Chemoenzymatic synthesis of a MUC1 glycopeptide carrying non-natural sialyl TF-β O-glycan

Article abstract of DOI:10.1271/bbb.60244

A MUC1 type of glycopeptide was synthesized by the 9- fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) protocol using benzyl and benzylidene-protected β-D-Gal-(1→3)-β-D-GalNAc- Ser/Thr (TF-β: a stereoisomer of the Thomsen-Friedenreich

Full text of DOI:10.1271/bbb.60244

Biosci. Biotechnol. Biochem., 70 (10), 2515–2522, 2006
Chemoenzymatic Synthesis of a MUC1 Glycopeptide Carrying
Non-Natural Sialyl TF-ꢀ O-Glycan
Eriko TANAKA, Yuko NAKAHARA, Yasuhiro KURODA, Yutaka TAKANO,
y
Naoya KOJIMA, Hironobu HOJO,y and Yoshiaki NAKAHARA
Department of Applied Biochemistry, Institute of Glycotechnology, Tokai University,
Kitakaname 1117, Hiratsuka, Kanagawa 259-1292, Japan
Received May 1, 2006; Accepted June 14, 2006; Online Publication, October 7, 2006
[doi:10.1271/bbb.60244]
A MUC1 type of glycopeptide was synthesized by the
9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide
synthesis (SPPS) protocol using benzyl and benzylidene-
protected ꢀ-D-Gal-(1!3)-ꢀ-D-GalNAc-Ser/Thr (TF-ꢀ:
a stereoisomer of the Thomsen-Friedenreich antigen).
The synthetic glycopeptide was released from the resin
with reagent K, and the resulting benzylated glycopep-
tide was deprotected under conditions of low-acidity
trifluoromethanesulfonic acid (TfOH). The glycopeptide
carrying duplicate non-natural O-glycans was dominant
in the products, but was accompanied by a considerable
amount of the glycopeptide missing one of the O-
glycans. In contrast, the native ꢁ-glycoside was rela-
tively stable under the acidic debenzylation conditions
as shown by a parallel experiment with the glycopeptide
involving ꢁ-D-GalNAc-Ser/Thr linkage. Enzymatic gly-
cosylation with CMP-NeuAc was successful with both
natural and non-natural O-glycans of the synthetic
glycopeptide.
forms of O-glycan such as Tn, sialyl-Tn, and T (TF:
Thomsen-Friedenreich) epitopes are known as the major
tumor-associated carbohydrate antigens. Such an altered
glycoform and the underglycosylated backbone peptide
would provide superior immunogens for specific immu-
notherapy of some cancer patients. However, more
investigations are necessary on the immune system,
since the dominant alterations in mucin are crucially
dependent upon the origin of the cancers. Due to the
difficulty in preparing homogeneous samples from
natural sources, we have studied the synthesis of
glycopeptides representing the partial structure of mucin
or the mucin-like domain of glycoproteins. Our strategy
has involved the synthesis of benzyl-protected glyco-
amino acid building blocks, Fmoc solid-phase synthesis,
acidic debenzylation, and enzymatic modification of the
O-glycans.7–13)
Chemical and enzymatic syntheses can also be used to
remodel the native structure into a modified functional
probe that will unveil information on glycopeptide-
receptor interaction, metabolic stability, or the pharma-
cological potential of a glycopeptide drug. As a part of
our synthetic project on non-natural glycans of glyco-
protein,14,15) reported here is a synthesis of the MUC1
tandem repeat icosapeptide carrying duplicate non-
natural trisaccharides. The synthetic target (1) was
designed to demonstrate consecutive ꢀ-D-Neu5Ac-
(2!3)-ꢁ-D-Gal-(1!3)-ꢁ-D-GalNAc-Thr and the -Ser
motif in the N-terminal region. The hydrophobic N-
Fmoc group was retained for easy purification and
detection by HPLC. Enzymatic sialylation was em-
ployed for the preparation of 1 because of the ready
availability of the sialyltransferase, with particular
interest in seeing if the enzyme would efficiently work
on a non-natural substrate. It is to be noted that the
Key words: core 1 O-glycan; glycopeptide; solid-phase
synthesis; MUC1; sialyltransferase
Mucins are extraordinarily large glycoproteins that
serve as a barrier to protect the epithelial cell surfaces
and participate in cell surface communication through
the attached oligosaccharide moieties. The characteristic
O-glycan clusters that cover the extracellular domain of
mucin proteins enhance interaction with such carbohy-
drate-recognition molecules as lectins. It has recently
been reported that carcinoma cells seemed to get rid of
the intrinsic apoptotic pathway by overexpressing
MUC1 mucin.1) On the other hand, unusual glycosyla-
tion in the site and in the glycoform has been frequently
observed for the carcinoma mucins.2–6) The truncated
y
To whom correspondence should be addressed. Tel: +81-463-50-2075; Fax: +81-463-50-2012; E-mail: yonak@keyaki.cc.u-tokai.ac.jp or
hojo@keyaki.cc.u-tokai.ac.jp
Abbreviations: BSA, bovine serum albumin; CMP-sialic acid, cytidine 50-(5-acetamido-3,5-dideoxy-D-glycero-ꢁ-D-galacto-non-2-ulopyranosy-
lonic acid monophosphate); DIEA, N,N-diisopropylethylamine; DMS, dimethylsulfide; Fmoc, 9-fluorenylmethoxycarbonyl; HBTU, O-benzotriazol-
1-yl-N,N,N0,N0-tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; MS, mass spectrometry; MAb, monoclonal antibody; NMP,
1-methyl-2-pyrolidinone; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; SPPS, solid-phase peptide synthesis; TFA, trifluoroacetic
acid;TF-ꢁ, ꢁ-linked Thomsen-Friedenreich antigen; TfOH, trifluoromethanesulfonic acid; Trt, triphenylmethyl

