Solid-phase modular synthesis of park nucleotide and lipids I and II analogues

Article abstract of DOI:10.1248/cpb.c17-00828

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. M

Full text of DOI:10.1248/cpb.c17-00828

84
Chem. Pharm. Bull. 66, 84–95 (2018)
Vol. 66, No. 1
Regular Article
Highlighted Paper selected by Editor-in-Chief
Solid-Phase Modular Synthesis of Park Nucleotide and Lipids I and II
Analogues
Akira Katsuyama,a Kousuke Sato,a,b Fumika Yakushiji,a,c Takanori Matsumaru,a,c and
,a,c
Satoshi Ichikawa*
a Faculty of Pharmaceutical Science, Hokkaido University; Kita-12, Nishi-6, Kita-ku, Sapporo 060–0812, Japan:
b Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Kanazawa 1757; Tobetsu, Hokkaido,
c
061–0293, Japan: and Center for Research and Education on Drug Discovery, Hokkaido University; Kita-12, Nishi-
6, Kita-ku, Sapporo 060–0812, Japan.
Received October 13, 2017; accepted October 26, 2017
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 mac-
romolecules to be modularly labeled. Multiple steps are used in a short time (4d) with a single purification
step to synthesize the molecules by solid-phase synthesis.
Key words peptidoglycan; natural product; solid-phase synthesis
Peptidoglycan is a component of bacterial cell walls and their amphiphilic properties as well as their molecular
that consists of repeating β-1-4-N-acetylglucosaminyl-N- size occasionally face difficulty in purification during synthe-
acetylmuraminic acid (β-1-4-GlcNAc-MurNAc) units, which sis. Solid-phase syntheses have been extensively developed
are further cross-linked with polypeptides. Peptidoglycan especially in the synthesis of biomacromolecules. Since syn-
plays a role as an extracellular skeleton to prevent bacterial thetic intermediates remain immobilized on the solid sup-
cells from lysis by intracellular high pressure and is a primary port, it is easy to handle a molecule with the abovementioned
defense from a variety of physiological, chemical and biologi- properties. Although Kurosu’s group reported the solid-phase
cal attacks outside the cells. Peptidoglycan biosynthesis con- synthesis of peptide fragments of Park nucleotide,20) the intro-
sists of several stages as shown in Fig. 1.1,2)
duction of the sugar domain and construction of the diphos-
Uridine-5′-diphospho-MurNAc-pentapeptide
(UDP- phate moiety on a solid-phase remains a challenge. Herein, a
MurNAc-pentapeptide, 1), which is also called as Park modular solid-phase synthesis of Park nucleotide (1), lipid I
nucleotide, is synthesized by a series of MurA-F enzymes in analogue 2 and lipid II analogue 3, is described.
cytoplasm. Phospho-MurNAc-pentapeptide transferase (MraY)
catalyzes the first reaction step of the lipid-linked cycle, where
Results and Discussion
Park nucleotide is attacked by undecaprenyl monophosphate in
The retrosynthetic analysis is illustrated in Chart 1. The
the bacterial cell membrane providing lipid I. Lipid I is fur- C-terminus of these molecules is immobilized onto a solid
ther glycosylated by N-acetylglucosamine transferase (MurG) support with suitable protection of the functional groups to
to afford lipid II. Lipid II in cytoplasm is then flipped out by give 4–6. Considering the lability of the diphosphate under
a flipase called MurJ, which was recently identified.3,4) The acidic conditions, base-removable protecting groups were cho-
lipid II in periplasm is then polymerized by a transglycosylase sen for all the functional groups. Accordingly, cleavage from
and a transpeptidase to form peptidoglycan. The biosynthesis the solid-phase was used under basic conditions. In addition,
of peptidoglycan is one of the major targets for antibacterial the hydrophilicity of the target molecules upon simultaneous
drug discovery and many drugs including β-lactams and van- deprotection under basic aqueous conditions was considered.
comycin are currently clinically used drugs. In addition to These considerations initiated the use of a 4-(hydroxymethyl)-
the fact that the analogues of Park nucleotide, lipids I and II, bezoylamidyl polyethyleneglycol (HMBA-PEG) resin, which
which are precursors to peptidoglycan, have the potential to be swells extensively in a wide range of solvents, including
inhibitors of peptidoglycan biosynthesis,5,6) they are used as water.21) The diphosphate moieties of 4–6 are disconnected to
biological tools to elucidate the biology of bacterial cell walls. give pentapeptidylglycosyl phosphates 7 and 8 as well as the
For example, dansylated Park nucleotide has been used as a corresponding uridine 5′-O-phosphate and neryl phosphate,
fluorescent substrate in an assay screening of MraY inhibitor,7) respectively. Regarding the disconnection of 7 and 8, it was
and lipids I and II analogues with short lipid tails, such as a reported by Ducho’s group that coupling of a pentapeptide
neryl group instead of a undecaprenyl group, have been used with a MurNAc derivative resulted in the formation of a dike-
in mechanistic studies of MurY and MurG8,9) (Fig. 2, 2 and 3). topiperazine consisting of L-Ala and D-Glu and that it is better
As a result, these molecules constitute an important class of to connect a tetrapeptide and a MurNAc derivative condensed
molecules in drug discovery and microbiology. The chemical with L-Ala (MurNAc-Ala).7) This observation was modeled for
synthesis of Park nucleotide, lipids I, II and their analogues the solid-phase synthesis to disconnect 7 and 8 between the
have previously been accomplished by solution-phase synthe- L-Ala and D-Glu residues to give tetrapeptide 9, MurNAc-Ala
sis.6,7,10–20) However, these molecules consist of amino acids, 10 and GlcNAc-MurNAc-Ala 11.
sugars, and diphosphates attached to uridine or the lipid chain
Both 10 and 11 were easily prepared from GlcNAc and
*To whom correspondence should be addressed. e-mail: ichikawa@pharm.hokudai.ac.jp
© 2018 The Pharmaceutical Society of Japan

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Fig. 1. Biosynthesis of Peptidoglycan
Fig. 2. Structures of Neryl Analogues of Lipids I and II

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Chart 1. Retrosynthetic Analysis of Target Molecules
D-glucosamine (GlcNH2) via known compounds 12 and 15, and iPr2NEt. After extensive investigations, it was found
respectively (Chart 2).22) Namely, the 4,6-O-benzylidene pro- that the conditions using (7-azabenzotriazol-1-yloxy)tripyr-
tecting group of 12, which was obtained from D-GlcNAc over rolidinophosphonium hexafluorophosphate (PyAOP), 1-hydro-
four steps, was converted to acetyl groups suitable for the fol- xy-7-azabenzotriazole (HOAt) and acridine in N,N-dimeth-
lowing synthetic route. The benzyl group at the O-1-position ylformamide (DMF)/CH2Cl2 completely suppressed the
of the resulting 13 was removed by hydrogenolysis and the epimerization. A single coupling resulted in incomplete reac-
liberated alcohol was phosphorylated by two steps to give tion and therefore a double-coupling was conducted to com-
O-1-α-diallylphosphate 14 in 39% yield over three steps. The pletely consume the amine. Deprotection of the allyl groups
phenylsulfonylethyl group at the Ala residue was removed by on the phosphate was conducted by the conditions using
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to afford the car- Pd(PPh3)4 and PhSiH3 to cleanly provide the phosphate 21. In-
boxylic acid 10. The glycosyl phosphates 11 was prepared by stead of 10, coupling of the disaccharide 11 to the tetrapeptide
the same procedure for the synthesis of 10.
amine gave 22.
