Design, Synthesis, and Photo-Responsive Properties of a Collagen Model Peptide Bearing an Azobenzene

: Collagen is a vital component of the extracellular matrix in animals. Collagen forms a characteristic triple helical structure and plays a key role in supporting connective tissues and cell adhesion. The ability to control the collagen triple helix structure is useful for medical and conformational studies because the physicochemical properties of the collagen rely on its conformation. Although some photo-controllable collagen model peptides (CMPs) have been reported, satisfactory photo-control has not yet been achieved. To achieve this objective, detailed investigation of the isomerization behavior of the azobenzene moiety in CMPs is required. Herein, two CMPs were attached via an azobenzene linker to control collagen triple helix formation by light irradiation. Azo-(PPG) 10 with two (Pro-Pro-Gly) 10 CMPs linked via a photo-responsive azobenzene moiety was designed and synthesized. Conformational changes were evaluated by circular dichroism and the cis -to- trans isomerization rate calculated from the absorption of the azobenzene moiety indicated that the collagen triple helix structure was partially disrupted by isomerization of the internal azobenzene.


Introduction
Collagen is the most abundant protein in mammals, and at least 29 different types of collagen proteins have been identified to date [1]. Collagen is one of the major components of the extracellular matrix in connective tissues, such as ligaments, bones, cartilage, tendons, and skin, and plays a vital role in providing mechanical strength to tissues and regulating cell adhesion and migration [2].
The collagen molecule comprises three polypeptide chains that assemble to form a right-handed triple helical structure [3]. This characteristic three-dimensional structure, the collagen triple helix, is responsible for the physicochemical properties of collagen [4]. The collagen triple helix collapses with warming until the temperature reaches the phase transition temperature (T m ), when the three polypeptide chains that form the three-dimensional structure are dissociated. This denaturation of the collagen molecule leads to a sol-gel transition [5]. The polypeptides in the collagen triple helix have a repetitive primary sequence of Gly-X aa -Y aa , where X aa is usually a proline and Y aa is often a (4R)-hydroxyproline [6]. In this typical sequence, the presence of the smallest amino acid, glycine, every third residue allows the three polypeptide chains to be in close proximity. The 4-Hydroxyproline, abbreviated as Hyp or O, has a greater stabilizing effect on the collagen triple helix compared with proline [7,8].
Collagen has attracted attention in protein conformation and interaction analysis, as a carrier molecule for drug delivery systems, and as a biomaterial for tissue engineering [9][10][11][12]. Although collagen is regarded as having high biocompatibility and low immunogenicity, there is a risk that natural collagen derived from animal tissues could potentially cause an undesired immune response, such as an allergy, and possible contamination with pathogens is also an issue. Therefore, the development of collagen-like peptides that are safer and have more functionality than natural collagen is desirable [13]. Currently, several collagen model peptides (CMPs), including (Pro-Hyp-Gly) 10 , (Pro-Pro-Gly) 10 , and other CMPs with similar sequences to these peptides, have been developed to mimic collagen [14].
