Synthesis and biological activities of lipid A analogs possessing β-glycosidic linkage at 1-position

c. Jpn.

Bull. Chem. Soc. Jpn., 76, 485–500 (2003)

485

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Headline Articles

Synthesis and Biological Activities of Lipid A Analogs Possessing

03

5

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SJA8

09-2673

297

β

-Glycosidic Linkage at 1-Position

Synthesis of Lipid A Analogs

K. Fukase et al.

Koichi Fukase,* Atsushi Ueno, Yoshiyuki Fukase, Masato Oikawa, Yasuo Suda,

and Shoichi Kusumoto*

Department of Chemistry, Graduate School of Science, Osaka University,

1-1 Machikaneyama, Toyonaka, Osaka 560-0043

02

(Received October 7, 2002)

New lipid A analogs having acidic groups β-glycosidically linked at the 1-position were synthesized in order to in-

vestigate the structural requirement for immunostimulating and endotoxic activity of lipid A. The β-(phosphonoxy)ethyl

(PE) and carboxymethyl (CM) analogs of Escherichia coli type having six acyl groups and those of the biosynthetic pre-

cursor type having four acyl groups were synthesized via a divergent synthetic route. The E. coli type β-(phosphonoxy)-

ethyl analog, which was previously reported to be not endotoxic, showed strong immunostimulating activity comparable

to the natural-type α-analog. The acidic functional groups are concluded to be essential but their strict spatial arrange-

ment is not required for expression of the biological activity.

An innate immune system is a phylogenetically ancient de-

fense mechanism against the invasion of microorganisms in-

cluding bacteria.^1,2This system recognizes common bacterial

components such as bacterial cell wall peptidoglycan (PGN),

lipopolysaccharide (LPS) of gram-negative bacteria, lipopro-

teins, and bacterial DNA to induce many kinds of mediators

such as cytokines, prostagrandins, platelet activating factor,

and NO. These mediators stimulate the immune system and

also cause clinical manifestations of bacterial infections such

as fever, inﬂammation, hypotension, and, in severe cases, le-

thal shock. LPS, also termed endotoxin, is a cell surface am-

phiphile characteristic of Gram-negative bacteria and has been

known as the most potent immunostimulant originating from

microorganisms.^3–8LPS consists of a glycolipid component

termed lipid A covalently bound to a polysaccharide chain.

Previously, we clearly demonstrated that lipid A is the chemi-

cal entity of endotoxic activity of LPS by the total synthesis of

Escherichia coli lipid A 1 (Fig. 1, the synthetic specimen is

termed 506).^9,10Lipid A of various bacterial origins were

shown to be structurally closely related and to consist of: 1)

β(1→6) disaccharide of D-glucosamines, 2) phosphono groups

at the reducing end and the 4-position of the non-reducing sug-

ar, and 3) long-chain acyl groups bound at 2,2ꢀ,3, and 3ꢀ posi-

tions.

Fig. 1. Structures of lipid A and analogs.

486 Bull. Chem. Soc. Jpn., 76, No. 3 (2003)

HEADLINE ARTICLES

Fig. 2. Structures of lipid A analogs possessing β-glycosidic linkage at 1-position.

It has been shown that both phosphono groups at the 1- and

possessing β-glycosidically linked acidic groups at the 1-posi-

tion, i.e., PE-analogs (β-PE-506: 7 and β-PE-406: 8) and CM-

analogs (β-CM-506: 9 and β-CM-406: 10) having E. coli-type

and biosynthetic precursor-type acylation pattern, respectively

(Fig. 2).

4ꢀ-positions and acyl groups are crucial for the biological ac-

tivities of lipid A. The tetraacyl lipid A 2 (the synthetic speci-

men is called 406) which lacks the dodecanoyl and tetrade-

canoyl moieties of 1 has been identiﬁed as a biosynthetic pre-

cursor of LPS. The biosynthetic precursor 2 shows weaker but

deﬁnite endotoxic activity against mice. Quite interestingly,

however, 2 acts as an antagonist to LPS or lipid A 1 in human

systems.^3,4,10–13Novel precursor-type lipid A analogs which

possess four (R)-3-hydroxyacyl moieties of shorter chain

length (C10) were synthesized. Their biological tests clearly

showed the critical importance of the chain length of the acyl

moieties in lipid A to the antagonistic activity.^14,15

As for the role of phosphate groups, lipid A analogs lacking

1- or 4ꢀ-phosphate showed considerably weaker activity than

lipid A 1 whereas a lipid A analog lacking both phosphates did

not show any activity.^{11–13,16–19}Since 1-O-glycosyl phosphate

in lipid A is chemically unstable under acidic conditions,

chemically stable and biologically active analogs of lipid A

had been required for the investigation of the action mecha-

nism as well as for clinical use. In 1990, the 2-(phospho-

nooxy)ethyl (PE) analogs 3 and 4 were reported by Kusama et

al. as chemically stable lipid A analogs. Both the E. coli-type

(3: PE-506) and the biosynthetic precursor-type (4: PE-406)

analogs showed indistinguishable activity from the activities of

the corresponding natural-type compounds.²⁰Kusama et al.

also reported another analog in which the phosphoryl group at

the 1-position is replaced with a carboxymethyl (CM) group,²¹

but the CM-analog 5 with the same acylation pattern as lipid A

1 was not synthesized. We hence synthesized both the E. coli-

type and the precursor-type analogs 5 (CM-506) and 6 (CM-

406), which also showed indistinguishable activity from those

of the corresponding natural-type compounds.^15,22Kusama

also prepared PE-analogs with β-conﬁguration at the 1-posi-

tion, whereas all the above analogs have the same α-conﬁgura-

tion at 1-position as the natural lipid A.^20aQuite interestingly,

they reported that β-PE analogs showed considerably weaker

activity than natural lipid A.^13,20aWe anticipated that the mo-

lecular conformation of β-PE analogs might be different from

those of natural lipid A and the α-PE analogs and the differ-

ence might be reﬂected in the difference in the biological activ-

ity. We therefore planned to synthesize four lipid A analogs

Results

Synthetic Plan. In our previous studies, we elaborated

divergent routes for the efﬁcient synthesis of lipid A ana-

logs.^14,15,22Precursor-type analogs having shorter acyl chains

were synthesized via a disaccharide 4ꢀ-phosphate as a common

synthetic intermediate and each acyl moiety was introduced

step by step to the respective position.^14,15We also described

another route by which E. coli lipid A, PE-506, and CM-506

were prepared from the same allyl glycoside via deprotective

removal or oxidative cleavage of the double bond.^22,23In the

present study, we employed a new divergent synthetic route as

shown in Fig. 3. The β-allyl glycoside of a disaccharide 4ꢀ-

phosphate 13 was constructed as a common synthetic interme-

diate and then transformed to E. coli-type and precursor-type

intermediates 14, 15. The β-PE- and β-CM-analogs for both

types were prepared from the corresponding intermediates.

Synthesis of the Glycosyl Donor 11. The common glyc-

osyl donor 11 was synthesized according to the method we re-

cently reported (Scheme 1).^14,24The allyl group of compound

16 was isomerized to the 1-propenyl group using an iridium

complex ([Ir(cod)(MePh₂P)₂]PF₆, H₂)²⁵and then an allyloxy-

carbonyl (Alloc) group was introduced to the 3-position. The

product 17 was subjected to the reductive benzylidene-ring

opening by using Me₂NH•BH₃and Et₂O•BF₃in CH₃CN to

give the 6-O-benzylated 18 in good yield with high regioselec-

tivity.^14,22,26The free 4-hydroxy group was treated with

Watanabe’s reagent and 1-H-tetrazole, and then with m-chlo-

roperbenzoic acid (mCPBA) to furnish the phosphate 19 in

99% yield.²⁷The 1-propenyl group was smoothly deprotected

with iodine and water to give 20 in 94% yield. Compound 20

was then converted in the presence of Cs₂CO₃to the trichloro-

acetimidate 11 in good yield.²⁸

Synthesis of the Glycosyl Acceptor 12. The acceptor 12

was prepared from N-acetyl glucosamine as shown in Scheme

2. The fully acetylated glucosamine 21 was treated with tri-

methylsilyl triﬂuoromethanesulfonate (TMSOTf) to give the

K. Fukase et al.

Bull. Chem. Soc. Jpn., 76, No. 3 (2003) 487

Fig. 3. Synthetic strategy for β-analogs via divergent route.

rivative with free hydroxy groups is reactive, glycosylation

proceeded smoothly at room temperature to give the allyl gly-

coside 23, which was converted to the 4,6-O-benzylidene 24 in

73% yield from 22. The 3-hydoxy group of 24 was then 4-

methoxybenzylated by the trichloroacetimidate method. The

product 25 was subjected to the reductive benzylidene-ring

opening by using Me₂NH•BH₃and Et₂O•BF₃in CH₂Cl₂.^14,26

In this case, the 4-O-benzylated 26 was obtained with high re-

gioselectivity. The 3-O-(4-methoxybenzyl) (MPM) group was

partially cleaved during the reduction by the action of

Et₂O•BF₃. The remaining 3-O-MPM group in 26 was also

cleaved with Et₂O•BF₃to give 27 quantitatively. The N-acetyl

group of 27 was then removed by treatment with Ba(OH)₂to

give 28, which was acylated with (R)-3-(4-triﬂuoromethylben-

zyloxy)tetradecanoic acid by using 1-hydroxybenzotriazole

(HOBt), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide

hydrochloride (WSCD•HCl) in CHCl₃.³⁰Compound 29 was

thus obtained in 40% yield from 27. The N-acetyl group of 25

or 26 having 3-O-MPM group was found to be not cleavable at

all by treatment with various bases. The 6-hydroxy group of

29 was temporarily protected with t-butyldimethylsilyl (TBS)

group and the 3-hydroxy group of the resulting 30 was acylat-

ed with (R)-3-(4-triﬂuoromethylbenzyloxy)tetradecanoic acid

and DCC in the presence of 4-(dimethylamino)pyridine

(DMAP). Removal of the TBS group of the product 31 by us-

ing HF–pyridine gave the glycosyl acceptor 12.

Scheme 1. a) [Ir(cod)(MePh₂P)₂]PF₆, H₂, THF; b) AllocCl,

pyridine, CH₂Cl₂; c) Me₂NH•BH₃, Et₂O•BF₃, CH₃CN; d)

N,N-Diethyl-1,5-dihydro-3H-2,4,3-benzodioxaphosphep-

in-3-amine, 1H-tetrazole, CH₂Cl₂; e) mCPBA, −20 °C; f)

I₂, water, THF; g) CCl₃CN, Cs₂CO₃, CH₂Cl₂.

oxazoline 22.²⁹The O-acetyl groups of 22 was then removed

by treatment with NaOAllyl in allyl alcohol. The solution was

acidiﬁed with p-toluenesulfonic acid. Since the oxazoline de-

488 Bull. Chem. Soc. Jpn., 76, No. 3 (2003)

HEADLINE ARTICLES

Scheme 3. a) TMSOTf (0.1 equiv), MS4A, CH₂Cl₂, −20 °C.

WSCD•HCl afforded 32. The Alloc group of 32 was readily

removed with [Pd(PPh₃)₄], HCOOH, and butylamine. The 3ꢀ-

O-acylation was then effected with (R)-3-(tetradecanoyloxy)-

tetradecanoic acid by using DCC and DMAP in CHCl₃. The

desired compound 14 was obtained in 56% yield. In this reac-

tion, undesired 33 was formed by β-elimination of the acyloxy

group in tetradecanoyloxytetradecanoic acid moiety. Dihy-

droxylation of the CwC double bond of 14 using catalytic

amounts of osmium(Ⅷ) oxide (OsO₄) and 4-methylmorpho-

line N-Oxide (NMO) in THF–t-BuOH–H₂O (10:10:1) fol-

lowed by lead(Ⅳ) acetate (Pb(OAc)₄) oxidation afforded the

aldehyde 34.

Reduction of the aldehyde 34 with sodium tetrahydroborate

(NaBH₄) gave the alcohol 35. Phosphorylation of 35 gave the

bisphosphate 36 in 99% yield (Scheme 5). The ﬁnal deprotec-

tion was accomplished by hydrogenolysis (13 kg cm⁻²of H₂)

with Pd-black in one-step. Puriﬁcation of the crude product

was effected by liquid–liquid partition column chromatogra-

phy using Sephadex^®LH-20 to give the desired β-PE-506 (7)

in 74% yield, whose structure was conﬁrmed by MS and NMR

spectra.

For the synthesis of β-CM-506 (9), the aldehyde 34 was

oxidized with sodium chlorite (NaClO₂) (Scheme 6). Succes-

sive deprotection of 37 under hydrogenolytic conditions fol-

lowed by puriﬁcation by liquid–liquid partition column chro-

matography afforded the desired β-CM-506 (9) in 69% yield.

The structure of 9 was conﬁrmed by MS and NMR spectra.

Scheme 2. a) TMSOTf, (CH₂Cl)₂, 50 °C; b) 0.1 M Allyl-

ONa, Allyl-OH; c) p-TsOH•H₂O, Allyl-OH; d) PhCH-

(OCH₃)₂, p-TsOH•H₂O, CH₃CN; e) 4-Methoxyphenyl-

methyl trichloroacetimidate, Sn(OTf)₂, THF; f) Me₂NH•

BH₃, Et₂O•BF₃, CH₂Cl₂; g) Et₂O•BF₃, CH₂Cl₂; h) Ba-

(OH)₂•(8H₂O), MeOH, 70 °C; i) (R)-3-(4-triﬂuoromethyl-

benzyloxy)tetradecanoic acid, HOBt, WSCD•HCl, CHCl₃;

j) t-butyldimethylsilyl chloride, imidazole, CH₂Cl₂; k) (R)-

3-(4-triﬂuoromethylbenzyloxy)tetradecanoic acid, DCC,

DMAP, CH₂Cl₂; l) HF/Pyridine.

Synthesis of the Common Allyl Glycoside Intermediate

13. In our previous studies, glycosylation of glucosamine α-

allyl glycosides with 3- and 6-hydroxy groups selectively gave

6-O-glycosylated product in good yields.^14,22,24By contrast, a

preliminary experiment showed that the glycosylation of the

corresponding β-allyl glycoside 29 gave a mixture of the de-

sired (1→6) disaccharide and the undesired trisaccharide

formed by over-glycosylation at the 3-position. Compound 12

whose 3-position was already acylated was therefore used for

the present synthesis. Coupling of the trichloroacetimidate 11

with the glycosyl acceptor 12 was carried out in CH₂Cl₂by us-

ing TMSOTf as a catalyst to give the desired disaccharide 13

in a good yield (Scheme 3).

β

Synthesis of -PE-406 (8) and -CM-406 (10) via the

Common Aldehyde Intermediate 39. The tetraacyl 406-

type analogs 8 and 10 were synthesized via the common alde-

hyde intermediate 39 in manners similar to the synthesis of the

corresponding hexaacyl analogs. Removal of the 2ꢀ-N-Troc

group of 13 followed by N-acylation with (R)-3-(4-triﬂuoro-

methylbenzyloxy)tetradecanoic acid gave 38. The Alloc group

of 38 was removed and the 3ꢀ-O-acylation with (R)-3-(4-triﬂu-

oromethylbenzyloxy)tetradecanoic acid gave the desired com-

pound 15 in 86% yield. The aldehyde 39 was obtained by di-

hydroxylation of 15 and subsequent Pb(OAc)₄oxidation in

85% yield (Scheme 7).

β

Synthesis of -PE-506 (7) and -CM-506 (9) via the

Common Aldehyde Intermediate 34. The common alde-

hyde intermediate 34 for the synthesis of hexaacyl 506-type

analogs 7 and 9 were prepared from 13, as shown in Scheme 4.

The 2ꢀ-N-Troc group of 13 was removed with Zn–Cu in acetic

acid followed by N-acylation with (R)-3-(dodecanoyloxy)tet-

radecanoic acid, 1-hydroxy-7-azabenzotriazole (HOAt), and

Reduction of the aldehyde 39 followed by phosphorylation

gave the protected β-PE-406 (41). The ﬁnal deprotection was

carried out in THF by hydrogenolysis (13 kg cm⁻²of H₂) with

Pd-black. As the hydrogenation proceeded, parts of the prod-

ucts precipitated and the reaction hence was not completed.

