for amino
CO2
NH3
N
N
R2
NO2
NO2
groups in precursors to 4-methoxy-7-nitroindoline derivatives of
L-
O
O
glutamate,13 and -alanine.6 For the eventual successful synthesis of
L
7, the problem was avoided by masking the amino group as an azide
that was converted to an amine at a late stage of the synthesis.
Nevertheless, in view of the successful application of the di-Boc
1 R1 = CH2CO2Me, R2 = H
2 R1 = H, R2 = OMe
3 R1 = Me, R2 = (CH2)3NH3
4 R1 = H, R2 = CH2NH3
protection strategy for synthesis of L-alanine, we felt it was
worthwhile to test the same methodology here. Using the
4-methoxy system, the route shown in Scheme 2 gave the di-Boc
precursor 10. Homogeneous nitration, as used by us previously
for related nitrations,3b,6,13 gave a mixture in which the major
component was the N-nitro species 11, isolated as a crystalline solid
and characterised by 1H NMR spectroscopy and elemental analysis
(see Supplementary data). Although 5- and 7-mononitro isomers
were also present in the reaction products, the substantial forma-
tion of 11 made this route unacceptable. It now appears that both
steric and electronic effects are important in the ability of the
double Boc protection to limit unwanted N-nitration, and we
therefore returned to the azide strategy used for 7.
Given the somewhat unpredictable outcome of results with only
minor changes in structure, we first tested the azide route by
preparing the methoxynitroindoline-caged glycine 15, as shown in
Scheme 3, both to validate the individual steps and to have a sample
of 15 available for pharmacological testing in comparison with our
intended target compound 8. Only two points of Scheme 3 deserve
specific comment. First, the nitration of the azidoacetylindoline 13
was carried out using a minor modification of a reported pro-
cedure14 (NaNO3 in place of LiNO3) in which scandium triflate acts
as a catalyst for nitration. In practice, for this substrate there was no
advantage in yield or regioselectivity over our previous claycop-
mediated nitration procedure,3b but the scandium triflate pro-
cedure was investigated as an alternative because of difficulties
encountered during the synthesis of 8 (see below) and because we
had previously found that the claycop procedure was ineffective
with indolines bearing a bulky substituent at the 4-position.10 The
second point of note relates to the Staudinger reduction of the azide
14 that was carried out in aq DMF, as had been done at the final
stage in synthesis of 8. In the latter case the initial product isolated
had been an iminophosphorane, which required mild but pro-
longed acid treatment (pH 1 at rt overnight) to release the required
amine.10 In contrast, the reduction of 14 under the same conditions
gave the amine directly upon work-up of the reaction mixture. As
discussed below, the susceptibility to hydrolysis of the initial imi-
nophosphorane product is remarkably idiosyncratic.
CO2Me
OMe
CO2
N
N
NH3
NH3
NO2
NO2
O
O
Me
5
6
photorelease at near-UV and 405 nm wavelengths, but brought
about a large reduction in the interaction of 7 with GABAA recep-
tors.11a Further detailed neurophysiological investigations11b
revealed the existence of a new class of miniature currents
(‘preminis’) in developing interneurons that reflect the activation
of presynaptic GABAA receptors following random vesicular release.
R
R
NH3
hν
+ H+ +
N
O
R
CO2
H2O
N
NH3
H
NO2
NO
R
Scheme 1. Photocleavage reaction of 7-nitroindoline-caged amino acids in aqueous
solution.
The successful reduction of adverse pharmacological properties
of 7 prior to photolysis opened the possibility that the same strategy
might be applied to the glycine analogue 8. Various caged glycine
derivatives have been reported by other workers12 but have suffered
from problems of instability or inadequate photolytic efficiency. We
had anticipated that synthesis of the diphosphate nitroindoline
(DPNI)-glycine 8 would closely follow the route described for 7. In
the event, a number of modifications were required, in part because
of greater hydrolytic sensitivity of the amide bond in the glycine
series of precursors. In addition, the method for introduction of the
two phosphate groups was substantially improved over that pre-
viously described in the synthesis of 7. In the course of developing
the synthesis, we also prepared the 4-methoxy compound 15 and in
the process obtained some further insight into side reactions that
occurred during nitration reactions of N-acylindolines.
