Aryldiazirine-modified pyroglutamates: Photoaffinity labels for glutamate

Article abstract of DOI:10.1055/s-2005-923595

The synthesis of an aryldiazirine-modified pyroglutamate is reported for potential application as a photoaffinity label at glutamate receptors. Detailed ¹³C NMR and ¹⁹F NMR spectroscopic characterisation data for these trifluoromethy

Full text of DOI:10.1055/s-2005-923595

LETTER
247
Aryldiazirine-Modified Pyroglutamates: Photoaffinity Labels for Glutamate
A
ryldiazirine-M
m
odifiedPyroglutam
i
ates lie L. Bentz, Hannah Gibson, Clare Hudson, Mark G. Moloney,* Debbie A. Seldon, Edwina S. Wearmouth
The Department of Chemistry, Chemistry Research Laboratory, The University of Oxford, Mansfield Road, Oxford, OX1 3TA, UK
Fax +44(1865)285002; E-mail: mark.moloney@chem.ox.ac.uk
Received 13 October 2005
thallium prompted the development of a Friedel–Crafts
Abstract: The synthesis of an aryldiazirine-modified pyro-
alkylation strategy for conversion of 3-methoxyphenyl-
glutamate is reported for potential application as a photoaffinity la-
bel at glutamate receptors. Detailed 13C NMR and 19F NMR
spectroscopic characterisation data for these trifluoromethyl-substi-
diazirine 1a to 4-aldehyde 1b or 4-hydroxymethyl deriva-
tive 1c (Scheme 1).21 The utility of this approach has been
tuted aryldiazirines has been determined and shown to be useful for demonstrated by the introduction of the 3-methoxyphen-
yldiazirine unit 1a into a range of biological probes.22–31
tracking this group during synthesis.
Of interest is the rapid photolysis of the diazirine function
in these compounds (typically t1/2 for photolysis with a 15
W UV lamp is 1.7 min).26 Although the facile preparation
of bromide 1d has also been reported,21 its application as
a diazirinyl carrier has not been widely applied, and so far
has only been used for the preparation of a photoactivat-
able phenylalanine analogue.32 We report here that
bromide 1d is applicable for the incorporation of aryldiaz-
irines into sterically hindered pyroglutamate systems of
relevance to glutamate receptor structural investigations.
Key words: glutamate, photoaffinity, diazirines
Glutamate is a key player in diverse biological processes,
including amino acid metabolism (e.g., where it is an im-
portant couple with a-ketoglutarate in deamination pro-
cesses) and the mammalian central nervous system
(where it acts on ionotropic and metabotropic receptors).1
As a consequence, the control of inter- and intracellular
glutamate concentration is of paramount importance;
membrane-bound glutamate transporters play a key role
in this regard. Much effort has therefore been invested in
the identification and structural elucidation of various
glutamate receptors, and although many of these are
membrane bound, structural details are beginning to
emerge.2,3 Caged glutamate has also been developed for
the controlled release of glutamate into biological sys-
tems,4 and photoaffinity labelling has been used for the
identification of amino acid residues at enzyme active
sites or receptor binding sites.5–7 Of the possible photopre-
cursors, diazirines exhibit excellent photolytic proper-
ties,8,9 but are less readily synthetically accessible than the
more commonly used azides or nitroaryl derivatives. We
have previously demonstrated the utility of aryldiazirines
as photaffinity labels readily activated by laser irradia-
tion,10 and have become interested in the application of
this approach for the determination of binding site infor-
mation for excitatory amino acid (EAA) receptors. Azido-
derived photoaffinity labels have recently been investigat-
ed for the NMDA receptor11,12 and KA receptors,13–17 and
there has been a recent report of a selective mGluR1 radio-
ligand,18 but there are no reports of the incorporation of
diazirine systems into EAA receptor agonists. Reports by
Hatanaka19 and Brunner20 indicated that 3-methoxy-
phenyldiazirines 1a could be thallated and further reacted
with a variety of electrophiles under mild conditions,
thereby permitting introduction of an aryldiazirine unit
intact into a substrate and avoiding a lengthy linear syn-
thesis. However, the obvious drawbacks with the use of
3-Methoxyphenyldiazirine 1a was readily prepared in
high overall yield in multigram quantities from 3-bro-
moanisole in 5 steps using our previously published pro-
tocol (Scheme 1);33 this route differs slightly from an
alternative literature method,21 in that commercially avail-
able methyl trifluoroacetate is used for the initial acylation
step and iodine/triethylamine for the final oxidation.
Friedel–Crafts alkylation followed by aqueous work-up
yielded aldehyde 1b (62%), along with a lesser amount of
isomer 2b (16%). The stereochemical assignment for each
of these was readily confirmed by NOESY analysis,
which exhibited the indicated correlations (Figure 1),
confirming the earlier assignment.21 Conversion of alde-
hyde 1b to bromide 1d via alcohol 1c was readily
achieved using the literature procedure.21 Of interest is
that electrophilic bromination of anisole 1a was also
found to be readily possible; thus, bromination (Br2,
TiCl4, CH2Cl2) of 1a gave a separable 3.2:1 mixture of
isomeric bromides 1e and 2e in an overall yield of 55%;
the stereochemistry of 2e was again determined by
NOESY analysis (Figure 1). Noteworthy is the difficulty
of demonstrating the presence of the diazirine unit in these
compounds, since its UV chromophore is very weak
(typically, e is less than 1000), and mass spectroscopic
analysis is unreliable due to the facile extrusion of N2; we
found that reliable indicators for the presence of the diaz-
irine subunit were the 13C (ca. 122 ppm) and 19F (ca. –65.0
2
ppm) chemical shift and JC–F (ca. 274 Hz) coupling
constant values for the adjacent CF3 unit, which could be
readily observed (Table 1). Mercuration [Hg(O2CCF3)2
in HO2CCF3 at r.t. for 30 h] of substrate 1a gave exclu-
sively derivative 2f, presumably as a result of chelation
control, but direct plumbation of the aromatic ring of 1a
SYNLETT 2006, No. 2, pp 0247–0250
0
1.
0
2.
2
0
0
6
Advanced online publication: 23.12.2005
DOI: 10.1055/s-2005-923595; Art ID: D31605ST
© Georg Thieme Verlag Stuttgart · New York