The precipitates contained the desired product accompanied

K. Fukase et al.

Bull. Chem. Soc. Jpn., 76, No. 3 (2003) 489

Scheme 4. a) Zn-Cu, AcOH; b) (R)-3-(dodecanoyloxy)tetradecanoic acid, HOBt, WSCD•HCl, CHCl₃; c) [Pd(PPh₃)₄], BuNH₂,

HCOOH, THF; d) (R)-3-(tetradecanoyloxy)tetradecanoic acid, DCC, DMAP, CHCl₃; e) OsO₄, NMO, THF/2-methyl-2-propanol/

H₂O (10:10:1); f) Pb(OAc)₄, benzene.

Scheme 6. a) NaClO₂, NaH₂PO₄, 2-methyl-2-butene, 2-

methyl-2-propanol/water (4:1); b) H₂(13 kg cm⁻²), Pd-

black, THF.

D₂O.^15,32However, β-PE-406 (8) gave analyzable spectra in

neither DMSO-d₆nor SDS-d₂₅/D₂O owing to signal broaden-

ing. These results indicate that compound 8 tends to aggregate

strongly in both organic and aqueous solvents. After several

solvent systems were examined, we found that an NMR spec-

trum of 8 in SDS-d₂₅/DMSO-d₆afforded clear signals. The

structure of 8 was thus conﬁrmed by NMR and MS spectra.

For the synthesis of β-CM-406 (10), the aldehyde 39 was

oxidized by the treatment with sodium chlorite (NaClO₂)

(Scheme 9). Successive deprotection under hydrogenolytic

conditions, followed by puriﬁcation by liquid–liquid partition

column chromatography, afforded the desired β-CM-406 (10)

in 54% yield. The structure of 10 was conﬁrmed by MS and

NMR spectra. ¹H NMR of β-CM-406 (10) gave well-resolved

spectra in CHCl₃–MeOH (1:1).

Scheme 5. a) NaBH₄; b) N,N-Diethyl-1,5-dihydro-3H-2,4,3-

benzodioxaphosphepin-3-amine, 1H-tetrazole, CH₂Cl₂;

c) mCPBA, −20 °C; d) H₂(13 kg cm⁻²), Pd-black, THF.

with substantial amounts of incompletely deprotected prod-

ucts. The latter still retained 4-triﬂuoromethylbenzyl groups,

whose complete removal was not successful. Puriﬁcation of

the crude product was effected by liquid-liquid partition col-

umn chromatography using Sephadex^®LH-20 to give the de-

sired β-PE-406 (8) in 51% yield (Scheme 8).³¹The solubility

of puriﬁed 8 was considerably lower (insoluble in CHCl₃–

MeOH (1:1) and water) than the solubilities of the crude prod-

uct and PE-406 (4). We recently found that the biosynthetic

precursor 406 (2), PE-406 (4), and CM-406 (6) gave well-

resolved ¹H NMR spectra in DMSO-d₆and in SDS-d₂₅/

Biological Activity. Biological activities of β-type ana-

logs (7, 8, 9, 10) were evaluated in comparison to the corre-

490 Bull. Chem. Soc. Jpn., 76, No. 3 (2003)

HEADLINE ARTICLES

Scheme 7. a) Zn-Cu, AcOH; b) (R)-3-(4-triﬂuoromethylbenzyloxy)tetradecanoic acid, HOAt, WSCD•HCl, CHCl₃; c) [Pd(PPh₃)₄],

BuNH₂, HCOOH, THF; d) (R)-3-(4-triﬂuoromethylbenzyloxy)tetradecanoic acid, DCC, DMAP, CHCl₃; e) OsO₄, NMO, THF/2-

methyl-2-propanol/H₂O (10:10:1); f) Pb(OAc)₄, benzene.

sponding α-type analogs (3, 4, 5, 6) and LPS (E. coli 0111:B4)

by measuring typical endotoxic activity such as Limulus activi-

ty and cytokine induction. Cytokine inducing activity of the

hexaacyl 506-type analogs and LPS was tested in human pe-

ripheral whole-blood cells.³³A mixture of a test sample and

heparinized human peripheral whole-blood collected from an

adult volunteer in RPMI 1640 medium (Flow Laboratories,

Irvine, Scotland) was incubated at 37 °C in 5% CO₂for 24 h.

The levels of cytokines, i.e., interleukin-6 (IL-6) and tumor

necrosis factor-α (TNF-α), in supernatant of incubation mix-

tures were measured by means of enzyme-linked immunosor-

bent assay (ELISA). Antagonistic activity of the tetraacyl 406-

type analogs was examined by inhibition to cytokine induction

by LPS as follows. A mixture of a test sample, LPS (2.5 ng

mL⁻¹) (E. coli 0111:B4; Sigma Chemicals Co.), and heparin-

ized human peripheral whole-blood was incubated and the lev-

els of cytokines were measured in the same manner as de-

scribed above. Since β-PE-406 (8) and β-CM-406 (10) were

hardly soluble in water, all samples were once dissolved in

DMSO and then sequentially diluted with water or saline.

Several 96-well plates were required for the measurement of

Scheme 8. a) NaBH₄; b) N,N-Diethyl-1,5-dihydro-3H-2,4,3-

the dose dependency, so that 2.5 ng mL⁻¹of LPS was used as a

benzodioxaphosphepin-3-amine, 1H-tetrazole, CH₂Cl₂;

c) mCPBA, −20 °C; d) H₂(13 kg cm⁻²), Pd-black, THF.

standard for each plate.

As can be clearly seen in Figs. 4 and 5, both β-PE and β-CM

506-type analogs showed cytokine inducing activity similar to

the corresponding α-analogs. On the other hand, β-CM-406

(10) showed deﬁnite but weaker antagonistic activity than the

α-counterparts. β-PE-406 (8) also showed antagonistic activi-

ty, but the potency was much weaker than α-PE-406 and α-

CM-406.

Limulus activity of the β-type analogs (7, 8, 9, 10) and LPS

was measured by the activation of factor C at various concen-

trations by means of Endospecy Test (Seikagaku Corporation,

Tokyo).³³The results are summarized in Table 1. All the sam-

ples were found to exhibit strong positive activity.

Discussions

We synthesized four lipid A analogs possessing β-glycosid-

ic linkage at the 1-position, i.e., PE-analogs, β-PE-506 (7) and

β-PE-406 (8), and CM-analogs, β-CM-506 (9) and β-CM-406

(10). The cytokine-inducing assay clearly demonstrated that

the hexaacyl β-analogs 7 and 9 of E. coli lipid A-type have

Scheme 9. a) NaClO₂, NaH₂PO₄, 2-methyl-2-butene, 2-

methyl-2-propanol/water (4:1); b) H₂(13 kg cm⁻²), Pd-

black, THF.

K. Fukase et al.

Bull. Chem. Soc. Jpn., 76, No. 3 (2003) 491

Fig. 4. IL-6 induction by 3, 5, 7, 9, and LPS and inhibitory effect of 4, 6, 8, 10 to IL-6 induction by LPS. The blood donor wasY. S.

Fig. 5. TNFα induction by 3, 5, 7, 9, and LPS and inhibitory effect of 4, 6, 8, 10 to TNFα induction by LPS. The blood donor was

Y. S.

492 Bull. Chem. Soc. Jpn., 76, No. 3 (2003)

HEADLINE ARTICLES

Table 1. Limulus Activity of 1, 3, 5, 7, 8, 9, 10, and LPS (E.

coli 0111:B4) as Tested by Endospecy Test^®(Seikagaku

Corporation, Tokyo).

shows much weaker inhibitory activity.

The Limulus activity of the β-analogs was somehow weaker

than that of the corresponding α-analogs. However, no signiﬁ-

cant difference was observed among the Limulus activities of

the hexa- and tetraacylated β-analogs. This fact seems to sug-

gest that activation of the Limulus enzyme, factor C, is not in-

ﬂuenced by the stage of aggregation of lipid A.

In the present study, we clearly demonstrated that the β-con-

ﬁgurated CM-and PE-analogs having E. coli acylation pattern

show comparable biological activity to the corresponding α-

analogs. This result indicates that the spatial arrangement of

acidic functional groups is not strictly required for the activity.

ED50/pg mL⁻¹

E. coli lipid A (1)

PE-506 (3)

CM-506 (5)

6*

2*

1*

β-PE-506 (7)

30

β-PE-406 (8)

30

20

10

3

β-CM-506 (9)

β-CM-406 (10)

LPS (E. coli 0111:B4)

*Data from previous study.²²

Experimental

All melting points are uncorrected. ¹H NMR was measured

with JEOL JNM-LA 600 or JEOL JNM-LA 500 spectrometers for

CDCl₃solutions unless otherwise noted. The chemical shifts are

given in δ values from tetramethylsilane (TMS) as an internal

standard. Mass spectra were obtained on a JEOL JMS-SX 102

mass spectrometer and a Mariner^TMBiospectrometry Workstation

(Applied Biosystems) mass spectrometer. Speciﬁc rotations were

measured on a Perkin-Elmer 241 polarimeter. Silica-gel column

chromatography was carried out using Kieselgel 60 (E. Merck,

0.040–0.063 mm) at medium-pressure (2–4 kg cm⁻²). Organic

solutions were washed with saturated aqueous NaHCO₃and brine,

dried over Na₂SO₄, and evaporated in vacuo unless otherwise

noted. An LPS specimen from E. coli 0111:B4 (Sigma Chemicals

Co) was used as a positive control for cytokine inducing assay and

Limulus assay.

strong immunostimulating activity comparable to the corre-

sponding α-type analogs and E. coli lipid A itself. Kusama et

al. reported that β-PE-506 (7) showed considerably weaker

activity than natural lipid A.^13,20The discrepancy might be ow-

ing to the solubility and aggregation problem rather than the

difference in the assay systems.

LPS, lipid A, and lipid A analogs form aggregates in aque-

ous solution. Lipopolysaccharide binding protein (LBP) in

serum is assumed to work as a shuttle that transfers LPS mono-

mer from LPS aggregates to CD14 on the cell surface mem-

brane of immunocompetent cells.^34,35

Takayama et al. claimed that a monomeric LPS artiﬁcially

prepared by sonication showed stronger activity than LPS

aggregates,³⁶which may suggest that monomeric LPS is more

readily transferred to immunocompetent cells than aggregates.

LPS is then recognized by the receptor complex consisting

in Toll like receptor 4 (TLR4), MD-2, CD14, and others.^3–5

CD14 and TLR4 are now considered as a capture receptor and

a signaling receptor of LPS, respectively. MD-2, which is es-

sential for LPS signaling, is an association protein to TLR4.

Several other proteins involved in the signaling of LPS were

also reported. Transfer of lipid A (or LPS) monomer from the

aggregates to immunocompetent cells seems to be one of the

critical steps for expression of endotoxic activity. It is, howev-

er, still unclear whether the receptor complex recognizes the

monomeric lipid A (or LPS) or the aggregated supramolecular

structure reconstructed on the cell membrane.

We thought β-analogs might form more rigid aggregates and

hence be more difﬁcult to be transferred than α-analogs.

Kusama et al. used usual aqueous solution for in vivo assays

and hence β-PE-506 (7) gave weaker response than α-PE-506

(3). In the present study, all the samples were once dissolved

in DMSO and then diluted with water or saline for the biologi-

cal assays. Under such conditions, aggregation property be-

tween α and β analogs might be similar. The biological activi-

ties of α and β analogs were hence not different in our in vitro

systems.

1-Propenyl 3-O-Allyloxycarbonyl-4,6-O-benzylidene-2-deoxy-

α

2-(2,2,2-trichloroethoxycarbonylamino)- -D-glucopyranoside

(17). To a degassed solution of allyl 4,6-O-benzylidene-2-

deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyrano-

side (16) (38.0 g, 78.7 mmol) in anhydrous THF (400 mL) was

added (1,5-cyclooctadiene)[bis(methyldiphenylphosphine)]irid-

ium(Ⅰ) hexaﬂuorophosphate (1 g, 1.18 mmol). After activation of

the iridium catalyst with hydrogen three times (each 30 s), the

mixture was stirred under nitrogen atmosphere at room tempera-

ture for 1 h and then the solution was concentrated in vacuo. To

the solution of the residue in CH₂Cl₂(100 mL) and pyridine (300

mL) was added allyl chloroformate (41.8 mL, 394 mmol) drop-

wise at 0 °C. The reaction mixture was stirred at room tempera-

ture for 5 h and concentrated in vacuo. EtOAc and water were

added to the residue. The aqueous layer was extracted with

EtOAc. The organic layer was worked up as usual. The crude

product was puriﬁed by silica-gel column chromatography (500 g,

toluene/EtOAc = 30:1) to give 17 as a white solid (37.1 g, 85%).

Mp 96.0–98.0 °C. [α]_D²⁴+65.9 (c 1.00, CHCl₃). Found: C, 48.79;

H, 4.53; N, 2.49%. Calcd for C₂₃H₂₆Cl₃NO₉: C, 48.74; H, 4.62; N,

2.47%. ¹H NMR (600 MHz, CDCl₃) δ 7.45–7.35 (m, 5 H, Ph–

CH), 6.15–6.07 (m, 1 H, OCHwCHCH₃), 5.92–5.84 (m, 1 H,

OCH₂CHwCH₂), 5.54 (d, J = 6.9 Hz, 1 H, Ph–CH), 5.40 (brd, 1

H, NH), 5.32 (dd, J = 17.3, 6.3 Hz, 2 H, OCH₂CHwCH₂), 5.27–

5.19 (m, 2 H, H-3 and OCHwCHCH₃), 5.11 (d, J = 3.4 Hz, 1 H,

H-1), 4.82 (d, J = 12.2 Hz, 1 H, CH₂of Troc), 4.71–4.60 (m, 3 H,

CH₂of Troc and OCH₂CHwCH₂), 4.29 (dd, J = 10.3, 4.4 Hz, 1 H,

H-6a), 4.17–4.13 (m, 1 H, H-2), 3.92 (ddd, J = 9.7, 9.7, 4.7 Hz, 1

H, H-6b), 3.80–3.75 (m, 2 H, H-4 and H-5), 1.68–1.56 (m, 3 H,

OCHwCHCH₃).

The biological tests of biosynthetic precursor-type analogs

support this assumption. As described, the solubility of β-PE-

406 (8) was very low in both aqueous and organic solvents. It

forms aggregates even in DMSO as indicated by NMR, where-

as other lipid A analogs do not aggregate in DMSO. Because

of its strong tendency to aggregation, β-PE-406 (8) is probably

difﬁcult to be transferred to the receptor complex and hence

1-Propenyl 3-O-Allyloxycarbonyl-6-O-benzyl-2-deoxy-2-

(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranoside

K. Fukase et al.

Bull. Chem. Soc. Jpn., 76, No. 3 (2003) 493

(18). To a solution of 17 (10.0 g, 17.6 mmol) in anhydrous

CH₃CN (90 mL) were added Me₂NH•BH₃(5.30 g, 88.2 mmol)

and Et₂O•BF₃(11.5 mL, 88.2 mmol) at 0 °C, successively. After

being stirred at 0 °C for 2 h, the solution was quenched with satu-

rated aqueous NaHCO₃, extracted with EtOAc, and worked up as

usual. The residue was puriﬁed by silica-gel column chromatog-

raphy (500 g, toluene/AcOEt/1,1,1,3,3,3-hexaﬂuoro–2-propanol

of Troc and OCH₂CHwCH₂), 3.83 (dd, J = 8.9, 1.9 Hz, 1 H, H-

6a), 3.75 (dd, J = 10.8, 5.7 Hz, 1 H, H-6b), 3.40 (s, 1 H, C₁–OH).

3-O-Allyloxycarbonyl-6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-

3-oxo-3 ⁵-3H-2,4,3-benzodioxaphosphepin-3-yl)-2-(2,2,2-tri-

λ

α

chloroethoxycarbonylamino)- -D-glucopyranosyl Trichloro-

acetimidate (11). To a solution of 20 (5.06 g, 7.12 mmol) in an-

hydrous CH₂Cl₂(10 mL) at room temperature were added mole-

cular sieves 4A, trichloroacetonitrile (7.1 mL, 71 mmol) and

Cs₂CO₃(1.16 g, 7.12 mmol). After being stirred for 2 h, saturated

aqueous NaHCO₃was added to the reaction mixture. The mixture

was extracted with EtOAc and the extract was worked up as usual

to give 11 as a pale yellow solid, which was used for the subse-

quent glycosylation without further puriﬁcation.

