Studies directed toward the synthesis of protein-bound GPI anchor

Article abstract of DOI:10.1016/S0040-4020(03)00615-X

Mannobioside-linked phosphoethanolamine 10, a prototype model of the GPI anchor, was synthesized via glycosidation of the monosaccharide donor and acceptor, and subsequent phosphorylation. In order to test the reactivity of the amino group involved in 10

Full text of DOI:10.1016/S0040-4020(03)00615-X

Tetrahedron 59 (2003) 4059–4067
Studies directed toward the synthesis of protein-bound
GPI anchor
*
Yasuko Tanaka, Yuko Nakahara, Hironobu Hojo and Yoshiaki Nakahara
Department of Applied Biochemistry, Institute of Glycotechnology, Tokai University, Kitakaname 1117, Hiratsuka,
Kanagawa 259-1292, Japan
Received 3 February 2003; accepted 10 April 2003
Abstract—Mannobioside-linked phosphoethanolamine 10, a prototype model of the GPI anchor, was synthesized via glycosidation of the
monosaccharide donor and acceptor, and subsequent phosphorylation. In order to test the reactivity of the amino group involved in 10 against
the activated amino acid esters, 10 was reacted with N-protected amino acid pentafluorophenyl esters in the presence of HOBt. The reactions
gave the aminoacylated products in moderate yields. When Fmoc-Ser-OPfp 12 and Fmoc-Cys(SBut)-OPfp 14 were reacted with 10,
byproducts 19, 20 and 21 derived from N- and O-acylation were produced. In contrast, reactions of 10 and N-protected amino acid thioesters
were promoted with AgNO3, HOSu, and DIEA to afford the coupling products without the undesired O-acylation. Peptidylation of 10 with
the synthesized oligopeptide thioesters 24 and 27 was also successful under the segment coupling conditions of the peptide thioester method
as well as those of the native chemical ligation. q 2003 Published by Elsevier Science Ltd.
1. Introduction
further been encouraged by combination with the protein-
splicing strategy.6 These modern peptide technologies are
promising not only in protein engineering but also in
synthesizing protein-bound GPIs. The synthesized protein-
bound GPIs will become indispensable probes to gain more
insight into the GPI-mediated signaling mechanism and also
will be used as potent devices for cell surface engineering.
Glycosylphosphatidylinositol (GPI) membrane anchors are
glycolipids, which anchor proteins in membranes to form
specific membrane microdomains by associating with
glycosphingolipids, cholesterol, and accessory proteins at
the cell surface. In addition to the anchoring function, GPIs
are thought to participate in transmembrane signaling.1 The
unique molecular architecture of GPI has prompted a
number of synthetic challenges, and the first total synthesis
was reported by Murakata and Ogawa in 1992.2,3 The major
subjects of those studies were concentrated on construction
of the common pentasaccharide [a-D-Man-(1!2)-a-D-
Man-(1!6)-a-D-GlcNH2-(1!6)-D-myo-Ins] backbone,
synthesis and strategic attachment of the branching
oligosaccharides, and conjugation with the phospholipid
moiety. However, little attention has been paid to the
question of how to reconstruct the linkage between the
protein C-terminal and the GPI anchor.
In this paper, we report our preliminary studies on the
construction of the peptide-GPI linkage. Coupling reactions
between some peptides and a mannobioside-linked phos-
phoethanolamine are demonstrated (Fig. 1).
2. Results and discussion
Our studies began with the preparation of known mannno-
bioside 77 by an alternative route. Phenyl 1-thio-a-D-
mannopyranoside 1 was regioselectively silylated with
t-BuMe2SiCl and imidazole. The resulting triol 2 was
benzylated to give 3 (92%). NIS/TfOH-promoted glycosi-
dation of 3 and 48 afforded a mixture of a-glycoside 5 and
b-glycoside 6 in 86% yield (5/6¼15/4). The anomeric
During the last decade, remarkable progress has been made
in the chemical synthesis of proteins, where the thioester
method4 and the native chemical ligation5 are of particular
value for large-molecule construction. It is noteworthy that
both methods now allow usage of the microbially expressed
segments for the total or semisynthesis of proteins. The
practical value of the native chemical ligation method has
1
configurations were assigned from the JCH coupling
constant. Compound 5 was desilylated with Bu4NF in
THF to give 7 (89%), to which a phosphoethanolamine
moiety was introduced in good yield by reaction with
amidite 82 followed by oxidation. The resultant phospho-
triester 9 was deprotected by treatment with NaOCH3/
CH3OH–ether and then hydrogenated to give 10 in 97%
yield.
Keywords: GPI anchor; mannobioside; peptide thioester; native chemical
ligation; CD 52.
*
Corresponding author. Tel./fax: þ81-463-50-2075;
e-mail: yonak@keyaki.cc.u-tokai.ac.jp
0040–4020/03/$ - see front matter q 2003 Published by Elsevier Science Ltd.
doi:10.1016/S0040-4020(03)00615-X

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Y. Tanaka et al. / Tetrahedron 59 (2003) 4059–4067
Figure 1. Structure of GPI anchors.
By employing compound 10 as a prototype of GPI, amide
linkage-forming reaction on the ethanolamine terminus was
tested. As the first model, a simple amino acid derivative,
N-Fmoc alanine pentafluorophenyl ester (Fmoc-Ala-OPfp:
2 equiv.) 11, was reacted with 10 in dry DMF in the
presence of HOBt (2.2 equiv.) at room temperature.
