A Comparison of the Antibacterial Efficacy of Carbohydrate Lipid-like (Thio)Ether, Sulfone, and Ester Derivatives against Paenibacillus larvae

Paenibacillus larvae is the causative agent of American foulbrood (AFB), the most serious bacterial disease affecting developing honeybee larvae and pupas. In this study, a library of 24 (thio)glycosides, glycosyl sulfones, 6-O-esters, and ethers derived from d-mannose, d-glucose, and d-galactose having C10 or C12 alkyl chain were evaluated for their antibacterial efficacy against two P. larvae strains. The efficacy of the tested compounds determined as minimal inhibitory concentrations (MICs) varied greatly. Generally, dodecyl derivatives were found to be more potent than their decylated analogs. Thioglycosides were more efficient than glycosides and sulfones. The activity of the 6-O-ether derivatives was higher than that of their ester counterparts. Seven derivatives with dodecyl chain linked (thio)glycosidically or etherically at C-6 showed high efficacy against both P. larvae strains (MICs ranged from 12.5 μM to 50 μM). Their efficacies were similar or much higher than those of selected reference compounds known to be active against P. larvae—lauric acid, monolaurin, and honeybee larval food components, 10-hydroxy-2-decenoic acid, and sebacic acid (MICs ranged from 25 μM to 6400 μM). The high efficacies of these seven derivatives suggest that they could increase the anti-P. larvae activity of larval food and improve the resistance of larvae to AFB disease through their application to honeybee colonies.


Introduction
American foulbrood (AFB) is initiated through the infection of young honeybee larvae with food contaminated by spores of the Gram-positive bacterium Paenibacillus larvae [1][2][3]. The pathological development of larval infections is associated with spore germination followed by the massive multiplication of the vegetative cells of the bacteria in the larval midgut. During this process, various substances are produced by pathogen cells, some of which help the cells penetrate through the intestinal epithelium into the larva hemocoel [4,5]. Here, the cells further multiply, leading to the death of the larva (or pupa), its decomposition, and the formation of billions of new spores. The spores are transmitted by worker bees into the food of other larvae, resulting in more and more of the latter becoming sick and dying, which finally causes the collapse of a diseased colony [6]. AFB disease is highly contagious and it spreads among colonies in several ways [7][8][9]. Its outbreaks are quite frequent in honeybee populations, causing large annual financial losses for beekeepers worldwide as well as for farmers dependent on crop pollination.
Honeybee colonies differ in resistance to AFB [3,6]. This resistance is associated with the immunity of individual larvae [10][11][12] and the social immunity mediated by many In the synthesis of compounds 18-21, the highly efficient oxidation of glycosidic sulfur with mCPBA yielding the corresponding protected sulfones preceded a saponification step. Decyl thioglucoside 22 was prepared by deacetylation of the compound 12 (Scheme 1). Glycosyl derivatives of fatty acids (capric, lauric, and 10-HDA) linked via their ωhydroxyl group to D-mannose and D-glucose were synthesized as follows. First, the glycosyl acceptors 25, 26, and 28 (ω-hydroxylated fatty acid methyl esters) were prepared in one step (Scheme 2). The 25 and 26 were obtained through a reduction of the free onies is discussed.

Synthesis
Glycolipid mimetics having C10 and C12 alkyl chains attached to D-mannose (1)(2)(3)(4), D-glucose (5)(6)(7)22), and D-galactose (8,9), in the form of O-and S-glycosides ( Figure 1, Scheme 1) and sulfones (Scheme 1) were designed as the first set of amphiphilic structures to be examined. These derivatives are easily available through a short sequence, i.e., through glycosylation of the corresponding (thio) alcohols with per-O-acetylated glycosyl donors, followed by saponification of the acetyl protective groups; most had been previously prepared [52,53,56]. Glycosyl derivatives of fatty acids (capric, lauric, and 10-HDA) linked via their ωhydroxyl group to D-mannose and D-glucose were synthesized as follows. First, the glycosyl acceptors 25, 26, and 28 (ω-hydroxylated fatty acid methyl esters) were prepared in one step (Scheme 2). The 25 and 26 were obtained through a reduction of the free In the synthesis of compounds 18-21, the highly efficient oxidation of glycosidic sulfur with mCPBA yielding the corresponding protected sulfones preceded a saponification step. Decyl thioglucoside 22 was prepared by deacetylation of the compound 12 (Scheme 1).
