Solid-Phase Modular Synthesis of Park Nucleotide and Lipids I and II Analogues

Abstract

A solid-phase synthesis of Park nucleotide as well as lipids I and II analogues, which is applicable to the synthesis of a range of analogues, is described in this work. This technique allows highly functionalized macromolecules to be modularly labeled. Multiple steps are used in a short time (4 d) with a single purification step to synthesize the molecules by solid-phase synthesis.

Peptidoglycan is a component of bacterial cell walls that consists of repeating β-1-4-N-acetylglucosaminyl-N-acetylmuraminic acid (β-1-4-GlcNAc-MurNAc) units, which are further cross-linked with polypeptides. Peptidoglycan plays a role as an extracellular skeleton to prevent bacterial cells from lysis by intracellular high pressure and is a primary defense from a variety of physiological, chemical and biological attacks outside the cells. Peptidoglycan biosynthesis consists of several stages as shown in Fig. 1.^1,2)

Fig. 1. Biosynthesis of Peptidoglycan

Uridine-5′-diphospho-MurNAc-pentapeptide (UDP-MurNAc-pentapeptide, 1), which is also called as Park nucleotide, is synthesized by a series of MurA-F enzymes in cytoplasm. Phospho-MurNAc-pentapeptide transferase (MraY) catalyzes the first reaction step of the lipid-linked cycle, where Park nucleotide is attacked by undecaprenyl monophosphate in the bacterial cell membrane providing lipid I. Lipid I is further glycosylated by N-acetylglucosamine transferase (MurG) to afford lipid II. Lipid II in cytoplasm is then flipped out by a flipase called MurJ, which was recently identified.^3,4) The lipid II in periplasm is then polymerized by a transglycosylase and a transpeptidase to form peptidoglycan. The biosynthesis of peptidoglycan is one of the major targets for antibacterial drug discovery and many drugs including β-lactams and vancomycin are currently clinically used drugs. In addition to the fact that the analogues of Park nucleotide, lipids I and II, which are precursors to peptidoglycan, have the potential to be inhibitors of peptidoglycan biosynthesis,^5,6) they are used as biological tools to elucidate the biology of bacterial cell walls. For example, dansylated Park nucleotide has been used as a fluorescent substrate in an assay screening of MraY inhibitor,⁷⁾ and lipids I and II analogues with short lipid tails, such as a neryl group instead of a undecaprenyl group, have been used in mechanistic studies of MurY and MurG^8,9) (Fig. 2, 2 and 3). As a result, these molecules constitute an important class of molecules in drug discovery and microbiology. The chemical synthesis of Park nucleotide, lipids I, II and their analogues have previously been accomplished by solution-phase synthesis.^6,7,10–20) However, these molecules consist of amino acids, sugars, and diphosphates attached to uridine or the lipid chain and their amphiphilic properties as well as their molecular size occasionally face difficulty in purification during synthesis. Solid-phase syntheses have been extensively developed especially in the synthesis of biomacromolecules. Since synthetic intermediates remain immobilized on the solid support, it is easy to handle a molecule with the abovementioned properties. Although Kurosu’s group reported the solid-phase synthesis of peptide fragments of Park nucleotide,²⁰⁾ the introduction of the sugar domain and construction of the diphosphate moiety on a solid-phase remains a challenge. Herein, a modular solid-phase synthesis of Park nucleotide (1), lipid I analogue 2 and lipid II analogue 3, is described.

Fig. 2. Structures of Neryl Analogues of Lipids I and II

Results and Discussion

The retrosynthetic analysis is illustrated in Chart 1. The C-terminus of these molecules is immobilized onto a solid support with suitable protection of the functional groups to give 4–6. Considering the lability of the diphosphate under acidic conditions, base-removable protecting groups were chosen for all the functional groups. Accordingly, cleavage from the solid-phase was used under basic conditions. In addition, the hydrophilicity of the target molecules upon simultaneous deprotection under basic aqueous conditions was considered. These considerations initiated the use of a 4-(hydroxymethyl)bezoylamidyl polyethyleneglycol (HMBA-PEG) resin, which swells extensively in a wide range of solvents, including water.²¹⁾ The diphosphate moieties of 4–6 are disconnected to give pentapeptidylglycosyl phosphates 7 and 8 as well as the corresponding uridine 5′-O-phosphate and neryl phosphate, respectively. Regarding the disconnection of 7 and 8, it was reported by Ducho’s group that coupling of a pentapeptide with a MurNAc derivative resulted in the formation of a diketopiperazine consisting of L-Ala and D-Glu and that it is better to connect a tetrapeptide and a MurNAc derivative condensed with L-Ala (MurNAc-Ala).⁷⁾ This observation was modeled for the solid-phase synthesis to disconnect 7 and 8 between the L-Ala and D-Glu residues to give tetrapeptide 9, MurNAc-Ala 10 and GlcNAc-MurNAc-Ala 11.

Chart 1. Retrosynthetic Analysis of Target Molecules

Both 10 and 11 were easily prepared from GlcNAc and D-glucosamine (GlcNH₂) via known compounds 12 and 15, respectively (Chart 2).²²⁾ Namely, the 4,6-O-benzylidene protecting group of 12, which was obtained from D-GlcNAc over four steps, was converted to acetyl groups suitable for the following synthetic route. The benzyl group at the O-1-position of the resulting 13 was removed by hydrogenolysis and the liberated alcohol was phosphorylated by two steps to give O-1-α-diallylphosphate 14 in 39% yield over three steps. The phenylsulfonylethyl group at the Ala residue was removed by 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to afford the carboxylic acid 10. The glycosyl phosphates 11 was prepared by the same procedure for the synthesis of 10.

Chart 2. Preparation of 10 and 11

The synthesis on the solid-phase was then investigated (Chart 3). First, Fmoc-D-Ala was immobilized onto the HMBA-PEG resin (0.39 mmol/g). Then, Fmoc-peptide synthesis was applied to give tripeptide 19. The protecting group of N-terminal peptide 19 was then switched to an Alloc group. After coupling of 19 with Alloc-D-Glu-OBn and the removal of the Alloc group of 20, the resulting amine was coupled with 10 to afford glycosyl peptapeptide 7. It should be noted that considerable epimerization occurred during the coupling with 10 using 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) and ⁱPr₂NEt. After extensive investigations, it was found that the conditions using (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), 1-hydroxy-7-azabenzotriazole (HOAt) and acridine in N,N-dimethylformamide (DMF)/CH₂Cl₂ completely suppressed the epimerization. A single coupling resulted in incomplete reaction and therefore a double-coupling was conducted to completely consume the amine. Deprotection of the allyl groups on the phosphate was conducted by the conditions using Pd(PPh₃)₄ and PhSiH₃ to cleanly provide the phosphate 21. Instead of 10, coupling of the disaccharide 11 to the tetrapeptide amine gave 22.

Chart 3. Solid-Phase Synthesis of 21 and 22

With 21 and 22 in hand, the key diphosphate formation reaction on the solid-phase was investigated. Generally, the diphosphate moiety is classically constructed by the condensation of a phosphate monoester with a phosphoroamidate upon activation by an activator, such as 1H-tetrazole (26).²³⁾ The reaction rate, however, was very slow even in the solution-phase synthesis and remained to be improved when applied to solid-phase synthesis. Thus, the reaction conditions were optimized using model substrates 23 and 24²⁴⁾ in the solution-phase, and the results are summarized in Table 1. Treatment of 24 with uridine-5′-phosphorylmorphoridate 23 in the presence of 26 as an activator, which is conventionally used in diphosphate coupling, gave desired 25 in 18% yield after 24 h and 42% yield after 72 h (Fig. 3). The activation ability of the phosphorylamidate correlates to the acidity of the activators. The activator acidity improves the chemical yields of 25, and the use of 29²⁵⁾ gave 25 in 58% yield.

Table 1. Optimization of Diphosphate Formation

Fig. 3. Time Course of Diphosphate Formation

The conditions were then applied to the coupling of 21 and 23 to complete the total synthesis of 1 (Chart 4a). Although the reaction catalyzed by 29 resulted in no reaction at room temperature, elevating the temperature to 50 °C accelerated the reaction rate to produce 4. Finally, cleavage from the resin as well as global deprotection by treating 4 with piperidine followed by aq. NaOH successfully afforded Park nucleotide (1) in 44% yield over 12 steps from 17 after purification by HPLC. In a manner similar to the synthesis of 1, neryl-lipid I (2), where the undecaprenyl group of lipid I was replaced with a short lipid (a neryl group) was synthesized in 20% yield over 12 steps from 17 (Chart 4b). Using 22 instead of 21, neryl-lipid II (3) was also synthesized (Chart 4c). This strategy allows for the modular labeling of highly functionalized macromolecules, which were obtained in multiple steps in a short time (4 d) using a single purification step by virtue of solid-phase synthesis.