Compound 10 was consumed after overnight stirring, the
reaction being monitored by reversed phase TLC (C-18)
with iodine coloration. The water-soluble product was
isolated by column chromatography on Sephadex LH-20
with H2O/MeOH (1/1) and then by preparative HPLC with a
reversed phase (C-8) column. The desired coupling product
15 was obtained in 64% yield and characterized by 1H NMR
and MALDI TOF MS. Coupling reactions with Fmoc-Ser-
OPfp 12, Fmoc-Thr-OPfp 13, and Fmoc-Cys(SBut)-OPfp
14, also proceeded to yield 16 (47%), 17 (65%), and 18
(61%), respectively. In addition to the desired products,
di-acylated products were produced in substantial quantities
in the preparation of 16 and 18. HPLC profiles of the
coupling products are exhibited in Figure 2. The byproducts
19 (4%), 20 (18%), and 21 (28%) most probably resulted
from N- and O-acylation showed their molecular ions
(þNa) at 1148.6, 1148.6, and 1356.0, respectively. It is to be
noted that amino acid Pfp esters have often been utilized in
solid-phase synthesis of the glycopeptides carrying
unmasked oligosaccharides on account of the presumed
poor O-acylation ability of the Pfp esters.9
In contrast, reaction of 10 with Fmoc amino acid thioesters
exclusively produced the N-acylated compounds under the
Figure 2. HPLC of the products from the reaction of 10 and 12 (a), the reaction of 10 and 14 (b), and the reaction of 10 and 22 (c). Column: Mightysil RP-8
(250£4.6 mm). Eluent A: distilled water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA. Flow rate: 1 ml/min. In the chromatogram (b), the peaks
at 13.2 min and at 18.7 min are Fmoc·Cys(SBut)·OH and Fmoc·Cys(SBut)·OMe probably generated during gel-permeation chromatography. In the
chromatogram (c), the peak at 11.9 min corresponds to Fmoc·Ser·OH.

Y. Tanaka et al. / Tetrahedron 59 (2003) 4059–4067
4061
Scheme 1. Synthesis of mannobioside-linked phosphoethanolamine 10 and the aminoacylated products.
conditions reported for the peptide thioester method.4 The
reaction with Fmoc-Ser-SCH2CH2CO2C2H5 2210 (2 equiv.)
smoothly proceeded in the presence of AgNO3, N-hydroxy-
succimide (HOSu), and diisopropylethylamine (DIEA) in
DMF and completed within 2 h at room temperature.
The reaction mixture was chromatographed on a gel-
permeation column, and then product 16 was isolated in
76% yield by preparative HPLC. Under similar conditions,
Fmoc-Thr-SCH2CH2CO2C2H5 2310 gave 17 in 76% yield.
These results suggest that the amino group on the GPI
molecule is as reactive as the peptide amine (Fig. 2 and
Scheme 1).
of lymphocytes, was chosen as a protein model.12 The
peptide thioester 24 was prepared on Rink amide MBHA
resin by Fmoc-based solid-phase synthesis according to the
reported protocol.13 The thioester, however, was produced
as a couple of diasteromers (24a/24b¼13/7, 20% overall
yield) probably generated by epimerization at the a carbon
center of the C-terminal serine residue (Fig. 3(a)). An
additional complication arose from the reaction of 10
(1.5 equiv.) with the separated diastereomer 24b. When the
reactants were treated with AgNO3, HOSu, and DIEA in
DMF, three products were generated. Two of them (25a
,25b) showed the desired molecular ion peaks at 1761.34
and 1761.47, respectively, in their mass spectra, whereas the
spectrum of the third one (26) exhibited m/z 1743.47
(Fig. 3(b)). Therefore, it seems likely that the former two
(25) were diastereomers to each other and the latter (26) was
derived from dehydration of the intermediate. Coupling
reaction between 10 and another isomer of the thioester
With the successful results on the chemoselective N-acyla-
tion of the phosphoethanolamine moiety with thioesters, we
next investigated the coupling reaction using a more
complex oligopeptide. A dodecapeptide representing the
peptide backbone of CD 52,11 a GPI-anchored glycoprotein

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Figure 3. HPLC of the synthesized peptide thioesters 24a and 24b (a), the products from the reaction of 10 and 24b (b), the products from the reaction of 10 and
27 (c), and the products from the reaction of 27 and 29 (d). Column: Mightysil RP-18 (150£4.6 mm). Eluent A: distilled water containing 0.1% TFA, B:
acetonitrile containing 0.1% TFA. Flow rate: 1 ml/min.
(24a) was also carried out under the same conditions to yield
a sole product but identical with the dehydrated molecule
(26: m/z 1743.61).
phosphate buffer at pH 8.0. The desired coupling product 30
was slowly but certainly produced as thioester 27 was
consumed. The progress of the reaction was readily
monitored by HPLC and MALDI TOF MS. After 10 days
running (Fig. 3(d)), the reaction was quenched by addition
of AcOH and the product was isolated by HPLC. The
compound 30 was obtained in 62% yield (78% based on the
consumed 27), while 20% of the starting thioester 27
remained unreacted. Elevated temperature (45–508C) had
little effect on acceleration of the total reaction. Further
studies will be necessary to optimize the conditions
(Scheme 2).
In order to avoid the cumbersomeness arising from the
labile serine thioester, a homologous peptide having
C-terminal glycine thioester was synthesized. Starting
with Fmoc-Gly-SCH2CH2CO2H and Rink amide MBHA
resin, tridecapeptide thioester 27 was prepared in 12%
overall yield. The thioester was allowed to react with 10
(1.5 equiv.) in the presence of the same promoters. The
reaction was run for 3 days with monitoring by HPLC and
MALDI TOF MS. The desired coupling product 28 was the
major component in the reaction mixture at the stage of one-
day running (Fig. 3(c)), and no further increase of the
product in the mixture was observed. By gel permeation
chromatography and subsequent preparative HPLC 28 was
isolated in 34% yield.
