Abstract

Introduction:

Microorganisms can destroy various materials that make up objects of cultural heritage. In particular, ancient tempera paintings are made with egg yolk, animal glue, and a number of other organic materials, which serve as a good breeding ground for the development of microorganisms. Recently, the range of traditional antiseptics used to protect tempera paintings from biodeterioration has been significantly reduced because of undesirable properties associated with their interaction with painting materials and toxicity. Therefore, it is necessary to develop a new generation of antiseptics that can effectively protect paintings from destructive microorganisms.

Methods:

To solve this challenging task and protect paintings from fungal damage, we used H-phosphinic analogs of natural amino acids. Twelve different H-phosphinic analogs of natural amino acids were screened on Czapek–Dox agar medium against 11 mold fungi belonging to the genera Aspergillus, Penicillium, Simplicillium, Microascus, Cladosporium, and Ulocladium. These mold fungi are responsible for the biodegradation of tempera paintings and are the dominant representatives of the microbiome of the State Tretyakov Gallery in Russia.

Results:

All the studied compounds at concentrations of 0.7–2.5 mM inhibited the mycelial growth of mold fungi. The supplementation of H-phosphinic analogs of alanine, aspartate, and valine resulted in the loss of characteristic pigmentation of Penicillium chrysogenum, which may be associated with inhibition of Ac-CoA and malonyl-CoA biosynthesis. The H-phosphinic analog of methionine protected mock layers with sturgeon glue more effectively than the other H-phosphinates and standard antiseptics, such as benzalkonium chloride or sodium pentachlorophenolate. The addition of H-phosphinic amino acid analogs to sturgeon glue did not significantly affect the spectral and surface properties of the glue applied on the layout but effectively inhibited the growth of the studied mold fungi on mock-up layers during long-term storage.

Conclusion:

Our data provide the first evidence of the successful use of nontoxic H-phosphinic analogs of natural amino acids for protecting paintings from biodeterioration.

Introduction

Molds are a major factor causing biodeterioration of cultural heritage objects, artwork, and historical artifacts (De Leo and Isola, 2022; Zucconi et al., 2022; Szczepanowska, 2023; Gadd et al., 2024). This is because these chemoorganotrophic organisms can use a wide variety of substrates for their development, with various organics as an energy source (Warscheid and Braams, 2000; Branysova et al., 2022; Leplat et al., 2025). During colonization, fungi are capable of causing both physical and chemical damage to cultural heritage objects (Scheerer et al., 2009; Negi and Sarethy, 2019; Zhang et al., 2019; Nitiu et al., 2020). Consequently, sharp-cut regulations have been developed for the conservation and restoration of cultural heritage objects, including the use of a variety of antiseptics that effectively affect molds (Palla and Barresi, 2017; Kakakhel et al., 2019; Avdanina and Zhgun, 2024). However, recently, the palette of antiseptics used to protect artworks from biodeterioration has been significantly reduced (Unger et al., 2001; Sterflinger and Piñar, 2013) because most biocides were not directly developed to protect heritage materials but were borrowed from medicine and agriculture (Franco-Castillo et al., 2021; Kosel et al., 2024). Unfortunately, toxicity and undesirable interactions of a number of these compounds with painting materials, leading to pigment fading and chemical and physical changes, have been revealed (Zhang et al., 2015; Palla and Barresi, 2017). Moreover, the range of biocides that can be used is limited by the Biocidal Products Regulation EU 528 (Sterflinger and Piñar, 2013). In addition, the widespread use of a limited number of antiseptics leads to the development of microbial resistance (Bastian et al., 2010; Martin-Sanchez et al., 2012). Therefore, a new generation of antiseptics that, on the one hand, exhibit targeted activity against microorganisms damaging works of art and, on the other hand, are inert to materials used in painting and non-toxic to restorers and museum visitors must be developed (Romero-Noguera et al., 2020; Alexandrova et al., 2021; Pinna, 2022; Isola et al., 2023; Zhu et al., 2023).