To use the physicochemical properties of collagen for conformational studies and in biomedical materials, the formation and denaturation of the collagen triple helix need to be controllable in a reversible manner. Light is an ideal external trigger because it is highly selective and harmless when correctly applied [15]. Incorporation of a photo-responsive organic molecule into a CMP can provide a reversible, light-controllable, collagen triple helical structure. Azobenzene is one of the most well-studied photochromic molecules [16][17][18]. Although the azobenzene molecule usually exists in the trans-form, it isomerizes to the cis-form upon ultraviolet (UV) irradiation at approximately 350 nm. The conversion from the cisto the trans-form can be also achieved using visible (Vis) light irradiation near 450 nm or by a temperature change (i.e., thermal effect) [19]. Because the structure of azobenzene can be easily converted from one form to another by light irradiation at specific wavelengths, azobenzene has been widely used as a photo-switch in biological applications [20][21][22]. Azobenzene derivatives that can be isomerized by Vis light irradiation, without the use of UV light, have been developed [23][24][25][26] with a wide range of applications. Reversible control of the major secondary structural motifs of peptides and proteins, such as helices [27][28][29][30][31][32] and hairpins [33,34], has been studied using azobenzene. Azobenzene has also been used in the functional regulation of proteins, including receptors [35], enzymes [36], ion channels [37], and motor proteins [38], and also nucleic acids [39].
Light-responsive CMPs bearing an azobenzene molecule on the side chain of the peptide have been reported [40][41][42]. Although these synthesized collagen peptides were photo responsive, the collagen triple helix structure could be only partially regulated by the light irradiation. Such side chain-modified (i.e., branched) CMPs tend to be relatively complicated to synthesize and it is difficult to alter the peptide sequence. In addition, the azobenzene moiety on the side chain may sterically disturb the formation of the triple helical structure, which results in the original physicochemical properties of the collagen peptide chain not being reproduced in CMPs incorporating azobenzene in this manner. Thus, estimation of the properties of CMPs may be difficult at the molecular design stage. Although azobenzene-terminated CMPs have been also developed [43,44], the structures formed by these short CMPs, which contained at most five of repeats of the POG unit, favored a random coil, over a triple helix, structure because of the trans-to-cis isomerization of the terminal azobenzene. This disruption was caused by the relatively loose triple helical structure because of the shorter CMP chain. Moreover, current interest in CMPs appears to be directed toward morphological observations and the preparation of various types of three-dimensional nanoarchitectures [45]. Few studies have reported on photo-responsive CMPs incorporating azobenzene in a linearly linked manner and few kinetics studies have investigated the trans-to-cis isomerization upon UV irradiation or thermal cis-to-trans isomerization of such peptides.
In the present study, a linear photo-responsive collagen peptide bearing an azobenzene molecule was designed and synthesized and the effect of the photo-isomerization of the azobenzene moiety on the collagen triple helix structure was evaluated (Figure 1a). A kinetic assay of the thermal cis-to-trans isomerization of the azobenzene moiety of the CMPs was also performed to investigate the isomerization when azobenzene was conjugated to the CMPs. The 4,4 -Diaminoazobenzene was used as the azobenzene moiety, and the collagen peptide (Pro-Pro-Gly) 10 , which is the simplest CMP, was conjugated at both ends of the azobenzene moiety via glycine linkers. The 9-Fluorenylmethoxycarbonyl (Fmoc) groups on the N α -termini of the CMPs were retained to provide the potential for further modification by terminal functionalization. The glycine linker was expected to act as a buffer to alleviate the steric strain caused by the linking via the azobenzene during collagen triple helix formation. Azo-(PPG) 10 should form a collagen triple helix structure and this conformation was expected to be disrupted by the isomerization of the internal azobenzene moiety induced by UV-Vis light irradiation or a thermal effect (Figure 1b). The disassembled CMP chains should re-aggregate to form the helix again on cooling, which means that this conformational change in the CMPs should be reversible.  10 and azobenzene linker unit Azo-Gly. (b) Expected mechanism for controlling the triple helical structure of Azo-(PPG) 10 .

