A bromocoumarin-based linker for synthesis of photocleavable peptidoconjugates with high photosensitivity

Article abstract of DOI:10.1039/b812058g

A new bromocoumarin-based bi-functional linker was developed for preparing photocleavable peptides and proteins with high photolytic efficiency, which have many potential applications in the study and engineering of biological systems. The Royal Society of Chemistry.

Full text of DOI:10.1039/b812058g

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
A bromocoumarin-based linker for synthesis of photocleavable
peptidoconjugates with high photosensitivityw
Kentaro Katayama,a Shinya Tsukiji,a Toshiaki Furutade and
Teruyuki Nagamune*abc
Received (in Cambridge, UK) 15th July 2008, Accepted 8th August 2008
First published as an Advance Article on the web 19th September 2008
DOI: 10.1039/b812058g
A new bromocoumarin-based bi-functional linker was developed
for preparing photocleavable peptides and proteins with high
photolytic efficiency, which have many potential applications in
the study and engineering of biological systems.
efficiencies. A photoreaction that proceeds as efficiently as
possible with UV light (ideally over 350 nm) is desired,
particularly for cell biological applications to minimize photo-
damage of cells. Thus, the development of new photocleavable
linkers with improved photolysis efficiency is of crucial im-
portance to expand the applicability of light-controlled pep-
tides/proteins. Here, we report the design, synthesis, and
photochemistry of a new photocleavable linker which is based
on a 6-bromo-7-hydroxycoumarin-4-ylmethyl (Bhc) group.
Bhc is a recently developed caging agent which has a photo-
sensitivity superior to that of NB-based photolabile groups.9,10
It has been previously used for preparing caged analogues of
various biologically active molecules, such as glutamic acid,10a
mRNA,10b cyclic nucleotides,10c protein synthesis inhibitors,10d
and phosphotyrosine peptides.10e Unfortunately, the original
Bhc is mono-functional and thus needs to be modified to be
introduced into polypeptides by conventional methods. Furuta
and coworkers recently reported that the 7-hydroxyl moiety of
the Bhc group can be alkylated without sacrificing the overall
photosensitivity to UV light.10f Based on this finding, we
decided to attach an additional functionality at the 7-position
of Bhc and designed a new 6-bromo-7-aminoethoxycoumarin-
4-ylmethoxycarbonyl (Bac) linker building block 1 which is
suitable for Fmoc-based solid-phase peptide synthesis (SPPS)
(Fig. 1). As shown in Fig. 1, during SPPS the Bac linker 1 can be
incorporated within a polypeptide chain at any position via
stable amide (the 7-position side) and carbamate (the 4-position
side) bonds. Upon light irradiation, the Bac linker-containing
Peptides, proteins, or their conjugates whose function can be
controlled by light are powerful tools for cell biology and
biotechnology.1 Light can be projected into samples, e.g. in test
tubes, on solid surfaces, or in live cells and tissues, at a desired
time and area, allowing us to trigger molecular and cellular
events with high spatiotemporal precision. Typically, they are
prepared by introducing a photoremovable protecting (caging)
group at the side chain of essential amino acid residue(s) to
abolish the function. The activity is then regained by releasing
the caging group with a flash of light. Another important
approach involves the incorporation of a photocleavable site
within the polypeptide backbone, so that a single peptide/
protein chain can be self-split into two fragments upon photo-
illumination. This ‘‘backbone-photocleavage’’ strategy is parti-
cularly useful for designing various types of light-controlled
molecules.2–6 For example, Bosques and Imperiali synthesized a
light-activatable self-assembling peptide by connecting an amylo-
idogenic sequence and a fibril-inhibitory unit via a photo-
cleavable linker.2 As well as activation,2,3 light-induced
inactivation of proteins4 and peptides5 has also been demon-
strated. In recent notable applications, Pellois and Muir6 and
Otaka et al.7 used the approach to control subcellular localiza-
tion of a protein and a peptide, respectively, by light.