Glycosyl derivatives of fatty acids (capric, lauric, and 10-HDA) linked via their ωhydroxyl group to D-mannose and D-glucose were synthesized as follows. First, the glycosyl acceptors 25, 26, and 28 (ω-hydroxylated fatty acid methyl esters) were prepared in one step (Scheme 2). The 25 and 26 were obtained through a reduction of the free carboxylic function of the corresponding dicarboxylic acid monomethyl esters (23 and 24) with BH 3· THF [57]. Acceptor 28 was prepared in a moderate yield via the esterification of 27 (10-HDA). These acceptors were reacted with per-O-benzoylated imidates 29 [58] and 30 [59] serving as glycosyl donors. The coupling reaction was promoted by TMSOTf and provided the conjugates 31-36 (Scheme 3). Then, all ester protective groups were easily removed following the Zemplen protocol (MeONa, MeOH, debenzoylation) in the first step, and their subsequent treatment with LiOH (fatty acid de-esterification) provided the target derivatives 37-42 in satisfactory overall yields. A synthetic sequence leading to the 6-O-ether and ester derivatives of methyl α-Dglycosides is shown in Scheme 4. Compounds 43 and 44 were synthesized using a strategy based on a selective deprotection of the least sterically hindered primary 6-OH group of the corresponding glycosides. In the case of mannoside 43 [60], the three-step sequence started with per-O-benzylation, followed by the selective acidic deprotection of the C-6 position by TFA/AcOH and the saponification of 6-O-acetyl derivative. Glucose unit 44 These acceptors were reacted with per-O-benzoylated imidates 29 [58] and 30 [59] serving as glycosyl donors. The coupling reaction was promoted by TMSOTf and provided the conjugates 31-36 (Scheme 3). Then, all ester protective groups were easily removed following the Zemplen protocol (MeONa, MeOH, debenzoylation) in the first step, and their subsequent treatment with LiOH (fatty acid de-esterification) provided the target derivatives 37-42 in satisfactory overall yields. These acceptors were reacted with per-O-benzoylated imidates 29 [58] and 30 [59] serving as glycosyl donors. The coupling reaction was promoted by TMSOTf and provided the conjugates 31-36 (Scheme 3). Then, all ester protective groups were easily removed following the Zemplen protocol (MeONa, MeOH, debenzoylation) in the first step, and their subsequent treatment with LiOH (fatty acid de-esterification) provided the target derivatives 37-42 in satisfactory overall yields.  A synthetic sequence leading to the 6-O-ether and ester derivatives of methyl α-Dglycosides is shown in Scheme 4. Compounds 43 and 44 were synthesized using a strategy based on a selective deprotection of the least sterically hindered primary 6-OH group of the corresponding glycosides. In the case of mannoside 43 [60], the three-step sequence started with per-O-benzylation, followed by the selective acidic deprotection of the C-6 position by TFA/AcOH and the saponification of 6-O-acetyl derivative. Glucose unit 44 A synthetic sequence leading to the 6-O-ether and ester derivatives of methyl α-Dglycosides is shown in Scheme 4. Compounds 43 and 44 were synthesized using a strategy based on a selective deprotection of the least sterically hindered primary 6-OH group of the corresponding glycosides. In the case of mannoside 43 [60], the three-step sequence started with per-O-benzylation, followed by the selective acidic deprotection of the C-6 position by TFA/AcOH and the saponification of 6-O-acetyl derivative. Glucose unit 44 [61,62] was also obtained over three steps, namely through tritylation followed by benzylation, and finally detritylation. [61,62] was also obtained over three steps, namely through tritylation followed by zylation, and finally detritylation.

Efficacy of Derivatives against P. larvae
The antibacterial efficacies of 24 glycolipid mimetics derived from D-mannose, D cose, and D-galactose, five reference compounds (Figure 2), and two antibiotics (cipro acin and tylosin tartrate) were evaluated against two P. larvae strains CCM 4483 (E genotype) and CCM 4486 (ERIC II genotype). The results are summarised in Table 1  The results showed that the derivatives exhibited a similar antibacterial ac against the P. larvae strains of ERIC I and ERIC II genotypes. Maximal 2-fold differ in activity were observed for nine out of fourteen decyl and dodecyl (thio)glycoside rivatives (1-9, 18-22). These were more effective against the P. larvae CCM 4486 tha P. larvae CCM 4483 strain. Similar compounds having the aglycone alkyl chain ca with the carboxylic group (37)(38)(39)(40)(41) were inactive against both strains in the tested ran activity. The compounds with a dodecyl chain attached to the saccharide C-6 positi either an ether or ester linkage (49)(50)(51)(52) showed the same activity against both strains

Efficacy of Derivatives against P. larvae
The antibacterial efficacies of 24 glycolipid mimetics derived from D-mannose, D-glucose, and D-galactose, five reference compounds ( Figure 2), and two antibiotics (ciprofloxacin and tylosin tartrate) were evaluated against two P. larvae strains CCM 4483 (ERIC I genotype) and CCM 4486 (ERIC II genotype). The results are summarised in Table 1.