The synthesis on the solid-phase was then investigated
With 21 and 22 in hand, the key diphosphate formation
(Chart 3). First, Fmoc-D-Ala was immobilized onto the reaction on the solid-phase was investigated. Generally, the
HMBA-PEG resin (0.39mmol/g). Then, Fmoc-peptide synthe- diphosphate moiety is classically constructed by the condensa-
sis was applied to give tripeptide 19. The protecting group of tion of a phosphate monoester with a phosphoroamidate upon
N-terminal peptide 19 was then switched to an Alloc group. activation by an activator, such as 1H-tetrazole (26).23) The
After coupling of 19 with Alloc-D-Glu-OBn and the removal reaction rate, however, was very slow even in the solution-
of the Alloc group of 20, the resulting amine was coupled phase synthesis and remained to be improved when applied
with 10 to afford glycosyl peptapeptide 7. It should be noted to solid-phase synthesis. Thus, the reaction conditions were
that considerable epimerization occurred during the coupling optimized using model substrates 23 and 2424) in the solution-
with 10 using 1-[bis(dimethylamino)methylene]-1H-1,2,3-tri- phase, and the results are summarized in Table 1. Treatment
azolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) of 24 with uridine-5′-phosphorylmorphoridate 23 in the pres-

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87
Chart 2. Preparation of 10 and 11
ence of 26 as an activator, which is conventionally used in yield over 12 steps from 17 (Chart 4b). Using 22 instead of 21,
diphosphate coupling, gave desired 25 in 18% yield after 24h neryl-lipid II (3) was also synthesized (Chart 4c). This strat-
and 42% yield after 72h (Fig. 3). The activation ability of the egy allows for the modular labeling of highly functionalized
phosphorylamidate correlates to the acidity of the activators. macromolecules, which were obtained in multiple steps in a
The activator acidity improves the chemical yields of 25, and short time (4d) using a single purification step by virtue of
the use of 2925) gave 25 in 58% yield.
solid-phase synthesis.
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
Conclusion
In conclusion, a solid-phase modular synthesis has been
temperature, elevating the temperature to 50 °C accelerated established for Park nucleotide and lipids I and II analogues.
the reaction rate to produce 4. Finally, cleavage from the resin These analogues could be useful as chemical probes for dis-
as well as global deprotection by treating 4 with piperidine covering novel antibacterial agents and elucidating detailed
followed by aq. NaOH successfully afforded Park nucleotide mechanistic studies on peptidoglycan biosynthesis.
(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
Experimental
General Experimental Methods All reactions except
with a short lipid (a neryl group) was synthesized in 20% that carried out in aqueous phase were performed under argon

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Chart 3. Solid-Phase Synthesis of 21 and 22
atmosphere, unless otherwise noted. Isolated yields were solvent peaks using JEOL ECA 500 (125MHz), JEOL ECS
calculated by weighing products. The weight of the starting 400 (100MHz) or JEOL ECX 400P (100MHz) spectropho-
materials and the products were not calibrated. Materials tometers. 31P-NMR were measured in CDCl3, DMSO-d6, or
were purchased from commercial suppliers and used without D2O solution, and referenced to H3PO4 (0.00ppm) using JEOL
further purification, unless otherwise noted. Solvents are dis- ECA 500 (202MHz), JEOL ECS 400 (162MHz) or JEOL
tilled according to the standard protocol. Analytical TLC was ECX 400P (162MHz) spectrophotometers. Abbreviations of
performed on Merck silica gel 60F254 plates. Normal-phase multiplicity were as follows; s: singlet, d: doublet, t: triplet,
column chromatography was performed on Merck silica gel q: quartet, sept: septet, m: multiplet, br: broad. Data were
5715 or Kanto Chemical silica gel 60N (neutral). High-flash presented as follows; chemical shift (multiplicity, integration,
1
column chromatography was performed on Fuji Sylysia silica coupling constant). Assignment was based on H–1H correla-
1
gel PSQ 60B. H-NMR were measured in CDCl3, dimethyl tion spectroscopy (COSY), heteronuclear multiple bond corre-
sulfoxide (DMSO)-d6, or D2O solution, and referenced to TMS lation (HMBC) and heteronuclear multiple quantum coherence
(0.00ppm) using JEOL ECA 500 (500MHz), JEOL ECS 400 (HMQC) NMR spectra. Optical rotations were determined on
(400MHz) or JEOL ECX 400P (400MHz) spectrophotom- JASCO P-1010-GT. Mass spectra were recorded on Thermo
eters, unless otherwise noted. 13C-NMR were measured in Scientific Exactive. The mass analyzer type used for the high
CDCl3, DMSO-d6, or D2O solution, and referenced to residual resolution (HR)-MS measurements was time-of-flight (TOF).

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89
Table 1. Optimization of Diphosphate Formation
1H, 2-NH, J2-NH,2=9.6Hz), 4.91 (d, 1H, H-1, J1,2=3.6Hz),
4.71 (d, 1H, Bn, J=11.9Hz), 4.47 (d, 1H, Bn, J=11.9Hz),
4.50–4.37 (m, 2H, PhSO2CH2CH2), 4.29 (dq, 1H, Ala-α-CH,
JAla-α-CH,Ala-NH=JAla-α-CH,Ala-β-CH=7.3Hz), 4.23 (ddd, 1H, H-2,
J2,1=3.6, J2,2-NH=9.6, J2,3=10.1Hz), 4.16 (q, 1H, Lac-α-CH,
JLac-α-CH,Lac-β-CH=6.9Hz), 3.83 (d, 2H, H-6, J6,5=3.2Hz),
3.76–3.65 (m, 2H, H-5, H-4), 3.58 (dd, 1H, H-3,
J3,2=J3,4=10.1Hz), 3.47–3.33 (m, 2H, PhSO2CH2CH2), 1.92 (s,
3H, NAc), 1.42 (d, 3H, Lac-β-CH, JLac-β-CH,Lac-α-CH=6.9Hz),
1.33 (d, 3H, Ala-β-CH, JAla-β-CH,Ala-α-CH=7.3Hz). This is a
known compound reported in ref. 14).
A mixture of benzyl-N-acetyl-muramyl-L-alanine phenyl-
sulfonylethyl ester (1.12g, 1.80mmol) in pyridine (20mL) was
treated with Ac2O (406µL, 4.32mmol) at room temperature
for 1d. Ac2O (102µL, 1.08mmol) was added to the mixture,
which was stirred for 24h. The reaction was quenched with
MeOH, then the resulting mixture was concentrated in vacuo.
The residue was partitioned between AcOEt and sat. aq.
Fig. 3. Time Course of Diphosphate Formation
Benzyl-N-acetyl-4,6-diacetylmuramyl-L-alanine Phenylsul- NaHCO3, and the organic phase was washed with 1M aq. HCl
fonylethyl Ester (13) and brine, dried (Na2SO4), filtered, and concentrated in vacuo.
Compound 12 (1.75g, 2.46mmol) was treated with 60% The residue was purified by high-flash silica gel column
AcOH/H2O (60mL) at 90°C for 1h. The mixture was concen- chromatography (50–85–100% AcOEt/hexane) to afford 13
trated in vacuo. The residue was crystalized by CHCl3/Et2O to (955mg, 1.35mmol, 75%) as a colorless foam.
afford benzyl-N-acetyl-muramyl-L-alanine phenylsulfonylethyl
ester (1.21g, 1.94mmol, 79%) as a white solid.