The choice of photocleavable linker is the key that deter-
mines photochemical properties of such peptides and proteins.
Although a number of new caging molecules with improved
efficiency of photolysis have been developed in the last
decade,8 currently available linkers are limited to classical
2-nitrobenzyl (NB) derivatives with relatively low photolytic
a Department of Chemistry and Biotechnology, School of Engineering,
The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo,
113-8656, Japan
b Department of Bioengineering, School of Engineering, The University
of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan.
E-mail: nagamune@bioeng.t.u-tokyo.ac.jp;
Fax: +81 (0)3 5841 8657; Tel: +81 (0)3 5841 7328
c Center for NanoBio Integration, The University of Tokyo, 7-3-1
Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan
d Department of Biomolecular Science, Toho University, 2-2-1
Miyama, Funabashi, 274-8510, Japan
e Research Center for Materials with Integrated Properties, Toho
University, 2-2-1 Miyama, Funabashi, 274-8510, Japan
w Electronic supplementary information (ESI) available: Full experi-
mental procedures, UV/vis spectrum of 5, and HPLC traces of
photolysis of 6 before and after photolysis. See DOI: 10.1039/
b812058g
Fig. 1 Schematic illustration of synthesis and photochemistry of
photocleavable peptidoconjugates containing the Bac linker.
ꢀc
This journal is The Royal Society of Chemistry 2008
Chem. Commun., 2008, 5399–5401 | 5399

View Article Online
peptides undergo self-cleavage into two fragments through the
photochemical decomposition of the carbamate linkage10a,f
with concomitant release of a bromocoumarin-4-ylmethanol
(BacOH)-attached N-terminal half peptide and another C-term-
inal half peptide with a free amine.
The synthesis of 1 was accomplished as shown in Scheme 1
(see ESIw for detailed procedures). The starting material 6-
bromo-7-hydroxycoumarin-4-ylmethanol 2 was prepared in
two steps as previously reported.10a The 7-hydroxyl group of
2 was selectively O-alkylated using 2-(tert-butoxycarbonyla-
mino)ethyl bromide and potassium carbonate in dimethylfor-
mamide (DMF), producing the compound 3 (50%). After
removal of the Boc protecting group in dichloromethane
(DCM) containing 10% trifluoroacetic acid (TFA), the amine
group was re-protected by an Fmoc group using 9-fluorenyl-
methyl succinimidyl carbonate (Fmoc-OSu) and N,N-diiso-
propylethylamine (DIEA) in DMF to yield compound 4 (93%
over two steps). Finally, the building block 1 was obtained by
reaction of 4 with 4-nitrophenyl chloroformate and DIEA in
DCM (91%). It should be noted that compound 1 is solid and
stable and can be stored for long periods at 4 1C without
significant decomposition.
Fig. 2 Synthesis and photoreaction of Bac-containing peptidoconju-
gates 5 and 6.
to that described above. Upon complete assembly of the pepti-
doconjugates, global deprotection and cleavage from the resin
was performed with TFA containing 5% ethanedithiol (EDT)
and 2.5% H2O. Both of the products were precipitated by Et2O
and purified to homogeneity by reversed-phase HPLC (RP-
HPLC). The new building block 1 proved resistant to the basic
conditions used for coupling and Fmoc removal reactions and
was also resistant to the acidic treatment necessary for the final
cleavage and deprotection steps.
