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Article

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

by
Veronika Šamšulová
1,
Mária Šedivá
2,
Juraj Kóňa
2,3,
Jaroslav Klaudiny
2,* and
Monika Poláková
2,*
1
Department of Organic Chemistry, Faculty of Science, Palacký University, 17. Listopadu 12, 779 00 Olomouc, Czech Republic
2
Center for Glycomics, Institute of Chemistry, Slovak Academy of Sciences, Dúbravská Cesta 9, 84538 Bratislava, Slovakia
3
Medical Vision, Civic Research Association, Záhradnícka 4837/55, 82108 Bratislava, Slovakia
*
Authors to whom correspondence should be addressed.
Molecules 2023, 28(6), 2516; https://doi.org/10.3390/molecules28062516
Submission received: 19 January 2023 / Revised: 23 February 2023 / Accepted: 7 March 2023 / Published: 9 March 2023
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Versions Notes

Abstract

:
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.

Graphical Abstract

1. 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 worker bees [13,14,15]. One of the immune social mechanisms is probably associated with the antibacterial properties of larval food [royal (RJ), worker (WJ), and drone (DJ) jelly] [16,17]. The food is produced by genetically heterogeneous populations of nurse bees in honeybee colonies and varies in constitutive contents of various antibacterial substances. These include proteinaceous compounds [18,19,20] and different derivatives of C8–C12 fatty acids (hydroxy and dicarboxylic) [21,22,23] many of which exhibit anti-P. larvae activity. These are peptide defensin1 [24,25], protein apalbumin2a [26], major RJ fatty acid 10-hydroxy-2-decenoic (10-HDA) [16], and abundant RJ acids sebacic [27] and 2-decene-1,10-dioic acids [28].
Beekeepers use several strategies to control AFB. These include various preventive precautions and some treatments [3,29,30] including the application of antibiotics to colonies [31,32]. Due to the appearance of resistant strains [33,34] and the possible contamination of honeybee products with them [35,36,37], their use was banned in many countries including EU member states. Instead, radical methods involving the burning of diseased colonies and contaminated hive materials are applied in these countries [6,38]. Other approaches for controlling AFB are therefore being investigated. These are based on the application of anti-P. larvae active substances including natural ones such as essential oils, plant extracts, propolis (its components), fatty acids, and probiotic bacteria [38,39,40,41,42], synthetic compounds such as indol analogs [43], and bacteriophages [44].
Fatty acids and some of their derivatives represent a large group of antibacterial substances. It has been reported that different fatty acids such as saturated C10–C14, monounsaturated C14, C16, C18, and some polyunsaturated C18–C22 fatty acids exhibit anti-P. larvae activity. The most active among them are lauric, myristoleic, palmitoleic [45], undecanoic, and homo-γ-linolenic acids [46]. Among the fatty acid derivatives, monolaurin, a glycerol monoester of lauric acid, was found to be effective against P. larvae [47]. Furthermore, it has also been shown that carbohydrate fatty acid esters are antibacterially active but mainly against Gram-positive bacteria [48,49,50]. The ester bond in these derivatives can, however, be cleaved by esterases in cells or guts leading to fatty acid and inactive sugar [51]. To avoid such hydrolysis, the ester linkage can be replaced by a (thio)ether bond which is resistant to esterases [52]. The activity of various ether derivatives has been found to be dependent on the alkyl chain length and the stereochemistry of the carbohydrate unit. The most efficient derivatives were those bearing decyl and dodecyl alkyl chains [52,53,54]. To the best of our knowledge, the inhibition effects of carbohydrate (thio)ether and ester derivatives against P. larvae have not been explored yet and this inspired us to perform the investigation presented here.
In this work, various lipid-like carbohydrate derivatives were studied for their antibacterial effects against two P. larvae strains possessing the ERIC I and ERIC II genotypes (ERIC- Enterobacterial Repeating Intergenic Consensus sequences). Only these two genotypes occur in field isolates of P. larvae. AFB disease caused by strains of the distinct genotypes exerts different time progression in infected larvae and in whole colonies, which is manifested by partially different symptoms in colonies [6,55]. The tested derivatives included (1) decyl and dodecyl (thio)glycosides, and glycosyl sulfones derived from d-mannose, d-glucose, and some from d-galactose; (2) glycosylated fatty acids (10-HDA, capric, and lauric acids) with d-mannose and d-glucose; and (3) 6-O-ethers and esters (with C12 alkyl chain) of methyl α-d-glycosides from d-mannose and d-glucose. Their antibacterial efficacies against P. larvae were determined and compared to reference compounds including fatty acids (lauric, sebacic, and 10-HDA) and their derivatives (monolaurin and monomethyl sebacate). The relations among chemical structures of the compounds and their anti-P. larvae efficacies were evaluated. Several new efficient compounds against the pathogen were identified. The potential of the most efficient compounds to increase the anti-P. larvae activity of larval food and improve the protection of larvae against AFB through their application (as individual substances) to honeybee colonies is discussed.

2. Results and Discussion

2.1. Synthesis

Glycolipid mimetics having C10 and C12 alkyl chains attached to d-mannose (1–4), d-glucose (5–7, 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].
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 ω-hydroxyl 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 BH3·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-d-glycosides 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.
The direct etherification or esterification of 43 and 44 with the corresponding alkyl bromides or acyl chlorides yielded the 6-O-subtituted glycosides 45, 46 [49], 47, and 48 [49]. In the last step, the removal of benzyl groups by catalytic hydrogenation provided the desired 6-O-ethers 49 and 51 [49] and 6-O-esters 50 [49] and 52 [49].

2.2. 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 (37–41) 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 (49–52) 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.
The thioglycosides (2, 4, 7, 22) were slightly more efficient than their O-analogs (1, 3, 5, 6). The oxidation of the glycosidic sulfur providing sulfones 18–21 led to a decrease in the inhibitory activity of the 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.

3. Materials and Methods

3.1. General

Thin layer chromatography (TLC) was performed on aluminium sheets precoated with silica gel 60 F254 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. 1H NMR and 13C 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, CDCl3 for 1H) or HOD (δ 4.87, CD3OD for 1H), and an internal CDCl3 (δ 77.00) or CD3OD (δ 49.00) for 13C. 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.

