A new method of carbohydrate synthesis in both solution and solid phases using a special hydroxy protecting group

DOI: 10.1002/ejoc.200500382

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European Journal of Organic Chemistry p. 5313 - 5329 (2005)

Update date:2022-08-04

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Authors:

Komba, Shiro

Kitaoka, Motomitsu

Kasumi, Takafumi

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Article abstract of DOI:10.1002/ejoc.200500382

A new carbohydrate synthesis method using a special hydroxy protecting group (uni-chemo protection = UCP) in both solution and solid phases was developed. The UCP group was comprised of polymerized amino acid derivatives. Each hydroxyl group was protected by a UCP group with a different degree of polymerization, which allowed them to be uniquely identified. To deprotect the UCP group, one cycle of Edman degradation was performed as follows: 1) removal of the amino protecting group; 2) phenyl isothiocyanate coupling; 3) removal of the N-terminal phenyl thiocarbamoyl mono-amino acid derivative by treatment with trifluoroacetic acid; and 4) re-protection of the newly exposed amino group with a Boc group. The hydroxyl groups were deprotected successively from the UCP group with the lowest to the highest degree of polymerization by repeating this Edman degradation cycle. First, commercially available N-α-f-Boc- sarcosine, N-α-t-Boc-N-α-methyl-L-alanine, and N-α-t-Boc-L- phenylglycine were examined as UCP groups. Despite successfully protecting and selectively deprotecting the hydroxyl groups, there were problems with stability or reactivity. To address these problems, N-α-1-ethylpropylglycine was chosen as the UCP group, and we successfully synthesized two sialyl-T antigen analogues. Tri-UCP and mono-UCP were attached to the 6- and 3-positions of the D-galactosamine (GalN) derivative, respectively, using cyanuric chloride. To selectively deprotect the mono-UCP group on the 3-position of the GalN derivative, one cycle of Edman degradation was performed. As a result of this cycle, the tri-UCP on the 6-position of the GalN derivative was degraded to di-UCP, and the mono-UCP on the 3-position was selectively deprotected to yield a GalN derivative with a free 3-position. Glycosylation of this selectively deprotected free 3-position GalN derivative with a suitably protected D-galactose (Gal) derivative in which the 3-position was protected by a mono-UCP group using N-iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH) in dichloromethane yielded the desired disaccharide in high yield in both a position- and stereoselective manner. By repeating one cycle of Edman degradation, the 3′-position-free disaccharide in which the 6-position was protected by a mono-UCP group was prepared selectively. Subsequent acetylation and a final cycle of Edman degradation (except the third and fourth steps) yielded the 6-position-free disaccharide selectively. Two disaccharides, one with a free 3′-position and another with a free 6-position, were coupled with a sialic acid donor using NIS and TfOH in acetonitrile to obtain sialyl (2→3) T and sialyl (2→6) T antigen derivatives, respectively. Solid-phase synthesis was demonstrated using polystyrene-type beads and a new linker, 2-{4-(hydroxymethyl)benzamido}acetic acid (HMBA-Gly), to synthesize Galβ (1→3) Gal from a suitably protected Gal derivative in which the 3-position was protected by mono-UCP. Solid-phase glycosylation was successfully monitored by measuring the removal of the Fmoc group, which protected the amino group on UCP, similar to the method for monitoring solid-phase Fmoc peptide synthesis. This UCP hydroxy protecting group was suitable for solid-phase synthesis, and will be the key technique in both automated oligosaccharide synthesis and oligosaccharide library synthesis. Wiley-VCH Verlag GmbH & Co. KGaA, 2005.

Full text of DOI:10.1002/ejoc.200500382

FULL PAPER

DOI: 10.1002/ejoc.200500382

A New Method of Carbohydrate Synthesis in Both Solution and Solid Phases

Using a Special Hydroxy Protecting Group

Shiro Komba,*^[a]Motomitsu Kitaoka,^[a]and Takafumi Kasumi^[a][‡]

Keywords: Hydroxy protecting group / UCP / Solid-phase carbohydrate synthesis / Solution-phase carbohydrate synthesis

A new carbohydrate synthesis method using a special hy-

droxy protecting group (uni-chemo protection = UCP) in both

solution and solid phases was developed. The UCP group

was comprised of polymerized amino acid derivatives. Each

hydroxyl group was protected by a UCP group with a dif-

ferent degree of polymerization, which allowed them to be

uniquely identified. To deprotect the UCP group, one cycle

of Edman degradation was performed as follows: 1) removal

of the amino protecting group; 2) phenyl isothiocyanate

coupling; 3) removal of the N-terminal phenyl thiocarbamoyl

mono-amino acid derivative by treatment with trifluoroacetic

acid; and 4) re-protection of the newly exposed amino group

with a Boc group. The hydroxyl groups were deprotected

successively from the UCP group with the lowest to the high-

deprotected to yield a GalN derivative with a free 3-position.

Glycosylation of this selectively deprotected free 3-position

GalN derivative with a suitably protected

D-galactose (Gal)

derivative in which the 3-position was protected by a mono-

UCP group using N-iodosuccinimide (NIS) and trifluoro-

methanesulfonic acid (TfOH) in dichloromethane yielded the

desired disaccharide in high yield in both a position- and

stereoselective manner. By repeating one cycle of Edman de-

gradation, the 3Ј-position-free disaccharide in which the 6-

position was protected by a mono-UCP group was prepared

selectively. Subsequent acetylation and a final cycle of Ed-

man degradation (except the third and fourth steps) yielded

the 6-position-free disaccharide selectively. Two disaccha-

rides, one with a free 3Ј-position and another with a free 6-

est degree of polymerization by repeating this Edman degra- position, were coupled with a sialic acid donor using NIS and

dation cycle. First, commercially available N-α-t-Boc-sarco-

sine, N-α-t-Boc-N-α-methyl- -alanine, and N-α-t-Boc-

phenylglycine were examined as UCP groups. Despite suc-

TfOH in acetonitrile to obtain sialyl (2Ǟ3) T and sialyl (2Ǟ6)

T antigen derivatives, respectively. Solid-phase synthesis

was demonstrated using polystyrene-type beads and a new

L-

cessfully protecting and selectively deprotecting the hy- linker, 2-{4-(hydroxymethyl)benzamido}acetic acid (HMBA-

droxyl groups, there were problems with stability or reactiv-

ity. To address these problems, N-α-1-ethylpropylglycine was

chosen as the UCP group, and we successfully synthesized

two sialyl-T antigen analogues. Tri-UCP and mono-UCP

Gly), to synthesize Galβ (1Ǟ3) Gal from a suitably protected

Gal derivative in which the 3-position was protected by

mono-UCP. Solid-phase glycosylation was successfully moni-

tored by measuring the removal of the Fmoc group, which

protected the amino group on UCP, similar to the method

for monitoring solid-phase Fmoc peptide synthesis. This UCP

hydroxy protecting group was suitable for solid-phase syn-

thesis, and will be the key technique in both automated oli-

gosaccharide synthesis and oligosaccharide library synthesis.

Germany, 2005)

were attached to the 6- and 3-positions of the

D-galactos-

amine (GalN) derivative, respectively, using cyanuric chlo-

ride. To selectively deprotect the mono-UCP group on the 3-

position of the GalN derivative, one cycle of Edman degrada-

tion was performed. As a result of this cycle, the tri-UCP on

the 6-position of the GalN derivative was degraded to di-

UCP, and the mono-UCP on the 3-position was selectively

must then be selectively deprotected for the next round of

reactions. At present, the best method for synthesis of oli-

gosaccharides is the orthogonal method.^[1,5,6]For orthogo-

nal synthesis of oligosaccharides, a hydroxy protecting

group that can be deprotected using a special reagent is

used. These special reagents do not influence other protect-

ing groups, and selectively react with and remove the de-

sired protecting group. However, increasing the number of

saccharide coupling steps also increases the number of or-

thogonal protecting groups. Unsurprisingly, because of the

limited number of such orthogonal protecting groups, these

can not be applied to complicated, branched oligosaccha-

ride synthesis. For this reason, there is no generally appli-

cable method for oligosaccharide synthesis. The develop-

1. Introduction

A large number of hydroxy protecting groups for carbo-

hydrate synthesis have been developed and used successfully

for oligosaccharide synthesis.^[1–4]Despite the availability of

such large numbers of protecting groups, oligosaccharide

synthesis is still quite laborious. For glycosylation reactions,

the hydroxyl groups on both the acceptor and donor must

be differentiated and protected selectively. The product

[a] Enzyme Laboratory, National Food Research Institute

2-1-12 Kannondai, Tsukuba 305-8642, Japan

E-mail: skomba@nfri.affrc.go.jp

[‡] Current address: Enzyme Laboratory, Nihon University,

1866 Kameino, Fujisawa 252-8510, Japan

Eur. J. Org. Chem. 2005, 5313–5329

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S. Komba, M. Kitaoka, T. Kasumi

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ment of an optimal hydroxy protecting group for solid- the hydroxyl group on the 4-position, decreasing its reactiv-

phase carbohydrate synthesis would facilitate both carbo- ity. To attack the sterically hindered 4-OH of the Gal deriv-

hydrate library synthesis and automated carbohydrate syn- ative, the reaction mixture was heated to 50 °C in pyridine.

thesis. To establish a general method for solid-phase carbo- The resultant Gal derivatives 5, 6, and 8, in which the 4-

hydrate synthesis, a new concept of hydroxy group protec- and 6-positions were protected with a mono- and a di-UCP

tion is required.

group, respectively, were treated with one cycle of Edman

Recently, Miranda and Meldal reported the concept of degradation (Scheme 2). One cycle of Edman degradation

uni-chemo protection (UCP).^[7]UCP is comprised of an was comprised of a total of four steps: 1) deprotection of

amino acid polymer, which attaches to and protects the the amino protecting group; 2) coupling of PITC (phenyl

amino group. The individual amino groups have a different isothiocyanate; Edman reagent) to all amino termini;

degree of amino acid polymerization. Deprotection of this 3) cleavage of the N-terminal phenyl thiocarbamoyl mono-

protecting group is performed by Edman degradation,^[8]amino acid derivative from all UCP groups; 4) re-protection

which removes only the N-terminal amino acid from all of all newly exposed N-terminal amino groups with Boc

UCP groups. Depending on the degree of polymerization groups. In the first step, three suitably protected Gal deriva-

of the amino acid protecting group, each amino group can tives 5, 6, and 8 were treated with TFA in CH₂Cl₂at room

be characterized and controlled. We applied this concept to temperature for 30 min, and the Boc group was completely

hydroxy protection for oligosaccharide synthesis. To protect removed. The reaction mixtures were then evaporated and

individual hydroxyl groups on the carbohydrate, amino ac- co-evaporated with toluene. In the second step, the result-

ids with different degrees of polymerization were attached ant three Gal derivatives with unprotected amino groups

by ester linkages. In addition, the polymerized amino acid were treated with phenyl isothiocyanate (PITC) and N-

protecting groups were removed successively from the N- methylmorpholine in DMF at room temperature for

terminus using the Edman degradation method. Each hy- 30 min. Subsequently, the organic layer was extracted with

droxyl group can be characterized and controlled using water and evaporated, and the phenylthiocarbamoyl com-

only one kind of protecting group and one deprotection

method.^[9]

To demonstrate the utility of this concept, three experi-

ments were performed. In the first experiment, we examined

the reactivity of the amino acid derivatives that comprise

the UCP group. In the second experiment, we examined the

synthesis of two kinds of sialyl-T antigens^[10]using a UCP

group. The third experiment was performed to examine so-

lid-phase oligosaccharide synthesis using a UCP group.

2. Results and Discussion

2.1. Examination of the Reactivity of the Amino Acid

Derivatives that Comprise the UCP Group

We used commercially available N-α-t-Boc-sarcosine, N-

α-t-Boc-N-α-methyl--alanine, and N-α-t-Boc--phenylgly-

cine as amino acids comprising the UCP group. Phenyl 2,3-

di-O-benzoyl-1-thio-β--galactose (1), which was prepared

from phenyl 4,6-O-benzylidene-1-thio-β--galactose (36)^[11]

in two steps, was chosen and used to protect the 4- and 6-

positions with a mono-UCP group and a di-UCP group,

respectively (Scheme 1). Each amino acid that was pro-

tected with a Boc group on the amine was selectively intro-

duced into the 6-position of the Gal residue by an ester

linkage using DIC and DMAP via symmetrical anhydride (Boc-N-Me-Ala-OH, DIC, CH₂Cl₂, 0 °C, 40 min.), DMAP, DMF,

or cyanuric chloride. Subsequently, the Boc group was re-

moved by TFA, and additional amino acid derivatives were

attached by amide linkages to the amino terminus of the

amino acid protected with 6-OH, using DIC and DMAP

via the same symmetrical anhydride. During this reaction,

the hydroxyl group on the 4-position of the Gal derivative

was also esterified by the amino acid, except in the case of

the phenylglycine derivative. The sterically large phenylgly-

Scheme 1. Reagents and conditions. 1, Boc-Sar-OH, cyanuric

chloride, pyr., room temp., 1 h, 97%; 1, symmetrical anhydride

room temp., 1 h, 78%; 1, symmetrical anhydride (Boc-Phg-OH,

DIC, CH₂Cl₂, 0 °C, 40 min.), DMAP, DMF, room temp., 1 h, 94%;

i), 2, TFA, CH₂Cl₂, room temp., 30 min. ii), symmetrical anhy-

dride (Boc-Sar-OH, DIC, CH₂Cl₂, 0 °C, 40 min.), DMAP, DMF,

room temp., 1 h, 73%; i), 3, TFA, CH₂Cl₂, room temp., 30 min.

ii), symmetrical anhydride (Boc-N-Me-Ala-OH, DIC, CH₂Cl₂,

0 °C, 40 min.), DMAP, DMF, room temp., 1 h, 75%; i), 4, TFA,

CH₂Cl₂, room temp., 30 min. ii), symmetrical anhydride (Boc-Phg-

OH, DIC, CH₂Cl₂, 0 °C, 40 min.), DMAP, DMF, room temp., 1 h,

92%; 7, symmetrical anhydride (Boc-Phg-OH, DIC, CH₂Cl₂,

cine protecting the 6-position of the Gal derivative covered 0 °C, 40 min.), DMAP, pyr., 50 °C, 5 h, 68 %.

5314 Eur. J. Org. Chem. 2005, 5313–5329

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A New Method of Carbohydrate Synthesis Using a Special Hydroxy Protecting Group

FULL PAPER

pounds 12, 13, and 14 were roughly separated by column protected with Boc groups using di-tert-butyl dicarbonate

chromatography. During this step, the mono-UCP group on in saturated aqueous NaHCO₃and DMF at room tempera-

the 4-position of the Gal derivatives was cleaved immedi- ture for 30 min. Subsequently, the organic layer was ex-

ately owing to the electron-withdrawing effect, except in the tracted with water and evaporated, and the selectively 4-

case of the phenyl glycyl compound; the phenyl group on position-deprotected Gal derivatives 18, 19, and 20, in

the phenylglycine residue neutralized this electron-with- which the 6-position was selectively protected by the mono-

drawing effect and stabilized the ester linkage. These results UCP group, were purified by column chromatography. The

were confirmed by ESI-MS spectroscopy in positive ion Boc re-protection of this final step was the most important

mode. In the third step, phenylthiocarbamoyl compounds step. Using a saccharide that was protected by amino-un-

were treated with TFA in CH₂Cl₂at room temperature for protected UCP for glycosylation yielded no glycosylated

30 min. The terminal phenylthiocarbamoyl mono-amino product because of problems with the stability of the UCP

acid derivatives were removed selectively in this step. In the containing a free amine (unpublished data). The yields of

fourth step, the resultant free amines 15, 16, and 17 were one cycle of Edman degradation for the three amino acid

Scheme 2. Reagents and conditions. 5, TFA, CH₂Cl₂, room temp., 30 min; 6, TFA, CH₂Cl₂, room temp., 30 min; 8, TFA, CH₂Cl₂,

room temp., 30 min; 9, PITC, NMM, DMF, room temp. 30 min; 10, PITC, NMM, DMF, room temp. 30 min; 11, PITC, NMM,

DMF, room temp. 30 min; 12, TFA, CH₂Cl₂, room temp., 30 min; 13, TFA, CH₂Cl₂, room temp., 30 min; 14, TFA, CH₂Cl₂, room

temp., 30 min; 15, Boc₂O, satd. aq. NaHCO₃, DMF, room temp., 30 min, 47% (4 steps); 16, Boc₂O, satd. aq. NaHCO₃, DMF, room

temp., 30 min, 62% (4 steps); 17, Boc₂O, satd. aq. NaHCO₃, DMF, room temp., 30 min, 69% (4 steps).

