Alicja
Kapuscinska
,
Anna
Olejnik
* and
Izabela
Nowak
Faculty of Chemistry, Adam Mickiewicz University in Poznań, Umultowska 89b, 61-614 Poznań, Poland. E-mail: annamar@amu.edu.pl; Tel: +48 61 829 15 80
First published on 12th September 2016
A novel biologically active compound, the conjugate of jasmonic acid and tetrapeptide, has been obtained. The newly synthesized conjugate was characterized using MS, NMR and FTIR spectroscopy. The purity and melting point of the conjugate were determined using a differential scanning calorimetry technique. The safety of topical application of the conjugate was theoretically estimated. The obtained compound may exhibit the activity of jasmonic acid as well as that of tetrapeptide, therefore it is expected to have a promising effect on the skin.
The synthesis of the JA–YPFF–NH2 conjugate performed by LipoPharm.pl is presented in Fig. 1. The peptide YPFF–NH2 was synthesized manually on the 0.3 millimole scale via a solid-phase method using AM RAM resin and the Fmoc/But procedure.16 The racemic jasmonic acid was purchased from TCI Europe N.V. and the amino-acid derivatives (Fmoc-L-Phe-OH, Fmoc-L-Pro-OH, and Fmoc-L-Tyr(tBu)-OH) were purchased from Sigma-Aldrich. The precise amounts of amino acid derivatives and jasmonic acid used in the conjugate synthesis were as follows: 585.1 mg of Fmoc-L-Phe-OH, 506.1 mg of Fmoc-L-Pro-OH, 689.3 mg of Fmoc-L-Tyr(tBu)-OH and 371.1 mg of jasmonic acid. Before the synthesis, the AM RAM resin was treated with a DCM/DMF solution (v/v, 9:1) for 1 h. After that the solvents were filtered. The synthesis of the conjugate began by adding to the AM RAM resin a mixture of HOBt and Fmoc-L-Phe-OH, previously dissolved in DCM/DMF (v/v, 9:1). The Fmoc-L-Phe-OH unit, which represents the C-terminus, was anchored to the rink amide resin by using DIC and DMAP in DMF. After capping (Ac2O/DIPEA in DMF), to protect unreacted hydroxyl groups of the resin, the Fmoc group was deprotected using a 20% solution of piperidine in DMF, then amide A was obtained.17 The addition of Fmoc-L-Phe-OH, Fmoc-L-Pro-OH, Fmoc-L-Tyr(tBu)-OH and jasmonic acid to A was performed using DIC as a coupling agent in the presence of HOBt. The capping was carried out using Ac2O/DIPEA in DMF and the N-Fmoc deprotection was performed using a 20% solution of piperidine in DMF. After each deprotection of the amino group, a chloranil test was performed to detect the amino groups. The result of the test was positive; dark blue to green beads of the resin implied that there were free amino groups and the deprotection was performed successfully. After careful washing of the resin, the JA–YPFF–NH2 conjugate was contemporarily cleaved from the resin, via acidic treatment using a mixture of TFA:H2O:TIS (v/v, 92.5:2.5:5) for 1.5 hour at 25 °C.18Fig. 1 presents the general synthesis scheme of the conjugate and does not include the stereoisomeric structures of jasmonic acid. However, there are two chiral centers at C-3 and C-4 and therefore four possible stereoisomeric forms of JA, (3S,4S)-JA or (+)-JA, (3R,4R)-JA or (−)-JA, (3S,4R)-JA, known as (−)-epi-JA, and (+)-(3R,4S)-JA, known as (+)-epi-JA, that may be generated. It was investigated that commercially available jasmonic acid is a racemic mixture containing approximately 3–5% each of (+)-epi-JA and (−)-epi-JA, and 45–47% each of (+)-JA and (−)-JA.19 No pure JA stereoisomers have been commercially available so far. Therefore it can be concluded that the obtained product is in the majority a mixture of (+)-JA and (−)-JA conjugates. Both forms are biologically active, however (−)-JA is believed to be the more biologically active compound.1
The desired product was purified using RP-HPLC. In this process a Phenomenex Luna column (5 μm, C18(2), 100 Å, 10 × 250 mm) was employed. As a mobile phase water and a mixture of acetonitrile and 0.1% of TFA were used in the gradient elution. The purity of the obtained conjugate was >97%. The conjugate of jasmonic acid and tetrapeptide YPFF-NH2 is a white powder with poor solubility in water, but a high solubility in organic solvents such as methanol and ethanol.
