Synthesis of lipid A monosaccharide analogues containing acidic amino acid: Exploring the structural basis for the endotoxic and antagonistic activities

Bioorganic & Medicinal Chemistry 14 (2006) 6759–6777

Synthesis of lipid A monosaccharide analogues containing acidic

amino acid: Exploring the structural basis for the endotoxic

and antagonistic activities

Masao Akamatsu,^aYukari Fujimoto,^aMikayo Kataoka,^aYasuo Suda,^b

Shoichi Kusumoto^a,ꢀand Koichi Fukase^a,*

^aDepartment of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan

^bDepartment of Nanostructure and Advanced Materials, Graduate School of Science and Engineering,

Kagoshima University, Kagoshima 890-0065, Japan

Received 23 March 2006; revised 25 May 2006; accepted 25 May 2006

Available online 7 July 2006

Abstract—For elucidation of the structural and conformational requirements on the endotoxic and antagonistic activity of lipid A

derivatives, we designed and synthesized lipid A analogues containing acidic amino acid residues in place of the non-reducing end

phosphorylated glucosamine. Deﬁnite switching of the endotoxic or antagonistic activity was observed depending on the diﬀerence

of the acidic groups (phosphoric acid or carboxylic acid) in the lipid A analogues.

1. Introduction

(Fig. 1). In contrast, the biosynthetic precursor 2 of

LPS with four acyl chains shows antagonistic activity

against cytokine induction stimulated with LPS in the

human system (Fig. 1).^16,18–20

The innate immune system is the ﬁrst line of the host de-

fense against invading microorganisms. It is a phyloge-

netically ancient mechanism found in essentially every

multicellular organism from plants to humans.¹Toll-like

receptors (TLRs) play a fundamental role in the molec-

ular recognition of microbial components, such as

lipopolysaccharide (LPS), peptidoglycan (PGN),

lipoprotein, bacterial DNA, and viral RNA.^2,3LPS is

sensed by a receptor complex consisting of TLR4^4–8

and an adaptor protein MD-2.^9,10LPS is a component

of the outer membrane of Gram-negative bacteria, and

is known as a potent immunostimulator or endotoxin

for its strong activity which sometimes causes lethal sep-

sis.^11–15It was previously demonstrated in our laborato-

ry that the terminal glycolipid part, lipid A, is the

chemical entity responsible for the biological activity

of LPS by our total synthesis of Escherichia coli lipid

A (1),^16,17which consists of a glucosamine disaccharide

having two phosphate groups and six acyl chains

Some monosaccharide lipid A analogues have been

reported to date. The non-reducing sugar moiety (com-

pound 411) of E. coli lipid A and most of its known ana-

logues having saturated acyl chains showed cytokine

(e.g., IL-8, TNF-a)-inducing activity.^21–23In contrast,

the reducing sugar moiety (compound 401, lipid X) 4

showed almost no activity for the cytokine induction,²¹

but has weak antagonistic activity against LPS.^16,24

The previous results on the structure–bioactivity rela-

tionships have shown the number and length of acyl

chains and also the acidic groups are important for the

bioactivity of lipid A.^25–28The NMR conformation

analysis of the tetraacylated lipid A 2 revealed that 2

takes a particular conformation and forms a character-

istic supramolecular assembly.²⁹These studies indicate

that hydrophobic interaction between acyl groups is

important for maintaining the conformation of lipid

A, which would be recognized by LPS receptors.

Keywords: Innate immunity; Lipid A; Cytokine induction; IL-6;

Endotoxin.

*

Corresponding author. Fax: +81 6 6850 5419; e-mail: koichi@chem.

sci.osaka-u.ac.jp

Hawkins reported weak LPS receptor agonists that have

a ﬂexible backbone without the disaccharide structure.³⁰

Their study indicated that the disaccharide backbone is

ꢀ

Present address: Suntory Institute for Bioorganic Research, Shi-

mamoto-cho, Mishima-gun, Osaka 618-8503, Japan

doi:10.1016/j.bmc.2006.05.051

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M. Akamatsu et al. / Bioorg. Med. Chem. 14 (2006) 6759–6777

Figure 1. The chemical structures of Escherichia coli lipid A (506) 1, its biosynthetic precursor (406) 2, triacylated analogues of lipid A with

4⁰-phosphate group 3a and with 3⁰-phosphate group 3b and the right part of E. coli lipid A (lipid X) 4.

not essential for the immunostimulatory activity but elu-

cidation of the endotoxic conformation is diﬃcult

because of the conformational ﬂexibility of their

agonists. These results prompted us to design new lipid

A analogues by molecular modeling on the basis of the

molecular mechanics calculation, which was used for

conformational analysis of 2. The bioactive conforma-

tion such as the relative spatial position of two acidic

moieties and the arrangement of the acyl groups can

be estimated by evaluation of the biological activity of

the analogues.

In this study, we thus designed a series of lipid A ana-

logues to investigate the structural requirements for the

biological activities based on the triacyl-type lipid A

analogue 3b(Fig. 1),²⁵which was synthesized in ourgroup

and revealed as an antagonist against LPS. The non-

reducing end of lipid A (3-phosphorylated glucosamine)

was substituted with an acidic amino acid such as aspartic

acid or phosphoserine (Fig. 2), and the acylation arrange-

ment was varied. We here synthesized 12 compounds of

acidic amino acid-substituted lipid A analogues and

observed the biological activities by such as the Limulus

test and human cytokine (IL-6) induction assay.

Recently, Miyake demonstrated that a potent LPS

receptor agonist such as E. coli LPS and lipid A induced

TLR4/MD-2 dimerization but antagonists did not. They

also revealed that maximal binding of the antagonistic

biosynthetic precursor analogue to TLR/MD-2 was

2-fold higher than that of the agonistic E. coli lipid A

analogue, suggesting two TLR4/MD-2 bind to a single

molecule of agonist 1, whereas one TLR4/MD-2 binds

to one antagonist 2.^31,32The size of the hydrophobic

moieties should determine the response of TLR4/MD-2

but the structural diﬀerence between LPS agonists and

antagonists is not clearly understood. Elucidation of

the structural requirements for these activities is another

objective of the present study.

2. Results and discussion

2.1. Design of the monosaccharide analogues of lipid A

In our previous study, we synthesized triacyl lipid A

analogues with a 4⁰-phosphate group (3a) and 3⁰-phos-

phate group (3b) which have shown antagonistic activity

against LPS.²⁵Therefore, we have designed novel lipid

A analogues based on 3b, in which the non-reducing

end (3-phosphorylated glucosamine) was substituted

with aspartic acid or phosphoserine containing ^D- or

L-conﬁgurations at the a-position of the amino acids,

M. Akamatsu et al. / Bioorg. Med. Chem. 14 (2006) 6759–6777

6761

Figure 2. The structural design of the monosaccharide analogues of lipid A containing acidic N-acyl-amino acids.

and three types of acyl group distributions were intro-

duced (Fig. 2). As it was also previously shown that

the 1-phosphate group can be replaced with a carb-

oxymethyl group without loss of the activity in the case

of E. coli type lipid A (506) and the biosynthetic precur-

sor,^33,34we thus used a more stable and synthetically

easy carboxymethyl group at the 1-position of

glucosamine.

2.2. Synthesis of the monosaccharide analogues of lipid A

For the synthesis of monosaccharide lipid A analogues

containing an acidic N-acyl-amino acid, aspartic acid

or phosphoserine, we ﬁrst synthesized the key intermedi-

ate 17 as shown in Scheme 1. First, the ^D-glucosamine

derivative 12 possessing a free hydroxyl group at the

3-position was prepared from ^D-glucosamine hydrochlo-

ride 11.³⁵Condensation of 12 and (R)-3-benzyloxytetra-

decanoic acid 18a with N,N⁰-dicyclohexylcarbodiimide

(DCC) and 4-(dimethylamino)pyridine (DMAP) in

CH₂Cl₂gave 13 in 91% yield. The trichloroethoxycar-

bonyl (Troc) group at the 2-position was reductively

cleaved with a Zn–Cu couple in acetic acid, followed

by introduction of 18a to obtain glycolipid 14 in 86%

yield for 2 steps. The anomeric allyl group of 13 was oxi-

dized with osmium (VIII) oxide to aﬀord a diol and suc-

cessive oxidative cleavage with lead (IV) acetate gave the

aldehyde 15 in 84% yield from 14. Further oxidation

with sodium chlorite and 2-methyl-2-butene under phos-

To predict conceivable conformations of each molecule

in water, the lower energy conformations of 12 newly

designed monosaccharide analogues of lipid A 5a, 5b,

6a, 6b, 7a, 7b, 8a, 8b, 9a, 9b, 10a, and 10b in water were

calculated by using the united atom AMBER* force-

ﬁeld. The results showed that all analogues tend to gath-

er the acyl chains to form a large hydrophobic domain

as in lipid A (506) 1 or a biosynthetic precursor (406)

2,²⁹though the relative spatial position of two acidic

moieties and acyl groups was diﬀerent depending on

the acylation pattern.

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M. Akamatsu et al. / Bioorg. Med. Chem. 14 (2006) 6759–6777

of the hydroxy group due to steric hindrance. We then

tried benzyloxycarbonylation with 1-(benzyloxycarbon-

yl)benzotriazole (ZOBt) to obtain 22a, 22b, 23a, and

23b, respectively. Treatment with triﬂuoroacetic acid

(TFA) in anhydrous dichloromethane to cleave the

tert-butyl group of the serine residue unexpectedly led

to hydrolysis of ester at the 6-position of glucosamine.

This side reaction was prevented by adding anisole as

a scavenger to yield 24a, 24b, 25a, and 25b in the range

of 80% to quantitative yields. Subsequent phosphoryla-

tion was successfully achieved with o-xylylene-N,N-diet-

hylphosphoramidite (XEPA) and 1H-tetrazole, followed

by oxidation with m-chloroperbenzoic acid (mCPBA) to

give 26a, 26b, 27a, and 27b in 40–59% yields.³⁸Finally,

catalytic hydrogenolysis aﬀorded the desired products

5a (^D-Ser(P)1011), 5b (L-Ser(P)1011), 6a (D-Ser(P)-

2011), and 6b (^L-Ser(P)2011), respectively.

Compounds 7a (^D-Ser(P)1111) and 7b (L-Ser(P)1111)

were synthesized as shown in Scheme 3. First, the Fmoc

groups of the serine of 19a and 19b were cleaved with

TBD–methyl polystyrene, and subsequent condensation

with two equivalents of fatty acid 18a to both the amino

group at the serine residue and the hydroxyl group at the

4-position of glucosamine simultaneously gave com-

pounds 28a and 28b by using 1-ethyl-3-(3⁰-dimethylami-

nopropyl)carbodiimide hydrochloride (WSCDÆHCl) and

1-hydroxy-7-benzotriazole (HOBt) together with

DMAP. After cleavage of the tert-butyl group with

TFA, introduction of a phosphate moiety to the result-

ing 29a and 29b gave 30a and 30b. Then, subsequent

hydrogenolysis gave 7a (^D-Ser(P)1111) and 7b (L-Ser

(P)1111).

Scheme 1. Reagents and conditions: (a) 18a, DCC, DMAP, CH₂Cl₂,

91%; (b) Zn–Cu couple, AcOH; (c) 18a, WSCDÆHCl, HOAt, CH₂Cl₂,

86% from 13; (d) OsO₄, N-methylmorpholine-N-oxide, THF, t-BuOH,

H₂O; (e) Pb(OAc)₄, benzene, 84% from 14; (f) NaClO₂, NaH₂PO₄,

2-methyl-2-butene, THF, t-BuOH, H₂O; (g) PhCHN₂, CH₂Cl₂, 0 ꢁC,

89% from 15; (h) 90% AcOH, 50 ꢁC, quant.

Six kinds of aspartic acid-substituted analogues 8a, 8b,

9a, 9b, 10a, and 10b were also synthesized with the pro-

cedures (Scheme 4) similar to the synthesis of phospho-

serine-substituted analogues 5a, 5b, 6a, and 6b. In this

synthesis, commercially available aspartic acid deriva-

tives, Boc-^D-Asp(OBn) and Boc-L-Asp(OBn), were con-

densed to the monosaccharide 17 to give 31a and 31b,

respectively. In this condensation, no racemization at

the chiral center of aspartic acid was observed. The

tert-butoxycarbonyl (Boc) groups of compounds 31a

and 31b were cleaved under acidic conditions, then each

fatty acid, (R)-3-benzyloxytetradecanoic acid 18a or (R)-

3-(tetradecanoyloxy)tetradecanoic acid 18b, was con-

densed to the liberated amines to obtain 32a, 32b, 33a,

and 33b. These compounds were catalytically hydroge-

nated in THF under 20 kgf/cm²of H₂atmosphere to

yield Asp1011s (8a and 8b) and Asp2011s (9a and 9b).

In the synthesis of Asp1111s (10a and 10b), after depro-

tection of the Boc group in 31a or 31b, two equivalents

of 18a was condensed to both the liberated amine at the

aspartic acid residue and the hydroxyl group at the 4-po-

sition of glucosamine. Finally, palladium-catalyzed

hydrogenolysis cleaved all the benzyl-type protecting

groups to yield the desired compounds, ^D-Asp1111

(10a) and ^L-Asp1111 (10b). The monosaccharide part

of the analogues (35) was also synthesized by hydrogen-

olysis of 17 with palladium-black catalyst (Scheme 5).

We thus achieved the synthesis of 12 analogues and ap-

plied them to a biological study.

phate-buﬀered solution yielded a 1-O-carboxymethyl

compound, which was then protected with a benzyl

group by treatment with phenyldiazomethane to give

16 in 89% yield over 2 steps from 15. Cleavage of the

4,6-O-benzylidene group was quantitatively achieved

by heating up to 50 ꢁC in 90% acetic acid solution to

give a common intermediate 17.