Chart 4. Completion of the Synthesis of 1–3

Conclusion

In conclusion, a solid-phase modular synthesis has been established for Park nucleotide and lipids I and II analogues. These analogues could be useful as chemical probes for discovering novel antibacterial agents and elucidating detailed mechanistic studies on peptidoglycan biosynthesis.

Experimental

General Experimental Methods

All reactions except that carried out in aqueous phase were performed under argon atmosphere, unless otherwise noted. Isolated yields were calculated by weighing products. The weight of the starting materials and the products were not calibrated. Materials were purchased from commercial suppliers and used without further purification, unless otherwise noted. Solvents are distilled according to the standard protocol. Analytical TLC was performed on Merck silica gel 60F254 plates. Normal-phase column chromatography was performed on Merck silica gel 5715 or Kanto Chemical silica gel 60N (neutral). High-flash column chromatography was performed on Fuji Sylysia silica gel PSQ 60B. ¹H-NMR were measured in CDCl₃, dimethyl sulfoxide (DMSO)-d₆, or D₂O solution, and referenced to TMS (0.00 ppm) using JEOL ECA 500 (500 MHz), JEOL ECS 400 (400 MHz) or JEOL ECX 400P (400 MHz) spectrophotometers, unless otherwise noted. ¹³C-NMR were measured in CDCl₃, DMSO-d₆, or D₂O solution, and referenced to residual solvent peaks using JEOL ECA 500 (125 MHz), JEOL ECS 400 (100 MHz) or JEOL ECX 400P (100 MHz) spectrophotometers. ³¹P-NMR were measured in CDCl₃, DMSO-d₆, or D₂O solution, and referenced to H₃PO₄ (0.00 ppm) using JEOL ECA 500 (202 MHz), JEOL ECS 400 (162 MHz) or JEOL ECX 400P (162 MHz) spectrophotometers. Abbreviations of multiplicity were as follows; s: singlet, d: doublet, t: triplet, q: quartet, sept: septet, m: multiplet, br: broad. Data were presented as follows; chemical shift (multiplicity, integration, coupling constant). Assignment was based on ¹H–¹H correlation spectroscopy (COSY), heteronuclear multiple bond correlation (HMBC) and heteronuclear multiple quantum coherence (HMQC) NMR spectra. Optical rotations were determined on JASCO P-1010-GT. Mass spectra were recorded on Thermo Scientific Exactive. The mass analyzer type used for the high resolution (HR)-MS measurements was time-of-flight (TOF).

Benzyl-N-acetyl-4,6-diacetylmuramyl-L-alanine Phenylsulfonylethyl Ester (13)

Compound 12 (1.75 g, 2.46 mmol) was treated with 60% AcOH/H₂O (60 mL) at 90°C for 1 h. The mixture was concentrated in vacuo. The residue was crystalized by CHCl₃/Et₂O to afford benzyl-N-acetyl-muramyl-L-alanine phenylsulfonylethyl ester (1.21 g, 1.94 mmol, 79%) as a white solid.

¹H-NMR (CDCl₃, 400 MHz) δ: 7.91 (d, 2H, o-PhSO₂, J_o,m=7.3 Hz), 7.69 (t, 1H, p-PhSO₂, J_p,m=7.3 Hz), 7.59 (dd, 2H, m-PhSO₂, J_m,o=J_m,p=7.3 Hz), 7.40–7.29 (m, 5H, Ph), 6.92 (d, 1H, Ala-NH, J_{Ala-NH, Ala-α-CH}=7.3 Hz), 5.04 (d, 1H, 2-NH, J_2-NH,2=9.6 Hz), 4.91 (d, 1H, H-1, J_1,2=3.6 Hz), 4.71 (d, 1H, Bn, J=11.9 Hz), 4.47 (d, 1H, Bn, J=11.9 Hz), 4.50–4.37 (m, 2H, PhSO₂CH₂CH₂), 4.29 (dq, 1H, Ala-α-CH, J_{Ala-α-CH, Ala-NH}=J_{Ala-α-CH, Ala-β-CH}=7.3 Hz), 4.23 (ddd, 1H, H-2, J_2,1=3.6, J_2,2-NH=9.6, J_2,3=10.1 Hz), 4.16 (q, 1H, Lac-α-CH, J_{Lac-α-CH, Lac-β-CH}=6.9 Hz), 3.83 (d, 2H, H-6, J_6,5=3.2 Hz), 3.76–3.65 (m, 2H, H-5, H-4), 3.58 (dd, 1H, H-3, J_3,2=J_3,4=10.1 Hz), 3.47–3.33 (m, 2H, PhSO₂CH₂CH₂), 1.92 (s, 3H, NAc), 1.42 (d, 3H, Lac-β-CH, J_{Lac-β-CH, Lac-α-CH}=6.9 Hz), 1.33 (d, 3H, Ala-β-CH, J_{Ala-β-CH, Ala-α-CH}=7.3 Hz). This is a known compound reported in ref. 14).

A mixture of benzyl-N-acetyl-muramyl-L-alanine phenylsulfonylethyl ester (1.12 g, 1.80 mmol) in pyridine (20 mL) was treated with Ac₂O (406 µL, 4.32 mmol) at room temperature for 1 d. Ac₂O (102 µL, 1.08 mmol) was added to the mixture, which was stirred for 24 h. The reaction was quenched with MeOH, then the resulting mixture was concentrated in vacuo. The residue was partitioned between AcOEt and sat. aq. NaHCO₃, and the organic phase was washed with 1 M aq. HCl and brine, dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was purified by high-flash silica gel column chromatography (50–85–100% AcOEt/hexane) to afford 13 (955 mg, 1.35 mmol, 75%) as a colorless foam.

¹H-NMR (CDCl₃, 500 MHz) δ: 7.92 (d, 2H, o-PhSO₂, J_o,m=6.9 Hz), 7.68 (t, 1H, p-PhSO₂, J_p,m=7.5 Hz), 7.59 (dd, 2H, m-PhSO₂, J_m,o=6.9, J_m,p=7.5 Hz), 7.41–7.56 (m, 5H, Ph), 6.85 (d, 1H, Ala-NH, J_{Ala-NH, Ala-α-CH}=6.9 Hz), 5.81 (d, 1H, 2-NH, J_2-NH,2=9.2 Hz), 5.07 (dd, 1H, H-4, J_4,3=J_4,5=9.8 Hz), 4.88 (d, 1H, H-1, J_1,2=4.0 Hz), 4.69 (d, 1H, Bn, J=11.5 Hz), 4.50 (d, 1H, Bn, J=11.5 Hz), 4.48–4.40 (m, 2H, PhSO₂CH₂CH₂), 4.38 (ddd, 1H, H-2, J_2,1=4.0, J_2,2-NH=9.2, J_2,3=9.8 Hz), 4.20 (dd, 1H, H-6, J_6,6=12.0, J_6,5=4.6 Hz), 4.13 (dq, 1H, Ala-α-CH, J_{Ala-α-CH, Ala-NH}=J_{Ala-α-CH, Ala-β-CH}=6.9 Hz), 4.03 (dd, 1H, H-6, J_6,6=12.0, J_6,5=2.3 Hz), 3.95 (q, 1H, Lac-α-CH, J_{Lac-α-CH, Lac-β-CH}=6.3 Hz), 3.91 (ddd, 1H, H-5, J_5,6=2.3, J_5,6=4.6, J_5,4=9.8 Hz), 3.64 (dd, 1H, H-3, J_3,2=J_3,4=9.8 Hz), 3.50–3.38 (m, 2H, PhSO₂CH₂CH₂), 2.11 (s, 3H, OAc), 2.07 (s, 3H, OAc), 1.88 (s, 3H, NAc), 1.30 (d, 3H, Lac-β-CH, J_{Lac-β-CH, Lac-α-CH}=6.3 Hz), 1.29 (d, 3H, Ala-β-CH, J_{Ala-β-CH, Ala-α-CH}=6.9 Hz); ¹³C-NMR (CDCl₃, 125 MHz) δ: 172.2, 171.8, 170.9, 170.4, 169.5, 139.3, 136.7, 134.2, 129.6, 128.9, 128.7, 128.5, 128.3, 97.2, 78.9, 78.6, 70.4, 69.5, 68.7, 62.3, 58.2, 55.1, 53.1, 48.1, 23.5, 21.0, 20.9, 18.7, 17.0; electrospray ionization (ESI)-MS-low resolution (LR) m/z 729.23 [(M+Na)⁺]; ESI-MS-HR Calcd for C₃₃H₄₂O₁₃N₂NaS 729.2230. Found 729.2299; [α]_D²⁰ +68.36 (c 0.23, CHCl₃).