3.2. Synthesis

Decyl 2,3,4,6-tetra-O-acetyl-1-thio-β-d-glucopyranoside (12).
To a stirred solution containing 1,2,3,4,6-penta-O-acetyl-d-glucopyrannose (1.5 g, 3.84 mmol) in anhydrous CH2Cl2 (15 mL), 1-decanethiol (0.8 g, 0.97 mL, 4.6 mmol) was added. The reaction mixture was stirred for 20 min, cooled down on an ice bath, and BF3OEt2 (0.82 g, 0.73 mL, 5.76 mmol) was added dropwise. The resulting mixture was stirred for 15 min, brought to rt, and stirred for 2 h. The reaction mixture was diluted with CH2Cl2 (100 mL) and poured into ice-cold water (150 mL) under stirring. The organic phase was separated, washed with saturated aqueous NaHCO3 (3 × 75 mL), water (70 mL), dried (Na2SO4), filtered, and concentrated. Purification by column chromatography (hexane:EtOAc 7:1→3:1) gave 12. Yield 0.66 g, 34%, yellowish oil, [α]D—31.2 (c 0.5, CHCl3). Analytical data are in agreement with Szabo et al. [67]. HRMS (ESI) m/z: calcd for C24H41O9S [M+H]+: 521.2376; found: 521.2415.
General procedure for thioglycoside oxidation (Method A).
To a stirred and 0 °C precooled solution containing corresponding thioglycoside (0.3 mmol) in CH2Cl2 (10 mL) mCPBA (0.9 mmol) was added. The reaction mixture was stirred at rt for 2 h, then diluted with CH2Cl2 (10 mL) and washed with saturated aqueous NaHCO3 (2 × 10 mL) and water (10 mL). The organic phase was dried (Na2SO4), filtered, and concentrated. The crude product was purified by column chromatography (hexane:EtOAc).
Decyl 2,3,4,6-tetra-O-acetyl-α-d-mannopyranosyl sulfone (14).
Treatment of 10 [53] (0.20 g, 0.38 mmol) as described in the general procedure (Method A) and purification by column chromatography (hexane:EtOAc 3:1→1:1) gave 14. Yield 0.18 g, 85%, colourless oil. [α]D + 33.8 (c 0.55, CHCl3). 1H NMR (400 MHz, CDCl3): δ 5.95 (dd, 1H, J2,3 3.7 Hz, H-2), 5.59 (dd, 1H, J 9.1 Hz, H-3), 5.30 (t, 1H, J4,5 9.4 Hz, H-4), 4.83 (d, 1H, J 2.3 Hz, H-1), 4.66 (ddd, 1H, H-5), 4.28 (dd, 1H, J5,6b 5.6 Hz, J6a,6b 12.5 Hz, H-6b), 4.15 (dd, 1H, J5,6a 2.4 Hz, H-6a), 3.11 (ddd, 2H, J 2.6 Hz, J 6.5 Hz, J 9.5 Hz, SO2CH2), 2.16, 2.10, 2.07, 2.01 (each s, each 3H, 4 × OCOCH3), 1.92–1.25 (m, 16H, 8 × CH2), 0.88 (t, 3H, J 6.8 Hz, CH3). 13C NMR (100 MHz, CDCl3): δ 170.5, 169.8, 169.4, 169.4 (4 × OCOCH3), 87.8 (C-1), 73.6 (C-5), 69.0 (C-3), 65.5 (C-4), 65.0 (C-2), 62.6 (C-6), 51.1 (SO2CH2), 32.0, 29.6, 29.4, 29.3, 29.1, 28.7, 22.8, 21.7 (8 × CH2), 20.8(3×), 20.7 (4 × OCOCH3), 14.2 (CH3). HRMS (ESI) m/z: calcd for C24H41O11S [M+H]+: 553.2274; found: 553.2290.
Dodecyl 2,3,4,6-tetra-O-acetyl-α-d-mannopyranosyl sulfone (15).
The reaction was carried out according to the general procedure (Method A) with thiomannoside 11 [53] (0.20 g, 0.36 mmol). Column chromatography (hexane:EtOAc 3:1→1:1) gave 15. Yield 0.17 g, 83%, colourless oil. [α]D + 31.5 (c 0.82, CHCl3). 1H NMR (400 MHz, CDCl3): δ 5.95 (dd, 1H, J 3.7 Hz, H-2), 5.59 (dd, 1H, J 9.1Hz, H-3), 5.29 (t, 1H, J4,5 9.4 Hz, H-4), 4.83 (d, 1H, J 2.2 Hz, H-1), 4.66 (ddd, 1H, H-5), 4.28 (dd, 1H, J5,6b 5.6 Hz, J6a,6b 12.5 Hz, H-6b), 4.15 (dd, 1H, J5,6a 2.4 Hz, H-6a), 3.11 (ddd, 2H, J 2.5 Hz, J 6.8 Hz, J 9.6 Hz, SO2CH2), 2.16, 2.09, 2.06, 2.01 (each s, each 3H, 4 × OCOCH3), 1.91–1.24 (m, 20H, 10 × CH2), 0.88 (t, 3H, J 6.8 Hz, CH3). 13C NMR (100 MHz, CDCl3): δ 170.5, 169.8, 169.4, 169.3 (4 × OCOCH3), 87.8 (C-1), 73.6 (C-5), 69.0 (C-3), 65.5 (C-4), 65.0 (C-2), 62.6 (C-6), 51.1 (SO2CH2), 32.0, 29.7(2×), 29.6, 29.5, 29.4, 29.1, 28.7, 22.8, 21.7 (10 × CH2), 20.8(3×), 20.7 (4 × OCOCH3), 14.2 (CH3). HRMS (ESI) m/z: calcd for C26H45O11S [M+H]+: 565.2677; found: 565.2695.
Decyl 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl sulfone (16).
The reaction was carried out according to the general procedure (Method A) with thiomannoside 12 [53] (0.30 g, 0.59 mmol). Column chromatography (hexane:EtOAc 3:1→1:1) gave 16. Yield 0.24 g, 75%, yellowish oil. [α]D—18.8 (c 0.6, CHCl3). 1H NMR (400 MHz, CDCl3): δ 5.48 (t, 1H, J2,3 9.6 Hz, H-2), 5.31 (t, 1H, J3,4 9.3 Hz, H-3), 5.12 (t, 1H, H-4), 4.42 (d, 1H, J1,2 9.9 Hz, H-1), 4.27 (dd, 1H, J5,6b 4.9 Hz, J6a,6b 12.6 Hz, H-6b), 4.20 (dd, 1H, J5,6a 2.4 Hz, H-6a), 3.82 (ddd, 1H, J4,5 10.1 Hz, H-5), 3.09 (dt, 2H, J 6.6 Hz, J 9.5 Hz, SO2CH2), 2.08, 2.06, 2.04, 2.02 (each s, each 3H, 4 × OCOCH3), 1.47–1.24 (m, 16H, 8 × CH2), 0.87 (t, 3H, J 6.8 Hz, CH3). 13C NMR (100 MHz, CDCl3): δ 170.5, 170.2, 169.5, 169.4 (4 × OCOCH3), 87.9 (C-1), 77.0 (C-5), 73.3 (C-3), 67.6 (C-4), 66.6 (C-2), 61.6 (C-6), 49.4 (SO2CH2), 32.0, 29.6, 29.4(2×), 29.3, 29.2, 28.7, 22.8 (8 × CH2), 20.9, 20.8(2×), 20.7 (4 × OCOCH3), 14.2 (CH3). HRMS (ESI) m/z: calcd for C24H41O11S [M+H]+: 537.2364; found: 537.2371.
Dodecyl 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl sulfone (17).
