Synthesis of a new hydrophilic o-nitrobenzyl photocleavable linker suitable for use in chemical proteomics

Article abstract of DOI:10.1016/j.tetlet.2005.09.077

Linkers currently used in solid phase synthesis are generally short and hydrophobic, limiting their usefulness in biological systems. Herein, we describe a facile synthesis of a long, hydrophilic, o-nitrobenzyl photocleavable linker, suitable for construc

Full text of DOI:10.1016/j.tetlet.2005.09.077

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 8241–8244
Synthesis of a new hydrophilic o-nitrobenzyl photocleavable
linker suitable for use in chemical proteomics
Andrew M. Piggott and Peter Karuso*
Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney NSW 2109, Australia
Received 8 July 2005; revised 1 September 2005; accepted 14 September 2005
Available online 6 October 2005
Abstract—Linkers currently used in solid phase synthesis are generally short and hydrophobic, limiting their usefulness in biological
systems. Herein, we describe a facile synthesis of a long, hydrophilic, o-nitrobenzyl photocleavable linker, suitable for constructing
affinity supports for use in chemical proteomics. The rates of photolysis of the linker on exposure to UV light emitting diodes are
reported.
ꢀ 2005 Elsevier Ltd. All rights reserved.
In chemical proteomics1 and reverse chemical proteo-
mics,2 a target molecule is linked to some form of tag
(such as biotin,3 a fluorophore4,5 or a solid support6),
which can be used to isolate a single protein or a family
of proteins from an entire proteome. Since Merrifieldꢀs
pioneering work on solid phase organic synthesis in
1963,7 there has been an exponential increase in the
range of cleavable linkers that are available to immobi-
lise small molecules onto solid supports.8,9 The incorpo-
ration of new photolabile,10 traceless11 and safety
catch12 linkers allows the orthogonal cleavage of differ-
ent reagents from the same solid phase, greatly increas-
ing the range of reactions possible. However, as these
linkers were designed for use in organic solvents, they
are generally hydrophobic, and are often quite short,
thereby limiting their usefulness in biological systems.
a statistical distribution of lengths, thereby increasing
chemical diversity. The o-nitrobenzyl grouphas been
incorporated in a wide variety of photolabile linkers14–16
and protecting groups17,18 due to its chemical stability
and rapid cleavage by exposure to UV light around
365 nm.
The rate limiting stepin the photolysis of the o-nitrobenz-
yl groupis the abstraction of a benzylic proton by the
photoactivated nitro group, so the presence of a methyl
groupon the benzylic carbon increases the acidity of this
proton and has been shown to increase the rate of pho-
tolysis considerably.19 Therefore, to set upa secondary
o-nitrobenzylic centre, 2-nitroterephthalic acid was
reduced to the diol 2 with BH3ÆTHF then oxidised to
the dialdehyde 3 with PCC (Scheme 1). Attempts to
methylate 3 using MeMgBr were unsuccessful, yielding
a mixture of products including methylation of the aro-
matic ring and nitro group, as reported previously in the
literature.20 MeTiCl3,21,22 which is more selective than
the standard Grignard reagents, gave the desired sec-
ondary diol 4 exclusively. Activation of 4 with N,N0-
disuccinimidyl carbonate to form the dicarbonate 5,
followed by coupling with mono-Boc protected TEG
diamine (Boc–NH–TEG–NH2) 1, gave the Boc-pro-
tected secondary o-nitrobenzyl linker 6, which is suitable
for long-term storage. When required, 6 was deprotected
with TFA, yielding the free diamine 7.
This letter describes the synthesis and characterisation
of a long, hydrophilic, photocleavable linker 7, suitable
for generating affinity supports for use in chemical
proteomics studies. Poly(ethylene glycol) (PEG) is an
ideal backbone for constructing linkers for use in aque-
ous media due to its hydrophilicity, chemical stability
and biocompatibility.13 Monodisperse PEGs, such as
tetra(ethylene glycol) (TEG), allow discrete, fixed-length
linkers to be synthesised and characterised easily, while
polydisperse PEGs can be used to generate linkers with
Keywords: Photocleavable; Photolabile; Linker; o-Nitrobenzyl; 2-
Nitrobenzyl; Chemical proteomics; Affinity support; UV LED.
A dilute solution of 6 in chloroform-d was exposed to
UV light (kmax = 365 nm) from a pyrex-filtered 16 W
mercury vapour fluorescent lamp and the photolysis
*
Corresponding author. Tel.: +612 9850 8290; fax: +612 9850 8313;
e-mail: peter.karuso@mq.edu.au
0040-4039/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.09.077