1H-NMR (CDCl3, 500MHz) δ: 7.92 (d, 2H, o-PhSO2,
Jo,m=6.9Hz), 7.68 (t, 1H, p-PhSO2, Jp,m=7.5Hz), 7.59 (dd, 2H,
1H-NMR (CDCl3, 400MHz) δ: 7.91 (d, 2H, o-PhSO2, m-PhSO2, Jm,o=6.9, Jm,p=7.5Hz), 7.41–7.56 (m, 5H, Ph), 6.85
Jo,m=7.3Hz), 7.69 (t, 1H, p-PhSO2, Jp,m=7.3Hz), 7.59 (dd, (d, 1H, Ala-NH, JAla-NH,Ala-α-CH=6.9Hz), 5.81 (d, 1H, 2-NH,
2H, m-PhSO2, Jm,o=Jm,p=7.3Hz), 7.40–7.29 (m, 5H, Ph), J2-NH,2=9.2Hz), 5.07 (dd, 1H, H-4, J4,3=J4,5=9.8Hz), 4.88 (d,
6.92 (d, 1H, Ala-NH, JAla-NH,Ala-α-CH=7.3Hz), 5.04 (d, 1H, H-1, J1,2=4.0Hz), 4.69 (d, 1H, Bn, J=11.5Hz), 4.50 (d, 1H,

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Chart 4. Completion of the Synthesis of 1–3
Bn, J=11.5Hz), 4.48–4.40 (m, 2H, PhSO2CH2CH2), 4.38 (ddd, 125MHz) δ: 172.2, 171.8, 170.9, 170.4, 169.5, 139.3, 136.7,
1H, H-2, J2,1=4.0, J2,2-NH=9.2, J2,3=9.8Hz), 4.20 (dd, 1H, H-6, 134.2, 129.6, 128.9, 128.7, 128.5, 128.3, 97.2, 78.9, 78.6, 70.4,
J6,6=12.0, J6,5=4.6Hz), 4.13 (dq, 1H, Ala-α-CH, JAla-α-CH,Ala-NH
=
69.5, 68.7, 62.3, 58.2, 55.1, 53.1, 48.1, 23.5, 21.0, 20.9, 18.7,
JAla-α-CH,Ala-β-CH=6.9Hz), 4.03 (dd, 1H, H-6, J6,6=12.0, 17.0; electrospray ionization (ESI)-MS-low resolution (LR)
J6,5=2.3Hz), 3.95 (q, 1H, Lac-α-CH, JLac-α-CH,Lac-β-CH=6.3Hz), m/z 729.23 [(M+Na)+]; ESI-MS-HR Calcd for C33H42O13N2NaS
3.91 (ddd, 1H, H-5, J5,6=2.3, J5,6=4.6, J5,4=9.8Hz), 3.64 (dd, 729.2230. Found 729.2299; [α]D20 +68.36 (c 0.23, CHCl3).
1H, H-3, J3,2=J3,4=9.8Hz), 3.50–3.38 (m, 2H, PhSO2CH2CH2),
2.11 (s, 3H, OAc), 2.07 (s, 3H, OAc), 1.88 (s, 3H, NAc),
Glycosyl Phosphate 14
A mixture of 13 (424mg, 0.60mmol) and 10% Pd/C
1.30 (d, 3H, Lac-β-CH, JLac-β-CH,Lac-α-CH=6.3Hz), 1.29 (d, (600mg) in MeOH (6mL) was vigorously stirred under H2
3H, Ala-β-CH, JAla-β-CH,Ala-α-CH=6.9Hz); 13C-NMR (CDCl3, atmosphere at room temperature for 2.5h. 10% Pd/C (600mg)

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91
was added to the mixture and vigorously stirred for 8h under J2-NH,2=8.0Hz), 6.86 (d, 1H, Ala-NH, JAla-NH,Ala-α-CH=6.3Hz),
H2 atmosphere. 10% Pd/C (300mg) was added to the mixture 5.98–5.88 (m, 2H, Hc), 5.72 (dd, 1H, H-1, J1,2=3.4, J1P=6.3Hz),
and vigorously stirred for 12h under H2 atmosphere. The cata- 5.39 (dd, 1H, Ha, JHa,Hc=16.6, JHa,1′=1.2Hz), 5.38 (dd, 1H, Ha,
lyst was filtered off through a Celite pad, and the filtrate was JHa,Hc=17.2, JHa,1′=1.2Hz), 5.30 (d, 2H, Hb, JHb,Hc=10.3 Hz),
concentrated in vacuo to afford a crude lactol. A mixture of 5.12 (dd, 1H, H-4, J4,3=J4,5=9.2Hz), 4.62–4.55 (m, 4H, H-1′),
the lactol and 5-(benzylthio)-1H-tetrazole (208mg, 1.08mmol) 4.37 (qd, 1H, Ala-α-CH, JAla-α-CH,Ala-β-CH=JAla-α-CH,Ala-NH=6.9
in CH2Cl2 (6mL) was treated with diallyl N,N-diisopro- Hz), 4.31–4.25 (m, 1H, H-2), 4.22–4.15 (m, 2H, Lac-α-CH,
pylphosphoramidite (238µL, 0.90mmol) at 0°C for 5min. The H-6), 4.09–4.04 (m 2H, H-5, H-6), 3.75 (dd, 1H, H-3,
mixture was warmed to room temperature and stirred for 1h. J3,2=J3,4=9.7Hz), 2.11 (s, 3H, OAc), 2.08 (s, 3H, OAc), 1.97 (s,
5-(Benzylthio)-1H-tetrazole (138mg, 0.72mmol) and diallyl 3H, NAc), 1.47 (d, 3H, Ala-β-CH, JAla-β-CH,Ala-α-CH=7.5Hz), 1.33
N,N-diisopropylphosphoramidite (159µL, 0.60mmol) was (d, 3H, Lac-β-CH, JLac-β-CH,Lac-α-CH=6.3Hz); 13C-NMR (CDCl3,
added to the mixture and stirred for 1h. The mixture was par- 125MHz) δ: 174.4, 173.4, 171.7, 170.7, 170.8, 169.4, 132.1,
titioned between CH2Cl2 and sat. aq. NaHCO3, and the organ- 132.0, 131.9, 131.8, 119.5, 119.2, 96.8, 96.8, 78.3, 76.7, 70.4,
ic phase was washed with H2O and brine, dried (Na2SO4), fil- 69.3, 69.2, 69.0, 69.0, 61.9, 53.5, 53.4, 48.5, 23.0, 20.9, 20.8,
tered, and concentrated in vacuo to afford a crude phosphite. 19.2, 17.5; 31P-NMR (CDCl3, 202MHz) δ −2.7; ESI-MS-LR
A mixture of the phosphite in tetrahydrofuran (THF) (6mL) m/z 631.19 [(M+Na)+]; ESI-MS-HR Calcd for C24H37O14N2NaP
was treated with 30% H2O2 (600 µL) at −78°C for 5min. The 631.1875. Found 631.1882; [α]D20 +63.23 (c 3.80, CHCl3).
mixture was warmed to room temperature and stirred for
Glycosyl Phosphate 16
2.5h. The reaction was quenched with sat. aq. Na2S2O3 at 0°C,
A mixture of 15 (497mg, 0.50mmol) and 10% Pd/C
and the mixture was partitioned between AcOEt and sat. aq. (600mg) in MeOH/EtOH=1/1 (10mL) was vigorously stirred
NaHCO3. The organic phase was washed with brine, dried under H2 atmosphere at room temperature for 24h. The cata-
(Na2SO4), filtered, and concentrated in vacuo. The residue lyst was filtered off through a Celite pad, and the filtrate was
was purified by high-flash silica gel column chromatography concentrated in vacuo to afford a crude lactol. A mixture
(50–100% AcOEt/hexane-0–1–2% MeOH/AcOEt) to afford 14 of the lactol and 1H-tetrazole (70mg, 1.0mmol) in CH2Cl2
(180mg, 0.23mmol, 39% over 3 steps) as a colorless foam.
