Syntheses of model compounds related to an antigenic epitope in pectic polysaccharides from Bupleurum falcatum L. (II)

Article abstract of DOI:10.1248/cpb.54.485

Stereocontrolled syntheses of model compounds related to a major antigenic epitope against antibupleurum 2IIc/PG-1-IgG from antiulcer pectic polysaccharide are described. A trisaccharide derivative (13) was prepared as a precursor and a novel and simple a

Full text of DOI:10.1248/cpb.54.485

April 2006
Chem. Pharm. Bull. 54(4) 485—492 (2006)
485
Syntheses of Model Compounds Related to an Antigenic Epitope in Pectic
Polysaccharides from Bupleurum falcatum L. (II)
Yuhua JIN,a Noriyasu HADA,a Junko OKA,a Osamu KANIE,b Shusaku DAIKOKU,b Yoshimi KANIE,b
Haruki YAMADA,c and Tadahiro TAKEDA*,a
a Kyoritsu University of Pharmacy; 1–5–30 Shibakoen, Minato-ku, Tokyo 105–8512, Japan: b Mitsubishi Kagaku Institute
c
of Life Sciences (MITILS); 11 Minamiooya, Machida, Tokyo 194–8511, Japan: and Oriental Medicine Research Center,
Kitasato Institute; 5–9–1 Shiroganedai, Minato-ku, Tokyo 108–8642, Japan.
Received October 12, 2005; accepted December 28, 2005
Stereocontrolled syntheses of model compounds related to a major antigenic epitope against antibupleurum
2IIc/PG-1-IgG from antiulcer pectic polysaccharide are described. A trisaccharide derivative (13) was prepared
as a precursor and a novel and simple approach for the rational design of a glycocluster and glycodendrimer was
developed, through the syntheses of the fluorescence-labeled glycocluster (2) and glycodendrimer (3).
Key words Bupleurum falcatum; glycocluster; glycodendrimer; b-alanine derivative; chemical synthesis
The roots of Bupleurum falcatum L. (Japanese name the presence of the antipolysaccharide antibody (antibup-
Saiko) have been used in Chinese and Japanese herbal medi- leuran 2IIc/PG-1-IgG). The mitogenic activity of bupleuran
cines for the treatment of chronic hepatitis, nephrosis syn- 2IIc was reduced with the addition of b-D-GlcAp-(1→6)-
drome, and autoimmune diseases. Yamada et al.1) and Sun et b-D-Galp-(1→6)-b-D-Galp or b-D-GlcAp-(1→6)-b-D-Galp,
al.2) reported that a potent antiulcer pectic polysaccharide which are a part of the epitopes of antibupleuran 2IIc/PG-1-
(Bupleuran 2IIc) was isolated from the hot-water extract of IgG.6) The proposed structure of the antigenic epitopes in
the roots. Bupleuran 2IIc consists of a galacturonan region, a PG-1 has been a target for the synthetic studies in our labora-
“ramified” region (PG-1) composed of a rhamnogalacturonan tory. Despite the specificity of the binding, it is known that
core with neutral sugar side chains, and a rhamnogalacturo- polysaccharide chains generally interact with their protein re-
nan II-like region3); the ramified region has been considered ceptors as a natural cluster. This explains why the binding
important for the expression of immunopharmacologic activ- affinity of a synthetic model compound to the active site is
ity (Fig. 1). We reported in our previous paper4) that the syn- low in various cases.7) Construction of a glycocluster aimed
thetic model compound shows specific activity. On the other at their bioactive augmentation is an important problem in
hand, a polyclonal antibody (antibupleuran 2IIc/PG-1-IgG) glycoscience.8) For this reason, we synthesized trivalent ana-
against the ramified region of bupleuran 2IIc (antibupleuran logue mono and trivalent analogues of b-D-GlcA4Me-(1→6)-
2IIc/PG-1-IgG) was prepared, and the antigenic epitopes b-D-Gal- and b-D-GlcA4Me-(1→6)-b-D-Gal-(1→6)-b-D-
were characterized to be 6-linked galactosyl chains with ei- Gal- in the hope of achieving a cluster effect.9) However, it
ther GlcA or 4-O-Me-GlcA as a nonreducing terminal.5) Bu- did not led to a marked augmentation (data not shown).
pleuran 2IIc has mitogenic activity in the murine spleen and Meanwhile, we developed new peptidic glycoclusters and a
Peyer’s patch cells, and the mitogenic activity was reduced in glycodendron, which consist of a b-alanine derivative and
Fig. 1. Structural Model of Bupleuran 2IIc and Its “Ramified” Region (PG-1)
∗ To whom correspondence should be addressed. e-mail: takeda-td@kyoritsu-ph.ac.jp
© 2006 Pharmaceutical Society of Japan

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Vol. 54, No. 4
Fig. 2. Structure of Synthetic Glycocluster and Glycodendron
Reagents: (a) NIS, TfOH, EtCN, MSAW-300; (b) TsOH, CHCl3–MeOH (2 : 1); (c) TMSOTf, MS4A, CH2Cl2; (d) i) H2, Pd–C, MeOH–THF
(2 : 1), ii) Ac2O, Pyr.; (e) Zn–AcOH
Chart 1
sugar unit.10—12) We report here the two types of cluster, 2 iodosuccinimide (NIS) and trifluoromethanesulfonic acid
and 3, carrying trisaccharide 1 (Fig. 2) and our attempts to (TfOH) in propionitrile (EtCN).14,15) Stereochemical control
achieve successful augmentation through the cluster effect.
was achieved by the solvent effect of nitrile16) to give the de-
sired b-glycoside 6 in 81% yield, and the a-glycoside was
not detected. The anomeric hydrogen atom of the galactose
Result and Discussion
Synthesis of Monovalent Trisaccharide Preparation of unit appeared as a signal at d 4.29 (d, Jꢀ7.9 Hz). Removal of
the designed trisaccharide derivative 1 was straightforward the tert-butyldimethylsilyl (TBDMS) group was achieved by
(Chart 1). Monosaccharide derivative 6 was obtained by the treatment of 6 with TsOH, giving the monosaccharide in-
condensation of phenyl 2,3,4-tri-O-benzyl-6-O-tert-butyl- termediate 7 quantitatively. The coupling reaction of 7 with 4
dimethylsilyl-1-thio-b-D-galactopyranoside (4), which was was carried out as described for the synthesis of 6 and gave
prepared by silylation and benzylation of phenyl-1-thio-b-D- compound 8. Compound 8 was formed as a mixture of
galactopyranoside,13) with the spacer 5 in the presence of N- anomers (70%) but could not be purified by silica gel column

April 2006
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Reagents: (a) dansyl glycine, DEPC, Et3N, DMF; (b) NaOMe, MeOH-1,4-dioxane; (c) 15, DEPC, Et3N, DMF; (d) Zn–AcOH; (e) 13, DEPC,
Et3N, DMF; (f) 50% TFA; (g) 17, DEPC, Et3N, DMF
Chart 2
chromatography. The structure of 8 was confirmed after re- ence of DEPC. Complete removal of the O-acyl groups and
moval of the TBDMS group (9, 75%; a : bꢀ1 : 7). The gly- esters provided the target compound 2 in 94% yield (Chart
cosylation of the acceptor 9b with the donor 109) was accom- 2).
plished using trimethylsilyl triflate (TMSOTf) and 4A MS in
Synthesis of a Glycodendrimer We chose the conver-
dichloromethane for 1 h at 0 °C, yielding the desired disac- gent approach using the bifunctional linker 2511) as the den-
1
charide 11 (91%), as evidenced by H-NMR spectroscopy dron core. In this approach, a coupling reaction of 13 with
(H-1ꢁ, 4.72 ppm, Jꢀ7.9 Hz). Removal of the benzyl groups dendron core 25 was carried out in the presence of DEPC to
from 11 by catalytic hydrogenolysis over 10% Pd–C in give 18 in 94% yield, which after removal of the Boc group
THF–MeOH and subsequent acetylation gave compound 12 gave dimer 19 (87%). Coupling of two equivalents of 19 with
(72%). Selective removal of the Troc group from 12 with 25 under the same conditions gave tetramer 26 in 86% yield.
Zn–AcOH gave the primary amine 13 (77%) (Chart 1). Com- The Boc group of 26 was removed to give amine 27 (76%),
pound 13 was condensed with dansyl glycine in the presence which was then treated with dansyl glycine in the presence of
of DEPC to give 14 (68%). The removal of all acyl groups DEPC to give 28 (73%). Finally, complete deacylation and
and esters with sodium methoxide afforded the monovalent the hydrolysis of methyl esters afforded the target compound
trisaccharide 1 in 83% yield (Chart 2).
