Systematic and Stereoselective Total Synthesis of Mannosylerythritol

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Systematic and Stereoselective Total Synthesis of Mannosylerythritol Lipids (MELs) and Evaluation of their Antibacterial Activity Junki Nashida, Nobuya Nishi, Yoshiaki Takahashi, Chigusa Hayashi, Masayuki Igarashi, Daisuke Takahashi, and Kazunobu Toshima J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b00032 • Publication Date (Web): 02 Mar 2018 Downloaded from http://pubs.acs.org on March 2, 2018

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The Journal of Organic Chemistry

Systematic and Stereoselective Total Synthesis of Mannosylerythritol Lipids (MELs) and Evaluation of their Antibacterial Activity 1

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Junki Nashida, Nobuya Nishi, Yoshiaki Takahashi, Chigusa Hayashi, Masayuki Igarashi, Daisuke Takahashi * and 1, Kazunobu Toshima * 1

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan 2 Institute of Microbial Chemistry (BIKAKEN), 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan ABSTRACT: The total synthesis of the twenty homogeneous members of mannosylerythritol lipids (MELs) with different alkyl chain lengths was effectively and systematically accomplished from a strategically designed common key intermediate that was stereoselectively constructed by the borinic acid catalyzed β-mannosylation reaction. In addition, their antibacterial activities against gram-positive bacteria were evaluated. Our results demonstrated that not only the length of the alkyl chains but also the pattern of Ac groups on the mannose moiety were important factors for antibacterial activity.

Mannosylerythritol lipids (MELs) are natural glycolipid biosurfactants, which are produced in high yield by yeast strains of genus Pseudozyma,1 such as Pseudozyma antarctica (renamed from Candida antarctica) T-34.2 Structurally, MELs are composed of two parts, one is a hydrophilic 4-O-β-D-mannopyranosyl-D-erythritol moiety and the other is a hydrophobic moiety containing a mixture of C6-C18 fatty acyl chains at C2’ and C3’ of the mannose moiety. In addition, MELs possess one or two Ac groups at C4’ and/or C6’ of the mannose moiety. MEL-A possesses two Ac groups, whereas MEL-B and C possess one Ac group at C4’ and C6’, respectively. MEL-D does not possess Ac groups, as shown in Figure 1. Although only one total synthesis of the diastereomer type3 MEL-A(C12) bearing the 1-O-β-D-mannopyranosyl-Derythritol structure and its 1:1 mixture with MEL-A(C12) 4 has been reported by Crich et al.,4 the total synthesis of homogeneous MELs bearing the 4-O-β-D-mannopyranosyl-D-erythritol structure has not yet been reported. MELs exhibit significant biochemical and physicochemical properties including low toxicity, biodegradability, biocompatibility, and surface activity. Thus, MELs have attracted much attention in the fields of food, cosmetics, and pharmaceutical industries.5 In addition, it has been reported that MEL-A and B show high antibacterial activities against gram-positive bacteria and weak activities against gram-negative bacteria. The MIC values of MEL-A and B against grampositive bacteria were found to be smaller than those of other glycolipid biosurfactants, such as sucrose monocaprate (SE 10), sorbitan monolaurate (Span 20), and rhamnolipids (RLs).5a, 6 Based on these reported results, we were interested in knowing whether MELs possessed high antibacterial activities even against multidrug-resistant gram-positive bacteria7a including methicillin-resistant Staphylococcus aureus (MRSA)7b and vancomycin (VCM)-resistant Enterococci (VRE),7c which have been recognized as serious problems in nosocomial infections worldwide.7d However, in order to carry out detailed structure-activity relationship studies, chemical synthesis of homogeneous and structurally well-defined MELs is essential because heterogeneity always exists in MELs obtained from microbial products, especially with respect to the chain length of the fatty acids. In this study, we focused on the systematic and stereoselective total synthesis of MELs 1-20

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(Figure 1) with different fatty acid chain lengths and different patterns of Ac groups at C4’ and C6’ of the mannose moiety (MEL-A type 1-5, MEL-B type 6-10, MEL-C type 11-15, and MEL-D type 16-20) utilizing our recently developed borinic acid catalyzed direct stereoselective β-mannosylation reaction8 as a key step. In addition, we investigated the antibacterial activity of the chemically synthesized MELs against several gram-positive bacteria. To the best of our knowledge, this is the first report of detailed SAR studies regarding antibacterial activity using chemically synthesized homogeneous MELs.

Figure 1. Chemical structures of MELs 1-20.

The retrosynthetic analysis of MELs 1-20 is shown in Scheme 1. Compounds 1-20 could be synthesized from our designed common key intermediate 21 by introduction of two fatty acyl chains and Ac groups to the appropriate positions as needed, followed by deprotection of the TBDPS and acetonide groups, to minimize the number of synthetic steps and to afford sufficient quantities of MELs for biological assays. The key intermediate 21 could be prepared by the βstereoselective mannosylation of 1,2-anhydro mannose donor 22 and erythritol derivative acceptor 23 using a bis(4fluorophenyl)borinic acid (24)9 catalyst. Compounds 22 and 23 could be prepared from D-mannose (25) and D-glucose (26), respectively.

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The Journal of Organic Chemistry Scheme 1. Retrosynthetic analysis of MELs 1-20.

The total synthesis of MELs 1-20 is shown in Scheme 2. Initially, we synthesized the 1,2-anhydro donor 22 and glycosyl acceptor 23. Known mannoside 2710 was prepared from D-mannose (25) according to the reported procedure in 3 steps. Removal of Ac groups in 27, followed by selective silylation using t-butyldimethylsilyl chloride (TBSCl) of the primary alcohol in the mannoside, gave the desired 28 in 71% yield in 2 steps. Selective protection of the 3-OH with a pmethoxybenzyl (PMB) group using nBu2SnO, followed by deprotection of the TBS group, gave 29. Benzylation of the 4and 6-OH groups and ring opening of orthoester 30 with trimethylsilyl chloride (TMSCl), followed by epoxide formation using tBuOK, gave 1,2-anhydro mannose 22 in high yield. Next, glycosyl acceptor 23 was synthesized from 31,11 which was prepared from D-glucose (26) in 3 steps. Selective reduction of the benzylidene acetal in 31 gave 32. Acetonide protection of the 1,2-diol in 32, followed by removal of the benzyl group, gave alcohol 23. Glycosylation of 22 and 23 was carried out using a catalytic amount of borinic acid 24 in MeCN at 0 oC for 2 h, which resulted in a 99% yield of desired β-mannoside 33 with excellent stereoselectivity. The anomeric configuration of 33 was determined by the 1JCH coupling constant, 155 Hz.12 Oxidative deprotection of the PMB group using CAN gave the common key intermediate 21 in high yield. Acylation of 21 using hexanoyl (C6), octanoyl (C8), decanoyl (C10), dodecanoyl (C12), or tetradecanoyl (C14) chloride, followed by deprotection of the benzyl groups, gave diols 34-38, respectively. Acetylation of the diols 34-38, followed by removal of the TBDPS and acetonide groups, gave MEL-A 1-5, respectively. The 1H and 13C NMR data for samples of synthetic MEL-A 1-5 were in good agreement with those reported.4, 13 Selective acetylation of the primary hydroxyl groups of 34-38, followed by removal of the TBDPS and acetonide groups, gave MEL-B 6-10, respectively. Alternatively, selective silylation of the primary hydroxyl groups 34-38, followed by acetylation and removal of the TBDPS and acetonide groups, gave MEL-C 1115, respectively. The 1H and 13C NMR data for samples of synthetic MEL-B and C were identical to the natural products.13 Finally, removal of the TBDPS and acetonide groups of 34-38 gave MEL-D 16-20, respectively. The 1H NMR data for samples of synthetic MEL-D were identical to the reported data.14

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Scheme 2. Total synthesis of MELs 1-20.

With chemically synthesized MELs 1-20 in hand, their antibacterial activities against gram-positive bacterial strains, Micrococcus luteus (IFO3333), Staphylococcus aureus (FDA209P and MS16526, MRSA), Enterococci faecalis (JCM5803 and NCTC12201, VRE) and Enterococci faecium (JCM5804 and NCTC12202, VRE), were evaluated by the conventional agar dilution method. In this screening, VCM and RLs were also used as controls. The minimum inhibitory concentration (MIC) values for the compounds tested are given in Table 1. In the cases of M. luteus and S. aureus (FDA209P), the results showed that MEL-A and MEL-B exhibited antibacterial activities as previously reported by Kitamoto et al.6 In addition, it was revealed for the first time that MEL-A(C10) 3 and MEL-B(C10) 8, which are the main components of MEL-A [C8 (17.5%), C10 (71.3%), C12 (10.1%), C14 (1.1%)] and MEL-B [C8 (26.6%), C10 (58.6%), C12 (13.3%), C14 (1.5%)] produced from soybean oil by a yeast strain of P. antarctica T-34,2 were most effective among MEL-A 1-5 and MEL-B 6-10, respectively. The shorter and longer alkyl chain length MEL-A and MEL-B were less effective, and the order of increasing antibacterial activity was C10 > C8 > C12 ≈ C14 ≈ C6. These results clearly indicated that the antibacterial properties were strongly influenced by the length of the alkyl chain. The same tendency was observed in MEL-C 11-15 and MEL-D 16-20, with MELD 18 showing the strongest antibacterial activity among MELs and another biosurfactant RLs tested. Moreover, although MELs 1-20 did not, unfortunately, show antibacterial activity against a MRSA strain, as in the case of RLs, MEL-D 18 was

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found to show high antibacterial activities against both VCM-sensitive Enterococci (VSE) and VRE strains, indicating that not only the length of the alkyl chain but also the pattern of Ac groups on the mannose moiety were important factors for the antibacterial activity.

Table 1. Antibacterial activity of MELs 1-20.