Some microorganisms have been shown to synthesize compounds with unusual phosphorus-carbon (P-C) bonds (Ju et al., 2015; Parkinson et al., 2019; Kayrouz et al., 2020; Shiraishi and Kuzuyama, 2021; Ju and Nair, 2022). The P-C bond is biochemically stable and can mimic a phosphate monoester, whereas the tetrahedral phosphorus-containing group with a P-C bond is a mimetic of the tetrahedral intermediate/transition state arising during carboxyl group transformations (Metcalf and Van Der Donk, 2009; Horsman and Zechel, 2017). Among these secondary metabolites with P-C bonds, substances with diverse biological activities have been reported. For example: (i) Fosmidomycin (Figure 1A), an antibiotic and a specific nanoM inhibitor of DXP reductoisomerase, a key enzyme in the non-mevalonate pathway of isoprenoid biosynthesis (Kuzuyama et al., 1998; Jawaid et al., 2009); and (ii) Fosfomycin (Figure 1A), an irreversible inhibitor of UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), catalyzing a key stage of the biosynthesis of cell wall peptidoglycan, thus preventing bacterial cell division (Hendlin et al., 1969; Kahan et al., 1974).

(A) Some biologically active natural products containing P-C or C-P-C bonds. (B) General structure of amino H-phosphinic acids.

Among the secondary metabolites with two phosphorus-carbon (C-P-C) bonds, L-Phosphinothricin (L-PT, Figure 1A) is a notable glutamate analog with a (C-P-CH3) group replacing the γ-carboxyl group (Metcalf and Van Der Donk, 2009). L-PT irreversibly inhibits glutamine synthetase, which catalyzes the ATP-dependent formation of glutamine from glutamate and ammonia (Gill and Eisenberg, 2001). Glutamine synthetase plays a key role in nitrogen assimilation, and its inhibition leads to the accumulation of toxic levels of ammonia, resulting in cell death (van Heeswijk et al., 2013). However, L-PT, like other aminophosphonates, poorly penetrates cells and is practically important as a tripeptide of L-PT, Bialaphos (L-alanyl-L-alanyl-L-phosphinothricin), which is among the top commercial herbicides (Leason et al., 1982). This naturally occurring tripeptide yields L-PT, an inhibitor of glutamine synthetase, upon cleavage in the cell. Bialaphos has excellent activity against E. coli (Hörömpöli et al., 2021). Recently, it was shown that Bialaphos and the dipeptide L-Leucyl-L-PT are effective against clinical isolates of Klebsiella pneumoniae, which are resistant to more than 20 commercial antibiotics of different classes (Demiankova et al., 2023).

Aminoalkyl H-phosphinic acids (AA-PH, Figure 1B), containing carbon-phosphorus-hydrogen (C-P-H) bonds, have been studied significantly less compared to aminophosphonates, but unlike the latter, they penetrate microorganisms and cells and have different biological activities. It is known that AA-PH can undergo substrate-like enzymatic transformations, yielding metabolites with a C-P-H bond, which are biologically active, and the targets of these metabolites are different from those of parent AA-PH (Table 1). These and other intracellular transformations of AA-PH are essential for understanding the biological effects of AA-PH and for reducing the risk of developing drug resistance.

SubstrateEnzymeProductTargetReferences Ala-PHAlanine aminotransferase Pyr-PHInhibition of Ac-CoA biosynthesisLaber and Amrhein (1987) Met-PHS-Adenosyl-methionine synthetase SAM-PHInhibition of some methyltransferase reactionsRudenko et al. (2024)DNA methyltransferase Dnmt3aMethylation of the CpG site Filonov et al. (2023), Filonov et al. (2025)DNA methyltransferase Dnmt1No methylation of the CpG site
Glu-γ-PHGlutamate decarboxylase GABA-PHPleiotropic effects on metabolismDe Biase et al. (2020) Glu-γ-PHGlutamate dehydrogenase α-KG-γ-PHFilonov et al. (2024) Asp-α-PHAspartate aminotransferase OAA-PHKhurs et al. (1989)

Substrate-like enzymatic transformations and metabolic targets of some amino H-phosphinic acids.