= 30:1:0.6) to give 18 as colorless oil (6.80 g, 68%). [α]_D²⁴

=

+58.7 (c 1.01, CHCl₃). Found: C, 48.28; H, 4.83; N, 2.47%.

1

Calcd for C₂₃H₂₈Cl₃NO₉: C, 48.56; H, 4.96; N, 2.46%. H NMR

(600 MHz, CDCl₃) δ 7.45–7.35 (m, 5 H, Ph-CH₂), 6.16–6.05 (m,

1 H, OCHwCHCH₃), 5.94–5.88 (m, 1 H, OCH₂CHwCH₂), 5.37–

5.26 (m, 3 H, NH and OCH₂CHwCH₂), 5.21-5.17 (m, 1 H,

OCHwCHCH₃), 5.09 (d, J = 10.0 Hz, 1 H, H-3), 4.94 (d, J = 3.4

Hz, 1H, H-1), 4.84 (d, J = 12.0 Hz, 1 H, CH₂of Troc), 4.64–4.53

(m, 5 H, CH₂of Troc, Ph–CH₂, and OCH₂CHwCH₂), 4.07–4.06

(m, 1 H, H-2), 3.91–3.79 (m, 3 H, H-4, H-5 and H-6a), 3.70–3.68

(m, 1 H, H-6b), 1.68–1.56 (m, 3 H, OCHwCHCH₃).

4ꢀ,5ꢀ-Dihydro-2ꢀ-methyloxazolo[5ꢀ,4ꢀ:1,2]-3,4,6-tri-O-acetyl-

α

1,2-dideoxy- -D-glucopyranose (22). To a solution of 1,3,4,6-

tetra-O-acetyl-2-deoxy-2-acetylamino-D-glucopyranose (21) in

ClCH₂CH₂Cl (20 mL) was added TMSOTf (1.39 mL, 7.71 mmol)

and the solution was stirred at 50 °C for 20 h. After the solution

was cooled to room temperature, Et₃N, EtOAc, and saturated

aqueous NaHCO₃were added. The aqueous layer was extracted

by EtOAc and the organic layer was worked up as usual. The resi-

due was puriﬁed by silica-gel column chromatography (100 g, tol-

uene/EtOAc/Et₃N = 100:200:1) to give 22 as pale yellow oil

(1.60 g, 95%).

1-Propenyl 3-O-Allyloxycarbonyl-6-O-benzyl-2-deoxy-4-O-

λ⁵

(1,5-dihydro-3-oxo-3 -3H-2,4,3-benzodioxaphosphepin-3-yl)-

α

2-(2,2,2-trichloroethoxycarbonylamino)- -D-glucopyranoside

(19). To a solution of 18 (12.0 g, 21.1 mmol) in anhydrous 1,2-

dichloroethane (40 mL) were added N,N-diethyl-1,5-dihydro-3H-

2,4,3-benzodioxaphosphepin-3-amine (5.05 g, 21.1 mmol) and

1H-tetrazole (3.80 g, 52.7 mmol). After the mixture was stirred at

room temperature for 30 min and then at −20 °C for 10 min,

mCPBA (3.77 g, 21.8 mmol) was added and stirring was contin-

ued for another 30 min. The solution was quenched with saturated

aqueous NaHCO₃, extracted with EtOAc, and worked up as usual.

The residue was puriﬁed by silica-gel column chromatography

(500 g, CHCl₃/acetone = 30:1) to give 19 as colorless crystals

(15.4 g, 99%). Mp 70.0–78.0 °C. [α]_D²⁴+56.7 (c 1.03, CHCl₃).

Found: C, 48.24; H, 4.58; N, 1.99%. Calcd for C₃₁H₃₅Cl₃NO₁₁P•

1.1H₂O: C, 48.31; H, 4.86; N, 1.82%. ¹H NMR (600 MHz, CD-

Cl₃) δ 7.37–7.19 (m, 9 H, Ph-CH₂and o-C₆H₄(CH₂O)₂P), 6.18–

6.10 (m, 1 H, OCHwCHCH₃), 5.90–5.87 (m, 1 H, OCH₂CHw

CH₂), 5.37-5.05 (m, 8 H, H-1, H-3, NH, OCHwCHCH₃and o-

C₆H₄(CH₂O)₂P), 4.85–4.76 (m, 3 H, CH₂of Troc, OCH₂CHwCH₂

and H-4), 4.69–4.56 (m, 2 H, CH₂of Troc, and OCH₂CHwCH₂),

4.13–4.11 (m, 1 H, H-2), 3.98 (m, 1 H, H-5), 3.79 (m, 2 H, H-

6ab), 1.91–1.55 (m, 3 H, OCHwCHCH₃).

β

Allyl 2-Acetylamino-4,6-O-benzylidene-2-deoxy- -D-glu-

copyranoside (24). A solution of 22 (0.18 g, 0.547 mmol) in 0.1

M Allyl-ONa in Allyl-OH (1.8 mL) was stirred at room tempera-

ture for 1 h. After addition of p-TsOH•H₂O (400 mg, 2.3 mmol),

the solution was stirred at room temperature for 15 min. The

crude allyl glycoside 23 was obtained by concentration. To a solu-

tion of 23 in CH₃CN (2 mL) was added benzaldehyde dimethyl

acetal (0.164 mL, 1.09 mmol) and the solution was stirred at room

temperature for 30 min. EtOAc and saturated aqueous NaHCO₃

were added. The aqueous layer was extracted with EtOAc and the

organic layer was worked up as usual. The residue was puriﬁed

by silica-gel column chromatography (10 g, CHCl₃/MeOH =

20:1) to give 24 as a white solid (0.140 g, 73%). [α]_D²⁴−76.9 (c

1.00, MeOH). ESI-MS (positive) m/z 350.2 [(M + H)⁺]. ¹H

NMR (600 MHz, CDCl₃) δ 7.50 (dd, J = 7.7, 2.2 Hz, 2 H, PhCH),

7.40–7.33 (m, 3 H, PhCH), 5.89 (m, 1 H, OCH₂CHwCH₂), 5.77

(d, J = 7.4 Hz, 1 H, NH), 5.58 (s, 1 H, PhCH), 5.32 (dd, J = 17.2,

1.4 Hz, 1 H, OCH₂CHwCH₂), 5.25 (d, J = 10.3 Hz, 1 H,

OCH₂CHwCH₂), 4.77 (d, J = 8.4 Hz, 1 H, H-1), 4.38–4.33 (m, 2H

OCH₂CHwCH₂and H-6a), 4.17 (dd, J = 9.1, 9.1 Hz, 1 H, H-3),

4.10 (dd, J = 12.8, 6.2 Hz, 1 H, OCH₂CHwCH₂), 3.80 (dd, J =

10.3, 10.3 Hz, 1 H, H-6b), 3.58 (dd, J = 9.1, 9.1 Hz, 1 H, H-4),

3.53–3.47 (m, 2H, H-2 and H-5), 2.05 (s, 3 H, CH₃).

3-O-Allyloxycarbonyl-6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-

3-oxo-3 ⁵-3H-2,4,3-benzodioxaphosphepin-3-yl)-2-(2,2,2-tri-

λ

α

chloroethoxycarbonylamino)- -D-glucopyranose (20).

To a

solution of 19 (4.40 g, 5.99 mmol) in THF (15 mL) and water (15

mL) was added iodine (3.04 g, 12.0 mmol) and the reaction mix-

ture was stirred at room temperature for 1 h. To the mixture was

added 5% aqueous Na₂S₂O₃and the mixture was extracted with

EtOAc. The extract was washed with 5% aqueous Na₂S₂O₃and

brine and worked up as usual. The crude product was puriﬁed by

silica-gel column chromatography (200 g, CHCl₃/acetone = 10:1)

to give 20 as colorless crystals (3.61 g, 86%). Mp 68.5–70.0 °C.

[α]_D²⁴+19.7 (c 1.14, CHCl₃). Found: C, 46.47; H, 4.30; N, 1.96%.

Calcd for C₂₈H₃₁Cl₃NO₁₂P•0.7H₂O: C, 46.48; H, 4.51; N, 1.94%.

FAB-MS (positive) m/z 711.3 [(M+H)⁺]. ¹H NMR (600 MHz,

CDCl₃) δ 7.38–7.26 (m, 5 H, Ph–CH₂), 7.21–7.17 (m, 4 H, o-

C₆H₄(CH₂O)₂P), 5.92–5.84 (m, 1 H, OCH₂CHwCH₂), 5.37 (d, J =

10.6 Hz, 1 H, OCH₂CHwCH₂), 5.33–5.30 (m, 2 H, H-1 and NH),

5.24 (d, J = 10.6 Hz, 1 H, OCH₂CHwCH₂), 5.23 (dd, J = 8.9, 8.9

Hz, 1 H, H-3), 5.18–5.05 (m, 4 H, o-C₆H₄(CH₂O)₂P), 4.82 (d, J =

12.1 Hz, 1 H, CH₂of Troc), 4.69–4.56 (m, 6 H, H-4, Ph–CH₂, CH₂

Allyl 2-Acetylamino-4,6-O-benzylidene-2-deoxy-3-O-(4-

β

methoxyphenylmethyl)- -D-glucopyranoside (25). To a solu-

tion of 24 (6.98 g, 20.0 mmol) and 4-methoxyphenylmethyl

trichloroacetimidate (8.01 mL, 40.0 mmol) in anhydrous THF

(200 mL) was added Sn(OTf)₂(833 mg, 2.00 mmol) at 0 °C. The

solution was stirred at 0 °C for 4 h and the reaction was quenched

by addition of saturated aqueous NaHCO₃. The mixture was ex-

tracted with CHCl₃and the organic layer was worked up as usual.

The residue was puriﬁed by silica-gel column chromatography

(300 g, CHCl₃/acetone = 10:1) to give 25 as a white solid (9.30 g,

99%). Mp 146–153 °C. [α]_D²²+1.3 (c 0.67, CHCl₃). Found: C,

65.79; H, 6.66; N, 2.96%. Calcd for C₂₆H₃₁NO₇•0.3 H₂O: C,

65.75; H, 6.71; N, 2.95%. ¹H NMR (600 MHz, CDCl₃) δ 7.50

(dd, J = 8.2, 1.7 Hz, 2 H, PhCH), 7.40–7.35 (m, 3 H, PhCH), 7.23

494 Bull. Chem. Soc. Jpn., 76, No. 3 (2003)

HEADLINE ARTICLES

(d, J = 8.4 Hz, 2 H, p-CH₃O–Ph–CH₂), 6.85 (d, J = 8.4 Hz, 2 H,

p-CH₃O–Ph–CH₂), 5.86 (m, 1H, OCH₂CHwCH₂), 5.59–5.58 (m, 2

H, NH and PhCH), 5.27 (d, J = 17.1 Hz, 1 H, OCH₂CHwCH₂),

5.19 (d, J = 10.3 Hz, 1 H, OCH₂CHwCH₂), 4.99 (d, J = 8.3 Hz, 1

H, H-1), 4.82 (d, J = 11.5 Hz, 1 H, p-CH₃O–Ph–CH₂), 4.59 (d, J

= 11.4 Hz, 1 H, p-CH₃O–Ph–CH₂), 4.35–4.30 (m, 2 H, OCH₂-

CHwCH₂and H-6a), 4.26 (dd, J = 9.4, 9.4 Hz, 1 H, H-3), 4.08

(dd, J = 12.8, 6.1 Hz, 1 H, OCH₂CHwCH₂), 3.81–3.76 (m, 4 H, p-

CH₃O–Ph–CH₂and H-6b), 3.67 (dd, J = 9.2, 9.2 Hz, 1 H, H-4),

3.52 (m, 1 H, H-5), 3.34 (dd, J = 17.8, 8.0 Hz, 1 H, H-2), 1.82 (s,

3 H, CH₃).

was worked up as usual to give amine 28. To the solution of 28

and (R)-3-(4-triﬂuoromethylbenzyloxy)tetradecanoic acid (3.40 g,

8.48 mmol) in anhydrous CHCl₃(30 mL) were added HOBt (1.56

g, 11.6 mmol) and 3-(3-dimethylaminopropyl)-1-ethylcarbodiim-

ide hydrochloride (WSCD•HCl) (2.95 g, 15.4 mmol) at 0 °C. The

reaction mixture was stirred at room temperature for 6 h. CHCl₃

and saturated aqueous NaHCO₃were added and the aqueous layer

was extracted with CHCl₃. The organic layer was worked up as

usual. The residue was puriﬁed by silica-gel column chromatog-

raphy (150 g, CHCl₃/acetone = 10:1) to give 29 as a white solid

(2.12 g, 40%). Mp 113–115 °C. [α]_D²⁵−11.6 (c 1.00, CHCl₃).

Found: C, 64.88; H, 7.78; N, 2.07%. Calcd for C₃₈H₅₄F₃NO₇•

0.5H₂O: C, 64.94; H, 7.89; N, 1.99%. ¹H NMR (600 MHz,

CDCl₃) δ 7.60 (d, J = 8.0 Hz, 2 H, CF₃PhCH₂–), 7.44 (d, J = 8.0

Hz, 2 H, CF₃PhCH₂–), 7.36–7.26 (m, 5 H, PhCH₂), 6.45 (d, J =

5.1 Hz, 1 H, NH), 5.75 (m, 1 H, OCH₂CHwCH₂), 5.21 (dd, J =

17.2, 1.4 Hz, 1 H, OCH₂CHwCH₂), 5.13 (dd, J = 10.3, 1.4 Hz, 1

H, OCH₂CHwCH₂), 4.96 (d, J = 11.2 Hz, 1 H, PhCH₂–), 4.72 (d,

J = 11.2 Hz, 1 H, PhCH₂–), 4.62–4.58 (m, 2 H, PhCH₂), 4.40 (d, J

= 8.1 Hz, 1 H, H-1), 4.22 (dd, J = 12.8, 5.3 Hz, 1 H,

OCH₂CHwCH₂), 3.95–3.91 (m, 2 H, OCH₂CHwCH₂and H-3),

3.88–3.83 (m, 2 H, H-6a and β-CH of C2-N-acyl), 3.72 (dd, J =

12.1, 4.6 Hz, 1 H, H-6b), 3.51–3.47 (m, 2 H, H-2 and H-4), 3.37

(m, 1 H, H-5), 2.55 (dd, J = 15.0, 3.8 Hz, 1 H, α-CH of C2-N-

acyl’s main chain), 2.44 (dd, J = 15.0, 7.3 Hz, 1 H, α-CH of C2-

N-acyl’s main chain), 1.69–1.25 (m, 20 H, CH₂× 10), 0.88 (t, J =

7.0 Hz, 3 H, CH₃).

Allyl 2-Acetylamino-4-O-benzyl-2-deoxy-3-O-(4-methoxy-

β

phenylmethyl)- -D-glucopyranoside (26). To a solution of 25

(16.6 g, 35.4 mmol) in anhydrous CH₂Cl₂(180 mL) were added

Me₂NH•BH₃(10.7 g, 177 mmol) and Et₂O•BF₃(11.5 mL, 88.4

mmol) successively at 0 °C. After the mixture was stirred for 2 h

at 0 °C, the reaction was quenched by addition of saturated aque-

ous NaHCO₃and extracted with CHCl₃and the organic layer was

worked up as usual. The residue was puriﬁed by silica-gel column

chromatography (CHCl₃/acetone = 3:1) to give 26 as white pow-

der (9.48 g, 57%) and 27 (2.32 g, 14%). [α]_D²⁸+5.9 (c 1.04,

CHCl₃). Found: C, 65.17; H, 7.13; N, 2.94%. Calcd for C₂₆H₃₃-

NO₇•0.4 H₂O: C, 65.23; H, 7.12; N, 2.93%. ¹H NMR (600 MHz,

CDCl₃) δ 7.35–7.29 (m, 5 H, PhCH₂), 7.22 (d, J = 8.2 Hz, 2 H, p-

CH₃O–Ph–CH₂), 6.85 (d, J = 8.0 Hz, 2 H, p-CH₃O–Ph–CH₂),

5.86 (m, 1 H, OCH₂CHwCH₂), 5.67 (d, J = 7.9 Hz, 1 H, NH),

5.25 (d, J = 17.2 Hz, 1 H, OCH₂CHwCH₂), 5.16 (d, J = 10.4 Hz,

1 H, OCH₂CHwCH₂), 4.85–4.83 (m, 2 H, PhCH₂and H-1), 4.76

(d, J = 11.2 Hz, 1 H, Ph–CH₂), 4.67 (d, J = 11.2 Hz, 1 H,

PhCH₂), 4.60 (d, J = 11.2 Hz, 1 H, Ph–CH₂), 4.29 (dd, J = 13.0,

5.3 Hz, 2 H, OCH₂CHwCH₂), 4.08–4.05 (m, 2 H, OCH₂CHwCH₂

and H-3), 3.86 (dd, J = 11.9, 2.8 Hz, 1 H, H-6a), 3.78 (s, 3 H, p-

CH₃O–Ph–CH₂) 3.72 (dd, J = 11.9, and 3.8 Hz, 1 H, H-6b), 3.58

(dd, J = 9.0, 9.0 Hz, 1 H, H-4), 3.44–3.40 (m, 2 H, H-2 and H-5),

1.88 (s, 3 H, CH₃).