With preparation of 15 readily achieved, we returned to the
synthesis of 8 and, following similar methods to those in Scheme 3,
readily obtained the azide 19 (Scheme 4). However, upon very brief
alkaline treatment (25 mM NaOH for 2 min), which was intended
to remove the acetate groups, a mixture of products was detected by
TLC and UVevis spectroscopy that indicated significant hydrolysis of
the amide linkage had taken place. This contrasts with experience in
the synthesis of the caged GABA 7, where hydrolysis of the corre-
sponding acetates was readily achieved under very similar condi-
tions, without detectable amide hydrolysis. Electron-withdrawal by
2-
2-
OPO3
OPO3
2-
2-
OPO3
OPO3
O
O
the a-azido substituentevidently makes the azidoacetyl amide bond
much more sensitive to base hydrolysis and we subsequently found
that the amide bond was w10% hydrolysed at pH 7.0 overnight at rt
(see details in Supplementary data).
To avoid the problem of amide hydrolysis, we instead carried out
the hydrolysis step before nitration, since the amide bond in the
non-nitrated indoline was expected to be much more stable. The
NH3
N
N
NH3
NO2
NO2
O
O
8
7

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G. Papageorgiou et al. / Tetrahedron 67 (2011) 5228e5234
OMe
OMe
NO2
OMe
OMe
c
a, b
d
Boc
N
N
N
O
N
N
N(Boc)2
NHBoc
NO2
H
O
O
10 (75%)
11 (37%)
9 (84%)
Scheme 2. Reagents: (a) NaBH3CNeAcOH; (b) EDCeHO2CCH2NHBoceMeCN; (c) (Boc)2OeEt3NeDMAPeCH2Cl2; (d) Cu(NO3)2eAc2OeCH2Cl2.
diacetate 18 was readily hydrolysed to the diol 20 (Scheme 5) and
we intended to introduce the two phosphate groups (in protected
form) at this stage. Phosphorylation of a related diol in the caged
GABA synthesis using phosphoramidite chemistry had been prob-
lematic because of low solubility of the diol in THF. A change to
dichloromethane solvent with pyridinium trifluoroacetate (PyTFA)
as the phosphitylation catalyst15 followed by oxidation with
m-CPBA gave the bis(di-tert-butyl phosphate) 21 in excellent yield.
Nitration of the protected bis-phosphate 21 was complicated by the
presence of the acid-sensitive tert-butyl esters, which underwent
partial deprotection under any of the conditions tried. After
numerous trial reactions, it was found that the nitration of
diphosphate 21 was best achieved with a mixture of acetic anhy-
dride, sodium nitrate and a 4-fold excess of trichloroacetic acid
(relative to the amount of NaNO3). As noted above, partial depro-
tection of the phosphate groups had taken place, indicated by the
fact that the compound had become water-soluble and behaved as
an anion on analytical anion-exchange HPLC (see Experimental
section). Rather than characterise the partially deprotected prod-
uct, although it appeared to be principally a single species, we felt it
OMe
OMe
OMe
a, b
c
N
N
N
Br
N3
H
O
O
12 (84%)
13 (96%)
d
OMe
OMe
e
N
N
N3
NH3
NO2
NO2
O
O
15 (64%)
14 (75%)
Scheme 3. Reagents: (a) NaBH3CNeAcOH; (b)EDCeBrCH2CO2HeMeCN; (c)NaN3eDMF;
(d) NaNO3eAc2OeSc(OTf)3eMeCN; (e) PPh3eaq DMF.
OAc
OAc
a, b
OAc
OAc
OAc
OAc
OAc
OAc
O
O
O
O
c
d
N
N
N
N
O
Br
N3
N3
H
NO2
O
O
17 (77%)
18 (90%)
19 (72%)
16
Scheme 4. Reagents: (a) NaBH3CNeAcOH; (b) EDCeBrCH2CO2HeMeCN; (c) NaN3eDMF; (d) Cu(NO3)2eAc2OeCH2Cl2.
2-
2-
OPO3
OPO3
OP(O)(OBut)2
OP(O)(OBut)2
d,e
OH
OH
2-
2-
OPO3
OPO3
O
O
O
O
f
b, c
N3
a
18
N
O
N
O
N
N3
NH3
N
N3
NO2
NO2
O
O
20 (85%)
21 (88%)
22 (55%)
8 (47%)
Scheme 5. Reagents: (a) NaOHeaq MeOH; (b) Et2NP(OtBu)2ePyTFAeCH2Cl2; (c) MCPBA; (d) NaNO3eAc2OeCCl3CO2HeMeCN; (e) TFA; (f) TCEPeaq DMF.