2516
E. TANAKA et al.
OH
OH
HO
O
HO
O
O
NH2
HN
O
NH
R
O
AcNH
H3C
HO
H3C
OH
CO2H
CH3
O
O
CH3
O
O
O
O
O
O
O
O
O
CH3
O
H
H
H
H
N
H
N
N
N
N
N
N
N
FmocNH
N
N
N
N
N
N
N
H
N
N
N
N
N
CO2H
H
H
H
H
H
H
H
H
O
O
O
CH3
O
O
O
O
CH3
OH
HO
CH3
HO
H3C
NH
AcNH
O
O
R
O
R
OH
GalNAc
O
O
HO
HO
OH
OH
HO
CO2H
CO2H
HO
AcNH
1
β
β
O
O
HO
2
H
OH
HO
HO
AcNH
3
4
α
α
HO
H
Fig. 1. Structures of the Synthetic MUC1 Glycopeptides.
Ph
Ph
O
O
O
O
OBn
O
OBn
O
CO2All
CO2All
NHFmoc
BnO
BnO
a, b
BnO
BnO
O
O
O
O
O
O
N3
AcNH
NHFmoc
OBn
OBn
R
R
5a: R = H
6a: R = H
5b: R = CH3
6b: R = CH3
Ph
O
Ph
O
O
OBn
O
BnO
BnO
O
O
OBn
O
CO2H
c
BnO
BnO
O
O
O
OBn
O
AcNH
R
NHFmoc
AcNH
OBn
R
O
7a: R = H
7b: R = CH3
FmocNH
CO2H
8a: R = H
8b: R = CH3
Scheme 1. Synthesis of Glycoamino Acid Building Blocks 7a and b, and Structures of 8a and b.
Reaction conditions: (a) Zn/AcOH/CH2Cl2, 0.5 h, (b) Ac2O/MeOH/CH2Cl2, 1.5 h, for 6a (71%) and 6b (79%); (c) Pd(Ph3P)4, dimedone,
THF, 2 h, for 7a (79%) and 7b (91%).
corresponding natural ꢀ-trisaccharide has previously
been synthesized by a fully chemical or chemoenzy-
matic method to use as building blocks for the solid-
phase synthesis of sialoglycopeptides by this group7,8)
and by others.16,17) On the other hand, the related
disaccharide, ꢁ-D-Gal-(1!3)-ꢁ-D-GalNAc-Ser/Thr (TF-
ꢁ), had been synthesized by us to use in the epitope
analysis of the colon carcinoma specific MAb, BW494,
exhibiting cross-reactivity with ꢁ-D-Gal-(1!3)-ꢁ-D-
GlcNAc (type 1 chain) and TF-ꢁ.18) In respect of the
MUC1 glycopeptides carrying natural O-glycans, sev-
eral groups have reported their synthetic efforts.19–21)
both of which had been produced as the minor isomer in
the syntheses of native core 1 O-glycans. Reduction of
the azide group with Zn-AcOH produced the amines,
which were subsequently acetylated to give 6a and 6b.
Selective cleavage of the allyl ester was performed with
a Pd(0) catalyst and 5,5-dimethyl-1,3-cyclohexanedione
to afford 7a and 7b.
Solid-phase synthesis of the glycopeptide and depro-
tection
The synthesis of glycopeptide 1 was commenced with
commercial Fmoc-Ala-CLEAR-acid resin 9 (0.1 mmol).
Among the sequence, the C-terminal fifteen amino acids
were assembled by using an automated peptide synthe-
sizer with the Fastmoc program, by which the N-
terminal Fmoc group was removed with 20% piper-
idine/NMP. The Fmoc amino acids were activated with
HBTU/HOBt in NMP before condensation. The side-
Results and Discussion
Synthesis of building blocks 7a and 7b
The disaccharide building blocks were obtained by
transformation of known compounds 5a22) and 5b,23)

Synthesis of a MUC1 Glycopeptide Carrying Non-Natural O-Glycan
2517
Fmoc-Ala-CLEAR-acid resin
9
a, b
Fmoc-Ala-Pro-Asp(OtBu)-Thr(tBu)-Arg(Pmc)-Pro-Ala-Pro-Gly-
Ser(tBu)-Thr(tBu)-Ala-Pro-Pro-Ala-CLEAR-acid resin
10
a, c - h
O-protected Gal
β 1-3GalNAc
β
O-protected Gal 1-3GalNAc
β
Fmoc-His(Trt)-Gly-Val-Thr-Ser-Ala-Pro-Asp(OtBu)-Thr(tBu)-
Arg(Pmc)-Pro-Ala-Pro-Gly-Ser(tBu)-Thr(tBu)-Ala-Pro-Pro-Ala-
CLEAR-acid resin
11: GalNAc
12: GalNAc
α
i, j
Gal β 1-3GalNAc
Gal β 1-3GalNAc
Fmoc-His-Gly-Val-Thr-Ser-Ala-Pro-Asp-Thr-Arg-Pro-Ala-Pro-Gly-
Ser-Thr-Ala-Pro-Pro-Ala-OH
2: GalNAc β
4: GalNAc α
Scheme 2. Solid-Phase Synthesis of the Glycopeptides.