Allyl 4-O-Benzyl-6-O-(t-butyldimethylsilyl)-2-deoxy-2-[(R)-

β

3-(4-triﬂuoromethylbenzyloxy)tetradecanoylamino]- -D-glu-

copyranoside (30). To a solution of 29 in anhydrous CH₂Cl₂(30

mL) were added t-butyldimethylsilyl chloride (1.30 g, 8.65 mmol)

and imidazole (589 mg, 8.65 mmol). After the solution was

stirred at room temperature for 30 min, EtOAc and water were

added. The organic layer was worked up as usual. The residue

was puriﬁed by silica-gel column chromatography (100 g, CHCl₃/

acetone = 10:1) to give 30 as colorless oil (2.30 g, 99%). [α]_D²⁵

−12.1 (c 1.00, CHCl₃). Found: C, 65.10; H, 8.54; N, 1.69%. Cal-

cd for C₄₄H₆₈F₃NO₇Si: C, 65.40; H, 8.48; N, 1.73%. ¹H NMR

(600 MHz, CDCl₃) δ 7.59 (d, J = 8.1 Hz, 2 H, CF₃PhCH₂–), 7.44

(d, J = 8.1 Hz, 2 H, CF₃PhCH₂–), 7.35–7.27 (m, 5 H, PhCH₂),

6.47 (d, J = 5.1 Hz, 1 H, NH), 5.70 (m, 1 H, OCH₂CHwCH₂),

5.20 (dd, J = 17.2, 1.3 Hz, 1 H, OCH₂CHwCH₂), 5.11 (dd, J =

10.3, 1.1 Hz, 1 H, OCH₂CHwCH₂), 4.93 (d, J = 11.4 Hz, 1 H,

PhCH₂–), 4.69 (d, J = 11.4 Hz, 1 H, PhCH₂–), 4.62–4.58 (m, 2 H,

PhCH₂), 4.40 (d, J = 8.1 Hz, 1 H, H-1), 4.21 (dd, J = 12.7, 5.1

Hz, 1 H, OCH₂CHwCH₂), 3.92 (dd, J = 12.7, 6.4 Hz, 1 H,

OCH₂CHwCH₂), 3.88-3.78 (m, 4 H, H-3, H-4, H-6a and β CH of

C2 N acyl), 3.54–3.50 (m, 2 H, H-2 and H-6b), 3.31 (m, 1 H, H-5),

2.53 (dd, J = 15.0, 3.8 Hz, 1 H, α-CH of C2-N-acyl’s main chain),

2.44 (dd, J = 15.0, 7.3 Hz, 1 H, α-CH of C2-N-acyl’s main chain),

1.34–1.25 (m, 20 H, CH₂× 10), 0.91–0.87 (m, 12 H, (CH₃)₃-

CSi(CH₃)₂and CH₃), 0.06–0.05 (m, 6 H, (CH₃)₃CSi(CH₃)₂).

Allyl 4-O-Benzyl-6-O-(t-butyldimethylsilyl)-2-deoxy-3-O-

[(R)-3-(4-triﬂuoromethylbenzyloxy)tetradecanoyl]-2-[(R)-3-(4-

β

Allyl 2-Acetylamino-4-O-benzyl-2-deoxy- -D-glucopyrano-

side (27). To a solution of 26 (6.10 g, 13.0 mmol) in anhydrous

CH₂Cl₂(60 mL) was added Et₂O•BF₃(1.97 mL, 15.6 mmol) at 0

°C and the solution was stirred at the same temperature for 1 h.

Saturated aqueous NaHCO₃and CHCl₃were added and the organ-

ic layer was worked up as usual. The residue was puriﬁed by sili-

ca-gel column chromatography (300 g, CHCl₃/MeOH = 30:1) to

give 27 as white powder (4.55 g, quant). [α]_D²²−30.7 (c 0.60,

MeOH). Found: C, 60.34; H, 7.11; N, 3.87%. Calcd for C₁₈H₂₅-

NO₆•0.5 H₂O: C, 59.99; H, 7.27; N, 3.89%. ¹H NMR (600 MHz,

CDCl₃) δ 7.45–7.26 (m, 5 H, PhCH₂), 5.90 (m, 1 H, OCH₂-

CHwCH₂), 5.72 (d, J = 7.9 Hz, 1 H, NH), 5.31 (dd, J = 17.2, 1.3

Hz, 1 H, OCH₂CHwCH₂), 5.24 (dd, J = 10.4, 1.3 Hz, 1 H,

OCH₂CHwCH₂), 4.96 (d, J = 11.2 Hz, 1 H, PhCH₂), 4.73 (d, J =

11.3 Hz, 1 H, PhCH₂), 4.54 (d, J = 8.2 Hz, 1 H, H-1), 4.35 (dd, J

= 12.8, 5.3 Hz, 1 H, OCH₂CHwCH₂), 4.10 (dd, J = 12.8 , 6.4 Hz,

1 H, OCH₂CHwCH₂), 3.92 (dd, J = 9.7, 8.4 Hz, 1 H, H-3), 3.88

(dd, J = 11.9, 2.8 Hz, 1 H, H-6a), 3.73 (dd, J = 12.0, 4.6 Hz, 1 H,

H-6b), 3.56−3.48 (m, 2 H, H-2 and H-4), 3.39 (m, 1 H, H-5), 2.02

(s, 3 H, CH₃).

β

triﬂuoromethylbenzyloxy)tetradecanoylamino]- -D-glucopy-

Allyl 4-O-Benzyl-2-deoxy-2-[(R)-3-(4-triﬂuoromethylbenzyl-

ranoside (31). To a solution of 30 (2.90 g, 3.59 mmol) and (R)-

3-(4-triﬂuoromethylbenzyloxy)tetradecanoic acid (2.17 g, 5.38

mmol) in anhydrous CH₂Cl₂(20 mL) were added DCC (1.48 g,

7.18 mmol) and DMAP (43.8 mg, 0.359 mmol) and the reaction

mixture was stirred at room temperature for 2 h. After insoluble

materials were removed by ﬁltration, EtOAc was added to the ﬁl-

β

oxy)tetradecanoylamino]- -D-glucopyranoside (29). To a so-

lution of 27 (2.70 g, 7.71 mmol) in MeOH (180 mL) was added

Ba(OH)₂•8H₂O (34.0 g, 108 mmol) and the reaction mixture was

stirred at 70 °C for 20 h. After the solvent was removed under re-

duced pressure, water and EtOAc were added. The organic layer

K. Fukase et al.

Bull. Chem. Soc. Jpn., 76, No. 3 (2003) 495

trate. The organic solution was worked up as usual. The residue

was puriﬁed by silica-gel column chromatography (150 g, tolu-

ene/EtOAc = 20:1) to give 31 as a white solid (4.30 g, 100%).

[α]_D²⁵−5.7 (c 1.06, CHCl₃). Found: C, 66.57; H, 8.60; N, 1.60%.

Calcd for C₆₆H₉₉F₆NO₉: C, 66.47; H, 8.37; N, 1.17%. ¹H NMR

(600 MHz, CDCl₃) δ 7.59 (d, J = 7.6 Hz, 2 H, CF₃PhCH₂–), 7.51

(d, J = 7.9 Hz, 2 H, CF₃PhCH₂–), 7.45 (d, J = 7.9 Hz, 2 H,

CF₃PhCH₂–), 7.36 (d, J = 7.6 Hz, 2 H, CF₃PhCH₂–), 7.22–7.15

(m, 5 H, PhCH₂), 5.98 (d, J = 9.3 Hz, 1 H, NH), 5.73 (m, 1 H,

OCH₂CHwCH₂), 5.18 (d, J = 17.2 Hz, 1 H, OCH₂CHwCH₂),

5.12–5.01 (m, 2 H, OCH₂CHwCH₂, H-3), 4.66–4.45 (m, 6 H,

PhCH₂), 4.30 (d, J = 8.1 Hz, 1 H, H-1), 4.21 (dd, J = 12.8, 4.5

Hz, 1 H, OCH₂CHwCH₂), 4.02 (ddd, J = 9.3, 9.3, 9.3 Hz, 1 H, H-

2), 3.93 (dd, 1 H, J = 12.8, 5.9 Hz, OCH₂CHwCH₂), 3.85–3.79

(m, 4 H, H-6a,b and β-CH of C3-O-acyl and C2-N-acyl), 3.72 (dd,

J = 9.1, 9.1 Hz, 1 H, H-4), 3.29 (d, 1 H, J = 9.1 Hz, 1 H, H-5),

2.54 (dd, J = 15.8, 7.5 Hz, 1 H, α-CH₂of C2-N-acyl’s main

chain), 2.44–2.29 (m, 3 H, α-CH₂of C3-O-acyl’s and C2-N-acyl’s

main chain), 1.86–1.24 (m, 40 H, CH₂× 20), 0.88 (t, J = 6.7 Hz,

6 H, CH₃× 2), 0.08–0.04 (m, 9 H, TBDMS).

CHCl₃/acetone = 25:1) to give 13 as a white solid (1.95 g, 91%).

Mp 123–127 °C. [α]_D²⁶−5.0 (c 1.00, CHCl₃). Found: C, 56.51; H,

6.39; N, 1.80%. Calcd for C₈₈H₁₁₄Cl₃F₆N₂O₂₀P•5H₂O: C, 56.79;

H, 6.72; N, 1.51%. ¹H NMR (600 MHz, CDCl₃) δ 7.59 (d, J =

8.2 Hz, 2 H, CF₃PhCH₂–), 7.48 (d, J = 8.2 Hz, 2 H, CF₃PhCH₂–),

7.44 (d, J = 8.2 Hz, 2 H, CF₃PhCH₂–), 7.36–7.26 (m, 10 H,

PhCH₂, CF₃PhCH₂– and o-C₆H₄(CH₂O)₂P), 7.22–7.15 (m, 6 H,

PhCH₂), 5.92–5.88 (m, 2 H, NH, CH₂CHwCH₂of Alloc), 5.75 (m,

1 H, OCH₂CHwCH₂), 5.36 (dd, J = 17.2, 1.3 Hz, 1 H, CH₂-

CHwCH₂of Alloc), 5.29–5.07 (m, 10 H, CH₂CHwCH₂of Alloc,

OCH₂CHwCH₂, H-3, H-3ꢀ, N-Hꢀ and o-C₆H₄(CH₂O)₂P), 4.73 (d, J

= 8.2 Hz, 1 H, H-1ꢀ), 4.64–4.47 (m, 13 H, PhCH₂× 4, CH₂-

CHwCH₂of Alloc, H-4ꢀ and CH₂of Troc), 4.38 (d, J = 8.2 Hz, 1

H, H-1), 4.23 (dd, J = 13.2, 4.6 Hz, 1 H, OCH₂CHwCH₂), 4.08 (d,

J = 9.9 Hz, 1 H, H-6a), 4.00–3.95 (m, 2 H, H-2 and OCH₂-

CHwCH₂), 3.86–3.78 (m, 3 H, H-6aꢀ,bꢀ and β-CH), 3.74–3.68 (m,

3 H, H-5ꢀ, H-6b, β-CH of C2ꢀ-N-acyl and β-CH of C3-O-acyl),

3.53 (dd, 1 H, J = 9.2, 9.2 Hz, 1 H, H-4), 3.53–3.50 (m, 2 H, H-2ꢀ,

and H-5), 2.54 (dd, J = 15.9, 7.8 Hz, 1 H, α-CH₂of C2-N-acyl’s

main chain), 2.41 (dd, J = 15.9, 4.4 Hz, 1 H, α-CH₂of C2-N-

acyl’s main chain), 2.29–2.27 (m, 2 H, α-CH₂of C3-O-acyl’s

main chain), 1.64–1.23 (m, 40 H, CH₂× 20), 0.88 (t, J = 6.8 Hz,

6 H, CH₃× 2).

Allyl 4-O-Benzyl-2-deoxy-3-O-[(R)-3-(4-triﬂuoromethyl-

benzyloxy)tetradecanoyl]-2-[(R)-3-(4-triﬂuoromethylbenzyl-

β

oxy)tetradecanoylamino]- -D-glucopyranoside (12). To a so-

lution of 31 (1.70 g, 1.43 mmol) in pyridine (15 mL) was added

pyridine•HF (0.15 mL) and the solution was stirred at room tem-

perature for 12 h. EtOAc and saturated aqueous NaHCO₃were

added to the solution. The aqueous layer was extracted with

EtOAc and the organic layer was washed with 1 M HCl solution

and worked up as usual. The residue was puriﬁed by silica-gel

column chromatography (100 g, CHCl₃/acetone = 20:1) to give

12 as a white solid (1.30 g, 84%). Mp 98–106 °C. [α]_D²⁵−8.4 (c

1.00, CHCl₃). Found: C, 66.89; H, 7.95; N, 1.42%. Calcd for

C₆₀H₈₅F₆NO₉: C, 66.83; H, 7.95; N, 1.30%. ¹H NMR (600 MHz,

CDCl₃) δ 7.53 (d, J = 7.8 Hz, 2 H, CF₃PhCH₂–), 7.41 (d, J = 7.8

Hz, 2 H, CF₃PhCH₂–), 7.38 (d, J = 7.9 Hz, 2 H, CF₃PhCH₂–),

7.29 (d, J = 7.8 Hz, 2 H, CF₃PhCH₂–), 7.16–7.12 (m, 5 H,

PhCH₂), 5.91 (d, J = 9.1 Hz, 1 H, NH), 5.66 (m, 1 H, OCH₂-

CHwCH₂), 5.14–5.08 (m, 2 H, OCH₂CHwCH₂, H-3), 5.02 (d, J =

10.4 Hz, 1 H, OCH₂CHwCH₂), 4.55–4.42 (m, 6 H, PhCH₂), 4.30

(d, J = 8.2 Hz, 1 H, H-1), 4.15 (dd, J = 13.0, 4.7 Hz, 1 H,

OCH₂CHwCH₂), 3.94 (ddd, J = 9.1, 9.3, 9.3 Hz, 1 H, H-2), 3.85

(dd, 1 H, J = 13.0, 5.9 Hz, OCH₂CHwCH₂), 3.84–3.72 (m, 3 H,

H-6a and β-CH of C3-O-acyl and C2-N-acyl), 3.65–3.58 (m, 2 H,

H-4 and H-6b), 3.30 (d, 1 H, J = 9.1 Hz, 1 H, H-5), 2.48 (dd, J =

16.0, 7.8 Hz, 1 H, α-CH₂of C2-N-acyl’s main chain), 2.35 (dd, J

= 16.0, 4.4 Hz, 1 H, α-CH₂of C2-N-acyl’s main chain), 2.24–

2.23 (m, 2 H, α-CH₂of C3-O-acyl’s main chain), 1.53–1.06 (m,

40 H, CH₂× 20), 0.80 (t, J = 6.7 Hz, 6 H, CH₃× 2).

Allyl 6-O-{3-O-Allyloxycarbonyl-6-O-benzyl-2-deoxy-4-O-

(1,5-dihydro-3-oxo-3 -3H-2,4,3-benzodioxaphosphepin-3-yl)-

λ⁵

β

2-[(R)-3-(dodecanoyloxy)tetradecanoylamino]- -D-glucopyra-

nosyl}-4-O-benzyl-2-deoxy-3-O-[(R)-3-(4-triﬂuoromethylben-

zyloxy)tetradecanoyl]-2-[(R)-3-(4-triﬂuoromethylbenzyloxy)-

α

tetradecanoylamino]- -D-glucopyranoside (32).

