p-Hydroxyphenacyl bromide as photoremoveable thiol label: A potential phototrigger for thiol-containing biomolecules

Article abstract of DOI:10.1016/S0040-4039(02)02194-9

p-Hydroxyphenacyl bromide is described as a photoremovable thiol protecting group on three biomolecules containing a free thiol group. The protecting group is efficiently incorporated by chemical coupling to the biomolecule in an ethanol-buffer mixture. The photofragmentations (λ=312 nm) were analyzed by UV, HPLC and MS methods, yielding over 70% of the free biomolecules. The concomitant formation of p-hydroxyphenylacetic thioesters derived from the corresponding thiols, as a sulfur-containing side-product, should not hinder the use of this protecting group for the caging of thiol-containing biomolecules.

Full text of DOI:10.1016/S0040-4039(02)02194-9

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 8947–8950
p-Hydroxyphenacyl bromide as photoremoveable thiol label:
a potential phototrigger for thiol-containing biomolecules
Alexandre Specht,a Sandra Loudwig,a Ling Pengb and Maurice Goeldnera,*
aLaboratoire de Chimie Bioorganique, UMR 7514 CNRS Faculte´ de Pharmacie, Universite´ Louis Pasteur Strasbourg,
BP24-67401 Illkirch cedex, France
bAFMB CNRS UMR 6098, De´partement de chimie, 163 Avenue de Luminy, F-13288 Marseille Cedex 09, France
Received 4 September 2002; accepted 27 September 2002
Abstract—p-Hydroxyphenacyl bromide is described as a photoremovable thiol protecting group on three biomolecules containing
a free thiol group. The protecting group is efficiently incorporated by chemical coupling to the biomolecule in an ethanol–buffer
mixture. The photofragmentations (u=312 nm) were analyzed by UV, HPLC and MS methods, yielding over 70% of the free
biomolecules. The concomitant formation of p-hydroxyphenylacetic thioesters derived from the corresponding thiols, as a
sulfur-containing side-product, should not hinder the use of this protecting group for the caging of thiol-containing biomolecules.
© 2002 Elsevier Science Ltd. All rights reserved.
The photochemical release of a biomolecule from an
inactive precursor represents a well-established method
for inducing a time- and space-controlled biochemical
or physiological response within functional proteins or
living cells respectively.1 Such masking of biomolecules
termed ‘caging’ requires the design of a photoreversible
group to label a chemical function on the biomolecule.
The photochemical blocking of thiol-containing
molecules has aroused considerable interest for bio-
chemical reasons (existence of numerous cysteine-con-
taining peptides and proteins) as well as for chemical
reasons inherent to the strong nucleophilic properties of
the thiol group, facilitating its chemical modification.
The photoreversible thiol labels mostly used were sub-
stituted o-nitrobenzyl bromide derivatives.2 The activity
of a series of cysteine-containing proteins could be
photoregulated using these reagents.3–5 Bayley and col-
leagues have extended this method by targeting cys-
teines incorporated by site-directed mutagenesis.6
Alternatively other photoreversible thiol labels have
been proposed such as 2-benzoylbenzoate thioesters7
and 2-nitro-1-(4,5-dimethoxy-2-nitrophenyl)ethyl which
was described to photoregulate the activity of b-
galactosidase.8
p-Hydroxyphenacyl groups have been developed as
powerful photoactivated protecting groups9 for car-
boxylate, phosphate and thiophosphate residues allow-
ing an efficient ‘caging’ of excitatory amino acids,10
nucleotides11 and thiophosphotyrosine peptides12
respectively.
The photolytic
reaction
of
p-
hydroxyphenacyl derivatives, besides allowing a fast
and efficient release of the biomolecule, generates pre-
dominantly low-wavelength excitation wavelengths
(uex>300 nm).9 Only one example of photoregulation of
the activity of protein phosphatase has been described
by masking the catalytic cysteine using p-substituted
phenacyl derivatives, as evidenced by MS-electrospray
experiments.13
Scheme 1. p-Hydroxyphenacyl bromide as photoreversible
thiol label.
The present article describes the syntheses and the
analyses of the photolytic reactions of p-
hydroxyphenacyl S-substituted N-Bz-cysteine-OMe
(A), 3%-thio-2%-deoxy- thymidine phosphate (B) and glu-
tathione (C) to demonstrate the usefulness of the p-
Keywords: caged compounds; photofragmentation reactions.
* Corresponding author. Tel.: +333 90244162; fax: +333 90244306;
e-mail: goeldner@bioorga.u-strasbg.fr
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)02194-9