In conclusion, the total synthesis of twenty homogeneous members of MELs, MEL-A, B, C, and D type, bearing 4-O-βD-mannopyranosyl-D-erythritol

structures with different alkyl chain lengths, was achieved effectively and systematically

from the common key intermediate 21, which was stereoselectively constructed by the borinic acid catalyzed βmannosylation reaction. Since our direct β-mannosylation method does not require the use of a donor with 2,6-, 3,6-, or 4,6-cyclic protection, which is essential for the most stereoselective β-mannosylation methods,15 the synthesis of different types of MEL derivatives would be feasible easily based on the present synthetic route. In addition, it was revealed for the first time that the length of the alkyl chain of the MELs significantly affected their antibacterial activities, and MELs with decanoyl groups, 3, 8, 13, and 18, exhibited the most effective activities against M. luteus and S. aureus (FDA209P). Furthermore, MEL-D 18 was found to be highly effective even against VRE strains tested in this study. Therefore, we anticipate that the results presented here will contribute to the development of novel MEL-based antibacterial agents against multidrug-resistant gram-positive bacteria. Although the mechanism of their antibacterial activity is still unclear at this stage, it has been previously reported that the physicochemical properties of biosurfactants are one of the factors responsible for the structure of microbial cells.16 Thus, investigation of their physicochemical properties and further structure-activity relationship studies on their antibacterial activity are currently underway in our laboratories.

EXPERIMENTAL SECTION General Experimental Methods. NMR spectra were recorded on a JEOL ECA-500 (500 MHz for 1H, 125 MHz for 13C) spectrometer. 1H NMR data are reported as follows; chemical shift in parts per million (ppm) downfield or upfield from

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CDCl3 (δ 7.26), CD3OD (δ 3.31), or tetramethyl silane (δ 0.00) integration, multiplicity (br = broad, s = singlet, d = doublet, t = triplet, q = quartet, and m = multiplet), and coupling constants (Hz). 13C NMR chemical shifts are reported in ppm downfield or upfield from CDCl3 (δ 77.0) or CD3OD (δ 49.0). For the measurement of the 1JCH coupling constant, undecoupled spectrum was obtained a gated decoupling technique; repetition time was 4.6 s, decoupling time 2.0 s. ESITOF Mass spectra were measured on a Waters LCT premier XE. High performance liquid chromatography (HPLC) was performed on JASCO apparatus with a Kaseisorb LC SIL-100-5 column (4.6 × 150 mm, Tokyo Chemical Industry Co., Ltd.). Detection of products was made by RI (refractive index) detector (JASCO, RI-2031 Plus). Melting points were determined on a micro hot-stage (Yanako MP-S3) and were uncorrected. Optical rotations were measured on a JASCO P-2200 polarimeter. Silica gel TLC and column chromatography were performed using Merck TLC 60F-254 and Silica Gel 60N (spherical, neutral, 63-210 µm) (Kanto Chemical Co., Inc.), respectively. Air- and/or moisture-sensitive reactions were carried out under an argon atmosphere using oven-dried glassware. Compound 28. To a solution of 2710 (3.70 g, 10.2 mmol, exo/endo = 92/8) in dry MeOH (37 mL) was added K2CO3 (141 mg, 1.02 mmol) at room temperature. After the reaction mixture was stirred for 2 h at the same temperature, the reaction mixture was concentrated in vacuo. To a solution of the residue in dry DMF (33.8 mL) were added 2,6-lutidine (5.07 mL, 43.4 mmol) and TBSCl (2.60 g, 17.2 mmol) at 0 °C. After the reaction mixture was stirred for 5 h at room temperature, the reaction was quenched by addition of sat. NaHCO3 aq. (5 mL). The resultant mixture was extracted with EtOAc (50 mL), and then the extracts were washed with sat. NaHCO3 aq. (30 mL), and brine (30 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (1/2 n-hexane/EtOAc) gave 28 (2.54 g, 7.25 mmol, 71% yield in 2 steps). White solid; Rf 0.62 (5/1 CHCl3/MeOH); [α]24D -8.2° (c 1.0, CHCl3); mp 104-105 oC; 1

H NMR (500 MHz, CDCl3) δ 5.45 (1H, d, J = 3.0 Hz), 4.50 (1H, dd, J = 3.0 and 4.0 Hz), 3.96 (1H, dd, J = 4.0 and 10.5 Hz),

3.88-3.74 (3H, m), 3.31 (3H, s), 3.30-3.25 (2H, m), 2.52 (1H, d, J = 7.0 Hz), 1.68 (3H, s), 0.90 (9H, s), 0.10 (6H, s); 13C NMR (125 MHz, CDCl3) δ 124.0, 97.5, 78.5, 73.0×2, 70.0, 64.2, 49.6, 25.8, 24.7, 18.2, -5.5, -5.6; HRMS (ESI-TOF) m/z [M+Na]+ calcd for C15H30O7NaSi 373.1659, found 373.1672. Compound 29. To a solution of 28 (2.99 g, 8.52 mmol) in dry toluene (85.3 mL) was added nBu2SnO (2.55 g, 10.2 mmol) at reflux. After the reaction mixture was stirred for 13 h, p-methoxybenzyl chloride (PMBCl) (1.39 mL, 10.2 mmol) and TBAI (4.73 g, 12.8 mmol) were added to the reaction mixture. The reaction mixture was stirred for 11 h at 90 °C, and then the reaction was quenched by addition of sat. NaHCO3 aq. (10 mL). The resultant mixture was extracted with EtOAc (50 mL), and then the extracts were washed with sat. NaHCO3 aq. (50 mL), and brine (50 mL), dried over anhydrous Na2SO4. After that, the resultant mixture was filtered through celite pad, and then concentrated in vacuo. To a solution of the residue in dry THF (42.6 mL) was added TBAF (25.6 mL, 1.0 M in THF, 25.6 mmol) at 0 °C. After the reaction mixture was stirred for 1 h at room temperature, the reaction was quenched by addition of sat. NaHCO3 aq. (5 mL). The resultant mixture was extracted with EtOAc (50 mL), and then the extracts were washed with sat. NaHCO3 aq. (50 mL), and brine (50 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (2/1 toluene/acetone) gave 29 (2.25 g, 6.31 mmol, 74% yield in 2 steps). Colorless syrup; Rf 0.29 (2/1 toluene/acetone); [α]24D -33.9° (c 0.83, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.35-7.30 (2H, m), 6.93-6.88 (2H, m), 5.42 (1H, d, J = 2.5 Hz), 4.75 and 4.61 (2H, ABq, J = 11.5 Hz), 4.44 (1H, dd, J = 2.5 and 3.5 Hz), 3.92-3.73 (6H, m), 3.54 (1H, dd, J = 3.5 and 9.5 Hz), 3.34 (1H, m), 3.28 (3H, s), 2.44 (1H, d, J = 2.0 Hz), 2.15 (1H, br-t, J = 6.5 Hz), 1.69 (3H, s); 13C NMR (125 MHz,

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CDCl3) δ 159.7, 129.7, 129.4, 123.9, 114.1, 97.9, 78.3, 75.9, 74.8, 71.5, 71.5, 66.7, 62.6, 55.3, 50.0, 24.1; HRMS (ESI-TOF) m/z [M+Na]+ calcd for C17H24O8Na 379.1369, found 379.1356. Compound 30. To a solution of 29 (2.24 g, 6.27 mmol) in dry DMF (31.3 mL) were added 60% NaH (1.50 g, dispersion in mineral oil, 37.6 mmol) at 0 °C under Ar atmosphere, followed by BnBr (4.48 mL, 37.6 mmol). After the reaction mixture was stirred for 12 h at room temperature, the reaction was quenched by addition of H2O (5 mL). The resultant mixture was extracted with n-hexane-EtOAc (1/1, v/v, 50 mL), and then the extracts were washed with brine (50 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (2/1 nhexane/EtOAc) gave 30 (2.34 g, 4.53 mmol, 71% yield). White solid; Rf 0.53 (8/1 toluene/acetone); [α]24D +32.4° (c 1.0, CHCl3); mp 125-126 oC; 1H NMR (500 MHz, CDCl3) δ 7.35-7.22 (12H, m), 6.88-6.84 (2H, m), 5.34 (1H, d, J = 2.5 Hz), 4.88 and 4.59 (2H, ABq, J = 11.0 Hz), 4.73 and 4.71 (2H, ABq, J = 11.5 Hz), 4.61 and 4.54 (2H, ABq, J = 12.0 Hz), 4.36 (1H, dd, J = 2.5 and 3.0 Hz), 3.90 (1H, dd, J = 9.5 and 9.5 Hz), 3.80 (3H, s), 3.76-3.68 (3H, m), 3.41 (1H, m), 3.29 (3H, s), 1.73 (3H, s); 13C NMR (125 MHz, CDCl3) δ 159.4, 138.2×2, 129.9, 129.7, 128.4, 128.3, 128.0, 127.7, 127.5, 123.9, 113.9, 97.5, 78.6, 77.2, 75.2, 74.2, 73.3, 72.0, 69.0, 55.3, 49.8, 24.4; HRMS (ESI-TOF) m/z [M+Na]+ calcd for C31H36O8Na 559.2308, found 559.2292. Compound 22. To a solution of 30 (2.32 g, 4.31 mmol) in dry CH2Cl2 (43.1 mL) was added TMSCl (821 µL, 6.47 mmol) at room temperature. After the reaction mixture was refluxed for 1 h, the reaction mixture was concentrated in vacuo. To a solution of the residue in dry THF (43.1 mL) was added tBuOK (1.45 g, 12.9 mmol) at -40 °C. After the reaction mixture was stirred for 30 min at -40 °C, the reaction mixture was poured into a solution of CHCl3/brine (= 2/1) (100 mL). The organic phase was washed with brine (30 mL), dried over anhydrous Na2SO4, and then filtered through celite pad. Purification of the residue by recrystallization (EtOAc/n-hexane) gave 22 (1.56 g, 3.36 mmol, 78% yield in 2 steps). White solid; Rf 0.38 (1/1 n-hexane/EtOAc); [α]24D +9.2° (c 1.0, CHCl3); mp 79-80 oC; 1H NMR (500 MHz, CDCl3) δ 7.35-7.25 (10H, m), 7.20-7.16 (2H, m), 6.88-6.84 (2H, m), 4.96 (1H, d, J = 2.5 Hz), 4.81 and 4.55 (2H, ABq, J = 10.5 Hz), 4.76 and 4.72 (2H, ABq, J = 11.0 Hz), 4.61 and 4.54 (2H, ABq, J = 12.5 Hz), 3.93-3.88 (2H, m), 3.80 (3H, s), 3.73 (1H, m), 3.67 (1H, dd, J = 2.0 and 11.0 Hz), 3.62 (1H, dd, J = 5.0 and 11.0 Hz), 3.30 (1H, dd, J = 2.5 and 3.0 Hz); 13C NMR (125 MHz, CDCl3) δ 159.4, 138.0×2, 130.0, 129.5, 128.4, 128.3, 128.0, 127.8×2, 127.6, 113.9, 78.9, 78.7, 78.1, 76.0, 75.1, 73.5, 71.8, 68.7, 55.3, 54.4; HRMS (ESI-TOF) m/z [M+H]+ calcd for C28H31O6 463.2121, found 463.2113. Compound 32. To a solution of 3111 (41.4 mg, 92.2 µmol) in dry CH2Cl2 (1.8 mL) were added Et3SiH (147 µL, 0.922 mmol) and trifluoroacetic acid (TFA) (70.6 µL, 0.922 mmol) at 0 °C. After the reaction mixture was stirred for 3 h at 0 °C, the reaction was quenched by addition of sat. NaHCO3 aq. (1 mL). The resultant mixture was extracted with CHCl3 (5 mL), and then the extracts were washed with sat. NaHCO3 aq. (5 mL), and brine (5 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (3/1 n-hexane/EtOAc) gave 32 (21.2 mg, 47.0 mmol, 51% yield). Colorless syrup; Rf 0.29 (2/1 n-hexane/EtOAc); [α]24D +4.1° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.69-7.62 (4H, m), 7.46-7.27 (11H, m), 4.54 (2H, s), 3.89-3.78 (3H, m), 3.73 (1H, m), 3.69 (1H, dd, J = 3.5 and 9.5 Hz), 3.63 (1H, dd, J = 6.0 and 9.5 Hz), 2.70 (1H, br-d, J = 5.5 Hz), 2.61 (1H, m), 1.06 (9H, s); 13C NMR (125 MHz, CDCl3)