Eur. J. Org. Chem. 2005, 5313–5329

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S. Komba, M. Kitaoka, T. Kasumi

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derivatives, sarcosine (5), N-methyl alanine (6), and phenyl- ize this UCP group, and successful coupling was obtained.

glycine (8), were 47%, 62%, and 69%, respectively. Despite The tert-butyldimethylsilyl 2-azido-4,6-O-benzylidene-2-de-

the success of one cycle of Edman degradation, these three oxy-β--galactopyranoside (30) was used as the GalN resi-

amino acid derivatives were not suitable for complex oligo- due in which the 1-position was protected by a tert-butyldi-

saccharide synthesis because of the low reaction yield due methylsilyl group for selective deprotection for future steps,

to poor stability (sarcosine), complicated NMR peaks from and the 2-position had an azido group to introduce an an-

the chiral center and the rotamers of carbamate (N-methyl omeric α-configuration for future coupling with serine or

alanine), and the reaction of the hydroxyl group being dis- threonine^[12](Scheme 4). The mono-UCP was selectively in-

turbed due to the sterically large phenyl group (phenylgly- troduced into the 3-position of the GalN residue using the

cine).

in situ activating reagent cyanuric chloride in pyridine at

40 °C. After deprotection of the benzylidene group, the 6-

position of GalN derivative was selectively esterified by the

tri-UCP group according to the same method. Sub-

sequently, the 4-position of the GalN derivative was pro-

tected by an acetyl group to prepare the suitably protected

GalN residue 34. Subsequent treatment with one cycle of

Edman degradation yielded the selectively deprotected 3-

OH GalN derivative 35, in which the 6-position was pro-

tected by the di-UCP group in high yield (four steps, total

yield of 69%). In comparison with previously tested com-

mercially available amino acids, sarcosine, N-methyl ala-

nine, and phenylglycine, this N-α-1-ethylpropylglycine was

highly stable and deprotected in high yield. The final step

of one cycle of Edman degradation was the Boc re-protec-

tion step. The reason we chose the Boc group instead of the

Fmoc group for amine protection was for the next round

2.2. Examination of the Synthesis of Two Kinds of Sialyl-T

Antigens Using a UCP Group

N-α-1-Ethylpropylglycine was chosen as the amino acid

for UCP. To achieve greater stability, a 1-ethylpropyl group

was introduced as an amino protecting group compared

with the methyl group in sarcosine or N-methyl alanine. As

the backbone amino acid, glycine was chosen because it has

no chiral center and no sterically large groups. Meldal et al.

used N-α-1-methylpropylglycine as the amino acid for the

UCP group.^[7]However, this amino protecting group has a

chiral carbon atom. To avoid the presence of a chiral center,

we chose a 1-ethylpropyl group as an amino protecting

group. However, the NMR spectrum was still complicated

due to the rotamers of carbamate. This N-α-1-ethylpro-

pylglycine was synthesized according to Meldal’s method^[7]

(Scheme 3). The N-α-1-ethylpropylglycine derivative was

prepared by the coupling of 3-aminopentane (21) with tert-

butyl bromoacetate (22). Subsequently, the amine was pro-

tected by the Fmoc group and the tert-butyl group was re-

moved, thus the Fmoc-protected mono-UCP 25 was pre-

pared. The coupling reagent PyBroP was used to polymer-

Scheme 4. Reagents and conditions. 25, cyanuric chloride, pyr.,

Scheme 3. Reagents and conditions. THF, room temp., 4 h, 98%;

40 °C, 2 h, 90 %; 80% aq. AcOH, 60 °C, 2 h, 79 %; 29, cyanuric

d e

Fmoc-Cl, 1,4-dioxane/satd. aq. NaHCO₃, room temp., 2 h, 86%;

chloride, pyr., 40 °C, 2 h, 65 %; Ac₂O, pyr., 40 °C, 2 h, 91 %; i),

20% piperidine in DMF, room temp., 20 min. ii), PITC, NMM,

room temp., 30 min. iii), TFA, CH₂Cl₂, room temp., 30 min. iv),

Boc₂O, satd. aq. NaHCO₃, DMF, room temp., 30 min, 69% (4

steps).

^c90% formic acid, 40 °C, 7 h, 90%; ^dDIPEA, PyBroP, DMF, room

temp., 30 min, 91%; ^e90% formic acid, 50 °C, 3 h, 93 %; ^fDIPEA,

PyBroP, DMF, room temp., 30 min, 85%; ^g90% formic acid, 50 °C,

2 h, 98%.

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Eur. J. Org. Chem. 2005, 5313–5329

A New Method of Carbohydrate Synthesis Using a Special Hydroxy Protecting Group

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of Edman degradation. If the di-UCP were protected by an

Fmoc group, subsequent treatment with piperidine in basic

solution to remove Fmoc would result in attack by the free

amino group on the carbonyl carbon at the C-terminus of

the UCP group, resulting in formation of cyclic di-UCP by

removal of the hydroxyl group. To avoid this cyclization,

the amino protecting group must be acid-cleavable, such as

a Boc group. Phenyl 4,6-O-benzylidene-1-thio-β--galacto-

pyranoside (36)^[11]was used as the Gal residue (Scheme 5).

To selectively protect the 3-position of the Gal derivative,

the mono-UCP was initially treated with cyanuric chloride

in pyridine at 40 °C for 30 min to convert it into the acti-

vated ester. This activated ester was added in a dropwise

manner to the Gal derivative in pyridine at 0 °C, and the

mixture was stirred for 1 h at 0 °C. Subsequently protected

by benzoyl group on the 2-position of the Gal residue, the

4, 6-protecting group was exchanged from a benzylidene

Scheme 5. Reagents and conditions. 25, cyanuric chloride, pyr.,

group to two acetyl groups. To match the amino protecting

group of UCP on the GalN derivative, the Fmoc group was

exchanged for a Boc group in high yield. The resultant Gal

donor 41 was selectively coupled with the 3-position on the

0 °C, 1 h, 73 %; BzCl, pyr., room temp., 2 h, 97%; 80% aq.

AcOH, 60 °C, 2 h, 65 %; Ac₂O, pyr., room temp., 2 h, 76%;

20% piperidine in DMF, room temp., 20 min then Boc₂O, satd. aq.

NaHCO₃, room temp., 1 h, 83% (2 steps).

Scheme 6. Reagents and conditions. ^aNIS, TfOH, MS 4 Å, CH₂Cl₂, –40 °C, 2 h, 89 %; ^bi), TFA, CH₂Cl₂, room temp., 30 min. ii), PITC,

NMM, room temp., 30 min. iii), TFA, CH₂Cl₂, room temp., 30 min. iv), Boc₂O, satd. aq. NaHCO₃, DMF, room temp., 30 min, 70% (4

steps); i), Ac₂O, pyr., room temp. 2 h, 99%. ii), TFA, CH₂Cl₂, room temp., 30 min. iii), PITC, NMM, room temp., 30 min, 98% (2

steps); NIS, TfOH, MS 3 Å, CH₃CN, –30 °C, 48 h, 13%; NIS, TfOH, MS 3 Å, CH₃CN, –30 °C, 10 h, 67% (α/β = 71:29).

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GalN₃acceptor (35) using N-iodosuccinimide (NIS) and pared by coupling of 4-(hydroxymethyl)benzoic acid

trifluoromethanesulfonic acid (TfOH) in CH₂Cl₂at –40 °C (HMBA) with glycine via a peptide linkage. This linker was

in a yield of 89% (Scheme 6). In the H-NMR spectrum of attached to the hydroxyl group on the solid support by an

the anomeric protons on the Gal residue, peaks were ob- ester linkage. The sugar anomeric position was attached via

served at δ (ppm) 4.87, 4.88, 4.91, and 4.92 (4d, 1 H, J_1,2

a hydroxyl group on the benzyl position of the linker by

7.9, 8.1, 7.9, and 7.8 Hz). The peaks were very complicated glycoside linkage formation so the sugar moiety could be

due to the rotamers of carbamate, but it was suggested to released from this linker by hydrogenolysis. Fmoc-protected

be in the β-configuration. Following treatment with one cy- glycine 49 was coupled to hydroxyl groups on the resin

cle of Edman degradation, the 3Ј-free disaccharide 43 in using DIC and DMAP in CH₂Cl₂at room temperature.

which the 6-position was protected by mono-UCP was pre- Fmoc loading was measured by 290 nm UV spectrometry

pared. This was an acceptor for the synthesis of sialyl (2– in 20% piperidine/DMF; the loading was estimated to be

3) T antigen. After protection by adding an acetyl group 0.449 mmol/g. Following cleavage of the Fmoc group using

to the 3Ј-position of the disaccharide, one cycle of Edman 20% piperidine/DMF, HMBA derivative 52, in which the

degradation was repeated, except the third step (TFA treat- hydroxyl was protected by a trityl group, was coupled to

ment to remove the N-terminal amino acid) and the fourth the amino terminus of the glycine on the resin using the

step (re-protecting the amino group with Boc). This pro- TBTU and NEM coupling method. After cleavage of the

duced the disaccharide 44 selectively deprotected at the 6- trityl group using TFA in H₂O, the suitably protected Gal

position in high yield. Disaccharide 44 was an acceptor for donor 40 was coupled using dimethyl(methylthio)sulfonium

the synthesis of sialyl (2–6) T antigen. These two disaccha- triflate (DMTST) as a promoter in CH₂Cl₂at room tem-

rides 43 and 44 were sialylated with the sialyl SPh donor perature for 1 d. The resin was washed with DMF, CH₂Cl₂,

45^[13]using NIS and TfOH as promoters in acetonitrile^[14,and Et₂O, dried in vacuo, and the glycosylation procedure

15]

at –30 °C. The sialyl (2–3) T antigen 46 was prepared in was repeated. To measure the coupling yield, aliquots of the

a yield of 13%. Unfortunately, the anomeric configuration resins were treated with 20% piperidine/DMF to cleave the

of sialic acid was shown to be β by NMR spectroscopy.^[16]Fmoc group protecting the UCP group on the 3-position

In this case, no α configuration was observed. On the other of Gal, and the eluate was examined by UV spectrometry

hand, sialyl (2–6) T antigen 47 was prepared in a yield of at 290 nm. For a single coupling, the loading was estimated

67% (α/β = 71:29 mixture).^[16]The ratio of the anomeric as 0.076 mmol/g, and after a double coupling, the loading

configuration was calculated from the integration of the was increased to 0.103 mmol/g. After the four steps (Fmoc

equatorial proton at the 3-position on the sialic acid resi- deprotection, Tr-HMBA coupling, Tr deprotection, and

due. Despite using acetonitrile as the solvent in both coup- glycosylation), the yields of single coupling and double

ling reactions, the α anomer was lost selectively because of coupling were estimated as 22% and 29%, respectively, with

steric problems (sialyl 2–3 T) and the high reactivity of the theoretical loading of 0.352 mmol/g. In addition, aliquots

primary hydroxyl group (sialyl 2–6 T), respectively. Never- of the double-coupled resin were treated with NaOMe in

theless, this new method for oligosaccharide synthesis using MeOH and CH₂Cl₂at room temperature for 4 h. In ad-

a UCP group is quite promising, because of the selective dition, following the addition of water, the reaction was

cleavage of the desired hydroxyl group and stepwise depro- continued for a further 4 h at room temperature. The eluate

tection using only one deprotection method.

of the aqueous layer was analyzed by analytical HPLC (Fig-

ure 1, A). The peaks were detected at 254 nm using a UV

detector. The yield was calculated from the area under the

peaks. The coupling yield of Gal was determined as 31%.

This demonstrated that Fmoc measurement allowed deter-

mination of the coupling yield. Following treatment with

acetic anhydride in pyridine and CH₂Cl₂at room tempera-

ture for 2 h, one cycle of Edman degradation was per-

2.3. Examination of Solid-Phase Oligosaccharide Synthesis

Using a UCP Group

Galβ (1Ǟ3) Gal was synthesized by a solid-phase reac- formed except for the third (TFA treatment) and fourth

tion using a UCP group (Scheme 7). N-Fmoc-protected N- (Boc reprotection) steps. The resultant Gal residue in which

α-1-ethylpropylglycine was chosen as the amino acid for the the 3-position hydroxyl group was free was glycosylated

UCP group. The previously prepared Gal derivative 40, in with the same Gal donor 40 in which the 3-position was

which the 3-position was protected by an Fmoc-protected protected by the Fmoc-protected mono-UCP group, using

mono-UCP group, was used and coupled together on a so- the same double coupling conditions. After single coupling,

lid support consisting of a polystyrene-type resin [ArgoPore the loading was estimated as 0.041 mmol/g. After double

OH (48); Argonaut Technologies Inc., Foster City, CA, coupling, the loading was increased to 0.046 mmol/g, as es-

USA].^[17]A new type of linker for solid-phase oligosaccha- timated from Fmoc measurement. The glycosylation yields

ride synthesis was designed, 2-[4-(hydroxymethyl)benz- of both single and double coupling, which included the

amido]acetic acid (HMBA-Gly), to make it easy to release UCP deprotection step, were estimated as 36% and 41%,

the carbohydrate-linker complex from the solid support un- respectively, with theoretical loading of 0.113 mmol/g. Ali-

der basic conditions. In addition, to allow detection of the quots of the double-coupled resin were treated with Na-

target compound by UV on HPLC, HMBA-Gly was pre- OMe in MeOH and CH₂Cl₂at room temperature for 4 h.

5318

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A New Method of Carbohydrate Synthesis Using a Special Hydroxy Protecting Group

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Water was then added, and the reaction was continued for was estimated as 4% over 8 steps (Fmoc deprotection, Tr-

a further 4 h at room temperature. The eluate of the aque- HMBA coupling, Tr deprotection, glycosylation, Fmoc de-

ous layer was analyzed by analytical HPLC (Figure 1, A). protection, UCP deprotection, glycosylation, and cleavage

The glycosylation yield was estimated as 42%. Again, Fmoc from the resin). These results demonstrated that this

measurement was confirmed to be a good method to deter- method is applicable to solid-phase oligosaccharide synthe-

mine yield. Cleavage of the disaccharide-HMBA-Gly com- sis, including solid-phase oligosaccharide library synthesis

plex from the resin was performed with NaOMe in CH₂Cl₂or solid-phase automated oligosaccharide synthesis.

and MeOH for one day at room temperature, water was

In conclusion, the results of the present study clearly

added, and the resultant mixture was mixed for one more demonstrated that this hydroxyl-protecting UCP method is

day.^[18]The cleaved disaccharide derivative was purified by a useful technique for oligosaccharide synthesis, which is

HPLC. The target disaccharide 58 was obtained in a yield applicable to both automated oligosaccharide synthesis and

of 1.8 mg, and the total yield of cleavage from the resin library synthesis.

Scheme 7. Reagents and conditions. 49, DIC, DMAP, CH₂Cl₂, room temp., 7 h, then Ac₂O, pyr., CH₂Cl₂, room temp., 2 h; 20%

piperidine in DMF, room temp., 22 min; ^c52, NEM, TBTU, DMF, room temp., 4 h; ^d90% TFA in H₂O, room temp., 3 h; ^e40, DMTST,

CH₂Cl₂, room temp., 1 d. This was repeated once more, then Ac₂O, pyr., CH₂Cl₂, room temp., 2 h; , i), 20% piperidine in DMF, room

temp., 22 min, ii), PITC, NMM, DMF, room temp., 2 h; ^g40, DMTST, CH₂Cl₂, room temp., 1 d, this was repeated once more; ^hNaOMe

in MeOH, CH₂Cl₂, MeOH, room temp., 1 d, then added H₂O, room temp., 1 d, 4% (8 steps from 50).

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Figure 1. HPLC traces: A) After double coupling of initial sugar to the resin. B) After double coupling of secondary sugar to the resin.

The yield was calculated by peak area.

Abbreviations Used: DIC: 1,3-diisopropylcarbodiimide, DMAP: 4-

(dimethylamino)pyridine, THF: tetrahydrofuran, tBu: tert-butyl,

Experimental Section

General Procedures: H- and ¹³C-NMR spectra were obtained on

Fmoc: fluorenylmethoxycarbonyl, Ac: acetyl, Bz: benzoyl, Boc: but-

oxycarbonyl, DIPEA: N,N-diisopropylethylamine, PyBroP^TM

a Bruker DRX-600 spectrometer in CDCl₃or D₂O. Chemical shifts

are expressed in parts per million relative to the signal of either

CHCl₃or Me₄Si in CDCl₃, adjusted to 7.24 or 0.00 ppm, respec-

tively, or MeOH in D₂O, adjusted to 3.30 ppm. ESI-FT-MS spectra

were recorded in the positive ion mode on a Bruker APEX II 70e

mass spectrometer. Optical rotations were determined with a JA-

SCO P-1020-GT polarimeter in CHCl₃or H₂O at ambient tem-

perature. Preparative chromatography was performed on silica gel

[silica gel 60 (230–400 mesh); Nacalai Tesque Inc., Kyoto, Japan]

with the solvent systems specified. Analytical normal-phase HPLC

separation was performed on a Shimadzu HPLC system, using a

TSK-gel Amide-80 column (4.6×250 mm, 5 µm, Tosoh Co. Ltd.),

with a flow rate of 1 mL min^–1and detection at 254 nm. The sol-

vent system was as follows: solvent A, TFA/acetonitrile/water

(1:900:100); solvent B, TFA/water (1:1000). All reagents and sol-

vents were of reagent grade.

bromo-Tris-pyrrolidino-phosphonium

hexafluorophosphate,

DMF: N,N-dimethylformamide, TBDMS: tert-butyldimethylsilyl,

pyr.: pyridine, Ac₂O: acetic anhydride, PITC: phenyl isothiocyan-

ate, TFA: trifluoroacetic acid, Boc₂O: di-tert-butyl dicarbonate,

SPh: thiophenyl, NIS: N-iodosuccinimide, TfOH: trifluorome-

thanesulfonic acid. MS: molecular sieves, room temp.: room tem-

perature, aq.: aqueous, satd.: saturated, GalN: -galactosamine,

Gal: -galactose, SA: sialic acid, NEM: N-ethylmorpholine,

NMM: N-methylmorpholine, TBTU: 2-(1H-benzotriazole-1-yl)-

1,1,3,3-tetramethyluronium tetrafluoroborate, DMTST: dimeth-

yl(methylthio)sulfonium triflate.

Phenyl 2,3-Di-O-benzoyl-6-O-{2-[tert-butoxycarbonyl(methyl)-

amino]acetyl}-1-thio-β-

D-galactopyranoside (2): To a solution of 1

(100 mg, 208 µmol) in pyridine (3 mL) was added N-α-t-Boc-sarco-

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A New Method of Carbohydrate Synthesis Using a Special Hydroxy Protecting Group

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sine (43 mg, 227 µmol) and cyanuric chloride (115 mg, 624 µmol),

and the mixture was stirred for one hour at room temp. After con-

centration, the product was purified by chromatography on a col-

umn of silica gel with 1:2 ethyl acetate/hexane to give 2 (131 mg,

97%). Owing to the rotamers of carbamate, some peaks of both

¹H NMR and ¹³C NMR appeared as double peaks. In addition,

all other compounds that had a tertiary amino group gave compli-

cated NMR signals because of their rotamers. [α]_D= 52.0 (c = 1.3,

H), 5.39, 5.42 [2 d, J_gem= 7.2, 7.2 Hz, 1 H, NCH(Ph)CO], 5.83,

5.84 (2 t, J_1,2= J_2,3= 10.0, 9.9 Hz, 1 H, 2-H), 7.29–8.00 [m, 20

H, 2 PhCOO, NCH(Ph)CO and SPh] ppm. ¹³C NMR (150 MHz,

CDCl₃): δ = 28.00, 28.05 (Me₃CO), 57.22, 57.56 [NCH(Ph)CO],

63.54, 63.76 (C-6), 66.74, 66.88 (C-4), 67.77 (C-2), 74.90 (C-3),

75.69, 75.73 (C-5), 79.90, 80.20 (Me₃CO), 86.28 (C-1), 126.89,

126.99, 127.76, 127.84, 128.13, 128.27, 128.41, 128.57, 128.66,

128.78, 129.15, 129.17, 129.52, 129.60, 129.84, 132.30, 132.48,

CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ = 1.40, 1.43 (2 s, 9 H, 133.00, 133.11, 133.16, 133.19 [2 PhCOO, NCH(Ph)CO and SPh],

Me₃CO), 2.90 (s, 3 H, MeN), 3.91, 3.93, 3.94, 4.05 (4 d, J_gem154.77, 156.36 (C=O Boc), 165.13, 165.52 (2 PhCOO), 170.00,

17.7, 17.4, 17.7, 17.7 Hz, 2 H, NCH₂CO), 3.99 (br. d, 1 H, 5-H), 1 7 1 . 2 6 [ N C H ( P h ) C O ] p p m . E S I - F T- M S : c a l c d . fo r

4.33, 4.35 (2 br. s, 1 H, 4-H), 4.47 (m, 2 H, 6-H), 4.96 (d, J_1,2

C₃₉H₃₉NO₁₀SNa⁺[M + Na⁺] 736.21869; found 736.21754.