As shown in Scheme 2, an N-9-ﬂuorenylmethoxycarbon-

yl (Fmoc)-protected serine derivative, Fmoc-^D-Ser(^tBu)

or Fmoc-^L-Ser(^tBu), was introduced to the 6-hydroxyl

group of monosaccharide 17 by using 1-(2-mes-

itylenesulfonyl)-3-nitro-1,2,4-triazole (MSNT) and N-

methylimidazole (NMI) in THF to give 19a (^D-serine)

or 19b (^L-serine), respectively.³⁶The Fmoc groups of

19a and 19b were cleaved with TBD (1,3,5-triazabicy-

clo[4.4.0]dec-5-ene)-methyl polystyrene³⁷resin to facili-

tate the work-up operation only by ﬁltration. Then,

the free amino group of the serine residue was acylated

with (R)-3-benzyloxytetradecanoic acid 18a for 20a and

20b, or with (R)-3-(tetradecanoyloxy)tetradecanoic acid

18b for 21a and 21b. In the next step, the 4-hydroxyl

group of 20a, 20b, 21a, and 21b was protected. An at-

tempt for the protection with a benzyl group by treat-

ment with benzyl bromide and silver oxide was

unsuccessful presumably because of the low reactivity

M. Akamatsu et al. / Bioorg. Med. Chem. 14 (2006) 6759–6777

6763

Scheme 2. Reagents and conditions: (a) Fmoc-D-Ser(^tBu) or Fmoc-L-Ser(^tBu), 1-(2-mesitylenesulfonyl)-3-nitro-1,2,4-triazole, N-methylimidazole,

THF, 85–91%; (b) TBD–methyl polystyrene, THF; (c) 18a or 18b, WSCDÆHCl, HOBt, CH₂Cl₂, 52–68%; (d) ZOBt, DMAP, THF, 50–60 ꢁC, 80%–

quant.; (e) triﬂuoroacetic acid, anisole, CH₂Cl₂, 80%–quant.; (f) XEPA, 1H-tetrazole, CH₂Cl₂, rt, then m-CPBA, ꢀ20 ꢁC, 40–59%; (g) palladium-

black, H₂(20 kgf/cm²), THF, 74%–quant.

Scheme 3. Reagents and conditions: (a) TBD–methyl polystyrene, THF; (b) 18a, WSCDÆHCl, HOBt, DMAP, CH₂Cl₂, 55–61%; (c) triﬂuoroacetic

acid, anisole, CH₂Cl₂, 82–83%; (d) XEPA, 1H-tetrazole, CH₂Cl₂, rt, then m-CPBA, ꢀ20 ꢁC, 49–81%; (e) palladium-black, H₂(20 kgf/cm²), THF,

67–93%.

2.3. Biological study

the result showed the Limulus activities of our synthetic

analogues were obviously aﬀected by the distribution of

the acyl chains (Fig. 3). The 2011-type compounds

(marked with circle) showed strongest Limulus activities

in three acylation patterns among the same amino

acid-substituted analogues. The 1011-type compounds

For detection of LPS, the Limulus test has been often

used. This test uses the reaction of Limulus amebocyte

lysate (LAL) of horseshore crab for quantitative

measurement of LPS.^39,40We carried out the test and

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M. Akamatsu et al. / Bioorg. Med. Chem. 14 (2006) 6759–6777

Scheme 4. Reagents and conditions: (a) Boc-D-Asp(OBn) or Boc-L-Asp(OBn), DCC, DMAP, CH₂Cl₂, 73–97%; (b) triﬂuoroacetic acid, CH₂Cl₂; (c)

(R)-3-benzyloxytetradecanoic acid 18a or 18b, WSCDÆHCl, HOBt, CH₂Cl₂, 64–77%; (d) palladium-black, H₂(20 kgf/cm²), THF, 82%–quant.; (e)

triﬂuoroacetic acid, CH₂Cl₂; (f) 18a, WSCDÆHCl, HOBt, CH₂Cl₂, 47–56%; (g) palladium-black, H₂(20 kgf/cm²), THF, 96–98%.

IL-6 induction by LPS, most of the analogues exhibit-

ed stronger inhibitory activities than the reducing end

part 35, whereas ^D-Asp2011 (9a) and D-Asp1011 (8a)

did not show the activity (Fig. 4B). The phosphoser-

ine-containing analogues showed stronger inhibitory

activities than the corresponding aspartic acid-contain-

ing analogues having the same acylation pattern in

general. Obvious trends were also observed between

the inhibitory activities and the acylation patterns.

In both cases of ^D- and L-phosphoserine analogues,

the 2011-acylation form exhibited the highest inhibito-

ry activity. The 1011-form showed slightly weaker

activity than the 2011-form but stronger activity than

the 1111-form. In the case of aspartic acid analogues,

the ^L-form showed stronger inhibition than the D-

form, while ^D- and L-phosphoserine analogues did

not exhibit deﬁnite diﬀerences. The weaker activity

of 1111-form might be caused by the diﬀerence of

the relative position of two acidic moieties and acyl

groups in 1111-form from that in 2011-form or

1011-form. Molecular mechanics calculations of ^D-

Asp1011 (8a), ^D-Asp2011 (9a), and D-Asp1111 (10a)

showed that 8a and 9a have similar spatial arrange-

ment of acidic moieties and acyl groups but 10a has

the diﬀerent spatial arrangement (Fig. 5). Probably,

the 4-acyl group inﬂuences the packing of acyl chains

and molecular conformation.

Scheme 5. Reagents: (a) palladium-black, H₂(20 kgf/cm²), THF,

quant.

(triangle) showed comparatively lower but still showed

deﬁnite activities, and 1111-type compounds (square)

showed little activities.

We also measured human cytokine induction and the

inhibitory activities against LPS. Cytokine-inducing

and inhibitory activities of the analogues and standard

LPS (E. coli O111:B4) were tested in human periphe-

ral whole-blood (hpwb) cells.⁴¹The stimulated levels

of cytokines, that is, interleukin-6 (IL-6), were mea-

sured by means of enzyme-linked immunosorbent as-

say (ELISA). The IL-6-inducing activities (A) and

the inhibitory activities (B) are shown in Figure 4.

Most of analogues exhibited no induction of IL-6,

but tetraacylated ^D-Asp2011 (9a) exhibited concentra-

tion-dependent induction at the level over 100 ng/

mL. ^L-Asp2011 (9b) also exhibited the IL-6 induction

at 10 lg/mL concentration, although the activity was

about a 100-fold weaker than that of ^D-Asp2011

(9a). In this assay, the monosaccharide analogue with-

out amino acid (the reducing end part) 35 induced no

IL-6 (Fig. 4A). As for the inhibitory activity against

3. Conclusion

In the present study, we synthesized acidic amino

acid-containing monosaccharide lipid A analogues

with aspartic acid and phosphoserine having three

kinds of acylation patterns. All the phosphoserine

analogues showed the antagonistic activity, whereas

D-Asp2011 and L-Asp2011 showed the immunostimu-

lating activity. The agonistic and antagonistic activities

were switched by structural change between the phos-

M. Akamatsu et al. / Bioorg. Med. Chem. 14 (2006) 6759–6777

6765

Figure 3. Limulus activities of synthetic lipid A analogues and LPS. (A) Aspartic acid-substituted analogues. (B) Phosphoserine-substituted

analogues.

phoryl group (e.g., D-Ser(P)2011, 6a) and the carbox-

ylic group (^D-Asp2011, 9a). We also observed that the

tetraacylated analogues showed stronger inhibitions

than the triacylated analogues. The results suggested

that the receptor protein of LPS/lipid A recognizes

second acidic groups on amino acid residues, and also

it seemed that the volume and shape of the hydropho-

bic part consisted with acyl chains were recognized by

the receptor. In the case of Limulus test, the tendency

was dramatically inﬂuenced by the acylation patterns.

The present study clearly showed that structural

requirements for expression of the Limulus activity

are diﬀerent from cytokine-inducing activities or its

antagonistic activities, since 1111-type analogues did

not stimulate Limulus test. We are now proceeding

to investigate more detailed biological activities and

the recognition mechanism by using the monosaccha-

ride lipid A analogues.

4. Experimental

4.1. General methods

NMR spectra were measured with a JEOL JNM-GX400

and a JEOL JMM-GX270 at 30 ꢁC unless otherwise

speciﬁed, and analyzed using Alice^ꢂprogram (version

2.0). The proton chemical shifts in CDCl₃are given in

d values from tetramethylsilane as an internal standard,

and the chemical shifts in other solvents or conditions

are given in d values from the residual proton signal of

the solvent. Mass spectra were obtained with ESI-TOF

mass spectrometer (Applied Biosystems, Mariner^TM).

Speciﬁc optical rotations were measured on a Perkin-

Elmer 241 polarimeter. Recycling preparative HPLC

was carried out with Japan Analytical Industry

LC908. Silica-gel chromatography was performed using

Kieselgel 60 (Merck, 0.040–0.063 mm) under medium

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M. Akamatsu et al. / Bioorg. Med. Chem. 14 (2006) 6759–6777

Figure 4. (A) IL-6 induction activity of synthetic lipid A analogues in comparison with LPS (Escherichia coli, O111:B4). (B) Antagonistic activity of

synthetic lipid A analogues to suppress IL-6 induction by LPS (Escherichia coli, O111:B4, 5 ng/mL). The induced level of IL-6 was determined by

ELISA. Average values of three repeated assays and deviations of individual experiments are given in the graph. In B, the dashed line shows the level

of the IL-6 induction of positive control (5 ng/mL of LPS).

Figure 5. The lower energy conformations of D-Asp2011 (brown), D-Asp1011 (blue), and D-Asp1111 (green), calculated by using the united atom

AMBER* forceﬁeld, with MontecCarlo-Low mode conformational search. Sugar moieties (light blue) and acidic moieties (pink) were highlighted.

pressure (2–4 kg/cm²) using the indicated solvent sys-

tems. Analytical and preparative thin-layer chromatog-

raphies (TLC) were performed on Kieselgel 60F₂₅₄

Plates (Merck, 0.25 mm thickness) and precoated Kie-

selgel 60F₂₅₄Plates (Merck, 0.5 mm thickness), respec-

tively. Nonaqueous reactions were carried out under

argon atmosphere unless otherwise noted. Anhydrous

dichloromethane (CH₂Cl₂) was prepared by distillation

M. Akamatsu et al. / Bioorg. Med. Chem. 14 (2006) 6759–6777

6767

from calcium hydride and phosphorus pentoxide. Anhy-

drous acetonitrile, tetrahydrofuran (THF), and toluene

were purchased from Kanto Chemicals Co. Distilled

water was purchased from Otsuka (Tokyo, Japan) or

prepared by a combination of Toray Pure LV-308 (Tor-

ay) and GSL-200 (Advantec, Tokyo, Japan). The water

was used as an eluent of liquid–liquid partition column

chromatography and a solvent of lyophilization. Molec-

PhCH), 5.95–5.85 (m, J = 17.1 Hz, J = 10.4 Hz,

J = 5.3 Hz, 1H, OCH₂CH@CH₂), 5.47 (s, 1H, PhCH),

5.44–5.39 (dd, J = 10.1 Hz, J = 9.9 Hz, 1H, H-3), 5.37–

5.34 (d, J = 10.1 Hz, 1H, NH), 5.34–5.29 (dd,

J = 17.2 Hz, J = 1.5 Hz, 1H, OCH₂CH@CH₂), 5.26–

5.23 (dd, J = 10.4 Hz, J = 1.5 Hz, 1H, OCH₂CH@CH₂),

4.94–4.93 (d, J = 3.7 Hz, 1H, H-1), 4.74–4.71 (d,

J = 12.1 Hz, 1H, OCH₂Ph), 4.60–4.57 (d, J = 12.1 Hz,

1H, OCH₂Ph), 4.52–4.49 (d, J = 12.1 Hz, 1H, CH₂of

Troc), 4.41–4.38 (d, J = 12.1 Hz, 1H, CH₂of Troc),

4.30–4.27 (m, J = 4.7 Hz, J = 10.2 Hz, 1 H, H-6a), 4.24–

4.19 (dd, J = 12.7 Hz, J = 5.3 Hz, 1H, OCH₂CH@CH₂),

4.10–4.00 (m, J = 3.7 Hz, J = 10.1, J = 10.1 Hz,

J = 12.8 Hz, J = 6.4 Hz, 2H, OCH₂CH@CH₂, H-2),

3.98–3.92 (ddd, J = 4.7 Hz, J = 9.9 Hz, 1H, H-5), 3.83–

3.68 (m, 3H, C^bH of acyl, H-6b, H-4), 2.70–2.64 (dd,

J = 15.3 Hz, J = 6.4 Hz, 1H, C^aH₂of acyl), 2.46–2.41

(dd, J = 15.3 Hz, J = 6.0 Hz, 1H, C^aH₂of acyl), 1.53–

1.41 (m, 2H, CH₂of acyl), 1.32–1.18 (CH₂of acyl),

0.90–0.86 (t, J = 7.2 Hz, 3H, CH₃of acyl); Found: C,

60.61; H, 6.70; N, 1.74. Calcd for C₄₀H₅₄O₉NCl₃: C,

60.11; H, 6.81; N, 1.75.

˚

ular sieves 4 A were activated in vacuo at 250 ꢁC for 3 h

before use. All other commercially obtained materials

were used as received.

4.2. Computational analysis

To predict the conformation of our synthetic compound

in water, we carried it out with the united atom Amber*

forceﬁeld in the environment of generalized Born/surface

area (GB-SA) continuum solvent model for water. The

molecular mechanics methods and conformational search

(MCMM (Monte Carlo Multiple Minimum) and Low

Mode calculations) calculation of lipid A analogues were

performed with MacroModel^TM8.1 (Schro¨dinger Inc.).

4.3. Biological assay

4.5. Allyl 4,6-O-benzylidene-3-O-((R)-3-benzyloxytetra-

decanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-2-

deoxy-a-^D-glucopyranoside (14)

We used commercially available standard LPS (E. coli

O111:B4, Sigma Chemical Co.) for each biological

assay. Biosynthetic precursor 2 used in this study was

synthesized in our laboratory.^18,42

To a solution of 13 (2.05 g, 2.57 mmol) in AcOH

(10 mL) was added zinc–copper couple (ca. 8.00 g).

The suspension was stirred at room temperature for

2 h. After the precipitate was ﬁltered oﬀ, the ﬁltrate

was coevaporated in vacuo with toluene. To the residue

was added saturated aqueous NaHCO₃(20 mL) and

then the mixture was extracted with AcOEt. The com-

bined extracts were washed with saturated aqueous

NaHCO₃and brine, dried over Na₂SO₄, and concentrat-

ed in vacuo to give crude amine which was used for the

following reaction without further puriﬁcation.

Limulus test was carried out using the Endospacy^TMES-

50M set (Seikagaku Kogyo, Tokyo, Japan). We per-

formed the test according to the procedure included in

the kit.

