Food Science and Technology Research
Online ISSN : 1881-3984
Print ISSN : 1344-6606
ISSN-L : 1344-6606
Technical Papers
Production Method for Cyclic Dipeptide Derived from Native Collagen
Fumitaka HayasakaShoko YamamotoYasuo Sakai
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2016 Volume 22 Issue 4 Pages 477-483

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Abstract

In this study, a new method for producing cyclo(-Gly-Pro) using collagen as a raw material was examined. First, collagen was enzymatically hydrolysed and purified to obtain collagen tripeptide (CTP), rich in “Gly-X-Y” tripeptides. After heating this product under atmospheric pressure in an aqueous solution at 95°C for 24 h, purification was achieved by reverse-phase column chromatography. The isolated component was confirmed to be cyclo(-Gly-Pro) through structural analysis by MS and NMR spectroscopies. Purity was determined to be 93.6%, and the recovery rate from CTP was 22%, indicating that much Gly-Pro-Y in CTP contributed to cyclization. The cyclization rate from Gly-Pro-Hyp or Gly-Pro-Ala was much higher than that of Gly-Pro, suggesting that cyclo(-Gly-Pro) was efficiently generated from the Gly-Pro-Y sequence. In summary, this is a simple, practical manufacturing method for producing cyclo(-Gly-Pro) from collagen at low cost with high efficiency.

Introduction

Cyclic dipeptides, also referred to as 2,5-diketopiperazines, are compounds formed by the condensation of two amino acids. They are regarded as secondary metabolites biosynthesized in various organisms such as fungi, bacteria, and mammals (Martins et al. 2007). Cyclic dipeptides have been reported to exhibit physiological activities such as anti-tumour (Arunrattiyakorn et al. 2007, He et al. 1999, van Loevezijn et al. 1999), anti-viral (Watson et al. 2005), and anti-bacterial (Houston et al. 2004, Park et al. 2006) effects. Cyclo(-Gly-Pro), a type of cyclic dipeptide, was reported to demonstrate physiological activities such as anti-amnesic effects (Gudasheva et al. 1996, Samonina et al. 2002) and neuroprotective actions (Guan and Peter 2009).

Conventional methods to produce cyclic dipeptides include their synthesis from amino acids followed by cyclization. In this method, dipeptide esters with protecting groups in an organic solvent such as toluene or xylene (Bull et al. 1998, Woodard 1985) undergo cyclic condensation in a solid phase using a base catalyst (Bray et al. 1994, Fridkin et al. 1965). This manufacturing process is complicated because of the necessity for deprotection and removal of the organic solvent and resin after the synthesis.

Collagen is a natural product that has a repeating sequence of “Gly-X-Y,” where glycine is found in every third residue. X and Y in the sequence may be any amino acid; however, the principal identity of X is proline. The data reported by Ramshaw et al. show that 28.1% of the sequence contains Gly-Pro (Ramshaw et al. 1998). Therefore, collagen can be considered to be a natural material rich in the Gly-Pro sequence, the raw material of cyclo(- Gly-Pro); however, the selective isolation of Gly-Pro from collagen requires many enzymes. Thus, we developed a technique to obtain tripeptide components (collagen tripeptide, CTP) of “Gly-X-Y” by specific hydrolysis of the N terminus of collagen's glycine residues (Sakai et al. 1998). CTP contains large amounts of tripeptides with a Gly-Pro-Y sequence, where Y is an amino acid such as Gly-Pro-Hyp or Gly-Pro-Ala, reflecting the primary sequence found in collagen.

In this study, we investigated a method for efficiently producing cyclo(-Gly-Pro) from collagen using multiple tripeptides with a Gly-Pro-Y sequence as an intermediate.

