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Research Article
Targeted synthesis of dipeptides containing derivatives of (S)-phenylalanine and study of their antifungal activity
expand article infoLala Stepanyan‡, Monika Israyelyan‡, Heghine Hakobyan‡, Nune Khachaturyan‡, Sona Gevorgyan‡, Anahit Hovhannisyan§, Silva Zhamharyan§, Tatevik Sargsyan§‡
‡ Scientific and Production Center “Armbiotechnology” NAS RA, Yerevan, Armenia
§ Yerevan State University, Yerevan, Armenia

Corresponding author: Lala Stepanyan ( lala_stepanyan@rambler.ru )

Academic editor: Georgi Momekov
© 2024 Lala Stepanyan, Monika Israyelyan, Heghine Hakobyan, Nune Khachaturyan, Sona Gevorgyan, Anahit Hovhannisyan, Silva Zhamharyan, Tatevik Sargsyan.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Stepanyan L, Israyelyan M, Hakobyan H, Khachaturyan N, Gevorgyan S, Hovhannisyan A, Zhamharyan S, Sargsyan T (2024) Targeted synthesis of dipeptides containing derivatives of (S)-phenylalanine and study of their antifungal activity. Pharmacia 71: 1-6. https://doi.org/10.3897/pharmacia.71.e121336
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Abstract

New dipeptides containing derivatives of phenylalanine, N-tert-butoxycarbonyl-(S)-alanyl-(S)-α-methylphenylalanine and N-tert-butoxycarbonyl-(S)-alanyl-(S)-α-methyl-4-fluorophenylalanine, were synthesized. The antifungal activity of initial non-protein amino acids, derivatives of phenylalanine, and synthesized dipeptides was studied by selecting widely spread pathogenic and conditionally pathogenic fungal strains as test fungi: Aspergillus versicolor 12134, A. flavus 10567, A. candidus 10711, Penicillium chrysogenum 8190, P. aurantiogriseum 12053, P. funiculosum 8258, Alternaria alternata 8126, Ulocladium botrytis 12027, and Aureobasidium pullulans 8269. The studies showed that the antifungal activity of (S)-α-methyl-phenylalanine amino acid was pronounced against P. aurantiogriseum 12053 and Ulocladium botrytis 12027 fungal strains when applied at a concentration of 0.177 mg/ml; the dipeptide containing the mentioned amino acid showed a high inhibitory effect on the P. aurantiogriseum 12053 strain, exhibiting the same degree of inhibition at concentrations of 0.232 mg/ml and higher. (S)-α-methyl-4-fluorophenylalanine amino acid most significantly suppressed the growth of P. aurantiogriseum 12053, P. funiculosum 8258, Ulocladium botrytis 12027, and Aureobasidium pullulans 8269 fungal strains when a 0.195 mg/ml solution was used. The growth of the P. aurantiogriseum 12053 strain was inhibited even at a lower concentration of 0.13 mg/ml; the dipeptide containing the amino acid significantly inhibited the growth of the P. aurantiogriseum 12053 strain when applied at a concentration of 0.369 mg/ml.

Keywords

peptides, antifungal, synthesis

Introduction

Fungi are an integral part of our lives and play an important role in various fields covering the human microbiota, the synthesis of biologically active substances, the food industry, etc. (Enaud et al. 2018; Kapitan et al. 2018; Mukherjee et al. 2018).

However, in addition to their beneficial properties, they are also causative agents of some diseases that can damage the skin, mucous membranes, internal organs, etc. (Kaushik et al. 2015). Such fungal infections are more dangerous for patients with weakened immune systems, HIV, autoimmune diseases, and cancer (Brown 2012; Rautemaa-Richardson and Richardson 2017). About 1.5 million mortalities annually are caused by these fungal diseases (Kuplińska and Rząd 2021). In recent years, this mortality rate has worsened due to the annual increase in resistant fungal strains, the scarcity and unavailability of therapeutic agents, and the inefficiency of several well-known medications (Billamboz et al. 2021). Fungal infections are numerous, but most of them are caused by strains of Aspergillus, Candida, Cryptococcus, and Pneumocystis fungi (Billamboz et al. 2012).

