Development and photo-responsive peptide bond cleavage reaction of two-photon near-infrared excitation-responsive peptide

Article abstract of DOI:10.1016/j.tetlet.2010.03.079

Two-photon near-infrared excitation-responsive amino acid was developed. It was incorporated into a peptide, and focused near-infrared pulsed laser irradiation-induced peptide bond cleavage reaction at the C-terminal position of the photo-responsive amino acid was observed.

Full text of DOI:10.1016/j.tetlet.2010.03.079

Tetrahedron Letters 51 (2010) 2868–2871
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Development and photo-responsive peptide bond cleavage reaction
of two-photon near-infrared excitation-responsive peptide
a
a
b
a,
Akira Shigenaga a, , Jun Yamamoto , Yoshitake Sumikawa , Toshiaki Furuta , Akira Otaka
*
*
a Institute of Health Biosciences and Graduate School of Pharmaceutical Sciences, The University of Tokushima, Shomachi, Tokushima 770-8505, Japan
b Department of Biomolecular Science and Research Center for Materials with Integrated Properties, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Two-photon near-infrared excitation-responsive amino acid was developed. It was incorporated into a
peptide, and focused near-infrared pulsed laser irradiation-induced peptide bond cleavage reaction at
the C-terminal position of the photo-responsive amino acid was observed.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 3 March 2010
Revised 18 March 2010
Accepted 19 March 2010
Available online 23 March 2010
Development of a method to control the function of peptide or
protein in a spatiotemporal manner is indispensable in the field of
chemical biology and drug delivery. Ultraviolet (UV) irradiation-in-
duced bond cleavage reaction or conformational change of peptide/
protein backbone has been successfully applied in order to control
function.1 However, UV irradiation sometimes causes serious dam-
age to living organisms, and one-photon UV photolysis occurs at a
whole optical path without three-dimensional spatial precision.
Near-infrared (NIR) two-photon photolysis can overcome these
problems because NIR, which has a wavelength longer than that
of UV, is less damaging to cells compared with UV, and simulta-
neous absorption of two photons occurs only at the point of focus
of a femtosecond pulsed laser with three-dimensional precision.2
To our knowledge, however, peptide bond cleavage reaction trig-
gered by NIR two-photon photolysis has yet to be reported. Previ-
ously, we reported a stimulus-responsive processing (peptide bond
cleavage) device and its application for controlling peptidyl func-
tion in living cells.3 In this Letter, we describe the development
and photo-reactivity of an NIR two-photon excitation (2PE)-
responsive processing device which induces a peptide bond cleav-
age reaction after exposure to a focused NIR pulsed laser.
In the UV one-photon excitation (1PE)-responsive system
developed by our group,3b,3c UV-induced removal of protective
group (PG) on the phenolic hydroxyl group of a trimethyl lock
moiety4 triggers a processing reaction as shown in Scheme 1 (PG =
o-nitrobenzyl). Therefore, we attempted to introduce a 2PE-
responsive protective group, which can be cleaved by NIR
two-photon absorption, at the PG position. NIR 2PE-responsive
protective groups such as Bhc group,5,6 quinoline derivatives,7
nitroindoline derivatives,8 and o-nitrobenzyl derivatives9,10 have
been extensively studied to date. A 4,5-dimethoxy-2-nitrobenzyl
group was originally developed by Patchornik et al. as a UV
1PE-responsive protective group,11 and it has recently been applied
as an NIR 2PE-responsive caged compound to control the function
of living cells.10 Because of its stability and ease of preparation, we
attempted to introduce a 4,5-dimethoxy-2-nitrobenzyl group at
the PG position.
Fmoc-protected 2PE-responsive amino acid 4 possessing a 4,5-
dimethoxy-2-nitrobenzyl group at the PG position was synthesized
as shown in Scheme 2. Phenol 13b was alkylated with 1-(bromo-
methyl)-4,5-dimethoxy-2-nitrobenzene12 in the presence of
K2CO3 to afford 4,5-dimethoxy-2-nitrobenzyl ether 2. After re-
moval of the TBS group of 2 under acidic conditions, the generated
hydroxyl group was oxidized with PDC to give aldehyde 3. After
oxidation of aldehyde 3 with sodium chlorite, the Boc group on
the obtained carboxylic acid derivative was replaced with Fmoc
group by acid treatment, followed by reaction with FmocOSu to
generate Fmoc-protected 2PE-responsive amino acid 4. The total
yield of amino acid derivative 4 amounted to 72% over six steps
beginning from phenol 1. Finally, amino acid 4 was incorporated
in model peptide 5 using Fmoc solid phase peptide synthesis (Fmoc
SPPS) to examine its photo-reactivity.
Next, the photo-responsive processing reaction of peptide 5 was
examined (Scheme 3). Prior to the two-photon NIR absorption
experiment, we performed a one-photon UV absorption experi-
ment to characterize processing products and to estimate photo-
physical properties of peptide 5. Diastereomerically purified
peptide 513 in phosphate buffer (pH 7.6, 20 mM) with 50% v/v ace-
tonitrile was irradiated by UV (>365 nm) for 3 min, and the reaction
mixture was incubated at 37 °C. Reaction progress was monitored
by HPLC, and the peptides were characterized by electrospray ion-
ization mass spectrometry (ESI-MS). After 4 h of incubation, pep-
tide 5 was completely converted to corresponding processing
products 6 and 7 as shown in Figure 1. Next, the time course for
photolysis of peptide 5 by irradiation with 365 nm UV light
(1.41 Â 1016 photon sÀ1) was monitored. As shown in Figure 2a,
the uncaging (photo-induced removal of 4,5-dimethoxy-2-nitro-
benzyl group) reaction of peptide 5 shows first-order dependence
* Corresponding authors.
E-mail addresses: ashige@ph.tokushima-u.ac.jp (A. Shigenaga), aotaka@ph.
tokushima-u.ac.jp (A. Otaka).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.03.079