In cytokine induction assay, a mixture of a test sample

and heparinized hpwb in RPMI 1640 medium (Flow

Laboratories, Irvine, Scotland) was incubated at 37 ꢁC

in 5% CO₂for 24 h. Otherwise, in cytokine inhibitory as-

say, a mixture of test sample, LPS (5 ng/mL), and hepa-

rinized hpwb was incubated and the level of cytokines

induced was measured in the same manner.

To the solution of the amine (R)-3-benzyloxytetradeca-

noic acid (18a) (0.94 g, 2.82 mmol) and HOBt (0.35 g,

2.57 mmol) in absolute CH₂Cl₂(25 mL) was added

WSCDÆHCl (1.48 g, 7.70 mmol) at room temperature un-

der Ar atmosphere. After stirring overnight, the mixture

was quenched with saturated aqueous NaHCO₃and

extracted with AcOEt. The combined extracts were

washed with saturated aqueous NaHCO₃and brine, dried

over Na₂SO₄, and concentrated in vacuo. The residue was

puriﬁed by silica-gel column chromatography (80 g, tolu-

4.4. Allyl 4,6-O-benzylidene-3-O-((R)-3-benzyloxytetra-

decanoyl)-2-deoxy-2-(2,2,2-trichloroethoxycarbonylami-

no)-a-^D-glucopyranoside (13)

To a solution of 12 (6.47 g, 13.0 mmol) and (R)-3-benzyl-

oxytetradecanoic acid (18a) (5.38 g, 15.6 mmol) in abso-

lute CH₂Cl₂(100 mL) were added DMAP (318 mg,

2.6 mmol) and DCC (4.02 g, 19.5 mmol) at room temper-

ature under N₂atmosphere. After stirring for 2 h, the mix-

ture was quenched with saturated aqueous NaHCO₃

(50 mL) and extracted with CHCl₃. The combined ex-

tracts were washed with aqueous 10% citric acid, saturat-

ed aqueous NaHCO₃, and brine, dried over MgSO₄, and

concentrated in vacuo. The residue was suspended with

AcOEt and the insoluble materials were removed. The

crude sample was puriﬁed by silica-gel column chroma-

ene/AcOEt = 50:1) to give 14 as a white solid (2.01 g,

23

D

84%). ½aꢁ +0.363 (c 1.0, CHCl₃); ESI-TOF (positive)

1

m/z = 962.7 [(M+Na)⁺]; H NMR (400 MHz, CDCl₃),

d = 7.42–7.20 (m, 15H, OCH₂Ph, PhCH), 6.30–6.28 (d,

J = 9.5 Hz,

1H,

NH),

5.78–5.68

(m,

1H,

OCH₂CH@CH₂), 5.46 (s, 1H, PhCH), 5.41–5.36 (dd,

J = 10.1 Hz, J = 9.9 Hz, 1H, H-3), 5.22–5.17 (dd,

J = 17.2 Hz, J = 1.5 Hz, 1H, OCH₂CH@CH₂), 5.14–

5.10 (dd, J = 10.4 Hz, J = 1.4 Hz, 1H, OCH₂CH@CH₂),

4.80–4.79 (d, J = 3.7 Hz, 1H, H-1), 4.56–4.48 (m,

J = 11.6 Hz, 3H, OCH₂Ph, OCH₂Ph), 4.43–4.36 (m,

J = 3.7 Hz, J = 10.5, J = 9.6 Hz, J = 11.6 Hz, 2H, H-2,

OCH₂Ph), 4.27–4.24 (dd, J = 4.7 Hz, J = 10.2 Hz, 1H,

H-6a), 4.06–4.00 (dd, J = 12.7 Hz, J = 5.5 Hz, 1H,

tography (500 g, toluene/AcOEt = 50:1) to give 13 as a

23

D

white solid (9.20 g, 89%). ½aꢁ +0.358 (c 1.0, CHCl₃);

ESI-TOF (positive) m/z = 822.3 [(M+Na)⁺]; H NMR

1

(400 MHz, CDCl₃) d = 7.42–7.21 (m, 10H, OCH₂Ph,

6768

M. Akamatsu et al. / Bioorg. Med. Chem. 14 (2006) 6759–6777

OCH₂CH@CH₂), 3.94–3.88 (ddd, J = 9.8 Hz, J = 4.6 Hz,

J = 9.8 Hz, 1H, H-5), 3.84–3.68 (m, 5H, OCH₂CH@CH₂,

C^bH of acyl, H-6b, H-4), 2.70–2.64 (m, J = 15.3 Hz,

J = 6.3 Hz, 1H, C^aH₂of acyl), 2.44–2.39 (dd,

J = 15.3 Hz, J = 6.1 Hz, 1H, C^aH₂of acyl), 2.37–2.27

(m, 2H, C^aH₂of acyl), 1.61–1.20 (m, 40H, CH₂of acyl),

0.90–0.86 (m, 3H, CH₃of acyl); Found: C, 73.81; H,

9.06; N, 1.50. Calcd for C₅₈H₈₅O₉N: C, 74.09; H, 9.11;

N, 1.49.

AcOEt. The combined extracts were washed with water

and brine, dried over Na₂SO₄, and concentrated in vacuo.

The residue was puriﬁed by silica-gel column chromatog-

raphy (40 g, toluene/AcOEt = 10:1) to give 16 as a white

23

solid (885.6 mg, 84%). ½aꢁ +0.348 (c 1.0, CHCl₃); ESI-

D

TOF (positive) m/z = 1070.7 [(M+Na)⁺]; ¹H NMR

(400 MHz, CDCl₃) d = 7.40–7.21 (m, 20H, OCH₂Ph,

PhCH), 6.71–6.69 (d, J = 9.5 Hz, 1H, NH), 5.44 (s, 1H,

PhCH), 5.43–5.38 (dd, J = 9.9 Hz, J = 10.1 Hz, 1H, H-

3), 5.12 (s, 2H, COOCH₂Ph), 4.80–4.79 (d, J = 3.7 Hz,

1H, H-1), 4.59–4.47 (m, 3H, OCH₂Ph, OCH₂Ph), 4.45–

4.39 (ddd, J = 3.7 Hz, J = 9.9 Hz, J = 9.5 Hz, 1H, H-2),

4.38–4.35 (d, J = 11.6 Hz, 1H, OCH₂Ph), 4.22–4.18 (dd,

J = 4.9 Hz, J = 10.4 Hz, 1H, H-6a), 4.01–3.92 (m, 3H,

1-O–CH₂–CO₂–, H-6b), 3.85–3.78 (m, 2H, C^bH of acyl),

3.74–3.67 (m, 2H, H-5, H-4), 2.70–2.64 (m, J = 15.1 Hz,

J = 6.6 Hz, 1H, C^aH₂of acyl), 2.43–2.37 (m,

J = 15.1 Hz, J = 5.8 Hz, 3H, C^aH₂of acyl), 1.48–1.43

(m, 40H, CH₂of acyl), 0.90–0.86 (m, 6H, CH₃of acyl);

Found: C, 72.94; H, 8.59; N, 1.36; Calcd for

C₆₄H₈₉O₁₁N: C, 73.32; H, 8.56; N, 1.34.

4.6. Formylmethyl 4,6-O-benzylidene-3-O-((R)-3-benzyl-

oxytetradecanoyl)-2-((R)-3-benzyloxytetradecanoylami-

no)-2-deoxy-a-^D-glucopyranoside (15)

To a solution of 14 (126.4 mg, 0.134 mmol) in THF/t-

BuOH/H₂O = 10:10:1 (20.1 mL) were added 4-meth-

ylmorpholine N-oxide (64.7 mg, 0.536 mmol) and aque-

ous OsO₄(1 M, 26.9 mL, 26.9 mmol) at room

temperature. After stirring for 8 h, to the mixture was

added 10% aqueous Na₂S₂O₃. The mixture was extract-

ed with AcOEt, washed with 10% aqueous Na₂S₂O₃,

and concentrated in vacuo to give the crude diol, which

was subjected to the following oxidation without further

puriﬁcation.

4.8. Benzyloxycarbonylmethyl 3-O-((R)-3-benzyloxytet-

radecanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-2-

deoxy-a-^D-glucopyranoside (17)

To the solution of the diol in anhydrous benzene (1.2 mL)

was added Pb(OAc)₄(93%, 76.8 mg, 0.161 mmol) at room

temperature under N₂atmosphere. After stirring for 1 h,

the mixture was ﬁltered with Celite and extracted with

AcOEt. The combined extracts were dried over Na₂SO₄,

and coevaporated with toluene three times. The residue

was puriﬁed by silica-gel column chromatography (10 g,

CHCl₃/acetone = 20:1) to give 15 as a white solid

To the suspension of 16 (56.0 mg, 53.4 lmol) in CH₂Cl₂/

H₂O = 50:1 (0.25 mL) was added the CH₂Cl₂solution of

triﬂuoroacetic acid (10%, 250 mL) at room temperature

under N₂atmosphere. After stirring for 40 min, the mix-

ture was quenched with aqueous saturated NaHCO₃

(1 mL) and extracted with AcOEt. The combined ex-

tracts were washed with water and brine, dried over

Na₂SO₄, and concentrated in vacuo. The residue was

puriﬁed by silica-gel column chromatography (5 g,

1

(109.2 mg, 86%). H NMR (400 MHz, CDCl₃) d = 9.41

(s, 1H, CHO), 7.37–7.23 (m, 15H, OCH₂Ph, CHPh),

6.58–6.55 (d, J = 9.5 Hz, NH), 5.45–5.35 (m, 2H, CHPh,

H-3), 4.73–4.72 (d, J = 3.5 Hz, 1H, H-1), 4.58-4.37 (m,

5H, OCH₂Ph, H-2), 4.24–4.20 (dd, J = 4.9 Hz,

J = 10.2 Hz, 1H, H-6a), 3.94–3.67 (m, 7H, H-5, CH₂of

formylmethyl, C^bH of acyl, H-6b, H-4), 2.70–2.65 (dd,

J = 14.8 Hz, J = 6.0 Hz, 1H, C^aH₂of acyl), 2.46–2.28

(m, 3H, C^aH₂of acyl), 1.47–1.18 (m, 40H, CH₂of acyl),

0.90–0.86 (t, 6H, CH₃of acyl).

CHCl₃/acetone = 10:1) to give 17 as a white solid

23

(46.2 mg, 90%). ½aꢁ +0.342 (c 1.0, CHCl₃); ESI-TOF

D

1

(positive) m/z = 982.7 [(M+Na)⁺]; H NMR (400 MHz,

CDCl₃) d = 7.44–7.20 (m, 15H, OCH₂Ph), 6.68–6.66

(d, J = 9.5 Hz, 1H, NH), 5.16–5.11 (dd, J = 10.7 Hz,

J = 9.0 Hz, 1H, H-3), 5.11 (s, 2H, COOCH₂Ph), 4.79–

4.78 (d, J = 3.7 Hz, 1H, H-1), 4.55–4.50 (m, 4H,

OCH₂Ph), 4.32–4.26 (ddd, J = 3.7 Hz, J = 10.7 Hz,

J = 9.5 Hz, 1H, H-2), 4.03–3.98 (d, J = 16.6 Hz, 1H,

1-O–CH₂–CO₂–), 3.97–3.93 (d, J = 16.6 Hz, 1H, 1-O–

CH₂–CO₂–), 3.90–3.84 (m, 1H, C^bH of acyl), 3.83–

3.79 (m, 1H, C^bH of acyl), 3.75–3.68 (m, 3H, H-5, H-

6a, H-6b), 3.65–3.60 (dd, J = 9.3 Hz, J = 9.3 Hz, 1H,

H-4), 2.64–2.59 (dd, J = 14.7 Hz, J = 8.1 Hz, 1H, C^aH₂

of acyl), 2.48–2.44 (dd, J = 14.7 Hz, J = 4.6 Hz, 1H,

C^aH₂of acyl), 2.38–2.37 (d, J = 5.9 Hz, 2H, C^aH₂of

acyl), 1.65–1.42 (m, 2H, C^cH₂of acyl), 1.30–0.90 (m,

36H, CH₂of acyl), 0.88–0.86 (t, J = 6.1 Hz, 6H, CH₃

of acyl); Found: C, 70.92; H, 8.94; N, 1.51. Calcd for

C₅₇H₈₅O₁₁N: C, 71.29; H, 8.92; N, 1.46.

4.7. Benzyloxycarbonylmethyl 4,6-O-benzylidene-3-((R)-

3-benzyloxytetradecanoyl)-2-((R)-3-benzyloxytetra-

decanoylamino)-2-deoxy-2-a-^D-glucopyranoside (16)

To a suspension of 15 (942.3 mg, 1.00 mmol), NaH₂PO₄

(120.0 mg,

1.0 mmol),

and

2-methyl-2-butene

(530 mL, 5.00 mmol) in t-BuOH/H₂O = 4:1 (10 mL)

was added NaClO₂(80% purity, 113.1 mg, 1.00 mmol)

at room temperature. After stirring overnight, the

mixture was neutralized with aqueous 1 M HCl and

freeze-dried to give the crude carboxylic acid, which

was subjected to the following reaction without

further puriﬁcation.

4.9. Benzyloxycarbonylmethyl 3-O-((R)-3-benzyloxytet-

radecanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-2-

deoxy-6-O-(N-(9-ﬂuorenylmethoxycarbonyl)-O-tert-

butyl-^D-seryl)-a-D-glucopyranoside (19a)

To the suspension of the carboxylic acid in CH₂Cl₂

(10 mL) was dropped the Et₂O solution of phenyl dia-

zomethane (0.46 M) at room temperature until the sus-

pension turned a pale red. The mixture was quenched

with AcOH (90% purity, 1 mL) and extracted with

To a solution of 17 (300 mg, 312 lmol) and Fmoc-^D-

Ser(^tBu) (136 mg, 344 lmol) in absolute tetrahydrofuran

M. Akamatsu et al. / Bioorg. Med. Chem. 14 (2006) 6759–6777

6769

(5 mL) were added 1-(2-mesitylenesulfonyl)-3-nitro-

1,2,4-triazole (284 mg, 937 lmol) and N-methyl imidaz-

ole (249 mL, 3.12 lmol) at room temperature under Ar

atmosphere. After stirring for 13 h, the mixture was

washed with H₂O and brine, and then extracted with

AcOEt. The organic layer was dried over Na₂SO₄and

concentrated in vacuo. The mixture was puriﬁed by sil-

ica-gel ﬂash column chromatography (30 g, toluene/

AcOEt = 7:1 to 5:1) to give 19a (349 mg, 85%). ESI-

TOF (positive) m/z = 1348.72 [(M+Na)⁺]; ¹H NMR

(400 MHz, CDCl₃) d = 6.66 (1H, d, J = 9.3 Hz, 2-NH),

5.66 (1H, d, J = 8.8 Hz, Ser-NH), 5.16 (1H, t,

J = 9.9 Hz, H-3), 5.09 (1H, s, –CO₂–CH₂–Ph), 4.75

(1H, d, J = 3.4 Hz, H-1), 4.51–4.48 (6H, m, H-6a, Ser-

C^aH, –O–CH₂–Ph of 2-O-, 3-O-acyl), 4.38 (2H, t,

J = 8.1 Hz, CH₂of Fmoc), 4.33–4.21 (3H, m, H-2, CH

of Fmoc, and H-6b), 3.96 (2H, d, J = 4.4 Hz, 1-O–

CH₂–CO₂–), 3.92–3.79 (4H, m, H-5, C^bH of 2-O-, 3-

O-acyl, and Ser-C^bHa), 3.62–3.55 (2H, m, Ser-C^bHb

and H-4), 3.12 (1H, d, J = 3.5 Hz, 4-OH), 2.63–2.35

(4H, m, C^aH₂of 2-O-, 3-O-acyl), 1.16 (9H, s, t-Bu),

0.89–0.87 (6H, m, –CH₃of 2-O-, 3-O-acyl).

over Na₂SO₄and concentrated in vacuo. The mixture

was puriﬁed by silica-gel ﬂash column chromatography

(10 g, toluene/AcOEt = 4:1) to give 20a (116 mg, 53%).