To confirm the molecular weight of the conjugate, the mass spectra were recorded using a hybrid QTOF instrument (AB Sciex, model 5600+). Ions were generated using electrospray ionization (ESI) under the following conditions: a flow rate of 10 mL min−1, a dry gas flow of 8 L min−1, a nebulizer pressure of 1.5 bar, a spray voltage of 5500 V, and a drying gas temperature of 250 °C. Analyst 1.6TF software (AB Sciex) was used to process the raw spectra. The MS spectrum was obtained in the positive mode. The conjugate of JA and tetrapeptide was successfully identified using mass spectrometry. The positive ion mode presents a peak of a protonated molecular ion [M + H]+ at m/z 765.39. Additionally, a molecular ion with associated sodium [M + Na]+ at m/z 787.37 was detected (Fig. 2).
Fourier-transform infrared spectroscopy was carried out using a Nicolet FTIR 200 spectrophotometer (Thermo Fisher Scientific, Inc., USA). The spectrum was collected in the wavelength range of 500–4000 cm−1. The typical chemical bonds of the synthesized compound were successfully characterized. FTIR measurements of JA–YPFF–NH2 and the identified functional groups are presented in Fig. 3. The band at about 3302 cm−1 corresponds to O–H stretching vibrations of the OH group. The band at about 3028 cm−1 is assigned to C–H stretching vibrations. Moreover, at 2964 cm−1 the stretching vibrations of C–H were identified. The CO group was manifested as a single band at about 1738 cm−1 region, whereas the amine group was identified at about 1653 cm−1 (C–N, stretch) and 3070 cm−1. The C–H and CC bonds from the aromatic ring gave the bands at 2964 cm−1 and 1518 cm−1, respectively.
To confirm the structure of JA–YPFF–NH2, its nuclear magnetic resonance (NMR) spectra were recorded using a NMR spectrometer (Bruker Avance 600 MHz; Bruker, UK). The conjugate sample was dissolved in chloroform-d (Sigma-Aldrich). The numbering of the atoms in the structure scheme (Fig. 4) was produced in order to present the NMR spectroscopy results. The 1H NMR shift for CDCl3 is 7.24 ppm and its 13C NMR shift is 77.0 ppm.
13C NMR (CDCl3, 600 MHz): δC 14.06 (C-10), 20.52 (C-9), 24.55 (C-23), 25.36 (C-6), 26.60 (C-5), 28.55 (C-24), 37.15 (C-14), 37.57 (C-11), 38.16 (C-37), 38.29 (C-28), 38.82 (C-4), 40.2 (C-11), 40.50 (C-1), 47.66 (C-22), 52.29 (C-13), 53.98 (C-36), 53.76 (C-3), 54.55 (C-27), 60.55 (C-25), 115.7 (C-17, C-19), 124.79 (C-7), 125.72–129.15 (C-30-C-34, C-39-C-43), 129.37 (C-15), 130.50 (C-16, C-20), 134.02 (C-8), 135.85 (C-29, C-37), 155.81 (C-18), 171.22 (C-26), 171.58 (C-35), 172.01 (C-21), 172.12 (C-44), 173.39 (C-12), 220.30 (C-2).
13C NMR (CDCl3, 600 MHz): δC 14.13 (C-10), 20.52 (C-9), 24.57 (C-23), 25.39 (C-6), 26.76 (C-5), 28.55 (C-24), 37.26 (C-14), 37.66 (C-11), 38.16 (C-37), 38.29 (C-28), 38.82 (C-4), 40.2 (C-11), 40.60 (C-1), 46.83 (C-22), 52.34 (C-13), 54.08 (C-36), 53.66 (C-3), 54.60 (C-27), 60.55 (C-25), 115.95 (C-17, C-19), 124.94 (C-7) 125.72-129.15 (C-30-C-34, C-39-C-43), 129.41 (C-15), 130.38 (C-16, C-20), 134.09 (C-8), 136.18 (C-29, C-37), 156.28(C-18), 171.22 (C-26), 171.58 (C-35), 172.01 (C-21), 172.12 (C-44), 173.52 (C-12), 220.1 (C-2).