δ 137.7, 135.5, 132.9, 132.8, 129.9, 128.5, 127.8×3, 73.6, 72.1, 71.5, 70.8, 65.0, 26.8, 19.2; HRMS (ESI-TOF) m/z [M+Na]+ calcd for C27H34O4NaSi 473.2124, found 473.2143. Compound 23. To a solution of 32 (111 mg, 0.245 mmol) in dry DMF (1.2 mL) were added 2,2-dimethoxypropane (1.11 mL, 9.02 mmol) and (+/-)-camphor-10-sulfonic acid (CSA) (5.70 mg, 24.5 µmol) at 0 °C. The reaction mixture was stirred for 2 h at room temperature, the reaction was quenched by addition of NEt3 (2 mL), and then concentrated in vacuo. To a

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solution of the residue in dry THF (817 µL) was added 20% Pd(OH)2/C (24.0 mg) at room temperature under H2 atmosphere (balloon). After the reaction mixture was stirred for 4 h at room temperature, the reaction mixture was filtered through celite pad, and then the filtrate was concentrated in vacuo. Purification of the residue by silica gel column chromatography (3/1 n-hexane/EtOAc) gave 23 (83.3 mg, 0.208 mmol, 85% yield in 2 steps). Colorless syrup; Rf 0.29 (4/1 nhexane/EtOAc); [α]24D +2.6° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.69-7.63 (4H, m), 7.48-7.37 (6H, m), 4.39 (1H, dd, J = 6.5 and 12.0 Hz), 4.29 (1H, m), 3.92 (1H, m), 3.87-3.78 (2H, m), 3.67 (1H, dd, J = 4.5 and 10.5 Hz), 2.76 (1H, dd, J = 6.5 and 7.5 Hz), 1.37 (3H, s), 1.33 (3H, s), 1.06 (9H, s); 13C NMR (125 MHz, CDCl3) δ 135.4, 132.5, 132.4, 129.9×2, 127.8, 108.3, 77.5, 76.6, 62.2, 61.0, 27.7, 26.7, 25.1, 19.0; HRMS (ESI-TOF) m/z [M+H]+ calcd for C23H33O4Si 401.2148, found 401.2154. Compound 33. To a solution of bis(4-fluoro)phenylborinic acid (24)9 (56.4 mg, 0.259 mmol) and 23 (518 mg, 1.29 mmol) in dry MeCN (15.5 mL) was added a solution of 22 (718 mg, 1.55 mmol) in dry MeCN (15.5 mL) at 0 °C under Ar atmosphere. After the reaction mixture was stirred for 2 h, the reaction was quenched by addition of 0.05 M NaBO3 aq. (0.285 mmol, 5.7 mL). To the resultant mixture was added sat. NH4Cl aq. (30 mL) and extracted with EtOAc (50 mL×3), and then the extracts were washed with brine (30 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. Purification of the residue by flash silica gel column chromatography (2/1 to 1/1 n-hexane/EtOAc) gave 33 (1.12 g, 1.29 mmol, 99% yield). Colorless syrup; Rf 0.35 (1/1 n-hexane/EtOAc); [α]24D -9.0° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.66-7.60 (4H, m), 7.45-7.34 (6H, m), 7.33-7.22 (12H, m), 4.88 (1H, d, J = 10.5 Hz), 4.69 and 4.59 (2H, ABq, J = 12.0 Hz), 4.63 (1H, d, J = 12.0 Hz), 4.53-4.42 (4H, m), 4.35 (1H, dd, J = 2.0 and 11.0 Hz), 4.20 (1H, m), 4.15 (1H, br-s), 3.96 (1H, dd, J = 9.5 and 9.5 Hz), 3.80 (3H, s), 3.76-3.63 (4H, m), 3.60 (1H, dd, J = 4.5 and 10.5 Hz), 3.53 (1H, dd, J = 3.0 and 9.5 Hz), 3.35 (1H, m), 2.40 (1H, s), 1.39 (3H, s), 1.33 (3H, s), 1.02 (9H, s); 13C NMR (125 MHz, CDCl3) δ 159.3, 138.4, 138.2, 135.6, 135.5, 133.0, 132.9, 129.9, 129.8×2, 129.5, 128.3×2, 128.0, 127.8, 127.7, 127.6, 127.5, 113.9, 108.8, 100.0 (1JCH = 155 Hz), 80.9, 76.5, 75.3, 75.1, 74.0, 73.5, 70.9, 68.7, 68.3, 68.0, 62.3, 55.2, 27.8, 26.8, 25.3, 19.1; HRMS (ESI-TOF) m/z [M+H]+ calcd for C51H63O10Si 863.4191, found 863.4171. Compound 21. To a solution of 33 (492 mg, 0.570 mmol) in MeCN/phosphate buffer (pH 7.2, 20 mM) (16.2 mL, 10/1) was added CAN (1.25 g, 2.28 mmol) at 0 °C. After the mixture was stirred for 2 h at the same temperature, the reaction mixture was quenched by addition of sat. NaHCO3 aq. (3 mL). The resultant mixture was extracted with EtOAc (20 mL), and then the extracts were washed with sat. NaHCO3 aq. (10 mL), and brine (10 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (1/1 n-hexane/EtOAc) gave 21 (355 mg, 0.477 mmol, 84% yield). Colorless syrup; Rf 0.13 (1/1 n-hexane/EtOAc); [α]24D -10.0° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.68-7.62 (4H, m), 7.45-7.21 (16H, m), 4.86 and 4.56 (2H, ABq, J = 11.5 Hz), 4.64 and 4.48 (2H, ABq, J = 12.0 Hz), 4.52 (1H, br-s), 4.46 (1H, m), 4.32 (1H, dd, J = 2.5 and 11.0 Hz), 4.21 (1H, m), 4.03 (1H, dd, J = 3.0 and 3.0 Hz), 3.80-3.65 (6H, m), 3.63 (1H, dd, J = 4.5 and 10.5 Hz), 3.34 (1H, m), 2.58 (1H, d, J = 9.0 Hz), 2.52 (1H, d, J = 3.0 Hz), 1.40 (3H, s), 1.34 (3H, s), 1.04 (9H, s); 13C NMR (125 MHz, CDCl3) δ 138.3, 138.1, 135.6, 133.0, 132.9, 129.8, 128.4, 128.3, 128.0, 127.9, 127.8, 127.6, 108.8, 99.9, 76.5, 75.8, 74.9, 74.8, 74.5, 73.5, 70.7, 68.7, 68.2, 62.3, 27.8, 26.8, 25.3, 19.1; HRMS (ESI-TOF) m/z [M+H]+ calcd for C43H55O9Si 743.3615, found 743.3619. General procedure for the synthesis of 34-38 from 21. To a solution of 21 (116-480 µmol, 1 equiv) in dry CH2Cl2 (1.24.8 mL) were added pyridine (0.696-2.88 mmol, 6 equiv), acyl chloride (hexanoyl, octanoyl, decanoyl, dodecanoyl, or tetradecanoyl chloride) (0.696-2.88 mmol, 6 equiv), and DMAP (23.2-96.0 µmol, 0.2 equiv) at room temperature. After the reaction mixture was stirred for 2 h at the same temperature, the reaction was quenched by addition of H2O (1 mL). The resultant mixture was extracted with CHCl3 (5 mL), and then the extracts were washed with H2O (4 mL), and brine (4