G. Papageorgiou et al. / Tetrahedron 67 (2011) 5228e5234
5231
was more convenient to remove the remaining tert-butyl groups
but found that prior removal of excess nitrate salt from the reaction
mixture was required before treatment with TFA. Without such
removal, treatment of the freeze-dried reaction mixture with TFA
resulted in an intractable dark mixture, and efforts to quench the
excess nitrate by addition of reactive electrophiles prior to work-up
were also ineffective. Thus the crude water-soluble reaction mix-
ture was subjected to preparative reverse-phase HPLC and the
isolated nitroindoline product, after lyophilisation, was successfully
deprotected with TFA to give 22 in 55% yield.
With 22 available, completion of the synthesis required only
reduction of the azide group, but Staudinger reaction using either
triphenylphosphine or tri-n-butylphosphine in aq DMF failed to
yield pure 8 even after prolonged mild acidic treatment of the re-
action mixture. It was evident from HPLC data (not shown) that an
iminophosphorane was formed, as in the case of the related caged
GABA synthesis,10 but it was much more resistant to hydrolysis.
This was particularly surprising since reduction of 14 under the
same conditions had given the amine 15 directly upon mild acidic
work-up (see above), implying ready hydrolysis of the imino-
phosphorane in that case. However, reduction of the azide 22 was
successfully accomplished using the water-soluble tris(2-
carboxyethyl)phosphine hydrochloride (TCEP) to give 8 in 47%
yield after purification by anion-exchange chromatography, pre-
cipitation as its barium salt and reconversion to the sodium salt.
The photochemistry of both 8 and its model analogue 15 was
similar to that of other N-acyl-7-nitroindolines. Continuous irra-
diation of separate solutions of 8 and 15, monitored by UVevis
spectroscopy, as described previously,2,3a,10 showed progressive
decrease of each starting compound and accumulation of the
nitrosoindole chromophore. Clear isosbestic points were formed as
for the progressive photolysis but extensive investigations (data not
shown) have not resolved this matter.
2.1. Neurophysiological recordings upon glycine release by
near-UV laser photolysis
Glycine is a major inhibitory fast neurotransmitter in the
mammalian spinal cord. To test the photorelease of glycine from
DPNI-glycine 8 and the effects of 8 itself upon neuronal prepara-
tions, experiments were done with laser photolysis in spinal cord
slices prepared from neonatal rats. Whole cell patch clamp re-
cordings were made under conditions of high intracellular chloride
ion concentration, generating inward membrane current in re-
sponse to activation of glycine receptors by glycine photoreleased
from 8. The morphology and identity of recorded neurons were
obtained by epifluorescence imaging of the fluorophore Alexa 488
included in the intracellular solution in the whole cell-recording
pipette. Photolysis was with laser pulses at 405 nm usually of
0.2 ms duration as described previously.11a The laser spot diameter
was 1 mm in the focal plane and could be directed to the soma or
neurites of recorded neurons with sub-micron accuracy. The ex-
perimental methods are described in Supplementary data.
Responses recorded in a motoneuron to concentrations of gly-
cine released at different laser intensities in 0.2 ms pulses from
DPNI-glycine 8, present at 2 mM, are shown in Fig. 1A. The ampli-
tude of the response increases with laser intensity (0.4e8.1 mW)
up to the point of saturation, as shown in the bar chart of Fig. 1D.
The 20e80% rise times increased from 1.2ꢂ0.2 to 2.8ꢂ0.5 ms with
increasing intensity of the pulse. For comparison, the rise time of
spontaneous post-synaptic currents (shown in Fig. 1B) is in the
0.3e1.1 ms range, over a population of 12 cells. As the laser in-
tensity was increased, also the decay time-constant increased, from
8.1ꢂ0.4 to 28.0ꢂ6.1 ms, compared with corresponding values for
spontaneous currents of between 4.2 and 9.1 ms. The slower rise
and fall of the responses to photolytically released glycine is ap-
parent in the normalised and superimposed traces of Fig. 1C. For
comparison, an average of spontaneous post-synaptic currents is
also shown. The slower kinetics may be attributed to the larger
photolysis progressed indicating
a
clean conversion (see
Supplementary data). Photolysis of 8 was also monitored by HPLC
and released glycine was measured by amino acid analysis, as de-
scribed for related compounds.2,3a Glycine recovery was in the
range 68e69%. This was significantly below the essentially stoi-
chiometric release reported for other 7-nitroindoline-caged amino
acids and appears inconsistent with the clean spectroscopic data
Fig. 1. Responses in spinal motorneurons under whole cell voltage clamp to laser-evoked photolysis of extracellular 2 mM DPNI-glycine 8 in a 1 mm focal spot. Negative deflections
show inward membrane current due to Clꢁ ion flux through receptor ion channels activated by glycine binding. (A) Increasing intensity of the laser pulse (duration 0.2 ms)
generates progressively larger amplitudes of response. Grey traces are superimposed individual events; black traces are the mean at each intensity. Laser power in mW is given at
the objective, with 2 mM of 8 present in the bath 50% of the light is absorbed by inner filtering. (B) For comparison spontaneous glycine synaptic responses and their mean are
shown on the same scale. (C) The time course of normalised responses to low (light grey), medium and high intensity (black) pulses are compared with synaptic responses (dashed
line) on the same time scale. The inset shows a time scale expansion of the rise times of the averaged response (continuous lines) and of spontaneous events (dashed line). (D, E, F)
Summary bar charts of data from 12 neurons of the peak amplitude (D), the 20e80% rise time (E) and the exponential time-constant of response decline (F). In panels (E) and (F) the
left column is data for spontaneous IPSCs due to synaptic glycine release in the same neurons, other columns show responses to glycine released by different laser intensities.