10.0 Hz, 1 H, 1-H), 5.36 (br. d, J_2,3= 10.0 Hz, 1 H, 3-H), 5.79 (t,

J_1,2= J_2,3= 9.9 Hz, 1 H, 2-H), 7.29–7.97 (m, 15 H, 2 PhCOO and

SPh) ppm. ¹³C NMR (150 MHz, CDCl₃): δ = 28.19 (Me₃CO),

35.45, 35.61 (MeN), 50.17, 50.71 (NCH₂CO), 63.02, 63.38 (C-6),

67.08, 67.27 (C-4), 67.72, 67.83 (C-2), 75.13 (C-3), 75.97 (C-5),

80.29 (Me₃CO), 86.54 (C-1), 127.92, 128.04, 128.31, 128.36, 128.78,

128.83, 128.90, 129.31, 129.69, 129.76, 132.40, 132.61, 133.14,

133.20, 133.31, 133.42 (2 PhCOO and SPh), 155.28, 156.07 (C=O

Boc), 165.24, 165.66 (2 PhCOO), 169.70 (NCH₂CO) ppm. ESI-

FT-MS: calcd. for C₃₄H₃₇NO₁₀SNa⁺[M + Na⁺] 674.20304; found

674.20387.

Phenyl 2,3-Di-O-benzoyl-6-O-[2-{2-[tert-butoxycarbonyl(methyl)-

amino]-N-methylacetamido}acetyl]-4-O-{2-[tert-butoxycarbonyl-

(methyl)amino]acetyl}-1-thio-β-D-galactopyranoside (5): To a solu-

tion of 2 (249 mg, 382 µmol) in dichloromethane (5 mL) was added

trifluoroacetic acid (1 mL). The mixture was stirred for 30 min at

room temp., and then concentrated (A). To a solution of N-α-t-

Boc-sarcosine (578 mg, 3.05 mmol) in dichloromethane (2 mL) was

added DIC (240 µL, 1.53 mmol) at 0 °C. The mixture was stirred

for 40 min at 0 °C, and then concentrated (B). To a solution of A

and B in DMF (3.2 mL) was added DMAP (5 mg, 41 µmol), and

the mixture was stirred for one hour at room temp. The work-up

as described for 3 yielded 5 (248 mg, 73%). [α]_D= 29.9 (c = 1.2,

Phenyl 2,3-Di-O-benzoyl-6-O-{2-[tert-butoxycarbonyl(methyl)-

amino]propionyl}-1-thio-β-

D-galactopyranoside (3): To a solution of

CHCl₃). H NMR (600 MHz, CDCl₃): δ = 1.35, 1.43, 1.47 (3 s, 18

N-α-t-Boc-N-α-methyl--alanine (127 mg, 625 µmol) in dichloro-

methane (500 µL) was added 1,3-diisopropylcarbodiimide (49 µL,

313 µmol) at 0 °C, and the mixture was stirred for 40 min at 0 °C.

After concentration, the product was dissolved in DMF (500 µL).

To the resultant solution was added 1 (100 mg, 208 µmol) and 4-

(dimethylamino)pyridine (3 mg, 25 µmol), and the mixture was

stirred for 1 h at room temp. After concentration, the product was

purified by chromatography on a column of silica gel with ethyl

acetate/hexane (1:1) to give 3 (108 mg, 78%). [α]_D= 38.8 (c = 1.2,

H, 2 Me₃CO), 2.71, 2.72, 2.75, 2.87, 2.87, 2.92, 2.93, 3.00, 3.02 (9

s, 9 H, 3 MeN), 3.89–4.27 (m, 6 H, 3 NCH₂CO), 4.20–4.40 (m, 2

H, 6-H), 4.21 (m, 1 H, 5-H), 5.01, 5.04, 5.07 (3 d, J_1,2= 9.3, 10.3,

9.9 Hz, 1 H, 1-H), 5.44–5.54 (m, 1 H, 3-H), 5.65, 5.67 (2 t, J_1,2

J_2,3= 10.0, 10.2 Hz, 1 H, 2-H), 5.71, 5.74 (2 d, J_3,4= 2.5, 2.4 Hz,

1 H, 4-H), 7.32–7.98 (m, 15 H, 2 PhCOO and SPh) ppm. ¹³C NMR

(150 MHz, CDCl₃): δ = 27.99, 28.08, 28.13, 28.22 (2 Me₃CO),

34.79, 35.17, 35.42 (3 MeN), 49.31, 49.56, 49.80, 49.85, 50.12, 50.37

(3 CH₂CO), 61.43, 61.57 (C-6), 67.59, 67.75 (C-2), 67.87, 68.03 (C-

4), 72.37, 72.69 (C-3), 74.04, 74.14 (C-5), 79.76, 80.03, 80.15 (2

CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ = 1.40 [d, J_CH3,CH

7.3 Hz, 3 H, NCH(Me)CO], 1.42, 1.46 (2 s, 9 H, Me₃CO), 2.81, MeCO), 86.51 (C-1), 128.30, 128.48, 128.62, 128.82, 128.85, 128.99,

2.85 (2 s, 3 H, MeN), 4.00 (br. s, 1 H, 5-H), 4.34 (br. d, 1 H, 4-H), 129.43, 129.58, 129.62, 132.73, 133.05, 133.23 (2 PhCOO and SPh),

4.36–4.54 (m, 2 H, 6-H), 4.43, 4.76, 4.82 [3 m, 1 H, NCH(Me)CO], 154.94, 155.55, 155.65, 156.10 (C=O 2 Boc), 165.02, 165.14, 165.18

4.96 (d, J_1,2= 10.0 Hz, 1 H, 1-H), 5.37, 5.40 (2 dd, J_2,3= 9.9,

10.0 Hz, J_3,4= 3.1, 3.2 Hz, 1 H, 3-H), 5.79 (t, J_1,2= J_2,3= 10.0 Hz,

(2 PhCOO), 168.30, 168.37, 168.94, 169.03, 169.18, 169.33 (3

NCH₂CO) ppm. ESI-FT-MS: calcd. for C₄₅H₅₅N₃O₁₄SNa⁺[M +

1 H, 2-H), 7.28–7.97 (m, 15 H, 2 PhCOO and SPh) ppm. ¹³C NMR Na⁺] 916.32970; found 916.32999.

(150 MHz, CDCl₃): δ = 14.49, 14.65, 14.97, 15.17 [NCH(Me)CO],

Phenyl 2,3-Di-O-benzoyl-4-O-{2-[tert-butoxycarbonyl(methyl)-

28.23 (Me₃CO), 30.51, 30.66, 31.09, 31.28 (MeN), 53.44, 53.68,

54.86, 55.10 [NCH(Me)CO], 63.08, 63.47 (C-6), 67.09, 67.30 (C-4),

67.83 (C-2), 73.03, 75.02 (C-3), 76.02 (C-5), 80.30 (Me₃CO), 86.64

(C-1), 127.88, 128.28, 128.84, 129.31, 129.69, 129.77, 132.24,

132.55, 133.14, 133.30 (2 PhCOO and SPh), 155.17, 156.02 (C=O

Boc), 165.24, 165.65 (2 PhCOO), 171.97, 172.17 [NCH(Me)CO]

ppm. ESI-FT-MS: calcd. for C₃₅H₃₉NO₁₀SNa⁺[M + Na⁺]

688.21869; found 688.21912.

amino]propionyl}-6-O-[2-{2-[tert-butoxycarbonyl(methyl)amino]-N-

methylpropanamido}propionyl]-1-thio-β- -galactopyranoside (6): To

a solution of 3 (217 mg, 326 µmol) in dichloromethane (5 mL) was

added trifluoroacetic acid (1 mL). The mixture was stirred for

30 min at room temp., and then concentrated (A). To a solution of

N-α-t-Boc-N-α-methyl--alanine (530 mg, 2.61 mmol) in dichloro-

methane (4 mL) was added DIC (204 µL, 1.30 mmol) at 0 °C. The

mixture was stirred for 40 min at 0 °C, and then concentrated (B).

Phenyl 2,3-Di-O-benzoyl-6-O-[2-(tert-butoxycarbonylamino)-2- To a solution of A and B in DMF (6 mL) was added DMAP (4 mg,

phenylacetyl]-1-thio-β- -galactopyranoside (4): To a solution of N- 33 µmol), and the mixture was stirred for one hour at room temp.

α-t-Boc--phenylglycine (345 mg, 1.37 mmol) in dichloromethane

(2 mL) was added DIC (108 µL, 690 µmol) at 0 °C, and the mixture

was stirred for 40 min at 0 °C. After concentration, the product was

dissolved in DMF (3 mL). To the resultant solution was added 1

The work-up as described for 3 yielded 6 (228 mg, 75%). [α]_D=

–19.1 (c = 0.5, CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ = 1.25–1.39

[m, 9 H, 3 NCH(Me)CO], 1.42, 1.47 (2 s, 18 H, 2 Boc), 2.49–2.93

(m, 9 H, 3 MeN), 4.12–4.39 (m, 2 H, 6-H), 4.17 (m, 1 H, 5-H),

(300 mg, 624 µmol) and DMAP (7.6 mg, 62 µmol), and the mixture 4.71–5.17 [m, 3 H, 3 NCH(Me)CO], 4.96 (br. d, J_1,2= 10.0 Hz, 1

was stirred for one hour at room temp. The work-up as described

H, 1-H), 5.36 (br. d, J_2,3= 9.5 Hz, 1 H, 3-H), 5.55 (br. t, J_1,2= J_2,3

= 9.6 Hz, 1 H, 2-H), 5.66 (br. s, 1 H, 4-H), 7.32–7.99 (m, 15 H, 2

for 3 yielded 4 (420 mg, 94%). [α]_D= 55.5 (c = 1.9, CHCl₃). ¹H

NMR (600 MHz, CDCl₃): δ = 1.43, 1.45 (2 s, 9 H, Me₃CO), 3.89, OBz and SPh). ¹³C NMR (150 MHz, CDCl₃): δ = 14.13, 14.39,

3.95 (2 t, J_5,6= J_5,6Ј= 6.2, 6.2 Hz, 1 H, 5-H), 4.13, 4.26 (2 br. s, 1

H, 4-H), 4.41–4.57 (m, 2 H, 6-H), 4.94 (d, J_1,2= 10.0 Hz, 1 H, 1-

H), 5.33, 5.36 (2 dd, J_2,3= 9.9, 10.0 Hz, J_3,4= 3.0, 3.1 Hz, 1 H, 3-

14.60, 14.67, 15.09, 15.59, 16.26 [3 NCH(Me)CO], 28.33, 28.39 (2

Me₃CO), 29.16, 29.33, 29.49, 29.66, 29.71, 30.00, 31.08 (3 MeN),

50.87, 52.48, 52.51, 52.77, 53.11, 53.19, 53.41, 53.55 [3 NCH(Me)-

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CO], 62.04, 62.35 (C-6), 67.25, 67.32, 67.40 (C-2), 67.79, 67.96 (C-

General Procedure for One Cycle of Edman Degradation: One cycle

4), 73.11, 73.17 (C-3), 74.30, 74.46 (C-5), 80.06, 80.33, 80.45 of Edman degradation is explained for the synthesis of compound

(Me₃CO), 85.64, 86.07 (C-1), 128.37, 128.49, 128.92, 129.66, 18. First step: deprotection of the amino protecting group (Boc

129.81, 133.41, 133.54, 133.79 (2 PhCO and SPh), 155.40, 155.77 or Fmoc). Second step: coupling of phenyl isothiocyanate (PITC).

(C=O 2 Boc), 165.23 (2 PhCOO), 171.07, 171.38, 172.19 [3

NCH(Me)CO]. ESI-FT-MS: calcd. for C₄₈H₆₁N₃O₁₄SNa⁺[M +

Na⁺] 958.37665; found 958.37740.

During this step, a mono-UCP group was removed, except for

phenylglycyl-UCP. Third step: removal of the N-terminal amino

acid derivative. Fourth step: Boc protection of the newly exposed

amino group.

Phenyl 2,3-Di-O-benzoyl-6-O-{2-[2-(tert-butoxycarbonylamino)-2-

phenylacetylamido]-2-phenylacetyl}-1-thio-β-D-galactopyranoside

Phenyl 2,3-Di-O-benzoyl-6-O-{2-[tert-butoxycarbonyl(methyl)-

amino]acetyl}-1-thio-β-D-galactopyranoside (18): To a solution of 5

(7): To a solution of 4 (321 mg, 450 µmol) in dichloromethane

(5 mL) was added trifluoroacetic acid (1 mL). The mixture was

stirred for 30 min at room temp., and then concentrated (A). To a

solution of N-α-t-Boc--phenylglycine (904 mg, 3.60 mmol) in

dichloromethane (9 mL) was added DIC (282 µL, 1.80 mmol) at

0 °C. The mixture was stirred for 40 min at 0 °C, and then concen-

trated (B). To a solution of A and B in DMF (10 mL) was added

DMAP (5.5 mg, 45 µmol), and the mixture was stirred for 6 h at

room temp. The work-up as described for 3 yielded 7 (349 mg,

(117 mg, 131 µmol) in dichloromethane (3 mL) was added tri-

fluoroacetic acid (600 µL), and the mixture was stirred for 30 min

at room temp. After concentration, the product (9) was dissolved

in DMF (1 mL). To the resultant solution was added phenyl isothi-

ocyanate (500 µL, 4.18 mmol) and N-methylmorpholine (120 µL),

and the mixture was stirred for 30 min at room temp. To the mix-

ture was added ethyl acetate, and the organic layer was washed with

water, dried (Na₂SO₄), and concentrated. Column chromatography

(ethyl acetate/hexane, 2:1) of the residue over silica gel gave phen-

ylthiocarbamoyl complex 12, which was dried in vacuo. To a solu-

tion of phenylthiocarbamoyl complex 12 in dichloromethane

(2 mL) was added trifluoroacetic acid (400 µL), and the mixture

was stirred for 30 min at room temp. After concentration in vacuo,

the product 15 was dissolved in DMF (1 mL). In addition, di-tert-

butyl dicarbonate (400 mg, 1.83 mmol) and saturated aqueous

NaHCO₃(1 mL) were added to the resultant solution, and the mix-

ture was stirred for 30 min at room temp. To the mixture was added

ethyl acetate and the organic layer was washed with water, dried

(Na₂SO₄), and concentrated. Column chromatography (ethyl ace-

tate/hexane, 1:1) of the residue over silica gel gave 18 (40 mg, 4

92%). [α]_D= 69.7 (c = 1.0, CHCl₃). H NMR (600 MHz, CDCl₃):

δ = 1.30, 1.31 (2 s, 9 H, Me₃CO), 3.81 (br. t, J_5,6= J_5,6Ј= 6.1 Hz,

1 H, 5-H), 4.13 (br. s, 1 H, 4-H), 4.31–4.40 (m, 2 H, 6-H), 4.85,

4.87 (2 d, J_1,2= 10.1, 10.6 Hz, 1 H, 1-H), 5.21, 5.24 (2 dd, J_2,3

9.9, 9.9 Hz, J_3,4= 3.0, 3.1 Hz, 1 H, 3-H), 5.34–5.56 [m, 2 H, 2

NCH(Ph)CO], 5.71, 5.73 (2 t, J_1,2= J_2,3= 10.0, 9.8 Hz, 1 H, 2-H),

7.14–7.95 [m, 25 H, 2 PhCOO, 2 NCH(Ph)CO and SPh] ppm. ¹³C

NMR (150 MHz, CDCl₃): δ = 28.09 (Me₃CO), 56.58, 56.92, 58.05

[NCH(Ph)CO], 63.69 (C-6), 66.79 (C-4), 67.77 (C-2), 74.94, 75.07

(C-3), 75.53, 75.67 (C-5), 80.48 (Me₃CO), 86.38, 86.52 (C-1),

127.07, 127.13, 127.14, 127.18, 127.96, 128.29, 128.32, 128.41,

128.79, 128.82, 128.84, 128.85, 128.90, 128.93, 129.05, 129.16,

129.45, 129.68, 129.73, 132.43, 132.67, 133.17, 133.32, 133.36 [2

PhCOO, 2 NCH(Ph)CO and SPh], 152.38, 152.40, 155.29, 155.34

(C=O Boc), 165.25, 165.27, 165.67 (2 PhCOO), 168.02, 170.08,

171.33, 172.30 [NCH(Ph)CO] ppm. ESI-FT-MS: calcd. for

C₄₇H₄₆N₂O₁₁SNa⁺[M + Na⁺] 869.27145; found 869.27256.

steps 47%). H NMR and ¹³C NMR signals were observed as de-

scribed for compound 2. ESI-FT-MS: calcd. for C₃₄H₃₇NO₁₀SNa⁺

[M + Na⁺] 674.20304; found 674.20350.

Phenyl 2,3-Di-O-benzoyl-6-O-{2-[tert-butoxycarbonyl(methyl)-

amino]propionyl}-1-thio-β-D-galactopyranoside (19): Using the gene-

ral procedure for one cycle of Edman degradation, compound 6

(50 mg, 53 µmol) was degraded to compound 19 (22 mg, 4 steps

62%) via compounds 10, 13, and 16. ¹H NMR and ¹³C NMR

signals were observed as described for compound 3. ESI-FT-MS:

calcd. for C₃₅H₃₉NO₁₀SNa⁺[M + Na⁺] 688.21869; found

688.21898.