Glycosyl Phosphate 14

A mixture of 13 (424 mg, 0.60 mmol) and 10% Pd/C (600 mg) in MeOH (6 mL) was vigorously stirred under H₂ atmosphere at room temperature for 2.5 h. 10% Pd/C (600 mg) was added to the mixture and vigorously stirred for 8 h under H₂ atmosphere. 10% Pd/C (300 mg) was added to the mixture and vigorously stirred for 12 h under H₂ atmosphere. The catalyst was filtered off through a Celite pad, and the filtrate was concentrated in vacuo to afford a crude lactol. A mixture of the lactol and 5-(benzylthio)-1H-tetrazole (208 mg, 1.08 mmol) in CH₂Cl₂ (6 mL) was treated with diallyl N,N-diisopropylphosphoramidite (238 µL, 0.90 mmol) at 0°C for 5 min. The mixture was warmed to room temperature and stirred for 1 h. 5-(Benzylthio)-1H-tetrazole (138 mg, 0.72 mmol) and diallyl N,N-diisopropylphosphoramidite (159 µL, 0.60 mmol) was added to the mixture and stirred for 1 h. The mixture was partitioned between CH₂Cl₂ and sat. aq. NaHCO₃, and the organic phase was washed with H₂O and brine, dried (Na₂SO₄), filtered, and concentrated in vacuo to afford a crude phosphite. A mixture of the phosphite in tetrahydrofuran (THF) (6 mL) was treated with 30% H₂O₂ (600 µL) at −78°C for 5 min. The mixture was warmed to room temperature and stirred for 2.5 h. The reaction was quenched with sat. aq. Na₂S₂O₃ at 0°C, and the mixture was partitioned between AcOEt and sat. aq. NaHCO₃. The organic phase was washed with brine, dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was purified by high-flash silica gel column chromatography (50–100% AcOEt/hexane-0–1–2% MeOH/AcOEt) to afford 14 (180 mg, 0.23 mmol, 39% over 3 steps) as a colorless foam.

¹H-NMR (CDCl₃, 500 MHz) δ: 7.93 (d, 2H, o-PhSO₂, J_o,m=7.4 Hz), 7.67 (t, 1H, p-PhSO₂, J_p,m=7.5 Hz), 7.60 (dd, 2H, m-PhSO₂, J_m,o=7.4, J_m,p=7.5 Hz), 6.76 (d, 1H, Ala-NH, J_{Ala-NH, Ala-α-CH}=6.9 Hz), 6.55 (d, 1H, 2-NH, J_2-NH, 2=8.6 Hz), 6.01–5.88 (m, 2H, Hc), 5.70 (dd, 1H, H-1, J_1,2=2.9, J_1, P=5.8 Hz), 5.40 (dd, 1H, H_a, J_Ha, Hc=17.2, J_Ha, 1′=1.2 Hz), 5.38 (dd, 1H, H_a, J_Ha, Hc=17.2, J_Ha, 1′=1.2 Hz), 5.31 (dd, 1H, H_b, J_Hb, Hc=10.3, J_Ha, 1′=1.2 Hz), 5.30 (dd, 1H, H_b, J_Hb, Hc=10.3, J_Ha, 1′=1.2 Hz), 5.14 (dd, 1H, H-4, J_4,3=10.3, J_4,5=9.8 Hz), 4.64–4.55 (m, 4H, H-1′), 4.53–4.44 (m, 2H, PhSO₂CH₂CH₂), 4.41–4.35 (m, 1H, H-2), 4.24 (dq, 1H, Ala-α-CH, 7.5, J_{Ala-α-CH, Ala-NH}=J_{Ala-α-CH, Ala-β-CH}=6.9 Hz), 4.20 (dd, 1H, H-6, J_6,6=12.0, J_6,5=4.0 Hz), 4.13–4.05 (m 2H, H-5, H-6), 4.03 (q, 1H, Lac-α-CH, J_{Lac-α-CH, Lac-β-CH}=6.3 Hz), 3.69 (dd, 1H, H-3, J_3,2=J_3,4=10.3 Hz), 3.52–3.41 (m, 2H, PhSO₂CH₂CH₂), 2.09 (s, 3H, OAc), 2.08 (s, 3H, OAc), 1.96 (s, 3H, NAc), 1.34 (d, 3H, Lac-β-CH, J_{Lac-β-CH, Lac-α-CH}=6.3 Hz), 1.33 (d, 3H, Ala-β-CH, J_{Ala-β-CH, Ala-α-CH}=7.5 Hz); ¹³C-NMR (CDCl₃, 125 MHz) δ: 172.3, 171.6, 170.9, 170.8, 169.3, 139.2, 134.3, 132.2, 132.2, 132.0, 132.0, 129.6, 128.2, 119.4, 119.3, 78.4, 77.0, 70.3, 69.1, 69.0, 69.0, 68.9, 61.8, 58.2, 55.0, 53.3, 53.3, 48.1, 25.1, 23.3, 20.9, 20.9, 18.9, 17.2; ³¹P-NMR (CDCl₃, 202 MHz) δ −1.8; ESI-MS-LR m/z 799.21 [(M+Na)⁺]; ESI-MS-HR Calcd for C₃₂H₄₅O₁₆N₂NaPS 799.2120. Found 799.2125; [α]_D²⁰ +49.99 (c 1.49, CHCl₃).

Glycosyl Phosphate 10

A mixture of 14 (212 mg, 0.27 mmol) in CH₂Cl₂ (3 mL) was treated with DBU (44.8 µL, 0.30 mmol) at room temperature for 40 min. The mixture was partitioned between AcOEt and 1 M aq. HCl, and the aqueous phase was extracted with AcOEt (×2). Combined organic phase was dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was purified by high-flash silica gel column chromatography (0–1–2% MeOH/CH₂Cl₂) to afford 10 (149 mg, 0.25 mmol, 90%) as a colorless foam.

¹H-NMR (CDCl₃, 500 MHz) δ: 7.47 (d, 1H, 2-NH, J_2-NH, 2=8.0 Hz), 6.86 (d, 1H, Ala-NH, J_{Ala-NH, Ala-α-CH}=6.3 Hz), 5.98–5.88 (m, 2H, Hc), 5.72 (dd, 1H, H-1, J_1,2=3.4, J_1 P=6.3 Hz), 5.39 (dd, 1H, H_a, J_Ha, Hc=16.6, J_Ha, 1′=1.2 Hz), 5.38 (dd, 1H, H_a, J_Ha, Hc=17.2, J_Ha, 1′=1.2 Hz), 5.30 (d, 2H, H_b, J_Hb, Hc=10.3 Hz), 5.12 (dd, 1H, H-4, J_4,3=J_4,5=9.2 Hz), 4.62–4.55 (m, 4H, H-1′), 4.37 (qd, 1H, Ala-α-CH, J_{Ala-α-CH, Ala-β-CH}=J_{Ala-α-CH, Ala-NH}=6.9 Hz), 4.31–4.25 (m, 1H, H-2), 4.22–4.15 (m, 2H, Lac-α-CH, H-6), 4.09–4.04 (m 2H, H-5, H-6), 3.75 (dd, 1H, H-3, J_3,2=J_3,4=9.7 Hz), 2.11 (s, 3H, OAc), 2.08 (s, 3H, OAc), 1.97 (s, 3H, NAc), 1.47 (d, 3H, Ala-β-CH, J_{Ala-β-CH, Ala-α-CH}=7.5 Hz), 1.33 (d, 3H, Lac-β-CH, J_{Lac-β-CH, Lac-α-CH}=6.3 Hz); ¹³C-NMR (CDCl₃, 125 MHz) δ: 174.4, 173.4, 171.7, 170.7, 170.8, 169.4, 132.1, 132.0, 131.9, 131.8, 119.5, 119.2, 96.8, 96.8, 78.3, 76.7, 70.4, 69.3, 69.2, 69.0, 69.0, 61.9, 53.5, 53.4, 48.5, 23.0, 20.9, 20.8, 19.2, 17.5; ³¹P-NMR (CDCl₃, 202 MHz) δ −2.7; ESI-MS-LR m/z 631.19 [(M+Na)⁺]; ESI-MS-HR Calcd for C₂₄H₃₇O₁₄N₂NaP 631.1875. Found 631.1882; [α]_D²⁰ +63.23 (c 3.80, CHCl₃).