ESI-TOF (positive) m/z = 1441.91 [(M+Na)⁺]; ¹H

NMR (400 MHz, CDCl₃) d = 7.04 (1H, d, J = 8.1 Hz,

Ser-NH), 6.65 (1H, d, J = 9.4 Hz, 2-NH), 5.16 (1H, t,

J = 10.3 Hz, H-3), 5.11 (1H, s, –CO₂–CH₂–Ph), 4.74–

4.70 (2H, m, H-1 and Ser-C^aH), 4.65–4.46 (7H, m, –

O–CH₂–Ph of 2-O-, 3-O-, Ser-acyl, and H-6a), 4.29

(1H, dt, J = 13.9, 5.0 Hz, H-2), 4.15 (1H, dd, J = 12.0,

2.1 Hz, H-6b), 3.96 (2H, d, J = 2.5 Hz, 1-O–CH₂–

CO₂–), 3.89–3.74 (5H, m, H-5, C^bH of 2-O-, 3-O-, Ser-

acyl, and Ser-C^bHa), 3.60–3.52 (2H, m, H-4 and Ser-

C^bHb), 3.22 (1H, d, J = 3.9 Hz, 4-OH), 2.74–2.35 (6H,

m, C^aH₂of 2-O-, 3-O-, Ser-acyl), 1.07 (9H, t,

J = 4.3 Hz, t-Bu), 0.89–0.87 (9H, m, –CH₃of 2-O-,

3-O-, Ser-acyl).

4.12. Benzyloxycarbonylmethyl 3-O-((R)-3-benzyloxytet-

radecanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-2-

deoxy-6-O-(N-((R)-3-(tetradecanoyloxy)tetradecanoyl)-

O-tert-butyl-^D-seryl)-a-D-glucopyranoside (20b)

4.10. Benzyloxycarbonylmethyl 3-O-((R)-3-benzyloxytet-

radecanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-2-

deoxy-6-O-(N-(9-ﬂuorenylmethoxycarbonyl)-O-tert-

butyl-^L-seryl)-a-D-glucopyranoside (19b)

In a manner similar to the synthesis of 20a, 19b (142 mg,

107 lmol) was deprotected and acylated with (R)-3-ben-

zyloxytetradecanoic acid (18a) (62 mg, 186 lmol) to give

20b (163 mg, 68%). ¹H NMR (400 MHz, CDCl₃)

d = 6.90 (1H, d, J = 8.1 Hz, Ser-NH), 6.62 (1H, d,

J = 9.3 Hz, 2-NH), 5.16 (1H, dd, J = 10.8, 9.3 Hz, H-

3), 5.10 (1H, s, –CO₂–CH₂–Ph), 4.74 (2H, dd, J = 6.3,

3.4 Hz, H-1 and Ser-C^aH), 4.58–4.46 (7H, m, H-6a

and –O–CH₂–Ph of 2-O-, 3-O-, Ser-acyl), 4.29 (1H,

ddd, J = 11.9, 8.3, 2.5 Hz, H-2), 4.15 (1H, dd, J = 12.2,

2.0 Hz, H-6b), 3.95 (2H, d, J = 5.1 Hz, 1-O–CH₂–

CO₂–), 3.90–3.80 (5H, m, H-5, C^bH of 2-O-, 3-O-, Ser-

acyl and Ser-C^bHa), 3.61–3.52 (2H, m, H-4 and Ser-

C^bHb), 3.09 (1H, s, 4-OH), 2.63–2.34 (6H, m, C^aH₂of

2-O-, 3-O-, Ser-acyl), 1.08 (9H, s, t-Bu), 0.88 (9H, dd,

J = 7.3, 6.4 Hz, –CH₃of 2-O-, 3-O-, Ser-acyl).

In a manner similar to the synthesis of 19a, 17 (255 mg,

266 lmol) was condensed with Fmoc-^L-Ser(^tBu)

(103 mg, 269 lmol) to give 19b (312 mg, 91%). ¹H

NMR (400 MHz, CDCl₃) d = 6.65 (1H, d, J = 9.2 Hz,

2-NH), 5.65 (1H, d, J = 8.5 Hz, Ser-NH), 5.17 (1H, t,

J = 10.2 Hz, H-3), 5.09 (2H, s, –CO₂–CH₂–Ph), 4.75

(1H, d, J = 3.5 Hz, H-1), 4.57–4.45 (6H, m, H-6a, Ser-

C^aH, and –O–CH₂–Ph of 2-O-, 3-O-acyl), 4.39 (2H, d,

J = 7.2 Hz, CH₂of Fmoc), 4.30 (1H, dt, J = 14.3,

5.3 Hz, H-2), 4.25–4.19 (2H, m, CH of Fmoc and H-

6b), 3.95 (2H, d, J = 5.2 Hz, 1-O–CH₂–CO₂–), 3.94–

3.80 (4H, m, H-5, Ser-C^bHa, and C^bH of 2-O-, 3-O-ac-

yl), 3.64–3.58 (2H, m, Ser-C^bHb and H-4), 3.07 (1H, d,

J = 4.2 Hz, 4-OH), 2.58–2.30 (4H, m, C^aH₂of 2-O-, 3-

O-acyl), 1.17 (9H, s, t-Bu), 0.88 (6H, t, J = 6.8 Hz,

–CH₃of 2-O-, 3-O-acyl).

4.13. Benzyloxycarbonylmethyl 3-O-((R)-3-benzyloxytet-

radecanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-2-

deoxy-6-O-(N-((R)-3-(tetradecanoyloxy)tetradecanoyl)-

O-tert-butyl-^D-seryl)-a-D-glucopyranoside (21a)

4.11. Benzyloxycarbonylmethyl 3-O-((R)-3-benzyloxytet-

radecanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-6-O-

(N-((R)-3-benzyloxytetradecanoyl)-O-tert-butyl-^D-seryl)-

2-deoxy-a-^D-glucopyranoside (20a)

In a manner similar to the synthesis of 20a, 19a (119 mg,

108 lmol) was deprotected and acylated with (R)-3-

(tetradecanoyloxy)tetradecanoic acid (18b) (49 mg,

108 lmol) to give 21a (77 mg, 52%). ESI-TOF (positive)

m/z = 1563.29 [(M+Na)⁺]; ¹H NMR (400 MHz, CDCl₃)

d = 6.68 (1H, d, J = 9.3 Hz, 2-NH), 6.53 (1H, d,

J = 8.2 Hz, Ser-NH), 5.22–5.12 (2H, m, H-3 and C^bH

of Ser-acyl), 5.11 (2H, s, –CO₂–CH₂–Ph), 4.76 (1H, d,

J = 3.5 Hz, H-1), 4.69 (1H, dt, J = 7.9, 3.0 Hz, Ser-

C^aH), 4.58–4.44 (7H, m, –O–CH₂–Ph of 2-O-, 3-O-,

Ser-acyl, and H-6a), 4.28 (1H, dt, J = 14.6, 5.1 Hz, H-

2), 4.17 (2H, d, J = 10.4 Hz, H-6b), 3.97 (2H, d,

J = 2.0 Hz, 1-O–CH₂–CO₂–), 3.92–3.85 (1H, m, H-5),

3.84–3.77 (3H, m, Ser-C^bHa and C^bH of 2-O-, 3-O-ac-

yl), 3.62–3.54 (2H, m, H-4 and Ser-C^bH₂), 3.32 (1H, d,

J = 3.8 Hz, 4-OH), 2.53–2.28 (8H, m, C^aH₂of 2-O-,

3-O-, Ser-acyl), 1.14 (9H, s, t-Bu), 0.88 (12H, t,

J = 6.8, –CH₃of 2-O-, 3-O-, Ser-acyl).

To a solution of 19a (330 mg, 249 lmol) in absolute tet-

rahydrofuran (10 mL) was added TBD–methyl polysty-

rene (1.3 g, 2.49 mmol) at room temperature. After

stirring for 2.5 d, the resin was ﬁltered oﬀ to give crude

amine.

To a solution of amine (170 mg, 154 lmol), (R)-3-ben-

zyloxytetradecanoic acid (18a) (57 mg, 169 lmol), and

HOBt (10 mg, 77.9 lmol) in absolute CH₂Cl₂was added

WSCDÆHCl (91 mg, 475 lmol) at room temperature

under Ar atmosphere. After stirring for 1 d, the mixture

was washed with aqueous saturated NaHCO₃and brine,

then extracted with AcOEt. The organic layer was dried

6770

M. Akamatsu et al. / Bioorg. Med. Chem. 14 (2006) 6759–6777

4.14. Benzyloxycarbonylmethyl 3-O-((R)-3-benzyloxytet-

radecanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-2-

deoxy-6-O-(N-((R)-3-(tetradecanoyloxy)tetradecanoyl)-

O-tert-butyl-^L-seryl)-a-D-glucopyranoside (21b)

dd, J = 10.7, 9.6 Hz, H-3), 5.09 (2H, d, J = 12.2 Hz,

CHH of Z), 5.09 (2H, s, –CO₂–CH₂–Ph) 4.97–4.88

(1H, m, H-4), 4.96 (1H, d, J = 12.2 Hz, CHH of Z),

4.74–4.69 (2H, m, H-1 and Ser-C^aH), 4.55–4.25 (8H,

m, –O–CH₂–Ph of 2-O-, 3-O-, Ser-acyl, H-2 and H-

6a), 4.13–3.98 (2H, m, H-6b and H-5), 3.91 (2H, d,

J = 2.4 Hz, 1-O–CH₂–CO₂–), 3.83–3.72 (4H, m, C^bH

of 2-O-, 3-O-, Ser-acyl and Ser-C^bHa), 3.49 (1H, dd,

J = 9.1, 3.5 Hz, Ser-C^bHb), 2.47–2.29 (6H, m, C^aH₂of

2-O-, 3-O-, Ser-acyl), 1.06 (9H, t, J = 2.8 Hz, t-Bu),

0.88 (9H, t, J = 6.6 Hz, –CH₃of 2-O-, 3-O-, Ser-acyl).

In a manner similar to the synthesis of 20a, 19b was

deprotected and acylated with (R)-3-(tetradecanoyl-

oxy)tetradecanoic acid (18b) to give 21b (68 mg, 55%).

ESI-TOF (positive) m/z = 1562.69 [(M+Na)⁺]; ¹H

NMR (270 MHz, CDCl₃) d = 6.63 (1H, d, J = 9.4 Hz,

2-NH), 6.46 (1H, d, J = 8.1 Hz, Ser-NH), 5.20–5.11

(4H, m, H-3, C^bH of Ser-acyl and –CO₂–CH₂–Ph),

4.75 (1H, d, J = 3.5 Hz, H-1), 4.72 (1H, dd, J = 6.8,

4.1 Hz, Ser-C^aH), 4.55–4.45 (5H, m, H-6a and –O–

CH₂–Ph of 2-O-, 3-O-acyl), 4.29 (1H, dt, J = 14.2,

5.4 Hz, H-2), 4.18 (1H, dd, J = 12.1, 2.0 Hz, H-6b),

3.95 (2H, d, J = 2.3 Hz, 1-O–CH₂–CO₂–), 3.89–3.80

(4H, m, H-5, C^bH of 2-O-, 3-O-acyl and Ser-C^bHa),

3.62–3.54 (2H, m, H-4 and Ser-C^bHb), 3.05 (1H, d,

J = 4.3 Hz, 4-OH), 2.59–2.25 (8H, m, C^aH₂of 2-O-, 3-

O-, Ser-acyl), 1.15 (9H, s, t-Bu), 0.88 (12H, t,

J = 6.6 Hz, –CH₃of 2-O-, 3-O-, Ser-acyl).

4.17. Benzyloxycarbonylmethyl 4-O-benzyloxycarbonyl-

3-O-((R)-3-benzyloxytetradecanoyl)-2-((R)-3-ben-

zyloxytetradecanoylamino)-2-deoxy-6-O-(N-((R)-3-(tet-

radecanoyloxy)tetradecanoyl)-O-tert-butyl-^D-seryl)-a-D-

glucopyranoside (23a)

In a manner similar to the synthesis of 22a, 21a (58 mg,

37.7 lmol) was protected by treatment with ZOBt

(146 mg, 542 lmol) to give 23a (50 mg, 80%). ESI-

TOF (positive) m/z = 1669.97 [(M+Na)⁺]; ¹H NMR

(400 MHz, CDCl₃) d = 6.61 (1H, d, J = 8.9 Hz, 2-

NH), 6.50 (1H, d, J = 8.3 Hz, Ser-NH), 5.34 (1H, t,

J = 10.2 Hz, H-3), 5.18 (1H, t, J = 6.1 Hz, C^bH of

Ser-acyl), 5.10 (1H, d, J = 12.1 Hz, CHH of Z), 5.10

(2H, s, –CO₂–CH₂–Ph), 4.97 (1H, d, J = 12.1 Hz,

CHH of Z), 4.94 (1H, t, J = 9.8 Hz, H-4), 4.75–4.71

(2H, m, H-1 and Ser-C^aH), 4.51–4.35 (4H, m, –O–

CH₂–Ph of 2-O-, 3-O-acyl), 4.34 (1H, dt, J = 14.6,

5.0 Hz, H-2), 4.23–4.10 (3H, m, H-6 and H-5), 3.92

(2H, dd, J = 23.0, 16.6 Hz, 1-O–CH₂–CO₂–), 3.81–

3.73 (3H, m, Ser-C^bHa and C^bH of 2-O-, 3-O-acyl),

3.55 (1H, dd, J = 9.0, 2.9 Hz, Ser-C^bHb), 2.58–2.23

(8H, m, C^aH₂of 2-O-, 3-O-, Ser-acyl), 1.13 (9H, s, t-

Bu), 0.88 (12H, dd, J = 6.9, 6.2 Hz, –CH₃of 2-O-, 3-

O-, Ser-acyl).