Phenyl 2,3-Di-O-benzoyl-4-O-[2-(tert-butoxycarbonylamino)-2-

phenylacetyl]-6-O-{2-[2-(tert-butoxycarbonylamino)-2-phenylacet-

amido]-2-phenylacetyl}-1-thio-β-D-galactopyranoside (8): To a solu-

tion of N-α-t-Boc--phenylglycine (119 mg, 474 µmol) in dichloro-

methane (1 mL) was added DIC (37 µL, 236 µmol) at 0 °C, and

the mixture was stirred for 40 min at 0 °C. After concentration, the

product was dissolved in pyridine. To the resultant solution was

added 7 (100 mg, 118 µmol) and DMAP (43 mg, 354 µmol), and

the mixture was stirred for 5 h at 50 °C. After concentration, the

product was purified by chromatography on a column of silica gel

with ethyl acetate/hexane (1:2) to give 8 (87 mg, 68%). [α]_D= 32.7

(c = 0.9, CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ = 1.37, 1.40, 1.41

(3 s, 18 H, 2 Me₃CO), 3.47, 4.03, 4.21, 4.38, 4.42 (5 m, 2 H, 6-H),

3.82–3.99 (m, 1 H, 5-H), 4.78, 4.80, 4.85 (3 d, J_1,2= 9.9, 9.9,

Phenyl 2,3-Di-O-benzoyl-6-O-[2-(tert-butoxycarbonylamino)-2-

phenylacetyl]-1-thio-β-D-galactopyranoside (20): Using the general

procedure for one cycle of Edman degradation, compound 8

(44 mg, 41 µmol) was degraded to compound 20 (20 mg, 4 steps

69%) via compounds 11, 14, and 17. ¹H NMR and ¹³C NMR

signals were observed as described for compound 4. ESI-FT-MS:

calcd. for C₃₉H₃₉NO₁₀SNa⁺[M + Na⁺] 736.21869; found

736.21800.

10.1 Hz, 1 H, 1-H), 5.22, 5.28, 5.50 [3 m, 3 H, 3 NCH(Ph)CO], tert-Butyl (1-Ethylpropylamino)acetate (23): A solution of tert-butyl

5.27, 5.35, 5.42 (3 m, 1 H, 3-H), 5.36, 5.58 (2 t, J_1,2= J_2,3= 9.9, bromoacetate (22, 5 mL, 33.9 mmol) in THF (20 mL) was added

10.1 Hz, 1 H, 2-H), 5.41, 5.44 (2 br. s, 1 H, 4-H), 7.17–7.97 [m, 30 dropwise to a cooled solution of 3-amino pentane (21, 8.7 mL,

H, 2 PhCOO, 3 NCH(Ph)CO and SPh] ppm. ¹³C NMR (150 MHz,

CDCl₃): δ = 28.04, 28.28 (2 Me₃CO), 56.91, 58.46 [3 NCH(Ph)CO],

62.07, 62.81 (C-6), 67.35, 67.45 (C-2), 68.52, 68.80 (C-4), 72.45 (C-

74.7 mmol) in THF (20 mL) over 5 min. The solution was stirred

for 4 h at room temp. and concentrated. The product was sus-

pended in diethyl ether; the residue of the hydrobromide salt was

3),74.20, 74.55 (C-5), 80.15, 80.30 (Me₃CO), 85.33, 85.55 (C-1), removed by filtration. The residue was washed with diethyl ether,

126.55, 127.14, 127.27, 127.37, 127.45, 128.21, 128.29, 128.35, and the filtrate was concentrated in vacuo to give 23 (6.7 g, 98%)

128.39, 128.52, 128.59, 128.86, 128.91, 129.08, 129.15, 129.20, as a colorless oil. [α]_D= –0.2 (c = 1.2, CHCl₃). ¹H NMR

129.70, 129.80, 133.13, 133.27, 134.35 [2 PhCOO, 3 NCH(Ph)CO

and SPh], 154.67, 155.13 (C=O Boc), 164.54, 165.24 (2 PhCOO),

1 69. 6 3 [ 3 NCH(P h)CO ] p pm . E SI -F T- MS : c al cd . for

C₆₀H₆₁N₃O₁₄SNa⁺[M + Na⁺] 1102.37665; found 1102.37514.

(600 MHz, CDCl₃): δ = 0.89 (t, J_CH3,CH= J_CH3,CHЈ= 7.5 Hz, 6 H,

2 CH₃CH₂), 1.42 (m, 4 H, 2 CH₃CH₂), 1.47 (s, 9 H, tBu), 1.67 (s,

1 H, NH), 2.35 [quint, J_CH,CHa= J_CH,CHaЈ= J_CH,CHb= J_CH,CHbЈ

= 5.9 Hz, 1 H, (CH₃CH₂)₂CH], 3.30 (s, 2 H, NHCH₂CO) ppm. ¹³

5322

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Eur. J. Org. Chem. 2005, 5313–5329

A New Method of Carbohydrate Synthesis Using a Special Hydroxy Protecting Group

FULL PAPER

NMR (150 MHz, CDCl₃): δ = 9.74 (2 CH₃CH₂), 25.73 (2 (Me₃CO), 43.25, 43.50, 43.81, 43.87 (2 NCH₂CO), 47.34, 47.42,

CH₃CH₂), 28.10 (Me₃CO), 49.46 (NHCH₂CO), 59.70 [(CH₃- 47.87 (C-9 Fmoc), 59.62, 59.95, 60.11, 60.23, 60.26 [2 (CH₃CH₂)₂-

CH₂)₂CH], 81.00 (Me₃CO), 172.08 (C=O) ppm. ESI-FT-MS: calcd.

CH], 67.17, 67.35 (CH₂Fmoc), 81.11, 81.18 (Me₃CO), 119.70,

119.74, 119.81, 119.83, 124.94, 125.21, 126.89, 126.96, 127.02,

127.39, 127.48, 127.81, 141.19, 141.29, 141.35, 144.21, 144.28,

144.41 (Ar Fmoc), 156.57, 156.89, 156.98, 157.02 (C=O Fmoc),

168.22, 168.50, 168.64, 168.76, 169.34, 169.81, 170.03 (2 NCH₂CO)

ppm. ESI-FT-MS: calcd. for C₃₃H₄₆N₂O₅Na⁺[M + Na⁺]

573.32989; found 573.33263.

for C₁₁H₂₄NO₂[M + H⁺] 202.18016; found 202.18082.

tert-Butyl [(1-Ethylpropyl)(9H-fluoren-9-ylmethoxycarbonyl)amino]-

acetate (24): A solution of 9-fluorenylmethyl chloroformate (9.4 g,

36.3 mmol) in 1,4-dioxane (100 mL) was added dropwise to a co-

oled solution of 23 (7.37 g, 36.6 mmol) in satd. aq. NaHCO₃

(100 mL) over 5 min. After stirring for 2 h at room temp., ethyl

acetate was added and the mixture was washed with water, dried

(Na₂SO₄), and concentrated. Column chromatography (ethyl ace-

tate/hexane, 1:4) of the residue on silica gel gave 24 (13.34 g, 86%).

[(1-Ethylpropyl){2-[(1-ethylpropyl)(9H-fluoren-9-ylmethoxycarbon-

yl)amino]acetyl}amino]acetic Acid (27): A solution of 26 (500 mg,

907.8 µmol) in 90% aq. formic acid (5 mL) was stirred for 3 h at

[α]_D= –1.0 (c = 1.1, CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ = 50 °C. The work-up as described for 25 gave 27 (403 mg, 93%).

0.77, 0.90 (2 t, J_CH3,CH= J_CH3,CHЈ= 7.4, 7.4 Hz, 6 H, 2 CH₃CH₂), [α]_D= –0.3 (c = 1.0, CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ =

1.25–1.50 (m, 4 H, 2 CH₃CH₂), 1.42, 1.45 (2 s, 9 H, tBu), 3.62, 0.75, 0.87, 0.88, 0.94 (4 t, 12 H, 4 CH₃CH₂), 1.25–1.59 (m, 8 H, 4

4.00 [2 ttt, J_CH,CHa= J_CH,CHb= 6.0, 5.6 Hz, J_CH,CHaЈ= J_CH,CHbЈ

CH₃CH₂), 3.37, 3.49, 3.62, 4.01 [4 m, 2 H, 2 (CH₃CH₂)₂CH], 3.80,

= 8.9, 9.1 Hz, 1 H, (CH₃CH₂)₂CH], 3.66, 3.67 (2 s, 2 H, NCH₂CO), 3.88, 3.88, 3.93 (4 s, 4 H, 2 NCH₂CO), 4.23 (m, 1 H, 9-H Fmoc),

4.22, 4.25 (2 t, J_9-H,CH= J_9-H,CHЈ= 7.0, 6.3 Hz, 1 H, 9-H Fmoc),

4.42, 4.51 (2 d, J_CH2,9-H= 7.0, 6.2 Hz, 2 H, CH₂Fmoc), 7.29–7.77

(m, 8 H, Ar Fmoc) ppm. ¹³C NMR (150 MHz, CDCl₃): δ = 11.00,

11.05 (2 CH₃CH₂), 25.75, 25.99 (2 CH₃CH₂), 28.00, 28.03

(Me₃CO), 44.48, 44.78 (NCH₂CO), 47.35, 47.50 (C-9 Fmoc), 59.58,

59.88 [(CH₃CH₂)₂CH], 67.18, 67.47 (CH₂Fmoc), 81.24, 81.47

4.46, 4.52 (2 d, J_CH2,9-H= 6.5, 6.1 Hz, 2 H, CH₂Fmoc), 7.27–7.76

(m, 8 H, Ar Fmoc) ppm. ¹³C NMR (150 MHz, CDCl₃): δ = 10.90,

11.11, 11.15, 11.18 (4 CH₃CH₂), 25.59, 25.84, 25.90, 26.18, 26.20

(4 CH₃CH₂), 43.40, 43.67, 43.74, 43.90, 43.99 (2 NCH₂CO), 47.30,

47.47 (C-9 Fmoc), 59.61, 60.18, 60.44, 60.86, 61.09 [2 (CH₃CH₂)₂-

CH], 67.13, 67.35, 67.46 (CH₂Fmoc), 119.78, 119.88, 124.57,

(Me₃CO), 119.87, 119.89, 124.88, 125.16, 126.98, 127.05, 127.56, 124.88, 124.95, 125.07, 125.19, 126.93, 127.02, 127.48, 127.58,

127.62, 141.28, 141.38, 144.11, 144.16 (Ar Fmoc), 156.71, 156.89

(C=O Fmoc), 168.86, 169.03 (NCH₂CO) ppm. ESI-FT-MS: calcd.

for C₂₆H₃₃NO₄Na⁺[M + Na⁺] 446.23018; found 446.22838.

141.24, 141.40, 144.10, 144.22 (Ar Fmoc), 156.80, 157.13, 157.35

(C=O Fmoc), 170.73, 171.16, 172.00, 172.28, 173.51 (2 NCH₂CO)

ppm. ESI-FT-MS: calcd. for C₂₉H₃₉N₂O₅[M + H⁺] 495.28535;

found 495.28670.

[(1-Ethylpropyl)(9H-fluoren-9-ylmethoxycarbonyl)amino]acetic Acid

(25): A solution of 24 (13.058 g, 30.8 mmol) in 90% aq. formic

acid (100 mL) was stirred for 7 h at 40 °C. After concentration, the

product was purified by chromatography on a column of silica gel

tert-Butyl [(1-Ethylpropyl){2-[(1-ethylpropyl){2-[(1-ethylpropyl)(9H-

fluoren-9-ylmethoxycarbonyl)amino]acetyl}amino]acetyl}-

amino]acetate (28): N,N-Diisopropylethylamine (603 µmol,

3.46 mmol) and PyBroP (1.2 g, 2.57 mmol) were added to a mixture

of 23 (522 mg, 2.59 mmol) and 27 (829 mg, 1.73 mmol) in DMF

with ethyl acetate/hexane (1:5) to give 25 (10.159 g, 90%). [α]_D

–0.4 (c = 0.8, CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ = 0.71, 0.85

(2 t, J_CH3,CH= J_CH3,CHЈ= 7.4 Hz, 6 H, 2 CH₃CH₂), 1.24–1.44 (m, (4 mL), and the mixture was stirred for 30 min at room temp. The

4 H, 2 CH₃CH₂), 3.56, 3.97 [2 m, 1 H, (CH₃CH₂)₂CH], 3.66, 3.77 work-up described for 26 gave 28 (975 mg, 85%). [α]_D= –0.2 (c =

(2 s, 2 H, NCH₂CO), 4.17, 4.24 (2 m, 1 H, 9-H Fmoc), 4.49, 4.58

(2 d, J_CH2,9-H= 6.2, 5.8 Hz, 2 H, CH₂Fmoc), 7.27–7.76 (m, 8 H,

1.2, CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ = 0.74–0.96 (m, 18

H, 6 CH₃CH₂), 1.26–1.52 (m, 12 H, 6 CH₃CH₂), 1.43, 1.44, 1.46,

Ar Fmoc) ppm. ¹³C NMR (150 MHz, CDCl₃): δ = 10.84, 10.93 (2 1.47, 1.47, 1.48 (6 s, 9 H, tBu), 3.38, 3.46, 3.68, 3.95 [4 m, 3 H, 3

CH₃CH₂), 25.71, 25.88 (2 CH₃CH₂), 43.09, 44.22 (NCH₂CO), (CH₃CH₂)₂CH], 3.60–3.96 (m, 6 H, 3 NCH₂CO), 4.18–4.28 (m, 1

47.32, 47.38 (C-9 Fmoc), 59.55, 60.24 [(CH₃CH₂)₂CH], 67.32,

67.53 (CH₂Fmoc), 119.89, 119.95, 124.76, 124.82, 127.02, 127.07,

127.57, 127.70, 141.34, 141.43, 143.84, 143.97 (Ar Fmoc), 156.50,

157.82 (C=O Fmoc), 173.78, 174.91 (NCH₂CO). ESI-FT-MS:

H, 9-H Fmoc), 4.37–4.50 (m, 2 H, CH₂Fmoc), 7.28–7.76 (m, 8 H,

Ar Fmoc) ppm. ¹³C NMR (150 MHz, CDCl₃): δ = 10.78, 10.82,

10.87, 10.93, 10.97, 11.02, 11.10, 11.24, 11.32, 11.56 (6 CH₃CH₂),

25.16, 25.24, 25.32, 25.44, 25.48, 25.67, 25.73, 26.44, 26.51 (6

CH₃CH₂), 27.90, 27.94, 27.99 (Me₃CO), 43.42, 43.50, 43.96 (3

NCH₂CO), 47.33, 47.49, 47.71 (C-9 Fmoc), 60.05, 60.12, 60.40,

60.46, 60.57, 60.77 [3 (CH₃CH₂)₂CH], 66.43, 67.19 (CH₂Fmoc),

81.28, 81.41, 81.75, 81.93, 82.52, 82.85 (Me₃CO), 119.59, 119.68,

119.78, 119.85, 125.02, 125.06, 125.26, 125.36, 126.86, 126.96,

127.36, 127.42, 127.53, 141.22, 141.26, 141.35, 141.37, 144.25,

144.44, 144.47 (Ar Fmoc), 156.92, 157.15 (C=O Fmoc), 168.50,

168.95, 169.34, 169.88, 169.91, 170.00 (3 NCH₂CO) ppm. ESI-FT-

calcd. for C₂₂H₂₆NO₄[M + H⁺] 368.18563; found 368.18691.

tert-Butyl [(1-Ethylpropyl){2-[(1-ethylpropyl)(9H-fluoren-9-yl-

methoxycarbonyl)amino]acetyl}amino]acetate (26): N,N-Diisopro-

pylethylamine (948 µmol, 5.44 mmol) and PyBroP (1.9 g,

4.08 mmol) were added to a mixture of 23 (1 g, 4.97 mmol) and 25

(1 g, 2.72 mmol) in DMF (5 mL), and the mixture was stirred for

30 min at room temp. The product was dissolved in chloroform

and the solution was washed with 2  HCl and water, dried

(Na₂SO₄), and concentrated. Column chromatography (ethyl ace-

tate/hexane, 1:3) of the residue on silica gel gave 26 (1.364 g, 91%).

[α]_D= –0.2 (c = 1.2, CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ =

MS: calcd. for C₄₀H₆₀N₃O₆[M + H⁺] 678.44766; found

678.44615.

[(1-Ethylpropyl){2-[(1-ethylpropyl){2-[(1-ethylpropyl)(9H-fluoren-9-

0.77–1.00 (m, 12 H, 4 CH₃CH₂), 1.24–1.53 (m, 8 H, 4 CH₃CH₂), ylmethoxycarbonyl)amino]acetyl}amino]acetyl}amino]acetic Acid

1.43, 1.43, 1.47, 1.49 (4 s, 9 H, tBu), 3.39, 3.45, 3.65, 3.91, 4.03 [5

m, 2 H, 2 (CH₃CH₂)₂CH], 3.42, 3.56, 3.70, 3.75, 3.89, 3.94, 3.95 (7

s, 4 H, 2 NCH₂CO), 4.25 (m, 1 H, 9-H Fmoc), 4.40, 4.47, 4.49 (3

(29): A solution of 28 (171 mg, 258.3 µmol) in 90% aq. formic acid

(2 mL) was stirred for 2 h at 50 °C. The work-up as described for

25 gave 29 (154 mg, 98%). [α]_D= –0.2 (c = 1.2, CHCl₃). ¹H NMR

d, J_CH2,9-H= 6.9, 6.3, 6.3 Hz, 2 H, CH₂Fmoc), 7.28–7.76 (m, 8 H, (600 MHz, CDCl₃): δ = 0.74–0.99 (m, 18 H, 6 CH₃CH₂), 1.21–1.63

Ar Fmoc) ppm. ¹³C NMR (150 MHz, CDCl₃): δ = 10.93, 10.95, (m, 12 H, 6 CH₃CH₂), 3.36, 3.44, 3.51, 3.65, 3.95, 4.01 [6 m, 3 H,

11.08, 11.18, 11.21, 11.25, 11.31, 11.35 (4 CH₃CH₂), 25.17, 25.30,

3 (CH₃CH₂)₂CH], 3.59–4.16 (m, 6 H, 3 NCH₂CO), 4.23 (m, 1 H,

25.62, 25.65, 25.87, 25.96, 26.46, 26.49 (4 CH₃CH₂), 27.92, 27.95 9-H Fmoc), 4.39–4.53 (m, 2 H, CH₂Fmoc), 7.27–7.77 (m, 8 H, Ar

Eur. J. Org. Chem. 2005, 5313–5329

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5323

S. Komba, M. Kitaoka, T. Kasumi

Fmoc) ppm. ¹³C NMR (150 MHz, CDCl₃): δ = 10.79, 10.83, 10.89, 74.97 (C-3), 97.65 (C-1), 119.98, 124.58, 124.69, 127.07, 127.28,

FULL PAPER

10.98, 11.05, 11.11, 11.16, 11.19, 11.21, 11.40, 11.50 (6 CH₃CH₂),

127.74, 127.82, 141.44, 141.50, 143.67 (Ar Fmoc), 158.12 (C=O

25.18, 25.26, 25.43, 25.53, 25.70, 25.81, 25.85, 26.07, 26.36 (6

Fmoc), 168.52 (NCH₂CO) ppm. ESI-FT-MS: calcd. for

CH₃CH₂), 42.02, 42.79, 44.03, 44.10 (3 NCH₂CO), 47.22, 47.26, C₃₄H₄₉N₄O₈Si⁺[M + H⁺] 669.33142; found 669.33028.

