Synthesis of Caffeoyl-Prolyl-Histidyl-Xaa Derivatives and Evaluation of Their Activities and Stability upon Long-Term Storage

Antioxidants play a critical role in the treatment of degenerative diseases and delaying the aging of dermal tissue. Caffeic acid (CA) is a representative example of the antioxidants found in plants. However, CA is unsuitable for long-term storage because of its poor stability under ambient conditions. Caffeoyl-Pro-His-NH2 (CA-Pro-His-NH2, CA-PH) exhibits the highest antioxidant activity, free radical scavenging and lipid peroxidation inhibition activity among the histidine-containing CA-conjugated dipeptides reported to date. The addition of short peptides to CA, such as Pro-His, is assumed to synergistically enhance its antioxidative activity. In this study, several caffeoyl-prolyl-histidyl-Xaa-NH2 derivatives were synthesized and their antioxidative activities evaluated. CA-Pro-His-Asn-NH2 showed enhanced antioxidative activity and higher structural stability than CA-PH, even after long-term storage. CA-Pro-His-Asn-NH2 was stable for 3 months, its stability being evaluated by observing the changes in its NMR spectra. Moreover, the solid-phase synthetic strategy used to prepare these CA-Pro-His-Xaa-NH2 derivatives was optimized for large-scale production. We envision that CA-Pro-His-Xaa-NH2 derivatives can be used as potent dermal therapeutic agents and useful cosmetic ingredients.


Introduction
Reactive oxygen species (ROS) include highly reactive free radicals, such as peroxide (ROO · ) and superoxide (O 2 · ). ROS induce oxidative stress in the human body via radical chain reactions, thereby causing damage to DNA or RNA, cancer, chronic disease and aging [1,2]. In addition, oxidative stress can cause degenerative diseases such as Alzheimer's disease [3][4][5].
Natural antioxidants found in various foods, including vegetables, meat, fruits and grains, inhibit the activity of ROS. They are considered suitable agents for scavenging radicals but are unsuitable for practical use as antioxidative nutrients or therapeutic agents because of their low concentration and/or activity [6,7]. Therefore, many synthetic antioxidants have been studied to prevent food corruption, delay the aging process, and treat degenerative diseases via scavenging ROS [8][9][10][11]. However, there are restrictions on the use of synthetic antioxidants, such as tertiary butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and bisphenol (BP), because they are potentially cytotoxic when used in high concentrations [12].
Phenolic compounds have been extensively studied as antioxidants because they provide a relatively stable free-radical structure after losing a hydrogen atom. Previous reports have shown that the antioxidant activity of phenolic compounds increases upon increasing the number of hydroxyl and methoxy groups on the phenyl ring [13]. Caffeic acid (CA, 3,4-dihydroxycinnamic acid) is a member of the hydroxycinnamic acid (HCA) family and is often found in nature. CA exhibits high antioxidant activity because of the delocalization of non-covalent electrons in its extended side chain [14]. In addition, the ortho-dihydroxyl group in CA forms a stable hydrogen bond after dissociation of the O-H bond [15][16][17]. To date, various CA derivatives, such as CA-triazole, CA-tyramine and CAamino phenol, have been studied. However, such CA derivatives have insufficient redoxactive catechol moiety or participate in side reactions, which decrease the antioxidative activity [18][19][20].
We have previously reported that the conjugation of CA to a peptide can enhance its stability. In particular, CA-Pro-His-NH 2 exhibits the highest antioxidant activity reported to date because the s-cis proline conformer contributes to the increased antioxidant activity of CA ( Figure 1) [21][22][23][24][25][26]. In this study, we expected that some of the CA-tripeptide derivatives would exhibit better antioxidant activity and stability than CA-Pro-His-NH 2 . Therefore, several CA-Pro-His-Xaa-NH 2 derivatives were synthesized as an extended library from CA-Pro-His-NH 2 , and their antioxidant activity and long-term storage stability were evaluated. the use of synthetic antioxidants, such as tertiary butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and bisphenol (BP), because they are potentially cytotoxic when used in high concentrations [12]. Phenolic compounds have been extensively studied as antioxidants because they provide a relatively stable free-radical structure after losing a hydrogen atom. Previous reports have shown that the antioxidant activity of phenolic compounds increases upon increasing the number of hydroxyl and methoxy groups on the phenyl ring [13]. Caffeic acid (CA, 3,4-dihydroxycinnamic acid) is a member of the hydroxycinnamic acid (HCA) family and is often found in nature. CA exhibits high antioxidant activity because of the delocalization of non-covalent electrons in its extended side chain [14]. In addition, the ortho-dihydroxyl group in CA forms a stable hydrogen bond after dissociation of the O-H bond [15][16][17]. To date, various CA derivatives, such as CA-triazole, CA-tyramine and CAamino phenol, have been studied. However, such CA derivatives have insufficient redoxactive catechol moiety or participate in side reactions, which decrease the antioxidative activity [18][19][20].
We have previously reported that the conjugation of CA to a peptide can enhance its stability. In particular, CA-Pro-His-NH2 exhibits the highest antioxidant activity reported to date because the s-cis proline conformer contributes to the increased antioxidant activity of CA ( Figure 1) [21][22][23][24][25][26]. In this study, we expected that some of the CA-tripeptide derivatives would exhibit better antioxidant activity and stability than CA-Pro-His-NH2. Therefore, several CA-Pro-His-Xaa-NH2 derivatives were synthesized as an extended library from CA-Pro-His-NH2, and their antioxidant activity and long-term storage stability were evaluated.  Figure 1. Antioxidant mechanism of CA-PH-NH2 with peroxyl radicals based on an H-atom transfer and single-electron transfer mechanism. Reproduced from [26], with permission of Springer Nature Limited.