8242
A. M. Piggott, P. Karuso / Tetrahedron Letters 46 (2005) 8241–8244
O
O
OH
HO
H
HO
c
a
b
OH
H
OH
NO2
O
NO2
O
NO2
NO2 OH
2
3
4
O
O
d
N
O
O
O
O
O
O
N
NO2
5
O
O
e
O
O
O
O
NH
HN
O
O
N
H
O
H
O
N
O
O
O
O
O
NO2
O
6
f
O
O
H2N
O
O
N
H
O
H
N
O
O
O
NH2
O
NO2
O
7
Scheme 1. Reagents and conditions: (a) BH3ÆTHF/THF, 0 ꢁC, 1 h then 40 ꢁC, 18 h, 92%, (b) PCC/DCM, 25 ꢁC, 4 h, 93%; (c) MeTiCl3/Et2O,
ꢀ30 ꢁC, 3 h, 65%; (d) DSC, Et3N, DMAP (cat.)/MeCN, 25 ꢁC, 18 h, 65%; (e) Boc–NH–TEG–NH2 1, TEA/MeCN, 25 ꢁC, 18 h, 82%; (f) 20% TFA/
CHCl3, 25 ꢁC, 30 min, quant.
Figure 1. Photolysis of 6 by exposure to UV light (kmax = 365 nm) from a pyrex-filtered 16 W mercury vapour fluorescent lamp, as followed by
1H NMR.
was followed by NMR spectroscopy (Fig. 1). The reac-
tion proceeded rapidly, with the majority of the linker
cleaved after 60 min of exposure. Analysis of the photo-
lysis products by NMR and MS revealed that the reac-
tion yields the expected nitrosoketone 8, as well as the
starting material 1, following spontaneous decarboxyl-
ation of the corresponding carbamate 9 (Scheme 2).
The data obtained were fitted to a first order exponential
equation (Fig. 2) using least squares regression and the
pseudo first order rate constant for the photolysis reac-
tion was found to be 1.0 · 10ꢀ3 sꢀ1, corresponding to a
half-life of 12 min.
Mercury vapour lamps emit high intensity radiation
over a wide range of wavelengths, including a consider-
able amount of heat and hence may cause undesirable
photooxidation and/or thermal damage to biological
systems. Conversely, UV light emitting diodes (LEDs),
which have recently become commercially available,
emit UV light in a focused beam over a narrow
(ꢁ30 nm) bandwidth, with virtually no dissipation of
heat. In addition, each LED is small enough to be posi-
tioned directly over one well of a 96-well microtitre
plate, allowing the UV light to be pointed directly at
the surface of the well or its contents. UV LEDs are

A. M. Piggott, P. Karuso / Tetrahedron Letters 46 (2005) 8241–8244
8243
6
0.14
a
0.12
O
O
O
N
O
N
O
H
H
3
0.10
0.08
0.06
0.04
O
8
NO
370 nm
395 nm
+
O
O
O
N
O
N
O
3
H
H
9
- CO2
O
0
1
2
3
4
O
Time (h)
H2N
N
O
3
H
Figure 3. Optical density of a solution of 6 at 240 nm after exposure to
UV light from either a 370 nm (1 mW) or a 395 nm (6 mW) UV LED.
1
Scheme 2. Reagents and conditions: (a) hm (365 nm)/CDCl3, 25 ꢁC,
2 h, quant.
In conclusion, we have described a facile synthesis of a
new hydrophilic o-nitrobenzyl photocleavable linker
with utility in many areas of chemical biology for the
reversible linking of macromolecules, combinatorial
libraries and natural products to solid phases, fluoro-
phores or other tags. We have also presented a novel
cleavage strategy using recently available UV diodes
that are small, give off no heat and have narrow emis-
sion profiles. These diodes are suitable for high through-
put devices due to their small size and wide range of
spectral characteristics.
100
80
60
40
20
0
Supplementary data
Supplementary data associated with this article can
be found, in the online version, at doi:10.1016/j.tetlet.
2005.09.077.
0
20
40
60
80
100
120
Time (min)
Figure 2. Rate of photolysis of
6 by exposure to UV light
(kmax = 365 nm) from a pyrex-filtered 16 W mercury vapour fluores-
cent lampwas determined by integrating the NMR signal correspond-
ing to H3 (d 7.90).
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now available in a range of different wavelengths from
350 to 400 nm, although power output is still quite
low at the shorter wavelengths. Therefore, two different
UV LEDs, with emission maxima of 370 nm (1 mW)
and 395 nm (6 mW), were investigated for their ability
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