47.45, 47.75 (C-9 Fmoc), 60.55, 60.66, 60.70 [3 (CH₃CH₂)₂CH],

67.36, 67.44, 67.74 (CH₂Fmoc), 119.66, 119.78, 119.86, 119.88,

124.88, 124.96, 124.99, 125.04, 125.24, 125.34, 126.92, 126.99,

127.01, 127.38, 127.43, 127.56, 127.59, 141.22, 141.36, 141.38,

tert-Butyldimethylsilyl 2-Azido-2-deoxy-6-O-{[(1-ethylpropyl){2-[(1-

ethylpropyl){2-[(1-ethylpropyl)(9H-fluoren-9-ylmethoxycarbonyl)-

amino]acetyl}amino]acetyl}amino]acetyl}-3-O-{[(1-ethylpropyl)(9H-

fluoren-9-ylmethoxycarbonyl)amino]acetyl}-β-D-galactopyranoside

(33): To a solution of 32 (100 mg, 149.5 µmol) in pyridine (2 mL)

was added 29 (100 mg, 165 µmol) and cyanuric chloride (138 mg,

748.3 µmol), and the mixture was stirred for 2 h at 40 °C. After

144.12, 144.18 (Ar Fmoc), 157.29, 157.34 (C=O Fmoc), 170.81,

171.57, 172.79 (3 NCH₂CO) ppm. ESI-FT-MS: calcd. for

C₃₆H₅₂N₃O₆[M + H⁺] 622.38506; found 622.38370.

tert-Butyldimethylsilyl 2-Azido-4,6-O-benzylidene-2-deoxy-3-O-{[(1- concentration, the product was purified by chromatography on a

ethylpropyl)(9H-fluoren-9-ylmethoxycarbonyl)amino]acetyl}-β- -ga- column of silica gel with ethyl acetate/hexane (1:1) to give 33

lactopyranoside (31): To a solution of 30^[12](496 mg, 1.22 mmol) in (122 mg, 65%). [α]_D= 7.9 (c = 1.5, CHCl₃). H NMR (600 MHz,

pyridine (9 mL) was added 25 (671 mg, 1.83 mmol) and cyanuric

chloride (1.12 g, 6.07 mmol), and the mixture was stirred for 2 h at

40 °C. After concentration, the product was purified by chromatog-

raphy on a column of silica gel with ethyl acetate/hexane (1:2) to

give 31 (830 mg, 90%). [α]_D= 19.8 (c = 0.9, CHCl₃). ¹H NMR

CDCl₃): δ = 0.16, 0.17, 0.17, 0.18 (4 s, 6 H, Me₂Si), 0.67–1.01 (m,

24 H, 8 CH₃CH₂), 0.95 (s, 9 H, Me₃CSi), 1.18–1.63 (m, 16 H, 8

CH₃CH₂), 3.49, 3.70 [2 m, 4 H, 4 (CH₃CH₂)₂CH], 3.61, 3.63, 3.81

(3 m, 8 H, 4 NCH₂CO), 3.63 (m, 1 H, 5-H), 3.66 (m, 1 H, 2-H),

4.15 (m, 1 H, 4-H), 4.23 (m, 2 H, 9-H 2 Fmoc), 4.33 (m, 2 H, 6-

(600 MHz, CDCl₃): δ = 0.16, 0.17, 0.17, 0.18 (4 s, 6 H, Me₂Si), H), 4.42, 4.47, 4.58 (3 m, 4 H, 2 CH₂Fmoc), 4.45 (m, 1 H, 3-H),

0.74, 0.89 (2 m, 6 H, 2 CH₃CH₂), 0.94, 0.95 (2 s, 9 H, Me₃CSi),

1.24–1.50 (m, 4 H, 2 CH₃CH₂), 3.35, 3.41 (2 s, 1 H, 5-H), 3.62,

4.53 (d, J_1,2= 7.7 Hz, 1 H, 1-H), 7.27–7.77 (m, 16 H, Ar 2 Fmoc)

ppm. ¹³C NMR (150 MHz, CDCl₃): δ = –5.26, –4.22 (Me₂Si),

4.03 [2 m, 1 H, (CH₃CH₂)₂CH], 3.78, 3.79 (2 dd, J_1,2= 7.6, 7.5 Hz, 10.74, 11.14, 11.19, 11.31 (8 CH₃CH₂), 17.94 (Me₃CSi), 25.48,

J_2,3= 11.1z, 11.0 Hz, 1 H, 2-H), 3.79, 3.88 (2 m, 2 H, NCH₂CO), 25.58, 25.85, 26.00 (8 CH₃CH₂), 25.63 (Me₃CSi), 42.50, 43.36,

3.88, 4.02, 4.18, 4.25 (4 dd, J_gem= 12.3 Hz, J_6,5= 1.4 Hz, 2 H, 6-

H), 4.06, 4.31 (2 d, J_3,4= 3.2, 3.7 Hz, 1 H, 4-H), 4.06, 4.36, 4.44,

4.49 (4 dd, J_gem= 10.7, 10.7, 10.7, 10.7 Hz, J_CH2,9-H= 7.5, 6.3, 6.8,

43.92, 44.57 (4 NCH₂CO), 47.17, 47.33 (C-9 2 Fmoc), 59.79, 60.02,

60.34, 60.48, 60.56 [4 (CH₃CH₂)₂CH], 62.63 (C-2), 63.27 (C-6),

64.56 (C-4), 67.21, 67.43 (CH₂2 Fmoc), 71.87, 71.92 (C-5), 74.74,

6.1 Hz, 2 H, CH₂Fmoc), 4.19 (m, 1 H, 9-H Fmoc), 4.60, 4.62 (2 74.83 (C-3), 97.57 (C-1), 119.69, 119.85, 119.98, 124.56, 124.71,

d, J_1,2= 7.6, 7.6 Hz, 1 H, 1-H), 4.66, 4.76 (2 dd, J_2,3= 10.9, 125.05, 125.34, 126.97, 127.07, 127.31, 127.37, 127.53, 127.74,

10.9 Hz, J_3,4= 3.6, 3.5 Hz, 1 H, 3-H), 5.08, 5.50 (2 s, 1 H, PhCH), 127.82, 141.25, 141.34, 141.44, 141.51, 143.65, 144.24 (Ar, 2 Fmoc),

7.23–7.77 (m, 13 H, Ar Fmoc and PhCH) ppm. ¹³C NMR

(150 MHz, CDCl₃): δ = –4.93, –4.16 (Me₂Si), 10.88, 10.92, 10.95,

157.18, 158.16 (C=O 2 Fmoc), 168.38, 168.44, 168.46, 169.57,

170.61 (4 NCH₂CO) ppm. ESI-FT-MS: calcd. for C₇₀H₉₈N₇O₁₃Si⁺

11.03 (2 CH₃CH₂), 17.99, 18.01 (Me₃CSi), 25.58, 25.69, 25.76, [M + H⁺] 1272.69864; found 1272.69712.

25.90, 26.00 (2 CH₃CH₂), 25.64, 25.65 (Me₃CSi), 43.66, 43.86

tert-Butyldimethylsilyl 4-O-Acetyl-2-azido-2-deoxy-6-O-{[(1-eth-

(NCH₂CO), 47.20, 47.25 (C-9 Fmoc), 59.57, 59.94 [(CH₃CH₂)₂-

CH], 62.60, 62.70 (C-2), 66.18, 66.28 (C-5), 67.21, 67.83 (CH₂

Fmoc), 68.95, 69.04 (C-6), 72.12, 72.34 (C-3), 72.47, 72.70 (C-4),

97.33, 97.40 (C-1), 100.85 (PhCH), 119.71, 119.86, 119.90, 119.96,

120.97, 124.79, 124.82, 124.98, 125.40, 126.15, 126.26, 126.96,

127.03, 127.55, 127.57, 128.08, 128.11, 129.01, 129.03 (Ar Fmoc

and PhCH), 156.50, 156.88 (C=O Fmoc), 169.39, 169.68

(NCH₂CO) ppm. ESI-FT-MS: calcd. for C₄₁H₅₃N₄O₈Si⁺[M + H⁺]

757.36272; found 757.36288.

ylpropyl){2-[(1-ethylpropyl){2-[(1-ethylpropyl)(9H-fluoren-9-ylmeth-

oxycarbonyl)amino]acetyl}amino]acetyl}amino]acetyl}-3-O-{[(1-eth-

ylpropyl)(9H-fluoren-9-ylmethoxycarbonyl)amino]acetyl}-β-D-galac-

topyranoside (34): To a solution of 33 (287 mg, 228.3 µmol) in pyri-

dine (3 mL) was added acetic anhydride (3 mL), and the mixture

was stirred for 2 h at 40 °C. The product was dissolved in chloro-

form and the solution was washed with 2  HCl and water, dried

(Na₂SO₄), and concentrated. Column chromatography (ethyl ace-

tate/hexane, 1:3) of the residue on silica gel gave 34 (269 mg, 91%).

tert-Butyldimethylsilyl 2-Azido-2-deoxy-3-O-{[(1-ethylpropyl)(9H- [α]_D= 0.5 (c = 1.0, CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ = 0.15,

fluoren-9-ylmethoxycarbonyl)amino]acetyl}-β-

-galactopyranoside

0.15, 0.16, 0.17 (4 s, 6 H, Me₂Si), 0.72–0.97 (m, 24 H, 8 CH₃CH₂),

0.93 (s, 9 H, Me₃CSi), 1.33–1.51 (m, 16 H, 8 CH₃CH₂), 2.04 (s, 3

H, OAc), 3.58 (m, 1 H, 2-H), 3.60, 3.66 [2 m, 4 H, 4 (CH₃CH₂)₂-

CH], 3.61, 3.70, 3.83 (3 m, 8 H, 4 NCH₂CO), 3.83 (m, 1 H, 5-H),

4.02–4.19 (m, 2 H, 6-H), 4.24 (m, 2 H, 9-H 2 Fmoc), 4.32–4.54 (m,

(32): A solution of 31 (780 mg, 1.03 mmol) in 80% aq. acetic acid

(50 mL) was stirred for 2 h at 60 °C. After concentration, the prod-

uct was purified by chromatography on a column of silica gel with

ethyl acetate/hexane (1:1) to give 32 (542 mg, 79%). [α]_D= 14.9 (c

= 1.3, CHCl₃). H NMR (600 MHz, CDCl₃): δ = 0.17, 0.17 (2 s, 6 4 H, CH₂2 Fmoc), 4.57, 4.58 (2 d, J_1,2= 7.6, 7.6 Hz, 1 H, 1-H),

H, Me₂Si), 0.69, 0.72 (2 t, J_CH3,CH= J_CH3,CHЈ7.4, 7.4 Hz, 6 H, 2

CH₃CH₂), 0.95 (s, 9 H, Me₃CSi), 1.18–1.41 (m, 4 H, 2 CH₃CH₂),

4.75, 4.80 (2 dd, J_2,3= 11.1, 11.1 Hz, J_3,4= 3.4, 3.4 Hz, 1 H, 3-H),

5.14–5.36 (m, 1 H, 4-H), 7.28–7.76 (m, 16 H, Ar 2 Fmoc) ppm.

3.49 [m, 1 H, (CH₃CH₂)₂CH], 3.51 (m, 1 H, 5-H), 3.64, 3.78 (2 d, ¹³C NMR (150 MHz, CDCl₃): δ = –5.15, –5.09, –4.41, –4.39

J_gem= 17.0, 17.0 Hz, 2 H, NCH₂CO), 3.66 (dd, J_1,2= 7.7 Hz, J_2,3(Me₂Si), 10.81, 10.89, 10.90, 10.98, 11.01, 11.08, 11.14, 11.18,

= 10.7 Hz, 1 H, 2-H), 3.78, 3.94 (2 m, 2 H, 6-H), 4.19 (d, J_3,4

2.3 Hz, 1 H, 4-H), 4.21 (t, J_9-H,CH= J_9-H,CHЈ= 5.5 Hz, 1 H, 9-H 21.05 (CH₃COO), 25.23, 25.42, 25.46, 25.69, 25.74, 25.89, 26.00,

Fmoc), 4.47 (dd, J_2,3= 10.7 Hz, J_3,4= 3.1 Hz, 1 H, 3-H), 4.55 (d, 26.30, 26.34, 26.38, 26.45 (8 CH₃CH₂), 25.58 (Me₃CSi), 42.44,

11.22, 11.33, 11.50 (8 CH₃CH₂), 18.00 (Me₃CSi), 20.57, 20.66,

J_1,2= 8.2 Hz, 1 H, 1-H), 4.57 (d, J_CH2,9-H= 5.5 Hz, 2 H, CH₂42.51, 43.32, 43.58, 43.88, 44.00 (4 NCH₂CO), 47.31 (C-9 2 Fmoc),

Fmoc), 7.30–7.77 (m, 8 H, Ar Fmoc) ppm. ¹³C NMR (150 MHz,

CDCl₃): δ = –5.18, –4.13 (Me₂Si), 10.74, 10.76 (2 CH₃CH₂), 17.93

59.69, 59.94, 60.06, 60.39, 60.48, 60.53, 60.59 [4 (CH₃CH₂)₂CH],

61.25, 61.37 (C-6), 63.16, 63.22 (C-2), 66.22, 66.29, 66.42, 66.57 (C-

(Me₃CSi), 25.61 (Me₃CSi), 25.87, 25.98 (2 CH₃CH₂), 44.48, 44.51, 4), 67.21, 67.32 (CH₂2 Fmoc), 70.56, 70.65, 70.76 (C-5), 71.33,

44.54 (NCH₂CO), 47.17 (C-9 Fmoc), 60.35 [(CH₃CH₂)₂CH], 62.45 71.39 (C-3), 97.38, 97.42, 97.49 (C-1), 119.69, 119.85, 119.91,

(C-6), 62.78 (C-2), 65.61 (C-4), 67.49 (CH₂Fmoc), 74.22 (C-5), 124.79, 124.83, 125.02, 125.13, 125.33, 126.96, 127.01, 127.36,

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Eur. J. Org. Chem. 2005, 5313–5329

A New Method of Carbohydrate Synthesis Using a Special Hydroxy Protecting Group

FULL PAPER

127.53, 127.61, 128.22, 129.03, 141.24, 141.27, 141.32, 141.34, 66.03, 66.26 (C-2), 67.88, 68.20 (CH₂Fmoc), 69.48, 69.62 (C-6),

141.37, 141.40, 143.99, 144.05, 144.11, 144.23, 144.38 (Ar, 2 Fmoc), 70.10, 70.14 (C-5), 73.89, 74.30 (C-4), 75.83, 76.35 (C-3), 87.33,

156.50, 156.87, 157.15 (C=O 2 Fmoc), 168.19, 168.22, 168.78, 87.85 (C-1), 101.29, 101.37 (PhCH), 120.35, 120.42, 125.21, 125.24,

168.89, 169.06, 169.62 (4 NCH₂CO), 170.07, 170.14 (C=O Ac)

ppm. ESI-FT-MS: calcd. for C₇₂H₁₀₀N₇O₁₄Si⁺[M + H⁺]

1314.70920; found 1314.70920.