5232
G. Papageorgiou et al. / Tetrahedron 67 (2011) 5228e5234
volume into which glycine is released by laser spot photolysis
compared with the volume of synaptic release.
4.2. 1-(2-Bromoacetyl)-4-(1,3-diacetoxypropan-2-yloxy)
indoline (17)
Direct interference between uncaged DPNI-glycine 8 and gly-
cine receptors was tested by measuring the amplitude of sponta-
neous synaptic currents in control and in the presence of 3 mM
DPNI-glycine. The mean event amplitudes as well as their rise
times did not change in the two conditions (see Supplementary
data Fig. S2 and text). However, following repetitive uncaging on
the same spot, a rundown of the light evoked current was consis-
tently observed (Fig. S2 and text). The origin of this rundown will
require further investigations, but it could be associated with the
observed incomplete stoichiometry obtained on photolysis of
DPNI-glycine.
A solution of 4-(1,3-diacetoxypropan-2-yloxy)indoline (2.93 g,
10 mmol; prepared from 16 as previously described10) in dry MeCN
(70 mL) was treated with 2-bromoacetic acid (2.08 g, 15 mmol),
followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hy-
drochloride (2.68 g, 14 mmol) and the mixture was stirred at rt
under nitrogen overnight. The solvent was evaporated and the
residue was taken up in EtOAc (80 mL), washed with dilute aq HCl,
saturated aq NaHCO3 and brine, dried and evaporated to pale
crystals. Recrystallisation (EtOAcehexanes) followed by flash
chromatography [EtOAcehexanes (2:3)] of the mother liquor gave
17 (3.17 g, 77%) as white crystals, mp 77e79 ꢃC (EtOAcehexanes);
3. Conclusions
1H NMR (600 MHz)
d
7.87 (d, J¼8.2 Hz, 1H), 7.19 (t, J¼8.2 Hz, 1H),
6.73 (d, J¼8.2 Hz, 1H), 4.71 (quintet, J¼5.2 Hz, 1H), 4.34 (dd, J¼5.7,
11.9 Hz, 2H), 4.29 (dd, J¼4.9, 11.9 Hz, 2H), 4.21 and 4.18 (2ꢀt,
J¼8.5 Hz, 2H, rotamers), 4.16 (s, 2H), 3.16 (t, J¼8.5 Hz, 2H), 2.07 (s,
6H). Anal. Calcd for C17H20NO6Br: C, 49.29; H, 4.87; N, 3.38. Found:
C, 49.53; H, 4.67; N, 3.63.
The synthesis of 8 was successfully accomplished but, despite its
similarity to the previously described caged GABA 7, significant
modification of the previously established synthetic route was re-
quired. The unexpected reactivity of the densely functionalised
intermediate compounds was surmounted by employing more ef-
fective reagents and appropriate reaction conditions. The con-
trolled nitration of indolines in synthesis of these caged
compounds remains subject to a need for individual optimisation,
while the improved phosphorylation method could usefully be
applied to a better synthesis of the caged GABA 7.