(5mL) was treated with diallyl N,N-diisopropylphosphorami-
1H-NMR (CDCl3, 500MHz) δ: 7.93 (d, 2H, o-PhSO2, dite (198µL, 0.75mmol) at 0°C for 5min. The mixture was
Jo,m=7.4Hz), 7.67 (t, 1H, p-PhSO2, Jp,m=7.5Hz), 7.60 (dd, warmed to room temperature and stirred for 1.5h. Diallyl
2H, m-PhSO2, Jm,o=7.4, Jm,p=7.5Hz), 6.76 (d, 1H, Ala-NH, N,N-diisopropylphosphoramidite (49.5µL, 0.19mmol) was
JAla-NH,Ala-α-CH=6.9Hz), 6.55 (d, 1H, 2-NH, J2-NH,2=8.6Hz), added to the mixture and stirred for 10min. The mixture was
t
6.01–5.88 (m, 2H, Hc), 5.70 (dd, 1H, H-1, J1,2=2.9, cooled to −50°C, and treated with 80% BuOOH (1mL) for
J1, P=5.8Hz), 5.40 (dd, 1H, Ha, JHa,Hc=17.2, JHa,1′=1.2Hz), 1h. The reaction was quenched with sat. aq. Na2S2O3, and the
5.38 (dd, 1H, Ha, JHa,Hc=17.2, JHa,1′=1.2Hz), 5.31 (dd, 1H, mixture was partitioned between AcOEt and sat. aq. NaHCO3.
Hb, JHb,Hc=10.3, JHa,1′=1.2Hz), 5.30 (dd, 1H, Hb, JHb,Hc=10.3, The organic phase was washed with H2O and brine, dried
J
Ha,1′=1.2Hz), 5.14 (dd, 1H, H-4, J4,3=10.3, J4,5=9.8Hz), (Na2SO4), filtered, and concentrated in vacuo. The residue
4.64–4.55 (m, 4H, H-1′), 4.53–4.44 (m, 2H, PhSO2CH2CH2), was purified by high-flash silica gel column chromatography
4.41–4.35 (m, 1H, H-2), 4.24 (dq, 1H, Ala-α-CH, 7.5, (0-1-2-5% MeOH/AcOEt) to afford 16 (271mg, 0.25mmol,
JAla-α-CH,Ala-NH=JAla-α-CH,Ala-β-CH=6.9Hz), 4.20 (dd, 1H, H-6, 50% over 2 steps) as a colorless foam.
J6,6=12.0, J6,5=4.0Hz), 4.13–4.05 (m 2H, H-5, H-6), 4.03 (q,
1H-NMR (CDCl3, 500MHz) δ: 7.93 (d, 2H, o-
1H, Lac-α-CH, JLac-α-CH,Lac-β-CH=6.3Hz), 3.69 (dd, 1H, H-3, PhSO2, Jo,m=7.5Hz), 7.70 (t, 1H, p-PhSO2, Jp,m=7.5Hz),
J3,2=J3,4=10.3Hz), 3.52–3.41 (m, 2H, PhSO2CH2CH2), 2.09 (s, 7.66–7.58 (m, 3H, m-PhSO2, 2-NH), 7.18 (d, 1H, Ala-NH,
3H, OAc), 2.08 (s, 3H, OAc), 1.96 (s, 3H, NAc), 1.34 (d, 3H, JAla-NH,Ala-α-CH=8.1Hz), 6.10 (d, 1H, 2′-NH, J2′-NH,2′=8.6Hz),
Lac-β-CH, JLac-β-CH,Lac-α-CH=6.3Hz), 1.33 (d, 3H, Ala-β-CH, 5.98–5.87 (m, 3H, Hc, H-1), 5.37 (dd, 1H, Ha, JHa,Hc=17.2,
JAla-β-CH,Ala-α-CH=7.5Hz); 13C-NMR (CDCl3, 125MHz) δ:
JHa,1′=1.2Hz), 5.35 (dd, 1H, Ha, JHa,Hc=17.2, JHa,1′=1.2Hz),
172.3, 171.6, 170.9, 170.8, 169.3, 139.2, 134.3, 132.2, 132.2, 5.26 (d, 1H, Hb, JHb,Hc=10.3Hz), 5.24 (d, 1H, Hb,
132.0, 132.0, 129.6, 128.2, 119.4, 119.3, 78.4, 77.0, 70.3, 69.1, JHb,Hc=10.3Hz), 5.20 (dd, 1H, H-3′, J3′,4′=9.8, J3′,2′=10.9 Hz),
69.0, 69.0, 68.9, 61.8, 58.2, 55.0, 53.3, 53.3, 48.1, 25.1, 23.3, 5.11 (dd, 1H, H-4′,
J4′,3′=J4′,5′=10.9Hz), 4.64 (q, 1H,
20.9, 20.9, 18.9, 17.2; 31P-NMR (CDCl3, 202MHz) δ −1.8; Lac-α-CH, JLac-α-CH,Lac-β-CH=6.3Hz), 4.61–4.54 (m, 3H, H-1′,
ESI-MS-LR m/z 799.21 [(M+Na)+]; ESI-MS-HR Calcd for H-1″), 4.54–4.48 (m, 4H, PhSO2CH2CH2, H-1″), 4.39 (dq,
C32H45O16N2NaPS 799.2120. Found 799.2125; [α]D20 +49.99 (c 1H, Ala-α-CH, JAla-α-CH,Ala-NH=8.1, JAla-α-CH,Ala-β-CH=7.5Hz),
1.49, CHCl3).
4.35–4.27 (m, 2H, H-6, H-6′), 4.23 (dd, 1H, H-6, JH-6,H-6=12.6,
J6,5=3.4Hz), 4.09 (dd, 1H, H-6′, J6′,6′=12.6, J6′,5′=2.3Hz),
Glycosyl Phosphate 10
A mixture of 14 (212mg, 0.27mmol) in CH2Cl2 (3mL) was 4.02–3.85 (m, 4H, H-2, H-2′, H-4, H-5), 3.68–3.63 (m, 1H,
treated with DBU (44.8µL, 0.30mmol) at room temperature H-5′), 3.55 (dd, 1H, H-3, J3,4=JH-3,H-2=10.1Hz), 3.51 (t, 2H,
for 40min. The mixture was partitioned between AcOEt and PhSO2CH2CH2, JPhSO CH CH ,PhSO CH CH =5.7Hz), 2.11 (s, 3H,
2
2
2
2
2
2
1M aq. HCl, and the aqueous phase was extracted with AcOEt OAc), 2.05 (s, 3H, OAc), 2.03 (s, 3H, OAc), 2.00 (s, 3H,
(×2). Combined organic phase was dried (Na2SO4), filtered, NAc), 1.99 (s, 3H, NAc), 1.96 (s, 3H, NAc), 1.38 (d, 3H,
and concentrated in vacuo. The residue was purified by high- Lac-β-CH, JLac-β-CH,Lac-α-CH=6.9Hz), 1.35 (d, 3H, Ala-β-CH,
flash silica gel column chromatography (0–1–2% MeOH/ JAla-β-CH,Ala-α-CH=7.5Hz); 13C-NMR (CDCl3, 125MHz) δ: 174.8,
CH2Cl2) to afford 10 (149mg, 0.25mmol, 90%) as a colorless 171.8, 171.4, 171.3, 171.2, 171.0, 170.7, 169.5, 139.1, 134.3,
foam.