To a solu-

tion of 13 (1.00 g, 565 µmol) in AcOH (20 mL) was added zinc-

copper couple (5 g) and the mixture was stirred at room tempera-

ture for 1 h. After the insoluble material was removed by ﬁltra-

tion, the ﬁltrate was concentrated in vacuo, and the residual sol-

vent was coevaporated with toluene three times. The crude prod-

uct was dissolved in EtOAc and worked up as usual to give the N-

deprotected product. The crude amine thus obtained was dis-

solved in anhydrous CHCl₃(15 mL). To this solution were added

(R)-3-(dodecanoyloxy)tetradecanoic acid (465 mg, 1.13 mmol),

HOBt (115 mg, 848 µmol), and WSCD•HCl (270 mg, 1.41

mmol). After the mixture was stirred at room temperature for 19

h, saturated aqueous NaHCO₃and EtOAc were added to the reac-

tion mixture. The organic layer was worked up as usual and the

residue was puriﬁed by silica-gel column chromatography (300 g,

CHCl₃/acetone/1,1,1,3,3,3-hexaﬂuoro-2-propanol = 10:1:0.6) to

give 32 as a white solid (724 mg, 64%). [α]_D²⁶−4.1 (c 1.00,

CHCl₃). Found: C, 65.80; H, 8.27; N, 1.68%. Calcd for C₁₁₁H₁₆₁

-

1

F₆N₂O₂₁P•H₂O: C, 65.92; H, 8.12; N, 1.39%. H NMR (600 MHz,

CDCl₃) δ 7.58 (d, J = 8.1 Hz, 2 H, CF₃PhCH₂–), 7.49 (d, J = 8.1

Hz, 2 H, CF₃PhCH₂–), 7.43 (d, J = 8.1 Hz, 2 H, CF₃PhCH₂–),

7.36–7.16 (m, 16 H, PhCH₂, CF₃PhCH₂– and o-C₆H₄(CH₂O)₂P),

5.98 (d, J = 7.7 Hz, 1 H, NHꢀ), 5.92–5.88 (m, 2 H, NH and

CH₂CHwCH₂of Alloc), 5.73 (m, 1 H, OCH₂CHwCH₂), 5.43 (dd, J

= 10.3, 8.9 Hz, 1 H, H-3ꢀ), 5.36 (dd, J = 17.3, 1.5 Hz, 1 H,

CH₂CHwCH₂of Alloc), 5.25–5.19 (m, 2 H, CH₂CHwCH₂of Alloc

and OCH₂CHwCH₂), 5.15–5.00 (m, 8 H, H-3, H-1ꢀ, OCH₂-

CHwCH₂β-CH of C2ꢀ-N-acyl and o-C₆H₄(CH₂O)₂P), 4.61–4.46

(m, 11 H, PhCH₂× 4, CH₂CHwCH₂of Alloc and H-4ꢀ), 4.37 (d, J

= 8.1 Hz, 1 H, H-1), 4.22 (dd, J = 13.1, 4.8 Hz, OCH₂CHwCH₂),

4.05–4.01 (m, 2 H, H-2 and H-6a), 3.94 (dd, J = 13.1, 6.1 Hz, 1

H, OCH₂CHwCH₂), 3.85–3.80 (m, 3 H, H-6aꢀ,bꢀ and β-CH of C3-

O-acyl), 3.75–3.71 (m, 3 H, H-5ꢀ, H-6b and β-CH of C2-N-acyl),

Allyl 6-O-[3-O-Allyloxycarbonyl-6-O-benzyl-2-deoxy-4-O-

λ⁵

(1,5-dihydro-3-oxo-3 -3H-2,4,3-benzodioxaphosphepin-3-yl)-

β

2-(2,2,2-trichloroethoxycarbonylamino)- -D-glucopyranosyl]-

4-O-benzyl-2-deoxy-3-O-[(R)-3-(4-triﬂuoromethylbenzyloxy)-

tetradecanoyl]-2-[(R)-3-(4-triﬂuoromethylbenzyloxy)tetra-

β

decanoylamino]- -D-glucopyranoside (13).

The imidate 11

(1.44 g, 1.69 mmol), the acceptor 12 (1.30 g, 1.21 mmol), and the

molecular sieves 4A in anhydrous CH₂Cl₂(10 mL) were stirred at

room temperature for 1 h. To this mixture was added TMSOTf

(21 µL, 0.12 mmol) and the mixture was stirred at −20 °C for 1 h.

The reaction mixture was neutralized with saturated aqueous

NaHCO₃, extracted with EtOAc, and worked up as usual. The

residue was puriﬁed by silica-gel column chromatography (100 g,

496 Bull. Chem. Soc. Jpn., 76, No. 3 (2003)

HEADLINE ARTICLES

3.60–3.51 (m, 3 H, H-2ꢀ, H-4 and H-5), 2.52 (dd, J = 16.0, 7.7 Hz,

1 H, α-CH₂of C2-N-acyl’s main chain), 2.44–2.38 (m, 2 H, α-

CH₂of C2-N-acyl’s main chain and α-CH₂of C2ꢀ-N-acyl’s main

chain), 2.31–2.24 (m, 5 H, α-CH₂of C2ꢀ-N-acyl’s main chain, α-

CH₂of C3-O-acyl’s main chain and α-CH₂of C2ꢀ-N-acyl’s side

chain), 1.60–1.48 (m, 8 H, CH₂× 4), 1.40–1.23 (m, 80 H, CH₂),

0.88 (m, 12 H, CH₃× 4).

up as usual. The crude diol thus obtained was dissolved in anhy-

drous benzene (1.5 mL). To this solution was added lead(Ⅳ) ace-

tate (Pb(OAc)₄) (30.1 mg, 60.1 µmol) and the mixture was stirred

at room temperature for 30 min. The mixture was ﬁltered through

a silica-gel column (2 g) using EtOAc as an eluent. After removal

of the solvent in vacuo, the residue was puriﬁed by silica-gel col-

umn chromatography (10 g, CHCl₃/acetone = 10:1) to give 34 as

a white solid (84 mg, 70%). ¹H NMR (600 MHz, CDCl₃) δ 9.50

(s, 1 H, CHO), 7.58 (d, J = 8.0 Hz, 2 H, CF₃PhCH₂–), 7.48 (d, J

= 8.0 Hz, 2 H, CF₃PhCH₂–), 7.44 (d, J = 8.0 Hz, 2 H, CF₃-

PhCH₂–), 7.42–7.15 (m, 16 H, PhCH₂× 2, CF₃PhCH₂– and o-

C₆H₄(CH₂O)₂P), 6.35 (d, J = 7.8 Hz, 1 H, NHꢀ), 6.19 (d, J = 8.9

Hz, 1 H, NH), 5.54 (dd, J = 9.3, 9.3 Hz, 1 H, H-3ꢀ), 5.28–4.99 (m,

8 H, H-3, H-1ꢀ, β-CH of C3ꢀ-O-acyl, β-CH of C2ꢀ-N-acyl and o-

C₆H₄(CH₂O)₂P), 4.63–4.46 (m, 9 H, PhCH₂× 4 and H-4ꢀ), 4.42

(d, J = 8.3 Hz, 1 H, H-1), 4.15–3.97 (m, 2 H, H-2 and H-6a),

3.84–3.76 (m, 4 H, H-6aꢀ,bꢀ and OCH₂CHO), 3.72–3.45 (m, 7 H,

H-4, H-5, H-6b, H-2ꢀ, H-5ꢀ, β-CH of C3-O-acyl and β-CH of C2-

N-acyl), 2.68–2.60 (m, 2 H, α-CH₂of C3ꢀ-O-acyl’s main chain),

2.55 (dd, J = 15.4, 7.0 Hz, 1 H, α-CH₂of C2-N-acyl’s main

chain), 2.43 (dd, J = 16.2, 4.6 Hz, 1 H, α-CH₂of C2-N-acyl’s

main chain), 2.40-2.21 (m, 8 H, α-CH₂of acyl), 1.58–1.43 (m, 12

H, CH₂× 6), 1.28–1.24 (m, 108 H, CH₂× 54), 0.88 (t, J = 6.8

Hz, 18 H, CH₃× 6).

Allyl 4-O-Benzyl-6-O-{6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-

3-oxo-3 ⁵-3H-2,4,3-benzodioxaphosphepin-3-yl)-2-[(R)-3-(do-

λ

decanoyloxy)tetradecanoylamino]-3-O-[(R)-3-(tetradecanoyl-

β

oxy)tetradecanoyl]- -D-glucopyranosyl}-2-deoxy-3-O-[(R)-3-

(4-triﬂuoromethylbenzyloxy)tetradecanoyl]-2-[(R)-3-(4-triﬂu-

β

oromethylbenzyloxy)tetradecanoylamino]- -D-glucopyrano-

side (14). To a solution of 32 (720 mg, 359 µmol) in anhydrous

THF (5 mL) were added n-BuNH₂(71 µL, 718 µmol), HCOOH

(27 µL, 718 µmol), and tetrakis(triphenylphosphine)palladium(0)

(41.5 mg, 35.9 µmol). After the mixture was stirred at room tem-

perature for 1 h, EtOAc was added. The organic layer was washed

with 1 M HCl, saturated aqueous NaHCO₃, and brine and worked

up as usual. The residue was puriﬁed by silica-gel column chro-

matography (35 g, CHCl₃/acetone = 10:1) to give 3ꢀ-hydroxy

compound. To a solution of the residue in anhydrous CHCl₃(3

mL) were added (R)-3-(tetradecanoyloxy)tetradecanoic acid (105

mg, 230 µmol), DCC (59.4 mg, 288 µmol), and DMAP (1.40 mg,

11.5 µmol). The mixture was stirred at room temperature for 3 h

and the insoluble materials were removed by ﬁltration. The ﬁl-

trate was worked up as usual. The residue was puriﬁed by silica-

gel column chromatography (250 g, CHCl₃/acetone/1,1,1,3,3,3-

hexaﬂuoro-2-propanol = 10:1:0.6) to give 14 as a white solid

(151 mg, 56%). [α]_D²⁶−3.5 (c 1.00, CHCl₃). Found: C, 69.19; H,

9.29%. Calcd for C₁₃₅H₂₀₉F₆N₂O₂₂P: C, 68.79; H, 8.94%. ESI-

MS (positive) m/z 2379.3 [(M + Na)⁺]. ¹H NMR (600 MHz,

CDCl₃) δ 7.58 (d, J = 8.0 Hz, 2 H, CF₃PhCH₂–), 7.49 (d, J = 7.1

Hz, 2 H, CF₃PhCH₂–), 7.43 (d, J = 7.4 Hz, 2 H, CF₃PhCH₂–),

7.37–7.18 (m, 16 H, PhCH₂× 2, CF₃PhCH₂– and o-C₆H₄-

(CH₂O)₂P), 6.14 (d, J = 7.1 Hz, 1 H, NHꢀ), 5.90 (d, J = 9.2 Hz, 1

H, NH), 5.76 (m, 1 H, OCH₂CHwCH₂), 5.50 (dd, J = 8.3, 8.3 Hz,

1 H, H-3ꢀ), 5.26–4.98 (m, 10 H, H-3, H-1ꢀ, OCH₂CHwCH₂, β-CH

of C3ꢀ-O-acyl, β-CH of C2ꢀ-N-acyl and o-C₆H₄(CH₂O)₂P), 4.61–

4.46 (m, 9 H, PhCH₂× 4 and H-4ꢀ), 4.38 (d, J = 8.1 Hz, 1 H, H-

1), 4.26 (d, J = 13.3 Hz, 1 H, OCH₂CHwCH₂), 4.08–3.95 (m, 3 H,

H-2, H-6a and OCH₂CHwCH₂), 3.82–3.68 (m, 5 H, H-6b, H-

6aꢀ,bꢀ, H-5ꢀ and β-CH of C3-O-acyl), 3.60–3.54 (m, 3 H, H-4, H-5

and β-CH of C2-N-acyl), 3.48 (dd, J = 16.3, 8.4 Hz, 1 H, H-2ꢀ),

2.65–2.61 (m, 2 H, α-CH₂of C3ꢀ-O-acyl’s main chain), 2.53 (dd, J

= 16.2, 7.7 Hz, 1 H, α-CH₂of C2-N-acyl’s main chain), 2.40 (d, J

= 16.2 Hz, 1 H, α-CH₂of C2-N-acyl’s main chain), 2.33–2.19 (m,

8 H, α-CH₂of acyl), 1.61–1.45 (m, 12 H, CH₂× 6), 1.34–1.09

(m, 108 H, CH₂× 54), 0.88 (t, J = 6.7 Hz, 18 H, CH₃× 6).

Formylmethyl 4-O-Benzyl-6-O-{6-O-benzyl-2-deoxy-4-O-

2-Hydroxyethyl 4-O-Benzyl-6-O-{6-O-benzyl-2-deoxy-4-O-

(1,5-dihydro-3-oxo-3 ⁵-3H-2,4,3-benzodioxaphosphepin-3-yl)-

λ

2-[(R)-3-(dodecanoyloxy)tetradecanoylamino]-3-O-[(R)-3-(tet-

β

radecanoyloxy)tetradecanoyl]- -D-glucopyranosyl}-2-deoxy-

3-O-[(R)-3-(4-triﬂuoromethylbenzyloxy)tetradecanoyl]-2-[(R)-

β

3-(4-triﬂuoromethylbenzyloxy)tetradecanoylamino]- -D-glu-

copyranoside (35). To a solution of 34 (25.0 mg, 10.6 µmol) in

2-propanol/MeOH/CH₂Cl₂(5:1:1, 1.4 mL) was added NaBH₄

(0.4 mg, 11 µmol) at 0 °C. After the mixture was stirred for 1h,

the reaction was quenched by the addition of saturated aqueous

NH₄Cl, and the mixture was extracted with EtOAc. The organic

layer was worked up as usual. The residue was puriﬁed by silica-

gel column chromatography (10 g, CHCl₃/acetone = 5:1) to give

35 as a white solid (21.0 mg, 84%). [α]_D²⁷−1.7 (c 0.59, CHCl₃).

Found: C, 67.87; H, 8.96; N, 1.22%. Calcd for C₁₃₄H₂₀₉F₆N₂O₂₃P:

C, 68.17; H, 8.92; N, 1.19%. ¹H NMR (600 MHz, CDCl₃) δ 7.58

(d, J = 8.2 Hz, 2 H, CF₃PhCH₂–), 7.49 (d, J = 8.2 Hz, 2 H,

CF₃PhCH₂–), 7.44 (d, J = 8.2 Hz, 2 H, CF₃PhCH₂–), 7.37–7.13

(m, 16 H, PhCH₂× 2, CF₃PhCH₂– and o-C₆H₄(CH₂O)₂P), 6.66

(d, J = 7.6 Hz, 1 H, NHꢀ), 6.02 (d, J = 9.2 Hz, 1 H, NH), 5.59 (dd,

J = 9.3, 9.3 Hz, 1 H, H-3ꢀ), 5.17–5.00 (m, 8 H, H-3, H-1ꢀ, β-CH of

C3ꢀ-O-acyl, β-CH of C2ꢀ-N-acyl and o-C₆H₄(CH₂O)₂P), 4.61–4.41

(m, 10 H, PhCH₂× 4, H-1 and H-4ꢀ), 4.12–4.03 (m, 2 H, H-2 and

H-6a), 3.84–3.67 (m, 5 H, H-6b, H-5ꢀ, H-6aꢀ, β-CH of C3-O-acyl

and β-CH of C2-N-acyl), 3.65–3.42 (m, 8 H, H-4, H-5, H-2ꢀ, H-

6bꢀ and –O(CH₂)₂OH), 2.64–2.61 (m, 2 H, α-CH₂of C3ꢀ-O-acyl’s

main chain), 2.53 (dd, J = 16.1, 7.7 Hz, 1 H, α-CH₂of C2-N-

acyl’s main chain), 2.45–2.24 (m, 9 H, α-CH₂of acyl), 1.57–1.41

(m, 12 H, CH₂× 6), 1.35–1.20 (m, 108 H, CH₂× 54), 0.88 (m, 18

H, CH₃× 6).