Materials and Methods

Chemicals    Cyclo(-Gly-Pro) (standard), Gly-Pro-Hyp, Gly-Pro-Ala, and Gly-Pro were purchased from Bachem AG (Bubendorf, Switzerland). High-performance liquid chromatography (HPLC) grade acetonitrile (ACN), trifluoroacetic acid (TFA), and dimethyl sulfoxide-d6 (DMSO-d6) were purchased from Wako Pure Chemical Industries Ltd. (Tokyo, Japan).

Synthesis and purification of cyclo(-Gly-Pro)    Porcine skin collagen was digested using a collagenase-type protease (Protease N, Nagase ChemteX Corporation, Osaka, Japan). The digest was deionised using an ion-exchange resin (DIAION-type SK, Mitsubishi Chemical Corp., Tokyo, Japan), passed through a 0.2-µm filter, and then subjected to ion-exchange column chromatography using Toyopearl DEAE-650 (TOSOH Corp., Tokyo, Japan). The tripeptide fraction (CTP) was isolated by reverse-phase HPLC. The mean molecular weight of CTP was approximately 300, and Gly-Pro-Hyp and Gly-Pro-Ala contents were 34% and 15%, respectively (Yamamoto et al. 2015). Thereafter, 500 mg of lyophilised CTP was dissolved in 5 mL of water and heated under normal pressure at 95°C for 24 h. The sample containing cyclo(-Gly-Pro) was purified by reverse-phase HPLC with a Triart C8 column (YMC Co., Ltd., Kyoto, Japan) at 25°C using a hyperpure water solution; thereafter, it was concentrated and lyophilised. After enzymatic decomposition, cyclization and purification of samples were accomplished by reverse-phase chromatography using the same method employed in the purity test.

Structural analysis    The structure of purified cyclo(-Gly-Pro) was confirmed by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and nuclear magnetic resonance (NMR) spectroscopy. Measurements were performed at the Research and Analytical Centre for Giant Molecules, Graduate School of Science, Tohoku University. For FT-ICR MS, solariX 9.4T (Bruker Daltonics K.K., Kanagawa, Japan) was used with electrospray ionisation (ESI). Water was used as the solvent. NMR analysis was performed using an ECA-800 (JEOL Ltd., Tokyo, Japan) to measure 1H NMR (800 MHz), 13C NMR (200 MHz), HSQC, and heteronuclear multiple-bond correlation (HMBC). DMSO-d6 was used as the solvent. For 1H NMR, the reference was the 1H shift (δ 2.50) of DMSO. The theoretical value of the molecular weight of cyclo(-Gly-Pro) was 154.074 based on a calculation using the following atomic weights: 12C: 12.0000, 1H: 1.0078, 14N: 14.0031, 16O: 15.9949.

Analysis of cyclo(-Gly-Pro) purity    The abovementioned standards were dissolved in water. The final concentrations of standard solutions were 10, 50, and 100 µg/mL. The cyclo(-Gly-Pro) sample was dissolved in water. The samples were injected into HPLC systems (LC-20AD and SPD-M20A, Shimadzu corp., Kyoto, Japan) with a TSKgel ODS-100V 5-µm column (4.6 mm × 250 mm, TOSOH Corp.). The HPLC gradient elution was performed with two eluents, A (0.1% (v/v) TFA/water) and B (0.1% (v/v) TFA/ACN), by linearly increasing eluent B: 0% (0 – 15 min), 0% – 50% (15 – 45 min), 50% – 0% (45 – 50 min), and 0% (50 – 60 min). The flow rate was 1.0 mL/min, and the column temperature was 30°C. Elution was monitored by absorbance at 214 nm.

Synthesis of cyclo(-Gly-Pro) from tripeptide/dipeptide reference standard    Gly-Pro-Hyp (71.4 µmol), Gly-Pro-Ala (83.1 µmol), and Gly-Pro (117.9 µmol) were dissolved in 200 µL of pure water, and the solution was heated under atmospheric pressure at 95°C for 24 and 48 h. After heating, the yield of cyclo(-Gly-Pro) was determined using the same method employed in the purity test. The generation rate was expressed as a percentage of the molar amount of cyclo(-Gly-Pro) with respect to that of standard peptides.