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mL), dried over anhydrous Na2SO4, and concentrated in vacuo. To a solution of the residue in dry THF (1.2-4.8 mL) was added 20% Pd(OH)2/C (100 wt%) at room temperature under H2 atmosphere (balloon). After the reaction mixture was stirred for 8 h at the same temperature, the reaction was filtered through celite pad, and the filtrate was concentrated in vacuo. Purification of the residue by silica gel column chromatography (2/1 to 1/1 n-hexane/EtOAc) gave 34-38. Compound 34. Yield 90% in 2 steps (79.0 mg, 104 µmol); colorless syrup; Rf 0.39 (1/1 n-hexane/EtOAc); [α]27D -32.4° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.70-7.61 (4H, m), 7.46-7.35 (6H, m), 5.52 (1H, br-d, J = 3.0 Hz), 4.91 (1H, dd, J = 3.0 and 10.0 Hz), 4.77 (1H, br-s), 4.36 (1H, m), 4.22 (1H, dd, J = 3.0 and 11.5 Hz), 4.19 (1H, m), 3.97 (1H, dd, J = 10.0 and 10.0 Hz), 3.89 (1H, dd, J = 3.5 and 12.0 Hz), 3.85 (1H, dd, J = 4.0 and 12.0 Hz), 3.77 (1H, dd, J = 8.5 and 11.5 Hz), 3.69 (1H, dd, J = 7.5 and 10.5 Hz), 3.62 (1H, dd, J = 4.5 and 10.5 Hz), 3.37 (1H, m), 2.36 (2H, t, J = 7.5 Hz), 2.29 (2H, t, J = 7.5 Hz), 1.68-1.55 (4H, m), 1.40 (3H, s), 1.33 (3H, s), 1.37-1.25 (8H, m), 1.05 (9H, s), 0.93-0.86 (6H, m); 13C NMR (125 MHz, CDCl3) δ 173.4, 172.8, 135.5, 132.9, 129.8, 127.7, 108.8, 98.7, 76.6, 75.7, 73.7, 68.8, 68.6, 65.9, 62.3, 62.2, 34.0, 31.1×2, 27.6, 26.7, 25.2, 24.7, 24.2, 22.2, 19.1, 13.9, 13.8; HRMS (ESI-TOF) m/z [M+NH4]+ calcd for C41H66NO11Si 776.4405, found 776.4377. Compound 35. Yield 81% in 2 steps (84.5 mg, 104 µmol); colorless syrup; Rf 0.43 (1/1 n-hexane/EtOAc); [α]27D -29.5° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.69-7.61 (4H, m), 7.47-7.35 (6H, m), 5.52 (1H, br-d, J = 3.0 Hz), 4.90 (1H, dd, J = 3.0 and 10.0 Hz), 4.78 (1H, br-s), 4.36 (1H, m), 4.22 (1H, dd, J = 3.0 and 11.5 Hz), 4.18 (1H, m), 3.98 (1H, dd, J = 10.0 and 10.0 Hz), 3.90 (1H, dd, J = 3.5 and 12.0 Hz), 3.86 (1H, dd, J = 4.0 and 12.0 Hz), 3.77 (1H, dd, J = 8.0 and 11.5 Hz), 3.69 (1H, dd, J = 7.0 and 11.0 Hz), 3.63 (1H, dd, J = 4.5 and 11.0 Hz), 3.37 (1H, m), 2.36 (2H, t, J = 7.5 Hz), 2.29 (2H, t, J = 7.5 Hz), 1.68-1.54 (4H, m), 1.40 (3H, s), 1.33 (3H, s), 1.34-1.22 (16H, m), 1.04 (9H, s), 0.92-0.85 (6H, m); 13C NMR (125 MHz, CDCl3) δ 173.4, 172.8, 135.5, 132.9, 129.8, 127.8, 108.8, 98.8, 76.6, 75.7, 73.7, 68.7, 68.6, 66.2, 62.3, 62.2, 34.0, 31.1×2, 27.7, 26.8, 25.2, 24.7, 24.2, 22.2, 19.1, 13.9, 13.8; HRMS (ESI-TOF) m/z [M+H]+ calcd for C45H71O11Si 815.4766, found 815.4801. Compound 36. Yield 80% in 2 steps (103 mg, 118 µmol); colorless syrup; Rf 0.46 (1/1 n-hexane/EtOAc); [α]24D -25.7° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.68-7.62 (4H, m), 7.47-7.36 (6H, m), 5.52 (1H, dd, J = 1.0 and 3.5 Hz), 4.89 (1H, dd, J = 3.5 and 10.0 Hz), 4.78 (1H, d, J = 1.0 Hz), 4.36 (1H, m), 4.22 (1H, dd, J = 2.5 and 11.5 Hz), 4.18 (1H, m), 3.98 (1H, dd, J = 10.0 and 10.0 Hz), 3.93-3.83 (2H, m), 3.77 (1H, dd, J = 8.0 and 11.0 Hz), 3.69 (1H, dd, J = 7.5 and 11.0 Hz), 3.62 (1H, dd, J = 5.0 and 11.0 Hz), 3.38 (1H, m), 2.36 (2H, t, J = 7.5 Hz), 2.29 (2H, t, J = 8.0 Hz), 1.67-1.55 (4H, m), 1.40 (3H, s), 1.33 (3H, s), 1.32-1.23 (24H, m), 1.04 (9H, s), 0.88 (6H, t, J = 7.5 Hz); 13C NMR (125 MHz, CDCl3) δ 173.5, 172.9, 135.5, 133.0, 129.8, 127.8, 108.8, 98.8, 76.6, 75.7, 73.9, 68.8, 68.7, 66.2, 62.3, 34.1, 31.9, 29.5, 29.4, 29.3×3, 29.1, 27.7, 26.8, 25.3, 25.1, 24.6, 22.7, 19.2, 14.1; HRMS (ESITOF) m/z [M+H]+ calcd for C49H79O11Si 871.5392, found 871.5413. Compound 37. Yield 80% in 2 steps (239 mg, 258 µmol); colorless syrup; Rf 0.50 (1/1 n-hexane/EtOAc); [α]27D -25.5° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.68-7.62 (4H, m), 7.47-7.36 (6H, m), 5.52 (1H, br-d, J = 3.0 Hz), 4.89 (1H, dd, J = 3.0 and 10.0 Hz), 4.78 (1H, br-s), 4.36 (1H, m), 4.22 (1H, dd, J = 3.0 and 11.5 Hz), 4.18 (1H, m), 3.98 (1H, m), 3.94-3.81 (2H, m), 3.77 (1H, dd, J = 8.0 and 11.5 Hz), 3.68 (1H, dd, J = 7.5 and 11.0 Hz), 3.63 (1H, dd, J = 4.5 and 11.0 Hz), 3.37 (1H, m), 2.36 (2H, t, J = 7.5 Hz), 2.29 (2H, t, J = 7.5 Hz), 1.68-1.55 (4H, m), 1.40 (3H, s), 1.33 (3H, s), 1.39-1.22 (32H, m), 1.04 (9H, s), 0.88 (6H, t, J = 7.5 Hz); 13C NMR (125 MHz, CDCl3) δ 173.5, 172.9, 135.5, 133.0, 129.8, 127.8, 108.8, 98.8, 76.6, 75.7, 73.9, 68.7×2, 66.2, 62.3, 34.1, 31.9, 29.6×2, 29.5×2, 29.3×2, 29.1, 27.7, 26.8, 25.2, 25.1, 24.6, 22.7, 19.1, 14.1; HRMS (ESI-TOF) m/z [M+NH4]+ calcd for C53H90NO11Si 944.6283, found 944.6313. Compound 38. Yield 79% in 2 steps (133 mg, 135 µmol); colorless syrup; Rf 0.54 (1/1 n-hexane/EtOAc); [α]24D -27.4° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.68-7.62 (4H, m), 7.48-7.35 (6H, m), 5.52 (1H, br-d, J = 3.0 Hz), 4.89 (1H, dd, J = 3.0

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and 10.0 Hz), 4.78 (1H, br-s), 4.36 (1H, m), 4.22 (1H, dd, J = 3.0 and 11.5 Hz), 4.18 (1H, m), 3.98 (1H, dd, J = 10.0 and 10.0 Hz), 3.94-3.81 (2H, m), 3.77 (1H, dd, J = 8.5 and 11.5 Hz), 3.69 (1H, dd, J = 7.5 and 11.0 Hz), 3.62 (1H, dd, J = 4.5 and 11.0 Hz), 3.37 (1H, m), 2.36 (2H, t, J = 7.5 Hz), 2.29 (2H, t, J = 7.5 Hz), 1.67-1.55 (4H, m), 1.40 (3H, s), 1.33 (3H, s), 1.33-1.22 (40H, m), 1.04 (9H, s), 0.88 (6H, t, J = 7.5 Hz); 13C NMR (125 MHz, CDCl3) δ 173.5, 172.9, 135.5, 133.0, 129.8, 127.8, 108.8, 98.8, 76.6, 75.7, 73.9, 68.7×2, 66.2, 62.3, 34.1×2, 31.9, 29.7×2, 29.6 29.5, 29.4, 29.3, 29.1, 27.7, 26.8, 25.3, 25.1, 24.6, 22.7, 19.2, 14.1; HRMS (ESI-TOF) m/z [M+H]+ calcd for C57H95O11Si 983.6644, found 983.6636. General procedure for the synthesis of MEL-A 1-5. To a solution of each compound 34-38 (15.2-33.9 µmol, 1 equiv) in dry pyridine (282-500 µL) were added Ac2O (282-500 µL) and DMAP (7.6-16.9 µmol, 0.5 equiv) at room temperature under Ar atmosphere, respectively. After the reaction mixture was stirred for 2 h at the same temperature, the reaction was quenched by addition of H2O (1 mL). The resultant mixture was extracted with EtOAc (2 mL), and then the extracts were washed with H2O (1 mL), and brine (1 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. To a solution of the each residue in dry MeCN (770-1425 µL) was added HF·py (76-170 µmol, 5 equiv) at room temperature under Ar atmosphere. After the reaction mixture was stirred for 24 h at the same temperature, 1 M HCl aq. (154-285 µL) was added to the reaction mixture and the mixture was stirred for 1 h. And then, the resultant mixture was quenched by addition of sat. NaHCO3 aq. (1 mL). The resultant mixture was extracted with EtOAc (2 mL), and then the extracts were washed with sat. NaHCO3 aq. (1 mL), and brine (1 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (2/1 to 1/1 toluene/acetone) gave a corresponding MEL-A. 4-O-(4,6-Di-O-acetyl-2,3-di-O-hexyl-β β-D-mannopyranosyl)-D-erythritol (1). Yield 80% in 2 steps (16.0 mg, 28.3 µmol); colorless syrup; Rf 0.36 (1/1 toluene/acetone); [α]27D -33.4° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 5.52 (1H, br-d, J = 3.0 Hz), 5.26 (1H, dd, J = 10.0 and 10.0 Hz), 5.08 (1H, dd, J = 3.5 and 10.0 Hz), 4.72 (1H, d, J = 0.5 Hz), 4.25 (1H, dd, J = 5.5 and 12.0 Hz), 4.20 (1H, dd, J = 2.5 and 12.0 Hz), 4.00 (1H, dd, J = 3.5 and 10.5 Hz), 3.85 (1H, dd, J = 6.0 and 10.5 Hz), 3.81-3.64 (5H, m), 3.01 (1H, br-s), 2.69 (1H, br-s), 2.49-2.38 (2H, m), 2.32 (1H, br-s), 2.28-2.19 (2H, m), 2.11 (3H, s), 2.04 (3H, s), 1.751.61 (2H, m), 1.60-1.50 (2H, m), 1.40-1.20 (8H, m), 0.93-0.86 (6H, m); 13C NMR (125 MHz, CDCl3) δ 173.4, 172.7, 170.7, 169.4, 99.3, 72.5, 72.3, 71.8, 71.2, 70.6, 68.5, 65.9, 63.6, 62.4, 34.1, 33.9, 31.1, 24.7, 24.3, 22.3, 22.2, 20.7×2, 13.9, 13.8; HRMS (ESI-TOF) m/z [M+H]+ calcd for C26H45O13 565.2860, found 565.2864. 4-O-(4,6-Di-O-acetyl-2,3-di-O-octyl-β β -D-mannopyranosyl)-D-erythritol (2). Yield 78% in 2 steps (10.1 mg, 16.3 µmol); colorless syrup; Rf 0.38 (1/1 toluene/acetone); [α]27D -30.3° (c 0.72, CHCl3); 1H NMR (500 MHz, CDCl3) δ 5.51 (1H, brd, J = 3.5 Hz), 5.25 (1H, dd, J = 10.0 and 10.0 Hz), 5.07 (1H, dd, J = 3.5 and 10.0 Hz), 4.72 (1H, br-s), 4.26 (1H, dd, J = 5.5 and 12.0 Hz), 4.20 (1H, dd, J = 2.5 and 12.0 Hz), 4.00 (1H, dd, J = 3.5 and 10.5 Hz), 3.85 (1H, dd, J = 6.0 and 10.5 Hz), 3.81-3.64 (5H, m), 3.02 (1H, br-s), 2.68 (1H, br-s), 2.49-2.38 (2H, m), 2.26-2.19 (2H, m), 2.11 (3H, s), 2.04 (3H, s), 1.75-1.59 (2H, m), 1.58-1.50 (2H, m), 1.39-1.21 (16H, m), 0.91-0.85 (6H, m); 13C NMR (125 MHz, CDCl3) δ 173.3, 172.7, 170.7, 169.4, 99.3, 72.5, 72.4, 71.8, 71.2, 70.5, 68.5, 65.9, 63.6, 62.4, 34.1, 34.0, 31.7, 31.6, 29.0×2, 28.9×2, 25.0, 24.7, 22.6×2, 20.7×2, 14.1; HRMS (ESI-TOF) m/z [M+NH4]+ calcd for C30H56NO13 638.3752, found 638.3757. 4-O-(4,6-Di-O-acetyl-2,3-di-O-decyl-β β -D-mannopyranosyl)-D-erythritol (3). Yield 79% in 2 steps (12.9 mg, 19.1 µmol, HPLC purity >99%); colorless syrup; Rf 0.43 (1/1 toluene/acetone); [α]27D -30.1° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3)