A. Shigenaga et al. / Tetrahedron Letters 51 (2010) 2868–2871
2869
Scheme 1. Photo-responsive processing system (PG: photo-removable protective group).
Scheme 2. Reagents and conditions: (i) 1-bromomethyl-4,5-dimethoxy-2-nitrobenzene,12 K2CO3, DMF, 97%; (ii) AcOH, H2O, THF, 96%; (iii) PDC, CH2Cl2, 82%; (iv) NaClO2, 2-
methyl-2-butene, NaH2PO4, acetone, tert-BuOH, H2O; (v) HCl, 1,4-dioxane; (vi) FmocOSu, Na2CO3, acetonitrile, H2O, 94% (three steps); (vii) Fmoc SPPS.
4,5-dimethoxy-2-nitrobenzyl group, the extinction coefficient of
peptide 5 at 365 nm (e365) is similar to that of 8, whereas quantum
yield of disappearance of 5 (U365) is higher than that of 8. The rea-
son for this high quantum yield is not clear at present; however, it
might be due to the influence of a peptide moiety and not by the
solvent effect.14 Actually, the photophysical parameters (e365 and
U365) of 8 in 50% v/v acetonitrile in phosphate buffer (pH 7.6,
20 mM) were estimated as 5063 MÀ1 cmÀ1 and 0.003, respectively.
These values are almost identical to that in K-MOPS buffer; there-
fore, no obvious solvent effect on the photophysical parameters
was observed under these conditions.
These results encouraged us to examine an NIR two-photon
absorption experiment. Two-photon excitation reaction of peptide
5 using a focused NIR pulsed laser was examined as shown in
Scheme 3b. Solution of peptide 5 in 50% v/v acetonitrile/phosphate
buffer (pH 7.6, 20 mM) was irradiated with a focused NIR pulsed
laser (740 nm, 3.48 Â 1012 photon sÀ1), and the time course con-
centration of peptide 5 was monitored by HPLC. Based on the decay
curve of peptide 5 depicted in Figure 2b, two-photon uncaging ac-
tion cross-section at 740 nm (du740) was estimated as 0.23 GM
(Goeppert-Meyer, 1 GM = 010À50 cm4 s photonÀ1) (Table 1). This
du740 value is higher than that reported for 4,5-dimethoxy-2-nitro-
benzyl derivatives including reference compound 8,6i,15 presum-
Scheme 3. Reagents and conditions: (a) UV irradiation (>365 nm), 50% v/v
acetonitrile in phosphate buffer (pH 7.6, 20 mM). (b) NIR pulsed laser irradiation
(740 nm), 50% v/v acetonitrile in phosphate buffer (pH 7.6, 20 mM).
on the concentration of peptide 5. Quantum yield of peptide 5 for
UV 1PE was then estimated based on the decay curve and the
extinction coefficient of 5; results are summarized in Table 1.
Compared with reference compound 8,6i which also possesses a