The melting point of the conjugate crystals was determined using differential scanning calorimetry (DSC) using a differential scanning calorimeter (Q2000; TA Instruments, USA). The conjugate sample was weighted on an aluminium pan which was then hermetically closed and placed into a DSC analyser. The initial and final temperatures of the measurement were 0 and 100 °C, respectively, and the temperature ramp was 5 °C min−1. The results of the DSC study are plotted with the percentage heat flow on the Y-axis and temperature on the X-axis. The DSC device is designed to maintain the test sample and the reference sample at the same temperature when they are heated. The precise and high quality data obtained from DSC provide information on the thermal stability of the samples in process development and in the formulation of potential therapeutics. Moreover, the knowledge of the melting point of the active compounds is essential in order to select the temperature for storage as well as the temperature for preparation of either the pharmaceutical or cosmetic formulations containing these compounds. The melting point of the conjugate was established using differential scanning calorimetry to be 52.42 °C. The change in enthalpy (ΔH) of the melting process of the conjugate crystals was determined. In order to determine ΔH, a line tangent to the DSC curve was drawn and the surface area of the peak was calculated. The change in melting enthalpy of the conjugate was 14.97 J g−1. The single peak in the DSC curve proves that the compound obtained does not contain any contaminations and that the purification method performed after the conjugate synthesis was effective. The DSC curve of JA–YPFF–NH2 is presented in Fig. 5.
The potential toxic hazard of JA–YPFF–NH2 was estimated using the application Toxtree. This application makes structure-based predictions for a number of toxicological endpoints. The conjugate is intended for topical applications. Therefore, it is important to investigate whether this substance may cause skin irritation before performing in vivo studies on volunteers. Toxtree analysis is based on the Threshold of Toxicological Concern (TTC) concept designed to establish a safety level of exposure to chemical compounds.20 The most popular approach for structuring chemicals in order to make a TTC estimation is the Cramer classification tree.21 The Toxtree software was evolved to implement diverse rule-based estimation approaches and was commissioned by the European Chemicals Bureau (ECB).22
Prior to in vitro and in vivo studies the potential risk of topical application of the conjugate was theoretically assessed. In order to estimate the potential toxic hazard of JA–YPFF–NH2, the open source application Toxtree was used. The results of this estimation show that the conjugate of jasmonic acid and tetrapeptide does not include skin sensitization reactivity domains and is not corrosive to the skin. Moreover, this estimation gave a negative response to both genotoxic and nongenotoxic carcinogenicity of the conjugate. Therefore, it can be concluded that JA–YPFF–NH2 may be used in topical applications and should cause no damage to skin appearance, condition and health. Therefore, it may be a promising active ingredient to add to topical formulations.
Since the conjugate is not highly soluble in water, we suggest that ethanol could be used as a solvent for the topical use of the active compound. This solvent is widely used in topical applications in cosmetology, dermatology and pharmacy. The excessive use of ethanolic solutions on the skin may cause dryness. Therefore, we suggest using a small amount of ethanol to dissolve the conjugate (e.g. 200 μl of EtOH per 5 mg of JA–YPFF–NH2) and to mix the obtained ethanolic solution of the conjugate with other components of pharmaceutical formulations. Moreover, ethanol is a well known skin penetration enhancer.23,24 Furthermore, the conjugate could also be encapsulated into nanocarriers or mixed with an oily phase of the pharmaceutical formulation, as long as the temperature during the preparation process does not exceed 35–40 °C (a higher temperature may cause the risk of conjugate degradation). Characterization of the newly obtained conjugate JA–YPFF–NH2 was carried out successfully. The theoretical mass of the obtained compound was confirmed using MS spectrometry. The structure of JA–YPFF–NH2 was confirmed using NMR spectroscopy and the typical chemical bonds of the conjugate were effectively characterized using FTIR spectroscopy. Moreover, the purity and melting point of the conjugate were determined using a DSC technique. Furthermore, the safety of topical application of the conjugate was theoretically estimated. It has also been shown that the obtained compound is not corrosive to skin and does not cause genotoxic and nongenotoxic carcinogenicity. Additionally, it is a promising compound that may exhibit a beneficial effect on the skin. The effect of this compound on the skin will be further studied in in vitro and in vivo tests.
Footnote |
† Electronic supplementary information (ESI) available: 1H NMR and 13C NMR spectra of the JA-YPFF-NH2 conjugate, HR-MS spectrum, CD spectrum and the results of Toxtree estimation of the properties of JA-YPFF-NH2. See DOI: 10.1039/c6nj01785a |
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