248
E. L. Bentz et al.
LETTER
(i) n-BuLi then CF3CO2Me (77%)
(ii) NH2OH·HCl, C5H5N (92%)
(iii) TsCl, C5H5N (93%)
(iv) NH3 (88%)
N
F3C
N
N
F3C
N
Br
+
R
(v) I2, Et3N, MeOH (73%)
OMe
OMe
OMe
R
1
2
a R = H
(i) TiCl4, Cl2CHOMe, CH2Cl2 (54%)
(ii) NaBH4 (91%)
(iv) TiCl4, Br2,
CH2Cl2
b R = CHO
c R = CH2OH
d R = CH2Br
e R = Br
(iii) PPh3, CBr4, Et2O (90%)
f R = HgO2CF3
Scheme 1
or attempted mercury–lead exchange of 1f gave an intrac- The utility of these substrates for inclusion in sterically
table mixture. The assignment of the regiochemistry of 1f hindered pyroglutamate-derived substrates was demon-
is based on a report that substitution of an aromatic ring strated by the successful alkylation of ethyl 2-oxo-
with mercury does not change chemical shift values of the cyclopentanecarboxylate with bromide 1d to give
remaining protons around the ring;34 the position of sub- cyclopentanone 3 in a yield of 54% using NaH as the base
stitution is therefore apparent by identification of the (Scheme 2). When this procedure was applied for the syn-
missing proton relative to the starting material.
thesis of lactam 4, prepared as a key intermediate in our
recently published synthesis kainoid analogues,35 with
aryl bromide 1d, adduct 5 was obtained as a single diaste-
reomer in a yield of 23%.36 The stereochemistry of this
F3C
N
F3C
N
N
F3C
H
N
N
N
O
O
1
compound was confirmed by comparison of H NMR
Br
H
H
H
H
chemical shift data for related compounds,35 and by
NOESY analysis, which clearly indicated the exo-orienta-
tion of the arylmethyl residue (see Scheme 2).36 This
product was elaborated to pyroglutamate 6 by acid-catal-
ysed deprotection, ester hydrolysis, oxidation and esterifi-
cation in good overall yield; in this compound, the 13C
NMR and 19F NMR characterisation data (see Table 1) of
the diazirine unit of 6 was consistent with that of its
H
OCH3
H
OCH3
H
OCH3
H
H
H
Figure 1
N
F3C
N
CF3
N
O
CO2Et
NaH
1d
N
OMe
CH2Br
O
MeO
CO2Et
3
EtO2C
O
CH2CO2t-Bu
(23%)
NaH
N
O
Ph
4
N
N
N
F3C
N
F3C
(i) CF3CO2H, CH2Cl2 (74%)
(ii) aq NaOH, then CF3CO2H,
then RuO4, NaIO4, MeCN,
CH2Cl2 then CH2N2 (24%)
OMe
OMe
CH2
N
CH2CO2t-Bu
EtO2C
O
CH2CO2Me
CO2Me
MeO2C
O
H
H
H
N
O
H
H
Ph
6
5 (arrows indicate
NOESY enhancements)
Scheme 2
Synlett 2006, No. 2, 247–250 © Thieme Stuttgart · New York

