Chemical Hybridization of Vizantin and Lipid A to Generate a Novel LPS Antagonist

Abstract

Lipopolysaccharide (LPS) antagonists have attracted considerable interest as promising candidates for the treatment of severe sepsis triggered by Gram-negative bacteria. In this article, we describe the development of a novel LPS antagonist based on chemical hybridization of vizantin and the hydrophobic molecular unit of LPS (lipid A). Vizantin, 6,6′-bis-O-(3-nonyldodecanoyl)-α,α′-trehalose, was designed as an immunostimulator from a structure–activity relationship (SAR) study with trehalose 6,6′-dicorynomycolate (TDCM). Our recent study indicated that vizantin displays adjuvant activity by specifically binding to the Toll-like receptor 4 (TLR4)/MD2 protein complex. Because lipid A unit (or LPS) is also known to trigger an inflammatory response via the same TLR4/MD2 complex as vizantin, we designed a hybrid compound of vizantin and lipid A with the aim of developing a novel biofunctional glycolipid. Focusing on the antagonism to Escherichia coli LPS in an in vitro model with human macrophages (THP-1 cells), we identified a potent LPS antagonist among the synthesized hybrid compounds. The novel LPS antagonist effectively inhibited LPS-induced release of tumor necrosis factor-alpha (TNF-α) in a dose-dependent manner with an IC₅₀ value of 3.8 nM, making it a candidate for the treatment drug of Gram-negative sepsis and/or septic shock.

Gram-negative sepsis and septic shock are serious conditions caused by the release of bacterial endotoxin.^1–4) Endotoxin, which is also called lipopolysaccharide (LPS), is a structural component of the outer membrane of Gram-negative bacteria that is released when the cells become disrupted.⁵⁾ LPS is known to activate immune cells, such as macrophages and monocytes, by binding of the lipid A (1) molecular unit (i.e., the hydrophobic domain of LPS) to the complex protein of Toll-like receptor 4 (TLR4) and MD2. This binding event results in the release of various cytokines, including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), from the immune cells.^6–8) While the immune response with lipid A brings about the onset of the host defense to Gram-negative infection, the sudden release of high levels of cytokines and cellular mediators can trigger many pathophysiological events including fever, sepsis, septic shock, disseminated intravascular coagulation (DIC) and organ failure.⁹⁾ These symptoms, especially Gram-negative sepsis and DIC, are irremediable even after administering potent antibiotics to combat the infection. Hence, LPS antagonists have been developed by several groups as promising candidates for the prevention and/or treatment of severe sepsis and excessive inflammatory response.^10–14)

We are interested in designing a safe adjuvant that displays immunological activity with no associated immunotoxicity. During the course of these investigations we recently developed vizantin (3)^15–17) based on a structure–activity relationship (SAR) study of trehalose 6,6′-dicorynomycolate (TDCM), which was first characterized in 1963 as a cell surface glycolipid of Corynebacterium spp. by Ioneda et al.¹⁸⁾ Vizantin also triggers the innate immune response through the specific binding to the same TLR4/MD2 protein complex as lipid A,¹⁹⁾ and effectively activates some types of immune system. However, the finding that vizantin induces almost no TNF-α production is clearly different from that of lipid A (or LPS). TNF-α is known to have significant high proinflammatory activity, which may lead to serious complications such as cytokine storm.²⁰⁾ Thus, vizantin induces no TNF-α production; a factor which contributes to its very low immunotoxicity. Vizantin also stimulates human macrophages, making it a promising candidate as an adjuvant in clinical applications. Lipid A and its analogues have also attracted considerable attention as lead compounds for adjuvant development, although as yet with only limited success^21–24) (Fig. 1).

Fig. 1. Lipid A, TDCM, Vizantin and 3NDDA

Vizantin possesses 3-nonyldodecanoic acid (4) (hereafter 3NDDA) as the fatty acid side-chain, which is a key structure for stimulating human macrophages without TNF-α production.¹⁵⁾ Thus, we reasoned that a new biofunctional glycolipid, which shows potent adjuvant activity without the associated production of TNF-α or antagonistic activity against LPS in human tissue, might be produced by introducing 3NDDA into the structure of lipid A in place of its acyl chains. Although the exact biological behavior of the hybrid compound was unclear, our approach might assist in developing the latent biological activity of 3NDDA.

Results and Discussion

Lipid A comprises two glucosamine units with attached acyl chains and normally contains one phosphate group on each sugar unit. A typical example is Escherichia coli lipid A (1) with four (R)-3-hydroxytetradecanoic acids directly attached to both the sugars, and C₁₄- and C₁₂-fatty acids further condensed with the β-hydroxyl group of one sugar unit.²⁵⁾ Because the number of acyl side-chains is known to affect the biological activity,²⁶⁾ we initially designed N,O-3NDDA-dicondensed 5, O-monocondensed 6 and N-monocondensed 7 by imitating the E. coli lipid A with six side-chains (Fig. 2).

Fig. 2. Hybrid Compounds Based on E. coli Lipid A

The synthesis of N,O-3NDDA-dicondensed 5 was effectively achieved through the coupling of sugar unit 10 and 14 (Chart 1A). Unit 10 was prepared from a glucosamine building block 8 and (R)-3-(benzyloxy)tetradecanoic acid (9) according to the reported procedures developed by the Kusumoto group.^27–29) Conveniently, unit 14 could also be prepared from a common 8. The C-3 hydroxy group of 8 was condensed with 3NDDA by the Keck procedure with 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride (EDCI) and N,N-dimethyl-4-aminopyridine (DMAP) as condensing agents, and 11 was treated with Et₃SiH and BF₃·Et₂O in CH₂Cl₂ at 0°C. The desired regioselective cleavage of the benzylidene acetal moiety in 11 proceeded smoothly, giving 12 as sole product in 90% yield. The resulting alcohol of 12 was phosphorylated with N,N-diethyl-1,5-dihydro-2,3,4-benzodioxaphosphepin-3-amine and 1H-tetrazole followed by in situ oxidation with m-chloroperbenzoic acid (mCPBA). Next, the allyl group of 13 was removed using a catalytic amount of [Ir(cod)(MePh₂P)₂]PF₆ and I₂ in tetrahydrofuran (THF) at room temperature, and then unit 14 was converted to the trichloroacetimidate derivative by treatment of CCl₃CN and Cs₂CO₃ in order to couple with unit 10. Using a catalytic amount of trimethylsilyl trifluoromethanesulfonate (TMSOTf) in the presence of molecular sieves 4 Å at −20°C, the coupling reaction was completed within 0.5 h, giving the desired β-linked disaccharide 15 in 82% yield with an excellent stereoselectivity. Next, the N-Troc group of 15 was removed using Zn–Cu couple dust in AcOH, and the resulting amino group of 16 was acylated with 3NDDA using EDCI. Deprotection of the allyl groups in 17 was accomplished in the same manner as that of 13, and the phosphorylation of 18 was achieved by treatment with n-BuLi and then [(BnO)₂PO]₂O. Finally, hydrogenolysis over 100% (w/v) palladium black resulted in the removal of the benzyl-protecting groups to give 5 as the desired compound. The synthesis of O-3NDDA-monocondensed 6 was also achieved with 16 and (R)-3-(dodecanoyloxy)tetradecanoic acid (19), which was readily prepared from a synthetic intermediate of 9 (Chart 1B). N-3NDDA-monocondensed 7 could be derived from known 22²⁸⁾ by the established four-step sequence for 5 (Chart 1C).

Chart 1. Syntheses of Hybrid Compounds 5, 6 and 7

Reagents and conditions: (a) EDCI, DMAP, MS 4 Å, CH₂Cl₂, reflux, 4 h; (b) Et₃SiH, BF₃·Et₂O, CH₂Cl₂, 0°C, 1 h; (c) C₆H₄(CH₂O)₂PNEt₂, 1H-tetrazole, (CH₂Cl)₂, −20°C, 10 min, then mCPBA, −20°C, 10 min; (d) [Ir(cod)(MePh₂P)₂]PF₆, H₂, THF, rt, 1 h, then I₂, rt, 10 min; (e) CCl₃CN, Cs₂CO₃, CH₂Cl₂, rt, 30 min, then 10, TMSOTf, MS 4 Å, CH₂Cl₂, −20°C, 45 min; (f) Zn–Cu alloy, AcOH, rt, 2 h; (g) 3NDDA, EDCI, MS 4 Å, CH₂Cl₂, rt, 11 h; (h) n-BuLi, THF, −78°C, 5 min, then [(BnO)₂PO]₂O, rt, 1 h; Pd/C, H₂, THF, rt, 4 h. (i) EDCI, MS 4 Å, CH₂Cl₂, rt, 12 h.

With the desired compounds in hand, our attention then shifted to exploring their biological properties. In the course of investigating the adjuvant properties of vizantin, we found that this compound effectively induced macrophage inflammatory protein-1β (MIP-1β) for human monocyte macrophage cells (THP-1 cells) without increasing TNF-α production.¹⁵⁾ Thus synthesized 5, 6 and 7 were initially evaluated by assessing their effect on the release of TNF-α and MIP-1β from THP-1 cells. Each sample (100 µM) was added to 5.0×10⁵ cells, and cell culture was then carried out at 37°C for 3 h. The presence of TNF-α and MIP-1β in the culture medium was measured using a commercial capture enzyme-linked immunosorbent assay (ELISA) kit.

Our assay results showed that the compounds 5, 6 and 7 did not stimulate the cells to release MIP-1β or TNF-α into the medium (Fig. 3). These findings suggested that the hybrid compounds might inhibit TNF-α and MIP-1β production triggered by LPS through blocking the binding site of the lipid A unit on the TLR4/MD2 complex. Thus, the antagonism of these hybrid compounds against E. coli LPS (10 ng/mL) was tested with THP-1 cells. Our group recently demonstrated that vizantin represses TNF-α production triggered by LPS in the same assay system.¹⁹⁾ Among the synthesized compounds, 5 effectively inhibited LPS-induced release of TNF-α in a dose-dependent manner (Fig. 4A) with an IC₅₀ value of 3.8 nM. This compound also displayed some inhibition of the release of MIP-1β triggered by LPS, albeit at a much greater IC₅₀ value (IC₅₀=0.6 µM). However, no such effect was observed for compounds 6 and 7 (Figs. 4B, C).

Fig. 3. TNF-α and MIP-1β Releasing Activities

The release of TNF-α and MIP-1β was determined using ELISA kits. E. coli LPS (10 ng/mL) was employed as a positive control.