Synthesis of Fmoc-PPG-OH
Synthesis of 2. Boc 2 O (7.86 g, 36.0 mmol, 1.2 equiv) was added to a mixture of H-Gly-OH (1, 2.25 g, 30.0 mmol, 1.0 equiv) and Et 3 N (6.30 mL, 45.0 mmol, 1.5 equiv) in H 2 O (30 mL) and 1,4-dioxane (30 mL) at 0 • C, and the reaction mixture was stirred overnight from 0 • C to room temperature. The reaction mixture was concentrated in vacuo, and the residue was diluted with 4% NaHCO 3 and washed with Et 2 O. The aqueous layer was neutralized with citric acid monohydrate, and the desired compound was extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous MgSO 4 , and filtered. The filtrate was concentrated in vacuo, and the desired compound was crystallized with petroleum ether, filtered, and dried under reduced pressure to give 2 as a white powder (5.16 g, 29.4 mmol, 98%).
Synthesis of 3. BnBr (4.10 mL, 35.3 mmol, 1.1 equiv) was added to a mixture of 2 (5.16 g, 29.4 mmol, 1.2 equiv) and Et 3 N (9.00 mL, 64.8 mmol, 2.2 equiv) in DMF (30 mL) at 0 • C, and the reaction mixture was stirred overnight from 0 • C to room temperature. Additional Et 3 N (4.50 mL, 32.4 mmol, 1.1 equiv) and BnBr (2.10 mL, 17.7 mmol, 0.6 equiv) were added, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated in vacuo, and the residue was diluted with EtOAc. The organic layer was washed with 4% NaHCO 3 and brine, dried over anhydrous MgSO 4 , and filtered. The filtrate was concentrated in vacuo, and the desired compound was crystallized with Et 2 O and petroleum ether, filtered, and dried under reduced pressure to give 3 as a white powder (4.86 g, 18.2 mmol, 62%).
Synthesis of 4. Compound 3 (5.31 g, 20.0 mmol, 1.0 equiv) was treated with 4 M HCl/1,4-dioxane (60 mL) at room temperature for 2 h. The reaction mixture was concentrated in vacuo, and the desired compound was crystallized with petroleum ether, filtered, and dried under reduced pressure to give 4 as a white powder (4.00 g, 19.8 mmol, 99%).
Synthesis of 6. Boc 2 O (7.86 g, 36.0 mmol, 1.2 equiv) was added to a mixture of H-L-Pro-OH (5, 3.45 g, 30.0 mmol, 1.0 equiv) and Et 3 N (6.30 mL, 45.0 mmol, 1.5 equiv) in H 2 O (30 mL) and 1,4-dioxane (30 mL) at 0 • C, and the reaction mixture was stirred overnight from 0 • C to room temperature. The reaction mixture was concentrated in vacuo, and the residue was diluted with 4% NaHCO 3 and washed with Et 2 O. The aqueous layer was neutralized with citric acid monohydrate, and extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous MgSO 4 , and filtered. The filtrate was concentrated in vacuo, and the desired compound was crystallized with petroleum ether, filtered, and dried under reduced pressure to give 6 as a white powder (6.11 g, 28.5 mmol, 95%). Synthesis of 7. Et 3 N (3.50 mL, 24.0 mmol, 1.2 equiv) was added to a solution of 4 (4.00 g, 19.8 mmol, 1.0 equiv) in DMF (40 mL) at 0 • C, and 6 (5.20 g, 24.0 mmol, 1.2 equiv), N,N -dicyclohexylcarbodiimide (DCC, 4.95 g, 24.0 mmol, 1.2 equiv), and HOBt·H 2 O (3.68 g, 24.0 mmol, 1.2 equiv) were added, and the reaction mixture was stirred overnight from 0 • C to room temperature. The reaction mixture was concentrated in vacuo, and the residue was diluted with EtOAc and 10% citric acid and filtered. After separation of the aqueous layer, the organic layer was washed with 10% citric acid, brine, 4% NaHCO 3 , and brine, dried over anhydrous MgSO 4 , and filtered. The filtrate was concentrated in vacuo, and the resulting yellow oil was purified by column chromatography [silica, CHCl 3 /MeOH (99/1)] to give 7 as a white foam (7.91 g, 19.8 mmol, quant.).
Synthesis of 9. Fmoc-OSu (11.1 g, 33.0 mmol, 1.1 equiv) was added to a mixture of H-L-Pro-OH (5, 3.45 g, 30.0 mmol, 1.0 equiv) and Na 2 CO 3 (15.9 g, 150 mmol, 5.0 equiv) in H 2 O (125 mL) and 1,4-dioxane (125 mL) at 0 • C, and the reaction mixture was stirred overnight from 0 • C to room temperature. The reaction mixture was concentrated in vacuo, and the residue was diluted with sat. Na 2 CO 3 and washed with Et 2 O. The aqueous layer was neutralized with citric acid monohydrate, and the desired compound was extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous MgSO 4 , and filtered. The filtrate was concentrated in vacuo, and the desired compound was crystallized with ethyl acetate and petroleum ether, filtered, and dried under reduced pressure to give 9 as a white powder (9.53 g, 28.2 mmol, 94%).
Synthesis of 11. A suspension of 10 (9.78 g, 16.6 mmol, 1.0 equiv) and 5% Pd/C powder in MeOH (200 mL) was stirred under an H 2 atmosphere at room temperature until the reaction was completed. The reaction mixture was filtered, and the filtrate was concentrated in vacuo. The desired compound was crystallized with petroleum ether, filtered, and dried under reduced pressure to give 11 as a white powder (6.20 g, 14.1 mmol, 85%).