The H-phosphinic analog of alanine (Ala-PH, Table 1) effectively inhibited anthocyanin synthesis in buckwheat hypocotyls and the growth of K. pneumoniae because it is transaminated intracellularly into the H-phosphinic analog of pyruvate (Pyr-PH), one of the most efficient inhibitors of pyruvate dehydrogenase (Laber and Amrhein, 1987). Ala-PH bleaches the mycelium of Pyricularia oryzae due to the intracellular formation of Pyr-PH, which decreases the levels of Ac-CoA and malonyl-CoA—precursors of fungal melanin (Zhukov et al., 2004b). The H-phosphinic analog of methionine (Met-PH, Table 1) inhibits the growth of L1210 cells, and the H-phosphinic analog of S-adenosylmethionine (SAM-PH, Table 1) was detected in these cells (Khomutov et al., 2000). Met-PH has superior fungicidal activity in field trials (equal to the Japanese fungicide, Fujione®) against rice blast disease caused by P. oryzae; however, the molecular mechanisms underlying this activity have not been studied (Zhukov et al., 2004a). The distal H-phosphinic analog of glutamate (L-Glu-γ-PH, Table 1) is a naturally occurring compound of this class (Murakami et al., 1992; Ju et al., 2015) and has antibacterial activity comparable to that of ampicillin against E. coli (De Biase et al., 2020). The metabolomic and proteomic analyses of E. coli treated with L-Glu-γ-PH demonstrated diverse effects of this glutamate analog (Giovannercole et al., 2024). Finally, L-Glu-γ-PH has negligible toxic effects when administered to rats and mice (Takara et al., 1982).

All of the above prompted us to study the antifungal activity of 12 H-phosphinic analogs of natural amino acids (Figure 2) against a panel of test cultures of mold fungi that destroy painting materials. The target of these AA-PHs will be determined by the structure of the side chain of the analog. Mold fungi were isolated from the paintings exhibited in the halls of the ancient Russian paintings of the State Tretyakov Gallery, Moscow (Zhgun et al., 2020). The sensitivity of these strains to traditional antiseptics used to protect paintings, such as benzalkonium chloride (BAC) or sodium pentachlorophenolate (NaPCP), has been previously studied, and some representatives of the genera Aspergillus and Cladosporium have been found to be resistant to these compounds (Alexandrova et al., 2024; Ermolyuk et al., 2024). All studied AА-PHs demonstrated varied antifungal activity in experiments on agarized Czapek–Dox medium. Ala-PH, Met-PH, Asp-α-PH, and the H-phosphine analog of valine (Val-PH) exhibited the best activity. In the experiments on mock layers with sturgeon glue, Met-PH was more active than traditional antiseptics (BAC and NaPCP) and overcame the drug resistance of Aspergillus and Cladosporium strains. This is the first application of water-soluble non-toxic H-phosphinic analogs of amino acids to prevent biodeterioration of painting materials.

Compounds studied in the current work as antiseptics protecting paintings from biodeterioration: H-phosphinic analogs of glycine—Gly-PH, valine—Val-PH, leucine—Leu-PH, isoleucine—Ile-PH, aspartic acid—Asp-α-PH, β-aspartic acid—Asp-β-PH, glutamic acid—Glu-γ-PH, homoserine—Hse-PH, homocysteine—Hcy-PH, methionine—Met-PH, and vitamin U (S-methylmethionine)—U-PH.