Synthesis of CA-PHX-NH2
Several CA-PHX-NH2 derivatives were synthesized on Rink amide aminomethyl polystyrene (AM PS) resin using Fmoc-chemistry-based solid-phase peptide synthesis Figure 1. Antioxidant mechanism of CA-PH-NH 2 with peroxyl radicals based on an H-atom transfer and single-electron transfer mechanism. Reproduced from [26], with permission of Springer Nature Limited.

Synthesis of CA-PHX-NH 2
Several CA-PHX-NH 2 derivatives were synthesized on Rink amide aminomethyl polystyrene (AM PS) resin using Fmoc-chemistry-based solid-phase peptide synthesis (SPPS) (Scheme 1). Prior to coupling CA to the peptides on the resin, the purity of the PHX-NH 2 synthesized on the resin was determined using high-performance liquid chro-matography (HPLC) after a small-scale cleavage reaction. The HPLC analysis showed that the purity of PHX-NH 2 synthesized using Rink amide AM PS resin was >93%. The effect of the resin on the purity of PHX-NH 2 was evaluated by synthesizing PHX-NH 2 using Rink amide 4-methylbenzhydrylamine (MBHA) resin, which is known to result in higher purity during SPPS [27]. Unexpectedly, the Rink amide AM PS resin gave an approximately two-fold higher yield than Rink amide MBHA resin, as well as a 3-7% higher crude purity for all the compounds studied (Table 1). The Rink amide MBHA resin released the fragment of Rink amide linker as well as the tripeptide products during the final cleavage step, whereas Rink amide AM PS did not release any compound except the tripeptide products. This affected the purity and yield of the tripeptide products observed during HPLC when the peptides were synthesized on Rink amide MBHA resin.
(SPPS) (Scheme 1). Prior to coupling CA to the peptides on the resin, the purity of the PHX-NH2 synthesized on the resin was determined using high-performance liquid chromatography (HPLC) after a small-scale cleavage reaction. The HPLC analysis showed that the purity of PHX-NH2 synthesized using Rink amide AM PS resin was >93%. The effect of the resin on the purity of PHX-NH2 was evaluated by synthesizing PHX-NH2 using Rink amide 4-methylbenzhydrylamine (MBHA) resin, which is known to result in higher purity during SPPS [27]. Unexpectedly, the Rink amide AM PS resin gave an approximately two-fold higher yield than Rink amide MBHA resin, as well as a 3-7% higher crude purity for all the compounds studied (Table 1). The Rink amide MBHA resin released the fragment of Rink amide linker as well as the tripeptide products during the final cleavage step, whereas Rink amide AM PS did not release any compound except the tripeptide products. This affected the purity and yield of the tripeptide products observed during HPLC when the peptides were synthesized on Rink amide MBHA resin. (h) treatment of dry resin with cleavage cocktail (88% TFA/5% phenol/5% water/2% TIPS) for 1 h and precipitation using diethyl ether. R: side chain of amino acid (serine, arginine, aspartic acid, glycine, asparagine, phenylalanine, lysine, glutamic acid, alanine and glutamine). Thus, several CA-PHX-NH2 derivatives were synthesized on Rink amide AM PS resin and obtained as white powders upon precipitation of the cleaved product using cold diethyl ether with crude purities in the range of 72-79%, as confirmed by ESI-MS (Table  2). Finally, the CA-PH-NH2 and CA-PHX-NH2 derivatives were obtained with >96% purity using preparative HPLC (Supplementary Figures S1-S11). The purified products were used for further investigation of their antioxidant activity. (h) treatment of dry resin with cleavage cocktail (88% TFA/5% phenol/5% water/2% TIPS) for 1 h and precipitation using diethyl ether. R: side chain of amino acid (serine, arginine, aspartic acid, glycine, asparagine, phenylalanine, lysine, glutamic acid, alanine and glutamine). Thus, several CA-PHX-NH 2 derivatives were synthesized on Rink amide AM PS resin and obtained as white powders upon precipitation of the cleaved product using cold diethyl ether with crude purities in the range of 72-79%, as confirmed by ESI-MS (Table 2). Finally, the CA-PH-NH 2 and CA-PHX-NH 2 derivatives were obtained with >96% purity using preparative HPLC (Supplementary Figures S1-S11). The purified products were used for further investigation of their antioxidant activity. The free-radical scavenging activity (RSA) of the purified CA-PHX-NH 2 derivatives was evaluated using a DPPH radical scavenging test. When the CA-PHX-NH 2 derivatives were in their radical form after donating a hydrogen atom to DPPH, the color of the DPPH solution in methanol (MeOH) changed from purple to yellow (Supplementary Figure S12), and the absorbance observed at 516 nm decreased. Figure 2  Results are presented as mean ± standard error.