δ 5.51 (1H, dd, J = 0.5 and 3.0 Hz), 5.25 (1H, dd, J = 10.0 and 10.0 Hz), 5.07 (1H, dd, J = 3.0 and 10.0 Hz), 4.72 (1H, d, J = 0.5 Hz), 4.25 (1H, dd, J = 5.5 and 12.0 Hz), 4.20 (1H, dd, J = 2.5 and 12.0 Hz), 4.00 (1H, dd, J = 3.5 and 10.5 Hz), 3.85 (1H, dd, J = 6.0 and 10.5 Hz), 3.81-3.63 (5H, m), 3.00 (1H, d, J = 6.5 Hz), 2.67 (1H, d, J = 5.5 Hz), 2.48-2.38 (2H, m), 2.25-2.20 (2H, m), 2.11

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(3H, s), 2.04 (3H, s), 1.70-1.61 (2H, m), 1.58-1.50 (2H, m), 1.40-1.21 (24H, m), 0.90-0.85 (6H, m); 13C NMR (125 MHz, CDCl3)

δ 173.4, 172.7, 170.7, 169.4, 99.3, 72.5, 72.4, 71.8, 71.2, 70.6, 68.5, 65.9, 63.6, 62.4, 34.1, 34.0, 31.9, 31.8, 29.4×2, 29.3×2, 29.2, 29.1, 29.0, 25.0, 24.7, 22.7, 20.7×2, 14.1; HRMS (ESI-TOF) m/z [M+K]+ calcd for C34H60O13K 715.3671, found 715.3657; tR = 7.46 min (HPLC conditions for Figure S67) . 4-O-(4,6-Di-O-acetyl-2,3-di-O-dodecyl-β β -D-mannopyranosyl)-D-erythritol (4). Yield 72% in 2 steps (8.1 mg, 11.1 µmol); colorless syrup; Rf 0.48 (1/1 toluene/acetone); [α]27D -25.7° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 5.51 (1H, br-d, J = 3.0 Hz), 5.25 (1H, dd, J = 10.0 and 10.0 Hz), 5.07 (1H, dd, J = 3.5 and 10.0 Hz), 4.71 (1H, br-s), 4.25 (1H, dd, J = 5.5 and 12.0 Hz), 4.21 (1H, dd, J = 2.5 and 12.0 Hz), 4.00 (1H, dd, J = 3.0 and 10.5 Hz), 3.86 (1H, dd, J = 6.0 and 10.5 Hz), 3.82-3.64 (5H, m), 2.94 (1H, br-s), 2.58 (1H, br-s), 2.49-2.38 (2H, m), 2.25-2.20 (2H, m), 2.11 (3H, s), 2.04 (3H, s), 1.69-1.50 (4H, m), 1.40-1.21 (32H, m), 0.88 (6H, t, J = 6.5 Hz); 13C NMR (125 MHz, CDCl3) δ 173.4, 172.7, 170.8, 169.4, 99.3, 72.6, 72.4, 71.8, 71.2, 70.5, 68.5, 65.9, 63.6, 62.5, 34.1, 34.0, 31.9×2, 29.6×2, 29.5, 29.4, 29.3×2, 29.2, 29.1, 29.0, 25.0, 24.7, 22.7, 20.7×2, 14.1; HRMS (ESI-TOF) m/z [M+H]+ calcd for C38H69O13 733.4738, found 733.4731. 4-O-(4,6-Di-O-acetyl-2,3-di-O-tetradecyl-β β -D-mannopyranosyl)-D-erythritol (5). Yield 83% in 2 steps (13.5 mg, 17.1 µmol); colorless syrup; Rf 0.54 (1/1 toluene/acetone); [α]27D -24.8° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 5.51 (1H, dd, J = 1.0 and 3.0 Hz), 5.25 (1H, dd, J = 10.0 and 10.0 Hz), 5.07 (1H, dd, J = 3.0 and 10.0 Hz), 4.72 (1H, d, J = 1.0 Hz), 4.25 (1H, dd, J = 6.0 and 12.0 Hz), 4.20 (1H, dd, J = 3.0 and 12.0 Hz), 4.00 (1H, dd, J = 3.0 and 11.0 Hz), 3.85 (1H, dd, J = 6.0 and 10.9 Hz), 3.81-3.64 (5H, m), 3.01 (1H, br-s), 2.67 (1H, br-s), 2.49-2.38 (2H, m), 2.27-2.16 (2H, m), 2.11 (3H, s), 2.04 (3H, s), 1.73-1.60 (2H, m), 1.58-1.49 (2H, m), 1.38-1.20 (40H, m), 0.88 (6H, t, J = 7.0 Hz); 13C NMR (125 MHz, CDCl3) δ 173.4, 172.7, 170.7, 169.4, 99.3, 72.5, 72.4, 71.8, 71.2, 70.6, 68.5, 65.9, 63.6, 62.4, 34.1, 34.0, 31.9, 29.7×3, 29.5×2, 29.3×2, 29.2, 29.1, 29.0, 25.0, 24.7, 22.7, 20.7×2, 14.1; HRMS (ESI-TOF) m/z [M+K]+ calcd for C42H80NO13 806.5630, found 806.5654. General procedure for the synthesis of MEL-B 6-10. To a solution of each compound 34-38 (17.8-25.8 µmol, 1 equiv) in dry CH2Cl2 (890-1290 µL) were added NEt3 (35.6-51.6 µmol, 2 equiv) and Ac2O (21.4-31.0 µmol, 1.2 equiv) at room temperature under Ar atmosphere. After the reaction mixture was stirred for 24 h at the same temperature, the reaction was quenched by addition of MeOH (500 µL), and then concentrated in in vacuo. To a solution of the each residue in dry MeCN (860-1035 µL) was added HF·py (89.0-129 µmol, 5 equiv) at room temperature under Ar atmosphere. After the reaction mixture was stirred for 24 h at the same temperature, 1 M HCl aq. (172-207 µL) was added to the reaction mixture and the mixture was stirred for 1 h. And then, the resultant mixture was quenched by addition of sat. NaHCO3 aq. (1 mL). The resultant mixture was extracted with EtOAc (2 mL), and then the extracts were washed with sat. NaHCO3 aq. (1 mL), and brine (1 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (2/1 to 1/1 toluene/acetone) gave a corresponding MEL-B. 4-O-(6-O-Acetyl-2,3-di-O-hexyl-β β-D-mannopyranosyl)-D-erythritol (6). Yield 63% in 2 steps (8.5 mg, 16.2 µmol); colorless syrup; Rf 0.29 (1/1 toluene/acetone); [α]27D -44.6° (c 0.92, CHCl3); 1H NMR (500 MHz, CDCl3) δ 5.51 (1H, br-d, J = 3.0 Hz), 4.92 (1H, dd, J = 3.0 and 10.0 Hz), 4.71 (1H, br-s), 4.46 (1H, dd, J = 5.0 and 12.0 Hz), 4.42 (1H, dd, J = 2.5 and 12.0 Hz), 3.99 (1H, dd, J = 3.0 and 10.5 Hz), 3.86 (1H, dd, J = 5.5 and 10.5 Hz), 3.83-3.63 (5H, m), 3.56 (1H, m), 3.06 (1H, br-s), 2.70 (2H, br-s), 2.40 (2H, t, J = 7.5 Hz), 2.35-2.25 (2H, m), 2.14 (3H, s), 1.73-1.53 (4H, m), 1.38-1.23 (8H, m), 0.91-0.85 (6H, m); 13C NMR (125 MHz, CDCl3) δ 173.5, 173.3, 171.6, 99.4, 74.6, 73.1, 72.2, 71.9, 71.2, 68.7, 65.6, 63.6, 63.1, 34.1, 34.0, 31.1, 24.7, 24.3, 22.3, 22.2, 20.8, 13.9×2; HRMS (ESI-TOF) m/z [M+H]+ calcd for C24H43O12 523.2755, found 523.2778.