Materials and methodsMaterials

1-Aminomethyl-H-phosphinic acid (Gly-PH) was synthesized as described by Grobelny (1989). 1-Aminoethyl-H-phosphinic acid (Ala-PH), l-amino-2-methylpropyl-H-phosphinic acid (Val-PH), l-amino-3-methylbutyl-H-phosphinic acid (Leu-PH), l-amino-2-methylbutyl-H-phosphinic acid (Ile-PH), and 1-amino-3-hydroxypropyl-H-phosphinic acid (Hse-PH) were prepared following Baylis et al. (1984); 1-amino-3-methylthiopropyl-H-phosphinic acid (Met-PH), 1-amino-3-thiopropyl-H-phosphinic acid (Hcy-PH), and l-amino-3-(dimethylthionia)propyl-H-phosphinic acid (U-PH) were prepared as described in Rudenko et al. (2024); l-amino-2-carboxyethyl-H-phosphinic acid (Asp-α-PH), and 2-amino-2-carboxyethyl-H-phosphinic acid (Asp-β-PH) were synthesized according to Khomutov et al. (1996); and 3-amino-3-carboxypropyl-H-phosphinic acid (Glu-γ-PH) was prepared following Khomutov et al. (2016).

Commercial antiseptic compounds used to protect painting materials: sodium pentachlorophenolate (NaPCP) was purchased from IndiaMART, India, and benzalkonium chloride (BAC, also known as alkyldimethylbenzylammonium chloride and by the trade name Katamin AB) was purchased from Neochemax, Russia. Materials for crafting mock layers: wooden plank—LLC Mytishchi Woodworking Plant (Mytishchi, Russia); canvas—LLC Belarusian Len-Ivanovo (Ivanovo, Russia); chalk—JSC Shebekinsky Chalk Plant (Shebekino, Belgorod region, Russia); sturgeon glue—LLC Condor (Moscow, Russia).

Strains

To determine the antifungal activity of the studied amino H-phosphinic acids, a panel of 11 strains of filamentous fungi, previously isolated from exhibits and in the halls of ancient Russian paintings in the State Tretyakov Gallery (STG-strains), was used (Zhgun et al., 2020). Aspergillus versicolor STG-25G (SRX7729174; MK260015.1) and Ulocladium sp. AAZ-2020a STG-36 (MW590700.1; SRX7729176) were isolated from the icon “The Church Militant” (dated 1550s). Cladosporium halotolerans STG-52B (SRX7729178; MK258720.1) was isolated from a bust fragment of the statue “Holy Great Martyr George the Victorious” (1,464, limestone, tempera). Aspergillus creber STG-57 (SRX7729151; MK266993.1) was isolated from the icon “Holy Great Martyr Demetrius of Thessaloniki” (dated 16th century). Aspergillus versicolor STG-86 (SRX7729182; MK262781.1), Aspergillus creber STG-93 W (SRX7729186; MW575292.1), Cladosporium parahalotolerans STG-93B (SRX7729188; MK262909.1), and Simplicillium lamellicola STG-96 (SRX7729192; MK262921.1) were isolated from the surfaces of hall No. 61. Microascus paisii STG-103 (SRX7729190; MW591474.1) was isolated from the hall No. 57. Aspergillus protuberus STG-106 (SRX7729192; MK268342.1) was isolated from the hall No. 56. Penicillium chrysogenum STG-117 (MW556011.1) was isolated from the surface of the icon “Prophet Solomon” (dated 1731).

Cultivation of fungal strains on agarized nutrient media and growth inhibition assay

Fungal cultures were cultivated on slant agarized Czapek–Dox (CDA) medium, as described previously (Hyvönen et al., 2020). To determine the toxic effect of AA-PH on mycelial growth, fungal cells were collected from agar slants; 3 μL of fungal spore suspension (5 × 105 CFU/mL) was inoculated as drops onto the center of Petri dishes containing CDA medium supplemented with the addition of AА-PH, BAC, or NaPCP at a concentration of 0.7 mM or without any additives (control). A drop of fungal cells was absorbed into the agar, which made it possible to observe the radial growth of the mycelium from the center of the Petri dishes or to record its absence in cases of 100% inhibition. To obtain an agar medium with additives, the CDA medium was autoclaved at 120°С for 1 h and cooled to 60–65 °С. Then, AA-PH, BAC, and NaPCP were sterilized by filtration (pore diameter 0.22 μm) and added to agar to reach a final concentration of 2.5 mM; 22.5 mL of agar was poured into each 90 mm Petri dish. Incubation was carried out for 40 days at 26°С. The inhibitory effect was measured every 5 days and evaluated by the ratio of mycelial growth on CDA medium with the relevant addition to the mycelial growth in the control. Fungal growth inhibition (FGI) was determined using the following formula: FGI % = [(Dc–Dt)/Dc] × 100, where Dc indicates the colony diameter in the control set, and Dt indicates the colony diameter in the treated set. The data were measured in triplicate and repeated at least three times.