Lipid Peroxidation (LPO) Test
The antioxidant activity was also measured using a lipid peroxidation (LPO) inhibition assay using Tween 20-emulsified linoleic acid (Supplementary Figure S13). The percentage of lipid peroxidation inhibition (%Pi) was calculated using the following equation:  Figure 3 shows that most of the CA-PHX-NH 2 derivatives were superior to CA-PH-NH 2 in the LPO inhibition assay. Hydrophilic DPPH and hydrophobic LPO tests showed different patterns for the antioxidant activity among the caffeoyl peptide derivatives studied. For example, CA-PHN-NH 2 showed the highest antioxidant activity in the DPPH test but had a lower activity than CA-PH-NH 2 in the LPO inhibition assay. These differences can be attributed to the different solubilities of the derivatives in the working solutions used.

Cytotoxicity of the CA-PHX-NH2 Derivatives
The MTT assay data exhibited no significant cytotoxicity for the CA-PHX-NH2 derivatives at low concentration (1 µM). Even at concentrations as high as 100 µM, most of the CA-PHX-NH2 derivatives were not cytotoxic, while some of the compounds, such as CA-PHA-NH2, CA-PHR-NH2 and CA-PHK-NH2, showed cell viabilities of <80%. In the case

Cytotoxicity of the CA-PHX-NH 2 Derivatives
The MTT assay data exhibited no significant cytotoxicity for the CA-PHX-NH 2 derivatives at low concentration (1 µM). Even at concentrations as high as 100 µM, most of the CA-PHX-NH 2 derivatives were not cytotoxic, while some of the compounds, such as CA-PHA-NH 2 , CA-PHR-NH 2 and CA-PHK-NH 2 , showed cell viabilities of <80%. In the case of CA-PH-NH 2 , CA-PHS-NH 2 , CA-PHN-NH 2 and CA-PHQ-NH 2 , cell viabilities exceeded 100%. We presume that those CA derivatives reduced the oxidative stress of the cells and promoted cell proliferation, overcoming cytotoxicity ( Figure 4). Thus, CA-PHX-NH 2 derivatives exhibiting low cytotoxicity have potential use as cosmetic ingredients.

Stability Evaluation Using 1 H-NMR Spectroscopy
1 H-NMR spectroscopy was used to evaluate the long-term structural stability of CA-PHN-NH2. The changes in the molar ratio of s-cis Pro, which demonstrates the stability of CA-PHN-NH2, were monitored upon storage at 25 °C under dark conditions over 3 months. The ratio of the s-cis form remained >93% for 3 months (Table 3, Supplementary Figure S14). The 1 H-NMR spectra show that CA-PHN-NH2 exhibited limited transition between the cis and trans conformations over 3 months, which is critical for maintaining its high antioxidation activity [26]. Therefore, CA-PHN-NH2 will likely maintain its antioxidant activity in hydrophilic solutions upon long-term storage.

Stability Evaluation
Using 1 H-NMR Spectroscopy 1 H-NMR spectroscopy was used to evaluate the long-term structural stability of CA-PHN-NH 2 . The changes in the molar ratio of s-cis Pro, which demonstrates the stability of CA-PHN-NH 2 , were monitored upon storage at 25 • C under dark conditions over 3 months. The ratio of the s-cis form remained >93% for 3 months (Table 3, Supplementary Figure S14). The 1 H-NMR spectra show that CA-PHN-NH 2 exhibited limited transition between the cis and trans conformations over 3 months, which is critical for maintaining its high antioxidation activity [26]. Therefore, CA-PHN-NH 2 will likely maintain its antioxidant activity in hydrophilic solutions upon long-term storage.