132.4, 129.6, 128.3, 118.8, 118.7, 99.8, 95.7, 95.7, 75.0, 74.0,
1H-NMR (CDCl3, 500MHz) δ: 7.47 (d, 1H, 2-NH, 72.2, 72.2, 71.2, 68.7, 68.6, 68.6, 68.6, 68.4, 62.2, 61.9, 58.4,

92
Chem. Pharm. Bull.
Vol. 66, No. 1 (2018)
55.1, 54.9, 53.9, 53.8, 48.1, 23.4, 23.1, 21.0, 20.8, 20.7, 18.9, 732.1072. Found 732.1060; [α]D20 +10.94 (c 0.60, DMSO-d6).
17.3; 31P-NMR (CDCl3, 162MHz) 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
Neryl Phosphoryl Imidazolide (30)
Neryl phosophate ammonium salt (2.9mg, 0.012mmol) was
co-evaporated with Et3N (20µL) in pyridine (1mL) twice. The
residue was co-evaporated with toluene (1mL) twice to afford
A mixture of 16 (127mg, 0.12mmol) in CH2Cl2 (1.5mL) neryl phosphate triethylamine salt. A mixture of the phosphate
was treated with DBU (19.4µL, 0.13mmol) at room tempera- salt in DMF was treated with 1,1′-carbonyldiimidazole (9.5mg,
ture for 40min. The mixture was partitioned between AcOEt 0.059mmol) at room temperature for 3h. The reaction was
and 1M aq. HCl, and the aqueous phase was extracted with quenched with MeOH (100µL) and stirred for 30min. The
AcOEt (×2). Combined organic phase was dried (Na2SO4), mixture was concentrated in vacuo and co-evaporated with
filtered, and concentrated in vacuo. The residue was purified toluene twice to afford 30. This compound was used without
by high-flash silica gel column chromatography (0–5–10% further purification.
MeOH/CH2Cl2) to afford 11 (100mg, 0.11mmol, 93%) as a 31P-NMR (DMSO-d6, 162MHz) δ: −9.6.
colorless foam.
Procedure for the Synthesis of 20
Each HMBA-PEG resin (150mg, 0.71mmol) was placed
1H-NMR (CDCl3, 500MHz) δ: 7.92 (d, 1H, 2-NH,
J2-NH,2=4.0Hz), 7.73 (d, 1H, Ala-NH, JAla-NH,Ala-α-CH=7.5Hz), in a 5mL polypropylene syringe fitted with a polyethylene
6.49 (d, 1H, 2′-NH, J2′-NH,2′=9.7Hz), 5.95–5.84 (m, 3H, Hc, filter disc. Each resin was agitated with CH2Cl2 (1.5mL, 1h),
H-1), 5.37 (d, 2H, Ha, JHa,Hc=17.2Hz), 5.25 (dd, 1H, Hb, After removal of CH2Cl2, a solution of Fmoc-D-Ala-OH·H2O
JHb,Hc=10.3, JHa,1′=1.2Hz), 5.24 (dd, 1H, Hb, JHb,Hc=10.3, (69mg, 0.21mmol) and N,N′-dissopropylcarbodiimide (33µL,
J
Hb,1′=1.2Hz), 5.12–5.05 (m, 2H, H-3′, H-4′), 4.71 (q, 0.21mmol) in DMF (1mL) was added at 0°C. Each mixture
1H, Lac-α-CH, JLac-α-CH,Lac-β-CH=6.3Hz), 4.59–4.52 (m, was agitated for 40min. 4-Dimethylaminopyridine (2.5mg,
3H, H-1′, H-1″), 4.52–4.45 (m, 2H, H-1″), 4.45 (dq, 1H, 0.021mmol) was added to the mixture at 0°C, which was
Ala-α-CH, JAla-α-CH,Ala-NH=JAla-α-CH,Ala-β-CH=7.5Hz), 4.31 (dd, warmed to room temperature. After agitation for 1h at room
1H, H-6, J6,6=12.6, J6,5=4.6Hz), 4.22 (d, 1H, H-6, J6,6=12.6, temperature, solvent and soluble reagents were removed by
J6,5=3.4Hz), 4.17–4.09 (m, 1H, H-2′), 4.08 (dd, 1H, H-6, suction. All resins were subjected to the following washing
J6,6=12.6, J6,5=2.3Hz), 3.97 (dd, 1H, H-4, J4,5=J4,3=9.7Hz), treatment with DMF (2mL×3), EtOH/CH2Cl2=1/1 (2mL×3),
3.92–3.86 (m, 1H, H-2), 3.77–3.71 (m, 1H, H-5), 3.66–3.58 (m, CH2Cl2 (2 mL×3) and DMF (2mL×3). The resins were treated
2H, H-5, H-3′), 2.09 (s, 3H, OAc), 2.04 (s, 3H, OAc), 2.02 (s, with Bz2O (48mg, 0.021mmol) in 20% pyridine/DMF (1mL)
3H, OAc), 2.01 (s, 3H, NAc), 2.00 (s, 3H, NAc), 1.96 (s, 3H, at room temperature for 1h, and the resins were washed
NAc), 1.48 (d, 3H, Ala-β-CH, JAla-β-CH,Ala-α-CH=7.5Hz), 1.35 (d, with DMF (2mL×3) and CH2Cl2 (2 mL×3) to afford 17. The
3H, Lac-β-CH, JLac-β-CH,Lac-α-CH=6.3Hz); 13C-NMR (CDCl3, amount of loading on the resin was determined as follow.
125MHz) δ: 175.7, 174.3, 171.8, 171.1, 171.1, 170.6, 169.5, Dried 17 (6.0mg) was agitated with DMF (2mL) for 30min,
132.1, 132.0, 132.0, 131.9, 119.1, 118.9, 99.3, 95.6, 95.5, 73.7, and DBU (40µL) was added to the mixture. The mixture was
73.0, 72.3, 72.0, 71.7, 69.1, 69.0, 68.9, 68.8, 68.5, 62.5, 61.9, agitated for 30min. The supernatant was diluted with DMF
54.7, 53.9, 53.9, 48.8, 23.3, 23.0, 20.9, 20.8, 20.7, 20.7, 18.7, and MeCN and subjected to UV measurement at 294nm.
16.8; 31P-NMR (CDCl3, 202MHz) δ: −3.2; ESI-MS-LR m/z The loading rate was determined to be 0.39mmol/g from the
918.29 [(M+Na)+]; ESI-MS-HR Calcd for C36H54O21N3NaP observed absorbance (0.172). The resins 17 were treated with
918.2880. Found 918.2885; [α]D20 +7.00 (c 0.21, CHCl3).
Diphosphate 25
A solution of 23 (16.5mg, 0.024mmol) and 24 (12.7mg, and CH2Cl2 (2 mL×3). A solution of Fmoc-D-Ala-OH·H2O
0.024mmol) in DMF (250µL) was treated with 29 (5.3mg, (94mg, 0.28mmol), HBTU (105mg, 0.28mmol) and Pr2NEt
piperidine/DMF (1:4, 5min, then 1:9, 15min) to remove the
Fmoc group, and the resins were washed with DMF (2mL×3)
i
0.024mmol) at 25°C for 24h. The mixture was concentrated (97 µL, 0.57mmol) in DMF (750µL) was added to the resins,
in vacuo. The residue was purified by high-flash ODS col- which were agitated for 2h. All the resins were washed with
umn chromatography (0–10% MeCN/25mM aq. AcOH·Et3N). DMF (2mL×3) and CH2Cl2 (2 mL×3) to afford 18. Kaiser
The product was further purified by reversed phase HPLC test indicated the completion of the all coupling reactions.