In conclusion, a synthetic strategy has been developed that
allows reconstruction of the protein-GPI anchor linkage
motif. Amino acid thioesters readily reacted with the
mannnobiose-linked phosphoethanolamine group by
activation with AgNO3 and HOSu, whereas amino acid
pentafluorophenyl esters resulted in not only N-amino
acylation but additional O-amino acylation in some
experiments. Peptide thioester involving the amino acid
sequence of CD 52 was reactive with the phosphoethano-
lamine in the presence of AgNO3 and HOSu to afford the
coupling product in 34% yield. The coupling reaction
designed in accordance with the native chemical ligation
procedure was successful to give the desired product in
higher yield. Although insertion of the Gly or Gly-Cys
residue generated the non-native peptide sequence, such
In addition, we attempted to couple thioester 27 and a GPI
anchor model under the conditions of the native chemical
ligation.5 The cystein-linked mannobiose 18 was
N-deprotected with NaOMe/MeOH to 29 in 74% yield.
Based on the expectation of in situ cleavage of the S–S
linkage under the ligation conditions, the S-protected
cysteinyl mannnobiose derivative was allowed to react
with tridecapeptide 27. A mixture of 27 and 29 (1/1) was
stirred at room temperature with thiophenol (22 equiv.) in a

Y. Tanaka et al. / Tetrahedron 59 (2003) 4059–4067
4063
Scheme 2. Synthesis of the peptidyl mannobiosides.
modification facilitated coupling of the peptide and the
phosphoethnolamine. The chemistry described here
will open the way for the total synthesis of the GPI-
anchored proteins of biological significance. Studies on
coupling reactions with a more complex mannoside
carrying the glycero-phospholipid moiety are currently
undertaken.
(100 MHz)] spectrometer. Chemical shifts are expressed in
ppm downfield from the signal for internal Me4Si for
solutions in CDCl3. MALDI·TOF mass spectra were
obtained with a Bruker AUTOFLEX-T spectrometer (2,5-
dihydroxybenzoic acid was used as a matrix). High
resolution Fab mass spectra were measured with JEOL
JMS HX-110 spectrometer (2,5-dihydroxybenzoic acid was
used as a matrix). All solid-phase reactions were performed
at room temperature in the capped polypropylene test tubes
with stirring on a vortex tube-mixer. HPLC was performed
using Mightysil RP-8, RP-18 (4.6£150 mm for analysis and
10£250 mm for preparation, Kanto Chemical Co.). Amino
acids were analyzed on a Hitachi L-8500 amino acid
analyzer. Fmoc Rink amide MBHA resin was purchased
from NOVA biochem.
3. Experimental
3.1. General
Optical rotations were determined with a Jasco DIP-370
polarimeter for solutions in CHCl3, unless noted otherwise.
Column chromatography was performed on silica gel PSQ
100B (Fuji Silysia). TLC and HPTLC were performed on
3.1.1. Phenyl 6-O-tert-butyldimethylsilyl-1-thio-a-D-
1
Silica Gel 60 F254 (E. Merck). H and 13C NMR spectra
mannopyranoside 2.
1.84 mmol), t-BuMe2SiCl (332 mg, 2.20 mmol) and
A
mixture of
1
(500 mg,
were recorded with a JEOL AL400 [1H (400 MHz), 13C

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Y. Tanaka et al. / Tetrahedron 59 (2003) 4059–4067
imidazole (375 mg, 5.51 mmol) in dry DMF (3 ml) was
stirred at 08C for 10 min. The reaction was quenched by
addition of ice-water, the mixture was extracted with
CHCl3, and the extract was washed with sat. NaHCO3 and
brine, dried (Na2SO4), and concentrated in vacuo. The
residue was chromatographed on silica gel with toluene/
EtOAc (1/4) to give 2 (512 mg, 72%). [a]D¼þ178.28 (c 1).
1H NMR: d 7.48–7.25 (m, 5H, Ar), 5.53 (s, 1H, H-1), 4.21
(s, 1H, H-2), 4.12–3.88 (m, 5H, H-3, H-4, H-5,H-6, H-60),
3.79, 3.68, and 3.40 (3s, OH), 0.90 (s, 9H, t-Bu), 0.01 (s, 6H,
Me2). Anal. calcd for C18H30O5SSi·0.3H2O: C, 55.15; H,
7.87; S, 8.18%. Found: C, 55.22; H, 7.77; S, 7.97%.
95.7 (1JCH¼166.7 Hz, C-1a), 98.7 (1JCH¼153.4 Hz, C-1b).
MALDI TOF MS: calcd for C63H76O11Si·Na; m/z 1059.51,
found; 1059.94. Anal. calcd for C63H76O11Si·0.5H2O: C,
72.18; H, 7.40%. Found: C, 72.38; H, 7.28%.
3.1.4. Allyl O-(2,3,4-tri-O-benzyl-a-D-mannopyranosyl)-
(1!2)-3,4,6-tri-O-benzyl-a-D-mannopyranoside 7. To a
solution of 5 (500 mg, 0.48 mmol) in freshly distilled THF
(10 ml) was added 1 M Bu4NF/THF (1.4 ml, 1.4 mmol) at
08C. The mixture was stirred at 08C–room temperature
overnight and concentrated in vacuo. The residue was
dissolved in CHCl3, washed with water and brine, dried
(Na2SO4), and concentrated in vacuo. The crude product
was chromatographed on silica gel with toluene/EtOAc
(5/1) to give 7 (397 mg, 89%).