Efficacy of Derivatives against P. larvae
The antibacterial efficacies of 24 glycolipid mimetics derived from D-mannose, D-glucose, and D-galactose, five reference compounds ( Figure 2), and two antibiotics (ciprofloxacin and tylosin tartrate) were evaluated against two P. larvae strains CCM 4483 (ERIC I genotype) and CCM 4486 (ERIC II genotype). The results are summarised in Table 1. The results showed that the derivatives exhibited a similar antibacterial activity against the P. larvae strains of ERIC I and ERIC II genotypes. Maximal 2-fold differences in activity were observed for nine out of fourteen decyl and dodecyl (thio)glycosides derivatives (1-9, 18-22). These were more effective against the P. larvae CCM 4486 than the P. larvae CCM 4483 strain. Similar compounds having the aglycone alkyl chain capped with the carboxylic group (37)(38)(39)(40)(41) were inactive against both strains in the tested range of activity. The compounds with a dodecyl chain attached to the saccharide C-6 position by either an ether or ester linkage (49)(50)(51)(52) showed the same activity against both strains. The The results showed that the derivatives exhibited a similar antibacterial activity against the P. larvae strains of ERIC I and ERIC II genotypes. Maximal 2-fold differences in activity were observed for nine out of fourteen decyl and dodecyl (thio)glycosides derivatives (1-9, 18-22). These were more effective against the P. larvae CCM 4486 than the P. larvae CCM 4483 strain. Similar compounds having the aglycone alkyl chain capped with the carboxylic group (37)(38)(39)(40)(41) were inactive against both strains in the tested range of activity. The compounds with a dodecyl chain attached to the saccharide C-6 position by either an ether or ester linkage (49)(50)(51)(52) showed the same activity against both strains. The findings suggest that the differences in activity against the strains of different ERIC genotypes occurring at some derivatives were likely caused by a genetic variability of the tested strains and not by the ERIC genotype of P. larvae. Out of 14 alkyl (thio)glycosides and sulfones, the compounds having a dodecyl aglycone were more active than decylated analogs. The effect of the aglycone length was more profound in glucosides and mannosides than in galactosides. Mannosides were generally more potent than glucosides. Among the decyl glycosides, thiomannoside 2 was the most efficient (MICs 100 µM and 50 µM for individual P. larvae strains). The most efficient dodecyl derivative was thiomannoside 4 (MICs 25 µM and 12.5 µM), which represented the most potent derivative of all the tested compounds.
Galactosides 8 and 9 showed a low antibacterial activity, confirming previous observations [52] that galacto-based derivatives inhibited some Gram-positive bacteria significantly weaker than analogous mannosides and glucosides. Therefore, no other galactose derivatives were studied.
Six glycosides 37-42 having an alkyl chain capped with an acid (capric, lauric, and 10-HDA) were inactive in the tested MIC range. This suggests that ω-glycosylation of the fatty acids was detrimental to their inhibitory activity against P. larvae. From another point of view, the termination of the alkyl aglycone of the O-glycosides with a carboxyl group resulted in a loss of inhibitory activity. This confirms that only a derivative with an amphiphilic nature comprising a hydrophilic (carbohydrate) moiety at one end and simultaneously a hydrophobic (aliphatic alkyl chain) moiety at the other end maintains the antibacterial activity.
The derivatives having an alkyl chain attached to the saccharide C-6 position showed high efficiency. However, their potency was slightly affected by a linkage (ether vs. ester) that connects the hydrophobic unit with the saccharide. Methyl 6-O-dodecyl α-D-glycosides 49 and 51 were slightly more potent against both P. larvae strains than the 6-O-acylated analogs 50 and 52.
In summary, an evaluation of the library of synthetic glycolipid mimetics revealed that the derivatives having alkyl units (thio)etherically linked either at the saccharide C-1 or the C-6 position exhibited a higher antibacterial effect than the corresponding C-6 esters or the C-1 ethers capped with a carboxylic group.
