FRET-based assay of the processing reaction kinetics of stimulus-responsive peptides: influence of amino acid sequence on reaction kinetics

Article abstract of DOI:10.1016/j.tet.2009.01.063

In the field of chemical biology, methods for controlling peptidyl function by a stimulus are attracting increasing attention. Recently, we reported a stimulus-responsive peptide, which can be cleaved after exposure to a stimulus. In this study, we develo

Full text of DOI:10.1016/j.tet.2009.01.063

Tetrahedron 65 (2009) 2212–2216
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FRET-based assay of the processing reaction kinetics of stimulus-responsive
peptides: influence of amino acid sequence on reaction kinetics
*
*
Akira Shigenaga , Jun Yamamoto, Hiroko Hirakawa, Keiko Yamaguchi, Akira Otaka
Institute of Health Biosciences and Graduate School of Pharmaceutical Sciences, The University of Tokushima, Shomachi, Tokushima 770-8505, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In the field of chemical biology, methods for controlling peptidyl function by a stimulus are attracting
increasing attention. Recently, we reported a stimulus-responsive peptide, which can be cleaved after
exposure to a stimulus. In this study, we developed a FRET-based assay system to estimate the kinetics of
the stimulus-induced processing (peptide bond cleavage) reaction. Based on the FRET system, it was
clarified that introduction of a sterically less-hindered or polar residue at the position adjacent to the
stimulus-responsive amino acid accelerates the processing reaction.
Received 5 December 2008
Received in revised form 12 January 2009
Accepted 13 January 2009
Available online 20 January 2009
Keywords:
FRET
Ó 2009 Elsevier Ltd. All rights reserved.
Stimulus responsive
Peptide bond cleavage
UV irradiation
1. Introduction
In the field of chemical biology, it is important to develop
technology to control the function of intracellular peptides and
proteins by an external stimulus. In this context, photo-responsive
bond cleavage reaction1 or conformational change2 of a peptide/
protein backbone has been successfully applied for spatiotemporal
control of its function. Whereas these stimulus-responsive peptides
and proteins can respond to specific stimuli, we developed a stim-
ulus-responsive peptide, which is applicable to arbitrary stimuli.
Based on the trimethyl lock system,3 we previously reported
a stimulus-responsive processing (peptide bond cleavage) device
with successful application for controlling peptidyl function that
induces processing reaction after exposure to a stimulus such as UV
irradiation or enzymatic reaction.4 When peptide 1 possessing the
stimulus-responsive processing device was exposed to stimulus,
removal of PG (protective group removable by a stimulus) followed
by nucleophilic involvement of the generated phenolic hydroxyl
group afforded processing products in good purity (Scheme 1). In
a previous report, we briefly mentioned that the rate of the pro-
cessing reaction was dependent on the amino acid adjacent to the
stimulus-responsive residue. In the present paper, we report details
of the influence of amino acid sequence on the kinetics of the
processing reaction.
Scheme 1. Stimulus-responsive processing system (PG: protective group removable by
a stimulus).
2. Results and discussions
2.1. Design of the FRET substrate
Fluorescence resonance energy transfer (FRET) is one of the
most widely used techniques to evaluate kinetics of the cleavage
reaction of a peptide.5 It was reported that a fluorescein-5-thio-
carbamoyl (FTC)-dabsyl (Dab) based FRET system is compatible
with a photo-removable o-nitrobenzyl (o-NB) group.6 Therefore,
we designed FTC-Dab based FRET substrate 2 possessing a photo-
responsive amino acid protected by an o-NB group (Scheme 2). If
* Corresponding authors. Tel.: þ81 88 633 7283; fax: þ81 88 633 9505.
E-mail addresses: ashige@ph.tokushima-u.ac.jp (A. Shigenaga), aotaka@ph.
tokushima-u.ac.jp (A. Otaka).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.01.063