4.15. Benzyloxycarbonylmethyl 4-O-benzyloxycarbonyl-

3-O-((R)-3-benzyloxytetradecanoyl)-2-((R)-3-ben-

zyloxytetradecanoylamino)-6-O-(N-((R)-3-benzyloxytet-

radecanoyl)-O-tert-butyl-^D-seryl)-2-deoxy-a-D-glucopyr-

anoside (22a)

To a solution of 20a (31 mg, 21.8 lmol) in absolute

CH₂Cl₂(5.0 mL) were added DMAP (21.5 mg,

176 lmol) and ZOBt (52.5 mg, 194 lmol) at room tem-

perature under Ar atmosphere. After stirring for 2.5 d,

the mixture was puriﬁed by preparative TLC

(200 · 100 · 1 mm, toluene/AcOEt = 3:1) to give 22a

(34 mg, quant.). ESI-TOF (positive) m/z = 1553.93

1

[(M+H)⁺]; H NMR (400 MHz, CDCl₃) d = 7.01 (1H,

d, J = 8.4 Hz, Ser-NH), 6.64 (1H, d, J = 9.3 Hz, 2-

NH), 5.34 (1H, dd, J = 10.6, 9.7 Hz, H-3), 5.10 (2H,

s, –CO₂–CH₂–Ph), 5.09 (1H, t, J = 6.0 Hz, CHH of

Z), 4.96 (2H, d, J = 6.0 Hz, CHH of Z), 4.94 (1H, t,

J = 10.7 Hz, H-4), 4.76 (1H, dt, J = 8.3, 3.1 Hz, Ser-

C^aH), 4.73 (1H, d, J = 3.5 Hz, H-1), 4.66–4.32 (7H,

m, –O–CH₂–Ph of 2-O-, 3-O-, Ser-acyl and H-2),

4.20–4.17 (2H, m, H-6), 4.10–4.07 (1H, m, H-5), 3.92

(2H, d, J = 3.1 Hz, –CO₂–CH₂–Ph), 3.82–3.73 (4H,

m, Ser-C^bHa and C^bH of 2-O-, 3-O-, Ser-acyl), 3.53

(1H, dd, J = 9.1, 3.2 Hz, Ser-C^bHb), 2.51–2.29 (6H,

m, C^aH₂of 2-O-, 3-O-, Ser-acyl), 1.07 (9H, t,

J = 4.2 Hz, t-Bu), 0.88 (9H, t, J = 6.7 Hz, –CH₃of 2-

O-, 3-O-, Ser-acyl).

4.18. Benzyloxycarbonylmethyl 4-O-benzyloxycarbonyl-

3-O-((R)-3-benzyloxytetradecanoyl)-2-((R)-3-ben-

zyloxytetradecanoylamino)-2-deoxy-6-O-(N-((R)-3-(tet-

radecanoyloxy)tetradecanoyl)-O-tert-butyl-^L-seryl)-a-D-

glucopyranoside (23b)

In a manner similar to the synthesis of 22a, 21b

(96 mg, 62.3 lmol) was protected by treatment with

ZOBt (252 mg, 935 lmol) to give 23b (83 mg, 80%).

ESI-TOF (positive) m/z = 1669.18 [(M+Na)⁺]; ¹H

NMR (270 Hz, CDCl₃) d = 6.62 (1H, d, J = 9.3 Hz,

2-NH), 6.46 (1H, d, J = 8.3 Hz, Ser-NH), 5.34 (1H,

dd, J = 10.7, 9.6 Hz, H-3), 5.17 (1H, t, J = 6.1 Hz,

C^bH of Ser-acyl), 5.10 (2H, s, –CO₂–CH₂–Ph), 5.09

(1H, d, J = 12.0 Hz, CHH of Z), 4.96 (1H, d,

J = 12.2 Hz, CHH of Z), 4.92 (1H, t, J = 12.4 Hz,

H-4), 4.75 (1H, d, J = 3.6 Hz, H-1), 4.68 (1H, dt,

J = 8.2, 3.1 Hz, Ser-C^aH), 4.53–4.27 (6H, m, –CH₂–

Ph of 2-O-, 3-O-acyl, H-2 and H-6a), 4.16–4.05 (2H,

m, H-5 and H-6a), 3.91 (2H, d, J = 2.3 Hz. 1-O–

CH₂–CO₂–), 3.85–3.74 (3H, m, C^bH of 2-O-, 3-O-acyl

and Ser-C^bHa), 3.51 (1H, dd, J = 9.1, 3.3 Hz, Ser-

C^bHb), 2.53–2.23 (8H, m, C^aH₂of 2-O-, 3-O-, Ser-ac-

yl), 1.12 (9H, s, t-Bu), 0.88 (12H, t, J = 6.6 Hz, –CH₃

of 2-O-, 3-O-, Ser-acyl).

4.16. Benzyloxycarbonylmethyl 4-O-benzyloxycarbonyl-

3-O-((R)-3-benzyloxytetradecanoyl)-2-((R)-3-ben-

zyloxytetradecanoylamino)-6-O-(N-((R)-3-benzyloxytet-

radecanoyl)-O-tert-butyl-^L-seryl)-2-deoxy-a-D-glucopyr-

anoside (22b)

In a manner similar to the synthesis of 22a, 20b (90 mg,

63.4 lmol) was protected by treatment with ZOBt

(146 mg, 542 lmol) to give 22b (86 mg, 88%). ¹H

NMR (270 MHz, CDCl₃) d = 6.83 (1H, d, J = 8.1 Hz,

Ser-NH), 6.62 (1H, d, J = 9.3 Hz, 2-NH), 5.33 (1H,

M. Akamatsu et al. / Bioorg. Med. Chem. 14 (2006) 6759–6777

6771

4.19. Benzyloxycarbonylmethyl 4-O-benzyloxycarbonyl-

3-O-((R)-3-benzyloxytetradecanoyl)-2-((R)-3-ben-

zyloxytetradecanoylamino)-6-O-(N-((R)-3-benzyloxytet-

radecanoyl)-^D-seryl)-2-deoxy-a-D-glucopyranoside (24a)

¹H NMR (400 MHz, CDCl₃) d = 6.63–6.61 (2H, m, 2-

NH and Ser-NH), 5.34 (1H, t, J = 10.1 Hz, H-3), 5.26–

5.23 (1H, m, C^bH of Ser-acyl), 5.09 (3H, d, J = 9.8 Hz,

–CO₂–CH₂–Ph and CHH of Z), 4.94 (1H, d,

J = 12.3 Hz, CHH of Z), 4.92 (1H, t, J = 9.8 Hz, H-

4), 4.75 (1H, d, J = 3.5 Hz, H-1), 4.64–4.60 (1H, m,

Ser-C^aH), 4.52–4.38 (4H, m, –O–CH₂–Ph of 2-O-, 3-

O-acyl), 4.35 (1H, dt, J = 15.0, 5.6 Hz, H-2), 4.29–

4.19 (1H, m, H-6), 4.17–4.14 (1H, m, H-5), 3.94–3.91

(3H, m, Ser-C^bHa and 1-O–CH₂–CO₂–), 3.86–3.72

(3H, m, Ser-C^bHb and C^bH of 2-O-, 3-O-acyl), 2.89

(1H, s, Ser-OH), 2.57–2.23 (8H, m, C^aH₂of 2-O-, 3-

O-, Ser-acyl), 0.88 (12H, t, J = 6.7 Hz, –CH₃of 2-O-,

3-O-, Ser-acyl).

The 20% TFA solution in CH₂Cl₂was prepared by

addition of neat TFA (1.0 mL) to absolute CH₂Cl₂

(4.0 mL) and MS4A under Ar atmosphere. To a solu-

tion of 22a (23 mg, 15.2 lmol) in 20% TFA solution

(1.0 mL) was added anhydrous anisole (20 lL,

184 lmol) at room temperature under Ar atmosphere.

After stirring for 18 h, the mixture was washed with

aqueous saturated NaHCO₃and brine, then extracted

with CHCl₃. The organic layer was dried over Na₂SO₄

and concentrated in vacuo. The mixture was puriﬁed

by silica-gel ﬂash column chromatography (10 g, tolu-

ene/AcOEt = 2:1) to give 24a (18.5 mg, 84%). ESI-

TOF (positive) m/z = 1497.92 [(M+H)⁺]; ¹H NMR

(400 MHz, CDCl₃) d = 7.00 (1H, d, J = 7.6 Hz, Ser-

NH), 6.61 (1H, d, J = 9.3 Hz, 2-NH), 5.33 (1H, dd,

J = 10.6, 9.5 Hz, H-3), 5.09–5.06 (3H, m, –CO₂–CH₂–

Ph and CHH of Z), 4.94–4.88 (2H, m, CHH of Z

and H-4), 4.70 (1H, d, J = 3.5 Hz, H-1), 4.68–4.65

(1H, m, Ser-C^aH), 4.61–4.38 (6H, m, –O–CH₂–Ph of

2-O-, 3-O-, Ser-acyl), 4.33 (1H, ddd, J = 11.8, 8.3,

2.5 Hz, H-2), 4.22 (2H, s, H-6), 4.11 (1H, dt,

J = 10.2, 3.2 Hz, H-5), 3.97–3.93 (1H, m, Ser-C^bHa),

3.89–3.72 (4H, m, Ser-C^bHb and C^bH of 2-O-, 3-O-,

Ser-acyl), 3.88 (2H, d, J = 6.0 Hz, 1-O–CH₂–CO₂–),

2.62 (1H, s, Ser-OH), 2.51–2.29 (6H, m, C^aH₂of 2-

O-, 3-O-, Ser-acyl), 0.88 (9H, dd, J = 8.5, 4.8 Hz,

–CH₃of 2-O-, 3-O-, Ser-acyl).

4.22. Benzyloxycarbonylmethyl 4-O-benzyloxycarbonyl-

3-O-((R)-3-benzyloxytetradecanoyl)-2-((R)-3-ben-

zyloxytetradecanoylamino)-2-deoxy-6-O-(N-((R)-3-(tet-

radecanoyloxy)tetradecanoyl)-^L-seryl)-a-D-glucopyrano-

side (25b)

In a manner similar to the synthesis of 24a, 23b (72 mg,

43.0 lmol) was deprotected to give 25b (62 mg, 90%).

ESI-TOF (positive) m/z = 1639.99 [(M+Na)⁺]; ¹H

NMR (270 MHz, CDCl₃) d = 6.60 (1H, d, J = 9.3 Hz,

2-NH), 6.54 (1H, d, J = 8.0 Hz, Ser-NH), 5.36 (1H,

dd, J = 10.7, 9.7 Hz, H-3), 5.20 (1H, t, J = 6.0 Hz,

C^bH of Ser-acyl), 5.10 (2H, s, –CO₂–CH₂–Ph), 5.05

(1H, d, J = 12.1 Hz, CHH of Z), 4.95 (1H, t,

J = 9.9 Hz, H-4), 4.89 (1H, d, J = 12.1 Hz, CHH of

Z), 4.74 (1H, d, J = 3.6 Hz, H-1), 4.67 (1H, td,

J = 5.3, 2.8 Hz, Ser-C^aH), 4.55–4.28 (6H, m, –O–

CH₂–Ph of 2-O-, 3-O-acyl, H-2 and H-6a), 4.21–4.12

(2H, m, Ser-C^bHa and H-5), 4.00–3.96 (1H, m, H-

6b), 3.92–3.74 (3H, m, Ser-C^bHb and C^bH of 2-O-,

3-O-acyl), 3.88 (2H, d, J = 6.8 Hz, 1-O–CH₂–CO₂–),

2.92 (1H, s, Ser-OH), 2.55–2.25 (8H, m, C^aH₂of 2-

O-, 3-O-, Ser-acyl), 0.88 (12H, t, J = 6.6 Hz, –CH₃of

2-O-, 3-O-, Ser-acyl).

4.20. Benzyloxycarbonylmethyl 4-O-benzyloxycarbonyl-

3-O-((R)-3-benzyloxytetradecanoyl)-2-((R)-3-ben-

zyloxytetradecanoylamino)-6-O-(N-((R)-3-benzyloxytet-

radecanoyl)-^L-seryl)-2-deoxy-a-D-glucopyranoside (24b)

In a manner similar to the synthesis of 24a, 22b (80 mg,

51.5 lmol) was deprotected to give 24b (70 mg, 91%). ¹H

NMR (270 MHz, CDCl₃) d = 6.92 (1H, d, J = 8.0 Hz,

Ser-NH), 6.60 (1H, d, J = 9.3 Hz, 2-NH), 5.35 (1H, t,

J = 10.2 Hz, H-3), 5.10 (2H, s, –CO₂–CH₂–Ph), 5.03

(1H, d, J = 12.1 Hz, CHH of Z), 4.95 (1H, t,

J = 9.9 Hz, H-4), 4.87 (1H, d, J = 12.1 Hz, CHH of Z),

4.74 (1H, d, J = 3.5 Hz, H-1), 4.69 (1H, dd, J = 6.9,

4.1 Hz, Ser-C^aH), 4.57–4.27 (8H, m, –O–CH₂–Ph of 2-

O-, 3-O-, Ser-acyl, H-2 and H-6a), 4.20–4.13 (2H, m,

Ser-C^bHa and H-5), 3.98 (1H, t, J = 6.1 Hz, H-6b),

3.88 (2H, d, J = 6.1 Hz, 1-O–CH₂–CO₂–), 3.84–3.74

(4H, m, C^bH of 2-O-, 3-O-, Ser-acyl and Ser-C^bHb),

2.79 (1H, s, Ser-OH), 2.55–2.30 (6H, m, C^aH₂of 2-O-,

3-O-, Ser-acyl), 0.88 (9H, t, J = 6.6 Hz, CH₃of 2-O-,

3-O-, Ser-acyl).