Fig. 4. Effects of 5 (A), 6 (B) and 7 (C) on TNF-α and MIP-1β Production upon Treatment with LPS

Cells were treated with sample compound and LPS at the same time. The release of TNF-α and/or MIP-1β was determined using an ELISA kit. Values represent the mean±S.E.M.; n=5; * p<0.01, ** p<0.005 compared with cells treated with LPS (10 ng/mL) plus vehicle (0.125% BSA).

The observation that of the tested compounds only 5 suppressed TNF-α and MIP-1β release is of great interest, and suggests that 5 acts as a highly active LPS antagonist.^14,30–33) Thus, its analog compounds with a variety of chain-lengths 25–28 were also prepared in order to elucidate the effect of the β-branched acyl chain (Fig. 5A). Compounds 25–28 were readily obtained by using the established synthetic route for 5.³⁴⁾

Fig. 5. Structures of 25–28 (A), and the Effect of 27 (B) and 28 (C) on TNF-α and MIP-1β Production in THP-1 Cells Treated with LPS

The antagonistic activities of 25–28 were evaluated in the same manner as described for 5. Surprisingly, short-chain analogues 25 and 26 showed significant cytotoxicity to THP-1 cells. In addition, long-chain analogues 27 and 28 were practically inactive in this assay (Figs. 5B, C). In the case of long-chain analogues, their low solubility may influence the biological activity. However, it is clear that chain length affects both cytotoxicity as well as the observed antagonism. Nonetheless, the introduction of 3NDDA is ideal for inducing antagonistic activity.

Docking simulation analysis of 5 and MD2 protein supports the suggestion that introduced 3NDDAs act as a key structure to induce its antagonistic activity. Figure 6 shows the most stable structure of 5 on the molecular electrostatic potential surface of MD2. Two introduced 3NDDA chains and one (R)-3-hydroxytetradecanoyl chain penetrate the cavity of MD2, forming a complex with stabilization energy of −43.17 kcal/mol. As indicated in the graphic abstract, the simulation of 5 with TLR4/MD2 complex also gave a similar result.

Fig. 6. Docking Simulation Analysis of 5 and MD2 Using MOE Software

The purple and green surfaces indicate the hydrophilic and hydrophobic surfaces, respectively. The gray, red, blue, and yellow in the compounds indicate carbon, oxygen, nitrogen, and phosphate, respectively.

Conclusion

Based on the known biological activity of lipid A and vizantin, we designed hybrid compounds of these glycolipids, and identified 5 as a potent endotoxin antagonist among a variety of the synthesized compounds. Specifically, 5 effectively inhibited the release of proinflammatory cytokines normally induced by E. coli LPS in an in vitro model with human immune cells (THP-1 cells). Thus, 5 may be useful in drug development for the treatment of Gram-negative sepsis and/or septic shock. Moreover, we anticipate a further enhancement in the antagonism efficiency of 5 by optimizing the moiety of the unmodified sugar unit. Experiments are currently underway to further enhance the remarkable biological activity of 5 for clinical utility by additional chemical modification combined with in vivo studies.

Experimental

General

Reactions were performed under argon unless otherwise stated. Commercially available chemicals were used as purchased. Special grade and dehydrated solvents were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). The progress of the reaction was monitored by TLC using precoated silica gel 60 F254 plates (Merck, Darmstadt, Germany). Spots were visualized under UV illumination (254 nm) after immersion in 2% p-anisaldehyde and 5% H₂SO₄ in EtOH, followed by heating on a hot plate. Solvents were removed under reduced pressure using a standard rotary evaporator. Flash column chromatography was performed using 63–210 mesh silica gel 60 (Kanto Chemical Co., Inc.). Specific rotations were determined on a JASCO P-1030 digital polarimeter using the sodium D line (λ=589 nm) at specific temperatures and are reported as follows: [α]_D^temp., concentration (c=g/100 mL), and solvent. Fourier transform (FT)-IR spectra were measured on a JASCO FT/IR-410 infrared spectrophotometer and reported as wavenumber (cm⁻¹) of significant peaks. ¹H- and ¹³C-NMR spectra were recorded on a Varian Mercury plus-300-4N spectrometer, a Varian 400-MR spectrometer, a Varian 500-MR spectrometer and a Varian Unity-600 spectrometer. The ¹H chemical shifts are reported in parts per million (ppm) from an internal standard of tetramethylsilane (TMS) (0.00 ppm), residual CHCl₃ (7.26 ppm), or C₅H₅N (7.19 ppm, right peak), and the ¹³C chemical shifts were reported using an internal standard of CDCl₃ (77.03 ppm, central peak) or C₅D₅N (123.50 ppm, central peak in right-side triplet). The ¹H-NMR spectra are reported as follows: chemical shift, multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, qui=quintet and br=broad), relative integral, coupling constants J (Hz). High-resolution (HR)-MS were recorded on a JEOL the Mstation JMS-700.

Allyl 4,6-O-Benzylidene-2-deoxy-3-O-(3-nonyldodecanoyl)-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranoside (11)

To a solution of 8²⁷⁾ (18.6 g, 39.0 mmol) in CH₂Cl₂ (130 mL) were added 3-nonyldodecanoic acid (16.6 g, 50.8 mmol), MS 4 Å (30.0 g), 4,4-dimethylaminopyridine (1.40 g, 11.7 mmol) and EDCI (11.2 g, 58.5 mmol) at room temperature. After the reaction mixture was stirred for 4 h at reflux, it was cooled to room temperature and then filtrated by Celite^®. The filtrate was diluted with CH₂Cl₂ and quenched by H₂O. The organic layer was separated, and the aqueous layer was extracted with CH₂Cl₂ three times. The combined organic layer was dried over MgSO₄, and concentrated in vacuo. The residue was purified by silica gel chromatography (hexane–AcOEt=10 : 1) to give 11 as colorless syrup (28.6 g, 36.1 mmol, 93%). 11: [α]_D²¹ +28.1 (c=1.0, CHCl₃); FT-IR (neat) 3323, 3065, 3036, 3013, 2954, 2924, 2852, 1742, 1722, 1647 cm⁻¹; ¹H-NMR (300 MHz in CDCl₃) δ: 0.88 (6H, m, CH₃×2), 1.24 (32H, m, CH₂×16), 1.80 (1H, m, β-CH), 2.23 (2H, t, J=6.6 Hz, α-CH₂), 3.74 (2H, m, H-4 and H-6b), 4.00 (3H, m, H-2, H-5 and OCH₂CH=CH₂), 4.22 (1H, dd, J=12.9, 5.4 Hz, OCH₂CH=CH₂), 4.29 (1H, dd, J=10.2, 4.8 Hz, H-6a), 4.66 (1H, d, J=12.0 Hz, CH₂ of Troc), 4.77 (1H, d, J=12.0 Hz, CH₂ of Troc), 4.94 (1H, d, J=3.9 Hz, H-1), 5.28 (2H, m, OCH₂CH=CH₂), 5.41 (2H, m, H-3 and NH), 5.53 (1H, s, PhCH(CO)₂), 5.89 (1H, m, OCH₂CH=CH₂), 7.39 (5H, m, PhCH(CO)₂); ¹³C-NMR (75 MHz in CDCl₃) δ: 14.16, 22.71, 26.39, 26.43, 29.37, 29.64, 29.67, 29.84, 29.87, 31.93, 33.40, 34.87, 38.95, 54.74, 63.03, 68.37, 69.79, 74.59, 79.26, 95.38, 97.02, 101.44, 118.55, 126.06, 128.15, 129.01, 133.03, 136.90, 154.31, 173.40; MS (FAB⁺) m/z 789 [M]⁺; HR-MS (FAB⁺) m/z Calcd for C₄₀H₆₂NO₈Cl₃: 789.3541. Found 789.3556.

Allyl 6-O-Benzyl-2-deoxy-3-O-(3-nonyldodecanoyl)-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranoside (12)

To a solution of 11 (10.0 g, 12.6 mmol) in CH₂Cl₂ (85 mL) were added triethylsilane (7.30 g, 63.0 mmol) and BF₃·Et₂O (8.90 g, 63.0 mmol) at 0°C. After the mixture was stirred for 1 h at 0°C, it was quenched with triethylamine and concentrated in vacuo. The residue was purified by silica gel chromatography (hexane–AcOEt=10 : 1) to give 12 as colorless syrup (9.00 g, 11.3 mmol, 90%). 12: [α]_D²¹ +38.6 (c=1.0, CHCl₃); FT-IR (neat) 3437, 3339, 3086, 3065, 3031, 2927, 2855, 1950, 1864, 1743, 1649 cm⁻¹; ¹H-NMR (300 MHz in CDCl₃) δ: 0.88 (6H, m, CH₃×2), 1.25 (32H, m, CH₂×16), 1.82 (1H, m, β-CH), 2.27 (2H, t, J=6.0 Hz, α-CH₂), 2.68 (1H, d, J=3.6 Hz, OH), 3.78 (4H, m, H-4, H-5 and H-6ab), 3.98 (2H, m, H-2 and OCH₂CH=CH₂), 4.19 (1H, dd, J=12.6, 5.1 Hz, OCH₂CH=CH₂), 4.57 (1H, d, J=12.0 Hz, CH₂ of Troc), 4.63 (1H, d, J=12.0 Hz, CH₂ of Troc), 4.65 (1H, d, J=12.0 Hz, PhCH₂), 4.74 (1H, d, J=12.0 Hz, PhCH₂), 4.91 (1H, d, J=3.6 Hz, H-1), 5.21 (4H, m, H-3, NH and OCH₂CH=CH₂), 5.87 (1H, m, OCH₂CH=CH₂), 7.34 (5H, m, PhCH₂); ¹³C-NMR (75 MHz in CDCl₃) δ: 14.11, 22.66, 26.35, 26.56, 29.32, 29.59, 29.63, 29.88, 29.89, 31.88, 33.51, 33.60, 34.82, 39.00, 53.79, 68.43, 69.59, 70.24, 70.46, 73.42, 73.64, 74.54, 95.37, 96.24, 118.21, 127.59, 127.75, 128.39, 133.18, 137.70, 154.18, 174.72; MS (FAB⁺) m/z 814 [M+H+Na]⁺; HR-MS (FAB⁺) m/z Calcd for C₄₀H₆₄NO₈Cl₃Na: 814.3595. Found 814.3601.

