Synthesis of cyclic peptides with antioxidant and skin-lightening properties

Synthesis of cyclic peptides with antioxidant and skin-lightening properties | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synthesis of cyclic peptides with antioxidant and skin-lightening properties Bo-Mi Kim, Ga-Hyun Kim This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7205295/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Cyclic peptides have emerged as a compelling class of therapeutic and bioactive compounds owing to their diverse and potent biological activities. Nevertheless, their commercialization has been hindered by considerable challenges in synthesis and elevated cost of production. Methods In this study, we employed a scalable and cost-effective chemical synthesis strategy to rapidly design and synthesize a linear peptide sequence with antioxidant and skin-lightening properties using solid-phase peptide synthesis (SPPS). Subsequently, the cyclic peptides Cys-Tyr-Gly-Ser-Arg (CR5) was developed using liquid-phase peptide synthesis (LPPS). Results The cytotoxicity of CR5 was evaluated via the WST-1 assay, revealing over 90% cell viability at concentrations up to 400 µg/mL, indicative of high cellular biocompatibility. Moreover, CR5 demonstrated more than 50% 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity with an IC₅₀ of 16.62 µg/mL, highlighting its notable antioxidant efficacy. In addition, CR5 showed 97.79% tyrosinase inhibition at 800 µg/mL, with an IC₅₀ of 30.77 µg/mL, confirming its strong skin-lightening potential. Conclusion The cyclic peptide CR5 developed in this study has high production scalability and dual functional properties in the form of antioxidant and skin-lightening activities. It is a promising candidate for future applications in the pharmaceutical and cosmetic industries. Cyclic peptides Antioxidant activity Skin-lightening Tyrosinase inhibition Cosmeceuticals Figures Figure 1 Figure 2 1. Introduction The rapid advancement of medical technology has significantly extended human life expectancy, leading to an increasing societal interest in healthy aging in general and skin aging in particular (Surber et al. 2015 ; Nagae et al. 2023 ). Age-related hyperpigmentation and oxidative stress affect both aesthetic appearance and overall health, thereby stimulating growing personal concern and active scientific research on their mitigation (Lee et al. 2024 ). The major mechanisms underlying skin aging include collagen degradation, melanin overproduction, inflammation, and accumulation of reactive oxygen species (ROS). Development of biomaterials capable of simultaneously modulating these multifactorial changes is a critical challenge in cosmetic and therapeutic science (Dan et al. 2024 ). Research focusing on functional peptides for skin health improvement has gained momentum in the life sciences amid the global trend toward population aging. A promising approach involves the application of bioactive peptides that exhibit antioxidant and skin-lightening activities (Shen et al. 2019 ; Thaha et al. 2021 ). Specific peptides exert their effects by mitigating ROS accumulation and inhibiting melanin synthesis through mechanisms such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging and tyrosinase inhibition (Yingchutrakul et al. 2022 ; Kriangkrai et al. 2024 ). Against this background, functional peptide technology has found active applications not only in the biotechnology and pharmaceutical fields but also, more recently, in the cosmetics industry, and is increasingly recognized as a next-generation platform for promoting skin health and preventing aging (Pintea et al. 2025 ). In addition to their role as simple auxiliary components, peptides are being reevaluated for their value as core functional agents with intrinsic bioactivity, and their scope of application continues to steadily expand (Hariri et al. 2021 ; Zhuang et al. 2025 ; Meng et al. 2025 ). Despite their advantages, linear peptides undergo rapid degradation in vivo, leading to a short half-life and poor bioavailability. To overcome these limitations, the development of novel cyclic peptides has become increasingly important (Tapeinou et al. 2015 ). Cyclic peptides have a stable three-dimensional conformation owing to their cyclic structure, which confers several advantages, including enhanced selectivity for specific receptor-binding sites, increased resistance to proteolytic degradation, and improved structural stability (Fagundez et al. 2018 ; Zhang and Chen 2022 ; Ji et al. 2024 ). The structural characteristics of cyclic peptides are strongly influenced by their ring size. When compared with their larger counterparts, smaller cyclic peptides typically exhibit enhanced conformational stability and superior drug-like properties. (Horton et al. 2008 ; Thakkar et al. 2013 ; Sarojini et al. 2019 ). In fact, cyclic peptides such as cyclosporin A, gramicidin S, and oxytocin have been utilized to treat several diseases (Tedesco and Haragsim 2012 ; Schulze 2014 ; Guan et al. 2019 ). However, synthesizing cyclic peptides is challenging because of the demanding reaction conditions, which can result in cyclization failure, side reactions, and dimer formation (Bechtler and Lamers 2021 ; Fang et al. 2024 ). Therefore, their production costs are higher than those of linear peptides, and the difficulty of scaling-up limits their economic feasibility for commercialization (Thapa et al. 2014 ). The synthesis methods for cyclization include microbial and chemical cyclizations. Microbial approaches include ribosomally synthesized and post-translationally modified peptide (RiPP) and non-ribosomal peptide synthetase (NRPS) systems. However, because these methods rely solely on enzymatic structures or predefined sequences, they offer lower design flexibility than chemical cyclization, and system construction remains technically challenging (Han and Won 2024 ; Huang et al. 2024 ). Several chemical cyclization strategies have been developed, including head-to-tail, side-chain-to-side chain, and O–S acyl shift methods. However, short cyclic peptides optimized for low-cost mass production need to be developed, as current strategies offer limited efficiency in this regard (Hayes et al. 2021 ; Buchanan et al. 2025 ). In the present study, we synthesized a short sequence of linear peptides with antioxidant and skin-lightening properties using solid-phase peptide synthesis (SPPS). Subsequently, a cyclization strategy based on liquid-phase peptide synthesis (LPPS) was employed to shorten the production time and enable high-purity mass production, thereby enhancing commercial applicability and improving the bioactivity of pharmaceutical and cosmetic materials. 