Glycosyl Phosphate 16

A mixture of 15 (497 mg, 0.50 mmol) and 10% Pd/C (600 mg) in MeOH/EtOH=1/1 (10 mL) was vigorously stirred under H₂ atmosphere at room temperature for 24 h. The catalyst was filtered off through a Celite pad, and the filtrate was concentrated in vacuo to afford a crude lactol. A mixture of the lactol and 1H-tetrazole (70 mg, 1.0 mmol) in CH₂Cl₂ (5 mL) was treated with diallyl N,N-diisopropylphosphoramidite (198 µL, 0.75 mmol) at 0°C for 5 min. The mixture was warmed to room temperature and stirred for 1.5 h. Diallyl N,N-diisopropylphosphoramidite (49.5 µL, 0.19 mmol) was added to the mixture and stirred for 10 min. The mixture was cooled to −50°C, and treated with 80% ^tBuOOH (1 mL) for 1 h. The reaction was quenched with sat. aq. Na₂S₂O₃, and the mixture was partitioned between AcOEt and sat. aq. NaHCO₃. The organic phase was washed with H₂O and brine, dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was purified by high-flash silica gel column chromatography (0-1-2-5% MeOH/AcOEt) to afford 16 (271 mg, 0.25 mmol, 50% over 2 steps) as a colorless foam.

¹H-NMR (CDCl₃, 500 MHz) δ: 7.93 (d, 2H, o-PhSO₂, J_o,m=7.5 Hz), 7.70 (t, 1H, p-PhSO₂, J_p,m=7.5 Hz), 7.66–7.58 (m, 3H, m-PhSO₂, 2-NH), 7.18 (d, 1H, Ala-NH, J_{Ala-NH, Ala-α-CH}=8.1 Hz), 6.10 (d, 1H, 2′-NH, J_{2′-NH, 2′}=8.6 Hz), 5.98–5.87 (m, 3H, Hc, H-1), 5.37 (dd, 1H, H_a, J_Ha, Hc=17.2, J_Ha, 1′=1.2 Hz), 5.35 (dd, 1H, H_a, J_Ha, Hc=17.2, J_Ha, 1′=1.2 Hz), 5.26 (d, 1H, H_b, J_Hb, Hc=10.3 Hz), 5.24 (d, 1H, H_b, J_Hb, Hc=10.3 Hz), 5.20 (dd, 1H, H-3′, J_3′,4′=9.8, J_3′,2′=10.9 Hz), 5.11 (dd, 1H, H-4′, J_4′,3′=J_4′,5′=10.9 Hz), 4.64 (q, 1H, Lac-α-CH, J_{Lac-α-CH, Lac-β-CH}=6.3 Hz), 4.61–4.54 (m, 3H, H-1′, H-1″), 4.54–4.48 (m, 4H, PhSO₂CH₂CH₂, H-1″), 4.39 (dq, 1H, Ala-α-CH, J_{Ala-α-CH, Ala-NH}=8.1, J_{Ala-α-CH, Ala-β-CH}=7.5 Hz), 4.35–4.27 (m, 2H, H-6, H-6′), 4.23 (dd, 1H, H-6, J_H-6,H-6=12.6, J_6,5=3.4 Hz), 4.09 (dd, 1H, H-6′, J_6′,6′=12.6, J_6′,5′=2.3 Hz), 4.02–3.85 (m, 4H, H-2, H-2′, H-4, H-5), 3.68–3.63 (m, 1H, H-5′), 3.55 (dd, 1H, H-3, J_3,4=J_H-3,H-2=10.1 Hz), 3.51 (t, 2H, PhSO₂CH₂CH₂, J_{PhSO2CH2CH2, PhSO2CH2CH2}=5.7 Hz), 2.11 (s, 3H, OAc), 2.05 (s, 3H, OAc), 2.03 (s, 3H, OAc), 2.00 (s, 3H, NAc), 1.99 (s, 3H, NAc), 1.96 (s, 3H, NAc), 1.38 (d, 3H, Lac-β-CH, J_{Lac-β-CH, Lac-α-CH}=6.9 Hz), 1.35 (d, 3H, Ala-β-CH, J_{Ala-β-CH, Ala-α-CH}=7.5 Hz); ¹³C-NMR (CDCl₃, 125 MHz) δ: 174.8, 171.8, 171.4, 171.3, 171.2, 171.0, 170.7, 169.5, 139.1, 134.3, 132.4, 129.6, 128.3, 118.8, 118.7, 99.8, 95.7, 95.7, 75.0, 74.0, 72.2, 72.2, 71.2, 68.7, 68.6, 68.6, 68.6, 68.4, 62.2, 61.9, 58.4, 55.1, 54.9, 53.9, 53.8, 48.1, 23.4, 23.1, 21.0, 20.8, 20.7, 18.9, 17.3; ³¹P-NMR (CDCl₃, 162 MHz) δ −2.3; ESI-MS-LR m/z 1086.31 [(M+Na)⁺]; ESI-MS-HR Calcd for C₄₄H₆₂O₂₃N₃NaPS 1086.3125. Found 1086.3120; [α]_D²⁰ +8.24 (c 1.24, CHCl₃).

Glycosyl Phosphate 11

A mixture of 16 (127 mg, 0.12 mmol) in CH₂Cl₂ (1.5 mL) was treated with DBU (19.4 µL, 0.13 mmol) at room temperature for 40 min. The mixture was partitioned between AcOEt and 1 M aq. HCl, and the aqueous phase was extracted with AcOEt (×2). Combined organic phase was dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was purified by high-flash silica gel column chromatography (0–5–10% MeOH/CH₂Cl₂) to afford 11 (100 mg, 0.11 mmol, 93%) as a colorless foam.

¹H-NMR (CDCl₃, 500 MHz) δ: 7.92 (d, 1H, 2-NH, J_2-NH, 2=4.0 Hz), 7.73 (d, 1H, Ala-NH, J_{Ala-NH, Ala-α-CH}=7.5 Hz), 6.49 (d, 1H, 2′-NH, J_{2′-NH, 2′}=9.7 Hz), 5.95–5.84 (m, 3H, Hc, H-1), 5.37 (d, 2H, H_a, J_Ha, Hc=17.2 Hz), 5.25 (dd, 1H, H_b, J_Hb, Hc=10.3, J_Ha, 1′=1.2 Hz), 5.24 (dd, 1H, H_b, J_Hb, Hc=10.3, J_Hb, 1′=1.2 Hz), 5.12–5.05 (m, 2H, H-3′, H-4′), 4.71 (q, 1H, Lac-α-CH, J_{Lac-α-CH, Lac-β-CH}=6.3 Hz), 4.59–4.52 (m, 3H, H-1′, H-1″), 4.52–4.45 (m, 2H, H-1″), 4.45 (dq, 1H, Ala-α-CH, J_{Ala-α-CH, Ala-NH}=J_{Ala-α-CH, Ala-β-CH}=7.5 Hz), 4.31 (dd, 1H, H-6, J_6,6=12.6, J_6,5=4.6 Hz), 4.22 (d, 1H, H-6, J_6,6=12.6, J_6,5=3.4 Hz), 4.17–4.09 (m, 1H, H-2′), 4.08 (dd, 1H, H-6, J_6,6=12.6, J_6,5=2.3 Hz), 3.97 (dd, 1H, H-4, J_4,5=J_4,3=9.7 Hz), 3.92–3.86 (m, 1H, H-2), 3.77–3.71 (m, 1H, H-5), 3.66–3.58 (m, 2H, H-5, H-3′), 2.09 (s, 3H, OAc), 2.04 (s, 3H, OAc), 2.02 (s, 3H, OAc), 2.01 (s, 3H, NAc), 2.00 (s, 3H, NAc), 1.96 (s, 3H, NAc), 1.48 (d, 3H, Ala-β-CH, J_{Ala-β-CH, Ala-α-CH}=7.5 Hz), 1.35 (d, 3H, Lac-β-CH, J_{Lac-β-CH, Lac-α-CH}=6.3 Hz); ¹³C-NMR (CDCl₃, 125 MHz) δ: 175.7, 174.3, 171.8, 171.1, 171.1, 170.6, 169.5, 132.1, 132.0, 132.0, 131.9, 119.1, 118.9, 99.3, 95.6, 95.5, 73.7, 73.0, 72.3, 72.0, 71.7, 69.1, 69.0, 68.9, 68.8, 68.5, 62.5, 61.9, 54.7, 53.9, 53.9, 48.8, 23.3, 23.0, 20.9, 20.8, 20.7, 20.7, 18.7, 16.8; ³¹P-NMR (CDCl₃, 202 MHz) δ: −3.2; ESI-MS-LR m/z 918.29 [(M+Na)⁺]; ESI-MS-HR Calcd for C₃₆H₅₄O₂₁N₃NaP 918.2880. Found 918.2885; [α]_D²⁰ +7.00 (c 0.21, CHCl₃).