The antibacterial activity of five reference compounds was examined in this study. Two of them, lauric acid and monolaurin, are known to be active against P. larvae. An agar diffusion method previously showed that lauric and myristoleic acids were the most active fatty acids against P. larvae among 38 different saturated and unsaturated fatty acids [45]. Lauric acid was found to be the second most efficient compound among 13 different natural compounds in tests with 10 P. larvae strains (MICs at individual strains were 25 µg or 50 µg/mL) [63]. The high efficacy of monolaurin (MIC 62.2 µg/mL) against four P. larvae strains has also been demonstrated in a previous study [47]. The MICs of lauric acid and monolaurin determined in this work were 2.5-5 and 4.5-9 times lower, respectively than those mentioned above. This could be explained by the use of a cultivation medium with a lower pH in our microdilution tests. An increase in antibacterial efficacy by lowering the pH has already been observed for some medium-chain fatty acids including the 10-HDA [16,64,65]. The lauric acid and monolaurin inhibited P. larvae with the same efficiency as derivatives 7, 50, and 52, but twice less than derivatives 3, 4, 49, and 51. The obtained results concerning the monolaurin activity are particularly important because the compound is used as a key ingredient in various antimicrobial food additives. The results confirmed its high antibacterial potential. Two honeybee larval food fatty acid reference compounds, 10-HDA and sebacic acid, inhibited the P. larvae strains much more weakly (MIC 6400 µM) than other active compounds, and the same activity was observed for the monomethyl sebacate 23 (MIC 6400 µM). The MIC of 10-HDA correlated well with those that we had determined previously [16]. The reference antibiotics used for confirming the sensitivity of experimental P. larvae strains, ciprofloxacin and tylosin tartrate, were shown to be 50-100 times more antibacterially potent than the most efficient synthetic glycolipid derivative, dodecyl thiomannoside 4.
In this study, a comparison of the antibacterial effect of glycolipid mimetics etherified either at the C-1 or C-6 position of the saccharide was performed for the first time. It showed that the antibacterial activity of some derivatives may be affected by the ether linkage position. In our case, dodecyl glucoside 6 (C-1 ether) exhibited lower efficacy than the glucoside 51 dodecyl etherified at C-6. The study further demonstrated that (thio)glycosides inhibited the Gram-positive strains of honeybee larval pathogen P. larvae with different efficacy. In our previous studies [52,53], other Gram-positive strains of S. aureus and E. faecalis were susceptible to some of these (thio)glycosides indicating that the nature of the saccharide units may affect their antibacterial efficacy. In line with the results of Smith et al. [49], methyl 6-O-dodecyl α-D-glucopyranoside 51 was a better inhibitor of the Gram-positive strain than its 6-O-lauroylated counterpart 52. Here, the same effect was observed for the mannoside analogs 49 and 50. Moreover, our observation that an optimal alkyl chain length for reaching a higher efficiency is C12 correlates with previous reports [49,53,54,66].
A very important result of this work was the identification of several compounds (3, 4,  7, 49, 50, 51, and 52) showing high efficacies against P. larvae. Their activities were either the same or 2-4 times higher (in dependence on tested P. larvae strain) than that of the reference compounds, lauric acid and monolaurin, and up to 128-356 times higher than the efficacies of 10-HDA and sebacic acid, the reference larval food compounds. Our previous findings suggested that the major RJ fatty acid 10-HDA together with abundant sebacic acid and other RJ fatty acids and proteins with anti-P. larvae properties could play a significant role in conferring antipathogenic activity to larval food and thus contribute to the resistance of individual larvae to P. larvae [16]. We assume that in honeybee colonies showing a higher resistance to AFB, the joint action of the antipathogenic compounds in the midguts of many infected young larvae may reach such high potency that this protects them from AFB. Such situations probably occur less frequently in the infected larvae in colonies showing a lower resistance to AFB. In this context, the fact that the most potent compounds identified were so many times more effective against P. larvae than the 10-HDA and sebacic acid was very important. Indeed, this suggests that the incorporation of only small amounts of any of these compounds into larval food could significantly contribute to increasing the constitutive anti-P. larvae activity of the food. Whether this increase could occur and whether it would suffice to provide larvae protection against AFB remains unknown and further research will be required to clarify this. We suppose that an efficient compound could mediate adequate antipathogenic action if: (1) it is not toxic or harmful to larvae or bees; (2) a suitable diet will be found in which it may be incorporated in adequate amounts; (3) it will be incorporated through nurse bees from the diet into larval food in such amounts that will contribute to the resistance of larvae to AFB.