2. Materials and Methods 2.1 Materials All reagents used in this study were purchased from GL Biochem (China), Sigma-Aldrich (USA), or TCI (Japan). 2.2 Synthesis of Cys-Tyr-Gly-Ser-Arg (CYGSR) using Fmoc-chemistry The CYGSR peptide used in this study was synthesized using SPPS. 2-Chlorotrityl chloride resin (0.5 g, 1.0 mmol/g) was stirred in 5.8 mL of dichloromethane (DCM) for 30 min. After swelling, the solvent was removed under vacuum, and the resin was washed twice with DCM. For the sequential coupling of amino acids, Fmoc-Arg(Pbf)-OH (2.0 eq, 0.6 g) was added to the reaction vessel along with 1-hydroxybenzotriazole (HOBt, 1.4 eq, 0.2 g), N,N-diisopropylethylamine (DIPEA, 5.0 eq, 0.4 mL), N,N′-diisopropylcarbodiimide (DIC, 3.0 eq, 0.2 mL), and N,N-dimethylformamide (DMF, 4.0 mL), and the mixture was stirred for 4 hours. Completion of the coupling reaction was then confirmed using the Kaiser test (Kaiser et al. 1970 ). Double coupling or capping was performed as necessary, based on the test results (Amblard et al. 2006 ). Amino acid coupling was performed following Fmoc deprotection using 20% piperidine in DMF (Fields and Noble 1990 ). The second amino acid, Fmoc-Ser(tBu), OH (2.0 eq, 0.4 g), the third, Fmoc-Gly-OH (2 eq, 0.3 g), the fourth, Fmoc-Tyr(tBu)-OH (2 eq, 0.5 g), and the fifth, Fmoc-Cys(Trt)-OH (2.0 eq, 0.6 g), were sequentially coupled using the same procedure as described for the initial amino acid. 2.3 Cleavage of CYGSR To cleave the synthesized CYGSR from the resin, 12.0 mL of a cleavage cocktail consisting of trifluoroacetic acid (TFA), triisopropylsilane, and H 2 O (95:2.5:2.5, v/v/v) was added to the reaction vessel and then stirred for 1 hour. The mixture was filtered, and the filtrate was then precipitated with 66.0 mL cold isopropyl ether to yield a white solid. 2.4 Cyclization of CYGSR The linear peptide, CYGSR, was cyclized via the formation of an intramolecular amide bond between the N-terminal amine and the C-terminal carboxylic acid. Lyophilized CYGSR (1.0 mmol) was dissolved in DMF to a final concentration of 1 mM. HOBt (2.5 eq, 0.3 g), N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl, 2.5 eq, 0.5 g), and DIPEA (5 eq, 0.9 mL) were sequentially added while stirring at 0°C. The reaction mixture was stirred under a nitrogen atmosphere at room temperature, and samples were collected at 4-hour intervals for monitoring by high-performance liquid chromatography (HPLC). After 12 hours, the reaction was quenched by adding 5 mL of cold distilled water. The product was extracted three times with 5.0 mL ethyl acetate, and the combined organic phases were dried over anhydrous sodium sulfate (Na₂SO₄) and concentrated under reduced pressure. The crude product was purified using reversed-phase high-performance liquid chromatography (RP-HPLC). 2.5 Isolation and purification of CYGSR Purification was performed using a preparative HPLC system (Waters 2545 Quaternary Gradient Module with 2998 Photodiode Array Detector, USA) after dissolving 0.05 g of the crude peptide in 5.0 mL of 0.1% TFA in water. A YMC-Pack ODS-A-HG C18 reversed-phase column (250 × 20.0 mm I.D., S-10 µm, 30 nm; YMC, Japan) was used. The mobile phases consisted of solvent A (0.1% TFA in water) and solvent B (0.1% TFA in acetonitrile), and separation was performed under the gradient conditions listed in Table 1 . Table 1 HPLC conditions Column YMC-Pack ODS-A-HG (250 × 20.0 mm I.D., 10 µm, 30 nm) Mobile Phase Solvent A: H 2 O0.1% TFA Solvent B: Acetonitrile + 0.1% TFA Time (min) Solvent A (%) Solvent B (%) 0.0 95 5 10.0 95 5 45.0 35 65 50.0 35 65 50.1 0 100 51.0 0 100 51.1 95 5 55.0 95 5 Flow Rate 20 mL/min Detection Wavelength 210 nm or 230 nm Injection Volume 5 mL Run Time 55 min 2.6 Cell culture The murine macrophage cell line RAW 264.7 was cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. Cells were maintained in 100 mm culture dishes in a 5% CO₂ incubator at 37°C. Upon reaching confluence, cells were subcultured using a cell scraper to maintain cell proliferation. A human-derived fibroblast cell line was cultured in Iscove’s Modified Dulbecco’s medium (IMDM) supplemented with 10% FBS and 1% penicillin–streptomycin in 150 mm culture dishes under the same incubation conditions. Upon confluence, the cells were passaged using mild trypsinization. 