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A. Shigenaga et al. / Tetrahedron Letters 51 (2010) 2868–2871
Table 1
Photophysical properties of photo-responsive compounds
5a
8b
c
e365
U365
U
5890
0.080
471
5200
0.005
26
d
e
e365
du740
f
0.23
0.03
a
Solvent: 50% v/v acetonitrile in phosphate buffer (pH 7.6, 20 mM).
Solvent: K-MOPS buffer (pH 7.2, 10 mM MOPS, 100 mM KCl). Values taken from
b
the literature.6i
Molar absorptivity at 365 nm (MÀ1 cmÀ1).
Quantum yield of disappearance of starting materials upon 365 nm irradiation.
Product of photolysis quantum yield and molar absorptivity at 365 nm
c
d
e
(MÀ1 cmÀ1).
f
Two-photon uncaging action cross-section at 740 nm (GM).
ably due to the high quantum yield of peptide 5 as mentioned
above. According to the literature,6i a du value exceeding 0.1 GM
is preferred for biological application of 2PE-responsive caged
compounds. Therefore, the NIR 2PE-responsive processing system
reported here can be potentially applicable for biological studies.
In conclusion, we developed a two-photon NIR excitation-
responsive peptide. The peptide bond at the C-terminal position of
the photo-responsive amino acid was successfully cleaved by irradi-
ation of a focused NIR pulsed laser at 740 nm to yield the processing
products, and the du740 value was sufficient for application in biolog-
ical studies. To our knowledge, this is the first example of a peptide
bond cleavage reaction triggered by NIR two-photon excitation.16
Applications of this unprecedented NIR 2PE-responsive processing
device for spatiotemporal control of peptide/protein function in liv-
ing cells are in progress.
Acknowledgments
This research was supported in part by a Grant-in-Aid for Scien-
tific Research (KAKENHI), Takeda Science Foundation, The Science
and Technology Foundation of Japan and Nagase Science and Tech-
nology Foundation.
Supplementary data
Figure 1. HPLC profiles (a) before and (b) after 3 min of UV irradiation followed by
4 h of incubation at 37 °C. Peptides were detected by UV absorbance at 220 nm.
Asterisked peak is 4,5-dimethoxy-2-nitrosobenzaldehyde derivative generated by
photolysis of 4,5-dimethoxy-2-nitrobenzyl group.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2010.03.079.
Figure 2. Time course for photolysis of peptide 5 under (a) UV at 365 nm (light intensity: 1.41 Â 1016 photon sÀ1). (b) NIR pulsed laser at 740 nm (light intensity:
3.48 Â 1012 photon sÀ1). Solid lines are least-squares fits of simple decaying exponentials.

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2871
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16. A referee pointed out that our approach brings together two previously known
things;
a a 2PE-
stimulus-responsive peptide bond cleavage device3 and
responsive protective group.10,11 However, we think that combination of these
two is novel, and the resulting 2PE-responsive peptide bond cleavage system
with potential applicability for biological studies is unprecedented.