3.1.2. Phenyl 2,3,4-tri-O-benzyl-6-O-tert-butyldimethyl-
silyl-1-thio-a-D-mannopyranoside 3. To a stirred mixture
of 2 (1.0 g, 2.6 mmol) and 60% NaH (0.47 g, 11.6 mmol) in
dry DMF (10 ml) was added BnBr (1.38 ml, 11.6 mmol) at
08C under Ar. The mixture was stirred at the temperature for
1 h before quenching the reaction with ice-water. The
product was extracted with ether, washed with water and
brine, dried (Na2SO4), and concentrated in vacuo. The
residue was chromatographed on silica gel with toluene to
1H NMR: d 7.51–7.14 (m, 30H, Ar), 5.86 (m, 1H,
CH2vCH–), 5.24 (brd, J¼17.1 Hz, CH2vCH–), 5.17
(brd, 1H, J¼10.2 Hz, CH2vCH–), 5.16 (brs, 1H, H-1b),
4.87 (brs, 1H, H-1a). MALDI TOF MS: calcd for
C57H62O11·Na; m/z 945.41, found; 945.70.
1
afford 3 (1.57 g, 92%). [a]D¼þ69.48 (c 1). H NMR: d
3.1.5. Allyl O-{2,3,4-tri-O-benzyl-6-O-[2-(N-benzyloxy-
carbonylamino)ethyl 2-cyanoethyl phosphonato]-a-D-
mannopyranosyl}-(1!2)-3,4,6-tri-O-benzyl-a-D-manno-
pyranoside 9. A mixture of 7 (458 mg, 0.50 mmol),
1H-tetrazole (70 mg, 0.99 mmol), 8 (982 mg, 2.48 mmol),
7.45–7.15 (m, 20H, Ar), 5.56 (d, 1H, J¼1.5 Hz, H-1), 4.95
(d, 1H, J¼11.0 Hz, PhCH2–), 4.63 (m, 5H, PhCH2–), 4.10
(m, 1H, H-5), 3.99–3.88 (m, 5H, H-2, H-3, H-4, H-6, H-60),
0.89 (s, 9H, t-Bu), 0.06 and 0.05 (2s, 6H, Me2). Anal. calcd
for C39H48O5SSi·0.5H2O: C, 70.34; H, 7.42; S, 4.82%.
Found: C, 70.30; H, 7.59; S, 4.82%.
˚
and dried molecular sieves 3 A (2 g) in dry CH3CN (20 ml)
was stirred for 4 h under Ar. Then another equivalent of
1H-tetrazole (35 mg, 0.49 mmol) was added to the mixture,
which was further stirred for 5 min. A solution of 5.5 M
t-BuOOH/nonane (1 ml, 5.5 mmol) was added to the
mixture. After stirring for 10 min, the reaction was
quenched with sat. NaHCO3, and the mixture was filtered
through Celite. The product in the filtrate was extracted with
CH2Cl2 and dried (Na2SO4), and concentrated in vacuo. The
residue was chromatographed on silica gel with toluene/
EtOAc (1/2) to afford 9 (553 mg, 90%) as a mixture of
diastereomeric phosphotriesters. 1H NMR: d 7.32–7.18 (m,
30H, Ar), 5.86 (m, 1H, CH2vCH–), 5.44 and 5.38 (2m, 1H,
NH), 5.24 (brd, 1H, J¼17.1 Hz, CH2vCH–), 5.17 (brd, 1H,
J¼10.4 Hz, CH2vCH–), 5.12 (brs, 1H, H-1b), 5.07 (brs,
2H, –CO2CH2Ph), 4.84 (brs, 1H, H-1a), 3.38 and 3.29 (2 m,
2H, ZNHCH2–), 2.48 and 2.25 (2m, 2H, –CH2CN). 13C
NMR: d 98.0 (C-1a), 99.6 and 99.7 (C-1b). HRMS: calcd for
C70H77N2O16PNa (MþNa)þ; m/z 1255.4908, found;
1255.4905. MALDI TOF MS: calcd for C70H77N2O16P·Na;
m/z 1255.49, found; 1255.66.
3.1.3. Allyl O-(2,3,4-tri-O-benzyl-6-O-tert-butyldimethyl-
silyl-a-D-mannopyranosyl)-(1!2)-3,4,6-tri-O-benzyl-a-
D-mannopyranoside 5 and allyl O-(2,3,4-tri-O-benzyl-6-
O-tert-butyldimethylsilyl-b-D-mannopyranosyl)-(1!2)-
3,4,6-tri-O-benzyl-a-D-mannopyranoside 6. A mixture of
3 (1.52 g, 2.3 mmol), 4 (0.95 g 1.9 mmol), NIS (0.71 g,
˚
2.9 mmol), and dried molecular sieves 3 A (5 g) in dry
CH2Cl2 (50 ml) was stirred at 08C under Ar for 15 min.
TfOH (25.6 ml, 0.3 mmol) was added to the mixture, which
was then stirred for 1 h before quenching with sat. NaHCO3.
The mixture was filtered through Celite, the filtrate was
extracted with CHCl3, and the extract was washed with 10%
Na2S2O3 and brine, dried (Na2SO4), and concentrated in
vacuo. The crude product was chromatographed on silica
gel with toluene/EtOAc (49/1) to give 5 (1.36 g, 68%) and 6
(0.36 g, 18%).