Solid-Phase Synthesis of CA-PHX-NH 2
Prolyl-histidyl-Xaa-NH 2 was synthesized on two types of resin using Fmoc/t-Bu chemistry. The tripeptides were synthesized on a Rink amide resin (300 mg) by repeating the coupling and deprotection steps: (a) in the coupling step, the resins were treated with a solution of Fmoc-amino acid (3.0 equiv.), HBTU (3.0 equiv.), HOBt (3.0 equiv.) and DIPEA (6.0 equiv.) in DMF (10 mL) for 2 h; (b) in the deprotection step, each resin was treated twice with 20% piperidine/DMF (v/v) solution (2 × 10 mL) for 5 and 10 min. The resins were washed with DMF, DCM and MeOH three times after each coupling and deprotection step. The completion of each amino acid coupling reaction used in the synthesis was confirmed using Kaiser's ninhydrin test.
The CA (3.0 equiv.) was dissolved in DMF (3 mL) in a glass vial. To this solution, HOBt (3.0 equiv.) and DIC (3.0 equiv.) were added, and the resulting mixture was stirred at room temperature for 30 min under dark conditions. The mixture was added to a reaction tube containing Pro-His-Xaa-NH 2 and DIPEA (6.0 equiv.) and incubated in a multirotator for 1 h at room temperature. The resin was washed three times with DMF, DCM and MeOH. The resin was treated with a cleavage cocktail comprising trifluoroacetic acid/phenol/triisopropylsilane/water (88:5:5:2, v/v/v/v; 3 mL) for 2 h. After removal of the resin, the resulting solution was precipitated upon the addition of diethyl ether. The supernatant was decanted after centrifugation, and the remaining pellet was washed three times with diethyl ether via centrifugation and dried in vacuo. The purities of the peptides and CA-PHX-NH 2 were analyzed using HPLC under the following conditions: gradient elution with A (0.1% TFA in water) and B (0.1% TFA in acetonitrile) from 10% to 90% B over 41 min; flow rate of 1.0 mL/min; UV detection at 230 and 214 nm; column temperature of 35.0 • C.

Cytotoxicity Assay for CA-PHX-NH 2
NIH 3T3 cells were seeded (100,000 cells/mL, 200 µL) in a 96-well plate and incubated for 1 day. Solutions of each CA-PHX-NH 2 derivative in RPMI 1640 media (1, 10, 50 and 100 µM) were prepared. The cell culture medium in each well was carefully aspirated, and the cells were treated with the CA-PHX-NH 2 solutions for 1 day in a CO 2 incubator. After removal of the solution, the cells were incubated in the presence of MTT solution (200 µL) for 3 h. After incubation, the MTT solution was discarded and DMSO was added to dissolve the formazan produced by the living cells. The mixtures in the 96-well plate were shaken in a shaking incubator for 20 min. The absorbance of the dissolved formazan was measured at a wavelength of 590 nm using a microplate reader. The percentage of cell viability was calculated by comparison with the number of standard cells which were not treated with the solutions of CA derivatives.

Long-Term Stability Test Using 1 H-NMR Spectroscopy
The sample of CA-PHN-NH 2 for 1 H-NMR spectroscopy was prepared by dissolving 10 mg of compound (±0.01 mg) in 1 mL of methanol-d 6 (MeOD). The resulting solution was transferred to a 5 mm standard NMR tube. The 1 H-NMR spectra were obtained on a Bruker AVANCE III-HD500 spectrometer operated at 500 MHz. Tetramethylsilane (TMS) was used as an internal standard for the analysis of the chemical shifts.

Conclusions
A library of caffeoyl tripeptides (CA-PHX-NH 2 ) was synthesized, and their antioxidant activities were evaluated to develop new CA-peptide derivatives with enhanced antioxidant capacity compared to CA-PH-NH 2 . The DPPH assay showed that CA-PHN-NH 2 exhibited the highest free RSA (92.5%), compared to CA-PHX-NH 2 and CA-PH-NH 2 . None of the CA derivatives exhibited significant cytotoxicity at concentrations up to 100 µM. 1 H-NMR spectroscopy showed that CA-PHN-NH 2 maintained its structural integrity for up to 3 months. Therefore, CA-PHN-NH 2 is expected to be used as a raw material for pharmaceutical or cosmetic ingredients because of its superior antioxidant ability and long-term stability.