Diphosphate 25

A solution of 23 (16.5 mg, 0.024 mmol) and 24 (12.7 mg, 0.024 mmol) in DMF (250 µL) was treated with 29 (5.3 mg, 0.024 mmol) at 25°C for 24 h. The mixture was concentrated in vacuo. The residue was purified by high-flash ODS column chromatography (0–10% MeCN/25 mM aq. AcOH·Et₃N). The product was further purified by reversed phase HPLC (YMC-Pack R&D ODS-A, 250×20 mm, 8% MeCN/25 mM aq. AcOH·t₃N) to afford 25 (6.9 mg, 0.0074 mmol, 31%) as a white powder, after freeze drying.

¹H-NMR (DMSO-d₆, 500 MHz) δ: 9.53 (d, 1H, 2″-NH, J_{2″-NH, 2″}=9.2 Hz), 7.93 (d, 1H, H-6, J_6,5=6.3 Hz), 5.75 (s, 1H, H-1′), 5.56 (d, 1H, H-5, J_5,6=7.5 Hz), 5.37–5.32 (m, 1H, H-1″), 5.08 (dd, 1H, H-3, J_3,2=9.2, J_3,4=9.7 Hz), 4.93 (dd, 1H, H-4, J_4,3=J_4,5=9.5 Hz), 4.19–3.91 (m, 9H, H-2′, H-3′, H-4′, H-5′, H-2″, H-5″, H-6″), 3.11–3.07 (m, 2H, CH₃CH₂N), 2.01 (s, 3H, OAc), 1.94 (s 3H, OAc), 1.85 (s, 3H, OAc), 1.83 (s, 3H, NAc), 1.17 (t, 3H CH₃CH₂N); ¹³C-NMR (DMSO-d₆, 125 MHz) δ: 170.1, 170.1, 169.6, 169.3, 163.2, 150.8, 140.8, 139.5, 101.7, 93.9, 82.8, 71.6, 67.4, 67.7, 68.4, 67.7, 61.5, 50.9, 47.7, 45.5, 22.3, 20.5, 20.4, 19.0, 11.0, 8.6; ³¹P-NMR (DMSO-d₆, 202 MHz) δ: −11.3 (d, J_P, P=26.3 Hz), −14.1 (d, J_P, P=26.3 Hz); ESI-MS-LR m/z 732.11 [(M–H)⁻]; ESI-MS-HR Calcd for C₂₃H₃₂O₂₀N₃P₂ 732.1072. Found 732.1060; [α]_D²⁰ +10.94 (c 0.60, DMSO-d₆).

Neryl Phosphoryl Imidazolide (30)

Neryl phosophate ammonium salt (2.9 mg, 0.012 mmol) was co-evaporated with Et₃N (20 µL) in pyridine (1 mL) twice. The residue was co-evaporated with toluene (1 mL) twice to afford neryl phosphate triethylamine salt. A mixture of the phosphate salt in DMF was treated with 1,1′-carbonyldiimidazole (9.5 mg, 0.059 mmol) at room temperature for 3 h. The reaction was quenched with MeOH (100 µL) and stirred for 30 min. The mixture was concentrated in vacuo and co-evaporated with toluene twice to afford 30. This compound was used without further purification.

³¹P-NMR (DMSO-d₆, 162 MHz) δ: −9.6.

Procedure for the Synthesis of 20

Each HMBA-PEG resin (150 mg, 0.71 mmol) was placed in a 5 mL polypropylene syringe fitted with a polyethylene filter disc. Each resin was agitated with CH₂Cl₂ (1.5 mL, 1 h), After removal of CH₂Cl₂, a solution of Fmoc-D-Ala-OH·H₂O (69 mg, 0.21 mmol) and N,N′-dissopropylcarbodiimide (33 µL, 0.21 mmol) in DMF (1 mL) was added at 0°C. Each mixture was agitated for 40 min. 4-Dimethylaminopyridine (2.5 mg, 0.021 mmol) was added to the mixture at 0°C, which was warmed to room temperature. After agitation for 1 h at room temperature, solvent and soluble reagents were removed by suction. All resins were subjected to the following washing treatment with DMF (2 mL×3), EtOH/CH₂Cl₂=1/1 (2 mL×3), CH₂Cl₂ (2 mL×3) and DMF (2 mL×3). The resins were treated with Bz₂O (48 mg, 0.021 mmol) in 20% pyridine/DMF (1 mL) at room temperature for 1 h, and the resins were washed with DMF (2 mL×3) and CH₂Cl₂ (2 mL×3) to afford 17. The amount of loading on the resin was determined as follow. Dried 17 (6.0 mg) was agitated with DMF (2 mL) for 30 min, and DBU (40 µL) was added to the mixture. The mixture was agitated for 30 min. The supernatant was diluted with DMF and MeCN and subjected to UV measurement at 294 nm. The loading rate was determined to be 0.39 mmol/g from the observed absorbance (0.172). The resins 17 were treated with piperidine/DMF (1 : 4, 5 min, then 1 : 9, 15 min) to remove the Fmoc group, and the resins were washed with DMF (2 mL×3) and CH₂Cl₂ (2 mL×3). A solution of Fmoc-D-Ala-OH·H₂O (94 mg, 0.28 mmol), HBTU (105 mg, 0.28 mmol) and ⁱPr₂NEt (97 µL, 0.57 mmol) in DMF (750 µL) was added to the resins, which were agitated for 2 h. All the resins were washed with DMF (2 mL×3) and CH₂Cl₂ (2 mL×3) to afford 18. Kaiser test indicated the completion of the all coupling reactions. The loading rate was determined as described above. Namely, dried 18 (4.9 mg) was agitated with DMF (2 mL) for 30 min, DBU (40 µL) was added to the mixture. The mixture was agitated for 30 min. The supernatant was diluted with DMF and MeCN and subjected to UV measurement at 294 nm. The yield was determined to be quantitative from the observed absorbance (0.135). The resins 18 were treated with piperidine/DMF (1 : 4, 5 min, then 1 : 9, 15 min) to remove Fmoc group, and the resins were washed with DMF (2 mL×3) and CH₂Cl₂ (2 mL×3). A solution of Alloc-L-Lys(Fmoc)-OH (96 mg, 0.21 mmol), HBTU (68 mg, 0.21 mmol) and ⁱPr₂NEt (72 µL, 0.43 mmol) in DMF (750 µL) was added to the resins, which were agitated for 2 h. All the resins were washed with DMF (2 mL×3) and CH₂Cl₂ (2 mL×3) to afford 19. Kaiser test indicated the completion of the all coupling reactions. The amount of loading on the resin was determined as follow. Namely, dried 19 (5.3 mg) was agitated with DMF (2 mL) for 30 min, DBU (40 µL) was added to the mixture. The mixture was agitated for 30 min. The supernatant was diluted with DMF and MeCN and subjected to UV measurement at 294 nm. The yield was determined to be 87% over 2 steps from the observed absorbance (0.107). The resins 19 were treated with a solution of BH₃·Me₂NH (25 mg, 0.43 mmol) in EtOH (600 µL) for 5 min, then a solution of Pd(PPh₃)₄ (16 mg, 0.014 mmol) in CH₂Cl₂ (1 mL) was added to the mixture. The mixture was agitated for 15 min. All the resins were washed with 0.5% ⁱPr₂NEt/CH₂Cl₂ (2 mL×3), 0.5%(w/v) sodium diethyldithiocarbamate/DMF (1 mL×8), MeOH (2 mL×3) and CH₂Cl₂ (2 mL×3). A solution of Alloc-D-Glu-OBn (68 mg, 0.21 mmol), HBTU (68 mg, 0.21 mmol) and ⁱPr₂NEt (72 µL, 0.43 mmol) in DMF (750 µL) was added to the resins, which were agitated for 2 h. All the resins were washed with DMF (2 mL×3) and CH₂Cl₂ (2 mL×3). Kaiser test indicated the completion of the all coupling reactions. The resin was dried in vacuo to afford 20 (194 mg, 0.055 mmol, 0.28 mmol/g, 89% over 2 steps). The yield was calculated by weighing resins.