Today, the search for substances with antifungal activity as well as their synthesis is urgent since this will allow us to obtain new, more effective, affordable antifungal medications with fewer side effects, a wide spectrum of activity, including a targeted one, and what is more resistant to pathogenic fungal strains. These substances include amino acids and peptides (Ullivarri et al. 2020), the number of which, according to 2016 data, reaches 1000 (Wang et al. 2016; De Lucca 2020).

Dozens of known antibacterial and antifungal agents are based on amino acids or peptides, since they are sometimes structural analogs of bacterial enzyme substrates, which often leads to enzyme inhibition, thereby inhibiting or stopping microbial growth (Fosgerau and Hoffmann 2015; Nowak et al. 2021). Currently, monoclonal antibodies, cytokine immunotherapy, vaccines, and antimicrobial peptides (AMPs) are being used as new medications for the prevention or treatment of fungal infections (Nicola et al. 2019). Studies have shown that structural analogs of the amino acid phenylalanine also exhibit antifungal activity (Niu et al. 2022).

Taking into account the structure of substances with antifungal activity, the structural units of peptides, as well as types of infections, new dipeptides containing structural analogs of phenylalanine, α-methyl-4-fluorophenylalanine, and α-methyl-phenylalanine were synthesized. An in vitro study of the antifungal activity of initial non-protein amino acids and synthesized dipeptides was carried out. Pathogenic and conditionally pathogenic species that cause widespread diseases in humans and animals were selected as test fungi: Aspergillus versicolor 12134, A. flavus 10567, A. candidus 10711, Penicillium chrysogenum 8190, P. aurantiogriseum 12053, P. funiculosum 8258, Alternaria alternata 8126, Ulocladium botrytis 12027, and Aureobasidium pullulans 8269. For example, the genus Aspergillus is one of the largest genera of fungi that causes various diseases (Barnes et al. 2006; Sugui et al. 2014). Some species of the genus Aspergillus, such as A. flavus, A. candidus, and A. versicolor, often cause diseases such as rhinosinusitis, cutaneous and subcutaneous aspergillosis, pulmonary infections, heart infections, otomycosis, etc. (Barnes and Marr 2006; Rudramurthy et al. 2019). Many species of Penicillium produce mycotoxins and volatile secondary metabolites, which are hazardous to health and contribute to the unpleasant taste of food (Salome et al. 2016). For example, P. chrysogenum is often the cause of types I and III allergies, which lead to the development of asthma (Geltner et al. 2013).

Alternaria alternata causes black spots on many fruits and vegetables. This is a latent fungus that develops during refrigerated storage of fruits and products removed from consumption (Troncoso-Rojas et al. 2014). U. botrytis was found in patients with allergic fungal sinusitis as well as onychomycosis, a fungal infection of the nail unit (Romano et al. 2004). Aureobasidium pullulans is a yeast-like fungus. In clinical practice, it has caused skin and soft tissue infections, meningitis, splenic abscesses, and peritonitis (Mittal et al. 2018). All fungi studied pose a threat to living organisms, and it is important to suppress their growth.

Materials and methods

Chemistry

Materials

All reagents were obtained from commercial sources and used without further purification. Thin-layer chromatography (TLC) was carried out on Merck aluminum foil-backed sheets precoated with 0.2-mm Kielselgel 60 F254. Column chromatography was performed on silica gel (60 × 120 mesh) on a glass column. Melting points (mp) were determined by “Elektrothermal.” 1H spectra were recorded on a “Varian Mercury 30000 300 MHz spectrometer using TMS as an internal standard. The NMR spectra were calibrated by solvent at 7.27 (CDCl3), 3.31 (CD3OD), 4.79 (D2O), and 2.50 ((CD3)2SO) for 1H. Elemental analysis was done by Euro EA3000.