1
Compound 5. [a]D¼þ18.38 (c 1). H NMR: d 7.33–7.15
(m, 30H, Ar), 5.87 (m, 1H, CH2vCH–), 5.24 (s, 1H, H-1b),
5.23 (brd, 1H, J¼17.0 Hz, CH2vCH–), 5.15 (d, 1H,
J¼10.5 Hz, CH2vCH–), 4.90 (d, 1H, J¼11.0 Hz,
PhCH2–), 4.86 (s, 1H, H-1a), 1.61 (s, 9H, t-Bu), 0.08 and
0.07 (2s, 6H, Me2). 13C NMR: d 98.2 (1JCH¼171.7 Hz,
C-1b), 98.6 (1JCH¼170.8 Hz, C-1a). MALDI TOF MS:
calcd for C63H76O11S i·Na; m/z 1059.51, found; 1059.94.
Anal. calcd for C63H76O11Si: C, 72.94; H, 7.38%. Found: C,
72.73; H, 7.41%.
3.1.6. n-Propyl O-[6-O-(2-aminoethyl phosphonato)-a-D-
mannopyranosyl]-(1!2)-a-D-mannopyranoside 10. To a
stirred solution of 9 (523 mg, 0.42 mmol) in anhydrous
MeOH/Et2O (1/1, 6 ml) was added 1 M NaOMe/MeOH till
the mixture became basic. Then the mixture was stirred for
45 min, before being neutralized with Amberlite IR 120
(Hþ), filtered, and concentrated in vacuo. The residue was
chromatographed on silica gel with CHCl3/MeOH (4/1)
containing 1% Et3N to afford de-cyanoethylated product as
the triethylammonium salt (527 mg, 97%), [a]D¼þ13.68 (c,
1). MALDI TOF MS: calcd for C67H74NO16P·Na; m/z
1202.46, found; 1202.15. The product (513 mg, 0.40 mmol)
was dissolved in 80% aq. MeOH (10 ml) with an additional
distilled THF, and hydrogenated with Pd(OH)2 (200 mg) for
1
Compound 6. [a]D¼237.68 (c 1). H NMR: d 7.54–7.10
(m, 30H, Ar), 5.89 (m, 1H, CH2vCH–), 5.28 (dd, 1H,
J¼1.5, 17.3 Hz, CH2vCH–), 5.20 (brd, 1H, J¼10.2 Hz,
CH2vCH–), 5.05 (brs, 1H, H-1a), 4.63 (brs, 1H, H-1b),
0.87 (s, 9H, t-Bu), 0.03 and 0.01 (2s, 6H, Me2). 13C NMR: d

Y. Tanaka et al. / Tetrahedron 59 (2003) 4059–4067
4065
3 days. The mixture was filtered through Celite, the filtrate
was concentrated in vacuo, and the residue was passed
through a column of Amberlite IR 120 (Naþ). The resulting
solution was lyophilized to quantitatively give 10 (237 mg).
15. HPLC purification of the product afforded 17 (10.4 mg,
65%). [a]D¼þ20.98 (c 5, H2O). 1H NMR (D2O; t-BuOH, d
1.24): d 7.72 (br, 2H, Ar), 7.60–7.55 (m, 2H, Ar), 7.34 (m,
4H, Ar), 5.00 (s, 2H, H-1a and H-1b), 1.50 (m, 2H,
–CH2CH2CH3), 1.06 (br, 3H, Thr-bH), 0.83 (brt, 3H,
J¼7.2 Hz, Me). MALDI TOF MS: calcd for C36H51N2O18-
P·Na; m/z 853.28, found; 853.20.
1
[a]D¼þ38.38 (c 1, H2O). H NMR (as Et3N salt, D2O): d
4.89 and 4.87 (2 brs, 2H, H-1a and H-1b), 1.45 (m, 2H,
–CH2CH2CH3), 0.76 (t, 3H, J¼7.0 Hz, Me). 13C NMR
(D2O): d 98.9 and 103.0 (C-1a and C-1b). MALDI TOF MS:
calcd for C17H34NO14P·Na; m/z 530.15, found; 529.65.
3.1.10. n-Propyl O-{6-O-[2-{[N-(9-fluorenylmethoxycar-
bonyl)-S-(tert-butylthio)-L-cysteinyl]amido}ethyl phos-
phonato]-a-D-mannopyranosyl}-(1!2)-a-D-manno-
pyranoside 18. Reaction of 10 (10 mg, 19 mmol) and 13
(22.5 mg, 38 mmol) was performed in the presence of HOBt
as described above for 15. Gel permeation chromatography
was done with MeOH. The product was purified by HPLC
using an RP-8 column eluted with a gradient of 50–70%/0–
20 min and 70–80%/20–25 min of CH3CN in H2O
containing 0.1% TFA to afford 18 (10.9 mg, 61%) and 21
(7.1 mg, 28%).
3.1.7. n-Propyl O-{6-O-[2-{[N-(9-fluorenylmethoxycar-
bonyl)-L-alanyl]amido}ethyl phosphonato]-a-D-manno-
pyranosyl}-(1!2)-a-D-mannopyranoside 15. A mixture
of 10 (10 mg, 19 mmol), 11 (18 mg, 38 mmol), and HOBt
(2.8 mg, 21 mmol) in dry DMF (2 ml) was stirred for 3.5 h at
room temperature under Ar. Then an additional amount
(2.8 mg) of HOBt was added to the mixture, which was
further stirred overnight. The reaction mixture was con-
centrated in vacuo to give the residue, which was
chromatographed on Sephadex LH 20 with 50% aq.