Reaction conditions: (a) 20% piperidine/NMP; (b) Fmoc amino acid, HBTU/HOBt/DMF, DIEA/NMP; (c) 7a, HBTU/HOBt/DMF, DIEA/
NMP, 50 ꢀC, overnight; (d) Ac2O/DIEA/HOBt/NMP; (e) 7b, HBTU/HOBt/DMF, DIEA/NMP, 50 ꢀC, overnight; (f) Fmoc-Val-OH, HBTU/
HOBt/DMF, DIEA/NMP, room temp., 1 h; (g) Fmoc-Glyl-OH, HBTU/HOBt/DMF, DIEA/NMP, room temp., 1 h; (h) Fmoc-His(Trt)-OH,
HBTU/HOBt/DMF, DIEA/NMP, room temp., 1 h; (i) reagent K, room temp., 1 h; (j) DMS/TFA/m-cresol/TfOH, ꢁ15 ꢀC, 2 h, for 2 (17%) and
4 (19%).
chain functional groups of the amino acids were masked
with t-Bu for serine, threonine and aspartic acid, with
Pbf for arginine, and with Trt for histidine. Half of
synthesized pentadecapeptide-bound resin 10 (0.05
mmol) was converted into the glycopeptide. The resin
was N-deprotected with 20% piperidine, and manually
condensed with 7a (2.4 equiv.) at 50 ꢀC for 15 h by
activation with HBTU/HOBt in NMP in a vortex mixer.
Complete condensation was judged by HPLC and MS
analyses of the product detached from the resin sample.
The employed cleavage and deprotective procedures
were same as those used for glycopeptide 2 (see below).
The resin was then treated with 20% piperidine, and
analogously reacted with 7b (2.4 equiv.). Condensation
with the second glycoamino acid proceeded without
difficulty. The peptide chain was successively further
elongated for the N-terminal three amino acids with
Fmoc-Val-OH, Fmoc-Gly-OH, and Fmoc-His(Trt)-OH
by a manual procedure. The glycopeptide thus synthe-
sized was released from resin 11 with simultaneous
cleavage of the acid-labile protecting groups by a
treatment with reagent K (TFA/phenol/water/thioani-
sole/ethanedithiol, 33:2:2:2:1) at room temperature for
1 h, and precipitated with ether. The crude product
was subjected to the previously established ‘‘low-
acidity TfOH’’ conditions (DMS/TFA/m-cresol/TfOH,
3:5:1:1) to remove the retained benzyl groups.9) The
HPLC profile of the resulting crude product is shown in
Fig. 2a. The major products were separated and charac-
terized by a MS analysis. Fraction 1 exhibited the
desired molecular weight for glycopeptide 2 (m=z
2839.96 for M þ Hþ). The yield of 2 was 17%. The
second fraction contained the glycopeptide missing one
of the galactose residues (m=z 2677.85 for M þ Hþ),
while the third fraction (8%) showed m=z 2474.35 which
is compatible with a molecule derived by the loss of
Gal-GalNAc. Scission between GalNAc and Ser/Thr
had never taken place in our previous investigations
for the syntheses of core 1 and core 2 O-glycans. The
structure of the glycopeptide in fraction 3 was further
analyzed by (MS)n spectrometry, after digestion with
endoproteinase Asp-N. The (MS)4 spectrum enabled the
molecular ion for disaccharide-linked Fmoc-His(1)-
Thr(4) to be detected together with the ion for the
Fmoc-tetrapeptide, suggesting that two glycopeptides
missing a disaccharide were included in the fraction. We
then decided to examine the acid stability of the natural
type ꢀ-glycoside on the same peptide, and glycopeptide
4 was synthesized by using building blocks 8a22) and
8b.23) The product was debenzylated under the same
conditions and analyzed by HPLC as shown in Fig. 2b.
The major peak represents desired glycopeptide 4. In

2518
E. TANAKA et al.
Fr. 1 (2)
Fr. 1 (4)
(a)
(b)
Fr. 2
Fr. 2
Fr. 3
45
35
25
45
35
25
15
5
15
5
0
0
10
Retention time (min)
10
Retention time (min)
20
20
30
30
Fig. 2. HPLC Profiles of Synthetic Glycopeptides 2 and 4.