The reaction was carried out according to the general procedure (Method A) with thiomannoside 13 [53] (0.35 g, 0.66 mmol). Column chromatography (hexane:EtOAc 4:1→2:1) gave 17. Yield 0.30 g, 80%, yellowish oil. [α]D—20.1 (c 0.5, CHCl3). 1H NMR (400 MHz, CDCl3): δ 5.48 (t, 1H, dd, J2,3 9.2 Hz, H-2), 5.31 (t, 1H, J3,4 9.3 Hz, H-3), 5.12 (t, 1H, J4,5 10.1 Hz, H-4), 4.42 (d, 1H, J1,2 10.0 Hz, H-1), 4.27 (dd, 1H, J5,6b 4.9 Hz, J6a,6b 12.6 Hz, H-6b), 4.21 (dd, 1H, J5,6a 2.5 Hz, H-6a), 3.82 (ddd, 1H, H-5), 3.09 (dt, 2H, J 6.6 Hz, J 9.5 Hz, SO2CH2), 2.09, 2.06, 2.04, 2.03 (each s, each 3H, 4 × OCOCH3), 1.92–1.78 (m, 2H, CH2), 1.46–1.24 (m, 18H, 9 × CH2), 0.87 (t, 3H, J 6.8 Hz, CH3). 13C NMR (100 MHz, CDCl3): δ 170.5, 170.2, 169.5, 169.4 (4 × OCOCH3), 87.9 (C-1), 77.0 (C-5), 73.3 (C-3), 67.6 (C-4), 66.6 (C-2), 61.6 (C-6), 49.4 (SO2CH2), 32.0, 29.7(2×), 29.6, 29.5, 29.4(2×), 29.2, 28.7, 22.8 (10 × CH2), 20.9, 20.8(2×), 20.7 (4 × OCOCH3), 14.3 (CH3). HRMS (ESI) m/z: calcd for C26H45O11S [M+H]+: 565.2677; found: 565.2689.
General procedure for deprotection (Method B).
The acetylated compound (0.25 mmol) was dissolved in anhydrous MeOH (9.5 mL), and MeONa (1M, 0.5 mL) was added. The reaction mixture was stirred for 16 h at rt, neutralized with Dowex 50 H+ form, filtered, and concentrated. The crude product was purified by column chromatography (EtOAc:MeOH).
Decyl α-d-mannopyranosyl sulfone (18).
The reaction was carried out according to the general procedure (Method B) with sulfone 14 (0.14 g, 0.25 mmol). Column chromatography (EtOAc:MeOH 0:1→7:1) gave 18. Yield 76.5 mg, 82%, colourless oil. [α]D + 47.5 (c 0.23, MeOH). 1H NMR (400 MHz, CD3OD): δ 4.95 (d, 1H, J1,2 1.5 Hz, H-1), 4.54 (dd, 1H, J2,3 3.8 Hz, H-2), 4.20–4.15 (m, 1H, H-5), 4.00 (dd, 1H, J3,4 9.3 Hz, H-3), 3.92 (dd, 1H, J5,6a 2.2 Hz, J6a,6b 12.1 Hz, H-6a), 3.77–3.65 (m, 2H, H-4, H-6b), 3.27–3.23 (m, 2H, SO2CH2), 1.89–1.81 (m, 2H, CH2), 1.55–1.31 (m, 14H, 7 × CH2), 0.94 (t, 3H, J 6.8 Hz, CH3). 13C NMR (100 MHz, CD3OD): δ 92.8 (C-1), 79.8 (C-5), 72.8 (C-3), 67.6 (C-4), 66.8 (C-2), 63.1 (C-6), 51.1 (SO2CH2), 33.0, 30.6, 30.5, 30.4, 30.2, 29.6, 23.7, 22.5 (8 × CH2), 14.4 (CH3). HRMS (ESI) m/z: calcd for C16H33O7S [M+H]+: 369.1942; found: 369.1949.
Dodecyl α-d-mannopyranosyl sulfone (19).
The reaction was carried out according to the general procedure (Method B) with sulfone 15 (0.14 g, 0.25 mmol). Column chromatography (EtOAc:MeOH 0:1→7:1) gave 19. Yield 79.3 mg, 80%, colourless oil. [α]D + 52.3 (c 0.22, MeOH). 1H NMR (400 MHz, CD3OD): δ 4.95 (d, 1H, J1,2 1.5 Hz, H-1), 4.53 (dd, 1H, J2,3 3.7 Hz, H-2), 4.20–4.14 (m, 1H, H-5), 4.00 (dd, 1H, J3,4 9.2 Hz, H-3), 3.92 (dd, 1H, J5,6a 2.2 Hz, J6a,6b 12.1 Hz, H-6a), 3.76–3.67 (m, 2H, H-4, H-6b), 3.27–3.22 (m, 2H, SO2CH2), 1.90–1.81 (m, 2H, CH2), 1.54–1.31 (m, 18H, 9 × CH2), 0.94 (t, 3H, J 6.8 Hz, CH3). 13C NMR (100 MHz, CD3OD): δ 92.8 (C-1), 79.8 (C-5), 72.8 (C-3), 67.7 (C-4), 66.8 (C-2), 63.2 (C-6), 51.1 (SO2CH2), 33.1, 30.6, 30.8, 30.7, 30.5, 30.5, 30.2, 29.6, 23.7, 22.5 (10 × CH2), 14.4 (CH3). HRMS (ESI) m/z: calcd for C18H37O7S [M+H]+: 397.2255; found: 397.2271.
Decyl β-d-glucopyranosyl sulfone (20).
The reaction was carried out according to the general procedure (Method B) with sulfone 16 (0.13 g, 0.25 mmol). Column chromatography (EtOAc:MeOH 0:1→ 6:1) gave 20. Yield 74.6 mg, 81%, yellowish solid. [α]D—11.2 (c 0.51, MeOH). 1H NMR (400 MHz, CD3OD): δ 4.43 (d, 1H, J1,2 9.5 Hz, H-1), 3.92 (dd, 1H, J5,6a 2.1 Hz, H-6a), 3.80 (t, 1H, J2,3 9.2 Hz, H-2), 3.71 (dd, 1H, J5,6b 6.0 Hz, J6a,6b 12.4 Hz, H-6b), 3.52–3.44 (m, 2H, H-3, H-5), 3.33–3.18 (m, 3H, H-4, SO2CH2), 1.90–1.82 (m, 2H, CH2), 1.54–1.32 (m, 14H, 7 × CH2), 0.93 (t, 3H, J 6.8 Hz, CH3). 13C NMR (100 MHz, CD3OD): δ 91.0 (C-1), 83.0 (C-5), 79.1 (C-3), 70.7, 70.6 (C-2, C-4), 62.6 (C-6), 52.0 (SO2CH2), 33.0, 30.6, 30.5, 30.4, 30.2, 29.6, 23.7, 22.2 (8 × CH2), 14.4 (CH3). HRMS (ESI) m/z: calcd for C16H33O7S [M+H]+: 369.1942; found: 369.1951.
Dodecyl β-d-glucopyranosyl sulfone (21).
The reaction was carried out according to the general procedure (Method B) with sulfone 17 (0.14 g, 0.25 mmol). Column chromatography (EtOAc:MeOH 0:1→ 6:1) gave 21. Yield 81.7 mg, 83%, yellowish solid. [α]D—20.1 (c 0.23, MeOH). 1H NMR (400 MHz, CD3OD): δ 4.44 (d, 1H, J1,2 9.5 Hz, H-1), 3.92 (dd, 1H, J5,6a 2.1 Hz, H-6a), 3.80 (t, 1H, J2,3 9.2 Hz, H-2), 3.71 (dd, 1H, J5,6b 6.0 Hz, J6a,6b 12.4 Hz, H-6b), 3.52–3.44 (m, 2H, H-3, H-5), 3.38–3.20 (m, 3H, H-4, SO2CH2), 1.90–1.83 (m, 2H, CH2), 1.54–1.31 (m, 18H, 9 × CH2), 0.94 (t, 3H, J 6.8 Hz, CH3). 13C NMR (100 MHz, CD3OD): δ 91.0 (C-1), 83.0 (C-5), 79.0 (C-3), 70.7, 70.6 (C-2, C-4), 62.6 (C-6), 52.0 (SO2CH2), 33.1, 30.7(2×), 30.6, 30.5(2×), 30.2, 29.6, 23.7, 22.2 (10 × CH2), 14.4 (CH3). HRMS (ESI) m/z: calcd for C18H37O7S [M+H]+: 397.2255; found: 397.2263.
Decyl 1-thio-β-d-glucopyranoside (22).
The reaction was carried out according to the general procedure (Method B) with thioglucoside 12 [67] (0.13 g, 0.25 mmol). Purification by column chromatography (EtOAc:MeOH 0:1→3:1) gave 22. Yield 65 mg, 75%, yellowish oil, [α]D—36.8 (c 0.83, MeOH). Analytical data are in agreement with Szabo et al. [67] HRMS (ESI) m/z: calcd for C16H33O5S [M+H]+: 337.2043; found: 337.2048.
Synthesis of acceptors 25, 26, and 28.
The acceptors methyl 10-hydroxydecanoate (25) and methyl 12-hydroxydodecanoate (26) were prepared according to the reported procedure [57] and their analytical data are in agreement with Yamamoto et al. [68].