4.23. Benzyloxycarbonylmethyl 4-O-benzyloxycarbonyl-

3-O-((R)-3-benzyloxytetradecanoyl)-2-((R)-3-ben-

zyloxytetradecanoylamino)-6-O-(N-((R)-3-benzyloxytet-

radecanoyl)-O-(1,5-dihydro-3-oxo-3H-2,4,3-k⁵-benzodi-

oxaphosphepin-3-yl)-^D-seryl)-2-deoxy-a-D-glucopyrano-

side (26a)

To a solution of 24a (30 mg, 20.0 lmol) in absolute

CH₂Cl₂(2.0 mL) were added N,N-diethyl-1,5-dihydro-

3H-2,4,3-benzodioxaphosphepin-3-amine

(24 mg,

100 lmol) and 1H-tetrazole (7 mg, 100 lmol) at room

temperature under Ar atmosphere. After stirring for

50 min, the mixture was cooled to ꢀ20 ꢁC and m-chlor-

operbenzoic acid (20 mg, 30.1 lmol) was added, then

stirring was continued for another 1.5 h. The mixture

was washed with aqueous saturated NaHCO₃and

brine, then extracted with CHCl₃. The organic layer

was dried over Na₂SO₄and concentrated in vacuo.

The mixture was puriﬁed by silica-gel ﬂash column

chromatography (5 g, toluene/AcOEt = 3:1) to give

26a (29 mg, 88%). ¹H NMR (400 MHz, CDCl₃)

d = 7.22–7.18 (1H, m, Ser-NH), 6.62 (1H, d,

J = 9.3 Hz, 2-NH), 5.33 (1H, dd, J = 10.6, 9.7 Hz, H-

4.21. Benzyloxycarbonylmethyl 4-O-benzyloxycarbonyl-

3-O-((R)-3-benzyloxytetradecanoyl)-2-((R)-3-ben-

zyloxytetradecanoylamino)-2-deoxy-6-O-(N-((R)-3-(tet-

radecanoyloxy)tetradecanoyl)-^D-seryl)-a-D-glucopyrano-

side (25a)

In a manner similar to the synthesis of 24a, 23a (42 mg,

25.1 lmol) was deprotected to give 25a (40 mg, quant.).

6772

M. Akamatsu et al. / Bioorg. Med. Chem. 14 (2006) 6759–6777

3), 5.13–5.02 (7H, m, CHH of Z and P–O–CH₂–Ph),

4.94–4.89 (3H, m, CHH of Z and Ser-C^aH), 4.67

(1H, d, J = 3.5 Hz, H-1), 4.62–4.32 (9H, m, –O–CH₂–

Ph of 2-O-, 3-O-, Ser-acyl, Ser-C^bH₂and H-2), 4.29–

4.19 (2H, m, H-6), 4.12 (1H, ddd, J = 10.2, 4.6,

2.4 Hz, H-5), 3.91 (2H, s, 1-O–CH₂–CO₂–), 3.85–3.73

(3H, m, C^bH of 2-O-, 3-O-, Ser-acyl), 2.51–2.30 (6H,

m, C^aH₂of 2-O-, 3-O-, Ser-acyl), 0.91–0.87 (9H, m,

–CH₃of 2-O-, 3-O-, Ser-acyl).

4.27. Carboxymethyl 3-O-((R)-3-hydroxytetradecanoyl)-

2-((R)-3-hydroxytetradecanoylamino)-6-O-(N-((R)-3-

hydroxytetradecanoyl)-O-phospho-^D-seryl)-2-deoxy-a-D-

glucopyranoside (^D-Ser(P)1011 (5a))

To a solution of 26a (14 mg, 8.33 lmol) in distilled THF

(1 mL) was added Pd-black (27 mg). The mixture was

stirred under 20 kgf/cm²of hydrogen at room tempera-

ture for 3 days. After removal of the Pd catalyst by ﬁl-

tration, the solution was concentrated with t-BuOH

and water then lyophilized to give 5a as a white solid

(7.2 mg, 80%). The purity was checked by TLC

(CHCl₃/MeOH/H₂O = 6:4:1) and ESI-MS ESI-TOF

(negative) m/z = 1081.6524 [(MꢀH)^ꢀ], 540.3447

4.24. Benzyloxycarbonylmethyl 4-O-benzyloxycarbonyl-

3-O-((R)-3-benzyloxytetradeca-noyl)-2-((R)-3-ben-

zyloxytetradecanoylamino)-6-O-(N-((R)-3-benzyloxytet-

radecanoyl)-O-(1,5-dihydro-3-oxo-3H-2,4,3-k⁵-benzodi-

oxaphosphepin-3-yl)-^L-seryl)-2-deoxy-a-D-glucopyrano-

side (26b)

[(Mꢀ2H)^2ꢀ];

HRMS

(ESI-TOF)

calcd

for

C₅₃H₉₇N₂O₁₈P/2 ([Mꢀ2H]^2ꢀ): 540.3237; found:

540.3239.

In a manner similar to the synthesis of 26a, 24b (65 mg,

43.4 lmol) was phosphorylated with N,N-diethyl-1,5-

dihydro-3H-2,4,3-benzodioxaphosphepin-3-amine

(52 mg, 217 lmol) and m-chloroperbenzoic acid (44 mg,

260 lmol) to give 26b (42 mg, 59%).

4.28. Carboxymethyl 3-O-((R)-3-hydroxytetradecanoyl)-

2-((R)-3-hydroxytetradecanoylamino)-6-O-(N-((R)-3-

hydroxytetradecanoyl)-O-phospho-^L-seryl)-2-deoxy-a-D-

glucopyranoside (^L-Ser(P)1011 (5b))

4.25. Benzyloxycarbonylmethyl 4-O-benzyloxycarbonyl-

3-O-((R)-3-benzyloxytetradecanoyl)-2-((R)-3-ben-

zyloxytetradecanoylamino)-2-deoxy-6-O-(N-((R)-3-(tet-

radecanoyloxy)tetradecanoyl)-O-(1,5-dihydro-3-oxo-3H-

2,4,3-k⁵-benzodioxaphosphepin-3-yl)-D-seryl)-a-D-gluco-

pyranoside (27a)

In a manner similar to the synthesis of 5a, 26b (15 mg,

8.93 lmol) was hydrogenated with Pd-black (143 mg,

1.34 mmol) to give 5b as a white solid (9.7 mg, quant.).

4.29. Carboxymethyl 3-O-((R)-3-hydroxytetradecanoyl)-

2-((R)-3-hydroxytetradecanoylamino)-6-O-(N-((R)-3-

(tetradecanoyloxy)tetradecanoyl)-O-phospho-^D-seryl)-2-

deoxy-a-^D-glucopyranoside (D-Ser(P)2011 (6a))

In a manner similar to the synthesis of 26a, 25a (32 mg,

19.8 lmol) was phosphorylated with N,N-diethyl-1,

5-dihydro-3H-2,4,3-benzodioxaphosphepin-3-amine

(24 mg, 99.9 lmol) and m-chloroperbenzoic acid

(10 mg, 59.3 lmol) to give 27a (14 mg, 40%). ESI-

TOF (positive) m/z = 1799.09 [(M+H)⁺]; ¹H NMR

(400 MHz, CDCl₃) d = 6.88 (1H, d, J = 7.8 Hz, 2-

NH), 6.61 (1H, d, J = 9.3 Hz, Ser-NH), 5.33 (1H, t,

J = 10.1 Hz, H-3), 5.25–5.14 (5H, m, C^bH of Ser-acyl

and P–O–CH₂–Ph), 5.09 (2H, s, –CO₂–CH₂–Ph), 5.07

(1H, d, J = 7.6 Hz, CHH of Z), 4.95–4.91 (2H, m,

CHH of Z and H-4), 4.86–4.82 (1H, m, Ser-C^aH),

4.70 (1H, d, J = 3.6 Hz, H-1), 4.56–4.30 (8H, m, Ser-

C^bH₂, –O–CH₂–Ph of 2-O-, 3-O-acyl H-2 and H-6a),

4.20–4.13 (2H, m, H-6b and H-5), 3.91 (2H, d,

J = 3.3 Hz, 1-O–CH₂–CO₂–), 3.81 (1H, t, J = 5.8 Hz,

C^bH of 2-O-acyl), 3.74 (1H, t, J = 5.6 Hz, C^bH of 3-

O-acyl), 2.59–2.26 (8H, m, C^aH₂of 2-O-, 3-O-, Ser-ac-

yl), 0.88 (12H, dd, J = 7.4, 6.3 Hz, –CH₃of 2-O-, 3-O-,

Ser-acyl).

In a manner similar to the synthesis of 5a, 27a

(9 mg, 5.00 lmol) was hydrogenated with Pd-black

(16 mg, 150 lmol) to give 6a as

(6.4 mg, 74%).

a white solid

ESI-TOF (negative) m/z = 1291.5998 [(MꢀH)^ꢀ],

645.3690 [(Mꢀ2H)^2ꢀ]; HRMS (ESI-TOF) calcd for

C₆₇H₁₂₃N₂O₁₉P/2 ([Mꢀ2H]^2ꢀ): 645.4229; found:

645.4233.

4.30. Carboxymethyl 3-O-((R)-3-hydroxytetradecanoyl)-

2-((R)-3-hydroxytetradecanoylamino)-6-O-(N-((R)-3-

(tetradecanoyloxy)tetradecanoyl)-O-phospho-^L-seryl)-2-

deoxy-a-^D-glucopyranoside (L-Ser(P)2011 (6b))

In a manner similar to the synthesis of 5a, 27b (18 mg,

10.0 lmol) was hydrogenated with Pd-black (32 mg,

300 lmol) to give 6b as a white solid (11.5 mg, 89%).

ESI-TOF (negative) m/z = 1290.6164 [(MꢀH)^ꢀ],

645.2721 [(Mꢀ2H)^2ꢀ].

4.26. Benzyloxycarbonylmethyl 4-O-benzyloxycarbonyl-

3-O-((R)-3-benzyloxytetradecanoyl)-2-((R)-3-ben-

zyloxytetradecanoylamino)-2-deoxy-6-O-(N-((R)-3-(tet-

radecanoyloxy)tetradecanoyl)-O-(1,5-dihydro-3-oxo-3H-

2,4,3-k⁵-benzodioxaphosphepin-3-yl)-L-seryl)-a-D-gluco-

pyranoside (27b)

4.31. Benzyloxycarbonylmethyl 3,4-O-((R)-3-benzyloxy-

tetradecanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-6-

O-(N-((R)-3-benzyloxytetradecanoyl)-O-tert-butyl-^D-ser-

yl)-2-deoxy-a-^D-glucopyranoside (28a)

In a manner similar to the synthesis of 26a, 25b (47 mg,

29.0 lmol) was phosphorylated with N,N-diethyl-1,

5-dihydro-3H-2,4,3-benzodioxaphosphepin-3-amine

(35 mg, 145 lmol) and m-chloroperbenzoic acid (15 mg,

87.1 lmol) to give 27b (33 mg, 64%).

To a solution of 19a (330 mg, 249 lmol) in absolute tet-

rahydrofuran (10 mL) was added TBD–methyl polysty-

rene (1.3 g, 2.49 mmol) at room temperature. After

stirring for 2.5 d, the resin was ﬁltered oﬀ to give crude

amine.

M. Akamatsu et al. / Bioorg. Med. Chem. 14 (2006) 6759–6777

6773

To a solution of amine (94 mg, 85.2 lmol), (R)-3-benzyl-

oxytetradecanoic acid (18a) (108 mg, 323 lmol), HOBt

(5.8 mg, 42.6 lmol), and DMAP (5.2 mg, 42.6 lmol) in

absolute CH₂Cl₂(2.0 mL) was added WSCDÆHCl

(122 mg, 636 lmol) at room temperature under Ar atmo-

sphere. After stirring for 11 h, the mixture was washed

with aqueous saturated NaHCO₃and brine, then extract-

ed with CHCl₃. The organic layer was dried over Na₂SO₄

and concentrated in vacuo. The mixture was puriﬁed by

silica-gel ﬂash column chromatography (10 g, toluene/

AcOEt = 8:1) to give 28a (80 mg, 61%). ESI-TOF (posi-

tive) m/z = 1737.62 [(M+H)⁺]; ¹H NMR (400 MHz,

CDCl₃) d = 6.99 (1H, d, J = 8.4 Hz, Ser-NH), 6.60 (1H,

d, J = 9.3 Hz, 2-NH), 5.32 (1H, t, J = 10.2 Hz, H-3),

5.10 (1H, t, J = 9.8 Hz, H-4), 5.10 (2H, s, –CO₂–CH₂–

Ph), 4.73–4.71 (2H, m, Ser-C^aH and H-1), 4.65–4.32

(9H, m, –O–CH₂–Ph of 2-O-, 3-O-, 4-O-, Ser-acyl and

H-2), 4.16 (1H, d, J = 10.7 Hz, H-6a), 4.07 (1H, dd,

J = 12.1, 5.3 Hz, H-6b), 4.00–3.98 (1H, m, H-5), 3.93

(2H, s, 1-O–CH₂–CO₂–), 3.77 (5H, m, C^bH of 2-O-, 3-

O-, 4-O-, Ser-acyl and Ser-C^bHa), 3.53 (1H, dd, J = 9.1,

3.2 Hz, Ser-C^bHb), 2.51–2.29 (8H, m, C^aH₂of 2-O-, 3-

O-, 4-O-, Ser-acyl), 1.06 (9H, t, J = 4.5 Hz, t-Bu), 0.88

(12H, t, J = 6.6 Hz, –CH₃of 2-O-, 3-O-, 4-O-, Ser-acyl).

(27 mg, 82%). ESI-TOF (positive) m/z = 1680.05

[(M+H)⁺]; ¹H NMR (400 MHz, CDCl₃) d = 6.87

(1H, d, J = 8.1 Hz, Ser-NH), 6.61 (1H, d, J = 9.3 Hz,

2-NH), 5.34 (1H, t, J = 10.2 Hz, H-3), 5.15 (1H, t,

J = 9.9 Hz, H-4), 5.11 (2H, s, –CO₂–CH₂–Ph), 4.73

(1H, d, J = 3.6 Hz, H-1), 4.68–4.65 (1H, m, Ser-

C^aH), 4.58–4.33 (9H, m, -O–CH₂–Ph of 2-O-, 3-O-,

4-O-, Ser-acyl, H-2), 4.20–4.16 (2H, m, H-6), 3.96

(1H, d, J = 10.2 Hz, H-5), 3.88–3.81 (5H, m, 1-O–

CH₂–CO₂– and C^bH of 2-O-, 3-O-, 4-O-acyl), 3.76

(2H, dt, J = 18.2, 6.3 Hz, Ser-C^bH₂), 3.64–3.59 (1H,

m, C^bH of of Ser-acyl), 3.04 (1H, s, Ser-OH), 2.52–

2.30 (8H, m, C^aH₂of 2-O-, 3-O-, 4-O-, Ser-acyl),

0.90–0.85 (12H, m, –CH₃of 2-O-, 3-O-, 4-O-, Ser-

acyl).