125.47, 125.91, 126.74, 126.88, 127.49, 127.50, 127.53, 127.56,

128.02, 128.09, 128.11, 128.13, 128.37, 128.43, 128.49, 128.73,

129.18, 129.43, 129.48, 131.45, 134.02, 134.16, 138.17, 141.80,

141.82, 144.32, 144.36 (Ar Fmoc, PhCH and SPh), 157.04, 157.82

(C=O Fmoc), 169.90, 170.45 (NCH₂CO) ppm. ESI-FT-MS: calcd.

for C₄₁H₄₄NO₈S⁺[M + H⁺] 710.27821; found 710.27645.

tert-Butyldimethylsilyl 4-O-Acetyl-2-azido-2-deoxy-6-O-{[{2-[tert-

butoxycarbonyl(1-ethylpropyl)amino]acetyl}(1-ethylpropyl)-

amino]acetyl}-β-D-galactopyranoside (35): To a solution of 34

(300 mg, 230.9 µmol) in DMF (1.6 mL) was added piperidine

(400 µL, 4.04 mmol), and the mixture was stirred for 20 min at

room temp. After cooling to 0 °C, phenyl isothiocyanate (1 mL,

8.36 mmol) and N-methylmorpholine (200 µL) were added to the

Phenyl 2-O-Benzoyl-4,6-O-benzylidene-3-O-{[(1-ethylpropyl)(9H-

fluoren-9-ylmethoxycarbonyl)amino]acetyl}-1-thio-β-D-galacto-

pyranoside (38): To a solution of 37 (521 mg, 733.9 µmol) in pyri-

dine (3 mL) was added benzoyl chloride (128 µL, 1.103 mmol), and

mixture, followed by stirring for 30 min at room temp. Sub- the mixture was stirred for 2 h at room temp. After adding ice to

sequently, work-up (extraction and purification), trifluoroacetic

acid treatment, amino group reprotection procedures, and work-

up (extraction and purification) were performed according to the

general procedure for one cycle of Edman degradation to yield

GalN derivative 35 (114 mg, 4 steps 69%). [α]_D= –2.0 (c = 2.1,

the mixture, the product was dissolved in chloroform and the solu-

tion was washed with 2  HCl and water, dried (Na₂SO₄), and con-

centrated. Column chromatography (ethyl acetate/hexane, 1:1) of

the residue on silica gel gave 38 (582 mg, 97%). [α]_D= –16.9 (c =

1.1, CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ = 0.46, 0.60, 0.64,

0.72 (4 t, J_CH3,CH= J_CH3,CHЈ= 7.4, 7.4, 7.4, 7.4 Hz, 6 H, 2

CHCl₃). H NMR (600 MHz, CDCl₃): δ = 0.16, 0.17, 0.17 (3 s, 6

H, Me₂Si), 0.83–1.00 (m, 12 H, 4 CH₃CH₂), 0.94, 0.94 (2 s, 9 H, CH₃CH₂), 0.85–1.31 (m, 4 H, 2 CH₃CH₂), 3.45, 3.55, 3.80, 3.82 (4

Me₃CSi), 1.33–1.53 (m, 8 H, 4 CH₃CH₂), 1.41, 1.46 (2 s, 9 H, Boc),

2.15, 2.16 (2 s, 3 H, OAc), 3.41, 3.53, 3.63, 3.96 [4 m, 2 H, 2

d, J_gem= 17.7, 18.3, 18.3, 17.7 Hz, 2 H, NCH₂CO), 3.48, 3.85 [2

m, 1 H, (CH₃CH₂)₂CH], 3.59, 3.61 (2 d, J_5,6= 1.1, 1.1 Hz, 1 H, 5-

(CH₃CH₂)₂CH], 3.46 (m, 1 H, 2-H), 3.60 (m, 1 H, 3-H), 4.52, 4.52 H), 3.92, 4.04 (2 dd, J_5,6= 1.5, 1.6 Hz, J_gem= 12.2, 12.3 Hz, 2 H,

(2 d, J_1,2= 7.6, 7.7 Hz, 1 H, 1-H), 5.29, 5.30 (2 d, J_3,4= 3.5, 3.6 Hz,

1 H, 4-H) ppm ¹³C NMR (150 MHz, CDCl₃): δ = –5.12, –4.31, –

4.29 (Me₂Si), 10.82, 11.21, 11.22, 11.25, 11.32, 11.33, 11.39, 11.60

6-H), 3.94, 4.24, 4.27, 4.33 (4 dd, J_CH2,9-H= 7.8, 6.7, 6.3, 6.4 Hz,

J_gem= 10.6, 10.6, 10.7, 10.6 Hz, 2 H, CH₂Fmoc), 4.04, 4.12 (2 t,

J_9-H,CH= J_9-H,CHЈ= 7.2, 6.2 Hz, 1 H, 9-H Fmoc), 4.21, 4.40 (2 dd,

(4 CH₃CH₂), 18.03 (Me₃CSi), 20.82, 20.84 (CH₃COO), 25.61, J_3,4= 3.4, 3.3 Hz, J_4,5= 0.8, 0.7 Hz, 1 H, 4-H), 4.85, 4.85 (2 d, J_1,2

25.67, 25.69, 25.92, 25.97, 26.26, 26.52, 26.59 (4 CH₃CH₂), 25.64,

25.65 (Me₃CSi), 28.25, 28.54 (Me₃CO), 42.55, 43.33, 43.65 (2

= 9.8, 9.8 Hz, 1 H, 1-H), 5.21, 5.49 (2 s, 1 H, PhCH), 5.23, 5.31 (2

dd, J_2,3= 9.9, 9.9 Hz, J_3,4= 3.4, 3.4 Hz, 1 H, 3-H), 5.55, 5.57 (2

NCH₂CO), 60.07, 60.24 [2 (CH₃CH₂)₂CH], 61.04, 61.82 (C-6), dd, J_1,2= 7.6, 7.6 Hz, J_2,3= 9.8, 9.8 Hz, 1 H, 2-H), 7.18–8.01 (m,

65.75, 66.21 (C-2), 68.50, 68.74 (C-4), 70.20, 70.61 (C-5), 70.71,

23 H, Ar Fmoc, PhCH, OBz and SPh) ppm. ¹³C NMR (150 MHz,

70.78 (C-3), 79.52, 79.79 (Me₃CO), 97.36 (C-1), 156.14, 156.49 CDCl₃): δ = 10.63, 10.71, 10.73, 10.76 (2 CH₃CH₂), 25.34, 25.47,

(C=O Boc), 168.76, 168.91, 169.41, 169.79 (2 NCH₂CO), 170.98,

171.17 (C=O Ac) ppm. ESI-FT-MS: calcd. for C₃₃H₆₂N₅O₁₀Si⁺[M

+ H⁺] 716.42605; found 716.42647.

25.82 (2 CH₃CH₂), 43.25, 43.44 (NCH₂CO), 47.06, 47.17 (C-9

Fmoc), 59.26, 59.56 [(CH₃CH₂)₂CH], 67.12, 67.39, 67.48, 67.59

(CH₂Fmoc and C-2), 68.93, 69.01 (C-6), 69.70, 69.78 (C-5), 73.17,

73.51 (C-3), 73.53, 73.68 (C-4), 85.07, 85.22 (C-1), 101.01, 101.03

(PhCH), 119.81, 119.83, 124.78, 124.94, 125.21, 126.43, 126.47,

126.89, 126.91, 126.94, 126.98, 127.51, 127.53, 127.61, 128.04,

128.11, 128.24, 128.32, 128.34, 128.70, 128.72, 129.06, 129.12,

129.73, 129.85, 133.13, 133.23, 133.77, 134.00, 137.19, 141.15,

141.19, 141.26, 143.70, 143.95 (Ar Fmoc, PhCH, PhCOO and

SPh), 156.34, 156.73 (C=O Fmoc), 164.72, 164.83 (C=O Bz),

169.31, 169.66 (NCH₂CO) ppm. ESI-FT-MS: calcd. for

C₄₈H₄₇NO₉SNa⁺[M + Na⁺] 836.28637; found 836.28760.

Phenyl 4,6-O-Benzylidene-3-O-{[(1-ethylpropyl)(9H-fluoren-9-yl-

methoxycarbonyl)amino]acetyl}-1-thio-β-D-galactopyranoside (37):

To a solution of 25 (1.12 g, 3.05 mmol) in pyridine (20 mL) was

added cyanuric chloride (2.56 g, 13.88 mmol), and the mixture was

stirred for 30 min at 40 °C. This mixture was added dropwise to a

cooled solution (0 °C) of 36^[11](1 g, 2.77 mmol) in pyridine (20 mL)

over 5 min. The combined mixture was stirred for a further 1 h at

0 °C. After adding ice to the mixture, the product was dissolved in

ethyl acetate, and the solution was washed with water, dried

(Na₂SO₄), and concentrated. Column chromatography (ethyl ace-

tate/hexane, 1:1) of the residue on silica gel gave 37 (1.439 g, 73%).

[α]_D= 11.4 (c = 1.0, CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ =

Phenyl 2-O-Benzoyl-3-O-{[(1-ethylpropyl)(9H-fluoren-9-ylmethoxy-

carbonyl)amino]acetyl}-1-thio-β-

D-galactopyranoside (39): A solu-

tion of 38 (432 mg, 530.7 µmol) in 80% aq. acetic acid (5 mL) was

0.55, 0.66, 0.81, 0.82 (4 t, J_CH3,CH= J_CH3,CHЈ= 7.3, 7.3, 7.3, 7.3 Hz, stirred for 2 h at 60 °C. After concentration, the product was puri-

6 H, 2 CH₃CH₂), 1.14–1.43 (m, 4 H, 2 CH₃CH₂), 3.49, 3.54 (br. 2 fied by chromatography on a column of silica gel with ethyl acetate/

s, 1 H, 5-H), 3.51, 3.96 [2 m, 1 H, (CH₃CH₂)₂CH], 3.66, 3.79 (2 d, hexane (1:1) to give 39 (250 mg, 65%). [α]_D= 15.0 (c = 1.3, CHCl₃).

J_gem= 17.2, 17.1 Hz, 2 H, NCH₂CO), 3.85, 3.97, 4.27, 4.33 (4 dd,

¹H NMR (600 MHz, CDCl₃): δ = 0.57, 0.61 (2 t, J_CH3,CH

J_5,6= 1.4, 1.5, 1.4, 1.5 Hz, J_gem= 12.3, 12.3, 12.5, 12.3 Hz, 2 H, 6- J_CH3,CHЈ= 7.4, 7.4 Hz, 6 H, 2 CH₃CH₂), 0.94–1.31 (m, 4 H, 2

H), 3.90, 3.90 (2 t, J_1,2= J_2,3= 9.7, 9.5 Hz, 1 H, 2-H), 4.05, 4.37, CH₃CH₂), 3.48, 3.61, 3.65, 3.71 (4 d, J_gem= 18.4, 17.1, 17.1,

4.44, 4.47 (4 dd, J_CH2,9-H= 7.6, 6.1, 6.7, 6.0 Hz, J_gem= 10.6, 10.7,

10.6, 10.7 Hz, 2 H, CH₂Fmoc), 4.13, 4.18 (2 t, J_9-H,CH= J_9-H,CHЈ

= 7.0, 7.3 Hz, 1 H, 9-H Fmoc), 4.17, 4.28 (2 d, J_3,4= 2.4, 2.7 Hz,

1 H, 4-H), 4.53, 4.61 (2 d, J_1,2= 9.5, 9.5 Hz, 1 H, 1-H), 4.90, 5.03

(2 dd, J_2,3= 9.8, 9.6 Hz, J_3,4= 3.4, 3.4 Hz, 1 H, 3-H), 5.06, 5.44

(2 s, 1 H, PhCH), 7.12–7.76 (m, 18 H, Ar Fmoc, PhCH and SPh)

17.8 Hz, 2 H, NCH₂CO), 3.51, 3.82 [2 m, 1 H, (CH₃CH₂)₂CH],

3.70 (m, 1 H, 5-H), 3.84, 3.87, 3.96, 4.02 (4 dd, J_5,6= 4.4, 4.6, 5.7,

6.4 Hz, J_gem= 11.9, 11.9, 11.8, 11.9 Hz, 2 H, 6-H), 4.04, 4.20 (2 t,

J_9-H,CH= J_9-H,CHЈ= 4.5, 5.9 Hz, 1 H, 9-H Fmoc), 4.36 (dd, J_3,4

3.1 Hz, J_4,5= 1.1 Hz, 1 H, 4-H), 4.45, 4.50 (2 dd, J_CH2,9-H= 6.2,

5.9 Hz, J_gem= 10.6, 10.6 Hz, 2 H, CH₂Fmoc), 4.85, 4.86 (2 d, J_1,2

ppm. ¹³C NMR (150 MHz, CDCl₃): δ = 11.21, 11.22, 11.41 (2 = 10.0, 10.0 Hz, 1 H, 1-H), 5.09, 5.18 (2 dd, J_2,3= 9.8, 9.7 Hz, J_3,4

CH₃CH₂), 26.18, 26.25, 26.40 (2 CH₃CH₂), 44.21, 44.64 = 3.1, 3.1 Hz, 1 H, 3-H), 5.53, 5.64 (2 t, J_1,2= J_2,3= 9.9, 9.9 Hz,

(NCH₂CO), 47.64 (C-9 Fmoc), 60.03, 60.50 [(CH₃CH₂)₂CH],

1 H, 2-H), 7.21–8.02 (m, 18 H, Ar Fmoc, OBz and SPh) ppm. ¹³C

Eur. J. Org. Chem. 2005, 5313–5329

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5325

S. Komba, M. Kitaoka, T. Kasumi

5.42, 5.51 (2 d, J_3,4= 3.3, 3.3 Hz, 1 H, 4-H), 5.50, 5.52 (2 t, J_1,2

FULL PAPER

NMR (150 MHz, CDCl₃): δ = 10.67, 10.71 (2 CH₃CH₂), 25.44,

25.79 (2 CH₃CH₂), 43.11, 44.26 (NCH₂CO), 47.15, 47.34 (C-9 J_2,3= 9.9, 10.0 Hz, 1 H, 2-H), 7.28–8.04 (m, 10 H, OBz and SPh)

Fmoc), 59.30, 59.88 [(CH₃CH₂)₂CH], 62.62 (C-6), 66.96, 67.15 (C- ppm. ¹³C NMR (150 MHz, CDCl₃): δ = 10.76, 10.96, 10.99, 11.06

4), 67.68 (CH₂Fmoc), 67.96, 68.26 (C-2), 74.81, 76.09 (C-3), 78.00,

78.28 (C-5), 86.52, 86.92 (C-1), 124.76, 124.90, 124.93, 127.04,

(2 CH₃CH₂), 20.69 (2 CH₃COO), 25.60, 26.09, 26.10 (2 CH₃CH₂),

27.97, 28.27 (Me₃CO), 43.09, 43.34 (NCH₂CO), 58.15, 59.82

127.08, 127.17, 127.65, 127.71, 127.79, 128.35, 128.40, 128.89, [(CH₃CH₂)₂CH], 61.74, 61.79 (C-6), 67.40, 67.52 (C-4), 67.80,

128.98, 129.56, 129.77, 129.86, 132.08, 132.59, 133.14, 133.27, 67.95 (C-2), 72.39, 72.50 (C-3), 74.63, 74.74 (C-5), 79.71, 79.84

141.34, 141.42, 143.66, 143.86 (Ar Fmoc, PhCOO and SPh), 157.69

(C=O Fmoc), 165.02, 165.12 (C=O Bz), 168.97, 169.12 (NCH₂CO)

(Me₃CO), 86.75, 86.84 (C-1), 128.19, 128.32, 128.41, 128.49,

128.83, 128.88, 129.10, 129.93, 130.06, 132.19, 132.88, 132.99,

ppm. ESI-FT-MS: calcd. for C₄₁H₄₃NO₉SNa⁺[M + Na⁺] 133.30, 133.57 (PhCOO and SPh), 155.57, 156.00 (C=O Boc),

748.25507; found 748.25643.

165.11, 165.30 (C=O Bz), 169.51, 169.63 (NCH₂CO), 170.01,

170.10, 170.40 (C=O 2 Ac) ppm. ESI-FT-MS: calcd. for

C₃₅H₄₆NO₁₁S⁺[M + H⁺] 688.27861; found 688.27911.

Phenyl 4,6-Di-O-acetyl-2-O-benzoyl-3-O-{[(1-ethylpropyl)(9H-

fluoren-9-ylmethoxycarbonyl)amino]acetyl}-1-thio-β-D-galacto-

pyranoside (40): To a solution of 39 (197 mg, 271.4 µmol) in pyri-

dine (3 mL) was added acetic anhydride (3 mL), and the mixture

was stirred for 2 h at room temp. The work-up as described for 34

tert-Butyldimethylsilyl O-{4,6-Di-O-acetyl-2-O-benzoyl-3-O-{[tert-

butoxycarbonyl(1-ethylpropyl)amino]acetyl}-β-

(1Ǟ3)-4-O-acetyl-2-azido-2-deoxy-6-O-{[{2-[tert-butoxycarbonyl-

-galac-

topyranoside (42): To a solution of 35 (90 mg, 125.7 µmol) and 41

D-galactopyranosyl}-

gave 40 (168 mg, 76%). [α]_D= 22.8 (c = 1.0, CHCl₃). ¹H NMR (1-ethylpropyl)amino]acetyl}(1-ethylpropyl)amino]acetyl}-β-

(600 MHz, CDCl₃): δ = 0.57, 0.69, 0.69, 0.88 (4 t, J_CH3,CH

J_CH3,CHЈ= 7.3, 7.4, 7.3, 7.3 Hz, 6 H, 2 CH₃CH₂), 0.99–1.42 (m, 4

H, 2 CH₃CH₂), 2.04, 2.06, 2.13, 2.14 (4 s, 6 H, 2 OAc), 3.51, 3.89

[2 m, 1 H, (CH₃CH₂)₂CH], 3.56, 3.61, 3.63, 3.67 (4 d, J_gem= 17.4,

(130 mg, 189 µmol) in dry dichloromethane (2 mL) was added 4-

Å molecular sieves (250 mg) and N-iodosuccinimide (85 mg, 377.7

µmol), and the mixture was stirred at –40 °C. Trifluoromethanesul-

fonic acid (5 µL, 56.5 µmol) was added, and the mixture was stirred

17.4, 18.1, 18.1 Hz, 2 H, NCH₂CO), 3.89, 4.11 (2 t, J_9-H,CH

J_9-H,CHЈ= 7.3, 6.5 Hz, 1 H, 9-H Fmoc), 3.99 (m, 1 H, 5-H), 4.01, for 2 h at –40 °C. The solid was filtered off and washed with chloro-

4.09, 4.19, 4.27 (4 dd, J_CH2,9-H= 7.4, 6.9, 6.2, 6.2 Hz, J_gem= 10.6,

10.6, 10.5, 10.6 Hz, 2 H, CH₂Fmoc), 4.16, 4.23 (2 dd, J_5,6= 6.2,

6.9 Hz, J_gem= 11.5, 11.4 Hz, 2 H, 6-H), 4.85, 4.86 (2 d, J_1,2= 9.9,

form. The combined filtrate and washings were washed with satd.

aq. Na₂S₂O₃and satd. aq. NaHCO₃, then dried (Na₂SO₄), and

concentrated. Column chromatography (ethyl acetate/hexane, 1:2)

of the residue on silica gel gave 42 (145 mg, 89%). [α]_D= 6.1 (c =

10.0 Hz, 1 H, 1-H), 5.32, 5.34 (2 dd, J_2,3= 10.0, 10.0 Hz, J_3,4

3.3, 3.3 Hz, 1 H, 3-H), 5.42, 5.49 (2 dd, J_3,4= 3.3, 3.4 Hz, J_4,5

1.0, CHCl₃). H NMR (600 MHz, CDCl₃): δ = 0.09, 0.10, 0.10 (3

1.0, 0.9 Hz, 1 H, 4-H), 5.49, 5.50 (2 t, J_1,2= J_2,3= 10.0, 10.0 Hz, s, 6 H, Me₂Si), 0.67, 0.69, 0.79, 0.97 (4 t, 18 H, 6 CH₃CH₂), 0.88,

1 H, 2-H), 7.10–7.99 (m, 18 H, Ar Fmoc, OBz and SPh) ppm. ¹³C

NMR (150 MHz, CDCl₃): δ = 10.79, 10.87, 11.07 (2 CH₃CH₂),

0.88 (2 s, 9 H, Me₃CSi), 1.03–1.56 (m, 12 H, 6 CH₃CH₂), 1.18,

1.33, 1.41, 1.46 (4 s, 18 H, 2 Boc), 2.07, 2.09, 2.10, 2.19, 2.19, 2.20

20.68, 20.70 (2 CH₃COO), 25.54, 25.79, 25.82 (2 CH₃CH₂), 43.22, (6 s, 9 H, 3 OAc), 3.37, 3.39 (2 t, J_1,2= J_2,3= 7.7, 7.7 Hz, 1 H, 2-

43.67 (NCH₂CO), 46.96, 47.19 (C-9 Fmoc), 59.49, 59.77 H GalN), 3.39–3.97 [m, 3 H, 3 (CH₃CH₂)₂CH], 3.45–3.95 (m, 6 H,

[(CH₃CH₂)₂CH], 61.65, 61.68 (C-6), 67.12, 67.84 (CH₂Fmoc), 3 NCH₂CO), 3.50 (m, 1 H, 3-H GalN), 3.74 (m, 1 H, 5-H GalN),

67.41, 67.49 (C-4), 67.71, 67.82 (C-2), 72.50, 72.69 (C-3), 74.59,

74.63 (C-5), 86.76, 86.89 (C-1), 119.80, 119.85, 124.85, 124.87,

3.92 (m, 1 H, 5-H Gal), 3.98, 4.16 (2 m, 4 H, 6-H GalN and 6-H

Gal), 4.44, 4.45 (2 d, J_1,2= 7.7, 7.7 Hz, 1 H, 1-H GalN), 4.87, 4.88,

125.01, 125.12, 126.85, 126.93, 126.95, 127.48, 127.51, 127.56, 4.91, 4.92 (4 d, J_1,2= 7.9, 8.1, 7.9, 7.8 Hz, 1 H, 1-H Gal), 5.26 (dd,

127.58, 128.17, 128.22, 128.29, 128.36, 128.63, 128.83, 128.85, J_2,3= 10.5 Hz, J_3,4= 3.5 Hz, 1 H, 3-H Gal), 5.28, 5.30, 5.31 (3 d,

129.32, 129.56, 129.98, 132.19, 132.46, 132.77, 132.95, 133.26, J_3,4= 3.4, 3.4, 3.4 Hz, 1 H, 4-H GalN), 5.37, 5.46 (br. 2 s, 1 H, 4-

141.19, 141.24, 141.34, 143.96, 144.00, 144.10, 144.22 (Ar Fmoc,

PhCOO and SPh), 156.38, 156.66 (C=O Fmoc), 164.97, 165.29

H Gal), 5.40 (dd, J_1,2= 7.9 Hz, J_2,3= 10.4 Hz, 1 H, 2-H Gal),

7.41–8.05 (m, 5 H, OBz) ppm. ¹³C NMR (150 MHz, CDCl₃): δ =

(C=O Bz), 169.08, 169.34 (NCH₂CO), 170.07, 170.21, 170.39 (C=O –5.20, –5.13, –4.39, –4.35 (Me₂Si), 10.75, 10.97, 11.06, 11.09, 11.21,

2 Ac) ppm. ESI-FT-MS: calcd. for C₄₅H₄₈NO₁₁S⁺[M + H⁺] 11.24, 11.31, 11.35, 11.45, 11.47, 11.53 (6 CH₃CH₂), 17.95

810.29426; found 810.29461.