Crafting of mock layers

The canvas was soaked in a 10% solution of sturgeon glue and placed on 8 mm thick birch boards. The materials were dried for 24 h at room temperature, and then three layers of gesso (a 7% solution of sturgeon glue and sifted chalk, 1:3 by volume) were applied and dried for 24 h, and the surface was leveled with sandpaper to prepare the workpieces. To introduce the studied compounds (AА-PH and antiseptics currently used to protect paintings) into the composition of the mock layers, so-called active mixtures were first prepared. For this purpose, 30 mM of compounds were added to a 7% solution of sturgeon glue, freshly prepared at 55–60 °C, to obtain active mixtures; to obtain the negative control, nothing was added to the sturgeon glue. The additives were AA-PH (individual compounds or cocktails based on them are listed in Table 2), BAC, and NaPCP. Seven types of these active mixtures were applied in three layers on the prepared workpieces to create seven types of mock layers.

Mock layer No.FeatureConcentration of added compoundsICocktail of AА-PH (components A-D)7.5 мМ Gly-PH
7.5 мМ Met-PH
7.5 мМ Asp-α-PH
7.5 мМ Asp-β-PHIICocktail component A30 мМ Gly-PHIIICocktail components B/C15 мМ Asp-α-PH
15 мМ Asp-β-PHIVCocktail component D30 мМ Met-PHVPositive control30 мМ BACVI30 мМ NaPCPVIINegative controlNo additions

Compounds and their concentrations are used to craft mock layers.

Fourier-transform infrared spectroscopy of selected materials and mock layers

Infrared spectra of mock layers containing sturgeon glue with and without AA-PH and standard antiseptics were acquired using a Nicolet™ iS50 Fourier transform infrared (FTIR) spectrometer (Thermo Fisher Scientific, Waltham, MA, United States), as described previously (Zhgun et al., 2022).

Atomic force microscopy of mock layers

The atomic force microscopy technique (AFM) was used to study the surface topography of the prepared mock layers containing sturgeon glue with and without AA-PH and standard antiseptics. The NTEGRA Prima microscope (NT MDT SI, Zelenograd, Russian Federation) was used in semi-contact mode with silicon cantilevers Etalon HA FM (TipsNano, Zelenograd, Russian Federation) having resonance frequencies of 77–114 kHz and force constants of 3.5–6.0 N/m; the images were processed and analyzed using the manufacturer’s software to extract surface roughness parameters and to compare topographical features between samples. The results were processed, and the statistical parameters were calculated using the Image Analysis P9 v.3.5.0.9900 program (NT-MDT SI, Zelenograd, Russian Federation). At least five AFM images (for each of the sizes: 2 × 2, 5 × 5, 10 × 10, and 20 × 20 μm) for each sample were used to calculate the root mean square surface roughness (Sq) and peak-to-valley height (St).

Scanning electron microscopy of mold fungus

The microstructure of the mold fungus growing on the control mock layers (without additives) was investigated using scanning electron microscopy (SEM); the images were acquired using a Carl Zeiss NVision-40 microscope (Carl Zeiss, Inc., Germany). Samples with mold fungus were collected from the control mock layers (1 month after inoculation), fixed on an aluminum objective table with conductive carbon tape, placed in a vacuum chamber of a microscope, and the operating pressure was adjusted to 5.5 × 10−6 mbar. An Everhart-Thornley secondary electron detector with a focal length of approximately 3.3 mm was used to study the material surfaces. In order to minimize the impact of the electron beam on the sample’s structure, its surface was scanned at a sufficiently low accelerating voltage (1 kV). Owing to the relatively low electrical conductivity of the studied specimens, the magnification was limited to 250–15,000 times.