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Page 12 of 17

4-O-(6-O-Acetyl-2,3-di-O-octyl-β β-D-mannopyranosyl)-D-erythritol (7). Yield 68% in 2 steps (8.8 mg, 15.2 µmol); colorless syrup; Rf 0.28 (1/1 toluene/acetone); [α]27D -39.0° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 5.50 (1H, br-d, J = 3.5 Hz), 4.92 (1H, dd, J = 3.5 and 10.0 Hz), 4.71 (1H, br-s), 4.46 (1H, dd, J = 5.0 and 12.0 Hz), 4.42 (1H, dd, J = 2.5 and 12.0 Hz), 3.99 (1H, dd, J = 3.0 and 10.5 Hz), 3.85 (1H, dd, J = 5.5 and 10.5 Hz), 3.83-3.63 (5H, m), 3.57 (1H, m), 3.03 (1H, d, J = 5.5 Hz), 2.73-2.60 (1H, m), 2.40 (2H, t, J = 7.5 Hz), 2.36-2.24 (2H, m), 2.14 (3H, s), 1.70-1.55 (4H, m), 1.38-1.21 (16H, m), 0.92-0.84 (6H, m); 13C NMR (125 MHz, CDCl3) δ 173.5, 173.3, 171.6, 99.4, 74.6, 73.1, 72.3, 71.9, 71.2, 68.7, 65.6, 63.6, 63.1, 34.2, 34.1, 31.7, 31.6, 29.0×3, 28.9, 25.0, 24.7, 22.6×2, 20.8, 14.0; HRMS (ESI-TOF) m/z [M+H]+ calcd for C28H51O12 579.3381, found 579.3380. 4-O-(6-O-Acetyl-2,3-di-O-decyl-β β-D-mannopyranosyl)-D-erythritol (8). Yield 72% in 2 steps (10.2 mg, 16.1 µmol, HPLC purity >99%); colorless syrup; Rf 0.37 (1/1 toluene/acetone); [α]27D -34.7° (c 0.74, CHCl3); 1H NMR (500 MHz, CDCl3)

δ 5.50 (1H, d, J = 3.0 Hz), 4.92 (1H, dd, J = 3.5 and 10.0 Hz), 4.72 (1H, s), 4.48-4.40 (2H, m), 3.99 (1H, dd, J = 3.0 and 10.5 Hz), 3.88-3.63 (6H, m), 3.58 (1H, m), 3.13 (1H, d, J = 4.5 Hz), 2.83 (1H, br-s), 2.70 (1H, d, J = 5.0 Hz), 2.40 (2H, t, J = 7.5 Hz), 2.352.25 (2H, m), 2.13 (3H, s), 1.68-1.54 (4H, m), 1.38-1.20 (24H, m), 0.91-0.85 (6H, m); 13C NMR (125 MHz, CDCl3) δ 173.5, 173.3, 171.6, 99.4, 74.6, 73.1, 72.2, 71.9, 71.2, 68.7, 65.6, 63.6, 63.1, 34.2, 34.1, 31.9, 29.5, 29.4, 29.3×3, 29.1×2, 25.1, 24.7, 22.7, 20.8, 14.1; HRMS (ESI-TOF) m/z [M+H]+ calcd for C32H59O12 635.4007, found 635.3992; tR = 6.43 min (HPLC conditions for Figure S68). 4-O-(6-O-Acetyl-2,3-di-O-dodecyl-β β -D-mannopyranosyl)-D-erythritol (9). Yield 72% in 2 steps (8.8 mg, 12.7 µmol); colorless syrup; Rf 0.38 (1/1 toluene/acetone); [α]26D -32.6° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 5.50 (1H, br-d, J = 3.0 Hz), 4.92 (1H, dd, J = 3.0 and 10.0 Hz), 4.72 (1H, br-s), 4.48-4.38 (2H, m), 3.99 (1H, dd, J = 2.5 and 10.5 Hz), 3.90-3.62 (6H, m), 3.57 (1H, m), 3.10 (1H, br-s), 2.71 (2H, br-s), 2.40 (2H, t, J = 7.5 Hz), 2.30 (2H, t, J = 7.0 Hz), 2.14 (3H, s), 1.70-1.51 (4H, m), 1.38-1.20 (32H, m), 0.88 (6H, t, J = 7.0 Hz); 13C NMR (125 MHz, CDCl3) δ 173.5, 173.3, 171.6, 99.4, 74.6, 73.1, 72.2, 71.9, 71.2, 68.7, 65.6, 63.6, 63.1, 34.2, 34.1, 31.9, 29.6×2, 29.5×2, 29.3×2, 29.1×2, 25.0, 24.7, 22.7, 20.8, 14.1; HRMS (ESI-TOF) m/z [M+H]+ calcd for C36H67O12 691.4633, found 691.4653. 4-O-(6-O-Acetyl-2,3-di-O-tetradecyl-β β -D-mannopyranosyl)-D-erythritol (10). Yield 73% in 2 steps (11.6 mg, 15.6 µmol); colorless syrup; Rf 0.43 (1/1 toluene/acetone); [α]29D -33.3° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 5.50 (1H, br-d, J = 3.5 Hz), 4.92 (1H, dd, J = 3.5 and 10.0 Hz), 4.71 (1H, br-s), 4.48-4.40 (2H, m), 3.99 (1H, dd, J = 3.0 and 10.5 Hz), 3.85 (1H, dd, J = 5.5 and 10.5 Hz), 3.83-3.63 (5H, m), 3.57 (1H, m), 3.07 (1H, br-s), 2.71 (1H, br-s), 2.68 (1H, d, J = 4.0 Hz), 2.40 (2H, t, J = 7.5 Hz), 2.35-2.27 (2H, m), 2.14 (3H, s), 1.68-1.54 (4H, m), 1.38-1.22 (40H, m), 0.88 (6H, t, J = 7.2 Hz); 13C NMR (125 MHz, CDCl3) δ 173.5, 173.3, 171.6, 99.4, 74.6, 73.1, 72.3, 71.9, 71.2, 68.7, 65.6, 63.6, 63.1, 34.2, 34.1, 31.9, 29.7×2, 29.6, 29.5, 29.4, 29.3, 29.1×2, 25.1, 24.7, 22.7, 20.8, 14.1; HRMS (ESI-TOF) m/z [M+K]+ calcd for C40H74O12K 785.4817, found 785.4845. General procedure for the synthesis of MEL-C 11-15. To a solution of each compound 34-38 (17.1-30.3 µmol, 1 equiv) in dry DMF (683-1212 µL) were added imidazole (68.4-121 µmol, 4 equiv) and TBSCl (61.6-109 µmol, 3.6 equiv) at 0 °C. After the reaction mixture was stirred for 1 h at room temperature, the reaction was quenched by addition of sat. NaHCO3 aq. (1 mL). The resultant mixture was extracted with EtOAc (2 mL), and then the extracts were washed with sat. NaHCO3 aq. (1 mL), and brine (1 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was passed through silica gel column chromatography (4/1 n-hexane/EtOAc) gave the crude product. To a solution of the each crude product in dry pyridine (286-600 µL) were added Ac2O (286-600 µL) and DMAP (8.55-15.2 µmol, 0.5 equiv) at room temperature under Ar atmosphere. After the reaction mixture was stirred for 2 h at the same temperature, the reaction was quenched by addition of H2O (1 mL). The resultant mixture was extracted with EtOAc (2 mL), and then the extracts were washed

12


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with H2O (1 mL), and brine (1 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. To a solution of the residue in dry MeCN (730-1555 µL) was added HF·py (85.5-152 µmol, 5 equiv) at room temperature under Ar atmosphere. After the reaction mixture was stirred for 24 h at the same temperature, 1 M HCl aq. (146-310 µL) was added to the reaction mixture and the mixture was stirred for 1 h. And then, the resultant mixture was quenched by addition of sat. NaHCO3 aq. (1 mL). The resultant mixture was extracted with EtOAc (2 mL), and then the extracts were washed with sat. NaHCO3 aq. (1 mL), and brine (1 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (2/1 to 1/1 toluene/acetone) gave a corresponding MEL-C. 4-O-(4-O-Acetyl-2,3-di-O-hexyl-β β-D-mannopyranosyl)-D-erythritol (11). Yield 52% in 3 steps (6.0 mg, 11.4 µmol); colorless syrup; Rf 0.14 (1/1 toluene/acetone); [α]27D -34.9° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 5.52 (1H, dd, J = 1.0 and 3.5 Hz), 5.21 (1H, dd, J = 10.0 and 10.0 Hz), 5.11 (1H, dd, J = 3.5 and 10.0 Hz), 4.75 (1H, d, J = 1.0 Hz), 4.04 (1H, dd, J = 3.0 and 10.0 Hz), 3.84-3.71 (5H, m), 3.71-3.64 (2H, m), 3.53 (1H, m), 3.01 (1H, br-s), 2.77 (1H, br-s), 2.59 (1H, br-s), 2.48-2.38 (2H, m), 2.28-2.17 (2H, m), 2.06 (3H, s), 1.70-1.52 (4H, m), 1.38-1.22 (8H, m), 0.93-0.86 (6H, m); 13C NMR (125 MHz, CDCl3)