A. Shigenaga et al. / Tetrahedron 65 (2009) 2212–2216
2213
Scheme 2. FRET-based assay system for the kinetics of the processing reaction (o-NB: o-nitrobenzyl).
peptide 2 was irradiated by UV (>365 nm) for 3 min, it should be
completely deprotected to generate intermediate 3 with weak
fluorescence. Afterward, intermediate 3 would be converted to
processing products 4 and 5 thereby emitting a strong fluorescence.
Based on the time course of fluorescence intensity, kinetics of the
processing reaction of intermediate 3 could be estimated.
residues such as Asn(Trt), Cys(Trt), and Ser(t-Bu) did not proceed
well, Cys(MPM) (MPM: p-methoxybenzyl) and side chain un-
protected Asn, Ser, and Thr were used for the synthesis of peptide 2
(Xaa1¼Gly; Xaa2¼Asn, Cys, Ser and Thr), respectively. This result
suggests that introduction of the photo-responsive amino acid on
a sterically hindered residue should be avoided for facile synthesis.
2.2. Synthesis of the FRET substrate
2.3. Kinetics measurement of the processing reaction
First, FRET substrates 2 were synthesized as shown in Scheme 3.
The peptide was elongated on NovaSynÒ TGR resin using Fmoc
solid phase peptide synthesis (Fmoc SPPS), and the peptide was
capped with dabsyl chloride to yield peptide resin 6. After removal
of the Alloc group on side chain of Lys, the regenerated amine was
reacted with fluorescein-5-isothiocyanate (FITC) to introduce
a fluorophore. Finally, peptide resin 7 was treated with a cleavage
cocktail (TFA/H2O/thioanisole¼95:2.5:2.5, v/v) to give FRET sub-
strate 2 that was purified as a diastereomeric mixture before sub-
sequent experiments.7 Because the coupling reaction of Fmoc
protected photo-responsive amino acid 84 on sterically hindered
First, we monitored the processing reaction of peptide 2
(Xaa1¼Gly; Xaa2¼Tyr) using HPLC, and processing products were
characterized by ESI-MS. The UV-induced processing reaction of
the FRET substrate is outlined in Scheme 2. Peptide 2 in phosphate
buffer (pH 7.6) with 30% MeCN was irradiated with UV light
(>365 nm) for 3 min and then incubated at 37 ꢀC. After 3 min of UV
irradiation, substrate 2 (Xaa1¼Gly; Xaa2¼Tyr) was completely
deprotected to yield intermediate 3 (Xaa1¼Gly; Xaa2¼Tyr) (data
not shown). As shown in Figure 1, intermediate 3 (Xaa1¼Gly;
Xaa2¼Tyr) was converted to processing products 4 (Xaa1¼Gly) and
5 (Xaa2¼Tyr) after 3 h of incubation.
Scheme 3. Reagents and conditions: (a) Fmoc SPPS; (b) dabsyl chloride, Et3N, DMF; (c) (Ph3P)4Pd, 1,3-dimethylbarbituric acid, 1,2-dichloroethane; (d) FITC, DIEA, DMF; (e) TFA/
thioanisole/H2O¼95:2.5:2.5 (v/v) (o-NB: o-nitrobenzyl).