Conditions: column, Mightysil RP-18 (5 mm), 4:6 ꢃ 150 mm; eluent A, distilled water containing 0.1% TFA; eluent B, acetonitrile containing
0.1% TFA; flow rate, 1 ml/min.
contrast with the preparation of 2, no by-product
resulting from the elimination of Gal-GalNAc was
detected. Accordingly, the benzyl-protection and acidic
debenzylation strategy would be useful for the synthesis
of glycopeptides with a natural ꢀ-GalNAc-Ser/Thr
linkage. Both glycopeptides 2 and 4 were readily
separable by preparative HPLC from the accompanying
mono-agalacto derivatives and subjected to the subse-
quent investigations on enzymatic sialylation.
In conclusion, the benzyl-protected disaccharide
building blocks of non-natural TF-ꢁ were introduced
into the N-terminal region of the synthetic MUC1
icosapeptide by the Fmoc solid-phase method. The
conditions for debenzylation with a combination of hard
TfOH and soft nucleophiles gave rise to cleavage of
Gal-GalNAc in part. In contrast, the natural ꢀ-linkage
was relatively stable under the debenzylation conditions,
as was confirmed by a parallel experiment with the
glycopeptide having a natural type of linkage. Both
synthetic glycopeptides carrying natural and non-natural
O-glycans were good substrates for recombinant ꢀ2,3-
(O)-sialyltransferase to afford disialoglycopeptides 1
and 3. The synthetic glycopeptides carrying natural and
non-natural O-glycan will be useful probes in the study
of mucin-mediated biological interactions, especially
with the endo-ꢀ-N-acetylgalactosaminidase-producing
pathogenic and non-pathogenic microbes. Cell surface
biology involving a glycosphingolipid will be another
candidate for the synthetic probes, since ꢀ-D-Neu5Ac-
(2!3)-ꢁ-D-Gal-(1!3)-ꢁ-D-GalNAc is the common
peripheral oligosaccharide found in the ganglio and
globo series of glycosphingolipids.
Sialylation of the synthetic glycopeptides
Sialylation of 2 was undertaken with commercially
available recombinant rat ꢀ2,3-(O)-sialyltransferase and
CMP-sialic acid (5 equiv.). Incubation in a cacodylate
buffer (pH 6.0) at 37 ꢀC with the addition of BSA
enabled transformation of 2 to proceed within 2 h to
afford desired disialoglycopeptide 1. HPLC and MS
analyses supported the successful transformation. Using
an ammonium acetate buffer-acetonitrile as the eluent,
the intermediary mono-sialo derivatives were separated
in the less-mobile fraction. Under the same conditions,
the conversion of glycopeptide 4 was more efficient to
exclusively give disialoglycopeptide 3 as expected. The
results of sialylation are shown in Fig. 3. It is important
to note that the enzymatic reaction was sufficiently
effective to transform non-natural TF-ꢁ as well as
natural TF-ꢀ into the corresponding ꢀ-(2!3)-sialylated
O-glycan at the glycopeptide level. Based on the slight
looseness in substrate specificity of the enzyme, we were
interested in the behavior of the stereoisomeric core 8 O-
glycans. However, the glycans of ꢀ-D-Gal-(1!3)-ꢀ-D-
GalNAc-Thr and -Ser on a synthetic glycopeptide12)
were entirely inactive to the enzymatic reaction.
Experimental
Optical rotation values were determined with a
Jasco DIP-370 polarimeter at 20 ꢂ 2 ꢀC for solutions
in CHCl3, unless noted otherwise. Column chromatog-
raphy was performed on silica gel PSQ 100B (Fuji
Silysia), while TLC and HPTLC were performed on
silica gel 60 F254 (E. Merck). 1H- and 13C-NMR spectra
were recorded with a Jeol AL400 spectrometer (1H at

Synthesis of a MUC1 Glycopeptide Carrying Non-Natural O-Glycan
2519
(a)
(b)
Fr. 1 (1)
3
Fr. 2
45
35
25
45
35
25
15
5
15
5
0
0
10
Retention time (min)
20
10
Retention time (min)
20
30
30
Fig. 3. HPLC Profiles of Synthetic Sialoglycopeptides 1 and 3.
Conditions: column; Mightysil RP-18 (5 mm), 4:6 ꢃ 150 mm. eluent A, ammonium acetate buffer (pH 5.8); eluent B, acetonitrile; flow rate,
1 ml/min.
400 MHz and 13C at 100 MHz). Chemical shifts are
concentrated with toluene in vacuo. The residue dis-
solved in a mixture of MeOH (1.4 ml) and CH2Cl2
(0.7 ml) was stirred with Ac2O (62 ml, 0.61 mmol) for
1.5 h. The mixture was concentrated in vacuo to a
residue which was chromatographed on silica gel with
CHCl3–EtOAc (1:1) to afford 6a (54 mg, 71%). ½ꢀꢄD ¼
expressed in ppm downfield from the signal for internal
Me4Si for solutions in CDCl3. In respect of the NMR
data, a and b are respectively used to represent GalNAc
and Gal residue. MALDI TOF mass spectra were
obtained with a PerSeptive Voyager-DE PRO spectrom-
eter (2,5-dihydroxybenzoic acid was used as a matrix).