Procedure for the Synthesis of 7

Resin-bound peptide 20 (150 mg, 0.042 mmol) was placed in a 5 mL polypropylene syringe fitted with a polyethylene filter disc. The resin was agitated with CH₂Cl₂ (1.5 mL, 1 h). After removal of CH₂Cl₂, the resin was treated with a solution of BH₃·Me₂NH (13 mg, 0.22 mmol) in EtOH (600 µL) for 5 min, then a solution of Pd(PPh₃)₄ (8.4 mg, 7.3 µmol) in CH₂Cl₂ (1 mL) was added to the mixture. The mixture was agitated for 15 min. The resin was washed with 0.5% ⁱPr₂NEt/CH₂Cl₂ (2 mL×3), 0.5% (w/v) sodium diethyldithiocarbamate/DMF (1 mL×8), MeOH (2 mL×3) and DMF/CH₂Cl₂=1/1 (2 mL×3). A solution of 10 (50 mg, 0.082 mmol), PyAOP (57 mg, 0.11 mmol), HOAt (7.5 mg, 0.055 mmol), and acridine (30 mg, 0.16 mmol) in DMF/CH₂Cl₂ (700 µL) was added to the resin which was agitated for 3 h. The resin was washed with DMF (2 mL×3), CH₂Cl₂ (2 mL×3) and DMF/CH₂Cl₂=1/1 (2 mL×3). A solution of 10 (50 mg, 0.082 mmol), PyAOP (57 mg, 0.11 mmol), HOAt (7.5 mg, 0.055 mmol), and acridine (30 mg, 0.16 mmol) in DMF/CH₂Cl₂ (700 µL) was added to the resin which was agitated for 3 h. The resin was washed with DMF (2 mL×3), CH₂Cl₂ (2 mL×3). The resin was treated with a solution of Ac₂O (16 µL, 0.16 mmol) and ⁱPr₂NEt (29 µL, 0.16 mmol) in DMF (800 µL) for 30 min. The resin was washed with DMF (2 mL×3), CH₂Cl₂ (2 mL×3). Kaiser test indicated the completion of the coupling reaction. The resin was dried in vacuo to afford 7 (189 mg, 0.042 mmol, 0.22 mmol/g, quantitative over 2 steps). The yield was calculated by weighing resin.

Procedure for the Synthesis of 8

Resin-bound peptide 20 (100 mg, 0.028 mmol) was placed in a 5 mL polypropylene syringe fitted with a polyethylene filter disc. The resin was agitated with CH₂Cl₂ (1.5 mL, 1 h). After removal of CH₂Cl₂, the resin was treated with a solution of BH₃·Me₂NH (18 mg, 0.33 mmol) in EtOH (600 µL) for 5 min, then a solution of Pd(PPh₃)₄ (13 mg, 0.011 mmol) in CH₂Cl₂ (1 mL) was added to the mixture. The mixture was agitated for 15 min. The resin was washed with 0.5% ⁱPr₂NEt/CH₂Cl₂ (2 mL×3), 0.5%(w/v) sodium diethyldithiocarbamate/DMF (1 mL×8), MeOH (2 mL×3) and DMF/CH₂Cl₂=1/1 (2 mL×3). A solution of 11 (49 mg, 0.055 mmol), PyAOP (38 mg, 0.073 mmol), HOAt (5.0 mg, 0.037 mmol), and acridine (20 mg, 0.11 mmol) in DMF/CH₂Cl₂ (600 µL) was added to the resin which was agitated for 3 h. The resin was washed with DMF (2 mL×3), CH₂Cl₂ (2 mL×3) and DMF/CH₂Cl₂=1/1 (2 mL×3). A solution of 11 (49 mg, 0.055 mmol), PyAOP (38 mg, 0.073 mmol), HOAt (5.0 mg, 0.037 mmol), and acridine (20 mg, 0.11 mmol) in DMF/CH₂Cl₂ (600 µL) was added to the resin which was agitated for 3 h. The resin was washed with DMF (2 mL×3), CH₂Cl₂ (2 mL×3). The resin was treated with a solution of Ac₂O (10 µL, 0.11 mmol) and ⁱPr₂NEt (19 µL, 0.11 mmol) in DMF (700 µL) for 30 min. The resin was washed with DMF (2 mL×3), CH₂Cl₂ (2 mL×3). Kaiser test indicated the completion of the coupling reaction. The resin was dried in vacuo to afford 8 (156 mg, 0.028 mmol, 0.18 mmol/g, quantitative over 2 steps).

Park Nucleotide (1)

Resin-bound peptide 7 (9.4 mg, 2.1 µmol) was placed in a 5 mL polypropylene syringe fitted with a polyethylene filter disc. The resin was agitated with CH₂Cl₂ (1 mL, 1 h). After removal of CH₂Cl₂, the resin was treated with a solution of Pd(PPh₃)₄ (1.9 mg, 1.6 µmol) and PhSiH₃ (8.4 µL, 69 µmol) in CH₂Cl₂ (400 µL) for 2 h. The resin was washed with 0.5% ⁱPr₂NEt/CH₂Cl₂ (2 mL×3), 0.5%(w/v) sodium diethyldithiocarbamate/DMF (1 mL×8), MeOH (2 mL×3), CH₂Cl₂ (2 mL×3) and DMF (2 mL×3). The resin was treated with a solution of 23 (7.6 mg, 11 µmol) and 29 (2.4 mg, 11 µmol) in DMF (300 µL) at 50°C for 3 d. The resin was washed with DMF (2 mL×3), CH₂Cl₂ (2 mL×3) to afford 4. The resin was treated with piperidine/DMF (1 : 4, 5 min, then 1 : 9, 15 min) to remove the Fmoc group, and the resin was washed with 0.5% ⁱPr₂NEt/CH₂Cl₂ (2 mL×3), DMF (2 mL×3), CH₂Cl₂ (2 mL×3) and THF (2 mL×3). The resin was treated with 2 M aq. NaOH/MeOH/THF=1/1/2 (400 µL) at 0°C, then warmed to room temperature and agitated for 2 h. The supernatant was neutralized by 1 M aq. HCl (220 µL) and purified with reversed phase HPLC (YMC-Pack R&D ODS-A, 250×20 mm, 1% MeCN/50 mM aq. NH₄HCO₃) to afford 1 (1.4 mg, 1.2 µmol, 44% over 12 steps) as a white powder, after freeze drying.

