Facile and Selective Determination of Dipeptides Using 3-Methylcatechol as a Novel Fluorogenic Reagent

  • Hasina Yasmin
  • Mohammed Shafikur Rahman
  • Takayuki Shibata


Selective determination of peptides in mixtures or biological samples requires specific techniques for analysis. Herein, we report 3-methylcatechol (3-MC) as a novel fluorogenic reagent for the selective determination of dipeptides by a simple fluorescence (FL) derivatization reaction. After extensive screening of 31 different catechol analogues, 3-MC was found to generate FL with peptides. The reaction was performed at 100 °C for 10 min in the presence of borate buffer (pH 7) and sodium periodate. The resulting FL intensities were measured by spectrofluorometer at excitation wavelengths of 380 nm and emission wavelengths of 500 nm. Different reaction conditions such as concentration of sodium periodate, reaction time and pH of the borate buffer were studied to determine the optimum reaction conditions. Linearity was obtained between FL intensity and peptide concentrations from 10 to 160 µM with a lower detection limit of 10 µM (S/N = 3). Dipeptides containing Ala, Phe, Leu and Val at the N-termini generated significant FL in comparison to the reagent blank (**p < 0.005, ***p < 0.0005). The reaction is simple, rapid, selective and sensitive which can be applied for the determination of the dipeptides as biomarkers or to determine enzyme activity.


Peptides 3-Methylcatechol Fluorescence Selectivity 



This work was partially supported by Grants-in-aid for Scientific Research form the Ministry of Education, Culture, Sports and Technology of Japan.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Research Involving Human and Animal Participants

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

10989_2018_9703_MOESM1_ESM.pptx (37 kb)
Supplementary Figure 1 Concentration range of AG used to determine the limit of detection (LOD) and the limit of linearity (LOL). Error bars represents standard uncertainty, n = 3 (PPTX 36 KB)