(YMC-Pack R&D ODS-A, 250×20mm, 8% MeCN/25mM aq. The loading rate was determined as described above. Namely,
AcOH·t3N) to afford 25 (6.9mg, 0.0074mmol, 31%) as a white dried 18 (4.9mg) was agitated with DMF (2mL) for 30min,
powder, after freeze drying.
DBU (40µL) was added to the mixture. The mixture was
1H-NMR (DMSO-d6, 500MHz) δ: 9.53 (d, 1H, 2″-NH, agitated for 30min. The supernatant was diluted with DMF
2″-NH,2″=9.2Hz), 7.93 (d, 1H, H-6, J6,5=6.3Hz), 5.75 (s, 1H, and MeCN and subjected to UV measurement at 294nm.
J
H-1′), 5.56 (d, 1H, H-5, J5,6=7.5Hz), 5.37–5.32 (m, 1H, H-1″), The yield was determined to be quantitative from the ob-
5.08 (dd, 1H, H-3, J3,2=9.2, J3,4=9.7Hz), 4.93 (dd, 1H, H-4, served absorbance (0.135). The resins 18 were treated with
J4,3=J4,5=9.5Hz), 4.19–3.91 (m, 9H, H-2′, H-3′, H-4′, H-5′, piperidine/DMF (1:4, 5min, then 1:9, 15min) to remove
H-2″, H-5″, H-6″), 3.11–3.07 (m, 2H, CH3CH2N), 2.01 (s, 3H, Fmoc group, and the resins were washed with DMF (2mL×3)
OAc), 1.94 (s 3H, OAc), 1.85 (s, 3H, OAc), 1.83 (s, 3H, NAc), and CH2Cl2 (2 mL×3). A solution of Alloc-L-Lys(Fmoc)-OH
i
1.17 (t, 3H CH3CH2N); 13C-NMR (DMSO-d6, 125MHz) δ: (96mg, 0.21mmol), HBTU (68mg, 0.21mmol) and Pr2NEt
170.1, 170.1, 169.6, 169.3, 163.2, 150.8, 140.8, 139.5, 101.7, 93.9, (72µL, 0.43mmol) in DMF (750µL) was added to the resins,
82.8, 71.6, 67.4, 67.7, 68.4, 67.7, 61.5, 50.9, 47.7, 45.5, 22.3, which were agitated for 2h. All the resins were washed with
20.5, 20.4, 19.0, 11.0, 8.6; 31P-NMR (DMSO-d6, 202MHz) δ: DMF (2mL×3) and CH2Cl2 (2 mL×3) to afford 19. Kaiser
−11.3 (d, JP, P=26.3Hz), −14.1 (d, JP, P=26.3Hz); ESI-MS-LR test indicated the completion of the all coupling reactions.
m/z 732.11 [(M–H)−]; ESI-MS-HR Calcd for C23H32O20N3P2 The amount of loading on the resin was determined as follow.

Vol. 66, No. 1 (2018)
Chem. Pharm. Bull.
93
Namely, dried 19 (5.3mg) was agitated with DMF (2mL) for (20mg, 0.11mmol) in DMF/CH2Cl2 (600 µL) was added to the
30min, DBU (40µL) was added to the mixture. The mix- resin which was agitated for 3h. The resin was washed with
ture was agitated for 30min. The supernatant was diluted DMF (2mL×3), CH2Cl2 (2 mL×3) and DMF/CH2Cl2=1/1
with DMF and MeCN and subjected to UV measurement at (2 mL×3). A solution of 11 (49mg, 0.055mmol), PyAOP
294nm. The yield was determined to be 87% over 2 steps (38mg, 0.073mmol), HOAt (5.0mg, 0.037mmol), and acridine
from the observed absorbance (0.107). The resins 19 were (20mg, 0.11mmol) in DMF/CH2Cl2 (600 µL) was added to the
treated with a solution of BH3·Me2NH (25mg, 0.43mmol) in resin which was agitated for 3h. The resin was washed with
EtOH (600µL) for 5min, then a solution of Pd(PPh3)4 (16mg, DMF (2mL×3), CH2Cl2 (2 mL×3). The resin was treated with
i
0.014mmol) in CH2Cl2 (1mL) was added to the mixture. The a solution of Ac2O (10µL, 0.11mmol) and Pr2NEt (19µL,
mixture was agitated for 15min. All the resins were washed 0.11mmol) in DMF (700µL) for 30min. The resin was washed
i
with 0.5% Pr2NEt/CH2Cl2 (2 mL×3), 0.5%(w/v) sodium di- with DMF (2mL×3), CH2Cl2 (2 mL×3). Kaiser test indicated
ethyldithiocarbamate/DMF (1mL×8), MeOH (2mL×3) and the completion of the coupling reaction. The resin was dried
CH2Cl2 (2 mL×3). A solution of Alloc-D-Glu-OBn (68mg, in vacuo to afford 8 (156mg, 0.028mmol, 0.18mmol/g, quanti-
i
0.21mmol), HBTU (68mg, 0.21mmol) and Pr2NEt (72µL, tative over 2 steps).
0.43mmol) in DMF (750µL) was added to the resins, which
Park Nucleotide (1)
were agitated for 2h. All the resins were washed with DMF
Resin-bound peptide 7 (9.4mg, 2.1µmol) was placed in a
(2 mL×3) and CH2Cl2 (2 mL×3). Kaiser test indicated the 5mL polypropylene syringe fitted with a polyethylene filter
completion of the all coupling reactions. The resin was dried disc. The resin was agitated with CH2Cl2 (1mL, 1h). After
in vacuo to afford 20 (194mg, 0.055mmol, 0.28mmol/g, 89% removal of CH2Cl2, the resin was treated with a solution of
over 2 steps). The yield was calculated by weighing resins.