(1,5-dihydro-3-oxo-3 ⁵-3H-2,4,3-benzodioxaphosphepin-3-yl)-

λ

2-[(R)-3-(dodecanoyloxy)tetradecanoylamino]-3-O-[(R)-3-(tet-

β

radecanoyloxy)tetradecanoyl]- -D-glucopyranosyl}-2-deoxy-

3-O-[(R)-3-(4-triﬂuoromethylbenzyloxy)tetradecanoyl]-2-[(R)-

3-(4-triﬂuoromethylbenzyloxy)tetradecanoylamino]- -D-glu-

2-(1,5-Dihydro-3-oxo-3 ⁵-3H-2,4,3-benzodioxaphosphepin-

β

λ

copyranoside (34). To a solution of the residue in THF/2-meth-

yl-2-propanol/water (10:10:1, 2.1 mL) were added 4-methylmor-

pholine N-oxide (NMO) (24.6 mg, 204 µmol) and OsO₄in water

(2.5%, 0.10 mL, 10 µmol) at room temperature. After the mixture

was stirred for 6 h, 10% aqueous Na₂S₂O₃was added, and the

mixture was extracted with EtOAc. The organic layer was worked

3-yloxy)ethyl 4-O-Benzyl-6-O-{6-O-benzyl-2-deoxy-4-O-(1,5-di-

hydro-3-oxo-3 -3H-2,4,3-benzodioxaphosphepin-3-yl)-2-[(R)-

3-(dodecanoyloxy)tetradecanoylamino]-3-O-[(R)-3-(tetradeca-

λ⁵

β

noyloxy)tetradecanoyl]- -D-glucopyranosyl}-2-deoxy-3-O-[(R)-

3-(4-triﬂuoromethylbenzyloxy)tetradecanoyl]-2-[(R)-3-(4-tri-

β

ﬂuoromethylbenzyloxy)tetradecanoylamino]- -D-glucopyrano-

K. Fukase et al.

Bull. Chem. Soc. Jpn., 76, No. 3 (2003) 497

β

side (36). To a solution of 35 (21.0 mg, 8.89 µmol) in anhydrous

CH₂Cl₂(1 mL) were added N,N-diethyl-1,5-dihydro-3H-2,4,3-

benzodioxaphosphepin-3-amine (6.38 mg, 26.7 µmol) and 1H-tet-

razole (1.87 mg, 26.7 µmol). After the mixture was stirred at

room temperature for 1 h, mCPBA (3.77 g, 21.8 mmol) was added

at −20 °C and stirring was continued for another 30 min. The re-

action was quenched with saturated aqueous NaHCO₃, extracted

with EtOAc, and worked up as usual. The residue was puriﬁed by

silica-gel column chromatography (15 g, CHCl₃/acetone = 10:1)

to give 36 as colorless oil (22.4 mg, 99%). ESI-MS (positive) m/z

2564.3 [(M + Na)⁺]. ¹H NMR (600 MHz, CDCl₃) δ 7.55 (d, J =

8.2 Hz, 2 H, CF₃PhCH₂–), 7.48 (d, J = 8.2 Hz, 2 H, CF₃PhCH₂),

7.43 (d, J = 8.2 Hz, 2 H, CF₃PhCH₂–), 7.37 (d, J = 7.1 Hz, 2 H,

CF₃PhCH₂–), 7.33–7.15 (m, 18 H, PhCH₂× 2, CF₃PhCH₂– and

o-C₆H₄(CH₂O)₂P), 6.78 (d, J = 8.0 Hz, 1 H, NHꢀ), 6.41 (d, J =

9.1 Hz, 1 H, NH), 5.52 (dd, J = 9.1, 9.1 Hz, 1 H, H-3ꢀ), 5.24–4.95

(m, 12 H, H-3, H-1ꢀ, β-CH of C3ꢀ-O-acyl, β-CH of C2ꢀ-N-acyl and

o-C₆H₄(CH₂O)₂P × 2), 4.61–4.44 (m, 10 H, PhCH₂× 4, H-1 and

H-4ꢀ), 4.24–4.20 (m, 2 H, OCH₂CH₂OP), 4.10 (ddd, J = 8.9, 8.9,

8.0 Hz, 1 H, H-2), 4.03 (d, J = 11.0 Hz, 1 H, H-6a), 3.87–3.67 (m,

7 H, H-6b, H-5ꢀ, H-6aꢀ,bꢀ, OCH₂CH₂OP, β-CH of C3-O-acyl and

β-CH of C2-N-acyl), 3.58–3.52 (m, 2 H, H-5 and H-2ꢀ), 3.05 (m, 1

H, OCH₂CH₂OP), 2.65–2.61 (m, 2 H, α-CH₂of C3ꢀ-O-acyl’s

main chain), 2.52 (dd, J = 16.3, 7.8 Hz, 1 H, α-CH₂of C2-N-

acyl’s main chain), 2.41–2.23 (m, 9 H, α-CH₂of acyl), 1.68–1.40

(m, 12 H, CH₂× 6), 1.35–1.20 (m, 108 H, CH₂× 54), 0.88 (t, J =

6.9 Hz, 18 H, CH₃× 6).

ylbenzyloxy)tetradecanoylamino]- -D-glucopyranosyloxy-

acetic Acid (37). To a solution of the aldehyde 34 (63.0 mg,

26.7 µmol), NaH₂PO₄(3.20 mg, 26.7 µmol), and 2-methyl-2-

butene (14.2 µL, 134 µmol) in water/2-methyl-2-propanol (1:4,

4.5 mL) was added NaClO₂(80%, 9.0 mg, 80 µmol) at room tem-

perature. After being stirred for 12 h, the reaction mixture was

acidiﬁed with hydrochloric acid (1 mol dm⁻³) and extracted with

EtOAc. The organic layer was worked up as usual. The residue

was puriﬁed by silica-gel column chromatography (20 g, CHCl₃/

acetone = 10:1) to give 37 as colorless oil (41.2 mg, 65%). ESI-

MS (positive) m/z 2396.3 [(M + Na)⁺]. ¹H NMR (600 MHz,

CDCl₃) δ 7.50 (d, J = 7.4 Hz, 2 H, CF₃PhCH₂–), 7.49 (d, J = 7.9

Hz, 2 H, CF₃PhCH₂–), 7.44 (d, J = 7.1 Hz, 2 H, CF₃PhCH₂–),

7.34–7.09 (m, 16 H, PhCH₂

× 2, CF₃PhCH₂– and o-

C₆H₄(CH₂O)₂P), 6.81 (brs, 1 H, NHꢀ), 6.35 (brs, 1 H, NH), 5.55

(brs, 1 H, H-3ꢀ), 5.25–4.89 (m, 8 H, H-3, H-1ꢀ, β-CH of C3ꢀ-O-

acyl, β-CH of C2ꢀ-N-acyl and o-C₆H₄(CH₂O)₂P), 4.60–4.47 (m, 10

H, PhCH₂× 4, H-1 and H-4ꢀ), 4.28–4.00 (m, 4 H, H-2, H-6a, H-

6aꢀ and OCH₂COOH), 3.81–3.65 (m, 7 H, H-6b, H-5ꢀ, H-6bꢀ, H-2ꢀ

OCH₂COOH, β-CH of C3-O-acyl and β-CH of C2-N-acyl), 3.58–

3.50 (m, 2 H, H-4 and H-5), 2.64-2.18 (m, 10 H, α-CH₂of acyl),

1.57–1.54 (m, 12 H, CH₂× 6), 1.45–1.29 (m, 108 H, CH₂× 54),

0.88 (td, J = 7.1, 2.0 Hz, 18 H, CH₃× 6).

2-Deoxy-6-O-{2-deoxy-2-[(R)-3-(dodecanoyloxy)tetradecan-

oylamino]-4-O-phosphono-3-O-[(R)-3-(tetradecanoyloxy)tetra-

decanoyl]- -D-glucopyranosyl}-3-O-[(R)-3-hydroxytetradeca-

noyl]-2-[(R)-3-hydroxytetradecanoylamino]- -D-glucopyrano-

syloxyacetic Acid ( -CM-506, 9). In a manner similar to the

β

2-(Phosphonooxy)ethyl 2-Deoxy-6-O-{2-deoxy-2-[(R)-3-(do-

decanoyloxy)tetradecanoylamino]-4-O-phosphono-3-O-[(R)-3-

synthesis of 7, 37 (20.0 mg, 8.42 µmol) was deprotected. The

crude product was puriﬁed by liquid–liquid partition column chro-

matography (20 g of Sephadex^®LH-20, CHCl₃/MeOH/water/2-

propanol = 7:7:6:1) to yield 9 as white powder (10.3 mg, 69%).

β

(tetradecanoyloxy)tetradecanoyl]- -D-glucopyranosyl}-3-O-

[(R)-3-hydroxytetradecanoyl]-2-(R)-3-hydroxytetradecanoyl-

β

amino]- -D-glucopyranoside ( -PE-506, 7). To a solution of

36 (20.0 mg, 7.86 µmol) in anhydrous THF (2 mL) was added Pd-

black (150 mg). The mixture was stirred under 13 kg cm⁻²of hy-

drogen at room temperature for 2 d. After removal of the Pd cata-

lyst by ﬁltration, the solvent was evaporated in vacuo. The crude

product was puriﬁed by liquid–liquid partition column chromatog-

raphy (20 g of Sephadex^®LH-20, CHCl₃/MeOH/water/2-propanol

= 15:15:13:2). The organic layer was the stationary phase, and

the aqueous layer was the mobile phase in this chromatography.

Removal of the solvent in vacuo followed by lyophilization gave 7

as a colorless solid (10.7 mg, 74%). ESI-MS (negative) m/z

1841.4 [(M − H)⁻]. ¹H NMR (500 MHz, CDCl₃: MeOH = 3:2)

δ 5.22 (m, 1 H, β-CH of C3ꢀ-O-acyl), 5.16 (m, 1 H, β-CH of C2ꢀ-

N-acyl), 5.10 (t, 1 H, H-3ꢀ), 5.05 (t, 1 H, H-3), 4.67 (m, 1 H, H-1),

4.53 (m, 1 H, H-1ꢀ), 4.27 (m, 1 H, H-4ꢀ), 4.18–3.89 (m, 9 H,

OCH₂CH₂OP, OCH₂CH₂OP, H-6a, H-6aꢀ, H-2, H-2ꢀ, β-CH of C3-

O-acyl and β-CH of C2-N-acyl), 3.88 (m, 1 H, H-6b), 3.78 (m, 1

H, H-6bꢀ), 3.67 (m, 1 H, H-4), 3.60 (m, 1 H, OCH₂CH₂OP), 3.50

(m, 1 H, H-5), 3.38 (m, 1 H, H-5ꢀ), 2.72 (dd, 1 H, α-CH₂of C3ꢀ-O-

acyl’s main chain), 2.65 (dd, 1 H, α-CH₂of C3ꢀ-O-acyl’s main

chain), 2.53–2.47 (m, 2 H, α-CH₂of C2-N-acyl’s main chain and

α-CH₂of C2ꢀ-N-acyl’s main chain), 2.44–2.37 (m, 2 H, α-CH₂of

C2-N-acyl’s main chain and α-CH₂of C2ꢀ-N-acyl’s main chain),

2.36–2.24 (m, 6 H, α-CH₂of acyl), 1.68–1.40 (m, 12 H, CH₂×

6), 1.39–1.12 (m, 108 H, CH₂× 54), 0.88 (t, 18 H, CH₃× 6).

4-O-Benzyl-6-O-{6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-

ESI-MS (negative) m/z 887.1 [(M − 2H)²⁻]. H NMR (500 MHz,

1

CDCl₃:MeOH = 1:1) δ 5.25–5.09 (m, 3 H, H-3ꢀ, β-CH of C3ꢀ-O-

acyl and β-CH of C2ꢀ-N-acyl), 5.01 (dd, J = 9.8, 9.8 Hz, 1 H, H-

3), 4.59–4.52 (m, 2 H, H-1 and H-1ꢀ), 4.28 (m, 1 H, H-4ꢀ), 4.22 (d,

J = 18.0 Hz, 1 H, OCH₂COOH), 4.11 (d, J = 18.0 Hz, 1 H,

OCH₂COOH), 4.05–3.96 (m, 3 H, H-2, H-6a and β-CH of C2-N-

acyl), 3.95–3.84 (m, 3 H, H-2ꢀ, H-6b and β-CH of C3-O-acyl),

3.77 (m, 1 H, H-6aꢀ), 3.63 (m, 1 H, H-4), 3.51–3.46 (m, 2 H, H-5

and H-6bꢀ), 3.38 (m, 1 H, H-5ꢀ), 2.72 (dd, J = 18.6, 7.3 Hz, 1 H,

α-CH₂of C3ꢀ-O-acyl’s main chain), 2.64 (dd, J = 18.6, 5.4 Hz, 1

H, α-CH₂of C3ꢀ-O-acyl’s main chain), 2.54–2.37 (m, 4 H, α-CH₂

of C2ꢀ-N-acyl’s main chain and α-CH₂of C2-N-acyl’s main

chain), 2.36–2.28 (m, 5 H, α-CH₂of acyl), 2.23 (dd, J = 14.7, 9.0

Hz, 1 H, α-CH₂of C3-O-acyl’s side chain), 1.68–1.40 (m, 12 H,

CH₂× 6), 1.39–1.20 (m, 108 H, CH₂× 54), 0.89 (t, J = 7.0 Hz,

18 H, CH₃× 6).

Allyl 6-O-{3-O-Allyloxycarbonyl-6-O-benzyl-2-deoxy-4-O-

(1,5-dihydro-3-oxo-3 ⁵-3H-2,4,3-benzodioxaphosphepin-3-yl)-

λ

β

2-[(R)-3-(4-triﬂuoromethylbenzyloxy)tetradecanoylamino]- -

D-glucopyranosyl}-4-O-benzyl-2-deoxy-3-O-[(R)-3-(4-triﬂuoro-

methylbenzyloxy)tetradecanoyl]-2-[(R)-3-(4-triﬂuoromethyl-

β

benzyloxy)tetradecanoylamino]- -D-glucopyranoside (38).

In a manner similar to the synthesis of 32, removal of Troc group

of the common synthetic intermediate 13 (80.3 mg, 45.2 µmol)

and subsequent acylation gave 38 as a white solid (68.8 mg, 77%).

[α]_D²⁵−3.5 (c 0.37, CHCl₃). Found: C, 64.27; H, 7.12; N, 1.14%.

Calcd for C₁₀₇H₁₄₄F₉N₂O₂₀P•1H₂O: C, 64.31; H, 7.36; N, 1.40%.