Release of glycine on spinal cord neurons by localised laser
photolysis of 8 showed fast activation of glycinergic currents with
time course in the millisecond range. Although slower than glycine
mediated synaptic currents recorded in the same neurons, the ki-
netics of activation by photoreleased glycine are nonetheless fast
enough to be useful in experiments to determine the distribution of
glycine sensitivity in neuronal compartments and to yield kinetic
data of glycine receptor function at synaptic sites in situ. A further
appealing feature of 8 is that, contrary to the block of GABA
4.3. 1-(2-Azidoacetyl)-4-(1,3-diacetoxypropan-2-yloxy)
indoline (18)
NaN3 (0.97 g, 15 mmol) was added to a solution of 17 (2.07 g,
5 mmol) in dry DMF (40 mL) and the mixture was stirred at rt
overnight. The solvent was removed under reduced pressure and
the residue was diluted with EtOAc (70 mL), washed with water
and brine, dried and evaporated to give 18 as white crystals (1.69 g,
90%), mp 64e65 ꢃC (Et2Oehexanes); 1H NMR (600 MHz)
d 7.89 (d,
J¼8.1 Hz, 1H), 7.19 (t, J¼8.1 Hz, 1H), 6.72 (d, J¼8.1 Hz, 1H), 4.71
(quintet, J¼5.2 Hz, 1H), 4.34 (dd, J¼5.8, 11.9 Hz, 2H), 4.29 (dd, J¼4.7,
11.9 Hz, 2H), 4.00 (t, J¼8.2 Hz, 2H), 3.97 (s, 2H), 3.14 (t, J¼8.2 Hz,
2H), 2.06 (s, 6H). Anal. Calcd for C17H20N4O6: C, 54.25; H, 5.36; N,
14.89. Found: C, 54.29; H, 5.52; N, 14.68.
(
g-aminobutyric acid) responses seen with the caged GABA ana-
logue DPNI-GABA 7, DPNI-glycine does not interact with the glycine
receptors. The origin of the rundown of responses seen with mul-
tiple pulses of DPNI-glycine photolysis applied consecutively to the
same sites was not elucidated in the experiments described here.
The present synthesis has provided enough material for further
pharmacological characterisation and these studies will be repor-
ted elsewhere in due course.
4.4. 1-(2-Azidoacetyl)-4-(1,3-diacetoxypropan-2-yloxy)-7-
nitroindoline (19)
To a solution of 18 (489 mg, 1.35 mmol) in a mixture of CH2Cl2
(13.5 mL) and acetic anhydride (27 mL) was added copper nitrate
hemipentahydrate (365 mg, 1.75 mmol) and the mixture was stir-
red at rt overnight. The solution was concentrated and the residue
was re-evaporated from toluene, diluted with EtOAc (50 mL) and
washed with saturated aq NaHCO3 and brine, dried and evaporated
to brown viscous oil. Flash chromatography [EtOAcehexanes (1:1)],
followed by trituration with ether, gave 19 (407 mg, 72%) as yellow
4. Experimental
4.1. General
crystals, mp 90e91 ꢃC (EtOAcehexanes); 1H NMR (800 MHz)
d 7.80
1H NMR spectra were determined on Varian Inova 600 MHz or
Varian Inova 800 MHz spectrometer in CDCl3 solution with TMS as
internal reference, unless otherwise specified. Elemental analyses
were carried out by MEDAC Ltd., Surrey, UK. Electrospray mass
spectra were recorded at the School of Pharmacy, University of
London. Merck 9385 silica gel was used for flash chromatography.
Analytical HPLC was performed on a 250ꢀ4 mm Merck Lichrospher
RP8 column or a 125ꢀ4 mm Whatman Partisphere SAX column.
Flow rate was 1.5 mL minꢁ1 with either column. Preparative HPLC
was carried out on a 2ꢀ30 cm column (Waters C18 packing, Cat. No.
20594) at 2 mL minꢁ1 flow rate. Details of mobile phases are given
at relevant points in the text. Preparative anion-exchange chro-
matography used a column of DEAE-cellulose (2ꢀ20 cm). Detection
for all analytical and preparative chromatography was at 254 nm.
Organic solvents were dried over anhydrous Na2SO4 and evaporated
under reduced pressure. Hexanes (bp 40e60 ꢃC) were redistilled
before use. Photolysis experiments were performed in a Rayonet
RPR-100 photochemical reactor fitted with 16ꢀ350 nm lamps.
(d, J¼9.0 Hz, 1H), 6.84 (d, J¼9.0 Hz, 1H), 4.82 (quintet, J¼5.3 Hz, 1H),
4.36 (dd, J¼5.9, 11.9 Hz, 2H), 4.31 (dd, J¼4.7, 11.9 Hz, 2H), 4.22 (t,
J¼8.1 Hz, 2H), 4.04 (s, 2H), 3.12 (t, J¼8.1 Hz, 2H), 2.07 (s, 6H). Anal.