Synthesis of N-tert-butoxycarbonyl-(S)-alanine

The synthesis was carried out by the previously developed method for activated esters of peptides. The authenticity, chemical purity, and optical purity of the resulted N-t-Boc-(S)-alanine-protected amino acid were checked and confirmed with the literature data. N-t-Boc-(S)-alanine. Specific rotation value: [α]D20Sample=-26.30 (c=2; acetic acid), [α]D20literatue=-26.00 (c=2; acetic acid). Melting temperature. Tmelt. sample = 86–89 °C; Tmelt. literature = 83–89 °C (Gershkovich 1987).

Synthesis of N-tert-butoxycarbonyl-(S)-alanyl-N-oxysuccinimide ester

An amount of 2.91 g (0.025 mol) of N-hydroxysuccinimide (2) dissolved in a mixture of 6 ml of 1.4 dioxane and 30 ml of methylene chloride was added to 4.11 g (0.023 mol) of N-Boc-Ala (1). 4.95 g (0.024 mol) of N,N´-dicyclohexylcarbodiimide (DCC) dissolved in 30 ml of 1.4 dioxane was added to the reaction mixture and stirred for ~ 2 h at 0 °C and 1 h at room temperature. The formed precipitate was filtered off, the solvent distilled off on a rotary evaporator, and crystallized in a mixture of ethyl acetate and hexane (1:2). The yield of the end product was 70%.

Synthesis of dipeptides

An amount of 0.0019 mol of non-protein amino acid was placed in a flat-bottomed flask with a magnetic stirrer and heated to 60 °C, dissolved in 6.75 ml of 0.5 M NaOH, then 0.00063 mol of NaHCO3 was added, and 0.0017 mmol of N-tert-butoxycarbonyl-(S)-alanyl-N-oxysuccinimide ester (3) dissolved in 2 ml dioxane was added at room temperature. After that, the reaction mixture was stirred for 3 hrs at room temperature, then transferred to a refrigerator at 5 °C. The next day, 5 ml of ethyl acetate, 3 ml of a 10% citric acid solution, and 0.2 g of NaCl were added to the reaction mixture and stirred for 15 min. Then the organic layer was separated, dried over sodium sulfate, and the solvent was removed under vacuum at 50 °C. The residue was recrystallized from the ethyl acetate-hexane mixture in a ratio of 1:3.

The course of the reaction was monitored by thin-layer chromatography, as a solvent system of chloroform, methanol, and ethyl acetate (3:2:1) was used. The reaction yield was between 50% and 73%.

N-tert-butoxycarbonyl-(S)-alanyl-(S)-α-methyl-phenylalanine (5a)

Found, %: C 58.06; H 6.93; N 8.07. C17H23FN2O5. Calculated, %: C 57.62; H 6.54; N 7.91. 1H NMR (300 MHz, DMSO) δ 7.29 – 6.18 (m, 5H, CHAr), 4.01 – 3.92 (m, 1H, CHCH3), 2.71 (d, J = 15.3 Hz, 2H, CH2Ph), 1.41 (s, 9H, (CH3)3), 1.39 (s, 3H, CCH3), 1.30 (d, J = 7.2 Hz, 1H, CHCH3).

N-tert-butoxycarbonyl-(S)-alanyl-(S)-α-methyl-4-fluorophenylalanine (5b)

Found, %: C 58.96; H 6.93; N 7.97. C18H25FN2O5. Calculated, %: C 58.68; H 6.84; N 7.60. 1H NMR (300 MHz, DMSO) δ 7.36 – 7.01 (m, 4H, CHAr), 4.01 – 3.94 (m, 1H, CHCH3), 2.67 (d, J = 15.2 Hz, 1H, CH2Ph), 2.56 (d, J = 15.2 Hz, 1H, CH2Ph), 1.41 (s, 9H, (CH3)3), 1.40 (s, 3H, CCH3), 1.30 (d, J = 7.3 Hz, 3H, CHCH3).

Biological activity

Study of antifungal activity

Aspergillus versicolor 12134, Aspergillus flavus 10567, Aspergillus candidus 10711, Penicillium chrysogenum 8190, Penicillium aurantiogriseum 12053, Penicillium funiculosum 8258, Alternaria alternata 8126, Ulocladium botrytis 12027, and Aureobasidium pullulans 8269 fungal strains were selected as objects under study.