3 in 80% yield (Chart 3).
Synthesis of a Glycocluster We first synthesized the
Structural Analysis of 2 and 3 The synthesized com-
conventional unit 16 from 15 and 13 to simplify the process. pounds 2 (glycocluster) and 3 (glycodendrimer) have the
The b-alanine derivative 15 was prepared according to the same molecular formula with Mw of 3148.24 but differ in
previously reported method.10) Elongation of the glycocluster the arrangement of the peptoide scaffolds. Collision-induced
was achieved by the iterative reactions of 1) peptide cou- dissociation (CID) experiments with 2 and 3 gave distinct
pling, 2) deprotection of the t-butoxycarbonyl (Boc) group, spectra showing characteristic daughter ions. The following
and 3) deprotection of the trichloroethyl ester (Tce) group. is a summary of CID MS/MS data obtained in the negative
Coupling of unit 15 with the sugar unit 13 in the presence of mode. Both parent ions (2P and 3P) were observed as
diethyl phosphorocyanidate (DEPC) in dry DMF gave the [Mꢂ4HꢃꢃNaꢃ]3ꢂ (m/zꢀ1055.7) (Tables 1, 2). One of the
2
glycocluster unit 16 in 92% yield. Subsequent removal of the daughter ions, D4 (m/zꢀ1094.9), observed for compound 2
Tce group with Zn–AcOH afforded 17 (80%). Coupling of consists of three trisaccharide units, which is not present in
17 with 13 gave the dimer derivative 18 in 89% yield. The compound 3, and thus it clearly explains the structure of 2.
Boc group of 18 was removed under acidic conditions with Also, the ion was found to be a major peak in the spectrum.
50% TFA, giving compound 19 (88%), which was subse- Other ions listed in the tables support both of the individual
quently subjected to the next cycle of elongation to give the structures. Additionally, interesting information was obtained
desired tetramer glycocluster derivative 23 (66%). Finally, in the experiments. Nitrogen atoms constituting dialkylated
dansyl glycine was introduced into tetramer 23 in the pres- amides are present in compounds 2 and 3 where one of the

488
Vol. 54, No. 4
Reagents: (a) 25, DEPC, Et3N, DMF; (b) 50% TFA; (c) 25, DEPC, Et3N, DMF; (d) dansyl glycine, DEPC, Et3N, DMF; (e) NaOMe, MeOH-1,4–dioxane
Chart 3
Table 1. Structural Analysis of Compound 2 by CID MS/MS
Ionic structure
Fomula
Exact mass
m/z (calculated)
m/z (observed)
2P
[M(2)ꢂ4HꢃꢃNaꢃ]3ꢂ
[F1ꢂHꢃ]ꢂ
[C129H205N9NaO77S]3ꢂ
[C28H46NO18]ꢂ
3167.2
684.3
1425.6
977.4
2189.8
2482.4
1448.6
1741.6
1055.7
684.3
712.8
1055.8
684.3
712.7
2D1
2D2
2D3
2D4
2D5
2D6
2D7
[F2ꢂ2Hꢃ]2ꢂ
[C58H95N3O37]2ꢂ
[F3ꢂHꢃ]ꢂ
[C41H61N4O21S]ꢂ
977.4
977.4
[F4ꢂ3HꢃꢃNaꢃ]2ꢂ
[F5ꢂ3HꢃꢃNaꢃ]2ꢂ
[F2ꢂ2HꢃꢃNaꢃ]ꢂ
[F6ꢂ2HꢃꢃNaꢃ]ꢂ
[C88H144N5NaO56]2ꢂ
[C101H157N8NaO59S]2ꢂ
[C58H95N3NaO37]ꢂ
[C71H110N6NaO40S]ꢂ
1094.9
1241.2
1448.6
1741.6
1094.9
1241.3
1448.6
1741.8
2P, parent ion; 2Dn, daughter ion of a fragment (Fn) from 2. 2D1 and 2D5 share a fragment structure but differ in the state of acid moieties.
alkyl groups corresponds to a substituted 3-carbonylethyl tively charged ion species were obtained. The CID MS/MS
group and the other corresponds to a substituted 2-carbonyl- of such ions resulted in preferential cleavages of the glyco-
methyl group. The former linkage tends to cleave under sidic linkages rather than the peptoide linkages (data not
given CID conditions in both 2 and 3, whereas no cleavage shown). These results indicate that the bond energies associ-
was observed for the latter. Furthermore, when the samples ated with a given ion depend very heavily on the charge state
were ionized from solutions containing formic acid, posi- of the ion. Based on the CID MS/MS results of compounds 2

April 2006
489
Table 2. Structural Analysis of Compound 3 by CID MS/MS
Ionic structure
Fomula
Exact mass
m/z (calculated)
m/z (observed)
3P
[M(3)ꢂ4HꢃꢃNaꢃ]3ꢂ
[F1ꢂHꢃ]ꢂ
[C129H205N9NaO77S]3ꢂ
[C28H46NO18]ꢂ
3167.2
684.3
1055.7
684.3
712.8
1055.7
684.1
712.7
3D1
3D2
3D3
3D4
3D5
3D6
3D7
[F2ꢂ2Hꢃ]2ꢂ
[C58H95N3O37]2ꢂ
1425.6
2932.1
2482.4
1394.5
1448.6
1741.6
[F3ꢂ4HꢃꢃNaꢃ]3ꢂ
[F4ꢂ3HꢃꢃNaꢃ]2ꢂ
[F5ꢂ2HꢃꢃNaꢃ]ꢂ
[F2ꢂ2HꢃꢃNaꢃ]ꢂ
[F6ꢂ2HꢃꢃNaꢃ]ꢂ
[C117H192N8NaO75]3ꢂ
[C101H157N8NaO59S]2ꢂ
[C55H93N3NaO36]ꢂ
[C58H95N3NaO37]ꢂ
[C71H110N6NaO40S]ꢂ
977.4
977.6
1241.2
1394.5
1448.6
1741.6
1241.2
1394.2
1448.3
1741.2
3P, parent ion; 3Dn, daughter ion of a fragment (Fn) from 3. 3D2 and 3D5 share a fragment structure but differ in the state of acid moieties.
CH2OH), 3.24 (2H, dd, NHCH2), 1.61—1.36 (8H, m, CH2ꢄ4). MALDI-
TOF-MS: Calcd for C9H16Cl3NO3Na: m/z 314.0 [MꢃNa]ꢃ. Found: m/z
314.3 [MꢃNa]ꢃ.
and 3, these cluster compounds are clearly a linear cluster
and a dendrimer.
In conclusion, efficient synthetic strategies in glycoconju-
gate chemistry were employed to obtain new glycoclusters.
The strategies allow changes in the length and pattern of the
core portion of the dendrimer. A variety of oligosaccharides
can be adopted in the structure. This method should find a
wide range of applications.
6-N-(2,2,2-Trichloroethoxycarbonyl)aminohexyl 2,3,4-tri-O-Benzyl-6-
O-tert-butyldimethylsilyl-b-D-galactopyranoside (6) To a solution of 4
(760 mg, 1.16 mmol) and 5 (315 mg, 1.08 mmol) in EtCN (10 ml) was added
MSAW-300 (800 mg), and the mixture was stirred for 2 h and then cooled to
ꢂ60 °C. NIS (391 mg, 1.74 mmol) and TfOH (5.2 ml, 57.9 mmol) were
added to the mixture, which was stirred for 1 h at ꢂ60 °C and then neutral-
ized with Et3N. The solids were filtered off and washed with CHCl3. The
combined filtrate and washings were successively washed with saturated
Na2S2O3 and water, dried (MgSO4), and concentrated. The product was puri-
fied on silica gel column chromatography (hexane : ethyl acetateꢀ8 : 1) to
Experimental
Optical rotations were determined with a Jasco digital polarimeter. 1H-
and 13C-NMR spectra were recorded with a JNM A 500 FT NMR spectrom-
eter with Me4Si as the internal standard for solutions in CDCl3 or CD3OD.
MALDI-TOF-MS was recorded on a Perceptive Voyager RP mass spectrom-
eter. ESI-QIT mass spectra were obtained using a Bruker Esquire 3000 plus.
TLC was performed on silica gel 60-F254 (Merck) with detection by
quenching of UV fluorescence and by spraying with 5% ninhydrin and 10%
H2SO4. Column chromatography was carried out on silica gel 60 (Merck).