2214
A. Shigenaga et al. / Tetrahedron 65 (2009) 2212–2216
position, rapid processing reaction was observed. Interestingly,
introduction of a polar residue at Xaa2 position also accelerated the
reaction regardless of its steric hindrance. The polar residue might
assist the processing reaction by hydrogen bond-mediated activa-
tion of the carbonyl group of the peptide bond and/or deprotona-
tion of the phenolic hydroxyl group. In the meantime, replacement
of Xaa1 with sterically less-hindered Gly or polar Glu or Lys also
accelerated the reaction; however, the polarity effect of the side
chain was smaller than that observed for Xaa2.
3. Conclusion
In conclusion, the half-lives of the stimulus-responsive peptides
were determined using a FRET-based assay system. Introduction of
a sterically less-hindered or polar residue at position Xaa2 signifi-
cantly accelerated the reaction. Replacement of Xaa1 with a steri-
cally less-hindered Gly or polar Glu or Lys also accelerated the
reaction whereas the polarity effect was smaller than that observed
for Xaa2. These results enable us to control the processing reaction
rate for different purposes by choosing an appropriate residue at
a position adjacent to the stimulus-responsive amino acid.
4. Experimental
4.1. General
Figure 1. HPLC profiles (a) before UV irradiation (b) after 3 min of UV irradiation
followed by 3 h of incubation at 37 ꢀC (Xaa1¼Gly; Xaa2¼Tyr). Diastereomeric mixture
of 2 was used. Peptides were detected by UV absorbance at 220 nm. Asterisked peak is
an o-nitrobenzyl derived small molecule.
Exact mass spectra were recorded on Bruker Esquire200T or
Waters MICROMASSÒ LCT PREMIERÔ. For HPLC separations,
a
Cosmosil 5C18-AR-II analytical column (Nacalai Tesque,
Next, we monitored the time course of the fluorescence in-
tensity of the reaction mixture at 37 ꢀC using a plate reader
4.6ꢁ250 mm, flow rate 1 mL/min) or a 5C18-AR-II semi-preparative
column (Nacalai Tesque, 10ꢁ250 mm, flow rate 3.0 mL/min) was
employed, and eluting products were detected by UV at 220 nm. A
solvent system consisting of 0.1% TFA in water (v/v, solvent A) and
0.1% TFA in MeCN (v/v, solvent B) was used for HPLC elution. Infinite
M200 flexible microplate reader (TECAN Austria GmbH) was used
for fluorescence measurements. Photolysis was performed using
Moritex MUV-202U with the filtered output (>365 nm) of
a 3000 mW/cm2 Hg–Xe lamp.
(lex¼495 nm; lem¼520 nm). Before UV irradiation, fluorescence
intensity of the reaction mixture was recorded as time¼0 min.
Then, the reaction mixture was irradiated by UV light (>365 nm)
for 3 min, and the fluorescence intensities were recorded at 3 (just
after UV irradiation), 5, 10, 15, 30, 60, 120, and 180 min. The per-
centage of remaining intermediate 3 was estimated based on rel-
ative fluorescence intensity. As shown in Figure 2, the processing
reaction of intermediate 3 (Xaa1¼Gly; Xaa2¼Tyr) shows first-order
dependence on the concentration of the intermediate, and the half
life of 3 (Xaa1¼Gly; Xaa2¼Tyr) was determined as 18.0 min.8
These results encouraged us to determine half-lives of the
stimulus-responsive peptides with various amino acids at a posi-
tion adjacent to the stimulus-responsive residue. FRET substrates 2
possessing various amino acid sequences were synthesized as
mentioned above. Their half-lives were estimated based on the
FRET assay system, and results are summarized in Figure 3. When
a sterically less-hindered amino acid had been introduced at Xaa2
4.2. General procedure for a preparation of FRET substrate 2
Elongation of peptides on NovaSynÒ TGR resin (0.25 mmol/g,
8.7 mg) was performed as described in previous report.4 For the
synthesis of peptide 2 (Xaa1¼Gly; Xaa2¼Asn, Cys, Ser or Thr),
Fmoc–Asn–OH, Fmoc–Cys(MPM)–OH, Fmoc–Ser–OH or Fmoc–
Thr–OH was used as a building block, respectively. Obtained resin
was reacted with dabsyl chloride (2.1 mg) and triethylamine
Figure 2. Kinetic measurement of the processing reaction of 3 (Xaa1¼Gly; Xaa2¼Tyr). (a) Change in fluorescence intensity in phosphate buffer (pH 7.6, 20 mM) with 30% MeCN at
37 ꢀC after UV irradiation (lex¼495 nm; lem¼520 nm). (b) First-order kinetic treatment of the data. Percentage of intermediate 3 was calculated as follow. 3 (%)¼100ꢁ(1ꢂrelative
fluorescence intensity).