(E)-methyl 10-hydroxydec-2-enoate (28).
The solution of 10-HDA 27 (0.10 g, 0.539 mmol) in anhydrous methanol (8 mL) was stirred with ion-exchange resin (Amberlite IR 120, H+ from) for 4 days at rt. The resin was filtered off and the solvent was evaporated. Purification of the residue by column chromatography (hexane:EtOAc 4:1→2:1) gave 28. Yield 78.5 mg, 73%, colorless oil. 1H NMR (400 MHz, CDCl3): δ 6.96 (dt, 1H, J 7.0 Hz, J 15.6 Hz, CH=), 5.81 (dt, 1H, J 1.6 Hz, J 15.6 Hz, CH=), 3.72 (s, 3H, OCH3), 3.63 (t, 2H, J 6.6 Hz, CH2OH), 2.21 (dd, 2H, J 1.6 Hz, J 7.1 Hz, CH2), 1.59–1.28 (m, 10H, 5 × CH2). 13C NMR (100 MHz, CDCl3): δ 167.3 (COOCH3), 149.8 (CH=), 121.0 (CH=), 51.5 (COOCH3), 63.1 (CH2OH), 32.8, 32.3, 29.3, 29.2, 28.1, 25.8 (7 × CH2). 13C NMR (400 MHz, CDCl3): δ 167.3 (COOCH3), 149.8 (CH=), 121.0 (CH=), 63.1 (CH2OH), 51.5 (COOCH3), 32.8, 32.3, 29.3, 29.2, 28.1, 25.8 (6 × CH2). HRMS (ESI) m/z: calcd for C11H20O3Na [M+Na]+: 223.1305; found: 223.1310.
General procedure for glycosylation (Method C).
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 CH2Cl2 (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 NaHCO3, the solid was filtered off through Celite and rinsed with CH2Cl2 (15 mL). The solvent was evaporated and the residue was purified by column chromatography (hexane:EtOAc).
Methyl 10-(2,3,4,6-tetra-O-benzoyl-α-d-mannopyranosyloxy)-decanoate (31).
Treatment of glycosyl donor 29 with acceptor 25 as described in the general procedure (Method C) and purification by column chromatography (hexane:EtOAc 6:1→3:1) afforded 31. Yield 0.56 g, 71%, colourless oil. [α]D—72.4 (c 0.96, CHCl3). 1H NMR (400 MHz, CDCl3): δ 8.10–8.05 (m, 4H, Harom), 7.97–7.95 (m, 2H, Harom), 7.85–7.82 (m, 2H, Harom), 7.61–7.25 (m, 12H, Harom), 6.09 (t, 1H, J4,5 10.0 Hz, H-4), 5.92 (dd, 1H, J2,3 3.3 Hz, J3,4 10.1 Hz, H-3), 5.69 (dd, 1H, H-2), 5.08 (d, 1H, J1,2 1.8 Hz, H-1), 4.69 (dd, 1H, J5,6a 2.5 Hz, H-6a), 4.49 (dd, 1H, J5,6b 4.6 Hz, J6a,6b 12.1 Hz, H-6b), 4.42 (ddd, 1H, H-5), 3.82 (dt, 1H, J 6.8 Hz, J 9.6 Hz, OCH2(CH2)8), 3.66 (s, 3H, COOCH3), 3.57 (dt, 1H, J 6.5 Hz, J 9.5 Hz, OCH2(CH2)8), 2.31 (t, 2H, J 7.5 Hz, CH2COOCH3), 1.72–1.60 (m, 4H, 2 × CH2), 1.42–1.24 (m, 10H, 5 × CH2). 13C NMR (100 MHz, CDCl3): δ 174.5 (COOCH3), 166.3, 165.7, 165.6, 165.5 (4 × C=O), 133.6, 133.5, 133.3, 133.2, 130.1, 130.0, 129.9, 129.8, 129.6, 129.3, 128.7, 128.6, 128.4 (Carom), 97.8 (C-1), 70.8 (C-2), 70.3 (C-3), 69.0, 68.9 (C-5, OCH2(CH2)8), 67.2 (C-4), 63.1 (C-6), 51.6 (COOCH3), 34.3, 29.6, 29.5(2×), 29.4, 29.3, 26.3, 25.1 (8 × CH2). HRMS (ESI) m/z: calcd for C45H48O12Na [M+Na]+: 803.3038; found: 803.3043.
Methyl 12-(2,3,4,6-tetra-O-benzoyl-α-d-mannopyranosyloxy)-dodecanoate (32).
Treatment of glycosyl donor 29 with acceptor 26 as described in the general procedure (Method C) and purification by column chromatography (hexane:EtOAc 6:1→3:1) afforded 32. Yield 0.65 g, 79%, colourless oil. [α]D—47.7 (c 1.0, CHCl3). 1H NMR (400 MHz, CDCl3): δ 8.10–8.05 (m, 4H, Harom), 7.97–7.95 (m, 2H, Harom), 7.85–7.82 (m, 2H, Harom), 7.61–7.25 (m, 12H, Harom), 6.09 (t, 1H, J4,5 10.0 Hz, H-4), 5.92 (dd, 1H, J2,3 3.3 Hz, J3,4 10.1 Hz, H-3), 5.69 (dd, 1H, H-2), 5.09 (d, 1H, J1,2 1.7 Hz, H-1), 4.69 (dd, 1H, J5,6a 2.5 Hz, H-6a), 4.49 (dd, 1H, J5,6b 4.6 Hz, J6a,6b 12.1 Hz, H-6b), 4.42 (ddd, 1H, H-5), 3.82 (dt, 1H, J 6.8 Hz, J 9.6 Hz, OCH2(CH2)10), 3.66 (s, 3H, COOCH3), 3.57 (dt, 1H, J 6.5 Hz, J 9.6 Hz, OCH2(CH2)10), 2.30 (t, 2H, J 7.6 Hz, CH2COOCH3), 1.74–1.60 (m, 4H, 2 × CH2), 1.44–1.25 (m, 14H, 7 × CH2). 13C NMR (100 MHz, CDCl3): δ 174.4 (COOCH3), 166.3, 165.6(2×), 165.5 (4 × C=O), 133.6, 133.5, 133.3, 133.2, 130.0(2×), 129.9(2×), 129.8, 129.6, 129.3, 128.7, 128.6, 128.4 (Carom), 97.7 (C-1), 70.8 (C-2), 70.3 (C-3), 69.0(2×) (C-5, OCH2(CH2)10), 67.2 (C-4), 63.1 (C-6), 51.6 (COOCH3), 34.2, 29.7(2×), 29.6, 29.5(2×), 29.4, 29.3, 26.3, 25.1 (10 × CH2). HRMS (ESI) m/z: calcd for C47H52O12Na [M+Na]+: 831.3351; found: 831.3356.
(E)-Methyl 10-(2,3,4,6-tetra-O-benzoyl-α-d-mannopyranosyloxy)dec-2-enoate (33).
Reaction of glycosyl donor 29 (0.25 g, 0.34 mmol) with acceptor 28 as described in the general procedure (Method C) and purification by column chromatography (hexane:EtOAc 6:1→3:1) afforded 33. Yield 0.22 g, 85%, yellowish oil. [α]D—58.3 (c 0.66, CHCl3). 1H NMR (400 MHz, CDCl3): δ 8.10–8.05 (m, 4H, Harom), 7.97–7.93 (m, 2H, Harom), 7.86–7.81 (m, 2H, Harom), 7.62–7.28 (m, 12H, Harom), 6.99 (dt, 1H, J 7.0 Hz, J 15.7 Hz, CH=), 6.09 (t, 1H, J4,5 10.0 Hz, H-4), 5.92 (dd, 1H, J2,3 3.4 Hz, J3,4 10.1 Hz, H-3), 5.84 (dt, J 1.6 Hz, J 15.6 Hz, CH=), 5.69 (dd, 1H, H-2), 5.08 (d, 1H, J1,2 1.8 Hz, H-1), 4.70 (dd, 1H, J5,6a 2.6 Hz, H-6a), 4.45 (dd, 1H, J5,6b 4.6 Hz, J6a,6b 12.0 Hz, H-6b), 4.42 (ddd, 1H, H-5), 3.82 (dt, 1H, J 6.7 Hz, J 9.7 Hz, OCH2(CH2)6), 3.72 (s, 3H, COOCH3), 3.57 (dt, 1H, J 6.7 Hz, J 9.7 Hz, OCH2(CH2)6), 2.25–2.19 (m, 2H, CH2), 1.74–1.66 (m, 2H, CH2), 1.50–1.12 (m, 8H, 4 × CH2). 13C NMR (100 MHz, CDCl3): δ 167.3, 166.4, 166.0, 165.3, 165.2 (5 × C=O), 149.8 (CH=), 133.6, 133.3, 133.2, 130.0(2×), 129.9, 129.2, 128.7, 128.6, 128.4 (Carom), 121.0 (CH=), 97.8 (C-1), 70.8 (C-2), 70.3 (C-3), 69.0, 68.9 (C-5, OCH2(CH2)6), 67.2 (C-4), 63.1 (C-6), 51.5 (COOCH3), 32.4, 29.5, 29.3, 29.2, 28.1, 26.2 (6 × CH2). HRMS (ESI) m/z: calcd for C47H50O12Na [M+Na]+: 829.3194; found: 829.3202.