Results

Structure determination by MS and NMR spectroscopies    Figure 1 shows chromatograms after enzymatic decomposition, cyclization, and purification. Peaks attributed to Gly-Pro-Hyp and Gly-Pro-Ala disappeared after cyclization, whereas the intensity of the peak attributed to cyclo-(-Gly-Pro) increased. The isolated component was subjected to structural analysis by ESI-MS and NMR spectroscopies. In the ESI mass spectrum, m/z 155.08149 with the strongest intensity was attributable to the protonated molecule [M + H+] (Fig. 2). Subtraction of the atomic weight of a proton (1.00782) provides a molecular weight of 154.07367, consistent with the molecular formulae of C7H10N2O2 and cyclo(-Gly-Pro).

Fig. 1.

Chromatogram after enzymatic decomposition, cyclization, and purification.

After enzymatic decomposition of collagen, cyclization, and purification, samples were analyzed by reverse-phase chromatography. Arrows A, B, and C indicate the retention times of Gly-Pro-Ala, Gly-Ala-Hyp, and cyclo(Gly-Pro) standards, respectively. Peaks A and B disappeared after cyclization, whereas the intensity of peak C increased. Furthermore, only peak C was detected after isolation.

Fig. 2.

ESI-MS spectrum of isolated components.

The m/z of the reference peak was 155.08149.

For the isolated component, 1H and 13C NMR spectroscopic data, heteronuclear multiple-quantum correlation, and HMBC are shown in Fig. 3. 1H NMR (DMSO-d6, 800 MHz) yielded the following data: δ 8.06 (s, 1 H), δ 4.11 (t, 1 H, J = 7.32 Hz), δ3.98 (m, 1 H), δ3.50 (dd, 1 H, J = 4.56, 16.4 Hz), δ3.41 (m, 1 H), δ 3.33 (m, 3 H), δ 2.13 (m, 1 H), δ 1.87 – 1.76 (m, 3 H). 13C NMR (DMSO-d6, 800 MHz) yielded the following data: δ 169.21, 163.78, 57.94, 45.87, 44.59, 27.82, 22.01. These data indicate that the product is a conjugate of glycine and proline; however, the amino group of glycine has one hydrogen atom (H), and according to HMBC, there is a correlation between the Hα of glycine and the carbonyl carbon of proline. Thus, product formation via the cyclizations of glycine and proline is consistent with cyclo(-Gly-Pro) (hexahydro-pyrrolo[1,2-a]pyrazine-1,4-dione).

Fig. 3.

NMR spectra of isolated components

“A” shows the 1H NMR (800 MHz, DMSO-d6) spectrum, “B” shows the 13C NMR (200 MHz, DMSO-d6) spectrum, “C” shows HMQC, and “D” shows HMBC. The circled area in “D” indicates the correlation between the Hα of Gly and the carbonyl carbon of Pro.

Fig. 3.

NMR spectra of isolated components

“A” shows the 1H NMR (800 MHz, DMSO-d6) spectrum, “B” shows the 13C NMR (200 MHz, DMSO-d6) spectrum, “C” shows HMQC, and “D” shows HMBC. The circled area in “D” indicates the correlation between the Hα of Gly and the carbonyl carbon of Pro.

Analysis of purity and yield    The purity of cyclo(-Gly-Pro) obtained by the heat treatment of CTP was 36.1%. After purification, the amount of obtained cyclo(-Gly-Pro) was 110 mg with a purity of 93.6% and a yield of 22%.