(Me₃CSi), 20.66, 20.74, 20.76 (3 CH₃COO), 25.54, 25.58, 25.68

(Me₃CSi), 26.11, 26.16, 26.41, 26.46, 26.56, 26.60 (6 CH₃CH₂),

27.98, 28.26, 28.34, 28.44, 28.55 (2 Me₃CO), 42.48, 42.70, 43.12,

43.40, 43.70 (3 NCH₂CO), 58.21, 58.26, 59.87, 60.09, 60.29, 60.43

[3 (CH₃CH₂)₂CH], 61.15, 61.19, 62.37, 62.51 (C-6 Gal), 65.12,

65.23 (C-2 GalN), 67.01, 67.13 (C-4 Gal), 68.20 (C-4 GalN), 69.33,

69.41 (C-2 Gal), 70.91, 70.98, 71.06 (C-5 Gal and C-3 Gal), 71.42

(C-5 GalN), 76.13, 76.30 (C-3 GalN), 79.48, 79.57, 79.72, 79.86 (2

Me₃CO), 97.42 (C-1 GalN), 101.41, 101.54 (C-1 Gal), 128.44,

128.51, 129.17, 129.70, 129.80, 133.41 (PhCOO), 155.61, 156.05,

156.12 (C=O 2 Boc), 165.13, 165.25 (C=O Bz), 168.75, 169.41,

169.62, 169.77, 169.99, 170.17, 170.30, 170.44 (3 NCH₂CO and

C=O 3 Ac) ppm. ESI-FT-MS: calcd. for C₆₂H₁₀₁N₆O₂₁Si⁺[M +

H⁺] 1293.67836; found 1293.67847.

Phenyl 4,6-Di-O-acetyl-2-O-benzoyl-3-O-{[tert-butoxycarbonyl(1-

ethylpropyl)amino]acetyl}-1-thio-β-D-galactopyranoside (41): To a

solution of 40 (500 mg, 617.3 µmol) in DMF (2.4 mL) was added

piperidine (600 µL, 6.06 mmol), and the mixture was stirred for

20 min at room temp. After cooling to 0 °C, di-tert-butyl dicarbon-

ate (1.72 g, 7.88 mmol) and satd. aq. NaHCO₃(3 mL) were added,

and the mixture was stirred for 1 h at room temp. The product was

dissolved in ethyl acetate and the solution was washed with water,

dried (Na₂SO₄), and concentrated. Column chromatography (ethyl

acetate/hexane, 1:2) of the residue on silica gel gave 41 (351 mg, 2

steps 83%). [α]_D= 17.1 (c = 1.0, CHCl₃). ¹H NMR (600 MHz,

CDCl₃): δ = 0.65, 0.66, 0.78, 0.85 (4 t, J_CH3,CH= J_CH3,CHЈ7.2, 7.3,

7.4, 7.4 Hz, 6 H, 2 CH₃CH₂), 0.88–1.33 (m, 4 H, 2 CH₃CH₂), 1.19,

1.34 (2 s, 9 H, Boc), 2.06, 2.06, 2.15, 2.15 (4 s, 6 H, 2 OAc), 3.50,

tert-Butyldimethylsilyl O-(4,6-Di-O-acetyl-2-O-benzoyl-β-D-galacto-

3.59 (2 s, 2 H, NCH₂CO), 3.58, 3.83 [2 m, 1 H, (CH₃CH₂)₂CH], pyranosyl)(1Ǟ3)-4-O-acetyl-2-azido-6-O-{[tert-butoxycarbonyl(1-

3.98, 4.01 (2 t, J_5,6= J_5,6Ј= 6.2, 6.2 Hz, 1 H, 5-H), 4.14, 4.17, 4.21, ethylpropyl)amino]acetyl}-2-deoxy-β- -galactopyranoside (43):

4.24, (4 dd, J_5,6= 6.1, 5.9, 6.8, 7.0 Hz, J_gem= 11.4, 11.4, 11.3, Using the general procedure for one cycle of Edman degradation,

11.5 Hz, 2 H, 6-H), 4.86, 4.89 (2 d, J_1,2= 10.0, 10.0 Hz, 1 H, 1-H), compound 42 (163 mg, 126 µmol) was degraded to compound 43

5.29, 5.33 (2 dd, J_2,3= 9.9, 9.9 Hz, J_3,4= 3.2, 3.3 Hz, 1 H, 3-H), (83 mg, 4 steps 70%) as a selectively 3Ј-position-free disaccharide.

5326

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Eur. J. Org. Chem. 2005, 5313–5329

A New Method of Carbohydrate Synthesis Using a Special Hydroxy Protecting Group

FULL PAPER

[α]_D= 2.7 (c = 1.3, CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ = 0.12, amino]acetyl}-2-deoxy-β-

D-galactopyranoside (46): To a solution of

0.12 (2 s, 6 H, Me₂Si), 0.90 (m, 6 H, 2 CH₃CH₂), 0.90, 0.90 (2 s, 9

H, Me₃CSi), 1.28–1.46 (m, 4 H, 2 CH₃CH₂), 1.40, 1.46 (2 s, 9 H,

Boc), 2.07, 2.08, 2.09, 2.09, 2.21, 2.21 (6 s, 9 H, 3 OAc), 3.44, 3.45

(2 dd, J_1,2= 7.5, 7.5 Hz, J_2,3= 10.5, 10.4 Hz, 1 H, 2-H GalN),

3.51, 3.51 (2 dd, J_2,3= 10.5, 10.4 Hz, J_3,4= 3.3, 3.3 Hz, 1 H, 3-H

GalN), 3.62, 3.66, 3.71 (d, d, s, J_gem= 17.6, 17.7 Hz, 2 H,

43 (10 mg, 10.6 µmol) and 45^[13](12.6 mg, 21.6 µmol) in dry aceto-

nitrile (150 µL) was added 3-Å molecular sieves (30 mg) and N-

iodosuccinimide (9.6 mg, 42.6 µmol), and the mixture was stirred

at –30 °C. Trifluoromethanesulfonic acid (0.6 µL, 6.7 µmol) was

added, and the mixture was stirred at –30 °C for 2 days. The solid

was filtered and washed with chloroform. The combined filtrate

NCH₂CO), 3.73, 3.95 [2 m, 1 H, (CH₃CH₂)₂CH], 3.74 (m, 1 H, 5- and washings were washed with satd. aq. Na₂S₂O₃and satd. aq.

H GalN), 3.87 (br. t, 1 H, 5-H Gal), 3.99 (m, 1 H, 3-H Gal), 4.03, NaHCO₃, dried (Na₂SO₄), and concentrated. Column chromatog-

4.20 (2 m, 2 H, 6-H GalN), 4.15 (m, 2 H, 6-H Gal), 4.46, 4.46 (2

d, J_1,2= 7.6 Hz, 7.5 Hz, 1 H, 1-H GalN), 4.84, 4.85 (2 d, J_1,2

7.8, 7.9 Hz, 1 H, 1-H Gal), 5.15 (dd, J_1,2= 7.9 Hz, J_2,3= 10.0 Hz,

1 H, 2-H Gal), 5.31, 5.32 (br. 2 d, J_3,4= 3.8, 4.1 Hz, 1 H, 4-H

raphy (ethyl acetate/hexane, 3:1) of the residue on silica gel gave 46

(2 mg, 13 %) as the β anomer selectively. [α]_D= 17.6 (c = 0.5,

CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ = 0.07, 0.11, 0.11, 0.12 (4

s, 6 H, Me₂Si), 0.89 (m, 6 H, 2 CH₃CH₂), 0.89, 0.89 (2 s, 9 H,

GalN), 5.36, 5.37 (br. 2 d, J_3,4= 4.0, 3.6 Hz, 1 H, 4-H Gal), 7.45– Me₃CSi), 1.28–1.44 (m, 4 H, 2 CH₃CH₂), 1.39, 1.45 (2 s, 9 H, Boc),

8.09 (m, 5 H, OBz) ppm. ¹³C NMR (150 MHz, CDCl₃): δ = 1.72 (br. t, 1 H, 3ax-H SA), 1.90, 1.90 (2 s, 3 H, NAc), 1.98, 1.99,

–5.18, –5.15, –4.33 (Me₂Si), 10.99, 11.00, 11.19 (2 CH₃CH₂), 17.97

(Me₃CSi), 20.64, 20.65, 20.74, 20.78, 20.83 (3 CH₃COO), 25.57, 2.43 (dd, J_3eq,4= 4.6 Hz, J_gem= 13.3 Hz, 1 H, 3eq-H SA), 3.11,

25.58 (Me₃CSi), 25.91, 25.95, 26.39 (2 CH₃CH₂), 28.25, 28.40 3.12 (2 s, 3 H, COOMe), 3.32, 3.33 (2 dd, J_1,2= 7.8, 7.8 Hz, J_2,3

2.02, 2.04, 2.05, 2.06, 2.07, 2.08, 2.09, 2.09, 2.12 (11s, 21 H, 7 OAc),

(Me₃CO), 43.54 (NCH₂CO), 58.32, 59.99 [(CH₃CH₂)₂CH], 61.52, 10.5, 10.4 Hz, 1 H, 2-H GalN), 3.50, 3.51 (2 dd, J_2,3= 10.7,

61.65 (C-6 Gal), 62.68, 62.90 (C-6 GalN), 65.20, 65.24 (C-2 GalN), 10.2 Hz, J_3,4= 3.5, 3.7 Hz, 1 H, 3-H GalN), 3.62, 3.65, 3.70 (d, d,

68.28, 68.37 (C-4 GalN), 69.38, 69.45 (C-4 Gal), 71.05, 71.10, s, 2 H, J_gem= 17.2, 17.4 Hz, NCH₂CO), 3.72, 3.95 [2 m, 1 H,

71.17, 71.23 (C-3 Gal and C-5 Gal), 71.58 (C-5 GalN), 73.26, 73.35 (CH₃CH₂)₂CH], 3.73 (m, 1 H, 5-H GalN), 3.83 (m, 2 H, 9-H SA),

(C-2 Gal), 76.22, 76.43 (C-3 GalN), 79.95, 80.05 (Me₃CO), 97.42, 3.97 (m, 1 H, 5-H SA), 3.99, 4.17 (2 m, 2 H, 6-H GalN), 4.06, 4.23

97.48 (C-1 GalN), 101.16, 101.20 (C-1 Gal), 128.55, 129.40, 129.42,

129.76, 133.43, 133.46 (PhCOO), 155.66, 156.31 (C=O Boc),

166.61, 166.66 (C=O Bz), 169.79, 169.86, 169.94, 170.08, 170.51,

(m, 2 H, 6-H Gal), 4.21 (m, 1 H, 5-H Gal), 4.47, 4.47 (2 d, J_1,2=

7.7, 7.8 Hz, 1 H, 1-H GalN), 4.65 (dd, J_5,6= 10.5 Hz, J_6,7= 1.5 Hz,

1 H, 6-H SA), 4.89, 4.89 (2 d, J_1,2= 7.9, 8.0 Hz, 1 H, 1-H Gal),

170.55, 170.98, 170.99 (NCH₂CO and C=O 3 Ac) ppm. ESI-FT- 4.94, 4.95 (2 dd, J_2,3= 10.1, 9.7 Hz, J_3,4= 3.5, 3.1 Hz, 1 H, 3-H

MS: calcd. for C₄₃H₆₆N₄O₁₇SiNa⁺[M + Na⁺] 961.40844; found

961.40827.

Gal), 5.04 (ddd, J_3ax,4= J_4,5= 11.0 Hz, J_3eq,4= 4.5 Hz, 1 H, 4-H

SA), 5.27 (m, 1 H, 8-H SA), 5.28 (br. d, J_3,4= 2.8 Hz, 1 H, 4-H

GalN), 5.31 (dd, J_1,2= 8.0 Hz, J_2,3= 10.3 Hz, 1 H, 2-H Gal), 5.37

(m, 1 H, 7-H SA), 5.34 (m, 1 H, 4-H Gal), 5.53, 5.53 (2 d, J_NH,5

10.2, 10.1 Hz, 1 H, NH), 7.45–8.05 (m, 5 H, OBz) ppm. ¹³C NMR

(150 MHz, CDCl₃): δ = –5.18, –4.35 (Me₂Si), 10.99, 11.01, 11.19 (2

CH₃CH₂), 17.97 (Me₃CSi), 20.52, 20.53, 20.66, 20.69, 20.83, 20.96,

20.99, 21.04, 21.06 (7 CH₃COO), 23.26 (CH₃CON), 25.58, 25.59

(Me₃CSi), 25.91, 25.96, 26.39 (2 CH₃CH₂), 28.25, 28.40 (Me₃CO),

37.26, 37.34 (C-3 SA), 43.54 (NCH₂CO), 48.57 (C-5 SA), 52.40

(COOMe), 58.31, 59.99 [(CH₃CH₂)₂CH], 60.95, 61.10 (C-6 Gal),

62.76 (C-6 GalN), 62.96, 63.01 (C-7 SA and C-9 SA), 64.95, 65.00

(C-2 GalN), 67.70 (C-4 SA), 68.41, 68.47 (C-4 GalN), 69.03, 69.06,

69.51, 70.01, 70.07, 70.12, 70.19, 70.34, 70.38 (C-4 Gal, C-5 Gal,

and C-3 Gal), 71.60, 71.62 (C-5 GalN), 71.71 (C-2 Gal), 72.54 (C-

8 SA), 73.16 (C-6 SA), 75.08, 75.24 (C-3 GalN), 79.95, 80.04

(Me₃CO), 97.55, 97.61 (C-1 GalN), 98.93 (C-2 SA), 100.90, 100.93

(C-1 Gal), 128.61, 129.81, 130.14, 130.17, 133.28, 133.31 (PhCOO),

155.66, 156.30 (C=O Boc), 165.71 (C=O Bz), 166.51 (C-1 SA),

169.77, 169.84, 169.86, 169.93, 170.08, 170.27, 170.67, 170.71,

171.17, 171.25, 171.27, 172.35, 172.38 (NCH₂CO and C=O 8 Ac)

ppm. ESI-FT-MS: calcd. for C₆₃H₉₃N₅O₂₉SiNa⁺[M + Na⁺]

1434.56177; found 1434.56135.

tert-Butyldimethylsilyl O-(3,4,6-Tri-O-acetyl-2-O-benzoyl-β-

D-

galactopyranosyl)(1Ǟ3)-4-O-acetyl-2-azido-2-deoxy-β- -galacto-

pyranoside (44): To a solution of 43 (53 mg, 56.4 µmol) in pyridine

(1 mL) was added acetic anhydride (1 mL), and the mixture was

stirred for 2 h at room temp. The work-up described for 34 gave an

acetylated intermediate (55 mg, 99%). Subsequent deprotection of

the amino protecting group and coupling of PITC as described in

the general procedure for one cycle of Edman degradation gave 44

(33 mg, 2 steps 98%) as a selectively 6-position-free disaccharide.