Calcd for C17H19N5O8: C, 48.46; H, 4.54; N, 16.61. Found: C, 48.12; H,
4.47; N, 16.37.
4.5. 1-(2-Azidoacetyl)-4-(1,3-dihydroxypropan-2-yloxy)
indoline (20)
A solution of 18 (1.51 g, 4 mmol) in MeOH (80 mL), water (8 mL)
and 1 M aq NaOH (9.6 mL, 9.6 mmol) was stirred at rt for 2 min,
quenched with 1 M aq citric acid (19.2 mL) and concentrated to
w40 mL. The solution was diluted with water (20 mL), extracted
with EtOAc (3ꢀ40 mL) and the combined organic extract was
washed with saturated aq NaHCO3 and brine, dried and evaporated
to give 20 (1.00 g, 85%) as white crystals, mp 110e112 ꢃC
(EtOAcehexanes); 1H NMR (800 MHz; DMSO-d6)
J¼8.1 Hz, 1H), 7.13 (t, J¼8.1 Hz, 1H), 6.78 (d, J¼8.1 Hz, 1H), 4.77 (t,
d 7.66 (d,

G. Papageorgiou et al. / Tetrahedron 67 (2011) 5228e5234
5233
J¼5.5 Hz, 2H), 4.26 (quintet, J¼5.5 Hz, 1H), 4.22 (s, 2H), 4.00
(t, J¼8.4 Hz, 2H), 3.58e3.61 (m, 2H), 3.33e3.56 (m, 2H), 3.04 (t,
J¼8.4 Hz, 2H). Anal. Calcd for C13H16N4O4: C, 53.42; H, 5.52; N,19.16.
Found: C, 53.69; H, 5.61; N, 18.80.
(Dowex 50, Naþ form); 1H NMR (600 MHz, D2O, acetone ref.)
d
7.85
(d, J¼9.0 Hz, 1H), 7.13 (d, J¼9.0 Hz, 1H), 4.92 (quintet, J¼4.7 Hz, 1H),
4.37 (s, 2H), 4.25 (t, J¼7.8 Hz, 2H), 4.09e4.17 (m, 4H), 3.23 (t,
J¼7.9 Hz, 2H); LRMS (ESI): calcd for (C13H13N5O12P2þ3H)ꢁ 496.0,
found: 495.9. The remainder of the solution was lyophilised to
a pale yellow solid and used in the next step.
4.6. 1-(2-Azidoacetyl)-4-[1,3-bis(di-tert-butoxyphosphoryl-
oxy)propan-2-yloxy]indoline (21)
4.8. 1-(2-Aminoacetyl)-4-[1,3-bis(dihydroxyphosphoryloxy)
propan-2-yloxy]-7-nitroindoline (8)
A solution of 20 (0.58 g, 2 mmol) in dry CH2Cl2 (60 mL) was
treated under nitrogen with pyridinium trifluoroacetate (1.16 g,
6 mmol) and di-tert-butyl N,N-diethylphosphoramidite (93% pu-
rity; 1.61 g, 6 mmol) and the mixture was stirred under nitrogen at
rt for 20 min. The solution was cooled to 0 ꢃC and treated dropwise
with a solution of m-chloroperbenzoic acid (77% peracid; 2.69 g,
12 mmol) in CH2Cl2 (60 mL). The solution was stirred at 4 ꢃC for
45 min, diluted with CH2Cl2 and washed successively with 10% aq
Na2S2O5, saturated aq NaHCO3 and brine, dried and evaporated.