The effect of the synthesized substances on the growth activity of the above-mentioned fungi was investigated. To investigate the antifungal activity, the studied compounds were dissolved in dimethylsulfoxide by obtaining 0.1 M solutions, which at concentrations of 0.3 ml, 0.6 ml, and 0.9 ml were added to the Czapek medium in the amount of 90 mg each with the following composition: sucrose (30.0), sodium nitrate (2.0), potassium hydrogen phosphate (1.0), magnesium sulfate (0.5), potassium chloride (0.5), and iron sulfate (0.01). The obtained mass was then evenly distributed into nine Petri dishes, with three strains in each dish and three replicates. Dishes were incubated at 28 °C for 5–7 days.

The results of the study were compared with the control, and the intensity of the growth of the fungi was observed with a magnifying glass. The data are presented in the results and discussion sections.

Results and discussion

Chemistry

For the synthesis of dipeptides containing non-protein amino acids (S)-alanyl-(S)-α-methyl-4-fluorophenylalanine and (S)-alanyl-(S)-α-methyl-phenylalanine, in the first stage the conversion of the N-t-Boc-(S)-alanine (1) to succinimide ester was done according to Scheme 1. The reaction was carried out according to the previously developed method, resulting in the synthesis of a stable N-tert-butoxycarbonyl-(S)-alanyl-N-oxysuccinimide ester (3) (Hakobyan et al. 2022).

Scheme 1. 

Synthesis of N-t-Boc-(S)- alanyl-N-oxysuccinimide ester.

Then, the non-protein amino acids (S)-α-methyl-phenylalanine and (S)-α-methyl-4-fluorophenylalanine were condensed with N-t-Boc-(S)-alanyl-N-oxysuccinimide. Condensation was carried out in various media using NaOH and Na2CO3 with different molar ratios. As a result of many tests, the best result was recorded when using a molar ratio of 3:1 (NaOH/Na2CO3=3/1). The experiment was also carried out in NaOH medium without Na2CO3, but the amount of by-products was doubled. As a result, N-tert-butoxycarbonyl-(S)-alanyl-(S)-α-methyl-phenylalanine and N-tert-butoxycarbonyl-(S)-alanyl-(S)-α-methyl-4-fluorophenylalanine (5a, 5b) dipeptides were synthesized (Scheme 2).

Scheme 2. 

Synthesis of dipeptides 5a, 5b.

Biological activity

The antifungal activity of 4a, 5a, 4b, and 5b was studied in relation to 9 strains of fungi from the Microbial Depository Center (MDC) of the SPC “Armbiotechnology” NAS RA: Aspergillus versicolor 12134, A. flavus 10567, A. candidus 10711, Penicillium chrysogenum 8190, P. aurantiogriseum 12053, P. funiculosum 8258, Alternaria alternata 8126, Ulocladium botrytis 12027, and Aureobasidium pullulans 8269.

The effect of 0.3 ml, 0.6 ml, and 0.9 ml of 0.1 M solutions of initial amino acids and synthesized dipeptides on the activity of the above-mentioned fungi was studied.

As a result of the conducted experiments, it was established that the synthesized dipeptides and initial amino acids affected the selected fungal strain, suppressing its growth (Figs 1, 2).

Figure 1. 

Antifungal activity of (S)-α-methyl-phenylalanine initial non-protein amino acid and N-tert-butoxycarbonyl-(S)-alanyl-(S)-α-methyl-phenylalanine dipeptide.

Figure 2. 

Antifungal activity of (S)-α-methyl-4-fluorophenylalanine, an initial non-protein amino acid, and N-tert-butoxycarbonyl-(S)-alanyl-(S)-α-methyl-4-fluorophenylalanine dipeptide.

In Figs 1, 2, the replicates according to appearance were performed without exception; the obtained data are presented in Table 1 below.

Table 1.
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Antifungal activity of synthesized dipeptides and initial amino acids on selected fungal strains.