δ 173.4, 172.7, 170.2, 99.2, 74.8, 71.9×2, 71.2, 70.5, 68.6, 66.1, 63.6, 61.4, 34.0×2, 31.1×2, 24.7, 24.4, 22.3, 22.2, 20.7, 13.9, 13.8; HRMS (ESI-TOF) m/z [M+H]+ calcd for C24H43O12 523.2755, found 523.2744. 4-O-(4-O-Acetyl-2,3-di-O-octyl-β β-D-mannopyranosyl)-D-erythritol (12). Yield 54% in 3 steps (9.0 mg, 15.6 µmol); colorless syrup; Rf 0.17 (1/1 toluene/acetone); [α]27D -33.7° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 5.51 (1H, dd, J = 0.5 and 3.5 Hz), 5.19 (1H, dd, J = 10.0 and 10.0 Hz), 5.11 (1H, dd, J = 3.5 and 10.0 Hz), 4.76 (1H, d, J = 0.6 Hz), 4.04 (1H, dd, J = 2.5 and 10.0 Hz), 3.83-3.70 (5H, m), 3.71-3.64 (2H, m), 3.53 (1H, m), 3.27 (1H, br-s), 3.04 (1H, br-s), 2.91 (1H, br-s), 2.71 (1H, br-s), 2.48-2.38 (2H, m), 2.28-2.19 (2H, m), 2.06 (3H, s), 1.68-1.58 (2H, m), 1.58-1.50 (2H, m), 1.38-1.20 (16H, m), 0.92-0.84 (6H, m); 13

C NMR (125 MHz, CDCl3) δ 173.5, 172.7, 170.2, 99.2, 74.9, 71.9, 71.1, 70.6, 68.6, 66.1, 63.5, 61.4, 34.1, 34.0, 31.7, 31.6, 29.0×2,

28.9×2, 25.0, 24.7, 20.7, 14.1, 14.0; HRMS (ESI-TOF) m/z [M+H]+ calcd for C28H51O12 579.3381, found 579.3397. 4-O-(4-O-Acetyl-2,3-di-O-decyl-β β-D-mannopyranosyl)-D-erythritol (13). Yield 50% in 3 steps (11.3 mg, 11.3 µmol, HPLC purity >99%); colorless syrup; Rf 0.22 (1/1 toluene/acetone); [α]30D -28.9° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3)

δ 5.51 (1H, d, J = 3.0 Hz), 5.19 (1H, dd, J = 10.0 and 10.0 Hz), 5.11 (1H, dd, J = 3.0 and 10.0 Hz), 4.76 (1H, s), 4.04 (1H, m), 3.833.63 (7H, m), 3.53 (1H, m), 3.29 (1H, br-s), 3.08 (1H, br-s), 2.93 (1H, br-s), 2.70 (1H, br-s), 2.48-2.38 (2H, m), 2.28-2.17 (2H, m), 2.06 (3H, s), 1.68-1.59 (2H, m), 1.59-1.49 (2H, m), 1.38-1.21 (24H, m), 0.91-0.85 (6H, m); 13C NMR (125 MHz, CDCl3)

δ 173.5, 172.7, 170.2, 99.2, 74.9, 71.9, 71.1, 70.6, 68.6, 66.1, 63.5, 61.4, 34.1, 34.0, 31.9, 31.8, 29.5, 29.4, 29.3×3, 29.2, 29.1, 29.0, 25.0, 24.7, 14.1; HRMS (ESI-TOF) m/z [M+H]+ calcd for C32H59O12 635.4007, found 635.3998; tR = 13.00 min (HPLC conditions for Figure S69). 4-O-(4-O-Acetyl-2,3-di-O-dodecyl-β β -D-mannopyranosyl)-D-erythritol (14). Yield 56% in 3 steps (11.2 mg, 16.2 µmol); colorless syrup; Rf 0.19 (1/1 toluene/acetone); [α]27D -28.0° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 5.51 (1H, dd, J = 1.0 and 3.0 Hz), 5.19 (1H, dd, J = 10.0 and 10.0 Hz), 5.11 (1H, dd, J = 3.0 and 10.0 Hz), 4.76 (1H, d, J = 1.0 Hz), 4.04 (1H, dd, J = 3.0 and 10.0 Hz), 3.83-3.70 (5H, m), 3.70-3.64 (2H, m), 3.53 (1H, m), 3.24 (1H, br-s), 3.01 (1H, br-s), 2.87 (1H, br-s), 2.63 (1H, brs), 2.48-2.37 (2H, m), 2.28-2.18 (2H, m), 2.06 (3H, s), 1.70-1.58 (2H, m), 1.58-1.49 (2H, m), 1.38-1.21 (32H, m), 0.90-0.86 (6H, m); 13C NMR (125 MHz, CDCl3) δ 173.4, 172.7, 170.2, 99.2, 74.8, 71.9, 71.8, 71.2, 70.5, 68.6, 66.1, 63.6, 61.4, 34.1, 34.0, 31.9×2, 29.6×2, 29.5×2, 29.3×2, 29.2, 29.1, 29.0, 25.0, 24.7, 22.7, 20.7, 14.1; HRMS (ESI-TOF) m/z [M+H]+ calcd for C36H67O12 691.4633, found 691.4642.

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4-O-(4-O-Acetyl-2,3-di-O-tetradecyl-β β -D-mannopyranosyl)-D-erythritol (15). Yield 55% in 3 steps (8.0 mg, 9.2 µmol); white solid; Rf 0.23 (1/1 toluene/acetone); [α]27D -25.6° (c 0.64, CHCl3); mp 105-106 oC; 1H NMR (500 MHz, CDCl3)

δ 5.51 (1H, dd, J = 0.5 and 3.0 Hz), 5.20 (1H, dd, J = 10.0 and 10.0 Hz), 5.11 (1H, dd, J = 3.0 and 10.0 Hz), 4.75 (1H, d, J = 0.5 Hz), 4.04 (1H, dd, J = 3.0 and 10.0 Hz), 3.84-3.63 (7H, m), 3.52 (1H, m), 3.08 (1H, br-s), 2.84 (1H, d, J = 3.0 Hz), 2.68 (1H, brs), 2.47-2.36 (2H, m), 2.28-2.18 (2H, m), 2.06 (3H, s), 1.70-1.58 (2H, m), 1.58-1.50 (2H, m), 1.36-1.21 (40H, m), 0.88 (6H, t, J = 7.5 Hz); 13C NMR (125 MHz, CDCl3) δ 173.5, 172.7, 170.2, 99.2, 74.9, 71.9×2, 71.1, 70.5, 68.6, 66.1, 63.5, 61.4, 34.1, 34.0, 31.9, 29.7, 29.5×2, 29.4×3, 29.3, 29.1×2, 25.0, 24.7, 22.7, 20.7, 14.1; HRMS (ESI-TOF) m/z [M+H]+ calcd for C40H78NO12 764.5524, found 764.5535. General procedure for the synthesis of MEL-D 16-20. To a solution of each compound 34-38 (17.6-35.9 µmol, 1 equiv) in dry MeCN (865-1565 µL) was added HF·py (88.0-180 µmol, 5 equiv) at room temperature under Ar atmosphere. After the reaction mixture was stirred for 24 h at the same temperature, 1 M HCl aq. (173-313 µL) was added to the reaction mixture and the mixture was stirred for 1 h. And then, the resultant mixture was quenched by addition of sat. NaHCO3 aq. (1 mL). The resultant mixture was extracted with EtOAc (2 mL), and then the extracts were washed with sat. NaHCO3 aq. (1 mL), and brine (1 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (2/1 to 1/1 toluene/acetone) gave a corresponding MEL-D. 4-O-(2,3-Di-O-hexyl-β β -D-mannopyranosyl)-D-erythritol (16). Yield 72% (6.6 mg, 13.8 µmol); colorless syrup; Rf 0.42 (4/1 CHCl3/MeOH); [α]27D -48.7° (c 0.57, CHCl3); 1H NMR (500 MHz, 9/1 CDCl3/CD3OD) δ 5.46 (1H, br-d, J = 3.0 Hz), 4.91 (1H, dd, J = 3.0 and 10.0 Hz), 4.73 (1H, s), 4.00 (1H, dd, J = 3.0 and 10.0 Hz), 3.91 (1H, dd, J = 3.0 and 12.0 Hz), 3.87-3.64 (6H, m), 3.59 (1H, m), 3.38 (1H, m), 2.39 (2H, t, J = 7.5 Hz), 2.35-2.23 (2H, m), 1.67-1.54 (4H, m), 1.37-1.23 (8H, m), 0.94-0.86 (6H, m); 13C NMR (125 MHz, 9/1 CDCl3/CD3OD) δ 173.7, 173.5, 98.9, 76.5, 73.1, 71.7, 71.6, 70.9, 69.1, 64.9, 63.3, 61.4, 33.9, 31.0, 24.5, 24.2, 22.1, 13.6; HRMS (ESI-TOF) m/z [M+NH4]+ calcd for C22H44NO11 498.2914, found 498.2917. 4-O-(2,3-Di-O-octyl-β β-D-mannopyranosyl)-D-erythritol (17). Yield 74% (10.4 mg, 19.4 µmol); colorless syrup; Rf 0.47 (4/1 CHCl3/MeOH); [α]27D -37.6° (c 0.77, CHCl3); 1H NMR (500 MHz, 9/1 CDCl3/CD3OD) δ 5.46 (1H, br-d, J = 3.0 Hz), 4.91 (1H, dd, J = 3.0 and 10.0 Hz), 4.74 (1H, s), 4.00 (1H, dd, J = 3.0 and 10.5 Hz), 3.91 (1H, dd, J = 3.0 and 12.5 Hz), 3.86-3.64 (6H, m), 3.58 (1H, m), 3.38 (1H, m), 2.39 (2H, t, J = 7.5 Hz), 2.35-2.23 (2H, m), 1.67-1.54 (4H, m), 1.37-1.22 (16H, m), 0.91-0.85 (6H, m); 13C NMR (125 MHz, 9/1 CDCl3/CD3OD) δ 173.7, 173.5, 98.9, 76.5, 73.1, 71.7, 71.6, 70.9, 69.1, 64.9, 63.4, 61.4, 34.0, 31.5×2, 28.9×2, 28.8×2, 24.9, 24.5, 22.5, 22.4, 13.8; HRMS (ESI-TOF) m/z [M+NH4]+ calcd for C26H52NO11 554.3540, found 554.3553. 4-O-(2,3-Di-O-decyl-β β -D-mannopyranosyl)-D-erythritol (18). Yield 73% (15.6 mg, 26.3 µmol, HPLC purity >99%); colorless syrup; Rf 0.48 (4/1 CHCl3/MeOH); [α]27D -47.1° (c 0.66, CHCl3); 1H NMR (500 MHz, 9/1 CDCl3/CD3OD) δ 5.46 (1H, br-d, J = 3.0 Hz), 4.90 (1H, dd, J = 3.0 and 10.0 Hz), 4.73 (1H, s), 4.00 (1H, dd, J = 3.0 and 10.5 Hz), 3.91 (1H, dd, J = 3.0 and 12.5 Hz), 3.87-3.64 (6H, m), 3.58 (1H, m), 3.39 (1H, m), 2.39 (2H, t, J = 7.5 Hz), 2.35-2.23 (2H, m), 1.67-1.54 (4H, m), 1.37-1.21 (24H, m), 0.91-0.85 (6H, m); 13C NMR (125 MHz, 9/1 CDCl3/CD3OD) δ 173.7, 173.5, 98.9, 76.5, 73.1, 71.7, 71.6, 70.9, 69.1, 64.9, 63.3, 61.4, 34.0, 31.7, 29.3×2, 29.2, 29.1×2, 29.0, 28.9, 24.9, 24.5, 22.5, 13.9; HRMS (ESI-TOF) m/z [M+H]+ calcd for C30H57O11 593.3901, found 593.3892; tR = 8.13 min (HPLC conditions for Figure S70). 4-O-(2,3-Di-O-dodecyl-β β -D-mannopyranosyl)-D-erythritol (19). Yield 65% (16.4 mg, 25.3 µmol); colorless syrup; Rf 0.50 (4/1 CHCl3/MeOH); [α]27D -31.6° (c 1.0, CHCl3); 1H NMR (500 MHz, 9/1 CDCl3/CD3OD) δ 5.46 (1H, br-d, J = 3.5 Hz), 4.90 (1H, dd, J = 3.5 and 10.0 Hz), 4.73 (1H, s), 4.00 (1H, dd, J = 3.0 and 10.5 Hz), 3.91 (1H, dd, J = 3.0 and 12.0 Hz), 3.87-3.64 (6H, m), 3.59 (1H, m), 3.40 (1H, m), 2.39 (2H, t, J = 7.5 Hz), 2.35-2.23 (2H, m), 1.67-1.54 (4H, m), 1.37-1.21 (32H, m), 0.91-0.85