2.7 Cell viability assay in RAW 264.7 and CCD-986sk cells In viable cells, mitochondrial dehydrogenases reduce water-soluble tetrazolium salt (WST-1) to form a chromogenic formazan product, which is quantified to assess cell viability. RAW 264.7 cells were seeded at a density of 1 × 10⁴ cells per well in 96-well culture plates and incubated for 24 hours in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin. After incubation, the medium was replaced with fresh DMEM containing the test samples at various concentrations, followed by a 24-hour treatment period. At the end of the treatment, the EZ-CYTOX reagent (DoGenBio, Korea) was added to each well, and the plates were incubated according to the manufacturer's instructions. Cell viability was determined by measuring the absorbance at 450 nm using a microplate reader. The percentage of viable cells was calculated using Eq. (1). CCD-986sk human dermal fibroblasts were seeded at a density of 3 × 10³ cells per well in 96-well plates and cultured for 24 hours in IMDM supplemented with 10% FBS and 1% penicillin–streptomycin. The medium was then replaced with serum-free IMDM (1% penicillin–streptomycin), and the cells were serum-starved for at least 6 hours. After starvation, the cells were harvested. The test compounds were dissolved in distilled water to prepare stock solutions and then diluted to the final concentrations using serum-free IMDM (1% penicillin–streptomycin). The culture medium was carefully aspirated and replaced with sample-containing medium. The cells were then incubated under standard conditions for 48 hours. After incubation, each well was treated with serum-free IMDM containing 10% EZ-CYTOX, and incubated for 2 hours. Cell viability was quantified by measuring the absorbance at 450 nm using a microplate reader and calculated according to Eq. (1). 2.8 DPPH radical-scavenging assay To assess the antioxidant activity of the test samples, serial dilutions of the synthesized peptides were prepared, using ascorbic acid as a positive control. Each reaction mixture consisted of 12.5 µL of the sample solution, 50 µL of ethanol, and 62.5 µL of 0.1 mM DPPH solution. The mixtures were incubated for 30 min at 4°C in the dark. After the reaction was performed, the absorbance was measured at 520 nm using a multimode microplate reader. Free radical-scavenging activity (%) was calculated using Eq. (2). a: Absorbance after reaction of stock solution b: Absorbance after reaction of sample solution a', b': Absorbance measured by substituting buffer for 0.1 mM DPPH 2.9 Tyrosinase inhibition assay To assess tyrosinase inhibitory activity, serial dilutions of the synthesized peptide and the positive control β-arbutin were prepared in duplicate. In a 96-well plate, 10 µL of sample solution was mixed with 110 µL of 0.1 M sodium phosphate buffer (pH 6.5), followed by the addition of 10 µL of mushroom tyrosinase solution (1,500 U/mL) and 20 µL of 1.5 mM L-tyrosine. The reaction mixture was then incubated at 37°C for 10 min. Absorbance was measured at an appropriate wavelength ( 475 nm) using a microplate reader, and the percentage of tyrosinase inhibition was calculated using Eq. (3). a : Absorbance after reaction of stock solution b : Absorbance after reaction of sample solution a', b' : Absorbance measured by substituting buffer for mushroom tyrosinase 3. Results and Discussion The cyclic peptide CYGSR was synthesized by cyclizing a linear pentapeptide composed of Cys–Tyr–Gly–Ser–Arg via intramolecular peptide bond formation. The resulting cyclic pentapeptide was then purified using HPLC, and its molecular weight was confirmed to be 567.0 Da by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), confirming successful synthesis. 3.1 Cell viability Mitochondrial dehydrogenases in viable cells reduce water-soluble tetrazolium salt (WST-1) to form the chromogenic compound formazan, which is used to assess cell viability. The viability of both RAW 264.7 macrophages and CCD-986sk fibroblasts was evaluated using this assay. CR5 exhibited no cytotoxicity at concentrations below 400 µg/mL in either cell line, with the cells maintaining viability above 90% (Fig. 1 a). 3.2 Antioxidant activity assay The antioxidant activity of the synthesized CR5 was evaluated using the DPPH radical scavenging assay, with ascorbic acid serving as the positive control (Figure S2b). CR5 exhibited a concentration-dependent scavenging effect in the range of 3.13 to 200 µg/mL. At concentrations above 25 µg/mL, CR5 showed more than 50% scavenging activity. Specifically, at 200 µg/mL, a scavenging rate of 83.18% was observed, indicating that CR5 exerted a noticeable antioxidant effect (Fig. 1 b). 3.3 Whitening activity assay The whitening activity of the synthesized pentapeptide CR5 was assessed based on its tyrosinase inhibitory effect. CR5 exhibited concentration-dependent inhibition over the range of 12.5 to 800 µg/mL. At the highest concentration tested (800 µg/mL), CR5 demonstrated a strong whitening effect with an inhibition rate of 97.79%, and the IC₅₀ was determined to be 130.77 µg/mL (Fig. 1 c). For comparison, β-arbutin—a widely used tyrosinase inhibitor—was also tested in the range of 31.25 to 500 µg/mL and showed an IC₅₀ of 160.12 µg/mL (Figure S2b). These results indicate that CR5 exhibited stronger tyrosinase inhibition than β-arbutin at comparable concentrations. 4. Conclusion In this study, the pentapeptide CYGSR, which exhibits antioxidant and whitening activities, was synthesized using SPPS. To enhance its stability and extend its biological half-life, CYGSR was cyclized to form CR5. The molecular weight of CR5 was confirmed by and MALDI-TOF MS, with a consistent [M + H] + peak at 567.0, corresponding to the theoretical mass and also confirming the successful synthesis. Evaluation of bioactivity revealed that CR5 possesses excellent safety and functional potential. Cell viability assays demonstrated over 90% viability of RAW 264.7 and CCD-986sk cells at CR5 concentrations up to 400 µg/mL, indicating low cytotoxicity. CR5 also exhibited strong antioxidant activity, achieving 83.18% DPPH radical scavenging at 200 µg/mL, and significant whitening activity, with a 97.79% tyrosinase inhibition rate at 800 µg/mL—outperforming the positive control, β-arbutin. Collectively, these results support CR5 as a promising cyclic peptide candidate with high biocompatibility and dual bioactivity. With further investigations into additional dermatological functions, such as anti-wrinkle or anti-inflammatory properties, CR5 holds strong potential as a multifunctional bioactive ingredient for pharmaceutical and cosmetic applications. Statements and Declarations Funding This research was supported by the Regional Innovation System & Education (RISE) initiative, funded by the Ministry of Education and administered by the National Research Foundation of Korea (NRF) Conflict of Interest The authors declare that they have no conflicts of interes References Akhigbe R, Ajayi A (2021) The impact of reactive oxygen species in the development of cardiometabolic disorders: A review. Lipids Health Dis 20:23. https://doi.org/10.1186/s12944-021-01435-7 Amblard M, Fehrentz JA, Martinez J, Subra G (2006) Methods and protocols of modern solid phase peptide synthesis. 