Determination of the antiseptic properties of amino H-phosphinic acids in mock layers

Mock layers (with AA-PH, BAC, and NaPCP and without additions) were transferred to sterile Petri dishes and saturated with 0.2 mL H2O/1.0 cm3 at 26 °C for 48 h. The sterile hydrophobic pads were used to avoid direct contact of the material with water. To determine the antiseptic properties of AA-PH in mock layers, the drop-dilution method was used with some modifications, as described previously (Dumina et al., 2013; Zhgun et al., 2019). Fungal cells were collected from CDA slants with 0.9% NaCl and diluted to 5 × 106 CFU/mL (designated as dilution 10−1); sequential tenfold dilutions were performed in 0.9% NaCl. Subsequently, 3 μL of each cell suspension at concentrations of 5 × 106, 5 × 105, and 5 × 104 CFU/mL was inoculated onto presaturated mock-up layers and incubated at 26 °C for 40 days. FGI was determined as described in the above section, Cultivation of Fungal Strains on Agarized Nutrient Media and Growth Inhibition Assay.

ResultsEffect of H-phosphinic analogs of amino acids (AA-PH) on the growth of fungal cells on agarized Czapek–Dox medium

To determine the potential of AA-PH as a new antiseptic, its effect on the growth of fungi that destroy paint and varnish materials was first studied. The target fungi were identified and isolated from the surfaces of artworks in the collections of the State Tretyakov Gallery (Moscow) (Zhgun et al., 2020). A total of 11 strains were used as test cultures; 5 belonged to the genus Aspergillus (A. versicolor STG-25G, A. creber STG-57, A. versicolor STG-86, A. creber STG-93 W, and A. protuberus STG-106); 2 belonged to the genus Cladosporium (C. halotolerans STG-52B; and C. parahalotolerans STG-93B), as well as one representative of each of the genera Penicillium (P. chrysogenum STG-117), Simplicillium (S. lamellicola STG-96), Microascus (M. paisii STG-103), and Ulocladium (Ulocladium sp. AAZ-2020a STG-36). This particular panel of fungi that destroys painting materials was chosen because these microorganisms are the dominant representatives of the microbiome of the Tretyakov Gallery, and the data obtained may have practical significance. Recently, the effects of two classes of biocides, such as alkyl nucleosides and chitosans (Alexandrova et al., 2022; Zhgun et al., 2022; Ermolyuk et al., 2024), were examined using this set of test cultures.

All tested AA-PH compounds demonstrated inhibitory effects on the mycelial growth of a panel of mold fungi on Czapek–Dox agar medium, with the degree of inhibition varying depending on the structure of the side chain of the analog and the fungal species (Figure 3).

The characteristic phenotype of fungal strains of the Tretyakov Gallery on Petri dishes on Czapek–Dox agar medium (cultivation for 20 days) with the addition of H-phosphinic analogs of some natural amino acids (2.5 mM) or without additives (control).

To quantify the effects of AA-PH, the dynamics of fungal growth inhibition (FGI) were studied for 40 days after inoculation of the test fungi on the experimental and control media. Fungal growth was analyzed every 5 days (Figure 4).

Effects of H-phosphinic analogs of natural amino acids and standard antiseptics on the growth of fungal strains of the Tretyakov Gallery on Czapek–Dox agar medium. Green, normal growth (no inhibition); red, complete inhibition. The degree of fungal growth inhibition was normalized to that of the control. BAC, benzalkonium chloride; NaPCP, sodium pentachlorophenolate. Data were acquired at 5, 10, 15, 20, 25, 30, 35, and 40 days after inoculation.