4.35. Benzyloxycarbonylmethyl 3,4-O-((R)-3-benzyloxy-

tetradecanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-6-

O-(N-((R)-3-benzyloxytetradecanoyl)-O-(1,5-dihydro-3-

oxo-3H-2,4,3-k⁵-benzodioxaphosphepin-3-yl)-D-seryl)-2-

deoxy-a-^D-glucopyranoside (30a)

In a manner similar to the synthesis of 26a, 29a

(37 mg, 22.0 lmol) was phosphorylated with N,

N-diethyl-1,5-dihydro-3H-2,4,3-benzodioxaphosphepin-

3-amine (22 mg, 91.2 lmol) and m-chloroperbenzoic

4.32. Benzyloxycarbonylmethyl 3,4-O-((R)-3-benzyloxy-

tetradecanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-6-

O-(N-((R)-3-benzyloxytetradecanoyl)-O-tert-butyl-^L-ser-

yl)-2-deoxy-a-^D-glucopyranoside (28b)

1

acid (18 mg, 104 lmol) to give 30a (20 mg, 49%). H

NMR (400 MHz, CDCl₃) d = 7.26 (1H, m, Ser-NH),

6.59 (1H, d, J = 9.3 Hz, 2-NH), 5.31 (1H, t,

J = 10.2 Hz, H-3), 5.12–5.00 (7H, m, P–O–CH₂–Ph,

H-4 and –CO₂–CH₂–Ph), 4.89–4.86 (1H, m, Ser-

C^aH), 4.67 (1H, d, J = 3.6 Hz, H-1), 4.62–4.32 (11H,

m, –O–CH₂–Ph of 2-O-, 3-O-, 4-O-, Ser-acyl, Ser-

C^bH₂and H-2), 4.23–4.17 (1H, m, H-6a), 4.14–4.09

(1H, m, H-6b), 4.02 (1H, m, H-5) 3.91 (2H, s, 1-O–

CH₂–CO₂–), 3.83–3.66 (4H, m, C^bH of 2-O-, 3-O-,

4-O-, Ser-acyl), 2.45–2.33 (4H, m, C^aH₂of 2-O-,

3-O-, 4-O-, Ser-acyl), 0.92–0.88 (12H, m, –CH₃of

2-O-, 3-O-, 4-O-, Ser-acyl).

In a manner similar to the synthesis of 28a, 19b was

deprotected and acylated with 18a to give 28b (47 mg,

55%). ESI-TOF (positive) m/z = 1759.24 [(M+Na)⁺].

4.33. Benzyloxycarbonylmethyl 3,4-O-((R)-3-benzyloxy-

tetradecanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-6-

O-(N-((R)-3-benzyloxytetradecanoyl)-^D-seryl)-2-deoxy-

a-^D-glucopyranoside (29a)

In a manner similar to the synthesis of 24a, 28a (72 mg,

43.0 lmol) was deprotected to give 29a (51 mg, 83%).

ESI-TOF (positive) m/z = 1702.0438 [(M+Na)⁺]; ¹H

NMR (400 MHz, CDCl₃) d = 6.99 (1H, d, J = 7.6 Hz,

Ser-NH), 6.57 (1 H, d, J = 9.3 Hz, 2-NH), 5.32 (1H, t,

J = 10.2 Hz, H-3), 5.10 (2H, s, –CO₂–CH₂–Ph), 5.08

(1H, t, J = 9.0 Hz, H-4), 4.69 (1H, d, J = 3.6 Hz, H-1),

4.66–4.62 (1H, m, Ser-C^aH), 4.61–4.37 (8H, m, –O–

CH₂–Ph of 2-O-, 3-O-, 4-O-, Ser-acyl), 4.33 (1H, ddd,

J = 11.8, 8.3, 2.4 Hz, H-1), 4.12 (2H, d, J = 2.6 Hz,

H-6), 3.99 (1H, dt, J = 10.3, 3.2 Hz, H-5), 3.94–3.86

(3H, m, Ser-C^bHa and 1-O–CH₂–CO₂–), 3.85–3.66

(5H, m, Ser-C^bHb and C^bH of 2-O-, 3-O-, 4-O-, Ser-ac-

yl), 2.67 (1H, d, J = 6.3 Hz, Ser-OH), 2.50–2.29 (8H, m,

C^aH₂of 2-O-, 3-O-, 4-O-, Ser-acyl), 0.90–0.86 (12H, m,

–CH₃of 2-O-, 3-O-, 4-O-, Ser-acyl).

4.36. Benzyloxycarbonylmethyl 3,4-O-((R)-3-benzyloxy-

tetradecanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-6-

O-(N-((R)-3-benzyloxytetradecanoyl)-O-(1,5-dihydro-3-

oxo-3H-2,4,3-k⁵-benzodioxaphosphepin-3-yl)-L-seryl)-2-

deoxy-a-^D-glucopyranoside (30b)

In a manner similar to the synthesis of 26a, 29b (10 mg,

5.95 lmol) was phosphorylated with N,N-diethyl-1,

5-dihydro-3H-2,4,3-benzodioxaphosphepin-3-amine

(7.5 mg, 31.4 lmol) and m-chloroperbenzoic acid

(8.2 mg, 47.5 lmol) to give 30b (8.9 mg, 81%). ESI-

TOF (positive) m/z = 1863.02 [(M+H)⁺]; ¹H NMR

(400 MHz, CDCl₃) d = 7.20 (1H, m, Ser-NH), 6.62

(1H, d, J = 9.4 Hz, 2-NH), 5.32 (1H, t, J = 10.2 Hz, H-

3), 5.17–4.89 (5H, m, H-4 and P–O–CH₂–Ph), 4.85–

4.83 (1H, m, Ser-C^aH), 4.68 (1H, d, J = 3.7 Hz, H-1),

4.61–4.34 (11H, m, –O–CH₂–Ph of 2-O-, 3-O-, 4-O-,

Ser-acyl, Ser-C^bH₂and H-2), 4.17–4.04 (2H, m, H-6),

4.02–3.98 (1H, m, H-5), 3.92 (2H, s, 1-O–CH₂–CO₂–),

3.90–3.65 (4H, m, C^bH of 2-O-, 3-O-, 4-O-, Ser-acyl),

2.52–2.29 (8H, m, C^aH₂of 2-O-, 3-O-, 4-O-, Ser-acyl),

0.88 (12H, t, J = 6.8 Hz, –CH₃of 2-O-, 3-O-, 4-O-,

Ser-acyl).

4.34. Benzyloxycarbonylmethyl 3,4-O-((R)-3-benzyloxy-

tetradecanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-6-

O-(N-((R)-3-benzyloxytetradecanoyl)-^L-seryl)-2-deoxy-

a-^D-glucopyranoside (29b)

In a manner similar to the synthesis of 24a, 28b

(35 mg, 20.2 lmol) was deprotected to give 29b

6774

M. Akamatsu et al. / Bioorg. Med. Chem. 14 (2006) 6759–6777

4.37. Carboxymethyl 3,4-O-((R)-3-hydroxytetradeca-

noyl)-2-((R)-3-hydroxytetradecanoylamino)-6-O-(N-((R)-

3-hydroxytetradecanoyl)-O-phospho-^D-seryl)-2-deoxy-a-

D-glucopyranoside (D-Ser(P)1111 (7a))

m/z = 1287.51 [(M+Na)⁺]; ¹H NMR (400 MHz, CDCl₃)

d = 6.67 (1H, d, J = 9.4 Hz, 2-NH), 5.45 (1H, d,

J = 7.9 Hz, Asp-NH), 5.17–5.05 (5H, m, H-3 and

–CO₂–CH₂–Ph), 4.71 (1H, d, J = 3.6 Hz, H-1), 4.60–

4.58 (1H, m, Asp-C^aH), 4.50 (4H, dd, J = 14.0,

11.6 Hz, –O–CH₂–Ph of 2-O-, 3-O-acyl), 4.41 (1H, dd,

J = 12.0, 4.2 Hz, H-6a), 4.30–4.22 (2H, m, H-2 and H-

6b), 3.96 (2H, d, J = 1.8 Hz, 1-O–CH₂–CO₂–), 3.92–

3.79 (3H, m, H-5 and C^bH of 2-O-, 3-O-acyl), 3.54

(1H, td, J = 9.6, 4.5 Hz, H-4), 3.09 (1H, d, J = 4.5 Hz,

4-OH), 2.96 (2H, ddd, J = 63.1, 17.1, 4.4 Hz, Asp-

C^bH₂), 3.60–2.35 (4H, m, C^aH₂of 2-O-, 3-O-acyl),

0.90–0.86 (6H, m, –CH₃of 2-O-, 3-O-acyl).

In a manner similar to the synthesis of 5a, 30a (14.5 mg,

7.79 lmol) was hydrogenated with Pd-black (25 mg,

234 lmol) to give 7a as a white solid (6.7 mg, 67%). ESI-

TOF (negative) m/z = 1307.6122 [(MꢀH)^ꢀ], 653.3762

[(Mꢀ2H)^2ꢀ]; HRMS (ESI-TOF) calcd for C₆₇H₁₂₅N₂O₂₀

P/2 ([Mꢀ2H]^2ꢀ): 653.4204; found: 653.4203.

4.38. Carboxymethyl 3,4-O-((R)-3-hydroxytetradeca-

noyl)-2-((R)-3-hydroxytetradecanoylamino)-6-O-(N-((R)-

3-hydroxytetradecanoyl)-O-phospho-^L-seryl)-2-deoxy-a-

D-glucopyranoside (L-Ser(P)1111 (7b))

4.41. Benzyloxycarbonylmethyl 3-O-((R)-3-benzyloxytet-

radecanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-6-O-

(N-((R)-3-benzyloxytetradecanoyl)-O⁴-benzyl-D-aspar-

tyl)-2-deoxy-a-^D-glucopyranoside (32a)

In a manner similar to the synthesis of 5a, 30b (23 mg,

12.3 lmol) was hydrogenated with Pd-black (197 mg,

1.85 mmol) to give 7b as a white solid (12.6 mg, 79%).

To a solution of 31a (40.8 mg, 32.2 lmol) in absolute

CH₂Cl₂(0.5 mL) was added TFA (125 lL) at 0 ꢁC under

Ar atmosphere. After stirring for 20 min at room tem-

perature, the mixture was quenched with aqueous satu-

rated NaHCO₃and brine, then extracted with AcOEt.

The organic layer was dried over Na₂SO₄, and concen-

trated in vacuo to give crude amine.

4.39. Benzyloxycarbonylmethyl 3-O-((R)-3-benzyloxytet-

radecanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-6-O-

(N-tert-butoxycarbonyl-O⁴-benzyl-D-aspartyl)-2-deoxy-

a-^D-glucopyranoside (31a)

To a solution of 17 (41.4 mg, 43.1 lmol) and N-t-butoxy-

carbonyl-O⁴-benzyl-L-aspartic acid (13.9 mg, 43.1 lmol)

in absolute CH₂Cl₂(0.4 mL) were added DMAP

(1.1 mg, 8.6 lmol) and DCC (10.7 mg, 51.7 lmol) at

room temperature under Ar atmosphere. After stirring

for 1 h, the mixture was washed with aqueous saturated

NaHCO₃and brine, then extracted with AcOEt. The

organic layer was dried over Na₂SO₄. The mixture was

puriﬁed by silica-gel ﬂash column chromatography (5 g,

CHCl₃/acetone = 6:1) to give 31a as a white solid

(42.5 mg, 78%). MALDI-TOF (positive) m/z = 1287.57

To the solution of crude amine (17.7 mg, 15.1 lmol) in

absolute CHCl₃(0.2 mL), (R)-3-benzyloxytetradecanoic

acid (18a) (8.8 mg, 26.3 lmol), and HOBt (4.1 mg,

30.3 lmol) was added WSCDÆHCl (5.8 mg, 30.3 lmol)

at room temperature under Ar atmosphere. After stir-

ring overnight, the mixture was puriﬁed with prepara-

tive TLC (200 · 100 · 0.5 mm, CHCl₃/acetone = 15:1)

to give 32a as a white solid (13.9 mg, 62%). ESI-TOF

(positive) m/z = 1503.11 [(M+Na)⁺]; ¹H NMR

(400 MHz, CDCl₃) d = 7.38–7.20 (m, 25H, OCH₂Ph),

7.12–7.10 (d, J = 7.6 Hz, 1H, Asp-NH), 6.66–6.64 (d,

J = 9.5 Hz, 1H, 2-NH), 5.15–5.10 (dd, J = 11.0 Hz,

J = 9.5 Hz, 1H, H-3), 5.09–5.03 (m, 4H, COOCH₂Ph),

4.87–4.83 (ddd, J = 4.6 Hz, J = 5.2 Hz, J = 7.9 Hz, 1H,

Asp-C^aH), 4.71–4.70 (d, J = 3.7 Hz, 1H, H-1), 4.58–

4.47 (m, 6H, OCH₂Ph), 4.39–4.35 (dd, J = 5.2 Hz,

J = 11.9 Hz, 1H, H-6a), 4.31–4.25 (ddd, J = 3.7 Hz,

J = 10.7 Hz, J = 9.5 Hz, 1H, H-2), 4.23–4.20 (dd,

J = 2.1 Hz, J = 11.9 Hz, 1H, H-6b), 3.99–3.94 (d,

J = 16.8 Hz, 1H, CHH of 1-O–CH₂–CO₂–), 3.94–3.89

(d, J = 16.8 Hz, 1H, CHH of 1-O–CH₂–CO₂–), 3.88–

3.77 (m, 4H, C^bH of acyl), 3.53–3.48 (dd, J = 9.5 Hz,

J = 10.7 Hz, 1H, H-4), 3.08 (s, 1H, 4-OH), 2.98–2.93

(dd, J = 17.1 Hz, J = 4.9 Hz, 1H, Asp-C^bH₂), 2.90–2.85

(dd, J = 17.0 Hz, J = 4.6 Hz, 1H, Asp-C^bH₂), 2.63–2.36

(m, 3H, C^aH₂of acyl), 1.59–1.25 (m, 60H, CH₂of acyl),

0.90–0.86 (t, J = 7.0 Hz, 9H, CH₃of acyl).