To investigate the versatility of building block 1, we synthesized
two model photocleavable peptidoconjugates, a linear form 5 and
a branched form 6, based on a FLAG epitope (DYKDDDDK)
(Fig. 2). The linear peptidoconjugate 5 contained the Bac linker
incorporated between a flexible GSGS sequence and the FLAG
motif, and a biotin tag was also introduced at the N-terminus. In
the branched peptidoconjugate 6, a heptaarginine sequence,
which is known as a cell-penetrating peptide,11 was attached to
the side-chain of the lysine residue in the FLAG motif via the Bac
linker. They were built on a Rink amide resin using a standard
Fmoc-based SPPS procedure according to the scheme shown in
Fig. 2. In the synthesis of 5, after assembly of protected FLAG
resin 7 and Fmoc deprotection, the Bac linker was installed by
treating the resin with 1 and DIEA in DMF. Complete incor-
poration of the linker was confirmed by the Kaiser test.12 Next the
Fmoc group was removed by a standard method using 20%
piperidine in DMF, which was followed by subsequent coupling/
deprotection cycles. In the synthesis of 6, following Fmoc depro-
tection and acetylation of protected FLAG resin 7, the acid-
sensitive methoxytrityl (Mtt) group on the side chain of the lysine
residue on position 3 was selectively removed with 2% TFA and
5% triisopropylsilane (TIS) in DCM. Subsequent incorporation
of 1 and chain elongation was achieved using a similar procedure
Next, we investigated the photochemical properties of 5 and 6
in K-MOPS solution (10 mM MOPS, 100 mM KCl, pH 7.2).
Both conjugates showed a characteristic absorption band with a
maximum at 330 nm (e330 = 10 080 MÀ1ÁcmÀ1), which is ascribed
to the Bac moiety (Fig. S1, ESIw). The absorption maximum was
almost identical to that of the (6-bromo-7-methoxycoumarin-4-
yl)methoxycarbonyl (Bmcmoc) group,10f indicating that the re-
placement of a methyl group with an amidoethyl group does not
affect the absorption properties of the 6-bromocoumarin photo-
trigger. The conjugate solutions were irradiated with UV light at
350 nm and analyzed by RP-HPLC as a function of time. 5 was
photocleaved into the corresponding two fragments, biotin-
GSGS-BacOH and FLAG motif with an N-terminus amine,
which were characterized by MALDI-TOF-MS analyses
(Fig. 3). 6 also underwent photocleavage to produce Ac-R7-
BacOH and an Ac-FLAG sequence as expected (Fig. S2, ESIw).
The photolytic consumption of the peptidoconjugates 5 and 6
followed a single exponential decay (Fig. 4). The quantum yields
for the photocleavage reaction were determined to be 0.26 for 5
and 0.16 for 6 using the previously reported method.10a In
addition, the overall photosensitivities, which can be expressed
as the product of the quantum yield of the photolysis (F) and
the molar absorption coefficient (e), were calculated to be
1445 MÀ1 cmÀ1 for 5 and 1139 MÀ1 cmÀ1 for 6. These values
are almost comparable with those of Bmcmoc-caged nucleobases,
thus also indicating that the photosensitivity of Bac is markedly
higher than that of other NB-based linkers (see ref. 10f). Moreover,
we confirmed that the peptidoconjugates were sufficiently stable
from hydrolysis in the absence of light (less than 10% of hydrolysis
at 37 1C even after one day incubation in K-MOPS solution).
Scheme 1 Route for synthesis of the Bac linker building block 1.
5400 | Chem. Commun., 2008, 5399–5401
ꢀc
This journal is The Royal Society of Chemistry 2008

View Article Online
and B. Imperiali, Trends Cell Biol., 2005, 15, 502–510; (e) S.
Loudwig and H. Bayley, in Dynamic Studies in Biology: Photo-
triggers, Photoswitches and Caged Biomolecules, ed. M. Goeldner
and R. S. Givens, Wiley-VCH, New York, 2005, pp. 253–304.
2 C. J. Bosques and B. Imperiali, J. Am. Chem. Soc., 2003, 125,
7530–7531.
3 M. Endo, K. Nakayama, Y. Kaida and T. Majima, Angew. Chem.,
Int. Ed., 2004, 43, 5643–5645.
4 P. M. England, H. A. Lester, N. Davidson and D. A. Dougherty,
Proc. Natl. Acad. Sci. U. S. A., 1997, 94, 11025–11030.