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Int J Pept Res Ther 20:545–551. https://doi.org/10.1007/s10989-014-9421-0 Yingchutrakul Y, Krobthong S, Choowongkomon K, Papan P, Samutrtai P, Mahatnirunkul T, Chomtong T, Srimongkolpithak N, Jaroenchuensiri T, Aonbangkhen C (2022) Discovery of a multifunctional octapeptide from Lingzhi with antioxidant and tyrosinase inhibitory activity. Pharmaceuticals (Basel) 15:684. https://doi.org/10.3390/ph15060684 Zhang H, Chen S (2022) Cyclic peptide drugs approved in the last two decades (2001–2021). RSC Chem Biol 3:18–31. https://doi.org/10.1039/D1CB00154J Zhuang Y, Lin H, Gao J, Cao W, Chen Z, Zheng H, Huang H (2025) A novel tyrosinase inhibitory peptide derived from Sipunculus nudus suppresses melanogenesis in B16F10 cells by down‑regulating TRP‑1/TRP‑2 expression. J Funct Foods 129:106872. https://doi.org/10.1016/j.jff.2025.106872 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7205295","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":491788388,"identity":"f557cdda-109a-41b7-aa3b-e799f0d1307e","order_by":0,"name":"Bo-Mi Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYBACNvb2A8Y/KmwYJCD8BMJa+HjOJBQznEkjQYucRILBZ8aWwyRoYWNISNxc2HA+ceaMBMYPPxjS8onQcvCw8cwdtxNnSyQwS/Yw5Fg2ENTC2JBmwHvmdu48iQQGaQaGCgPCtjAzmP/gbTsH0sL8mzgtbAwGxrxtB3KBDmMD2pJDhBYengTDGWeS62f2PGyz7DFII6xFfv7zAwYfKuyMJY4nH77xoyKZsBYkwNjAwECShlEwCkbBKBgFOAEASWs5f96XL4QAAAAASUVORK5CYII=","orcid":"","institution":"Wonkwang University","correspondingAuthor":true,"prefix":"","firstName":"Bo-Mi","middleName":"","lastName":"Kim","suffix":""},{"id":491788389,"identity":"d4312d6d-a60e-4295-ba28-dccb72a6c4c7","order_by":1,"name":"Ga-Hyun Kim","email":"","orcid":"","institution":"Wonkwang University","correspondingAuthor":false,"prefix":"","firstName":"Ga-Hyun","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2025-07-24 11:53:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7205295/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7205295/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87819686,"identity":"bb792db8-83bd-4b3f-a30f-8da25ff4c382","added_by":"auto","created_at":"2025-07-29 10:42:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":17257,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Structure of the synthesized cyclic peptide CR5 (b) MALDI-TOF mass spectrometry spectrum of the synthesized cyclic peptidesCR5\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7205295/v1/3e616e9bc013f7ab39dd09a3.png"},{"id":87819687,"identity":"0173fc1c-7abe-472a-934d-1737e1175f17","added_by":"auto","created_at":"2025-07-29 10:42:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":22619,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Effect of CR5 on viability of RAW 264.7 and CCD-986sk cells, estimated using the WST method (b) Antioxidant effect of CR5 by DPPH method (IC₅₀ of 16.52 μg/mL) (c) Tyrosinase inhibitory activity of CR5\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7205295/v1/f8d34c4ababc05b91370696f.png"},{"id":102164507,"identity":"9618fb1a-536b-495c-ba52-98a27c9b9723","added_by":"auto","created_at":"2026-02-09 01:39:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":593801,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7205295/v1/94307b8f-fe45-4449-bab6-0c38694687fc.pdf"},{"id":87819688,"identity":"03797820-1767-4e05-a9ff-5ce707ffbe3a","added_by":"auto","created_at":"2025-07-29 10:42:18","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":352404,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7205295/v1/2bbf0a47139613e074ff0583.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synthesis of cyclic peptides with antioxidant and skin-lightening properties","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe rapid advancement of medical technology has significantly extended human life expectancy, leading to an increasing societal interest in healthy aging in general and skin aging in particular (Surber et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Nagae et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Age-related hyperpigmentation and oxidative stress affect both aesthetic appearance and overall health, thereby stimulating growing personal concern and active scientific research on their mitigation (Lee et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe major mechanisms underlying skin aging include collagen degradation, melanin overproduction, inflammation, and accumulation of reactive oxygen species (ROS). Development of biomaterials capable of simultaneously modulating these multifactorial changes is a critical challenge in cosmetic and therapeutic science (Dan et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eResearch focusing on functional peptides for skin health improvement has gained momentum in the life sciences amid the global trend toward population aging. A promising approach involves the application of bioactive peptides that exhibit antioxidant and skin-lightening activities (Shen et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Thaha et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Specific peptides exert their effects by mitigating ROS accumulation and inhibiting