Procedure for the Synthesis of 7
Pd(PPh3)4 (1.9mg, 1.6µmol) and PhSiH3 (8.4 µL, 69µmol)
in CH2Cl2 (400 µL) for 2h. The resin was washed with 0.5%
Resin-bound peptide 20 (150mg, 0.042mmol) was placed iPr2NEt/CH2Cl2 (2 mL×3), 0.5%(w/v) sodium diethyldithiocar-
in a 5mL polypropylene syringe fitted with a polyethylene bamate/DMF (1mL×8), MeOH (2mL×3), CH2Cl2 (2 mL×3)
filter disc. The resin was agitated with CH2Cl2 (1.5mL, 1h). and DMF (2mL×3). The resin was treated with a solution of
After removal of CH2Cl2, the resin was treated with a solu- 23 (7.6mg, 11µmol) and 29 (2.4mg, 11µmol) in DMF (300µL)
tion of BH3·Me2NH (13mg, 0.22mmol) in EtOH (600µL) at 50°C for 3d. The resin was washed with DMF (2mL×3),
for 5min, then a solution of Pd(PPh3)4 (8.4mg, 7.3µmol) in CH2Cl2 (2 mL×3) to afford 4. The resin was treated with pi-
CH2Cl2 (1mL) was added to the mixture. The mixture was peridine/DMF (1:4, 5min, then 1:9, 15min) to remove the
i
i
agitated for 15min. The resin was washed with 0.5% Pr2NEt/ Fmoc group, and the resin was washed with 0.5% Pr2NEt/
CH2Cl2 (2 mL×3), 0.5% (w/v) sodium diethyldithiocarba- CH2Cl2 (2 mL×3), DMF (2mL×3), CH2Cl2 (2 mL×3) and
mate/DMF (1mL×8), MeOH (2mL×3) and DMF/CH2Cl2=1/1 THF (2mL×3). The resin was treated with 2M aq. NaOH/
(2 mL×3). A solution of 10 (50mg, 0.082mmol), PyAOP MeOH/THF=1/1/2 (400µL) at 0°C, then warmed to room
(57mg, 0.11mmol), HOAt (7.5mg, 0.055mmol), and acridine temperature and agitated for 2h. The supernatant was neu-
(30mg, 0.16mmol) in DMF/CH2Cl2 (700µL) was added to tralized by 1M aq. HCl (220µL) and purified with reversed
the resin which was agitated for 3h. The resin was washed phase HPLC (YMC-Pack R&D ODS-A, 250×20mm, 1%
with DMF (2mL×3), CH2Cl2 (2 mL×3) and DMF/CH2Cl2=1/1 MeCN/50mM aq. NH4HCO3) to afford 1 (1.4mg, 1.2µmol,
(2 mL×3). A solution of 10 (50mg, 0.082mmol), PyAOP 44% over 12 steps) as a white powder, after freeze drying.
(57mg, 0.11mmol), HOAt (7.5mg, 0.055mmol), and acridine
1H-NMR (D2O, 500MHz) δ: 7.96 (d, 1H, H-6, J6,5=8.0Hz),
(30mg, 0.16mmol) in DMF/CH2Cl2 (700µL) was added to the 5.99 (d, 1H, H-1′, J1′,2′=5.2Hz), 5.97 (d, 1H, H-5, J5,6=8.0Hz),
resin which was agitated for 3h. The resin was washed with 5.47 (dd, 1H, H-1″, J1″,2″=2.9, J1″,P=7.5Hz), 4.39–4.30 (m, 3H,
DMF (2mL×3), CH2Cl2 (2 mL×3). The resin was treated with Ala-α-CH, H-2′, H-3′), 4.30–4.09 (m, 9H, H-4′, H-5′, H-2″,
i
a solution of Ac2O (16µL, 0.16mmol) and Pr2NEt (29µL, Ala-α-CH, Lac-α-CH, Lys-α-CH, D-Glu-α-CH), 3.98–3.94
0.16mmol) in DMF (800µL) for 30min. The resin was washed (m, 1H, H-5″), 3.88 (dd, 1H, H-6″, J6″,6″=12.6, J6″,5″=2.3Hz),
with DMF (2mL×3), CH2Cl2 (2 mL×3). Kaiser test indicated 3.84 (dd, 1H, H-6″, J6″,6″=12.6, J6″,5″=4.0Hz), 3.80 (dd, 1H,
the completion of the coupling reaction. The resin was dried H-3″, J3″,4″=J3″,2″=10.3Hz), 3.65 (dd, 1H, H-4″, J4″,3″=10.3,
in vacuo to afford 7 (189mg, 0.042mmol, 0.22mmol/g, quan-
titative over 2 steps). The yield was calculated by weighing 2.31 (t, 2H, D-Glu-γ-CH, JD
J
4″,5″=9.7Hz), 3.01 (t, 1H, Lys-ε-CH, JLys-ε-CH, Lys-δ-CH=7.5Hz),
-Glu-β-CH=8.0Hz), 2.20–2.12
D
-Glu-γ-CH,
resin.
Procedure for the Synthesis of 8
(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
Resin-bound peptide 20 (100mg, 0.028mmol) was placed (m, 2H, Lys-δ-CH), 1.52–1.41 (m, 2H, Lys-γ-CH),
in a 5mL polypropylene syringe fitted with a polyethylene 1.45 (d, 3H, Ala-β-CH, JAla-β-CH,Ala-α-CH=7.5Hz), 1.41
filter disc. The resin was agitated with CH2Cl2 (1.5mL, 1h). (d, 3H, Ala-β-CH, JAla-β-CH,Ala-α-CH=6.9Hz), 1.37 (d, 3H,
After removal of CH2Cl2, the resin was treated with a solu- Ala-β-CH, JAla-β-CH,Ala-α-CH=6.9Hz), 1.34 (d, 3H, Lac-β-CH,
tion of BH3·Me2NH (18mg, 0.33mmol) in EtOH (600µL) for JLac-β-CH,Lac-α-CH=6.9Hz); 31P-NMR (D2O, 162MHz) δ: −10.9
5min, then a solution of Pd(PPh3)4 (13mg, 0.011mmol) in (d, JP, P=21.7Hz), −12.6 (d, JP, P=21.7Hz); ESI-MS-LR m/z
CH2Cl2 (1mL) was added to the mixture. The mixture was 573.67 [(M−2H)2−]; ESI-MS-HR Calcd for C40H63O26N9P2
i
agitated for 15min. The resin was washed with 0.5% Pr2NEt/ 573.6685. Found 573.6694; [α]D20 +13.35 (c 0.08, MeOH).
CH2Cl2 (2 mL×3), 0.5%(w/v) sodium diethyldithiocarba-
The analytical data for synthetic 1 were in good agreement
mate/DMF (1mL×8), MeOH (2mL×3) and DMF/CH2Cl2=1/1 with the previously reported data.6)
(2 mL×3). A solution of 11 (49mg, 0.055mmol), PyAOP
(38mg, 0.073mmol), HOAt (5.0mg, 0.037mmol), and acridine
Neryl-lipid I (2)
Resin-bound peptide 7 (9.4mg, 2.1µmol) was placed in a

94
Chem. Pharm. Bull.
Vol. 66, No. 1 (2018)
5mL polypropylene syringe fitted with a polyethylene filter tralized by 1M aq. HCl (220µL) and purified with reversed
disc. The resin was agitated with CH2Cl2 (1mL, 1h). After phase HPLC (YMC-Pack R&D ODS-A, 250×20mm, 15%
removal of CH2Cl2, the resin was treated with a solution of MeCN/50mM aq. NH4HCO3) to afford 3 (0.8mg, 0.64µmol,
Pd(PPh3)4 (1.9mg, 1.6µmol) and PhSiH3 (8.4 µL, 69µmol) 21% over 12 steps) as a white powder after freeze drying.