MeOH. The product was purified by HPLC using an RP-8
column eluted with a gradient of 30–50%/0–20 min and
50–80%/20–25 min of CH3CN in H2O containing 0.1%
TFA to afford 15 (9.9 mg, 64%). [a]D¼þ25.18 (c 0.5,
MeOH). 1H NMR (D2O; t-BuOH, d 1.24): d 7.75 (brd, 2H,
J¼6.8 Hz, Ar), 7.58 (brd, 2H, J¼6.4 Hz, Ar), 7.46–7.41 (m,
4H, Ar), 5.00 (brs, 2H, H-1a and H-1b), 1.50 (m, 2H,
–CH2CH2CH3), 1.08 (br, 1H, Ala-bH), 0.82 (t, 3H,
J¼7.3 Hz, Me). MALDI TOF MS: calcd for C35H49N2O17-
P·Na; m/z 823.27, found; 823.58.
Compound 18. [a]D¼-16.08 (c 1.3, H2O). 1H NMR
(CD3OD): d 7.78 (d, 2H, J¼7.6 Hz, Ar), 7.69 (d, 2H,
J¼7.3 Hz, Ar), 7.40–7.30 (m, 4H, Ar), 5.00 (d, 1H,
J¼1.4 Hz) and 4.98 (1s, 1H) (H-1a and H-1b), 4.45 (m,
1H, Cys-aH), 3.20 (dd, 1H, J¼4.9, 13.4 Hz, Cys-bH), 2.99
(dd, 1H, J¼8.8, 13.4 Hz, Cys-bH), 1.58 (m, 2H,
–CH2CH2CH3), 1.34 (s, 9H, t-Bu), 0.93 (t, 3H, J¼7.3 Hz,
Me). MALDI TOF MS: calcd for C39H57N2O17PS2·Na; m/z
943.27, found; 943.20.
Compound 21. 1H NMR (CD3OD): d 7.68 (brd, 4H,
J¼7.5 Hz, Ar), 7.57 (brd, J¼7.3 Hz, Ar), 7.30–7.18 (m,
8H, Ar), 4.90 and 4.89 (2 brs, 2H, H-1a and H-1b), 1.47 (m,
2H, –CH2CH2CH3), 1.23 (s, 18H, t-Bu), 0.82 (t, 3H,
J¼7.3 Hz, Me). MALDI TOF MS: calcd for C61H80N3O20-
PS4·Na; m/z 1356.39, found; 1356.00.
3.1.8. n-Propyl O-{6-O-[2-{[N-(9-fluorenylmethoxycar-
bonyl)-L-seryl]amido}ethyl phosphonato]-a-D-manno-
pyranosyl}-(1!2)-a-D-mannopyranoside 16. Reaction
of 10 (10 mg, 19 mmol) and 12 (18.6 mg, 38 mmol) was
performed in the presence of HOBt as described above for
15. HPLC purification of the product afforded 16 (7.5 mg,
47%), 19 (0.8 mg, 4%) and 20 (4.0 mg, 18%).
3.1.11. Reaction of 10 with 22 (alternative synthesis of
16). A mixture of 10 (10 mg, 19 mmol), 22 (16.7 mg,
38 mmol), HOSu (43.3 mg, 376 mmol), AgNO3 (19.2 mg,
113 mmol), and DIEA (20 ml, 113 mmol) in dry DMF (2 ml)
was stirred for 3 h in the dark at room temperature under Ar,
and then concentrated n vacuo. The residue was dissolved in
50% aq. MeOH, and insoluble material was filtered off
through Celite and a disk of membrane filter. The filtrate
was concentrated in vacuo to give the residue, which was
chromatographed on Sephadex LH 20 with 50% aq. MeOH.
The product was purified by HPLC as described above to
give 16 (12.1 mg, 76%).
1
Compound 16. [a]D¼þ20.18 (c 0.4, H2O). H NMR (D2O;
t-BuOH, d 1.24): d 7.83 (brd, 2H, J¼7.1 Hz, Ar), 7.65–7.60
(m, 2H, Ar), 7.44–7.38 (m, 4H, Ar), 4.84 (brs, 2H, H-1a and
H-1b), 1.52 (m, 2H, –CH2CH2CH3), 0.85 (t, 3H, J¼7.2 Hz,
Me). MALDI TOF MS: calcd for C35H49N2O18P·Na; m/z
839.26, found; 839.07.
Compound 19. 1H NMR (CD3OD): d 7.79 (brd, 4H,
J¼7.6 Hz, Ar), 7.68 (m, 4H, Ar), 7.41–7.30 (m, 8H, Ar),
5.04 and 5.00 (2s, 2H, H-1a and H-1b), 1.58 (m, 2H,
–CH2CH2CH3), 0.93 (t, 3H, J¼7.4 Hz, Me). MALDI TOF
MS: calcd for C53H64N3O22P·Na; m/z 1148.36, found;
1148.62.
3.1.12. Reaction of 10 with 23 (alternative synthesis of
17). Coupling reaction of 10 (10 mg, 19 mmol) and 23
(17.2 mg, 38 mmol) was performed in an analogous
procedure as described above to produce 17 (12.2 mg,
76%).
Compound 20. 1H NMR (CD3OD): d 7.82 (m, 4H, Ar), 7.70
(brs, 4H, Ar), 7.43–7.35 (m, 8H, Ar), 5.07 and 5.04 (2 brs,
2H, H-1a and H-1b), 1.62 (m, 2H, –CH2CH2CH3), 0.98 (t,
3H, J¼7.3 Hz, Me). MALDI TOF MS: found; 1148.62.