melanin synthesis through mechanisms such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging and tyrosinase inhibition (Yingchutrakul et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kriangkrai et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Against this background, functional peptide technology has found active applications not only in the biotechnology and pharmaceutical fields but also, more recently, in the cosmetics industry, and is increasingly recognized as a next-generation platform for promoting skin health and preventing aging (Pintea et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In addition to their role as simple auxiliary components, peptides are being reevaluated for their value as core functional agents with intrinsic bioactivity, and their scope of application continues to steadily expand (Hariri et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhuang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Meng et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Despite their advantages, linear peptides undergo rapid degradation in vivo, leading to a short half-life and poor bioavailability. To overcome these limitations, the development of novel cyclic peptides has become increasingly important (Tapeinou et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Cyclic peptides have a stable three-dimensional conformation owing to their cyclic structure, which confers several advantages, including enhanced selectivity for specific receptor-binding sites, increased resistance to proteolytic degradation, and improved structural stability (Fagundez et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhang and Chen \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ji et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe structural characteristics of cyclic peptides are strongly influenced by their ring size. When compared with their larger counterparts, smaller cyclic peptides typically exhibit enhanced conformational stability and superior drug-like properties. (Horton et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Thakkar et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Sarojini et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In fact, cyclic peptides such as cyclosporin A, gramicidin S, and oxytocin have been utilized to treat several diseases (Tedesco and Haragsim \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Schulze \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Guan et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, synthesizing cyclic peptides is challenging because of the demanding reaction conditions, which can result in cyclization failure, side reactions, and dimer formation (Bechtler and Lamers \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Fang et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, their production costs are higher than those of linear peptides, and the difficulty of scaling-up limits their economic feasibility for commercialization (Thapa et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The synthesis methods for cyclization include microbial and chemical cyclizations. Microbial approaches include ribosomally synthesized and post-translationally modified peptide (RiPP) and non-ribosomal peptide synthetase (NRPS) systems. However, because these methods rely solely on enzymatic structures or predefined sequences, they offer lower design flexibility than chemical cyclization, and system construction remains technically challenging (Han and Won \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Several chemical cyclization strategies have been developed, including head-to-tail, side-chain-to-side chain, and O\u0026ndash;S acyl shift methods. However, short cyclic peptides optimized for low-cost mass production need to be developed, as current strategies offer limited efficiency in this regard (Hayes et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Buchanan et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the present study, we synthesized a short sequence of linear peptides with antioxidant and skin-lightening properties using solid-phase peptide synthesis (SPPS). Subsequently, a cyclization strategy based on liquid-phase peptide synthesis (LPPS) was employed to shorten the production time and enable high-purity mass production, thereby enhancing commercial applicability and improving the bioactivity of pharmaceutical and cosmetic materials.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Materials\u003c/h2\u003e\n \u003cp\u003eAll reagents used in this study were purchased from GL Biochem (China), Sigma-Aldrich (USA), or TCI (Japan).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Synthesis of Cys-Tyr-Gly-Ser-Arg (CYGSR) using Fmoc-chemistry\u003c/h2\u003e\n \u003cp\u003eThe CYGSR peptide used in this study was synthesized using SPPS. 2-Chlorotrityl chloride resin (0.5 g, 1.0 mmol/g) was stirred in 5.8 mL of dichloromethane (DCM) for 30 min. After swelling, the solvent was removed under vacuum, and the resin was washed twice with DCM. For the sequential coupling of amino acids, Fmoc-Arg(Pbf)-OH (2.0 eq, 0.6 g) was added to the reaction vessel along with 1-hydroxybenzotriazole (HOBt, 1.4 eq, 0.2 g), N,N-diisopropylethylamine (DIPEA, 5.0 eq, 0.4 mL), N,N\u0026prime;-diisopropylcarbodiimide (DIC, 3.0 eq, 0.2 mL), and N,N-dimethylformamide (DMF, 4.0 mL), and the mixture was stirred for 4 hours. Completion of the coupling reaction was then confirmed using the Kaiser test (Kaiser et al. \u003cspan class=\"CitationRef\"\u003e1970\u003c/span\u003e). Double coupling or capping was performed as necessary, based on the test results (Amblard et al. \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e). Amino acid coupling was performed following Fmoc deprotection using 20% piperidine in DMF (Fields and Noble \u003cspan class=\"CitationRef\"\u003e1990\u003c/span\u003e). The second amino acid, Fmoc-Ser(tBu), OH (2.0 eq, 0.4 g), the third, Fmoc-Gly-OH (2 eq, 0.3 g), the fourth, Fmoc-Tyr(tBu)-OH (2 eq, 0.5 g), and the fifth, Fmoc-Cys(Trt)-OH (2.0 eq, 0.6 g), were sequentially coupled using the same procedure as described for the initial amino acid.