in CH2Cl2 (400 µL) for 2h. The resin was washed with 0.5%
iPr2NEt/CH2Cl2 (2 mL×3), 0.5%(w/v) sodium diethyldithiocar- H-1, H-2″), 5.22–5.17 (m, 1H, H-6″), 4.62 (d, 1H, H-1′,
bamate/DMF (1mL×8), MeOH (2mL×3), CH2Cl2 (2 mL×3) 1′,2′=8.6Hz), 4.45 (dd, 2H, H-1″, J1″,2″=J1″,P=6.9Hz), 4.33
1H-NMR (D2O, 500MHz) δ: 5.47–5.41 (m, 2H,
J
and DMF (2mL×3). The resin was treated with a solution (q, 1H, Ala-α-CH, JAla-α-CH,Ala-β-CH=7.5Hz), 4.28–4.12 (m,
of 30 (11 µmol) and 29 (2.4mg, 11µmol) in DMF (300µL) 5H, Ala-α-CH, Lys-α-CH, D-Glu-α-CH, H-2), 4.11 (q,
at 50°C for 2d. The resin was washed with DMF (2mL×3), 1H, Lac-α-CH, JLac-α-CH,Lac-β-CH=7.5Hz), 3.97–3.88 (m,
CH2Cl2 (2 mL×3) to afford 5. The resin was treated with pi- 4H, H-4, H-6, H-6′), 3.81 (dd, 1H, H-3, J3,4=J3,2=9.7Hz),
peridine/DMF (1:4, 5min, then 1:9, 15min) to remove the 3.77–3.69 (m, 3H, H-5, H-2′, H-6′), 3.55 (dd, 1H, H-3′,
i
Fmoc group, and the resin was washed with 0.5% Pr2NEt/
J3′,4′=J3′,2′=8.6Hz), 3.44–3.38 (m, 2H, H-4′, H-5′), 3.00
CH2Cl2 (2 mL×3), DMF (2mL×3), CH2Cl2 (2 mL×3) and (t, 1H, Lys-ε-CH, JLys-ε-CH, Lys-δ-CH=7.5Hz), 2.31 (t, 2H,
THF (2mL×3). The resin was treated with 2M aq. LiOH/ D-Glu-γ-CH, JD -Glu-β-CH=8.6Hz), 2.20–2.10 (m, 5H,
D
-Glu-γ-CH,
MeOH/THF=1/1/2 (400µL) at 0°C, then warmed to room D-Glu-β-CH, H-4″, H-5″), 2.05 (s, 3H, NAc), 1.99 (s, 3H,
temperature and agitated for 2h. The supernatant was neu- NAc), 1.93–1.85 (m, 1H, D-Glu-β-CH), 1.85–1.75 (m, 2H,
tralized by 1M aq. HCl (210µL) and purified with reversed Lys-β-CH), 1.77 (s, 3H, 3″-Me), 1.74–1.65 (m, 2H, Lys-δ-CH),
phase HPLC (YMC-Pack R&D ODS-A, 250×20mm, 15% 1.69 (s, 3H, 7″-Me), 1.62 (s, 3H, 7″-Me), 1.51–1.41 (m, 2H,
MeCN/50mM aq. NH4HCO3) to afford 2 (0.6mg, 0.55µmol, Lys-γ-CH), 1.45 (d, 3H, Ala-β-CH, JAla-β-CH,Ala-α-CH=7.5Hz),
20% over 12 steps) as a white powder after freeze drying.
1.44 (d, 3H, Ala-β-CH, JAla-β-CH,Ala-α-CH=6.9Hz), 1.37 (d, 3H,
1H-NMR (D2O, 500MHz) δ: 5.47–5.42 (m, 2H, H-1, Ala-β-CH, JAla-β-CH,Ala-α-CH=7.5Hz), 1.33 (d, 3H, Lac-β-CH,
H-2′), 5.20 (t, 1H, H-6′, J6′,5′=6.9Hz), 4.49–4.43 (m, 2H, JLac-β-CH,Lac-α-CH=6.9Hz); 31P-NMR (D2O, 202MHz) δ: −10.2
H-1′), 4.33 (q, 1H, Ala-α-CH, JAla-α-CH,Ala-β-CH=6.9Hz), 4.26 (d, JP, P=21.4Hz), −12.7 (d, JP, P=21.4Hz); ESI-MS-LR m/z
(q, 1H, Ala-α-CH, JAla-α-CH,Ala-β-CH=7.5Hz), 4.23–4.18 (m, 630.24 [(M−2H)2−]; ESI-MS-HR Calcd for C49H82O26N8P2
2H, Ala-α-CH, Lys-α-CH), 4.17–4.08 (m, 3H, D-Glu-α-CH, 630.2413. Found 630.2425; [α]D20 +10.00 (c 0.08, MeOH).
Lac-α-CH, H-2), 3.98–3.93 (m, 1H, H-5), 3.91–3.86 (m,
The analytical data for synthetic 3 were in good agreement
1H, H-6), 3.84 (dd, 1H, H-6, J6,6=12.6, J6,5=4.0Hz), 3.80 with the previously reported data.14)
(dd, 1H, H-3, J3,4=J3,2=9.7Hz), 3.64 (dd, 1H, H-4, J4,3=9.7,
J4,5=10.3Hz), 3.00 (t, 1H, Lys-ε-CH, JLys-ε-CH, Lys-δ-CH=7.5Hz),
Acknowledgments The authors wish to thank Ms. S. Oka
2.31 (t, 2H, D-Glu-γ-CH, JD-Glu-γ-CH, -Glu-β-CH=8.0Hz), 2.20–2.10 (Center for Instrumental Analysis, Hokkaido University) for
D
(m, 5H, D-Glu-β-CH, H-4′, H-5′), 2.00 (s, 3H, NAc), 1.93–1.85 measurement of the mass spectra. This research was support-
(m, 1H, D-Glu-β-CH), 1.85–1.75 (m, 2H, Lys-β-CH), 1.77 ed by the JSPS Grant-in-Aid for Scientific Research (B) (SI,
(s, 3H, 3′-Me), 1.73–1.66 (m, 2H, Lys-δ-CH), 1.69 (s, 3H, Grant Number 25293026), The Ministry of Education, Cul-
7′-Me), 1.62 (s, 3H, 7′-Me), 1.51–1.42 (m, 2H, Lys-γ-CH), ture, Sports, Science and Technology through the Program for
1.45 (d, 3H, Ala-β-CH, JAla-β-CH,Ala-α-CH=6.9Hz), 1.41 Leading Graduate Schools (Hokkaido University “Ambitious
(d, 3H, Ala-β-CH, JAla-β-CH,Ala-α-CH=6.9Hz), 1.37 (d, 3H, Leader’s Program”), and the Platform Project for Supporting
Ala-β-CH, JAla-β-CH,Ala-α-CH=7.5Hz), 1.33 (d, 3H, Lac-β-CH, Drug Discovery and Life Science Research (Platform for Drug
JLac-β-CH,Lac-α-CH=7.5Hz); 31P-NMR (D2O, 202MHz) δ: −10.2 Discovery, Informatics and Structural Life Science).
(d, JP, P=21.4Hz), −12.7 (d, JP, P=21.4Hz); 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)
Conflict of Interest The authors declare no conflict of
interest.
Resin-bound peptide 8 (12.4mg, 2.2µmol) was placed in a
Supplementary Materials The online version of this ar-
5mL polypropylene syringe fitted with a polyethylene filter ticle contains supplementary materials.
disc. The resin was agitated with CH2Cl2 (1mL, 1h). After
removal of CH2Cl2, the resin was treated with a solution of
Pd(PPh3)4 (2.0mg, 1.7µmol) and PhSiH3 (8.9 µL, 73µmol)
in CH2Cl2 (300 µL) for 2h. The resin was washed with 0.5%
iPr2NEt/CH2Cl2 (2 mL×3), 0.5%(w/v) sodium diethyldithiocar-
bamate/DMF (1mL×8), MeOH (2mL×3), CH2Cl2 (2 mL×3)
and DMF (2mL×3). The resin was treated with a solution
of 30 (12 µmol) and 29 (2.6mg, 12µmol) in DMF (300µL)
at 50°C for 3d. The resin was washed with DMF (2mL×3),
CH2Cl2 (2 mL×3) to afford 6. The resin was treated with pi-
peridine/DMF (1:4, 5min, then 1:9, 15min) to remove the
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