Methyl 10-(2,3,4,6-tetra-O-benzoyl-β-d-glucopyranosyloxy)-decanoate (34).
Reaction of glycosyl donor 30 with acceptor 25 as described in the general procedure (Method C) and purification by column chromatography (hexane:EtOAc 6:1→3:1) afforded 34. Yield 0.56 g, 71%, yellowish oil. [α]D—6.7 (c 0.78, CHCl3). 1H NMR (400 MHz, CDCl3): δ 8.04–7.99 (m, 2H, Harom), 7.97–7.94 (m, 2H, Harom), 7.91–7.88 (m, 2H, Harom), 7.84–7.82 (m, 2H, Harom), 7.55–7.28 (m, 12H, Harom), 5.90 (t, 1H, J3,4 9.7 Hz, H-3), 5.67 (t, 1H, J4,5 9.7 Hz, H-4), 5.51 (dd, 1H, J2,3 7.8 Hz, H-2), 4.83 (d, 1H, J1,2 7.9 Hz, H-1), 4.63 (dd, 1H, J5,6a 3.4 Hz, J6a,6b 12.1 Hz, H-6a), 4.51 (dd, 1H, J5,6b 5.2 Hz, H-6b), 4.16 (ddd, 1H, H-5), 3.91 (1H, J 6.2 Hz, J 9.7 Hz, OCH2(CH2)8), 3.66 (s, 3H, COOCH3), 3.53 (dt, 1H, J 6.5 Hz, J 9.5 Hz, OCH2(CH2)8), 2.31 (t, 2H, J 7.5 Hz, CH2), 1.56–1.49 (m, 4H, 2 × CH2), 1.21–1.06 (m, 10H, 5 × CH2). 13C NMR (100 MHz, CDCl3): δ 174.4 (COOCH3), 166.3, 166.0, 165.4, 165.2 (4 × C=O), 133.5, 133.4, 133.30, 133.2, 130.0, 129.9, 129.8, 129.6, 129.0, 128.6, 128.5, 128.4 (Carom), 101.5 (C-1), 73.1 (C-3), 72.3, 72.1 (C-2, C-5), 70.5 (OCH2(CH2)8), 70.1 (C-4), 63.4 (C-6), 51.6 (COOCH3), 34.2, 29.5, 29.4, 29.3, 29.2(2×), 25.9, 25.1 (8 × CH2). HRMS (ESI) m/z: calcd for C45H48O12Na [M+Na]+: 803.3038; found: 803.3041.
Methyl 12-(2,3,4,6-tetra-O-benzoyl-β-d-glucopyranosyloxy)-dodecanoate (35).
Reaction of glycosyl donor 30 with acceptor 26 as described in the general procedure (Method C) and purification by column chromatography (hexane:EtOAc 6:1→3:1) afforded 35. Yield 0.53 g, 65%, yellowish oil. [α]D—40.3 (c 0.78, CHCl3). 1H NMR (400 MHz, CDCl3): δ 8.03–8.00 (m, 2H, H arom), 7.97–7.94 (m, 2H, Harom), 7.91–7.88 (m, 2H, Harom), 7.84–7.81 (m, 2H, Harom), 7.54–7.28 (m, 12H, Harom), 5.90 (t, 1H, J3,4 9.7 Hz, H-3), 5.67 (t, 1H, J4,5 9.7 Hz, H-4), 5.51 (dd, 1H, J2,3 7.8 Hz, H-2), 4.83 (d, 1H, J1,2 7.9 Hz, H-1), 4.63 (dd, 1H, J5,6a 3.4 Hz, J6a,6b 12.1 Hz, H-6a), 4.51 (dd, 1H, J5,6b 5.2 Hz, H-6b), 4.17 (ddd, 1H, H-5), 3.91 (1H, J 6.3 Hz, J 9.7 Hz, OCH2(CH2)10), 3.66 (s, 3H, COOCH3), 3.54 (dt, 1H, J 6.5 Hz, J 9.6 Hz, OCH2(CH2)10), 2.30 (t, 2H, J 7.5 Hz, CH2), 1.65–1.55 (m, 4H, 2 × CH2), 1.31–1.08 (m, 14H, 7 × CH2). 13C NMR (100 MHz, CDCl3): δ 174.5 (COOCH3), 166.3, 166.0, 165.4, 165.2 (4 × C=O), 133.5, 133.4, 133.3, 133.2, 130.0, 129.9, 129.8, 129.6, 129.0, 128.6, 128.5, 128.4 (Carom), 101.5 (C-1), 73.1 (C-3), 72.3, 72.1 (C-2, C-5), 70.5 (OCH2(CH2)10), 70.1 (C-4), 63.4 (C-6), 51.6 (COOCH3), 34.3, 29.6(2×), 29.5, 29.4, 29.3, 29.2(2×), 25.9, 25.1 (10 × CH2). HRMS (ESI) m/z: calcd for C47H52O12Na [M+Na]+: 831.3351; found: 831.3359.
(E)-Methyl 10-(2,3,4,6-tetra-O-benzoyl-β-d-glucopyranosyloxy)dec-2-enoate (36).
Reaction of glycosyl donor 30 (0.25 g, 0.34 mmol) with acceptor 28 as described in the general procedure (Method C) and purification by column chromatography (hexane:EtOAc 6:1→3:1) afforded 36. Yield 0.19 g, 71%, oil. [α]D—20.8 (c 0.65, CHCl3). 1H NMR (400 MHz, CDCl3): δ 8.03–7.99 (m, 2H, Harom), 7.97–7.95 (m, 2H, Harom), 7.91–7.89 (m, 2H, Harom), 7.84–7.81 (m, 2H, Harom), 7.56–7.27 (m, 12H, Harom), 6.91 (dt, 1H, J 7.0 Hz, J 15.6 Hz, CH=), 5.90 (t, 1H, J3,4 9.7 Hz, H-3), 5.77 (dt, J 1.6 Hz, J 15.6 Hz, CH=), 5.67 (t, 1H, H-4), 5.52 (dd, 1H, J2,3 9.8 Hz, H-2), 4.83 (d, 1H, J1,2 7.8 Hz, H-1), 4.63 (dd, 1H, J5,6a 3.3 Hz, H-6a), 4.51 (1H, J5,6b 5.2 Hz, J6a,6b 12.1 Hz, H-6b), 4.15 (ddd, 1H, J4,5 9.5 Hz, H-5), 3.91 (dt, 1H, J 6.1 Hz, J 9.7 Hz, OCH2(CH2)6), 3.73 (s, 3H, COOCH3), 3.53 (dt, 1H, J 6.6 Hz, J 9.8 Hz, OCH2(CH2)6), 2.11–2.05 (m, 2H, CH2), 1.54–1.44 (m, 2H, CH2), 1.34–1.04 (m, 10H, 5 × CH2). 13C NMR (100 MHz, CDCl3): δ 167.3, 166.3, 166.0, 165.4, 165.2 (5 × C=O), 149.8 (CH=), 133.6, 133.4, 133.2, 130.0, 129.9(2×), 129.8, 129.6, 129.0, 129.0, 128.5, 128.5, 128.4 (Carom), 120.9 (CH=), 101.5 (C-1), 73.1 (C-3), 72.3 (C-5), 72.1 (C-2), 70.4 (OCH2(CH2)6), 70.0 (C-4), 63.4 (C-6), 51.5 (COOCH3), 32.3, 29.5, 29.1(2×), 28.0, 25.8 (6 × CH2). HRMS (ESI) m/z: calcd for C47H50O12Na [M+Na]+: 829.3194; found: 829.3200.
General procedure for deprotection (Method D).
To a protected glycoside (0.5 g) in methanol (17 mL) 1M MeONa (340 µL) was added and the reaction was stirred at rt for 1 h. The solvent was evaporated. Water (2 mL) was added and THF was dropped into the cloudy solution until it became clear. LiOH (170 mg) was added and the reaction mixture was stirred at rt for 1 h (TLC EtOAc:MeOH 4:1) and neutralized with Amberlite IR120 (H+ form) to pH 6. The resin was filtered off, rinsed with THF and the solvent was evaporated. The residue was diluted with water (20 mL), freeze-dried (2×) from water (2 × 20mL), and purified by column chromatography (CHCl3:MeOH).
10-(α-d-Mannopyranosyloxy)-decanoic acid (37).