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(6H, m); 13C NMR (125 MHz, 9/1 CDCl3/CD3OD) δ 173.7, 173.5, 98.9, 76.5, 73.1, 71.7, 71.6, 70.9, 69.1, 64.9, 63.3, 61.4, 34.0×2, 31.8, 29.5×2, 29.4, 29.3, 29.2×2, 29.0, 28.9×2, 24.9, 24.5, 22.5, 13.9; HRMS (ESI-TOF) m/z [M+H]+ calcd for C34H65O11 649.4527, found 649.4518. 4-O-(2,3-Di-O-tetradecyl-β β -D-mannopyranosyl)-D-erythritol (20). Yield 90% (11.1 mg, 15.7 µmol); white solid; Rf 0.55 (4/1 CHCl3/MeOH); [α]27D -26.0° (c 1.0, CHCl3); mp 81-82 °C; 1H NMR (500 MHz, 9/1 CDCl3/CD3OD) δ 5.45 (1H, br-d, J = 3.0 Hz), 4.90 (1H, dd, J = 3.5 and 10.0 Hz), 4.73 (1H, s), 4.00 (1H, dd, J = 3.0 and 10.5 Hz), 3.91 (1H, dd, J = 2.5 and 12.5 Hz), 3.873.63 (6H, m), 3.58 (1H, m), 3.38 (1H, m), 2.38 (2H, t, J = 7.5 Hz), 2.35-2.23 (2H, m), 1.67-1.54 (4H, m), 1.37-1.21 (40H, m), 0.910.85 (6H, m); 13C NMR (125 MHz, 9/1 CDCl3/CD3OD) δ 173.7, 173.5, 98.9, 76.5, 73.1, 71.7, 71.6, 70.9, 69.1, 64.9, 63.3, 61.4, 34.0, 31.8, 29.5×2, 29.4, 28.9, 24.9, 24.5, 22.5, 13.9; HRMS (ESI-TOF) m/z [M+NH4]+ calcd for C38H76NO11 722.5418, found 722.5406. Strains. Reference strains Enterococcus faecalis JCM5803 and Enterococcus faecium JCM5804, vancomycin-susceptible Enterococcus (VSE), were purchased from Japan Collection of Microorganisms. E. faecalis NCTC 12201 and E. faecium NCTC 12202, vancomycin-resistant Enterococcus (VRE), purchased from National Collection of Type Cultures. Micrococcus luteus IFO 3333, Staphylococcus aureus FDA 209P, meticillin-susceptible S. aureus (MSSA), and S. aureus MS16526, meticillin-resistant S. aureus (MRSA), were exploited from the culture collection at the Institute of Microbial Chemistry. Determination of the MICs by the agar dilution method against Gram-positive bacteria. The MICs were examined by a serial agar dilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines17 except for the media, which was Nutrient Broth (Difco) containing with 18 g/L agar (Wako), final pH 6.9. The testorganism suspension was prepared at approximately 104 CFU per spot using a MIC-2000 (Dynatech Laboratories, Inc.) inoculum replicating apparatus. The MIC was defined as the lowest concentration of antibiotic that inhibited development of visible growth on the agar after 18 h of incubation at 37 °C. Compounds were dissolved in dimethyl sulfoxide except for vancomycin, which was dissolved in water. Appropriate dilutions were made with the required culture medium immediately before testing. The MIC value was calculated from the geometric means from the value obtained by measuring the MIC three times.

ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website at DOI: 1

H and 13C NMR spectra for all new compounds (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. *E-mail: [email protected].

ORCID Daisuke Takahashi: 0000-0003-3222-6648

Notes The authors declare no competing financial interests.

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ACKNOWLEDGMENTS This research was supported in part by the JSPS KAKENHI Grant Numbers JP16H01161 in Middle Molecular Strategy and JP16K05781 in Scientific Research (C), and TOBE MAKI Scholarship Foundation.

REFERENCES (1)

(a) Morita, T.; Konishi, M.; Fukuoka, T.; Imura, T.; Kitamoto, H.; Kitamoto, D. FEMS Yeast Res. 2007, 7, 286. (b) Review: Yu, M.; Liu, Z.; Zeng, G.; Zhong, H.;Liu, Y.; Jiang, Y.; Li, M.; He, X.; He, Y. Carbohydr. Res. 2015, 407, 63. (c) Review: Morita, T.; Fukuoka, T.; Imura, T.; Kitamoto, D. J. Oleo Sci. 2015, 64, 133.

(2) Kitamoto, D.; Akiba, S.; Hiroki, C.; Tabuchi, T. Agric. Biol. Chem. 1990, 54, 31. (3) Morita, T.; Takashima, M.; Fukuoka, T.; Konishi, M.; Imura, T.; Kitamoto, D. Appl. Microbiol. Biotechnol. 2010, 88, 679. (4) Crich, D.; de la Mora, M. A.; Cruz, R. Tetrahedron 2002, 58, 35. (5) (a) Kitamoto, D.; Isoda, H.; Nakahara, T. J. Biosci. Bioeng. 2002, 94, 187. (b) Arutchelvi, J. I.; Bhaduri, S.; Uppara, P. V.; Doble, M. J. Ind. Microbiol. Biotechnol. 2008, 35, 1559. (6) Kitamoto, D.; Yanagishita, H.; Shinbo, T.; Nakane, T.; Kamisawa, C.; Nakahara, T. J. Biotechnol. 1993, 29, 91. (7) (a) WHO. WHO Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics, 2017, available at http://www.who.int/medicines/publications/WHO-PPL-Short_Summary_25Feb-ET_NM_WHO.pdf?ua=1 (b) Chambers, H. F.; DeLeo, F. R. Nat. Rev. Microbiol. 2009, 7, 629. (c) Arias, C. A.; Murray, B. E. 2012, 10, 266. (d) Muto C. A.; Jernigan J. A.; Ostrowsky B. E.; Richet H. M.; Jarvis W. R.; Boyce J. M.; Farr. B. M. SHEA guideline for preventing nosocomial transmission of multidrug-resistant strains of staphylococcus aureus and enterococcus. Infection Control and Hospital Epidemiology, 2003, 24, 362. (8) (a) Tanaka, M.; Nashida, J.; Takahashi, D.; Toshima, K. Org. Lett. 2016, 18, 2288. (b) Tanaka, M.; Takahashi, D.; Toshima, K. Org. Lett. 2016, 18, 5030. (9) Mori, Y.; Kobayashi, J.; Manabe, K.; Kobayashi, S. Tetrahedron, 2002, 58, 8263. (10) Julien, B.; James, T.; Chang, H. W.; Benson, M. K.; Liam, R. C. J. Org. Chem. 2004, 69, 6341. (11) Marco, J. A.; Carda, M.; Gonzàlez, F.; Rodriguez, S.; Murga, J. Liebigs Ann. 1996, 1801. (12) Bock, K.; Pedersen, C. J. Chem. Soc., PerkinTrans. 2 1974, 293. (13) Kitamoto, D.; Ghosh, S.; Ourisson, G.; Nakatani, Y. Chem. Commun. 2000, 861. (14) Fukuoka, T.; Yanagihara, T.; Imura, T.; Morita, T.; Sakai, H.; Abe, M.; Kitamoto, D. Carbohydr. Res. 2012, 351, 81. (15) Sasaki, K.; Tohda, K. Tetrahedron Lett. 2018, 59, 496. (16) Abalos, A.; Pinazo, A.; Infante, M. R.; Casals, M.; García, F.; Manresa, A. Langmuir 2001, 17, 1367. (17) Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard. 8th ed. Wayne, PA: CLSI; 2009. Document M07-A8.

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GRAPHICAL ABSTRACT

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Systematic and Stereoselective Total Synthesis of Mannosylerythritol

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