Amino acids and peptides ml mmol/l Names and numbers of strains according to MDC
Aspergillus versicolor 12134 A. flavus 10567 A. candidus 10711 Penicillium chrysogenum 8190 P. aurantiogriseum 12053 P. funiculosum 8258 Alternaria alternata 8126 Ulocladium botrytis 12027 Aureobasidium pullulans 8269
(S)-α-methyl-phenylalanine 0.3 0,166 + + + ++ + ++ – + –
0.6 0,331 + ++ ++ ++ ++ ++ ++ ++ –
0.9 0,495 ++ ++ ++ ++ +++ ++ ++ +++ ++
N-tert-butoxycarbonyl-(S)-alanyl-(S)-α-methyl-phenylalanine 0.3 0,166 + + + + ++ ++ + + –
0.6 0,331 ++ ++ ++ ++ +++ ++ + + –
0.9 0,495 ++ ++ ++ ++ +++ ++ ++ ++ –
(S)-α-methyl-4-fluorophenylalanine 0.3 0,166 + – – + ++ ++ – + ++
0.6 0,331 + + + + +++ ++ + ++ ++
0.9 0,495 + ++ + ++ +++ +++ + +++ +++
N-tert-butoxycarbonyl-(S)-alanyl-(S)-α-methyl-4-fluorophenylalanine 0.3 0166 + + + + ++ + – – –
0.6 0,331 + + + + ++ ++ + + –
0.9 0,495 + ++ ++ + +++ ++ + + –

Note: +++ - active suppression of mycelial growth and sporulation, ++ - moderate suppression of mycelial growth and sporulation, + - suppression of sporulation, – - lack of antifungal activity.

As can be seen from the table, the antifungal activity of the initial 4a amino acid is pronounced in P. aurantiogriseum 12053 and Ulocladium botrytis 12027 fungal strains; 5a dipeptide containing the amino acid has shown a high inhibitory effect in the case of the P. aurantiogriseum 12053 strain. The same degree of inhibition in a 0.6 ml (0.232 mg/ml) solution containing the dipeptide was observed, and the initial amino acid showed the same degree of inhibition in a 0.9 ml (0.177 mg/ml) solution. 4b Amino acid most clearly suppressed the growth of P. aurantiogriseum 12053, P. funiculosum 8258, Ulocladium botrytis 12027, and Aureobasidium pullulans 8269 fungal strains in a 0.9 ml (0.195 mg/ml) solution; 5b dipeptide containing the amino acid significantly inhibited the growth of P. aurantiogriseum 12053 strain in a 0.9 ml (0.369 mg/ml) solution. Thus, the studied substances—derivatives of phenylalanine and dipeptides constructed on their basis—can be an active component in the process of suppressing the growth of various pathogenic and conditionally pathogenic fungi.

Conclusion

N-tert-butoxycarbonyl-(S)-alanyl-(S)-α-methyl-4-fluorophenylalanine and N-tert-butoxy-carbonyl-(S)-alanyl-(S)-α-methyl-phenylalanine dipeptides, undescribed in the literature, were synthesized by the activated ester method for classical peptide synthesis. The structure of the synthesized dipeptides was confirmed by NMR spectroscopic analysis. The antifungal activity of phenylalanine derivatives of initial amino acids and dipeptides constructed on their basis was investigated. As a result of the research, it was found that both the initial non-protein amino acids and synthesized peptides inhibited the growth of the selected fungal strains differently. These compounds mostly suppressed the growth of Penicillium chrysogenum 8190, P. aurantiogriseum 12053, and P. funiculosum 8258 fungal strains. Among the other studied fungal strains, in some cases, fungal growth inhibition was not observed when applying small amounts of substances, but growth inhibition zones were observed when the concentration of the active substance was increased. N-tert-butoxycarbonyl-(S)-alanyl-(S)-α-methyl-4-fluorophenylalanine dipeptide had no effect on the Aureobasidium pullulans 8269 strain, even when varying the concentration. Thus, almost all the studied compounds showed pronounced antifungal activity. Consequently, these compounds can be an active component in the process of suppressing the growth of various pathogenic and conditionally pathogenic fungi.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

The work was supported by the Science Committee of RA in the framework of research project No. 22YR-1D007.

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