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Cleavage of CYGSR\u003c/h2\u003e\n \u003cp\u003eTo cleave the synthesized CYGSR from the resin, 12.0 mL of a cleavage cocktail consisting of trifluoroacetic acid (TFA), triisopropylsilane, and H\u003csub\u003e2\u003c/sub\u003eO (95:2.5:2.5, v/v/v) was added to the reaction vessel and then stirred for 1 hour. The mixture was filtered, and the filtrate was then precipitated with 66.0 mL cold isopropyl ether to yield a white solid.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Cyclization of CYGSR\u003c/h2\u003e\n \u003cp\u003eThe linear peptide, CYGSR, was cyclized via the formation of an intramolecular amide bond between the N-terminal amine and the C-terminal carboxylic acid. Lyophilized CYGSR (1.0 mmol) was dissolved in DMF to a final concentration of 1 mM. HOBt (2.5 eq, 0.3 g), N-ethyl-N\u0026prime;-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC\u0026middot;HCl, 2.5 eq, 0.5 g), and DIPEA (5 eq, 0.9 mL) were sequentially added while stirring at 0\u0026deg;C. The reaction mixture was stirred under a nitrogen atmosphere at room temperature, and samples were collected at 4-hour intervals for monitoring by high-performance liquid chromatography (HPLC). After 12 hours, the reaction was quenched by adding 5 mL of cold distilled water. The product was extracted three times with 5.0 mL ethyl acetate, and the combined organic phases were dried over anhydrous sodium sulfate (Na₂SO₄) and concentrated under reduced pressure. The crude product was purified using reversed-phase high-performance liquid chromatography (RP-HPLC).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 Isolation and purification of CYGSR\u003c/h2\u003e\n \u003cp\u003ePurification was performed using a preparative HPLC system (Waters 2545 Quaternary Gradient Module with 2998 Photodiode Array Detector, USA) after dissolving 0.05 g of the crude peptide in 5.0 mL of 0.1% TFA in water. A YMC-Pack ODS-A-HG C18 reversed-phase column (250 \u0026times; 20.0 mm I.D., S-10 \u0026micro;m, 30 nm; YMC, Japan) was used. The mobile phases consisted of solvent A (0.1% TFA in water) and solvent B (0.1% TFA in acetonitrile), and separation was performed under the gradient conditions listed in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eHPLC conditions\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eColumn\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eYMC-Pack ODS-A-HG (250 \u0026times; 20.0 mm I.D., 10 \u0026micro;m, 30 nm)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMobile Phase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eSolvent A: H\u003csub\u003e2\u003c/sub\u003eO0.1% TFA\u003c/p\u003e\n \u003cp\u003eSolvent B: Acetonitrile\u0026thinsp;+\u0026thinsp;0.1% TFA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTime (min)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSolvent A (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSolvent B (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e51.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e51.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFlow Rate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e20 mL/min\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDetection Wavelength\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e210 nm or 230 nm\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInjection Volume\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e5 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRun Time\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e55 min\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6 Cell culture\u003c/h2\u003e\n \u003cp\u003eThe murine macrophage cell line RAW 264.7 was cultured in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin\u0026ndash;streptomycin. Cells were maintained in 100 mm culture dishes in a 5% CO₂ incubator at 37\u0026deg;C. Upon reaching confluence, cells were subcultured using a cell scraper to maintain cell proliferation. A human-derived fibroblast cell line was cultured in Iscove\u0026rsquo;s Modified Dulbecco\u0026rsquo;s medium (IMDM) supplemented with 10% FBS and 1% penicillin\u0026ndash;streptomycin in 150 mm culture dishes under the same incubation conditions. Upon confluence, the cells were passaged using mild trypsinization.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7 Cell viability assay in RAW 264.7 and CCD-986sk cells\u003c/h2\u003e\n \u003cp\u003eIn viable cells, mitochondrial dehydrogenases reduce water-soluble tetrazolium salt (WST-1) to form a chromogenic formazan product, which is quantified to assess cell viability. RAW 264.7 cells were seeded at a density of 1 \u0026times; 10⁴ cells per well in 96-well culture plates and incubated for 24 hours in DMEM supplemented with 10% FBS and 1% penicillin\u0026ndash;streptomycin. After incubation, the medium was replaced with fresh DMEM containing the test samples at various concentrations, followed by a 24-hour treatment period. At the end of the treatment, the EZ-CYTOX reagent (DoGenBio, Korea) was added to each well, and the plates were incubated according to the manufacturer\u0026apos;s instructions. Cell viability was determined by measuring the absorbance at 450 nm using a microplate reader. The percentage of viable cells was calculated using Eq.\u0026nbsp;(1).