Phenyl 2,3,4-tri-O-benzyl-6-O-tert-butyldimethylsilyl-1-thio-b-D-ga-
lactopyranoside (4) To a solution of phenyl 6-O-tert-butyldimethylsilyl-1-
thio-b-D-galactopyranoside (4 g, 10.4 mmol) in DMF (10 ml) was added
NaH in oil (2.5 g, 62.2 mmol) and BnBr (7.4 ml, 62.2 mmol). The reaction
mixture was stirred for 2 h at 0 °C, and then methanol was added to elimi-
nate excess NaH. The reaction mixture was poured into ice water and ex-
tracted with ethyl acetate. The extract was washed with water, dried
(MgSO4), and concentrated. The product was purified on silica gel column
chromatography (hexane : ethyl acetateꢀ20 : 1) to give 4 (4.9 g, 72%). [a]2D3
ꢃ6.3° (cꢀ5.0, CHCl3). 1H-NMR (500 MHz, CDCl3): d 7.58—7.18 (15H, m,
Ar-H), 4.99 (1H, d, H-1), 4.79—4.62 (6H, m, benzylmethylene), 3.97—3.93
(2H, m, H-2, H-4), 3.77—3.71 (2H, m, H-6a, H-6b), 3.61 (1H, dd, H-3),
3.45 (1H, t, H-5). 13C-NMR (125 MHz, CDCl3): d 138.9, 138.4, 138.3,
134.3, 128.7, 128.4, 128.3, 128.1, 127.7, 127.61, 127.58, 127.3, 126.9, 87.7,
84.2, 78.9, 75.6, 74.4, 73.5, 72.8, 61.5, 18.2. MALDI-TOF-MS: Calcd for
C39H48Cl5NO8SSiNa: m/z 679.3 [MꢃNa]ꢃ. Found: m/z 679.4 [MꢃNa]ꢃ.
N-(2,2,2-Trichloroethoxycarbonyl)hexanolamine (5) To a solution of
2,2,2-trichloroethylchloroformate (4.7 ml, 0.03 mol) in 5 ml of dioxane was
added at 0 °C a mixture of hexanolamine (5 g, 0.04 mol) and MgO (3 g) in
dioxane (25 ml) and H2O (25 ml). The reaction mixture was stirred for 16 h
at room temperature. Then, ethylacetate was added, the solids were filtered
off and washed with 5% HCl, aqueous NaHCO3, and water, dried (MgSO4),
and concentrated. The product was purified on silica gel column chromatog-
raphy (chloroform : methanolꢀ200 : 1) to give 5 (8 g, 64.3%). 1H-NMR
(500 MHz, CDCl3): d 5.03 (1H, s, NH), 4.72 (2H, s, CH2CCl3), 3.65 (2H, t,
1
give 6 (736 mg, 81.2%). [a]2D3 ꢃ4.5° (cꢀ0.7, CHCl3). H-NMR (500 MHz,
CDCl3): d 4.92—4.58 (8H, m, benzyl methyleneꢄ3, COOCH2CCl3), 4.29
(1H, d, Jꢀ7.9 Hz, H-1), 3.88, 3.48—3.41 (3H, m, H-3, OCH2), 3.82 (1H, d,
H-4), 3.76 (1H, dd, H-2), 3.65 (2H, m, H-6a, 6b), 3.32 (1H, t, H-5), 3.13
(2H, dd, NHCH2). 13C-NMR (125 MHz, CDCl3): d 104.0, 95.8, 82.2, 79.7,
75.2, 75.1, 74.6, 74.5, 73.7, 73.4, 73.1, 69.7, 61.7. MALDI-TOF-MS: Calcd
for C42H58Cl3NO8SiNa: m/z 860.3 [MꢃNa]ꢃ. Found: m/z 860.2 [MꢃNa]ꢃ.
6-N-(2,2,2-Trichloroethoxycarbonyl)aminohexyl 2,3,4-tri-O-Benzyl-b-
D-galactopyranoside (7) To a solution of 6 (1.1 g, 1.31 mmol) in 2 : 1
CHCl3–MeOH (12 ml) was added p-toluenesulfonic acid (113 mg). The re-
action mixture was stirred for 1 h at room temperature. After completion
of the reaction and neutralization with Et3N, the mixture was concentrat-
ed and purified on silica gel column chromatography (toluene : acetoneꢀ
10 : 1) to give 7 (983 mg, quantitative). [a]2D3 ꢃ2.7° (cꢀ1.0, CHCl3). 1H-
NMR (500 MHz, CDCl3): d 4.97—4.65 (8H, m, benzyl methyleneꢄ3,
COOCH2CCl3), 4.35 (1H, d, H-1), 3.91—3.48 (7H, m, H-4, 2, 3, 6a, 6b,
OCH2), 3.37 (1H, t, H-5), 3.17 (2H, dd, NHCH2). MALDI-TOF-MS: Calcd
for C36H44Cl3NO8Na: m/z 746.2 [MꢃNa]ꢃ. Found: m/z 746.4 [MꢃNa]ꢃ.
6-N-(2,2,2-Trichloroethoxycarbonyl)aminohexyl 2,3,4-tri-O-benzyl-6-
O-tert-butyldimethylsilyl-a/b-D-galactopyranosyl)-(1→6)-2,3,4-tri-O-
benzyl-b-D-galactopyranoside (8) To a solution of 4 (156 mg, 0.24 mmol)
and 7 (143 mg, 0.20 mmol) in EtCN (1.5 ml) was added MSAW-300
(200 mg), and the mixture was stirred for 2 h and then cooled to ꢂ60 °C.
NIS (80 mg, 0.36 mmol) and TfOH (2.1 ml, 23.4 mmol) were added to the
mixture, which was stirred for 1 h, cooled to ꢂ60 °C, and then neutralized
with Et3N. The solids were filtered off and washed with CHCl3. The com-
bined filtrate and washings were successively washed with saturated
Na2S2O3 and water, then dried (MgSO4) and concentrated. The product was
purified on silica gel column chromatography (hexane : ethyl acetateꢀ5 : 1)
to give 8 as a mixture of anomers (177 mg, 70.0%). MALDI-TOF-MS:
Calcd for C69H86Cl3NO13SiNa: m/z 1292.5 [MꢃNa]ꢃ. Found: m/z 1292.7

490
Vol. 54, No. 4
[MꢃNa]ꢃ.
was extracted with CHCl3, washed with water, dried (MgSO4), and concen-
trated. The product was purified on silica gel column chromatography
(CHCl3 : MeOHꢀ50 : 1) to give the dansyl derivative 14 (11.6 mg, 68.1%).
[a]2D3 ꢃ8.2° (cꢀ0.3, CHCl3). 1H-NMR (500 MHz, CDCl3): d 8.58—7.20
(16H, m, C10H6 of dansyl glycine, Ar-H), 6.40 (1H, t, NH), 5.73 (1H, t,
NHCH2CO of dansyl glycine), d 5.60 (1H, t, H-3ꢁ), 5.36—5.30 (3H, m, H-
2ꢁ, H-4, H-4ꢅ), 5.20—5.09 (2H, dd, dd, H-2, H-2ꢅ), 5.03—4.91 (2H, br dd,
H-3, H-3ꢅ), 4.77 (1H, d, H-1ꢁ), 4.49—4.45 (2H, t, H-1, H-1ꢅ), 4.09 (1H, d,
H-5ꢁ), 3.97—3.38 (17H, m, H-4ꢁ, H-5, H-5ꢅ, H-6a, H-6b, H-6aꢅ, H-6bꢅ,
COOCH3, NHCH2CO of dansyl glycine, OCH2 of sugar unit, OCH3), 3.09
(2H, s, NCH2 of sugar unit), 2.89 (6H, s, N(CH3)2 of dansyl glycine), 2.10—
1.95 (18H, m, COOCH3ꢄ6), 1.66 [8H, s, (CH2)ꢄ4]. 13C-NMR (125 MHz,
CDCl3): d 170.2, 170.1, 170.3, 169.7, 169.4, 168.5, 167.7, 165.5, 165.0,
152.3, 133.4, 133.3, 131.1, 130.2, 130.0, 129.8, 129.5, 129.2, 129.1, 128.9,
128.5, 123.2, 118.2, 115.4, 101.2, 101.0, 100.7, 78.7, 74.2, 74.1, 72.05,
71.97, 71.8, 71.1, 70.9, 69.9, 69.2, 68.8, 67.6, 67.4, 66.9, 60.4, 52.8, 45.9,
45.4, 39.3, 29.7, 29.1, 29.0, 26.4, 25.6, 20.8, 20.74, 20.66, 20.59. MALDI-
TOF-MS: Calcd for C66H81N3O28SNa: m/z 1418.5 [MꢃNa]ꢃ. Found: m/z
1418.6 [MꢃNa]ꢃ.