Synthesis of Fmoc-(PPG) 10 -OH
Synthesis of 13. Wang-PEG resin (12, 2.00 g, 0.48 mmol, 1.0 equiv) was swelled with CH 2 Cl 2 . To a suspension of the resin in CH 2 Cl 2 (15 mL), Fmoc-Gly-OH (0.210 g, 0.74 mmol, 1.5 equiv.) and N,N -diisopropylcarbodiimide (DIC, 0.200 mL, 1.40 mmol, 3.0 equiv) were added. After stirring at room temperature for 15 min, 4-dimethylaminopyridine (DMAP, 30.0 mg, 0.24 mmol, 0.5 equiv) was added, and the suspension was stirred overnight at room temperature. After removal of the reaction solution, the resin was washed with CH 2 Cl 2 , CH 2 Cl 2 /EtOH (1/1), EtOH, CH 2 Cl 2 , and Et 2 O and dried under reduced pressure to give 13 with uncapped OH groups as a yellow resin. Two resin samples (2.00−3.00 mg) were each treated with 20% piperidine/DMF (10 mL) at room temperature for 30 min. According to the absorbance of each reaction solution at 290 nm derived from the Fmoc moiety, the quantity of Fmoc-Gly-OH loaded onto the resin was estimated to be 0.230 mmol/g resin. For capping of the unreacted sites on the resin, Fmoc-Gly-Wang PEG resin was swelled with CH 2 Cl 2 . To a suspension of this resin in CH 2 Cl 2 (15 mL), was added DIEA (0.240 mL, 1.40 mmol, 3.0 equiv) and Ac 2 O (0.130 mL, 1.40 mmol, 3.0 equiv), and the suspension was stirred at room temperature for 2 h. After removal of the reaction solution, the resin was washed with CH 2 Cl 2 , DMF, CH 2 Cl 2 , and Et 2 O and dried under reduced pressure to give acetyl-capped 13 as a yellow resin.
Synthesis of 14. Compound 13 (2.00 g, 0.460 mmol) was swelled with DMF, and was treated with 20% piperidine/DMF (30 mL) at room temperature for 30 min. After removal of the reaction solution, the resin was washed with DMF, i-PrOH, and DMF. To a suspension of the resulting H-peptidyl-Wang PEG resin in DMF (15 mL), Fmoc-amino acid (1.40 mmol, 3.0 equiv), HBTU (0.350 g, 0.920 mmol, 2.0 equiv), HOBt·H 2 O (0.140 g, 0.920 mmol, 2.0 equiv), and DIEA (0.320 mL, 1.80 mmol, 4.0 equiv) were added, and the suspension was stirred at room temperature for 2 h. After removal of the reaction solution, the resin was washed with DMF, i-PrOH, and CH 2 Cl 2 . This deprotection and coupling cycle was repeated until the desired peptide sequence was synthesized. The resulting resin was finally washed with Et 2 O and dried under reduced pressure to give 14 as a yellow resin.
Synthesis of 15. Compound 14 was swelled with DMF, and was treated with 20% piperidine/DMF (30 mL) at room temperature for 30 min. After removal of the reaction solution, the resin was washed with DMF, i-PrOH, and DMF. To a suspension of resulting H-peptidyl-Wang PEG resin in DMF (15 mL), Fmoc-PPG-OH (11, 0.560 g, 1.15 mmol, 2.5 equiv), HBTU (0.350 g, 0.920 mmol, 2.0 equiv), HOBt·H 2 O (0.140 g, 0.920 mmol, 2.0 equiv), and DIEA (0.320 mL, 1.80 mmol, 4.0 equiv) were added, and the suspension was stirred at room temperature for 2 h. After removal of the reaction solution, the resin was washed with DMF, i-PrOH, and CH 2 Cl 2 . This deprotection and coupling cycle was repeated until the desired peptide sequence was synthesized. The resulting resin was finally washed with Et 2 O and dried under reduced pressure to give 15 as a yellow resin (2.48 g).
Synthesis of 16. Compound 15 (1 g, 0.240 mmol) was treated with TFA (30 mL) at room temperature for 2 h. The suspension was filtered, and the resin was washed with CH 2 Cl 2 . The filtrate was concentrated in vacuo with a small amount of H 2 O, and the partial residue (0.134 g, 50.0 µmol) was purified by gel filtration column chromatography (Sephadex G-25, 25% AcOH) and lyophilized to give 16 as a white powder (60.