Flash chromatography (EtOAc) gave 21 (1.19 g, 88%) as white crys-
A solution of 22 (191 mmol) in water (3 mL) was diluted with
DMF (27 mL) and treated with tris(2-carboxyethyl)phosphine hy-
drochloride (TCEP) (383 mg, 1.34 mmol) and the mixture was stir-
red at rt under nitrogen for 28 h. The solution was concentrated
under reduced pressure and the residue was diluted with water
(50 mL) and washed with EtOAc (3ꢀ40 mL). Analysis by reverse-
phase HPLC [mobile phase 25 mM Na phosphate, pH 6.0eMeCN
(25:1), tR 1.8 min], and by anion-exchange HPLC [mobile phase
100 mM Na phosphate, pH 6eMeCN (5:1), 1.5 mL/min, tR 5.2 min]
confirmed complete reduction of the azide. The solution was then
adjusted to pH 6.0 with 1 M aq NaOH and quantified by UVevis
tals, mp 78e80 ꢃC (Et2Oehexanes); 1H NMR (600 MHz)
d 7.87 (d,
J¼8.2 Hz, 1H), 7.18 (t, J¼8.2 Hz, 1H), 6.75 (d, J¼8.2 Hz, 1H), 4.70
(quintet, J¼4.9 Hz, 1H), 4.18 (d, J¼5.2 Hz, 2H), 4.17 (d, J¼5.2 Hz, 2H),
3.98 (t, J¼8.4 Hz, 2H), 3.97 (s, 2H), 3.18 (t, J¼8.4 Hz, 2H), 1.47 (s,
18H), 1.45 (s, 18H). Anal. Calcd for C29H50N4O10P2: C, 51.47; H, 7.45;
N, 8.28. Found: C, 51.87; H, 7.591; N, 8.18.
spectroscopy (51 mL, 3.7 mM, 189 mmol). The solution was diluted
with water (250 mL) to conductivity 5.3 mS cmꢁ1 and subjected to
anion-exchange chromatography using a linear gradient formed
from 10 and 500 mM NaOAc, pH 6.0 (each 1000 mL). Fractions
containing the product, which eluted at w300 mM NaOAc, were
analysed as above, combined (144 mL) and quantified by UV
4.7. 1-(2-Azidoacetyl)-4-[1,3-bis(dihydroxyphosphoryloxy)
propan-2-yloxy]-7-nitroindoline (22)
spectroscopy (0.678 mM, 98 mmol). The solution was concentrated
To a solution of 21 (237 mg, 0.35 mmol) in dry MeCN (3.5 mL)
were added acetic anhydride (357 mg, 3.5 mmol), sodium nitrate
(59.5 mg, 0.7 mmol) and trichloroacetic acid (457 mg, 2.8 mmol)
and the mixture was stirred under nitrogen at rt for 24 h. TLC
[MeOHeEtOAc (1:19)] confirmed that all starting material was
consumed. The reaction mixture was diluted with 200 mM NaOAc,
pH 6.0 (70 mL), the pH was raised from 4.8 to 5.5 with 1 M aq NaOH
and the solution was washed with Et2O (3ꢀ50 mL). Analysis of the
aqueous phase by reverse-phase HPLC [mobile phase 25 mM Na
phosphate, pH 6.0eMeCN (25:1), tR 5.8 min], and by anion-
exchange HPLC [mobile phase 100 mM Na phosphate, pH
6.0eMeCN (5:1), tR 5.2 min], confirmed that all starting material
was nitrated but partial deprotection of phosphate esters took
place. The solution was loaded onto the preparative HPLC column,
which was pre-equilibrated with 200 mM NaOAc, pH 6.0, and
subsequently eluted with the same buffer for further 1.5 h. The
column was then washed with water and the compound began to
elute after about 1 h, when the conductivity of the eluate had
lowered to that of water. The fractions containing the compound
were combined, concentrated to about 25 mL and lyophilised. The
pale yellow residue was dissolved in TFA (15 mL) and stirred at rt
for 1 h. The solvent was evaporated, the residue was dissolved in
25 mM triethylammonium phosphate, pH 6.0 (75 mL) and the pH
was raised from 2.6 to 6.0 with 1 M aq NaOH. HPLC analysis as
above confirmed full deprotection of the phosphate esters. The
solution was loaded onto the preparative HPLC column, which was
pre-equilibrated with 25 mM triethylammonium phosphate, pH
6.0, and subsequently eluted with the same buffer for further 1 h.
The column was then washed with water and the compound began
to elute after about 50 min, when the conductivity of the eluate had
lowered to that of water. Fractions containing the compound were
analysed by reverse-phase HPLC [mobile phase 100 mM Na phos-
phate, pH 6.0eMeCN (25:1), tR 3.6 min], and by anion-exchange
HPLC [mobile phase 100 mM Na phosphate, pH 6.0eMeCN (5:1),
tR 5.8 min], combined and concentrated. The residue was taken
to about 4 mL, diluted with water to 8 mL, treated with 2 M
Ba(OAc)2 (2 mL) and EtOH (4 mL) and allowed to stand at 4 ꢃC
overnight. The mixture was centrifuged and the supernatant was
analysed by UVevis spectroscopy (9% of original concentration, i.e.,
91% precipitation). The precipitate was washed with watereEtOH
(1:1) (5ꢀ15 mL) by resuspension and subsequent centrifugation
after each wash cycle. The final precipitate was dissolved in water
(15 mL) and mixed with Dowex 50 (Naþ form; 4 g) for 2 h. The resin
was filtered off, washed with water (20 mL) and the combined fil-
trates were adjusted from pH 8.4 to 6.8 with 1 M aq HCl. The filtrate
was passed through a 0.2
residue was dissolved in water (4 mL) and quantified by UVevis
spectroscopy to give 8 (22.3 mM, 89
mol, 47%) as the Naþ salt
mm membrane filter, lyophilised and the
m
[containing a small amount of tris(2-carboxyethyl)phosphine oxide
in approximately 4:1 ratio]; 1H NMR (500 MHz, D2O, acetone ref.)