Allyl 6-O-Benzyl-2-deoxy-3-O-(3-nonyldodecanoyl)-4-O-(3-oxido-1,5-dihydrobenzo[e][1,3,2]dioxaphosphepin-3-yl)-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranoside (13)

To a solution of 12 (2.10 g, 2.70 mmol) in 1,2-dichloroethane (5.4 mL) were added MS 4 Å (1.50 g), N,N-diethyl-1,5-dihydro-2,3,4-benzodioxaphosphepin-3-amine (0.8 mL, 3.70 mmol) and 1H-tetrazole (476 mg, 6.80 mmol) at room temperature. After the mixture was stirred for 10 min at 0°C, m-chloroperoxybenzoic acid (contain 55%) (467 mg, 2.70 mmol) was added to the mixture at −20°C. After the mixture was stirred for 10 min at −20°C, it was filtrated by Celite^®. The filtrate was diluted with AcOEt and quenched with aqueous NaHCO₃ and Na₂S₂O₃. The combined organic layer was dried over MgSO₄, and concentrated in vacuo. The residue was purified by silica gel chromatography (hexane–AcOEt=4 : 1) to give 13 as colorless syrup (2.61 g, 2.67 mmol, 99%). 13: [α]_D²² +29.2 (c=1.0, CHCl₃); FT-IR (neat) 3437, 3331 3064, 3029, 2927, 2855, 1958, 1864, 1804, 1747, 1647 cm⁻¹; ¹H-NMR (300 MHz in CDCl₃) δ: 0.87 (3H, t, J=6.6 Hz, CH₃), 0.88 (3H, t, J=6.6 Hz, CH₃), 1.24 (32H, m, CH₂×16), 1.82 (1H, m, β-CH), 2.34 (2H, d, J=6.6 Hz, α-CH₂), 3.73 (1H, dd, J=10.8, 4.8 Hz, H-6a), 3.80 (1H, dd, J=10.8, 2.1 Hz, H-6b), 4.01 (3H, m, H-2, H-5 and OCH₂CH=CH₂), 4.21 (1H, dd, J=12.9, 5.4 Hz, OCH₂CH=CH₂), 4.55 (1H, d, J=12.3 Hz, CH₂ of Troc), 4.58 (1H, d, J=12.3 Hz, CH₂ of Troc), 4.66 (1H, d, J=12.3 Hz, PhCH₂), 4.74 (1H, m, H-4), 4.81 (1H, d, J=12.3 Hz, PhCH₂), 4.95 (1H, d, J=3.9 Hz, H-1), 5.10 (4H, m, o-C₆H₄(CH₂O)₂P), 5.30 (4H, m, H-3, NH and OCH₂CH=CH₂), 5.88 (1H, m, OCH₂CH=CH₂), 7.18–7.40 (9H, m, o-C₆H₄(CH₂O)₂P and PhCH₂); ¹³C-NMR (75 MHz in CDCl₃) δ: 14.14, 22.69, 26.47, 26.61, 29.36, 29.66, 29.73, 30.02, 31.91, 33.38, 33.62, 34.33, 38.69, 54.13, 68.28, 68.59, 68.72, 69.84, 70.75, 73.62, 74.42, 74.64, 95.29, 96.06, 118.47, 127.59, 127.85, 128.33, 128.41, 128.49, 128.95, 133.02, 134.68, 137.89, 154.03, 173.86; MS (FAB⁺) m/z 996 [M+Na]⁺; HR-MS (FAB⁺) m/z Calcd for C₄₈H₇₁NO₁₁Cl₃PNa: 996.3728. Found 996.3715.

6-O-Benzyl-2-deoxy-3-O-(3-nonyldodecanoyl)-4-O-(3-oxido-1,5-dihydrobenzo[e][1,3,2]dioxaphosphepin-3-yl)-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranose (14)

To a solution of 13 (2.50 g, 2.60 mmol) in THF (52 mL, degassed with Ar) was added [Ir(cod)(MePh₂P)₂]PF₆ (60.0 mg, 0.20 mmol) at room temperature. The mixture was stirred for 1 h under bubbling H₂ gas at room temperature. H₂O (32.5 mL) and I₂ (660 mg, 2.60 mmol) were added to the mixture at room temperature. After the mixture was stirred for 10 min at room temperature, it was diluted with AcOEt and quenched with saturated Na₂S₂O₃ solution. The organic layer was separated and washed with saturated NaHCO₃ solution and then brine. The combined organic layer was dried over MgSO₄, and concentrated in vacuo. The residue was purified by silica gel chromatography (hexane–AcOEt=2 : 1) to give 14 as colorless syrup (2.00 g, 2.14 mmol, 80%). 14: [α]_D²² +6.6 (c=1.0, CHCl₃); FT-IR (neat) 3374, 3109, 3066, 3026, 2955, 2925, 2853, 2737, 1949, 1910, 1742, 1725 cm⁻¹; ¹H-NMR (500 MHz in CDCl₃) δ: 0.87 (3H, t, J=7.0 Hz, CH₃), 0.87 (3H, t, J=6.5 Hz, CH₃), 1.25 (32H, m, CH₂×16), 1.80 (1H, m, β-CH), 2.32 (2H, t, J=6.0 Hz, α-CH₂), 3.69 (1H, dd, J=11.0, 6.0 Hz, H-6a), 3.78 (1H, dd, J=11.0, 2.0 Hz, H-6b), 3.95 (1H, dt, J=10.5, 3.5 Hz, H-2), 4.24 (1H, m, H-5), 4.55 (1H, d, J=12.0 Hz, CH₂ of Troc), 4.58 (1H, d, J=12.0 Hz, CH₂ of Troc), 4.60 (2H, m, H-4 and PhCH₂), 4.75 (1H, d, J=12.0 Hz, PhCH₂), 5.04 (4H, m, o-C₆H₄(CH₂O)₂P), 5.25 (1H, d, J=3.5 Hz, H-1), 5.39 (2H, m, H-3 and NH), 7.16–7.37 (9H, m, o-C₆H₄(CH₂O)₂P and PhCH₂); ¹³C-NMR (75 MHz in CDCl₃) δ: 14.14, 22.70, 26.46, 26.62, 29.37, 29.68, 29.75, 29.99, 30.04, 31.92, 33.38, 33.61, 34.28, 38.68, 54.33, 68.32, 68.72, 69.17, 70.40, 74.70, 91.45, 95.24, 127.85, 128.14, 128.43, 128.52, 129.00, 134.58, 134.63, 137.46, 154.06, 173.78; MS (FAB⁺) m/z 956 [M+Na]⁺; HR-MS (FAB⁺) m/z Calcd for C₄₅H₆₉NO₁₁Cl₃PNa: 956.3415. Found 956.3415.

Allyl 6-O-{6′-O-Benzyl-2′-deoxy-3′-O-(3-nonyldodecanoyl)-4′-O-(3-oxido-1,5-dihydrobenzo[e][1,3,2]dioxaphosphepin-3-yl)-2′-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosyl}-2-deoxy-3-O-[(R)-3-(benzyloxy)tetradecanoyl]-2-O-[(R)-3-(benzyloxy)tetradecanoylamino]-α-D-glucopyranoside (15)

To a solution of 14 (1.00 g, 1.00 mmol) in CH₂Cl₂ (50 mL) were added Cs₂CO₃ (160 mg, 0.450 mmol) and trichloroacetonitrile (1.40 g, 10.0 mmol) at room temperature. After the mixture was stirred for 30 min at room temperature, it was quenched with saturated NaHCO₃ solution. The organic layer was separated, and the aqueous layer was extracted with CH₂Cl₂ three times. The combined organic layer was dried over MgSO₄, and concentrated in vacuo to afford the imidate derivative as a yellow syrup. The imidate derivative was dried over P₂O₅ under vacuum for 1 d, and it was dissolved in CH₂Cl₂ (20 mL). 10²⁶⁾ (866 mg, 1.00 mmol) and MS 4 Å (4.0 g) were added to the solution of imidate derivative at room temperature. Next, TMSOTf (20.0 µL, 0.100 mmol) was slowly added to the mixture at −20°C, which was stirred for 45 min at −20°C. The reaction was quenched with saturated NaHCO₃ solution at 0°C. The organic layer was separated and washed with saturated NaHCO₃ solution and then brine. The organic layer was dried over MgSO₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (hexane–AcOEt=2 : 1) to give 15 as colorless syrup (1.47 g, 0.820 mmol, 82%). 15: [α]_D²¹ +14.3 (c=1.0, CHCl₃); FT-IR (neat) 3376, 3064, 3029, 2925, 2854, 1741 cm⁻¹; ¹H-NMR (500 MHz in CDCl₃) δ: 0.87 (12H, m, CH₃×4), 1.26 (72H, m, CH₂×36), 1.82 (1H, m, β-CH of C₃′-O-acyl), 2.30 (2H, m, α-CH₂ of N-acyl), 2.35 (2H, m, α-CH₂ of C₃′-O-acyl), 2.45 (1H, dd, J=15.5, 5.0 Hz, α-CH₂ of C₃-O-acyl), 2.63 (1H, dd, J=15.5, 8.5 Hz, α-CH₂ of C₃-O-acyl), 3.70 (10H, m, H-2, H-4, H-5, H-6a, H-2′, H-6′ab, β-CH×2 and OCH₂CH=CH₂), 4.02 (1H, dd, J=12.5, 5.5 Hz, H-6b), 4.08 (1H, d, J=10.0 Hz, OCH₂CH=CH₂), 4.27 (1H, dt, J=9.5, 3.5 Hz, H-5′), 4.50 (4H, m, PhCH₂×2), 4.68 (5H, m, H-4′, PhCH₂ and CH₂ of Troc), 4.76 (1H, d, J=4.0 Hz, H-1), 5.09 (9H, m, H-3, H-1′, o-C₆H₄(CH₂O)₂P, OCH₂CH=CH₂ and TrocNH), 5.43 (1H, t, J=10.0 Hz, H-3′), 5.72 (1H, m, OCH₂CH=CH₂), 6.24 (1H, d, J=9.5 Hz, acylNH), 7.16–7.38 (19H, m, o-C₆H₄(CH₂O)₂P and PhCH₂×3); ¹³C-NMR (100 MHz in CDCl₃) δ: 14.12, 22.67, 25.05, 25.27, 26.50, 29.35, 29.61, 29.64, 29.67, 31.91, 33.39, 33.58, 33.98, 34.03, 34.38, 38.62, 39.81, 41.73, 51.32, 56.28, 68.27, 68.39, 68.59, 68.65, 68.85, 70.74, 71.06, 71.38, 71.49, 73.62, 74.11, 74.18, 74.54, 74.70, 74.76, 75.73, 76.29, 95.24, 96.55, 100.83, 117.93, 127.56, 127.60, 127.63, 127.66, 127.91, 127.94, 128.32, 128.35, 128.38, 128.42, 128.51, 128.97, 133.35, 134.56, 134.60, 137.79, 138.19, 138.40, 153.98, 171.01, 172.58, 173.41; MS (FAB⁺) m/z 1805 [M+K]⁺; HR-MS (FAB⁺) m/z Calcd for C₉₆H₁₄₆N₂O₁₉Cl₃PK: 1805.8960. Found 1805.8953.