A. Shigenaga et al. / Tetrahedron 65 (2009) 2212–2216
2215
Figure 3. Half-lives of intermediates 3 in the processing reaction. Ser(Ac) is an O-acetyl serine.
(1.8
m
L) in DMF (70
m
L) for 3 h. After washing with DMF followed by
the peptide with TFA/thioanisole/H2O (95:2.5:2.5, v/v) afforded
a crude product. After trituration with diethyl ether, the precipitate
was purified by HPLC to give FRET substrate 2 as a diastereomeric
mixture in 10–20% yield. Analytical HPLC conditions: linear gradi-
ent of solvent B in solvent A, 30–90% over 30 min. Semi-preparative
HPLC conditions: linear gradient of solvent B in solvent A, 40–70%
over 30 min (Table 1).
dichloromethane, Alloc group was removed by a treatment with
(Ph3P)4Pd (4.0 mg) and 1,3-dimethylbarbituric acid (3.4 mg) in 1,2-
dichloroethane (172
dichloromethane followed by DMF. To obtained resin were added
fluorescein-5-isothiocyanate (FITC, 2.0 mg) and DIEA (10 L) in
DMF (50 L), and the reaction was performed overnight. After fil-
tration, the resin was treated with FITC (2.0 mg) and DIEA (0.88 L)
L) for additional 24 h. Finally, global deprotection of
mL) overnight. Then the resin was washed with
m
m
m
in DMF (50
m
4.3. Reaction monitoring of the processing reaction by using
HPLC
Table 1
The experiment was performed as described in previous report.4
Analytical HPLC conditions: linear gradient of solvent B in solvent
A, 30–90% over 30 min. ESI-MS: Compound 4 (Xaa1¼Gly) calcd for
[MþH]þ 564.2, obsd 563.9. Compound 5 (Xaa2¼Tyr) calcd for
[MþH]þ 698.2, obsd 697.9. Retention time¼23.5 or 5.4 min, re-
spectively, for 4 (Xaa1¼Gly) or 5 (Xaa2¼Tyr).
2
ESI-MS ([Mþ2H]2þ
)
Xaa1
Xaa2
Calcd
Obsd
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Glu
Ile
Ala
652.7
695.5
674.4
674.7
668.7
681.3
681.8
645.7
685.8
673.8
673.8
681.3
682.8
690.8
665.8
660.7
681.8
667.8
710.3
698.8
666.8
734.8
726.5
734.3
652.5
695.1
674.4
674.7
668.9
681.6
682.1
645.5
686.0
673.9
673.6
681.4
682.9
690.9
666.0
660.9
681.9
667.9
710.4
699.0
666.7
735.4
727.0
734.5
Arg
Asn
Asp
Cys
Gln
Glu
Gly
His
Ile
Leu
Lys
Met
Phe
Pro
Ser
4.4. Kinetics measurement based on FRET system
Fluorescence intensity of FRET substrate
tration¼8 M) in phosphate buffer (pH 7.6, 20 mM) with 30% MeCN
was measured before photolysis and it was defined as time¼0 min
2 (final concen-
m
(
lex¼495 nm; lem¼520 nm). Then, the reaction mixture was irra-
diated by UV light (>365 nm) for 3 min and the fluorescence of the
reaction mixture at 37 ꢀC was recorded at 3, 5, 10, 15, 30, 60, 120,
and 180 min. Percentage of remaining intermediate 3 was esti-
mated based on relative fluorescence intensity. Remaining in-
termediate 3 (%)¼100ꢁ(1ꢂrelative fluorescence intensity).
Ser(Ac)a
Thr
Trp
Tyr
Val
Tyr
Tyr
Tyr
Acknowledgements
This work was supported in part by a Grant-in-Aid for Scientific
Research (KAKENHI), Mitsubishi Chemical Corporation Fund and
Takeda Science Foundation.
Lys
a
Ser(Ac)¼O-acetyl serine.

2216
A. Shigenaga et al. / Tetrahedron 65 (2009) 2212–2216
2. Renner, C.; Morroder, L. ChemBioChem 2006, 7, 868–878 and references therein.
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7. Racemic 8 was used for a synthesis of FRET substrates 2.
8. Because pH dependence of the processing reaction kinetics was observed, ki-
netics of this reaction should be described by pseudo-first-order (half life of 3
(Xaa1¼Gly, Xaa2¼Tyr) in 30% MeCN/phosphate buffer (pH 5.5): 26.9 min).