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A. Specht et al. / Tetrahedron Letters 43 (2002) 8947–8950
hydroxyphenacyl group to mask thiol-containing
biomolecules (Scheme 1). We used an N-Bz substituted
cysteine derivative to facilitate UV detection for HPLC
analysis during the photolytic reaction.
Finally, hydrolysis of the thioester 5 gave the desired
3%-thio dTMP 6. This nucleotide monophosphate
analog was masked by treatment with p-
hydroxyphenacyl bromide using a 1/1 ethanol/HEPES
buffer mixture. Probe B was synthesized in 21% overall
yields from the lyxofuranosyl thymidine derivative 1.
Direct introduction of the p-hydroxyphenacyl group as
a protecting group by converting the mesylate 2 with
p-hydroxyphenacyl mercaptan could not be used due to
non-compatible properties of the phenacyl group with
the subsequent phosphorylation conditions.
The chemical modification of thiol-containing
molecules can be achieved by direct treatment with
p-hydroxyphenacyl bromide (Scheme 1). The coupling
yields are usually high and the reagent can be used in
1/1 ethanol/aqueous buffer (1N HEPES pH 7.0) mix-
tures for the reaction of water soluble substrates (A:
80%; B 90%; C: 92%). It should be expected that
selective modification of cysteines13 can be obtained by
direct treatment of a protein with p-hydroxyphenacyl
bromide.
The photolytic reactions of A, B and C (u=312 nm in
Tris–HCl buffer; pH 7.2) were analyzed by UV spec-
troscopy and HPLC as well as by MS for the glu-
tathione derivative C (Fig. 1). All three compounds
showed a decrease in absorbance at 290 and 340 nm
corresponding to the disappearance of the starting com-
pounds (umax 290 and 340 nm). The presence of
isobestic points at 270 nm was indicative of homoge-
neous photolytic reactions. The HPLC analyses of these
reactions depicted the formation of the unmasked thiol
derivatives, the expected side-products: p-hydroxy-
phenylacetic acid together with p-hydroxy acetophen-
one18 as well as a new compound: the p-hydroxy-
phenylacetic thioester19 derived from the biomolecules.
Table 1 summarizes the photochemical results on com-
pounds A, B and C, respectively.
3%-Thio-2%-deoxythymidine phosphate (3%-thio-dTMP)
represents a substrate analog14 for thymidilate mono
phosphate kinase (TMPK) which trans-phosphorylates
a phosphate group from ATP to dTMP forming ADP
and dTDP. The synthesis of the 3%-thio analog of
dTMP is outlined in Scheme 2. 5%-O-Methoxymethyl-
trityl-lyxofuranosyl thymidine 1 was synthesized from
thymidine using a described method.15 This four-step
synthesis ensured the desired configuration of the 3%-
thio group by two successive inversions at this position.
A thioester group (compound 3), precursor of the thiol
group, was stereospecifically introduced16 at the 3%-posi-
tion by reaction of sodium thiobenzoate with the mesyl-
ate 2. The monophosphate group was incorporated at
the 5%-OH position by reaction with phosphorus oxy-
chloride after removal of the protecting group.17
Quantum yield of the photolytic reaction for B was
determined by actinometry using azobenzene as
actinometer20 at 313 nm. A quantum yield of 0.085 for
Scheme 2. Synthesis of p-hydroxyphenacyl thioether of 3%-thio dTMP (B). Reagents and conditions: (i) MsCl, pyridine, 0°C, v22
h, z=98%; (ii) sodium thiobenzoate, DMF, 100°C, 22 h, z=55%; (iii) AcOH, rt, 24 h, z=85% ; (iv) 1. POCl3, trimethylphos-
phate, 0°C, 22 h, 2. triethylammonium formate 0.2N pH 7, 0°C 30 min and C18 HPLC reversed phase purification, z=58%; (v)
NaOH, EtOH, 0°C, 70 min, C18 HPLC reversed phase purification, z=88%; (vi) p-hydroxybromophenacyl 1N HEPES buffer pH
7/EtOH: 1/1, rt, 2 h, C18 HPLC reversed phase purification, z=90%.