Treatment of 31 as described in the general procedure (Method D) and purification by column chromatography (CHCl3:MeOH 9:1→3:1) afforded 37. Yield 0.20 g, 92%, colourless oil. [α]D + 25.1 (c 0.58, MeOH). 1H NMR (400 MHz, CD3OD): δ 4.73 (d, 1H, J1,2 1.7 Hz, H-1), 3.82 (dd, 1H, J5,6a 2.4 Hz, J6a,6b 11.7 Hz, H-6°), 3.78 (dd, 1H, J2,3 3.3 Hz, H-2), 3.75–3.68 (m, 3H, H-3, H-6b, OCH2(CH2)8), 3.61 (t, 1H, J3,4 9.5 Hz, J4,5 9.5 Hz, H-4), 3.55–3.50 (m, 1H, H-5), 3.41 (dt, 1H, J 6.3 Hz, J 9.6 Hz, OCH2(CH2)8), 2.24 (t, 2H, J 7.5 Hz, CH2COOH), 1.63–1.56 (m, 4H, 2 × CH2), 1.40–1.30 (m, 10H, 5 × CH2). 13C NMR (100 MHz, CD3OD): δ 170.0 (COOH), 101.6 (C-1), 74.6 (C-5), 72.7, 72.3 (C-2, C-3), 68.7(2×) (C-4, OCH2(CH2)8COOH), 62.9 (C-6), 35.0, 29.2(2×), 29.1, 29.0(2×), 25.9, 25.3 (8 × CH2). HRMS (ESI) m/z: calcd for C16H30O8Na [M+Na]+: 373.1833; found: 373.1843.
12-(α-d-Mannopyranosyloxy)-dodecanoic acid (38).
Treatment of 32 as described in the general procedure (Method D) and purification by column chromatography (CHCl3:MeOH 9:1→3:1) afforded 38. Yield 0.21 g, 91%, colourless oil. [α]D + 25.5 (c 0.6, MeOH). 1H NMR (400 MHz, CD3OD): δ 4.72 (d, 1H, J1,2 1.7 Hz, H-1), 3.81 (dd, 1H, J5,6a 2.4 Hz, J6a,6b 11.7 Hz, H-6°), 3.77 (dd, 1H, J2,3 3.3 Hz, H-2), 3.74–3.66 (m, 3H, H-3, H-6b, OCH2(CH2)10), 3.61 (t, 1H, J3,4 9.5 Hz, J4,5 9.5 Hz, H-4), 3.54–3.50 (m, 1H, H-5), 3.40 (dt, 1H, J 6.3 Hz, J 9.6 Hz, OCH2(CH2)10), 2.23 (t, 2H, J 7.5 Hz, CH2COOH), 1.61–1.54 (m, 4H, 2 × CH2), 1.37–1.28 (m, 14H, 7 × CH2). 13C NMR (100 MHz, CD3OD): δ 169.9 (COOH), 101.5 (C-1), 74.6 (C-5), 72.7, 72.3 (C-2, C-3), 68.6(2×) (C-4, OCH2(CH2)10), 62.9 (C-6), 36.2, 30.6(3×), 30.5(2×), 30.4, 29.4, 27.3, 26.6 (10 × CH2). HRMS (ESI) m/z: calcd for C18H34O8Na [M+Na]+: 401.2146; found: 401.2152.
(E)-10-(α-d-Mannopyranosyloxy)-dec-2-enoic acid (39).
Treatment of 33 (0.20 g, 0.26 mmol) as described in the general procedure (Method D) and purification by column chromatography (CHCl3:MeOH 9:1→3:1→1:2) afforded 39. Yield 72 mg, 81%, oil. [α]D + 15.5 (c 0.32, MeOH). 1H NMR (400 MHz, CD3OD): δ 6.74 (dt, 1H, J 7.0 Hz, J 15.6 Hz, CH=), 5.81 (dt, 1H, J = 1.5 Hz, J 15.5 Hz, CH=), 4.73 (d, 1H, J1,2 1.7 Hz, H-1), 3.83 (dd, 1H, J5,6a 2.4 Hz, J6a,6b 11.7 Hz, H-6°), 3.78 (dd, 1H, J2,3 3.4 Hz, H-2), 3.75–3.67 (m, 3H, H-3, H-6b, OCH2(CH2)6), 3.59 (t, 1H, J3,4 9.5 Hz, J4,5 9.5 Hz, H-4), 3.52 (ddd, J5,6b 5.7 Hz, H-5), 3.42 (dt, 1H, J 6.3 Hz, J 9.7 Hz, OCH2(CH2)6), 2.20–2.14 (m, 2H, CH2), 1.61–1.28 (m, 10H, 5 × CH2). 13C NMR (100 MHz, CD3OD): δ 170.2 (COOH), 146.6 (CH=), 126.8 (CH=), 101.6 (C-1), 74.6 (C-5), 72.7, 72.3 (C-2, C-3), 68.7, 68.6 (C-4, OCH2(CH2)6), 62.9 (C-6), 33.0, 30.6, 30.3, 30.2, 29.8, 29.5 (6 × CH2). HRMS (ESI) m/z: calcd for C18H32O8Na [M+Na]+: 399.1989; found: 399.1995.
10-(β-d-Glucopyranosyloxy)-decanoic acid (40).
Treatment of 34 as described in the general procedure (Method D) and purification by column chromatography (CHCl3:MeOH 9:1→2.5:1) afforded 40. Yield 0.18 g, 81%, colourless oil. [α]D—5.8 (c 0.53, MeOH). 1H NMR (400 MHz, CD3OD): δ 4.26 (d, 1H, J1,2 7.8 Hz, H-1), 4.92–4.85 (m, 2H, H-6°, OCH2(CH2)8), 3.68 (dd, 1H, J5,6b 5.3 Hz, J6a,6b 11.9 Hz, H-6b), 3.54 (dt, 1H, J 6.3 Hz, J 9.6 Hz, OCH2(CH2)8), 3.36–3.26 (m, 3H, H-3, H-4, H-5), 3.18 (dd, 1H, J2,3 9.0 Hz, H-2), 2.23 (t, 2H, J 7.5 Hz, CH2COOH), 1.66–1.58 (m, 4H, 2 × CH2), 1.41–1.31 (m, 10H, 5 × CH2). 13C NMR (100 MHz, CD3OD): δ 170.1 (COOH), 104.4 (C-1), 78.2 (C-3), 77.9 (C-5), 75.1 (C-2), 71.7 (C-4), 70.9 (OCH2(CH2)8), 62.8 (C-6), 36.9, 30.8, 30.6(2×), 30.5(2×), 27.1, 26.8 (8 × CH2). HRMS (ESI) m/z: calcd for C16H30O8Na [M+Na]+: 373.1833; found: 373.1839.
12-(β-d-Glucopyranosyloxy)-dodecanoic acid (41).
Treatment of 35 as described in the general procedure (Method D) and purification by column chromatography (CHCl3:MeOH 9:1→2.5:1) afforded 41. Yield 0.17 g, 72%, yellowish oil. [α]D—5.1 (c 0.51, MeOH). 1H NMR (400 MHz, CD3OD): δ 4.26 (d, 1H, J1,2 7.8 Hz, H-1), 4.93–4.85 (m, 2H, H-6°, OCH2(CH2)10), 3.67 (dd, 1H, J5,6b 5.3 Hz, J6a,6b 11.9 Hz, H-6b), 3.54 (dt, 1H, J 6.3 Hz, J 9.6 Hz, OCH2(CH2)10), 3.35–3.26 (m, 3H, H-3, H-4, H-5), 3.18 (dd, 1H, J2,3 9.0 Hz, H-2), 2.27 (t, 2H, J 7.4 Hz, CH2COOH), 1.66–1.58 (m, 4H, 2 × CH2), 1.41–1.31 (m, 14H, 7 × CH2). 13C NMR (100 MHz, CD3OD): δ 170.2 (COOH), 104.4 (C-1), 78.1 (C-3), 77.9 (C-5), 75.1 (C-2), 71.7 (C-4), 70.9 (OCH2(CH2)10), 62.8 (C-6), 35.3, 30.8, 30.7(2×), 30.6(2×), 30.4, 30.3, 27.1, 26.2 (10 × CH2). HRMS (ESI) m/z: calcd for C18H34O8Na [M+Na]+: 401.2146; found: 401.2150.
(E)-10-(β-d-Glucopyranosyloxy)-dec-2-enoic acid (42).
Treatment of 36 (0.17 g, 0.22 mmol) as described in the general procedure (Method D) and purification by column chromatography (CHCl3:MeOH 10:1→5:1→1:2) afforded 42. Yield 57 mg, 75 %, oil. [α]D—3.2 (c 0.41, MeOH). 1H NMR (400 MHz, CD3OD): δ 6.86 (dt, 1H, J 7.0 Hz, J 15.6 Hz, CH=), 5.82 (dt, 1H, J 1.5 Hz, J 15.5 Hz, CH=), 4.26 (d, 1H, J1,2 7.8 Hz, H-1), 3.92 (dt, 1H, J 6.7 Hz, J 9.5 Hz, OCH2(CH2)6), 3.87 (dd, 1H, J5,6a 1.8 Hz, J6a,6b 11.8 Hz, H-6°), 3.68 (dd, 1H, J5,6b 5.3 Hz, J6a,6b 11.9 Hz, H-6b), 3.55 (dt, 1H, J 6.7 Hz, J 9.5 Hz, OCH2(CH2)6), 3.36–3.27 (m, H-3, H-4, H-5), 3.18 (dd, 1H, J2,3 9.0 Hz, H-2), 2.21 (dq, 2H, J 1.5 Hz, J 7.2 Hz, CH2), 1.68–1.61 (m, 2H, CH2), 1.50–1.36 (m, 8H, 4 × CH2). 13C NMR (100 MHz, CD3OD): δ 172.8 (COOH), 148.8 (CH=), 124.9 (CH=), 104.4 (C-1), 78.1 (C-3), 77.9 (C-5), 75.1 (C-2), 71.7 (C-4), 70.8 (OCH2(CH2)6), 62.8 (C-6), 33.0, 30.7, 30.3, 30.2, 29.4, 27.0 (6 × CH2). HRMS (ESI) m/z: calcd for C18H32O8Na [M+Na]+: 399.1989; found: 399.1996.