General
Thin layer chromatography (TLC) was performed on aluminium sheets precoated with silica gel 60 F 254 from Merck (Darmstadt, Germany). Flash column chromatography was carried out on silica gel 60 (0.040-0.060 mm) from Merck (Darmstadt, Germany) with distilled solvents (hexanes, ethyl acetate, chloroform, methanol). The anhydrous solvents (dichloromethane, methanol, DMF, and pyridine), monolaurin, lauric acid, sebacic acid, and monomethyl sebacate were purchased from Aldrich. 10-HDA with a 98% purity was purchased from AK Scientific, Inc. (Union City, CA, USA), and ciprofloxacin from Salutas Pharma GmbH (Barleben, Germany). Tylosin tartrate was obtained as a Tylancel veterinary preparation from Bares (Nitra, Slovakia). All reactions containing sensitive reagents were carried out under an argon atmosphere. 1 H NMR and 13 C NMR spectra were recorded at 25 • C with a Bruker AVANCE III HD 400 spectrometer. Chemical shifts were referenced to either TMS (δ 0.00, CDCl 3 for 1 H) or HOD (δ 4.87, CD 3 OD for 1 H), and an internal CDCl 3 (δ 77.00) or CD 3 OD (δ 49.00) for 13 C. Optical rotations were measured on a Jasco P2000 polarimeter at 20 • C. High-resolution mass determination was performed by electrospray ionisation mass spectrometry (ESI-MS) on a Thermo Scientific Orbitrap Exactive instrument operating in positive mode. All the tested compounds were lyophilised before their use.
A mixture of trichloroacetimidate 29 [58] or 30 [59] (1.0 mmol), corresponding acceptor 25, 26 or 28 (1.10 mmol), and 4 Å molecular sieves (100 mg/1 mmol of the donor) were stirred in anhydrous CH 2 Cl 2 (10 mL) for 30 min. at rt. The reaction mixture was cooled to 0 • C and TMSOTf (0.10 mmol) was added. Then the reaction mixture was stirred for 20 min at rt. After neutralisation with solid NaHCO 3 , the solid was filtered off through Celite and rinsed with CH 2 Cl 2 (15 mL). The solvent was evaporated and the residue was purified by column chromatography (hexane:EtOAc). A broth microdilution method in 96-well microplates was used to determine the MICs of the tested compounds. First, stock solutions with 20 mM concentrations of individual compounds were prepared in methanol and stored at -25 • C. Before performing the tests, working solutions with suitable concentrations of compounds were prepared from those using two-fold serial dilutions. Stock and working solutions of antibiotics were prepared in dimethyl sulfoxide (DMSO). Overnight bacterial cultures of the examined strains were prepared on an orbital shaker and diluted in a cultivation medium to a final concentration of 1 × 10 5 CFU/mL. Aliquots of the diluted cultures (147 µL) were pipetted into the wells of sterile polystyrene microplates. Then, 3 µL of the working solutions of the tested compounds were added to the wells to reach final concentrations of the compounds and the antibiotics ranging from 12.5 µM to 6400 µM and 0.125-6.25 µM, respectively. Each compound was pipetted into wells in triplicate. The microplates were shaken on a microplate shaker Biofil (Merci, Paris, France) at 1200 rpm for 5 min and then left to incubate under stationary conditions for 43 h. The shaking of the microplates was repeated after 18 h and at the end of the cultivation. Bacterial growth was determined spectrophotometrically by measuring the absorbance at 630 nm using a Mithras 2 LB 943 microplate reader (Berthold Technologies). The positive and negative controls of bacterial growth contained 3 µL of methanol or DMSO. The antibacterial sensitivity of the used P. larvae strains was evaluated with ciprofloxacin and tylosin tartrate antibiotics. The MIC of each compound was determined by three independent tests.

Conclusions
This work is the first study evaluating the susceptibility of P. larvae strains of distinct ERIC genotypes to synthetic carbohydrate lipid-like compounds. These compounds consisted of non-toxic, biodegradable, and eco-friendly alkyl, fatty acid, and carbohydrate units. The study confirmed that the structure of the sugar units and the length of the alkyl chains had an impact on the antibacterial efficacy of the derivatives. The incorporation of an alkyl unit to the saccharide at the C-6 position by ether linkage was shown to be more beneficial than the ester function between these units. The thioglycosides were generally more active than their O-counterparts and sulfones. The C-1 saccharides conjugated with fatty acids, including 10-HDA, were shown to be inactive in the performed tests. This demonstrated that a polarity of the functional group terminating the alkyl chain was another important factor modulating the antibacterial effects of the sugar-based amphiphiles. It can be concluded that some carbohydrate-based amphiphiles with appropriate sugar cores and dodecyl alkyl chains may act as efficient inhibitors of the honeybee pathogen P. larvae. The most potent anti-P. larvae derivatives identified in this work represent potential candidates that could be examined for their ability to improve larval protection against AFB.