¹H-NMR (D₂O, 500 MHz) δ: 7.96 (d, 1H, H-6, J_6,5=8.0 Hz), 5.99 (d, 1H, H-1′, J_1′,2′=5.2 Hz), 5.97 (d, 1H, H-5, J_5,6=8.0 Hz), 5.47 (dd, 1H, H-1″, J_1″,2″=2.9, J_1″, P=7.5 Hz), 4.39–4.30 (m, 3H, Ala-α-CH, H-2′, H-3′), 4.30–4.09 (m, 9H, H-4′, H-5′, H-2″, Ala-α-CH, Lac-α-CH, Lys-α-CH, D-Glu-α-CH), 3.98–3.94 (m, 1H, H-5″), 3.88 (dd, 1H, H-6″, J_6″,6″=12.6, J_6″,5″=2.3 Hz), 3.84 (dd, 1H, H-6″, J_6″,6″=12.6, J_6″,5″=4.0 Hz), 3.80 (dd, 1H, H-3″, J_3″,4″=J_3″,2″=10.3 Hz), 3.65 (dd, 1H, H-4″, J_4″,3″=10.3, J_4″,5″=9.7 Hz), 3.01 (t, 1H, Lys-ε-CH, J_{Lys-ε-CH, Lys-δ-CH}=7.5 Hz), 2.31 (t, 2H, D-Glu-γ-CH, J_{D-Glu-γ-CH, D-Glu-β-CH}=8.0 Hz), 2.20–2.12 (m, 1H, D-Glu-β-CH), 2.02 (s, 3H, NAc), 1.92–1.85 (m, 1H, D-Glu-β-CH), 1.85–1.74 (m, 2H, Lys-β-CH), 1.74–1.65 (m, 2H, Lys-δ-CH), 1.52–1.41 (m, 2H, Lys-γ-CH), 1.45 (d, 3H, Ala-β-CH, J_{Ala-β-CH, Ala-α-CH}=7.5 Hz), 1.41 (d, 3H, Ala-β-CH, J_{Ala-β-CH, Ala-α-CH}=6.9 Hz), 1.37 (d, 3H, Ala-β-CH, J_{Ala-β-CH, Ala-α-CH}=6.9 Hz), 1.34 (d, 3H, Lac-β-CH, J_{Lac-β-CH, Lac-α-CH}=6.9 Hz); ³¹P-NMR (D₂O, 162 MHz) δ: −10.9 (d, J_P, P=21.7 Hz), −12.6 (d, J_P, P=21.7 Hz); ESI-MS-LR m/z 573.67 [(M−2H)²⁻]; ESI-MS-HR Calcd for C₄₀H₆₃O₂₆N₉P₂ 573.6685. Found 573.6694; [α]_D²⁰ +13.35 (c 0.08, MeOH).

The analytical data for synthetic 1 were in good agreement with the previously reported data.⁶⁾

Neryl-lipid I (2)

Resin-bound peptide 7 (9.4 mg, 2.1 µmol) was placed in a 5 mL polypropylene syringe fitted with a polyethylene filter disc. The resin was agitated with CH₂Cl₂ (1 mL, 1 h). After removal of CH₂Cl₂, the resin was treated with a solution of Pd(PPh₃)₄ (1.9 mg, 1.6 µmol) and PhSiH₃ (8.4 µL, 69 µmol) in CH₂Cl₂ (400 µL) for 2 h. The resin was washed with 0.5% ⁱPr₂NEt/CH₂Cl₂ (2 mL×3), 0.5%(w/v) sodium diethyldithiocarbamate/DMF (1 mL×8), MeOH (2 mL×3), CH₂Cl₂ (2 mL×3) and DMF (2 mL×3). The resin was treated with a solution of 30 (11 µmol) and 29 (2.4 mg, 11 µmol) in DMF (300 µL) at 50°C for 2 d. The resin was washed with DMF (2 mL×3), CH₂Cl₂ (2 mL×3) to afford 5. The resin was treated with piperidine/DMF (1 : 4, 5 min, then 1 : 9, 15 min) to remove the Fmoc group, and the resin was washed with 0.5% ⁱPr₂NEt/CH₂Cl₂ (2 mL×3), DMF (2 mL×3), CH₂Cl₂ (2 mL×3) and THF (2 mL×3). The resin was treated with 2 M aq. LiOH/MeOH/THF=1/1/2 (400 µL) at 0°C, then warmed to room temperature and agitated for 2 h. The supernatant was neutralized by 1 M aq. HCl (210 µL) and purified with reversed phase HPLC (YMC-Pack R&D ODS-A, 250×20 mm, 15% MeCN/50 mM aq. NH₄HCO₃) to afford 2 (0.6 mg, 0.55 µmol, 20% over 12 steps) as a white powder after freeze drying.

¹H-NMR (D₂O, 500 MHz) δ: 5.47–5.42 (m, 2H, H-1, H-2′), 5.20 (t, 1H, H-6′, J_6′,5′=6.9 Hz), 4.49–4.43 (m, 2H, H-1′), 4.33 (q, 1H, Ala-α-CH, J_{Ala-α-CH, Ala-β-CH}=6.9 Hz), 4.26 (q, 1H, Ala-α-CH, J_{Ala-α-CH, Ala-β-CH}=7.5 Hz), 4.23–4.18 (m, 2H, Ala-α-CH, Lys-α-CH), 4.17–4.08 (m, 3H, D-Glu-α-CH, Lac-α-CH, H-2), 3.98–3.93 (m, 1H, H-5), 3.91–3.86 (m, 1H, H-6), 3.84 (dd, 1H, H-6, J_6,6=12.6, J_6,5=4.0 Hz), 3.80 (dd, 1H, H-3, J_3,4=J_3,2=9.7 Hz), 3.64 (dd, 1H, H-4, J_4,3=9.7, J_4,5=10.3 Hz), 3.00 (t, 1H, Lys-ε-CH, J_{Lys-ε-CH, Lys-δ-CH}=7.5 Hz), 2.31 (t, 2H, D-Glu-γ-CH, J_{D-Glu-γ-CH, D-Glu-β-CH}=8.0 Hz), 2.20–2.10 (m, 5H, D-Glu-β-CH, H-4′, H-5′), 2.00 (s, 3H, NAc), 1.93–1.85 (m, 1H, D-Glu-β-CH), 1.85–1.75 (m, 2H, Lys-β-CH), 1.77 (s, 3H, 3′-Me), 1.73–1.66 (m, 2H, Lys-δ-CH), 1.69 (s, 3H, 7′-Me), 1.62 (s, 3H, 7′-Me), 1.51–1.42 (m, 2H, Lys-γ-CH), 1.45 (d, 3H, Ala-β-CH, J_{Ala-β-CH, Ala-α-CH}=6.9 Hz), 1.41 (d, 3H, Ala-β-CH, J_{Ala-β-CH, Ala-α-CH}=6.9 Hz), 1.37 (d, 3H, Ala-β-CH, J_{Ala-β-CH, Ala-α-CH}=7.5 Hz), 1.33 (d, 3H, Lac-β-CH, J_{Lac-β-CH, Lac-α-CH}=7.5 Hz); ³¹P-NMR (D₂O, 202 MHz) δ: −10.2 (d, J_P, P=21.4 Hz), −12.7 (d, J_P, P=21.4 Hz); ESI-MS-LR m/z 528.70 [(M−2H)²⁻]; ESI-MS-HR Calcd for C₄₁H₆₉O₂₁N₇P₂ 528.7016. Found 528.7025; [α]_D²⁰ +20.42 (c 0.06, MeOH).

Neryl-lipid II (3)

Resin-bound peptide 8 (12.4 mg, 2.2 µmol) was placed in a 5 mL polypropylene syringe fitted with a polyethylene filter disc. The resin was agitated with CH₂Cl₂ (1 mL, 1 h). After removal of CH₂Cl₂, the resin was treated with a solution of Pd(PPh₃)₄ (2.0 mg, 1.7 µmol) and PhSiH₃ (8.9 µL, 73 µmol) in CH₂Cl₂ (300 µL) for 2 h. The resin was washed with 0.5% ⁱPr₂NEt/CH₂Cl₂ (2 mL×3), 0.5%(w/v) sodium diethyldithiocarbamate/DMF (1 mL×8), MeOH (2 mL×3), CH₂Cl₂ (2 mL×3) and DMF (2 mL×3). The resin was treated with a solution of 30 (12 µmol) and 29 (2.6 mg, 12 µmol) in DMF (300 µL) at 50°C for 3 d. The resin was washed with DMF (2 mL×3), CH₂Cl₂ (2 mL×3) to afford 6. The resin was treated with piperidine/DMF (1 : 4, 5 min, then 1 : 9, 15 min) to remove the Fmoc group, and the resin was washed with 0.5% ⁱPr₂NEt/CH₂Cl₂ (2 mL×3), DMF (2 mL×3), CH₂Cl₂ (2 mL×3) and THF (2 mL×3). The resin was treated with 2 M aq. LiOH/MeOH/THF=1/1/2 (400 µL) at 0°C, then warmed to room temperature and agitated for 2 h. The supernatant was neutralized by 1 M aq. HCl (220 µL) and purified with reversed phase HPLC (YMC-Pack R&D ODS-A, 250×20 mm, 15% MeCN/50 mM aq. NH₄HCO₃) to afford 3 (0.8 mg, 0.64 µmol, 21% over 12 steps) as a white powder after freeze drying.