Methyl 2,3,4-tri-O-benzyl-6-O-dodecanyl-α-d-mannopyranoside (45).
Compound 43 [60] (0.21 g, 0.45 mmol) was dissolved in anhydrous DMF (3 mL), the solution was cooled to 0 °C and sodium hydride (60% in mineral oil, 21.6 mg, 0.9 mmol) was added. After 15 min, dodecyl bromide (0.17 g, 0.16 mL, 0.67 mmol) was added and the resulting mixture was brought to rt. The stirring was continued for 16 h. The reaction was quenched with methanol (2 mL), diluted with EtOAc (20 mL), and washed with water (5 × 10 mL). The organic phase was dried (Na2SO4), filtered, and concentrated. Purification by column chromatography (hexane:EtOAc 30:1→10:1) gave 45. Yield 0.21 g, 75%, oil. [α]D + 30.4 (c 0.22, CHCl3). 1H NMR (400 MHz, CDCl3): δ 7.39–7.27 (m, 15H, Harom), 4.93 (d, 1H, J 10.9 Hz, OCH2Ph), 4.76 (d, 1H, J1,2 1.8 Hz, H-1), 4.72 (br s, 2H, OCH2Ph), 4.63–4.60 (m, 3H, 2 × OCH2Ph), 3.95 (dd, 1H, H-3), 3.87 (dd, 1H, J3,4 9.4 Hz, H-4), 3.78 (dd, 1H, J2,3 2.5 Hz, H-2), 3.71–3.65 (m, 3H, H-5, H-6a, H-6b), 3.54 (dt, 1H, J 6.6 Hz, J 9.3 Hz, OCH2(CH2)10CH3), 3.42 (dt, 1H, J 6.9 Hz, J 9.3 Hz, OCH2(CH2)10CH3), 3.31 (s, 3H, OCH3), 1.62–1.25 (m, 20H, 10 × CH2), 0.88 (t, 3H, J 6.8 Hz, CH3). 13C NMR (100 MHz, CDCl3): δ 138.9, 138.7, 138.5, 128.5, 128.4, 128.0, 128.0, 127.7(2×), 127.6 (Carom), 99.1 (C-1), 80.4 (C-4), 75.2(2×) (OCH2Ph, C-3), 74.7 (C-2), 72.6(2×) (2 × OCH2Ph), 71.9, 71.7 (OCH2(CH2)10CH3, C-5), 70.2 (C-6), 54.8 (OCH3), 32.1, 29.9, 29.8(3×), 29.7(2×), 29.5, 26.4, 22.8 (10 × CH2), 14.3 (CH3). HRMS (ESI) m/z: calcd for C40H57O6 [M+H]+: 633.4150; found: 633.4158.
Methyl 2,3,4-tri-O-benzyl-6-O-lauroyl-α-d-mannopyranoside (46).
To a solution of 43 (0.21 g, 0.45 mmol) in anhydrous pyridine (3 mL), DMAP (10 mg) and lauroyl chloride (0.20 g, 0.21 mL, 0.90 mmol) were added. The solution was stirred at rt for 24 h and then concentrated. Purification of the residue by column chromatography (hexane:EtOAc 30:1→8:1) gave 46. Yield 0.23 g, 79%, oil. Analytical data are in agreement with Smith et al. [49].
Methyl 2,3,4-tri-O-benzyl-6-O-dodecanyl-α-d-glucopyranoside (47).
Treatment of 44 [61] (0.42 g, 0.9 mmol) by the same procedure as described for 45 gave 47 (0.54 g, 94 %), oil. 1H NMR (400 MHz, CDCl3): δ 7.37–7.27 (m, 15H, Harom), 4.97 (d, 1H, J1,2 10.8 Hz, H-1), 4.88 (d, 1H, J 10.9 Hz, OCH2Ph), 4.82 (d, 1H, J 11.2 Hz, OCH2Ph), 4.79 (d, 1H, J 12.8 Hz, OCH2Ph), 4.66 (d, 1H, J 12.1 Hz, OCH2Ph), 4.62–4.58 (m, 2H, OCH2Ph), 3.98 (t, 1H, J3,4 9.2 Hz, H-3), 3.73–3.45 (m, 6H, H-2, H-4, H-5, H-6a, H-6b, OCH2(CH2)10CH3), 3.36 (s, 3H, OCH3), 3.36–3.31 (m, 1H, OCH2(CH2)10CH3), 1.59–1.55 (m, 2H, CH2), 1.32–1.22 (m, 18H, 9 × CH2), 0.87 (t, 3H, J 6.8 Hz, CH3). HRMS (ESI) m/z: calcd for C40H57O6 [M+H]+: 633.4150; found: 633.4154.
Methyl 2,3,4-tri-O-benzyl-6-lauroyl-α-d-glucopyranoside (48).
Treatment of 44 (0.47 g, 1.02 mmol) by the same procedure as described for 46 gave 48 (0.61 g, 94 %), oil. Analytical data are in agreement with Smith et al. [49].
Methyl 6-O-dodecanyl-α-d-mannopyranoside (49).
To a solution of 45 (0.17 g, 0.27 mmol) in MeOH (20 mL) 10% Pd/C (0.1 g) was added. The reaction mixture was stirred under an H2 atmosphere for 4 h. The catalyst was filtered off and the filtrate was evaporated. The residue was purified by column chromatography (hexane:EtOAc 9:1→0:1) to afford 49. Yield 77.2 mg, 79%, oil. [α]D + 36.2 (c 0.46, CHCl3). 1H NMR (400 MHz, CDCl3): δ 4.73 (d, 1H, J1,2 1.6 Hz, H-1), 3.92 (dd, 1H, J2,3 3.0 Hz, H-2), 3.80–3.65 (m, 5H, H-3, H-4, H-5, H-6a, H-6b), 3.53 (dt, 1H, J 6.7 Hz, J 9.4 Hz, OCH2(CH2)10CH3), 3.49 (dt, 1H, J 6.9 Hz, J 9.4 Hz, OCH2(CH2)10CH3), 3.38 (s, 3H, OCH3), 3.20 (br s, 1H, OH), 2.86 (br s, 1H, OH), 2.63 (br s, 1H, OH), 1.62–1.55 (m, 2H, CH2), 0.87 (t, 3H, J 6.7 Hz, CH3). 13C NMR (100 MHz, CDCl3): δ 100.9 (C-1), 72.4 (OCH2(CH2)10CH3), 71.8(2×) (C-3, C-6), 70.6 (C-2), 70.5 (C-4), 69.3 (C-5), 55.2 (OCH3), 32.1, 29.8(3×), 29.7(2×), 29.6, 29.5, 26.2, 22.8 (10 × CH2), 14.3 (CH3). HRMS (ESI) m/z: calcd for C19H39O6 [M+H]+: 363.2741; found: 363.2745.
Methyl 6-O-lauroyl-α-d-mannopyranoside (50).
Treatment of 46 (0.27 g, 0.42 mmol) by the same procedure as described for 49 gave 50. Yield 83.8 mg, 72%, oil. Analytical data are in agreement with Smith et al. [49].
Methyl 6-O-dodecanyl-α-d-glucopyranoside (51).
Treatment of 47 (0.54 g, 0.85 mmol) by the same procedure as described for 49 gave 51 (0.26 g, 82 %) white solid. Analytical data are in agreement with Smith et al. [49].
Methyl 6-O-lauroyl-α-d-glucopyranoside (52).
Treatment of 48 (0.55 g, 0.85 mmol) by the same procedure as described for 49 gave 52 (0.31 g, 98 %) white solid. Analytical data are in agreement with Smith et al. [49].