d
8.00 and 7.86 (2ꢀd, J¼9.2 Hz, 1H, rotamers), 7.90 and 7.20 (2ꢀd,
J¼9.2 Hz, 1H, rotamers), 4.89 and 4.55 (2ꢀquintet, J¼5.0 Hz, 1H,
rotamers), 4.27 and 4.21 (2ꢀt, J¼7.9 Hz, 2H, rotamers), 4.14 (s, 2H),
4.00e4.07 (m, 4H), 3.47 and 3.27 (2ꢀt, J¼7.9 Hz, 2H, rotamers);
LRMS (ESI): calcd for (C13H16N3O12P2þ2H)ꢁ 470.0, found: 470.0.
Complete separation from tris(carboxyethyl)phosphine oxide was
not achieved in the purification, as shown by signals in the 1H NMR
spectrum at
d 2.38e2.43 [m, P(O)(CH2CH2CO2)3], 2.09e2.14 [m,
P(O)(CH2CH2CO2)3], with intensities corresponding to w20 mol %
of the concentration of 8.
4.9. Quantitative photolysis and product analysis for (8)
Separate solutions of 8 (0.47 mM in 25 mM Na phosphate, pH 7.0
containing 5 mM dithiothreitol) were irradiated for varying times
(20 or 25 s) in 1 mm path length cells (Rayonet Photochemical
Reactor). The solutions were analysed by anion-exchange HPLC
(mobile phase as in Section 4.8) and the extent of photolysis of each
solution was determined by comparison of peak heights with those
of non-irradiated controls. Aliquots of the photolysed solutions
were also subjected to quantitative amino acid analysis. Measured
glycine concentrations were 68e69% of the expected values from
the extent of photolysis and were not affected by the concentration
up in water (35.5 mL), passed through a 0.2
quantified by UVevis spectroscopy to give pure 22 (5.47 mM,
194
mol, 55%). An aliquot (0.6 mL) was exchanged to the Naþ salt
mm membrane and
m

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G. Papageorgiou et al. / Tetrahedron 67 (2011) 5228e5234
Marriott, G., Ed.; Academic: New York, NY, 1998; Vol. 291; (e) Pelliccioli, A.;
Wirz, J. Photochem. Photobiol. Sci. 2002, 1, 441e458.
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of dithiothreitol. The stock solution (20.9 mM) was also subjected
to quantitative amino acid analysis and the measured free glycine
contamination was 0.35%.
121, 6503e6504.
3. (a) Papageorgiou, G.; Corrie, J. E. T. Tetrahedron 2000, 56, 8197e8205; (b) Pa-
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In a further experiments, separate solutions of 8 and 15
(0.51 mM) in 25 mM Na phosphate, pH 7.0 were irradiated for in-
creasing times (0e90 s) in a 1-mm path length cell and the extent
of photolysis was monitored after each irradiation interval by
UVevis spectroscopy.
6. Zhang, Z.; Papageorgiou, G.; Corrie, J. E. T.; Grewer, C. Biochemistry 2007, 46,
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Acknowledgements
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We are grateful to Dr. J.E.T. Corrie for useful discussions and to
Dr. G. Kelly for recording NMR spectra. We thank the MRC for fi-
nancial support (project number U117592730) and the MRC Bio-
medical NMR Centre for access to facilities. DO gratefully
acknowledges support from the CNRS, ANR, Foundation Recherche
Medicale (FRM), Federation pour la Recherche sur le Cerveau (FRC),
and the EU (Photolysis LSHM-CT-2007-037765). MB is a Royal So-
ciety University Research Fellow.
Supplementary data
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2002, 1, 960e969.
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Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2011.05.045. These data in-
clude MOL files and InChiKeys of the most important compounds
described in this article.
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