A. Specht et al. / Tetrahedron Letters 43 (2002) 8947–8950
8949
Table 1. Product yields generated by 312 nm irradiation
of 0.1 mM solutions of A, B and C in 100 mM Tris–HCl
buffer pH 7.2, 1 mM DTT
Figure 1. The caged 3%-thio-2%-deoxythymidine phosphate (0.1
mM in 100 mM Tris–HCl buffer pH 07.2, 1 mM DTT) was
exposed to the 312 nm line of a 1000 W Hg lamp. (Left) UV
spectrum of the caged 3%-thio-2%-deoxythymidine phosphate
(upper trace) and difference UV-spectra recording during
photolysis (lower traces). (Right) HPLC analysis. B has a
retention time of 16.6 min. The appearing peaks at 13.8, 15.3,
17 and 18.2 min correspond to the 3%-thio dTMP, the p-
hydroxy-phenylacetic acid, the thioester and the p-hydroxy
acetophenone, respectively.
B was evaluated in 0.1 M phosphate buffer pH 7.2.
This value is lower than the ones described for the
photolyses of p-hydroxyphenacyl-derived carboxylate21
or phosphate esters.11
The formation of the thioester derived from p-hydroxy-
phenylacetic acid is an undesired side-product for the
use of p-hydroxyphenacyl bromide as a photochemical
thiol protecting group; nevertheless such side-reactions
do not necessarily occur within a protein binding site
where the different reaction intermediates do not
remain in vicinity to the unmasked thiol. Therefore, this
reagent remains an interesting ‘caging’ group for
biomolecules as demonstrated by the efficient photoreg-
ulation of the activity of a tyrosine phosphatase.13
Concerning the formation of sulfur-containing
molecules, these photolytic reactions generate about
70% of the thiols together with 30% of the thioesters.
The photochemical fragmentation mechanism of p-
hydroxyphenacyl derivatives is not fully established; a
triplet diradical intermediate has been proposed to gen-
erate a hypothetical spirocyclopropanone intermediate
whose hydrolysis does explain the formation of the
p-hydroxyphenylacetic acid (Scheme 3).11,22 Thiols are
sufficiently acidic (pKa about 8.4 in water) to allow such
fragmentations to occur while their strong nucleophilic
properties could explain the formation of the thioesters
by nucleophilic opening of the spirocyclopropanone.
Acknowledgements
The authors thank Professor Jakob Wirz and Anna
Paola Pelliccioli for assistance in quantum yield deter-
mination and Raymond Hueber and Dr. Alain Van
Dorsselaer for MS experiments. This work was sup-
ported by the Centre National de la Recherche Scien-
tifique, the Re´gion Alsace and the Universite´ Louis
Pasteur, Strasbourg.
Alternative radical mechanisms are indeed possible, to
account in particular for the formation of p-hydroxy-
acetophenone which is formed in about 10% yield.
Scheme 3. Hypothetical photochemical fragmentation mechanism of p-hydroxyphenacyl thioethers.

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A. Specht et al. / Tetrahedron Letters 43 (2002) 8947–8950
12. Zou, K.; Miller, T.; Givens, R. S.; Bayley, H. Angew.
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