3.3. Biology

Paenibacillus larvae strains.
The CCM 4483 strain with an ERIC I genotype and CCM 4486 with an ERIC II genotype were obtained from the Czech collection of microorganisms (CCM).
Bacterial cultivation.
The bacterial cultivations were performed in an MYPGP P. larvae cultivation medium [69]. The pH values of the media were adjusted to values of 6.6, i.e., lower than that of the original medium (7.2–7.4). The lower pH corresponded better with the pH conditions in the midguts of young larvae where P. larvae reproduce naturally [16]. The cultivations were performed at 35 °C, a temperature typical for honeybee hives.
Determination of minimal inhibitory concentrations.
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 × 105 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 Mithras2LB 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.

4. 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.

Author Contributions

Conceptualization, M.P., J.K. (Jaroslav Klaudiny) and J.K. (Juraj Kóňa); methodology, V.Š., M.Š., M.P. and J.K. (Jaroslav Klaudiny); formal analysis, V.Š., M.Š., M.P. and J.K. (Jaroslav Klaudiny); investigation, V.Š., M.Š. and M.P.; writing—original draft preparation, M.P., M.Š., J.K. (Jaroslav Klaudiny); writing—review and editing J.K. (Juraj Kóňa), M.P. and J.K. (Jaroslav Klaudiny); supervision, M.P. and J.K. (Jaroslav Klaudiny); project administration, J.K. (Jaroslav Klaudiny), M.P and J.K. (Juraj Kóňa). All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Scientific Grant Agency of the Ministry of Education of the Slovak Republic and the Slovak Academy of Sciences (the projects VEGA-2/0164/19 and 2/0010/23), the Slovak Research and Development Agency (the project APVV-19-0376) and the project implementation CEMBAM (Centre for Medical Bio-Additive Manufacturing and Research, ITMS2014+: 313011V358 supported by the Operational Programme Integrated Infrastructure funded by the European Regional Development Fund).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Molecules 28 02516 g001 550
Figure 1. The previously synthesized nonionic glycolipids.
Figure 1. The previously synthesized nonionic glycolipids.
Molecules 28 02516 g001
Molecules 28 02516 sch001 550
Scheme 1. Synthesis of the sulfones 18–21 and thioglucoside 22. Reagents and conditions: (a) mCPBA, CH2Cl2, 2 h, rt, 85% for 14, 83% for 15, 75% for 16, 80% for 17; (b) MeONa, MeOH, 16 h, rt, 82% for 18, 80% for 19, 81% for 20, 83% for 21, 75% for 22.
Scheme 1. Synthesis of the sulfones 18–21 and thioglucoside 22. Reagents and conditions: (a) mCPBA, CH2Cl2, 2 h, rt, 85% for 14, 83% for 15, 75% for 16, 80% for 17; (b) MeONa, MeOH, 16 h, rt, 82% for 18, 80% for 19, 81% for 20, 83% for 21, 75% for 22.
Molecules 28 02516 sch001
Molecules 28 02516 sch002 550
Scheme 2. Synthesis of the glycosyl acceptors 25, 26, and 28. Reagents and conditions: (a) 1M BH3.THF, THF, 16 h, rt, 78% for 25, 80% for 26; (b) MeOH, Amberlite IR 120, H+ form, 96 h, rt, 73%.
Scheme 2. Synthesis of the glycosyl acceptors 25, 26, and 28. Reagents and conditions: (a) 1M BH3.THF, THF, 16 h, rt, 78% for 25, 80% for 26; (b) MeOH, Amberlite IR 120, H+ form, 96 h, rt, 73%.
Molecules 28 02516 sch002
Molecules 28 02516 sch003 550
Scheme 3. Synthesis of ω-O-glycosylated fatty acids 37–42. Reagents and conditions: (a) 25, 26, or 28, TMSOTf, CH2Cl2, 20 min, rt, 71% for 31, 79% for 32, 85% for 33, 71% for 34, 65% for 35, 71% for 36; (b) MeONa, MeOH, 1 h, rt, then LiOH, 1h, rt, 92% for 37, 91% for 38, 81% for 39, 81% for 40, 72% for 41, 75% for 42.
Scheme 3. Synthesis of ω-O-glycosylated fatty acids 37–42. Reagents and conditions: (a) 25, 26, or 28, TMSOTf, CH2Cl2, 20 min, rt, 71% for 31, 79% for 32, 85% for 33, 71% for 34, 65% for 35, 71% for 36; (b) MeONa, MeOH, 1 h, rt, then LiOH, 1h, rt, 92% for 37, 91% for 38, 81% for 39, 81% for 40, 72% for 41, 75% for 42.
Molecules 28 02516 sch003
Molecules 28 02516 sch004 550
Scheme 4. Synthesis of 6-O-substituted glycosides 49–52. Reagents and conditions: (a) C12H25Br, NaH, DMF, 16 h, rt, 75% for 45, 94% for 47; C11H23COCl, DMAP, pyridine, 24 h, rt, 79% for 46, 94% for 48; (b) Pd/C, H2, MeOH, 4 h, rt, 79% for 49, 72% for 50, 82% for 51, 98% for 52.
Scheme 4. Synthesis of 6-O-substituted glycosides 49–52. Reagents and conditions: (a) C12H25Br, NaH, DMF, 16 h, rt, 75% for 45, 94% for 47; C11H23COCl, DMAP, pyridine, 24 h, rt, 79% for 46, 94% for 48; (b) Pd/C, H2, MeOH, 4 h, rt, 79% for 49, 72% for 50, 82% for 51, 98% for 52.
Molecules 28 02516 sch004
Molecules 28 02516 g002 550
Figure 2. The reference compounds.
Figure 2. The reference compounds.
Molecules 28 02516 g002
Table
Table 1. Antibacterial efficacy of carbohydrate lipid-like derivatives and the reference compounds against two P. larvae strains.
Table 1. Antibacterial efficacy of carbohydrate lipid-like derivatives and the reference compounds against two P. larvae strains.
Compound P. larvae CCM 4483 (a)P. larvae CCM 4486 (b)
MIC (µM)MIC (µg/mL)MIC (µM)MIC (µg/mL)
1C10Man400128.220064.1
2SC10Man10033.65016.8
3C12Man258.7258.7
4SC12Man259.112.54.6
5C10Glc800256.3800256.3
22SC10Glc400134.6400134.6
6C12Glc10034.85017.4
7SC12Glc5018.2259.1
8C10Gal800256.4400128.2
9C12Gal800278.8800278.8
18SO2C10Man400147.4400147.4
19SO2C12Man10039.75019.8
20SO2C10Glc800294.8400147.4
21SO2C12Glc10039.75019.8
37ManC10acid>6400>2242.6>6400>2242.6
38ManC12acid>6400>2242.1>6400>2242.1
39Man10-HDA>6400>2229.7>6400>2229.7
40GlcC10acid>6400>2242.6>6400>2242.6
41GlcC12acid>6400>2422.1>6400>2422.1
42Glc10-HDA>6400>2229.7>6400>2229.7
49MeMan6-Dod259.1259.1
50MeMan6-Lau5018.85018.8
51MeGlc6-Dod259.1259.1
52MeGlc6-Lau5018.85018.8
23Monomethyl sebacate64001384.164001384.1
2710-HDA6400119264001192
Sebacic acid 64001294.464001294.4
Lauric acid 50105010
Monolaurin 5013.7256.9
Ciprofloxacin <0.25<0.1<0.25<0.1
Tylosin tartrate <0.25<0.3<0.25<0.3
(a) ERIC I strain, (b) ERIC II strain, reference compounds are highlighted by bold letters. MIC (minimal inhibitory concentration) was determined by three independent tests which provided the same result for each compound
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Šamšulová, V.; Šedivá, M.; Kóňa, J.; Klaudiny, J.; Poláková, M. A Comparison of the Antibacterial Efficacy of Carbohydrate Lipid-like (Thio)Ether, Sulfone, and Ester Derivatives against Paenibacillus larvae. Molecules 2023, 28, 2516. https://doi.org/10.3390/molecules28062516

AMA Style

Šamšulová V, Šedivá M, Kóňa J, Klaudiny J, Poláková M. A Comparison of the Antibacterial Efficacy of Carbohydrate Lipid-like (Thio)Ether, Sulfone, and Ester Derivatives against Paenibacillus larvae. Molecules. 2023; 28(6):2516. https://doi.org/10.3390/molecules28062516

Chicago/Turabian Style

Šamšulová, Veronika, Mária Šedivá, Juraj Kóňa, Jaroslav Klaudiny, and Monika Poláková. 2023. "A Comparison of the Antibacterial Efficacy of Carbohydrate Lipid-like (Thio)Ether, Sulfone, and Ester Derivatives against Paenibacillus larvae" Molecules 28, no. 6: 2516. https://doi.org/10.3390/molecules28062516

APA Style

Šamšulová, V., Šedivá, M., Kóňa, J., Klaudiny, J., & Poláková, M. (2023). A Comparison of the Antibacterial Efficacy of Carbohydrate Lipid-like (Thio)Ether, Sulfone, and Ester Derivatives against Paenibacillus larvae. Molecules, 28(6), 2516. https://doi.org/10.3390/molecules28062516

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