Synthesis of Azo-Gly and Azo-(PPG) 10
Synthesis of 18. DCC (45.0 mg, 0.220 mmol, 2.2 equiv) was added to a solution of Boc-Gly-OH (2, 38.0 mg, 0.220 mmol, 2.2 equiv) in CH 2 Cl 2 , and the reaction mixture was stirred at room temperature for 30 min. To the mixture, 4,4 -diaminoazobenzene (17, 21.0 mg, 0.100 mmol, 1.0 equiv) was added, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated in vacuo, and the residue was diluted with EtOAc and 10% citric acid and filtered. After separation of aqueous layer, the organic layer was washed with 10% citric acid, brine, 4% NaHCO 3 , and brine, dried over anhydrous

Mesurements and Analysis
General. All sample solutions were preincubated overnight at 4 • C to form the triple helix structure. Circular dichroism (CD) spectra were recorded on a J-820 spectropolarimeter (JASCO Co., Ltd., Hachioji, Japan) equipped with a thermostatic cell holder coupled with a thermo supplier EZL-80F (TAITEC Co., Ltd., Koshigaya, Japan) under a N 2 atmosphere. Experiments were performed with a 1-mm quartz cell over 190-300 nm. UV-Vis spectra were acquired using a Ultrospec 3300 pro (GE Healthcare Co., Ltd., Illinois, U.S.) or V-570 UV/VIS/NIR spectrophotometer (JASCO Co., Ltd., Hachioji, Japan) with a 1-cm quartz cell. For photoisomerization, a Handy UV Lamp SLUV-4 at 365 nm, 4 W (AS ONE Co., Ltd., Osaka, Japan) was used for UV irradiation, and Vis light at 436 nm was produced using an Ultrospec 3300 pro (GE Healthcare Co., Ltd., Illinois, U.S.). The temperature of the sample solutions during absorption measurements was controlled using a thermo bath (AGC TECHNO GLASS Co., Ltd., Haibara, Japan).

CD Measurements
Each sample solution for CD spectroscopy was prepared in water to a concentration of 100 µM and measured over 190-300 nm. To estimate the T m for Azo-(PPG) 10 , the sample solution was adjusted to 100 µM with water. Thermal denaturation was monitored by measuring the change in dichroic intensities at 227 nm as a function of increasing temperature from 4 to 60 • C. The heating rate was 0.2 • C/min −1 .

Photo-Induced Denaturation of Collagen Triple Helix
To investigate the light-induced helix denaturation of Azo-(PPG) 10 , a series of sample solutions were prepared in water at a concentration 100 µM and the spectral data over 190-300 nm were obtained. The incubation of the sample solution at room temperature was carried out in the dark for 1 h. UV irradiation for 1 h was executed with a UV lamp in the dark at room temperature. UV-Vis irradiation for 1 h was performed with UV irradiation for 5 min and subsequent irradiation by a UV/Vis spectrophotometer for 5 min, for six cycles. The helix content was calculated from the maximal peak intensities in the CD spectra according to the following formula: where [θ] is the maximal mean residue molar ellipticity of the sample solution under the corresponding conditions and [θ] 0 is the maximal mean residue molar ellipticity of the the intact helix in the sample solution.

Photo-and Thermal-Reversibility of Azobenzene Moiety
Photo-reversibility was investigated using an aqueous solution of Azo-(PPG) 10 (11.1 µM) in water and Azo-Gly (11.7 µM) in 5% MeOH/water because it is less soluble in water than Azo-(PPG) 10 . After measuring the first absorbance data, UV irradiation for 1 min was carried out with a UV lamp in the dark, and then the absorbance data were re-measured. Subsequently, the samples were stood for 5 min at room temperature in the dark, and the absorption was measured.
For the kinetic assay of the cis-to-trans isomerization caused by a thermal effect, an aqueous solution of 50 µM Azo-(PPG) 10 was prepared. In the case of the measurement for Azo-(PPG) 10 at 40 • C, the concentration was adjusted to 11.1 µM. A solution of Azo-Gly (10 µM) was prepared in 5% MeOH in water. Each sample solution was incubated in a water bath in the dark and the absorption was chronologically measured. From the first-order reaction, the kinetic constant was calculated according to the absorbance at 364 nm followed by fitting the curve to minimize the error between the measured value and the regression curve equation with the following formula: where [cis-form] t is the concentration of the cis-form according to the absorbance at 364 nm when measured, [cis-form] 0 is the initial concentration of the cis-form regarded as the sample concentration, k is the first order rate constant, and t is the measuring time.
The half-life t 1/2 was calculated using the rate constant k as follows:

Synthesis of Photo-Responsive CMP
For the synthesis of the CMP 16, a convergent strategy in which Fmoc-protected Pro-Pro-Gly fragments prepared in solution were coupled to a solid-supported Pro-Pro-Gly fragment was adopted because of the reduced number of couplings required [46]. The component Pro-Pro-Gly tripeptides for the fragment condensation were synthesized by solution-phase methods (Scheme 1). After preparing the Fmoc-Pro-Pro-Gly tripeptide anchored to the resin via one-by-one peptide chain elongation, Fmoc-(PPG) 10  For the synthesis of the CMP containing an azobenzene moiety, both amino groups of 4,4 -diaminoazobenzene 17 were reacted with tert-butyloxycarbonyl (Boc)-protected glycine 2 (Scheme 2). After deprotecting the Boc groups to provide Azo-Gly (Supplementary Figure S1), two molecules of CMP 16 were conjugated to obtain Azo-(PPG) 10 . Although the mass of Azo-(PPG) 10 could not be determined by mass spectrometry techniques, such as fast atom bombardment mass spectrometry, electrospray ionization time-of-flight, or matrix assisted laser desorption/ionization, because of the large molecular weight and intrinsic aggregation property, Azo-(PPG) 10 displayed a characteristic CD curve and the absorption properties of azobenzene, and a single peak was present in liquid-chromatography with a gel permeation column (Supplementary Figure S2). Therefore, we concluded that Azo-(PPG) 10 was successfully synthesized. Scheme 2. Synthesis of the azobenzene linker Azo-Gly and the photo-responsive CMP Azo-(PPG) 10 .

Investigation of Collagen Triple Helical Properties
The ability of Azo-(PPG) 10 to form a collagen triple helix in water was examined using a CD assay with the positive control, Fmoc-(PPG) 10 -OH (16), to examine the extent of the helix content. The CD spectrum of Azo-(PPG) 10 showed a maximal peak at 227 nm, as was observed for 16, which indicated that Azo-(PPG) 10 formed a collagen triple helical structure ( Figure 2). The slightly lower maximal peak height for Azo-(PPG) 10 compared with 16 was because of the distortion of the collagen terminus caused by the insertion of the azobenzene. However, the peak height of Azo-(PPG) 10 was comparable to that of Fmoc-(PPG) 8 -OH (data not shown), which indicated that the triple helical content of Azo-(PPG) 10 was similar to that of Fmoc-(PPG) 8 The T m value of Azo-(PPG) 10 was estimated to be approximately 35 • C by monitoring the maximal peak height in the CD spectrum at 227 nm while heating ( Figure 3). The T m value of (Pro-Pro-Gly) 10 ranges from 27 to 41 • C, depending on the solvent [47,48], which indicated that Azo-(PPG) 10 has adequate stability toward heating.