LETTER
Aryldiazirine-Modified Pyroglutamates
249
simpler precursors.37 In contrast to the successful
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This route provides direct access to novel aryldiazirines,
and we anticipate that this sequence will facilitate the
development novel photoaffinity labels suitable for EAA
receptors.
Table 1 13C NMR and 19F NMR Chemical Shift and Coupling
Constants for the CF3 Unit in some Trifluoromethyldiazirines
Compound 13C (ppm)
JC–F (Hz)
274.7
274.8
274.9
274.4
274.7
274.7
275.6
274.7
19F (ppm)
–66.94
–64.68
–68.70
–65.16
–64.99
–
1a
1b
2b
1c
1d
1e
2e
6
122.1
121.7
121.7
122.1
122.1
121.9
121.7
122.1
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Acknowledgment
We wish to acknowledge the use of the EPSRC Chemical Database
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Obtained as a colourless oil (0.111 g, 23%); Rf = 0.20
[PE(40–60 °C)–EtOAc, 5:1]. 1H NMR (400 MHz, CDCl3):
d = 1.30 (3 H, t, J = 7.0 Hz, CH3CH2), 1.47 [9 H, s, C(CH3)3],
2.66 (2 H, d, J = 7.6 Hz, CH2CO2t-Bu), 3.09–3.15 [1 H, m,
H(6)], 3.23 (2 H, dd, J = 13.9 Hz, CH2Ph), 3.33 (3 H, s,
OCH3), 3.60–3.66 [1 H, m, H(5)], 3.76–4.26 [2 H, m, H(4)],
4.30–4.34 (2 H, m, CH3CH2), 6.21 [1 H, s, H(2)], 6.39 (1 H,
s, ArH ortho to OMe)], 6.47 (1 H, d, J = 8.0 Hz, ArH para
to OMe), 7.23 (1 H, d, J = 8.0 Hz, ArH meta to OMe), 7.27–
7.44 (5 H, m, PhH). 13C NMR (400 MHz, CDCl3): d = 14.1
(CH3CH2), 28.1 [C(CH3)3], 28.3 (CH2Ph), 33.9 (CH2CO2t-
Bu), 45.9 [C(6)], 54.7 (OCH3), 61.9 [C(5)], 62.0
(CO2CH2CH3), 63.9 [C(7)], 72.8 [C(4)], 86.9 [C(2)], 107.8
(ArC ortho to OMe), 118.3 (ArH para to OMe), 126.0,
126.5, 128.4, 128.7, 128.9 (all ArCH), 127.4 (q, J = 280.5
Hz, CF3), 126.1, 129.1 (ArC), 132.6 [C(15)], 138.3 [(C(13)],
157.6 [C(11)], 170.6 (lactam C=O), 170.8 (ethyl ester C=O),
171.7 (t-Bu ester C=O). IR (thin film): nmax = 2981 (s,
aliphatic C–H), 1728 (br s, ester and lactam C=O), 1261 (s,
C–O), 1039 (s, C–F) cm–1.
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E. L. Bentz et al.
LETTER
(37) Data for Lactam 6.
OMe), 118.7 (ArC para to OMe), 122.0 (q, J = 274.7 Hz,
CF3), 126.2 (ArC ortho to OMe), 129.3 (ArC), 131.7 (ArC),
157.6 [C(8)], 170.3, 170.7, 171.4 (C=O esters), 173.0 (C=O
lactam). IR (thin film): nmax = 3020, 2956 (m, C–H), 2401
(m, N=N), 1740 (s, C=O), 1577, 1515 (m, Ar C=C), 1216 (s,
C–O), 1039 (s, C–F) cm–1.
Obtained as a colourless oil (0.0198 g, 24%). Rf = 0.23
[EtOAc–PE (40–60 °C), 6:5]. 1H NMR ( 400 MHz, CDCl3):
d = 2.73–2.85 (2 H, m, CH2CO2Me), 3.22 (2 H, dd, J = 14.7
Hz, CH2Ph), 3.52–3.62 [1 H, m, H(3)], 3.70, 3.74, 3.77, 3.79
(4 × 3 H, s, OCH3), 3.69–3.76 [1 H, m, H(2)], 6.31 (1 H, s,
NH), 6.55 (1 H, s, ArH ortho to OMe), 6.69 (1 H, d, J = 7.9
Hz, ArH para to OMe), 7.37 (1 H, d, J = 8.0 Hz, ArH meta
to OMe). 13C NMR (400 MHz, CDCl3): d = 27.2 (CH2Ph),
28.6 [C(3)], 32.3 (CH2CO2Me), 43.9 [C(2)], 52.0, 52.8, 53.0
(OCH3), 55.2 (ArOCH3), 58.6 [C(4)], 108.0 (ArC ortho to
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Comput. Sci. 1996, 36, 746.
Synlett 2006, No. 2, 247–250 © Thieme Stuttgart · New York