Compound 1 To a solution of compound 14 (11.0 mg, 11.9 mmol) in
1 : 5 MeOH–H2O (1.2 ml) was added NaOMe (30 mg), and the mixture was
stirred for 14 h at room temperature and then neutralized with Amberlite IR-
120 (Hꢃ) resin. The resin was filtered off and washed with MeOH–H2O. The
filtrate and washings were combined and concentrated. Column chroma-
tography (MeOH : H2Oꢀ3 : 1) of the residue on Sephadex LH-20 gave 1
(6 mg, 82.7%). [a]2D3 ꢂ19.9° (cꢀ0.2, H2O). 13C-NMR (125 MHz, 1 : 1
CD3OD–D2O): d 152.8, 135.1, 131.7, 130.9, 130.6, 130.4, 130.1, 124.9,
120.0, 117.0, 104.8, 104.3, 104.0, 83.5, 77.8, 76.7, 75.0, 74.9, 74.5, 74.2,
74.1, 72.1, 71.4, 70.2, 70.1, 69.91, 69.88, 61.0, 50.0, 46.3, 46.1, 40.2, 30.1,
29.3, 27.1, 26.1. MALDI-TOF-MS: Calcd for C39H59N3O20SNa: m/z 944.3
[MꢃNa]ꢃ. Found m/z 944.3 [MꢃNa]ꢃ.
Compound 16 To a solution of 13 (120 mg, 0.11 mmol) and b-alanine
derivative 15 (49 mg, 0.13 mmol) in DMF (2 ml) were added triethylamine
(27 ml, 0.02 mmol) and DEPC (22 ml, 0.02 mmol). The reaction mixture was
stirred for 16 h at room temperature. After completion of the reaction, the
mixture was extracted with chloroform, washed with water, dried (MgSO4),
and concentrated. The product was purified on silica gel column chromatog-
raphy (toluene : acetoneꢀ3 : 1) to give 16 (147 mg, 92.4%). [a]2D3 ꢃ2.4°
(cꢀ1.1, CHCl3). 1H-NMR (500 MHz, CDCl3): d 7.99—7.38 (10H, m, Ar-
H), 6.28 (1H, s, NH), 5.60 (1H, t, H-3ꢁ), 5.36—5.29 (3H, m, H-2ꢁ, H-4, H-
4ꢅ), 5.19—5.10 (2H, dd, dd, H-2, H-2ꢅ), 5.01—4.92 (2H, br dd, H-3, H-3ꢅ),
4.77 (1H, d, H-1ꢁ), 4.74 (2H, s, Tce), 4.45—4.43 (2H, br d, br d, H-1, H-1ꢅ),
4.08 (1H, d, H-5ꢁ), 3.97—3.40 (19H, m, H-4ꢁ, H-5, H-5ꢅ, H-6a, H-6b, H-
6aꢅ, H-6bꢅ, COOCH3, NCH2CO of b-alanine, OCH2 of sugar unit, NCH2 of
b-alanine, OCH3), 3.24 (2H, s, NCH2 of sugar unit), 2.77 (2H, t, COCH2 of
b-alanine), 2.10—1.96 (18H, m, COOCH3ꢄ6), 1.77—1.33 [17H, m, t-Bu,
(CH2)ꢄ4]. 13C-NMR (125 MHz, CDCl3): d 170.1, 170.03, 169.92, 169.4,
169.3, 169.2, 168.4, 165.4, 164.9, 137.8, 133.3, 129.7, 129.1, 129.0, 128.4,
128.2, 125.2, 101.2, 100.9, 100.6, 94.7, 81.1, 78.6, 74.1, 72.0, 71.9, 71.7,
71.0, 70.8, 69.9, 69.0, 68.7, 67.4, 67.3, 67.2, 66.7, 60.4, 52.7, 52.5, 44.9,
39.3, 33.2, 33.1, 29.5, 29.4, 29.3, 29.2, 28.4, 28.2, 26.6, 25.5, 21.4, 20.7,
20.7, 20.6, 20.54, 20.51. MALDI-TOF-MS: Calcd for C64H83Cl3N2O30Na:
m/z 1487.4 [MꢃNa]ꢃ. Found: m/z 1487.8 [MꢃNa]ꢃ.
6-N-(2,2,2-Trichloroethoxycarbonyl)aminohexyl 2,3,4-tri-O-Benzyl-
a/b-D-galactopyranosyl)-(1→6)-2,3,4-tri-O-benzyl-b-D-galactopyra-
noside (9) To a solution of 8 (234 mg, 0.18 mmol) in 2 : 1 CHCl3–MeOH
(3 ml) was added p-toluenesulfonic acid (80 mg). The reaction mixture was
stirred for 1 h at room temperature. After completion of the reaction and
neutralization with Et3N, the mixture was concentrated and purified on silica
gel column chromatography (toluene : acetoneꢀ10 : 1) to give 9a (23 mg,
10.8%) and 9b (159 mg, 74.7%). 9a: 1H-NMR (500 MHz, CDCl3): d 4.98—
4.57 (15H, m, benzyl methyleneꢄ6, COOCH2CCl3, H-1ꢅ), 4.31 (1H, d,
J1,2ꢀ7.5, H-1), 4.06—3.40 (14H, m, H-2, H-2ꢅ, H-3, H-3ꢅ, H-4, H-4ꢅ, H-5,
H-5ꢅ, H-6a, H-6b, H-6aꢅ, H-6bꢅ, OCH2), 3.16 (2H, dd, NHCH2). 9b: [a]2D3
ꢃ2.7° (cꢀ1.0, CHCl3). 1H-NMR (500 MHz, CDCl3): d 4.95—4.63 (14H, m,
benzyl methyleneꢄ6, COOCH2CCl3), 4.35, 4.28 (2H, d, d, H-1, H-1ꢅ),
3.84—3.45 (12H, m, H-2, H-2ꢅ, H-3, H-3ꢅ, H-4, H-4ꢅ, H-6a, H-6b, H-6aꢅ,
H-6bꢅ, OCH2), 3.35 (2H, m, H-5, 5ꢅ), 3.12 (2H, dd, NHCH2). MALDI-TOF-
MS: Calcd for C63H72Cl3NO13Na: m/z 1178.4 [MꢃNa]ꢃ. Found: m/z 1178.7
[MꢃNa]ꢃ.
6-N-(2,2,2-Trichloroethoxycarbonyl)aminohexyl [methyl(2,3-di-O-ben-
zoyl-4-O-methyl-b-D-glucopyranosyl)uronate]-(1→6)-2,3,4-tri-O-benzyl-
b-D-galactopyranosyl)-(1→6)-2,3,4-tri-O-benzyl-b-D-galactopyranoside
(11) To a solution of 9b (159 mg, 0.14 mmol) and 109) (95 mg, 0.17
mmol) in CH2Cl2 (2 ml) was added MS4A (500 mg), and the mixture was
stirred for 2 h at 0 °C. TMSOTf (3 ml, 16.6 mmol) was added to the mixture,
which was stirred for 1 h at 0 °C and then neutralized with Et3N. The solids
were filtered off and washed with CHCl3. The combined filtrate and wash-
ings were successively washed with water, dried (MgSO4), and concentrated.
The product was purified on silica gel column chromatography
(toluene : acetoneꢀ15 : 1) to give 11 (196 mg, 90.9%). [a]2D3 ꢃ24.5° (cꢀ0.5,
CHCl3). 1H-NMR (500 MHz, CDCl3): d 5.63 (1H, t, H-3ꢁ), 5.35 (3H, m, H-
2ꢁ), 4.72 (1H, d, Jꢀ7.9 Hz, H-1ꢁ), 4.70 (2H, s, –COOCH2Cl3), 4.29—4.25
(2H, d, H-1, H-1ꢅ), 3.76 (3H, s, COOCH3), 3.40 (3H, s, OCH3). MALDI-
TOF-MS: Calcd for C85H92Cl3NO21Na: m/z 1590.5 [MꢃNa]ꢃ. Found: m/z
1590.8 [MꢃNa]ꢃ.