Tandem mass spectra were measured with a Kratos
AXIMA-QIT spectrometer (2,5-dihydroxybenzoic acid
was used as a matrix). Automated solid-phase peptide
synthesis was performed with an Applied Biosystems
433A peptide synthesizer. Manual solid-phase reactions
were undertaken in capped polypropylene test tubes
equipped with a filter and three-way stopcock by stirring
on an EYELA CM-1000 vortex mixer. HPLC was
performed in Mightysil RP-8 and RP-18 columns
(150 ꢃ 4:6 mm for analysis and 250 ꢃ 10 mm for prep-
aration, Kanto Chemical Co.). Amino acids were
analyzed by a Hitachi L-8500 amino acid analyzer.
The Fmoc-Ala-CLEAR-acid resin was purchased from
Peptide International and ꢀ2,3-(O)-Sialyltransferase, rat,
recombinant was purchased from Calbiochem. The
yields of sialylated glycopeptides were determined by
an amino acid analysis after each sample had been
hydrolyzed in a sealed tube with 20% HCl and 0.5%
phenol at 150 ꢀC for 2 h.
1
þ27:7ꢀ (c 1). Rf 0.40 (1:1 toluene–EtOAc). H-NMR
(DMSO-d6) ꢂ: 7.88 (brd, 2H, J ¼ 7:6 Hz, Ar), 7.77 (d,
1H, J ¼ 9:0 Hz, GalNAc-NH), 7.71 (m, 2H, Ar), 7.43–
7.21 (m, 30H, Ser-NH, Ar), 5.88 (m, 1H, –CH=CH2),
5.50 [s, 1H, PhCH(O–)2], 5.30 (dd, 1H, J ¼ 1:7,
17.3 Hz, –CH=CH2), 5.17 (dd, 1H, J ¼ 1:4, 10.5 Hz,
–CH=CH2), 4.77 (d, 2H, J ¼ 12:2 Hz, –CH2Ph), 4.74
(d, 1H, J ¼ 12:0 Hz, –CH2Ph), 4.65 (d, 1H, J ¼ 12:0
Hz, –CH2Ph), 4.60–4.45 (m, 8H, –CH2CH=CH2,
–CH2Ph, H-1a, H-1b), 1.55 (s, 3H, Ac). 13C-NMR
(DMSO-d6) ꢂ: 99.7 (C-1a), 100.7 [PhCH(O)2], 104.5
(C-1b). MALDI TOF MS m=z (Mþ þ Na): calcd. for
þ
.
C70H72N2O15 Na, 1203.48; found, 1203.99; m=z (M þ
.
K): calcd. for C70H72N2O15 K, 1219.46; found, 1219.99.
Anal. Found: C, 70.99; H, 6.19; N, 2.35%. Calcd. for
C70H72N2O15: C, 71.17; H, 6.14; N, 2.37%.
N-(9-Fluorenylmethoxycarbonyl)-O-[2,3,4,6-tetra-O-
benzyl-ꢁ-D-galactopyranosyl-(1!3)-2-acetamido-4,6-
O-benzylidene-2-deoxy-ꢁ-D-galactopyranosyl]-L-threo-
nine allyl ester (6b). Azide 5b (216 mg, 0.18 mmol) was
reduced with Zn and AcOH, and then treated with Ac2O
as described for 6a. The crude product was purified by
chromatography on silica gel with toluene–EtOAc (1:1)
to give 6b (170 mg, 79%). ½ꢀꢄD ¼ þ17:3ꢀ (c 1). Rf 0.36
(1:1 toluene–EtOAc).1H-NMR (DMSO-d6) ꢂ: 7.93 (d,
2H, J ¼ 7:6 Hz, Ar), 7.86 (d, 1H, J ¼ 8:8 Hz, GalNAc-
NH), 7.79 (brd, 2H, J ¼ 6:5 Hz, Ar), 7.49–7.26 (m, 29H,
N-(9-Fluorenylmethoxycarbonyl)-O-[2,3,4,6-tetra-O-
benzyl-ꢁ-D-galactopyranosyl-(1!3)-2-acetamido-4,6-
O-benzylidene-2-deoxy-ꢁ-D-galactopyranosyl]-L-serine
allyl ester (6a). A mixture of 5a (70 mg, 60 mmol),
AcOH (50 ml, 0.83 mmol) and powdered Zn (248 mg,
3.79 mmol) in CH2Cl2 (1.5 ml) was stirred for 0.5 h. The
mixture was filtered through Celite, and the filtrate was

2520
E. TANAKA et al.
Ar), 6.71 (d, 1H, J ¼ 8:8 Hz, Thr-NH), 5.95 (m, 1H,
–CH=CH2,), 5.55 [s, 1H, PhCH(O–)2], 5.38 (dd, 1H,
J ¼ 1:5, 17.3 Hz, –CH=CH2), 5.20 (dd, 1H, J ¼ 1:5,
10.5 Hz, –CH=CH2), 4.80 (d, 1H, J ¼ 12:2 Hz,
–CH2Ph), 4.76 (d, 1H, J ¼ 10:7 Hz, –CH2Ph), 4.74 (d,
1H, J ¼ 11:3 Hz, –CH2Ph), 4.65 (d, 1H, J ¼ 12:2 Hz,
–CH2Ph), 4.63–4.45 (m, 8H, H-1b, H-1a, –CH2CH=
CH2, –CH2Ph), 1.69 (s, 3H, Ac), 1.18 (d, 3H, J ¼
6:1 Hz, Thr-ꢃH). 13C-NMR (DMSO-d6) ꢂ: 99.0 (C-1a),
99.7 [PhCH(O)2], 104.5 (C-1b). MALDI TOF MS
1H, J ¼ 10:7 Hz, –CH2Ph), 4.46 (brs, 2H, –CH2Ph),
1.63 (s, 3H, Ac), 1.13 (d, 3H, J ¼ 6:4 Hz, Thr-ꢃH). 13C-
NMR (DMSO-d6) ꢂ: 99.3 (C-1a), 99.6 [PhCH(O)2],
104.4 (C-1b). Anal. Found: C, 69.72; H, 5.95; N, 2.17%.