¹H NMR (600 MHz, CDCl₃) δ 7.60 (d, J = 8.1 Hz, 2 H,

CF₃PhCH₂–), 7.58 (d, J = 8.1 Hz, 2 H, CF₃PhCH₂–), 7.48 (d, J =

⁵-3H-2,4,3-benzodioxaphosphepin-3-yl)-2-[(R)-3-(dodecanoyl-

oxy)tetradecanoylamino]-3-O-[(R)-3-(tetradecanoyloxy)tetra-

λ

3

β

decanoyl]- -D-glucopyranosyl}-2-deoxy-3-O-[(R)-3-(4-triﬂuo-

romethylbenzyloxy)tetradecanoyl]-2-[(R)-3-(4-triﬂuorometh-

498 Bull. Chem. Soc. Jpn., 76, No. 3 (2003)

HEADLINE ARTICLES

8.1 Hz, 2 H, CF₃PhCH₂–), 7.45 (d, J = 8.1 Hz, 2 H, CF₃PhCH₂–),

7.42 (d, J = 8.1 Hz, 2 H, CF₃PhCH₂–), 7.34–7.14 (m, 16 H,

PhCH₂, CF₃PhCH₂– and o-C₆H₄(CH₂O)₂P), 6.34 (d, J = 8.1 Hz, 1

H, NHꢀ), 5.88–5.82 (m, 2 H, NH and CH₂CHwCH₂of Alloc), 5.70

(m, 1 H, OCH₂CHwCH₂), 5.36–5.22 (m, 2 H, H-3ꢀ and CH₂-

CHwCH₂of Alloc), 5.20–5.17 (m, 2 H, CH₂CHwCH₂of Alloc,

OCH₂CHwCH₂), 5.11–5.04 (m, 6 H, H-3, N-Hꢀ and o-C₆H₄-

(CH₂O)₂P), 4.82 (d, J = 8.2 Hz, 1 H, H-1ꢀ), 4.62–4.48 (m, 13 H,

PhCH₂× 5, CH₂CHwCH₂of Alloc and H-4ꢀ), 4.30–4.19 (m, 2 H,

H-1 and OCH₂CHwCH₂), 4.04–3.96 (m, 2 H, H-2 and H-6a), 3.90

(dd, J = 13.2, 6.1 Hz, 1 H, OCH₂CHwCH₂), 3.83–3.76 (m, 3 H,

H-6aꢀ,bꢀ and β-CH of C2-N-acyl), 3.75–3.62 (m, 5 H, H-2ꢀ, H-5ꢀ,

H-6b and β-CH × 2), 3.53 (dd, 1 H, J = 8.9, 8.9 Hz, 1 H, H-4),

3.46 (m, 1 H, H-5), 2.51 (dd, J = 16.0, 7.7 Hz, 1 H, α-CH₂of C2-

N-acyl’s main chain), 2.39 (dd, J = 16.0, 4.6 Hz, 1 H, α-CH₂of

C2-N-acyl’s main chain), 2.36–2.33 (m, 2 H, α-CH₂of C2ꢀ-N-

acyl’s main chain), 2.27–2.25 (m, 2 H, α-CH₂of C3-O-acyl’s

main chain), 1.55–1.22 (m, 60 H, CH₂× 30), 0.88 (t, J = 6.9 Hz,

9 H, CH₃× 3).

(d, J = 8.7 Hz, 1 H, NHꢀ), 5.92 (d, J = 9.0 Hz, 1 H, NH), 5.45 (dd,

J = 9.4, 9.4 Hz, 1 H, H-3ꢀ), 5.06-4.89 (m, 5 H, H-3 and o-

C₆H₄(CH₂O)₂P), 4.66–4.41 (m, 14 H, PhCH₂× 6, H-4ꢀ and H-1ꢀ),

4.20 (d, J = 8.6 Hz, 1 H, H-1), 4.07 (q, J = 9.1 Hz, 1 H, H-2),

4.00–3.80 (m, 6 H, H-6a, H-6aꢀ,bꢀ, H-2ꢀ and β-CH × 2), 3.70-3.65

(m, 5 H, H-6b, H-5ꢀ OCH₂CHO and β-CH), 3.59–3.32 (m, 3 H, H-

4, H-5 and β-CH), 2.73 (dd, J = 17.0, 7.7 Hz, 1 H, α-CH₂of

acyl), 2.58 (dd, J = 17.0, 4.1 Hz, 1 H, α-CH₂of acyl), 2.51 (dd, J

= 16.1, 7.7 Hz, 1 H, α-CH₂of acyl), 2.40 (dd, J = 16.1, 4.6 Hz, 1

H, α-CH₂of acyl), 2.35–2.22 (m, 3 H, α-CH₂of acyl × 3), 1.92

(d, J = 12.6 Hz, 1 H, α-CH₂of acyl), 1.62–1.15 (m, 80 H, CH₂×

40), 0.88 (m, 12 H, CH₃× 4).

2-Hydroxyethyl 4-O-Benzyl-6-O-{6-O-benzyl-2-deoxy-4-O-

(1,5-dihydro-3-oxo-3 ⁵-3H-2,4,3-benzodioxaphosphepin-3-yl)-

λ

3-O-[(R)-3-(4-triﬂuoromethylbenzyloxy)tetradecanoyl]-2-[(R)-

β

3-(4-triﬂuoromethylbenzyloxy)tetradecanoylamino]- -D-glu-

copyranosyl}-2-deoxy-3-O-[(R)-3-(4-triﬂuoromethylbenzyloxy)-

tetradecanoyl]-2-[(R)-3-(4-triﬂuoromethylbenzyloxy)tetrade-

β

canoylamino]- -D-glucopyranoside (40).

In a manner similar

Allyl 4-O-Benzyl-6-O-{6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-

to the synthesis of 35, the aldehyde 39 (89.0 mg, 39.0 µmol) was

reduced with NaBH₄to give 40 as a white solid (74.8 mg, 84%).

[α]_D²⁶−2.2 (c 0.81, CHCl₃). Found: C, 64.56; H, 7.49; N, 1.24%.

Calcd for C₁₂₄H₁₇₁F₁₂N₂O₂₁P•1H₂O: C, 64.68; H, 7.57; N, 1.22%.

¹H NMR (600 MHz, CDCl₃) δ 7.58 (d, J = 8.1 Hz, 4 H,

CF₃PhCH₂–), 7.50 (d, J = 13.1 Hz, 4 H, CF₃PhCH₂–), 7.37–7.10

(m, 21 H, PhCH₂× 2, CF₃PhCH₂× 2 and o-C₆H₄(CH₂O)₂P),

6.78 (d, J = 7.4 Hz, 1 H, NHꢀ), 6.74 (d, J = 7.4 Hz, 1 H, o-

C₆H₄(CH₂O)₂P), 5.77 (d, J = 9.2 Hz, 1 H, NH), 5.50 (dd, J = 9.2,

9.2 Hz, 1 H, H-3ꢀ), 5.05–4.90 (m, 5 H, H-3 and o-C₆H₄(CH₂O)₂P),

4.70 (d, J = 8.3 Hz, 1 H, H-1ꢀ), 4.66–4.39 (m, 13 H, PhCH₂× 6

and H-4ꢀ), 4.19 (d, J = 8.5 Hz, 1 H, H-1), 4.04–3.98 (m, 2 H, H-2

and H-6a), 3.84–3.70 (m, 8 H, H-6aꢀ, O(CH₂)₂OH, H-2ꢀ, H-5ꢀ and

β-CH), 3.58–3.42 (m, 6 H, H-6b, H-6bꢀ, H-5ꢀ and β-CH × 3), 3.30

(dd, J = 9.0, 9.0 Hz, 1 H, H-4), 2.71 (dd, J = 17.0, 7.8 Hz, 1 H, α-

CH₂of acyl), 2.53 (dd, J = 17.0, 4.3 Hz, 1 H, α-CH₂of acyl), 2.51

(dd, J = 16.5, 7.6 Hz, 1 H, α-CH₂of acyl), 2.39 (dd, J = 16.0, 4.6

Hz, 1 H, α-CH₂of acyl), 2.31–2.27 (m, 3 H, α-CH₂of acyl × 3),

2.21 (dd, J = 14.7, 4.1 Hz, 1 H, α-CH₂of acyl), 1.70–1.24 (m, 80

H, CH₂× 40), 0.87 (t, J = 6.9 Hz, 12 H, CH₃× 4).

3-oxo-3 ⁵-3H-2,4,3-benzodioxaphosphepin-3-yl)-3-O-[(R)-3-(4-

λ

triﬂuoromethylbenzyloxy)tetradecanoyl]-2-[(R)-3-(4-triﬂuoro-

β

methylbenzyloxy)tetradecanoylamino]- -D-glucopyranosyl}-2-

deoxy-3-O-[(R)-3-(4-triﬂuoromethylbenzyloxy)tetradecanoyl]-

β

2-[(R)-3-(4-triﬂuoromethylbenzyloxy)tetradecanoylamino]- -

D-glucopyranoside (15). In a manner similar to the synthesis of

14, 3ꢀ-O-alloc group in 38 (220 mg, 111 µmol) was removed to

give the 3ꢀ-hydroxy compound (210 mg, 100%), which was then

acylated to give 15 as a white solid (197 mg, 86%). Mp 110–113

°C. [α]_D²⁶−6.5 (c 1.00, CHCl₃). Found: C, 65.97; H, 7.67; N,

1.55%. Calcd for C₁₂₅H₁₇₁F₁₂N₂O₂₀P: C, 65.83; H, 7.56; N,

1.23%. ESI-MS (positive) m/z 2303.0 [(M + Na)⁺]. ¹H NMR

(600 MHz, CDCl₃) δ 7.60–7.57 (m, 4 H, CF₃PhCH₂–), 7.51–7.42

(m, 10 H, CF₃PhCH₂–), 7.34–7.11 (m, 15 H, PhCH₂× 2,

CF₃PhCH₂– and o-C₆H₄(CH₂O)₂P), 6.75 (d, J = 7.3 Hz, 1 H, o-

C₆H₄(CH₂O)₂P), 6.09 (d, J = 8.8 Hz, 1 H, NHꢀ), 5.83 (d, J = 9.4

Hz, 1 H, NH), 5.70 (m, 1 H, OCH₂CHwCH₂), 5.32 (dd, J = 9.3,

9.3 Hz, 1 H, H-3ꢀ), 5.20 (dd, J = 17.4, 1.6 Hz, 1 H, OCH₂-

CHwCH₂), 5.07–4.65 (m, 6 H, H-3, OCH₂CHwCH₂and o-C₆H₄-

(CH₂O)₂P), 4.63–4.45 (m, 14 H, PhCH₂× 6, H-4ꢀ and H-1ꢀ), 4.27

(d, J = 8.2 Hz, 1 H, H-1), 4.23 (dd, J = 16.9, 3.7 Hz, 1 H,

OCH₂CHwCH₂), 4.01 (dd, J = 9.3, 9.3 Hz, 1 H, H-2), 3.95–3.85

(m, 7 H, OCH₂CHwCH₂, H-6a, H-6aꢀ,bꢀ, H-2ꢀ and β-CH × 2),

3.72–3.61 (m, 3 H, H-6b, H-5ꢀ and β-CH), 3.53–3.44 (m, 3 H, H-

4, H-5 and β-CH), 2.72 (dd, J = 16.8, 7.7 Hz, 1 H, α-CH₂of

acyl), 2.57-2.48 (m, 2 H, α-CH₂of acyl), 2.44 (dd, J = 15.1, 7.1

Hz, 1 H, α-CH₂of acyl), 2.31–2.17 (m, 3 H, α-CH₂of acyl), 1.94

(d, J = 9.4 Hz, 1 H, α-CH₂of acyl), 1.68–1.04 (m, 80 H, CH₂×

40), 0.87 (m, 12 H, CH₃× 4).

2-(1,5-Dihydro-3-oxo-3 ⁵-3H-2,4,3-benzodioxaphosphepin-

λ

3-yloxy)ethyl 4-O-Benzyl-6-O-{6-O-benzyl-2-deoxy-4-O-(1,5-

dihydro-3-oxo-3 ⁵-3H-2,4,3-benzodioxaphosphepin-3-yl)-3-O-

λ

[(R)-3-(4-triﬂuoromethylbenzyloxy)tetradecanoyl]-2-[(R)-3-(4-

β

triﬂuoromethylbenzyloxy)tetradecanoylamino]- -D-glucopy-

ranosyl}-2-deoxy-3-O-[(R)-3-(4-triﬂuoromethylbenzyloxy)tetra-

decanoyl]-2-[(R)-3-(4-triﬂuoromethylbenzyloxy)tetradecano-

β

ylamino]- -D-glucopyranoside (41). In a manner similar to the

synthesis of 36, the alcohol 40 (72.0 mg, 31.5 µmol) was phos-

phorylated to give 41 as colorless oil (76.2 mg, 98%). ESI-MS

(positive) m/z 2488.2 [(M + Na)⁺]. ¹H NMR (600 MHz, CDCl₃)

δ 7.58–7.54 (m, 4 H, CF₃PhCH₂–), 7.49–7.10 (m, 30 H, PhCH₂×

2, CF₃PhCH₂× 3, o-C₆H₄(CH₂O)₂P × 2 and NHꢀ), 6.70 (d, J =

7.4 Hz, 1 H, o-C₆H₄(CH₂O)₂P), 6.06 (d, J = 9.2 Hz, 1 H, NH),

5.46 (dd, J = 9.3, 9.3 Hz, 1 H, H-3ꢀ), 5.23–4.84 (m, 9 H, H-3 and

o-C₆H₄(CH₂O)₂P × 2), 4.83 (d, J = 8.3 Hz, 1 H, H-1ꢀ), 4.64–4.44

(m, 13 H, PhCH₂× 6 and H-4ꢀ), 4.23 (m, 1 H, OCH₂CH₂OP),

4.18 (d, J = 8.2 Hz, 1 H, H-1), 4.05–4.00 (m, 2 H, H-2 and H-6a),

3.87–3.78 (m, 8 H, H-6aꢀ,bꢀ, H-6b, H-2ꢀ and β-CH × 4), 3.74–

3.68 (m, H-5ꢀ and OCH₂CH₂OP), 3.65 (m, 1 H, OCH₂CH₂OP),

3.60 (dd, J = 11.2, 6.5 Hz, 1 H, H-5), 3.49 (m, 1 H, H-4), 3.06 (m,

1 H, OCH₂CH₂OP), 2.70 (dd, J = 17.0, 7.9 Hz, 1 H, α-CH₂of

Formylmethyl 4-O-Benzyl-6-O-{6-O-benzyl-2-deoxy-4-O-

λ⁵

(1,5-dihydro-3-oxo-3 -3H-2,4,3-benzodioxaphosphepin-3-yl)-

3-O-[(R)-3-(4-triﬂuoromethylbenzyloxy)tetradecanoyl]-2-[(R)-

β

3-(4-triﬂuoromethylbenzyloxy)tetradecanoylamino]- -D-glu-

copyranosyl}-2-deoxy-3-O-[(R)-3-(4-triﬂuoromethylbenzyloxy)-

tetradecanoyl]-2-[(R)-3-(4-triﬂuoromethylbenzyloxy)tetrade-

β

canoylamino]- -D-glucopyranoside (39).

In a manner similar

to the preparation of 34, the allyl glycoside of 15 (33.0 mg, 14.5

µmol) was oxidatively cleaved to give colorless oil 39 (26.4 mg,

80%). ¹H NMR (600 MHz, CDCl₃) δ 9.52 (s, 1 H, OCH₂CHO),

7.60–7.11 (m, 29 H, PhCH₂× 2, CF₃PhCH₂× 4 and o-

C₆H₄(CH₂O)₂P), 6.74 (d, J = 7.4 Hz, 1 H, o-C₆H₄(CH₂O)₂P), 6.40

K. Fukase et al.

Bull. Chem. Soc. Jpn., 76, No. 3 (2003) 499

acyl), 2.53 (dd, J = 17.0, 4.1 Hz, 1 H, α-CH₂of acyl), 2.49 (dd, J

= 16.1, 7.7 Hz, 1 H, α-CH₂of acyl), 2.38–2.30 (m, 3 H, α-CH₂of

acyl), 2.30–2.22 (m, 2 H, α-CH₂of acyl), 1.82–1.23 (m, 80 H,

CH₂× 40), 0.87 (t, J = 6.9 Hz, 12 H, CH₃× 4).

β-CH of acyl), 3.68 (m, 2 H, H-2), 3.61 (m, 1 H, H-6bꢀ), 3.35 (m,

1 H, H-4), 3.26 (m, 1 H, H-5), 3.25 (m, 1 H, H-5ꢀ), 2.66–2.01 (m,

8 H, α-CH₂of acyl), 1.51–1.00 (m, 80 H, CH₂× 40), 0.88–0.77

(m, 12 H, CH₃× 4).

2-(Phosphonooxy)ethyl 2-Deoxy-6-O-{2-deoxy-3-O-[(R)-3-

hydroxytetradecanoyl]-2-[(R)-3-hydroxytetradecanoylamino]-

Cytokine Induction in Human Peripheral Whole-Blood Cell

Cultures. An aqueous 5% DMSO solution (50 µg/mL) of each

sample was prepared by dilution of DMSO solution (1 mg/mL)

with saline (Otsuka Pharmaceuticals Co., Ltd.). Each sample was

further diluted stepwise with saline on a 96-well plastic plate

(#2870-096, Iwaki Glass Co., Ltd). The mixture consisting of test

sample (25 µL), RPMI 1640 medium (75 µL; Flow Laboratories,

Irvine, Scotland), and heparinized human peripheral whole-blood

(25 µL) collected from an adult volunteer was incubated in tripli-

cate at 37 °C in 5% CO₂for 24 h. The plate was centrifuged at

300 × g for 2 min. The levels of TNFα and IL-6 in culture super-

natant were measured by means of enzyme-linked immunosorbent

assay (ELISA) as described in the previous paper.