Allyl 6-O-{2′-Amino-6′-O-benzyl-2′-deoxy-3′-O-(3-nonyldodecanoyl)-4′-O-(3-oxido-1,5-dihydrobenzo[e][1,3,2]dioxaphosphepin-3-yl)-β-D-glucopyranosyl}-2-deoxy-3-O-[(R)-3-(benzyloxy)tetradecanoyl]-2-[(R)-3-(benzyloxy)tetradecanoylamino]-α-D-glucopyranoside (16)

Compound 15 (1.10 g, 0.620 mmol) was dissolved in acetic acid (16 mL). Zinc-copper couple powder (4.40 g, 400% (w/w)) was added to the mixture, which was stirred for 2 h at room temperature. After the suspension was filtrated by Celite^®, the filtrate was cooled in ice bath. One molar KOH solution was added dropwise to the mixture at 0°C. The mixture was diluted with AcOEt, and the organic layer was separated and washed with saturated NaHCO₃ solution and then brine. The combined organic layer was dried over Na₂SO₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (hexane–AcOEt–NEt₃=1 : 4 : 0.01) to give 16 as colorless syrup (959 mg, 0.601 mmol, 97%). 16: [α]_D²¹ +14.1 (c=1.0, CHCl₃); FT-IR (neat) 3370, 3063, 3029, 2925, 2854, 1738 cm⁻¹; ¹H-NMR (300 MHz in CDCl₃) δ: 0.88 (12H, m, CH₃×4), 1.25 (72H, m, CH₂×36), 1.94 (1H, m, β-CH of C₃′-O-acyl), 2.31 (2H, m, α-CH₂ of N-acyl), 2.39 (2H, d, J=6.9 Hz, α-CH₂ of C₃′-O-acyl), 2.45 (1H, dd, J=15.0, 4.8 Hz, α-CH₂ of C₃-O-acyl), 2.63 (1H, dd, J=15.0, 7.5 Hz, α-CH₂ of C₃-O-acyl), 2.90 (1H, dd, J=10.2, 8.1 Hz, H-2′), 3.77 (8H, m, H-2, H-4, H-5, H6′ab, OCH₂CH=CH₂ and β-CH×2), 4.03 (1H, dd, J=12.6, 5.4 Hz, H-6a), 4.12 (1H, m, OCH₂CH=CH₂), 4.31 (2H, m, H-6b and H-5′), 4.53 (5H, m, H-4′, PhCH₂×2), 4.56 (1H, d, J=12.0 Hz, PhCH₂), 4.65 (1H, d, J=12.0 Hz, PhCH₂), 4.78 (1H, d, J=3.6 Hz, H-1), 5.08 (9H, m, H-3, H-1′, H-3′, o-C₆H₄(CH₂O)₂P and OCH₂CH=CH₂), 5.72 (1H, m, OCH₂CH=CH₂), 6.26 (1H, d, J=9.6 Hz, acylNH), 7.15–7.39 (19H, m, o-C₆H₄(CH₂O)₂P and PhCH₂×3); ¹³C-NMR (125 MHz in CDCl₃) δ: 14.13, 22.68, 25.10, 25.29, 26.40, 26.60, 29.35, 29.62, 29.67, 30.00, 31.91, 33.60, 33.79, 33.99, 34.12, 34.59, 39.00, 39.80, 41.71, 51.26, 56.07, 68.18, 68.23, 68.33, 68.57, 68.64, 68.96, 69.07, 69.38, 70.84, 71.04, 71.40, 73.59, 74.25, 74.29, 74.95, 75.00, 75.76, 76.29, 76.77, 96.57, 104.58, 117.89, 127.59, 127.60, 127.62, 127.86, 127.91, 128.33, 128.36, 128.47, 128.91, 128.92, 133.33, 134.59, 134.63, 137.95, 138.17, 138.39, 171.06, 172.57, 173.64; MS (FAB⁺) m/z 1616 [M+Na]⁺; HR-MS (FAB⁺) m/z Calcd for C₉₃H₁₄₅N₂O₁₇PNa: 1616.0152. Found 1616.0352.

Allyl 6-O-{6′-O-Benzyl-2′-deoxy-3′-O-(3-nonyldodecanoyl)-2′-(3-nonyldodecanoylamino)-4′-O-(3-oxido-1,5-dihydrobenzo[e][1,3,2]dioxaphosphepin-3-yl)-β-D-glucopyranosyl}-2-deoxy-3-O-[(R)-3-(benzyloxy)tetradecanoyl]-2-[(R)-3-(benzyloxy)tetradecanoylamino]-α-D-glucopyranoside (17)

To a solution of 16 (924 mg, 0.580 mmol) in CH₂Cl₂ (62 mL) were added 3-nonyldodecanoic acid (400 mg, 1.20 mmol), MS 4 Å (10.0 g) and EDCI (240 mg, 1.20 mmol) at room temperature. After the reaction mixture was stirred for 11 h at room temperature, it was filtrated by Celite^®. The filtrate was diluted with CH₂Cl₂ and quenched by H₂O. The organic layer was separated and washed with brine. The combined organic layer was dried over MgSO₄, and concentrated in vacuo. The residue was purified by silica gel chromatography (hexane–AcOEt=2 : 1) to give 17 as colorless syrup (550 mg, 0.290 mmol, 50%). 17: [α]_D²¹ +10.9 (c=1.0, CHCl₃); FT-IR (neat) 3514, 3311, 3063, 3031, 2954, 2922, 2852, 1736, 1651 cm⁻¹; ¹H-NMR (300 MHz in CDCl₃) δ: 0.88 (18H, m, CH₃×6), 1.25 (104H, m, CH₂×52), 1.81 (2H, m, β-CH of N′-acyl and β-CH of C₃′-O-acyl), 2.01 (2H, d, J=7.2 Hz, α-CH₂ of N-acyl), 2.31 (4H, m, α-CH₂ of N′-acyl and α-CH₂ of C₃′-O-acyl), 2.43 (1H, dd, J=15.3, 5.4 Hz, α-CH₂ of C₃-O-acyl), 2.63 (1H, dd, J=15.3, 7.2 Hz, α-CH₂ of C₃-O-acyl), 3.77 (10H, m, H-2, H-4, H-5, H-6a, H-2′, H-6′ab, OCH₂CH=CH₂, β-CH of N-acyl and β-CH of C₃-O-acyl), 4.01 (2H, m, H-6b and OCH₂CH=CH₂), 4.28 (1H, dt, J=10.5, 3.3 Hz, H-5′), 4.45 (1H, d, J=11.4 Hz, PhCH₂), 4.50 (2H, s, PhCH₂), 4.57 (1H, d, J=11.4 Hz, PhCH₂), 4.58 (3H, m, H-4′ and PhCH₂), 4.76 (1H, d, J=3.6 Hz, H-1), 4.85 (1H, d, J=8.4 Hz, H-1′), 5.06 (7H, m, H-3′, o-C₆H₄(CH₂O)₂P and OCH₂CH=CH₂), 5.39 (1H, dd, J=10.5, 9.3 Hz, H-3), 5.46 (1H, d, J=8.4 Hz, N′H), 5.71 (1H, m, OCH₂CH=CH₂), 6.21 (1H, d, J=9.3 Hz, NH), 7.15–7.34 (19H, m, o-C₆H₄(CH₂O)₂P and PhCH₂×3); ¹³C-NMR (75 MHz in CDCl₃) δ: 14.14, 22.71, 25.18, 25.28, 25.62, 26.41, 26.47, 26.55, 26.59, 29.39, 29.43, 29.72, 29.78, 30.07, 30.14, 31.95, 33.26, 33.47, 33.71, 34.09, 38.17, 38.75, 39.78, 41.86, 51.62, 54.59, 67.98, 68.21, 68.43, 68.94, 70.01, 71.14, 71.43, 72.00, 73.61, 73.79, 74.08, 74.72, 75.66, 76.30, 96.52, 100.79, 117.78, 127.53, 127.62, 127.68, 127.82, 128.02, 128.27, 128.33, 128.39, 128.46, 128.53, 128.97, 133.37, 134.64, 134.69, 137.68, 138.39, 138.46, 148.87, 160.52, 171.00, 172.39, 173.15, 173.52; MS (FAB⁺) m/z 1940 [M+K]⁺; HR-MS (FAB⁺) m/z Calcd for C₁₁₄H₁₈₅N₂O₁₈PK: 1940.2997. Found 1940.3008.