.
Calcd. for C68H70N2O15 H2O: C, 69.61; H, 6.19; N,
2.39%.
N-(9-Fluorenylmethoxycarbonyl)-L-histidyl-L-glycyl-
L-valyl-O-[ꢁ-D-galactopyranosyl-(1!3)-2-acetamido-
2-deoxy-ꢁ-D-galactopyranosyl]-L-threonyl-O-[ꢁ-D-galac-
topyranosyl-(1!3)-2-acetamido-2-deoxy-ꢁ-D-galacto-
pyranosyl]-L-seryl-L-alanyl-L-prolyl-L-aspartyl-L-threo-
nyl-L-arginyl-L-prolyl-L-alanyl-L-prolyl-L-glycyl-L-seryl-
L-threonyl-L-alanyl-L-prolyl-L-prolyl-L-alanine (2). Com-
mercial Fmoc-Ala-CLEAR-acid resin (270 mg, 0.1
mmol) was subjected to an automated synthesis of the
peptide to produce a pentadecapeptide (APDTRPAPG-
STAPPA)-resin by the Fastmoc program of the synthe-
sizer, using HBTU/HOBt as the condensing agent and
20% piperidine/NMP for N-deprotection. Half of the
peptide resin (0.05 mmol) was transferred into a poly-
propylene test tube, N-deprotected by stirring on a vortex
mixer with 20% piperidine/NMP (2.5 ml) for 10 min,
and then thoroughly washed with NMP. This procedure
was conducted ten times to complete the N-deprotection.
Glycoamino acid 7a (137 mg, 0.12mmol) was pre-
activated with 0.45 M HBTU/HOBt/DMF (267 ml, 0.12
mmol) and 2 M DIEA/NMP (72 ml, 0.14 mmol) at room
temperature for 10 min, and added to the reaction vessel
of the N-deprotected peptide resin with NMP (0.5 ml).
The mixture was stirred overnight on the vortex mixer at
50 ꢀC in an oven. After filtration of the soluble reactants,
the resin was washed with NMP and treated with 0.5M
Ac2O/0.125 M DIEA/0.015 M HOBt/NMP (2 ml) at
room temperature for 3 h to acetylate the unreacted
amino moiety. The resin was washed with NMP and
N-deprotected with 20% piperidine/NMP as already
mentioned. Glycoamino acid 7b (139 mg, 0.12 mmol)
was then condensed in a similar manner. The remaining
N-terminal three amino acid residues were manually
attached. Condensation was performed at room temper-
ature for 1 h with the same activating agents, using
Fmoc-Val-OH (68 mg, 0.2mmol), Fmoc-Gly-OH (59
mg, 0.2 mmol), and Fmoc-His(Trt)-OH (124 mg, 0.2
mmol), respectively. After completing the peptide
elongation process, the resin was successively washed
with NMP and CH2Cl2, and dried in vacuo to afford 9
(260 mg). Experiments to isolate the synthetic glycopep-
tide were undertaken with part of resin 9 (50 mg). To the
resin was added reagent K (TFA/phenol/water/thio-
anisole/ethanedithiol, 33:2:2:2:1, 1 ml), and the mixture
was stirred with the vortex mixer at room temperature for
1 h. The resin was filtered off and washed with CH2Cl2.
The volatile materials in the combined filtrate and
washings were evaporated in vacuo. Ether was added
to the residue to precipitate the product, which was
separated by centrifugation. The precipitate was washed
several times by suspending in ether and then centrifug-
m=z (Mþ þ Na): calcd. for C71H74N2O15 Na, 1217.50;
.
found, 1217.46; m=z (Mþ þ K): calcd. for C71H74N2O15
.
K, 1233.47; found, 1233.42. Anal. Found: C, 71.06; H,
6.31; N, 2.30%. Calcd. for C71H74N2O15: C, 71.34; H,
6.24; N, 2.34%.
N-(9-Fluorenylmethoxycarbonyl)-O-[2,3,4,6-tetra-O-
benzyl-ꢁ-D-galactopyranosyl-(1!3)-2-acetamido-4,6-
O-benzylidene-2-deoxy-ꢁ-D-galactopyranosyl]-L-serine
(7a). A mixture of 6a (254 mg, 0.21 mmol), Pd(Ph3P)4
(36 mg, 28 mmol), and 5,5-dimethyl-1,3-cyclohexane-
dione (883 mg, 6.32 mmol) in freshly distilled THF
(30 ml) was stirred at room temperature for 2 h under Ar.