6-N-(2,2,2-Trichloroethoxycarbonyl)aminohexyl [methyl(2,3-di-O-ben-
zoyl-4-O-methyl-b-D-glucopyranosyl)uronate]-(1→6)-2,3,4-tri-O-acetyl-
b-D-galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-b-D-galactopyranoside
(12) A solution of 11 (533 mg, 0.34 mmol) in MeOH (8 ml) and THF
(4 ml) was hydrogenated over 10% Pd–C (450 mg) for 2 h at room tempera-
ture, filtered through Celite, and the residue was washed with MeOH and
concentrated. The residue was acetylated with Ac2O (4 ml) in pyridine
(6 ml) for 3 h at room temperature. The reaction mixture was poured into
ice water and extracted with CHCl3. The extract was washed sequentially
with 5% HCl, aqueous NaHCO3, and water, dried (MgSO4), and concen-
trated. The product was purified on silica gel column chromatography
(toluene : acetoneꢀ5 : 1) to give 12 (311 mg, 71.5%). [a]2D3 ꢃ3.2° (cꢀ0.5,
CHCl3). 1H-NMR (500 MHz, CDCl3): d 5.60 (1H, t, H-3ꢁ), 5.36—5.29 (3H,
m, H-2ꢁ, H-4, H-4ꢅ), 5.19—5.10 (2H, dd, dd, H-2, H-2ꢅ), 5.01—4.92 (2H,
br dd, H-3, H-3ꢅ), 4.77 (1H, d, H-1ꢁ), 4.72 (2H, s, –COOCH2Cl3), 4.44 (2H,
d, H-1, H-1ꢅ), 4.08 (1H, d, H-5ꢁ), 3.97—3.40 (15H, m, H-4ꢁ, H-5, H-5ꢅ, H-
6a, H-6b, H-6aꢅ, H-6bꢅ, COOCH3, OCH2 of sugar unit, OCH3), 3.20 (2H, s,
NCH2 of sugar unit). 13C-NMR (125 MHz, CDCl3): d 101.2, 101.0, 100.7,
78.7, 74.2, 72.1, 72.0, 71.8, 71.1, 70.9, 69.9, 69.1, 68.7, 67.5, 67.40, 67.39,
66.8. MALDI-TOF-MS: Calcd for C55H68Cl3NO27Na: m/z 1302.3 [MꢃNa]ꢃ.
Found: m/z 1302.0 [MꢃNa]ꢃ.
Compound 17 To a solution of 16 (147 mg, 0.10 mmol) in acetic acid
(2 ml) was added zinc powder. The reaction mixture was stirred for 1 h at
room temperature. After completion of the reaction (TLC monitoring), the
mixture was filtered through Celite. The filtrate was concentrated and puri-
fied on silica gel column chromatography (chloroform : methanolꢀ30 : 1) to
6-Aminohexyl [methyl(2,3-di-O-benzoyl-4-O-methyl-b-D-glucopyra-
nosyl)uronate]-(1→6)-2,3,4-tri-O-acetyl-b-D-galactopyranosyl)-(1→6)-
2,3,4-tri-O-acetyl-b-D-galactopyranoside (13) To
a solution of 12
give 17 (107 mg, 79.8%). [a]2D3 ꢃ1.7° (cꢀ1.9, CHCl3) H-NMR (500 MHz,
1
(292 mg, 0.23 mmol) in acetic acid (6 ml) was added zinc powder (500 mg).
The reaction mixture was stirred for 16 h at room temperature. After com-
pletion of the reaction (TLC monitoring), the mixture was filtered off and
washed with CHCl3. The filtrate was concentrated and purified on silica gel
column chromatography (chloroform : methanolꢀ10 : 1) to give 13 (194 mg,
CDCl3): 7.99—7.38 (10H, m, Ar-H), 6.59 (1H, s, NH), 5.61 (1H, t, H-3ꢁ),
5.36—5.29 (3H, m, H-2ꢁ, H-4, H-4ꢅ), 5.19—5.09 (2H, dd, dd, H-2, H-2ꢅ),
5.03—4.93 (2H, br d, H-3, H-3ꢅ), 4.77 (1H, d, H-1ꢁ), 4.46—4.44 (2H, br d,
br d, H-1, H-1ꢅ), 4.09 (1H, d, H-5ꢁ), 3.97—3.40 (19H, m, H-4ꢁ, H-5, H-5ꢅ,
H-6a, H-6b, H-6aꢅ, H-6bꢅ, COOCH3, NCH2CO of b-alanine, OCH2 of sugar
unit, NCH2 of b-alanine, OCH3), 3.30—3.23 (2H, m, NCH2 of sugar unit),
2.57 (2H, t, COCH2 of b-alanine), 2.10—1.96 (18H, m, OAcꢄ6) 1.77—1.33
[17H, m, t-Bu, (CH2)ꢄ4]. 13C-NMR (125 MHz, CDCl3): d 170.1, 168.5,
165.5, 165.0, 133.3, 129.8, 129.1, 128.4, 101.2, 100.9, 100.6, 78.6, 74.1,
72.0, 71.8, 70.98, 70.90, 70.0, 69.2, 68.7, 67.5, 67.3, 66.8, 60.4, 52.8,
29.2, 28.1, 26.5, 25.5, 20.8, 20.7, 20.6. MALDI-TOF-MS: Calcd for
C62H82N2O30Na: m/z 1357.5 [MꢃNa]ꢃ. Found: m/z 1358.2 [MꢃNa]ꢃ.
Compound 18 To a solution of 17 (49 mg, 36.7 mmol) and 13 (41 mg,
37.1 mmol) in DMF (2 ml) were added triethylamine (7.7 ml, 40.4 mmol)
and DEPC (6.1 ml, 40.4 mmol). The reaction mixture was stirred for 16 h at
76.9%). [a]23 ꢂ2.8° (cꢀ0.2, CHCl3). H-NMR (500 MHz, CDCl3): d 5.61
1
.D
(1H, t, H-3ꢁ), 5.36—5.30 (3H, m, H-2ꢁ, H-4, H-4ꢅ), 5.17—5.08 (2H, dd, dd,
H-2, H-2ꢅ), 5.02—4.93 (2H, br dd, H-3, H-3ꢅ), 4.78 (1H, d, H-1ꢁ), 4.47—
4.45 (2H, d, H-1, H-1ꢅ), 4.10 (1H, d, H-5ꢁ), 3.96—3.40 (15H, m, H-4ꢁ, H-5,
H-5ꢅ, H-6a, H-6b, H-6aꢅ, H-6bꢅ, COOCH3, OCH2 of sugar unit, OCH3), 2.95
(2H, s, NCH2 of sugar unit). MALDI-TOF-MS: Calcd for C52H67NO25Na:
m/z 1128.4 [MꢃNa]ꢃ. Found: m/z 1129.1 [MꢃNa]ꢃ.
Compound 14 To a solution of 13 (13.5 mg, 12.7 mmol) and dansyl
glycine (5.7 mg, 18.3 mmol) in DMF (1 ml) were added triethylamine (2.6 ml,
18.3 mmol) and DEPC (1.1 ml, 13.4 mmol). The reaction mixture was stirred
for 16 h at room temperature. After completion of the reaction, the mixture

April 2006
491
room temperature. After completion of the reaction, the mixture was ex- 169.60, 169.57, 169.3, 168.5, 168.4, 165.5, 164.9, 133.33, 133.28, 131.77,
tracted with chloroform, washed with water, dried (MgSO4), and concentrat- 129.8, 129.1, 128.4, 101.7, 101.1, 100.5, 93.3, 78.6, 75.6, 74.1, 73.3, 72.0,
ed. The product was purified on silica gel column chromatography (chloro- 71.9, 71.7, 71.0, 70.9, 70.3, 69.93, 69.89, 69.2, 68.7, 67.5, 67.3, 66.9, 66.8,
form : methanolꢀ40 : 1) to give 18 (80 mg, 89.3%). [a]2D3 ꢃ4.5° (cꢀ0.7,
66.5, 65.9, 60.0, 59.8, 52.8, 39.5, 29.7, 29.3, 29.2, 26.6, 25.6, 20.8, 20.7,
CHCl3). 1H-NMR (500 MHz, CDCl3): d 7.99—7.38 (20H, m, Ar-H), 5.60 20.62, 20.56. MALDI-TOF-MS: Calcd for C166H212N5O79Na: m/z 3562.3
(2H, t, H-3ꢁꢄ2), 5.36—5.29 (6H, m, H-2ꢁꢄ2, H-4ꢄ2, H-4ꢁꢄ2), 5.18—5.09 [MꢃNa]ꢃ. Found: m/z 3562.4 [MꢃNa]ꢃ.