6-O-{6′-O-Benzyl-2′-deoxy-3′-O-(3-nonyldodecanoyl)-2′-(3-nonyldodecanoylamino)-4′-O-(3-oxido-1,5-dihydrobenzo[e][1,3,2]dioxaphosphepin-3-yl)-β-D-glucopyranosyl}-2-deoxy-3-O-[(R)-3-(benzyloxy)tetradecanoyl]-2-[(R)-3-(benzyloxy)tetradecanoylamino]-α-D-glucopyranose (18)

To a solution of 17 (500 mg, 0.260 mmol) in THF (43 mL, degassed with Ar) was added [Ir(cod)(MePh₂P)₂]PF₆ (20.0 mg, 0.0260 mmol) at room temperature. After the mixture was stirred for 10 min under bubbling H₂ gas at room temperature, it was stirred for another hour. H₂O (10 mL) and I₂ (66.0 mg, 0.540 mmol) were added to the mixture, which was stirred for 30 min at room temperature. The mixture was diluted with AcOEt and quenched with saturated Na₂S₂O₃ solution. The organic layer was separated and washed with saturated NaHCO₃ solution and then brine. The combined organic layer was dried over MgSO₄, and concentrated in vacuo. The residue was purified by silica gel chromatography (hexane–AcOEt=2 : 1) to give 18 (421 mg, 0.226 mmol, 87%). 18: [α]_D¹⁸ −1.9 (c=1.0, CHCl₃); FT-IR (neat) 3409, 3327, 3063, 3030, 2954, 2925, 2853, 1742, 1650 cm⁻¹; ¹H-NMR (300 MHz in CDCl₃) δ: 0.86 (18H, m, CH₃×6), 1.23 (104H, m, CH₂×52), 1.79 (2H, m, β-CH of N′-acyl and β-CH of C₃′-O-acyl), 2.00 (2H, d, J=6.9 Hz, α-CH₂ of N-acyl), 2.31 (4H, m, α-CH₂ of N′-acyl and α−CH₂ of C₃′-O-acyl), 2.42 (1H, dd, J=14.7, 4.8 Hz, α-CH₂ of C₃-O-acyl), 2.59 (1H, dd, J=14.7, 7.8 Hz, α-CH₂ of C₃-O-acyl), 3.30 (2H, m, β-CH of N-acyl and β−CH of C₃-O-acyl), 3.74 (6H, m, H-4, H-5, H-6a, H-2′ and H-6′ab), 3.98 (3H, m, H-2, H-6b and H-5′), 4.52 (7H, m, PhCH₂×3 and H-4′), 5.01 (6H, m, H-1, H-1′ and o-C₆H₄(CH₂O)₂P), 5.42 (1H, d, J=8.1 Hz, N′H), 5.60 (2H, m, H-3 and H-3′), 6.21 (1H, d, J=9.3 Hz, NH), 7.16–7.37 (19H, m, o-C₆H₄(CH₂O)₂P and PhCH₂×3); ¹³C-NMR (100 MHz in CDCl₃) δ: 14.13, 22.70, 25.03, 25.10, 25.22, 26.35, 26.42, 26.54, 26.62, 26.70, 29.36, 29.41, 29.64, 29.66, 29.69, 29.72, 29.79, 29.94, 30.04, 30.09, 30.11, 30.19, 31.92, 31.95, 33.29, 33.37, 33.48, 33.73, 33.86, 33.91, 34.07, 34.17, 34.39, 35.10, 38.72, 39.78, 41.90, 51.59, 56.30, 68.28, 68.62, 68.71, 69.70, 70.98, 71.43, 71.56, 71.65, 73.57, 73.77, 74.70, 75.05, 75.11, 75.81, 76.61, 91.56, 99.57, 127.74, 127.81, 127.86, 127.89, 127.98, 128.03, 128.12, 128.35, 128.38, 128.41, 128.46, 128.53, 128.99, 134.66, 137.62, 138.11, 138.44, 171.20, 172.70, 173.13, 174.15; MS (FAB⁺) m/z 1900 [M+K]⁺; HR-MS (FAB⁺) m/z Calcd for C₁₁₁H₁₈₁N₂O₁₈PK: 1900.2684. Found 1900.2678.

6-O-{2′-Deoxy-3′-O-(3-nonyldodecanoyl)-2′-(3-nonyldodecanoylamino)-β-D-glucopyranosyl}-2-deoxy-3-O-[(R)-3-hydroxytetradecanoyl]-2-[(R)-3-hydroxytetradecanoylamino]-α-D-glucopyranose 4,4′-Diphosphate (5)

Compound 18 (370 mg, 0.200 mmol) was dissolved in THF (20 mL), and then 1.6 M solution of n-BuLi (0.16 mL in hexane, 0.260 mmol) was added to the solution at −78°C. The mixture was stirred for 5 min at −78°C, and pyrophosphate (130 mg, 0.240 mmol) was added to the mixture at −78°C. After stirring for 30 min at room temperature, the reaction was quenched with saturated NaHCO₃ solution. The organic layer was separated and dried over MgSO₄, and it was concentrated to afford the crude product as a yellow syrup. Next, it was dissolved in THF (20 mL), and then Pd-Black (300 mg, 95% (w/w)) was added to the solution at room temperature. The mixture was stirred for 4 h under H₂ (10.0 kg/cm²) at room temperature. Triethylamine was added to the mixture at room temperature, and Pd/C was removed by filtration. The filtrate was concentrated in vacuo and the residue was purified by reverse phase silica gel chromatography (MeOH only) to give 5 (213 mg, 0.136 mmol, 68%).

N,O-3NDDA-Dicondenced Analog 5

[α]_D²⁰ +23.2 (c=1.0, CHCl₃); FT-IR (neat) 3360, 3062, 2957, 2933, 2855, 2710, 2500, 1739, 1731, 1660 cm⁻¹; ¹H-NMR (300 MHz in C₅D₅N) δ: 0.87 (18H, m, CH₃×6), 1.23 (106H, m, CH₂×52, β-CH of N′-acyl and β-CH of C₃′-O-acyl), 2.23 (1H, m, α-CH₂ of N′-acyl), 2.36 (1H, m, α-CH₂ of C₃′-O-acyl), 2.69 (6H, m, α-CH₂ of N-acyl, α-CH₂ of C₃-O-acyl, α-CH₂ of N′-acyl and α-CH₂ of C₃′-O-acyl), 3.68 (1H, d, J=9.3 Hz, H-5′), 4.04 (1H, t, J=9.6 Hz, H-4), 4.13 (1H, d, J=12.0 Hz, H-6′a), 4.42 (4H, m, H-6ab, β-CH of N-acyl and β-CH of C₃-O-acyl), 4.57 (1H, d, J=12.0 Hz, H-6′b), 4.73 (1H, q, J=9.0 Hz, H-2′), 4.90 (2H, m, H-2 and H-5), 5.01 (1H, q, J=9.9 Hz, H-4′), 5.72 (1H, d, J=8.7 Hz, H-1′), 5.96 (2H, m, H-3 and H-3′), 6.30 (1H, dd, J=7.8, 3.0 Hz, H-1), 8.28 (1H, d, J=9.0 Hz, NH), 9.39 (1H, d, J=9.0 Hz, N′H); ¹³C-NMR (125 MHz in C₅D₅N) δ: 8.94, 14.29, 14.32, 22.94, 22.97, 23.00, 26.25, 26.79, 27.14, 27.19, 29.63, 29.70, 29.75, 29.77, 29.87, 29.94, 30.01, 30.05, 30.09, 30.11, 30.15, 30.21, 30.30, 30.40, 30.52, 30.60, 30.70, 32.14, 32.18, 32.23, 33.83, 34.02, 34.10, 34.15, 34.81, 35.63, 38.05, 38.61, 39.90, 41.91, 43.98, 45.56, 45.65, 53.13, 53.69, 54.79, 61.44, 68.24, 68.43, 69.53, 71.97, 74.86, 75.22, 75.35, 77.33, 93.67, 101.76, 172.74, 173.39, 173.46; MS (FAB⁻) m/z 1568 [M−H]⁻; HR-MS (FAB⁻) m/z Calcd for C₈₂H₁₅₇N₂O₂₁P₂: 1568.0754. Found 1568.0743.

Allyl 6-O-{6′-O-Benzyl-2′-deoxy-3′-O-(3-nonyldodecanoyl)-2′-[(R)-3-(dodecanoyloxy)tetradecanoylamino]-4′-O-(3-oxido-1,5-dihydrobenzo[e][1,3,2]dioxaphosphepin-3-yl)-β-D-glucopyranosyl}-2-deoxy-3-O-[(R)-3-(benzyloxy)tetradecanoyl]-2-[(R)-3-(benzyloxy)tetradecanoyl Amino]-α-D-glucopyranoside (20)

Compound 20 (750 mg, 56%) was prepared from 16 (1.30 g, 0.67 mmol) and (R)-3-(tetradecanoyloxy)tetradecanoic acid (19) (600 mg, 1.30 mmol) according to the procedure described for compound 17. 20: [α]_D²⁰ +13.0 (c=1.0, CHCl₃); FT-IR (neat) 3514, 3308, 3087, 3065, 3031, 2955, 2925, 2853, 1739, 1653 cm⁻¹; ¹H-NMR (500 MHz in CDCl₃) δ: 0.88 (18H, m, CH₃×6), 1.25–1.58 (112H, m, CH₂×56), 1.81 (1H, m, β-CH of C₃′-O-acyl), 2.31 (8H, m, α-CH₂ of N-acyl, α-CH₂ of C₃′-O-acyl and N′HCOCH₂CHROCOCH₂R), 2.42 (1H, m, α-CH₂ of C₃-O-acyl), 2.63 (1H, dd, J=15.5, 7.5 Hz, α-CH₂ of C₃-O-acyl), 3.77 (10H, m, H-2, H-4, H-5, H-6a, H-2′, H-6′ab, OCH₂CH=CH₂, β-CH of N-acyl and β-CH of C₃-O-acyl), 4.02 (2H, m, OCH₂CH=CH₂ and H−6b), 4.28 (1H, dt, J=10.5, 3.5 Hz, H-5′), 4.45 (1H, d, J=11.5 Hz, PhCH₂), 4.50 (5H, m, PhCH₂×2 and H−4′), 4.54 (1H, d, J=11.5 Hz, PhCH₂), 4.77 (1H, d, J=3.5 Hz, H-1), 4.86 (1H, d, J=8.0 Hz, H-1′), 5.11 (8H, m, H-3′, OCH₂CH=CH₂, o-C₆H₄(CH₂O)₂P and β-CH of N′-acyl), 5.39 (1H, dd, J=10.5, 9.0 Hz, H-3), 5.71 (1H, m, OCH₂CH=CH₂), 6.00 (1H, d, J=8.0 Hz, N′H), 6.22 (1H, d, J=9.5 Hz, NH), 7.16–7.38 (19H, m, o-C₆H₄(CH₂O)₂P and PhCH₂×3); ¹³C-NMR (100 MHz in CDCl₃) δ: 14.12, 22.69, 24.95, 25.19, 25.26, 25.29, 26.55, 29.25, 29.38, 29.54, 29.58, 29.65, 29.70, 29.78, 30.00, 30.08, 30.11, 31.92, 31.94, 33.41, 33.64, 34.09, 34.32, 34.43, 34.48, 34.57, 38.61, 39.75, 41.88, 42.03, 45.61, 51.62, 51.92, 54.96, 58.72, 67.22, 67.84, 68.26, 68.61, 70.80, 71.17, 71.21, 71.44, 71.90, 73.59, 73.90, 74.14, 74.82, 75.62, 76.29, 96.63, 100.69, 117.77, 127.50, 127.53, 127.62, 127.65, 127.79, 127.95, 128.26, 128.34, 128.38, 128.46, 128.52, 128.96, 129.00, 133.41, 134.63, 134.69, 137.79, 138.45, 170.08, 171.01, 172.35, 173.44, 174.26; MS (FAB⁺) m/z 2040 [M+K]⁺; HR-MS (FAB⁺) m/z Calcd for C₁₁₉H₁₉₃N₂O₂₀PK: 2040.3521. Found 2040.3530.