The mixture was concentrated in vacuo, and the
resulting residue was chromatographed on silica gel
with CHCl3–MeOH–AcOH (20:1:0.2). The product was
further purified in a column of Sephadex LH-60 with
CHCl3–MeOH (3:2) to afford 7a (189 mg, 79%). ½ꢀꢄD ¼
þ25:4ꢀ (c 1). Rf 0.24 (20:1:0.2 CHCl3–MeOH–AcOH).
1H-NMR (DMSO-d6) ꢂ: 7.88 (brd, 2H, J ¼ 7:6 Hz, Ar),
7.78 (d, 1H, J ¼ 7:8 Hz, GalNAc-NH), 7.72 (brt, 2H,
J ¼ 7:1 Hz, Ar), 7.42–7.20 (m, 30H, Ser-NH, Ar), 5.49
[s, 1H, PhCH(O–)2], 4.78–4.72 (m, 3H, –CH2Ph), 4.65
(d, 1H, J ¼ 12:0 Hz, –CH2Ph), 4.56 (d, 1H, J ¼ 7:6 Hz,
H-1b), 4.52–4.45 (m, 5H, H-1a, –CH2Ph), 1.54 (s, 3H,
Ac). 13C-NMR (DMSO-d6) ꢂ: 99.8 (C-1a), 100.8
[PhCH(O)2], 104.5 (C-1b). Anal. Found: C, 70.01; H,
.
6.03; N, 2.31%. Calcd. for C67H68N2O15 0.5H2O: C,
69.96; H, 6.04; N, 2.43%.
N-(9-Fluorenylmethoxycarbonyl)-O-[2,3,4,6-tetra-O-
benzyl-ꢁ-D-galactopyranosyl-(1!3)-2-acetamido-4,6-
O-benzylidene-2-deoxy-ꢁ-D-galactopyranosyl]-L-threo-
nine (7b). Compound 6b (159 mg, 0.13 mmol) was
deallylated with Pd(Ph3P)4 (15 mg, 13 mmol) and 5,5-
dimethyl-1,3-cyclohexanedione (373 mg, 2.67 mmol) in
THF (13 ml) as described for 7a. Chromatography of
the crude product afforded 7b (136 mg, 91%). ½ꢀꢄD ¼
þ14:5ꢀ (c 1). Rf 0.34 (20:1:0.2 CHCl3–MeOH–AcOH).
1H-NMR (DMSO-d6) ꢂ: 12.8 (br, 1H, –CO2H), 7.88 (d,
2H, J ¼ 7:6 Hz, Ar), 7.83 (d, 1H, J ¼ 9:0 Hz, GalNAc-
NH), 7.75 (brt, J ¼ 8:0 Hz, Ar), 7.44–7.21 (m, 29H, Ar),
6.42 (d, 1H, J ¼ 8:8 Hz, Thr-NH), 5.50 [s, 1H,
PhCH(O–)2], 4.82 (d, 1H, J ¼ 12:2 Hz, –CH2Ph), 4.78
(d, 1H, J ¼ 10:7 Hz, –CH2Ph), 4.75 (d, 1H, J ¼ 11:2
Hz, –CH2Ph), 4.66 (d, 1H, J ¼ 12:0 Hz, –CH2Ph), 4.59
(d, J ¼ 7:6 Hz, H-1b), 4.55 (d, 1H, J ¼ 12:2 Hz,
–CH2Ph), 4.51 (d, 1H, J ¼ 8:6 Hz, –CH2Ph), 4.77 (d,

Synthesis of a MUC1 Glycopeptide Carrying Non-Natural O-Glycan
2521
ing to give a crude product (20 mg) which was dissolved
in a mixture of TFA/DMS/m-cresol (5:3:1, 250 ml) and
cooled at ꢁ15 ꢀC. To the stirried mixture was added
TfOH (28 ml). The reaction was continued for 2 h, before
being terminated by the addition of ether. The mixture
was centrifuged to separate the debenzylated product,
which was washed five times with ether and centrifuged
as already mentioned to give a precipitate (7.0 mg). The
precipitate was dissolved in 10% CH3CN/H2O contain-
ing 0.1% TFA and purified by preparative HPLC. The
major fraction was collected and lyophilized to afford
glycopeptide 2 (3.4 mg, 17% overall yield). MALDI-
TOF MS m=z (Mþ þ H): calcd. for C123H184N27O50,
2839.27; found 2839.96. The second fraction was only
separated and analyzed: m=z (Mþ þ H): calcd. for
supernatant was subjected to reversed-phase HPLC on
a column of C-18 silica gel with gradient elution by an
acetonitrile/ammonium acetate buffer (pH 5.8). The
fraction containing glycopeptide 1 was collected, and
part of the sample was used in an amino acid analysis
to estimate the yield (0.13 mg, 37%). MALDI-TOF MS
m=z (Mþ þ H): calcd. for C145H218N29O66, 3421.46;
found, 3420.90. The second fraction was collected and
analyzed by MS. MALDI-TOF MS m=z (Mþ þ H):
calcd. for C134H201N28O58, 3130.36; found, 3130.95.