Cyclization rate from reference standard    We examined the efficiency of the cyclizations of Gly-Pro-Hyp and Gly-Pro-Ala, which constitute the main components of CTP, by heating in the same manner as that done for CTP. The same test was also performed for Gly-Pro. The generation efficiency of cyclo(-Gly-Pro) from Gly-Pro-Hyp was found to be 75.4% in 24 h and 80.4% in 48 h. In the case of Gly-Pro-Ala, the generation efficiency was found to be 81.8% in 24 h and 80.7% in 48 h. In contrast, the generation efficiency from Gly-Pro was 19.6% in 24 h and 34.9% in 48 h, lower than those of the two tripeptides (Table 1).

Table 1. Generation rate of cyclo(-Gly-Pro) in the case of heat treatment of standard tripeptides and dipeptides.
Standard peptides Generation rate (%)
24 hr 48 hr
Gly-Pro-Hyp 75.4 80.4
Gly-Pro-Ala 81.8 80.7
Gly-Pro 19.6 34.9

Standard peptides were subjected to heat treatment under atmospheric pressure at 95°C for 24 h and 48 h. The value was expressed as a percentage of the molar amount of cyclo(Gly-Pro) to that of standard peptides.

Discussion

In this study, we examined a method for producing cyclo(-Gly-Pro) from collagen using the tripeptide Gly-Pro-Y sequence as an intermediate. We degraded collagen to the tripeptide component, heated it, and then purified the product by column chromatography. All steps were conducted in an aqueous solution. According to the results of MS and NMR spectroscopies, the isolated component was identified as cyclo(-Gly-Pro). We have also confirmed that the MS and NMR data were consistent with the cyclo(-Gly-Pro) standard (data not shown).

In the past, the cyclization of dipeptides with proline in the C terminal position has been widely known in the field of peptide synthesis; however, this study is the first to report that cyclic dipeptides can be formed from the unprotected tripeptide Gly-Pro-Y. The efficiency study relative to the cyclizations of dipeptides and tripeptides indicates that the cyclizations of Gly-Pro-Hyp and Gly-Pro-Ala are much higher than that of Gly-Pro. The cyclization of tripeptides was found to be more advantageous than that of dipeptides for obtaining a product from unprotected linear peptides. Cyclization by the dissociation of the tripeptide bond at high-temperature, which occurs more easily than the desorption of the hydroxyl group on the C terminus of the dipeptide, is a likely explanation for this difference. The yield of cyclo(-Gly-Pro) from 500 mg of CTP was 110 mg, whereas the theoretical yield was 139 mg if Gly-Pro-Hyp and Gly-Pro-Ala, which occupied 49% of CTP and became cyclo(-Gly-Pro), showing cyclo(-Gly-Pro) was efficiently obtained from CTP. However, the product may also include cyclo(-Gly-Pro) derived from other Gly-Pro-Y formed by the desorption of the amino acid Y, similar to Gly-Pro-Hyp and Gly-Pro-Ala.

Conventional methods for producing cyclic dipeptides, based on a build-up technique, from amino acids prove to be complicated because of the need to remove the organic solvent and resin used for solid-phase synthesis. Moreover, complications include the need for a multistage reaction, including protection and deprotection, in an organic solvent. In contrast, the present method is a breakdown technique starting from a natural material; moreover, cyclization was achieved by the simple application of heat to CTP in an aqueous solution, precluding the need for protection and deprotection of amino acids. Processes from enzymatic decomposition to cyclization can also be performed in one-pot production, which offers a unique methodology with a high practical value. In the purification process by column chromatography, high purity can be attained using water as the solvent. In summary, this method is inexpensive and highly efficient for the production of cyclic peptides. We anticipate the development of cyclic dipeptide use in functional foods and pharmaceutical materials using this technology.

Acknowledgements    A part of this work was supported by “Nanotechnology Platform” of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, at the Center for Integrated Nanotechnology Support, Tohoku University. The author would like to thank Dr. Eunsang Kwon and Shinichiro Yoshida for performing the NMR analysis.

References
 
© 2016 by Japanese Society for Food Science and Technology
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