6-O-{6′-O-Benzyl-2′-deoxy-3′-O-(3-nonyldodecanoyl)-2′-[(R)-3-(dodecanoyloxy)tetradecanoylamino]-4′-O-(3-oxido-1,5-dihydrobenzo[e][1,3,2]dioxaphosphepin-3-yl)-β-D-glucopyranosyl}-2-deoxy-3-O-[(R)-3-(benzyloxy)tetradecanoyl]-2-[(R)-3-(benzyloxy)tetradecanoyl Amino]-α-D-glucopyranose (21)

Compound 21 (430 mg, 87%) was prepared from 20 (500 mg, 0.25 mmol) according to the procedure described for compound 18. 21: [α]_D²² +15.4 (c=0.26, CHCl₃); FT-IR (neat) 3417, 3331, 3087, 3063, 3030, 2954, 2924, 2853, 1741, 1732, 1652 cm⁻¹; ¹H-NMR (500 MHz in CDCl₃) δ: 0.88 (18H, m, CH₃×6), 1.25–1.58 (112H, m, CH₂×56), 1.81 (1H, m, β-CH of C₃′-O-acyl), 2.29 (8H, m, α-CH₂ of N-acyl, α-CH₂ of C₃′-O-acyl and N′HCOCH₂CHROCOCH₂R), 2.43 (1H, m, α-CH₂ of C₃-O-acyl), 2.61 (1H, dd, J=15.0, 7.5 Hz, α-CH₂ of C₃-O-acyl), 3.38 (1H, t, J=9.0 Hz, β-CH of N-acyl), 3.55 (1H, q, J=8.5 Hz, β-CH of C₃-O-acyl), 3.79 (6H, m, H-4, H-5, H-6a, H-2′ and H-6′ab), 4.01 (2H, m, H-2 and H-6b), 4.19 (1H, dt, J=10.0, 3.5 Hz, H-5′), 4.53 (7H, m, H-4′ and PhCH₂×3), 5.07 (7H, m, H-1, H-1′, o-C₆H₄(CH₂O)₂P and β-CH of N′-acyl), 5.17 (1H, t, J=8.5 Hz, H-3′), 5.46 (1H, t, J=9.0 Hz, H-3), 6.11 (1H, d, J=8.0 Hz, N′H), 6.29 (1H, d, J=9.5 Hz, NH), 7.14–7.41 (19H, m, o-C₆H₄(CH₂O)₂P and PhCH₂×3); ¹³C-NMR (125 MHz in CDCl₃) δ: 14.10, 22.68, 24.94, 25.11, 25.21, 25.24, 25.59, 26.56, 29.22, 29.35, 29.49, 29.57, 29.65, 29.68, 29.71, 29.77, 30.01, 30.09, 30.12, 31.92, 33.44, 33.67, 34.13, 34.35, 34.45, 34.49, 38.62, 39.79, 41.89, 42.09, 51.65, 55.57, 67.95, 68.06, 68.28, 68.62, 68.72, 69.56, 70.98, 71.26, 71.52, 71.74, 71.81, 73.59, 74.13, 74.53, 74.98, 75.79, 76.50, 91.60, 99.86, 127.60, 127.66, 127.77, 127.81, 127.85, 127.93, 127.99, 128.33, 128.38, 128.44, 128.49, 128.93, 128.97, 134.67, 134.73, 137.81, 138.16, 138.48, 170.40, 171.18, 172.68, 173.32, 174.58; MS (FAB⁺) m/z 2000 [M+K]⁺; HR-MS (FAB⁺) m/z Calcd for C₁₁₆H₁₈₉N₂O₂₀PK: 2000.3208. Found 2000.3236.

6-O-{2′-Deoxy-3′-O-(3-nonyldodecanoyl)-2′-[(R)-3-(dodecanoyloxy)tetradecanoylamino]-β-D-glucopyranosyl}-2-deoxy-3-O-[(R)-3-hydroxytetradecanoyl]-2-[(R)-3-hydroxytetradecanoylamino]-α-D-glucopyranose 4,4′-Diphosphate (6)

Compound 6 (120 mg, 45%) was prepared from 21 (320 mg, 0.16 mmol) according to the procedure described for compound 5.

O-3NDDA-monocondenced Analog 6

[α]_D²⁰ +31.0 (c=1.0, CHCl₃); FT-IR (neat) 3361, 3064, 2955, 2924, 2853, 2685, 1732, 1660 cm⁻¹; ¹H-NMR (300 MHz in C₅D₅N) δ: 0.87 (18H, m CH₃×6), 1.26 (111H, m, CH₂×55, β-CH of C₃′-O-acyl), 2.22 (1H, m, α-CH₂ of C₃′-O-acyl), 2.75 (8H, m, α-CH₂ of N-acyl, α-CH₂ of C₃-O-acyl, α-CH₂ of C₃′-O-acyl and NHCOCH₂CHROCOCH₂R), 3.21 (1H, dd, J=14,7, 5.1 Hz, N′HCOCH₂CHROCOCH₂R), 3.71 (1H, d, J=9.9 Hz, H-5′), 3.99 (1H, t, J=9.6 Hz, H-4), 4.16 (1H, d, J=12.0 Hz, H-6′a), 4.45 (5H, m, H-6ab, H-6′b, β-CH of N-acyl and β-CH of C₃-O-acyl), 4.71 (1H, q, J=9.9 Hz, H-2′), 4.90 (2H, m, H-2 and H-5), 5.05 (1H, q, J=9.3 Hz, H-4′), 5.78 (1H, d, J=7.8 Hz, H-1′), 5.87 (1H, t, J=5.4 Hz, N′HCOCH₂CHROCOCH₂R), 5.97 (2H, m, H-3 and H-3′), 6.34 (1H, m, H-1), 8.37 (1H, d, J=7.5 Hz, NH), 9.69 (1H, d, J=8.4 Hz, N′H); ¹³C-NMR (125 MHz in C₅D₅N) δ: 8.73, 14.29, 18.67, 22.94, 22.96, 22.99, 23.68, 25.48, 25.82, 26.05, 26.24, 26.77, 27.06, 29.55, 29.63, 29.70, 29.75, 29.82, 29.92, 29.95, 29.99, 30.02, 30.12, 30.20, 30.22, 30.50, 30.60, 30.77, 32.14, 32.18, 32.21, 33.75, 34.07, 34.80, 38.04, 38.45, 39.83, 41.31, 43.92, 45.32, 45.64, 53.45, 54.87, 61.36, 68.21, 68.47, 69.44, 71.52, 72.06, 74.73, 75.23, 77.22, 93.73, 101.52, 170.49, 172.71, 173.35, 173.45; MS (FAB⁻) m/z 1668 [M−H]⁺; HR-MS (FAB⁻) m/z Calcd for C₈₇H₁₆₅N₂O₂₃P₂: 1668.1278. Found 1668.1261.

Allyl 6-O-{6′-O-Benzyl-2′-deoxy-3′-O-[(R)-3-(tetradecanoyloxy)tetradecanoyl]-2′-(3-nonyldodecanoylamino)-4′-O-(3-oxido-1,5-dihydrobenzo[e][1,3,2]dioxaphosphepin-3-yl)-β-D-glucopyranosyl}-2-deoxy-3-O-[(R)-3-(benzyloxy)tetradecanoyl]-2-[(R)-3-(benzyloxy)tetradecanoylamino]-α-D-glucopyranoside (23)

Compound 23 (1.01 g, 73%) was prepared from 22²⁶⁾ (1.40 g, 0.740 mmol) and 3NDDA (500 mg, 1.5 mmol) according to the procedure described for compound 17. 23: [α]_D²³ +18.9 (c=1.0, CHCl₃); FT-IR (neat) 3517, 3305, 3087, 3063, 3030, 2922, 2852, 1736, 1651 cm⁻¹; ¹H-NMR (300 MHz in CDCl₃) δ: 0.88 (18H, m, CH₃×6), 1.25 (116H, m, CH₂×58), 1.82 (1H, m, β-CH of N′-acyl), 2.03 (2H, m, α-CH₂ of N-acyl), 2.27 (4H, m, α-CH₂ of N′-acyl and OCOCH₂CHROCOCH₂R), 2.43 (1H, dd, J=15.3, 5.4 Hz, α-CH₂ of C₃-O-acyl), 2.63 (3H, m, α-CH₂ of C₃-O-acyl and OCOCH₂CHROCOCH₂R), 3.74 (10H, m, H-2, H-4, H-5, H-6a, H-2′, H-6′ab, OCH₂CH=CH₂, β-CH of N-acyl and β-CH of C₃-O-acyl), 4.03 (2H, m, H-6b and OCH₂CH=CH₂), 4.28 (1H, dt, J=10.8, 3.9 Hz, H-5′), 4.46 (1H, d, J=11.4 Hz, PhCH₂), 4.53 (1H, d, J=11.4 Hz, PhCH₂), 4.55 (5H, m, H-4′ and PhCH₂×2), 4.77 (1H, d, J=3.6 Hz, H-1), 5.12 (8H, m, H-1′, H-3′, OCH₂CH=CH₂, o-C₆H₄(CH₂O)₂P and OCOCH₂CHROCOCH₂R), 5.44 (1H, dd, J=10.2, 9.0 Hz, H-3), 5.72 (1H, m, OCH₂CH=CH₂), 5.99 (1H, d, J=7.5 Hz, N′H), 6.23 (1H, d, J=9.3 Hz, NH), 7.17–7.38 (19H, m, o-C₆H₄(CH₂O)₂P and PhCH₂×3); ¹³C-NMR (75 MHz in CDCl₃) δ: 14.13, 22.70, 25.09, 25.19, 25.23, 25.28, 26.39, 26.44, 29.21, 29.37, 29.43, 29.59, 29.68, 29.72, 29.78, 30.04, 30.11, 31.94, 33.24, 33.47, 34.08, 34.58, 34.80, 39.76, 41.61, 41.86, 51.62, 55.51, 68.22, 68.54, 68.85, 70.26, 70.99, 71.17, 71.43, 72.82, 73.61, 73.80, 75.67, 76.31, 96.54, 100.72, 117.77, 127.53, 127.62, 127.65, 127.82, 127.97, 128.27, 128.32, 128.38, 128.49, 128.78, 129.04, 133.38, 134.72, 134.79, 137.74, 138.37, 138.45, 170.35, 171.00, 172.41, 173.60, 173.80; MS (FAB⁺) m/z 2068 [M+K]⁺; HR-MS (FAB⁺) m/z Calcd for C₁₂₁H₁₉₇N₂O₂₀PK: 2068.3835. Found 2068.3826.