(4H, m, H-2ꢄ2, H-2ꢅꢄ2), 5.01—4.92 (4H, br d, H-3ꢄ2, H-3ꢅꢄ2), 4.77
Compound 22 Compound 22 was synthesized from 21 according to the
(2H, d, H-1ꢁ), 4.45—4.43 (4H, br d, br d, H-1ꢄ2, H-1ꢅꢄ2), 4.08 (2H, d, H- procedure described for the synthesis of 20. Yield: 65 mg (96.7%). [a]2D3
5ꢁꢄ2), 3.96—3.45 (28H, m, H-4ꢁꢄ2, H-5ꢄ2, H-5ꢅꢄ2, H-6aꢄ2, H-6bꢄ2, ꢃ2.6° (cꢀ0.8, CHCl3). 1H-NMR (500 MHz, CDCl3): d 7.92—7.31 (40H, m,
H-6aꢅꢄ2, H-6bꢅꢄ2, COOCH3ꢄ2, NCH2CO, NCH2 of b-alanine, OCH2 of Ar-H), 5.53 (4H, t, H-3ꢁꢄ4), 5.29—5.22 (12H, d, m, H-2ꢁꢄ4, H-4ꢄ4, H-
sugar unitꢄ2), 3.40 (6H, s, OCH3ꢄ2), 3.23—3.19 (4H, m, NCH2 of sugar 4ꢅꢄ4), 5.11—5.02 (8H, m, H-2ꢄ4, H-2ꢅꢄ4), 4.94—4.85 (8H, br d, H-3ꢄ4,
unitꢄ2), 2.40 (2H, m, COCH2 of b-alanine), 2.10—1.95 (36H, m,
OAcꢄ6ꢄ2), 1.60—1.33 [25H, m, t-Bu, (CH2)ꢄ4ꢄ2]. 13C-NMR (125 MHz,
CDCl3) d 170.2, 170.0, 169.9, 169.5, 169.3, 168.4, 165.4, 164.9, 133.33,
H-3ꢅꢄ4), 4.71 (4H, d, H-1ꢁ), 4.37 (8H, dd, H-1ꢄ4, H-1ꢅꢄ4), 4.01 (4H, d,
H-5ꢁꢄ4), 3.89—3.33 (72H, m, H-4ꢁꢄ4, H-5ꢄ4, H-5ꢅꢄ4, H-6aꢄ4, H-
6bꢄ4, H-6aꢅꢄ4, H-6bꢅꢄ4, COOCH3ꢄ4, NCH2CO of b-alanineꢄ3, NCH2
133.26, 129.8, 129.1, 128.4, 101.2, 100.9, 100.6, 80.7, 78.6, 74.1, 72.0, of b-alanineꢄ3, OCH2 of sugar unitꢄ4, OCH3ꢄ4), 3.12 (8H, m, NCH2 of
71.9, 71.8, 71.0, 70.9, 70.0, 69.1, 68.7, 67.5, 67.3, 67.2, 66.8, 60.4, 52.7,
39.6, 39.4, 29.7, 29.3, 28.2, 26.7, 26.6, 25.6, 20.8, 20.7, 20.61, 20.56.
MALDI-TOF-MS: Calcd for C114H147N3O54Na: m/z 2444.9 [MꢃNa]ꢃ.
Found: m/z 2445.4 [MꢃNa]ꢃ.
sugar unitꢄ4), 2.77—2.16 (3H, m, COCH2 of b-alanineꢄ3ꢄ1/2), 2.03—
1.76 (75H, COOCH3ꢄ6ꢄ4, COCH2 of b-alanineꢄ3ꢄ1/2), 1.48—1.19
[41H, m, t-Bu, (CH2)ꢄ4ꢄ4]. 13C-NMR (125 MHz, CDCl3): d 170.1, 170.0,
169.9, 169.5, 169.3, 168.4, 165.4, 164.9, 133.33, 133.26, 130.9, 129.8,
Compound 19 To
a solution of 18 (71 mg, 29.3 mmol) in di- 129.1, 128.8, 128.4, 101.2, 100.9, 100.7, 78.6, 74.1, 72.0, 71.9, 71.83,
chloromethane (1 ml) was added trifluoroacetic acid (1 ml). The reaction 71.75, 71.0, 70.8, 69.9, 69.1, 68.7, 67.5, 67.3, 67.2, 66.8, 66.2, 60.4, 52.7,
mixture was stirred for 1 h at room temperature. After completion of the re-
action, the mixture was concentrated and purified on silica gel column chro-
39.5, 29.6, 29.3, 29.2, 28.22, 28.19, 26.7, 25.61, 25.6, 20.8, 20.7, 20.61,
20.56. MALDI-TOF-MS: Calcd for C228H291N7O108Na: m/z 4877.7
matography (chloroform : methanolꢀ20 : 1) to give 19 (60 mg, 88.1%). [a]2D3 [MꢃNa]ꢃ. Found: m/z 4877.4 [MꢃNa]ꢃ.
ꢃ2.7° (cꢀ1.0, CHCl3). 1H-NMR (500 MHz, CDCl3): d 7.99—7.38 (20H, m,
Compound 23 Compound 23 was synthesized from 22 according to the
Ar-H), 6.22 (1H, s, NH), 5.60 (2H, t, H-3ꢁꢄ2), 5.36—5.28 (6H, m, H-2ꢁꢄ2, procedure described for the synthesis of 21. Yield: 42 mg (66.0%). [a]2D3
H-4ꢄ2, H-4ꢅꢄ2), 5.18—5.09 (4H, m, H-2ꢄ2, H-2ꢅꢄ2), 5.03—4.93 (4H, m ꢃ2.9° (cꢀ0.8, CHCl3). 1H-NMR (500 MHz, CDCl3): d 7.91—7.31 (40H, m,
H-3ꢄ2, H-3ꢅꢄ2), 4.79 (2H, d, H-1ꢁ), 4.46 (4H, br d, br d, H-1ꢄ2, H-1ꢅꢄ2), Ar-H), 5.53 (4H, t, H-3ꢁꢄ4), 5.29—5.22 (12H, d, m, H-2ꢁꢄ4, H-4ꢄ4, H-
4.09 (2H, d, H-5ꢁꢄ2), 3.97—3.40 (30H, m, H-4ꢁꢄ2, H-5ꢄ2, H-5ꢅꢄ2, H-
4ꢅꢄ4), 5.09—5.02 (8H, m, H-2ꢄ4, H-2ꢅꢄ4), 4.98—4.86 (8H, br d, H-3ꢄ4,
6aꢄ2, H-6bꢄ2, H-6aꢅꢄ2, H-6bꢅꢄ2, COOCH3ꢄ2, OCH3ꢄ2, OCH2 of H-3ꢅꢄ4), 4.71 (4H, d, H-1ꢁ), 4.38 (8H, dd, H-1ꢄ4, H-1ꢅꢄ4), 4.02 (4H,
sugar unitꢄ2), 3.26—3.21 (4H, m, NCH2 of sugar unitꢄ2), 3.01 (2H, t, d, H-5ꢁꢄ4), 3.92—3.33 (60H, m, H-4ꢁꢄ4, H-5ꢄ4, H-5ꢅꢄ4, H-6aꢄ4,
NCH2CO of b-alanine), 2.52 (2H, m, NCH2 of b-alanine), 2.10—1.95 (38H,
H-6bꢄ4, H-6aꢅꢄ4, H-6bꢅꢄ4, COOCH3ꢄ4, OCH2 of sugar unitꢄ4,
m, OAcꢄ6ꢄ2, COCH2 of b-alanine), 1.56—1.33 [16H, (CH2)ꢄ4ꢄ2]. 13C- OCH3ꢄ4), 3.10 (8H, m, NCH2 of sugar unitꢄ4), 2.10—1.89 (78H,
NMR (125 MHz, CDCl3): d 170.0, 169.6, 169.3, 168.4, 165.4, 164.9, OAcꢄ6ꢄ4, COCH2 of b-alanineꢄ3), 1.48—1.18 [32H, m, (CH2)ꢄ4ꢄ4].
133.33, 133.26, 129.8, 129.13, 129.10, 128.4, 101.2, 100.92, 100.87, 100.7, 13C-NMR (125 MHz, CDCl3): d 170.2, 170.05, 169.98, 169.6, 169.3, 168.4,
100.6, 78.6, 74.12, 74.09, 72.0, 71.93, 71.88, 71.8, 71.01, 70.98, 70.9, 70.0,
165.5, 164.9, 133.35, 133.28, 129.9, 129.7, 129.1, 128.4, 101.2, 100.9,
69.9, 69.14, 69.09, 68.7, 67.5, 67.4, 67.3, 66.9, 66.8, 60.4, 52.8, 45.4, 39.4, 100.6, 78.6, 74.12, 74.09, 72.0, 71.9, 71.7, 71.0, 70.9, 69.9, 69.1, 68.7,
39.2, 29.32, 29.28, 29.23,29.19, 26.6, 25.6, 25.5, 20.8, 20.7, 20.62, 20.56. 67.5, 67.3, 66.8, 60.4, 52.8, 39.5, 29.6, 29.3, 26.7, 25.5, 22.6, 20.8, 20.7,
MALDI-TOF-MS: Calcd for C109H140N3O52Na: m/z 2345.8 [MꢃNa]ꢃ. 20.62, 20.56. MALDI-TOF-MS: Calcd for C223H284N7O106Na: m/z 4778.7
Found: m/z 2345.5. [MꢃNa]ꢃ.