\u003c/p\u003e\n \u003cp\u003eCCD-986sk human dermal fibroblasts were seeded at a density of 3 \u0026times; 10\u0026sup3; cells per well in 96-well plates and cultured for 24 hours in IMDM supplemented with 10% FBS and 1% penicillin\u0026ndash;streptomycin. The medium was then replaced with serum-free IMDM (1% penicillin\u0026ndash;streptomycin), and the cells were serum-starved for at least 6 hours. After starvation, the cells were harvested. The test compounds were dissolved in distilled water to prepare stock solutions and then diluted to the final concentrations using serum-free IMDM (1% penicillin\u0026ndash;streptomycin). The culture medium was carefully aspirated and replaced with sample-containing medium. The cells were then incubated under standard conditions for 48 hours. After incubation, each well was treated with serum-free IMDM containing 10% EZ-CYTOX, and incubated for 2 hours. Cell viability was quantified by measuring the absorbance at 450 nm using a microplate reader and calculated according to Eq.\u0026nbsp;(1).\u003c/p\u003e\n \u003cdiv id=\"Equa\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8 DPPH radical-scavenging assay\u003c/h2\u003e\n \u003cp\u003eTo assess the antioxidant activity of the test samples, serial dilutions of the synthesized peptides were prepared, using ascorbic acid as a positive control. Each reaction mixture consisted of 12.5 \u0026micro;L of the sample solution, 50 \u0026micro;L of ethanol, and 62.5 \u0026micro;L of 0.1 mM DPPH solution. The mixtures were incubated for 30 min at 4\u0026deg;C in the dark. After the reaction was performed, the absorbance was measured at 520 nm using a multimode microplate reader. Free radical-scavenging activity (%) was calculated using Eq.\u0026nbsp;(2).\u003c/p\u003e\n \u003cdiv id=\"Equb\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\u003cimg 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\"\u003e\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003ea: Absorbance after reaction of stock solution\u003c/p\u003e\n \u003cp\u003eb: Absorbance after reaction of sample solution\u003c/p\u003e\n \u003cp\u003ea\u0026apos;, b\u0026apos;: Absorbance measured by substituting buffer for 0.1 mM DPPH\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9 Tyrosinase inhibition assay\u003c/h2\u003e\n \u003cp\u003eTo assess tyrosinase inhibitory activity, serial dilutions of the synthesized peptide and the positive control \u0026beta;-arbutin were prepared in duplicate. In a 96-well plate, 10 \u0026micro;L of sample solution was mixed with 110 \u0026micro;L of 0.1 M sodium phosphate buffer (pH 6.5), followed by the addition of 10 \u0026micro;L of mushroom tyrosinase solution (1,500 U/mL) and 20 \u0026micro;L of 1.5 mM L-tyrosine. The reaction mixture was then incubated at 37\u0026deg;C for 10 min. Absorbance was measured at an appropriate wavelength ( 475 nm) using a microplate reader, and the percentage of tyrosinase inhibition was calculated using Eq.\u0026nbsp;(3).\u003c/p\u003e\n \u003cdiv id=\"Equc\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003ea : Absorbance after reaction of stock solution\u003c/p\u003e\n \u003cp\u003eb : Absorbance after reaction of sample solution\u003c/p\u003e\n \u003cp\u003ea\u0026apos;, b\u0026apos; : Absorbance measured by substituting buffer for mushroom tyrosinase\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cp\u003eThe cyclic peptide CYGSR was synthesized by cyclizing a linear pentapeptide composed of Cys\u0026ndash;Tyr\u0026ndash;Gly\u0026ndash;Ser\u0026ndash;Arg via intramolecular peptide bond formation. The resulting cyclic pentapeptide was then purified using HPLC, and its molecular weight was confirmed to be 567.0 Da by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), confirming successful synthesis.\u003c/p\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cem\u003e3.1 Cell viability\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eMitochondrial dehydrogenases in viable cells reduce water-soluble tetrazolium salt (WST-1) to form the chromogenic compound formazan, which is used to assess cell viability. The viability of both RAW 264.7 macrophages and CCD-986sk fibroblasts was evaluated using this assay. CR5 exhibited no cytotoxicity at concentrations below 400 \u0026micro;g/mL in either cell line, with the cells maintaining viability above 90% (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Antioxidant activity assay\u003c/h2\u003e\n \u003cp\u003eThe antioxidant activity of the synthesized CR5 was evaluated using the DPPH radical scavenging assay, with ascorbic acid serving as the positive control (Figure S2b). CR5 exhibited a concentration-dependent scavenging effect in the range of 3.13 to 200 \u0026micro;g/mL. At concentrations above 25 \u0026micro;g/mL, CR5 showed more than 50% scavenging activity. Specifically, at 200 \u0026micro;g/mL, a scavenging rate of 83.18% was observed, indicating that CR5 exerted a noticeable antioxidant effect (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Whitening activity assay\u003c/h2\u003e\n \u003cp\u003eThe whitening activity of the synthesized pentapeptide CR5 was assessed based on its tyrosinase inhibitory effect. CR5 exhibited concentration-dependent inhibition over the range of 12.5 to 800 \u0026micro;g/mL. At the highest concentration tested (800 \u0026micro;g/mL), CR5 demonstrated a strong whitening effect with an inhibition rate of 97.79%, and the IC₅₀ was determined to be 130.77 \u0026micro;g/mL (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e\n \u003cp\u003eFor comparison, \u0026beta;-arbutin\u0026mdash;a widely used tyrosinase inhibitor\u0026mdash;was also tested in the range of 31.25 to 500 \u0026micro;g/mL and showed an IC₅₀ of 160.12 \u0026micro;g/mL (Figure S2b). These results indicate that CR5 exhibited stronger tyrosinase inhibition than \u0026beta;-arbutin at comparable concentrations.