Denaturation of the Collagen Triple Helix Structure by Light Irradiation
Next, the effect of light irradiation on the collagen triple helix was investigated by CD. Incubation of the triple helical solution of Azo-(PPG) 10 at room temperature for 1 h resulted in an 8% decrease in the collagen triple helix content (Figure 4). UV irradiation for 1 h denatured 9% of the helical content. These results indicated that single trans-tocis isomerization of the azobenzene moiety did not disrupt the collagen triple helix. In contrast, six cycles of UV-Vis irradiation over 1 h decreased the helical content by 19%, which demonstrated that repetitive isomerization of the azobenzene moiety by UV-Vis irradiation considerably induced the unfolding the triple helix structure of Azo-(PPG) 10 . The completely unfolded Azo-(PPG) 10 could be re-formed to the triple helical structure on cooling. The destabilization, misfolding, or delayed folding of collagen fibers caused by a sequence variant, or mechanical damage of the collagen molecules, is related to several human diseases [49][50][51][52]. Although our results indicated that 75% of the helical structure remained, this partial disruption of the structure (i.e., slow unfolding) has the potential to be used in elucidating the mechanism of the folding/unfolding processes, which may lead to better understanding of diseases related to collagen and the design of treatments for such diseases. Figure 2. CD spectra of Azo-(PPG) 10 and the positive control Fmoc-(PPG) 10 -OH (16). The inset shows the spectra over the range of the characteristic maximal peak for the collagen triple helix structure.   10 upon irradiation with light. a Measurement was carried out before light irradiation. b Sample solution was incubated at room temperature in the dark, and then measured. c The sample solution was continuously irradiated with UV light (365 nm) for 1 h, and then measured. d The sample was alternatively irradiated with UV (365 nm, 5 min) and Vis (436 nm, 5 min) light six times, and then measured.

Isomerization of the Azobenzene Moiety by Light Irradiation or Thermal Effect
To investigate the slow unfolding of Azo-(PPG) 10 upon UV irradiation, and the thermal effect, the isomerization behavior of the internal azobenzene was analyzed. Initially, the reversibility of the trans-to-cis isomerization upon UV irradiation and the cis-to-trans isomerization at room temperature (i.e., thermal effect) was evaluated at 20 and 40 • C. The absorption at approximately 350 nm of the trans-form of azobenzene is greater than that of the cis-form, which enables monitoring of the isomerization of the azobenzene moiety. The azobenzene moiety of Azo-(PPG) 10 at 20 • C showed reversibility of the isomerization, and at 40 • C the reversibility was slightly greater ( Figure 5). In comparison with Azo-Gly, less reversibility in the conformation of Azo-(PPG) 10 was observed because of the interactions between the CMP chains. In the kinetic assays for the cis-to-trans isomerization of the azobenzene moiety, changes in the percentage of the cis-form were calculated according to the chronological absorption measurements at 364 nm. This isomerization could be considered to be a first-order reaction, and the kinetic constant k and half-life t 1/2 were calculated by curve fitting (Figure 6). The cis-to-trans recovery rate of the azobenzene moiety in Azo-(PPG) 10 at 20 • C was sixfold slower than that of Azo-Gly at same temperature (Table 1). For Azo-(PPG) 10 at 40 • C, the rate was approximately five-fold faster than at 20 • C because of the loose triple helical structure. At a lower temperature (4 • C) compared with 20 • C, the rate of the cis-to-trans isomerization of Azo-(PPG) 10 was halved. These results are encouraging for the application of Azo-(PPG) 10 not only in investigating the folding/unfolding mechanism of CMPs but also for understanding the behavior of the azobenzene moiety in photo-controllable biomolecules because the kinetics of the CMP folding/unfolding and the isomerization of the azobenzene moiety can be controlled by a combination of light irradiation and heating.

Conclusions
In summary, Azo-(PPG) 10 with an azobenzene conjugated at both termini to a (Pro-Pro-Gly) 10 chain was successfully designed and synthesized. The light-responsive Azo-(PPG) 10 displayed a collagen triple helix structure with good stability (T m = 35 • C), and this helical structure could be reversibly folded and unfolded depending on the temperature. According to the CD spectral measurements, approximately one-fifth of the triple helical content of Azo-(PPG) 10 was denatured by repetitive UV-Vis irradiation for 1 h. Although the azobenzene moiety in Azo-(PPG) 10 showed reversibility upon UV irradiation and thermal changes, the rate of cis-to-trans recovery of Azo-(PPG) 10 was relatively slower than that of the azobenzene linker Azo-Gly that does not have CMP chains. Higher temperatures induced improvements in the reversibility and kinetic profile of Azo-(PPG) 10 because of the looser helical structure at higher temperatures. The rates of folding/unfolding of the triple helical structure and isomerization of the azobenzene moiety were adjustable by a combination of light irradiation and heating, indicating that Azo-(PPG) 10 may be useful for developing photo-responsive CMPs and for understanding the assembly/disassembly of CMP chains.