3.1.13. S-(N-Acetyl-L-glycyl-L-glutaminyl-L-asparagi-
nyl-L-a-aspartyl-L-threonyl-L-seryl-L-glutaminyl-L-
threonyl-L-seryl-L-seryl-L-proryl-L-seryl)-3-thiopropio-
namide 24. A mixture of Fmoc-Ser(But)-OH (2.0 g,
5.2 mmol), HOSu (0.72 g, 6.3 mmol), and WSCI (1.6 g,
8.4 mmol) in dry DMF (20 ml) was stirred at 08C–room
temperature for 9 h. The mixture was concentrated in vacuo
3.1.9. n-Propyl O-{6-O-[2-{[N-(9-fluorenylmethoxycar-
bonyl)-L-threonyl]amido}ethyl phosphonato]-a-D-man-
nopyranosyl}-(1!2)-a-D-mannopyranoside 17. Reaction
of 10 (10 mg, 19 mmol) and 12 (19.1 mg, 38 mmol) was
performed in the presence of HOBt as described above for

4066
Y. Tanaka et al. / Tetrahedron 59 (2003) 4059–4067
to the residue, which was extracted with EtOAc, washed
with 5% aq. KHSO4 and brine, dried (Na2SO4), and
concentrated in vacuo. The residue was dissolved in dry
DMF (20 ml), and stirred with 3-mercaptopropionic acid
(350 ml, 4.0 mmol) and DIEA (350 ml, 2.0 mmol) at room
temperature for 8 h. The mixture was concentrated in vacuo,
the residue was extracted with EtOAc, and the extract was
washed with 5% aq. KHSO4 and brine, dried (Na2SO4), and
concentrated in vacuo. The crude product was chromato-
graphed on C-18 silica gel with MeOH/H2O (3/1) to give
Fmoc-Ser(But)-SCH2CH2CO2H (2.0 g, 83%), which was
used for the following solid-phase synthesis. All the solid-
phase reactions were performed in a polypropylene tube
equipped with a coarse filter and three-way stopcock by
stirring with a vortex mixer.
filtered through a membrane filter and then through a C-18
cartridge. The crude product was chromatographed on
Sephadex LH 20 with MeOH/H2O (1/1), and further
purified by HPLC on a C-18 column eluted with a gradient
of 5–10%/0–20 min of CH3CN in H2O containing 0.1%
TFA to give 25a (1.1 mg, 8%), 25b (2.5 mg, 19%).
Compound 26 was also obtained in the less mobile fraction
(20%).
On the other hand, reaction of 10 and 24a performed in an
analogous conditions exclusively gave 26.
3.1.15. S-(N-Acetyl-L-glycyl-L-glutaminyl-L-aspara-
ginyl-L-a-aspartyl-L-threonyl-L-seryl-L-glutaminyl-L-
threonyl-L-seryl-L-seryl-L-proryl-L-seryl-L-glycyl)-3-
thiopropionamide 27. Tridecapeptide thioester 27 was
synthesized starting from Fmoc-Gly-OH in a similar manner
as described for 24. Purification of the product by reversed-
phase HPLC with a gradient of 5–10%/0–30 min of
CH3CN in H2O containing 0.1% TFA afforded 27 (12%).
MALDI TOF MS: calcd for C52H83N17O26S·Na; m/z
1416.53, found; 1416.44.
Commercial Fmoc Rink amide MBHA resin (274 mg,
0.2 mmol) was stirred with 20% piperidine/NMP (5 ml) for
5 min. After filtration the resin was again stirred with 20%
piperidine/NMP (5 ml) for 15 min to complete N-deprotec-
tion. The resin was washed several times with NMP (5 ml)
and then stirred with Fmoc-Ser(But)-SCH2CH2CO2H
(377 mg, 0.8 mmol), 1 M DCC/NMP (0.88 ml,
0.88 mmol), and 1 M HOBt/NMP (0.88 ml, 0.88 mmol) in
NMP (5 ml) for 1 h. The mixture was filtered and the
resultant resin was washed successively with NMP and
MeOH/CH2Cl2 (1/1). The unreacted amino group on the
resin was masked by acetylation with 10% Ac2O–5%
DIEA/NMP (5 ml). After washing with NMP, the resin was
stirred with a mixture of 25% 1-methylpyrrolidine–2%
hexamethylene imine–2% HOBt/NMP-DMSO (1/1,
5 ml)13 for 3 min in order to selectively remove the Fmoc
group. After filtration, the de-Fmoc procedure was again
repeated but for 10 min to complete N-deprotection. The
resin thoroughly washed with NMP was used for further
peptide assembling with Fmoc-Pro-OH, Fmoc-Ser(But)-
OH, Fmoc-Thr(But)-OH, Fmoc-Gln(Trt)-OH, Fmoc-
Asp(OBut)-OH, Fmoc-Asn(Trt)-OH, and Fmoc-Gly-OH.
Fmoc amino acids (4 equiv.), DCC (4.4 equiv.), and HOBt
(4.4 equiv.) were used for each coupling reaction. N-Depro-
tection with the above mixture (3 and 18 min) and acetyl
capping procedures (5 min) were performed as described
above. The dodecapeptide thus prepared was finally
transformed into the N-terminally acetylated peptide and
cleaved from the resin by treatment with reagent K (82.5%
TFA, 2.5% ethaneditiol, 5% phenol, 5% thioanisole, 5%
H2O) for 2 h. The resin was filtered off, and the product was
precipitated from the filtrate by adding Et2O. The precipitate
was washed with Et2O, dissolved in aq. CH3CN, and
purified by HPLC with a reversed-phase (C-18) column
eluted with a gradient of 7–14%/0–20 min of CH3CN in
H2O containing 0.1% TFA to give 24a and 24b as two
diastereomers in 13% (34 mg) and 7% (18 mg) overall
yields, respectively. MALDI TOF MS: calcd for C50H80-
N16O25S·Na; m/z 1359.51, found; 1359.51 (for 24a) and
1359.39 (for 24b).