[α]_D= 1.8 (c = 0.5, CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ = 0.09

(s, 6 H, Me₂Si), 0.87 (s, 9 H, Me₃CSi), 1.92, 2.07, 2.19, 2.19 (4 s,

12 H, 4 OAc), 3.40 (dd, J_5,6= 8.4 Hz, J_gem= 11.0 Hz, 1 H, 6-H

GalN), 3.44 (dd, J_1,2= 6.6 Hz, J_2,3= 10.5 Hz, 1 H, 2-H GalN),

3.47 (dd, J_2,3= 10.4 Hz, J_3,4= 3.2 Hz, 1 H, 3-H GalN), 3.52 (t,

J_5,6= J_5,6Ј= 7.9 Hz, 1 H, 5-H GalN), 3.57 (m, 1 H, 6Ј-H GalN),

3.96 (ddd, J_4,5= 1.2 Hz, J_5,6= J_5,6Ј= 6.6 Hz, 1 H, 5-H Gal), 4.09,

4.13 (2 dd, J_5,6= 6.7, 6.5 Hz, J_gem= 11.3, 11.3 Hz, 2 H, 6-H Gal),

4.44 (d, J_1,2= 6.4 Hz, 1 H, 1-H GalN), 4.82 (d, J_1,2= 7.9 Hz, 1 H,

1-H Gal), 5.21 (dd, J_2,3= 10.6 Hz, J_3,4= 3.4 Hz, 1 H, 3-H Gal),

5.23 (d, J_3,4= 2.3 Hz, 1 H, 4-H GalN), 5.42 (dd, J_3,4= 3.4 Hz, J_4,5

= 1.1 Hz, 1 H, 4-H Gal), 5.45 (dd, J_1,2= 7.9 Hz, J_2,3= 10.6 Hz, 1

H, 2-H Gal), 7.44–8.05 (m, 5 H, OBz) ppm. ¹³C NMR (150 MHz,

CDCl₃): δ = –5.17, –4.33 (Me₂Si), 17.91 (Me₃CSi), 20.56, 20.69,

20.84 (4 CH₃COO), 25.53 (Me₃CSi), 59.91 (C-6 GalN), 61.17 (C-

6 Gal), 65.18 (C-2 GalN), 66.98 (C-4 Gal), 69.18, 69.21 (C-4 GalN

and C-2 Gal), 70.53 (C-3 Gal), 70.92 (C-5 Gal), 73.47 (C-5 GalN),

77.70 (C-3 GalN), 97.50 (C-1 GalN), 102.13 (C-1 Gal), 128.52,

129.33, 129.68, 133.37 (PhCOO), 165.20 (C=O Bz), 170.19, 170.23,

1 7 0 . 4 5 , 1 7 2 . 6 2 ( C = O 4 A c ) . E S I - F T- M S : c a l c d . fo r

C₃₃H₄₇N₃O₁₅SiNa⁺[M + Na⁺] 776.26687; found 776.26714.

tert-Butyldimethylsilyl O-(3,4,6-Tri-O-acetyl-2-O-benzoyl-β-

galactopyranosyl)(1Ǟ3)-O-[(methyl 5-acetamido-4,7,8,9-tetra-O-

acetyl-3,5-dideoxy- -glycero-α,β- -galacto-2-nonulopyranosylo-

nate)(2Ǟ6)]-4-O-acetyl-2-azido-2-deoxy-β- -galactopyranoside

D-

(47): To a solution of 44 (23 mg, 30.5 µmol) and 45 (27 mg, 46.2

µmol) in dry acetonitrile (400 µL) was added 3-Å molecular sieves

(40 mg) and N-iodosuccinimide (21 mg, 93.3 µmol), and the mix-

ture was stirred at –30 °C. Trifluoromethanesulfonic acid (1.2 µL,

13.5 µmol) was added and the mixture was stirred at –30 °C for

10 h. The solid was filtered and washed with chloroform. The com-

tert-Butyldimethylsilyl O-(Methyl 5-acetamido-4,7,8,9-tetra-O-ace-

tyl-3,5-dideoxy-

(2Ǟ3)-O-(4,6-di-O-acetyl-2-O-benzoyl-β-

(1Ǟ3)-4-O-acetyl-2-azido-6-O-{[tert-butoxycarbonyl(1-ethylpropyl)-

-glycero-β-

-galacto-2-nonulopyranosylonate)- bined filtrate and washings were washed with satd. aq. Na₂S₂O₃

-galactopyranosyl)- and satd. aq. NaHCO₃, then dried (Na₂SO₄), and concentrated.

Gel filtration column chromatography (chloroform/methanol, 1:1)

Eur. J. Org. Chem. 2005, 5313–5329

www.eurjoc.org

5327

S. Komba, M. Kitaoka, T. Kasumi

FULL PAPER

Table 1.¹H NMR data for trisaccharide 47.

of the residue on Sephadex LH-20 gave the crude product, which

was then purified by column chromatography on silica gel with 4:1

ethyl acetate/hexane, to give 47 (25 mg, 67%, α/β = 71:29). The α

to β ratio was calculated from the 3-H equatorial proton on the

sialic acid residue determined by ¹H NMR spectroscopy. ¹H NMR

(600 MHz, CDCl₃, α/β = 71:29): δ = 0.09, 0.13, 0.15 (3 s, Me₂Si),

0.87, 0.90 (2 s, Me₃CSi), 1.87, 1.92, 1.93, 1.94, 1.97, 2.01, 2.02,

2.03, 2.03, 2.06, 2.06, 2.10, 2.11, 2.14, 2.16, 2.18, 2.19, 2.29 (18s, 8

nected to a suction flask through a Teflon tube with a manual

two-way Teflon valve; excess reagents, DMF etc., were removed by

applying a vacuum. The resin was derivatized with the HMBA-

Gly-linker. Briefly, the resin was swelled with dichloromethane

(6 mL). N-α-Fmoc-glycine (49; 358 mg, 1.20 mmol), DMAP

(98 mg, 0.80 mmol), and DIC (157 µL, 1.00 mmol) were added to

the resin in dichloromethane, and then agitated. After 7 h, the solu-

tion was removed. Subsequently, dichloromethane (3 mL), acetic

OAc and NAc), 3.78, 3.79 (2 s, COOMe), 7.44–8.05 (m, OBz) ppm. anhydride (3 mL) and pyridine (1 mL) were added to the resin, and

For the remaining ¹H NMR data, see Table 1. ¹³C NMR then agitated. After 2 h, the solution was removed, and the resin

(150 MHz, CDCl₃): δ = –5.28, –4.18 (Me₂Si), 17.89, 17.99

(Me₃CSi), 20.57, 20.65, 20.69, 20.72, 20.77, 20.81, 20.85, 20.93, (5×10 mL), and diethyl ether (3×10 mL), then dried in vacuo (ge-

21.08, 21.37 (8 CH₃COO), 23.11, 23.20 (CH₃CON), 25.49, 25.65 neral washing and drying procedure). The Fmoc group was cleaved

(Me₃CSi), 37.20 (C-3 SA β), 37.63 (C-3 SA α), 47.89 (C-5 SA β), from the Gly residue, and the loading of ArgoPore resin was deter-

was washed thoroughly with DMF (10×10 mL), dichloromethane

49.33 (C-5 SA α), 52.60 (COOMe β), 52.85 (COOMe α), 60.15 (C-

6 GalN β), 60.62 (C-6 Gal β), 60.92 (C-6 Gal α), 61.91 (C-9 SA

β), 62.28 (C-6 SA α and C-9 SA α), 63.57 (C-6 GalN α), 65.29 (C-

mined as 0.449 mmol/g from the UV absorbance of the eluate. N-

α-Fmoc deprotection was effected by treatment of the resin for

22 min with 20% piperidine in DMF (28 mL). 4-(Trityloxymethyl)

2 GalN), 66.82 (C-4 Gal β), 66.92 (C-4 Gal α), 67.11 (C-7 SA α), benzoic acid (52; Tr-HMBA, 419 mg, 1.06 mmol), NEM (270 µL,

67.63 (C-8 SA α), 67.76 (C-8 SA β), 67.97 (C-7 SA β), 68.42 (C-4

GalN α), 68.53 (C-4 GalN β), 68.93 (C-4 SA β), 69.04 (C-4 SA α),

69.40 (C-2 Gal α), 69.54 (C-2 Gal β), 70.58 (C-3 Gal), 70.70 (C-5

2.12 mmol), and TBTU (329 mg, 1.02 mmol) were dissolved in

DMF, and after 3 min, the solution was added to the resin, and

then agitated. After 4 h, the solution was removed, and the resin

Gal β), 70.74 (C-5 Gal α), 71.40 (C-5 GalN β), 71.98 (C-6 SA β), was washed with DMF (6×10 mL), and a solution (10 mL) of ace-

72.86 (C-5 GalN α), 75.90 (C-3 GalN α), 77.58 (C-3 GalN β), 97.36 tic anhydride/DMF, (1:7) was added. After 20 min, the resin was

(C-1 GalN α), 97.53 (C-1 GalN β), 98.45 (C-2 SA α), 98.75 (C-2

SA β), 101.32 (C-1 Gal α), 101.71 (C-1 Gal β), 128.53, 128.57,

washed and dried as described in the general washing and drying

procedure. Deprotection of the trityl group from the resin was per-

129.33, 129.37, 129.65, 129.69, 133.35, 133.39 (PhCOO), 165.11 formed using TFA (10.8 mL) and H₂O (1.2 mL) at room tempera-

(C=O Bz α), 165.25 (C=O Bz β), 167.28 (C-1 SA β), 167.75 (C-1 SA

α), 169.49, 170.09, 170.19, 170.23, 170.27, 170.29, 170.38, 170.40,

170.46, 170.62, 170.72, 170.95, 171.79 (C=O 9Ac) ppm. ESI-FT-

MS: calcd. for C₅₃H₇₅N₄O₂₇Si⁺[M + H⁺] 1227.43825; found

1227.43891.

ture. After agitation for 3 h, the resin was washed and dried as

described in the general washing and drying procedure to obtain

hydroxy-functionalized HMBA-Gly-ArgoPore resin (54).

The hydroxy-functionalized HMBA-Gly-ArgoPore resin (54;

171 mg) was packed into a 5-mL disposable chromatography col-

umn (mini column, Sarstedt, Nümbrecht, Germany). This hydroxy-

functionalized resin was glycosylated with thiophenyl galactose (40;

100 mg, 0.12 mmol) in which the 3-position was protected by an

Fmoc-protected mono-UCP group using DMTST (111 mg,

2-{4-[Methyl O-β-D-galactopyranosyl(1Ǟ3)-O-β-D-galacto-

pyranoside]benzamido}acetic Acid (58): ArgoPore OH resin (48,

550 mg) was packed into a 20 mL disposable chromatography col-

umn (Econo-Pac, Bio-Rad, Hercules, CA). The column was con-

5328

www.eurjoc.org

Eur. J. Org. Chem. 2005, 5313–5329

A New Method of Carbohydrate Synthesis Using a Special Hydroxy Protecting Group

FULL PAPER

0.43 mmol) in dichloromethane (1.5 mL) at room temperature. Af-

ter agitation for 1 d, the resin was washed and dried as described

in the general washing and drying procedure. To determine the

coupling yield, aliquots of the resin were taken and treated with a

solution of 20% piperidine in DMF (14 mL) for 22 min. The eluate

was examined by UV at 290 nm and the Fmoc loading and yield

were determined to be 0.076 mmol/g and 22%, respectively. Glyco-

sylation coupling was repeated once more according to the same

procedure. After double coupling, the glycosylation yield was deter-

mined to be 29% by Fmoc UV measurement. To confirm the accu-

racy of the yield from Fmoc UV measurement, the sugar was

cleaved from the resin and analyzed by HPLC. Aliquots of the resin

were taken, and treated with a solution of dichloromethane/MeOH/

25 wt.-% NaOMe in MeOH (360 µL/40 µL/8 µL). After agitation

for 4 h, H₂O (400 µL) was added, and agitation was continued for

4 h. After filtration of the resin, the water layer was analyzed by

normal-phase HPLC. Initially, isocratically 100% solvent A was

used at 1 mL/ min for 10 min. Then, a linear gradient of 0 to 100%

solvent B was used at 1 mL/ min for 30 min. The peaks were de-

tected by UV at 254 nm. The glycosylation yield was determined

to be 31%, which was calculated from the area under the peaks.

4 H, 6_a-H and 6_b-H), 3.74 (dd, J_2,3= 9.9 Hz, J_3,4= 3.3 Hz, 1 H,

3_a-H), 3.86 (d, J_3,4= 3.4 Hz, 1 H, 4_b-H), 4.01 (s, 2 H, NCH₂CO),

4.14 (d, J_3,4= 3.0 Hz, 1 H, 4_a-H), 4.48 (d, J_1,2= 7.9 Hz, 1 H, 1_a-

H), 4.55 (d, J_1,2= 7.7 Hz, 1 H, 1_b-H), 4.80 (d, J_gem= 12.0 Hz, 1

H, CH-Ph), 4.97 (d, J_gem= 12.4 Hz, 1 H, CHЈ-Ph), 7.54 (d, J_o,m

8.2 Hz, 2 H, Ph_o), 7.79 (d, J_o,m= 8.1 Hz, 2 H, Ph_m) ppm. ¹³C NMR

(150 MHz, D₂O): δ = 43.12 (NCH₂CO), 61.04, 61.10 (C-6_aand C-

6_b), 68.58 (C-4_a), 68.71 (C-4_b), 70.06 (C-2_a), 70.69 (CH₂-Ph), 71.17

(C-2_b), 72.64 (C-3_b), 74.99, 75.20 (C-5_aand C-5_b), 82.46 (C-3_a),

101.66 (C-1_a), 104.50 (C-1_b), 127.62 (Ph_o), 128.77 (Ph_m), 133.12

(Ph-1), 141.08 (Ph_p), 170.68 (Ph-CO-N), 175.87 (CH₂COOH);

HMBC NMR (600 MHz, D₂O): δ = 4.55Ǟ82.46 (1_b-HǞC-3_a).

ESI-FT-MS: calcd. for C₂₂H₃₁NO₁₄Na⁺[M + Na⁺] 556.16368;

found 556.16422.

Acknowledgments

This work was supported by the Program for Promotion of Basic

Research Activities for Innovative Biosciences (PROBRAIN). We

thank Dr. H. Ono and his staff for NMR measurements, and Dr.

M. Kameyama and her staff for ESI-FT-MS measurements. In ad-

dition, we also thank Ms. J. Maru and Ms. S. Sasaki for technical

assistance.

The unreacted hydroxyl group on the HMBA-Gly linker was sub-

jected to acetyl capping with acetic anhydride and pyridine in

dichloromethane for 2 h, to selectively deprotect the 3-position of

Gal by one cycle of Edman degradation without the TFA treatment

and Boc reprotection steps. The resin was treated with a solution

of 20% piperidine in DMF (4 mL). After agitation for 22 min, the

solution was removed, and the resin was washed with DMF

(10×4 mL), followed by dissolving in DMF (2.5 mL), and addition

of PITC (1.25 mL) and N-methylmorpholine (0.3 mL), and the

mixture was then agitated. After 2 h, the resin was washed and

dried as described in the general washing and drying procedure to

give selectively 3-position-free Gal acceptor (56).

[1] P. Sears, C.-H. Wong, Science 2001, 291, 2344–2350.

[2] S. L. Flitsch, G. M. Watt, Glycopeptides and Related Com-

pounds, Dekker, New York, 1997, p. 207.

[3] P. H. Seeberger, W.-C. Haase, Chem. Rev. 2000, 100, 4349–

4394.

[4] B. Ernst, G. W. Hart, P. Sinaÿ, Carbohydrates in Chemistry and

Biology, Vol. 1, Wiley-VCH, Weinheim, 2000.

[5] Y. Ito, O. Kanie, T. Ogawa, Angew. Chem. Int. Ed. Engl. 1996,

35, 2510–2512.

[6] C.-H. Wong, X.-S. Ye, Z. Zhang, J. Am. Chem. Soc. 1998, 120,

7137–7138.

This selectively free hydroxyl group on the 3-position of Gal was

glycosylated with thiophenyl galactose (40; 48 mg, 59.2 µmol) using

DMTST (53 mg, 0.21 mmol) in dichloromethane (1.5 mL) at room

temp., according to the same procedure as described for the pre-

vious Gal coupling. After double coupling, the glycosylation yield

was determined to be 41% by Fmoc UV measurement. HPLC mea-

surement indicated the glycosylation yield to be 42%.

[7] L. P. Miranda, M. Meldal, Angew. Chem. Int. Ed. 2001, 40,

3655–3657.

[8] P. Edman, Acta Chem. Scand. 1950, 4, 283–293.

[9] S. Komba, M. Kitaoka, T. Kasumi, Tetrahedron Lett. 2004, 45,

2759–2762.

[10] O. J. Finn, K. R. Jerome, R. A. Henderson, G. Pecher, N. Do-

menech, J. Magarian-Blander, S. M. Barratt-Boyes, Immunol.

Rev. 1995, 145, 61–89.

The final cleavage of the disaccharide from the resin was performed

by treatment with a solution of dichloromethane/MeOH/25 wt.-%

NaOMe in MeOH (2 ml/200 µL/10 µL). After agitation for 1 d,

H₂O (50 µL) was added and the mixture was agitated for 1 more

day. After neutralization with acetic acid, the reaction mixture was

filtered. The resin was washed with CH₂Cl₂/MeOH, (1:1; 5×4 mL),

MeOH (5×4 mL), and H₂O (5×4 mL), and the combined filtrate

and washings were concentrated. The disaccharide was purified by

analytical HPLC using the same column and solvent system as de-

scribed above to obtain 1.8 mg of the target disaccharide (58). The

total yield was determined to be 4% for eight steps from compound

50. [α]_D= –0.4 (c = 0.14, H₂O). ¹H NMR (600 MHz, D₂O): δ =

[11] U. Ellervik, G. Magnusson, J. Org. Chem. 1998, 63, 9314–9322.

[12] G. Grundler, R. R. Schmidt, Liebigs Ann. Chem. 1984, 1826–

1847.

[13] A. Marra, P. Sinaÿ, Carbohydr. Res. 1989, 187, 35–42.

[14] Murase, H. Ishida, M. Kiso, A. Hasegawa, Carbohydr. Res.

1988, 184, c1–c4.

[15] A. Hasegawa, T. Nagahama, H. Ohki, K. Hotta, M. Kiso, J.

Carbohydr. Chem. 1991, 10, 493–498.

[16] G. J. Boons, A. V. Demchenko, Chem. Rev. 2000, 100, 4539–

4565.

[17] O. J. Plante, E. R. Palmacci, P. H. Seeberger, Science 2001, 291,

1523–1527.

[18] F. Roussel, M. Takhi, R. R. Schmidt, J. Org. Chem. 2001, 66,

8540–8548.

3.55 (dd, J_1,2= 7.7 Hz, J_2,3= 9.9 Hz, 1 H, 2_b-H), 3.61 (dd, J_2,3

9.9 Hz, J_3,4= 3.4 Hz, 1 H, 3_b-H), 3.62–3.66 (m, 2 H, 5_a-H and 5_b-

H), 3.69 (dd, J_1,2= 7.9 Hz, J_2,3= 9.9 Hz, 1 H, 2_a-H), 3.68–3.77 (m,

Received: May 27, 2005

Published Online: November 2, 2005

Eur. J. Org. Chem. 2005, 5313–5329

www.eurjoc.org

5329

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