5 (a) L. L. Parker, J. W. Kurutz, S. B. H. Kent and S. J. Kron,
Angew. Chem., Int. Ed., 2006, 45, 6322–6325; (b) M. Toebes, M.
Coccoris, A. Bins, B. Rodenko, R. Gomez, N. J. Nieuwkoop, W.
van de Kasteele, G. F. Rimmelzwaan, J. B. A. G. Haanen, H. Ovaa
and T. N. M. Schumacher, Nat. Med., 2006, 12, 246–251; (c) G. M.
Grotenbreg, N. R. Roan, E. Guillen, R. Meijers, J.-H. Wang, G.
W. Bell, M. N. Starnbach and H. L. Ploegh, Proc. Natl. Acad. Sci.
U. S. A., 2008, 105, 3831–3836.
6 J.-P. Pellois and T. W. Muir, Angew. Chem., Int. Ed., 2005, 44,
5713–5717.
7 A. Shigenaga, D. Tsuji, N. Nishioka, S. Tsuda, K. Itoh and A.
Otaka, ChemBioChem, 2007, 8, 1929–1931.
8 (a) G. Mayer and A. Heckel, Angew. Chem., Int. Ed., 2006, 45,
4900–4921; (b) G. C. R. Ellis-Davies, Nat. Methods, 2007, 4, 619–628.
9 T. Furuta, in Dynamic Studies in Biology: Phototriggers, Photo-
switches and Caged Biomolecules, ed. M. Goeldner and R. S.
Givens, Wiley-VCH, New York, 2005, pp. 29–54.
Fig. 3 HPLC traces of 5 before (top) and after (bottom) UV photolysis
(350 nm, 2 min). Conditions: 9 mM in K-MOPS buffer. HPLC was
performed on an ODS column with a linear gradient of acetonitrile
containing 0.1% TFA (solvent A) and 0.1% aqueous TFA (solvent B).
(A) Detection at 220 nm. Gradient: A/B = 4 : 90 to 10 : 60 for 15 min.
Peak
(H-DYKDDDDK-NH2). (B) Detection at 325 nm. Gradient: A/B =
10 : 90 to 40 : 60 for 15 min. Peak K denotes Biotin-GSGS-BacOH
J denotes FLAG motif with an N-terminus free amine
.
10 (a) T. Furuta, S. S.-H. Wang, J. L. Dantzker, T. M. Dore, W. J.
Bybee, E. M. Callaway, W. Denk and R. Y. Tsien, Proc. Natl.
Acad. Sci. U. S. A., 1999, 96, 1193–1200; (b) H. Ando, T. Furuta,
R. Y. Tsien and H. Okamoto, Nat. Genet., 2001, 28, 317–325; (c) T.
Furuta, H. Takeuchi, M. Isozaki, Y. Takahashi, M. Kanehara, M.
Sugimoto, T. Watanabe, K. Noguchi, T. M. Dore, T. Kurahashi,
M. Iwamura and R. Y. Tsien, ChemBioChem, 2004, 5, 1119–1128;
(d) M. Goard, G. Aakalu, O. D. Fedoryak, C. Quinonez, J. St.
Julien, S. J. Poteet, E. M. Schuman and T. M. Dore, Chem. Biol.,
2005, 12, 685–693; (e) T. Kawakami, H. Cheng, S. Hashiro, Y.
Nomura, S. Tsukiji, T. Furuta and T. Nagamune, ChemBioChem,
2008, 9, 1583–1586; (f) T. Furuta, T. Watanabe, S. Tanabe, J.
Sakyo and C. Matsuba, Org. Lett., 2007, 9, 4717–4720.
11 P. A. Wender, D. J. Mitchell, K. Pattabiraman, E. T. Pelkey, L.
Steinman and J. B. Rothbard, Proc. Natl. Acad. Sci. U. S. A., 2000,
97, 13003–13008.