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this study, the pentapeptide CYGSR, which exhibits antioxidant and whitening activities, was synthesized using SPPS. To enhance its stability and extend its biological half-life, CYGSR was cyclized to form CR5. The molecular weight of CR5 was confirmed by and MALDI-TOF MS, with a consistent [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e peak at 567.0, corresponding to the theoretical mass and also confirming the successful synthesis.\u003c/p\u003e\u003cp\u003eEvaluation of bioactivity revealed that CR5 possesses excellent safety and functional potential. Cell viability assays demonstrated over 90% viability of RAW 264.7 and CCD-986sk cells at CR5 concentrations up to 400 \u0026micro;g/mL, indicating low cytotoxicity. CR5 also exhibited strong antioxidant activity, achieving 83.18% DPPH radical scavenging at 200 \u0026micro;g/mL, and significant whitening activity, with a 97.79% tyrosinase inhibition rate at 800 \u0026micro;g/mL\u0026mdash;outperforming the positive control, β-arbutin.\u003c/p\u003e\u003cp\u003eCollectively, these results support CR5 as a promising cyclic peptide candidate with high biocompatibility and dual bioactivity. With further investigations into additional dermatological functions, such as anti-wrinkle or anti-inflammatory properties, CR5 holds strong potential as a multifunctional bioactive ingredient for pharmaceutical and cosmetic applications.\u003c/p\u003e"},{"header":"Statements and Declarations ","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This research was supported by the Regional Innovation System \u0026amp; Education (RISE) initiative, funded by the Ministry of Education and administered by the National Research Foundation of Korea (NRF)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u0026nbsp; The authors declare that they have no conflicts of interes\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAkhigbe R, Ajayi A (2021) The impact of reactive oxygen species in the development of cardiometabolic disorders: A review. 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Mar Drugs\u003cem\u003e \u003c/em\u003e22:374. https://doi.org/10.3390/md22080374\u003c/li\u003e\n\u003cli\u003eMeng F, Wang Y, Liu Y, Hao Z, Li F, Wang Y, Ding Y, Li Y, Jiang Y (2025) Naturally occurring new peptides from velvet antler of Cervus nippon Temminck: Structural characterization and organic synthesis, antioxidant and anti‑melanogenic effects using in vitro cell models and in vivo zebrafish models. Chem Biodivers:e00288. https://doi.org/10.1002/cbdv.202500288\u003c/li\u003e\n\u003cli\u003eNagae M, Mitsutake T, Sakamoto M (2023) Impact of skin care on body image of aging people: A quasi-randomized pilot trial. Heliyon 9:e13230. https://doi.org/10.1016/j.heliyon.2023.e13230/\u003c/li\u003e\n\u003cli\u003ePintea A, Manea A, Pintea C, Vlad R-A, B\u0026icirc;rsan M, Antonoaea P, R\u0026eacute;dai EM, Ciurba A (2025) Peptides: Emerging candidates for the prevention and treatment of skin senescence: A review. 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RSC Chem Biol\u003cem\u003e \u003c/em\u003e3:18\u0026ndash;31. https://doi.org/10.1039/D1CB00154J\u003c/li\u003e\n\u003cli\u003eZhuang Y, Lin H, Gao J, Cao W, Chen Z, Zheng H, Huang H (2025) A novel tyrosinase inhibitory peptide derived from Sipunculus nudus suppresses melanogenesis in B16F10 cells by down‑regulating TRP‑1/TRP‑2 expression. J Funct Foods 129:106872. https://doi.org/10.1016/j.jff.2025.106872\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cyclic peptides, Antioxidant activity, Skin-lightening, Tyrosinase inhibition, Cosmeceuticals","lastPublishedDoi":"10.21203/rs.3.rs-7205295/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7205295/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e\u003cp\u003eCyclic peptides have emerged as a compelling class of therapeutic and bioactive compounds owing to their diverse and potent biological activities. Nevertheless, their commercialization has been hindered by considerable challenges in synthesis and elevated cost of production.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eIn this study, we employed a scalable and cost-effective chemical synthesis strategy to rapidly design and synthesize a linear peptide sequence with antioxidant and skin-lightening properties using solid-phase peptide synthesis (SPPS). Subsequently, the cyclic peptides Cys-Tyr-Gly-Ser-Arg (CR5) was developed using liquid-phase peptide synthesis (LPPS).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe cytotoxicity of CR5 was evaluated via the WST-1 assay, revealing over 90% cell viability at concentrations up to 400 \u0026micro;g/mL, indicative of high cellular biocompatibility. Moreover, CR5 demonstrated more than 50% 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity with an IC₅₀ of 16.62 \u0026micro;g/mL, highlighting its notable antioxidant efficacy. In addition, CR5 showed 97.79% tyrosinase inhibition at 800 \u0026micro;g/mL, with an IC₅₀ of 30.77 \u0026micro;g/mL, confirming its strong skin-lightening potential.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThe cyclic peptide CR5 developed in this study has high production scalability and dual functional properties in the form of antioxidant and skin-lightening activities. It is a promising candidate for future applications in the pharmaceutical and cosmetic industries.\u003c/p\u003e","manuscriptTitle":"Synthesis of cyclic peptides with antioxidant and skin-lightening properties","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-29 10:42:13","doi":"10.21203/rs.3.rs-7205295/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bec76c19-c4c7-46ff-9c02-51e5c0de7f23","owner":[],"postedDate":"July 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-09T01:39:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-29 10:42:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7205295","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7205295","identity":"rs-7205295","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}