6-O-{6′-O-Benzyl-2′-deoxy-3′-O-[(R)-3-(tetradecanoyloxy)tetradecanoyl]-2′-(3-nonyldodecanoylamino)-4′-O-(3-oxido-1,5-dihydrobenzo[e][1,3,2]dioxaphosphepin-3-yl)-β-D-glucopyranosyl}-2-deoxy-3-O-[(R)-3-(benzyloxy)tetradecanoyl]-2-[(R)-3-(benzyloxy)tetradecanoylamino]-α-D-glucopyranose (24)

Compound 24 (490 mg, 82%) was prepared from 23 (600 mg, 0.300 mmol) according to the procedure described for compound 18. 24: [α]_D²³ −10.5 (c=1.0, CHCl₃); FT-IR (neat) 3412, 3320, 3087, 3064, 3031, 2954, 2924, 2852, 1738, 1725, 1649 cm⁻¹; ¹H-NMR (500 MHz in CDCl₃) δ: 0.88 (18H, m, CH₃×6), 1.25 (116H, m, CH₂×58), 1.79 (1H, m, β-CH of N′-acyl), 2.02 (2H, t, J=6.0 Hz, α-CH₂ of N-acyl), 2.29 (4H, m, α-CH₂ of N′-acyl and OCOCH₂CHROCOCH₂R), 2.44 (1H, dd, J=15.0, 5.0 Hz, α-CH₂ of C₃-O-acyl), 2.58 (1H, dd, J=13.0, 7.5 Hz, OCOCH₂CHROCOCH₂R), 2.62 (1H, dd, J=12.0, 7.5 Hz, OCOCH₂CHROCOCH₂R), 2.66 (1H, dd, J=15.0, 4.0 Hz, α-CH₂ of C₃-O-acyl), 3.27 (1H, q, J=8.0 Hz, β-CH of N-acyl), 3.37 (1H, t, J=9.5 Hz, β-CH of C₃-O-acyl), 3.77 (6H, m, H-4, H-5, H-6a, H-2′ and H-6′ab), 4.02 (2H, m, H-2, H-6b), 4.20 (1H, dt, J=9.5, 3.0 Hz, H-5′), 4.55 (7H, m, H-4′ and PhCH₂×3), 5.06 (7H, m, H-1, H-1′, H-3′ and o-C₆H₄(CH₂O)₂), 5.27 (1H, m, OCOCH₂CHROCOCH₂R), 5.59 (1H, t, J=9.0 Hz, H-3), 5.68 (1H, d, J=8.5 Hz, N′H), 6.25 (1H, m, NH), 7.17–7.41 (19H, m, o-C₆H₄(CH₂O)₂P and PhCH₂×3); ¹³C-NMR (125 MHz in CDCl₃) δ: 14.11, 22.69, 22.71, 25.09, 25.12, 25.23, 26.34, 26.58, 29.23, 29.37, 29.42, 29.56, 29.59, 29.64, 29.66, 29.68, 29.72, 29.78, 30.06, 30.15, 31.93, 31.95, 33.40, 33.52, 34.12, 34.21, 34.62, 34.81, 34.94, 39.80, 40.26, 41.77, 41.96, 51.65, 57.03, 67.97, 68.35, 68.69, 69.74, 70.61, 70.98, 71.59, 71.98, 72.61, 73.64, 73.73, 74.87, 75.15, 75.85, 76.54, 91.61, 99.17, 127.62, 127.67, 127.72, 127.82, 127.89, 127.97, 128.03, 128.10, 128.35, 128.36, 128.41, 128.48, 128.78, 129.01, 134.75, 134.81, 137.73, 138.16, 138.49, 170.12, 171.19, 172.75, 173.72, 174.65; MS (FAB⁺) m/z 2028 [M+K]⁺; HR-MS (FAB⁺) m/z Calcd for C₁₁₈H₁₉₃N₂O₂₀PK: 2028.3521. Found 2028.3512.

6-O-{2′-Deoxy-3′-O-[(R)-3-(tetradecanoyloxy)tetradecanoyl]-2′-(3-nonyldodecanoylamino)-β-D-glucopyranosyl}-2-deoxy-3-O-[(R)-3-hydroxytetradecanoyl]-2-[(R)-3-hydroxytetradecanoylamino]-α-D-glucopyranose 4,4′-Diphosphate (7)

Compound 7 (120 mg, 35%) was prepared from 24 (390 mg, 0.200 mmol) according to the procedure described for compound 5.

N-3NDDA-monocondenced Analog 7

[α]_D²³ +22.3 (c=1.0, CHCl₃); FT-IR (neat) 3376, 2954, 2929, 2853, 2699, 1739, 1659, 1652 cm⁻¹; ¹H-NMR (300 MHz in C₅D₅N) δ: 0.86 (18H, m, CH₃×6), 1.25 (115H, m, CH₂×57, β-CH of N′-acyl), 2.33 (1H, m, α-CH₂ of N′-acyl), 2.60 (7H, m, α-CH₂ of N-acyl, α-CH₂ of C₃-O-acyl, α-CH₂ of N′-acyl and OCOCH₂CHROCOCH₂R), 3.04 (1H, m, OCOCH₂CHROCOCH₂R), 3.30 (1H, dd, J=16.5, 6.6 Hz, OCOCH₂CHROCOCH₂R), 3.70 (1H, m, H-5), 4.01 (1H, t, J=9.6 Hz, H-4), 4.16 (1H, d, J=12.0 Hz, H-6′a), 4.38 (4H, m, H-6a, H-6′b, β-CH of N-acyl and β-CH of C₃-O-acyl), 4.56 (1H, d, J=12.3 Hz, H-6b), 4.77 (1H, dd, J=9.3 Hz, H-2′), 4.91 (2H, m, H-2 and H-5), 5.05 (1H, q, J=9.6 Hz, H-4′), 5.77 (2H, m, H-1′ and OCOCH₂CHROCOCH₂R), 5.96 (2H, m, H-3 and H-3′), 6.33 (1H, dd, J=7.5, 3.0 Hz, H-1), 8.34 (1H, d, J=9.3 Hz, NH), 9.44 (1H, d, J=8.7 Hz, N′H); ¹³C-NMR (75 MHz in C₅D₅N) δ: 8.72 11.57, 14.26, 14.41, 22.78, 22.93, 25.48, 25.63, 25.79, 26.21, 27.12, 27.22, 29.61, 29.74, 29.83, 29.98, 30.09, 30.25, 30.36, 30.50, 30.66, 30.84, 31.99, 32.12, 32.22, 33.83, 33.97, 34.07, 34.39, 34.57, 34.70, 34.89, 35.57, 38.03, 38.34, 38.47, 39.35, 39.83, 41.72, 41.88, 42.06, 43.42, 43.57, 43.90, 45.32, 45.54, 45.69, 51.25, 53.48, 54.50, 54.71, 61.42, 68.12, 68.23, 68.47, 68.57, 68.80, 69.47, 69.82, 70.49, 71.94, 72.97, 74.83, 75.20, 76.12, 76.40, 77.14, 93.12, 93.81, 101.25, 101.64, 170.88, 172.71, 173.28, 173.34, 173.54; MS (FAB⁻) m/z 1696 [M−H]⁻; HR-MS (FAB⁻) m/z Calcd for C₈₉H₁₆₉N₂O₂₃P₂: 1696.1591. Found 1696.1578.

6-O-{2′-Deoxy-3′-O-(3-pentyloctanoyl)-2′-(3-pentyloctanoylamino)-β-D-glucopyranosyl}-2-deoxy-3-O-[(R)-3-hydroxytetradecanoyl]-2-[(R)-3-hydroxytetradecanoylamino]-α-D-glucopyranose 4,4′-Diphosphate (25)

Compound 25 was synthesized by using 3-pentyloctanoic acid instead of 3NDDA in accordance with the procedure described for compound 5. 25: [α]_D¹⁹ +24.0 (c=1.0, CHCl₃), FT-IR (neat) 3360, 2955, 2931, 2855, 2678, 1738, 1666 cm⁻¹; ¹H-NMR (500 MHz in C₅D₅N) δ: 0.86 (18H, m, CH₃×6), 1.25 (74H, m, CH₂×36, β-CH of N′-acyl and β-CH of C₃′-O-acyl), 2.15 (1H, m, α-CH₂ of N′-acyl), 2.28 (1H, m, α-CH₂ of C₃′-O-acyl), 2.67 (6H, m, α-CH₂ of N-acyl, α-CH₂ of C₃-O-acyl, α-CH₂ of N′-acyl and α-CH₂ of C₃′-O-acyl), 3.64 (1H, d, J=10.0 Hz, H-5′), 4.05 (1H, t, J=9.5 Hz, H-4), 4.10 (1H, d, J=11.5 Hz, H-6′a), 4.40 (4H, m, H-6ab, β-CH of N-acyl and β-CH of C₃-O-acyl), 4.54 (1H, d, J=11.5 Hz, H-6′b), 4.65 (1H, q, J=9.0 Hz, H-2′), 4.86 (2H, m, H-2 and H-5), 4.93 (1H, dd, J=10.0, 9.5 Hz, H-4′), 5.66 (1H, d, J=8.5 Hz, H-1′), 5.86 (1H, t, J=9.5 Hz, H-3′), 5.95 (1H, t, J=10.0 Hz, H-3), 6.25 (1H, dd, J=8.0, 3.0 Hz, H-1), 8.26 (1H, d, J=8.5 Hz, NH), 9.30 (1H, d, J=9.0 Hz, N′H); ¹³C-NMR (75 MHz in C₅D₅N) δ: 8.78, 14.24, 14.31, 14.34, 22.90, 22.96, 23.00, 23.06, 23.07, 26.20, 26.23, 26.28, 26.66, 26.68, 26.71, 29.58, 29.90, 29.95, 29.98, 30.06, 30.08, 32.09, 32.51, 32.53, 32.57, 32.66, 33.60, 33.82, 33.91, 33.95, 34.64, 35.46, 38.00, 38.39, 39.70, 41.75, 43.92, 45.21, 45.66, 53.40, 53.45, 54.72, 61.30, 68.13, 68.40, 69.25, 71.94, 74.82, 74.86, 75.20, 77.14, 93.84, 101.80, 172.67, 173.24, 173.30, 173.33; MS (FAB⁻) m/z 1343 [M−H]⁻; HR-MS (FAB⁻) m/z Calcd for C₆₆H₁₂₅N₂O₂₁P₂: 1343.8250. Found 1343.8238.