[MꢃNa]ꢃ. Found: m/z 4778.3 [MꢃNa]ꢃ.
Compound 20 To a solution of 19 (34 mg, 14.6 mmol) and 17 (59 mg,
Compound 24 Compound 24 was synthesized from 23 according to the
44.2 mmol) in DMF (2 ml) were added triethylamine (7.3 ml, 52.4 mmol) procedure described for the synthesis of 14. Yield: 28.4 mg (86.4%). [a]2D3
and DEPC (6.2 ml, 40.9 mmol). The reaction mixture was stirred for 16 h at
room temperature. After completion of the reaction, the mixture was ex- (6H, m, C10H6 of dansyl glycine), 7.91—7.31 (40H, m, Ar-H), 5.53 (4H, t,
tracted with chloroform, washed with water, dried (Na2SO4), and concentrat- H-3ꢁꢄ4), 5.29—5.21 (12H, d, m, H-2ꢁꢄ4, H-4ꢄ4, H-4ꢅꢄ4), 5.10—5.02
ed. The product was purified on silica gel column chromatography (chloro- (8H, m, H-2ꢄ4, H-2ꢅꢄ4), 5.00—4.85 (8H, br d, H-3ꢄ4, H-3ꢅꢄ4), 4.71
(4H, d, H-1ꢁ), 4.36 (8H, dd, H-1ꢄ4, H-1ꢅꢄ4), 4.02 (4H, d, H-5ꢁꢄ4), 3.90—
ꢃ1.2° (cꢀ1.0, CHCl3). 1H-NMR (500 MHz, CDCl3): d 8.47—8.16, 7.11
form : methanolꢀ30 : 1) to give 20 (66 mg, 71.4%). [a]2D3 ꢃ0.4° (cꢀ0.9,
CHCl3). 1H-NMR (500 MHz, CDCl3): d 7.99—7.38 (30H, m, Ar-H), 5.60 3.32 (72H, m, H-4ꢁꢄ4, H-5ꢄ4, H-5ꢅꢄ4, H-6aꢄ4, H-6bꢄ4, H-6aꢅꢄ4, H-
(3H, t, H-3ꢁꢄ3), 5.36—5.29 (9H, m, H-2ꢁꢄ3, H-4ꢄ3, H-4ꢅꢄ3), 5.18—5.09 6bꢅꢄ4, COOCH3ꢄ4, NCH2CO of b-alanineꢄ3, NCH2 of b-alanineꢄ3,
(6H, m, H-2ꢄ3, H-2ꢅꢄ3), 5.01—4.92 (6H, br d, H-3ꢄ3, H-3ꢅꢄ3), 4.77 OCH2 of sugar unitꢄ4, OCH3ꢄ4), 3.11 (8H, m, NCH2 of sugar unitꢄ4),
(3H, d, H-1ꢁ), 4.45—4.43 (6H, br d, br d, H-1ꢄ3, H-1ꢅꢄ3), 4.08 (3H, d, H- 2.81 (6H, s, NC2H6 of dansyl glycine), 2.64—2.24 (3H, m, COCH2 of b-
5ꢁꢄ3), 3.97—3.40 (53H, m, H-4ꢁꢄ3, H-5ꢄ3, H-5ꢅꢄ3, H-6aꢄ3, H-6bꢄ3, alanineꢄ3ꢄ1/2), 2.10—1.89 (75H, m, OAcꢄ6ꢄ4, COCH2 of b-
H-6aꢅꢄ3, H-6bꢅꢄ3, COOCH3ꢄ3, NCH2COꢄ2, NCH2ꢄ2 of b-alanine, alanineꢄ3ꢄ1/2), 1.48—1.18 [32H, m, (CH2)ꢄ4ꢄ4]. 13C-NMR (125 MHz,
OCH2 of sugar unitꢄ3, OCH3ꢄ3), 3.22 (6H, m, NCH2 of sugar unitꢄ3), CDCl3): d 170.1, 170.05, 169.97, 169.6, 169.5, 169.3, 168.4, 165.5, 164.9,
2.70—2.40 (4H, m, COCH2ꢄ2 of b-alanine), 2.10—1.96 (54H, m,
OAcꢄ6ꢄ3), 1.55—1.26 [33H, m, t-Bu, (CH2)ꢄ4ꢄ3]. 13C-NMR (125 MHz,
CDCl3): d 170.1, 170.0, 169.9, 169.52, 169.47, 169.3, 168.4, 165.4, 164.9,
133.33, 133.26, 129.8, 129.1, 128.4, 101.2, 100.89, 100.85, 100.6, 78.6,
74.1, 72.0, 71.9, 71.8, 71.0, 70.8, 69.9, 69.1, 68.7, 67.5, 67.3, 67.2, 66.8,
60.4, 52.7, 29.32, 29.29, 26.7, 25.6, 25.5, 20.8, 20.7, 20.61, 20.56. MALDI-
133.35, 133.28, 129.8, 129.6, 129.2, 128.4, 101.2, 100.9, 100.7, 78.6, 74.13,
74.11, 72.0, 71.9, 71.8, 71.0, 70.9, 69.9, 69.1, 68.7, 67.5, 67.3, 66.8, 60.4,
52.8, 45.4, 39.64, 39.56, 29.7, 29.3, 26.7, 26.6, 25.59, 25.56, 20.8, 20.7,
20.62, 20.57. MALDI-TOF-MS: Calcd for C237H297N9O109SNa: m/z 5067.8
[MꢃNa]ꢃ. Found: m/z 5069.5 [MꢃNa]ꢃ.
Compound 2 Compound 2 was synthesized from 24 according to the
TOF-MS: Calcd for C171H219N5O81Na: m/z 3661.3 C114H147N3O54Na. Found: procedure described for the synthesis of 1. Yield: 6.8 mg (94.0%): [a]2D3
m/z 3661.4 [MꢃNa]ꢃ.
ꢂ17.1° (cꢀ0.2, H2O). 13C-NMR (125 MHz, 1 : 1 CD3OD–D2O): d 104.5,
104.1, 103.8, 83.3, 77.5, 76.5, 74.8, 74.3, 73.94, 73.89, 71.9, 71.4, 69.9,
69.7, 60.9, 49.8, 46.0, 40.4, 31.1, 30.0, 29.6, 27.1, 26.0.
Compound 21 Compound 21 was synthesized from 20 according to the
procedure described for the synthesis of 19. Yield: 49 mg (76.3%). [a]2D3
ꢃ5.0° (cꢀ0.6, CHCl3). 1H-NMR (500 MHz, CDCl3): d 7.99—7.38 (30H, m,
Ar-H), 5.60 (3H, t, H-3ꢁꢄ3), 5.36—5.29 (9H, m, H-2ꢁꢄ3, H-4ꢄ3, H-
4ꢅꢄ3), 5.17—5.09 (6H, m, H-2ꢄ3, H-2ꢅꢄ3), 5.03—4.93 (6H, br d, H-3ꢄ3, 0.22 mmol) and DEPC (24 ml, 0.16 mmol). The reaction mixture was stirred
H-3ꢅꢄ3), 4.78 (3H, d, H-1ꢁ), 4.47—4.45 (6H, m, H-1ꢄ3, H-1ꢅꢄ3), 4.10— for 16 h at room temperature. After completion of the reaction, the mixture
Compound 18 To a solution of 13 (160 mg, 0.14 mmol) and 25
(14.3 mg, 57.9 mmol) in DMF (2 ml) were added triethylamine (30 ml,
4.08 (3H, m, H-5ꢁꢄ3), 3.97—3.40 (49H, m, H-4ꢁꢄ3, H-5ꢄ3, H-5ꢅꢄ3, H-
was extracted with chloroform, washed with water, dried (MgSO4), and con-
6aꢄ3, H-6bꢄ3, H-6aꢅꢄ3, H-6bꢅꢄ3, COOCH3ꢄ3, NCH2CO, NCH2 of b- centrated. The product was purified on silica gel column chromatography
alanine, OCH2 of sugar unitꢄ3, OCH3ꢄ3), 3.22 (6H, m, NCH2 of sugar (chloroform : methanolꢀ40 : 1) to give 18 (132 mg, 94.1%).