Inhibition Assay in Cytokine Induction. The mixture con-

sisting of test sample (25 µL) prepared in the same manner as

above, LPS (25 µL, E. coli 0111:B4; Sigma Chemicals Co.),

RPMI 1640 medium (75 µL; Flow Laboratories, Irvine, Scotland),

and heparinized human peripheral whole-blood (25 µL) collected

from an adult volunteer was incubated in triplicate at 37 °C in 5%

CO₂for 24 h. The plate was centrifuged at 300 × g for 2 min.

The levels of TNFα and IL-6 in culture supernatant were mea-

sured by means of enzyme-linked immunosorbent assay (ELISA)

as described in the previous paper.

β

4-O-phosphono- -D-glucopyranosyl}-3-O-[(R)-3-hydroxytetra-

β

decanoyl]-2-[(R)-3-hydroxytetradecanoylamino]- -D-glucopy-

ranoside ( -PE-406, 8). In a manner similar to the synthesis of

β

7, all benzyl-type protective groups of 41 (20.0 mg, 8.12 µmol)

were removed by catalytic hydrogenation. The crude product was

puriﬁed by liquid-liquid partition column chromatography (20 g

of Sephadex^®LH-20, CHCl₃/MeOH/water/2-propanol = 10:8:

8:1) to yield 8 as white powder (5.99 mg, 51%). ESI-MS (nega-

tive) m/z 1447.98 [(M − H)⁻]. ¹H NMR (600 MHz, 8 (1 mg) and

SDS-d₂₅(19.3 mg) were dissolved in DMSO-d₆(0.7 mL)) δ 7.87

(m, 1 H, NH), 7.84 (m, 1 H, NHꢀ), 5.10 (t, 1 H, H-3ꢀ), 4.87 (t, 1 H,

H-3), 4.58 (m, 1 H, H-1ꢀ), 4.53 (m, 1 H, H-1), 4.13 (m, 1 H,

OCH₂CH₂OP), 4.09 (m, 1 H, H-4ꢀ), 4.04 (m, 1 H, H-6a), 3.85 (m,

1 H, OCH₂CH₂OP), 3.83–3.80 (m, 2 H, H-2ꢀ and H-6aꢀ), 3.76 (m,

1 H, OCH₂CH₂OP), 3.75–3.74 (m, 2 H, β-CH of acyl), 3.74 (m, 1

H, H-6bꢀ), 3.70 (m, 1 H, H-2), 3.69 (m, 1 H, H-6b), 3.65 (m, 2 H,

β-CH of acyl), 3.48 (m, 1 H, H-4), 3.46 (m, 2 H, OCH₂CH₂P and

H-5ꢀ), 3.44 (m, 1 H, H-5), 2.40–1.96 (m, 8 H, α-CH₂of acyl),

1.37–0.98 (m, 80 H, CH₂× 40), 0.78–0.64 (m, 12 H, CH₃× 4).

4-O-Benzyl-6-O-{6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-

oxo-3 ⁵-3H-2,4,3-benzodioxaphosphepin-3-yl)-3-O-[(R)-3-(4-

λ

triﬂuoromethylbenzyloxy)tetradecanoyl]-2-[(R)-3-(4-triﬂuoro-

Limulus Assay. DMSO solution of each sample (1 mg/mL)

was diluted stepwise with distilled water (Otsuka Pharmaceuticals

Co., Ltd.) on a 96-well plastic plate (#2870-096, Iwaki Glass Co.,

Ltd). Limulus activity was measure by means of Endospecy Test^®

(Seikagaku Kogyo).

β

methylbenzyloxy)tetradecanoylamino]- -D-glucopyranosyl}-

2-deoxy-3-O-[(R)-3-(4-triﬂuoromethylbenzyloxy)tetradecanoyl]-

β

2-[(R)-3-(4-triﬂuoromethylbenzyloxy)tetradecanoylamino]- -

D-glucopyranosyloxyacetic Acid (42). In a manner similar to

the synthesis of 37, the aldehyde 39 (47.0 mg, 20.6 µmol) was

oxidized with NaClO₂to give 42 (36.2 mg, 77%). ESI-MS (posi-

tive) m/z 2320.1 [(M + Na)⁺]. ¹H NMR (600 MHz, CDCl₃) δ

7.58–7.19 (m, 29 H, PhCH₂× 2, CF₃PhCH₂× 4 and o-C₆H₄-

(CH₂O)₂P), 6.94 (brs, 1 H, o-C₆H₄(CH₂O)₂P), 6.82 (brs, 1 H,

NHꢀ), 6.14 (brs, 1 H, NH), 5.58 (brs, 1 H, H-3ꢀ), 4.93–4.86 (m, 5

H, H-3 and o-C₆H₄(CH₂O)₂P), 4.63–4.44 (m, 14 H, H-1ꢀ, PhCH₂

× 6 and H-4ꢀ), 4.28 (brs, 1 H, H-1), 4.14 (brs, 1 H, OCH₂COOH),

4.06–3.74 (m, 10 H, H-2, H-6a, H-6aꢀ,bꢀ, OCH₂COOH, H-2ꢀ, H-5ꢀ

and β-CH × 3), 3.65–3.56 (m, 2 H, H-5 and β-CH), 3.41 (brs, 1

H, H-4), 3.35 (brs, 1 H, H-6b), 2.69 (dd, J = 16.7, 7.6 Hz, 1 H, α-

CH₂of acyl), 2.55–2.24 (m, 7 H, α-CH₂of acyl), 1.52–1.18 (m, 80

H, CH₂× 40), 0.88 (t, J = 6.9 Hz, 12 H, CH₃× 4).

This work was supported in part by Grants-in-Aid for Scien-

tiﬁc Research on Priority Areas No. 06240105, No. 08245229,

and No. 09231228 and by Grant-in-Aid for International Sci-

entiﬁc Research No. 09044086 from the Ministry of Educa-

tion, Science and Culture by “Research for the Future” Pro-

gram No. 97L00502 from the Japan Society for the Promotion

of Science, and by a research fund from Kyowa Hakko Kogyo

Co., Ltd.

References

2-Deoxy-6-O-{2-deoxy-3-O-[(R)-3-hydroxytetradecanoyl]-

1

(2002).

2

R. Medzhitov and C. A. Janeway, Jr., Science, 296, 298

β

2-[(R)-3-hydroxytetradecanoylamino]-4-O-phosphono- -D-glu-

copyranosyl}-3-O-[(R)-3-hydroxytetradecanoyl]-2-(R)-3-hydro-

B. Pulendran, K. Palucka, and J. Banchereau, Science, 293,

β

xytetradecanoylamino]- -D-glucopyranosyloxyacetic Acid ( -

CM-406, 10). In a manner similar to the synthesis of 7, all ben-

zyl-type protective groups of 42 (25.0 mg, 10.9 µmol) were re-

moved by catalytic hydrogenation. The crude product was puri-

ﬁed by liquid–liquid partition column chromatography (20 g of

Sephadex^®LH-20, CHCl₃/MeOH/water/2-propanol = 10:9:8:1)

to yield 10 as white powder (8.1 mg, 54%). ESI-MS (negative)

253 (2001).

3

A. J. Ulmer, E. Th. Rietschel, U. Zähringer, and H. Heine,

Trend. Glycosci. Glycotechnol., 14, 53 (2002).

4

C. Alexander and U. Zähringer, Trend. Glycosci. Glycote-

chnol., 14, 69 (2002).

5

C. Alexander and E. Th. Rietschel, J. Endotoxin Res., 7,

167 (2001).

1

m/z 690.5 [(M − 2H)²⁻]. H NMR (600 MHz, 10 (1 mg) and SDS-

6

“Endotoxin in Health and Disease,” ed by H. Brade, S. M.

d₂₅(19.3 mg) were dissolved in DMSO-d₆(0.7 mL)) δ 7.80 (NHꢀ

and NH), 5.05 (t, 1 H, H-3ꢀ), 4.85 (t, 1 H, H-3), 4.52 (m, 1 H, H-1),

4.50 (m, 1 H, H-1ꢀ), 4.15 (m, 1 H, H-6a), 4.03 (m, 2 H, CH₂COOH

and H-6b), 4.02 (m, 1 H, H-4ꢀ), 3.97 (m, 1 H, H-6aꢀ), 3.80 (m, 2 H,

β-CH of acyl), 3.76 (m, 2 H, H-2ꢀ and CH₂COOH), 3.74 (m, 2 H,

Opal, S. N. Vogel, and D. C. Morrison, Marcel Dekker, New

York•Basel (1999).

7

E. Th. Rietschel, T. Kirikae, F. U. Schade, U. Mamat, G.

Schmidt, H. Loppnow, A. J. Ulmer, U. Zähringer, U. Seydel, F. Di

Padova, M. Schreier, and H. Brade, FASEB J., 8, 217 (1994).

500 Bull. Chem. Soc. Jpn., 76, No. 3 (2003)

HEADLINE ARTICLES

8

(1992).

9

E. Th. Rietschel and H. Brade, Scientiﬁc American, 267, 54

(1991).

22 W.-C. Liu, M. Oikawa, K. Fukase, Y. Suda, and S.

Kusumoto, Bull. Chem. Soc. Jpn., 72, 1377 (1999).

23 K. Fukase, T. Kirikae, F. Kirikae, W.-C. Liu, M. Oikawa, Y.

Suda, M. Kurosawa, Y. Fukase, H. Yoshizaki, and S. Kusumoto,

Bull. Chem. Soc. Jpn., 74, 2189 (2001).

24 Y. Fukase, S.-Q. Zhang, K. Iseki, M. Oikawa, K. Fukase,

and S. Kusumoto, Synlett, 2001,1693.

25 J. J. Oltvoort, C. A. A. v. Boeckel, J. H. d. Koning, and J.

H. v. Boom, Synthesis, 1981, 305.

26 M. Oikawa, W.-C. Liu, Y. Nakai, S. Koshida, K. Fukase,

and S. Kusumoto, Synlett, 1996, 1179.

27 Y. Watanabe, Y. Komoda, K. Ebisuya, and S. Ozaki, Tetra-

hedron Lett., 31, 255 (1990).

28 F. J. Urban, B. S. Moore, and R. Breitenbach, Tetrahedron

Lett., 31, 4421 (1990).

29 S. Nakabayashi, C. D. Warren, and R. W. Jeanloz, Carbo-

hydrate Res., 150, C7 (1986).

30 An unsubstituted benzyl group used for protection of the

hydroxy function on the 3-hydroxyacyl residue is prone to air-oxi-

dation and gradually transformed into the corresponding benzoyl

group. We recently found the 4-(triﬂuoromethyl)benzyl group at

this position is resistant to oxidation but readily removable by

conventional hydrogenolysis, a) Y. Sakai, M. Oikawa, H.

Yoshizaki, T. Ogawa, Y. Suda, K. Fukase, and S. Kusumoto,

Tetrahedron Lett., 41, 6843 (2000). b) H. Yoshizaki, N. Fukuda, K.

Sato, M. Oikawa, K. Fukase, Y. Suda, and S. Kusumoto, Angew.

Chem., Int. Ed., 40, 1475 (2001).

31 M. Oikawa, A. Wada, H. Yoshizaki, K. Fukase, and S.

Kusumoto, Bull. Chem. Soc. Jpn., 70, 1435 (1997).

32 M. Oikawa, T. Shintaku, H. Yoshizaki, K. Fukase, S.

Adachi, K. Lee, and S. Kusumoto, Bull. Chem. Soc. Jpn., 74, 1455

(2001).

33 Y. Suda, H. Tochio, K. Kawano, H. Takada, H. Yoshida, S.

Kotani, and S. Kusumoto, FEMS Immun. Med. Microbiol., 12, 97

(1995).

34 a) P. A. Detmers, N. Thieblemont, T. Vasselon, R.

Pironkova, D. S. Miller, and S. D. Wright, J. Immunol., 157, 5589

(1996). b) B. Yu and S. D. Wright, J. Biol. Chem., 271, 4100

(1996). c) E. Hamilton, H. S. Lichenstein, M. M. Wurfel, D. S.

Miller, D. A. Johnson, M. Kelley, L. A. Busse, M. M. Zukowski,

and S. D. Wright, J. Exp. Med., 179, 269 (1994).

a) M. Imoto, H. Yoshimura, M. Yamamoto, T. Shimamoto,

S. Kusumoto, and T. Shiba, Tetrahedron Lett., 25, 2667 (1984). b)

M. Imoto, H. Yoshimura, M. Yamamoto, T. Shimamoto, S.

Kusumoto, and T. Shiba, Bull. Chem. Soc. Jpn., 60, 2197 (1987).

10 a) M. Imoto, H. Yoshimura, M. Yamamoto, N. Sakaguchi,

S. Kusumoto, and T. Shiba, Tetrahedron Lett., 26, 1545 (1985). b)

M. Imoto, H. Yoshimura, T. Shimamoto, N. Sakaguchi, S.

Kusumoto, and T. Shiba, Bull. Chem. Soc. Jpn., 60, 2205 (1987).

11 H. Loppnow, H. Brade, I. Durrbaum, C. A. Dinarello, S.

Kusumoto, E. Th. Rietschel, and H.-D. Flad, J. Immunol., 142,

3229 (1989).

12 E. Th. Rietschel, T. Kirikae, F. U. Schade, A. J. Ulmer, O.

Holst, H. Brade, G. Schmidt, U. Mamat, H.-D. Grimmecke, S.

Kusumoto, and U. Zähringer, Immunobiol., 187, 169 (1993).

13 T. Kirikae, F. U. Shade, U. Zähringer, F. Kirikae, H. Brade,

S. Kusumoto, T. Kusama, and E. Th. Rietschel, FEMS Immunol.

Med. Microbiol., 8, 13 (1994).

14 K. Fukase, Y. Fukase, M. Oikawa, W.-C. Liu, Y. Suda, and

S. Kusumoto, Tetrahedron, 54, 4033 (1998).

15 K. Fukase, M. Oikawa, Y. Suda, W.-C. Liu, Y. Fukase, T.

Shintaku, H. Sekljic, H. Yoshizaki, and S. Kusumoto, J. Endotoxin

Res., 5, 46 (1999).

16 H. Takada, S. Kotani, M. Tsujimoto, T. Ogawa, I.

Takahashi, K. Harada, C. Katsukawa, S. Tanaka, T. Shiba, and S.

Kusumoto, Infect. Immun., 48, 219, (1985).

17 S. Kotani, H. Takada, I. Takahashi, T. Ogawa, M.

Tsujimoto, H. Shimauchi, T. Ikeda, H. Okamura, T. Tamura, K.

Harada, S. Tanaka, T. Shiba, S. Kusumoto, and T. Shimamoto,

Infect. Immun., 54, 673 (1986).

18 H. Loppnow, L. Brade, H. Brade, E. Th. Rietschel, S.

Kusumoto, T. Shiba, and H.-D. Flad, Eur. J. Immunol., 16, 1263

(1986).

19 M. Yoshida, M. Hirata, K. Inada, N. Tsunoda, T. Kirikae, T.

Onodera, Y. Ishikawa, O. Sasaki, T. Shiba, and S. Kusumoto,

Microbiol. Immunol., 33, 797 (1989).

20 a) T. Kusama, T. Soga, E. Shioya, K. Nakayama, H.

Nakajima, Y. Osada, Y. Ono, S. Kusumoto, and T. Shiba, Chem.

Pharm. Bull., 38, 3366 (1990). b) T. Kusama, T. Soga, Y. Ono, E.

Kumazawa, E. Shioya, Y. Osada, S. Kusumoto, and T. Shiba,

Chem. Pharm. Bull., 39, 1994 (1991). c) A. J. Ulmer, A. H.

Heine, W. Feist, S. Kusumoto, T. Kusama, H. Brade, F. U. Schade,

E. Th. Rietschel, and H.-D. Flad, Infect. Immun., 60, 3309 (1992).

21 T. Kusama, T. Soga, Y. Ono, E. Kumazawa, E. Shioya, K.

Nakayama, K. Uoto, and Y. Osada, Chem. Pharm. Bull., 39, 3244

35 T. Gustman, N. Haberer, S. F. Carroll, U. Seydel, and A.

Wiese, Biol. Chem., 382, 425 (2001).

36 K. Takayama, Z. Z. Din, P. Mukerjee, P. H. Cooke, and T.

N. Kirkland, J. Biol. Chem., 265, 14023 (1990).

Article Doi

Synthesis and biological activities of lipid A analogs possessing β-glycosidic linkage at 1-position

DOI: 10.1246/bcsj.76.485

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