3.1.16. n-Propyl O-[6-O-{2-[(N-acetyl-L-glycyl-L-gluta-
minyl-L-asparaginyl-L-a-aspartyl-L-threonyl-L-seryl-L-
glutaminyl-L-threonyl-L-seryl-L-seryl-L-proryl-L-seryl-
L-glycyl)amido]ethyl phosphonato}-a-D-mannopyrano-
syl]-(1!2)-a-D-mannopyranoside 28. Coupling reaction
of 10 (5.6 mg, 11 mmol) and 27 (10 mg, 7 mmol) was
performed by stirring with HOSu (16.3 mg, 142 mmol),
AgNO3 (7.2 mg, 43 mmol), and DIEA (7.4 ml, 43 mmol) in
dry DMF (4 ml) for 3 days and worked up as described for
25. Purification of the product by reversed-phase HPLC
with elution of a gradient of 5–10%/0–20 min of CH3CN in
H2O containing 0.1% TFA afforded 28 (4.4 mg, 34%).
MALDI TOF MS: calcd for C66H110N17O39P·Na; m/z
1817.67, found; 1818.53.
3.1.17. n-Propyl O-{6-O-[2-{[S-(tert-butylthio)-L-
cysteinyl]amido}ethyl phosphonato]-a-D-mannopyrano-
syl}-(1!2)-a-D-mannopyranoside 29. To a solution of
18 (36 mg, 38 mmol) in MeOH (2 ml) was added 1 M
NaOMe/MeOH (378 ml, 378 mmol). The mixture was
stirred for 2 h at room temperature and concentrated in
vacuo. The residue was chromatographed on Sephadex LH
20 with MeOH/H2O (1/1) and then on silica gel with CHCl3/
MeOH/H2O (12/7/1) to give 29 (20 mg, 74%).[a]D¼þ32.98
(c 1, MeOH). 1H NMR (CD3OD): d 5.00 (brs, 1H) and 4.98
(d, 1H, J¼1.5 Hz) (H-1a and H-1b), 3.59 (brt, 1H,
J¼9.5 Hz) and 3.45 (dt, 1H, J¼6.3, 9.5 Hz) (–OCH2Et),
3.23 (dd, 1H, J¼5.7, 14.1 Hz) and 3.06 (dd, 1H, J¼7.8,
14.1 Hz) (Cys-bH), 1.60 (m, 2H, –CH2CH2CH3), 1.37 (s,
9H, t-Bu), 0.95 (t, 3H, J¼7.6 Hz, Me). 13C NMR: d 99.7 and
103.9 (C-1a and C-1b). HRMS: calcd for C24H47N2O15PS2
(MþH)þ; m/z 699.2234, found; 699.2153. MALDI TOF
MS: calcd for C24H46N2O15PS2·Na; m/z 721.21, found;
721.46.
3.1.14. Coupling reaction of 10 and 24. A mixture of 10
(5.9 mg, 11 mmol), 24b (10 mg, 38 mmol), HOSu (17 mg,
148 mmol), AgNO3 (7.5 mg, 44 mmol), and DIEA (7.7 ml,
44 mmol) in dry DMF (2 ml) was stirred overnight in the
dark at room temperature under Ar, and then concentrated in
vacuo. The residue was dissolved in MeOH/H2O (1/1), and
3.1.18. n-Propyl O-[6-O-{2-[(N-acetyl-L-glycyl-L-gluta-
minyl-L-asparaginyl-L-a-aspartyl-L-threonyl-L-seryl-L-
glutaminyl-L-threonyl-L-seryl-L-seryl-L-proryl-L-seryl-
L-glycyl-L-cysteinyl)amido]ethyl phosphonato}-a-D-
mannopyranosyl]-(1!2)-a-D-mannopyranoside
30.

Y. Tanaka et al. / Tetrahedron 59 (2003) 4059–4067
4067
Green, L. G.; Hahn, M. G.; Ince, S. J.; Ley, S. V. Chem. Eur. J.
2000, 6, 172–186, and references cited therein. (b) For the
latest total synthesis, Pekari, K.; Schmidt, R. R. J. Org. Chem.
2003, 68, 1295–1308.
Compound 29 (2.6 mg, 3.6 mmol) was dissolved in
minimum volume of a mixture (1/1) of MeOH/0.1 M
phosphate buffer (pH 8.0). To the solution was added
thioester 27 (5.0 mg, 3.6 mmol), and the mixture was diluted
with the phosphate buffer (70 ml). Then thiophenol (2.4 ml,
23.4 mmol) was added. The mixture was stirred at room
temperature on a vortex mixer for 2 days. Since more than
half of starting 27 had remained as judged by HPLC
analysis, the mixture was allowed to stir with an additional
thiophenol (5.5 ml, 53.6 mmol) for further 8 days. Then the
reaction was quenched by addition of AcOH (50 ml) and the
mixture was centrifuged. The supernatant solution was
submitted to the purification by HPLC on a C18 column
eluted with a gradient of 5–25%/0–20 min of CH3CN in
H2O containing 0.1% TFA, affording 30 (4.3 mg, 62%) in
addition to the recovered 27 (1.0 mg). MALDI TOF MS:
calcd for C69H115N18O40PS·Na; m/z 1921.69, found;
1921.45.
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Acknowledgements
This work was supported by Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports,
Science, and Technology of Japan [Exploratory Reseach
14656048]. The authors also acknowledge Tokai University
for a grant-aid for high-technology research.
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