unitꢄ3), 2.70—2.40 (4H, m, COCH2ꢄ2 of b-alanine), 2.10—1.96 (58H,
m, OAcꢄ6ꢄ3, NCH2CO, NCH2 of b-alanine), 1.55—1.26 [24H, m,
Compound 26 To a solution of 19 (45 mg, 19.4 mmol) and 25 (1.9 mg,
7.7 mmol) in DMF (1 ml) were added triethylamine (4 ml, 29 mmol)
(CH2)ꢄ4ꢄ3]. 13C-NMR (125 MHz, CDCl3): d 170.2, 170.05, 169.97, and DEPC (3.2 ml, 21.3 mmol). The reaction mixture was stirred for 16 h at

492
Vol. 54, No. 4
room temperature. After completion of the reaction, the mixture was ex- 20.66, 20.6. MALDI-TOF-MS: Calcd for C237H297N9O109SNa: m/z 5067.8.
tracted with chloroform, washed with water, dried (MgSO4), and concentrat- Found: m/z 5069.5 [MꢃNa]ꢃ.
ed. The product was purified on silica gel column chromatography (chloro-
Compound 3 Compound 3 was synthesized from 28 according to the
form : methanolꢀ40 : 1) to give 26 (32 mg, 85.7%). [a]2D3 ꢃ3.8° (cꢀ0.8, procedure described for the synthesis of 1. Yield: 4.5 mg (80%). [a]2D3 ꢂ8.5°
CHCl3). 1H-NMR (500 MHz, CDCl3): d 7.99—7.38 (40H, m, Ar-H), 5.60 (cꢀ0.2, H2O). 13C-NMR (125 MHz, 1 : 1 CD3OD–D2O): d 173.8, 171.3,
(4H, t, H-3ꢁꢄ4), 5.36—5.28 (12H, d, m, H-2ꢁꢄ4, H-4ꢄ4, H-4ꢅꢄ4), 5.15— 167.4, 167.4, 167.3, 152.5, 135.5, 131.8, 131.3, 130.50, 130.45, 130.4,
5.09 (8H, m, H-2ꢄ4, H-2ꢅꢄ4), 5.01—4.92 (8H, br d, H-3ꢄ4, H-3ꢅꢄ4), 4.77 130.2, 124.5, 119.9, 116.6, 103.9, 101.6, 75.5, 75.4, 74.5, 74.0, 73.4, 72.9,
(4H, d, H-1ꢁ), 4.45—4.42 (8H, br d, br d, H-1ꢄ4, H-1ꢅꢄ4), 4.08 (4H, d, H- 72.1, 71.9, 70.7, 70.6, 70.1, 61.7, 46.4, 45.8, 40.0, 29.8, 29.2, 26.9, 26.0.
5ꢁꢄ4), 3.96—3.40 (72H, m, H-4ꢁꢄ4, H-5ꢄ4, H-5ꢅꢄ4, H-6aꢄ4, H-6bꢄ4,
CID-MS/MS Experiments of Compounds 2 and 3 All CID experi-
H-6aꢅꢄ4, H-6bꢅꢄ4, COOCH3ꢄ4, NCH2COꢄof b-alanineꢄ3, OCH2 of ments were performed on a Bruker Daltonics Esquire 3000 plus (Bruker
sugar unitꢄ4, NCH2 of b-alanineꢄ3, OCH3ꢄ4), 3.16 (8H, m, NCH2 of Daltonics GmbsH, Bremen, German), a quadruapole ion-trap mass spec-
sugar unitꢄ4), 2.50 and 2.42 (3H, m, COCH2 of b-alanine), 2.10—1.95
trometer equipped with an ESI source. The samples were introduced into the
(75H, m, OAcꢄ6ꢄ4, COCH2 of b-alanineꢄ3ꢄ1/2), 1.54—1.26 [41H, m, t- ion source via infusion (flow rate, 120 ml/h); He pressure; 4.86ꢄeꢂ6 mbar;
Bu, (CH2)ꢄ4ꢄ4]. 13C-NMR (125 MHz, CDCl3): d 170.2, 170.1, 170.0,
CID time, 40 ms, Other parameters: dry temperature, 250 °C; nebulizer gas
169.5, 169.3, 168.4, 165.5, 164.9, 133.35, 133.28, 129.8, 129.2, 128.4, (N2), 10 psi; drying gas (N2), 6.0 l/min; sample solutions, prepared in a
128.3, 101.2, 100.9, 100.7, 78.6, 74.1, 72.0, 71.93, 71.85, 71.8, 71.0, 70.9, mixed solution of MeOH/water (200 : 1) where concentrations were in the
69.9, 69.1, 68.7, 67.5, 67.3, 66.8, 60.4, 52.8, 39.5, 31.9, 29.7, 29.4, 29.32, range of pmol/ml; smart frag, off; scan range, 50—1300 m/z; compound sta-
29.26, 28.2, 26.7, 26.64, 26.58, 25.61, 25.57, 22.6, 20.8, 20.7, 20.62, 20.57. bility, 100%; ion charge control target, 5000; and maximum accumulation
MALDI-TOF-MS: Calcd for C228H291N7O108Na: m/z 4877.7 [MꢃNa]ꢃ. time, 200 ms. The average of 10 spectra was used as the mass spectra.
Found: m/z 4877.3 [MꢃNa]ꢃ.
Compound 27 Compound 27 was synthesized from 26 according to the
Acknowledgments This work was supported under the High-Tech Re-
procedure described for the synthesis of 19. Yield: 32 mg (76.0%). [a]2D3 search Center project of the Ministry of Education, Culture, Sports, Science
ꢃ6.4° (cꢀ0.7, CHCl3). 1H-NMR (500 MHz, CDCl3): d 7.99—7.38 (40H, m, and Technology of Japan. We gratefully acknowledge financial support in
Ar-H), 5.60 (4H, t, H-3ꢁꢄ4), 5.36—5.28 (12H, d, m, H-2ꢁꢄ4, H-4ꢄ4, H- the form of a Sasagawa Scientific Research Grant.
4ꢅꢄ4), 5.17—5.09 (8H, m, H-2ꢄ4, H-2ꢅꢄ4), 5.02—4.93 (8H, br d, H-3ꢄ4,
H-3ꢅꢄ4), 4.78 (4H, d, H-1ꢁ), 4.45 (8H, dd, H-1ꢄ4, H-1ꢅꢄ4), 4.09 (4H, d, References
H-5ꢁꢄ4), 3.96—3.40 (71H, m, H-4ꢁꢄ4, H-5ꢄ4, H-5ꢅꢄ4, H-6aꢄ4, H-
6bꢄꢄ4, H-6aꢅꢄ4, H-6bꢅꢄ4, COOCH3ꢄ4, NCH2CO of b-alanineꢄ3,
OCH2 of sugar unitꢄ4, NCH2 of b-alanineꢄ3, OCH3ꢄ4), 3.16 (8H, m,
NCH2 of sugar unitꢄ4), 2.10—1.95 (75H, m, OAcꢄ6ꢄ4, COCH2 of b-ala-
nineꢄ3ꢄ1/2). 13C-NMR (125 MHz, CDCl3): d 170.1, 170.0, 169.9, 169.3,
168.4, 165.4, 164.9, 133.33, 133.26, 129.7, 129.1, 128.4, 101.2, 100.9,
100.7, 78.6, 74.14, 74.09, 72.0, 71.9, 71.8, 71.0, 70.9, 70.0, 69.1, 68.7, 67.5,
67.3, 67.2, 60.4, 52.7, 29.6, 29.3, 29.2, 26.7, 20.8, 20.7, 20.61, 20.56.
MALDI-TOF-MS: Calcd for C223H284N7O106Na: m/z 4778.7 [MꢃNa]ꢃ.
Found: m/z 4779.4 [MꢃNa]ꢃ.
Compound 28 To a solution of 27 (22 mg, 4.6 mmol) and dansyl glycine
(2.3 mg, 7.5 mmol) in DMF (1 ml) were added triethylamine (1.1 ml,
7.9 mmol) and DEPC (0.8 ml, 5.3 mmol). The reaction mixture was stirred for
16 h at room temperature. After completion of the reaction, the mixture was
extracted with chloroform, washed with water, dried (MgSO4), and concen-
trated. The product was purified on silica gel column chromatography
(CHCl3 : MeOHꢀ20 : 1) to give the dansyl derivative 28 (17 mg, 73%). [a]2D3
ꢂ2.5° (cꢀ0.3, CHCl3). 1H-NMR (500 MHz, CDCl3): d 8.02—7.39 (40H, m,
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