N-(9-Fluorenylmethoxycarbonyl)-L-histidyl-L-glycyl-
L-valyl-O-[(5-acetamido-3,5-dideoxy-D-glycero-ꢀ-D-ga-
lacto-2-nonulopyranosylonic acid)-(2!3)-ꢁ-D-galacto-
pyranosyl-(1!3)-2-acetamido-2-deoxy-ꢀ-D-galactopy-
ranosyl]-L-threonyl-O-[(5-acetamido-3,5-dideoxy-D-gly-
cero-ꢀ-D-galacto-2-nonulopyranosylonic acid)-(2!3)-
ꢁ-D-galactopyranosyl-(1!3)-2-acetamido-2-deoxy-ꢀ-D-
galactopyranosyl]-L-seryl-L-alanyl-L-prolyl-L-aspartyl-
L-threonyl-L-arginyl-L-prolyl-L-alanyl-L-prolyl-L-glycyl-
L-seryl-L-threonyl-L-alanyl-L-prolyl-L-prolyl-L-alanine
(3). Glycopeptide 4 (0.28 mg, 100 nmol) was incubated
with ꢀ2,3-(O)-sialyltransferase and CMP-sialic acid as
described for the synthesis of 1. The product was
separated by HPLC to give 3 (0.22 mg, 65%). MALDI-
TOF MS m=z (Mþ þ H): calcd. for C145H218N29O66,
3421.46; found, 3421.37.
C117H174N27O45, 2677.22; found 2677.07. The third
fraction gave the disaccharide-missing glycopeptide
(1.8 mg, 8%), m=z (Mþ þ H): calcd. for C109H161N26O40,
2474.14; found, 2474.31.
N-(9-Fluorenylmethoxycarbonyl)-L-histidyl-L-glycyl-
L-valyl-O-[ꢁ-D-galactopyranosyl-(1!3)-2-acetamido-2-
deoxy-ꢀ-D-galactopyranosyl]-L-threonyl-O-[ꢁ-D-galac-
topyranosyl-(1!3)-2-acetamido-2-deoxy-ꢀ-D-galacto-
pyranosyl]-L-seryl-L-alanyl-L-prolyl-L-aspartyl-L-threo-
nyl-L-arginyl-L-prolyl-L-alanyl-L-prolyl-L-glycyl-L-seryl-
L-threonyl-L-alanyl-L-prolyl-L-prolyl-L-alanine (4). The
natural type of glycopeptide was synthesized in a solid-
phase by using 10 (0.025 mmol), 8a (68 mg, 0.06 mmol),
and 8b (69 mg, 0.06 mmol) as described for the synthesis
of 2. The crude product was purified by preparative
HPLC to afford 4 (3.5 mg, 19%). MALDI-TOF MS m=z
(Mþ þ H): calcd. for C123H184N27O50, 2839.27; found
2839.35. The subsequent fraction was also collected
and analyzed by MS, m=z (Mþ þ H): calcd. for
Acknowledgments
This work was supported by grant-aid for creative
scientific research (17GS0420) from Japan Society for
the Promotion of Science and in part by NEDO (New
Energy and Industrial Technology Development Organ-
ization, Japan). We thank Yamasa Corporation for
providing CMP-sialic acid, and Dr. T. Chihara and his
staff at Riken for the combustion analyses. We also
thank Tokai University for support by grant-aid for high-
technology research.
C
117H174N27O45, 2677.22; found 2677.43.
N-(9-Fluorenylmethoxycarbonyl)-L-histidyl-L-glycyl-L-
valyl-O-[(5-acetamido-3,5-dideoxy-D-glycero-ꢀ-D-galac-
to-2-nonulopyranosylonic acid)-(2!3)-ꢁ-D-galactopy-
ranosyl-(1!3)-2-acetamido-2-deoxy-ꢁ-D-galactopyra-
nosyl]-L-threonyl-O-[(5-acetamido-3,5-dideoxy-D-glyc-
ero-ꢀ-D-galacto-2-nonulopyranosylonic acid)-(2!3)-ꢁ-
D-galactopyranosyl-(1!3)-2-acetamido-2-deoxy-ꢁ-D-
galactopyranosyl]-L-seryl-L-alanyl-L-prolyl-L-aspartyl-
L-threonyl-L-arginyl-L-prolyl-L-alanyl-L-prolyl-L-glycyl-
L-seryl-L-threonyl-L-alanyl-L-prolyl-L-prolyl-L-alanine
(1). To an ice-cooled polypropylene micro-tube con-
taining synthetic glycopeptide 2 (0.28 mg, 100 nmol)
were added BSA (10 ml; 5 mg/ml), 50 mM cacodylate
buffer (78 ml, pH 6.0), 0.1 M CMP-sialic acid (10 ml, 1
mmol), and ꢀ2,3-(O)-sialyltransferase (2 ml, 5.4 mU;
875 mU/ml). The mixture was incubated while mechan-
ically shaking at 37 ꢀC for 2 h, before the reaction was
quenched with 10% acetonitrile/water containing 0.1%
TFA (100 ml). A 9% acetonitrile/ammonium acetate
buffer (100 ml) was then added to the mixture, which was
centrifuged at 12000 rpm for 5 min. The resulting
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