12 E. Kaiser, R. L. Colescott, C. D. Bossinger and P. I. Cook, Anal.
Biochem., 1970, 34, 595–598.
Fig. 4 Time courses of photolysis of peptidoconjugates 5 (K, 9 mM)
and 6 (’, 8 mM) in K-MOPS buffer.
13 We have also successfully prepared a cyclic analogue of 16-residue
peptide derived from a Bcl-2 homology 3 (BH3) domain of Bak
(GQVGRQLAIIGDDINR) by connecting the N- and C-terminus
through the Bac linker. The Bak peptide is known to interact with
Bcl-xL and induce apoptosis (M. Sattler, H. Liang, D. Nettesheim,
R. P. Meadows, J. E. Harlan, M. Eberstadt, H. S. Yoon, S. B.
Shuker, B. S. Chang, A. J. Minn, C. B. Thompson and S. W. Fesik,
Science, 1997, 275, 983–986). In preliminary experiments, it was
confirmed that the cyclic form has no affinity toward Bcl-xL, but
recovers its binding ability upon light-induced conversion to a
linear peptide. These results demonstrate the feasibility of control-
ling the function of peptides by constraining the structure with
photocleavable linkers, which should become a new design strategy
for light-activatable peptides/proteins. More details and cell bio-
logical applications of the cyclized Bak peptide will be reported
elsewhere in due course.
14 (a) T. W. Muir, D. Sondhi and P. A. Cole, Proc. Natl. Acad. Sci.
U. S. A., 1998, 95, 6705–6710; (b) T. Ando, S. Tsukiji, T. Tanaka
and T. Nagamune, Chem. Commun., 2007, 4995–4997; (c) H. Mao,
S. A. Hart, A. Schink and B. A. Pollok, J. Am. Chem. Soc., 2004,
126, 2670–2671; (d) T. Tanaka, T. Yamamoto, S. Tsukiji and T.
Nagamune, ChemBioChem, 2008, 9, 802–807.
15 (a) G. T. Hermanson, in Bioconjugate Techniques, Academic Press,
San Diego, CA, 1996; (b) D. W. Romanini and M. B. Francis,
Bioconjugate Chem., 2008, 19, 153–157.
In summary, we have developed a new bromocoumarin-
based linker for the synthesis of photocleavable peptido-
conjugates with high photosensitivity. The building block 1
is versatile and can in principle be incorporated into any
peptide sequence at any position by SPPS.13 It will also be
feasible to integrate Bac-containing peptides with larger pro-
teins using protein ligation14 or chemical modification techni-
ques15 to yield photocleavable semisynthetic proteins. This
work will enhance the applications of light-controlled peptide/
protein tools for investigating and/or artificially manipulating
biological processes. Moreover, the strategy described here to
design the Bac linker may also be used to create new photo-
cleavable linkers with different reactive functionalities such as
maleimide, which could be used in preparing photoactivatable
gene-targeting compounds.16
This work was supported by the Center for NanoBio
Integration.
Notes and references
16 (a) X. Tang and I. J. Dmochowski, Angew. Chem., Int. Ed., 2006,
45, 3523–3526; (b) X. Tang, S. Maegawa, E. S. Weinberg and I. J.
Dmochowski, J. Am. Chem. Soc., 2007, 129, 11000–11001; (c) I. A.
Shestopalov, S. Sinha and J. K. Chen, Nat. Chem. Biol., 2007, 3,
650–651.
1 Selected reviews: (a) K. Curley and D. S. Lawrence, Curr. Opin.
Chem. Biol., 1999, 3, 84–88; (b) D. S. Lawrence, Curr. Opin. Chem.
Biol., 2005, 9, 570–575; (c) M. E. Hahn and T. W. Muir, Trends
Biochem. Sci., 2005, 30, 26–34; (d) D. M. Rothman, M. D. Shults
ꢀc
This journal is The Royal Society of Chemistry 2008
Chem. Commun., 2008, 5399–5401 | 5401