¹H-NMR (D₂O, 500 MHz) δ: 5.47–5.41 (m, 2H, H-1, H-2″), 5.22–5.17 (m, 1H, H-6″), 4.62 (d, 1H, H-1′, J_1′,2′=8.6 Hz), 4.45 (dd, 2H, H-1″, J_1″,2″=J_1″, P=6.9 Hz), 4.33 (q, 1H, Ala-α-CH, J_{Ala-α-CH, Ala-β-CH}=7.5 Hz), 4.28–4.12 (m, 5H, Ala-α-CH, Lys-α-CH, D-Glu-α-CH, H-2), 4.11 (q, 1H, Lac-α-CH, J_{Lac-α-CH, Lac-β-CH}=7.5 Hz), 3.97–3.88 (m, 4H, H-4, H-6, H-6′), 3.81 (dd, 1H, H-3, J_3,4=J_3,2=9.7 Hz), 3.77–3.69 (m, 3H, H-5, H-2′, H-6′), 3.55 (dd, 1H, H-3′, J_3′,4′=J_3′,2′=8.6 Hz), 3.44–3.38 (m, 2H, H-4′, H-5′), 3.00 (t, 1H, Lys-ε-CH, J_{Lys-ε-CH, Lys-δ-CH}=7.5 Hz), 2.31 (t, 2H, D-Glu-γ-CH, J_{D-Glu-γ-CH, D-Glu-β-CH}=8.6 Hz), 2.20–2.10 (m, 5H, D-Glu-β-CH, H-4″, H-5″), 2.05 (s, 3H, NAc), 1.99 (s, 3H, NAc), 1.93–1.85 (m, 1H, D-Glu-β-CH), 1.85–1.75 (m, 2H, Lys-β-CH), 1.77 (s, 3H, 3″-Me), 1.74–1.65 (m, 2H, Lys-δ-CH), 1.69 (s, 3H, 7″-Me), 1.62 (s, 3H, 7″-Me), 1.51–1.41 (m, 2H, Lys-γ-CH), 1.45 (d, 3H, Ala-β-CH, J_{Ala-β-CH, Ala-α-CH}=7.5 Hz), 1.44 (d, 3H, Ala-β-CH, J_{Ala-β-CH, Ala-α-CH}=6.9 Hz), 1.37 (d, 3H, Ala-β-CH, J_{Ala-β-CH, Ala-α-CH}=7.5 Hz), 1.33 (d, 3H, Lac-β-CH, J_{Lac-β-CH, Lac-α-CH}=6.9 Hz); ³¹P-NMR (D₂O, 202 MHz) δ: −10.2 (d, J_P, P=21.4 Hz), −12.7 (d, J_P, P=21.4 Hz); ESI-MS-LR m/z 630.24 [(M−2H)²⁻]; ESI-MS-HR Calcd for C₄₉H₈₂O₂₆N₈P₂ 630.2413. Found 630.2425; [α]_D²⁰ +10.00 (c 0.08, MeOH).

The analytical data for synthetic 3 were in good agreement with the previously reported data.¹⁴⁾

Acknowledgments

The authors wish to thank Ms. S. Oka (Center for Instrumental Analysis, Hokkaido University) for measurement of the mass spectra. This research was supported by the JSPS Grant-in-Aid for Scientific Research (B) (SI, Grant Number 25293026), The Ministry of Education, Culture, Sports, Science and Technology through the Program for Leading Graduate Schools (Hokkaido University “Ambitious Leader’s Program”), and the Platform Project for Supporting Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics and Structural Life Science).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

References

• 1) Bouhss A., Trunkfield A. E., Bugg T. D. H., Mengin-Lecreulx D., FEMS Microbiol. Rev., 32, 208–233 (2008).
• 2) Bugg T. D. H., Walsh C. T., Nat. Prod. Rep., 9, 199–215 (1992).
• 3) Sham L.-T., Butler E. K., Lebar M. D., Kahne D., Bernhardt T. G., Ruiz N., Science, 345, 220–222 (2014).
• 4) Ruiz N., Proc. Natl. Acad. Sci. U.S.A., 105, 15553–15557 (2008).
• 5) Dumbre S., Derouaux A., Lescrinier E., Piette A., Joris B., Terrak M., Herdewijn P., J. Am. Chem. Soc., 134, 9343–9351 (2012).
• 6) Chen K.-T., Chen P.-T., Lin C.-K., Huang L.-Y., Hu C.-M., Chang Y.-F., Hsu H.-T., Cheng T.-J. R., Wu Y.-T., Cheng W.-C., Sci. Rep., 6, 31579 (2016).
• 7) Wohnig S., Spork A. P., Koppermann S., Mieskes G., Gisch N., Jahn R., Ducho C., Chem. Eur. J., 22, 17813–17819 (2016).
• 8) Siricilla S., Mitachi K., Skorupinska-Tudek K., Swiezewska E., Kurosu M., Anal. Biochem., 461, 36–45 (2014).
• 9) Ye X.-Y., Lo M.-C., Brunner L., Walker D., Kahne D., Walker S., J. Am. Chem. Soc., 123, 3155–3156 (2001).
• 10) VanNieuwenhze M. S., Mauldin S. C., Zia-Ebrahimi M., Winger B. E., Hornback W. J., Saha S. L., Aikins J. A., Blaszczak L. C., J. Am. Chem. Soc., 124, 3656–3660 (2002).
• 11) Liu C.-Y., Guo C.-W., Chang Y.-F., Wang J.-T., Shih H.-W., Hsu Y.-F., Chen C.-W., Chen S.-K., Wang Y.-C., Cheng T.-J. R., Ma C., Wong C.-H., Fang J.-M., Cheng W.-C., Org. Lett., 12, 1608–1611 (2010).
• 12) Hitchcock S. A., Eid C. N., Aikins J. A., Zia-Ebrahimi M., Blaszczak L. C., J. Am. Chem. Soc., 120, 1916–1917 (1998).
• 13) Shih H.-W., Chen K.-T., Cheng T.-J. R., Wong C.-H., Cheng W.-C., Org. Lett., 13, 4600–4603 (2011).
• 14) Mitachi K., Mohan P., Siricilla S., Kurosu M., Chem. Eur. J., 20, 4554–4558 (2014).
• 15) VanNieuwenhze M. S., Mauldin S. C., Zia-Ebrahimi M., Aikins J., Blaszczak L. C., J. Am. Chem. Soc., 123, 6983–6988 (2001).
• 16) Schwartz B., Markwalder J. A., Wang Y., J. Am. Chem. Soc., 123, 11638–11643 (2001).
• 17) Eid C. N., Nesler M. J., Zia-Ebrahimi M., Wu C.-Y. E., Yao R., Cox K., Richardson J., J. Labelled Compd. Rad, 41, 705–716 (1998).
• 18) Meng F.-C., Chen K.-T., Huang L.-Y., Shih H.-W., Chang H.-H., Nien F.-Y., Liang P.-H., Cheng T.-J. R., Wong C.-H., Cheng W.-C., Org. Lett., 13, 5306–5309 (2011).
• 19) Ueda T., Feng F., Sadamoto R., Niikura K., Monde K., Nishimura S., Org. Lett., 6, 1753–1756 (2004).
• 20) Li K., Kurosu M., Heterocycles, 76, 455–469 (2008).
• 21) Grøtli M., Gotfredsen C. H., Rademann J., Buchardt J., Clark A. J., Duus J. Ø., Meldal M., J. Comb. Chem., 2, 108–119 (2000).
• 22) Saha S. L., VanNieuwenhze M. S., Hornback W. J., Aikins J. A., Blaszczak L. C., Org. Lett., 3, 3575–3577 (2001).
• 23) Feld B. K., Weiss G. A., Bioorg. Med. Chem. Lett., 16, 1665–1667 (2006).
• 24) Bosco M., Massarweh A., Iatmanen-Harbi S., Bouhss A., Chantret I., Busca P., Moore S. E. H., Gravier-Pelletier C., Eur. J. Med. Chem., 125, 952–964 (2017).
• 25) Sekine M., Ohkubo A., Seio K., J. Org. Chem., 68, 5478–5492 (2003).