The effectiveness of the studied compounds significantly depends on the structure of the side radical of AA-PH. Among the tested compounds, Ala-PH and Asp-α-PH exhibited the best antifungal activity, inhibiting the growth of all the studied strains (Figure 4). Moreover, Ala-PH completely inhibited the growth of STG-36, STG-52B, and STG-93B throughout the cultivation period (Figure 4). Among the studied compounds, Val-PH, Met-PH, and U-PH showed high activity, inhibiting the growth of more than 80% of the test cultures throughout the experimental period. Simultaneously, Gly-PH, Asp-β-PH, and Glu-γ-PH exhibited high activity, mostly against Cladosporium (Figure 4).

Among the analogs of aliphatic amino acids, Ala-PH exhibited the highest activity, followed by Val-PH. Interestingly, among the H-phosphinic analogs of branched-chain amino acids, only Val-PH, but not Leu-PH and Ile-PH, exhibited good activity (Figure 4). This structure–activity relationship confirms that the nature of the side radical of the H-phosphinic amino acid analogs is important for antifungal activity. The highest activity of Ala-PH is most likely related to its intracellular transamination, yielding the H-phosphinic analog of pyruvate—one of the most effective inhibitors of pyruvate dehydrogenase (PDH). This has been for buckwheat hypocotyls (Laber and Amrhein, 1987) and Pyricularia oryzae (Zhukov et al., 2004b). In the first case, inhibition of PDH resulted in the inhibition of Ac-CoA-dependent anthocyanin biosynthesis, and in the second, melanin biosynthesis was inhibited, and the fungi’s mycelium became colorless.

A comparison of the activities of two analogs of aspartic acid, which differ in the position of the H-phosphinic substituent (α and β positions), is of interest. Asp-α-PH is more active than Asp-β-PH against all fungi studied, and only against STG-52B are their activities comparable (Figure 4). The H-phosphinic analog of glutamate (Glu-γ-PH) has a similar inhibition dynamic profile to Asp-α-PH but has slightly weaker activity (Figure 4).

Methionine is one of the key compounds in sulfur metabolism. This may explain why its H-phosphinic analog (Met-PH) was among the most active AA-PH (Figure 4). Homocysteine is a direct metabolic precursor of methionine, and we assumed that Hcy-PH may have fungicidal activity close to that of Met-PH. However, this assumption was not confirmed, as the effect of Hcy-PH on the growth of fungal cells was significantly weaker than that of Met-PH (Figure 4). It is possible that in fungal cells, Hcy-PH is not metabolized to Met-PH and is, therefore, less effective in the metabolic fluxes of the cell. In this regard, the data obtained for the analog of another important compound involved in sulfur metabolism, vitamin U, is of interest. The activity profile of U-PH against a panel of test cultures was similar to that of Met-PH. The dynamics of inhibition of various fungal strains were comparable for both analogs; however, Met-PH exhibited somewhat higher activity.

Overall, AA-PH most effectively inhibited the growth of fungi belonging to the classes Dothideomycetes and Sordariomycetes, whereas some representatives of the class Eurotiomycetes belonging to the genus Penicillium showed moderate resistance to AA-PH (Figure 4). The most resistant to AA-PH were fungi of the genus Aspergillus. Among Aspergillus strains, the most resistant to AA-PH were strains A. creber STG-57, A. creber STG-93 W, and A. protuberus STG-106. However, when considering the endpoint of cultivation on day 40, the STG-36 strain was able to completely overcome the toxic effect of all AA-PH, except for Ala-PH, Asp-α-PH, and Met-PH. At this cultivation period, the STG-93 W and STG-106 strains also completely overcame the toxic effects of 9 out of 12 AA-PH studied. STG-93 W remained sensitive to Ala-PH, Val-PH, and Met-PH, and STG-106 remained sensitive to Ala-PH, Val-PH, and Asp-α-PH.

Effect of AA-PH on the pigmentation of Penicillium chrysogenum STG-117 on agarized Czapek–Dox medium

It has been shown that during the cultivation of Penicillium strains on agarized nutrient media, in the period after the transition from the trophophase to the idiophase stage, a characteristic greenish-yellow color develops. This pigmentation is due to the biosynthesis of chrysogenin and sorbicillin (secondary metabolites), which color the