1

[(M+Na)⁺]; H NMR (400 MHz, CDCl₃) d = 7.38–7.20

(20H, m, OCH₂Ph) 6.67–6.65 (1H, d, J = 9.5 Hz, 2-

NH), 5.47-5.45 (1H, d, J = 8.5 Hz, Asp-NH), 5.16–5.07

(5H, m, H-3 and –CO₂–CH₂–Ph), 4.74–4.73 (1H, d,

J = 3.7 Hz, H-1), 4.58–4.50 (5H, m, Asp-C^aH and –O–

CH₂–Ph of 2-O-, 3-O-acyl), 4.41–4.37 (1H, dd, J = 12.0,

4.6 Hz, H-6a), 4.32–4.23 (2H, m, H-2 and H-6b), 4.01–

3.97 (1H, d, J = 16.5 Hz, 1-O–CH₂–CO₂–), 3.95–3.91

(1H, d, J = 16.6 Hz, 1-O–CH₂–CO₂–), 3.89–3.80 (3H,

m, H-5 and C^bH of 2-O-, 3-O-acyl), 3.57–3.52 (1H, ddd,

J = 9.3, 9.3, 4.1 Hz, H-4), 3.12–3.10 (1H, d, J = 4.1 Hz,

4-OH), 3.03–2.98 (1H, dd, J = 17.0, 4.6 Hz, Asp-C^bH₂),

2.90–2.85 (1H, dd, J = 17.0, 4.6 Hz, Asp-C^bH₂), 2.64–

2.36 (4H, m, C^aH₂of 2-O-, 3-O-acyl), 1.59–1.17 (49 H,

m, CH₃of Boc and CH₂of 2-O-, 3-O-acyl), 0.90–0.84

(6H, t, –CH₃of 2-O-, 3-O-acyl).

4.40. Benzyloxycarbonylmethyl 3-O-((R)-3-benzyloxytet-

radecanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-6-O-

(N-tert-butoxycarbonyl-O⁴-benzyl-L-aspartyl)-2-deoxy-

a-^D-glucopyranoside (31b)

4.42. Benzyloxycarbonylmethyl 3-O-((R)-3-benzyloxytet-

radecanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-6-O-

(N-((R)-3-benzyloxytetradecanoyl)-O⁴-benzyl-L-aspar-

tyl)-2-deoxy-a-^D-glucopyranoside (32b)

In a manner similar to the synthesis of 31a, 17 (100 mg,

104 lmol) was condensed with N-tert-butoxycarbonyl-

O⁴-benzyl-L-aspartic acid (34 mg, 105 lmol) to give

31b as a white solid (128 mg, 97%). ESI-TOF (positive)

In a manner similar to the synthesis of 32a, 31b (74 mg,

58.5 lmol) was dissolved in 50% CH₂Cl₂solution of

TFA to give crude amine (62 mg).

M. Akamatsu et al. / Bioorg. Med. Chem. 14 (2006) 6759–6777

6775

The crude amine (27.8 mg, 23.9 lmol) was condensed

with (R)-3-benzyloxytetradecanoic acid (18a) (8.0 mg,

23.9 lmol) to give 32b (27.3 mg, 77%). ESI-TOF (posi-

tive) m/z = 1503.11 [(M+Na)⁺]; ¹H NMR (400 MHz,

CDCl₃) d = 7.04 (1H, d, J = 8.0 Hz, Asp-NH), 6.65

(1H, d, J = 9.4 Hz, 2-NH), 5.16–4.96 (5H, m, H-3,

–O–CH₂–Ph), 4.90–4.85 (1H, m, Asp-C^aH), 4.69 (1H,

d, J = 3.6 Hz, H-1), 4.52–4.43 (6H, m, –O–CH₂–Ph of

2-O-, 3-O-, Asp-acyl), 4.37 (1H, dd, J = 12.0, 4.8 Hz,

H-6a), 4.30–4.20 (2H, m, H-2 and H-6b), 3.95 (2H, s,

1-O–CH₂–CO₂–), 3.90–3.73 (3H, m, H-5 and C^bH of

2-O-, 3-O-, Asp-acyl), 3.52 (1H, td, J = 9.7, 3.6 Hz, H-

4), 3.10 (1H, d, J = 4.0 Hz, 4-OH), 2.92 (2H, ddd,

J = 66.9, 17.1, 4.7 Hz, Asp-C^bH₂), 2.62–2.36 (6H, m,

C^aH₂of 2-O-, 3-O-, Asp-acyl), 0.88 (9H, dd, J = 7.0,

6.3 Hz, –CH₃of 2-O-, 3-O-, Asp-acyl).

4.45. Carboxymethyl 3-O-((R)-3-hydroxytetradecanoyl)-

2-((R)-3-hydroxytetradecanoylamino)-6-O-(N-((R)-3-

hydroxytetradecanoyl)-^D-aspartyl)-2-deoxy-a-D-gluco-

pyranoside (^D-Asp1011 (8a))

To a solution of 32a (7.63 mg, 5.14 lmol) in dis-

tilled THF (0.2 mL) was added Pd-black (10.6 mg).

The mixture was stirred under 7 kgf/cm²of hydro-

gen at room temperature overnight. After removal

of the Pd catalyst by ﬁltration, the solution was

concentrated in vacuo. The residue was puriﬁed

by liquid–liquid partition column chromatography

(Sephadex^ꢂLH-20, 5 g; solvent, CHCl₃/MeOH/H₂O/

i-PrOH = 8:12:9:1), and then added distilled water

and t-BuOH. The suspension was lyophilized to

give 8a as a white powder. The purity was checked

by TLC (CHCl₃/MeOH/H₂O = 6:4:1) and ESI-MS

ESI-TOF (negative) m/z = 1030.6 [(MꢀH)^ꢀ], 514.3

[(Mꢀ2H)^2ꢀ].

4.43. Benzyloxycarbonylmethyl 3-O-((R)-3-benzyloxytet-

radecanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-2-

deoxy-6-O-(N-((R)-3-(tetradecanoyloxy)tetradecanoyl)-

O⁴-benzyl-D-aspartyl)-a-D-glucopyranoside (33a)

4.46. Carboxymethyl 3-O-((R)-3-hydroxytetradecanoyl)-

2-((R)-3-hydroxytetradecanoylamino)-6-O-(N-((R)-3-

hydroxytetradecanoyl)-^D-aspartyl)-2-deoxy-a-L-gluco-

pyranoside (^L-Asp1011 (8b))

In a manner similar to the synthesis of 32a, 31a

(40.8 mg, 32.2 lmol) was dissolved in absolute CH₂Cl₂

(0.5 mL) and TFA (125 lL) to give crude amine.

To a solution of 32b (32 mg, 21.6 lmol) in distilled THF

(2 mL) was added Pd-black (69 mg, 648 lmol). The mix-

ture was stirred under 20 kgf/cm²of hydrogen at room

temperature for 3 days. After removal of the Pd catalyst

by ﬁltration, the solution was concentrated with

t-BuOH and water then lyophilized to give 8b as a white

solid (18 mg, 82%). The purity was checked by TLC

(CHCl₃/MeOH/H₂O = 6:4:1) and ESI-MS ESI-TOF

(negative) m/z = 1029.6400 [(MꢀH)^ꢀ], 514.3377

[(Mꢀ2H)^2ꢀ].

The crude amine (17.7 mg, 15.1 lmol) was condensed

with (R)-3-(tetradecanoyloxy)tetradecanoic acid (18b)

(13.5 mg, 29.7 lmol) to give 33a (27.3 mg, 77%).

1

MALDI-TOF (positive) m/z = 1623.07 [(M+Na)⁺]; H

NMR (400 MHz, CDCl₃) d = 7.73–7.21 (m, 20H,

OCH₂Ph), 6.72–6.70 (d, J = 7.9 Hz, 1H, Asp-NH),

6.68–6.66 (d, J = 9.5 Hz, 1H, NH), 5.16–5.10 (m,

6H, H-3,–CO₂CH₂Ph), 4.86–4.82 (ddd, J = 4.6 Hz,

J = 4.9 Hz, J = 7.9 Hz, 1H, Asp-C^aH), 4.73–4.73 (d,

J = 3.7 Hz, 1H, H-1), 4.51–4.50 (m, 4H, OCH₂Ph of

2-O-,

3-O-acyl),

4.38–4.34

(dd,

J = 4.9 Hz,

4.47. Carboxymethyl 3-O-((R)-3-hydroxytetradecanoyl)-

2-((R)-3-hydroxytetradecanoylamino)-6-O-(N-((R)-3-

(tetradecanoyloxy)tetradecanoyl)-^D-aspartyl)-2-deoxy-a-

D-glucopyranoside (D-Asp2011 (9a))

J = 12.2 Hz, 1H, H-6a), 4.31–4.25 (m, 2H, H-2, H-

6b), 4.00–3.96 (d, J = 16.8 Hz, 1H, CH of 1-O–CH₂–

CO₂–), 3.95–3.91 (d, J = 16.8 Hz, 1H, CH of 1-O–

CH₂–CO₂–), 3.89–3.80 (m, 3H, C^bH of acyl, H-5),

3.56–3.51 (dd, J = 9.5 Hz, J = 9.5 Hz, 1H, H-4),

3.04–2.99 (dd, J = 17.4 Hz, J = 4.9 Hz, 1H, Asp-

C^bH₂), 2.92–2.86 (dd, J = 17.0 Hz, J = 4.6 Hz, 1H,

Asp-C^bH₂), 2.65–2.39 (m, 4H, C^aH₂of of 2-O-, 3-O-

acyl), 2.30–2.26 (t, J = 7.9 Hz, 2H, C^aH₂of 2-O-, 3-

O-acyl), 1.77–1.25 (m, 82H, CH₂of 2-O-, 3-O- acyl),

0.90–0.86 (t, J = 7.0 Hz, 12H, –CH₃of 2-O-, 3-O-,

Asp-acyl).

To a solution of 33a (9.67 mg, 6.14 lmol) in distilled

THF (0.2 mL) was added Pd-black (9.2 mg). The mix-

ture was stirred under 7 kgf/cm²of hydrogen at room

temperature overnight. After removal of the Pd cata-

lyst by ﬁltration, the solution was concentrated in

vacuo. The residue was added distilled water and

t-BuOH, and the suspension was lyophilized to give

9a as a white powder (7.82 mg). The purity was

checked by TLC (CHCl₃/MeOH/H₂O = 6:4:1) and

ESI-MS ESI-TOF (negative) m/z = 1239.90 [(MꢀH)^ꢀ],

619.5 [(Mꢀ2H)^2ꢀ].

4.44. Benzyloxycarbonylmethyl 3-O-((R)-3-benzyloxytet-

radecanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-2-

deoxy-6-O-(N-((R)-3-(tetradecanoyloxy)tetradecanoyl)-

O⁴-benzyl-D-aspartyl)-a-D-glucopyranoside (33b)

4.48. Carboxymethyl 3-O-((R)-3-hydroxytetradecanoyl)-

2-((R)-3-hydroxytetradecanoylamino)-6-O-(N-((R)-3-

(tetradecanoyloxy)tetradecanoyl)-^L-aspartyl)-2-deoxy-a-

L-glucopyranoside (L-Asp2011 (9b))

In a manner similar to the synthesis of 32a, 31b (74 mg,

58.5 lmol) was dissolved in 50% CH₂Cl₂solution of

TFA to give crude amine (62 mg).

In a manner similar to the synthesis of 8b, 33b (23 mg,

14.4 lmol) was hydrogenated with Pd-black (46 mg,

431 lmol) to give 9b as a white solid (18 mg, quant.).

ESI-TOF (negative) m/z = 1239.8854 [(MꢀH)^ꢀ],

619.4328 [(Mꢀ2H)^2ꢀ].

The crude amine (36 mg, 30.9 lmol) was condensed with

(R)-3-(tetradecanoyloxy)tetradecanoic

acid

(18b)

(14.0 mg, 30.9 lmol) to give 33a (31 mg, 64%). ESI-

TOF (positive) m/z = 1624.70 [(M+Na)⁺].

6776

M. Akamatsu et al. / Bioorg. Med. Chem. 14 (2006) 6759–6777

4.49. Benzyloxycarbonylmethyl 3,4-O-((R)-3-benzyloxy-

tetradecanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-2-

deoxy-6-O-(N-((R)-3-(tetradecanoyloxy)tetradecanoyl)-

O⁴-benzyl-D-aspartyl)-a-D-glucopyranoside (34a)

tory Institute for Bioorganic Research for MS measure-

ments, and Mr. S. Adachi for NMR measurements, and

Ms. K. Hayashi and Ms. T. Ikeuchi for elemental analysis

at Osaka University. The present work was ﬁnancially

supported in part by Grants-in Aid for Scientiﬁc Research

(No. 17035050, No. 17310128) from the Japan Society for

the Promotion of Science, grants from Suntory Institute

for Bioorganic Research (SUNBOR Grant), the Houan-

sha Foundation, and Hayashi Memorial Foundation

for Female Natural Scientists (Y.F.).

Compound 31a (56 mg, 44.2 lmol) was dissolved in 50%

TFA solution in CH₂Cl₂(2 mL) at room temperature.

After being stirred for 11 h, the mixture was washed

with saturated aqueous NaHCO₃and NaCl, then

extracted with AcOEt. The organic layer was dried over

Na₂SO₄and concentrated in vacuo to give crude amine

(51 mg).

References and notes

To the solution of crude amine in absolute CH₂Cl₂

(2 mL) and (R)-3-benzyloxytetradecanoic acid (18a)

(72 mg, 214 lmol) were added HOBt (6 mg, 42.9 lmol),

DMAP (5 mg, 40.9 lmol) and WSCDÆHCl (99 mg,

515 lmol) at room temperature under Ar atmosphere.

After stirring for 16 h, the mixture was puriﬁed by sili-

ca-gel ﬂash column chromatography (5 g, toluene/

AcOEt = 10:1) to give 34a (37 mg, 47%).

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Article Doi

Synthesis of lipid A monosaccharide analogues containing acidic amino acid: Exploring the structural basis for the endotoxic and antagonistic activities

DOI: 10.1016/j.bmc.2006.05.051

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