  1. Baggerman G, Verleyen P, Clynen E, Huybrechts J, De Loof A et al (2004) Pept J Chromatogr B 803:3–16CrossRefGoogle Scholar
  2. Bajpai K, Singh VK, Sharan R, Yadav VS, Haq W et al (1998) Immunomodulating activity of analogs of noninflammatory fragment 163–171 of human interleukin-1β. Immunopharmacol 38(3):237–245CrossRefGoogle Scholar
  3. Chikuma T, Ogura Y, Kasamatsu M, Taguchi K, Mitsui K et al (1999) High-performance liquid chromatographic-fluorometric assay for cathepsin A (lysosomal protective protein) activity. J Chromatogr B 728(1):59–65CrossRefGoogle Scholar
  4. Dallas DC, Underwood MA, Zivkovic AM, German JB (2012) Digestion of protein in premature and term infants. J Nutr Disord Ther 2:1–9CrossRefGoogle Scholar
  5. Goldman A, Garza C, Schanler R, Goldblum R (1990) Molecular forms of lactoferrin in stool and urine from infants fed human milk. Pediatr Res 27:252–255CrossRefPubMedGoogle Scholar
  6. Gozdowska M, Kulczykowska E (2004) Determination of arginine–vasotocin and isotocin in fish plasma with solid-phase extraction and fluorescence derivatization followed by high-performance liquid chromatography. J Chromatogr B 807(2):229–233CrossRefGoogle Scholar
  7. Ishida J, Kai M, Ohkura Y (1986) High-performance liquid chromatography of tyrosine containing peptides by pre-column derivatization involving formylation followed by fluorescence reaction with 1,2-diamino-4,5-dimethoxybenzene [4,5-dimethoxy-o-phenylenediamine]. J Chromatogr 356:171–177CrossRefPubMedGoogle Scholar
  8. Kabashima T, Yu Z, Chang T, Nakagawa Y, Okumura K et al (2008) Selective fluorescence reaction for peptides and chromatographic analysis. Peptides 29(3):356–363CrossRefPubMedGoogle Scholar
  9. Kai M, Ohkura Y (1986) Selective determination of N-terminal tyrosine containing peptides by a novel fluorescence reaction with borate, hydroxylamine and cobalt(II). Anal Chim Acta 182:177–183CrossRefGoogle Scholar
  10. Kai M, Miyuzaki T, Sakomoto Y, Ohkura Y (1985) Use of benzoin as pre-column fluorescence derivatization reagent for the high-performance liquid chromatography of angiotensins. J Chromatogr 322:473–477CrossRefPubMedGoogle Scholar
  11. Khan RS, Yu C, Kastin AJ, He Y, Ehrensing RH, Hsuchou H, Stone KP, Pan W (2010) Brain activation by peptide Pro-Leu-Gly-NH2 (MIF-1). Int J. Google Scholar
  12. Kobb KA, Novotny MV (1992) Selective determination of arginine-containing and tyrosine-containing peptides using capillary electrophoresis and laser-induced fluorescence detection. Anal Biochem 200(1):149–155CrossRefGoogle Scholar
  13. Kojima E, Ohba Y, Kai M, Ohkura Y (1993) Phenylglyoxal and glyoxal as fluorogenic reagents selective for N-terminal tryptophan-containing peptides. Anal Chim Acta 280:157–162CrossRefGoogle Scholar
  14. Ling BL, Dewaele C, Baeyens WRG (1991) Micro liquid chromatography with fluorescence detection of thiols and disulfides. J Chromatogr 553:433–439CrossRefGoogle Scholar
  15. Morihara K, Tsuzuki H (1970) Thermolysin: kinetic study with oligopeptides. Eur J Biochem 15:374–380CrossRefPubMedGoogle Scholar
  16. Myers RD (1994) Neuroactive peptides: unique phases in research on mammalian brain over three decades. Peptides 15(2):367–381CrossRefPubMedGoogle Scholar
  17. Nakamura H, Zimmerman CL, Pisano JJ (1979) Analysis of histidine-containing dipeptides, polyamines, and related amino acids by high-performance liquid chromatography: application to guinea pig brain. Anal Biochem 93:423–429CrossRefPubMedGoogle Scholar
  18. Ortega-Velasquez R, Dies-Marques ML, Ruiz-Torres MP, Gonzalez-Rubio M, Rodriguez-Puyol M et al (2003) Arg-Gly-Asp-Ser (RGDS) peptide stimulates transforming growth factor 1 transcription and secretion through integrin activation. FASEB J 17(11):1529–1531CrossRefGoogle Scholar
  19. Puche JE, Castilla-Cortázar I (2012) Human conditions of insulin-like growth factor-I (IGF-I) deficiency. J Transl Med 10:224CrossRefPubMedPubMedCentralGoogle Scholar
  20. Rayan CA, Gregory P, Scheer J, Moura DS (2002) Polypeptide hormones. Plant Cell 14(Suppl):s251–s264CrossRefGoogle Scholar
  21. Roberti ML, Ricottini L, Capponi A, Sclauzero E, Vicenti P et al (2014) Immunomodulating treatment with low dose interleukin-4, interleukin-10 and interleukin-11 in psoriasis vulgaris. J Bio Regul Homeost Agents 28(1):133–139Google Scholar
  22. Sforza S, Cavatorta V, Lambertini F, Galaverna G, Dossena A et al (2012) Cheese peptidomics: a detailed study on the evolution of the oligopeptide fraction in Parmigiano–Reggiano cheese from curd to 24 months of aging. J Dairy Sci 95:3514–3526CrossRefPubMedGoogle Scholar
  23. Theodorescu D, Schiffer E, Bauer HW, Douwes F, Eichhorn F et al (2008) Discovery and validation of urinary biomarkers for prostate cancer. Proteom Clin Appl 2:556–570CrossRefGoogle Scholar
  24. Toyo’oka T, Ishibashi M, Terao T (1994) Sensitive determination of N-terminal prolyl peptides by high-performance liquid chromatography with laser-induced fluorescence detection. J Chromatogr A 661(1):105–112CrossRefPubMedGoogle Scholar
  25. Ueno T, Tanaka M, Matsui T, Matsumoto K (2005) Determination of antihypertensive small peptides, Val-Tyr and Ile-Val-Tyr by fluorometric high-performance liquid chromatography combined with a double heart-cut column-switching technique. Anal Sci 21:997–1000CrossRefPubMedGoogle Scholar
  26. Voelter W, Zech K (1975) High-performance liquid chromatographic analysis of amino acids and peptide-hormone hydrolysates in the picomole range. J Chromatogr A 112:643–649CrossRefGoogle Scholar
  27. Weidman SW, Kaiser ET (1966) The mechanism of the periodate oxidation of aromatic systems. iii. a kinetic study of the periodate oxidation of catechol. J Am Chem Soc 88(24):5820–5827CrossRefGoogle Scholar
  28. Yang J, Cohen Stuart MA, Kamperman M (2014) Jack of all trades: versatile catechol crosslinking mechanisms. Chem Soc Rev 43:8271–8298CrossRefPubMedGoogle Scholar
  29. Yang J, Saggiomo V, Velders AH, Cohen Stuart MA, Kamperman M (2016) Reaction pathways in catechol/primary amine mixtures: a window on crosslinking chemistry. PLoS ONE 11(12):1–17Google Scholar
  30. Yasmin H, Shibata T, Rahman MS, Kabashima T, Kai M (2012) Selective and sensitive determination of peptides using 3,4-dihydroxyphenylacetic acid as a fluorogenic reagent. Anal Chim Acta 721:162–166CrossRefPubMedGoogle Scholar
  31. Yasmin H, Rahman MS, Shibata T, Kabashima T, Kai M (2014) Novel fluorometric method for the selective determination of Pro-Gly and Pro-Gly-Pro. Int J Pept Res Ther 20:441–446CrossRefGoogle Scholar
  32. Yasmin H, Rahman MS, Shibata T, Kabashima T, Kai M (2015) Sensitive and selective determination of peptides, PG and PGP, using a novel fluorogenic reagent 4-chlorobenzene-1,2-diol. Chem Pap 69(4):504–509CrossRefGoogle Scholar
  33. Zurbig P, Dihazi H, Metzger J, Thongboonkerd V, Vlahou A (2011) Urine proteomics in kidney and urogenital diseases: moving towards clinical applications. Proteom Clin Appl 5:256–268CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of PharmacyBRAC UniversityDhakaBangladesh
  2. 2.Department of PharmacyDaffodil International UniversityDhakaBangladesh
  3. 3.Faculty of Pharmaceutical Sciences, Graduate School of Biomedical SciencesNagasaki UniversityNagasakiJapan

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