6-O-{2′-Deoxy-3′-O-(3-heptyldecanoyl)-2′-(3-heptyldecanoylamino)-β-D-glucopyranosyl}-2-deoxy-3-O-[(R)-3-hydroxytetradecanoyl]-2-[(R)-3-hydroxytetradecanoylamino]-α-D-glucopyranose 4,4′-Diphosphate (26)

Compound 26 was synthesized by using 3-heptyldecanoic acid instead of 3NDDA in accordance with the procedure described for compound 5. 26: [α]_D¹⁹ +21.3 (c=1.0, CHCl₃), FT-IR (neat) 3370, 2955, 2925, 2854, 2728, 1737, 1658 cm⁻¹; ¹H-NMR (500 MHz in C₅D₅N) δ: 0.85 (18H, m, CH₃×6), 1.28 (90H, m, CH₂×44, β-CH of N′-acyl and C₃′-O-acyl), 2.19 (1H, m, α-CH₂ of N′-acyl), 2.32 (1H, m, α-CH₂ of C₃′-O-acyl), 2.70 (6H, m, α-CH₂ of N-acyl, α-CH₂ of C₃-O-acyl, α-CH₂ of N′-acyl and α-CH₂ of C₃′-O-acyl), 3.68 (1H, d, J=9.0 Hz, H-5′), 4.03 (1H, t, J=9.5 Hz, H-4), 4.14 (1H, d, J=13.0 Hz, H-6′a), 4.38 (4H, m, H-6ab, β-CH of N-acyl and β-CH of C₃-O-acyl), 4.54 (1H, d, J=11.5 Hz, H-6′b), 4.74 (1H, m, H-2′), 4.90 (2H, dd, J=9.0, 8.0 Hz, H-2 and H-5), 5.04 (1H, dd, J=10.0, 9.5 Hz, H-4′), 5.72 (1H, d, J=8.5 Hz, H-1′), 5.99 (2H, m, H-3 and H-3′), 6.34 (1H, dd, J=7.5, 3.0 Hz, H-1), 8.44 (1H, d, J=8.5 Hz, NH), 9.38 (1H, d, J=9.0 Hz, N′H); ¹³C-NMR (125 MHz in C₅D₅N) δ: 8.61, 14.25, 14.28, 14.31, 22.90, 22.96, 22.98, 26.22, 26.70, 26.86, 27.03, 27.09, 27.22, 29.60, 29.65, 29.68, 29.71, 29.74, 29.79, 29.82, 29.91, 29.97, 30.20, 30.34, 30.37, 30.42, 30.54, 32.10, 32.15, 32.19, 32.25, 33.76, 33.98, 34.05, 34.72, 35.53, 38.06, 38.16, 38.32, 39.77, 41.91, 43.92, 45.09, 45.66, 53.31, 53.37, 54.69, 61.32, 68.14, 68.21, 68.49, 68.53, 69.33, 72.37, 72.41, 74.63, 75.14, 77.01, 94.11, 94.14, 101.78, 172.64, 173.29, 173.39, 173.44; MS (FAB⁻) m/z 1455 [M−H]; HR-MS (FAB⁻) m/z Calcd for C₇₄H₁₄₁N₂O₂₁P₂: 1455.9502. Found 1455.9531.

6-O-{2′-Deoxy-3′-O-(3-undecyltetradecanoyl)-2′-(3-undecyltetradecanoylamino)-β-D-glucopyranosyl}-2-deoxy-3-O-[(R)-3-hydroxytetradecanoyl]-2-[(R)-3-hydroxytetradecanoylamino]-α-D-glucopyranose 4,4′-Diphosphate (27)

Compound 27 was synthesized by using 3-undecyltetradecanoic acid instead of 3NDDA in accordance with the procedure described for compound 5. 27: [α]_D²⁰ +17.4 (c=1.0, CHCl₃); FT-IR (neat) 3367, 2955, 2932, 2855, 2737, 2622, 2605, 2530, 2498, 1738, 1665 cm⁻¹; ¹H-NMR (500 MHz in C₅D₅N) δ: 0.88 (18H, m, CH₃×6), 1.25 (122H, m, CH₂×60, β-CH of N′-acyl and β-CH of C₃′-O-acyl), 2.24 (1H, m, α-CH₂ of N′-acyl), 2.36 (1H, m, α-CH₂ of C₃′-O-acyl), 2.64 (6H, m, α-CH₂ of N-acyl, α-CH₂ of C₃-O-acyl, α-CH₂ of N′-acyl and α-CH₂ of C₃′-O-acyl), 3.67 (1H, d, J=9.0 Hz, H-5′), 4.06 (1H, dd, J=10.0, 9.0 Hz, H-4), 4.13 (1H, d, J=12.0 Hz, H-6′a), 4.41 (4H, m, H-6ab, β-CH of N-acyl and β-CH of C₃-O-acyl), 4.57 (1H, d, J=12.0 Hz, H-6′b), 4.74 (1H, dd, J=9.5, 8.5 Hz, H-2′), 4.90 (2H, m, H-2 and H-5), 5.01 (1H, q, J=10.0 Hz, H-4′), 5.49 (1H, d, J=8.5 Hz, H-1′), 5.97 (2H, m, H-3 and H-3′), 6.31 (1H, dd, J=8.0, 3.0 Hz, H-1), 8.31 (1H, d, J=9.0 Hz, NH), 9.41 (1H, d, J=8.5 Hz, N′H); ¹³C-NMR (125 MHz in C₅D₅N) δ: 8.61, 14.28, 14.30, 22.94, 22.97, 26.24, 26.26, 26.78, 27.13, 29.63, 29.67, 29.70, 29.95, 30.01, 30.04, 30.06, 30.08, 30.13, 30.17, 30.24, 30.32, 30.51, 30.55, 30.60, 30.71, 32.13, 32.15, 32.19, 33.80, 34.01, 39.86, 41.94, 43.97, 45.32, 45.64, 53.50, 54.74, 61.36, 68.24, 68.48, 69.40, 72.19, 74.69, 75.17, 75.27, 77.17, 79.83, 93.92, 101.85, 172.71, 173.34, 173.47; MS (FAB⁺) m/z 1704 [M+Na]⁺; HR-MS (FAB⁺) m/z Calcd for C₈₇H₁₇₄N₂O₂₁P₂Na: 1704.1982. Found 1704.1995.

6-O-{2′-Deoxy-3′-O-(3-tridecylhexadecanoyl)-2′-(3-tridecylhexadecanoylamino)-β-D-glucopyranosyl}-2-deoxy-3-O-[(R)-3-hydroxytetradecanoyl]-2-[(R)-3-hydroxytetradecanoylamino]-α-D-glucopyranose 4,4′-Diphosphate (28)

Compound 28 was synthesized by using 3-tridecylhexadecanoic acid instead of 3NDDA in accordance with the procedure described for compound 5. 28: [α]_D²⁰ +16.9 (c=1.0, CHCl₃); FT-IR (neat) 3373, 2924, 2853, 2679, 2498, 1739, 1659 cm⁻¹; ¹H-NMR (500 MHz in C₅D₅N) δ: 0.88 (18H, m, CH₃×6), 1.25 (138H, m, CH₂×68, β-CH of N′-acyl and β-CH of C₃′-O-acyl), 2.24 (1H, m, α-CH₂ of N′-acyl), 2.37 (1H, m, α-CH₂ of C₃′-O-acyl), 2.69 (6H, m, α-CH₂ of N-acyl, α-CH₂ of C₃-O-acyl, α-CH₂ of N′-acyl and α-CH₂ of C₃′-O-acyl), 3.68 (1H, d, J=9.0 Hz, H-5′), 4.05 (1H, dd, J=10.0, 9.5 Hz, H-4), 4.14 (1H, d, J=12.5 Hz, H-6′a), 4.42 (4H, m, H-6ab, β-CH of N-acyl and β-CH C₃-O-acyl), 4.57 (1H, d, J=12.5 Hz, H-6′b), 4.75 (1H, q, J=9.0 Hz, H-2′), 4.91 (2H, m, H-2 and H-5), 5.03 (1H, q, J=10.0 Hz, H-4′), 5.74 (1H, d, J=8.0 Hz, H-1′), 5.98 (2H, m, H-3 and H-3′), 6.33 (1H, dd, J=8.0, 3.0 Hz, H-1), 8.33 (1H, d, J=8.5 Hz, NH), 9.42 (1H, d, J=9.0 Hz, N′H); ¹³C-NMR (125 MHz in C₅D₅N) δ: 8.74, 14.42, 23.07, 23.09, 26.09, 26.38, 26.90, 27.25, 29.76, 29.80, 30.08, 30.10, 30.14, 30.17, 30.22, 30.23, 30.28, 30.33, 30.36, 30.39, 30.45, 30.64, 30.67, 30.74, 30.84, 32.27, 32.29, 33.92, 34.14, 34.24, 34.89, 35.71, 38.21, 38.57, 39.99, 42.07, 44.07, 45.42, 45.80, 53.60, 54.87, 61.46, 68.36, 68.60, 69.51, 72.29, 74.83, 75.22, 75.37, 77.26, 79.46, 79.72, 79.93, 79.98, 94.06, 101.99, 172.84, 173.47, 173.61; MS (FAB⁺) m/z 1816 [M]⁺; HR-MS (FAB⁺) m/z Calcd for C₉₈H₁₉₀N₂O₂₁P₂: 1816.3234. Found 1816.3251.

Docking Simulation Analysis

The docking study of 5 was carried out using Molecular Operating Environment software (MOE 2014.09, Chemical Computing Group, Montreal, Canada). The X-ray crystallographic structures of MD2 (PDB ID: 2E59) and TLR4/MD2 complex (PDB ID: 3FXI) were used. The minimizations of MD2 and TLR4/MD2 complex were performed using MMFF94s force-field. The active sites of MD2 and TLR4/MD2 complex were identified using MOE a Site Finder. The docking simulations with 5 and these proteins (Fig. 6 and graphic abstract) were performed using ASE Dock program (Ryoka Systems, Tokyo, Japan).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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